Olivier

The ethnobotany and chemistry of South African traditional
tonic plants
by
Denise Karola Olivier
THESIS
submitted in fulfillment of the requirements for the degree
PHILOSOPHIAE DOCTOR
in
BOTANY
in the
FACULTY OF SCIENCE
at the
UNIVERSITY OF JOHANNESBURG
Supervisor
: Prof. B.-E. van Wyk
Co-supervisors : Prof. F. R. van Heerden
Dr. C. F. Albrecht
APRIL 2012
TABLE OF CONTENTS
Section
Page
Table of contents
ii
Abstract
vii
CHAPTER 1: Introduction and hypothesis
CHAPTER 2: The concept of herbs as tonics
2.1
Tonics and related concepts (definitions)
2.1.1
Tonics
2.1.2
Adaptogens
2.1.3
Bitter tonics
2.1.4
Alteratives
2.1.5
Adjuvants
2.1.6
Stimulants, immunostimulants and immunomodulators
2.2
10
Tonics in traditional healing cultures
10
2.2.1
11
Europe and Asia

2.2.1.1 Islamic medicine (North African and Middle Eastern traditional
medicine)
11
2.2.1.2 Ancient Jewish medicine
12
2.2.1.3 European medicine
13
2.2.1.4 Northern Eurasian medicine (Slavic countries and Russia)
15
2.2.1.5 Southern and South-eastern Asian traditional medicine
17
2.2.1.6 East Asian traditional medicine
20
Oceania and the Americas
24
2.2.2.1 Bush medicine (Australian traditional medicine)
24
2.2.2.2 Rongo Mori (New Zealand traditional medicine)
25
2.2.2.3 North American Indian and eclectic medicine
26
2.2.2.4 Meso and South American traditional medicine
26
Africa
28
2.2.3.1 Islamic medicine (North Africa)
37
2.2.3.2 Yorb medicine
38
2.2.3.3 Bantu traditional medicine
38
2.2.3.4 Khoi-San and Cape Dutch medicine
41
2.3
The concept tonic in the southern African context
42
2.4
Conclusions
43
2.2.2
2.2.3
ii
CHAPTER 3: Materials and methods
45
3.1
Introduction
45
3.2
Plant materials
45
3.3
Screening protocol for classes of phytochemicals
46
3.3.1
Qualitative colour tests
46
3.3.2
Alkaloids
58
3.3.3
Water extractable compounds
59
3.3.3.1 Alcohol precipitable solids
60
3.3.3.2 Amino acids
60
3.3.3.3 Sugars
60
3.3.3.4 Phenolic glycosides
61
3.3.3.5 Triterpenoids and steroids
62
Organic extractable compounds
63
3.3.4.1 Mono-, sesqui- and diterpenes
63
3.3.4.2 Flavonoid aglycones (Agathosma species)
63
3.3.4
3.3.5
3.4
Cyanogenic glycosides
64
Quantitative Analytical Chromatography
65
3.4.1
Liquid chromatography
65
3.4.1.1 High performance liquid chromatography (HPLC)
65
3.4.1.2 Liquid chromatography-mass spectrometry (LC-MS)
67
High resolution gas chromatography-mass spectrometry (HR-GC-MS)
67
3.4.2.1 Amino acids
67
3.4.2
3.4.2.2 GC-MS analyses of Arctopus kaurenes (performed by Witte

& De Castro, unpublished data)
3.5
3.6
3.7
73
Isolation of compounds
74
3.5.1
Column chromatography (CC)
74
3.5.2
Preparative thin layer chromatography
75
Structure elucidation
76
3.6.1
Nuclear magnetic resonance spectroscopy (NMR)
76
3.6.2
Mass spectrometry (MS)
76
3.6.3
Optical rotation
77
Bitterness taste testing
78
iii
CHAPTER 4: Ethnobotany
82
4.1
Introduction
82
4.2
The ethnobotany of South African traditional tonic plants
83
4.2.1 Agathosma betulina
83
4.2.2 Aloe species
86
4.2.3 Arctopus species
91
4.2.4 Artemisia afra
93
4.2.5 Balanites maughamii
95
4.2.6 Dicoma species
97
4.2.7 Harpagophytum procumbens
100
4.2.8 Hypoxis hemerocallidea
103
4.2.9 Muraltia heisteria
104
4.2.10 Sutherlandia frutescens
105
4.2.11 Vernonia oligocephala
107
4.2.12 Warburgia salutaris
109
4.2.13 Withania somnifera
111
4.2.14 Ziziphus mucronata
113
Conclusions
114
CHAPTER 5: Chemistry
116
5.1
Introduction
116
5.2
The chemistry of South African traditional tonic plants
117
5.2.1 Agathosma species
117
5.2.2 Aloe species
126
132
142
146
149
5.2.7 Harpagophytum species
155
160
163
5.2.10 Sutherlandia species
166
171
4.3
iv
CHAPTER 5: Chemistry (continued)

176
179
183
5.3
Bitterness value
187
5.4
Ionic fluids
189
5.5
Conclusions
190
CHAPTER 6: Pharmacology
194
6.1
Introduction
194
6.1.1
Classes of compounds and their biological activities
194
6.1.2
Synergism
198
6.1.3
Pharmacological and physiological effects of tonics
200
6.1.3.1 Stress and adaptogens
201
6.1.3.2 Immunity and bitter tonics
206
6.1.3.3 The placebo effect
209
Plant use in traditional healing and drug development
210
6.1.4
6.2
6.3
Biological activities of South African traditional tonic plants
211
211
6.2.2 Aloe species
215
217
218
221
222
223
226
229
229
233
234
236
240
Conclusions
243
v
CHAPTER 7: Conclusions
249
7.1
General conclusions
249
7.2
Summary of main findings
252
7.3
Implications of findings
253
References
255
Acknowledgments
312
Appendixes
Appendix 1:
Bitterness taste testing Application to ethics committee (1A), and

Informed Consent form completed by willing participants (1B)
Appendix 2:
Table of ethnobotanical anecdotes and uses (cited verbatim from the

referenced texts) for the 14 main traditional tonic plants in southern
Africa, in alphabetical order (2A-2M)
Appendix 3:
HPLC chromatograms (Agathosma species) and quantification data

(Sutherlandia species), to confirm TLC chemical variation
Appendix 4:
NMR data for compounds isolated from Arctopus
Appendix 5:
Scientific papers resulting from this study
vi
Abstract
The most well-known tonic plants in South Africa have been used traditionally for the
treatment of a great variety of ailments but aspects of their ethnobotany and chemistry
remain poorly studied. Possible relationships between their ethnobotany and pharmacology
are mostly speculative. In this study, literature reviews of the ethnobotany of these plants
were combined with phytochemical screening studies and bitterness taste testing results in
order to establish constituent patterns which may contribute to a scientific rationale for the
claimed tonic (stimulating) properties of these plants.
The tonic concept and definitions of terms associated with it are often used incorrectly and
ambiguously. An analysis of literature on the traditional healing systems across the globe was
used to establish the historical and cultural aspects relevant to tonics. This analysis revealed
that sickness/illness is usually considered to be a result of imbalance in many cultures,
whether this imbalance is between the patient and the environment or due to a lack of
homeostasis in the body. In several healing cultures substances or mixtures of substances
are used to rectify these imbalances through proposed effects on several bodily systems
concurrently. According to some cultures, as in Eastern and Indian traditional medicine, tonic
plants are considered superior to other medicinal plants in that they impart health, strength
and a general sense of well-being, as well as being prophylactic. This definition of a tonic
plant is consequently broad, but excludes plants merely used as multipurpose medicines.
Where these tonics exhibit a specific mode of action, further classification is required, i.e. as
bitter, adaptogenic, alterative, adjuvant or stimulant tonics.
The South African traditional tonic plants studied were Agathosma species (Rutaceae), Aloe
species (Asphodelaceae), Arctopus species (Apiaceae), Artemisia afra (Asteraceae),
Balanites
maughamii
(Balanitacae),
Dicoma
species
(Asteraceae),
Harpagophytum
procumbens (Pedaliaceae), Hypoxis hemerocallidea (Hypoxidaceae), Muraltia heisteria

(Polygalaceae), Sutherlandia species (Fabaceae), Vernonia oligocephala (Asteraceae),
Warburgia salutaris (Canellaceae), Withania somnifera (Solanaceae) and Ziziphus
mucronata (Rhamnaceae). A detailed compendium of medicinal applications was compiled
following a thorough, in-depth scrutiny of the historical and medicinal ethnobotany of each of
these species. Such ethnobotanical data is important in understanding the cultural aspects of
healing in southern Africa, and provides valuable direction and focus with regards to the
phytochemical and pharmacological research of these plants. Ethnobotanical information
vii
may provide, for example, insight into aspects such as genetic, seasonal or geographical
variation (based on harvesting techniques and reported efficacy), possible classes of
contained compounds, in which plant part the active compounds accumulate and the
pharmacological properties responsible for their claimed efficacy (e.g. the most bitter
compounds in Aloe species are responsible for its laxative properties and are obtained from
the leaf exudate), as well as possible synergistic interactions between species combined in
prescribed preparations.
All the tonic plants studied were found to be chemically complex and therefore contained
compounds from several classes including alcohol precipitable solids (APS), terpenes
(monoterpenes, diterpenes, sesquiterpenes, iridoids, triterpenes and saponins), phenolic
compounds (anthrones, chromones, phenolic acids, flavonoids, coumarins), amino acids,
cyanogenic glycosides and alkaloids. These were studied using analytical methods for
variation studies including TLC, GC-MS, HPLC (PDA) and LC-MS. In cases where the major
compounds were unknown (i.e. Sutherlandia and Arctopus), column chromatography was
used for isolation, followed by 1D and 2D NMR spectroscopic analyses for structure
elucidation. HR-LC-MS and optical rotation was used for complete characterization of the
identified structures. Two publications resulted from this research: Three triterpenoid
saponins (SU1, SU2 and SU3) were identified from Sutherlandia and the structure of SU3
was published as novel. From Arctopus, three diterpenes and three phenolic acids were
identified for the first time. The structures of the phenolic acids were published. As all the
studied plants were reported to be bitter and/or have stomachic properties as shown in the
ethnobotanical literature, and contained bitter principles such as sesquiterpene lactones,
anthrones, flavonoids and/or alkaloids, a taste testing experiment was conducted to
determine bitterness values. This type of study on tonic plants is the first of its kind in South
Africa. All the plants were found to be bitter, providing scientific grounding for these plants
exerting an amarum effect. Furthermore, the discovery of ionic fluids shed new light on the
importance of simple molecules such as small sugars, amino acids and organic acids in the
solubility of substances of medium polarity normally not soluble in water or lipids.
A literature review on the pharmacological properties of the listed tonics revealed that,
possibly due to the chemical complexity of these plants, they elicited several biological
effects. The compounds seem to act on a variety of receptors simultaneously, and are
probably able to impart gentle, tonic and healing effects. Tonics are not considered harmful to
the body as the concentrations of the active principles in prescribed doses are low, and in
many instances, synergistic interaction between these compounds may result in a stronger
effect than what would be the observed with an isolated compound. Furthermore, it was
viii
found that the plants exerting the highest bitterness values were also those used traditionally
for stress related ailments. To this effect, the musa-pelo concept (tonics which impart
strength and are able to turn the heart around) was addressed. Although chemical and
pharmacological testing is important, it became clear that a reliable scientific rationale for the
determination of tonic properties of the studied plants (especially properties such as
adaptogenic and immunostimulant activities) may only be found through placebo-controlled
double-blinded clinical trials.
In conclusion, the tonic concept, as it is understood in the healing cultures of southern Africa,
is shared by healing systems globally, in the sense that healing and health is brought about
by rectifying imbalances internally and with the environment. Tonics may be defined as a
substance or mixtures of substances that are able to impart healing and health by aiding in
the re-establishment of homeostasis and improving strength in a gentle, non-toxic way. To
this effect the South African tonics, i.e. those used as general strengthening mixtures or
adaptogens (known as imbizas and musa-pelo), were found to be chemically complex,
resulting in many possible biological activities contributing simultaneously to improve general
health and instill a feeling of well-being and vigour.
ix
CHAPTER 1
Introduction and hypothesis
Medicinal plants are traditionally used in various healing cultures around the world to improve
general health and wellbeing (Schulz et al., 2001; Van Wyk & Wink, 2004). Herbs used for this
purpose are rather vaguely referred to as tonics, bitter tonics, adaptogens, alteratives,
roborants, adjuvants, stimulants, immune stimulants and strengthening mixtures. These terms
are often used interchangeably and imprecisely. Tonic herbs are considered to support and
tone either specific organs or the whole body by strengthening and stimulating the immune
system, the nerve function or the hormonal system. Herbs used as tonics are also believed to
exert balancing actions in organ systems and biochemical processes of the body (Mowrey,
1998), and protect the body against environmental factors that cause stress (Panossian &
Wagner, 2005). Internationally, the most famous tonic plants include Panax ginseng C.A. Mey.
(ginseng), Withania somnifera (L.) Dunal, (Indian ginseng, also known as ashwagandha in
Ayurveda), Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (Siberian ginseng),
Astragalus membranaceus Bunge (astragalus), Glycyrrhiza glabra L. (licorice or liquorice) and
Artemisia absinthium L. (wormwood).
The use of tonics is a well-established practice in southern African traditional medicine. Many
early records of multi-purpose, cure-all plants are found in Khoi-San medicine, and later in
records of Cape Dutch medicine (Smith, 1888, 1895; Smith, 1966; Hutchings et al., 1996; Van
Wyk & Gericke, 2000; Van Wyk, 2008a; Van Wyk et al., 2008; Van Wyk et al., 2009). There is
a large variety of these strengthening plants, as individual or combination preparations, which
are believed to play a significant role in improving health and general wellbeing (Van Wyk et
al., 2009). Various traditional terms are used to describe southern African tonics. The Zulu term
imbiza refers to plant mixtures that impart strength, health and vigour (Ngubane, 1977). The
term is derived from the traditional Zulu cooking pot, known as the imbiza (L. Posthumus,
personal communication to B-E van Wyk, 2011). These decoctions are used extensively as
general medicines but also as cleansing enemas (ukuchatha) or as purifying ritual emetics (to
ukuphalaza or ukughabha). Imbizas that are specifically used during pregnancy are called
isihlambezo (Van Wyk & Gericke, 2000). In the Sotho culture, the term musa-pelo is used for
1
Chapter 1
Introduction and hypothesis
medicine made from a diversity of shrubby legumes (Fabaceae) that is used to treat anxiety,
stress and grief. Musa-pelo literally means to turn the heart around (Moteetee & Van Wyk,
2007), perhaps suggesting the tonic and adaptogenic properties of these Sotho plants. A wellknown example is Sutherlandia frutescens (L.) R.Br.
Van Wyk and Gericke (2000) and Van Wyk et al. (2009) listed 14 medicinal plant species that
are commonly used as tonics in southern Africa. These are Agathosma Willd. species
(Rutaceae), Aloe species (Asphodelaceae), Arctopus L. species (Apiaceae), Artemisia afra
(Asteraceae),
Balanites
(Asteraceae),
Harpagophytum
maughamii
Sprague
procumbens
(Balanitacae),
DC.
ex
Meisn.
Cass.
species
(Pedaliaceae),
Hypoxis
Dicoma
hemerocallidea Fisch., C.A.Mey. & Av-Lall. (Hypoxidaceae), Muraltia heisteria
(L.) DC.
(Polygalaceae), Sutherlandia R.Br. in W.T.Aiton species (Fabaceae), Vernonia oligocephala

(DC.) Sch.Bip. (Asteraceae), Warburgia salutaris (G.Bertol.) Chiov. (Canellaceae), Withania
somnifera (Solanaceae) and Ziziphus mucronata Willd. (Rhamnaceae).
The purpose of this study is to explore the ethnobotany and phytochemistry of these species in
order to relate the data to known pharmacological effects, thus attempting to explain the
traditional tonic uses of the plants. The hypothesis to be tested is that southern African tonic
plants are typically characterised by chemical complexity, resulting in a wide range of possible
biological activities. Each tonic not only accumulates a diversity of individual compounds but
also a diversity of classes of compounds, all potentially contributing to additive and synergistic
effects.
Traditionally, phytochemists are focused on describing new compounds and rarely report on
the presence of well-known constituents in the plant species they have studied. As a result, the
full chemical complexity of medicinal plants (especially in southern Africa) has remained
unexplored. In this study, comparative screening procedures will be used to investigate the
presence of various classes of compounds, including terpenoids (mono-, di-, sesqui- and
triterpenes, as well as saponins and iridoids) and steroids, nitrogen-containing compounds
(alkaloids, amino acids and cyanogenic glycosides), phenolic compounds (anthracenes,
coumarins, flavonoids, phenolic acids and quinones), as well as alcohol precipitable solids
(polysaccharides and glycoproteins). The ultimate purpose of this investigation is to find a
possible scientific rationale for the wide spectrum of uses of southern African tonic plants and
to contribute towards a more profound understanding of principles and practices relating to the
use of tonic plants in traditional medicine systems.
2
CHAPTER 2
The concept of herbs as tonics
2.1 Tonics and related concepts (definitions)
As early as 1500 BC, the Ebers papyrus and the Jewish Torah (520 BC) listed single plants
used for treating various ailments, which were also able to lift the spirit and give a feeling of
general wellbeing, i.e. vitality medicines (Hamdard Pharmacopoeia of Eastern Medicine,
1970). Dioscorides compiled the first pharmacopoeia (78 AD), listing many plants used for
general health purposes (Osbaldeston & Wood, 2000). Galen (129 217 AD) recommended
Mithridates Treacle, a syrup made from vinegar and raisins, used to increase resistance to a
wide variety of debilitating circumstances, to purify the blood, expand the spirit and gladden it
and to strengthen the heart and give it power (Grant, 2000). Maimonides (1135 1204, in
Rosner, 1998), who wrote extensively on disease prevention and tonic medicines, included
food plants as important vehicles to healing (Gordon, 1958), a principle also included in
traditional Chinese medicine (Fu, 2005). Gerards Herbal (1633) listed plants mentioned by all
the contributors to medicine from the Old World, as well as plants used in traditional Chinese
medicine, Ayurveda, Arabic medicine and from native America. These plants improved general
health through improved digestion, increased resistance and a better state of mind (Gerard,
1633). In popular, more modern literature, tonics are referred to as substances that maintain
and restore health and vigour (Van Wyk & Wink, 2004). Tonics help reduce reactions to stress
and help prevent exhaustion, burnout, chronic fatigue, loss of concentration, depression and
degenerative diseases (Hobbs, 1998; Mowrey, 1998; Tharakan & Manyam, 2006). Tonics are
believed to support and tone either specific organs or the whole body by strengthening and
stimulating the immune system, the nerve function or the hormonal system (Mowrey, 1998) and
also seem to protect the body against environmental/external factors that cause stress
(Panossian & Wagner, 2005).
Chapter 2
These herbs that make new again, help restore ones youthful state of mental and physical
health or help expand a state of happiness exist in most healing cultures across the world.
This concept, as applied in different cultures, is summarised in this chapter. A variety of other
terms also used to describe this concept or its various effects, often being confused with one
another and cited ambiguously, are included (in bold print) and clarified.
2.1.1 Tonics
The term tonic originates from the Latin word tonicus, which means tension or tone; and from
the Greek words tonikos capable of extension, or tonos a stretching. According to Harper
(2011), the term was used for the first time in 1649, relating to or characterised by muscular
tension. The meaning "maintaining the healthy firmness of tissues" was recorded from 1684,
and was extended to "having the property of restoring to health" in 1756. The noun, meaning "a
tonic medicine", has been in use since 1799. It is a collective term that could be used for plants
described by Gerard (1633) as having the ability to improve digestion, refresh the spirits,
exhilarate the mind, comfort and increase the liveliness of the senses. The term tonic might
have originated within the European medicinal system and might have been influenced by
Egyptian, Jewish and Greco-Roman medicine. Synonyms for tonic include:
(a)
Roborant a substance that strengthens a patient suffering from exhaustion or who is
in a state of recovery (convalescence) (Burger & Wachter, 1998); a word used in the English
language sometime before 1828 (Webster, 1828); from the Latin words roborre, which means
to strengthen, and rbur, an oak (Collins English Dictionary, 2003).
(b)
Curative an old French word (curatif, adjective) used since the early 1500s, meaning
healing, from the Latin word curat- (stem of curare), which means to cure. Its use as a noun
has been attested from 1857 (Harper, 2011).
(c)
Restorative (hui fu in Mandarin) as a noun, it indicates a substance that is able to
strengthen and invigorate, and as an adjective, tending to impart new life and vigour, strength
and vitality, promoting recuperation (WordNet 3.0, 2003-2008). Panax ginseng, for example, is
a traditional Chinese all-healing herbal tonic also known as a restorative (Liu & Xiao, 1992).
In Greek, pan means all and akos means healing (Gillis, 1997).
(d)
Elixir a medieval Latin (ca. 700 1500) word for the "philosopher's stone", a
substance believed by alchemists to change base metals into gold and/or to cure diseases and
prolong life; from the Arabic word al-iksir, which means miracle substance. During the 1590s
the term was used in a general sense for "strong tonic"; from the1630s it was used for quack
medicines (Harper 2011).
4
Chapter 2
(e)
Panacea a synonym for elixir. Panacea is a Latin word, adopted from the Greek word
panakeia "cure-all (e.g. Panax ginseng, see restorative), spelt panace in early English
(1513). From 1548, it was used to describe any all-healing herb (Harper, 2011).
(f)
Preventive [from the Latin word praevent- (stem of praevenire) + -ive, used in general
from the 1630s, but as a medical term from the 1670s (Harper, 2011), or preventative [also
used from the 1650s as an adjective and as a noun from 1774 (Harper, 2011)]; a synonym is
prophylactic (from the Greek word prophylaktikos, that means "precautionary", or from
prophylassein, which means to keep guard before, ward off), used from the 1570s to describe
medicines "that tend(s) to prevent disease". From the 1640s, the noun has been attested as "a
medicine or treatment to prevent disease (Harper, 2011). The purpose of such a medicine is
thus to prevent, rather than treat or cure a disease. A division is made between primary
prophylaxis (to prevent the development of a disease) and secondary prophylaxis (whereby the
disease has already developed and the patient is protected against worsening of the process).
These substances may include vaccines, antibiotics or tonic medicines.
Examples of tonics plants representative of the traditional healing cultures of the world are
provided in Section 2.2. Terms such as adaptogen, amarum, alterative, immune
stimulant/modulator and adjuvant describe substances with specific tonifying effects, and are
discussed below (Paragraphs 2.1.2 2.1.6).
2.1.2 Adaptogens
The concept of adaptogen has its origin with a Russian researcher, Nikolai Lazarev. He studied
the effects that chemically synthesised compounds, i.e. benzimidazole derivatives (also called
dibazoles), had on the ability of animals to resist stressors such as cold, toxins and bacteria
(Lazarev, 1947). During 1959, he published his findings that these substances were
responsible for a bodily state of non-specifically increased resistance (sostoyanie
povyshennoi nespetsificheskoi soprotivlyaemosti in Russian) (Davydov & Krikorian, 2000;
Jaremenko, 2005). Lazarev called such substances adaptogens [the word stems from the
Latin word adaptare, which means to adjust and make suitable, and from the Greek word
genes, which means to produce (Davydov & Krikorian, 2000)]. Adaptogens are said to be
innocuous, increase resistance to a wide range of stressors in a non-specific way, and have a
normalising effect irrespective of the direction of the preceding pathological changes
(Brekhman & Dardymov, 1969a). Heightened resistance in a healthy organism allows it to
5
Chapter 2
withstand extraordinary challenges better and to adapt to physical and mental stresses more
effectively. Plant adaptogens are consequently used for prevention or remediation of
heightened stress sensitivity and health problems resulting from stress. Examples of
adaptogens indigenous to Russia are Eleutherococcus senticosus (Siberian ginseng); Rhodiola
rosea L. and Schisandra chinensis Bail. (Panossian & Wagner, 2005). Today, the most wellknown adaptogens in the world also include Panax ginseng and Astragalus membranaceus
(Fisch.) Bge. from China (Wu, 2005), as well as Withania somnifera (Indian ginseng).
European adaptogenic tonics, which include Acorus calamus L., Angelica archangelica L. and
Cnicus benedictus L. [reclassified as Centaurea benedicta (L.) L.] were adopted from early
Arabic, Jewish and Greco-Roman medicine (Gerard, 1633). Ayurvedic rasayana (i.e.
rejuvenating) herbs that have been tested for adaptogenic properties include Tinospora
cordifolia (Willd.) Hook.f. & Thomson, Asparagus racemosus Willd., Emblica officinalis Gaertn.,
Piper longum Blume. and Terminalia chebula Retz. (Rege et al., 1999). Baccharis trimera
(Less.) DC. and Davilla rugosa Poir. are known adaptogens from South America (Mendes et
al., 2007), while Panax quinquefolius L. is known as American ginseng and Pfaffia paniculata
Kuntze. as Brazillian ginseng, suggesting that their healing properties resemble those of
Chinese, Indian and Siberian ginseng. Examples of adaptogens used in Africa include Ocimum
tenuiflorum L. from the tropical regions (Kuhn & Winston, 2007), Griffonia simplicifolia Bail.
from West Africa (Zmilacher et al., 1988), Caesalpinia bonduc (L.) Roxb. from East Africa
(Kannur et al., 2006) and Sutherlandia frutescens from southern Africa (Van Wyk & Albrecht,
2008).
2.1.3 Bitter tonics

According to Burger and Wachter (1998), an amarum (Latin origin) or bitter tonic is a bittertasting drug or extract with intensely bitter active substances, which cause amplified reflex
gastric secretion and increased appetite. Increased secretion, motility and absorption within the
gastrointestinal tract upon ingestion (Sternini, 2007), which in turn increase appetite and
improve digestion through stimulation of acid production by the gastric mucosa in the stomach
(Gebhardt, 1997), together with an increase in bile flow (a cholagogic effect) (ztrk et al.,
1998), is known as the amarum effect. Bitter tonics are classified as amara pura (bitter drugs in
which only the bitter substances are active), amara aromatica (bitter drugs containing essential
oils), amara mucilaginosa (bitter drugs containing polysaccharides) and amara adstringentia
(bitter drugs containing tannins which are astringent) (Burger & Wachter, 1998).
6
Chapter 2
The use of bitter tonics is an ancient practice. Hyssopus officinalis L., Artemisia absinthium L.
and Commiphora myrrha L., for instance, were mentioned in the Old Testament of the Bible,
originating from the Jewish Torah (520 BC) (Hamdard Pharmacopoeia of Eastern Medicine,
1970). Apart from being bitter, these plants have numerous other medicinal uses [Michie &
Cooper, 1991; Brooke, 1998; Bradley, 2006; Bora & Sharma, 2010(a) and 2010(b); Kizil et al.,
2010; Su et al., 2011], contributing to their tonic status. Gentiana lutea L. was listed as a bitter
remedy for a variety of digestive tract ailments by Dioscorides in the first century (Osbaldeston
& Wood, 2000) and was used as traditional Chinese medicine for the same indications even
before this time (Wu, 2005). Other renowned bitter tonics include Angelica archangelica and
Centaurea benedicta from Europe (Gerard, 1633), as well as Andrographis paniculata (Burm.f.)
Wall. ex Nees, Nyctanthes arbortristis L., Filipendula ulmaria (L.) Maxim., and Grewia
asiatica L. from Asia, Australia and New Zealand (Sharma et al., 2010). The bitter tonics native
to the Americas include Annona muricata L., Hydrastis canadensis L., and Cinchona
pubescens Vahl., Vernonia species [V. amygdalina Del., V. fasciculata Michaux and V.
colorata (Willd.) Drake] are often used in Africa as well as Aloe ferox Mill. and Harpagophytum
procumbens, which are indigenous to southern Africa (Kokwaro, 1993; Van Wyk & Gericke,
2000; Wichtl & Bisset, 2000; Long, 2005).
Schultz et al. (2001) indicated that bitter substances are able to lower the heart rate, and
Zimmermann et al. (1986) proposed that there was a link between bitterness and the
stress/immune-modulating properties of these tonics. Bitter tonics are consequently not only
purgatives or laxatives, but also impart general tonic (strengthening) properties.
2.1.4 Alteratives
The term alterative comes from the Old French word altrer or the Italian word alterare,
meaning to change or worsen. It is a derivative of Medieval Latin alter (meaning other) and tvus (likely or able to produce), i.e. altertvus means to produce alteration. It was used from
1350 1400 in Middle English for drugs that were able to restore normal health, or used
empirically to alter the course of an ailment favourably (Dictionary.com, 2011). During the early
1900s, alteratives were described as agents that alter the course of morbid conditions by
modifying the nutritive processes while promoting waste, and thus indirectly curing many
chronic diseases (Crellin & Philpott, 1997). Fulder (1998) describes alteratives (syn. blood
purifiers) as western herbal medicines used for treating acute and chronic infections and
7
Chapter 2
inflammations, with the purpose of promoting circulation of lymph and body fluids, in order to
increase immune response and long-term immune function. Both terms (alterative and blood
purifier) are out-dated as medical terms (Van Wyk & Wink, 2004), but are still used when
describing traditional medicine. Currently, more specific terms may be used to describe the
pharmacological actions of a blood-purifying agent or alterative. Fulder (1998), for instance,
refers to Arctium lappa L. (burdock), Salvia officinalis L. and S. sclarea L. (sage and clary sage)
as well as Commiphora myrrha (Nees) Engl. (myrrh) as the most commonly-known western
alteratives. Van Wyk and Wink (2004), however, refer to Arctium lappa as a diuretic, to the
Salvia species as antiseptic, antispasmodic, carminative and astringent, and to
Commiphora myrrha as astringent, antiseptic and antiinflammatory. Other examples include
cholagogues, which promote the flow of bile, by either increasing the bile production in the
liver (choleretic) (e.g. Curcuma longa L.) or the demand of gallbladder emptying
(cholekinetic) (e.g. Harungana madagascariensis Lam. ex Poiret), as well as stomachics, i.e.
digestive tonics that stimulate digestion in the stomach and relieve stomach aches through
increased stomach secretions (e.g. Anethum graveolens L.) (Van Wyk & Wink, 2004). Gastric
secretion demanding substances may include amara (see bitter tonic), antacids and
carminatives (Burger & Wachter, 1998).
Bitter tonics seem to differ from alteratives with regard to taste but their effects may be similar
otherwise, i.e. both bitter tonics and alteratives are referred to as stomachics. In traditional
Chinese medicine, blood purifiers such as Rehmannia glutinosa (Gaertn.) Libosch., Polygonum
multiflorum Thunb. and Glycine max (L.) Merr., are tonics that may have a sweet, bitter or
pungent taste, associated with cooling the liver, i.e. hepatoprotective and hepatostimulative (a
build-up of poisons due to ineffective detoxification may lead to fever). Blood purifiers may also
act on the spleen, stomach and/or kidney meridians to re-establishing the flow of q (Wu,
2005). In this instance, many herbs used in various Asian healing systems have been shown to
have alterative/blood purifying as well as antipyretic properties (Sharma, et al., 2010).
Improved blood circulation also benefits the skin, i.e. many alterative herbs such as Arctium
lappa are indicated for treatment of infection, skin eruptions and chronic degenerative
conditions (Chan et al., 2011). In popular literature and in marketing materials, terms such as
"digestive alterative", "cardiac alterative" or nutritive are sometimes used to indicate specific
modes of action (http://herbsorganic.co.za/GLOSSARY.htm, 2011).
Chapter 2
2.1.5 Adjuvants
An adjuvant is said to be a substance that aids in removing or preventing a disease, especially
as a substance added to a prescription drug to aid the effect of the main ingredient. The term is
Latin (stem of adjuvns or adjuvre, equivalent to ad- + juv- (stem of juvre, meaning to help) +
-ant) and originated in the 1600s (Dictionary.com, 2011). In most examples listed by webbased medical dictionaries, the term is used to describe substances that enhance the body's
immune response to an antigen. A synonym for adjuvant is adjunct. Derived from the Latin
term adjunctus, it was used as an adjective (to be closely connected, joined, united) and as a
noun (a characteristic, essential attribute) since the 1850s. The McGraw-Hill Concise
Dictionary of Modern Medicine (2002) describes it as a supplementary addition rather than an
essential ingredient, while Mosby's Medical Dictionary (2009) describes it as an additional
substance, treatment, or procedure used for increasing the efficacy or safety of the primary
substance, treatment, or procedure or for facilitating its performance. Consequently, adjuvants
(or adjuncts) are pharmacological or immunological agents that may modify the effect of other
agents, such as drugs or vaccines, while not necessarily being biologically active by itself.
From the definitions, it can be deduced that adjuvants or adjuncts are thus not essentially
tonics. However, when tonic extracts are combined with chemosynthetics or antibiotics, the
overall effect is usually greater than that which may be obtained when the drugs are added
together in equivalent quantities, i.e. synergism is observed (Wagner, 2006). Recent advances
in
treatment
for
cancer
include
the
use
as
herbal
tonic
adjuvants
to
western
chemotherapeutics, where the general health of patients with cancer is increased, and the
probability of metastasis (development of secondary tumours) is reduced. The debilitating
effects of radiotherapy and chemotherapy are also reduced, therefore providing a possibility for
higher dosages. Results where Panax ginseng, Astragalus membranaceus and Curcuma longa
were applied as adjuncts, included prompt recovery after serious operations, shorter
hospitalisation periods and fewer complications afterwards (Qi et al., 2010). Where
Eleutherococcus senticosus was administered as adjuvant to other medicines, prompting the
cure of chronic conditions such as pneumonia, tuberculosis and vascular dystonia was
observed (Fulder, 1980).
Chapter 2
2.1.6 Stimulants, immunostimulants and immunomodulators

A stimulant is an agent that arouses organic activity, strengthens the action of the heart,
increases vitality, and promotes a sense of well-being (The American Heritage Medical
Dictionary, 2007). It comes from the Latin word stimulantem and has been used as an
adjective since 1772, and as a noun since 1794. The verb (stimulation, from Latin
stimulationem, from stimulare "prick, goad, urge,") has been used since the 1520s to indicate
the "act of pricking or stirring to action" (Harper, 2011).
The term modulation has been used since the 1530s, to describe an "act of regulating
according to measure or proportion" (Harper, 2011). An immunomodulator is consequently a
substance that alters the immune response by augmenting (by immune-stimulation) or
reducing (by immune-suppression) the ability of the immune system to produce antibodies or
sensitised cells that recognise and react with the antigen that initiated its production. Many
adaptogens are immunomodulatory, i.e. they may stimulate or depress immune responses in
order to establish immunostasis [e.g. Gentiana lutea, Panax ginseng, Rhodiola rosea, Withania
somnifera and Eleutheroccocus senticosus (Tharakan and Manyam, 2006)], where an
immunotonic, such as Echinacea purpurea (L.) Moench. only stimulates (Van Wyk & Wink,
2004; Naik et al., 2010).
2.2 Tonics in traditional healing cultures

While the term tonic dates back to the 1600s, the concept is well known in many ancient
traditional healing systems. In order to understand and be able to compare the various
traditional healing systems of the world, one has to take the development of the cultures within
which they are practiced into consideration, as it is dependent on the history of the particular
culture. Kokwaro (1993) summarises traditional medicine stating that it is the sum of all the
knowledge and practices, whether explicable or not, used in diagnosis, prevention and
elimination of physical, mental or social imbalance and relying exclusively on practical
experience and observation handed down from generation to generation, whether verbally or in
writing. This section outlines the development of traditional healing and tonic medicine, and
highlights aspects, concepts or philosophies thereof.
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Even though Egypt forms part of Africa, Ancient Egyptian and Arabian Medicine played a major
role in the development of European medicine. Consequently, these healing cultures (Islamic
medicine) are discussed together with other traditional systems pertaining to Europe. Over the
centuries, much emigrational movement was evident between Europe and Asia, resulting in
mutual exchanges between healing cultures of resident peoples within the Old World, and is
consequently grouped together. The native peoples of Oceania (Australia and New Zealand),
the Americas and Africa place great emphasis on the contribution of ancestral spirits in healing.
Oceania and the Americas are consequently grouped together, while Africa is treated
separately, as special emphasis is placed on southern African traditional healing and its tonic
medicines, which is the focus of this study. The various traditional healing systems are
indicated in bold print.
2.2.1 Europe and Asia

2.2.1.1 Islamic medicine (North African and Middle Eastern traditional medicine)
The root of Islamic medicine possibly lies in Ancient Egyptian medicine. While several
existing papyri provide evidence of the advanced medicinal practices followed by the Egyptians
at the time, the Ebers papyrus (1550 BC, in Chauncey, 1952) contains recipes classified
according to diseases, including remedies for internal diseases and a formulary for various
conditions among others. Many of the recipes deal with conditions where the cause is not
evident. It contains more than 500 plants included in more than 700 remedies. Some of the
plants listed as vitality medicines are Artemisia vulgaris L., Aloe socotrina Lam., Gentiana
lutea, Cuminum cyminum L., Mentha L. species, Foeniculum vulgare Mill. and Commiphora
myrrha (Hamdard Pharmacopoeia of Eastern Medicine, 1970). While incantation and charms
were important vehicles of healing in Egyptian medicine, the Ebers papyrus does not contain
much information on such methods (Chauncey, 1952).
Over 600 cuneiform tablets, some dating back to 668 BC, were discovered in the ancient
Assyrian Ashurbanipal library in Nineveh (modern-day Iraq) containing traditional Assyrian
(2500612 BC) and Babylonian (612539 BC) medicinal practices in ancient Mesopotamia.
Diseases were believed to be curses of spirits, gods or ghosts, although healing was brought
about by treatment with herbal remedies or by means of surgery. Lists of plants used as
remedies are provided, but plant identification is challenging as metaphorical names were
used. It can, however, be deduced that remedies were plant extracts, resins, or spices, and
that some species, such as mint, were used for several ailments (Wilson, 2005).
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Ancient Iranian Medicine (i.e. medicine in Persia from 539336 BC) was strongly influenced
by Egyptian and Jewish medicine at the time, as King Darius (ca. 520 BC) conquered Egypt,
and instructed the redaction of the Jewish Torah (the first five books of the Bible, which contain
valuable information on historical Jewish medicinal plant use) (Hamdard Pharmacopoeia of
Eastern Medicine, 1970). Much later (ca. 860 AD), the Persian scientist Ali ibn Sahl Rabban alTabari, authored the first encyclopaedia of medicine in Arabic, entitled Firdous al-Hikmah
("Paradise of Wisdom"), which contained aspects of Sushruta and Chanakya (Ayurveda,
paragraph 2.2.1.5) (Selin, 1997). Muhammad ibn Zakarya Rzi (known as Rhazes), also a
Persian physician and chemist, and student of Ali ibn Sahl, wrote the al-Hawi, or Large
Comprehensive, in the 9th century, containing novel accurate descriptions of diseases such as
measles and small-pox, and criticisms on the works of Aristotle, Plato and Galen (Deming,
2010). This work was translated into Latin in 1279, affording it considerable influence in
European medicine (Savage-Smith, 1996).
Islamic medicine developed during the Islamic Golden Age (7501258) and was based on
theoretical and practical applications of traditional medicine in Arabia, together with the
knowledge of the Greco-Roman physicians (Hippocrates, Dioscorides and Galen). Other
influences include Ayurveda, Ancient Iranian medicine and Unani (paragraph 2.2.1.5) (Saad et
al., 2005). While several works on ethical practice of medicine and surgery were written,
Avicenna (or Ibn-Sina, 9801037) was the first to establish the use of scientific experiments in
medicine. He described the progression of cancers and other diseases as well as the treatment
of early tumours by complete removal in his medical textbook Canon of Medicine (on which
Unani was based, see South Asian traditional medicine), but also introduced clinical trials and
provided protocols for drug discovery and experimental testing of the efficacy of drugs. Ibn AlBaitar (11881248), a botanist/herbalist from Spain, collected ca 1 500 plants on his way from
Spain to Syria, and wrote the most complete Arabic herbal (Corpus of Simples) (Selin, 1997).
2.2.1.2 Ancient Jewish Medicine

Recorded aspects of Ancient Jewish Medicine appear in the Bible (see redaction of Torah,
Ancient Iranian Medicine). These were influenced by the Assyrians and Babylonians, as they
were captured by these tribes in 722 BC and 586 BC, respectively (Thiele, 1983). Under the
rule of the Roman Empire (27 BC1453 AD), Jerusalem was destroyed (70 AD) and the Jews
spread through Europe, Turkey and Arabia. Oral teaching of the Torah was no longer practical,
and the rabbis started drafting the Talmud (in 200 AD), based on subject matter rather than
biblical verse. The largest contribution to Jewish medicine was made by Maimonides (or Moses
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Chapter 2
ben-Maimon, also known as Ms ibn Maymn in Arabic) (11351204 AD), an important

Jewish physician and biblical scholar. He was born in Spain, but was forced to evacuate to
Morocco with the invasion of the Almohad (Moroccan Berber-Muslim dynasty). During this
period (1158), Maimonides wrote the Mishnah (a study on Jewish oral traditions or Torah). He
and his family then briefly lived in Israel before moving to Fostat (Egypt), where he worked as a
physician to the royal family. In his writings (A Treatise on Asthma, A Treatise on Poisons,
Haemorrhoids, and Cohabitation, Essays on Sexual Intercourse, Preservation of Youth, The
Causes of Symptoms, Medical Aphorisms Volumes 1 and 2, and Glossary of Materia Medica)
he described many conditions including asthma, diabetes, hepatitis, and pneumonia, and
emphasised moderation and healthy living (Selin, 1997). He was knowledgeable about Greek
and Arabic medicine, and followed the principles of humorism in the tradition of Galen, had a
critical view on traditional medicine and added his own observations and experience. He wrote
extensively on disease prevention and tonic medicines, including Allium sativum L.,
Cinnamomum zeylanicum Breyne., Mentha viridis L. (syn. M. spicata L.), Sesamum indicum L.,
Glycyrrhiza glabra and Coriandrum sativum L. (Maimonides, 1958, Gordon, 1958, Rosner,
1998). Interestingly, he also included food plants as important vehicles to healing, such as,
Eruca sativa (L.) Mill. (rocket or arrugula), Vitis vinifera L. (grapes), Ficus carica L. (figs),
Asparagus officinalis L. (asparagus), Portulaca oleracea L. (pigweed) and Citrullus lanatus
(Thunb.) Mansf. (watermelon) (Maimonides, 1958).
2.2.1.3 European Medicine

Hippocrates, the great Greek physician (ca. 460377 BC), can probably be considered the
father of Ancient Greek medicine, European healing systems and western medicine as it is
today (Chast, 2003). Other great contributors include Aristotle (384322 BC), a Greek
philosopher who also studied biology and wrote pieces containing research from Hippocrates
(Nuland, 1988) and Theophrastus (370287 BC), the Greek successor to Aristotle, who studied
more than 500 plants and wrote several books on botany (Theophrastus, 1916). Ancient Greek
medicine was influenced by Ancient Egyptian, Iranian and Indian medicine as Alexander the
Great (Greek Macedonian king) conquered Egypt in 332 BC and the Persian Empire in 331
BC, after which he invaded India in 326 BC (Roisman & Worthington, 2010).
Dioscorides (ca. 4090 AD), the Greek surgeon of the Roman Emperor during the rule of the
Roman Empire (27 BC1453 AD), was a great contributor to Ancient Roman medicine. During
his travels with the Roman army through Italy, Greece, Spain, Asia Minor and France, he
collected 600 plants and described their medicinal uses in De Materia Medica ("Regarding
13
Chapter 2
Medical Materials"), the first pharmacopeia (78 AD) (Osbaldeston & Wood, 2000). The
accomplished medical researcher, Galen (129217 AD), was a Greco-Roman physician,
surgeon and philosopher, and first in developing drug compounds. Many books were written
regarding this research (Nuland, 1988).
The rule of the Byzantine (Eastern Roman) Empire (4001453 AD) followed the Dark ages
(480800 AD), when the Western Roman Empire fell into Germanic hands. In Byzantine
medicine, medical knowledge was compiled into textbooks (e.g. the Medical Compendium in
Seven Books, written by the leading physician Paul of Aegina) (Aegineta, 1846). During
medieval times, patients sought medical treatment in monasteries. Hildegard of Bingen (1098
1179), a German Benedictine abbess, wrote several texts on theology, botany and medicine.
Among the herbs she prescribed were Plantago spp. (psyllium), Aloe spp., Marrubium vulgare
L. (horehound), Geranium robertianum L., Foeniculum vulgare (fennel) and Petroselinum
crispum (Mill.) A.W. Hill (Chast, 2003).
At the beginning of the Renaissance (13001700 AD), after the Ottoman Empire conquered
the Byzantine Empire, ancient Greco-Roman texts were translated back into European
languages. Some of the authors who made medicinal plants known to the public during this
time, was: Leonhart Fuchs, a German physician who wrote Historia Stirpium (1542) and New
Kreuterbuch (1543), Matthias de Lobel, a Flemish botanist with his Kruydtboeck (1581), John
Gerard with Gerards Herball (1633) and Nicholas Culpeper with The English Physitian (1652)
(Van Wyk & Wink, 2004). Gerards Herball, for instance, contained many references and
philosophies with regard to health maintenance and disease prevention of both Greek and
Roman authors (Gerard, 1633). Medicinal plants used in Traditional Chinese Medicine,
Ayurveda and Arabic medicine were included. Once again, populations were concentrating
around urban centres, and infectious disease was an increasing concern. Gerard prescribed
remedies that could increase resistance against infectious diseases, taken by the Turks at
Constantinople and the Tartars, medicines from the north applicable from the time of the dark
ages, plants native to the Mediterranean region, and north from Portugal to southern France
and east to Iran. These plants include Aconitum napellus Thunb., Anethum graveolens,
Pimpinella anisum L., Prunus dulcis (Mill.) D.A.Webb var. amara (De Candolle) H.E.Moore,
Juniperus communis L., Vitis vinifera, Cetraria islandica (L.) Ach., Gentiana officinalis Harry
Sm., Elettaria cardamomum Maton. and Cyperus rotundus L. (Gerard, 1633, Stephenson &
Churchill, 1879, Osbaldeston & Wood, 2000).
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Chapter 2
2.2.1.4 Northern Eurasian medicine (Slavic countries and Russia)

During 300500 AD, Slavic speaking tribes formed part of the many multi-ethnic confederacies
of Eurasia, such as the Sarmatian, Hun and Gothic empires who migrated and settled in the
areas of the former Western Roman Empire, i.e. they migrated from Kiev towards present-day
Suzdal and Murom, and from Polotsk towards Novgorod and Rostov (Christian, 1998).
Consequently the medicine used was analogous to that of the primitive European peoples, i.e.
the Germans and Celts. Medicinal knowledge stemmed from Greek, Latin and Arabic sources
through Arabic merchants and Christian missionaries (Grmek, 1959). Slavic, Turkic and other
tribes migrated further to Eastern Europe, and from the 7th century onwards, the East Slavs
constituted the bulk of the population in Western Russia and gently assimilated the native
Finno-Ugric tribes. There was a simultaneous westward movement of Germans and the Huns,
Avars and Bulgars from Siberia and Eastern Europe. They settled in areas of present day
Austria, the Pannonian plain and the Balkans, and northwards along the upper Dnieper river.
The Vikings from Scandinavia raided all over Europe from the 8th century and settled in many
places, including Normandy, the northern part of England, Scotland and Ireland (Christian,
1998). Until the 11th century, medicine was largely in the hands of folk healers regarded as
pagan cult leaders (Zguta, 1980).
The period between 862 and 1237 marks the traditional beginning of Russian history with the
establishment of Kievan Rus', the first united East Slavic state, centred in Novgorod. The state
adopted Christianity from the Byzantine Empire in 988, beginning the synthesis of Byzantine
and Slavic cultures that defined Russian culture (Christian, 1998). Monasteries were erected
with hospitals on the same premises. The Pantocrator monastery in Constantinople for
example, had two hospitals attached to it: a six-bed unit for sick monks, and a larger facility for
the city. It also served as a medical education centre for Russian clergy. Treatment methods
basically followed Galen, but staff physicians also prescribed their own treatments. The
Pecherski Monastery near Kiev was home to three monks, Nestor, Simon and Polikarp,
authors of the very valuable Kievo-Pecherskij Paterik, which was included in The Primary
Chronicle. Kievo-Pecherskij Paterik is a collection/synthesis of written and oral medical
practices and was the single most important source on monastic medicine in Kievan Rus' at the
time. It mentions the treatment of leprosy, epilepsy, fever, urinary obstruction, kidney
dysfunction and several other unspecified illnesses. The prevailing therapy was herbal,
accompanied by prayer. Hydrotherapy, in the form of a steam bath, was applied especially for
gout and arthritic conditions. Other medical texts that would have been available to the
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Chapter 2
Pecherskij monks would have included the Izbornik Svyatoslava (an encyclopaedic work
covering hygiene, diet and medical botany), the Theology of Saint John Damascene (a work by
John, the Exarch of Bulgaria, which included astronomy, the four elements and the four
humours), the Shestodnev (also by John, the Exarch of Bulgaria, with sections on theology,
anatomy, physiology and materia medica). In the latter work, John drew extensively from
Aristotle, Dioscorides, Theophrastos, Hippocrates and Galen. A fourth Byzantine source was
the Fiziolog (containing animal stories as well as medico-biological information). The KievoPecherskij Paterik does, however, indicate a tremendous rivalry between secular court
physicians, the monk physicians and folk healers (Zguta, 1980).
From 12371240, the Mongol invasion of Rus' resulted in the disintegration of Kievan Rus' as a
state, and from 13401547, Moscow came to dominate Rus through the leadership of several
successive rulers called The Muscovy (Curtis, 1998). During this time, the power of Moscow
expanded and its territory stretched from the Polish-Lithuanian Commonwealth eastwards to
the Pacific Ocean. This expansion transformed Russia into a multi-ethnic, multi-confessional
state. During this time, the Trinity-Sergius Monastery was erected (mid-14th century) and was
particularly known for its library, which served as repository for medical knowledge. These texts
include works from the late 12th/early 13th century to the 15th century and included five copies
of the Paleja (a biblical history that also includes an unusual explanation of nature, human
embryology, anatomy and information from various herbals and lapidaries), a copy of the
Pchela (four of its 70 chapters are devoted to hygiene and medicine), a 15th century anthology
of selected writings by John Damascene and other authors (on bloodletting, the zodiac, moon
phases, illness and astrology, overviews of Galen and Hippocrates's physiology, and the
classic humoral theory of disease, Byzantine history, and the Gromnik) (Zguta, 1980).
Since the proclamation of Tsardom of Russia, starting with the rule of Ivan IV (Ivan the Terrible)
(15331584), followed by the Russian Empire under Peter the Great (16821725), until the
start of the Russian Revolution in 1917, great advances had been made in the development of
medical practices, with frequent exchange of physicians between Russia and Europe.
Unfortunately, medical care for most of Russia remained in the hands of unskilled people or
country clergy, due to the vast extent of territory, with its scattered distribution of villages. Apart
from the distribution challenges presented, the peasants were unable to afford the medicine.
The years preceding the Revolution indicated a slow improvement in medical provision
together with a spread of hygienic and preventive propaganda, but it still remained inadequate
16
Chapter 2
(Garrison, 1931). Russian traditional medicine, herbal lore transferred through generations,
describes tonic remedies as toniziruyuzhie sredstva (directly translated, something like
stimulating remedy) (Davydov & Krikorian, 2000), such as Eleutherococcus senticosus,
Rhodiola rosea, Schisandra chinensis and Rhaponticum carthamoides (Willd.) Iljin, (Brekhman
& Dardymov, 1969a). An amalgamated medical tradition, based on translated Greek medical
texts and native materia medica, was used well into the 17th century and beyond. Even though
Russian physicians followed European healing practices, they also had access to a large
variety of phytomedicines and herbal traditions through their international relations (e.g. with
America, through Turkey and Norway to Korea, Japan, Spain and Mexico) as well as those
from their own territory spanning over eleven time zones (even from Far East Russia through
the relations of the early informant tribes with the Chinese). Consequently, Russia maintained
a high usage of herbs for healing (Domarew et al., 2002).
2.2.1.5 Southern and South-eastern Asian traditional medicine

Siddha medicine (south Indian Tamil traditional medicine) is the oldest medicinal system in
India, with the other systems, e.g. Ayurveda and Unani, being rooted in its philosophies and
practices. It is believed that the Hindu God, Lord Shiva, and Goddess Parvathi handed this
medicinal system over to 18 of their followers, i.e. ancient supernatural spiritual saints of India
called the Siddhar, with Agathiyar being the first recipient (5th6th century BC). Siddha medicine
is derived from alchemy, with Chinese alchemy having the greatest influence, involving
aurifaction, elixirs of gold and mercuric sulfide (cinnabar), and herbs of deathlessness or
immortality (Needham, 1974). It is based on five elements Earth, Water, Fire, Air and Space
(or Ether), called the fundamental Bhutas. These five elements are present in all matter, also
the human body and are responsible for its proper functioning. The bodily functions are
supported by the Tridosha (three forces, i.e. physical, emotional and mental) Karpam, Pitham
and Vatham, functioning together harmoniously. A disturbance in the equilibrium of the
Tridosha results in disease. A healthy diet and lifestyle is believed to play a key role in health
and healing. Medicines used include herbal products, animal products or inorganic substance,
with much emphasis on the latter. These drugs are classified based on taste, character,
potency, class and action. To restore the balance in the five elements present in the human
body, the drug(s) of choice would consist predominantly of five elements opposite to each
other in nature (Shukla et al., 2011).
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Chapter 2
Ancient Indian knowledge and wisdom were passed on through generations by means of
memorised songs and poems. These songs and hymns were written in four canonical sacred
Hindu texts called the Vedas (Sanskrit word veda means knowledge), i.e. Rig Veda, Yajur
Veda, Sama Veda and Atharva Veda. The Indo-Aryan language used in the Rig Veda text
dates back to ca. 2000 BC (Mallory & Adams, 1997). Ayurveda originated from the Atharva
Veda (ca. 12001000 BC), which mostly pertains to the Hindu religion and contains 114 hymns
relating to the treatment of diseases. However, the principle classic reference for Ayurveda is
considered to be the Charaka Samhita, authored by Charaka (ca. 700 BC). It provided a
rational approach to cause and cure of disease, and an introduction to objective methods for
clinical examination. Charaka described approximately 50 plants in this book, classified in
groups of 10 plants each (Chattopadhyya, 1982). A further principle contribution to Ayurveda
was made by Sushruta (ca. 800 BC), the father of surgery, who wrote the Sushruta Samhita.
This work contained all the major concepts of Ayurvedic medicine and consisted of 184
chapters, describing 1120 illnesses, 700 medicinal plants, 64 preparations from mineral
sources and 57 preparations from animal origin (Dwivedi & Dwivedi, 2007). Ayurveda is based
on Siddha medicine (see above), but with a Galenic approach where it also includes bodily
humours (dosas) and suggests an inner life force (prana). The dosas are classified as somatic
(vata, pitta and kapha) and psychic (rajas and tamas), and an imbalance in the dosas results in
disease. Health and healing is achieved through good digestion and mental activity (i.e.
through maintenance of the prana), which is linked to six tastes (sweet, sour, salt, bitter,
pungent, astringent). Herbal remedies are formulated according to these tastes in such a way
that the tastes are balanced (Clements, 2006). Ayurvedic medicine has four basic components
in which disease prevention is a central focus: rejuvenation (the use of drugs to increase
immunity, resistance, improve mental function, and increase vitality), personal hygiene (daily
routines, including bathing, exercising, meals and sleep), virility enhancement (the use of
aphrodisiacs), and the practice of yoga. Medicines that promote health and longevity, i.e.
rejuvenating or tonic medicines, are called rasayana. It may be used singly or in combinations
that are often complex formulae prepared in a complex manner. Rasayana includes Withania
somnifera, Asparagus racemosus, Balsamodendron mukul Hook (syn Commiphora mukul
(Hook ex Stocks) Engl., Bassia latifolia Roxb. (syn Madhuca indica J.F. Gmel.), Berberis
asiatica Roxb., Cinnamomum camphora (L.) Nees & Eberm, Curcuma longa, Piper longum,
Wedelia calendulacea Less. and Zingiber officinale Rosc. (Dutt, 1877, Chopra et al., 2006).
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Unani medicine (also known as Unani-tibb or Yunani Medicine (in Arabic, Hindi-Urdu and
Persian, meaning "Greek medicine") originated as a form of traditional medicine widely
practiced in southern Asia. It is based on a tradition of Graeco-Arabic medicine, which has its
roots in the teachings of Hippocrates [the four humours: Phlegm (Balgham), Blood (Dam),
Yellow bile (afr') and Black bile (Saud')] and Galen, and was developed into an elaborate
medical system by Arab and Persian physicians, such as Rhazes, Avicenna, Al-Zahrawi and
Ibn Nafis (see Islamic Medicine above) (Saad et al., 2005). The greatest contribution to this
medicinal system stems from the Canon of Medicine, written by Avicenna, which relates to the
date of origin of this medicinal system (1025 AD). Unani first arrived in India with the
establishment of Delhi Sultanate (12061527) and during Muslim rule over North India (Zillur
Rahman, 2011).
Cinnamon (Cinnamomum species) is native to Sri Lanka, was recorded to be found in Ancient
Egypt as early as 1500 BC, suggesting early trade between Egypt and Sri Lanka. Sri Lankan
traditional medicine consequently incorporates several systems, including Siddha, Ayurveda
and Unani from India, as well as Arabic medicine, together with indigenous Sinhala traditional
medicine and Desheeya Chikitsa (indigenous medicine). Prince Vijaya, exiled from eastern
India with 700 of his followers (543 BC), is said to have been the father of Sinhala traditional
medicine (Kirk, 1976). Sri Lanka was introduced to Buddhism from India in the 3rd century BC,
and this played a vital role in invocatory healing ceremonies (Bali). Some medicaments used
are mixtures of several species and heal all diseases, e.g. the Bodhisattva pill containing
ginger (Zingiber officinale) and chilli (Capsicum species), among other substances
(Liyanaratne, 2001).
Jamu (formerly Djamu) is traditional Indonesian medicine, said to have originated from the
Hindu-Bhuddist Mataram kingdom (7521045) (Soekmono, 1973). Although heavily influenced
by Ayurveda, Jamu incorporates the rich spectrum of medicinal plants in Indonesia not
indigenous to India, but rather to Australia. Jamu is also the term used for tonifying remedies in
the traditional Malaysian and Indonesian healing systems (Davydov & Krikorian, 2000).
Medicines are mostly distributed as powdered material, predominantly prepared from herbs,
but may also contain animal products. Generally, the Jamu tradition is passed on orally from
generation to generation and practiced mostly by women who prepare the mixtures and sell it
on the street. Redaction of Jamu handbooks was mostly done by European physicians who
studied the tradition (Pols, 2009). Jacobus Bontius, a Batavian (Jakarta) physician wrote De
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medicina Indorum in 1642, Georgius Everardus Rumphius, a German born botanist, wrote
Herbaria Amboinesis (The Ambonese Herbal (6 volumes) from 17411750, and Carl Waitz, a
German physician, made several valuable contributions, but in Praktische waarnemingen over
eenige Javaansche geneesmiddelen (1829) he listed medicinal herbs used in Jamu, including
those used as tonics such as Curcuma longa (turmeric), Zingiber officinale (ginger) and
Myristica fragrans L. (nutmeg). Mrs J Kloppenburg-Versteegh, a Dutch lady concerned with
health in India, wrote Wenken en Raadgevingen Betreffende het Gebruik Van Indische
Planten, Vruchten Enz. (Guidance and Advice Regarding the Use of Indies Plants, Fruits), two
volumes, (1907), which became an important reference for self-medication in the Dutch East
Indies. Pols (2009) provides a detailed list of many other pharmacological researchers who
studied Jamu and their resulting publications.
2.2.1.6 East Asian traditional medicine

Traditional Chinese Medicine (TCM) shares many commonalities with Ayurveda. The
emphasis is on the patient rather than the disease. Health and healing is approached
holistically and diet and quality-of-life carry much emphasis, as well as the five elements of
matter (Patwardhan et al., 2005). In TCM the latter, collectively known as Wu Xing, are Water,
Earth, Metal, Wood and Fire. Yin and yang are the two main antithetic aspects (opposites, i.e.
positive and negative energy) affecting homoeostasis within the five elements. Wu Xing and
the Yin-Yang principle are ancient Chinese concepts, believed to be more than 5 000 years
old. The Yellow Emperors Inner Classic (recorded by Huang Di Nei Jing during the Han
Dynasty from 206 BC220 AD) is a work composed of two texts, based on these two theories,
which systematically describes medical physiology, anatomy and acupuncture. This work is
considered to be the most important book of traditional Chinese medicine, as it integrates the
four humours (q; blood; bodily fluid; vitality), with 12 zang-fu organ systems (heart, lung,
kidney, spleen, liver, small intestine, large intestine, bladder, stomach, gall bladder, triple
heater, pericardium) all playing an integral role in balancing the yin and yang in the human
body. The term meridian channel refers to the route of qi-flow through the body. Q is literally
translated as air, breath or gas, but means life force or energy flow, and is similar to prana in
Ayurveda. Vitality is related to mental health, i.e. mental activities and emotions, and is
dependent on the proper function of the zang-fu organ systems (Wu, 2005). The second most
important work in TCM is the Shanghan Zabing Lun' or Treatise on Cold Pathogenic and
Miscellaneous Diseases, a Chinese medical treatise compiled by Zhang Zhongjing (150
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219 AD), a physician from the Zhang family, at the end of the Han dynasty. It contained
knowledge on how to treat epidemic infectious diseases causing fevers prevalent during that
era, as well as a compendium of clinical experiences. During the Chin (221206 BC) and Han
(206 BC220 AD) dynasties, many scholars were dedicated to the collection of pharmaceutical
data. The Shen Nung Pen Tsao Ching or Divine Husbandman's Materia Medica was written,
said to be based on the findings of Shen Nong (ca. 3000 BC), the Yan Emperor, also known as
the first herbalist in Chinese tradition. It is presumed to be the earliest Chinese work on
pharmacology, listing properties and effects of 252 botanical, 67 zoological and 45 mineral
entries. It also includes topics such as harmony of the seven emotions, identification between
the real and the false (i.e. psychological aspects of healing), ingredients with mutual
reinforcement and assistance properties (i.e. tonic properties), the properties of drugs and their
origins, various types of medicaments, compatibility and incompatibility in the clinical
application of drugs (i.e. early findings on additive, synergistic or antagonistic interactions),
doses, the correct time for taking medicine, and the conditioning relationship among medicines
(i.e. physiological effects of drugs) (Hamdard Pharmacopoeia of Eastern Medicine, 1970). Two
texts, the Collection on Commentaries on the Shen Nong Herbal (or Shen Nong Ben Cao Jing
Ji Zhu) by Tao Hong-Jing, written during the Liang dynasty (456536 AD), and Materia Medica
of Medicinal Properties (or Yao Xing Lun, 581907 AD), introduced categorisation of various
herbs into upper (nourishing life), middle (nourishing constitutional types) and lower (used to
expel disease) grades. Discussions of the concepts of taste (Wei) and temperature (Qi),
toxicity, harvesting, selection and processing of herbs, herbal combinations, functions, primary
clinical application and reactions are included (Wu, 2005). The upper grade or superior
herbs (Mowrey, 1998) are tonics that create balance while nourishing the adrenal glands, that
are major energy managers, powering the human body (Gilbert, 1998; Cheng, 2000; Wu,
2005). Furthermore, these tonics are grouped by Zhang Yuan-Su (ca. 11511234) in two of his
works, i.e. Origins of Medicine (or Yi Xue Qi) and Pouch of Pearls (or Zhen Zhu Nang) (Bensky
et al., 2003, Wu, 2005) into:
(a) those that tonify q and vitality (called zi bu) (e.g., Astragalus membranaceus, Panax
ginseng, Codonopsis pilosula (Franch.) Nannf., Glycyrrhiza uralensis Fisch., Pseudostellaria
heterophylla (Miq.) Pax ex Pax et Hoffm. and Ziziphus jujuba Mill.) (Wu, 2005),
(b) those that tonify the blood and bodily fluids, and
(c) those that tonify yin and yang respectively (see bitter tonics).
Li Shi-Zhen (15181593) was the author of the impressive Compendium of Materia Medica
(Ben Cao gang Mu) or Grand Materia Medica (52 volumes containing 1892 medical
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substances of which 1173 were botanical), in which he systemised folk medicine of all the
previous generations through his own investigation of the listed medicinal substances. Lastly,
the importance of food plants in the holistic healing approach afforded the establishment of
several specialised recipes used for specific ailments and the inclusion of medicinal plants in
food preparation (Wu, 2005).
Traditional medicine from Thailand, Japan (Kampo), Mongolia, Tibet and Korea was influenced
strongly by or is based on Traditional Chinese Medicine. The history of Korean herbology (or
Hanbang) is integrated into the history of the Chinese herbology, i.e. until after the Chinese
Tang dynasty (618907 AD). Most of the traditional Korean medicine knowledge was based
on the work of a few Chinese doctors. Buddhism was introduced in the 4th century (Baker,
1994). During the Goryeo (or Kory) dynasty, from where Korea gets its name (9181392 AD),
Choi Chong-jun produced a book named The Concise Prescriptions of Royal Doctors (1226)
and the medical text First Aid Prescriptions Using Native Ingredients (or Hyangyak
Gugeupbang) in 1245 (Baker, 2008). The Classified Collection of Medical Prescriptions was a
book written by Kim Ye-mong and other Korean official doctors (14431445) and contained
more than 50 000 prescriptions from 152 medical works of ancient China before the fifteenth
century, together with prescriptions from The Concise Prescriptions of Royal Doctors.
Dongeui Bogam or Mirror of Eastern Medicine was written by Heo Jun after the Japanese
invasion in 1592 (Kyung, 2009). It consisted of 25 volumes, divided into five chapters: Internal
Medicine, External Medicine, Miscellaneous Diseases, Remedies and Acupuncture. This work
integrated Korean and Chinese medicine and was influential in Chinese, Japanese and
Vietnamese medicine at the time. Heo Jun emphasised the balance of energies, resulting in
good bodily health. The second physician, Yi Je-ma, systematically theorised the Metabolism
Theory or Sasang typology in his book, "The Principle of Life Preservation in Oriental
Medicine". He found that even if patients suffered the same illness, they needed to use
different herbal applications to treat the same illness due to the different metabolisms of the
individuals. Sasang typology focuses on the individual differences of patients based on body
shape and characteristics. The illness was to be treated by finding the root cause through
proper diagnosis, by first determining the metabolism type of each patient (Leem & Park,
2007). From 19101945, several other Korean physicians contributed to the body of medicinal
knowledge, until early 20th century, before the last Japanese occupation. An important Korean
material medica from this time is the Pangyak happyn (Kim, 2010).
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Even though much of the knowledge upon which traditional Tibetan medicine is based, was
obtained from China during the reign of Namri Songtsen (gNam-ri srong-btsan, also known as
"Namri Lntsen"), the 32nd King of Tibet (Yarlung dynasty), and founder of the Tibetan Empire
(7th century), curative practises existed in the prebuddhistic era in Tibet, and was developed
more than 2 500 years ago (Kletter & Kriechbaum, 2001). The Bon religion, which preceeded
Bhuddism, was founded by gshen rab mi bi che (also known as Tnpa Shenrab Miwoche, born
657 BC), in which four elements were described, and the microcosm and macrocosm of the
world exhibited qualities of these elements. The essence of this religion was compiled in a
work called gso rig mdo dgu bum bshi (708 AD) by the founders son. It comprised four
Tantras and is considered to be the fundamental Tibetan medical treatise (Bauer, 2000). With
the contribution of many scholars, this text was edited and expanded over a period of 1 200
years. The 33rd King, Songtsen Gampo (617650 AD), for instance, invited physicians and
scientists from various countries such as China, Buddhist India, Byzantium and Central Asia to
share their knowledge with Tibetan physicians and scientists for the first time. Each physician
wrote a treatise that was later incorporated into a text called Mijigpe-Tsoncha (A Fearless
Weapon), which contained seven volumes (Martin, 2011).
Shivago Komarpaj (Jvaka Komarabhcca) (ca. 600500 BC), the Buddha's physician, is
considered the founder of traditional Thai medicine (Cousins, 1996). Buddhism, and the
teachings of Shivago, arrived in Thailand between 400 and 600 AD. Traditional Thai medicine
originated from a combination of influences from Indian, Chinese and Southeast Asian
traditions of medicine and applies similar healing principles, i.e. holistic healing and humoral
homeostasis as a basis of vitality, longevity and health (Salguero, 2007). It uses four systems
of treatment, i.e. traditional herbal medicine, psychological treatment, traditional massage and
physical therapy. Two herbs often used for several indications, both as bitter tonics, are
Andrographis paniculata and Tinospora crispa L. (Disayavanish & Disayavanish, 1998). The
Khamphi prathom chinda was the first publication of herbal texts (1908), and was translated to
English, Herbal Medicine in Paediatrics: Translation of a Thai Book of Genesis, by Mulholland
(1989). Currently, traditional Thai medicine is highly diverse, as practiced by traditional doctors
(mo boran or mo phaen boran), who are practicing these healing arts by means of knowledge
gained from traditional texts such as Ayurveda/studies not based on science (elite medicine),
but also by laymen trained orally and rooting their medicine on indigenous Tai animism
(ancient ethnic group scattered throughout south-eastern Asia), Buddhist ritual, Chinese
medicine and Tantric cabalism (Salguero, 2007).
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Kampo, for instance, means the way of the Chinese, where kan is indicative of Chinese
substances, and po means way or method, brought to Japan between the 7th and 9th
centuries. Kampo also uses acupuncture, massage therapy and dietary therapy as with TCM,
but is primarily concerned with the study of herbs (Terasawa, 2004). Similar plants are used in
Japan and China. Astragalus membranaceus, Angelica archangelica and Panax ginseng are
also used in immunomodulatory mixtures (Borchers et al., 2000).
Traditional Mongolian medicine, with its own unique culture and beliefs, developed among
the Mongolian people under Genghis Khan, since the establishment of the Mongol Empire in
1162 (Ratchnevsky, 1991). Mongolian medicine includes mainly herbs, but also minerals in the
form of powdered metals or stones, as well as water (for medicine, not only a solvent). Other
aspects of Traditional Mongolian medicine include moxibustion (burning mugwort over
acupuncture points), bone setting (a form of physiotherapy) and Dom (tradition of household
cures, mostly superstitions) (Lane, 2006).
2.2.2 Oceania and the Americas

2.2.2.1 Bush medicine (Australian traditional medicine)
Bush medicine is the traditional medicinal knowledge and practices used by Aboriginal people
in mainland Australia, Tasmania, and some of the other adjacent islands and are still relevant.
The Aboriginal people are known to be semi-nomadic hunter-gatherers. A second group of
indigenous Australians are called the Torres Strait Islanders, who are indigenous to the Torres
Strait Islands (at the northern-most tip of Queensland near Papua New Guinea). These
Islanders possess a heritage and cultural history distinct from Aboriginal traditions. It is the
general belief among all that the Aboriginal healing system is holistic, personal and social, and
that illness may result from natural or supernatural causes, i.e. physiological and psychological
diseases respectively, due to social and spiritual dysfunction. Natural causes would be treated
with natural remedies, and supernatural illnesses could only be treated with a spiritual cure
(through the tribe's medicine man or ngangkari) (Saethre, 2007). In this instance, Bush
medicine prescribes that the indigenous population should cover themselves from head to toe
in ochre, (white coloured clay used in ceremonies) and ash, as a spiritually medicinal way of
warding off the evil spirits, such as viruses and diseases. This practice came into being after
small-pox, measles and tuberculosis epidemics, brought on by initial European contact with
indigenous populations (17881880), which resulted in the decimation of the Aboriginal
population in Post Settlement Australia (Warren, 2007).
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Different language groups have different bush medicine knowledge, mostly dictated by the
geographical availability of the plant and raw materials required for each individual 'cure'. Bush
medicine is traditionally used in a preventative way, as with rongoa (see New Zealand
traditional medicine), and consists of plant materials such as bark, leaves and seeds, but may
also include animal products, such as emu and goanna fat. The term bush tucker (i.e. bush
food) is used along with Bush medicine and refers to any native fauna/flora used for culinary
and/or medicinal purposes, regardless of the continent or culture it originates from. Healing
practices include steaming, smoking and heating, or topical application by means of rubbing,
i.e. remedies are rarely taken internally (Saethre, 2007). Plants typically used for multiple
indications include Eucalyptus species, i.e. E. camaldulensis Dehnh. and E. terminalis F.Muell.,
Melaleuca species, i.e. M. viridiflora Sol. ex Gaertn., Eremophila longifolia (R.Br.) F.Muell.,
several Cymbopogon Spreng. spp., Mentha australis R.Br., Morinda citrifolia L., Beyeria
lechenaultii (DC.) Baill. and Cochlospermum fraseri Planch. (Lassak & McCarthey, 1983;
Clarke, 2007).
2.2.2.2 Rongo Mori (New Zealand traditional medicine)

Rongo Mori is the traditional healing system of the Mori people, where Rongoa is the Mori
term for native herbal medicines used preventatively and as remedies for several ailments
(Williams, 1996; Jones, 2000). Some of these include Macropiper excelsum (Forst.f.) Miq.,
Pomaderris kumeraho A.Cunn. ex Fenzl., Leptospermum scoparium J.R. & G. Forst. and
Kunzea ericoides A. Rich. (Brooker et al., 1987; Harvey & Waring, 1987; Williams, 1996;
Jones, 2000). If someone is sick, the tohunga (expert practitioner of any skill) would first
determine what imbalance had occurred (by incorporating the spiritual dimension in the
assessment), before the illness could be treated both spiritually and physically (Jones, 2000).
In Mori medicine, muri (spark or life force), wairua (spirit), and tapu (natural law) is involved
in healing, and must be kept in balance. Tapu and noa are concepts concerning the prevention
of illness in the community or individual by obeying the natural law and order of the Mori
world. Tapu is considered the strongest force in Mori life, and transgressing it could result in
sickness, mental illness or death. The connection and imbalance that occurs between the
elements and sickness or medical problems is the key, and is determined by the tohunga
(healer). The illness could then be treated both spiritually and physically by using mirimiri
(massage), rongo (herbal treatments), karakia (spiritual prayer) and wai tapu (water therapy,
including suffusions, steam and heat applications) (Williams, 1996; Jones, 2000).
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2.2.2.3 North American Indian and eclectic medicine

Native Americans are also known as First Nations, Indians, Red Indians and American Indians
(or Amerindians) (Jacobs, 2002). The Iroquois, also known as the Haudenosaunee or the
"People of the Longhouse" are associated with several indigenous tribes people of North
America (since ca.1000) (Frey & Meyers, 2010). Their philosophy of healing centres around a
power called Orenda, present in animals, plants, humans, ancestors, elements and objects.
Orenda manifests in natural phenomena such as thunder, wind and storm. Life is visualised as
a constant dynamic interchange between one Orenda and another. When both powers have
equal strength, there is a balance of power, i.e. a most fortunate occurrence and concentration
of strength. Sickness is caused by an imbalance in power, but also by the presence of a bad
spirit. For this reason, American Indian medicine men, or women, may be both healers and
priests (shamans) who inherited their professions. Treatment is holistic, i.e. the spirits are
consulted, and treatments may involve herbal cures, washing or cleansing with water,
massages or casting out of spirits (Baier-Kleinow, 1992). Traditionally, herbal remedies were
prepared from locally available ingredients, with tribes developing their own herbal remedies,
often by observing ailing animals to see what plants they would eat to heal and get well (Bial,
1999). Some plants, such as Achillea millefolium L., Asclepias syriaca L., Ipomoea pandurata
G.Mey and Malva neglecta Wallr., are considered to be powerful panacea, used to treat
physical and spiritual ailments, while Cimicifuga racemosa L., Hieracium pilosella L.,
Lycopodium digitatum Dill. ex A.Braun, Ranunculus acris L. and Solidago canadensis L. are
not classified as powerful, but are used for a diversity of ailments (Frey & Meyers, 2010).
Eclectic medicine is a combination of Native American and European herbal medicine,
proposed by Wooster Beech (ca. 1830). Famous examples of plants promoted by this system
are Echinacea purpurea and Hydrastis canadensis (Van Wyk & Wink, 2004).
2.2.2.4 Meso and South American traditional medicine

Ayahuasca (or ayawaska) means "spirit vine" or "vine of the souls" in the Quechua language.
It comprises any of various psychoactive infusions or decoctions prepared from the vines of
Banisteriopsis species, usually mixed with other plant species, and is used for divinatory and
healing purposes by the native peoples of Amazonian Colombia (Ecuador, Bolivia and Peru,
and to a lesser extent, Brazil) (McKenna et al., 1998). While Ayahuasca is claimed to be able
to cure mental and physical illness, the effects are mainly hallucinogenic (the active principle in
Banisteriopsis caapi is harmaline, an alkaloid recognised as a scheduled narcotic drug in most
countries Callaway et al., 1999) and does not appear to have any tonic effects.
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Maya is a Meso-American civilisation established between ca. 2000 BC250 AD. This
civilisation is characterised by a high degree of interaction and cultural diffusion over a vast
and varied landscape, from the elevated terrain in Guatemala and the Chiapas highlands in the
south, to the southern lowlands just north of the highlands, incorporating northern Guatemala,
Belize, and the southern portions of Mexico, and to the northern lowlands, covering the
remainder of the Yucatn Peninsula and the Puuc hills in Mexico. In ancient Maya healing
systems, medicine was practiced only by a select few shamans known as ah-men, who
generally inherited their positions and received extensive education (Kunow, 2003). The Maya
equated sickness with the captivity of ones soul by supernatural beings, angered by some
perceived misbehaviour. For this reason, curing a sickness involved elements of ritual,
cleansing, and often herbal remedy by means of a codified divinatory system, which translates
the behavioural, biological, and physical conditions in which a patient finds him/herself, and
how it relates to the universe (Kunow, 2003; Winkelman & Peek, 2004). Herbal remedies were
eaten, drank, smoked, snorted, rubbed on the skin, and even used as enemas to force rapid
absorption of a substance into the blood stream. Cleansing techniques included fasting,
sweating, and purging flushed substances out of the body (Winkelman & Peek, 2004). The
Mayan doctors would, however, also employ specialists for specific healing techniques such as
bone-setting and child birth, or perform surgery where necessary (Kunow, 2003).
Aztec medicine was practiced since the establishment of the Aztec Empire (13001600 AD)
(Nahuatl-speaking ethnic groups of central Mexico), and dominated large parts of MesoAmerica (from central Mexico to Belize, Guatemala, El Salvador, Honduras, Nicaragua, and
Costa Rica). According to the Nahua people, the universe is divided into four realms (i.e. the
sky, earth, underworld, and water place). Myriads of spirits occupy all four realms and affect
human behaviour in varying degrees. Everything, including plants, animals, humans (both dead
and alive) and certain objects, have a yolotl (life force) and is consequently connected to a
universal deity. Only human beings have both a yolotl, and a tonali (roughly explained as a
talent or aptitude, closely related to individual energy). Religion and divination are closely
related in Nahua medicine, and, as with other native American medicine systems, the Nahua
shaman (or Curandero) would tend to all types of illnesses, whether physical and spirituallyrelated, with the purpose to determine the causes of illness, i.e. disharmony or imbalance that
has occurred between the patient and his or her physical and social surroundings. Ailments
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beyond the scope of the Curanderos would be treated by Hueseros (bone-setters), Parteras
(midwives) or Sobadores (healers through massage therapy) (Aguilar-Moreno, 2007).
Plants used for various indications by the indigenous healers of the Meso-American healing
systems, i.e. Peruvian Indians, Maya or Aztec medicine, include Lepidium meyenii L.,
Capsicum annuum L., Erythroxylum coca Lam., Agave americana L., Lophophora williamsi
(Lem.) J.M.Coult., Calliandra anomala (Kunth) Macbride, Lycopodium clavatum L. and L.
complanatum L., Casimiroa sapota Oerst. and C. edulis Llave. (Morton, 1987; Ratsch, 1998;
Kunow, 2003; Gonzales et al., 2006).
Traditional Brazilian medicine is a combination of native South American and African
practices. It is used by indigenous groups and the black Native American mestizo population
(people of mixed European and Native American descent, classified as a group since 1582),
from the Northeast coast and in nearly all interior regions, including Amazon regions,
savannahs, rainforest, foothills, and Pantanal (tropical wetland). Plants used include edible
foods, such as cashew nuts, peppers, mangosteen and coconut, while the medicines comprise
the inedible parts of these foods, such as the fruits, leaves, husks and bark (Botsaris, 1997).
Plants specifically indicated as tonics include Achyrocline satureioides (Lam.) DC., Bowdichia
virgilioides Kunth, Drimys winteri J.R.Frost and Senna occidentalis (L.) Link (Brando et al.,
2008), while other such as Ipomoea cairica (L.), Pfaffia paniculata Kuntze., Baccharis trimera
(Less.) DC., Davilla rugosa Poir. and Euterpe oleracea Mart., are used for multiple indications
(Plotkin & Balick, 1984; Zardini, 1984; Vieira, 1999; Mendez et al., 2007).
2.2.3 Africa
Traditional African Medicine is one of the oldest healing systems, encompassing great
biological and cultural diversity, which is unfortunately poorly recorded. In general, it shows
much similarity to traditional healing in South America, in that disease is a result of wrongdoing or imbalance, and treatment incorporates consultation of (ancestral) spirits.
Neuwinger (2000) produced a useful literature review of all African medicinal plants and their
uses. Many plants have multiple uses and may therefore serve as tonics, but given here are
only plant species explicitly listed as tonics:
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Abrus precatorius L.
Acacia dudgeon Craib; A. etbaica Schweinf.
Acalypha psilostachya Hochst. ex A.Rich
Achyranthes aspera L.
Acokanthera schimperi (A.DC.) Schweinf.
Acridocarpus plagiopterus Guill. & Perr.
Adenia venenata Forssk.
Aeschynomene multicaulis Harms
Aframomum alboviolaceum (Ridley) K.Schum
Afzelia bella Harms
Ageratum conyzoides L.
Agrocharis incognita (Normann) Heywood & Jury
Albizia tanganyicensis Baker f.
Albizia zygia (DC.) J.F. Macbr.
Alchornea cordifolia (Schumach. & Thonn.) Mll. Arg.; A. floribunda Mll-Arg.
Allophylus chaunostachys Gilg.
Amaranthus graecizans L. Lam. subsp. graecizans
Ampelocissus multistriata (Baker) Planch.
Anacardium occidentale L.
Ancylanthos rubiginosus Desf.
Angylocalyx oligophyllus (Baker) Baker f.
Annona arenaria Thonn.; A. senegalensis Pers.
Anogeissus leiocarpus (DC.) Guill. & Perr.
Anthospermum rigidum Eckl. & Zeyh. subsp. rigidum
Apium graveolens L.
Aptosimum albomarginatum Marloth & Engl.
Asclepias physocarpa (E.Mey.) Schltr.
Asparagus aethiopicus L.
Barteria fistulosa Mast.
Basella alba L.
Biophytum helenae Buscal. & Muschl.
Borassus aethiopum Mart.
Bothrioclyne longipes (Oliv. & Hiern) N.E.Br.
Brachylaena discolor DC.
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Bridelia micrantha (Hochst.) Baill.

Bryophyllum pinnatum (Lam.) Oken
Buchholzia coriacea Engl.
Buddleja polystachya Fresen.; B. salviifolia (L.) Lam.
Calantia cerasiflora Tul.
Callilepis leptophylla Harv.
Calotropis procera (Aiton) Aiton f.
Calpurnea aurea (Aiton) Benth.
Canarium schweinfurthii Engl.
Cannabis sativa L.
Capparis afzelii Pax
Capsicum annuum L.
Caralluma dalzielii N.E. Br.
Carapa procera DC.
Carduus nyassanus (S.Moore) R.E.Fr.
Carissa edulis (Forssk.) Vahl
Cassia abbreviata Oliv. subsp. beareana (Holmes) Brenan
Catha edulis (Vahl) Forssk. ex Endl.
Ceiba pentandra (L.) Gaertn.
Celosia argentea L.
Centella asiatica (L.) Urb.
Chamaecrista mimosoides (L.) Greeme
Chasmanthera dependens Hochst.
Chytranthus atroviolaceus Hutch. & Dalziel
Cichorium intybus L.
Cissus doeringii Gilg & Brandt; C. leonardii Dewit
Citropsis articulata (Spreng.) Swingle & Kellerm.
Clausena anisata (Willd.) Hook. f.
Cleistachne sorghoides Benth.
Clematis simensis Fresen.
Clematopsis scabiosifolia (DC.) Hutch.
Clerodendrum johnstonii Oliv.; C. myricoides (Hochst.) Vatke
Clutia abyssinica Jaub. &Spach
30
Chapter 2
Cola acuminata (P.Beauv.) Schott & Endl.; C. anomala K. Schum.; C. gigantean var.
glabrescens Brenan & Keay; C. hispida Brenan & Keay; C. nitida (Vent.) Schott & Endl.;
C. verticillata (Thonn.) Stapf ex A.Chev.
Combretum kraussii Hochst.; C. platypterum (Welw.) Hutch. & Dalziel
Corchorus olitorius L.
Cordia dewevrei De Wild.
Cordyla pinnata (Lepr. ex A.Rich.) Milne-Redh.
Corynanthe pachyceras K.Schum.
Crossoperyx febrifuga (Afzel.) Benth.
Crotalaria agatiflora Schweinf. subsp. imperalis (Taub.) Polhill
Croton dichogamus Pax; C. longiracemosus Hutch.; C. machrostachyrus Hochst. ex Delile;
C. mubango Mll.Arg.
Cryptolepis sanguinolenta (Lindl.) Schltr.
Cryptosepalum maraviensis Oliv.
Culcasia scandens P.Beauv.
Cussonia arborea Hochst. ex A.Rich.
Cuviera longiflora Hiern
Cymbopogon nervatus (Hochst.) Chiov.
Cyphostemma serpens (A.Rich.) Desc. subsp. serpens
Dennettia tripetala Baker f.
Detarium senegalense G.F.Gmel.
Dichapetalum lugae T.Durand & De Wild.
Dichrostachys cinerera (L.) Wight & Arn.
Dicoma zeyheri Sond. and subspecies
Diospyros mespiliformis A.DC.
Discopodium penninervium Hochst.
Dissotis pterocaulos Wickens
Dombeya goetzenii K.Schum.
Dracaena usambarensis Engl.
Drypetes gossweileri S.Moore
Echinops giganteus A.Rich
Elaeodendron buchananii (Loes.) Loes.
Elionurus ciliaris Kunth
31
Chapter 2
Ellataria cardamomum Maton

Embelia schimperi Vatke
Emilia caespitosa Oliv.; E. coccinea (Sims) G.Don
Entada abyssinica Steud. ex A.Rich; E. africana Guill. & Perr.; E. rheedei Spreng.
Entandrophragma utile (Dawe & Sprague) Sprague
Eriosema montanum Baker f.; E. rhynchosioides Baker
Erythrina senegalensis DC.
Erythrophleum ivorense A.Chev.
Euadenia eminens Hook f.
Euclea divinorum Hiern; E. divinorum Hiern subsp keniensis (R.E.Fr.) De Wit
Faurea saligna Harv.
Ferula communis L.
Ficus asperifolia Miq.; F. exasparata Vahl.; F. scott-elliotii Mildb. & Burret
Flueggea virosa Baill.
Foeniculum vulgare Mill.
Garcinia huillensis Welw. ex Oliv.
Gardenia ternifolia Schumach. & Thonn.; G. ternifolia subsp. jovis-tonantis (Welw.) Verdc. var.
goetzei (Stapf & Hutch.) Verdc.
Gerbera piloselloides (L.) Cass.
Gisekia phanaceoides L.
Gladiolus daleni Van Geel
Gnaphalium luteoalbum L.
Gossypium herbaceum L.
Gouania longispicata Engl.
Grewia carpinifolia Juss.; G. tenax (Forssk.) Fiori
Guizotia scabra (Visc.) Chiov.
Gutenbergia cordifolia Benth. ex Oliv.
Hagenia abyssinica (Bruce) J.F.Gmel.
Hallea rubrostipulata (K.Schum.)J.-F.Leroy
Harpagophytum procumbens DC. ex Meisn. subsp. procumbens
Heisteria zimmereri Engl.
Helichrysum forskahlii (J.M.Gmel.) Hilliard; H. gerberifolium Sch. Bip.; H. odoratissimum (L.)
Sweet; H. schimperi (Sch.Bip.) Moeser
32
Chapter 2
Helinus integrifolius (Lam.) Kuntze

Heteromorpha trifoliata (H.L.Wendl.) Eckl. & Zeyh.
Hibiscus fuscus Garcke; H. sabdariffa L.; H. tiliaceus L.
Holoptelea grandis (Hutch.) Mildbr.
Hypericum revolutum Vahl.
Hypoxis hemerocallidea Fisch. & C.A.Mey.
Hyptis spicigera Lam.
Indigofera atriceps Hook. f.; I. simplicifolia Lam.
Irvingia grandiflora (Engl.) Engl.
Isolona cooperi Hutch. & Dalziel ex Cooper & Record
Jateorhiza palmata (Lam.) Miers
Jatropha curcas L.
Jaundea pinnata (P.Beauv.) Schellenb.
Khaya senegalensis (Desr.) A.Juss.
Kleinia longiflora DC.
Macrosphyra longistyla (DC.) Hook. f.
Maerua trichophylla Gilg
Maesa lanceolata Forssk. var. mildbraedii (Gilg & Schellenb.) Taton
Manilkara multinervis (Baker) Dubard
Maranthes glabra (Oliv.) Prance; M. kerstingii (Engl.) Prance; M. robusta (Oliv.) Prance
Marantochloa leucantha (K.Schum.) Milne-Redh.
Margaritaria discoidea (Baill.) G.L.Webster
Maytenus acuminata (L. f.) Loes.; M. senegalensis (Lam.) Exell.; M. undata (Thunb.) Blakelock
Melianthus comosus Vahl.
Microglossa pyrifolia (Lam.) Kuntze
Micromeria purtschelleri Grke
Micilia excelsa (Welw.) C.C.Berg
Mitragyna inermis (Willd.) Kuntze
Momordica foetida Schumach. & Thonn.
Monodora myristica (Gaertn.) Dunal
Morinda morindoides (Baker) Milne-Redh.
Mussaenda tenuifloa Benth.
Myrianthus serratus (Trcul) Benth. & Hook.
33
Chapter 2
Myrica kilimandscharica Engl.; M. salicifolia Hochst. ex A.Rich.

Nicotiana tabacum L.
Ocimum americanum L. var. americanum
Okoubaka michelsonii J.Lonard & Troupin
Olea woodiana Knobl.
Oncoba spinosa Forssk.
Opilia celtidifolia (Guill. & Perr.) Endl. ex Walp.
Osyris tenuifolia Engl.
Oxalis corniculata L.
Oxytenanthera abyssinica (A.Rich.) Munro
Palisota hirsuta (Thunb.) K.Schum.
Panda oleosa Pierre
Pappea capensis Eckl. & Zeyh.
Parinari excelsa Sabine
Parkia biglobosa (Jacq.) R.Br. ex Don; P. oliveri J.F.Macbr.
Passiflora foetida L.
Paullinia pinnata L.
Pauridiantha canthiiflora Hook. f.
Pausinystalia johimbe (K.Schum.) Pierre
Pavetta lanceolata Eckl.; P. ternifolia (Oliv.) Hiern.
Pentaclethra macrophylla Benth.
Pentanisia ouranogyne S.Moore
Pentas zanzibarica (Klotzsch) Vatke
Pentopetia alba Jum. & H.Perrier
Pericopsis angolensis (Baker) Meeuwen
Periploca linearifolia Quart-Dill. & A.Rich.
Petersianthus macrocarpus (P.Beauv.) Liben
Phoenix reclinata Jacq.
Phyllanthus nummulariifolius Poir.
Physena sessiliflora Tul.
Phytolacca dodecandra LHr.
Piper cubeba L. f.; P. guineense Schumach. & Thonn.; P. nigrum L.; P. umbellatum L.
Pittosporum spathicalyx De Wild.
34
Chapter 2
Plantago palmata Hook. f.

Platostoma africanum P.Beauv.
Plectranthus lanuginosus (Benth.) Agnew
Polygonum nepalense Meisn.
Pseuderanthenum tunicanum (Afzel.) Milne-Redh.
Psophocarpus palustris Desv.
Pulicaria scabra (Thunb.) Druce
Pycnostachys erici-rosenii R.E.Fr.
Quassia africana (Baill.) Baill.
Ravensara anisata Danguy
Reissantia indica (Willd.) N.Hall var. loeseneriana (Hutch. & M.B.Moss) N.Hall
Rhabdophyllum welwitschii Van Tiegh.
Rhamnus prinoides LHr.
Rhynchosia luteola (Hiern) K.Schum.; R. minima (L.) DC. var. prostrata (Harv.) Meikle
Ricinodendron heudelotii subsp. africanum (Mll. Arg.) J.Lonard
Ricinus communis L.
Rothmannia octomera (Hook.) Fagerl.
Rubus apetalus Poir.
Rytigynia monantha (K.Schum.) Robyns
Salvadora persica L.
Sansevieria liberica Gerard & Labr.
Sapium ellipticum (Hochst.) Pax
Schizogya coffeoides Baill.
Schrebera alata (Hochst.) Welw.
Securidaca longepedunculata Fresen.
Senecio latifolius DC.
Senna didymobotrya (Fresen.) H.S.Irwin & Barneby
Sesamum angustifolium (Oliv.) Engl.
Sigesbeckia abyssinica (Sch.Bip.) Oliv. & Hiern.
Silene burchellii Otth. var. burchellii
Smeathmannia laevigata Sol. ex R.Br.
Smilax anceps Willd.
Solanum aculeastrum Dunal var. albifolium (C.H.Wright)
35
Chapter 2
Spermacoce princeae (K.Schum.) Verdc.

Sphaeranthus bullatus Mattf.; S. suaveolens (Forssk.) DC.
Spilanthes acmella (L.) Murill
Stachys spathulata Burch. ex Benth.
Stachytarpheta jamaicensis (L.) Vahl.
Stephania abyssinica (Dill. & A.Rich.) Walp.
Sterculia appendiculata K.Schum.; S. quinqueloba (Garcke) K.Schum.
Strophanthus sarmentosus DC.
Stylosanthes erecta P.Beauv.; S. fruticosa (Retz) Alston
Sutherlandia frutescens (L.) R.Br. ex Aiton
Swartzia madagascariensis Desv.
Syzygium guineense (Willd.) DC.; S. rowlandii Sprague
Tabernaemontana crassa Benth.
Terminalia macrocarpa Guill. & Perr.; T. sericea Burch. ex DC.
Tetrapleura tetraptera (Schumach. & Tonn.) Taub.
Teucrium africanum Thunb.; T. incanum Aitch. & Hemsl.; T. kraussii Codd.
Thomandersia laurifolia (T.Anderson ex Benth.) Baill.
Thunbergia alata Bojer ex Sims
Treculia africana Decne.
Trema orientalis (L.) Blume
Tricalysia pallens Hiern.
Trichoscypha acuminata Engl.
Trilepisium madagascariense DC.
Turraea heterophylla Sm.
Uncaria africana G.Don
Vernonia aemulans Vatke; V. colorata (Willd.) Drake and subspecies; V. lasiopus O.Hoffm.;
V. nudicaulis Less.; V. abyssinica Fresen.
Vicia paucifolia Baker
Vismia guineensis (L.) Choisy
Vitex madiensis Oliv.; V. rivulatis Grke
Voacanga africana Stapf
Withania somnifera (L.) Dunal
36
Chapter 2
Xylopia buxifolia Baill.

Xysmalobium undulatum (L.) Aiton f.
Zanthoxylum chalybeum Engl.; Z. davyi (Verd.) P.G.Waterman
Zehneria scabra (L. f.) Sond.
Zingiber zerumbet Sm.
Ziziphus mucronata Willd.
The long list of species indicates that the concept of African tonic plants is complicated and
that it is in need of scientific study. Normally the tonics are used as strengthening medicines
and stimulating agents, sometimes aimed at specific outcomes such as curbing fatigue,
improving general health (during or after illness), to calm nerves and to enrich the blood.
They are traditionally prescribed for women (as uterotonics, for pregnant women and after
delivery), for men (also as aphrodisiacs) and for children of all ages. A variety of plant parts
(i.e. root, bark, leaves and fruit) are used as tonics. Preparations may be applied topically or as
enemas, or the tonics may be taken orally without prior extraction, as decoctions or infusions or
together with food or other plants.
2.2.3.1 Islamic medicine (North African):

The most important contributions to Islamic medicine during the Islamic Golden Age (750
1258 AD) are discussed in Paragraph 2.2.1.1. Colonisation of North Africa started from Europe
and Western Asia by Greeks and Phoenicians at this time (570 BCca. 600 AD). After several
rulerships, including Roman and Byzantine rule, North Africa eventually fell to the Arabs in the
7th century, who introduced the Islamic religion, its medicine and the Arabic language (see
Fig. 2.1). Islamic religion (and medicine) is mostly practiced among people in the Afro-Asiatic
language groups, but has spread to much of the Nilo-Saharan and Niger-Congo language
groups as well. Under colonial rule, the practice of traditional healing and the selling of herbal
medicines were declared illegal by the colonial authorities, as it was considered to be witchcraft
(Helwig, 2001). From 600 AD, Arab trade with sub-Saharan Africa resulted in a gradual
colonisation of East Africa, around Zanzibar and other bases. Trans-Saharan trade resulted in
a small number of West African cities developing Arab quarters. Islamic medicine consequently
spread, as colonisation took place, and plants often used for several ailments included species
of European and North African origin such as Artemisia herba-alba Asso, Ballota nigra L.,
Chamomilla recutita (L.) Rauschert, Cichorium intybus L., Geranium robertianum L., Lavandula
stoechas L., Marrubium vulgare L., Rubia tinctorum L., Salvia officinalis L. and Ziziphus lotus
(L.) Lam. (Boulos, 1983).
37
Chapter 2
2.2.3.2 Yorb medicine

Yorb or Orisha medicine (egbogi) originated from a religious text, the Ifa Corpus, revealed by
the prophet Orunmilla in the ancient city of Ile-Ife (ca 2000 BC), situated in a cultural region in
Nigeria, Benin and Togo, called Yarubaland (see Fig. 2.1). It is claimed that Orunmilla not only
advised his people on spiritual baths, meditation and herbal medicine, but also taught the
customs of divination, prayer, dance, symbolic gestures, personal, and communal elevation.
Slave trade from West Africa to the Americas afforded the spread of Yorb medicine to the
Caribbean from 16501860, and it is now practiced generally in West Africa and the Caribbean
(Falola & Usman, 2009). It deviates from Traditional African Medicine (see Bantu medicine in
the section that follows), as the Yorb people believe that tiny, invisible "germs" or insects
(kokoro) and worms (aron) inhabit small bags within the body. These germs and worms
perform useful functions in the healthy body, aiding among others, digestion and fertility.
However, if these tiny organisms become too powerful, they cause illnesses and must be
controlled by killing or expelling them from the body with bitter-tasting plants, contained in
medicines. These views of the human body and its reaction to illness are derived from the
image of a cooking pot, susceptible to overflowing (Buckley, 1985). According to tradition,
comparable to that of the Nahuatl-speaking ethnic groups in South America, there are many
deities or divinities (Orishas) that play a significant role in the life of the Yorb people.
Osanyin or Osain (the whispering genie) is deemed one of the more important deities, as he
dominates over all wild herbs, and is considered as the greatest herbalist that ever lived by
most practitioners. Furthermore, plants and herbs carry "mystical value" apart from their
"medicinal value", and may consequently also be used by a Yorb herbalist (Onisegun) for
incantation (ofo) to bring good luck (awure). Also typical of native American traditional healing,
a holistic approach is followed, with an investigation directed at the spiritual and physical
causes of the illness. While curative prescriptions include herbal remedies to address
symptoms, the complete treatment is holistic in nature (Buckley, 1985). Important Yorb
medicines include Pleioceras barteri Baill., Calotropis procera, Emilia coccinea, Euadenia
trifoliolata (Schumach. & Thonn.) Oliv., Byrsocarpus coccineus Thonn. ex Schumach., Telfaria
occidentalis Hook. f. and Baphia nitida Lodd. (Nigeria Natural Medicine Development Agency,
2005).
2.2.3.3 Bantu traditional medicine

The Bantu languages (spoken by more than 400 ethnic groups, including Zulu, Xhosa,
Ndebele, Sotho, Swazi, Shona and Swahili), a traditional sub-branch of the NigerCongo
38
Chapter 2
languages, are spoken in central Africa, east Africa, and southern Africa (see Fig. 2.1, Falola &
Usman, 2009). The original proto-Bantu language group is approximately 2 5003 000 years
old and migrated eastwards and southwards, where agriculture is suggested to have played a
major role in their populating the Sub-Saharan region.
Treatment of illnesses involves a consultation with a herbalist first, and if the illness cannot be
identified easily or treated effectively, the patient is referred to a diviner, who would then come
in contact with the spirit world, in order to provide a diagnosis and cure, with the aid of
medication, charms and/or sacrifices (Onwuanibe, 1979). Midwives and bone setters are also
regarded as traditional healers (Van Wyk & Gericke, 2000). Diviners, such as the South African
sangomas or sanusi (in Zulu), amaquira (in Xhosa) or dingaka ta ditaola (in Sotho), are
traditional Nguni healers, able to practice herbal medicine, divination and also act as
counsellors. They may be graded as high, medium or low classes of diviners, based on their
experience and abilities. Their social and political roles in the community also include finding
lost cattle, protecting warriors, counteracting witches, and narrating the history, cosmology, and
myths of their tradition (Kale, 1995). Vusamazulu Credo Mutwa is a famous example of a wellrespected sangoma, who have authored several books on his experiences (Mutwa et al.,
2001). Diviners are distinguished from prophets, spiritual healers (Zulu: abathandazi),
witchdoctors (Zulu: abathakathi), spirit mediums, intuitives and dreamers (Van Wyk &
Gericke, 2000; Ndhlala et al., 2011). An example is the inyanga, a traditional herbal healer
(inyanga is a Zulu word literally meaning "the man of the trees", plural: izinyanga; ixwele in
Xhosa; dingaka ta dihlare in Sotho; Nanga in Shona (Zimbabwe); bossiedokter or kruiedokter
in Afrikaans Bryant, 1966; Shaw, 1993; Van Wyk & Gericke, 2000), who heals by using
herbs, but often does not involve divination when doing so. Variations of the term inyanga are
used in other countries, i.e. in Swahili, mganga refers to a qualified physician or traditional
healer, in Haiti the Bantu term nganga indicates a voodoo high priest, and in Cuba, Angola and
the Democratic Republic of the Congo (DRC) it refers to a mixture prepared in an iron pot into
which several items are placed, or may refer to the spirit of the dead that resides there (Ballard,
2006). In the Sotho culture, diviners are automatically also herbalists, and those who are
merely medicine suppliers (dingaka te dithupa) are distinguished from herbalists (Kriel,
1998). Many traditional medicinal practitioners have no formal western education, and inherited
knowledge of medicinal plants, appropriate methods of collecting the plants and preparing
remedies, as well as their effects on the human body, from their forebears, orally or in
apprenticeship with an established practitioner (Onwuanibe, 1979).
39
Chapter 2
Fig. 2.1 Distribution of African language families over Africa (adapted from Ruhlen, 1991).
Herbal medicines prescribed by healers are called muti (or muthi), a term derived from a Zulu
word for "tree," used in South Africa and as far north as Lake Tanganyika (a large lake of Africa
shared by Burundi, DRC, Tanzania and Zambia).The term is also used loosely in Afrikaans or
South African English for general medicine. Muti may be employed in healing either bodily or
spiritual conditions, according to the opinion of the healer. Traditional medicine makes
extensive use of botanical products, but may include other formulations, such as those from
zoological origin or mineral composition, and may be used for topical application, as internal
medicines, aromatics to be inhaled, or charms to be worn on the body (imfingo), or to be
displayed/sprinkled (intelezi) in or around the homestead (Bryant, 1966; Ndhlala et al., 2011.
Kokwaro (1993) listed muti plants used as tonics in East Africa, among others Centella
asiatica, Euclea divinorum, Helichrysum gerberifolium and H. nudifolium, Heteromorpha
trifoliata, Myrica salicifolia and Pentanisia ouranogyne. Sotho medicines include those used for
a specific physiological function (e.g. an analgesic), those that strengthen the spirit (seriti) and
those that cleanse, purify or cool (misfortune, evil and certain illnesses are associated with
heat) (Kriel, 1998). Maatla (or amandla in Nguni) is the vital force present in all living
organisms, and may be transferred from one to the other, i.e. from the root of a medicinal plant
(where maatla is concentrated) to a sick person (Vorster, 2001). An example of a cooling
medicine is the bulb of Rhoicissus tridentata (L.f.) Wild & R.B.Drumm. (Sotho: mopidikwa; Zulu:
isinwazi), used to treat various ailments, including fever, gastro-intestinal problems, gonorrhea
and epilepsy (Van Wyk & Gericke, 2000).
40
Chapter 2
2.2.3.4 Khoi-San and Cape Dutch medicine

Khoi-San is a unifying name describing two completely separate ethnic groups in southern
Africa, which are distinct from the Bantu. The |Xam, |Kung and other groups are examples of
the San or Bushman, the first inhabitants of southern Africa, and are considered one of the
oldest (70 000100 000 years old) existing populations on earth, based on genetic studies
(Tishkoff et al., 2007). These hunter-gatherers now live mainly in the Kalahari Desert in
Namibia, Botswana and in Angola (see Fig. 2.1). The Khoi are thought to have originated from
the northern area of Botswana and migrated southwards over time. Approximately 2 000 years
ago they were practicing their pastoral agriculture in the Cape region. When the southward
migrating Bantu reached the area (ca. 300 AD), they were forced to more arid regions where
they sometimes intermarried the San (Barnard, 1992).
The religion of the San people consists of a spiritual and a material world. Gu|e (or Tsui Goab,
|Kaang, |Kaggen, Khub or Nanub according to the Khoi) (| here represents the various clicking
sounds commonly used in the spoken language) is the supreme being, the creator god, who
gives life and has taught the Khoi-San which veldfood to eat, i.e. which are poisons and which
are medicines. This knowledge is passed on verbally from generation to generation. Any
wrong-doing, including taking from nature without purpose or taking more than what meets the
needs of the community, is punished by sickness or death. An alternative Khoi sky god was
Utixo (or |oa in San religion), also known to the Xhosa. The Khoi also have Ga-gorib (god or
evil spirit) who is the bringer of fate and death, but is submissive to Tsui Goab (Heinz, 1975;
Cotterell, 1979). The Nama (a group within the Khoi) has three main categories of healers: the
diviner (|gua aup), often female, who is able to treat serious ailments, the herbalist
(bossiedokter), who uses herbs to treat minor and chronic ailments, and the poison or snake
doctor, who treats snake or scorpion bites. While many Khoi-San have a sound knowledge of
herb usage for general first aid, the term bossiedokter indicates a highly skilled and
experienced herbalist. Clear differentiation is made between the medical treatment of men and
women (Van Wyk, 2008a). Animal parts/products are included in many preparations, e.g. milk,
fat, dried intestines, dung or urine (Laidler, 1928; Archer, 1990). Massage and aromatherapy,
accompanied with a trance dance, is also applied in healing (Winkelman & Dobkin de Rios,
1989). Three of the tonic plant species used by the Khoi-San, as suggested by archaeological
evidence, are Harpagophytum procumbens (Wilman, 1968), Aloe ferox Mill. (Reynolds, 1950)
and Hypoxis species (Wells, 1965).
41
Chapter 2
While the early Dutch settlers introduced European medicinal plants initially, geographical
isolation necessitated the use of local plants. A unique medicinal culture, called Cape Dutch
medicine, was the result. This medicinal system is still widely used in the Cape region of South
Africa and has been reviewed by Van Wyk (2008a), who called the system Cape Herbal
Medicine for the first time. Van der Stel, Thunberg, Burchell, Harvey and Sonder, and later
Marloth, were pioneers in contributing to the recording of the indigenous ethnobotany and
materia medica of southern Africa (Van Wyk, 2008a). Pappe (1847, 1850, 1857), Smith (1895)
and Kling (1923) authored the first books focusing on South African medicinal plants. Some of
the plant species listed as tonics by these authors include Agathosma betulina (P.J. Bergius)
Pillans and A. crenulata (L.) Pillans, Artemisia afra Jacq. ex Willd., Dicoma capensis Less.,
Dodonaea angustifolia L.f., Oncosiphon suffruticosum L., Osmitopsis asteriscoides (P.J.
Bergius) Less., Sutherlandia frutescens, Teucrium africanum and Withania somnifera.
2.3 The concept tonic in the southern African context

In southern Africa, the tonic concept has been mostly influenced by the Khoi-San, Cape Dutch,
Zulu, Sotho and Xhosa cultures. In the Zulu culture, three types of muti may be used to treat an
ailment, depending on its seriousness (Bryant, 1966): imiThi emnyama (black medicines,
tshidi in Sotho), indicative of their colour, are drastic in nature with great potency to expel the
evil responsible for the ailment; imiThi emhlophe (white medicines), also indicative of their
colour, are to be administered after black medicines as tonics or sedatives to counter any
damaging effects caused by the ailment or the black medicine and restore health completely;
amaKhambhi (green medicines) are freshly gathered herbs and roots. The latter is used most
commonly and includes the largest variety of medicines (Bryant, 1966). In the Sotho culture,
red medicines are also very important, as red is associated with blood and good health. In the
treatment of weakness, the San people believe that plants with red parts are used for anaemia,
weakness (i.e. to strengthen the blood) and repeatedly for fevers (Laidler, 1928). Hence, the
importance of blood purification, where plant parts with a red colour, or those that produce
red extracts, are used, e.g. Elephantorrhiza elephantina (Burch.) Skeels has a reddish root and
Pterocarpus angolensis DC. bark produces red sap (Vorster, 2001).
The first reference to plants as tonics in the South African literature was made by Smith (1895),
when he described Artemisia afra and Sutherlandia frutescens as tonics. Tonic plants are
known as imbizas in traditional medicine in southern Africa (Van Wyk & Gericke, 2000).
42
Chapter 2
According to Ngubane (1977), imbiza is the generic Zulu name for plant mixtures that impart
strength, health and vigour, normally as herbal preparations of a single plant or mixtures of
plants which are administered orally for a purgative action, or as enemas. Van Wyk and
Gericke (2000) define imbizas as strengthening plant combinations which are believed to play
a significant role in maintaining health and vigour, consequently describing the tonic properties
of these mixtures. The Zulu word imbiza refers to the traditional Zulu cooking pot in which
decoctions were prepared (L. Posthumus, personal communication to B.-E. van Wyk, 2011). In
the Sotho culture, the term musa-pelo is used for 17 different species of shrubby legumes
(Fabaceae), from which medicine is prepared, including Sutherlandia frutescens. These plants
are used to treat anxiety, stress and grief; musa-pelo literally means to turn the heart around
(Moteetee & Van Wyk, 2007). Ndhlala et al. (2011) found that herbal mixtures sold as general
tonics throughout KwaZulu-Natal, often bear names associated with the strength of powerful
animals, such as ingwe izifozonke, literally translated as leopard all diseases, indicating that
the medicine is as strong as a leopard and can cure numerous conditions. The names of
mixtures can also indicate the method of administration (e.g. imbiza ephuzwato means a
medicine to drink) or an indication of what the mixture is intended for (e.g. umzimba omubi
means bad skin).
In this study, the tonic properties of 14 medicinal plant species and their close relatives, all
indigenous to southern Africa, were investigated with regard to their ethnobotany and chemical
compositions. These species were explicitly listed by Van Wyk & Gericke (2000) as tonic
plants, based on the experience of these authors after studying traditional medicine in southern
Africa for many years. The 14 species are: Agathosma betulina, Aloe ferox, Arctopus
echinatus,
Artemisia
afra,
Balanites
maughamii,
Dicoma
capensis,
Harpagophytum
procumbens, Hypoxis hemerocallidea, Muraltia heisteria, Sutherlandia frutescens, Vernonia

oligocephala, Warburgia salutaris, Withania somnifera and Ziziphus mucronata.
2.4 Conclusions
While modern technology has certainly infiltrated traditional systems, these systems still centre
around the common view that health and disease are affected by a balance between a person
and his/her environment, and that foods, herbs and medicines are classified according to their
ability to impart this natural homeostasis. Also, a single disease affects various physiological
processes simultaneously, and a chemically complex mixture exerting a wide variety of
43
Chapter 2
biological properties would be a better suited medicine than a single compound magic bullet
medicine. Furthermore, certain medicines are considered to be able to re-establish
homeostasis by acting on various ailments at the same time, therefore healing while exerting a
toning, balancing effect on several physiological processes, ultimately also improving the
immune response of the body. This type of holistic healing/treatment is imparted by a class of
medicines referred to as tonics, i.e. superior herbs in TCM, rasayana in Ayurveda and
imbizas or musa pelo in South African traditional healing cultures. Specific terms such as
adaptogen (Russian origin) or bitter tonic are used with reference to a certain mode of tonic
action, such as increasing resistance to stress, or enhancing the function of the digestive
system, respectively.
While some of these healing concepts are foreign or may seem unscientific to practitioners of
western medicine, there is a renewed public interest in alternative and complementary
medicines due to newly emerging diseases, lack of curative treatment for several chronic
diseases, microbial resistance against synthesised drugs, their side-effects and high cost. In
countries such as South Africa, the healing evidence obtained from the use of traditional
medicines (uses reported for traditional practices of Khoi-San people and Cape Dutch settlers
even before 1650) has also encouraged commercialisation of some of these traditional herbal
products. Unfortunately, commercialised herbs used for general enhancement of quality of life
are often rather vaguely and often incorrectly referred to as tonics, bitter tonics, adaptogens,
alteratives, roborants, adjuvants, elixirs, stimulants, immunostimulants and strengthening
mixtures. In this chapter, these terms were defined, explained and contextualised, relative to
the term tonic.
44
CHAPTER 3
Materials and Methods
3.1
Introduction
This chapter describes the materials used and methods followed in order to investigate
which classes of compounds present in the 14 chosen tonic plants. By subjecting all the
plants to the same analytical protocols, the aim was to find similarities between biological
diversity and chemical composition among plants of the same species, as well as chemical
characters that are similar or different among species of a genus. It is known that relative
quantities and even the presence or absence of phytochemicals within a certain species
may vary. Although the biochemical synthesis of phytochemicals within a plant is regulated
genetically, external factors such as climate, geographical location, season and growth
conditions vary (Franz et al., 1985). Hence, in cases where a variety of plant samples were
available, geographical variation studies were done. Comparisons between phytochemical
content of different plant parts is also necessary as traditional uses of these plants are partspecific in many instances. Methods of preparation of tinctures, infusions, decoctions and
lotions necessitated investigation into the use of a variety of extraction solvents to optimally
extract the major compounds from the plant parts used. Where little was known of the
chemical composition of some of the plants, the main compounds were isolated and
identified. In order to obtain these results, a variety of qualitative (preliminary colour tests,
TLC) and quantitative (HPLC, GC-MS, NMR, bitterness taste testing) methods of analysis
were incorporated.
With the obtained phytochemical knowledge of each plant and its parts, the traditional uses
of the selected tonic plants (in Chapter 4) and their respective pharmacological activities
(Chapter 6) can be better understood and explained in a scientific context.
3.2
Plant materials
Table 3.1 is a summary of all the plant material subjected to chemical study, which
consisted of qualitative colour tests and selective extractions for alkaloids, water extractable
compounds and organic extractable compounds. In some cases, bulk material was
45
Chapter 3
obtained for isolation of major compounds as with Arctopus and Sutherlandia (the details of
this material will be given with the discussions of these plants in Chapter 5, also referring to
Table 3.1). Samples representative of all 14 species were further extracted for use in the
bitterness taste testing experiment. A table with these samples is given in Chapter 5.
Voucher specimens were deposited in the herbarium of the Department of Botany and
Plant Biotechnology at the University of Johannesburg (JRAU) see Table 3.1. In cases
where bulk plant material was not available from the JRAU herbarium, plants were collected
in such a way as to represent possible geographical variation. Only dried plant material was
used for extraction purposes. Where fresh material was obtained, the plants were cut into
smaller pieces and dried at 40 C in a drying oven. All plant material was milled before
extraction by means of a rotary beater mill or a bench coffee mill.
3.3
Screening protocol for classes of phytochemicals
The screening protocol entails qualitative wet chemistry tests to identify classes of
compounds to start off with. The presence or absence of these classes of compounds was
then confirmed by qualitative TLC and lastly quantitative chromatography (GC-MS, HPLC
or LC-MS) were used to quantify major compounds. These procedures are discussed in this
section. The aim of these protocols were intended to show the classes of compounds within
the chosen tonic plants, and highlight possible similarities or variation between species by
using chemical composition as a distinguishing character.
3.3.1 Qualitative colour tests

Extracts were prepared by mixing the specified mass of dry, ground plant material with the
method-specific volume of solvent, and leaving it at room temperature for 12 h. The
suspensions were filtered through cotton wool and then subjected to the respective colour
tests below.
Test for saponins: One gram of plant material was boiled with 5 mL of distilled water.
Another 3 mL of distilled water was added and the mixture shaken vigorously for 5 minutes.
Frothing which persisted on warming or for 5 minutes, indicated the presence of saponins
(Ashokkumar et al., 2010).
46
Chapter 3
Table 3.1:
Plant species used for TLC screening with voucher specimen numbers (small alphabet letters indicate different plants of the
same species), provenances with collection dates and sample numbers used throughout the study (abbreviations in brackets
indicate plant parts extracted: L = leaves; S = stems; B = bark; R = root)
Plant species
Voucher
Specimens#
Provenances (date obtained*)
Sample
number
DO 51a-f
All DO 51 samples were commercial samples obtained from Grassroots

Natural Products CC, Gouda, Western Cape Province
DO 53
BMVW 4310 a-c
Cultivated by W. Eigenhuys, Grahamstown district, seed from Piketberg

Cederberg type, cultivated (15/12/2008)
AB 51a L
AB 51b L
AB 51c L
AB 51d L
AB 51e L
AB 51f L
AB 53 L
AB 4310a L
AB 4310b L
AB 4310c L
Agathosma
-
A. betulina
A. ovata
DO 55
BMVW 4039 a-c
Wild sample obtained from W. Eigenhuys, Grahamstown district

Piketberg type (basterboegoe), cultivated (15/12/2008)
AO 55 L
AO 4309a L
AO 4309b L
AO 4309c L
A. crenulata
DO 54
DO s.n.
Cultivated by W. Eigenhuys, Grahamstown district, seed from Tulbagh

Herb garden, Walter Sisulu National Botanical Garden (10/2008)
AC 54 L
AC 10 L
47
Chapter 3
Aloe
-
A. ferox
DO 60
Cnr University & Kingsway Avenues, Auckland Park (28/08/2006)
AF 60 L
A. marlothii
DO 61
Alida street, Northcliff, Gauteng (28/08/2006)
AMa 61 L
A. aborescens
DO 62
Chiselhurst Dr, Auckland Park, Gauteng (28/08/2006)
AAb 62 L
A. vera
DO 65
9 Rolina Str, Florida Glen, Gauteng (12/2006)
AV 65 L
PW & BEVW 170

ARM & JSB 15
BEVW s.n.
BEVW 4128a-c
Top of Du Toits Kloof Pass

Hermanus (27/11/2004)
Leipoldts grave, Pakhuis Pass (11/10/1993)
Nieuwoudtville (10/09/2004)
ARM & JSB 4

DO 66a-b
Rondeberg
Port Alfred, southern residential area (27/06/2005)
AE 1 R
AE 2 R
AE 3 R
AE 4a R
AE 4b R
AE 4c R
AE 5 R
AE 6a R
AE 6b R
Arctopus
-
A. echinatus
A. dregei
ARM & JSB 31

ARM & JSB 2a-c
Malmesbury (06/12/2004)
Rondeberg (05/09/2004)
AD 7 R
AD 8a R
AD 8b R
AD 8c R
A. monacanthus
BEVW 3522
BEVW 4161a
BEVW 4141a
Gifberg (13/10/1993)
Gifberg (26/10/2004)
Elandskloof Pass, Citrusdal (23/10/2004)
AMo 9 R
AMo 10 R
AMo 11 R
48
Chapter 3
Artemisia afra
Commmercial
DO 34
DO 35a
AAf com L
AAf 34 L
AAf 35a L
DO 67
BioAfrica, South Africa

Johannesburg Botanical Garden (10/03/2005)
East side of the garden at 9 Chiselhurst Drive, Auckland Park
(17/03/2005)
West side of the garden at 9 Chiselhurst Drive, Auckland Park
(17/03/2005)
Herb garden, Walter Sisulu National Botanical Garden (25/03/2005)
Ohrigstad Dam Nature Reserve (04/0402005)
On bank of Orange River, Breakaway Trails resort, 20 km from Rouxville
(12/2005)
Golden Gate Nature Reserve, 5 km before entrance (15/01/2008)
DO 68
Golela, Nelspruit district (09/2006)
DO 69
Mpaka coal mine, Nelspruit district (09/2006)
DO 70
Kruger National Park gate (09/2006)
BM 68 L
BM 68 B
BM 69 L
BM 69 B
BM 70 L
BM 70 B
BEVW 4032a-c
Turn-off from N3 to Frankfort (01/05/1999)
DO 35b
DO 38
DO 42
DO 59
Balanites maughamii
AAf 35b L
AAf 38 L
AAf 42 L
AAf 59 L
AAf 67 L
Dicoma
-
D. anomala
DA 4032a R
DA 4032a S
DA 4032b L
DA 4032b S
DA 4032c L
DA 4032c S
49
Chapter 3
D. capensis
D. schinzii
D. zeyheri
DO 58a-c
Hoons farm, Williston (06/2005)
BEVW 4035
Netnou 127
Northern Cape
Aggeneys, Swartberg
BEVW s.n.
Intersection of Koffiefontein and Luckhoff roads, 1 km from Luckhoff
BEVW 4017a-d
52 km north-west of Tosca on the road to Terra Firma (01/05/1998)
BEVW s.n.
BEVW 4046
Kalahari
Kingswood (20/11/1999)
Netnou 47
Netnou 36
Mothogoang
Kalahari
DC 58a L
DC 58b L
DC 58c L
DC 4035 L
DC 127 L
DC 127 S
DC KL L
DS 4017b L
DS 4017b S
DS 4017c L
DS 4017c S
DS 4017d L
DS 4017d S
DS K L
DS 4046 L
DS 4046 S
47 L
NN 36 L
NN 36 R
50
Chapter 3
Harpagophytum
-
H. procumbens
H. procumbens
subsp. procumbens
BEVW 3807c
BEVW 3807d
BEVW 3807e
BEVW 3807f
BEVW 3807g
BEVW 3807i
BEVW s.n. a
GO a-b
85 km north-west of Vryburg on the Tosca road, Northern Cape

(10/04/1996)
BEVW s.n. a
BEVW s.n. b
Molapo village
Makalamabedi
Kuruman
BEVW s.n. c
D. zeyheri
BEVW s.n. f
BEVW s.n. l
Bray
BEVW s.n. a
BEVW s.n. i
BEVW s.n. j
Waterberg
HP 3807c R-3
HP 3807d R-2
HP 3807e R-2
HP 3807f R-2
HP 3807g R-2
HP 3807i R-3
HP Ma R
HP GOa R
HP GOb R
HP MVa R-1
HP MVb R-1
HP MVb R-2
HP MVc R-1
HP MVc R-2
HP MVc R-3
HP Bf R
HP Bl R
HZ Wa R
HZ Wi R
HZ Wj R
51
Chapter 3
Hypoxis hemerocallidea
DO 71a-e
Farraday Muti Market, Johannesburg
HH 71a R
HH 71b R
HH 71c R
HH 71d R
HH 71e R
Muraltia heisteria
BEVW 4189
Middelburg Pass, Elandskloof (26/11/2006)
BEVW 4194
Signal Hill, Kramat (28/11/2006)
MH 4189 L
MH 4189 S
MH 4194 L
MH 4194 S
Palmer 1b, g
Olifantshoek (22/11/1991)
Palmer 2a-b
Gamsberg (22/11/1991)
Palmer 6b
DM & BEVW & AdC
18a-c
Vanrhynsdorp
Camps Bay
Palmer 9c
Palmer 12 a-b
Aurora (28/11/1991)
Blouberg Strand (28/11/1991)
DM & BEVW & AdC

16a-c
Blouberg Strand (20/11/1996)
Sutherlandia
- S. frutescens
S. frutescens var.
incana
SF 1b L
SF 1g L
SF 2a L
SF 2b L
SF 6b L
SF 18a L
SF 18b L
SF 18c L
SFI 9c L
SFI 12a L
SFI 12b L
SFI 16a L
SFI 16b L
SFI 16c L
52
Chapter 3
S. humilis
S. microphylla
S. montana
Palmer 14 a-b
Hout Bay
Palmer 18a-c
Struisbaai
BEVW & AdC

3668 b-c
Pearly Beach
Palmer 17a
Palmer 25c
Barrydale
Uniondale
Commercial
Palmer 3c
Palmer 5e
Palmer 7a
Palmer 24b
Palmer 24d
Palmer 26a
DM & BEVW & AdC
20a
BEVW s.n. a, d-e
CA Albrecht
Kamiesberg
Bitterfontein
Vanrhynsdorp
Klein Sleutelfontein
Unionpoort
Leeuberg Pass (21/11/2006)
BEVW 3800a-c
Reitz (01/06/1996)
Anon. s.n. (PRE 5a)
Wolkberg
Colesberg (16/9/1996)
SFI 14a L
SFI 14b L
SFI 18a L
SFI 18b L
SFI 18c L
SFI 3668b L
SFI 3668c L
SH 17a L
SH 25a L
SMi comm. L
SMi 3c L
SMi 5e L
SMi 7a L
SMi 24b L
SMi 24d L
SMi 26a L
SMi 20a L
SMi Ca L
SMi Cd L
SMi Ce L
SMo 3800a L
SMo 3800b L
SMo 3800c L
SMo PRE 5a L
53
Chapter 3
DM & BEVW & AdC

22a-c
Reitz (22/11/1996)
BEVW 2771
Golden Gate
SMo 22a L
SMo 22b L
SMo 22c L
SMo 2771 L
S. speciosa
Palmer 4c
Germishuizen 4724
Kamiesberg
Richtersveld, Eksteenfontein
SS 4c L
SS 4724 L
S. tomentosa
Palmer 13a-h
Blouberg Strand
Palmer 19e
BEVW & AdC 3669
b,f
Witsand
Still Bay (16/09/1996)
ST 13a L
ST 13c L
ST 13e L
ST13f L
ST 13g L
ST 13h L
ST 19e L
ST 3669b L
ST 3669f L
DO 45 a-c
SANBI Pretoria (24/04/2005)
DO 48 a-e
North of Anglo Gold Mines sport ground, Carletonville (27/04/2005)
Vernonia oligocephala
VO 45a S
VO 45a L
VO 45b S
VO 45b L
VO 45c S
VO 45c L
VO 48a S
VO 48a L
VO 48b S
VO 48b L
54
Chapter 3
VO 48c S
VO 48c L
VO 48d L
VO 48e L
VO 57 L
VO 63a S
VO 63a L
VO 63d L
VO 64 S
VO 64 L
DO 57
DO 63 a, d
Suikerbosrand Nature Reserve (21/05/2005)

N14 between Krugersdorp and Pretoria, at Zwartkop off-ramp
(01/10/2006)
DO 64
Opening next to Krediet Street, Wilgeheuwel
Commercial sample
Commercial samples
Roger Stewart (05/2005)

Roger Stewart (2006)
Commercial sample
Faraday Muti Market, Johannesburg
Withania somnifera
DO 37a, b
Walter Sisulu Botanical Garden, Roodepoort
WSo 37a R
WSo 37a S
WSo 37a L
WSo 37b R
WSo 37b S
WSo 37c L
Ziziphus mucronata
DO 36 a-c
187 Hartebeestfontein, on Bultfontein road, Hekpoort (21/03/2005)
ZM 36a L
ZM 36a R
ZM 36a B
ZM 36b L
ZM 36b R
ZM 36b B
Warburgia salutaris
Wsa RSa L
WSa RSb B
WSa RSb L
WSa F B
55
Chapter 3
DO 39 a, b
Sudwala Caves, Mpumalanga (03/04/2005)
DO 40 b
DO 43
DO 44
DO 46 a, b
Lowveld National Botanical Garden, Nelspruit (04/04/2005)

Next to Bultfontein road, Hekpoort (17/04/2005)
Close to Groenkloof Nature Reserve entrance, Pretoria (24/04/2005)
Walter Sisulu Botanical Garden, Roodepoort (25/04/2005)
DO 47
BDV 32
ARM & JSB s.n.
North of gholf course, Anglo Gold Mines, Carletonville (27/04/2005)

Mount Sanctuary Nature Reserve
Rustenburg
ZM 36c L
ZM 36c R
ZM 36c B
ZM 39a L
ZM 39b L
ZM 40b L
ZM 43 L
ZM 44 L
ZM 46a L
ZM 46b L
ZM 47 L
ZM 32 L
ZM AJ R1
ZM AJ R2
Abbreviations of the names of collectors:

AdC = A de Castro; ARM = AR Magee; BDV = B de Villiers; BEVW = B-E van Wyk; DM = D Moshe; DO = D Olivier; GO = G
Olivier; JSB = JS Boatwright; NN = NC Netnou; PW = P Winter.
Where available
56
Chapter 3
Liebermann-Burchard test for steroids, terpenoids and bitter principles: A MeOH extract
was prepared by extracting 10 g of plant material with 40 mL of solvent, filtering and drying
the filtrate by means of a vacuum dryer at a maximum temperature of 40 C. Two milliliters
of acetic acid was added to 0.2 g of extract, the solution was cooled and conc. H2SO4 was
added drop-wise. Colouration from violet to blue or bluish-green indicated the presence of
the glycones of steroidal rings, terpenoids or bitter principles (Wagner et al., 1984;
Ashokkumar et al., 2010).
(a)
(b)
(c)
(d)
Fig. 3.1 Positive result for Hypoxis hemerocallidea (HH 71a R ) in the LiebermannBurchard test. Photos were taken at five minute intervals from (a) to (d).
Shinodas test for flavonoids: 0.5 g of MeOH extract was dissolved in 3 mL of EtOH,
warmed and filtered. Three magnesium chips were added to the filtrate followed by a few
drops of conc. HCl. A pink, orange or red to purple colouration indicated the presence of
flavonoids (Ashokkumar et al., 2010).
Test for alkaloids: A spatula tip of dry MeOH extract was stirred in 5 mL of 1% aqueous HCl
for 1 hour and filtered. One milliliter of Mayers reagent was added to 1 mL of the extract. A
resulting cream or pale yellow extract indicated the presence of alkaloids (Ashokkumar et
al., 2010). Mayers reagent is prepared by dissolving 1.358 g of mercury(II) chloride in 60
mL of distilled water. Five grams of potassium iodide is dissolved in 10 mL of distilled water.
The two solutions are added together and the total volume adjusted to 100 mL with distilled
water (Santiaworn et al., 2005).
The results for these tests are given in Chapter 5.
57
Chapter 3
3.3.2 Alkaloids
Alkaloids are nitrogen containing compounds and can hence be extracted by using their
basic properties. These compounds are normally insoluble in water, but upon treatment
with an acid, a salt is formed which is soluble in water. The method described here was
designed for the specific extraction of alkaloids, but contaminants with similar properties
may also be extracted if only trace amounts of alkaloids are available in the plant material
(De Wet et al., 2004). Therefore it is vital to do TLC of the alkaloid extracts to ascertain if
alkaloids are present or not.
Glass columns through which the extract is filtered are prepared in advance. Each glass
column (27 x 2.5 cm) were packed with coarse grade celite-577 as shown in Fig. 3.2. Sand
was mixed to the celite and placed on top of the column to prevent the extensive formation
of bubbles in the stationary phase when it was washed with solvents of different polarity
and viscosity. The stationary phase needed to be packed firmly in order to prolong the
interaction of alkaloids with solvent. Anhydrous sodium sulfate (Na2SO4) was added at the
bottom of the column in order to dry the organic solvent that was eluted from the column
(De Wet, 2006).
Fig. 3.2
Glass-column set-up used for
micro-scale alkaloid extraction
Micro-extraction involved the stirring of ca. 1 g of powdered dry plant material in 15 mL of

0.05 M H2SO4 for 1 hr. Most alkaloids present (not necessarily quaternary alkaloids) will
consequently have been converted to water soluble salts and will have dissolved in the acid
58
Chapter 3
solution. The extract was loaded on top of the glass columns by filtering through Whatman
no. 4 filter paper in a funnel. The reaction flask was rinsed with another 5 mL of 0.05 M
H2SO4 which was loaded with the filtrate onto the column. Concentrated ammonia (25%
NH3) (ca. 4 mL) was added on top of the column to neutralise the acid (i.e. adjust the pH to
7). By doing so, the alkaloid salt was converted back to its water insoluble counterpart.
Dichloromethane (100 mL) was then used to wash all non-polar compounds from the celite.
The solvent was evaporated under reduced pressure at 40 C and the remaining extract
was washed into a weighed vial with methanol, dried in the fume hood and the yield
calculated (De Wet, 2006). The presence or absence of alkaloids in all 14 species, with
alkaloid extract yields are given in Chapter 5.
Glass-backed silica gel F254 plates (Machery-Nagel 20 x 20 cm, 0.25 mm) were used for
TLC. The alkaloid extracts were reconstituted in 1:1 MeOH:CH2Cl2 and 50 L applied per
spot (spots were placed 1.5 cm from the bottom of the plate, 1 cm apart). A hairdryer was
used on low heat to dry the extract after application before adding more in order to load the
full amount onto 0.5 cm diameter spots. The solvent system used is a screening system
suitable for most alkaloids (CHCl3:cyclohexane:ethylamine in a 4:5:1 ratio) (Wagner &
Bladt, 2001). The plates were removed from the tank after development and left to dry (until
no smell of ethylamine could be detected), before visualising the spots by means of UV 254,
UV365 and with acidified iodoplatinate spray reagent (150 mg potassium hexachloroplatinate
and 3 g potassium iodide dissolved in 100 mL 5 M HCl, refrigerated after preparation).
Many alkaloids show considerable quenching of fluorescence in UV254 and show blue, bluegreen or violet fluorescence in UV365. Alkaloid zones appear brown, blue or visibly white on
a blue-grey background after spraying with the iodoplatinate reagent (Wagner & Bladt,
2001).
3.3.3 Water extractable compounds

Water-extractable compounds are soluble in boiling water and include polysaccharides and
glycoproteins (alcohol precipitable solids APS), amino acids, monosaccharides and other
small sugars (alditols and cyclitols), saponins and other glycosylated terpenes as well as
flavonoids and other glycosylated phenolic compounds. All these compounds were
extracted by mixing ca. 0.3 g of ground dry plant material with 23 mL boiling water in a test
tube, vortexing the mixture for one minute and leaving it overnight. If notable amounts of
saponins were extracted, a foam layer was formed on top of the extract (after vortexing for
one minute) which was then recorded (see test for saponins Paragraph 3.3.1). The
mixture was consequently filtered through a Pasteur pipette with cotton wool into a preweighed centrifuge tube. At this stage the APS was removed (see Paragraph 3.3.3.1), both
the yield of APS and alcohol-soluble polar compounds calculated upon drying and the polar
59
Chapter 3
extracts examined for amino acids, EtOH-soluble sugars, terpenes and phenolic
compounds by means of thin-layer chromatography (TLC) (plates consisted of silica gel 60
F254, 1 mm thickness, on aluminum sheets obtained from Merck). The combined mass of
the APS and polar extracts constituted the water-extractable solids.
3.3.3.1 Alcohol-precipitable solids

Polysaccharides, glycoproteins, organic acids and metal ions might be soluble in boiling
water, but precipitate when the solution contains more than 75% EtOH (OBrien, 2005).
After the boiling water extracts were prepared and filtered into pre-weighed centrifuge
tubes, three times the extract volume of EtOH was added to the tubes in order to constitute
a solution with an EtOH content of approximately 75%. In some cases the mixture turned
milky at once as an indication of the rapid precipitation of APS. The mixtures were left to
precipitate for an hour upon which the tubes were centrifuged for 15 minutes at low speed.
The supernatant was decanted into a pre-weighed vial and contained the compounds
soluble in alcohol (polar extract). The polar extracts were dried and the extract weights
obtained. The precipitate (APS) was dried in the centrifuge tube using the same centrifuge
under vacuum at maximum speed and weighed. The identity of the compounds in the APS
was not determined. APS yields are given per species in Chapter 5.
2.3.3.2 Amino acids

Free amino acids are extracted with boiling water and remain dissolved in 75% EtOH. The
polar fraction was reconstituted in 1 mL MeOH:H2O (1:1) and used for the TLC screening of
amino acids. TLC plates were prepared (50 L of extract per spot) and were developed in
the amino acid solvent system (n-BuOH:CH3COOH:H2O in a 4:1:2 ratio), sprayed with 0.3%
ninhydrin spray reagent (30 mg ninhydrin dissolved in 10 mL BuOH and 0.3 mL of glacial
acetic acid added) and baked for 510 minutes. Amino acids appear violet-red to red-brown
with a low Rf value. Yellow staining is unusual except for proline (Wagner & Bladt, 2001).
The TLC plates for the respective species are shown in Chapter 5.
Quantification of amino acids was done by using HR-GC-MS-TOF, as discussed in
Paragraph 3.4.2.
3.3.3.3 Sugars
While polysaccharides are only soluble in water, mono- and disaccharides, cyclitols and
alditols are soluble in water and in 75% EtOH. The polar fraction could hence be used for
TLC of the smaller sugars (50 L of the reconstituted polar extract in Paragraph 3.3.3.2 was
applied per spot). The plates were developed in a solvent system (ACN:CS2:H2O:formic
60
Chapter 3
acid in a 85:5:10:0.5 ratio) developed for monosaccharides in our laboratory (OBrien,

2005). After developing a plate, it was dried and developed again to ensure good resolution
as these sugars are very polar and all exhibit low Rf values. The resolved sugars were
charred to a brown-black colour by spraying the plates with chromic acid and heating them
at 120 C for 5 minutes. Extracts were run against sugar standards which were prepared by
dissolving 8 mg of sugar in 1 mL of water and applying 5 L per spot. The respective TLC
plates are shown in Chapter 5.
3.3.3.4 Phenolic glycosides

Many phenolic compounds that occur in plants are products from the shikimic parthway
which originates in the formation of shikimic acid from glucose. Shikimic acid is
consequently metabolised to prephenic acid which is the precursor of the aromatic ring
present in all phenolic compounds (Davies et al., 1964). According to Harborne (1988),
phenolic compounds may largely be classified as those with one phenolic ring
(phenylpropanoids, where a three-carbon side chain is attached to the aromatic ring), those
with two phenolic rings (flavonoids, anthrones, chromones and chalcones) and the
polyphenols (i.e. tannins). Phenylpropanoids stem from the aromatic amino acid
phenylalanine which is synthesized from prephenic acid, and is derivatised in turn to form phydroxycinnamic acid. Three other closely related hydroxycinnamic acids are well known
secondary metabolites: caffeic, ferulic and sinapic acid. Caffeic acid regularly occurs as the
quinic acid ester, chlorogenic acid, and derivatives with sugars (caffeoylglucose) and
organic acids (rosmarinic acid) are also well known (see Chapter 5 Arctopus; Olivier et
al., 2008). Flavonoids and chalcones contain diarylpropane units whereby the two phenyl
rings are connected by means of a propane chain (C6-C3-C6). In the case of the flavonoids,
the C3-unit is oxygenated and closed to form a pyran ring, whereas the C3-unit contains a
ketone group with the chalcones and is not closed to form a ring. The flavonoids are
classified based on the functionalisation of the C3-unit into flavanones, flavones, flavonols,
flavanonol and isoflavones (Mabry et al., 1971). Glycosylated phenolic compounds exhibit
polar to medium polar properties depending on the amount of hydroxyl functional groups
the compound contains and the amount of sugars attached. These compounds are
extracted by means of boiling water and are soluble in the 75% EtOH fraction. They may
also be successfully extracted with MeOH and in some cases with a 1:1 MeOH:CHCl3
mixture.
TLC screening for flavonoids or polar phenolic acids by TLC also entailed 50 L spots (of
reconstituted polar extract see Paragraphs 3.3.3.2 and 3.3.3.3), and development of the
plates in different solvent systems: more polar compounds showed good separation in the
61
Chapter 3
Sutherlandia solvent system [CHCl3:MeOH:H2O:CH3COOH in a 60:30:8:6 ratio adapted

from the saponin system in Wagner and Bladt (2001)], while less polar compounds could be
separated best with CHCl3:MeOH in a 6:3 ratio. In cases where considerable streaking
prevented good resolution, the amino acid system (n-BuOH:CH3COOH:H2O in a 4:1:2 ratio)
worked well as the high concentration of acid in the system curbed streaking. The plates
were visualised under different wavelengths before development with spray reagents as
flavonoids are known to quench fluorescence at UV-254 and appear as dark blue zones.
UV-365 may be used to differentiate between flavonoids which fluoresce yellow and
phenolic acids which fluoresce blue. After spraying the dry TLC plates with 5% ethanolic
H2SO4 followed by 1% ethanolic vanillin and then baking them for 510 minutes, the
phenolic compounds appeared yellow to orange or brown. Anisaldehyde-sulfuric acid may
also be used for similar results (Wagner & Bladt, 2001).
As phenolic compounds show excellent response factors upon analysis with HPLC (PDA)
and LC-MS, identification and quantification was relatively easy (see Paragraph 3.4.1.1).
2.3.3.5 Triterpenoids and steroids

Terpenoids are classified by the number of C5-isoprene units they comprise of. The classes
are: monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids
(C25), steroids and triterpenoids (C30) and lastly carotenoids (C40). Most of the terpenoids
have cyclic structures. The mono- and sesquiterpenes are often volatile or essential oils
and are pleasant smelling compounds. Diterpenes may be liquids or solids. These three
classes of terpenes are almost insoluble in water but readily soluble in organic solvents,
see Paragraph 3.3.4. Steroids and triterpenoids are often present in plants as saponins, i.e.
they have one or more sugar molecules attached to them, making them water soluble. The
presence of saponins is evident from the foam which forms when the aqueous extract is
vigorously shaken (Paragraph 3.3.1), and they often cause the aqueous extracts to be
viscous or slimy.
Steroids and triterpenoids may have high to medium polarity depending on the level of
glycosylation. For this reason, they elute with flavonoids and phenolic acids, in the same
TLC solvent systems (mainly CHCl3:MeOH:H2O:CH3COOH in a 60:30:8:6 ratio, see
Paragraph 3.3.3.4). Saponins are also visualized by spraying the dry plates with the
vanillin-sulfuric acid reagent as with flavonoids and phenolic acids, where blue, blue-violet,
and sometimes red or yellow-brown zones represent saponins (Wagner & Bladt, 2001).
62
Chapter 3
3.3.4 Organic extractable compounds

3.3.4.1 Mono-, sesqui- and diterpenes
Monoterpenoids and sesquiterpenes are the aromatic major components of complex
mixtures called essential oils, which form the basis of the perfume and flavouring industries.
Many diterpenoids are wood resin products which are there to protect the plant from
grazing animals as many diterpenes are bitter, as are sesquiterpene lactones and some
iridoids (Wagner & Bladt, 2001). Commercially, diterpenes are used in varnishes and resin
soaps (Hanson, 2003).
These non-polar components were extracted by adding 3 mL CHCl3:MeOH (1:1) to
ca. 0.3 g of dried, ground plant material, vortexing to mix and leaving the suspension
(closed) overnight. After filtration, the extract was taken to dryness and the yield calculated.
For TLC purposes, the extract was reconstituted in 1 mL of CHCl3:MeOH (1:1) and 50 L
loaded per spot. Each extract was evaluated in two different solvent systems: CHCl3:MeOH
(6:3) for more polar, and diethyl ether:hexane (2:3) for less polar compounds. All the plates
were visualised by using the vanillin-sulfuric acid reagent as in Paragraphs 3.3.3.4 and
3.3.3.5. The TLC plates and yields are given in Chapter 5, per species.
3.3.4.2 Flavonoid aglycones (Agathosma species)

According to Wollenweber and Graven (1992), oval leaf buchu (Agathosma crenulata)
contained flavonoid aglycones in the waxy layer that covers the leaves of the plant. The
intention was thus to use this as a distinguishing character between A. betulina and
A. crenulata.
Ten grams of whole leaves were washed with 200 mL acetone (AR) by swirling the mixture
for 1 minute. The acetone was decanted and the volume reduced to almost complete
dryness. The extract was defatted by adding 2 mL MeOH (AR) and 2 mL hexane (AR) and
shaking the mixture. The top hexane layer was removed, and the bottom MeOH layer was
cleaned by eluating it through an SPE C18 (end-capped) cartridge. The cartridge was
washed with 1 mL MeOH (AR) and the complete extract was taken to dryness before
calculating the yield (Wollenweber & Graven, 1992). The extracts were submitted for HPLC
(PDA) analyses only.
63
Chapter 3
3.3.5 Cyanogenic glycosides

The Feigl-Anger HCN test (Feigl & Anger, 1966) was used for screening of cyanogenic
glycosides. Indicator paper was prepared by dipping filter paper into a mixture of 1% (w/v)
N,N,N',N'-tetramethyl-4,4'-diaminediphenylmethane in CHCl3 and 1%
(w/v)
copper
ethylacetoacetate in CHCl3 (the solutions were prepared separately before adding equal
volumes together), drying it overnight and cutting it into thin strips. For the cyanogenic
glycoside test, 1 g of dried, ground plant material was placed in a glass vial, ca. 10 drops of
water was added to moisten the plant material and the prepared indicator strip placed
inside the vial immediately and the vial sealed quickly (the test is sensitive to HCN gas, and
slow action could result in a false negative result due to the gas escaping before reacting
with the indicator paper). Care should be taken that the indicator strip does not touch the
sample or sides of the container as to not get wet unnecessarily. This is accomplished by
folding the strip in a Z-shape prior to insertion (see Fig. 3.3 for an example of positive and
Warburgia
salutaris
(bark)
Warburgia
salutaris
(leaves)
Prunus
laurocerasus
Harpagophytum
procumbens
Dicoma
capensis
Dicoma
Anomala
Agathosma
betulina
Prunus
laurocerasus
negative results).
Fig. 3.3: Cyanogenic glycoside test results with Prunus laurocerasus L. as control:
(a) negative; (b) positive
64
Chapter 3
3.4
Quantitative Analytical Chromatography
Chromatography is intended to separate component molecules in the extracts applied.

While TLC is able to give qualitative information, quantitative information is obtained
through GC and HPLC coupled with a means of characterization and identification of these
natural molecules. Variation studies were conducted on GC instruments coupled with mass
spectrometers (MS), while the HPLC instruments were coupled with photodiode array
(PDA) detectors which were able to scan the ultraviolet (UV) to the visible (vis) regions with
wavelengths between 190 nm and 800 nm. Structural elucidation of isolated compounds
was based on different nuclear magnetic resonance (NMR) spectroscopy experiments, and
accurate mass determination through high-resolution LC-MS. Optical rotations were
determined for novel compounds.
3.4.1 Liquid chromatography

3.4.1.1 High-performance liquid chromatography (HPLC)
Variation studies (Sutherlandia, Arctopus, Agathosma): A Shimadzu 10A HPLC instrument
with a Phenomenex RP C18 column (150 x 4.6 mm, 5 m), with a guard column and
cartridge with the same packing material as the column, a binary gradient system and a
photodiode array (PDA) detector was used for routine analyses. UV spectra were recorded
between 210 and 400 nm. Specific conditions varied for the extracts investigated as shown
in Table 3.2.
Sample preparation for Sutherlandia (selective separation of flavonoids and triterpenoids):
The EtOH extract obtained after APS have been removed, was used (see Paragraph 3.3.3).
The dry extract was reconstituted in 1 mL 50% MeOH, and loaded onto a prepared SPE
cartridge (Strata, C18 end-capped, 500 mg/3 mL, manufactured for Phenomenex). The
cartridge was washed with 2 mL 50% MeOH (until all the flavonoids have been removed visible as a yellow line), followed by 4 mL 100% MeOH to strip the column from the
triterpenoids retained. The samples were consequently injected on the HPLC (20 L).
Sample preparation for Arctopus (selective separation of phenolic acids and triterpenoids):
As with Sutherlandia, the EtOH extract obtained after APS have been removed, was used.
Here the dry extract was reconstituted in 1 mL 30% MeOH, and loaded onto a prepared
SPE cartridge as before. The cartridge was rinsed twice with 1 mL 30% MeOH (to
selectively remove the phenolic acids), followed by 3 mL 90% MeOH to remove the
retained triterpenoids without stripping the more non-polar compounds still on the column.
The phenolic acid fractions were then injected on the HPLC (20 L).
65
Chapter 3
Table 3.2
Two different sets of high performance liquid chromatography (HPLC)

conditions for resolution of phenolic compounds and triterpenes in
Agathosma, Arctopus and Sutherlandia extracts.
Methods
Sutherlandia flavonoids
Instrument settings
Sutherlandia triterpenoids/
Arctopus phenolic acids/
Agathosma flavonoids
Solvent system
Gradient eluation
Diode Array Detector

channels (wavelength
scanning)
A: MeOH
A: MeOH
B: 1% CH3COOH/H2O
B: 1% CH3COOH/H2O
30% A for 2 min
40% A for 2 min
30% A100% A over 22 min
40% A100% A over 22 min
100% A for 2 min
100% A for 2 min
100% A5% A over 1 min
5% A for 1 min
5% A for 1 min
30% A for 1 min
40% A for 1 min
A: 250 40 nm
A: 250 40 nm
B: 340 50 nm
B: 340 50 nm
Sample preparation for Agathosma (flavonoids within leaves and on leaf surface): As with
the previous two species, the EtOH extract obtained after APS have been removed, was
used to determine flavonoid variation within the leaves. Here the dry extract was
reconstituted in 1 mL 50% MeOH, and loaded onto a prepared SPE cartridge as before. In
this case, no selective separation was required and the prepared cartridge was rinsed with
1 mL 50% MeOH, followed by 3 mL 100% MeOH to strip the column completely. The
samples were then injected on the HPLC (20 L). For the leaf surface flavonoids, the
acetone extracts (Paragraph 3.3.4.2) were reconstituted in 2 mL MeOH and 20 L injected
into the HPLC.
66
Chapter 3
3.4.1.2 Liquid chromatography-mass spectrometry (LC-MS)

LC-MS quantitative analyses of the Arctopus and Alepidea kaurenoic acids were conducted
with extracts prepared by soaking 0.3 g of dried, ground root material overnight in
CHCl3:MeOH (1:1), filtering them and evaporating the filtrates to dryness. These extracts
were reconstituted in 1 mL of MeOH, of which 5 L was injected with a Waters API Quatro
Micro instrument. A Waters SunFire column (C18 3.5 m, 4.6 x 150 mm, with SunFire C18
3.5 m, 4.6 x 20 mm guard cartridge) was used for the separation of the components by
using gradient elution: 70100% of solvent B (solvent A is 2% formic acid, 10 mM
ammonium formate; solvent B is acetonitrile) over a period of 50 min at a flow rate of
1 mL/min at 35 C. An APCI MS source was used in positive mode (settings included
Corona voltage: 7 V; cone voltage: 20; RFI: 40; source: 120 C; desolvation temperature:
450 C; desolvation gas: 55 L/h; cone gas: 50 L/h).
3.4.2 High-resolution gas chromatography-mass spectrometry (HR-GC-MS)

3.4.2.1 Amino acids
It is virtually impossible to distinguish between amino acids on TLC. Identification and
quantification thus necessitated the use of GC-MS. Amino acids are normally not volatile
enough to inject on GC due to their high polarity. Consequent derivatisation provided
increased volatility, thermal stability and an adequate detector response for such analysis. It
has also been shown that derivatisation may improve resolution and reduce tailing
(Braithwaite & Smith, 1996).
An EZ faast-Free Amino Acid Phenomenex analysis kit (obtained from Separations) was
used for the GC-MS analysis. A 100
L sample of polar extract (re-suspended in 1 mL
MeOH:H2O 1:1) and 100 L of an internal standard (0.2 mM norvaline) was pipetted onto
a solid phase extraction (SPE) sorbent tip with a strong affiliation for amino acids. Norvaline
was used to correct variations in the injected volumes and the peak retention times of the
amino acids. The adsorbed extract was consequently washed with 200 L of n-propanol to
ensure that compounds such as proteins, lipids and inorganic salts that could interfere with
the chromatographic analyses were removed. The extracted amino acids were
deprotonated and washed from the stationary phase in the sorbent tip by means of 200 L
sodium hydroxide and n-propanol (3:2) solution. A chloroform mixture containing the
derivitising agent (50 L) (an unidentified alkyl chloroformate) was consequently added to
the extracted amino acids and the mixture vortexed and left for a minute or more to react.
(The derivatisation reaction is illustrated in Fig. 3.4). A 100 L of iso-octane was added to
67
Chapter 3
the mixture in order to extract the derivatised amino acids. The formation of two layers was
observed at this stage with the organic top layer containing the derivatised amino acids.
This layer (50 L) was removed with a pipette into a GC vial and dried under a stream of
nitrogen. The dry amino acid extracts were consequently reconstituted in 100
L of 80%
iso-octane in chloroform for injection into the GC-MS. This method of preparation and
analyses compares well with a method suggested in literature (Oh et al., 1995).
O
R
Cl
O
H2N
+
OH
O CATALYST
2
OR'
OR'
NH
R'O
2HCl + CO2
Fig. 3.4: Amino acid derivitisation reaction as per EZ faast-Free Amino Acid analysis kit
Calibration standards containing mixtures of amino acids used for quantification purposes
were also provided in the kit. Four calibration levels were used. Different volumes of
standard solution were pipetted together with 100
L of an internal standard (0.2 mM
Norvaline) onto a solid phase extraction (SPE) sorbent tip as was the case with the
extracts. The same method of derivatization was followed as with the sample mixtures. The
volumes of standard solution used to prepare the four concentrations for calibration is given
below:
Calibration level 1 50 nmoles/mL:
25 L of standard solutions 1, 2 and 3 respectively
Calibration level 2 100nmoles/mL:
Calibration level 3 200nmoles/mL:
A GC capillary column and GC-MS amino acid library was also provided in the amino acid
kit. The GC-MS method proposed by the kit and used for analyses is outlined in Table 3.3.
An Agilent GC with a LECO Pegasus high-throughput time-of-flight mass spectrometer
(HR-GC-MS-TOF) instrument was used for the amino acid analyses. The instrument was
optimized using the conditions prescribed by EZ faast (Table 3.3). Analyses were done in
duplicate and the mean values were reported. Fig. 3.5 is a chromatogram of the amino acid
standard used for calibration to show the retention times of the respective amino acids
68
Chapter 3
while Table 3.4 provides their structures and major ions used for identification. It should be
noted that the peaks that are not numbered are urine amino acids (proline-hydroxyproline,
hydroxylysine, glycine-proline, cystathionine and -aminopimelic acid), which are included
in the standard mixture, but is not relevant for a plant study. The quantification of amino
acids in the tonic plants are given in Chapter 5.
Table 3.3:
EZ: faast Free (Physiological) Amino acid analysis method by GC-MS
Column type
Zebron ZB-AAA GC 10 m x 0.25 mm for amino acid analysis
Injector
Split 1:15, 250 C, 1.5 L
Carrier gas
Helium, constant flow
Flow rate
1.1 mL/min
Oven program
Increase temperature at 30 C/min from 110 to 320 C (hold 2 min)
Scan range
45450 m/z
Sampling rate
3.5 scans
Fig. 3.5: HR-GC-MS-TOF chromatogram of amino acid standards [area (Total Ion Count) x
retention time (sec)], numbers correspond to the amino acids in Table 3.4.
69
Chapter 3
Table 3.4: Quantified amino acids (numbers in Fig. 3.5 correspond with structures, molecular
masses, retention times and major ions used for identification
Peak Name of
Retention time (sec) RT Structure of amino acid
No.
amino acid
Molar mass (g/mol) M
Major ions m/z#
O
1
Alanine
RT:
55.1
M:
89.09
m/z: 130, 158
OH
Arginine
NH 2
Not included in standard

mixture
NH
H2N
OH
Asparagine
RT:
M:
m/z:
112.2
132.11
69, 113, 129, 141,
155
NH 2
H2N
OH
O
Aspartic acid
RT:
M:
m/z:
145.8
132.09
88, 99, 114, 130,
156, 174, 184,
216, 244
NH 2
HO
OH
O
Cysteine
Glutamine
Glutamic acid
Glycine
RT:
M:
m/z:
335.5
118.06
74, 104, 116, 132,
146, 174, 188, 206,
216, 248
RT:
M:
m/z:
205.5
146.13
84, 100, 114, 142,
155, 187, 215
RT:
M:
m/z:
167.8
146.12
84, 100, 128, 142,
155, 170, 188, 215,
230, 258
RT:
M:
m/z:
60.7
75.06
116, 162
SH
NH 2
O
OH
NH 2
O
OH
H2N
NH 2
OH
HO
O
H2N
NH 2
OH
70
Chapter 3
Histidine
RT:
M:
m/z:
257.6
155.14
81, 94, 110, 121,
136, 153, 180, 196,
224, 282, 310
O
OH
N
NH
10
Isoleucine*
10a
Allo-isoleucine*
11
Leucine
RT:
M:
m/z:
RT:
M:
m/z:
87.5
131.17
130, 143, 157, 172
85.7
131.17
130, 143, 157, 172
RT:
M:
m/z:
84.3
131.17
172
NH 2
O
OH
NH 2
O
OH
12
Lysine
RT:
M:
m/z:
246.6
147.18
170, 185, 198, 213,
240, 300
NH 2
H2N
OH
NH 2
13
14
15
16
Methionine
Phenylalanine
Proline
Serine
RT:
M:
m/z:
RT:
M:
m/z:
147.0
146.18
61, 75, 88, 101,
114, 129, 143, 156,
175, 190, 203, 217,
277
168.0
165.19
74, 91, 103, 120,
131, 148, 164, 190,
206, 234
RT:
M:
m/z:
105.9
114.12
70, 114, 156
RT:
M:
m/z:
102.5
105.09
60, 74, 101, 116,
146, 174, 203
OH
NH 2
O
OH
NH 2
OH
O
OH
HO
NH 2
71
Chapter 3
17
18
Threonine
Tryptophan
RT:
M:
m/z:
RT:
M:
m/z:
OH
100.2
119.12
101, 119, 143,
160, 203
O
OH
NH 2
291.0
205.22
130, 143, 229, 332
O
OH
NH 2
HN
19
Tyrosine
RT:
M:
m/z:
274,4
181.19
107, 164, 156, 169,
200, 257
OH
NH 2
HO
20
Valine
RT:
M:
m/z:
72.0
117,14
116, 143, 158
OH
21
-Aminoadipic
acid
RT:
M:
m/z:
186.4
161.16
98, 124, 142, 169,
184, 244, 272
NH 2
HO
OH
O
22
-Aminobutyric
acid
RT:
M:
m/z:
NH 2
66.6
103.12
144. 172
OH
23
24
-Aminobutyric
acid (GABA)
-Aminoisobutyric
acid
RT:
M:
m/z:
102.2
103.12
130, 144, 172, 231
RT:
M:
m/z:
75.9
103.12
116, 130, 143,
172, 231
NH 2
H2N
OH
H2N
OH
72
Chapter 3
25
L-Canavanine
Not included in standard

mixture
NH
H2N
O
N
OH
26
Norvaline
(internal standard)
RT:
79.4
M: 117.13
NH 2
OH
27
Ornithine
RT:
M:
m/z:
230.9
133.16
156, 171, 184, 226,
286
NH 2
O
OH
H2N
NH 2
major ions are underlined

L-Allo-isoleusine and L-isoleucine are stereoisomers and have different C-3 configurations
The first 20 amino acids are incorporated into proteins, of which eight (isoleucine, leucine,
lysine, methionine, phenylalanine, threonine, tryptophan and valine) cannot be produced in
the human body, and are considered essential amino acids to be supplemented through a
balanced diet (Cseke et al., 2006). The other seven amino acids are not incorporated into
proteins, but fulfill important roles in immune response, for example, i.e. L- canavanine, an
analog of L-arginine, is incorporated in the place of L-arginine and is consequently able to
disrupt RNA and DNA metabolism as well as protein synthesis of cancerous cells (see
Sutherlandia frutescens in Chapter 5 and 6; Crooks & Rosenthal, 1994). GABA (aminobutyric acid), for example, is an inhibitory neurotransmitter, which may play a role in
the treatment of anxiety and stress, and inhibits tumour cell migration (Ortega, 2003).
3.4.2.2 GC-MS analyses of Arctopus kaurenes (performed by Witte & De Castro,

unpublished data)
Due to similar polarities of these diterpenes, separation by means of TLC or column
chromatography presents a hugh challenge. Even though they are non-polar, their acidic
character prevents easy volatilization. For this reason, esterification was done by means of
methylation with diazomethane prior to screening with GC-MS. A J & W DB-1 fused silica
capillary column (30 m x 0.25 mm i.d.) was used with He as carrier gas. The conditions
were as follows: injector 230 C; detector (FID) 310 C; oven programme 200300 C at
4 C/min; split ratio 30:1; injection volume 1 L. Selected samples were studied by GC-MS
under the following conditions: DB-1 fused silica capillary column (30 m x 0.32 mm i.d.; He
as carrier gas; column temperature 150300 C at 6 C/min; split ratio 20:1; injection
73
Chapter 3
volume 1 L). The preliminary identification of the components was based on their retention
times as linear retention indices (RI) relative to the series of n-hydrocarbons, and on
computer matching against home-made library mass spectra built from pure substances
and components of known compounds and MS literature data (Mitscher et al., 1983;
Metzger & Hazebroek, 1989; Morris et al., 2005). Data obtained were coded on the basis of
presence or absence, with the relative proportions of the components being percentages
obtained by FID peak-area normalization.
3.5
Isolation of compounds
3.5.1 Column chromatography (CC)

This technique was applied to separate constituent compounds in order to isolate them.
Two types CC were used: initially to separate classes of compounds roughly into high to
low polarity fractions, a bulk C18 reverse phase (Chromabond, Machery Nagel) column
(21 x 2.5 cm, ca. 100 mL gel) was packed and prepared with MeOH. A crude MeOH plant
extract to be separated was adsorbed onto the column, and the column eluted with a
gradient of MeOH:H2O, normally 50100% MeOH with 100 mL increments. More polar
compounds would exit first and least polar compounds last. The fractions with similar
compounds were combined and reduced in volume. Further separation was then conducted
on silica gel columns (Kieselgel GF254, bead size 0.0400.063 mm, pore size15 m, Merck)
varying in size depending on the amount of sample to be separated. The fractions were
impregnated on the silica before loading on the column to reduce band width. Solvent
systems used for optimum separation was tested in small scale on TLC first.
In the case of the Arctopus kaurenoic acids, manool (55.2 mg), ent-trachyloban-19-oic acid
(151.9 mg) and ent-kauren-19-oic acid (18.4 mg) (Fig. 5.10) were isolated by extracting
24.6 g of dry ground root material of A. monacanthus overnight in 250 mL MeOH. The
mixture was filtered and the volume of the filtrate reduced under vacuum to yield a sticky
oily mixture. A phase separation was done by rinsing the extract with 50% MeOH/H2O to
dissolve and consequently remove the polar compounds leaving only the sticky non-polar
mixture containing the diterpenes. Flash column chromatography with gradient elution
[100% hexane, increasing the diethyl ether component incrementally with addition of 5%
diethyl ether until 100% diether ether until 100% was reached, each increment being
100 mL of mixed solvent] was applied for separation. Fractions of 20 mL were collected.
Fraction 5 of the first separation yielded a pure sample of manool. Repeated flash column
chromatography of fraction 6 with a hexane:diethyl ether mixture (3:2) afforded a purified
sample of ent-trachyloban-19-oic acid. Manool, ent-trachyloban-19-oic acid and ent-kauren19-oic acid are the three major components in the non-polar extract and have very similar
74
Chapter 3
polarities. Consequently these diterpenes are very challenging to separate. For this reason
ent-kauren-19-oic acid could not be isolated successfully, but an impure fraction was also
subjected to NMR spectroscopy together with the other isolated diterpenes to determine a
possible structure for it. Methyl-16-hydroxy-ent-kaur-11-en-19-oate was obtained from
fraction 8 of the first separation and purified by means of preparative TLC in hexane:diethyl
ether (3:2). The physical characters of the isolated compounds are given as:
Manool: Colourless crystalline solid; positive APCI-VTOF-MS: m/z = 273.2571 [M + H+
H2O]+ (calc. for C20H33: 273.2582); 1H- and
13
C-NMR (1H: 300 MHz;
13
C:75 MHz, CDCl3):
see Appendix 4; GC-MS, m/z = 290 [M H] derivatized to give the methyl ester (base peak
= 137, RI = 2031, see Table 5.9) calculated for C20H34O: 290.4880 (underivatized).
Ent-trachyloban-19-oic acid: Colourless crystalline solid; positive APCI-VTOF-MS: m/z =
303.2299 [M + H]+ (calc. for C20H31O2: 303.2324); 1H- and
13
C-NMR (1H: 300 MHz;
13
C:75
MHz, CDCl3): see Appendix 4; GC-MS, m/z = 316 [M H] derivatized to give the methyl
ester (base peak = 105, RI = 2200, see Table 5.9) calculated for C20H30O2: 302.4558
(underivatized).
Kauren-19-oic acid: Impure; colourless crystalline solid; positive APCI-VTOF-MS: m/z =
301.2121 [M + H]+ (calc. for C20H29O2: 301.2168); GC-MS, m/z = 314 [M H] derivatized to
give the methyl ester (base peak = 131, RI = 2144, see Table 5.9), calculated for C20H28O2:
300.4400 (underivatized).
Methyl 16-hydroxy-ent-kaur-11-en-19-oate: Colourless crystalline solid; positive APCIVTOF-MS: m/z = 315.2317 [M + H H2O]+ (calc. for C21H31O2: 315.2324); 1H- and 13C-NMR
(1H: 300 MHz;
13
C:75 MHz, CDCl3): see Appendix 4; GC-MS: m/z = 332 [M H] naturally
occurring as the methyl ester (base peak = 121, RI = 2360, see Table 5.9), calculated for
C21H32O3: 332.4820.
3.5.2 Preparative thin layer chromatography

When small quantities of compound needed to be purified, preparative TLC was used.
Plates consisted of silica gel 60 F254, 1.5 mm thickness, on glass sheets obtained from
Merck). The sample was applied in a thin line across the bottom of the plate, 1.5 cm from
the edge, with a Pasteur pipette. The best suited solvent was used to develop the plates,
sometimes twice or thrice, with drying in between, to obtain the best resolution. The
resolved compounds were visualized under UV and by using the relevant spray reagents
(1 cm on a vertical side of the plate) and then scraped from the plate with a spatula. The
impregnated silica that was removed, was stirred in hot EtOH to remove the adsorbed
compounds, vacuum filtered through Whatman no. 4 filter paper and the filtrate taken to
dryness to produce the isolated product.
75
Chapter 3
3.6
Structure elucidation
3.6.1 Nuclear magnetic resonance spectroscopy (NMR)

NMR is a form of absorption spectroscopy. A molecule can absorb electromagnetic
radiation in the radio frequency region under appropriate conditions when placed in a
magnetic field. This absorption is a function of for instance the carbon and hydrogen atom
nuclei in the molecule (Silverstein & Webster, 2005). When energy is applied as radio
frequency radiation, of for instance 300 MHz, in a stationary magnetic field of strength (B0)
of 7.5 T for a proton, the system is said to be at resonance. The proton absorbs energy,
raising it to a higher energy state, and thus a spectrum is recorded as a function of
quantified energy absorption. Carbon atoms need lower radio frequencies to become
excited (75 MHz in a magnetic field of 7.5 T). 1D-NMR gives information about the number
of each type of hydrogen and carbon atom, as well as the nature of the immediate
molecular environment of each atom. 2D-NMR (COSY, HSQC, HMBC and DEPT) gives
information about the connectivity of fragments within a molecule.
1
H-NMR and
13
C-NMR experiments (1D and 2D) were performed on a Varian Unity Inova
spectrometer (at WITS) operating at 300 MHz (1H) and 75 MHz (13C) in DMSO-d6 for the
more polar (R)-3-O--D-glucopyranosylrosmarinic acid from Arctopus, and in CD3OD for
rosmarinic acid (from Arctopus) and SU1, SU2 and SU3 (from Sutherlandia) of medium
polarity. The non-polar Arctopus kaurenes were analysed in CDCl3 on a Bruker Avance 300
spectrometer, also operating at 300 MHz (1H) and 75 MHz (13C). The solvents were used as
internal standards in all the cases (see Appendix 4). Some of the triterpenoids from
Arctopus were also isolated and submitted for NMR, but the structures are yet to be solved.
3.6.2 Mass spectrometry (MS)

For full characterization of isolated compounds, accurate masses are required. The
analytical instrumentation applied is dependent on the polarity and size of the molecules.
Sutherlandia triterpenoids: ESI-TOF-MS measurements were obtained on a Waters API QTOF Ultima instrument in positive-ionization mode. Data was acquired by scanning from
m/z 100 to 1999, at a scan rate of 1 second per scan in the continuum acquisition. The
785.8426 ion of GFP was used as lock mass for accurate mass work. The instrument is
situated in the Department of Chemistry, University of Stellenbosch.
76
Chapter 3
Arctopus phenolic acids: A Waters Acquity Ultra Preformance LC system equipped with
PDA, a Waters LCT Premier mass spectrometer and an Acquity UPLC BEH C18 column
(17 m, 2.1 x 100 mm) was used. ESI in negative-ionization mode was applied. The
instrument is situated on the Modderfontein campus of the CSIR.
Arctopus kaurenes: Accurate masses of the isolated compounds were obtained by means
of a Waters SYNAPT HDMS system with a QTOF design equipped with a lock spray
interface to improve mass accuracy. A Waters Acquity BEH C18 UPLC column (C18
1.7 m, 2.1 x 100 mm) was used in all experiments. The column was kept at 40 C. The
chromatographic procedure entailed 0.1 minute of 60% methanol in water (v/v), followed by
a 6 minute linear gradient to 90% methanol in water (v/v), kept for 1 minute before a
1 minute linear gradient back to the starting conditions (60% methanol in water) followed by
2 min equilibration to prepare the system for the next injection. The flow rate was kept
constant at 0.3 mL/min and the total analysis time was 10 minutes. A PDA detector was
placed in tandem before the mass detector and was programmed to scan 200500 nm with
a scan rate of 20 spectra per second and a resolution of 1.2 nm. Ionisation of all the
compounds could be achieved with chemical ionisation in positive mode. The ionisation of
the compounds was obtained with the following source settings: capillary voltage of 2.5 kV,
sample cone voltage of 20 V, extraction cone voltage of 4 V and carona pin voltage to 20 V.
The source temperature was set at 120 C, the desolvation temperature at 350 C and the
desolvation gas flow at 500 L/hr (nitrogen gas). Leucine enkephalin was used as a lock
mass calibrant at 50 pg/mL and was continuously infused at 10 uL/min. Sampling of the
lock mass signal was done every 30 seconds. Data collection was done using Waters
MassLynx software (version 4.1). All empirical formulae calculations were done using the
embedded software tools.
3.6.3 Optical rotation

Optical rotations were measured with a Jasco DIP-370 digital polarimeter using a sodium
lamp with wavelength 589 nm (Department of Chemistry, WITS). The instrument was
temperature controlled, and zeroed on the same solvent in which the isolated compounds
were dissolved. Specific rotations were calculated relative to the concentration of the
sample.
77
Chapter 3
3.7
Bitterness taste testing
The hypothesis is that one of the many typical characteristics of a tonic plant is that it will
have a bitter taste, according to the imbiza (Zulu) and musa-pelo (Sotho) concepts (see
Appendix 1A as well as Chapters 2 and 4). A bitter taste is responsible for the amarum
effect to stimulate the taste buds and promote the flow of saliva, gastric juices and bile
(Van Wyk & Wink, 2004), which in turn enhances the function of the digestive system, and
ultimately boosts the immune system (Zimmerman et al., 1986) by means of a more
efficient uptake of nutrients or some other unknown mechanism. The aim of this experiment
is to establish if these plants traditionally classified as amara, imbiza or musa-pelo, are
indeed bitter as per definition.
Bitterness is usually quantified with a so-called bitterness value, which is available for many
European traditional tonics such as gentian and absinth (Wagner & Wiesenauer, 1995;
European Pharmacopoeia, 2005b). The European Pharmacopoeia (2005a) defines
bitterness value as the reciprocal of the dilution of a compound, a liquid or an extract that
still has a bitter taste. It is determined by comparison with quinine hydrochloride, of which
the bitterness value is set at 200 000.
H
HO
H
O
Fig. 3.5:
The structure of quinine
The procedure used here is an adaptation from the methods prescribed by the World
Health Organisation (2002) and given in the European Pharmacopoeia (2005a), and was
approved by the Ethics Committee of the University of Johannesburg. Healthy male or
female volunteers over the age of 18 years were used. All potential participants were
screened first for their ability to taste bitterness using 0.100 g of quinine hydrochloride R
(obtained from Merck) dissolved in Bonaqua still drinking water and diluted to 100 mL.
Bonaqua still is high quality commercial drinking water purified by reverse osmosis and
reconstituted with normal salts found in drinking water. This solution was diluted 100 times
(5 mL diluted to 500 mL) and constituted the stock solution (S1) with a concentration of
0.01 mg/mL. Nine different dilutions ranging from low to high were prepared (in tubes) from
78
Chapter 3
solution S1 according to Table 3.5, and were used to obtain the bitterness tasting ability of
the enrolled participants.
All participants had to taste the solutions from the respective tubes, (starting from 1,
proceeding to taste from consecutive tubes until a bitter taste sensation was obtained) by
following the tasting procedure given below. A person who did not manage to appreciate a
bitter sensation when tasting the solution from tube 9 (highest concentration of quinine
hydrochloride at 0.058 mg/10 mL), was not a suitable participant for continuing the
experiment.
Table 3.5: Serial dilutions of quinine hydrochloride R to determine bitterness tasting ability,
given in mg of quinine hydrochloride R per 10 mL water. S1 is the stock solution,
prepared by dissolving 0.100 g of quinine hydrochloride R in 100 mL of pure
water and further diluted 100 times (5 mL diluted to 500 mL).
Tube number
1
S1 (mL)
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
Safe drinking water

(mL)
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
mg quinine HCl in
10 mL of solution
0.042 0.044 0.046 0.048 0.050 0.052 0.054 0.056 0.058
The bitterness taste testing of the 15 medicinal plant extracts (Dicoma anomala was added)
was conducted in a similar way: 1 g of dried ground plant sample was extracted overnight
by bringing 100 mL of Bonaqua drinking water to boiling point, adding this to the plant
sample, vortexing the mixture for 1 minute and leaving it to stand overnight. The mixture
was filtered through cotton wool and the filtrate diluted 100 times (5 mL diluted to 500 mL
with Bonaqua drinking water) for the very bitter plants (see Table 3.6). This stock solution
(S2) with a concentration of 0.1 mg/mL has a dilution factor (DF) of 10 000. Nine different
dilutions ranging from low to high were prepared (in tubes) from solution S2 according to the
table below, and were used together with solution S2 (undiluted) to obtain the bitterness
value through bitterness tasting by participants who were able to taste bitterness (as
described in the previous paragraph).
Some of the plant extracts were not found to be bitter at a concentration of 0.1 mg/mL
(Tube 10). In these cases the original extracts were prepared by extracting 1 g of dried
ground plant sample in 100 mL of Bonaqua drinking water overnight as before, but was
diluted only 20 times (25 mL diluted to 500 mL with Bonaqua drinking water) instead of 100
times (see Table 3.6 less bitter plants). These stock solutions (S2) had a concentration of
79
Chapter 3
0.5 mg/mL instead of 0.1 mg/mL. Therefore the DF of S2 for the less bitter plants was
2 000.
Table 3.6: Determination of bitterness value: serial dilution of very bitter and less bitter
plant extracts tested. Very bitter: Aloe ferox, Artemisia afra, Dicoma anomala,
D. capensis, Hypoxis hemerocallidea, Sutherlandia microphylla, Vernonia
oligocephala and Ziziphus mucronata. Less bitter: Agathosma betulina,
Arctopus monacanthus, Balanites maughamii, Harpagophytum procumbens,
Muraltia heisteria, Warburgia salutaris and Withania somnifera.
Tube number
1
10
S2 (mL)
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.0
Safe drinking water

(mL)
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Final concentration
(mg/mL)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
S2 (mL)
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.0
Safe drinking water

(mL)
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Final concentration
(mg/mL)
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Very bitter
Less bitter
To save time, the person tasting the S3 dilutions would start with tube 5. If a bitter taste
sensation was experienced, the person would then continue tasting tubes 1 to 4, to
determine the smallest concentration at which the bitter sensation is obtained. If the person
could not taste bitterness in tube 5, he/she would then continue with tubes 6 to 10 to find
the smallest concentration at which bitterness is experienced. If bitterness was tasted in
tube 1 already, the content of tube 1 was then diluted the same way as S2 in order to obtain
10 more tubes starting with a concentration of 0.001 mg/mL and ending with a
concentration of 0.01 mg/mL for the very bitter plants, and with a concentration of
0.005 mg/mL and ending with a concentration of 0.05 mg/mL for the less bitter plants.
The tasting procedure entailed the following: After rinsing the mouth with Bonaqua drinking
water, 10 mL were tasted of test solution from the tubes provided (lowest concentration
80
Chapter 3
first) by swirling the solution in the mouth mainly near the base of the tongue for
30 seconds. If a bitter sensation was not felt after this time, the solution was spat out and
the participant had to wait for 1 minute to ascertain if there might be a delayed sensitivity. If
not, the mouth was rinsed with Bonaqua drinking water and the participant had to wait
10 minutes before tasting the tube with the next highest concentration.
The expression used for calculating bitterness value incorporates the dilution of the extract
that still exhibits a bitter taste, and relates this to the concentration of quinine hydrochloride
standard used for calibrating the taste perception of a particular participant. The World
Health Organisation (2002) expresses bitterness value as units per g, where 1 g of quinine
hydrochloride in 2000 mL has a bitterness value of 2 000 units/g. In the European
pharmacopoeia (2005a) the bitterness value of quinine hydrochloride is set at 1:200 000 (as
a dilution) and is incorporated in the formula used for calculation below. As each individual
has a different level of bitterness tasting ability (that may even vary from day to day), a
correction factor, k, is used for each member of the panel:
k = n/5.00
(n = mL of S1 in the 10 mL tube)
Bitterness value is calculated for each panel member from the expression:
DFS2 x k
mL S2 in tube which still has a bitter taste x 0.1
According to the European pharmacopoeia (2005a), an average value obtained from six
participants is sufficient for an accurate result. A summary of the results obtained for the
species subjected to bitterness taste testing in Chapter 5, including the amount of taste
results obtained per plant in some cases six results were not sufficient as some taste
results returned as not bitter in the same sitting where results of very bitter was obtained.
In these cases, more samples were subjected for taste testing, until at least six repeatable
results were obtained. One person never tasted the same species twice.
81
CHAPTER 4
Ethnobotany
4.1
Introduction
The mixture of the informal traditional medicine systems of the indigenous Khoi-San, Nguni
(Xhosa, Zulu, Swati, Hlubi, Phuthi and Ndebele) and Sotho-speaking peoples, together with
Cape Dutch medicine, makes southern African traditional medicine a unique, rich blend of
cultural and botanical diversity. It is estimated that South Africa has approximately 3 000
indigenous medicinal plant species (Van Wyk & Gericke, 2000). Approximately 60% of the
South African population (almost 27 million consumers) consult indigenous traditional healers,
of which there are approximately 200 000 in the country (Van Wyk et al., 2009).
Apart from Cape Dutch medicine, the majority of the other indigenous traditional medicine
systems have not yet been systematised, and are merely passed on by word of mouth from
generation to generation. Unfortunately, it is mainly elderly people who has knowledge of
herbal lore (Van Wyk et al. 2009), and much of the historical ethnobotany (a multi-disciplinary
study based on the interactions between people and plants Jones, 1941; Ford, 1978; Veilleux
& King, 1996) is lost without being recorded, due to ineffective verbal transmission and cultural
changes. Indigenous medicine systems are dynamic and adaptive, i.e. recent influences (over
the past century Van Wyk, 2008a) in southern African traditional medicine include Ayurvedic
medicine, Traditional Chinese Medicine, European and homoeopathic medicine, and allow for
the incorporation of exotic plants into some southern African traditional medicine practices
(Watt & Breyer-Brandwijk, 1962). This presents a complex mixture of remedies available for
self-medication (Dold & Cocks, 1999; Van Wyk et al., 2009). Consequently, it is important to
conserve the intellectual property of the vast body of knowledge rooted in past traditions. Also,
it will only be ethically correct to ensure that the people from whom the ethnobotany was
obtained originally, be the keepers of the intellectual property they have shared (International
Society of Ethnobiology, 2006; Thring & Weitz, 2006; Crouch et al., 2008; Scott & Hewett,
2008). Ethnobotanists who have made worthy contributions with regard to conserving such
indigenous knowledge in the past 40 years, include Ben-Erik van Wyk, renowned for his
reviews of southern African medicinal plants, with particular emphasis on the Khoi-San and
Cape Dutch medical ethnobotany and materia medica (Van Wyk & Gericke, 2000; Van Wyk,
82
Chapter 4
Ethnobotany
2008a,b; Van Wyk & Albrecht, 2008; Van Wyk et al., 2008; Van Wyk et al., 2009; De Beer &
Van Wyk, 2011). In addition, Anne Hutchings (Hutchings & Johnson, 1986; Hutchings et al.,
1996), Johannes van Staden (Hutchings & Van Staden, 1994; Jger et al., 1996; Kelmanson et
al., 2000; Ndhlala et al., 2011) and Annah Moteetee (Moteetee & Van Wyk, 2007; 2011) are
well known for their contributions to ethnobotany, phytochemistry and pharmacological
properties of Zulu, Sotho and Xhosa medicinal plants.
An ethnobotanical overview of 14 of the most well-known tonic plants in South Africa is
provided in this chapter, together with a botanical background and geographical distribution of
these plants and their close relatives. The full details of recorded ethnobotanical uses can be
found in Appendix 2. Aspects relating to their tonic properties are highlighted. According to
Smith (1895) plants which have medical virtues have a recognisable peculiarity of taste,
usually bitter, but otherwise pungent, aromatic, astringent, or acid, and they frequently though
not always have a peculiar smell. The ethnobotany in this chapter and the bitterness taste
testing results in Chapter 5, will test this statement. Together with a thorough scrutiny of the
phytochemistry (Chapter 5) and biological activity (Chapter 6) of these plants, one will be able
to ascertain whether these plants can indeed be regarded as tonics.
4.2
The ethnobotany of South African traditional tonic plants

Agathosma species (previously Barosma Pillans, 1950; Spreeth, 1976) are from the
Rutaceae family. They are perennial shrubs with woody branches and small flat gland-dotted
leaves, and are all aromatic (Greek agathos means good, osma means aroma Rood,
1994). These species are elements of typical fynbos (vegetation found in the Western Cape
Province of South Africa) and are abundant in mountainous areas in the Cape (Van Wyk &
Gericke, 2000).
While the Khoi-San word for buchu [or boechoe, boekoe, boggoa, bookoo, bouchou, bugu,
buccho, bucchuu, bucco (Smith, 1966)] indicates any aromatic plant that can be dried and
powdered (Moolla & Viljoen, 2008), the modern term buchu refers to Agathosma betulina
[buchu, boegoe, letulina or bookoo in Khoi (Van Wyk, 2008a); round-leaf buchu in English;
ibuchu in Xhosa (Van Wyk et al., 2009), French and Italian; bucco in German (Van Wyk &
Wink, 2004)]. This Cape endemic species is well adapted to dry sunny conditions and are
found on rocky sandstone slopes in the north western Cape region [Fig. 4.1(b)]. A. crenulata L.
83
Chapter 4
Ethnobotany
Pillans [buchu or boegoe in Khoi (Van Wyk, 2008a); oval/long-leaf buchu; anysboegoe (Van
Wyk et al., 2009), bosboegoe, bergboegoe, fonteinboegoe, olifantsboegoe (Rood, 1994),
regteboegoe or rivierboegoe (Smith, 1966) in Afrikaans] on the other hand, grows on the more
damp middle to bottom of slopes and valleys [Fig. 4.1(d)]. Mainly A. betulina and A. crenulata
are used medicinally.
A. betulina and A. crenulata are often confused, but can be distinguished by means of leaf
morphology: A. betulina has leaves of about 20 mm in length, that are less than twice as long
as broad, while A. crenulata leaves are more than twice as long as they are broad [Pillans,
1950; Spreeth, 1976, see Fig. 4.1(a) and (c)]. Chemical characters may also be used to
distinguish these two species, as hybridisation between the two species is common. See
Chapter 5 (Paragraph 5.2.1) for a detailed discussion on this topic.
A. ovata (Thunb.) Pillans [Fig. 4.1(e)], also known as false buchu, basterboegoe or
rivierboegoe in Afrikaans (Smith, 1966), is a perennial variable shrub that may grow up to 3 m
tall and is known to spread. Its distribution is much wider than that of A. betulna and
A. crenulata: from sea level to altitudes of 2000 m [Fig. 4.1(f)], as it is famous for its adaptability
in various climates. Where A. betulina and A. crenulata have white to light pink, star-shaped
flowers, A. ovata has white, pink or purple flowers similarly shaped, also borne towards the tips
of the branches. A. ovata has small ovate leaves (8 mm broad, 1015 mm long) dotted with oil
glands, the same as the other two species (Moolla, 2005).
Buchu leaves (mostly A. betulina and A. crenulata) have a long ethnobotanical history (see
Appendix 2A), and was regarded as an important medicine in the Khoi-San repertoire (Van
Wyk, 2008a), long before its discovery by the Cape Dutch settlers Thunberg (17721775)
noted its topical use (Forbes, 1986), and Burchell (18221824) recorded a variety of other
uses, including its tonic properties, for the first time. Rood (1994) stated that boegoe was used
for almost every known illness in the olden days, and was listed six times as a general health
tonic. Its popularity as general medicine was evident from 34 different recipes for remedies
(boererate) for a great variety of ailments listed by Dykman (1908) where buchu is an
ingredient. From the 29 citations, buchu is mostly used as analgesic and antiinflammatory for
rheumatism and related ailments (20), urinary tract infections, i.e. mostly as diuretic (27),
externally for wounds, sprains and bruises (29), and for stomach ailments as a bitter digestive
tonic, appetite stimulant and anti-spasmodic (23). Not only is it indicated for dyspepsia, but also
as both laxative and anti-diarrhoeal, suggesting a balancing, tonic effect. Its mentioned use for
84
Chapter 4
Ethnobotany
serious (respiratory tract) infections such as tuberculosis, diphtheria and colera is noteworthy,
while several references include treatment of cough or whooping cough. Furthermore, its use
as a treatment for cancer was recorded by Thring and Weitz (2006).
(b)
(a)
(b)
(c)
(d)
(c)
(e)
(f)
Fig. 4.1: (a) Agathosma betulina (round leaf buchu)*; (b) Geographical distribution of
A. betulina in the western Cape#; (c) A. crenulata (oval leaf buchu)* ;
(d) Geographical distribution of A. crenulata in the western Cape#; (e) A. ovata
flowers and leaves* ; (f) Geographical distribution of A. ovata along the South African
coast#
[* = Van Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
85
Chapter 4
Ethnobotany
The wide variety of medicinal uses and the importance thereof in the flavour industry,
consequently rendered buchu one of the top commercially important medicinal plants in
southern Africa (Van Wyk, 2008b), as well as internationally (Van Wyk & Wink, 2004; Moolla &
Viljoen, 2008). The traditional uses suggest that Agathosma betulina is a possible general
health tonic, panacea and stimulant that promotes a general feeling of wellbeing (even by
means of the uptake of oils through the skin) and increases resistance (against infections in
gastro-intestinal and respiratory tracts). The anecdotes also suggest that it probably has amara
aromatica (a bitter tonic containing essential oils) and alterative (diuretic, anti-spasmodic,
stomachic) properties.
4.2.2 Aloe species

South Africa boasts a great diversity of Aloe (Asphodelaceae) species (Reynolds, 1950; Bruce,
1975; Glen & Hardy, 2000; Van Wyk & Smith, 2003). All Aloes are perennial leaf-succulent
xerophytes, characterised by their thick, fleshy, leaves, adapted for water storage in mucilage.
The leaf cuticles are thick and covered with a wax layer (Berger, 1908; Reynolds, 1950).
Medicinal species have groups of pericyclic cells associated with the vascular bundles in the
leaves that store and secrete a yellow exudate when the leaves are cut or broken. This sap is
boiled and cooled to form a very bitter black solid called bitters, lump or aloes [Marloth,
1915; Krger & Beyers, 1977; Robertson, 1979; Forbes, 1986; see Fig. 4.3(d)]. The word
aloe has an Arabic origin (alloeh), meaning shining, bitter substance, referring to the above
exudates (Dagne, et al., 2000).
The history of uses of medicinal Aloe species in several healing cultures across the globe date
back as far as 2 100 BC (a Mesopotamian clay tablet), listed in the Ebers papyrus (1 500 BC)
as well as the Jewish Torah (ca. 520 BC), and noted in texts by Aristotle (384322 BC),
Dioscorides (ca. 4090 AD) and Galen (129217 AD) (see Chapter 2). While the Egyptians
used aloes as cosmetics (for chapping and hair loss), it was also used topically as an antiseptic
for treating wounds, genital ulcers, haemorrhoids, boils and mouth ulcers, and as an insecticide
(Boulos, 1983). In small doses, it was effective as a digestive tonic, stomachic and cholagogue,
while large doses were intended as powerful purgatives, emmenagogues and anthelmintics
(Oronzo-Barocio et al., 1999). Aloes were used for eczema and sinusitis in the East since the
seventh century, and indicated efficacy for the treatment of chronic bronchial asthma (Mascolo
et al., 2004). Currently, a variety of uses and indications are also known for whole leaf extracts
86
Chapter 4
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or juice (a confusing term that might refer to the latex expressed from the leaves or the
mucilage), including the treatment of bacterial and viral infections, high cholesterol and even
cancer, with proposed immunomodulatory, antiinflammatory and anti-tumor properties (OronzoBarocio et al., 1999; Mascolo et al., 2004; Steenkamp & Stewart, 2007). The four most well
known medicinal Aloe species in South Africa are Aloe arborescens Mill., A. ferox Mill., A.
marlothii A. Berger and A. vera L. Burm.f. (also known as A. barbadensis Mill.). Grace et al.
(2008) reviewed the therapeutic uses of Aloe in southern Africa, including recorded references
of the four mentioned species from as early as 1854 (details included in Appendix 2B from
references where specific uses were provided).
A. arborescens is a multi-stemmed Aloe with leaves of about 40 cm long, borne in apical
rosettes and re-curved with teeth on the leaf margins [Reynolds, 1950, 1982; Van Wyk &
Smith, 2003; [see Fig. 4.3(a)]. The latter researchers, however, reported a type with no spines
on the leaves. These succulent shrubs can grow taller than two metres, and is very easy to
propagate (Reynolds, 1950, 1982; Van Wyk & Smith, 2003). A. arborescens is commonly
known as krantz aloe, kransaalwyn (Afrikaans), inkalane (Zulu) (Van Wyk & Smith, 2003),
inhlaba lencane (siSwati) (Amusan et al., 2007) or unomaweni (Xhosa) (Pooley, 2003). In
Japan it is well known as Japan aloe or kidachi aloe, as it was used to treat nuclear irradiation
burn wounds of people injured during the bombing of Hiroshima in the Second World War (Van
Wyk et al., 2009). It was also used to treat constipation and dermatological ailments in
Japanese folklore medicine (Kampo) and is a known health and nutrition food in Japan (Sato et
al., 1991; Yagi, 2004). Leaves are widely used in animal and human medicine (see Appendix
2B), and may be spray-dried or freeze-dried to produce commercial leaf or gel powder (Ni &
Tizard, 2004; Ni et al., 2004; Standards South Africa, 2007).
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(a)
(b)
(c)
(d)
Fig. 4.2: Habits*, flowers* and distributions# of (a) Aloe arborescens, (b) A. ferox and
(c) A. marlothii; (d) A. vera*; A. vera cosmetic products* and Aloe lump* [* = Van
Wyk & Smith, 2003; Van Wyk & Wink, 2004; Van Wyk et al., 2009; # =
http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
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The difference between A. ferox and other Aloes is that it is single-stemmed with
characteristic erect racemes. It can grow up to 5 m tall and has persistent dry leaves on the
bottom part of the stem [see Fig. 4.3 (b)]. The epiphet ferox stems from the word
ferocious, indicating the sharp, dark brown spines along the leaf margins and sometimes on
the lower surface of the leaves (Reynolds, 1950, 1982; Van Wyk & Smith, 2003). This Aloe
species has a long history of medicinal uses in southern Africa. It was found depicted in San
rock paintings as plants held by humans (Reynolds, 1950), and export of the dried latex
known as Cape aloes to Europe began as early as 1761 (Marloth, 1915; Hodge, 1953;
Krger & Beyers, 1977; Robertson, 1979; Forbes, 1986). A. ferox has a wide distribution and
is traditionally used as a Cape Dutch remedy, as well as in the Bantu cultures of southern
Africa (Hutchings et al., 1996; Van Wyk, 2008a), as can be found in Appendix 2B. Common
names for A. ferox include red aloe, bitter aloe, aalwyn, bitteraalwyn, Kaapse aalwyn (Van
Wyk, 2008a; Van Wyk et al., 2009), tapaalwyn, goreebosch, makalwyn, kraalalwyn,
(op)regte-aalwyn, Swellendamsalwee (Smith, 1966) in Afrikaans, iNlaba (Thomas and Grant,
2011), umHlaba (Van Wyk et al., 2009) or inkalane (Van Wyk & Smith, 1996) in Zulu, iKhala,
uNomaweni or umHhlaba in Xhosa (Bhat & Jacobs, 1995), i-umHlaba in Siswati (Thomas &
Grant, 2011), and lekhala la quthing (Bryant, 1966; Smith, 1966; Bhat & Jacobs, 1995),
lekhala-le-leholo (Maliehe, 1997; Moteetee & Van Wyk, 2011) or lekhala-la-thaba in seSotho.
A. marlothii can be divided into two subspecies: A. marlothii A.Berger subsp. marlothii, which
has solitary stems, taller than 2 m, and leaves with many surface prickles, and A. marlothii
subspecies orientalis, which has stems in clumps, shorter than 2 m and leaves with few
surface prickles (Glen & Hardy, 2000). A. marlothii species is exceptionally common in
KwaZulu-Natal, Botswana and Mozambique [see Fig. 4.3(c)]. Common names include
mountain aloe, snuifaalwyn (Thomas & Grant, 2011), bergaalwyn or boomaalwyn in
Afrikaans, umHlaba or imiHlaba in Zulu, sekgopha in Nothern Sotho (Thomas & Grant, 2011),
kgopha in seSotho (Van Wyk & Smith, 2003), mhanga in Changana (Ribeiro et al., 2010),
mhanga (Thomas & Grant, 2011) or mhangani (Luseba et al., 2007) in Tsonga, mokgopha or
mogwapha in Tswana (Thomas & Grant, 2011), bindamutsho (Thomas & Grant, 2011) or
tshikopa (Luseba et al., 2007) in Venda, mokgopa (Luseba et al., 2007) in Setswana and
inHlaba (Thomas & Grant, 2011) or inhaba lenkhulu (Amusan et al., 2007) in siSwati. This
Aloe species is medicinally important for human and animal treatment (see Appendix 2B).
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A. vera is the most well-known Aloe in the world, particularly for its valuable medicinal
contribution in the cosmetics industry, as well as being an important ingredient in health
drinks and tonic preparations (Van Wyk & Wink, 2004). This stemless Aloe has thick, fleshy
leaves with small teeth along the leaf margins [Van Wyk & Smith, 2003; see Fig. 4.3(d)]. A
summary of its ethnobotanical value is given in Appendix 2B.
From more than 70 references (Appendix 2B), it became apparent that A. ferox (47
references) and A. vera (19 references) are medicinally of more importance than all the other
Aloe species in southern Africa. Notably Traditional Chinese Medicine does not differentiate
between these two species and indicate them interchangeably for the same ailments (Wu,
2005), but because A. ferox is indigenous to South Africa, it is consequently more widely
spread and commonly available. For both A. ferox and A. vera, large percentages (at least
70%) of indications are focused on the skin (including wound healing, venereal diseases,
ulcers, and a hydrating effect, which is particularly important in the cosmetics industry). Also
most pronounced (for all four species) is the bitter taste of the leaf exudate, historically known
for a laxative effect, but notably indicated as bitter tonic, and used for gastric ulcers, which
points to internal wound healing properties, particularly for A. ferox and A. vera. Of interest
also, is the frequency of indications mentioned for these two species, related to infections,
stress or old age, i.e. arthritis, rheumatism (analgesic and antiinflammatory properties),
diabetes, high cholesterol, high blood sugar, psoriasis and eczema, tuberculosis, asthma,
cancer, HIV/Aids [anti-viral and immunomodulatory effects (Tizard & Ramamoorthy (2004)],
as well as hypertension. Several mentionings are made of the gel as a general health tonic,
claimed to stimulate the immune system. A. arborescens (13 references) seems to be
recognised for its outstanding wound-healing properties, with one report (Amusan et al.,
2007) indicating its use as anti-diabetic, analgesic and for increased circulation, as well as a
single mention of its use as tonic drink for general health and for the prevention and cure of
cancer (Van Wyk et al., 1997, 2009). A. marlothii (15 references), on the other hand, shows a
greater diversity of uses, five as anthelmintic, six as an ingredient of snuff, six as analgesic,
five as antiinflammatory or for rheumatism, and two for malaria. A single mention is made of
its use in the treatment of heart (Amusan et al., 2007), liver and biliary disorders (Ribeiro et
al., 2010). The exudate undoubtedly has an amarum effect, and from this review, possible
immunomodulatory or immunostimulatory properties became evident.
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Chapter 4
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The name Arctopus means spiny bears foot, aptly given by Linnaeus, due to the simple
leaves borne flat on the ground, armed with large inflexed spines (Theodore, 1972) [see
Fig. 4.4 (a) and (b)]. One of its common names, (Kaapse) platdoring, given by Thunberg
(Theodore, 1972; Forbes, 1986), is indicative of the large inflexed spines on the leaves,
which were a nuisance to the bare-footed slaves in the Cape (Smith, 1966). Sieketroos(t)
(Marloth, 1917) or pokkiesdoring [Thunberg, 1823; Thunberg (1785), cited in Scott & Hewett
(2008)] in Afrikaans, both signify the medicinal used of the plant. All three common names
can apply to all three species (A. dregei Sond., A. echinatus L. and A. monacanthus
Carmicheal ex Sond., Apiaceae), which are all endemic to the Cape Floristic region of South
Africa (Goldblatt & Manning, 2000). A. echinatus [See Fig. 4.4 (e)] is the most widely spread
of the three species and most commonly used. Fig. 4.4 (a) and (b) show the aerial parts of
the three species, with few obvious morphological differences between the species. White to
pinkish flowers are borne on separate male and female plants [see Fig. 4.4 (c)]. The spiny
fruits are dispersed by animals and humans. These plants have substantial underground
tuberous roots; the only part of the plant used medicinally (Thunberg, 1823). The roots can
be used to prepare decoctions, infusions or tinctures, while the resinous exudate of freshly
cut root can be applied topically (Van Wyk et al., 2009). Magee et al. (2007) reviewed the
ethnobotany of the Arctopus species (also see Appendix 2C).
Sieketroos means comfort to the sick, possibly referring to it being used as a general
medicine for many ailments (see Appendix 2C). During 1827, Dr James Barry, assistant
surgeon to the Forces, reported that it produces moderate excitement and steadily, gradually
augments and accelerates the force of circulation without inducing nervous irritability or
depressing effects consequent to the stimuli, and that under exhibition of the Arctopus,
the disease (syphilis) gave way, the ulcers healed, and the patient became fat, strong and
sound (Theodore, 1972). These descriptions are typical of substances classified as
restoratives and possibly also adaptogens (see Chapter 2). The tonic properties of Arctopus
most likely result from an induced amarum or alterative effect (gastro-intestinal tract)
[A. echinatus is one of many plants also known as bitterwortel (Smith, 1966)] and is used for
blood purification (urinary tract). Its uses to treat tuberculosis, cough and inflammation (Van
Wyk & Gericke, 2000), together with its reported antibacterial properties (Magee et al., 2007)
suggests an ability to increase resistance against infection.
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Chapter 4
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(a)
(b)
(d)
(c)
(e)
(f)
(g)
Fig. 4.4: (a) Arctopus echinatus (left) and A. monacanthus (right) side by side*;
(b) A. dregei*; (c) Male A. echinatus*; (d) Female A. echinatus*; (e) Distribution of
A. dregei#; (f) Distribution of A. echinatus#; (g) Distribution of A. monacanthus#
[* = from B-E van Wyk; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
92
Chapter 4
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The medicinally used species of the genus Alepidea F. Delaroche (known as ikhathazo in
Zulu) is closely related to the Arctopus species. Alepidea is as important as Arctopus in
southern African traditional medicine, and is used widely to treat respiratory ailments during
the winter period (De Castro & Van Wyk, 1994; Hutchings et al., 1996; Van Wyk & Gericke,
2000; Magee et al., 2007). Smoking the roots also causes mild sedation and vivid dreams
(Van Wyk et al., 2009).The dry rhizome and roots are smoked or powdered and taken as
snuff by diviners and healers to assist in divination and communication with ancestors
(Hutchings et al., 2006; Van Wyk et al., 2009). Rhizomes of Alepidea cordifolia B.-E. van Wyk
and Alepidea amatymbica Eckl. & Zeyh. (these two species are often confused, see Fig. 4.5)
are commonly sold at traditional muti markets, especially in KwaZulu-Natal. Interestingly,
Arctopus and Alepidea are similar to each other and belong to the subfamily Saniculoideae of
the family Apiaceae (Magee et al., 2008; Magee et al., 2010). The similar medicinal uses of
these species are ascribed to the presence of similar phytochemicals (Holtzapfel et al., 1995;
Van Wyk & Gericke., 2000; Olivier et al., 2008) (see Chapter 5).
(a)
(b)
Fig. 4.5: (a) Alepidea cordifolia (b) Alepidea amatymbica [Photos from B-E van Wyk]

Artemisia afra (Asteraceae), the only Artemisia species among 350 that is indigenous to
Africa (Sibernagel et al., 1990), is an aromatic medicinal herb used across the world (Van
Wyk & Wink, 2004). It is commonly known as African wormwood or wild wormwood, but is
also called armoise dAfrique (French), Afrikanischer Wermut (German) (Van Wyk & Wink,
2004), wildeals, wilde-als, als, alsem (Marloth, 1917; Van Wyk & Gericke, 2000; Van Wyk,
93
Chapter 4
Ethnobotany
2008a; Van Wyk et al., 2009), bitterals (Smith, 1966) (Afrikaans); lanyana (Liu et al., 2009) or
lengana in Sotho (Phillips, 1917; Watt & Brandwijk, 1927; Jacot Guillarmod, 1971; Schmitz,
1982; Hutchings & Van Staden, 1994; Maliehe, 1997; Moteetee & Van Wyk, 2011) and
Tswana (Van Wyk et al., 2009), and umhlonyane (siSwati, Xhosa and Zulu) (Amusan et al.,
2007; Van Wyk et al., 2009). Its distribution covers the eastern parts of Africa, from northern
Ethiopia, southwards to tropical East Africa (Kenya, Zimbabwe, Tanzania, Angola), to South
Africa (see Fig. 4.6b) (Kokwaro, 1993; Van Wyk & Wink, 2004), where it typically flourishes at
altitudes of between 2 0002 400 m. This multi-stemmed perennial herb appears to be a
thick, bushy, untidy mass, and can grow as tall as 2 m. The basal stems are thick, firm and
woody, in contrast to the upper parts. The finely divided feather-like leaves are dark green
above and covered with small white hairs below. Small inconspicuous, cream-coloured flower
heads are borne along the branch ends towards the end of summer. During cold periods, the
branches die back, but rapidly regenerate from the base in spring (Hilliard, 1977; Botha &
Herman, 1980).
(a)
(b)
Fig. 4.6: (a) Artemisia afra leaves and flower heads (Van Wyk & Wink, 2004) (b) A. afra
distribution in South Africa (http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
A. afra is one of the oldest and best-known indigenous medicines in southern Africa, as KhoiSan and Cape Dutch medicine, but also used in the Nguni, Sotho and other Bantu cultures
(Kokwaro, 1993; Van Wyk, 2008a) (see Appendix 2D). Its exceptionally wide variety of
recorded uses, resembling those of its European counterpart, A. absinthium (Marloth, 1917),
contributes to its fame as one of the most important commercial medicinal herbal products in
South Africa (Van Wyk, 2008b). Leaf infusions are known to be extremely bitter and are often
sweetened with sugar or honey, while vapours from boiling leaves are known to release the
volatile oils responsible for decongestion in the case of colds (Watt & Breyer-Brandwijk,
1962). Of the 47 references listed (Appendix 2D), seven classified A. afra as a tonic, with
94
Chapter 4
Ethnobotany
Pappe (1847) being the first. Indications as strengthening agent appeared seven times, and
listings of properties typical of an amarum, 24 times. Although the ailments treated by buchu
certainly compare with those treated by A. afra, as proposed by Rood (1994), it seems that
buchu has a more pronounced wound-healing effect than A. afra. The use of A. afra as blood
purifier, and related ailments, appear eight times, with a similar emphasis on its
antiinflammatory, anti-diabetic and anti-malarial properties. From the 1990s, its use for
cardiopathy (Von Koenen, 2001), stress and nerves (Cilli, 1992 Appendix 2), with three
consequent mentionings, became evident, but it is warned that overdose may be poisonous
(Rood, 1994), and that prolonged use might affect the heart (Roberts, 1992).
A. afra is used extensively to treat infections, i.e. respiratory ailments and fever, which
suggests an ability to strengthen resistance. This, together with its apparent effectiveness in
reducing stress, and its mentioned amara aromatica properties, may afford A. afra tonic
status as per the definition in Chapter 2.

Balanites Delile species (Balanitaceae) are spiny trees found in tropical climates. Balanites
maughamii [torch fruit tree (Smith, 1966) or torchwood, fakkelhout or groendoring (Van Wyk
et al., 2009) in Afrikaans; mandur or nulo (Portugese); mkonga or mguguni in Swahili; ipamu,
ugobendlovu, umgobendlovu (Van Wyk et al., 2009), uMandulo, iPhambo (Raymond, 2005),
uGobandlovu, umNulu, iPhamba, iPhamu (Hutchings et al., 1996; Raymond, 2005) or
umDlulamithi (Corrigan et al., 2011) in Zulu; umnulu (Van Wyk et al., 2009) or liphambo
(Dlamini, 1981) in Swazi; nulu or nulo in Changana (Ribeiro et al., 2010)] are found in open
savanna, and often along rivers (Van Wyk et al., 2009; Ribeiro, et al., 2010), but can also be
found in arid and sometimes saline conditions (Coates Palgrave, 1977; Hutchings et al.,
1996). In South Africa, the species can only be found in the extreme eastern parts of
KwaZulu-Natal [see Fig. 4.6(b)], but have a much wider distribution in tropical Africa (Coates
Palgrave, 1977; Sands, 2001). Torchwood trees can grow to a height of up to 25 m and have
a smooth, grey bark. They have distinctive unevenly forked, green thorns, as well as paired,
ovate leaflets, which have a velvety texture when young, but have a smooth upper surface
when mature. Inconspicuous greenish-yellow flowers in spring make way for edible, yellowish
date-like fruit in summer. The fruit have a spongy, fibrous, dark and oily flesh on the inside,
which has a pleasant sweet scent and taste, but becomes bitter in time. The kernel contains
clear, yellow edible oil (about 60%) that is slightly bitter and odourless, but flammable (from
95
Chapter 4
Ethnobotany
there the name torchwood) (Sprague, 1913; Palmer & Pitman, 1961; Coates Palgrave, 1977;
Van Wyk, et al., 1995).
While B. maughamii trees yield large straight logs of valuable hard timber, the stem and root
bark is mainly used medicinally (see Appendix 2E). Of the 24 ethnobotanical references
used, the majority referred to the use of the fruit as arrow or snail poison. Medicinal uses
included mostly the stem bark used as emetic (10), for malaria (two), cough (one) and
nervous complaints (one), including six reports of its application as a general tonic and
panacea, in strengthening and exhilirating baths or as subcutaneous implantations to
strengthen the body. Many of the uses of B. maughamii correspond to those of B. aegyptiaca,
possibly due to similar active principles in both species (Van Wyk et al., 2009) (see
Chapter 5).
(b)
(a)
(c)
(d)
(e)
Fig. 4.7: (a) Balanites maughamii*; (b) Distribution in South Africa#; (c) Leaves and thorny
branches*; (d) Fruit*; (e) B. maughamii trunk from which bark has been harvested*
(* = Van Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
96
Chapter 4
Ethnobotany
The common names of B. aegyptiaca [i.e. desert date, soapberry tree, Jerico balsam, simplethorned torchwood or corona di Jesus (Spanish, meaning Christs crown)] point to the bittersweet fleshy fruit, high levels of saponins in fruit extracts, oily extracts from the kernels and
its conspicuous, stout, yellow or green thorns of approximately 8 cm in length, respectively
(Sands, 2001). The ripe fruit is eaten to treat liver and spleen ailments, but is also used to
control the snail hosts of schistosomiasis (bilharzia) in Sudan (Van Wyk & Gericke, 2000).
The seed kernels and bark are known to have anthelmintic and emetic activity (Sands, 2001).
The bark is also reported to have antiinflammatory, sedative, antioxidant, antipyretic,
astringent and tonic properties, and is used to treat mental diseases, epilepsy, jaundice,
yellow fever and syphilis (Anis et al., 2000; Speroni et al., 2005; Yadav & Panghal, 2010). In
India, B. aegyptiaca bark and fruit are used to treat dysentery, mouth ulcers, skin diseases,
whooping cough and sleeping sickness, while the leaves are used to clean malignant
wounds, and also being effective for the treatment of anthrax, bronchitis and dyspepsia (Anis
et al., 2000; Yadav & Panghal, 2010). The roots are known to have purgative and
anthelmintic properties and are used to treat abdominal pains. Chest complaints are treated
by eating maize meal porridge mixed with molten gum from the wood (Kokwaro, 1993).
Not much is known about the phytochemistry of B. maughamii (see Chapter 5), but its use as
a strengthening agent, general tonic and panacea certainly warrants further investigation.

The genus Dicoma Cass. (Asteraceae) contains about 30 species of herbs, shrubs and small
trees of which most are from tropical and southern Africa, as well as Madagascar (Santiago &
Netnou, 2005). The four most important medicinal Dicoma species in southern Africa
(Hutchings et al., 1996; Van Wyk et al., 2009; see Fig 4.7) are D. capensis Less, which grows
in dry, sandy soils, mostly along seasonal streams (Pope, 1992), D. schinzii O.Hoffm, which
is not endemic to South Africa, and prefers dry, sandy soils, D. anomala Sond, with a wide
sub-Saharan distribution (Kokwaro, 1993; Hutchings et al., 1996) and D. zeyheri Sond, which
is indigenous to the Natal coast. Three varieties (subspecies) have been identified for
D. anomala including var. , sonderi Harv., var. , cirsioides Harv. and var. , microcephala
Harv. (syn. D. gerrardii) (Harvey, 1865). However, no distinction was made between these
subspecies for the ethnobotanical, phytochemical and pharmacological reviews featuring
D. anomala in this study.
97
Chapter 4
Ethnobotany
D. capensis [koorsbossie or wilde karmedik (Van Wyk & Gericke, 2000; Van Wyk et al.,
2009), karmedik, bietou, melktou (Smith, 1966), hen-en-kuikens, dermbos, teringbos (De
Beer & Van Wyk, 2011), vrpis or vyfpondbos (Van Wyk et al., 2008) in Afrikaans is a Capeendemic herbal panacea, regarded as a particularly important Khoi-San and Cape Dutch
medicine used in the Karoo (Van Wyk, 2008a). It is a small perennial plant with creeping
branches spreading from a woody rootstock (Pope, 1992), which produces milky latex (Smith,
1966). The grayish-green leaves are densely covered with short, white hairs, and are variable
in shape, while the flowers are pale mauve and inconspicuous [Fig. 4.8 (c)]. On the surface,
the plant is superficially similar to its medicinally well known European namesake, Centaurea
benedicta (syn. Cnicus benedictus L.), commony known as holy thistle and also karmedik
(Van Wyk & Wink, 2004; Van Wyk et al., 2009). It should be noted that several sources of
ethnobotanical data relating to medicinal plants refer to the use of karmedik only, without an
indication of whether D. capensis or Centaurea benedicta is used. In such cases, the uses of
karmedik are listed (e.g. Dykman, 1908; Oupa & Ouma se boererate, 1962 Appendix 2F).
D. anomala [swartstorm(bos) (Smith, 1966; Van Wyk et al., 2009), maagwortel,
maagbitterwortel, kalwerwortel (Van Wyk & Gericke, 2000), gryshout, kalwerbossie or
wurmbossie
(Smith,
1966),
korsbossie
(Roberts,
1990),
aambeibos,
koorsbossie,
maagbossie and vyfaartjies (Van der Merwe, 2008) in Afrikaans; fever bush or stomach bush
in English (Van der Merwe, 2008); hloenya (Phillips, 1917; Jacot Guillarmod, 1971; Schmitz,
1982; Hutchings & Van Staden, 1994; Maliehe, 1997; Moteetee & Van Wyk, 2011), kloenya,
nyingwane (Mahlaba, 1999) or mohlatsetse (Van Wyk & Gericke, 2000) in Sotho; hlwejane in
Southern Sotho (Robert, 1990); thlongati or thlonya in Setswana (Roberts, 1990);
inyongwane or inyongwana in Swazi and Xhosa (Van der Merwe, 2008); chiparurangomo in
Manyika (Roberts, 1990); chifumuro in Shona (Van der Merwe, 2008); umuna (Van Wyk et
al., 2009) or isihlabamakhondlwane in Zulu (Van Wyk & Gericke, 2000)] has simple, narrow,
linear leaves, with bright green upper leaf surfaces and larger flower heads compared to D.
capensis and D. schinzii (see Fig. 4.8(a); Hutchings et al., 1996; Van Wyk et al., 2009). The
latter is commonly known as vaalplatblaar in Afrikaans (Fig. 4.8 (e); Smith, 1966). D. zeyheri
has spine-tipped leaves, covered densely with hairs on the lower surface, long, tubular,
purple to yellow-brown florets and a thickened rootstock [Fig. 4.8 (g)]. The leaves, twigs or
roots of all four Dicoma species have medicinal uses, as summarised in Appendix 2F.
98
Chapter 4
Ethnobotany
(b)
(a)
(c)
(e)
(g)
(d)
(f)
(h)
Fig. 4.8: (a) Dicoma anomala*; (c) D. capensis*; (e) D. schinzii; (g) D. zeyheri;
Geographical distribution of (b) D. anomala, (d) D. capensis, (f) D. schinzii and
(h) D. zeyheri in southern Africa# [* = Van Wyk et al., 2009; # =
http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1; = Photo by Mercia Komen http://operationwildflower.org.za/index.php?option=com_content&task=view&id=15
8&limitstart=1, retieved 26 September 2011); = http://sophy.u3mrs.fr/Afriqsud/HI109.HTM, retrieved 26 Sept 2011]
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In many instances, D. anomala and D. capensis are used for the same indications (Van Wyk
et al., 1997, 2009). However, due to the wider distribution of D. anomala across several
countries in Africa, more ethnobotanical data pertaining to it is available (21 references for
D. anomala, 14 for D. capensis). Smith (1895) was the first to report on D. anomala, and
Dykman (1908) on D. capensis, indicating uses for various medicinal purposes, and that its
infusions were very bitter in each case. Consequently, all the references included treatment
of various stomach ailments, with colic and diarrhea/dysentery being the most prevalent.
D. anomala was labled as stomach tonic by Smith (1966), and D. capensis as a bitter tonic by
Van Wyk (2008a), suggesting an amarum effect upon ingestion of both species. Both species
were indicated for treatment of diarrhea as well as constipation by the same references,
indicating a possible balancing effect in the digestive system. Furthermore, Van Wyk &
Gericke (2000) described D. anomala as a cardiac tonic to increase circulation, and karmedik
was indicated for the heart and circulation (Oupa en Ouma se boererate, 1962 Appendix 2).
Both D. anomala and D.capensis were also mentioned as treatment for high blood pressure.
While both D. anomala and D. capensis seem to be important treatments for fever,
respiratory ailments [mostly cough, colds and influenza, few of pneumonia, and one early
(1908) mention of tuberculosis and diphtheria for D. capensis only], venereal diseases and
haemorrhoids, it was also indicated as panacea (twice), to treat unspecified diseases, all
ailments and cancer, as well as insanity, hypertension, epilepsy, dizziness or migraine.
Furthermore, D. capensis is classified as a liver tonic (De Beer & Van Wyk, 2011). Therefore,
both D. anomala and D. capensis show adaptogenic tonic potential. While D. schinzii and
D. zeyheri have also been indicated for stomach ailments, D. schinzii is particularly known for
treating fever, and D. zeyheri for its strengthening ability indicated for the treatment of weak
limbs after a long illness, as blood strengthener after giving birth, and for anemic patients.

Harpagophytum procumbens (Pedaliaceae) is a weedy, perennial herb (Henderson &
Anderson, 1966) with creeping stems spreading as wide as 1.5 m from a thick, carrot-like
primary root with several secondary roots extending from it (Merxmller & Schreiber, 1968;
Ihlenfeldt & Hartmann, 1970; Dyer, 1975; Ihlenfeldt, 1988). The stems die back during cold
months or drought periods, but emerge after the first rains (Stewart & Cole, 2005). Its leaves
are grayish-green and irregularly lobed. Tubular yellow and violet, or uniformly dark violet
flowers make way for its characteristic thorny fruits (Merxmller & Schreiber, 1968; Ihlenfeldt
& Hartmann, 1970; Dyer, 1975; Ihlenfeldt, 1988), from which the names originated, i.e. in
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Greek, harpago means a hook (Mills & Bone, 2000). Traditionally, this species has many
common names in southern African, i.e. devils claw, grapple plant, woolspider (Burchell,
1822-1824; Marloth, 1917), grapple thorn (Smith, 1966), duiwelsklou (Van Wyk & Gericke,
2000; Van Wyk et al., 2009), beesdubbeltjie, sanddoring, sandbur, sanddubbeltjie,
skerpioendubbeltjie, bobbejaandoring, bobbejaandubbeltjie, bobbejaanklou, leeudoring,
duiwelsdoring, dubbeltjiedoring, duiwelshaak(doring), duiwelsnaels, haakdoring, katnaels,
noorsdoring, sandnoors, veldspinnekop (Smith, 1966), rankdoring, toutjie, kloudoring
(Marloth, 1917) in Afrikaans, sengaparile (Van Wyk & Gericke, 2000; Van Wyk et al., 2009) or
kanako (Iwu, 1993) in Tswana, and kamangu (Stewart & Cole, 2005). Most names refer to
the shape of the fruit (Smith, 1966) or the method of dispersal by clinging to animals (Van
Wyk et al., 2009) [see Fig. 4.9 (a) and (b)]. The medicinal value lies in the secondary roots,
which are mostly sliced and dried, to be used when required (Van Wyk et al., 2009). Another
species, H. zeyheri Decne, is also used medicinally, but has a variable chemical composition,
making its suitability doubtful (Czygan & Krger, 1977; Van Wyk, 2008b) (see Appendix 2G
for the medicinal ethnobotanical data on both species). These two species are distinguished,
based on the fruit morphology: the thorny arms on the H. procumbens fruit are longer than
the width of the fruit, while those of H. zeyheri are the same length or shorter than the width
of the fruit (Van Wyk & Wink, 2004). Several subspecies of H. procumbens and H. zeyheri
have been identified (Ihlenfeldt & Hartman, 1970), but the users of the plant do not seem to
differentiate between the subspecies (Van Wyk, 2008b).
(a)
(b)
(c)
Fig. 4.9: (a) Harpagophytum procumbens leaves and flowers*; (b) H. procumbens fruit*;
(c) Distribution of H. procumbens in southern Africa# [* = Van Wyk & Wink, 2004;
#
= http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
H. procumbens is an important traditional medicine and tonic of the indigenous people in and
around the Kalahari Desert and Namibia [Van Wyk et al., 2009; see Fig. 4.9 (c)]. While some
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archeological records of H. procumbens were found in the form of rock engravings (Wilman,
1968), few historical records of it exist (Van Wyk, 2008b). It was first collected and described
by European scientists in 1820, but its potential therapeutic uses were reported for the first
time when a German farmer in Namibia, GH Menhert, recorded its uses by the indigenous
population (1907) and sent plant samples to Germany to be studied by B. Zorn at the
University of Jena in the 1950s (Wegener, 2000). During that time, it became an important
remedy in Europe, where it was known as griffe du diable (French), Afrikanische teufelskralle
(German), artiglio del diavolo or arpagofito (Italian) (Van Wyk & Wink, 2004). It is thus not
regarded as part of the Cape healing culture (Van Wyk, 2008a), as is evident from
Appendix 2G, due to the lack of recorded uses before the second half of the 1900s.
From the 31 (mostly European and American) references for H. procumbens in Appendix 2G,
it is evident that this species has already been the topic of much research, due to its
effectiveness in the treatment of musculoskeletal illnesses (antiinflammatory, antiarthritic,
antirheumatic and analgesic). It is also claimed to relieve menstrual and labour related pain
(12 references) and backache (six references). Many of these health claims may however be
motivated by commercial gain without any traditional anecdotes or scientific evidence.
Furthermore, it is renowned for its bitter tonic (13 references) and digestive stimulant
properties (stomachic, poor appetite even anorexia, dyspepsia, peptic ulcers, heartburn,
constipation and diarrhea). H. procumbens is indicated for the treatment of ailments
pertaining to the pancreas, liver, gall bladder, kidneys and urinary tract, thus stimulating the
whole lymphatic system (12 references). Noteworthy, is mention of its use for the treatment of
various ailments, as a general health tonic (eight times), with anti-ageing properties (twice),
and its use for hypertension or stress (five times). It is apparently sedative (four references)
with consequent application in the treatment of epilepsy, childhood convulsions and
headache (five references). Furthermore, mention is made of its use for lowering blood
pressure, cholesterol and uric acid, while also being an antibiotic used for treating infectious
diseases such as tuberculosis, as well as skin diseases such as syphilis, gonorrhea and skin
cancer. Amidst the vast array of indications, the possible tonic, amarum, and adaptogenic
properties contribute to the global commercial value of devils claw (Van Wyk, 2008b). Its
bitter tonic properties, for example, resulted in its inclusion in Schweden-Bitter
(PharmaNatura), the well known digestive tonic mixture (Van Wyk et al., 2009).
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Hypoxis species are tuberous perennials from the Hypoxidaceae family with long, strapshaped leaves and yellow, star-shaped flowers. H. hemerocallidea (previously H. rooperi
S.Moore) has broad, slightly hairy leaves, arranged one above the other to form three distinct
groups, spreading outward from the centre of the tuberous rootstock (or corm). The flowers
are borne on long, slender stalks (Singh, 1999). The corm is dark brown to black on the
outside with yellow flesh on the inside [see Fig. 4.9 (a) and (b)]. This plant is widely
distributed in the grasslands of South Africa [see Fig. 4.9 (c)], extending further north to
Lesotho, Zimbabwe, Mozambique and parts of East Africa (Fabian & Germishuizen, 1997;
Singh, 1999).
(a)
(b)
(c)
Fig. 4.9: (a) Hypoxis hemerocallidea leaves and flowers*; (b) H. hemerocallidea corms used
for medicinal purposes*; (c) H.hemerocallidea distribution in South Africa# (* = Van
Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
African potato [(common name, not used traditionally, referring to the tuber that is used
medicinally; also known as star flower or sterblom in Afrikaans (Van Wyk et al., 2009)] has
become a commercially important phytomedicine (see Appendix 2I for ethnobotanical
medicinal uses), also known internationally (Van Wyk, 2008b). The earliest reference (Watt &
Breyer-Brandwijk, 1962) of its traditional use is fairly recent (and 54% of reported
ethnobotanical data in Appendix 2H dates from the year 2000), the historical use of this plant
by the Zulu, Sotho and Xhosa people inhabiting areas where it grows naturally, has not been
recorded. Traditionally, H. hemerocallidea is known in Sotho as lotsane or moli-kharatsa, in
siSwati as lilaba-tseka or zifozonke (the latter meaning a plant that can be used for many
diseases) (Amusan et al., 2007), and Zulu cultures as inkomfe (Crouch et al., 2006; Van
Wyk et al., 2009), ilabatheka (Crouch et al., 2006; Drewes et al., 2008), isidumo or inongwe
(Grierson & Afolayan, 1999). From Appendix 2H (24 references included), it indeed becomes
evident that hypoxis is used for a large variety of ailments (mentioned thrice), as a general
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tonic (mentioned twice) and as a strengthening tonic (thrice). Its apparent immune system
stimulating ability is mentioned five times, with nine consequent indications of it use for
HIV/Aids and related ailments, as well as tuberculosis (eight references). While no evidence
exist for it being a cure for these diseases, cancer (mostly internal and prostate related,
17 accounts) or diabetes (four reports), it does seem to improve the quality of life for such
sufferers (with an added three accounts of its use as convalescent), and prolongs life.
According to 80% of the anecdotes, hypoxis is used to treat insanity, dizziness, anxiety,
depression, hypertension (associated with headaches), several central nervous system
disorders, heart diseases, palpitations, as well as psoriasis (a stress-related skin disease,
seven anecdotes). Furthermore, it is claimed to have a stimulating muscular and hormonal
effect, together with antiarthritic, antiinflammatory and analgesic properties, useful for
treatment of arthritis, rheumatoid arthritis and chronic fatigue syndrome (i.e. yuppie flu) (11
accounts in total). Hypoxis is also indicated for bladder (urinary tract infections, 13 accounts),
kidney and liver problems, (gastric and duodenal) ulcers, diarrhea and as purgative (mostly
for expelling internal parasites, possibly for haemorrhoids). The ethnobotanical evidence
provided in this study does not indicate that H. hemerocallidea is an immunostimulant or
possible adaptogenic tonic. These claims date from recent times and have their origin in
recent commercial developments and marketing materials.

While Muraltia heisteria (Polygalaceae) shares many characters with the other Muraltia
species (ca. 115), the highly variable leaf characters that vary according to its habitat, present
a taxonomical challenge within the genus (Levyns, 1954), i.e. Muraltia heisteria was first
known as Polygala heisteria. Being a fynbos plant [see Fig. 4.10(c); (Adamson & Salter,
1950; Maytham Kidd, 1983; Van Wyk & Gericke, 2000)], it grows in nutrient-poor, acidic soils,
strong wind, high temperatures and summer drought, and can be found on flats and lower
rocky slopes (mainly on sandstone), from sea level to over 1 700 m (Goldblatt & Manning,
2000). M. heisteria is commonly known as purple gorse [kastybos or persblommetjie (Van
Wyk & Gericke, 2000) in Afrikaans]. It is a loosely upright, rigid, spiny shrublet that usually
grows about 1 m high. The leaves are 512 mm in length with sharply pointed tips ending in a
spine. Young growth and the undersides of leaves are covered in soft hairs, and the leaf
margins are fringed with hairs. Many small purple, pink or white flowers are scattered among
the leaves and can be present for several months of the year (Fig. 4.10). The fruit is a
capsule with four elongated, slender, horn-like outgrowths, and contains two seeds. The
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seeds have membranous lobes on either side that allow wind dispersal (Levyns, 1954;
Leistner, 2000).
The only ethnobotanical medicinal record that exists for M. heisteria is from Van Wyk and
Gericke (2000): flowering twigs are used as an appetite stimulant in the Nieuwoudtville area
(Willem Steenkamp provided this anecdote). Other medicinal species are M. muraltioides
(Eckl. & Zeyh.) Levyns [pyn-in-die-sy-bossie in Afrikaans, used for relieving neuralgic pains
(Smith, 1966)], M. beiliana Harv. [pijn-in-zijdebos (Marloth, 1917)] and M. lancifolia Harv.
(roots are used as traditional medicines in KwaZulu-Natal) (Smith, 1966; Hutchings et al.,
1996). In-depth ethnobotanical, phytochemical and pharmacological studies may reveal the
tonic properties of M. heisteria.
(a)
(b)
(c)
Fig. 4.10: (a) Muraltia heisteria plant*; (b) M. heisteria leaves and flowers*; (c) M. heisteria
distribution in South Africa# (* = http://www.fernkloof.com/searchflower.mv?search,
retrieved 28 Sept 2011; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
4.2.10
Sutherlandia frutescens
The genus Sutherlandia (Fabaceae) is an intricate species complex with many regional
forms, genotypes and chemotypes, and has resulted in three revisions of the genus (Phillips
& Dyer, 1934; Moshe, 1998; Goldbladt & Manning, 2000), explained in summary by Van Wyk
and Albrecht (2008). For the purpose of this study, Sutherlandia frutescens (L.) R.Br. [syn.
Lessertia frutescens (L.) Goldbladt & JC Manning] will refer to all the various inland species
(including the typical, hairy, dwarf and high altitude forms of S. frutescens subsp. frutescens,
as well as S. frutescens subsp. speciosa and S. frutescens subsp. microphylla) (Moshe,
1998). For traditional users, cancer bush [or kankerbos in Afrikaans, Marloth (1917)] is
recognised as an erect, perennial shrub that can grow between 0.22.5 m tall [users
distinguish between the species as the short one (S. frutescens subsp. frutescens), and the
long one (Van Wyk et al., 2008), grootgansies(bos) or grootkankerbos (Smith, 1966)
(S. frutescens subsp. microphylla), or alternatively, no distinction is made between the two
species (Van Wyk & Gericke, 2000)]. The silvery, compound leaves are borne towards the
105
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tips of the stems, and have eight pairs of opposite, oblong leaflets with one terminal leaflet.
Some common names for S. frutescens such as blasenstrauch, krebsbusch (German)
(Smith, 1966; Powrie, 2004), or common Afrikaans names such as gansies, belbos (Van Wyk
& Gericke, 2000), blaasbossie, rooi-ertjie(bos), kleinkankerbossie or kleingansies(bossie)
(specifically referring to S. frutescens subsp. humilis, the hairy form), eendjies, gansies,
hoenderbelletjie, kalkoenbelletjie (Marloth, 1917), gansbossie, gansiebos, gansieskeur(tjie),
jantjiebarend (or Jantjie Barend Marloth, 1917), (rooi)keurtjie, kiepiebos, kipkippers,
kipkippies, klapper(s), klapperbos(sie) (Smith, 1966), kalkoenblom, particularly refer to the
shape and colour of the bright red flowers [Fig. 4.11 (a)] and inflated, bladdery pods that carry
numerous brown, kidney-shaped seeds. Morphological variation with geographical
distribution can be seen in height, presence or absence of hairs on stems and leaves, as well
as the shapes of flowers, pods and seeds. S. frutescens is distributed widely across southern
Africa, the largest part of Lesotho, the extreme southern corner of Botswana and southern
parts of Namibia [see Fig. 4.11 (b)] (Van Wyk, 2008a).
(a)
(b)
Fig. 4.11: (a) Sutherlandia frutescens leaves and flowers*; (b) Distribution of S. frutescens
sensu lato (including all various subspecies) in South Africa# [* = Van Wyk et al.,
2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
S. frutescens is a known cure-all tonic plant in South Africa, and is used in the Nguni and
Sotho traditional healing cultures, as well as those of the Khoi-San and Cape Dutch
(Moteetee & Van Wyk, 2007; Van Wyk, 2008a). Tinctures, infusions and decoctions of the
leaves and young stems are effective treatment for a myriad of indications (see Appendix 2I,
quoted verbatim from Van Wyk & Albrecht (2008), with recent data added). The adaptogenic
tonic uses of Sutherlandia (Van Wyk & Wink, 2004) are reflected in Appendix 2I, as well as
the Sesotho names for S. frutenscens, i.e. musa-pelo, musa-pelo-oa-nka or motlepelo (Van
Wyk et al., 2009), and musa-pelo-o-moholo-oa-noka (the high-altitude form, also known as
S. montana E.Phillips & R.A.Dyer; Moteetee & Van Wyk, 2007). These names all have one
106
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Ethnobotany
meaning in common, i.e. to turn the heart around (Moteetee & Van Wyk, 2007),
emphasising its use for treatment of dropsy of the heart (four records) and stress-related
ailments such as nervousness, anxiety, shock, trauma, severe depression, heart palpitations,
increased blood pressure and fits (21 records altogether). The name phetola in Setswana
(Van Wyk et al., 2009), means it changes, implying that it changes the course of various
illnesses (six reports) to a favourable outcome (Mncwangi & Viljoen, 2007), indicating
Sutherlandia as a general health and strengthening tonic (11 accounts). The Zulu name,
insiswa, means the one who dispels darkness (see Van Wyk & Albrecht, 2008), similar to
the definition of an alterative, i.e. an agent that alters the course of morbid conditions
(Chapter 2), such as cancer (26 accounts, 83% of all anecdotal records; hence the names
cancerbush or kankerbos in Afrikaans), diabetes (nine anecdotes) and even Aids (Van Wyk &
Wink, 2004). Sutherlandia is called lerumo-lamadi in northern Sotho, which means the spear
of the blood, emphasising its use as blood-purifier (Mncwangi & Viljoen, 2007) (seven
anecdotes), synonymous with bladder, kidney and liver-related ailments (16 reports
altogether). The Afrikaans name bitterblaar (Smith, 1966) refers to its bitter taste, but also its
bitter tonic properties (six accounts), indicated by its use for stomach ailments (33 accounts
altogether) such as lack of appetite, digestive disorders, diarrhea and dysentery, peptic
ulcers, abdominal pain and nausea. Sutherlandia is also claimed to increase resistance to
infections such as tuberculosis, chickenpox, cold and influenza symptoms, bronchitis and
asthma (44 accounts altogether), together with anecdotal antipyretic (15 reports),
antiinflammatory and analgesic (i.e. use for rheumatism, backache, general aches and pains,
25 accounts altogether) properties. Topical treatment of wounds, burns, ulcers, piles and
rash, as well as eye ailments and haemorrhoids are also listed. This vast array of medicinal
uses and the importance of Sutherlandia as a possible adaptogenic tonic undoubtedly
contribute to its popular use (Van Wyk, 2008b).

Vernonia oligocephala (the name has recently changed to Hilliardiella oligocephala (DC.)
H.Rob.) (Asteraceae) is a herbaceous perennial with its upright flowering branches growing
to about 300800 mm tall, from a woody rootstock. The elliptic leaves are borne on short
stalks, pointed sharply, dark green and smooth on top, but densely haired and silvery
underneath (Van Wyk et al., 2009). This characteristic leaf morphology distinguishes
V. oligocephala from its relatives, especially V. natalensis Sch.Bip., also used medicinally
(Van Wyk et al., 2009), which has the same distribution and flowering period, but narrower
leaves with similar textures on both sides (Hilliard, 1977). Violet coloured flower heads are
107
Chapter 4
Ethnobotany
borne in large groups at the branch tips, and the seeds are light with hairy parachutes,
adopted to wind dispersal. V. oligocephala has a widespread distribution in the wooded,
rocky grassland areas of southern Africa [Fig. 4.12 (c)], Zimbabwe and Zambia.
(Germishuizen, 1997; Van Wyk & Malan, 1997; Pooley, 1998; Arnold et al., 2002).
The Afrikaans common names for V. oligocephala include groenamara (Van Wyk & Gericke,
2000; Van Wyk et al., 2009), (groen)amarabossie, bitterbos(sie), bloutee(bitter)bos (Smith,
1966), maagbossie, wildetee or maagtee (Van Wyk & Malan, 1997; Pooley, 1998), and are all
indicative of the bitter areal parts (Van Wyk & Gericke, 2000). Other common names include
mofefa-bana (Schmitz, 1982; Maliehe, 1997; Moteetee & Van Wyk, 2011) or mofolotsane
(Van Wyk & Gericke, 2000; Van Wyk et al., 2009) in southern Sotho, sefafatse in Tswana,
lihlunguhlungu in siSwati (Amusan et al., 2007) and ihlambihloshane (Van Wyk et al., 2009)
or uhlungu-hloshana in Zulu (Van Wyk & Malan, 1997; Pooley, 1998).
(a)
(b)
(c)
Fig. 4.12: (a) Vernonia oligocephala leaves and flower heads*; (b) V. oligocephala
stems and leaves used for medicinal purposes*; (c) V. oligocephala
distribution in southern Africa# (* = Van Wyk et al., 2009; # =
http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
From the ethnobotanical medicinal uses of V. oligocephala (Appendix 2J; 20 references), it
can be deduced that the general health tonic properties (Van Wyk & Gericke, 2000) of
groenamara is almost exclusively (all but five references) due to its bitterness (mentioned
seven times; by means of the amarum effect, see Chapter 2), as the name suggests. The
stomach ailments that are treated, include abdominal pain, colic, ulcerative colitis,
constipation, diarrhea, dysentery (note the apparent balancing effect between purgative and
anti-diarrheal actions), poor appetite, nausea and worms. When considering the frequency of
anecdotes for debilitating diseases such as diabetes (10) and rheumatism (nine), as well as
anecdotes for improvement/treatment of the heart and circulation, hysteria and epilepsy, the
plant may well have strengthening and tonic properties.
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Warburgia salutaris (syn. W. ugandensis Sprague; W. breyeri Pott.) is a medium-sized tree
from the Canellaceae family that grows to a height of about 10 m. Its rough, mottled bark or
root bark is reddish on the inner side and has a strong peppery taste (Coates Palgrave,
1977), is used medicinally, and affords the name pepper-bark tree [peperbasboom in
Afrikaans, ishibaha (Gordon, 1953), mulanga or manaka (Van Wyk & Gericke, 2000) in
Venda, shibaha (Van Wyk & Gericke, 2000) or xibaha (Drewes et al., 2001) in Tsonga,
molaka in Sotho (Drewes et al., 2001), and isibhaha (Van Wyk & Gericke, 2000; Van Wyk et
al., 2009), isibaha (Hutchings et al., 1996), isiBhaha or amaZwecehlabayo (Hutchings et al.,
1996; Raymond, 2005) in Zulu]. The leaves are about 60 mm in length, glossy green above,
paler green underneath, covered with gland dots, and also have a peppery taste (Verdcourt,
1955, 1990; Codd, 1976; Coates Palgrave, 1977). Currently, the leaves are harvested from
cultivated plants for medicinal use, instead of the bark, in an attempt to conserve this locally
endangered species (Zschocke et al., 2000; Drewes et al., 2001). The flowers are small,
greenish-yellow and are borne between the leaves on the stem. Green to black spherical
berries with a leathery skin are produced between October and January [see Fig. 4.13 (b)].
These tropical forest trees prefer evergreen montane forest or sandveld forest along the
coastline, but can also be found in secondary bushland, wooded ravines and in grasslands
[Coates Palgrave (1977); see Fig. 4.13 (c)]. They are found at altitudes between 1 0002 200
m, in regions with an average rainfall of between 4001 750 mm across Africa (Codd, 1976;
Coates Palgrave, 1977; Orwa et al., 2009). There has been much debate about whether
W. ugandensis subsp. ugandensis (found naturally in eastern and central Africa) is indeed
the same as W. salutaris (Hollmann & Van der Schijff, 1996), and the name W. ugandensis is
currently still in use. For this reason, ethnobotanical data was collected and considered
relevant for W. salutaris under both names (Appendix 2K).
Salutaris is a Latin word meaning "healthful", derived from salus, i.e. "good health" (Harper,
2011). This is indeed the case when considering that it is indicated for various diseases
(four times), called a powerful muti (Hollmann & Van der Schijff, 1996) and is classified as
panacea (thrice) and a general tonic (thrice) in Appendix 2K (35 references included). It is a
particulary important remedy for combating infection against malaria (27 anecdotes), as well
as respiratory ailments (28 references) and skin diseases (sores, ulcers; 17 anecdotes) as
antibiotic (four references), while also reducing pain [i.e headache (12 accounts), toothache
109
Chapter 4
Ethnobotany
(14 accounts), general body pain (five accounts)] and fever (16 indications), simultaneously.
The chest ailments W. salutaris is indicated for include coughs, colds, influenza, bronchitis,
asthma and sinusitis, with inevitable later application for tuberculosis (the first was Wube et
al., 2005; followed by three other references) and HIV/Aids (Lamorde et al., 2010). Other
infections for which it is often used include oral thrush, mouth sores, throat infections,
venereal diseases, penile irritations and sores, as well as urethritis. Its claimed effectiveness
for treating internal ulcers (12 references) is also noteworthy, together with its treatment of
abdominal pains (15 anecdotes), constipation (13 anecdotes) and diarrhea (mentioned
thrice). Rheumatism (11 anecdotes), and related ailments such as muscle pains, backache,
lumbago and weak joints, as well as inflammation are also treated with W. salutaris. The first
of two anecdotes for cancer was recorded by Van Wyk and Gericke (2000). The
ethnobotanical data thus provides indications that W. salutaris may help to improve
resistance against infection, and is able to support the healing process by reducing pain and
inflammation.
(a)
(b)
(c)
(d)
Fig. 4.13: (a) Warburgia salutaris*; (b) W. salutaris leaves and fruit*; (c) W. salutaris bark
used for medicinal purposes*; (d) W. salutaris distribution in South Africa# (* =
Van Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
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Ethnobotany

Withania somnifera (Solanaceae) is commonly known as Indian ginseng or winter cherry,
geneesblaarbossie (Van Wyk et al., 2009), geneesblare (Marloth, 1917), koorshout,
vernietbossie, vuilsiektebossie or bitterappelliefie (Smith, 1966) in Afrikaans, mofera-ngope
(Phillips, 1917; Watt & Breyer-Brandwijk, 1962; Jacot Guillarmod, 1971; Moteetee & Van
Wyk, 2011) or bofepha (Van Wyk & Gericke, 2000) in Sotho, ubuvuma in Xhosa (Van Wyk &
Gericke, 2000), ubuVimbha in Zulu (Van Wyk et al., 2009), ashwagandha in Hindi or
Schlafbeere in German (Van Wyk & Wink, 2004). This erect, perennial shrublet stems from a
large woody rootstock. The stems and oblong, pale green leaves are densely covered with
short hairs giving it a velvety texture. Small flowers are produced in short clusters, and are
yellowish to white in colour. The fruits are small, round, orange-red berries of about 8 mm in
diameter, enclosed in brown papery, bladdery structures (i.e. remains of the sepals)
(Henderson & Anderson, 1966). W. somnifera has a wide distribution in Africa, southern
Europe and Asia, and while it is considered to be indigenous to South Africa [Fig. 4.14 (c)], it
is considered a weed in some places (Henderson & Anderson, 1966; Dold & Cocks, 2000).
(a)
(b)
(c)
Fig. 4.14: (a) Withania somnifera*; (b) W. somnifera bright red fruit with characteristic
papery calyx covering it*; (c) W. somnifera distribution in southern Africa# [* = Van
Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1]
Somnifera refers to the narcotic properties (mentioned thrice in Appendix 2L) of
W. somnifera, but ashwagandha is the Sanskrit name used in Ayurveda (asva means horse;
gandha is smell, i.e. smell of a horse, because the root of the infected plant has the smell of
horse urine), generally meaning that which gives the energy and sexual vitality of a horse
(Scartezzini & Speroni, 2000). In Appendix 2L, it is listed as an aphrodisiac, along with a
treatment for impotency and sterility (17 times), and for geriatric ailments (four times).
According to Bhattacharya and Muruganandam (2003), ashwagandha is considered a
rasayana drug [a group of plant-derived drugs reputed for promoting health and longevity by
enhancing defense against disease, slowing down the ageing process, revitalising the body
in debilitating conditions, increasing resistance against adverse environmental factors, and
111
Chapter 4
Ethnobotany
creating a sense of mental wellbeing (Weiner & Weiner, 1994), see Chapter 2], which has
been used in Ayurvedic medicine for 2 500 years or more. In South Africa, geneesblare also
has a very old history of medicinal use in the Khoi-San and Cape Dutch healing systems (first
mentioned by Smith in 1895), and has since been adopted as medicine in the Nguni, Sotho
and Zulu cultures (Hutchings et al., 1996; Van Wyk, 2008a). From the 45 references included
in Appendix 2L, it became evident that the use of leaves is mostly topical for a wide variety of
skin conditions, and decoctions, infusions or tinctures of the roots (sometimes together with
leaves, or the whole plant) are mostly taken orally for a myriad of indications. The anecdotal
evidence alone, including its use for various ailments or general ill health (mentioned six
times), for dyspepsia, general debility or fatigue (15 anecdotes altogether), with
strengthening, preventative, rejuvenating and restorative properties (seven anecdotes),
qualifies its use as a tonic (13 references). It is also described as an adaptogenic tonic (five
times), based on its apparent ability to increase resistance to stress, i.e. it is indicated for
anxiety, depression, hysteria, insanity, epilepsy, sleeping disorders, nervous exhaustion,
paralysis, palpitations and dropsy (22 anecdotes altogether), with mood stabilising, sedative
and hypnotic properties (17 anecdotes altogether). Furthermore, it is used as a brain tonic,
and is particularly helpful in cases of learning problems and memory loss (five anecdotes).
This ability to increase resistance is also evident in its use to treat infection, be it respiratory
[colds, influenza, bronchitis, coughs (26 anecdotes altogether), tuberculosis (mentioned five
times), asthma (19 anecdotes)], internal [upset stomach (seven anecdotes), abdominal pain
(eight anecdotes), diarrhea and dysentery (seven anecdotes), typhoid fever (mentioned four
times), anthrax (seven anecdotes), parasites (10 anecdotes), genital sexually transmitted
diseases (STDs) such as syphilis and gonorrhea (32 anecdotes), HIV (Reddy et al., 2009)] or
topical [skin infections such as erysipelas (five anecdotes), wounds, sores, ulcers, boils, rash,
smallpox, worms, as well as conjunctivitis, otitis, gingivitis (65% of listed references)],
accompanied with fever (15 anecdotes). The rejuvenating, restorative effect of W. somnifera
is evident in its use for musculoskeletal conditions [such as arthritis (four anecdotes),
rheumatism (24 anecdotes), together with inflammation (14 anecdotes) and pain
(12 anecdotes)], but also with liver (eight anecdotes), kidney and bladder (nine anecdotes)
ailments, blood-related conditions (such as high blood pressure, low blood circulation,
anemia and blood purification, six anecdotes), as well as an indication for emaciation (severe
loss of body mass, mentioned thrice) and cancer (four anecdotes). The international
recognition of this species as medicinally important in many healing cultures is consequently
not surprising (Van Wyk & Wink, 2004).
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Chapter 4
Ethnobotany

Ziziphus mucronata subsp. mucronata [buffalo-thorn, mokgalo in Sotho and Tswana (Van
Wyk & Gericke, 2000), blinkblaar-wag-n-bietjie(doringboom), blinkblaar(boom), buffelsdoring
(Marloth, 1917), haakdoring, haak-en-steek-wag-n-bietjie, skynblaar-wag-n-bietjie, wag-nbietjie(-doring) (Smith, 1966) in Afrikaans, umPhafa in Xhosa or Zulu (Bryant, 1966;
Hutchings et al., 1996; Raymond, 2005) or umLahlankosi (Raymond, 2005; Van Wyk et al.,
2009), umHlahlankosi (Hutchings et al., 1996; Raymond, 2005), imBhufa or umHllabantu
(Raymond, 2005) in Zulu, literally translated as come-and-Ill-kiss-you (Smith, 1966),
mutshetshete in Venda, and mokgalo in Setswana (Luseba et al., 2007)] is a small to
medium-sized tree which mostly grows to a height of about 5 m (Palmer & Pitman, 1961;
Coates Palgrave, 1977; Van Wyk, 1995). It is distributed all over South Africa [Fig. 4.15 (c)],
due to its adaptability to a wide variety of habitats, such as open woodland, alluvial soils
along rivers, frequently on termite mounds, apparently indicative of underground water
(Palmer & Pitman, 1961; Coates Palgrave, 1977). The young, reddish-brown stems have
distinctive pairs of spines (one hooked sharply downwards, the other curved slightly
upwards), that may be absent from mature trees. The three-veined, ovate leaves are shiny,
green and hairless on top and paler green underneath. Small, yellowish, inconspicuous
flowers make way for small, shiny reddish-brown fruit (Coates Palgrave, 1977), which are
edible (Smith, 1966).
The roots, bark and leaves are all used in traditional medicine, in combination or separately
(Van Wyk et al., 2009) for a wide variety of indications (Appendix 2M, 38 references
included), affording Z. mucronata the title of panacea (two anecdotes) and tonics (Neuwinger,
2000). It is used for a wide variety of skin disorders, including boils, carbuncles, abscesses,
ulcers, wounds and (septic) swelling (33 anecdotes altogether), and also includes scrofula (a
tuberculosis-related infliction exhibited by means of glandular swellings in the neck, 23
anecdotes). Noteworthy is its claimed effectiveness in the treatment of respiratory infections
[chest ailments (15 anecdotes), coughs (16 anecdotes), asthma and pheumonia (three
anecdotes altogether)], digestive tract infections causing diarrhea and dysentery (12
anecdotes each) in particular, as well as infectious diseases such as scarlet fever, measles,
gonorrhea, bilharzia (schistosomiasis), malaria and smallpox (23 anecdotes altogether).
Z. mucronata also seems to have sedative and possible antiinflammatory properties as it is
indicated for general body pain (10 anecdotes), lumbago, back pain, rheumatism, muscular
pain, headache, toothache, abdominal pain and earache (22 anecdotes altogether).
Furthermore, it is an important treatment for female problems [i.e. sterility/infertility,
113
Chapter 4
Ethnobotany
dysmenorrhea, hypermenorrhea, threatened abortion (18 anecdotes altogether) (eventhough

there is anecdotal evidence of its abortifacient ability)], and urinary inconvenience (i.e. urinary
incontinence, hematuria, dysuria, oliguria, urethral discharge) with diuretic and blood
purification properties. From Appendix 2M, one can deduce that Z. mucronata may indeed be
a panacea with an ability to increase the bodys resistance to infection, with possible tonifying
effects on the female hormonal system and urinary tract.
(a)
(c)
(b)
(d)
Fig. 4.14: (a) Ziziphus mucronata*; (b) Characteristic thorns of Z. mucronata*;

(c) Z. mucronata bark and leaves*; (d) Z. mucronata distribution in southern Africa#
(* = Van Wyk et al., 2009; # = http://sibis.sanbi.org/faces/Mapping/Map.jsp?1=1)
4.3
Conclusions
Table 4.1 summarises the multiple uses and tonic properties of all the plants that were
researched. It is a useful tool, in combination with the phytochemical results (Chapter 5) and
pharmacological evidence (Chapter 6), to ascertain whether their chemical complexity and
wide range of biological activities do indeed provide a scientific rationale for these anecdotal
tonic properties.
114
Chapter 4
Ethnobotany
Ethnobotany
Table 4.1: Summary of ethnobotanical uses for 14 traditional South African tonic plants (discussed in Paragraphs 4.2.14.2.14 and
given in detail in Appendix 2)
Uses
Type of
1
tonic
Stress2
related
Heart and
circulation
Urinary
tract
Liver
Digestive
tract
Chest
GT
BT, GT
Arctopus species
GT
Artemisia afra
BT
Balanites maughamii
GT, P
Dicoma species
BT, P
Harpagophytum
procumbens
Musculo4
skeletal
Infectuous/
debilitating
5
diseases
BT, GT
GT
Muraltia heisteria
BT
Sutherlania frutescens
A, BT, GT
BT, GT
Warburgia salutaris
GT, P
Withania somnifera
Ziziphus mucronata
GT, P
Plants
Agathosma betulina
Aloe species
1
2
3
4
5
A = adaptogenic tonic; BT = bitter tonic; GT = general health tonic; P = panacea (given as anecdotes).
Including anxiety, insomnia, depression, hysteria, insanity, fatigue, psoriasis,and epilepsy (sedative and hypnotic).
Including colds, influenza, bronchitis, pneumonia, tuberculosis or coughs.
Including arthritis, rheumatism, and lumbago (antiinflammatory and analgesic).
Including bilharzia, cancer, diabetes, HIV/Aids, malaria, smallpox, measles, scarlet fever, typhoid fever, venereal diseases, and
fever.
115
CHAPTER 5
Chemistry
5.1
Introduction
The main hypothesis to be tested in this thesis is that tonic plants contain a wide diversity of
chemical compounds. While not all phytochemicals are necessarily biologically active, they
may play a nutritional role or aid in the biological activity of other compounds, i.e. a tonic
mixture may also contain vitamins, trace elements, appetite stimulants (bitter compounds)
and amino acids (Burger & Wachter, 1998). These phytochemicals may also act together
additively or synergistically to enhance the over-all effect, and a variety of structurally
different classes of compounds may act on several different receptors. Furthermore, plants
classified as tonics also do not necessarily contain the same classes of compounds or
function in the same way, as found by Davydov and Krikorian (2000), who compared Panax
ginseng and Eleutherococcus senticosus (both from the family Araliaceae, both profound
traditional tonic plants). They found that even though the habit and gross morphology of the
two species show similarities, they differ considerably where their chemical composition
and pharmacological actions are concerned. Adaptogenic tonic ability, for instance, could
be brought about by many different substances, from very different origins and most
probably different modes of action, but they may all exhibit the same pharmacological effect
[Brekhman & Dardymov, 1969(b)].
Further variations in these proposedly complex phytochemical mixtures include the
presence and relative quantities of phytochemicals within a given species: although the
biochemical synthesis of phytochemicals within a plant is regulated genetically, external
factors such as climate, geographical location, season and growth conditions vary (Franz et
al., 1985).
The results presented here, per plant in alphabetical order, show the different classes of
compounds (APS, numerous types of terpenes, flavonoids and phenolic acids, amino acids,
small sugars, alkaloids and cyanogenic glycosides) contained in the chosen tonic plants.
The chemical variation among plants of the same species due to possible genetic,
geographical distribution or climatic effects, as well as chemical characters that are similar
among species of a genus (chemotaxonomy) is also given. A further comparison between
116
Chapter 5
Chemistry
phytochemistry of plant parts used for traditional medicine provide a possible scientific
rationale for preference of certain plant parts above others, or strengthen proposals for
alternative plant part use in cases where species are being destroyed due to overharvesting. The amino acid analyses may indicate nutritional value, while bitterness value
determinations show scientific evidence for the use of the chosen plants as digestive tonics.
The process of isolation, characterization and identification of compounds are shown in
cases where little were known about the major compounds (see Arctopus and
Sutherlandia). The phytochemistry of each species may consequently provide a link
between its ethnobotanical medicinal history (Chapter 4) and its pharmacological activities
(Chapter 6).
5.2
The chemistry of South African traditional tonic plants
5.2.1 Agathosma species

Buchu is known mostly for its volatile or essential oils comprised of mainly monoterpenes,
some of which are sulfur-containing. Analyses have shown a total of 69 of these
compounds for A. betulina and 70 compounds for A. crenulata [Viljoen et al., 2006(a)].
Fluck et al. (1961) were the first to identify limonene, menthone, isomenthone, diosphenol,
-diosphenol, l-pulegone and isopulegone as the main monoterpenes from these two
species (see Fig. 5.1). While the leaf dimensions are useful characters to distinguish
between A. betulina and A. crenulata (Blommaert & Bartel, 1976), hybridisation between
the two species is common. Requirements for high quality oils by the international markets
consequently necessitate GC (or GC-MS) fingerprinting and UV (colour) analysis of oils as
quality control measures. In such instances, A. betulina oils are known to contain high
quantities of diosphenol, -diosphenol, and have a golden colour, whereas A. crenulata
produces high amounts of pulegone and has a pale coloured oil (Blommaert & Bartel,
1976). A. betulina has < 10% pulegone in its oil while A. crenulata has > 30% (Fluck et al,
1961; Kaiser et al, 1975; Collins et al., 1996; Posthumus et al., 1996). Two chemotypes of
A. betulina may further be distinguished based on the relative diosphenol and -diospenol
contents of their oils (Collins et al., 1996). Commercial Agathosma essential oils are known
for their unique sulfur-containing compounds which have organoleptic (sensory perception)
properties: A. betulina has a strong sweetish, peppermint-like or citrus odour, and
A. crenulata is pungent with a sharp pulegone note (Kpke et al., 1994; Collins et al., 1996;
Moolla, 2006). Lamparsky and Schudel (1971) were the first to isolate and identify these
menthan-3-one monoterpenes (e.g. 8-mercapto-p-menthan-3-one, see Fig. 5.1), which are
naturally derived from pulegone (Kaiser et al., 1975). These rare bi- and trifunctional
monoterpenes (diosphenols, hydroxylated diosphenols and several hydroxymenthones) are
117
Chapter 5
Chemistry
characteristic of buchu oils, even though some other common monoterpenes such as 1,8cineol, borneol, linalool, eugenol, menthol and -pinene, also occur in these oils (Kaiser et
al., 1975; Viljoen et al., 2006). While A. ovata is not sought after in the flavour and
fragrance industry, it is also used medicinally. The initial identification of mycrene, ocimene,
terpineol, -pinene, -pinene and linalool in its oil by Moran et al. (1975) was confirmed by
Moolla (2006), who also determined sabinene to be the major compound in the oil.
Terpinen-4-ol, -pinene, -pinene, p-cymene and linalool seems common to all three
species (Moolla, 2006). Essential oils are extracted by means of hydrodistillation with a
Clevenger apparatus and analysed by means of GC or GC-MS. As these oils have been
studied exhaustively for buchu, it was not repeated in this study. The Liebermann-Burchard
colour test did however indicate positive results for terpenes.
Apart from the essential oils, buchu also contains mucilage, resins and flavonoids, which
are all likely to play a role in its activity (Wicht & Bisset, 2000). This is evident from
Table 5.1 where it is shown that the non-polar fractions are larger than the polar fractions in
most instances, but that APS (i.e. mucilage) are also present in notable quantities.
Table 5.1: Extract yields for four Agathosma species
Extract yields (% g extract/g dry sample)
Agathosma
species
Sample
number
TLC
track
APS
Polar
A. betulina
AB 51a L
1.5
1.8
3.3
4.5
A. betulina
AB 51b L
4.1
3.0
7.0
14.9
0.082
A. betulina
AB 51c L
1.7
5.1
6.8
9.9
0.085
A. betulina
AB 51d L
1.6
7.2
8.8
12.0
0.072
A. betulina
AB 51e L
1.4
3.1
4.5
9.8
0.107
A. betulina
AB 51f L
0.9
1.8
2.8
7.6
0.133
A. betulina
AB 53 L
3.8
9.4
13.1
9.2
0.067
A. betulina
AB 4310a L
0.038
A. betulina
AB 4310b L
0.039
A. betulina
AB 4310c L
10
0.072
A. crenulata
AC 54 L
11
2.0
7.6
9.5
9.8
0.065
A. crenulata
AC 10 L
12
0.114
A. ovata
AO 55 L
13
2.5
10.7
13.2
11.2
0.117
A. ovata
AO 4309a L
14
0.056
A. ovata
AO 4309b L
15
0.059
A. ovata
AO 4309c L
16
0.036
Total water
extractable
Non-polar
Acetone
leaf
surface
118
Chapter 5
Chemistry
Monoterpenes:
Of more than 300 volatile compounds identified from 17 Agathosma species, these are the
main constituents of A. betulina and A. crenulata essential oils [Viljoen et al., 2006(a)].
OH
OH
O
SH
(+)-Menthone
Diosphenol
-Diosphenol
(-)-Pulegone
(-)-8-Mercapto-pmenthan-3-one
Flavonoids:
Flavanones and flavones identified from A. betulina and A. crenulata (Hegnauer, 1973;
Wollenweber and Graven, 1992; El-Shafae & El-Domiaty, 2001).
5'
6'
8
HO
O
1
7
6
OH
2
3
OCH3
OH
4'
3'
1'
2'
RO
OR'
OH
OR
OH
Quercetin:
R = H; R = H
Quercetin 3-methyl ether:
R = CH3; R = H
Quercetin 3,3-dimethyl ether: R = CH3; R = CH3
Rutin:
R = rhamnoglucosyl; R = H
Diosmetin R = H
Diosmin R= rhamnoglucosyl
OCH3
HO
OH
CH3
OH
OR
O
HO
HO
HO
OH
OR
OH
OH
OH
Hesperidin
Kaempferol:
R=H
Kaempferol 3,4-dimethyl ether: R = CH3
Coumarins:
O
O
O
Fig. 5.1:
Puberulin*
Monoterpenes and phenolic compounds (flavonoids and coumarins) identified

from Agathosma betulina, A. crenulata and A. ovata; * = identified in A. ovata by
Moolla (2006)
119
Chapter 5
Chemistry
The presence of flavonoids was confirmed in all three species studied by means of the
Shinoda colour test, TLC (Fig. 5.2) and HPLC (Table 5.2) analyses. Diosmin, hesperidin,
rutin and quercetin-3,7-diglucoside (Fig. 5.1) are known flavonoids from A. betulina and
A. crenulata (Hegnauer, 1973; El-Shafae & El-Domiaty, 2001). Wollenweber and Graven
(1992) isolated four flavonoid aglycones (kaempferol 3-methyl ether, kaempferol 3,4dimethyl ether, quercetin 3-methyl ether, quercetin 3,3-dimethyl ether Fig. 5.1) from leaf
surface extracts of A. crenulata, but were unable to establish whether these flavonoids were
dissolved in the essential oils produced in the oil glands in the mesophyll or if they were
epicuticular deposits. TLC of the polar, non-polar and acetone leaf surface extracts show
that the investigated Agathosma species contain flavonoids, both glycones and aglycones
(see Fig. 5.2). The flavonoids typically fluoresces blue under UV and are yellow after
visualisation with vanillin/H2SO4 (Wagner & Bladt, 2001). It is obvious from TLC that the
acetone leaf surface extracts and polar MeOH extracts contain different types of flavonoids
in variable concentrations. According to Mabry et al. (1970), rutin, quercetin 3-methyl ether,
diosmin and hesperidin shown deep purple zones under UV (four zones are indicated under
UV, for AB 4310c).
Fig. 5.2:
TLC of Agathosma species: (a) non-polar extracts in MeOH:CHCl3:acetic acid

(60:30:1) after visualisation with vanillin/H2SO4 spray reagents; (b) polar
extracts as compared with the leaf surface extracts under UV-254; (c) polar
extracts as compared with the leaf surface extracts after visualisation with
vanillin/H2SO4 spray reagents. The track numbers correspond with the
respective sample numbers as given in Table 5.2. (Q = quercetin standard)
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Chapter 5
Chemistry
Flavones and flavanones show two major absorption peaks between 240400 nm with UV
analysis, i.e. Band I (300380 nm) represents the B-ring (cinnamoyl system) and Band II
(240280 nm) represents the A-ring (benzoyl system). With flavanones, such as hesperidin,
and it aglycone hesperetin, the saturation between C2 and C3 causes little or no
conjugation between the two ring systems with a consequent low intensity for Band I and an
intense Band II typically between wavelengths 270295 nm. The other flavonoids
previously identified in buchu are flavones and exhibit strong intensities for Band I (normally
between 304350 nm) with a significant shift in cases of hydroxylation at C3 (352385 nm),
i.e. kaempferol, quercetin and rutin. Twelve major peaks were detected by HPLC (Fig. 5.3,
Table 5.2, Appendix 3), where ten show characteristics of flavonoids. Diosmin and
hesperidin were present in the polar extracts but also appear in the leaf surface acetone
extracts. It may be suggested that some of the Agathosma flavonoids are indeed dissolved
in the essential oils produced in the oil glands in the mesophyll. HPLC analyses of the nonpolar (MeOH:DCM, 1:1) leaf extracts will probably confirm the results obtained from the
acetone leaf surface extracts. The UV spectral data (in nm) of the phenolic compounds
compare well with those reported (in MeOH) by Mabry et al. (1970), i.e. hesperidin max =
283, 326sh; diosmin max = 240sh, 252, 267, 291sh, 344; rutin max = 259, 266sh, 299sh,
359; quercetin max = 255, 269sh, 301sh, 370; quercetin 3-methyl ether max = 257, 269sh,
294sh, 358; kaempferol max = 253sh, 266, 294sh, 322sh, 367. Proper identification and
quantification will be possible by LC-MS analyses. The HPLC results presented by
Moolla (2006) do however confirm the HPLC findings reported here, and also contained
large amounts of the first unknown peak. The first two polar compounds correspond with
the TLC zones at Rf 0.30 and Rf 0.36. According to the position and colouration (charring
when heated with an acidic spray reagent), these may be heavily glycosylated, and could
contribute to the mucilage in the polar leaf extracts. Development of the polar extracts in
ACN:CS2:H2O:formic acid (85:5:10:0.5) with visualisation by means of heating in the
presence of chromic acid also indicated the presence of sugars.
Many Agathosma species were investigated for the presence of coumarins (Finkelstein &
Rivett, 1976; Campbell et al., 1986; Moolla, 2006), and puberulin was previously identified
in A. ovata (Moolla, 2006). Such simple coumarins show distinct fluorescence quenching
under UV at 254 nm and intense blue or blue-green fluorescence at 365 nm (Wagner &
Bladt, 2001), as well as maximum absorption in the UV spectrum at max = 229, 293 and
343 nm (Moolla, 2006), but were not detected in the samples presented here.
121
Chapter 5
Chemistry
mAU
600
400
400
200
200
mAU
227
600
Hesperidin, 8.73 min
1200
283
Det 168-350nm
Hesperidin 1.25mg per mL 40-100%MeOH
8.983
Det 168-340nm
BEVW A betulina 2008
Retention Time
1000
0
200
800
220
240
260
280
300
320
340
360
380
nm
500
10.300
9.717
200
225
250
275
300
325
350
375
400
425
21.850
20.417
20.700
21.067
19.517
19.900
20.050
18.833
17.917
18.333
17.067
17.450
16.083
16.500
15.617
14.750
13.883
14.083
12.267
9.500
12.850
13.167
nm
10.767
11.033
11.333
11.683
8.617
7.517
7.817
8.117
5.950
6.200
6.583
6.883
5.283
500
Rutin, 10.12 min
400
2.350
2.617
2.767
3.100
3.533
3.750
4.083
4.533
4.767
1000
mAU
mAU
1000
200
354
257
1500
226
600
10.067
mAU
1500
10
12
14
16
18
20
22
24
26
Minutes
Retention Time
Det 168-280nm
A. betulina 51b (polar) gradient 40%
A. betulina 51b (polar) gradient 40%.dat
400
Hesperidin
Retention Time
300
mAU
175
300
175
200
200
150
283
150
100
2.217
100
419
125
200
125
400
200
100 450
mAU
400
20.950
50
20.983
354
20.333
20.617
19.367
19.667
17.600
25
100
0
250
300
350
400
450
nm
-25
2
10
11
12
13
14
15
16
17
18
-25
19
20
21
Minutes
Fig. 5.3:
HPLC chromatogram of the polar Agathosma betulina extracts: top AB 4310a,

to show the presence of hesperidin and possibly rutin; bottom AB 51(b) to
show the presence of hesperidin and diosmin
122
mAU
19.683
20.317
20.600
18.183
16.450
300
201
17.167
257
15.650
16.483
15.583
14.633
14.967
mAU
14.633
14.183
14.183
13.733
12.833
12.833
13.150
100
75
200
18.383
11.333
11.950
11.950
12.233
12.217
200
400
Diosmin
300
11.300
9.917
10.217
10.500
10.517
10.783
10.817 10.900
9.883
8.650
6.350
6.317
6.800
6.767
7.183
7.183
7.533
7.550
7.883
7.900
8.167
8.233
5.350
5.350
5.717
5.783
4.667
4.700
5.000
4.067
4.050
3.467
3.5503.717
25
Diosmin, 9.17 min
2.217
Hesperidin, 8.73 min
2.567
50
350
400
13.433
75
300
nm
9.150
9.150
8.650
250
2.567
2.833
3.050
mAU
100
mAU
Det 168-340nm
200
Chapter 5
Table 5.2:
Chemistry
Chemistry
HPLC results for four Agathosma species (chromatograms shown in Fig. 5.3 and Appendix 3A, 3B and 3C)
Retention times with relative quantities per peak
Agathosma
species
Sample
number
Unknown polar
compounds
2.27
3.32
A. betulina
AB 51a L
A. betulina
AB 51b L
A. betulina
AB 51c L
+++
A. betulina
AB 51d L
+++
A. betulina
AB 51e L
++
A. betulina
AB 51f L
++
A. betulina
AB 53 L
+++
A. betulina
AB 4310a L
A. betulina
Polar flavonoids
(whole leaf MeOH extracts*)
8.32
8.73
Hespiridin
9.17
Diosmin
Non-polar flavonoids
(leaf surface acetone extracts*)
10.10
13.52
13.98
14.25
16.10
16.31
19.18
Na
na
na
na
+ (++)
+ (+)
++
+ (+)
t (t)
++
++
++
Na
na
na
na
na
na
(++)
t (t)
+ (t)
++
(++)
t (+)
t (t)
++
+++
++
na
na
(t)
++
AB 4310b L
na
na
(t)
++
A. betulina
AB 4310c L
na
na
(t)
++
A. crenulata
AC 54 L
+++
A. crenulata
AC 10 L
A. ovata
AB 55 L
A. ovata
A. ovata
A. ovata
(++)
+++
na
na
+
+++
+
Na
na
na
na
na
na
(t)
na
(t)
na
(t)
+++
AO 4309a L
na
na
AO 4309b L
na
AO 4309c L
na
++
na
* all the flavonoids detected in the acetone leaf surface extracts are indicated in italics; where the same polar flavonoids as were detected in the MeOH
extracts were found, results are given in red (and in brackets and italics).
t = trace amounts (peak height 0-50 mAU); + = peak height 50-150 mAU; ++ = peak height 150-300 mAU; +++ = peak height > 300 mAU; na = not analysed
1
2
3
4
Possibly rutin; possibly hesperetin; possibly diosmetin; possibly kaempherol or a quercetin ether as based on UV spectra
123
Chapter 5
Chemistry
Campbell et al. (1987) analysed 42 taxa from the tribe Diosmeae for alkaloids, of which
A. ovata was one, but A. betulina and A. crenulata was not included. In these findings, the
result for A. ovata was negative. The qualitative colour test for alkaloids in this study, as well
as alkaloid extraction returned negative results for all three species.
The amino acid content of the three Agathosma species is reported here for the first time.
While the results seem variable, proline, isoleucine, its steoisomer allo-isoleucine,
tryptophan and tyrosine were present in all the samples analysed. Proline, glutamine and
asparagine were present in the highest concentrations.
Fig. 5.4:
HR-GC-MS-TOF amino acid chromatogram of Agathosma betulina aqueous

extract AB4310a [area x total ion count (TIC)] as an example of the amino acid
chromatograms produced for all the samples in which amino acids were
quantified.
In summary, this phytochemical investigation of A. betulina, A. crenulata and A. ovata

revealed that some of the polar flavonoid glycosides (hespiridin and diosmin), may be found
on the leaf surface and not only within the leaf. An LC-MS study of both polar and non-polar
flavonoids would confirm these results and reveal the identities of all of these flavonoids. No
alkaloids were detected in any of the three species. The amino acid content of A. betulina, A.
crenulata and A. ovata were determined for the first time where proline, glutamine and
asparagine occurred in the highest yields, even though the results were very variable.
124
Chapter 5
Table 5.3:
Chemistry
Chemistry
Amino acid content of Agathosma betulina, A. crenulata and A. ovata leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
-Aminoisobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT (s)
54.9
85.5
186.1
66.4
101.3
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257.0
87.3
84.1
246.0
146.5
230.1
167.7
105.6
56.5
101.6
99.3
290.2
274.1
71.8
t = trace amounts (< 0.2 mg/g)
Concentration (mg/g dry weight)

A. betulina
AB51a AB51b AB51c AB51d
17.5
t
3.6
3.0
6.3
3.7
4.9
5.1
4.0
0.7
t
t
0.5
1.8
3.2
2.5
3.5
2.0
10.1
3.6
6.4
2.4
2.0
17.9
1.0
7.9
1.8
6.0
2.4
9.2
12.5
94.3
72.8
0.3
9.8
1.9
5.2
9.5
5.2
8.9
9.3
63.3
424.4
9.2
1.4
0.4
8.5
1.3
8.6
8.1
4.0
8.1
224.9
3.8
7.5
117.0
AB51e
2.5
t
1.2
5.2
0.4
1.4
0.5
1.0
2.4
8.2
7.1
89.0
9.0
15.7
6.3
2.7
AB51f
4.3
5.6
t
1.6
8.6
8.1
8.5
44.0
t
12.9
5.7
1.5
11.0
4.5
1.5
10.6
4.6
1.5
10.4
4.2
10.8
4.9
2.3
AB4310a
27.5
20.6
3.6
0.9
7.6
4.9
121.2
20.1
16.6
37.0
4.6
15.4
4.8
16.1
6.7
10.3
25.4
205.0
35.7
20.7
17.8
14.2
12.4
AB53
5.1
4.5
1.0
1.7
2.8
t
A. ovata
AO55
5.4
3.6
2.6
t
t
2.7
9.2
A. crenulata
AC10
AC54
35.5
6.6
8.4
8.3
1.3
t
12.8
3.7
4.8
75.8
5.0
7.2
4.4
9.7
4.2
17.5
2.1
16.8
2.6
1.8
8.6
700.0
0.2
9.0
1.4
2.0
8.3
7.8
8.5
86.4
7.8
8.3
160.7
12.7
5.2
4.9
17.6
20.2
0.9
9.7
10.4
5.2
1.9
11.0
4.9
1.8
20.6
8.0
16.3
1.1
8.9
1.5
10.0
313.5
12.6
5.8
2.8
125
Chapter 5
Chemistry
5.2.2 Aloe species

A variety of phenolic compounds, alcohol precipitable solids, amino acids, enzymes and
sterols in Aloe contribute to many of the known medicinal properties (Chapter 4; Appendix 2;
Bruneton, 1995; Viljoen, 1999; Dagne et al., 2000; Wichtl & Bisset, 2000; Reynolds, 2004;
OBrien, 2005; Chen et al., 2012). The main classes of compounds in leaf exudates (i.e.
bitters or Aloe lump) of Aloe species are anthrones and chromones (Viljoen, 1999). These
compounds have medium to high polarity and are soluble in water, MeOH or EtOH
(Steenkamp & Stewart, 2007). In an extensive chemotaxonomic study of the aloe exudates,
Viljoen (1999) listed references reporting novel isolation and identification of these molecules
and their derivatives from the genus Aloe. The main compounds in the four Aloe species
studied here, are hydroxyanthracene derivatives, aloin A and B, which are 10-C--D-glucosyl
diastereomers of aloe-emodin (Dagne et al., 2000; Fig 5.5(a); Fig. 5.6) (aloe-emodin is an
anthrone formed after reductive cleavage of aloin in the colon, see Paragraph 6.2.2). These
bitter substances contribute 1227% of the lump in A. ferox, as equal amounts of aloin A
and B (Wichtl & Bisset, 2001). Other anthrones in A. ferox which occur in small, varying
amounts are aloinosides A and B as well as 5-hydroxyaloin, together with chromone
derivatives, i.e. mainly aloeresins A and B (2540%), with small amounts of aloeresin C (Van
Wyk et al., 1995; Fig. 5.6). Medium-polar, dichloromethane extractable minor compounds
from A. ferox exudates include molecules based on naphthalene (which are fused with
cyclohexane rings to form tetrahydoanthracenes) and tetralin (i.e. 1-methyltetralins, such as
feroxidin and feroxins A and B) nuclei, as well as p-hydroxybenzenaldehyde, phydroxyacetophenone,
pyrocatechol,
three
decanoic
acid
derivatives,
three
methylnaphtofuranones and methyl p-coumarate [Dagne et al., 2000; Wichtl & Bisset, 2001;
Chen et al., 2012; Fig. 5.5(b) and (c)]. As with A. ferox, the main constituents of A. vera lump
are the aloins A and B (2540%), together with 7-hydroxyaloin A, B (Fig. 5.6) as well as its
6-p-coumaroyl and feruloyl esters (34%). Its constituent chromones include aloeresin B (up
to 30%) with smaller amounts of aloesone and aloeresins A and C (Wichtl & Bisset, 2001).
Aloenin is a bitter phenylpyrone characteristic of A. arborescens (Viljoen, 1999; Wichtl &
Bisset, 2001; Fig. 5.6). Other compounds from A. arborescens include furans (3furanmethanol, 2,3-dihydrobenzofuran), chromones (2-O-feruloylaloesin), aloenin acetal and
-sitosterol (Dagne et al., 2000). Aloin A and B, as well as homonataloin A and B show
erratic occurrence in variable amounts in A. marlothii without geographical correlation
(Viljoen, 1999). Other compounds identified in A. marlothii include 7-O-methylaloesin A, 5hydroxyaloin A 6-O-acetate and aloesin (Bisrat et al., 2000).
TLC (Wagner & Bladt, 2001) and HPLC (Chen et al., 2012) are analytical methods used in
quality control of aloe bitters. Aloenin and its derivatives, for example, are characteristic of
126
Chapter 5
Chemistry
A. arborescens, but are sometimes found in A. ferox bitters, indicating adulteration or

hybridisation. Extracts of A. ferox and A. vera can also be identified based on
presence/absence of the 5- or 7-hydroxyaloins. With TLC (ethyl acetate:MeOH:water
100:13.5:10; without chemical treatment under UV-365 nm), aloinosides (Rf 0.250.3), aloin
(Rf 0.5) and aloe-emodin (solvent front) show characteristic yellow to orange fluorescent
zones, where aloeresins such as aloeresin A and aloesin (Rf 0.55 and Rf 0.25 respectively)
fluoresce blue (Wagner & Bladt, 2001). Treatment with a base affords visible yellow, pink,
red or violet zones, where treatment with ethanolic vanillin and sulfuric acid returns yellow
zones typical of phenolic substances [Fig. 5.5(a)]. The UV-spectra of anthrones and
chromones are significantly different, where anthrone structure typically exhibit max in MeOH
at approximately 268, 296 and 355 nm, and chromones give peaks at max 215, 234, 265 as
shouldered peaks together with one large peak at 297 nm (Viljoen, 1999; Dagne et al.,
2000). Flavonoids (apigenin, dihydroisorhamnetin, naringenin and isovitexin) have been
identified from several Aloe species, but were not detected in the four species studied here
(Viljoen et al., 1998).
Fig. 5.5:
TLC of Aloe: (a) MeOH extracts visualised with ethanolic vanillin/H2SO4, plate
developed in CHCl3:MeOH:H2O:CH3COOH (60:30:8:6)]; (b) MeOH:CHCl3 (1:1)
extracts developed in CHCl3:MeOH (6:3) and visualised with ethanolic
vanillin/H2SO4 (c) MeOH:CHCl3 (1:1) extracts developed in diethyl ether:hexane
(2:3) visualised with ethanolic vanillin/H2SO4
127
Chapter 5
Chemistry
Anthrone-C-glycosides:
OH
8
OH
OH
OH
Anthraquinone
OH
OR3
10
4
5
2
Aloin A*: R = 1-C--D-glucosyl; R = H; R = H

Aloe-emodin
1
2
3
Aloin B*: R = H; R = 1-C--D-glucosyl; R = H
1
2
3
Aloinoside A*: R = 1-C--D-glucosyl; R = H; R = 1-O--L-rhamnopyranosyl
1
2
3
Aloinoside B*: R = H; R = 1-C--D-glucosyl; R = 1-O--L-rhamnopyranosyl
OH
OH
OMe
R1
OH
HO
OH
2
HO
HO
OH OH
OH OH
OH
OH
5-Hydroxyaloin A*: R = H; R = OH
1
2
7-Hydroxyaloin A: R = OH; R = H
Homonataloin A & B
Chromones:
OH
HO
OH
1
RO
R2O
Aloeresin A*: R = 2-p-coumaroyl; R = H
Aloeresin B (= Aloesin*): R = H; R = H
1
Aloeresin C*: R = 2-p-coumaroyl; R = 2-O--D-glucoside
O
O
Aloesone: aglycone of aloeresins A, B, C
Phenylpyrones:
1-Methyl tetralin derivatives
Gluc
HO
OH
O
OH
OH
O
HO
OR
HO
OH
O
Aloenin A
Fig. 5.6:
Feroxidin*:
Feroxin A*:
R=H
R = 3-O-glucoside
Ferarolide*
Main classes of phenolic compounds known (with a few examples of each) from
Aloe arborescens, A. ferox, A. marlothii and A. vera according to Van Wyk et al.
(1995), Van der Bank et al. (1995), Viljoen (1999), Dagne et al. (2000) as well as
Wichtl and Bisset (2001); * = the major characteristic compounds of A. ferox, as
listed in a detailed review on the phytochemistry of A. ferox also giving all other
compounds identified from this species (Chen et al., 2012).
128
Chapter 5
Chemistry
Yaron (1993) reported that free monosaccharides may make up as much as 30% of A. vera
leaf pulp. OBrien (2005) found that the main sugar in the four species included here is
glucose, ranging from 0.1 mg/mL to 3.3 mg/mL in the gel, with A. ferox having the lowest
and A. vera having the highest glucose contents. A. ferox gel, however, is known to also
contain an appreciable amount of fructose (Chen et al., 2012). According to the WHO
(1999), Aloe gel is known to have a high water content (> 95%) and APS, which includes a
complex mixture of
(a)
polysaccharides,
i.e.
pectins,
hemicelluloses,
glucomannan,
acemannan
(polysaccharides containing highly acetylated mannan units with an approximate molecular

weight of 1000 kDa) and mannose derivatives; at least five saccharides are present, i.e.
arabinose, galactose, glucose, mannose and xylose (Davis, 1997); A. ferox polysaccharides
have been determined to be mostly arabinogalactans or rhamnogalacturonans (Mabusela et
al., 1990);
(b) approximately 30 amino acids in A. ferox (Ishikawa et al., 1987; Van Wyk & Gericke,
2000) and 22 in A. vera (Davis, 1997) (see Table 5.5 and the accompanying discussion);
(c) minerals (as ions), i.e. high levels of Ca, also Zn, Cu, Cr, Mn, Fe, K, Na and Mg, as well
as trace elements such as Se (Van Wyk & Gericke, 2000); A. vera and A. arborescens have
high levels of K, Ca and Mg (Byun et al., 1997; Femenia et al, 1999);
(d) organic acids malic acid and quinic acid are the two major organic acids in A. ferox gel
(Chen et al., 2012); lactic acid content, relative to malic acid content, determines the
freshness of the gel sample, as lactic acid is a microbial derivitisation product of malic acid
(OBrien, 2005).
(e) lipids, where A. vera and A. arborescens are known to contain lauric, myristic, palmitic,
palmitoleic, stearic, oleic, linoleic and linolenic fatty acids (Byun et al., 1997);
(f) sterols (lupeol, campesterol and -sitosterol) (Davis, 1997),
(g) vitamins (B1, B2, B6, B12, C and others) (Davis, 1997)
(h) tannins and
(i) enzymes.
The level of APS in Aloe gel is reportedly indicative of the biological activity of the gel: the
higher the APS content, the higher the activity (OBrien, 2005). Of the four species studied
here, A. vera has been shown to have the highest levels of APS (ca. 34%), and A. marlothii
the second highest (2433%) in the gel (OBrien 2005). It should be noted that the APS
values reflected in Table 5.4 are lower than these as whole leaves were used for this study,
where OBrien analysed the gel fillets only. Polysaccharides can be hydrolysed and the ratio
of resulting monosaccharides determined. All the species studied here, except for A. vera,
contains mainly glucose, with the latter containing mainly mannose (OBrien, 2005).
Galactose may occur in A. ferox polysaccharides and its level relative to that of glucose is
129
Chapter 5
Chemistry
used to distinguish three chemotypes within the species. A. arborescens polysaccharides

may also contain galactose, arabinose and/or mannose together with glucose depending on
climatic temperature, rainfall or leaf age (Beppu et al., 2004). Xylose may be present in A.
ferox polysaccharides in trace amounts but may be a more prominent sugar in A. marlothii
polysaccharides. Significant population and plant-to-plant variation in terms of hydrolysable
sugars exist (OBrien, 2005). When whole-leaf samples are used instead of gels, this result
is not evident (Table 5.4), however relatively high levels of APS were obtained. The
qualitative saponin foam test was positive for all four species tested, confirming the high
levels of mucilage (APS) as well as the presence of lipids and sterols in the leaf exudates
contained in the dry leaf extracts (Table 5.5).
Table 5.4: Extract yields for four medicinal Aloe species
Aloe species
Sample
number

APS
Polar
Total water
extractable
Non-polar
Alkaloids
A. arborescens
AAb 62 L
19.3
8.1
27.4
7.6
0.061
A. ferox
AF 60 L
13.8
4.2
18.1
5.3
0.059
A. marlotthii
AMa 61 L
6.9
3.8
10.7
2.4
0.090
A. vera
AV 65 L
8.6
11.3
19.9
4.5
0.077
Asparagine, aspartic acid, threonine and tryptophan are amino acids which are common to
all aloes, while alanine, phenylalanine, leucine and valine are said to occur in selected
species only (Hutchings et al., 1996). The amino acid content for the four studied Aloe
species have been determined and is compared with those from A. arborescens and A. vera
in literature (Byun et al., 1997; Table 5.5). From several other studies, Reynolds (2004)
reported that asparagine, glutamine, alanine and histidine were most abundant in A. ferox,
that alanine, proline, lysine and glutamic acid were the main amino acids in A. arborescens,
and that aspartic acid, glutamic acid and serine were noted from A. vera, amongst at least
17 amino acids. From the 25 amino acids identified here, only glutamine and proline are
present in high levels (i.e. > 100 mg/g dry weight). The results obtained indicate some level
of agreement with those from literature, which are also variable due to analyses from a
variety of matrixes (juice, pulp or whole leaf) being reported.
Other minor compounds identified from Aloe include coniine alkaloids, but were not detected
in the species investigated here (Reynolds, 2005). The qualitative colour test results were
confirmed negative results for alkaloids for three of the Aloe species under investigation
(A. arborescens was not tested). Furthermore, Magwa et al. (2006) reported 21 volatile
components in A. ferox leaves of which 3-cyclohexene-1-acetaldehyde, 2,4-decadien-1-ol, 2130
Chapter 5
Chemistry
heptanol and bornylene were detected in the highest yields. Lastly, anthraquinones may also
occur in Aloe roots. Two types of anthraquinones have been identified from A. ferox, i.e.
aloesaponarin I type and chrysophanol type, where the latter type may also be detected in
the leaves. Examples of these A. ferox root anthraquinones include aloesaponarin I and II,
aloesaponol I and II, asphodelin and chrysophanol (Chen et al., 2012). While the roots are
not medicinally important (see Chapter 4, Appendix 2), and consequently have no impact on
the commercial value of Aloe, these compounds play an important chemotaxonomic role.
Table 5.5: Amino acid content of Aloe arborescens, ferox, A. marlothii and A. vera whole
leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leusine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.3
246
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8

A. arborescens
A. ferox A. marlothii
Lit.*
AF 60
Ama 61
AAb 62
14.0
9.4
2.1
0.9
t
nd
20.1
5.6
12.7
12.5
2.8
0.5
t
1.4
nd
3.7
3.3
5.4
21.0
353.0
0.8
11.8
5.6
6.3
6.7
1.0
10.1
7.6
10.6
9.2
3.5
1.5
2.9
5.2
4.4
0.3
4.7
11.3
162.1
10.1
10.7
7.6
6.9
16.0
8.2
17.7
Byun et al., 1997; nd = not determined
2.2
11.9
7.2
10.5
3.1
3.0
2.7
2.4
3.5
26.6
10.1
4.4
0.5
1.1
3.9
nd
5.6
39.4
199.3
1.0
14.0
2.8
10.6
4.7
13.5
385.3
0.7
4.4
16.1
14.0
10.4
A. vera
Lit.*
AV 65
1.2
3.8
4.7
1.9
0.7
1.2
2.4
1.8
1.3
1.8
1.4
1.0
1.8
41.1
12.2
1.5
0.6
0.9
1.8
Nd
14.0
2.7
15.9
92.3
2.8
12.5
3.8
11.2
5.5
4.9
11.3
1155.7
20.9
4.6
16.8
8.9
0.3
The phytochemical assessment of A. arborescens, A. ferox, A. marlothii and A. vera showed

the large amount of research that have already been conducted on aloes. The great diversity
of chemical compounds identified from these four species are indicated for an equally
diverse collection of biological activities (Paragraph 6.2.2), giving rise to a wider range of
traditional uses than simply wound-healing and as laxative (Paragraph 4.2.2, Appendix 2).
131
Chapter 5
Chemistry

After his first encounter with Arctopus, Pappe (1847) wrote: A. echinatus root contains an
alkaloid, which, combined with acids, assumes the form of neutral salts. Thus Arctopium
sulphuricum (the sulfate) consists of small scaly white crystals, which are astringent in taste,
and which in half grain doses produce coagulation of the saliva within the mouth. Dr. James
Barry, assistent surgeon the Forces (in Theodore, 1972), reported that the roots contained
gum resin in abundance, a moderate proportion of arctopine (possibly an alkaloid; the
sulphate of arctopine has strong, bitter, permanently styptic taste half a grain quickly and
powerfully excites the salivary glands, leaving a peculiar sensation and unpleasant metallic
taste long impressed on the tongue and fauces) was present, together with small amounts
of essential oils, extractive narcotic matter, considerable amounts of tannin, a bitter
aromatic acrid principle, a very small amount of Feculae Saccharine matter and lignum.
Much later (Watt, 1967; Watt & Breyer-Brandwijk, 1962), the following chemical
characteristics were added to the description: the root yields sucrose, resin, a glucoside, a
brown volatile oil (containing no sulfur and which yields two fractions volatilizing at 90
100 C and 180190 C), and two crystalline products (melting at 172173 C and 149
152 C). Polar phenolic acids were recently isolated and identified from aqueous extracts
from the roots of A. echinatus, A. monacanthus and A. dregei (Olivier et al., 2008; Fig 5.7;
Appendix 5A). Van Wyk et al. (1997, 2009) suggested that the root exudate of Arctopus
species contained kaurenes similar to those from Alepidea (Rustaiyan & Sadjadi, 1987;
Holtzapfel et al., 1995; Somova et al., 2001) based on TLC and GC-MS comparisons and
the phylogenetic relationship between the two genera (Calvio & Downie, 2007). The
identities of these non-polar Arctopus compounds had not previously been determined by
spectroscopic methods until now.
From the qualitative colour test results, the presence of steroids, terpenoids or bitter
principles (Burchard test) as well as saponins could be established, but was negative for
alkaloids. Further screening involved extraction with solvents of different polarities followed
by TLC of the extracts obtained in different solvent systems (Table 5.6; Fig. 5.8). High yields
in APS and polar extracts were obtained, even for the leaves, where TLC of the polar
extracts confirmed the presence of large terpenoids (such as saponins) and phenolic
substances. Both the non-polar fractions and sticky root exudate were soluble in low polarity
solvents such as chloroform, predicting that the exudate mainly contains non-polar terpenes,
and was confirmed by means of TLC. Low yields were obtained after micro-alkaloid
extraction, returning negative results after TLC investigation for alkaloids, confirming the
qualitative tests.
The phenolic compounds showed unexpected behaviour by distinct pale yellow colouration
on TLC after treatment with ninhydrin, developed in a highly viscous, acidic solvent system
132
Chapter 5
Chemistry
to curb streaking and consequently improve resolution (see Fig. 5.8). This class of
compounds could also be separated from the other polar compounds easily by means of
preliminary solid phase extraction. They were isolated by means of column chromatography
(Section 3.5.1), followed by identification with LC-MS (Paragraph 3.4.1.2; Fig. 5.9) and 2DNMR (Paragraph 3.6.1), and quantification on HPLC-PDA (Paragraph 3.4.1) (Olivier et al.,
2008). The three major phenolic acids identified in the Arctopus species were (R)-3-O--Dglucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid (Olivier et al., 2008; Fig 5.7).
These acids were also identified in Alepidea species, but the highest yields for rosmarinic
acid and its glucoside were obtained from A. echinatus (15.3 and 15.5 mg/g dry root weight
respectively), and the highest yield for caffeic acid was obtained from A. monacanthus
(3.6 mg/g dry root weight). The occurrence of these phenolic compounds in both Arctopus
species and Alepidea species confirmed the suggested taxonomic relationship between the
genera (Van Wyk et al., 1997, 2009; Calvio & Downie, 2007).
Table 5.6: Extract yields obtained for Arctopus echinatus, A. dregei and A. monacanthus
Arctopus species
Sample
number

APS
Polar
Total water
extractable
Non-polar
Alkaloids
A. echinatus
AE 1 R
1.7
5.0
6.8
11.8
0.049
A. echinatus
AE 2 R
2.8
5.1
7.9
Ns
0.020
A. echinatus
AE 3 R
1.2
5.0
6.3
7.6
0.046
A. echinatus
AE 4a R
1.2
4.1
5.2
4.4
0.059
A. echinatus
AE 4b R
2.0
9.3
11.3
12.9
0.068
A. echinatus
AE 4c R
2.3
9.3
11.7
10.7
0.069
A. echinatus
AE 5 R
2.4
7.0
9.4
Ns
0.087
A. echinatus
AE 6a R
3.4
5.5
9.0
3.4
0.040
A. echinatus
AE 6a L
2.4
9.2
11.6
Ns
Ns
A. echinatus
AE 6b R
1.9
7.9
9.7
3.2
0.060
A. echinatus
AE 6b L
2.7
7.4
10.0
Ns
Ns
A. dregei
AD 7 R
1.4
2.8
4.3
9.0
0.090
A. dregei
AD 8a R
4.5
6.5
11.0
Ns
Ns
A. dregei
AD 8b R
2.9
5.8
8.6
Ns
0.108
A. dregei
AD 8c R
3.2
8.6
11.7
Ns
Ns
A. monocanthus
AMo 9 R
2.4
7.7
10.1
11.1
0.090
A. monocanthus
AMo 10 R
4.3
12.6
17.0
17.4
0.100
A. monocanthus
AMo 11 R
3.3
9.9
13.1
21.6
0.129
ns = no sample
133
Chapter 5
Chemistry
Phenolic acids*:
O
2'
7'
3'
8'
9'
1'
4'
6'
HO
5'
OH
OH
OH
3
2
HO
OH
OH
(R)-3-O--D-Glucopyranosylrosmarinic acid:
R = glucopyranosyl
R=H
Rosmarinic acid:
Caffeic acid
Diterpenes:
14
12
11
16
9
10
2
3
4
18
20
OH
20
1
12
15
13
17
8
Manool
16
14
14
OH
15
9
8
15
7
6
19COOH
18
19
13
17
13
11
10
12
17
16
11
COOMe
Ent-trachyloban-19-oic acid
Methyl 16-hydroxy-ent- kaur-11-en-19-oate
OH
COOH
Ent-kauren-19-oic acid
Ent-kauren-16-ol
Triterpenes:
OR
OR
OH
RO
Fig. 5.7:
Barrigenol type triterpenes

R = glycopyranosyls or H
Main classes of compounds (with all known examples) present in Arctopus

species (all data from this study). * = as published in Olivier et al., 2008
134
Chemistry
AE 1R
AE 2R
AE 3R
AE 4aR
AE 4bR
AE 4cR
AE 5R
AE 6aR
AE 6bR
AE 6aL
AE 6bL
AD 7R
AD 8aR
AD 8bR
AD 8cR
AMo 9R
AMo 10R
AMo 11R
(c)
Fig. 5.8:
(b)
AE 1R
AE 2R
AE 3R
AE 4aR
AE 4bR
AE 4cR
AE 5R
AE 6aR
AE 6bR
AE 6aL
AE 6bL
AD 7R
AD 8aR
AD 8bR
AD 8cR
AMo 9R
AMo 10R
AMo 11R
Arginine
AE 1R
AE 2R
AE 3R
AE 4aR
AE 4bR
AE 4cR
AE 5R
AE 6aR
AE 6bR
AE 6aL
AE 6bL
AD 7R
AD 8aR
AD 8bR
AD 8cR
AMo 9R
AMo 10R
AMo 11R
(a)
(d)
AE 1R
AE 2R
AE 3R
AE 4aR
AE 6aR
AE 6aL
AD 8aR
AMo 10R
Arabinose
Fructose
Galactose
Glucose
Lactose
Maltose
Mannose
Rhamnose
Ribose
Sucrose
Xylose
Chapter 5
TLC of Arctopus species: (a) amino acids developed in n-BuOH:CH3COOH:

H2O (4:1:2), visualised with ninhydrin (note the phenolic acids brown spots in
the upper part of the plate); (b) non-polar terpenes developed in diethyl
ether:hexane (2:3), visualised with ethanolic vanillin/H2SO4; (c) polar extract after
removal of phenolic compounds developed in CHCl3:MeOH:H2O:CH3COOH
(60:30:8:6), visualised with ethanolic vanillin/ H2SO4; sugars developed in
ACN:CS2:H2O:formic acid (85:5:10:0.5), visualised through charring with chromic
acid.
TLC of the polar extracts also revealed the presence of triterpenoids, confirming the positive
findings in the saponin test. HR-LC-MS for polar extracts (Fig. 5.9) showed that the PDA
response for these triterpenes are negligible compared to those of the phenolic acids, but
confirmed the TLC results by indicating the presence of at least 15 of these triterpenes.
Although isolation of some of these compounds by means of column chromatography was
successful, the structures could not be solved with the NMR and accurate mass data
obtained. It is possible that the compounds are not sufficiently pure resulting in obscuring
peaks. The parent structure of several of these triterpenes could be barrigenol. Saponins
such as these, structurally related to barrigenol (see Fig. 5.7), have been isolated from the
medicinally important Pittosporum viridiflorum Sims. (Van Wyk et al., 1997) with
considerable glycosylation also being evident from the high masses of the compounds and
135
Chapter 5
Chemistry
NMR data obtained. These saponins may contribute to the slightly bitter taste of polar
Arctopus extracts (bitterness value of 1 60021 600, see Paragraph 5.3).
Fig. 5.9:
Data obtained from high-resolution liquid chromatography mass spectrometry

(HR-LC-MS) for polar Arctopus monacanthus extracts
The presence and character of the non-polar kaurenes were established from unpublished
comparative TLC [as in Fig. 5.8(b)] and GC-MS studies (Witte & De Castro, Department of
Botany and Biotechnology, University of Johannesburg) in which air-dried material of
Arctopus echinatus (A1), A. monacanthus (B1) and Alepidea amatymbica (D1, could
possibly also be Alepidea cordifolia according to its place of origin) were ground to fine
powders and extracted with CH2Cl2 (5 g in 50 mL) (see Table 5.7 for provenances, voucher
numbers and yields). After TLC analyses, the extracts were filtered through celite and the
solvent evaporated under vacuum, followed by derivatisation with diazomethane to produce
volatile esters suitable for GC-MS analysis (see Paragraph 3.4.2.2; results given in
Table 5.8). To confirm these results, dry ground plant material (0.3 g) of Arctopus echinatus
(A2), A. monacanthus (B2), A. dregei (C) and Alepidea amatymbica (D2) were extracted in
3 mL CHCl3:MeOH (1:1) overnight, filtered and the filtrates evaporated to dryness. The dry
extracts were reconstituted in 1 mL of MeOH, and analysed by means of TLC and LC-MS
(results given in Table 5.9).
TLC [Fig. 5.8 (b)] of these non-polar extracts (Arctopus and Alepidea species) showed
several poorly resolved main compounds as blue and violet zones with similar Rf-values. A
few minor diterpene components were also detected, indicating similarity amongst Arctopus
species but marked differences from the Alepidea samples. The preliminary GC-MS
136
Chapter 5
Chemistry
comparison between Arctopus echinatus, Arctopus monacanthus and Alepidea amatymbica

reiterated these findings and revealed similar major diterpenes for the two genera based on
retention times and masses (Table 5.8). The major compounds were subsequently isolated
and their identities determined by means of 2D-NMR (Paragraph 3.6.1; Appendix 4),
together with UPLC/HR-APCI-MS accurate mass determination (see Paragraph 3.4.1; even
though kauren-19-oic acid was impure, good resolution between all the constituents allowed
the determination of its accurate mass; partial structure elucidation with NMR together with
accurate mass determination confirmed its proposed structure) as manool, ent-trachyloban19-oic acid and kauren-19-oic acid, while methyl 16-hydroxy-ent-kaur-11-en-19-oate is a
minor diterpene (see Paragraph 3.5 for physical characters of the compounds).
Table 5.7: Provenances, voucher specimens and sample numbers of the rhizome and root
material of Arctopus and Alepidea species used for extraction and analysis. The
relative yields of non-polar compounds are also given. Voucher specimens were
collected by A de Castro (AdC), AR Magee (ARM), B-E Van Wyk (BEVW), JS
Boatwright (JSB) and PJD Winter (PW).
Species and voucher specimens
Sample
Locality or
% yield
reference provenance
(g/g dry
number
weight)
Arctopus echinatus L.
- BEVW s.n.
A1
Pakhuis Pass
7.6
- PW & BEVW 170
A2
Du Toits Kloof Pass
11.8
Arctopus monacanthus Sond.
- BEVW 3522
B1
Gifberg
11.1
- BEVW 4141(a)
B2
Citrusdal, Elandskloof
21.6
Arctopus dregei Sond.
- ARM & JSB 31
C
Malmesbury
8.9
Alepidea amatymbica Eckl. & Zeyh.
- ADC 404
D1
Johannesburg Muti
13.8
Market
Alepidea cordifolia B.-E.Van Wyk
- PW 252
D2
Mphendle
21.6
Manool (Fig 5.7; Table 1 Appendix 4) is a well-known labdane-type diterpene
characterised by a
13
C NMR spectrum containing 20 carbon signals resulting from four
tertiary methyl carbons (C 14.4, 21.7, 27.7 and 33.6), three quaternary carbons of which
one is oxygenated (C 33.6, 39.9 and 73.7) and four sp2 carbons (C 106.5, 111.6, 145.3 and
148.7). Apart from clear correlation between the H-14 methine proton (H 5.84, dd, J = 10.8,
17.4) and the protons on C-15 in the COSY spectrum, they were also mutually spin coupled
(H-15a 4.98, dd, J = 1.2, 10.8; H-15b 5.13, dd, J = 1.2, 10.8), indicative of a vinyl group. The
two H-17 protons at H 4.44 (br s) and H 4.75 (d, J = 1.2) showed long-range 1H-13C
correlation with C-7 and C-9, which clarified the position of the end methylene group. Its is
difficult to distinguish manool from its enantiomer, 13-epimanool, by NMR (Rowe &
137
Chapter 5
Chemistry
Scroggins, 1964), but according to Barrero et al. (1993) this signal of H-17a may be used as
the distinguishing factor (H 4.46 (our value 4.44) for manool, H 4.50 for 13-epimanool). The
other 1H- and 13C NMR data of manool is in agreement with those in literature (Barrero et al.,
1993; Lu et al., 1995; Rowe & Scroggins, 1964). UPLC/HR-APCI-MS analysis produced a
molecular ion peak at m/z 273.2571 suggesting a molecular formula of C20H33, indicative of
the loss of the hydroxyl group, but confirming unsaturation for the compound. Conclusively,
the spectral data suggested that manool was a bicyclic compound.
Trachylobanes (pentacyclic) such as ent-trachyloban-19-oic acid (Table 1 Appendix 4;
Leong & Harrison, 1997; Mitscher et al., 1983; Morris et al., 2005; Takahashi et al., 2001)
and kaurane-type diterpenes (tetracyclic) such as methyl-16-hydroxy-ent- kaur-11-en-19oate (Table 1 Appendix 4; Batista et al., 2007; Croft et al., 1974) and ent-kauren-19-oic
acid are all derivatives of pimarane (a tricyclic precursor derived from labdane) (Fig. 5.7;
Otto & Wilde, 2001). Ent-trachyloban-19-oic acid and ent-kauren-19-oic acid both contain 20
carbons in their respective
13
C spectra, while methyl 16-hydroxy-ent- kaur-11-en-19-oate is
an ester derivative showing the presence of 21 carbons. The
13
C spectra of ent-trachyloban-
19-oic acid, methyl 16-hydroxy-ent- kaur-11-en-19-oate and ent-kauren-19-oic acid showed

only three tertiary methyl carbons in each case in comparison with four methyl carbons for
manool. Long-range HMBC experiments for ent-trachyloban-19-oic acid, methyl 16hydroxy-ent- kaur-11-en-19-oate and ent-kauren-19-oic acid revealed a carbonyl carbon (C19), typical of carboxylic acids or esters at C 185.6, 184.9 and 177.9 respectively,
correlating with the methyl protons of C-18, and also with the methylene protons of C-3 in
the case of ent-kauren-19-oic acid. Ent-trachyloban-19-oic acid was saturated, while methyl
16-hydroxy-ent- kaur-11-en-19-oate and ent-kauren-19-oic acid both had a pair of sp2hybridized carbons each. The spectral data obtained for ent-trachyloban-19-oic acid
(summarized in Table 1 Appendix 4) corresponds well with those reported for enttrachyloban-19-oic acid (Mitscher et al., 1983; Leong & Harrison, 1997; Takahashi et al.,
2001; Morris et al., 2005). The DEPT spectrum for methyl 16-hydroxy-ent- kaur-11-en-19oate showed two monoprotonated sp2 carbons (C 127.0 and 132.5), while the coupling
constants for the respective protons (H-11 5.50, J 8.8 and H-12 5.88, J 8.9) indicated a cisorientation. A long-range HMBC coupling was also observed between C-9 and H-11, with
further COSY correlation between C-11 and C-12 and between C-12 and C-13 clarifying the
position of the double bond. C-16 was deshielded (C 83.8), indicative of oxygenation. The
signal for C-17 (25.8 ppm) corresponds to the literature value for that of a -epimer, where
an -epimer would exhibit a C-17 signal at about 32.5 ppm (Croft et al., 1974; St. Pyrek,
1984). Furthermore, HMBC correlation was observed between C-19 and the methoxyprotons on C-21 of methyl 16-hydroxy-ent- kaur-11-en-19-oate distinguishing it from 16methoxy-ent-kaur-11-en-19-oic acid, reportedly isolated from Alepidea amatymbica (Somova
et al., 2001).
138
Chapter 5
Chemistry
Table 5.8: The distribution of major diterpenoids in samples of Arctopus echinatus (A1), A.
monacanthus (B1) and Alepidea amatymbica (D1) as determined by GC-MS
(after derivatisations, single measurements)*,#
Compound
GC-MS reference data
Isopimaradiene
Manoyl oxide
Manool (1)
Kaurenoic acid
Kauranol (5)
RI
272
109
1948
290
81
1996
290
137
2031
+++
+++
239
2105
314
131
2144
+++
+++
135
2147
314
239
2169
++
++
290
316
Ent-trachyloban-19-oic acid (2)

Trachylobanoic acid isomer
B1
Base peak
314
A1
Ent-kauren-19-oic acid (3)

Kaurenoic acid
Kaurenoic acid
++
++
++
123
2188
105
2200
+++
+++
+++
92
2217
316
316
D1
91
2231
314
316
105
2246
++
++
316
991
2248
++
++
288
81
2301
332
121
2360
Methyl hydroxy-dehydro-kaurenoate
c
isomer
330
201
2372
Methyl 16-hydroxy-ent-kaur-11c
en-19-oate (4)
332
274
2379
++
++
c
isomer
330
300
2389
c
isomer
330
121
2415
314
105
2416
Methyl hydroxylkauranoate
334
121
2430
Unknown 1
328
328
2473
Unknown 2
372
57
2593
++
Dehydro-manoyloxide isomer
Methyl hydroxy-kaurenoate isomer
Kaurenoic acid
++
Relative quantities [t = trace amounts; + = minor compound (ca. 1-5%); ++ = major compound (6
20%); +++ = dominant compound (more than 20% of total peak area)]; compounds indicated in bold
are the isolated major compounds
#
Preliminary identification based on GC-MS spectral libraries (see Paragraph 3.4.2.2)
a
Isopimarane-type diterpenoid
b
Labdene-type diterpenoid
c
Kaurene-type diterpenoid
D
Derivatized compound (methyl ester of carboxylic acid)
I
Derivatised trachylobanoic acid and underivatized wedelia seco-kaurenolide are isomers
139
Chapter 5
Chemistry
The pure compounds were used as standards in LC-MS quantification of diterpenoids from
the three Arctopus species and Alepidea cordifolia (Table 5.9). By comparing the GC-MS
and LC-MS results, it became evident that there is good agreement between the tentative
identifications and also the relative quantities of main compounds shown in Tables 5.8 and
5.9, respectively. The LC-MS results (Table 5.9) showed that the three Arctopus species all
contain small amounts of the minor kaurene ester derivative (methyl 16-hydroxy-ent-kaur11-en-19-oate) while it is absent in Alepidea cordifolia. The preliminary GC-MS results did
however indicate its presence in Alepidea amatymbica. A general overview of all the
chromatographic data and non-polar extract yields shows variable quantities of diterpenes
even within samples of the same species.
Table 5.9: A quantitative comparison of isolated major compounds in samples of Arctopus
echinatus (A2), A. monacanthus (B2), A. dregei (C) and Alepidea cordifolia (D2)
as performed by LC-MS (without derivatisation, single measurements) using
known amounts of isolated compounds, calculated as % (g/g dry extract x 100)
LC/MS reference data
A2 (%)
B2 (%)
C (%)
D2 (%)
Compound
Manool
290
34.13
16.27
1.10
1.38
Ent-kauren-19-oic acid
314
16.55
0.96
7.74
2.66
4.47
Ent-trachyloban-19-oic
acid
316
21.77
3.13
24.90
29.57
Methyl 16-hydroxy-entkaur-11-en-19-oate
332
9.19
0.06
0.77
0.25
Rt (mins)
Both the patterns of main diterpenoids and phenolic acids show that Arctopus and Alepidea,
despite their obvious morphological differences, are indeed chemically closely related.
Diterpenoids such as the ones described here (a labdane, manool, and derivatives of
pimarane-type diterpenes, ent-trachyloban-19-oic acid, methyl 16-hydroxy-ent- kaur-11-en19-oate and ent-kauren-19-oic acid) (Otto & Wilde, 2001) are widely distributed in the Plant
Kingdom (including the Apiaceae, e.g. Elaeoselinum W.D.J.Koch ex DC. see Mongelli et
al., 2002) but have, however, not yet been reported in the Saniculeae except for Alepidea
and Arctopus (Holtzapfel et al., 1995; Rustaiyan & Sadjadi, 1987; Somova, et al., 2001),
unlike the case with the reported phenolic acids. A wider survey of other taxa is therefore
desirable, especially since Alepidea and Arctopus are successively sister (in a cladistic
sense) to the rest of the tribe, which includes Actinolema Fenzl, Astrantia L., Hacquetia
Neck. ex DC., Sanicula, Eryngium and Petagnaea Caruel (Magee et al., 2010). Some
Eryngium species, i.e. E. bourgatii Gouan and E. glaciale Boiss. contain phyllocladene-type
tetracyclic diterpenes (Pal-Pal et al., 2005a, 2005b) but these are derived from
isopimaranes (Otto & Wilde, 2001).
140
Chapter 5
Chemistry
Table 5.10: Amino acid content of Arctopus dregei and A. echinatus root extracts
Amino acids
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
R.T. (s)
54.9
85.5
186.1
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.1
246.0
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8

A. dregei
A. echinatus
AD 7R
AE 4b R
AE 6a R
2.2
14.1
5.4
4.7
5.0
1.3
3.1
4.4
6.0
0.6
3.4
3.1
2.9
347.7
4.3
0.2
19.0
2.2
3.2
16.0
6.6
7.6
4.5
11.2
1.6
11.9
1.6
10.8
1.5
10.7
11.0
10.2
5.0
9.0
21.5
4.8
8.2
240.4
4.7
8.5
11.3
3.5
13.8
6.7
1.9
13.4
6.8
2.4
12.5
5.5
1.5
No previous results on the amino acid content of Arctopus species exist. From the results
presented here, it is evident that while there is considerable variation between the relative
amounts of the 19 amino acids identified from A. echinatus and A. dregei (A. monacanthus
was not analysed), one of the two extracts of the former showed high levels of asparagine
and proline.
In summary, three phenolic acids and three diterpenes have been successfully isolated and
characterised from A. dregei, A. echinatus and A. monacanthus for the first time in this
study. The phenolic acids exhibit a wide range of biological activities including antioxidant,
antiinflammatory, antimutagenic and antimicrobial properties (Olivier et al., 2008). TLC and
HPLC studies also revealed the presence of labdane, trachylobane and kaurene type
diterpenoids as well as triterpene saponins, and the absence of alkaloids. A total of 19 amino
acids have been identified in A. dregei and A. echinatus for the first time. The presence of
GABA (-aminobutyric acid) and especially the hypotensive diterpenes, ent-trachyloban-19oic acid and ent-kaur-16-en-19-oic acid, are noteworthy, as compounds in Arctopus extracts
have been reported to bind effectively at the GABAA-benzodiazepine receptor complex
which is involved in sedation, epilepsy and convulsions (Stafford et al., 2005; Paragraphs
4.2.3 and 6.2.3).
141
Chapter 5
Chemistry

As with Agathosma betulina, the medicinal value of A. afra is partly due to the volatile
constituents contained in the aromatic oil in the leaves. The compositions of the oils are
highly variable, depending on the geographical origin of the plants, genetic variation and
method of extraction. Liu et al. (2009) reviewed the phytochemistry of A. afra and reported
131 volatile components identief from the essential oil, including artemisyl acetate, camphor,
borneol, -pinene, artemisia ketone, 1,8-cineol (or eucalyptol), -thujone and -thujone, with
the latter four monoterpenes being the most abundant (Viljoen et al., 2006; see Fig 5.11).
Other classes of non-volatile secondary metabolites reported include sesquiterpenoids and
their lactone counterparts, guaianolides, glaucolides, triterpenes, long chain alkanes,
coumarins, organic acids, glycosides and flavonoids (Jakupovic et al., 1988; Liu et al., 2009,
and references therein; Gakuba, 2009; see Fig 5.11).
A. afra samples from seven different localities were screened qualitatively, where the
Liebermann-Burchard test indicated the presence of steroids, terpenoids and bitter
principles, with positive results for the saponin test as well [an essential oil investigation was
not done, as this topic has been thoroughly investigated and reviewed (Viljoen et al., 2006;
Liu et al., 2009)]. High yields were obtained for APS and polar extracts (Table 5.11), with
lower levels of non-polar compounds. The absence of alkaloids was notable in both the
qualitative colour tests and from TLC of the alkaloid extracts.
Table 5.11: Extract yields for seven Artemisia afra samples from different locations
Artemisia afra
samples
Sample
number
A. afra

APS
Polar
Total water
extractable
Non-polar
Aaf comm L
3.7
12.3
16.0
5.6
A. afra
Aaf 34 L
2.9
12.9
15.8
5.8
A. afra
Aaf 35a L
2.7
17.5
20.3
6.3
A. afra
Aaf 35b L
2.9
11.9
14.8
7.2
A. afra
Aaf 38 L
3.1
12.2
15.3
4.5
A. afra
Aaf 42 L
3.2
16.7
19.8
5.9
A. afra
Aaf 59 L
2.4
22.9
25.3
5.9
According to literature, 12 different flavonoids were isolated from A. afra, some of them
aglycones (Gakuba, 2009; Liu et al., 2009), as well as four different triterpenes (Liu et al.,
2009). The yellow (flavonoid) and blue (triterpene) zones on the polar extract TLC (Fig. 5.10,
left) are consequently not surprising. However, it is evident that the composition of wild herbs
from harsher climates (Aaf 42 and 59, containing higher amounts of the more polar
142
Chapter 5
Chemistry
glycosylated compounds) vary markedly from cultivated garden plants (AAf commercial, 34,
35, 38). Also, in agreement with literature, large amounts of sesquiterpene lactones with
medium to low polarity are evident from the CHCl3:MeOH (1:1) extracts (Fig. 5.10, right).
Gakuba (2009) isolated several sesquiterpene lactones (including taurin, artesin, maritimin,
artemin, and three santolinifolide derivatives), a coumarin (scopoletin), and two
acetophenones (4-hydroxyacetophenone and 2,4-dihydroxy-6-methoxyacetophenone) from
two different chemotypes of A. afra (Fig. 5.11). It was indicated that the acetophenones
(yellow zone on TLC after visualisation with acidic anisaldehyde or vanillin) are the least
polar, followed by artesin, then artemin (both indicated with blue zones on TLC) and lastly
scopoletin (a yellow-green zone) as the most polar of the four compounds. These four
compounds were indicated as the main medium polarity substances in the two chemotypes
studied (Gakuba, 2009). The presence of coumarins are typically detected on TLC through
their characteristic fluorescence quenching of UV-254 nm, and simple coumarins such as
scopoletin exhibits intense blue fluorescence at UV-365 nm (Wagner & Bladt, 2001). From
the TLC results, three major sesquiterpene lactones are visible, with little variation
[Fig. 5.10(b)], as well as several coumarins [as green zones, see Fig. 5.10(a)]. The extreme
bitterness of the aqueous extracts (bitterness value 7 20084 000, see Paragraph 5.3) is
thus not questionable, as sesquiterpene lactones, some flavonoids and triterpenoids are
known to be bitter (Van Wyk & Wink, 2004).
Fig. 5.10: TLC of leaves of seven Artemisia afra plants: (a) polar leaf extracts developed
in CHCl3:MeOH:H2O:CH3COOH (60:30:8:6), visualised with ethanolic
vanillin/H2SO4; (b) non-polar extracts developed in diethyl ether:hexane (2:3),
visualised with ethanolic vanillin/H2SO4
143
Chapter 5
Chemistry
Monoterpenes:
Of the 131 volatile components in A. afra essential oil (Liu et al., 2009), these are the four
major monoterpenes (Viljoen et al., 2006).
O
1,8-Cineole
Artemisia ketone
/-Thujone
Sesquiterpenes:
22 Non-polar sesquiterpenes, such as germacrene D, were identified in the essential oil (Liu
et al., 2009), while medium polar sesquiterpene lactones (including guaianolides, glaucolides
and eudesmanolides), such as artesin and artemin, are extracted by means of MeOH or
dichloromethane (Gakuba, 2009).
OH
OH
OAc
OAc
HO
Germacrene D
OH
1-Hydroxyafraglaucolide
Coumarins:
Artesin
Artemin
Acetophenones:
HO
HO
Scopoletin
OH
2,4-Dihydroxy-6- methoxyacetophenone
Flavonoid aglycones:
Mostly flavones (e.g. apigenin, luteolin and
diosmetin*) and flavonols (e.g. kaempferol*)
(Liu et al., 2009)
Triterpenes:
OH
HO
OH
Apigenin
Friedelin
Fig. 5.11: Six classes of compounds from Artemisia afra. All the compounds known to date
from A. afra have been listed by Liu et al. (2009) and Gakuba (2009);
#
= identified by Gakuba, 2009; * = as in Agathosma species, see Fig 5.1)
144
Chapter 5
Chemistry
Polar extracts of A. afra seem to be rich in amino acids, as is seen here for the first time.
Variation is slight, and high levels of asparagine, aspartic acid and proline were detected.
Valine, phenylalanine, alanine (three essential amino acids) and allo-isoleusine are also
present in noteable amounts, while two sporadical instances of high concentrations in
glutamine and glutamic acid were evident. The presence of -aminobutyric acid (GABA) is
important in the light of the sedative properties ascribed to the species (Stafford et al., 2005),
but the levels were quite low. It should be noted however that small volatile terpenes (such
as the thujones) may also cross the blood brain barrier where they act as non-competitive
blockers of the GABA gated chloride channel (Sirisoma et al., 2001) contributing to these
sedative effects.
Table 5.12: Amino acid content of Artemisia afra leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
R.T. (s)
54.9
85.5
186.1
66.4
101.5
75.7
111.7
145.3
335
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
102.0
99.3
290.2
274.1
71.8

AAf
AAf
AAf
com
AAf 34
35a
35b
72.4
38.0
59.2
62.2
39.0
15.8
39.5
27.7
3.6
3.3
8.6
3.5
1.2
0.7
2.3
1.8
3.3
0.7
1.3
2.2
1.7
2.5
4.1
120.2
347.8
921.3
60.2
495.0
279.4
290.9
518.2
76.4
222.3
14.7
13.5
31.5
51.1
2.6
8.8
10.5
16.3
66.7
9.1
7.7
13.3
10.6
17.8
51.1
16.7
4.6
10.9
10.7
9.4
13.0
AAf
42
36.7
33.5
3.6
0.9
2.2
2.9
185.6
48.8
4.6
41.4
21.0
8.3
11.4
26.2
18.2
13.6
15.0
18.6
12.2
9.9
41.5
466.2
9.5
31.4
176.4
9.8
75.1
178.9
9.2
36.9
79.7
9.8
32.7
147.6
9.4
31.5
99.1
9.2
11.1
305.9
63.0
34.0
17.3
29.8
44.8
27.1
12.5
17.5
13.7
18.5
123.0
52.1
25.4
22.5
104.9
66.8
24.4
15.0
18.3
44.0
55.8
14.5
18.8
15.0
28.5
36.2
30.0
20.5
19.3
52.4
1.9
102.6
1.7
13.6
12.3
22.8
AAf
38
45.8
17.4
3.3
1.5
4.2
2.3
116.4
444.1
AAf
59
31.4
9.5
2.4
0.4
1.6
26.4
5.1
8.4
1.2
10.6
2.9
18.8
10.4
7.1
A. afra is used traditionally for similar indications as Agathosma betulina (Paragraphs 4.2.1
and 4.2.4; Appendix 2), and it is interesting to note the chemical similarity (i.e. monoterpenes
such as 1,8-cineole, limonene and linalool, as well as flavonoid aglycones such as
kaempferol and diosmetin), suggesting the same biological activities (Paragraphs 6.2.1 and
6.2.4). Sesquiterpene lactones, some flavonoids and triterpenes are known to be bitter, and
are probably responsible for the extremely bitter taste of A. afra (Appendix 2; Paragraph 5.3)
145
Chapter 5
Chemistry
resulting in the amarum effect upon ingestion (Paragraph 6.1.3). While high levels of amino
acids may contribute to the nutritional value of A. afra, they may also aid in increasing the
solubility of the great variety of medium polar compounds in the cell medium, and especially
also when A. afra is prepared with sugar and used as a syrup (see ionic fluids, Paragraph
5.4).

Many of the uses of B. maughamii correspond to those of B. aegyptiaca, possibly due to
similar active principles [such as saponins and steroidal glycosides (Yadov & Panghal,
2010); see Fig. 5.13] in both species (Van Wyk et al., 2009). From the qualitative colour tests
and extract yields (Table 5.13), it was consequently found that both leaf and bark extracts
contain steroids, terpenoids and saponins, and that polar compounds occur in higher yields.
No alkaloids were found in the samples presented here, contradictory to B. aegyptiaca,
where two tyramine alkaloids were isolated from the stem-bark (Sarker et al., 2000).
However, the phytochemical composition of B. maughamii seems very variable (Fig. 5.12)
warranting a further investigation involving plant material from a greater geographical
distribution.
From Fig. 5.12, it is evident that the bark mainly contain saponin triterpenoids (yellow-green
zones), supposedly steroidal glycosides derived from diosgenin and yamogenin as isolated
from Balanites species (Dictionary of Natural Products, 1996; Yadov & Panghal, 2010). The
leaves contain flavonoids [yellow zones; six flavonoids have been identified from
B. aegyptiaca leaves (Maksoud & El-Hadidi, 1988)] and other terpenes (blue/purple zones)
(Fig. 5.12).
Fig. 5.12: TLC of Balanites maughamii: polar leaf (L) and bark (B) extracts developed in
n-BuOH:CH3COOH:H2O (4:1:2), visualised with ethanolic vanillin/H2SO4;
146
Chapter 5
Chemistry
Triterpenes:
Saponins
Sapogenins
H
H
H
H
RO
H
H
HO
Balanitins 17: yamogenin glycosides

(R = branched tri- or tetraglycosides)
Diosgenin
Hydroxy cholestenones
O
OH
HO
Cryptogenin
Phenolic compounds:
Flavonoids
RO
Organic acids
OH
O
O
OH
OR
OH
OH
HO
OH
HO
O
Quercitin 3,7-diglucoside: R = glucoside
Vanillic acid
Syringic acid
Furanocoumarins
O
HO
Marmesin
Bergapten
Fig. 5.13: Six classes of compounds from Balanites aegytiaca [Yadov & Panghal (2010)
reviewed all compounds known from this species], suggested to be similar to
those from B. maughamii (Van Wyk et al., 2009)
147
Chapter 5
Chemistry
Table 5.13: Extract yields of six Balanites maughamii leaf and bark samples
Balanites maughamii
samples
Sample
number
APS
Polar
B. maughamii
BM 68 L
7.9
8.8
16.7
3.1
B. maughamii
BM 68 B
0.9
5.3
6.1
1.8
B. maughamii
BM 69 L
4.1
7.8
11.9
4.1
B. maughamii
BM 69 B
1.3
6.4
7.7
2.5
B. maughamii
BM 70 L
8.4
8.2
16.6
1.9
B. maughamii
BM 70 B
2.9
13.2
16.1
2.8
Total water
extractable
Non-polar
Table 5.14: Amino acid content of Balanites maughamii leaf and bark extracts
Amino acid
R.T. (s)
Lit*
L

BM 68 BM 68 BM 69 BM 69 BM 70
L
B
L
B
L
Alanine
54.9
18.0
52.3
6.6
56.7
14.4
40.6
Allo-isoleucine
85.5
40.5
8.4
25.5
17.5
40.1
-Aminoadipic acid
186.1
3.0
2.9
2.9
3.7
-Aminobutyric acid
66.4
0.4
0.6
0.4
-Aminobutyric acid
101.4
2.4
0.4
17.0
0.4
75.7
3.6
1.6
3.6
4.0
4.1
Arginine
42.0
nd
nd
nd
nd
nd
Asparagine
111.7
53.5
29.8
269.4
38.1
Aspartic acid
145.3
78.6
19.5
6.4
20.5
39.8
56.7
Cysteine
335
7.9
4.8
108.0
Glutamic acid
167.3
61.2
3.2
t
14.0
84.4
Glutamine
205.1
30.1
5.2
Glycine
60.5
96.5
14.0
5.4
1.2
2.7
Histidine
257
28.3
11.4
10.0
12.5
10.9
Isoleucine
87.3
35.0
20.2
3.2
7.8
7.2
22.4
Leucine
84.1
62.3
Lysine
246
45.1
17.8
10.4
19.2
12.3
11.5
Methionine
147.2
7.3
3.0
Ornithine
230.1
10.0
8.9
9.2
9.8
9.7
Phenylalanine
167.7
48.0
23.6
9.1
19.7
12.0
18.0
109.3
Proline
105.6
18.5
28.8
72.9
13.7
32.6
Sarcosine
56.5
Serine
101.4
20.1
23.3
4.0
18.0
9.3
13.1
Threonine
99.3
28.8
21.8
1.1
3.0
3.3
12.9
Tryptophan
290.2
15.8
12.0
15.5
13.1
12.3
Tyrosine
274.1
31.6
21.5
6.5
14.5
10.1
15.7
Valine
71.8
40.7
28.7
5.1
14.8
12.3
32.9
* Amino acid content in B. aegyptiaca leaves as reported by Kubmarawa et al. (2008)
nd = not determined
BM 70
B
158.0
33.8
5.9
0.8
9.3
5.0
nd
308.1
9.4
61.4
18.5
10.0
12.5
33.5
29.2
21.1
2.6
9.7
32.2
99.1
26.7
14.2
29.3
51.7
148
Chapter 5
Chemistry
The amino acid content for B. maughamii is reported here for the first time, and compared
well with that reported for the leaves of B. aegytiaca, used as a vegetable in Nigeria
(Kubmarawa et al., 2008), indicating its nutritional value. In general, it seems that
B. maughamii contains high levels of amino acids with that of the leaves being higher than
that for the bark, which is to be expected. Of the 26 amino acids listed (Table 5.14), the
highest concentrations were found for alanine, aspartic acid and proline. Lysine,
phenylalanine, valine (three essential amino acids), serine, tyrosine, aspartic acid and
alanine were present in all the samples tested.
From the TLC analysis, the phytochemical composition of B. maughamii presents a large
diversity of compounds which are as yet unidentified. It also shows considerable variation in
the presence of compounds and classes of compounds warranting a wide geographical
variation study. Such a study should be extended into tropical Africa, as this species is
limited to extreme eastern parts of KwaZulu-Natal in South Africa (see Paragraph 4.2.5). As
with B. aegytiaca, the high levels of saponins in B. maughamii may afford its insecticidal
properties, and its amino acid content contributes to its nutritional value as fodder plant.

The four Dicoma species included here (D. anomala, D. capensis, S. schinzii and D. zeyheri)
are all used as traditional medicines in southern Africa. Their main constituents are bitter
sesquiterpene
lactones,
including
eudesmanolides,
germacronolides,
melampolides,
guaianolides and pseudoguaianolides (Bohlman & Le Van, 1978; Bohlmann et al., 1982;
Zdero & Bohlmann, 1989; Tselanyane 2007; Van der Merwe, 2008; see Fig. 5.15).
Examples of sesquiterpenes identified from D. anomala and D. zeyheri include -humulene,
-farnesine, germacrene D (as in Artemisia afra, see Fig. 5.11), albicolide, urospermol A and
-hydroxy dehydrozaluzanin C (Bohlmann & Le Van 1978; Bohlmann et al., 1982; Van der
Merwe, 2008). The presence of melampolides, germacranolides and eudesmanolides has
been reported in D. capensis (Zdero & Bohlmann, 1990), while isobutyrates with germacrane
skeletons were reported for D. schinzii (Van der Merwe, 2008). Dehydrobrachylaenolide and
a guaianolide dimer are sequiterpenes recently isolated from D. anomala (Van der Merwe,
2008). Furthermore, lupenone and lupeol (triterpenes), stigmasterol, -sitosterol and several
taraxasterols (phytosterols) have been reported from D. anomala (Bohlman & Le Van, 1978;
Bohlmann et al. 1982; Fig 5.15). Several acetylenic (cyclic and acyclic) compounds (e.g.
phytol) have been identified in D. zeyheri and D. anomala (Bohlman & Le Van, 1978;
Bohlmann et al., 1982; Fig. 5.15), and phenolic compounds such as caffeic acid (Bohlman &
Le Van, 1978; see Arctopus, Fig. 5.7) as well as cirsimaritin and scutellarein (flavonoids;
Zdero and Bohlmann, 1990) have been identified from D. anomala. Other Dicoma species
are known to contain kaempferol, quercetin or apigenin glycosides and aglycones [Hutchings
149
Chapter 5
Chemistry
et al. (1996); see Agathosma betulina (Fig. 5.1), Artemisia afra (Fig. 5.11) and Balanites
maughamii (Fig. 5.13) for structures].
From the qualitative colour tests and TLC analysis, the presence of steroids/terpenoids/bitter
principles, saponins and flavonoids were evident, but variable, in all four Dicoma species.
The qualitative tests (colour tests and TLC) for alkaloids were negative. From Table 5.15, it
is clear that the four Dicoma species contain high levels of alcohol precipitable solids (APS)
and polar compounds, more than in the non-polar extracts.
Table 5.15: Extraction yields from four Dicoma species
Dicoma
species
Sample
number
D. anomala
DA 4032a R
17.7
8.5
26.2
2.5
D. anomala
DA 4032a S
2.2
6.2
8.3
2.6
D. anomala
DA 4032a L
3.4
5.7
9.1
8.9
D. anomala
DA 4032b S
2.5
11.0
13.4
3.1
D. anomala
DA 4032c L
4.7
8.8
13.5
8.0
D. anomala
DA 4032c S
1.3
8.0
9.3
2.2
D. capensis
DC 58a L
4.4
7.4
11.8
6.8
D. capensis
DC 58b L
2.0
7.1
9.1
7.3
D. capensis
DC 58c L
2.7
9.2
11.9
5.9
D. capensis
DC 4035 L
3.3
6.3
9.6
7.2
D. capensis
DC 127 L
1.2
5.1
6.3
9.9
D. capensis
DC 127 S
0.7
6.1
6.8
5.0
D. capensis
DC KL L
2.5
8.1
10.6
7.1
D. schinzii
DS 4017b L
2.6
ns
--
4.8
D. schinzii
DS 4017b S
2.2
5.3
7.4
3.1
D. schinzii
DS 4017c L
1.6
5.7
7.3
4.5
D. schinzii
DS 4017c S
5.9
11.6
17.5
3.6
D. schinzii
DS 4017d L
2.9
7.2
10.1
3.7
D. schinzii
DS 4017d S
2.8
11.2
14.0
2.4
D. schinzii
DS K L
3.3
4.3
7.6
2.6
D. zeyheri
DZ 4046 L
3.0
4.9
7.9
4.4
D. zeyheri
DZ 4046 S
5.3
6.3
11.7
8.2
D. zeyheri
DZ 47 L
3.6
13.3
16.9
5.4
D. zeyheri
DZ 36 L
2.7
1.9
4.6
3.6
D. zeyheri
DZ 36 R
16.4
7.5
23.9
5.3
APS
Polar
Total water
extractable
Non-polar
ns = no sample
150
Chapter 5
Chemistry
TLC analysis of the polar extracts [Fig. 5.14 (a)] shows that there is at least one heavily
glycosylated compound (possibly a saponin) with high polarity, based on the charring in acid
and streaking nature of the grey zone. Several different flavonoids (yellow zones) and
medium polarity terpenoids (blue-purple) zones can be seen, with great variation
(geographically and amongst the species). The glycosylation of these polar terpenes and
flavonoids is confirmed by TLC for sugars, where compounds are presented in a similar
pattern as in [Fig. 5.14 (a)], and show charring with chromic acid. At least ten non-polar
terpenes (probably sesquiterpene lactones) can be recognised from the non-polar extracts
[Fig. 5.14 (b)], with slight variation in concentration.
Fig. 5.14: TLC of Dicoma capensis (DC), D. schinzii (DS), D. zeyheri (DZ) and D. anomala
(DA) (a) polar extracts developed in n-BuOH:CH3COOH:H2O (4:1:2); (b) nonpolar extracts developed in diethyl ether:hexane (2:3); both plates visualised with
ethanolic vanillin/H2SO4
151
Chapter 5
Chemistry
Sesquiterpene lactones:
Eudesmanolides
Guaianolides
H
Germacronolides
OH
OH
HO
O
O
3-Hydroxy-1,4(15),11(13)#
eudesmatrien-12,6-olide
OH
HO
9-Hydroxydehydrozaluzanin C*
14-Acetoxydicomanolide*
Triterpenes:
H
H
H
H
HO
H
H
Lupeol*
HO
Stigmasterol*
Unsaturated carbon chains:

OH
Phytol*
OR
C
O
Allenic acid and its ester*
R = H or Me
Fig. 5.15: Four classes of compounds from Dicoma anomala and D. capensis (all known
compounds identified from D. anomala, D. capensis, D. schinzii and D. zeyheri
were reported by Bohlman & Le Van, 1978; Bohlmann et al., 1982; Zdero &
Bohlmann, 1989; Tselanyane 2007; Van der Merwe, 2008); caffeic acid (see
Arctopus, Fig. 5.7) has also been identified from D. anomala (Bohlman & Le Van,
1978); # = identified from D. capensis; * = identified from D. anomala.
152
Chapter 5
Chemistry
Chemistry
Table 5.16(a): Amino acid content of Dicoma anomala and D. capensis root, stem and leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
R.T.
(s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8
t = trace amounts (<0.2 mg/g)
D. anomala
DA
DA
4032aR 4032aS
25.7
6.6
8.7
7.7
8.4
DA
4032bL
6.7
DA
4032bS
1.0
8.9
T
10.1
5.0
2.3
27.7
41.3
4.7
4.6
117.7
2.1
14.8
25.0
8.1
15.7
1.5
1.7
1.5
2.5
5.3
13.2
87.5
1.6
11.7
3.6
t
3.5
1.6
11.6
8.1
389.3
9.1
531.4
124.2
18.8
16.8
9.5
16.5
10.1
2.3
13.1
7.2
5.1
0.8

D. capensis
DA
DA
DC
DC
4032cL 4032cS 58aL
58bL
0.4
1.1
21.3
8.3
12.2
12.8
20.6
10.9
4.2
3.0
0.3
0.5
0.5
0.4
T
t
5.7
0.8
6.7
3.0
3.2
3.1
5.4
69.0
4.3
11.6
2.3
0.4
2.9
4.3
315.4
68.0
DC
127L
1.4
4.7
1.8
DC
127S
8.1
6.0
DC KL
L
10.8
6.4
0.6
0.4
5.8
19.6
0.9
3.7
4.5
12.7
2.6
0.7
10.6
7.9
12.6
16.8
11.0
3.0
10.8
13.7
9.3
5.2
10.2
9.8
12.9
9.7
10.2
9.7
9.8
6.6
2.1
3.8
11.1
3.2
4.3
10.4
6.9
80.9
8.2
278.4
9.3
434.9
5.1
9.1
521.3
10.1
16.5
95.5
5.0
7.9
190.1
10.1
10.9
54.9
5.0
6.5
117.3
7.0
351.4
5.1
10.5
835.8
0.6
11.6
5.4
2.6
1.8
T
12.8
6.4
20.3
12.8
5.7
10.5
12.4
6.9
22.7
13.6
15.0
23.2
2.0
0.5
12.0
7.2
10.4
3.4
1.0
12.8
8.1
8.7
1.1
DC
4035L
15.5
10.4
5.5
7.2
625.8
4.8
10.6
3.4
5.1
1.5
1.1
2.2
0.7
12.1
5.0
1.3
12.3
5.1
3.6
2.8
3.9
14.6
11.8
7.9
153
Chapter 5
Chemistry
Chemistry
Table 5.16 (b): Amino acid content of D. schinzii and D. zeyheri root, stem and leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT
(s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8
D. schinzii
DS
DS
4017bS 4017cL
4.6
1.8
6.3
5.3
2.4
0.3
1.6
0.1
32.3
15.4
9.3
6.1
t
0.8
10.1
1.9
DS
4017cS
3.2
4.0
t
2.3
2.7
1.1
2.7
DS
4017dL
1.6
9.7
DS K
L
9.1
6.7
2.4
T
5.5
3.3
64.1
73.9
DS
4046L
8.4
7.3
5.4
2.4
10.1
2.0
4.9
2.6
2.5
9.9
2.8
4.9
9.4
8.3
407.7
6.9
2.1
5.2
DS
4017dS
2.1
8.5
0.3
7.0
3.9
0.4
1.7
2.0
9.7
9.0
1.4
2.4
8.6
9.8
7.4
107.4
6.5
249.8
4.4
5.9
16.8
0.5
0.6
1.0
12.6
5.9
3.6
11.7
5.5
2.3
10.5
4.7
2.2
1.9
3.2
t
13.2
6.7
3.2
t
0.7
3.0
14.3
10.9
DS
4046S
19.5
5.6
7.4
t
0.8
3.1
76.4
51.2
D. zeyheri
DZ
DZ
47 L
36 L
22.2
t
10.1
4.1
2.6
0.5
1.3
t
3.9
1.5
71.7
4.9
0.3
10.2
2.8
19.6
7.7
0.6
10.8
4.3
31.6
160.2
2.9
13.7
5.6
9.7
9.7
10.1
4.9
6.9
563.7
9.7
7.6
15.7
8.2
399.3
0.5
1.9
11.7
6.0
6.0
11.7
5.2
3.8
3.8
2.0
12.0
6.7
6.7
DZ 36
R
17.2
5.2
0.3
1.0
3.4
254.3
37.0
9.7
13.9
1.3
2.3
9.7
0.8
12.3
5.1
3.8
11.0
4.8
8.1
360.5
10.2
8.6
749.5
5.0
6.5
8.8
5.4
9.2
273.9
9.1
4.6
12.5
8.8
4.3
21.8
10.4
17.5
11.7
20.7
12.0
4.6
14.1
8.4
6.1
16.0
5.2
1.5
154
Chapter 5
Chemistry
With the amino acid analyses (TLC and HR-GC-MS-TOF), great variation was observed
geographically and amongst species. As with most of the other tonic plants, proline was
present as the major amino acid in all the Dicoma samples. The essential amino acids
valine, tryptophan, lysine, phenylalanine and iso-leusine were present in almost all the
samples analysed [Tables 5.16 (a, b)].
Even though a great diversity of compounds are evident from the TLC analyses, further
research is required to establish the identities and variation patterns of these compounds.
No research has yet been focussed on phenolic compounds or polar triterpenes of
D. capensis, as most compounds known from South African medicinally used Dicoma
species have been identified from D. anomala. Considerable variation is observed (TLC and
HR-GC-MS amino acid analyses) among species and provenances, so that more detailed
studies (including the use of metabolomics) may yield interesting results.
5.2.7 Harpagophytum species

H. procumbens is known to contain several classes of compounds, with iridoid glycosides
being of main medicinal interest (ca. 3% of the dry weight of the root; Fig. 5.16) as these
iridoids are considered to contribute to most of the biological activities ascribe to this
species. These compounds include harpagoside (content may vary between 0.52%), as
well as smaller amounts of harpagide and procumbide, with their respective cinnamic or
coumaric acid esters [Qi et al., 2006, see Fig. 5.17(a)]. Harpagide seems to be a breakdown
product of harpagoside (Van Wyk & Wink, 2004). Other compounds in the root include
phenolic glycosides [i.e. acetoside, isoacetoside and derivatives (Munkombwe, 2003)],
phenolic acids [i.e. caffeic (also from Arctopus Fig. 5.7, and Dicoma), chlorogenic and
cinnamic acid (Fig. 5.16)], flavonoids [i.e. kaempferol (also from Agathosma Fig. 5.1, and
Artemisia) and luteolin glycosides (Fig. 5.16)], phytosterols [stigmasterol (see Dicoma,
Fig. 5.15) and -sitosterol (also identified from Aloe, Hypoxis and Sutherlandia], triterpenoids
[i.e. ursolic and oleanolic acid (Fig.5.16) with their derivatives], alkaloids [i.e. quinine
(Fig. 3.5) and harpagoquinone] and sugars [i.e. the monosaccharides glucose, galactose,
fructose, myo-inositol, the oligosaccharides sucrose, raffinose and stachylose, the main
sugar component constituting as much as 46% of the dried root material (Ziller & Franz,
1979)]. Furthermore, Clarkson et al. (2003, 2006) reported at least 11 compounds from the
non-polar (petroleum ether) fraction of H. procumbens, of which several were totarane (e.g.
maytenoquinone), abietane (e.g. ferruginol) and chinane-type diterpenes [see Fig.5.17(b)
and Fig 5.16]. Several amino acids, carotenoids (Ludwig-Mller et al., 2008), n-alkanes, fats
and waxes (Wichtl & Bisset, 2000) have also been reported.
155
Chapter 5
Chemistry
Terpenoids:
Iridoids:
HO
HO
HO
OH
OH
OH
O
H
O
O
HO
H
Glucosyl
Procumbide
Glucosyl
Glucosyl
Harpagide
R'
Harpagoside: R = H; R = H
8-(4-Coumaroyl) harpagide: R = OH; R = H
8-Feruloylharpagide: R = OH; R = OMe
Non-polar diterpenes:
Triterpenes:
R'
O
OH
OH
H
O
H
HO
OH
Maytenoquinone
Ferruginol
Ursolic acid:
R = Me; R = H
Oleanolic acid: R = H; R = Me
Phenolic compounds:
Acetosides:
R''O
R'O
O
O
HO
HO
OH Acetoside:
R = H; R = caffeyol; R = H
Isoacetoside: R = H; R = H; R = caffeoyl
2-O-Acetylacetoside: R = acetyl; R = R = H
OH 6-O-Acetylacetoside: R = R = H; R = acetyl
OR
OH
Flavonoids:
Phenolic acids:
OH
HO
HO
OH
COOH
OH
HO
O
OH
Luteolin
OH
Cinnamic acid
OH
OH
Chlorogenic acid
Fig. 5.16: Six classes of compounds (with examples of the main compounds in each class)
from Harpagophytum procumbens as reviewed by Kikuchi et al. (1983), Bradley
(1992), Wichtl & Bisset (2000) and Clarkson et al., (2006).
156
Chapter 5
Chemistry
According to the qualitative colour tests for 17 H. procumbens and three H. zeyheri samples,
steroids/terpenoids, saponins, phenolic compounds and alkaloids were present. These
results were confirmed through TLC analyses [Fig. 5.16(a, b and c)], where the alkaloids
were detected as white zones on a purple background. For quality control purposes, the
medicinally important iridoids may be selectively extracted with less polar solvents according
to standardised methods (MeOH:CHCl3, 1:1) and the extracts subjected to TLC or HPLC
analyses, upon which the iridoid yields are compared to international standards) (Fig. 5.15;
Pourrat et al., 1986). Furthermore, Ziller and Franz (1979) reported a ca. 6063% total water
extractable fraction, of which 8.83% was alcohol precipitable. These values compare well
with those obtained for H. procumbens as shown in Table 5.17.
Table 5.17: Extract yields for Harpagophytum species
APS
Polar
Total
water
extractable
Nonpolar
Alkaloids
HP 3807c R-3
3.5
44.0
47.4
3.2
0.100
H. procumbens
HP 3807d R-2
4.0
20.2
24.2
5.3
ns
H. procumbens
HP 3807e R-2
5.5
21.4
26.9
4.6
ns
H. procumbens
HP 3807f R-2
6.2
35.4
41.6
4.9
ns
H. procumbens
HP 3807g R-2
4.0
18.1
22.1
6.3
ns
H. procumbens
HP 3807i R-3
8.2
24.3
32.5
2.5
ns
H. procumbens
HP Ma R
4.5
45.9
50.4
3.7
0.100
H. procumbens
HP GOa R
3.7
39.3
43.0
2.7
0.059
H. procumbens
HP GOb R
4.6
26.2
30.8
5.0
0.057
H. proc. subsp. procumbens
HPP MVa R
5.6
19.8
25.4
4.7
ns
HPP MVb R-1
5.0
4.7
9.7
4.2
ns
HPP MVb R-2
3.8
39.3
43.1
5.0
ns
HPP MVc R-1
4.6
6.1
10.6
4.7
0.149
HPP MVc R-2
3.8
7.3
11.1
5.5
0.109
HPP MVc R-3
8.4
33.8
42.2
4.6
0.079
HPP Bf R-1
5.7
39.3
45.1
6.3
0.050
HPP Bl R-1
3.6
46.1
49.7
5.1
ns
H. zeyheri
HZ Wa R
4.1
22.0
26.1
5.2
0.085
H. zeyheri
HZ Wi R
2.0
25.0
27.0
4.1
ns
H. zeyheri
HZ Wj R
1.5
24.9
26.4
6.3
ns
Harpagophytum species
Sample
number
H. procumbens
ns = no sample
157
Chapter 5
Chemistry
Fig. 5.17: TLC of Harpagophytum roots: (a) medium polar extracts developed in
CHCl3:MeOH (6:3); (b) non-polar extracts developed in diethyl ether:hexane
(2:3); both plates (a) and (b) visualised with ethanolic vanillin/H2SO4; (c) alkaloid
extracts developed in CHCl3:cyclohexane:ethylamine (4:5:1), visualised with
acidified iodoplatinate spray reagent.
Even though 26 amino acids were identified in the polar extracts of the H. procumbens and
H. zeyheri, the presence and concentrations are variable and no amino acid occurs in high
levels throughout. However, five essential amino acids (lysine, phenylalanine, tryptophan,
tyrosine and valine), as well as glycine and proline were found in all the samples. LudwigMller et al. (2008) identified glycine, lysine, proline and valine in H. procumbens, which, to
some extent, confirms the results obtained here.
158
Chapter 5
Table 5.18:
Chemistry
Chemistry
Amino acid content of Harpagophytum procumbens and H. zeyheri root extracts

Amino acid
RT (s)
H. procumbens
3807a
3807d
R-3
R-2
3807e
R-2
3807
f R-2
3807i
R-3
3807g
R-2
MVa
R-3
Alanine
54.9
89.8
37.4
159.9
61.8
109.3
25.4
10.6
Allo-Isoleucine
-Aminoadipic
acid
-Aminobutyric
acid
-Aminobutyric
acid
-Aminoisobutyric
acid
85.5
8.1
5.6
6.9
6.1
6.4
3.4
6.2
186.1
46.7
60.0
30.9
105
66.4
0.9
0.4
101.4
6.6
3.1
2.7
4.8
75.7
4.6
3.7
Asparagine
111.7
90.8
7.1
Aspartic acid
145.3
272.4
21.2
335
9.7
Cysteine
167.3
61.7
9.0
Glutamine
205.1
379.9
3.0
2.8
1.3
31.4
8.9
6.7
5.7
MVc
R-2
5.1
MVc
R-3
Bf
R
Ma
R
35.8
42.0
46.9
53.5
31.3
25.8
4.7
5.9
6.0
5.9
5.0
5.2
56.7
35.7
122.7
32.7
21.4
1.8
3.9
2.1
4.9
1.3
3.9
Bl R
4.1
1.7
2.6
1.8
2.7
3.2
3.4
3.3
4.1
3.5
5.6
6.0
35.7
7.0
7.7
14.2
73.0
4.0
26.3
36.7
30.6
56.7
2.5
17.2
2.3
8.2
45.7
13.7
1.2
0.9
3.4
15.6
15.1
71.2
GOa
R
GOb R
12.2
45.4
52.8
63.1
3.2
60.5
4.6
0.7
2.1
257
15.0
11.3
11.8
10.9
Isoleucine
87.3
6.3
1.9
4.1
2.6
Leucine
84.1
2.8
3.7
3.7
12.3
93.1
32.1
151.0
Lysine
246
7.7
41.2
3.4
81.0
40.3
198.2
47.0
1.2
Methionine
146.5
Ornithine
230.1
10.3
Phenylalanine
167.7
9.6
Proline
105.6
13.2
10.9
2.7
4.6
11.6
2.8
0.2
0.6
2.3
0.3
0.3
2.1
0.7
1.2
10.0
10.4
11.9
10.7
10.1
10.6
10.4
18.4
10.9
11.4
10.3
1.6
2.2
2.9
1.8
2.0
2.4
2.5
2.8
3.0
3.2
3.5
1.4
1.6
0.2
4.5
12.2
H. zeyheri
Wa
Wj
R
R
9.0
Histidine
Serine
MVc
R-1
1.0
Glycine
Sarcosine
4.6
MVb
R-2
2.6
9.8
Glutamic acid
MVb
R-1
11.9
10.2
9.7
1.3
4.3
10.5
10.2
11.1
10.5
10.9
9.4
10.7
10.3
14.0
10.5
10.8
10.8
11
5.2
10.2
9.7
9.6
9.7
9.4
9.2
9.7
9.4
10
8.7
9.5
9.3
9.0
9.0
8.0
8.4
8.2
9.6
8.8
8.5
7.6
8.4
8.2
8.7
7.9
8.8
8.1
8.2
7.3
6.9
5.9
5.7
3.2
6.5
2.5
9.3
3.0
2.9
6.6
7.2
6.4
11.4
2.3
5.9
2.1
1.7
6.8
0.8
2.5
8.0
3.4
7.0
4.3
6.9
0.4
4.1
0.9
2.5
14.9
2.0
9.8
56.5
9.3
0.9
101.4
25.8
2.2
Threonine
99.3
25.3
0.8
1.5
Tryptophan
290.2
13.2
12.7
14.5
12.4
12.3
11.8
12.1
12.3
14.2
12.6
12.7
12.1
12.2
11.9
12.9
12.1
13.4
12.3
13
Tyrosine
274.1
10.1
5.9
7.2
6.7
6.3
5.5
6.0
5.0
8.1
6.3
5.5
5.4
5.9
5.4
6.3
5.1
6.0
5.5
5.3
71.8
1.8
3.0
3.9
4.3
4.6
1.7
3.2
1.0
3.5
1.5
1.1
1.0
4.4
4.0
1.0
4.1
4.2
1.0
1.2
Valine
6.0
2.8
159
Chapter 5
Chemistry
H. procumbens extracts contain a large diversity of chemical compounds of which the

identities are mostly known. Apart from the biological properties reported for the iridoids
(antiinflammatory and analgesic), the other classes of compounds also contribute to the wide
range activities of devils claw (Paragraph 6.2.7), i.e. the phenolic compounds are
antioxidants, and the non-polar diterpenes are antiplasmodial. Quinine is one of the most
bitter alkaloids known, i.e. it was used in this study as bitter standard against which
bitterness taste was measured (see Paragraph 5.3). Anecdotes for the bitter taste of
H. procumbens extracts (Paragraph 4.2.7; Appendix 2G) are thus not surprising, as the
iridoids are also known to be bitter. The resulting amarum effect on the digestive tract
possibly lead to its inclusion in bitter tonic mixtures such as Swedish bitters (Paragraphs
6.1.3.2 and 6.2.7). The high sugar content in the root may also play a valuable role in not
only masking the bitter taste resulting from the alkaloids, and possibly the flavonoids and
triterpenoids, but also aiding in the solubility of the compounds of medium polarity such as
the iridoids (see ionic fluids, Paragraph 5.4).

Six norlignans have been isolated from H. hemerocallidea (Pegel, 1973; Laporta et al., 2007;
Drewes et al., 2008; Fig. 5.18), of which hypoxoside (Marini Bettolo et al., 1982) is the main
constituent. The glucose moieties of hypoxoside may be removed by hydrolysis with glucosidase, or through a similar reaction in the gastro-intestinal system, yielding rooperol
(Drewes et al., 1984), known for its powerful antioxidant activity (Laporta et al., 2007; see
Chapter 6). High levels of phytosterols, such as -sitosterol (Fig. 5.18) and its glycone,
sitosterolin (in a 100:1 ratio), are also present (Pegel, 1973). Furthermore, monoterpenes
have been reported (Pegel, 1973; Wagner & Wiesenauer, 1995), but the identities of these
compounds are unknown. Van Staden (1981) identified the purine zeatin (Fig. 5.18), along
with its riboside and glucoside derivatives. In a seasonal variation study, Ncube et al. (2011)
reported that the phenolic compound and saponin content in hypoxis corms were the highest
in winter, while that of gallotannins and tannins were normally high, peaking in autumn. Low
levels of steroidal saponins were reported all year round. The phenolic compounds
(norlignans) and steroidal saponins are said to be the main active principles in the corm and
elevated levels of these compounds correspond with increased antibacterial and antifungal
activity (Ncube et al., 2011; see Chapter 6).
The qualitative colour tests and TLC (Fig. 5.19) confirmed the presence of steroids,
terpenoids and saponins in all five tubers of hypoxis obtained from the Farraday muti market.
No alkaloids were detected. From TLC, it is clear that norlignans produce different
colouration (dirty yellow) after visualisation with ethanolic vanillin/H2SO4 than flavonoids
160
Chapter 5
Chemistry
(yellow to orange) would [Fig. 5.19(a)]. The sterols and sterolins are visualised as bluepurple zones after visualisation with ethanolic vanillin/H2SO4 and quench UV-365 nm light
[Fig. 5.19(a) and (c)]. The presence of tannins is evident as orange zones on the origin
without visualisation [Fig. 5.19(b)]. High yields were obtained for APS (containing
gallotannins and tannins) and water extractable extracts (with sterols and norlignans)
(Table 5.19), typical of storage organs such as tubers. The geographical origin and season
of harvest is unknown for material bought on a muti market. The small variation in yields
(Table 5.19) and relative concentrations of constituents in the extracts as seen from TLC
(Fig 5.19) show that external factors have little influence on presence or absence of
compounds in the tuber.
Norlignans:
R1
R3
R
1
Hypoxoxside:
Dehydroxyhypoxoside:
Bis-dehydroxyhypoxoside:
Rooperol:
Dehydroxyrooperol:
Bis-dehydroxyrooperol
R
-OH
-H
-H
-OH
-H
-H
H
2
R
-O--D-glucopyranosyl
-OH
-OH
-OH
Sterols:
R
-OH
-OH
-H
-OH
-OH
-H
R
-OH
-OH
-OH
Purines:
N
N
H
H
H
HO
-Sitosterol
HO
N
NH
Zeatin
Fig. 5.18: Three classes of compounds from Hypoxis hemerocallidea, i.e. all known
norligans (Laporta et al., 2008), -sitosterol [together with its glycone sitosterolin
originally reported by Pegel (1984; 1997)], and zeatin [together with zeatin
riboside and zeatin glucoside, reported by Van Staden (1981)].
161
Chapter 5
Chemistry
Fig. 5.19: TLC of five polar corm extracts of Hypoxis hemerocallidea developed in
CHCl3:MeOH:H2O:CH3COOH (60:30:8:6), (a) visualised with ethanolic
vanillin/H2SO4; (b) before visualisation; (c) under UV-365 nm
Table 5.19: Extract yields for five Hypoxis hemerocallidea tubers
H. hemerocallidea
tubers
Sample
number
H. hemerocallidea

APS
Polar
Total water
extractable
Non-polar
HH 71a R
8.2
20.9
29.1
14.1
H. hemerocallidea
HH 71b R
6.4
13.5
19.9
9.8
H. hemerocallidea
HH 71c R
10.7
18.3
29.0
11.9
H. hemerocallidea
HH 71d R
9.1
20.0
29.1
14.2
H. hemerocallidea
HH 71e R
8.1
15.2
23.3
11.6
From TLC and HR-GC-MS-TOF (Table 5.20) it became clear that H. hemerocallidea corms
are not rich in amino acids. Again variation is slight, and of the 19 identified amino acids, 13
are present in all five corms extracted, of which five are essential amino acids (isoleucine,
lysine, phenylalanine, tryptophan, and valine). Although in very low levels, the presence of
GABA (-aminobutyric acid) is relevant especially in the light of the use of hypoxis for
treatment of convulsions (Chapter 4) and the fact that extracts show anti-convulsant activity
(Chapter 6).
Although hypoxis grows easily, it is difficult to propagate. Consequently, protecting this
popular traditional medicinal plant is proving to be a problem, as it is the corm that is mostly
used to make infusions and decoctions for treatment (Van Wyk et al., 2009). Fortunately the
major active principles (-sitosterol, sitosterolin, rooperol and hypoxoside) have been
162
Chapter 5
Chemistry
identified and can be synthesized or extracted from other sources, as an alternative for
extraction from H. hemerocallidea rootstock (Drewes et al., 1984; Pegel, 1997).
Table 5.20: Amino acid content of Hypoxis hemerocallidea corm extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
R.T. (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8

HH 71a HH 71b HH 71c HH 71d
4.5
6.0
3.1
6.1
6.2
5.3
4.6
5.7
1.5
0.4
1.5
0.7
1.5
1.1
0.7
2.6
HH 71e
13.1
6.8
t
3.4
0.9
0.8
1.4
2.3
0.6
6.1
1.3
2.3
3.7
3.6
10.3
1.9
10.4
1.6
9.3
1.6
9.8
1.7
2.3
10.0
9.8
8.9
9.4
10.4
9.3
8.9
3.1
4.6
8.6
2.8
4.1
7.8
1.6
9.0
7.2
4.8
9.7
4.6
3.9
t
12.2
6.3
3.3
3.0
2.9
3.0
12.3
5.5
2.2
11.2
5.0
1.8
11.6
5.9
2.6
4.6
t
12.8
7.5
4.5

Very little is known about the phytochemical components of M. heisteria apart from the fact
that they may be bitter, due to the fact that M. heisteria is known as an appetite stimulant
(Van Wyk & Gericke, 2000; Chapter 4). In this study, a M. heisteria aqueous extract was
found to exhibit a bitterness value of 1 68014 400 (Paragraph 5.16). This bitterness may be
caused by several triterpene saponins (acylated presenegenin type saponins; Fig. 5.20)
previously isolated from M. heisteria (Elbandy et al., 2002a; 2004). This type of saponin is
also present in other genera within Polygalaceae (Polygala, Carpolobia and Securidaca
species), so that acylated presenegenin type saponins seems to be useful chemotaxonomic
markers for the family (Lacaille-Dubois & Mitaine-Offer, 2005). Medicagenic acid type
saponins have also been isolated from M. ononidifolia (Elbandy et al., 2002b). No other
compounds are known from M. heisteria.
163
Chapter 5
Chemistry
Saponins:
O
OH
HO
OH
HO
HO
OR'
RO
OH
OH
HOOC
Api
HO
R1
OH
Ara
O
HO
HO
O
OH
Gal
OH
O
HO
OH
OH
HO
OH
Ara
O
O
OH OH
Xyl
R2
Rha
OH
OH
OH
HO
O
Gal
Xyl
HO O
Rha
OH
OH
HO
OH
OCH3
OCH3
2
S1
S
H3CO
OCH3
OCH3
OCH3
O
Presenegenin trans-3,4,5-trimethoxycinnamoyl glycoside 1:

Presenegenin cis-3,4,5-trimethoxycinnamoyl glycoside 2:
Presenegenin trans-3,4,5-trimethoxycinnamoyl glycoside 3:
Presenegenin cis-3,4,5-trimethoxycinnamoyl glycoside 4:
R = R ; R = S
1
1
R = R ; R = S
2
2
R = R ; R = S
2
1
R = R ; R = S
Fig. 5.20: Structures of all four known acylated presenegenin type saponins in polar
extracts of Muraltia heisteria (Elbandy et al., 2002).
Through the qualitative colour tests and TLC of the leaves and stems of two plants, the
presence of steroids, terpenoids, saponins and flavonoids were confirmed, but no alkaloids
were detected. Low levels of APS are observed for all the samples, and variation in polar
and non-polar extract yields (Table 5.21) is also reflected in TLC, when comparing both
presence and relative quantity of the mentioned classes of compounds, i.e. polar extracts
[Fig. 5.21(a,b)] and non-polar extracts [Fig. 5.21(c)]. Glycosylation of the polar compounds is
confirmed by charring of such zones with chromic acid. Many of the non-polar terpenes
seem to show bright blue zones under UV-254 nm [Fig. 5.21(d)].
164
Chapter 5
Chemistry
Table 5.21: Extract yields of Muraltia heisteria leaves and stems

M. heisteria
samples
Sample
number
M. heisteria
MH 4189 L
1.1
14.6
15.7
6.7
M. heisteria
MH 4189 S
0.4
6.5
7.0
5.2
M. heisteria
MH 4194 L
0.1
17.6
18.7
15.3
M. heisteria
MH 4194 S
0.8
9.5
10.2
9.3
APS
Polar
Total water
extractable
Non-polar
Fig. 5.21: TLC of Muraltia heisteria leaves and stems: (a) polar extracts developed in
CHCl3:MeOH:H2O:CH3COOH (60:30:8:6); (b) polar extracts developed in nBuOH:CH3COOH:H2O (4:1:2); (c) non-polar extracts developed in CHCl3:MeOH
(6:3) under UV-254; plates (a), (b) and (c) were visualised with ethanolic
vanillin/H2SO4
The amino acid content seems low and slightly variable. Six of the essential amino acids
(isoleucine, lysine, phenylalanine, threonine, tryptophan and valine), as well as GABA (aminobutyric acid) are among those always present.
From this study, it is clear that more phytochemical research is required in order to establish
constituent variation patterns within the species, but also to identify the phenolic and other
unknown compounds from the complex mixture shown in TLC analyses. Its value as
possible tonic plant may be found in the presence of saponins [normally bitter according to
Van Wyk & Wink (2004)] having an amarum effect, six essential amino acids enhancing its
nutritional value and the presence of GABA, an inhibitory neurotransmitter, which may have
165
Chapter 5
Chemistry
a sedative effect and thus relevant to the treatment of stress and anxiety (see Arctopus and
Sutherlandia, Paragraphs 6.2.3 and 6.2.10 respectively).
Table 5.22: Amino acid content of Muraltia heisteria leaf and stem extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
R.T. (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8

4189 L 4189 S 4194 L 4194 S
16.5
1.9
5.7
8.3
3.4
5.4
1.2
0.6
1.0
3.0
1.3
2.8
3.8
2.3
2.8
34.1
20.4
26.3
9.7
4.6
4.2
1.5
3.9
2.1
2.6
3.1
8.6
3.4
15.6
4.1
5.8
10.8
3.5
0.9
16.1
1.1
3.5
16.3
1.7
0.9
17.4
1.1
11.1
15.8
17.0
17.3
10.0
49.0
8.1
7.2
10.1
65.0
8.8
12.8
15.5
4.3
12.9
7.9
6.1
4.1
t
16.4
8.3
1.2
7.8
4.5
16.1
10.9
2.8
6.5
0.5
21.1
9.1
1.3

The major compounds in the polar extracts of S. frutescens are three cycloartane type
triterpenoid glycosides (Fig. 5.22), i.e. SU1 and SU2 (Van Wyk & Albrecht, 2008) [also
known as sutherlandiosides B and A respectively (Fu et al. 2008)], as well as
sutherlandioside C (a 3,24-dihydroxycycloartane-1,11-dione glucoside; Fu et al. (2008)].
Sutherlandioside D, a dihydroxycycloart-2-en-1-one glucoside also identified by Fu et al.
(2008), may be an acid hydrolysis derivative of SU1, which do not naturally occur in
Sutherlandia (see Appendix 5B, a publication on SU1 and SU2, which was accepted with
changes by Journal of Natural Products in August 2007, but did not get published due to
delay from the corresponding author). A fourth, more polar saponin (SU3) was later isolated
as main saponin from S. humilis (see Figures 5.22 and 5.23 (TLC of track SH 17a); Olivier et
al., 2009). While the presence of at least six flavonoids had been noted in the respective
Sutherlandia species (Moshe, 1998; D. Olivier, unpublished research), four flavonoids were
166
Chapter 5
Chemistry
isolated from S. frutescens and their identities determined as 3-hydroxy-3-methylglutaroylcontaining flavonol glycosides, i.e. sutherlandins A-D (Yang et al., 2010; Fig. 5.22). Other
compounds also identified from S. frutescens include high levels of free amino acids
(asparagine, proline, L-arginine, L-canavanine and -aminobutyric acid, see Table 3.4 for
structures), a cyclitol sugar (pinitol; Fig. 5.22), hexadecanoic acid, -sitosterol, sigmast-4-en3-one, at least three long chain fatty acids and high levels of polysaccharides (Van Wyk &
Albrecht, 2008). No alkaloids have been reported for Sutherlandia and were also not
detected in this study.
Table 5.23: Extract yields of Sutherlandia species to show geographic and chemical variation
Sutherlandia species
Sample
number
S. frutescens

APS
Polar
Total water extractable
SF 1g L
4.5
18.5
23.0
S. frutescens
SF 2a L
7.1
14.3
21.4
S. frutescens
SF 6b L
6.7
18.5
25.2
S. frutescens
SF 18c L
5.7
19.3
25.0
S. frutescens var. incana
SFI 3668b L
8.0
8.0
16.0
SFI 9c L
11.4
13.0
24.4
SFI 14a L
9.4
13.1
22.5
SFI 16b L
6.7
9.7
16.4
SFI 18a L
11.8
7.8
19.6
S. humilis
SH 25c L
11.1
19.2
30.3
S. microphylla
SMi Comm L
3.1
13.5
16.6
S. microphylla
SMi 3c L
7.9
20.5
28.4
S. microphylla
SMi 5e L
7.5
12.9
20.4
S. microphylla
SMi 7a L
9.4
14.7
24.1
S. montana
SMo 3800a L
8.5
14.3
22.8
S. speciosa
SS 4c L
12.8
13.8
26.6
S. tomentosa
ST 13a L
7.4
5.3
12.7
S. tomentosa
ST 3669b L
3.8
11.1
14.8
The ethnobotany of S. frutescens (Chapter 4) was treated in a broad sense, including all its
various inland forms (i.e. the typical, hairy, dwarf and high altitude forms of S. frutescens
subsp. frutescens, as well as S. frutescens subsp. speciosa and S. frutescens subsp.
microphylla) under S. frutescens, separated from S. tomentosa (Moshe, 1998; Van Wyk &
Albrecht, 2008). However, to make the geographical variation study simpler and to establish
whether there is chemical variation within the various forms, a narrow species concept was
followed using the names of the various forms (Phillips & Dyer, 1934; see Table 5.23).
167
Chapter 5
Chemistry
Saponins:
OH
OH
26
21
23
22
11
19
16
10
5
4
15
R = glucose
8
7
6
28
R = glucose
30
OH
HO
29
OR
OH
17
13
14
OR
27
12
25
24
20
18
HO
SU1 (Sutherlandioside B)
SU2 (Sutherlandioside A)
OR
OH
OR
OR
R = glucose
R = glucose
HO
Sutherlandioside C
OH
Flavonols:
SU3
OH
HO
OH
HO
O
OH
O
O
HOOC
HO
O
c
Sutherlandin A : R = OH
c
Sutherlandin C : R = H
OH
OH
O
HO
OH
HOOC
HO
OH
HO
HO HO
OH
OH
OH
Sutherlandin B : R = OH
c
Sutherlandin D : R = H
Sugars:
Sitosterols:
OH
OH
CH 3
OH
OH
OH
d
Pinitol
OH
-sitosterol
Stigmast-4-en-3-one
Fig. 5.22: Three classes of compounds from Sutherlandia frutescens including all four
known saponins, all four known flavonoids, pinitol and sterols. The triterpene
saponins and flavonoids are the main compounds in the polar extract. (a = Fu et
al., 2008; b = Olivier et al., 2008; c = Yang et al., 2010; d = Van Wyk & Albrecht,
2008.)
168
Chapter 5
Chemistry
Fig. 5.23: TLC showing triterpenoid variation in six Sutherlandia species, all developed in
CHCl3:MeOH:EtOAc (65:20:15), visualised with ethanolic vanillin/H2SO4
Positive results for some of the qualitative tests (i.e. those for steroids, terpenoids, bitter
principles, and saponins), together with TLC (Fig. 5.21) and HPLC (Appendix 3) confirmed
the presence of a great diversity of triterpenoids, where two of the three major blue zones in
TLC correspond with SU1 (Rf = 0.68) and SU2 (Rf = 0.75), and the third zone (Rf = 0.57)
may very well correspond with sutherlandioside C (as mentioned above). The TLC solvent
system is less polar than that used for the flavonoids, showing that the saponins are less
polar than the flavonoids, even though both classes of compounds are glycosylated. The
high yields of polar extracts obtained (Table 5.23) confirm that the saponins and flavonoids
are some of the main compounds of Sutherlandia. An attempt at isolation lead to the
identification of an as yet unpurified flavonoid roughly indicated to be a flavonol glycoside,
possibly a sutherlandin, as those identified by Yang et al. (2010). When determining
variation of these compounds among the various morphological forms of Sutherlandia, it
should be noted that, when the saponins are analyzed on HPLC, the triterpenoid photo diode
array (PDA) response factors are not a true reflection of the relative quantities of this class of
compounds (Appendix 3D, Table 3D-2), as is the case for flavonoids (Appendix 3D,
Table 3D-1). This is due to the variable unsaturation and/or conjugation in triterpene
backbone structures. From TLC and HPLC (Appendix 3D) variation studies, presence and
relative quantity of at least seven detected flavonoids seem variable amongst the species.
However, Aluva et al. (2010) showed the applicability of LC-MS and LC-UV/ELSD for
quantitative determination of the flavonoids and saponins in the aerial parts of S. frutescens.
169
Chapter 5
Table 5.24:
Chemistry
Chemistry
Amino acid content of Sutherlandia species leaf extracts
Amino acid
RT (s)

S. frutescens
var. incana
S. frutescens
S. humilis
S. microphylla
SF 1g
SH 1*
SMi 1*
SF 1-4*
SFI 1-4 L*
S. montana
SMo
SMo
PRE5a
1*
S. speciosa
SS
SS 4c
4724
SS
1*
S. tomentosa
ST
ST
13a
3669b
ST 1-7*
Alanine
54.9
148.8
0.8 - 10.7
0.4 - 6.3
0.2
6.5
1.9
30.7
5.1
4.9
25.2
0.7
Allo-isoleucine
85.5
4.1
nm
nm
nm
nm
15.4
nm
18.1
5.8
nm
28.0
6.7
Nm
-Aminoadipic acid
186.1
3.3
0 - 1.8
0.4 - 37.4
5.6
2.0
7.9
5.1
3.4
1.2
2.9
0.7 - 3.3
-Aminobutyric acid
66.4
6.9
0 - 0.2
0 - 8.9
1.2
0.1
3.2
2.4
1.0
-Aminobutyric acid
101.4
5.4
0 - 7.5
0 - 17.7
2.0
1.3
5.9
75.7
8.0
0 - 4.6
0 - 0.8
1.7
2.4
9.7
0.1
8.6
3.8
nm
0.4 - 11.6
0.4 - 9.0
11.0
6.3
nm
5.1
Nm
Nm
Arginine*
4.5 - 14.7
0.1 - 0.6
1.6
1.5
3.9
3.7
1.7 - 17.5
0 - 1.2
8.9
nm
nm
1.7 - 14.5
Asparagine
111.7
116.6
0.9 - 60.8
44.3 - 70.8
6.6
5.9
92.6
61.2
329.5
6.8
24.0
12.1
24.5 - 109.0
Aspartic acid
145.3
7.6
0.4 - 46.3
0.8 - 20.4
4.1
16.5
164.2
11.0
68.2
9.5
31.6
6.8
11.8
9.7 - 42.9
nm
5.6 - 60.3
2.0 - 56.0
8.1
3.3
nm
42.2
Nm
Nm
2.2
nm
nm
2.0 - 58.7
0 - 3.2
0 - 0.8
3.3
11.0
5.8 - 14.3
Canavanine*
Cysteine
335.5
Glutamic acid
167.3
Glutamine
205.1
Glycine
21.5
2.3
0 - 6.9
5.5 - 9.9
1.3
2.9 - 25.0
4.8 - 35.9
0.8
8.1
0.1
481.8
3.8
0.2
4.9
41.5
25.1
60.5
17.0
0.3 - 4.3
1.2 - 5.6
1.7
Histidine
257
14.8
0.3 - 4.5
0.3 - 4.2
0.7
Isoleucine
87.3
85.4
0.1 - 6.5
0.2 - 6.3
1.1
Leucine
84.1
78.8
0.1 - 8.0
0.4 - 9.7
0.6
1.5
Lysine
246
14.7
0.3 - 22.0
0.2 - 3.5
0.7
1.6
28.4
25.8
17.7
3.0
1.5 - 17.3
6.1
0.8
3.0
0.8
2.6
5.1
1.0
1.2 - 3.9
1.6
28.8
3.4
22.2
11.5
2.0
10.8
10.9
2.3 - 13.2
3.0
5.5
0.8
10.5
2.1
5.2
14.7
2.8
2.6 - 10.1
12.6
10.9
0.6 - 5.7
1.4
0.7
8.6
11.4
12.9
10.0
1.3
9.7
9.7
0 - 1.4
Methionine
146.5
0.1 - 3.3
0.4 - 10.0
0.7
Ornithine
230.1
10.6
0 - 0.2
0 - 0.2
0.1
0.7
Phenylalanine
167.7
61.5
0.2 - 13.3
0.1 - 23.9
0.2
0.9
32.7
6.2
78.8
19.8
6.9
23.2
12.0
7.2 - 27.5
Proline
105.6
35.5
0.2 - 36.8
2.1 - 28.4
0.1
1.9
338.3
18.5
270.8
13.1
6.5
41.0
18.2
2.7 - 65.9
56.5
0 - 0.2
0 - 1.2
101.4
0.2 - 6.7
3.8 - 19.2
1.2
2.8
5.2
0.7
36.6
1.3
5.1
16.0
3.3
6.1 - 19.8
14.1 - 41.3
6.7
17.8
2.2
14.3
42.5
2.6
26.8
19.6
3.8
5.7 - 87.8
2.7 - 9.7
Sarcosine
Serine
0.5
3.0
0 - 17.0
15.0
0.1
1.6
0.4 - 2.5
3.7
0 - 5.7
Threonine
99.3
1.3
1.9 - 28.0
Tryptophan
290.2
16.4
0.4 - 7.5
0 - 9.9
3.3
2.0
33.2
3.5
27.4
14.5
2.5
12.7
12.8
Tyrosine
274.1
79.8
0.2 - 8.4
0.2 - 5.2
10.0
6.8
22.3
1.6
16.2
13.4
4.0
20.4
8.0
0.5 - 8.7
0.1
9.2
5.0
83.8
4.3
7.4
24.7
5.1
7.8 - 28.0
122.1
Valine
71.8
0.1 - 10.4
0.1 - 22.5
*
Determined by Moshe (1998)
nm = not measured; t = trace amounts (< 0.2 mg/g dry weight, for this study only)
170
Chapter 5
Chemistry
With amino acid analyses, 28 amino acids were shown to be present in Sutherlandia
species, and 12 of these appear in all the samples tested (Table 5.24). Amongst these, five
(isoleucine, lysine, phenylalanine, threonine and tryptophan) are essential amino acids, and
two (GABA and L-canavanine) are medicinally important non-protein amino acids. The latter
is a potent L-arginine antagonist said to have anticancer and antiviral properties, and the
GABA is an inhibitory neurotransmitter, which may contribute to the use of Sutherlandia for
the treatment of stress and anxiety (Paragraph 6.2.10). Even though no trends in relative
quantities are evident, sporadic high values were obtained for alanine, asparagine, aspartic
acid, glutamic acid and proline. Reported ranges for asparagine (5.9329.5 mg/g), arginine
(0.411.6 mg/g) and proline (0.1338.3 mg/g) were higher than those reported by Van Wyk
and Albrecht (2008), i.e. 1.635.0 mg/g, 0.56.7 mg/g and 0.77.5 mg/g respectively.
In summary, succesful isolation and characterisation of the main triterpenoids from
S. frutescens (SU1 and SU2, Appendix 5B) and a third minor saponin from S. humilis (SU3)
was done here. The structure of SU3 was published as novel as mentioned previously
(Appendix 5C). Where variation studies are concerned, it was evident that quantitative study
by means of LC-MS provided more comprehensive data about the identities of the
respective compounds, especially that of the saponins as well as the level and type of
glycosylation of the compounds. Metabolomic profiling, as done by Mncwangi (2009), is also
a useful tool in determining geographic and genetic variation in terms of phytochemistry.
Furthermore, the Sutherlandia saponins are structurally related to those of Astragalus
membranaceus, which are not only known to be bitter and exhibit an amarum effect
(Paragraphs 5.3), but may also have immunostimulatory properties (Paragraphs 6.1.3.2 and
6.2.10). Of particular interest is the proven in vivo chemopreventive activity of the
cycloartanes (Kikuchi et al., 2007, see Paragraph 6.2.10). In addition, the flavonoids
contribute to the antioxidant potential of Sutherlandia, while pinitol is a known anti-diabetic
agent (Paragraph 6.2.10).

Very little is known about the phytochemistry of V. oligocephala (syn. Hilliardiella
oligocephala), apart from the sesquiterpene lactones [a hirsutinolide, i.e. 8-(2hydroxymethyl acryloyloxy)-hirsutinolide-13-O-acetate, guaiatrienolides and glaucolides]
(Bohlmann et al., 1978, 1983, 1984; Fig. 5.24). This is not surprising, as sesquiterpenes,
also including enol lactones, eudesmane derivatives and sesquiterpenoid aldehydes
(Hutchings et al., 1996) are the main chemical characters of the genus Vernonia (Dictionary
of Natural Products, 1996). Flavonoids, glycosides, polyphenols, saponins and steroids have
also been indicated in the root extracts of V. oligocephala used traditionally in Swaziland for
171
Chapter 5
Chemistry
the treatment of diabetes (Amusan et al., 2007). The flavonoids normally occur as aglycones
on leaf surfaces and accumulate as glycones internally in Vernonia species (Igile et al.,
1994; Hutchings et al., 1996). Hesperidin (a flavanone, see Agathosma Fig. 5.1),
fasciculation, as well as derivatives of luteolin (see Harpagophytum procumbens Fig. 5.16)
and velutin (all flavones) are commonly found in Vernonia species (Dictionary of Natural
Products, 1996). Triterpenes such as stigmastane, lupenol, taraxerenol and oleanenol
derivatives are known from Vernonia species (Dictionary of Natural Products, 1996), while
bitter saponins [i.e. vernoniosides A1, A2, A3 and B1 (Jisaka et al., 1992) as well as
vernoniosides D and E (Igile et al., 1995)] have been isolated from V. amygdalina, also
medicinally used in southern Africa. Additional compounds identified from other southern
African Vernonia species include diterpenes (Bohlmann et al., 1984), alkaloids, cyanogenic
glycosides, sugars (inositol and scyllitol) and vernolic acid (Hutchings et al., 1996).
Table 5.25: Extract yields of 18 Vernonia oligocephala leaf samples
V. oligocephala
samples
Sample
number
V. oligocephala
VO 45a S
1.4
8.1
9.5
5.9
0.050
V. oligocephala
VO 45a L
1.0
7.4
8.4
3.9
0.072
V. oligocephala
VO 45b S
1.3
7.5
8.7
4.7
ns
V. oligocephala
VO 45b L
1.9
7.4
9.3
3.8
ns
V. oligocephala
VO 45c S
1.7
11.1
12.8
5.7
ns
V. oligocephala
VO 45c L
1.0
8.5
9.6
6.9
ns
V. oligocephala
VO 48a S
2.8
7.5
10.2
5.5
0.059
V. oligocephala
VO 48a L
3.7
14.9
18.6
7.6
0.123
V. oligocephala
VO 48b S
1.9
6.9
8.8
5.2
ns
V. oligocephala
VO 48b L
1.8
12.7
14.5
13.4
ns
V. oligocephala
VO 48c S
1.7
5.0
6.7
11.9
ns
V. oligocephala
VO 48c L
1.7
9.6
11.3
7.8
ns
V. oligocephala
VO 57 L
2.1
8.7
10.8
9.2
0.063
V. oligocephala
VO 63a S
1.0
4.0
5.0
2.7
ns
V. oligocephala
VO 63a L
2.1
11.7
13.8
4.8
0.084
V. oligocephala
VO 63d L
1.1
10.0
11.1
2.0
ns
V. oligocephala
VO 64 S
1.4
4.6
6.0
7.4
ns
V. oligocephala
VO 64 L
0.8
2.5
3.3
7.2
0.062
APS
Polar
Total water
extractable
Non-polar
Alkaloids
ns = no sample
172
Chapter 5
Chemistry
Sesquiterpene lactones:
Glaucolidesa
OH
O
O
O
OAc
OH
OAc
OR
OAc
17,18-Epoxyvernonatolide*
Stilpnotomentolide-8-O[4-hydroxymethacrylate]*
2-Oxo-2-desacetoxyglaucolide
Guaiatrienolidesb
Hirsutinolides
OH
OAc
OH
OH
HO
H
O
O
O
O
8-Senecioyloxyvanillosmin*
8-Acetoxy-10-hydroxyhirsutinolide
Saponins:
HO
HO
OH
O
H
HO
OH
OH
GlcO
OH
Vernonioside D
Vernonioside A3
Diterpenes:
Long chain acids:

COOH
AcO
AcO
12-Acetoxy--bergamotene 12-Acetoxy--santalene
Fig. 5.24:
Vernolic acid
Three types of terpenes from Vernonia species, including sesquiterpene

lactones (* = from V. oligocephala; a = Bohlmann et al., 1984; b = Bohlmann et
al., 1978; c = Jakupovic et al., 1986), saponins (vernoniosides A1, A2, A3 and B1
Jisaka et al., 1992; vernoniosides D and E Igile et al., 1995) and diterpenes
(Bohlmann et al., 1984), as well as a long chain fatty acid (Hutchings et al.,
1996).
173
Chapter 5
Chemistry
From the extract yields (Table 5.25) it is evident that V. oligocephala has a larger polar
component than non-polar. This was confirmed when the qualitative colour tests of five of
the leaf samples indicated the presence of steroids, terpenoids, bitter principles, saponins
and flavonoids. From TLC analyses (Fig. 5.25), it was ascertained that the majority of polar
compounds were indeed comprised of a variety of flavonoids, saponins and some medium
polarity terpenes. The sugar TLC plate provided an indication of the glycosylation of these
polar compounds as they are charred by chromic acid. It seems that the stems contain lower
concentrations of these compounds, and only slight geographical variation was observed
with regards to relative amounts of compounds rather than their presence or absence. No
cyanogenic glycosides were detected (see Paragraph 3.3.5), in contrast with other Vernonia
species.
Fig. 5.25: TLC of polar Vernonia oligocephala extracts developed in

CHCl3:MeOH:H2O:CH3COOH (60:30:8:6), (a) visualised with ethanolic
vanillin/H2SO4 (b) under UV-365 nm before visualisation; (c) under UV-254
before visualisation
V. oligocephala seems to be rich in amino acids (Table 5.26). Asparagine and proline is
present in high amounts, and six (threonine, valine, phenylalanine, isoleucine, lysine and
tryptophan) of the eight essential amino acids are present, giving this plant a high nutritional
value. The amino acid content compares well with that of another medicinally important
southern African Venonia species, V. amygdalina, but the latter also contains methionine
(5.0 mg/g), leucine (16.4 mg/g) and arginine (11.7 mg/g) (Eleyinmi et al., 2008).
In summary, it is evident that the phytochemistry of V. oligocephala needs to be further
explored, as the flavonoids, most of the terpenes of medium and high polarity, as well as the
compounds in the large non-polar fraction are unknown. The bitterness of this species
(bitterness value of 8 00012 000, Paragraph 5.3) may however be explained by the
presence of many sesquiterpene lactones, and the suggested bitter triterpenes.
Furthermore, this initial variation study also shows that there is relatively little chemical
variation among plants distributed over the Gauteng Province of South Africa.
174
Chapter 5
Table 5.26:
Chemistry
Amino acid content of Vernonia oligocephala leaf and stem extracts
Amino acid
RT
(s)
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
54.9
85.5
186.1
66.4
101.5
75.7
111.7
145.3
335
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.7
99.3
290.2
274.1
13.5
71.8
Valine
Chemistry
Lit*
45b
S
45b
L
22.0
7.3
2.5
1.0
33.3
30.7
3.3
3.1
3.6
155.2
5.0
106.8
18.9
22.0
16.7
26.1
5.7
16.5
4.6
10.1
45c
S
45c
L
48a
S
16.7
13.1
2.8
1.1
2.5
4.0
20.1
7.5
11.7
10.4
2.6
31.0
48.6
9.2
2.7
1.2
5.5
4.1
23.7
6.7
9.5
3.3
22.8
8.0
3.3
t
10.0
6.5
5.2
11.8
31.4
0.5
11.1
9.5
2.9
12.2
14.9
14.4
10.1
16.1
10.9
12.7
9.0
10.4
24.0
10.0
161.9
6.1
12.0
7.7
9.4
46.5
31.3
t
76.6
t
17.6
7.2
17.9
13.9
80.0
10.2
8.3
19.9

48a
48b
48b
48c
48c
L
S
L
S
L
3.6
7.3
13.3
10.1
4.2
5.6
1.2
3.1
27.6
3.1
1.4
63a
S
3.9
11.7
2.5
2.5
8.2
3.3
30.8
6.7
1.0
3.2
13.8
1.6
21.5
15.1
3.1
1.3
3.7
4.1
23.3
8.3
1.5
2.8
5.3
6.2
16.0
5.5
22.0
9.8
3.0
7.3
10.7
9.5
1.1
9.5
2.3
2.2
10.9
3.7
t
9.0
2.3
16.7
11.8
21.1
10.8
13.4
9.6
23.5
38.6
8.7
11.5
267.8
12.8
5.9
21.2
3.7
3.5
13.5
0.6
31.1
20.7
2.7
0.8
7.4
3.5
35.7
7.9
9.3
32.5
57 L
63a
L
63d
L
2.7
3.3
115.4
21.7
35.2
7.6
2.0
4.3
3.8
11.7
42.0
58.2
81.6
6.1
7.6
6.4
20.1
73.7
3.1
18.2
4.6
12.0
17.3
7.2
10.4
6.4
0.6
10.3
5.6
3.1
10.5
19.7
7.3
10.8
24.0
0.7
9.7
2.1
3.0
10.4
3.8
9.7
15.6
11.6
10.3
12.5
17.3
9.6
11.4
9.0
23.5
12.1
9.3
11.0
168.4
11.0
43.9
8.9
33.2
279.4
9.1
62.4
20.9
8.6
9.1
159.4
9.0
25.8
22.9
3.2
3.0
11.5
6.1
0.9
6.0
14.5
6.9
4.7
8.8
17.9
489.5
9.2
35.1
11.0
119.7
27.8
17.8
6.1
4.4
1.1
11.3
6.1
7.8
3.2
14.5
9.8
2.3
0.8
10.8
4.5
t
16.2
6.0
2.7
6.1
13.6
5.6
6.7
7.5
12.7
8.0
25.1
17.4
6.7
5.9
3.9
12.0
10.3
28.0
11.4
78.5
36.3
22.5
17.9
36.5
3.8
15.7
2.3
4.7
3.3
43.8
7.8
14.0
57.3
104.3
0.7
5.2
2.4
64 L
20.0
22.4
5.9
1.5
4.9
2.8
361.1
8.1
8.6
124.0
* Amino acid content in V. amygdalina leaves (Eleyinmi, 2008)

64 S
7.3
10.1
2.7
0.8
2.9
3.1
249.8
18.7
175
Chapter 5
Chemistry

W. salutaris (syn. W. ugandensis) have yielded many biologically active compounds of which
the major constituents are from the drimane sesquiterpene lactone class (Drewes et al.,
2001). At least twelve of these compounds have been identified from the leaves and bark,
including warburganal, polygodial, mukaadial, salutarisolide, isopolygodial, muzigadial and
ugandensidial (Kioy et al., 1990; Wube et al., 2005; Clarkson et al., 2007; Fig. 5.27). Due to
irresponsible harvesting of its bark, and threatening of its natural habitat, W. salutaris is now
locally endangered in South Africa. As a protective conservation measure, W. salutaris is
currently cultivated for the production of leaves, as warburganal and polygodial occur in both
the leaves and bark (Drewes et al., 2001). Furthermore, several kaempferol and quercetin
type flavonols have been identified from the leaves (Manguro et al., 2003; Fig. 5.27; also see
Agathosma, Artemisia, Balanites, Dicoma and Sutherlandia). According to Watt and BreyerBrandwijk (1962) the bark contains tannin and mannitol, but no alkaloids. Wube et al. (2005)
also reported linoleic acid (Z,Z) to be present in the bark.
Fig. 5.26: TLC of Warburgia salutaris extracts: (a) non-polar extracts developed in
CHCl3:MeOH (6:3); (b) polar extracts developed in CHCl3:MeOH:H2O:CH3COOH
(60:30:8:6); both plates were visualised with ethanolic vanillin/H2SO4
Qualitative screening confirmed the presence of steroids, terpenoids, bitter principles and
saponins, but also cyanogenic glycosides (see Paragraph 3.3.5). Both qualitative screening
and TLC returned negative results for alkaloids, confirming the findings in the literature. The
levels of APS, polar and non-polar compounds are lower for bark compared to leaves
(Table 5.27), as expected, with APS not contributing significantly to the total water
extractable component. TLC revealed that flavonoids are only present in leaf samples, while
saponins are present in higher quantities in the bark. Kioy et al. (1990) compared the
sesquiterpenes and leaf volatile oils patterns of W. salutaris with that of W. stuhlmannii and
176
Chapter 5
Chemistry
found no consistent differences. It would consequently seem that, while the compounds
within the leaves vary considerably from that from the bark, there is little geographical
variation.
Drimane sesquiterpenes:
At least 12 drimane and coloratane type sesquiterpenes are known from W. salutaris (Wube
et al., 2005)
Drimane type
Coloratane type
O
H
R
O
O
R'
Polygodial:
Warburganal:
Mukaadial:
HO
R = R = H
R = OH; R = H
R = R = OH
Muzigadial:
R = OH
9-Deoxymuzigadial: R = H
Other sesquiterpenes
Salutarisolide
Warburgiadione
Warburgin
Flavonoids:
A total of 15 flavonoids are known from W. salutaris including myricetin, kaempferol and
derivatives, and quercetin and derivatives (Manguro et al., 2003)
3
OR
R2O
OR1
OH
OH
Kaempferide 3-O--glucoside:
1
R = xylosyl (12) glucose
2
R =H
3
R = Me
Kaempferol 3-O--rhamnoside-7,4-di-O--galactoside:
1
R = rhamnose
2
3
R = R =glucose
Kaempferol 3,7,4-tri-O-glucoside:
1
2
3
R = R = R = glucose
OH
2
RO
OR
OH
Quercetin 3-O-[-glucosyl (12)][-rhamnosyl(16)]-glucoside-7-O--rhamnoside:

1
R = [rhamnosyl (16)][glucosyl (12)]glucoside
2
R = rhamnose
3
R = Me
Fig. 5.27: Two classes of compounds (sesquiterpenes and flavonoid glycosides)

representing the main compounds from Warburgia salutaris leaves and bark.
177
Chapter 5
Chemistry
Table 5.27:
Extract yields for Warburgia salutaris bark and leaf samples
W. salutaris
samples
Sample
number
W. salutaris
WSa RSa L
2.3
22.3
24.6
6.2
W. salutaris
WSa RSb L
2.1
18.3
20.4
6.2
W. salutaris
WSa RSb B
1.0
9.7
10.7
2.4
W. salutaris
WSa F B
0.3
5.0
5.3
3.5

APS
Polar
Total water
extractable
Non-polar
W. salutaris leaves and bark are not particularly rich in amino acids. Of the 20 amino acids
identified, only six (allo-isoleucine, lysine, phenylalanine, tryptophan, tyrosine and valine)
appeared in all the samples tested. However, the latter five are essential amino acids.
Asparagine and aspartic acid are present in the highest levels. The presence of GABA (aminobutyric acid) may be relevant, since W. salutaris is traditionally used to treat headache
(Chapter 4).
Table 5.28: Amino acid content of Warburgia salutaris leaf and bark extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.6
99.3
290.2
274.1
71.8

WSa RSa L WSa RSb L
WSa RSb B
WSa F B
14.5
3.0
13.7
7.1
5.4
6.8
4.4
1.4
1.4
3.5
26.2
12.6
1.7
3.6
116.9
39.2
0.9
3.7
34.5
17.3
8.9
6.8
9.6
t
10.5
3.9
2.4
0.6
10.6
3.9
10.2
10.4
10.4
10.2
10.0
7.0
8.9
11.1
9.7
8.2
2.0
0.6
13.1
7.2
3.1
7.6
2.1
12.6
8.0
6.5
12.7
5.3
0.8
3.5
11.7
6.4
1.1
12.7
7.7
6.3
178
Chapter 5
Chemistry
While numerous reports from literature are based on single samples, none report on
variations between populations and only one between species. Based on these preliminary
results, such a study would be valuable. The two main classes of compounds
(sesquiterpenes and flavonoids) known for W. salutaris seem to contribute most to the
biological activities of this medicinal plant, where the sesquiterpenes show antimicrobial,
curative and prophylactic ability and the flavonoids have antioxidant properties
(Paragraph 6.2.12).

By April 2012, 177 compounds had already been isolated and identified from Withania
species, featuring W. somnifera (Dictionary of Natural Products, 2012). These mostly include
triterpene lactones (such as withanolides, withaferins and coagulins, i.e. C28-steroidal
lactones based on an intact or rearranged ergostane framework, as well as
ashwagandhanolide, a dimeric thiowithanolide) and alkaloids (such as nicotine, tropane,
choline, withasomnine, anaferine, anahygrine and the withanamides) (Fig. 5.28; Atta-urRahman et al., 1993; Jayaprakasam et al., 2004; Mirjalili et al., 2009). Withanolides occur in
all the parts of W. somnifera (Jayaprakasam et al., 2004), but withaferin A and withanolide D
have been suggested as markers in the different plant parts used medicinally, i.e.
withaferin A is completely absent from all plant parts except the leaves (Mirjalili et al., 2009).
HPLC may consequently be applied successfully as a tool in quality assessment of
commercially available products (Ganzera et al., 2003). A recent variation study provided
insight into the polypeptide and withanolide diversity within W. somnifera and discrete
chemotypes were identified (Chauraslya et al., 2009). The roots were recorded to
accumulate ca. 0.130.31% tropane alkaloids, but the leaves were found to produce higher
yields than this by extracting with 45% alcohol (Gupta & Rana, 2007). Other compounds also
identified include starch, reducing sugars, hentriacontane, glycosides, dulcitol and withanicil
from the roots, chlorogenic acid (see Harpagophytum procumbens, Fig. 5.16), calystegines
[nitrogen containing polyhydroxylated heterocyclic compounds Bekkouche et al., (2001),
Fig. 5.28], withanone, tannins and flavonoids from the leaves, as well as amino acids and
withanamides from the fruit (Mirjalili et al., 2009). The fruits are also said to be rich in
saponins and were used as substitute for soap (Schmelzer & Gurib-Fakim, 2008). Palmitic,
oleic and linoleic acid, as well as porphyrines, carotenoids, pheophytines and sterols
including campesterol, stigmasterol and -sitosterol (see Hypoxis hemerocallidea, Fig. 5.18)
have recently been identified in leaf and root hexane extracts (Chatterjee et al., 2010).
Furthermore, elements such as N, P, Na, K, Ca, Mg, Zn, Mn, Cu and Fe have been
quantified from W. somnifera, where high levels of Fe (from a minimum of 280 ppm in the
stems to a maximum of 945 ppm in the roots) were reported (Krishnamurthy & Sarala,
2010).
179
Chapter 5
Chemistry
Ergostane-type steroidal lactones:

2
OR
R1
H O
O
O
Withaferin A: R1= H; R2 = OH
Withanolide D: R1 = OH; R2 = H
Sitoinoside IX: R1 = H; R2 = 1-O-glucosyl
OH
Coagulin F: R= H
Withanolide coagulin: R = OH
Ergostane-type sterols and saponins:
Flavones:
OH
HO
OR
HO
H
OH
OH
RO
Ergosta-5,24-dien-3-ol: R = H
Sitoindoside VII: R = 3-O-(6-O-hexadecanoyl-D-glucopyranoside)
Caycopteretin 3-rutinoside:
R = 3-O-[-L-rhamnopyranosyl-(16)-D-glucopyranoside
Alkaloids:
H
Anaferine
Anahygrine
L-Nicotine
R
OH
HO HO
HN
OH
O
O
HO HO
O
OH
N
H
Withasomnine
Withanamide A: R = H2C
Calystegnine B2: R = H
Calystegnine C1: R = OH
HN
HO
R
OH OH
OH
HO
OH
Withanamide B: R =
H2C
Withanamide C: R = H2C
OH
Fig. 5.28: Examples of compounds from three classes identified from Withania somnifera
of which the steroidal lactones and alkaloids are the main components (177
compounds are known from this species Dictionary of Natural Products, 2012)
180
Chapter 5
Chemistry
Table 5.29: Extract yields for roots, stems and leaves of two samples of W. somnifera
W. somnifera
samples
Sample
number
W. somnifera
WSo37a R
0.7
15.0
W. somnifera
WSo37a S
2.0
W. somnifera
WSo37a L
W. somnifera
APS
Polar
Total water
extractable
Non-polar
Alkaloids
15.7
4.0
0.078
9.5
11.5
1.9
0.049
8.6
7.7
16.3
8.3
0.097
WSo37b R
3.7
25.4
29.1
6.7
0.078
W. somnifera
WSo37b S
0.3
5.5
5.8
2.3
0.048
W. somnifera
WSo37b L
2.6
18.0
20.6
7.0
0.164
From Table 5.29 it is clear that the roots accumulate the highest levels of water extractable
compounds, as would be expected. Higher yields of polar compounds are found in all the
plant parts when compared to non-polar compound yields. Secondary metabolite patterns
vary considerably between the plant parts studied, providing a scientific rationale for the
traditional combination of different plant parts in preparation of certain remedies
(Appendix 2). The black zones exhibited during TLC of the polar root extracts [Fig. 5.29(a)]
indicate the presence of saponins such as the sitoinosides (Fig. 5.28) in high quantities. The
largest concentrations of triterpene lactones and non-polar terpenes seem to be present in
the leaves [blue/purple zones in Fig. 5.29(a), (b) and (c)], i.e. 0.0010.5% of dry leaf weight
constitutes withanolides (Mirjalili et al., 2009). As much is known about the alkaloids of
W. somnifera, and the qualitative colour test for alkaloids was positive for all the samples
tested, a detailed study on the topic was not attempted.
Fig. 5.29: TLC of leaves, stems and roots of Withania somnifera: (a) polar extracts
developed in CHCl3:MeOH:H2O:CH3COOH (60:30:8:6); (b) non-polar extracts
developed in CHCl3:MeOH (6:3); (c) non-polar extracts developed in diethyl
ether:hexane (2:3); all plates visualised with ethanolic vanillin/H2SO4
181
Chapter 5
Chemistry
Table 5.30: Amino acid content of Withnania somnifera root, stem and leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335
167.3
205.1
60.5
257
87.3
84.1
246
146.6
230.1
167.7
105.6
56.5
101.4
99.3
290.2
274.1
71.8
37a R
112.7
37.6
1.6
t
9.3
9.5
1395.4
152.4
9.8
145.1
443.5
8.5
18.6
23.4
20.9

37a S
37a L
37b R
37b S
34.0
74.0
41.7
6.4
132.8
41.0
8.6
8.3
6.8
5.2
1.3
3.1
0.6
2.6
T
13.2
11.2
T
2.5
11.5
9.8
5.1
5.6
2283.9
91.2
302.6
286.7
307.9
49.4
21.7
9.6
9.6
86.9
68.1
12.5
3.1
183.3
51.2
17.7
17.9
24.8
18.7
1.4
t
30.2
32.9
12.3
11.4
120.3
73.2
2.4
3.3
39.7
24.4
37.6
10.9
10.6
9.9
22.0
557.1
62.5
309.9
10.2
138.8
339.7
60.4
40.8
16.6
23.0
53.0
270.9
139.4
18.6
23.6
289.0
154.3
118.8
60.5
84.0
168.6
37b L
96.0
28.7
11.1
6.5
21.3
11.0
9.3
11.2
145.0
9.3
11.2
606.3
119.1
9.8
105.0
279.8
18.5
44.5
47.8
26.5
31.8
2.6
10.2
108.8
687.4
13.5
10.0
6.6
10.2
8.4
13.6
7.4
7.4
134.8
116.9
80.9
52.5
106.8
W. somnifera has high levels of amino acids (Table 5.30), contributing to its nutritional value.
All eight essential amino acids are present, and high levels of asparagine, aspartic acid,
glutamic acid, glutamine, proline, serine, threonine and valine were obtained. Four nonprotein amino acids [-amino adipic acid, -aminoisobutyric acid, -aminobyturic acid
(GABA) and ornithine] were also detected.
Withania somnifera has been a well-known adaptogenic tonic with memory-enhancing
capability (Paragraph 6.2.13) across the world for many centuries, and its phytochemistry
has consequently been well researched. Its constituent compounds, in particular the
withanolides and alkaloids [e.g. serotonin, a neurotransmitter, is the aglycone analogue of
the withanamides Jayaprakasam et al. (2004)], have been shown to have a variety of
effects on the central nervous system (CNS) and the immune system. These two classes of
compounds are the main constituents and may also contribute to the bitter taste (bitterness
value of 1 600 14 400, Paragraph 5.3) as steroidal lactones and alkaloids are known to be
bitter.
182
Chapter 5
Chemistry

Most of the phytochemical research done on Z. mucronata involves alkaloids as a major
class of compounds. They are primarily cyclopeptide alkaloids, collectively known as
mucronines (Barboni et al., 1994) and abyssenines, together with a polar isoquinoline
alkaloid (N-methylcoclaurine) (Tschesche et al., 1974). Mucronines A H have all been
isolated from the bark and are characterised by the presence of a styrylamine group in a 15membered cyclopeptide alkaloid (13-membered in mucronine D) (Fig. 5.30). The alkaloid
fraction from the roots yielded 0.05%, and after development on TLC the alkaloids were
visible as fluorescent zones under UV-254 nm and as faint orange zones when sprayed with
Dragendorffs reagent. Mucronine D was reported as the most abundant alkaloid in the roots,
together with two other structurally related alkaloids (Barboni et al., 1994). Furthermore, the
root bark and stem bark are said to contain anthocyanins and small quantities of quinones
(Mpiana et al., 2008), while the bark reportedly contains 1215% tannins (Van Wyk, 1974).
Apart from alkaloids, steroids, terpenoids, bitter principles, saponins and flavonoids were
also detected in the qualitative colour tests. High alcohol precipitable solid (APS) yields were
obtained for most samples (Table 5.31), especially in leaves and bark (possibly due to large
amounts of tannins), and the samples seemed to contain more polar than non-polar
compounds. TLC analyses confirmed the presence of alkaloids, flavonoids and polar
terpenes (Fig. 5.31). Variation in flavonoid and triterpenoid presence and concentration
seemed to be geographical rather than between parts of the same plant, which was
interesting. Very little variation was evident when comparing leaf alkaloid extracts
[Fig. 5.31(a)]. Several triterpenes and saponins are known from Ziziphus, e.g. damaranetype saponins such jujuboside A and B (Kawai et al., 1974; jujubogenin in Fig. 5.30 is the
aglycone of jujuboside B), together with oleanonic acid, colubrinic acid, ceanothic acid,
betulinic acid, sitosterol and their glycones (Dictionary of Natural products, 1996). Betulinic
acid (Fig. 5.30) and proanthocyanidins have been suggested as chemotaxonomic markers
for the genus Ziziphus (Weinges & Schick, 1995). Various flavonoids have been isolated
from the Chinese medicine, Z. jujuba, including swertish, puerarin, as well as spinosin,
isovexitin and apigenin glycones (Cheng et al., 2000; Fig. 5.30). From sugar TLC analyses, it
was evident that some of the polar flavonoids and triterpenoids may indeed be glycosylated
based on the presence of small charred zones of lower polarity. Other phenolic compounds
known from Ziziphus species include (+) and (-)-epigallocatechin (Weinges & Schick, 1995)
and epicatechin [Choi et al., 2011(a)]. Alkaloid colouration after visualisation with iodoplatinate is normally reddish, brown or purple, but a colourless zone was also evident on the
alkaloid TLC, indicative of piperidine ring systems (as in Lobelia species) or steroidal
alkaloids (C-nor-C-homo-cholestanes as in Sabadillae semen) (Wagner & Bladt, 2001).
From the sugar TLC, it would seem that free sugars are present in the leaves, particularly
fructose and mannose, but no positive identification can be made based on TLC.
183
Chapter 5
Chemistry
Alkaloids:
Cyclopeptide alkaloids
Isoquinoline alkaloids
O
Coclaurine:
R1
O
O
O
HN
NH
HN
R2
NH
NH
R = R = H
R = OH
R = OMe
R'''
N
HN
NH
R''
R'
Mucronine E: R1 = -OMe;R2 =
HO
Juziphine:
Abyssenine E: R1 = H; R2 =
Mucronine D
R = Me
R = OH
R = OMe
R = H
Porphine alkaloids
Laurelliptine:
Asimilobine:
3
R =H
2
4
R = R = OH
3
5
R = R = OMe
1
2
3
R =R =R =H
4
R = OMe
5
R = OH
R2
Other phenolic compounds:

Flavonoids
RO
HO
Anthraquinones
OH
OH
COOH
OH
O
OH
OH
HO
OH
O
OH
Swertish:
R = CH3
Apigenine glucoside: R = H
Laccaic acid D
Triterpenoids:
OH
H
COOH
O
HO
Jujubogenin
HO
Betulinic acid
Fig. 5.30: Four classes of compounds (alkaloids, flavonoids, anthroquinones and

triterpenoids) from Ziziphus species. Mucronines A-H and abyssenines A-H are
the main compounds identified from Z. mucronata (Dictionary of Natural
Products, 1996)
184
Chapter 5
Chemistry
Table 5.31: Extract yields of 13 root, bark and leaf samples of Ziziphus mucronata
Z. mucronata
samples
Sample
number
Z. mucronata

APS
Polar
ZM 36a L
5.3
5.9
Z. mucronata
ZM 36a B
0.5
Z. mucronata
ZM 36a R
Z. mucronata
Total water
extractable
Non-polar
Alkaloids
11.2
5.9
0.59
7.6
8.1
3.8
0.04
0.8
2.1
2.9
4.7
0.05
ZM 36b B
0.7
8.4
9.1
ns
ns
Z. mucronata
ZM 36c L
7.3
6.8
14.1
ns
ns
Z. mucronata
ZM 36c B
1.9
8.1
10.0
ns
ns
Z. mucronata
ZM 36c R
0.9
1.7
2.6
ns
ns
Z. mucronata
ZM 39a L
11.3
9.6
20.9
3.8
0.13
Z. mucronata
ZM 39b L
8.9
8.7
17.6
5.7
0.14
Z. mucronata
ZM40b L
3.5
9.4
12.9
6.9
0.15
Z. mucronata
ZM 43 L
4.1
15.3
19.4
5.5
0.22
Z. mucronata
ZM 44 L
3.4
10.1
13.5
7.6
0.16
Z. mucronata
ZM46a L
7.3
16.1
23.4
5.2
0.15
Z. mucronata
ZM46b L
2.7
1.8
4.5
13.4
0.16
Z. mucronata
ZM 47 L
9.5
6.9
16.4
7.8
0.15
Z. mucronata
ZM 32 L
4.1
5.8
9.9
11.9
0.12
Z. mucronata
ZM AJ R1
3.8
5.3
9.1
9.2
0.12
ns = no sample
Fig. 5.31: TLC of Ziziphus mucronata leaf extracts (a) alkaloids, developed in
CHCl3:cyclohexane:ethylamine (4:5:1), visualised with acidified iodoplatinate
spray reagent; (b) polar extracts, developed in CHCl3:MeOH:H2O:CH3COOH
(60:30:8:6), visualised with ethanolic vanillin/H2SO4
185
Chapter 5
Table 5.32:
Chemistry
Chemistry
Amino acid content of Ziziphus mucronata root, bark and leaf extracts
Amino acid
Alanine
Allo-isoleucine
-Aminoadipic acid
-Aminobutyric acid
-Aminobutyric acid
-Aminoisobutyric
acid
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Ornithine
Phenylalanine
Proline
Sarcosine
Serine
Threonine
Tryptophan
Tyrosine
Valine
RT (s)
54.9
85.5
186.1
66.4
101.4
75.7
111.7
145.3
335.5
167.3
205.1
60.5
257
87.3
84.1
246
146.5
230.1
167.7
105.6
56.5
101.5
99.3
290.2
274.1
71.8

36a
36a
36b
36c
36c
R
B
B
R
B
t
1.6
t
0.8
4.0
3.8
4.0
4.2
4.3
2.5
2.0
0.9
2.2
2.0
36c
L
19.4
3.3
2.5
0.5
2.6
4.0
467.9
101.2
2.8
17.0
3.5
3.0
107.3
86.9
2.8
3.0
64.9
21.7
3.2
58.8
9.2
19.2
27.5
10.0
1.0
9.3
1.6
1.3
1.2
9.7
8.7
9.5
75.7
8.5
7.4
139.1
8.2
7.5
36.3
11.1
4.5
0.6
6.8
t
11.5
4.9
0.9
1.4
4.8
1.0
39a
L
24.6
3.6
2.7
0.6
7.8
39b
L
32.0
9.9
3.3
0.7
6.2
40b
L
39.4
3.6
2.8
0.5
3.4
43
L
36.2
11.1
44
L
21.0
12.4
46a
L
40.4
15.4
1.2
t
1.8
3.9
32.9
3.9
81.8
3.0
3.9
62.1
2.0
3.1
39.6
5.6
1.8
7.6
19.2
t
11.6
5.0
0.8
2.0
4.9
47
L
13.7
6.0
32
L
13.2
6.5
AJ
R1
8.9
5.1
3.5
t
0.8
0.9
1.7
2.9
90.2
8.5
2.8
42.8
t
12.3
2.8
5.6
3.0
23.5
2.8
0.5
t
10.1
4.6
2.1
10.6
5.5
2.8
10.3
6.7
1.4
2.1
10.5
2.2
1.5
9.9
11.2
10.3
10.2
9.5
10.2
9.9
8.4
3.0
9.0
8.6
51.0
9.5
22.0
9.3
8.5
6.4
0.5
12.5
6.9
2.3
12.2
5.2
1.3
1.9
0.6
11.0
3.0
7.6
11.1
1.4
10.7
4.7
9.7
t
9.9
3.3
7.0
9.5
12.1
10.2
3.5
7.3
10.0
9.2
7.8
52.9
8.7
7.7
253.9
9.8
10.1
38.3
9.3
12.7
46.2
9.1
8.7
92.8
11.3
41.3
9.6
14.1
27.2
9.2
12.0
62.0
1.9
3.9
t
11.6
6.3
5.8
1.9
4.1
13.5
7.2
6.9
9.7
13.7
12.2
7.8
6.2
4.9
12.3
6.3
5.8
3.1
2.0
12.2
8.8
6.6
7.6
4.9
12.7
12.6
6.1
5.8
2.8
12.3
11.2
8.4
11.9
5.0
0.8
46b
L
0.5
12.1
5.9
1.0
11.9
5.9
2.1
186
Chapter 5
Chemistry
Of the 24 amino acids identified in the leaves, bark and root of Z. mucronata, only alloisoleucine, -aminobutyric acid (GABA), proline, tyrosine and valine are present in all the
samples analysed. Z. mucronata contains seven of the eight essential amino acids (histidine,
isoleucine, leucine, lysine, phenylalanine, threonine and tryptophan). While these results are
not comparable to those obtained from Z. jujuba fruit pulp and seeds, the same trend was
recognized, i.e. high levels of asparagine and proline were present [Choi et al., 2011(a)].
A great diversity of compounds has been identified from the genus Ziziphus. This is also
evident from the phytochemical analysis of Z. mucronata, which is still largely unexplored.
The only compounds isolated and characterised from this species are peptide alkaloids
(mucronines A-H and abyssenines A-C), while the presence of tannins (Van Wyk, 1974),
anthocyanins and small quantities of quinones (Mpiana et al., 2008) have been reported.
The presence of the amino acids, reported here for the first time, along with elements such
as Ca, Na, Zn, Mn, K and Cu (Hassen et al., 2009) makes Z. mucronata highly nutritious,
justifying its use as fodder plant (Van Wyk, 1974). The bitterness (bitterness value 8 400
12 000, Paragraph 5.3) of this plant is probably due to its alkaloids. However, damaranetype triterpenoids such as those from Z. jujuba, also used medicinally, are known to act as
sweetness inhibitors (Suttisri et al., 1995) and tannins cause astringency. All these
compounds consequently have an effect on the taste of the extracts, masking the bitter taste
(three results were returned as not bitter, see Table 5.33).
5.3
Bitterness value
Bitterness is associated with various classes of compounds, including terpenoids such as

sesquiterpene lactones and triterpenes, as well as iridoids, flavonoids, and alkaloids (Van
Wyk and Wink, 2004). The physiological effects associated with bitter taste are summarised
as the amarum effect, has to do with digestive stimulation and are discussed in Chapter 6.
All 14 tonic plants investigated were indicated for the treatment of digestive disorders
(Chapter 4; Appendix 2). The aim was then to obtain bitterness values for these plants in
order to find a rationale for their digestive properties. The plant samples chosen for this
experiment, were based on maximum relative percentage yield obtained for aqueous
extracts during variation studies.
From the results presented in Table 5.33, it seems that all of the species tested are
sufficiently bitter to be called bitter tonics. The high bitterness values of Aloe ferox, Artemisia
afra, Dicoma anomala, D. capensis, Sutherlandia microphylla and Vernonia oligocephala
come as no surprise, as infusions and decoctions of these species are well known to be very
bitter (Van Wyk et al., 2009; Appendix 2). However, this is the first time that any attempt is
made to quantify the bitterness. The only species for which comparative data on bitterness
187
Chapter 5
Chemistry
could be obtained, is Harpagophytum procumbens, with a bitterness value of 4 359

[ca. 6 000 according to Hnsel and Tyler (2001)]. The leaves of Artemisia afra has a very
high bitterness value of 7 20084 000, comparable to Artemisia absinthium, which has a
reported bitterness value of 10 00025 000 (Wagner & Wiesenauer, 1995; European
Pharmacopoeia, 2005a; Lachenmeier, 2007). It should be noted that the bitterness value of
Aloe ferox is much higher because the dried leaf exudate was tested, and not leaf or root
extracts as in all other species. Purified bitter compounds or exudates have much higher
bitterness values than crude extracts. In gentian, for example, the bitter principles are
concentrated in the root (28% dry weight). One of the secoiridoid glycosides affording
Gentiana lutea extracts their bitter taste (bitterness value of 10 00030 000), is
amarogentine, the most bitter compound known (bitterness value of 58 million), contributing
to only 0.0250.4% of the extract yield (Wagner & Wiesenhauer, 1995; Wichtl & Bisset,
2000). The actual bitterness would, however, depend on the dose and dosage form, so that
the bitterness value is merely an indication that the plant is sufficiently bitter to impart a bitter
taste and thus induce the amarum effect.
Table 5.33: Bitterness values for 15 South African tonic plants. The average was calculated
from all the taste results obtained per plant, where not bitter results were
assigned a zero value for bitterness value calculation.
Bitterness values obtained
Plants
Sample
number
Highest
Not bitter
Results
Total
no. of
taste
results
Mean
Bitterness
Value
Lowest
Agathosma betulina
AB 53 L
1440
14400
11
4859
Aloe ferox (lump)
AF comm.*
11556
720000
13
78254
Arctopus monacanthus
AMo 11 R
1600
21600
5407
Artemisia afra
AAf 35a L
7200
84000
11
26393
Balanites maughamii
BM 70 B
1800
14400
4211
Dicoma anomala
DA 4032a R
8000
12000
8330
Dicoma capensis
DC 58a L
8000
72000
14531
Harpagophytum
procumbens
HP Ma R
1440
14400
4359
HH 71e R
1800
14400
10
2615
Muraltia heisteria
MH 4194 L
1680
14400
11
5594
SMi comm. L
9333
37333
14123
Sutherlania microphylla
VO 48a L
8000
12000
10032
Warburgia salutaris
WSa RSa L
1600
8400
3425
Withania somnifera
WSo 37b R
1600
14400
13
2456
8400
12000
6410
Ziziphus mucronata
ZM 46b L
* = lump used
L= leaves; B = bark; R = root
188
Chapter 5
Chemistry
The very wide ranges in bitterness values in Table 5.33 are noteworthy and reflect the
difficulty to obtain repeatable results. Six consistent bitterness values for Withania somnifera
and Aloe ferox could be obtained only after 13 taste experiments (i.e. 13 participants) in
each case. Warburgia salutaris, Vernonia oligocephala and Harpagophytum procumbens on
the other hand, generated similar bitter sensations in the first six participants it was offered
to and show narrow ranges of bitterness values. The bitter taste resulting from these
molecules may however be masked by other compounds present in the extracts responsible
for tastes such as saltiness, sweetness, sourness and astringency, all competing for the
available receptor sites (Reinberger, 2006; Ley, 2008; Slack et al., 2010). It is possible that
the species with less variable bitterness values have less interfering substances masking the
bitterness. Further influences include genetic (Bufe et al., 2005; Behrens & Meyerhof, 2010),
physiological (Prutkin et al., 2000; Bufe et al., 2005) and hormonal (Prutkin et al., 2000)
variations within the participants. Taste preferences may be learnt through cultural familiarity
with certain foods, herbs and beverages (Reinberger, 2006), and sensitivity to bitterness
may vary for the same person from day to day because of fatigue, smoking, or after eating
strongly flavoured food (WHO, 2002). As mentioned in Chapter 3, a correction factor is
applied for each participant to attempt minimization of these influences. However, it is
evident that bitterness taste testing does not provide absolute values and must therefore be
reported as ranges as suggested by Wagner and Wiesenauer (1995). Correlation between
the relative bitterness values of the tested species and their main bitter compounds, as done
in alcoholic beverage industries (Lachenmeier, 2007; Intelmann et al., 2009), would provide
interesting results.
5.4
Ionic fluids
Choi et al. (2011b) recently suggested that high levels of choline, organic acids (e.g. citric
acid, maleic acid and malic acid), certain amino acids (e.g. proline) and simple sugars (i.e.
glucose, fructose and sucrose) in plants in arid regions or growing in highly variable climatic
conditions, are not coincidental. It has been shown that combinations of these solids in
simple ratios (e.g. glucose:fructose 1:1) produce liquids through hydrogen bonding
interaction amongst the compounds. Such liquids are known as Natural Deep Eutectic
Solvents (NADES). They do not evaporate, even at high temperatures over long periods,
and are able to dissolve compounds which are neither soluble in water nor lipids, suggesting
that these may be the solvents in which biochemical reactions occur within living cells.
Harpagophytum procumbens, a plant known to occur in the arid, harsh conditions of
Namibia, presents a good example in that its roots contain up to 46% sugars together with
some organic acids and small amounts of proline (see Paragraph 5.8). Interestingly, all the
189
Chapter 5
Chemistry
species investigated in this study contain proline, and Aloe, Artemisia, Dicoma, Sutherlandia,
Vernonia as well as Withania exhibited proline contents higher than 300 mg/g dry weight.
These species are all well adapted to harsh (cold and/or arid) conditions.
The discovery of NADES has a twofold bearing on this study. Firstly, it clarifies the biological
involvement of many of the identified substances of medium polarity. Rutin, in Agathosma for
example, is a flavonoid exhibiting low solubility in water, and is insoluble in non-polar
solvents. Yet it contributes to the antioxidant capacity of a myriad of plants, and has been
shown to be soluble in NADES mixtures comprised of 1:1 combinations between sucrose,
fructose, glucose, malic acid or aconitic acid with choline chloride (Choi et al., 2001b).
Secondly, the ethnobotanical popularity of the use of syrups as solvents may now be
understood as a vehicle of ingestion and metabolic assimilation for biologically important
substances which are insoluble in water or fats/oils (Chapter 4, Appendix 2). Previously,
syrups may only have been considered useful as expectorants due to their ability to reduce
surface tension.
5.5
Conclusions
The hypothesis to be tested in this study was that southern African tonic plants are typically
characterised by chemical complexity, resulting in a wide range of possible biological
activities. The results in this chapter show a wide diversity of chemical compounds identified
from each of the 14 tonic plants and their related species. These compounds represented
seven main classes of compounds which include terpenoids, phenolic compounds, alkaloids,
amino acids, sugars, alcohol precipitable solids and cyanogenic glycosides (Table 5.34). The
standard methods of chromatography which were used here seem inadequate for studying
such a great diversity of compounds in these plants. More modern analytical techniques
such as LC-MS and HR-GC-MS produced reliable, reproducible results when conducting
variation studies. The subsequent use of LC-MS, GC-MS and possibly also NMR (the
metabolomic approach) would be relevant and more appropriate tools for studying chemical
complexity. Furthermore, the wide diversity of compounds in these tonic plants presents the
possibility of an equally wide diversity of pharmacological activities (Chapter 6).
Bitterness determination confirmed the presence of bitter compounds (these include
flavonoids, chromones, anthrones and various terpenoids which were detected by
chromatographic means) in all 14 species. The discovery of ionic fluids sheds new light on
the presence of seemingly unimportant sugars, organic acids and amino acids as
constituents of plant extracts. Not only does this contribute to a better understanding of plant
physiology, but also the traditional practices of combining plants or substances in herbal
190
Chapter 5
Chemistry
medicine preparations. Harpagophytum procumbens, as an example, illustrates the potential

value of seemingly unimportant molecules in high levels. High levels of sugars (ca. 46%) in
the root possibly increase the solubility of sesquiterpene lactones of medium polarity which
are only slightly soluble in aqueous medium, thus improving metabolic assimilation.
Sesquiterpene lactones are known to be very bitter, but their bitterness may have been
masked by the high sugar content in the H. procumbens root, resulting in a relatively
medium bitterness value when compared to 14 other species in this study.
191
Chapter 5
Table 5.34:
Chemistry
Chemistry
Summary of classes of compounds in 14 tonic plants (detailed information in Sections 5.2.1 5.2.14) (relative quantities given as
+++ = major compounds; ++ = present in appreciable amounts; + = present, even as traces; = not present)
Species
Agathosma
A. betulina
A. crenulata
A. ovata
Aloe
A. arborescens
A. ferox
A. marlothii
A. vera
Arctopus
A. dregei
A. echinatus
A. monacanthus
Artemisia
A. afra
Balanites
B. maughamii
Dicoma
D. anomala
D. capensis
D. schinzii
D. zeyheri
Harpagophytum
H. procumbens
Terpenes
Phenolic
compounds
Alkaloids
Amino
acids
APS
Cyanogenic
glycosides
Monoterpenes (+++)
Medium polar (+)
Glycosides (+)
Flavonoids (++)
--
++
--
Sterols (+)
Anthrones (+++)
Chromones (+++)
Benzenes (+)
Naphthalenes (+)
Furans (+)
Tannins (+)
--
++
+++
--
Diterpenes (+++)
Saponins (++)
Phenolic acids (++)
--
++
--
Monoterpenes (+++)
Sesquiterpene lactones (+)
Triterpenes (+)
Flavonoids (++)
Coumarins (+)
Acetophenones(+)
--
+++
++
--
Steroidal glycosides (+++)

Medium polar terpenes (+)
Flavonoids (++)
--
++
++
--
Sesquiterpene lactones (+++)

Saponins (++)
Sitosterols (+)
Flavonoids (++)
Phenolic acids (+)
--
++
++
--
Diterpenes (+)
Iridoids (+++)
Triterpenes (+)
Sitosterols (+)
Flavonoids(+)
Acetosides (++)
++
++
--
Other compounds
Monosaccharides
vitamins, minerals,
organic acids,
enzymes
Long chain alkanes;

organic acids
Sugars
Carotenoids
Organic acids
192
Chapter 5
Species
Hypoxis
H. hemerocallidea
(tubers)
Muraltia
M. heisteria
Sutherlandia
S. frutescens
Vernonia
V. oligocephala
Warburgia
W. salutaris
Withania
W. somnifera
Ziziphus
Z. mucronata
Chemistry
Chemistry
Terpenes
Phenolic
compounds
Alkaloids
Amino
acids*
APS
Cyanogenic
glycosides
Other compounds
Monoterpenes (+)
Sterols (+++)
Norligans (+++)
Tannins (++)
--
+++
--
Saponins (+++)
Flavonoids (+++)
--
--
Saponins (+++)
Sitosterol (+)
Flavonoids (++)
--
++
+++
--
Sesquiterpene lactones (++)

Triterpenoids (++)
Flavonoids (+++)
--
++
--
Monoterpenes (+)
Sesquiterpene lactones (++)
Triterpenoids(+)
Flavonoids (++)
Tannins (+)
--
Mannitol
Monoterpenes (+)
Steroidal lactones (+++)
Tripenes (++)
Coumarins (+)
Tannins (+)
Flavonoids (+)
+++
+++
++
--
Sugars
Fatty acids
Hydric alcohols
Saponins (+++)
Flavonoids (+++)
+++
++
++
--
Pinitol
Long chain
acids
fatty
193
CHAPTER 6
Pharmacology
6.1
Introduction
Pharmacology is the science that deals with the origin, nature, chemistry, effects, and uses of
drugs (Dorland's Medical Dictionary for Health Consumers, 2007). The word pharmacology
has a Greek origin (pharmakon, i.e. drug and logos, i.e. science), and also deals with the
preparation, properties and actions of drugs (Mosby's Medical Dictionary, 2009). When a
pharmacological study is based on chemicals from natural sources such as plants, animals,
fungi, molds or yeasts to be used as drugs or in the preparation of drugs, it is referred to as
pharmacognosy (Mosbys Medical Dictionary, 2009). The actions of the drug on the body is
called its pharmacodynamics (from the Greek words pharmakon, and dynamis, i.e. power), and
includes the pharmacologic response, duration and magnitude of response observed relative to
the concentration of the drug at an active site in the organism (Mosby's Medical Dictionary,
2009). On the other hand, the actions of the body on an administered drug is called
pharmacokinetics (from the Greek words pharmakon and kinesis, i.e. motion), and includes
studies of the mechanisms of drug absorption, distribution, metabolism, and excretion; onset of
action; duration of effect; biotransformation; and effects and routes of excretion of the
metabolites of the drug (Mosby's Medical Dictionary, 2009).
This chapter highlights the pharmacognosy and presumed pharmacological properties of 14
chosen plants, traditionally considered to be tonics. By combining this anecdotal information
with the pharmacodynamic and pharmacokinetic evidence obtained from in vitro, in vivo and
clinical tests on the extracts or isolated compounds, one may assess the validity of the
ascribed tonic properties of these plants.
6.1.1
Classes of compounds and their biological activities
Considering that each tonic plant contains several classes of compounds, often with a large
number of compounds in each class (Chapter 5), a wide range of biological activities is likely.
By considering the water extractable component (representing a traditionally used tea or
194
Chapter 6
Pharmacology
infusion made with hot or cold water, respectively), alcohol precipitable solids, i.e. tannins,
polysaccharides and glycoproteins (see Aloe, Paragraph 5.3), and other polar to medium polar
compounds such as the larger terpenes, phenolic compounds, amino acids and sugars, would
be included. Medium to non-polar components, i.e. aglycones of the mentioned polar
compounds and smaller compounds such as iridoids, sesqui- and monoterpenes, together with
alkaloids would be extracted by preparing alcoholic tinctures, while volatile components would
be released when using a steam bath or smoke of a tonic plant.
Alcohol precipitable solids (APS) contain macromolecules which are soluble in water, but
precipitate in 75% alcohol (Paragraph 3.3.3.1). Polysaccharides and glycoproteins are the
main constituents of APS. Apart from the potential energy source (i.e. amylase and
amylopectin) found in polysaccharides, cellulose is a structural component in plants that serve
as fibre in the human diet. Polysaccharides have also been suggested to have wound-healing
(Bruneton, 1995; Aloe is a good example see Paragraphs 5.2.2 and 6.2.2) and
immunostimulatory properties (Qui et al., 1999; Pugh et al., 2001). Glycoproteins consist of
carbohydrate chains on certain amino acids such as hydroxyproline, proline, lysine or tyrosine,
responsible for keeping cell walls intact (Cseke et al., 2006). Plants which produce mucilage
usually also
have
high
levels
of
APS,
e.g.
Agathosma,
Aloe
and
Sutherlandia
(Paragraphs 5.2.1, 5.2.2 and 5.2.10 respectively). Medicinal plants rich in mucilage are often
prescribed to treat coughs, improve digestion and for topical application to treat inflamed skin
(Van Wyk & Wink, 2004; see Agathosma as example Paragraph 4.2.1 and Table 5.1).
Another class of macromolecules are the tannins, which are bitter, water-soluble oligomers rich
in phenolic groups and capable of binding to or precipitating water-soluble proteins (Cseke et
al., 2006). Tannin-rich roots and bark are traditionally used to treat diarrhea because of the
ability of tannins to act as non-specific protein poisons. Condensed tannins are comprised of
polymeric networks of flavanols, while the hydrolysable tannins consist of sugar esters
combined with a small ratio of trihydroxybenzenecarboxylic acids. These highly bitter
macromolecules
are
anti-feedants,
but
have
also
shown
activity
against
human
immunodeficiency virus by inhibiting reverse transcriptase (Notka et al., 2004).

Terpenes are hydrophobic molecules which interact with biomembranes. They affect the fluidity
of the membranes through conformational changes in the membrane proteins (e.g. ion
channels and transporters) (Van Wyk & Wink, 2004). Where Na+, K+ or Ca2+ channels are
affected, signal transduction could be disturbed, e.g. small terpenes (mono- and
sesquiterpenes which are normally volatile and are collectively known as the essential oil
195
Chapter 6
Pharmacology
component of an aromatic plant) may cross the blood brain barrier where they act as noncompetitive blockers of the GABA gated chloride channel (Sirisoma et al., 2001). Mono- and
sesquiterpenes may consequently cause sedation or may be effective in the treatment of
spasms and pain in cases of dyspeptic disorders (see Agathosma Paragraphs 4.2.1 and
6.2.1, as well as Artemisia afra - Paragraphs 4.2.4 and 6.2.4). Cell death may result from
unspecific terpene interaction with cell membranes that leads to uncontrolled efflux of ions,
metabolites and receptors. Many antimicrobial and cytotoxic properties may consequently be
ascribed to terpenes (all the plants in this study contain terpenes Table 5.34, and all are used
as treatment for infections, except Muraltia heisteria Tables 4.1 and 6.3). The largest
terpenoids are the tetraterpenes or carotenoids. They are pigments used by the plant to attract
pollinators and seed dispersers, but also have antioxidant properties useful in the human diet
(Cseke et al., 2006). Among the triterpenes are sterols, sterolins, cardiac glycosides and
saponins. Due to their structures resembling those of steroids, they may exhibit corticomimetic
properties (e.g. Sutherlandia Van Wyk and Albrecht (2008); Smith & Myburgh, 2004), and
may consequently play an important part in hormone regulation. Saponins are glycosylated
sterols, steroid alkaloids or triterpenes which typically have detergent properties, thus affording
antiparasitic ability for example (aqueous extracts containing saponins produce foam when
shaken vigorously Paragraph 3.3, e.g. Balanites maughamii Paragraphs 5.2.5 and 6.2.5).
Saponins (such as those in Sutherlandia) may contribute to the synergistic effects of a
phytomedicine by increasing the permeability of cell membranes, thus allowing for a higher
level of absorption than would normally be possible. Triterpenes and sesquiterpene lactones
are usually bitter (see bitterness value Paragraph 5.3; e.g. Dicoma, Sutherlandia and
Vernonia oligocephala Table 5.33), giving rise to the amarum effect (Paragraph 6.1.3.2). The
non-polar character of the diterpenes and iridoids often result in their occurrence as resins, and
they have also been associated with antimicrobial and antiinflammatory properties among
others (see Arctopus and Harpagophytum, Paragraphs 6.2.3 and 6.2.7).
A wide diversity of compounds are classified as phenolic and includes flavonoids, coumarins,
anthrones, chromones, phenylpropanoids and phenolic acids, as well as quinones (Chapters 3
and 5). Catechins are structurally related to flavonoids, and may polymerise to form
procyanidins. Where these phenolic compounds are highly hydroxylated, the hydroxyl groups
may strongly interact with proteins resulting in the formation of hydrogen and ionic bonds, or
even covalent bonds (Van Wyk & Wink, 2004). Such interactions with proteins may inhibit
receptors and enzymes which may in turn be effective in treatments for blood disorders and as
insecticides, e.g. Ziziphus mucronata which exhibits antisickling activity and has anti-amoebic
196
Chapter 6
properties,
Pharmacology
both
ascribed
to
the
presence
of
highly
hydroxylated
anthocyanidins
(Paragraph 6.2.14). Furthermore, the conjugated electronic structures of all these phenolic
compounds are not only likely to exhibit antioxidant properties, but also antimicrobial,
antiinflammatory, estrogenic (Cseke et al., 2006) and bitter properties (Paragraph 5.3).
Alkaloids are nitrogen-containing, alkaline compounds mostly derived from amino acids, such
as phenylalanine, tyrosine and tryptophan, from which neurotransmitters such as serotonin,
GABA, glutamic acid and histamine are also derived (Van Wyk & Wink, 2004). Alkaloids are
consequently structurally related to these neurotransmitters resulting in their ability to compete
for the same receptor sites, and thus also have CNS-related effects such as sedation, moodand memory enhancement e.g. Withania somnifera (Paragraphs 4.2.13, 5.2.14 and 6.2.13).
Unfortunately such CNS-related activities may be habit-forming or toxic. Alkaloids are also
known to be bitter (Paragraph 5.3), where the taste may be affected by the molecular structure.
Alkaloids are grouped by the base structure (ring system), i.e. indolizidine and quinolizidinebased systems, or quinoline, quinazoline and acridone-based systems (Herbert, 2003).
Amino acids are also important in the polar plant extracts. The essential amino acids
(threonine, valine, tryptophan, isoleucine, leucine, lysine, phenylalanine and methionine)
cannot be produced by the human body and must be assimilated through a healthy diet. Nonprotein amino acids are also biologically active and may contribute to the biological activity of
the crude mixture. Examples include L-canavanine, an L-arginine antimetabolite, have
anticancer properties, while -aminobutyric acid (GABA) binds selectively to the GABAbenzodiazepine receptor to act as sedative (see Sutherlandia in Paragraph 6.2). Amino acids
contribute to muscle maintenance and possible immunostimulation (e.g. Sutherlandia, which is
used to treat patients with wasting diseases such as Aids and cancer, Paragraph 6.2.10).
Carbohydrates provide energy and structural material for plants. However, it is when these
mono- and oligosaccharides are incorporated into the structures of the mentioned classes of
compounds to form glycosides, or when they form Natural Deep Eutectic Solvents (NADES,
see Paragraph 5.17) with amino acids or organic acids, that they play fundamentally important
roles in cell metabolism. In the former instance, the optical activity of the sugars and their
positions on the parent molecule, play a role in the enzymatic activation/deactivation of the
glycoside it is bonded to, and consequently the measure of activity of the said glycoside. Other
small sugars such as cyclitols (e.g. pinitol in Sutherlandia frutescens) may play an important
role in the provision of cellular energy (Van Wyk & Albrecht, 2008).
197
Chapter 6
6.1.2
Pharmacology
Synergism
Paul Ehrlich, a pharmacologist who introduced the magic bullet concept, inspired scientists to
develop powerful synthetic therapeutic substances with clearly defined targets (Bosch &
Rosich, 2008), such as the anticancer drug etoposide, synthetically modified from a cytotoxic
lignan extracted from the mayapple (Podophyllum peltatum L.) (Cseke et al., 2006). However,
the approval rate for new drugs is declining despite a global increase in spending on drug
discovery and development, because of toxicity and undesirable side effects of these drugs
(Morrow et al., 2010). Furthermore, effective drug-target interactions would involve the
modulation of whole networks rather than only single targets. To this effect, traditional
phytotherapy employs the use of plant extracts or mixtures of extracts, an ancient practice,
developed over thousands of years, referred to in Egyptian medicine (the Ebers papyrus),
Traditional Chinese Medicine, Ayurveda as well as in De Materia Medica by Dioscorides, to
name a few (Patwardhan et al., 2008; also see Chapter 2). In African traditional medicine the
use of herbal mixtures is not an uncommon practice either (see Appendix 2), suggesting that
the mixture makes the medicine (Gertsch, 2011), and that synergistic effects have been
discovered through the trial and error over many generations of traditional healers. It is only
with recent scientific research that an understanding is gradually being developed for this
concept.
Unfortunately, ethnopharmacology is not only based on molecular concepts, but also include
metaphysical elements and beliefs (such as the doctrine of signatures), incorporation of
spiritual healing and magic, which are beyond the scope of empirical science. Added to this, in
vitro and in vivo results often give opposing results, which are discouraging to many a
researcher in this field. However, several examples exist, in Traditional Chinese Medicine for
instance, where mixtures of plant extracts are traditionally used for multiple indications, and
show superior effects over monodrugs (Eng, 2010; Lam et al., 2010), and where plant extracts
are administered together with chemosynthetic drugs in order to facilitate an overall effect
which is usually greater than that which may be obtained when administering only the synthetic
drug, even at elevated, often more toxic doses (Cseke et al., 2006; Gertsch, 2011). This is an
example of a type of potentiation resulting from a pharmacological synergistic interaction in
which the effect of two drugs given simultaneously is greater than the sum of the effects of
each drug given separately (Berenbaum, 1978, 1980).
198
Chapter 6
Pharmacology
Synergism is grounded in four proposed mechanisms based on recent pharmacological,

molecular-biological and clinical research (Wagner & Ulrich-Merzenich, 2009):
(a) Synergistic multi-target effects where a single component in a mono-extract or multi-extract
combination can affect multiple targets and bring about an over-additive or potentiated,
synergistic effect.
(b) Synergistic pharmacokinetic or physiochemical effects could be a result of enhanced
solubility, resorption rates or bioavailability of the active compounds, brought about by other
agents in the extract which need not necessarily be biologically active.
(c) Interactions of agents with the resistance mechanisms of bacteria can enhance antibacterial
effects synergistically. This result can be obtained when an agent intervenes at the binding
site, i.e. it attacks the cell wall of the bacteria, when an agent protects antibacteria against
deactivation which in turn results in prolonged activity, or when an agent blocks the pumping
systems put in place by the bacteria to prevent agents from penetrating it.
(d) Synergism could result from the respective elimination or neutralization of adverse effects
caused by compounds contained in the extract, by adding these agents to the extract or
affecting the extract by heating it. An overall better effect is consequently achieved with these
additions or manipulations than without it.
Phytochemical synergism is an evolutionary principle where a plant develops pathways to
produce complex mixtures of molecules to be solely advantageous for the fitness of the plant
in which it is biosynthesized (Cseke et al., 2006). There are more than 5 000 distinct natural
product scaffolds (i.e. chemical skeleta) (Cordell & Colvard, 2005) from which millions of
secondary metabolites with multiple functionalities and bioactivities are biosynthesised, and
through which plants gain resistance against internal and external stresses they are subjected
to over time. This evolutionary benefit is applied in the use of these plants (and their
constituent mixtures) as medicines, where the biological activity may not necessarily be
ascribed to high concentrations of any one biologically active secondary metabolite, but rather
as synergy between a range of weakly active compounds to bring about an effect that is
beyond their meaning response (Wink, 2003; Gertsch, 2011). Volatile essential oils for
example, may be highly variable within a taxon, reflecting environmental adaptation and
ongoing molecular evolution, but are promising agents for treatment of pain and inflammation
(Gertsch, 2011, also see Agathosma betulina Appendix 2A as well as Chapter 5).
199
Chapter 6
6.1.3
Pharmacology
Pharmacological and physiological effects of tonics
As seen in Chapter 2, a differentiation is made between stimulant tonics and modulatory tonics.
The first, also known as stimulants, gently stimulate functional activity by quickening and
enhancing the physiological function of the body (Hobbs, 1998). Panossian and Wagner (2005)
suggested that single doses of stimulating tonics are able to trigger the sympatho-adrenalsystem, which activates a rapid response mechanism to provide an acute reaction when a
stress is applied to an organism. Furthermore, uni-directional modes of action are described for
stimulants. Hydrastis canadensis (goldenseal), for example, exhibits a strong, uni-directional
antibiotic function which stimulates production of white blood cells, and is mainly used for
colds, influenza and infections (Naik et al., 2010). Consequently, stimulating tonics are
effective in long-term treatment of illnesses related to chronic fatigue, for instance Chronic
Fatigue Syndrome, Parkinsons disease, Multiple sclerosis, amyotrophic lateral sclerosis as
well as fatigue accompanying ageing, depression, cancer and HIV infection. The modulatory
type of tonic nourishes (i.e. it provides vitamins, minerals, amino acids and other nutrients
required for proper tissue health and organ function) and has a beneficial effect on the cellular
foundations of the human immune response (Hobbs, 1998). For example, Astragalus
membranaceus is a traditional tonic (Van Wyk & Wink, 2004) known to enhance various types
of immune responses such as antiinflammation, phagocytic activity on macrophages, T-cell
activity and cytotoxicity of natural killer or lymphokine-activated killer cells (Zhoa et al., 1990).
Modulatory tonics such as adaptogens exhibit bi-directional modes of action (Naik et al. 2010,
see Paragraph 6.1.2.1).
Rangel (2005) suggested a further scientific rationale for the tonic mode of action when he
postulated that there are three essential factors that enhance the life of any living system. The
first is energy, which comprises physiological mechanisms associated with ATP synthesis.
Secondly, biological intelligence is responsible for regulating neuro-endocrine, biochemical,
immune and cellular processes. Lastly, there is organization, which relates to organ function
and structure. Consequently, it is proposed that the general health of a human will increase
with an increase of any or all three these factors. A tonic, according to Rangel (2005), is thus a
substance which can provide negative entropy, and can bring about an increase in order, an
increase in energy availability and ample information flow within the body.
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6.1.3.1
Pharmacology
Stress and adaptogens
Any living organism has to able to adapt to its environment in order to survive. Any type of
demand on the organism, whether internal or external, elicits a specific or non-specific reaction
from the organism (Selye, 1983). Selye (1946) defined such a non-specific response as
stress and the demand on the organism as the stressor. He described an organisms
attempt to adapt the General Adaptation Syndrome, which constituted three stages, namely
an Alarm Reaction, followed by a State of Resistance and then a State of Exhaustion (Selye,
1946, 1950).
The Alarm Reaction is initiated immediately after the organism has been exposed to a stressor
it has not previously been adapted to (Selye, 1950). This reaction is firstly categorized by a
phase of shock, in which the body requires a surge of energy needed to react to the stressor.
The energy is a result of heightened adrenalin concentrations, which is produced through
activation of the sympathetic nervous system, causing the hypothalamus-pituitary-adrenal
gland axis to be stimulated. The adrenal gland consequently reduces in mass as ascorbic acid
and cholesterol stored in the gland are converted to adrenaline and corticosterols. More blood
is transported to the muscles, the liver produces more detoxification and other protective
chemicals (with a resulting decrease in weight of the liver), and the body supplies more energyproviding compounds (catabolism of body fat) (Fulder, 1980). Other physical changes include
loss of weight of the thymus, spleen, lymphatic glands, accompanied by acute damage to the
digestive tract, the development of ulcers and hypothermia. After the phase of shock, the initial
effects of the Alarm Reaction listed above, is reversed in a phase of counter-shock, mainly due
to an increasing amount of corticosterols in the blood stream (Panossian et al., 1999).
The phase of counter-shock brings about the advancement of the organism to the State of
Resistance if the presence of the harmful stressor persists for several days (Selye, 1950).
Increased resistance results in normalization of physiological functions: catabolism is replaced
with anabolism, adrenal lipids are deposited instead of being consumed, and blood sugar
levels and blood pressure levels are normalized - the body functions return to those of a
normal resting state (Panossian et al., 1999). Selye (1950) found that an animal in the State of
Resistance could not sustain prolonged over-exposure of a specific stressor. Eventually the
animal reaches a State of Exhaustion and symptoms of the Alarm Reaction re-appear. Organ
damage becomes evident once more, which eventually results in disease and possible death.
Head and Kelly (2009) reviewed prolonged effects of chronic stress on the human body:
natural killer (NK) cell cytotoxicity activity declines, which depletes the immune system
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surveillance capacity for viruses and cancer cells; lower capacity for production of secretory
immunoglobulin A (sIgA), an antibody contributing to immunity in mucosal secretions in the
digestive tract, mouth, lungs, urinary tract and other body cavities; imbalance in microflora in
the digestive tract required bifidobacteria and lactobacilli levels drop, while unwanted E. coli
and enterobacteria levels are raised. According to Selye (1950), the length of the State of
Resistance is directly related to the amount of adaptation energy the organism has. This view
is consistent with that of Rangel (2005) who suggested that energy is one of the three essential
factors that enhance the life of any living system, and that one of the characteristic modes of
action of tonics is to facilitate an increase in this energy to the body (see the previous section
on tonic mode of action).
Fulder (1981) proposed that the primary reaction to stress starts with secretion of
adrenocorticotrophic hormones (ACTH) from the hypothalamus and pituitary glands, which then
produces a cascade of subsidiary effects in the body. Some steroids and ACTH are able to act
directly in the brain as psychohormones and promote motivation, alertness and sensitivity
within a patient. Fulder (1980) suggested that the saponins in Panax ginseng (also see bidirectionality above; Sengupta et al., 2004) and Eleutherococcus senticosus might be
responsible for such a corticomimetic effect, as these adaptogens are able to help expand a
state of happiness (Davydov & Krikorian, 2000). This notion was reiterated in a review by
Panossian and Wikman (2009) on the relationship between fatigue, mental performance and
stress, focusing mainly on Rhodiola rosea and Eleutherococcus senticosus as model
adaptogens. They suggested that adaptogenic tonics act as challengers or mild stressors
(stress-mimetics) giving rise to the adaptive stress-protective effects affected through the
hypothalamus-pituitary-adrenal-axis, which then cascades to the nervous, cardiovascular,
endocrine, gastointestinal and immune systems. Enhanced mental performance is thus the
result of resistance to stress through adaptogen induced up-regulation of heat shock protein
(Hps70, molecular chaperons involved with adaptation to repeated exposure to an initial
stressor) which triggers stress induced JNK-1 and DAF-16-mediated pathways (Panossian &
Wikman, 2009; 2010). Apart from these antistress properties, antioxidant, anticarcinogenic,
immunomodulatory, hypocholesteroletic, hypoglycemic and choleretic activities are also
ascribed to these two herbs (Davydov & Krikorian, 2000). It seems that the greater the stress
applied, the stronger the protective effect of the adaptogenic tonic (Fulder, 1980).
Adaptogenic tonics, then, are a class of tonics which support health and prevent disease in
both sick and healthy individuals through nonspecific effects, which neutralize various
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environmental and physical stressors while remaining relatively safe and free of side effects,
as it is concisely defined by Bleakney (2008) (see Paragraph 2.1.2). The concept of the
adaptogen has its origin with a Russian researcher, Nikolai Lazarev (1947, 1959) with
substances responsible for a bodily state of non-specifically increased resistance. Brekhman
expanded the adaptogen concept considerably in the twenty years after Lazarevs findings
were published. He focused on the physiological effects plants, traditionally used as tonics, had
on the human body [Brekhman, 1968; Brekhman & Dardymov, 1969(a)]. Plants studied
included Panax ginseng, Rhodiola rosea and Eleutheroccocus senticosus. Brekhman and
Dardymov (1969a) noted that these herbs had the capacity of returning the body to the state of
homeostasis, or to prevent stress. Consequently, more detail was added to the initial definition
of an adaptogen (Brekhman, 1968). Firstly, an adaptogen should be innocuous (harmless, have
no adverse effect), causing minimal disturbance to the normal physiological functioning of the
organism. (see the bi-directional properties of tonics in the previous paragraph). Secondly, it
must have a broad spectrum of therapeutic action, i.e. not be applicable for specific indications
only (see synergism in Paragraph 6.1.2). Thirdly, an adaptogen should be a non-specific
protective, i.e. it should raise the bodys resistance to physical, chemical, or biological stresses,
and even more so when the organism is threatened with more intense stressors. The research
showed that adaptogens seemed to prevent damage of organs normally associated with the
Alarm Reaction (Selye, 1950), and increased non-specific resistance to the applied stresses.
Bu et al. (2005) showed that Eleutherococcus senticosus had a neuroprotective effect by
inhibiting inflammation and activating the microglia (macrophages that phagocytize pathogens
within the central nervous system, i.e. the first and main active immune defense in the central
nervous system). Adaptogens also mostly exhibits antioxidant capacity, which is important in
preventing cancer and generally enhances immunity (European Medicines Agency, 2007).
Lastly, an adaptogen should be able to enhance recovery through homeostasis, i.e. it should
normalize the physiological functioning of organisms irrespective of the direction of the
pathological changes. These conclusions were made based on research on cellular level. It
was found that when erythrocytes (red blood cells) were treated with the mentioned
adaptogens, they were not damaged by radiation as expected and they showed increased
protein synthesis as well as increased DNA replication. The effects were mostly ascribed to the
presence of saponins (eleutherosides and panaxosides) in the extracts [Brekhman &
Dardymov, 1969(b)].
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Panossian et al. (1999) showed that adaptogens were able to regulate the production of a
variety of physiologically required substances the body needs in reaction to stress. In doing so,
it is able to regulate homoeostasis through a series of switch on and switch off responses to
stressors, a mode of action also referred to as bi-directionality (Mowrey, 1998). The adaptogen
may switch on the defense reaction to stress by producing stress-markers (such as nitric
oxide,
catecholamines,
leukotrienes
and
cytokines)
which
activate
the
psychoneuroimmonulogic system. Adaptogens may then also trigger a switch off response to
stressors increasing the bodys ability to endure higher stress levels while preventing hyperreactivity that might cause damage to the body. Deactivating stress-markers include
corticosteroids and prostaglandin E2 which act as endogenous mediators of intercellular
communication (Panossian et al., 1999; European Medicines Agency, 2007). Panax ginseng,
for example, reportedly has such (contradictory) hypertensive and hypotensive properties (Han
et al., 2005; Siegel, 1980), and has also been found to exhibit both wound-healing (Morisaki et
al., 1995) and antitumor activity (Sato et al., 1994), by means of opposing effects on
angiogenesis (development of new blood vessels). Sengupta et al. (2004) ascribed these
apposing actions to two types of triterpenoids isolated from the same P. ginseng extract. Both
types of triterpenes have damarene backbones (see jujubogenin as example, Fig 5.30), but
differ in their level of hydroxylation and structures of their side-chains: Rg1 ginsenosides
(protopanaxatriols) lead to angiogenesis, whereas Rb1 ginsenosides (protopanaxadiols) have
the opposite effect. Furthermore, Rg1 molecules promote mitosis and stimulate the nervous
system, while Rb1 molecules inhibit mitosis in human lymphocytes and calm or soothe the
nervous system (Kim et al., 2002; Scott et al., 2001).
Assessment of adaptogenic ability includes the effects a herb has on the central nervous
system, its role in preventing fatigue, and consequently also in enhancing the immune system
(Kimura & Sumiyoshi, 2004; Pannosian & Wikman, 2009). Fulder (1980) and Asano et al.
(1986) summarized the effects Eleutherococcus senticosus had on Russian athletes: It
increased stamina and performance, with far fewer side-effects than the known stimulants at
the time (only occasional and transient rise of blood pressure was evident). An increase in
endurance, reflexes and concentration was also observed, particularly with athletes doing
longer races. Furthermore, the athletes could tolerate more training without injury. Mendez et
al. (2007) explained the validation of adaptogenic activity during and after ingestion of herbal
extracts by laboratory animals through tests performed which include:
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(a) Stress by immobilization and cold, to measure protection against ulceration (Alarm Reaction
Selye, 1950).
(b) Physical resistance is tested by letting the animal run on a treadmill until it is exhausted,
after which it is evaluated for grip or prehension strength, motor coordination and locomotor
activity. A positive outcome would be increased physical resistance or reduced fatigue (State of
Resistance and State of Exhaustion Selye, 1950).
(c) Evaluation of learning in a T-maze by old animals, where adaptogens are known to enhance
cognitive functions (State of Resistance Selye, 1950).
(d) The measurement of blood viscosity, as a decrease in blood viscosity has been shown to
improve cognition.
(e) Measurement of antioxidant capacity in vitro will indicate whether a herb is able to prevent
oxidative stress and damage resulting from cellular oxidation. This parameter, however, may
also be applicable for plants that are not adaptogens (European Medicines Agency, 2007).
Apart from these tests, the effects of immobilization, radiation, toxic drugs and chemicals as
well as diseases such as cancer and malaria may be assessed (Fulder, 1980; Mendez et al.,
2007). Animals that were treated with adaptogens prior to subjection to these stresses showed
less secretion of adrenalin, increase capacity to produce hormones by the adrenal glands, as
well as less damage to the stomach, spleen and thymus (Fulder, 1980). This indicates a faster
response to stress and a more rapid return to normality (see the Alarm Reaction and State of
Resistance Selye, 1950) (Fulder, 1980). Panossian et al. (1999) ascribed this to the
adaptogens being able to prevent the stress-induced increase of nitrogen oxide, associated
with a decline in ATP (energy) production.
While the term and concept of the adaptogen was coined by Lazarev, herbs have been used
as adaptogens since antiquity, especially in Traditional Chinese Medicine (TCM) and
Ayurveda, as well as the medicinal systems developed from these traditions. In both systems
the focus is on the patient and his/her environment rather than on the incurred disease (Gilbert,
1998; Cheng, 2000), and both systems are so sophisticated that medicines are divided into
subgroups to address specific aspects and symptom combinations related to different aspects
of the body. Adaptogens are known as superior herbs in TCM or rasayana in Ayurveda and
help balance the meridians while nourishing the adrenal glands, which are the major managers
of energy powering the human body (Gilbert, 1998; Cheng, 2000; Wu, 2005). Furthermore,
these adaptogens are grouped into several classes (Wu, 2005) among which are:
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(a) those that tonify qi(literally translated to air, breath or gas, but means life force or energy
flow in TCM Yellow Emperors Classic; prana in the Ayurvedic Vedas (1 5001000 BC, see
Chapter 2) and vitality (referring to mental activities and emotions; it is dependent on the
proper function of the five zang organs and vice versa),
(b) those that tonify blood (which has a nourishing and moistening function and has an effect
on mental health) and bodily fluids (such as intestinal juices and excretions from viscera,
tissues and organs also has a moistening and nourishing function in the body, as with blood)
(c) those that tonify yin and yang.
TCM and Ayurveda both suggest that when the body is in balance through proper functioning
of the organs and flow of qi and the other fluids, the body is able to heal itself. The terms are
explained through understanding of these two healing systems in which there are five
environmental elements considered are water, earth, metal, wood and fire. Yin and yang are
the two main antithetic aspects (opposites, i.e. positive and negative energy) in TCM affecting
homoeostasis in the patient relative to his/her environment. Four humors (q, blood, bodily
fluid and vitality) together with 12 zang organ systems (heart, lung, kidney, spleen, liver, small
intestine, large intestine, bladder, stomach, gall bladder, triple heater and pericardium) play
important roles in balancing the yin and yang in the human body. The term channel refers to
the route of q-flow through the body. These channels are associated with the zang organ
systems. The formation of vitality, blood and body fluid is dependent on the food, drink and
energy (qi) it is supplied with. Blood and bodily fluid pertain to yin and govern stillness, i.e. it
needs qi essence to move and fulfil its functions. Meridian channel tropism is a specific effect
of a substance on a certain part of the body, where a substance may have an effect on one or
several of these meridian channel systems.
6.1.3.2
Immunity and bitter tonics
The use of bitter tonics is an ancient practice believed to have beneficial effects on appetite
and digestion. Early mention of such bitter plants includes those from the Bible, such as
hyssop (Hyssopus officinalis), myrrh (Commiphora myrrha) and wormwood (Artemisia
absinthium). Hyssop is a stomachic with tonic, carminative, antispasmodic, sedative and
nervine properties, among others (Brooke, 1998; Bradley, 2006; Kizil et al., 2010). Myrrh is a
known bitter stomachic with carminative effects that acts as a tonic in combination with other
ingredients (Michie & Cooper, 1991; Su et al., 2011). Wormwood is mentioned several times in
the Bible as being very bitter, but also has antispasmodic properties and is used to treat
dysentery and neurodegenerative diseases (Bora & Sharma, 2010a,b).
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The 15 tonic plant species included in this study are all known as imbizas [i.e. a single plant
or mixtures of plants given orally or as enemas to improve general health Ngubane (1977), or
strengthening plant combinations which are believed to play a significant role in maintaining
health and vigour Van Wyk and Gericke (2000)] or to be incorporated in their preparation. All
of these tonic plants are traditionally known to be bitter, some more than others (Chapter 4,
Appendix 2). Furthermore, the term musa-pelo is used for 17 different species of shrubby
legumes (Fabaceae) in the Sotho culture, from which medicine is made, including Sutherlandia
frutescens, and are known to be bitter. These 17 plants are used to treat anxiety, stress and
grief; musa-pelo literally means to turn the heart around (Moteetee & Van Wyk, 2007).
Consequently, an attempt was made in this study for the first time to investigate the
relationship between the bitterness of 15 chosen species and their use as tonics
(Paragraphs 3.7 and 5.3).
Bitter tonics, digestive bitters or amara stimulate the taste buds and promote the secretion of
saliva, gastric juices and bile through taste stimuli via the nervus vagus (Van Wyk & Wink,
2004). Gentiana lutea, for instance, is one of the most bitter plants known, and has a bitterness
value of 10 00030 000 (Wagner & Wiesenhauer, 1995). It was listed as a bitter remedy for a
variety of digestive tracks ailments by Dioscorides as early as the first century (Osbaldeston &
Wood, 2000) and was being used as traditional Chinese medicine for the same indications
even before this time (Wu, 2005). It is a typical amarum (Leslie, 1978; Borgia, 1981), but is also
able to generate integrated responses such as secretion, motility and absorption within the
gastrointestinal tract upon ingestion (Sternini, 2007). Furthermore, G. lutea increases appetite
and stimulates digestion by stimulating acid production by the gastric mucosa in the stomach
(Gebhardt, 1997). Consequently food is better digested and immune disorders resulting from
food intolerance or dietary antigen leakage are improved. Increases in digestive secretions
also increase the tone of the gastro-esophageal sphincter, thereby preventing reflux of
corrosive stomach contents into the esophagus in 'heartburn', hiatus hernia, or esophageal
inflammation.
Extreme bitterness is not necessarily a requirement for a stomachic to be effective: of the 15
species tested, Withania somnifera, Hypoxis hemerocallidea, Warburgia salutaris, Balanites
maughamii, Arctopus monacanthus, Muraltia heisteria, Ziziphus mucronata and Agathosma
betulina returned lower bitterness values (Table 5.33), but these species are all used to treat
digestive disorders (Appendix 2; Chapter 4). This is possibly because bitter compounds are
suspected to be able to regulate metabolic and digestive functions via gene expression in the
gastrointestinal tract itself as well, and not only in the mouth (Behrens & Meyerhof, 2010).
Furthermore, bitter tonics are not necessarily digestive stimulants but may also have a bi207
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directional function, i.e. species of Hoodia are traditionally used in southern Africa as food
items as well as to reduce hunger and thirst, but can also be used to stimulate appetite (Van
Wyk & Gericke, 2009).
Additional effects such as antibacterial, antifungal, antioxidant and hepatoprotective properties,
have also been observed for bitter substances, apart from digestive stimulation (Kondo et al.,
1994; Kusar et al., 2006). Furthermore, bitters are postulated to have a potential immunological
influence as well by decreasing the sIgA-level in the saliva of patients with inflammable gastrointestinal diseases (Zimmermann et al., 1986). According to TCM, bitter substances have
draining and drying functions, i.e. they are used for clearing Heat, purging Fire, treating
constipation, resolving dampness, or lowering the rebelling q, and may preserve Yin (Wu,
2005). Heat is a term used to describe irritability and other aggressive behaviours, which
could lead to increased heart rate due to stress. Schulz et al. (2001) reiterated this finding when
he reported that bitter substances act on the cardiovascular system, decreasing the heart rate
and cardiac stroke volume. Bitter substances also aid in reducing fevers, inflammation and
infection (e.g. Centaurium erythraea (common centaury) (Berkan et al., 1991). Astragalus
membranaceus, Panax ginseng and Eleutherococcus senticosus are all bitter, and are known
immunostimulants and adaptogens (Van Wyk & Wink, 2004; Wu, 2005). To this effect, it should
be noted that 12 of the 15 bitter plants are traditionally used for stress or anxiety, and/or are
general health tonics (Appendix 2; Chapter 4). Of these 12 species, Sutherlandia frutescens
probably has the most profound tonic properties [Van Wyk 2008 (a),(b); Van Wyk & Albrecht,
2008]. The genus Sutherlandia is closely related to Astragalus, and contains structurally similar
bitter-tasting triterpenoids as are found in A. membranaceus (Van Wyk & Wink, 2004; Fu et al.,
2008.; Van Wyk & Albrecht, 2008; Olivier et al., 2009). Apart from being bitter, these triterpenes
may also have coticomimetic effects, responsible for immunostimulating effects not necessarily
triggered through bitter taste perception (Bedir et al., 2000; Prevoo et al., 2008). Other bittertasting compounds such as iridoids and flavonoids (the latter is also present in Sutherlandia Van Wyk & Albrecht, 2008) are powerful antioxidants, also contributing to increased immune
responses (Kondo et al., 1994; Kusar et al., 2006), while alkaloids are known mood and mind
drugs which addresses stress and immune response through improved mental health (as seen
in TCM where tonifying q has a direct impact in improved vitality) (Van Wyk & Wink, 2004; Wu,
2005). In view of the similarity between Sutherlandia and Astragalus, the bitterness of the
extracts tested (and knowledge on constituent compounds) as well as the musa-pelo and
imbiza uses of these plants in general (Van Wyk & Gericke, 2000; Moteetee & Van Wyk, 2007;
Van Wyk et al., 2009), it seems that there is a link between bitterness and the
stress/immunomodulating properties (Zimmermann et al., 1986) of tonics.
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6.1.3.3
Pharmacology
The placebo effect
A placebo (Latin for "I will please" Jacobs, 2000) is "a substance or procedure that is
objectively without specific activity for the condition being treated" (Moerman & Jonas, 2002).
These substances or procedures may include pills, creams, inhalants, injections, the use of
certain medical devices, and many more. Under this definition, traditional rituals or cultural
aspects pertaining to plant use are certainly also regarded as placebos (Moerman, 2000). The
placebo effect, a phenomenon where the use of an inert substance results in the medical
improvement of a patient, has however been a point of contention in the medicinal arena for
many years. Moerman and Jonas (2002) suggested the use of the term "meaning response",
instead of placebo effect, correlated with the meaning that the brain associates with the
placebo, which in turn causes a physiological placebo effect.
The placebo effect is based upon expectations and conditioning. To this effect, the
administration of traditional or plant based medicine is often accompanied by rituals (e.g. by
spiritual healers) or apt marketing (Ndhlala et al, 2011, Chapter 2), which most certainly
contributes to an appreciable placebo effect. Apart from the presentation of a medicine, the
information given about the medicine is also responsible for creating the said expectation with
the user. For example, where placebos have been prescribed as muscle relaxants, such
effects resulted (Flaten et al, 1999) and, when presented as stimulants, effects on heart rhythm
and blood pressure were observed (Kirsch, 1997). On the other hand, the effect disappears
when a patient is informed that the vehicle of medication was merely through a placebo
(Montgomery & Kirsch, 1997). The question then would be to what extent the South African
tonics described here would elicit a placebo effect, as all medicines assessed in clinical trials
reportedly show a placebo effect. One such an example was presented by Beedie and Foad
(2009), who reviewed the magnitudes of placebo effects on the performance of athletes, and
found the results to vary between -1.9% and 50.7%. Furthermore, they concluded that the
placebo effect observed in an individual may be correlated with the psychological potential of
the said individual to enhance the attainment of the desired/expected outcome proposed for
the administered medicine. When the medicine thus also contains nitrogen-compounds such
as amino acids or alkaloids, known to exhibit psychological effects (Van Wyk & Wink, 2004),
the magnitude of the placebo effect may even be elevated.
Placebo effects associated with herbal tonics thus play a positive role in the efficacy of these
plants. Examples have been recorded where beneficial end organ functioning and overall
health have been observed after administering placebos, suggesting a possible improvement
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in the immunity of an individual (Pacheco-Lpez et al., 2006). To this effect, much of what has
been presented in this study, is based on conjecture and speculation because there is almost
no clinical evidence to support the efficacy of tonics and their claimed pharmacological effects.
Unfortunately, it is extremely difficult to obtain supporting data because of the chemical
complexity of the tonic products and the large number of potential pharmacological effects and
benefits of tonics. Therefore, the only scientifically accepted method of proving beneficial
effects is through double-blinded placebo-controlled clinical studies.
6.1.4
Plant use in traditional healing and drug development
As per definition, tonics (imbizas) are strengthening mixtures of several plants, or of plants with
other components of mineral or animal origin (Ngubane, 1977; Ndhlala et al., 2011). The use of
traditional medicine and tonic mixtures for self-medication in South Africa is becoming more
prevalent, not only by indigenous people, but also in the western markets. In many instances
the choice of natural remedies is financially driven, where western pharmaceuticals may be too
expensive and often unattainable in remote areas. On the other hand, rapid growth and
urbanization of the black population, inflates the demand for plant medicines from muti markets
and other informal markets at an alarming rate. This may also be fuelled by the HIV/Aids and
tuberculosis pandemics in southern Africa (Mander & Le Breton, 2006). The plant material
traded informally and products produced thereof, are from plants wild-harvested by muti sellers
themselves. In most cases these traders have no training in harvesting plants and are not
ecologically responsible, as trained traditional healers are (Steenkamp, 2005). The huge
demand for medicinal plants or herbal preparations obtained by untrained traders and healers
as well as doubtful storage of this wild harvested plant material and prepared remedies, lead to
variations in the concentration of active compounds. As a result of this lack of quality control
and standardization, these treatments may be ineffective (due to too low a dosage), harmful
(due to too high a dosage or the presence of toxic co-extracted compounds), and may even
result in death (Drewes et al., 2006; Gertsch, 2011). Furthermore, most herbal products
marketed and sold in the western markets in South Africa, do not have proper scientific
(clinical) proof of efficacy. Additionally, it has been suggested that over-dose of uni-directional
stimulant tonics may result in severe imbalances (this cannot be the case with bi-directional,
modulatory or adaptogenic tonics as they exert homeostasis) (Mowrey, 1998). Certification of
plant material and herbal products is however becoming more important in the western market,
where higher standards in environmental management, production and quality control is
required (Mander & Le Breton, 2006). In Africa and in South Africa, product quality is variable
and often of questionable quality due to the absence or neglect of regulatory control.
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Research and regulation with regards to herbal medicinal products are consequently crucial,
not only for human safety, but also for ecological conservation purposes (Cunningham, 1988;
Williams, 1996; Crouch et al., 2008).
6.2
Biological activities of South African traditional tonic plants
The data given for the 14 selected tonic plants is a mere account of the various biological
activities of these plants reported in the literature and not a validation of the quality of results,
as this is beyond the scope of this chapter. Correlation between the ethnobotanical,
phytochemical and pharmacological information for each plant is a valuable tool in finding a
plausible scientific rationale for the said tonic properties of these plants. While the toxicity and
spectrum of therapeutic actions of tonic plants may be assessed through in vitro testing, their
actual effects on the ability to decrease physical, biological or chemical stress and enhance
immune response may only be measured when tested in vivo by means of placebo-controlled
double-blinded clinical trials (see Paragraph 6.1.3). Such research or the lack thereof is
highlighted for each of the species. The only example among the species studied here, where
applicable clinical and in vivo research focussed on tonic properties, is Withania somnifera
(Paragraph 6.2.13), an internationally acclaimed tonic.

The Agathosma species have been popular commercially for many years in the flavour and
fragrance industry due to their interesting essential oils (Moolla & Viljoen, 2008), which have
been the topic of phytochemical and pharmacological study more frequently than the polar
compounds. Antispasmodic carminative properties (Van Wyk & Gericke, 2000) of these oils
account for the uses related to stomach ailments such as flatulence, colic and stomach ache.
Lis-Balchin et al. (2001) showed, through in vitro investigation, that A. betulina has greater
spasmolytic ability than A. crenulata, with an initial smooth muscle spasmogenic activity
followed by spasmolysis. Spasmolytic action was dose related with an IC50 of 8 x 10-6 for
A. betulina. Antibacterial activity (Lis-Balchin et al., 2001; Viljoen et al., 2006(a); Moolla &
Viljoen, 2008) exhibited by the essential oils relate to uses for diarrhea and nausea, respiratory
ailments and treatment of wounds, where diosphenol is considered to be one of the
compounds responsible for the activity (Wichtl & Bisset, 2000). Essential oil activity against
Candida albicans, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus (Moolla et
al., 2007; Viljoen et al, 2006(a); see Table 6.1) is possibly due to monoterpene constituents
such as 1,8-cineol, borneol, linalool, eugenol, -pinene, limonene (Viljoen et al, 2006).
211
Chapter 6
Pharmacology
Agathosma species In general seem to show greater activity toward gram-positive bacteria
than toward gram-negative bacteria, and A. ovata seems to have the best activity against
Staphylococcus aureus and Bacillus cereus when compared to other South African Agathosma
species (Moolla et al., 2007).This enhanced activity of A. ovata might be due to the presence of
puberulin, a coumarin (Moolla et al., 2007). Lis-Balchin et al. (2001) however, found the
antimicrobial activity of A. betulina and A. crenulata essential oils (undiluted at 10 L/well) to be
weak against Escherichia coli, S. aureus, and Saccharomyces cerevisiae, while no activity was
shown against Enterococcus hirae or Pseudomonas aeruginosa. Moolla (2006) ascribed poor
activity to quenching interactions with the medium constituents, or insolubility of the oil in the
growth medium used for these experiments. Furthermore, essential oils with high levels of
monoterpene hydrocarbon are said to be active against bacteria, but not against fungi (Moolla,
2006). A. betulina and A. crenulata oils have also been shown to have significant
antiinflammatory activity, and while inflammation is a very complex process with possible
interaction at various sites, limonene is thought to play a major role (Viljoen et al., 2006(a);
Table 6.1). This activity is in agreement with the use of buchu for treatment of rheumatism,
arthritis and inflamed skin conditions (Chapter 4; Appendix 2).
Apart from the essential oils, the mucilage and flavonoids in the polar extracts also play a role
in the activity of buchu (Wicht & Bisset, 2000). When comparing in vitro antibacterial activity of
water, DCM:MeOH and MeOH A. betulina extracts against seven different pathogenic
microorganisms (Listeria monocytogenes, P. aeruginosa, C. albicans, E. coli, Proteus vulgaris,
S. aureus and Enterococcus faecalis), the DCM:MeOH extracts show the greatest activity with
MIC values ranging between 3 and 6 mg/mL (Sandasi, 2008). This activity was confirmed for
two of the pathogens (Moolla et al., 2007; Moolla & Viljoen, 2008), and the same study with
A. ovata MeOH:DCM (1:1) extracts showed that this species exhibited the highest activity of
the three species (Table 6.1; Moolla & Viljoen, 2008), possibly due to the presence of puberulin
in A. ovata only (Moolla, 2006). When subjecting the different A. betulina extracts to simulated
stomach and intestinal fluid conditions, the antimicrobial activity of the extracts seemed
unaffected except for the water extract which showed an increase in activity after exposure
(Viljoen et al., 2007). Evidence of synthetic derivitisation of the compounds in the extracts was
also found after subjecting the resulting intestinal fluid to LC-UV-MS analysis (Viljoen et al.,
2007). In the same study, ca. 100 % of the water extract was found to be transported across a
Caco-2 cell monolayer in vitro model, indicating almost complete absorption and bioavailability
of the extract to the body through the intestines. The mucilage may provide a mild laxative
effect, and the diuretic properties may be a result of the flavonoids or terpinen-4-ol in the
212
Chapter 6
Pharmacology
extracts and oil respectively (Wichtl & Bisset, 2000). Despite the presence of flavonoids, the
antioxidant activity of these Agathosma species is not particularly significant (see Chapter 5.2;
Wollenweber & Graven, 1992; Moolla, 2006; Moolla et al., 2007). However, over 100
references can be found on the Academic Search Complete databases of EBSCO Host for
titles containing hesperidin or hesperetin respectively (search done on 19 December 2011), not
to mention the other flavonoids identified in buchu species. Among these references are the
cardioprotective effects of hesperetin (Trivedi et al., 2011), and the restoration of impaired
immune and nutritional function in irradiated mice by hesperidin (Lee et al., 2011), which both
have direct bearing on the tonic properties of buchu. The use of buchu as aperitif (Van Wyk
and Gericke, 2000) is probably due to the slight bitter taste of the water extract (bitterness
value of 1 44014 400, see Paragraph 5.16), and also contribute to restoration in nutritional
function.
Buchu presents an example of synergistic enhancement of activity among its constituents.
Cineol is a stimulant which may be absorbed by inhalation or through the skin, with the latter
being more effective (Cseke et al., 2006). The same is true for limonene, -pinene and
camphor, all of which are able to cross the blood-brain barrier and act as weakly uncompetitive
reversible inhibitors of human erythrocyte acetylcholinesterase. When combined in an essential
oil, these monoterpenes show significant inhibitory activity on acetylcholinesterase (Perry et al.,
2000), which helps preserve brain levels of choline and acetylcholine and curb the occurrence
of Alzheimers disease (Cseke et al., 2006). Furthermore, hesperetin and quercitin showed IC50
values of 18 and 10.4 g/mL respectively against breast cancer cells, but showed significantly
lower values when combined, also indicating synergy (So et al., 1996).
It should however be noted that cis-isopulegone or R-(+)-pulegone contained in buchu
essential oils are toxic [Table 6.1; Moolla 2006, Viljoen et al., 2006(a)]. R-(+)-Pulegone itself is
hepatotoxic, causing the oil to irritate the gastrointestinal tract and kidneys (Moolla, 2006).
However, toxicity assessment of MeOH:DCM extracts of the three species showed that
A. betulina and A. crenulata are not toxic (Moolla et al., 2007).
In summary, in vitro testing have provided a possible scientific rationale for the antispasmodic
and antimicrobial properties of the three Agathosma species. However, there is no clinical or in
vivo evidence available yet to confirm the tonic properties of these species even though some
of the compounds have been subjected to such tests. An example is the treatment of irradiated
mice with hesperidin which resulted in restoration of impaired immune and nutritional function.
213
Chapter 6
Table 6.1:
Pharmacology
The in vitro biological activity of extracts and essential oils from Agathosma betulina, A. crenulata and A. ovata
Species
(sample
tested)
S. aureus
(MIC mg/mL)
B. cereus
(MIC mg/mL)
K.
pneumonia
(MIC mg/mL)
Activity tested
C. albicans
Antiinflammatory
1
(MIC mg/mL) activity
(IC50 g/mL)
Cytotoxicity
(IC50 mg/mL)
A. betulina
(essential oil)
32
50.37
< 0.0001
24
59.15
< 0.0001
16
36.83
< 0.0001
32
4
4
4
8
4
8
2
> 100
> 100
> 100; > 100; 37.75
24
2
3
2
8
4
4
2
> 100
> 100
> 100; > 100; 33.32
1.00
0.16
1.06
0.13
7
8
6
4
81.15
25.20
51.45; 24.71
> 100; 46.81
A. crenulata
(essential oil)
A. ovata
(essential oil)
A. betulina
(essential oil)
(extract)
A. crenulata
(essential oil)
(extract)
A. ovata
(hook leaf)
(round leaf)
1
Antioxidant
(g/mL)
(DPPH; ABTS)
Refs
Viljoen et
al., 2006
Moolla et
al., 2007;
Moolla &
Viljoen
2008
by means of inhibition of 5-lipoxygenase enzyme; 2 by means of MTT cellular viability assay
214
Chapter 6
Pharmacology
6.2.2 Aloe species

Whole Aloe leaves, the exudate and the gel have medicinal applications (Appendix 2;
Chapter 4; Grace et al., 2008; Joseph & Raj, 2010). Aloe lump is mainly used as stimulant
laxative and cathartic, where specifically 1,8-dihydroxyanthracene glucosylated derivatives
such as aloin A and B, are responsible for the effect (Van Wyk et al., 1995; Blumenthal,
1998). These -glycosides are prodrugs which are poorly absorbed through the gastrointestinal tract. They are, however, degraded in the colon through reductive cleavage by
bacterial enzymes into aloe-emodin anthrones, which are responsible for the laxative effect
(Blumenthal et al., 1998). The anthrones irritate the mucous membrane, resulting in
increased mucous secretion and peristalsis, with simultaneous inhibition of water and
electrolyte absorption (Wichtl & Bisset, 2000). Cape aloes are included in Swedish bitters,
together with Aurantii pericarpium (bitter orange peel), Croci stigma (stigma of Crocus sativus
L.), Galangae rhizome, Myrrha, Rhei radix, Sennae folium and Sennae fructus, which is
made into a tincture with alcohol, taken dropwise for indigestion, as laxative and as panacea
(Wichtl & Bisset, 2000). Aloe ferox is also an ingredient of Lewensessens (Aspen
Pharmacare), one of the best-selling traditional herbal products in South Africa (Van Wyk et
al., 2009). Hydroxyanthracenes (such as aloin) and phenylpyrones (such as aloenin) add to
the bitterness of these mixtures (Dagne et al., 2000; Wichtl & Bisset, 2000), where A. ferox
lump returned a bitterness value of 11 556720 000 (Paragraph 5.16). It should be noted,
however, that Aloe exudate is an example of a uni-directional stimulant which may upset
homeostasis of the digestive tract upon improper use. It is suggested that the use of
exudates as laxative should be restricted to acute constipation (0.06-0.17 g in adults and
children over 10 years of age, corresponding to 10-30 mg hydroxyanthracenes per day; or
100 mg taken as a single dose in the evening) (Blumenthal, 1998; ESCOP, 2003), as it may
upset the electrolyte balance and cause damage to the heart or kidneys (Newall et al., 1996;
Wichtl & Bisset, 2000). It is consequently contraindicated during pregnancy, menstruation,
breast-feeding, as well as for inflamed intestinal diseases such as ulcerative colitis.
Chromones (aloeresins I and H, as well as aloesin) from A. vera and A. ferox exhibit
antiinflammatory (Speranza et al., 2005; Fawole et al., 2010; Mwale & Masika, 2010),
acetylcholinesterase inhibiting and antioxidant properties (Hu et al., 2003; Fawole et al.,
2010), which may contribute to the prophylactic, analgesic (Mwale & Masika, 2010) and
curative effect of the exudates on gastric lesions, irritable bowel syndrome and inflamed
bowel disease (Steenkamp & Stewart, 2007). Two dihydrocoumarin derivatives from Aloe
215
Chapter 6
Pharmacology
vera have also been shown to exhibit antioxidant activity against superoxide and hydroxyl
radicals, and one of the compounds exhibited possible immunomodulatory activity in relation
to increasing the phagocytic activity and stimulating the production of superoxide anions in
the oxygen respiratory burst of rat peritoneal macrophages (Zhang et al., 2006). The
immunomodulating activity of A. arborescens was suggested to be due to the presence of
aloctin A (Imanishi, 1993). A. vera gel has also been shown to cause restoration of cellular
immune response of immunosuppressed mice (Oronzo-Barocio et al., 1999), possibly due to
the presence of the polysaccharide, aloeride (Pugh et al., 2001). Acemannan specifically has
been associated with this activity with particular relevance in inhibition of the Aids virus
(Kahlon et al., 1991), but these results are now controversial. Furthermore, A. vera has been
shown to decrease oxidative damage of the brain, memory impairment and motor dysfunction
in diabetic mice (Parihar et al., 2004).
Furthermore, anti-diabetic properties have been described for A. arborescens, A. ferox and
A. vera (extracts, gel or leaf pulp) in vivo in rats and mice (Okyar et al., 2001; Rajasekaran et
al., 2005; Beppu et al., 2006; Tanaka et al., 2006; Prez et al, 2007; Loots et al., 2011).
Ghannam et al. (1986) produced clinical evidence of the hypoglycaemic effect of aloe sap.
The antitumor properties of aloes have been ascribed to the antitumor activity of aloe-emodin
(Chen et al., 2012), or polysaccharides (Wang et al., 2001). Tumor regression and disease
control have also been observed for patients (240 randomized patients with solid metastatic
tumors) treated with A. arborescens as adjunct to chemotherapy (Lissoni et al., 2009).,
Estrogenic (Steenkamp & Stewart, 2007), hypotensive (Steenkamp & Stewart, 2007),
hepatoprotective (Arosio et al., 2000; Can et al., 2004), radioprotective (Pande et al., 1998),
antiviral (Kambizi et al., 2007) and antibacterial activitiesKambizi et al., 2005; Kambizi &
Afolayan, 2008) Additionally, antibacterial properties were described for A. arborescens and
A. marlothii; (McGaw et al., 2000; Luseba et al., 2007)].,
Aloe gel has important wound-healing and antiinflammatory properties (Lindsey et al., 2002;
Diallo et al., 2001), while anti-allergic, antibacterial, anticancer, fungicidal, immunostimulant
and anti-arthritic properties have also been reported for A. ferox and A. vera (Newall et al.,
1996; Blumenthal, 1998; Reynolds & Dweck, 1999; Wichtl & Bisset, 2000; ESCOP, 2003;
Boudreau & Beland, 2006; Steenkamp & Stewart, 2007). The wound-healing properties of
A. ferox gel is ascribed to its hydrating, insulating and protective effects (Bruneton, 1995),
where polysaccharides (Qui et al., 1999) and mineral ions (i.e. K, Na, Ca and Mg) in Aloe gel
have been reported to play a key role in enzymatic reactions responsible for cell growth and
216
Chapter 6
Pharmacology
maintenance (Motykie et al., 2004). -Sitosterol reportedly increases collagen activity and
suppression of contact hypersensitivity (Steenkamp & Stewart, 2007), while malic acid
acylate carbohydrates (such as veracylglucans A, B and C in A. vera gel) have been found to
possess antiinflammatory properties and affect cell growth (Esua & Rauwald, 2006). The
polysaccharides also exhibit an adjuvant activity on specific antibody production and
enhancement of cytokine release (Steenkamp & Stewart, 2007), which may contribute to the
immunostimulant properties of the gel. Dagne et al. (2000) suggests that the wide range of
biological activities of Aloe gel is due to synergistic action between its components.
When assessing the biological activity of Aloe species in relation to possible tonic properties,
the low toxicity (in vivo and clinical results) and a wide spectrum of therapeutic actions (in
vitro) that have been reported in numerous scientific papers is noteworthy. An enhanced
resistance to chronic stress is suggested by in vivo results in rats and mice when A.
arborescens, A. ferox and A. vera (extracts, gel or leaf pulp) are tested for anti-diabetic and
antitumor properties. Protective tonic properties are indicated by the antiinflammatory,
analgesic hypotensive, hepatoprotective, radioprotective and immunomodulating activities of
Aloe. Surprisingly, convincing clinical evidence of tonic ability is still lacking.

The medicinal value of the polar phenolic acids in Arctopus species, i.e. caffeic acid,
commonly found in medicinal plants, (R)-3-O--D-glucopyranosylrosmarinic, and especially
rosmarinic acid, are ascribed to antioxidant, antiphlogistic, astringent, antiinflammatory,
antimutagenic, antibacterial and antiviral activities (Burger & Wachter, 1998; Parnham &
Kesselring, 1985; Petersen & Simmonds, 2003; Olivier et al., 2008). The triterpenes (possibly
barrigenol sapogenins, see Paragraph 5.2.3) may contribute antiinflammatory, analgesic and
antibiotic properties to Arctopus species, as suggested for Pittosporum viridiflorum from
which it has been identified (Van Wyk et al., 1997). Corticomimetic and amarum effects (polar
Arctopus extracts are bitter, see Paragraph 5.3) may also be found, as are typical from
saponins.
Stafford et al. (2005) reported that ethanolic Arctopus extracts stimulated the binding of
flumazenil to the GABAA-benzodiazepine receptor in a dose-dependent way, indicating that
the species has sedative and potentially CNS-acting ability. This activity was ascribed to the
presence of diterpenes in the said extract. Somova et al. (2001) further reported that extracts
of Alepidea amatymbica, which is genetically related to the Arctopus species, and the
217
Chapter 6
Pharmacology
diterpenes isolated from it (including ent-trachyloban-19-oic acid and ent-kaur-16-en-19-oic

acid) displayed substantial hypotensive effects, decreasing the heart rate. It also had diuretic
activity. Ambrioso et al. (2006) listed analgesic properties for ent-kaur-16-en-19-oic acid, also
adding its ability to induce hyperthermia and its activity on vascular smooth muscle
contractility by inhibiting the in vitro contractility of the rat aorta at 20.0 g/mL. The C-19
carboxyl group is key to the inhibitory ability and methylation of this carboxyl group reduces
but does not abolish the antispasmodic activity (Ambrosio et al., 2004). A study directed at
the antimicrobial activity of the root extracts, showed good to moderate activity against nine
different pathogens, including three Gram-positive and four Gram-negative bacterial strains,
as well as two yeasts (Magee et al., 2007). The highest activity was reported against
Staphylococcus epidermidis and S. aureus. The activity may be ascribed to the diterpenes
present, as manool has been found to exhibit antibacterial activity against S. aureus as well
as antifungal activity against Candida albicans (Ulubelen et al., 1994). The antimicrobial
activity of ent-trachyloban-19-oic acid is reportedly comparable to that of ent-kaur-16-en-19oic acid (Zgoda-Pols et al., 2002), and include antibacterial, antifungal as well as
molluscicidal activity (Garca et al., 2007). Kaurenoic acid (Ghisalberti, 1997), enttrachyloban-19-oic acid (Das-Viciedo et al., 2008) and manool (Yang et al., 2010) also
showed antiinflammatory activity. Anticancer properties have further been described for
kaurenoic acid (it displayed antiproliferative action in tumor cells, including human breast
cancer, human colon cancer and leukemia cancer Ambrosio et al., 2006; Fatope & Audu,
1996) and manool (it exhibited cytostatic activities against human malignant cell strains,
specifically against HT-29 human colon adenocarcinoma cells, without affecting normal
human cells Pratsinis et al., 2010). Lastly, synergistic trypanomicidal activity (trypanosome
infection from tsetse flies causes sleeping sickness in humans) has been observed when entkaur-16-en-19-oic acid is combined with kauran-16-ol (Batista et al., 2007). The latter is a
hydroxylated kaurene from Alepidea (Holtzapfel et al., 1995), possibly also present in
Arctopus (see Chapter 5).
The traditional uses of Arctopus species (as general tonics, treatment of infections and for
relief of epilepsy, see Appendix 2 and Chapter 4) are strongly supported by the confirmed
presence of relatively high levels of phenolic acids, diterpenes, triterpenes and GABA in the
rhizomes and tubers (i.e. the parts that are used medicinally), together with reports of
antimicrobial activity. However, no clinical or in vivo research is available to confirm the tonic
properties of Arctopus.
218
Chapter 6
Pharmacology

In two recent reviews featuring A. afra as a commercially important South African medicinal
plants, Van Wyk (2008b) and Liu et al. (2009) listed many references showing the high
antimicrobial efficacy, as well as narcotic, analgesic, anti-histaminic, spasmolytic, antioxidant,
antinematodal, antimalarial, anti-convulsive, hypotensive cardiovascular and sedative
properties. An in vivo (rabbit model) study showed that the aqueous extract (at doses of 10
45 mg/kg) has a hypotensive effect and a dose-dependent biphasic effect on rabbit heart
tissue in vitro. Lower doses initially induced cardiostimulation, followed by cardiodepression,
whereas higher doses were mainly cardiodepressant. This effect was ascribed to scopoletin
which, at a dose of 1.02.5 mg, induced a dose-dependent decrease in inotropic activity
together with an appreciable decrease in chronotropic effects, especially at higher dose
levels (Guantai & Addae-Mensah, 1999). Van Wyk et al. (2008) also noted that Artemisia afra
may be used interchangeably with Agathosma betulina for treatment of the same ailments.
Comparison between the phytochemical constituents of the two plants (see Chapter 5) shows
that some components are indeed the same for both species, i.e. 1,8-cineol, borneol and pinene (monoterpenes) in the volatile component, as well as quercitin, diosmetin and
kaemferol (flavonoids) in the polar extracts. Both A. afra and A. betulina also contain
coumarins, suggested to enhance the antimicrobial activity exhibited by the essential oils
(Moolla, 2006). Rabe and Van Staden (1997) reported that antimicrobial activity was evident
from MeOH rather than from aqueous extracts, indicating that the antimicrobial components
may be monoterpenes as well as the sesquiterpene lactones, flavonoids and coumarins of
medium polarity. The latter two classes of compounds together with the isolated
acetophenones, should have antioxidant capability due to their phenolic structures, but Burits
et al. (2001) showed that the volatile oils and a sesquiterpene, chamazulene, were also
responsible for radical scavenging. Scopoletin, a coumarins also has antioxidant capacity,
antiinflammatory,
antimicrobial,
antithyroid,
antihyperglycemic
(Gakuba,
2009),
immunomodulatory (Manuele et al., 2006) and hepatoprotective (Kang et al., 1998) properties
in vitro. Borneol, 1,8-cineol and -pinene have been shown to be absorbed through the skin
or through inhalation, pass the blood brain barrier and inhibit acetylcholinesterase (Cseke et
al., 2006), while taurin has been shown to decrease the incidence and severity of
audiogenically induced convulsions in vivo (Gakuba, 2009). This, together with the presence
of flavonoids and GABA (see Chapter 5), may be the reason for the anti-convulsive, sedative
and GABAA-benzodiazepine receptor-binding activity of A. afra (Stafford et al., 2005). To this
effect, the ethanolic extract (in which GABA is extracted) exhibited a concentration-related
effect on the serotonin transporter protein, showing the potential of depression treatment
using A. afra (Nielsen et al., 2004).
219
Chapter 6
Pharmacology
Even though A. afra is does not contain artemisinin (Van der Kooy et al., 2008), the
antimalarial component of several other Artemisia species, it still exhibits antimalarial
properties. This activity has been ascribed to flavonoids (two acacetin derivatives, genkwanin
and
apigenin)
and
sesquiterpene
lactones
(two
rupicolin
derivatives
and
dihydroxybishopsolicepolide) (Kraft et al., 2003). These antimalarial components are all of

medium to low polarity, explaining why extracts from lipophilic (non-polar) solvents such as
petroleum ether and DCM (Weenen et al., 1990; Kraft et al., 2003), and MeOH (Gathirwa et
al., 2007) show high activity against Plasmodium falciparum.
As with Agathosma betulina, Artemisia afra is also a good example of synergistic interaction.
Cseke et al. (2006) described a synergistically improved inhibition of acetylcholinesterase
when the monoterpenes mentioned are administered in combination rather than isolated (see
Paragraph 6.1.2). Viljoen et al. [2006(b)] also described significant antibacterial activity
against K. pneumonia and C. neoformans only when the major A. afra monoterpene
constituents were used in combination. Van Vuuren (2007) reported synergistic interaction
between the volatile oils of A. afra and Lippia javanica against K. pneumonia, which is
significant because these plants are used in combination for the treatment of colds and
influenza (Appendix 2D). Kraft et al. (2003) indicated that the antiplasmodial activity is
synergistically, or at least additively, better where the active principles are combined. Cseke
et al. (2006) reiterated this synergistic antimalarial activity in other Artemisia species by
mentioning examples where extracts were more potent than the active principle (artemisinin)
by itself.
There are indications that A.afra may be toxic when used in high doses or for prolonged
periods. The potential toxicity is due to an unidentified sesquiterpene lactone (Jennet-Siems
et al., 2002) and thujone [(Viljoen et al., 2006(b)]. Thujone is known to cause confusion,
convulsions and even coma (Van Wyk, 2008b) but fortunately it is poorly soluble in polar
solvents (i.e. it will not be present at high concentrations in aqueous extracts used
traditionally). The volatile oil composition is variable [(Viljoen et al., 2006(b)], so that
chemotypes producing oils with low thujone content can be cultivated for commercial use.
Mukinda and Syce (2007) did however perform a chronic toxicity assay in rats and mice
where no significant changes in general behaviour, body weight, haematological and
biochemical parameters, organ weight or morphology of the organs occurred with oral
administration of doses of 0.1 or 1 g/kg/day of extract for three months.
220
Chapter 6
Pharmacology
The only in vivo evidence of biological activity is that of hypotensive effects ascribed to
scopoletin. There is as yet no direct evidence for any tonic properties but the wide range of
biological activities reported in in vitro studies suggests that A. afra may have some value as
a general tonic.

Very little is known with regards to the pharmacognosy and phytochemistry of B. maughamii.
Prozesky et al. (2001) reported that the antiplasmodial activity of DCM bark extracts of
B. maughamii against Plasmodium falciparum was five times greater than that of
B. aegyptiaca. Various insecticidal properties of B. aegyptiaca have been described (i.e.
larvicidal, anthelmintic and molluscicidal properties Yadov & Panghal, 2010). These
activities are mostly ascribed to the saponins contained in the root, bark, seed kernel and fruit
of the trees, and which exhibit the larvicidal activity by means of a disarranging interaction
with the cuticle membranes of the larvae, thus interfering with their aquatic breathing. The
kernel and pulp of ripe fruit are toxic to snails at concentrations of 25 mg/ml, and
molluscicidal activity is retained in powdered material for up to 122 days. The use of
B. maughamii for its molluscicidal properties was first proposed in the 1930s when it was
observed that proliferation of bilharzia snail-vectors were inhibited when the fruits fell in
infested water (Pretorius et al., 1988). The same effect is observed with guinea-worm, even if
the tree grows next to the affected water (Sands, 2001). It is postulated that yamogenin, the
steroidal sapogenin to which molluscicidal activity is attributed in B. aegyptiaca, is also
present in B. maughamii, but in higher concentrations (Kloos & McCullough, 1982).
Unfortunately, B. maughamii, has been found to be mutagenic (i.e. it causes DNA damage
and chromosomal aberrations Taylor et al., 2003)
A wide range of biological activities have been demonstrated (in vitro and in vivo) for B.
aegyptiaca but thus far not for B. maughamii. These include antiinflammatory, antinociceptive, antioxidant (Speroni et al., 2005), sedative and anti-convulsant (Bum et al.,
2005), antibacterial, antifungal, hepatoprotective, anticancer as well as anti-diabetic (Yadov &
Panghal, 2010) properties. No clinical data exist to support the biological activities (or tonic
uses) ascribed to B. maughamii and B. aegyptiaca.
221
Chapter 6
Pharmacology

The leaf and root hexane extracts of D. anomala have shown strong in vitro antiinflammatory
activity (> 80% inhibition), with similar results for MeOH leaf extracts (Shale et al., 1999). The
activity may be due to sesquiterpene lactones, as they are soluble in hexane and MeOH
(Paragraph 5.2.6). D. anomala has shown 75% inhibition at 33 M (in vitro) of recombinant
GSTP-1 (i.e. an enzyme, glutathione transferase P1-1, which is over expressed in some
cancer cells, contributing to detoxification of anticancer drugs, which in turn leads to drugresistant tumors) (Mukanganyama et al., 2011). Van der Merwe (2008) reported the
anticancer properties of two isolated sesquiterpenes from D. anomala, both more active than
the crude extract. Dehydrobrachylaenolide showed activity (GI50 = 17.9 M) in a 60-cell line
panel comparable to parthenolide, a sesquiterpene currently used in clinical development as
an amino derivative. The dimer showed the highest activity (IC50 = 0.68 g/mL) against
Chinese hamster ovarian cancer. Other Dicoma terpenes exhibiting anticancer properties
include -humulene (also a sesquiterpene Pichette et al., 2008), lupeol and -sitosterol.
Lupeol is a triterpene with known antiinflammatory and anticancer properties (Saleem, 2009),
where anticancer efficacy was demonstrated for skin cancer (Saleem et al., 2004) and
prostate cancer (Siddique et al., 2011) in vivo. -Sitosterol is a phytosterol reported to inhibit
growth and metastasis of prostate, colon and breast cancer in vitro (Awad & Fink, 2000).
D. capensis has shown pronounced cytotoxicity by inhibiting proliferation of DU-145 (human
prostrate carcinoma), MCF-7 (breast cancer) and MCF-12A (non-malignant breast cancer)
cells (Steenkamp & Gouws, 2006)..
D. anomala and D. capensis both exhibited high bitterness values (8 00012 000 and 8 000
72 000 respectively, Paragraph 5.3), which may be due to its saponins(s), flavonoids and/or
sesquiterpene lactones, the latter of which are known to be very bitter (Hutchings et al.,
1996). These high bitterness values will undoubtedly contribute to an amarum effect upon
ingestion. The flavonoid scutellarein has been shown to have effects on pathological changes
to the heart caused by hypertension, i.e. reverse ventricular remodelling, improve
cardiovascular stiffness and protect the cardiac muscle (Zhou et al., 2002). Interestingly, both
the aspect of bitterness and hypertension is addressed in the musa-pelo concept, where
bitter plants are indicated to turn the heart around (Moteetee & Van Wyk, 2011;
Paragraph 6.1.2.2).
Furthermore, D. anomala exhibits in vitro and in vivo antimalarial (Matsabisa, 2001;
Tselanyane, 2007; Van der Merwe, 2008) as well as anthelmintic activity (Mlgaard et al.,
2001). Tselanyane (2007) ascribed to the antiplasmodium activity to two unidentified dimeric
222
Chapter 6
sesquiterpene
brachylaenolide
Pharmacology
lactones.
and
These
sesquiterpenes
guaianolide
dimer,
were
where
later
the
identified
former
was
as
dehydro-
more
active
[IC50(D10) = 0.38 g/mL; IC50(KI) = 0.06 g/mL] (Van der Merwe, 2008). A moderate in vitro
anthelmintic effect is seen at a concentration of 31.0 mg root/mL after 1 hr, and continues for
slightly more than 24 hrs, where a negligible anthelmintic in vivo effect was seen.
The literature presented here does not include clinical results for any of the Dicoma species.
However, the presence of compounds exhibiting antiinflammatory, anticancer and cardiac
properties is encouraging when considering the use of D. anomala and D. capensis as
cardiac tonics (Paragraph 4.2.6). The high bitterness values for these plants are also
significant as D. capensis is known as a bitter tonic (Paragraph 4.2.6; Van Wyk (2008a).

H. procumbens is mainly known as treatment for internal and external pain, but is also
applied for gastrointestinal problems (Chapter 4, Appendix 2). Most of the research reported
to date has been directed at the antiinflammatory and analgesic properties of H. procumbens
with regards to the treatment of osteoarthritic, rheumatic and other forms of musculoskeletal
pain, with several reviews on in vitro, in vivo and clinical research (Brien et al., 2006; Grant et
al., 2007). Its antiinflammatory properties have been assessed in vivo through treatment of
carrageenin-induced rat paw oedema, adjuvant-induced arthritis and fresh egg albumininduced acute inflammation in animals (rats, mice and guinea pigs) where the effects were
variable, but some indicated equivalent efficacy to that of phenyl butazone, a conventional
anti-arthritic drug (Grant et al., 2007). Inhibition of inflammation was shown to be dosedependent (Lanhers et al., 1992) in a chronic rather than acute treatment (Newall et al.,
1996). Schultz et al. (2001) showed that the anti-rheumatic and antiphlogistic properties of
H. procumbens are comparable to that of pyrazole. Other antiinflammatory tests include
measuring of responses in deeper tissues through post-transcutaneous delivery when the
extract is applied to the skin (ex vivo tests on freshly excised porcupine skin (Quitas & Heard,
2009) and measuring the in vivo protective effect of H. procumbens on cartilage in rabbits
(Wachsmuth et al., 2011). Writhing/latency of paw tests as well as chemically and thermally
induced pain were used to measure the analgesic efficacy of H. procumbens, where a
protective effect against pain was reported in both acute and chronic experimental conditions
(Andersen et al., 2004). Furthermore, the antioxidant effects of H. procumbens on rats
reported upon analyzing the frontal cortex and striatum after exposure to alcoholic extracts
for one, seven or 14 days, showed an increase in protective enzymes leading to the reduction
of free radicals (Battacharya & Battacharya, 1998). Since oxidative free radicals are over-
223
Chapter 6
Pharmacology
expressed in patients suffering from inflammation (Halliwell et al., 1988), the antioxidant
potential of H. procumbens was reported to affect its antiinflammatory properties (Grant et al.,
2009). The clinical effectiveness and safety of H. procumbens in the treatment of
osteoarthritic and rheumatic articulation of the hip or knee, as well as chronic non-specific
back pain and various other forms of musculoskeletal pain in terms of pain and motility have
also been reviewed (Chrubasik et al., 2003; Gagnier et al., 2004; Grant et al., 2004). Results
from open, double-blind placebo controlled and double-blind comparative tests were listed
and showed variable results. Furthermore, several different clinical studies with isolated
iridoid
glycosides
and
their
metabolites
indicated
that
these
compounds
have
antiinflammatory and slight analgesic properties due to their ability to inhibit cyclooxigenase
(Wegener, 2000). It was shown that isolated harpagoside had no effect on inflammation, but
contributed to peripheral analgesia (Lanhers et al., 1992).
Where the use for gastrointestinal ailments are concerned, H. procumbens has been shown
to be a bitter tonic with a bitterness value of 5 00012 000 (comparable to gentian root),
which is ascribed to the bitter iridoid glycosides (0.51.6 %) in the polar extracts (Wichtl &
Bisset, 2000; Schulz et al., 2001). It has also been indicated as a successful treatment for
diseases of the upper duodenum in which the pancreas is involved, accompanied by
disturbances of cholekinesis and choleresis improved enterohepatic circulation and bile
activation have been indicated (Iwu, 1993; Wichtl & Bisset, 2000). No spasmolytic activity
was evident.
Activities for H. procumbens which address adaption to stress include the lowering of arterial
blood pressure in rats, a decrease in heart rate in rabbits and protection against arrhythmias
caused by aconitine, reperfusion, adrenalin/chloroform or calcium chloride (Iwu, 1993; Newall
et al., 1996). The latter effect is said to be based on the antioxidant capacity of the species
(Beresford, 2006). Apart from the antioxidant capacity of the phenyl ethanoids, fractions
containing these compounds have also been shown to strongly inhibit cholinesterases (even
better than the galanthamine control used), indicating possible application in treatment of
Alzheimers disease (Georgiev et al., 2011). H. procumbens is also used to treat neuralgia
and headaches, and has shown anti-convulsant activity in mice through suppression of the
CNS (Mahomed & Ojewole, 2006). In cases of patients with hypercholesterolemia, it was
found that serum cholesterol, uric acid and natural fat levels are lowered upon treatment with
H. procumbens (Tyler et al., 1981). For cancer treatment, in vitro studies have shown that an
ethanolic extract (i.e. 2.9% harpagoside) inhibited the release of tumour necrosis factor (TNF). Isolated harpagoside and harpagide were found ineffective in the same study, indicating
224
Chapter 6
Pharmacology
synergism amongst constituent phytochemicals (Fiebich et al., 2001; Kaszkin et al., 2004).
Significant hypoglycaemic effects were evident when a dose-dependent (50800 mg/kg i.p.)
reduction of blood glucose was observed in both fasted normal and fasted diabetic rats
(Mahomed & Ojewole, 2004).
Other activities include weak antifungal activity of extracts against Penicillium digitatum and
Botrytis cenerea (Newall et al., 1996), and antiplasmodial activity ascribed to abietane and
totarane diterpenes isolated from H. procumbens (Clarkson et al., 2003). Immunomodulatory
effects (in vitro) of H. procumbens have also been reported (Spelman et al., 2006).
Acute and subacute toxicity testing of polar and non-polar crude extracts as well as isolated
harpagoside on rats and mice, indicated low toxicity (LD50 < 510g/kg body weight for the
extracts; LD50 = 13g/kg body weight for harpagoside) (Schulz et al., 2001), and no adverse
reaction was observed in rats after high doses of harpagoside were administered
(Whitehouse et al., 1983). The use of H. procumbens is however contraindicated for patients
with gastric or duodenal ulcers (as bitter substances stimulate stomach acid secretion), in
patients with gallstones (as H. procumbens tends to increase bile production in the liver), for
patients using blood thinning agents (H. procumbens may increase the blood thinning effects)
(Stewart & Cole, 2005), and for diabetics (it may cause a hypoglycaemic action) as well as
pregnant or lactating patients (oxytoxicity have been observed in animals) (Newall et al.,
1996). Furthermore, reported non-transferable effectiveness from one product to another
(Whitehouse et al., 1983; Chrubasik et al., 2003), may be explained through variable
composition of H. procumbens preparations and extracts (as seen in Chapter 5) with
subsequent standardisation of products to contain at least 50 mg of harpagoside per daily
dose (Chrubasik et al., 2003), or simply ascribed to deactivation of active principles by
stomach acid (Soulimani et al., 1994), in which case topical application would be more
effective.
In summary, most pre-clinical (animal) and clinical research done on H. procumbens has
been directed at its antiinflammatory and analgesic activity related to musculoskeletal
ailments. In vivo results have, however, shown a decrease in blood pressure and heart rate
as well as hypoglycaemic and anti-convulsant effects, which may be considered relevant to
the claimed tonic properties of this plant. A decrease in serum cholesterol, uric acid and
natural fats in hypercholesterolemic patients, together with in vitro Immunomodulatory effects,
suggests that the tonic properties of Harpagophytum may extend beyond those of a digestive
bitters. Direct in vivo and clinical evidence for tonic properties are still lacking.
225
Chapter 6
Pharmacology

Hypoxoside and rooperol have a central pentenyne (norlignan) system, affording it
antioxidant capacity, markedly higher in rooperol than in hypoxoside.activity (Drewes et al.,
2008). Rooperol has four unprotected phenolic groups compared to two in hypoxoside, which
enhances antioxidant capacity significantly (Drewes et al., 2008), i.e. rooperol and
H. hemerocallidea extracts showed radical scavenging activity similar to that of quercitin (Nair
et al., 2007). To this effect, aqueous extracts of hypoxis were shown to strengthen the
antioxidant system under normal conditions and protect the body against chloroquine
induced oxidative stress in albino rats (Chaturvedi & Mwape, 2011). Due to the in vivo
cytotoxic and antimutagenic actions of hypoxoside and rooperol (Albrecht, 1996), and more
recent results showing inhibition of MCF-7 cell growth (Steenkamp & Gouws, 2006), much
research has been done to ascertain the possibility of using rooperol as a prodrug in cancer
therapy (Smit et al., 1995), but convincing clinical test results are yet to be produced
(Albrecht, 1996). Anticancer properties have also been reported for -sitosterol in the
treatment of prostate, colon and breast cancer (see Dicoma, Paragraph 6.2.6).
Apart from possible anti-HIV activity proposed by Albrecht (1996), other activities such as in
vitro antiphlogistic (Weiss, 1985), bacterostatic and bactericidal (Drewes & Liebenberg, 1982)
effects as well as in vitro and in vivo antiinflammatory, anti-nociceptive, anti-diabetic
(Ojewole, 2002, 2006), antineoplastic and antimetastatic (Owira & Ojewole, 2009) properties
have also been identified. Antiinflammatory activity was ascribed to inhibition of COX-1
and/or COX-2 enzymatic activity by ethanolic or aqueous extracts (Steenkamp et al., 2006),
where whole extracts presented better results than partially purified fractions (Gaidamashvili
& Van Staden, 2006), i.e. indicating a possible synergistic action between extract
constituents. Liu et al. (2010) suggested prophylactic treatment of inflammation with
methanolic
hypoxis
extracts
based on the fact
that
it
ameliorated
Brachyspira
hyodysenteriae-induced mice typhlocolitis. Anti-diabetic action was proposed to be by

stimulating insulin release, leading to increased cellular glucose uptake and consequently an
induction of hypoglycaemia in rats (Mohamed & Ojewole, 2003; Ojewole, 2006). All the
mentioned biological activities are thought to be due to the presence of hypoxoside and
rooperol (Drewes et al., 2008).
One of the major ethnobotanical uses of H. hemerocallidea is for urinary and prostrate
problems (Chapter 4; Appendix 2). To this effect, the high levels of -sitosterol and sterolin
(in a 100:1 ratio) in the corm were found to be active against prostatic adenoma, as in
226
Chapter 6
Pharmacology
pumpkin oil, which is marketed in Europe for the treatment of benign prostate hypertrophy
(Bruneton, 1995). The activity of sitosterols is ascribed to enzymatic effects (inhibition of 5reductase and aromatase), or to decreased binding of dihydro-testosterone within the
prostate (Bruneton, 1995). Some positive results were reported for the treatment of benign
prostatic hyperplasia (BPH) with -sitoterol. Three randomized, double-blind, placebo
controlled clinical trials involving 80 (in 1993), 200 (in 1995) and 177 (in 1997) men
respectively, all with mild to moderate BPH, showed significant improvement relative to the
placebo after daily treatment with -sitoterol (Wilt et al., 2000). In an earlier clinical study, 177
men with the same symptoms were treated with 0.30 mg -sitosterol--D-glucoside (the
equivalent of what is present in hypoxis), and no effect was observed (Kadow & Abrams,
1986). There were claims that -sitoterol and sterolin have in vitro immunomodulatory
properties (Bouic et al., 1996) but the validity of these results has been questioned. It should
be noted that 150300 mg sitosterols are ingested in a normal daily diet, which is more than
what is contained in H. hemerocallidea (Van Wyk & Wink, 2004).
Reports where hypoxis extracts were tested for biological activity include aqueous extracts of
the corm showing anti-convulsant activity [Ojewole, 2008(a)], where doses of 100800
mg/kg i.p. antagonised picrotoxin (PCT) and pentylenetetrazole (PTZ)-induced seizures in
mice, and delayed the onset of the latter. Risa et al. (2004) confirmed these results in the in
vitro GABAA-benzodiazepine assay, and found ethanolic extracts to be slightly more potent.
Furthermore, in vitro (myocardial contractile performance on guinea-pig isolated arterial
muscle strips) and in vivo (hypotensive effect examined in hypertensive Dahl salt-sensitive
rats) research on the cardiovascular effects of corm aqueous extracts showed the
concentration-dependent cardiodepressant and transient hypotensive properties of hypoxis
(Ojewole et al., 2006).
Ncube et al. (2011) tested seasonal variation of antimicrobial activity of hypoxis corm and
leaves, and found that elevated levels of phenolic and sapogenic compounds correlated with
elevated antibacterial and antifungal activity of the corms (see Table 6.2), especially in
autumn and winter (when human immune response is normally lower). Moderate anthelmintic
activity (minimum lethal concentration (MLC) of 1.044.17 mg/mL against Caenohabditis
elegans) was reported (Aremu et al., 2010).
227
Chapter 6
Pharmacology
Table 6.2: Seasonal variation of antimicrobial activity* of different extracts of Hypoxis

hemerocallidea corms (* extracted from Ncube et al., 2011; activities lower than
1 mg/mL)
Extract Summer
Antibacterial
B.s.
EtOH
0.78
H2O
0.78
Autumn
Winter
E.c.
S.a.
E.c.
S.a.
B.s.
K.p.
S.a.
0.78
0.39
0.78
0.78
0.78
0.39
0.78
0.20
0.78
MIC
MFC
0.39
0.39
PE
Antifungal
MIC
MFC
DCM
0.78
EtOH
0.78
0.78
H2O
0.39
0.39
Gram positive: B.s. = Bacillus subtilis; S.a. = Stapphylococcus aureus ;

Gram negative: E.c. = Eschericia coli; K.p. = Klebsiella pneumonia
MIC = minimum inhibitory concentration; MFC = minimum fungicidal concentration
Taylor et al. (2003) indicated possible genotoxicity for MeOH/water extracts of hypoxis corms
at levels between 1002 500 ppm in an in vitro micronucleus assay, but Laporta et al. (2007)
reported no toxicity with single dose (2 000 mg/kg, equated to an LD50 value of 900 mg/kg
of hypoxoside) administration over a period of two weeks. However, with the acute and
chronic treatment of rats with hypoxis aqueous extracts, it has been found that these extract
may be harmful to the kidneys (Musabayane et al., 2005). Concurrent treatment of HIV/Aids
with hypoxis corm extracts and antiretroviral drugs were also discouraged by Owira and
Ojewole (2009) as patients may be at risk of developing adverse effects which may lead to
treatment failure, viral resistance and/or drug toxicity. Aqueous extracts may also cause
bradycardia and brief hypotension (Ojewole et al., 2006).
The diversity of activities ascribed to hypoxis shows the possibility that it may have tonic
properties, with special reference to positive in vivo (pre-clinical) results for its
antiinflammatory, anti-diabetic, anti-convulsant and hypotensive activity. The reported
immunostimulating effects are questionable. No other in vivo or clinical tests have been
conducted to establish the tonic value of hypoxis.
228
Chapter 6
Pharmacology

Polygalaceae (tribe Polygalae) includes four West and South African genera, i.e. Polygala,
Muraltia, Carpolobia and Securidaca, all known to contain presegenin type saponins
(Lacaille-Dubois & Mitaine-Offer, 2005). In this review, in vitro effects on the central nervous
system together with lymphoproliferative properties, as well as in vivo immunoadjuvant
properties have been described for Polygalaceae, where in vivo hypoglycaemic and in vitro
immunomodulatory activities have been ascribed to the presegenin saponins from the genus
Polygala. Furthermore, acylated presenegenin type saponins, as those from M. heisteria
(Fig. 5.20) showed in vitro cytotoxicity toward human colon cancer cells, but were unable to
potentiate in vitro cisplatin cytotoxicity [Elbandy et al., 2002(a)]. From the phytochemical
screening of M. heisteria, the presence of flavonoids and medium polar terpenes has also
been detected. The former may have an antioxidant effect, and both the triterpenes and
flavonoids may contribute to the bitterness (bitterness value of 1 68014 400; see
Paragraph 5.3) of the plant (Van Wyk & Wink, 2004). The bitterness is known to promote the
amarum effect, which is directed at the ethnobotanical use as bitter tonic.
Other species of the tribe Polygalae have been shown to exhibit CNS related effects, such as
an in vivo inhibition of toxin-induced neuronal death (i.e. a model for Parkinsons disease)
(Polygala tenuifolia Choi et al., 2011) and in vitro antidepressant properties, possibly due to
the presence of oligosaccharide esters (Polygalae radix Liu et al, 2010).
Clinical results are available for mixtures containing Polygalae radix but the pure drug has
apparently not yet been subjected to clinical studies. No published in vivo studies could be
found for Muraltia or related genera but some evidence exist that presegenin type saponins
have immunomodulatory effects.

Based on its common name, cancer bush, and a myriad of ethnobotanical anecdotes, the use
of S. frutescens for treatment and prevention of cancer is an age-old practice (Van Wyk,
2008(a),(b); Chapter 4; Appendix 2). Van Wyk and Albrecht (2008) recently reviewed the
pharmacological evidence for these claims, the first of which were reported by Tai et al.,
(2004), were in vitro results showed dose dependant inhibition of proliferation of MCF7, MDAMB-468, Jurkat and HL60 cells. In the same period, methanol extracts of Sutherlandia were
found to inhibit DNA binding of activated NF-B in MCF10A human breast epithelial cells,
229
Chapter 6
Pharmacology
also in a dose dependant fashion, which may enhance the chemopreventive or

chemoprotective activity in Sutherlandia (Na et al., 2004). Aqueous whole plant extracts were
found to induce apoptosis in neoplastic cervical carcinoma and Chinese hamster ovary cell
lines (Chinkwo, 2005). This topic was extended by Stander (2009), showing that Sutherlandia
extracts have different in vitro effects on cell numbers, morphology, cell cycle progression
and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line. Polar and
non-polar extracts were shown to be able to inhibit growth of two human prostate tumour cell
lines (Chen, 2007). Steenkamp and Gouws (2006) however, reported contradictory results
where no cytotoxicity against human DU-145 prostate cancer cells, malignant MDA-MB-468
and MCF7 breast cancer cells or non-malignant MCF-12A breast cells. Recent in vitro
apoptosis-promoting effects of S. frutescens extracts on normal human lymphocytes were
also reported (Korb et al., 2010), which has particular bearing on the treatment of HIV/Aids.
Many of these activities have been ascribed to the presence of L-canavanine, the potent Larginine antagonist, commonly found in legumes (Fabaceae) (Van Wyk & Albrecht, 2008),
even though it occurs in low levels (0.4214.5 mg/g) in leaves of Sutherlandia species
(Moshe, 1998). Anticancer and antiviral activity (Crooks & Rosenthal, 1994), as well as
inhibition of the influenza virus and retroviruses (Green, 1988), have been documented for Lcanavanine. A suggested mode of action of canavanine is dose-dependent inhibition of nitric
oxide synthase, which presents potential for treatment of septic shock, associated with
advanced stages of Aids (Van Wyk & Albrecht, 2008). Even though canavanine is said to be
toxic, especially at levels high enough for in vivo apoptosis of cancer cells, S. frutescens was
found to be non-toxic (Seier et al., 2002). One would consequently suspect synergistic
potentiation (see Paragraph 6.1.2) of the apoptotic effect by other compounds also present in
the polar extracts (e.g. triterpenoids). Another non-protein amino acid, -aminobutyric acid
(GABA), has further been found to inhibit tumour cell migration (Ortega, 2003). Additionally,
arginine, a non-essential free amino acid, is said to attenuate the antiproliferative activity of
canavanine (Van Wyk & Albrecht, 2008). Furthermore, the cycloartane type triterpenoids,
isolated from the polar extracts of S. frutescens, share mutual features with the
astragalosides from Astragalus species, related to Sutherlandia and used for similar
indications in Traditional Chinese Medicine. These triterpenes, particularly those with C-24
hydroxylation and a 3-oxo group, have been shown to have chemopreventive activity and
exhibited powerful in vivo inhibition effects in a mouse skin carcinogenesis test (Kikuchi et al.,
2007).
230
Chapter 6
Pharmacology
GABA is also an inhibitory neurotransmitter, found in low levels [017.7 mg/g, or 0.23
0.85 mg/g according to Van Wyk and Albrecht (2008)] in S. frutescens, and may contribute to
the use of the species as treatment of stress and anxiety. Furthermore, Ojewole (2008b)
reported significant delay in the onset as well as antagonism of pentylenetetrazole (PTZ)induced seizures in mice by Sutherlandia aqueous extracts, comparable to phenobarbitone
and diazepam, to the extent where it was suggested as supplementary remedy in the
management, control and/or treatment of childhood convulsions and epilepsy. The mode of
action was suggested to be like GABA or by enhancing GABA neurotransmission and/or its
action in the brain. Van Wyk and Albrecht (2008) also suggested that the possible
corticomimetic effects of the Sutherlandia saponins may contribute in the treatment of stressrelated ailments as these are linked to the endocrine system. To this effect, Sutherlandia
extracts were shown to significantly reduce corticosterone levels by using a model of chronic
intermittent immobilisation stress in rats (Smith & Myburgh, 2004). Chloroform extracts were
also shown to have better inhibitory effects on progesterone and pregnenolone metabolism
than methanol extracts, indicating triterpenoid contribution almost exclusively (see
Paragraph 5.2.10 i.t.o. triterpenoid polarity). Prevoo et al. (2008) reported steroidogenesis as
a possible mode of action by which Sutherlandia is able to reduce glucocorticoid levels and
consequently also symptoms of stress.
The Sutherlandia saponins are also said to be bitter (Van Wyk & Wink, 2004), contributing to
its extreme bitterness (bitterness value 9 33337 333, see Paragraph 5.3), and consequently
also the amarum effect it exerts (Chapter 4, Appendix 2). Apart from claimed stimulation of
appetite, the triterpenoids are also suspected to have a corticomimetic effect (Van Wyk &
Wink, 2004). They may also have immunostimulative potential as they are structurally similar
to those from Astragalus membranaceus, claimed to have such activity (WHO, 1999). Based
on this immunostimulative potential, it was suggested that nevirapine and S. frutescens be
used concurrently for the treatment of HIV/Aids. However, Brown et al. (2008) reported that
both Sutherlandia extracts and canavanine increased the in vitro bioavailability of nevirapine,
which may lead to adverse effects of this drug in HIV/Aids patients. Minocha et al. (2011)
investigated the effects of nevirapine (6 mg/kg) when administered orally alone (control) and
with co-administration of Sutherlandia in the short term (12 mg/kg single dose) and long term
(12 mg/kg, once a day for 5 days). Although no significant difference in the pharmacokinetic
parameters of nevirapine was found upon short-term co-administration of Sutherlandia, a
50% decrease in the AUC and C values of nevirapine was observed after 5 days of chronic
231
Chapter 6
Pharmacology
exposure. Furthermore, a 23-fold increase in the hepatic and intestinal mRNA expression of
CYP3A2, relative to vehicle control, was demonstrated. To ascertain the clinically relevancy
of the pharmacokinetic interaction in patients, the effect on LS-180 cells were tested an in
vitro induction model for human CYP3A4. Results obtained ninety-six hours after treatment
with Sutherlandia extract (300 g/mL), were comparable to positive control rifampicin (25
M), and showed elevated m-RNA expression levels and functional activity of CYP3A4
(human homologue of rodent CYP3A2) in LS-180 cells. These results imply that a positive
drugherb interaction exist between nevirapine and S. frutescens, but still needs to be
investigated in a clinical setting.
Pinitol is a known anti-diabetic agent that may have application in treatment of wasting in
cancer and AIDS (Ostlund & Sherman, 1996). It plays a role in regulating cellular energy by
increasing the availability of glucose for cell metabolism, consequently lowering blood sugar
in an insulin-like manner (Bates et al., 2000). Several in vivo reports show such an increased
sugar uptake upon administering Sutherlandia extracts to rats (Ojewole, 2004; Van Wyk &
Albrecht, 2008). Canavanine and arginine were also suggested to play a role in the antidiabetic effects of Sutherlandia, along with pinitol, either directly or via antiinflammatory
activity and nitric oxide inhibition (Sia, 2004). Insulitis of autoimmune diabetes may
consequently be counter-acted by protecting the pancreas against oxygen radicals such as
nitric oxide. Recently, S. frutescens has also been found to limit the development of insulin
resistance by decreasing plasma free fatty acid levels , which is important considering that
the intake of high caloric food induces raised plasma free fatty acids, which in turn may lead
to insulin resistance and Diabetes mellitus type 2 (MacKenzie et al., 2009). Pinitol also
augments the retention of creatinine by muscle cells (Greenwood et al., 2001). Sutherlandia
contains up to 14 mg/g pinitol in the leaves (Moshe, 1998), which, along with the amino acids
and triterpenonoids, provide a scientific rationale for its use in treatment of wasting in cancer
and Aids.
Flavonoids are known to have antioxidant properties. Polar and non-polar extracts of
S. frutescens, also containing flavonol glycosides, have consequently been shown to have
such properties (Van Wyk & Albrecht, 2008), where Fernandes et al. (2004) not only
indicated superoxide and hydrogen peroxide scavenging capability for hot water extracts (at
concentrations as low as 10 g/mL), but also linked antioxidant potential with
antiinflammatory activity. Kundu et al. (2005) proposed that such activity takes place through
inhibition of COX-2 and suggested several mechanisms for this, as well as a link between the
232
Chapter 6
Pharmacology
antiinflammatory and chemopreventive activities of Sutherlandia. Apart from in vivo

antiinflammatory activity, Ojewole (2004) further recorded analgesic properties of
Sutherlandia.
Several studies on toxicity of Sutherlandia, including tests related to acute and chronic use,
as well as a phase 1 clinical study, showed no adverse effects (Van Wyk & Albrecht, 2008). A
few side effects have been noted, such as dryness of the mouth, occasional mild diarrhoea or
mild diuresis, as well as dizziness in cachectic patients (Mills et al., 2005). It is also
suggested to be avoided during pregnancy or lactation (Van Wyk & Gericke, 2000).
S. frutescens extracts at high concentrations appear to be harmful to the kidneys as it
increases oxidative stress, alters mitochondrial membrane integrity, and promotes apoptosis
in renal tubule epithelia (Phulukdaree et al., 2010). A phase I clinical study (randomized,
double-blind, placebo-controlled) have however shown no side-effects in 25 healthy adults
arising with the use of Sutherlandia (Johnson et al., 2007).
In summary, the diversity of pharmacological activities is as wide as the diversity of chemical
compounds reported for S. frutescens. As its common name (cancer bush), ethnobotanical
history and anecdotal evidence (Paragraph 4.2.10, Appendix 2) suggest, it has been shown
to exhibit in vivo anticancer, antitumor, cytotoxic, antiproliferative and chemopreventive
properties. Apart from pre-clinical evidence for antiinflammatory and analgesic activity, it also
shows anti-diabetic properties, and its extreme bitterness has been confirmed and quantified
(Paragraph 5.3). Amarum and immunostimulative effects have consequently been suggested,
but no clinical evidence has yet been produced to this effect. Furthermore, Sutherlandia has
been shown to have in vivo anti-convulsant properties and to reduce corticosterone levels in
an immobilisation stress rat model. Based on the pre-clinical findings presented here, it is
likely that the tonic and adaptogenic properties of Sutherlandia will in due course be proven
through clinical studies.

The common name for V. oligocephala, groenamara, is indicative of its extremely bitter
leaves (bitterness value of 8 00012 000, Paragraph 5.3). Sesquiterpene lactones (Ganjian
et al., 1983) and triterpenes such as steroid glucosides (Jisaka et al., 1992) are responsible
for this bitterness and are consequently not only insect feeding deterrents, but also have an
amarum effect. For this reason, some of the main traditional uses of V. oligocephala are
related to digestive tract ailments (Chapter 4, Appendix 2). Hirsutinolide-type sesquiterpene
233
Chapter 6
Pharmacology
lactones commonly occur in southern African Vernonia species (Bohlmann et al., 1983), and
are said to be responsible for the antimalarial properties of V. oligocephala [DCM:MeOH (1:1)
leaf extracts showed a IC50 of 3.5 g/mL; Pillay et al., 2008). Vernonia sesquiterpenes have
also been shown to have cytotoxic activity (Chukwujekwu et al., 2009), while the flavonoids
have been indicated as antioxidants (Igile et al., 1994).
Several reports of pharmacologically tested properties of V. amygdalina, the medicinally used
South African counterpar of V. oligocephala, have been recorded. An account of the preclinical (in vivo) and clinical results include in vivo hepatoprotective, antioxidant (Iwalokun et
al., 2006), cytotoxic (Omoregie et al., 2010), antiplasmodial and analgesic activity (Njan et al.,
2008), while its hypoglycaemic (Okolie et al., 2010) and antimalarial (Calland & Willcox,
2009) properties have been subjected to clinical testing with positive results.
Unlike V. amygdalina, no pharmacological testing has been conducted with V. oligocepala
and it possible tonic value. The phytochemistry of V. oligocephala is still poorly explored.

The strong peppery taste of warburganal is responsible for its insect anti-feedant and
molluscicidal properties (Drewes et al., 2001). To this effect, a wide range of toxicological
activities against fish, crustaceans, mosquito larvae and freshwater snails have been
recorded for aqueous leaf extracts, pointing to the fact that it contains similar compounds
than the bark (see Chapter 5). Were et al. (2010) reported in vitro antiplasmodial activity
(IC50 = 3.14 g/mL) for chloroform extracts of W. salutaris, and provided evidence of in vivo
curative (69.15%) and prophylactic (49,18%) capacity of these extracts in mice. Furthermore,
significant in vivo (Ngure et al., 2009) and in vitro (Githinji et al., 2010) activity against
Leishmania major were recorded, where aqueous stem bark extracts exhibited a mean
Leishmania mortality rate of 76% at a concentration of 1000 g/mL. The extracts were said to
interfere with the normal growth and development of the parasites, either causing quick death
of extracellular parasites before they were phagocytosed or through impairment of the
phagocytosis process. In vitro anti-trypanosomal activity was also determined for a crude
extract and isolated compounds from W. salutaris where one of the isolated compounds
showed activity and the other was toxic (Kioy et al., 1998). The crude extract was inactive, as
was the case with a subsequent in vivo study on mice.
234
Chapter 6
Pharmacology
W. salutaris is described as a natural antibiotic (Chapter 4, Appendix 2). The Warburgia

drimane sesquiterpenoids (especially polygodial, warburganal and muzigadial) exhibit broad
spectrum antimicrobial properties, against pathogens such as Gram-positive bacteria, yeasts
and filamentous fungi (Kubo & Taniguchi, 1988, Rabe & Van Staden, 1997; Mwambo et al.,
2009). Similar antimicrobial properties were reported for both the leaves and bark, with the
latter producing slightly better results for Gram-positive bacteria (Zschocke et al., 2000).
Muzigadial have shown activity against Stapphyllococcus aureus, S. epidermidis, Bascillus
subtilis, Escherichia coli and M. luteus at ranges between 12.5100 g/mL (Rabe & Van
Staden, 2000). Jansen and De Groot (1991) suggested that the intensity of the taste of a
particular sesquiterpene corresponds with its potency in biological activity, and is due to the
enal functionality as well as the -orientation of the C-9 aldehyde. In a study on the antifungal
activity of polygodial (e.g. MIC of 1.56 g/mL against Candida utilis), the most active of the
three, Taniguchi et al. (1983) reported its irreversible reaction with the sulfhydryl group of Lcysteine, for example, a reaction also occurring with isopolygodial. It was also found that
enal-aldehydes preferentially formed adducts with amino groups rather than sulfhydryl
groups, and that 9-isomers exhibited higher reactivity (DIschia et al., 1982). Furthermore,
the mode of action suggested for polygodial, was to disrupt the structural integrity of the cell
membrane, in a fungicidal rather than a fungistatic way (Kubo & Taniguchi, 1988). It also
exhibited a far greater potential when used as adjunct to antibiotics such as actinomycin D, in
acidic conditions. Consequently it was suggested that polygodial synergistically facilitated
entrance of the antibiotic into the fungus cells by initial disruption the plasma membrane.
Polygodial is thus a potentially useful adjuvant to treatment with antibiotics and antifungals
that have poor membrane permeability (Iwu, 1993). Madikane et al. (2007) determined that
the antimycobacterial activity of Warburgia salutaris is due to the inhibition of mycobacterial
arylamine N-acetyltransferase, and muzigadial and linoleic acid (Z,Z), are said to be
responsible for this activity, i.e. they exhibited good antimycobacterial activity against four
different pathogens, i.e. MIC values for the latter were comparable to that of the antibiotic
isoniazid ranging between 4 and 16 g/mL (Wube et al., 2005).
W. salutaris extracts exhibited a prophylactic and therapeutic action against crystalline silicainduced lung injury by suppressing H2O2-induced membrane peroxidation, DNA damage and
lipid peroxidation, by means of oxidant neutralisation (Leshwedi et al., 2008). Based on these
results, its ability to protect cells against cytotoxicity and genotoxicity has also been
predicted. As the production of free radicals is linked to the inflammatory process (Schinella
et al., 2002), the ability of W. salutaris to reduce oxidative stress is suggested to be linked to
235
Chapter 6
Pharmacology
its use as antiinflammatory agent. Although inflammation is a complex process affecting

many biochemical pathways, Jger et al. (1996) have reported low (14% for aqueous extracts
and 11% for EtOH extracts) prostaglandin-synthesis inhibition. Immunomodulatory activity
was also proposed as the extracts stimulated considerable NO production (112.3% more
than the negative control, and at higher levels than that of pentostam, a known
immunomodulator), which supposedly kill foreign bodies indirectly (Githinji et al., 2010).
Interestingly, methanol extracts shown no or poor results in all these reports, possibly as a
result of interaction between the aldehyde functionalities of the active drimane
sesquiterpenes and the alcohol group of methanol, leading to the formation of inactive
adducts (Kubo & Taniguchi, 1988).
While W. salutaris have not shown genotoxicity (Taylor et al., 2003) and no other reports on
its possible toxicity could be found, it should be noted that cyanogenic glycosides may be
present (Chapter 5). As no variation study was done, these results may be sporadic.
In summary, antimicrobial, antiinflammatory and immunomodulatory properties have been
indicated for W. salutaris showing its tonic potential but these are all in vitro results (in vivo
studies are limited to molluscicidal, antiplasmodial and antiparasitic activities).

As an adaptogenic, cognition-enhancing and memory-improving tonic, W. somnifera and its
constituent compounds have been shown to have a variety of effects on the central nervous
system (CNS). To this effect, Mirjalili et al. (2009) reviewed:
(a) anxiolytic properties, where the withanolides cause a decrease in tribulin (an endocoid
marker of anxiety) in a rat brain;
(b) antidepressant properties in several rat models, where the number and severity of chronic
stress-induced ulcers decreased, and a reverse in chronic stress-induced male sexual
behaviour as well as in stress-induced immunosupression was observed when withanolides
were administered; a reduction in ulcer index, volume of gastric secretion as well as free and
total acid were also reported for methanol extracts;
(c) memory and cognition enhancing effects were obtained through sitoindosides VII-X and
withaferin induced enhancement of cortical muscarinic acetylcholine receptor capacity;
extracts were observed to improve scopolamine-induced memory deficits in mice; methanol
extracts induced neurite extention by means of neuritic regeneration and synaptic
reconstruction together with a preventive effect on dentritic atrophy;
236
Chapter 6
Pharmacology
(d) an antiparkinsonian effect, whereby haloperidol or reserpine induced catalepsy is

inhibited; reversed oxidative stress in a 6-hydroxydopamine rat model was observed,
indicating a protective effect on neuronal tissue through increased antioxidant activity in the
frontal cortex and striatum of the brain;
(e) a prolonged hypotensive, bradycardic, respiratory-stimulant effect of W. somnifera
alkaloids in dogs, where the former was suggested to be through autonomic ganglion
blocking or through a depressant action on higher cerebral centres
Kulkarni and Dhir (2008) also described anti-epileptic and anti-convulsant properties through
an effect on the GABAA receptor system, applicable in the treatment of acute and chronic
epilepsy.
The anti-stress activity of W. somnifera was further illustrated when a withanolide-free
aqueous root extract was administered to rats and mice in a dose-related fashion (Singh et
al., 2001). The tests performed to measure such activity, as discussed in Paragraph 6.1.3.1,
included hypoxia time, antifatigue effects, swimming performance time, gastric ulceration
induced through swimming, immobilisation or hypothermia, autoanalgesia, and measurement
of biochemical change in the adrenal glands. The extracts showed significant anti-stress
capacity in all the parameters tested, as well as a good margin of safety in acute toxicity
testing. In a subsequent study (Singh et al., 2003), the antiinflammatory activity of
withanolide-free aqueous root extracts were found to be dose for dose more effective than
phenylbutazone in the treatment of carrageenan-induced pedal rat oedema. It also exhibited
immunostimulant activity with a significant influence on primary antibody synthesis, as well as
protection against chemical or swimming stress-induced impaired hepatic function. The
immunoregulatory potential of W. somnifera was consequently ascribed to an observed
increased capacity in experimental animals to tolerate non-specific stress, as it aids in the
restoration of a large number of parameters with no interference in the normal physiological
functions of the body. Bhattacharya and Muruganandam (2003) illustrated the adaptogenic
activity of a standardised root extract in a chronic stress rat model where symptoms such as
hyperglycaemia, glucose intolerance, increased plasma corticosterone levels, gastric
ulcerations, male sexual dysfunction, cognitive deficits, immunosuppression and mental
depression, were attenuated by W. somnifera, administered an hour before footshock for 21
days. The Withania withanolides, on the other hand, serve as hormone precursors which may
be converted to human physiological hormones as required. As such, it is suggested that, in
cases of hormonal excess, these plant-based hormones would occupy relevant cell
membrane receptor sites, thus preventing hormone binding, and consequently exert inhibition
237
Chapter 6
Pharmacology
of hormonal action (Withania somnifera monograph, 2004). This amphoteric ability to regulate
physiological processes contributes to the adaptogen capacity of W. somnifera.
Winters (2006) reviewed the role of W. somnifera in integrative oncology. Polar leaf and root
extracts as well as their constituent compounds have been included in in vitro and in vivo
testing of their respective antineoplastic actions. In this instance, in vitro testing returned IC50
values of 0.24, 0.36, 0.28 and 0.27 g/mL against lung, colon, CNS and breast tumour lines
respectively, while in vivo experiments with root extracts showed slower tumour growth and
increased survival time. Furthermore, withaferin A has exhibited antitumor (Yang et al., 2007)
and apoptosis inducing (Sen et al., 2007) properties, while a withanolide fraction was
reported to completely suppress the NF-B activation pathway (Winters, 2006). As NF-B is
activated by carcinogens, tumour promoters and inflammatory agents, this finding puts the
antitumor capacity of W. somnifera extracts into perspective. It was also suggested that these
antitumor and chemopreventive effects (Prakash et al., 2002) exhibited by W. somnifera
would be linked with its potent antioxidant capacity. Such capacity is associated with
increased levels of the major free-radical scavenging enzymes (superoxide dismutase,
catalase and glutathione peroxidise) and ascorbic acid, as well as decreased lipid peroxidase
(Bhattacharya et al., 1997). An observed decrease in proliferation and higher susceptibility to
the effects of oxidative stress upon in vitro subjection of osteogenic sarcoma and breast
carcinoma cells to aqueous leaf extracts W. somnifera, together with its immunostimulatory
properties mentioned previously, indicate a sensitization to radiation and chemotherapy
treatment (Winters, 2006). Furthermore, treatment of ascetic sarcoma-bearing mice with an
extract mixture (W. somnifera : Tinospora cordifolia, 80:20) and alkaloid-free polar fractions of
W. somnifera, resulted in chemo and immunoprotection based on a significant increase in
white blood cells, as well as hemaglutinating and haemolytic antibody titers (Diwanay et al.,
2004). These results were also obtained after treatment with root extract, together with an
increase in bone marrow cellularity and -esterase positive cell number, along with an
enhancement in plaque forming cells in the spleen. With a further inhibited delayed type
hypersensitivity reaction in mice and an enhancement in phagocytic activity of peritoneal
macrophages, all these results confirm the immunomodulatory property of W. somnifera
extracts (Davis & Kuttan, 2000). As adaptogen, it consequently not only protects during
chemotherapy, but also has the capacity to reduce some of the most common side-effects
exhibited in chemotherapy (Winters, 2006).
238
Chapter 6
Pharmacology
Inflammation, a symptom of arthritis, is a result of oxidative stress. In this instance, Parihar et

al. (2004) reported the ability of W. somnifera to prevent the susceptibility of hippocampus
and cerebral cortex to oxidative damage in streptozotocin treated mice. Mishra et al. (2000)
also reviewed the antiinflammatory properties of W. somnifera extracts and constituents as
shown in several in vitro and in vivo tests, as well as clinical trials, where the analgesic effect
of W. somnifera is also apparent. More recently, Rasool and Varalakshmi (2006) reported
that increased lipid peroxidase and glycoprotein levels corresponding with decreased
antioxidant status and bone collagen levels typical of arthritis, which were reversed upon
treatment with W. somnifera extracts (1 000 mg/kg/day for eight days after adjuvant-induced
arthritis in rats). Crude ethanol extracts have also been found to suppress the production of
pro-inflammatory molecules in vitro, and it was suggested that inhibition of NF-B and AP-1 in
human peripheral blood and synovial fluid mononuclear cells by the withanolides may
contribute to such antiinflammatory activity (Singh et al., 2007). In an anatomical asthmatic
BALB/c mouse model study, the antiinflammatory action of W. somnifera was illustrated to
improve the stability of fibrin networks in the lungs, while selenium, an antioxidant
supplement normally taken by asthmatic patients to prevent deficiency, improved platelet
stability (Pretorius et al., 2009).
Several reports on the antimicrobial activity of W. somnifera exist (Ashokkumar et al, 2010).
Some specific examples include the in vitro study against six strains Neisseria gonorrhoea
and nine strains of Candida albicans, the extracts showed activity against N. gonorrhoea at
concentrations of ranging from 0.5 (methanol extract) to 10 (water extract) mg/mL. For
C. albicans, only the methanol extract was effective at 20 mg/mL (Kambizi & Afolayan, 2008).
While methanol extracts of leaves show activity against Salmonella typhimurium, hexane
extracts of leaves and roots inhibit Escherichia coli. Added to Tribim (a combination of
rifampicin and isoniazid), the antibiotic activity of methanol extracts against S. typhimurium,
and hexane extracts against E. coli is synergistically improved in both roots and leaves (Arora
et al., 2004). Aqueous extracts have also shown 100% cytotoxicity of herpes simplex virus
type 1, 96 hours after infection, at concentrations ranging between 7.8 g/mL and 500 g/mL
(Kambizi et al., 2007).
In their review, Mishra et al. (2000) added that W. somnifera has also been associated with
stimulation of thyroidal activity, as shown in a mouse model where a significant increase in
blood serum levels of thyronines T3 and T4 were observed upon administration of root
extract. It was further established from several clinical trials that W. somnifera is useful in
younger and older populations as general health tonic, where its use resulted in increased
body weight, level of haemoglobin, serum iron and hand grip, to name but a few.
239
Chapter 6
Pharmacology
While W. somnifera is considered safe, large doses have been noted to have aborticacient
properties and cause gastrointestinal upset, diarrhoea and vomiting. Prescribed doses are 3
6 g dried root per day, or 300500 mg standardised extract equated to 1.5% withanolides. It
is a mild CNS depressant and use with alcohol, sedatives and other anxiolytics should be
avoided (Withania somnifera monograph, 2004).
Pre-clinical and clinical evidence show that W. somnifera is a true adaptogenic tonic
according to the definition (Paragraph 6.1.3.1):
(a)
It is innocuous (Mishra et al., 2000; Withania somnifera monograph, 2004).
(b)
It has a broad spectrum of therapeutic actions [as summarized here, and as shown in a
clinical study focussing on the hypoglycemic, diuretic and hypocholesterolemic effects
on six diabetic and six mild hypercholesterolemic subjects Andallu & Radhika (2000)].
(c)
It should be a non-specific protective in that it should be able to raise the bodys

resistance to physical, chemical, or biological stresses (e.g. a clinical study subjecting
40 young healthy individuals to exercise in order to measure the effects of
W. somnifera on physical performance and cardiorespiratory endurance Sandhu et
al., 2010; a clinical study on the effects of W. somnifera on breast cancer in
combination with chemotherapy (Biswal et al., 2012); several accounts given here of in
vivo experiments performed to assess the effects of W. somnifera on adaptation to
chronic or a variety of non-specific stresses, showing immunomodulatory and
immunostimulant as well as chemopreventive capacity.
(d)
It should normalize the physiological functioning of organisms irrespective of the

direction of the pathological changes in order to enhance recovery through
homeostasis. Mishra et al. (2000) for example, have listed clinical trials in which
W. somnifera showed positive effects on several parameters associated with general
health.

The main traditional uses are due to microbial or parasitic infections, with an important
analgesic or antiinflammatory component (Chapter 4, Appendix 2). To this effect, several
pharmacological studies were aimed at the antiparasitic properties of Z. mucronata. Variable
results were obtained from testing anthelmintic ability: Mlgaard et al. (2001) found the root
bark to be more active against Hymenolepis diminuta cestodes than the leaves or roots,
where a concentration of 1.6 mg/mL was lethal after one hour, and a concentration of
240
Chapter 6
Pharmacology
0.02 mg/mL was lethal after 24 hours. No effect was seen from any of the extracts with
Schistosoma mansoni schistosomules, however. Aqueous extracts of the root was found to
be moderately lethal to schistsomula of Bulinus africanus snails at a concentration of
25 mg/mL after 1 hour (Sparg et al., 2000). Waterman et al. (2010) tested Tanzanian
Z. mucronata sample extracts against a levomisole-resistant strain of Caenorhabditis elegans
worms, and found organic extracts of the twigs and aqueous extracts of the fruit to be most
effective with 69.5% and 40.6% worms dead (higher than the control), respectively. This
activity was ascribed to the presence of tannins and phenolic compounds due to their proteincoagulating ability, or to alkaloids. McGaw et al. (2000), on the other hand, showed no
anthelmintic activity against C. elegans for hexane, methanol or water extracts of bark or
leaves. Z. mucronata also has antimalarial activity, i.e. Prozesky et al. (2001) reported
antimalarial MIC values of 73% at a concentration of 50 g/mL for an acetone stem bark
extract, together with an antiplasmodial IC50 value of 4.13 g/mL. Clarkson et al. (2004)
reported an IC50 of 12 g/mL for DCM leaf extracts.
The effective treatment of pain and lumbago suggested analgesic, antiinflammatory and/or
sedative properties for Z. mucronata. In this instance, Luseba et al. (2007) showed moderate
in vitro prostaglandin synthesis inhibition by methanolic bark extracts (68.5% for COX-1 and
66.2% for COX-2), and suggested that the flavonoids might contribute to the said activity.
These results are corroborated by even higher inhibition exhibited by ethanolic leaf extracts
(89%; Jger et al., 1996). Furthermore, the strong sedative effects exhibited by Z. jujuba and
Z. vulgaris are said to be due to the presence of the alkaloid frangufoline (syn. sanjoinine A),
which is structurally closely related to those extracted from Z. mucronata (Van Wyk et al.,
1997). Flavonoids such as swertish and spinosin from Z. jujuba are also known to exhibit
sedative activity (Cheng et al., 2000).
Treatment of many respiratory infections such as bronchitis, colds, influenza and fever, have
lead to the determination of its in vitro activity of Z. mucronata against two strains of
Mycobacterium tuberculosis, one sensitive to first-line drugs and the other resistant to these
drugs, which returned MIC values of 50 g/mL for acetone bark extracts with both virus
strains (Green et al., 2010). In another in vitro experiment, ethanol bark extracts have been
shown to be inactive toward M. tuberculosis and M. smegmatis, but were cytotoxic toward
Vero cells instead (IC50 = 2.7 g/mL) (Mativandlela et al., 2008). In an evaluation of inhibitory
properties against human immunodeficiency virus type 1 reverse transcriptase and integrase,
241
Chapter 6
Pharmacology
aqueous and methanol leaf extracts showed poor or no activity (Bessong et al., 2005).
Z. mucronata also does not exhibit strong antibacterial activity, i.e. in vitro antibacterial
results for DCM and 90% MeOH bark extracts (Luseba et al., 2007) as well as for hexane,
methanol and water extracts of both leaves and bark (McGaw et al., 2007) were shown to be
poor. Ethanol leaf extracts also exhibited poor anti-amoebic activity (IC50 > 5.0 mg/mL) and
water extracts were inactive (MgGaw et al., 2000). These poor results are surprising as
mucronines F-H have reported bacteriostatic and fungicidal activities (Barboni et al., 1994).
The use of Z. mucronata for treating a variety of blood disorders lead to a study on its
antisickling activity, where aqueous and ethanolic extracts of both stem and root bark were
found to exhibit high activity in the emmel test using blood samples from known drepanocitary
adolescent patients (> 70%; Mpiana et al., 2008). This activity was ascribed to the
anthocyanins contained in the mentioned extracts.
Unfortunately Z. mucronata was found to be genotoxic: neither DCM nor 90% MeOH extracts
of the bark (Luseba et al., 2007), roots or twigs (Elgorashi et al., 2003) show mutagenicity,
but 90% MeOH leaf extract were found to be mutagenic in the Ames assay, with metabolic
activation (Elgorashi et al., 2003). These results were confirmed through the micronucleus
test and comet assay (Taylor et al., 2003; Verschaeve & Van Staden, 2008), i.e.
Z. mucronata leaves are able to cause DNA damage and chromosomal aberrations. In a
brine shrimp toxicity test, however, all the leaf extracts were non-toxic except for the hexane
extract, which had a low toxicity of LC50 = 0.9 mg/mL (McGaw et al., 2007).
Anticancer potential for Z. mauritiana (Mishra et al., 2011) and the inhibitory effect of
jujuboside A on the glutamate-mediated excitatory signal pathway in the hippocampus by
Z. jujuba (Zhang et al., 2003) have been shown through in vivo models, and may also be
applicable to Z. mucronata (if it contains the same or similar compounds). The antisickling,
analgesic, antiinflammatory, sedative and antimicrobial results given here do indicate
possible tonic properties for Z. mucronata, but are all in vitro accounts. Pre-clinical and clinic
evidence are yet to be obtained.
242
Chapter 6
6.3
Pharmacology
Conclusions
The literature reviewed for the 14 tonic plants are summarised in Table 6.3. The
pharmacological activities were grouped together and classified as antimicrobial, antioxidant,
antiinflammatory, anticancer and antiparasitic properties, those that have an effect on the
central nervous system, and those indicating toxicity. Where activities were ascribed to
certain extracts or compounds, it was indicated as such.
From these results, it is important to take cognisance of the fact that while in vitro activity of
extracts or compounds certainly give an indication of biological activity, the true
pharmacodynamics (actions of a drug on the body) and pharmacokinetics (actions of the
body on an administered drug) may be understood only through in vivo and clinical testing.
Interactions (synergistic, additive or antagonistic) amongst constituent compounds within an
extract, or amongst extracts/drugs used together in mixtures, as well as toxicity should also
be taken into account. The placebo effect probably plays an important role in the use of
tonics and it is therefore important that clinical studies should be done to prove efficacy and
to ensure that commercial marketing material of products and associated health claims are
based on scientifically validated data.
243
Chapter 6
Table 6.3:
Pharmacology
Summary of biological activity of 14 tonic plants (pharmacological details to be found in Paragraph 6.2)
Agathosma
A. betulina
A. crenulata
A. ovata
Aloe
A. arborescens
A. ferox
A. marlothii
A. vera
Arctopus
A. echinatus
A. dregei
A. monacanthus
Artemisia
A. afra
Intestinal
Cardiac
Antioxidant
Antiinflam.
++
Spasmolytic
Diuretic
Laxative;
+ (bitter)
+ (oil)
++;
++ (bitter)
++ (lump)
++
Anti-arthritic
++
(caffeic
acid)
++ (oils,
chamazulene,
scopoletin)
+
Spasmolytic
Diuretic
(diterpenes*)
+ (bitter)
+
Hepatoprotective;
Spasmolytic
++ (bitter)
++
(scopoletin)
CNS-related
Anticancer
Antimicrobial
Antiparasitic
Immune
function
+
Breast(*,s)
antibacterial
(+++)
antifungal (+)
++
Hypotensive
Analgesic
ACE inhibitor
++
+++
Antibacterial
Antiviral
Antifungal
++
Stimulant ();
Anti-allergic
Hypoglycaemic
Estrogenic
++
(caffeic acid,
diterpenes)
+
Analgesic
(terpenes);
Hypotensive
(kaurenes)
++
Antimutagenic
(caffeic acid);
Colon, breast,
leukemia
(diterpenes)
++
Antibacterial
Antiviral
Antifungal
Antibiotic
(terpenes;
caffeic acid)
+
Trypanomicidal
Moluscicidal
(diterpenes)
+
(triterpenes)
+
(scopoletin)
++
Narcotic;
Analgesic;
Anticonvulsive ();
Hypotensive;
Sedative;
Antidepressant
++
(terpenes,
flavonoids,
scopoletin)
+
Antimalarial
(flavonoids);
Antinematodal
(polar extract)
+
Anti-histaminic;
Immunomodulatory;
Antihyperglycemic;
Anti-thyroidal
(scopoletin)
244
Chapter 6
Pharmacology
Intestinal
Cardiac
Antioxidant
Antiinflam.
CNS-related
Anticancer
Antimicrobial
Antiparasitic
Immune
function
Balanites
B. maughami
++
Antidiarrhoeal
(bark)
Antiplasmodial
(DCM bark
extract);
+ (bitter)
Larvicidal,
anthelmintic,
molluscicidal
(saponins)
Dicoma
D. anomala
++ (bitter)
++ (hexane);
++ (*,)
++ (MeOH)
Antimalarial
-- (water)
Anthelmintic
(sesquiterp.)
D. capensis
++ (bitter)
++
Cytotoxic, antiproliferative
(prostrate &
breast)
Harpagophytum
H. procumbens
++ ()
++ (, c)
++ ()
+ (*, s)
++ ()
Improves
enterohepatic
circulation &
bile
activation
(iridoids);
decrease
blood
pressure &
heart rate;
protect
against
arrhythmia
(phenyl
ethanoid)
Antirheumatic;
Analgesic;
inhibits ACE;
anticonvulsant
Inhibition of
TNF release
Antifungal
Antiplasmodial
Decreases
cholesterol, uric
acid, natural
fat;
hypoglycaemic
Antiphlogistic;
(iridoids)
+ (bitter)
245
Chapter 6
Pharmacology
Intestinal
Hypoxis
H.
hemerocallidea
+ (bitter)
Muraltia
M. heisteria
+ (bitter)
Sutherlandia
S. frutescens
++ (bitter)
Cardiac
Antioxidant
Antiinflam.
CNS-related
Anticancer
Antimicrobial
Antiparasitic
++
(rooperol)
++ ()
(hypoxoside
& rooperol);
prophylactic
++ (*,)
Antinociceptive;
anticonvulsant
(polar extract)
++ (*,)
Cytotoxic; antimutagenic;
antiphlogistic;
antineoplastic;
antimetastatic
(prostate &
BPH;
hypoxoside,
rooperol, sitosterol)
++ (*,)
Bacteriostatic;
bactericidal;
antifungal
+
Anthelmintic
Immune
function
++
Anti-diabetic
+ (*)
Cytotoxicity
(colon;
saponins)
++ (*,)
(aq.
extract)
++ (*,)
(aq. extract)
++
Delays onset
& antagonizes
seizures
(GABA)
++ (*,s):
Antiproliferative
(breast,
prostate);
Chemopreventive;
Chemoprotective;
antineoplastic
(cervical);
Inhibits tumor
cell migration
(, GABA);
inhibits
carcinogenisis
(triterpenes)
+
Antiviral
(canavanine)
++
Promotes
lymphocyte
apoptosis (*,
canavanine);
immunostimulant &
inhibits HIV
target enzymes
(corticomimetic
saponins-);
anti-diabetic &
anti-wasting
(pinitol)
246
Chapter 6
Pharmacology
Intestinal
Vernonia
V. oligocephala
++ (bitter)
Warburgia
W. salutaris
+ (bitter)
Withania
W. somnifera
+ (bitter)
Cardiac
Antioxidant
Antiinflam.
CNS-related
+
(flavonoids)
++
Bradycardiac
Anticancer
Antimicrobial
+
Cytoxic
(sesquiterp)
++ (*,,c)
(EtOH root
extr)
++ (*,,
adaptogen)
Anti-epileptic;
anxiolytic;
memory
enhancing;anti
-Parkinsonian;
CNSdepressant;
anticonvulsive;
hypotensive;
analgesic
Antiparasitic
Immune
function
++
Antimalarial
(sesquiterp)
++
Cytotoxic &
haemolytic
(warburganla)
++ (s,
adjuvant)
Fungicidal
(warburganal
&polygodial);
Antibacterial
(polar bark/
leaf extr);
antimycobacterial
(muzigodial)
++ (*,)
Antineoplastic
(lung, colon,
CNS, breast;
polar leaf &
root extr);
Antitumor;
apoptosis
inducing
(withaferin A &
withanolides)
++
Antifungal;
antiviral
++ (*,)
Molluscicidal
(warburganal)
Antimalarial
(stem & root
bark);
Antileishmanial
(aq. bark extr)
++
Immunomodulatory
(sesquiterp)
++
Respirastimulant;
immunostimulant &
modulatory;
chemo &
immunoprotective
247
Chapter 6
Pharmacology
Intestinal
Cardiac
Antioxidant
Antiinflam.
CNS-related
++
(flavonoids
in MeOH
bark extr)
Anticancer
Antimicrobial
Antiparasitic
Immune
function
+ (*)
++
Sedative
(alkaloids)
Antibacterial
(MeOH leaf/
bark extr);
antiviral
(acetone
bark extr)
Anti-amoebic;
anthelmintic
(tannins,
phenolic
compounds,
alkaloids);
antimalarial
(medium
polar bark/
leaf extr)
Antisickling
(anthocyanins
in aq & EtOH
bark extr)
Ziziphus
Z. mucronata
++ (bitter)
++ = very active; + = weak or moderately active; -- = not active

* = in vitro; = in vivo; = human clinical trial; s = synergism
ACE = acetylcholine esterase; BPH = benign prostatic hyperplasia; CNS = central nervous system; aq. = aqueous; MeOH = methanol; EtOH =
ethanol; extr. = extract
248
CHAPTER 7
Conclusions
7.1
General conclusions
The purpose of this study was to explore the ethnobotany and phytochemistry of 14 medicinal
plant species and their close relatives that are commonly used as tonics in southern Africa, i.e.
three Agathosma species, four Aloe species, three Arctopus species, Artemisia afra, Balanites
maughamii, four Dicoma species, two Harpagophytum species, Hypoxis hemerocallidea,
Muraltia heisteria, two Sutherlandia species, Vernonia oligocephala, Warburgia salutaris,
Withania somnifera and Ziziphus mucronata. Relating the ethnobotanical information to the
phytochemistry and pharmacological effects was aimed at validating and/or understanding the
traditional tonic uses of these plants.
The hypothesis to be tested was that southern African tonic plants are typically characterised
by chemical complexity, resulting in a wide range of possible biological activities. Each tonic
would then accumulate a diversity of individual compounds as well as a diversity of classes of
compounds, all potentially contributing to possible additive and synergistic effects.
The ultimate purpose was consequently to find a possible scientific rationale for the wide
spectrum of uses of southern African tonic plants and to contribute towards a more profound
understanding of principles and practices relating to the use of tonic plants in traditional
medicine systems.
From an ethnobotanical point of view, all the plants were anecdotally recognized as tonics,
whether it was as general health tonics, bitter tonics, panaceas or adaptogenic tonics. Only
Sutherlandia frutescens and Withania somnifera were indicated as adaptogens, and in both
cases the plants were used for all the classes of ailments, including stress related ailments,
heart and circulation conditions, urinary tract, liver, digestive tract, chest and musculo-skeletal
ailments, as well as infectious or debilitating diseases. All but Muraltia heisteria were indicated
for infectious or debilitating diseases, and 12 of the 14 species (not Muraltia heisteria or
Vernonia oligocephala) were used for chest ailments. This is significant when considering the
possible immunostimulatory capacity of tonics. Furthermore, plants used for stress related
249
Chapter 7
Conclusions
ailments were also indicated for treatment of heart and circulation conditions, in accordance
with the musa-pelo concept, where plants used to treat anxiety, stress and grief; are able to to
turn the heart around.
From the chemical variation study, it was evident that all the plants were chemically complex
with terpenoids, phenolic compounds, nitrogen-containing compounds and alcohol precipitable
solids being present in all the plants. Agathosma betulina and Artemisia afra are aromatic
plants traditionally used to treat the same ailments, and were found to contain the same or
similar bioactive compounds (i.e. mostly monoterpenes and flavonoids). On the other hand, the
chemical compositions of Sutherlandia and Withania somnifera are incomparable and yet both
of these species exert adaptogenic tonic properties, as is the case with Panax ginseng and
Eleutherococcus senticosus. The highest levels of alcohol precipitable solids were recorded in
Aloe species (6.919.3%), Dicoma species (0.717.7%), Sutherlandia species (3.112.8%),
Ziziphus mucronata (0.511.3%) and Hypoxis hemerocallidea (6.410.7%), which may be
correlated with (internal) wound healing potential. High amino acid content was evident in
almost all the species, pointing to possible nutritional aspects of tonics, but more importantly to
the possibility that these plants can contribute to the formation of ionic fluids (NADES), either
as an adaptive means in harsh climatic conditions, or to provide a means of solubility where
bioactive compounds which are not soluble in water or lipids may be assimilated into cell
metabolism. This also provides a rationale for combination of plants in herbal remedy
preparation (e.g. when used in combination with other plants high in organic acids), or
preparing syrups for oral administration (e.g. combining amino acids and sugars). This aspect
deserves further study in order to evaluate the potential impact of ionic fluids on the solubility of
particular active compounds that would normally be considered insoluble. Similarly the
presence of sugars and the wide-spread use of syrups need to be investigated.
The use of all 14 plants for digestive tract ailments was corroborated by their bitterness,
exerting an amarum effect through stimulation of the nervus vagus. The bitterness values for
13 of the plants were recorded for the first time and Aloe ferox, Artemisia afra, Dicoma
anomala, D. capensis, Sutherlandia microphylla and Vernonia oligocephala were found to be
most bitter. Harpagophytum procumbens returned a bitterness value comparable with
international standards. Compounds known to contribute to the bitterness (i.e. sesquiterpene
lactones, triterpenes, iridoids, flavonoids, anthrones and alkaloids) were identified in all the
tonic plants in accordance with the findings. A further investigation into the actual bitterness of
these compounds would yield interesting results. Furthermore, bitter substances are said to act
250
Chapter 7
Conclusions
on the cardiovascular system, decreasing the heart rate and cardiac stroke volume, and all of
these highly bitter species were also indicated anecdotally for heart and circulation conditions.
Pharmacological proof of this phenomenon was reported for Agathosma, Aloe, Artemisia afra,
Harpagophytum procumbens and Withania somnifera. Pre-clinical (in vivo) and clinical results
for the other species would thus be useful.
The tonic plants studied were found to exhibit:
-
Antioxidant and antiinflammatory properties: in many instances, the antiinflammatory

activity was found to be related to or dependent on the antioxidant activity, suggesting that
the biological pathways of these processes are linked. Plants traditionally used for
musculoskeletal illnesses, respiratory ailments and infections exerted antiinflammatory
properties, significantly combined with analgesic properties.
Immunostimulant/modulatory properties: reported for Aloe, Artemisia afra, Hypoxis

hemerocallidea, Sutherlandia frutescens, Warburgia salutaris and Withania somnifera,
Stomachic/digestive properties are in turn related to the amarum effect elicited by bitter
tasting extracts, which leads to increased absorption and consequently an improved
immune system as suggested by TCM theory. However, these results for Artemisia afra,
Hypoxis hemerocallidea, Sutherlandia frutescens and Warburgia salutaris were obtained
through in vitro experiments, so that in vivo confirmation is required.
Anti-stress properties: Sutherlandia and Withania somnifera are known as adaptogens,

and exhibited anti-stress properties consistent with the definition for adaptogens.
Substances or extracts which affect the central nervous system (i.e. as anti-epileptic,
anxiolytic, CNS-depressant, anti-convulsant or hypotensive) were indicated to contain
saponins or steroids which may act in a corticomimetic fashion, or small molecules such as
monoterpenes and free amino acids able to cross the blood-brain barrier and act via
inhibition of cholinesterase enzyme or binding to GABA-benzodiazepine receptors.
However, no clinical results have been provided yet for Sutherlandia to show whether it is
able to enhance resistance to psychological stress in humans.
Anticancer properties: a great variety of compounds have been indicated as effective in the
treatment or prevention of cancer through antitumour, antimutagenic, antiproliferative,
antineoplastic,
antimetastatic,
chemopreventive,
chemoprotective
or
cytotoxic
mechanisms. It is indeed expected that efficacy in cancer therapy is unlikely to result from
a single compound, but more likely from a chemically complex plant or mixture of plants.
-
Other activities such as antimicrobial and antiparasitic were shown in almost all the
species, which may be relevant to the overall efficacy of the plants as tonics.
251
Chapter 7
Conclusions
Furthermore, the biological potency ascribed to certain plant extracts (Agathosma species,
Artemisia afra, Harpagophytum procumbens, Hypoxis hemerocallidea, Sutherlandia species,
Warburgia salutaris and Withania somnifera) could not be justified by the activity of a certain
active principle only. The efficacies of such supposedly active compounds were often found to
be disappointing, and consequently synergy (or at least an additive effect) amongst
component molecules were suggested and/or proven. Also, the need for placebo-controlled
double-blinded clinical trials is undeniable when scientific evidence is required for the actual
efficacy of these tonics. As nitrogen-containing compounds (amino acids and alkaloids) have
been identified in all the tonic plants, a possible agreement between the mood enhancing
effect of such compounds and the magnitude of the placebo effect should be considered.
7.2
Summary of main findings
All the tonic plants were found to be chemically complex (with a diversity of individual
compounds and classes of compounds) leading to possible additive and synergistic effects
(summarized in Table 5.34):
(a)
Phenolic compounds flavonoids, tannins, lactones, acids, anthrones, chromones,

coumarins
(b)
Terpenoids mono-, di-, sequi- and triterpenoids, saponins, iridoids
(c)
Nitrogen-containing compounds alkaloids, amino acids
(d)
Alcohol precipitable solids polysaccharides, glycoproteins
(e)
Bitter substances alkaloids, terpenoids, some phenolics
(f)
Sugars mono-, di and oligosaccharides, cyclitols, alditols
All the tonic plants have multiple biological effects in the human body (summarized in
Table 6.3), including:
(a)
immunostimulatory/regulatory or corticomimetic effects amino acids, sesquiterpenes,

steroidal triterpenes/saponins
(b)
Adaptogenic, stress-releasing, mood enhancing and mind-altering (possibly contributing

to a placebo effect?) alkaloids, amino acids
(c)
Bitter (amarum) effect bitter substances
(c)
Respiratory antimicrobial and expectorant compounds
(d)
Digestive and carminative effect essential oils, and others
(e)
Internal topical effects polysaccharides, glycoproteins

252
Chapter 7
Conclusions
The data thus support and validate the traditional use of these tonic plants as general
strengthening mixtures (imbizas), panaceas or adaptogens (musa-pelo).
The hypothesis that southern African tonic plants are chemically complex can thus not be
rejected even though further phytochemical variation and identification studies are required for
many of the species studied. The hypothesis that the chemical complexity of these tonic plants
inevitably lead to a wide diversity of possible biological activities, can also not be denied based
on the pharmacological literature review for each plant. The available phytochemical and
pharmacological knowledge presented here provides a possible scientific rationale for the wide
spectrum of uses of these southern African tonic plants. It also highlights the need for novel,
innovative clinical research approaches to unequivocally prove the efficacy of the plants in
improving general health in a non-specific way.
7.3
Implications of findings
Complete, verbatim recording of ethnobotanical information is important for the conservation of

indigenous knowledge and the cultural heritage of a traditional healing system, but more
importantly gives indications of possible geographical or seasonal variation, plant parts
containing most active/less toxic compounds, or additive/synergistic/NADES interactions
among constituent compounds or amongst plants used together in preparations.
In depth phytochemical research may help to gain a better understanding of the traditional
uses of plants. While the Agathosma, Aloe, Harpagophytum, Hypoxis, Warburgia and Withania
species are well investigated, important species such as Artemisia afra, Balanites maughamii,
Dicoma anomala, D. capensis, Muraltia heisteria and Vernonia oligocephala may still be further
scrutinised. Metabolomics and LC-MS may be employed as more efficient and practical means
of investigating the full chemical complexity of medicinal plants than standard chromatographic
methods.
Even though phytochemical and in vitro biological testing are valuable and give an indication
as to why certain species are applied as tonic plants, true tonic/ adaptogenic/ immunostimulant
or regulatory capacity can be ascertained only through biological activity in living systems and
cannot be simulated in the laboratory. Despite inadequate in vivo data and clinical trials, a
preliminary classification of these South African tonics based on the given reviews is presented
here:
253
Chapter 7
(a)
Conclusions
All the plants investigated are traditionally known as tonics, and are considered to be of
value in maintaining and improving general health, possible through a complex process
involving nutritive, enzymatic, hormonal and mind-altering effects. Are these tonic plants
useful in maintaining and improving general health? Clinical studies are needed in
which improved bodily functions are measured in a rigorous way to generate
scientifically sound comparative data (as has already been done for Withania).
(b)
Sutherlandia frutescens and Withania somnifera are adaptogens, based on their ability
to increase the bodily resistance to stress non-specifically. Can Sutherlandia reduce
stress and improve the immune system? Clinical studies are needed (as has already
been done for Withania).
(c)
All the species investigated are bitter tonics i.e. they contain intensely bitter
substances, which lead to amplified reflex gastric secretion and increased appetite,
through stimulus of the nervus vagus. Agathosma betulina, A. crenulata, A. ovata and
Artemisia afra may be further classified as amara aromatica based on the high levels
of volatile oils they contain. Which substances are responsible for the bitter taste
recorded during this study in crude extracts? Studies are needed to isolate the bitter
compounds and to determine their pharmacological properties (as has already been
done for Gentiana).
(d)
Agathosma betulina, A. crenulata, A. ovata, Arctopus dregei, A. echinatus,

A. monacanthus, Artemisia afra and Harpagophytum procumbens are alteratives, i.e.
even though they are bitter, they are able to alter the course of morbid conditions
through modifying the nutritive processes while promoting waste. Are these plants
useful to enhance elimination of waste products from the body? Clinical studies are
needed to test liver and kidney function as well as to quantify diuretic activity.
(e)
Sutherlandia frutescens, Warburgia salutaris and Withania somnifera have been

indicated as adjuvants to chemotherapeutic treatments and antibiotics (by aiding in the
removal or prevention of a disease). Are Sutherlandia and Warburgia of value as
adjuvants in the treatment of serious ailments? Clinical studies are needed (as has
already been done with Withania).
(f)
Aloe, Artemisia afra, Sutherlandia frutescens, Warburgia salutaris and Withania

somnifera have been shown to have in vitro immunostimulatory and/or modulatory
capacity. These plants may therefore arouse organic activity, strengthen the action of
the heart, increase vitality, and promote a sense of well-being. Do Sutherlandia and
other tonics affect the immune system in humans? Innovative new approaches are
needed to obtain plausible clinical data showing in vivo immunostimulation.
254
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Acknowledgements
Hereby I would like to thank:
My promoter, Prof B-E van Wyk, for his continued guidance and his talent to
develop the full potential of his students, so that they may produce work of
excellent standard by following a logical, practical and applicable thought
process.
My co-promoters, Dr C Albrecht and Prof F van Heerden, for their support,
expert advice and motivation.
The NRF, URC and Mr P Olivier for financial support.
Coca Cola (Pty.) Ltd. for sponsoring the Bonaqua still drinking water used for the
bitterness taste testing experiment.
All the students, colleagues and friends who participated in the bitterness taste
testing experiment.
Prof L Greyling and Mrs E Diedericks for language reviewing the thesis.
Mr C Diedericks for formatting the figures in the thesis.
On a more personal note, I would like to thank:

God, for purpose, perseverance and perspective, renewed each day.
My husband (Pieter), parents (Deon and Liz Greyling), siblings (Elsa and Willie), parentsin-law (Henning and Hester Olivier) for their continued patience, support and motivation,
appreciated more than words could describe.
My children, Divan and Deirdre, for their unconditional love.
Prof Rui Krause, for his encouragement and support as a mentor, colleague and friend.
My friends, family, colleagues and fellow-students, for bearing with me patiently through
tough times.
312
Appendix 1
Bitterness Taste Testing
APPENDIX 1: Bitterness taste testing Application to ethics

committee
(1A),
and
Informed
Consent
Form
completed by willing participants (1B)

Appendix 1A:
Application to ethics committee for bitterness taste testing
Faculty of Science
Ethics Committee
APPLICATION FOR APPROVAL OF INVESTIGATION INVOLVING THE USE OF HUMAN/
ANIMAL SUBJECTS
1.
2.
Name of Applicant
Denise K Olivier
Title
Mrs
Student Number
909121668___________________________
Department
Botany and Plant Biotechnology
Qualifications
MSc
Tel. no.
011 559 6158
Cell no.
082 570 8674
Is this for your thesis, dissertation or minor dissertation?

Yes X
No
If yes, provide the following:

Supervisor: Prof Ben-Erik van Wyk___________Tel: 011 559 2412
Co-supervisor Dr Carl Albrecht
Tel: 084 208 5150___
Appendix 1
3.
Title of the project:

Botanical, ethnobotanical and chemotaxonomic aspects of African tonics
4.
Total project period:
5.
Has this study previously been submitted to any Ethics review committee?
Yes
6.
8 years
No X
Is the project supported by the external funding agency?

Yes
No X
If yes, sponsors name and identification:
7.
Description of human subjects:

Number
Age
above 18 years
Sex:
7b
minimum = 6; maximum = 84
F X
M X
Description of experimental subjects N/A

Species:
Number:
8.
Included copies of all pertinent attachments including:
Questionnaires N/A
Informed consent(s)
Letters of approval from cooperating institutions, etc. N/A
Permits N/A
Yes X
No
If NO, explain below:
Appendix 1
9.
Brief description of proposed research: include major hypotheses research

design.
The hypothesis is that one of the many typical characteristics of a tonic plant is that it
will have a bitter taste which is responsible for the amarum effect to stimulate the
taste buds and promote the flow of saliva, gastric juices and bile (Van Wyk B-E, Wink
M. Medicinal plants of the world. Pretoria: Briza publications, 2004: p 61, 155, 313, 400)
which in turn enhances the function of the digestive system, and ultimately boosts the
immune system by means of a more efficient uptake of nutrients or some other
unknown mechanism .
10.
Describe the source(s) of subjects and the selection criteria. Specifically, when
did you obtain the names of potential subjects? Where and how will you contact
them?
As a lecturer in the Department of Chemical Technology, with a least 30 post-graduate
students at any given time of the year, and at least 15 members of staff, I wanted to
engage these students and colleagues in the department in the taste testing experiment
as the logistics around the experiment is simplified if all the candidates are from one
department on one campus, but also to promote Natural Products Research in our
department. Furthermore, these are all people who would attempt the tasting in a
responsible way, meet the requirements as stated in paragraph 15 of this document
and paragraph 4 of the informed consent form attached.
11.
Procedures: Provide a step-by-step description of procedure, including the

frequency, duration and location of each procedure.
The scientific procedure to obtain bitterness values for herbal tonics was obtained from
the World Health Organisation, Geneva, (2002) A.I.T.B.S. Publishers & Distributors
(Regd.) Delhi-51, Quality control methods for medicinal plant materials, page 3840. A similar method can be found in the European Pharmacopoeia (2005), 5, p.221222.
The method entails the following basic steps:
Step 1: Screen all available candidates ability to taste bitterness
0.100 g of quinine hydrochloride R is dissolved in Bonaqua still drinking water
(This water is produced by the internationally known Coca-Cola Company; it is
tap water purified by reverse osmosis and reconstituted with normal salts found
in drinking water. This is an international formula and should release molecules
from the plant material as would be the case with drinking water from a tap) and
diluted to 100 mL. This solution is diluted 100 times (5 mL diluted to 500 mL
with Bonaqua drinking water) and constitutes the stock solution (S1) with a
concentration of 0.01 mg/mL.
Appendix 1
Nine different dilutions ranging from low to high is prepared (in tubes) from
solution S1 according to Table 1 below, and is used to obtain the bitterness
tasting ability of the enrolled candidates.
Table 1: Serial dilution of solution S1 to determine bitterness tasting ability
S1 (mL)
Safe drinking water
(mL)
Mg quinine
hydrochloride in 10 mL
of solution
1
4.2
5.8
2
4.4
5.6
3
4.6
5.4
4
4.8
5.2
Tube number
5
6
5.0
5.2
5.0
4.8
7
5.4
4.6
8
5.6
4.4
9
5.8
4.2
0.042 0.044 0.046 0.048 0.050 0.052 0.054 0.056 0.058
All candidates must then taste the solutions from the respective tubes, (starting
from 1, proceeding to taste from consecutive tubes until a bitter taste sensation
is obtained) by following the procedure given below. A person who does not
manage to appreciate a bitter sensation when tasting the solution from tube 9
(highest concentration of quinine hydrochloride at 0.058 mg/10 mL), is not a
suitable candidate for continuing this experiment.
Step 2: Bitterness taste testing of 15 medicinal plant extracts
0.100 g of dried ground plant sample will be extracted overnight by bringing 100
mL of Bonaqua drinking water to boiling point, adding this to the plant sample
and leaving it to stand overnight. The mixture will be filtered through cotton wool
and the filtrate diluted 100 times (5 mL diluted to 500 mL with Bonaqua drinking
water). This solution constitutes the stock solution (S2) with a concentration of
0.01 mg/mL. Nine different dilutions ranging from low to high is prepared (in
tubes) from solution S2 according to the table below, and is used together with
solution S2 (undiluted) to obtain the bitterness value through bitterness tasting
by candidates who passed step 1.
Table 2: Determination of bitterness value: serial dilution of plant extracts tested
1
S2 (mL)
1.00
Safe drinking 9.00
water (mL)
Concentration 0.01
of S2 (mg/mL)
2
2.00
8.00
3
3.00
7.00
Tube number
4
5
6
4.00 5.00 6.00
6.00 5.00 4.00
0.02
0.03
0.04
0.05
0.06
7
7.00
3.00
8
8.00
2.00
9
10
9.00 10.0
1.00 -
0.07
0.08
0.09 0.10
To save time, the person tasting the S2 dilutions will start with Tube 5. If a bitter
taste sensation is experienced, the person would then continue tasting Tubes 1
to 4, to determine the smallest concentration at which the bitter sensation is
obtained.
4
Appendix 1
If the person cannot taste bitterness in Tube 5, he/she will then continue with
Tubes 6 to 10 to find the smallest concentration at which bitterness is
experienced. If bitterness is tasted in Tube 1 already, the content of Tube 1 will
then be diluted the same way as S2 in order to obtain 10 more tubes starting
with a concentration of 0.001 mg/mL and ending with a concentration of 0.01
mg/mL.
Tasting procedure:
After rinsing the mouth with Bonaqua drinking water, taste 10 mL of test solution from
the tubes provided (lowest concentration first) by swirling the solution in the mouth
mainly near the base of the tongue for 30 seconds. If a bitter sensation is not felt after
this time, spit out the solution and wait for 1 minute to ascertain if there is a delayed
sensitivity. If not, rinse with Bonaqua drinking water, wait 10 minutes and taste the tube
with the next highest concentration.
Each candidate should ascertain their bitterness tasting ability prior to tasting the plant
extract dilutions of each plant by obtaining the minimum concentration of quinine
hydrochloride provoking a bitter sensation for that day first, as is given in Table 1 (find a
value for c each time a certain plant is tested).
There is no safety concern as the plants extracts will not be ingested. Furthermore, the
plants are all popular traditional medicines that are widely used and ingested, with no
indications of toxicity having been reported to date.
12.
Informed consent:
documents.
Describe the consent process and attach all consent
All candidates willing to participate in this experiment will have to fill out and sign the
attached informed consent form before proceeding with the process described in
section 11 above. This form provides background about tonic plants, the amarum effect
and a brief outline of the procedure followed to obtain the bitterness value of a given
sample. It also contains questions pertaining to the physical condition of the candidate
and explains that the candidate will be chosen based on the answers of these
questions together with the ability of the candidate to be able to taste bitterness. The
possible risks when participating in this experiment, minimal as they may be, will be
highlighted to the candidates. Consequently, each candidate must also indicate a
promise not to swallow the samples offered for tasting and will follow the protocol
stipulated in the method supplied with each tasting session to minimise any possible
risk. Finally, the candidate is asked for consent to publish his/her results as part of a
scientific publication, to use his/her name in the acknowledgements in this publication
and if the candidate would like personal feedback about this experiment.
Appendix 1
13.
Subject confidentiality: Describe the confidentiality process.

The confidentiality aspect is addressed in the consent form attached and in section 12
above. If the candidate wishes to remain anonamous, care will be taken to only use the
bitterness tasting results.
14.
Benefits: Describe the anticipated benefits to the subjects, and the importance of
the knowledge that may reasonably be expected to result.
Main benefits in survey have been included in the informed consent form
Yes X
15.
No
Risks:
Describe the risks involved with these procedures (Physical,
psychological, and/or social) and the precautions you have taken to minimized
these risks.
The risks are minimal as all 15 plants used in the experiment are widely used by
traditional healers across southern Africa for medicinal purposes. No indication of
toxicity has ever been recorded for these 15 species. All samples offered to the
candidates will have been prepared by extracting with boiling Bonaqua drinking water
and diluted further, by using Bonaqua drinking water. So, even if some of the sample is
ingested, the quantities would be minimal and we do not expect any adverse effects on
the candidates.
Precautions taken would include a preliminary screening of candidates based on
physical health (candidates older than 18, using no chronic medication, who are not
pregnant), the signing of an undertaking by each candidate not to swallow the samples
and discontinuing with the tasting experiment by a candidate if any adverse effects are
experienced by that candidate. A qualified health practitioner will also be present during
all tasting periods to assist in the unlikely event of someone being hypersensitive to any
of the solutions.
16.
Abstract or summary of the proposed research

In traditional Chinese medicine tonic plants are used to strengthen and stimulate the
immune functions of the body, thereby increasing its resistance to all kinds of assaults.
These plants are non-toxic and help the body to detoxify, while strengthening it at the
same time. Tonics are categorized as either stimulating tonics (gently stimulates
functional activity, like building muscle), or nourishing tonics (provides vitamins,
minerals, other nutrients and building blocks for proper tissue health and organ
function).
Fifteen known South African tonic plants have been subjected to screening studies
where seven classes of chemical compounds (phytochemicals) have been scrutinized
in order to establish a constituent pattern that might explain the (stimulating) tonic
properties of these plants. All these plants have been used traditionally for the
Appendix 1
treatment of a great variety of ailments from stomach to chest problems and many
more.
The plants include Agathosma (Rutaceae) or buchu, Aloe (Asphodelaceae) or bitter
aloe, Arctopus species (Apiaceae) or sieketroos, Artemisia afra (Asteraceae) or African
wormwood, Balanites maughamii (Balanitacae) or torchwood, Dicoma anomala
(Asteraceae) or maagbitterwortel, Harpagophytum procumbens (Pedaliaceae) or devils
claw, Hypoxis hemerocallidea (Hypoxidaceae) or African potato, Muraltia heisteria
(Polygalaceae) or persblommetjie, Sutherlandia (Fabaceae) or cancer bush, Vernonia
oligocephala (Asteraceae) or groenamara, Warburgia salutaris (Canellaceae) or
pepper-bark tree, Withania somnifera (Solanaceae) or winter cherry and Ziziphus
mucronata (Rhamnaceae) or buffalo-thorn. By conducting the bitterness taste testing
experiment, we would like to confirm that these tonic plants are indeed bitter, and to
quantify the bitterness, so that direct comparisons can be made with other well-known
bitter tonics described in European and other pharmacopoeias. The novelty of this work
is that it will represent a very first study of bitterness in South African medicinal plants.
A variety of phytochemicals may be responsible for the bitter taste sensation
responsible for the amarum effect (see section 9) synthesized by the plant as a defence
mechanism against grazing animals. These compounds may include saponins
(triterpene molecules bound to glycosides), flavonoids (polyphenolic antioxidants) or
alkaloids (nitrogen-containing molecules that are not amino acids). While the
taxonomic, chemotaxonomic and chemical studies have revealed the identities of many
different phytochemicals within each of the plant species, we would now like to explore
possible links between the bitterness of these tonic plants and their ethno-botanical
uses.
17.
Additions to or changes in procedures involving human subjects as well as any

problems connected with the use of human/ experimental animals subjects once
the project has begun will be brought to the attention of the ethics committee. I
will ensure that the rights and welfare of the human / experimental animals
subjects are properly protected. I understand that I cannot initiate any contact
with human subjects/ start experiments with animals before I have received
approval and/or complied with all contingencies made in connection with the
approval.
Signature of Principal Investigator
Date
Appendix 1
18a.
Declaration and approval by Department Chair (Department of Botany and Plant

Biotechnology)
ETHICAL:
Acceptable
Not acceptable
Remarks:
Signature of the Department Chair

18b.
Date
Declaration and approval by Department Chair (Department of Chemical

Technology)
ETHICAL:
Acceptable
Not acceptable
Remarks:
Signature of the Department Chair
19.
Declaration
and
approval
ETHICAL:
Acceptable
Date
by Representative:
Not acceptable
Faculty Ethics
Committee
Remarks:
Representative: Faculty Ethics Committee
20.
Date
Declaration and approval by the Dean

ETHICAL:
Acceptable
Not acceptable
Remarks:
Signature of the Dean
Date
8
Appendix 1
Appendix 1B:
Ethics informed consent form completed by willing participants
`
BITTERNESS TASTE TESTING
Approved by the Faculty of Science
Supported by the Department of Chemical Technology
PLEASE READ THE INFORMATION BELOW CAREFULLY AND ANSWER THE
QUESTIONS THAT FOLLOW IN ORDER TO GIVE YOUR INFORMED CONSENT FOR THE
EXPERIMENT YOU ARE ABOUT TO PARTICIPATE IN. IF THE ANSWER IS NO TO ANY
OF THE QUESTIONS, THEN THE CANDIDATE WILL BE EXCLUDED FROM
PARTICIPATION
1.
Name of participant:
Title
Student/Staff Number :
3.
Department
Tel. no.
Cell no.
________________________________
Information about this experiment:

In traditional Chinese medicine tonic plants are used to strengthen and stimulate the
immune functions of the body, thereby increasing its resistance to all kinds of assaults.
Tonics are categorized as either stimulating tonics (gently stimulates functional activity,
like building muscle), or nourishing tonics (provides vitamins, minerals, other nutrients
and building blocks for proper tissue health and organ function). Fifteen known South
African tonic plants have been subjected to screening studies where seven classes of
chemical compounds (phytochemicals) have been scrutinized in order to establish a
constituent pattern that might explain the (stimulating) tonic properties of these plants.
Appendix 1
All these plants have been used traditionally for the treatment of a great variety of
ailments from stomach to chest problems and many more. Despite their regular use in
traditional medicine, there are no reports of any adverse side-effects or toxicity for any
of the species.
The hypothesis for the outcome of this specific experiment is that one of the many
typical characteristics of a tonic plant is that it will have a bitter taste which is
responsible for the amarum effect to stimulate the taste buds and promote the flow of
saliva, gastric juices and bile (Van Wyk B-E, Wink M. Medicinal plants of the world.
Pretoria: Briza publications, 2004: p 61, 155, 313, 400) which in turn enhances the
function of the digestive system.
Bitterness values have been determined for many of the traditional bitter tonic plants
used in Europe and other parts of the world (e.g. gentian Gentiana lutea). This study
will have novelty value as it is the first attempt at determining the bitterness values for
South African traditional tonic plants. The results will be of no direct commercial value
but will have considerable scientific value.
I understand the background against which this experiment is performed.
Yes
3.
No
Procedure:
This scientific procedure to obtain bitterness value was obtained from the World Health
Organisation, Geneva, (2002) A.I.T.B.S. Publishers & Distributors (Regd.) Delhi-51,
Quality control methods for medicinal plant materials, page 38-40. A similar
method can be found in the European Pharmacopoeia (2005), 5, p.221-222.
The method entails the following basic steps:
Step 1: Screen all available candidates ability to taste bitterness
Nine different dilutions of a known bitter substance, quinine hydrochloride, are
prepared in Bonaqua still drinking water. These dilutions ranging from low to
high is offered to you, the enrolled candidate, and is used to obtain your
bitterness tasting ability:
You must taste the solutions from the respective tubes by following the
procedure summarised below under Tasting procedure. If you not manage to
appreciate a bitter sensation when tasting the solution containing the highest
concentration of quinine hydrochloride, you are not a suitable candidate for
continuing this experiment.
10
Appendix 1
Step 2: Bitterness taste testing of 15 medicinal plant extracts

Plant extracts will be prepared by extracting the dry plant material in boiling
Bonaqua drinking water, filtered and diluted further in Bonaqua drinking water.
Once your bitterness tasting ability has been established, you will then
proceed to taste the extract dilutions from lowest to highest until a bitter taste
sensation is obtained.
Tasting procedure:
After rinsing your mouth with Bonaqua drinking water, you will taste 10 mL of test
solution from the tubes provided (lowest concentration first) by swirling the solution in
the mouth for 30 seconds. If a bitter sensation is not felt after this time, you will spit out
the solution and wait for 1 minute to ascertain if there is a delayed sensitivity. If not,
you will rinse with Bonaqua drinking water, wait 10 minutes and taste the tube with the
next highest concentration.
You will have to ascertain your bitterness tasting ability prior to tasting the plant extract
dilutions of each plant by obtaining the minimum concentration of quinine hydrochloride
provoking a bitter sensation for that day first, as in Step 1 above.
Despite no documented records of toxicity, it is important that the extracts are
not ingested.
Duration of experiment:
The tasting experiment should not take longer than 2 hours maximum per sample (1
sample per candidate per day). A total of 150 samples need to be tasted, which implies
that this experiment might take a few days in total. Once your bitterness tasting ability
is established, it would be greatly appreciated if you will help with the assessment of
several samples over the total time it takes to complete the experiment.
I understand the procedure explained above and will follow the exact tasting procedure
as it is outlined here, and which will be provided in more detail during the tasting
experiment:
Yes
4.
No
Risks:
The risks are minimal as all 15 plants used in the experiment are widely used by
traditional healers across Southern Africa for medicinal purposes. No indication of
toxicity has ever been recorded for these 15 species. All samples offered to the
candidates will have been prepared by extracting with boiling Bonaqua water and
diluted further, by using Bonaqua drinking water. So, even if some of the sample is
ingested, the quantities would be minimal and we do not expect any adverse effects on
the candidates.
11
Appendix 1
I agree to the following precautions taken to minimise the risk taken by myself during
my participation in this experiment:
a) I am in good physical health (I am older than 18, use no chronic medication, and is
not pregnant):
Yes
No
b) I will not swallow any of the samples offered to me for bitterness taste testing:
Yes
No
c) I will discontinue my participation in this experiment if I experience any adverse

effects due to tasting the samples I am offered to taste:
Yes
5.
No
Confidentiality:
a) My identity may be used in the acknowledgements given in the scientific presentation
of the results of this experiment:
Yes
No
b) I want personal feedback on the summarized results obtained from this experiment:
Yes
6.
No
Benefits:
The results of the experiment will be of no direct commercial value but will be of
considerable scientific interest. It will present novel data to the international scientific
community on the bitterness of South African tonic plants and this insight may
contribute towards a better understanding of the amarum effect and the potential
benefits to be derived from bitter tonics.
I know that I will gain no financial benefit from my participation in this experiment.
However, Ms D Olivier, the principal investigator, undertakes to sponsor a social event
(with refreshments) in order to show her gratitude. Feedback on the main results of the
experiment will be presented at this function.
I understand the benefits described above:
Yes
No
Signature of Participant
Date
12
Appendix 2
Ethnobotanical Anecdotes and Uses
APPENDIX 2: Table of ethnobotanical anecdotes and uses (cited

verbatim from the referenced texts) for the 14 main
traditional
tonic
plants
in
southern
Africa,
in
alphabetical order (2A-2M)

Appendix 2A: Agathosma betulina, A. crenulata and A. ovata (* = information
based on personal interviews, initials given in text, full names at
end of the appendix)
Pappe, 1847, 1850, 1857:
1847, page 5: A. crenulata a slightly astringent and bitter taste, and, used in the form of an
infusion, promotes perspiration. Hence, its utility in chronic rheumatism, gout, and other
maladies, caused by the sudden suppression of cutaneous action highly beneficial in
diseases of the bladder, especially incatarrh of the bladder,spasmodic strictures, and
similar complaints, dropsy all such cachectic or hydropical cases, as these originate in
suppression of the perspiration of urine; leaves used in the form of a bath (Kruiden bad) in
rheumatism; buchu vinegar (Buccho-azyn) and the tincture (Buccho-brandewyn), are excellent
embrocations in sugillations, sprains, and contusions.
1850, page 5: A. crenulata as in the 1847 edition, with the treatment of Cholera Morbus,
Haematuria, Calculus, and in suppuration of the vesica, urethra, and prostrate gland; stimulant
to the stomach, indigestion, increased appetite; herb bath, buchu vinegar and tincture also
used for rheumatic pains and luxations.
1857, pages 7-8: as in the 1850 edition.
Dykman, 1908:
The author does not provide the botanical identity of boegoe:
Cough page 113: one cup each of sugar, dry figs, boegoe, kruisement, anise, pounded
together, added to bottle brandy, take a kelkievol thrice daily; pages 113, 142 prepare a tea
of one handful each of boegoe, hottentotskooigoed and kattekruie, together with a teaspoon of
aniseed, take 2-3 times daily; coughing (for young children) pages 142: one teaspoonful of
boegoebrandewyn with a few drops kopifa.
Whooping cough page 147: boil together one cup each of als, boegoe, wynruit, sugar as well
as a clove of garlic and a piece of camphor with a bit of water until syrupy; whooping cough
and hoarseness pages 29, 147: mix one spoon of wilde-als syrup, one spoon of
boegoebrandewyn, one spoon of soetolie and an egg yolk use one teaspoonful three times
daily.
Appendix 2
Tuberculosis, cough with blood in sputum pages 113, 165: one cup each of vinegar, old
wine, strong boegoebrandewyn and brown sugar, boiled together, remove from heat and add
half a cup of camphor brandy, drink a teaspoonful when coughing; pages 164-165: two bottles
of bronkorssap, one bottle of hottentotsvyesap and a whole lemon cooked together until it
curdles, add a little mak salie and as much boegoe, filter, add half a pound sugar , six spoons
of honey, two spoons of soetoilie and six cloves, boil to reduce the volume until the contents
will fill one bottle, take half a kelkie twice daily; mix and grind together brandnetels, boegoe,
bels and karmedik, add one teaspoon of ginger for every cup of powder and a good helping of
sugar, mix and take a teaspoon of powder at a time; throat tuberculosis pages 146, 165:
prepare a brandy tincture with a handful of salie, thyme and buchu each, 7 branches wynruit,
one piece of ginger, seven figs, half a cup of halved raisins and a pint of sugar, drink a little bit
thrice daily.
Phlem in chest for children page 115: add a handful each of wynruit, boegoe and als to three
bottles of water and boil together until the volume is halved, filter, add one pound of sugar and
boil together until syrupy, drink a teaspoonful four times daily.
Chest ailments pages 131-132: one pound of sugar, finely cut figs, one cup of peperwortels,
one spoon of als, two spoons of boegoe and one pound of raisins in two bottles of brandy, left
in the sun for two days, take half a kelkievol twice daily; a handful boegoe, half a cup of sugar,
30-40 dried figs added to a bottle of good wine, take a kelkievol mornings and evenings.
Heavy coughing page 142: a brandy tincture made of a cupful each of peperwortel, dried
figs, kruisement, dried boegoe, taken now and then; page 143: make a brandy tincture with
one cup of sugar and one each of dried figs, boegoe, kruisement or wilde salie and
peperwortel, take half a kelkie thrice daily with sugar.
Hoarseness page 142: mix a teaspoon of raw honey, a spoon of sugar, a teaspoon of
crushed ginger, three spoons of boegoebrandewyn, use often.
Infected wounds page 120: mix together one cup of good brandy, one cup of boegoe-asyn,
two cups of water and a tablespoon of salt, wet a cloth with the mixture and dress the wound,
keep the cloth wet with the mixture, reputed remedy for inflammation, used in war; bruises
page 146: a small handful of boegoe in a bottle of vinegar, used externally as a compress and
taken orally; poultices for wounds page 156: prepare an ointment of pounded boegoe fried
with wax and soetolie; pages 157-158: prepare a plaster by adding two spoons each of wax,
soetoilie, kasterolie, lynolie, boegoebrandewyn and boerseep, boil to dryness and add a
teaspoon of balsam sulfirus while still luke warm; page 172: mix together one cup each of
water, vinegar, wine, brandy and sugar, with one spoon each of camphor brandy, sugar and
boegoe, use as wash; internal wounds page 172: mix a strong tea of a handful chamomile
together with one spoon each of boegoe-asyn, boegoebrandewyn, camphor brandy and spirit
vinegar, with a teaspoon of saltpeter or a tablespoon of salt, sufficient to fill a deep plate, take
one tablespoon of the mixture (luke warm) thrice daily and wet clothes to put on sore.
2
Appendix 2
Haemorrhoids page 127: prepare a brandy tincture of boegoe, bels, als, renostertoppe,
kruisement, karmedik and pimpernel, take a sopie mornings and evenings.
As a laxative page 138: add together one bottle of pontakwyn, a small bottle of
kraamdruppels, a small bottle of red lavender, half a small bottle of Jamaica ginger, powder
kina and half a cup of finely pounded boegoe; page 139: boegoebrandewyn.
Nose bleed or blood in sputum page 153: add one cup each of finely cut raisins and
peperwortel, one finely cut, dry fig, half a pound of sugar and one spoon each of aniseed,
boegoe and wilde-als, mixed together in a flask of good brandy, take three kelkies daily.
Persi page 155: powder together a spoonful each of boegoe and anys, mix with a spoonful of
honey and take orally.
Plaster page 156: crushed boegoe fried with wax and soetolie; plaster for all wounds page
157: mix together two spoons each of wax, soetolie, castor oil, lynolie, boegoebrandewyn and
boerseep as well as one spoonful of camphor brandy, boil together until dry, add one teaspoon
of balsem sulfirus when slightly cooled and mix.
Sinken pains page 158: prepare an ointment by mixing one tablespoon of terpentyne, one
each of boegoebrandewyn and bontseep (bought from the pharmacy), one teaspoon of
soetolie, one tablespoon of hartshoorn (optional) together, rub onto painful parts, also good for
buiksiekte.
Sore nipples page 166: mix a kelkie of boegoebrandewyn with black sugar, rub onto nipples
thrice daily.
Water retention page 167: mix one cup each of boegoe and prywortels with a jar of jenewer,
take three half kelkies daily.
Sores in throat (diphteria) page 170: mix a tablespoon each of boegoe-asyn, camphor
brandy, burned sugar and honey, with a knife tip of aluin and three spoons of hot water, use to
gargle.
Kling, 1923:
Reference is made to Boegoe only, without any distinction between species:
Page 11: diarrhoea (in children), acid in stomach and convulsions (stuipe) damp, heated
leaves (boegoeblare) placed as warm compresses on stomach; page 17: topical treatment for
rheumatism, sciatica, gout, backache, lumbago boegoe and bellisbossie infused as an
embrocation and rubbed in.
Page 17: backache and kidney ailments a mixture of boegoe, sandolyn, hottentotskooigoed,
bergsalie and droedaskruie, possibly used as tincture
Page 18: bladder pains and colic apply a damp, hot compress made of boegoeblare
(leaves), koblare and allteeblare or take a mixture of boegoe, chamomile and alltee possibly
Appendix 2
used astincture; catarrh in the bladder or urinary troubles and swelling of the extended gland
take boegoe tea freely or apply a damp, warm compress on bladder.
Page 19: Cholera Morbus in case of inflammation of the bladder, use warm boegoe tea and
boegoe compresses.
Page 21: sprains, bruises or swelling apply cold boegoe-asyn as a compress.
Laidler, 1928:
Page 434: Buchu roots are bitter, and cause the same reaction as Radix Calumbae.
A. betulina:
page 441: powder used for dusting (fire and sun burns), roasted with oil or fat
and dropped into the ear for earache (first mixed with mothers milk before dropping into
babies ears).
Pereira, 1857:
Pages 399-401: Buchu (Barosma crenulata and other species); warm, mint-like taste; aromatic
stimulant tonic; in moderate dosages promotes appetite, relieves nausea and flatulence,
used as a diuretic and diaphoretic; buchu brandy chronic stomach and bladder diseases;
buchu chronic urino-genital maladies of the organs, chronic inflammation of the mucous
membrane of the bladder, irritable conditions of the urethra, gleet, lithiasis (increased secretion
of uric acid buchu, administer together with alkali-like liquor potassae), prostatic affections,
rheumatism, skin diseases, dyspepsia; dosage forms are powder in wine, or Infusum Buchu
(infusion in water where the leaves are macerated for 1 4 hours and strained through calico)
used as a tonic, sudorific or diuretic, Tinctura Buchu (tincture of leaves in Proof Spirit
digested for seven days,and the clear liquor filtered, or percolated, or macerated).
Watt & Breyer-Brandwijk, 1962:
Agathosma spp.:
page 909: kidney problems take a leaf infusion; page 910: buchu
brandy or vinegar used as liniment or embrocation for kidney and urinary tract diseases, and
local application to bruises and relief of rheumatic pains and gout (infusion stimulates
perspiration), used for almost every disease that afflicts mankind; leaf preparations taken for
cholera, bladder diseases (especially chronic catarrh), haematuria, calculus, infections of the
bladder, urethra, prostrate gland; buchu produces diaeresis and imparts a characteristic odour
to urine; it has a slightly astringent bitter taste; used for dropsy; is a stimulant and stomachic
bitter; added to the bath as a relief for rheumatism.
A. betulina:
page 910: commercially important volatile oil, due to diuretic and anti-septic
properties.
Appendix 2
Anonymous, 1962. Oupa en Ouma se boererate (Grandfather and Grandmothers traditional

remedies). Tafelberg-Uitgewers, Kaapstad. pp. 146:
Inflammation page 22: add half a cup of boegoe-asyn, half a cup of rou lynolie, one cup of
castor oil, half a cup of kanferbrandewyn, one tablespoon of bloekomolie, one tablespoon of
lamp oil and one tablespoon of terpentyne , shaken together. Take one tablespoonful in the
morning and evening or half a teaspoonful to 10 drops for children depending on age; page 23:
add one small bottle each of soetoilie, balsemsulfuris, Hoffmannsdruppels, versterkdruppels,
rou lynolie, castor oil, camphor and turpentine, together with one cup of boegoe-asyn, two
tablespoons of vinegar and one cup of brandy, mixed together. One tablespoonful thrice daily
for adults.
Water retention page 24: one cup of preiwortels (dried in shade) and one cup of boegoe
leaves, prepare a strong brandy tincture, take a kelkievol twice daily.
Sprains page 43: boegoe-asyn for sprained ankles.
Respiratory tract page 92: take one handful each of boegoe, hottentotskooigoed and
kattekruie together with one teaspoonful of anise seed, prepare an infusion as a tea, take twice
or thrice daily; page 93: prepare a strong tincture of one bottle of fransbrandewyn, half a cup of
finely cut, dried figs, half a cup boegoe, one pound of teesuiker (black is best) and one
tablespoon of aniseed. Take a kelkievol thrice daily before meals.
Digestive system page 131: prepare a strong tincture of one packet of boegoe and one bottle
of vinegar in a luke warm oven, add one cup of rou lynolie and shake before use, take one
tablespoon every morning; page 138: one mespunt ground boegoe leaves in water taken for
an upset stomach.
Kidney ailments page 143: make a tea of boegoe and hotnoskooigoed, take one cup of the
hot mixture and add a pinch of cream of tartar, stir well and take before going to bed; prepare a
tea of bergboegoeblare, take thrice daily.
Gall page 151: prepare a weak tea of a handful of boegoe leaves in three cups of boiling
water, take one tablespoonful thrice daily.
Headache page 189: prepare a tincture of a few boegoe leaves and half a bottle of brandy,
filter and wet the head with the tincture. Close the head with a cloth and lie still for a while,
covering the head with cushions.
Joints and muscles page 198: add big handfuls each of boegoe, ysterblare, wilde-als, wilde
kopiva, chamomile, gansies and bitterbossie, to three big spoons of honey and a packet of
Engelse sout (Epsom salts) in an enamel pot. Add four cups of boiling water and leave
overnight. Transfer liquid (even the condensated liquid formed in the lid, which is most
powerful) to the bottle, and shut cork. Take a big spoonful thrice daily after meals.
Appendix 2
Anonymous, date and publisher unknown. The Khoikhoi:

Page 9: buchu (Agathosma spp.) leaves dried and powdered used by the Khoikhoi as a
dusting powder for skin conditions and wounds; also for stomach complaints, rheumatism,
indigestion, kidney and bladder ailments.
Batten & Bokelman, 1966:
Page 92 (A. ovata): medicine a tea from plant; good for nerve disorders.
Smith, 1966:
Pages 135-139: the San women sprinkled powdered leaves on their heads when participating
in a tribal dance, and it was used in marriage rites; dried leaves were chewed as medicine for
stomach complaints, or powdered and mixed with fat to anoint the body as a perfume and
protection from the sun and weather; buchu brandy (boegoebrandewyn brandy poured onto
leaves and a tincture prepared) used as a pick-me-up tonic and used; buchu brandy and
(preferably) buchu vinegar (boegoe-asyn leaves steeped in vinegar at length, becoming
almost a mucilage) used to treat kidney and bladder ailments as a diuretic, rheumatism,
arthritis and gout, as a bitter digestive tonic, for external local application to bruises,
contusions, fractures and sprains.
Page 181: buchu-asyn used for swellings, bruises, etc., or used to cleanwounds, or as an
embrocation for treating sprains, rheumatism and arthritis, etc.; buchu brandewyn used
internally to stimulate perspiration in rheumatic afflictions, for digestive complaints and as a
general diuretic for bladder and kidney diseases.
Page 281: brandy or vinegar tincture of A. betulina was used for sprains, etc.
Courtenay-Latimer, et al., 1967:
Plate 41 (A. ovata): used for a number of medicinal remedies.
Palmer 1985:
Page 49: Agathosma spp. used as a Hottentot perfume leaves mixed with fat from fat-tailed
sheep; page 87: dried, powdered buchu leaves used for wounds, stomach and other
complaints; most widely used medicine by early Cape Colonists; as a brandy or wine tincture,
sometimes as a tea for kidney and bladder complaints, digestion, rheumatism and gout; a
vinegar embrocation used for fractures, sores and swellings; oil known to have anti-eptic
properties.
Page 87: Burchell (late 1700s) wrote that he had treated a wound on his servants hand with
buchu vinegar and brandy, and rinsed it with wilde-als afterwards.
Page 98: rheumatism A. crenulata or buchu used for infusion taken orally, sometimes
prepared by adding blousalie, wild mints or willow leaves; buchu used as an embrocation or for
a bath.
6
Appendix 2
Forbes, 1986 [i.e. Thunberg (1772 1775)]:

Buchu [any of various Agathosma species, i.e. A. ovata (page 77), A. pulchella (page 312)],
after mixing the powdered, dry leaves with sheep fat, used to anoint the body, possibly for
protection against the heat of the sun, or the cold of winter, for cosmetic reasons and antibiotic protection.
Page 174: buchu used to powder the body, or as an ointment.
Page 182: buchu leaves first dried in the shade and then over the fire, before it was pulverised;
the powder was used as a cosmetic by the Hottentots; buchu is a generic term for various
species from the Rutaceae family; mostly A. crenulata or A. betulina.
Cilli AM, 1992. Kruie op witblits, rate, resepte en feite (Herbs on witblits, traditional remedies,
recipes and facts), p 43. Unpublished notes, Worcester Museum:
Page 14: tinctures made of leaves in witblits or vinegar is put in the sun to age used as a
herbal bath for rheumatism; buchu vinegar is good for bruises, sprains and broken bones, as
well as kidney and bladder problems, causes perspiration and is a digestive tonic. A herbal
wine can be prepared by adding 1% buchu and 15% wilde-als to sweet white wine.
Pages 21-23: used for haemorrhoids (page 25: a brandy tincture made of buchu, bels, als,
renosterbostoppe, kruisement, karmedik and pimpernel take some in the morning and
evening), chest ailments (page 26: mix sugar, figs, peperwortel, als, buchu, raisins and
brandy, leave it in the sun for two days take half a glass thrice daily), poisoning (page 27:
treat topically with boegoebrandewyn, mixed with ground kruitjieroemeniet), coughing (page
28: mix one cup of fine sugar, dry figs, buchu, kruisement or wilde salie, crushed peperwortel
and crushed aniseed each, prepare a tincture with brandy take one glass thrice daily),
whooping cough, bruises (page 29: rub in boegoebrandewyn - K), rheumatism, sores,
sinkpyn (page 32: add together one tablespoon each of turpentine, boegoebrandewyn, bonte
seep and soetolie, to rub onto painful areas, can also be used for stomach illnesses
buiksiekte); nipples (page 33: boegoebrandewyn with sugar, applied topically thrice daily),
diphtheria, wounds (page 34: for infected wounds, use good boegoe-asyn mixed with water).
Page 29: tuberculosis.
Rood, 1994:
Page 25: headaches place bruised branches of wilde-als on the head and wet with boegoeasyn.
Pages 89 90: Boegoe used for almost every known illness in the olden days; rheumatism
place leaves in warm water as a herbal bath; also cholera, bloedwatering (blood in urine),
graweelstene (kidney stones) and water retention; boegoebrandewyn prepared by adding
leaves to brandy and leaving the closed container in the sun until it ferments, during which time
an oily or fatty liquid is formed that makes the mixture look like honey; boegoebrandewyn is
7
Appendix 2
applied to bruises, wounds and many external ailments; has several functions, like causing
sweating in the case of a rheumatism attack, used for digestive system disorders, as well as a
diuretic for bladder and kidney ailments; A. crenulata used to prepare boegoe vinegar leaves
are placed in a bottle of cold vinegar and left until it has converted into a plant slyme
(planteslym); used for intestinal pains (colic) take a spoonful of leaf infusion in warm water;
boegoe vinegar and brandy used by the Khoi khoi for the treatment of muscle pain (verrekte
spiere), rheumatism, gout or joint inflammation; Recipe for boegoe syrup used for stomach
ailments: boil together 250ml or more boegoe in 2 litres of water, filter through cloth. Add 1kg of
brown (or yellow) sugar, two lengths of drop (liquorice, bought from the pharmacy, cut into
pieces, melted in boiling water before adding) and 500ml of good vinegar to the boegoe water,
boil until syrupy. Before removing from the heat, add two small bottles of Turlington and one
small bottle of Paragoric, mix and boil through.
Hutchings et al., 1996:
Page 152:
A. ovata roots used in Zulu medicine, but unclear for which indications;
Agathosma spp. used for kidney and urinary tract diseases, applied locally to bruises and
rheumatic pains.
Newall et al., 1996:
Page 51: Buchu used as a urinary antiseptic, diuretic, also for cystitis (especially for acute
catarrhal cystitis), urethritis and prostatitis.
Ody, 1996:
Page 28: Buchu used for backache associated with kidney weakness; page 48: buchu is
diuretic; page 110: oval, long and round leaf buchu have identical medicinal uses, i.e. diuretic,
diaphoretic, stimulant, tonic, urinary antieptic, warming effect on kidneys, used for urinary
infections, cystitis and fluid retention; example of remedy is Frank Roberts Buchu Backache
Compound.
Simpson, 1998:
A. betulina leaves for rheumatism and gout, prostatitis, kidney and bladder ailments (including
haematuria and calculus) as well as for wounds, sprains, bruises and contusions.
Neuwinger, 2000:
A. betulina: for stomach problems; wounds; diuretic, kidney and urinary tract diseases;
rheumatism and bruises.
A. ovata: for kidney and urinary tract diseases, rheumatism and bruises used as with A.
betulina.
Appendix 2
Van Wyk & Gericke, 2000:

A. betulina:
pages 139-140: a general health tonic; leaves can be chewed fresh or dried, or
infusions and brandy tinctures (boegoebrandewyn) can be prepared; diuretic; mild urinary antiseptic; mild laxative effect (SM) (possibly due to a high mucilage content); a stimulant (taken
for hangovers); taken to stimulate kidney function; to treat mild cystitis and prostatitis; in small
dosages it can be an appetite stimulant, is a good digestive, carminative and anti-spasmodic;
remedy for treating coughs, colds, influenza, gout and rheumatism.
Wichtl & Bisset, 2000:
A. betulina:
page 103: for inflammation of the urinary tract used as a disinfectant and
diuretic, boiling water poured over ca. 1g (one teaspoon) of drug, covered and allowed to stand
for 10 minutes, strain, take several times daily as a diuretic.
Schwegler, 2003:
Page 8: A. betulina and A. crenulata used medicinally; buchu spp. used in cosmetics, soaps,
perfumes, food colouring and favouring; buchu oils are very strong and should be used with
care as they could cause sensitivity of the skin to sunlight.
Van Wyk & Wink, 2004:
A. betulina: treatment for kidney and urinary tract diseases; symptomatic relief of rheumatism;
external applications to wounds and bruises; minor digestive disturbances; a known diuretic,
diaphoretic and stimulant tonic.
Thring & Weitz, 2006:
A. betulina:for pain, dried leaves placed in a cloth and sprinkled with brandy or vinegar before
applying to sprains and pains (also arthritic pain); or a tea from a handful of fresh leaves can
be used for bladder, kidney and stomach pain (take a small cupful thrice daily); other ailments
include backache, fever and prevention of cancer.
Van Wyk et al., 2008*:
A. ovata was also indicated as a remedy for colds (AS) and asthma (KS), treatment for
backache and kidney failure (KS).
Van Wyk et al., 2009:
A. betulina:
page 34: tinctures or infusions of dried leaves used, where a cup of boiling water
is poured on 1g of the drug and allowed to stand for 10 minutes before straining, the infusion is
taken thricedaily; powdered, dried leaves mixed with sheep fat used to anoint the body;
stomach ailments the leaves are chewed or boegoebrandewyn taken orally; buchu vinegar
(boegoe-asyn) is very effectively used to wash and clean wounds and bruises; also for
symptomatic relief of rheumatism, urinary tract infections (mild urinary anti-septic) and kidney
ailments, used as a diuretic.
9
Appendix 2
Wileman L, no date. The uses of our Karoo plants in bygone times. Unpublished notes,
Worcester Museum:
A. betulina:
Page 6: dried leaves either used by the early Hottentots as a medicine for
stomach complaints, or powdered after drying and in an admixture with sheep fat, used for
anointing their bodies; Tulbagh (Governer at the Cape since 1752) sent buchu specimens to
Linnaeus Latrobe in 1816 stating that it was justly esteemed for its healing properties. Its
leaves are steeped in brandy or vinegar, and the bottle placed in the heat of the sun, until it
emits an unctuous juice, with the fluid rendered as thick as honey and applied particularly for
the healing of contusions, sores and all external complaints. Buchu had much vogue in the
treatment of kidney and bladder diseases, for stimulating perspiration whentreating rheumatism
and gout, or as a digestive tonic, for external local application to bruises, contusions, sprains,
fractures, etc.
Vergoes Houwens NF, no date. Medicine from the Veld. Unpublished notes, Worcester
Museum:
A. betulina:
page 2: prepare a tea or tincture and use for digestive, kidney, bladder and
chest problems. A vinegar tincture for external applications in cases of rheumatism, gout,
swellings, bruises and wounds.
Author, date and publisher unknown. Herbal Remedies of Montagu Museum:
A. crenulata: bruises, kidney, bladder draw in vinegar with rooisteelrabas for a few days.
Mostert H, Van Elfen, J, no date. Helse Rate van Hennie Hoed Blits en Ander. (Hellish
remedies from Hennie Hoed Blits and others.) Unpublished notes, from the Gamkaskloof,
Oudtshoorn, Prince Albert region:
Page 3: boegoe bladder and digestive system ailments, e.g. boegoebrandewyn for
indigestion; Mist Pot. Cit & Buchu (calcium citrate and buchu mixture) female bladder
problems.
Page 4: A. crenulata (langblaarboegoe) as a vinegar tincture, mixed with warm cow dung,
used for sprains (to be kept away from cuts and open wounds).
Pages 5-6: San and Khoi applied buchu mixed with animal fat as a cosmetic and wound
healing ointments; leaves chewed and infusions of leaves used for stomach and bladder
illnesses; boegoebrandewyn for indigestion and boegoe-asyn on cloth for swelling and pain
due to sprains, as well as insect bites.
Page 11: snake bites.
10
Appendix 2
Ellis, D, no date. Veldkruie. n Gids tot die kruietuin in Kirstenbosch. (Field herbs. A guide to
the herb garden in Kirstenbosch).Booklet published by the National Botanical Gardens:
Used for almost all ailments, including chronic rheumatism, gout (jig), Cholera morbus, bladder
ailments, haematuria, kidney stones, ulcers in bladder (verswering van die blaas), urethra,
prostate problems; boegoe vinegar and boegoe brandy used as rubbing lotions for rheumatic
pains, sprains and bruises; a weak diuretic, rinary anti-septic, infusion added in preparations
for treatment of infections of the urinary tract; A. betulina and A. crenulata leaves and stems
are used for similar indications, but A. betulina can also be used as a flavouring agent.
De Beer & Van Wyk, 2011*:
Leaves (sometimes dried) used to treat colds and stomach aches (JB); often mixed with fat and
used for sores (rou sere) (JB); an infusion of leaves taken for treating ulcers (JB).
Based on interviews with the community, indications directly quoted, names of

interviewees abbreviated as follows: AS = Andries Salmon; JB = Jan Baadjies; K = JFD
Kriel (stories and anecdotes on radio tape, Worcester Museum); KS = Kiewiet (Hottie)
Steenkamp; SM = Solomon Mahlaba.
11
Appendix 2
Appendix 2B: Aloe arborescens, A. ferox, A. marlothii and A. vera

(* = information based on personal interviews, initials given in text,
full names at end of the appendix)
Pappe 1847, 1850, 1857:
1847 (page 12); 1850 (page 28); 1857 (page 41): A. ferox Cape aloes are bitter and used as
a purgative.
Smith (1888), 1895:
Pages 120-121 (1895): A. ferox laxative (lump is an irritant when used as is, should rather be
mixed with soap and melted together to prepare an aloetic tablet; leaf sap used as eye drops
for conjunctivitis and bad cases of ophthalmia; used to treat scab in sheep, due to possible
germ-killing properties; sweet nectar is a snack favoured by children, but causes intoxication
and weakening joints).
Marloth, 1915:
A. ferox: page 96: juice is bitter, lump preparation.
Phillips, 1917:
A. ferox: one of the ingredients for the horn.
Kling, 1923:
A. ferox: page 11: a laxative.
Laidler, 1928:
A. ferox: for digestive discomfort juice in warm broth.
Cawston, 1933:
Aloe spp.: snuff prepared from a mixture of tobacco and burned leaves of aloes.
Hodge, 1953:
Aloe spp.: a laxative; also A. vera.
Morton, 1961:
A. ferox: leaf pulp applied directly to skin to treat ringworm
12
Appendix 2

A. arborescens: page 680: a leaf decoction administered just before parturition to assist the
process; a cold infusion used as a drench for sick calves; juice used to treat X-ray burns; an
extract used in first and second degrees of hypophiseal dystrophy in infants.
A. ferox: pages 681-684: a purgative for humans and animals mixed with meal or clay, or
administered on its own; ophthalmia leaf sap dropped into the eye; juice applied to sheep
with scab; children sucking sweet nectar from flowers experienced persistent weakness of the
joints, i.e. nectar is said to be narcotic; for venereal sores (such as syphilis) a leaf decoction
applied; ticks plant juice applied to feeding stock; leaf used to make jam; washing hair
using preparation from leaves; the whole plant regularly used as an ingredient in witch doctors
medicine horns.
A. marlothii: pages 684-685: tapeworm chopped-up leaves boiled in water with sage, taken
orally; roundworm decoction of leaves and roots taken orally or as an enema; to hasten
weaning rub leaf pulp over nipples; stomach problems decoction of the shoots taken orally
(large quantities produce emesis); horse sickness a leaf decoction used; powdered leaf, or
its ash used used as snuff, but can be hazardous contains carcinogens comparable to those
in cigarette smoking; pages 982-983: dried leaf ground and included in snuff.
A. vera: page 680: juice used to treat X-ray burns.
Aloe species: page 686: painful menstruation associated with sterility a decoction taken
orally and inserted into uterus; meningitis leaf pulp applied locally to relieve symptoms;
cancer sap of dead plant used; insect repellent for grain ash of leaf mixed with grain;
anthelmintic and fish poison; seedlings are food to ostriches and other animals; skin ulcers
fresh juice applied; impotence and sterility foam from stirring powdered root taken orally;
tuberculosis leaf decoction taken orally and in porridge; colic and constipation juice of a leaf
in water taken orally; toothache a drop of leaf juice applied to hollow tooth; prevention of
seasonal deaths of fowls water in which leaves have been soaked administered orally; page
982: dried leaf ground and included in snuff.
remedies). Tafelberg-Uitgewers, Kaapstad. p. 146:
Liver ailments page 150: mix one bottle of white muskadelwyn, two bottles of miltessens and
one teaspoonful of alwynpoeier, leave for 24 hrs, take a tablespoonful before a meal.
Bryant, (1909), 1966:
A. ferox: page 59 (1966): leaves used for treatment of venereal diseases such as syphilis.
Aloe spp. page 64 (1966): roots (called uMpondonde in Zulu) used for impotency and
barrenness.
13
Appendix 2
Smith, 1966:
Aloe species: page 53: In Natal, the dried Aloe leaves pounded into a powder by the Bantu
who use it as a snuff invigorator.
A. ferox: page 103: is known as bitteraalwyn, principle source of bitter aloes, the sap is no
more or less bitter than that of other species tapped for the medicinal product; page 457:
leaves used for making a preserve, the bitter sap used as a wound dressing and has been
used more generally as a purgative.
Courtenay-Latimer et al., 1967:
Plate 7 (A. arborescens): used to heal X-ray burns, for stomach complaints and to ward off evil.
Jacot Guillarmod, 1971:
A. ferox: one of the ingredients for the horn.
Bouquet & Debray, 1974:
A. arborescens: for burns (also those from radiation, cicatrisation; used as A. buettneri
A.Berger) topical application of fresh leaf cut in lengths or dried leaf powder used with
moisture rich Grewia L. leaves, or a very effective wash is prepared by decocting the leaves
with bark sap of Cola cordifolia R.Br. and foam produced when grinding Terminalia
glaucescens Planch. ex Benth.
Bruce, 1975:
A. ferox: leaves and roots are boiled in water and used for the treatment of arthritis, eczema
and conjunctivitis, wounds, venereal sores (syphilis), stomach ache, toothache, as a gargle, as
well as for hypertension and stress.
Coates Palgrave, 1977:
A. ferox: page 80: leaves used as fodder for livestock in times of drought and famine; jam
made from leaves; Cape aloes used as a laxative; leaves dried and burnt to repel insects;
planted around cattle kraals.
A. marlothii: page 81: dried leafs burned, ash mixed with snuff; roundworm leaf decoctions
used; to hasten weaning of babies bitter leaf pulp and juice rubbed on breasts.
Robertson, 1979:
A. ferox, A. marlothii, A. vera: unspecified medicinal value for humans and animals.
Reynolds, 1950, 1969, 1974, 1982:
A. arborescens: pages 411-412 (1969): X-ray burns pulp used as a dressing; labour a leaf
decoction administered to women just before parturition to assist in the process.
14
Appendix 2
A. ferox: pages 465-466 (1969): jam prepared from fleshy portion of leaf; exudate used in
preparation of Cape Aloes.
A. marlothii: pages 482-483 (1969): roundworm infections a decoction of green leaf and root
taken orally or as an enema; snuff leaf ash mixed with tobacco; to hasten weaning of children
green leaf pulp rubbed over breasts; stomach problems a decoction of shoots taken orally.
Arnold and Gulumian, 1984:
A. marlothii:
blood in stool a leaf decoction is eaten with porridge.
Palmer, 1985:
A. ferox: pages 108, 118 juice (containing active principle Cape Aloes) used as a purgative
and for healing; inflammation of the eyes fresh juice used; peeled leaves used to prepare
jam.
Cunningham, 1988:
Page 125 (A. marlothii): dried leaves ground for snuff, A. ferox also used in this way; Aloe snuff
is carcinogenic.
Hutchings, 1989(a):
A. arborescens: gynaecological indications; used as a charm.
A. ferox: for skin, eye, ear and nose ailments.
A. marlothii: used for the treatment of newly born infants; gastro-intestinal ailments as an
anthelmintic, also adminstered to infants and children.
Pujol, 1990:
Page 144 (Aloe): laxative, vermifuge, antiseptic leaves used; skin diseases leaf juice used
externally.
Cilli AM, 1992. Kruie op witblits, rate, resepte en feite (Herbs on witblits, traditional remedies,
recipes and facts), pp 43. Unpublished notes, Worcester Museum:
A. ferox: pages 19, 23: bitter sap used for wounds and as a laxative; if sap is applied to burn
wounds immediately, less pain will be experienced and no blisters will form.
Iwu, 1993:
A. ferox: pages 115-116: Cape aloes used as a purgative.
A. vera: pages 114-115: vertigo, wound dressing fresh leaves used orally and topically;
guinea worm infestation exudates from freshly cut leaves used; colds, asthma, bruises
exudates mixed with egg white, taken orally; stomach ulcers exudates mixed with seawater,
15
Appendix 2
taken orally; severe constipation dried exudates used; also menstrual irregularities and
functional sterility; has healing, moisturising and emollient properties useful for a wide range of
cosmetic applications; used as humectants in skin preparations such as moisturisers, suntan
lotions, aftershave creams; applied to severely chapped skin.
Rood 1994:
A. ferox: pages 64-65: a laxative sap used, or clean water with part of a leaf in it; vertigo and
gall sap diluted with water; to prevent nail biting rub sap on nails; sunburn, small burn
wounds, insect bites, skin irritations, cuts and abrasions cut leaves rubbed over skin; swollen
feet place a thin, flat piece of leaf under the sole of the foot inside the shoe.
A. marlothii: page 64: rheumatism and prevention of malaria aloe pills; mix drops of sap into
boermeel and mix to form a pill, pills dried, two pills taken in the mornings and evenings every
second day until the patient is better or healed.
Bhat & Jacobs, 1995:
A. ferox: a laxative the mucilaginous sap from the leaves mixed with hot water and taken
orally, or a decoction of leaves and root taken; stomach ache leaves boiled, cooled, the
decoction filtered and half a cup taken orally; toothache infusion taken orally as a gargle.
Roberts, 1995:
Page 14:
A. ferox or A. vera juice: to expel worms, relieve constipation and indigestion (not for use when
suffering from piles as it draws blood to the large intestine); applied to burns, scalds, rashes
and heat rash, mouth ulcers shave off the spiked borders of the leaf, cut the leaf across,
apply fresh, sticky juice directly; juice very bitter.
A. ferox: juice is a purgative 2-3 pinches of dried juice in 150ml of warm milk, sweetened with
honey or molasses, or mix juice with mealie-meal or clay and take as a purge; veterinary
juice used to get rid of ticks on cattle and dogs, used to treat scab on sheep.
A. vera: cosmetic sap used to prepare moisturising cream; mix sap into shampoos to sooth
dry or itchy scalp, or add to suntan creams; chapped skin, skin disorders, burns crush leaves
and apply as a poultice.
Van der Bank et al., 1995:
A. ferox: most important use as a laxative (Cape aloes); also cosmetic (skin lotions and hair
care products) or as food (leaves used to prepare jam).
A. marlothii: for the treatment of roundworm infections; powdered dry leaves used in snuff,
stomach troubles, to hasten weaning of children by rubbing green leaf pulp over breasts.
16
Appendix 2

Pages 32-34:
A. arborescens: leaf decoctions used during childbirth; pounded leaf infusions used to sprinkle
protective charms against storms; stomach ache cold water leaf infusions used, leaves are
purgative, leaf sap relieves X-ray burns.
A. ferox: cathartic; ophthalmia fresh leaf juice applied; venereal sores leaf powder from
charred, ground leaves or decoction applied; a purgative; for syphilis; overdose can cause
gastritis, diarrhoea, pelvic congestion and nephritis; should be avoided by pregnant women.
A. marlothii: for roundworm infestation leaf and root decoction administered orally or as an
enema; chewed roots used as an enema for babies; to hasten weaning leaf sap applied to
mothers breasts; stomach ailments shoot decoctions used; snuff powdered leaves and ash
used.
A. vera: used as a purgative, for skin healing, cathartic.
Maliehe, 1997:
A. ferox: leaf decoction for constipation, high blood pressure; (leaves and roots applied
topically, sometimes mixed with animal fat, or taken internally to treat eczema, dermatitis and
acne); enema for animals.
Blumenthal, 1998:
Pages 80-81: A. barbadensis (Curaao) and A. capensis (Cape aloe) a herbal stimulant;
laxative drugs used as a powder, aqueous and aqueous-alcoholic extracts for oral use.
Grierson & Afolayan, 1999:
A. ferox: The sap is applied as a dressing for wounds.
Reynolds & Dweck, 1999:
A. vera: gel ingredient in health care and cosmetic products; treatment for wounds, burns,
frostbite; adjuvant in arthritis treatment; gastro-intestinal problems, ulcers; diabetes; cancer;
high cholesterol; psoriasis; as an immune system stimulant.
Sachedina & Bodeker, 1999:
Aloe used mainly for epithelium and to boost the immune system.
A. ferox: bitters used in the medicinal and beverage industries, and as a laxative;
mucilaginous gel for skin preparations and cosmetics.
A. vera: skin products and health drinks.
17
Appendix 2
World Health Organisation, 1999:

Cape Aloes from A. ferox and Curaao from A. vera: page 34 tastes somewhat sour,
nauseating and very bitter; page 37: used for short-term treatment of occasional constipation;
seborrhoeaic dermatitis, peptic ulcers, tuberculosis, fungal infections, high blood sugar.
A. vera gel: page 43 odourless, colourless, a slightly bitter taste; page 45 freshly prepared;
external treatment of minor wounds and inflammatory skin disorders; minor skin irritations (first
and second degree thermal and radiation burns, bruises, abrasions); used as a hydrating agent
in cosmetics, i.e. liquids, creams, sun lotions, shaving creams, lip balms, healing ointments,
face packs; page 46 treatment of acne, haemorrhoids, psoriasis, anemia, glaucoma, peptic
ulcers, tuberculosis, blindness, seborrheaic dermatitis, fungal infections.
Glen & Hardy, 2000:
A. ferox:infection.
A. marlothii:
poison, digestion, inflammation; used during pregnancy.
Neuwinger, 2000:
A. arborescens: for burns or skin rashes.
A. ferox: a purgative; for stomach pain; conjunctivitis and ophthalmia; frontal sinusitis; arthritis;
wounds from venereal diseases.
A. marlothii: for the treatment of blood in stool; stomach problems; worms; to stop milk flow.
A. arborescens: leaves used to treat wounds, burns and various skin conditions, i.e. eczema,
skin irritations and bruises.
A. ferox: a general health tonic, leaf gel eaten as konfyt (preserve); Cape aloes applied
topically to sores caused by viral infections such as warts, herpes and shingles.
Van der Merwe et al., 2000:
A. marlothii: bitter taste; wounds (animals) leaves crushed, juice applied.
Cape Aloes from A. ferox and Curaao from A. vera: page 59 bitter taste; page 60 a
powerful colonic laxative.
A. vera gel: page 61 used in cosmetic preparations due to humectants, anti-inflammatory
and anti-bacterial properties; in drinks as a firmness preparation.
Pachter et al., 2002:
A. vera: for the treatment of asthma juice in alcoholic preparation rubbed onto chest.
18
Appendix 2
Schwegler, 2003:
Pages 8-9: (A. arborescens): for burns, wounds split or crushed leaves and commercial gel
extracted from leaves used; leaf extracts are healing, anti-inflammatory, anti-ulcer, antibacterial, anti-cancer and anti-diabetic.
Van Wyk & Smith, 1996, 2003:
A. arborescens: pages 78-79: first aid for minor cuts and burn wounds (also X-ray burns) and
abrasions.
A. ferox: pages 56-57: Cape aloes (lump) is purgative, used as a treatment for arthritis, eczema
and wound-healing; fresh juice used as a first-aid treatment for burn wounds; gel used to make
jam and a range of skin and hair care products.
A. marlothii: pages 62-63: an exudate (Natal Aloes) previously used and exported as medicine;
dry leaves ground and mixed with snuff.
Kleinschmidt, 2004:
A. ferox: promotes digestion; gel contributes to health benefits of beverages and fortified foods.
A. ferox: page 40: bitters a laxative (one small crystal, i.e. 0.05-0.2 g, taken orally) and as a
tonic; included in bitter tonics; half a laxative dose taken for arthritis; fresh bitter sap
administereded directly to treat conjunctivitis and sinusitis.
Non-bitter gel in/as a tonic (health drink), for wound-healing, eczema, and in cosmetics.
A. vera: page 41: bitters a stimulant laxative; bitter tonic (amarum).
Non-bitter gel wound-healing, skin care (burns and other skin disorders), infections,
inflammation, diabetes, high cholesterol, cancer, as a tonic drink (to stimulate the immune
system).
Kambizi et al., 2005:
A. ferox: various STD infections such as syphilis and gonorrhea infusions of fresh or dried
leaves taken orally, or juice squeezed from fresh leaves and directly applied to penile sores, or
leaves pulverised and mixed with Vaseline to form a paste that is directly applied to sores.
Smith et al., 2005:
A. ferox and A. marlothii used as snuff.
Crouch et al., 2006:
A. ferox: page 101: a purgative; bitter exudates; leaf gel used for skin and hair treatment; cut
leaves applied directly to burns, insect bites, sores and sunburn; traditional preparations used
19
Appendix 2
for toothache, arthritis, sinusitis, conjunctivitis, venereal sores and stomach pain; dried leaves
mashed and ground for inclusion in snuff.
Luseba & Van der Merwe, 2006:
A. marlothii: bitter; wounds (animals) leaves crushed, juice applied.
A. ferox: Skin problems treated by applying leaf juice to the affected areas; also used as a
laxative; for arthritis; eczema; conjunctivitis; hypertension; stress.
Other Aloe spp.: sap used to treat irritations, burns, bruises, ophthalmia, venereal sores
(syphilis), used as a purgative.
Amusan et al., 2007:
A. arborescens: for diabetes a leaf decoction used.
A. marlothii: cardiac problems leaf decoction used.
Lans et al., 2007
A. vera: gel used for internal injuries (animals); sap used to purge chickens.
Shackleton & Gambiza, 2007:
A. ferox: major use as a laxative; medicinal, veterinary and nutritional value.
Grace et al., 2008:
A. arborescens: used as medicine, for circulation, digestion and injuries; treatment of pain,
used during pregnancy.
A. ferox: used as medicine, for circulation, digestion, genitor-urinary ailments; infections,
inflammation, injuries and muscular-skeletal illnesses; nutritional; used for pain; sensory and
skin ailments.
A. marlothii: used as medicine, for digestion and infection; indicated for pregnancy.
Schmelzer & Gurib-Fakim, 2008:
A. arborescens: pages 63-65: for wounds, burns split or crushed leaves used; to ease
childbirth a leaf decoction given; constipation, to accelerate gastric secretion, purgative,
dermatological use leaves used, included in diet as vegetables and processed into candies.
A. ferox: pages 68-69: Cape aloes a purgative; fresh exudates ophthalmia, syphilis; gel
similar uses as for A. vera, including skin afflictions (burns, wounds, abrasions, irritations),
used as poultice on contusions, refrigerant, hair wash (to promote hair growth and as a cure for
dandruff), hydrating and skin-protecting (to improve the complexion and smoothen the skin), as
jam, refreshing and nutritive ingredient of food and drinks; leaves ash used as insect
repellant, dried and crushed in a decoction used to make a herbal tea.
20
Appendix 2
A. marlothii: page 88: for stomach ache, internal worms a leaf decoction taken with porridge;
to wean children leaf pulp rubbed on breasts; dried leaves mixed with snuff.
A. vera: pages 82-85: lump (Aloes) a laxative, purgative, vermifuge; fresh exudates
laxative, purgative, used externally as a refrigerant and to treat acne and cuts, asthma,
coughing, dysentery, kidney problems, dyspepsia (taken orally, mixed with other ingredients to
mask the bitter taste), bittering agent for food and beer; gel skin (burns, wounds, abrasions,
skin diseases, irritations, sprains, muscle pain, callosities of feet), as hair wash (to promote hair
growth and as a cure for dandruff), hydrating and skin-protecting (to improve the complexion,
smoothen the skin and other skin problems), poultice on contusions, refrigerants, on
haemorrhoids, to prevent/minimise radiation-induced skin reactions in cancer patients, sore
throat, coughing, as a mild laxative (gel eaten), to facilitate digestion, improve blood and
lymphatic circulation, improve kidney, liver and gall bladder function (taken as a food
supplement); A. vera products used for Aids, arthritis, other chronic and debilitating conditions.
Van Wyk, 2008a:
A. ferox: juice taken as a laxative.
Van Wyk, 2008b:
A. ferox: bitters a laxative, bitter tonic, anti-arthritic, anti-inflammatory; gel wound-healing,
health (tonic) drinks, cosmetics (skin care).
A. ferox: for constipation (AS, PT) sap mixed with water (short or young aloe is said to be
best (PT); to clean the stomach dry leaves soaked in a bottle of water and a tablespoonful
taken in the morning (EW, AW, SG, JO); stomach pills (maagpille) processed from the dry
sap (KS).
Van Wyk et al., 1997, 2009:
A. arborescens: page 40 (2009): fresh leaves or fresh or processed leaf juice and leaf gel
used; in human and animal medicine; for wounds, burns and various skin ailments (eczema,
skin irritations and bruises leaf sap rubbed in); fresh or processed gel added to many
cosmetic products; a tonic prepared from the leaves and used to treat and prevent cancer;
fresh gel or juice used as tonic drinks.
A. ferox: page 42 (2009): Aloe gel used in hair and skin care products; bitters used as a
laxative ingredient of Lewensessens and Schweden bitters, a small crystal of crude drug
(Cape aloes/lump) about the size of a match head or a decoction of leaves and roots taken
orally (not to be used during pregnancy); arthritis half a laxative dose of Aloe lump is taken
orally; for conjunctivitis and sinusitis fresh bitter sap instilled directly;used to a great extent as
human and livestock medicine; other uses include treatment of eczema, hypertension and
stress.
21
Appendix 2
Museum:
A. ferox: page 2: juice tapped from leaves, used as a purgative. Alternatively, dissolve two
pieces of hardened dehydrated aloe juice in some cold water. Take a dessertspoonful
mornings and evenings. Also good for rheumatism, high blood pressure and as a blood purifier.
Anonymous, date and publisher unknown. The Khoikhoi:
A. ferox: page 9: sap used as a poultice for wounds.
A. ferox: page 2: for constipation bitter, black aalwyn crystals or pills (processed by dripping
the sap in flour). Take when needed as a laxative; gall due to eating too much fatty food use
aalwyn-pills; page 5: powdered or crystalline sap used as a laxative, a blood purifier and for
gall elimination, cosmetic gel and refreshing drinks prepared from inner leaf gel.
Ribeiro et al., 2010:
A. marlothii: biliary disorders, malaria sap from leaves taken orally; wounds sap from leaves
applied topically; toothache roots and leaves applied directly onto the tooth or a decoction
taken orally; liver disorders roots and leaves taken orally as a decoction.
A. ferox: Take an infusion of leaves for kidneys and bladder (JB); stomach problems (JT, HG);
backache (RT); place leaves in drinking water of chickens for lice and chicken diseases (piep)
(DB, EB, HG, BB).
Moteetee & Van Wyk, 2011:
A. ferox: a leaf decoction for constipation; one of the ingredients for the horns; a leaf and root
decoction for diabetes; also used for high blood pressure; used as an enema for animals.
Thomas & Grant, 1998, 2004, 2008, 2011:
A. ferox: pages 170-171 (2011): Cape aloes a laxative; gel high in minerals and trace
elements, used as an ingredient in jam, for cosmetics and a soothing balm for skin irritations,
eczema, burns, bites and ulcers; dried leaves a protective ring around the kraal, burned and
crumbled to use for snuff.
A. marlothii: pages 172-173 (2011): leaves ash mixed with snuff, a decoction used for
roundworm; roots a remedy for stomach ailments.
*
based on interviews with the community, indications directly quoted, names of
interviewees abbreviated as follows: AS = Andries Salmon; AW = Abraham Wessels;
BB = Bertus Baadjies; DB = Danster Baadjies; EB = Elizabeth Baadjies; EW = Ernest Williams;
HG = Hendrik Gouws; JB Jan Baadjies; JO = Jan Oormeyer; JT = Jakob Thys; KS = Kiewiet
(Hottie) Steenkamp; PT = Poppie Teo; RT = Rachel Tromp; SG = SallyGoliath.
22
Appendix 2
Appendix 2C: Chronological list of ethnobotanical anecdotes and uses for Arctopus
echinatus (similar to A. dregei and A. monacanthus), as cited verbatim from
the referenced texts (* = information based on personal interviews, initials
given in text, full names at end of the appendix)
Pappe 1847, 1850, 1857:
1847 (pages 7-8): Platdoorn a demulcent, diuretic, root decoction prescribed for lues, lepra,
or cutaneous chronic eruptions of all types.
1850 (page 13): Platdoorn or Zieke-troost
1857 (page 19): similar to the above.
Dykman, 1891, 1908:
Pages 137138 (1908): for Fuilsiikte (vuilsiekte), Sifilis, of Fenussiikte (siekte) (i.e. syphilis) a
decoction of siekentroost or platdoorn; use material from plants in flower (species not
specified).
Kling 1923:
Page 18: for bladder ailments, gravel (graweel bladder stones), bladder catarrh, extended
glandular swellings use as an infusion or boiled; works on other organs (ingewande) at the
same time, a remedy to count on in times of need (noodanker).
Page 19: for the treatment of water retention boiled with water.
Page 20: for the treatment of lupus mixed with bitterblare [any of many species, including S.
frutescens subsp. microphylla (Smith, 1966)], boiled and used for blood purification.
Page 22: syphilis roots used as a tincture or cooked.
Page 1034: for epilepsy, syphilis a decoction of roots with potassium nitrate, causes
sleepiness; bladder ailments, gravel, catarrh of the bladder, swelling of glands, hypochondria,
skin irritations and clearing the skin of lupus, gonorrhea, as a purgative, demulcent, diuretic;
venereal diseases (African use) plant infusions used internally or locally; used as a blood
purifier (Hottentot, Xhosa and European use) a root decoction, tincture or infusion; the plant
has an astringent taste and makes a pleasant beverage.
Smith, 1966:
Page 375: pokkiesdoring refers specifically to its use for the treatment of venereal diseases
(syphilis); page 416: Sieketroos means comfort to the sick.
Watt, 1967:
Pages 4-5: for the treatment of epilepsy a decoction of plant material with potassium nitrate,
causes drowsiness; venereal diseases.
23
Appendix 2
Theodore, 1972 (Dr James Barry, 1827):

Plant collection preferably from October to December when plants are mature, selected from a
dry, sandy soil for higher yields of resin in the roots; only roots used medicinally; syphilis,
gonorrhea, lepra, elephantiasis, cutaneous diseases incident to the climate decoction,
tincture or pills [of gum resin, obtained abundantly from horisontal sections of fresh roots; 8-12
grains twice or thrice daily; a drachm of tincture added to the decoction has benefits; slight
acidification of a decoction with sulphuric acid improves potency (probably to contain a portion
of Arctopine in the solution)]; ulcers and blotches wash and dress once or twice daily with the
decoction; gum resin is anti-syphilitic, increases secretion of saliva, a strong diaphoretic,
stimulates the vascular system; the decoction is diuretic, slightly refrigerant, a highly permanent
tonic by stimulating cutaneous exhalant vessels, rather than by immediate action on the
sanguiferous system (no increased heart rate was observed).
Forbes, 1986 (Thunberg 1772 1775):
Page 44: soft textured root contains a very white and pure gum; used in the form of a
decoction, as a blood purifier and remedy for gonorrhea.
Van Wyk & Gericke, 2000*:
Page 140: Infusions, decoctions and tinctures prepared from the root and taken orally as a
sedative and to treat epilepsy, as well as a blood purifier for the treatment of syphilis and
gonorrhoea, urinary calculi, cystitis and lymphadenopathy, or applied topically for inflamed skin
disorders, sores and ulcers (DB, MB); the resin from the root is applied topically for the
treatment of ringworm (WS); the root is chewed and the juice swallowed for cough production
of purulent sputum (DB) and tuberculosis (MB, PK).
Schwegler, 2003:
Pages 9-10 (A. echinatus): roots, or a white, resinous gum that oozes from it has medicinal
use; used for numerous diseases; venereal diseases decoctions, infusions or tinctures from
the root used locally or taken internally; epilepsy, bladder ailments decoctions taken orally;
also used as a diuretic, demulcent (soothing), purgative, and to treat skin irritations.
Van Wyk et al., 1997, 2009:
Page 46: root decoctions, infusions or tinctures used (or the white resinous gum that oozes
from it); used for venereal diseases; epilepsy; bladder ailments; as a diuretic, demulcent,
purgative, treatment for skin irritations.
*

interviewees abbreviated as follows: DB = Dawid Bester; D&MB = Dawid and Mrs.
Bester; MB = Mrs. Bester; PK = Phillip Kubukhele; WS = Willem Steenkamp
24
Appendix 2
Appendix 2D: Chronological list of ethnobotanical anecdotes and uses for

A. afra, as cited verbatim from the referenced texts
(* = information based on personal interviews, initials given in
text, full names at end of the appendix)
Pappe 1847, 1850, 1857:
1847 (page 9): Alsem the whole plant has a strong balmy smell, and a bitter, aromatic that
causes nausea This herb is a tonic, anti-spasmodic and anthelmintic, very useful in
debilitating the stomach, visceral obstructions, jaundice, hypochondriasis, or similar evilsa
vermifuge; used as an infusion, decoction, and tincture; pounded leaves and stalks applied
externally as a discutient in edema and suggilations.
1850 (page 16): similar to 1847, with a strong infusion used externally as Collyrium for
weakness of the eyes
Smith 1895:
Page 96: epidemic influenza mix wormwood with blue gum leaves for external and
internal use (an alsobe used separately).
Pages 99-100: prophylactic for malaria, various forms of chest complaints, colds, influenza,
catarrh decoction, or other preparations made (blue gum leaves can be included); coughing
with febrile symptoms decoction sweetened with sugar or honey (especially for children), or
inhale the steam from a strong decoction of leaves while covered with a blanket to promote
profuse perspiration and immediate relief.
Dykman 1908:
For phlem in chest for children page 115: add a handful each of wynruit, boegoe and als to
three bottles of water and boil together until the volume is halved, filter, add one pound of
sugar and boil together until syrupy, take a teaspoonful four times daily; bad chest infection
page 132: one pound each of sugar and finely cut figs, one cup of peperwortel, one spoon of
als, two spoons of boegoe and one pound of raisins in two bottles of brandy, left in the sun
for two days, take half a kelkievol twice daily; colds page 136: boil a handful of als with half
a pound of sugar to form a syrup, take a teaspoonful every now and then, a good remedy;
coughng and hoarseness page 142: add one handful each of karmedik and wilde als to
three large cups of water, boil until half the volume, filter, then add sugar to prepare a syrup,
take often; whooping cough p147: boil together one cup each of als, boegoe, wynruit,
sugar, as well as a clove of garlic and a piece of camphor, with a bit of water until syrupy;
whooping cough and hoarseness p29, 147: mix one spoon wilde-als syrup, one spoon of
boegoebrandewyn, one spoon of soetolie and an egg take one teaspoonful thrice daily.
For haemorrhoids page 127: prepare a brandy tincture of boegoe, bels, als, renostertoppe,
25
Appendix 2
For Erysipelas (belroos type of skin rash) page 128: grind together one tablespoon of als,
rye or wheat bran and camphor (as much as the size of a nut) in a thin cloth and apply as a
cold poultice.
For fever page 149: powder together slang wortels and dry als and take orally.
Heartburn in children pages 118, 163: boil together a handful each of als and wynruit in half
a bottle of water, filter, add sugar and rabarber powder, boil together until a thick syrup is
formed, administer one teaspoonful once or twice daily; weak stomach page 151: prepare a
tincture of kruisement, wynruit, roosmaryn, pimpernel and als in a flask of brandy; make a
tincture of a bit of kwassihout with als and wynruit in old wine, take thrice daily before meals;
wear a small bag containing fresh wilde als on the stomach, replace when dry, continue
treatment until all that is eaten or drunk tastes bitter, as a sign that the stomach is healed;
stomach drops page 152: dry a few handfuls of flowers and leaves together with
peppermint leaves in the shade, cut in small pieces and wet with brandy, leave to infuse next
to a fire or in the warming oven, filter into bottle, add fresh leaves and add sufficient brandy to
fill the bottle, take a glassful now and then to strengthen the stomach, useful for weak
stomachs, not good for young energetic people.
For nose bleed or blood in sputum page 153: add one cup each of finely cut raisins and
peperwortel, one finely cut, dry fig, half a pound of sugar and one spoon each of aniseed,
boegoe and wilde als together in a flask of good brandy, take three kelkies daily.
For sore eyes page 154: dip als in boiling water and then put on eyes.
For Sinken pains (possibly neuralgia) page 158: prepare an ointment by frying together
pounded als and a cup of fresh butter, filter through cloth and add two spoons of sulphur,
apply for three days in the morning and evening, follow with half a cup of lemon soup and two
spoonfuls of castor oil every morning for four days.
For sleeplessness page 161: put als under the head.
For the treatment of water retention page 167: prepare a brandy tincture with wegeblare,
kruisement, dagga, brandnetels, sydissels, als, wynruit and kerwelblom, take mornings and
evenings.
Marloth, 1917:
Page 5: similar to A. absinthium, and used for similar purposes.
Phillips, 1917:
Page 142: For treating children with constipation a decoction administered as an enema;
the whole plant used to prepare a lotion for washing the body.
Kling, 1923:
Page 11: for Typhoid fever and enteric fever (maagkoors and ingewandskoors) bruise the
leaves and make a strong aqueous infusion, take often for thirst and fever.
Page 19: for eye ailments use aqueous extract of leaves as a strengthening eye wash.
26
Appendix 2
Watt & Brandwijk, 1927:

Pages 97-100: for treating colds, influenza, chills, coughing leaves boiled in water and a
decoction taken (doses of a wineglassful, or two tablespoonfuls sometimes mixed with
brandy) only, or by using together with a steam bath whereby the leaves are placed in a pot
of boiling water, and the patient is covered over the pot with a blanket; acute coryza (with
blockage of the nose) leaves inserted in the nose, leaves boiled and the decoction taken
internally with a little sugar; colds, coughing, bronchitis and other respiratory conditions an
infusion or decoction is taken, sometimes with the addition of brandy, sugar, ginger, thyme,
rosemary, mint and chamomile; headache, loss of appetite, bad taste in the mouth two
tablespoonfuls taken, sometimes mixed with brandy; earache leaf infusion instilled into
ears; umKhuhlane (meaning any feverish condition, often associated with malaria) an
infusion is made from a double handful of leaves in a quart of hot water and administered as
a clyster (enema) or emetic; also used for constipation and as a lotion for washing the body;
dyspepsia, poor appetite taken as a bitter tonic; sour stomach and other gastric conditions
in children, to promote sweating when sleeping, as a lotion for haemorrhoids hot infusions
taken; can be mixed with other plants to process medicine, such as an infusion made from a
mixture of A. afra, Asmitopsis asteriscoides Cass. and blue gum; to bring out the rash in the
exanthemata the whole plant used, and leaves used as a local application; blood
poisoning, inflammatory conditions on the surface of the body a poultice of boiled leaves is
applied; neuralgia leaves placed between two pieces of thin cloth, heated and applied to
the affected area; a tincture used as vermifuge.
Cawston, 1933:
Men and women use the leaves to insert into the nostrils to protect them during dust storms
and for treating catarrh and bronchitis; placed under the pillow to repel insects.
Bally, 1937:
For inflamed throat and fever in children use warmed plant as a dressing.
Pages 199-202:
For coughing, colds, chills, dyspepsia, loss of appetite, stomachache and other gastric
derangements, colic, croup, whooping cough, gout, as a purgative infusions or decoctions
bitter, often made syrupy by adding sugar (especially when used for bronchial trouble).
Haemorrhoids, to bring out the rash in measles an infusion or decoction used as a lotion to
bathe in (hot bath in case of measles), earache an infusion or decoction in ear; gumboils
an infusion or decoction held in mouth to ease pain and hasten bursting; menstrual chill and
after childbirth genitalia steamed; toothache leaves inserted into hollow tooth; neuralgia,
for swellings in mumps a leaf poultice applied; eye ailments leaves and stalks used as a
discutient, an infusion used as a lotion.
For treating fever (resulting from measles and malaria), blood-poisoning an infusion or
decoction taken; sweating of feet place leaves in socks; diaphoretic an infusion taken.
27
Appendix 2
For respiratory affections, sore throat in case of scarlet fever vapour from boiling leaves
inhaled, or leaves smoked; scarlet fever an infusion gargled; colds, headaches fresh leaf
tips inserted into nostrils; miners phthisis an infusion used; coughing in children a
decoction used; throat inflammation warmed herbs applied to painful spot.
For infantile colic a leaf poultice moistened with brandy applied to abdomen; colic brandy
tincture taken orally; vermifuge a tincture used; emetic, an enema for constipation, a blood
purifier, blood tonic, stomachaches, pimples, anthelmintic a leaf decoction used.
South African Herbal Wine 1% Agathosma and 1.5% A. afra.
Pages 202, 910: for diabetes an infusion taken regularly.
Page 252: for influenza plant used on its own or with Eucalyptus spp.
Page 519: for digestive disorders accompanied with fever an infusion from A. afra,
Eucalyptus and Leonotis microphylla Skan.
Page 674: a diaphoretic A. afra with Allium sativum L. and Fagara capensis Thunb.
Page 886: for colds a decoction of A. afra with Agrimonia eupatoria L.
Page 919: for febrile conditions decoctions or infusions together with Fagara capensis.
Page 982: dried leaves used as snuff.
Page 1051: for fever, influenza, measles, used as a prophylactic against lung inflammation
an infusion of A. afra and Lippia javanica Spreng.
remedies). Tafelberg-Uitgewers, Kaapstad. Pp. 146:
For water retention page 25: a small handful of kruisement, dagga, brandnetels, sydissels,
wilde-als, wynruit and kerwelblom prepared into a brandy tincture (one bottle of brandy), take
one spoonful in water in the morning and evening; a large pinch of wynruit and wilde-als
each in a pot, prepare an infusion by covering it with water, take thrice daily, cleans the
kidneys as well.
For joints and muscles page 198: place large handfuls each of boegoe, ysterblare, wildeals, wilde kopiva, chamomile, gansies and bitterbossie, together with three big spoons of
honey and a packet of Epsom salts in an enamel pot. Add four cups of boiling water and
leave overnight. Transfer liquid (even the condensated liquid formed in the lid, which is most
powerful) to a bottle, and shut cork. Take a big spoonful thrice daily after meals.
Bryant, 1966:
Page 53: leaves used for febrile complaints.
Smith 1966:
Page 60: Alsies can refer to a medicinal decoction brewed from A. afra.
Page 104: widely used medicinally, an extremely bitter aqueous extract is prepared from the
leaves.
Page 502: a tonic and anthelmintic (usually used as a tincture); a strong solution was said to
be good for weakness of the eyes.
28
Appendix 2
Jacot Guillarmod 1971:

A leaf decoction for flu; colds; fever; coughing; decongestant; stomach ailments; an enema
administered to children with constipation.
Gelfand et al., 1985:
For pneumonia, amenorrhoea, pain during labour a root infusion is taken; fever; for
extended labour pains leaf decoctions taken; edema legs washed with a root decoction;
to dilate birth canal root powder mixed with salt and soot and taken with porridge; shortness
of breath and delirium roots burned and the smoke inhaled; anorexia and loss of appetite
root infusions taken orally.
Palmer, 1985:
Page 45: used as an insect repellent.
Page 87: used for many ailments; effective treatment for wounds, due to its healing and
disinfectant properties.
Pages 95-99: for loss of appetite an infusion or decoction taken; colds, influenza vapour
from boiling water and leaves inhaled; coughing a tea prepared with a little honey; eye
ailments an infusion used as a lotion; fever used as a cooling bath, or hot bath to bring
out rash with measles, or an infusion or decoction taken orally, or a warm infusion used as
emetic or an enema; headache soft green branch tips inserted into nostrils; indigestion,
flatulence an infusion taken; itching use twigs in bath; plantains bruised leaves rubbed
on skin; mumps used as a poultice on swelling; piles an infusion or decoction used
externally; sore throat leaves smoked; wounds an infusion used as wash.
Cunningham 1988:
Page 122: for fever (umKhuhlane) leaf infusions administered as clysters or emetics; leaves
and stalkes contain toxic oil (0.3%).
Hutchings, 1989(a):
For gastro-intestinal disorders; a tonic; febrile complaints snuffed or inhaled; used as a
charm.
Hutchings, 1989(b):
For influenza, colds a plant infusion taken orally, or leaves inserted into nostrils; aromatic;
bitter taste; anti-histamine; narcotic; analgesic.
29
Appendix 2
Cilli AM, 1992. Kruie op witblits, rate, resepte en feite (Herbs on witblits, traditional
remedies, recipes and facts), p 43. Unpublished notes, Worcester Museum:
Page 10: a moth repellent.
Page 19: a tonic; strong infusions considered good for weak eyes and used as anthelmintic; a
tincture made with witblits, sugar, thyme, ginger, rosemary, wilde-als and mint used for colds,
bronchitis and stomach ailments.
Pages 21-23: for chest ailments, whooping cough, fever, stomach ailments, rheumatism
(page 32: use a brandy tincture K), stress/nerves, water retention.
Page 25: haemorrhoids (a brandy tincture prepared of buchu, bels, als, renosterbostoppe,
kruisement, karmedik and pimpernel take some in the morning and evening), asthma.
Page 26: for chest ailments (mix sugar, figs, peperwortels, als, buchu, raisins and brandy, let
it stand in the sun for two days take half a glass three times daily); page 30: coughing
(tincture in brandy); page 34: water retention (a brandy tincture prepared with wegeblare,
kruisement, dagga, brandnetels, sydissel, als and kerwelblom take in the morning and
evening).
Roberts, 1992:
Page 87: an insect repellent rub fresh leaves on skin (beware of sensitivity); skin ailments
use for a wash, or apply warmed leaves to draw pimples and boils, or take the brew orally to
cleanse skin; anti-septic, vermifuge and narcotic; pain, fever, constipation; coughing, sore
throat; influenza; jaundice; poor appetite; indigestion; diarrhoea; dysentery; female
complaints such as morning sickness; earache or ear infection add three 15cm sprigs to
600ml boiling water, sweeten with honey to disguise bitterness, drink 10ml in the morning and
at night for four days at most (if taken for too long, it will dilate the blood vessels and have an
effect on the heart; toothache place a leaf on the tooth).
Iwu, 1993:
Page 121: for bronchial diseases a leaf decoction used; coughing, colds, dyspepsia,
stomachache, gout, constipation ingredient in preparations used for various treatments;
anti-pyretic, malaria a decoction prepared with leaves and lemon grass; sore throat in
children fermentation with heated herb administered; indigestion; worms; anti-inflammatory;
skin infections external use; measles a hot bath; haemorrhoids local application; colic
a weak leaf infusion administered.
Kokwaro, 1976, 1993:
Page 69 (1993): for sore throats in children, fever fermentation of heated herb taken orally;
indigestion the whole plant used; worms roots boiled, a decoction taken orally 2-3 times
daily; emetic leaves chewed and juice swallowed; an expectorant: abdominal pain the
whole plant dried and ash used.
30
Appendix 2
Hutchings & Van Staden, 1994*:

For head colds, influenza leaves inserted into nostrils (JM), or steam inhaled; toothache,
earache.
Rood, 1994:
Pages 24 26: a potent strengthening agent and remarkable appetite stimulant; infusions
and decoctions effective for any ailment; fresh and dried plants equally effective. For the
promotion of stomach juice and gall secretion, bloatedness, worms, bad breath caused by
stomach ailments harvest leaves and flowers, dry in sun, add 250ml boiling water to 5ml
powdered plant (fresh or dry), leave for 5 10 min, take decoction thrice daily, or 12.5ml cold
leaf tea taken four hourly (a little bit of brandy can be added for the treatment of colic);
constipation a decoction of roots taken orally, or mixed with sugar and boiled until syrupy;
indigestion, poor appetite; colic in babies place a hot leaf poultice on babys tummy;
stomach pain, children suffering from vomiting and diarrhoea a branch with leaves placed
between two cloths, dampened with brandy, heated and applied locally on stomach; excess
gall drink as a tea; as anthelmintic, anti-septic, anti-spasmodic, diuretic, stimulant of liver,
gall bladder and blood circulation, for loss of appetite, heartburn an infusion used; worms
powder of crushed, dried flowerheads used to make a tea and taken.
For coughing a decoction of roots taken orally; coughing chew leaves and swallow juice;
colds, influenza, fever infusion, or leaf tea, filtered and 12.5ml taken every four hours, or a
decoction of Eucalyptus (broad leaves), wilde-als and ginger boiled together and filtered, a
heated glassful taken in the evenings before bedtime; chest ailments and persistent coughing
boil tips of branches and young leaves, add 25ml honey; chronic coughing and bronchitis in
children leaves in flanel bag on chest; blocked nose, asthma, bronchitis inhale vapour of
boiling als; colds and headache insert fresh leaves into the nostrils; sore throat boil tips of
branches and young leaves, add 25ml of honey, with brandy, use as a gargle, a good
remedy; croup and whooping cough in children, chest ailments the decoction mixed with
sugar and boiled until syrupy; scarlet fever throat steamed with boiling infusion; asthma a
tea of dried leaves taken.
For ulcers (bloedvinte and pitswere) a decoction of the whole plant taken as a blood
purifier; sinkings, protruding nerve on head, swollen glands, mumps a leaf poultice applied
locally; sore feet feet are scraped and then washed with the decoction; gout a decoction
mixed with sugar and boiled until syrupy; to hasten the outbreak of measles on skin an
infusion or decoction used to bath in; measles, fever, malaria an infusion used; sprains
mixed with vinegar; haemorrhoids sit in warm wilde-als bath; skin irritations, sprains,
bruises paste prepared with tea applied.
For toothache put fresh leaf tips in the hollow of sore tooth; mouth ulcers a decoction kept
in the mouth to ease pain; headaches bruised leaves splashed with boegoe-asyn and
placed on head.
31
Appendix 2
An eye wash use a strong decoction.

Klisma (enema), a blood purifier powdered plant material suspended in water or milk; blood
poisoning an infusion taken.
For sleeplessness a cushion stuffed with leaves and slept on.
Used for similar ailments as those for which boegoe is used.
Dosage forms: infusion 12.5ml broken leaves or flowerheads in 250ml boiling water, leave
for one week, take 125ml daily in 5ml quantities; tincture 8-10 drops twice or thrice daily; oil
2-5 drops twice or thrice daily (warning: pure oil is very poisonous and an overdose can
cause poisoning, no harm caused by reasonable dosages); powder 1-2ml once or twice
daily (very bitter).
Shearing & Van Heerden, 1994:
Page 154: used as an anthelmintic, a tonic and tincture.
Bhat & Jacobs, 1995:
For colds and influenza inhaling the vapours from a handful of leaves in boiling water, or
squeezing a handful of leaves and inhaling the fumes; measles a hot infusion of fresh
leaves mixed with goat droppings, applied locally.
Page 117: for colds a decoction of A. afra with Agrimonia bracteata E.Mey. ex C.A.Mey.
Page 263: for fever, measles weak stem and leaf infusions of A. afra and Lippia javanica
(Burm. F.) Spreng.
Page 327: for febrile complaints leaf infusions taken as teas or administered as enemas or
emetics; headaches, colds, influenza crushed leaves or steam from infusions inhaled; also
used for diabetes, measles and fevers, including malaria; constipation or intestinal worms in
children plant material ground and suspended in water or milk, administered as an enema;
a blood purifier to treat acne and boils a decoction taken; precaution against colds insert
fresh leaves into the nostrils; constipation (as an enema), lotion or body wash decoctions
used; various respiratory and gastro-intestinal ailments including croup, whooping cough,
dyspepsia, loss of appetite, colic; as anthelmintics (roots used) and emetics; other ailments
treated include gout, in diabetes to keep urine free from sugar, menstrual chill after childbirth,
haemorrhoids (used to bath in), gumboils, earache, toothache (a leaf poultice on the tooth);
pimples, boils, mumps, sprains, colic in infants (wrapped around the stomach) warmed
leaves applied as a poultice; fever, sore throat, indigestion whole plants used; pneumonia
roots used; overdose of essential oil is poisonous.
Maliehe, 1997:
A leaf decoction for flu; colds; fever; coughing; decongestant.
32
Appendix 2

Leaf infusions used to clean wounds.
Neuwinger, 2000:
For colds; influenza; bronchitis; pneumonia; coughing; whooping cough; an expectorant;
blocked nose; inflamed throat in children; fever; scarlet fever; measles; headache; earache;
nerve pain; sprains; toothache; amenorrhoea; pain during pregnancy; extended labour pains;
heartburn; dysentery; to stimulate appetite; anorexia; constipation; colic; worms; malaria;
edema; to dilate birth canal; shortness of breath; delirium; diabetes; acne; skin ulcers;
pimples; gumboils; wounds; haemorrhoids; mumps swellings; used sas a blood purifier,
emetic and enemas.
Schlage et al., 2000:
For treating malaria leaf extracts used.
Page 142: fresh and dry leaves of young stems are mostly used as infusions, decoctions and
tinctures: a tonic for fever, colds, influenza, sore throats, coughing, asthma, pneumonia and
headaches infusions taken orally or inhaling the vapours from a steam bath; gastritis,
indigestion, loss of appetite, colic and flatulence, intestinal worms, constipation, gout,
diabetes (plants from different areas have different potencies CM); powdered leaves iused
as snuff for headaches.
Von Koenen, 2001:
Pages 79-80 (2001): plant has bitter taste and eliminatory, purgative, anthelmintic and
diaphoretic properties; used for diabetes, fever, upper and lower respiratory tract infections,
kidney and stomach ailments and wound healing; respiratory problems, colds, coughing,
pertussis, croup, diphtheria steam from a leaf decoction inhaled and the extract taken orally
or gargled; toothache, gumboils mouthwash of a leaf decoction; haemorrhoids, to bring out
the rash of measles a concentrated leaf decoction is added to bath water, or used to swab
haemorrhoids; earache, gout, rheumatism plant used internally or externally; wounds,
stomachache, colic, mumps, neuralgia, eye conditions crushed leaves used as a compress;
sweaty feet leaves inserted into socks; acne, pimples tea acts internally as a skin and
blood-purifying agent; to clean lesions and bite wounds, used as ear drops a concentrated
leaf decoction used; colds, coughing, heartburn, digestive upsets, stomach cramps an
alcoholic tincture used; blocked nose, bronchitis steam from boiling leaves inhaled; to draw
pimples and ulcers, painful areas affected by neuralgia, swellings of mumps hot leaf
fomentations applied; a moth repellent.
33
Appendix 2
Kraft et al., 2003:

A bitter essential oil; used for colds and respiratory complaints; a diversity of symptoms from
gastric derangements to febrile conditions; anti-malarial.
Page 55: for intestinal worms, tonic, coughing, colds, influenza, loss of appetite, colic, fever,
headaches, earache, malaria, constipation, diabetes, used as a blood purifier, to treat acne
and boils, analgesic, a bitter tonic.
Erasto et al., 2005:
For diabetes leaves or roots boiled, sugar added to the infusion to mask the bitter taste.
Page 103: fresh or dry leaves and young stems used to prepare a tea, often with alcohol
added to it; roots sometimes used; indications include upper respiratory tract infections, fever,
colds, influenza, sore throats, coughing, asthma, pneumonia, headache, gastritis, indigestion,
poor appetite, flatulence, colic, intestinal worms, constipation, gout, malaria; toothache
insert fresh leaves into the hollow of the tooth; gum infections hold a decoction in the mouth
to relieve pain.
For bladder and kidney problems a tea prepared from a handful of leaves, taken daily;
coughing a syrup prepared by boiling the leaves with sugar; inflammation and fever
leaves prepared into a poultice with brandy or vinegar and wrapped around the inflamed part,
or around the stomach in case of fever; diabetes small amounts of tea taken twice daily
continuously; other ailments include colds, influenza, heart problems, headaches,
rheumatism, convulsions, stomach disorders and worms.
Viljoen et al., 2006:
Uses include treatment for respiratory ailments, i.e. coughing, colds, influenza, to clear
bronchial passages, aerial parts, often used as an inhalant; digestive complaints; skin
ulcerations.
For coughing a leaf infusion used.
34
Appendix 2

Treatment for colds (JO, AS, PT) tea leaves (JO), or as a tea from the leaves to which
honey can be added (KS, EW, AW, SG), and intended as strengthening medicine (PT), also
for infants where fresh, bruised leaves are taken as a tea in the morning and evening (PT);
chest problems for infants a flannel bag with fresh leaves can be worn around the babys
neck (JO); stomach problems (AS).
The exotic counterpart of African wormwood, namely A. absinthium L., is grown in some
gardens in the Murraysburg township, of which the bright green, very bitter tea (of leaves) is
used to treat diarrhoea.
Liu et al., 2009:
Indicated for a variety of ailments including coughing, whooping cough, asthma, colds,
influenza, headaches, toothache, menstrual chills, fever, convulsions, dyspepsia, loss of
appetite, gastric derangements, as a purgative, (infantile) colic, croup, gout, malaria,
diabetes, bladder and kidney disorders, heart inflammation and rheumatism; neuralgia;
swellings; mumps; preparations often made in combination with brandy, sugar, ginger, thyme,
rosemary, mint, chamomile, Osmitopsis asteriscoides, Eucalyptus globulus, Lippia
asperifolia, Agrimonia bracteata, Tetradenia riparia or salt.
Van Wyk et al., 1997, 2009:
Page 48 (2009): mainly leaves are used, but sometimes also the roots; honey or sugar can
be added to infusions or decoctions to mask the bitter taste; coughing, colds, influenza, fever,
loss of appetite, colic, headaches, earache, malaria, intestinal worms; blocked nasal
passages.
Leaves used to treat colds and a tight chest, together with wynruit (CB, JB, FB, RT); pains
(especially backache) (RT, KB, EB, MT); stomach pain (BB); kidneys (JT, HG).
A leaf decoction for flu; colds; fever; coughing; decongestant; stomach ailments; an enema
for children with constipation; a leaf infusion for earache and toothache.
Museum:
Page 7: an infusion makes a good vermifuge to bathe the eyes, haemorrhoids and wounds.
Infusions also used for coughing, colds, whooping cough, fever, headaches, colic and gout.
Sprigs of leaves inserted into the nostrils to relieve headaches and a blocked nose, while a
piece on a tooth will relieve toothache.
35
Appendix 2
Page 4: children with whooping cough put a bag containing dry leaves around the neck.
Page 9: leaves, sometimes roots, used to prepare infusions and sugar or honey added to
prepare a syrup, taken orally for coughing and asthma, or the vapour inhaled for blocked
nose.
Page 11: mixed and burned together with brandy, figs, thyme and sugar, take one
teaspoonful three or four times daily for asthma; a few handfuls of flowers and leaves dried
together with peppermint, cut smaller, brandy added and then filtered into a bottle, add fresh
leaves and fill to the top with brandy take a glassful every now and then for strengthening of
the stomach.
Used for the treatment of coughing, colds, indigestion, loss of appetite, colic, croup, whooping
cough, gout, earache and as a laxative, haemorrhoids an infusion or decoction used as a
wash; measles added to a hot bath to make the rash appear quicker; mouth sores an
infusion kept in the mouth to relieve pain; fever, blood purification an infusion taken; also
used for ginecological applications and as a tonic.

interviewees abbreviated as follows: AS = Andries Salmon; AW = Abraham Wessels;
BB = Bertus Baadjies; CB = Christien Baadjies; CM = Credo Mutwa; EB = Elizabeth
Baadjies; EW = Ernest Williams; FB = Frans Baadjies; HG = Hendrik Gouws; JB =
Jan Baadjies; JM = J Mhlongo; JO = Jan Oormeyer; JT = Jakob Thys; K = JFD Kriel
(stories and anecdotes on radio-tape, Worcester Museum); KB = Kato Baadjies; KS =
Kiewiet (Hottie) Steenkamp; MT = Martiens Thys; PT = Poppie Teo; RT = Rachel
Tromp; SG = Sally Goliath.
36
Appendix 2
Appendix 2E: Chronological list of ethnobotanical anecdotes and uses for

B. maughamii, as cited verbatim from the referenced texts
text, full names at end of the appendix)
Pages 1065-1067: ritual emetics roots are popular ingredients; fruits are lethal to freshwater snails, certain small fish and tadpoles soak fruit in water for 3-4 days, still active after
10 days, but loses potency after that, and has been recommended for the eradication of
bilharzia.
Smith, 1966:
Page 466: Witch doctors use the fruit in their bone-throwing ceremonies.
Van Wyk, 1974:
Vol.1, pages 230-232: fruit used as fish poison, fatal to Bilharzia snails, tadpoles, small fish
and young mosquito larvae, toxicity due to continuous exudation of a toxic substance from
fruit when placed in water, with the effect lasting for 10 days, poisonous only to organisms
without lungs, thus edible to man and land animals.
Coates-Palgrave, 1977, 2002:
Page 339 (1977); page 407 (2002): the fruitis lethal to snails and some other forms of
aquatic life; evil spirits are warded off a froth is prepared by beating together roots and
bark soaked in water with certain other ingredients, the froth is licked three times daily, and
the remains is poured out on the roof of the house so that it spills onto the entrance; a bath in
water in which the bark has been infused, is exhilarating and stimulating; emetics are
prepared from decoctions of the bark.
Kloos & McCullough, 1982:
Molluscicidal.
Pretorius et al., 1988:
Molluscicidal.
Hutchings, 1989(a):
For the treatment of snake bites; growths.
Ngwenya, 1994:
Page 13: roots are popular ingredients of ritual emetics.
37
Appendix 2

Page 151: for protection roots and bark are ingredients of infusions used by traditional
healers as a protection against evil spirits, or thorns used as protective charms; exhilarating
bath bark used; emetic bark decoctions used; arrow poison fruit used; fruit eaten by
humans and animals, but skin and fruit pulp lethal for aquatic fauna and small fish.
Neuwinger, 2000:
Page 62: used as an emetic a bark decoction taken orally.
Page 142: a paste of the bark cooked and taken orally as a general tonic and panacea (in
Mozambique); cooked with beans to treat haematuria; decoctions of bark and roots used as
emetics; bark infusions used to prepare refreshing baths.
Prozesky et al., 2001:
An emetic, arrow poison, molluscicidal, anti-malarial stem bark used.
Page 104: known as Sangomas Tree roots used for ceremonial purposes; security
chopped bark sprinkled around the homestead to confuse evil-doers; to strengthen the body
chopped bark implanted in the skin (mgaba); fish poison, leathal for bilharzia snails,
tadpoles and fresh water aquatic life in general fruits used.
Page 58: stem or root bark used; root bark mixed with other ingredients for use as emetics
(no details available); bark applied in the form of cutaneous implantations to strengthen the
body; also recommended for the eradication of bilharzia; used to treat coughing.
Ribeiro et al., 2010:
For malaria root scrapings or root infusions taken orally.
Corrigan et al., 2011*:
Good luck (GM, SF) if you own a tuck shop, the root bark is used to attract customers.
*

interviewees abbreviated as follows: GM = Goodenough (Sgwili) Mdluli; SF =
Sibusiso Falakhe.
38
Appendix 2
Appendix 2F: Chronological list of ethnobotanical anecdotes and uses for

D. anomala, D. capensis, D. schinzii and D. zeyheri, as cited
verbatim from the referenced texts (* = information based on
personal interviews, initials given in text, full names at end of
the appendix)
Smith, 1895:
D. anomala pages 64-65: leaves are intensely bitter; the whole plant used for various
purposes; colic administer a little of the powdered root in cold water; if someonemust go to
a strange place, s/he chews a piece of the root, so that if s/he receives any poisonous food,
s/he will vomit immediately.
Dykman, 1908:
For haemorrhoids page 127: prepare a brandy tincture of boegoe, bels, als, renostertoppe,
For coughing and hoarseness page 142: add one handful each of karmedik and wilde-als
to three large cups of water, boil until half the volume, filter, then add sugar to prepare a
syrup, take often; chest and throat (brand in die bors of keel) page 156: wine or brandy
tincture of karmedik is very good.
For weak stomachs page 151: take some brandy tincture mornings and evenings;
nauseous feeling before or after a meal take half a kelkie of a tincture made with good old
wine an hour before a meal, extra good remedy.
For female illnesses page 160: fry karmedik with soetolie and wax, and rub on painful spot.
For shingles page 156: wash with a strong brandy tincture of karmedik, take now and
thento reduce inflammation (dryf die vuur uit), or use as a dressing of dry powder in a thin
cloth on the wound.
For treating tuberculosis page 165: mix and grind together brandnetels, boegoe, bels and
karmedik, add one teaspoon of ginger for every cup of powder and a good helping of sugar,
mix and take a teaspoon of powder at a time.
For the treatment of Diphteria page 170: prepare a weak tea of karmedik, pour out excess
water, add honey to water for taste, take half a teaspoon regularly.
Phillips, 1917:
Page 170 (D. anomala): an infusion of roots taken for colic; toothache.
Kling, 1923:
D. capensis: page 9: for diarrhoea (so-called morning diarrhoea or chronic diarrhoea) and
weak stomachs a decoction or infusion prepared from the whole plant. taken early in the
morning and later in the day.
39
Appendix 2

Pages 85-86 (D. anomala): for venereal diseases roots crushed and prepared into a
decoction, strained, taken cold; bilious attacks plant material mixed with Berkheya setifera
DC.; a purgative; blood purifier a decoction of roots taken internally or used as an enema;
scabby sores on childrens heads burned roots powdered and applied; colic a
tablespoonful of powdered roots mixed with a cup of water taken orally; an emetic a little of
piece of root is chewed when a strange kraal is visited, will cause one to vomit if poisonous
food is eaten; dysentery roots used as a powder or in a decoction.
Bally, 1937:
D. anomala: for dysentery a root decoction taken orally.
D. anomala: pages 222-223: diarrhoea, dysentery, colic, used as a purgative a root
decoction used, or roots chewed and saliva swallowed; worms, toothache, colic powdered
roots used; emetic chew a small piece of root, so that if any poisonous food is eaten, it will
be expelled immediately; venereal disease, gall-sickness in cattle a root decoction taken
orally; sterility the whole plant used; cold in nose root bark ground and snuffed, causes
lachrymation, sneezing and coughing; coughing, colds a plant decoction taken orally;
ringworm a plant decoction applied locally; children suffering from blood disorders a root
decoction as an enema or taken orally; scabby heads in children charred root paste
applied locally; haemorrhoids root decoctions in gin taken orally; fever a root decoction
with melkbos taken orally; to make voice clear and high root powder in hot water taken
orally.
D. capensis: page 223: for febrile conditions, used as a diaphoretic leaf infusions taken.
D. zeyheri: page 223: for umlundagazi (meaning a disease, possibly any ailment) a root
infusion used; backache, lumbago, anemia a root decoction taken.
remedies). Tafelberg-Uitgewers, Kaapstad. Pp. 146:
For heart and circulation problems page 79: prepare a tincture by adding two tablespoonfuls
each of kruisement, pimpernel, karmedik and boegoebels, as well as six renosterkoppe to 1
bottle of good brandy. Take a tablespoonful thrice daily (very good).
Smith, 1966:
D. anomala: page 270: a laxative for calves; page 327: roots very bitter and used as a
stomach tonic and for treating colic aqueous extracts prepared from pounded roots;
40
Appendix 2
decoctions of powdered root used as a remedy for dysentery, diarrhoea, intestinal worms and
gall-sickness in cattle; powdered roots used as snuff for nasal problems, and for the
treatment of wounds and sores; roots of Dicoma spp. used for stomach problems, dysentery
and diarrhoea (page 451: i.e. the vernacular name swartstormbos means that the bark of the
root is black and causes a stormy purgative action when taken as a decoction)
D. anomala: an infusion of roots taken for colic, diarrhoea, constipation, flatulency; powdered
flowers used as an ointment for wounds and sores, toothache; mixed with Scabiosa
columbaria Willk. ex Willk. & Lange for period pains; mixed with Cymbopogon Spreng. spp.
for gall-sickness.
Schmitz, 1980:
D. anomala: powdered flowers used as an ointment for wounds and sores; snuff for nasal
congestion; mixed with Cymbopogon spp. for gall-sickness; vermifuge.
Chinemana et al., 1985:
D. anomala: for diarrhoea preparations of root taken as a powder in food or orally as an
extract.
D. anomala: for abdominal pain, vomiting, cataracts, coughing, dizziness, gonorrhoea a
root infusion taken orally; uterine pain root powder introduced into the vagina; pneumonia
root powder eaten or rubbed into scarifications; throat problems root powder taken with
porridge and salt; body pain, as a panacea and to calm the insane the body is washed with
a root infusion; extended labour pain tuber infused together with the soot and roots of
Aspilia pluriseta Schweinf. ex Engl. and taken orally.
Palmer, 1985:
Page 135 (D. anomala): used mostly as a decoction to cure a wide variety of ailments
including colds, colic, toothache, blood disorders, ringworm, sterility; dysentery, diarrhoea
the root chewed and the saliva swallowed.
Page 154 (D. zeyheri): a decoction of roots used for lumbago, backache and anaemia,
among other ailments.
41
Appendix 2
Lucas & Pike, 1987:

Page 98 (D. zeyheri): for abdominal problems, lumbago, backache, anaemia root stock
used.
Baerts & Lehmann, 1989:
D. anomala: for coughing a root decoction taken orally; colic a leaf decoction or root
powder taken orally; intestinal parasites a leaf decoction taken orally; abscesses,
dermatosis, yaws leaf ash eaten or sniffed; scabies leaves eaten; insanity, possession
a leaf decoction taken orally.
Hutchings 1989 (a):
D. anomala: gynaecological indications; for gastro-intestinal ailments, including anthelmintic
and diarrhoea; a tonic also for children; respiratory ailments; dental and skin indications;
used as an insecticide.
D. zeyheri: for respiratory ailments.
Kokwaro, 1976, 1993:
D. anomala: page 74 (1993): for stomach complaints roots used in preparation of medicine.
Rwangabo, 1993:
D. anomala: for colic, stomach pain extract of leaves or roots taken orally; worms a root
extract taken orally; coughing a leaf extract taken orally; yaws preparations of roots and
leaves rubbed in; malaria, fever, epilepsy, constipation an extract of leaves and twigs taken
orally; haemorrhoids preparations of leaves and twigs used externally; snake bite, migraine
leaves used (no details).
Archer, 1994:
D. capensis leaf infusions used for all ailments, especially stomachaches and coughing.
Rood, 1994, 2008:
D. anomala: page 30(1994):
For the treatment of fever an infusion of the whole plant or roots; coughing, colds an
infusion of plant material used; nose ailments, sores, wounds dried, powdered root used as
snuff.
For venereal illnesses, worms (douwurm), sores (kruipsere) an infusion of the plant used;
haemorrhoids an infusion of roots mixed with jenewer, taken orally; children with rough
scalp ailments (skurwe kopvelsiektes) burned roots crushed to paste and rubbed onto the
head.
42
Appendix 2
For toothache an infusion of the plant used; ears with puss oozing out (sweerore) roots
used.
For constipation, colic, an infusion of the plant used; dysentery, diarrhoea, worms
infusions or decoctions of dried, powdered roots; diarrhoea the root is chewed and sap
swallowed.
For blood illnesses a root infusion applied as a klisma (an enema), or taken orally by
children .
Roots are bitter, pounded before use; too much of strong root infusion taken orally can lead
to vomiting; the swartstorm has similar properties and a similar appearance as the
maagbossie (the latter statement by the researcher is peculiar, as she assigned both these
common names to D. anomala).
Shearing, 1994:
Page 148 (D. capensis): for upset stomachs.
D. anomala: page 334: for coughing, respiratory complaints and dysentery; blood disorders in
children; topical application on head sores or ringworm; used as a purgative, in mixtures for
sterility, intestinal worms, colic, diarrhoea, dysentery, toothache, gall sickness in stock
animals; sores and wounds on horses; to induce vomiting after eating food thought to be
poisoned; root bark used as snuff for nose colds; also colds, fever, venereal diseases, colic
and as a purgative a root decoction taken orally or a piece of root chewed and sap
swallowed; haemorrhoids root decoctions in gin taken orally; to make voice clear and high
pitched root powder in hot water taken orally.
D. zeyheri: page 335: unspecified plant material used for coughing and chest ailments;
decoctions used as blood strengtheners after child birth; backache, lumbago, anemia,
weakness of the limbs a root decoction used; strengthener after a long illness a plant
extract used as a body wash; ulcers powder of burned flowers and fruit applied topically;
blood strengthener after child birth a decoction taken; sores burned flowers and fruit
applied; infertility and early infant deaths burned flowers and fruit taken.
Maliehe, 1997:
D. anomala: an infusion of roots taken for diarrhoea, constipation, flatulency, heartburn;
powdered flowers used as an ointment for wounds and sores; backache; diabetes; mixed
with Helichrysum caespititium, Scabiosa columbaria and Zantedeschia albomaculata to treat
venereal diseases; vermifuge.
43
Appendix 2
Pooley, 1998:
Page 220 (D. anomala): used to treat coughing, dysentery, toothache and sterility.
Page 446 (D. zeyheri): used to treat chest ailments and as blood strengtheners to mothers
after a long, difficult labour.
Mahlaba (Dr Solomon), 1999:
D. anomala: root used as effective treatment of respiratory disorders and asthma.
Shale et al., 1999:
D. anomala: an infusion of roots taken for diarrhoea; powdered flowers used as an ointment
for wounds and sores.
Neuwinger, 2000:
D. anomala: page 180: for coughing; chest complaints; colds; pneumonia; fever; throat
problems; to make voice pitch high and clear; colic; stomach pain; vomiting; general stomach
problems; diarrhoea; dysentery; constipation (i.e. as purgative); malaria; internal parasites
(worms), such as ringworm in children; wounds; abscesses; dermatosis; yaws; (head) ulcers;
haemorrhoids; venereal disease (gonorrhoea); scabies; cataracts; dizziness; insanity or
possession (to calm the insane); epilepsy; snake bite; migraine; uterine pain; extended labour
pains; body pain; panacea.
D. capensis: page 180: for treating fever; febrile conditions; stomach (gastro-intestinal)
disorders; coughing.
D. schinzii: page 181: for treating fever; chills.
D. zeyheri: page 181: for backache; lumbago; anemia; weakness of the legs; used as a tonic
after illness; ulcers; coughing; chest ailments.
D. anomala: page 144: roots (fresh, dry, used as decoctions or tinctures) are regarded as a
panacea, and is also taken orally to treat fever, coughing, colds, sore throats, colic,
abdominal pain, diarrhoea, dysentery, constipation, intestinal worms, gonorrhoea and other
venereal diseases, as a purgative for haemorrhoids; pneumonia the powdered root is taken
with porridge; roots are said to be a cardiac tonic and good for circulation; a root decoction is
administered to children with unspecified diseases; preparations are applied topically for the
treatment of toothache and ringworm.
D. capensis: page 144: for fever, colds, influenza, hypertension, diarrhoea, cancer.
D. schinzii: page 144: for febrile convulsions (referred to as arm movements by the San,
possibly resulting from fever) in infants, as well as coughing and sore throats.
44
Appendix 2
Von Koenen, 1977, 1996, 2001:

Page 111 (2001) (D. anomala): for stomach complaints, colic, constipation a root decoction
or pulverised roots used, or the root is chewed (healing effect on gastro-intestinal activity);
(head) colds, coughing pulverised bark causes sneezing, coughing and watery eyes; the
plant used as a cardiovascular tonic; diarrhoea, dysentery two dessertspoonfuls of a
decoction taken; intestinal worms two spoonfuls are taken on an empty stomach in the
morning and twice more during the day, treatment continued for three days; wounds, ulcers,
ringworm, dandruff in children dried pulverised leaves, stems and roots used; haemorrhoids
gin extract of roots used; fever a tea taken; sore throat, colds, upset stomach root
chewed; plant acts on heart and stimulates circulation.
Page 111 (2001) (D. capensis): for treating fever a root decoction taken orally and a vapour
bath taken; coughing the plant boiled briefly and taken orally; gastro-intestinal problems a
leaf tea taken.
Pages 111-112 (2001) (D. schinzii): plant material can be used on its own or with D.
tomentosa; fever, shivering vapour of the whole plant inhaled in a vapour bath, or a plant
extract used as an enema.
Steenkamp & Gouws, 2006:
D. capensis:
for the treatment of cancer, high blood pressure, fever.
Van Wyk 2008a:

D. capensis: abitter tonic; diuretic; for kidney and bladder ailments; backache, nausea;
influenza; colds; cancer; diarrhoea.
D. capensis: for backache a tea prepared of the whole plant is taken orally twice daily (AS);
back and kidney problems (EW, AW, SG) (indicated for mostly men PT); to clean kidneys
and treat back problems (JO); backache and kidneys, rheumatism and nausea (KS);
influenza and colds, bladder and kidneys (PC) [used to be very expensive (PC) and very
bitter (EW, AW, SG, PC)]; infusion very bitter (PT); treatment for all ailments and cancer
(WdT).
Van Wyk et al., 1997, 2009:
D. anomala: page 118 (2009): used for similar conditions as D. capensis; aerial parts; roots
ground and snuffed as a treatment for colds, or a decoction with gin has been used to treat
haemorrhoids and fever.
D. capensis: page 118 (2009): leaves and twigs mainly used, but roots often included; for
fever and upset stomachs an infusion of leaves used (no precise details available); other
indications include influenza, high blood pressure, diarrhoea, cancer.
D. schinzii: page 118 (2009): used for febrile convulsions in infants.
45
Appendix 2

D. capensis: for colds and influenza decoctions of leaves and roots taken as a tea (JB, RT,
AT, EB); high blood pressure (JB); stomach pains (CB, FB, HG); liver tonic (FB); backache
(FB, KB, HB).
D. anomala: an infusion of roots taken for diarrhoea, constipation, flatulency, heartburn;
powdered flowers used as an ointment for wounds and sores; mixed with Scabiosa
columbaria Willk. ex Willk. & Lange for period pains; mixed with Helichrysum caespititium
Sond. ex Harv. & Sond., Scabiosa columbaria Willk. ex Willk. & Lange and Zantedeschia
albomaculata Baill. to treat venereal diseases; vermifuge; other indications include gall
sickness; bilious attacks; toothache; backache; diabetes; nasal congestion; colic.
Ramathal & Ngassapa, 2011:
D. anomala: known as umwanzuranya in Tanzania and Rwanda; for intestinal worm
infestations, chest infections fresh herb is crushed, and its decoction used; intestinal worm
infestations, chest infections, various stomach complaints roots crushed, and its decoction
used.

interviewees abbreviated as follows: AS = Andries Salmon; AT = Abraham Thys; AW
= Abraham Wessels; CB = Christien Baadjies; EB = Elizabeth Baadjies; EW = Ernest
Williams; FB = Frans Baadjies; HB = Hendrik Baadjies; HG = Hendrik Gouws; JB =
Jan Baadjies; JO = Jan Oormeyer; KB = Kato Baadjies; KS = Kiewiet (Hottie)
Steenkamp; PC = Piet Cupido; PT = Poppie Teo; RT = Rachel Tromp; SG = Sally
Goliath; WdT = Willem du Toit.
46
Appendix 2
Appendix 2G: Chronological list of ethnobotanical anecdotes and traditional

uses for H. procumbens and H. zeyheri# roots, as cited verbatim
from the referenced texts (#all ethnobotanical data is of
H. procumbens unless H. zeyheri is mentioned) (* = information
based on personal interviews, initials given in text, full names at
end of the appendix)
Page 830: for indigestion, a purgative roots taken orally; for fever, blood disease a root
infusion taken orally; a bitter tonic; pain in pregnant women 0.25g of dried tuber powder
taken thrice daily, can be taken post-partum in smaller dosages; in anticipation of a difficult
birth fresh roots preparedas an ointment and applied to abdomen; sores, ulcers, boils,
external cancerous growths fresh root ointment applied.
Kgathi, 1988:
For various ailments; stomach disorders; wounds powder applied onto wounds; arthritis,
rheumatism.
Van den Eynden, et al., 1992:
For stomach or post-natal pain, fever, lack of appetite, indigestion, diabetes a decoction of
rootstock taken; ulcers, boils, external cancerous growths an ointment of root applied.
Iwu, 1993:
Page 188: an astringent taste; a general analgesic; a bitter tonic (bitterness value 6 000); a
digestive aid; anti-phlogistic, anti-inflammatory taken internally or applied topically;
rheumatism, arthritis, low backache due to spondylosis, rheumatoid arthritis, neuralgia,
headaches, diseases of the upper duodenum with pancreatic involvement, enterohepatic
circulation, bile acid activation, reduction in cholesterol and natural fat levels as a tea taken
orally, ampoules for parenteral medication, an ointment for external use; fever, blood
diseases infusion taken; pain during pregnancy, post-partum pain; to facilitate labour
fresh roots applied as an ointment; sprains, sores, boils an ointment applied; to be avoided
during pregnancy, due to oxytocic properties.
Foster & Tyler, 1993, 1999:
Page 139 (1999): used for a wide variety of conditions; diseases of the liver, kidneys,
bladder; allergies; arteriosclerosis; lumbago; rheumatism; arthritis; gastro-intestinal
disturbances; menstrual difficulties; neuralgia; headaches; climatic (change of life) problems;
heartburn; nicotine poisoning.
47
Appendix 2
Rood 1994:
Pages 76-77: for rheumatism, diabetes, haemorrhoids, indigestion, fever, constipation, gall
and liver ailments, high blood pressure, heartburn, a variety of ailments roots cut in slices,
dried, powdered, used to make a tea, by diluting 12.5ml tea with 500ml with boiling water,
take thrice daily before meals (very bitter, treatment continued for 3-6 weeks); sores, ulcers,
other skin ailments, cancerous growths an ointment made of fresh roots, externally applied;
in-growing toe nails, alopecia areata (stress syndrome with hairloss as one of the symptoms)
roots used abroad; also exported to Germany, where capsules are marketed as
Treufelskrallen.
Bown, 1995:
Pages 29, 230: used for various ailments; bitter; stringent; a sedative; for painkilling; reduces
inflammation; stimulates digestive and lymphatic systems; arthritis, rheumatoid arthritis,
spondylosis, neuralgia, digestive problems involving the gall bladder and pancreas taken
orally; arthritic and rheumatic joints external application; not administered to patients with
gastric or duodenal ulcers.
Newall et al., 1996:
Page 98: anti-inflammatory, anti-rheumatic, analgesic, a sedative, diuretic; used for arthritis,
gout, myalgia, fibrositis, lumbago, pleurodynia and rheumatic diseases.
Ody, 1996:
Page 136: analgesic; a sedative; bitter; digestive stimulant; anti-inflammatory; anti-rheumatic;
constant use for at least six weeks significantly improved movement of arthritic joints and
reduced swelling; externally applied to muscles and painful joints; often sold in simple rather
than complex preparations; stimulates urine contraction (to be avoided during pregnancy).
Blumenthal, 1998:
Pages 120-121: for loss of appetite 1.5g of crude drug daily; dyspepsia, supportive therapy
of degenerative disorders of the locomotor system; all other indicated ailments 4.5g of
crude drug daily (contra-indicated for gastric and duodenal ulcers); choleretic, anti-phlogistic,
mildly analgesic.
Leung & Foster, 1999:
Pages 59-60: anti-inflammatory; analgesic; anti-rheumatic; a sedative.
Wagner 1999:
Page 411: for arthritis and rheumatism treatment by anti-phlogistic, analgesic action a tea
or injection; intestinal disorders (due to bitterness, of which the value should be 5 000 12
000).
48
Appendix 2
World Health Organisation, 1999:

Pages 184-185: for pain associated with rheumatic conditions, a supportive treatment for
rheumatism, painful arthrosis, tendonitis; loss of appetite and dyspeptic conditions; allergies;
boils and sores; diabetes; liver conditions.
Neuwinger, 2000:
H. procumbens: used in European medicine for rheumatic problems, arthrosis pain and
diseases of the liver, gall bladder, kidneys, pancreas, stomach and intestines; also fever,
blood diseases, blood purification, lumbago, arthritis, arthrosis, pain, pain in pregnant women,
coughing, digestive disorders, diarrhoea, constipation, lack of appetite, syphilis, gonorrhoea,
ulcers and wounds.
H. zeyheri: for snake bite; gum bleeding; lumbago.
Page 146: used as infusions, decoctions, tinctures and extracts of the roots taken orally:
treatment of osteo-arthritis, fibrositis, rheumatism, small joint disease (particulary effective),
diabetes (IM), hypertension, peptic ulcers, gout, fever; taken as a bitter tonic to stimulate the
appetite, for indigestion and as a laxative (taken on a regular daily basis); as a tonic for
infectious diseases such as tuberculosis (M); in small dosages to treat menstrual cramps and
higher dosages to expel the retained placenta; post-partum as an analgesic and to keep the
uterus contracted.
Topical application: dry powdered tuber applied directly as a wound dressing, or mixed with
animal fat or Vaseline and applied as wound-healing and burn-healing ointments. Minor
muscle aches and pains as well as painful joints can be treated by applying commercial
ointment and creams.
Wegener, 2000:
Anti-inflammatory and analgesic properties.
Pages 248-250: slightly analgesic, anti-phlogistic, anti-arthritic, anti-inflammatory; stomachic,
a bitter tonic a tea made by pouring 300ml of boiling water over ca. 4.5g (one teaspoon) of
powdered root and allowing to stand for 8 hours before straining, three 100ml portions taken
during a day, repeated over several days; febrifuge (questioned whether the frequent
occurrence of this property together with the bitterness could be a doctrine of signatures);
complaints during pregnancy; stimulates appetite and is therefore useful in dyspeptic
complaints; liver, bile, kidney and bladder complaints; allergies; general manifestation of
ageing; anti-diabetic.
49
Appendix 2
Chevallier, 2001:
Pages 42, 101: anti-inflammatory, analgesic, a bitter tonic, digestive stimulant; for digestive
problems; gall; arthritis, rheumatism; gout; backache; fibrositis; rheumatoid arthritis; fever; an
ointment for sores, ulcers and boils.
Schulz et al., 2001:
Pages 316-318: a bitter tonic (bitterness value 6 000); anti-pyretic; analgesic; antiinflammatory; for anorexia; indigestion; a supportive treatment for degenerative
musculoskeletal disorders.
Von Koenen, 1977, 1996, 2001*:
Page 126 (2001) (H. procumbens): dosage forms include teas, powders, ointments, dilutions
and potentiated medicines; indicated for diseases of the liver, gall bladder, pancreas, kidneys
(overdose could cause damage, even bleeding kidneys), stomach, intestines; arthritic
ailments, rheumatism tubers applied as an ointment; coughing, diarrhoea, constipation,
syphilis, gonorrhoea tubers are chopped finely, immersed in a little cold water and left
standing to draw well, two tablespoons of the extract taken daily (too much often leads to
diarrhoea KB); blood purification, acting via the kidneys an infusion taken (HH); pain a
tuber tea taken; lumbago dried, chopped tuber together with dried roots of Clerodendrum
uncinatum Schinz is laid on a potsherd with coals, back is smoked, the patient exposed to the
smoke, and the ash mixed with fat and rubbed onto the affected area, a hot water extract
prepared from the two plants to administer daily as an enema (MF); wounds, external cancer
an ointment applied; to prevent difficult births an ointment applied to the abdomen; pain
relief for pregnant women powder taken orally.
Pages 126-127 (2001) (H. zeyheri): for snake bite boiled tubers applied; gum bleeding a
tuber decoction used as mouthwash; lumbago dried tuber mixed with warm water and used
as an enema, or fine tuber powder together with root powder of Clerodendrum uncinatum
heated on a sherd of pottery, the back of the patient exposed to the smoke, and the ash
mixed with fat or oil and rubbed in.
Chrubasik et al., 2003:
Anti-inflammatory and analgesic properties.
ESCOP, 2003:
Page 233: symptomatic relief of painful oste-oarthritis 2-5g of drug or an equivalent
aqueous/hydro-alcoholic extract per day; relief of low backache 4.5-9g of drug as a dry
extract or an equivalent of 30-100mg of harpagoside; dyspepsia, poor appetite 0.5g of drug
in a decoction thrice daily, or 3ml tincture (1:10, 25% ethanol).
50
Appendix 2
Gagnier et al., 2004:

Anti-inflammatory and analgesic properties; a general health tonic used as an adjuvant in the
treatment of the mentioned disorders that degenerate the locomotor system, including
headache and backache.
H. procumbens: -age 165: a bitter tonic, anti-inflammatory, anti-rheumatic, a weak analgesic;
traditional tonic, general medicine for a wide variety of ailments, stomachic (digestive
complaints, lack of appetite); arthritis; pain (during and after labour); sores, ulcers, boils an
ointment made from roots is applied; dosage of up to 9g dried roots per day, taken as an
infusion (1-3g per dosage), also available in standardised extracts and capsules.
H. zeyheri: page 165: also used medicinally, but its variable chemical constitution makes it
less suitable.
Stewart & Cole, 2005:
H. procumbens and H. zeyheri: a general tonic; for hypertension; diabetes; analgesic; antiinflammatory; anti-rheumatic; for arteriosclerosis; arthritis; osteo-arthritis; fibrosis; gout;
sprains; post-partum pain; abortifacient; digestive disorders; laxative; diarrhoea; peptic ulcers;
dyspepsia; gall bladder diseases and gallstones; intestinal disorders; kidney disease; urinary
tract complaints; blood diseases; anti-biotic; fevers; tuberculosis; venereal diseases; sores,
ulcers and boils.
Mahomed & Ojewole, 2006:
Treatment, management and/or control of epilepsy and childhood convulsions; known to
decrease cholesterol and uric acid; a mild laxative with regular daily dosage; used for the
treatment of anorexia, indigestion, infectious diseases, allergies, diabetes, skin cancer,
hypertension, gout, osteo-arthritis, fibrositis, rheumatism, fever, menstrual cramps, postpartum pain, to expel placenta after birth and contract the uterus; wounds and burns.
Grant et al., 2007:
For various conditions; anti-inflammatory and analgesic for post-partum pain,
dysmenorrhoea, rheumatic and general musculoskeletal pain; for rheumatoid arthritis, osteoarthritis, tendonitis, headache, backache; sprains, sores, ulcers, boils an ointment applied;
bitter; for symptomatic relief from gastro-intestinal disturbances such as dyspepsia; blood
disorders an infusion used; dosage forms include infusions, ethanol tinctures or a dry
extract as coated tablets or capsules.
European Medicines Agency, 2009:
Page 4: for painful arthritis, tendonitis; loss of appetite, dyspeptic complaints.
51
Appendix 2

Page 162: the thick, fleshy secondary roots sliced and dried an infusion of 1.5 g of
powdered material prepared in a cup of boiling water, strained and can be taken daily;
standardised extracts available in the form of capsules, tablets, tinctures and ointments; used
in Germany as a supportive therapy for degenerative disorders of the locomotor system, for
lack of appetite and dyspeptic problems; also as treatment for rheumatism, arthritis, digestive
disorders; a general health tonic; analgesic (especially during pregnancy, and after labour);
an ointment prepared from the roots applied to sores, ulcers and boils.
*

interviewees abbreviated as follows: HH = Hilde Hester; IM = Isaac Mayeng; KB =
Klaus Binding; M = Matambo; MF = Maria Fisch.
52
Appendix 2
Appendix 2H: Chronological list of ethnobotanical anecdotes and uses for H.

hemerocallidea (syn. H. rooperi), as cited verbatim from the
referenced texts (* = information based on personal interviews,
initials given in text, full names at end of the appendix)
H. hemerocallidea: page 39: one of the ingredients of a medicine used as a charm against
thunder and storms.
H. rooperi: page 41: a tonic for weak children a decoction administered orally (produces a
purgative action); burns juice of corm applied; headache the corm is shaped into a small
hollow receptacle in which some blood from the afflicted persons forehead is collected, when
the receptacle is buried, the headache is cured; general medication.
Hypoxis spp.: page 41: internal parasiticide, a purgative an infusion prepared together
with other ingredients, taken orally; for delirium; wounds corm ash applied; when a baby
drank milk from a woman who has conceived again the child is taken off the breast and
given a corm decoction to drink, twice daily for two weeks; for (bilious) vomiting, loss of
appetite, abdominal pain, fever sprinkle salt on the cut surface of the corm, lick and
swallow the bitter juice that forms (large dosages cause vomiting).
Bryant, 1966:
Page 21: a distinction made between Hypoxis species called inKomfe or inKomfe enkula in
Zulu, where the former was considered poisonous, as it caused delirium [possibly H.
colchicifolia Baker (Watt and Breyer-Brandwijk, 1962)], while the latter was used to expel
internal parasites.
Page 30: H. hemerocallidea used as a parasiticide and a purgative a small handful of the
corm pounded and infused like a tea, a cupful taken when cool.
Hutchings, 1989(a):
H. rooperi for headaches; Hypoxis spp.: gastro-intestinal ailments, intestinal worms,
nervous disorders, skin ailments.
Hutchings, 1989(b):
Hypoxis spp. (inkomfe): to treat heart palpitations, cancer, hysteria, ulcers a corm extract
used.
Pujol, 1990:
Page 63: magic potions (Intelezis), medicines used to treat various diseases a corm used;
for weaning children from mothers milk stems and leaves used; dizziness, apoplexy dried
corm reduced to fine powder, administered in dosages of half a cup twice daily; prostate
53
Appendix 2
gland ailments stems and leaves mixed with Imbabazane roots and Unsukumbili leaves in
equal amounts; for insanity, bladder disorders corm and leaves used as an emetic; for fever
and coughing in children leaves used.
Page 78: to calm or sedate a tuber or one teaspoon of powder boiled in 500ml water, one
tablespoonful taken twice daily.
Vinesi et al., 1990:
H. rooperi: 6-9 patents registered, commercialisation of the extract under the trade name
Harzol for the treatment of prostate adenoma.
Albrecht, 1996:
Hypoxis spp.: used as a tonic and rejuvenator; treatment for prostatic hypertrophy, testicular
tumors, internal cancers, urinary diseases.
Pages 55-56: for dizziness, mental disorders corm infusions administered; symptoms of
benign prostate hypertrophy hot aqueous extracts of fresh or dried corms used; patients
who cannot speak, possibly due to shock ground corm decoctions administered orally or as
enemas; a tonic for weak children a plant decoction administered; burns juice from
rootstock applied topically; headache corms shaped into receptacles into which some blood
from the forehead of the patient is placed and then buried; plant decoctions have purging
effects; ulcers, impetigo, as anthelmintic corm pounded and mixed with pork fat.
Louw, 1999:
For the convalescent; enlarged prostrate; an improved immune system (used by cancer and
HIV/Aids patients); cancer (prolonging life, reducing side-effects such as nausea after
chemotherapy; prolonged life and strengthening with pancreatic (WV) and prostate cancer
(KL); tuberculosis; psoriasis; chronic fatigue syndrome; improved quality of life with motorneuron illness (VZ); improvement in movement and anti-inflammatory treatment for
rheumatoid arthritis (MR).
Pooley, 1998:
Page 234: used to treat headaches, dizziness, mental disorders, cancers, inflammation and
HIV.
The juice from sliced tubers applied directly to sores; fresh or dried leaves and corms
extracted and used as a wash for wounds; dried leaf-powder sprinkled on wounds.
54
Appendix 2
Powers, 1999*:
Corm sold for: tuberculosis; flu; psoriasis; a possible immune booster; treatment of many
ailments including cancer, HIV, kidney and liver problems (AaM).
Several Hypoxis spp. are toxic; can induce vomiting and diarrhoea.
Singh, 1999:
Hypoxis spp: for intestinal parasites; infertility; impotency; urinary infections; heart weakness;
coughing; nausea; vomiting; palpitations; nervous disorders; anxiety; insanity; corms boiled or
roasted as food in times of famine; sterols and sterolins from corms boost immunity.
H. hemerocallidea product (Moducare) tablet or extract (since 1997), claimed to boost
immunity; used for prostrate hypertrophy, Aids, tuberculosis, arthritis, psoriasis (H.
hemerocallidea; sterols have since been removed from the product).
Neuwinger, 2000:
Page 268: used as an emetic for dizziness, mental disorders and bladder problems; urinary
tract infection; enlarged prostrate; prostrate adenoma; a strengthening tonic; purgative;
burns; headache.
Page 146: weak infusions and decoctions of the corms are administered as convalescent and
strengthening tonics to children and adults with wasting diseases (AM), such as tuberculosis
and cancer taken thrice daily over a period of one month; a laxative and vermifuge; anxiety,
palpitations and depression (AsM); rheumatoid arthritis fresh plant tinctures.
Page 176: a general tonic decoctions (of ca. 2-4 g dry root per day, equivalent to 30-60 mg
pure phytosterols) traditionally administered to weak children, currently commercially
available; for prostrate hyperplasia/hypertrophy; burns a juice applied; for prostrate
problems, urinary infections stems and leaves mixed with other ingredients.
Page 124: headaches, dizziness, mental disorders, cancers, inflammation, HIV/Aids
tuberous rootstock used; a convalescent, strengthening tonic a weak infusion of bulb used;
for urinary complaints; testicular tumors; a laxative and emetic.
Ojewole, 2006:
Corm applied for several ailments; used as muthi; for HIV/Aids-related diseases; yuppie flu;
hypertension; diabetes mellitus; cancer; psoriasis; gastric and duodenal ulcers; tuberculosis;
urinary tract infections; asthma; some central nervous system disorders such as epilepsy and
55
Appendix 2
childhood convulsions; anti-inflammatory; hypoglycemic; for management and/or control of

painful, arthritic and inflammatory conditions.
For ulcers, HIV/Aids-related illnesses and used as multiple-use medicine infusions or
decoctions of dried corm.
Koduru et al., 2007:
For treating cancer corms pulverised, boiled in water and taken orally until signs of relief
are obvious.
Laporta et al., 2007:
Corm of H. hemerocallidea: for various diseases; colds; flu; hypertension; adult-onset of
diabetes mellitus; psoriasis; urinary infections; testicular tumors; prostate hypertrophy;
internal cancer; HIV/Aids; some central nervous system disorders.
Hydroalcoholic extract of H. rooperi patented for: anti-inflammatory; anti-biotic; anti-arthritic;
anti-atherosclerotic; diuretic; a stimulant of muscular and hormonal activities.
Commercially available dietary supplements and pharmaceutical products: mainly for benign
prostatic hyperplasia.
Drewes et al., 2008:
Indicated for urinary infections; hypertension (headaches); a variety of inflammatory
conditions; testicular tumors and some other cancers; HIV/Aids.
Historical traditional uses with Zulu names: for cardiac diseases (izifo zenhiliziyo); bad
dreams (amaphupho amabi); impotency (ukuphela kwaqmandla esithweni sangasese);
apprehension (uvalo); insanity (ukuhlanya); barrenness (ukungazali); intestinal parasites
(izinkelemu).
Recent uses with Zulu names: for cancer (umdlavusa); headaches (ikanda elibuhlungu);
dizziness (unzululwane); to boost the immune system (isandulela-ngculaza); testicular cancer
(amathumba asesithweni sangasese somuntu wesilisa); prostate hypertrophy (isifo sendlala
eseseithweni somuntu wesilisa sangasese); burns (ukuska); ulcers (izilonda ezibasesiswini
ngaphakathi); as an emetic (umuthi wokuphalaza); an enema (umuthi wokuchata).
Commercially available on its own or in mixtures with aloe and Sutherlandia; product names
include Vuselela, Vikelela, Down to Earth, Smart Hypoxis, Nutriherb; preparations
normally in capsules containing 60-600mg plant extract per capsule; descriptions on labels
include properties such as immune-active, used for immunity for cancer patients, HIV,
haemorrhoids, Herpes simplex, varicose ulcers, asthma, allergies, high blood pressure,
diabetes, impotence, enlarged prostate; skin cream preparations together with tea tree oil,
aloe, comfrey, evening primrose oil, or vitamin E indicated for clearing skin blemishes, with a
claimed anti-viral and anti-fungal function; a food supplement known as Hypo-plus marketed
as an energy booster and immunity modulator.
56
Appendix 2
Van Wyk, 2008b:

An emetic to treat bladder disorders, dizziness and insanity; a traditional tonic; for prostrate
hyperplasia.
Ojewole et al., 2009:
HIV/Aids-related diseases; arthritis; yuppie flu; hypertension; diabetes; cancer; psoriasis;
gastric and duodenal ulcers; tuberculosis; asthma; some central nervous system disorders;
urinary tract infections; diarrhoea; with anti-inflammatory, analgesic, anti-oxidant,
antibacterial, anti-cancer and anti-convulsant properties.
Owira & Ojewole, 2009:
Management and/or control of various ailments; cancer; nervous disorders; immune-related
illnesses; heart weakness; urinary tract infections corm washed with clean water, chopped
into small pieces and boiled for 20 min, a decoction taken orally (dose correlating to ca. 20g
corm/250ml daily); also used as an extract, powder and infusion.
Van Wyk et al., 1997, 2009:
Page 178 (2009): infusions of the corm and/or leaves are used; as an emetic, laxative,
vermifuge and a tonic to weak children; to treat bladder disorders, dizziness, insanity, burns;
prostrate hypertrophy, urinary infections; extracts used commercially in prostrate preparations
and in various tonics and so-called immune-boosting preparations.
*

interviewees abbreviated as follows: AaM = Aaron Mathenjwa (traditional healer);
AsM = Ashley Mashigo; KL = Koos Louw; MR = Michael Reed; VZ = Vivienne Zilberg;
WV = Willem Visser.
57
Appendix 2
Appendix 2I:
Chronological list of ethnobotanical anecdotes and uses for

S. frutescens. Those from 1847 to 2008 were taken verbatim
from Van Wyk and Albrecht (2008) (* = information based on
personal interviews, initials given in text, full names at end of
the appendix).
Thunberg, 1772-1775 (in Forbes, 1986):

For the -tTreatment of eye problems - bathing the eyes in decoctions of roots and leaves.
Pappe, 1847, 1850, 1857:
Page 6 (1847): dried and pulverised roots and leaves used for eye diseases.
Smith, 1895:
Page 62: preparation of leaves as a tonic; page 66: a pinch of leaves in boiling water taken
twice or thrice daily for extreme weakness and sinking of the stomach; page 86: blood
purifier; pages 116-117: leaf decoctions for dysenteric diarrhoea; pages 138-139: curing of
malignant tumors, cancerous in appearance; also used as a blood purifier and tonic to
delay the progress of true cancer and promote a much prolonged life; page 228: also
indicated for nervous dyspepsia and gangrenous sores; leaves are intensely bitter.
Dykman, 1908:
Page 145: an infusion of bark and leaves for cancer, stomach ailments and blood purification
one cup thrice daily until it starts working, then a little less.
Marloth, 1917:
Page 18: a much esteemed remedy for various purposes among the natives. Clinical
experiments have not shown any specific action on cancer.
Kling, 1923:
Page 20: leaf juice mixed with fat or (leaf-) lard as blister-plaster (treksalf) for infections,
pustules.
Laidler, 1928:
Page 443: decoctions for cleaning wounds; administered for fever; also used for
consumption, chickenpox, etc. the etc. implies a wide range of ailments, i.e. general
medicine or tonic use.
58
Appendix 2

For liver ailments page 146: a knife tip of powdered dry leaves after each meal for
lewerkwaal.
For joints and muscles page 198: add big handfuls each of boegoe, ysterblare, wilde-als,
wilde kopiva, chamomile, gansies and bitterbossie, together with three big spoons of honey
and a packet of Engelse sout (Epsom salts) in an enamel pot. Add four cups of boiling water
and leave overnight. Transfer liquid (even the condensated liquid formed in the lid, which is
most powerful) to a bottle, and shut the cork. Take a big spoonful thrice daily after meals.
Page 649: infusions or decoctions of the leaf and bark used for influenza, stomach
complaints, internal cancers, uterine troubles, liver diseases, rheumatism, inflammation,
haemorrhoids, dropsy, backache, and as a tonic; infusions are taken for amenorrhoea, used
as a blood tonic and cancer prophylactic; powdered leaves in syrup used to treat coughing;
weak infusions taken before meals seem to act as a bitter tonic to improve the appetite and
digestion; can cause sweating, can be slightly purgative and emetic if too strong.
High altitude form (S. montana): dried roots snuffed for headaches
Batten & Bokelman, 1966:
Page 85: used as a blood purifier and tonic; to delay the progress of cancer; chest colds.
Smith 1966:
Page 106: S. frutescens subsp. microphylla leaves have a very bitter taste; Pages 275-276:
leaves when crushed are aromatic and very bitter; leaf decoctions used to clean wounds and
taken internally for fevers; very successful treatment of chicken pox and especially for cancer;
also for the treatment of eye problems.
Palmer, 1985:
Pages 97, 124 (S. frutescens, S. microphylla, S. tomentosa): for eye ailments powdered
roots and leaves used in a decoction to bathe the eyes; for cancer; used as a blood purifier
take half a cup of the leaf infusion or decoction thrice daily.
Van Breda & Barnard, 1986:
Pages 1-2: dried leaves and flowers used as snuff for head cancer, and an infusion of leaves
used for stomach cancer.
59
Appendix 2
Anonymous, undated. Kruieresepte van die Montagu Museum (i.e. Herbal recipes from the
Montagu Museum). Based on indigenous knowledge collected by Mr Kobus Kriel
(unpublished notes):
Page 2: for cancer, wounds, fever, blood purification dried, pounded plant used as a
tincture.
Archer, 1990:
Page 966: an infusion of leaves and flowers used for stomach complaints; page 967: a basic
ingredient in most medicines in Namaqualand mostly for stomach ailments, also listed
specifically for influenza, wounds, pains, aches and skin disorders.
Cilli AM, 1992. Kruie op witblits, rate, resepte en feite (Herbs on witblits, traditional
remedies, recipes and facts), pp 43. Unpublished notes, Worcester Museum:
Page 17: leaves steeped in brandy used for back and kidney ailments; a leaf infusion (tea)
made from the whole plant used to treat cancer and also to clean wounds.
Rood, 1994:
S. frutescens: pages 52-53: a decoction of leaves for cancer growths; leaf infusions used for
influenza, stomach complaints, cancer, chickenpox, and the common cold; dried, powdered
leaves snuffed for head and nose cancer; leaf infusions used for diabetes and as an eye
wash for weak eyes; flowers in a strong decoction for fever and to clean wounds; a decoction
also taken internally for stomach nerves and varicose veins; an infusion of bark and leaves
used as a tea, mainly to cure haemorrhoids.
S. microphylla: page 53: for stomach and digestive system ailments, internal cancer, bladder
ailments, influenza, liver ailments, rheumatism, inflammation, haemorrhoids, water retention
and backache, used as a tonic or strengthening agent any amount of a leaf infusion or
decoction used; coughing powdered leaves mixed with syrup; a purgative, to make
someone sweat an infusion used (if too strong, it can cause vomiting); for wounds,
tuberculosis, fever, chickenpox a decoction used as a wash; bitter strengthening agent
(tonic) for increased appetite and digestion a diluted infusion taken before meals.
Page 82: for cleaning wounds, fevers, chickenpox and cancer.
Maliehe, 1997:
For fits, circulation problems, stress-related ailments and chronic illness.
60
Appendix 2
Pooley, 1998:
Page 58: used as a tonic; treatment for eye ailments, pain and influenza; delays the spread of
cancer.
Neuwinger, 2000:
For colds; influenza; chickenpox; diabetes; internal cancer; inflammation; rheumatism;
backache; liver problems; stomach complaints; bladder diseases; fever; wounds;
haemorrhoids; varicose veins; used as a tonic and appetite stimulant.
Page 148: a tonic to treat fever, poor appetite, indigestion, gastritis, oesophagitis, peptic
ulcers, dysentery, cancer (prevention and treatment, e.g. significantly beneficial in the
treatment of pancreatic and other cancers, improved quality of life in patients with terminal
metastatic breast cancer), diabetes, colds, influenza, coughing, asthma, chronic bronchitis,
kidney and liver conditions, urinary tract infections, stress and anxiety, rheumatism, heart
failure, rheumatoid arthritis (relief of pain and inflammation); while preparations can be taken
on a chronic basis when necessary, it should not be taken during pregnancy, as
teratogenicity and abortion are known to have occurred (D&MB).
Von Koenen, 2001:
Pages 182-183: for internal cancer, diseases of the bladder and uterus, stomach conditions,
backache a leaf tea used; chickenpox to reduce fever also as an additive to bath water
and a leaf tea taken; eye ailments a leaf tea used as eye drops; influenza, rheumatism, liver
problems a leaf tea used internally in diluted form; haemorrhoids a leaf tea used internally
in diluted form and for external application in concentrated form; taken as a tonic before
meals to stimulate appetite and digestion; fever, influenza flowers, leaves and seeds
added to bath water.
Schwegler, 2003:
Page 34: mainly leaves used, but all aerial parts are usually included; a bitter tonic; general
medicine; used for stomach problems, internal cancers (hairy costal form particularly
effective), colds, influenza, chickenpox, diabetes, varicose veins, piles, inflammation, liver
problems, backache, rheumatism; wounds a decoction used as a wash; fever and other
ailments a decoction taken internally; prolapse of the uterus the plant used as douche; to
expel retained blood from the uterus after labour, post-partum pain, healing and resolution of
the uterus a decoction taken orally; for extreme pain in cancer and HIV/Aids patients seed
and leaves smoked; not to be used during pregnancy; a tea taken orally and used as a
soothing bath or lotion for blisters one cup of leaves added to one one litre of boiling water,
apply directly for external use, take a quarter to half a cup orally every half an hour for
internal use; influenza, rheumatism, liver and other alments take a weak infusion prepared
with two leaves in a cup of boiling water; used as an eye wash a tea prepared from roots
and leaves.
61
Appendix 2

Page 313: an adaptogenic, bitter tonic; for colds; influenza; coughing; asthma; bronchitis;
fever; indigestion; poor appetite; gastritis; peptic ulcers; dysentery; diabetes; liver conditions;
rheumatism; urinary tract infections; tuberculosis; stress and anxiety; treatment and
prevention of cancer; immune stimulatory properties for treatment of cancer and Aids;
extracts used topically for burns, wounds and inflammatory skin conditions.
Bessong et al., 2005:
For colds, influenza, chickenpox, diabetes, varicose veins, piles, backache, rheumatism,
physical and mental stress leaves and twigs used.
Page 137: used for internal cancers; to stimulate appetite and wound-healing; fever;
chickenpox; influenza; diabetes; tuberculosis; ulcers; haemorrhoids; stomach complaints;
bladder diseases; liver problems and varicose veins; dried leaves marketed as a general
health tonic.
Thring and Weitz, 2006:
Tea (an infusion) from 3 teaspoons/a small handful of fresh or dried leaves and stems infused
in one litre of boiling water, 25ml is taken in the morning and at night to treat backache,
bladder and kidney problems, cancer, colds, influenza, liver problems, diabetes, fever and
stomach complaints.
High altitude form (S. montana): an infusion of leaves used for dropsy of the heart, as a
sedative and to treat stress-related ailments, shock, trauma, fits and severe depression; root
decoctions taken for heart palpitations.
Museum:
Page 2: a weak infusion of leaves, flowers and pods taken for internal cancers; also taken for
fever, as a blood purifier and to clean wounds; a root and leaf infusion used as an eye bath.
Wileman L, no date. The uses of our Karoo plants in bygone times. Unpublished notes,
Worcester Museum:
Page 7: for cleaning wounds, fevers, chickenpox, cancer, an eye wash.
62
Appendix 2
Youthed G (Ed.), no date. Dictionary of South African traditional medicinal plants of the
Eastern Cape. Pharmaceutical Society of South Africa, Johannesburg (unpublished):
Page 158: leaves, stems and roots used for blood purification, as a tonic, for chest colds
powdered leaf mixed with sugar and abdominal complaints; said to delay the course of
cancer.
B-E van Wyk (unpublished data):
70g of leaves, stems and pods boiled in two litres of water until reduced to 1.5 litres (a twoweek supply, i.e. the daily dose is ca. 5g in the decoction the recipe from local Khoi-San
sheepshearers). Four good swings taken in the morning and midday (not in the evening) for
high blood pressure, obesity and improved energy. (No side-effects noted after six years of
continuous use, except for insomnia when taken in the evening).
C Albrecht (unpublished data):
A 65-year old male was clinically diagnosed with cancer of the head and pancreas, due to
jaundice, CT-scanning, enlarged gall bladder, epigastric mass, endoscopic retrograde
cholangiopancreatography (ERCP) and laparotomy by an experienced surgeon at a teaching
hospital. He received a bypass to relieve the jaundice and was discharged, after whichhe
decided to self-medicate and chose the pods of S. frutescens because he remembered that
this was from the kankerbos (cancer bush). It happened in December 1992, when
Sutherlandia plants had most of their leaves and numerous pods. He made a water decoction
and drank the extract of 1g dry weight daily for 45 days, after which he felt so good that he
returned to work. Four years later he was re-examined at the teaching hospital by the same
surgeon with CT-scans, and no detectable sign of the epigastric mass could be found. He
lived five years post-diagnosis and died of kidney failure, unrelated to his pancreas
pathology.
Stomach problems drink as a tea, the short one is female, and used for male problems,
while the long one is male, and used for female problems (EW, AW, SG); high blood
pressure take powdered leaf as a tea (can be taken often, but kortbeenwildekeur (S.
frutescens) is better than wildekeur (S. frutescens subsp. microphylla) (AS, JO, KS, PC).
Wildekeur is used to treat: backache and kidney pain (as a general cleanser or blood purifier)
leaves of three twigs steeped in a small pot and taken after a meal (not on an empty
stomach) (JO); backache or stomach pain (KS); a general medicine taken with honey (PC);
cancer (for improvement, not as a cure, but no personal experience) an infusion of dry or
fresh leaves of two twigs per cup, not poisonous) (PT); cancer and all ailments (WdT).
63
Appendix 2
Van Wyk et al., 1997, 2009:

Page 280 (2009): strong decoctions or alcoholic tinctures of mainly leaves are taken; the
hairy coastal form (S. frutescens var. incana) is said to be particularly effective against
cancer; also a remedy for stomach problems, internal cancers, colds, influenza, chickenpox,
diabetes, varicose veins, piles, inflammation, liver problems, backache, rheumatism, stressrelated ailments, shock, trauma, fits and severe depression; a bitter tonic; used topically for
wounds and eye ailments.
For eye ailments leaves used, also for stomach ailments.
Page 7: the Khoi use it to clean wounds and for many other ailments; brandy tinctures of
leaves are bitter tonics used for colds, influenza, backache, stomach problems, liver and
kidney ailments and cancer.
Used for numerous ailments, e.g. nausea leaf infusions taken (JB); diabetes/treating
sugar (JB, CB, FB); high blood pressure (JB); kidney stones, gall, to cleanse the bladder
(JB, AT, KB, HB, DB, EB); toothache chewed and placed on tooth (JB, CB, FB); rashes on
skin (JB); backache (JB, CB, FB, HG, MT); stomach problems (EB, BB, AnT); colds and
influenza (JT).
High altitude form (S. montana): for dropsy of the heart; a sedative; for heart palpitations;
headaches.
As cited in Van Wyk and Albrecht (2008).
Based on interviews with the community, indications directly quoted, names of

interviewees abbreviated as follows: AnT = Andreas Thys; AS = Andries Salmon; AT
= Abraham Thys; AW = Abraham Wessels; BB = Bertus Baadjies; CB = Christien
Baadjies; DB = Danster Baadjies; D&MB = Dawid and Mrs. Bester; EB = Elizabeth
Baadjies; EW = Ernest Williams; FB = Frans Baadjies; HB = Hendrik Baadjies; HG =
Hendrik Gouws; JB = Jan Baadjies; JO = Jan Oormeyer; JT = Jakob Thys; KB = Kato
Baadjies; KS = Kiewiet (Hottie) Steenkamp; MT = Martiens Thys; PC = Piet Cupido;
PT = Poppie Teo; SG = Sally Goliath; WdT = Willem du Toit.
64
Appendix 2
Appendix 2J: Chronological list of ethnobotanical anecdotes and uses for

V. oligocephala, as cited verbatim from the referenced texts
text, full names at end of the appendix).
Phillips, 1917:
Decoctions of the whole plant used for syphilis.
Dykman, 1908:
Page 119: used to stop vomering (vomitting) administer half a teaspoon of soetolie with 510 drops of groen amara, if expelled, administer once again.
Kling, 1923:
Page 11: For the treatment for constipation.
Gerstner, 1939:
The roots can be used for treating hysteria (in the Zulu culture).
Pages 297-298: for abdominal pain during pregnancy, a stomachic bitter a decoction
administered; for dysentery, colic plant material used; rheumatism, diabetes a decoction
used; a purgative a leaf infusion used; serious chronic illness, ulcerative colitis roots
used; used as a charm to divert a hailstorm plant material tied to a stick and waved at the
approaching storm, or smoke from a fire made of the whole plant is used.
For heart and circulation problems page 79: make an infusion as a tea by adding one bottle
of boiling water to a handful of leaves, when dark green, filter through a cloth and store in the
bottle to which one small bottle of lewensessens is added, shut cork. Take a good mouthful
every morning on an empty stomach, as a laxative. Take less if the effect is too severe.
For digestive system problems page 138: for children, 11 drops of kraamdruppels and 11
drops of groenamara, also good for adults in larger dosages; for maag wat nie kan stil nie,
the bitterbossie is dug out and prepared as a tea, to be taken with water.
For treating joints and muscles page 198: add big handfuls each of boegoe, ysterblare,
wilde-als, wilde kopiva, chamomile, gansies and bitterbossie, to three big spoons of honey
and a packet of Epsom salts in an enamel pot. Add four cups of boiling water and leave
overnight. Transfer liquid (even the condensated liquid formed in the lid, which is most
powerful) to a bottle, and shut cork. Take a big spoonful thrice daily after meals.
65
Appendix 2
Smith 1966:
Page 61: Decoctions are bitter and used for stomach disorders.
For abdominal pain a flower decoction taken orally.
Palmer 1985:
Page 95: for poor appetite leaves or young twigs chewed.
Page 154: for stomach pain, dysentery, rheumatism, diabetes, as a purgative a tea (bitter)
taken; fevers root bark and stems used; scurvy leaves used; boiled as a vegetable (less
bitter than when raw).
Hutchings, 1989(a):
For stomach ailments; used as a charm.
Rood 1994:
Page 39: the plant infusion is very bitter; used for labour pains, a stomach bitter, for
dysentery, diabetes a plant infusion used; for constipation, colic a concentrated infusion
(obtained after lengthy boiling or afkooksel) taken orally; rheumatism a concentrated
infusion applied externally.
Page 312: for intestinal complaints a root infusion used; abdominal pain in pregnancy,
dysentery, stomach bitters, diabetes unspecified plant sections used; rheumatism a plant
decoction taken orally; abdominal pain flower decoctions taken; ulcerative colitis roots
used; magical power used as a charm against hailstorms.
Pooley, 1998:
Page 498: leaves used as a tea; treatment for intestinal and other complaints.
Grierson & Afolayan (1999):
Dried leaves and stems soaked in warm water and applied to wounds as a lotion.
Shale et al., 1999:
Decoctions of the whole plant used for syphilis and gonorrhoea.
Neuwinger, 2000:
For intestinal complaints; abdominal pain; ulcerative colitis; used as a purgative; for
rheumatism; diabetes.
66
Appendix 2
Van Wyk and Gericke, 2000*:

Page 148: a general health tonic; appetite stimulant; used for the treatment of general
malaise, abdominal pain, diarrhoea, dysentery, constipation, colitis, diabetes (anecdotal antidiabetic activity has been scientifically confirmed TGF); and rheumatism.
Von Koenen, 2001:
Page 191 (2001): for abdominal pain, dysentery, diarrhoea, constipation, septic colitis,
diabetes, rheumatism a tea prepared from the plant; for slimming and blood-purification
leaves eaten as a vegetable.
Erasto et al., 2005:
For -dDiabetes fresh leaves, twigs and roots pulverised, an infusion taken orally.
A tea is prepared from a small handful of leaves infused in boiling water, used for stomach
complaints, diabetes and worms; V. oligocephala can act as a substitute for A. afra,
depending on availability, in cases where the listed ailments need to be treated.
For diarrhoea a root decoction used.
Artemisia absinthium can also be referred to as groenamara and used to treat diarrhoea.
Van Wyk et al., 1997, 2009:
Page 304 (2009): leaves and twigs used, rarely roots; leaf infusions are extremely bitter,
suggested similarity with real amara, Quassia amara L.; uses include: as stomach bitters for
abdominal pain and colic, dysentery, ulcerative colitis; rheumatism; diabetes.
Museum:
Page 6: an infusion prepared from leaves and finely chopped roots. Take as a tea for
rheumatism, stomach ailments, dysentery and diabetes.
For syphilis; gonorrhoea.
*
TGF = based on interview with Dr TG Fourie.
67
Appendix 2
Appendix 2K: Chronological list of ethnobotanical anecdotes and uses for W.

salutaris (and syn. W. ugandensis), as cited verbatim from the
referenced texts (* = information based on personal interviews,
initials given in text, full names at end of the appendix).
Bryant, 1909:
For colds, chest complaints bark taken orally with cold water; rheumatism bark used.
Pott, 1918:
For treating malarial fever bark used.
Gerstner, 1939:
For coughing, chest complaints cooked roots taken as an expectorant.
Gordon, 1953:
A remedy for colds root bark smoked.
Pages 158-159 (W. ugandensis): a purgative bark used; common cold stem or root bark
used as an expectorant, often smoked; for chest complaints and aphthae fumes from
powdered root bark sprinkled on hot embers inhaled, or powdered bark taken orally with
water; for fever, malaria stem or root bark extracts taken orally.
Glover et al., 1966:
For fever fruit chewed; for malaria, used as an emetic bark and leaves boiled and the
decoction taken orally.
Lindsay, 1978:
For coughing, an expectorant smoke of burned plant inhaled.
Arnold & Gulumian, 1984:
For coughing bark maceration taken orally; gastric ulcers pulverised bark taken with
porridge; skin diseases pulverised bark rubbed in; for backache, an aphrodisiac, for throat
problems a bark decoction taken orally.
A panacea; for venereal diseases, abdominal pain bark decoctions taken orally, or
pulverised bark taken with porridge; headaches pulverised bark rubbed into scarifications
on temples; abortifacient bark decoctions taken orally.
68
Appendix 2
Hutchings, 1989 (a):

For gastro-intestinal ailments used as an enema; renal and skin ailments; respiratory
ailments used as snuff or inhaled; febrile complaints used as an emetic or enema; for
sprains and fractures.
Jansen & Mendes, 1990:
For treating inflammation of gums; throat infections bark used; angina root bark used.
Pujol, 1990:
Page 189: for influenza, chest congestion, headaches, malaria.
Iwu, 1993:
Pages 258-259: for common colds; toothache; fever; a general tonic; remedy for a variety of
illnesses; dried stem bark or root is chewed and the juice swallowed; for stomach ache,
constipation, muscle pains, weak joints, general body pains inner bark used; diarrhoea a
decoction of roots, or a soup prepared from roots; skin diseases leaves used in the
preparation of lotions; to clear sinuses powdered bark used as snuff; malaria aqueous
extract of bark used; chest complaints smoke of burning bark inhaled.
Kokwaro, 1976, 1993:
Page 56 (1993): for coughing, fever, stomachache, constipation, weak joints and general
body pains (including toothache and muscle pains) dried bark chewed and the juice
swallowed, or small amounts of dry, ground bark can be added to a cup of tea (very bitter)
and taken twice or thrice daily; malaria a decoction of bark and leaves (induces violent
vomiting); a variety of unspecified diseases leaves boiled in bathing water; diarrhoea
roots included as an ingredient in a special soup.
Hutchings & Van Staden, 1994:
Bark used for headaches, chest congestion, coughing, colds, influenza, malaria, toothache.
Rood, 1994:
Page 20: for colds inner bark chewed; chest ailments smoke of burning bark inhaled;
malaria bark boiled in water.
Mander et al., 1995:
For colds, sinusitis, influenza and other chest complaints a bark decoction taken orally;
used as snuff, application to sores powdered bark used; malaria a bark preparation used.
69
Appendix 2
Hollmann & Van der Schijff, 1996:

Powerful muti; magical and aphrodisiac properties; used to treat various ailments, including
stomach ulcers, sore throats, coughing, colds, chest complaints, blood disorders, skin sores,
toothache, backache, headaches, venereal diseases, rheumatism, nightmares, malaria;
contains anti-feedant compounds with insect repellant and antibacterial properties; red inner
bark smells like cinnamon and tastes burny when chewed; bark can be ground and mixed
with water or milk, taken as snuff, shaped into small capsules, or applied externally as a
paste.
Pages 204-205: for febrile complaints, rheumatism and ailments (isibhobo or amanxeba, that
seem to be mainly inter-costal neuralgia, rheumatism or symptoms of the liver) thought to be
caused by sorcery bark used as emetics or purgatives; a dry cough a pinch of pulverised
bark taken orally with a spoonful of water, or bark mixed with Cannabis sativa smoked, or
boiled roots eaten; colds, coughing, malaria root bark or stem bark used as an expectorant,
or smoked; penial irritation powdered bark mixed with fat used as an ointment; inflammation
of the urethra an ointment prepared from leaves and stalks together with Hibiscus
surratensis L. applied to penis; sores, skin irritations an ointment iprepared from pulverised
leaves and stalks, mixed with bark and any kind of fat, and rubbed on; used as a purgative
and aphrodisiac, for toothache, malaria, colds, chest complaints, venereal diseases,
backache, skin complaints, stomach ulcers bark used; also to make dogs and bees
aggressive and thus protected; influenza, fevers, pains, stomachache and other gastrointestinal complaints bark and roots used.
Felhaber, 1997:
Page 110: for scabies preparations used that incorporate W. salutaris.
Mashimbye et al., 1999:
For sores; ulcers; anti-fungal; insect anti-feedant; molluscicidal.
Neuwinger, 2000:
Page 558: for coughing; used as an expectorant; colds; chest complaints; aphthous oral
infection; throat problems; urethritis; inflammation; backache; muscular pain; toothache;
headaches; constipation; stomach pain; gastric ulcers; used as an emetic; malaria; fever; skin
diseases; venereal diseases; used as an aphrodisiac, abortifacient and panacea.
Rabe & Van Staden, 2000:
For colds, influenza, sinusitis and other respiratory complaints; inflammation, sores, and skin
irritations; malaria.
70
Appendix 2

Page 150: bark mostly used, taken orally in a powdered form, as infusions or decoctions, a
tonic for all health conditions: fever, malaria, colds and influenza, used as an expectorant for
coughing, as a natural anti-biotic for chest infections productive of purulent sputum (SM), for
venereal diseases, abdominal pain, constipation, stomach ulcers, cancer (IM), rheumatism;
for headaches topical application to cuts made on the temples; used as an aphrodisiac.
Drewes et al., 2001:
A panacea; for headaches; throat infections; venereal diseases; abdominal pain; rheumatism;
chest complaints; malaria leaves or bark used; malarial fever; skin complaints; stomach
ulcers; inflammation of gums, mouth sores, angina root bark used.
Coates Palgrave, (1977) 2002:
Pages 618-619 (1977); 741-742 (2002): the inner bark is reddish, bitter and peppery and has
a variety of applications, particularly for the treatment of the common cold - dried, ground
inner bark used as a snuff to clear the sinuses, chewed as a remedy for chest complaints, or
the smoke from burning the bark inhaled; the inner bark boiled in water together with the
roots effective against malaria.
Page 345: an anti-biotic; general tonic; powdered leaf in the form of a tablet or powdered and
licked from the hand, swallowed with some water; recommended dosage for supportive
treatment of infections three tablets (200mg) daily; a remedy for coughing and colds cold
water infusions of powdered bark taken orally as expectorants, or the bark smoked; for chest
complaints, bronchial infections, influenza, headaches, oral thrush, toothache, malaria,
rheumatism, venereal diseases, gastric ulcers.
Wube et al., 2005:
For stomach pain, constipation, toothache, coughing, fever, muscle pain, weak joints, general
body pain, tuberculosis.
Page 141: bark, stems, roots and leaves used to treat colds and respiratory complaints as a
natural anti-biotic for such infections; a tonic for all health conditions; fever; malaria;
influenza; coughing; venereal diseases; abdominal pain; constipation; cancer; rheumatism;
stomach ulcers; topical application for headaches in cuts on temples; used as an aphrodisiac.
Kiringe, 2006:
Bark used for stomach pain, bloatedness, loss of appetite, internal wounds, lung and chestrelated ailments, coughing, malaria.
71
Appendix 2
Iwalewa et al., 2007:

For malaria, painful back/chest, rheumatism bark used.
Ssegawa and Kasenene, 2007:
For malaria dried, powdered bark swallowed; possession dried powdered bark burned
and the smoke inhaled.
Ngure et al., 2009:
For skin diseases a leaf decoction used for a bath; visceral and cutaneous leishmaniasis
stem bark taken orally in boiled water or soup; also used for stomach pain, constipation,
coughing, fever, toothache, muscle pain, weak joints, general body pain, venereal diseases,
malaria.
Orwa et al., 2009:
Chest complaints treated by inhaling the smoke from burning bark; dried, ground bark
inserted into the nostrils to clear the sinuses.
Van Wyk et al., 1997, 2009:
Page 308 (2009): bark or root bark is traditionally used, currently also leaves; cold water
infusions of bark taken orally as expectorants for coughing (production of purulent sputum);
bark smoked as cough and cold remedy; tablets prepared from leaves used as a natural antibiotic, said to be effective against oral and oesophageal thrush; also for chest complaints,
influenza, malaria, venereal diseases, headaches, toothache and gastric ulcers.
Green et al., 2010:
For colds, sinusitis, influenza, other chest complaints, tuberculosis, used as an anti-parasitic
powder on sores leaves used.
Lamorde et al., 2010:
For HIV/Aids, coughing.
Tabuti et al, 2010:
For tuberculosis and related diseases stem bark used.
Were et al., 2010:
For tooth decay, asthma, bronchitis, malaria hot or cold decoctions from leaves and stem
bark taken orally.
72
Appendix 2

To make dogs aggressive a mixture of bark and other plants administered to dogs (GM); for
coughing and flus root bark ground and boiled for a short while, plant material removed
and remaining water taken (SF); to relieve toothache powdered roots are placed inside the
holes of the teeth (SF); to make food more enjoyable take a leaf decoction to make it more
enjoyable (SF); leaves are added to food as spice to give it a peppery taste (G).
*

interviewees abbreviated as follows: G = Mr Gumede; GM = Goodenough (Sgwili)
Mdluli; IM = Isaac Mayeng SF = Sibusiso Falakhe; SM = Solomon Mahlaba.
73
Appendix 2
Appendix 2L: Chronological list of ethnobotanical anecdotes and uses for

W. somnifera, as cited verbatim from the referenced texts
text, full names at end of the appendix).
Dymock et al., 1890:
Page 468: Fruit used as an emetic; a remedy for dyspepsia arising from chronic liver
diseases (smaller dosages used).
Smith, 1895:
Page 83: for wounds, sores boil leaves in lard and apply as an ointment; leaves for ulcers
and wounds; root bark used as a tonic; page 100: asthma a decoction of root bark used;
chest complaints a root bark decoction of W. somnifera root bark and um-Tumana taken
orally when still hot; page 133: ringworm green berries bruised and rubbed in; pages 145146: syphilis used externally as a leaf paste; page 157: black gall sickness used as a
decoction of roots; pages 166-167: sores on horses green berries, leaves and twigs
moistened and pounded into a paste, applied to sores, covered up and kept moist (the plant
is a sedative and possibly anti-septic).
Phillips, 1917:
Page 210: Small reptile (nohana, introduced through witchcraft) in the stomach a
decoction of the plant used as an enema or taken orally to expel this pest, or ash from the
plant is powdered, mixed with fat and rubbed over the arms.
Pages 83-84: for colds, chills, to tone up the uterus where a woman habitually miscarries, to
remove retained conception products a root decoction used, prepared by chopping up the
root, washing it and boiling it until the root is tasteless, the decoction is poured off periodically
and a half to full cup of the decoction is taken every half an hour to hour as required; feverish
infants decorticated root steeped in warm water administered as an enema; gangrenous
rectitis a warm infusion prepared from a small handful of roots with the same quantity of
Pentanisia variabilis and sufficient water to form a clyster; syphilis a leaf paste applied, or
other preparations (not specified) used; the plant is anti-septic; anthrax in humans leaves
moistened and juice squeezed onto pustules, that may not be interfered with in any other
way; anthrax infected meat the plant used; wounds, sores leaves boiled in fat and used
as an ointment; asthma, chest complaints root bark decoction used; ringworm green
berries bruised and rubbed on infected areas.
74
Appendix 2
Bally, 1937:
An aphrodisiac 2-3 finger-sized pieces of root pounded in milk and taken orally; for
conjunctivitis leaf sap dropped into the eye; abortifacient a root extract taken orally.
Nadkarni, 1954:
Pages 1292-1294: a tonic; an aphrodiasiac; used for consumption, emaciation, debility,
dyspepsia, rheumatism roots used; for colds, chills a root decoction used; syphilis plant
used; asthma a decoction of root bark administered.
Simons, 1957:
A remedy against intestinal parasites, asthma, chest problems; syphilis, sepsis, piles,
erysipelas.
Irvine, 1961:
For rheumatism, fever, colic pounded fresh leaves rubbed on body; fever, chills, colic an
extract of leaves and root taken orally; inflammatory or chronic swellings fomentation (warm
compresses) of leaves applied; fever, tonic a leaf infusion taken orally.
Pages 1010-1012: for colds, chills, to tone the uterus in case of habitual miscarriage, to
remove retained conception products, abortion a root decoction used; asthma, chest
complaints a bark infusion or root bark decoction taken orally; bedsores a bark infusion
applied as an ointment; for intestinal parasites introduced through witchcraft, typhoid fever, a
disinfectant for anthrax-infected meat plant material used; feverish infants decorticated
root used as an enema; gangrenous rectitis an infusion of roots, with Pentanisia variabilis
used as an enema; syphilitic sores a leaf paste applied; infections, eruptive diseases such
as smallpox a root and leaf decoction used; anthrax pustules, pinkeye, conjunctivitis fresh
leaf juice applied; wounds, sores leaves boiled in fat and applied as an ointment; ringworm
green fruit pounded and rubbed in; black gall sickness a root decoction used; erysipelas
a leaf paste applied; haemorrhoids a leaf decoction applied internally and externally; an
aphrodisiac, for diarrhoea, proctitis roots used; nausea, rheumatism leaves used; eating
berries cause severe gastro-intestinal upset; emphysema, alcoholism, pulmonary
tuberculosis an alcoholic infusion used.
Bryant, 1966:
Page 42: for gangrenous rectitis a small handful of roots and sufficient water used to
prepare a clyster; page 59: venereal diseases (syphillis) roots used; page 73: skin diseases
(wounds and sores) leaves used.
75
Appendix 2
Glover et al., 1966:

For body pain heated leaves applied; upset stomach, gonorrhoea a root decoction taken
orally.
Smith, 1966:
Page 106: fruit is juicy and bitter, formerly used in rural medicine, but not eaten; page 226:
leaves can be used for preparing an ointment; page 483: leaves applied directly as
trekpleister (poultice) for bringing sores to a head; leaf decoctions used for the treatment of
typhoid fever; page 489: plants used to treat syphilis.
Watt, 1967:
Page 12: Ayurveda: the plant is considered a sedative with hypnotic properties.
Anonymous. The Wealth of India, 1969:
Page 582: plant used for the treatment of ulcers, rheumatism, coughing, dropsy,
consumption, senile debility.
A root decoction for colds and chills, intestinal ailments, wounds; used as an enema to get rid
of small snake in the stomach.
Lindsay, 1978:
A purgative, an anthrax decoction taken orally; pain heated leaves applied as a dressing;
skin diseases, ulcers ash of burned leaves rubbed in; gingivitis heated roots rubbed in.
Palmer 1985:
Page 95: bed sores leaves used to prepare an ointment.
Page 121: used as a disinfectant and healer; for wounds, sores, syphilitic sores, boils fresh
leaf juice used, or an ointment prepared of leaves; rheumatism leaves pounded and rubbed
on body; asthma a bark decoction used; ringworm bruised berries used; to disinfect
anthrax-infected meat plant used, probably by boiling leaves with meat.
Baerts & Lehmann, 1989:
For purulent otitis leaf sap dropped into the ear; skin diseases a leaf decoction taken
orally and used for bathing; insanity, possession a leaf decoction or sap of leafy twigs taken
orally.
76
Appendix 2
Hutchings 1989 (a):

For veneral diseases; gastro-intestinal ailments (also indicated for infants and children); used
as charms; respiratory ailments; febrile complaints (also used for infants and children);
nervous complaints includes use as an enema; skin ailments.
Pujol, 1990:
Page 76: for various infected sores and abscesses leaf poultices (Izithobo) applied
externally, or root ointments prepared by boiling with fat of a python snake or a crocodile
applied; page 78: to tone the system one teaspoonful of root powder boiled in 500ml of
water, one cup taken twice daily.
Samuelsson et al., 1991:
For intestinal pain fresh roots ground, mixed with a glass of water and the filtrate taken
orally; gonorrhoea a 15cm piece of root boiled in a glass of water and the filtrate taken
orally; muscular pain a handful of fresh leaves ground, mixed with 5g of resin from
Commiphora myrrha and rubbed in.
Adjanohoun et al., 1993:
For peptic ulcers two large teaspoons of dried, powdered leaves mixed with a cup of water,
two teaspoons taken every two hours; threadworm leaves, together with Chenopodium
ambrosioides, macerated with water, left overnight and 250ml taken orally; recurrent abortion
root bark steeped in water, half a cup taken orally thrice daily.
Balagizi & Chifundera, 1993:
Abortifacient fresh leaves together with the same amount of fresh leaves from Vernonia
kirungae, Conyza volkensii, Solanum nigrum, Carduus leptacanthus, roots of Microglossa
pyrifolia, Carica papaya, Musa paradisiacal and Catharanthus roseas, pounded together and
one glass of the sap taken daily until the abortion occurs.
Iwu, 1993:
Pages 259-260: roots and leaves used internally; for fever, chills, rheumatism, various other
uses freshly pounded leaves (internal and external use); anthelmintic, ritual plant against
witchcraft whole plant used; hyperpyrexia in infants an enema of decorticated root;
anthrax, meat disinfectant whole plant used; eruptive diseases such as smallpox a
decoction of leaf and root; also for asthma, bronchial diseases, syphilis and other venereal
infections, black gall sickness, used as a general anti-septic and wound dressing.
77
Appendix 2
Kokwaro, 1976, 1993:

Page 225 (1993): for liver and spleen-related ailments, general stomach ache (mainly for
constipation and gastric ulcers) root sap produced by pounding the root; skin rashes, ulcers
and gonorrhoea, labour pains, colds in children, excess bile and for general ill health a root
decoction used (prepared by drying and grinding it into a fine powder. A teaspoon of powder
is taken in a cup of tea or with honey, once or twice daily. It has no pronounced taste, but a
slight smell); pain heated leaves applied externally.
Leaf poultices are applied externally for the treatment of open cuts, abscesses, wounds,
inflamed parts, haemorrhoids, rheumatism and syphilis.
Rood 1994:
Pages 97-98: leaves and stems seem to have anti-biotic and anti-viral properties; plant
known to have a calming and hypnotic effect; for injury, infection, inflammation of skin (e.g.
rash and wound shingles), haemorrhoids, conjunctivitis, ringworm an infusion of leaves
applied; asthma, chest ailments roots used; diarrhoea, gall, rheumatism a decoction of
leaves and root bark used.
Page 132: for colds, chills, ringworm, syphilitic sores, erysipelas and many other ailments; an
insecticide.
Pages 273-274: for fever in infants decorticated roots administered as an enema; colds,
chills, used as a uterine tonic after a miscarriage a root decoction taken orally; asthma,
suspected anthrax poisoning a root bark decoction taken orally; gangrenous rectitis warm
water maceration of a handful of roots, together with a handful of roots of Pentanisia
prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp., used as an enema; sores, wounds, to
disinfect anthrax-infested meat, used as sexual stimulants and abortifacients, for fever,
convulsions, rheumatic pains leaves used; infected sores, abscesses root powder boiled
with fat and applied; syphilis root bark decoctions taken orally and a leaf paste applied as
an ointment; ringworm bruised green fruit applied; face rashes ground leaves applied;
sores leaves boiled in lard and applied as an ointment; dysuria a root decoction made
with Solanum capense L. used; for cancer, intestinal parasites, diarrhoea, proctitis, chest
complaints, fever, black gall sickness, nausea, rheumatism, conjunctivitis unspecified
sections used; haemorrhoids a leaf decoction applied internally and externally; open sores
leaves and fruit applied externally; rheumatism, pain, inflammation, swelling leaves
applied in poultices or emollients with olive oil; painful swellings, carbuncles, ulcers bruised
leaves and ground roots applied; roots used as a tonic, have narcotic and anti-epileptic
effects; sap has diuretic and emmenagogic effects.
78
Appendix 2
Pooley, 1998:
Page 72: used to treat fever, intestinal infestations, asthma and to heal sores.
Leaf poultices used on open cuts, abscesses and wounds.
Dold & Cocks, 2000:
For coughing a large woody rootstock peeled, pieces of raw soft wood chewed, but not
swallowed; fever, syphilis, asthma, ringworm, rashes the root used; asthma, bed sores
the bark used; chest complaints, bronchitis, typhoid fever, anthrax, wounds, sores, syphilis,
gangrene, ringworm, rheumatism, pain, swellings the leaves used; sores leaves and fruit
applied locally; boils, parasites (internal and external, e.g. ringworm), anthrax, haemorrhoids,
syphilis whole plant used; black gall sickness, gangrenous rectitis, venereal diseases,
fatigue, learning disorders, memory retention unspecified plant sections used.
Mishra et al., 2000:
An ingredient in many formulations a variety of musculoskeletal conditions (e.g. arthritis,
rheumatism), a general tonic to increase energy, improve overall health and longevity, to
prevent disease in athletes, the elderly and during pregnancy; a multi-purpose medicinal
agent; anti-inflammatory, anti-stress, anti-tumour, anti-oxidant, immune modulatory,
hemopoetic and rejuvenating properties; beneficial to the endocrine, cardiopulmonary and
central nervous systems.
Neuwinger, 2000:
Pages 558-559: used as a tonic, abortifacient, aphrodisiac, used as an enema: purgative;
anthrax (pustules and infected meat); for muscular pain; rheumatism; inflammation; swelling;
labour pains; to expel the placenta; skin diseases; furuncles; wounds; gangrenous proctitis;
abscesses; face rashes; smallpox; haemorrhoids; syphilis; gonorrhoea; ulcers; gingivitis;
conjunctivitis; purulent otitis; erysipelas; dysuria; ringworm; threadworm; upset stomach;
stomach pain; gastric (peptic) ulcers; colic; diarrhoea; meteorism; bladder diseases; fever;
colds; chills; asthma; coughing; bile vomiting; epilepsy; insanity and possession; recurrent
abortion; a snake and scorpion repellant.
Scartezzini & Speroni, 2000:
In Ayurveda: used as a tonic, aphrodisiac, sedative, for geriatric problems and as an agent
for promoting learning and good memory; roots, leaves and seeds used, leaves can be
poisonous.
79
Appendix 2

Page 150: infusions, decoctions and tinctures of fresh or dry roots are used, and with larger
woody roots, only the soft outer cortex is used: an adaptogenic tonic, for fever, colds,
influenza, asthma, general ill health and debility, any infections, syphilis, abdominal
discomfort, diarrhoea, typhus, typhoid, proctitis, worms, used as a sedative; hypnotic
properties, an aphrodisiac, for womens health; applied topically for many skin conditions;
also used as a (brain) tonic, amoebicide, anti-spasmodic treatment, and for cancer, candida,
coughing, bronchitis, dyspepsia, inflammation, rheumatism, tuberculosis, fatigue, to improve
learning and memory retention, anxiety, and mild to moderate depression.
Von Koenen, 2001:
Page 193 (2001): to expel the placenta, coughing, upset stomach, malaise, bladder diseases
a root decoction taken orally; for wounds root pulp used as a dressing; meteorism, gastrointestinal disorders, diarrhoea root chewed or a root extract taken orally; open wounds on
legs, poorly healing wounds, ulcers root bark, young roots or leaves dried, pulverised and
sprinkled on; snake and scorpion repellant roasted roots placed around the house; typhoid
fever, sedation and calming the whole plant used; erosive proctitis a root decoction
infused rectally; lesions, infections, inflammation of the skin, decubitus ulcers, erysipelas,
syphilitic ulcers, rashes related to infectious diseases, haemorrhoids, conjunctivitis, ringworm
leaves prepared into fresh sap and applied, or berries used, due to their anti-viral and antibiotic properties; asthma, chest complaints roots used; as a stimulant roots crushed in
milk, taken orally; abortifacient roots used.
Arora et al., 2004:
For a large number of ailments, including mental disease, asthma, inflammation, arthritis,
rheumatism, tuberculosis, infections, fever, male sexual disorders, cancer.
Page 346: a sedative; adaptogenic tonic (roots); wound-healing (leaves or the whole plant)
leaf poultices applied to treat open cuts, wounds, abscesses, inflammation, haemorrhoids,
rheumatism, syphilis, or prepared into an ointment with fat or oil, the whole plant can also be
used; a hypnotic; general tonic; effective against a large variety of ailments; roots taken orally
as a decoction, infusion or tincture (3-6g per day).
Njoroge & Bussmann, 2006:
For asthma roots boiled and taken orally.
Kambizi et al., 2007:
For the treatment of arthritis, tuberculosis, cancer, sexually transmitted illnesses such as
Herpes simplex.
80
Appendix 2
Sharma et al., 2007:

Promotes immunity in adulthood and functions of the reproductive system; adaptogenic,
cognition promoting; anti-stress, mood stabilising, anti-inflammatory and rejuvenating
properties; used for osteo-arthritis; sleep disorders administered as a mixture with oil
(Shirodhara); for hypertension and related symptoms such as headaches, disturbed sleep,
fatigue giddiness, palpitations administered as a mixture with Bacopa monnieri (L.) Pennell,
Convolvulus pluricaulis Choicy, Nardostachys jatamansi DC. and Hyoscyamus niger L., all in
equal quantities.
Schmelzer & Gurib-Fakim, 2008:
Pages 629-632:
In India: a rasayana medicine in Ayurveda used to improve overall physical and mental
health, and to increase longevity and vitality by rejuvenating the body; narcotic, hypnotic, and
aphrodisiac properties, a liver tonic, purgative and diuretic properties; used for tuberculosis,
senile debility, nervousness, rheumatism, furuncles, sores, dropsy, coughing, hiccups, to
induce abortion, wounds and bed sores ointment from leaves applied, antidote for cobra
poison plant extract applied to the skin, alternative for Panax ginseng (the mode of action is
different).
In Africa: a blood purifier a leaf infusion taken; diuretic properties, for gonorrhoea, coughing,
asthma, epilepsy several plant preparations used; slow blood circulation plant burned and
smoke wafted over patient; abortifacient leaves/roots pounded together with other plants, or
roots used ( in some parts, the roots are administered to enhance fertility where there is a
history of miscarriages, or to regulate menstruation); a purgative, for general body pains
leaves administered; ulcers, gingivitis ash of leaves rubbed in; erysipelas, haemorrhoids
leaves or a leaf decoction administered; sores, abscesses, smallpox leaf/root powder boiled
with fat and applied as an ointment; anthrax pustules leaf sap applied, anthrax-infected
meat boiled in water with leaves; purulent otitis leaf sap used as ear drops; poorly healing
open wounds leaves/roots used in several different preparations; asthma a plant infusion
or root bark decoction taken; rheumatic limbs a poultice from fresh leaves and roots
applied; tonic, skin problems the whole plant used; as an aphrodisiac the whole plant
used, or root powder mixed with milk taken orally; ringworm pounded roots or root powder
used to prepare medicines; dysuria, gonorrhoea, upset stomach a root decoction taken
orally; gangrenous inflammation of the rectum maceration of roots used as an enema;
alcoholism, tuberculosis, emphysema alcohol extract has a hypnotic effect; children with
fever or disturbed sleep (nightmares) a root decoction administered, or rubbing
preparations of crushed leaf applied; snake and scorpion repellant roasted roots placed
around the house; insect repellant, vegetable leaves, but can be poisonous; an emetic
fruit; to curdle milk and make cheese seeds used.
81
Appendix 2

For the treatment of fever mixed with other plants (AS; PT), e.g. the root is mixed with
bitterkoud [(an important medicine, causes perspiration (JO)], or an infusion of the root caan
be taken (PT, WdT); used as an emetic (to clean the stomach) (EW); kidney failure
(nierstuipe) and inflammation work with dawidjie (also used for magic, i.e. toorgoed) (KS);
cancer used as a decoction of koorshout, boegoe, dawidjie and wynruit (KS).
Reddy et al., 2009:
HIV 100g root powder in a glass of water administered twice daily for six months (no
intercourse during this time) (Note: the high dosage is questionable, the use of the word
administer is ambiguous, i.e. internally or externally).
Mirjalili et al., 2009:
An aphrodisiac; sedative; rejuvenative; for life prolonging; a general energy enhancing tonic
for geriatric problems; to promote youthful vigour, endurance, strength, health; to increase
production of vital fluids, muscle fat, blood, lymph, semen, cells; to counteract chronic fatigue,
weakness, dehydration, bone weakness, loose teeth, thirst, impotency, premature ageing,
emaciation, debility, muscle tension; leaves bitter, used as an anthelmintic, as an infusion
for fever; bruised leaves and fruit applied locally to tumours, tubercular glands, carbuncles,
ulcers; roots used as a nutrient and health restorative for pregnant women and old people,
sterility in woman (a decoction of root boiled with milk and ghee), constipation, senile debility,
rheumatism, general debility, nervous exhaustion, memory loss, loss of muscular energy,
spermatorrhoea.
Page 310: for wound-healing (treatment of open cuts, wounds, abscesses, inflammation,
haemorrhoids, rheumatism, syphilis); also for the treatment of asthma and as an adaptogenic
tonic, sedative and hypnotic treatment.
Teklehaymanot & Giday, 2010:
For diarrhoea, amoebic dysentery in blood, stomach pain, boils, abscesses, swelling, chest
pain roots used, crushed (with water in some instances).
Used as an enema to get rid of small snake in stomach; other indications include colds and
chills; intestinal ailments; wounds; to tone uterus.
82
Appendix 2
Page 5: porridge prepared from leaves and applied to wounds, sores, haemorrhoids,
rheumatism and syphilis; decoctions of roots used for asthma and as a sedative, a root
tincture in brandy is strengthening.
*

interviewees abbreviated as follows: AS = Andries Salmon; EW = Ernest Williams; JO
= Jan Oormeyer; KS = Kiewiet (Hottie) Steenkamp; PT = Poppie Teo; WdT = Willem
du Toit.
83
APPENDIX 2
Ethnobotany
Appendix 2M: Chronological list of ethnobotanical anecdotes and uses for

Z. mucronata, as cited verbatim from the referenced texts
(* = information based on personal interviews, initials given in text,
full names at end of the appendix).
Smith, 1895:
Page 88: for scrofulous diseases, swollen glands in the neck a root decoction used; glandular
swellings apply a paste of leaves; page 136: lumbago use a decoction of roots.
Palmer & Pitman, 1961:
Pages 259-261: fruit has a slightly sweet taste, but is not very palatable, used as a coffee
substitute during the Boer War, can prepare a porridge from ripe fruit; pain of any kind a
poultice of meal and a root decoction, or powdered baked root applied, poultice eaten
afterwards.
Page 66: plant juice an ingredient of arrow poison; Page 884: chest problems powdered
leaf and bark in water used as an emetic, or a decoction of bark used; as an expectorant for
coughing a hot infusion of bark used; for boils, carbuncles, other septic swellings of the skin
a poultice of leaves applied; diarrhoea and dysentery roots chewed and sap swallowed, or
root infusions taken orally; tubercular glandular swellings (scrofulous swellings), lumbago,
swollen glands in the neck a decoction of root taken orally and a paste of leaves applied;
general pain a poultice of meal prepared with a root decoction or with powdered baked root
applied, and eaten afterwards; measles, scarlet fever a decoction of leaves and shoots
gargled, and vapours inhaled; a remedy for gonorrhoea; magical a person hiding under this
tree will not be harmed by lightning; if it is cut down after the first summer rain, drought will
follow; thorny branches used in burial rites to be placed over the grave, probably as protection.
Bryant, 1966:
Page 50: bark used for chest complaints (emetic for chronic coughing).
Smith 1966:
Page 113: a decoction of the gelatinous roots is taken internally by the Bantu for scrophulous
disease and glandular swellings in the neck; the root chewed as an anthelmintic; the thorny
green branches used in burial rites to protect the corpse against exhumation by carnivores,
and used as fences; the presence of the trees are said to indicate sub-terranean water
supplies as it comes into seasonal leaf and flower independence of rainfall.
Teichler, 1971:
if blood is full of water; for pain in the whole body; cold feet; black spots in the face.
84
APPENDIX 2
Ethnobotany
Coates Palgrave, (1977) 2002:

Pages 550-551 (1977), page 667 (2002): the seeds used as rosary beads; poultices prepared
from powdered and baked roots are remedies for almost any pain, once the poultice is
removed from the affected area, it is eaten; boils and skin infections are treated with a paste
prepared from the leaves; a leaf paste and a dosage of a root decoction is used to treat
tubercular gland swellings; dysentery and lumbago treated by chewing the roots or taking a
root infusion; powdered leaf and bark used to relieve chest complaints, a bark infusion used to
cure coughing.
Kerharo & Adam, 1974:
For bed-wetting, urinary incontinence 48 hour maceration of pounded fruit with seeds, or
seed maceration, taken orally; a diuretic, for haematuria, schistosomiasis, infection with
gonococcus, dysuria, oliguria a root extract taken orally.
Malan & Owen-Smith, 1974:
For all types of skin diseases, ulcers pulverised bark sprinkled on, or a bark extract used as a
dressing.
Van Wyk, 1974:
Vol. 2, pages 365-367: for carbuncles, boils, swollen glands root infusions and/or pastes of
roots and leaves used internally and externally; chest complaints infusions of bark and leaves
used; dysentery, lumbago, general pains root extracts used; fruits edible, but unpalatable,
used as a substitute for coffee and maize meal, possibly poisonous; leaves highly nutritional.
Lindsay, 1978:
For rheumatism bark decoctions, together with Z. mauritiana Lam., taken orally.
Haxaire, 1979:
For stomach complaints a bark infusion taken orally.
Arnold & Gulumian, 1984:
For hypermenorrhoea root maceration, together with Terminalia sericea, or a root decoction,
taken orally; general body pain a root decoction taken with porridge; sterility root powder,
together with Tabernaemontana elegans, Euclea natalensis and Ximenia caffra, taken with
porridge.
Burkill, 1985-1997:
An anthelmintic leaves chewed; for blood purification in long-standing syphilis patients,
urinary inconvenience, urinary tract diseases, urethral discharge leaf decoctions taken orally;
lumbago, muscular pain a root decoction taken orally and applied as a dressing; toothache
fruit pulp applied or fruit chewed; abscesses, adenitis, tumors leaf pulp applied.
85
APPENDIX 2
Ethnobotany
Chinemana et al., 1985:

For diarrhoea pulverised roots taken in food or the extract taken orally; ulcers ground
leaves applied as a dressing; headaches leaves steeped in water and liquid rubbed into
scarifications on temples; depressed fontanelle roots burned, ash mixed with cooking oil or
Vaseline applied to fontanelle and inside the roof of the mouth (palate).
For ulcers pulverised fruit and leaves sprinkled on; diarrhoea, schistosomiasis, cholemesis,
venereal diseases, threatening abortion, abdominal pain, hypermenorrhoea a root infusion
taken orally; female sterility, dysmenorrhoea root powder eaten with porridge; pneumonia a
decoction of leaves and roots taken orally and rubbed in; snake bite antidote pulped roots
applied; edemas pulverised roots rubbed into scarifications on the body; wounds pulverised
leaves sprinkled onto the wound; bladder weakness a leaf decoction taken orally; panacea
a root decoction taken orally.
Rodin, 1985:
For the treatment of malaria, fever, diarrhoea in children one teaspoon of a leaf decoction or
cold water maceration taken orally; dysentery, bloody diarrhoea young roots boiled until the
water is red, one teaspoon taken orally every hour; ophthalmia eyes bathed with a leaf
extract.
Hutchings 1989 (a):
For the treatment of growths; respiratory ailments including use as an emetic; pain; dental
application; skin ailments; sprains and fractures.
Kokwaro, 1976, 1993:
Page 198 (1993): for snake bite roots used; rheumatism, stomach troubles a decoction of
bark used.
Archer, 1994:
Hakiesdoring leaves, roots and bark used, specific uses not provided.
For inflammation leaves taken as oral decoctions.
Rood 1994:
Page 86: for the treatment of bloody dysentery thin roots cut in lengths of 2.5cm, boiled in
water until the water is red, 18ml consumed mildly warm every hour; gout (lendejig), boils,
sores and glandular swellings decoctions of roots and leaves applied externally as a paste ;
constipation a root infusion used; painful eyes leaves steeped in water, the infusion dripped
into eyes; an emetic powdered leaves and roots in water; coughing - warm bark infusions
used; measles, scarlet fever steam of a decoction of leaves and young stems inhaled; blood
blisters (bloedblaas) leaves pounded until juicy, applied to affected area.
86
APPENDIX 2
Ethnobotany
Makgakga, 1995:
Warm bark infusions (leaves or roots can be added) used as expectorants and emetics for
coughing and chest problems; root infusions used as remedies for diarrhoea and dysentery;
chewed leaves applied externally to boils, sores and glandular swellings to promote healing
and reduce pain.
Page 193: used as emetics for chronic coughing warm infusions of pounded bark (a large
dishful) in a quart (ca. 1.2 litres) water, taken orally; respiratory ailments leaf and bark
infusions taken as emetics; chest complaints, rheumatism, stomach ailments a bark
decoction used; to purify the complexion bark used in a steam bath; toothache, scrofula,
infertility, for purification roots used; boils, glandular swellings a leaf paste used; boils,
carbuncles a leaf poultice used; dysentery a root infusion taken; general pain leaves and
roots used, or a poultice prepared from powdered, baked roots or meal mixed with a root
decoction applied, the poultice eaten afterwards; measles, scarlet fever a decoction of leaves
and shoots gargled; gonorrhoea, bloody diarrhoea, dysmenorrhoea, urogenital complaints
unspecified plant sections used; pain, delayed or irregular menstruation a root decoction
taken orally; colds, diarrhoea, used as carminative leaves, fruit and stems used; fruits are
edible and used as a coffee substitute, but considered poisonous by some; flowers used as
fish poison.
Rabe & Van Staden, 1997:
For the treatment of boils, abscesses, septic swellings leaves used as a poultice; wounds a
leaf powder applied; pneumonia a leaf decoction taken orally.
Neuwinger, 2000:
Pages 571-572: for the treatment of sterility, smallpox stem bark or roots boiled and the
decoction taken orally; severe chest colds dried pulverised roots smoked in a pipe; used as a
tonic a root decoction taken orally; threatening abortion, stomach pain leaf sap and a root
decoction taken orally; earache dried pulverised fruit inserted into the ear; blood in urine a
bark decoction taken orally.
Page 152: roots, bark and leaves used to treat diarrhoea, dysentery, coughing, chest problems
and other ailments; infusions and decoctions of the root considered a panacea in Zimbabwe.
Prozesky et al., 2001:
For the treatment of anti-plasmodial activity; also chronic coughing, respiratory ailments, pain,
infertility, measles, scarlet fever, boils, carbuncles, dysentery, gonorrhoea, a treatment for
snake bite, rheumatism and stomach ailments.
87
APPENDIX 2
Ethnobotany
Van der Merwe et al., 2000:

For the treatment of boils, sores, glandular swelling (animals) a root decoction applied
externally.
Page 350: leaves and bark applied to infected wounds and boils to promote healing and for its
sedative effect.
Bessong et al., 2005:
For the treatment of diarrhoea and dysentery (anti-peristaltic), stomach ulcers, fever, skin
diseases, infertility, hypermenorrhoea, anti-inflammatory treatment leaves used.
Luseba & Van der Merwe, 2006:
For the treatment of boils, sores, glandular swelling (animals) a root decoction applied
externally.
Iwalewa et al., 2007:
For the treatment of chronic coughing, boils, rheumatism, swellings, toothache leaves, stem
bark and roots used.
Mativandlela et al., 2008:
For the treatment of coughing, chest pains, fever bark used.
Mpiana et al., 2008:
For gonorrhoea, feminine sterility, dysmenorrhoea a root bark decoction used.
Van Wyk et al., 1997, 2009:
Page 286 (1997): roots, bark or leaves are used separately or in combination; warm infusions
taken orally as a tea; warm bark infusions (sometimes together with roots or leaves) used as
expectorants (and emetics) for coughing and chest problems; root infusions used as remedies
for diarrhoea and dysentery; root and leaf decoctions (or chewed leaves) applied externally to
boils, sores and glandular swellings to promote healing and relieve pain.
Waterman et al., 2010:
An anthelmintic twigs and fruit; for infection, parasitic diseases, diseases of the digestive tract
fruit used.
Green et al., 2010:
For the treatment of boils, sores, glandular swellings, diarrhoea, dysentery, coughing (as
expectorants and emetic), chest problems bark, leaves or roots used; tuberculosis-related
diseases or symptoms bark used.
88
APPENDIX 2
Ethnobotany

A twig is taken along on a journey to attract the spirit of a dead person (GM, TN); a leaf put on
a wound to extract a thorn (SF); fruit eaten (T); also for the treatment of chronic coughing,
respiratory ailments, to purify the complexion, toothache, scrofula, boils, glandular swellings.
Thomas & Grant, 1998, 2004, 2008, 2011:
Pages 194-195 (2011): fruit used to prepare porridge and meal; used medicinally for stomach
ailments, skin ulcers and chest problems; burial tree of Zulu and Sotho, planted around the
body, branches used to attract ancestral spirits from one dwelling to the next.

interviewees abbreviated as follows: GM = Goodenough (Sgwili) Mdluli; SF = Sibusiso
Falakhe; TN = Themba Nxumalo.
89
Appendix 3
HPLC chromatograms
APPENDIX 3: HPLC
chromatograms
(Agathosma
species)
and
quantification data (Sutherlandia species), to confirm

TLC chemical variation
Appendix 3A:
HPLC chromatograms and UV spectra obtained with
Det 168-350nm
Diosmin 1.25mg per mL MeOH
30
Det 168-350nm
9.633
Retention T ime
14.267
Agathosma standard analysis
25
20
8.667
mAU
15
24.867
23.517
24.000
24.383
21.600
21.783
20.533
20.883
19.417
19.750
18.383
18.683
17.383
17.700
16.200
16.383
16.733
15.567
14.833
15.100
11.300
8.250
7.417
6.717
5.650
6.100
4.500
1.883
2.067
2.483
2.850
3.333
3.567
10.550
12.100
12.317
12.533
12.783
13.017
13.433
10.083
10
10
12
14
16
18
20
22
24
26
28
Minutes
Fig 3A-1: HPLC chromatogram showing peaks for hesperidin (green) and diosmin
(black)
Appendix 3
HPLC chromatograms
(a)
3000
371
255
3000
1000
mAU
2000
223
1000
212
mAU
2000
200
250
300
350
400
450
nm
(b)
227
283
600
400
400
200
200
mAU
mAU
600
200
220
240
260
280
300
320
340
360
380
nm
(c)
1000
1000
mAU
262
365
2000
206
mAU
2000
200
225
250
275
300
325
350
375
400
425
nm
Fig 3A-2: UV spectra obtained for standards (a) quercitin; (b) hesperidin; (c) kaempherol
Appendix 3
HPLC chromatograms
(a)
13.98 Min
A. betulina DO51-b (LSF) 40-100%MeOH
300
235
287
Lambda Max
Lambda Min
300
mAU
200
mAU
200
100
412
100
0
200
225
250
275
300
325
350
375
400
425
nm
400
264
200
200
mAU
Lambda Max
Lambda Min
350
400
mAU
(b)
16.10 Min
A. betulina DO53 (LSF) 40-100%MeOH
225
250
275
300
325
350
375
400
425
nm
16.31 Min
300
300
(c)
Lambda Max
Lambda Min
200
mAU
mAU
345
251
200
100
100
0
200
225
250
275
300
325
350
375
400
425
nm
(d)
19.18 Min
A. betulina DO53 (LSF) 40-100%MeOH
Lambda Max
Lambda Mi n
200
200
351
mAU
400
254
mAU
400
225
250
275
300
325
350
375
400
425
nm
Fig. 3A-5:
UV spectra obtained for peaks eluating at (a) 13.98 min; (b) 6.10 min;
(c) 16.21 min; (d) 19.18 min
Appendix 3
HPLC chromatograms
Appendix 3B: HPLC chromatograms of Agathosma polar extracts

Det 168-340nm
A. betulina 51a (polar) gradient 40%
A. betulina 51a (polar) gradient 40%.dat
225
Retention Time
225
Retention Time
200
175
175
2.233
200
125
100
100
21.017
50
25
19.350
19.667
19.700
19.850
20.350
20.617
19.083
18.200
18.200
17.600
17.183
17.583
16.183
16.367
15.517
14.633
14.650
13.117
13.333
12.583
12.567
11.950
11.933
12.217
12.217
11.300
11.400
7.433
7.650
7.667
7.983
6.583
6.400
6.567
7.017
5.900
5.917
5.083
5.167
5.433
3.933
3.950
4.283
4.383
4.700
2.583
2.583
3.017
3.033
3.367
3.533
25
10.867
50
9.167
9.167
9.383
9.383
9.833
10.333
10.317
8.517
75
8.517
75
2.233
125
-25
-25
2
10
11
12
13
14
15
16
17
18
19
20
21
HPLC-PDA chromatogram for the whole leaf extract of Agathosma betulina

(sample no. AB 51a L). The black chromatogram was recorded at 340 nm and
the red chromatogram was recorded at 280 nm.
Det 168-340nm
200
Retention Time
Det 168-280nm
200
Retention Time
175
150
150
2.217
175
125
20.950
50
20.983
20.333
20.617
20.317
20.600
19.683
19.367
19.667
18.383
18.183
17.167
17.600
16.450
16.483
15.583
15.650
14.183
14.183
14.633
14.633
14.967
13.433
13.733
12.833
12.833
13.150
11.950
11.950
12.233
12.217
11.300
11.333
9.917
10.217
10.500
10.517
10.783
10.817 10.900
9.883
6.350
6.317
6.800
6.767
7.183
7.183
7.533
7.550
7.883
7.900
8.167
8.233
5.350
5.350
5.717
5.783
4.667
4.700
5.000
4.067
4.050
3.467
3.5503.717
25
2.567
2.833
3.050
2.567
50
75
2.217
75
9.150
9.150
100
8.650
100
8.650
mAU
Minutes
Fig. 3B-1:
125
mAU
150
mAU
150
mAU
Det 168-280nm
A. betulina 51a (polar) gradient 40%
A. betulina 51a (polar) gradient 40%.dat
25
-25
-25
2
Fig. 3B-2:
10
11
12
13
14
15
16
17
18
19
20
21
Minutes

(sample no. AB 51b L). The black chromatogram was recorded at 340 nm and
4
Appendix 3
HPLC chromatograms
Det 168-340nm
A. betulina 51c (polar) gradient 40%
A. betulina 51c (polar) gradient 40%.dat
450
Det 168-280nm
A. betulina 51c (polar) gradient 40%
A. betulina 51c (polar) gradient 40%.dat
2.217
Retention Time
450
Retention Time
350
350
300
300
250
250
200
2.217
mAU
400
mAU
400
200
19.033
19.400
19.700
19.700
19.933
20.333
20.633
20.633
21.100
21.017
18.200
18.450
17.850
16.450
16.450
16.633
17.050
17.183
15.550
15.667
9.217
9.250
9.467
9.467
9.883
10.217
9.950
10.183
10.417
10.417
8.600
6.000
6.000
6.467
6.467
6.700
6.817
7.050
7.083
7.417
7.317
7.683
7.700
5.167
5.117
3.3503.533
3.950
3.950
4.417
4.467
2.517
2.517
2.783
2.967
50
14.617
14.617
100
13.350
13.333
13.750
13.817
100
10.90010.983
11.317
11.333
11.633
11.650
11.950
11.950
12.250
12.300
12.750
150
8.600
150
50
11
12
13
14
15
16
17
18
19
20
21
Minutes
Fig. 3B-3:

(sample no. AB 51c L). The black chromatogram was recorded at 340 nm and
Det 168-340nm
A. betulina 51d (polar) gradient 40% 13 Nov
A. betulina 51d (polar) gradient 40% 13 Nov.dat
800
10
Retention Time
Det 168-280nm
A. betulina 51d (polar) gradient 40% 13 Nov
A. betulina 51d (polar) gradient 40% 13 Nov.dat
800
700
600
600
500
500
400
400
mAU
700
2.267
mAU
2.267
Retention Time
300
9.067
9.067
300
100
19.950
20.333
20.633
20.633
21.033
21.033
21.283
19.067
19.383
19.717
16.500
16.700
17.083
17.183
17.533
17.617
17.850
18.217
15.500
15.483
14.383
14.633
14.650
12.783
12.817
13.117
13.267
13.133
11.967
12.217
12.217
10.467
10.467
10.750
10.900
11.333
11.200
9.867
8.583
7.033
7.033
7.450
7.333
7.700
7.717
8.067
8.117
5.950
5.950
6.433
6.433
6.650
4.400
4.650
4.633
5.117
5.050
3.933
4.000
2.533
2.967
3.017
3.350
3.517
2.533
100
200
8.583
200
0
2
Fig. 3B-4:
10
11
12
13
14
15
16
17
18
19
20
21
Minutes

(sample no. AB 51d L). The black chromatogram was recorded at 340 nm and
Appendix 3
Retention Time
Det 168-280nm
A. betulina 51e (polar) gradient 40%
A. betulina 51e (polar) gradient 40%.dat
400
Retention Time
350
300
300
2.267
350
250
200
200
2.267
250
150
9.100
9.100
150
50
19.933
20.333
20.633
20.650
20.950
21.033
18.433
18.750
19.117
19.200
19.717
17.900
18.217
17.383
17.533
16.483
16.633
14.667
14.650
15.133
15.133
15.467
13.133
13.233
12.233
12.583
11.667
11.967
11.950
10.883
11.317
11.233
9.867
10.183
10.133
10.483
10.500
10.750
8.567
100
8.567
7.700
7.717
8.117
6.583
6.433
6.783
6.783
7.017
5.950
5.950
4.350
5.100
4.983
5.400
2.550
2.550
2.983
3.050
3.317
3.500
3.917
3.883
100
50
mAU
Det 168-340nm
A. betulina 51e (polar) gradient 40%
A. betulina 51e (polar) gradient 40%.dat
400
mAU
HPLC chromatograms
-50
-50
2
10
11
12
13
14
15
16
17
18
19
20
21
Minutes
Fig. 3B-5:

(sample no. AB 51e L). The black chromatogram was recorded at 340 nm and
Det 168-340nm
A. betulina 51f (polar) gradient 40%
A. betulina 51f (polar) gradient 40%.dat
300
Retention Time
Det 168-280nm
A. betulina 51f (polar) gradient 40%
A. betulina 51f (polar) gradient 40%.dat
300
Retention Time
250
200
150
150
100
100
Fig. 3B-6:
10
12
14
16
18
20.617
20.750
21.017
21.000
21.133
19.483
19.700
19.667
19.917
20.317
19.050
18.950
18.217
18.317
16.500
16.633
17.083
17.183
17.583
15.000
15.000
15.500
12.517
12.983
13.017
13.383
13.267
11.967
12.250
12.233
11.283
11.200
9.083
9.083
9.850
10.183
10.467
10.467
10.700
10.883
6.350
6.550
6
6.983
6.983
7.350
7.283
7.650
7.667
8.017
8.083
8.533
8.533
5.833
5.883
5.100
4.950
5.383
3.933
3.767
3.933
4.117
4.550
4.550
3.333
3.517
50
2.567
2.567
2.983
50
mAU
200
2.267
mAU
2.267
250
20
22
Minutes

(sample no. AB 51f L). The black chromatogram was recorded at 340 nm and
6
Appendix 3
1000
HPLC chromatograms
Det 168-340nm
A. betulina 53 (polar) gradient 40%
A. betulina 53 (polar) gradient 40%.dat
1000
Det 168-280nm
A. betulina 53 (polar) gradient 40%
A. betulina 53 (polar) gradient 40%.dat
Retention Time
Retention Time
800
700
9.067
700
500
400
2.250
400
600
9.067
500
100
21.800
20.767
21.100
21.150
20.050
20.400
19.450
19.750
18.700
19.067
18.217
18.250
16.483
16.850
17.100
17.217
15.550
15.733
15.733
13.933
14.267
14.350
14.650
14.650
13.283
13.150
11.650
11.967
11.967
12.200
12.200
10.467
10.467
10.967
10.933
11.300
11.200
8.600
8.617
7.100
7.267
7.433
7.733
7.783
8.083
7.967
6.550
6.733
100
6.067
6.100
200
5.317
5.167
200
3.833
4.017
4.500
4.533
4.717
4.750
300
2.533
300
2.783
2.967
3.317
3.367
3.567
mAU
600
0
2
10
14
16
18
20
22

(sample no. AB 53 L). The black chromatogram was recorded at 340 nm and the
red chromatogram was recorded at 280 nm.
Det 168-340nm
A. crenulata 54 (polar) gradient 40%
A. crenulata 54 (polar) gradient 40%.dat
Retention Time
Det 168-280nm
A. crenulata 54 (polar) gradient 40%
A. crenulata 54 (polar) gradient 40%.dat
Retention Time
10.317
1600
1400
10.317
2.300
1400
1200
12
Minutes
Fig. 3B-7:
1600
mAU
800
900
2.250
900
1200
800
800
600
2.300
mAU
1000
mAU
1000
600
21.667
20.700
21.083
21.083
19.983
20.383
19.400
19.750
19.100
18.250
18.317
17.217
17.567
16.483
16.667
15.567
15.917
14.600
14.600
13.867
14.150
12.867
13.183
13.167
200
12.250
12.300
11.333
11.333
11.667
11.667
8.450
8.450
8.717
8.717
9.217
9.217
9.467
9.467
9.833
7.350
7.333
7.717
7.717
8.017
7.933
5.817
5.850
6.367
6.333
6.733
4.900
4.917
5.417
3.517
3.483
3.933
4.033
4.333
0.883
200
400
2.567
400
0
2
Fig. 3B-8:
10
12
14
16
18
20
Minutes
HPLC-PDA chromatogram for the whole leaf extract of Agathosma crenulata

(sample no. AC 54 L). The black chromatogram was recorded at 340 nm and the
7
Appendix 3
HPLC chromatograms
Det 168-340nm
A. ovata 55 (polar) gradient 40%
A. ovata 55 (polar) gradient 40%.dat
Det 168-280nm
A. ovata 55 (polar) gradient 40%
A. ovata 55 (polar) gradient 40%.dat
Retention Time
Retention Time
1000
2.300
1000
600
600
400
400
2.567
9.033
2.300
9.017
mAU
800
mAU
800
21.750
20.800
20.683
21.133
21.050
19.717
19.767
19.950
20.367
19.100
18.217
16.633
16.633
17.050
17.200
15.500
15.483
15.683
14.467
14.600
14.617
13.950
10.983
11.283
11.167
11.650
11.667
11.933
12.150
12.117
12.450
12.467
12.667
12.683
13.000
13.117
9.867
9.883
10.300
10.317
10.433
8.550
8.550
7.350
7.633
7.667
8.000
8.100
6.350
6.583
6.583
6.783
5.833
5.883
4.333
4.367
4.717
3.350
5.100
5.167
5.400
3.517
200
2.983
200
0
2
10
12
14
16
18
20
22
Minutes
Fig. 3B-9:
350
HPLC-PDA chromatogram for the whole leaf extract of Agathosma ovata

(sample no. AO 55 L). The black chromatogram was recorded at 340 nm and the
Det 168-350nm
9.033
Det 168-340nm
DO A crenulata 2008
Retention Time
300
250
150
10.500
21.917
19.533
19.917
20.067
20.433
20.733
21.117
18.283
18.617
18.867
16.283
16.550
16.767
17.167
17.367
14.733
15.200
15.617
15.800
13.200
13.733
13.933
14.133
11.700
12.083
12.300
12.600
11.267
10.833
9.550
9.767
10.000
8.233
8.683
6.783
7.167
7.500
7.867
2.467
2.817
3.050
3.250
1.883
1.117
50
5.100
4.133
3.733
100
5.967
6.317
4.433
mAU
200
Fig. 3B-10:
10
12
14
16
18
20
22
24
26
28
Minutes
HPLC-PDA chromatogram for the whole leaf extract of Agathosma crenulata

(sample no. AC 10 L). The dark green chromatogram represents AC 10 L at
340 nm and the light green chromatogram represents a hesperidin standard at
350 nm.
8
Appendix 3
HPLC chromatograms
Appendix 3C: HPLC chromatograms of Agathosma leaf surface acetone extracts

Det 168-350nm
A. betulina DO51-c (LSF) 40-100%MeOH
Det 168-350nm
16.150
Det 168-350nm
Retention Time
8.350
250
200
26.600
25.017
25.217
24.067
24.200
24.450
22.633
22.933
21.567
22.167
20.700
21.017
18.717
20.017
20.217
16.817
17.267
17.600
17.800
18.033
14.567
15.033
15.350
12.733
13.133
13.550
13.833
12.267
9.767
10.067
9.250
6.900
7.117
6.000
5.033
2.417
2.633
3.150
3.383
3.817
4.117
4.517
50
11.133
11.367
11.783
10.533
8.800
100
15.667
14.300
19.183
mAU
150
10
12
14
16
18
20
22
24
26
28
Minutes
Fig. 3C-1: HPLC-PDA chromatogram for the leaf surface acetone extract of Agathosma
betulina at 350 nm. The black chromatogram represents sample AB 51b L, the blue
chromatogram represents sample AB 51c L and the green chromatogram
represents a hesperidin standard.
Det 168-350nm
A. betulina DO51-f (LSF) 40-100%MeOH
Det 168-350nm
16.100
Det 168-350nm
A. betulina DO51-e (LSF) 40-100%MeOH
Retention Time
250
8.283
200
19.167
mAU
14.233
150
26.617
24.033
24.433
24.633
25.033
25.233
22.600
22.900
20.683
20.983
21.233
21.533
21.917
22.133
20.000
20.200
18.733
17.233
17.583
18.017
16.450
15.317
15.617
13.800
13.150
13.517
12.233
12.700
11.117
11.350
11.767
10.017
10.483
8.750
9.217
6.850
7.067
5.933
4.983
3.383
3.800
4.100
4.483
2.400
2.683
50
16.800
100
10
12
14
16
18
20
22
24
26
28
Minutes
betulina at 350 nm. The black chromatogram represents sample AB 51e L, the blue
chromatogram represents sample AB 51f L and the green chromatogram
represents a hesperidin standard.
Appendix 3
HPLC chromatograms
Det 168-350nm
A. betulina BMVW 4309-b (40-100% MeOH)
Det 168-350nm
A. betulina BMVW 4309-c (40-100% MeOH)
Det 168-350nm
16.217
Det 168-350nm
A. betulina BMVW 4309-a (40-100% MeOH)
90
Retention Time
16.867
80
70
60
40
26.633
24.000
24.350
24.533
24.717
25.067
25.267
25.700
26.100
22.167
22.667
23.033
21.600
19.950
20.267
20.733
17.267
17.583
18.033
18.300
18.567
18.833
19.083
19.267
14.367
13.117
13.350
13.917
11.183
11.850
12.067
6.917
7.117
6.033
5.550
4.467
4.983
3.317
3.450
3.683
3.917
1.900
2.133
2.417
2.833
10
9.500
9.850
8.983
20
10.533
10.133
8.400
30
15.433
15.750
mAU
50
-10
2
10
12
14
16
18
20
22
24
26
28
Minutes
Fig. 3C-3: HPLC-PDA chromatogram for the leaf surface acetone extract of Agathosma ovata
at 350 nm. The black chromatogram represents sample AO 4309a L, the blue
chromatogram represents sample AO 4309b L, the brown chromatogram
represents sample AO 4309c L and the green chromatogram represents a
hesperidin standard.
Det 168-350nm
A betulina BMVW 4310-b (40-100%MeOH)
Det 168-350nm
A betulina BMVW 4310-c (40-100%MeOH)
16.200
Det 168-350nm
A. betulina BMVW 4310-a (40-100% MeOH)
Retention Time
225
Det 168-350nm
200
175
150
mAU
125
14.350
19.233
100
75
26.617
25.050
25.250
24.567
23.983
22.633
22.800
23.250
23.467
20.883
21.033
21.400
21.583
21.950
22.133
19.933
20.250
18.817
17.700
18.067
18.317
16.850
15.417
15.733
13.100
13.300
12.050
11.150
11.517
9.617
9.817
10.117
10.550
8.383
8.833
6.933
7.133
5.650
6.033
4.967
2.117
2.400
2.617
25
3.350
3.800
4.200
4.433
50
10
12
14
16
18
20
22
24
26
28
Minutes
betulina at 350 nm. The black chromatogram represents sample AB 4310a L, the
blue chromatogram represents sample AB 4310b L, the brown chromatogram
represents AB 4310c L and the green chromatogram represents a hesperidin
standard.
10
Appendix 3
HPLC chromatograms
Appendix 3D: HPLC analyses of Sutherlandia polar leaf extracts

Table 3D-1:
Flavonoid variation in Sutherlandia species as determined by HPLC-PDA
SAMPLE
S. frutescens
SF 1(b)
SF 1(g)
SF 2(a)
SF 2(b)
SF 6(b)
SF 18(b)
SF 18(c)
S. humilis
SH 25(c)
SFI 18(a)
SFI 18(b)
SFI 14(a)
SFI 14(b)
SFI 12(b)
SFI 12(c)
SFI 9(c)
SFI 3668(b)
SFI 3668(c)
S. microphylla
SMi 5(e)
SMi 3(c)
SMi 7(a)
SMi 24(b)
SMi 24(d)
SMi 26(a)
SMi 20(a)
SMi C(a)
SMi C(d)
SMi C(e)
S. Montana
SMo 22(a)
SMo 22(b)
SMo 22(c)
S. tomentosa
ST 13(a)
ST 13(c)
ST 13(e)
ST 13(f)
ST 13(g)
ST 13(h)
ST 3669(b)
ST 3669 (f)
11.35
12.38
RETENTION TIMES (Rf)(minutes)

12.75
12.83
13.55
14.14
2
3
2
4
2
5
3
1
1
1
5
5
2
2
2
2
1
2
1
3
3
2
2
3
1
2
5
5
6
5
5
5
5
1
1
1
1
1
1
1
1
1
4
2
2
1
1
1
1
1
5
5
5
3
3
3
2
5
6
6
7
1
3
2
3
3
1
4
3
3
3
3
3
3
4
3
3
1
2
1
3
3
3
2
2
2
2
6
6
5
6
6
1
3
1
1
2
3
3
3
2
3
2
3
1
2
4
4
4
1
4
4
4
2
4
4
5
5
2
5
5
4
2
5
4
4
4
3
4
4
1
1
4
3
4
4
4
2
3
3
4
4
5
4
3
3
3
1
1
1
2
2
2
6
4
4
4
4
5
1
2
2
3
3
1
4
4
4
4
5
5
5
2
7
7
6
5
4
14.46
Relative quantities of flavonoids given as numbers (1-7) where 1 = highest level, i.e. main flavonoid, and
7 = present, in lowest quantity relative to the other flavonoids in the extract
11
Appendix 3
Table 3D-2:
HPLC chromatograms
Triterpene variation (qualitative only) determined from polar extracts of Sutherlandia species by means of HPLC-PDA
Species / Locality
Isolated:
16.32
16.55
16.72
SU1
16.82
17.67
17.90
SU4
18.05
Retention time (min)

SU5
SU6
18.27 18.47 19.28
SU2
19.58
19.75
19.92
20.18
20.35
22.50
22.87
S. speciosa
Namibia Aus
Richtersveld
Kamiesberg
S. frutescens
Olifantshoek (Northern
Cape)
Gamsberg (Northern Cape)
Vanrhynsdorp
Camps Bay
S. humilis
Uniondale
Barrrydale
S. frut. var. incana
Struisbaai
Houtbaai
Bloubergstrand
Aurora
Pearly Beach
S. microphylla
Murrysburg
Bitterfontein
Vanrhynsdorp
Kamiesberg
Unionpoort
Colesberg
S. montana
Wolkberg
Golden Gate
Reitz
S. tomentosa
Bloubergstrand
Stilbaai
12
23.15
23.30
Appendix 4
NMR data
Appendix 4:
NMR data for compounds isolated from Arctopus
Appendix 4A:
Table of 1H and 13C NMR data for manool, ent-trachyloban-19-oic acid and methyl-16-hydroxyent- kaur-11-en-19-oate
position
1
C, mult.
17.7
42.2
4
5
6
33.6
55.6
24.4
38.4
8
9
10
11
148.7
57.2
39.9
19.4
12
41.4
13
14
73.7
145.3
15
111.6
16
17
27.7
106.5
39.1
Compound 1
H (J = Hz)
0.92 (dd, J 12.3, 4.2)

1.69 (m)
1.28 (m)
1.51 (m)
1.10 (m)
1..31 (m)
1.01 (dd, J 12.6, 2.7 Hz)
1.24 (m)
1.65 (m)
1.89 (td, J 12.9, 4.8)
2.31 (ddd, J 12.6, 3.9, 2.4)
1.50 (m)
1.47 (m)
1.21 (m)
1.65 (m)
5.84 (dd, J 17.4, 10.8)
4.98 (dd, J 10.8, 1.2)
5.13 (dd J 17.4, 1.2)
1.20 (s)
4.44 (br s)
4.75 (d J 1.2)
0.80 (s)
0.73 (s)
0.61 (s)
18
33.6
19
21.7
20
14.4
21
* Obscured by overlapping peak
Compound 2
C, mult.
H (J = Hz)
C, mult.
17.2
1.48 (m)
19.0
38.2
1.42 (m*)
38.0
47.2
50.3
22.9
1.23 (d, J 7.8 Hz)

1.43 (m)
38.6
55.8
21.8
37.0
1.67 (m)
41.3
38.4
40.9
53.2
40.9
19.5
20.5
24.2
33.4
50.2
22.4
16.2
20.5
185.6
14.9
1.42 (m*)
1.20 (m)
1.64 (m)
1.86 (ddd, J 14.6, 11.3 and 3.0 Hz)
0.55 (d, J 7.2)
0.81 (dd, J 8.1 and 6.5)
1.13 (m*)
1.13 (d, J 11.4)
2.00 (d, J 11.7)
1.27 (m)
1.37 (m)
1.11 (s)
1.10 (s)
0.94 (s)
40.0
43.2
61.2
43.9
127.0
Compound 4
H (J = Hz)
0.99 (m*)
1.82 (m)
1.41 (m*)
1.85 (dd J 12.4, 3.0)
0.99 (m)
2.15 (m*)
1.07 (br d J 8.2)
1.72 (m*)
1.47 (m)
1.73 (m)
1.39 (d J 3.0)
5.50 (dd J 8.8, 2.9)
132.5
5.88 (t J 8.9)
50.1
34.4
58.6
2.18 (dd J 6.1, 2.9)

1.58 (dd J 12.2, 5.8)
1.83 (dd J 5.8, 3.0)
1.47 (s)
83.8
25.8
1.29 (s)
28.7
177.9
15.3
51.2
1.15 (s)
0.70 (s)
3.60 (s)
Appendix 4
Appendix 4B:
NMR data
NMR spectra for manool
Fig. 4B-1: 1H-spectrum for manool
Fig. 4B-2: 13C-spectrum for manool

2
Appendix 4
NMR data
Fig. 4B-3: HSQC for manool
Fig. 4B-4: HMBC for manool

3
Appendix 4
NMR data
Fig 4B-5: COSY for manool
Fig 4B-6: NOESY for manool

4
Appendix 4
Appendix 4C:
NMR data
NMR spectra for ent-trachyloban-19-oic acid
Fig. 4C-1: 1H-spectrum for ent-trachyloban-19-oic acid
Fig. 4C-2: 13C-spectrum for ent-trachyloban-19-oic acid
Appendix 4
NMR data
Fig 4C-3: HSQC for ent-trachyloban-19-oic acid
Fig 4C-4: HMBC for ent-trachyloban-19-oic acid

6
Appendix 4
NMR data
Fig 4C-5: COSY for ent-trachyloban-19-oic acid
Appendix 4
Appendix 4D:
NMR data
NMR spectra for methyl-16-hydroxy-ent- kaur-11-en-19-oate
Fig. 4D-1: 1H-spectrum for methyl-16-hydroxy-ent- kaur-11-en-19-oate
Fig. 4D-2: 13C-spectrum for methyl-16-hydroxy-ent- kaur-11-en-19-oate

8
Appendix 4
NMR data
Fig. 4D-3: HSQC for methyl-16-hydroxy-ent- kaur-11-en-19-oate
Fig. 4D-4: HMBC for methyl-16-hydroxy-ent- kaur-11-en-19-oate

9
Appendix 4
NMR data
Fig. 4D-5: COSY for methyl-16-hydroxy-ent- kaur-11-en-19-oate
Fig. 4D-6: NOESY for methyl-16-hydroxy-ent- kaur-11-en-19-oate
10
APPENDIX 5
Scientific papers from this study
APPENDIX 5: Scientific papers resulting from this study

Appendix 5A:
Olivier DK, Van Wyk B-E, Van Heerden FR, 2008. The
chemotaxonomic and medicinal significance of phenolic
acids in Arctopus and Alepidea (Apiaceae subfamily
Saniculoideae). Biochemical Systematics and Ecology. 36,
274-279
Appendix 5B:
Olivier DK, Gabrielse VS, Albrecht CF, Van Wyk B-E, Van
Heerden FR, Triterpenoids from Sutherlandia microphylla,
submitted to Journal of Natural Products, accepted with
changes by Journal of Natural Products in August 2007, not
published
Appendix 5C:
Olivier DK, Van Wyk B-E, Albrecht CF, Van Heerden FR, 2009.
SU3, an oxocycloartane diglucoside from Sutherlandia
humilis. Phytochemistry Letters. 2, 123125
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Author's personal copy

Biochemical Systematics and Ecology 36 (2008) 724729
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco
The chemotaxonomic and medicinal signicance of phenolic acids in

Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)
Denise K. Olivier a, *, Ben-Erik van Wyk a, Fanie R. van Heerden b
a
b
Department of Botany and Plant Biotechnology, University of Johannesburg, PO Box 524, Auckland Park, 2006, Johannesburg, South Africa
Department of Chemistry, University of KwaZulu-Natal, Private Bag X01, Scottsville, 3209, Pietermaritzburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2008
Accepted 26 July 2008
The occurrence of (R)-30 -O-b-D-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic

acid in two important South African medicinal plants is reported for the rst time. (R)-30 O-b-D-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identied in
several samples from three species of the genus Arctopus L. (sieketroos) and three species of
the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the
subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-30 -O-b-D-glucopyranosylrosmarinic acid per g dry wt) in
roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as
the identied compounds are all known for their antioxidant activity, with rosmarinic acid
further contributing to a wide range of biological activities. Furthermore, we conrm the
idea that (R)-30 -O-b-D-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker
for the subfamily Saniculoideae.
2008 Elsevier Ltd. All rights reserved.
Keywords:
(R)-30 -O-b-D-Glucopyranosylrosmarinic acid
Rosmarinic acid
Caffeic acid
Saniculoideae
Apiaceae
Arctopus
Alepidea
Antioxidant
1. Introduction
The discovery of (R)-30 -O-b-D-glucopyranosylrosmarinic acid by Le Claire et al. (2005), together with other phenolic acids
such as rosmarinic acid and chlorogenic acid in many Eryngium species, as well as in Sanicula europaea L. (all from the
Apiaceae subfamily Saniculoideae), led to the proposal that the glycoside may be a chemotaxonomic marker for the Saniculoideae. While rosmarinic acid is commonly found in a wide range of species from different families, its glycosylated
derivative, (R)-30 -O-b-D-glucopyranosylrosmarinic acid, seems to be unique to the Saniculoideae. It is not known from any
other higher plants but has been isolated from cell cultures of a hornwort, Anthoceros agrestis Paton (Vogelsang et al., 2006).
Rosmarinic acid (2) occurs in numerous medicinal plants, especially of the Boraginaceae, Lamiaceae and Apiaceae, and is
known for its antioxidant, antiphlogistic, astringent, anti-inammatory, antimutagenic, antibacterial and antiviral activities
(Burger and Wachter, 1998; Parnham and Kesselring, 1985; Petersen and Simmonds, 2003; Tewtrakul et al., 2003).
Based on molecular evidence, the southern African genera Alepidea and Arctopus were recently shown to be basally
o and Downie, 2007). It was shown that Alepidea and Arctopus
divergent within a broadened concept of Saniculoideae (Calvin
are the rst two (earliest) lineages within the phylogeny of the subfamily. Species of both genera are very important in
traditional medicine (see later). Roots of Alepidea and Arctopus contain kaurene-type diterpenoids (Holzapfel et al., 1995),
known for their antimicrobial, anti-parasitic and anti-inammatory activity (Ghisalberti, 1997). No other compounds have
hitherto been identied from these genera.
* Corresponding author. Tel.: 27 11 559 6158; fax: 27 11 559 6425.

E-mail address: [email protected] (D.K. Olivier).
0305-1978/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2008.07.003

D.K. Olivier et al. / Biochemical Systematics and Ecology 36 (2008) 724729
725
During routine chromatographic analyses of various medicinal plants used as traditional tonics, extracts of Arctopus and
Alepidea showed similar and highly characteristic compounds in HPLC chromatograms. The purpose of this paper is to report
on the presence of phenolic acids in samples of the two genera. We also present supporting evidence for the chemotaxonomic
value of (R)-30 -O-b-D-glucopyranosylrosmarinic acid (1) in the subfamily Saniculoideae.
2. Materials and methods
2.1. General procedures
TLC analysis of the phenolic acids was performed with silica gel 60 F254 plates (Merck) and detected with 5% ethanolic
sulfuric acid and 1% ethanolic vanillin (heated to 100 C). A Shimadzu 10A HPLC instrument with a Phenomenex RP C18
column (150 4.6 mm, 5 mm), a binary gradient system and a photodiode array (PDA) detector was used for routine analyses.
UV spectra were recorded between 210 and 400 nm. Solid phase extraction was used for sample preparation (Chromabond
sorbens (ec f), Machery Nagel). C18 reversed-phase column chromatography was performed on a Machery Nagel Chromabond
ec f column (2.5 cm 21.0 cm). Kieselgel GF254 (15 mm, Merck) was used for ash chromatography. 1H-NMR and 13C-NMR
experiments were performed on a Varian Unity Inova spectrometer operating at 300 MHz (1H) and 75 MHz (13C) in DMSO-d6
for 1 and in CD3OD for 2, with the solvents used as internal standards in both cases. The UPLC-MS instrumentation consisted
of a Waters Acquity Ultra Performance LC system equipped with PDA, a Waters LCT Premier mass spectrometer and an
Acquity UPLC BEH C18 column (17 mm, 2.1 100 mm). ESI in negative ionization mode was used to determine the accurate
masses of the isolated phenolic acids (1 and 2), and to conrm the identity of compound 3.
2.2. Plant material

Voucher specimens were deposited in the herbarium of the Department of Botany and Plant Biotechnology at the
University of Johannesburg (JRAU): AdC, A. de Castro; ARM, A.R. Magee; BEVW, B.-E. van Wyk; DKO, D.K. Olivier; JSB, J.S.
Boatwright; PW, P. Winter. The samples studied are listed in Table 1 together with provenances: Arctopus echinatus: ARE1, PW
& BEVW 170; ARE2, ARM & JSB 15; ARE3, BEVW s.n.; ARE4, BEVW 4128(ac); ARE5, ARM & JSB 4; ARE6, DKO 66(a,b). Arctopus
dregei: ARD7, ARM & JSB 31; ARD8, ARM & JSB 2(ac). Arctopus monacanthus: ARM9, BEVW 3522; ARM10, BEVW 4161(a);
ARM11, BEVW 4141(a). Alepidea amatymbica: ALA12, PW 252. Alepidea comosa: ALC13, AdC 357. Alepidea longifolia: ALL14, AdC
357(a,b).
2.3. Phenolic acid variation study

Air-dried samples were extracted with boiling water (0.3 g in 3 mL), treated with ethanol to remove alcohol precipitable
solids and centrifuged. The dry supernatant was re-suspended in 1 mL of 50% MeOH/H2O and loaded onto a SPE C18RP
cartridge. The rosmarinic acid and its derivatives were selectively extracted with 2 mL of 30% MeOH/H2O.
The presence of the phenolic acids was conrmed by TLC in (a) n-BuOH/H2O/AcOH (4:1:2) and (b) EtOAc/MeOH/H2O/
AcOH (60:15:15:1) (Le Claire et al., 2005). Rf-values of 0.23 and 0.50 for 1 and 2 (system b) corresponded to those of Le Claire
et al. (2005). The approximate concentrations of the acids were determined by HPLC analyses (ow rate 1 mL/min, linear
gradient of 40100% MeOH/1% AcOH over 30 min). Three well-resolved peaks were observed at 5.67 min, 8.23 min and
10.13 min for 3, 1 and 2, respectively (see Table 1).
2.4. Identication of compounds 1, 2 and 3

Compounds 1 (5.2 mg) and 2 (13.3 mg) were isolated from root material of A. monacanthus (ARM9, 24.6 g), using phase
separation (30% MeOH/H2O, absorbed on a C18 reversed-phase column, eluted with a MeOH/H2O gradient (50100%)), followed by repeated ash column chromatography (n-BuOH/MeOH/H2O (4:1:1)). Compound 1 is unstable (Le Claire et al.,
2005) and required further purication.
The structures of 1 and 2 were determined by utilizing 1D 1H-NMR and 13C-NMR experiments and COSY, HMBC and HSQC
experiments. The spectroscopic data are summarized in Table 2.
The identity of compound 3 was determined by means of comparison of the mass and UV spectra, as well as the retention
time of the compound with that of an authentic sample of caffeic acid (3). HPLC with both PDA and MS detection were used for
this purpose. A gradient solvent system was used for LC/MS consisting of (A) acetonitrile and (B) 0.1% aqueous formic acid.
Gradient: start, A 5%, 3 min; A 50%, 10 min; A 95%, 10 min; A 5%, 10 min; hold for 2 min.
2.4.1. (R)-30 -O-b-D-Glucopyranosylrosmarinic acid (1)
LC-online UV: lmax 329 nm, 251 nm. HR-TOF-ESIMS, m/z 521.1284 [M H], Calculated for C24H25O13: 521.1295. 1Hand 13C-NMR (1H 300 MHz; 13C 75 MHz, DMSO-d6): see Table 2.

726
Table 1
Presence and approximate levels (single measurements) of (R)-30 -O-b-D-glucopyranosylrosmarinic acid (1), rosmarinic acid (2) and caffeic acid (3) in
different species of Arctopus and Alepidea as determined by HPLC
Sample no.
Arctopus echinatus
ARE1a
Locality
1 (mg/g)
2 (mg/g)
3 (mg/g)
2.6
15.5
0.6
0.7
8.4
0.2
1.7
0.2
2.5
0.2
2.4
n.d.c
15.3
1.3
10.1
2.8
3.2
0.8
4.0
5.6
1.1
7.9
3.3
0.8
0.3
1.1
0.4
0.4
0.4
0.2
0.3
0.5
0.5
ARE2a
ARE3a
ARE4(a)a
ARE4(b)a
ARE4(c)a
ARE5a
ARE6(a)b
ARE6(a)a
ARE6(b)b
ARE6(b)a
Du Toits
Kloof Pass
Hermanus
Pakhuis Pass
Nieuwoudtville
Nieuwoudtville
Nieuwoudtville
Rondeberg
Port Alfred
Port Alfred
Port Alfred
Port Alfred
Arctopus dregei
ARD7a
ARD8(a)a
ARD8(b)a
ARD8(c)a
Malmesbury
Rondeberg
Rondeberg
Rondeberg
0.5
4.0
3.1
6.5
0.3
11.3
5.0
14.4
0.4
1.8
1.5
1.2
Arctopus monacanthus
ARM9a
ARM10a
ARM11a
Gifberg
Gifberg
Citrusdal
3.9
3.2
2.4
13.1
1.9
2.9
0.4
3.6
1.3
Alepidea amatymbica
ALA12b
ALA12a
Mphendle
Mphendle
n.d.
0.5
7.3
9.6
0.4
0.4
Alepidea comosa
ALC13a
Drummond
0.9
6.5
0.3
Alepidea longifolia
ALL14b
ALL14a
Gods Window
Gods Window
0.6
0.5
9.5
14.0
1.0
0.9
a
b
c
Roots used for extraction.

Leaves used for extraction.
Not detected at a detection limit of 0.1 mg/g.
2.4.2. Rosmarinic acid (2)

LC-online UV: lmax 330 nm, 248 nm. Negative HR-TOF-ESIMS, m/z 359.0772 [M H]. Calculated for C18H15O8:
359.0767. 1H- and 13C-NMR (1H 300 MHz; 13C 75 MHz, CD3OD): see Table 2.
2.4.3. Caffeic acid (3)
LC-online UV: lmax 323 nm, 245 nm. Negative ESIMS: m/z 179 [M H].
3. Results and discussion

3.1. Isolation and identication of main phenolic compounds
The presence of rosmarinic acid (2), its glucoside (1), as well as caffeic acid (3) in Arctopus and Alepidea is reported here for
the rst time. The initial TLC screening of aqueous extracts of the powdered dried rhizomes of three Arctopus species collected
in different localities revealed three pink zones, indicating the presence of phenolic compounds. Rosmarinic acid (2) and its
glucosylated derivative (1) were successfully isolated. Their identities were determined as (R)-30 -O-b-D-glucopyranosylrosmarinic acid (1) and its aglycone, rosmarinic acid (2) (see Fig. 1) by means of 1D and 2D NMR which compared
favorably with the NMR data from the literature (Le Claire et al., 2005; Woo and Piao, 2004). Long-range 1H,13C correlations
observed in a HMBC experiment conrmed the identity of 1 as (R)-30 -O-b-D-glucopyranosylrosmarinic acid and not the (R)-3O-b-D-glucopyranosylrosmarinic acid isomer by clear correlation between the anomeric proton of the glucopyranosyl unit
and the oxygen bearing C-30 on the dihydroxyphenyllactic acid ring. LC/MS and HPLC-PDA analyses proved that compound 3,
contrary to expectation (Le Claire et al., 2005), was caffeic acid (m/z 179 [M H]; UV lmax 323 nm, 245 nm) and not
chlorogenic acid.
The yields of the three compounds in the different species vary greatly within populations and between localities.
Compound 2 is the major constituent in most of the aqueous extracts, followed by 1, and 3 being the minor constituent in

727
Table 2
1
H and 13C NMR spectroscopic data for (R)-30 -O-b-D-glucopyranosylrosmarinic acid (1) and rosmarinic acid (2)
Position
R-()-30 -O-b-D-Glucopyranosyl rosmarinic

acid (1)
dH (DMSO-d6)
Rosmarinic acid moiety
1
2
7.05 (d, J 1.5 Hz)
3
4
5
6.75 (d, J 9.0 Hz)
6
6.99 (dd, J 9.0 and 1.5 Hz)
7
7.45 (d, J 15.0 Hz)
8
6.25 (d, J 15.0 Hz)
9
10
6.74 (brs)
20
30
40
7.01 (d, J 9.0 Hz)
50
6.65 (dd, J 7.5 and 1.5 Hz)
60
3.04 (dd, J 13.5 and 3.0 Hz)
70
2.95 (dd, J 13.5 and 9.0 Hz)
5.07 (dd, J 7.5 and 6.0 Hz)
80
90
Glucose moiety
100
200
300
400
500
600
a
4.63 (d, J 6.0 Hz)

3.28 (m)
3.28 (m)
3.13 (m)
3.28 (m)
3.75 (m)a
3.45 (m)a
Rosmarinic acid (2)
dC (DMSO-d6)
125.6
114.9
148.9
146.3
116.0
122.1
146.5
113.4
166.2
131.4
116.5
145.8
144.4
117.2
120.5
36.3
72.9
171.1
dH (MeOH-d4)
7.05 (d, J 1.8 Hz)
6.76 (d, J 8.1 Hz)

6.99 (dd, J 8.4 and 2.1, Hz)
7.44 (d, J 15.9 Hz)
6.23 (d, J 15.6 Hz)
6.67 (d, J 2.1 Hz)
6.63 (d, J 8.1 Hz)

6.52 (dd, J 8.1 and 2.1 Hz)
2.99 (dd, J 14.4 and 4.2 Hz)
2.88 (dd, J 14.4 and 8.4 Hz)
5.01 (dd, J 8.4 and 4.2 Hz)
dC (MeOH-d4)
125.5
114.9
145.8
148.8
115.9
121.8
148.8
113.5
166.1
127.7
116.8
145.1
144.1
115.2
120.2
36.3
73.2
171.3
102.3
73.5
76.0
70.0
77.3
60.9
Obscured by solvent peak.
most cases. A comparison of leaves and roots from the same plant of Arctopus echinatus (ARE6(a) and (b)) showed that the
glycoside 1 is practically absent from the leaves.
3.2. Chemotaxonomic signicance of phenolic acids
Le Claire et al. (2005) suggested that the rosmarinic acid glucoside (1) may be a chemotaxonomic marker for the subfamily
Saniculoideae of the family Apiaceae as this compound was detected in several species of the genus Eryngium L. and in
Sanicula europaea L., both members of the tribe Saniculeae of the subfamily Saniculoideae. Compound 1 has not been reported
from any other higher plants (but curiously, it was also found in cell cultures of a hornwort; Vogelsang et al., 2006). This
compound, together with compounds 2 and 3, were present in root samples of all three Arctopus species and three Alepidea
species. While rosmarinic acid (2) and caffeic acid (3) are commonly found in medicinal plants, the occurrence of 1 in both
o and Downie, 2007) that the two genera are
Arctopus and Alepidea agrees with molecular systematic evidence (e.g. Calvin
related. (R)-30 -O-b-D-Glucopyranosylrosmarinic acid is thus conrmed to be a chemotaxonomic marker for the Saniculoideae,
tribe Saniculeae. It may be interesting to extend this study to other genera of this tribe for which data on the presence of 1 is
o and Downie, 2007).
currently not available, as well as for the newly described tribe Steganotaeniae (Calvin
3.3. Medicinal signicance of phenolic acids
Species of Arctopus and Alepidea are very important in southern African traditional medicine (De Castro and Van Wyk,
1994; Hutchings et al., 1996; Magee et al., 2007; Van Wyk et al., 2000). Rhizomes of Alepidea amatymbica Eckl. & Zeyh. (known
as ikhathazo in Zulu) and other species are widely used to treat respiratory ailments during the winter period and are
commonly sold at traditional muthi markets (De Castro and Van Wyk, 1994; Hutchings et al., 1996). Tubers of Arctopus
echinatus and A. monacanthus (sieketroost or platdoorn in Dutch) are well known as important Khoi-San and Cape Dutch
traditional medicines and general tonics, used for a wide range of ailments that includes venereal diseases and respiratory
ailments (Magee et al., 2007). They are also used by epilepsy sufferers and have sedative properties (Stafford et al., 2005). The
medicinal uses of Arctopus and Alepidea species were hitherto ascribed to the presence of kaurene-type diterpenoids
(Holzapfel et al., 1995; Van Wyk et al., 2000) so that the presence of phenolic acids adds a new dimension to our understanding of the medicinal value and possible efcacy of these plants. As mentioned in Section 1, the medicinal value of

728
O
2'
7'
3'
8'
9'
1'
4'
HO
6'
5'
OH
HO
HO
OH
1''
2''
3''
3
2
5''
4''
HO
OH
4
8
7
6''
1: R =
OH
2: R = H
OH
HO
OH
O
Fig. 1. Structures of phenolic acids: 1, (R)-30 -O-b-D-glucopyranosylrosmarinic acid; 2, rosmarinic acid; 3, caffeic acid.
phenolic acids (and especially rosmarinic acid) are ascribed to antioxidant, antiphlogistic, astringent, anti-inammatory,
antimutagenic, antibacterial and antiviral activities (Burger and Wachter, 1998; Parnham and Kesselring, 1985; Petersen and
Simmonds, 2003). The traditional uses of Alepidea and Arctopus species (especially as general tonics and for treating respiratory ailments) are therefore strongly supported by the conrmed presence of relatively high levels of phenolic acids in the
rhizomes and tubers (i.e., the parts that are used medicinally). It is interesting to note that the European Sanicula europaea,
Eryngium campestre L. and Eryngium planum L. are used in much the same way (Burger and Wachter, 1998; Van Wyk and
Wink, 2004). In the case of Sanicula europaea (sanicle herb, saniculae herba), the leaves are mainly used medicinally and it is
therefore noteworthy that Le Claire et al. (2005) reported high levels of rosmarinic acid and its glycoside in leaves of this
species. The German Commission E allows for sanicle herb the indication mild cattarhs of the respiratory tract (Blumenthal
et al., 1998). The medicinal members of the subfamily Saniculoideae therefore represent an interesting example of related and
chemically similar plants that are used in different parts of the world for the same or similar treatments.
4. Conclusion
The presence of phenolic acids, and especially rosmarinic acid and its glucoside, are useful chemotaxonomic markers for
members of the tribe Saniculeae (subfamily Saniculoideae of the Apiaceae). The close relation between the morphologically
anomalous Arctopus and Alepidea and the genera Eryngium and Sanicula suggested by molecular systematic evidence is
supported by the unique presence of the rosmarinic acid glycoside in these genera. Phenolic acids were not previously known
from Arctopus and Alepidea, so that their presence provides new supporting evidence for the traditional medicinal uses of
these African plants, especially when the similarity of uses of the European members of the same tribe and subfamily are
considered.
Acknowledgements
This research was nancially supported by the National Research Foundation and the University of Johannesburg.
References
Blumenthal, M., Busse, W.R., Goldberg, A., Gruenwald, J., Hall, T., Riggins, C.W., Rister, R.S. (Eds.), 1998. The Complete German Commission E Monographs.
American Botanical Council, Austin, TX, p. 200.
Burger, A., Wachter, H. (Eds.), 1998. Hunnius Pharmazeutisches Worterbuch, eighth ed. Walter de Gruyter, Berlin, pp. 482, 1199, 1222.
o, C.I., Downie, S.R., 2007. Circumscription and phylogeny of Apiaceae subfamily Saniculoideae based on chloroplast DNA sequences. Mol. Phylogenet.
Calvin
Evol. 44, 175191.
De Castro, A., Van Wyk, B.-E., 1994. Diagnostic characters and geographical distribution of Alepidea species used in traditional medicine. S. Afr. J. Bot. 60,
345350.
Ghisalberti, E.L., 1997. The biological activity of naturally occurring kaurane diterpenes. Fitoterapia 4, 303325.
Holzapfel, C.W., Van Wyk, B.-E., De Castro, A., Marais, W., Herbst, M., 1995. A chemotaxonomic survey of kaurene derivatives in the genus Alepidea
(Apiaceae). Biochem. Syst. Ecol. 23, 799803.

729
Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants. Natal University Press, Pietermaritzburg, South Africa, p. 223.
Le Claire, E., Schwaiger, S., Banaigs, B., Stuppner, H., Gafner, F., 2005. Distribution of a new rosmarinic acid derivative in Eryngium alpinum L. and other
Apiaceae. J. Agric. Food Chem. 53, 43674372.
Magee, A.R., Van Wyk, B.-E., Van Vuuren, S.F., 2007. Ethnobotany and anti-microbial activity of sieketroos (Arctopus species). S. Afr. J. Bot. 73, 159162.
Parnham, M.J., Kesselring, K., 1985. Rosmarinic acid. Drugs Future 10, 756757.
Petersen, M., Simmonds, M.S.J., 2003. Molecules of interest: rosmarinic acid. Phytochemistry 62, 121125.
Stafford, G.I., Jager, A.K., Van Staden, J., 2005. Activity of traditional South African sedative and potentially CNS-acting plants in the GABA-benzodiazepine
receptor assay. J. Ethnopharmacol. 100, 210215.
Tewtrakul, S., Miyashiro, H., Nakamura, N., Hattori, M., Kawahata, T., Otake, T., Yoshinaga, T., Fujiwara, T., Supavita, T., Yuenyongsawad, S., Rattanasuwon, P.,
Dej-Adisai, S., 2003. HIV-1 integrase inhibitory substances from Coleus parvifolius. Phytotherapy Res. 17, 232239.
Van Wyk, B.-E., Wink, M., 2004. Medicinal Plants of the World. Briza Publications, Pretoria, South Africa, p. 288.
Van Wyk, B.-E., Van Oudtshoorn, B., Gericke, N., 2000. Medicinal Plants of South Africa, second ed. Briza Publications, Pretoria, South Africa, pp. 38-39,
42-43.
Vogelsang, K., Schneider, B., Petersen, M., 2006. Production of rosmarinic acid and a new rosmarinic acid 30 -O-b-D-glucoside in suspension cultures of the
hornwort Anthoceros agrestis Paton. Planta 223, 369373.
Woo, E.R., Piao, M.S., 2004. Antioxidative constituents from Lycopus lucidus. Arch. Pharm. Res. 27, 173176.
Phytochemistry Letters 2 (2009) 123125
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
SU3, an oxocycloartane diglucoside from Sutherlandia humilis

Denise K. Olivier a,*, Carl F. Albrecht a, Ben-Erik van Wyk a, Fanie R. van Heerden b
a
b
Department of Botany and Plant Biotechnology, University of Johannesburg, Johannesburg, P.O. Box 524, Auckland Park, 2006, South Africa
School of Chemistry, University of KwaZulu-Natal, Private Bag X01, Scottsville, 3209, Pietermaritzburg, South Africa
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 2 February 2009
Received in revised form 24 March 2009
Accepted 24 March 2009
Available online 7 April 2009
The structure of a new cycloartane-type triterpene glycoside was determined as 24,25-O-b-Ddiglucopyranosyl-6a-hydroxycycloart-3-one (SU3) by spectroscopic methods. This is the rst
cycloartane diglycoside reported from the genus Sutherlandia (an important South African traditional
medicine and general tonic known as cancer bush). It was isolated from a dwarf form of S. frutescens,
currently known as Sutherlandia humilis.
2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Sutherlandia humilis
Cancer bush
Saponin
Oxocycloartane diglucoside
NMR
1. Introduction
Sutherlandia humilis E.Phillips & R.A.Dyer is one of six closely
related species of the genus Sutherlandia R.Br. ex W.T.Aiton (family
Fabaceae) that is endemic to southern Africa and is commonly
known as cancer bush (Phillips and Dyer, 1934; Germishuizen and
Meyer, 2003). Cancer bush is traditionally used for a myriad of
indications, ranging from poor appetite to the prevention and
treatment of cancer. The taxonomy, ethnobotany, chemistry and
pharmacological properties of cancer bush was recently thoroughly revised and summarized by Van Wyk and Albrecht (2008).
There are six species of cancer bush which will soon be reduced to
two (Moshe, 1998) and S. humilis is often considered to merely
represent a dwarf form of the variable S. frutescens (L.) R.Br. Users of
cancer bush often do not differentiate between species.
Free amino acids, the cyclitol pinitol and avonoid glycosides
have been detected in commercially used S. microphylla Burch. ex
DC (Van Wyk and Albrecht, 2008). We have previously isolated
(Gabrielse, 1996) and later identied two major cycloartane
glycosides from this taxon which we called SU1 (Fig. 1) and SU2
(Olivier, Van Heerden, Van Wyk and Albrecht, unpublished work).
Whilst Van Wyk and Albrecht (2008) did not give the exact
stereochemistry of the hydroxyl groups on C-7 and C-24 of SU1,
Olivier claried this along with the structure of SU2, and found it to
be the same as the structures recently published as Sutherlandio-
* Corresponding author. Tel.: +27 11 559 6158; fax: +27 11 559 6425.
E-mail address: [email protected] (D.K. Olivier).
sides B and A, respectively (Fu et al., 2008). Herewith, we report on

the isolation and characterization of a novel triterpenoid diglucoside, SU3, from S. humilis (Fig. 1).
2. Results and discussion
TLC analysis of the methanolic leaf extract of S. humilis revealed
the presence of triterpenoids SU1 (1) and SU2, also known as
Sutherlandiosides B and A, respectively (Fu et al., 2008). These two
compounds seem to be present in variable concentrations in many,
but not all, Sutherlandia samples, as was evident from a TLC and
HPLC variation study on methanol extracts of a large number of
geographically representative provenances of all the species.
However, the major compound isolated from S. humilis was a
triterpenoid of higher polarity, called SU3 (2).
The ESI-TOF-MS spectrum of SU3 (2) exhibited a [M + Na]+ peak
at m/z 821.4665, corresponding to a molecular formulae of
C42H70O14. The NMR data gave evidence for the presence of two
glucopyranosyloxy moieties and a triterpene aglycone containing
six tertiary methyls, one secondary methyls, a ketone, two
secondary and one tertiary oxygen-bearing carbons and two
methylene protons (dH 0.68, 0.49 J = 3.9 Hz) characteristic of the
19-CH2 of a cycloartane. Correlations observed between the latter
two protons and neighbouring carbons in a CIGAR2J3J experiment
enabled us to assign C-1, 5, 8, 9, 10 and 11 in the 13C NMR spectrum.
The methyl protons assigned to C-28 and C-29 showed long range
1
H, 13C coupling to each other, C-5 and the ketone carbon, thereby
establishing a 3-oxo functionality. Correlations observed for H-5
(dH 2.08, d, J = 9.9 Hz) conrmed the assignments of C-1, 4, 10, 19,
1874-3900/$ see front matter 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2009.03.005
D.K. Olivier et al. / Phytochemistry Letters 2 (2009) 123125
124
Fig. 1. Chemical structures of compounds 1 and 2.
28 and 29 and the presence of a hydroxyl function on C-6. The

assignment of the 6eq-OH is corroborated by correlations observed
in the COSY spectrum and the coupling pattern observed for H-6.
The NMR assignments of the A and B rings of SU3 (2) are in good
agreement with literature data for 3-oxocycloartanes (Inanda
et al., 1995) and 6a-hydroxycycloartanes (Radwan et al., 2004),

respectively.
The C-17 side chain of SU3 (2) is closely related to that of SU1 (1)
(Fu et al., 2008), the difference being that in 2 both the hydroxyls at
C-24 and C-25 are glucosylated. HMBC correlations between the
Table 1
NMR spectroscopic data (300 MHz, methanol-d4) for SU1 (1) and SU3 (2).
Position
SU1 (1)
dC, mult.
1
213.0, qC
48.7, CH2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
78.8,
40.1,
38.6,
30.8,
69.3,
51.2,
30.6,
40.4,
29.0,
33.8,
46.4,
50.5,
34.4,
28.7,
17
18
19
53.0, CH
15.6, CH3
24.6, CH2
20
21
22
23
24
25
26
27
28
29
30
10
20
30
40
50
60
38.0,
19.3,
35.1,
29.3,
79.5,
81.9,
23.8,
21.3,
24.8,
21.7,
18.9,
98.6,
75.1,
78.1,
71.6,
77.7,
62.6,
100
200
300
400
500
600
a
CH
qC
CH
CH2
CH
CH
qC
qC
CH2
CH2
qC
qC
CH2
CH2
CH
CH3
CH2
CH2
CH
qC
CH3
CH3
CH3
CH3
CH3
CH
CH
CH
CH
CH
CH2
SU3 (2)
dH (J = Hz)
a: 3.01 dd (14.3, 4.5)

b: 2.41 dd (14.3, 4.5)
3.75 t (4.8)
2.48
1.90
3.75
2.12
dd (14.0, 3.6)
m, 1.28 m
m
d (3.6)
1.88 m
1.67 m, 1.53 d (3.0)
1.39 td (10.5, 2.1)

1.90 m
1.68 td (9.3, 2.2)
1.01 s
a: 0.78 d (4.5)
b: 1.47 d (4.5)
1.48 m
0.96 d (6.3)
1.90 m
1.68 m
3.36 br d (9.3)
1.29 s
1.26 s
1.04 s
0.97 s
1.01 s
4.56 d (7.8)
3.21 br t (7.8)
3.35 t (8.6)
3.38 m
3.32 m
a: 3.69 dd (11.9, 5.1)
b: 3.87 dd (11.7, 2.1)
dC, mult.
dH (J = Hz)
COSYa
CIGARa
32.7
1.42, m
2.14, m
2.44 td (14.4, 12.0)
2.72 td (14.1, 11.1)
1b
1a
2b
2a
5, 19a, 19b
36.4
220.0
51.5
54.3
70.7
38.3
49.6
22.3
29.3
27.1
34.0
46.4
49.6
36.9
29.1
53.9
20.0
31.5
37.6
18.9
34.3
29.1
87.5
83.4
24.8
20.0
28.6
20.4
18.9
98.3
75.7
77.8
71.8
77.7
62.8
104.7, CH
75.2, CH
78.5, CH
72.0, CH
77.7, CH
63.3, CH2
COSY and CIGAR correlations are from proton(s) stated to the indicated carbon.
2.08
3.48
1.49
3.36
d (9.9)
td (9.9, 3.9)
m
m
6
5, 7a, 7b
1.13 m
1.72 br d (3.6)
12
11
1.42 m
a: 1.43 m
b: 2.08 br d (9.9)
1.66 d (3.9)
1.05 s
a: 0.49 d (3.9)
b: 0.68 d (3.9)
1.45 t (5.7)
0.94 d (6.3)
1.74 m
1.37 m
3.63 dd (9.0, 1.5)
16 a, 16b
15
15
16a, 16b
17, 21, 22a, 22b

20
20
24
23
1.30 s
1.35 s
1.37 s
1.24 s
1.06 s
4.57 d (7.8)
3.23 t (9.3)
3.35 m
3.32 m
3.31 m
a: 3.68 dd (4.8, 3.3)
b: 3.88 br d (10.5)
4.81 d (7.8)
3.23 t (9.3)
3.51 dd (8.1, 3.9)
3.32 m
3.31 m
a: 3.72 dd (4.8, 3.3)
b: 3.91 br d (9.6)
20
10 , 3 0
40
30 , 50
40 , 60 a, 60 b
50
50
200
100 , 300
400
300 , 500
400 , 600 a, 600 b
500
500
2a, 2b, 28, 29

5, 28, 29
19a, 19b, 28, 29
5, 7
6
7, 18, 19a, 19b
7, 19a, 19b
2a, 2b, 5, 7, 19a, 19b
19a, 19b
18
18, 30
18, 30
30
18, 21
5
21
26, 27, 100

26, 27, 100
27
26
29
5, 28
20
30
20
30 , 50
10 , 40 , 60 a, 60 b
200
300
200
300 , 500
100 , 400 , 600 a, 600 b
D.K. Olivier et al. / Phytochemistry Letters 2 (2009) 123125
anomeric protons of the b-D-glucopyranose units with C-24 and C25 conrmed the position of these sugars. The anomeric 1H NMR
doublet of the C-24 glucosyl moiety is substantially broadened,
most likely because of restricted rotation around the sterically
hindered C-24 glycosidic bond. Therefore, the structure of SU3 was
established as shown in 2.
SU3 (2) is similar in structure to those of SU1 and SU2 (the
Sutherlandiosides B and A previously isolated from S. frutescens)
(Fu et al., 2008) and also to various triterpenoids isolated from
zipek et al., 2005).
Astragalus species (Bedir et al., 1998, 2000; O
These triterpenoids are responsible for the bitter taste and the
amarum effect (Van Wyk and Wink, 2004) of Sutherlandia and may
also provide a rationale for the numerous other medicinal uses that
have been recorded. It was also reported that cycloartanes with
hydroxylation at C-24 and a 3-oxo group (the latter is true for SU3)
exhibit a powerful inhibitory ability toward skin carcinogenesis
(Kikuchi et al., 2007). Further studies on the triterpenoid variation
and biological activity of Sutherlandia triterpenoids will undoubtedly yield interesting results.
3. Experimental section
3.1. General experimental procedures
ESI-MS measurements were obtained on a Waters API Q-TOF
Ultima instrument in positive ionization mode. Optical rotations
were measured with a Jasco DIP-370 digital polarimeter. 1H NMR
and 13C NMR experiments were performed on a Varian Unity Inova
300. C18 reverse phase gel (Chromabond, Machery Nagel) and
silica gel (Kieselgel GF254 15 mm, Merck) were used for column
chromatography and silica gel 60 F254 on aluminum sheets (Merck)
for thin-layer chromatography (TLC). The solvent system used for
TLC was chloroform:methanol:water:acetic acid (60:30:8:6).
Plates were sprayed with 5% ethanolic sulfuric acid followed by
1% ethanolic vanillin and baked for 5 min at 100 8C.
3.2. Plant material
The plant material was collected near Barrydale, South Africa in
September 1996 and identied by B.-E. van Wyk. A voucher
specimen (Palmer 17a) was deposited in the Herbarium, Department of Botany and Plant Biotechnology at the University of
Johannesburg (JRAU).
3.3. Extraction and isolation
SU3 (2) was isolated from a methanolic extract obtained by
stirring 2.8 g of ground dried leaf material in 30 mL of methanol for
18 h. After ltration, the extract was dried under reduced pressure
125
at 40 8C to yield 36.4 mg of extract. This extract was subjected to

ash column chromatography and eluted with a gradient system of
chloroform:methanol:ethyl acetate (65:20:15 and 40:50:10). The
fraction containing 2 was subjected to further ash column
chromatography, this time using chloroform:methanol:water
(60:20:2.5) as solvent system, to yield 2.7 mg of the pure
amorphous compound (Rf: 0.45).
3.4. Compound 2 (SU3)
24,25-O-b-D-Diglucopyranosyl-6a-hydroxycycloart-3-one (2):

white amorphous solid; aD
25 50:0 (ca. 0.28 g/100 mL, MeOH);
positive ESI-TOF-MS: m/z = 821.4665 [M + Na]+ (calc. for
C42H70O14Na: 821.4663); 1H and 13C NMR data, (1H: 300 MHz;
13
C: 75 MHz, CD3OD) see Table 1.
Acknowledgements
Financial support was provided by the National Research
Foundation and the University of Johannesburg.
References
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glycosides from the roots of Astragalus brachypterus and Astragalus microcephalus. J. Nat. Prod. 61, 14691472.
Bedir, E., Pugh, N., Calis, I., Pasco, D.S., Khan, A., 2000. Immunostimulatory effects of
cycloartane-type triterpene glycosides from Astralagus species. Biol. Pharm.
Bull. 23, 827834.
Fu, X., Li, X.-C., Smillie, T.J., Carvalho, P., Mabusela, W., Syce, J., Johnson, Q., Folk, W.,
Avery, M.A., Khan, I.A., 2008. Cycloartyne glycosides from Sutherlandia frutescens. J. Nat. Prod. 71, 17491753.
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The ethnobotany and chemistry of South African traditional

CHAPTER 1: Introduction and hypothesis

CHAPTER 2: The concept of herbs as tonics

Tonics and related concepts (definitions)

Stimulants, immunostimulants and immunomodulators

Tonics in traditional healing cultures

Europe and Asia

2.2.1.2 Ancient Jewish medicine

2.2.1.3 European medicine

2.2.1.4 Northern Eurasian medicine (Slavic countries and Russia)

2.2.1.5 Southern and South-eastern Asian traditional medicine

2.2.1.6 East Asian traditional medicine

Oceania and the Americas

2.2.2.1 Bush medicine (Australian traditional medicine)

2.2.2.2 Rongo Mori (New Zealand traditional medicine)

2.2.2.3 North American Indian and eclectic medicine

2.2.2.4 Meso and South American traditional medicine

2.2.3.1 Islamic medicine (North Africa)

2.2.3.2 Yorb medicine

2.2.3.3 Bantu traditional medicine

2.2.3.4 Khoi-San and Cape Dutch medicine

The concept tonic in the southern African context

CHAPTER 3: Materials and methods

Screening protocol for classes of phytochemicals

Qualitative colour tests

Water extractable compounds

3.3.3.1 Alcohol precipitable solids

3.3.3.2 Amino acids

3.3.3.4 Phenolic glycosides

3.3.3.5 Triterpenoids and steroids

Organic extractable compounds

3.3.4.1 Mono-, sesqui- and diterpenes

3.3.4.2 Flavonoid aglycones (Agathosma species)

Quantitative Analytical Chromatography

3.4.1.1 High performance liquid chromatography (HPLC)

3.4.1.2 Liquid chromatography-mass spectrometry (LC-MS)

High resolution gas chromatography-mass spectrometry (HR-GC-MS)

3.4.2.1 Amino acids

3.4.2.2 GC-MS analyses of Arctopus kaurenes (performed by Witte

Column chromatography (CC)

Preparative thin layer chromatography

Nuclear magnetic resonance spectroscopy (NMR)

Mass spectrometry (MS)

Bitterness taste testing

The ethnobotany of South African traditional tonic plants

4.2.1 Agathosma betulina

4.2.2 Aloe species

4.2.3 Arctopus species

4.2.4 Artemisia afra

4.2.5 Balanites maughamii

4.2.6 Dicoma species

4.2.7 Harpagophytum procumbens

4.2.8 Hypoxis hemerocallidea

4.2.9 Muraltia heisteria

4.2.10 Sutherlandia frutescens

4.2.11 Vernonia oligocephala

4.2.12 Warburgia salutaris

4.2.13 Withania somnifera

4.2.14 Ziziphus mucronata

The chemistry of South African traditional tonic plants

5.2.1 Agathosma species

5.2.2 Aloe species

5.2.3 Arctopus species

5.2.4 Artemisia afra

5.2.5 Balanites maughamii

5.2.6 Dicoma species

5.2.7 Harpagophytum species

5.2.8 Hypoxis hemerocallidea

5.2.9 Muraltia heisteria

5.2.10 Sutherlandia species

5.2.11 Vernonia oligocephala

CHAPTER 5: Chemistry (continued)

5.2.13 Withania somnifera

5.2.14 Ziziphus mucronata

Classes of compounds and their biological activities

Pharmacological and physiological effects of tonics