CHAPTER I

PHARMACOGNOSY

1.1 INTRODUCTION

The use of plants as medicine is as old as human civilization. Men of all ages

in both developing and undeveloped countries use plants in an attempt to cure various

diseases and to get relief from physical sufferings. India is known as the “Emporium

of Medicinal plants” due to the prevalence of several thousands of medicinal plants in

different bioclimatic zone (Yoganarasimhan, 2000). The peninsular India probably

has the richest and varied flora than other tracks of equal area in India, possible in the

world (Gamble, 1967), a phenomenon contributed by its combined effects of its

geographical situation and topography. The Western Ghats have been designated as

one of the hot spots of global biodiversity. A rich depository of flora with high

endemism is found in the Western Ghats. The immense taxonomic diversity of the

country throws a challenge to the Indian chemists and biologist to transform the

enormous bioresource into economic wealth and intellectual property. The state Tamil

Nadu is endowed with a very rich flora due to the various physiographic features and

physiognomic factors and different types of vegetation exist in the state. A total of

5640 species of the flowering plants (including 6 gymnosperms) are reported either

naturally occurring or cultivated in the state (Nair and Henry, 1983). Among them

numerous plants have been reported to be medicinal plants. However, our knowledge

of medicinal plants has mostly been inherited traditionally. Our ancestors had a

profound knowledge of these medicinal plants and they knew innumerable remedies,

a fact indicated in the writings of Siddhars of Tamil Nadu. Had their expertise

documented properly it would help the modern man to find more effective

prophylactic use of these herbs.

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 Modernity has brought us several new things and we have discarded certain

old beliefs and practices little realizing the adverse impact on our physical well being.

Now the side effects of most effective life saving allopathic drugs have recently

opened the eyes to look behind for effective non-toxic safe drugs. The modern

pharmacologists now strive to learn more about ancient systems of medicine and the

vast treasure of the herbal wealth of our country (Pandey et al., 1995). Even today,

years after the introduction of modern medicine in our country, the Indian systems of

traditional medicine continue to provide medical relief to nearly 80% of our total poor

people (Saxena and Tripathi, 1989). It has also been estimated that as many as 90%

of the world’s rural population rely on herbal drugs for their primary health care

(Yoganarasimhan, 2000).

Thus, research in plants, scientific evaluation and standardization of the new

and little known herbals have gained their drive since the upsurge of interest in the

molecular aspects of the drugs.

Meliaceae or the Mahogany family is a flowering plant family of mostly trees

and shrubs (and a few herbaceous plants, mangroves) in the order Geraniales.

Meliaceae is a woody family widely distributed throughout the tropics and subtropics,

with only slight penetration into temperate zones; they occur in a variety of habitats

from rain forests and mangrove swamps to semi-deserts. It consists of about 50 genera

and about 500 species (Pennington and Styles, 1975). About 20 genera and over 75

species of Meliaceae are reported from India. Common Indian species include

Azadirachta indica (Neem), Melia azedarach and Toona ciliata. The family contains

many important medicinal, timbers and ornamental trees. Other uses of Meliaceae

comprise shade and street trees, fruit trees and sources of biologically active

compounds (Mabberley et al., 1995). Among other secondary metabolites, Meliaceae

2
synthesize and accumulate bitter and biologically active nortriterpenoids, which are

also known as limonoids and meliacins. These and other compounds have aroused

considerable commercial interest due to their insect-antifeedant (Simmonds et al.,

2001), insect-repellent (Shukla, et al., 1997), insecticidal (Greger et al., 2001),

molluscicidal (Singh et al., 1998), antifungal (Engelmeier et al., 2000), bactericidal

(Aboutabl et al., 2000), and anti viral (Singh et al., 1988) activities as well as their

numerous medicinal effects in humans and animals (Benencia et al., 2000).

The genus Naregamia of Meliaceae is represented by only one species in India

(Hooker, 1872), Naregamia alata Wight & Arn. It is commonly called as Goanese

Ipecacuanha. It is a rare under shrub, 15-45 cm tall, found mainly on rocky or grassy

slopes in western peninsular India upto 1000m height. It has been used in traditional

medicines in India and elsewhere in the treatment of rheumatism, itch, malarial and

chronic fevers, wounds, anaemia, enlarged spleen, ulcers, vitiated conditions of pitta

and vata, halitosis, cough, asthma, splenomegaly, scabies, pruritis, dysentery,

dyspepsia and catarrh. In Southern India, a small dose of the plant is used as an

expectorant. The plant is acrid, sweet, cooling, aromatic alexeteric, emetic,

expectorant and depurative (Biju, 2014). The root of Naregamia alata is used to cure

asthma, bronchitis, biliousness and ulcers. Naregamia alata has been reported to be a

constituent of an Ayurvedic drug ‘Pittapapda’ (Rajopadhye and Upadhye, 2013).

The genus Walsura Roxb., belonging to the family Meliaceae comprises 10

species in India (Hooker, 1872) but Willis (1955) reports the occurrence of 15 Indo-

malayan species of the genus. Walsura trifoliata (A. Juss.) Harms. (Syn: Walsura

piscidia Roxb., Heynea trifoliata A.Juss.) is an evergreen tree distributed widely in

the tropical areas of Asia, such as Southern China, India, Malaysia, and Indonesia

(Chetty et al., 2008). It grows on dry deciduous forests of 200 to 300m height. This

3
plant is well reputed in traditional system of medicine and used by tribal people to

treat various diseases like skin allergies, astringent and diarrhoea (Pullaiah and Rani,

1999). The bark of the plant is reported to posses stimulant, expectorant,

emmenagogue and emetic properties. The fruit pulp is used as fish poison

(Anonymous, 1976).

Pharmacognosy literally means knowledge of drugs or pharmaceuticals, which

deals with the drugs of vegetable, animal and mineral origin. It may be defined as an

applied science that deals with “biological, biochemical and economical features of

natural drugs and their constituents”. Pharmacognosy helps to study the identification

of the source of the material forming drug, description of its morphology and

anatomy, investigation of its potency, purity and freedom from admixture, devising

the methods of cultivation, prescribing the details of collection and preparation

processes and studying the constituents of the drug and investigation of their physico-

chemical properties (Wallis, 1985).

The pharmacognostical studies of the herbal drugs have become imperative for

several reasons. As per the WHO norms, every drug has to undergo botanical

standardization, particularly macroscopic and microscopic characterization which

constitutes the major part of pharmacognosy. This primary step enables the

researcher in phytodrugs to affirm. The botanical standardization is based upon the

tenet that certain microscopic characters are specific and restricted in distribution

(Metcalfe and Chalk, 1979). Microscopic parameters, though limited in their

application under certain circumstances, have still highly reliable diagnostic values

and play appreciable role in the herbal drugs.

A perusal of literature on the much-valued medicinal plants Naregamia alata

and Walsura trifoliata of Meliaceae reveals a lacuna in the pharmacognosy and other

4
parameters. This fact induced the present investigation of the micro morphological

standardization of the folklore drug to put forth a protocol of anatomical features.

1.1.1. AIM AND SCOPE OF STUDY

Microscopical analysis of plant is a well established and useful criterion for

the identification and authentication of medicinal plants (Jackson and Snowdon,

1990). It has the advantage of requiring only small quantities of material, economical

and can provide a rapid tentative identification, prior to confirmation by analytical

techniques. Microscopic specimens prepared can be compared to microscopic

descriptions present in the monographs, or in a visual atlas of plant material.

Most of the species of Meliaceae have medicinal properties. Currently

however, no such description or standardization is available for Naregamia alata and

Walsura trifoliata. A microscopic illustration describing diagnostic characteristics on

such plants will be helpful fulfilling a need for information on plant identification and

quality control.

In the present study, the time renowned microtechnical procedures were

employed and data pertaining to morphological and anatomical characteristics of the

selected taxa were retrieved. Every essential observation was supplemented by

supporting photographs. Customary parameters of pharmacognosy such as powder

drug analysis and powder microscopy were given due importance. These studies will

offer the scope for easy and accurate identification of the specimen either in

incomplete or fragmentary form.

1.1.2. PLANT PROFILE:

The following plant species were selected for the present investigation.

The plant names, localities and dates of collection are furnished below:

5
1.1.2.1. Naregamia alata Wight & Arn.

Vernacular Names :

Tamil : Nilanarai

Malayalam : Nilanarakam

English : Goanese Ipecac, Goanese ipecacuanh

Locality : Petchiparai, Kanyakumari District, TamilNadu.

Date of Collection : September, 2010

Botanical descriptions:

Habit : Rare shrub (Fig. 1.1), 15-45 cm tall

Leaves : Leaves are divided into three leaflets (Fig.1.2), each of

which is wedge-shaped-obovate, quite entire, and

stalkless. Petiole 1-3cm long winged; leaflets oblong-

obovate, base acute, inequilateral, apex acute; nerves 5-

6 pairs, petiolule inconspicuous.

Inflorescence /flower : White flowers (Fig. 1.4), 2.5-5 cm long, arise solitary in

leaf axils. Sepals five, 5 mm long, calyx persistent.

Petals are 5, very long, strap-shaped, distinct, and free

from the stamen tube. Filaments are united into a long

tube, inflated and spherical at the tip (Fig. 1.3).

Fruit & seed : Capsule is 3-cornered, 3-valved, valves circular, 8 mm

long. Seeds 2 in each cell, curved. Flowering: April-

May.

Ecology : Found mainly on rocky or grassy slopes in western

peninsular India.

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1.1.2.2. Walsura trifoliata(A. Juss.) Harms. :

Synonym : Heynea trifolia A. Juss.

Walsura piscida Roxb.

Walsura tabulata Hiern.

Vernacular names :

Tamil : Walsura, Cheddavokko, Kanjimaram

Malayalam : Perillapacha; Perilla ppichu

Kannada : Male sagade

Locality : Karaiyar - Papanasam, Tirunelveli.

Date of Collection : October, 2010

Botanical descriptions:

Habit : Trees up to 15 m tall (Fig. 8.1).

Trunk & Bark : Bark pale brown, shallowly fissured, lenticels rusty

brown; blaze pink (Fig. 8.6).

Branches and

branchlets : Branchlets slender, terete, lenticellate, glabrous.

Leaves : Leaves compound, trifoliate (Fig. 8.2), alternate, spiral;

rachis up to 5 cm long, triangular, pulvinate, glabrous;

petiolule of side leaflets 0.4-1 cm long and middle

leaflet with 1.3-3 cm long, swollen at both ends, angled

or subterete; lamina 4-15 x 2.5-5 cm, variable in shape,

narrow oblong to elliptic or narrow obovate, apex

acuminate with retuse tip or rounded with retuse, base

acute to cuneate, margin entire, chartaceous to

subcoriaceous, glaucous beneath; glabrous; midrib flat

7
 above; secondary nerves 7-13, gradually curved; tertiary

nerves broadly reticulate, slender.

Inflorescence /

Flower : Inflorescence terminal or axillary panicles (Fig. 8.5);

flowers greenish-yellow. Flowering: January-March.

Fruit and Seed : Berry ovoid (Fig. 8.4), to 1.3 cm long; seeds 1-2, pale

brown, enclosed in a white fleshy aril.

Ecology : Understorey trees in wet evergreen to semi- ever

green forests upto 1200 m.

Distribution : India and Sri Lanka; in the Western Ghats – South

and Central Sahyadris.

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 1.2. REVIEW OF LITERATURE

Pharmacognostic studies on Indian medicinal plants have gained momentum

in recent years following an awareness of the values of indigenous and traditional

systems of medicine. Since the members of Meliaceae possess valuable medicinal

properties, pharmaceutical chemists and medicinal botanists have started paying

attention to the members of this family.

The presence of unicellular, multicellular and glandular trichomes with

varying shapes, single layer of palisade and difference in vascular bundle shape have

been observed as common characters of family Meliaceae (Metcalfe and Chalk,

1957).

Presence of cylindrical or single crescent shaped vascular bundle, various

types and sizes of secretory cells and solitary/clustered crystals have been observed as

common characters of petiole of family Meliaceae by Alexandra et al., 2008.

Ahmad et al., 2010 recognized the polygonal cells with smooth walls in leaf

epidermis in Melia azedarach.

Jafari et al., 2013 studied the peltate trichomes in the leaf epidermis of

Azadirachta indica and Melia azedarach. Fragments of lignified vessels with spiral

thickening are found in the leaves of both plants.

Mensah, 2012 reported the occurrence of numerous stomata on lower

epidermis in Azadirachta indica. The epidermis is relatively thin. The palisade

mesophyll is composed of two layers of elongated closely packed cells. The spongy

mesophyll is composed of loosely arranged cells with air spaces.

Lagos et al., 2007 studied the leaf of Trichilia catigua (Meliaceae), and

reported that the leaf midrib is convex and traversed by one collateral vascular bundle,

encircled by a complete sclerenchymatic sheath. The stem bark is distinguished into

9
periderm, consisting of suber, phellogen and various layers of phelloderm. The fibres

form small bands and contain many calcium oxalate prisms.

Attarde et al., 2010 reported the presence of more amounts of cluster type

calcium oxalate crystals in the leaflets of Soymida febrifuga.

Ibrahim et al., 2006 examined the powdered bark of Khaya species. This

examination revealed different types and shapes of lignified sclereids, abundant

distribution of prismatic calcium oxalate crystals, druses, lignified fibres, medullary

rays and parenchyma cells which may contain starch grains.

Singh et al., 2012 studied the powdered bark material of Toona ciliata and

showed the presence of pointed lignified fibres, scattered stone cells, few in groups

and multicomponent starch grains.

Bhat, 1994 studied the vascular tissue of Aglaia barberi and according to him

the phloem rays were 1 to 3 seriate more commonly biseriate ranging from 100 to

450µm in height and 10 to 35 µm in width. Phloem rays were closely spaced and

heterogenous with usually a single marginal row of square cells. Ray heterogeneity

was sometimes less pronounced.

The present proposed study is justified since it may throw immense light on

the anatomical and phytochemical dimensions of the two selected species. The study

will provide a protocol for the pharmacognostic standardization of the two species.

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 1.3. MATERIALS AND METHODS

1.3.1. Collection of specimens:

The plant specimens were collected from the southern Western Ghats, and

identified and checked by Dr.Chelladurai, Retired Research Officer, Botany, Scientist

C cadre, Central council for Research in Ayurveda and Siddha, Government of India.

The Flora of presidency of Madras (Gamble, 1997) and Flora of Tirunelveli Hills

(Manickam et al., 2008) were referred for the identification of the chosen plants. The

taxonomic identity of the medicinal plants were confirmed by comparing the collected

voucher specimens with those of known identity and voucher specimens deposited in

the Herbarium (SJCH 935 & 936) at PG and Research department of Botany,

St.John’s College, Palayamkottai, Tamilnadu.

Preparation of specimens

Healthy plants and normal organs were collected carefully. The required

samples of different organs were cut and removed from the plant and fixed in FAA

(Formalin-5ml+ Acetic acid-5ml + 70% Ethyl alcohol-90ml). After 24 hrs of fixing,

the specimens were dehydrated with graded series of Tertiary Butyl alcohol (TBA) as

per the schedule given by Sass, 1940. Infiltration of the specimens was carried by

gradual addition of paraffin wax (melting point 58-60oC) until TBA solution attained

super saturation. The specimens were cast into paraffin blocks.

Sectioning

The paraffin embedded specimens were sectioned with the help of Rotary

Microtome. The thickness of the sections was 10-12 µm. Dewaxing of the sections

was carried out by customary procedure (Johansen, 1940). The sections were stained

with Toluidine blue (O’Brien et al., 1964). Since Toluidine blue is a polychromatic

stain, the staining results were remarkably good; some cytochemical reactions were

11
also obtained. The dye rendered pink colour to the cellulose walls, blue to the

lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies

and other necessary sections were also stained with safranin and Fast-green and

KI(for Starch).

For studying the stomatal morphology, venation pattern and trichome

distribution, paradermal sections (sections taken parallel to the surface of leaf) as well

as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial

maceration employing Jeffrey’s maceration fluid (Sass, 1940) were prepared.

Glycerine mounted temporary preparations were made for macerated/cleared

materials. Powdered materials of different parts were cleared with NaOH and

mounted in glycerine medium after staining. Different cell components were studied

and measured.

Photomicrographs

Microscopic descriptions of tissues were supplemented with micrographs

wherever necessary. Photographs of different magnifications were taken with Nikon

labphoto 2 microscopic Unit. For normal observations bright field was used. For the

study of crystals, starch grains and lignified cells, polarized light was employed. Since

these structures have birefringent property, under polarized light they appear bright

against dark background. Magnifications of the figures are indicated by the scale-bars.

Descriptive terms of the anatomical features were given as in the standard Plant

Anatomy books (Esau, 1964).

1.3.2. Physicochemical parameters:

The following physico-chemical parameters were determined by standard

methods (Anonymous, 1996).

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Ash values

Ash values are indicative to some extent, of care taken in collection and

preparation of drug for market and of foreign matter content of natural drug. The

purpose of ash preparation is to remove all traces of organic material interfering in an

analysis of inorganic elements.

a. Total ash:

Total ash was determined as formulated by the Association of official

analytical chemists (Horwitz, 1980). 2 gm of the sample was taken in a silica crucible

which had been previously ignited and cooled before weighing. The ignition was

repeated until constant weight was obtained.

b. Water soluble ash:

The ash was boiled with 25ml of water and was filtered through an ashless

filter paper (Whatman 41). It was followed by washing with hot water. The filter

paper was ignited in the silica crucible, cooled and the water insoluble ash was

weighed. The water soluble ash was calculated by subtracting the water insoluble ash

from the total ash.

c. Acid insoluble ash:

It was determined by boiling the water insoluble ashes with 25ml dilute HCl

for five minutes and filtering through an ashless paper (Whatman 41). The filter

paper was ignited in the silica crucible, cooled and acid insoluble ash was weighed.

d. Loss on Drying:

Loss on drying is the loss in weight in percent w/w resulting from loss of

water and volatile matter of any kind that can be driven off under specific conditions.

5 gm of powdered sample was weighed and placed in a crucible of silica. The crucible

was cleaned and dried and weight of empty dried crucible was taken. The powder was

13
spread as a thin uniform layer. The crucible was placed in the oven at 105oC. The

powder was dried for 2 hours and cooled in a desiccator to room temperature and the

weight of the cooled crucible plus powder was noted.

1.3.3. Solubility:

Solubility was determined by standard methods (Anonymous, 1958).

a. Alcohol soluble extractive values

5gm of air dried, macerated and coarsely powdered sample was soaked with

100ml of alcohol in a closed flask for twenty four hours, shaking frequently for six

hours and was allowed to stand for eighteen hours. It was filtered rapidly, taking

precautions against the loss of alcohol. 20ml of the filtrate was evaporated to dryness

in a tared flat-bottomed shallow dish. Again it was dried at 105oC to obtain constant

weight. The percentage of alcohol soluble extractive was calculated with reference to

the air dried sample.

b. Water soluble extractive values

It was proceeded as directed for alcohol soluble extractive, using water instead

of alcohol.

1.3.4. Extractive values (Successive extraction):

The freshly collected plant material was cut into small pieces. It was dried

and coarsely powdered. The powdered material of 5gm was taken in Soxhlet

apparatus and successively extracted with n-hexane, petroleum ether, chloroform, and

alcohol till the extracts became colourless. Nearly 80% of the solvent was collected

and distilled over boiling water bath. The extracts so obtained were further dried in

vacuum desiccators and the yield of the extracts was weighed.

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1.3.5. Thin Layer Chromatography:

2gm of the sample was soaked in 20 ml of distilled Ethyl alcohol, kept

overnight, boiled for 10 minutes and filtered. The filtrate was concentrated and made

upto 5 ml in a graduated test tube. 10 and 12µl of this solution was applied on Merck

aluminium plate 60 F254 precoated with silica gel of 0.2mm thickness and the plate

was developed in Toluene: Ethyl acetate 1:1. After drying, the plate was visualized

under UV 254 and 366 nm and photographs were taken. The plate was dipped in

vanillin-sulphuric acid reagent and kept in oven at 105oC till the colour of the spots

appeared. The photograph was taken.

1.3.6. Fluorescence analysis:

Fluorescence of the drug was observed in day light and UV light (265nm &

365nm) using various solvent extracts of the drug. The powder was treated with

neutral solvents like hexane, petroleum ether, chloroform, ethyl alcohol, ethyl acetate,

distilled water, acid like 1N HCl and alkaline solution like 1 N NaOH (Chase and

Pratt, 1949).

1.3.7. Qualitative organic analysis:

The 90% alcoholic extract of the aerial part of the plant was subjected to

qualitative analysis (Overton, 1963 and Harborne, 1973).

The following tests were carried out.

a. Liebermann-Burchard test: (Steroid)

A few mg of the substance in chloroform was treated with a few drops of

acetic acid, acetic anhydride, two drops of con.H2SO4 and heated gently. Blue (or)

green colour showed the presence of steroid.

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b. Noller’s test: (Triterpenoid)

A few mg of the substance in a dry test tube was treated with a bit of tinfoil,

0.5ml of thionyl chloride and heated gently. Pink colour showed the presence of

triterpenoid.

c. Alkaloid:

A few mg of the substance in acetic acid was treated with two drops of

Dragendorff’s reagent. Red or orange precipitation indicated the presence of alkaloid.

Excess reagent was avoided.

d. Phenol:

A few mg of the substance in alcohol was treated with alcoholic Ferric

chloride. Any coloration indicated the presence of phenolic compounds.

e. Sugars/Glucosides:

A few mg of the substance was mixed with equal quantity of Anthrone and

treated with two drops of con.H2SO4. It was then heated gently on a waterbath. Dark

green colour indicated the presence of sugar/glucosides.

f. Quinone:

A few mg of the substance was treated with con. H2SO4 or aqueous NaOH.

Colourisation indicated the presence of quinoid compounds.

g. Coumarin:

A few mg of the substance in alcohol was treated with alcoholic NaOH.

Yellow colour indicated the presence of coumarin.

h. Shinoda test: (Flavonoid)

A few mg of the substance in alcohol was treated with magnesium turnings

and few drops of con.HCl. Red or Pink colour indicated the presence of flavonoid.

16
i. Ehrlich’s test: (Furanoid)

A few mg of the substance in alcohol was treated with a pinch of

paradimethylamine benzaldehyde and a few drops of con.HCl. Red or pink colour

indicated the presence of furanoids.

j. Tannin:

A few mg of the substance in alcohol was treated with a few drops of lead

acetate. Precipitation indicated the presence of tannins.

k. Test for saponin

About 2gm of the powdered sample was boiled in 2ml of distilled water in a

water bath and filtered. 10ml of the filtrate was mixed with 5ml of distilled water and

shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops

of olive oil and shaken vigorously then observed for the formation of emulsion.

17
 1.4. OBSERVATION

Microscopic features

1.4.1. NAREGAMIA ALATA Wight & Arn.

1.4.1.1. Leaf:

The leaf has thick midrib which is projecting on both adaxial and abaxial

sides. The lamina is thin and dorsiventral (Fig. 1.5). The midrib has thick, short and

cylindrical adaxial hump and fairly wide and thick abaxial part. The epidermal layer

of the midrib consists of squarish, thick walled cells with prominent cuticle. The

ground tissue is parenchymatous and the cells are angular, thin walled and compact

(Fig. 1.6).

The vascular system consists of two vascular strands: one strand is small,

circular and collateral with prominent sclerenchyma cap. It is adaxial in position.

The abaxial strand is larger, bowl-shaped in outline and collateral. It includes several

short, parallel lines of narrow angular thick walled xylem elements and small groups

of phloem elements located at the lower end of each xylem strand (Fig. 1.6). A thin

layer of fibres occurs all along the abaxial part of the vascular strand.

Lamina:

The lamina is smooth and even on adaxial and abaxial sides (Fig. 2.1). The

adaxial epidermal layer consists of thick, rectangular cells with thick and smooth

cuticle. The abaxial epidermal layer is comparatively smaller, squarish or cylindrical

in shape; the cuticle is thin. The mesophyll tissue is differentiated into a thin adaxial

row of short conical compact palisade mesophyll cells and abaxial zone of five or six

layers of spherical or lobed, loosely arranged spongy mesophyll cells. The lamina is

120µm thick.

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Leaf Margin:

The marginal part of the lamina is slightly dilated and bent down. It consists

of small squarish epidermal cells with very prominent cuticle (Fig. 2.2). The size of

the epidermal cells becomes gradually reduced in size in the abaxial side of the leaf

margin. The differentiation of the mesophyll tissues remains unchanged in the leaf

margin. The marginal part is 100µm thick.

Epidermal cells and stomata:

The stomata were studied from the surface view of the paradermal sections

(Fig. 2.3 and 2.4). The epidermal cells are small and polyhedral in outline. Their

anticlinal walls are thin and straight. The stomata are diffuse in distribution. The

stomata are cyclocytic type: each stoma will have six or more subsidiary cells which

will radiate in all directions from the guard cells. The stomatal complex will appear

star shape in surface (Fig. 2.4). The guard cells are 15×20µm in size.

Venation pattern (Fig. 2.5 and 2.6):

The lamina has distinct intra marginal veins and distinct reticulate venation

system. The lateral veins are thick and prominent. The vein lets are thin and less

prominent. They form vein islets of various shape and size. Within the islets occur

the vein terminations. The terminations are either unbranched or branched once or

twice. They are long, slender and wavy (Fig. 2.6).

Petiole:

The leaf has decurrent leaf base and in cross sectional view of the petiole there

are two lateral wide wings. The petiole consists of adaxial thick, short adaxial hump

and wide and thick abaxial part (Fig. 3.1). The petiole is 900µm thick, the adaxial

hump is 250µm wide and the abaxial part of the petiole consists of small, squarish

epidermal cells with thick cuticle which consists of minute cuticular spines. The

19
ground tissue is circular, less compact and some of them contain tannin bodies. The

vascular system of the petiole includes two prominent strands of which one is adaxial

and semi circular and the other is wide and bowl shaped. The adaxial strand consists

of several, short parallel rows of small, angular thick walled xylem elements and a

prominent horizontal band of phloem elements on the adaxial side. The strand being

protected by a thick arc of lignified fibres. Abaxial strand is wide and thicker it is

also collateral with upper xylem and lower arc of phloem elements. The xylem

elements are in short, parallel rows comprising narrow, thick walled xylem elements

and phloem arc includes small isolated clusters of sieve elements (Fig. 3.2).

1.4.1.2. Stem:

The stem is circular in cross sectional view. It includes thick superficial

periderm, thick cortex, narrow phloem cylinder and dense solid xylem cylinder (Fig.

3.3 and 3.4). The periderm is enclosed within an intact darkly stained epidermal cell.

The periderm is 100µm thick. It includes about eight layers of tabular, homocellular

suberized cells arranged in thick radial row. The cortex is wide and includes large,

thick walled less compact parenchyma cells of various shape and size. Secondary

phloem occurs in continuous circle around the xylem cylinder. It includes small

clusters of sieve elements mixed with phloem parenchyma. The sieve elements have

distinct companion cells (Fig. 3.5). Secondary xylem is compact dense cylinder and

includes vessels, xylem fibres and xylem rays. The vessels are fairly wide and

circular or narrow and not very distinct from wide fibres. The vessel walls are thick

and lignified. The fibres are highly thick walled with wide lumen and the xylem rays

are prominent and occur in straight prominent lines. The ray cells are also wide,

thick walled and lignified. The vessels are 20µm in diameter and the narrow vessels

are 10µm wide (Fig. 3.6).

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1.4.1.3. Root:

Both thin and thick roots were studied.

Thin root:

A thin root measuring about 2.35mm thick was studied (Fig. 4.1). The thin

root is circular in cross section with undulate outline. The epidermis and a few

cortical cells are crushed forming dark surface layer. The cortical zone is wide and

the cortical cells are large, lobed and compact (Fig. 4.2). The secondary phloem

consists of a thin continuous layer running around the xylem cylinder (Fig 4.2). The

xylem cylinder is 120µm in diameter and it consists of scattered, solitary, circular

vessels which are thick walled (Fig. 4.2). The vessels are upto 25µm in diameter.

The fibres are angular, thick walled and lignified. The fibres occur in compact regular

radial rows. The fibre walls are thick and lignified and the lumen is fairly wide. The

xylem rays are straight and narrow and their cells are also lignified.

Thick root:

The thick root is highly wavy in outline due to the presence of thick and wide

fissures. The thick root is 4mm thick (Fig. 5.1). The root consists of very thick

periderm which is deeply folded and grooved. The periderm is differentiated into

outer thick wavy cylinder of suberized phellem cells and inner equally thick

phelloderm layers. The phelloderm cells are squarish in shape with thin walls and

deeply stained. The cortical zone is very prominent and thick. The cells are

parenchymatous variously shaped and compact (Fig. 5.2). The secondary phloem is

quite thick and surrounds the xylem cylinder all around. It consists of collapsed outer

phloem and inner narrow non collapsed phloem (Fig. 5.3). In the collapsed phloem

the parenchyma cells and phloem rays are dilated and the sieve elements are crushed

into dark tangential lines (Fig. 5.4). The non collapsed phloem is very narrow and

21
includes a few layers of intact sieve elements. In the phloem parenchyma cells have

dense accumulation of starch grains (Fig. 5.6). The starch grains are also seen in the

cortical parenchyma.

The secondary xylem is circular, dense and compact. It includes circular lines

of narrow, thick walled, solitary vessels and thick walled lignified compact radial

lines of xylem fibres. The vessels are narrow or wide. The vessel elements are upto

30µm in diameter (Fig. 5.3 and 5.5).

1.4.1.4. Powder microscopy:

The powder preparation of the material exhibits the following inclusions:

I) Fragments of adaxial epidermis were seen in surface view. The epidermis is

apostomatic. The epidermal cells are small, and their anticlinal walls are thick

and highly wavy (Fig. 6.1).

II) The abaxial epidermal peeling was also seen in the powder. The epidermis is

stomatiferous. The stomata are cyclocytic type. A stoma is surrounded by two

polar subsidiary cells and two lateral subsidiary cells. Some of the stomata

have more than four subsidiary cells which encircle the guard cells (Fig. 6.2

and 6.3). The anticlinal walls of the epidermal cells are thick and wavy. The

guard cells are 15×20µm in size.

III) Foliar sclereids: Long, filiform, branched thread like foliar sclereids are

wide spread in the lamina. The sclereids run along with the veins and at

certain places they come out of the veins and penetrate into the vein islets

(Fig. 6.4, 6.5, 7.1 and 7.2). The sclereids are 10µm thick and unlimited in

length.

IV) Epidermal trichomes: Epidermal trichomes are seen either attached on the

lamina or free from the lamina (Fig. 6.5 and 6.6). The trichomes are

22
 unicellular, unbranched, pointed at the tip and highly thick walled. The

trichomes are nonglandular type. The trichome measures 400µm long and

20µm thick.

V) Xylem fibres: Xylem fibres are abundant in the powder. The fibres are

narrow, thick walled and tapering at the end. The cell lumen is fairly wide.

No pits are evident. The fibres are upto 550µm long and 15µm thick (Fig. 7.4,

7.5 and 7.7).

VI) Vessel elements: Vessel elements are equally abundant in the powder. Most

of the vessel elements are long, narrow and fibre like in appearance. They

have simple, oblique elliptical perforations at the end. The pits are circular,

multiseriate and bordered. Some of the vessel elements have long narrow tails

(Fig. 7.3, 7.4, 7.5 and 7.6).

VII) Epidermal cells: The epidermal cells of the stem were seen in surface view.

The cells are vertically elongated, rectangular and run parallel to each other.

The cells have thick walls and circular wide simple pits (Fig. 7.8).

1.4.2. WALSURA TRIFOLIATA (A. Juss.) Harms. :

1.4.2.1. Leaf:

The leaf in sectional view exhibits smooth adaxial surface and prominent

midrib with adaxial concavity (Fig. 9.1). The midrib is concavo-convex in sectional

view, having wide shallow adaxial concavity and thick convex on the abaxial side.

The midrib 500µm in vertical section and 500µm in horizontal plane; it is 450µm in

vertical plane (Fig. 9.2).

The epidermal layer of the midrib consists of small, highly thick walled

squarish cells with thick cuticle. The ground tissue includes angular, thin walled

23
compact parenchyma cells; some of the ground parenchyma cells possess dense

accumulation of tannin (Fig. 9.2).

The vascular system includes two circular masses of collateral strands located

in the adaxial side and one wide bowl shaped abaxial strand (Fig. 9.3). Both adaxial

and abaxial strands are collateral and their xylem strands are just opposed. Phloem

occurs on the outer part of the xylem. The xylem elements are wide, elliptical in

outline and occur in short or long radial chains. Phloem elements are in small groups,

located on their outer part of the xylem mixed with parenchyma cells and fibre sheath.

The fibre sheath extends cellular growth on the adaxial and abaxial surfaces of the

vascular strands (Fig 9.3).

Lamina:

The lamina is dorsiventral with distinct differentiation of the dorsal and

ventral surfaces. The adaxial (Ventral) surface of the lamina consists of prominent

vertically oblong epidermal cells with thick cuticle. The abaxial epidermis includes

small, squarish thick walled cells with finger like epidermal trichome arising from

every epidermal cell, the lamina is 260µm thick (Fig. 10.1). The palisade cells occur

in one or two compact dense layer of cells on the adaxial side. The spongy mesophyll

includes lobed small cells interconnected with each other and forming wide air

chambers. Vascular strands are seen in the middle part of the mesophyll.

Leaf margin:

As seen in T.S view the leaf margin is slightly bent down and it measures

about 200µm thick. The basic structure of the leaf margin is similar to that of lamina

region; it includes adaxial palisade zone abaxial reticulate spongy mesophyll tissue

and small vascular strand located in the mesophyll tissue. The extreme margin of the

lamina includes a small compact mass of thick walled cells (Fig. 10.2)

24
Epidermal cells and stomata:

The epidermal cells and stomata were studied in surface view of the

paradermal sections. The epidermal cells are small polygonal with thick straight

anticlinal walls. The stomata are deeply sunken in the epidermal layer. The guard

cells are surrounded by 9 to 11 radiating subsidiary cells. Thus the stoma appears to

be stellate stomata (Fig. 10.3, 10.4 and 10.5). The guard cells are broadly elliptical

measuring 20×20µm in size. The stomatal aperture is narrow and slit like (Fig. 10.4

and 10.5).

Venation pattern of the lamina:

The veins and vein lets are thick and straight. They form fairly wide vein

islets with well defined thick and straight vein boundaries (Fig. 10.6). Almost all vein

islets have vein terminations. There may be more than one termination in vein islet.

The terminations are either unbranched or branched once. They are short and thick

and curved (Fig. 10.7).

Petiole:

The petiole is circular with short two lateral wings. It is 1mm thick. The

petiole consists of thin epidermal layer which is often broken due to growth in

diameter of the petiole (Fig.11.1). There is a narrow less prominent periderm. The

cortical zone is parenchymatous the cell being small and circular. The inner boundary

of the cortex is marked by a thin cylinder of discontinuous masses of fibres. The

vascular cylinder consists of outer wide cylinder of xylem and phloem and central

mass of tangentially oblong vascular bundle. The outer cylinder includes outer thick

cylinder of secondary phloem in which the phloem elements occur in radial compact

files. The secondary xylem includes several radial lines of xylem elements and thick

walled xylem fibres. The central strand consists of a few compact lines of xylem

25
elements mixed with phloem fibres. The phloem elements occur in the form of wide

hollow cup on the lower arc of the xylem strand (Fig. 11.2).

1.4.2.2. Stem:

The stem is circular in outline with epidermis, cortex, sclerenchyma cylinder,

secondary phloem and secondary xylem. The stem is 2.35mm thick (Fig. 11.3). The

stem consists of an epidermal layer which has undergone periclinal divisions

producing narrow periderm zone. The cells of the epidermis have thick walls with

spiny cuticle. The periderm cells are in 4 or 5 layers of rectangular cells and

suberized. Inner to the periderm is wide cortex which includes small, compact

parenchyma cells of various shape and size. The cortical zone is about 50µm in

thickness. The boundary layer of the cortex is marked by a few isolated irregular

masses of sclerenchymatous cells (Fig. 11.4). The secondary phloem quite thick

comprises two or three layers of phloem sclerenchyma alternating phloem elements.

The outer part of the secondary xylem includes collapsed sieve elements and inner

part includes non collapsed intact sieve elements. Secondary xylem cylinder is

circular and lobed. It includes several radial lines of vessels and xylem fibres. Xylem

rays are well marked and they are thin and straight, running from secondary xylem to

secondary phloem. The vessels are circular and the secondary xylem vessels are upto

30µm wide. The pith includes both thin walled parenchyma and thick walled fibres.

These two types of cells are mixed with each other (Fig 11.5).

Bark:

T.S of bark of the stem:

The surface of the stem bark is highly fissured with remnants of peeling of

surface fissures. There are deep and wide fissures left by scaling off the periderm

(Fig. 12.1). The periderm seems to be deeper in origin arising from several layers of

26
inner secondary phloem. The periderm includes larger portion of inner periderm

which has arised from outer part of the secondary phloem. This periderm consists of

several radial files of rectangular thin walled suberized phellem cells. Inner to this

periderm zone is the collapsed phloem which consists of several tangential bands of

sclerenchyma segments alternating with collapsed phloem tissue (Fig. 13.1 and 13.2).

The rays are also dilated in the collapsed phloem zone (Fig. 12.2). The non collapsed

phloem is sharply distinguished from the collapsed phloem by the absence of wide

dilated rays and intact sieve elements. In the non collapsed phloem the phloem fibres

are seen in several tangential blocks alternating with non collapsed phloem cells (Fig.

13.3 and 13.4). The phloem rays are narrow, and do not expand crushing the sieve

elements. In the non collapsed phloem the sieve elements are located in radial lines

and the cells are rectangular with lateral companion cells (Fig. 13.4).

Calcium oxalate crystals are abundant along the boundaries of sclerenchyma

segment (Fig. 13.5 and 13.6).

TLS view of the phloem:

The phloem rays are non-storied. They are long and spindle shaped. The ends

of the rays are located at different levels. The individual rays are multiseriate,

biseriate or uniseriate. The multiseriate rays are heterocellular and they possess

middle squarish cells and terminal upright cells. The end wall plate of sieve element

is simple and vertically oriented (Fig. 14.1 and 14.2). Calcium oxalate prismatic

crystals are abundant in the phloem rays and phloem parenchyma. They are seen

arranged in continuous vertical rows. The crystals are rhomboidal or prismatic type

(Fig 14.3 and 14.4).

27
1.4.2.3. Powder microscopy:

The powder preparation of the material shows epidermal peeling in surface

view. The adaxial surface of the epidermis exhibits square shaped cells which are

compact two or three layered and wide circular simple pits are seen on the walls of the

epidermal cells (Fig. 15.1 and 15.2)

Vessels and fibres are abundant in the powder (Fig 15.3). The fibres are long,

thin needle shaped. A fibre is 500µm long and 10µm thick. Some of the fibres have

highly reduced lumen and thick secondary walls (Fig 15.4).

Vessel elements:

Long cylindrical thick vessel elements are found in the powder. They have

simple slightly oblique end wall perforations (Fig. 15.6). Some of the vessel elements

have short tails and well developed pits (Fig. 15.7). The vessel elements are upto

250µm long and 40µm wide.

1.4.3. Results of analytical studies:

The physicochemical parameters of Naregamia alata and Walsura trifoliata

were studied by determining the total ash value, alkalinity, water soluble ash, acid

insoluble ash, loss on drying, water, alcohol soluble extractive and successive

extractive values in various solvents like hexane, petroleum ether, chloroform and

ethanol. The observations are tabulated in Table 1.

The TLC profile of ethanolic extracts of the two plants (Fig. 16) is tabulated in

Table 2 and 3. Fluorescence characters of the plants were studied under daylight and

ultra violet light (265nm and 365nm) in different treatments such as hexane,

petroleum ether, chloroform, ethylacetate, ethylalcohol, distilled water, 1N NaOH and

1N HCl. The observations are tabulated in Table 4.

Qualitative organic contents of the alcoholic extracts were studied and the

presence or absence of some organic matters is tabulated in Table 5.

28
 1.5. DISCUSSION

The family Meliaceae contains more than 50 genera and about 500 species all

over the world. These are mainly distributed in the tropics and warm temperate

regions. In India, this family is represented by 20 genera 113 species (Hooker, 1872).

It is observed that, in Naregamia alata, flowers occur in solitary form, the

fruits are capsule and petioles are winged. In Walsura trifoliata, flowers occur in

corymbose panicles and the fruits are berry and the petioles are not winged. These

morphological characters play a key role in distinguishing Naregamia alata from

Walsura trifoliata.

While discussing the habit of Meliaceae, Hooker (1872) distinguishes woody

species and shrub species. In Walsura trifoliata, the upper epidermal cells are

vertically oblong with thick cuticle. In shruby species (Naregamia alata), epidermal

cells are thick with smooth cuticle. Both the species are characterized by cyclocytic

stomata.

Of the two species studied during the present investigation the correlation of

the epidermal cell walls, parenchyma distribution of the plants could not be

satisfactorily explained with their habitat. In Walsura trifoliata, which is a tree

inhabiting dry-deciduous habitats the anticlinal walls of the epidermis are straight;

stomata is sunken; the palisade tissue is 1-2 layered; spongy mesophyll cells are

interconnected to one another forming wide airchambers. This feature may be

visualized as dry-deciduous environmental impact. In Naregamia alata, the palisade

layers are short, conical and compact ones. Spongy mesophyll cells are loosely

arranged. These characters exhibit mesomorphic features as far as the palisade

structures are concerned.

29
 The vascular system in the leaves of Meliaceae differs widely from one

another in such details as massiveness, development of parenchyma versus

sclerenchyma in the bundle sheath and vein endings. Thus the leaf vascular system

appears to be a prominent source of systematically valuable criteria (Webster 1957).

In the two species studied adaxial vascular bundle of Naregamia alata is small,

circular and collateral with prominent sclerenchymatous cap. In Walsura trifoliata

the midrib bundle is circular, collateral without the sclerenchymatous cap (Fig. 9.3).

Walsura trifoliata has more distinct vein islets and vein terminations are short, thick

and curved. In Naregamia alata intra marginal veins and vein islets are thin and less

prominent, and vein terminations are long slender and wavy (Fig. 2.6).

The structural diversity of petiolar anatomy and its application in systematics

have been recognized by the earlier anatomists. Grew (1675), De Candolle (1879)

and Hare (1943) are the pioneers in the study of petiole and have proposed extensive

terminology and structural variations of the petiole. The two species of the present

study have distinctive structures of the petiole. Naregamia alata has two prominent

vascular strands, the adaxial side with semicircular and in abaxial side bowl shaped

vascular cylinder which is protected by thick arc of lignified fibre. Walsura trifoliata

has characteristic wide cylinder on adaxial side and abaxial side oblong vascular

cylinder. Thus the two species of the present study have distinctive features in the

petiole.

Wood anatomy of the two species of the present study has the distinctive

anatomical features suitable for the diagnostic purpose. The stem woods of

Naregamia alata and Walsura trifoliata have mostly radial and multiples of vessels.

In Naregamia alata, vessels are long, narrow fibre like with oblique elliptical

perforations at the end with long narrow tails. The pits are circular, multiseriate. In

30
Walsura trifoliata, the wood has long, cylindrical, thick vessels. The vessels have

simple, slightly oblique end wall perforations with short tail and well developed pits

(Fig. 15.6 and 15.7). A comparison of salient features of two taxa is presented in

Table 6.

Physicochemical parameters are important in detecting adulteration and are

adopted to confirm the purity and quality of the drug.

The present study reveals that the loss on drying does not vary much for the

two plants (7.87 and 7.01%w/w) (Table 1). Its dried form is expected to have a long

shelf-life with reduced chance of microbial growth due to its relatively low moisture

content. Insufficient drying favours spoilage by moulds and bacteria make possible

the enzymatic destruction of active principles. It is observed that the drug is properly

dried and properly stored.

Total ash value for the two plants (5.82 and 4.97%w/w) indicates low

inorganic components in the plants (Table 1). Total ash is particularly important in

the evaluation of purity of drugs i.e., the presence or absence of foreign matter such as

metallic salts or silica.

The amount of acid insoluble siliceous matter present in the two plants is 1.04

and 0.34%w/w. The water soluble ashes are found to be 1.85 and 1.17%w/w(Table 1).

These parameters are used to detect the presence of foreign material exhausted by

water. As the ash values of the crude drugs lie within the fair limit which signify its

quality and purity and gives idea about the total inorganic content.

The water soluble extractive values are found to be 14.63 and 15.10%w/w

(Table 1). The alcohol soluble extractive values for the two plants are 12.31 and

10.09%w/w which indicates the presence of polar constituents like phenols,

31
glycosides and flavonoids. The water soluble extractive is found to be signifying that

the large amounts of constituents of the plants are soluble in water than in alcohol.

Extractive values of the plant with different solvents give a preliminary picture

of the percentage of the compounds extracted. Extraction with ethyl alcohol gives the

highest yield in the successive extraction (5.4 and 4.18%w/w). So ethyl alcohol is the

best solvent for extraction among the five solvents used (Table 1).

In the TLC profile, the extracts of the two plants in ethyl acetate and Toluene

(1:1) solvent system confirm the presence of diverse potent biomolecules in these

plants. In Walsura trifoliata it shows maximum seven bands under UV 366nm (Fig.

16.2). In Naregamia alata maximum seven bands are observed with vanillin

sulphuric acid spray reagent (Fig. 16.1). TLC analysis provides an idea about the

polarity of various chemical constituents, in a way such that compound showing high

Rf value has low polarity and with less Rf value has high polarity (Table 2 and 3).

These potent biomolecules can be further used for the development of different drugs

in future.

Fluorescence analysis is an essential parameter for first line standardization of

crude drug. The fluorescent light is always of greater wavelength than the exciting

light. Light rich in short wavelength is very active in producing fluorescence and for

this reason UV light produces fluorescence in many substances which do not visible

in day light. Plants usually contain both fluorescent and non-fluorescent compounds.

When powdered samples of these plants are exposed to UV light of shorter (265nm)

and longer wavelengths (365nm) they emit specific colours depending on the

wavelengths of light. However, the colours will be constant for any given extract.

This procedure helps to standardize the fluorescent colour characteristic for a drug

(Table 4).

32
 For qualitative tests, ethyl alcohol extract was used for the two plants. The

extracts were positive for triterpenoid, alkaloid, phenol, flavonoid, coumarin, quinone,

furan, tannin and sugars. Steroid and saponins are absent in the two plants under

study (Table 5).

Thus, simple chemical analysis of crude drugs of the two plants helps in two

ways. It gives a preliminary idea for the prospects of the chemical compounds

present in the plants and it gives some chemical parameter for determining the quality

and purity of the raw drugs obtained from the market.

33
 TABLE 1

PHYSICO CHEMICAL CONSTANTS

Naregamia alata value (%w/w) Walsura trifoliata value (%w/w)

S.No Parameter
(Whole plant) (Aerial parts)
1. Total ash value 5.82 4.97
2. Water soluble ash 1.85 1.17
3. Alkalinity of water soluble ash 0.48ml of 0.1N HCl/g 0.6ml of 0.1N HCl/g
4. Acid insoluble ash 1.04 0.34
5. Loss on drying at 105oC 7.87 7.01
Solubility
6. Water soluble extractive 14.63 15.10
7. Alcohol soluble extractive 12.31 10.09
8. Extractive value(Successive extraction)
a. Hexane 1.6 1.3
b. Petroleum ether 2.7 2.52
c. Chloroform 3.2 2.73
d. Ethyl alcohol 5.4 4.18

34
 TABLE 2

TLC PROFILE OF WHOLE PLANT ETHANOLIC EXTRACT OF NAREGAMIA ALATA

WITH VANILLIN SULPHURIC

UV 254nm UV 366nm
S.NO. ACID SPRAY REAGENT
COLOUR RF COLOUR RF COLOUR RF
1. Green 0.08 Red 0.05 Grey 0.15
2. Red 0.10 Grey 0.48
3. Red 0.14 Grey 0.57
4. Blue 0.44 Grey 0.61
5. Red 0.49 Grey 0.69
6. Red 0.86 Grey 0.75
7. Red Grey 0.90

TABLE 3

TLC PROFILE OF AERIAL PARTS ETHANOLIC EXTRACT OF WALSURA TRIFOLIATA

WITH VANILLIN SULPHURIC

UV 254nm UV 366nm
S.NO. ACID SPRAY REAGENT
COLOUR RF COLOUR RF COLOUR RF
1. Green 0.05 Red 0.03 Grey 0.39
2. Green 0.77 Red 0.08 Grey 0.63
3. Blue 0.43 Grey 0.70
4. Red 0.48 Grey 0.86
5. Red 0.57
6. Red 0.76
7. Red 0.84

35
 TABLE 4

FLUORESCENCE ANALYSIS

Naregamia alata (Whole plant) Walsura trifoliata (Aerial parts)

S.
Treatment Under ordinary Under UV light Under UV light Under ordinary Under UV light Under UV light
No
light (265nm) (365nm) light (265nm) (365nm)
1. Powder Green Yellowish green Brown Pale green Palegreen Reddish brown
2. Powder+Petroleumether Pale yellow Green Brown Pale green Dark green Dark brown
3. Powder+Hexane Yellowish green Green Brown Pale green Pale green Blackish brown
4. Powder+Chloroform Yellowish green Light green Brown Dark green Brown Black
5. Powder+Ethylacetate Dark yellow Yellowish green Dark brown Pale green Dark green Black
6. Powder+Ethyl alcohol Yellow Pale green Brown Orange Light green Black
7. Powder+Distilled water Pale green Pale yellow Brown Light brown Pale green Brown
8. Powder+1N NaOH Yellow Light green Brown Reddish brown Dark green Blackish brown
9. Powder+1N HCL Pale yellow Pale green Brown Light brown Pale green Dark brown

36
 TABLE 5

QUALITATIVE ORGANIC ANALYSIS

Naregamia alata Walsura trifoliata

S.No Contents of the alcoholic extract
(Whole plant) (Aerial parts)
1. Steroid - -
2. Triterpenoid + +
3. Alkaloid + +
4. Phenol + +
5. Flavonoid + +
6. Coumarin + +
7. Quinone + +
8. Furan + +
9. Tannin + +
10. Glucoside/Sugars + +
11. Saponin - -

37
 TABLE 6
COMPARISON OF SALIENT FEATURES OF THE TWO TAXA
S.NO CHARACTERS NAREGAMIA ALATA WALSURA TRIFOLIATA
1. Habit Under shrub Tree
2. Habitat Moist deciduous forests Dry ever green forests
3. Leaf Trifoliate, cuneate-obovate Trifoliate, elliptic
4. Fruits Capsule Berry
5. Stomatal type Cyclocytic stomata Cyclocytic stomata
6. Subsidiary cells Six or more Nine to eleven
7. Guard cells 15×20µm in size 20×20µm in size
8. Lamina 120µm thick 260µm thick
9. Veinlets Thin and less prominent Thick and straight
10. Vein terminations Long, slender and wavy. Either unbranched or Short, thick and curved. Either unbranched or
branched once or twice branched once
11. Petiole Two lateral wide wings Two lateral short wings
12. Stem Occurs in continuous circle around the xylem Quite thick, comprising 2 or 3 layers of
Secondary phloem cylinder. phloem sclerenchyma, alternating phloem
elements.
13. Periderm Eight layers of tabular, suberized cells Four or five layers of rectangular, suberized
cells
14. Powder microscopy Abundant, narrow, thick walled and tapering Equally abundant, long, thin, needle shaped
Fibres at the end, upto 550µm long and 15µm thick. upto 500µm long and 10µm thick.
15. Vessel elements Abundant, long, narrow and fibre like, they Abundant, long, cylindrical, thick, they have
have simple, oblique, elliptical perforations at simple slightly oblique end wall perforations,
the end, long narrow tails. Pits are circular, short tails and well developed pits.
multiseriate and bordered.
16. Sclerenchyma Present in the leaf vascular strand. Present in the stem cortex.
17. Spiny cuticle Present in the petiole epidermis. Present in the stem epidermis.
18. Epidermal trichomes Present in the leaf epidermis(lamina) (Powder Present in the leaf epidermis (lamina T.S).
microscopy)

38