Vol. 7(8), pp.

369-373, August 2013

DOI: 10.5897/AJPS2013.1003
ISSN 1996-0824 2013 Academic Journals
http://www.academicjournals.org/AJPS

African Journal of Plant Science

Full Length Research Paper

In vitro anthelminthic activities of four medicinal plants

against Haemonchus contortus
Abdi Mohammed1, Abebe Wossene2, Mirutse Giday2, Getachew Tilahun2 and Nigatu Kebede2*
1

Faculty of Veterinary Medicine, Addis Ababa University, Debre Zeit, Ethiopia.

Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.

Accepted 10 June, 2013

In vitro experiments were conducted to determine the anthelmintic effects of crude aqueous extracts of
the leaves of Carissa spinarum and Azadrichta indica, fruits of Phytolacca dodecandra and stem bark of
Acacia tortilis on eggs and adults of Haemonchus contortus using egg hatch assay and mortality of
adult parasite. Extracts of the leaves of C. spinarum and A. indica inhibited hatching of egg at
concentration less than or equal to 1 mg/ml. Low egg hatch inhibition were observed for extracts of A.
tortilis (100%) and P. dodecandra (99.4%) at the maximum concentration tested (2 mg/ml). Of the plants
tested, extracts of C. spinarum and A. indica showed very good activity against the adult worms of H.
contortus, mortality raised to the levels of 96.8 and 93.9%, respectively, at concentration of 4 mg/ml. P.
dodecandra and A. tortilis produced mortality of 68.1 and 53.03% of adult H. contortus at 4 mg/ml
concentration, respectively. Albendazole killed the parasites in a dose dependant manner and all the
worms were dead at a concentration of 0.5 mg/kg within 24 h. The overall findings of the current study
indicated that most of the plants have potential anthelmintic effect warranting further in vitro and in
vivo evaluation.
Key words: Anthelmintic, Haemonchus contortus, in vitro experiment, plant extracts.

INTRODUCTION
Helminthosis play a crucial role in small ruminant production leading to enormous economic losses particularly in
areas where extensive grazing is practiced (Tembely et
al., 1994; Waller, 1997). It causes loss of production
directly and indirectly. The direct loss is manifested
through mortality, loss of blood and plasma protein by
blood sucking behavior of the parasites and leakages into
gastrointestinal tract, depression of mineral level and
diarrhea, all contributing to weight loss, reduced milk and
wool production (Soulsby, 1986). The indirect economic
impact is manifested by increased cost of control strategies (anthelmintics, labor, drenching equipments) and
other parasite-related penalties such as delay in achieving target weights, increased feed requirements to achieve
target weight and reduced quality of carcass and

predisposition to other diseases (Kassai, 1999).

Haemonchus contortus, the causative agent of haemonochosis, is one of the most pathogenic and highly prevalent nematode parasites of small ruminants particularly in
the tropics and subtropics. Haemonchosis is characterized by anemia attributable to blood loss via blood
sucking activities of worms in the abomasums (Soulsby,
1986); causing acute disease and high morality in all
classes of livestock (Allonby and Urquhart, 1975). It is
one of the top 10 constraints of sheep and goat production in East Africa (Perry et al., 2002). In Kenya, haemonchosis alone was estimated to cause an annual loss of 26
million dollar in sheep and goat production (Allonby and
Urquhart, 1975).
Commercial anthelmintics have been used for some

*Corresponding author. E-mail: [email protected]. Fax: 251-11-2755296. Tel. +251112763091.

370

Afr. J. Plant Sci.

decades throughout the world to minimize the losses

caused by helminth infections (Waller, 1997). The threats
of anthelmintic resistance, risk of residue, availability and
high cost, especially to farmers of low income in developing countries, have led to the notion that sustainable
helminth control can not be achieved with commercial
anthelmintics alone. Other alternative options like biological control, vaccine and traditional medicinal plants are
being examined in different corners of the world (Bain,
1999; Chandrawathani et al., 2003; Githiori, 2004; Waller
and Thamsborg, 2004). Evaluation of the activities of
medicinal plants claimed for anthelmintic property is
getting attention these days (Gathuma et al., 2004;
Githiori, 2004). Rich literature is available on ethno veterinary use of medicinal plants as anthelmintics. Traditionally in Ethiopia, the leaves, root and bark of Carissa
spinarum are used against GIT parasites and ring worm
(Sory, 1999; Hayatu, 2003). The leaves and fruits of
Phytolacca dodecandra are used against endo-parasites
(Belay, 2004), and the leaves as antiseptic (Haylessielassie,
2004). The root, bark and inner bark of Acacia tortilis are
used to treat diarrhea (Sory, 1999; Beyecha, 2004) and
the bark is used topically to treat ring worm (Hayatu,
2003). The roots, leaves and bark of Azadrichta indica
used against endo- and ecto parasites (Sory, 1999;
Beyecha, 2004). However, most of the reports did not
provide detailed information on the part of the plants used
and method of preparation. Often, no validation of the
effect against the disease conditions is provided.
The present study was, therefore, carried out to assess
the anthelmintic activities of aqueous extracts of four
Ethiopian plants, C. spinarum L. (Apocynaceae), A. indica
A. Juss (Meliaceae), P. dodecandra LHerit (Phytolaccaceae) and A. tortilis (Forssk) Hayne (Fabaceae), in vitro
using eggs and adults of live H. contortus. Aqueous
extracts of these plants have not previously been evaluated for their activity against H. contortus.
MATERIALS AND METHODS
Plant collection
Selection of plants was based on literature survey on traditional
uses in Ethiopia and other parts of the world. The leaves root and
bark of C. spinarum are used against GIT parasites and ring worm
(Sory, 1999; Hayatu, 2003). The leaves and fruits of P. dodecandra
are used against endoparasites (Belay, 2004), and the leaves as
antiseptic (Haylessielassie, 2004). The root, bark and inner bark of
A. tortilis are used to treat diarrhea (Sory, 1999; Beyecha, 2004)
and the bark is used topically to treat ring worm (Hayatu, 2003).
The roots, leaves and bark of A. indica used against endo- and
ectoparasites (Sory, 1999; Beyecha, 2004). For the in vitro test,
leaves of C. spinarum and A. indica, fruits of P. dodecandra and
stem bark of A. tortilis were collected from different localities of the
country between November 2007 and January 2008. All the plants
were identified by a plant taxonomist and voucher specimens of
each species were deposited at the Aklilu Lemma Institute of
Pathobiology (ALIPB), Addis Ababa University. The collected plant
parts were air-dried at room temperature, ground and stored until
extraction.

Extraction method
Aqueous extracts were produced at the Aklilu Lemma Institute of
Pathobiology (ALIPB). Fifty grams of the dry powder of each plant
sample was socked in distilled water and shaken for 24 h by electric
shaker. The suspension was filtered using filter paper and the filtrate kept in deep freezer (-70C) for 24 h, and then lyophilized
using lyophilizer. The lyophilized extract (freeze-dried) dry powder
was then collected, weighed and kept in a dry place to avoid
absorption of water until being used for the test.

Parasites
Adult female parasites of H. contortus were collected from the
abomasums of infected sheep obtained from Addis Ababa
Abattoirs Enterprise. H. contortus is reported to have developed
resistance to most anthelmintics used in the country according to
unpublished reports of the Ethiopian Ministry of Agriculture and
Rural Development. The worms were washed and crushed to liberate eggs. The eggs were then cultured in a glass jar filled with
autoclaved horse faeces for eight days at room temperature. At the
end of 8th day, infective larvae were harvested by rinsing the side of
the culture jar with a drop of water. About 3000 larvae were then
orally inoculated to worm free sheep (5 g of feaces was taken from
the rectum for floatation and concentration techniques to make sure
that the sheep were worm free) of ages between 4 and 6 months,
kept in-door in separate house in the animal facilities of the ALIPB
throughout the study period. These sheep served as H. contortus
egg donors for subsequent in vitro trials.

In vitro experiments
Collection of eggs
Briefly, faecal pellets were collected from the rectum of donors
sheep and placed in small bucket. Warm water was slowly added to
the faeces and the pellets stirred until a relatively uniform homogenate was obtained, liquid suspension was obtained. The suspension was filtered through sieve with 3 mm aperture. The resulting
suspension was again made to pass through a sieve of 150 m
pore size. The suspension was then poured into 15 ml test tubes
and centrifuged for 2 min at 377 g and the supernatant decanted.
The tube was agitated by vortex mixer to loosen the sediment.
Saturated sodium chloride was then added to the test tube until the
meniscus forms above the test tube on which the cover slip was
placed. After 3 to 5 min, the cover slip was carefully taken off the
tube and eggs washed into glass centrifuge tubes filled with water
and centrifuged for 2 min at 377 g. Most of the water was then
decanted and the number of eggs per ml was determined before
diluting it to the required concentration for use in egg hatch assay.
Egg hatch assay
The egg hatch assay was conducted according to World Association for the Advancement of Veterinary Parasitology (WAAVP)
guidelines (Coles et al., 1992). The eggs used for this test were
aged less than 3 h. Aqueous extracts of the plant materials were
used as the active treatment. Albendazole (99.3% pure standard
reference) obtained from the Ethiopian Drug Administration and
Control Authority (DACA) was used as positive control while
untreated eggs in water were used as negative control. The test
was conducted in 4 ml test tubes. In the assay, approximately 100
to 120 eggs in 1.5 ml water were placed in each test tube. Each
plant extract was serially diluted in a total volume of 2 ml distilled
water to make concentrations of 0.0625, 0.125, 0.25, 0.5, 1 and 2
mg/ml together with water containing the eggs. Albendazole origin-

Mohammed et al.

371

nally dissolved in dimethyl sulfoxide (DMSO) and distilled water at

the concentrations of 0.0625, 0.125, 0.25, 0.5, 1 and 2 g/ml was
used. The test tubes were then covered and kept in incubator at
27C for 48 h. The experiment was conducted in duplicates for
each concentration and replicated three times. Hatched larvae
(dead or alive) and unhatched eggs were then counted under
dissecting microscope at 40X magnification.

P. dodecandra produced 68.1% mortality at the same

concentration. Albendazole, on the other hand, brought
about 100% parasite mortality at a concentration of 0.5
mg/kg within 24 h (Table 2).

In vitro effect of plant extracts on adult worms

In this study, significant variation in the yield of extracts

among the different medicinal plants was observed. The
fruits of P. dodecandra gave the highest yield while the
lowest was observed for the leaves of C. spinarum. Apart
from the difference in species, plant part used, age,
harvest season and habitat could also contribute to the
variation in biochemical profiles and yields (Habtemariam
et al., 1994). Several in vivo and in vitro techniques have
been developed to detect anthelminitic resistance in
nematodes (Craven et al., 1999). However, in vivo tests
are not the best model to screen plants extracts with
anthelmintic activity, since these tests are time-consuming, expensive and present low precision and reproducibility due to inter-animal variation and pharmacodynamics of the drugs in the host (Lacey et al., 1990).
Although, the adult nematode is the major target for the
chemotherapy, gastrointestinal nematode parasites cannot yet be raised in continuous culture (Geary et al.,
1999). Thus, the lack of a culture system yielding adult of
any nematode parasite prevents a preliminary study of
the effect of medicinal plants on this stage. On the other
hand, the in vitro tests using free living stages of parasitic
nematodes offer a means of evaluating the anthelmintic
activity of new plant compounds, as already reported by
various authors (Asase et al., 2005). Most of the plant
extracts in the current study inhibited 100% egg hatchability at low concentration (2 mg/ml) as compared to other
plants studied previously. For example, 7.1 mg/ml of
aqueous extract of Annona senegslensis, 2.5 mg/ml of
essential oil of Ocimum gratissimum and 50 mg/ml
methanol extract of Spigelia anthelmia inhibited 11.5%
(Alawa et al., 2003), 96.94% (Pessoa et al., 2002) and
97.4% (Asase et al., 2005) of egg hatchability, respecttively.
Plant materials evaluated in the current study had been
identified from various sources to serve as anthelmintic
agents by traditional healers or farmers in different parts
of Ethiopia. The leaf, root and bark of C. spinarum is
used against GIT parasites and ring worm (Sory, 1999;
Hayatu, 2003). This finding is confirmed by the current
study that the leaves of C. spinarum have a higher effect
on the survival of the parasite showing 100% inhibition of
eggs hatchability at concentration of 0.5 mg/ml and
98.8% adult mortality of H. contortus at concentration of 4
mg/ml. The roots, leaves and bark of A. indica are used
traditionally against endo- and ecto-parasites (Sory,
1999; Beyecha, 2004) and the present study also showed
that the aqueous extract from the leaves of this plant
exhibited 100% inhibition of egg hatchability at concentration of 1 mg/ml and 93.9% of adult mortality of H.
contortus at concentration of 4 mg/ml. The leaves and

Adult H. contortus were collected from abomasums of the sheep

slaughtered at the Addis Ababa Abattoirs enterprise. Immediately
after slaughtering, the abomasums were collected and transported
to ALIPB laboratory. The collected parasites were washed and kept
in phosphate buffered saline (PBS). The test was performed in 5
cm diameter plastic petridish. Nine to eleven worms were placed in
petri dishes filled with 0.25, 0.5, 1, 2, 4 and 8 mg/ml of the extract of
the plant material in PBS and those with PBS alone (to serve as a
control group) in total volume of 4 ml. Albendazole dissolved in
DMSO at the concentration of 0.0625, 0.125, 0.25, 0.5, 1 and 2
mg/ml was also used as a positive control. Each concentration was
tested in triplicate. After 24 h, the extract was washed away and the
parasites were re-suspended in PBS for 30 min for possible recovery of the parasite motility. Finally, the number of motile (alive) and
immotile (dead) worms were counted under dissecting micro-scope
and recorded for each concentration. A mortality index was calculated as the total number of dead worms divided by the total
number of worms per petri dish.
Data management and statistical analysis
Comparison of mean percentages of egg hatch inhibition and adult
mortality, at different concentrations with the control, was performed
by one-way ANOVA. All statistical analysis was performed by SPSS
version 13.0. The Post Hoc statistical significance test employed
was list square difference (LSD), the difference between the means
were considered significant at p <0.05.

RESULTS
In vitro experiments
Egg hatch assay
All the four extracts of the plants exhibited good activities
against eggs of H. contortus; although, there was variation in doses required for each type of extract. In all plant
extracts, the maximum concentration used in the study (2
mg/ml) induced nearly 100% egg hatch inhibition. Extract
of C. spinarum induced 100% egg hatch inhibition at a
concentration of 0.5 mg/ml, the least concentration
among all plant extracts tested to bring about the same
effect, while P. dodecandra was the weakest plant that
gave 99.4% inhibition at 2 mg/ml concentration.
Albendazole induced 100% egg hatch inhibition at a
concentration of 0.25 g/ml (Table 1).
In vitro effects of plant extract on adult parasites
All the extracts showed inhibitory effect on the survival of
H. contortus in a dose dependant manner. C. spinarum,
A. indica and A. tortilis produced mortality of adult H.
contortus significantly to the level of 96.8, 93.9 and
53.03%, respectively, at a concentration of 4 mg/ml while

DISCUSSION

372

Afr. J. Plant Sci.

Table 1. Mean percentage inhibition of egg hatching after 48 h exposure of H. contortus to different concentrations of plant extracts
(mg/ml) and Albendazole (g/ml).

Plant type
Albendazole
Carissa spinarum
Phytolacca dodecandra
Acacia tortilis
Azadrichta indica

0.0
0.290.5
6.10.61
0.610.6
0.290.9
0.610.6

0.0625
15.71.4
12.51.7
2.80.12
5.80.97
4.92.6

Mean SE at different concentrations

0.125
0.25
0.5
63.21.9
1000
1000
36.610.2
99.70.5
1000
16.41.6
36.83.7
72.50.5
39.41.3
68.12.5
86.82.8
14.31.2
73.21.8
98.80.8

1
1000
1000
99.40.6
99.70.3
1000

2
1000
1000
99.40.6
1000
1000

Table 2. Mean percentage mortality of adult parasites after 24 h exposure of H. contortus to different concentrations of plant extracts
and Albendazole (mg/ml).

Control
Albendazole

Plants
Carissa spinarum
Phytolacca dodecandra
Acacia tortilis
Azadrichta indica

0.0625
37.82.2

0.125
67.63.9

Mean SE at different concentrations

0.25
0.5
89.90.6
1000

1
1000

2
1000

0.0
21.82.8
9.8o.6
16.13.1
36.6

0.25
21.71.7
26.73.3
19.40.6
24.12.1

Mean SE at different concentrations

0.5
1.0
2.0
62.16.6
89.63.3
89.60.37
38.810.2
5.80.97
39.41.3
25.62.9
4.92.6
14.31.2
36.61.9
89.93.3
78.83.3

4.0
96.83.3
68.12.5
53.031.5
93.93.1

8.0
1000
86.82.8
51.91.9
1000

fruits of P. dodecandra are traditionally used against

endo-parasites (Belay, 2004) and the leaves as antiseptic
(Haylessielassie, 2004). This is evident from the current
study which showed 99.4% inhibition of egg hatchability
by the plant extract at a concentration of 0.1 mg/ml and
68.1% of adult mortality of H. contortus at concentration
of 4 mg/ml. The root, bark and inner bark of A. tortilis
were reported to treat diarrhea (Sory, 1999; Beyecha,
2004) and the bark was used topically to treat ring worm
(Hayatu, 2003).
In the current study, aqueous extract of A. tortilis
exhibited 99.7% inhibition of egg hatchability at 0.1 mg/ml
and 53.03% of adult mortality of H. contortus at
concentration of 4 mg/ml. Based on the ability of plant
extract to inhibit egg hatchability, the most potent extracts
in a decreasing order were that of C. spinarum, A. indica,
A. tortilis and P. dodecandra. And based on the ability of
adult mortality, the most potent extracts were that of C.
spinarum, A. indica, P. dodecandra and A. tortilis. In this
study, all the four extracts showed very good effect (72.5
to 100%) in inhibiting egg hatchability at concentrations
between 0.5 and 2 mg/ml. Whereas extracts of two (C.
spinarum and A. indica) out of the four medicinal plants
showed a very good adult mortality (89.6 to 100%) of H.
contortus at concentrations between 1 and 8 mg/ml. On
the other hand, A. tortilis showed weak effect on adult
mortality of H. contortus at all concentrations used in the
study.

Conclusion
In the current study, extracts of all the study plants (C.
spinarum, A. indica, A. tortilis and P. dodecandra) have
shown promising in vitro anthelmitic activity against eggs
of H. contortus. Extracts from C. spinarum and A. indica
have shown promising adult mortality, while extracts of A.
tortilis did not demonstrate appreciable result. Based on
the aforementioned facts, the following recommendations
are forwarded: plants that demonstrated promising
activities in vitro for their different parts in the current
study should be further evaluated in vivo. Other types of
extracts and in vitro evaluation must be conducted for
those plant parts and extracts not showing promising
results. Phytochemical screening and toxicological evaluation should be performed for those that exhibited
promising results in the in vitro test.
ACKNOWLEDGEMENTS
The authors are grateful to Mr. Engidawork Muleta, Mr.
Hailu Getu and Mr. Nega Nigussie for their technical
assistance. Financial support was obtained from Research
and Graduate Studies of the Addis Ababa University.
REFERENCES
Alawa CB, Adamu, AM Getu JO, Ajansui O J, Abdu PA, Chiezey NP,
Alawa JN, Bowman DD (2003). In vitro screening of two Nigerian

Mohammed et al.

medicinal plants (Vernonia amygdalina and Annona senegalensis) for

anthelmintic activity. Vet. Parasitol. 113:73-81.
Allonby EW, Urquhart M (1975). The Epidemiology and the Pathogenic
Significance of Haemonchosis in Merino Folk in Eastern Africa. Vet.
Parasitol. 1:129-143.
Asase A, Oteng-Yeboah AA, Odamtten GT, Simmonds MS (2005).
Ethnobotanical study of some Ghanaian anti-malarial plants. J.
Ethnopharmacol. 99:273-279.
Bain RK (1999). Irradiated vaccines for helminth control in livestock. Int.
J. Parasitol. 29:185-191.
Belay K (2004). A Study on Ethnoveternary Medical Practices in East
Showa Zone, Ethiopia and the Anthelmenthic Effect of Withania
somnifea against H. contortus, DVM thesis. Addis Ababa University,
Faculty of Veterinary Medicine, Debrezeit, Ethiopia.
Beyecha K (2004). A Study on Ethinoveternary Knowledge and
Practices in Moyale Wereda of Liben Zone, Somali, DVM thesis.
Addis Ababa University, Faculty of Veterinary Medicine, Debrezeit,
Ethiopia.
Chandrawathani P, Jamnah O, Waller PJ, Larsen M, Gillespie AT,
Zahari WM (2003). Biological control of nematode parasites of small
ruminants in Malaysia using the nematophagous fungus
Duddingtonia flagrans. Vet. Parasitol. 117:173-183.
Coles GC, Bauer C, Borgsteede FH, Geerts S, Klei TR, Taylor MA,
Waller PJ (1992). World Association for Advancement of Veterinary
Parasitology (WAAVP) methods for detection of anthelmintic
resistance in nematodes of veterinary importance. Vet. Parasitol.
44:35-44.
Craven J, Bjorn H, Barnes EH, Henriksen SA, Nansen P (1999). A
comparison of in vitro tests and feacal egg count reduction test in
detecting anthelmintic resistance in horse strongyles. Vet. Parasitol.
85:49-59.
Gathuma JM, Mbaria JM, Wanyama J, Kaburia HF, Mpoke L, Mwangi
JN (2004). Efficacy of Myrsine africana, Albizia anthelmintica and
Hilderbrantia sepalosa herbal remedies against mixed natural sheep
helminthosis in Samburu district, Kenya. J. Ethnopharmcol. 91:7-12.
Geary TM, Sangster NC, Thompson DP (1999). Frontiers in anthelmintc
pharmacology. Vet. Parasitol. 84:275-295.
Githiori JB (2004). Evaluation of Anthelmintic Properties of Ethnoveterinary Plant Preparations Used as Livestock Dewormers by
Pastoralists and Small Holder Farmers in Kenya. Doctoral thesis.
Swedish University of Agricultural Sciences, Uppsala, Sweden.

373

Habtemariam S, Gray AI, Waterman PG (1994). Diterpenes from the

leaves of Leonotis ocymifolia var. raineriana. J. Nat. Prod. 57:15701574.
Hayatu A (2003). A study on Ethinoveternary Knowledge and Practice
in Low Lands of Borena Pastoral System, DVM thesis. Addis Ababa
University, Faculty of Veterinary Medicine, Debrezeit, Ethiopia.
Haylessielassie M (2004). Survey and Preliminary Screening of
Selected Medicinal Plants for Treatment of Bovine Mastitis and Skin
Diseases in Tigray, Northern Ethiopia. DVM thesis. Addis Ababa
University, Faculty of Veterinary Medicine, Debrezeit, Ethiopia.
Kassai T (1999). Veterinary Helminthology. Read Educational and
Professional publishing LTD, London.
Lacey E, Redwin JM, Gill JH, Demargheriti VM, Waller PJ (1990). A
larval development assay for the simultaneous detection of broad
spectrum anthelmintic resistance. In: Boray, J. C., Martin, P. J.,
Roush, R. T. (Eds.), Resistance of Parasites to Antiparasitic Drugs.
MSD Agvet, Rajway, Ny, pp.177-184.
Perry B, Randolph T, McDermott J, Sones K, Tornton PK (2002).
Investing in Animal Health Research to Alleviate Poverty,
International Livestock Research Institute, Nairobi, Kenya, pp.148.
Pessoa LM, Morias SM, Bevilaqua CML, Lucano JHS (2002). Anthelmitic activity of essential oil Ocimum gratissimum Linn. and eugenol
against Haemonchus contortus. Vet. Parasitol. 109:59-63.
Sory T (1999). Ethnoveterinatry Practices of the Borena Range Land
Pastoral System. DVM thesis. Addis Ababa University, Faculty of
Veterinary Medicine, Debrezeit, Ethiopia.
Soulsby EJ (1986). Helminths, Arthropods and Protozoa of Domesticated Animals. Balliere, Tindall, London.
Tembely S, Bengaly K, Berchmoes W (1994). Effect of strategic anthelmintic treatment on growth performance and survival rate of native
lambs in the sub humid environment in Mali, small ruminant research
and development in Africa. Proceeding of the Second Biennial
Conference of African Small Ruminant Research Net work, AICC
Arusha, Tanzania.
Waller P (1997). Sustainable helminth control of ruminants in
developing Countries. Vet. Parasitol. 71:195-207.
Waller PJ, Thamsborg SM (2004). Nematode control in green Ruminant
production system, Trends Parasitol. 20:493-497.