Anthelmintic activity of crude water extract of neem (azdirachta indica) leaf and stem bark against nematode parasites of smal

Animal Research International (2006) 3(3): 549 – 552 549
IN-VITRO ANTHELMINTIC EFFICACY OF CRUDE AQUEOUS EXTRACTS OF
NEEM (Azadirachta indica) LEAF, STEM AND ROOT ON NEMATODE
NWOSU, Chukwunyere Okwudiri, YAKUBU, Samaila, SALEH, Ummate Abba and ABDULLAHI, Gipaja
Department of Veterinary Microbiology and Parasitology, University of Maiduguri, PMB 1069, Maiduguri,
Corresponding Author: Nwosu, C. O., Department of Veterinary Microbiology and Parasitology, University of
Maiduguri, PMB 1069, Maiduguri, Borno State, Nigeria. Phone: (+234) 0803-4535510. Email:

ABSTRACT

The anthelmintic efficacy of the aqueous extracts o neem (Azadirachta indi
ca) leaf and stem and
root barks against the hatching of eggs and the survival of larvae of nematode parasites of small
ruminants were studied. The results of the in vitro egg hatch assay showed that the aqueous
extracts of the leaf and stem bark produced significant anthelmintic effect through reduction in
nematode egg hatch. The reduction in egg hatch was concentration dependent being highest (51
% and 50 % for the leaf and stem bark extracts respectively) at the highest concentration (100
mg/ml) of the extracts but inferior to those produced by albendazo e (10
0 % at 40 mg/ml).
Aqueous extracts of the leaf and root bark produced significant reduction in larval survival within
60 minutes at ambient temperature (30 – 35 ºC). Larval death was similar in both extracts and
concentration dependent, increasing with increasing concentration of the leaf and root bark
extracts. The reduction in larval survival due to the extracts was similar to that produced by
albendazole. In general, the aqueous extract of neem leaf was more efficacious in limiting
nematode larvae survival and in-vitro egg hatch. The results confirm the folkloric claims that neem
has anthelmintic effect and thus suggest its possible usefulness as an anthelmintic.

Keywords: Anthelmintic efficacy, Aqueous extract, Leaf, Stem, Root, Bark, Azadirachta indica

INTRODUCTION
(Ekanem, 1978; Ivbijaro, 1987; Khalid et al., 1989; Dwivedi, 1999), promote cutaneous wound healing Gastrointestinal helminthiasis, especially parasitic following mange infestation as well as act as fly- gastroenteritis (PGE) constitutes a major set back in repellent against haematophagous insects (ITDG and the productivity of small ruminant livestock in Nigeria IIRR, 1996; Nwosu, 2001). However, it is evident and other tropical countries (Schillhorn van Veen, that only a small proportion of the possible medicinal 1973; Akerejola et al., 1979; Chiejina, 1987; Nwosu usefulness of the plant in veterinary practice has et al., 1996 ab). The control of PGE is usually been exploited (Nwosu, 2001). In this paper, we achieved through anthelmintic medication and evaluated the anthelmintic efficacy of aqueous grazing management in cattle (Chiejina and Emehelu, extracts of neem leaf, root and stem barks against in- 1986; Chiejina, 1987) and goats (Nwosu et al., vitro hatching of eggs and the survival of the larvae 1996b). However, extensive use of anthelmintics has of nematode parasites of small ruminants. resulted in drug resistance for many nematodes of sheep, goats and cattle (Jackson, 1993; Pritchard, MATERIALS AND METHODS
1994). Since newer anthelmintics are not being brought into the market, there has been increasing Neem: Fresh leaves, stem and root barks of neem
search for novel, environmentally friendly and more were collected from adult plant within the University sustainable drugs for control of helminthosis. of Maiduguri, Nigeria. A botanist in the Department Consequently, several plants traditionally said to have of Biological Sciences, University of Maiduguri, medicinal properties are being investigated for their Nigeria, where voucher specimen of the plant was potency. The plant, Azadirachta indica commonly deposited, confirmed the identity of the plant. The known as neem is a good candidate for such stem and root barks were pealed off the plant using a sharp knife while the leaves were hand-cut. The All parts of the plant including the leaves, samples were collected into polythene bags and bark, fruits, seed, oil and sap have been shown to transported to the laboratory for processing. have medicinal properties and contain over ten The neem leaves, stem and root barks were different active components with azadirachtin as the sun-fried for 10 days at 8 hours per day. They were most potent and widely studied (Nwosu, 2001). separately ground into powder using a pestle and Neem is very popular in traditional medicine and neem-derived medicinal preparations have been exhaustively soxhlet extracted using the aqueous shown to be efficacious against a wide range of method (Mittal et al., 1981; WHO, 1992). The animal diseases including bacterial (ITDG and IIRR, extracts were concentrated in a conical flask 1996), protozoal and other parasitic conditions ISSN: 159-3115 ARI 2006 3(3): 549 – 552 www.zoo-unn.org Table 1: In-vitro anthelmintic efficacy of crude aqueous stem bark and leaf extracts of neem
(Azadiracta indica) against strongylid nematodes of small ruminants
Extract/Drug
No. of samples with
No. of larvae hatched
Reduction in egg
concentration
egg hatch*
Mean ± S.D.
hatch (%)
Water control
Stem bark extract
100 mg/ml
Leaf extract
100 mg/ml
Albendazole
t l cultures was used as standard ( .ie. 0 % reduction in egg hatch); abc Figures in same column with different superscrip s
Table 2: Survival of infective nematode larvae following incubation in albendazole or neem leaf and
root bark extracts for 60 minutes at room temperature
Extract/Drug
Number of surviving larvae
Percent larval death after
concentration
Mean ± S.D.
60 minutes
Water control
Leaf extract
100 mg/ml
Root bark extract
100 mg/ml
Albendazole
12.5 mg/ml
- 6.25 mg/ml
*Larval survival in water (control) was used as standard (i.e. 0% larval death; abc Figures in same column with different superscripts are significantly different (P<0.05) Each extract was diluted in three concentrations (25, identified using standard parasitological criteria 50 and 100 mg/ml). The in-vitro anthelmintic efficacy of the various concentrations of the leaf and stem bark extracts was evaluated against the Albendazole: Albendazole, containing 250 mg of
hatching of nematode eggs using the egg hatch Albendazole B. P. (Sam Pharmaceutical, Nigeria assay (Kelly et al., 1981) while the survival of Limited) was used for the study. Three dilutions (25, infective larvae in various concentrations of the leaf 12.5 and 6.25 mg/ml) of the drug were used for the and root bark was evaluated by culturing a known study based on previous studies (Onyeyili et al., 2001 number of larvae in the solutions for 60 minutes. In all cases, the proportion of unhatched eggs or dead larvae, at each concentration of the extracts was Data Analysis: The results were summarized as
calculated by relating the number of hatched eggs or means + Standard Error while differences between surviving larvae to the total number of eggs or larvae the means were analysed at the 5 % level of significance using the one way analysis of variance Faecal Samples: Faecal samples were collected
directly from the rectum of trade sheep and goats during slaughter at the Maiduguri Metropolitan Abattoir. Faecal egg counts were determined by the The results of the egg hatch assay using the aqueous modified McMaster technique using saturated sodium extract of neem leaf and stem bark are presented in chloride solution as the floating medium (MAFF, Table 1. Compared to the control (water cultures), 1977). Only samples with counts of at least 500 eggs both the leaf and stem bark extracts showed per gram of faeces were used in the test. Faecal significant reduction in nematode egg hatch. In both culture and larval recovery were done using the test cases, the reduction in egg hatch was concentration tube filter paper method described by Harada and dependent with the greatest reduction in egg hatch Mori (1955). Nematode eggs and larvae were at the highest concentrations (100 mg/ml) of the extracts used in the study. The reduction in egg as well as purify the extract, determine the active hatch was similar (P > 0.05) with the leaf and stem components, their lethal dose, appropriate route of bark extracts at the various concentrations tested but administration as well as ascertain which particular these were significantly less (P < 0.05) effective than parasite species and/or developmental stage are most susceptible to the effect of the extracts so as to The survival of strongyle larvae cultured for further enhance their anthelmintic usefulness. When 60 minutes in water or various concentrations of these studies have been carried out, a new albendazole and the aqueous extracts of the leaf and anthelmintic that will be readily available and root bark of neem are presented in Table 2. The acceptable to the rural farmers may be produced. results showed that both the leaf and root bark Meanwhile, this study highlights the possible extracts caused larval death in a similar manner (P > anthelmintic usefulness of neem extracts in the 0.05). Larval survival in both extracts was also control of nematode infections of small ruminants. concentration dependent, decreasing with increasing concentration of the extracts. The reduction in larval REFERENCES
survival caused by the leaf and root bark extracts was similar (P > 0.05) to that produced by albendazole at AKEREJOLA, O. O., SCHILLHORN VAN VEEN, T. W. caprine diseases in Nigeria: a review of DISCUSSION
economic losses. Bulletin of Animal Health The results of the study revealed that neem leaf, root CHIEJINA, S. N. (1984). Bovine parasitic and stem bark extracts have some anthelmintic gastroenteritis in Nsukka. Worm egg output properties against strongylid nematodes of sheep and and herbage infestation following dry season goats since they significantly limited the hatching of nematode eggs and the survival of nematode larvae during their first grazing season. Tropical cultured in them. These findings confirm the folkloric claims regarding the anthelmintic efficacy of neem CHIEJINA, S. N. (1987). Parasitic gastroenteritis in against intestinal helminths. Previous studies have cattle and small ruminants: pathogenesis, shown that neem extracts similarly affected the survival of salmonella (ITDG and IIRR, 1996) CHIEJINA, S. N. and EMEHELU, C. O. (1986). 1978; Ivbijaro, 1987; Khalid et al., 1989). That the Evaluation of three strategic anthelmintic leaf, stem or root bark extract showed similar effects in reducing egg hatch or larval survival suggest that gastroenteritis in cattle in eastern Nigeria. the extracts contain similar active components Tropical Animal Health and P oduction, 18: possibly in similar concentrations and probably possess the same mechanisms of action. These DWIVEDI, S. K. (1999). Evaluation of indigenous observations suggest the possible usefulness of the herbs as anti-trypanosomal agents. Pages aqueous extracts of neem leaf, stem and root barks 10 – 15. In: MATHIAS, E., RANNEKAR, D. V. in the treatment of nematode infections in both man Ethnoveterinary medicine: alternatives for At the various concentrations used in this study, the efficacy of the extracts was concentration- dependent as they showed graded effect that was highest at the 100 mg/ml concentration. However, at EKANEM, J. (1978). Dongo-Yaro, does it work? this concentration (100 mg/ml), the effects of the extracts were significantly (P < 0.05) inferior to GRAPHPAD INSTAT (2000). GraphPad InStat version albendazole in preventing nematode egg hatch but 3.00 for Windows, GraphPad Software, San similar in preventing nematode larval survival. The efficacy of the extracts or drug in limiting egg hatch HARADA, Y. and MORI, O. (1955). A new method for has a direct bearing on the effective penetration of their active components through the eggshell to reach the larvae. This may explain the greater IBRAHIM, M. A., NWUDE, N. J., ALIU, V. O. and efficacy of the extracts and the drug in limiting larval survival than egg hatch at the various concentrations Previous studies suggested that the anti- trypanosomal activity of neem extracts was associated with the alkaloids or other ingredients and ITDG and IIRR, (1996). Ethnoveterinary medicine in had effect in-vitro but became degraded or Kenya: a field manual of traditional health metabolised and thus ineffective when introduced into the host animal (Dwivedi, 1999). Consequently, there is need for further studies, especially in-vivo Institute of Rural Reconstruction, Nairobi, studies, in order to confirm the present observations IVBIJARO, M. F. (1987). Prospects for neem. Pages NWOSU, C. O., OGUNRINADE, A. F. and FAGBEMI, B. ASCHER, K. R. S. (Eds.). Natural pesticides changes in the gastro-intestinal helminths of from the neem tree (Azadirachta indica A Nigerian goats. Journal of Helminthology, Agency for Technical Co-operation (GTZ), NWOSU, C. O., HARUNA, N. K., OGUGBUAJA, V. O., JACKSON, F. (1993). Anthelmintic resistance – the (2001). Anthelmintic efficacy of ethanolic state of play. British Veterinary Journal, stem bark extract of Sacoglottis gabonensis (Baill) against strongyline nematodes of KELLY, J. D., WEBSTER, J. L. and GRIFFIN, D. L. anthelmintics in equine strongyle. Australian NWOSU, C. O., MOBEE, K. M., GULANI, I. G., KHALID, S. A., DUDDECK, H. and GOUZALEZ-SIERRA, M. (1989). Isolation and characterization of aqueous extracts of Garcina kola seed and stem bark against strongylid nematodes of small ruminants in Nigeria. Nigerian Journal MAFF. (1977). Manual of Veterinary Parasitological ONYEYILI, P. A., NWOSU, C. O., AMIN, J. D. and Laboratory Techniques. Technical Bulletin JIBIKE, G. I. (2001a). Anthelmintic activity No. 18, Ministry of Agriculture, Fisheries and of crude aqueous extract of Nauclea latifolia MITTAL, G. C., AGUWA, C. N., EZEIRU, V. U. and AKUBUE, P. I. (1981). Preliminary ONYEYILI, P. A., AMIN, J. D., H. I. GAMBO, NWOSU, pharmacological studies on antivenom action C. O. and JIBIKE, G. I. (2001b). Toxicity and of Diodia scandens leaves. Nigerian Journal anthelmintic efficacy of ethanolic stem bark extract of Nauclea latifolia sm. Nigerian NWOSU, C. O. (2001). Formulation of neem-derived biopesticides as alternative to persistent PRICHARD, R. K. (1994). Anthelmintic resistance. organic pollutants (POPS) for the control of Veterinary Parasitology, 54: 259 – 268. pests of livestock. Paper presented at the SCHILLHORN VAN VEEN, T. W. (1973). Small Utilization of Neem Tree in Northern Nigeria, Nigeria with emphasis on the helminthiasis. Dutse Jigawa State, 18 – 19th September, Nigerian Veterinary Journal, 2: 26 - 31. SOULSBY, E. J. L. (1982). Helminths, Arthropods and NWOSU, C. O., OGUNRINADE, A. F. and FAGBEMI, B. edition, Baillere Tindall, London. 809 pp. Haemonchus species in Red Sokoto (maradi) WHO (1992). Quality Control Methods for Medicinal Plants Materials. World Health Organization,

Source: http://www.zoo-unn.org/ARI%20vols/vol3/vol3-3/Nwosu.pdf