ANTHELMINTIC ACTIVITY OF ESSENTIAL OILS OF CYMBOPOGAN NARDUS

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1 ANTHELMINTIC ACTIVITY OF ESSENTIAL OILS OF CYMBOPOGAN NARDUS AND AZADIRACHTA INDICA ON FASCIOLA GIGANTICA N. Jeyathilakan*, K. Murali¹, A. Anandaraj², B.R. Latha and S. Abdul Basith Department of Veterinary Parasitology Madras Veterinary College, Chennai ABSTRACT The efficacy of essential oils of Cymbopogan nardus (citronella) and Azadirachta indica (neem) on Fasciola gigantica was assessed in vitro. The essential oil of citronella showed a potential anthelmintic activity whereas neem oil did not show any significant effect. Study revealed that 1% citronella oil showed anthelmintic effect on live Fasciola gigantica. The in vitro anthelmintic effect was evaluated in comparison with control and score index was made based on gross motility. The morphological variation and histopathological changes in treated and control fluke was studied. Key words: Citronella oil, Neem oil, Anthelmintic activity, Fasciola gigantica INTRODUCTION Fasciolosis has been recognized as an important helminthic disease of livestock causing significant loses to livestock owners, on account of poor growth and lower productivity of domestic ruminants. It is estimated that 300 million bovines and 2.4 million people are exposed to Fasciolosis world wide causing enormous loss, amounting to more than US $ 3 billion per annum. The disease is mainly caused by Fasciola gigantica in animals in India (Dalton, 1999). Effective control of this parasite includes strategic use of anthelmintic drugs and control of snail intermediate host. However problems have emerged with the use of chemical drugs, notably the development of resistance in parasite, chemical residues and toxicity problem as well as cost of drugs for treatment in animals. For these various reasons, screening of medicinal plants for their anthelmintic activities remains of great scientific interest. A number of medicinal plants have been used in man and animals (Akthar et al., 2000). Citronella oil is used in traditional medicine as an antiseptic, bactericidal, deodorant, tonic, stimulant, weedicide and acaricide (Thorsell et al., 2006). Neem oil is also used in traditional medicine as fly repellant (Sharma et al., 1993). Kushwaha et al. (2004) studied the effect of extracts of carioca, papaya, Mallotus phillipiensis and Azadirachta indica on Fasciola gigantica in vitro. Alcoholic extracts of Allium sativum and Piper longum showed inhibitor effect on F. gigantica (Singh et al., 2007). However effect of plant oils on F. gigantica was not studied elsewhere. Under these circumstances, the present study is taken up to evaluate the anthelmintic property of the herbal plant oils in vitro in comparison with flukicide drug oxyclozanide against F.gigantica. ¹ ²UG scholars *Corresponding author, drjthilakan@yahoo.com 204

2 Jeyathilakan et al.,. MATERIALS AND METHODS Collection of Liver flukes Fasciola gigantica were collected from the bile duct of infected cattle at the slaughterhouse in Perambur in Chennai. They were kept in normal saline and transferred to the laboratory of department of Veterinary Parasitology. After washing the flukes several times with normal saline, the healthy ones with normal microscopic structure and good motility were selected. They were kept in a RPMI 1640 medium(gibco,usa) until the experiment began. Source of plant oils Citronella oil was obtained from dried grass of Cymbopogan nardus (Graminae). The oil was extracted by steam distillation process of finely chopped fresh dried plant. Neem oil was extracted from the dried seeds of Azadirachta indica. Experimental design Anthelmintic activity was studied by in vitro petri dish method as described by Jiraungkoorskul et al. (2005) and Githiori et al. (2006). Ten flukes were taken for each group in petridishes with RPMI-1640 medium and exposed to each of the following treatment group at room temperature at a concentration of 1% of Citronella oil, neem oil, oxyclozanide drug powder treated control and normal RPMI-1640 control. The inhibition of motility and / or mortality of flukes were observed after 3,12,15 hrs and score index was made.the morphological and histopathological variation of flukes was studied after the experiment. The experiment was repeated thrice with fresh flukes. Motility criteria The motility was scored using the following criteria (Jiraungkoorskul et al., 2005) Score 3 - Moving whole body Score 2 - Moving only parts of the body Score1 - Immobile but alive Score 0 - Died Specimen preparation for light microscopic analysis Carmine staining After death of flukes, five flukes from each group were prepared for carmine staining. The flukes were washed thoroughly with 0.1M phosphate buffered saline, ph 7.4 and pressed in between two slides, tied both sides with rubber band and immersed in 10% formalin for at least 12 hrs for fixing. Then they were washed overnight in running tap water to remove the formalin. The washed flukes were dehydrated with ascending grades of alcohol three times and stained with acetic alum carmine stain overnight. The flukes were destained with 1% acid alcohol, washed in ammonia water, dehydrated with graded series of ethanol, cleared in xylene and mounted with DPX. They were examined for abnormalities using Olympus TL3, S2-ST dissection microscope (Tokyo, Japan) and photographed using a Leica dissection photomicroscope. Haematoxylin and eosin staining A further four flukes from each group were set for paraffin embedding. They were fixed in 10% formalin for 24 hrs, dehydrated with ascending series of ethanol and cleared with xylene. They were then embedded in paraffin, sectioned longitudinally at thickness of 5 micrometers using a rotary microtome and stained with haematoxylin and eosin.they were examined for abnormalities using an Olympus CH 40 light microscope (Tokyo, Japan) and photographed RESULTS AND DISCUSSION Gross visual motility The movement of the treated and control flukes were observed and motility score index was 205

3 Anthelmintic activity of... Fig 1 Control fluke uterus showing fully yolk packed eggs Fig 2. Citronella treated fluke uterus showing shrinkage of yolk material with various shapes in egg Fig 3. Control fluke showing tegument with spin Fig 4. Neem oil treated fluke tegument shows breakage of spines Fig 5. Vacuolation of parenchyma and sloughing of spines in citronella treated fluke Fig 6. Intestinal caeca with villi of control fluke. 206

Fig 7 Shrinkage of intestinal caeca with complete desquamation of villi in citronella oil treated fluke Jeyathilakan

Oxyclozanide treated fluke showing massive shrinkage of smooth muscle and disintegration of testis Fig 10.

The flukes (100%) in control and neem oil treated group remained active with whole body movements from 0 to 3 hrs

Fourty per cent of neem oil treated flukes showed motility by moving only parts of the body (score 2) and remaining

4 Fig 7 Shrinkage of intestinal caeca with complete desquamation of villi in citronella oil treated fluke Jeyathilakan et al.,. Fig 8 Erosion of intestinal villi in neem oil treated fluke Fig 9. Oxyclozanide treated fluke showing massive shrinkage of smooth muscle and disintegration of testis Fig 10. Control fluke showing normal testes prepared (Table.1). The flukes (100%) in control and neem oil treated group remained active with whole body movements from 0 to 3 hrs (score 3). Citronella oil and oxyclozanide treated flukes were dead within 3hrs (score 0). Fourty per cent of neem oil treated flukes showed motility by moving only parts of the body (score 2) and remaining 60% were died (score 0) at 12 hrs. The RPMI-1640 normal control flukes (80%) showed motility by moving only parts of the body (score 2) and remaining 20% flukes died (score 0). All the neem oil treated and RPMI-1640 control flukes were died at 15hrs (score 0) 207

5 Anthelmintic activity of... Groups % of Motile flukes Table.1 Motility Score index After 3 hrs After 12 hrs After 15 hrs Citronella oil 100% 80% 20% 40% 60% Neem oil Oxyclozanide control Normal control 100% 100% 80% 20% 100% Light microscopic studies The gross changes in carmine stained flukes were observed under light microscope. Grossly deformation of the body shape was observed in treated groups after death. Disintegration of Intestinal, uterine and testicular branches was noticed in citronella oil treated group. Eggs were blackened inside uterus and sloughing of spines from the cuticle was also observed. In case of neem oil group, spines of the flukes sloughed off from the tegument. Testicular branches were also separated. Spiny eruptions on the surface of the body and shrinkage of the fluke were noticed in oxyclozanide control group. The flukes were sectioned longitudinally and were explored for any histopathological changes in their organs under microscope. Morphological changes in the tegument layer and spines were observed in most of oil treated groups. Total disappearance of spines from cuticle (Fig, 5), complete desquamation of gut epithelial cells and absence of villi in intestinal caeca (Fig.7) were 208 noticed in citronella oil treated flukes. They also showed disintegration of eggs in uterus with half moon shaped shrinkage of yolk (Fig.2). The neem oil treated flukes showed the blebbing of teguments, breakage of spines (Fig.4) and erosion of intestinal villi (Fig.8). Vacuole formation was noticed in oxyclozanide control group (Fig.9) From the in vitro trials, it was concluded that citronella oil showed flukicidal effect similar to that of oxyclozanide. It affects the tegument, intestinal caeca and uterus. However comparatively neem oil showed less effect on flukes than citronella oil. Literature showed that, these oils were tried for controlling various ectoparasites (Abdul- Shafy and Zayed, 2002; Kim et al., 2004). However the effect of these oils on endoparasites was not studied earlier. The mechanism of action of these oils was not clearly understood. This in vitro study may act as a base for evaluation of effect of these oils in vivo against Fasciola gigantica. Toxicological studies of these oils also are carried out though

6 Jeyathilakan et al.,. Chenopodium oil was used for many years in UK to treat nematode parasite infections in monogastric animals including humans and mustard oil was tried for treating helminthic parasites (Gibson, 1965; Ketzis et al., 2002). These oils may be used to enrich feedstuffs like silage and hay and also incorporated in feeds as a binder for the preparation of feed pellets to treat fasciolosis in animals REFERENCES Abdul-Shafy, S. and Zayed, A.A. (2002). In vitro acaricidal effect of plant extracts of neem seed oil (Azadirachta indica) on egg, immature, adult stages of Hyalomma anatolicum excavatum (Ixodoidea:Ixodidae). Veterinary Parasitology,106: Akthar, M.S., Iqbal, Z., Khan,M.N and Lateef.,M.(2000). Anthelmintic activity of medicinal plants with particular reference to their use in animals in the Indo-Pakistan subcontinent. Small Ruminant Research, 38: Dalton, J. P. (1999). Fasciolosis. CABI publications. Wallingford, U.K., pp Gibson, T.E. (1965).Veterinary anthelmintic medication, second ed. Technical communication No.33 of the Commonwealth Bureau of Helminthology,St Albans, Herts, pp 206. Githiori, B., Athanasiadou, S and Thamsborg, M. (2006). Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants. Veterinary Parasitology, 139: Jiraungkoorskul, W., Sahaphong, S., Tansatit, T.,Kanwanrangsan,N and Pipatshukiat,S. (2005). Eurytrema pancreaticum: The in vitro effect of praziquantel and triclabendazole on the adult fluke. Experimental Parasitology, 111: Ketzis, J.K., Taylor, A., Bowman, D.D., Brown D.L., Warwick, L.D. and Erb,H.N.(2002). Chenopodium ambrosioides and its essential oil as treatment for Haemonchus contortus and mixed adult nematode infections in goats. Small Ruminant Research, 44: Kushwaha, D.S., Kumar, D., Tripathi, H.C. and Tandan, S.K. (2004). Effect of some indigenous medicinal plants on Fasciola gigantica in vitro. Indian Journal of Animal Science, 74: Sharma, V.P., Ansari,M. and Razdan, R.K.(1993).Mosquito repellent action of neem (Azadirachta indica) oil. Journal of American Mosquito Control Association, 9: Singh, T.U., Kumar, D. and Gupta P.K.(2007). Inhibitory effect of alcoholic extract of Allium sativum and Piper longum on gross visual motility and glucose uptake of Fasciola gigantica and Gigantocotyle explanatum. Journal of Veterinary Parasitology, 21: Kim,S.I., Yi,J.H., Tak,J.H and Ahn Y.Y.(2004). Acaricidal activity of plant essential oils against Dermanyssus gallinae. (Acari: Dermanyssidae).Veterinary Parasitology, 120; Thorsell, W., Mikiver, A. and Tunon, H. (2006). Repelling properties of some plant materials on the tick Ixodes ricinus Lin. Phytomedicine, 13:

Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum

Research Article Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum T.U. Singh, D. Kumar, S.K. Tandan ABSTRACT Division of Pharmacology