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ISSN: 0974-0376 NSave Nature to Survive : Special issue, Vol. 1; 87-92 QUARTERLY www.theecoscan.

1 ISSN: NSave Nature to Survive : Special issue, Vol. 1; QUARTERLY INHIBITION OF ACETYLCHOLINESTERASE AND PHOSPHATASES BY ACTIVE MOLLUSCICIDAL COMPONENTS OF SARACA ASOCA AND THUJA ORIENTALIS IN THE NERVOUS TISSUE OF LYMNAEA ACUMINATA Arundhati Singh et al. KEYWORDS Fascioliasis AChE Phosphatases Saraca asoca Thuja orientalis Saponin Thujone Paper presented in 3rd International Conference on Climate Change, Forest Resource and Environment (ICCFRE, 2011) December 09-11, 2011, Thiruvananthapuram, organized by Department of Environmental Sciences, University of Kerala in association with National Environmentalists Association, India 87

2 NSave Nature to Survive QUARTERLY ARUNDHATI SINGH, PRIYA SRIVASTAVA 1, P. KUMAR, D. K. SINGH AND V. K. SINGH* Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur , INDIA 1 Department of Zoology, St. Xavier s College, Ranchi , Jharkhand, INDIA vksingh_gpu@yahoo.co.in ABSTRACT Sub-lethal (40% and 80% of 24h LC 50 ) in vivo treatments of column purified Saraca asoca leaf and Thuja orientalis fruit and their active molluscicidal component saponin and thujone significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ ALP) activities in the nervous tissue of Lymnaea acuminata. The snail is the intermediate host of liver fluke Fasciola gigantica which endemic caused fascioliasis in the eastern Uttar Pradesh. Fascioliasis is the referred most prevalent parasitic disease in the world in terms of morbidity, socio-economic and public health importance. Column purified fraction of S. asoca leaf and saponin caused more inhibition of AChE than ACP/ALP. Among all the treatments highest inhibition (52.41% of control) in ACP activity was observed in the nervous tissue of snail exposed to 80% 24h LC 50 of S. asoca leaf column purified fraction. In withdrawal experiment there was a significant (p<0.05) recovery in AChE, ACP and ALP activity in the nervous tissue of L. acuminata. Thus results from the present study suggest that inhibition of AChE, ACP, and ALP by Saraca asoca and Thuja orientalis, and their active component in the snail Lymnaea acuminata may be the cause of snail death. INTRODUCTION Fascioliasis is one of the most debilitating zoonotic diseases. The world health organization (WHO) has estimated that 2.4 million people are infected with Fasciola, and a further 180 million are at risk of infection (WHO, 2007). Snails (Lymnaea acuminata, Indoplanorbis exustus, Bulinus, Passaria) serve as intermediate hosts for parasitic flat worm of Fasciola species (Osman et al., 2007). This disease ranks as major causes of morbidity and mortality both in man and live-stock and contribute to socioeconomic problem (Mas-Coma et al., 2005). Human infection has been reported in 51 different countries from 5 continents (Estaben et al., 1998; WHO, 2007). Due to higher funding priorities such as SARS, AIDS and malaria and research in immunological approaches to worm control, there is a little current interest in snail control as a means of managing fascioliasis/schistosomiasis (Kumar et al., 2011). Worldwide losses in animal productivity due to fascioliasis were conservatively estimated at over US$ 3.2 billion per annum (Spithill et al., 1999). Use of molluscicides to eradicate the snail vector is considered the method of choice to eliminate fascioliasis. One of the possible approaches to eradicate or control this problem is to interrupt the life cycle of the parasitic trematodes by eliminating the snail, which is essential to life cycle. A number of chemically diverse plant molluscicide have been isolated and identified (Singh et al., 1996). Advantages of natural products over synthetic have eco-friendly, biodegradable and hence are less likely to accumulate in the environment. Recently, it has been reported that Saraca asoca leaf and Thuja orientalis fruit and their active molluscicidal component saponin and thujone have potent molluscicidal activity against Lymnaea acuminata (Singh and Singh, 2009). The mechanism by which they cause snail death is not known. The present study is to explore the effect of the active moieties on the different enzymes namely, acetylcholinesterase (AChE), acid phosphatase (ACP), and alkaline phosphatase (ALP) in the nervous tissue of snail Lymnaea acuminata. MATERIALS AND METHODS *Corresponding author Test materials Fresh leaves and bark of Saraca asoca Roxb. (Asoke, family- Caesalpiniaceae) and leaves and fruits of Thuja orientalis Linn. (Morpankhi, family- Cupressaceae) were collected from the University campus. Saponin (Sapogenin ~ 10%) and Thujone (1-isopropyl, 4, methylcyclo [3.1.0] hexan-3-one were procured from Sigma Chemical Co. USA. Bioassay Adult snail Lymnaea acuminata (2.30 ± 0.25 cm in length) were collected locally and allowed to acclimatize in laboratory condition for 72h. Twenty experimental snails were kept in a glass aquarium containing 3 L of dechlorinated tap water at 22 24ºC. Six aquaria were set up for each concentration. Snails were exposed to sublethal concentrations 40% and 80% of 24h LC 50 of different column purified 88

3 INHIBITION OF ACETYLCHOLINESTERASE fraction of S. asoca and T. orientalis and their active components saponin and thujone for 24h (Table 1). Six batches were prepared for each concentration. Control aquarium contained only equal volume of dechlorinated tap water without treatment. After 24h of the treatment snails were washed with water and nervous tissue was quickly taken out for the measurement of different enzyme activity such as AChE, ACP and ALP. Nervous tissue was removed and placed on ice cubes. Afterwards, the nervous tissue was placed on filter paper to remove the adherent water and weight. Enzyme activity was performed in treated as well as control group of test animals. In withdrawal experiments the snails were transferred from 96h exposure of 80% of 24h LC 50 of different active components into the fresh water then after 96h enzyme assays were estimated. Acetylcholinesterase Acetylcholinesterase activity was measured according to the method of Ellman et al. (1961) as modified by Singh et al. (1982). Fifty miligram of nervous tissue of Lymnaea acuminata was taken around the buccal mass and homogenized in 1.0mL of 0.1M phosphate buffer ph 8.0 for 5 minutes in an ice bath then centrifuged at 1000 g for 30 minutes at 4 C. The supernatant was used as an enzyme source. Enzyme activity was measured in a 10mm path length cuvette using an incubation mixture consisting of 0.1mL of enzyme source, 2.9mL of 0.1M buffer ph 8.0, 0.1mL of chromogenic agent DTNB (5, 5-dithio-bis-2-nitrobenzoic acid), and 0.02mL of freshly prepared ATChI (aetylthiocholine iodide) solution in distilled water. The change in optical density at 412nm was recorded for 3 minutes after every 30 second interval at 25 C. Enzyme activity has been expressed as μmole SH hydrolyzed min/ mg/ protein. Acid Phosphatase Acid phosphatase (ACP) activity in the nervous tissue of Lymnaea acuminata was measured by the method of Bergmeyer (1967) as modified by Singh and Agarwal (1989). Tissue homogenate (2%, w/v) was prepared in ice cold 0.9% NaCl and centrifuged at 5000 g for 15 minutes at 4ºC. The supernatant was used as an enzyme source. 0.2mL of enzyme source was added to 1.0 ml of acid buffer substrate (0.41 g citric acid, g sodium citrate, and 165 mg 4-nitrophenyl phosphate sodium salt to 100mL of double distilled water) pre-incubated at 37ºC for 10 minutes. The incubation mixture was mixed thoroughly and incubated for 30 minutes at 37 C. 4.0 ml of 0.1 N NaOH was then added to the incubation mixture. The yellow colour, developed due to the formation of 4-nitrophenol, was determined by spectrophotometer at 420 nm. Standard curves were drawn with different concentrations of 4-nitrophenol. The ACP activity has been expressed as μmole substrate hydrolyzed/30 min/ mg protein. Alkaline Phosphatase Alkaline phosphatase (ALP) activity in the nervous tissue of Lymnaea acuminata was measured by the method of Bergmeyer (1967) as modified by Singh and Agarwal (1989). Tissue homogenate (2%, w/v) was prepared in ice cold 0.9% NaCl and centrifuged at 5000 g for 15 minutes at 4ºC. The supernatant was used as an enzyme source. 0.1mL of enzyme source was added to 1.0mL of alkaline buffer substrate (375 mg glycine, 10mg MgCl2.6H 2 O, 165 mg 4-nitrophenyl phosphate disodium salt in 42mL of 0.1N NaOH and a mixture was made up to 100mL with double distilled water). The incubation mixture was mixed thoroughly and incubated for 30 minutes at 37ºC. 10mL of 0.02N NaOH was then added to the incubation mixture. The yellow colour, developed due to the formation of 4-nitrophenol, was determined by spectrophotometer at 420 nm. Standard curves were drawn with different concentrations of 4-nitrophenol. The ALP activity has been expressed as μmole substrate hydrolyzed/30 min/ mg protein. Protein Protein was estimated in the enzyme source supernatant by the method of Lowry et al. (1951). 1.0 ml of enzyme supernatant was taken and mixed in 5.0 ml of 5% of TCA and centrifuged at 6000 g for 20 minutes. The precipitate was washed with 5.0 ml of 5% TCA and centrifuged at the same speed for 20 minutes. The precipitate was dissolved in 4.0 ml of NaOH and used for the determination of protein. Standard curves were prepared with different concentration of bovine serum albumin. Values have been expressed as μg protein/ mg nervous tissue. Statistical analysis Each experiment was replicated at least six times and results were expressed as mean ±SE of six replicates. Student s t-test was applied between control and treated groups to locate significant (p < 0.05) variations (Sokal and Rohlf, 1973). RESULTS In in vivo sublethal treatment with 40% and 80% of 24h LC 50 of column purified of S. asoca leaf and T. orientalis fruit powder and their active component saponin and thujone for 24h caused significant inhibition in AChE, ACP and ALP in the nervous tissue of snail L. acuminata (Table 2 and 4). In control group of snails acetylcholinesterase, acid and alkaline phosphatases activity in the nervous tissue of L. acuminata were 0.58 μ mole SH hydrolysed/ min/ mg protein, and μmole substrate hydrolysed/ 30min/ mg protein, respectively. Maximum inhibition in AChE (51.72% of control), ACP (52.41% of control) and ALP (55.09% of control) activity was observed in the snail exposed to 80% of 24h LC 50 of column purified fraction of S. asoca leaf (Table 2 and 4). In withdrawal experiment there was a significant (p<0.05) recovery in AChE, ACP and ALP activity in the nervous tissue of L. acuminata. Maximum recovery 84.21%, 91.48% and 85.63% of control in AChE, ACP and ALP activity, respectively were noted in the nervous tissue of snail withdrawn from 24h treatment of 80% of 24h LC 50 of saponin for next 96h (Table 2 and 4). DISCUSSION It is evident from the result section that in vivo sublethal 24h exposure to 40% and 80% of 24h LC 50 of different treatments of column purified fraction of S. asoca leaf and T. orientalis fruit and their active component saponin and thujone caused a significant inhibition of acetylcholinesterase (AChE), acid (ACP) and alkaline phosphatase (ALP) activity in the nervous 89

4 ARUNDHATI SINGH et al., tissue of Lymnaea acuminata. Key enzymes AChE, ALP and ACP are inhibited by most of the plant as well as synthetic molluscicides in the treated snails (Singh et al., 1982; 2005, Table 1: Sublethal concentrations (40% and 80% of 24h LC 50 ) of active components and column purified fractions of S. asoca, and T. orientalis against L. acuminata Treatments 24h LC 50 40% of 24h 80% of 24h (mg/l) LC 50 (mg/l) LC 50 (mg/l) Saraca asoca (leaf-cp) Saraca asoca (Saponin) Thuja orientalis (fruit-cp) Thuja orientalis(thujone) Abbreviation: CP- Column purified Jaiswal et al., 2010). Column purified fraction of S. asoca leaf, their active component saponin caused more inhibition in AChE than ALP and ACP. Acetylcholinesterase (AChE) inhibition results in accumulation of acetylcholine at the nerve synapses, so that the post synaptic membrane is in a state of permanent stimulation producing paralysis, ataxia, and general lack of co-ordination in neuromuscular system and eventual death (Singh and Agarwal, 1982a, b; 1983; 1986; Matsumura, 1985). Saponin causes haemolysis of red blood cells (George et al., 2002). It also acts as anticarcinogens (Rao and Sung, 1995). Thuja orientalis fruit column purified fraction caused more inhibition of ALP than AChE and ACP. The inhibition of alkaline phosphatase (ALP) may result in reduction in protein Table 2: In vivo effect of 24h exposure to sublethal concentrations (40% and 80% of 24h LC 50 ) of active components and column purified fractions on acetylcholinesterase (AChE) activity in nervous tissue of L. acuminata Treatment Concentrations (mg/l) AChE-μ mol SH hydrolyzed Withdrawal 80% % of 24h LC 50 / min / mg protein of 24h LC 50 to96h Control ±0.004(100) 0.57±0.038(100) S. asoca(leaf-cp) 40%(3.18 mg/l) 0.31±0.002*(53.44) - 80%(6.37 mg/l) 0.30±0.004*(51.72) 0.42±0.002 (73.68) Saponin 40%(2.81 mg/l) 0.45±0.005*(77.58) - 80%(5.63 mg/l) 0.436±0.007*(74.13) 0.48±0.061 (84.21) T. orientalis(fruit-cp) 40%(3.48 mg/l) 0.34±0.007* (58.62) - 80%(6.96 mg/l) 0.44±0.008*(75.58) 0.46±0.078 (80.70) Thujone 40%(3.21 mg/l) 0.457±0.006*(73.68) - 80% (6.42 mg/l) 0.38±0.008* (65.51) 0.45±0.009 (78.94) Values are mean±se of six replicates. Values in parentheses indicate percent enzyme activity with control taken as 100%. Concentrations (w/v) are expressed as final concentration in aquarium water. Acetylcholinesterase activity: μ mol SH hydrolyzed / min/ mg protein. (*) Significant (p<0.05) when student s t test was used for locating differences between experimental and control groups of animals Table 3: In vivo effect of 24h exposure to sublethal concentrations (40% and 80% of 24h LC 50 ) of active components and column purified fractions on acid phosphatase (ACP) activity in nervous tissue of L. acuminata Treatment Concentrations (mg/l) ACP-μ mol substrate hydrolyzed Withdrawal80% of % of 24h LC 50 / 30min / mg protein 24h LC 50 to96h Control ±0.240 (100) 17.96±0.225 (100) S. asoca(leaf-cp) 40% (3.18 mg/l) 11.34±0.132* (62.96) - 80% (6.37 mg/l) 9.44±0.101* (52.41) 12.01±0.196 (66.87) Saponin 40% (2.81 mg/l) 17.51±0.134* (97.22) - 80% (5.63 mg/l) 16.03±0.170* (89.95) 16.43±0.184 (91.48) T. orientalis(fruit-cp) 40% (3.48 mg/l) 11.22±0.169* (80.89) - 80% (6.96 mg/l) 10.57±0.174* (74.01) 11.72±0.312 (65.25) Thujone 40% (3.21 mg/l) 14.57±0.544* (62.29) - 80% (6.42 mg/l) 13.33±0.375* (58.68) 13.87±0.341 (77.22) Values are mean±se of six replicates. Values in parentheses indicate percent enzyme activity with control taken as 100%. Concentrations (w/v) are expressed as final concentration in aquarium water. Acid phosphatase (ACP) activity: μ mol substrate hydrolyzed /30 min/ mg protein. (*) Significant (p<0.05) when student s t test was used for locating differences between experimental and control groups of animals Table 4: In vivo effect of 24h exposure to sublethal concentrations (40% and 80% of 24h LC 50 ) of active components and column purified extracts on alkaline phosphatase (ALP) activity in nervous tissue of L. acuminata Treatment Concentrations (mg/l) ALP-μ mol substrate hydrolyzed Withdrawal80% of % of 24h LC 50 / 30min / mg protein 24h LC 50 to96h Control ±0.532(100) 16.01±0.621 (100) S. asoca(leaf-cp) 40% (3.18 mg/l) 10.42±0.069*(64.16) - 80% (6.37 mg/l) 9.110±0.052*(55.09) 9.91±0.061 (61.89) Saponin 40% (2.81 mg/l) ±0.410*(86.82) - 80% (5.63 mg/l) 13.53±0.410*(83.31) 13.71±0.396 (85.63) T. orientalis(fruit-cp) 40% (3.48 mg/l) 10.50±0.167*(79.31) - 80% (6.96 mg/l) 10.23±0.221* (70.01) 11.91±0.301 (74.39) Thujone 40% (3.21 mg/l) 12.88±0.959* (64.65) - 80% (6.42 mg/l) 11.37±0.125* (62.99) 12.21±0.213 (76.26) Values are mean±se of six replicates. Values in parentheses indicate percent enzyme activity with control taken as 100%. Concentrations (w/v) are expressed as final concentration in aquarium water. Alkaline phosphatase (ALP) activity: μ mol substrate hydrolyzed /30 min/ mg protein. (*) Significant (p<0.05) when student s t test was used for locating differences between experimental and control groups of animals 90

5 INHIBITION OF ACETYLCHOLINESTERASE level (Singh and Agarwal, 1989) as it is play a critical role in protein synthesis (Pilo et al., 1972), shell formation (Timmermans, 1969) and other secretary activities (Ibrahim et al., 1974) in gastropod. It plays an important role in the transport of metabolites across the membrane (Vorbrodt, 1959). Thujone the active component of T. orientalis is reported to be toxic to both brain and liver cells and could cause convulsions. It inhibit GABA receptor activation, neurons may fire more easily which can cause muscle spasms and convulsions (Hold et al., 2000, Naser et al., 2005). Among all the treatments highest inhibition (52.41% of control) in ACP activity was observed in the nervous system of snail exposed to 80% of 24h LC 50 of S. asoca leaf column purified fraction. Acid phosphatase (ACP), is a lysosomal enzyme (Aruna et al., 1979) which plays an important role in catabolism, pathological necrosis, autolysis, and phagocytosis (Abou-Donia, 1978). ACP was also inhibited by column purified fraction of S. asoca leaf and T. orientalis fruit and their active component saponin and thujone. It seems that alternation in AChE, ALP and ACP activity in the nervous tissue by the active components present in these plants may be one of the causes of snail death. It has been noted that thujone is neurotoxic substance (Bonkovsky et al., 1992) it inhibit the ALP and ACP activity up to same extent as AChE. In the present study plants viz. S. asoca and T. orientalis have shown that they are potential candidates for control of snail L. acuminata. Withdrawal of snails from 80% of 24h LC 50 for next 96h in untreated water caused trend of recovery in AChE, ALP and ACP activity indicate that treatment of different preparations of S. asoca, and T. orientalis plants caused reversible inhibition of these enzymes. REFERENCES Abou-Donia, M. B Increased acid phosphatase activity in following an oral dose of Leptophos. Toxicology Ltrs. 2: Aruna, P., Chetty, C. S., Naidu, R. C. and Swami, K. S Acid phosphatase activity in Indian apple snail Pila globosa (Swainson), during aestivation and starvation stress. Proc. Ind. Acad. Sci. 88: Bergmeyer, U. H Methods of enzymatic analysis. New York, Academic Press. p Bonkovsky, H. L., Cable, E. E., Cable, J. W., Donohue, S. E., White, E. C., Greene, Y. J., Lambrecht, R. W., Srivastava, K. K. and Arnold, W. N Porphyrogenic properties of the terpenes camphor, pinene, and thujone. Biochem. Pharmacol. 43(11): Ellman, G. L., Courtney, K. D., Andres, V. and Featherstone, R. I A new rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 7: Estaban, J. G., Bargues, M. D. and Mas-Coma, S Geographical distribution, diagnosis and treatment of human fascioliasis: A review. Res. Rev. Parasitol.58: George, F., Kerem, Z., Harinder, P., Makkar, S. and Becker, K The biological action of saponins in animal systems: a review. British J. Nutrition. 88(6): Hold, K.M., Sirisoma, N. S., Ikida, T., Narahashi, T. and Casida, J. E Alpha-thujone (the active component of absinthe):gammaaminobutyric acid type a receptor modulation metabolic detoxification. Proc. Natl. Acad. Sci. USA 97(8): Ibrahim, A. M., Higazi, M. G. and Demian, E. S Histochemical locolization of alkaline phosphatase activity in the alimentary tract of the snail Marisa cornuarieties (L.). Bull. Zoological Society of Egyp. 26: Jaiswal, P., Kumar, P., Singh, V. K. and Singh, D. K Enzyme Inhibition by Molluscicidal Components of Myristica fragrans Houtt. in the Nervous Tissue of Snail Lymnaea acuminata. Enzyme Research Vol 2010, doi: /2010/ Kumar, P., Singh, V. K. and Singh, D. K Combination of molluscicides with attractant carbohydrates and amino acids in bait formulation against the snail Lymnaea acuminata. European Review for Medical and Pharmacological Sciences. 15: Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J Protein measurement with the folin phenol reagents. J. Biol. Chem. 193: Mas-Coma, S., Bargues, M. D. and Valero, M. A Fascioliasis and other plant- borne trematode zoonose. Int. Parasitol. 35: Matsumura, F Toxicology of Insecticides, 2 nd Ed. Plenum Press, New York. Naser, B., Bodinet, C., Tegtmlier, M. and Lindequist, U Thuja occidentalis (Arbor vitae): A review of its Pharmaceutical, pharmacological and Clinical properties. Evidenced Based Complimentary and Alternative Medicine. 2(1): Osman, E. A., Mohamed, E. M., Abu Elreesh, B. I. and Elegami, A. A Molluscicidal activity of Combretum glutinosum. International J. Mole. Med. And Adv. Sci. 3(4): Pilo, B., Asnani, M. V. and Shah, R. V Studies on wound healing and repair in pigeon. III: Histochemical studies on acid and alkaline phosphatase activity during the process. J. Animal. Morpho. Physiol. 19: Rao, A. V. and Sung, M. K Saponins as anticarcinogens. In: J Nutr.25: Singh, A. and Singh, V. K Molluscicidal activity of Saraca asoca and Thuja orientalis against the fresh water snail Lymnaea acuminata. Veterinary Parasitology. 164: Singh, A., Singh, D. K., Mishra, T. N. and Agarwal, R. A Molluscicide of Plant origin. Bio. Agric. and Horti. 13: Singh, D. K. and Agarwal, R. A. 1982a. In vitro inhibition of acetyl cholinesterase by carbamate and organophosphorous pesticide in the snail L. acuminata. Comp. Physiol. Ecol. 7: Singh, D. K. and Agarwal, R. A. 1982b. Cholinesterase in the snail Lymnaea acuminata. Zool. J. Physiol. 86: Singh, D. 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6 ARUNDHATI SINGH et al., Sokal, R. R. and Rohlf, F. J Introduction to Biostatistics. Freemann, W. H., San Francisco. p.368. Spithill, T. W., Smooker, P. M. and Copeman, D. B Fasciola gigantica: epidemiology, control, immunology and molecular biology. In: Dalton, J. P. (Ed) Fasciolosis. CAB International Publishing, Wallingford. pp Timmermans, L. P. M Studies on shell formation in molluscs Neth. J. Zool. 19: Vorbrodt, A The role of phosphate in intracellular metabolism, Postepy. Hig. Med. DOSW. 13: WHO Report of the WHO Informal Meeting on Use of Triclabendazole in Fascioliasis Control, WHO Headquarters, Geneva. Switzerland, October. WHO/CDS/NTD/PCT/ WHO, Geneva. 92

J. Sci. I. R. Iran Vol. 11, No. 3, Summer S. Singh and D. K. Singh 1*

J. Sci. I. R. Iran Vol. 11, No. 3, Summer 2000 EFFECT OF ACTIVE MOLLUSCICIDAL COMPONENETS OF ABRUS PRECATORIUS, ARGEMONE MEXICANA AND NERIUM INDICUM ON CERTAIN ENZYMES IN THE NERVOUS TISSUE OF LYMNAEA