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Indian Journal of Pharmacology, Vol. 40, No. 2, March-April, 2008, pp. 64-68 Research Article Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum Singh TU, Kumar D, Tandan SK Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar - 243 122, UP Date of Submission: 20-Aug-2007 Code Number: ph08025 Abstract Objective: To investigate the effects of alcoholic extract of Allium sativum and Piper longum on the muscular activity of a parasitic amphistome, Gigantocotyle explanatum.Materials and Methods: Amphistomes were isometrically mounted to record the spontaneous muscular activity by using Chart 4 software program (Power Lab, AD Instruments, Australia) and to examine the effects of cumulative doses (100, 300, 1000, and 3000 µg/ml) of the plant extracts on the amplitude (g), frequency (per 10 min), and baseline tension (g) of the spontaneous muscular activity of the amphistome. Results: Alcoholic extract of A. sativum produced significant reduction in the frequency and amplitude of contractile activity of the amphistome at 1000 and 3000 µg/ml bath concentrations. Complete paralysis of the amphistome was observed after 15 min of addition of 3000 µg/ml concentration. Alcoholic extract of P. longum also caused paralysis following 15-20 min exposure of the amphistome to 3000 µg/ml concentration. In both the cases the amphistomes did not recover from paralysis following 2-3 washes. Conclusion: The observations demonstrate the paralytic effect of alcoholic extract of A. sativum and P. longum on G. explanatum. Keywords: Alcoholic extract, Allium sativum, Gigantocotyle explanatum, Piper longum, spontaneous muscular activity Parasitic infection is a major health problem throughout the world and is responsible for considerable economic losses to the livestock industry, particularly to poor livestock owners in developing countries. Other adverse effects of these parasites include loss of meat, wool, and egg production. Amongst helminths, infection caused by trematodes like amphistomes ( Gigantocotyle explanatum and Gastrothylax crumenifer ) and Fasciola sp. is more serious than that due to round worms. The prevalence of G. explanatum is very high in ruminants in the Indian subcontinent. These parasites cause hemorrhages and connective tissue proliferation at the site of attachment, vacuolar degeneration in the liver, and hyperplasia in the bile duct, thereby seriously affecting the health and productivity of infected animals. [1] Chemotherapy is the only efficient and effective tool to cure and control the helminth infection, as efficacious vaccines against helminths have not been developed so far. Indiscriminate use of synthetic anthelmintics in domestic animals has resulted in the development of resistance in helminth parasites. [2],[3],[4] Further, residual toxicity, adverse reactions, high cost, and inaccessibility to the rural farmers are problems associated with these agents. Consequently, there is an urgent need to develop newer, selective, and eco-friendly agents to control helminth infections. Plant-based anthelmintics offer an alternative to overcome some of these problems and they can be both sustainable and environmentally acceptable. Unlike synthetic anthelmintics, plant-based anthelmintics with different modes of action could be of value in preventing the development of resistance. [5] Herbal drugs have been in use since ancient times for the treatment of a variety of acute and chronic parasitic diseases, both in human and in veterinary medicine. [6],[7] The use of an extract of male fern ( Dryopteris felix mas ) against cestodes and trematodes and that of arecoline (from Areca catechu ) against tapeworms of dogs and poultry has also been reported. [8] In the search for plant-based anthelmintics, extracts of different medicinal plants have been tested for action against flatworms and roundworms in vitro and in vivo and have been found to possess anthelmintic activity. For example, an alcoholic extract of Mallotus philippinensis caused complete paralysis of F. gigantica in vitro , [9] garlic protected mice against Schistosoma infection, A. sativum has shown anthelmintic action in vitro against Heterakis gallinae and Ascaridia galli , [10] Haemonchus contortus, [11] a free-living nematode of Rhabditis sp., larvae of Nippostrongylus brasiliensis , and eggs of Ascaris summ. [12] In vivo A. sativum has demonstrated activity against strongyloids in donkeys. [13] P. longum has been reported to produce paralysis of Ascaris lumbricoides. [14] Moreover, extracts of the plants used in the present study, A. sativum and P. longum , have shown good hepatoprotective activity in rats. [15],[16] Although A. sativum and P. longum have shown activity against roundworms, to the best of our knowledge, there is no study on the effects of these plants on G. explanatum . The present study evaluates the effects of alcoholic extract of A. sativum and P. longum on spontaneous muscular activity of G. explanatum . Materials and Methods Collection of parasites Preparation of alcoholic extract of the plant material Preparation of plant extract suspension Isometric mounting of G. explanatum and mechanical recording of the spontaneous muscular activity The isometrically mounted parasite was equilibrated without any tension for 30 min, following which 300 mg tension was applied and spontaneous muscular activity was recorded using Chart 4 software program (Power Lab, AD Instruments, Australia). Control recordings were made for 15 min before the addition of a drug. During the equilibration period, the bath fluid was changed once every 10 min. Three parameters, namely, frequency (total number of contractions in 10 min), amplitude (average of all peaks per 10 min or average tension) of spontaneous muscular contractions, and baseline tension (average of all minimum levels of contractions used for measuring amplitude) of the isometrically mounted G. explanatum were measured. Measurements were made for a period of 10 min immediately before the application of a dose of the extract or before a wash following application of a dose. Effect of cumulative concentrations of plant extracts on spontaneous muscular activity of isometrically mounted G. explanatum Statistical analysis Results Recording of spontaneous muscular activity (SMA) of G. explanatum Effect of cumulative concentrations of alcoholic extract of A. sativum on SMA of G. explanatum Effect of cumulative concentrations of alcoholic extract of P. longum on the SMA of G. explanatum Discussion The main finding from this investigation was that alcoholic extracts of A. sativum and P. longum produced complete paralysis of G. explanatum after 15 min of addition of 3000 µg/ml concentration of the extracts and the amphistomes did not recover from paralysis even after 2-3 washes. Chemotherapeutic agents available for treatment of helminth infection act mainly through three different mechanisms, viz, by disruption of the neuromuscular physiology, by blocking the energy metabolism, and by disturbing the highly efficient reproductive system of the parasites. [18] Several important anthelmintics cause paralysis of helminth parasites by disrupting one or the other aspect of their neuromuscular system. [8],[19] The muscular activity of helminth parasites can be appreciated as SMA, which can be recorded mechanically with the help of a physiograph. The SMA can be quantified in terms of frequency, amplitude of rhythmic contractions, and baseline tension and these parameters can be measured before and following drug treatment and the values compared. [20] The changes produced in the SMA of isometrically mounted amphistome by a drug, shows the involvement of the neuromuscular system on account of rapidity of action. Rapid and marked change in the SMA of an isometrically mounted parasite by a drug indicates that the neuromuscular system of the parasites has been affected. Thus, SMA of isometrically mounted parasite can be used to evaluate anthelmintic activity of new compounds in vitro . In the present study, the SMA of G. explanatum is grossly similar to that of G. crumenifer, [17] S. mansoni , [21] and F. hepatica. [22] We have observed in a separate study that alcoholic extracts of A. sativum and P. longum inhibited the gross (visually assessed) motility of G. explanatum at 4 h of incubation (under publication). In the present study both the extracts induced marked decrease in the amplitude and frequency of rhythmic contractions at 1000 µg/ml concentration and produced complete paralysis of the amphistome within 15 min recording in presence of their highest concentration (3000 µg/ml). It can be appreciated from the recording of the effect of alcoholic extract of A. sativum that the inhibitory effect was dose-dependent from 100 to 3000 µg/ml bath concentrations, although same could not be verified statistically. In case of P. longum also, a dose-dependent effect could be seen. Alcoholic extract of P. longum also demonstrated concentration-dependent inhibitory effect on the SMA of G. crumenifer from 1000 to 3000 µg/ml bath concentrations. Thus, the paralysis of G. explanatum is the effect of the alcoholic extracts of A. sativum and P. longum . The chemotherapeutic value of both the extracts is also evident from an earlier study, [23] wherein P. longum (fruits), along with Azadirachta indica (bark), Butea frondosa (seeds), and Nigella sativa (seeds), produced broad spectrum anthelmintic action against roundworms ( H. contortus and Oesophagostomum columbianum ) and flukes ( Paramphistomum cervi ) in calves. In vitro , P. longum produced paralysis of A. lumbricoides. [14] Furthermore, A. sativum has also been shown to possess anthelmintic action in vitro against H. gallinae and A. galli, [10] H. contortus, [11] etc., and in vivo against strongyloides in donkeys. [13] The present observations provide evidence for the paralytic effect of alcoholic extracts A. sativum and P. longum on amphistomes. In conclusion, the observations demonstrate a paralytic effect of alcoholic extract of A. sativum and P. longum on G. explanatum by progressive reduction in the SMA, which may be associated with their inhibitory effect on the neuromuscular system of the amphistome. Acknowledgment The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar (U.P.), for providing the necessary facilities to complete this work.References
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