J. Bangladesh Agril. Univ. 3(2):271-276, 2005 ISSN 1810-3030 In vitro effects of some indigenous against gastrointestinal of goats

M. Rahman', M. Shahiduzzaman2, M.K. Islam2, M.M.H. Monde Department of Pathology and 2 Parasitology, Dinajpur Government Veterinary College, Basherhat, Dinajpur Department of Parasitology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Abstract

Aqueous and ethanol extracts of twelve indigenous plants were screened for in vitro anthelmintic effect against adult gastrointestinal nematodes of goat origin (Haemonchus contortus, Trichostrongylus spp., Trichuris spp., Strongyloides papillosus, Oesophagostomum columbianum, Cooperia spp. and Buno.stomum trigonocephalum) and their infective larval stage (L3) obtained from in vitro culture. Aqueous extract of the plants showed potent (90-100%) effects against adult g/i nematodes at a concentration of 100 mg/ml whereas in ethanol extracts all plants except Amrul (Oxalis comiculata) were potent only at 50mg/m1 concentration. Ethanol extracts were found to be more effective than aqueous extract against both adult and infective larval stage (L3) comparing at different concentrations. In both cases in vitro activities against adults were much higher than that of infective larvae. Two patent drugs Deminthe (Morantel citrate) and Helmex0 (Albendazole) were included as positive control showed 100% effective against adult worms in vitro. Keywords: Indigenous plants, In vitro anthelmintic effects, Gastrointestinal nematodes, Goat Introduction

The kingdom is a rich source of botanical , antibiotics and insecticides (Satyavati et al., 1976; Lewis and Elvin-Lewis, 1977). Goats have been cOnsidered most important part of livestock throughout the world. Helminths, especially gastrointestinal nematodes are recognized as a major constraint to goat production in Bangladesh (Rahman and Razzak, 1973; Qadir, 1981). Among the gastrointestinal nematodes Haernonchus contortus (65.63%) is most prevalent in goat in Bangladesh (Shahiduzzaman et at, 2003). Imported manufactured anthelmintics, particularly administration of benzimidazole and Probenzimidazole groups, have long been considered the only effective way of controlling Parasitic infections. Due to unavailability, high price and indiscriminate use of some anthelmintics there is development of resistance to helminths to various anthelmintic compounds (Waller and Prichard, 1985). For these various reasons, interest in the screening of medicinal plants for their anthelmintic activity remains of great scientific interest despite extensive use of synthetic chemicals in modern clinical practices all over the world (Akhtar et at, 2000). However very little works (Mostofa, 1983; Begum, 1997) has been performed in our country to investigate the anthelmintic properties of medicinal plants. Therefore the present work was undertaken to develop a simple protocol for in vitro screening of aqueous and ethanol extracts of plants to evaluate the anthelmintic properties of the plants against infective larval stage (L3) and adult gastrointestinal nematodes of goat origin and to select only those plants having great potential comparing with synthetic drugs. Materials and Methods

This study was conducted during the period from January, 2001 to April, 2002 in the laboratory of the Department Qf Parasitology, Bangladesh Agricultural University (BAU), Mymensingh. 272 Gastrointestinal nematodes of goats

The test parasites

Gastrointestinal (g/i) nematodes used as the test parasites in this study were collected from the viscera of goats slaughtered in local K.R. market of Bangladesh Agricultural University, Mymensingh. The selected g/i nematodes of goats are Haemonchus contortus, Trichostrongylus spp., Trichuris spp., Strongyloides papillosus, Oesophagostomum columbianum, Cooperia spp. and Bunostomum trigonocephalum.

Preparation of plant extract

The plant and plant materials (Table 1) used in this experiment were procured from BAU campus and its surrounding rural areas, and identified by a botanist. The common names and scientific names of the used plants and the portion of the plants extracted for the present experiment are given in Table 1. The plant materials were dried at 55-60°C for 48-72 hours and prepared dust by pulverising with manual grinder. Ten gram of each category of dust was taken in a 500 ml beaker and separately mixed with 100 ml of either ethanol or distilled water. Then the mixture was stirred for 30 minutes by a magnetic stirrer (6000 rpm) and left stand for next 24 hrs. The mixture was then filtered through a fine cloth and again through filter paper (Whatman No. 1). The filtered materials were taken into a round bottom flask and then condensed by evaporation of solvent from filtrate in a water bath at 50°C and 60° C for ethanol and water respectively up to final volume of 10 ml. After the evaporation of solvent from filtrate, the condensed extracts were preserved in tightly corked labelled bottle and stored in a refrigerator until their screening for anthelmintic property.

Table 1. Name of the plants and part used for extraction Common name Scientific name Part used Jute Corchorus olitorious Leaves Pineapple Ananas comosus Leaves Amrul Oxalis corniculata Leaves Biskathali leaves Polygonum hydropiper Leaves _ Padmagulancha Tinospora tornentosa Stem Karolla Momordica charantia Whole part Neem Azaairachta indica Leaves ____ Hatishur _ Heliotropium indicum Root Katakhura Amaranthus spinosus Leaves Lazzabati Mimosa pudica Leaves Garlic Allium sativum Whole part

The test drugs

Two patent drugs Deminte; Morantel citrate (Reneta Bangladesh Limited) and Helmee; Albendazole,(Reneta Bangladesh Limited) were used as positive control. Solution of different extract Aqueous solutions of 25, 50 and 100 mg/m1 and ethanol solution of 10, 25 and 50 mg/ml were prepared by adding distilled water to the stock solutions. All the solutions were prepared at the day of experiment. Rahman etal. 273

In vitro culture of infective larvae Adult nematodes were collected from the abomasi of goats according to the procedure described by Taylor (1934), Bell (1957) and Rahrnan (1969). An in vitro culture system has already been established in our laboratory for harvesting the infective third stage larvae (L3) of gastrointestinal nematodes (Islam and Begum, 2001). Briefly, the collected worms were washed for several times with phosphate buffer saline (PBS). Then uteri of gravid females were dissected out, crushed gently in a petri dish to release eggs. A known volume of PBS was added to eggs and incubated at room temperature (25-30 C)for about 72 hrs and then transferred to a 100 ml beaker and incubated further until development of L3 had occurred. During cultivation the culture media were monitored every morning for observing the development of larvae towards L3 stage.

The L3 were washed several times in PBS through centrifugation at 2000 rpm for 7 minutes and finally counted and suspended in a 100 ml beaker. The L3 were maintained in the laboratory by incubating them at 37°C in sterile condition. In vitro screening of plant extracts Aqueous and ethanol plant extracts at various concentration (10, 25, 50, 100 mg/ml) levels were screened by using both adult worms and L3 stage larvae. A 200 pl PBS containing 25 adult worms (both male and female) and 100 L3 was pipetted on to separate petri-dishes and a 800 pl of extracts of each concentration was then added. Following a 3 hrs treatment period at room temperature, the percent non- motile (dead) L3 was calculated from the dead adult and larvae killed by adding a drop of lugols iodine. Statistical analysis Data were analysed by Student t-test to determine the significant differences among the variables (Steel and Torrie, 1980). Results and Discussion Aqueous extract of all the plants were much more effective (92-100%) against adults selected worms than L3 (Table 2). Out of 12 plants, 9 showed 100% efficacy against adult worms and 3 showed 92-96% efficacy at a concentration of 100 mg/ml. The efficacy of aqueous extract of different plants at a concentration level of 25 mg/ml and 50 mg/ml was much lower than that of concentration of 100 mg/ml (Table 2). Two patent drugs (Deminth0 and Helmexe, Renata Ltd.) screened as positive control were also 100% effective against adult g/i nematodes and L3 in vitro except in two cases where Helmex0 showed only 21 and 32% effectiveness at concentration of 25 and 50, respectively against the L3 larvae. The plants, which had highly significant activity against adults, were not so active against L3 (Table 2). However, aqueous extract of different plants were not 100% effective against L3 larvae even at the highest concentration used in the experiment. In this study in vitro anthelmintic properties of some indigenous plants against g/i nematodes of goats were reported for the first time in Bangladesh. In vitro anthelmintic effects of several plants have been demonstrated against g/i worms of animals and birds in various parts of the world ( Jawale et al., 2000; Akhtar et al., 2000; Dano and Bogh, 1999, Shilashkar and Parashar, 1989; Kaushik et aL, 1981, Agarwal et al., 1979). The present results of in vitro screening showed that larvae L3 are more resistant with few exceptions than adult worms at the same concentration because they are naturally adapted to environmental condition (Table 2 and 3). These findings indicate that the adult g/i nematodes are more vulnerable to indigenous plants and patent anthelmintic like albendazole and morantel citrate. Ethanol extracts were more effective against both adults and L3 stage even at lower concentration 274 Gastrointestinal nematodes of goats level (Table 3). The higher effect of ethanol might be due to extraction of large amount of plant ingredient dissolved in alcohol. Similar results were found in a study of Mefod et aL (1996) who stated that alcohol extract is highly effective than aqueous extract. Various published reports for indigenous plants of the world indicated the potential effect of several medicinal plants against g/i nematodes in vitro [Neogi et aL (1964), Shrisvastava and Singh (1967), Sharma et al. (1971), Lal et aL (1976)].

Table 2. Percent non-motile adult (A) and infective larvae (L3) of g/i nematodes exposed to different increasing concentrations of aqueous extracts in vitro SI. Name of Plants Percent non-motile Adult and L3 in different No. concentration of plant extracts (mg/ml) Common name Scientific name 25 50 100

A L3 A L3 A 3 01 Jute leaves Corchorus olitorious 32 21 60 , 50 92" 84 02 Pineapple Ananas comosus 48 12 72 22 100* 53 03 Amrul _ Oxalis comiculata 32 7 76 11 100" 18 04 _ Biskathali Polygonum hydropiper 44 5 76 8 100* 15 05 Padmagulancha Tinospora tomentosa 42 30 70 65 96" 82 06 Karolla Momordica charantia 24 16 80 39 100* 70 07 Neem Azadirachta indica , 16 10 40 i 9 92* 36 08 Hatishur Heliotropium indicum 32 19 72 34 100* 67 09 Katakhura Amaranthus spinosus 48 _ 5 80 6 100* 10 10 Lazzabati - Mimosa pudica 20 8 36 10 _ 100* 26 11 Pan Piper belle 25 17 52 23 100* 54 12 Garlic Allium sativum 52 14 68 29 100* 56 13 Deminth (Morantel 100 100 100 100 100* 100* citrate), positive control 14 Helmex (Albendazole), _ 100 21 100 32 . 100* 100* positive control 15 PBS nesative control - - 04 04 "Considered strong wormicidal and larvicidal (P<0.05) Table 3. Percent non-motile (dead) Adult (A) & infective larvae (L3) when exposed to an increased concentration of ethanol extract of indigenous plants in vitro SI. Indigenous plants Percent non-motile L3 in different concentration of plant No. extracts (mg/ml) Common name Scientific _ name 10 25 50 A' L3 A I-3 A L3 01 Jute leaves Corchorus olitorious 59 14.25 82 71.0 100* 82.0 02 Pineapple leaves Ananas comosus 63 31.0 85 80.75 , 100* 95.75* 03 Amrul Oxalis comiculata 24 15.5 36 21.0 88 47.75 04 Biskathali leaves Polygonum hydropiper 34 18.5 69 29.0 92* 49.0 05 , Padmagulancha Tinospora tomentosa 71 53.5 91 90.75 100" , 100* , 06 Karolla whole Momordica part charantia 72 55.0 92 91.0 100* 100" 07 Neem leaves Azadirachta indica 72 59.25 96 . 92.75 100* 100* 08 Hatishur Heliotropium , indicum 59 32.5 77 70.5 96* 80.0 09 .Katakhura Amaranthus spinosus 50 25.75 83 60.75 _ 100* 80.75 10 Lazzabati Mimosa pudica 34 14.25 68 47.75 90* 60.75 11 Pan ..Piper betle 42 29.25 80 61.0 100* 90.0 12 Garlic whole Allium sativum 69 , 21.75 92 47.75 100* 81.0 13 Deminth (Morantel 100* 50 100* 100* 100* 100* citrate), positive control 14 Helmex (Albendazole), 50 15 100* 20 100* 32 positive control . 15 PBS(Control) 0 _ 0 05 0 07 03.25 "Considered strong wormicidal and larvicidal (P<0.05) Rahman etal. 275

Structural alterations of adult worms were found during in vitro screening of both extracts. Grossly the extracts caused inhibition of spontaneous motility of worm, this was characterized by initial, short lasting, small increases in amplitude and tone of contractions followed by paralysis. These findings also substantiated by Rajindar et al. (1997). Tandon et aL (1997) and Bishnupada et al. (1997) reported that scanning electron microscopy (which was not performed in this study) of parasites exposed to plant extracts ( vestita, Cannabis sativa) revealed structural alteration in the integument/surface architecture particularly of the papillated surface. Deep scars were also observed on the dorsal and ventral surfaces. In the present study we observed, the complete cessation of motility and finally mortality (percent death) of worm and larvae which depends on the type and concentration of the extracts used. The time for complete cessation of motility and ultimate death was found to be reduced as the concentrations of the extracts increased. This finding suggests that mortality Percentage of g/i nematodes varied significantly (P<0.05) among the plants, solvents used and doses. In this study, PBS was used for incubation of adult and infective larvae treated With plant extract but test would be more accurate/reliable if incubation the adult and infective larvae could-be done in abomasal or rumen fluid as described by Molan et aL (2000). Further studies are needed to understand the precise role of the indigenous plants as phytochemicals against g/i nematodes to be cultured in vitro in serum free and serum supplemented tissue culture media at 27°C. Acknowledgements The Authors cordially acknowledge the support rendered by the Ministry of Science, Information and Communication Technology, Government of the People's Republic of Bangladesh. References Agarwal, R., Kharya, M.D. and Shrivastava, R. 1979. Antibacterial and anthelmintic activity of the essential oil of Nigella sativa Linn. Indian Journal of Experimental Biology, 17 (11): 1264-1265. Akhtar, M.S., lqbal, 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 : 99- 107. All, S.M. and Mehta, R.K. 1970. Preliminary pharmacological and essential oil of the Piper betle L. Indian Journal of Pharmacology, 32 : 132-133. Bell, R.R., 1957. Survey of gastrointestinal parasites of cattle in North California. American Journal of Veterinary Research, Vii: 294. Begum, T. 1997. Comparative efficacy of some indigenous plants (Bironja, Turmeric and Veranda) with that of patent drug Nilzan against trematodiasis and nematodiasis in sheep. M. S. in Pharmacology, a thesis submitted to the Department of Pharmacology, Bangladesh Agricultural University, Mymensingh. Bishnupada, R., Venna, T., Roy, B. and Tandon, V. 1997. In vitro flukicidal effect of leaf extracts of Cannabis sativa Linn. on the trematodes Fsciolopsis buski. Indian Journal of Experimental Biology, 35(1): 80-82. Dano, A.R., and Bogh, H.O. 1999. Use of herbal medicine against helminth in livestock, renaissance of an old tradition. In : World Animal Review,(The FAO Journal on Animal Health, Production and products), 2: 60- 67. Garg, S.C., Rajshree, J. and Jain, R. 1992. Biological activity of essential oil of Piper belle L. Journal of Essential Oil Research, 4(6): 601-606. Islam, M.K. and Begum, N. 2001. Studies on in vitro culture, isolation and survivability of free living infective larvae of gastrointestinal nematodes of ruminants and their application in screening pesticides. Repot submitted to the Bangladesh Agricultural University Research System (BAURES), Mymensingh-2202, pp 1-18. Jawale, R.Y., Bhojne, G.R., Dak Shinkar, N.P. and Jangde, C.R. 2000. In vitro anthelmintic activity of some indigenous medicinal plants against of Haemonchus contortus. Indian Veterinary and Medical Journal, 24: 338-340. 276 Gastrointestinal nematodes of goats

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