ISSN: 0258-3216 Vol. 5, No. 2, 2012 RESEARCH ARTICLE PUJ In Vitro Evaluation of Antihelminthic Activity of Allium sativum Against Adult Cotylophoron cotylophorum (Paramphistomidae) Nahla A. Radwan, Amal I. Khalil, Amera E. Wahdan Department of Zoology, Faculty of Science, University of Tanta, Tanta, Egypt Received: March, 2012 Accepted: May, 2012 ABSTRACT Background: Plant-based anti-helminthics have been reported for their safety and eco-friendly properties. They are used as alternatives for toxic chemical drugs. Allium sativum (garlic) has shown antihelminthic action in vitro and in vivo against many helminthes. Objective: The present study was designed to evaluate the in vitro antihelminthic activity of A. sativum in comparison with two commonly used antihelminthic drugs; albendazole and niclosamide against the cattle amphistome, Cotylophoron cotylophorum. Material and Methods: Live adult C. cotylophorum worms were collected from the rumen and reticulum of slaughtered cattle. Twelve media (natural calf serum, RPMI 1640 in ten formulas and normal saline) were evaluated to choose the most favorable one for incubation of worms during the study. Different concentrations of albendazole, niclosamide and alcoholic extract of A. sativum were tested in vitro against C. cotylophorum. Treated worms were examined after 2, 4, 8, 12 and 24 hours and the mortality rate was calculated in all experiments. The effect of the sub-lethal concentrations of albendazole and A. sativum on the tegument of the worm was evaluated by scanning electron microscopy (SEM). Results: Albendazole was highly effective against adult C. cotylophorum, where it exhibited dose-dependent lethal activity at different concentrations. Niclosamide was less effective, with high mortality rates recorded at relatively higher concentrations than albendazole. SEM revealed remarkable changes in the tegument and muscles of adult C. cotylophorum treated with a sub-lethal concentration of albendazole (1.5 mg/L) for eight hours. The whole tegument was deformed and papillae were hardly seen as swollen bodies. Numerous blebs and crater-like structures covered the whole tegument. A. sativum alcoholic extract gave high mortality rate at a relatively low concentration. SEM of adult worms treated with a sub-lethal concentration of A. sativum alcoholic extract (1.0 mg/L) revealed moderate changes in the tegument and a more severe effect on the muscle integrity. The mouth appeared flaccid and slightly retracted and the tegument surrounding it appeared feebly damaged and corrugated. The papillae lost their uniform structure. The acetabulum appeared retracted and lost its specific shape. Conclusion: The present study revealed that A. sativum alcoholic extract has an antihelminthic effect against adult C. cotylophorum at low concentrations. Garlic-based antihelminthic product may be used against rumen cattle amphistomes as an alternative for the commonly used chemical drugs. Keywords: Cotylophoron cotylophorum, Albendazole, Niclosamide, Allium sativum, In Vitro, Tegument, SEM. Corresponding author: Amal I. Khalil, [email protected] INTRODUCTION genital atrium of cotylephron type surrounded by Cotylophoron cotylophorum is a digenetic trematode that muscularized genital sucker in the first third of the body. parasitizes the rumen and reticulum of livestock. It has The pathogenic effect of the worm is associated with a bean-shaped body that measures about 12±1.68 mm the intestinal phase of infection. Young flukes are plug- length and 4±0.7 mm width(1). The body is characterized feeders causing severe erosions of the duodenal mucosa. by a well developed subterminal acetabulum and Adults destroy part of rumen and reticulum lining and Personal non-commercial use only. PUJ copyright © 2011. All rights reserved PUJ; 2012, 5(2): 135-146 Allium sativum against Adult C. cotylophorum cause inflammation of the intestine. In heavy infections, to albendazole and niclosamide in order to find a safe enteritis, edema, hemorrhage and ulceration may occur. plant-based antihelminthic as an alternative to the toxic Paramphistomiasis is a disease of prime economic chemotherapeutic agents. The antihelminthic activity importance to the animal industry, causing serious loss in was evaluated by scanning electron microscopy (SEM) wool, meat and milk production(2). to detect ultrastructural morphological changes of treated Antihelminthics are commonly used against rumen worms. flukes. Some of these chemotherapeutic agents are effective against immature flukes, others are effective MATERIALS AND METHODS against mature stages and few are lethal to both. The most commonly used in vivo antihelminthics against Collection of worms: Live C. cotylophorum worms paramphistomes are niclosamide, oxyclosanide, were collected from the rumen and reticulum of cattle triclabendazole and albendazole(3-9). Albendazole, immediately after being slaughtered. Live worms were marketed as Albenza, Eskazole, Zentel and Andazol, kept in 0.9% sodium chloride solution (normal saline) for is a member of the benzimidazoles indicated for the further investigations. treatment of a variety of worm infestations. Its efficiency Selection of the appropriate medium for maintenance against rumen amphistomes in general and against C. of worms during the study experiments: The collected cotylophorum infection in cattle was reported(10-12). worms were divided into groups (8 worms each) The high incidence of resistance to antihelminthic drugs and incubated in different media to choose the most of livestock, in addition to the relative toxicity and side appropriate for in vitro maintenance of worms during effects of many of these drugs, urge the necessity of the study experiments. Twelve media (Table 1) were finding alternative safe and eco-friendly agents against tested including normal saline, calf serum, RPMI 1640 helminthes. This applies to plant-based antihelminthics medium, and nine formulas prepared by modifications that have been used to destroy and expel parasitic worms of RPMI 1640 medium (Sigma-Aldrich company, USA). (13,14) from the gastrointestinal tract . Calf serum was separated by centrifugation from blood Allium sativum (garlic) contains components that show samples obtained from calves in slaughter house, and antiparasitic effects(14). A recent review analyzed the kept at 0ºC for further use(17). Ten ml of each medium active components of different herbs, including A. together with 2 ml of sterilization solution (streptomycin sativum that could be used as drug targets in parasitic and penicillin) were applied to each group of worms diseases(15). Alliin found in the intact bulb is easily in sterile Petri-dishes, and incubated at 37º C and pH converted to allicin (diallyl thiosulphinate) which is 7.4 for 24 hours. The worms were observed visually responsible for the characteristic flavor ofA. sativum and for activity and by mechanical stimulation using a (16) its antimicrobial properties . dissecting needle(18). The mortality rate was calculated In the present study, C. cotylophorum, collected in all experiments according to the following equation: from cattle, was chosen as a model to study the Mortality rate = (Number of dead worms/Total number antihelminthic activity of Allium sativum in comparison of cultivated worms)X100(19). Table (1): Concentrations of different media used to select the appropriate one for maintenance of worms during the study experiments. Composition Media PRMI% Normal saline%* Natural Calf serum% 1 Normal saline* 0 100 0 2 Natural calf serum 0 0 100 3 RPMI 1640 Formula 1 100 0 0 4 RPMI 1640 Formula 2 90 10 0 5 RPMI 1640 Formula 3 80 20 0 6 RPMI 1640 Formula 4 70 30 0 7 RPMI 1640 Formula 5 60 40 0 8 RPMI 1640 Formula 6 50 50 0 9 RPMI 1640 Formula 7 40 60 0 10 RPMI 1640 Formula 8 10 90 0 11 RPMI 1640 Formula 9 50 0 50 12 RPMI 1640 Formula 10 70 0 30 * 0.9% Sodium chloride solution 136 Radwan et al., Antihelminthic agents: The in vitro activity of A. sativum Statistical analysis: All values were expressed as mean alcoholic extract on C. cotylophorum was studied in ± standard deviation (SD). comparison to that of albendazole (Methyl 5-propylthio- 2-benzimidazolecarbamate) and niclosamide (5-chloro- RESULTS N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide) (Adwia Company, Egypt). Dried bulbs of A. sativum Incubation of C. cotylophorum in different media showed were pounded and extracted with 70% ethanol (10 that natural calf serum was the most appropriate medium gm/L) for preparation of alcoholic extract (14). Different as observed by the high activity and low mortality rates concentrations of albendazole (0.05, 0.25, 0.5, 1.5 and of the worms during the exposure period (24 hours) 2.5 mg/L), niclosamide (0.25, 0.5, 1.5, 2.5, 3.5, 4.5and when compared with other media (Table 2). Accordingly, 5.5mg/L) and A. sativum alcoholic extract (0.1, 0.2, 0.3, natural calf serum was selected for worm incubation to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/L) were prepared study the in vitro effect of antihelminthics against C. in distilled water to be tested against C. cotylophorum cotylophorum. worms maintained in the selected medium. In vitro antihelminthic effect of albendazole and Evaluation of antihelminthic activity of albendazole, niclosamide: The antihelminthic effect of different niclosamide and A. sativum alcoholic extract against concentrations of albendazole and niclosamide against adult C. cotylophorum: Ten ml of each concentration of adult C. cotylophorum for 24 hours exposure is shown the antihelminthic agents were applied to a group of 8 in table (3). A high albendazole concentration of 1.5 worms of the same length (10-12 mm) maintained in 10 mg/L showed high mortality rate (95.5%) after 8 hours ml of the selected medium and 2 ml of the sterilization exposure. On the other hand, lower concentrations solution. Each experiment was performed in triplicate (0.05, 0.25 and 0.5 mg/L) allowed worms to survive at optimal temperature (37ºC) and pH 7.4(18). Non- longer, where the mortality rates reached 87.5, 97.2 and treated control groups were included with different 97.5%, respectively after 24 hours.