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. Niclosamide was less experiments. All worms were examined after 2, 4, 8, 12 effective than albendazole as 91.1% and 95 % mortality and 24 hours and the mortality rate was calculated for each experiment. To detect the sub-lethal concentration rates were observed at high concentrations of 4.5 and 5.5 of each drug the percentage of dead worms in each group mg/L, respectively after 12 hours. Lower concentrations was recorded together with the magnitude of the dose of 0.25, 0.5, 1.5 and 3.5 mg/L gave relatively low that brought about that percentage. The percentage of mortality rates (54, 58, 70, and 87.5% respectively) at death was converted into probits(20) and plotted with the the same exposure time (Table 3). corresponding dose logarithm using linear regression In vitro antihelminthic effect of A. sativum alcoholic analysis(21). The sub-lethal dose was directly deduced extract: Alcoholic extract of A. sativum showed a high from the curve. mortality rate of 97% at a concentration of 1 mg/L In vitro effect of sub-lethal concentration of drugs: after 8 hours exposure time. On the other hand, low The in vitro effect of both albendazole and niclosamide concentrations (0.1, 0.3, and 0.5 mg/L) gave lower were evaluated. The lowest effective sub-lethal mortality rates (12.5, 20.1, and 41.18%) at the same concentration of albendazole was applied for both drugs. exposure time (Table 4). Three groups (8 worms each) in addition to the 4th Scanning electron microscopy: The body of adult C. control group were used for each experiment. Ten ml of cotylophorum is bean-shaped (Figure 1) with slightly the sub-lethal concentration was applied to each group corrugated surface. Dome-shaped papillae are distributed of worms maintained in 10 ml of the selected medium on the tegumental surface, sometimes in groups of 3-15 and 2 ml of the sterilization solution. Eight hours post- or discrete in different parts of the body (Figure 2). The incubation, live worms were collected, washed in 0.9% phosphate buffered saline solution and prepared for oral opening is terminal and is surrounded by wrinkled, scanning electron microscopic examination. extensively ridged tegument that forms concentric elevated rings encircling the oral opening (Figures 3 Scanning electron microscopy (SEM)(22): Treated and and 4). The tegument covering these rings is corrugated control worms were fixed for 24 hours in a mixture of into small tubercle-like structures that are more distinct buffered formaldehyde and gluteraldehyde (4:1) at 4ºC around the oral opening, but gradually diminish and pH 7.4. They were washed three times in phosphate posteriorly (Figure 5). The mid-ventral genital sucker is buffered saline solution and post fixed in 1% osmium surrounded by a tire-shaped elevation of the tegument tetraoxide for 2 hours at 4ºC. Fixed specimens were (Figure 6). The tegument covering the genital opening is washed three times in PBS, dehydrated through a graded series of ethanol and critical point dried. Specimens irregularly corrugated and lacks papillae (Figure 7). The were mounted, coated with gold and examined using tegument covering the outer border of the subterminal JEOL JSM 5300 scanning electron microscope at an acetabulum is somewhat smooth, while that close to the accelerated voltage of 30k.v, in the Electron Microscope center is corrugated in a bee-comb like structure (Figure Unit, Alexandria University. 8). 137 Allium sativum against Adult C. cotylophorum Table (2): Activity and mortality rates of C. cotylophorum worms incubated in different media for 24 hours. ACTIVITY% MORTALITY% Exposure time (Hours) Exposure time (Hours) Media 2 4 8 12 24 2 4 8 12 24 RPMI 1640 Formula 1 100 100 75 62.5 25 0 0 25 37.5 75 RPMI 1640 Formula 2 100 100 75 62.5 12.5 0 0 25 37.5 87.5 RPMI 1640 Formula 3 100 100 62.5 50 12.5 0 0 37.5 50 87.5 RPMI 1640 Formula 4 100 100 50 37.5 0 0 0 50 62.5 100 RPMI 1640 Formula 5 100 75 37.5 25 0 0 25 62.5 75 100 RPMI 1640 Formula 6 100 50 12.5 0 0 0 50 87.5 100 100 RPMI 1640 Formula 7 100 50 0 0 0 0 50 100 100 100 RPMI 1640 Formula 8 50 25 0 0 0 50 75 100 100 100 RPMI 1640 Formula 9 25 0 0 0 0 75 100 100 100 100 RPMI 1640 Formula 10 100 100 100 87.5 75 0 0 0 12.5 25 Normal saline 100 75 0 0 0 0 25 100 100 100 Natural calf serum 100 100 100 100 100 0 0 0 0 0 Table (3): In vitro antihelminthic effect of different concentrations of albendazole and niclosamide against C. cotylophorum incubated for 24 hours showing the sub-lethal concentration of each drug. Exposure ALBENDAZOLE NICLOSAMIDE Conc. Time No. of dead worm Mortality No. of dead worm Mortality (mg/L) (Hours) Mean ± SD rate% Mean ± SD rate% 2 0 0 - - 4 0 0 - - 0.05 8 3.33± 0.50 41.6 - - 12 4.67± 0.58 58.3 - - 24 7± 0 87.5 - - 2 0.67± 0.58 8.3 1± 0 12.5 4 2± 1 29.2 1.33 ± 0.58 16.7 0.25 8 5± 1 62.5 2.67 ± 0.58 33.3 12 6.33± 0.57 79.2 4.33 ± 1.57 54 24 8± 0 97.2 7 ± 1 87.5 2 3± 0 12.5 1.67 ± 0.58 20.8 4 4.33± 0.58 54.2 2 ± 1 25 0.5 8 5.67± 0.58 70.8 3 ± 1 37.5 12 7.67± 0.58 87.5 4.67 ± 1.55 58 24 8± 0 97.5 7± 1 87.5 2 7.67± 0.58 87.5 2.67 ± 0.58 33.3 4 7.67± 0.58 89.1 3± 1 37.5 1.5 8 7.81± 0.58 95.5* 4.33 ± 1.1 54 12 8± 0 100 5.67 ± 0.58 70 24 8± 0 100 8 ± 0 100 2 7.67± 0.58 95.5 3 ± 0 37.5 4 8± 0 100 4 ± 0 50 2.5 8 8± 0 100 5.33 ± 0.58 66.1 12 8± 0 100 7 ± 1 87.5 24 8± 0 100 8 ± 0 100 2 - - 4.67 ± 0.58 54.1 4 - - 5 ± 0 62.5 3.5 8 - - 6.33 ± 1.17 66.1 12 - - 7 ± 0 87.5 24 - - 8 ± 0 100 2 - - 5 ± 0 62.5 4 - - 5.33 ± 0.58 70.8 4.5 8 - - 6.67 ± 0.58 82.3 12 - - 7.33 ± 0.58 91.1* 24 - - 8 ± 0 100 2 - - 6 ± 0 75 4 - - 6.67 ± 0.58 83.3 5.5 8 - - 7 ± 1 87.5 12 - - 7.67 ± 0.58 95* 24 - - 8 ± 0 100 * The sub-lethal concentration
138 Radwan et al.,
Table (4): In vitro antihelminthic effect of A. sativum alcoholic extract against C. cotylophorum incubated for 24 hours showing the sub-lethal concentration. Concentration Exposure Time No. of dead worm Mortality rate (%) (mg/L) (Hours) Mean ± SD 2 0 0 4 0 0 0.1 8 1 ± 0 12.5 12 1.67 ± 0.58 20.8 24 2.67 ± 0.58 33.3 2 0 0 4 0.67 ± 0.58 0.83 0.2 8 1.33 ± 0.58 16 12 2.33 ± 0.58 29 24 3.67 ± 0.58 45.5 2 1 ± 0 12.5 4 1.33 ± 0.58 16.7 0.3 8 1.67 ± 0.58 20.1 12 3 ± 0 37.1 24 3.67 ± 0.58 45.8 2 1.33 ± 0.58 16.7 4 1.67 ± 0.58 20.8 0.4 8 2.67 ± 0.58 33.3 12 3.67 ± 0.58 45.8 24 4 ± 1 50 2 1.33 ± 0.58 16.7 4 2.33 ± 0.58 29.2 0.5 8 3.33 ± 0.58 41.1 12 5 ± 0 62.5 24 6.67 ± 1.15 83.3 2 2.33 ± 0.58 29.1 4 3.67 ± 0.58 45.8 0.6 8 4 ± 0 50 24 6 ± 1 83.3 2 3 ± 0 37.5 4 4 ± 0 50 0.7 8 5.33 ± 0.58 66 12 7.67 ± 1.33 95 24 8 ± 0 100 2 3.67 ± 0.58 45.8 4 4 ± 1 50 0.8 8 5.67 ± 0.58 70 12 8 ± 0 100 24 8± 0 100 2 5.33 ± 0.58 54 4 5 ± 1 62 0.9 8 6 ± 0 75 12 8 ± 0 100 24 8± 0 100 2 4 ± 0 50 4 5.33 ± 0.58 62 1 8 7.33 ± 0.58 97* 12 8± 0 100 24 8 ± 0 100 * The sub-lethal concentration
139 Allium sativum against Adult C. cotylophorum
Figures (1-8): Ultrastructure of adult Cotylophoron cotylophorum Figure (1): Whole bean-shaped body of adult C. cotylophorum. Figure (2): Body surface is slightly corrugated with dome-shaped papillae (DB) arranged in groups. Figure (3): Anterior half of the body showing terminal oral opening (OP) and genital sucker (GS) in the anterior third of the body. Figure (4): Terminal oral sucker surrounded by wrinkled tegument forming concentric elevated rings (arrows) encircling the mouth opening. Figure (5): Tegument surrounding the mouth is corrugated with tubercle-like structures. Figure (6): Genital sucker (GS) appears as ovoid tyre-shaped elevation of the tegument. Figure (7): Tegument covering genital sucker is irregularly corrugated and lacks papillae. Figure (8): Tegument covering outer border of the acetabulum is smooth (arrows), while that close to the center is corrugated in a bee-comb like structures (arrowheads).
140 Radwan et al., Ultrastructural changes of the tegument of in vitro unorganized tegumental ridges (Figure 9). Papillae were treated C. cotylophorum: hardly seen as swollen bodies on the tegument around
Sub-lethal concentration (LC95.5) of albendazole: SEM the mouth (Figure 10). The smooth tegument covering of the tegument of adult C. cotylophorum, treated in vitro the acetabulum was deformed into a highly corrugated with a sub-lethal concentration (LC95.5) of albendazole structure with numerous blebs (Figures 11 and 12). The (1.5 mg/L) for 8 hours, revealed remarkable changes in deformation of the tegument covering the body varied the tegument integrity. The oral opening was retracted and in severity from few blebs and crater-like structures to deformed and the concentric tegumental rings encircling complete tegumental deformation covering the whole the mouth were no longer seen, but were substituted by surface (Figures 13 and 14).
Figures (9-14): Ultrastructure of Cotylophoron cotylophorum in vitro treated with LC95.5 albendazole Figure (9): Oral opening (OP) is retracted and deformed. The concentric tegumental rings encircling the mouth are no longer seen. Figure (10): Papillae around oral opening are hardly seen as swollen bodies. Figure (11): Smooth tegument covering the acetabulum (Ac) is deformed into highly corrugated structure with numerous blebs (B). Figure (12): Numerous blebs (B) and crater-like structures (arrowheads) covering the tegument of acetabulum. Figure (13): Severe deformation of the body tegument showing bleb (B) formation and crater-like structures (arrowheads). Figure (14): Blebs (B) and crater-like structures (arrowheads) cover the whole body tegument.
141 Allium sativum against Adult C. cotylophorum
Sub-lethal concentration (LC97) of A. sativum alcoholic was less damaged, corrugated and the papillae lost their extract: Adult C. cotylophorum treated in vitro with a sub- uniform structure (Figures 15 and 16). The acetabulum lethal concentration (LC97) of A. sativum alcoholic extract appeared retracted and lost its specific shape (Figure 17). (1.0 mg/L) for 8 hours showed moderate changes in the Although the tegument covering the acetabulum was tegumental integrity. Treatment seems to have a greater less damaged than in albendazole treated worms, yet the effect on the muscle integrity than the tegumental surface. smooth outer and bee comb-like inner tegument changed This was revealed by changes in the form and integrity of into an undifferentiated corrugated structure with few blebs different surface structures. The mouth appeared flaccid (Figures 17 and 18). The body tegument was also affected and slightly retracted (Figure 15). Compared with changes where it appeared more corrugated with many blebs and the induced by albendazole, the tegument surrounding the mouth papillae were no longer seen (Figures 19 and 20).
Figures (15-20): Ultrastructure of Cotylophoron cotylophorum treated with LC97 Allium sativum alcoholic extract Figures (15 and 16): Flaccid and slightly retracted oral opening surrounded by corrugated tegument and disorganized papillae. Figure (17): Acetabulum (Ac) appeared retracted and lost its specific shape. Figure (18): The smooth outer and bee comb-like inner tegument covering acetabulum changed into undifferentiated corrugated structures with few blebs (B). Figure (19): Tegument in the middle part, corrugated and papillae not clearly seen. Figure (20): Higher magnification (X 350) of figure 19 showing corrugated tegument.
142 Radwan et al.,
DISCUSSION like structures covering the tegument of treated worms An antihelminthic drug, which could cause adverse result from bleb rupturing and extensive contraction changes to a parasite but not the host, is said to be a of the whole body. The origin and mechanism of bleb potential antihelminthic drug. Antihelminthic drugs formation is controversial and not clearly understood. impair the vital activities of the worm and consequently Nerve bulbs under the sensory papillae in Clonorchis lead to worm death. The most common effect of sinensis and Paragonimus westermain were suggested to antihelminthic drugs is paralysis of parasite musculature be the origin of bleb formation(29). On the other hand, this either by inhibiting neuro-muscular transition or enzymes suggestion was denied in another report(23) based on the involving energy production(6,23). Drugs may also damage finding of intact papillae. Bleb formation and muscular the body wall(3,17,18) allowing partial digestion or rejection contraction of drug-treated worms was suggested to be by host immune system. In general, benzimidazoles are calcium dependent. In confirmation, praziquantel was known to disturb the energy metabolism of parasites(24,25). found to induce increase in permeability of Opisthorchis In the present study the exposure time, at which the drug viverrini tegument to calcium binding or transport (31) brought about the best result, was chosen according to across the membrane resulting in bleb formation . the reported half life time (duration of action) of the The depletion of calcium in S. mansoni cultivation drug, being 8 hours for albendazole(25) and 12 hours for medium was shown to inhibit tegumental bleb formation (32) niclosamide(26). In comparison with albendazole, the and block muscular contraction of the worm . It was effect of A. sativum alcoholic extract was evaluated after shown that the increase of calcium uptake by the parasite (33) 8 hours exposure time. is directly related to tegumental vacuolization . In addition, the immediate tetanic contraction of the worm The present results showed that albendazole at a dose after treatment with albendazole, was reported to be a of 1.5 mg/L brought about a high mortality rate (95.5%) unique feature in many trematode and cestode parasites compared to niclosamide which only induced high and muscular or nervous paralysis may be one of the mortality rates (91.1% and 95%) at concentrations of 4.5 direct causes of worm death(29). mg/L and 5.5 mg/L respectively. Allium sativum alcoholic extract proved effective at a concentration of 1 mg/L. Several antihelminthic agents, including certain plant extracts, have been reported to cause considerable Albendazole has a broad spectrum activity against all structural alterations against paramphistomes(34,35). classes of parasitic helminthes. This drug has been However, the nature of the changes is highly specific reported to cause direct disruption of the tegumental and different for each agent. Previous reports indicate and muscle layers by binding specifically to β-tubulins, that A. sativum (garlic) possesses antimicrobial, thereby inhibiting polymerization and functioning antihelminthic, anti-protozoal, anti-fungal, anti- (27,28) of the cellular motor proteins . In the present carcinogenic, hypo- and hyperglycemic and insecticidal study, albendazole showed a potential in vitro effect properties(14,36). It is known that garlic contains many against C. cotylophorum. This finding contradicts the components that show antiparasitic effects(13), and its oil results of previous studies which reported that oral acts as a chemotherapeutic agent for poultry parasites(37). administration of albendazole at different doses failed The present study showed that 1 mg/L of alcoholic to control both adult and immature C. cotylophorum extract of A. sativum had a sublethal effect (97%) on and Paramphistomum spp.(4,29). Moreover, the present C. cotylophorum after 8 hours exposure. Comparable work showed that when albendazole was administered results were reported where the alcoholic extract of A. in vitro at a dose of 1.5 mg/L (LC95) for 8 hours, the sativum caused pronounced effect on muscle integrity of tegument of adult worms was severely damaged, where Gigantocotyle explanatum worm(14). tegumental blebs appeared especially around the oral The physiological activity of dietary A. sativum is opening and acetabulum. Similar findings were reported attributed to a number of organosulphate compounds, for Fasciola hepatica treated in vitro with 10 μg/ml of one of which is the amino acid “alliin” found in the intact (30) albendazole for 12 hours . However, only roughening bulb(38). When the bulb is cut or crushed, the C-slyase and thickening of the tegument without bleb formation enzymatic system called allinse converts alliin to allicin were observed, but after a shorter exposure time of 6 (diallyl thiosulphinate) which is responsible for the hours(5,6). Bleb formation and tegumental thickening were characteristic flavor of A. sativum and its antimicrobial also reported for C. cotylophorum treated in vitro with properties(38,39). In addition, A. sativum is the best source other antihelminthics of the benzimedazole group namely of selenium and the sulfur compound allicin produced mebendazole, febendazole(5), levamizole(6), and even by crushing or chewing of garlic, in turn produces other drugs of other category such as praziquantel(18). Crater- sulfur compounds; ajoene and vinyldithiins(36). Allicin
143 Allium sativum against Adult C. cotylophorum can disable two groups of enzymes; dehydrogenase and 8. Sahoo N, Mohanty TN, Samal S. Prevalence of cysteine proteinases which provide infection agents with gastrointestinal helminthic infection among grazing and the means to damage and invade tissues(38). Allicin was stall-fed cattle in a rained district of Orissa. J Vet Parasitol; also reported to inhibit the activity of acetylcholinesterase, 2002, 16(1): 61- 62. (39) lactic dehydrogenase and alkaline phosphatase . In 9. Galdhar CN, Roy S. Effect of steno bolus: Antihelminthic our study, in addition to the garlic induced tegumental treatment on working performance of bullocks during changes, the death of the worm might have resulted bovine paramphistomiasis. Pashudhan; 2004, 19:4-7. from dysfunction of the muscular and nervous tissues, in 10. Gevrey J, Bourdoiseau G. Treating paramphistomiasis in addition to impairment of the overall metabolic activity ruminants. Point Veterinaire; 1998, 29(194):807-10. of the worm. 11. Rolf P F, Boray J C. Chemotherapy of paramphistomosis In conclusion, Allium sativum alcoholic extract in sheep. Aust Vet J; 1988, 65(5):148-50. possesses a remarkable antihelminthic activity against 12. Veerakumari L, Paranthaman D. Light and scanning C. cotylophorum. It may serve as an alternative for electron microscopic studies of the effect of niclosamide antihelminthic chemotherapeutic agents to avoid their and oxyclosanide on Cotylophoron cotylophorum. J Vet toxic side effects and development of resistance in a safe Parasitol; 2004, 18(1):1-12. and eco friendly manner. Further in vivo studies should be done to evaluate the efficacy of the active gradients of 13. Soffar SA, Mokhtar GM. Evaluation of the antiparasitic garlic on paramphistomiasis. effect of aqueous garlic (Allium sativum) extract in hymenolepiasis nana and giardiasis. J Egypt Soc Parasitol; Author contribution: NA Radwan performed SEM, 1991, 21(2):497-502. supervised data analysis and writing of manuscript. 14. Singh T U, Kumar D, .Tandan S K Paralytic effect of AI Khalil suggested the study point, supervised data alcoholic extract of Allium sativum and Piper longum on analysis, wrote part of the manuscript and did the liver amphistome, Gigantocotyle explanatum. Indian J proof reading. AE Wahdan collected the material and Pharmacol. 2008 Mar-Apr; 40(2): 64–68. performed the experiments. 15. Etewa SE, Abaza SM. Herbal medicine and parasitic REFERENCES diseases (Review article). PUJ; 2011; 4(1):4-14. 16. Chybowski J. Study of the anthelmintic activity of garlic 1. Wahdan AE. Morphological and the therapeutic studies on extracts. Herba Polonica. 1997;43:383–7. some paramhistomes of ruminants. M.Sc. Thesis, Tanta 17. Jensen DV, Stirewalt MA, Walters M. Growth of University, Faculty of Science, 2012. Schistosoma mansoni cercaria under purpose dialysis 2. De Alwis MCL, Subasinghe DHA, Horadagoda NU. Water membrane in Rose multi-purpose chambers. Exp Parasitol; buffalo in Asia: IV Diseases of Buffalo. Publication of 1965, 17: 15-9. the SAREC/NARESA Buffalo Information Dissemination Program; 1999, pp 1-27. 18. Andrews P, Thomas H, Weber H. The in vitro uptake of C14 praziquantel by cestodes, trematodes and nematodes. 3. Madhekar DR, Ramanathan S, Khan MA, Roy RM. Studies on the activity of Terenol: A new antihelminthic J Parasitol; 1980, 66:920-5. against Paramphistomum species in ruminants. Livestock 19. Formulas and definitions of calculating rates (on line) Adviser; 1987, 12(7):5-13. Accessed June 03, 2009 at http://www.howany.com/how- 4. El-Shehawy I. Some morphobiological studies on to-calculate-mortality-rate . paramphistomidea in Kafr El-Sheikh Governorate. M.V.Sc 20. Finney D J. Laboratory and feild evaluation of the Thesis, Tanta University, Faculty of Veterinary Medicine, molluscidal properties of phytolacca dodecandra. Bulletin 1999. of the world health organization; 1970, 42: 597-612. 5. Veerakumari L, Munuswamy N. In vitro studies on 21. Clarke G M. Statistics and experimental design: the effects of some antihelminthics on Cotylophoron Contemporary biology. (J.Wilis and Sleigh edition) cotylophorum (Digenea, Paramphistomidae): A structural Edward arnold Ltd. London, V.K; 1980. analysis. Cytobios J; 1999, 98(387):39-57. 22. Lee RE. Scanning Electron Microscopy and X-ray 6. Veerakumari L, Munuswamy N. In vitro effect of some antihelminthics on lactate dehydrogenase Microanalysis. Prentice Hall, Englewood Cliffs, New activity of Cotylophoron cotylophorum (Digenea: Jersey, 1993. Paramphistomidae). Vet Parasitol; 2000, 91(1/2):129-40. 23. Manger BR. Antihelminthic veterinary applied 7. Chaudhri SS. Reduced responsiveness of oxyclosanide and pharmacology and Therapeutic. Fifth edition, Brander tetramisole hydrochloride combinations to gastrointestinal GC, Pugh DM, Bywater RJ, Jenkins WL (Editors), ELBS/ parasites of sheep. Indian J Ani Sci; 2000, 70(4): 396-8. Bailliere Tindall; 1991, pp. 513-48.
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24. Zia G, Nizami WA. Biochemical and immunotoxic effects Naunyn Schmiedebergs Arch Pharmacol; 1978, of mebendazole and oxyclosanide on two amphistomes 304(3):309-15. of buffaloes. J Vet Parasitol; 1998, 12(1): 9-16. 33. Seo B S, Chai J Y, Hong S J, Lee SH. Studies on intestinal 25. Venkatesan P. Albendazole. J Antimicrob Chemother; trematodes in Korea. XIX. Light and scanning electron 1998, 41:145-7. microscope of Fibricola seoulensis collected from albino rats treated with Praziquantel. Korean J Parasitol; 1985, 26. FAO.org/ag/AGP/AGPP/Pesticid/Specs/docs/Pdf/new/ 23: 47-57. niclosam.pdf. 34. Korshom M, Abd El-Moghney A, Mandour A. 27. Lacey E. The role of the cytoskeletal protein tubulin in Biochemical and parasitological evaluation of Nigella the mode of action and mechanism of drug resistance to sativa against rumen flukes (Paramphistomum) in benzimidazoles. Int J Parasitol; 1988, 18:885-936. sheep as compared with trematocide "Hepadex". Assuit 28. Ahmed M, Nizami WA. In vitro effect of mebendazole Vet Med J; 1998, 39(78):238-44. on the carbohydrate metabolism of Gigantocotyle 35. Chinnaperumal K, Abdul Abdul R, Anita M, Asokan explanatum (Trematoda:Digenea). Indian J Parasitol; B, Gandhi E. Insecticidal and larvicidal activities of 1991, 15: 19-26. medicinal plant extracts against mosquitoes. Parasitol 29. Tinar R, Coskun S Z, Dogan H, Demir S, Akyol VC. Res; 2010, 21:33-41. Efficacy of albendazole against nematode and trematode 36. Koch H P, Lawson L D (Editors). Garlic: The science and infection in sheep. Veteriner Fakultesi Dergisi Uludag therapeutic application of Allium sativum L and related Univ; 1988, 7:117-23. species. Baltimore, Med: Williams & Wilkins; 1996, 221- 30. Buchanan JF, Fairweather I, Brennan GP, Trudgett A, 9. Hoey E M. Fasciola hepatica: Surface and internal 37. Hagras HG. Efficacy of some herbs: Antihelminthic tegumental changes induced by treatment in vitro with drugs and immunomdulating agents against Ascridia the sulphoxide metabolite of albendazole (Valbazen). galli infection in poultry. Ph.D. Thesis, Department of Parasitol; 2003, 126:141-5. Zoology, Faculty of science, Tanta Univeristy, 2004. 31. Ruenwongsa P, Hutadilok N, Thamavit W, Suchart E. 38. Velisek J, Kubee R, Davidek J. Chemical composition Liver procollagen prolyl hydroxylase in Opisthorchis and classification of culinary and pharmaceutical A. viverrini infected hamsters after praziquantel sativum based products Z Lebensm Unters Forsch A; administration. Parasitol; 1983, 9(4):289-95. 1997, 204(2):161-4. 32. Pax R, Bennett JL, Fetterer R. A benzodiazepine 39. Singh VK, Singh DK. Inhabitation by Allicin, the derivative and praziquantel: Effects on musculature molluscicidal Agent of Allium sativum L (Garlic). of Schistosoma mansoni and Schistosoma japonicum. Phytother Res; 1996, 10(5): 383-6.
145 Allium sativum against Adult C. cotylophorum
تقييم الفاعلية الدوائية لنبات الثوم معمليا علي طفيلي كوتيليفرون كوتيليفروم )بارامفيستوماتيدي(
نهله عبد العليم رضوان، آمال اسكندر خليل، اميره السيد وهدان قسم علم الحيوان، كلية العلوم، جامعة طنطا
مقدمة: تعتبر النباتات ذات الفاعلية الدوائية لمعالجه الديدان الطفيليه بدائل آمنة لالدوية الكيميائية ذات التأثيرالضار علي العائل والبيئة. وقد اثبتت الدراسات السابقة ان لنبات الثوم ) ايليم ساتيفم( تأثير دوائي علي العديد من الديدان الطفيلية. هدف البحث: تهدف الدراسة الي تقييم الفاعلية الدوائية لنبات الثوم بالمقارنة باثنين من العقاقير المضادة للطفيليات وهي االلبندازول والنيكلوزاميد علي الدودة المعدية لالبقار كوتيليفرون كوتيليفروم معمليا. الطرق المستخدمة: تم تجميع الديدان البالغة الحية لطفيلي كوتيليفرون كوتيليفروم من معدة األبقار المصابة بمجازر الغربية. ولتقييم انسب االوساط الغذائية العاشه الطفيلي معمليا، تم استخدام اثنا عشرة وسطا غذائيا وهي مصل صغار االبقار وعشرة تركيبات للوسط الغذائي المصنع ) RPMI 1640(، باالضافة الي محلول كلوريد الصوديوم الملحي بتركيز0.9% عند درجه حراره 37 درجه مئويه ودرجه اس هيدروجيني 7.4. وقد تم اختبار تركيزات مختلفة لكل من االلبندازول والنيكلوزاميد والمستخلص الكحولي لبصيالت نبات الثوم معمليا ضد الطفيلي في وسط االعاشة االمثل البقاء الطفيلي لمده 24 ساعه وتم فحص الديدان المعالجة بعد2، 4، 8، 12 و24 ساعه وحساب نسبة الديدان النافقة تحت تأثير كل من التركيزات المستخدمة، ومنها تم تحديد الجرعة تحت المميتة لكل من االلبندازول والمستخلص الكحولي لبصيالت نبات الثوم واستخدامهما لدراسه التأثير الدوائي لكليهما معمليا علي التركيب الدقيق للديدان المعالجة باستخدام الميكروسكوب االلكتروني الماسح. النتائج: اثبتت الدراسة ان عقار االلبندازول له تأثير دوائي اكثر فاعليه من عقار النيكلوزاميد الذي اعطي نتائج مماثلة )95%( عند تركيزات دوائية عالية )5.5 مجم/لتر( بالمقارنة بااللبندازول )1.5 مجم/لتر(. وقد اوضح استخدام المجهر االلكتروني الماسح ان معالجة الديدان بالجرعة تحت المميتة 1.5 مجم/لتر لاللبندازول لمده 8 ساعات احدثت تأثيرا مرضيا علي االهاب وطبقه العضالت. حيث حدث تشوه في شكل االهاب وبات من الصعب التعرف علي الحلمات السطحية المنتشرة عليه كما ظهرت العديد من األنتفاخات المتكورة بعضها لها فتحات بركانية. أما في الديدان المعالجة بالجرعة تحت المميتة )1 مجم/لتر( من المستخلص الكحولي لبصيالت نبات الثوم في نفس الفترة الزمنية لتأثير الدواء، فكان تأثير النبات اكثر تركيزا علي طبقة العضالت حيث ظهرت فتحه الفم مترهلة، واصبحت طبقة االهاب المحيطة بها معرجة كما فقدت الحلمات السطحية شكلها المميز، كذلك ظهر الممص البطني منقبضا وفقد شكله المميز. االستنتاجات: ثبت ان نبات الثوم له فاعلية دوائية ضد طفيلي كوتيليفرون كوتيليفروم معمليا عند تركيزات منخفضة يمكن مقارنتها بتلك التي يحدثها عقار االلبندازول، ولذلك يمكن استخدام المشتقات الدوائية لنبات الثوم لطرد والقضاء علي الدودة المعدية لالبقار كبدائل آمنة للعقاقير الكيميائية الشائعة االستخدام مما يساعد على التخلص من آثارها الجانبية أو حدوث مقاومة الديدان لها.
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