RESEARCH ARTICLE PUJVol. 5, No. 2, 2012ISSN: 0258-3216

Personal non-commercial use only. PUJ copyright © 2011. All rights reserved PUJ; 2012, 5(2): 135-146

In Vitro Evaluation of Antihelminthic Activity of Allium sativum Against Adult Cotylophoron cotylophorum (Paramphistomidae)

Nahla A. Radwan, Amal I. Khalil, Amera E. WahdanDepartment 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, amalikhalil@hotmail.com

INTRODUCTION

Cotylophoron cotylophorum is a digenetic trematode that parasitizes the rumen and reticulum of livestock. It has a bean-shaped body that measures about 12±1.68 mm length and 4±0.7 mm width(1). The body is characterized by a well developed subterminal acetabulum and

genital atrium of cotylephron type surrounded by muscularized genital sucker in the first third of the body.The pathogenic effect of the worm is associated with the intestinal phase of infection. Young flukes are plug-feeders causing severe erosions of the duodenal mucosa. Adults destroy part of rumen and reticulum lining and

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cause inflammation of the intestine. In heavy infections, enteritis, edema, hemorrhage and ulceration may occur. Paramphistomiasis is a disease of prime economic importance to the animal industry, causing serious loss in wool, meat and milk production(2).

Antihelminthics are commonly used against rumen flukes. Some of these chemotherapeutic agents are effective against immature flukes, others are effective against mature stages and few are lethal to both. The most commonly used in vivo antihelminthics against paramphistomes are niclosamide, oxyclosanide, triclabendazole and albendazole(3-9). Albendazole, marketed as Albenza, Eskazole, Zentel and Andazol, is a member of the benzimidazoles indicated for the treatment of a variety of worm infestations. Its efficiency against rumen amphistomes in general and against C. cotylophorum infection in cattle was reported(10-12).

The high incidence of resistance to antihelminthic drugs of livestock, in addition to the relative toxicity and side effects of many of these drugs, urge the necessity of finding alternative safe and eco-friendly agents against helminthes. This applies to plant-based antihelminthics that have been used to destroy and expel parasitic worms from the gastrointestinal tract(13,14).

Allium sativum (garlic) contains components that show antiparasitic effects(14). A recent review analyzed the active components of different herbs, including A. sativum that could be used as drug targets in parasitic diseases(15). Alliin found in the intact bulb is easily converted to allicin (diallyl thiosulphinate) which is responsible for the characteristic flavor of A. sativum and its antimicrobial properties(16).

In the present study, C. cotylophorum, collected from cattle, was chosen as a model to study the antihelminthic activity of Allium sativum in comparison

to albendazole and niclosamide in order to find a safe plant-based antihelminthic as an alternative to the toxic chemotherapeutic agents. The antihelminthic activity was evaluated by scanning electron microscopy (SEM) to detect ultrastructural morphological changes of treated worms.

MATERIALS AND METHODS

Collection of worms: Live C. cotylophorum worms were collected from the rumen and reticulum of cattle immediately after being slaughtered. Live worms were kept in 0.9% sodium chloride solution (normal saline) for further investigations.

Selection of the appropriate medium for maintenance of worms during the study experiments: The collected worms were divided into groups (8 worms each) and incubated in different media to choose the most appropriate for in vitro maintenance of worms during the study experiments. Twelve media (Table 1) were tested including normal saline, calf serum, RPMI 1640 medium, and nine formulas prepared by modifications of RPMI 1640 medium (Sigma-Aldrich company, USA). Calf serum was separated by centrifugation from blood samples obtained from calves in slaughter house, and kept at 0ºC for further use(17). Ten ml of each medium together with 2 ml of sterilization solution (streptomycin and penicillin) were applied to each group of worms in sterile Petri-dishes, and incubated at 37º C and pH 7.4 for 24 hours. The worms were observed visually for activity and by mechanical stimulation using a dissecting needle(18). The mortality rate was calculated in all experiments according to the following equation: Mortality rate = (Number of dead worms/Total number 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.

CompositionMedia PRMI% Normal saline%* Natural Calf serum%

1 Normal saline* 0 100 02 Natural calf serum 0 0 1003 RPMI 1640 Formula 1 100 0 04 RPMI 1640 Formula 2 90 10 0

5 RPMI 1640 Formula 3 80 20 06 RPMI 1640 Formula 4 70 30 07 RPMI 1640 Formula 5 60 40 08 RPMI 1640 Formula 6 50 50 09 RPMI 1640 Formula 7 40 60 010 RPMI 1640 Formula 8 10 90 011 RPMI 1640 Formula 9 50 0 5012 RPMI 1640 Formula 10 70 0 30

* 0.9% Sodium chloride solution

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Antihelminthic agents: The in vitro activity of A. sativum alcoholic extract on C. cotylophorum was studied in comparison to that of albendazole (Methyl 5-propylthio-2-benzimidazolecarbamate) and niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide) (Adwia Company, Egypt). Dried bulbs of A. sativum were pounded and extracted with 70% ethanol (10 gm/L) for preparation of alcoholic extract (14). Different concentrations of albendazole (0.05, 0.25, 0.5, 1.5 and 2.5 mg/L), niclosamide (0.25, 0.5, 1.5, 2.5, 3.5, 4.5and 5.5mg/L) and A. sativum alcoholic extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg/L) were prepared in distilled water to be tested against C. cotylophorum worms maintained in the selected medium.

Evaluation of antihelminthic activity of albendazole, niclosamide and A. sativum alcoholic extract against adult C. cotylophorum: Ten ml of each concentration of the antihelminthic agents were applied to a group of 8 worms of the same length (10-12 mm) maintained in 10 ml of the selected medium and 2 ml of the sterilization solution. Each experiment was performed in triplicate at optimal temperature (37ºC) and pH 7.4(18). Non-treated control groups were included with different experiments. All worms were examined after 2, 4, 8, 12 and 24 hours and the mortality rate was calculated for each experiment. To detect the sub-lethal concentration of each drug the percentage of dead worms in each group was recorded together with the magnitude of the dose that brought about that percentage. The percentage of death was converted into probits(20) and plotted with the corresponding dose logarithm using linear regression analysis(21). The sub-lethal dose was directly deduced from the curve.

In vitro effect of sub-lethal concentration of drugs: The in vitro effect of both albendazole and niclosamide were evaluated. The lowest effective sub-lethal concentration of albendazole was applied for both drugs. Three groups (8 worms each) in addition to the 4th control group were used for each experiment. Ten ml of the sub-lethal concentration was applied to each group of worms maintained in 10 ml of the selected medium and 2 ml of the sterilization solution. Eight hours post-incubation, live worms were collected, washed in 0.9% phosphate buffered saline solution and prepared for scanning electron microscopic examination.

Scanning electron microscopy (SEM)(22): Treated and control worms were fixed for 24 hours in a mixture of buffered formaldehyde and gluteraldehyde (4:1) at 4ºC and pH 7.4. They were washed three times in phosphate buffered saline solution and post fixed in 1% osmium tetraoxide for 2 hours at 4ºC. Fixed specimens were washed three times in PBS, dehydrated through a graded series of ethanol and critical point dried. Specimens were mounted, coated with gold and examined using JEOL JSM 5300 scanning electron microscope at an accelerated voltage of 30k.v, in the Electron Microscope Unit, Alexandria University.

Statistical analysis: All values were expressed as mean ± standard deviation (SD).

RESULTS

Incubation of C. cotylophorum in different media showed that natural calf serum was the most appropriate medium as observed by the high activity and low mortality rates of the worms during the exposure period (24 hours) when compared with other media (Table 2). Accordingly, natural calf serum was selected for worm incubation to study the in vitro effect of antihelminthics against C. cotylophorum.

In vitro antihelminthic effect of albendazole and niclosamide: The antihelminthic effect of different concentrations of albendazole and niclosamide against adult C. cotylophorum for 24 hours exposure is shown in table (3). A high albendazole concentration of 1.5 mg/L showed high mortality rate (95.5%) after 8 hours exposure. On the other hand, lower concentrations (0.05, 0.25 and 0.5 mg/L) allowed worms to survive longer, where the mortality rates reached 87.5, 97.2 and 97.5%, respectively after 24 hours. Niclosamide was less effective than albendazole as 91.1% and 95 % mortality rates were observed at high concentrations of 4.5 and 5.5 mg/L, respectively after 12 hours. Lower concentrations of 0.25, 0.5, 1.5 and 3.5 mg/L gave relatively low mortality rates (54, 58, 70, and 87.5% respectively) at the same exposure time (Table 3).

In vitro antihelminthic effect of A. sativum alcoholic extract: Alcoholic extract of A. sativum showed a high mortality rate of 97% at a concentration of 1 mg/L after 8 hours exposure time. On the other hand, low concentrations (0.1, 0.3, and 0.5 mg/L) gave lower mortality rates (12.5, 20.1, and 41.18%) at the same exposure time (Table 4).

Scanning electron microscopy: The body of adult C. cotylophorum is bean-shaped (Figure 1) with slightly corrugated surface. Dome-shaped papillae are distributed on the tegumental surface, sometimes in groups of 3-15 or discrete in different parts of the body (Figure 2). The oral opening is terminal and is surrounded by wrinkled, extensively ridged tegument that forms concentric elevated rings encircling the oral opening (Figures 3 and 4). The tegument covering these rings is corrugated into small tubercle-like structures that are more distinct around the oral opening, but gradually diminish posteriorly (Figure 5). The mid-ventral genital sucker is surrounded by a tire-shaped elevation of the tegument (Figure 6). The tegument covering the genital opening is irregularly corrugated and lacks papillae (Figure 7). The tegument covering the outer border of the subterminal acetabulum is somewhat smooth, while that close to the center is corrugated in a bee-comb like structure (Figure 8).

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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 75RPMI 1640 Formula 2 100 100 75 62.5 12.5 0 0 25 37.5 87.5RPMI 1640 Formula 3 100 100 62.5 50 12.5 0 0 37.5 50 87.5RPMI 1640 Formula 4 100 100 50 37.5 0 0 0 50 62.5 100RPMI 1640 Formula 5 100 75 37.5 25 0 0 25 62.5 75 100RPMI 1640 Formula 6 100 50 12.5 0 0 0 50 87.5 100 100RPMI 1640 Formula 7 100 50 0 0 0 0 50 100 100 100RPMI 1640 Formula 8 50 25 0 0 0 50 75 100 100 100RPMI 1640 Formula 9 25 0 0 0 0 75 100 100 100 100RPMI 1640 Formula 10 100 100 100 87.5 75 0 0 0 12.5 25Normal saline 100 75 0 0 0 0 25 100 100 100Natural 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.

Conc.(mg/L)

Exposure Time

(Hours)

ALBENDAZOLE NICLOSAMIDENo. of dead worm

Mean ± SDMortality

rate%No. of dead worm

Mean ± SDMortality

rate%

0.05

2 0 0 - -4 0 0 - -8 3.33± 0.50 41.6 - -12 4.67± 0.58 58.3 - -24 7± 0 87.5 - -

0.25

2 0.67± 0.58 8.3 1± 0 12.54 2± 1 29.2 1.33 ± 0.58 16.78 5± 1 62.5 2.67 ± 0.58 33.312 6.33± 0.57 79.2 4.33 ± 1.57 5424 8± 0 97.2 7 ± 1 87.5

0.5

2 3± 0 12.5 1.67 ± 0.58 20.84 4.33± 0.58 54.2 2 ± 1 258 5.67± 0.58 70.8 3 ± 1 37.512 7.67± 0.58 87.5 4.67 ± 1.55 5824 8± 0 97.5 7± 1 87.5

1.5

2 7.67± 0.58 87.5 2.67 ± 0.58 33.34 7.67± 0.58 89.1 3± 1 37.58 7.81± 0.58 95.5* 4.33 ± 1.1 5412 8± 0 100 5.67 ± 0.58 7024 8± 0 100 8 ± 0 100

2.5

2 7.67± 0.58 95.5 3 ± 0 37.54 8± 0 100 4 ± 0 508 8± 0 100 5.33 ± 0.58 66.112 8± 0 100 7 ± 1 87.524 8± 0 100 8 ± 0 100

3.5

2 - - 4.67 ± 0.58 54.14 - - 5 ± 0 62.58 - - 6.33 ± 1.17 66.112 - - 7 ± 0 87.524 - - 8 ± 0 100

4.5

2 - - 5 ± 0 62.54 - - 5.33 ± 0.58 70.88 - - 6.67 ± 0.58 82.312 - - 7.33 ± 0.58 91.1*24 - - 8 ± 0 100

5.5

2 - - 6 ± 0 754 - - 6.67 ± 0.58 83.38 - - 7 ± 1 87.512 - - 7.67 ± 0.58 95*24 - - 8 ± 0 100

* The sub-lethal concentration

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Table (4): In vitro antihelminthic effect of A. sativum alcoholic extract against C. cotylophorum incubated for 24 hours showing the sub-lethal concentration.

Concentration (mg/L)

Exposure Time (Hours)

No. of dead wormMean ± SD Mortality rate (%)

0.1

2 0 04 0 08 1 ± 0 12.512 1.67 ± 0.58 20.824 2.67 ± 0.58 33.3

0.2

2 0 04 0.67 ± 0.58 0.838 1.33 ± 0.58 1612 2.33 ± 0.58 2924 3.67 ± 0.58 45.5

0.3

2 1 ± 0 12.54 1.33 ± 0.58 16.78 1.67 ± 0.58 20.112 3 ± 0 37.124 3.67 ± 0.58 45.8

0.4

2 1.33 ± 0.58 16.74 1.67 ± 0.58 20.88 2.67 ± 0.58 33.312 3.67 ± 0.58 45.824 4 ± 1 50

0.5

2 1.33 ± 0.58 16.74 2.33 ± 0.58 29.28 3.33 ± 0.58 41.112 5 ± 0 62.5

24 6.67 ± 1.15 83.3

0.6

2 2.33 ± 0.58 29.14 3.67 ± 0.58 45.88 4 ± 0 5024 6 ± 1 83.3

0.7

2 3 ± 0 37.54 4 ± 0 508 5.33 ± 0.58 6612 7.67 ± 1.33 9524 8 ± 0 100

0.8

2 3.67 ± 0.58 45.84 4 ± 1 508 5.67 ± 0.58 7012 8 ± 0 10024 8± 0 100

0.9

2 5.33 ± 0.58 544 5 ± 1 628 6 ± 0 7512 8 ± 0 10024 8± 0 100

1

2 4 ± 0 504 5.33 ± 0.58 628 7.33 ± 0.58 97*12 8± 0 10024 8 ± 0 100

* The sub-lethal concentration

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Allium sativum against Adult C. cotylophorum

Figures (1-8): Ultrastructure of adult Cotylophoron cotylophorumFigure (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).

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Ultrastructural changes of the tegument of in vitro treated C. cotylophorum:Sub-lethal concentration (LC95.5) of albendazole: SEM of the tegument of adult C. cotylophorum, treated in vitro with a sub-lethal concentration (LC95.5) of albendazole (1.5 mg/L) for 8 hours, revealed remarkable changes in the tegument integrity. The oral opening was retracted and deformed and the concentric tegumental rings encircling the mouth were no longer seen, but were substituted by

unorganized tegumental ridges (Figure 9). Papillae were hardly seen as swollen bodies on the tegument around the mouth (Figure 10). The smooth tegument covering the acetabulum was deformed into a highly corrugated structure with numerous blebs (Figures 11 and 12). The deformation of the tegument covering the body varied in severity from few blebs and crater-like structures to complete tegumental deformation covering the whole surface (Figures 13 and 14).

Figures (9-14): Ultrastructure of Cotylophoron cotylophorum in vitro treated with LC95.5 albendazoleFigure (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.

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Sub-lethal concentration (LC97) of A. sativum alcoholic extract: Adult C. cotylophorum treated in vitro with a sub-lethal concentration (LC97) of A. sativum alcoholic extract (1.0 mg/L) for 8 hours showed moderate changes in the tegumental integrity. Treatment seems to have a greater effect on the muscle integrity than the tegumental surface. This was revealed by changes in the form and integrity of different surface structures. The mouth appeared flaccid and slightly retracted (Figure 15). Compared with changes induced by albendazole, the tegument surrounding the mouth

was less damaged, corrugated and the papillae lost their uniform structure (Figures 15 and 16). The acetabulum appeared retracted and lost its specific shape (Figure 17). Although the tegument covering the acetabulum was less damaged than in albendazole treated worms, yet the smooth outer and bee comb-like inner tegument changed into an undifferentiated corrugated structure with few blebs (Figures 17 and 18). The body tegument was also affected where it appeared more corrugated with many blebs and the papillae were no longer seen (Figures 19 and 20).

Figures (15-20): Ultrastructure of Cotylophoron cotylophorum treated with LC97 Allium sativum alcoholic extractFigures (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.

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DISCUSSION

An antihelminthic drug, which could cause adverse changes to a parasite but not the host, is said to be a potential antihelminthic drug. Antihelminthic drugs impair the vital activities of the worm and consequently lead to worm death. The most common effect of antihelminthic drugs is paralysis of parasite musculature either by inhibiting neuro-muscular transition or enzymes involving energy production(6,23). Drugs may also damage the body wall(3,17,18) allowing partial digestion or rejection by host immune system. In general, benzimidazoles are known to disturb the energy metabolism of parasites(24,25).

In the present study the exposure time, at which the drug brought about the best result, was chosen according to the reported half life time (duration of action) of the drug, being 8 hours for albendazole(25) and 12 hours for niclosamide(26). In comparison with albendazole, the effect of A. sativum alcoholic extract was evaluated after 8 hours exposure time.

The present results showed that albendazole at a dose of 1.5 mg/L brought about a high mortality rate (95.5%) compared to niclosamide which only induced high mortality rates (91.1% and 95%) at concentrations of 4.5 mg/L and 5.5 mg/L respectively. Allium sativum alcoholic extract proved effective at a concentration of 1 mg/L.

Albendazole has a broad spectrum activity against all classes of parasitic helminthes. This drug has been reported to cause direct disruption of the tegumental and muscle layers by binding specifically to β-tubulins, thereby inhibiting polymerization and functioning of the cellular motor proteins(27,28). In the present study, albendazole showed a potential in vitro effect against C. cotylophorum. This finding contradicts the results of previous studies which reported that oral administration of albendazole at different doses failed to control both adult and immature C. cotylophorum and Paramphistomum spp.(4,29). Moreover, the present work showed that when albendazole was administered in vitro at a dose of 1.5 mg/L (LC95) for 8 hours, the tegument of adult worms was severely damaged, where tegumental blebs appeared especially around the oral opening and acetabulum. Similar findings were reported for Fasciola hepatica treated in vitro with 10 μg/ml of albendazole for 12 hours(30). However, only roughening and thickening of the tegument without bleb formation were observed, but after a shorter exposure time of 6 hours(5,6). Bleb formation and tegumental thickening were also reported for C. cotylophorum treated in vitro with other antihelminthics of the benzimedazole group namely mebendazole, febendazole(5), levamizole(6), and even drugs of other category such as praziquantel(18). Crater-

like structures covering the tegument of treated worms result from bleb rupturing and extensive contraction of the whole body. The origin and mechanism of bleb formation is controversial and not clearly understood. Nerve bulbs under the sensory papillae in Clonorchis sinensis and Paragonimus westermain were suggested to be the origin of bleb formation(29). On the other hand, this suggestion was denied in another report(23) based on the finding of intact papillae. Bleb formation and muscular contraction of drug-treated worms was suggested to be calcium dependent. In confirmation, praziquantel was found to induce increase in permeability of Opisthorchis viverrini tegument to calcium binding or transport across the membrane resulting in bleb formation(31). The depletion of calcium in S. mansoni cultivation medium was shown to inhibit tegumental bleb formation and block muscular contraction of the worm(32). It was shown that the increase of calcium uptake by the parasite is directly related to tegumental vacuolization(33). In addition, the immediate tetanic contraction of the worm after treatment with albendazole, was reported to be a unique feature in many trematode and cestode parasites and muscular or nervous paralysis may be one of the direct causes of worm death(29).

Several antihelminthic agents, including certain plant extracts, have been reported to cause considerable structural alterations against paramphistomes(34,35). However, the nature of the changes is highly specific and different for each agent. Previous reports indicate that A. sativum (garlic) possesses antimicrobial, antihelminthic, anti-protozoal, anti-fungal, anti-carcinogenic, hypo- and hyperglycemic and insecticidal properties(14,36). It is known that garlic contains many components that show antiparasitic effects(13), and its oil acts as a chemotherapeutic agent for poultry parasites(37). The present study showed that 1 mg/L of alcoholic extract of A. sativum had a sublethal effect (97%) on C. cotylophorum after 8 hours exposure. Comparable results were reported where the alcoholic extract of A. sativum caused pronounced effect on muscle integrity of Gigantocotyle explanatum worm(14).

The physiological activity of dietary A. sativum is attributed to a number of organosulphate compounds, one of which is the amino acid “alliin” found in the intact bulb(38). When the bulb is cut or crushed, the C-slyase enzymatic system called allinse converts alliin to allicin (diallyl thiosulphinate) which is responsible for the characteristic flavor of A. sativum and its antimicrobial properties(38,39). In addition, A. sativum is the best source of selenium and the sulfur compound allicin produced by crushing or chewing of garlic, in turn produces other sulfur compounds; ajoene and vinyldithiins(36). Allicin

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Allium sativum against Adult C. cotylophorum

can disable two groups of enzymes; dehydrogenase and cysteine proteinases which provide infection agents with the means to damage and invade tissues(38). Allicin was also reported to inhibit the activity of acetylcholinesterase, lactic dehydrogenase and alkaline phosphatase(39). In our study, in addition to the garlic induced tegumental changes, the death of the worm might have resulted from dysfunction of the muscular and nervous tissues, in addition to impairment of the overall metabolic activity of the worm.

In conclusion, Allium sativum alcoholic extract possesses a remarkable antihelminthic activity against C. cotylophorum. It may serve as an alternative for antihelminthic chemotherapeutic agents to avoid their toxic side effects and development of resistance in a safe and eco friendly manner. Further in vivo studies should be done to evaluate the efficacy of the active gradients of garlic on paramphistomiasis.

Author contribution: NA Radwan performed SEM, supervised data analysis and writing of manuscript. AI Khalil suggested the study point, supervised data analysis, wrote part of the manuscript and did the proof reading. AE Wahdan collected the material and performed the experiments.

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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 وهدانقسم 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 االلبندازولوالنيكلوزاميد 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 ( )، 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%) عندان الماسح االلكتروني المجهر استخدام اوضح وقد (1.5 مجم/لتر). بااللبندازول بالمقارنة (5.5 مجم/لتر) عالية دوائية تركيزات 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.

RPMI 1640