Evaluation of the Ethanolic Extracts of Three Plants

International Journal of Basic and Applied Sciences Huseein et.al., Vol. 1 No.3 ISSN: 2277-1921

(A peer reviewed International Journal) Evaluation of The Ethanolic Extracts Of Three Plants For Their Molluscicidal Activities Against Snails Intermediate Hosts of Schistosoma Mansoni And Fasciola International Journal of Basic and Applied Sciences, Vol. 1 No. 3. pp. 235-249 2277-1921. 2012

ISSN 2277 – 1921 Article type Full Length Research Article Submission date 24 June 2012 Acceptance date 30 June 2012 Publication date 15 July 2012 Article URL http://www.crdeep.com/category/ijbas

Authors Abdalla A. Hassan, Abeer E. Mahmoud, Rasha A. H. Attia and *Enas A. M. Huseein

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Online version available at: www.crdeep.com International Journal of Basic and Applied Sciences Huseein et.al., Vol. 1 No.3 ISSN: 2277-1921

Full Length Research Paper

Evaluation of The Ethanolic Extracts Of Three Plants For Their Molluscicidal Activities Against Snails Intermediate Hosts of Schistosoma Mansoni And Fasciola

Abdalla A. Hassan, Abeer E. Mahmoud, Rasha A. H. Attia and *Enas A. M. Huseein Department of Parasitology, Faculty of Medicine, Assiut University, Egypt.

*Corresponding Author: Enas A. M. Huseein

Abstract Schistosoma mansoni and fascioliasis are worldwide parasitic diseases infecting 83 and 17 million people respectively causing significant morbidity and mortality. The present work was carried out to evaluate the molluscicidal activities of the ethanolic extract of three plant species namely Euphorbia aphylla, Ziziphus spina-christi and Enterolobium contortisiliquum against Biomophalaria alexandrina and Lymnaea cailliaudi (natalensis), the snails intermediate hosts of Schistosoma mansoni and Fasciola respectively. The highest molluscicidal activity was recorded for Euphorbia aphylla on both snails’ species. The least activity was recorded for Enterolobium contortisiliquum which gave negative results against both snail species up to1000 ppm. The LC50 and LC90 of the ethanolic extract of

Euphorbia aphylla against Biomophalaria alexandrina snails were 87.6 ppm and142.5ppm respectively while the LC50 and LC90 of

Euphorbia aphylla against Lymnaea cailliaudi snails were 0.66 ppm and 0.88 ppm respectively. The LC50 and LC90 of Ziziphus spina- christi ethanolic extract against Lymnaea cailliaudi snails were 311ppm and 500 ppm respectively. However it showed no molluscicidal activity againist B. alexandrina snails up to 1000 ppm. The histopathological changes in hermaphrodite gland of B. alexandrina and

Lymnaea cailliaudi snails 2 weeks post exposure to LC25 of the ethanolic extract Euphorbia aphylla and the histopathological effect of LC25 of the ethanolic extract Ziziphus spina- christi on Lymnaea cailliaudi snails were studied. Degenerative changes in hermaphrodite acini and their contents of ova and sperms were observed. The purification of active compounds present in Euphorbia aphylla and Ziziphus spina- christi plants may offers an alternative tool for the control of snails’ intermediate hosts of Schistosoma mansoni and Fasciola. Further investigations for Enterolobium contortisiliquum may increase its potential use as plant molluscicide are recommended.

Key words: Euphorbia aphylla, Ziziphus spina-christi, Enterolobium contortisiliquum, molluscicides, Biomphalaria and Lymnaea.

Introduction of Fasciola requires high or multiple doses of drugs with Schistosoma mansoni is endemic in 54 countries. It is frequent side effects (Ismail et al., 1999 and Abdul-Samie et estimated to infect more than 83 million people worldwide al., 2010). Therefore snail control is considered essential in (Standley et al., 2010 and Mitiku et al., 2010). In the Schistsoma and Fasciola control (Mello-Silva et al., 2006 and developing world including Egypt, intestinal schistosomiasis Jigyasu and Singh, 2010). Due to the hazardous enviromental is common, recurrent and long-lasting health problem (Sadek affects of niclosamide, its toxicities to non-target organisms et al., 2008). Biomophalaria alexandrina as specific and even man, the search for alternative safe molluscicides is intermediate host of Schistosoma mansoni is prevalent in both still ongoing (WHO, 2002 and Abdelrazek et al., 2007). Upper and Lower Egypt (WHO, 2002). During recent years much attention has drawn for the use of molluscicides of plant origin. The use of plants with Fascioliasis is a worldwide zoonotic disease infects over 17 molluscicidal properties appears to be a simple, inexpensive million people causing significant morbidity and mortality and safe alternative (Tantawy et al., 2004, Bakry and Hamdi, (Mas-Coma et al., 2005 and WHO, 2006). In Egypt, 2007, Al-Daihan, 2010 and Singh and Singh, 2010). fascioliasis becomes hyperendemic and problematic where animal reservoir and snail vector are available (Rashed et al., In Egypt, several local plant species screened and proved to 2008). Nearly 24 million Egyptians are at risk and about 800 have molluscicidal properties against different snail species 000 suffering from fasciolosis (WHO, 1995 and Haseeb et al., e.g. Ambrosia maritime and Commiphora molmo) against 2002). Human infection causes serious hepatic pathological Lymnaea cailliaudi snails (Abou Basha et a1., 1994 and Allam consequences (Soliman, 2008). et al., 2001), Solanum species, Guayacum officinalis, Calatropis procera and Euphorbia splendens against Treatment of Schistosoma and Fasciola infections remains Biomophalaria alexandrina snails (Tantawy et al., 2000 and highly problematic. Praziquantel developed drug resistance to Bakry, 2009) and Commiphora molmol against Biomphalaria Schistosoma strains with serious side effects while treatment alexandrina, Biomphalaria arabica, Bulinus truncatus and

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Lymnaea cailliaudi snails (Allam et al., 2001, Al-Mathal and Schistosome Biological Supply Program Unit (SBSP), Fouad, 2006 and Abd-Allah et al. 2009). Theodor Bilharz Research Institute (TBRI), Giza, Egypt were used. It is now well established that in many plants the molluscicidal activity is due to the presence of saponin contents (Rawi et al., Plant Material 1996, Osman et al., 2007 and Singh and Singh 2010) and The plants used in this study were Euphorbia aphylla (Family alkaloid components (Melendez and Capriles, 2002, El- Euphorbiaceae), Ziziphus spina-christi (Family Rhamnaceae) Ansary et al., 2001, Ahmed and Rifaat 2005, Silva et al., 2005 and Enterolobium contortisiliquum (Family Fabaceae). The and Singh et al., 2010). Euphorbia aphylla (Euphorbiaceae), plant materials were collected locally from Faculty of Ziziphus spina-christi (Rhamnaceae) and Enterolobium Agriculture, Assiut University. Plant species was kindly contortisiliquum (Fabaceae) have been described as plants rich identified and extracted by Prof. Dr. Zedan Z. Ibraheim, in saponin and /or alkaloids (Hostettmann et al., 1982, Shahat Pharmacognacy Department, Faculty of Pharmacy, Assiut et al., 2001, Mimaki et al., 2004, Anthony, 2005, Osman et University.Voucher specimens of each plant were kept in the al., 2007 and Siddiqui et al., 2009). Museum of Pharmacognacy Department, Faculty of Pharmacy. Euphorbia (Euphorbiaceae) is the largest genus of flowering plants in the Egyptian flora (El-Karemy, 2008). Over the past The aerial parts of Euphorbia aphylla, Ziziphus spina- christi twenty years, they have received considerable phytochemical and the mature ripe fruit of Enterolobium contortisiliquum and biological attention (Wu et al., 2009). According to were cleaned. Each plant was cut into small pieces and dried Mwine (2011) a good number of Euphorbia species are in shade then grounded using blender. About 250 g of air actually potent as medicinal plants and their extracts have dried powdered plant material was extracted with ethanol been isolated and patented as modern drugs for the treatment (70%), filtered and distilled off under vacuum at temperature of intestinal parasites (Appendino and Szallasi, 1997 and Shi not exceeding 50 °C and the residues were stored in dry glass et al., 2008). They also possess antiamoebic (Tona et al., bottles according to (Bakry, 2009). 2000), anti-plasmodial (Tona et al., 2004) and anti-leishmanial activity (Ahmed et al., 2006). Earlier studies indicated that the Determination of molluscicidal activity euphorbiales have molluscicidal activity (Tantawy et al., The efficacy of the ethanolic extracts of the plants was 2004, Sermsart et al., 2005, Bakry 2009 and Singh and Singh, primarily determined against the adult snails using the 2010). standered method of (WHO, 1965 and Singab et al., 2006). Thus one liter of each plant material at a concentration of 20 Ziziphus spina-christi (Rhamnaceae) is one of the plants most ppm was prepared and 10 snails were added. commonly used in Egyptian folk for treatment of different diseases (El-Rigal et al., 2006 and Nawash and Al-Horani, The snails were maintained in the solution for 24 hours at 2011). Different extracts of Ziziphus spina-christi showed 25°C ± 2°C. After exposure the snails were thoroughly anti-schistosomal activity (Aly and Aly, 2006 and El-Rigal et washed and transferred to dechlorinated fresh water for al., 2006) and anti-leishmanial activities (Tonkal et al., 2005). another 24 hours for recovery. Three replicates were carried out and three groups of snails Enterolobium contortisiliquum is an important species of the were maintained in dechlorinated fresh water under the same family Fabaceae. The essential oil of Enterolobium experimental conditions as negative control. contortisiliquum seeds had been reported to have an antimicrobial activity (Shahat et al., 2008). The Three plant extracts were retested by the same method using various concentrations to give mortalities between 0-100 The present study aims to evaluate the molluscicidal activity percent. Snails were considered dead if they probed and of the ethanolic extracts of Euphorbia aphylla remained motionless or if the shell looked discoloured. (Euphorbiaceae), Ziziphus spina-christi (Rhamnaceae) and Mortality rates were recorded at the end of recovery period. Enterolobium contortisiliquum (Fabaceae) against Biomophalaria alexandrina and Lymnaea cailliaudi Probit regression analysis (SPSS version 7) was used for (natalensis), the snails’ intermediate hosts of Schistosoma calculating LC50 and LC90 values as well as their 95 % mansoni and Fasciola species respectively, as well as their confidence limits according to Finney (1971). histopathological effect on hermaphrodite glands of the previously mentioned snails in a trial to apply these plant Histolopathological Study extracts as eco-friend molluscicides. The histopathological changes in the hermaphrodite gland of Biomphalaria alexandrina snails exposed to LC25 (58.7ppm) Materials and Methods of the ethanol extract of Euphorbia aphylla for 2 weeks were Snail materials studied. Also Lymnaea cailliaudi (natalensis) snails exposed Laboratory bred uninfected adult Biomphalaria to LC25 of Euphorbia aphylla (0.55ppm) and Ziziphus spina- alexandrina snails (6-8mm in diameter) and Lymnaea christi (212 ppm) separately for 2 weeks. Three groups 10 cailliaudi (natalensis) (8-10mm in shell length) from snails each were used for tested plants. Another three groups

Online version available at: www.crdeep.com 236 International Journal of Basic and Applied Sciences Huseein et.al., Vol. 1 No.3 ISSN: 2277-1921 were kept in dechlorinated fresh water for the same period as negative controls (Bakry, 2009). The hermaphrodite gland of The probit mortality showed that the response of the two snail treated and control snails were removed from their shells and species illustrated a linear relationship with the different processed for histopathological study (Harris, 1990). concentrations (dose∕ppm) (fig.1, 2.).

Results Molluscicidal activity of Ziziphus spina- christi Molluscicidal activity of Euphorbia aphylla Molluscicidal effect of the ethanolic extract of the aerial The effect of various concentrations of the ethanolic extract of portion of Ziziphus spina- christi on B. alexandrina snails the aerial portion of Euphorbia aphylla on adult snails of gave negative results up to 1000 ppm. Biomophalaria alexandrina and Lymnaea cailliaudi (nalatensis) snails after 24 hour exposure were evaluated The effects of various concentrations of ethanol extract of the (tables 1and 2). aerial portion of Ziziphus spina- christi on Lymnaea cailliaudi (nalatensis) snails after 24 hour exposure were evaluated The LC50 and LC90 of this extract against Biomophalaria (table 3). The LC50 and LC90 of the ethanolic extracts Ziziphus alexandrina snails after 24 hour exposure were 87.6 and 142.5 spina- christi on L. cailliaudi snails were 311ppm and 500 ppm respectively. While the LC50 and LC90 of the same extract ppm respectively (table 4). The probit mortality showed that against Lymnaea cailliaudi (nalatensis) snails after 24 hour the response of L. cailliaudi snails illustrated a linear exposure were 0.66 and 0.88 ppm respectively (table 4). relationship with the concentrations (dose ∕ ppm) (fig. 3).

There was a significant difference between molluscicidal Molluscicidal activity of Enterolobium contortisilicuum activities of the ethanolic extract of Euphorbia aphylla against The ethanolic extract of the mature ripe fruit of Enterolobium both snails. Lymnaea cailliaudi (nalatensis) snails were more contortisilicuum gave negative results against both snail sensitive to Euphorbia aphylla extract than Biomophalaria species up to1000 ppm. alexandrina snails.

Table 1: Mortality rates of ethanolic extract of Euphorbia aphylla against Biomophalaria alexandrina snails.

Conc. Number of Number of Mortality (ppm) tested snails dead snails rates (%)

150 30 30 100 100 30 12 40 50 30 9 30 40 30 6 20 20 30 0 0

Probit Transformed Responses

Figure 1: Dose ∕ probit regression line of Euphorbia aphylla on Biomophalaria alexandrina snails.

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Table 2: Mortality rates of the ethanolic extract of Euphorbia aphylla against Lymnaea cailliaudi ( nalatensis) snails.

Conc. Number of Number of Mortality (ppm) tested snails dead snails rates (%)

1 30 30 100 0.80 30 24 80 0.75 30 18 60 0.60 30 12 40 0.50 30 6 20 0.25 30 0 0

Probit Transformed Responses

Concentrations (ppm) Figure 2: Dose ∕ probit regression line of Euphorbia aphylla on Lymnaea cailliaudi (nalatensis) snails.

Table 3: Mortality rates of the ethanolic extract of Ziziphus spina- christi against Lymnaea cailliaudi (nalatensis) snails.

Conc. Number of Number of Mortality (ppm) tested snails dead snails rates (%)

800 30 30 100 600 30 27 90 400 30 24 80 300 30 20 66.66 200 30 5 16.66 100 30 0 0

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Probit Transformed Responses

Figure 3: Dose ∕ probit regression line of Ziziphus spina-christi on Lymnaea cailliaudi (natalensis) snails.

Table 4: LC50 , LC90 and confidence limits of the ethanolic extract of tested plants on B. alexandrina and Lymnaea cailliaudi (nalatensis) snails.

Plant Molluscicidal Activity (ppm) Snail LC50 (95% C.l) LC90 (95% C.l) L .l U.l L l. U.l

Euphorbia aphylla 87.6 39.99 332.5 142.5 98.3 1092.3 B. alexandrina Euphorbia aphylla 0.66 0.62 0.7 0.88 0.82 0.97 L. cailliaudi (nalatensis) Ziziphus spina- 311 163.8 465.7 500 384 1089.7 L. cailliaudi christi (nalatensis)

C.l= confidence limit, L .l=Lower limit, U.l = Upper limit

Histopathological study Hermaphrodite glands of control (normal) Biomphalaria almost damaged (plate 2, 5 fig. A). The acinar epithilum alexandrina and Lymnaea cailliaudi (natalensis) snails. showed necrotic changes in the form of invagination (plate 2, The hermaphrodite gland of the adult Biomphalaria 5 fig. B, A) and partial destruction (plate 5 fig. C). alexandrina and Lymnaea cailliaudi (natalensis) snails as that Degenerative changes were observed in most of the ova, of any other pulmonate snail consists of number of vesicles where some of them have faint staining nuclei and others lost known as acini (plate1, 3 fig. A) separated from each other by their nucleous (plate 2, 5 fig. C). Reduction in the number of thin vascular connective tissue (plate1, 3 fig. B). Each acinus sperms was also observed (plate 2 fig. B). Some acini appear is enveloped in a sheath of squamous epithelium. In each more or less evacuated (plate 5 fig. B). acinus both male and female reproductive gametes are produced where mature ova are located at the periphery of the Regarding the effect of Euphorbia aphylla on Lymneae acini and bundles of male sperms are arranged in the center cailliaudi snails, most of the examined sections showed sever (plate1 fig. B, C & plate 3 fig. C). necrotic changes where the acini completely lost their normal architechture (plate 4 fig. A), became reduced in number and Histopathological Effects shrinked (plate 4 fig. B). Marked degenerative changes of Effects of Euphorbia aphylla on Biomphalaria alexandrina sperms and ova were also observed. Shrinkage and/or snails and Ziziphus spina-christi on Lymneae cailliaudi snails cytoplasmic evacuation were recognized in some ova. Other were more ore less similar. The acini lost their normal ova were swollen and lost their normal shape (plate 4 fig. C). architechture and their separating connective tissues are

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Some acini seemed to be empty and completely evacuated Commiphora molmol (Myrrh) is a plant recommended as safe from both sperms and ova (plate 4 fig. B). molluscicides (Massoud et al., 2004 and Al-mathal and Fouad., 2006) and has been licensed for medical use in Egypt Discussion and several countries as a fasciolicidal and schistosomicidal Consideration of cost and environmental effect of synthetic drug with high efficacy and safety (Aly and Aly, 2006 and molluscicides makes plants the focus of attention as an ideal Abdul-Samie et al., 2010). Also this activity is much higher source of cheap, safe and effective molluscicidal agents than that of Meryta denhamii (LC50 26.4 and LC90 70.8 ppm) (WHO, 1993, 2003 and Aladesanmi, 2007). against Lymnaea cailliaudi (Hassan et al., 2010).

Molluscicidal activity of Euphorbia aphylla: In the present study, Lymnaea cailliaudi snails have been Molluscicidal activity is widespread in the family found to be more susceptible than Biomophalaria alexandrina Euphorbiaceace, although activity varies greatly from species snails to the molluscicidal activity of Euphorbia aphylla with to species (Al-Zanbagi, 2005 and Sharma et al., 2009). Mello- the latter requiring high concentrations as lethal doses. This Silva et al. (2006), Bakry (2009) and Sharma et al. (2009) observation is in accordance with the findings of Allam et al. revealed the molluscicidal activity of different Euphorbia (2001) and Hassan et al., (2010) using Commiphora molmol species with varying degrees of potency. (Myrrh) oil and Meryta denhamii against Lymnaea cailliaudi and Biomophalaria alexandrina snails. In the present study, the molluscicidal effect of the ethanol extract of Euphorbia aphylla against B. alexandrina snails and The difference in susceptibility of the two snails to the lethal Lymneae cailliaudi (natalensis) snails after 24 hour exposure effect of the same extract could be attributed to the natural showed considerable activity with LC50 87.6 ppm and LC90 resistance of different snail’s genera and that the molluscicides 142.5 ppm for B. alexandrina snails and 0.66 ppm and may vary in their toxicological effects according to the species 0.88ppm for Lymneae cailliaudi (natalensis) snails. Lymneae of the snails’ used (Bakry and Hamdi, 2007 and Osman et al., cailliaudi (natalensis) snails were much more senestive to the 2007). extract than B. alexandrina snails. Beside its considerable molluscicidal potency, Euphorbia In the present study, the LC50 and LC90 values of Euphorbia aphylla also presents some very interesting characteristics for aphylla against B. alexandrina are promising in comparison an ideal plant molluscicide. It is cosmopolitan and perennial with some previously studied plants. Euphorbia schimperiana plant. It is not edible to animals and easily cultivable (its and Euphorbia helioscopia caused no mortality up to 100 ppm multiplication is done by means of asexual reproduction which on Biomphalaria pfeifferi (Al- Zanbagi 1999). The aqueous does not require frequent watering or application of pesticides extract from Jatropha curcas L. (Euphorbiaceae) performed or fertilizer) (Baptista et al., 1997 and Brickell and Cathey, poorly against snails transmitting Schistosoma mansoni as 500 2008). ppm caused 50% mortality (Rug and Ruppel, 2000). Molluscicidal activity of Ziziphus spina-christi: The molluscicidal activity of the ethanol extract of Euphorbia In the present study, the LC 50 and LC90 of ethanol extract of aphylla is better than Atriplex stylosa (Chenopediaceae), Ziziphus spina-christi against L. cailliaudi after 24 hours Guayacum officinalis (Zygophyllaceae) and Calatropis exposure were 311 and 500 ppm respectively. This activity is procera (Apocynaceae) with LC50 ranging from 94 to 243 much higher than that reported for the Egyptian plant, ppm and LC90 ranging from 180 to 360 ppm against B. Ambrosia maritima (damsissa) (LC90 3000 ppm) against alexandrina snails. On the other hand this activity is lower Egyptian Lymnaea cailliaudi snails (Abou Basha et al., 1994). than the activity of E. milii (LC5019 ppm and LC90 38 ppm) (Bakry et al., 2004), E. splendens (LC50 40 ppm and LC90 73 Ziziphus spina-christi showed less potent molluscicidal ppm) (Bakry, 2009) and Commiphora molmol (Myrrh) oil activity than Euphorbia aphylla against Lymnaea cailliaudi (LC50 and LC90 155 and 195 ppm) (Allam et al. 2001) aginst which can be attributed to the differences in each plant active the same snail species. These differences in potency can be ingredients, their mode of action and method of penetration of attributed to several factors including the locality of the plant the snails (Rawi et al., 1996 and Hassan et al., 2010). species, time of collection of the plant sample, part used, storage conditions, method of extraction and solvents type In the present study, Ziziphus spina-christi gave no (Brackenbury and Appleton, 1997 and Hassan et al., 2010). molluscicidal activity against B. alexandrina snails up to 1000 ppm. The possibility for the same plant extract to have The current study was extended to evaluate the molluscicidal molluscicidal activity against certain snail species and absence effect of the ethanol extract of Euphorbia aphylla on L. of activity against other species were recorded by Al-Zanbagi cailliaudi. The LC50 and LC90 were 0.66 ppm and 0.88 ppm (2005) who found that Euphorbia schimperiana and respectively. This activity is higher than the latex of E. hirta Euphorbia helioscopia (Family Euphorbiaceae) have a against Lymnaea acuminate (LC50 1.29 ppm) (Yadav and relatively high toxicity against the snails Bulinus wrighti while Singh, 2011) and Commiphora molmol (Myrrh) oil (LC50 and they gave negative results against the snails Biomphalaria LC90 50 and 85 ppm respectively) (Allam et al. 2001). pfeifferi.

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Molluscicidal activity of Enterolobium contortisiliquum: The same results obtained by Mossalem (2003) post B. Enterolobium contortisiliquum is rich in saponins (Mimaki et alexandrina snails exposure to the LC50 of the plants al., 2004), saponins have haemolytic properties and toxic Dyzygotheca kerchoveana, Solanum nigrum and Panicum effect on most cold-blooded animals including snails and are repens for 24 and 48 hours. Also, the histolopathological proved to have molluscicidal activity (Herlt et al., 2002, effect of Guayacum officinalis, Atriplex stylosa and Euphorbia Osman et al., 2007 and Singh and Singh, 2009). However no splendens methanol extract on the hermaphrodite gland of molluscicidal activity of the ethanol extract of Enterolobium Biomphalaria alexandrina snails were studied by Bakry contortisiliquum mature fruits on both snail species up to 1000 (2009) who obtained the same observations post two weeks of ppm was detected. This could be attributed to the fact that exposure to LC25 of the tested plant’s extract. saponins responsible for its activity are extracted in greater measures with other solvents. Supporting this explanation the Conclusion results obtained by Abdel-Rahman and Hassan (2008) who  The use of the ethanolic extract of Euphorbia found that the butanol fraction of Hedera canariensis (Family aphylla as a molluscicide may play a vital role in Araliaceae) has molluscicidal activity against Biomophalaria controlling Schistosoma mansoni and Fasciola. alexandrina and Lymnaea cailliaudi. While ethyl acetate Phytochemical investigations to identify the bioactive extract of the same plant was inactive. On the contrary, ingredient(s) responsible for the molluscicidal potency are Hassan et al. (2010) found that the butanol fraction of Meryta recommended. denhamii flowers which belongs to family Araliaceae was  Toxicological studies on man, fauna and flora of inactive and ethyl acetate extract was active against the fresh water are needed. Biomophalaria alexandrina and Lymnaea cailliaudi.  Further laboratory tests to search for the active component of Ziziphus spina- christi plant may In this study, selection of the hermaphrodite glands of B. increase its potential use as plant molluscicide. alexandrina snails and Lymnaea cailliaudi snails based on the  Further investigations using different solvents to fact that it is a target tissues for molluscicides (Bode et al., search for other active ingredients of Enterolobium 1996). In addition, previous studies have shown that it is contortisiliquum may increase its potential use as strongly affected by molluscicides (El-feky et al., 2009 and plant molluscicide. Rawi et al., 2011).  The application of LC25 of the ethanol extracts of Euphorbia aphylla, and Ziziphus spina- christi may The present study demonstrated histolopathological changes be helpful in snail control as they resulted in in the hermaphrodite glands of Biomphalaria alexandrina evacuations in many gonads’ cells which severely snails exposed to the LC25 of Euphorbia aphylla and Lymnaea suppressed the snails’ capacity for egg-laying. cailliaudi snails intoxicated with Euphorbia aphylla and

Ziziphus spina- christi. The histolopathological changes in these snails were more or less similar showing evacuations Acknowledgement and reduction in number of acini, marked degenerative The authors are grateful to Prof. Dr. Zedan Z. Ibraheim, changes of ova and decrease in numbers of sperms. However Pharmacognacy Department, Faculty of Pharmacy, Assiut sever necrotic changes of the acini with more marked University for identifying , extracting the plant materials and degenerative changes in ova and sperms were detected after for his valuable advice and continuous care to this work. the exposure of Lymnaea cailliaudi snails to Euphorbia aphylla extract. These findings agree with Rizk (1998) who References recorded severe changes in the sperms and ova besides Abd-Allah, K. F., Negm-Eldin, M. M., Saleh, M. H., El- degeneration in the gonadal acini structure of B. alexandrina Hamshary, A. M., El-Gozamy, B.M. and Aly, N. S. (2009). snails post exposure to sublethal concentrations of the plant A study on biological control of six freshwater snails of Sesbania sesban. According to (El-Hommossany and El- medical and veterinary importance. J. Egypt. Soc. Parasitol., Sherbibni, 2011) evacuations in many gonads’ cells severely 39(1): 121-39. suppressed the snails’ capacity for egg-laying. Abdel-Rahman, E. H. and Hassan, S. H. 2008. Molluscicidal saponins from leaves of Hedera canariensis. The differences in histolopathological changes detected in B. J. Egypt. Soc. Parasitol., 38: 293-304. alexandrina and Lymnaea cailliaudi snails using the same Abdelrazek, M. F., Michael1, A. F. and El-Mahrouky, F. S. plant extract (Euphorbia aphylla) may be attributed to the fact (2007). Synthesis and molluscicidal activity of some new that molluscicides may vary in their toxicological effects substituted-furan and furo [2,3-d] pyrimidine derivatives. according to the species of the snails’ used (Bakry and Hamdi, Int. J. Phys. Sci., 2(8): 212-216. 2007 and Osman et al., 2007). Abdul-Samie, R. E., Soliman, E. O., El-Nemr, H. and Masou, A. (2010). Study of Il b, Il , Il and IgE before and 1 4 5 Atlam (2000) found harmful effects in the hermaphrodite after Mirazid Therapy in children with intestinal gland cells and gametogenic stages of B. alexandrina snails schistosomiasis and fasc sioliasis. New York Science and Lymnaea cailliaudi (natalensis) snails treated with Journal, 3(12): 116-122. sublethal concentrations of Euphorbia peplus.

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Plate( 1 )

Plate (1): Transverse sections (T.S.) in normal hermaphrodite gland of B. alexandrina snails (control) Fig. A. Showing normal acinar architecture (arrows) (X100). Fig.B. Showing thin vascular connective tissue (head arrow) and sperms (arrow) (X200). Fig. C. Showing the ova (arrows) (X400).

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Plate (2)

Plate (2): T.S. in hermaphrodite gland of B. alexandrina snails after exposure to LC25 of the ethanolic extract of Euphorbia aphylla Fig. A. Showing loss of normal acinar architecture and damaged separating connective tissue (arrows) (X100). Fig. B. Showing invaginated epithelium (arrows) and reduced number of sperms (head arrow) (X200). Fig. C. Showing partial destruction of acinar epithelium (head arrow), ova lost their nucleous and others has faint staining nucleous (arrows) (X400).

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Plate (3)

Plate (3) T.S. of hermaphrodite gland of normal Lymneae cailliaudi (natalensis) (control). Fig. A. Showing normal acinar architecture (arrows) (X100). Fig. B. Showing separating thin vascular connective tissue (arrow) (X200). Fig. C. Showing ova (arrow) and sperms (head arrow) (X400).

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Plate (4)

Plate (4) T.S. of hermaphrodite gland of Lymneae cailliaudi (natalensis) after exposure to LC25 of the ethanolic extract of Euphorbia aphylla. Fig.A. Showing complete loss of acinar architecture (X100). Fig. B. Showing shrinkage of acini, reduction of their number and some acini appeared to be empty (arrows) (X200) Fig. C. Showing distortion in the shape of some ova with cytoplasmic evacuation (arrows) (X400).

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Plate (5)

Plate (5) T.S. of hermaphrodite gland of Lymneae cailliaudi (natalensis) after exposure to LC25 of the ethanolic extract of Ziziphus spina- christi. Fig. A. Showing loss of normal acinar architecture, damaged separating connective tissue and invagination of acinar epithilium (arrow) (X100). Fig. B. Showing disruption of the acini and some of them appeared more or less evacuated (arrows) (X200). Fig. C. Showing partial destruction of acinar epithilium (head arrow), ova lost their nucleous and others has faint staining nucleous (arrows) (X400).

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