In-Vitro Schistosomicidal and Molluscicidal Activities of the Plant Extract Euphorbia Aphylla

Schistosomicidal and molluscicidal activities of ethanol extract of Euphorbia aphylla (Euphorbiaceae) on Schistosoma mansoni and Schistosoma haematobium worms, their free living stages and snail vectors

Rasha A. H. Attia, Abeer E. Mahmoud and *Samia El Bardicy

Department of Parasitology, Faculty of Medicine, Assiut University.

*Department of Medical Malacology,Theodor Bilharz Research Institute, Imbaba

Abstract

The ethanol extract of Euphorbia aphylla (Family Euphorbiaceae) was evaluated for molluscicidal activities against Biomphalaria alexandrina and Bulinus truncatus, the snail vectors of Schistosoma mansoni and Schistosoma haematobium in Egypt, respectively. It was also tested for the in-vitro schistosomicidal activity against adult worms and the free- living stages (eggs, miracidia and cercariae) of both species.

This study showed that this extract has a considerable molluscicidal activity on both snail spp. (LC50 7.6 and 0.013 ppm and LC90 16.6 and 0.038 ppm respectively). B. truncatus is much more susceptible to E. aphylla ethanol extract than B. alexandrina. On the other hand, the conventional synthetic molluscicide niclosamide assayed by the same procedure showed LC50 0.2 ppm and LC90 0.6 ppm for both snails respectively. Consequently the molluscicidal effect of this extract against B. truncatus is much higher than that of niclosamide.

The in vitro assay of this extract at concentration of 100 ug/ml showed lethal effect on adult worms of S. mansoni and S. haematobium after 3 days. The LC50 was found to be 60.4 ug/ml and 50.8 ug/ml and LC90 to be 90.4 ug/ml and 88.7 ug/ml for these species respectively. However, this effect is still much less than that of the reference drug praziquantel which gave LC50 0.2 ug/ml for both species and LC90 0.6 ug/ml for both species.

The corresponding molluscicidal LC50 of ethanol extract of E. aphylla killed all S. mansoni free living stages (eggs, miracidia and cercariae) after 30, 20 and 40 minutes respectively and killed the same stages of S. haematobium after 20, 15 and 30 minutes respectively. Consequently the possibility of utilizing E. aphylla extract as an alternative natural molluscicide and as a potential source for anti-schistosomal drug especially for S. haematobium should be considered for further investigations.

Keywords: Euphorbia aphylla, Schistosoma mansoni, Schistosoma haematobium, molluscicidal, In-vitro schistosomicidal.

Introduction

Schistosomiasis is a major source of morbidity and mortality in developing countries including Africa, South America and Asia. However, with the increase in immigration from and tourism to these endemic areas, cases of schistosomiasis are now occurring throughout the developed world (Chitsulo et al., 2000). The World Health Organization (WHO, 2002) estimates that 200-300 million people are infected with schistosomiasis. The use of molluscicides of water masses serving as transmission sites is considered an important element in an integrated strategy for the control of the disease. Chemotherapy with praziquantal (PZQ) provides for morbidity control of schistosomiasis by preventing chronic liver disease or bladder cancer. However, there has been recent arguement about the possibility of development and spread of Schistosoma strains that are resistant to PZQ (Kabatereine et al., 2003). Moreover the high cost of the synthetic molluscicides, together with concerns about their toxicity to non-target organisms, this necessitates searching for a new safe and effective molluscicide (Abo-Madyan et al., 2004).

Euphorbia is an enormous genus of flowering plants with approximately 2,000 species belonging to family Euphorbiaceae. Ten species of this family are found in Egypt (Abd El-Ghani and El-Sawaf, 2004). Several spp. of Euphorbia are used in herbal medicine to treat asthma, bronchitis, cough, and throat spasms. Many Euphorbiaceae members including the genus Euphorbia, contain latex which is used as a simple topical treatment for certain skin cancers, such as basal cell and squamous cell carcinomas. The latex extract of these plants showed also high molluscicidal potency against snails and are promising alternatives to niclosamide (Vasconcellos and Schall, 1986). Moreover, E. aphylla is known to possess antihelminthic properties and to be useful in treating intestinal worms (McGuffin et al., 1997). In-vitro schistosomicidal and molluscicidal experiments of some plant extracts had been investigated by many authors. Dos Santos et al. (2007) reported that the latex of Euphorbia conspicua showed lethal activity against Biomphalaria glabrata which is much higher than those reported for extract of other Euphorbia species. De-Carvalho et al. (1998) found that E. milii latex is slightly toxic to S. mansoni free living stages at concentrations much higher than those which are sufficient to kill their intermediate snail host.

In Egypt, Abdel-Hamid (2003) reported that the latex extract of E. royleana showed the highest toxicity against B. alexandrina snails while the latex extract of E. mouritanica showed the lowest toxicity against the same snail. He also reported that the latex extract of E. royleana showed a high percentage of in-vitro schistosomicidal mortality against S.mansoni adult worms. Yousif et al. (2007) screened many plant species including Euphorbia for their potential schistosomicidal activities. Tadros et al. (2008) showed that the saponin fraction from the methanol extract of Dracaena fragrans (Family Agavaceae) had considerable molluscicidal activity on B. alexandrina and B. truncatus. The extract killed all Schistosoma miracidia and cercariae and showed also in vitro lethal effect on Schistosoma adult worms.

The aim of the present work is to carry out in-vitro bioassay of the ethanol extract of E. aphylla on S. mansoni and S. heamatobium adult worms, eggs, miracidia, cercariae as well as to investigate its molluscicidal effect on their respective snail vectors (Biomphalaria alexandrina and Bulinus truncatus).

Material and Methods

• Plant material:

The plant material used in this study is cultivated specimens of Euphorbia aphylla collected from the botanical garden, Faculty of Pharmacy, Assiut University. The plants were cut and left to dry in shade. The plant material was kindly identified and extracted by Prof. Dr. Zedan Z. I. Ibraheim, Pharmacognosy Department, Faculty of Pharmacy, Assiut University according to (El-Menshawi, 2003). Voucher specimen is kept in the Museum of Pharmacognosy of the same faculty.

The dried plant material was powdered and kept in a glass container in the dark till being used. 500 g of the powdered total herb were extracted with ethanol (70%) using maceration and percolation for 24 hours. The process of extraction was repeated twice and the total alcohol extract was pooled together and evaporated under reduced pressure till free from the solvent. The alcohol free residue yielded 48.30 g of the dried residue (9.66%).

• Parasite and snail material:

The parasite material was various stages of S. mansoni and S. haematobium (adult worms, eggs, miracidia and cercariae). The snail material was Biomphalaria alexandrina and Bulinus truncatus adult snails. All materials were procured and obtained from the Schistosome Biological Supply Center (SBSC) at Theodor Bilharz Research Institute, Egypt. All the experiments were done in Theodor Bilharz Research Institute, Egypt.

The adult worms were obtained from laboratory-bred hamsters and rapidly placed in culture medium RPMI 1640 according to (Yousif et al., 2007).

The Schistosoma eggs were obtained from infected hamsters in physiological saline (0.9%). The miracidia were obtained in small amounts of dechlorinated tap water. The cercariae were obtained from experimentally infected B. alexandrina and B. truncatus snails at 25 °C ± 2 °C.

1. Molluscicidal test:

The efficacy of the ethanol extract of E. aphylla was primarily determined against adult snails using the standard method of the World Health Organization (WHO, 1965). Thus, one liter of the plant material at a concentration 20 ppm was prepared and ten snails were added. The snails were maintained in the solution for 24 hrs at 25 ◦C ± 2 ◦C. After exposure, the snails were thoroughly washed and transferred to fresh water for another 24 hrs for recovery. Three replicates were carried out and three groups of snails were run in fresh dechlorinated water under the same experimental conditions as negative control. The conventional molluscicide (Niclosamide) was used as positive control. E. aphylla extract was then retested by the same method using various concentrations to determine LC50 and LC90. The statistical program SPSS package version 7 was used for calculation.

2. Schistosomicidal test:

E. aphylla ethanol extract was in vitro tested on S. mansoni and S. haematobium adult worms according to (Yousif et al., 2007). The freshly obtained worms were cleaned from blood in small sieves 20 µ mesh size using phosphate buffer. Then, they were rapidly washed in the culture medium for more sterilization inside a sterilized laminar flow.

A stock solution (10 mg/mL) of the plant extract was prepared in DMSO (dimethylsulfoxide) diluted with RPMI to produce 3mL test solution of 100 µg/mL final concentration in a 10-mL vial for the screening.

The culture medium was RPMI 1640 containing 20% fetal calf serum, 300 mg streptomycin, 300 units penicillin, and 160 µg gentamicin/100mL medium. The worms were exposed to this concentration in sterilized tissue culture plates 24 wells. Two replicates were used and three pairs of Schistosoma worms, males and females equally represented were placed in each well using sterilized forceps. Positive and negative controls were concurrently used. The reference drug praziquantel 0.2 µg/ml was used as positive control. Test and control wells were examined daily for 3 days for worm viability using a stereomicroscope. Worms which did not show any sign of motility for one minute were considered dead. The activity of the plant extract was measured by calculating the number of dead worms relative to the total number of worms and compared with the negative (DMSO) and positive (PZQ) controls. For determination of LC50 and LC90 the same experiment was repeated several times using various concentrations of the extract and the viability of worms was followed up for three days. The worm mortality was recorded in each case after three days. LC50 and LC90 were determined using SPSS statistical program.

3. Biocidal effect on eggs, miracidia and cercariae:

Ten ml of water containing 100 fresh eggs or hatched miracidia or freshly shed cercariae were put in small graduated Petri dish. The concentrations of molluscicidal LC50 of the extract of both species were tested. Equal number of eggs, miracidia or cercariae in 20 ml of dechlorinated water were used as control. Two replicates were run in each case. Microscopical observation of the viability of these stages, as indicated by hatching or motility was performed after five minutes exposure. Miracidia and cercariae were considered dead when motion ceased completely (Tadros et al., 2008).

Results

The molluscicidal LC50 and LC90 of the extract against B. alexandrina and B. truncatus adult snails after 24 h exposure are given in Table (1). There was difference in susceptibility between the two species; the extract had much stronger lethal effect against B. truncatus than Biomphalaria showing LC50 0.013 ppm and LC90 0.038 ppm in case of Bulinus, in comparison with LC50 7.6 ppm and LC90 16.6 ppm in case of Biomphalaria. The activity of the reference molluscicide ‘Niclosamide’ was determined using the same assay procedure and gave LC50 0.2 ppm and LC90 0.6 ppm for both snails.

The biocidal effect of the ethanol extract of E. aphylla on S. mansoni and S. haematobium adult worms after exposure for 3 days are listed in Table (2). The extract showed worm mortality with concentration 100 ug/ml. LC50 was found to be 60.4 µg/ml and 50.8 µg/ml for S. mansoni and S. haematobium worms respectively. LC90 was found to be 90.4 µg/ml and 88.7 µg/ml for S. mansoni and S. haematobium worms respectively. On the other hand, the activity of the reference drug Praziquantel determined using the same assay gave LC50 0.2 ug/ml and an LC90 0.3 ug/ml for both species of worms.

Schistolarvicidal activities of E. aphylla on S. mansoni and S. haematobium free-living stages are shown in Table (3). Total death rate was reached when Schistosoma free-living stages (eggs, miracidia and cercariae) were exposed to LC50 of the corresponding molluscicidal concentration after variable periods of time ranging between 15-40 minutes.

Table (1): The molluscicidal effect of ethanol extract of E. aphylla on Biomphalaria alexandrina and Bulinus truncatus snails.

Table (2): In vitro schistosomicidal activity of ethanol extract of E. aphylla on S. mansoni and S. haematobium adult worms (after 3 days exposure).

Table (3): Schistolarvicidal activity of ethanol extract of E. aphylla on free- living stages of S. mansoni and S. haematobium using the corresponding molluscicidal LC50.

Discussion

Molluscicides have a history of success and failure in the control of schistosomiasis. The high cost of synthetic compounds, along with increasing concern over the possibility of snail resistance to these compounds and their toxicity to non-target organisms, have given a new impetus to the study of plant molluscicides (Al-Zanbagi, 1999). The use of plants with molluscicidal properties is a simple, inexpensive and has appropriate technology for control of the snail vector (Hostettmann, 1984). Furthermore, investigation of plants used in traditional medicine or recorded in the ethnopharmacological literature provides a ready means of increasing the diversity of available molluscicides and simplifying the choice of selective, ecologically safe snail-killing compounds (Farnsworth et al., 1987). Moreover, in the last decades, plant extracts were widely used for the treatment of Schistosoma infection (Sparg et al., 2000 and Molgaard et al., 2001).

The present study was performed to evaluate the molluscicidal and in vitro schistosomicidal effects of ethanol extract of E. aphylla. This extract showed considerable molluscicidal activity on both B. alexandrina and B. truncatus snails. B. truncatus was more susceptible to the extract than B. alexandrina. On the other hand, niclosamide which is the conventional synthetic molluscicide commonly used nowadays was assayed in the same procedure and gave LC50 0.2 ppm and LC90 0.6 ppm for both snails. The molluscicidal effect of the extract against B. truncatus is much higher than that of niclosamide. This molluscicidal activity of E. aphylla extract is higher than that given by other authors when they studied other species of the family Euphorbiaceae on the same or other snail species (Abdel- Hamid, 2003). Molluscicidal activity is known to be widespread in the family Euphorbiaceae. This activity varies greatly with respect to species, plant part, and even method of extraction, a number of species possess activities significant enough to warrant further investigation as potential alternative molluscicides (Dos Santos et al., 2007). Pereira et al. (1978) studied the molluscicidal activity of Euphorbia cotinifolia against Biomphalaria glabrata and found it lethal for the eggs and snails at concentration of 20 ppm. Schall et al. (1998) described the action of the latex of Euphorbia milli on different Schistosoma snails and they estimated LC90 ranging from 0.13-4.0 ppm. They stated that this material has proved to be one of the most potent and specific plant molluscicide discovered thus far, presenting advantages in terms of application so that it could be used in programs involving community participation in endemic areas in both Brazil and Africa.

Al-Zanbagi (2000) showed the comparative susceptibility of the snail B. pfeifferi to the action of plant extracts from E. helioscopia and E. schimperiana. They were promising from the molluscicidal point of view with LC50 values between 10-100 ppm. Bayluscide was assayed in the same work and the results were LC50 0.08 and LC90 0.2 ppm. The results in this study showed higher activities than those obtained by Abdel-Hamid (2003) who indicated that the latex extract of E. royleana and the acetonitril extract of Jatropha carcus showed the highest toxicity against Biomphalaria alexandrina snails ( LC50 were 11.7 ppm for E. royleana and 10.6 ppm for Jatropha carcus respectively). In a study made by Dos Santos et al. (2007) the latex of E. conspicua showed a sufficient activity against B. glabrata snails that is much higher than those reported for extracts of others Euphorbia species by Pereira et al. (1978), Ahmed et al. (1984), Shoeb and El-Sayed (1984) and Al-Zanbagi (2000) and considerably higher than minimum level recommended by WHO for plants to be considered for use as molluscicidal agents (WHO 1983).Vasconcellos and Schall (1986) and Baptista et al. (1994) used the latex of E.milli and recorded a high molluscicidal activity against B. glabrata snails.

There are limited options available for the chemotherapeutic treatment of Schistosoma infection with the drug of choice being praziquantel (WHO 1993, 1999). Unfortunately, the long-term worldwide application of the drug coupled with the recent discovery of praziquantel-tolerant schistosomes has generated concern over the development of drug-resistant Schistosoma strains. So, for combating schistosomiasis there is an urgent need to develop new drug alternative to praziquantel (Shengliang et al., 2001 and Appiah and Devlas 2002). In the present study E. aphylla showed a strong lethal effect on S. mansoni and S. haematobium adult worms after 3 days with a concentration of 100 ug/ml. It showed LC50 60.4ug/ml and 50.8ug/ml respectively. However, this effect is still much less than that of the reference drug praziquantel which gave LC50 0.2 ug/ml.

Our results are in accordance with that given by Abdel-Hamid, (2003) who showed that the latex extract of E. royleana against S. mansoni adult worms had lethal effect at concentrations of 30-100 ug/ml. On the other hand, Mohamed et al. (2005) in his study on Nigella sativa proved that crushed seeds have a strong lethal effect against S. mansoni worms showing LC50 40 µg/ml, 100% of the parasites were dead at concentration of 100 ug/ml.

Since the overall value of a molluscicide is increased if it also shows toxicity towards the free living transmission stages of the parasite (Marston and Hostettmann, 1991), the present study was extended to evaluate the biocidal effect of the ethanol extract of E. aphylla on S. mansoni and S. haematobium free living stages (eggs, miracidia and cercariae). The corresponding molluscicidal LC50 of E. aphylla killed all free living stages (eggs, miracidia and cercariae) of S. mansoni after 30, 20 and 40 minutes and S. haematobium after 20, 15 and 30 minutes respectively. The lethal activity against S. mansoni free living stages in the present study is higher than that given by other authors, the latex of E. conspicua was lethal at 100 µg/ml to cercariae of S. mansoni after 1–2 hours exposure (Dos Santos et al., 2007), while De-Carvalho et al. (1998) proved that E. milii latex has a narrower spectrum of biocidal action as it did not show any inhibitory effect on S. mansoni eggs hatching. Furthermore, latex proved to be only slightly toxic to miracidia and cercariae, ineffective in achieving a 100% mortality rate at 100 ug/mL after 4 h exposure. Thus, E. milii molluscicidal latex does not present the additional advantage of affecting the viability of Schistosoma miracidia and cercariae.