Indian Journal of Natural Products and Resources Vol. 2(4), December 2011, pp. 452-457

Molluscicidal and ovicidal activity of euphorginol against two harmful freshwater gastropods

Saroj Chauhan and Ajay Singh* Natural Product Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur-273 009, Uttar Pradesh,

Received 23 November 2010; Accepted 23 May 2011

Two freshwater gastropods acuminata and exustus are the intermediate of , which causes endemic fascioliasis disease in cattle and and thus cause immense harm to man and domestic in northern part of India. Thousands of the chemicals used for controlling these snails cause serious environmental pollution. The present investigation deals with molluscicidal potential of active compound euphorginol isolated from the stem bark of a plant Euphorbia tirucalli Linn. against the above harmful freshwater snails in laboratory as well as in pond. The toxicity of euphorginol was time as well as dose dependent and there was a significant negative correlation between LC values and exposure periods. Thus, the LC50 values of euphorginol was decreased from 1.64 mg/l (24h) >1.32 mg/l (48h) >0.71 mg/l (72h) >0.46 mg/l (96h) in case of L. acuminata and from 1.91 mg/l (24h) >1.78 mg/l (48h) >0.88 mg/l (72h) >0.56 mg/l (96h) in the case of I. exustus in laboratory. Similar pattern of toxicity of euphorginol was found in the pond against both the snails but toxicity of euphorginol was reduced about four times in pond than laboratory. The sub lethal doses (20% and 40% of 24h LC50) of euphorginol were exposed to the eggs of both snails and effect was observed up to 28 days to assess its ovicidal activity under laboratory condition. It was observed that sub lethal doses of euphorginol significantly reduced the hatchability of both the snails and maximum reduction was found at the higher doses. It also reduced the survivability of hatchlings to zero percent in the case of L. acuminata only after 21 days of hatching than control group. The LC90 (24h) doses of euphorginol against snails have no apparent killing in non target organism i.e. fish Channa puctatus in the treatment of mixed population of snails and fish which shares the habitat of these snails. Thus, the active compound euphorginol is toxic against both the snails and can be helpful in management of fascioliasis eco-friendly.

Keywords: Euphorbia tirucalli, Euphorginol, Fascioliasis, Flukes, Gastropods, Indoplanorbis exustus, , Molluscicidal, Ovicidal, Snails. IPC code; Int. cl. (2011.01)—A61K 36/47, A61K 129/00, A01P 9/00

Introduction has given very satisfactory results5,6. Vasconcelos 7 Two species of harmful freshwater snails, Lymnaea et al stated that the search for new molluscicides acuminata and Indoplanorbis exustus are intermediate derived from plant extracts is growing in importance hosts of Trematodes (Flukes), Fasciola hepatica and because synthetic products are expensive and present F. gigantica which causes endemic fascioliasis in difficulties regarding application and transport and are cattle and livestock1. Today, fascioliasis is recognized also less likely to cause environmental contamination as an emerging and re-emerging vector-borne disease because they contain biodegradable compounds. 8 with the widest latitudinal, longitudinal and altitudinal According to WHO , effective molluscicides should distribution known for any zoonotic disease2. This destroy cercariae and kill all or nearly all the snail disease entails the highest economic losses on cattle hosts and snails eggs. A number of medicinal production as well as severely impacting public plants belonging to different families, viz. Thevetia health3. One way to tackle the problem of fascioliasis peruviana (Pers.) Merrill, Azadirachta indica A. Juss., is to destroy the carrier snail and thus remove the link Euphorbia royleana Boiss., Jatropha gossypifolia in the life cycle of parasite4. The use of molluscicides Linn., Croton tiglium Linn., Codiaeum variegatum Blume, Nerium indicum Mill., etc. were found to ——————— have toxicological action against vector snail9-14. *Correspondent author: E-mail: [email protected]; The steroidal saponin-containing fraction from Phone: 0551-2201171 methanolic extract of Dracaena fragrans Ker.-Gawl. CHAUHAN & SINGH: MOLLUSCICIDAL & OVICIDAL ACTIVITY OF EUPHORGINOL AGAINST FRESHWATER SNAILS 453

(Family—Agavaceae) was reported for molluscicidal of increasing polarity contaning hexane-chloroform and ovicidal activities against Biomphalaria and finally with chloroform-methanol. The fraction alexandrina and truncatus15. obtained with chloroform-methanol (9.8:0.2) afforded In the present investigation the toxic activity triterpene euphorginol through preparative TLC of active compound euphorginol, isolated from the using solvent system chloroform-ether-methanol stem bark of Euphorbia tirucalli Linn. (Family— (9:0.8:0.2). Euphorginol crystallized from methanol, ), was estimated in the laboratory as m.p. 168-170°C. well as in pond. The sub lethal doses (20 and 40% of Experimental animals LC50 of 24h) of euphorginol was exposed to the fresh The two harmful freshwater snails, Lymnaea eggs of both snails and growth and development was acuminata (3.65±1.00 cm total shell length) and recorded up to 28 days to assess its ovicidal activity Indoplanorbis exustus (0.85±0.037 cm in shell under laboratory condition. length) were collected from the freshwater bodies of Gorakhpur district (India). The collected animals Materials and Methods were kept in a plastic tank containing de-chlorinated

Plant materials tap water for 72 h for acclimatization under The stem bark of the plant E. tirucalli was collected laboratory conditions. The fresh eggs of both the locally from Gorakhpur, India. It is identified by Prof. experimental snails i.e. L. acuminata and I. exustus S.K. Singh, Department of Botany, DDU Gorakhpur were collected from the adult reproducing snails, University, Gorakhpur where the voucher specimen reared in laboratory in plastic tank containing de- is deposited. chlorinated water at room temperature, to evaluate ovicidal activity. Extraction and isolation Statistical analysis Euphorginol (C30H50O, Fig. 1) was isolated by the method of Khan16 from the alcoholic extract of stem The effective doses (LC values), upper and bark of E. tirucalli. The stem bark was extracted four lower confidence limits, slope value, ‘t’ ratio and heterogeneity were calculated through the POLO Plus times with ethanol. The combined ethanolic extracts 17 were evaporated at reduced pressure to afford a gummy computer programme of Robertson et al (2007) . residue. This residue was portioned between ethyl ‘Student’s t’ test was applied to determine the acetate and water. The former fraction was further significant (P<0.05) differences between treated divided into hexane soluble and insoluble portions. The and control animals. Product moment co-relation crude terpenes obtained from hexane insoluble fraction coefficient was applied in between exposure time and lethal concentrations18. were chromatographed over activated silica gel column and the elution was carried out with solvent gradient Toxicity experiments Toxicity experiment was performed by the method of Singh and Agarwal19. Thirty animals were kept in glass aquaria containing 3l de-chlorinated tap water in the laboratory. Snails were exposed for 24, 48, 72 or 96h at four different concentrations of euphorginol. Each set of experiments were replicated six times. Mortality was recorded after every 24h during the observation period of 96 hours. The experiment was also conducted in ponds, 29.28 m3 in area and 9.19 m3 in water volume. Each pond was stocked with 100 snails with a size difference not greater than 1.5 times20. The experimental animals were exposed continuously for 96h to four different concentrations of euphorginol. Contraction of the snail body within the shell and

no response to a needle probe were taken as evidence Fig. 1—Chemical structure of Euphorginol of death of snails. Dead animals were removed to 454 INDIAN J NAT PROD RESOUR, DECEMBER 2011

prevent the decomposition of body in experimental Table 1—Toxicity (LC50 values) of euphorginol against aquarium. Control animals were kept in similar L. acuminata and I. exustus at different condition without any treatment. time intervals in laboratory Exposure LC50 Limits (mg/l) Slope ‘t’ ratio Hetero- Exposure of non-target organism to euphorginol periods Value value geneity LCL-UCL The LC90 (24h) doses of euphorginol against snails (mg/l) (L. acuminata or I. exustus) were also tested against L. acuminata non target organism i.e. fish Channa puctatus which 24h 1.64 1.16-1.72 4.72±1.21 5.23 0.06 shares the habitat of these snails in the treatment 48h 1.32 0.70-1.43 3.53±1.47 4.53 0.01 of mixed population of snails (L. acuminata or 72h 0.71 0.67-.94 3.56±1.35 4.25 0.03 I. exustus) and fish both in the laboratory as well 96h 0.46 0.32-0.69 5.64±1.01 5.21 0.12 as in pond. I. exustus 24h 1.91 1.65-2.32 3.21±0.23 4.41 0.02 Ovicidal activity 48h 1.78 1.33-3.12 4.41±0.27 7.32 0.05 The eggs of the snail L. acuminata (number of eggs 72h 0.88 0.46-1.35 5.43±0.32 6.32 0.08 were about 301±1 in each set) and I. exustus (number 96h 0.56 0.39-0.71 2.34±0.28 6.63 0.12 of eggs were about 232±1 in each set) were taken There was no mortality in the control group. separately, and exposed to sub lethal doses i.e. 20% Regression coefficient showed that there was significant (P<0.05) negative correlation between exposure time and different LC values. of LC50 (24h) (0.32 mg/l) and 40% of LC50 (24h) LCL-Lower confidence limit; UCL-Upper confidence limit.

(0.64 mg/l) against L. acuminata and 20% of LC50 Table 2—Toxicity (LC50 values) of euphorginol against (24h) (0.38 mg/l) and 40% of LC50 (24h) (0.76 mg/l) freshwater snail L. acuminata and I. exustus at different against I. exustus of euphorginol at room temperature time intervals in pond in 1 litre de-chlorinated tap water in glass aquarium, Exposure LC50 Limits (mg/l) Slope ‘t’ ratio Hetero- control group was free from any treatment. The periods Value value geneity hatched young snails were fed with leaf powder of (mg/l) LCL-UCL Nelumbo nucifera Gaertn. The number of the hatched L. acuminata snails was recorded and survivability of hatched snails 24h 6.72 5.36-7.25 2.49±0.48 4.12 0.05 was studied after every 7 days up to 28 days after 48h 5.23 5.11-6.38 2.30±0.46 6.21 0.02 hatching. Strict hygienic condition was maintained at 72h 2.72 2.14-5.81 2.72±0.46 5.44 0.01 96h 1.87 1.01-3.61 3.25±0.47 5.62 0.08 the time of experiment. Disintegration of embryos or I. exustus absence of movement of the embryo was considered 24h 7.87 5.77-8.95 2.65±0.22 5.41 0.04 for calculating the percent mortality of eggs. 48h 6.89 6.33-8.41 2.64±0.32 5.67 0.01 ‘Student’s t test’ is applied between treated and 72h 3.45 2.15-4.75 2.55±0.24 5.82 0.02 control groups and hatched eggs and survivability of 96h 2.68 1.12-2.84 3.21±0.34 4.65 0.12 hatchlings in corresponding treated groups. There was no mortality in the control group. Regression coefficient showed that there was significant (P<0.05) Results negative correlation between exposure time and different LC values. LCL-Lower confidence limit; UCL-Upper confidence limit. Toxicity activity >2.72mg/l (72h) >1.87mg/l (96h) in case of The toxicity of euphorginol was time and dose- L. acuminata and from 7.87mg/l (24h) >6.89mg/l dependent against both the snails, L. acuminata and (48h) >3.45mg/l (72h) >2.68mg/l (96h) in the case of I. exustus in laboratory as well as in pond. There was I. exustus (Table 2). a significant negative correlation between LC50 values and exposure periods (Table 1 and 2). Thus with Ovicidal activity increase in exposure periods the LC50 value of After exposure to sub lethal doses (20 and 40% euphorginol was decreased from 1.64 mg/l (24h); of LC50 of 24h) of euphorginol against eggs of >1.32 mg/l (48h); >0.71 mg/l (72h); >0.46 mg/l (96h) L. acuminata, the number of hatched eggs was against L. acuminata; and from 1.91 mg/l (24h); > significantly reduced to 65 and 51% of control, 1.78 mg/l (48h); > 0.88 mg/l (72h); > 0.56 mg/l (96h) respectively (Table 3). After 20% treatment the in the case of I. exustus, respectively in laboratory survival of hatched young snails was significantly (Table 1). The toxicity of euphorginol in pond was reduced to 42% after 7 days, 31% after 14 days, 22% decreased from 6.72mg/l (24h) >5.23mg/l (48h) after 21 days and no survival was recorded after CHAUHAN & SINGH: MOLLUSCICIDAL & OVICIDAL ACTIVITY OF EUPHORGINOL AGAINST FRESHWATER SNAILS 455

Table 3—Number of eggs treated, duration of hatching (in days), percent hatchability after exposure of eggs of L. acuminata and I. exustus with sub lethal doses (20% and 40% of 24h of LC50) of euphorginol. L. acuminata

Control 20% of 24h LC50 (0.32 mg/l) 40% of 24h LC50 (0.64 mg/l) No. of eggs treated 301.50±1.08 301.50±0.83 301.10±1.24 Duration of hatching (in days) 10-13 11-16 12-16 Hatchability (in %) 301.3±0.96 194.50±0.83* 153.10±0.83* (100) (65) (51) Survivability of hatchlings After 7 days 300±21 82.11±25** 57.15±55** (100) (42) (37) After 14 days 296.55±42 60.51±23** 30.63±83** (98) (31) (20) After 21 days 289.87±33 42.30±83** - (96) (22) After 28 days 287.33±62 - - (95) I. exustus

Control 20% of 24h LC50 (0.38 mg/l) 40% of 24h LC50 (0.76 mg/l) No. of eggs treated 232.50±1.08 232.50±0.83 232.10±1.24 Duration of hatching (in days) 9-12 11-16 12-16 Hatchability (in %) 232.3±0.96 194.50±0.83* 153.10±0.83* (100) (72) (53) Survivability of hatchlings After 7 days 231.55±41 98.37±86** 66.22±83** (100) (51) (43) After 14 days 229.61±23 91.41±64** 55.13±23** (99) (47) (36) After 21 days 225.30±33 48.20±83** 6.76±33** (97) (25) (4) After 28 days 222.65±83 - - (96) Values are means ± SE of six replicates. Values in parentheses are percentages of the corresponding value with control taken as 100%. *, Significant (P<0.05), when Student’s‘t’ test was applied between control and treated groups. **, Significant (P<0.05), when Student’s t test applied between number of hatched eggs and survivability of hatchlings in corresponding treated groups. -, shows no survival. 28 days after hatching of control, similarly after 40% Exposure of non-target organism to euphorginol treatment the survival of hatchlings was reduced to In the treatment of mixed population of snails 37% after 7 days, 20% after 14 days and no survival (L. acuminata or I. exustus) and fishes (C. punctatus) was recorded after 21 days after hatching of control, with the LC90 (24h) doses of euphorginol used against respectively (Table 3). snails, caused no mortality amongst non-target Similar trends of reduction in hatchability was organism fish C. punctatus which shares the habitat recorded after exposure to sub lethal doses (20 and with these snails. 40% of LC50 of 24h) of euphorginol against eggs of I. exustus and it was 72 and 53% of control, Discussion respectively (Table 3). After 20% treatment the From above results it is clear that there was a survival of hatched young snails was significantly significant positive correlation between exposure reduced to 51, 47, 25 and zero percent after 7, 14, 21, period and mortality. The increase in mortality with and 28 days of control after hatching, respectively and increase in exposure period could be due to several after 40% treatment the survival of hatchlings was factors, which may be acting separately or conjointly. significantly reduced 43, 36, 4 and zero percent after The uptake of euphorginol could be time dependent 7, 14, 21 and 28 days after hatching of control, leading to a progressive increase in the titre of the respectively (Table 3). active ingredients and its effects on the snails19,21. It is 456 INDIAN J NAT PROD RESOUR, DECEMBER 2011

clear that the LC50 value of euphorginol against freshwater harmful snails and act as good ovicide and L. acuminata is lower than I. exustus, therefore it can thus can be helpful in eco-friendly management of be said that L. acuminata being more susceptible than fascioliasis because it does not affect the non-target those of I. exustus. It is also clear from results that the organism fish which shares the habitat with toxicity of euphorginol was reduced about four times these snails. in pond than laboratory, it may be due to fact that under natural conditions many factors such as Acknowledgement temperature, sunlight, adsorption by soil particles, etc. One of the authors (Saroj Chauhan) is thankful to influences on toxicity and toxicant degradation22. Indian Council of Medical Research, New Delhi Statistical analysis of the data on toxicity brings out (59/24/2006/BMS/TRM) for financial assistance several important points. 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