Malays. Appl. Biol.

(2008) 37(1): MOLLUSCICIDAL 4146 ACTIVITY OF CERTAIN PLANTS

41

TOXICITY OF PIPER CUBEBA, PIPER LONGUM AND TRIBULUS TERRESTRIS AGAINST THE SNAIL LYMNAEA ACUMINATA
JITENDRA, K.P. and SINGH, D.K.* Malacology Laboratory Department of Zoology, DDU Gorakhpur University, Gorakhpur, 273 009, U.P. India E-mail:dksingh_gpu@yahoo.co.in

ABSTRACT
The toxic effect of dried berries powder of Piper cubeba, dried fruit powder of Piper longum and Tribulus terrestris against snail Lymnaea acuminata was studied. Toxicity of these plant products were time and concentration dependent. The toxicity of Piper longum fruit powder (96h LC50- 48.99 mg/l) was more effective than fruit powder of Piper cubeba (96h LC50- 54.01 mg/l) and fruit powder of Tribulus terrestris (96h LC50- 83.49 mg/l). Ethanol extracts of these plants were more effective than other organic extracts. 96h LC50 of column purified fraction of Piper cubeba against L. acuminata was 3.57 mg/l, whereas 96h LC50 of the column purified fraction of Piper longum and Tribulus terrestris was 5.03 mg/l and 13.53 mg/l, respectively. These plants may be used as potent source of molluscicide against the snail Lymnaea acuminata.

Key words: Lymnaea acuminata; Fascioliasis; cubebene; piperine; harmane; harmine

INTRODUCTION Some of the fresh water snails are the vector of digenean trematode (Fasciola hepatica and Fasciola gigantica) larvae, which causes endemic fascioliasis to man and his domestic cattles (Malek and Cheng, 1974; Godan, 1983; Singh et al., 1996a; Kumar and Singh, 2006). Lymnaea acuminata and Indoplanorbis exustus are acknowledged intermediate hosts of these liver flukes (Agarwal and Singh, 1988; Singh et al., 1996a). The best method to control trematode infection is to control the population of vector snail (Singh et al., 1996a; Allam, 2000; Tripathi and Singh, 2001; Singh et al., 2005). Several attempts have been made to reduce the incidence of fascioliasis by using synthetic pesticides and plant derived products against transmitting snails (Marston and Hostettmann, 1985; Agarwal and Singh, 1988; Singh et al., 1996a; Kumar and Singh, 2006). Uses of plant products as molluscicide are ecologically sound and culturally more acceptable than synthetic one. A large number of plant products have been identified as molluscicide against harmful snails (Singh et al., 1996b; Rao and Singh, 2001; Kumar and Singh, 2006). In the present study common medicinal
* To whom correspondence should be addressed.

plants such as Piper cubeba (Family-Piperaceae), Piper longum (Family-Piperaceae) and Tribulus terrestris (Family-Zygophyllaceae) were tested as molluscicides against the snail Lymnaea acuminata, to explore its molluscicidal activity.

MATERIALS AND METHODS Animals Adult Lymnaea acuminata (2.25 0.20 cm in length) were collected locally. The animals were acclimatized for 72h in laboratory condition. The pH of the water was 7.1-7.3 and dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 6.5-7.1 mg/l, 5.2-6.2 mg/l and 102-104 mg/l respectively. Ten experimental animals were kept in glass aquaria containing 3l of dechlorinated tap water (tap water was kept for 72h in laboratory condition to remove the chlorine) at 22C to 24C. Dead animals were removed immediately from the aquaria to avoid any contamination. Plants Berries of Piper cubeba, dried fruit of Piper longum and spiny fruit of Tribulus terrestris were collected from local area and identified by Prof. S.K. Singh, Plant Taxonomist from the Department

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MOLLUSCICIDAL ACTIVITY OF CERTAIN PLANTS

of Botany, D.D.U. Gorakhpur University, Gorakhpur, (UP) India. Crude Plant Product Stalked berries of Piper cubeba, dried fruit of Piper longum and dried spiny fruit of Tribulus terrestris were pulverized separately with a grinder and the crude powder, thus obtained were used for the toxicity experiments. Organic solvent extracts Five grams stalked berries of Piper cubeba, dried fruit of Piper longum and dried spiny fruit of Tribulus terrestris were mixed with 100 ml each of 95% ethanol, 98% ether, 99.7% chloroform and 98% acetone at room temperature. After 24h in each extracts solvent was removed under vacuum and remaining dried parts were used for determination of molluscicidal activity. Piper cubeba - ethanol-580 mg, chloroform-630 mg, acetone-430 mg, ether-380 mg, Piper longum ethanol-630 mg, acetone-670 mg, chloform-570 mg, ether-510 mg, Tribulus terrestris- ethanol-710 mg, chloroform-520 mg, acetone-620 mg, ether-540 mg were obtained after extraction. Column purification 100 ml of ethanol extract fraction of stalked berries of Piper cubeba, dried fruit of Piper longum and dried spiny fruit of Tribulus terrestris were subjected to silica gel (60-120 mesh, Qualigens Glass, Precious Elctrochemindus private limited, Mumbai, India) chromatography through a 5x45 cm column. 5 ml fractions with ethanol (95%) were collected. Ethanol was evaporated under vacuum and remaining solids were used for determination of molluscicidal activity. Pure compounds Piperine, cubebene, Harmine (7-methoxy-1methyl-9H-pyrido [3,4b] indole), Harmane (Aribine, 1-methyl-9H-pyrido [3,4b] indole) were purchased from Sigma Chemical Co. USA. Thin layer chromatography Molluscicidal components present in berries of Piper cubeba, dried fruit of Piper longum and dried spiny fruit of Tribulus terrestris were identified by thin layer chromatography (TLC). TLC was done on 20x20 cm. pre coated silica gel (Precious Electro Chemical Industry, Pvt. Limited, Mumbai). The solvent was benzyne ethyl acetate (90:10). Comigration of column purified fraction of the plant with pure component cubebene, harmane harmine and piperine were done for identification. TLC plates were developed by iodine vapour.

Treatment of animals Toxicity experiments were performed by method of Singh and Agarwal (1984). Six aquaria were set for each concentration of plant derived molluscicides. Ten experimental animals were kept in each glass aquarium containing 3l of dechlorinated tap water. Snails were exposed to different concentrations of P. cubeba, P. longum and T. terrestris (Table 1). Concentrations of plant products were determined by testing different range of concentrations against L. acuminata. After that four concentrations of plant products were taken which can caused snail mortality between 10% to 100%. Mortality was recorded at 24h intervals up to 96h exposure periods. Control animals were kept in an equal volume of dechlorinated water under similar condition but without treatment. Lethal Concentration (LC50), lower and upper confidence limits (LCL and UCL), slope values, tratio, g- values and heterogeneity factors were calculated by using the POLO computer programme (probit) of Russell et al. (1977). The regression coefficient between exposure time and different values of LC50 was determined by the method of Sokal and Rohlf (1973).
Table 1. Different concentration of Piper cubeba , Piper longum and Tribulus terrestris used against the snail Lymnaea acuminata Test concentration (mg/l) 30, 50, 70, 90 20, 25, 35, 50 10, 15, 20, 25 30, 35, 50, 70 5, 10, 15, 20 3, 5, 7, 9 1, 3, 5, 7 30, 50, 70, 90 15, 20, 25, 50 25, 35, 50, 70 35, 50, 70, 90 10, 15, 20, 25 5, 7, 9, 12

Material used Fruit powder of Piper cubeba Chloroform extract Acetone extract Ether extract Ethanol extract Column extract Cubebene Fruit powder of Piper longum Chloroform extract Acetone extract Ether extract Ethanol extract Column extract

Piperine
Fruit powder of Tribulus terrestris Chloroform extract Acetone extract Ether extract Ethanol extract Column extract Harmane Harmine

1, 3, 5, 7
70, 90, 110, 150 25, 30, 35, 50 25, 35, 50, 60 50, 70, 90, 110 20, 25, 30, 50 10, 15, 20, 30 1, 3, 5, 7 1, 2, 5, 7

MOLLUSCICIDAL ACTIVITY OF CERTAIN PLANTS

43

Table 2. Toxicity of fruit powder of Piper cubeba and its different organic solvents and purified fraction against snail Lymnaea acuminata Limits Exposure Period 24 h Tested materials LC50 (mg / l) 120.72 061.96 089.04 031.64 032.56 009.89 013.45 137.15 046.42 063.87 028.71 019.92 007.86 008.29 082.24 036.13 047.07 016.89 012.32 005.56 004.84 054.01 025.76 038.86 013.55 007.49 003.57 001.81 LCL UCL Slope value t-ratio g-value Heterog eneity 0.30 0.28 0.29 0.21 0.26 0.12 0.25 0.18 0.16 0.18 0.20 0.18 0.23 0.15 0.18 0.14 0.18 0.23 0.14 0.22 0.13 0.11 0.17 0.17 0.24 0.30 0.33 0.20

Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Cubebene Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Cubebene Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Cubebene Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Cubebene

96.49 48.97 70.85 25.72 22.98 07.96 08.39 92.67 40.36 54.58 23.09 15.46 06.51 05.49 66.58 31.31 41.06 14.54 09.89 04.63 03.24 41.78 21.24 28.06 11.43 05.45 02.64 01.04

200.63 109.19 153.29 051.49 080.65 016.00 045.31 691.39 058.85 086.49 050.20 033.55 011.14 022.18 128.48 044.44 055.49 019.77 015.89 006.67 010.21 068.34 029.51 047.57 015.29 009.17 004.25 002.50

3.070.68 2.720.64 2.980.68 3.080.71 2.020.49 2.270.53 1.410.35 1.550.51 3.410.61 2.830.61 2.390.62 1.730.40 2.110.49 1.220.29 1.910.49 2.640.56 2.810.59 2.690.57 1.780.38 2.250.48 0.930.27 1.770.47 2.790.56 1.910.58 3.230.59 1.980.38 2.550.49 1.230.27

4.53 4.26 4.35 4.35 4.05 4.30 4.05 3.03 5.59 4.64 3.88 4.31 4.31 3.91 3.94 4.69 4.75 4.71 4.69 4.74 3.50 3.79 4.86 3.27 5.51 5.18 5.15 4.63

0.19 0.21 0.20 0.20 0.23 0.21 0.23 0.42 0.12 0.18 0.26 0.21 0.21 0.25 0.25 0.18 0.17 0.17 0.18 0.17 0.31 0.27 0.16 0.36 0.13 0.14 0.15 0.18

48 h

72 h

96 h

Batches of 10 snails were exposed to different concentration. Mortality was determined every 24h up to 96h. Each set of experiment was replicated six times. Concentrations given are the final concentration (mg/l, w/v) in the aquarium water. Abbreviation: LCL= lower confidence limits; UCL= upper confidence limits. Significant negative regression (P<0.05) was observed between exposure time and LC50 of treatments. Ts testing significant of the regression coefficient Piper cubeba (fruit powder) 2.57+; chloroform extract 13.20+; acetone extract 5.37+; ether extract 13.09++; ethanol extract 6.93++; column extract 51.23+; cubebene 10.99+ . +: linear regression between x and y; ++: non-linear regression between log x and log y.

RESULTS The toxicity of crude powder of berries of P. cubeba, fruit of P. longum, and T. terrestris and their organic solvent extracts against L. acuminata were time and concentration dependent. There was a significant negative regression in between the exposure period and LC50 of all the treatments (P<0.05). The LC50 of stalked berries of P. cubeba, fruit of P. longum and T. terrestris powder at 24h was 120.72, 135.20, 174.76 mg/l and at 96h was 54.01, 48.99, 83.49 mg/l, respectively (Table 2, 3, 4). Among all the organic solvent extracts, the ethanol extract of all the three medicinal plants were more toxic (Table 2, 3, 4). The 24h LC50 of ethanol extracts of berries of P. cubeba, fruit of P. longum

and T. terrestris against L. acuminata were 32.56, 42.24 and 41.53 mg/l, respectively. The column purified fractions of three plant products were highly toxic. The 24h LC50 of the column purified fractions of berries of P. cubeba, fruit of P. longum and T. terrestris were 9.89, 13.29 and 33.89 mg/l, respectively. The 96h LC 50 of column purified fraction of T. terrestris (13.53 mg/l) was higher than P. longum dried fruit (5.03 mg/l) and P. cubeba fruit powder (3.57 mg/l). TLC analysis demonstrated that the Rf value of harmane (0.98) and harmine (0.16) was equivalent to the Rf value of two spots in column purified fraction of T. terrestris (0.98 and 0.16). The Rf value of piperine (0.54) was equivalent to the Rf value of column-purified fraction in P. longum (0.54). The Rf value of

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MOLLUSCICIDAL ACTIVITY OF CERTAIN PLANTS

Table 3. Toxicity of fruit powder of Piper longum and its different organic solvents and purified fraction against snail Lymnaea acuminata Limits Exposure Period 24 h Tested materials LC50 (mg / l) 135.20 060.37 117.80 065.25 042.24 013.29 006.48 095.38 035.85 087.39 051.56 030.01 010.31 003.13 064.00 024.03 055.96 039.52 021.35 007.39 002.36 048.99 017.93 045.75 027.64 014.95 005.03 001.44 LCL UCL Slope value t-ratio g-value Heterog eneity 0.17 0.30 0.15 0.16 0.31 0.24 0.29 0.17 0.24 0.18 0.20 0.14 0.14 0.15 0.26 0.25 0.25 0.12 0.11 0.14 0.20 0.20 0.20 0.32 0.20 0.19 0.25 0.19

Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Piperine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Piperine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Piperine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Piperine

98.86 44.68 92.69 55.19 30.52 11.15 05.05 77.24 29.79 73.46 44.27 23.89 08.84 02.35 56.30 20.02 49.90 32.54 17.99 06.16 01.61 43.03 14.67 40.14 21.46 12.55 03.71 00.88

327.02 121.74 213.89 088.39 123.26 019.39 009.69 150.15 048.86 124.06 064.51 054.76 013.85 004.11 074.59 028.73 062.41 046.91 029.71 008.61 003.31 054.77 020.59 050.56 032.12 017.15 005.88 001.94

2.180.57 2.060.47 2.690.65 2.730.54 2.760.79 3.170.69 1.460.27 2.260.52 2.200.44 2.580.58 2.500.51 2.420.63 2.560.62 2.560.47 3.020.51 2.270.45 3.650.58 2.290.51 2.230.56 2.560.60 1.350.26 3.540.51 2.920.52 4.090.61 2.990.55 2.770.57 3.190.66 1.530.27

3.81 4.37 4.16 5.03 3.50 4.60 4.86 4.34 4.96 4.45 4.88 3.86 4.17 4.12 5.97 5.06 6.33 4.54 3.87 4.26 4.82 6.95 5.67 6.77 5.40 4.84 4.86 4.20

0.27 0.20 0.22 0.15 0.31 0.18 0.18 0.20 0.16 0.19 0.16 0.26 0.22 0.21 0.11 0.15 0.09 0.18 0.26 0.21 0.16 0.08 0.12 0.08 0.13 0.16 0.16 0.16

48 h

72 h

96 h

Batches of 10 snails were exposed to different concentration. Mortality was determined every 24h up to 96h. Each set of experiment was replicated six times. Concentrations given are the final concentration (mg/l, w/v) in the aquarium water. Abbreviation: LCL= lower confidence limits; UCL= upper confidence limits. Significant negative regression (P<0.05) was observed between exposure time and LC50 of treatments. Ts testing significant of the regression coefficient Piper longum (fruit powder) 8.30++; chloroform extract 18.13++; acetone extract 40.94+; ether extract 6.95+; ethanol extract 9.77+; column extract 26.58+; piperine 10.66++. +: linear regression between x and y; ++: non-linear regression between log x and log y.

cubebene is (0.49) equivalent to the Rf value of column-purified fraction (0.49) in P. cubeba. 24h LC50 of harmane, harmine, piperine and cubebene were 8.87, 10.08, 6.48 and 13.45 mg/l, respectively (Table 2, 3, 4).

DISCUSSION The present result clearly indicates that the berries of P. cubeba , dried fruit of P. longum and T. terrestris are potent source of botanical molluscicides. Their toxic effects are time as well as concentration dependent. Higher toxicity of ethanol extract among other organic extract indicate that the molluscicidal component present in these plants are more soluble in ethanol. It is evident from the

TLC that the molluscicidal activity of P. cubeba fruit powder may be due to the cubebene, where as in P. longum and T. terrestris it may be due to the presence of piperine and harmane, harmine, respectively. The time dependent effect of these plant products may be due the absorption of active molluscicidal component, in snail body which increases progressively with increase in exposure period or, it may be possible that the active components could change in to more toxic form in aquarium water or, in snail body by the action of different enzymes. It has been reported that if piperine is given at doses of 5-10 mg /kg body weight for 30 days resulted in significant reduction in the weights of testis and accessory sex organs and damage the semniferous tubules (Malini et al ., 1999). The toxicity of harmine present in T.

MOLLUSCICIDAL ACTIVITY OF CERTAIN PLANTS

45

Table 4. Toxicity of fruit powder of Tribulus terrestris and its different organic solvents and purified fraction against snail Lymnaea acuminata Limits Exposure Period 24 h Tested materials LC50 (mg / l) 174.76 053.03 119.01 068.94 041.53 033.89 008.87 010.08 141.07 044.97 094.85 051.58 038.52 023.57 007.29 005.06 106.98 038.21 084.78 043.82 028.53 018.92 003.56 002.75 083.49 029.91 065.23 035.29 022.22 013.53 001.37 001.33 LCL UCL Slope value t-ratio g-value Heterog eneity 0.17 0.38 0.21 0.33 0.56 0.31 0.40 0.27 0.19 0.22 0.14 0.18 0.27 0.19 0.28 0.20 0.11 0.22 0.23 0.16 0.24 0.21 0.19 0.18 0.20 0.16 0.22 0.29 0.34 0.24 0.33 0.33

Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Harmane Harmine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Harmane Harmine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Harmane Harmine Fruit powder Chloroform extract Ether extract Acetone extract Ethanol extract Column purified Harmane Harmine

145.26 046.56 103.45 059.31 036.91 027.65 006.75 006.86 121.66 039.87 084.58 045.99 033.43 020.36 005.03 003.78 094.35 034.41 075.19 038.31 024.75 016.38 002.39 002.04 070.98 026.17 054.69 029.86 018.66 011.07 000.72 000.73

271.83 068.12 157.80 092.46 049.73 051.16 012.05 022.28 194.22 056.57 113.68 061.07 048.79 029.57 016.24 007.92 124.53 044.50 099.08 051.47 032.69 022.39 005.63 003.65 092.83 032.93 073.43 040.06 024.89 015.57 001.94 001.86

3.370.79 4.620.88 3.830.79 3.910.77 3.840.62 2.870.58 2.040.40 1.480.31 2.990.72 3.660.78 3.440.69 3.520.63 2.830.58 2.750.51 1.170.29 1.370.27 3.080.69 3.600.77 3.170.67 2.860.59 2.960.59 2.680.49 1.020.26 1.390.26 3.690.73 3.960.79 3.220.67 2.990.59 3.680.66 2.780.51 1.290.27 1.270.26

4.28 5.24 4.83 5.09 6.19 4.92 5.05 4.82 4.17 4.67 4.96 5.62 4.86 5.36 4.09 5.12 4.42 4.69 4.72 4.88 5.01 5.37 3.86 5.42 5.06 4.95 4.82 5.11 5.55 5.44 4.81 4.86

0.21 0.14 0.17 0.15 0.10 0.16 0.15 0.17 0.22 0.18 0.16 0.12 0.16 0.13 0.23 0.15 0.19 0.18 0.17 0.16 0.15 0.13 0.26 0.13 0.15 0.16 0.17 0.15 0.13 0.13 0.17 0.16

48h

72 h

96 h

Batches of 10 snails were exposed to different concentration. Mortality was determined every 24h up to 96h. Each set of experiment was replicated six times. Concentrations given are the final concentration (mg/l, w/v) in the aquarium water. Abbreviation: LCL= lower confidence limits; UCL= upper confidence limits. Significant negative regression (P<0.05) was observed between exposure time and LC50 of treatments. Ts testing significant of the regression coefficient Tribulus terrestris (fruit powder) 17.19+; chloroform extract 37.25+; acetone extract 10.92++; ether extract 9.42+; ethanol extract 7.40+; column extract 2.15+; harmane 9.42+; harmine 6.68++. +: linear regression between x and y; ++: non-linear regression between log x and log y.

terrestris is very high against L. acuminata (96h LC50- 1.33 mg/l). The fruits of T. terrestris are sweet, cooling, diuretic, aphrodisiac (Gauthaman et al ., 2002). The fruit of P. cubeba has antiinflammatory, and antioxidant property (Choi et al ., 2003; Karthikeyan et al ., 2003). The main constituent of P. longum is an alkaloid, piperine (Kulkarni et al ., 2001). P. longum has antiinflammatory, and antioxidant property (Majumdar et al ., 1990). A comparison of molluscicidal activity of the column-purified fraction of all the three medicinal plants with synthetic molluscicide clearly indicates that the

purified fractions are more effective. Thus 96h LC50 values of carbaryl (14.4 mg/l), phorate (15.0 mg/l) and formothion (8.5 mg/l) against L. acuminata are higher than those of P. cubeba (7.49 mg/l), P. longum (5.03 mg/l) and T. terrestris (13.53 mg/l). 96h LC50 of crude powder of P. cubeba (54.01 mg/ l), P. longum (48.99 mg/l), and T. terrestris (83.49 mg/l) against L. acuminata are lower than crude powder of Allium sativum bulb (271.06 mg/l), Zingiber officinale rhizome (273.80 mg/l) and Cinnamomum tamala bark (445.22 mg/l) (Singh and Singh, 1995; Singh et al., 1997; Srivastava, 2004). It is evident from steep slope value that a small

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MOLLUSCICIDAL ACTIVITY OF CERTAIN PLANTS

increase in the concentration of different plant products causes a marked mortality in snails. A tratio greater than 1.96 indicates that regression is significant. Heterogeneity factor values lower than 1.0 denote that in the replicate tests of random samples the concentration response curves would fall with in the 95% confidence limits and thus, the model fits over data adequately. The index of significance of the potency estimation g-value less than 0.5 indicates that the value of the mean is with in the limits of all the probability limits. In conclusion our study demonstrate that the powder of berries of P. cubeba, dried fruit of P. longum and fruit of T. terrestris may be used as effective molluscicides against vector snail L. acuminata.

REFERENCES Agarwal, R.A. & Singh, D.K. 1988. Harmful gastropods and their control. Acta Hydrochemistry Hydrobiology, 16: 113-138. Allam, A.F. 2000. Evaluation of different means of control of snail intermediate host of Schistosoma mansoni. Journal of the Egyptian Society of Parasitology, 30: 441-450. Choi, E.M. & Hwang, J.K. 2003. Investigations of anti-inflammatory and antinociceptive activities of Piper cubeba, Physalis angulata and Rosa hybrida. Journal of Ethnopharmacology, 89: 171-175. Gauthaman, K., Adaikan, P.G. & Prasad, R.N. 2002. Aphrodisiac properties of Tribulus terrestris extract (Protodioschin) in normal and castrated rats. Life Science, 71: 1385-1396. Godan, D. 1983. Pest slugs and snails, Biology and control. Dora Godan (eds). translated by Shelia Gruber, Springer verlog, Heidelberg, New York. Karthikeyan, J. & Rani, P. 2003. Enzymatic and non enzymatic antioxidants in selected Piper species. Indian Journal of Experimental Biology, 42: 135-140. Kulkarni, D., Apte, P., Mary, F. & Sana, R.T. 2001. Zhigh performance thin layer chromatographic method for the determination of piperine from Piper nigrum. Indian Drugs, 38: 323-326. Kumar, P. & Singh, D.K. 2006. Molluscicidal activity of Ferula asafoetida , Syzygium aromaticum and Carum carvi and their active components against the snail Lymnaea acuminata. Chemosphere, 63: 1568-1574. Majumdar, A.M., Dhuley, J.N. & Deshmukh, V.K. 1990. Anti-inflammatory activity of piperine. Japanese Journal of Medical Science and Biology, 433: 95-100.

Malek, E.A. & Cheng, T.C. 1974. Medical and economic malacology. Academic Press. New York. Malini, T., Manimaran, R.R, Arunakaran, J. & Govindarulu, P. 1999. Effect of piperine on testis of albino rats. Journal of Ethnopharmacology, 64: 219-225. Marston, A. & Hostettmann, K. 1985. Plant molluscicides. Phytochemistry, 24: 639-652. Rao, I.G. & Singh, D.K. 2001. Combination of Azadirachta indica and cedrus deodara oil with Piperonyl butoxide, MGK-264 and Embelia ribes against Lymnaea acuminata. Chemosphere, 14: 1691-1695. Russell, R.M., Robertson, J.L. & Savin, N.E. 1977. POLO; A new computer programme for probit analysis. Bulletin of Entomological Society of America, 23: 209-213. Singh, A., Singh, D.K., Mishra, T.N. & Agarwal, R.A. 1996a. Molluscicide of Plant origin. Biological Agriculture and Horticulture, 13: 205-252. Singh, D.K. & Agarwal, R.A. 1984. Correlation of the anti- cholinesterase and molluscicidal activity of the latex of the Euphorbia royleana Bioss on Lymnaea acuminata. Journal of Natural Products, 47: 702-705. Singh, K., Singh, A. & Singh, D.K. 1996b. Molluscicidal activity of neem (Azadirachta indica A. Juss). Journal of Ethnophormacology, 52: 35-40. Singh, P., Singh, V.K. & Singh, D.K. 2005. Effect of binary combination of some plant derived molluscicides with MGK- 264 or, Piperonyl butoxide on the reproduction of the snail Lymnaea acuminata. Journal of Pest Management Science, 61: 204-208. Singh, S., Singh, V.K. & Singh, D.K. 1997. Molluscicidal activity of some common spice plants. Biological Agriculture & Horticulture, 13: 237-249. Singh, V.K. & Singh, D.K. 1995. Characterization of allicin as a molluscicidal agent in Allium sativum (Garlic). Biological Agriculture & Horticulture, 12: 119-131. Sokal, R.R. & Rohlf, F.J. 1973. Introduction to biostatistics .WH. Freeman and co. San Francisco, 271-273 pp. Srivastava, P. 2004. Molluscicidal activity of Piper nigrum and Cinnamomum tamala and their combination against harmful snails. Ph.D. Thesis. D.D.U. Gorakhpur University, Gorakhpur, UP India. Tripathi, S.M. & Singh, D.K. 2001. Molluscicidal activity of Punica granatum and Canna indica combination with plant derived molluscicides against harmful snails. Malaysian Applied Biology, 30: 25-31.