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International Journal of Traditional and Natural Medicines International Journal of Traditional and Natural Medicines, 2016, 6(1): 52-60 International Journal of Traditional and Natural Medicines ISSN: 2167-1141 Journal homepage:www.ModernScientificPress.com/Journals/IJTNM.aspx Florida, USA Article Toxicity of Two Common Euphorbiales Effect on Metabolism and Enzyme System of Freshwater Snail Lymnaea acuminata Ram P. Yadav and Ajay Singh* Natural Product Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur – 273 009 (U.P.), INDIA * Author to whom correspondence should be addressed; E-Mail:[email protected] Article history: Received 2April 2016, Received in revised form 5 July2016, Accepted 28 July 2016, Published6 August 2016. Abstract: Toxicity of aqueous bark extracts of Codiaeum variegatum and Croton tiglium were studied on the acetylcholinesterase activity and metabolism in the nervous tissue of freshwater harmful snail Lymnaea acuminata. Sub-lethal doses of aqueous extracts of both the plants significantly alter the total protein, total free amino acid, glycogen, pyruvate, lactate level and also significant affect the activity of acetylcholinesterase (AChE) and lactic dehydrogenase (LDH), Alterations in all the cases were dose dependent. This study also shows that there is partial recovery in these parameters in the snail after 7th day of the withdrawal of the treatment, which supports the view that plant products are safer in use as molluscicidesfor controlling the snails in aquatic bodies Keywords: Euphorbiales, Lymnaea acuminata, Lactic dehydrogenase, Acetylcholinesterase and Metabolism 1. Introduction Earlier studies indicate that the aqueous extracts of Euphorbiales have potent molluscicidal activity against the freshwater snails Lymnaeaa cuminataand Indoplanorbis exustus (Singh and Agarwal, 1988; 1990; 1992; 1995; Yadav and Singh, 2001). These snails are the intermediate hosts of Copyright © 2016 by Modern Scientific Press Company, Florida, USA Int. J. Trad. Nat. Med.2016, 6(1): 52-60 53 liver-flukes Fasciola hepatica and Fasciola gigantica. This causes endemic fascioliasis in Terai region (wet part) of Northern part of India (Singh and Agarwal, 1981). Most of studies have carried out in this area however suffer from one common drawback. While there is much information on the toxicity and lethal doses of these plant molluscicides, very little literature is available on their mode of action in the organism. The aim of this paper is to present a biochemical and pharmacological information about two potent molluscicidal euphorbious plants, Croton tiglium and Codiaeum variegatum on snail pest. In the present study, the effects of sub-lethal doses of aqueous stem bark extracts of both the plants are given on the biochemical changes occurred by these extracts in the body of this snail. 2. Materials and Methods The stem bark of medicinalplant Croton tiglium and Codiaeum variegatum were collected around Gorakhpur from their natural habitat in winter season. These plants were identified by the taxonomist, Botany Department, D.D. U Gorakhpur University, Gorakhpur (U.P), India. Adult Lymnaea acuminata(2.6±0.3 cm in total shell height) were collected from Ramgarh Lake of Gorakhpur district and maintained in plastic tank for acclimatization to laboratory condition. The acclimatized animals were treated with stem extracts of Croton tiglium and Codiaeum variegatum according to the method of Singh and Agarwal (1988).The experimental animals were treated with sub-lethal doses (40% and 80% of LC50) of the stem bark extracts of Croton tiglium and Codiaeum variegatum for 96h exposure period. Six aquaria were set up for each dose and each aquarium contained after 20 snails in 3L de-chlorinated tap water. The fresh stem bark were mined in 5.0 ml of distilled water, homogenized for 5 min and centrifuged at 1000 g for 10 min. The supernatant was used as a water extract for the biochemical activity. The LC50 of Croton tiglium and Codiaeum variegatum against snail Lymnaea acuminata was exposed to 40% (2.44 mg/L) and 80% (4.88 mg/L) of 96h LC50 of stem bark extracts of Croton tiglium and Codiaeum variegatum 40% (4.97 mg/L) and 80% (9.93 mg/L) of 96h LC50 these doses were based on LC50 values reported by (Yadav and Singh, 2001). After completion of treatment the test animals were removed from the aquaria and washed with water. The nervous tissue of Lymnaea acuminata was excised and used for biochemical analysis. Control animals were kept under similar conditions without any treatment. In order to see the effect of withdrawal from the treatment, the experimental animals were exposed for 96h in case of L. acuminata to sub-lethal doses of aqueous extracts, following which test animals were transferred to freshwater. Copyright © 2016 by Modern Scientific Press Company, Florida, USA Int. J. Trad. Nat. Med.2016, 6(1): 52-60 54 Each experiment was replicated at least six times and the values have been expressed as mean ±SE of six replicates. Student’s‘t’ test and analysis of variance were applied to locate significant changes (Sokal and Rohlf, 1973). 2.1. Biochemical Estimation Protein- Protein levels were estimated according to the method of Lowry et al. (1951) using bovine serum albumin as standard. Homogenates (5 mg/mL, w/v) were prepared in 10%TCA. Total free amino acids- Estimation of total free amino acid was made according to the method of Spices (1957). Homogenates (10 mg/mL, w/v) were prepared in 95% ethanol, centrifuged at 6000 xg and used for amino acid estimation. Glycogen- Glycogen was estimated by the Anthrone method of Van Der Vies (1954). In present experiment 50 mg of tissue was homogenised with 5 mL of cold 5%TCA. The homogenate were filtered and 1.0 mL of filtrate was used for assay. Pyruvate- Pyruvate level was measured according to Friedemannand Haugen (1943). Homogenate (50 mg/mL, w/v) was prepared in 10% TCA. Sodium pyruvate was taken as standard. Lactate-Lactate was estimated according to Barkerand Summerson (1941), modified by Huckabee (1961). Homogenate (50 mg/mL, w/v) was prepared in 10% cold TCA. Sodium lactate was taken as standard. Lactic dehydrogenase- Lactic dehydrogenase activity was measured according to the method of Anonymous(1984). Homogenates (50 mg/mL, w/v) were prepared in 1 mL of 0.1 M phosphate buffer, pH 7.5 for 5 min in an ice bath. Enzyme activity has been expressed as nano mol of pyruvate reduced/min/mg protein. Acetylcholinesterase- Acetylcholinesterase was estimatedby the method of Ellman et al. (1961) as in 0.1 M phosphate buffer in ice bath. Optical density was measured at 412 nm at 25C. Enzyme activity expressed in mol ‘SH’ hydrolysed/min/mg protein. 3. Results and Discussion 3.1. Results Experimental conditions of water determined by the method of APHA/WPCF (1998). Atmospheric and water temperature was ranging from 30.5-31.5 ºC and 27.0-28.0ºC, respectively.pH of water was 7.3-7.5, while dissolved oxygen, free carbondioxide and biocarbonate alkalinity were ranging from 6.8-7.6 ppm, 4.4-6.5 ppm and 105.0-109.0 mg/L respectively, during experiments. Exposure to extracts of stem bark of both the plant against freshwater snail Lymnaea acuminata, Behavioural changes appear with 5 to 10 min of exposure. The initial 30-40 min was a Copyright © 2016 by Modern Scientific Press Company, Florida, USA Int. J. Trad. Nat. Med.2016, 6(1): 52-60 55 period of hyperactivity during which, sluggish snails moved rapidly in the aquarium water. After some time they started crawling on each other. As the poison enters in the snails body a muscular twitching and the snails become spirally twisted, which resulted ataxia, convulsion, paralysis and finally death of snails. Prior to death, there was complete withdrawal of the body inside the shell that indicates the symptoms of nerve poisoning. Exposure of snails to 40% and 80% of LC50 (96h) of aqueous extracts of stem bark of Croton tiglium and Codiaeum variegatum for 96h caused significant dose dependent reduction in the total protein, glycogen, pyruvate level and significant inhibit the acetylcholinesterase and lactic dehydrogenase activity while increased the amino acids and lactate levels (Table 1 and 2). Table 1:Change in total protein, total free amino acids (g/mg) and glycogen, lactate (mg/g), pyruvate (mol/g), AChE (mol ‘SH’ hydrolysed/min/mg protein) and LDH (mol/mg protein/h) activity in nervous tissue of L .acuminata after 96h exposure to 40% and 80% of LC50 (96h) of stem-bark extract of Croton tiglium. th Parameter Control 40% of LC50 (96h) 80% of LC50 (96h) 7 days withdrawal (2.44 mg/L) (4.88 mg/L) Protein 64.5±0.18 (100) 41.90±0.18 (61) 30.30±0.80 (45)+ 65.56±0.02 (97)+ Aminoacids 33.4±1.12 (100) 42.40±1.00 (127) 45.00±1.00 (135) + 34.07±0.31 (102) + Glycogen 7.9±0.03 (100) 4.2±0.02 (53) 2.8±0.04 (35) + 6.78± 0.02 (86) + Lactate 2.18±0.07 (100) 3.39±0.15 (156) 4.06±0.19 (186) + 2.32± 0.10 (106) + Pyruvate 0.698±0.034 (100) 0.416±0.024 (60) 0.289±0.023(41) + 0.67± 0.15 (88) + LDH 0.072±0.008 (100) 0.045±0.006 (62) 0.028±0.002 (38) + 0.648± 0.15 (90) + AChE 0.071±0.004 (100) 0.051±0.003 (72) 0.039±0.003 (55) + 0.0646± 0.21 (91) + +, Significant (P<0.05) Student’s ‘t’ test was applied between 80% of LC50 (24h) and withdrawal groups. Values are mean ±SE of six replicates. Values in parenthesis are percent change with control taken as 100%. Protein levels were reduced to 61% and 62% of controls after treatment with 40% of LC50 (96h) of stem bark extracts of Croton tiglium and Codiaeum variegatum in the nervous tissues.
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