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

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.

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 25C. 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

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. The maximum decrease in protein level (45% of controls) was observed in nervous tissue of snail at high

doses 80% of LC50 (96h) of stem bark of Croton tiglium (Table 1). The total free amino acids levels in the nervous tissue increased to 127% and 158% of controls

after treatment with 40% of LC50 (96h) of stem bark extracts of Croton tiglium and Codiaeum variegatum (Table1 and 2). Similarly higher increase was also observed after treatment with 80% of

LC50 (96h) of the aqueous stem bark extracts of Codiaeum variegatum (Table 1). The maximum

enhancement in amino acids levels (166% of controls) was found after treatment with 80% of LC50 (96h) of aqueous stem bark extracts of Codiaeum variegatum (Table 2).

Table 2: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 Codiaeum variegatum.

th Parameter Control 40% of LC50 (96h) 80% of LC50 (96h) 7 days of withdrawal (4.97 mg/L) (9.93 mg/L) Protein 64.5±0.18 (100) 39.99±0.18 (62) 34.18±1.64 (53) + 63.85± 1.32 (99) + Amino acids 32.4±1.12 (100) 51.19±1.22 (158) 53.78±0.84 (166) + 34.02± 2.21 (105) + Glycogen 7.9±0.02 (100) 4.9±0.02 (62) 3.0±0.01 (38) + 6.96± 3.11 (88) + Lactate 2.23±0.14 (100) 4.17±0.17 (185) 4.14±0.16 (185) + 2.37± 2.22 (106) + Pyruvate 0.694±0.036 (100) 0.398±0.018 (56) 0.261±0.013 (37) + 0.611± 0.02 (88) + LDH 0.072±0.008 (1000 0.041±0.003 (57) 0.027±0.003 (38) + 0.067± 0.12 (92) AChE 0.071±0.004 (100) 0.040±0.003 (56) 0.024±0.003 (33) + 0.064± 0.11 (90) +

Changes in the levels of glycogen, pyruvate in the nervous tissue of Lymnaea acuminata following exposure of stem bark extracts of Croton tiglium and Codiaeum variegatum are given in (Table 1 and 2). In the AChE activity was reduced to 72% to 56% of stem bark extracts of Croton

tigliumand Codiaeum variegatum 40% of LC50 (96h) (Table 1 and 2). While lactic dehydrogenase is

reduced up to 62% to 57% in case of 40% of LC50 (96h) of Croton tigliumand Codiaeum variegatum

and maximum reduction is 38 % in 80% of LC50 (96h) in stem bark extracts of Croton tiglium (Table 1). The lactate is significantly increased the level of bark extracts of Croton tiglium and Codiaeum

variegatum which is significant increased 156% and 185% of 40% of LC50 (96h) of Croton tiglium and Codiaeum variegatum (Table 1 and 2). The maximum enhancement of the lactate level of 186% of

80% of LC50 (96h) of stem bark extracts of Croton tiglium (Table 1).

3.2. Discussion

It is clear from the result presented here that both the plants i.e. Croton tiglium and Codiaeum variegatum are highly toxic to freshwater snail Lymnaea acuminata. The aqueous stem bark of both the plants significantly alters the protein, total free amino acids, glycogen, pyruvate level and also activity of acetylcholinesterase and lactic dehydrogenase. The alteration caused by protein, glycogen, pyruvate levels and enhancement in amino acids level and also caused by activity of acetylcholinesterase, lactic dehydrogenase of both euphorbiales found to be dose dependent. The action of Pharmacological of Croton tiglium is due to the presence of alkaloids (Rizk, 1987). The alkaloids are naturally occurring organic bases which contain at least one nitrogen atom

Copyright © 2016 by Modern Scientific Press Company, Florida, USA Int. J. Trad. Nat. Med.2016, 6(1): 52-60 57 either in the heterocyclic ring or linked to an aliphatic skeleton. They are found in vascular plants and rarely occur in gymnosperms cryptogams and monocotyledons. Okunji and Iwu (1988), screened several plants of different families for molluscicidal properties and suggested that the toxic properties of these plants may be due to the presence of alkaloids. Toxicity in Codiaeum spp. is due to the presence of tanin in the latex (Wealth of India 1985). Tanin are complex phenolic compounds, divided in to two groups (i) The hydrolysable tanins, which one esters of garlic acid and also glycosides of these esters and (ii) The condensed tanins, which are polymers derived from various flavonoids. The molluscicidal activity was found to be related to the free phenolic groups of the tanins Mommesen and Walsh (1992) reported that protein play important role in the architecture and physiology of the cell and also in cell metabolism. Carbohydrate represents the principal and immediate energy for animals exposed to stress condition. While, protein is the energy source to spore during chronic of stress. Animal exposed to sub-lethal concentrations of plant extracts, experienced greater stress during the process of detoxification of given plant extracts on the metabolic level of animals. The metabolic rate of animal reduced at different concentration of pesticides was greater than that of animals reared in freshwater Aranachalem et al. (1980). Protein depletion in treated animals was the physiological strategy played by the animal to adopt itself to the changed metabolic system. This leads to degradative processes like proteolysis and utilization of degraded products for increased metabolism. The decreased in protein level observed in present investigation may be due to their degradation and also to their possible utilization for metabolic purpose. Bradbury et al. (1987) pointed out that the decreased protein content might also be attributed to the destruction or necrosis of cells and consequent impairment in protein synthesis machinery. The quantity of protein is dependent on the rate of protein synthesis, or on the rate of its degradation. The quantity of protein may also be affected due to impaired incorporation of amino acids in to polypeptide chain (Singh et al., 1996). Enhanced protease activity and decreased protein level have resulted in marked elevation of free amino acid content in nervous tissue of Lymnaea acuminata. The accumulation of free amino acids can also be attributed to lesser use of amino acids Seshagiri Rao et al. (1987) and their involvement in the maintenance of an acid base balance Moorthy et al. (1984). It has been suggested that the stress condition in general induce elevation in the transamination pathway Natarajan (1985). Reductions in glycogen level supposed to be the results of greater stress, the organs experienced during the process of detoxification of active moieties and their metabolites. Several reports are available on the effect of muscular exercise on liver glycogen energy reserves in fish, which get depleted glycogen levels (Nath and Kumar, 1987).

In the present study, in the nervous the glycogenolysis seems to be the result of increased secretion of catecholamines due to stress of plant extracts treatment, because the aqueous stem bark extracts of Codiaeum variegatum and Croton tiglium have potent anti-AChE activity. Umminger (1977) reported carbohydrate represents the principal and immediate energy source for animals exposed to stress conditions carbohydrate reserves to meet energy source for animals exposed to stress conditions carbohydrate reserve depleted to meet energy demand. The rate production of pyruvate by glycolysis exceeds the rate of oxidation of pyruvate by the citric acid cycle in contracting skeletal muscle, under anaerobic conditions as during vigorous exercise. Under these conditions moreover the rate of formulation of NADH by glycolysis is greater than the rate of its oxidation by the respiratory chain (Sambasiva Rao, 1999). Lactate is a dead end in metabolism. It must be converted back in to pyruvate before it can be metabolized. The only purpose of the reduction of pyruvate to lactate is to regenerate NAD+ so that glycolysis can proceed in active skeletal muscle and erythrocytes (Siva Prasad Rao, 1980). Withdrawal experiments were performed to see whether biochemical alteration caused by aqueous extracts of both the plants moiety would return normal, if the treatment were discontinued. There was nearly complete recovery of total protein, total free amino acid, lactate, pyruvate level and in the activity of lactic dehydrogenase and acetylcholinesterase a partial recovery of glycogen level in the nervous tissues of freshwater snail L. acuminata.

4. Conclusion

From present study, it is believed that the extracts of Codiaeum variegatum and Croton tiglium may be used as potent source of molluscicides, because plant products are less expensive, easily available, and easily soluble in water and also have low toxicity to non-target animals.

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