Effect of ethanolic extract of Phyllanthus emblica on captan induced oxidative stress in vivo

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1 South Indian Journal Of Biological Sciences 2016; 2(1); ONLINE ISSN: Research Article Effect of ethanolic extract of Phyllanthus emblica on captan induced oxidative stress in vivo Nusrath Noorudheen 1, Dhanya K.Chandrasekharan 1, * 1 Department of Microbiology, St Mary s College, Thrissur, Kerala , India * Corresponding author: Dhanya K.Chandrasekharan; E mail: dhanuchandra@yahoo.com; Tel.: Received 26 June 2015; Revised 28 September 2015; Accepted 30 September 2015; Published 2 January 2016 Abstract Pesticides include all classes of chemicals used for killing or repelling insects, fungi, vegetation, and rodents. Eventhough they are in a way beneficial since they can protect against forest and farm crop losses aid in more efficient food production and are instrumental in controlling insect vector borne diseases, their increased use has several drawbacks including health effects, loss of bio diversity and irreversible changes and degradation of natural ecosystems. In the present study, the nutraceutical, Phyllanthus emblica are evaluated for their ability to protect against Captan, a fungicide, induced genotoxicity and oxidative stress, in vivo. Chronic exposure to pesticides adversely affects various organ systems and theses effects have been attributed to increased oxidative stress and cellular DNA damage. Oxidative stress induced by Captan was studied in vivo by analyzing the extent of lipid peroxidation and levels of GSH in the gill tissue of fish (guppy) grown in water containing Captan in presence or absence of gooseberry extracts. The genotoxicity of Captan and the genoprotective action of gooseberry was studied using comet assay on gill tissue of fish (guppy) grown in water containing Captan in presence or absence of the extracts. The results clearly indicated that the extracts could mitigate captan induced oxidative stress and genotoxicity which could be due to the efficient free radical scavenging activity by the extracts. Keywords: Pesticides, Fungicide, Genotoxicity, Oxidative stress, Lipid peroxidation, GSH 1. Introduction Pesticides are a group of chemicals used in agriculture and against vectors for vector borne disease control and thus they are frequently being released into the environment. Persons, especially farmers with prolonged exposure to low doses of pesticides eventually develop health effects, for example, immune suppression, hormone disruption, diminished intelligence neurobehavioral abnormalities (Stephens et al., 1996), adverse reproductive outcomes and increased cancer incidence (Ritter et al., 1997) due to increased chromosome aberrations and significantly abnormal DNA repair responses (Au 95

2 et al., 1999; Antonisamy et al., 2015; Balamurugan 2015; Barathi and Agastian 2015; Rathi et al., 2015; Nandhini and Stella Bai 2015). A number of in vitro, animal and human studies have shown that insecticides alter DNA integrity of different cell types (Lieberman 1998). Molecules having antioxidant activity can mitigate the formation of free alkyl radicals in the initiation step and disrupt the free radical chain reactions during lipid peroxidation and thus they could be used against the genotoxic effects of pesticides. The present study demonstrate the ability of the ethanolic extract of Phyllanthus emblica (Gooseberry) to offer protection to cellular DNA and to ameliorate oxidative stress induced by a commercially available fungicide, Captan, under in vivo conditions. 2. Materials and methods 2.2 Preparation of ethanolic extract The plant part used in our study is the dried fruits of gooseberry (Phyllanthus emblica). Hundred grams of the gooseberry was purchased from the local market, ground to get the powder which was then extracted using 95% ethanol. The concentrated extract was then evaporated to dryness, weighed and stored for further use Phytochemical analysis The phytochemical analysis of the extracts were carried out for the presence of biomolecules such as: coumarins, saponins, quinones, cardiac glycosides terpenoids, acids, phlobotannins, anthraquinones, steroids, phytosteroids, tannins, phenols and flavanoids using the standard qualitative procedures Determination of anti oxidant activity The free radical scavenging potetntial of the extracts were studied in terms of DPPH free radical and hydroxyl free radical scavenging assay (Rejiniemon et al., 2015) Studies on the toxicity of captan on fish To study the toxicity of Captan, fish (Guppy) (Poecilia reticulata) was selected as the experimental animal. Fish (3 fish per group) were kept in water containing different concentrations of the pesticide, Captan as detailed below. Group I 3 Guppy + water Group II Group III Group IV 3 Guppy ppm captan 3 Guppy ppm captan 3 Guppy + 1 ppm captan 96

3 At 24 h, fish were were euthanized in ice, gill tissue obtained, homogenized in ice cold phosphate buffered saline (ph 7.4) and homogenate (10 %) was prepared and extent of lipid peroxidation (Buege and Aust (1978)) and level of GSH and protein were determined by following standard method (Moron et al., 1979; Lowry et al., 1951). The dosage of captan for further studies was determined (0.1 ppm captan) based on the results Effect of gooseberry extract on captan induced tissue toxicity and genotoxicity To study the effect of gooseberry extract on captan induced tissue toxicity in terms of oxidative stress ans cellular DNA damage, Guppy were kept in presence or absence of captan and/or Gooseberry extract as detailed below. Group I Group II Group III Group IV 15 Guppy + water 9 Guppy ppm captan 9 Guppy mg/ml Gooseberry extract 9 Guppy ppm captan mg/ml Gooseberry extract At 24 h, 48 h and on 72 nd h, fish (3 per group) were euthanized in ice, gill tissue obtained and extent of lipid peroxidation and level of GSH and protein were determined. Comet assay was performed on the gill tissues of fish kept in Captan containing water in presence or absence of Gooseberry extract for a period of 24 h. Fish were euthanized in ice, gill tissue obtained, washed with ice cold phosphate buffered saline and kept at 20 o C. The tissue was homogenized in ice cold phosphate buffered saline (ph 7.4) and 10 % of homogenate was prepared. It was stored at 20 o C till analysis Determination of reduced glutathione (GSH) in the tissue homogenate Reduced glutathione in tissue homogenate was determined according to the method of Moron et al. (1979) with minor modifications. Reduced glutathione forms a yellow colored complex with DTNB with an absorbance at 412 nm. 100 μl of the tissue homogenate was mixed with 63 μl of 25% TCA and cooled on ice for 5 minutes followed by further dilution of the mixture with 300 μl of 5% TCA and these were then subjected to centrifugation at 3000 g for 5 minutes to settle down the precipitate. 150 μl of the supernatant was mixed with 350 μl of sodium phosphate buffer (0.2M, ph 8.0) and 1.0 ml of DTNB (0.6 mm in 0.2M, ph 8.0 phosphate buffer). The yellow color obtained was measured at 412 nm. A standard graph was prepared using different concentrations (10 50 nmoles) of GSH. The GSH content of the sample was calculated from the standard graph and expressed as n mol/mg protein Determination of lipid peroxidation in the tissue homogenate The level of lipid peroxidation was measured as malondialdehyde (MDA) according to the method of Buege and Aust (Buege and Aust 1978). The malondialdehyde (MDA) is formed mainly from the peroxidation of PUFAs. MDA is a TBA reacting substance (TBARs) and the product formed between the reaction of MDA and TBA is estimated at 532 nm. Briefly, the reaction mixture contained 1 ml of tissue homogenate( 100 ml of 10 % tissue homogenate ml distilled water) and 1mL of TBA 97

4 reagent (which contains 0.375% thiobarbituric acid, N HCl, 15% trichloroacetic acid and 6.0 mm EDTA). The reaction mixture was heated at 90 o C for 30 min, cooled and centrifuged at 10,000 xg for 10 m. The amount of TBARS in the supernatant was estimated by measuring the absorption at 532 nm. The lipid peroxidation values are expressed as n moles of MDA per mg protein.1, 1, 3, 3 tetraethoxypropane was used as the standard Determination of the total protein in the tissue homogenate Protein content in the tissue was determined according to the method of Lowry et al. (1951). The tyrosine and tryptophan residues of proteins cause reduction of the phosphomolybdate and phosphotungstate components of Folin Ciocalteau reagent in an alkaline medium to give a bluish purple colour with absorbance at 660 nm. Ten μl of the homogenate was mixed with 990 μl of distilled water, 5 ml of alkaline CuSO4 (0.5 % CuSO4 in 1 % sodium potassium tartarate and 2% Na2CO3 in 0.1 N NaOH mixed in the ratio 1:50) was kept for 10 min at room temperature. 0.5 ml of 1 N Folin Ciocalteau reagent was added and absorbance was measured after 30 min at 660 nm against the reagent blank. Protein content was calculated from the standard graph plotted using different concentrations (0 500 μg/ml) of bovine serum albumin (BSA) Comet assay (Single cell gel electrophoresis or microgel assay) The DNA strand breaks in the cells of tissue homogenate of fish were measured using alkaline single cell gel electrophore sis method (Singh 2000). Microscopic slides were coated with normal melting point agarose and 200 μl of 0.8% low melting point agarose containing 50 μl of treated cells (containing cells) were added onto the slide and the slides were kept at 4 o C.After solidification the slides were immersed in prechilled lysing solution contain ing 2.5 M NaCl, 100 mm Na2EDTA, 10 mm Tris HCl, ph10, 1% DMSO, 1% TritonX and kept for 1h at 4 C. After lysis, slides were drained properly and placed in a hori zontal electrophoretic apparatus filled with freshly prepared electrophoresis buffer containing 300 mm NaOH, 1 mm EDTA, 0.2% DMSO, ph 13. The slides were equi librated in buffer for 20min and electrophoresis was carried out for 30 min at 20 V, 300 ma. After electrophoresis the slides were washed gently with 0.4 mm Tris HCl buffer, ph 7.4 followed by distilled water, dried an flourescent staining was performed. 3. Results and discussion 3.1. Phytochemical screening and antioxidant activity of Embilica officianalis The percentage yield of extracts was found to be 34.6% respectively for gooseberry (Embilica officianalis). The gooseberry extract contained coumarins, quinones, cardiac glycosides, tannins, phenols and flavanoids. The free radical scavenging activity of the extracts are given in figure 1. 98

5 Fig. 1. Percentage inhibition of DPPH free radical and hydroxyl radical by different concentrations of gooseberry extract. Captan was analysed for its toxicity by growing fish (guppy) in the presence of this fungicide. After 24 h the fish were euthanized and their gill tissue was obtained and analyzed for extent of lipid peroxidation and levels of tissue GSH. Lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to action of free radicals. The resulting reaction, known as lipid peroxidation was found to increase with increased concentrations of Captan. When cells are exposed to increased levels of oxidative stress, GSSG (Glutathione) will accumulate and the ratio of GSH (reduced glutathione) to GSSG will decrease. The increased Captan concentration was found to increase the oxidative stress in the fish as evidenced by the decreased levels of the GSH in gill tissues. From the toxicity studies, the dosage of captan for further studies was selected to be 0.1 ppm Effect of gooseberry extract to offer protection to cellular DNA in fish gill tissue against Captan Comet assay was performed with the cells of gill tissues of fish kept in Captan containing water (0.1 ppm) in presence or absence of Gooseberry extract for a period of 24 h (Fig.2). Fig.2. Protection effect of gooseberry extract to cellular DNA in fish gill tissue against Captan. 99

6 In comet assay, the cells embedded in agarose on a microscope slide are lysed with detergent and salt, electrophoresed and the slides were observed under flourescent microscope after propidium iodide staining. When the slides were observed by fluorescence microscopy after the assay, the cells obtained from fish treated with Captan alone showed a comet like appearance indicating cellular DNA damage while the gill cells of fish treated with or Gooseberry extract alone remained circular indicating the non genotoxic nature of the extract. The cells of fish treated with Gooseberry extract along with Captan were found to be protected from captan induced genomic DNA damage as indicted by the comet assay results in those cells Effect of gooseberry extract to offer protection against Captan induced oxidative stress The effect of gooseberry extract on Captan induced oxidative stress was analyzed by measuring the levels of GSH and lipid peroxidation on gill tissue of fish kept in presence of Captan, and/ or gooseberry extract for different time durations. The results are given in table 1 and 2. Table 1. Effect of gooseberry extract against Captan induced oxidative stress as measured in terms of levels of GSH in gill tissue of Guppy kept in water containing Captan and/ or gooseberry extract for different time durations. (n=3 per group). GSH (nanomoles/mg Protein) 24 th h 48 th h 72 nd h Control ± ± ± ppm Captan ± ± ± mg/ml GE ± ± ± ppm Captan+0.01 mg/ml GE ± ± ±43.87 Table 2. Effect of gooseberry extract against Captan induced oxidative stress as measured in terms of levels of lipid peroxidation in gill tissue of Guppy kept in water containing Captan and/ or gooseberry extract for different time durations. (n=3 per group). MDA (nanomoles/mg Protein) 24 th h 48 th h 72 nd h Control 12.55± ± ± ppm Captan 23.34± ± ± mg/ml GE 6.13± ± ± ppm Captan+0.01 mg/ml GE 13.25± ± ±

7 Guppy kept in presence of captan along with GE showed near normal levels of GSH which indicate the protective ability of these extracts against captan induced oxidative stress. The gooseberry extract possess antioxidant activity which helped them to scavenge the oxygen radicals responsible for the lipid peroxidation therefore the extent of captan induced lipid peroxidation decreased when guppy were kept in the presence of GE or TE. Our previous works has demonstrated the genotoxicity of this commonly and extensively used contact fungicide, Captan, under in vitro conditions. It was also observed that Gooseberry extract possessed significant antioxidant activity and can significantly minimize the genotoxic effect of the fungicide, Captan, while not interfering with its antifungal activity. In the present study, genotoxicity of Captan and the ability of the extracts to offer genoprotection under in vivo conditions were evaluated using the technique of alkaline single cell gel electrophoresis or comet assay on gill tissue of fish (guppy) grown in water containing Captan in presence or absence of the extracts. The oxidative stress induced by Captan was studied in vivo by analyzing the extent of lipid peroxidation and GSH level in the gill tissue of fish (guppy). The results clearly indicated that captan induced genotoxicity and oxidative stress could be mitigated by the extracts of gooseberry. To meet the dietary requirement for the ever increasing population of our country, crop productivity is to be enhanced for which a sustained use of pesticides is indispensible. Pesticide is considered as an integral input for crop production during the green revolution regime and the application of pesticides justified due to social and economic consideration, when food security was the major concern (Von Gadow et al., 1997; Devi 2010). 4. Conculsion In the current scenario when the use of a toxic pesticide is restricted, it is getting replaced by another pesticide which may still be toxic. Thus proper management techniques to ameliorate toxicity resulting from pesticide accumulation are essential in the current scenario. Conflict of interest statement We declare that we have no conflict of interest. References 1. Antonisamy P, Duraipandiyan V, Ignacimuthu S, Kim J H. (2015). Anti diarrhoeal activity of friedelin isolated from Azima tetracantha Lam. in Wistar rats. South Indian Journal of Biological Sciences, 1(1), Au WW, Carlos H. Sierra Torres, Nohelia Cajas Salazar, Shipp BK and Legator MS. (1999). Cytogenetic effects from exposure to mixed pesticides and the influence from genetic susceptibility. Environmental Health Perspectives, 107, Balamurugan R. (2015). Smilax chinensis Linn. (Liliaceae) root attenuates insulin resistance and ameliorate obesity in high diet induced obese rat. South Indian Journal of Biological Sciences, 1(1), Barathi KK, Agastian P. (2015). In vitro regeneration of a rare antidiabetic plant Epaltes divaricata L. South Indian Journal of Biological Sciences, 1(1), Buege AJ, Aust SD. (1978). Microsomal lipid peroxidation. Methods in Enzymoogy, 52,

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