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Biochemical Alterations Induced by Sodium Arsenate in Momordica Charantia L

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Biochemical Alterations Induced by Sodium Arsenate in Momordica Charantia L Central International Journal of Plant Biology & Research Bringing Excellence in Open Access Research Article *Corresponding author Sarmistha Sen Raychaudhuri, Department of Biophysics, Molecular Biology and Bioinformatics, University of Biochemical Alterations Calcutta, India, Tel: 91(0) 332-3508386 (Ext: 324); Fax: 91(0) 332-8661573; Email: [email protected]; Induced by Sodium Arsenate in Submitted: 17 February 2017 Accepted: 14 March 2017 Published: 18 March 2017 Momordica charantia L. Seeds ISSN: 2333-6668 Copyright In vitro © 2017 Raychaudhuri et al. OPEN ACCESS Suman Kumar Ray and Sarmistha Sen Raychaudhuri* Department of Biophysics, Molecular Biology and Bioinformatics, University of Keywords Calcutta, India • Momordica charantia L • Sodium arsenate • Chlorophyll Abstract • Total antioxidant • Free radical scavenging activity Momordica charantia L. (Family: Cucurbitaceae) is a well known medicinal plant and widely distributed and cultivated in many parts of the world. Momordica is a powerful, nutrient-dense plant, composed of a complex array of beneficial compounds. Seeds were collected from arsenic free area and were propagated in tissue culture media in presence of different concentration of sodium arsenate along with control one. The aim of the present study was to investigate some biochemical parameters of extracts of Momordica under the influence of arsenic. Chlorophyll and carotenoid content was affected by arsenic maximum (150 µM) in Momordica. Arsenic induced alteration in free radical scavenging activity, total polyphenolic content, lipid peroxidation, antioxidant activity and flavonoid content were determined in this study. INTRODUCTION MATERIALS AND METHODS Momordica charantia L. (bitter melon) is a vegetable crop Collection of seeds and in vitro propagation plant of the family Cucurbitaceae [28,29,38] widely grown Seeds of M. charantia were collected from Burdwan town, in India, South Asia, China, Africa and the Caribbean [2]. It is a District Burdwan, West Bengal, India. Seeds were propagated medicinally important species which is also used as foodstuff by proper tissue culture technique [23]. Seeds were germinated [37]. Popularity of in various systems of traditional M. charantia in sodium arsenate heptahydrate (Na2HAsO4.7H2O) (Himedia, medicine for several ailments focused the investigator’s attention Mumbai, India) supplemented agar sucrose media. Different on this plant [23]. concentration of sodium arsenate i.e. 10 µM, 25 µM, 50 µM, 75 µM, 100 µM, 125 µM, 150 µM, 175 µM and 200 µM were chosen Arsenic (As) ranks twentieth most abundant of elements in for the experiment along with control one (no treatment) in vitro the earth’s crust and fourteenth in sea water. As is the twelfth (LD50 was determined as 200 µM). of most abundant element usually found in human body [18]. Arsenic is an element that is nonessential for and toxic to plants Preparation of extracts [42]. Large areas of Bangladesh, West Bengal and other states in Plant extracts were prepared by taking 100 mg of roots and India and Vietnam rely on arsenic contaminated ground-water shoots from each of the samples (control and sodium arsenate for irrigation [1,3]. Arsenic originates from geochemical and anthropogenic sources. Arsenic is known to induce oxidative ethanol (Merck, Germany) using mortar and pestle. The mixture wastreated). then The ultrasonicated tissues were for finely 20 min,crushed followed and dissolved by centrifugation. in 1 ml of stress [8] resulting a wide range of responses in plants, including The supernatant was collected and prepared for the experiments. readjustment of transport and metabolic processes, growth The plant extracts were used for determination of 1, 1-diphenyl- inhibition [12] and biochemical changes in plants. The present 2-picryl hydroxyl (DPPH) radical scavenging activity, polyphenol alteration such as chlorophyll, carotenoid, free radical scavenging study was designed to specifically investigate biochemical activity, total polyphenolic activity, lipid peroxidation, total content,Estimation total antioxidantof chlorop activity,hyll and and carotenoid total flavonoid content assay. Chlorophyll and carotenoid (carotene + xanthophyll) contents antioxidant, total flavonoid content etc. Cite this article: Ray SK, Raychaudhuri SS (2017) Biochemical Alterations Induced by Sodium Arsenate in Momordica Charantia L. Seeds In vitro. Int J Plant Biol Res 5(1): 1060. Raychaudhuri et al. (2017) Email: Central Bringing Excellence in Open Access were determined following the methods of Lichtenthaler [15] treated and control seedlings of M. charantia) were taken and trichloroaceticas malondialdehyde acid (TCA) (MDA) and content centrifuged. and measuredThe supernatant [10]. Root was werewith minorcrushed modifications.1 in a mortar and mg pestle leaves with from acetone seedlings and adjusted(arsenic collectedand shoot and tissue mixed samples with were4 ml TCA–thiobarbituriccrushed 0.1 ml of 0.1%acid (TBA)(w/v) to 5ml. After homogenization the extract is centrifuged and the solution. The mixture was heated at 95°C for 30 min and then clear supernatant was diluted and used for spectrophotometric quickly cooled on ice. The absorbance of the supernatant was analysis. Absorbance for Chlorophyll a and Chlorophyll b read at 532 nm. The concentration of lipid peroxides together was taken at 662 nm and 645 nm wavelengths respectively. Absorbance for carotenoids (Carotene and Xanthophyll) was measured at 470 nm. Chlorophyll and carotenoid content was Lwith-1 cm oxidative and expressed modified as proteins µmol L-1 gof plants were thus quantified calculated using the following equations: in terms−1 of MDA level using an extinction−1 coefficient of 155 mmol Estimation of total antioxidant FW. ) – 2.04 (A ) 662 645 Antioxidant activity of the plant extracts was determined Chlorophyll a (μg/ml) = 11.24(A 645) – 4.19 (A662) using phosphomolybdenum assay method [26]. The assay is ) + 18.71 (A ) based on the reduction of Mo (VI)-Mo (V) by the plant extracts Chlorophyll b (μg/ml) = 20.13 (A 663 646 and then formation of a green phosphate-Mo(V) complex at Total Chlorophyll (μg/ml) = 7.15 (A acidic pH. Best results were obtained at 695 nm absorbance. 0.3 ml of each samples were mixed with 3ml reagent solution. The Carotenoids(1000 A470-1.90 (Carotene+Xanthophyll) Chlorophyll (a)-63.14 (μg/ml) Chlorophyll = (b) reaction mixture was incubated at 950C for 90 min. Absorbance 214 was taken at 695 nm in UV–visible spectrophotometer .The antioxidant activity was expressed as grams of ascorbic acid The chlorophyll and carotenoid content was finally expressed ascorbic acid calibration curve. Standard curve was prepared asFree mg/g radical fresh weight scavenging (FW). assay withequivalents a range (AAE) of standard per Kg ascorbic FW tissue acid by(Sigma comparing Aldrich, with StLouis, the MO, USA) concentrations from 0 to 100 µg ml-1. Brand-Williams et al. [4], Estimation of total flavonoids content reactFree with radical DPPH scavenging and reduce assayit to DPPH-H was done which using is yellowthe method in color of where as the original colorwith of freesome radical, modifications. DPPH is Antioxidantsred in color. chloride (AlCl ) method [16]. 1 ml root and 1 ml shoot extract Thus the degree of decolorization indicates scavenging activity 3 The total flavonoid content was determined using aluminium of the antioxidants using their hydrogen-donating ability. 150 µl of each sample with control one was taken. Samples were mixed with 0.1 ml AlCl , 0.1 ml of potassium acetate and 1.8 ml deionized extract of each sample was allowed to react with 2.850 ml DPPH 3 solution and kept for 1h in the dark. DPPH is a stable free radical with a characteristic absorption at 517 nm, was used to study water. The flavonoid contact was expressed as grams of rutin the radical-scavenging effects of grown in different the rutin (Sigma Aldrich, StLouis, MO, USA) standard curve. Momordica equivalent (RE) per kilogram of FW of tissue by comparing with concentration of arsenic in vitro. The antioxidant capacity was Standard curve was prepared with rutin concentration from calculated using the following formula: . −1 0-100Data analysisμg mL control - Asample control] ×100%. The data were presented as the mean ± standard error DPPH radical scavenging activity = [(A )/A of mean (SE). The experiments were performed with three Acontrol is the absorbance of the control (without any extract) replicates. The statistical analysis was carried out by using one- and Asample is the absorbance of extract. way analysis of variance (ANOVA). Data represent the mean ± Estimation of total polyphenol . SE. Asterisks indicate significant differences at p < 0.05 (*) or p < 0.01RESULTS (**) or p AND <0.001 DISCUSSION (***) compared to respective controls PlantTotal extract polyphenol (shoots contentand roots) was of determined each of the bysamples Folin-Ciocalteu’s was mixed phenol reagent (FCR) using the method of Singleton et al. [33], To evalu ate the impact of abiotic stress induced by sodium The mixture was mixed well and incubated at room temperature arsenate chlorophyll content was measured in Momordica. forwith 30 250µl min in FCR dark and condition. 750µl of The 10% absorbance sodium carbonatewas then measured solution. Changes in pigment content are associated with photosynthetic at 760 nm in a UV–visible spectrophotometer. Total polyphenol content was expressed as grams of gallic acid equivalents (GAE) by the effect of arsenic toxicity. As a result, decrease of
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