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QUANTITATIVE PHYTOCHEMICAL AND ANTIOXIDANT STUDIES IN LEAF EXTRACTS OF MANGROVE GRASS MYRIOSTACHYA WIGHTIANA

M. Kiran Kumar, M. B. K. Priyanka, S. J. Mounika, Pola Sudhakar, B. V. Sandeep Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India. ARTICLE INFO ABSTRACT Article history Myriostacya wightiana is a perennial mangrove grass belongs to Poaceae family, occurs in Received 15/02/2016 India, Bangladesh and extending to Myanmar, Malaysia and Vietnam. Due to more salinity in Available online its habitat Myriostacya wightiana possess high antioxidant potential. The present study 29/02/2016 evaluates the phytochemicals, antioxidants and free radical scavenging activities of the Myriostacya wightiana leaf extracts. Specific extraction and estimation methods were used to Keywords quantify the phytochemicals and antioxidants. The results of in vitro studies were analysed Antioxidants, with Mean ± Standard Deviation (SD) obtained from three independent experiments. Among Carotenoids, the phytochemicals flavonoids were found in more concentration. i.e., 416±8.2mg/gm. Free Radicals, highest enzymatic antioxidant activity was showed by catalase (32.7±1.555 U/mg protein) Mangroves, whereas lowest activity was showed by peroxidase. Carotenoids occupies the major Phytochemicals. proportion among the non enzymatic antioxidant i.e., 90.16±0 mg/g and ascorbate has least. Leaf extracts of Myriostacya wightiana shows highest percent of scavenging activity with superoxide radicals (80±6.4%). The present study revealed that Myriostacya wightiana had valuable phytochemicals, antioxidants such as enzymatic and non enzymatic which prevents oxidative damage. Because of these properties Myriostacya wightiana is referred as rich medicinal source.

Corres ponding author M. Kiran Kumar Research Scholar, Department of Biotechnology, Andhra University, Visakhapatnam – 530003. Andhra Pradesh. India. [email protected]

Please cite this article in press as M. Kiran Kumar et al. Quantitative Phytochemical and Antioxidant Studies in Leaf Extracts of Mangrove Grass Myriostachya wightiana. Indo American Journal of Pharmaceutical Research.2016:6(02).

C opy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical 4472

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INTRODUCTION Mangroves are the large plant community with divergent families being adapted to saline environment and grow in estuarine tropical world especially in continuous exposure of tidal influence. Due to more saline in its habitat, mangroves possess high phytochemicals and antioxidant potential. Phytochemicals and antioxidants protect cells and biomolecules from oxidative damage. In

developing countries people eat rice which is not a complete food, it may cause for nutrient deficiency. In India also people were suffering with many diseases because of malnutrition. Oryza sativa possess very low content of phytochemicals and antioxidants when compared to mangroves like Porteresia coarctata. Mangrove plants are rich in phytochemicals such as alkaloids, flavonoids, phenolics, and tannins, etc., which have medical uses [1]. Stress tolerant plants produce enzymatic and non enzymatic antioxidants [26]. Zizania aquatica is a mangrove associate grass show significant phytochemical and antioxidant activities [19]. Along with commercial uses like timber and fire wood they also provide non timber products such as tannin, fish poison medicine, food fodder, etc [3]. Myriostachya wightiana is a perennial grass belongs to Poaceae family with 1-2 m height, leaves are linear with 1-1.5 m length and smooth surface. It has raceme type inflorescence, ascending solitary spikelet and laterally compressed ovoid shaped fruit. Myriostachya grows in inter-tidal mangrove swamps of Ganges Delta of India. In India it is mainly distributed in east coast of the Bay of Bengal, South India [34]. The presence of ample phytochemicals and antioxidants protect plants from oxidative stress. Naturally adapted salt tolerant plants provide an excellent material for investigating the adaptive mechanism they use to encounter salinity. Myriostacya wightiana showed a large range of anatomical adaptive features and it is used as a fodder grass, thatching material [29] [31]. The present study aimed to evaluate the medicinal quality of Myriostachya wightiana by quantifying the phytochemicals like phenolics, flavonoids, tannins, alkaloids and assess enzymatic antioxidants such as superoxide dismutase, peroxidase, catalase, polyphenol oxidase and quantitatively analyse non enzymatic antioxidants such as reduced glutathione, Carotenoids, lycopenes, ascorbate and also determine the free radical scavenging activities.

MATERIALS AND METHODS Sample collection Healthy leaves of Myriostachya wightiana were collected from Bhavanapadu creek, Tekkali, Andhra Pradesh, India. Leaves were cleaned under tap water and used for evaluation of phytochemicals, antioxidants.

Chemicals Analytical grade chemicals and reagents used for evaluation of phytochemicals and antioxidants were purchased from Merck. The experiments were performed at room temperature, otherwise stated.

Phytochemical analysis Methanolic extract of fresh leaves was prepared according to modified method of [14]. The total alkaloid content was estimated by the method of [36]. Standard solution was prepared by dissolving 5 gm of boldine in 5 ml distilled water. The results of alkaloids were expressed as mg of boldine equivalents per gm extract. Total flavonoids content was quantified by the Calorimetric method [4]. Quercetin was used for standard calibration curve and the results were expressed as mg of quercetin equivalents per gm extract. Total phenolics were determined according to the method of Folin-Ciocalteu [17]. and the results were expressed as mg of gallic acid equivalents per gm of extract as calculated from standard gallic acid graph. The total tannins were estimated by the method of [16]. The results were calculated and expressed as mg of tannic acid equivalents per gm of extract from standard tanninc acid graph.

Enzymatic Antioxidants Young leaves of Myriostachya wightiana were macerated in pre cooled homogenizer with extraction buffer containing 1 M sucrose, 0.2 M tris Hcl, 0.056 M β-mercaptoethanol and 0.1 g PVP. (pH-8.5). The extracts were centrifuged at 10,000 rpm for 20 min at 40C. Supernatant was stored at -200C and used for enzymatic antioxidant assays. The activity of superoxide dismutase was determined by the method of [5]. based on the principle reduction of nitroblue tetrazolium. Units of SOD were expressed as amount of enzyme required for inhibiting the reduction of 50% NBT. The enzyme activity was expressed in terms of Units per mg of protein. Catalase activity was determined by the titrimetric method of [7]. Units of enzyme activity were expressed as ml of 0.1 N potassium

permanganate equivalents of H2O2 decomposed per mg protein per min. Assay of Peroxidase was performed by spectrophotometry [20]. Polyphenol oxidase activity was quantified by the spectrophotometry [12]. Results observed were expressed in Units/mg of protein.

Nonenzymatic antioxidants Ascorbic acid was extracted using the methodology of [10]. Dried plants were homogenized in a 2% Meta phosphoric acid and centrifuged at 2500 rpm for 15 min. the supernatant was used to estimate the ascorbic acid by the procedure of [18]. Reducing glutathione was estimated by the [22] method. Carotenoids and Lycopenes were evaluated by the procedure of [39]. 0.5 gm of leaves 0 were macerated and saponified with 12% alcoholic KOH for 30 min at 60 C. The extract was mixed with petroleum ether in separating funnel. The aqueous and petroleum ether layers were separated. The upper layer was collected, after repeated extractions the aqueous layer becomes colourless and anhydrous sodium sulphate was added to remove excess moisture. Final volume of petroleum ether was noted. The absorbance was read at 450 nm and 503 nm for Carotenoids and lycopenes respectively. Results were expressed in mg/gm. 4473

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Estimation of free radical scavenging activities Superoxide scavenging activity was determined by the methodology of [38] by using NBT. DMSO was used instead of enzyme extract in control. According to the [21] method DPPH activity was determined in leaf extracts. ABTS radical scavenging activity of leaf extract was measured by using [33] method. Hydrogen peroxide radical scavenging activity estimated with the

procedure of [30]. All the results were expressed in % of inhibition.

RESULTS AND DISCUSSION Phytochemicals The phytochemicals show large variation in Myriostachya wightiana. The concentration of phenols in Myriostachya wightiana were 103±5.1 mg/gm whereas flavonoids were 416±8.2 mg/gm. 171.3±6.11 mg/gm of tannins were present in the leaf extract of Myriostachya wightiana. Alkaloids were observed as 130±5.1 mg/gm. Among all the phytochemicals, flavonoids have highest concentration. The results were showed in Fig.1. Increased concentrations of phytochemicals were observed when compared to Suaeda monaica [23]. Phenolic content of Rhizophora mangle found higher than Myriostachya wightiana [15]. High phenolic content is found in a halophyte Mesembryanthemum edule, which is used for treat diseases by giving it as food [13]. Our results collinear with the earlier reports. Zizania aquatica is mangrove associated grass showing similar results [19]. Polyphenols acts as reducing agents, hydrogen donators and singlet oxygen quenchers. Hence Polyphenols considered as potential preservatives for consumer benefits food industries use natural preservatives instead of chemical preservatives [25]. [6] Reported that the potential health benefits of Polyphenols have increased enormously.

Fig: 1. Concentrations of different phytochemicals in the leaf extracts of Myriostachya wightiana.

Enzymatic antioxidants Significant amounts of enzymatic antioxidants were found in Myriostachya wightiana leaf extracts. The activities of superoxide dismutase, catalase, peroxidase and polyphenol oxidase were found as 3.2±0.277, 32.7±1.555, 4.14±0.011, 2.5±0.05 U/mg protein respectively. Among the enzymatic antioxidants catalase showed highest activity. The results were showed in fig.2. Our findings show similarity with the earlier reports. Activities of antioxidants like superoxide dismutase increases with the stress [35]. Observations of [2] in Sonneratia alba have similarity with the result of Superoxide dismutase in Myriostachya wightiana but Avicennia marina shows less enzymatic antioxidant activity than Myriostachya wightiana. The results of the present study shows collinear with the findings of [19] in Zizania aquatica. Enzymatic antioxidants such as peroxidase and polyphenol oxidase shows salinity related responses during adaptive reactions and tissue damage [24] [11].

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Fig: 2. Different enzymatic antioxidant activities of Myriostachya wightiana leaf extracts.

Non enzymatic antioxidants The concentration of reduced glutathione in the extracts of Myriostachya wightiana leaves was 6.8±0.057mg/g. 2.8±0.005mg/g of Ascorbate was found in Myriostachya wightiana leaf extracts. Amount of lycopenes present in the extract were 37.31±0mg/g. Carotenoids present in M.wightiana with the concentration of 90.16±0 mg/g. M.wightiana possess Carotenoids with highest concentration among the non enzymatic concentration. Results were shown in Fig.3. Non enzymatic antioxidants like Carotenoids eliminate free radicals [32]. Hence foods with Carotenoids and lycopenes can reduce oxidative damage of DNA [28]. Glutathione and ascorbate scavenge the free radicals which are generated during stress conditions [8].

Fig: 3. Concentrations of Nonenzymatic antioxidants in the leaf extracts of Myriostachya wightiana.

Free radical scavenging Activity The leaf extracts of Myriostachya wightiana were tested for free radical scavenging activity and the percentage of scavenging activity with DPPH was found to be 14.5±0.75%,scavenging activity of ABTS was found to be 47.6±6.58%,superoxide scavenging

activity was found to be 80±6.4% and hydrogen peroxide scavenging activity was found to be 53±5.49%. Results were showed in Fig.4. Avicennia marina has shown similar radical scavenging activity with Myriostachya wightiana [27]. Hydrogen peroxide scavenging activity of Excoecaria agallocha has shown difference from Myriostachya wightiana [9]. ABTS scavenging activity of Myriostachya wightiana have similarity with the Avicennia officinalis [37]. In the present study, superoxide and hydrogen peroxide scavenging activities were found to be high due to high phenolic content. Maximum content of phenolic compounds are observed in

mangroves which are responsible for free radical scavenging activity [25].

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Fig: 4. Percentage of different free radical scavenging activities of Myriostachya wightiana leaf extracts.

CONCLUSION The present study revealed that Myriostachya wightiana had valuable phytochemicals, Antioxidants such as enzymatic and nonenzymatic which play a role in prevent oxidative damage of biomolecules and cell. Due to high content of alkaloids and flavonoids Myriostachya wightiana had medicinal value.

ACKNOWLEDGMENTS The authors acknowledge the facilities made available by the Department of Biotechnology, Andhra University, Visakhapatnam and University Grants Commission, Govt. of India for The financial support.

Abbreviations NBT -Nitroblue tetrazolium DPPH -1-1-phenyl 2-dipicryl Hydrazyl ABTS -2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

Conflict of interest The authors declare that no conflict of interest.

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