Focus on Estimation and Antioxidant Studies of Salacia Species
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International Journal of Scientific Research in ______________________________ Research Paper . Biological Sciences Vol.6, Issue.1, pp.65-74, February (2019) E-ISSN: 2347-7520 DOI: https://doi.org/10.26438/ijsrbs/v6i1.6574 Focus on Estimation and Antioxidant Studies of Salacia Species G. Priya1, M. Gopalakrishnan2, E. Rajesh3 and T. Sekar4* 1PG and Research Department of Botany, Pachaiyappa’s College, Chennai, Tamil Nadu, India 2PG and Research Department of Botany, Pachaiyappa’s College, Chennai, Tamil Nadu, India 3PG and Research Department of Botany, Pachaiyappa’s College, Chennai, Tamil Nadu, India 4PG and Research Department of Botany, Pachaiyappa’s College, Chennai, Tamil Nadu, India *Corresponding authors email: [email protected] Available online at: www.isroset.org Received: 11/Jan/2019, Accepted: 10/Feb/2019, Online: 28/Feb/2019 Abstract - Salacia is a valuable medicinal genus found to be composed of various secondary metabolites. Hence, the present study was aimed to estimate three important secondary metabolites such as phenol, flavonoid and tannin. Antioxidant assays such as DPPH assay and ABTS assay and Total Antioxidant Capacity were also studied for this medicinal genus. Seven species of Salacia were considered for this present study. They are Salacia beddomei Gamble, Salacia chinensis L, Salacia fruticosa Heyne ex Lawson, Salacia gambleana Whiting & Kaul, Salacia macrosperma Wight, Salacia malabarica Gamble, and Salacia oblonga Wall. Various concentrations such as 20µg, 40µg, 60 µg, 80 µg and 100 µg were taken for all studies and the values are entered in terms of Mean±SD. In case of DPPH assay and ABTS assay, IC50 values are calculated using ANNOVA and Total Antioxidant Capacity was calculated using a calibration curve. Key Words - Salacia, phenol, flavonoid, tannin, Total Antioxidant Capacity, DPPH, ABTS. I. INTRODUCTION distributed in various plants especially in forest trees. The flavonols(e.g., quercetin) and hydroxyicinnamicacids (e.g., Salacia is a medicinally important genus belongs to Caffeic and Ferulic acids) were determined to be more the family Hippocrateaceae, which was formerly known as potent antioxidants than ascorbic acid. Hence, this present Celastraceae, Class Magnoliopsida and Order Celastrales study was mainly concentrated on estimation of phenol, [1]. It comprises of about 407 different species and they are flavonoid, tannin, total antioxidant capacity and antioxidant widely distributed in South-West India, Peninsular region of assays such as DPPH and ABTS. India, Sri Lanka, Vietnam, China, Indonesia, Brazil, South The present work was mainly aimed to estimate Africa, Malaysia, Thailand and Philippines [2,3]. The rich three important secondary metabolites such as Phenols, biodiversity of medicinal plants make them a treasure house Flavonoids, Tannins, Total Antioxidant Capacity etc, also it for obtaining novel compounds either themselves as drugs or was focused on the Antioxidant study by carrying out two pilot molecules for invention of new drugs with different assays like DPPH and ABTS assays. mechanism of action. Medicinal plants are found to be composed of II. MATERIALS AND METHODS various phytochemicals also known as secondary Extraction of Plant Materials metabolites which are used to treat many diseases [4]. The secondary metabolites such as alkaloids, flavonoids, Leaves of seven species of Salacia such as S. terpenoids, saponins, tannins, glycosides, coumarinsand beddomei, S.chinensis, S.fruticosa, S. gambleana, phenolic compounds are important natural chemical S.macrosperma, S. malabarica and S.oblongawere collected constituents [5,6,7]. These medicinal plants with from Wayanad district of Kerala. They are washed phytochemicals can be used as a supplement for human thoroughly, and shade dried. Plant materials were coarsely diseases by acting as a natural antioxidants [8]. powdered and soaked in 300ml of Methanol for 48hrs. The Saurai 2015 [9], Lopez-Alarcon et al 2013 [10] and extract was filtered using WhattmannNo.1 filter paper and Martin-Sanchez et al 2014 [11] were studied that the filtrate was concentrate under reduced pressure in polyphenols, such as flavonoids, hydroxycinnamic acids and vacuum at 40º C for 25min using a rotary evaporator and proanthocyanidins, are considered as a powerful kept into lyophilisation to remove the traces of solvent. antioxidants and these antioxidants are found to be Estimation Studies © 2019, IJSRBS All Rights Reserved 65 Int. J. Sci. Res. in Biological Sciences Vol. 6(1), Feb 2019, ISSN: 2347-7520 Total Phenolic Content M sulfuric acid, 28 mM sodium phosphate and 4 mM Total phenols were assayed according to Dewanto ammonium molybdate were added in 20 ml of distilled et al 2002 [12] with some modifications. Various water and made up volume to 50 ml by adding distilled concentrations such as 20µg, 40µg, 60 µg, 80 µg and 100 µg water. The Chloroform and ethanolic extracts of Pouzolzia were taken and an aliquot of diluted extract was added to 0.5 zeylanica and Tribulus terristris were taken in different mL of distilled water and 0.125 mL of Folin–Ciocalteu concentration ranging from 100 l to 500 l were added to reagent. The mixture was shaken and allowed to stand for 6 each test tube individually containing 3 ml of distilled water min, before addition of 1.25 mL of 7% Na2CO3. The and 1 ml of Molybdate reagent solution. These tubes were solution was then adjusted with distilled water to a final kept incubated at 95 C for 90 min. After incubation, these volume of 3 mL and mixed thoroughly. After incubation in tubes were normalized to room temperature for 20-30 min the dark, absorbance at 760 nm was read versus a prepared and the absorbance of the reaction mixture was measured at blank. The total phenol content of plant parts was expressed 695 nm. Total Antioxidant activity was expressed in terms as milligrams of gallic acid equivalents per gram of dry of Mm of Ascorbic acid/g of extract and was calculated weight (mg GAE/g DW) from a calibration curve with gallic using calibration curve. acid. All samples were analyzed in three replicates. a. Scavenging effect on 1,1 - diphenyl- 2-picrylhydrazyl radicals (DPPH) Total Flavonoid Content The free radical scavenging activity of methanolic Total flavonoid content in leaf extracts of S. leaf extract of selected species of Salacia was analysed with molesta was determined by aluminum chloride colorimetric stable 1,1 - diphenyl - 2 - picryl hydrazyl radical (DPPH) method [13]. 0.5 ml of extracts of at a various (Sigma - Aldrich) photo spectrometrically as stated by [16]. concentrations such as 20µg, 40µg, 60 µg, 80 µg and 100 µg Stock solution was prepared with Methanol solution for each were taken and the volume was made up to 3 ml with plant extract at a concentration of 1 mg/ml. From the stock methanol. Then, 0.1 ml AlCl3 (10%), 0.1 ml of potassium solution. Various concentrations such as 20µg, 40µg, 60 µg, acetate and 2.8 ml distilled water were added sequentially. 80 µg and 100 µg were taken and mixed with equal volume The test solution was vigorously shaken. Absorbance was of Methanolic solution of DPPH (0.1 mM). 0.5 ml of each recorded at 415 nm after 30 minutes of incubation. A sample in Methanol solution was mixed with 2.5 ml of 0.5 standard calibration plot was generated at 415 nm using mM of Methanolic DPPH solution. The mixture was known concentrations of quercetin. The concentrations of vortexed vigourously and incubated for 30 minutes in a dark flavonoid in the test samples were calculated from the room under room temperature. The absorbance was calibration plot and expressed as mg quercetin equivalent measured at 517 nm against a blank using a UV (QE)/g of sample. All samples were analyzed in three Spectrophotometer (DU 800; Beckman Coulter, Fullerton, replicates. CA, USA). Ascorbic acid was used as the positive control. The experiments were conducted in triplicates. Total Tannin Content The tannins were determined by Folin - Ciocalteu b. Scavenging effect on ABTS radical cation (2,20 method. Various concentrations such as 20µg, 40µg, 60 µg, azinobis-(3-ethylbenzthiazoline -6-sulphonic acid) 80 µg and 100 µg were taken. About 0.1 ml of the sample The ABTS cation radical scavenging activity of the extract was added to a volumetric flask (10 ml) containing extracts was determined according to the modified method 7.5 ml of distilled water and 0.5 ml of Folin-Ciocalteu of Re et al. (1999) [17]. A stock solution of ABTS was phenol reagent, 1 ml of 35 % Na2CO3 solution and dilute to produced by mixing 7 mM aqueous solution of ABTS with 10 ml with distilled water. The mixture was shaken well and 2.45 mM of potassium persulfate in the dark at ambient kept at room temperature for 30 min. A set of reference temperature for 12–16 h before use. Various concentrations standard solutions of gallic acid were prepared in the same such as 20µg, 40µg, 60 µg, 80 µg and 100 µg were taken. manner as described earlier. Absorbance for test and The radical cation solution was further diluted until the standard solutions were measured against the blank at 725 initial absorbance value of 0.7± 0.005 at 734 nm was nm with an UV/Visible spectrophotometer. The tannin reached. For assaying test samples, 0.98 mL of ABTS content was expressed in terms of mg of GAE /g of extract solution was mixed with 0.02 mL of the plant extracts. The [14]. decrease in absorbance was recorded at 0 min. and after 6 min. Ascorbic acid was used as the positive control. The Antioxidant Study experiments were conducted in triplicates. Total Antioxidant Capacity Phosphomolybdenum assay (PM) Statistical analysis Total antioxidant activity was estimated by The data was statistically analyzed by one-way phosphomolybdenum assay [15]. Various concentrations ANNOVA using SPSS 17.0. The difference was considered such as 20µg, 40µg, 60 µg, 80 µg and 100 µg were used.