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Free Radical Scavenging Activity and Reducing Power of Gnidia Glauca (Fresen.) Gilg

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Free Radical Scavenging Activity and Reducing Power of Gnidia Glauca (Fresen.) Gilg Journal of Applied Pharmaceutical Science Vol. 3 (06), pp. 203-207, June, 2013 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2013.3634 ISSN 2231-3354 Free radical scavenging activity and reducing power of Gnidia glauca (Fresen.) Gilg Smitha B. Rao, M. Jayanthi, R. Yogeetha, H. Ramakrishnaiah*and J. Nataraj Department of P.G. Studies and Research in Biotechnology, Government Science College, Bangalore 560 001, India. ARTICLE INFO ABSTRACT Article history: Received on: 11/05/2013 The present study describes the free radical scavenging activity and reducing power of methanolic leaf extract of Revised on: 05/06/2013 Gnidia glauca (Fresen.) Gilg.using ABTS [2, 2' azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)], FRAP (Ferric Accepted on: 20/06/2013 reducing antioxidant power) and nitric oxide scavenging assays. Total antioxidants were determined by phosphomolybdenum method. The results have indicated that the total phenolic content of the extract was 203.3 Available online: 27/06/2013 GAE/g and showed significant antioxidant activity with an IC50 value of 16.3µg/mL in ABTS and 360.8µg/mL in nitric oxide scavenging assays respectively. A FRAP value of 993.7 µm TE/mg of leaf extract was recorded at 30 Key words: Gnidia glauca, minutes. The total antioxidant activity was 142.5 mg AAE/g of extract. The results strongly establish the antioxidant, free radical, significant antioxidant property of G. glauca owing to its high phenolic content. ABTS assay, FRAP. INTRODUCTION (BHA) and butylatedhydroxytoluene (BHT) get accumulated in the body and cause liver damage beside acting as carcinogens Plants represent a major source of biologically active (Jiangninget al., 2005). Further, most of these synthetic molecules and only a small fraction of plants with medicinal antioxidants show moderate antioxidant activity, low solubility activity have been explored. The rich traditional knowledge of (Barlow, 1990; Branen, 1975) and therefore natural antioxidants mega biodiversity nations like India and China on medicinal plants from plant sources are considered as viable alternatives. The and health care has been the basis of research and development by extracts of medicinal plants and natural products have become a pharmaceutical companies to develop novel drugs for major great source of antioxidant and anti-ageing compounds (Sumazian, ailments. The chain reaction set up by the unstable free radicals is 2010). Gnidia glauca (Fresen.) Gilg (Thymelaeaceae), is reported effectively terminated by antioxidants. Various free radicals are to be used widely in folkloric medicine. The genus Gnidia known to cause pathological conditions such as ischemia, anemia, possesses a variety of traditional phytomedicinal uses and asthma, arthritis, inflammation, neurodegeneration, Parkinson's agrochemical applications. It is used in traditional African medicine disease, mongolism, ageing process and perhaps dementias to treat cancer, sore throat, abdominal pain, wounds, burns and (Polterait, 1990). These free radicals exert oxidative stress on the snake bites. It is also used as molluscicidal, insecticidal, piscicidal, body cells, rendering each cell susceptible to 10 000 oxidative hits (Borris and Cordell, 1984; Franke et al., 2002; Amarajeewa et al., per second (Lata and Ahuja, 2003). The free radicals are highly 2007) homicidal agent and as a source of arrow poison by reactive species capable of damaging DNA, proteins and lipids in tribals.Leaves of Gnidia glauca have been used for the treatment of the cells. Dietary antioxidants have an important role as contusions, swellings, back ache and joint aches (Kareru et al., nutraceuticals due to their ability to protect the body from 2007). The species Gnidia glauca has been studied for its cascading ill effects of free radicals (Vijaykumar et al., 2013). At antihelminthic, antifungal and antidiabetic properties (Nethravathi present, most of the antioxidants used are manufactured et al., 2010; Kharde et al., 2010; Ghosh et al., 2012). Though the synthetically which are known to have adverse side effects in vivo free radical scavenging ability of the plant extracts has been (Ramamoorthy and Bono, 2007). Studies have reported that reported against DPPH radical, no systematic study has been the most widely used antioxidants like butylatedhydroxyanisole . conducted to assess the antioxidant potential of Gnidia glauca * Corresponding Author which forms the basis of this research work. Mail id: [email protected] © 2013 Smitha B. Rao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlike Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 204 Rao et al. / Journal of Applied Pharmaceutical Science 3 (06); 2013: 203-207 MATERIALS AND METHODS 5-10 minutes prior to use. In a 96 well plate, 175µL of FRAP reagent was added and the plate was pre read at 593 nm. To each Plant material well, 25 µL of sample solution was added and contents were Fresh leaves of Gnidia glauca were collected during mixed well using a pipette. Absorbance was measured at 593 nm August 2012 – February 2013 from Kuppali and Jaipura regions of at 4, 15, 30 and 60 minutes, using methanol as blank. Change in Shringeri, Western Ghats, India. The specimen was identified absorbance at different time intervals was then translated into a using Flora of Coorg (Keshavamurthy and Yoganarasimhan, 1990) FRAP value using a standard trolox curve. and further authenticated by the National Ayurveda Dietetics Research Institute (Central Council for Research in Ayurveda and Total antioxidant activity Siddha) with reference number Drug authentication/ SMPU/ The total antioxidant activity of methanolic leaf extract NADRI/ BNG/2012-13/1007. was determined by using phosphomolybdenum (Prieto et al., 1999). An aliquot of 0.4 mL of methanolic leaf (1 mg/mL) was Source of chemicals mixed with 4 mL of the phosphomolybdenum reagent (0.6 M All the chemicals used in this study were procured from sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium Sigma, Himedia, SD Fine and Merck and all were analytical grade. molybdate). The tubes were capped and incubated in water bath at 95º C for 90 minutes, cooled and the absorbance was read at 695 Preparation of plant extract nm. The antioxidant activity of the sample was determined using a Fresh leaves were washed repeatedly to remove adhered standard curve of ascorbic acid. The reducing capacity of the dust particles, blotted, shade dried, powdered using an electric extract was expressed as mg of ascorbic acid equivalents (AAE)/g blender and stored in air tight container. About 300g of the leaf of extract. powder was subjected to methanolic extraction under reflux for 1 hour. The procedure was repeated thrice until complete extraction Nitric oxide scavenging assay was achieved. About 1 mg of the methanolic leaf extract was dissolved in 20µL of methanol and the volume was made up to 1 mL with Estimation of total phenolic content distilled water. Further dilutions were done to final concentrations The total phenolic content of the methanolic leaf extract of 25, 50, 100, 250, and 500µg/mL using vehicle buffer. was determined by Folin–Ciocalteau reagent (Singelton and Rossi, Nitric oxide was generated from sodium nitroprusside and was 1965). Briefly, 40 µl of the methanol solution of the extract (1 measured by the Griess reagent. The reaction mixture contained 50 mg/ml), 200 µl of Folin–Ciocalteau reagent was mixed with 2.4 ml L of 10 mM sodium nitroprusside, 50 L of distilled water and of distilled water and the contents were mixed thoroughly. After 1 test solution of various concentrations. The reaction mixture was min, 600 µl of sodium carbonate (20% Na CO ) was added and the 2 3 pre-incubated at room temperature for 15 minutes in the presence volume was made up to 4.0 ml with distilled water. After 2 h of of light, followed by the addition of 125 LGriess reagent and incubation at room temperature, the reduction of the Folin– incubated for 10 minutes at room temperature. The absorbance Ciocalteau reagent by phenolic compounds under alkaline was measured at 546 nm (Sreejayan et al., 1997). conditions resulted in the development of a blue colour, the absorbance was measured at 765 nm and compared to a gallic acid RESULTS AND DISCUSSION calibration curve. Results were expressed as mg of gallic acid equivalents (GAE) /g of extract. Total phenolic content The mean phenolic content of the methanolic leaf extract ABTS assay was 203.3 mg GAE/g of extract. This indicates that Gnidia The 2, 2' azinobis 3-ethylbenzothiazoline-6-sulphonate glaucais significantly rich in phenolic contents. Presence of (ABTS) assay was performed as per Auddy et al., (2003) where in flavonoid as a major phytoconstituent has been reported in Gnidia radical cations were produced by reacting ABTS with ammonium glauca (Nethravathi et al., 2009). It is well established that persulphate and incubating the mixture at room temperature in flavonoids containing hydroxyl functional groups contribute to the dark for 16 hours. The total reaction volume contained 20μL of antioxidant potential of the plant extracts (Das and Pereira, 1990; 10mM PBS pH 7.4, test solutions of various concentrations and Younes, 1981). 230μL of ABTS radical solution (0.238mM). The reagents were Hakkim et al., (2008) reported lower total phenolic mixed and the absorbance was immediately read at 734nm using contents of 168.2 mg and 123.1 mg GAE/g respectively in blank run without test sample. Ocimum gratissimum and Ocimum americanum. This indicates that Gnidia glaucais significantly rich in phenolic contents. Ferric reducing antioxidant power (FRAP) assay Phenolic compounds such as phenolic acids, polyphenols and The FRAP assay was performed according to Benzie et flavonoids are commonly found in plants and have been reported al., (1996) with slight modifications suggested by Firuzi and to have multiple biological effects, including antioxidant activity Lancana (2005). FRAP reagent was prepared and kept at 37oC for (Brown and Rice-Evans, 1998).
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