A 96 WELL-MICROTITER PLATE ABTS BASED ASSAY FOR ESTIMATION OF ANTIOXIDANT ACTIVITY IN GREEN LEAFY VEGETABLES

Abstract

A 96 well-microtiter plate-ABTS based decolorization assay was developed for the screening of antioxidant activity of various extracts such as food extracts, plasma antioxidants and synthetic antioxidants. Oxidation of 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with potassium persulfate generated cation radical ABTS•+and reduction followed in the presence of hydrogen-donating antioxidants. Optimization was carried out for different parameters like concentration, incubation time, volume of the reagent and sample, reagent preparation, pH using the buffer. 96 well-microtiter plate-ABTS based method denotes very good intra- and inter-assay analysis. IC50 values of standards and methanolic extracts of selected green leafy vegetables showed per cent coefficient of variation (CV) 4.1% and 5% for inter- and intra-day assay results respectively and was a significant achievement for the sample volume maintained at 5μl. The inter-day and intra-day accuracy were proved by the low values relative standard deviations (RSD) did not exceed 4.5%, indicating high reproducibility and precision of the method. This 96 well-microtiter plate-ABTS based decolorization assay method has advantages over the techniques like ferryl myoglobin/ABTS assay, such as no involvement of an intermediary radical, suppression of rapidly reacting antioxidants, low volume of sample, reaction at desired pH, and analysis of huge number of sample in a single run.