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.
