An end-point method for estimation of the total antioxidant activity in plant material

The 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase. This ABTS radical (ABTS·+), a chromogen, is stable at room temperature but is unstable above 35°C and/or at pH values of above 7.5. Nevertheless, the most important factor in its stability is the ABTS/ABTS·+ concentration ratio in the medium. The radical reacts with the antioxidant, L-ascorbic acid, with a high rate constant, the stoichiometry of the reaction being 1 mol of L-ascorbic acid per 2 mol of ABTS·+ reduced. Based on these considerations, a spectrophotometric end-point method has been developed to evaluate L-ascorbic acid in aqueous media, and this represents an improvement over the lag-method previously reported. Under optimal conditions of temperature, pH and reagent concentration, the end-point method was capable of determining L-ascorbic acid with a limit of quantification of 0.38 nmol. In the assay described here, this ability is used to evaluate the total antioxidant activity of commercial citrus juices, in which ascorbic acid is a principal component. In our opinion this procedure can quickly provide useful information on the antioxidant content of foods and plant extracts.