A number of assays have been introd

A number of assays have been introduced for the measurement of the total antioxidant activity of body fluids [1], [2], [3], [4], [5] and [6], food extracts [7], [8], [9], [10] and [11], and pure compounds [7], [12], [13], [14], [15] and [16]. Each method relates to the generation of a different radical, acting through a variety of mechanisms and the measurement of a range of end points at a fixed time point or over a range (reviewed in refs [13] and [17]). Two types of approach have been taken, namely, the inhibition assays in that the extent of the scavenging by hydrogen- or electron-donation of a pre-formed free radical is the marker of antioxidant activity, as well as assays involving the presence of antioxidant system during the generation of the radical.

Generation of the ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation [18] forms the basis of one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of solutions of pure substances [12], [19] and [20], aqueous mixtures and beverages [7] and [8]. The original ABTS•+ assay was based on the activation of metmyoglobin with hydrogen peroxide in the presence of ABTS to produce the radical cation, in the presence or absence of antioxidants. This has been criticized on the basis that the faster reacting antioxidants might also contribute to the reduction of the ferryl myoglobin radical. A more appropriate format for the assay is a decolorization technique in that the radical is generated directly in a stable form prior to reaction with putative antioxidants.

The improved technique for the generation of ABTS•+ described here involves the direct production of the blue/green ABTS•+ chromophore through the reaction between ABTS and potassium persulfate. This has absorption maxima at wavelengths 645 nm, 734 nm and 815 nm, as reported previously [1], [13] and [17], as well as the more commonly used maximum at 415 nm. Addition of antioxidants to the pre-formed radical cation reduces it ABTS, to an extent and on a time-scale depending on the antioxidant activity, the concentration of the antioxidant and the duration of the reaction. Thus the extent of decolorization as percentage inhibition of the ABTS•+ radical cation is determined as a function of concentration and time and calculated relative to the reactivity of Trolox as a standard, under the same conditions. The method is applicable to the study of both water-soluble and lipid-soluble antioxidants, pure compounds, and food extracts.