Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method

In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid>epicatechin>quercetin≈catechin>α-tocopherol>rosmarinic acid>trolox>caffeic acid>ascorbic acid.