Crocin bleaching antioxidant assay revisited: Application to microplate to analyse antioxidant and pro-oxidant activities

The crocin bleaching assay (CBA) is a common method for evaluating the antioxidant activity of hydrosoluble samples. It is criticised due to its low reproducibility, problematic quantification of results, differences in reagent preparation, doubtful need for a preheating phase and sensitivity to factors such as temperature, pH, solvents and metals. Here, the critical points of the method were extensively revised, and a highly reproducible procedure for microplate readers redeveloped. The problems of using quantification procedures, disregarding kinetic considerations, are discussed in detail and a model is proposed for quantifying simultaneously anti- and pro-oxidant activities as function of concentration and time. Thus, the combined use of a reproducible procedure and robust mathematical modeling produced consistent and meaningful criteria for comparative characterization of any oxidation modifier, taking into account the dose-time-dependent behaviour. The method was verified by characterising several commercial antioxidants and some metal compounds using the parametric values of the proposed models. The activity of the tested antioxidants decreased in the order ETX > TR > PG > AA > TBHQ > BHA. Others, such as the lipophilic antioxidants of BHT and α-Tocopherol did not show any activity. Interference from metals were for Fe2+, Fe3+, Cd2+, Ni2+, Mg2+, Zn2+ and Sr2+, slightly antioxidant for Cu1+ and Cu2+, and strongly antioxidant for Mn2+. None of the tested metals showed a pro-oxidant activity.