Reactive oxygen radical-scavenging activity assay by photolysis of azo-radical initiator without exposing samples to ultraviolet light: A preliminary feasibility study of a modified assay

We previously developed an electron paramagnetic resonance (EPR)-based oxygen radical absorbance capacity (ORAC-EPR) assay that can be used to evaluate the radical-scavenging activity (RSA) of samples by employing the established EPR spin trap method. In this assay, since reactive oxygen radicals are generated by the photolysis of the azo-radical initiator by ultraviolet (UV) light irradiation, the sample in the assay mixture exposed to UV light may also get photolysed. In the present study, irradiation of the sample was avoided; a novel radical-generation method was adopted in the ORAC-EPR assay, and the feasibility of the modified assay was assessed. RSA was evaluated in the modified as well as in the conventional assay with three antioxidants. Although the RSA values normalized to the spin trap reagent obtained with the modified assay were approximately 20% lower than those obtained with the conventional assay, the maximum difference between the RSA values of the two methods was 15% when this data were normalized to the standard antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Elucidation of the generation mechanism of the oxygen radicals and the reasons for lower RSA is necessary prior to application of the new radical-generation method coupled to ORAC-EPR assay.