Evaluation of ORAC methodologies in determination of antioxidant capacity of binary combinations of quercetin and 3-(3,4,5-trihydroxybenzoyl) coumarin derivatives

Abstract

Antioxidant capacity is used to refer to ability of compounds to react with free radicals, it is also described as ability to inhibit oxidation processes. There are different methods that evaluate the antioxidant capacity of compounds of natural origin or extracts. ORAC methodologies measure the ability to transfer hydrogen atoms to RO·/ROO· radicals generated by the AAPH thermolysis, in presence of a probe that accounts for oxidation of antioxidant. Despite extensive use of these methods to assess antioxidant capacity, they have been questioned by type of radical generated and information they can deliver, especially if it is a mixture of compounds. In this work, antioxidant capacity of binary combinations of quercetin with synthetic 3-phenylcoumarins was evaluated through ORAC-FL, PGR and quantification techniques after oxidation kinetics via free radicals, through HPLC. It was found through ORAC-FL that derived 3-(3,4,5-trihydroxybenzoyl) coumarin have greater antioxidant capacity than Trolox: 1 < 2 < 3 < quercetin. Determination of ORAC-FL indices of combination showed an antagonistic effect among the antioxidants. Also, ORAC-PGR method did not allow determination of antioxidant capacity of compounds, separately, indicated high reactivity of compounds. However, unusual behaviors were observed in combinations, unable to explain antagonistic effect observed in ORAC-FL. HPLC oxidation kinetics analysis showed that in the combination the consumption of the most reactive antioxidant dominated the antioxidant capacity and followed a similar trend as observed by ORAC-FL. Therefore, ORAC methodologies would not be useful in characterizing antioxidant capacity of mixture in relation to reactivity of metabolites present therein, but in relation to amount of hydroxyl groups available.