Accuracy and Validity of AREC (Alkoxy Radical Elimination Capacity) Assay in Evaluating the Antioxidant Abilities of Various Biosubstances

Abstract

Abstract The AREC (alkoxy radical elimination capacity) assay was developed to observe the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin adducts of free radicals produced by thermal decomposition of AAPH (2,2′-azobis(2,4-amidinopropane) dihydrochloride) using a flow-injection ESR (FI-ESR) system. The γ50 value is defined as [DMPO]0/ID50 = kS/k1, where k1 is the rate constant of spin-trapping of the alkoxy radical by DMPO, kS, that of alkoxy radical-elimination by the substrate, and ID50, 50% inhibition of the alkoxy radical by the substrate, and the AREC value is the ratio of γ50 value of the substrate to that of Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid, TRX). The γ50 and AREC values show fairly wide variations and can be determined for most biosubstances. The highest AREC values were observed for sinapic acid, l-glutathione, caffeic acid, and chlorogenic acid, followed by TRX, syringic acid, trans-ferulic acid, and homogentisic acid. Little correlation is observed between the AREC values and the hydroxy and superoxide radical-elimination abilities. The AREC values of 4-hydroxycinnamic acid derivatives (HCAs) are linearly related to the aryloxy radical-elimination abilities, which indicate that the alkoxy radical-elimination by HCAs is mainly caused by hydrogen-atom transfer. The newly defined AREC value is applicable for various biosubstances, and is far superior and a more reliable indicator than the oxygen radical absorption capacity (ORAC) value determined by the ORAC-fluorescein assay. Thus, the AREC value is an excellent indicator to characterize the antioxidant activities of a wide range of biologically important antioxidants present in fruits, vegetables, and beverages.