Evaluation of pure antioxidative capacity of antioxidants: ESR spectroscopy of stable radicals by DPPH and ABTS assays with singular value decomposition

Background: Among the antioxidant reactions, no correlation (R-2 = 0.5468) has been observed between the logarithm of the reciprocal of EC50 obtained by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, which can measure single electron transfer reactions. Therefore, we attempted to investigate the radical scavenging reaction of each antioxidant in the DPPH and ABTS assays by using electron spin resonance (ESR), and to study an analytical method that can eliminate other than that factor.Results: Singular value decomposition (SVD) of the spectra that were obtained by ESR measurements was used to separate the components and to determine the detailed changes of each component. We calculated the EC50 from the changes in the spectra of the first and second components owing to the change in the antioxidant concentration in the DPPH and ABTS assays to identify which component contributed more to the radical scavenging reaction. The results demonstrated a positive correlation (R-2 = 0.8806) between the EC(50)s of the first component of each of the DPPH and ABTS assays. In this study, common spectral changes were observed for all compounds with radical scavenging activity, so the first component was considered to be radical scavenging activity. Conclusions: Through this study, we could calculate the EC(50 )that significantly contributed to the radical scavenging reaction by decomposing the spectra that were obtained from ESR measurements of the reactions by the DPPH and ABTS assays into specific values.

Automated summary

This study investigated the radical scavenging reaction of antioxidants in DPPH and ABTS assays using ESR and developed an analytical method. The results demonstrated that the first component in both assays contributed significantly to the radical scavenging reaction.