Simultaneous detection of superoxide anion radicals and determination of the superoxide scavenging activity of antioxidants using a N,N-dimethyl-p-phenylene diamine/Nafion colorimetric sensor

Abstract

The widely used nitroblue tetrazolium (NBT) colorimetric method for superoxide anion radical (O₂˙⁻) detection suffers from limitations such as formazan solubility and interference from similar electron donors. This work reports the development of a novel assay for superoxides, where O₂˙⁻ generated by a NADH/PMS/O₂ system oxidizes the N,N-dimethyl-p-phenylene diamine (DMPD) reagent to pink-colored DMPD-semiquinone (DMPDQ) cationic radicals retained by a cation exchange Nafion® membrane and measured colorimetrically. Antioxidants exhibiting superoxide radical scavenging activity (SRSA) cause less DMPDQ radical production, resulting in an attenuated color intensity on the Nafion® membrane, the absorbance difference (ΔA) at 514 nm being proportional to the antioxidant concentration. The developed method was applied to 14 antioxidant compounds including trolox, phenolic and hydroxycinnamic acids, flavonoids, and thiols, and the linear concentration ranges, calibration equations (as ΔA versus concentration), and trolox-equivalent antioxidant capacities of these antioxidants were established. The results were discussed in the light of structure–activity relationships. This method was used for measuring the SRSA of antioxidant mixtures and a green tea extract. The Nafion® membrane-contacted green tea extract was transferred to an online-HPLC post-column CUPRAC system to measure unconsumed catechins. The percentage inhibitions for green tea found with the developed method were compared to those measured by the reference NBT method.