A novel antioxidant assay of ferric reducing capacity measurement using ferrozine as the colour forming complexation reagent

Abstract

Since antioxidants are health-beneficial compounds capable of removing reactive species, assay of total antioxidant capacity (TAC) by simple and low-cost methods is important. The magenta-coloured iron(II)-ferrozine (Fe(II)-FZ) complex showing an absorbance maximum at 562 nm has previously been utilized for iron-binding assays, but not for antioxidant determination. Ferrozine is a highly ferrous-stabilizing ligand such that ferric ion in the presence of ferrozine easily oxidizes antioxidants and is itself reduced to Fe(II)-FZ, yielding a very high molar absorptivity and thus enhanced sensitivity for most antioxidants. The hierarchic order of antioxidant power for common antioxidants was in accordance with known structure–activity relationships. The Fe(III)-FZ assay was applied to synthetic antioxidant mixtures to yield additive absorbance values, which is a prerequisite for precise determination of antioxidant capacity of complex mixtures. The calibration curves (lines) of trolox and quercetin individually and in herbal infusions—by using the method of standard additions—were parallel, confirming that the herbal antioxidants and trolox did not chemically interact among each other so as to cause apparent deviations from Beer's law. The proposed method was applied to medicinal plant infusions for total antioxidant capacity assay as trolox-equivalents, and the results were compared to those found with CUPRAC (cupric reducing antioxidant capacity), FRAP (ferric reducing antioxidant power) and Folin total phenols assays, the highest correlation being achieved with CUPRAC. In short, a novel ferric reducing assay for food antioxidants was introduced, which was superior to FRAP in regard to its realistic pH, enhanced sensitivity, faster kinetics, and absence of free Fe(II)—which can cause Fenton-type oxidations—in the reaction products.