The 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ) radical cation-based assays are among the most abundant antioxidant capacity assays, together with the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical-based assays according to the Scopus citation rates. The main objective of this review was to elucidate the reaction pathways that underlie the ABTS/potassium persulfate decolorization assay of antioxidant capacity. Comparative analysis of the literature data showed that there are two principal reaction pathways. Some antioxidants, at least of phenolic nature, can form coupling adducts with ABTS •+ , whereas others can undergo oxidation without coupling, thus the coupling is a specific reaction for certain antioxidants. These coupling adducts can undergo further oxidative degradation, leading to hydrazindyilidene-like and/or imine-like adducts with 3-ethyl-2-oxo-1,3-benzothiazoline-6-sulfonate and 3-ethyl-2-imino-1,3-benzothiazoline-6-sulfonate as marker compounds, respectively. The extent to which the coupling reaction contributes to the total antioxidant capacity, as well as the specificity and relevance of oxidation products, requires further in-depth elucidation. Undoubtedly, there are questions as to the overall application of this assay and this review adds to them, as specific reactions such as coupling might bias a comparison between antioxidants. Nevertheless, ABTS-based assays can still be recommended with certain reservations, particularly for tracking changes in the same antioxidant system during storage and processing.

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This report explores the antioxidant interaction of combinations of flavonoid–glutathione with different ratios. Two different 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS•+)-based approaches were applied for the elucidation of the antioxidant capacity of the combinations. Despite using the same radical, the two approaches employ different free radical inflow systems: An instant, great excess of radicals in the end-point decolorization assay, and a steady inflow of radicals in the lag-time assay. As expected, the flavonoid–glutathione pairs showed contrasting results in these two approaches. All the examined combinations showed additive or light subadditive antioxidant capacity effects in the decolorization assay. This effect showed slight dilution dependence and did not change when the initial ABTS•+ concentration was two times as high or low. However, in the lag-time assay, different types of interaction were detected, from subadditivity to considerable synergy. Taxifolin–glutathione combinations demonstrated the greatest synergy, at up to 112%; quercetin and rutin, in combination with glutathione, revealed moderate synergy in the 30–70% range; while morin–glutathione appeared to be additive or subadditive. In general, this study demonstrated that, on the one hand, the effect of flavonoid–glutathione combinations depends both on the flavonoid structure and molar ratio; on the other hand, the manifestation of the synergy of the combination strongly depends on the mode of inflow of the free radicals.

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