Assays of total antioxidant capacity (TAC) of complex materials bring no information on the composition of antioxidants present in a sample. As the thermodynamic condition for a redox reaction is that redox potential of the oxidant must be higher than that of a reductant (antioxidants), it seemed to be of interest whether it is possible to estimate the content of antioxidants of various ranges of redox potentials using a set of assays employing oxidants/indicators of different values of redox potentials. Antioxidant activities of nine antioxidants and TAC of an aqueous garlic extract were estimated using nine assays of E_o′ of oxidants/indicators ranging from 0.11 to 1.15 V. The antioxidant activities were expressed in mol Trolox equivalents/mol compound. The thermodynamic conditions made some antioxidants unreactive with indicators of sufficiently low E_o′, but otherwise, no dependence between the antioxidant activities and redox potentials of oxidants/indicators and reactivities of antioxidants was observed. TAC of the garlic extract did not show any regular dependence on the redox potential of the oxidant/indicator, being the highest in the test of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS^•) decolorization. These results indicate that kinetic factors play a primary role in determining the antioxidant activities of antioxidants and TAC in various assays.

The reaction of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS^●) with proteins (bovine serum albumin, blood plasma, egg white, erythrocyte membranes, and Bacto Peptone) leads not only to a reduction of ABTS^● but also to the appearance of a purple color (absorption maximum at 550–560 nm). The aim of this study was to characterize the formation and explain the nature of the product responsible for the appearance of this color. The purple color co-precipitated with protein, and was diminished by reducing agents. A similar color was generated by tyrosine upon reaction with ABTS^●. The most feasible explanation for the color formation is the addiction of ABTS^● to proteins’ tyrosine residues. The product formation was decreased by nitration of the bovine serum albumin (BSA) tyrosine residues. The formation of the purple product of tyrosine was optimal at pH 6.5. A decrease in pH induced a bathochromic shift of the spectra of the product. The product was not a free radical, as demonstrated by electrom paramagnetic resonance (EPR) spectroscopy. Another byproduct of the reaction of ABTS^● with tyrosine and proteins was dityrosine. These byproducts can contribute to the non-stoichiometry of the antioxidant assays with ABTS^●. The formation of the purple ABTS adduct may be a useful index of radical addition reactions of protein tyrosine residues.

Phenolics are the main components of plant extracts contributing to their antioxidant activity. However, they autoxidize, generating hydrogen peroxide. This study aimed to examine the correlation between the phenolic content, total antioxidant capacity (TAC), and the amount of H₂O₂ generated in extracts of 18 various plant materials. A significant correlation was found between the phenolic content and TAC measured by ABTS^● decolorization, CUPRAC, FRAP, and DPPH^● decolorization methods (correlation coefficients r of 0.94, 0.93, 0.90, and 0.78, respectively). However, the correlation between the phenolic content and H₂O₂ amount generated upon brewing (r = 0.25) and after 1 h incubation (r = −0.37) was low or negative. The correlation between the phenolic content and the change of H₂O₂ concentration during 1 h incubation of the extracts was negative (r = −0.61). Examination of three phenolics (pyrogallol, gallic acid, and quercetin) showed that all compounds generate but also scavenge H₂O₂. Therefore, the H₂O₂ concentrations in phenolic-containing extracts represent net results of the rates of generation and scavenging of H₂O₂, which may differ depending on the composition of phenolics in the extracts, do not always increase with the increase in time and concentration of phenolics, and cannot serve as an index of the phenolic content.

It has been estimated and demonstrated that the antioxidant capacity of proteins is increased as a result of digestion in the gastrointestinal tract, which can be contributed by denaturation and digestion. This study aimed to evaluate the effect of denaturation and proteolytic digestion on the antioxidant activity of bovine serum albumin (BSA) and chicken egg white proteins in model systems. Denaturation with an anionic detergent (sodium dodecyl sulfate) and digestion with papain and trypsin increased the antioxidant activity/capacity of the proteins, apparently due to the increased exposure of amino acid residues responsible for the antioxidant activity of proteins (tyrosine, tryptophan, cysteine, histidine, arginine, and cystine in the ABTS^● decolorization assay; cysteine, tryptophan, tyrosine, and cystine in the FRAP assay). As the increase in the protein antioxidant activity/capacity was limited in extent, it does not invalidate the use of the antioxidant capacity of proteins to be consumed as a rough measure of their antioxidant capacity after modifications in the gastrointestinal tract.

Interactions between antioxidants are of interest, mainly for understanding their action in complex biological and food systems. This study aimed to evaluate interactions between the anthocyanin-rich aqueous red cabbage extract and several natural (ascorbic acid, gallic acid, and glutathione) and synthetic (Trolox and TEMPOL) antioxidants as a function of reaction time, concentration, and extract/antioxidant ratio in the ABTS^• decolorization and FRAP antioxidant activity assays. The measured interaction type showed dependence on assay type, reaction time, and the extract/antioxidant ratio, but no general dependence on the concentrations of the extract and the antioxidants over a 6-fold concentration range. In the ABTS^• decolorization assay, the interactions between the red cabbage extract and exogenous antioxidants were additive (Trolox), weakly antagonistic (ascorbic acid, gallic acid, and glutathione), and definitely antagonistic for TEMPOL. In the FRAP assay, the interactions were additive (ascorbic acid and Trolox), weakly antagonistic (gallic acid and TEMPOL), and definitely synergistic for glutathione. These results suggest the need for a series of assays covering a range of conditions to demonstrate a deviation from additivity in the interactions between antioxidants. The synergy of the interaction of glutathione with the extract in the FRAP assay indicates the possibility of a higher reactivity of this compound in Fe³⁺ reduction in complex systems than in an assay of the pure compound.

The biological role of the fluorescence of flowers is a matter of debate. Anthocyanins are a group of compounds that are weakly fluorescent; their fluorescence in flowers has been rarely studied. This study aimed to compare the absorption and fluorescence spectra of anthocyanins extracted from several anthocyanin-containing autumn flowers and examine changes in these spectra during the storage of petals at cold-room and room temperatures and during the storage of dried petals. Petals of red clover Trifolium pratense, pink petunia Petunia × hybrida, Pelargonium horatum, Pelargonium. zonale, Pelargonium. peltatum, red and pink Begonia semperflorens, Buddleja japonica, and purple Chrysanthemum were studied. The results demonstrate that it is possible to distinguish between petals of various flowers based on the absorption spectra of petal extracts and the fluorescence spectra of petal extracts and intact petals. Spectral changes during storage were not always unidirectional and progressive; the most common one was the increase in the intensity of the fluorescence band at 500–560 nm at the excitation wavelength of 460 nm. These results point to the possibility of using fluorescence measurements to identify and estimate the freshness of petal-based material in herbalism, forensic analysis, and the food industry. The measurement of the spectra of whole petals or their fragments by front-face fluorimetry, including common plate readers, may be especially useful due to its simplicity and rapidity.