Desenvolvimento e aplicação de um novo ensaio para a determinação eletroquímica da capacidade antioxidante de compostos modelo e de matrizes complexas / Development and application of a new assay for the electrochemical determination of the antioxidant capacity of model compounds and of complex matrices

Rafael de Queiroz Ferreira

This work describes the development and practical applications of a novel and simple electrochemical methodology for the determination of the antioxidant capacity of specific model molecules and/or some complex food samples currently consumed in Brazil. Other systems having either theoretical or technological interest were also investigated. The method is based on the use of a known amount of an inorganic ion as the oxidant and in the chronoamperometric determination of its remaining concentration after reaction with the chosen antioxidant species. However, initial tests for different commercial brands of orange juices using Fe3+ as the oxidant (modified FRAP assay) were only successful when the antioxidant has a totally irreversible electrochemical behavior as, for example, ascorbic acid. To overcome this problem, the assays were then performed using Ce4+ as the oxidant (the CRAC assay) since its reduction after reaction can be carried out at 0.8 V vs Ag/AgCl, a potential region where the reduction of species formed by the reversible or quasi-reversible oxidation of the antioxidant does not occur. Due to the high anodic potentials required when using Ce4+, it was necessary to have a boron-doped diamond film as the working electrode. After a rigorous characterization of the electrochemical systems, measurements of the antioxidant capacity of eight standard compounds (ascorbic acid, gallic acid, tannic acid, BHA, catechin, quercetin, rutin and trolox) were carried out using the CRAC assay. The results showed a satisfactory correlation with those reported in the literature using other more complex assays and these studies were then applied to a set of industrialized fruit juices showing maximum values almost one order of magnitude higher than that of the reference compound (trolox) and following the antioxidant capacity sequence: cashew>guava>grape>mango>orange>passion fruit. Considering that the local "cachaça" industry is looking for alternative woods to the use of oak barrels, CRAC assays were carried out using four ethanol extracts of Brazilian woods [Pequi (Caryocar brasiliense), Imbuia (Octea porosa), Cabreúva (Myrocarpus frondosus) e Cabreúva-vermelha (Myroxylon balsamum)] as well as an Oak (Quercus sp) extract, for comparison. The results indicate an increasing antioxidant capacity in the order presented above and, although the best sample (Cabreúva-vermelha) has only 60% of the capacity shown by oak, its local availability and price makes it interesting for further research. A comparative evaluation of the results obtained using the CRAC and the DPPH assays was carried out for methanol extracts of sugar cane juice and passion fruit pulp. That comparison revealed a quantitative difference between the assay values but the hierarchy was maintained for each set of results. Such effect was attributed to differences in the prevailing mechanism for radical deactivation, as well as, the experimental conditions used for each assay. The correlation between structure and antioxidant activity of model flavonoid molecules under investigation was related to the presence of certain groups in the diphenilpyrene structure. The activity hierarchy for them was established as: OH(C2´C4´) >OH(C4´) ~ OH(C3´C4´) >C2=C3 + 4-oxo >OH(C3,C5) + 4-oxo >OH(C3) + 4-oxo >OH(C5) + 4-oxo >OH(C3,C5). The complex formation between flavonoids and metal ions, such as Fe2+, has a strong effect on the antioxidant capacity and CRAC assay showed that for morin, quercetin and fisetin the increase was 15.3, 31.8 and 27.9%, respectively. On the other hand, for catechin and chrysin the increase was only 1.8 and 7.8%, respectively. These increases were related to the presence of, at least, one of three types of active sites in the polyphenolic molecule that can interact with metal ions. All these findings confirm that the CRAC assay is simple and convenient tool for the determination of the antioxidant capacity of a variety of practical systems and model molecules.

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