Avaliação da capacidade antioxidante de cachaças envelhecidas em barris de madeira nativa do Brasil e desenvolvimento de uma metodologia de sequestro de ácido hipocloroso em sistema ex-vivo

Abstract

Cachaça is a typically Brazilian drink and can be aged in barrels of different types of wood in order to add value to the final product. The aging process of cachaça in wooden barrels leads to the extraction of several compounds, mostly with antioxidant properties, which depend on the type of wood and the aging time. Thus, the antioxidant capacity of cachaça aged in barrels of different wood species was evaluated as a function of time (2 years) and barrel reuse. The aged cachaças were evaluated for the content of total phenolic compounds (Folin-Ciocalteau method) and the antioxidant capacity was measured using the ABTS•+, DPPH• and •NO radical scavenging methods, in addition to the ability to reduce Fe(III) ) (FRAP assay). As a control system, the results were compared to non-aged cachaça. Gallic acid, quercetin and Trolox were used as standards and the results expressed in their respective equivalents (mg L-1). The cachaças aged in umburana, balsam and Jatobá showed the highest concentration of total phenolic compounds, and for all cachaça the concentration of these compounds was proportional to the aging time. The process of reusing the barrels led to a reduction in the concentration of extracted phenolic compounds. Similar behavior was obtained for the antioxidant capacity by the ABTS•+, DPPH• and FRAP methods. The •NO radical scavenging capacity was about 70% after 150 min for all samples. The results of ABTS•+ were superior to those of DPPH• indicating a higher concentration of hydrophilic antioxidant compounds. Finally, the type of wood, the time and the processes involved in the aging of the beverage are determinant in the antioxidant profile of the cachaça. The development of methods to determine the total antioxidant capacity (TAC) and reactive oxygen and nitrogen species has been a challenge given the complexity of the samples. Thus, the development of methods that mimic physiological conditions using biologically active molecules as probes is required. Since high concentrations of HOCl can be associated with inflammatory processes, the second step of this work aimed to develop a simple, fast, and sensitive method to determine the HOCl scavenging capacity (Csc) of different compounds and anti-inflammatory drugs based on the inhibition of HOCl-mediated thiamine to thiochrome oxidation under simulated physiological conditions. The method is based on the inhibition of thiochrome formation (ex = 370 nm e em = 440 nm), a product of the oxidation of thiamine by HOCl. Species that can react with HOCl reduce the formation of thiochrome and, therefore, the intensity of the fluorescence signal. Gallic acid was used as standard, and the percentage of fluorescence inhibition was used as an analytical response. The probe selectivity was analyzed against seven biologically active reactive species and the parameter evaluated was the analytical sensitivity (S = uaF μmolL-1), with the results obtained: HOCl (23.4 ± 1.2), •NO (1.4 ± 0.2), H2O2 (1.8 ± 0.2), HO• (4.2 ± 0.2), terc-BuOOH (0.6 ± 0.1), terc-BuOO• (0.70 ± 0 .05), 1O2 (0.50 ± 0.07), O2•- (0.100 ± 0.007) and ONOO- (0.30 ± 0.03). The probe used was selective for HOCl. The method allowed evaluating the Csc of 25 compounds from different classes: phenolics (n = 14); thiolics (n = 5) and non-steroidal anti-inflammatory drugs (n = 6). The probe was applied to human neutrophils, under inflammatory conditions, showing selective HOCl and allowing to determine the Csc of compounds in cell media. Thus, the proposed method proved to be selective and suitable for evaluating the scavenging ability of HOCl against natural and synthetic compounds.