Desenvolvimento e aplicação de métodos analíticos para avaliação da capacidade antioxidante em amostras de bebidas e própolis vermelha de Alagoas

Abstract

The determination of antioxidant capacity (CAO ) is a recent demand in strategic areas such as food quality control, drug development and evaluation of biologically active substances. In this way, this work aims to develop an analytical method for CAO determination in drinks samples based on the inhibition of the thiochrome (λexc = 370 nm e λem = 440 nm), the product of thiamine oxidation by K3Fe(CN)6 (HCF), and the evaluation of the antioxidant profile of the hydroalcoholic extract of red propolis from Alagoas (HAERP). For the thiamine method development, gallic acid (GA) was employed as the reference and the inhibition percentage (%IF) as an analytical response. The first parameter evaluated was the order of reagent addition. When the GA+HCF+ thiamine order was evaluated, there was a linear reduction of the signal dependent on the GA concentration. Kinetic assay showed that the reaction was stable at 10 min. Medium pH was optimized by the evaluation of five buffer solutions: phosphate (pH 6.5-8.0), Tris (pH7.0-8.5), Carbonate (pH 8.0-11.0), borate (8.0-10) and Britton-Robinson (pH 9.0-12.0). Better results were obtained with carbonate buffer (CB) 0.1 mol L-1 at pH 8 and phosphate buffer (PB) 0.01 mol L-1 at pH 7.4. The ionic strength influence was evaluated in PB, with better results with NaCl at 0.1 mol L-1. The [HCF/thiamine] ratio was investigated and the best ratio was determined as 15. For the optimized conditions employing CB, the method showed LOD (3σ) = 0.008 mg L-1, RSD ≤ 3.4% correspondent to the curve %IF = 218(±6)CAO + 2.06 (±0.96) (r = 0.9984); PB showed LOD (3σ) = 0.002 mg L-1, RSD ≤ 7.1% correspondent to the curve %IF = 1435(± 95)CAO + 1.60(±1,35) (r = 0.9894). The proposed method, using CB, was employed for CAO determination in different samples: teas and infusions (n=7) and wines (n=6). The obtained results were compared with the Folin-Ciocalteau (FC), ABTS+ and DPPH methods. The correlation among the results for different methods indicates a greater consistence for FC and ABTS+ with the proposed method. Applying ANOVA, it was not observed significant difference among the obtained results for the methods at 95% confidence interval. The proposed method can be described as a fast, sensible and simple method for determination of CAO in beverages, and the results agreed with the established methods. Then, the HAERP CAO was evaluated utilizing FC, ABTS+ and DPPH methods, ●NO sequestration capacity, Fe(II) complexation capacity and FRAP ( Ferric Reducing Antioxidant Power). Preliminary studies such as liquid chromatography identified 14 phenolic compounds in the HAERP. Based on this result, quercetin (QC), caffeic acid (CA) and Trolox (TR) were selected as references. For FC, the following results were obtained: 110.6 (QC), 63.1 (CA), 494.4(TR) in mg g-1 HAERP. According to the ABTS+ results, it was observed that EC50 (mg mL-1): QC < CA < TR < HAERP and for DPPH EC50 (mg mL-1): QC<CA<HAERP<TR. The HAERP showed reduction of ~74% of ●NO generated in the medium. The extract was able to reduce Fe(III), however, in a minor proportion than the references. The HAERP was not able to complex Fe(II) efficiently. Results indicate that HAERP has a great CAO, and may be applied in the prevention of oxidative stress related diseases.