In 125-ml Erlenmeyer flasks, 50 ml of distilled water and 250 mg of
each sample of tea were combined. The extraction NLG919 chemical structure of compounds from green tea was performed in a water bath, at 100 °C, for 30 min. The samples were filtered on filter paper, and the extracts were freeze-dried. The resulting powder, referred to as dried tea extract, was used for antioxidant assays (Cao, Sofic, & Prior, 1996). As an identified representative polyphenol from green tea, standard commercial epigallocatechin gallate (EGCG, 95%) was used as a control. This sample was tested and treated with tannase using the same procedures as were employed for the tea extract. The extracts obtained from the green tea and the commercial control samples were used as substrates for enzymatic hydrolysis by tannase isolated from P. variotii ( Battestin et al., 2008). The dried tea extract (5 mg) was dissolved in 1 ml of phosphate buffer (pH 7.4, 75 mM) and incubated with 5 mg of tannase, at 40 °C, for 30 min. The hydrolysis process was stopped by placing the reaction in an ice bath
for 15 min. The biotransformed tea was used for the antioxidant assay after suitable dilution with the same phosphate buffer (pH 7.4, 75 mM) for ORAC and with a 70% methanol solution for DPPH. For the cellular assays, the samples were diluted with DMEM. C59 wnt research buy ORAC assays were performed using fluorescein (FL) as the fluorescent probe, as described by Macedo et al. (2011). The automated ORAC assay was carried out on a NovoStar Microplate reader (BMG LABTECH, Germany) with fluorescence filters for an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The measurements were made in a COSTAR 96 plate. The reaction was performed at 37 °C, the reaction was started by thermal decomposition of AAPH in a 75 mM phosphate buffer (pH 7.4) due to the sensitivity of FL to pH. The measurements were performed in triplicate. ORAC values were defined as the difference Methane monooxygenase between the area under the FL decay curve and the blank (net AUC). Regression equations between net AUC and antioxidant concentration were calculated
for all of the samples. A tannase control was performed, and the ORAC value obtained was subtracted from the samples treated with the enzyme. ORAC-FL values were expressed as μmol of Trolox equivalent/mg of tea extract (Cao & Ito, 2004). The potential antioxidant activity of the tea extract was assessed based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, as described by Macedo et al. (2011). The measurements were performed in triplicate, and anti-radical activity was calculated using the linear regression equation determined by plotting the anti-radical activity of Trolox solutions of known concentrations. Antiradical activity was expressed as μmol of Trolox equivalent/mg of tea extract.
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