Three μL of each sample were analyzed on an Acquity UPLC system (Waters, Milford,MA, USA) using a UPLC BEH column (2.1 × 50 mm, 1.7 μm particle size) at a temperature of 30 °C. A gradient of (A) deionized purified water Trichostatin A in vivo with 1% formic acid and (B) methanol (Tedia, Brazil) starting with 5% B and ramping to 100% B at 8 min, maintained till 8.50 min, then returning to initial conditions and stabilizing by 10 min. Detection in negative ion modes was achieved on an Acquity TQD mass spectrometer (Micromass Waters, Milford, MA, USA) with capillary – 3000 V, Cone – 30 V, source temperature 150 °C; desolvation temperature 350 °C. The antioxidant
activity of samples was assessed on the basis of scavenging activity of the stable 2, 2-diphenylpicrylhydrazyl free radical
(DPPH ). 750 μL of a methanolic solution of DPPH (0.02 mg/L) were added to 0.1 μmol of test samples in acetate buffer 0.3 M, pH 3.8 or 120 μL of methanol, in the case of control. Flasks were incubated at 25 °C for 25 min and absorbance was determined at 517 nm. All assays were performed in triplicate. A solution of rutin in acetate buffer 0.3 M, pH 3.8 (0.75 mg/mL) was used to calibrate the equipment. The scavenging capacity of DPPH radical check details was calculated using the following equation: DPPHscavenging effect(%)=(Ac-As/Ac)×100,where Ac and As are absorbance values of control reaction and test samples, respectively. The effect on lipid peroxidation inhibition was determined in a β-carotene-oleic acid system according to the Miller (1971) method with adaptations. A mixture containing 50 μL of β-carotene (2 mg/mL in chloroform), 40 μL of oleic acid standard, 1 mL of chloroform
and 400 mg of Tween 40 was prepared. Chloroform was removed under nitrogen atmosphere and 50 mL of aerated redistilled water were added. The mixture was then subjected to vigorous shaking. In screw-top glasses, 225 μl of β-carotene/oleic acid solution was added to 0.1 μmol of samples in acetate Phospholipase D1 buffer (0.3 M, pH 3.8) or 100 μL of methanol (blank assay). Absorbance was read at 470 nm, at 0 min (immediately after emulsion addition) and after 120 min of incubation at 45 °C (induction of thermal oxidation). Peroxidation leads to the bleaching of the β-carotene molecule, so the higher the absorbance the higher the antioxidant activity. The degradation rate was calculated according to zero order reaction kinetics. Inhibition of lipid peroxidation was calculated according to the following equation: %inhibition=[1-(A0-A1)/(A0′-A1′)]×100,where A 0 is the absorbance of sample at zero time, A 1 is the absorbance of sample after incubation (120 min) at 45 °C, A0′ is the absorbance of control at zero time and A1′ is the absorbance of control after incubation (120 min) at 45 °C. Xanthine oxidase activity was determined by measuring the formation of uric acid from xanthine. Xanthine in phosphate buffer 0.1 M, pH 7.4 (1.3 μmol) was incubated with 100 μL of ethanol and test samples (90 μM).