, 2009) The CL was measured by adding 4 ml of AAPH dissolved in

, 2009). The CL was measured by adding 4 ml of AAPH dissolved in glycine buffer to a glass scintillation vial. Then, luminol was added and the CL was measured until reached constant light intensity. After this stabilization time, the Trolox solutions or the sample was added and the CL was measured in a liquid scintillator counter. The last count before the addition of Trolox or samples was considered as 100%. The count time was 10 s, and the CL emission was monitored for 3000 s after the addition of Trolox or samples. Graphs were

obtained by plotting percentage of counts per minute (%cpm) versus time (s) of instantaneously generated values of CL inhibition and area under curve (AUC). The total antioxidant reactivity (TAR) was calculated Selleckchem Trametinib as the ratio of light intensity in absence of samples (I0)/light intensity right after ATR addition

(I) and expressed as percent of inhibition. AUC and radical basal production were acquired by software GraphPad Prism software 5.0. TBARS (thiobarbituric acid reactive species) assay was employed to quantify lipid peroxidation (Draper and Hadley, 1990) and an adapted TBARS method was used to measure the antioxidant MLN8237 capacity of ATR using egg yolk homogenate as lipid rich substrate (Silva et al., 2007). Briefly, egg yolk was homogenized (1% w/v) in 20 mM phosphate buffer (pH 7.4), 1 ml of homogenate was sonicated and then homogenized with 0.1 ml of ATR at different concentrations. Lipid peroxidation was induced by addition of 0.1 ml of AAPH solution (0.12 M). Control mafosfamide was incubation medium without AAPH. Reactions were carried out for 30 min at 37 °C. Samples (0.5 ml) were centrifuged with 0.5 ml of trichloroacetic acid (15%) at 1200g for 10 min. An aliquot of 0.5 ml from supernatant was mixed with 0.5 ml TBA (0.67%) and heated at 95 °C for 30 min. After cooling, samples absorbance was measured using a spectrophotometer at 532 nm. The results were expressed as percentage of TBARS formed by

AAPH alone (induced control). The formation of OH (hydroxyl radical) from Fenton reaction was quantified using 2-deoxyribose oxidative degradation (Lopes et al., 1999). The principle of the assay is the quantification of the 2-deoxyribose degradation product, malondialdehyde, by its condensation with 2-thiobarbituric acid (TBA). Briefly, typical reactions were started by the addition of Fe2+ (FeSO4 6 mM final concentration) to solutions containing 5 mM 2-deoxyribose, 100 mM H2O2 and 20 mM phosphate buffer (pH 7.2). To measure ATR antioxidant activity against hydroxyl radical, different concentrations of ATR were added to the system before Fe2+ addition. Reactions were carried out for 15 min at room temperature and were stopped by the addition of 4% phosphoric acid (v/v) followed by 1% TBA (w/v, in 50 mM NaOH).

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