Mice were sacrificed by cervical dislocation, and the liver, kidneys, and brain were quickly removed, placed on ice, and homogenized in 10 volumes of cold, Tris buffer (10 mM, pH 7.4). The homogenates were centrifuged at 4000×g at 4 °C for 10 min to yield a low-speed supernatant fraction (S1) for each tissue (liver, kidney and brain) that was used for SNP-induced lipid peroxidation

and H2DCF-DA assays. The antioxidant effect of the PCs was evaluated against production of SNP (5 μM)-induced thiobarbituric acid reactive substances (TBARS), using vehicle, dimethyl sulfoxide (DMSO), or PCs (1–100 μM). The S1 was pre-incubated for 1 h at 37 °C in a buffered medium with the PCs in the presence or absence of SNP. TBARS formation was determined spectrophotometrically

selleckchem at 532 nm, using malondialdehyde (MDA) as a standard, according to Ohkawa et al. (1979). In this work we used the SNP as a mechanism of toxicity, in a concentration of 5 μM according to previously described (Puntel et al., 2009). In fact, sodium nitroprusside (SNP) is a good chemical inducer of lipid peroxidation in mice tissues (Rauhala et al., 1998), since it release in a short-lasting time NO in tissue preparations. The antioxidant effect of the PCs was evaluated against basal production of thiobarbituric acid reactive substances (TBARS), using vehicle, dimethyl sulfoxide (DMSO), selleck compound library or PCs (1–100 μM). The S1 was pre-incubated for 1 h at 37 °C in a buffered medium with the PCs. TBARS formation was determined spectrophotometrically at 532 nm, using malondialdehyde (MDA) as a standard, according to Ohkawa et al. (1979). S1 was used for the 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA)

oxidation assay to evaluate levels of RS (reactive species). S1, in Tris buffer (10 mM, pH 7.4) was incubated with different PCs at concentrations of 1, 5, 10, 50, and 100 μM at 37 °C. After 1 h, aliquots were removed, H2DCF-DA (7 μM) was added to the medium, and incubation was continued for 1 h in the dark. Fluorescence was determined using 488 nm for excitation and 520 nm for emission. A standard curve was created using increasing concentrations of 2,6-dicloroindophenol sodium salt hydrate (DCF) incubated in parallel (Pérez-Severiano GNAT2 et al., 2004). The results were analyzed as a percentage value in relation to the control group. The protein content was determined according to Lowry et al. (1951), using bovine serum albumin (BSA) as a standard. The scavenging of NO was assessed by incubating SNP (5 mM, in potassium buffer) with different PC concentrations at 25 °C. After 120 min, 0.5 mL of incubation solution was sampled and mixed with 0.5 mL of Griess reagent (Green et al., 1981), and the absorbance was measured at 550 nm. The amount of nitrite was calculated using different concentrations of sodium nitrite.