Phenolic compounds from fermented rice bran were extracted with methanol at a ratio of 1:10 (w/v), following the method described by Souza, Oliveira, Rocha, and Furlong (2010). Samples of 5 g were subjected to orbital shaking (150 rpm) at room temperature for 3 h with methanol and the extract obtained was filtered through filter paper (Whatman n° 4) into a separating funnel and washed three times with 10 mL of hexane. The methanolic extract was evaporated on a rota-evaporator at 50 °C under reduced pressure and the phenolic compounds were resuspended with 10 ml of distilled water in an ultrasonic bath for 10 min. The resulting extract was clarified with 5 ml of 0.1 M ZnSO4

and 5 mL of 0.1 M Ba(OH)2, and allowed to rest for 20 min. After centrifugation (10 min, 25 °C, 3200g,) the supernatant containing the phenolic

compound was collected, lyophilized and quantified Bcl-2 inhibitor spectrophotometrically selleck products at 750 nm with Folin–Ciocalteau reagent (Qell, Brazil) using ferulic acid (Sigma, Japan) as standard (2–20 μg/ml). Phenolic extracts were resuspended in water and methanol (1:1), and 20 μL aliquots injected into a chromatograph (Shimadzu, Tokyo, Japan, CLASS-M10A) at a flow rate of 0.7 mL/min at 35 °C. The separation of the phenolic acids was accomplished using a C18 column (4.6 × 250 mm, 5 μm, Discovery®, USA) and a gradient isocratic solvent consisting of methanol and acidified water (1% v/v acetic acid) at a 20:80 ratio during 25 min, with UV detection at 280 nm until 15 min and 320 nm until 25 min. Phenolic acids were identified by comparison of retention times and absorption spectrum with different standards of phenols present in rice bran (caffeic, chlorogenic, p-coumaric,

ferulic, gallic, p-hydroxybenzoic, protocatechuic, syringic and vanillin, obtained from Sigma–Aldrich, USA) as described in the literature ( Mira et al., 2008 and Pourali et al., 2010). The detection limit (LOD) was calculated by the background Protein kinase N1 noise signal (solution containing the solvents used in the extraction of phenolic compounds) at 3:1. The determination limit (LOQ) was established as three times the amount of the LOD ( Ribani, Bottoli, Collins, Jardim, & Melo, 2004). The phenolic antioxidant activity of the extracts was determined according to the methods described by Rufino et al., 2009 and Sánchez-Moreno et al., 1998 and Brand-Wiliams, Cuvelier, & Berset, 1995 measured by the reduction in free radical 1,1-diphenyl-2-picrihidrazil (DPPH). This method is based on the transfer of electrons from one antioxidant substance to a free radical, DPPH, which loses its purple colour upon reduction, becoming yellow. Different concentrations of solutions of ascorbic acid (0.01–0.1 mg/mL), ferulic acid (0.01–1 mg/ml), fermented and unfermented rice bran (0.01–0.