Extraction of free/conjugated phenolics
Free/conjugated phenolics were extracted following the method reported by Li et al. (2008) with minor modification. Briefly, rice flour (1 g) was extracted twice with 80% methanol (20 mL) using a weight to volume ratio of 1:20. After concentrating the supernatants, 40 ml of 4 M NaOH was added for hydrolysis for 2 h under nitrogen. After digestion, the mixture was adjusted to a pH of 1.5–2.0 using concentrated HCl. The solution was defatted once using hexane (10 mL) and extracted thrice with ethyl acetate (70 mL). The ethyl acetate fractions were pooled and rotary evaporated at 35 °C. The dried extracts were dissolved in 50% methanol (5 mL) and used as free/conjugated phenolics extracts.
2.5. Extraction of bound phenolics
After extracting the free/conjugated phenolics, the residue was washed with distilled water prior to extraction of bound phenolics (Zhang et al., 2010). The residue (about 2 g) was digested with 4 M NaOH at room temperature for 2 h while shaking under nitrogen. The mixture was then adjusted to pH 1.5–2.0 with concentrated HCl. After centrifugation at 15,300 × g for 30 min, the supernatant was defatted three times using 25 mL hexane and extracted thrice with ethyl acetate (70 mL). The ethyl acetate fractions were combined and evaporated to dryness at 35 °C. The dried extracts were dissolved in 50% methanol and used as bound phenolic extracts.