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 1.5e2.0 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.