Determination of total proanthocyanidin content
Total proanthocyanidin content was determined with the
vanillin/hydrochloric acid method. Rice bran (100 mg) was
mixed with acetone/water/acetic acid (70:29.5:0.5, v/v/v)
solution (2 mL) and sonicated at 37C for 30 minutes. After
centrifugation (10,000 g, 10 minutes, 20C), the supernatant
was decanted. The solid was extracted again with the above
procedures. The supernatant was combined and evaporated
to dryness by a rotary evaporator at 40C, and the residue
was redissolved with 2 mL of 30% methanol. The appropriate
diluted sample (10 mL) was mixed with 4% vanillin
methanol solution (0.6 mL) and concentrated HCl (0.3 mL),
and stood for 15 minutes at room temperature in the dark.
After mixing, the absorbance was measured at 500 nm. To
eliminate the interference from ACNs, the blank assay was
made with a mixture of 10 mL of diluted extract, 0.6 mL of
methanol, and 0.3 mL of concentrated HCl. Proanthocyanidins
concentration of the extract was determined against
external standard of cetachin. Total proanthocyanidin
content of rice bran was expressed as catechin equivalent
(mg CE/g DM).