2.3.4. Extraction of phenolics
Free and bound phenolics were extracted according to the
method developed by Vitali, Dragojevic', and Sebecic (2009) with
slight modifications. Extractions of free and bound phenolics were
carried out in triplicate samples of each flour type. For free phenolics,
sample (2.0 g dw) was mixed with 20 mL of HClconc/methanol/
water (1:80:10,v/v) mixture and shaken by using a rotary
shaker (JB50-D; China) at 250 rpm for 2 h at 20 C, and then the
mixture was centrifuged at 3500g for 10 min at 4 C in a centrifuge
(Sigma 3K 30). The supernatants (free phenolics) were stored
at 20 C until used. For bound phenolics, after free phenolic
extraction, the residue which combined with 20 mL of methanol/
H2SO4conc (10:1) mixtures was placed in water bath at 85 C
for 20 h and then cooled at room temperature. The mixtures were
centrifuged at 3500g for 10 min at 4 C in a centrifuge
(Sigma 3K 30). The supernatants (bound phenolics) were stored
at 20 C until used.