Anthocyanin Analysis. Total anthocyanin was determined using the
spectrophotometric method adapted from that of Abdel-Aal and Huel
(21). A 0.5 g corn sample was weighed in a 50 mL centrifuge tube,
and 10 mL of chilled, acidified methanol (95% methanol and 1 N HCl
85:15, v/v) was added. The tube was flushed with nitrogen gas, agitated
for 30 min, and then centrifuged at 3000g for 10 min, and the
supernatant was collected. Absorbance of the solution was measured
immediately at 535 and 700 nm. Anthocyanin content was calculated
by
where C is the concentration of total anthocyanin (mg of cyanidin
3-glucoside equivalents per g of sample), is the molar absorptivity
(cyanidin-3-glucoside ) 25965/cm/M), and MW is the molecular weight
of cyaniding-3-glucoside, 449.2 (21).