using 24 mL of methanol acidified with 1.0 N HCl (85:15, v/v) at a
stirring speed of 1000 rpm for 60 min at room temperature. The pH
of the mixture was kept at pH 1 during extraction. The mixtures
were centrifuged at 10,000g for 20 min at 4 C, and the supernatant
was refrigerated at 4 C for 2 days to precipitate insoluble
matters. The extract was re-centrifuged at 10,000g for 20 min
at 4 C. Supernatants were brought up to 50 mL with acidified
methanol. The anthocyanin content, extractable phenolic content
and antioxidant activities were determined. Forty milliliters of
supernatant was concentrated under a stream of nitrogen. The precipitates
formed during N2-flushing were separated by centrifugation
at 10,000g for 20 min at 4 C. The concentrated extracts were
adjusted to 4 mL with acidified methanol and vigorously mixed
and filtered through a 0.45-lm Nylon Acrodisc syringe filter membrane
for determination of individual anthocyanins and extractable
phenolic acids by HPLC.
2.4.