Determination of anthocyanins
Anthocyanin contents in the samples were determined using
the method described by Fuleki and Francis (1968) with some
modifications. Briefly, 2 g of samples were homogenised with a
Polytron homogeniser for 2 min at medium speed with acidified
organic solvent (10 ml). A 95% ethanol:1.5 N HCl (85:15, v:v)
extracting solvent was used. The pH of the solvent was adjusted
as necessary to obtain a final pH of 1.0. The sample was transferred
quantitatively to a 250 ml Erlenmeyer flask, and extracted using
approximately 50 ml of extracting solvent for 24 h in an electrical
shaker at 4 C. The samples were extracted twice and filtered
through Whatman No. 1 paper (Whatman, Maidstone, UK). The
flask, as well as residue on the filter, was washed repeatedly with
the extracting solvent until 100 ml of extract was collected in the
volumetric flask and made up to volume. Absorbance was measured
at 535 nm. Total anthocyanin contents of the samples were
calculated as mg total anthocyanin per 1 g of sample using an
extinction coefficient of E1%
1 cm = 982.