Bound phenolic acids were extracted according to Mattila,
Pihlava, and Hellström (2005) with slight modifications. The residue
remaining after anthocyanin extraction was mixed with
42 mL of a mixture of acidified methanol and 10% acetic acid
(85:15 v/v). The mixture was homogenized (Polytron PT3100,
Lucerne, Switzerland) at 10,000 rpm for 5 min and sonicated for
30 min. The volume was brought to 132 mL with deionized water.
Subsequently, 30 mL of 10 N NaOH was added, and the homogenate
was stirred for 24 h at 25 C and was adjusted to a pH of 2.
Bound phenolic compounds were extracted by adding a mixture
of cold diethyl ether and ethyl acetate (1:1) at an extract to solvent
ratio of 1:2. The extraction was performed 3 times, and the organic
layers were combined, evaporated to dryness under N2 gas and dissolved
in methanol before HPLC analyses for bound phenolic
compounds.