To obtain the free and conjugated phenolic fractions, a 1.5 mL
aliquot of the crude extract (containing all extractable compounds)
was evaporated to dryness using a Savant Speed Vac Concentrator
attached to a Labconco CentriVap Cold Trap, and re-suspended in
0.5 mL acidified water (pH 2 with HCl). This was partitioned with
1 mL of diethyl ether/ethyl acetate (DE/EA, 1:1, v/v), repeated three
times, and the combined organic layer, the free phenolics (FP) fraction,
was evaporated under N2 and reconstituted in 70% methanol,
and filtered using Phenex-NY 4 mm 0.2 lm syringe filters (Sigma–
Aldrich Co., St. Louis, MO) prior to HPLC analysis.