2.5. Total phenol content
The total phenol content (TPC) was determined using Folin–Ciocalteu
reagent (Singleton & Rossi, 1965). About 0.3 mL of the ethanol
or methanol:acetone solutions of the different samples (30 g/L)
were introduced into test tubes followed by 2.5 mL of Folin–Ciocalteu
reagent (diluted 10 times with water) and 2 mL of sodium carbonate
(7.5% w/v). The tubes were vortexed; covered with parafilm
and incubated at 50 C for 5 min. Absorption at 760 nm was measured
with an HP 8451 spectrophotometer (Hewlett Packard, Cambridge,
UK) and compared to a gallic acid calibration curve. The
results were expressed as mg gallic acid equivalents (GAE)/g dry
mass, as mean of three replicates.