2.4. Determination of total phenolics
The concentration of total phenols in the extracts was determined
by using Folin–Ciocalteu reagent and external calibration with caffeic
acid. Briefly, 0.2 mL of extract solution and 0.2 mL of Folin–Ciocalteu
reagent were added and the contents mixed thoroughly. After 4 min,
1 mL of 15% Na2CO3 was added, and then the mixture was allowed to
stand for 2 h at room temperature. The absorbance was measured at
760 nm using a HITACHI, U-2000 spectrophotometer. The concentration
of the total phenolics was determined as mg of caffeic acid
equivalent by using an equation obtained from caffeic acid calibration
curve. The estimation of phenolic compounds in the fractions was
carried out in triplicate and the results were averaged.