Total phenolic content in the extracts was measured by a colorimetric assay based on procedures described by Singleton and Rossi (1965) with some modifications. An aliquot of the extracts
(200 L) was mixed with a diluted Folin–Ciocalteu’s phenol reagent
(800 L). After 10 min, 7.5% (w/v) sodium carbonate solution (2 mL)
was added to the mixture and incubated in the dark for 90 min. The
absorbance at 725 nm was measured using an UV–vis spectrometer
(BIOMATE 3S, Thermo Scientific, MA). To calculate TPC, gallic acid
solutions at different concentrations (100–500 g/mL) were used
as standards. When NaOH was used for extraction, the alkalinity
itself affected the absorbance during the analysis, especially when
the phenolic content in the sample was low. So accurate measurement was difficult for the low phenolic content in the NaOH extracts
prepared at 25 ◦C.