Total phenolics content was determined by the Folin–
Ciocalteu method, which was adapted from Swain and
Hillis (1959). The 150 mL of extract, 2400 mL of nanopure
water, and 150 mL of 0.25 N Folin–Ciocalteu reagent were
combined in a plastic vial and then mixed well using a
Vortex. The mixture was allowed to react for 3 min then
300 mL of 1 N Na2CO3 solution was added and mixed well.
The solution was incubated at room temperature (23 1C) in
the dark for 2 h. The absorbance was measured at 725 nm
using a spectrophotometer (Hewlett Packard 8452A, Diode
Array, USA) and the results were expressed in gallic acid
equivalents (GAE; mg/100 g fresh mass) using a gallic acid
(0–0.1 mg/mL) standard curve. Additional dilution was
done if the absorbance value measured was over the linear
range of the standard curve.