2.4. Determination of total phenolics

The total phenolic contents of the extracts were determined using the modified Folin–Ciocalteu method as described by Wolfe and Islam et al. (Islam et al., ²⁰¹³; Wolfe et al., ²⁰⁰³). Here, 500 µg/ml of the extract was mixed with 2 ml Folin–Ciocalteu reagent (previously diluted with water 1:10 v/v) and 2 ml (75 g/L) of sodium carbonate. The tubes were vortexed for 15 s and allowed to stand for 20 min at 25°C for color development. Absorbance was then measured at 760 nm UV‐spectrophotometer (Shimadzu). Total phenolic contents were expressed in terms of gallic acid equivalent, GAE (standard curve equation: y = 0.102x + 0.02687, R2 = 0.9999), and mg of GA/g of dry extract. Gallic acid (GA) stock solution (5 mg/1 ml methanol) was diluted in five concentrations (0.156–25.00 μg/ml) to obtain standard curve, where the range of absorbance values was 1.56–2.57. Moreover, there were five fractions of sample with a concentration of 0.5 mg/ml and the range of the absorbance values was 0.448–2.890. The experiment was repeated three times at each concentration.