Determination of total phenolic content (TPC)

The procedure to determine TPC was based on that of Cheung et al. TPC was expressed in milligram of gallic acid equivalents per gram dry weight of leaves (mg GA/g dry weight). Freshly prepared 7.5% sodium carbonate solution was made by placing 7.5 g Na₂CO₃ in a volumetric flask and adjusting the volume to 100 ml with distilled water. A standard reference solution (gallic acid solution) was prepared by dissolving 100 mg of gallic acid in distilled water to a final volume of 100 ml. From this, a serial dilution was performed to obtain solutions of gallic acid at 100, 70, 50, 40, and 10 μg/ml). The stock solutions of the fractions from leaves were prepared by dissolving 100 mg plant extract in distilled water, adjusted to a total volume of 100 ml. Reaction mixtures were prepared by mixing 0.5 ml of each fraction solution with 2.5 ml 10% Folin-Ciocalteu’s reagent, which was dissolved in water with 2.5 ml 7.5% sodium bicarbonate. The sample tubes were incubated for 45 min at 45 °C. Then, the absorbance of each was measured in a spectrophotometer at wavelength 765 nm. The working samples were prepared in triplicate for each analytic trial, from which the mean and standard deviation values were calculated [21].