2.4. Determination of total flavonoid content and total phenolic content

The total phenolic content (TPC) of the A. occidentale leaf extract and its fractions was measured using a modified Folin–Ciocalteu method (Encarnação et al., ²⁰¹⁶) with some minor modifications. Briefly, 1 mL of the extract was mixed with 7 mL of diluted Folin–Ciocalteu reagent (1:10, v/v, in water) and 2 mL of 7.5% sodium carbonate. The reaction mixture was then incubated at room temperature for 1 h before measuring the absorbance at 765 nm using a spectrometer (V‐730 UV–Vis Spectrophotometer, Jasco). Gallic acid (50–250 μg/mL) was used to establish a standard curve (y = 0.003x – 0.0134; R ²  = .9982) for the calculation of the TPC. The TPC was expressed as milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM).

The total flavonoid content (TFC) of the crude extract and its fractions was estimated using a modified spectrophotometric method (Medina‐Medrano et al., ²⁰¹⁹). Briefly, the extracts (2 mL) were mixed with 5% AlCl₃ (0.5 mL) and 1 M potassium acetate solution (0.5 mL). The mixture was then incubated at room temperature for 15 min before measuring the absorbance at 415 nm using a V‐730 UV–Vis spectrophotometer (Jasco). Quercetin (20–100 μg/mL) was used as a standard to establish a calibration curve (y = 0.01x + 0.017; R ²  = .9967) for the calculation of the TFC. The TFC was shown as milligrams of quercetin equivalents per gram of dry matter (mg QE/g DM).