Quantification of Total Flavonoids

TF was quantified by UV-visible spectrophotometry, according to [57 (link)]. A volume of 1 mL of the diluted extract was placed in a 15 mL tube; 4 mL of distilled water was added, and the mixture was homogenized. Then, 0.3 mL of 5% sodium nitrite (w/v) and 0.3 mL of 10% aluminum chloride (w/v) were added, and allowed the sample to stand for 5 min after the addition of each reagent. Finally, 2 mL of 1N NaOH was added, and the volume was made up to 10 mL with distilled water. This reaction formed a pink chromophore, its absorbance was measured at 490 nm with a Shimadzu UV-VIS model 2600 spectrophotometer (Shimadzu, Kyoto, Japan). Five extraction cycles were needed to reach 100% TF recovery. Results are expressed as milligrams of catechin equivalents per gram of dry sample (mg cat g⁻¹ DW).