Model 2600

Total flavonoid content was measured using a UV-visible spectrophotometer by the method of Zhishen et al. [39 (link)]. The diluted extract (1 mL) was placed in a 15 mL tube with 4 mL of distilled water and the mixture homogenized. Next, 0.3 mL of 5% sodium nitrite (w/v) and 0.3 mL of 10% aluminum chloride (w/v) were added; the sample was left to rest for 5 min after the addition of each reagent. Finally, 2 mL of 1N NaOH was added to distilled water to a volume of 10 mL. The absorbance was measured in the pink chromophore at 490 nm using a UV-VIS spectrophotometer, model 2600 (Shimadzu, Kyoto, Japan). Five extraction cycles were required to recover 100% of the flavonoid content. The quantification was performed by the interpolation of the corresponding absorbance value of each sample on a calibration curve made with (+)—catechin, 0–100 mg (+)—catechin/L. Results were expressed as mg of catechin equivalents per gram of dry sample (mg catechin g⁻¹ DW).

Ferric reducing power (FRAP) was also measured to estimate the AA [38 (link)]. The diluted extract (1 mL) was placed in a 15 mL test tube and 2.5 mL of phosphate buffer at pH 6.6 and 2.5 mL of a 1.0% potassium ferrocyanide solution were added. The mixture was shaken and incubated at 50 °C for 20 min, then 2.5 mL of 10% trichloroacetic acid with 2.5 mL of water and 0.5 mL of 1% FeCl₃ were added. The mixture was homogenized in a vortex (Mistral Multi-Mixer, Melrose Park, IL, USA). The solution was rested for 30 min in the dark and a green complex (ferrous chloride–potassium ferrocyanide) was formed. The absorbance was measured at 700 nm using a UV-VIS spectrophotometer, model 2600 (Shimadzu, Kyoto, Japan). The AA and results were estimated as for the ABTS method.

Total polyphenol content quantification was carried out by UV-visible spectrophotometry [38 (link)]. The diluted extract (1 mL) was placed in a 15 mL test tube and 6 mL of distilled water and 1 mL of Folin–Ciocalteau reagent added, then the mixture was rested for 3 min. After that, 2 mL of 20% Na₂CO₃ (w/v) was added and heated to 40 °C for 2 min. The absorbance of the blue chromophore was measured at 760 nm using a UV-VIS spectrophotometer, model 2600 (Shimadzu, Kyoto, Japan). Five extraction cycles were necessary to obtain total polyphenol content recovery. Quantification was carried out by the interpolation of the corresponding absorbance value of each sample on a calibration curve made with gallic acid at 0–100 mg gallic acid/L. Results were expressed as mg of gallic acid equivalents per gram of dry sample (mg GAE g⁻¹ DW).