3.2.3. The Determination of the Total Flavonoids Content (TFC)

The assessment of total flavonoid content in the analyzed extracts was performed spectrophotometrically using aluminum nitrate nonahydrate by method adopted by Matejić et al. with modifications [79]. According to this method and procedure described by Nizioł-Łukaszewska et al. [80], a reaction mixture was prepared containing 80% C₂H₅OH, 10% Al(NO₃)₃ × 9 H₂O and 1M C₂H₃KO₂. Then, 2400 μL of the previously prepared reaction mixture was mixed with 600 μL of tested extract sample in various concentrations. The deionized water was used for dilution and a control sample. After incubation for 40 min at room temperature, the absorbance was measured at λ = 415 nm with UV/VIS spectrophotometer AquamateHelion (Thermo Scientific, Waltham, MA, USA). Quercetin was used as a standard for calibration curve and the results were expressed as Quercetin equivalents (Qu/g) of extract averaged from three independent measurements.