4.4. Total Phenolic, Flavonoid Content and DPPH Assay

The total phenolic content (TPC) of each extract was evaluated according to the previous method [49,50]. Each extract was prepared at 1 mg mL⁻¹ concentration. 3.16 mL of distilled water, 1 mL of methanol and 200 μL of Folin–Ciocalteu reagent were added to 300 μL of this solution taken into a tube. Then, after incubation at room temperature, 600 μL of a sodium carbonate solution was added and the tube was covered with aluminium foil and incubated in a water bath at 40 °C for 30 min. A blank was prepared using the same procedure, but using an equal volume of methanol instead of the plant extract. The absorbance of the extracts was determined at 765 nm. The standard curve of Gallic acid was obtained using the same procedure. The total flavonoid content (TFC) of each extract was evaluated using a previous protocol [51]. In a test tube, first 300 µL of extract, 3.4 mL of methanol (30%), then 150 µL of sodium nitrite solution (0.5 M) and after 150 µL of aluminium chloride solution (0.3 M) were added. After 5-min incubation, 1 mL of sodium hydroxide solution (1 M) was added and the contents mixed well. Afterwards, measurements were made against the blind tube at a wavelength of 506 nm. Results were calculated as rutin equivalents. The radical scavenging activity of the extracts was measured by means of the DPPH test. DPPH analysis was performed according to the Chu method with minor modifications [50,52]. The extracts were added to 0.01% DPPH at various concentrations (0–1 mg/mL) and incubated at room temperature for 30 min. Absorbance was measured at 490 nm and the DPPH radical scavenging activity was calculated as IC₅₀ values for each extract.