3.4. Determination of Total Phenolics, Flavonoids Content, and Antioxidant Capacity (DPPH and FRAP) in Aqueous Buffer Extracts

Total phenolics, total flavonoids, and antioxidant capacity were measured in the aqueous extracts (in triplicate, twice) with methods adapted for 96-well plates, and the absorbance was measured in a UV/vis microplate reader (Sunrise, Tecan Austria) against blanks.

Total phenolic content was determined with the Folin–Ciocalteau reagent method [57,58] at 620 nm and is expressed as mg of gallic acid equivalents (GAE) per g of dry weight (D.W.) of plant material.

Total flavonoid content was determined with the aluminum chloride (AlCl₃) method [59] at 405 nm and the results are expressed as mg of quercetin equivalents (QE) per g of D.W. of plant material. In detail, 75 µL of ddH₂O were mixed with 5 µL of CH₃COOH 1Μ, 16 µL extract or standard (quercetin) in ethanol 60% (v/v), 40 µL ethanol 95% (v/v), and 5 μL of AlCl₃ 10% w/v incubated at RT for 45 min.

The antioxidant activity was evaluated with two different assays—ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The FRAP method [60] measures the ability of antioxidants to reduce the [Fe_III(TPTZ)₂]³⁺ to [Fe_II(TPTZ)₂]²⁺. In detail, 80 µL of FRAP solution (15 mL of a solution of 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl, 15 mL of 20 mM FeCl₃.6H₂O, and 75 mL of 300 mM acetate buffer solution, pH 3.6), were mixed with 55 µL of acetate buffer and 60 µL extract or standard (FeSO₄.7H₂O) and incubated at room temperature for 5 min. Absorbance was measured at 592 nm and the results are expressed as mmol Fe²⁺ per g of D.W.

In addition, the antioxidant activity was determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [61,62], which measures the ability of antioxidants to scavenge the stable organic nitrogen radical DPPH•. Absorbance was measured at 540 nm and the results are expressed as IC₅₀ g of D.W. of plant material. In detail, 195 µL of 0.1 mM DPPH (in methanol) were mixed with 5 µL of extract or BHT (butylated hydroxytoluene) used as standard (both diluted in methanol 50% v/v) and incubated at RT for 30 min.