3.6.2. Ferric Reducing Antioxidant Power Test (FRAP Assay)

The antioxidant activity of the crude extracts, expressed as mg Fe²⁺ ion equivalent per gram of dry weight (mg Fe²⁺ g⁻¹ DW) and essential oils, expressed as mg Fe²⁺ ion equivalent per milliliter of essential oil (mg Fe²⁺ mL⁻¹ EO), was determined by a standard spectrophotometric method with slight modifications [77]. In short, the daily fresh reagent FRAP was prepared by mixing acetate buffer (pH = 3.60), 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl₃ 6H₂O in the volume ratio 10:1:1 at 37 °C [78]. Working solutions of Fe²⁺ ions in the concentration range 10–300 mg L⁻¹ were prepared by diluting 20 mM Fe²⁺ standard solution with ultrapure water. For the measurements, 4950 µL of the reagent FRAP was mixed with 50 µL of properly diluted crude extracts/properly diluted essential oil or Fe²⁺ working solutions or properly diluted crude extract or properly diluted essential oil. After 30 min of incubation at 37 °C, the absorbances at 593 nm were measured against FRAP reagent as a blank value.