2.4. Determination in Vitro of Antioxidant Properties

The DPPH free radical scavenging assay was carried out for the evaluation of the antioxidant activity. This assay measures the free radical scavenging capacity of the investigated extract. DPPH is a molecule containing a stable free radical. In the presence of an antioxidant, which can donate an electron to DPPH, the purple color typical for free DPPH radical decays and the absorbance change is measured at 517 nm. The antiradical activity of the plant extract was examined based on the scavenging effect of the stable DPPH free radical activity [23]. Briefly, 2 mL of DPPH (0.1 mM prepared in methanol) was introduced into a test tube containing 0.5 mL of extract (0.1 to 1 mg/mL). Then the mixture was stirred well for 5 min and incubated in the dark for 60 min at room temperature (20°C). For the control tube, methanol was used in place of the extract. The reference used was ascorbic acid at concentrations of 0.1 mg/mL to 1 mg/mL. A calibration curve was drawn from this reference. The antioxidant activity of the plant extract was expressed as a percentage inhibition following the relationship:

The IC₅₀ value (µg/mL) is the effective concentration at which DPPH radicals were scavenged by 50% and the value was obtained by interpolation from linear regression analysis.

The assessment of ferric-reducing antioxidant power (FRAP) was performed based on the ability of the tested substance to reduce ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form (intense blue color) at low pH by using a modified method of Benzie and Strin [24]. The solution of TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) was obtained by diluting TPTZ (10 mM) in 10 ml of HCl (400 mM diluted with distilled water) and 10 mL of 10 mM iron chloride (FeCl₃) solution was prepared in distilled water. FRAP reagent was obtained by mixing 100 mL of acetate buffer (pH 3.6) with 10 ml of TPTZ solution and 10 ml of iron chloride solution. In test tubes containing 2 mL of FRAP reagent, 75 μL of sample (extract/catechin) was added and the mixture was stirred and incubated for 15 minutes. Optical densities were read at the wavelength of 593 nm against white.

To determine ABTS radical scavenging assay, the method described by Re et al. [25] was used. Radical ABTS was obtained as follows: in an Erlenmeyer flask, 0.0384 g of ABTS and 0.00662 g of potassium persulfate (K₂S₂O₈) were weighed and then 10 mL of distilled water was added. The mixture was then solubilized for 5 min and incubated for 16 h at room temperature (20°C) in the dark before use. For the actual analysis, the ABTS solution was diluted with ethanol to an absorbance of 1.3 (±0.02) at 734 nm and stable at 30°C (initial OD). Then in a test tube, 1.8 mL of this diluted ABTS solution and 0.2 mL of extract (1 mg/mL) were introduced and shaken well. The absorbance reading was taken at 734 nm and the values considered were those that remained stable at room temperature for approximately 1 minute. Ascorbic acid was used as the reference antioxidant at the same concentrations as extract. The results were expressed as a percentage of inhibition and calculated according to the following formula: