*3.4. Antioxidant Activity Estimation*

The antioxidant activity of the extracted EO was assessed by its ability to reduce the free radicals 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, Germany) and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich, Germany). According to Miguel [53], a range of concentrations of the EO (50–400 mg L−1) were prepared in MeOH. This range was selected based on the observed scavenging percentage, i.e., to be suitable to determine the IC50 (the concentration of EO necessary to scavenge the radical by 50%) [3]. In glass tubes, 2 mL of each concentration was mixed vigorously with 2 mL of freshly prepared 0.3 mM DPPH. Negative control was performed using MeOH treated with DPPH-like treatment. The reaction mixtures were kept in dark conditions at room temperature for 30 min, and the absorbance was measured immediately at 517 nm by spectrophotometer (Spectronic 21D model).

For confirmation, the scavenging of ABTS was performed following the method of Re et al. [54]. Briefly, the ABTS radical was prepared by mixing about 7 mM ABTS (1/1, *v*/*v*) with 2.45 mM potassium persulfate, and this mixture was stored in dark conditions at 25 ± 2 ◦C for 16 h. In glass tubes, 2 mL of the prepared ABTS radical and 0.2 mL of each EO concentration (50–400 mg L−1) were mixed well and kept for 6 min at room temperature. The absorbance was measured by a spectrophotometer at 734 nm. Moreover, the antioxidant activity of ascorbic acid as a standard antioxidant was determined following the same procedures for DPPH and ABTS using a range of 20–100 mg L<sup>−</sup>1. The scavenging activity was calculated according to the following equation:

$$\text{Racional scanning activity (\%)} = 100 \times \left[1 - \left(\text{Absorbance}\_{\text{sample}} / \text{Absorbance}\_{\text{control}}\right)\right]^{\frac{1}{2}}$$
