*2.9. Total Phenolics and Antioxidant Activity of the Essential Oil*

Total phenolics in the EOs was determined using the Folin-Ciocalteu method, as described previously [26]. Results were expressed as milligrams of gallic acid equivalents per gram of EO.

The DPPH assay was used to evaluate the scavenging activity of the EO as described by Oke et al. [27]. A volume of 400 μL of serial EO dilutions (0–100 μg/mL) or of the methanol control was mixed with 100 μL of 0.2 mM DPPH solution (in methanol). The mixture was incubated in the dark for 30 min and the absorbance was measured at 517 nm. Butylatedhydroxytoluene (BHT) was used as a positive control. Results were expressed as IC50 values.

The 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) method was also assayed [28]. The ABTS radical was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulfate and incubated for 16 h in the dark. The solution was then diluted with 80% methanol until the absorbance at 734 nm was 0.700 ± 0.02. Therafter, 1 mL of the diluted ABTS solution was mixed with 100 μL of the EO dilutions (0–100 μg/mL) and dilutions of the positive control, ascorbic acid (0–250 μg/mL). The absorbance was recorded after 5 min at 743 nm. The ABTS radical scavenging activity was expressed as IC50 values.

The ability of the *S. perfoliata* oil to reduce Fe3<sup>+</sup> was performed according to Bettaieb Rebey et al. [29]. Dilutions of essential oils (0–100 μg/mL) and ascorbic acid (0–250 μg/mL) as positive control, were mixed with 500 μL of 200 mM sodium phosphate buffer (pH 6.6) and 500 μL of 1% potassium ferricyanide (K3Fe(CN)6). The mixture was incubated at 50 ◦C for 20 min. Then 500 μL of 10% trichloroacetic acid was added and the reaction was centrifuged at 650× *g* for 10 min. A volume 500 μL of the supernatant was then mixed with 500 μL of distilled water and 100 μL of 0.1% ferric chloride. The absorbance was then measured at 700 nm. The EC50 (effective concentration) was defined as the concentration at which the absorbance was 0.5 for the reducing power.

The EO was tested for the bleaching of β-carotene in linoleic acid system, by measuring the coupled autoxidation of β-carotene and linoleic acid [30]. Briefly, 5 mg of β-carotene were dissolved in 50 mL of chloroform. A 3 mL volume of the mixture was added to 40 mg of linoleic acid and 400 mg of Tween 20. Chloroform was removed using nitrogen gas, after which 100 mL of distilled water was added to the emulsion and mixed well. Thereafter, 1.5 mL of the emulsion was added to a test tube containing 20 μL of the essential oil or BHT. Absorbance was measured immediately (t = 0) at 470 nm, after an incubation time of 1 h in a water bath at 50 ◦C (t = 60). The antioxidant activity was expressed as percentage inhibition compared with the negative control (0.02 mL of methanol instead of EO) using the following equation:

$$\text{Antioxidation activity } (\%) = 100 \times \frac{\left(\text{DR}\_{\text{C}} - DR\_{\text{S}}\right)}{\text{DR}\_{\text{C}}}$$

where DRc is the degradation rate of the control, DRs is the degradation rate of the sample. DRC and DRS were calculated using the following equation:

$$\text{Depradation rate (DR)} = \left\lceil \frac{(\ln a/b)}{60} \right\rceil$$

where a = absorbance at t = 0 and b = absorbance at t = 60 min.
