*3.5. Antioxidant Capacity Assays*

The antioxidant capacity of the *O. suffruticosum* EO was studied by the following four antioxidant assays to cover both HAT and ET mechanisms.

#### 3.5.1. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay

The DPPH assay was performed according to the method previously described by Bondet et al. [65] with slight modifications. In a clear 96-well plate, 275 μL of DPPH reagent (Sigma-Aldrich®, Cat no. D9132) (absorbance of 2.0 ± 0.1 at 517 nm) was added to 25 <sup>μ</sup>L of EO sample and Trolox® (Sigma-Aldrich®, Cat no. 238831) positive control (2.0, 1.0, and 0.5 mg/mL). For the blank, ethanol was added instead of the sample. The total volume of the assay was 300 μL. The absorbance was read at 517 nm and 37 ◦C at the 6 min time point. The EO/Trolox® sample was read in triplicate (n = 3). The percentage radical scavenging activity (% RSA) of the samples was calculated using Equation (1).

$$\% \, RSA\_{6\text{ }min} = 1 - \frac{Abs\_{sample}}{Abs\_{blank}},\tag{1}$$

where *Abssample* is the absorbance signal of the EO sample and *Absblank* is the absorbance signal of the DPPH solution (ethanol in place of the sample) at 517 nm after 6 min. The results were expressed as the mean percentage of triplicate measurements (±standard deviation, SD).

#### 3.5.2. 2,2 -Azino-bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) Assay

The ABTS assay was performed according to Re et al. [66] with slight modifications. The ABTS radical cation (ABTS•+) (Sigma-Aldrich®, Cat no. A1888) stock reagent was produced by reacting 5 mL of freshly prepared 7 mM ABTS solution with 88 μL of a freshly prepared 140 μM K2S2O8 (Merck, Cat no. 105091) then allowing the mixture to sit overnight for 16 h in the dark at room temperature. In a clear 96-well plate, 275 μL of ABTS•+ reagent (absorbance of 2.0 ± 0.1 at 734 nm) was added to 25 μL of each ethanolic Trolox® working standard (50 μM, 100 μM, 150 μM, 250 μM, and 500 μM) and EO sample (2.0, 1.0, and 0.5 mg/mL). Gallic acid (Sigma-Aldrich®, Cat no. G7384) was used as a positive control. For the blank, ethanol was added instead of the sample. The total volume of the assay was 300 μL. The absorbance was read at 734 nm and 37 ◦C at the 6 min time point. The EO sample, working standard, and gallic acid sample were read in triplicate (n = 3). The percentage of radical scavenging activity (% RSA) of each EO or positive control working solution was calculated using Equation (1), where Abssample is the absorbance signal of the EO sample/positive control and Absblank is the absorbance signal of the ABTS•+ solution (ethanol in place of the sample) at 734 nm. The results were expressed as the mean percentage of triplicate measurements (±standard deviation, SD). The Trolox® equivalent capacity assay (TEAC) values were reduced from the linear regression (R<sup>2</sup> = 0.9980) of Trolox® concentrations (μM) and the absorbance readings at 734 nm at 6 min and expressed as mean (±SD) of triplicate measurements in <sup>μ</sup>mol Trolox® equivalents per liter of the sample tested (μmol TE/L).
