*2.2. Antibacterial Activity: Minimum Inhibitory Concentration (MIC) Using the Broth Microdilution Method*

The evaluation of the cutaneous antibacterial effect of *O. suffruticosum* essential oil was assessed against three bacterial strains, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Escherichia coli*, in the broth microdilution susceptibility assay. The results were taken as the lowest concentration inhibiting visible bacterial growth as detected by the *p*-iodonitrotetrazolium chloride (INT) reagent and expressed in mg/mL as presented in Table 2.

**Table 2.** MICs (mg/mL) of *O. suffruticosum* EO and control.


\*R=resistant.

The MIC of *O. suffruticosum* EO was detected as 12.8 mg/mL for *S. aureus* and *E. coli*, whereas it was found twice as lower for *P. aeruginosa* and detected as 6.4 mg/mL. According to Van Vuuren [16], essential oils with an MIC ≤ 2 mg/mL can be taken as effective. Therefore, according to these results, *O. suffruticosum* EO may be classified to possess low to moderate antibacterial activity. These findings correlate well with the chemical composition of this EO. Indeed, it is known that the chemical structure of terpenoids parallels their activity [28], whereby the presence of an oxygen function in the framework enhances their antimicrobial properties [29]. The phenol and aldehydes are often characterized by the highest antibacterial activity [30] followed by the alcohols which are usually bactericidal rather than bacteriostatic, then the ketones and the terpene hydrocarbons which have weak activities [29]. In the EO of *O. suffruticosum*, phenols were not detected and only one aldehyde terpene was detected, 2-ethylidene-6-methyl-3,5-heptadienal, as 5.71%. The predominant functional moieties were ketones, alcohols, and terpene hydrocarbons by 78.93% which could explain the lower bacterial inhibitory activity.

#### *2.3. Antioxidant Capacities*

Free radicals chain reactions culminate in oxidative stress when the number of free radicals surpasses the number of systemic defenses, the antioxidants, in the target cell [31]. Oxidative stress in the skin is expressed by blotchy pigmentation, sagging skin, and wrinkles [32]. The strength of the antioxidative potential of *O. suffruticosum* essential oil was evaluated by four in vitro antioxidant capacity assays. The selection of the assays considered covering electron transfer (ET)- and hydrogen atom transfer (HAT)-based mechanisms. The ET-based methods selected were the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Ferric reducing antioxidant power (FRAP) assays although DPPH and ABTS can involve both HAT and ET mechanisms [33,34]. The HAT-based method selected was Oxygen radical absorbance capacity (ORAC) assay [35,36]. The results are summarized in Table 3.

In the DPPH assay, the values of % radical scavenging activity (% RSA) of the essential oils were found to be extremely poor, 10.03 ± 1.02%, 8.38 ± 0.24%, and 7.06 ± 0.20% at 2, 1, and 0.5 mg/mL, respectively. Additionally, they were significantly lower than that of Trolox® positive control found as 94.94 ± 0.02%, 94.78 ± 0.06%, and 94.45 ± 0.04% at the respective concentrations tested. In the ABTS assay, the % RSA's were found to be higher and comparable to the gallic acid positive control. The values were found to be 87.17 ± 0.76% to 71.46 ± 0.04% for the EO vs. 97.97 ± 0.13% to 98.05 ± 0.03% for the positive control in the 2 to 0.5 mg/mL concentration range. The higher performance of the EO in the ABTS assay was expected as ABTS•+ are more reactive than DPPH radicals [34]. However, the difference in antioxidant strength between the EO and gallic acid was evident in the discrepancy in Trolox® equivalent values which were 100-fold higher for gallic acid than those of the EO. Moreover, the EO was found to be −505.8 ± 80.8 μmol (AAE)/L at 2 mg/mL in the FRAP assay against being 635,500 ± 4070.9 μmol AAE/L for gallic acid positive control, and 6701.8 ± 57.2 μmol TE/L in the ORAC assay at the same concentration against 26,904 ± 328.2 μmol TE/L for EGCG positive control. The antioxidant capacity of a substance assesses the amount of antioxidant which reacts with the oxidant [37]. Overall, the EO was found to exhibit a much weaker performance than the reference controls. Therefore, the results indicated that the EO possesses poor to moderate antioxidant capacity.

**Table 3.** Antioxidant capacities of *O. suffruticosum* essential oil in the DPPH, ABTS, FRAP, and ORAC assays.


\* Average values of triplicate measurements (n = 3); RSA: radical scavenging activity; SD = standard deviation; RSD = relative standard deviation; TE: Trolox® equivalent; AAE: ascorbic acid equivalent. \*\* EGCG: (-)-epigallocatechin gallate.
