3.5.4. Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was conducted as recommended by Benzie and Strain [68] with slight adjustments. Firstly, the fresh blue FRAP reagent was achieved by mixing 30 mL of acetate buffer, 3 mL of 2,4,6-tris[2-pyridyl]-s-triazine (TPTZ) (Merck, Cat no. T1253) with 3 mL of FeCl3 solution and 6.6 mL of distilled water. Then, an L-ascorbic acid (Sigma-Aldrich®, Cat no. A5960) standard series of 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM was

prepared from a 1 mM of L-ascorbic acid stock in distilled water. Lastly, in a clear 96-well plate, 300 μL of the FRAP reagent was added to 10 μL of L-ascorbic acid working standard solutions and EO sample (2.0 mg/mL) in triplicate (n = 3). Gallic acid was used as a positive control. For the blank, the phosphate buffer (pH 3.6) was added instead of the sample. The total volume of the assay was 310 μL. The absorbance of TPTZ-Fe (II) in the samples was read at 593 nm at 37 ◦C for 30 min. The results were calculated using the linear regression (R2 = 0.9965) of the L-ascorbic acid (AA) standard series concentrations (μM) and absorbance signals expressed as mean (±SD) of triplicate measurements in μmol L-ascorbic acid equivalents per liter of the sample tested (μmol AAE/L).

#### *3.6. Antityrosinase Assay*

3.6.1. Essential Oils Samples and Positive Control Preparation

A total of 10 mg/mL of EO working solution was prepared with a DMSO: Tween®20 (1:1) solution to facilitate dispersion of the oils then further diluted to 1 mg/mL working solutions with methanol. A 10 mg/mL kojic acid working solution was made up with 100% DMSO and then diluted to 1 mg/mL with methanol.

### 3.6.2. Tyrosinase Inhibition Assay

The tyrosinase inhibition assay was performed as described previously by Popoola et al. [69] and Cui et al. [70] with slight modifications. The concentrations of EO sample and kojic acid chosen, 200 μg/mL and 50 μg/mL, were achieved by setting up the 96-well plate in the following order: 70 μL of the sample (1 mg/mL) then 30 μL of tyrosinase enzyme (500 U/mL). Each concentration of the sample and positive control was set up in two different wells, whereby one of the wells received enzyme and the other well had no enzyme volume added. All volume deficits were compensated by adding excess buffer. The negative controls, 10% *v*/*v* of 1:1 DMSO: Tween®20 in methanol for the EO and 10% *v*/*v* DMSO in methanol for kojic acid were treated the same way. Then, the plate was incubated at 37 ◦C (±2.0 ◦C) for 5 min. Thereafter, the reaction was initiated by adding 110 μL of L-tyrosine (2 mM) and subsequently incubated at 37 ◦C (±2.0 ◦C) for 30 min. The absorbance of L-DOPA was read at 490 nm on a Multiskan™ spectrum plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Two independent experiments were carried out in triplicate and the percentage tyrosinase inhibition was calculated using Equation (2).

$$\text{Tyrosinase inhibition (\%)} = \frac{(A - B) - (C - D)}{(A - B)} \times 100,\tag{2}$$

where *A* is the negative control with an enzyme, *B* is the negative control without enzyme, *C* is the EO sample or kojic acid with enzyme and *D* is the EO sample or kojic acid without enzyme. The inhibition percentages were expressed as the mean (±standard deviation) of duplicate measurements. One-way ANOVA was used to compare the absorbance values of the two groups (*p* < 0.05).

#### *3.7. Sun Protection Factor (SPF)*

The protocol used for this assay was conducted as per Kaur and Saraf [71]. The solubility of the EO in different ratios of ethanol and water was tested by taking 10% to 50% of ethanol in distilled water. The maximum solubility was detected at ethanol: water in a 40:60 ratio above which turbidity developed. Thereafter, an initial stock solution of 1% *v*/*v* was prepared by making up 10 μL of the EO to 1 mL of ethanol: water (40:60). Then out of this stock, 0.1% *v*/*v* was prepared. Subsequently, 100 μL of the EO aliquot and the blank (ethanol: water, 40:60) were injected in the 96-well plate and read in triplicate (n = 3) over the 290–320 nm range at 5 nm interval. The SPF value of the essential oil was calculated following the method by Mansur et al. [43]. The mean of the observed absorbance values was multiplied by their respective erythemogenic effect times solar

intensity at wavelength *λ* values, *EE* (*λ*) × *I* (*λ*), then their summation was obtained and multiplied with the correction factor (=10). The calculation is described as Equation (3).

$$\text{SPF}\_{\text{spectrophic} \text{mometric}} = \text{CF} \times \sum\_{290}^{\\$20} \text{EE}\left(\lambda\right) \times I\left(\lambda\right) \times A \text{bs}\left(\lambda\right), \tag{3}$$

where *CF* is the correction factor (=10), *EE* (*λ*) is the erythemogenic effect of radiation at wavelength *λ*, *I* (*λ*) is the solar intensity at wavelength *λ,* and *Abs (λ*) represents the spectrometric absorbance value at wavelength *λ*. The values of *EE* (*λ*) × *I* (*λ*) are constant values that were determined by Sayre et al. [44] as shown in Table 6.


**Table 6.** Relationship between erythemogenic effect and radiation intensity.

#### **4. Conclusions**

The present work is the first report to investigate the chemical composition of *O. suffruticosum* essential oil and its biological activities to explore its cosmeceutical potential in selected biological activities of dermatological relevance. The GC-MS analysis served to identify sixteen constituents (**1**–**8**, **11**–**15**, **17**, **18**, **20**) totaling 85.09% of the composition. The monoterpenoids predominated the chemical composition of the essential oil by 84.64%. The major compounds were found to be ketone and alcohol monoterpenes, camphor (**12**) 31.21%, filifolone (**8**) 13.98%, chrysanthenone (**11**) 8.72%, 1,8-cineole (**6**) 7.85%, and terpinen-4-ol (**14**) 7.39%. According to the in vitro biological evaluations conducted, *O. suffruticosum* essential oil possessed low tyrosinase inhibitory activity, low to moderate antibacterial and antioxidant activity, but a promising sun protection ability as per the calculated SPF value. It is further proposed that the therapeutic properties of this essential oil can be improved by the application of nanotechnologies such as nanoencapsulation and nanostructured lipid carriers. This study establishes that the *O. suffruticosum* essential oil can be used as a complementary ingredient to boost the performance of cosmeceuticals with a prominent potential to be used in sunscreen formulations.

**Author Contributions:** All the authors have participated and contributed substantially to this manuscript. S.O.A.: methodology, investigation, data curation, writing the original draft. R.S.: supervision, validation, writing—review & editing. C.W.J.A.: methodology, validation, resourcesbiology-antioxidants. J.L.M.: methodology, validation, resources—biology-antioxidants. A.A.H.: conceptualization, methodology, supervision, resources, writing—review & editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by National Research Foundation, South Africa (grant number 106055), and "The APC was funded by the Cape Peninsula University of Technology and University of the Western Cape".

**Acknowledgments:** Thanks to the Cape Flat Natural Reserve for the collection of the plant materials.

**Conflicts of Interest:** The authors declare no conflict of interest.
