*2.4. Tyrosinase Inhibition*

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase, is a copper-containing enzyme that has a central role in the production of melanin, the pigment responsible for the color of the skin. It catalyzes the first two steps of the multiphase process of melanogenesis, the biosynthesis of melanin. Today, tyrosinase inhibitors are increasingly prevalent cosmeceuticals' ingredients aiming to treat hyperpigmentation problems caused by the overproduction of melanin in the skin [38]. In the present work, the *O. suffruticosum* essential oil was tested in the tyrosinase inhibition assay exploring the monophenolase activity of the enzyme by monitoring the absorbance of L-DOPA (λ<sup>490</sup> nm) using L-tyrosine as a substrate. The essential oils were tested at 200 μg/mL and 50 μg/mL and compared to kojic acid, a standard tyrosinase inhibitor used in cosmetics, at the same concentrations. The results were obtained as presented in Table 4.

**Table 4.** Summary of the tyrosinase inhibition assay results of *O. suffruticosum* EO at 200 μg/mL and 50.


*O. suffruticosum* EO exhibited significantly lower tyrosinase inhibition values than kojic acid at both concentrations tested. At 200 μg/mL, the EO was found to be 61.46 ± 11.00% against 96.24 ± 3.62% for kojic acid and at 50 μg/mL, the EO was found to be 26.14 ± 3.74% against 98.34 ± 0.80% for kojic acid. These values indicate that the enzyme inhibition is concentration dependent, and *O. suffruticosum* EO is a relatively weak tyrosinase inhibitor.

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

Solar UV rays are recognized as the main contributor to extrinsic cutaneous aging in humans [39–41]. Chronic exposure to ultraviolet radiation (UVR) induces various dermatological problems including skin cancer [42]. Herein, the SPF of *O. suffruticosum* essential oil was determined by measuring the absorbance of a dilute hydroalcoholic solution of EO (0.1% *v*/*v*) at 290–320 nm at 5 nm interval then calculated using the equation given by Mansur et al. [43]. The results are presented in Table 5.

**Table 5.** Spectrophotometric absorbances of hydroalcoholic aliquots of *O. suffruticosum* essential oil and its calculated SPF.


\* Values represent mean absorbance values ± standard deviation of triplicate measurements, n = 3; \*\* constant values erythemogenic effect (EE) of radiation with wavelength λ x solar intensity (I) at wavelength λ determined by Sayre et al. [44].

According to the study, the essential oil of *O. suffruticosum* was found to possess an SPF value of 2.299. It has been reported that an SPF value above 2 is noteworthy [45,46]. Such a substance may block UV radiation by approximately 57% [45–47]. Therefore, the results establish *O. suffruticosum* EO as an important cosmeceutical for sunscreen formulation.

In an attempt to compare the biological activities of the plants which are rich in the major components found in the *O. suffruticosum* EO, the essential oils of *Cinnamomum camphora*, *Artemisia herba-alba*, *Eucalyptus globulus*, and *Melaleuca alternifolia* were selected as representative examples with camphor, chrysanthenone, 1,8-cineole, and terpinen-4-ol as respective major components. The results from the literature search indicated that mainly these essential oils have been studied for their antibacterial and antioxidant properties and they showed variable degree of activities.

The *C. camphora* essential oil contains camphor as the main constituent. The sample collected from Pantnagar, India, was effective against *Pasturella multocida* but not against *Salmonella enterica enterica* and *Escherichia coli* [48]. *C. camphora* oil from Nepal also showed marginal activity against *B. cereus* and *S. aureus*, with a MIC = 313 μg/mL [49]. During an antioxidant study by the DPPH assay, the IC50 value of the *C*. *camphora* essential oil was found to be 31.85 μL/mL, whereas that for the reference butylated hydroxytoluene (BHT) was reported to be 7.6 μg/mL [50].

Essential oil of *A. herba-alba* from Makther Seliana, Tunisia having camphor and chrysanthenone as major components displayed MIC (μg/mL) values of 100, 50, and >100 against *S. aureus*, *E. coli*, and *P. aeruginosa*, respectively [51]. The antioxidant activity of *A. herba-alba* EO by DPPH assay showed an IC50 of 2.66 μg/mL whereas that for the synthetic antioxidant butylated hydroxyanisole (BHA) was 1.66 μg/mL [52]. At a concentration of 1 mg/mL, the *A. herba-alba* EO exhibited a tyrosinase inhibition of 31.35%, which was much lower than that of the standard inhibitor kojic acid (87.54% at 0.05 mg/mL) [53].

EOs of *E. globulus* collected from Skoura, Morocco, presented excellent activity on *E. coli* in the agar disc diffusion assay with inhibition zone diameter (izd) = 48.15 mm compared to *S. aureus* (izd = 13.5 mm) and *S. intermedius* (izd = 10.26). The MIC for *E. coli* corresponded to 0.15 mg/mL while for *S. aureus* and *S. intermedius* the values corresponded to 0.75 mg/mL and 1.08 mg/mL, respectively [54]. The main component of *E. globulus* EOs is 1,8-cineole and it has been demonstrated that this compound has antimicrobial activity against several microorganisms including *S. aureus* and *E. coli* [55]. In an antioxidant study of this plant with the DPPH method, its methanolic extract exhibited the strongest free radical-scavenging activity with an IC50 value of 23 μg/mL, followed by the ethyl acetate extract (IC50 = 29 μg/mL) and hexane extract (IC50 = 65 μg/mL). However, the essential oil did not show any noticeable activity with the DPPH method [56]. This activity may be attributed to the high content of phenolic compounds (542.42 mg GAE/g) in methanol extract from *E. globulus*.

Essential oil from *M*. *alternifolia* is referred to as tea tree oil, the major component of which is terpinen-4-ol present at least 30% of total oil [57]. *M. alternifolia* essential oil obtained from a commercial source in Germany inhibited the growth of *S. aureus*, *E. coli,* and *P. aeruginosa* at a concentration of 5% *w*/*v* [58]. An antioxidant activity study by the DPPH method indicated that *M. alternifolia* EO at a concentration of 10 μL/mL produced 80% free radical scavenging activity which was equivalent to that of 30 mM BHT [59].

No tyrosinase inhibition studies are reported for essential oil of these plants except *A. herba-alba*. There was also no report in the literature regarding the SPF studies of the essential oils of these plants. As per the above discussed results from the literature, no direct correlation could be ascertained among the biological activity and the major component of the essential oil, suggesting that the biological activity of the essential oils is because of the synergism among the components of the essential oil rather than any one of the major constituents.

#### **3. Materials and Methods**

#### *3.1. Plant Material*

The plant material (3.0 kg) was wildly harvested from the Cape Flats Nature Reserve of the University of the Western Cape in December 2018. A voucher specimen was authenticated by Hlokane Mabela and deposited at the Horticultural Sciences Department of the Cape Peninsula University of Technology.
