JOURNAL OF COSMETIC SCIENCE 76 EFFECT OF SESAMOL ON CELL CYTOTOXICITY To test the possibility of a clinical use for sesamol as a skin application, the cytotoxicity of sesamol was investigated in vitro in both normal (Vero) and melanoma (SK-MEL2) cell lines. The Vero cell line was used to represent normal cells. Cytotoxicity was evaluated using a colorimetric NR assay (20). Viable cells accumulate and bind neutral red within the lysosomes, but dead cells cannot because of the fragility of the lysosomal membrane and irreversible molecular alterations in the dead cells (21). Figure 3 shows that sesamol barely affected cell viability. Although as much as 800 μg/ml (5792 μM) sesamol was used, the cytotoxicity to Vero cell was only 22% after 48 h treatment. Similar non- cytotoxicity to the Vero cell line was observed for the positive control β-arbutin and kojic acid, which possessed a cytotoxicity of only 4% and 7%, respectively. The SK-MEL2 cell line was used to represent melanocytes. The cytotoxicity of the test compounds on the growth of SK-MEL2 was found to be non-cytotoxic when using concentrations up to 400 μg/ml (Figure 3). A 40% cytotoxicity was observed in the sesamol-treated SK-MEL2 cell line at concentration of 600 μg/ml (4344 μM) (Figure 3), while β-arbutin and kojic acid did not possess any cytotoxicity. Sesamol thus possesses a cytotoxicity IC50 value of 4406.5 μM in the SK-MEL2 cell line (Figure 3). Notwithstanding, the concentration used to create the tyrosinase inhibition effect in vitro was much lower than the concentra- tion used in this cellular assay. Sesamol therefore may not be cytotoxic at the effective whitening concentrations used in the melanoma cell line. Further experiment on the normal melanocyte cells should be conducted to confi rm non-toxicity of sesamol. Figure 2. Inhibition effects of kojic acid sesamol and β-arbutin on cellular tyrosinase enzyme. Data are pre- sented as mean ± S.D. (n = 3). Figure 3. Effect of sesamol, kojic acid and β-arbutin on cytotoxicity of the SK-MEL2 and Vero cell lines after 48 h treatment. Data are presented as mean ± S.D. (n = 3).

WHITENING AND ANTIAGING EFFECT OF SESAMOL 77 DISCUSSION Oxidative stress is induced by various factors leading to oxidation of biomolecules (e.g., lipids, proteins, and DNA) and pathological status (22). Under certain physiological conditions, when the level of ROS increases more than the intracellular antioxidant ca- pacity, oxidative stress will occur. The tyrosinase enzyme in melanogenesis also increases the oxidative risk in physiological systems (23). Therefore, antioxidant is benefi cial for antiaging. Our study demonstrates that sesamol can play an important role as an anti- oxidant because it possesses radical scavenging, lipid peroxidation prevention, and reduc- ing power. Our results confi rm the radical scavenging of sesamol as reported by Suja et al. (24) and Hayes et al. (25). Moreover, sesamol has the strongest lipid peroxidation preven- tion among our tested compounds. Occurring in cell membranes, lipid peroxidation re- leases arachidonic acid, which is involved in the infl ammatory response (26). Sesamol can hereby protect the cell membrane which is composed of a lipid bilayer from lipid peroxi- dation and might also prevent the infl ammatory process from decreasing arachidonic acid. Chen and Ahn (27) reported that sesamol inhibited lipid oxidation in UV-induced lipid oxidation in the following order: quercetin rutin = caffeic acid = ferulic acid = sesamol catechin. The advantage of a reducing ability is the capacity to neutralize free radicals and to stabilize and stop harmful chain reactions (17). In this study, sesamol pos- sessed the reducing ability according to the results of the FRAP assay. Melanogenesis is a natural mechanism—the product of melanocytes. Melanin is a pigment for preventing UV-induced skin damage and that acts as a photoprotectant (28). The abnor- mal accumulation of melanin leads to hyperpigmentation which is a main concern in cos- metics. Anti-tyrosinase agents are therefore important ingredients in cosmeceuticals for skin whitening. Skin aging can be classifi ed as intrinsic and photo albeit both processes induce skin wrinkling. UV radiation generates ROS that can induce a transcription factor that promotes collagen destruction by upregulating enzymes, matrix metalloproteinases (MMPs). These MMPs induce collagen destruction, resulting in wrinkle formation. Laxity and fragility of the skin are also caused by ROS-activated hyaluronidase and elastase, which block hyaluronan and elastin formation, respectively (29). Since ROS causes photo and in- trinsic aging, protection from UV radiation and antioxidant homeostasis is crucial. Based on the results of this study, sesamol can supplement antioxidants, which can inhibit ROS via multiple mechanisms, and it acts as photoprotection. So, sesamol can be successfully used against the skin wrinkling associated with photoaging. In this study, cellular toxicity was determined in the human melanoma cell line (SK- MEL2) and the normal (Vero) cell line by using the NR assay after treating them with various compounds for 48 h. The known whitening agents—kojic acid and β-arbutin— and sesamol had negligible cytotoxicity on the SK-MEL2 and Vero cell lines. It should be noted that the concentration of sesamol that can inhibit cellular tyrosinase was much lower than the cytotoxic concentration. This study demonstrated that sesamol inhibits tyrosinase activity against mushroom ty- rosinase and human melanoma tyrosinase in a dose-dependent manner. These data were found to be in agreement with the previous studies regarding mushroom tyrosinase inhi- bition of sesamol (30). Tyrosinase inhibition of sesamol at the fi rst and second step of melanin biosynthesis is reportedly due to competitive and non-competitive inhibition, respectively (31). β-Arbutin was previously reported to have no inhibition effect on mush- room tyrosinase but had an inhibition effect against melanoma tyrosinase (32). It has