JOURNAL OF COSMETIC SCIENCE 382 RESULTS INHIBITION OF TYROSINASE ACTIVITY AND MELANIN BIOSYNTHESIS BY JKTM-12 The effect of the JKTM-12 EtOH extract (JKTM-12) on tyrosinase activity was exam- ined. As shown in Figure 2, JKTM-12 inhibited tyrosinase activity in a dose-dependent manner. Kojic acid is a well known as tyrosinase inhibitor (11), so we used kojic acid as a positive control. Because tyrosinase is the rate-limiting enzyme for melanin, recent stud- ies have demonstrated that the inhibitory activity of compounds toward mushroom ty- rosinase is correlated with their inhibition of melanin synthesis in melanocytes in vitro (8,9,12). In this study, we examined cell viability and melanin biosynthesis by JKTM-12 in B16F10 murine melanoma cells. The cells were incubated with 50, 100, 500, 1000 μg/ml of JKTM-12 for 48 h. Fifty microgram/milliliter JKTM-12 had no cytotoxicity on B16F10 cells. Although 100 μg/ml JKTM-12 slightly decreased cell viability, it was not signifi cant. However, high concentrations of JKTM-12 (500 and 1000 μg/ml) decreased B16F10 cell viability by 67.83% and 57.57%, respectively (Figure 3A). Next, we investi- gated whether JKTM-12 prevented melanin biosynthesis by the indicated concentrations Figure 3. Effects of JKTM-12 on (A) viability and (B) melanin contents in B16F10 cells. (A) Cells were cultured in 96-well plates (1 × 104 cells/well) with the indicated concentrations of JKTM-12 for 24 h and then processed for the analysis of viability. (B) Cells were cultured in 6-well plates (2 × 105 cells/well) with the indicated concentration for 1 h and then exposed to 100 nM α-MSH for 48 h. Data are presented as the mean ± SEM of three individual experiments, performed in duplicate. Values are signifi cantly different by comparison with untreated control (*p 0.05) and only α-MSH-treated control (#p 0.05).

NELUMBO NUCIFERA AND INHIBITED TYROSINASE ACTIVITY AND MELANOGENESIS 383 without cytotoxicity. Indicated concentrations of JKTM-12 (10, 50, and 100 μg/ml) were pretreated in B16F10 cells for 1 h, and then 100 nM α-MSH was added to the cells to stimulate melanin biosynthesis in the cells. After 48 h, melanin level was detected using melanin assay described in the experimental section. Melanin was increased by 100 nM α-MSH treatment in B16F10 cells however, pretreatment of 50 μg/ml and 100 μg/ml JKTM-12 inhibited the melanin level (Figure 3B). Moreover, 100 μg/ml JKTM-12 in- hibited the cellular tyrosinase activity and tyrosinase protein expression by western blot- ting (Figure 6). These result demonstrated that JKTM-12 inhibits melanin biosynthesis without cell cytotoxicity. INHIBITION OF MELANIN BIOSYNTHESIS AND TRP-1, TRP-2 mRNA EXPRESSION BY HYPEROSIDE AND ASTRAGALIN We tested tyrosinase activity using the components of JKTM-12 ethanol extract. Inter- estingly, the receptables of N. nucifera inhibited tyrosinase activity more strongly than the Figure 4. Effects of hyperoside and astragalin on (A) viability and (B) melanin contents in B16F10 cells. Cells were cultured with the indicated concentrations of hyperoside and astragalin and then processed for the analysis of viability and melanin contents. Data are presented as the mean ± SEM of three individual experi- ments, performed in duplicate. *p 0.05 versus the only α-MSH-treated control values.