NELUMBO NUCIFERA AND INHIBITED TYROSINASE ACTIVITY AND MELANOGENESIS 381 peroxidase antibody for 30 min. After the membrane was washed four times every 15 min, the bends of bound antibodies were detected by enhanced chemiluminescence re- agents, and the images of protein expression were obtained using imaging system (Li-Cor Inc., Lincoln, NE). TRP-1, TRP-2 mRNA ASSAY Total RNA was isolated with the Trizol-Reagent (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer. Reverse transcription reactions were per- formed with SuperScript III reverse transcriptase (Invitrogen) following the manufac- turer’s instructions, using 2 μg of total RNA. Real-time PCR reactions were performed using Taqman gene expression assays by the 7500 Real Time PCR system (AB Applied Biosystems, Foster City, CA). The mRNA expression levels of TRP-1 (assay ID: Mm00453201_m1) and TRP2 (assay ID: Mm01225584_m1) were normalized to Hprt1 (assay ID: Mm00446968_m1) as loading controls. ANALYTICAL HPLC OF JKTM-12 The purity of the two compounds was confi rmed by analytical HPLC using a diode array detector. Analytical HPLC was run on a Supelco discovery C18 reverse phase column, 25 cm × 4.6 mm, 5 μm. The mobile phase consisted of two solvents, water (0.05% formic acid) and acetonitrile. The run started with 100% water and then increased to 100% acetonitrile in 35 min the fl ow rate was 1 ml/min. STATISTICAL ANALYSIS The results were analyzed statistically using one-way analysis of variance (ANOVA), and the least significant differences (p 0.05) were determined according to Duncan’s t-test. Figure 2. Effect of JKTM-12 on tyrosinase activity. Mushroom tyrosinase was incubated with various con- centrations of JKTM-12 and L -DOPA as substrates. Kojic acid was used as positive control. Data are pre- sented as the mean ± SEM of three individual experiments, performed in triplicate. Values are signifi cantly different by comparison with untreated control. *p 0.05, **p 0.001.

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).