NELUMBO NUCIFERA AND INHIBITED TYROSINASE ACTIVITY AND MELANOGENESIS 379 were purchased from Cell Signaling Technology (Denver, MA). Anti-mouse horseradish peroxidase-conjugated immunoglobulin G (IgG) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ECL solution was purchased from Millipore Cor- poration (Billerica, MA). All other chemicals and solvents used in this study were of the analytical grade. PREPARATION OF JKTM-12 N. nucifera was harvested from the Muan-Gun area, South Korea, in August 2010 to Feb- ruary 2011. JKTM-12 (240 g) was composed of the fl owers (46 g), seeds (54 g), roots (60 g), and receptacles (80 g) of N. nucifera, respectively. JKTM-12 was prepared by boiling at 70°C with 70% EtOH for 3 h three times. It was then fi ltered and concentrated with a vacuum evaporator (N-3000, EYELA, Tokyo, Japan). After freeze-drying, the powder was stored at −4°C. The weight of the EtOH extract powder of JKTM-12 was 24.2 g (yield: 10.1%). JKTM-12 (EtOH extract powder) was dissolved in DMSO when used. MUSHROOM TYROSINASE INHIBITORY ASSAY The assay was performed using relevant methods (9). JKTM-12 each sample was dis- solved in DMSO to a fi nal concentration of 200 mg/ml. This extract stock solution was then diluted to 100 μg/ml in 100 mM potassium phosphate buffer (pH 6.8). Serial dilu- tions were made to attain fi ve concentrations. Kojic acid was used as a positive control. In a 96-well plate, 20 μl of each extract serial dilution was combined with 20 μl of mush- room tyrosinase (250 Units/ml in phosphate buffer) and 100 μl of 100 mM potassium phosphate buffer in triplicate. After incubation at room temperature for 5 min, 40 μl of the substrate (100 unit L -DOPA) was added to each well. The fi nal concentrations of the ex- tract samples ranged from 100 to 2000 μg/ml. After incubation at 37°C for 15 min, optical densities of the reaction mixtures in the wells were recorded at 490 nm using a BIO-TEK Power Wave XS multi-well plate reader (Bio-Teck Instruments, Winooski, VT). The activity rate was calculated by the following equation: Mushroom tyrosinase activity (%) = Absorbance of sample/Absorbance of control × 100. CELL CULTURE B16F10 murine melanoma cells were supplied by KCLB (Seoul, South Korea). They were cultured in DMEM supplemented with 10% heat-inactivated FBS, 10 U/ml of penicillin, 10 μg/ml of streptomycin in a 37°C, 5% CO2, 95% air humidifi ed atmosphere. ASSAY OF CELL VIABILITY B16F10 cell viability by the samples was measured by MTS assay according to the in- structions provided by the manufacturer. Briefl y, B16F10 cells were plated in 96-well culture plates (1 × 104 cells per well). After 24 h, the media were changed and the cells were treated with the samples at various concentrations for 24 h. Ten microliters of MTS (5.0 μg/μl) was added to each well for an additional 4 h of incubation at 37°C.

JOURNAL OF COSMETIC SCIENCE 380 Absorbance was read with a microplate reader (MultiskanMK3, Thermo Scientifi c, Waltham, MA) at 490 nm. Cell viability rate (%) of the samples against the proliferation of B16F10 was calculated using the following equation Cell viability rate (%) = Absorbance of well with samples/Absorbance of well without samples × 100 MELANIN ASSAY B16F10 cells (2 × 105 cells) were cultured in DMEM with 10% FBS. After 12 h, the cells were treated with various concentrations of the samples or media only as a blank for 1 h. Following treatment, 100 nM α-MSH was added to the cells and incubated at 37°C with 5% CO2 in a humidified atmosphere. After 48 h, the cells were washed with phosphate- buffered saline (PBS) and harvested (5000 rpm × 10 min). The pellets containing a known number of cells were dissolved in 1 N NaOH solution containing 10% DMSO and sonicated for 1 h. The amount of melanin content was then monitored by a micro- plate reader at 490 nm. Data are expressed in terms of melanin synthesis inhibitory activ- ity compared to the control. Inhibitory activity (%) was calculated using the following equation. Inhibitory activity (%) = [1 − (Absorbance of sample − Absorbance of blank)/Absorbance of control] × 100. CELLULAR TYROSINASE ACTIVITY ASSAY Cellular tyrosinase activity was determined based on a modifi cation of a previously de- scribed method (8). As many as 2 × 104 cells/cm2 were treated with the samples for 1 h, followed by addition of 100 nM α-MSH. After 48 h, the incubated cells were harvested and washed twice with ice-cold PBS by centrifugation at 1000 × g for 5 min. The har- vested cells were lysed in 1% Triton X-100 and 0.1 mM PMSF in PBS. The total protein was collected by centrifugation at 10,000 × g for 25 min at 4°C. The reaction mixture consisted of 100 μl 1 mg/ml L -DOPA solution and 50 μl cell-extracted protein. Dopach- rome formation at 37°C for 30 min was measuring the absorbance at a wavelength of 475 nm using a microplate reader. WESTERN BLOT ASSAY B16F10 cells were treated with the samples for 1 h, and α-MSH was added. After 48 h, cells were collected and lysed in a RIPA cell lysis buffer 3 containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.2% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS (Enzo Life Sciences, Inc., Plymouth, PA). The lysates were denatured at 95°C for western blot assay. Proteins were separated using 10% SDS-poly- acrylamide gel electrophoresis running gel. The resolved proteins were transferred to ni- trocellulose membranes and then were blocked using 5% skim milk in Tris HCl buffer. Membranes were incubated with tyrosinase and GAPDH antibodies. And the membranes were washed three times every 15 min then incubated with anti-mouse horseradish