JOURNAL OF COSMETIC SCIENCE 178 for the assay was purchased from Sigma-Aldrich. All test compounds dissolved in DMSO to 500 μM. Two hundred and twenty microliters of 0.1 M phosphate buffer (pH 6.5), 20 μl of sample solution, and 20 μl of mushroom tyrosinase (2000 units/ml in the same buffer) were mixed in a 96-well microplate, and the mixture was preincubated at 37°C for 5 min. Then, 40 μl of 1.5 mM L-tyrosine was added. After incubation at 37°C for 10 min, the absorbance was determined at 490 nm using an ELISA plate reader. % Inhibition = {(A − b)/A} × 100, where A = OD at 490 nm without sample and B = OD at 490 nm with sample. CELL LINES AND CELL CULTURE The mouse melanoma cell line, B16F10, was obtained from Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were cultured in DMEM containing 10% FBS, 50 μg/ml penicillin, and streptomycin at 37°C in the humidifi ed atmosphere of 5% CO2. CYTOTOXICITY TEST Cell viability was determined with a modifi ed version of the method published by Tsukahara et al. (14) using reduction by thiazolyl blue tetrazolium bromide (MTT). B16F10 cells were seeded in DMEM with 10% FBS in separate 96-well plates at the density of 1 × 104 cells/well before being incubated for 24 h. The test compounds were then added to separate wells, and the cells were incubated for another 24 h, after which 100 μl MTT (300 μg/ml) was added to each well. The cells were incubated for 4 h, and 150 μl DMSO was added to dissolve the formazan crystals. The absorbance of the formazan complexes was measured at 540 nm using an ELISA plate reader. IN-VITRO MELANIN SYNTHESIS INHIBITION ASSAY The melanin content of cultured B16F10 cells was determined using a published proce- dure (7) with slight modifi cations. The cells were seeded into six-well plates at the den- sity of 3 × 105 cells/well and incubated for 48 h. Each test sample was then added to the cells, which were subsequently incubated for an additional 24 h. The cells were collected by incubation in trypsin-EDTA followed by centrifugation. Cell pellets were dried, dis- solved in 1 N NaOH, and boiled for 10 min. After cooling, the absorbance was measured at 400 nm using an ELISA plate reader. The absorbance values of a series of known con- centrations of pure melanin were used to construct a calibration curve to determine the amount of melanin produced by the cells. WESTERN BLOTTING B16F10 cell lysates were separated by SDS-PAGE (10% polyacrylamide gels) and transferred to polyvinylidene fl uoride (PVDF). The membranes were then probed with tyrosinase, TRP- 1, TRP-2, and tyrosinase antibodies. Briefl y, the cultured B16F10 cells were washed with phosphate-buffered saline and incubated with a RIPA lysis buffer (1% Nonidet P-40, 1%

EFFECT OF CYCLOHEXANE AND BENZENE DIESTER ON MELANOGENESIS 179 sodium deoxycholate, 0.1% SDS, 0.15 NaCl, 0.01 M sodium phosphate [pH 7.2], 2 mM EDTA, 50 mM sodium fl uoride, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fl uoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) for 30 min on ice. Following incubation, the cell lysates were cleared by centrifugation at 15,000 rpm for 30 min, and the resultant supernatants were collected and used to estimate protein concentration by a Bradford assay (15). Then, 10 μg of total protein lysates were resolved on 10% sodium dodecyl sulfate poly- acrylamide gels (SDS-PAGE) the separated proteins were transferred to the PVDF mem- brane. The level of protein expression in each sample was detected using specifi c primary antibodies, diluted in a TBST solution (Tris-Bufferd Saline and Tween 20) containing 5% (w/v) of skim milk or 3% (w/v) of bovine serum albumin. The membranes were incubated with a specifi c HRP-conjugated secondary antibody and developed using the enhanced che- miluminescent substrate from West zol-plus. Then, they were stripped and reproved with a β-actin primary antibody as a protein loading control. REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION The total RNA was extracted from B16F10 cells by using the Trizol reagent (Invitrogen, Carlsbad, CA). After the synthesis of cDNA with oligo d(T)15 as a reverse transcriptase primer with the extracted RNA as template, PCR amplifi cation was performed using a GenePro Thermal Cycler (Bioer Technology Co., Ltd, Hangzhou, China). The oligonucleotide primers included (i) tyrosinase: 5ʹ-GGG CCC AAA TTG TAC AGA GA-3ʹ (upstream) and 5ʹ-GGC AAA TCC TTC CAG TGT GT-3ʹ (downstream) (ii) TRP-1: 5ʹ-AGG AAT CTG GCT TGG GAT TT-3ʹ (upstream) and 5ʹ-ATG AGC CAC AAG GGT CAG TC-3ʹ (downstream) (iii) TRP-2: 5ʹ-AGC AGA CGG AAC ACT GGA CT-3ʹ (upstream) and 5ʹ-CAG GTA GGA GCA TGA TAG GC-3ʹ (downstream) and (iv) GAPDH: 5ʹ-AAC TTT GGC ATT GTG GAA GG-3ʹ (upstream) and 5ʹ-ACA CAT TGG GGG TAG GAA CA-3ʹ (downstream). The yields of cDNA represented approximately 3 μg of the total input RNA. The reaction was carried out for 35 cycles for 30 s at 94°C, 30 s at 52°C (TRP-1, GAPDH)/53 (TRP-2, tyrosinase), and 30 s at 72°C. The reaction mixtures were analyzed by electrophoresis using 1% agarose gels that were further stained with ethidium bromide. The intensity of the bands was measured using GeneTools software (Syngene, Cambridge, UK). STATISTICAL ANALYSIS Mean ± SEM (standard error of the mean) were calculated. Statistical analyses of the results were performed using the t-test for independent samples. p 0.05 was considered signifi cant. A TYROSINASE INHIBITOR MODEL FOR STRUCTURE-BASED DRUG DESIGN Structure-based drug design is used in assessing changes in protein structure and the interac- tions (docking) between small-molecule organic compounds. This technique is also employed in the development of new drugs through the use of molecular dynamics. This study investigated the interactions and the mechanism of whitening by using a tyrosinase inhibitor model and a whitening-derivatives model, the latter being selected based on its whitening effect at the cellular level.