INHIBITION OF MELANOGENESIS BY P. ACIDULA EXTRACT 43 (5%) incubator. The cells were seeded at an appropriate cell density in a 24-well or a six- well plate. After 1 d of incubation, the cells were treated with various concentrations of the drugs in the absence or presence of a stimulation agent (100 μM of IBMX) for another 6 h (qRT-PCR), 16 h (tyrosinase activity assay and western blot), or 48 h (melanin deter- mination). Thereafter, the cells were harvested and used for various assays. MEASUREMENTS OF CELL VIABILITY An MTT assay was performed to examine the viability of cells. After the cells were incubated with the samples for 48 h, the culture medium was removed and replaced with 1 mg/ml of MTT solution dissolved in phosphate-buffered saline (PBS) and incubated for an additional 2 h. The MTT solution was then removed and DMSO was added, following which the absorbance of the dissolved formazan crystals was determined at 570 nm by a spectrophotometer. DETERMINATION OF MELANIN CONTENT At the end of cell cultivation, the cells were harvested and washed twice with PBS. The pelleted cells were homogenized in lysis buffer containing 20 mM of sodium phosphate (pH 6.8) and 1% Triton X-100 at 4°C with 30 strokes in a Dounce homogenizer. After centrifugation at 15,000g for 15 min, the melanin pellets were dissolved in 1 N NaOH containing 20% DMSO for 1 h at 95°C. The absorbance at 490 nm was measured, and the melanin content was measured using the authentic standard of synthetic melanin. MEASUREMENT OF CELLULAR TYROSINASE ACTIVITY To determine the tyrosinase activity in the crude extract, a source of crude cellular tyros- inase was obtained by homogenizing drug-treated or untreated cells in 20 mM of sodium phosphate (pH 6.8), 1% Triton X-100, and 1 mM of PMSF at 4°C with 30 repeated strokes in a Dounce homogenizer. Detergent was used to release the membrane-bound tyrosinase from the melanosomes. The lysates were centrifuged at 15,000 rpm for 15 min to obtain the supernatant as the source of crude cellular tyrosinase. The protein content in the supernatant was determined using a Bradford assay with BSA as the protein stan- dard. Tyrosinase activity was then determined as follows: 1 ml of the reaction mixture contained 50 mM of phosphate buffer (pH 6.8), 2.5 mM of l-dopa, and 500 μg of the supernatant protein, and was incubated at 37°C for 15 min, following which the dopa- chrome formation was monitored by measuring absorbance at a wavelength of 475 nm. WESTERN BLOT ANALYSIS The cells were washed three times in ice-cold PBS, and lysed in cold lysis buffer (20 mM of sodium phosphate (pH 6.8), 1% Triton X-100, 1 mM of PMSF, and 1 mM of EDTA) containing protease inhibitor cocktail (Abcam, Cambridge, UK). An aliquot of the lysate

JOURNAL OF COSMETIC SCIENCE 44 was used to determine the protein content with a Bradford assay, using BSA as the stan- dard. The proteins (100 μg) were separated using 10% SDS-polyacrylamide gel electro- phoresis and blotted onto polyvinyl difl uoride (PVDF) membranes (MP Biomedicals Co., Irvine, CA). The membranes were blocked with 5% non-fat skim milk in TBS-T buffer. Tyrosinase and β-actin (as an internal control) were detected using rabbit polyclonal an- tibodies and mouse monoclonal anti-β-actin antibodies, respectively. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody. All bound antibodies were then detected using an Amersham ECL system (Amersham Phar- macia Biotech, Piscataway, NJ). The signal intensity of each band was quantifi ed with a GS-700 densitometer system (Bio-Rad, CA) equipped with an integrator, and normal- ized with that of the internal control. QUANTITATIVE REAL-TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (qRT-PCR) A quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA) using Fast SYBR® Green Master Mix (Applied Biosystems). Total RNA was extracted using an RNeasy® mini Kit (Qiagen, CA) according to the manufacturer's instructions. The quality of the total RNA sample was evaluated by determining the OD260/OD280 ratio. To prepare a cDNA pool from each RNA sample, total RNA (2 μg) was reverse transcribed at 42°C for 90 min in the presence of oligo(dT) primers (MD Bio. Co., Taipei, Taiwan) and reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany). The oligonucleotide primers for mouse tyrosinase (forward, 5′-GGCCAGCTTTCAG- GCAGAGGT-3′ reverse, 5′-TGGTGCTTCATGGGCAAAATC-3′) and mouse glyc- eraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (forward, 5′-ACCACAGTCCATGCCATCAC-3′ reverse, 5′-TCCACCACCCTGTTGCTGTA-3′) were used. After the initial incubation of 2 min at 50°C, the cDNA was denatured at 95°C for 10 min followed by 40 cycles of PCR (95°C, 15 s, 60°C, 60 s). All results were obtained from at least three independent experiments. The mRNA level of tyrosinase was normalized using GAPDH as an internal control. STATISTICAL ANALYSIS All the data in the present study were obtained as averages of experiments that were per- formed in triplicate and are expressed as means ± S.D. Statistical analysis was performed by the Student's t-test. A value of p 0.05 was considered to be statistically signifi cant. RESULTS AND DISCUSSION EFFECT OF PAE ON CELL VIABILITY Safety is an important criterion for a skin-lightening drug. Therefore, before investigat- ing the effect of PAE on the melanogenesis of mouse B16 melanoma cells, the concentra- tion range of the extract that is nontoxic to the cells should be determined. The viability of drug-treated cells was determined by the MTT method. Arbutin was used as the