JOURNAL OF COSMETIC SCIENCE 42 Therefore, several studies have focused on the inhibition of tyrosinase activity and the prevention of abnormal pigmentation (2,3). A previous study has reported that some cytokines and growth factors play important regulatory roles in melanogenesis (4). α-Melanocyte stimulating hormone (α-MSH) is the most well-studied hormone. This hormone binds to its receptor, melanocortin recep- tor 1 (MC1R), on the membrane of melanocytes and stimulates melanogenesis via the GPCR (G protein-coupled receptor)-cAMP-MITF (microphthalmia-associated transcrip- tion factor) pathway where the melanogenesis-related enzymes, including tyrosinase and tyrosinase-related proteins 1 and 2 (TRP1 and TRP2) are up-regulated. In addition to α-MSH, other intracellular cAMP elevation agents such as forskolin or 3-isobuty1-1- methylxanthin (IBMX) also stimulate melanogenesis through the same signal pathway as does α-MSH. Accordingly, agents blocking the signal pathway would exhibit depigmen- tation against melanocytes (5,6). In the present study, we screened more than 200 crude extracts of traditional Chinese medicinal herbs to identify their applicability as skin-lightening agents. The P. acidula extract (PAE) was found to have strong inhibitory activity on melanogenesis in mouse B16 melanoma cells. The inhibitory effect of the extract on melanogenesis of the cells was investigated in advance. MATERIALS AND METHODS PREPARATION OF PAE The dried powder of the bark (235.0 g) of P. acidula was extracted with one liter of water at room temperature overnight four times, followed by fi ltration at the end of each extrac- tion. The fl ow-through extracts were concentrated in a vacuum and combined to yield a black syrup (14.3 g). CHEMICALS AND ANTIBODIES Arbutin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, phenylmethylsulfonyl fl uoride (PMSF), l-dopa, dimethyl sulfoxide (DMSO), trypsin/EDTA, synthetic melanin, and IBMX were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-tyrosinase antibodies (#62914) and protease inhibitor cocktail were obtained from Abcam (Cambridge, MA). Anti-β-actin antibodies (#3662) were pur- chased from Bio Vision Inc. (Irvine, CA). All other chemicals were obtained from Tokyo Chemical Industry Co. (Tokyo, Japan) and were of analytic reagent grade. CELL CULTURES AND DRUG TREATMENTS Mouse B16 melanoma cells (4A5) were obtained from the Bioresources Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC). The cells were cultured in Dulbecco’s modifi ed Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum at 37°C in a humidifi ed, CO2-controlled

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