J. Cosmet. Sci., 62, 41–48 (January/February 2011) 41 Inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells HSIOU-YU DING, TE-SHENG CHANG, CHIEN-MIN CHIANG, and SORGAN SHOU-KU TAI, Institute of Cosmetics Science, Chia Nan University of Pharmacy and Science (H.-Y.D.), Department of Biological Science and Technology, National University of Tainan (T.-S.C.), Department of Biotechnology, Chia Nan University of Pharmacy and Science (C.-M.C.), and Department of Biotechnology, National Formosa University (S.S.-K.T.), Taiwan, R.O.C. Accepted for publication November 3, 2010. Synopsis The inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells was investigated. The results showed that the P. acidula extract (PAE) inhibited melanogenesis in 3-isobutyl- 1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner, with an IC50 value of 33.5 μg/ml. In addition, PAE also inhibited cellular tyrosinase activity. Moreover, western blot and real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses respectively confi rmed that PAE down-regulated levels of tyrosinase protein and its mRNA in IBMX-stimulated B16 cells. These results demonstrated that PAE inhibits melanogenesis of B16 cells by reducing tyrosinase gene expression. From the present study, PAE is proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation. INTRODUCTION Skin pigmentation is produced by dermal melanocytes. Melanogenesis has been defi ned as the entire process leading to the formation of dark macromolecular pigments, i.e., melanin, which are formed by a combination of enzymatically catalyzed and chemical reactions (1). Melanogenesis is initiated in melanosomes, the special organelles of mela- nocytes, with the fi rst step of l-tyrosine oxidation to l-dopa (l-3,4-dihydroxyphenylala- nine) and then to dopaquinone, which is catalyzed by tyrosinase. This is a rate-limiting step in melanin synthesis because the remainder of the reaction sequence can proceed spontaneously at a physiological pH value. Although melanin mainly plays a photopro- tective role, the accumulation of abnormal amounts of melanin in different parts of the skin, which results in pigmented patches of skin, might become an esthetic problem. Address all correspondence to Sorgan Shou-Ku Tai at 64 Wun-Hua Road, Huwei, Yunlin, Taiwan, R.O.C. (sorgan@nfu.edu.tw).
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
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