Inhibitory Effects of .ALPHA.-Arbutin on Melanin Synthesis in Cultured Human Melanoma Cells and a Three-Dimensional Human Skin Model

Abstract

Hyperpigmentation in the epidermis is caused by excessive melanin synthesis, and tyrosinase (EC 1.14.18.1) is a key-enzyme in melanin synthesis. It catalyzes the first two steps in melanin synthesis: the hydroxylation of tyrosine to 3-(3,4-dihydroxyphenyl)-alanine (DOPA) and the oxidation of DOPA to dopaquinone. Several tyrosinase inhibitors have been used as skin-lightening agents in the cosmetic industry.4-Hydroxyphenyl a-D-glucopyranoside (a-arbutin; Fig. 1) was enzymatically synthesized from hydroquinone and saccharides, [1][2][3][4] and its inhibitory activity against tyrosinases from mushroom, B16 mouse melanoma and HMV-II human melanoma cells has been examined previously.2,5,6) a-Arbutin specifically inhibited mammalian tyrosinases, and its effect on human tyrosinase was the strongest among the hydroquinone-glycosides that we have studied so far. 6) In this study, we examined the inhibitory effects of a-arbutin on melanin biosynthesis in cultured human melanoma cells and in a three-dimensional human skin model. MATERIALS AND METHODSCell Culture Human malignant melanoma cells, HMV-II 7) were provided by the Cell Resource Center for Biomedical Research, Tohoku University. The cells were cultured in F12/DMEM (GIBCO) supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO 2 in air.The three-dimensional cultured human skin model, MelanoDerm, (MEL312B; a co-culture of normal human melanocytes (NHM) from black donors and normal human keratinocytes (NHK)) was purchased from Kurabo Co. (Osaka, Japan). The human skin model was maintained according to the manufacturer's instructions.Reagents L-DOPA and synthetic melanin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Medium for the human skin model [long life maintenance medium (LLMM)] was supplied with the MEL-312B kit. Other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka).Preparation of a a-Arbutin a-Arbutin was prepared by the method described by Nishimura et al.1) a-Amylase from Bacillus sp. strain X-23 was added to 100 ml of 50 mM sodium acetate buffer solution (pH 5.0) containing 5% hydroquinone and 20% maltopentaose. After incubation at 40°C for 16 h, glucoamylase from Aspergillus niger was added, and the mixture was incubated at 40°C for 4 h. a-Arbutin was purified from the reaction mixture by extraction with ethyl acetate and charcoal column chromatography.Assay of Melanin Synthesis by HMV-II Cells HMV-II cells (1.0ϫ10 6 ) were plated with 10 ml of medium in a 100-mm dish and grown for 10 d. On days 1, 4 and 7, the medium was changed to fresh medium containing various concentrations of a-arbutin.Determination of Melanin Content The melanin content of cells was measured by the method of Hosoi et al. -free phosphate-buffered saline [PBS(Ϫ)]. After being treated with 0.25% trypsin, the cells were harvested by centrifugation at 1000ϫg for 10 min and sonicated in 0.5 ml of 1% Triton X-100/PBS(Ϫ) on ice. In contrast, the human skin model was harvested and sonicated in 0.5 ml of PBS(Ϫ). The lys...

Human Tyrosinase Inhibitor in Rum Distillate Wastewater

“…On the 10th day, the cells were scraped from the tissue culture plate in the presence of Mg 2+ and Ca 2+ free phosphate buffered saline [PBS ( )], sonicated in PBS ( ) on ice for 1 h, then the mixture was centrifuged at 10000 g for 30 min. The supernatant, which contained tyrosinase, was used for measurements of inhibitory effects 18) .…”

Peptides-derived from Scales of Branchiostegus japonicus Inhibit Ultraviolet B-induced Oxidative Damage and Photo-aging in Skin Cells

“…따라서 미백효과를 보기 위해서는 tyrosinase 의 억제효과를 봄으로써 melanin 생성의 억제효과를 입증하 게 된다. 미백제로 잘 알려진 arbutin은 이러한 tyrosinase 억 제제인 hydroquinone과 유사한 hydroquinone 부분을 갖고 있다 [1,23,24]. 하지만 안정성의 문제로 인해 사용이 제한되고 있다.…”

Molecular Profiling of A375 Human Malignant Melanoma Cells Treated with Kojic Acid and Arbutin

“…Arbutin, a natural compound of hydroquinone beta-D-glucopyranoside, is widely used as an ingredient in skin care products 13 . It is effective in the treatment of various cutaneous hyperpigmentations and inhibits melanogenesis in melanoma cells 14 . However, recent findings have raised serious concerns regarding both the safety and side effects of arbutin.…”