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1College of Pharmacy, Yonsei University, Incheon 406-840
2College of Pharmacy, Gachon University, Incheon 406-799
3National Institute of Crop Science, Rural Development Administration, Iksan 570-080
4Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-799
5Gachon Medical Research Institute, Gil Medical Center, Inchon 405-760, Republic of Korea
Resveratrol is a polyphenolic compound found in various natural products such as grapes and berries and possesses anti-cancer, anti-hyperlipidemia, and anti-aging properties. Recently, it has been reported that resveratrol inhibits α-melanocyte-stimulating hormone signaling, viability, and migration in melanoma cells. However, these effects have not been confirmed
Melanin is the predominant contributing to skin pigmentation, and is also found in the hair, eyes, ears, and brain. A primary function of melanin is to counteract skin damage caused due to ultraviolet radiation (UVR) (Moon
Melanin is produced by melanocytes in the basal layer of the epidermis. Melanocytes contain granules within the cytoplasm called melanosomes. These melanosomes have melanin pigments, which can be transferred out to adjacent keratinocytes in the epidermis (Riley, 1997). Melanin synthesis, or melanogenesis, is affected by many enzymes and is induced by the α-melanocyte-stimulating hormone (α-MSH) and UVR. Tyrosinase is the predominant enzyme in melanogenesis, as it is involved in two melanin synthesis processes. The first step is the conversion of tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA), which is subsequently converted to dopaquinone (Gillbro and Olsson, 2011). Melanin is a polymer synthesized by tyrosinase-related protein (TRP)-2 that converts DOPA chrome to 5,6-dihydroxyindole 2-carboxylic acid (DHICA). One of another enzymes, TRP-1, can oxidize DHICA to indole-5-6-quinone carboxylic acid. Thus, they regulate the synthesis of brownish 5,6-dihydroxyindole-2-carboxylic acid-rich melanins. These proteins are activated by microphthalmia-associated transcription factor (MITF), the key transcriptional regulator in melanogenesis (Bertolotto
Resveratrol (3’,4’,5’-trihydroxy-trans-stilbene) is a polyphenolic compound found in various natural products such as grapes and berries and has been reported to possess biological activities such as antioxidation (Goldberg, 1996), anticancer (Song
B16F10 mouse melanoma cells were cultured in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin in air containing 5% CO2 at 37°C.
The melanin content was determined according to the modified methods of Hosoi
The cell proliferation assay was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). B16-F10 cells were cultured in 5×104 cells/well in 48-well plates. After 24 h, the cells were treated with various concentrations of samples (1, 10, and 100 μM) for 48 h. At the end of the incubation, 50 μl of MTT solution (1 mg/ml in PBS) was added to each well. After incubation at 37°C for 1 h, the medium was gently removed and 150 μl of dimethyl sulfoxide (DMSO) was added. The absorbance of each well was measured at 570 nm using a spectrophotometer.
Five-week-old male guinea pigs (KIWA:A1) (weight, 272–297 g; n=4) were obtained from Japan Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of Korea Conformity Laboratories (IA13-00229). The guinea pigs were housed in a temperature-and humidity-controlled room (22 ± 1°C, 60 ± 5% humidity) with 12 h light/dark cycles. After 1 week of quarantine, the guinea pigs were acclimated to individual cages. During the experimental period, food and water were given ad libitum.
The UV source was supplied by a closely spaced array of five Sankyo Denki sun lamps, which have peak irradiance at 310 nm (Kanagawa, Japan). Bulbs were positioned 15 cm above the guinea pigs. The irradiation (0.1 mW/cm2) was measured with an IL1700 Research Radiometer (International Light, Inc., Newburyport, MA, USA) equipped with a UVB sensor. The guinea pigs were exposed to 390 mJ/cm2 UVB radiation three times per week for two weeks. UVB irradiation was applied to the dorsal skin of the guinea pigs after hair removal.
Resveratrol was dissolved in a mixture of ethanol and propylene glycol (3:7, v/v). The sample solution was applied topically to the dorsal skin once a day for 12 days after the final UVB treatment. Post treatment, 1% resveratrol (200 μl; 10 mg/ ml) was applied to 2 cm2 of skin.
To analyze changes in the melanin content of the skin tissue, the guinea pigs were sacrificed after the final treatment and biopsies were obtained from the dorsal skin, which were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections, approximately 4 μm thick, were stained with Fontana-Masson staining solution (IHC WORLD, GA, USA). The stained slides were examined under a light microscope.
Changes in the levels of proteins related to melanin production, including tyrosinase, TRP-1, TRP-2, and MITF in the B16F10 cells and skin of guinea pigs were evaluated by Western blotting. Prepared samples were homogenized and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and was centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was collected and assayed for protein concentration using the protein assay kit (Bio-Rad, CA, USA). Lysates containing 40 μl of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 10% resolving and 3% acrylamide stacking gel and were transferred to nitrocellulose sheets. Blocking was performed in tris-buffered saline containing 5% skim milk powder and 0.1% Tween-20. The primary antibodies used for Western blotting include mouse anti-a-tubulin antibody, goat anti-tyrosinase, and goat anti-TRP-1 and -TRP-2 (Santa Cruz Biotechnology Inc., CA, USA), and mouse anti-MITF (Neomarkers, CA, USA). Finally, immune complexes were detected with a ChemiDocXRS+ imaging system (Bio-Rad, CA, USA).
Data were expressed as mean ± SD from at least three independent experiments. Student’s
In Fig. 1B, the melanin contents were increased in α-MSH (100 ng/ml)-stimulated control cells as compared to the nonstimulated normal cells. However, resveratrol treatment significantly decreased melanin production by 28.2 ± 1.9% without cell toxicity at 100 μM after 48 h treatment in α-MSH stimulated B16F10 cells.
Chen
Inhibitory effects of resveratrol have not yet been confirmed in an UVB-induced brownish guinea pigs
To investigate the mechanism of resveratrol on depigmentation, we determined the protein levels of melanogenesisregulated proteins including tyrosinase, TRP-1, TRP-2, and MITF by Western blot analysis in skin tissue. As seen in Fig. 4, the expression of tyrosinase, TRP-1, TRP-2, and MITF were increased by UVB exposure. However, the protein levels of tyrosinase, TRP-1, TRP-2, and MITF were decreased by resveratrol treatment. Thus, we confirmed that resveratrol regulated melanogenesis-related protein in the dorsal skin of UVB-irradiated guinea pigs. In addition, we assessed melanin production in the skin tissues by the Fontana-Masson staining technique. Melanin synthesis of skin epidermis was increased by UVB exposure. Based on the histological study in Fig. 5, resveratrol treatment decreased melanin production, but not to the levels observed for the normal group.
Recently, interest in the various activities of resveratrol has risen along with the well-being trend. Resveratrol is widely present in natural sources such as grapes, berries, and nuts and possesses various activities including anticancer properties, extension of lifespan (Baur and Sinclair, 2006), and antihyperpigmentation (Wu
Although previous studies have reported the inhibitory role of resveratrol
Our study suggests that resveratrol significantly inhibits melanin synthesis