Blue-Bio Industry Regional Innovation Center, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan 614-714, Korea.
Blue-Bio Industry Regional Innovation Center, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan 614-714, Korea. ; Department of Food Science and Nutrition, College of Natural Science, Dong-Eui University, Busan 614-714, Korea.
Nutr Res Pract. 2014 Oct;8(5):509-15. doi: 10.4162/nrp.2014.8.5.509. Epub 2014 Aug 30.
BACKGROUND/OBJECTIVES: The root of Vitis amurensis Ruprecht, a sort of wild-growing grape, has been used in oriental medicine for treatment of skin ailments; however, its dermatological activity is not sufficiently understood. The aim of this study was to investigate tyrosinase inhibitory and anti-melanogenic activities of V. amurensis Ruprecht root methanol extract (VARM) in B16F10 mouse melanoma cells and to attempt to isolate and identify the active compound issued from VARM.
MATERIALS/METHODS: Anti-melanogenic activity of VARM was analyzed in α-melanocyte stimulating hormone (MSH)-stimulated B16F10 cells through evaluation of antioxidative activity as well as inhibited tyrosinase activity and melanin contents compared with those of kojic acid and arbutin. After anti-melanogenic analysis of VARM, serial fractionation, nuclear magnetic resonance (NMR), and thin layer chromatorgraphy (TLC) were applied for identification of active compounds contained in VARM.
VARM significantly inhibited oxidative stress and tyrosinase activity and attenuated α-MSH-induced melanin production in B16F10 cells. For isolation of active compounds, VARM was fractionated using a series of organic solvents, including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Among fractions showing anti-melanogenic activity, the CH2Cl2 fraction induced the most potent attenuation of melanogenesis without cytotoxicity and the major compound in the CH2Cl2 fraction was identified as betulinic acid. Betulinic acid isolated from the CH2Cl2 fraction of VARM significantly attenuated α-MSH-induced melanogenesis in a dose dependent manner, which was stronger than that of arbutin used as a positive control.
These results indicate that VARM inhibits oxidative stress, tyrosinase activity, and α-MSH-induced melanogenesis in B16F10 cells, due primarily to the active compound, betulinic acid, in the CH2Cl2 fraction.
背景/目的：山葡萄是一种野生葡萄，其根在东方医学中被用于治疗皮肤病；然而，其皮肤科活性尚未得到充分理解。本研究的目的是研究山葡萄根甲醇提取物（VARM）在 B16F10 小鼠黑素瘤细胞中的酪氨酸酶抑制和抗黑色素生成活性，并试图从 VARM 中分离和鉴定活性化合物。
材料/方法：通过评估抗氧化活性以及与曲酸和熊果苷相比抑制酪氨酸酶活性和黑色素含量，分析 VARM 对 α-促黑素细胞激素（MSH）刺激的 B16F10 细胞的抗黑色素生成活性。在对 VARM 进行抗黑色素生成分析后，采用一系列有机溶剂（包括二氯甲烷（CH2Cl2）、乙酸乙酯（EtOAc）和正丁醇（n-BuOH））对 VARM 中的活性化合物进行了连续分级分离、核磁共振（NMR）和薄层色谱（TLC）分析。
VARM 显著抑制 B16F10 细胞中的氧化应激、酪氨酸酶活性和α-MSH 诱导的黑色素生成。为了分离活性化合物，VARM 用一系列有机溶剂进行了分级分离，包括二氯甲烷（CH2Cl2）、乙酸乙酯（EtOAc）和正丁醇（n-BuOH）。在显示抗黑色素生成活性的馏分中，CH2Cl2 馏分在没有细胞毒性的情况下诱导最强的黑色素生成衰减，并且 CH2Cl2 馏分中的主要化合物被鉴定为白桦脂酸。从 VARM 的 CH2Cl2 馏分中分离出的白桦脂酸显著地呈剂量依赖性地减弱α-MSH 诱导的黑色素生成，其强度强于用作阳性对照的熊果苷。
这些结果表明，VARM 主要通过 CH2Cl2 馏分中的活性化合物白桦脂酸抑制 B16F10 细胞中的氧化应激、酪氨酸酶活性和α-MSH 诱导的黑色素生成。
