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  • 學位論文

白頭翁素抑制人類正常黑色素細胞內酪氨酸酵素活性與酪氨酸相關酵素表現

Anemonin inhibits the tyrosinase activity and expression oftyrosinase-related proteins in human epidermal melanocytes

指導教授 : 李美賢
共同指導教授 : 黃彥華(Yen-Huang Huang)
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摘要


在黑色素生成步驟裡酪氨酸相關酵素共同參與其反應過程,包含酪氨酸酵素、酪氨酸相關酵素1和酪氨酸相關酵素2。酪氨酸酵素是個含銅的單氧酶,在生成黑色素中進行羥基化與氧化反應此為合成黑色素的速率決定步驟。 本實驗的20種純化合物由天然物分離出來,包含植物、黴菌、海洋細菌與海藻,拿來篩選對黑色素細胞內酪氨酸酵素的活性與抑制黑色素生成的效果。在實驗過程中,一開始化合物在50 µM的濃度時處理正常人類黑色素細胞24小時,得到結果為10種化合物的細胞存活率>80%。此10種化合物接下來也以50 µM進行對酪氨酸酵素活性的探討。在此步實驗結果顯示,白頭翁素(Anemonin)對酪氨酸酵素的抑制活性能大於50%。 由上述的篩選過程中發現白頭翁素(Anemonin)由厚葉鐵線蓮(Clematis crassifolia Bentham)萃取而來,具有抑制黑色素細胞內酪氨酸酵素的能力其IC50為43.5 µM,當細胞加藥處理48小時,酪氨酸酵素抑制率可高達60%以上。在抑制黑色素生成方面,加藥24小時後對黑色素的生成並無明顯的影響,但持續加藥48小時後黑色素生成可將低約70%左右。 因此在確定黑色素被抑制後進一步的討論白頭翁素對黑色素細胞體內機制的探討,在西方點墨法的結果酪氨酸酵素與相關酵素2的蛋白表現在24小時後都被明顯的抑制,但酪氨酸相關酵素1的蛋白表現在48小時後才有明顯的抑制。以細胞免疫染色法加以觀測細胞內此3種酵素,酪氨酸酵素與酪氨酸相關酵素2在細胞體內的表現是降低的。最後利用即時定量聚合酵素連鎖反應觀察3種酵素的基因表現,白頭翁素確實能減少酪氨酸及其相關蛋白的基因表現。 本實驗結果討論,白頭翁素在影響黑色素的生成原因可能是經由抑制酪氨酸及其相關酵素的基因表現,因此能調控此3種酵素的蛋白表現。白頭翁素對黑色素細胞在不產生細胞毒性之下能夠抑制黑色素的生成,但完整的作用機制尚未明確,希望在未來可以實際運用在化妝品美白的用途上,做為新一代皮膚美白劑。

並列摘要


Melanin synthesis is a highly cooperative process carried out by tyrosinase family proteins, including tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). Tyrosinase is a copper-containing monooxygenase. Tyrosinase catalysis reaction including hydroxylation and oxidation steps which are the rate-limiting steps in melanin production. In our study, 20 compounds isolated from natural products, including plants, fungus, ocean bacteria and seaweed, were evaluated their ability to inhibit cellular tyrosinase activity and reduce melanin synthesis in human epidermal melanocyte (HEMn). Among the compounds, cell viabilities of 10 compounds were>80%, when cells were treated with 50 µM of different compounds for 24 hours. They were further evaluated the tyrosinase activity. Among these compounds, Anemonin isolated from Clematis crassifolia Bentham showed the most efficacious in inhibition of tyrosinase activity. When treated with Anemonin for 48 hours, the inhibition of tyrosinase activity was above 60%. Melanin synthesis was reduced to 70% when sustained administration for 48 hours. But it showed no variation in melanin synthesis when administration for 24 hours. The cellular mechanism in melanocyte was further investigated. The protein expression of tyrosinase and TRP2 were significantly Inhibited in 24 hours but the protein expression of TRP1 were inhibited until 48 hours later. Decreased of these protein expression were further confirmed by immunocytochemistry. Anemonin suppressed the expression of tyrosinase and it’s related proteins as demonstrated by quantitative real-time PCR (qRT-PCR). In conclusion, Anemonin may down-regulated gene encoding of tyrosinase and it’s related proteins (TRP2 and TRP2) and resulted in reducing melanin synthesis. Anemonin can inhibit melanin synthesis without cytotoxicity in human melanocyte although cellular mechanism was still unclear. Our results suggest that Anemonin can be used as whitening agent for therapeutic intervention of hyperpigmentation on cosmetics.

參考文獻


27.陳克君. 金絲草成分之研究. 臺北醫學大學生藥學研究所碩士論
28.林怡沛. 台灣蘋果及雞血藤之抗氧化活性成分探討. 臺北醫學大
24.吳政億. 蘭嶼山欖葉部成分之研究. 臺北醫學大學生藥學研究所
26.黃俐瑋. 蘭嶼肉桂葉部成分之研究. 臺北醫學大學生藥學研究
45.Min Kyung Cho, Young P. J., Young C. K., Sang G. K.

被引用紀錄


黃靜儀(2010)。(一) 樟葉槭之成分研究 (二) 兒茶酚衍生物於人類黑色素細胞活性探討〔碩士論文,臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-0308201012344400

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