行政院國家科學委員會專題研究計畫成果報告 - 嘉南藥理大學.docVIP

嘉南藥理科技大學專題研究計畫成果報告 蕃茄酪氨酸酶抑制劑之 蕃茄酪氨酸酶抑制劑之 一、中文摘要 本實驗所利用之酪胺酸酶抑制劑是由蕃茄（學名Lycopersicon esculentum Mill）直接以水萃取出，所萃取出蕃茄酪胺酸酶粗抽液在-18℃及4℃儲存六個月後，其相對殘留抑制活性分別為80﹪、70﹪，該蕃茄酪胺酸酶抑制劑粗抽液再經由Sephdex G-25管柱層析進行純化步驟，收集抑制劑活性部分以進行冷凍乾燥濃縮，而半抑制濃度(IC50)為2.46mg/ml。 以Superdex peptide HR管柱層析測量此酪胺酸酶抑制劑的分子量約為0.2KDa；此抑制劑和酪胺酸酶作用0分鐘時的抑制活性為0%，而抑制效果最佳達100%時是在作用了5小時後，為緩慢結合型。蕃茄酪胺酸酶抑制劑之最適作用溫度為50℃、最適作用酸鹼值為pH4到pH5之間，而在pH4到pH9之間都 很穩定。在動力學方面，以L-DOPA當基質時，蕃茄酪胺酸酶抑制劑屬於mix type抑制作用。根據Molish, Barford’s , Seliwanoff’s, Schiff’s 和Ninhydrin試驗皆為正反應，於TLC試驗中蕃茄酪胺酸酶抑制劑與醣苷類呈色劑結合，推測出蕃茄酪胺酸酶抑制劑中可能為醣苷化合物。 Abstract The tyrosinase inhibitor from tomato （Lycopersicon esculentum Mill）was extracted by water. The inhibitor was then stored at –18℃and 4℃, the relative inhibition activity of tyrosinase were 80﹪and 70﹪after 6 month storage, respectively. The tyrosinase inhibitor was partially purified with Sephadex G-25 column chromatography. The IC50 of partially purified tyrosinase inhibitor from tomato was 2.46 mg/ml. The molecular mass of tyrosinase inhibitor was 0.2 KDa examined by Superdex Peptide HR column.The inhibition activity of tyrosinase was 0 ﹪when preincubated with the inhibitor for 0 minute, while 100﹪ for 5 hour, indicating a slow binding behavior. The optimal temperature of the inhibition activity of tyrosinase treated with the inhibitor was 50℃；optimal pH was 4-5.The inhibitor was stable at pH4-9. A kinetic analysis show that the inhibitor is a mixed type inhibitor using L-DOPA as substrate. The tyrosinase inhibitor from tomato was probably a glycosidyl compound, according to the positive reaction with Molish, Barford’s , Seliwanoff’s, Schiff’s and Ninhydrin ,TLC test. Keywords: tyrosinase inhibitor ,tomato ,partially purify, 二、緣由與目的 由於酪胺酸酶（monophenol，o-diphenol-oxygen oxidoreductase；EC ）為黑色素生成之關鍵酵素，酪胺酸酶又稱多酚氧化酶，為一種含銅酵素（Sanchez-Ferrer，1995），不僅對蔬果、穀類外觀有顯著影響，且降低了食品品質及價值（Friedman M.，1996），於人類若生理失調或過量紫外線作用會造成黑色素生產過多，使皮膚暗沉或生成雀斑、黑斑（Jae KyungNo， 1999），更嚴重的話形成黑變病或惡性黑色素腫瘤皮膚癌（C.Murray et al.，1999），而酪胺酸酶抑制劑可調控黑