臺灣博碩士論文加值系統

酪胺酸酶 (tyrosinase) 為一含銅的酵素，在黑色素 (melanin) 生合成途徑中扮演重要的催化角色。其作用是催化受質酪胺酸 (tyrosine) 羥基化 (hydroxylation) 後產生多巴 (Dopa)，並將多巴氧化成多巴醌 (Dopaquinone)，經由一連串的反應後形成黑色素，進而決定哺乳動物的皮膚及毛髮顏色。先前實驗室以虛擬篩選的方式選出9個化合物，進行酵素活性測試。其中以5-CPD 抑制酪胺酸酶的能力較熊果素 (Arbutin) 佳。因此以5-CPD結構為主選出57個相關衍生物，並從中挑選9個化合物進行酵素活性測試。實驗結果發現仍以5-CPD抑制酪胺酸酶的效果為最佳。本次研究是以分子對接 (Docking)方式加上做結構分群，在剩餘的48個5-CPD相關衍生物中選出26個化合物，以進行酪胺酸酶活性測試。實驗結果顯示有7個化合物無法容於有機溶劑中、1個化合物不具抑制效果、6個化合物的抑制效果較熊果素佳，而以A16-CPD 為最好。其抑制效果比5-CPD及熊果素佳且為一天然化合物。故將以A16-CPD為研究重心，找尋與A16-CPD性質相似且對酪胺酸酶有抑制效果的天然化合物。首先，先將1個與A16-CPD性質相似的化合物進行酪胺酸酶活性測試實驗，結果顯示抑制效果較 A16-CPD 佳；另外利用實驗定出市售熊果素、麴酸 (kojic acid) 及A16-CPD 的IC50值，分別為1.25 mM、0.029 mM 和0.46 mM，驗證A16-CPD 抑制酪胺酸酶的能力比熊果素好。之後將針對另外25個與A16-CPD 性質相似的化合物進行酪胺酸酶活性測試實驗的篩選，並將有抑制效果的化合物定出IC50值。

Tyrosinase is a copper containing enzyme and key player in biosynthetic pathway of melanin. It catalyzes the reaction of the hydroxylation of tyrosine to dopa and the oxidation of dopa to dopaquinone. Synthesis of melanin is after a series of reactions. In mammals, melanin determins the color of their skin and hair. The nine compounds was chosen from the study of virtual screening in our lab and carried on the test of tyrosinase activity. According to the test, the inhibitory effect of 5-CPD is better than Arbutin. Based on the structure of 5-CPD, we selected for 57 compounds of 5-CPD related derivatives. Then picked up nine compounds to carry on the test of tyrosinase activity from it. According to the test of tyrosinase activity, we found the 5-CPD get the best inhibitory effect of tyrosinase in experiments result. In our present study, we use the virtual screening by Docking and structure cluster to choose twenty-six from forty-eight compounds of 5-CPD related derivatives. Then carry on the test of tyrosinase activity. The results show that seven compounds are insolubility, one is no inhibition, and inhibitory effect of six compounds are better than Arbutin. The inhibitory effect of A16-CPD is best than 5-CPD and Arbutin and it is a natural compound. We depend on A16-CPD and look for the natural compounds of quality similar to A16-CPD that could inhibit the activity of tyrosinase. First of all, we test inhibitory effect of a natural compound from A16-CPD analog. The results show that inhibition of A16-CPD analog is better than A16-CPD. Besides, we utilize the test of tyrosinase activity to determine IC50 about Arbutin, Kojic acid and A16-CPD, the value are 1.25 mM, 0.029 mM and 0.46 mM, respectively. We confirmed that inhibition of A16-CPD is better than Arbutin. We will aim at twenty-five compounds from A16-CPD analog to carry out the screening of tyrosinase activity and determine IC50 that if have inhibitory effect.

英文摘要 4
中文摘要 5
第一章 緒論
1-1黑色素的來源及功能 6
1-2黑色素的生合成路徑 6
1-3 酪胺酸酶的催化作用機制 7
1-4 酪胺酸酶的影響 7
1-5 酪胺酸酶抑制劑的應用 8
1-6 鏈黴菌酪胺酸酶結構 8
1-7 電腦輔助藥物設計 9
1-8 研究動機 10
第二章 實驗材料與方法
2-1 In silico本研究所使用的軟體
2-1-1 分子對接的準備 12
2-2 In vitro 酪胺酸酶活性測試
2-2-1 藥品來源 12
第三章 結果
3-1 In silico 虛擬篩選
3-1-1 挑選化合物 13
3-2 In vitro 酪胺酸酶活性測試
3-2-1 酪胺酸酶活性測試實驗條件 13
3-2-2 A1-CPD~A26-CPD對酪胺酸酶活性測試篩選結果 13
3-2-3 RY3酪胺酸酶活性測試 15
3-3 IC50 的測定
3-3-1 熊果素 IC50 的測定 15
3-3-2 麴酸 IC50的測定 16
3-3-3 A16-CPD IC50 的測定 16
第四章 討論
4-1 化合物的可溶性 17
4-2 結構分群篩選分析 17
4-3 虛擬篩選分析 18
4-4 篩選後續抑制劑的選擇 19
4-5 熊果素 IC50 的測定 19
4-6 麴酸IC50的測定 20
4-7 A16-CPD IC50的測定 20
第五章 結論與未來方向 21
第六章 參考文獻 38

圖表目錄
圖一. 黑色素的生合成途徑 22
圖二. 酪胺酸酶的催化機制 22
圖三. 酪胺酸酶活化位中心的催化機制 23
圖四. 不同濃度的DMSO對酪胺酸酶活性測試 23
圖五. 酪胺酸酶剩餘活性百分比 24
圖六. 不同抑制劑濃度處理下，酪胺酸酶剩餘活性百分比 24
圖七. 熊果素對於酪胺酸酶活性的抑制作用 25
圖八. 麴酸對於酪胺酸酶活性的抑制作用 25
圖九. A16-CPD對於酪胺酸酶活性的抑制作用 25
圖十. 熊果素在分子對接的分析 26
圖十一. 麴酸在分子對接的分析 26
表一.26個化合物實驗系統條件 27

1.LaBonne, C., and Bronner-Fraser, M. (1998) Induction and patterning of the neural crest, a stem cell-like precursor population, J Neurobiol 36, 175-189.
2.Sulaimon, S. S., and Kitchell, B. E. (2003) The biology of melanocytes, Vet Dermatol 14, 57-65.
3.Kim, Y. J., and Uyama, H. (2005) Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future, Cell Mol Life Sci 62, 1707-1723.
4.Kobayashi, T., Vieira, W. D., Potterf, B., Sakai, C., Imokawa, G., and Hearing, V. J. (1995) Modulation of melanogenic protein expression during the switch from eu- to pheomelanogenesis, J Cell Sci 108 ( Pt 6), 2301-2309.
5.Strothkamp, K. G., Jolley, R. L., and Mason, H. S. (1976) Quaternary structure of mushroom tyrosinase, Biochem Biophys Res Commun 70, 519-524.
6.Decker, H., and Tuczek, F. (2000) Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism, Trends Biochem Sci 25, 392-397.
7.Olivares, C., Garcia-Borron, J. C., and Solano, F. (2002) Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle, Biochemistry 41, 679-686.
8.Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., and Sugiyama, M. (2006) Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis, J Biol Chem 281, 8981-8990.
9.Parvez, S., Kang, M., Chung, H. S., and Bae, H. (2007) Naturally occurring tyrosinase inhibitors: mechanism and applications in skin health, cosmetics and agriculture industries, Phytother Res 21, 805-816.
10.Xu, Y., Stokes, A. H., Roskoski, R., Jr., and Vrana, K. E. (1998) Dopamine, in the presence of tyrosinase, covalently modifies and inactivates tyrosine hydroxylase, J Neurosci Res 54, 691-697.
11.Asanuma, M., Miyazaki, I., and Ogawa, N. (2003) Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson's disease, Neurotox Res 5, 165-176.
12.Friedman, M. (1996) Food Browning and Its Prevention: An Overview, American Chemical Society 44.
13.Xue, C. B., Zhang, L., Luo, W. C., Xie, X. Y., Jiang, L., and Xiao, T. (2007) 3D-QSAR and molecular docking studies of benzaldehyde thiosemicarbazone, benzaldehyde, benzoic acid, and their derivatives as phenoloxidase inhibitors, Bioorg Med Chem 15, 2006-2015.
14.Tae Joung Ha, M. S. Y., Dae Sik Jang, Sang Uk Choi,† and Ki Hun Park*. (2001) Inhibitory Activities of Flavanone Derivatives Isolated from Sophora flavescens for Melanogenesis, Bull. Korean Chem. Soc. 22.
15.Piao, L. Z., Park, H. R., Park, Y. K., Lee, S. K., Park, J. H., and Park, M. K. (2002) Mushroom tyrosinase inhibition activity of some chromones, Chem Pharm Bull (Tokyo) 50, 309-311.
16.Lin, Y. P., Hsu, F. L., Chen, C. S., Chern, J. W., and Lee, M. H. (2007) Constituents from the Formosan apple reduce tyrosinase activity in human epidermal melanocytes, Phytochemistry 68, 1189-1199.
17.Hori, I., Nihei, K., and Kubo, I. (2004) Structural criteria for depigmenting mechanism of arbutin, Phytother Res 18, 475-479.
18.Chang, T. S., Ding, H. Y., and Lin, H. C. (2005) Identifying 6,7,4'-trihydroxyisoflavone as a potent tyrosinase inhibitor, Biosci Biotechnol Biochem 69, 1999-2001.
19.Ha, Y. M., Chung, S. W., Song, S., Lee, H., Suh, H., and Chung, H. Y. (2007) 4-(6-Hydroxy-2-naphthyl)-1,3-bezendiol: a potent, new tyrosinase inhibitor, Biol Pharm Bull 30, 1711-1715.
20.Rangkadilok, N., Sitthimonchai, S., Worasuttayangkurn, L., Mahidol, C., Ruchirawat, M., and Satayavivad, J. (2007) Evaluation of free radical scavenging and antityrosinase activities of standardized longan fruit extract, Food Chem Toxicol 45, 328-336.

國內論文
1.曾天生Virtual High Throughput Screening of Tyrosinase Inhibitors from PubChem and Chinese-Herbal-Medicine databases.
2.陳翊庭 3D-QSAR Pharmacophore Identification for Tyrosinase Inhibitors using Molecular Docking and Catalyst.
3.吳佳穎 The kinetics and the screening of tyrosinase inhibitors.