臺灣博碩士論文加值系統

酪胺酸酶抑制劑是具有治療皮膚相關疾病如黑色素過度沉著的分子
，同時應用於化妝品作為去黑色素之原料。雖然許多化合物作為酪胺酸酶抑制劑已於體外細胞實驗或利用酵素活性測試獲得證實，然而這些分析方法須花費較高的成本，且有時功效無法反應於活體。斑馬魚因具黑色素生成時間快速、小分子吸收滲透能力好與容易操作的優點，因此可有效作為篩選黑色素調節分子的生物模式試驗。本實驗以斑馬魚胚胎評估30種科學中藥的黑色素抑制程度。實驗使用已知的黑色素抑制劑，PTU (N-Phenylthiourea) 與熊果素 (arbutin) 作為正對照組。樣品浸泡三天後的試驗結果顯示，水萃之白豆蔻 (編號 A7) 與款冬花 (編號A23) 科學中藥具有黑色素生成的抑制效果，於1.5 mg/ml與1.7 mg/ml分別為最佳處理濃度，浸泡處理後存活率分別是88.7 %與96.3 %。A7與A23的黑色素生成量下降36.4 %與75.3 %，酪胺酸酶活性與控制組相較分別下降了37.4 %與67.8 %。這些結果說明了由A7和A23的中藥萃取液具有降低黑色素合成功效，並具有美白產品發展應用的潛力。

Tyrosinase inhibitors are potential useful molecules for the treatment of some dermatological diseases associated with melanin hyperpigmentation and in cosmetics for depigmentation. Although several compounds as tyrosinase inhibitors have been proved in vitro cell culture or enzymatic assays, however these would be high price and none of physiological relevance. Zebrafish with the advantages such as rapid pigmentation process, permeability to small molecules and ease of handling, thus the feasibility of zebrafish model system for screening melanogenic regulatory agents was evaluated in this study. In the present study, zebrafish was used to evaluate the melanin inhibitory potent of thirty Chinese herbal ingredients. The melanogenic inhibitors PTU and arbutin were used as positive control group and distilled water was used as a negative control. Results revealed that extracts of Eletteria cardamomum Maton (No. A7) and Tussilago farfara (No. A23) exhibited potency to inhibit melanin synthesis. The optimal concentrations for immersion treatment for A7 and A23 are 1.5 mg/ml and 1.7 mg/ml, respectively. The survival rates in the presence of the optimal concentration of A7 and A23 were 88.7% and 96.3% respectively. The tyrosinase activity and the amount of melanin after A7 and A23 treatment were decreased 36.4% and 75.3%, and 37.4 % and 67.8% respectively compare to that in negative control. These results elucidate that Chinese herbal ingredients A7and A23 can potency of blocking melanin synthesis indicating both compounds potentially can be applied in skin whitening products.

摘 要 II
Abstract III
謝 誌 V
第一章 文獻回顧 1
一、中草藥應用於美白化妝品之市場潛力 1
二、黑色素細胞 (melanocyte) 與黑色素體 (melanosomes) 2
三、黑色素形成機制 4
四、黑色素之生成調控 6
4.1 紫外光對於黑色素細胞影響因子 7
4.2 間接性紫外光影響黑色素生成 8
4.3 MITF與環腺苷酸 (cAMP) 為酪胺酸酶轉錄重要的角色 8
4.4 酪胺酸酶 (tyrosinase) 10
五、已知常用黑色素抑制劑 10
5.1 維生素C (ascorbic acid) 11
5.2 對苯二酚 (hydroquinone) 11
5.3 熊果素 (arbutin) 12
5.4 麴酸 (kojic acid) 12
六、以斑馬魚作為動物模式試驗 12
七、研究動機 13
第二章 材料與方法 14
一、實驗架構 14
二、實驗動物與受試之科學中藥 15
三、實驗方法 16
3.1 斑馬魚產卵方法 16
3.2 酪胺酸酶抑制劑篩選 16
3.3 科學中藥浸泡與表觀觀察 17
3.4 魚卵毒性評估 17
3.5 魚卵黑色素含量測定 17
3.6 魚卵酪胺酸酶活性測定 18
3.7 統計分析 18
第三章 結果 19
一、篩選具有抑制斑馬魚黑色素生成能力之科學中藥 19
二、科學中藥白豆蔻 (A7) 與款冬花 (A23) 最適處理濃度及其對黑色素生成的抑制效果 21
2.1科學中藥A7與A23之最佳處理濃度 21
2.2 黑色素生成抑制觀察 23
2.3 毒性測試 25
三、中藥成分白豆蔻 (A7) 與款冬花 (A23) 對斑馬魚黑色素合成影響與酪胺酸酶活性 27
3.1 黑色素合成測定 27
3.2 酪胺酸酶活性測定 27
第四章 討論 30
第五章 參考文獻 33
作者簡介 42
圖目錄
圖 1、斑馬魚胚胎測試浸泡在於稀釋8倍之A4、A5、A7、A9、A14、A15、A17、A19、A22、A23、A25、A28與A30中藥萃取液中，觀察其黑色素的抑制能力 20
圖 2、科學中藥對斑馬魚黑色素生成之影響 24
圖 3、科學中藥對斑馬魚心跳之影響 26
圖 4、科學中藥對斑馬魚黑色素生成量之影響 28
圖 5、科學中藥對斑馬魚酪胺酸酶活性之影響 29

表目錄
表 1、30種單方科學中藥 15
表 2、浸泡A7和A23萃取液三天後對於斑馬魚死亡率與孵化率之影響 22

附圖目錄
附圖 1、人體表皮層示意圖 3
附圖 2、黑色素小體與核內體之關係 4
附圖 3、酪胺酸酶與其相關蛋白DCT調節調節黑色素生成路徑 6
附圖 4、紫外光能經由直接或是間接誘發的方式使黑色素細胞產生黑色素進而釋放至角質層中 7
附圖 5、cAMP調控MITF、酪胺酸酶及其相關蛋白之分子機制 10

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