臺灣博碩士論文加值系統

佛手柑（Citrus medica var. sarcodactylis (Noot.) Swingle）為芸香科(Rutaceae)柑橘屬(Citrus)的常綠小喬木，果實為常用中藥材之ㄧ。本研究以佛手柑果皮為材料，進行成分之單離純化，所得到之純化合物，經鑑定為limettin，並探討此成分對B16黑色素細胞中酪胺酸酶活性的影響及在化妝品上的應用價值。實驗結果顯示，由佛手柑乙酸乙酯的粗萃物(0.5 g/20 mL)具有抑制酪胺酸酶活性，從乙酸乙酯萃出物中分離出limettin，由MTT細胞毒性測試顯示，超過300 μM的濃度造成B16細胞存活率只剩56.78％（400 μM）和43.14％（500 μM），再由細胞型態存活測試可以知道在黑色素生成試驗中，limettin濃度400 μM和500 μM黑色素的產生變少是因為濃度太高導致B16細胞死亡，沒辦法產生黑色素所以才會下降，而不是確實抑制B16細胞產生黑色素。因此limettin對B16細胞合理的濃度為200 μM，能夠抑制酪胺酸酶的活性但是不會對細胞造成毒害，且由酵素免疫染色證明，limettin除了可以抑制酪胺酸酶的活性，也能抑制下游蛋白TRP-2的產生。

摘 要 ………………………………………………………………………..I
Abstract ………………………………………………………………………II
誌謝…………………………………………………………………………III
目錄…………………………………………………………………………IV
圖表索引………………………………………………………………… VIII

壹、前言……………………………………………………..……………… 1
貳、文獻回顧…………………………………………………..…………… 3
一、佛手柑 ……………………………………………………..……………3
（一）植株性狀……………………………………………….…………… 3
（二）生長特性與栽培…………………………………………………… 3
（三）栽培品種………………………………………………..…………… 3
（四）佛手柑成分與藥理活性……………………………….…………… 4
1. 佛手柑成分………………………………………………….…………… 4
2. 藥理活性…………………………………………………….…………… 4
二、Limettin………………………………………………………………… 7
三、黑色素與黑色素細胞……………………………………….………… 8
（一）黑色素細胞的分布……………………………………….………… 8
（二）黑色素的生成…………………………………………….………… 9
（三）黑色素種類…………………………………………………………...10
（四）酪胺酸酶在細胞中之作用和降解……………………………… 11
（五）表皮細胞中參予黑色素生成的蛋白質………………………… 12
（六）美白機制的介紹………………..……………………………… 14
參、材料與方法………………………………………………………… 16
一、材料………………………………………………………………… 16
（一）藥材……………………………………………………………… 16
（二）細胞株…………………………………………………………… 16
（三）溶劑及試藥……………………………………………………… 16
（四）儀器……………………………………………………………… 16
二、方法………………………………………………………………… 17
（一）佛手柑粗萃物…………………………………………………… 17
（二）佛手柑中limettin之定量分析……………………………………17
1. 檢品溶液之製備……………………………..….…………………… 17
2. 標準品溶液之配製…………………………..….…………………… 17
3. 檢量線之製作…………..…………………………………………… 18
4. 高效能液相層析（HPLC）分析條件……………………………… 18
（三）細胞培養………………………………………………………… 19
1. 細胞培養液………………..………………………………………… 19
2. 培養條件…………………………………..………………………… 20
3. 繼代培養…………………………………...………………………… 20
（四）細胞毒性分析…………………………………………………… 20
（五）細胞型態存活測試……………………………………………… 21
（六）細胞黑色素含量測定…………………………………………… 21
（七）細胞蛋白質萃取………………………………………………… 21
（八）SDS膠體製備…………………………………………………… 22
1. 試劑……………………………………..……….…………………… 22
2. 鑄膠………………………………………………………………….. 23
（九）酪胺酸酶活性染色法…………………………………………… 23
（十）酪胺酸活性抑制法…………………………………………… 24
（十一）西方墨點法（Western blot）………………………………… 24
肆、結果………………………………………………………………… 25
一、佛手柑中limettin之定量分析…………………………………… 25
二、佛手柑粗萃物酪胺酸酶活性染色………………………………… 25
三、酪胺酸酶活性抑制法……………………………………………… 25
四、細胞毒性分析……………………………………………………… 26
五、細胞型態存活測試………………………………………………… 26
六、細胞黑色素含量測定……………………………………………… 26
七、300 μM limettin酪胺酸酶活性染色……………………………… 26
八、西方墨點法（Western blot）……………………………………… 27
伍、討論……………………………………………………………… 36
陸、結論……………………………………………………………… 38
參考文獻……………………………………………………………… 39
作者簡介……………………………………………………………… 46

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