臺灣博碩士論文加值系統

近年來，民間盛傳荖葉具有去除黑色素的功效，但是又有部分民眾在敷完臉後造成色素沉澱及花白斑點的情況。由於相關之研究仍缺乏，本研究以黑色素瘤細胞B16-F1為模式系統，探討氧化前後之荖葉多元酚粗萃物對細胞黑色素之生成、釋放，及其對酪胺酸酶酵素活性、dopa的含量、細胞內外的氧化狀態的影響。
由試驗結果可知，本實驗所使用之荖葉多元酚粗萃物(＜50μg/ml)並不會造成細胞的傷害，在早期48小時的培養可以有效抑制細胞黑色素之生成與釋放，但時間增長時，雖然細胞內黑色素生的生成仍然被抑制，但釋放至細胞外的黑色素卻有增加的情形。荖葉多元酚粗萃物會影響酪胺酸酶的活性，但未隨荖葉多元酚粗萃物的濃度而變化，荖葉多元酚粗萃物在最初的24小時對酪胺酸酶活性具有些微的抑制效果，但隨時間的增長，反而有促進活性的功用；另外荖葉多元酚粗萃物在培養的前48小時，可能是延緩dopa的自氧化速度因而可觀測到細胞內dopa含量較高，但培養至72小時卻可能會促進dopa自氧化，使在35mg/ml的劑量處理下之細胞內的dopa含量迅速減少。
荖葉多元酚粗萃物與細胞共同培養後，細胞內外的氧化狀態都較對照組低，然而荖葉多元酚之氧化產物（37℃，72hr）不但未能抑制黑色素生成與釋放，反而還有促進的效果，此兩種截然不同的現象可能是因為荖葉多元酚粗萃物之氧化產物所造成的。

The facial leukomelanosis not only affects the appearance but also possibly does damage to health. In Taiwan, steamed Piper betle leaf was used as a bleaching agent. Dermatologists documented a kind of severe facial leukomealnosis to the use of dressing with washed and steamed Piper betle leaf. The bleaching effect was evident sometimes, but some cases showed hyperpigmentation after such treatment. The underlying mechanisms for the bleaching and hyperpigmentation need to be understood. In this study, melanoma cells will be used as a model system to realize the role of Piper betle leaf on the formation of melanin.
Results clearly showed that Piper betle leaf polyphenol extracts inhibited the formation and secretion of melanin by melanoma cells in a dose-dependent manner (10~50 g/mL) after forty-eight hours incubation. However, hyperpigmentation (secretion into medium) was found after seventy-two hours and longer incubation. The tyosinase activity was strongly inhibited by Piper betle polyphenol extracts in vitro, but promotion was found in melanoma cells after longer incubation (> 48 hours). Dopa level was used to evaluate the function of sample on melanoma cells and formation of melanin. Piper betle polyphenol extracts increased dopa levels within 48hrs. But dopa contents were greatly decreased after 72hr incubation. To further understand the role of Piper betle leaf polyphenol extracts on the hyperpigmentation and promotion of tyrosinase, the oxidized Piper betle leaf polyphenol extracts were used to evaluate again. Results showed the oxidation of Piper betle leaf greatly affect the performance of melanin formation and secretion by melanoma cells.

表次‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥•‥‥‥‥‥‥‥‥‥‥I
圖次‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥II
中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥Ⅳ
英文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥Ⅴ
壹、 前言‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
貳、 文獻探討‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥2
一、 荖葉•‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥…‥2
二、 黑色素•‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥6
1. 黑色素之生合成••‥‥‥‥‥‥‥‥‥‥‥‥‥‥6
2. 酪胺酸酶與黑色素之生合成•‥•‥‥‥‥‥‥‥9
3. 人體荷爾蒙跟黑色素細胞••‥‥‥‥‥‥‥‥‥9
三、 荖葉與黑色素‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥10
四、 皮膚‥‥‥‥‥•‥‥•‥•‥‥‥‥‥‥‥‥‥‥‥11
1. 皮膚的組成構造‥‥‥‥•‥‥•‥‥‥‥‥‥‥11
2. 皮膚顏色之決定因素‥‥‥‥‥‥‥‥‥‥•‥‥12
參、 研究目的‥‥‥‥‥‥•‥‥‥‥‥‥‥‥‥•‥‥‥‥‥13
肆、 試驗設計‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥14
伍、 材料與方法‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
一、 試驗材料‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥15
（ㄧ）荖葉‥‥‥‥‥‥‥‥‥‥‥•‥‥‥‥‥‥‥‥‥‥15
1. 荖葉來源‥‥‥‥‥‥‥‥‥‥‥‥‥•‥‥‥‥15
2. 荖葉多元酚粗萃物的製備‥‥‥‥‥‥•‥‥‥‥15
（二）試驗細胞株‥‥‥‥‥‥‥‥‥‥‥‥•‥‥‥‥‥‥15
二、 試驗用藥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16
三、 試驗儀器‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥19
四、 試驗方法‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥21
（ㄧ）細胞培養‥‥‥‥‥‥‥•‥‥‥‥‥‥‥‥‥‥‥‥21
1. PBS製備‥‥‥•‥‥‥‥•‥‥‥‥‥‥‥‥‥‥21
2. DMEM培養液的製備‥‥‥‥‥‥‥‥‥‥•‥‥21
3. 冷凍細胞活化‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥21
4. 細胞培養‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥22
5. 細胞冷凍保存‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥22
6. 細胞計數‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
（二）荖葉多元酚粗萃物對黑色素生成之影響‥‥‥‥‥‥‥23
1. 黑色素含量測定‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
A. 蛋白質定量‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
B. 細胞內黑色素含量測定‥‥‥‥‥‥‥‥‥‥24
C. 細胞外黑色素含量測定‥‥‥‥‥‥‥‥‥‥24
2. 酪胺酸酶活性測定‥‥‥•‥•‥‥•‥•‥‥‥‥‥25
A. 細胞外‥‥‥‥‥‥‥•‥‥‥‥‥‥•‥‥‥25
I. 總吸光度之測定方法‥‥•‥•‥‥‥•‥‥25
II. 酪胺酸酶空白吸光度之測定方法•‥•‥•‥25
III. 多元酚粗萃物空白吸光度之測定方法•‥•25
IV. 總空白吸光度之測定方法‥•‥‥•‥•‥‥26
V. 抑制酪胺酸酶活性之抑制率計算方法••‥•26
B. 細胞內‥‥‥‥‥•‥‥‥‥•‥‥‥‥‥‥•‥26
3. 萃取物對dopa氧化還原的影響‥‥•‥•‥‥•‥‥27
A. 高效能液相層析‥‥‥‥‥•‥‥‥•‥‥‥‥27
B. dopa以高效能液相層析定量•‥‥‥‥‥‥‥27
4. 荖葉多元酚粗萃物對黑色素細胞生長之影響‥••‥29
（三）細胞內外氧化之測定‥‥•‥‥•‥•‥‥‥•‥‥‥‥‥29
1. 細胞內TBARS測定‥‥‥•‥‥‥•‥‥•‥‥‥•‥29
2. 細胞外總抗氧化測定‥‥‥•‥‥•‥‥‥••‥‥‥29
（四）荖葉多元酚粗萃物氧化產物對黑色素瘤細胞之影響•‥30
五、 統計分析‥‥‥‥‥‥‥‥‥‥•‥‥‥‥•‥‥‥‥30
陸、 結果與討論‥‥‥‥‥‥‥‥‥‥•‥‥‥‥‥‥•‥‥‥31
一、 荖葉多元酚粗萃物對B16-F1黑色素瘤細胞生長之影響31
（ㄧ）B16-F1黑色素瘤細胞型態之影響‥‥‥‥‥‥‥•‥‥31
（二）B16-F1黑色素瘤細胞數量之影響‥‥‥‥‥‥‥•‥‥31
（三）B16-F1黑色素瘤細胞之蛋白質定量‥‥‥‥•‥‥‥‥35
二、 荖葉多元酚粗萃物對B16-F1黑色素瘤細胞黑色素生成與
釋放之影響‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥35
（ㄧ）B16-F1黑色素瘤細胞內黑色素生成之影響‥•‥‥•‥35
（二）B16-F1黑色素瘤細胞黑色素釋放之影響‥‥‥•‥•‥39
三、 荖葉多元酚粗萃物對細胞內外氧化狀態之影響‥•‥‥42
（ㄧ）B16-F1黑色素瘤細胞內氧化狀態之影響‥‥‥•‥‥‥42
（二）B16-F1黑色素瘤細胞外氧化狀態之影響‥‥‥‥‥•‥42
四、 荖葉多元酚粗萃物對酪胺酸酶活性之影響‥‥•‥‥•45
（ㄧ）酪胺酸酶（in vitro）活性之影響‥‥‥•‥‥•‥•‥45
（二）B16-F1黑色素瘤細胞內酪胺酸酶活性之影響‥••‥‥47
五、 荖葉多元酚粗萃物對細胞內dopa含量之影響‥‥••‥47
六、 荖葉多元酚粗萃物之氧化產物對B16-F1黑色素瘤細胞內
外黑色素含量之影響‥‥‥‥‥‥‥‥‥‥‥‥‥‥50
（ㄧ）B16-F1黑色素瘤細胞內黑色素含量之影響‥‥‥‥‥‥50
（二）B16-F1黑色素瘤細胞外黑色素含量之影響‥‥‥‥•‥52
七、 荖葉多元酚粗萃物之氧化產物對細胞內外氧化狀態之影
響‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥•‥‥‥‥•‥‥52
（ㄧ）B16-F1黑色素瘤細胞內氧化狀態之影響‥•‥‥•‥‥52
（二）細胞外氧化狀態之影響‥‥‥‥•‥‥‥‥‥‥‥‥‥55
八、 荖葉多元酚粗萃物之氧化產物對B16-F1黑色素瘤細胞內
酪胺酸酶活性之影響‥‥•‥‥‥‥‥•‥‥‥•‥‥‥57
九 、 荖葉多元酚粗萃物之氧化產物對B16-F1黑色素瘤細胞內
dopa含量之影響‥‥•‥‥‥‥‥‥‥•‥‥‥•‥‥‥59
柒、 結論‥‥‥‥‥•‥‥‥•‥‥‥‥‥‥‥•‥‥‥•‥‥‥61
捌、 參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥62

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