臺灣博碩士論文加值系統

中文摘要
　　本研究主要利用有機合成方法，將原兒茶酸(protocatechuic acid)及咖啡酸(caffeic acid)進行酯化或醯胺化，得到一系列之酯類(編號1 ~ 10)及醯胺類(編號11 ~ 16)衍生物，經由清除DPPH自由基能力的測試、抑制酪胺酸酶(Tyrosinase)活性的測試、MTT細胞毒性試驗及人體之有效性評估，探討其在化粧品上的應用價值。
　　在清除DPPH自由基結果方面發現：原兒茶酸之酯化衍生物(編號1 ~ 7)比醯胺化衍生物(編號11 ~ 13)，對DPPH自由基有較佳之清除效果，其中以Compound 3 (Propyl 3,4-dihydroxybenzoate)之IC50 = 2.48 μg/ml為最低。且原兒茶酸衍生物之抗氧化效果整體而言皆優於咖啡酸之衍生物。
　　在酪胺酸酶活性抑制的結果方面發現：原兒茶酸之含苯酯化衍生物(編號5~7)的抑制效果最好，其中以Compound 6 (2-phenyl- ethyl 3,4-dihydroxybenzoate)之IC50 = 13.61 μg/ml最低；而Compound 7 (3-phenylpropyl 3,4-dihydroxybenzoate)之IC50 = 16.41 μg/ml次之。此兩種化合物之抑制效果較原兒茶酸、咖啡酸、維生素C甚至麴酸更好。
　　在細胞毒性實驗結果方面發現：以Compound 1 ~ 4及11之存活率較高(IC50 = 102.74，103.67，98.24，95.92及93.07 μg/ml，皆大於或接近於100 μg/ml)。
　　在人體有效性試驗方面：經由DPPH抗氧化試驗、酪胺酸酶活性抑制試驗及MTT細胞毒性試驗中，挑選出具有抗氧化、美白且安全性高之化合物進行實際塗抹測試，發現以Compound 11 (N-hexyl 3,4-dihydroxybenzamide)之效果最好。
　　因此由本研究所合成之Compound 11 (N-hexyl 3,4-dihydroxy- benzamide)，同時具有低毒性、美白和抗氧化等特性，其合成步驟簡單，可進一步大量開發成具有美白兼抗氧化之複合幼藺吨帖~，因此在消費巿場上極具商品化之潛力。

Abstract
　　This research utilizes the method of organic synthesis to put protocatechuic acid and caffeic acid into esterification or amidation process so as to produce a series of esters (No. 1 ~ 10) and amides (No. 11 ~ 16). By way of tests on DPPH antioxidant capability, inhibit- tive capability on the vitality of tyrosinase, MTT cell toxicity test and appraisal of validity of human body, their practical values in cosmetics will be further discussed.
　　The experiment on antioxidant capability shows that: the alkyl ester esterification derivatives of protocatechuic (No. 1 ~ 7) have better effects to clean DPPH than amidation derivatives of proto- catechuic (No. 11 ~ 13), and IC50 = 2.48 μg/ml of Compound 3 (Pro- pyl 3,4-dihydroxybenzoate) is the lowest especially. And the antioxidant capability of the alkyl ester derivatives of protocatechuic are better than that of caffeic acid.
The experiment on inhibitive capability on the vitality of tyrosinase shows that: the alkyl ester esterification derivatives with benzene of protocatechuic acid (No. 5 ~ 7) have better inhibitive capability on the vitality, and IC50 = 13.61 μg/ml of Compound 6 (2-phenylethyl 3,4-dihy- droxybenzoate) is the lowest especially; IC50 = 16.41 μg/ml of Compound 7 (3-phenylpropyl 3,4-dihydroxybenzoate) is the inferior one. The inhibitive capability of these two compounds are better than protocatechuic acid, caffeic acid, vitamin C even kojic acid.
The experiment on cell toxicity shows that: survival percentage of Compounds 1 ~ 4 and 11 is the highest (IC50 = 102.74, 103.67, 98.24, 95.9 and 93.07 μg/ml，are larger than or approach to 100 μg/ml).
The experiment of validity on human body shows that: by way of DPPH antioxidant capability, inhibitive capability on the vitality of tyrosinase and MTT cell toxicity test, to pick the compounds that have anti-aging, whitening functions and high safety to carry on the practical test by spreading. It is found that the effect of Compound 11 (N-hexyl 3,4-dihydroxybenzamide) is best.
It is found that the Compound 11 (N-hexyl 3,4-dihydroxybenzamide) made in this research can be easily synthesized and has high rate of production, low toxicity, whitening and anti-aging functions, which can be further developed into cosmetics with whitening and anti- aging functions and therefore has great potential in consumer market.

本 文 目 錄
中文摘要……………………………………………………………………… I
英文摘要……………………………………………………………………… III
謝誌…………………………………………………………………………… V
本文目錄……………………………………………………………………… VII
表目錄………………………………………………………………………… X
圖目錄………………………………………………………………………… X I

一、緒論……………………………………………………………………… 1
1-1 前言…………………………………………………………………. 1
1-2 研究動機……………………………………………………………. 1
1-3 研究目的……………………………………………………………. 2
1-4 研究架構…………………………………………………………..... 3
二、文獻回顧………………………………………………………………… 4
2-1 自由基簡介…………………………………………………………. 4
2-1-1 氧屬自由基(ROS)………………………………………….. 4
2-1-2 氮屬自由基(RNS)…………………………………………... 7
2-1-3 自由基傷害…………………………………………………. 7
2-2 抗氧化簡介…………………………………………………………. 9
2-2-1 抗氧化之作用機轉………………………………………….. 10
2-2-2 皮膚之抗氧化保護………………………………………….. 11
2-3 黑色素簡介…………………………………………………………. 12
2-3-1 人體膚色…………………………………………………….. 12
2-3-2 黑色素的形成機轉………………………………………….. 13
2-3-3 黑色素體的形成及轉移…………………………………….. 15
2-3-4 影響酪胺酸酶活性之因子………………………………….. 16
2-3-5 影響皮膚顏色深淺之決定因子…………………………….. 17
2-4 美白簡介……………………………………………………………. 18
2-4-1 美白作用機轉……………………………………………….. 19
2-4-2 行政衛生署公告之美白成分……………………………….. 21
2-5 美白及抗氧化成份開發趨勢………………………………………. 21
三、實驗……………………………………………………………………… 24
3-1 實驗材料……………………………………………………………. 24
3-1-1 藥品來源…………………………………………………….. 24
3-1-2 溶液配製…………………………………………………….. 26
3-1-3 實驗儀器…………………………………………………….. 26
3-2 實驗方法……………………………………………………………. 27
3-2-1 原兒茶酸與咖啡酸之酯化及醯胺化反應………………….. 27
3-2-2 清除DPPH自由基能力之測試………………………….…. 28
3-2-3 抑制酪胺酸酶活性測試………………………………….…. 29
3-2-4 細胞存活率測試………………………………………….…. 30
3-2-5 人體之美白有效性評估………………………………….…. 31
四、結果與討論……………………………………………………………… 34
4-1 合成之化合物………………………………………………………. 34
4-1-1 合成protocatechuic acid之衍生物………………………… 34
4-1-2 合成caffeic acid之衍生物..................................................... 36
4-2、清除DPPH自由基能力之探討………………………………….. 37
4-2-1 清除DPPH自由基能力之實驗結果…………………………. 37
4-2-2 原兒茶酸烷鏈酯化衍生物清除DPPH自由基之能力…….. 39
4-2-3 原兒茶酸含苯酯化衍生物清除DPPH自由基之能力…….. 40
4-2-4 原兒茶酸醯胺化衍生物清除DPPH自由基之能力……….. 41
4-2-5 咖啡酸含苯酯化衍生物清除DPPH自由基之能力……….. 42
4-2-6 咖啡酸醯胺化衍生物清除DPPH自由基之能力………….. 43
4-3 抑制酪胺酸酶活性之探討…………………………………………. 45
4-3-1 抑制酪胺酸酶活性測試結果………………………………. 45
4-3-2 原兒茶酸烷類酯化衍生物抑制酪胺酸酶之活性…………. 47
4-3-3 原兒茶酸含苯酯化衍生物抑制酪胺酸酶之活性………. 48
4-3-4 原兒茶酸醯胺化衍生物抑制酪胺酸酶之活性……………. 49
4-3-5 咖啡酸含苯酯化衍生物抑制酪胺酸酶之活性……………. 50
4-3-6 咖啡酸醯胺化衍生物抑制酪胺酸酶之活性……………. 51
4-4 細胞毒性-MTT細胞存活率之探討……………………………….. 53
4-4-1 MTT細胞存活率測試結果…………………………………. 53
4-4-2 原兒茶酸烷類酯化衍生物之細胞存活率…………………. 55
4-4-3 原兒茶酸含苯酯化衍生物之細胞存活率…………………. 57
4-4-4 原兒茶酸醯胺化衍生物之細胞存活率…………………. 58
4-4-5 咖啡酸含苯酯化衍生物之細胞存活率…………………. 60
4-4-6 咖啡酸醯胺化衍生物之細胞存活率………………………. 61
4-5 皮膚有效性之探討…………………………………………………. 63
4-5-1 皮膚有效性評估……………………………………………. 63
4-5-2 L*值………………………………………………………….. 65
4-5-3 a*值………………………………………………………....... 67
4-5-4 b*值…………………………………………………………... 69
五、結論……………………………………………………………………… 71
六、參考文獻………………………………………………............................ 72
七、附錄……………………………………………………………………… 79

表目錄
表一、抑制黑色素的成分與機制類型……………………………………… 20
表二、材料及供應商………………………………………………………… 24
表三、O/W型乳化配方……………………………………………………… 31
表四、色差的分類等級……………………………………………………… 33
表五、不同濃度樣品之DPPH自由基清除能力…………………………… 37
表六、不同濃度樣品對酪胺酸酶活性之抑制率…………………………… 45
表七、不同濃度樣品之細胞存活率………………………………………… 53
表八、測試期間之皮膚色澤變化……………………………………………. 63
表九、志願者使用前後皮膚色澤之變化量…………………………………. 64

圖目錄
圖1、研究流程圖……………………………………………………………. 3
圖2、自由基之氧化還原過程………………………………………………... 6
圖3、自由基與皮膚老化的機轉……………………………………………. 9
圖4、生物體內之抗氧化防禦機轉…………………………………………. 10
圖5、黑色素形成的反應機轉………………………………………………. 14
圖6、黑色素形成及移轉之示意圖…………………………………………. 15
圖7、人體內黑色素細胞調節因子之簡圖…………………………………. 17
圖8、Protocatechuic acid之抗氧化機轉…………………………………….. 22
圖9、色差的標示值(ΔE) …………………………………………………… 32
圖10、原兒茶酸衍生物之結構……………………………………………… 35
圖11、咖啡酸衍生物之結構………………………………………………… 36
圖12、合成衍生物清除DPPH自由基能力之IC50………………………… 38
圖13、原兒茶酸烷鏈酯化衍生物清除DPPH自由基之能力……………… 39
圖14、原兒茶酸含苯酯化衍生物清除DPPH自由基之能力……………… 40
圖15、原兒茶酸醯胺化衍生物清除DPPH自由基之能力………………… 41
圖16、咖啡酸含苯酯化衍生物清除DPPH自由基之能力………………… 42
圖17、咖啡酸醯胺化衍生物清除DPPH自由基之能力…………………… 43
圖18、合成衍生物抑制酪胺酸酶活性之IC50……………………………… 46
圖19、原兒茶酸烷類酯化衍生物抑制酪胺酸酶之活性…………………… 47
圖20、原兒茶酸含苯酯化衍生物抑制酪胺酸酶之活性…………………… 48
圖21、原兒茶酸醯胺化衍生物抑制酪胺酸酶之活性……………………… 49
圖22、咖啡酸含苯酯化衍生物抑制酪胺酸酶之活性……………………… 50
圖23、咖啡酸醯胺化衍生物抑制酪胺酸酶之活性………………………… 51
圖24、合成衍生物之細胞存活率比較圖…………………………………… 54
圖25、原兒茶酸烷類酯化衍生物之細胞存活率比較圖…………………… 56
圖26、原兒茶酸含苯酯化衍生物之細胞存活率比較圖………………… 57
圖27、原兒茶酸醯胺化衍生物之細胞存活率比較圖……………………… 59
圖28、咖啡酸含苯酯化衍生物之細胞存活率比較圖…………………… 60
圖29、咖啡酸醯胺化衍生物之細胞存活率比較圖………………………… 61
圖30、實驗期間△L值變化之比較………………………………………… 66
圖31、△L值之比較………………………………………………………… 66
圖32、實驗期間△a值變化之比較……………………………….………… 68
圖33、△a值之比較…………………………………………………………. 68
圖34、實驗期間△b值變化之比較………………………………………… 69
圖35、△b值之比較…………………………………………………………. 70

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