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研究生:洪惠琳
研究生(外文):Hung, Hui-Lin
論文名稱:添加寡胜肽之漢方七白加減方之 美白效果
論文名稱(外文):The Effect of Oligopeptide Additives on the Skin-whitening Efficiency of Modified, Traditional Seven-white Skin-whitening Formula
指導教授:林鳳蘭黃吉法
指導教授(外文):Lin, Feng-LangHwang, Jyi-Faa
口試委員:施承典林鳳蘭王澤川黃吉法黃郁婷
口試委員(外文):Shih, Cheng-DeanLin, Feng-LangWang, Tzer-ChuanHwang, Jyi-FaaHuang, Yuh-Tyng
口試日期:2015-06-27
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:製藥科技研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:61
中文關鍵詞:中藥七白抗氧化酪胺酸酶抑制劑寡胜肽
外文關鍵詞:Seven-white Chinese herbal formullatrionantioxidationtyrosinase inhibitorsoligopeptide
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本論文目的在於探討如何以傳統七白之加減方為基礎,從其水萃出物及25%及50%乙醇萃出物中,篩選出較有潛力之美白及抗氧化之配方,接著單獨及合併添加幾種不同胜肽,探討是否能造成美白之加乘效果。本研究採用四、五、六、八和九胜肽天加入七白加減方,研究其對漢方七白美白效果之影響。
本研究之另一目的在建立具美白效果之化妝品劑型的功能性評估平台。因此,本研究也以B16黑色素瘤細胞為模型,評估添加胜肽之七白加減方萃出物對B16細胞之毒性及對其黑色素合成之抑制效果。
結果發現,整體而言,添加胜肽之七白傳統方及加減方之水萃物之酪胺酸酶抑制能力遠不如其乙醇萃出物。在添加四、五、六、八、九共五種寡胜肽之七白複方及其加減方乙醇萃出物中,以添加八、九胜肽之加減方之25及50%乙醇萃出物之酪胺酸酶抑制及美白效果較顯著。
就體外酪胺酸酶抑制率而言,添加3%胜肽之200 g/mL七白加減方之25%和50%乙醇萃出物之酪胺酸酶抑制率以添加桑白皮的F2-25-8P、F2-25-9P、F2-50-8P、F2-50-9P配方及添加兩倍量白芨之F5-25-8P、F5-25-9P、F5-50-8P、F5-50-8P之體外酪胺酸酶抑制率較佳 (酪胺酸酶抑制率 78.42 ~ 94.12%),且優於七白傳統方之25%和50%乙醇出物F1-25 及F1-50,酪胺酸酶抑制率依序為70.46和66.37%。美白效果最佳之F5-50-8P配方之酪胺酸酶抑制IC50 ~ 110 g/mL 與熊果素(IC50 ~ 112 g/mL)相當接近。
在十二位年齡19 ~ 45歲之女性自願受測者連續四週塗抹F5-50-8P萃出物面膜貼片(200 g 萃出物/mL +3%胜肽)之手臂皮膚貼布測試中,依Fitzpatrick皮膚膚色分類判斷,57%受測者的皮膚呈現有顯著的美白效果。
本研究中,美白效果最佳之配方F5-50-8P之酵素動力學參數測定結果顯示其Michaelis Menten常數KM ~ 0.25 mM,比α-熊果素之0.30 mM稍低,而其Michaelis Menten酵素抑制動力學圖形與無添加酪胺酸酶抑制劑者相比,顯示F5-50-8P屬於非競爭型酪胺酸及酪胺酸酶抑制劑。表示在F5-50-8P萃出物影響下,相對較少的基質(tyrosine)就可造成相同的皮膚致黑速率,換言之,F5-50-8P之酪胺酸酶抑制效果比α-熊果素稍弱。
在B16黑色素細胞瘤活性抑制及其黑色素合成抑制之初期試驗中,F5-50-8P萃出物顯現延緩B16黑色素瘤細胞成長之明顯效果。在抑制B16黑色素瘤細胞中黑色素合成之試驗中,添加F5-50-8P (100-1000 g/mL)配方之細胞酪胺酸酶活性抑制效果達到控制組(維生素C,200 g/mL)的75%,本研究認為結合體外及細胞酪胺酸酶抑制試驗、細胞毒性試驗及人體皮膚貼布試驗是有效的化妝品美白效果評估方法。

The objective of this study is to establish the skin-whitening efficiency of various oligo-peptide additives on modified, traditional seven-white skin-whitening formula. The possibility of synergistic effect of incorporating oligopeptide was explored.
A secondary objective of this study was to establish a platform for assessing the functional properties of cosmetic dosage forms made of Chinese herbal medicines. Therefore, a B16 melanoma cell model was used to assess the efficiency of herbal medicine extract with oligopeptides on cellular tyrosinase activity.
Our findings indicated that the tyrosinase ibhibitory activity of the peptide-incorporated aqueous extract of conventional and modified seven-white formula is generally inferior to its ethanolic extract counterparts. Among the tetra-, penta-, hexa-, octa- and nona-peptide incorporated, the octapeptide and nonapeptide significantly improved the skin-whitening and antityrosinase activity of 25% and 50 % ethanolic extract of modified seven-white formula.
The following 25% and 50% ethanolic extract of modified seven-white formulae with added morus alba L. (桑白皮): F2-25-8P, F2-25-9P, F2-50-8P and F2-50-9P,and the following formulae with doubled amount of bletilla striata (白芨): F5-25-8P, F5-25-9P, F5-50-8P and F5-50-8P demonstrated tyrosinase inhibition rate between 78.42% and 94.12% that are better than that of the 25% and 50% ethanolic extract of conventional seven-white formula, namely, F1-25 and F1-50 that exhibited tyrosinase inhibition rate of 70.46% and 66.37%, respectively. The tyrosinase inhibition IC50 of the best performed formula F5-50-8P with octapeptide is 110 µg/mL (cf. arbutin IC50 ~112 µg/mL)
During the four weeks skin patch test which involved 12 female volunteers, age 19 ~ 45, that applied our candidate extract in facial mask form (200 g/mL extract with 3% peptide), 57% of the tested volunteers showed improved skin tone according to Fitzpatrick’s skin typing.
The tyrosinase kinetic parameter of F5- 50-8P (50% ethanol extract of F5 formulations, containing octapeptide) revealed a Michaelis Menten constant KM of about 0.25 mM, slightly smaller than 0.30 mM of α-Arbutin, indicating that the affinity of F5-50-8P extract with tyrosine is considerably good. Furthermore, the pattern of its Michaelis Menten plot, compared with that without tyrosinase inhibitor, suggests that F5-50-8p is a non-competitive inhibitor of tyrosine and tyrosinase.
During preliminary test, the treatment of B16 melanoma cells with F5-50-8P formula showed considerable efficacy in retarding the growth of B16 melanoma cell, and in suppressing melanin synthesis within B16 melanoma cells. The F5-50-8P formula (200 g/mL extract with 3% peptide) demonstrated inhibitory effect equivalent to 70% that of the control standard vitamin C (200 g/mL) against melanin synthesis within B16 melanoma cells. This seems to suggest that the combination of in vitro and cellular tyrosinase inhibition test, cellular toxicity and human skin patch test is a useful platform to evaluate the skin-whitening efficiency of cosmetics.

中文摘要 III
Abstract VII
致謝 VIII
目錄 IXX
表目錄 XIII
圖目錄 XIII
第一章 緒言 1
第二章 文獻回顧 5
第一節. 胜肽化妝品的發展趨勢 5
第二節. 美白機制 11
第三節. 七白傳統方及加減方之中藥成分簡介 13
第三章 材料與方法 19
第一節. 實驗流程 19
第二節. 實驗材料 21
第三節. 實驗步驟與方法 25
(一). 中藥七白傳統方及加減方之萃取 25
(二). 添加胜肽之中藥七白加減方配方 25
(三). 七白傳統方及加減方萃出物之製備 26
(四). 七白傳統方及加減方萃出物之抗老化能力評估 26
(五). 美白的有效性試驗 28
(六). B16黑色素瘤細胞培養 29
(七).七白加減方萃出物對B16黑色素瘤細胞之存活率及細胞中黑色素生成抑制之影響 31
(1). 添加胜肽之七白加減方萃出物對黑色素細胞B16存活率之影響 ( MTT assay) 31
(2). 添加胜肽之七白加減方萃出物對抑制B16黑色素瘤細胞中黑色素生成之影響 32
(八). 酪胺酸酶酵素抑制動力學 32
(九). 含中藥萃取物之面膜美白效果評估之人體試驗 35
(1). 添加胜肽之七白加減方萃出物之不織布面膜配方製作 35
(2). 添加胜肽之七白加減方萃出物之美白有效性評估測試 36
(3). 面膜貼布試用效果評估方式 36
第四章 結果與討論 37
第一節. 七白傳統方及加減方中單一藥材於不同溶劑下之萃取產率 37
第二節. 添加胜肽之七白加減方萃出物之酪胺酸酶活性抑制能力及DPPH自由基捕捉能力評估 38
第三節. 添加胜肽之七白加減方萃出物於B16黑色素瘤細胞中黑色素生成抑制效果之評估 46
第四節. 添加胜肽之中藥七白加減方萃取物之細胞存活率試驗(MTT assay) 47
第五節. 酪胺酸酶酵素抑制動力學 48
第六節. 添加胜肽之七白加減方之美白劑型探討及有效性評估 50
第五章 總結論 54
第六章 參考文獻 56

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