臺灣博碩士論文加值系統

表面素（Surfactin）是由水產微生物－枯草桿菌（Bacillus subtilis）所產出之
二次代謝產物，經由非核醣體合成機制所產出，為一環型脂胜肽並具有兩性分子
的特色，其疏水端容易與細胞膜作用，具有抗菌、抗病毒、抗腫瘤、抗凝血及皮
下助滲劑等生物活性。本研究之目的為利用自泰國魚池中所分離之枯草桿菌
（Bacillus subtilis TH）以半固態式發酵法所生產的表面素，對小鼠胚胎纖維母細
胞及人類皮膚纖維母細胞之抗老化影響，進一步探討以半固態式發酵之表面素純
化方法、皮下滲透功能及其安全性試驗。在表面素活化抗老化功能之實驗中，結
果顯示添加25 μM 表面素可有效提高sirtuin I 及sirtuin III 抗老化基因表現，而
添加50 μM 表面素可使纖維母細胞於紫外線照射下活存率提高，並以氧化壓力
緊迫處理下，添加75 μM 表面素可有效提高細胞存活率與修復能力。在細胞增
生的實驗中，分別以75 μM 及100 μM 之表面素處理後，可使小鼠胚胎纖維母細
胞及人類皮膚纖維母細胞有明顯增生的現象，並發現於小鼠胚胎纖維母細胞中其
膠原蛋白之蛋白表現量提高，並可抑制膠原蛋白分解酶－基質金屬蛋白脢9 型。
在皮下助滲試驗中，添加50 ppm 表面素可提升奈米金吸收效果。在經皮吸收實
驗中，表面素於2 小時作用下有9.7 ％之皮下滲透率。在物質安全性試驗中，表
面素對細胞毒性分類為2 級，在皮膚敏感性及刺激性上皆未發現不良效果。本研
究表明由半固態式發酵法所生產之表面素純度與商用標準純品比較其純度約為
35 ％，且以質譜儀定性結果分析，推測本實驗室所生產的表面素其主要分子量
為1022 Da，由7 個胺基酸與C14 脂肪酸碳鏈構成。由本研究結果發現表面素可
有效提高細胞抗老化功能及細胞基質增生，並協助奈米金進入皮層中，且表面素
可作為抗老化活化劑、皮下助滲劑，未來可將應用於抗老藥妝品及醫療藥品上。

Surfactin is an amphipathic cyclic lipopeptide molecule derived from aquatic
probiotics, Bacillus subtilis, secondary metabolites. This simple molecule also looked
very promising as an anti-bacteria, anti-virus, anti-tumor, anti-coagulant and
transdermal penetration enhancer. The objectives of present study are used surfactin
produced by Bacillus subtilis derived from fishpond in Thailand, 1） to know the
surfactin anti-aging function and the effects of mouse embryo fibroblast and human
skin fibroblast aging mechanism, 2） to new purification for surfactin products from
semi-solid state fermentation method, 3）to study the function of transdermal
penetration enhancer and safety test. The present results showed that surfactin can
up-regulated the expression of sirtuin I and sirtuin III effectively and proliferation of
fibroblast by treatment with 25 μM to 100 μM surfactin with culture medium.
Surfactin also can increased cell survival rate and repair ability after oxidative stress
and UV-A damage, and induced collagen and inhibited in MMP-9 of fibroblast. Using
surfactin as a transdermal penetration enhancer, the results for permeability assay
demonstrated that 50 ppm purified surfactin was capable in enhancing the
permeability of gold-nanoparticle. The result for percutaneous absorption indicated
that 9.7 ％ subcutaneous penetration with treated surfactin to 2 hours. Surfactin
safety test showed that the degree of cytotoxicity was 2, having weak allergenic
potential and no skin irritation. These results indicated that surfactin could induce
anti-aging function and extracellular matrix by efficacious stimulation on the
fibroblast. Surfactin consists of a heptapeptide headgroup with the sequence
Glu-Leu-D-Leu-Val-Asp-D-Leu-Leu closed to a lactone ring by a C14 β-hydroxy fatty.
Surfactin also enhancing the penetration of Au-NPs. Surfactin serve as anti-aging
activator and transdermal penetration enhancer, and it might be applied in anti-aging
cosmeceutical and pharmaceutical industry.

謝辭................................................................................................................................. i
中文摘要....................................................................................................................... iii
Abstract ........................................................................................................................ iv
目錄................................................................................................................................ v
表目錄.......................................................................................................................... vii
圖目錄......................................................................................................................... viii
壹、前言........................................................................................................................ 1
一、藥妝品簡介與全球市場導向分析 .................................................................... 1
二、人類皮膚結構及其老化因子 ............................................................................ 2
三、人類抗老化蛋白之探討 .................................................................................... 6
四、表面素（Surfactin）之介紹 ............................................................................. 7
五、表面素之生物活性 ............................................................................................ 9
六、胜肽於化妝品之應用 ...................................................................................... 13
七、本研究之目標 .................................................................................................. 14
貳、實驗材料 ............................................................................................................. 16
參、實驗方法 ............................................................................................................. 18
一、表面素生產菌株之培養 .................................................................................. 18
二、以半固態發酵生產表面素 .............................................................................. 18
三、表面素之純化及分析 ...................................................................................... 18
四、細胞培養及分選採樣分析 .............................................................................. 19
五、表面素對人類皮膚纖維母細胞及小鼠胚胎纖維母細胞之抗老化功能試驗
.................................................................................................................................. 21
六、表面素對小鼠胚胎纖維母細胞之膠原蛋白增生效果測定 .......................... 24
七、表面素對皮膚穿透之影響 .............................................................................. 25
八、表面素安全性測試 .......................................................................................... 26
肆、結果...................................................................................................................... 28
一、Bacillus subtilis TH 表面素定性及定量分析 ................................................. 28
二、以二氯甲烷萃取法之表面素其二氯甲烷殘留量分析 .................................. 28
三、表面素乙醇純化萃取法之粗產物分析 .......................................................... 28
四、Bacillus subtilis TH 生產之表面素對人類皮膚纖維母細胞以及小鼠胚胎纖
維母細胞抗老化基因sirtuin 1 與人類皮膚纖維母細胞sirtuin 3 基因表現
量之影響 .......................................................................................................... 29
五、Bacillus subtilis TH 生產之表面素對人類皮膚纖維母細胞以及小鼠胚胎纖
維母細胞抗光老化修護能力之影響 .............................................................. 29
vi
六、Bacillus subtilis TH 生產之表面素對小鼠胚胎纖維母細胞抗氧老化修護能
力之影響 .......................................................................................................... 30
七、Bacillus subtilis TH 生產之表面素對人類皮膚纖維母細胞以及小鼠胚胎纖
維母細胞其細胞存活率之影響 ...................................................................... 30
八、Bacillus subtilis TH 生產之表面素對小鼠胚胎纖維母細胞膠原蛋白增生之
影響 .................................................................................................................. 31
九、Bacillus subtilis TH 生產之表面素對皮膚吸收奈米金（2 nm goldnanoparticles）
之影響 .............................................................................................................. 31
十、Bacillus subtilis TH 生產之表面素經皮吸收率分析 ..................................... 31
十一、Bacillus subtilis TH 生產之表面素對細胞毒性、皮膚刺激性以及皮膚敏
感性試驗 .......................................................................................................... 32
伍、討論...................................................................................................................... 33
一、Bacillus subtilis TH 表面素定性及定量 ......................................................... 33
二、半固態式發酵法生產之表面素其乙醇純化萃取法 ...................................... 33
三、Bacillus subtilis TH 生產之表面素刺激抗老化基因 sirtuin 1 與sirtuin 3 基
因表現量 .......................................................................................................... 34
四、Bacillus subtilis TH 生產之表面素提高細胞抗光、氧老化修護能力 ......... 34
五、Bacillus subtilis TH 生產之表面素對細胞存活率之效果 ............................. 35
六、Bacillus subtilis TH 生產之表面素對增生細胞膠原蛋白之效果 ................. 35
七、Bacillus subtilis TH 生產之表面素增加皮膚物質滲透率及經皮吸收之效果
.................................................................................................................................. 36
八、Bacillus subtilis TH 生產之表面素其物質安全性 ......................................... 36
陸、參考文獻 ............................................................................................................. 37
柒、圖表...................................................................................................................... 45
附錄.............................................................................................................................. 76

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