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研究生:姜櫳宣
研究生(外文):Lung-Hsuan Chiang
論文名稱:奈米/次微米山藥對人類皮膚細胞膠原蛋白生合成與雌激素活性表現之影響
論文名稱(外文):Effects of nano/submicron yam on secretion of collagen and expression of estrogenic activity
指導教授:葉安義葉安義引用關係
口試委員:盧訊張永和黃青真蔣丙煌
口試日期:2013-01-04
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:130
中文關鍵詞:山藥介質研磨膠原蛋白雌激素活性
外文關鍵詞:yammedia millingcollagen secretionestrogenic activity
相關次數:
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山藥為薯蕷屬多年生蔓生性根莖類植物,富含生理活性成分(如固醇類皂素與植物固醇),為合成部份固醇類荷爾蒙的重要前驅物。山藥皮是廢棄物,卻有高量的薯蕷皂配基(diosgenin),可調節皮膚老化現象,將其製成奈米/次微米懸浮液,不僅可增加活性成分的吸收與利用,同時減少廢棄物的產生。
以介質研磨方法製備奈米/次微米山藥懸浮液,山藥之粒數平均粒徑小於100 nm,以掃描式電子顯微鏡確認奈米粒子的存在。山藥未去皮,可大幅提升山藥中膳食纖維與有效成分(diosgenin、stigmasterol與beta-sitosterol)之含量。奈米/次微米山藥經過基因毒性試驗,證實其對S. typhimurium TA98及TA100不具毒性,也不具有致突變性,對於添加4-NQO致突變劑誘導致突變之菌株也具有抗致突變效果。利用奈米/次微米山藥處理皮膚纖維母細胞WS1,發現其膠原蛋白分泌量增加,奈米/次微米山藥可促進細胞分泌hTGF beta與EGF,促進膠原蛋白的分泌,並減少MMP-1分泌,減少膠原蛋白分解。奈米/次微米山藥可被REA酵母菌利用,影響雌激素活性之表現。奈米/次微米磨山藥之雌激素當量為4.87×10-8 micro g/g乾重,為細碎山藥之1.22倍,且可造成MCF-7細胞凋亡與壞死,具有抑制乳癌細胞MCF-7與子宮內膜癌細胞Ishikawa增生之功效。

Nano/submicrometer-scaled yam particles have been prepared by using media-milling. The particle size of media-milled yam was determined by the laser light scattering method and was confirmed by scanning electron microscopy. Influences of nano/submicron yam on skin fibroblast cells (WS1) were evaluated. The size reduction did not significantly alter the proximate composition, and the presence of nanoparticles was not toxic to WS1 cells. Nano/submicron yam does not have mutagenicity as well but exhibits antimutagenicity ability. The contents of bioactive compounds (diosgenin, stigmasterol and β-sitosterol) were significantly increased by media-milling, which enhanced the secretion of hTGF-β and inhibited the formation of MMP-1. Thus, the collagen secretion from WS1 was significantly increased by size reduction. Diosgenin was employed as a positive control. Nevertheless, media-milled yam exhibited greater effects on WS1 cells than diosgenin. It appeared that both diosgenin and size reduction were helpful for enhancing the secretion of collagen by WS1 cells. In addition, the irritancy of yam was eliminated by media-milling. The effect of nano/submicron yam on estrogenic activity of REA yeast was also discussed. Nano/submicron yam inhibited proliferation of human breast cancer cell MCF-7 and human endometrial adenocarcinoma Ishikawa cells though increasing cell apoptosis and necrosis.
In conclusion, reducing size into nano/submicron scale of yam retained diosgenin and fiber for functional food/neutricosmetics and minimized the quantity of waste.


目錄 CONTENTS
摘要 I
ABSTRACT II
目錄 CONTENTS IV
研究架構 XIII
研究目的 XIV
第壹章 文獻回顧 1
一、 研究背景 1
二、 山藥 2
1. 簡介 2
2. 品種與產地分佈 2
3. 栽種條件 3
4. 一般成分與特殊保健成分 5
三、 奈米科學與技術 9
1. 奈米材料特性: 9
2. 奈米材料的製備 10
3. 奈米技術於食品與化妝保養品之應用 11
4. 奈米材料之毒性研究 14
四、 皮膚老化與病變 18
1. 正常皮膚結構 18
2. 膠原蛋白 22
3. 皮膚老化理論 27
4. 雌激素與皮膚老化 31
第貳章 材料與方法 36
一、 材料與方法 36
1. 基隆山藥 36
2. Ames test使用菌株 36
3. WS1細胞株 36
4. 微生物與細胞培養藥品 37
5. 實驗設備 38
6. 奈米/次微米山藥懸浮液之製備 39
7. 粒徑分布分析 39
8. 型態觀察 40
9. 奈米/次微米山藥基因致突變性評估 40
10. 奈米/次微米山藥細胞毒性評估 42
(1) 細胞存活率測試: 43
(2) AlamarBlue (AB) assay 43
11. 基本成分分析 44
12. 薯蓣皂配基與植物固醇含量分析 47
13. 第一型膠原蛋白生合成 48
14. 生長因子分析 49
15. MMP-1分泌測定 51
16. 奈米/次微米山藥對於基因改造酵母菌表現雌激素活性之影響 52
(1) 基因重組酵母菌 52
(2) 鑑定用Primers104: 52
(3) 培養藥品 52
(4) PCR鑑定 53
(5) 酵母菌生長曲線 54
(6) 酵母菌雌性激素測試法 (Rikilt Yeast Estrogen bioassay) 54
(7) 雌激素當量(estradiol equivalent) 55
17. 奈米/次微米山藥抑制MCF-7乳癌細胞與Ishikawa子宮內膜癌細胞增生試驗 55
18. 統計分析: 58
第參章 結果與討論 59
一、 奈米/次微米山藥製備與安全性評估 59
1. 型態觀察 59
2. 粒徑分布 62
3. 致突變與抗致突變性 64
4. 細胞存活率 70
5. 成分分析 73
二、 人類皮膚纖維母細胞膠原蛋白生合成之影響 76
1. 膠原蛋白分泌量 76
2. 生長因子與基質金屬蛋白酶MMP-1分析 80
三、 雌激素活性研究 84
1. 酵母菌雌激素試驗 84
2. 樣品對MCF-7細胞與Ishikawa細胞增生之影響 91
3. MCF-7細胞凋亡分析 99
結論 102
參考文獻 103



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