臺灣博碩士論文加值系統

天然萃取保養品深受女性所青睞，其中減少黑色素生合成是近年來的重要美學議題。色素沉澱產生是用於保護皮膚不受到 UV 所造成 DNA 損傷的正常機制，但卻會造成膚色增深、膚色不均、色斑沉著等肌膚問題，是許多想要肌膚白皙、透亮等愛美女性的困擾。本研究探討的植物為一年生藤蔓植物─落葵，其嫩枝葉可作蔬菜，全株可作藥用，涼血，外敷治癰毒，其含有豐富的多醣，具有保濕功效，但在美白及抵禦紫外線照射功效卻未被探討。本研究以小鼠 B16-F0 黑色素細胞及 WI-38 纖維母細胞，作為體外實驗模式，觀察落葵萃取物抑制黑色素、防禦紫外線的效果及其黑色素生合成路徑。體內動物模式，以斑馬魚美白動物模式，觀察落葵萃取物抑制斑馬魚黑色素生成之變化。本實驗目的，旨在於探討落葵萃取物是否能開發作為天然保養品，並進一步觀察在透過黑色素刺激荷爾蒙 (α-melanocyte-stimulating hormones; α-MSH) 介導作用下，其萃取物是否能抑制黑色素的生合成。結果顯示在體外實驗中，熱水或酒精萃取物對纖維母細胞均無細胞毒殺性，並具有抵禦 UV 照射能力且能降低介白素 6 (IL-6) 與腫瘤壞死因子 (TNF-α)；另外在黑色素瘤細胞中，熱水與酒精萃取皆能抑制黑色素生成及抑制酪胺酸酶之活性，在降低膠原蛋白及 MMP-1 表現量中則以酒精萃取較佳。未來可將落葵萃取物運用於化妝品、保養品添加，作為一種新的抗黑色素生合成及抵禦紫外線的機能性成分。

Reduce melanin biosynthesis is a major aesthetic issues in recent years. It is a skin problem for many beauty-conscious women. Pigmentation protects the skin from UV caused by the normal mechanism of DNA damage, but it will cause increased to discolorations, blotchiness, and sun and age spots, pigmentation spots. The Basella alba is an annual vine, tender leaves as a vegetable; whole plant for medicinal, refreshing and cooling, and topical treatment, which is rich in polysaccharides, has a moisturizing effect, but in the whitening effect is unknown. In this study, as a experimental model in vitro, first, the α-melanocyte-stimulating hormones (α-MSH) treated the mice B16-F0 melanoma cells to observe the effective of extract of Basella alba inhibits melanin biosynthesis; second, to explore the effective of UV resistance, use the extract of Basella alba to the WI-38 fibroblasts cell. In vivo, the zebrafish as an animal models to whitening, observe the extract of Basella alba inhibit melanogenesis. Therefore, the purpose of this experiment is to explore whether the extract of Basella alba can inhibit the α-MSH signal pathway and the biosynthesis of melanin. Finally, the hot water extracts or ethanol extracts has effective of UV resistance and down regulates the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in fibroblast cell; inhibited tyrosinase activity and melanogenesis of melanoma cell. Moreover, the ethanol extracts increases expression of collagen I and reduced expression of MMP-1. In the future, the polysaccharides plant extract can be used for applications from cosmetics to the pharmaceutical industry, as the new anti-melanin biosynthesis ingredients.

目錄
摘要 I
Abstract IV
目錄 VII
附圖次 X
附表次 XI
圖次 XII
表次 XIII
縮寫表 XIV
第一章 導論 1
第一節 皮膚黑色素生成之機轉 1
1.1 前言 1
1.2 黑色素生成之路徑 2
1.3 抑制酪胺酸酶活性 3
第二節 抑制黑生素生成之路徑 5
第三節 基質金屬蛋白酶-1 (Matrix Metalloproteinase-1, MMP-1) 與膠原蛋白第一型 (Collagen I) 7
第四節 發炎性相關蛋白質：介白素-6 (interleukin-6, IL-6) 及 腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α) 8
第五節 落葵 (Basella alba) 9
第二章 研究動機 10
第一節 研究背景 10
第二節 研究目的 10
第三章 實驗材料 11
第一節 材料 11
1.1 實驗儀器 11
1.2 藥品試劑 12
第四章 實驗方法與步驟 14
第一節 實驗流程 14
第二節 落葵萃取 15
1. 萃取流程 15
2. 多醣含量檢測法 - 酚-硫酸比色法(Phenol-sulfuric acid assay) 16
第三節 細胞培養 17
1. 解凍細胞 17
2. 細胞培養 17
3. 冷凍細胞 17
4. 細胞計數 18
第四節 細胞存活率分析 (MTT assay) 18
第五節 黑色素含量檢測 (Measurement of Melanin Content) 19
第六節 細胞激素 TNF-α、IL-6 之分析 19
第七節 防禦 UV 照射之檢測 19
第八節 自由基清除活性分析 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Assay 19
第九節 總抗氧化能力測試 (TEAC Assay)： 20
第十節 西方墨點法 (Western blot) 21
10.1 細胞蛋白質萃取 21
10.2 樣本蛋白質濃度的定量分析 21
10.3 聚丙烯醯胺膠體電泳 22
10.4 西方墨點分析法 22
第十一節 抑制能力確認測試： 22
第十二節 斑馬魚動物模式 23
第十三節 統計分析 23
第五章 實驗結果 24
第一節 落葵以熱水萃取及酒精萃取多醣含量之比較 24
第二節 落葵對纖維母細胞 (WI-38) 細胞存活率之影響 24
第三節 落葵於纖維母細胞 (WI-38) 抵禦 UV 照射之檢測 24
第四節 落葵於細胞激素 IL-6、TNF-α之分析 24
第五節 落葵於纖維母細胞 (WI-38) 膠原蛋白增生檢測 (Collagen I hyperplasia assay) 25
第六節 落葵於纖維母細胞 (WI-38)基質金屬蛋白酶-1 (Matrix Metalloproteinase-1, MMP-1) 抑制率分析 25
第七節 落葵於黑色素瘤細胞 (B16-F0) 細胞存活率之影響 25
第八節 落葵於黑色素瘤細胞 (B16-F0) 黑色素含量之檢測 25
第九節 落葵萃取物於酪胺酸酶抑制率、清除DPPH自由基能力、總抗氧化能力試驗 (TEAC Assay) 之影響 26
第十節 落葵萃取物於斑馬魚黑色素生成之影響 26
第六章 討論 27
第一節 落葵萃取物應用於美白實驗之探討 27
第二節 落葵萃取物應用於斑馬魚黑色素生成之探討 29
第七章 結論與未來展望 31
第八章 圖表 32
参考文獻 40

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