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研究生:吳明謙
研究生(外文):Ming-Chang Wu
論文名稱:高粱酒糟水萃物之美白功效暨其對小鼠黑色素腫瘤細胞凋亡之影響
論文名稱(外文):Effects of Sorghum Distillery Residue Water Extracts on Whitening and Apoptosis of Mus musculus Melanoma Cell
指導教授:徐源泰徐源泰引用關係
口試委員:曾文聖劉育姍陳增蔚王鐘毅
口試日期:2015-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
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:95
中文關鍵詞:高粱酒糟小鼠黑色素腫瘤細胞式細胞儀細胞凋亡酪胺酸酶
外文關鍵詞:Sorghum distillery residueMus musculus Melanoma Cellflow cytometrytyrosinase
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  高粱(Sorghum bicolor)為禾本科高粱屬,全球主產區有美國、非洲與南亞,糧食作物種植面積為世界排名第五位,在加工釀造過程產生大量的副產品-高粱酒糟,亦稱高粱酒粕。如何將酒糟再利用是許多學者研究的方向,過去最主要的再利用方式為堆肥和畜牧飼料,像是以高粱酒糟取代黃豆參合在飼料中餵食吳郭魚,可使魚體具有抗寒之能力;過去已有研究指出高粱酒糟萃取物,具抗氧化之能力及對酪胺酸酶具抑制作用;部份研究亦指出高粱中萃取之單寧對黑色素腫瘤細胞具生理上之影響,因此高粱酒糟若是可抑制黑色素腫瘤細胞,不但能提升加工副產品價值也能進一步提供抑制黑色素腫瘤之研究方向。本研究除了分析高粱酒糟水萃物 (SWE) 之生物活性物質,亦將萃取物施予B16F10小鼠黑色素腫瘤細胞,探討造成細胞凋亡之現象。SWE中總酚含量為21.50 ± 0.051 mg gallic acid equivalent (GAE)/g;總類黃酮含量為26.60 ± 0.013 mg querectin equivalent (QE)/g;DPPH捕捉自由基能力的IC50值為0.44 ± 0.011 mg/ml。而體外酪胺酸脢抑制試驗的IC50值約1.790 ± 0.042 mg/ml,SWE施於B16F10小鼠黑色素腫瘤細胞,以MTS assay檢驗在8 mg/ml達到76.65%細胞存活率。以不同濃度SWE處理細胞濃度為1x105 cell/ml的黑色素腫瘤細胞並檢測其胞內黑色素含量,當萃取物濃度為10 mg/ml時可達到37.54%之黑色素生成抑制率。此外,以流式細胞儀分析annexin-V/PI雙染試驗,在水萃物10 mg/ml時有最高細胞早期凋亡率25.57%和最高細胞晚期凋亡率63.91%,在2.5 mg/ml時有最高細胞壞死率4.47%。隨後以PI染色分析不同時期之DNA含量,顯示高粱酒糟水萃物對B16F10具有生長停滯(cell arrest)現象,以10 mg/ml SWE處理24小時sub-G1 phase比例升高至7.9%。以濃度10、5、2.5、1.25、0.625 mg/ml施予B16F10小鼠黑色素腫瘤細胞,並在12小時、24小時、48小時測量live-cell protease、dead-cell protease、caspase 3/7活性,結果顯示在48小時最高濃度10 mg/ml時會有最高caspase3/7活性、dead-cell protease活性。

  Sorghum bicolor, Poaceae Family, Sorghum species, occupies the fifth most cultivated area of cereal crops of the world. The several major cultivated regions include the North of America, Africa and South of Asia. In China, Sorghum is used to produce liquor by fermenting and distilling. After liquor processing, tremendous amount of sorghum distillery residues are remained, and applications of making great value of these byproducts have been an important issue. In this study, to confirm the skin whitening ability of sorghum distillery residues, several basic bioactive assays of sorghum distillery residue water extracts (SWE) were conducted. Total phenolic acid content was 21.50 mg Gallic acid equivalent (GAE) /g; Total Flavonoids content was 26.60 mg Querectin equivalent (QE) /g; DPPH scavenging ability concentration of IC50 was 0.4421 mg/ml; Vitro Tyrosinase inhibition concentration of IC50 was 1.790 mg/ml. Malignant melanoma cells are originated from melanocytes, pigmented cells, which are health-threaten cancer of skin. In this research, B16F10 melanoma cell from Mus musculus were treated with different concentration of SWE from 0.3125 to 10 mg/ml. Afterwards, MTS assay and melanin contents were performed, and Annexin-V/PI staining apoptosis and PI staining cell cycle were both carried out by using FC500 Flow Cytometry. The results indicates at 8 mg/ml SWE treatment, cell viability reach 76.65%, and SWE inhibit melanin synthesis by 37.54% at 10 mg/ml. As for apoptosis and cell cycle analysis, SWE treatment has highest early apoptosis rate 25.57%, late apoptosis rate 63.91% at 10 mg/ml, and numbers of cell in Sub-G1 phase (cell arrest) increase from 3.0 % (vehicle control) to 7.9% (10 mg/ml SWE). Assay of live-cell protease, dead-cell protease & caspase-3/7 by Fluorescence & luminescence. The results indicated that after treated with SWE 48hrs had highest caspase activity, and after treated with 24hrs had highest dead-cell protease activity.

第一章 前言 1
第二章 前人研究 2
第一節 高粱 2
第二節 高粱生物活性成分 2
一、酚酸 2
二、單寧 3
三、花青素 3
第三節 流式細胞儀 13
一、流式細胞儀原理 13
二、流體系統 13
三、光學與電子系統 13
四、訊號分析 14
五、流式細胞儀沿革 14
第四節 流式細胞儀與細胞凋亡之研究 20
一、細胞凋亡之概述 20
二、以流式細胞儀分析細胞凋亡情形 20
第五節 皮膚與黑色素腫瘤細胞 24
一、黑色素與黑色素細胞 24
二、黑色素生成途徑 24
三、黑色素與自由基生成 25
四、美白效果評估 26
五、美白成分 27
第七節 研究目的 36
第三章 實驗材料與方法 37
第一節 實驗架構 37
第二節 樣品製備 38
一、材料萃取前製備 38
二、材料萃取 38
第三節 樣品基本生物活性分析 38
一、高粱酒糟水萃物總酚含量測定 38
二、總類黃酮含量測定 39
三、DPPH清除能力測定 40
第四節 黑色素生成相關試驗 40
一、體外酪胺酸酶含量測定 40
二、抑制細胞黑色素生成能力試驗 41
第五節 細胞培養 43
一、細胞繼代 43
二、 細胞低溫保存 44
三、細胞活化 45
四、細胞計數 45
第六節 高粱酒糟水萃物於B16F10小鼠黑色素瘤細胞凋亡之影響 46
一、B16F10黑色素腫瘤細胞型態觀察 46
二、B16F10黑色素腫瘤細胞活性測定 47
三、B16F10黑色素腫瘤細胞annexin-V/PI雙染分析 49
四、B16F10黑色素腫瘤細胞PI染色細胞週期分析 50
五、B16F10黑色素腫瘤細胞凋亡蛋白Caspase3/7活性測定 51
第七節 統計分析 53
第四章 結果與討論 53
第一節 高粱酒糟萃取物基本生化活性試驗 53
一、萃取物之萃取率 53
二、SWE總酚含量 53
三、SWE總類黃酮含量 53
四、萃取物DPPH清除能力 54
第二節 黑色素生成相關試驗 54
一、萃取物體外酪胺酸酶活性抑制能力 54
二、不同濃度萃取物抑制B16F10黑色素腫瘤細胞黑色素生成能力 55
第三節 高粱酒糟萃取物於B1610小鼠黑色素瘤細胞凋亡之影響 55
一、萃取物影響B16F10黑色素瘤細胞形態觀察 55
二、B16F10黑色素腫瘤細胞活性測定 56
三、B16F10黑色素腫瘤細胞凋亡測定 56
四、B16F10黑色素腫瘤細胞週期分析 57
五、B16F10黑色素腫瘤細胞凋亡蛋白分析 58
第五章 結論 86
第六章 引用文獻 87


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