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研究生:曹婕鈴
研究生(外文):CAO, JIE-LING
論文名稱:以田口設計優化馬勃菇之萃取及功效研究
論文名稱(外文):Optimization of the Extraction and the Efficacy from Puffball Mushroom by Taguchi Design
指導教授:黃淑玲黃淑玲引用關係
指導教授(外文):HUANG, SHU-LING
口試委員:黃淑玲林永昇邱智東
口試委員(外文):HUANG, SHU-LINGLIN, YUNG-SHENGCHIU, CHIH-TUNG
口試日期:2020-07-15
學位類別:碩士
校院名稱:國立聯合大學
系所名稱:化學工程學系碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:142
中文關鍵詞:馬勃菇田口設計抗氧化LC-MS/MS抑菌性抗癌
外文關鍵詞:Puffball mushroomTaguchi designantioxidantLC-MS/MSantibacterialanti-cancer
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本研究利用田口設計萃取兩個成長階段的馬勃菇,分別為白色子實體(White fruiting bodies, WFB)與褐色孢子(Brown spores, BS),以田口直交表優化提取萃取物,並比較馬勃菇之成分分析及功效性評估。實驗結果顯示,由化學檢定分析中,BS具有高的總酚含量(47.37 ± 0.43 mg GAE/ g)與總蛋白質含量(538.27 ± 2.38 mg/ g)。在LC-MS/MS儀器分析中,鑑定出BS具有沒食子酸含量(0.304 mg/g)。抗氧化試驗中,以BS具有極佳之抗氧化能力,對DPPH自由基清除能力之半最大效應濃度(Concentration for 50% of maximal effect, EC50)為0.63 ± 0.01 mg/mL、ABTS陽離子自由基清除能力(EC50 = 1.23 ± 0.01 mg/mL)、還原能力(A700 = 1.323)及超氧化物歧化酶(EC50 = 0.29 ± 0.01 mg/mL)具有顯著之功效,和馬勃菇之成分分析有正相關。此外,WFB對金黃色葡萄球菌與綠膿桿菌具有好的抑菌性,BS對以上兩種菌皆無抑菌效應。抗癌試驗中,BS於5000 ppm下對抗人類結腸癌細胞(HT-29)之細胞存活率(21.31 ± 3.20%)與腫瘤化癌藥物5-FU(17.03 ± 3.20%)之抗癌效果相似。WFB在濃度5000 ppm以上對人類結腸癌細胞(HT-29)具有極強之毒殺性,HT-29細胞存活率約只有5%。因此,證實馬勃菇在天然抗氧化保健食品和降低大腸癌症風險具有很大的應用潛力。
In this study, Taguchi design method was used to find the best extraction conditions of puffball mushroom with two growth stages including white fruiting bodies (WFB) and brown spores (BS). The extraction of samples was optimized by a Taguchi orthogonal array to compare the component analysis and the efficacy evaluation for WFB and BS samples. The results show that the BS has high total phenolic content (47.37 mg GAE/g) and the total protein amount (538.27 ± 2.38 mg/ g). Using LC-MS/MS instrument analysis identified the gallic acid content of BS with 0.304 mg/g. In the anti-oxidation test, BS has better antioxidant ability than of those WFB. And the EC50of DPPH free radical scavenging ability was 0.63 ± 0.01 mg/mL, ABTS cationic free radical scavenging ability (EC50 = 1.23 ± 0.01 mg/mL), reducing ability (A700 = 1.323) and superoxide dismutase activity (EC50 = 0.29 ± 0.01 mg/mL) for BS. The anti-oxidation properties show a significant effect and the positive correlation with the composition analysis of puffball mushroom. In addition, WFB has a good antibacterial effect on S. aureus and P. aeruginosa, but that BS hasn’t antibacterial effect on the above two bacteria. The cell survival rate (21.31 ± 3.20%) of BS against human colorectal cancer cells (HT-29) at a concentration of 5000 ppm is similar to the anti-cancer drug 5-FU (17.03 ± 3.20%). WFB shows a very high toxic for human colon cancer cells (HT-29) at above 5000 ppm, and the survival rate of HT-29 cells is only about 5%. Therefore, the puffball mushroom has a great potential for application in the natural health foods of antioxidant and reduceing the risk of cancer.
誌謝 I
摘要 II
ABSTRACT III
目錄 IV
圖目錄 VIII
表目錄 XI

第一章 緒論 1
1.1 前言 1
1.2 研究目標 4

第二章 文獻回顧與原理 5
2.1 馬勃菇之相關研究 5
2.2 實驗設計 12
2.3 天然植物之成分分析 15
2.3.1 化學檢測定分析 15
2.3.2 LC-MS/MS儀器分析 16
2.4 植物性與動物性蛋白質 19
2.5 抗氧化機制(Antioxidant mechanism) 21
2.5.1 自由基(Free radicals) 21
2.5.2 抗氧化劑種類 24
2.5.3 抗氧化功效評估方法 32
2.6 美白能力 37
2.7 保濕機制 40
2.8 抗生素之殺菌與抑菌機制 45
2.9 細胞毒性(Cytotoxicity) 51
2.10 天然植物之抗癌機制 56

第三章 材料與方法 60
3.1 實驗架構圖 60
3.2 實驗器材與儀器 62
3.2.1 化學藥品 62
3.2.2 儀器設備 64
3.2.3 菌株 65
3.2.4 細胞株 65
3.3 田口設計 66
3.3.1 樣品前處理 66
3.3.2 田口直交表設計 66
3.4 成分分析 68
3.4.1 化學檢定分析 68
3.4.2 LC-MS/MS儀器分析 69
3.5 功效性評估 71
3.5.1 抗氧化試驗 71
3.5.2 美白能力 72
3.5.3 保濕能力 73
3.5.4 抗菌測定 73
3.5.5 抗癌測定 74

第四章 優化馬勃菇之萃取條件與成分分析 75
4.1 田口設計分析 75
4.1.1 白色子實體(WFB)與褐色孢子(BS)之粗萃分析 75
4.1.2 BS之田口直交表分析 76
4.2 以化學檢定分析成分 79
4.2.1 總酚含量測定 79
4.2.2 總黃酮含量測定 80
4.2.3 總蛋白質含量測定 81
4.3 以LC-MS/MS分析沒食子酸成分 83
4.4 結論 84

第五章 馬勃菇之功效性評估 86
5.1 抗氧化試驗 86
5.1.1 DPPH自由基清除能力測定 86
5.1.2 ABTS陽離子自由基清除能力測定 88
5.1.3 還原能力測定 90
5.1.4 超氧化物歧化酶測定 92
5.2 美白能力 93
5.2.1 抑制酪胺酸酶能力測定 93
5.3 保濕能力 95
5.4 抗菌測定 98
5.5 抗癌測定 102
5.5.1 體外細胞毒性分析 102
5.5.2 人類結腸癌細胞(HT-29)之抗癌分析 105
5.6 結論 108

第六章 總結與未來展望 110
6.1 總結 110
6.1.1 馬勃菇之成分分析 110
6.1.2 馬勃菇之功效評估 110
6.2 未來展望 112

參考文獻 113
個人資料表 128
個人學術著作 129

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