臺灣博碩士論文加值系統

摘 要
近年來許多研究報告指出，過多的自由基會對人體的健康造成危害，如動脈粥狀硬化症、高血壓和癌症的發生等，陸續也有研究證實蔬果中之抗氧化物質，可防禦自由基的攻擊，降低對人體疾病的發生率；本研究以冬瓜為研究對象，對冬瓜各部位作機能性之比較探討。冬瓜(Wax gourd)學名為Benincasa hispida Cogn, 屬於葫蘆科植物。本文研究方向有四，一是藉由多重檢測方法來評估冬瓜之抗氧化效能，由AAPH誘發亞麻油酸氧化並以碘化鉀呈色法來評估抑制亞麻油酸氧化情形、利用DPPH法評估冬瓜清除自由基能力、以TBARS法評估LDL氧化情形、藉由Folin Ciocalteu試劑分析總多酚化合物之含量並利用SOD活性染色法，鑑定SOD活性之表現；其二是評估冬瓜萃取液對ACE酵素活性之抑制能力研究；其三為藉由Muller Hiton洋菜培養基培養不同的菌株，由抑菌環的之測量結果，判定冬瓜萃取液之抑菌能力；其四是採用MTT法檢定冬瓜萃取液刺激纖維母細胞後，檢定細胞存活數。實驗結果指出，在碘化鉀呈色法中，不同部位之冬瓜萃取液在抑制50% 11.45mM亞麻油酸氧化率之效果最佳者為冬瓜籽，冬瓜芯、冬瓜皮次之。在清除DPPH自由基的測定結果顯示，以水萃取之冬瓜籽清除自由基效果最好，由TBARS法評估LDL經銅離子誘發氧化之結果顯示，以50%甲醇萃取之冬瓜籽抑制LDL氧化的表現效果最佳，以新鮮萃取之冬瓜芯對LDL氧化抑制氧化效果最差；新鮮冬瓜各部位之總多酚物質的測定，以每克新鮮冬瓜肉中含相對於沒食子酸之當量濃度（gallic acid equivalent, GAE）最多，冬瓜芯最少。從SOD的活性染色結果證實，冬瓜籽中的SOD活性的表現最強，冬瓜皮最弱；於ACE酵素活性抑制試驗中，新鮮萃取液中冬瓜肉汁對於抑制ACE酵素活性有顯著的效果，達89%(p<0.05)，在乾燥濃縮物萃取物中，冬瓜籽對於ACE之抑制效果，較另三者佳；在抑菌能力試驗上，不管是新鮮萃取或是濃縮的冬瓜萃取物，對菌株皆具有不具擴散性之抑菌效果；在細胞存活檢定結果顯示，新鮮過濾之冬瓜汁在促進細胞存活能力上沒有顯著性結果。因此，實驗結果證實，冬瓜萃取物具有清除自由基及抑制脂質過氧化的效果、降低ACE 酵素活性之作用和抑制細菌生長的作用等，但無法由此判斷其對上皮細胞增生之效果。

Abstract
It is well know that free radical represents in the cell and tissue danger thus leading to various disease. Many results have been shown that plant-derived antioxidants could prevent and delay oxidation damage in cell and tissue.
In this study, we try to investigate the potential health benefits of different component of wax gourd extract have not been studied. The objective of this study was divided to four parts. First, to assess the antioxidant properties of these materials, we have used a range of established assays to analysis the total antioxidants and capability of prevention lipid peroxidation, KI and DPPH assay were used to determine the total antioxidant activity. The total polyphenolic compounds were determined according to the Folin-Ciocalteu’s procedure. TBARS assay used human LDL as an index of antioxidants against status in vitro, and SOD activity stain assay to verify SOD in wax gourd. Second, used ACE inhibition assay to verify whether the wax gourd content ACE inhibitor. Third, cultured different bacterias on the different Muller Hitione agar medium and measured antibacterial activity of wax gourd extract by paper disk diffusion. Fourth, used MTT assay to check fibroblast cell proliferation rate offer cell stimulated by fresh wax gourd extract.
In the KI assay, wax gourd seed had highest antioxidant capability, then wax gourd core and wax gourd peel. Water-extracted wax gourd seed had best performance in scavenging DPPH radical assay , wax gourd seed also had best result in inhibition of LDL oxidation in TBARS assay, and fresh wax gourd core extract had lowest inhibition rate of LDL oxidation. Fresh pulp part of wax gourd had highest polyphenol content 169.1 GAE（gallic acid equivalent concentration, mM）, then peel part of wax gourd 151.1 GAE, seed part 148.0GAE and core 109.5GAE. The seed part of wax gourd expressed the highest SOD activity than the core, pulp and peel part of wax gourd. All the parts of wax gourd had positive result in ACE inhibitor assay, but the wax gourd seed show the strongest result than others. In antimicrobial assay, neither fresh sample extract nor condense wax gourd sample extract had diffusible antimicrobial result. There was no significant result in MTT assay. These results suggested that wax gourd may prevented lipid peroxidation, provided good antioxidants source, prevented hypertension, and antimicrbials ability to the human.

目 錄
頁數
中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ-Ⅲ
一、緒論
1. 自由基………………………………………………………….1
1.1 何謂自由基…………………………………………….1
1.2 自由基的種類………………………………………….1
1.3 體內自由基的產生與作用…………………………….2-3
1.4 自由基對人體的傷害………………………………… 3-5
1.5 自由基與疾病的發生………………………………….5-7
2. 抗氧化物質…………………………………………………….8
2.1 抗氧化物質之功能……………………………………..8
2.2 抗氧化物質之來源與分類……………………………..8-9
2.3 抗氧化劑之相關研究………….………………………9-10
2.4 抗氧化物質與疾病治療之關係…...………………….10-11
3. 天然抗氧化物質研究…………………………………………11
3.1 蔬果類與抗氧化………………………………………11-12
3.2 蔬果與疾病關係………………………………………12-13
3.3 其他物質……………………………………………..13-14
4. 研究動機……………………………………………………..14
4.1 冬瓜之簡介…………………………………………..14
4.2 瓜類之相關醫學研究………………………………..15-16
5. 研究架構……………………………………………………..17
二、材料與方法………………………………………………………18
1. 實驗材料……………………………………………………..18
1.1 藥品……………………………………………………..18
1.2 菌種……………………………………………………..18-19
1.3 細胞……………………………………………………..19
1.4 微生物培養基…………………………………………..19
1.5 細胞培養基……………………………………………..19-20
1.6 試劑與緩衝液…………………………………………..20-21
2. 實驗方法………………………………………………………21
2.1 樣品製備………………………………………………21
2.2 抗氧化試驗分析………………………………………22
亞麻油酸氧化分析法.…………………..……………..22-23
DPPH自由基清除率檢測法……………….…………23-24
低密度脂蛋白氧化分析法………….………...………24-26
總多酚含量分析法………………….…….………..…26-27
超氧歧化脢活性染色鑑定法………………….………27
2.3 抑制ACE的活性測定………………………………..28
2.4 抑菌能力試驗…………………………………………29
2.5 細胞存活檢定法………………………………………29
細胞續代培養…………...…………………………….29
MTT細胞存活檢定法………………………..………29-30
2.6 統計分析……………………………………………….30
三、結果……………………………………………………………….31
1. 冬瓜各部份之水分含量……………………………………….31
2. 冬瓜各部位之抗氧化活性比較……………………………31
2.1 抑制亞麻油酸氧化之活性比較法測定…..……………31-32
2.2 清除DPPH自由基能力測定………………………….32-33
2.3 人體低密度脂蛋白過氧化分析………………………..33
2.4 總多酚類化合物含量測定……………………………..33-34
2.5 超氧歧化酶活性染色鑑定……………………………...34
3. 對於抑制ACE酵素活性之測定結果…………………………34
3.1 新鮮冬瓜萃取液對ACE活性之抑制結果…………….34
3.2 乾燥後冬瓜萃取液對ACE活性之抑制結果………….34-35
4. 冬瓜萃取液之抑菌能力研究…………………………………35
4.1 新鮮冬瓜萃取液對抑菌能力影響……………………...35
4.2 乾燥濃縮5倍之冬瓜萃取液對抑菌之分析……………36
5. 不同濃度的新鮮冬瓜汁液對纖維母細胞之影響…………….36
四、討論………………………………………………………………..37-42
五、結論………………………………………………………………..43-44
圖表………………………………………………………………..45-67
六、參考文獻…………………………………………………………..68-82
圖目錄
圖一、血管細胞中自由基來源……………………………………………45
圖二、在免疫機制中誘發動脈粥狀動脈硬化之假說……………………46
圖三、細胞訊息傳遞和ROS/RNS間之作用及發展為動脈粥狀硬
化症之關係…………………………………………………47
圖四、氧化型低密度脂蛋白與心血管疾病發生之關係…………....48
圖五、類黃酮類物質之基本結構圖………………………………………49
圖六、多酚物質與金屬離子鍵結呈穩定之結構….………………………50
圖七、類黃酮清除ROS之機制圖……..….……………………………....51
圖八、SOD活性染色原理…..…………………………………………….52
圖九、冬瓜之體組成百分比……………………………………………….53
圖十、超氧岐化酶之活性染色分析.………………………………………54
圖十一、新鮮冬瓜萃取液在ACE酵素活性抑制之表現…………………..55
圖十二、抑菌實驗中抑菌環大小之判定………………………………..56
圖十三、冬瓜汁對纖維母細胞之存活能力影響………………………….57
表目錄
頁數
表一、多酚物質之主要類別……………………………………………58
表二、食物中所含類黃酮之分類………………………………………59
表三、冬瓜之各部位水份含量測定……………………………………60
表四、不同部位、不同萃取方式之冬瓜萃取液在碘化鉀呈色分析法
之結果……………………………………………………….61
表五、不同部位、不同萃取方式的冬瓜萃取液對清除DPPH自由基
能力測定結果……………………………………………..62
表六、不同部位、不同萃取方式之冬瓜萃取液對人體低密度脂蛋白
之氧化分析……………………………………………….63
表七、每克新鮮冬瓜樣品之總多酚物質相對於GAE當量濃度測定….64
表八、乾燥濃縮之冬瓜萃取液在ACE酵素活性抑制之表現………….65
表九、新鮮冬瓜之抑菌能力分析………………………………………..66
表十、冷凍乾燥濃縮之冬瓜抑菌能力分析…………………………..…67

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