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研究生:王懷德
研究生(外文):Huai-Der Wang
論文名稱:大豆屬一條根抑制15-LOX成分之鑑定
論文名稱(外文):Identification of Anti 15-LOX Compounds in I-Tiao Gung (Glycine tomentella Hayata)
指導教授:孫寶年孫寶年引用關係
指導教授(外文):Bonnie Sun Pan, Ph. D.
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
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:2008
畢業學年度:96
語文別:中文
論文頁數:89
中文關鍵詞:一條根15-脂氧合?
外文關鍵詞:I-Tiao Gung15-lipoxygenase
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摘要
大豆屬一條根 (Glycine tomentella Hayata) 具有抑制不飽和脂肪酸自氧化、清除自由基、螯合鐵離子之能力及延緩低密度脂蛋白氧化。於吳郭魚低溫緊迫試驗中,可降低吳郭魚血液流動之起始應力、黏稠度及血小板 5-, 12-, 15- 脂氧合?活性。在一條根 95% 乙醇萃取物 (GTE) 中,確定含有 daidzein、daidzin、malonyldaidzin、genistein 及 glycitin,但尚有較強於 異黃酮類之抗氧化功效成分未被鑑定。本研究為找出 GTE 抗氧化力最高之成分,以抑制 15-脂氧合?之活性能力為篩選依據,並檢測其對環氧合?之抑制功效。
GTE 經乙酸乙酯萃取、silica gel 管柱層析後可得 5 個區分,以第三區分 (GTE-EA-III) 抑制 15-脂氧合?之能力最高,於濃度 2 μg/mL 之抑制率約為 92.76%。GTE-EA-III 經 silica gel 管柱層析得 GTE-EA-III-A~J 共十區分物,其中 GTE-EA-III-C於濃度 2 μg/mL 對 15-LOX 抑制率約為 98.70%,高於 genistein 且與脂氧合?抑制劑 (nordihydroguaiaretic acid, NDGA) 於濃度為0.2 μg/mL 之抑制率相似。GTE-EA-III-C 經 silica gel 管柱層析得 GTE-EA-III-C-a~m,共十三區分,經檢測抑制 15-脂氧合?活性,得知以 GTE-EA-III-C-f & g 區分抗氧化力較強,而其中以 GTE-EA-III-C-g 區分回收量較 f 區分高,因此進一步以半製備型 RP-HPLC分離 GTE-EA-III-C-g ,共分 GTE-EA-III-C-g-1~4 四區分物,GTE-EA-III-C-g-3 抑制 15-脂氧合?活性之 IC50 為 0.3 μg/mL,劑量為 NDGA 的 7.9倍。
GTE-EA-III-C-g-3的紫外光光譜中於 245 nm 與 345 nm 有吸收波峰,在 306 nm 有小肩峰;再以 LC/MS 分析,發現陽離子質量為 464 Da,以核磁共振光譜分析,初步推測為 prenylate alkaloids。
目錄
摘要 I
Abstract III
目錄 IV
圖目錄 VIII
表目錄 X
壹、前言 1
貳、文獻回顧 4
一、豆科植物之異黃酮 (isoflavone) 成分 4
二、一條根生理活性 5
1. 一條根之來源 5
2. 一條根的藥理作用 5
3. 一條根之成份及藥理活性 6
4. 闊葉大豆之毒性試驗 7
三、黃酮類與異黃酮化合物之結構 8
四、黃酮類對心血管之影響 10
1. 預防紅血球溶血 11
2. 預防心血管疾病 13
五、異黃酮類之生理活性 14
1. 減緩更年期症候群 15
2. 預防骨質疏鬆症 16
3. 預防癌症 16
4. 預防心血管疾病 17
六、黃酮類之抗氧化機制 18
1. 清除活性氧自由基 18
2. 螯合金屬離子 19
3. 抑制脂質氧化 20
七、發炎及過敏的發生機制 20
八、Lipoxygenase (LOX) 生成物及其生理活性 22
1. 5-Lipoxygenase (5-LOX) 之氧化產物及發炎反應 23
2. 12-Lipoxygenase (12-LOX) 氧化作用及相關疾病(Ref) 23
3. 15-Lipoxygenase (15-LOX) 氧化作用及對血管的影響 24
九、Cyclooxygenase (COX) 區分及氧化產物 25
十、黃酮類抑制 LOX 及 COX 之作用 26
?、 實驗流程 27
一、 一條根 28
二、 方法 28
1. 一條根萃取物 (GTE) 之製備 28
2. 一條根萃取物之分離 29
2.1 乙酸乙酯極性區分 29
2.2 silica gel 管柱層析 29
2.3 薄層層析分析 30
2.4 分離物之 HPLC 分析 31
2.5 半製備型HPLC分析 32
3. 抗氧化性分析測定脂氧合?總活性測定 32
4. LC/MS/MS測定分析 33
5. 核磁共振光譜 33
6. 統計分析 33
伍、結果與討論 34
一、一條根抗氧化成分之分離 34
1. 一條根經 95% 乙醇萃取 34
2. GTE經乙酸乙酯極性劃分 35
3. Silica gel 60 管柱層析 37
3.1 GTE-EA 之區分產率及對 15-LOX 抑制活性 37
3.2 GTE-EA-III 之區分產率及對 15-LOX 抑制活性 40
3.3 GTE-EA-III-C 之區分產率及對 15-LOX 抑制活性 43
4. GTE-EA-III-C-g半製備型 HPLC 之區分及對 15-LOX 抑制活性 46
二、 一條根區分物之鑑定 49
1. 異黃酮之 HPLC 圖譜檢測 49
1.1 GTE 及 GTE-EA 之RP-HPLC 圖譜比較 49
1.2 GTE-EA-I~V 區分之 RP –HPLC 圖譜 51
1.3 GTE-EA-III-A~J 區分之 RP-HPLC 圖譜 53
1.4 genistein之 LC/MS 圖譜與定量 59
1.5 GTE-EA-III-C-a~m 區分之 RP-HPLC 圖譜 65
2. 類黃酮分類之鑑定 70
陸、結論 82
柒、參考文獻 83
柒、參考文獻
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