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研究生:李懿純
研究生(外文):Lee Yi Chun
論文名稱:山藥對同半胱胺酸血症之抗氧化活性研究
論文名稱(外文):Study on the antioxidative activity of Dioscores in hyperhomocysteinema
指導教授:張素瓊張素瓊引用關係
指導教授(外文):Chang Sue Joan
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:同半胱胺酸同半胱胺酸血症甲硫胺酸山藥
外文關鍵詞:homocysteinhyperhomocysteinemiamethionineDioscores
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近來許多研究顯示,血漿同半胱胺酸(homocysteine, Hcy)稍微升高是造成心血管疾病(cardiovasular disease, CVD)的獨立危險因子。人類血漿Hcy正常範圍為5-15 mM,大於15 mM即為同半胱胺酸血症(hyperhomocysteinemia, HHcy)。本研究室近年發現,國人CVD患者血漿Hcy濃度顯著高於正常人(12.2±0.3 vs. 8.5±0.4 mM)。然而,HHcy導致CVD的機制未明,HHcy促進血管內活性氧生成所產生的氧化壓力導致低密度脂蛋白(low density lipoprotein)及內皮細胞(endothelial cell)膜脂質過氧化可能為主要原因。此外,過氧化現象亦會促進血小板吸附(adhesion)能力及血小板活性進而造成血栓症。故本研究利用餵食甲硫胺酸(methionine)誘發大白鼠HHcy後,餵以山藥(Dioscores)來探討其對HHcy及氧化壓力之影響。餵以出生六週的大白鼠(Sprague-Dawley)食用Met(1 g/Kg body wt./day)十週誘發HHcy。將HHcy大白鼠隨機分為五組:Met 組、Met+vitamin C組(200 mg/Kg body wt./day)及三個Met+山藥組(D1、D2及D3)。山藥劑量分別為1、2.5及5 g/Kg body wt./day。分別於食用山藥及vitamin C八週與十二週時抽血並犧牲老鼠。血漿以7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F)衍生化後,利用高效能液相層析儀(HPLC)分析血漿Hcy濃度。以血小板凝集儀(aggregometer)偵測thrombin(3.2 U/ml)誘發性血小板凝集反應。利用thiobarbituric acid reactive substances (TBARS)反應,以malondialdehyde(MDA)表示脂質過氧化。利用luminal當探針,以化學冷光偵測器(chemiluminescent)分析肝臟ROS含量。利用酵素動力學分析肝臟中抗氧化酵素。肝臟glutathione(GSH)和 Glutathion disulfide(GSSG)以O-Phthalaldehyde衍生化後,利用螢光分光光度計(Fluorescence spectrophotometer)分析。
結果發現, Met fed十週後,血漿Hcy濃度由6.7±1.0 mM顯著增加至 21.7±4.5 mM。食用三個劑量山藥八~十二週後,血漿Hcy濃度顯著比未食山藥(Met)組低,且與控制組相近。食用中及高劑量山藥十二週後,血小板凝集反應顯著比未食山藥(Met)組小。食用三個劑量山藥十二週後,也使血漿MDA濃度及肝臟ROS含量顯著比未食山藥(Met)組低,並與控制組相似。當大白鼠食用中及高劑量山藥十二週後,肝臟之catalase活性顯著比未食山藥(Met)組高。然而,食用山藥對肝臟GSH及GSSG沒有顯著影響。
本實驗顯示,食用山藥可以恢復因Met所誘發的HHcy。此外,HHcy可藉由攝取山藥而減少血小板凝集、脂質過氧化及氧化壓力。本研究結論,山藥對高蛋白飲食(高Met攝取),可能誘發的HHcy與氧化壓力導致的相關疾病具有保護功能,值得推廣山藥成為保健食品。
Recently, a moderately elevated plasma homocystein (Hcy) level has been identified as an independent risk factor for cardiovascular disease (CVD). Normal range of Hcy levels is 5-15 mM in human plasma. Plasma concentration of Hcy > 15 mM is hyperhomocysteinemia (HHcy). In this laboratory, we found that patients with CVD had significantly higher plasma Hcy concentration than normal person (12.2 ± 0.3 vs. 8.5± 0.4 mM). However, the mechanism by which HHcy leads to CVD is not known. HHcy-induced oxidative stress due to the production of reactive oxygen species (ROS) resulted in oxidation of low density lipoprotein (LDL) and endothelial membrane may contribute to the possible mechanism. In addition, peroxidation in cell membrane could activate platelet and promote adhesion leading to thrombosis. Therefore, the purpose of this study was to investigate the antioxidative effects of Dioscores in methionine (Met)-induced HHcy animals. HHcy was induced by oral feeding Met (1 g/Kg body wt./day) for 10 weeks in Sprague-Dawley rats at the age of 6 weeks. HHcy rats were randomly divided into five groups: Met group without Dioscorea feeding, Met+vitamin C group supplemented with vitamin C 200 mg/Kg body wt./day, and 3 Dioscores groups (D1, D2 and D3) supplemented with freeze-dried Dioscorea powder 1, 2.5 and 5 g/Kg body wt./day, respectively. Animals were sacrificed after 8 or 12 weeks of Dioscorea feeding. Plasma levels of Hcy were assayed based on HPLC of the fluorescent 7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F) derivative. Platelet aggregation induced by thrombin (3.2 U/ml) was analyzed with an aggregometer. Plasma lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) expressed in malondialdehyde (MDA) equivalents. ROS generation was monitored with luminometer using luminal as the probe. Activities of antioxidative enzymes were measured by kinetics of enzymes using UV spectrophotometer. Hepatic concentrations of glutathione and glutathion disulfide were quantified by fluorescence spectrophotometer after derivatized with O-Phthalaldehyde.
The results of this study showed that plasma Hcy levels were significantly increased from 6.7±1.0 to 21.7±4.5 mM after 10 weeks of Met feeding. Eight and 12 weeks after Dioscorea feeding, plasma Hcy of D1, D2, and D3 were significantly lower than that of Met, and similar to the basal level prior to Met feeding. Twelve weeks after Dioscorea feeding, thrombin-induced platelet aggregation of D2 and D3 were significantly lower than that of Met group. Plasma MDA levels and hepatic ROS of D1, D2, and D3 were similar to that of Ctl group and significantly lower than that of Met group after 12 weeks of Dioscorea feeding. The activities of hepatic catalase in D2 and D3 groups were significantly elevated compared to Met group at 12 weeks. However, feeding Dioscorea 8 or 12 weeks did not significantly change hepatic levels of GSH and GSSG.
Results of our study indicated that HHcy induced by Met could be reversed by Dioscorea feeding. In addition, Dioscorea feeding could alleviate platelet aggregation, lipid peroxidation, and oxidative stress in HHcy. The protective effects of Dioscores feeding in oxidative stress induced by high protein (Met) intake. Suggested Dioscores as a valuable functional food.
目 錄
頁數
中文摘要………………………………………………………… Ⅰ
英文摘要………………………………………………………… Ⅳ
致謝……………………………………………………………… Ⅶ
目錄……………………………………………………………… Ⅷ
表目錄…………………………………………………………… Ⅸ
圖目錄…………………………………………………………… Ⅹ
縮寫符號………………………………………………………..ⅩⅢ
第一章、 前言…………………………………………………… 1
第二章、 文獻探討……………………………………………… 4
第三章、 實驗材料及方法……………………………………… 16
第四章、 結果…………………………………………………… 36
第五章、 討論…………………………………………………… 47
參考文獻………………………………………………………… 57
表次……………………………………………………………… 69
圖次……………………………………………………………… 80
自述………………………………………………………………100
表 目 錄
頁數
表一、實驗動物飼料組成……………………………………… 69
表二、食用Methionine十週大白鼠的體重變化量…………… 71
表三、食用山藥八週大白鼠的體重變化量……………………. 72
表四、食用山藥十二週大白鼠的體重變化量…………………. 73
表五、不同山藥劑量與不同時間處理對變異數之影響………. 74
表六、不同山藥劑量處理對大白鼠血漿生化值及肝臟
酵素活性之比較…………………………………………. 75
表七、不同時間處理對大白鼠血漿生化值及肝臟酵素
活性之比較………………………………………….…… 75
表八、食用山藥八、十二週對大白鼠肝臟中
GSH/GSSG比率…………………………………………. 76
表九、Hcy與血漿生化值及肝臟酵素活性的相關性…………. 77
表十、Platelet Aggregation與血漿生化值及肝臟酵素
活性的相關性……………………………...……………. 78
表十一、MDA與血漿生化值及肝臟酵素活性的相關性……… 78
表十二、ROS與血漿生化值及肝臟酵素活性的相關性……… 79
圖 目 錄
頁數
圖一、Homocysteine代謝圖……………...……………….…… 80
圖二、抗氧化酵素作用……………...…………………….…… 81
圖三、Homocysteine造成動脈硬化機制…………………..…… 82
圖四、血漿Homocysteine層析圖譜...…………………….…… 83
圖五、血漿Vit. C層析圖譜………...…………………….…..… 83
圖六、食用Methionine十週前後大白鼠血漿中Hcy濃度…..… 84
圖七、食用山藥八週後大白鼠血漿中Hcy濃度……….………. 84
圖八、食用山藥十二週後大白鼠血漿中Hcy濃度…….………. 85
圖九、不同山藥劑量處理第八週及第十二週大白鼠
血漿Hcy濃度………………………...………………… 86
圖十、食用山藥八週後,大白鼠thrombin誘發性
血小板凝集反應………………………………………… 86
圖十一、食用山藥十二週後,大白鼠thrombin誘發性
血小板凝集反應…………………………………...…. 87
圖十二、不同山藥劑量處理第八週及第十二週大白鼠
thrombin誘發性血小板凝集反應…...……………….. 87
頁數
圖十三、食用山藥八週後,大白鼠血漿中MDA含量………… 88
圖十四、食用山藥十二週後,大白鼠血漿中MDA含量……… 88
圖十五、不同山藥劑量處理第八週及第十二週大白鼠
血漿中MDA含量………………………………….…. 89
圖十六、食用山藥八週後,大白鼠肝臟中ROS含量…………… 89
圖十七、食用山藥十二週後,大白鼠肝臟中ROS含量…..…… 90
圖十八、不同山藥劑量處理第八週及第十二週大白鼠
肝臟中ROS含量………………………..………….…. 90
圖十九、食用山藥八週後,大白鼠血漿中Vit. C濃度…….…. 91
圖二十、食用山藥十二週後,大白鼠血漿中Vit. C濃度…….…. 91
圖二十一、食用山藥八週後,大白鼠肝臟中SOD活性….……. 92
圖二十二、食用山藥十二週後,大白鼠肝臟中SOD活性….…. 92
圖二十三、食用山藥八週後,大白鼠肝臟中Catalase活性.…. 93
圖二十四、食用山藥十二週後,大白鼠肝臟中Catalase活性…. 93
圖二十五、不同山藥劑量處理第八週及第十二週
大白鼠肝臟中Catalase活性...…..…………….…… 94
圖二十六、食用山藥八週後,大白鼠肝臟中
Glutathione Peroxidase活性………………………. 94
頁數
圖二十七、食用山藥十二週後,大白鼠肝臟中
Glutathione Peroxidase活性………...…..………… 95
圖二十八、不同山藥劑量處理第八週及第十二週
大白鼠肝臟中Glutathione Peroxidase活性………. 95
圖二十九、食用山藥八週後,大白鼠肝臟中
Glutathione reductase活性……………..……..……. 96
圖三十、食用山藥十二週後,大白鼠肝臟中
Glutathione reductase活性……………………...…….. 96
圖三十一、食用山藥八週後,大白鼠肝臟中
Glutathione濃度.…………………………………… 97
圖三十二、食用山藥八週後,大白鼠肝臟中
Glutathione disulfide濃度……...…………………… 97
圖三十三、食用山藥十二週後,大白鼠肝臟中
Glutathione濃度…..…………………………...…… 98
圖三十四、食用山藥十二週後,大白鼠肝臟中
Glutathione disulfide濃度……...…………………… 98
圖三十五、不同山藥劑量處理第八週及第十二週
大白鼠肝臟中Glutathione 濃度…………………… 99
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