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研究生:曾惠君
研究生(外文):Hui-Chun Tseng
論文名稱:苦瓜乙醇萃取物可能藉由抗氧化及抗發炎之作用機轉而具有改善小鼠酒精性脂肪肝之功效
論文名稱(外文):Liver Protective Function of Ethanol Extracts from Bitter Melon Against Alcoholic Fatty Liver in Mice by Attenuating Oxidative Stress and Inflammatory Response
指導教授:沈立言沈立言引用關係
指導教授(外文):Lee-Yan Sheen
口試委員:李宗貴邱智賢鍾景光黃青真
口試委員(外文):Chong-Kuei LiiChih-Hsien ChiuJing-Gung ChungChing-Jang Huang
口試日期:2013-07-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
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:2013
畢業學年度:101
語文別:中文
論文頁數:124
中文關鍵詞:酒精性脂肪肝酒精性肝臟疾病氧化壓力苦瓜抗氧化抗發炎
外文關鍵詞:alcoholic fatty liveralcoholic liver diseaseoxidative stressbitter melonantioxidant capacityinflammation
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肝臟為代謝酒精之主要器官,若過量攝取酒精會導致酒精性肝臟疾病(alcoholic liver disease, ALD),依其病程可分成三階段:第一階段為酒精性脂肪肝(alcoholic fatty liver),第二階段為酒精性肝炎及肝纖維化,最後第三階段形成肝硬化,其發病機制與酒精代謝所造成之氧化壓力及發炎反應密切相關。苦瓜(Momordica charantia L.)已有千年食用歷史,傳統用於改善糖尿病,並具有多種生理功效。文獻指出,苦瓜乙醇萃取物富含多酚類物質,相較於其他溶劑之萃取物,具較高之抗氧化能力。因此,本研究以Lieber-DeCarli酒精液態飼料誘導小鼠酒精性脂肪肝之動物模式,進行四種不同苦瓜品系乙醇萃取物之護肝功效評估。實驗分成六組,分別為正常控制組、酒精誘導脂肪肝負對照組及酒精誘導脂肪肝以苦瓜「花蓮1號」乙醇萃取物、苦瓜「花蓮2號」乙醇萃取物、苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物處理組,餵食劑量皆為500 mg/kg bw/day。結果顯示,餵食苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物可有效改善小鼠肝臟中脂肪堆積情形,並降低血清中肝臟生化功能指標酵素天門冬胺酸轉胺酶(aspartate transaminase, AST)及丙胺酸轉胺酶(alanine transaminase, ALT)之數值。且在餵食苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物後,可回復小鼠肝臟中抗氧化物質麩胱甘肽(glutathione, GSH)含量,與提升抗氧化酵素麩胱甘肽過氧化酶(glutathione peroxidase, GPx)、麩胱甘肽過還原酶(glutathione reductase, GRd)、過氧化氫酶 (catalase, CAT)及超氧化物歧化酶(superoxide dismutase, SOD)之活性,並改善肝臟脂質過氧化情形及降低肝臟發炎反應。進一步以西方墨點法評估相關蛋白表現量,結果顯示,給予苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物後,小鼠肝臟中SREBP-1c、FAS及ACC之蛋白表現量皆顯著降低,由此結果推論,苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物可能藉由改善肝臟發炎反應,而減少SREBP-1c表現量,進而降低FAS及ACC等酵素活性,因此可減緩酒精所導致之脂質堆積情形。而苦瓜「花蓮1號」乙醇萃取物及苦瓜「花蓮2號」乙醇萃取物亦具有減緩肝臟氧化壓力及發炎反應之能力,但改善肝臟脂肪堆積之效果不顯著。此外,以化學分析法檢測四種苦瓜之乙醇萃取物中抗氧化物質含量,結果顯示,苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物中,總酚化合物(total phenol)之含量皆顯著高於苦瓜「花蓮1號」乙醇萃取物及苦瓜「花蓮2號」乙醇萃取物。綜合上述之結果,苦瓜「花蓮3號」乙醇萃取物及苦瓜「花蓮4號」乙醇萃取物可能是藉由降低肝臟抗氧壓力及發炎反應而具有改善酒精性脂肪肝之功效。

The liver plays an essential role in metabolizing ingested ethanol, if excessive alcohol ingestion would lead to alcoholic liver disease (ALD). ALD results in alcoholic fatty liver, inflammation and fibrosis, and development of cirrhosis. The important roles in the pathogenesis of ALD are oxidative stress and inflammatory responses. Bitter melon (Momordica charantia L.) is an edible vegetable that has been used as a folk medicine to treat diabetes in India. And, it exhibits several biological effects, such as regulation of glucose metabolism, blood lipids, antioxidant potential, anti-inflammation, anti-cancer, and antimicrobial activities. In addition, it has been demonstrated that ethanol extract of bitter melon, containing abundant of polyphenols, possesses outstanding antioxidant capacity compared to other solvent extracts. Therefore, this study aims to investigate the liver protective capability of ethanol extract of bitter melon from four different cultivars in C57BL/6 mice which fed Lieber-DeCarli ethanol-containing liquid diet. Mice were fed with normal liquid diet, ethanol-containing liquid diet, or ethanol-containing liquid diet treated with Hualien No.1’ bitter melon extract (H1E), Hualien No.2’ bitter melon extract (H2E), Hualien No.3’ bitter melon extract (H3E), and Hualien No.4’ bitter melon extract (H4E) at 500 mg/kg bw/day for 4 weeks. Treatment with H3E and H4E significantly attenuated the increased level of hepatic triglyceride accumulation and serum transaminases (aspartate aminotransferase and alanine aminotransferase). Furthermore, the content of glutathione (GSH) and the activities of antioxidative enzymes glutathione peroxidase (GPx), glutathione reductase (GRd), catalase (CAT) and superoxide dismutase (SOD) in the liver were elevated in H3E and H4E treated groups, and reduced the levels of hepatic lipid peroxidation and inflammatory response. Moreover, to further elucidate the hepatoprotectve mechanisms of H3E and H4E, we performed Western blotting. The results showed that the expressions of CYP2E1, SREBP-1c, FAS and ACC in the liver were decreased in mice treated with H3E and H4E. The other two ethanol extracts, H1E and H2E, also have the ability to improve oxidative stress and inflammatory response but the effects are not significant. Additionally, we also determined the content of antioxidants in the ethanol extracts. The total phenolic content of H3E and H4E were significantly higher than H1E and H2E. All of the findings suggested that the Hualien No.3’ bitter melon extract (H3E) and Hualien No.4’ bitter melon extract (H4E) may have the hepatoprotective functions against alcoholic fatty liver disease by attenuating oxidative stress and inflammatory response.

目錄 I
中文摘要 VII
英文摘要 IX
表次 VIII
圖次 IX
第一章 前言 1
第二章 文獻回顧 2
第一節 肝臟 2
一、肝臟之生理構造 2
二、肝臟之生理功能 2
三、肝功能指數(liver function index) 5
第二節 酒精性肝損傷 7
一、酒精性肝臟疾病 7
二、人體之酒精代謝 8
三、酒精造成肝臟損傷之作用機轉 10
四、酒精造成肝臟脂肪堆積之作用機轉 12
五、誘導酒精性肝損傷之動物模式 17
第三節 自由基與氧化壓力 19
一、自由基及活性氧屬之定義 19
二、氧化壓力與疾病 20
三、生物體之抗氧化防禦系統 20
四、抗氧化物質 21
第四節 苦瓜(Momordica charantia L.) 23
一、命名 23
二、植物性狀 23
三、生產與分佈 23
四、品系 23
五、特性 25
六、營養成分 25
七、苦瓜之植物化學成份(phytochemicals) 25
八、苦瓜之生理效用 25
第三章 研究假說與研究目的 29
第一節 研究假說 29
第二節 研究目的 29
第四章 實驗架構 30
第一節 樣品萃取 30
第二節 動物實驗 31
第四章 實驗材料與方法 32
第一節 實驗材料 32
一、實驗動物 32
二、苦瓜 32
三、實驗藥品 33
四、實驗儀器 35
第二節 方法 36
一、試驗樣品製備 36
二、苦瓜乙醇萃取物之劑量篩選 37
三、苦瓜乙醇萃取物之樣品成分分析 38
四、苦瓜乙醇萃取物之抗氧化物質含量分析 39
五、酒精液態飼料誘導酒精性脂肪肝之動物模式 40
六、西方墨點法 41
第三節 實驗步驟 45
一、肝臟組織病理學觀察 45
二、血液生化值測定 45
三、肝臟之蛋白質測定 46
四、肝臟之麩胱甘肽(glutathione, GSH)含量測定 46
五、肝臟之三酸甘油酯(triglyceride, TG)含量分析 47
六、肝臟之麩胱甘肽過氧化酶(glutathione peroxidase, GPx)活性測定 47
七、肝臟之麩胱甘肽過還原酶(glutathione reductase, GRd)活性測定 48
八、肝臟之過氧化氫酶(catalase, CAT)活性測定 48
九、肝臟之超氧化物歧化酶(superoxide dismutase, SOD)活性測定 49
十、肝臟之脂質過氧化程度(lipid peroxidation, LPO)分析 49
十一、肝臟中細胞激素(cytokines)含量分析 50
十二、統計方法 50
第五章 結果 51
第一節 酒精液態飼料誘導小鼠酒精性脂肪肝實驗 51
一、平均體重變化及平均攝食量 51
二、肝臟相對重量 51
三、肝臟組織病理切片觀察 52
四、血清中AST(aspartate transaminase)及ALT(alanine transaminase)值 52
五、血清中三酸甘油酯及總膽固醇含量 53
六、肝臟中三酸甘油酯含量 53
七、肝臟中抗氧化物質麩胱甘肽含量 53
八、肝臟抗氧化酵素活性 54
九、肝臟脂質過氧化程度 55
十、肝臟發炎反應相關之細胞激素含量 55
十一、以西方墨點法探討苦瓜乙醇萃取物改善酒精性脂肪肝之作用機轉 56
第二節 苦瓜乙醇萃取物之樣品分析實驗 58
一、苦瓜乙醇萃取物之成分分析 58
二、抗氧化物質含量分析 60
第六章 討論 61
第一節 酒精液態飼料誘導小鼠酒精性脂肪肝實驗 61
一、平均體重變化及平均攝食量 61
二、評估苦瓜乙醇萃取物對酒精液態飼料誘導小鼠酒精性脂肪肝之保護效果 62
三、以西方墨點法探討苦瓜乙醇萃取物改善酒精性脂肪肝之作用機轉 65
第二節 苦瓜乙醇萃取物之樣品分析實驗 67
一、苦瓜乙醇萃取物之成分分析 67
二、苦瓜乙醇萃取物之抗氧化物質含量分析 68
第三節 未來研究 69
第七章 結論 70
第八章 圖表 71
第九章 參考文獻 108
第十章 附錄 122


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