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研究生:楊蒨旻
研究生(外文):Chien-Min Yang
論文名稱:小飛揚草之抗氧化及抗皰疹病毒活性評估
論文名稱(外文):Evaluation of the Antioxidant and Antiherpes Viruses Activities of Euphorbia thymifolia Linn.
指導教授:林俊清林俊清引用關係
指導教授(外文):Chun-Ching Lin
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:天然藥物研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:小飛揚草脂質過氧化作用Cytochrome C 還原比色法電子自旋共振法分劃層和純化合物細胞毒性自由基及病毒XTT法測試抗皰疹病毒活性
外文關鍵詞:Euphorbia thymifoliaAntiherpes Viruses ActivitiesAntioxidant ActivitiesElectron Spin ResonanceESRCytotoxicity AssayXTTCytochrome C Test.Xanthine Oxidase Inhibition Test
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生藥小飛揚草為大戟科植物千根草(Euphorbia thymifolia Linn.)。根據文獻上記載,小飛揚草具有清熱、 利濕、 消腫、解毒、治瘧疾、 痢疾、 泄瀉、 濕疹、 乳癰、 痔瘡的作用。近年來的研究認為許多疾病的形成與體內產生過度的自由基有關。另有文獻指出,病毒的感染會造成自由基含量的升高。因此,為了評估小飛揚草與自由基及病毒之間的關係,著者以下列實驗模式進行測試:
1.FeCl2-Ascorbic acid誘發脂質過氧化作用
2.Cytochrome C 還原比色法測試自由基之清除能力。
3.Xanthine Oxidase Inhibition Test測試自由基之抑制能力。
4.電子自旋共振法(ESR, electron spin resonance)測試超氧陰離子自由基及氫氧自由基之清除能力。
5.XTT法測試抗皰疹病毒活性。
本次實驗結果顯示:
一、FeCl2-Ascorbic acid誘發脂質過氧化作用
小飛揚草各分劃層和純化合物具抗脂質過氧化作用。其中正丁醇分劃層、氯仿分劃層、乙酸乙酯分劃層、水粗抽物層、甲醇分劃層、有機水層、Rugosin B、1, 3, 4, 6-tetra-O-galloyl-β-D-glucose和3-O-galloyl-4, 6-(s)-HHDP-D-glu- cose的IC50分別為2.81mg/ml、4.23mg/ml、4.27mg/ml、5.69mg/ml、7.63mg/ml、>20mg/ml、3.69mM、5.10mM和5.72mM。
二、Cytochrome C 還原比色法測試自由基之清除能力
小飛揚草各分劃層和純化合物具有良好的超氧陰離子自由基清除能力。其中正丁醇分劃層、乙酸乙酯分劃層、甲醇分劃層、氯仿分劃層、有機水層、水粗抽取物層、Rugosin B、3-O-galloyl-4, 6-(s)-HHDP-D-glucose和1, 3, 4, 6-tetra-O-galloyl-β-D-glucose的IC50值分別為0.013mg/ml、0.019mg/ml、0.054mg/ml、0.069 mg/ml、0.386mg/ml、 0.410mg/ml、0.023mM、0.077mM和3.650mM。
三、Xanthine Oxidase Inhibition Test測試自由基之抑制能力
小飛揚草各分劃層和純化合物具有抑制超氧陰離子自由基的形成。其中水粗抽物層、乙酸乙酯分劃層、甲醇分劃層、氯仿分劃層、正丁醇分劃層、有機水層、Rugosin B、1, 3, 4, 6-tetra-O-galloyl-β-D-glucose和3-O-galloyl-4, 6-(s)-HHDP-D-glu- cose的IC50值分別<為0.03 mg/ml 、0.71 mg/ml、0.76 mg/ml、1.31 mg/ml、2.18 mg/ml、>20 mg/ml、0.36 mM、0.60 mM和2.33 mM。
四、電子自旋共振法(ESR, electron spin resonance)測試超氧陰離子自由基及氫氧自由基之清除能力。
小飛揚草水粗抽物及其三種純化合物具SOD-like之作用,其活性強弱次序分別為3-O-galloyl-4, 6-(s)-HHDP-D-glucose (IC50為1.21mg/ml),Rugosin B (IC50為1.93mg/ml)、1, 3, 4, 6-tetra-O-galloyl-β-D-glucose (IC50為4.95mg/ml)及小飛揚草水粗抽物(IC50為10.44mg/ml)。另外小飛揚草水粗抽物及3-O-galloyl-4, 6-(s)-HHDP-D-glucose清除氫氧自由基的活性不顯著。
五、XTT法測試抗皰疹病毒活性
在抗HSV-2分析中,只有3-O-galloyl-4, 6-(s)-HHDP-D-glucose及乙酸乙酯分劃層具有抗HSV-2活性,其IC50值分別為7.49±0.73μM及7.72±0.15μg/ml。另外細胞毒性實驗証明了3-O-galloyl-4, 6-(s)-HHDP-D-glucose及乙酸乙酯分劃層之抗病毒活性不是由於藥物本身對細胞毒性所引起的。
上述實驗結果顯示了小飛揚草具有抑制脂質過氧化、清除超氧陰離子自由基、抑制超氧陰離子自由基形成、SOD-like活性和抗皰疹病毒第二型的作用。

Euphorbia thymifolia Linn. (Euphorbiaceae) is a medicinal herb used as a diuretic and a laxative in Taiwan. In literature record, this crude drug has detumescence, anti-diarrhea, anti-malaria, anti-rash, anti-dysentery, anti-carbuncle, detoxify and anti-hemorrhoids. Recent studies showed that the causes of many diseases are related with excess free radicals in body, while viruses infection will accomplish with the production of free radicals. To evaluate the antioxidant and anti-HSV activities of E. thymifolia, the following studies will be evaluated:
1.FeCl2-Ascorbic Acid Test.
2.Cytochrome C Test.
3.Xanthine Oxidase Inhibition Test.
4.Electron Spin Resonance(ESR)
5.Antiviral Assay and Cytotoxicity Assay.
The results showed that Euphorbia thymifolia Linn. exhibited an antioxidant and anti-HSV-2 activity. The range of IC50 for fractions during FeCl2-Ascorbic acid test, Xanthine Oxidase Inhibition Test and Cytochrome C Test were 2.81~7.63, 0.03~32.13 and 0.013~0.410 mg/ml, while for pure compounds were 3.69~5.72, 0.36~2.33 and 0.023~3.650 mM, respectively. ESR test results showed that water fraction and pure compounds of E. thymifolia Linn. possessed SOD-like activity. However, the hydroxyl radical scavenger activities of E. thymifolia was weak. Besides antioxidant activities, EtOAc fraction and 3-O-galloyl-4, 6-(s)-HHDP-D-glucose also exhibited anti-HSV-2 activity. None of the fractions and pure compounds of E. thymifolia possessed anti-HSV-1 activity.
The results show that E. thymifolia has exhibited anti-lipid peroxidation, superoxide anion radical scavenger, superoxide anion radical formation inhibition, SOD-like and anti-HSV-2 activities.

中文摘要……………………………………………………………Ι
英文摘要……………………………………………………………Ш
第一篇 小飛揚草成份之抗氧化作用………………………………1
第一章 小飛揚草之簡介、文獻記載、來源、製備及化學結構…1
第一節 小飛揚草的簡介……………………………………………1
第二節 小飛揚草的文獻記載………………………………………3
第三節 小飛揚草的來源與製備……………………………………4
第四節 小飛揚草成分之化學結構…………………………………8
參考文獻……………………………………………………………10
第二章 抗脂質過氧化活性測試……………………………………11
第一節 前言…………………………………………………………11
第二節 實驗材枓與方法……………………………………………13
第三節 結果與討論…………………………………………………17
參考文獻……………………………………………………………22
第三章 活性氧清除作用活性測試…………………………………24
第一節 前言…………………………………………………………24
第二節 實驗材枓與方法……………………………………………25
第三節 結果與討論…………………………………………………28
參考文獻……………………………………………………………31
第四章 自由基生成抑制作用活性測試……………………………32
第一節 前言…………………………………………………………32
第二節 實驗材枓與方法……………………………………………34
第三節 結果與討論…………………………………………………37
參考文獻……………………………………………………………41
第五章 電子自旋共振法……………………………………………43
第一節 前言…………………………………………………………43
第二節 實驗材枓與方法……………………………………………45
第三節 結果與討論…………………………………………………52
參考文獻……………………………………………………………64
第二篇 小飛揚草成份之抗單純皰疹病毒作用……………………65
第一章 緒論…………………………………………………………65
第一節 單純皰疹病毒………………………………………………65
第二節 抗病毒藥物…………………………………………………67
第三節 天然物的抗病毒活性………………………………………69
第四節 XTT實驗原理……...………………………………………71
第二章 小飛揚草成分之抗單純皰疹病毒作用……………………72
第一節 實驗材料及儀器……………………………………………72
第二節 實驗培養液及試劑的配製…………………………………74
第三章 細胞、病毒之培養及病毒之定量…………………………78
第一節 細胞之繼代培養……………………………………………78
第二節 病毒之培養…………………………………………………78
第三節 病毒之定量…………………………………………………78
第四章 實驗方法……………………………………………………80
第一節 抗病毒活性之實驗方法……………………………………80
第二節 細胞毒性之實驗方法………………………………………81
第三節 選擇指數之評估……………………………………………82
第五章 結果…………………………………………………………83
第一節 抗病毒活性…………………………………………………83
第二節 細胞毒性……………………………………………………83
第三節 選擇指數……………………………………………………83
第六章 討論…………………………………………………………84
參考文獻……………………………………………………………96
總結…………………………………………………………………104
投稿與學會發表論文………………………………………………105

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