臺灣博碩士論文加值系統

香椿(Toona sinensis Roem.)為楝科植物，其葉和根莖部分在亞洲傳統用藥上具有治療腸炎、腹瀉、疼痛和疥瘡的效用。現有研究已知香椿葉中主要成分為methyl gallate(MG)與gallic acid(GA)等酚酸，且具有抗發炎、抗氧化、清除自由基能力。本實驗室亦證實在細胞模式中MG誘發HepG2解毒酵素作用。本研究目的是進一步探討香椿萃取純化物在動物模式中對誘發解毒酵素與抗氧化酵素之影響。初期實驗將香椿葉中萃取的MG及GA單一成份餵食及腹腔注射小鼠後取其血液組織再藉由High performance liquid chromatography(HPLC)探討其藥物動力學。將MG與GA餵食小鼠進行代謝動力分析，血液中MG短時間內被代謝與轉換成GA且在血液中濃度迅速降低。GA則在血液中維持較長時間穩定低濃度。其結果指出消化道影響gallic acid與methyl gallate吸收差異。藥物分布得知methyl gallate存在肝組織中。以RT-PCR探討methyl gallate誘發小鼠肝解毒酵素表現方面，Nrf2、γ-GCS、GST等mRNA在200mg/kg body weight劑量下明顯被誘發。在抗病毒實驗中50μg/ml的香椿甲醇粗萃物具有直接作用抑制50％的腸病毒數能力。

關鍵字： Toona sinensis Roem、Methyl gallate、Gallic acid、解毒酵素。

Toona sinensis is a broadleaf tree. The leaves and toung shoots have been used as a vegetable in China for thousands of years. The leaves and stems of this plant have been used for treatment of enteritis, dysentery and itch in oriental medicine. The main components of Toona sinensis are methyl gallate(MG) and gallic acid(GA). These components have significant biological properties such as anti-inflammatory, anticarcinogenic and free radical scavenging activity. In the previous data,we have shown that MG can induce de-toxification enzymes in HepG2 cells. In this study, we administered mice with GA and MG through oral or intrapeitonealrout. Pharmacokintic analysis of GA and MG in mice using HPLC was conducted. The results showed that MG was absorbed immediatedy and converted into GA. Detoxified enzyme induced, by methyl gallate, were further studied using Reverse TranscrI.Ption PCR (RT-PCR) to measure mRNA expression.The expression of GSH synthesis related gene, Nrf2 mRNA, increased significantly with increasing methyl gallate concentration.

Key words: Toona sinensis Roem, Methyl gallate, Gallic acid, Phase II.

中文摘要 I
Abstract II
謝誌 IV
目錄....................................................................................................V
圖表目錄 VII
第1章 前言 1
第2章 文獻回顧 3
2.1 香椿 3
2.2 多酚 5
2.3 自由基 7
2.4 肝臟解毒作用 8
2.4.1 生物轉換代謝系統 8
2.4.2 Phase I enzymes 8
2.4.3 Phase II enzymes 10
2.4.4 Glutathione之生合成 10
2.4.5 Phase II enzymes mRNA的誘發 14
2.5 抗病毒藥物作用機制 15
2.6 腸病毒 15
第3章 材料與方法 16
3.1 實驗設計 16
3.1.1 實驗動物 16
3.1.2 細胞株 16
3.1.3 病毒株 17
3.2 儀器設備 17
3.3 藥品試劑 17
3.4 實驗方法 18
3.4.1 香椿葉萃取物製備 18
3.4.2 標準品的配置 18
3.4.3 HPLC分析 18
3.5 Methyl gallate藥物動力學 19
3.5.1 動物飼養 19
3.5.2 實驗設計 19
3.5.3 樣品收集 20
3.5.4 HPLC血清樣品液製備 20
3.5.5 HPLC組織樣品液製備 20
3.5.6 HPLC分析 20
3.6 香椿粗萃餵食小鼠對Phase II酵素mRNA誘發分析 21
3.6.1 實驗設計 21
3.6.2 犧牲與臟器收集 21
3.6.3 Total RNA萃取 21
3.6.4 RNA電泳（Electrophoresis） 22
3.6.5 核醣核酸濃度定量 22
3.6.6 反轉錄作用 22
3.6.7 引子序列 23
3.6.8 聚合酶鏈鎖反應（PCR，Polymerase chain reaction） 23
3.6.9 DNA電泳（Electrophoresis） 24
3.7 香椿粗萃物對腸病毒71型生存能力的影響 24
3.7.1 培養基之配製 24
3.7.2 細胞培養 24
3.7.3 細胞解凍 24
3.8 腸病毒71型效價測定(Titration of enterovirus 71) 25
3.9 統計分析 26
第4章 結果 27
4.1 香椿嫩葉粗萃物HPLC分析結果 27
4.2 Methyl gallate之藥物動力學分析結果 27
4.2.1 腹腔注射投藥之藥物動力結果 29
4.2.2 插管餵食投藥之藥物動力結果 29
4.3 Methyl gallate對小鼠誘發Phase II enzymes劑量安全性評估 31
4.4 Methyl gallate對小鼠誘發Phase II enzymes mRNA之影響 31
4.5 香椿粗萃物之抗病毒活性試驗 32
第5章 討論 33
5.1 香椿嫩葉粗萃物HPLC分析 33
5.2 Methyl gallate之藥物動力學分析 33
5.3 藥物吸收 33
5.4 藥物代謝 34
5.5 藥物組織分布 34
5.6 Methyl gallate對誘發Phase II enzymes 35
5.7 抗病毒 35
第6章 結論 58
參考文獻 59
作者簡介 65

圖表目錄
圖 1、 香椿中多酚類抗氧化物結構 4
圖 2、 Gallic acid衍生物結構 6
圖 3、 化學防護劑藉由不同機制對抗致癌物的作用 9
圖 4、 Glutathione的結構組成 11
圖 5、 GSH接合作用途徑 12
圖 6、 Glutathione 的抗氧化功能 13
圖 7、 Nrf2誘發Phase II enzymes路徑 14
圖 8、 實驗設計 16
圖 9、 標準品圖譜 36
圖 10、 香椿嫩葉粗萃物HPLC分析 37
圖 11、 腹腔注射methyl gallate投藥後血液HPLC定性分析 38
圖 12、 插管餵食methyl gallate投藥後血液HPLC定性分析 39
圖 13、 腹腔注射gallic acid投藥後血液HPLC定性分析 40
圖 14、 插管餵食gallic acid投藥後血液HPLC分析 41
圖 15、 腹腔注射與插管餵食gallic acid後血液分析 42
圖 16、 腹腔注射與插管餵食methyl gallate後血液分析 43
圖 17、 腹腔注射methyl gallate投藥後血液分析 44
圖 18、 插管餵食methyl gallate投藥後血液分析 45
圖 19、 插管餵食methyl gallate投藥後腎組織樣品分析 46
圖 20、 插管餵食methyl gallate投藥後肝組織分析 47
圖 21、 插管餵食methyl gallate後肌肉組織分析 48
圖 22、 餵食7天methyl gallate後小鼠臟器重 49
圖 23、 餵食7天methyl gallate後小鼠腎HE染色切片 50
圖 24、 餵食7天methyl gallate後小鼠肝HE染色切片 51
圖 25、 餵食7天methyl gallate後小鼠肝指數值 52
圖 26、 電泳分析小鼠肝之Nrf2 mRNA表現量之差異 53
圖 27、 電泳分析小鼠肝之γ-GCS mRNA表現量之差異 54
圖 28、 電泳分析小鼠肝之GST-α2 mRNA表現量之差異 55
圖 29、 電泳分析小鼠肝之GST-μ2 mRNA表現量之差異 56
圖 30、 不同濃度香椿粗萃物對腸病毒的抑制作用 57

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