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研究生:歐貴仁
研究生(外文):Kuei-Ren Ou
論文名稱:樟芝的保肝功效與保肝有效成分純化之研究
論文名稱(外文):Purification and Characterization of the Hepatoprotective Constituents from Antrodia camphorata
指導教授:李益謙李益謙引用關係
指導教授(外文):Eric Li
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:76
中文關鍵詞:樟芝保肝
外文關鍵詞:liver protectionAntrodia camphorata
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樟芝(Antrodia camphorata) 是台灣民間廣為流傳的保健植物。過去研究指出,樟芝具有顯著抗氧化效果,可以減輕由四氯化碳引發動物的肝纖維化程度與抗氧化酵素的下降。然而樟芝可能的作用模式仍不明,並且尚未發現其有效成分為何。
本研究先測試樟芝過濾發酵液(the fermented filtrate from Antrodia camphorata, AC-FF)的保肝功效。在連續給予八週四氯化碳造成肝損害的動物模式中,AC-FF的確有改善肝損害的效果-無論是血清中穀草轉氨酶(GOT)、穀丙轉氨酶(GPT)等臨床常用指標,以及肝臟抗氧化酵素,肝臟組織切片與肝纖維的氫脯氨酸(hydroxyproline)含量評估-均有顯著的改善。並且與臨床上常用的水飛薊效果相仿。
我們接著將AC-FF進一步做有機萃取分離與篩選,在AC-FF的乙酸乙酯萃取層中,發現一個近似純性物質,命名為AC-X。AC-X只需比AC-FF較低的劑量具有同效之保肝效用。在為期八週的四氯化碳肝損害的動物模式中,所有觀察指標,相對於不給予治療組,有顯著的改善。
肝臟星狀細胞(HSCs)是否活化是造成肝臟纖維化的一個重要步驟,而reactive oxygen species(ROS)的氧化壓力存在,是促進HSCs活化的一個重要因子。故一般保肝藥物效果評估,常以抗自由基能力為主。從動物模式中,我們觀察到AC-FF與AC-X具有效提升肝臟抗氧化酵素的能力,提示樟芝有促進清除自由基的功效;HSCs活化時會大量表現的α-smooth muscle actin (α-SMA)表現量也下降。同樣的在HSCs細胞模式中,亦觀察到ROS表現明顯下降。其他HSCs活化指標,例如:分泌metalloproteinase-2 (MMP-2)的能力,無論在活體或細胞中,均有減少趨勢。
我們證明樟芝的活性成分能降低肝功能異常生化指標,並增強肝臟抗氧化能力,降低ROS產生,減少HSCs活化,來達到保護肝臟效果。
Antrodia camphorata (AC) is a unique fungus found only in the natural habitat in Taiwan and has been used by indigenous people as a medicinal botanical for a variety of illness. Previous researches indicate the AC possesses some antioxidant activities, capable of reducing toxic effect of carbon tetrachloride (CCl4), including anti-oxidative stress as indicated by enzyme assays and anti-fibrosis as indicated by histochemical staining. However, the active ingredient of AC and its action mechanism is still unknown. The present investigation aims at studying the hepatoprotective activity and principle from the fermented filtrate of AC (AC-FF). Using a cultured hepatic stallate cell (HSCs) line, an animal model with an eight-week treatment schedule of CCl4, we found that AC-FF indeed can protect liver from damage exerted by CCl4 as judged by (1) serum enzyme assays: GPT and GOP; (2) hepatic enzymes assays, such as catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase; (3) histochemical staining of collagen and α-smooth muscle actin (α-SMA) and assay of hydroxyproline, all these three are used as indicators of liver fibrosis. We investigated further by purifying to almost homogeneity from AC-FF an ingredient, named AC-X, which possesses the same hepatoprotecive activities as AC-FF but at a much lower concentration. One of the major indicators of liver fibrosis is the activation of HSCs, which is activated by the oxidative press exerted by reactive oxygen species (ROS), which in turn is generated by damaged hepatocyte upon the treatment of CCl4. Using these parameters we found that both AC-FF and AC-X act to protect hepatocytes by (1) suppressing serum GPT and GOT; (2) down regulating ROS; (3) down regulating α-SMA which is expressed following the activation of HSCs; and (4) reducing the expression of metalloproteinase-2 (MMP-2), another indicator of HSCs activation. The results combined suggest strongly that Antrodia camphorata possesses some liver protection principles worthy of further detailed investigation.
考試合格證明
中文摘要.........................................................................................................Ⅰ
英文摘要.........................................................................................................Ⅱ
致謝.................................................................................................................Ⅲ
目錄.................................................................................................................Ⅴ
表目錄..............................................................................................................Ⅶ
圖目錄..............................................................................................................Ⅷ
縮寫表..............................................................................................................Ⅸ
第一章 前言.....................................................................................................1
第一節 肝病簡介.............................................................................................1
第二節 肝炎與肝纖維化.................................................................................3
第三節 肝臟抗氧化系統.................................................................................6
第四節 生藥保肝模式.....................................................................................7
第五節 樟芝.....................................................................................................8
第六節 肝纖維化動物模式.............................................................................9
第二章 材料與方法.........................................................................................10
第一節 實驗材料.............................................................................................10
壹、 樟芝製備.................................................................................................10
貳、 實驗動物與分組.....................................................................................10
參、 儀器.........................................................................................................12
肆、 試劑與藥品.............................................................................................12
伍、 細胞株.....................................................................................................15
第二節 實驗方法.............................................................................................15
壹、 四氯化碳誘導小鼠肝纖維化方法.........................................................15
貳、 血清肝功能生化值測定.........................................................................15
參、 小鼠犧牲與組織取得.............................................................................16
肆、 肝組織切片與染色.................................................................................16
一、 hematoxylin與eosin雙重染色.................................................................16
二、 Masson’s trichrome三重纖維染色.......................................................16
三、 α-平滑肌動蛋白組織免疫化學染色.....................................................19
伍、 肝組織纖維化評估—羥脯胺酸含量測定............................................21
陸、 肝組織抗氧化酵素測定........................................................................22
一、 肝均質液製備........................................................................................22
二、 過氧化氫脢(Catalase) ...........................................................................23
三、 超氧化物歧化酶(Superoxide Dismutase, SOD).....................................24
四、 穀胱甘肽過氧化酶(Glutathione peroxidase, GSH Px)..........................25
五、 穀胱甘肽還原酶(Glutathione reductase, GSH Rd)................................27
柒、 金屬基質蛋白脢測定...........................................................................29
捌、 肝星狀細胞培養...................................................................................31
玖、 肝星狀細胞ROS含量測定....................................................................33
一、 免疫螢光染色.......................................................................................33
二、 細胞分析儀...........................................................................................33
第三節 統計分析...........................................................................................34
第三章 結果....................................................................................................35
第一節 樟芝深層發酵過濾液........................................................................35
第二節 樟芝乙酸乙酯層X-分層....................................................................38
第三節 肝臟星狀細胞實驗............................................................................41
第四章 討論.....................................................................................................43
參考文獻.........................................................................................................46
附表.................................................................................................................52
附圖.................................................................................................................55
自敘.................................................................................................................76
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