牛樟芝(Antrodia cinnamomea)是一種傳統的藥用真菌類，對腹瀉、腹痛、高血壓、糖尿病、尿蛋白、肝硬化、肝癌等症狀或疾病具有非常良好的療效。因此本實驗以固態培養之牛樟芝菌絲體為主題，利用管柱層析、薄層層析、高效能液相層析儀及核磁共振質譜儀等對其主要成分做鑑定和定量。由分析結果整理，固態培養牛樟芝之主要成分為Cytosine (0.21 μg/mg)、Uracil (0.15 μg/mg)、Cytidine (0.76 μg/mg)、Uridine (1.24 μg/mg)、Adenine (0.06 μg/mg)、Inosine (1.61 μg/mg)、Guanosine (0.71 μg/mg)、Adenosine (0.61 μg/mg)、Deoxyadenosine (1.03 μg/mg)、Dehydroeburicoic acid (0.95 μg/mg)、Dehydrosulphurenic acid (0.91 μg/mg)、3-Isobutyl-4-furan-2,5-dione (1.31 μg/mg)、Antroquinonol (1.78 μg/mg) 和4-Acetyl-antroquinonol B (1.48 μg/mg)等。
在生物活性的部分，本論文則分為兩個主題，第一個主題是牛樟芝的抗發炎活性，首先我們以牛樟芝菌絲體之粗萃物對LPS誘導體內發炎之ICR小鼠做體內抗發炎活性的分析，可以明顯觀察牛樟芝菌絲體抽出物可以有效的抑制由LPS誘導發炎所活化之NF-κB路徑及其下游之iNOS、COX-2蛋白的表現。接著續以生物活性為導向配合小鼠巨噬細胞RAW264.7做抗發炎反應分析，我們從牛樟芝菌絲體萃取物中獲得了能夠有效抑制發炎反應的化合物4-Acetyl-antroquinonol B，並在體外試驗證實，4-Acetyl-antroquinonol B能有效透過抑制NF-κB路徑的表現，進而抑制下游iNOS蛋白表現合一氧化氮自由基的產生。因此我們得知無論在體內試驗還是體外試驗，牛樟芝菌絲體都能夠提供優良的抗發炎能力。
另一方面本論文透過了動物試驗及細胞試驗證實固態培養之牛樟芝菌絲體粗萃物，具有保護動物肝臟因酒精誘導產生病變之活性。在動物試驗中，本研究使用乙醇 (80 % v/v) 來誘導ICR公鼠之肝臟病變。而由一些與乙醇誘導肝臟病變相關之細胞激素，包括MDA、ALT和AST等的表現得知，牛樟芝菌絲體萃取物可以顯著地對肝臟細胞產生保護作用並呈現劑量相關性。此外，牛樟芝菌絲體萃取物可增加肝臟細胞在酒精傷害後穀胱甘

Antrodia cinnamomea is endemic medicine fungus of Taiwan. It has been claimed for having significant effects on diarrhea, abdominal pain, high blood pressure, diabetes, urinates the protein, liver cirrhosis and liver cancer. In this study, we choose Antrodia cinnamomea mycelium producing by solid-state culture was selected as the target for investigate. First, the composition of extract prepared from mycelium was analyzed by HPLC. The results revealed that the principal components of mycelium is cytosine (0.21 μg/mg), uracil (0.15 μg/mg), cytidine (0.76 μg/mg), uridine (1.24 μg/mg), adenine (0.06 μg/mg), inosine (1.61 μg/mg), guanosine (0.71 μg/mg), adenosine (0.61 μg/mg), deoxyadenosine (1.03 μg/mg), dehydroeburicoic acid (0.95 μg/mg), dehydrosulphurenic acid (0.91 μg/mg), 3-isobutyl-4-furan-2,5-dione (1.31 μg/mg), antroquinonol (1.78 μg/mg) and 4-acetyl-antroquinonol B (1.48 μg/mg).
As regard to the bioactivity study, there are two subjects, anti-inflammatory and anti-oxidative activities, were evaluated in this study. The model of LPS-induced acute inflammation in ICR mice was conducted to the anti-inflammatory activity of Antrodia cinnamomea (EMAC) in vivo. According to the results obtained from protein expression assay, EMAC presented a significant inhibitory activity for NF-κB, as well as its downstream proteins, iNOS and COX-2. Furthermore, an anti-inflammation compound, 4-acetyl-antroquinonol B was obtained from EMAC by using LPS-induced inflammation on macrophage RAW264.7 cell model. 4-acetyl-antroquinonol B revealed the strong activity to inhibit NF-κB and iNOS expression, by this reducing NO production.
On the other hand, the heapprotective effect of EMAC on mice liver damaged by oxidative stress was investigated in this study. The animal model of alcohol-induced liver damage in ICR mice was used to evaluate the activity of EMAC. When the mice pretreated EMAC, the liver oxidative damaged related cytokines, including MDA, ALT and AST were significantly reduced with dose-dependent manner comparing without EMAC pretreated mice. Besides, the level of glutathione (GSH) was reduced after the mice received EMAC. Moreover, the EMAC treated groups could increase the HO-1 and Nrf-2 proteins expression. The anti-inflammation compound, 4-acetyl-antroquinonol B, also revealed the hepaprotective effect on HepG2 cell after alcohol-induced damage.

目次 …………………………………………………………… Ⅰ
表目次 ………………………………………………………… Ⅴ
圖目次 ………………………………………………………… Ⅵ
摘要 …………………………………………………………… 1
Abstract ……………………………………………………… 4
壹、前言 ……………………………………………………… 6
貳、文獻回顧 ………………………………………………… 8
一、牛樟芝及其活性研究進展 ……………………………… 8
(一)牛樟芝子實體之抗癌活性研究 ………………………… 9
(二)子實體抗發炎及保肝活性 ……………………………… 13
(三)牛樟之菌絲體之生物活性 ……………………………… 15
二、發炎反應機制 …………………………………………… 19
三、肝臟保護機制 …………………………………………… 22
參、材料與方法 ……………………………………………… 24
一、試驗材料 ………………………………………………… 24
二、固態培養牛樟芝菌絲體粉末之成分萃取與分離純化 … 24
(一)牛樟芝菌絲體粉末芝成分萃取 ………………………… 24
(二)液相─液相分離(Liquid-liquid partion) …………… 24
(三)管柱層析分離(Column chromatography) ……………… 25
(四)薄層層析分離(Thin layer chromatography) ………… 26
三、固態培養牛樟芝菌絲體之主要成分鑑定及定量 ……… 26
(一)牛樟芝菌絲體主要成分之HPLC指紋圖譜分析條件 ……… 26
(二)成分分析與定量 ………………………………………… 27
四、固態培養牛樟芝菌絲體之抗發炎活性研究 …………… 28
(一)試驗動物處理 …………………………………………… 28
(二)小鼠血液中一氧化氮自由基抑制試驗 ………………… 29
(三)小鼠肝臟蛋白之萃取與西方墨點法分析 ……………… 29
(四)細胞培養及細胞處理 …………………………………… 30
(五)小鼠巨噬細胞中一氧化氮自由基抑制試驗 …………… 30
(六)細胞存活率測試 ………………………………………… 31
(七)細胞蛋白萃取和西方墨點法分析 ……………………… 31
五、固態培養牛樟芝菌絲體之肝臟病變之保護作用機制探討… 33
(一)試驗動物處理 …………………………………………… 33
(二)小鼠血液中總抗氧化能力試驗 ………………………… 34
(三)細胞培養及細胞處理 …………………………………… 34
(四)ALT及AST生成量測定 …………………………………… 35
(五)脂質過氧化及GSH測定 ………………………………… 35
(六)細胞蛋白萃取和西方墨點法分析 ……………………… 36
肆、結果與討論 ……………………………………………… 37
一、固態培養牛樟芝菌絲體之主要成份分析鑑定及定量 … 37
二、固態培養牛樟芝菌絲體之抗發炎活性研究 …………… 41
(一)體內試驗 ………………………………………………… 41
1. 小鼠體內一氧化氮自由基抑制試驗 …………………… 41
2. 小鼠肝臟蛋白表現之分析 ……………………………… 43
(二)體外試驗 ………………………………………………… 46
1. 牛樟芝菌絲體抽出物以小鼠巨噬細胞之一氧化氮自由基抑制活性為導向之成分分離 ………………………………………………46
2. 小鼠巨噬細胞RAW264.7之蛋白表現之分析 ……………… 50
三、固態培養牛樟芝菌絲體之肝臟病變之保護作用機制探討… 52
(一)動物試驗 ………………………………………………… 52
1. 灌食牛樟芝菌絲體乙醇萃取物之小鼠體重及臟器重量之變化 …………………………………………………………………… 52
2. 小鼠血液中總抗氧化能力分析 ………………………… 54
3. 小鼠血液中ALT及AST測試 ……………………………… 56
4. 小鼠血液中GSH及MDA測試 ……………………………… 59
5. 小鼠肝臟蛋白表現之分析 ……………………………… 62
(二) 4-Acetyl-antroquinonol B於體外試驗之肝細胞保護活性 ………………………………………………………… 65
1. 4-Acetyl-antroquinonol B之MTT測試 ………………… 65
2. 4-Acetyl-antroquinonol B對乙醇誘導HepG2細胞損傷之ALT、AST、GSH及MDA之影響 ………………………………………… 67
3. 4-Acetyl-antroquinonol B對乙醇誘導HepG2細胞中HO-1及Nrf-2蛋白的影響 …………………………………………………… 72
伍、結論 ……………………………………………………… 74
陸、參考文獻 ………………………………………………… 76

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