謝兆樞臺灣大學：農藝學研究所許文綺Hsu, Wen-ChiWen-ChiHsu2007-11-282018-07-112007-11-282018-07-112005http://ntur.lib.ntu.edu.tw//handle/246246/59181牛樟芝 (Antrodia cinnamomea) 為台灣特有的藥用蕈類，生長 於台灣特有的保育類樹種牛樟樹之中空腐朽心材上，其子實體生長速度十分緩慢，因此造成醫療及研究上材料不足。本研究目的是希望在短時間內，以人工培養方式培養出優質又高產的菌絲體，在培養基中加入樟樹萃取液可以達到此目的。本實驗使用五種菌株，分別為35396、35398、B71、B85 及B86。結果顯示五種牛樟芝菌株其菌絲體的產率皆因為添加樟樹萃取液在培養基中而增加，其中以菌株35396 產率最高，每公升培養基中可得5.96 ± 0.19 公克的菌絲乾重。在二次代謝物方面，添加樟樹萃取液在培養基中亦會使所偵測的三種三萜類化合物， dehydrosulphurenic acid 、15α-acetyl -dehydrosulphurenic acid 及dehydroeburicoic acid 的含量增加而與子實體的含量接近，其中菌株B86 的dehydrosulphurenic acid 含量每克菌絲中可高達13.10 ± 0.44 毫克，顯著高於子實體中的每克含量9.49 ± 0.10 毫克。而且添加樟樹萃取液在培養基中的確會使具有抗發炎活性及抑制DNA 聚合酶 (DNA polymerase) α、β 和DNA topoisomerase 的作用之dehydroeburicoic acid 成分增加。此外亦會使具有使血管舒張的活性和可防止神經細胞細胞凋亡的adenosine成分增加，由此可知，在培養基中加入樟樹萃取液的確是會得到優質又高產的牛樟芝菌絲體。在培養基中加入樟樹萃取液會使菌絲多醣體高分子量的部分所佔比例增加，而且對子宮頸癌細胞的毒殺效果大都比用基礎培養基培養的菌絲多醣體來得好。牛樟芝多醣體也會使靜脈內皮細胞 (human vascular endothelial cell, ECV cell) 內反應逆境的調控轉錄抑制子 (transcriptional repressor) ， activating transcription factor 3 (ATF-3) 蛋白的量產生變化。Antrodia cinnamomea (Niu-Chan-Chi) is a peculiar medicinal fungus in Taiwan, which grows on the moribund inner cavity heart wood of the endemic species, Cinnamomum kanehirae. Because of its slow growth-rate in nature and limited host plant, the material is insufficient on medical treatment and research. Liquid culture to mass produce the fungus is the aim of this study. Five A. cinnamomea strains 35396, 35398, B71, B85 and B86 were used.The results showed that A. cinnamomea treated with the water-extract of C. camphora exhibited higher growth promotion effect on the biomass than that of control. Strain 35396 showed the highest dry weight, being 5.96 ± 0.19mg/l medium. All contents of dehydrosulphurenic acid, 15α-acetyl-dehydrosulphurenic acid and dehydroeburicoic acid were increased with the addition of water-extract of C. camphora and the contents were approximately close to fruiting body. It also increased the contents of dehydroeburicoic acid and adenosine, which exhibited biological activity. Polysaccharides were extracted from cultured mycelia of five A. cinnamomea strains. Characterization of polysaccharide by high-performance anion-exchange chromatography, showed varying yields and composition of neutral sugars. The polysaccharide profiles revealed that the addition of water-extract of C. camphora increased the production of the highest molecule weight part of polysaccharide and resulted in a direct effect on the cytotoxicity of Hela cell. Activating transcription factor 3 (ATF-3) is a transcriptional repressor that is rapidly induced in cells exposed to a wide range of stress stimuli. Polysaccharides extracted from A. cinnamomea can affect the ATF-3 expression in the protein level.致謝..................................................................................................................1 目錄..................................................................................................................2 縮寫字全名對照................................................................................................5 中文摘要..........................................................................................................7 英文摘要..........................................................................................................8 圖表目錄..........................................................................................................9 緒論................................................................................................................11 前人研究........................................................................................................13 1. 牛樟芝的介紹.............................................................................13 1.1. 名稱變革...............................................................................13 1.2. 分類地位...............................................................................13 1.3. 型態構造與分佈.....................................................................14 2. 牛樟芝的成分.............................................................................17 2.1. ㄧ次代謝產物........................................................................17 2.1.1. 粗脂質...........................................................................17 2.1.2. 粗纖維...........................................................................17 2.1.3. 碳水化合物....................................................................17 2.1.4. 蛋白質...........................................................................17 2.1.5. 胺基酸...........................................................................18 2.1.6. 脂肪酸...........................................................................18 2.1.7. 多醣體...........................................................................18 2.2. 二次代謝產物........................................................................18 2.2.1. 長鏈酯類 (long chain ester)..........................................19 2.2.2. 固醇類 (steroids) ..........................................................19 2.2.3. 木脂素 (lignans) ...........................................................20 2.2.4. 酚類 (phenolics) ...........................................................21 2.2.5. 萜類 (terpenoids) .........................................................23 3. 牛樟芝的生物活性......................................................................28 3.1. 菌絲體、濾液及醱酵液..........................................................28 3.2. 水萃取物...............................................................................29 3.3. 多醣體 (polysaccharides, PS) ..............................................30 3.4. 乙醇萃取物............................................................................30 3.5. 甲醇萃取物............................................................................31 3.6. 其他.......................................................................................31 3.7. 純化合物...............................................................................32 3.8. 安全性評估............................................................................33 3.8.1. 急毒性試驗....................................................................33 3.8.2. 毒性試驗.......................................................................33 3.8.3. 亞急毒性試驗................................................................33 3.8.4. 畸胎試驗.......................................................................33 材料與方法.....................................................................................................34 1. 材料...........................................................................................34 1.1. 菌株.......................................................................................34 1.2. 樟樹來源...............................................................................34 1.3. 藥品及試劑............................................................................34 1.4. 儀器及設備............................................................................37 2. 方法...........................................................................................39 2.1. 培養基之配製方法.................................................................39 2.2. 牛樟芝菌絲培養方法..............................................................39 2.3. 生長曲線之測定.....................................................................40 2.4. 菌絲體萃取物之製備..............................................................40 2.5. 牛樟芝中dehydrosulphurenic acid 、15 α-acetyl-dehydro sulphurenic acid 及dehydroeburicoic acid 成分之定量..........................41 2.6. 牛樟芝乙醇萃取物中cytidine、adenosine、inosine 及thymidine 成分之定量.............................................................................................42 2.7. 多醣體分子量分佈分析..........................................................43 2.8. 水解及單醣的組成分析..........................................................44 2.9. 細胞培養方法........................................................................45 2.10. MTT 分析..........................................................................45 2.11. 西方墨點法........................................................................45 結果................................................................................................................47 1. 生長曲線....................................................................................47 2. 菌絲外表型態.............................................................................52 3. 菌絲乾重產率及多醣體含量比較................................................54 4. 牛樟芝中三萜類dehydrosulphurenic acid、15α-acetyl-dehydro sulphurenic acid 及dehydroeburicoic acid 成分之定量與比較...................57 5. 牛樟芝乙醇萃取物中adenosine 及inosine 成分之定量.............69 6. 多醣體分子量分析......................................................................79 7. 多醣體單醣組成分析..................................................................91 8. 多醣體對Hela 細胞的毒殺能力分析...........................................95 9. 多醣體對ECV 細胞內的ATF-3 蛋白質表現影響........................97 討論................................................................................................................99 結論..............................................................................................................104 參考文獻......................................................................................................1051876711 bytesapplication/pdfen-US牛樟芝多醣體Antrodia cinnamomeapolysaccharidesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59181/1/ntu-94-R91621107-1.pdf