臺灣博碩士論文加值系統

牛樟芝為台灣特有的藥用真菌，其子實體生長緩慢且稀少。近年來，牛樟芝已被證實具有治療肝病及其他如抗腫瘤、抗氧化能力、抗發炎等等功效。目前以非椴木種植方式培養出牛樟芝子實體仍有困難。本研究的目的為設計出一種結合液態培養和固態培養的方法，輔以微生物誘導，以期尋找出樟芝菌絲出菇形成子實體的條件，並測定其對肝癌細胞株（HepG2）之影響。將樟芝菌絲塊以液態搖瓶培養兩週後，將菌絲塊移至培養皿上並添加麥芽萃取物培養基（MEB）。四週後可看見菌絲塊邊緣有菌絲堆積的現象，且其乙醇萃取物在HPLC分析下，發現與子實體圖譜相似，惟含量甚低。若提高MEB的濃度能使菌絲的顏色加深，但卻不會產生堆積的結構。從樟芝的伴存菌中分離且篩選出一株細菌和兩株真菌，分別標示為B01、F01和F02。在靜置時加入三種微生物的菌液皆能促使樟芝菌絲出現堆積的現象，而若F02菌液濃度提升至50% (v/v)則能促使菌絲塊產生類似子實體密孔狀結構的菇化現象，且經乙醇萃取、HPLC分析後，發現其圖譜與野生子實體在20至100分鐘之波峰相當類似。將培養出之菌絲塊用乙醇萃取後，利用HepG2細胞測試其細胞毒殺活性。實驗顯示樟芝子實體和培養出之菌絲塊對肝癌細胞的存活率分別為48.58%和52.42%，結果顯示利用此方法培養之樟芝具有與子實體類似之細胞毒殺效果。

Taiwanofungus camphoratus is a kind of medical fungus that only occurring in Taiwan, it is rare and grows slowly. Recently, it has been reported for treatment of liver diseases and has some other beneficial functions for health, such as anti-tumor, anti-oxidant and anti-inflammation capability, etc. At present, it still has the difficult to raise T. camphoratus fruiting bodies by non-woodblock cultivation. The aim of this study was to investigate the formation of T. camphoratus fruiting bodies by combining liquid-solid cultivation and microbial induction and to determine anti-cancer effect on human hepatocellular carcinoma cell lines (HepG2). Agar block culture (ABC) containing mycelia of T. camphoratus were prepared by shake flask cultivation for two weeks, then moved the ABC to petri dish and added 10 ml malt extract broth (MEB). Four weeks later, mycelia on the ABC periphery were found the phenomenon of mycelia aggregation. The HPLC profile showed the ethanol extracts of ABC were similar to the fruiting bodies, but the contents of 20 to 100 minutes elutes peaks were only in low level. Increasing the concentration of MEB caused mycelia color became deepened but there were no mycelia aggregation occurred. One bacterium and two fungi, named as B01, F01 and F02, were isolated from symbiosis with T. camphoratus fruiting body. Adding these three kinds of microbial culture filtered-broth into MEB, there were found all three tested microorganisms could push mycelia of ABC to aggregate individually. When increasing F02 culture filtered-broth concentration to 50% (v/v) could further stimulate the formation of micro-pore structure on the surface of ABC similar to fruiting body. HPLC profile showed the ethanol extracts of the above ABC were similar to the fruiting body between 20 to 100 minutes elutes peaks. Using HepG2 to test the cytotoxic activity of ethanol extract derived from fruiting body and ABC, the results showed that the cell viability of HepG2 treated by ethanol extract of fruiting body and ABC extract were 48.58% and 52.42%, separately. This result demonstrated that the T. camphoratus cultivated by this way had the cancer-inhibition effects closely to the fruiting bodies.

中文摘要 I
Abstract III
目錄 V
圖目錄 VII
表目錄 IX
壹、前言 1
貳、文獻回顧 3
一、牛樟芝簡介 3
（一）、植物學分類 3
（二）、生長特徵 5
二、牛樟芝成份分析 6
（一）一般成份 6
（二）生理活性成份 6
（二）菌絲體與子實體成份比較 14
三、牛樟芝生理活性功能 18
（一）抗癌 19
（二）抗發炎 22
（三）抗氧化 23
（四）免疫調節 23
（五）保護肝臟 24
（六）抗高血壓 24
（七）抗高血脂 24
（八）保護神經 25
（九）抗病毒 26
（十）抗血管新生 26
四、牛樟芝的安全性 26
五、子實體誘導研究 27
參、研究目的 31
肆、實驗材料與儀器 33
一、實驗材料 33
二、實驗耗材 34
三、實驗儀器 34
伍、實驗方法 37
一、基礎培養條件試驗 37
二、改變靜置培養液濃度試驗 38
三、伴存菌分離純化 38
四、添加細菌B01菌液試驗 38
五、添加真菌F01菌液試驗 39
六、添加真菌F02菌液試驗 41
七、HPLC分析 42
八、細胞培養與測試 42
陸、結果與討論 49
一、基礎培養條件試驗 49
二、改變靜置培養液濃度試驗 49
三、伴存菌分離純化 50
四、添加細菌B01菌液試驗 50
五、添加真菌F01菌液試驗 51
六、添加真菌F02菌液試驗 52
七、細胞存活率測試（MTT assay） 53
柒、結論與未來展望 83
參考文獻 85
附錄 97

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