臺灣博碩士論文加值系統

樟芝 (Antrodia cinnamomea )和其宿主牛樟樹 (Cinnamomum kanehirae )為台灣本土的特有種。樟芝為一種昂貴的藥用真菌，自然野生生長的牛樟樹腐朽後，樟芝會生長於其樹幹內。就目前而言，對於樟芝的高度需求，而大量的砍伐牛樟樹以收集樟芝子實體，已威脅到台灣特有種牛樟樹的生存。因此學術和業界的學者積極的在實驗室開發樟芝的產品，以培養出樟芝的菌絲體及其化學成份中的二次代謝物為主，在這方面的研究能有效的縮短時間至七到十四天，比人工培養基兩的月的生長時間短的多，不過其成份和野生的子實體相去甚遠。因此，人工培養樟芝子實體仍然有進一步研究的價值，本論文研究營養源的減少，以促進子實體的生成的影響。把標準的MEA (malt extract agar)配方視為100%，對其做一系列的稀釋，用來培養樟芝，在培養的過程，發現子實體的形成，以肉眼和顯微鏡判斷其多孔性狀的生成，再以HPLC進行分析。本次實驗的結果以50% MEA培養的樟芝出現多孔性狀最為明顯，此50% MEA培養的樟芝，在HPLC圖譜的分析上出現了在2006年Lin之參考文獻，中所指出的指標性波峰。另外，分別抽取菌絲體和子實體total RNA 合成cDNA後做real-time PCR分析。取得四條基因與老化有關的序列分別為：cytochrome P450、glutathione-S- transferase、peroxiredoxin 和manganese superoxide dismutase。在這四條基因當中，以cytochrome P450表現量的變化為1.49為最大，而perxoiredoxin、glutathion- S-transferase和manganese superoxide dismutase在子實體和菌絲體間表現差異不大。

Antrodia cinnamomea and its host, Cinnamomum kanehirae, are both native species of Taiwan. A. cinnamomea is an expensive medicinal fungus that only grows inside the rotten stem of the native tree species, C. kanehirae, in the field. The high demand of A. cinnamomea is causing a serious conservation issue since people have massively harvested wild A. cinnamomea fruiting body by cutting off the C. kanehirae trunk and endangering the unique tree species in Taiwan. Therefore, scientists from academics and pharmaceutical industry have been intensively working on to develop the products of A. cinnamomea in laboratories. One major approach of the laboratory studies is to culture the fungus in hypha forms and optimize the chemical composition, especially production of secondary metabolites, by culturing the hypha. This approach greatly shortened the cultural time to around 7~14 days rather than two months for fruiting body culture in agar medium, but its medicinal components were quite different from those of field collected A. cinnamomea fruiting body. Therefore, in vitro culture of A. cinnamomea fruiting body is still of value and needed further investigation. This thesis studies the effects of nutrient deficiency on the growth of A. cinnamomea fruiting body. The malt extract agar (MEA) was the basic medium used in this study. The standard MEA formula was designated as 100%, and a series of dilution of the MEA were made to culture A. cinnamomea. During the culture period, we determine the formation of fruiting body by visual and microscopic observation on A. cinnamomea’s porous morphogenesis, and HPLC analysis. The results showed A. cinnamomea has the best porous morphology in 50% MEA. The fruiting body from 50% MEA culture also showed strong peaks reported only in fruiting body when subjected to HPLC analysis. In addition, the cultural mycelium and fruiting body were subjected to total RNA isolation, cDNA preparation, and real-time PCR analysis. We studied four genes relating to senescence. They are cytochrome P450, glutathione-S-transferase, peroxiredoxin and manganese superoxide dismutase. We found cytochrome P450 was expressed 1.49 fold in fruiting body than in mycelium, and perxoiredoxin, glutathion-S-transferase, and manganese superoxide dismutase were found with similar expression in both fruiting body and mycelium.

中文摘要....................................................................................Ⅰ
英文摘要....................................................................................Ⅲ
研究目的....................................................................................Ⅴ
目錄....................................................................................Ⅵ
圖、表目錄....................................................................................Ⅸ
第一章 文獻回顧
一. 樟芝介紹----------------------------------------------------------------1
1. 樟芝的命名---------------------------------------------------------1
2. 樟芝宿主------------------------------------------------------------2
3. 樟芝型態------------------------------------------------------------3
4. 樟芝成份分析------------------------------------------------------4
(1) 一次代謝物---------------------------------------------------4
(2) 多醣體的簡介------------------------------------------------4
(3) 二次代謝物---------------------------------------------------5
二. 樟芝的療效及其研究-------------------------------------------------6
1. 抗氧化功效---------------------------------------------------------6
2. 保肝作用------------------------------------------------------------7
3. 毒殺癌細胞能力---------------------------------------------------8
4. 增進免疫能力-----------------------------------------------------9
5. 抗病毒能力-------------------------------------------------------10
6. 抗發炎反應-------------------------------------------------------10
7. 降血醣能力-------------------------------------------------------10
8. 安全性-------------------------------------------------------------11
第二章 樟芝的栽培----------------------------------------------------------13
一. 前言-------------------------------------------------------------------13
1. 培養基-------------------------------------------------------------13
2. 添加物對樟芝生長的影響------------------------------------15
二. 研究方法-------------------------------------------------------------16
三. 結果和討論----------------------------------------------------------18
第三章 甲醇萃取物的分析-------------------------------------------------23
一. 前言-------------------------------------------------------------------23
二. 研究方法-------------------------------------------------------------25
三. 結果和討論----------------------------------------------------------27
第四章 即時定量PCR------------------------------------------------------31
一. 前言----------------------------------------------------------------31
1. 蛋白質功能的介紹---------------------------------------------32
(1) Cytochrome P450簡介-----------------------------------32
(2) Glutathione-S-transferase簡介--------------------------33
(3) Peroxiredoxin和Manganese superoxide dismutase--33
(3.1) Manganese superoxide dismutase簡介---------34
(3.2) Peroxiredoxin簡介---------------------------------34
二. 研究方法-------------------------------------------------------------36
三. 結果和討論----------------------------------------------------------38
第五章 總結-------------------------------------------------------------------41

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