含子實體特有三?類的轉殖牛樟芝菌絲體之篩選與其大量培養系統之建立

Analysis of Fruiting Bodies-Specific Triterpennoids in Activation-Tagged Antrodia cinnamomea and Establishment of Its Large-Scale Production System

王宣芸(Hsuan-Yun Wang)

指導教授 ： 蔡新聲 ； 陳靖棻

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2011年)

摘要

牛樟芝(Antrodia cinnamomea)為台灣知名傳統中藥藥用真菌，常用於治療癌症、高血糖、抗氧化、抗發炎等。牛樟芝所含具有抗癌、抗發炎等之三?大多存於其子實體中，然而子實體的取得又十分不易。由於本研究室已成功利用主動誘變方法得到含較高抗發炎三?類dehydroeburicoic acid and dehydrosulpurenic acid之牛樟芝菌絲體H、J-1與J-2，故本試驗試圖從前述主動誘變轉殖牛樟芝菌絲體中，尋找含於牛樟芝子實體中抗發炎(antcin A)、抗副交感神經作用(antcin B)以及抗膽鹼(antcin C)之三?類之菌絲體。首先，使用牛樟芝子實體進行分層萃取(partition)以取得antcin A、B及C標準品，接著將3個轉殖牛樟芝菌絲體H、J-1與J-2品系進行萃取並分析，結果顯示在三個轉殖系牛樟芝菌絲體中皆可測得antcin A、B、C成分，其中轉殖系在antcin A的含量比未轉殖牛樟芝菌絲體高3-40倍，antcin B則更多出20-60倍，另外轉殖系H所含之antcin A、B及C成分較其它轉殖系高。由於轉殖系牛樟芝菌絲體含有子實體特有之成份，因此更進一步建立牛樟芝轉殖系菌絲體的量產條件有其必要，為此，本研究利用紗布、牙籤等輔助材料當作培養之基質進行培養，其中由於以牙籤培養之菌絲生長較豐厚飽滿，因此選用為最佳培養之基質材料。經HPLC分析後，發現轉殖牛樟芝菌絲體大量培養後所含的antcin A和B分別是未轉殖牛樟芝菌絲體的1-1.7倍以及1.7倍，而dehydrosulphurenic acid 及dehydroeburicoic acid則是2-3.5倍及5-16倍。此結果明顯比小量培養差，故利用牙籤大量培養之建立無助於量產前述含高量antcin A、B及C成份之轉殖牛樟芝菌絲體。最後，由於在H、J-1與J-2等3個轉殖牛樟芝菌絲體中可測得antcin A、B及C三?生合成路徑上，重要酵素sterol 14α-demethylase之表現量較高，故推測有可能為主動誘變影響了相關三?生合成相關基因之表現。

關鍵字

三?類 ； 大量培養 ； 牛樟芝 ； 麥角甾烷

並列摘要

Antrodia cinnamomea, a medicinal fungi, is well known in Taiwan as source of traditional Chinese medicine for the treatment of cancer, hyperglycemia, anti-oxidation and anti-inflammation. Triterpenoids with anti-cancer or anti-inflammatory effect are mainly found in the rare and valuable fruiting bodies of A. cinnamomea. Therefore an activation-tagging method was established previously in our lab to obtain A. cinnamomea mycelia with enhanced anti-inflammatory t riterpenoids (dehydroeburicoic acid and dehydrosulphurenic acid). The activation-tagged mycelia H, J-1and J-2 are used in this study to verify possible content of fruiting bodies specific triterpenoids: antcin A (anti-inflammatory), antcin B (anti-parasympathetic) and antcin C (anticholinergic). In order to obtain antcin A, B and C standards, the fruiting bodies of A. cinnamomea were extracted through partition. On the other hand, selected transgenic lines H, J-1 and J-2 were treated with standard extraction. After HPLC analysis, the results showed that all transgenic lines contain antcin A, B, C (antcin A is 3-40 times higher than the wild type, antcin B is 20-60 fold), specially H line. Based on this result, a large scale production system was developed by using gauze, toothpicks and other supporting materials as the matrix for culture. Toothpick was proved to be the best one for mycelia growth and was then used for large scale production of H, J-I and J-2. After HPLC analysis, the result showed that antcin A and B in transgenic lines H, J-1 and J-2 are 7-17.2 times and 3-7 times higher than wild-type respectively after large scale production, and dehydrosulphurenic acid and dehydroeburicoic acid are 2-3.5 times and 5-16 times higher than wild-type. Compared to the previous data, the toothpick-based large scale production system is not ideal one for the production of transgenic lines. And as sterol 14α-demethylase, key enzyme of triterpenoid biosynthesis, was detected with higher expression in all three transgenic lines, this may indicate a possible activation-tagged insertion altered the expression of genes involved in triterpenoid biosysthesis.

並列關鍵字

Antrodia cinnamomea ； ergostane ； large-Scale ； Triterpennoids

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