柑橘類果皮添加對樟芝代謝生理活性物質之影響

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Title:	柑橘類果皮添加對樟芝代謝生理活性物質之影響
Other Titles:	Effect of citrus peel addition on the formation of bioactive metabolites of Antrodia cinnamomea
Authors:	馬德威 Ma, Te-Wei
Contributors:	楊芳鏘 Yang, Fan-Chiang 東海大學化學工程與材料工程學系
Keywords:	樟芝;三?類;平板式固態培養;柑橘類果皮;檸檬烯 Antrodia cinnamomea;triterpenoids;plate solid state culture;citrus peel;limonene
Date:	2012
Issue Date:	2013-01-03T05:59:16Z (UTC)
Abstract:	樟芝已廣泛應用在台灣傳統醫藥，主要有效成份來自於大分子的多醣體、小分子的三?類化合物、半?內酯化合物及固醇類，樟芝的生理活性成分及功能仍是目前研究重點。除多醣體外，近年來最受重視的生理活性物質就是三?類。本研究目的為評估樟芝深層培養添加柑橘類果皮精油及樟芝平板式固態培養添加柑橘類果皮粉末提高生理活性代謝產物的可行性。兩階段溶氧培養(在第21天靜置)對樟芝菌絲體三?類代謝有良好的促進功效，在培養28天後三?類含量達22.30 mg/g DW，為控制組(10.48 mg/g DW)的2.13倍。橘子果皮精油添加對樟芝菌絲體形成生理活性代謝產物促進效果最佳，在第7天添加橘子果皮精油4% (v/v)，三?類含量和產量達到最高值，為117.17 mg/g DW和1417.77 mg/L，分別為控制組的11倍及14倍。檸檬烯為柑橘類果皮精油促進樟芝菌絲體生成三?類的重要成分之ㄧ，檸檬烯進入細胞的質體內，抑制MCC路徑的單?類生成，改由MVA路徑合成三?類。蕎麥平板式固態培養，菌絲體只能在基質表面上成長，因此取樣時可以將菌絲體與基質分離，完整分析菌絲體生理活性。葡萄柚果皮粉末添加能有效提升生理活性代謝物產量，當添加4 g/plate葡萄柚果皮粉末時，第30天三?類的含量與產量分別為47.10 mg/g DW和166.68 mg/plate，為控制組(9.66 mg/g DW和32.26 mg /plate)的4.88倍與5.17倍。此外由HPLC圖譜中，發現經添加葡萄柚果皮粉末平板式固態培養的樟芝，含有代表子實體形成有關的5種麥角甾烷三?類(ergostanes: antcins C and K, and zhankuic acids A, B, and C)，因此可以證明，此種培養在短時間(30天)所得到的三?類化合物與野生樟芝子實體成份大致相同，換句話說，此培養得到的應該為樟芝子實體。 Basidiomes of Antrodia cinnamomea are used as a traditional medicine in Taiwan. Bioactive compounds found in A. cinnamomea include: polysaccharide, triterpenoids, sesquiterpene lactone, and steroids. The bioactivity and efficacy of A. cinnamomea are still the main focus in many researches. In addition to polysaccharide, triterpenoids have recently been considered as the most biologically active components. The aim of this study was to evaluate the feasibility of adding citrus peel extract in the submerged culture or adding citrus peel powder on the plate solid state culture of A. cinnamomea to enhance the formation of bioactive metabolites. A two-stage fermentation process was proposed to enhance triterpenoids content by combining the conventional shake-flask fermentation (on the 21th day) with static culture (on the 7th day). The content of triterpenoids rose from 10.48 mg/g DW of the control to 22.30 mg/g DW on the 28th day. Adding tangerine peel extract was the most effective way to enhance bioactive metabolite production. With an addition of 4% (v/v) on the seventh day, the content and production of triterpenoids were 117.17 mg/g DW and 1417.77 mg/L, which were 10-fold and 13-fold higher than the control, respectively, on the 28th day. Limonene was an important ingredient of citrus peel extract to enhance the formation of triterpenoids in the submerged culture of A. cinnamomea. When limonene entered the mitochondria, MCC pathway was inhibited which synthesizes monoterpenes in the mitochondria. Thus MVA pathway was then enhanced to synthesize triterpenes in the cytoplasm. Mycelia of the plate solid state culture only grew on the substrate surface so that it was easy to separate mycelia from the substrate surface. Grapefruit was the most effective element in enhancing bioactive metabolite production. With an addition of 4 g powder/plate, the content and production of triterpenoids rose from 9.66 mg/g DW and 32.26 mg/plate of the control to 47.10 mg/g DW and 166.68 mg/plate, respectively, on the 30th day. Moreover, this study also demonstrates that the plate solid state culture with an additional grapefruit powder of A. cinnamomea could contain the 5 ergostanes (antcins C and K, and zhankuic acids A, B, and C). The production of ergostanes is related to basidiomatal formation of A. cinnamomea. This result indicates that mycelia of this culture could be basidiomes of A. cinnamomea.
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