樟屬植物之牛樟芝菌絲體生長促進因子

Promotion of hyphal growth in Antrodia camphorata by host factors from the genus Cinnamomum

張怡潔(Yi-Chieh Chang)

指導教授 ： 徐鳳麟 ； 盧美光

臺北醫學大學/藥學院/生藥學研究所/碩士(2003年)

摘要

牛樟芝（Antrodia camphorata）為台灣特有的一種真菌，生長於本土特有牛樟樹之中空腐朽心材上，其子實體生長速度非常緩慢。本研究的主要目的為，在所收集到的五種樟屬植物中，尋找促使牛樟芝生長之天然物成分。本研究利用添加不同樟屬植物萃取物之液態培養方式，並分別測量牛樟芝菌絲體乾重累積量、菌絲體胞內酵素之代謝能力、蛋白質及碳水化合物含量、捕捉1,1-diphenyl-2-picrylhydrazyl （DPPH）自由基能力，及利用高效能液相層析儀（HPLC）定性分析菌絲體之三萜類化合物、二維電泳分析菌絲體之蛋白質等電點分佈及分子量表現。由實驗結果顯示，樟樹之水萃取物對菌絲體產量及生長代謝力呈現最佳的促進效果。其萃取物中多醣分離部分，對菌絲體之產率（5.19 g/L），與基礎培養基所得產率（2.88 g/L）相比，有顯著的促進效果（p＜0.01）。其甲醇萃取物在濃度為2 mg/ml下，對DPPH自由基有85.10 %的清除力，且隨培養時間及劑量的增加，清除能力有增強的效果。樟樹之多醣部經酸水解後，以高效陰離子交換層析法分析，其組成含量佔多醣部百分比由高至低分別為mannose （10.964 %）、galN（5.316 %）、glucose（3.195 %）、myo-inositol（2.62 %）、sorbitol（1.912 %）、fucose（1.551 %）、galactose（1.229 %）及fructose（0.072 %）。添加樟樹水萃取物所培養之菌絲體，培養至第七天時，以HPLC分析，經紫外光全光譜掃描，已有三萜類的化合物產生，其含量及種類隨培養時間增加而提高，添加樟樹水萃取液培養21天的菌絲體，進一步與子實體所得之化合物比對，證實菌絲體含有相同的化合物15-Acetyl-dehydrosulphurenic acid。菌絲體蛋白質經二維電泳膠片圖譜分析，添加樟樹萃取物之菌絲體蛋白質，等電點集中於pH 4-7，以基礎培養基培養，則集中於pH 5-8，且比基礎培養基的蛋白質多一個分子量為21.5 KDa的酸性蛋白質。

關鍵字

牛樟芝 ； 樟屬 ； 三萜類 ； 二維電泳 ； DPPH ； HPAEC ； HPLC ； 菌絲體

並列摘要

Antrodia camphorata is an indegenous fungus that parasitic on the inner cavity heart wood of the endemic species Cinnamomum kanehirae Hay. The fruiting body grow extremely slow in the nature. The aim of this research was to investigate the hyphal growth factors of Antrodia camphorata (Niu-Chan-Chi) from natural products of the Cinnamomum Schaeffers (Cinnamomum kanehirae, Cinnamomum micranthum, Cinnamomum osmophloeum, Cinnamomum camphora and Cinnamomum kotoense). The effects of treated with different extracts from Cinnamomum Schaeffers on the biomass, growth-related metabolic activity, protein and total carbohydrate content, scavenging activity on 1,1-diphenyl-2- picrylhydrazyl (DPPH), High-performance liquid chromatography （HPLC）analysis and electrophoresis on the A. camphorata B85 were determined. The results showed that A. camphorata feeding with the water-extract fraction of C. camphora exhibited the highest growth promotion effect on the biomass accumulation at the dosage of 5.19 g/L than that of control (2.88 g/L). The metabolic activity also showed a higher degree than that of control at the significant level of ? < 0.01. Likewise, the scavenging activity on DPPH exhibited a dose-dependent manner. At dosage 2 mg/ml of feeding with polysaccharide from C. camphora on mycelia have 85.10 % scavenging activity. The high-performance anion-exchange chromatography (HPAEC) analysis was used to characterize the crude polysaccharide of C. camphora. Compositional analysis showed that the polysaccharide fraction composed of mannose (10.964%), galactosamine (5.316%), glucose (3.195%), myo-inositol (2.62 %), sorbitol (1.912%), fucose (1.551%), galactose (1.229 %) and fructose (0.072 %). The chromatogram of A. camphorata mycelia feeding with C. camphora water-extract showed triterpenoid-signal with prolonged cultivation, the signal gets higher. The triterpenoids, 15?-Acetyl- ehydrosulphurenic acid , one of the compounds in fruiting bodies of A. camphorata was also detected in the 21-day-cultured mycelia. Two-dimensional gel electrophoresis of the proteins showed that the spots were mainly localized in the pI range value of 4-7. Whereas, the proteins isolated from basal medium cultured A. camphorata were localized in the pI range value of 5-8.

並列關鍵字

Antrodia camphorata ； Cinnamomum Schaeffer ； Triterpenoids ； Two-dimensional electrophoresis ； 1 ； 1-diphenyl-2-picrylhydrazyl ； High-performance anion-exchange chromatography ； High-performance liquid chromatography ； mycelia

參考文獻

林文川、郭淑卿、吳岳文。樟芝菌株(CCRC 93032)發酵液對大鼠28天餵食的毒性作用，J. Chin Med. 2001, 12(4):293-303.

陳啟楨、蘇慶華、藍明煌。樟芝固體栽培其生物活性研究，Fung. Sci., 2001, 16 (1, 2): 65-72.

林志彬。靈芝的現代研究(第二版)，北京醫科大學出版社，2001, P. 172. 吳昇原。牛樟抽出物對樟芝生長影響之探討，國立台灣大學森林學研究所碩士論文，2001. 高曉薇。台灣靈芝屬新種樟芝之三萜類成分研究，台北醫學院天然物醫學研究所碩士論文，1992. 張東柱。靈芝與熱帶靈芝子實體型態與菌絲純培養之比較，林業試驗所研究報告季刊，1991, 6(4): 397-406.

Boyle, C. D., Kropp, B. R. Development and comparison of methods for measuring growth of filamentous fungi on wood. Can. J. Microbiol. 1992, 38: 1053-1060.

Bradford, M. M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254.