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研究生:詹世豪
研究生(外文):Shih-Hao Zhan
論文名稱:牛樟芝多醣體之免疫活性分析及結構鑑定
論文名稱(外文):Structural determination of immunoactive polysaccharides from Antrodia cinnamomea
指導教授:吳世雄吳世雄引用關係
指導教授(外文):Shih-Hsiung Wu
口試委員:花國鋒趙國評廖志中王賢達
口試日期:2011-06-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:56
中文關鍵詞:牛樟芝多醣免疫活性膠體過濾氣相層析質譜儀腫瘤壞死因子一氧化氮
外文關鍵詞:Antrodia cinnamomeapolysaccharidesimmunoactivitygel filtratonGC-MSTNFNO
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牛樟芝傳統上多用來做為中藥用,先前的研究也指出,牛樟芝多醣體擁有免疫調節上的活性,而對於它的多醣的結構鑑定尚未明瞭。
  首先利用氰化甲烷以及冷熱水來萃取牛樟芝菌絲體,之後將水層所得到的多醣再經由膠體過濾層析法來分離,得到了三個不同分子量大小的多醣體,依序為 HMPS、LT70和GT70。根據生物活性以及多醣產量的結果,我們選擇了LT70並利用離子交換樹脂層析法來做進一步的純化萃取並且獲得了三部分的多醣樣本(QA、QB和QC)。
  在本篇研究中,牛樟芝多醣刺激了一氧化氮(NO)產生,並且也進一步測試了牛樟芝參與老鼠巨噬細胞在腫瘤壞死因子-α(TNF-α)以及細胞介質素-6(IL-6)的產生。由氣相層析質譜儀的分析結果上,QB的主要醣類成分組成為甘露糖和半乳糖且兩者比例約為4:1。同時也利用氣相層析質譜儀分析在QB樣本中含有:鍵結terminal mannopyranose,1,2 linked galactopyranose,1,3 linked mannopyranose,以及 1,2,6 linked mannopyrnose。同時並利用化學以及光譜分析來鑑定由牛樟芝所分離出的活性多醣QB結構,初步推測應為:
  而單醣D應是接在A或C的6號位置的一個分支醣基。然而完整的結構待需進一步的分析來鑑定。


Antrodia cinnamomea is used as traditional Chinese medicine. In previous studies, polysaccharides from A. cinnamomea possess immune-modulating activity, and the structure determination is still not clear.
Mycelia of A. cinnamomea were first extracted with acetonitrile and followed with hot and cold water, in which the extract was further separated by gel filtration chromatography to give three fractions containing polysaccharides HMPS, LT70, and GT70. Due to the biological activities and amounts of polysaccharides, LT70 was selected for further purification and identification. By using ion exchange chromatography, LT70 was separated into three polysaccharide fractions; QA, QB, and QC.
In the present study, A. cinnamomea polysaccharides participated in the stimulation of nitric oxide(NO) production and stimulation of murine wild-type macrophages predominantly in tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) production.
From sugar analysis by gas-chromatography mass spectrum(GC-MS), the major components of A. cinnamomea polysaccharides QB are mannose and galactose in the molar ratio of 4:1. The main glycosidic linkages were also characterized by GC-MS. The results showed that there are terminal mannopyranose, 1,2 linked galactopyranose, 1,3 linked mannopyranose, and 1,2,6 linked mannopyranose. The structures of bioactive polysaccharides from A. cinnamomea were determined by chemical and NMR methods, and we preliminarily presume that the structure is as following:
And D might be a 6 linked monosaccharide to A or C as a branched terminal residue.The completed structure must be determined by further analysis.


謝誌 ii
中文摘要 iii
Abstract v
Chapter 1 Introduction 1
1-1 Polysaccharides 1
1-1-1 Structural polysaccharides 1
1-1-2 Storage polysaccharides 2
1-2 Application of polysaccharides- as Food and Biomedicals 3
1-2-1 Food polysaccharides 3
1-2-2 Biomedical polysaccharides 4
1-3 Antrodia cinnamomea 4
1-3-1 Characteristic of Antrodia cinnamomea 4
1-3-2 Origination of Antrodia cinnamomea 5
1-3-3 Circumstance of Antrodia cinnamomea 6
1-3-4 Usages and bioactive effects of Antrodia cinnamomea
7
1-4 Fungal studies: Polysaccharides in stimulation of biological 7
activities and immune system 7
Chapter 2 Materials and Methods 10
2-1 Extraction of Polysaccharides 10
2-2 Chemicals and Antibodies 10
2-3 Purification of Antrodia cinnamomea polysaccharides
11
2-4 Sugar compositions and Linkages 11
2-5 Nuclear magnetic resonance spectroscopy 12
2-6 Cell Cultures 13
2-7 Nitric oxide detection 13
2-8 ELISA 13
Chapter 3 Results 15
3-1 Extraction of Antrodia cinnamomea polysaccharides 15
3-2 Separation of Antrodia cinnamomea polysaccharides 15
3-3 Purification by ion exchange chromatography Mono-Q 16
3-4 Immunoactive analysis of Antrodia cinnamomea polysaccharides 16
3-5 Sugar compositions and linkage analysis 17
3-6 NMR analysis 19
Chapter 4 Discussion 21
4-1 Immunoactive properties in Antrodia cinnamomea 21
4-2 Structure determination of immunoactive Antrodia cinnamomea polysaccharides 23
Figure 25
Figure.1 Gel filtration chromatography 25
Figure. 2 Result of Reflex Index detector 26
Figure.3 Purified polysaccharides LT70 visualized by TLC
27
Figure.4 NO production of extracted Antrodia cinnamomea mycelia polysaccharides 28
Figure.5 TNF-α production of extracted Antrodia cinnamomea mycelia polysaccharides 29
Figure.6 IL-6 production of extracted Antrodia cinnamomea mycelia polysaccharides 30
Figure.7 TNF-α production of purified Antrodia cinnamomea polysaccharides 31
Figure.8-(A) 1H NMR spectrum of Antrodia cinnamomea polysaccharides LT70 32
Figure.8-(B) 1H NMR spectrum of Antrodia cinnamomea polysaccharides GT70 33
Figure.9 1H NMR spectrum of Antrodia cinnamomea polysaccharides QA, QB, and QC 34
Figure.10 1H NMR spectrum of Antrodia cinnamomea polysaccharides QB at five different temperatures 35
Figure.11 1H NMR spectrum of Antrodia cinnamomea polysaccharides QB at 308K 36
Figure.12 13C NMR spectrum of Antrodia cinnamomea polysaccharides QB 37
Figure.13 DEPT-135 NMR spectrum compared with 13C NMR spectrum of Antrodia cinnamomea polysaccharides QB 38
Figure.14-A 1H/1H COSY correlation of Antrodia cinnamomea polysaccharides QB 39
Figure.14-B Chemical shifts 3.5 to 4.4 enlarged drawing of COSY spectrum 40
Figure.15 1H/13C HSQC correlation of Antrodia cinnamomea polysaccharides QB 41
Figure. 16 1H/13C HMBC correlation of Antrodia cinnamomea polysaccharides QB 42
Figure.17 1D TOCSY spectra of Antrodia cinnamomea polysaccharides QB 43
Figure.18 Preliminary presumed structure of Antrodia cinnamomea polysaccharides QB 44
Table 45
Table. 1 Extraction flow chart of Antrodia cinnamomea polysaccharides and the yields 45
Table.2 Sugar content percentage of extracted Antrodia cinnamomea polysaccharides 46
Table.3 Results of linkage analysis of extracted Antrodia cinnamomea polysaccharides 47
Table.4 Chemical shift assignments of 1H NMR and 13C NMR spectra of QB on the basis of HSQC, HMBC, COSY and TOCSY
49
References 51


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