翁啟惠Wong, Chi-Huey臺灣大學:化學研究所簡政Chien, ChengChengChien2010-06-302018-07-102010-06-302018-07-102008U0001-0102200815264800http://ntur.lib.ntu.edu.tw//handle/246246/187420霍山石斛經由冷水萃取得到O-acetylglucomannan多醣。此黏液多醣是經由DEAE離子交換樹酯及SEC管柱層析純化分離而得。這由霍山石斛中分離出的黏液多醣經由核磁共振儀、質譜儀及高效陰離子交換色譜-脈衝安培儀分析其主幹為 1 ,4連結的β-D-甘露糖和1 ,4連結的β-D-葡萄糖比例為10:1。經由核磁共振儀分析，在部分甘露糖的二號及三號位置上含有乙醯取代基比例為34﹪不規則的散佈在O-acetylglucomannan上。利用高效液相色譜及核磁共振儀並用已知分子量的普魯蘭多醣為標準品分析其分子量為1.0×104 。小鼠脾臟細胞及人類周邊單核血球細胞的實驗模式中，從霍山石斛中萃取得到的O-acetylglucomannans可以誘發幾種細胞激素的產生，包含有IL-6、IL-10、IFN-γ、G-CSF、M-CSF和GM-CSF。其中，G-CSF在此黏液多醣誘發幾種細胞激素的產生最顯著的生物性指標。相對的再黏液多醣去除乙醯取代基後，多醣誘導小鼠脾臟細胞激素產生的活性消失。Water-soluble O-acetylglucomannan was isolated from Dendrobium huoshanense by cold water extraction. This mucilaginous polysaccharide was purified by chromatography on DEAE-cellulose and SEC columns. These mucilage fractions of Dendrobium huoshanense were characterized by NMR, Mass, HPAEC-PAD and analysis to have a backbone of β-D-(1→4)-mannopyranosyl units and β-D-(1→4)-glucopyranosyl units in a ratio of 10:1. Its molecular weight was estimated to be 1.0×104 by HPLC and NMR, using pullulan of known molecular weight as standards. The mannopyranosyl residues were partially acetylated at C-2 and C-3 with approximately 34% degree of acetylation appeared randomly from water extracted fractions as shown by the NMR analysis. The extract of O-acetylglucomannans of D. houshanense can induce several cytokines in mice splenocytes and PBMC models, including IL-6, IL-10, IFN-γ, G-CSF, M-CSF and GM-CSF. Among these cytokines, the G-CSF is the most obvious biomarker this mucilage fraction. In contrast, the mucilage after deacetylation shows no cytokines induction in mice.Chapters hapter 1 Introduction1 1~ 8.1 Dendrobium huoshanense 1.2 O-Acetylglucomannan 2.3 Nuclear magnetic resonance 3.4 Gas chromatography-mass spectroscopy 4.5 HPAEC-PAD 5.6 Enzymatic assays 6.7 Strategy for structural analysis of polysaccharide 7hapter 2 Results and Discussion 9~43.1 Isolation of the mucilage polysaccharide from Dendrobium huoshanense 9.2 Glycosyl composition 11.3 NMR analysis of fraction 4-2 13.4 The α and β configuration of glycosidic linkages 20 (1) Nuclear magnetic resonance spectroscopy 20 (2) Enzymatic assays 21.5 De-esterification of the O-acetylglucomannan 25.6 Molecular weight 29 (1) Diffusion-ordered NMR analysis 29 (2) Size exclusion HPLC 31.7 Difference between crude extract and fraction 4-2 33.8 Biological Assay 36hapter 3 Conclusion44~45hapter 4 Experimental Section 46~53.1 Materials and source of sample 46.2 Monosaccharide analysis 46.3 Analysis via methylation 46.4 De-esterification of the O-acetylglucomannan 47.5 Digestion with α-mannosidase 47.6 Digestion with β-mannosidase 48.7 Digestion with endo-β-mannase 48.8 Fractionation 481) Ion-exchange column chromatography 482) Gel-filtration chromatography 49.9 Desalting 491) Dialysis 492) Gel-filtration chromatography 49.10 NMR analysis 49.11 Phenol-sulfuric acid assay 50.12 Protein assay 50.13 Gas chromatography-mass spectrometry 51.14 HPAEC-PAD analysis 51.15 Molecular weight determination by DOSY 52.16 Size exclusion HPLC 53.17 Biological Method 53eferences 54~561556221 bytesapplication/pdfen-US石斛多醣Dendrobiumpolysaccharidethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187420/1/ntu-97-R94223074-1.pdf