| Objective: It has been proven that the polysaccharide extracted from Dendrobium nobile Lindl is the active ingredient.To date,the concrete structure and related activity of Dendrobium nobile polysaccharide are still not very clear.So,the thorough study of its structure and bioactivity could lay the foundation for material basis of Dendrobium nobile polysaccharide,quality control and related innovative drug development.Methods: In this thesis,the crude polysaccharide was extracted through hot waterextracted and hot water-extracted after enzymolysis.The crude polysaccharide was further fractionated by anion-exchange chromatography on a DEAE Sepharose Fast Flow.Then the inhomogenous polysaccharide was purified by Sephacryl S-100 HR to obtain homogenous polysaccharide.The molecular weight was estimated by HPGPC.The sugar composition was determined by GC analysis as alditol acetates and by HPLC as polysaccharide was derived by PMP.Structural features of polysaccharide were investigated by a combination of chemical and instrumental analysis,including FTIR,GC,GC–MS,methylation analysis,partial acid hydrolysis,and NMR spectroscopy.JCS1 was sulfated by sulphur trioxide pyridine complex and was acelyted by acetic anhydride.The PC-12 cells were used to examined neuritogenesis induced effect of acetylated polysaccharide.The human microvascular endothelial cells(HMEC-1)was used to evaluate the cell migration and tube formation assay.Chick chorioallantoic membrane test was used to evaluate antiangiogenic activity of polysaccharides in vivo.Results: The crude polysaccharide JCS(47.96 g,yield 1.20%)was the hot water extract from the dried stems of Dendrobium nobile Lindl.JCS was isolated and purified to obtain two water-soluble homogeneous polysaccharide JCS0(122.0 mg,yield 0.25%),JCS1(4.50 g,yield 9.38%)and heterogeneous polysaccharide JCS2(661.0 mg, yield 1.38%).JCS2 was furtherly purified by Sephacryl HR-100 to obtain a homogeneous polysaccharide JCS2S1(174.5 mg,yield 0.36%).The stems were extracted by Na OH to obtain crude polysaccharide JCN(4.03 g,yield 0.08%)after it was extracted by hot water.JCN was isolated and purified to obtain a homogeneous polysaccharide JCN1-1(151.0 mg,yield 3.75%).In another extraction method,the polysaccharide was extract through hot water-extracted after enzymolysis to obtain crude polysaccharide AJCS(33.45 g,yield 0.67%).AJCS was purified by anion exchange column DEAE Sepharose Fast Flow to obtain two fractions AJCS1A(1.42 g,yield 4.20%)and AJCS1B(1.20 g,yield 3.60%).1AE was from AJCS1 A after enzymatic hydrolysis.1AE(470.0 mg,yield 33.10%)was furtherly purified to obtain two homogeneous polysaccharides 1AE1(71.0 mg,yield 15.10%)and 1AE2(41.0 mg,yield 8.75%).AJCS1 B was purified to obtain homogeneous polysaccharide B1(117.0 mg,yield 9.75%).The high performance gel chromatography(HPGPC)analysis for the above homogeneous polysaccharides showed that the molecular weights of JCS0,JCS1,JCS2S1,JCN1-1,1AE1,1AE2 and B1 were 14.3 k D,12.3 k D,54.0 k D,20.3 k D,89.0 k D,37.1 k D and 94.8 k D,respectively.There was no acid sugar in JCS0,JCS1,JCN1-1,1AE1,1AE2 and B1.Actually,they are neutral polysaccharides,while JCS2S1 contained acid sugar.JCS0 consisted of Man and Glc in the molar ratio of 4.6:1.0.JCS1 consisted of Man,Glc,Gal,Xyl and Ara in the molar ratio of 40.2:6.1:4.8:2.9:1.9.JCS2S1 consisted of Man,Rha,Glc A,Gal A,Glc,Gal and Ara in the molar ratio of 3.1:2.5:1.0:1.1:4.4:5.7:1.5.JCN1-1 consisted of Rha,Ara,Xyl,Glc and Gal in the molar ratio of 1.8:1.0:16.6:1.2:6.6.1AE1 contained Rha,Ara,Xyl,Man,Glc and Gal in the molar ratio of 1.0:4.3:1.0:2.4:25.6:17.3.1AE2 contained Rha,Ara,Xyl,Man,Glc and Gal in the molar ratio of 1.0:9.4:3.2:1.3:12.0:18.7.B1 contained Glc,Gal and Ara in the molar ratio of 1.0:1.9:1.5.Among of them,three homogeneous polysaccharides JCS1,B1 and 1AE2 were completely methylated and analyzed by GC-MS to determine linkage type and the molar ratio.The results showed that JCS1 contained nine linkage types: 1,4-Glcp,1,6-Glcp,T-Glcp,T-Galp,1,4,6-Glcp,1,4-Manp,1,4-Xylp,1,3,6-Glcp and T-Araf in the molar ratio of 34.2:2.0:2.6:1.2:3.2:2.2:1.6:1.4:1.0.B1 contained seven linkage types: 1,4-Glcp,1,6-Glcp,1,6-Galp,T-Glcp,1,3,6-Galp,1,3-Galp and T-Araf in the molar ratio of 3.7:5.5:1.3:1.0:6.2:4.0:7.3.1AE2 contained six linkage types: 1,6-Glcp,T-Glcp,T-Galp,1,3,6-Galp,1,3-Galp and T-Araf in the molar ratio of 5.2:1.3:1.0:2.5:3.0:1.3.The further structure analysis of JCS1 shown that JCS1 had a backbone consisting of(1?4)-linked ?-D-Glcp with branches at C-6 of(1?4)-linked ?-D-Glcp.There were three kinds of branches: the first kind was that the C-6 of(1?4)-linked-Glcp was linked 1,6-?-Glcp,the C-6 of 1,6-?-Glcp was linked 1,4-?-Manp,the C-4 of 1,4-?-Manp was linked T-?-Glcp.The second kind was similar with the first kind,the only difference was the terminal linkage type was T-?-Galp.The third kind was that the C-6 of 1,4-?-Glcp was linked 1,3,6-?-Glcp,the C-3 of 1,3,6-?-Glcp was linked 1,4-?-Xylp,the C-4 of 1,4-?-Xylp was linked T-?-Araf,the C-6 of 1,3,6-?-Glcp was linked T-?-Glcp.Bioactive research on polysaccharides showed that YJCS1,the acetylated derivative of JCS1 could induce neuritogenesis of PC12 cells at low concentration(5.56 ?M)and high concentration(55.56 ?M).JCS1S2,the sulfated derivative of JCS1 might inhibit migration and tube formation of human microvascular endothelial cells(HMEC)at very low concentration(0.2 ?M).Further study indicated that JCS1S2 impeded angiogenesis using in vivo Chick chorioallantoic membrane(CAM)model.Conclusion: Six kinds of neutral polysaccharides(JCS0,JCS1,JCN1-1,1AE1,1AE2,B1)and an acidic polysaccharide(JCS2S1)were isolated and purified from the stems of Dendrobium nobile Lindl.Among of them,JCS1,a galactomannoglucan,had a backbone consisting of(1?4)-linked ?-D-Glcp.YJCS1,the acetyl derivative of JCS1 could induce neuritogenesis on PC-12 cells.JCS1S2,the sulfated derivative of JCS1 exhibited a significant inhibition on migration and tube formation of human microvascular endothelial cells(HMEC),and angiogenesis in vivo. |
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