Isolation, Purification, Structural Elucidatuion, And Chemical Modification Of Polysaccharides From The Fruting Bodies Of Phellinus Baumii Pilát

Phellinus baumii Pil(?)t is a well-known fungus of the genus Phellinus in the family of Hymenochaetaceae and has been used as a traditional herb medicine for years in oriental countries. Polysaccharides extracted from P. baumii Pil(?)t were reported to have antitumor and immunostimulatory properties, as well as anti-mutagenicity activity, hypoglycemic, anti-oxidant and free radical scavenging properties. However, structural characterization and structure-activity relationship of the polysaccharides from P. baumii Pil(?)t were not investigated in detail. Thus, in order to study the polysaccharides of this fungus, and investigate the structure-function relationship of polysaccharides, the extraction, isolation, purification, structure elucidation and chemical modification were performed on the polysaccharides from the fruiting bodies of P. baumii Pil(?)t.There are three parts in this dissertation. Studies on isolation, purification, physic-chemical character, structure elucidation and chemical modification of polysaccharides from the fruiting bodies of P. baumii Pil(?)t were discussed in this dissertation.The crude polysaccharides were isolated and purified using DEAE Sepharose Fast Flow and gel-filtration chromatography (Sephacryl S) repeatedly. Six water-soluble homogeneous polysaccharides (PBF2-PBF7) were obtained. No absorption at 280 nm and 260 nm by UV scanning (PBF2-PBF6) indicated that they contained no protein and nucleic acid. PBF7 contained 0.53% protein by Lowry method. The HPLC profile showed a single and symmetrical peak, indicating each of them was homogeneous polysaccharide. Correlation with the calibration curve of dextran standards, their molecular weight were 2.0×10⁶Da,2.3×10⁵Da,1.41×10⁶ Da,1.74×10⁶ Da,3.23×10⁵ Da and 1.42×10⁴ Da from PBF2 to PBF7, respectively.2 Studied on structures of the homogeneous polysaccharide from the fruiting bodies of P. baumii Pil(?)tTheir structures were detailedly identified on the basis of chemical methods (sugar composition analysis, glycosyl residue linkage analysis, etc.), and spectral technologies (IR, GC-MS, ID NMR: ¹H NMR,¹³C NMR, DEPT spectra, 2D NMR:¹H-¹H COSY, TOCSY, HMQC, NOESY and HMBC spectra, etc.).1.Structural analysis of PBF2Sugar analysis showed this polysaccharide was composed of fucose,mannose and glucose in a molar ratio of 1.0:2.3:10.7. Methylation analysis showed it contained terminal fucose, 1,3,6 mannose linkage, 1,6-, 1,3,6- and terminal glucose linkage. NMR analysis further showed that it consisted of aβ-(1→6)-D-glucopyranosyl backbone with a fucosyl unit on 0-3 of the 3,6-di-O- substituted-D-glucosyl units and it also contained a minor (1→3,6)-β-D-mannose residue and terminal glucose residues.2. Structural analysis of PBF3Sugar analysis showed this polysaccharide was composed of glucose only. The m-hydroxydiphenyl method revealed that it contained 16.85% uronic acid. Methylation analysis showed it contained 1,3-,1,4-,1,3,6- and terminal glucose linkage in a molar ratio of 2:4:1:1.NMR analysis further indicated that it is a more branches glucan consisting mainly of a backbone of (1,4)-linked P-D-glucopyranosyl residues and (1,3)-linkedβ-D-glucopyranosyl residues and substituted at O-6 either byβ-D-glucopyranosyl residues. 3. Structural analysis of PBF4Sugar analysis showed this polysaccharide was composed of fucose and glucose in a molar ratio of 1:4.Methylation analysis showed it contained 1,2- fucose linkage,1,4-, 1,4,6- and terminal glucose linkage. NMR analysis further showed that PBF4 was a fucoglucan consisted of a a-(1→4)-D-glucopyranosyl backbone with some insertions ofα-(1→2)-L-Fucp residues and also contained a minorβ-(1→4,6)-linked D-glucopyranosyl andβ-glycosidically linked nonreducing-end D-glucopyranosyl residues with repeating units as follows:4. Structural analysis of PBF5Sugar analysis showed this polysaccharide was composed of mannose and glucose in a molar ratio of 1:5.It contained 6.5% uronic acid. Methylation analysis revealed 1,4-linked Manp,1,4-linked Glcp,1,3,4-linked Glcp and 1-linked Glcp in a molar ratio of 1.0:3.6:0.4:1.0.NMR analysis further showed that the structure consist mainly of a backbone of (1,4)-linkedα-D-glucopyranosyl residues and (1,4)-linkedβ-D-mannopyranosyl residues. Glucopyranosyl side chains were present as terminal residue on the 3-position of 4-linked glucopyranosyl residues. PBF5 consists of repeating units with the following structure:5. Structural analysis of PBF6Sugar analysis showed this polysaccharide was composed of glucose only and contained 8.02% uronic acid. Methylation analysis revealed 1,6-linked Glcp and 1,3-linked Glcp in a molar ratio of 1.0:2.0. NMR analysis further showed that the structure consist mainly of a backbone of (1,3)-linked P-D-glucopyranosyl residues and (1,6)-linkedβ-D-glucopyranosyl residues with the following structure: 6. Structural analysis of PBF7Sugar analysis showed this polysaccharide was composed of glucose only and contained 10.51% uronic acid. Methylation analysis revealed 1,6-linked G\cp and 1,3-linked Glcp in a molar ratio of 1.0:1.0. NMR analysis further showed that the structure consist mainly of a backbone of (1,3)-linkedβ-D-glucopyranosyl residues and (1,6)-linkedβ-D-glucopyranosyl residues.3 Studied on sulfated polysaccharide from the fruiting bodies of P. baumii Pil(?)tWater-insoluble polysaccharide PBFA2 was extracted from the fruiting bodies of P. baumii Pil(?)t. A water-soluble sulfated derivative SPBFA was obtained from PBFA2 with pyridine sulfur trioxide complex, and purified by DEAE-Sepharose F. F. ion exchange chromatography and Sephacryl S-400 High Resolution column chromatography. Its sulfate content was 14.7% and average molecular weight was 1.28×10⁴Da.The IR spectrum showed the characteristic absorptions of sulfate ester bond at 1240 cm^-1 and 810 cm^-1.Sugar analysis showed it was composed of glucose only. Methylation analysis showed it contained 1,3-, and terminal glucose linkage. NMR analysis further showed that SPBFA was a water-solubleα-(1→3)-D-glucan.