Isolation, Purification, Structural Elucidation, Chemical Modification And Biological Activities Of Polysaccharides From The Fruting Bodies Of Hericium Erinaceus

Hericium erinaceus was a well known edible and medicinal fungus which has beenused traditionally in china to treat gastric ulcer, chronic gastritis and other digestive tractrelated diseases. Both the fruiting bodies and mycelia of the fungus have been reported tocontain some bioactive polysaccharides. It has been shown that the polysaccharides fromHericium erinaceus exhibit such various pharmacological activities as enhancement ofimmune system, antitumor, hypoglycemic and anti-aging, etc. Most of these studies wereperformed on basis of the crude polysaccharides. In order to investigate the chemicalstructure and biological activities of the polysaccharides in this fungus, the extraction,isolation, purification, structure characterization and pharmacological tests ofpolysaccharides from the fruiting bodies of Hericium erinaceus were carried out.There are four parts in this dissertation. Studies on physic-chemical character, sugaranalysis, structural analysis, chemical modification and biological activities ofpolysaccharides from the fruiting bodies of Hericium erinaceus were discussed in thisdissertation.1 Studies on isolation, purification and physic-chemical character from the fruitingbodies of Hericium erinaceusThe polysaccharide fractions from water extract of Hericium erinaceus were isolatedand purified using DEAE Sepharose F. F. and gel-filtration chromatography (SephacrylS-100-500) repeatedly. Five water-soluble homogeneous polysaccharides (HEPF1-HEPF5)were obtained. Lack of absorption at 280 nm and 260 nm by UV scanning indicated thatthey contained no protein and nucleic acid. IR spectra showed that the sugar characteristicgroups were existed. HPLC producing a single symmetrical peak, indicated each of themwas homogeneous and their molecular weight were 1.94×10⁴ Da, 1.66×10⁴ Da, 1.90×10⁴Da, 2.03×10⁴ Da and 4.23×10⁵ Da from HEPF1 to HEPF5, respectively.2 Studied on structure of the polysaccharide (HEPF1-HEPF5) from the fruitingbodies of Hericium erinaceus Their structures were investigated using chemical (Sugar analysis, Methylation analysis,partial acid hydrolysis, etc.), and spectroscopic methods (1D NMR: ¹H NMR, ¹³C NMR,DEPT spectra; 2D NMR: ¹H-¹H COSY, TOCSY, HMQC, NOESY and HMBC spectra; etc.)to confirm the sugar composition, the number of sugar residues, the form of glycosyllinkage, the configuration of anomeric center, the sequence of sugar residues.2.1 Structural analysis of HEPF1Sugar analysis showed this polysaccharide was composed of fucose, galactose andglucose in the ratio of 1.00:4.03:1.04 and a minor of 3-O-methyl-rhamnose. Methylationanalysis showed it contained terminal of 3-O-methyl-rhamnose and fucose linkage, 1,6,1,2,6galactose linkage, terminal,1,6,1,3,6 glucose linkage. NMR analysis further showed thatits main chain was composed ofα-D-1,6,α-D-1,2,6 linked galactosyl, side chain wascomposed of terminalα-L-fucose andβ-1,6-D-glucose and it also contains a minorterminal 3-O-methyl-rhamnose.The proposal structure as follows:2.2 Structural analysis of HEPF2Sugar analysis showed this polysaccharide was composed of fucose, galactose andglucose in the ratio of 1.00:4.03:1.04 and a minor of 3-O-methyl-rhamnose. Methylationanalysis showed it contained terminal,1,4,1,6 glucose linkage, terminal,1,6, 1,2,6 galactoselinkage, terminal fucose and a minor of 3-O-methyl-rhamnose. Partial hydrolysis andacetolysis indicated that its side chain was composed of 1,4, 1,6 linked glucosyl residue and1,6 linked galactosyl residue, attached to the O-2 of main chain with terminal fucosylresidue or a minor of terminal gulcosyl and galactosyl residues. NMR spectra showed 1,4linked glucose isβconfiguration, 1,6, 1,2,6 linked galactose are a configuration, terminalfucose is a configuration.2.3 Structural analysis of HEPF3Sugar analysis showed this polysaccharide was composed of fucose, galactose and in the ratio of 1.00:4.12 and a minor of 3-O-methyl-rhamnose. Methylation analysis showedterminal fucose linkage, 1,6,1,2,6 galactose linkage in the molar ratio of 1.00:2.94:1.2 andalso contains a minor of terminal 3-O-methyl-rhamnose linkage. NMR analysis furthershowed that its main chain was composed of pentasaccharide repeating unit ofα-D-1,6linkedα-D-galactan backbone and branches composed of terminalα-L-fucose, and alsocontains a minor proportion of 3-O-methyl-rhamnose which is thought to terminate thepolymer main chain with the following structure:2.4 Structural analysis of HEPF4Sugar analysis showed this polysaccharide was composed of fucose, galactose andglucose in the ratio of 1.00:3.27:0.28 and a minor of 3-O-methyl-rhamnose. Methylationanalysis showed terminal fucose linkage, 1,6, 1,2,6 galactose linkages in the molar ratio of1.00:2.12:1.07 and also contains a minor of terminal glucose and 3-O-methyl-rhamnoselinkages. NMR analysis further showed that its main chain was composed oftetrasaccharide repeating unit ofα-D-1, 6 linkedα-D-galactan backbone and branchescomposed of terminalα-L-fucose, and also contains a minor proportion of3-O-methyl-rhamnose and glucose residues with the following structure:2.5 Structural analysis of HEPF5Sugar analysis showed HEPF5 was composed of glucose. Methylation analysis showedterminal, 1, 3, 4 glucose linkages in the molar of 1:2.82. NMR analysis further indicatedthat it is a more branchs glucan with tetrasaccharide repeating unit with the following structure:3 Studied on structure and conformation behavior of sulfated polysaccharide from thefruiting bodies of Hericium erinaceusWater-insoluble polysaccharide HEPFA was extracted with 1 mol/L NaOH from thefruiting bodies of Hericium erineceus. A water-soluble sulfated derivative S-HEPFA wasobtained from HEPFA with chlorosulfonic acid-Pyridine, and purified by DEAE-SepharoseFast Flow ion exchange chromatography and Sephacryl S-500 High Resolution columnchromatography. Its sulfate content was 19.5% and average molecular weight was 5.38×10⁵Da. The IR spectrum of S-HEPFA showed the characteristic absorptions of sulfate esterbond at 1236 cm^-1 and 820 cm^-1. The S-HEPFA consisted of only glucose by ionchromatogram. Methylation analysis of the S-HEPFA revealed 1-linked glucose, 1,3-linkedglucose and 1,3,6-linked glucose in the ratios 2.07:1:1.83. The Congo red test showed thatS-HEPFA in neutral or weak base solution takes a triple-helical conformation.4 Studied on biological activities of the polysaccharide from the fruiting bodies ofHericium erinaceusFive kinds of Hericium erinaceus extractions derived from the fruiting bodies wereused to treat injured stomachal mucosa of rat. The results indicated that the medicinaleffect of the crude polysaccharide of the water extracts was the most significant when theamount of the crude drug of five extractions was equal, and that the ulcer inhibitory ratewas up to 60.03%. while the ulcer inhibitory rates of ethanol extracts,HEPF30,HEPF60,HEPF85,positive contrast,negative contrast were 47.1%, 52.62%, 56.40%, 54.36%,43.93%, 24.94 % respectively.