Isolation, Purification, Structure Elucidation And Biological Activities Of Polysaccharides From The Mycelium Of Coprinus Comatus

Coprinus comatus is a kind of edible and medicinal fungi. When cooked, it has tenderflesh with good appearance, a delicate flavor and pleasant fragrance. As an herb, Coprinuscomatus is good to spleen and stomach and can relieve heart burden and treat hemorrhoid.It is reported that Coprinus comatus exhibited especial activities such as hypoglycemic,enhancement of immune system, antitumor, restrain bacteria, etc.At present, researches on Coprinus comatus has been developed, but most of themwere focused on the planting method and nutritional element. Although some researcheswere done on pharmacology, they were mostly on the basis of crude extracts. There are twoways to obtain polysaccharides. One is to extract polysaccharide from fruiting bodies, theother is to Obtain them from fermentation products. The cycle culture time of fermentationis shorter than that of planting and the cost of fermentation is cheaper than that of planting.In order to investigate the chemical structure and bioactivity of polysaccharides fromCoprinus comatus, the isolation, purification, structural elucidation and the immunity testsof the polysaccharides from the mycelium of Coprinus comatus were carried out.There are four parts in this dissertation. Study on physic-chemical character, sugaranalysis, structural analysis, chemical modification and biological activities ofpolysaccharids from the mycelium of Coprinus comatus were discussed in this dissertation.1 Studies on submerged fermentation of Coprinus comatusThis chapter has compared of growth rate and fermentation metabolic curve of CN andCS mycelium. We selected CN as fermentation material because of excellent growth rate,high biomass and yield of intracellar polysaccharides.In 3 L bioreactor, agitation rate 180 rpm and airflow 2.4 L air min^-1 are optimized according to high biomass and yield of intracellar potysaccharides in agitation rate 120～180rpm and airflow 1.2～2.4 L air min^-1.In 30 L bioreactor, fermentation process controls were as follows: temperature 26℃,operational volume factor 2/3, inoculation 10%, agitation rate 150 rotation per minute andairflow 10 L air min^-1 in 24 hours, gitation rate 200 rotation per minute and airflow 20 L airmin^-1 afer 24 hours, cultured time 4 days. The highest yield of mycelium biomass is 17.26g/L.2 Studies on isolation, purification and physic-chemical character of thepolysaccharids from the mycelium of Coprinus comatusThe mycelium of Coprinus comatus were defatted by 95% ethanol, followed byextraction with hot water and alkali successively, the polysaccharide fractions from waterextract (C30, C60, C80, C80s) and alkali extract (CAP) were obtained. Usinganion-exchanged chromatography (DEAE-Sepharose Fast Flow), gel filtrationchromatography (Sephacryl S100～500) to isolate and purify, fourteen homogeneouspolysaccharides were obtained from the C30, C60 and C80.The polysaccharide yield of water extract fractions (C30, C60, C80) from themycelium of Coprinus comatus is 15.6%, which is higher 2.4 times than that from fruitingbodies. Sugar component analysis revealed that polysaccharides from the mycelium ofCoprinus comatus mainly contained glucose, galactose and fucose.3 Studied on structures of three homogeneous polysaeeharides from the myeelium ofCoprinus comatusThe structures of C30-FA3, C60-FA1 and C60-FB1 were investigated by compositionanalysis, methylation analysis, IR, NMR spectroscopic methods (1D NMR: ¹H NMR, ¹³CNMR, 2D NMR: COSY, TOCSY, HMQC, HMBC and NOESY spectra).â‘ Structural analysis of C30-FA3C30-FA3, with a molecular mass of 1.03×10⁴ Da, contained galactose and fucose inthe ratio 1:4.02. Methylation analysis revealed 1-1inked Fuc, 1,6-linked Gal and1,2,6-linked Gal in the ratio 1.15:2.88:1. NMR analysis further showed that the structureconsist mainly of a backbone of (1,6)-linkedα-D-galactopyranosyl residues and substitutedat O-2 either byα-L-fucopyranosyl residue. C30-FA3 consists of pentasaccharide repeatingunits with the following structure: The structure of C30-FA3 is same as that of CC30w-4 from fruiting bodies.â‘¡Structural analysis of C60-FA1C60-FA1, with a molecular mass of 1.03×10⁴ Da, contained galactose, fucose andglucose in the ratio 1:3.04:0.53. Methylation analysis revealed 1-linked Fuc, 1,6-linkedGal and 1,2,6-linked Gal in the ratio 1:2.04:0.91. NMR analysis further showed that thestructure consist mainly of a backbone of (1,6)-linkedα-D-galactopyranosyl residues andsubstituted at O-2 either byα-L-fucopyranosyl residue. C60-FA1 consists of tetrasacchariderepeating units with the following structure:â‘¢C60-FB1, with a molecular mass of 16.69×10⁴ Da, contained glucose. Methylationanalysis revealed 1,4-1inked Glc. C60-FB1 consists of repeating units with the followingstructure: 4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4 Studied on immunocompetence of the polysaccharides from the mycelium ofCoprinus comatusIn the immunocompetence tests, all the crude fractions C30, C60, C80, except for CS0sand CAP, showed effect on the proliferation of lyrnphocytes. The result of the crudefractions is similar to that of fruiting bodies. Of all the homogeneous polysaccharides,C30-FB1,C30-FB2,C60-FB2,C60-FB3,C80-FA2,CS0-FB1,C80-FB2 showed theeffect of the proliferation of lymphocytes under some concentration. C30-FA2,C60-FA1has lightly effects of stimulating the proliferation of lymphocytes. C30-FA3,C60-FA2,C60-FB1,CS0-FA1 has no obvious effects of stimulating the proliferation of lymphocytes.The immunocompetence results of the crude fractions indicated that it is feasibility polysaccharide extraction substituting mycelia for fruiting bodies of Coprinus comatus.