Isolation,Purification,Structure Elucidation And Immunomodulatory Activity Of Polysaccharides From The Cell Wall Of Ganoderma Sinense

Ganoderma is a precious edible and medicinal fungi which has a long history in use by Chinese people and shares lots of benefits to human beings.Ganoderma sinense is an important member of Ganoderma spp.It was recorded as an official medicinal substance in Chinese Pharmacopoeia(2000 edition)together with Ganoderma lucidum.Polysaccharides,as the main bioactive components in Ganoderma sinense,possess lots of pharmacological effects,such as antitumor and immunoregulation.Most of the research on polysaccharides from G.sinense were focused on the mycelium polysaccharides and water soluble intracellular polysaccharides of fruiting boldies.However,studies have shown that the polysaccharides in the cell wall of edible fungi also presented strong immune activity.Thus,in order to improve the utilization of G.sinense,the cell wall polysaccharides were extracted by superfine pulverized powder of hot water extraction residue of G.sinense fruit bodies,after separation and purification,7 homogeneous polysaccharides were obtained.The physicochemical properties,chemical structure and immune activity of the polysaccharides were investigated,which provided scientific data for the further development and utilization of polysaccharides from the cell walls of G.sinense.The main results of this paper are presented as follows:Three polysaccharide fractions,GSCW30E,GSCW50E and GSCW70E were obtained by stepwise ethanol precipitation with final concentration of 30%,50%and70%,respectively.The supernatant fraction of 70%ethanol precipitation was coded as GSCW70EU.The yields,polysaccharide contents,monosaccharide compositions and molecular weight distributions of four fractions were studied.Results showed that the total yield of the extracts was 2.0%,and the polysaccharide contents of four fractions were between 75.33%and 98.99%.The most abundant fractions were centralized in GSCW30E,GSCW70E and GSCW70EU.They can be divided into high molecular weight fraction(Mw~10~6 g/mol)and low molecular weight fractions(10~3~10~4 g/mol).GSCW30E possessed the highest polysaccharide content with the value of 98.99%and was mainly composed of glucose.The polysaccharide content of GSCW70E and GSCW70EU were 97.17%and 75.33%respectively.The monosaccharide compositional analysis showed that glucose was the main component in low molecular weight fractions,while some other monosaccharides including xylose,mannose,galactose,glucuronic acid,fucose and glucosamine were also detected in the low molecular weight fractions.Seven homogeneous polysaccharides were obtained by purification of crude extracts through 20%ethanol precipitation,ion exchange chromatography and gel filtration chromatography.A high molecular weight(155.2?10~4 g/mol)glucan GSCW30E-20E was purified from GSCW30E by 20%ethanol precipitation.GSCW70E-W(2.2?10~4 g/mol)and GSCW70E-S(2.5?10~4 g/mol)were two purified fractions—water-eluted fraction and salt-eluted fraction of GSCW70E separated by ion exchange chromatogarphy.And salt-eluted fraction GSCW70E-S showed immune activities in vitro.Through ion exchange chromatography and gel filtration chromatography,we got 4 homogenous polysaccharides named GSCW70EU-W2(5.5?10~4 g/mol)、GSCW70EU-W4(0.8?10~4 g/mol)、GSCW70EU-S1(55.1?10~4g/mol)、GSCW70EU-S2(3.2?10~4 g/mol).The main fraction-GSCW70EU-W4exhibited the activity of stimulating macrophage to release NO and THP-1 to release cytokines TNF-α,IL-1βand IL-6 in vitro.The structure of 4 homogeneous polysaccharides were characterized by infrared spectroscopy,methylation analysis and nuclear magnetic resonance.The structure of high molecular weight polysaccharide GSCW30E-20E was elucidated.Resulted showed GSCW30E-20E is a glucan with both alpha and beta configurations.Every nine beta-(1,3)-glucose residues connects an alpha-(1,4)-glucose residues constitute the main chain structure,while every three beta-(1,3)-glucose residues are linked to a glucose residue as a branch chain by a beta-(1,6)-glycoside bond.Small molecular weight components GSCW70E-W and GSCW70EU-W4 have both alpha and beta configurations,while GSCW70E-S showed beta configurations.The main chains of the three low molecular weight components are mainly beta(1,3),beta(1,4)and beta(1,6),and the branched chains are beta(1,3,4)and beta(1,3,6).