Structural Feature Of Polysaccharide From Hericium Erinaceus Fruiting Body And Its Auxiliary Protection Function On Gastric Mucosa

Gastric ulcer,a common type of peptic ulcer,is afflicting millions of individuals worldwide.Treatment of gastric ulcer mostly depends on the usage of synthetic drugs,while it triggers diverse side-effects even toxic effects.In this regard,seeking for more effective and safe anti-gastric ulcer substitutes has attracted much attention.Recently,Hericium erinaceus(H.erinaceus)fruiting body and its water-extracts have been demonstrated to possess the auxiliary protective effect on gastric mucosa.However,there was insufficient evidence that which bioactive compound had this effect.Polysaccharide is one main bioactive compound of natural product,and polysaccharide is an important ingredient of H.erinaceus.Thus,the polysaccharide from H.erinaceus fruiting body was selected as the research subject.The object of this study is to explore polysaccharide fractions which have auxiliary protective effect on gastric mucosal lesion.The polysaccharide from H.erinaceus fruiting body was obtained by hot water extraction,and it was purified by gradual ethanol precipitation method.The structural features of the purified polysaccharides were characterized by methylation and 1D/2D NMR analyses.Ethanol-induced gastric lesions,pylorus ligation-induced acute gastric ulcer and acetic acid-induced chronic gastric ulcer models were used to ascertain whether the polysaccharide from H.erinaceus fruiting body exert auxiliary protection on gastric mucosa or not.The hydrogen peroxide-induced human gastric mucosal epithelial cell line GES-1 cells injury model,was applied to screen the characteristic fraction from the purified polysaccharides,of which had the relatively stronger protective effect on GES-1 cells.The major contents and findings are showed as follows:1.Purification of the polysaccharide from H.erinaceus fruiting body was carried out.Polysaccharide from H.erinaceus fruiting body was obtained by hot water extraction and ethanol precipitation methods,and it was purified by gradual ethanol precipitation means.The basic physicochemical properties of it and its purified fractions were determined.Results showed that HERP was polysaccharide which had a little of proteins.It was mainly composed by fucose(Fuc),galactose(Gal)and glucose(Glc),and it exhibited smooth flakiness structures on the surface morphologies.Five polysaccharide fractions(RP-50,RP-60,RP-70,RP-80 and RP-S)with good homogeneity were successfully purified from HERP.The yield of RP-50,RP-60,RP-70,RP-80 and RP-S was 23.9%,23.4%,16.7%,8.6%and 6.8%,respectively.The total sugar content of them was 93.5%,91.5%,93.1%,81.3%and71.8%.2.The primary structure of RP-50 was characterized.Methylation and 1D/2D NMR tools were mainly carried out to study the structural feature of RP-50.Results showed that the relatively average molecular weight of RP-50 was 20.7 k Da.It was composed of Fuc,Gal and Glc in the mass percentage of 15.6:55.2:10.7.Scanning electron microscopy(SEM)observation reflected that RP-50 behaved as smooth flakiness structures.RP-50 mainly consisted of four sugar residues including?-Fucp-(1?(18.0%),?6)-?-Glcp-(1?(11.6%),?6)-?-Galp-(1?(45.6%)and?2,6)-?-Galp-(1?(15.1%).It had a backbone composed of?6)-?-Galp-(1?,and a?-Fucp-(1?branch was linked to the O-2 position of?6)-?-Galp-(1?.It might also consist of?6)-?-Glcp-(1?6)-?-Glcp-(1?fragment.3.The primary structure of RP-60 was investigated.Methylation and 1D/2D NMR means were mainly conducted to determine the structural characteristic of RP-60.Results indicated that the relatively average molecular weight of RP-60 was17.4 k Da,and it was made up of Fuc,Gal and Glc in the mass percentage of 8.8:34.6:35.9.SEM observation reflected that RP-60 exhibited flakiness,smooth and blocky structures.RP-60 was composed of Fucp-(1?(8.9%)?Glcp-(1?(13.8%)?Galp-(1?(3.7%)??3)-Galp-(1?(6.6%)??6)-Glcp-(1?(32.0%)??6)-Galp-(1?(17.2%)??3,6)-Glcp-(1?(9.7%)and?2,6)-Galp-(1?(7.8%)sugar residues.The main chain of RP-60 consisted of?6)-?-Glcp-(1?and?6)-?-Galp-(1?.A?-Glcp-(1?side chain was substituted at the O-3 position of?6)-?-Glcp-(1?,and a?-Fucp-(1?branch was linked to the O-2 position of?6)-?-Galp-(1?.Its might also contain?-Galp-(1?3,6)-?-Glcp-(1?or?3)-Galp-(1?3,6)-?-Glcp-(1?fragment.4.The primary structure of RP-70 was analyzed.The structural feature of RP-70was primarily ascertained by methylation and 1D/2D NMR technologies.Results reflected that the relatively average molecular weight of RP-70 was 9.0 k Da,and it was mainly made up of Gal(8.3%)and Glc(51.1%).SEM observation reflected that RP-70 behaved as flakiness,blocky,rodlike and globular structures.RP-70 was mainly composed of?-Glcp-(1?(15.0%),?3)-?-Glcp-(1?(13.8%),?6)-?-Glcp-(1?(45.9%)and?3,6)-?-Glcp-(1?(16.3%).RP-70 possessed a main chain consisted of?6)-?-Glcp-(1?,and had a?-Glcp-(1?3)-?-Glcp-(1?side chain at the O-3 position.5.The primary structure of RP-80 was explored.The structural property of RP-80 was primarily identified by methylation and 1D/2D NMR methods.Results implied that the relatively average molecular weight of RP-80 was 4.4 k Da,and it was mainly composed of Glc.In the SEM image,RP-80 showed flakiness and rodlike structures.RP-80 was composed of four sugar residues including?-Glcp-(1?(16.6%),?3)-?-Glcp-(1?(11.9%),?6)-?-Glcp-(1?(57.2%)and?3,6)-?-Glcp-(1?(14.3%).It had?6)-?-Glcp-(1?backbone,which branched at the O-3 position by a side chain composed of?-Glcp-(1?3)-?-Glcp-(1?structure.6.The primary structure of RP-S was proposed.Combination of methylation and1D/2D NMR analysis was mainly performed to investigate the structural property of RP-S.Results showed that the relatively average molecular weight of RP-S was 2.1k Da,and it was mainly made up of Glc.In the SEM image,RP-S had blocky structures which aggregated together.RP-S mainly consisted of?-Glcp-(1?(24.4%),?6)-?-Glcp-(1?(51.5%)and?3,6)-?-Glcp-(1?(19.9%),and it contained small amounts of?3)-?-Glcp-(1?(4.1%).The backbone of it was composed of?6)-?-Glcp-(1?,and that was branched with a side chain made up of?-Glcp-(1?units.7.The alleviative effect of polysaccharide from the fruiting body against acute gastric lesions and acute gastric ulcer was evaluated.Two models,including ethanol-induced acute gastric lesions in rats and pylorus ligation-induced acute gastric ulcer in rats,were applied to evaluate the auxiliary protection function of the polysaccharide on gastric mucosa.Results implied that polysaccharide from the fruiting body could reduce the gastric lesion index of rats induced by ethanol,and HERP was more effective than HECP.In the pylorus ligation-induced gastric ulcer model,pre-treatment of the H.erinaceus polysaccharide reduced gastric ulcer index,alterations of macroscopic and histopathological morphologies,volume of gastric juice,pepsin activity and output of gastric acid,and increased p H value of gastric juice and level of gastric mucus.Moreover,the levels of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)and activity of myeloperoxidase(MPO)in serum of rats were decreased.Meanwhile,increments in activities of superoxide dismutase(SOD)and glutathion peroxidase(GPx)and contents of nitric oxide(NO),prostaglandin E2(PGE2)and epidermal growth factor(EGF),and reduction in histamine(HIS)were observed in gastric tissues in rats.Furthermore,at the dose of200 mg/kg bw,the auxiliary protective effect of HERP on the gastric ulcer of rats induced by pylorus ligation was stronger than HECP.Above results suggested that the action mechanism of the polysaccharide in alleviating pylorus ligation-induced gastric ulcer might be regulation of gastric secretion,reduction of inflammatory response,improvement of antioxidant status and modulation of the releases of gastric mucosal defensive and offensive factors.8.The accelerative effect of polysaccharides from the fruiting body on healing of chronic gastric ulcer was studied.Acetic acid induced chronic gastric ulcer in rats was utilized to explore the accelerative effect of the polysaccharide on the healing of gastric ulcer.Results indicated that the size of ulcer circle and the loss of gland structures in gastric tissues were decreased when ulcer rats treated with polysaccharide.Moreover,at the doage of 400 mg/kg bw,HERP had stronger accelerative effect than HECP on the healing of chronic gastric ulcer of rats.Meanwhile,polysaccharide treatment could enhance catalase activity of serum and SOD activity of gastric tissues,decline the levels of interleukin-6(IL-6),TNF-?and malondialdehyde(MDA)and the activity of MPO in gastric tissues,increase the releases of defensive factors in gastric tissues including NO,PGE2,EGF,vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(b FGF).Furthermore,immunohistochemical analysis indicated that the protein expressions of phosphoinositol 3 kinase(PI3K)and protein kinase B(AKT)in gastric tissues of rats were increased.These results suggested that the action of polysaccharides from the fruiting body in promoting the healing of gastric ulcer was related to reduction of inflammatory response,increment of antioxidant capacity,facilitation of gastric mucosal defensive factors releases and increase of the expressions of PI3K and AKT.9.The purified fraction from HERP with the relatively higher inhibitive effect on cells injury of GES-1 was screened.H₂O₂-induced GES-1 cells damage model was used to evaluate the protective effect of HERP and its different fractions on GES-1cells.Results reflected that HERP and its fractions could increase the cell viability of GES-1 cells.Among them,RP-S exerted the relatively higher auxiliary protective effect.Thus,RP-S was selected for further study,and relevant biochemical indexes were determined.Pre-treatment of RP-S could decrease lactate dehydrogenase(LDH)activity and cell apoptosis of GES-1 cells induced by H₂O₂,lower the levels of MDA and reactive oxygen(ROS)and increase the activities of SOD and GPx in the GES-1cells supernatant.These results implied that RP-S pre-treatment could elevate the antioxidant capacity of GES-1 cells.