Purification And Biological Activity Of Carboxymethyl Pachyman

Poria cocos,a common traditional Chinese medicine with high dietary supplement and medicinal value,are widely used in food,health care products and medicine.Pachymaran is the main pharmacodynamic component of poria cocos,and the poor water solubility severely limited its clinical applications.In this dissertation,the homemade carboxymethyl crude polysaccharide with 0.52 degree of substitution from Poria cocos was used as experimental material,which was purified by columns of ion-exchange chromatography and glucan gel chromatography.Hereafter,the structure of purification components was identified,and their antioxidant activity,anti-proliferation activity and anti-inflammation activity were studied in vitro.In addition,the acute colitis model induced by TNBS/ethanol was sucessessfully established,and the purified component CMP33 was used to treat the model mice by the way of gastric irrigation to test the anti-inflammation activity was studied in vivo.Moreover,proteomics and metabolomics techniques were used to screen proteins and metabolites to explore the possible anti-inflammation mechanism of inflammation bowel disease（IBD）.The main results of this study were as follows:（1）Three carboxymethyl polysaccharide components from Poria cocos were obtained,named CMP11,CMP33 and CMP44 with polysaccharides content of 97%,99%and 99%respectively,and the average relative molecular weight were 31.38×10⁴ Da,15.23×10⁴ Da and 20.96×10⁴ Da respectively.None of the three components contained proteins and nucleic acids confirmed by the mothod of UV scan.The infrared spectrum analysis showed that all these three components contained carboxymethyl groups and possessed characteristic infrated absorption of polysaccharides.Results from the Gas Chromatography（GC）,high iodate oxidation and Smith degradation,NMR and Congo red experiment showed that the monosaccharide from all these components was D-glucose.CMP11 contained 87.89%（1→3）-βglycosides,5.12%（1→6）glycosides and 6.99%（1→2）glycosides,without tripe helix structure.CMP33 contained 78.62%（1→3）-βglycosides,4.83%（1→6）glycosides and16.55%（1→2）glycosides,with tripe helix structure.CMP44 contained 79.71%（1→3）-βglycosides,8.13%（1→6）glycosides and 12.16%（1→2）glycosides,with tripe helix structure.（2）Antioxident activity in vitro experiment showed that,all these three components possessed certain reduction ability,hydroxyl radical（·OH）,ABTS radical（ABTS⁺）,DPPH radical（DPPH·）and superoxide anion radical（·O₂^-）eliminating capability in a dose-dependent way,and the removal of hydroxyl radical capability was strongest（CMP33>CMP44>CMP11）,showing EC₅₀ values of 2.31 mg/mL,2.94 mg/mL and 5.36 mg/m L;the removal of ABTS radical capability was good（CMP33>CMP44>CMP11）,showing EC₅₀values of 3.55 mg/mL,4.83 mg/mL,6.33 mg/mL;the removal of DPPH radical capability was good（CMP33>CMP44>CMP11）,showing EC₅₀ values of 3.62 mg/m L,4.85 mg/mL,7.50 mg/m L;and the removal of superoxide anion radical capability was worst（CMP33>CMP44>CMP11）,showing scavenging rate of 36.87%,36.07%and 33.12%on the concentration of 10 mg/m L.（3）Anti-proliferation activity in vitro experiment showed that,all these three components possessed certain anti-proliferation effect on HT-29,HepG-2,MCF-7,SGC-7901 and A549cells.The inhibitory effect on SGC-7901 cells by these three components was best（CMP33>CMP44>CMP11）,showing IC₅₀ values of 79.56μg/m L,102.5μg/mL and 146.0μg/m L;on HT-29 cells was better（CMP33>CMP44>CMP11）,showing IC₅₀ values of 113.3μg/m L,140.5μg/m L and 207.4μg/mL;on HepG-2 cells was good（CMP44>CMP33>CMP11）,showing IC₅₀ values of 264.3μg/m L,282.7μg/m L and 385.4μg/mL;on A549 cells was good as also（CMP33>CMP44>CMP11）,showing IC₅₀ values of 204.1μg/m L,313.2μg/mL and378.9μg/m L;but on SGC-7901 cells was worst（CMP44>CMP33>CMP11）,showing IC₅₀values of 256.4μg/mL,385.8μg/mL and 487.1μg/m L.（4）Anti-inflammatory activity in vitro experiment showed that,all these three components possessed good inhibitory effect on NO,IL-6,TNF-αand IL-1βrelease in LPS-induced RAW264.7 cells,which showed that they had significant anti-inflammatory activity,and the CMP33 had best inhibitory effect in a dose-dependent way.Moreover,all these three components can activate RAW264.7 cells to secrete NO,IL-6,TNF-αand IL-1βwithout LPS-induced,which proved that they had certain immunocompetence.（5）Animal experiments showed that,CMP33 treated by lavaged method to the TNBS-induced mice can effectively improve the macro performance,reduce the pathological histology grade,myeloperoxidase（MPO）activity and malondialdehyde（MDA）content in colon,and the CMP33 higher doses treated group has the most significant inhibitional effect.Besides,CMP33 showed a certain regulation effect on the release of related inflammatory cytokines both in colon and blood from TNBS-induced model mice,reduced the production of Th1 related cytokines as IL-1β,IL-2,IFN-γ,I L-12 and TNF-α,Th2 related cytokines as IL-6,and Th17 related cytokines as IL-17,but increased Th2 related cytokines as IL-4 and IL-10.（6）Proteomics analysis showed that,the related differentional proteins between CMP33higher doses group and model group have been identified 194 numbers of differental proteins,with 83 numbers were increased and 107 numbers were reduced significantly,such as Ebp,mt-Co3,S100g,Slpi,Rps27,H2-Aa,S100a14,Hbb-b2,Hmgcs2,Hp and Rb1cc1have significant expression,and the main KEGG pathways these differential proteins participated in were fatty acid metabolism,valine,leucine and isoleucine degradation,TypeⅠdiabetes mellitus,metabolism of xenobiotics by cytochrome P450,graft-versus-host disease,PPAR signaling pathway,drug metabolism and ECM-receptor interaction.The related differentional proteins between CMP33 lower doses group and model group have been identified 306numbers of differental proteins,with 144 numbers were increased and 162 numbers were reduced significantly,such as Ggh,Spink1,Slpi,B3gnt6,Pyy,Insl5,Hbb-b2,Hp and Rb1cc1have significant expression,and the main KEGG pathways these differential proteins participated in were PPAR signaling pathway,fatty acid metabolism,antigen processing and presentation,cell adhesion molecules,valine,leucine and isoleucine degradation,TypeⅠdiabetes mellitus,viral myocarditis,O-Glycan biosynthesis,propanoate metabolism,butanoate metabolism,retinol metabolism,metabolism of xenobiotics by cytochrome P450,drug metabolism,and so on.（7）Metabolomics analysis showed that,44 numbers of differental metabolites were identified between CMP33 higher doses group and model group,with 31 numbers were increased metabolites and 13 numbers were reduced metabolites,such as dihydrotesto,4-hydroxybutyrate,androsterone,glutathione,mannose,oleic acid and hexadecane have significant expression,which were mainly participated in glutathione metabolism,glyoxylate and dicarboxylate metabolism,phenylalanine metabolism,steroid hormone biosynthesis,propanoate metabolism,fructose and mannose metabolism,glycerolipid metabolism,and so on.19 numbers of differental metabolites were identified between CMP33 lower doses group and model group,with 16 numbers were increased metabolites and 3 numbers were reduced metabolites,such as uccinic acid,maleic acid,erone,dihydrotesto,D-erythronolactone,mannose and oleic acid have significant expression,which were mainly participated in propanoate metabolism,citrate cycle（TCA cycle）,fructose and mannose metabolism,amino sugar and nucleotide sugar metabolism,and so on.