Study Of Extraction,Isolation,Structural Characterization And Effect On Osteoblasts Of Cynomorium Songaricum Polysaccharides

Objective:The extraction process of crude Cynomorium songaricum polysaccharide（CSP）was optimized by complex enzyme extraction;Isolation and purification of homogeneous polysaccharides from crude polysaccharides;To study the physicochemical properties and structural characteristics of CSP;To investigate the biological activity of CSP on osteoblasts.Methods:（1）Single factor investigation and orthogonal test were used to optimize the optimal enzyme addition and process conditions for extracting of CSP.（2）CSP was prepared by water boiling method and ethanol fractionation precipitation method,and crude polysaccharide was separated and purified by cellulose DEAE 52 ion exchange column chromatography and Sephadex G10 gel column chromatography,Phenol sulfuric acid method,m-hydroxybiphenyl method and BCA method were used to determine the theoretical properties,PMP pre column derivatization high performance liquid chromatography was used to determine the composition of monosaccharides,and gel permeation chromatography was used to determine the molecular weight.（3）The structure of polysaccharides was studied by methylation combined with GC-MS,IR,and Congo red staining.（4）The proliferative effect of CSP on osteoblasts was detected using CCK8method,and the effects of CSP on the secretion of alkaline phosphatase（ALP）,osteocalcin（OCN）,and type I collagen（COL-Ⅰ）by MC3T3-E1 cells were measured using enzyme linked immunosorbent assay（Elisa）;The effect of CSP on the mineralization of MC3T3-E1cells was detected by alizarin red staining.Results:（1）The optimal ratio of complex enzymes for extracting CSP is 9000 U/g pectinase,800 U/g cellulase,and 3200 U/g papain;The optimal process conditions were enzymatic hydrolysis time of 2.5 h,p H 5.0,enzymatic hydrolysis temperature of 40℃,and material liquid ratio of 1:50 g/m L.Under these conditions,the extraction rate was 39.65%±0.13%.（2）After extraction,separation and purification,uniform polysaccharides CSPⅠ₁and CSPⅡ₁were obtained,with polysaccharide mass fractions of 83.27%and 79.43%,and molecular weights of 5.17×10⁴Da and 2.15×10⁴Da.CSPⅠ₁is mainly composed of Man,Glc,Gal,and Ara,with a molar ratio of 0.06:0.21:0.11:1.00.CSPⅡ₁is mainly composed of Man,Glc,and Ara,with a molar ratio of 0.09:0.51:1.00.（3）The IR experiment results showed that there were furans,pyranoses andα-Epimer in CSPⅠ₁and CSPⅡ₁.The results of methylation experiments indicate that CSPⅠ₁and CSPⅡ₁are branched polysaccharides mainly composed of arabinose and have multiple glycosidic bond connections,and there are differences in their branching degrees.The results of Congo red staining experiment showed that there was a triple helix structure in CSPⅠ₁,while there was no triple helix structure in CSPⅡ₁.（4）In vitro cell activity experiments found 50～200μg/m L of CSPⅠ₁and CSPⅡ₁have proliferative effects on MC3T3-E1 cells in both normal and high glucose environments;5、100～200μg/m L of CSPⅠ₁and 50～100μg/m L CSPⅡ₁can increase the content of ALP,OCN,and COL-Ⅰin cells,promote the differentiation of MC3T3-E1 cells,and alleviate the inhibitory effect of high glucose environment on the differentiation of MC3T3-E1 cells;100～200μg/m L of CSPⅠ₁and 100μg/m L of CSPⅡ₁can promote mineralization of MC3T3-E1 cells in normal environments,100～200μg/m L of CSPⅠ₁and 50～100μg/m L of CSPⅡ₁can alleviate the inhibitory effect of high glucose environment on mineralization of MC3T3-E1 cells.Conclusion:The best extraction process of CSP by compound enzyme extraction method was determined through single factor investigation and orthogonal test analysis.CSP was extracted and purified by water extraction and ethanol precipitation,cellulose DEAE 52 ion exchange column chromatography and Sephadex G10 gel column chromatography,Homogeneous heteropolysaccharides CSPⅠ₁and CSPⅡ₁were obtained,both of which were presentα-Epimer,a branched chain polysaccharide dominated by arabinose;CSP can promote the proliferation,differentiation,and mineralization of MC3T3-E1 cells,alleviate inhibitory effect of high glucose environment on the proliferation,differentiation,and mineralization of MC3T3-E1 cells,and provide a certain theoretical basis for subsequent research on the role of CSP in the treatment of osteoporosis.