Purification,Structure Characterization Of Polysaccharide From Panax Notoginseng And Its Protective Effect On Myelosuppression Induced By Cyclophosphamide

Objectives:In order to establish method of isolation,purification polysaccharides form medicinal residue of Panax notoginseng and investigate its structural,protective effect on myelosuppression induced by cyclophosphamide.Methods:CPPN was extracted by water extraction and alcohol precipitation method,ecolorization in AB-8 macroporous resin column process,and further purified by anion exchange chromatography and gel filtration coiumn chromatography.The neutral sugar content and uronic acid content was determined by anthranone sulfate method and m-hydroxydiphenyl.Its molecular weight was determined by HPGPC,monosaccharide composition was determined by HPAEC.The structure of polysaccharides was analyzed by methylation analysis,ATR FT-IR,scanning electron microscope(SEM).Model mice resulted from myelosuppression by cyclophosphamide.Investigate the spleen index,thymus index,haematological indexes of myelosuppression model treated with NPPN.Apoptosis rate and cell cycle of nucleated cells in bone marrow was detected by flow cytometry.Serum levels GM-CSF?TPO?EPO in myelosuppression were detected by ELISA.Results:CPPN was extracted by water extraction and alcohol precipitation method,yield rate was 3.49%.Decolorization of CPPN with AB-8 macroporous adsorption resins.It shows that the decoloration rate was 45.26%,polysaccharides holding rate was 65.58%.CPPN was purified by DEAE Sepharose Fast Flow anion exchange chromatography and NPPN,APPN??APPN??APPN ? were obtained.,the yield of polysaccharides were 5.72%,3.98%,31.57%,20.32%,respectively.APPN ? was further purified by gel filtration column chromatography with Sephadex G-50,APPN 1I-A and APPN ?I-B were obtained,and the yield of polysaccharides was 27.20%,58.80%.APPN ? was further purified by gel filtration column chromatography with Sephadex G-75,APPN ?-A and APPN ?-B were obtained,and the yield of polysaccharides was 15.30%,73.40%.Chemical composition and structure of NPPN?APPN??APPN ?-A?APPN ?-B?APPN ?-A and APPN ?-B were determined by anthranone sulfate method,m-hydroxydiphenyl method,HPGPC,HPAEC,methylation analysis,ATR FT-IR and SEM.Results were as follows:NPPN is a neutral polysaccharide,total sugar content was 96.40%,the number-average weight was 29120 Da,weight-average molecular molecular was 232500 Da and dispersion coefficient was 7.984.NPPN consist of Ara,Gal,Glc and Man,the molar ratio was 1:4.12:17.04:0.18.NPPN is a a-pyranoside and have low branching coefficient.The methylation analysis showed that the main chain of NPPN is 1,4-Glcp and contains both 1,4-Manp,1,6-Glcp,I,6-Galp sugar residues.Meanwhile,the branched chain of NPPN consist of Galp 1?3,4-Galp and 1?4,6-Galp sugar residues.APPN ? is an acid polysaccharide,the content of neutral sugar,uronic acid and total sugar content was 80.87%,15.02%,95.89%,respectively.The number-average weight was 19720 Da,weight-average molecular molecular was 489900 Da,and dispersion coefficient was 24.85.The monosaccharide composition was measured by ion chromatography.The result showed that APPN ? consist of Ara,Gal,Glc,Man,Gal-AC and Glc-AC,the molar ratio was 1:2.53:3.16:0.17:0.38:0.15.APPN ? is a a-pyranoside and have low branching coefficient The methylation analysis showed that the main chain of APPN ? is 1,4-Glcp and contains both T-Araf,T-Glcp,T-Glc-ACp,T-Glcp,1,3-Galp,1,4-Gal-ACp,1,6-Galp sugar residues.Meanwhile,the branched chain of APPN ? consist of 1?4,6-Glcp and 1?3,6-Galp sugar residues.APPN ?-A is an acid polysaccharide,the content of neutral sugar,uronic acid and total sugar content was 70.46%,20.17%,90.63%,respectively.The number-average weight was 91340 Da,weight-average molecular molecular was 450100 Da,and dispersion coefficient was 4.928.The monosaccharide composition was measured by ion chromatography,The result showed that APPN ?-A consist of Ara,Gal,Glc,Gal-AC and Glc-AC,the molar ratio was 1:2.99:3.00:0.49:0.18.APPN ?-A is a ??pyranoside and have low branching coefficient In addition,APPN ?-A have bulbous structure.The methylation analysis showed that the main chain of APPN ?-A is 1,4-Glcp and contains both T,Araf,T-Glcp,T-Araf,T-Glcp,T-Glc-ACp,T-Galp,1,3-Galp,1,4-Galp,1,4-Glcp,1,4-Glc-ACp,1,6-Galp sugar residues.Meanwhile,the branched chain of APPN ?-A consist of 1?3,4-Glcp,1?4,6-Glcp,1 ? 3,6-Galp sugar residues.APPN ?-B is an acid polysaccharide,the content of neutral sugar,uronic acid and total sugar content was 6.28%,85.34%,91.62%,respectively.The number-average weight was 12510 Da,weight-average molecular molecular was 28600 Da,and dispersion coefficient was 2.287.The monosaccharide composition was measured by ion chromatography.The result showed that APPN ?-B 'consist of Ara,Gal,Glc and Gal-AC,the molar ratio was 1:0.92:1.40:48.97.APPN ?-B is a homogalacturonan pectin as well as a ?-pyranoside.APPN ?-B have low branching coefficient.The methylation analysis showed that the main chain of APPN ?-B is 1,4-Gal-ACp and contains both T-Gal-ACp,1,4-Glcp sugar residues.Meanwhile,the branched chain of APPN ?-A consist of 1?3,4-Gal-ACp,1?4,6-Gal-ACp sugar residues.APPN ?-A is an acid polysaccharide,the content of neutral sugar,uronic acid and total sugar content was 39.86%,30.69%,70.56%,respectively.The number-average weight was 89430 Da,weight-average molecular molecular was 336100 Da,and dispersion coefficient was 3.758.The result of monosaccharide composition showed that APPN ?-A consist of Fuc,Ara,Gal,Glc Xyl,Man,Gal-AC and Glc-AC,the molar ratio was 0.16:1:3.46:1.46:0.12:0.15:2.18:0.29.APPN ?-A linked by ? glycosidic bond and had linear and bulbous structure.APPN ?-B is an acid polysaccharide,the content of neutral sugar,uronic acid and total sugar content was 5.89%,77.07%,82.96%,respectively.The number-average weight was 25110 Da,weight-average molecular molecular was 56280 Da,and dispersion coefficient was 2.241.The monosaccharide composition was measured by ion chromatography.The result showed that APPN ?-B consist of Ara,Gal,Glc and Gal-AC,the molar ratio was 1:1.04:1.98:59.80.APPN ?-B is a homogalacturonan pectin as well as a a-pyranoside.APPN ?-B have low branching coefficient.The methylation analysis showed that the main chain of APPN ?-B is 1,4-Gal-ACp and contains both T-Gal-ACp,1,4-Glcp sugar residues.Meanwhile,the branched chain of APPN ?-A consist of 1?3,4-Gal-ACp sugar residues.The marrow protection effect of NPPN on myelosuppression mice were studied,Low and high dosage NPPN could improve spleen index(P<0.050 or P<0.01).Low,high dosage NPPN,rh G-CSF and diyushengbai table had same efficacy in improvement spleen indexAfter treatment,7 blood count indicators showed significant increases up to the levels of the model group.High dosage NPPN could increase red blood cell number.Hemoglobin concentration,white blood cell,neutrophil number(P<0.05 or P<0.01),neutrophil number returned to normal level.The effect of improve red blood cell and hemoglobin concentration had no significant difference between high dosage NPPN and rh G-CSF(P>0.05).Low,Medium and high dosage NPPN could increase packed cell volume(P<0.01).The level of platelet increased in medium and high dosage NPPN(P<0.01),the therapeutic effect of medium NPPN was better than diyushengbai table(P<0.05).The effect of improve white blood cell number had no significant difference between high dosage NPPN and diyushengbai table(P>0.05).Medium dosage NPPN and diyushengbai table had same efficacy in improvement lymphocyte number.Low,medium and high dosage NPPN could obviously reduce apoptosis rate of bone marrow cell(P<0.01),the therapeutic effect of medium NPPN was better than diyushengbai table(P<0.05).Meanwhile,medium dosage NPPN and rh G-CSF had same efficacy in reduction 1 apoptosis rate of bone marrow cell(P>0.05).The cell ration of bone marrow cell in S phase was significantly decreased.After NPPN administration,implied that NPPN can active bone marrow cell stasis into cell cycling In addition,NPPN could increase the level of serum EPO,TPO,GM-CSF(P<0.05 or P<0.01),promote the proliferation of hematopoietic cells,which will contribute to the recovery of the hematopoietic function.The effect of improvement GM-CSF serum level had no significant difference between high dosage high dosage NPPN and diyushengbai table(P<0.05)as well as had same efficacy with rh G-CSF(P>0.05).The effect of improve EPO serum level had no significant difference between high dosage NPPN,rh G-CSF and diyushengbai table(P>0.05).Conclusions:The current study established a method of isolation,purification polysaccharides form medicinal residue of Panax notoginseng.In this study,CPPN was extracted by water extraction and alcohol precipitation method and further purification by DEAE Sepahrose Fast Flow anion exchange chromatography to prepare a netural polysaccharide(NPPN)and three acide polysaccharides(APPN ?,APPN ?,APPN ?)from CPPN.APPN ? was fiurther purified by Sephadex G-75 gel filtration coiumn chromatography and two components APPN ?-A and APPNII-B were got.APPN ?was further purified by Sephadex G-100 gel filtration Goiumn chromatography and two components APPN ?-A and APPN ?-B were got.Primarily elucidated the structural features of neutral polysaccharide(NPPN),five acid polysaccharides(APPN I,APPN?-A,APPN?-B,APPN ?-A,and APPN ?-B),The results confirmed that NPPN,APPN ? and APPN ?-A are a glycan with 1,4 main chain,APPN ?-A is a glycan,APPN ?-B and APPN ?-B are a homogalacturonan pectin with 1,4 main chain.This study demonstrated that NPPN had a bone marrow hematopoietic function protective role in cyclophosphamide induced myelosuppression mice NPPN could relieve cell cycle arrest,reduce marrow cell apoptosis rate,improve GM-CSF?TPO?EPO serum level which contribute to promote the proliferation of hematopoietic cells.