Study On Isolation,purification,structure Characterization And Anti-inflammatory Activity Of Polysaccharide From Codonopsis Tangshen Oliv.

Object:In this study,water extraction and alcohol precipitation method were used to obtain polysaccharides from Codonopsis tangshen Oliv..In order to obtain homogeneous polysaccharides,we used anion exchange column chromatography and gel column chromatography to purify the refined polysaccharides from Codonopsis tangshen Oliv.and identified the structure of homogeneous polysaccharide.The anti-inflammatory activity was screened by lipopolysaccharide-induced RAW264.7 inflammation model in order to obtain a homogeneous polysaccharide with anti-inflammatory activity and well-defined structure.1.Extraction of refined polysaccharide from Codonopsis tangshen Oliv.The crude polysaccharides were extracted by water extraction and alcohol precipitation method,and the protein was purified by sevag method to obtain the refined polysaccharides of Codonopsis tangshen Oliv.and the content of sugar of crude polysaccharides and refined polysaccharides was determined by phenol-concentrated sulfuric acid method.2.Separation and Purification of Refined Polysaccharides from Codonopsis tangshen Oliv.Extracted polysaccharide from Codonopsis tangshen Oliv.was fractionated by DEAE Sepharose Fast Flow gel.The fraction was further purified by dextran G150 and G100 gel,and the final component was subjected to furfural determination of acid content.3.Structural characterization of a homogeneous polysaccharide The purity and molecular weight of homogenous polysaccharides were determined by UV chromatography and high performance gel permeation chromatography.The monosaccharide components of homogenous polysaccharides were analyzed by complete acid hydrolysis and thin layer chromatography.Finally,structural characterization of homogeneous polysaccharides was performed using a variety of spectroscopic techniques(eg,infrared,nuclear magnetic,mass spectrometry,etc.)and methylation analysis.4.Activity screening of homogeneous polysaccharides The lipopolysaccharide-induced RAW264.7 cell inflammatory model in mouse macrophages was used to detect the release of NO in culture medium and the expression of IL-6,TLR4,NF-?B and TNF-? m RNA in cells active screening.Results: 1.9kg medicine was obtained by water extraction and alcohol precipitation of 1.3kg light brown powder CCOP,the yield was 14.4%,the sugar content was 63.78%.After removing protein by sevage method,300 g CCOP obtained 154.6g of light yellow powdery RCOP with a yield of 51.5%,a total yield of 9.3% and a sugar content of 92.20%.2.Two polysaccharide fractions,RCOP1-2-1 and RCOP3-1-1,were isolated and purified from RCOP.The yield of RCOP1-2-1 and RCOP3-1-1 were 28.9% and 9.3%,respectively.Among them,RCOP1-2-1 did not contain uronic acid.The uronic acid content of RCOP3-1-1 was 76.30%.3.The components RCOP1-2-1 and RCOP3-1-1 have no UV absorbance at both 280 and 260 nm and their HPLC shows a single symmetrical peak.The relative molecular mass of the component RCOP1-2-1 is 25.8 k Da,the monosaccharide composition is fructose,the chemical structure of which is composed of 2?1-?-D-fructose residue and the backbone of 2-linked ?-D-Fructose contains both linear oligosaccharides with a small amount of terminal-linked ?-D-glucose.The component RCOP3-1-1 has a relative molecular mass of 49.5 k Da and is composed of arabinose,rhamnose,galactose,and galacturonic acid.4.RCOP,RCOP1-2-1 and RCOP3-1-1 could significantly reduce the NO content in LPS-induced RAW264.7 cell culture medium,and the expression levels of TLR4,NF-?B,TNF-?,IL-6 are significantly reduced(P <0.05,P <0.01).Conclusion: 1.Adopting water extraction and alcohol precipitation to extract polysaccharides from Codonopsis tangshen Oliv.,the extraction conditions are mild,the operation is convenient,the extraction rate is high,and the structure and activity of the plate polysaccharide are not affected.The sevege method is used to remove proteins,the conditions are mild,the glycosidic bond properties of the polysaccharides do not change after treatment,and the protein transfer rate is high.The phenol-concentrated sulfuric acid method was used to determine the sugar content.The operation was simple,rapid,sensitive,with good color stability and good repeatability.It is the most widely used method for the determination of polysaccharide content.2.From the elution curves of the components RCOP1-2-1 and RCOP3-1-1,the elution peaks are single symmetry with high purity,suggesting that the two polysaccharides are homogeneous polysaccharides.According to the separation principle of anion exchange chromatography and the determination of uronic acid content of each component,it is presumed that the component RCOP1-2-1 is a neutral polysaccharide and the component RCOP3-1-1 is an acidic polysaccharide.3.None of the components RCOP1-2-1 and RCOP3-1-1 contain proteins and nucleic acids.Component RCOP1-2-1 is a neutral homogeneous polysaccharide composed of fructose with a relative molecular mass of 25.8 k Da.Its chemical structure is composed of 2 ? 1-?-D-fructose residues with a 2-terminal Linked ?-D-fructose contains a small amount of terminal-linked ?-D-glucose linear levan;Component RCOP3-1-1 is a homopolysaccharide composed of arabinose,rhamnose,galactose,and galacturonic acid,and is an acidic heteropolysaccharide with a relative molecular mass of 49.5 k Da.4.RCOP,RCOP1-2-1 and RCOP3-1-1 have good anti-inflammatory effects.And its anti-inflammatory effect may be through inhibiting the activation of TLR4 / NF-?B pathway to achieve,and may also be related to inflammatory cytokines TNF-?,IL-6.