Structural Characterization And Immunomodulatory Activity Of Codonopsis Polysaccharide CPC

Objective:Modern research indicates that polysaccharide is one of the important bioactive components of Codonopsis Radix.At present,although studies on the physicochemical properties and immunomodulatory effect of Codonopsis Radix polysaccharides have been reported,it is still not systematic and clear whether the immunomodulatory effect of Codonopsis Radix polysaccharides is related to its molecular weight and structural characteristics.Therefore,in this study,Codonopsis Radix was used as material,and the structure of CPC was analyzed after the crude polysaccharides was extracted and purified.The immunomodulatory activity of RAW264.7 cells in vitro was systematically evaluated,so as to provide theoretical basis for the development and utilization of Codonopsis Radix polysaccharide resources.Methods:(1)Isolation and purification of polysaccharide CPC from Codonopsis Radix:In this study,the extraction of polysaccharides from Codonopsis Radix was carried out according to the patent of Codonopsis Radix polysaccharides extraction obtained by the research group.The dried Codonopsis Radix powder(10 kg)was refluxed with distilled water according to the solid-liquid ratio of 1:10,and extracted three times for1 h each time.After the extracting solution was concentrated to 1/10 volume,95%ethanol was added until the concentration of alcohol precipitation was 80%,standing at 4℃ for 12 h,and then the precipitation was washed with anhydrous ethanol,acetone and petroleum ether for 3 times respectively.After freeze-drying,the total polysaccharides of Codonopsis Radix was obtained.The total polysaccharides of Codonopsis Radix(600 g)was dissolved in proper amount of distilled water and subjected to 5000,3000 and 1000 grade ultrafiltration membrane to obtain CPC(120g)after freeze-dried.(2)Structure analysis of polysaccharide CPC from Codonopsis Radix: The molecular weight and homogeneity of CPC were determined by high performance gel permeation chromatography(HPGPC).Ultraviolet spectrum(UV),infrared spectroscopy(IR),acid hydrolysis,methylation analysis,gas chromatography-mass spectrometry(GC-MS)and NMR spectroscopy were used to identify the chemical structure of CPC.(3)Study on the immune activity of polysaccharide CPC in vitro: In this study,RAW264.7 cells were used as the model.The proliferation of macrophages was detected by CCK-8 method,and the phagocytic activity was measured by neutral red staining method.The level of reactive oxygen species(ROS)released by cells was detected by DCFH-DA method,and the release level of nitric oxide(NO)in cell supernatant was measured by Griess method.Also,the secretion and gene expression of cytokines(iNOS,TNF-α,IL-6,IL-1β,IL-10)in macrophages were detected by enzyme-linked immunosorbent assay(ELISA)and real-time fluorescent quantitative PCR(RT-qPCR).Results:(1)Structure analysis of polysaccharide CPC from Codonopsis Radix: HPGPC showed that CPC was a polysaccharide with uniform molecular weight and the molecular weight of CPC was about 1698 Da.Then,the basic structure of CPC was analyzed by acid hydrolysis,IR,methylation analysis,GC-MS and NMR analysis.The results demonstrated that CPC was composed of glucose,and the main glycosidic bond type was β-D-Glcp-(1→,→4)-α-Glcp-(1→ and →4,6)-α-Glcp-(1→ in the molar ratio of 1.7:6.4:1.7.(2)Study on the immune activity of polysaccharide CPC in vitro: In this study,the immunomodulatory effect of CPC was explored at cellular and molecular levels in the immune activity evaluation test with mouse macrophage as mode.The results showed that CPC in the concentration range of 250～1000 μg/ml could enhance the Immunological activity by enhancing the proliferation of RAW264.7 cells,enhancing the phagocytic ability of macrophages,promoting the release of NO and ROS,promoting the secretion of iNOS,TNF-α,IL-6,IL-1β,IL-10 and up regulating the gene expression of cytokines.Conclusion:CPC isolated from total polysaccharides of Codonopsis Radix is a kind of homogeneous glucan,which showed good immunoregulatory activity and is expected to become a new immunomodulator.This lays a foundation for further research and application of Codonopsis pilosula polysaccharides.