The Primary Structure And Biological Activities Comparisons Of Polysaccharide From Panax Notoginseng Residues

Panax notoginseng((Burk.)F.H.Chen)is a traditional Chinese herb whose main active ingredient is saponins.In most cases,The residue was discarded after saponins of Panax notoginseng was introduced into the medicine.But some study estimated that 3 to 5 percent of the bioactive ingredients in every ton of residue were still unutilized.At present,there are few researches on the active ingredients contained in traditional Chinese medicine residues.From the perspective of secondary development and utilization of resources,we carried out researches on the main polysaccharide components in Panax notoginseng residues.Panax notoginseng polysaccharide,as a kind of natural active polymer compounds,has many pharmacological activities.At present,most of the researches on Panax notoginseng polysaccharide are directly extracted and separated from the medicinal materials of Panax notoginseng,and the composition structure and pharmacological activity of Panax notoginseng polysaccharide in the pharmaceutical residue after alcohol extraction of saponins are rarely studied.This topic is to conduct a comparative study on the primary structure and biological activity of the main polysaccharide components in the residue of Panax notoginseng,and try to find the correlation between structure and activity.The results provided some reference for the development of polysaccharide from Panax notoginseng residues and further study on their pharmacological activities.The main research contents and results of this paper include:1.The crude polysaccharide(PNPS)was extracted by hot water and was precipitated out by alcohol,and purified by DEAE Fast Flow and Sephadex G-50glucan gel column.The purified polysaccharide components were detected by ultraviolet spectrophotometry without macromolecular impurities.According to the elution mobile phase,they were named as PNPS-0,PNPS-0.2 and PNPS-0.3,with the yield of 10.26%,32.15%and 23.27%,respectively.2.The molecular weight and purity of polysaccharides were determined by HPGPC after column chromatography.SEM,FT-IR,HPLC-derivatization,GC-MS and other methods were used to characterize the primary structure of single polysaccharide fragments separated and purified.The results showed that PNPS-0,PNPS-0.2 and PNPS-0.3 were homogeneous polysaccharides with high purity,Molecular weights(Mw)were 29152,173876 and 104201,respectively.The surface of each polysaccharide component was similar,and there was no obvious morphological change.FT-IR analysis showed that PNPS-0 was neutral sugar and the PNPS-0.2 and PNPS-0.3 spectra revealed that they were acidic sugars.The monosaccharide composition and proportion of each polysaccharide component were different.The main component of PNPS-0 was D-glucose(52.28%)and D-galactose(34.14%).PNPS-0.2 was mainly composed of D-galactose(40.89%),D-arabinose(19.38%)and D-glucose(12.25%).PNPS-0.3 was mainly composed of D-glucuronic acid(40.43%),D-galactose(22.61%)and D-glucose(18.32%).The difference of glycosidic bond composition and proportion was similar to that of monosaccharides.Comprehensive analysis of the primary structure data showed that although the three were from the same Panax notoginseng residue,there were obvious differences in the primary structure.3.The total reducing capacity and in vitro antioxidant capacity of polysaccharides were determined.The oxidative damage model of RAW264.7macrophages was established.SOD,CAT and GSH-Px kits were used to determine the effect of polysaccharide on the antioxidant enzyme index of cell fluid.The protective effect of polysaccharide on the oxidative damage of RAW264.7 cells was qualitatively and quantitatively analyzed by laser confocal microscope and multifunctional enzyme spectrometer.By comparing the antioxidant activities of polysaccharide components,the results showed that the antioxidant capacity of PNPS-0,PNPS-0.2 and PNPS-0.3 was concentration dependent.Among them,PNPS-0.3 reveled the best antioxidant,the highest total reducing power,the highest scavenging activity against DPPH free radicals,OH~-free radicals,ABTS free radicals and superoxide anion free radicals,and the best protective effect against oxidative damage of RAW 264.7 cells.4.The target pathway of macrophages affected by polysaccharide components was predicted based on network pharmacology,and it was concluded that each polysaccharide component may polarize macrophages towards M1 type,and induce macrophages to release TNF-α,i NOS and other inflammatory factors by activating TLRs of cell membrane and nuclear transcription factor NF-kappa B signaling pathway,etc.,to play an immune effect.5.Fluorescent probe labeling,q RT-PCR,Immunofluorescence and Western blot were used to study the immune activity of polysaccharide components in vitro.The M1/M2 model of RAW264.7 macrophages was established to investigate the polarization trend of polysaccharide components on RAW264.7 macrophages.The activity was verified by fluorescence probe experiment in zebrafish.The results showed that polysaccharide could increase the production of NO and ROS in RAW264.7 macrophage culture.q RT-PCR and Western blot analysis showed that polysaccharide components significantly increased the expression levels of TNF-αand i NOS genes and proteins,and the results were as follows:PNPS-0.3>PNPS-0.2>PNPS-0;Polysaccharide components can change the polarization trend of macrophages,significantly increase the gene expression of macrophage marker CCR7of M2 phenotype,and effectively polarize macrophages into M1 phenotype with immunoactivation properties.Immunofluorescence and Western blot experiments verified the above experimental results.Further experiments showed that polysaccharide components could increase the expression level of macrophage membrane protein gene TLR2,TLR4,TLR7 pathway and downstream nuclear transcription factor NF-kβgene.Zebrafish in vivo activity experiment verified that the levels of NO and ROS in zebrafish embryos treated with polysaccharide components were significantly increased,indicating that polysaccharide components could activate the immune system of zebrafish embryos by stimulating the production of ROS and NO.In summary,in this study,three natural homogeneous polysaccharides were isolated from the residues of Panax notoginseng,and their primary structures were identified.The results showed that these three polysaccharide components showed good antioxidant capacity in vitro,and could significantly increase the exxpression of cellular antioxidant enzymes.Cell and zebrafish animal experiments showed that the three polysaccharides could polarize macrophages towards M1 phenotype,activate the immune system and have good immunomodulatory activities.Although the three polysaccharides come from the same raw material,there are great differences in structure and activity.We analyzed this difference in structure and activity,which has a far-reaching influence on the establishment of the structure-activity relationship of polysaccharide.The results provided some reference for the development of polysaccharide and further study on their pharmacological activities.