Characterization Of Polysaccharides Extracted From Platycodon Grandiflorus(Jacq.) A.DC.and Their Immunological Activity In Chicken Peritoneal Macrophages

The immunopotentiator can enhance or regulate the immune system.In clinical practice,it is often used in combination with various vaccines to improve immunity,enhance the body's resistance,prevent or improve immune suppression,and have a positive effect on the prevention and control of infectious diseases.Many pharmacological and clinical studies show that the majority of Chinese medicine can enhance the immunity.The polysaccharide as the research and application of immune enhancing agent is one of the active ingredients.It has attracted the great importance attaches attention of livestock and poultry breeding.In this study,Platycodon polysaccharides as the research object were extracted,separated and purificated,and then its structure were analysised.The best immune-enhancing activity of Platycodon grandiflorum Polysaccharides?PGPS?was screened in vitro activity tracking,which provided a theoretical basis for the development of new type of immune enhancing agent.In this research,the crude total PGPS?PGPStc?and crude two fractional PGPS(PGPS₆₀c and PGPS₈₀c)were extracted by one-step or stepwise ethanol precipitation method respectively.The three polysaccharides' structure were analysised after separated and purificated.Their immune-enhancing activities were compared and three polysaccharides with better efficacy were selected.The detection index included macrophage phagocytosis of chicken,cytokine and NO secretion and cell surface expression of costimulatory molecules.The three crud polysaccharides(PGPStc,PGPS₆₀ c and PGPS₈₀c)were extracted by water-decocting and one-step or stepwise ethanol precipitation method.The three polysaccharides were purified by chromatography through DEAE sephadex A-25 to get purified PGPS₈₀,PGPS₆₀ and PGPSt.The carbohydrate and protein contents of all polysaccharides were determinated recpectively by phenol-sulfuric acid and coomassie brilliant blue G-250 methods.Identification of the polysaccharides structural by fourier transforms infrared spectroscopy?FTIR?,gas chromatography-mass spectrometry?GC-MS?,gel permeation chromatography?GPC?and nuclear magnetic resonance spectroscopy?NMR?analysis.The results showed that the extraction rate of PGPS₆₀ was the hightest up to 4.331%.The carbohydrate content of PGPSt was the highest up to 89.96%.The protein contents of all polysaccharides were lower.Polysaccharides had characteristic absorption peak in infrared spectrum;molecular weight distribution showed that three polysaccharides all had two different retention time,indicating the existence of a narrow symmetrical peak and the polysaccharides were homogeneous with higher purity.The average molecular weight ofPGPS₈₀,PGPS₆₀ and PGPSt were 4.14×103¹.01×105 Da,5.69×103¹.12×105 Da and 2.05×103².67×105Da,respectively;monosaccharide composition analysis showed that three polysaccharides were all composed of glucose,mannose,arabinose,galactose,xylose and rhamnose.PGPS₈₀ was mainly composed of glucose?54.322%?,arabia?16.801%?,galactose?15.013%?,PGPS₆₀ was mainly composed of glucose?62.603%?,arabinose?13.925%?,PGPSt was mainly composed of glucose?55.397%?,mannose?22.305%?;1H-NMR and13C-NMR showed that PGPS₈₀ and PGPSt linked mainly by?1?3?-?-D-Glcp-?1?6?-?-D-Glcp residues.Different concentrations of PGPS₈₀,PGPS₆₀ and PGPSt were added into the chicken peritoneal macrophages and the safe concentration were determined by MTT method.The polysaccharides under the safe concentration added to chicken peritoneal macrophages in culture after 4h accession to the LPS,continue culturing for 44 h,proliferation changes of chicken macrophages were determination.The results showed that the proliferation rate of PGPSt was 132% as the highest at 15.625 ?g mL-1,and the proliferation rate of PGPS₈₀ and PGPS₆₀ at the same concentrate was 128% and 117%,respectively.The assay of phagocytic activity of macrophages was examinedusing the method of FITC-labeled E.coli.Different concentrations of PGPS₈₀,PGPS₆₀ and PGPSt were added into the chicken peritoneal macrophages for 4h.LPS was used as mitogens for macrophages of chicken for 24 h,and then incubated with FITC-E.Coli and Triton X-100 for 2 h with black.The cells were scraped by cell scraper and washed with PBS.Finally,flow cytometry determined phagocytic rate.Results showed that PGPS₈₀,PGPS₆₀ and PGPSt all promoted the phagocytic uptake capacity of macrophage towards FITC-labeled E.coli.PGPSt markedly enhanced the phagocytosis ratio up to 23.04% on 15.625 ?g mL-1,at the same concentrate PGPS₈₀ increased the ratio to 19.60%,PGPS₆₀ elevated the ratio to 16.96%?P< 0.05?and followed by 8.24% of LPS while control only 5.14%NO secretion was detected by the method of Griess.Different concentrations of PGPS₈₀,PGPS₆₀ and PGPSt were added into the chicken peritoneal macrophages for 4h.LPS was used as mitogens for macrophages of chicken for 24 h.According to the operating instructions NO kit,OD values at 540 nm were determined by enzyme immunoassay instrument.Compared with LPS group,chicken macrophages groups treated with 7.813-31.25 ?g m L-1of PGPS₈₀,PGPS₆₀ and PGPSt had shown statistical difference in cell viability,especially in the 15.625?g mL-1,the secretion of NO was the most significantly.Different concentrations of PGPS₈₀,PGPS₆₀ and PGPSt were added into the chicken peritoneal macrophages for 4h.LPS was used as mitogens for macrophages of chicken for 24 h.According to the operating instructions ELISA kit,OD values at 450 nm were determined by enzyme immunoassay instrument.The results showed that at the concentration of 31.25 ?g m L-1,PGPS₈₀,PGPS₆₀ and PGPSt could significantly promote chicken macrophages to release TNF-?,IL-1 and IL-6?.Different concentrations of PGPS₈₀,PGPS₆₀ and PGPSt were added into the chicken peritoneal macrophages for 4h.LPS was used as mitogens for macrophages of chicken for 24 h.The cells were scraped by cell scraper and washed with PBS staining buffer containing chicken serum,room temperature closed.After CD80-FITC?or CD86-FITC?added,the cell were washed with PBS.Finally,flow cytometry determined relative fluorescence intensity.The results showed thatThree polysaccharides groups and LPS group all enhanced CD80 and CD86 expression levels compared with unstimulated control cells.When stimulated by PGPSt at 15.625 ?g m L-1 CD80 and CD86 were the most significance,increased 3.89 and 3.31 folds.In summary,PGPSt can effectively activate chicken peritoneal macrophages at a concentration of 15.625?g mL-1,while the polysaccharide characterization has a closely special relationship with its immune activity.