Purification,Structual Characterization Of Polysaccharides From Platycondon Grandiflorum And Their Immunological Activity On Chicken Peripheral Blood Lymphocytes

With the development of animal production, animal husbandry has become more and more intensive. It is unable to establish a good immune environment for livestock and poultry breeding, thus the immune prevention was affected. Polysaccharide from plants, animals and microorganisms is common. It is a category of natural biological macromolecule, performing a variety of functions. Pharmacological studies found that polysaccharide from Chinese medicine had extensive immunomodulatory and promoting effects, and it is a good biological regulator with efficacy of security, stability, and little side effects.In our study, the total Platycodon grandiflorum polysaccharides?PGPStc? and two fractional PGPSs were extracted by one-step and stepwise ethanol precipitation method,respectively. The immune-enhancing activities of three PGPSs were compared. We compared the effects of lymphocyte cell cycle distribution and the percentages of CD⁴⁺ and CD⁸⁺ T cells in vitro.The crude total PGPS?PGPStc? and two crude fractional PGPSs?PGPS60c, PGPS80c?were extracted by water-decocting and one-step or stepwise ethanol precipitation method,respectively. They were purified by chromatography through Sephadex G-75 gel-filtration chromatography to get purified PGPStp. The carbohydrate and protein contents of all polysaccharides were determined respectively by phenol-sulfuric acid and Coomassie brilliant blue methods. Identification of the polysaccharides structure was determined by fourier transforms infrared spectroscopy?FTIR?.The results showed that extraction rate of PGPS80 c was the highest. The carbohydrate content of PGPS60 c, PGPS80 c, PGPStc and PGPStp was64% ? 53.7%? 63.5% and 73%, respectively. The protein contents of all polysaccharides were lower. The results indicated that the carbohydrate content of polysaccharides was increased after purified and the polysaccharides had characteristic absorption peak in infrared spectrum. PGPS60 c, PGPS80 c, PGPStc and PGPStp were added into the cultivating system of chicken peripheral blood lymphocyte. MTT method was used to test the safety concentration.The results indicated that the A570 values of PGPS60 c and PGPS80 c in 500?g mL-1, PGPStc in250 ?g mL-1 and PGPStp in 62.5?g mL-1 were just not significantly less than control group, sothe concentration can be as their biggest security levels. To facilitate comparison, we set the unified biggest safe concentration as 62.5?g mL-1.In order to select the immune-enhancing active site of PGPS, four PGPSs at five concentrations in safe concentration were added into chicken peripheral blood lymphocyte singly or synergistically with PHA. The peripheral blood lymphocyte proliferation was determined by MTT method. The results exhibited that four PGPSs could significantly stimulate lymphocyte proliferation singly, and the lymphocyte proliferation rate of PGPStc at31.25?g mL-1 was the highest. When affected with PHA, PGPS60 c at 15.625³1.25 ?g mL-1,PGPStc at 3.907¹5.625 ?g mL-1could significantly stimulate lymphocyte proliferation. The lymphocyte proliferation rate of PGPS60 c at 31.25 ?g mL-1 was the highest. In general evaluation, PGPStc possessed best action and maybe the immune-enhancing active site of PGPSs, followed by PGPS60 c.The cell cycle of T lymphocyte was detected by flow cytometry?FCM?. Within 72 h the control group cells were most in G0/G1-phase, the change was not obvious. Compared with the PHA group, PGPS60 c and PGPStc groups reduced the percentage of cells in G0/G1-phase and increased the percentage of cells in S-phase in 24 h, 48 h.The percentages of CD⁴⁺ and CD⁸⁺ T lymphocytes cells were determined by flow cytometry.The results testified that after treated 48 h,the percentages of CD⁴⁺ and CD⁸⁺ T lymphocytes in PGPStc group were significantly higher than PHA group.