The Structural Feature And Immune-Enhancing Activity Of Ep5imedium Polysaccharide

Epimedium is the dried leaves of Berberidaceae epimedium and a tonic drug of traditional Chinese medicine,which can tonify the liver and kidney,strong bones and muscles,regulate immunity,anti-oxidation,anti-aging,anti-tumor.Epimedium Polysaccharide is one of epimedium main active ingredient,can immune regulation,antiviral,anti-aging,anti-oxidation,anti radiation.In this study,we extracted the polysaccharides by water decoction and alcohol precipitation method,Purified the polysaccharides by column chromatography method.Infrared spectrum and ultraviolet spectrum was used to analysis its structure,scanning electron microscope was used to observe the morphology,the study focused on the determination of the effect of EPS on the proliferation of chicken peripheral blood lymphocyte,the secretion of cytokine,the function of dendritic cells,the effect of Newcastle disease vaccine.The purpose of this study was to investigate the structural characteristics and immune enhancing activity of theEPS.The experiment included four parts:Experiment 1.Preparation and structural feature observation of EPS Extracted the crude epimedium polysaccharides(EPSct)by the method of water extraction and alcohol precipitation,removed protein by the method of three chloride acetic acid,purified polysaccharide by the method of DEAE-52 column chromatography,finally,get the purified epimedium polysaccharides(EPS).The content of polysaccharide was measured by phenol sulfuric acid method and the structure of polysaccharides was observed by IR and UV method.The results showed that extract rate was 5.12%,the sugar content of EPS was 8.36%;the infrared spectra showed,there was absorption peaks at 1621.68 cm-1 and 1413.48 cm-1 and that may contain uronic acid,there was no absorption peak for at 810 cm-1 and 870 cm-1 and that may not containing mannose;the UV spectra showed that there was no obvious absorption peaks in 100-500 cm-1 range,indicated that may not contain protein and nucleic acid;scanning electron microscopy showed that the surface of EPSct was rough,presented in a patchy state,the binding is more dense,the surface of EPS was smooth,showed the state of granular aggregation,the particles can be seen as a state of a sheet stack and has the holes inside it at high magnification.Experiment 2.Effect of EPS on proliferation of chicken peripheral blood lymphocytes Determined the safe concentrations of EPS on chicken peripheral blood lymphocytes by MTT assay,the five concentrations(1.22-19.53 ?g·mL-1)within the safe concentration were selected to join in the chicken peripheral blood lymphocytes.Determinated the effects of EPS on proliferation of chicken peripheral blood lymphocytes by MTT assay and the effects of EPS on the secretion of IL-2,IL-4,IFN-y,IL-12,TNF-a by ELISA kit.The results showed that:when EPS stimulated lymphocytes alonely,A570 value of five concentration group of EPS were significantly higher than cell control group,concentration of IL-2 and IL-4 at 1.221-19.53 ?g·mL-1 concentration group,concentration of IFN-y and IL-12 at 19.53 ?g·mL-1 concentration group,concentration of TNF-a at 2.441?19.53 ?g·mL-1 concentration group,were significantly higher than the cell control group;when EPS stimulated lymphocytes with PHA,A570 value of five concentration group of EPS were significantly higher than PHA group,concentration of IL-2 and IFN-? at 9.766?19.53 ?g·mL-1 concentration group,concentration of IL-4 at 19.53 jig·mL-1 concentration group,concentration of TNF-? and IL-12 at 2.441-19.53 ?g·mL-1 concentration group were significantly higher than the PHA group.The results showed that EPS could significantly promote the proliferation of lymphocyte and the secretion of cytokine in the appropriate concentration,thus enhancing cellular immunity.Experiment 3.Effect of EPS on function of chicken bone marrow dendritic cells Separated the chBM-DCs from the femur and tibia 3-4 week old chickens;studied the effect of EPS on proliferation and phagocytosis of chBM-DCs by MTT assay and flow cytometry;then add 25 ng·mL-1 rmG-CSF and rmIL-4 to induce culture,observed the dynamic changes of morphology by inverted microscope,when chBM-DCs that cultured for 6 d were stimulated with EPS for 48 h,determined the expression of the surface molecules of CD11c,MHC II,CD40 and CD86 by flow cytometry and confocal microscopy,determined effect of chBM-DCs on the proliferation of allogeneic lymphocytes promoted by MTT assay and changes of secretion ability of cytokines by ELISA kit.The results showed that EPS can promote proliferation and phagocytosis of chBM-DCs;the average expression of CD11c and MHC ? were respectively 78%and 65.3%;EPS can upregulate the expression of CD40 and CD86,enhance the ability of chBM-DCs to stimulate the proliferation of lymphocytes cell,promote secretion of IL-1?,IFN-y,TNF-a,IL-12p70,RANTES by chBM-DCs and inhibiting its secretion of IL-10.These results indicate that EPS can promote maturation of chBM-DCs and activation of its function,thereby enhanced immunity.Experiment 4.Effect of EPS on immune response of chicken Newcastle disease vaccine Effects of EPS on immune effect of newcastle disease vaccine were determined.One hundred and fifty 14-day-old chickens were divided randomly into 5 groups,thirty wings in each group.The chickens except blank control group were vaccinated with ND vaccine,repeated vaccination at 28 days old.At the same time of the vaccination,the chickens in three EPS groups were drinked with high(12 mg-kg-1),middle(6 mg-kg-1),low(3 mg kg-1)dose of EPS,the BC group and VC group were without adding drug and drinking water normally.On days 14,21,28 and 35 after the first vaccination,selected 6 wing randomly in each group to carry out the venous blood sampling,detected the serum ND-HI antibody titer and the content of IFN-?,IL-2 and IL-4;On days 14 and 28 after the first vaccination,selected 6 wing randomly in each group to carry out cardiac blood sampling,detected the proliferation of lymphocytes,separated duodenum,detected the content of IL-17 and sIgA in the small intestine lotion.The results show that high,medium dose group of EPS at 5 time points,the serum antibody titer,IL-2,IL-4 and IFN-y content was significantly higher than VC group;high,medium dose group of EPS at 2 time points,A570 values were significantly higher than VC group;three dose group of EPS at 2 time points,the content of IL-17 and sIgA in the small intestine lotion was significantly higher than VC group.The results show that EPS can significantly increase serum antibody titer and promote lymphocyte proliferation and secretion of IFN-?,IL-2,IL-4,L-17,enhanced immune effect of Newcastle disease vaccine,and effect of high and medium dose is remarkable.