Study On Immunological Enhancement Activity Of Achyranthes Bidentata Polysaccharides Liposome

Aehyranthes bidentata,also named as Niu Ke Xi,which belongs to the Achyranthes L.,Amarantaceae family.Aehyranthes bidentata can make the liver and kidney nutritious,,dissipate thromboxane and broken physical problems.Achyranthes bidentata polysaccharides（ABPS）is a water soluble bioactive polysaccharide with small molecules,which is extraction ofAehyranthes bidentata.ABPS can enhancebody immunity,prohibit the growth of tumors and promote the number of WBC.Besides,the chemical structure of Achyranthes bidentata polysaccharides has been clear:the glycosyl group consists of fructose and glucose,and ratio of glycosyl group is 8.7:1.0,contains 4～21 sugar units,and average molecular weight is 1400.ABPS has smaller molecular weight among the polysaccharide family,better water-solubility,comprehensive herb resource,either oral way or injection is acceptable.So it has broad prospect in the medicine development in the future.Liposome is a superfine spherical carrier,which was made from the entrapment of the drug by the thin films formed by the lipid bilayer.The liposome can reduce the toxicity,enhance the drug stability,alleviate the allergic and immunological reaction,delay the drug release,slow down internal elimination speed,change the internal distribution of the drug and deliver the drug targeted.Encapsulating ABPS into the liposome can increase the production availability and clinical utilization,provide a referred formulation and,and provide the further research more reference.Therefore,the objectives in this study are obtained a well characterized novel Achyranthes bidentata polysaccharides liposome（ABPSL）formulation and to verify its activity of immunological enhancement.Prepare ABPSL by film dispersion method and optimize the preparation condition of ABPSL by Response Surface Methodology（RSM）.In vitro,the effects of ABPSL and ABPS on T lymphocyte and B lymphocyte proliferation and the peritoneal macrophages function were discussed.In vivo.The details are as following:Experiment 1 Preparation and optimization of preparation condition of ABPSLABPSL was prepared by film dispersion method and optimize its preparation condition of ABPSL through Response Surface Methodology（RSM）.Separate the lipsome with the unencapsulated drug through the microcolumn centrifugation method（MCM）,determine the concentration of ABPS through 5%phenol-sulfuric acid colorimetric method and calculate the encapsulation efficiency of the liposome.In order to optimize the preparation conditions of ABPSL,with the encapsulation efficiency and the drug-loading capacity as the indexes,four single-factors were determined including ratio of lipid to drug,ratio of soybean phospholipid to cholesterol,ratio of lipid to tween-80 and ultrasound time.According to the single-factor experiment,ratio of lipid to drug,ratio of lipid to tween-80 and ultrasound time was studied by response surface methodology（RSM）.The data was analyzed with Design-Expert software and the verification of experiment was rechecked on these modified optimal conditions:the ratio of lipid to drug（w/w）is 26:1,ratio of lipid to tween-80 is 6:1,and the ultrasound time is 14min.A mean value of EE is 80.885±1.145%,and the relative error of prediction value 79.90%is 1.23%.Experiment 2 Effect of ABPSL on lymphocyte proliferation of mice in vitro Determine the growth safety concentration of the lymphocytes in peripheral blood of mice of ABPSL and ABPS.From the concentration of 2mg·mL^-1,double-dilute ABPSL and ABPS to 11 concentrations,and add them into the separated cells.The lymphocytes with drugs were respectively incubated in vitro for 48h.Determining the lymphocyte proliferation with MTT method,and select the maximum safe concentrations which are not significantly lower than the control group.Based on the safety concentration,five concentrations of ABPSL,ABPS and blank liposome（BL）were singly or synergistically with PHA and LPS added into mice’s peripheral blood lymphocytes,and were respectively incubated in vitro for 48h.Determine the values through MTT method,and observe effect of ABPSL on the proliferation of splenic lymphocyte,T and B cells.Experiment 3 Effect of ABPSL on T lymphocyte subpopulations of mice This experiment is to study the effect of ABPSL,ABPS,BL and BC on the analysis of CD3⁺,CD4⁺ and CD8⁺ of spleen T lymphocyte subpopulations.Add 0.5mL ABPSL,ABPS and BL(31.25μg·mL^-1)into the spleen lymphocyte suspension,repeat 3 holes and set the Cell Control.After culturing for a while,add CD3⁺,CD4⁺ and CD8⁺ hybrid antibody,and mark the spleen lymphocyte with double-staining method,and detect the changes of the concentration of CD4⁺ and CD8⁺ with flow cytometer.The result shows that the concentration of CD8⁺ of ABPSL group is significantly lower than the ABPS group and BL group;the concentration of CD4⁺ and the ratio of CD4⁺/CD8⁺ of ABPSL group are both significantly higher than the values of other groups,which indicates that ABPSL’s immunological effect is better than ABPS and BL.In sum,the ABPS encapsulated with liposome can effectively enhance the immune functions of organism.Experiment 4 Effect of GPSL on the peritoneal macrophages of mice in vitro This study will further research the influence on the nonspecific immunity of ABPSL.Take the peritoneal macrophages of mice as the objects,we will research the influence of ABPSL on the activities and functions of macrophages.The result shows that the concentration of 1L-1β stimulated by ABPSL high-concentration group is significantly higher than the other groups;LPS group and ABPSL medium-concentration group are secondary.The concentration of IFN-γ stimulated by ABPSL high-concentration group ’is significantly higher than the other groups,and the ABPS medium-concentration group is the second.Concentration of TNF-α stimulated by LPS group is higher than ABPSL high-concentration group,and they both higher than the other groups.