Preparation And Effect Of Astragalus Polysaccharide Nanoemulsion On Immune Adiuvant

This study was used to reform the dosage formunation of the Chinese traditionalmedicine Astragalus Polysaccharide with nanoemulsion delivery system as the carrier. On thebase of the establishment of blank nanometer emulsion, through the Pseudo-ternary phasediagrams, the Astragalus polysaccharide nanoemulsion (APS-NE) delivery system wasprepared. The study had researched the quality stability, security and pharmacodynamicproperties of the APS-NE, and the effect on the adjuvant and its mechanism.Part One: Nanoemulsion is a multicomponent system, which will be affected by manyfactors, such as surfactant, cosurfactant, oil phase, surfactant and cosurfactant mass ratio(Km) and drug concentration. This study observed the appearance and properties ofjudgment of the formunation of the nanoemulsion, combined with the Pseudo-ternary phasediagrams, screened out the formulation of the W/O astragalus polysaccharide nanoemulsion.The optimum composition was: Tween80/Span80/ethanol/liquid paraffin/water solution,and Tween80: Span80(M: m)=3:1, Km=4:1.Part Two: Based on the APS-NE size, thermodynamic stability, drug release in vitro andso on, the APS-NE prescription was determined Tween80: Span80: ethanol: liquid paraffin:water=31:11:12:22:24(mass ratio). The APS-NE samples prepared with the mass ratiowere pale yellow transparent. The APS-NE was easy to be dyed red by oil soluble dyetonyred, and could maintain clear and transparent state, without flocculation and delamination,by liquid paraffin dilution or by high speed centrifugation. The APS-NE was proved a W/Onanoemulsion. APS-NE droplet was uniform size spherical, particle diameter less than100nm, no adhesion, good dispersibility, average particle diameter of63.7nm measured bylaser particle size instrument, and narrow range of particle size distribution. In addition,APS-NE was pH6.71, point74.1C, viscosity of168.5mPa s, APS content9.6mg/mL. Byillumination test, accelerated test and long term stability test, it showed that APS-NE couldkeep stable light yellow transparent state, no delamination, no turbidity, no flocculation andno drug precipitation phenomenon.Part Three: the muscle average stimulus response levels of blank nanometer emulsionand APS-NE were all lower than1. pyrogen inspection was qualified. LD₅₀was2.41mL and 2.09mL (respectively equivalent to24.1times and20.9times of the normal dosage in mice),and the95%confidence intervals were2.31².54mL and1.94².18mL. long term toxicitytest showed that the blank nanoemulsion and APS-NE in20.27mL/Kg body weight (in ratbody weight74g calculation) range of long-term continuous use on rats were no obvioustoxic injury. A certain concentration range (less than300μg/mL) on L929cells was no toxiceffect. This study shows that APS-NE has very low toxicity, good security.Part Four: This study was conducted to evaluate the effects of Astragalus polysaccharide(APS) nanoemulsion (NE) on the production of the serum Newcastle disease antibody invaccinated chickens. The results showed that APS and APS-NE at high (H) dosage and low(L) dosage all significantly improved the serum ND HI antibody titer of the chickenscompared with the control of medicine-free chickens after d14(P≤0.05). The serum ND HIantibody titers of APSH, APSL-NE and APSH-NE were significantly increased contrast to thatof APSL(P≤0.05). But, it was not significant between APSL-NE and APSH, APSH-NE andAPSH, APSH-NE and APSL-NE. In addition, we found that the curves of APSL-NE andAPSH-NE were higher from d14to d42and from d42to d63, than that of the control, APSL,even APSH. The results indicated that APS and APS-NE, especially APS-NE, could promotespecific antibody production earlier and maintain it longer, and thus improve the immuneeffect of the vaccine. Because of the similar composition of nanoemulsion to vaccine,APS-NE would be a possibility adjuvant of the vaccine. In summary, this research confirmsthat APS-NE displayed better effects on improvement the serum ND HI antibody titer inchickens and would be expected to be effective as component drugs of a novel vaccineadjuvant.Part Five: To study the APS-NE on immune function of mechanism, this chapter usedflow cytometric to detect the population and dynamics of chickens peripheral blood CD4⁺and CD8⁺T lymphocyte. The results showed that APS-NE and APS were able to raise thepopulation of chickens peripheral blood CD4⁺and CD8⁺T lymphocyte, reduce the increasingdynamics of the peripheral blood CD4⁺/CD8⁺ratio caused by the vaccine in chicken.APS-NE and APS both enhanced the body of an immune response to an antigen, throughup-regulation the peripheral blood CD4⁺and CD8⁺T lymphocyte levels in chickens, thusenhanced the body's immunity and resistance. While the effect of APS-NE was more obviousbecause of the characteristics of nanoemulsion.Part Six: The study observed the effects of APS-NE on the culture of chicken spleniclymphocytes, NO class, Th1cell factor (IFN-γ, IL-2) and Th2cell factor (IL-4) by culturedchicken splenic lymphocyte proliferation in vitro. The results showed that APS-NE and APScould significantly stimulate lymphocyte proliferation(P≤0.05), promote chicken splenic lymphocytes to produce NO, NOS, iNOS and IL-2, IL-4, IFN-γ(P≤0.05), and various indexesof APS-NE were more significant role than APS(P≤0.05). In addition, with the increasementof the drug dose, the regulate effect of the drugs became stronger on chicken spleniclymphocytes in vitro, especially two dose groups of APS-NE differences between bothreached a significant level (P≤0.05). The results showed that APS-NE promoted thepopulation of the chicken splenic lymphocytes on the generation of NO, by enhancement ofchicken splenic lymphocytes on NOS and iNOS activity, and stimulated further secretion ofcytokines of the different subsets T cells, such as IL-2, IL-4, IFN-γ, thus played betterimmune regulation effects than APS.In addition, the secrete of NO and cytokines on culturedlymphocytes had certain dose-effect relationship to the APS-NE, and in the study,300μg/mL(high dose group) was the best.