Study On The Preparation Of W/O/W Type Ginseng Stem And Leaf Saponin Multiple Emulsion And Its Adjuvant Effect

Ginseng,a valuable traditional Chinese medicine herb,has the characteristic of warm,flat,sweet and bitter.It has beneficial effects on spleen,stomach,lung,spirit and intelligence.The primary active ingredients of ginseng are generally attributed to ginsenosides.Ginsenosides exert varying effiects on animal health including anti-inflammatory,antimicrobial,antimicrobial,anti-parasitic,antineoplastic and antiviral.Modern pharmacology studies showed that Ginseng stem-leaf saponin(GSLS)has the pharmacological activity similar to ginsenosides,and less expensive.The synergistic effect between the Ginsenosides and the water-in-oil vaccine,which can improve the body’s immune response,was found by Hu Songhua et al.W/O/W type multiple emulsion is a kind of multiple emulsion which can package water-soluble active substances.The water-soluble active substances in the multiple emulsion could be isolated,protected,controlled release,etc.The aim of the present study was to prepare a W/O/W multiple emulsion adjuvant of GSLS.The antigen and GSLS could be controlled releasefrom water phase which is the mix liquor of antigen and GSLS in it to reduce the amount of vaccine,the number of immunization and improve the immune effect.For developing a new vaccine adjuvant with quality control and good effect of immune,the main contents of this study included optimization of GSLS extraction process,preparation of W/O/W type GSLS multiple emulsion,the research on improving the encapsulation efficiency of GSLS and adjuvant effect of W/O/W type GSLS multiple emulsion.Experiment I Extraction and Purification of GSLS.GSLS has the pharmacological activity similar to ginsenosides,and less expensive.Boiling method is a traditional extraction method of Chinese medicine.This experiment disposed the GSLS extracts which obtained by boiling method with four methods:directly drying,purified with 30%ethanol,purified with 60%ethanol and the extract with macroporous adsorption resin column purification method.The yield of each method is 26.2%,24.3%,17.7%and 5%respectively.The purity of GSLS obtained by High Performance Liquid Chromatography(HPLC)is 18.37%,25.86%,38.54%and 49.99%respectively.Considered the cause of the cost,the yield and etc,the GSLS obtained by purified with 60%ethanol in the subsequent study.Experiment II Preparation and optimization of W/O/W Type GSLS Multiple Emulsion.The W/O/W type GSLS multiple emulsion was prepared as the optimized condition.Firstly,the particle size of the W/O/W type GSLS multiple emulsion was measured by particle size analyzer,while the viscosity and the stability of GSLS multiple emulsion was measured.Secondly,the encapsulation efficiencies of GSLS in the multiple emulsion were determined by HPLC.Finally,with orthogonal experimental test,the optimal process conditions was selected using the encapsulation efficiency as evaluation index,while the mass ratio between oil phase and water phase,the emulsification time and the emulsion strength as major factors.The results displayed that the grain size of GSLS multiple emulsion is epigranular in the range from 0.086 μm to 0.115 μm and the average is 110 nm the viscosity and the stability are all normal.The content rang of ginsenoside Rgl in GSLS multiple emulsion is 17.82～23.68 μg/mL,that of ginsenoside Re in it is 27.82～33.68μg/mL and that of ginsenoside Rd in it is 16.82～23.08 μg/mL.The result of orthogonal experimental showed that the conditions of the highest encapsulation efficiency:the mass ratio between oil phase and water phase is 46:54,the emulsification time is 30 min and the emulsion strength is 300 rpm.Experiment III Effect of W/O/W type GSLS multiple emulsion on the immune response in mice immunized with FMDV.For exploring the effect of W/O/W type GSLS multiple emulsion on the immune response in mice immunized with FMDV,96 6-weeks-old ICR male mice were randomly divided into six groups and immunized respectively with saline,ISA206,blank W/O/W type multiple emulsion,extract of GSLS,blank W/O/W type multiple emulsion added extract of GSLS,W/O/W type GSLS multiple emulsion by subcutaneous injection in this experiment.Blood was respectively gained from the eyeballs of mice at the 14,21,28,42,49,56 d after immunization to determine the change of concentration in FMDV-specific IgG antibodies and cytokines IL-4,IFN-γ.The levels of mouse spleen lymphocyte proliferation were measured by MTT assay.Results indicated that the concentration of IgG,IL-4 and IFN-y in serum was increased observably while the mouse’s spleen lymphocyte proliferated significantly by W/O/W type GSLS multiple emulsion.It indicates that the adjuvant activity of W/O/W type GSLS multiple emulsion is very good and the immune response can be enhanced effectively in mice.