| Gynostemma pentaphyllum,also known as sweet tea shoots,had a reputation of"Southern Ginseng".Modern researches have shown that the effective components in gynostemma pentaphyllum saponins?Gypenosides,GPS?,have effects of hypolipidemic,hpyerglycemic,antifatigue,immunoregulation,antioxidant,hepatic,anti-ulcer and so on.However,GPS taste bitter and were resulting in hemolysis and less easily absorbed which becomes a limiting factor in its clinical application.The new form of GPS should be promoted.Liposome,typically composed of amphiphilic phospholipids,is a lip id-based vesicle made from biodegradable and nontoxic molecules.As a highly biocompatible and biodegradable drug carrier,there are lots of characteristics of liposome,such as drug-targeting,slow-re lease property,non-immunogenicity,reducing the toxicity of drugs,increasing the stability and prolonging the effect time of drugs.Encapsulating GPS into liposome as oral drugs could avoid the adverse reaction effectively.Therefore,the objectives in this study are obtained a well characterized novel gypenosides liposome?GPSL?formulation and to verify its acticity of immunological enhancement.Prepare GPSL by ammonium sulfate gradient method and optimize the preparation condition of GPSL by Response Surface Methodology?RSM?.In vitro,the effects of GPSL and GPS on T and B lymphocyte proliferation and the peritoneal macrophages function were discussed.In vivo,the immunological adjuvant activity of GPSL against Newcastle disease vaccine was studied.The details were divided into four parts as follows:Experiment1 Preparation and optimization of the preparation condition of gypenosides liposome Gypenosides liposome was prepared by ammonium sulfate gradient method.With the encapsulation efficiency and the drug-loading capacity as the indexes,six single-factors were determined.Based on the single-factor experiment,ratio of lipid to drug,ratio of soybean phospholipid to cholesterol,temperature and time of incubation of drug in water bath on the encapsulation efficiency was studied by response surface methodology?RSM?.The data was analyzed with Design-Expert 8.0.6 software and the verification of experiment was rechecked on these modified optimal conditions:the ratio of lipid to drug?w/w?of 8:1,the ratio of soybean phospholipid to cholesterol?w/w?of 6:1,the temperature of incubation of drug in water bath of 55?,the time of incubation of drug of 11 min.A mean value of EE is 90.17±0.38%?n=3?that demonstrated the validation of the RSM model.The trehalose is chosen as cryoprotectant of GPSL,the trehalose and phospholipids ratio is 4:1.Experiment 2 Effect of GPSL on lymphocyte proliferation on chickens In vitro Based on the safety concentration determination,five concentrations of GPSL,GPS,blank liposome?BL?were singly or synergistically with PHA and LPS added into chicken's peripheral blood lymphocytes and splenic lymphocyte.The lymphocytes with drugs were respectively incubated in vitro for 48 h.Then the lymphocyte proliferation was determined with MTT method.The results showed that the maximal safety concentration of GPSL was higher than that of GPS.In concentration of 62.5 ?g·mL-1,GPSL in single stimulation on lymphocyte proliferation was most obvious.In concentration of 31.25 ?g·mL-1,GPSL in synergistical stimulation with PHA on lymphocyte proliferation was obvious.In concentration of 62.5 ?g·mL-I,GPSL in synergistical stimulation with LPS on lymphocyte proliferation was obvious.At 125-7.813 ?g·mL-1,GPSL could significantly enhance T and B lymphocytes proliferation singly or synergistically with PHA and LPS and the efficacy were superior to those of gypenosides?GPS?and blank liposome?BL?at most of concentrations.Experiment 3 Effect of GPSL on the peritoneal macrophages of mice in vitro In order to determine the effect of GPSL activated macrophages,phagocytosis activity and cytokine secretion were experimented.In the test of the neutral red phagocytosis,five concentrations of GPSL,GPS,blank liposome?BL?were added into the peritoneal macrophages of mice.After incubating for 48h in vitro,three concentrations were chosen for further experiment.The cell culture supernatant were collected and analyzed TNF-?,IL-1?,IL-6,IL-12,IFN-? production by using the ELISA kits.The macrophages with GPSL treated were taken in Fluorescein-labeled E.coli,detected the fluorescence intensity and calculated the efficiency ratio.The efficiency ratio of the positive fluorescence cells with GPSL stimulating was higher than other control groups.At 100?g·mL-1,the macrophage phagocytosis of GPSL and GPS were obvious.At 100 ?g·mL-1,50 ?g·mL-1,the efficiency ratio of macrophage stimulated by GPSL was significantly higher than that of GPS.After GPSL stimulation,macrophages to secrete various cytokines with immune function were significantly increased.As the result,the GPSL had significantly enhanced phagocytosis activity and the secretion of cytokines of the peritoneal macrophages of mice in vitro.Experiment 4 Effect of GPSL on immune response of Newcastle disease?ND?vaccine in chickens In this experiment,GPSL was to assess the effect of humoral immunity and cellular immunity in chicken against ND vaccine.Three hundred and fifty 14-day-old chickens were assigned to 7 groups randomly and vaccinated with ND vaccine.Simultaneously,the chickens in experimental groups were respectively oral administration with the GPSL at three doses,GPS and BL.The results showed that GPSL could significantly enhance lymphocyte proliferation,increase antibody titer,and promote cytokine secretion in vitro and in vivo.Moreover,the adjuvant activity of GPSL was better than those of GPS and BL.In conclusion,GPSL not only significantly enhanced lymphocyte proliferation and a specific antibody against ND vaccine,but also promoted the concentrations of IFN-y,IL-2,and IL-4 in serum.Therefore,GPSL has immunological adjuvant activity by regulating cellular immunity and humoral immunity. |
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