Study On Immunological Enhancement Activity Of Rehmannia Glutinosa And Its Liposome

Rehmannia glutinosa(RG),a widely used traditional Chinese herb,belongs to the family of Scrophulariaceae family.Rehmannia refers to the root of RG,and was recorded in Chinese medical classics "Shennong's Herba".Many clinical and experimental studies show that RG possesses various pharmacological properties.It has effects on the blood system,immune system,endocrine system,cardiovascular system and nervous system,etc.In recent decadesthere have been many more and more reports of the pharmacological functions and activities of Rehmannia glutinosa and its active principles.Rehmannia glutinosapolysaccharide(RGP)is the main active ingredient related to the immune-enhancement of the herb.However,RGP,as one of the big molecule polysaccharide,has many disadvantages,such as a fast-metabolism,short action time,non-focused action scope and large clinical usage,among others.Thus,it is necessary to increase the bioavailability of RGP in clinical applications.Liposome are artificial microscopic vesicles with an aqueous core enclosed in one or more phospholipid bilayers.One or more active pharmaceutical ingredients can be encapsulated in its aqueous core and/or the membrane.Liposomes,as novel drug delivery systems and promising adjuvants,have been the subject of intensive investigations for the past few decades.They can transport drugs for site-specific delivery to enhance efficacy and bioavailability,sustain slow-release,decrease adverse reactions and improve patient compliance.In the research RGP was encapsulated in the liposome using the reverse-phase evaporation method,and the preparation conditions of RGPL were optimized with Response surface methodology(RSM).Experiments in vivo and vitro were done to determine the immunological enhancement activity of RGPL.The effects of RGPL on animal lymphocyte proliferation,antibody titers,cytokines in vivo and that on mouse peritoneal macrophages and bone marrow-derived dendritic cells in mice(BMDCs)in vitro were researched.The details were divided into seven parts as follows:Experiment 1 Immunoenhancement effect of rehmannia glutinosa polysaccharide on lymphocyte proliferation and dendritic cellsThe aim of this study is toinvestigate immunomodulatory effect of rehmannia glutinosa polysaccharide(RGP)on murine splenic lymphocyte and bone marrow derived dendritic cells(DCs).Splenic lymphocytes obtained from mice were co-cultured with RGP for 48 h and then harvested for analyzing with MTT method.The cytokine production of T lymphocytes was measured by ELISA.Effects of RGP treatment on DCs were investigated and assessed by MTT method.The results showed RGP significantly stimulated lymphocyte proliferation and the growth rate of T cell was more significant.The IL-2 and IFN-?production of T lymphocyte were significantly upregulated after being stimulated with RGP.DCs stimulating on proliferation of T cells and the ability of antigen presenting of DCs have been enhanced under the stimulation of RGP.In conclusion,these findings provided valuable information thatRGP possessed strong immunoenhancementactivity,which provided the theoretical basis for the further experiment.Experiment 2 Optimization on the preparation conditions of Rehmannia glutinosa polysaccharideliposome The aim of this study was to investigate the optimizing preparation conditions of Rehmannia glutinosa polysaccharide liposome(RGPL).Reverse-phase evaporation method was chosen to prepare RGPL among four different methods.Based on the single-factor experiments on ratio of soybean phosphatide to drug,soybean phosphatide to cholesterol,soybean phosphatide to tween 80,soybean phosphatide to chloroform and methanol,chloroform to methanol,and evaporating temperature,four factors were chosen from them according to their effects on entrapment rate(EE),which were soybean phosphatide to cholesterol ratio,chloroform to methanol ratio,soybean phosphatide to tween 80,and the temperature.These results showed that the optimum preparation conditions were:a soybean phosphatide to cholesterol ratio of 8:1,a chloroform to methanol ratio of 3:5,a soybean phosphatide to tween 80 ratio of 10:1,a temperature(?)of 66? and an EE of72.753±0.318%.Experiment 3 Quality evaluation on RGPL and its effect on lymphocyte proliferation In this study,synthetical evaluation was made on the RGPL.Physicochemical character such as morphology and size,and the release rule were all investigated.The results showed that RGPLpresented as spherical particles with uniform particle sizewhich was 118nm.From the release experiment in vitro,RGPL exhibited excellent controlled realease properties.Experiment 4Effects of RGPL on immune response of ND vaccine in chickensThe purpose of the study was to investigate the effects of RGPL on immune response to ND vaccine in chickens.7 groups of 14-day-old chickens were vaccinated with ND vaccine,and received repeated vaccination on 28-day-old.At the same time with the first vaccination,chickens of the experimental groups got intramuscular injection withRGPL(8,4 and 2 mg·mL-1),RGP(8 mg·mL-1)and blank liposome(BL),respectively.The immune control group and the blank control group were injected with PBS at the same volume.On the 7th,14th,21th,28th,35th and 42th day after the first vaccination,blood samples were collected from both cardiac and brachial vein.The former was used to determine peripheral T lymphocyte proliferation in vivoby MTT method,and the later was used to test serum HI antibody by ?-micro-method.The results indicated thatRGPL could significantly enhance valence of serum antibody,and promote peripheral T lymphocyte proliferation.Thus,RGPL could improve the adjuvanticity and drug action of RGP.