Study On Preparation Of Glycyrrhiza Polysaccharide Liposome And Its Immunological Adjuvanticity

Radix Glycyrrhizae is one of the common herbs used in the prescriptions of traditional Chinese medicine.The effective components of Glycyrrhiza include flavonoids,polysaccharides,triterpenoid sapogenins and triterpenoid saponins.The polysaccharide has numerous pharmacological effects such as immune modulation,antivius,anti-inflammatory,antioxidant and antitumor.Recent researches revealed that the liposones of polysaccharides can significantly increase their biological activities,especially their immune-enhancement ability.In this study,Glycyrrhiza polysaccharide(GP)as the target was extracted by water and precipitated by alcohol with gradient concentrations,then deproteinized by trichloroacetic acid.The deproteinized GP with the best immune-enhancement effect detected by the experiment of peripheral lymphocyte proliferation in vitro was chosen to prepare the glycyrrhiza polysaccharide liposome(GPL).Then experiments in vivo and vitro were performed to determine the immunological enhancement activity of GP and GPL.The purpose of this study is to investigate the enhancing activity of glycyrrhiza polysaccharide liposome for chicken immune system to provide theoretical support for the liposome applications in Chinese medicine polysaccharides and vaccine adjuvants application and provide a theoretical basis.The study was divided into six parts as follows:Test 1.Extraction of GPs The total glycyrrhiza polydsaccharides(GPt)and three fractional GPs,including GP30?GP50 and GP80,were extracted by water and precipitated by gradient alcohol.The totalsugar contents of these polysaccharides were determined by phenol-sulfuric acid method and the protein contents were determined by Bradford method.The results show that the extraction rate of GPt in the four polysaccharides was the highest of 4.13%.The highest extraction yield of GP50 was 2.16%,with sugar content of 54.9%,which indicated that GP50 was the main part of polysaccharide content.Test 2.The immune-enhancing activity comparison of GPs Firstly,the safe concentration of four polysaccharides(GPt,GP30,GP50 and GP80)were determined for the peripheral blood lymphocytes,then GPs at five concentrations within safe concentration were added into the peripheral blood lymphocytes,respectively,singly or synergistically with PHA.The values of peripheral blood proliferation were tested by MTT assay.The results showed that the four polysaccharides in the appropriate concentrations could stimulate lymphocytes proliferation singly and synergistically with PHA,and GP50 with the besteffectwas considered as the optional immune-enhancing activity site of GP.Test 3.Preparation of GPL Firstly,the purified GP50 was encapsulated to be GPL by reverse evaporation method,ethanol injection method,thin-film dispersion method and ammonium sulfate gradient method,respectively.A modified mini-column centrifugation was applied to determine the encapsulation efficiency(EE)of GPL with Sephadex G-50.Reverse-phase evaporation method was chosen to prepare GPL among four different methods.Based on the single-factor experiments on ratio of soybean phosphatide to drug,soybean phosphatide to cholesterol,evaporating temperature,ultrasonic time and soybean phosphatide to tween 80,three factors were selected according to their effects on EE and drug-loading rate,which were soybean phosphatide to drug ratio,evaporating temperature and ultrasonic time.The optimum preparation conditions were optimized by response surface method,and the optimal preparation conditions were verified.The data was analyzed with Design-Expert software to give the optimum preparation conditions as follows:Soybean phosphatide to GP ratio of 24:1,the temperature of incubation of drug in water bath of 46?,the ultrasound time of 16 min and an EE of 78.33±0.25%with spherical shape and uniform sizes.Test 4.The comparison of GP and GPL in promoting chicken peripheral lymphocytes proliferation GPL,GP and blank liposome(BL)were diluted to thirteen concentrations with RPMI-1640 then were added into the peripheral lymphocytes to give the maximal safety concentrations.Then,GPL,GP and BL at five concentrations within safe concentration were added into the peripheral lymphocytes,respectively,singly or synergistically with PHA and LPS.The data of peripheral blood proliferation were determined by MTT assay.These results showed that GPL and GP in certain concentrations can significantly promoted the proliferation of lymphocytes singly,or significantly promote the proliferation of T lymphocytes and B lymphocytes synergistically with PHA and LPS respectively.Furthermore,the effects of GPL were significantly better than those of GP,which indicated that GP encapsulated liposome could significantly improve GP in the immune-enhancing activity.As to dendritic cells,cells were isolated from the bone marrow of legs of chickens,then GP and GPL with series of concentrations were added to the DCs,then incubated.The proliferations of cells and the effect of DCs on the stimulation of lymphocytes were tested by MTT assay,while the IL-2 and IFN-y secretions were analized by ELISA kits.The results showed that GP and GPL can significantly promote the proliferation of dendritic cells,increase the effects on proliferations of T cellsand enhance the secretions of IL-2 and IFN-y.Moreover,the GPL had better effects than GP.Test 5.The comparison of GP and GPL in promoting proliferation and function of chicken dendritic cell This experiment was conducted to study the effects of GPL and GP on proliferation of dendritic cells,mature and function.GPL and GP were respectively added in chicken bone marrow derived dendritic cells,the MTT assay was used to detect the GPL and GP of chicken bone marrow derived dendritic cell proliferation effect,dendritic cells promote T cell proliferation and the effect on IL-2 and IFN-y factor secretion of dendritic cells.These results showed that GP can significantly promote the proliferation of dendritic cell precursors;the GP role of dendritic cells,promoting the proliferation of T cells and antigen presenting function were improved significantly;after dendritic cells were stimulated by GP,the secretion of IL-2 and IFN-y were significantly up-regulated,and GP encapsulated liposomes,the function improved significantly.Test 6.The comparison of GP and GPL on immune response of ND vaccine in vivo The purpose of this section was to investigate the activities of GP and GPL in vivo.Seven groups of 12-day-old chickens were vaccinated with ND vaccine,and re-vaccinated on the 28thday of birth.At the same time with the first vaccination,chickens of the experimental groups got intramuscular injection with GPL(4,2 and 1 mg·mL-1),GP(4 mg·mL-1)and blank liposome(BL),respectively.The immune control group and the blank control group were injected with physiological saline of the same volume.One time each day,lasted for 3 days.On the 7th,14th,21th,28th,35th,42th day after the first vaccination,blood samples were collected from brachial vein;On the 14th,28th,42th day after the first vaccination,blood samples were collected from cardiac for determination of peripheral T lymphocyte proliferation in vivo by MTT method,at the same time,weights of chickens were weighed.On 42th day after the first vaccination,organic index of thymus,spleen and bursa were analized.These results showed that the GPL and GP can significantly improve the immune effect of Newcastle disease vaccine,and GP encapsulated by liposomes,can significantly improve immune-enhancing activity than GP.