Preparation Of Salidroside Liposome And Its Application Of Immune Adjuvant Activity

Rhodiola is a kind of perennial herbs or sub shrubs plants,belonging to the Crassulaceae rhodiola.Both root and stem can be used as medicine.Actually,rhodiola is a kind of Chinese traditional medicine.In recent years,rhodiola has received broad attention of domestic scholars and experts,and much research was done on it.There have been more than 40 kinds of chemical constituents and pharmacological active ingredient isolated from Rhodiola.According to pharmacological studies,it was confirmed that salidroside,casein Savi,flavonoids and Rhodosin are pharmacologically active ingredients.As the main active ingredient,salidroside's main pharmacological effects are anti-aging,anti-fatigue,anti-anoxia,anti-tumor and anti-radiation.In addition,it has a moderating effect on the immune system,and it has a protective effect on vessel.However,the shortcomings of low bioavailability resulted in salidroside clinical dosage is too large.Liposome is vesicle of bilayer membrane,composed of phospholipids and other synthetic materials,having biodegradability,biocompatibility,low toxicity and other characteristics.Lipophilic drug and hydrophic drugs can be encapsulated in liposome.Since liposome may enhance the pharmacological effects of drugs,and has a good affinity with the cell membrane,liposome has become a very valuable drug delivery system.As an immune adjuvant,liposome adjuvant has its own characteristics and advantages,which include changing the type of immune response,safety,non-toxic side effects,targeting and increase drug stability.This study was conducted on the basis of salidroside could enhance the immune function of animals.Firstly,porcine circovirus type 2 was encapsulated in liposome using repeated freezing and thawing method.As a consequence,liposome was used as the drug delivery system.Secondly,porcine circovirus type 2 salidroside liposome was prepared.Salidroside was encapsulated in liposome by ammonium sulfate gradient method.Thirdly,immune effect of liposome adjuvant was studied by animal experiments of mouse model.It could provide new research ideas and methods for developing a new generation porcine circovirus vaccine.The study is divided into the following sections:Experiment 1 Study on preparation of liposome salidroside The main purpose of this chapter is to test the preparation of high encapsulation efficiency of salidroside liposome.Phospholipids,cholesterol and salidroside,as the main material,was used to prepared salidroside liposome by ammonium sulfate gradient method.Encapsulation efficiency and drug loading as test indicators,Main factors affecting encapsulation efficiency were filtered out from salidroside concentration,temperature,ethanol injection rate,ethanol volume,membrane ratio and ammonium sulfate concentration by single factor test method.The main factors were the concentration of ammonium sulfate,ethanol injection volume,temperature and ethanol injection rate.With the entrapment efficiency selected as the response value,preparation process of salidroside liposome is further optimized by response surface method.Experimental data obtained was analyzed by Design-Expert 7.0 software.The optimum combination of test conditions,i.e.,ammonium sulfate concentration of 0.26 mol/mL,ethanol for 6.5 mL,drug loading temperature of 43 ?,ethanol injection speed 3 mL/min,and the predicted encapsulation efficiency was 95.422%.Verification test results showed that the obtained encapsulation efficiency of salidroside liposome was 94.527±0.682%,consistent with the predicted value.Experiment 2 Study on preparation of inactivated vaccine of porcine circovirus type 2 salidroside liposome The main purpose of this chapter is to prepare porcine circovirus type 2 salidroside liposome with high encapsulation efficiency.Both repeated freezing and thawing method and freeze drying method were selected for screening the optimal conditions preparing the porcine circovirus type 2 salidroside liposome.The morphology,size distribution,encapsulation efficiency and other characteristics were detected.The test results showed that when the liposome and antigen volume ratio was 6:1,porcine circovirus type 2 salidroside liposome could be obtained with a high encapsulation efficiency of 85%using repeated freeze-thaw method.Liposome vesicles were observed by TEM,and the morphological was nearly circular.The particle size was about 171 nm,which was analyzed by Malvern particle size analyzer.Experiment 3 Study on immunological activity of inactivated vaccine of porcine circovirus type 2 salidroside liposome The purpose of this chapter is to prepare three different concentrations of porcine circovirus type 2 salidroside liposome,and to study on how the three different liposome affecting the immune response.According to the method mentioned in Chapter ?,ree different concentrations of porcine circovirus type 2 salidroside liposomes were prepared.The morphological,size,encapsulation efficiency and other characteristics were detected.The drug release test was conducted,since it was considered as stability of the liposome.After the animal experiments,the antibody level in mice was measured and analyzed.The results showed that salidroside liposome as adjuvant could enhance the immune response in mice significantly,and the lowest concentration of salidroside liposome had the most obvious effect.