Study On The Preparation And The Immunomodulatory Effect Of Pseudostellaria Polysaccharide Injection

ObjectivePseudostellaria Polysaccharide（PP）is the main active ingredient of Pseudostellaria heterophylla,a traditional Chinese medicine which has long been used to tonify the qi and spleen.The aim of this paper is to prepare the injection and evaluate its immunomodulatory effects on different animal models.Firstly,the extraction process of PP was optimized.Then,the molecular weight and monosaccharide components of PP were determined.After that,PP injection was prepared and its quality standard was defined.Finally,the immunomodulatory effects of PP injection were evaluated against cyclophosphamide induced immunodepressed mince and chickens immunized with infectious bursal disease inactivated vaccine.Methods（1）Extraction of PP.The extraction process of PP optimized by single factor experiment and orthogonal test.The alcohol precipitation process was also investigated.（2）Analysis and detection of PP.The monosaccharide composition and molecular weight of PP were determined by high performance liquid chromatography（HPLC）and gel permeation chromatography（GPC）.（3）Prepare PP Injection and study on its quality and control.The PP Injection was prepared.The content of the polysaccharide was determined according to the2015 edition of Chinese Veterinary Pharmacopoeia.（4）Immunomodulatory effect study of PP injection in immunosuppression mice.KM mice were used as experimental animals and astragalus polysaccharides as positive drugs.Immunosuppressive model was established by intraperitoneal injection of CTX at 80mg/kg·bw.The PP injection was intraperitoneally given to mice at 50mg/kg·bw、100mg/kg·bw、200mg/kg·bw for 10 days.The general performances of animals were recorded very day.The immune organ index and serum levels of IgA,IgG,IgM,IL-2,IL-4,IL-6 and IFN-γwere measured at the end of the experiment.（5）Effect of PP Injection on chickens immunized with infectious bursal disease inactivated vaccine.Chinkens were used as experimental animals and astragalus polysaccharides as positive drugs.All chickens at age of 15 days were immunized with infectious bursal disease inactivated vaccine.The general performance,immune organ index and infectious bursal disease（IBD）antibody titer were measured after intramuscular injection of different doses of PP injection.Results（1）The optimum conditions to extract PP were at 90℃,add 9 times of water and extract3 times,each time for 3h.The concentration of extract solution was to 2.0g·mL^-1 and add 95%ethanol to the final concentration of ethanol to 80%.The precipitate was harvested by filtration.Then the precipitate was dissolved using 10 times of water and filted again.The polyssachride in the filtrate were precipitated again by adding 95%ethanol to the final concentration of ethanol to 80%and harvested by filtration.Repeat this process two times.（2）The PP is water soluble.Polysaccharide content is 56.83%.Reducing sugar content is3.17%.Protein content is 0.33%.The results of HPLC showed that the PP contained 41.37%galactose,14.25%glucose,1.06%lactose,0.32%rhamnose,0.18%glucuronic acid,0.15%D-xylose.The molecular weight distribution of PP measured was from 1000 to 5000 kDa.（3）The PP Injection is a light yellow clear liquid.The pH value is 5.92.Polysaccharide content is 11.19 mg/ml.The results of protein,resin,tannin,oxalate,potassium ion,heat source,biological activity,abnormal toxicity and hemolysis test all conform to the provisions of Chinese Veterinary Pharmacopoeia.The results of TLC showed that the hydrolysate of polysaccharid showed the same color spots with glucose and galactose at the same position.The results of HPLC showed that per ml injection contains 9.20 mg galactose,2.95mg glucose,0.20mg lactose,0.07mg rhamnose,0.03mg glucuronic acid and 0.03mg D-xylose.According to the methodological study,the quality standard of PP Injection was established.（4）Compared with the model group,the weight and average weight gain of mice in the low dose group significantly increased（P?0.05）,and the feed weight gain ratio decreased.The immune organ index of mice in each dosage group of PP was higher than model group.The spleen index and thymus index of mice in model group were 2.58±0.62mg/g,1.17±0.48 mg/g.The spleen index of mice in the high dose group of PP was 3.04±0.32mg/g.It was significantly higher than the model group（P?0.05）.Compared with the negative control group,the contents of immunoglobulin and cytokines in the model group decreased.The levels of IgG,IgM,IL-2,IL-6 and IFN-γsignificantly decreased（P?0.05）.Compared with the model group,the contents of immunoglobulin and cytokines in each dose group of PP increased.The levels of IgM,IL-2 and IL-6 in serum of mice in low dose group increased significantly（P?0.05）,and the levels of IgG,IFN-γvery significantly increased（P?0.01）.The levels of IgM,IL-2,IL-4,IL-6 and IFN-γin serum of mice in middle dose group increased significantly（P?0.05）,and the levels of IgG very significantly increased（P?0.01）.The levels of IgG、IgM、IL-2 and IFN-γin serum of mice in high dose group increased significantly（P?0.05）,and the levels of IL-6 very significantly increased（P?0.01）.The results showed that intraperitoneal injection of different doses of PP improved the growth performance of mice,increase the index of immune organs and the content of immunoglobulin and cytokines in serum of mice,but there was no good dose dependence.（5）Compared with the normal group,there was no significant difference in IBD antibody titer among the groups,but the spleen index and bursa index of all dosage groups of PP were increased.The spleen index and bursa index of normal chickens were 1.85±0.17mg/g,4.60±1.18mg/g.The spleen index and bursa index of chickens in low dose group were2.20±0.40mg/g,6.04±1.13mg/g which was significantly higher than the normal group（P?0.05）.The bursa index of chickens in middle dose group was 5.80±1.43 mg/g which was significantly higher than the normal group（P?0.05）.The results showed that PP Injection had certain effects on the immune function of chickens.ConclusionOptimized water extraction and ethanol precipitation described in the current paper is an effective method to extract and purify polysaccharide from Pseudostellaria heterophylla.The extraction rate was 11.09%.The content of polysaccharide was 54.94%the extract.The molecular weight distribution of PP measured was from 1000 to 5000 kDa.The main components of PP are galactose and glucose.The quality of PP injection prepared in current study was in accordance with the provisions of Chinese Veterinary Pharmacopoeia 2015edition.Injection of PP can restore the clinical performance and immune organ index of immunosuppressed mice,promote the secretion of immunoglobulin and cytokines in serum of immunosuppressed mice,and increase the immune organ index of chickens immunized with infectious bursal disease inactivated vaccine.These results indicated that the PP injection enhanced the immune function.