Optimization Of Extraction Process Of Polysaccharides From Metaplexis Japonica Makino Nutshell And Research Of Pharmacological Activity

ObjectiveTo optimize the best extraction process of polysaccharides from Metaplexis Japonica Makino nutshell(MJNP)and discuss its antioxidant in vitro and immune activity carries on the preliminary study.MethodsThe suitable range of the main four factors(the ratio of raw materia to water,temperature,time,number)affecting the polysaccharides extraction yield were selected through the single factor tests preliminary;Based on the results of single-factor tests,the response surface methodology(RSM)was employed to set up the center of the three levels of the four factors combination Design scheme(BBD).Experimental schemes were designed by the Design-Expert8.0.6 statistical analysis software,and the best extraction condition of polysaccharides extraction yield were obtained through the data from regression analysis;Antioxidant activity of polysaccharides was primarily evaluated in vitro by determining the ability of scavenging free radicals(DPPH�,�OH,O2-);The mice were lavaged from low to high concentration of 5kinds of polysaccharides solution,then basal metabolism and biochemical indexes of mice were investigated compared with blank group and model group;By producing low immune model in mice,the following experiments such as chicken red blood cell to gobble up,spleen lymphocyte proliferation by CCK-8method,enzyme standard instrument to detect cytokine IL-2,IL-6 and IL-4,determination of flow cytometry cell subsets CD3~+,CD4~+,CD8~+and CD4~+/CD8~+ratio,laser confocal detection of spleen lymphocytes calcium ion concentration were investigated to evaluate the effects of MJNP on the immune low immunity in mice.ResultsThe optimal extraction conditions of MJNP were as follow: the ratio of water to raw material at 1:18(g/m L),extraction temperature at 86 ?,extraction time at 3 h and number of extraction at 3 times.Validation experiments showed that the average extraction yield and crude content under above these conditions were 11.22% and 26.91% respectively,the total polysaccharide reached99.21% after purification.The results of oxidation experiments showed that MJNP had a strongest super oxide anion scavenging effect,the highest reached80.61%,the DPPH�clearance rate was 73.01%,and the hydroxyl free radical scavenging effect was not significant.There's no non-toxic side effects on mice body in 100,200,400 mg/kg doses of MJNP.MJNP can enhance the ability of phagocytes eating chicken red blood cells in mice(P<0.01),effect on lymphocyte proliferation significantly(P<0.01),and promote the cytokines IL-2and the secretion of IL-6(P<0.01),but there is no change of IL-4;Polysaccharides can increase cell subsets CD3~+,CD4~+ and CD8~+ percentage(P<0.01),and increase the ratio of CD4~+ / CD8~+(P<0.01);Polysaccharides can also increase intracellular Ca2~+content,extremely significant difference compared with model group(P<0.01),no significant difference compared with the blank group.ConclusionsThe result of optimize the extraction process by RSM showed that the polysaccharides of MJNP extraction yield and content were increased.MJNP has antioxidant activity in vitro and the function of the immune enhancement to immunosuppressive mice.In this paper,the study found that MJNP can be used as a immune regulator on the body's cellular immunity(nonspecific immunity and nonspecific immune)and humoral immunity function,the mechanism of itsaction to further explore direction and theoretical basis.