Optimization Of The Process For Purifying Icariin And Its Biological Activity Analysis And Inhibition Role In Pancreatic Cancer Progression

ObjectivesThis article intend to make effort to do something about the purification and biological activity of icariin,a traditional Chinese medicine monomer.And then,we did the study of the regulation of Icariin on tumor microenvironment to observe its inhibition of pancreatic cancer.At last,in this article,taking this study as example,we want to illustrate the potential of integrated of traditional Chinese and Western medicine(ITCWM)treatment of tumors.Methods(1)Icariin purification by macroporous resin:determine the adsorption capacity,adsorption rate and desorption rate of different macroporous resins to choose the highest efficiency one.Complete the dynamic adsorption-desorption test of macroporous resin by screening the dynamic leakage amount,desorption ethanol concentration,and desorption ethanol dosage.The crude extract of icariin was purified according to the optimal purification conditions.(2)Potential biological activity of icariin:evaluate the pharmacokinetics(ADME)of icariin through the TCMSP database,predict the icariin Target by the Swiss Target Prediction tool,construct and analyze the Target protein interaction network by GeneMANIA database,and use WebGestalt analysis tool to do enrichment analysis through GO and KEGG database.(3)Direct killing effect of icariin on pancreatic cancer cells(in vitro):observe the healing of scratch of Panc 02 cells after treatment with icariin for 24h and 48h;determine the apoptosis rate of Panc 02 cells treated with icariin for 48h with Annexin v-fitc/PI apoptosis assay kit.(4)Effect of icariin in mice pancreas tumor microenvironment(in vivo):establish the mice pancreatic cancer model,three days later,do lavage treatment(corn oil or icariin of 120 mg/kg)everyday,11 days later,measure the tumor size and weight,detect the ratio of CD4+T cells,CD8+T cells,PMN-MDSC,M2 macrophages in the spleen and tumor.(5)The mechanism of M2 macrophages polarization:induce mice bone marrow cells to M2 macrophage polarization by IL-4,treatment with icariin for 24 h;detect the M2 macrophages proportion induced by icariin and IL-4 separate;detect the specific molecular markers(ARG1 and MRC1)expression;detect the expression of p-STAT6 in the process of RAW 264.7 cells to M2 macrophages.Results(1)Icariin purification:static screening results showed that HPD300 was the optimal macroporous resin,and HPD300 was selected for dynamic test.EBM extract was purified by HPD300 resin and the purity of icariin was 30.54%.Further crystallization results in a purity of 95.49%.(2)Icariin bioactivity analysis:the results of enrichment analysis showed that icariin is likely to regulate cell migration,cell movement,cell communication,peptide acyl-modified amino acids,protein phosphorylation,intracellular signal transduction and cell proliferation and it also can regulate signaling pathways of many tumors and other diseases,such as the AGE-RAGE signal pathway,Relaxin signal pathway,etc.(3)In vitro experiment:the scratch healing ability of Panc02 treated with 100μM and 150μM icariin was significantly lower than that of the group with 0μM icariin.Compared with the cells treated with 0μM icariin for 48h,the apoptosis rate of cells treated with icariin at 100μM,150μM and 200μM increased significantly.(4)In vivo experiment:the tumor size and mass of icariin treatment group were significantly lower than that of control group;The proportion of M2 macrophages and PMN-MDSC cells in tumor in icariin treatment group decreased significantly.There was no significant difference between CD4+T cells and CD8+T cells in the two groups.(5)Mechanism study:after treatment with 20μM and 80μM icariin,the proportion of IL-4-mediated M2 macrophages and the expressions of ARG1 and MRC1 was significantly reduced.After treatment with 80μM icariin,the expression of p-stat6 in RAW 264.7 cells mediated by IL-4 decreased significantly.ConclusionThe method of extracting and purifying icariin by HPD300 macroporous resin is simple and effective.The ligand-based target prediction approach reveals that icariin is predicted to target multiple proteins and pathways that shape a network which can exert systematic pharmacological effects.In vitro and in vivo experiments have shown that icariin can inhibit the progression of pancreatic cancer by inhibiting the migration of Panc02 cells and inducing apoptosis,and icariin can also regulate the proportion of M2 macrophages and PMN-MDSCs in the immune microenvironment by down-regulating the STAT6 pathway.In summary,icariin can be used as a potential antitumor and immunoregulatory agent for pancreatic cancer.