The Extraction And Separation Of Iridiods In Scrophularia Ningpoensis Hemsl. And Prediction In The Action Targets For Anti-cerebral Ischemia Based On Network Pharmacology

ObjectiveThe Iridiods in Scrophularia ningpoensis Hemsl.have the activity of anti-cerebral ischemia,but the chemical constitution and mechanism of action are not very clear.In order to solve these problems,I designed three parts of experiments:（1）To explore the Optimization of the hot reflux extraction of iridoid glycoside in Radix Scrophulariae for as many structural types as possible;（2）to investigate the chemical constituents in Radix Scrophulariae,and in order to get some new iridoid glycoside;（3）to predict the main targets of iridiod in Scrophulariae against cerebral ischemia and explore the active mechanism.Methods（1）Optimization of the hot reflux extraction of iridoid glycoside in Radix Scrophulariae by response surface methodThe extraction temperature,extraction time and liquid ratio were chosen as the independent variables by single factor study,then use Box-Behnken to build the model.The extraction process was optimized by the total peak area of Catalpol、Aucubin、Harpagoside、Harpagoside.（2）The separation and identification of chemical constituents of Radix ScrophulariaeThe chemical constituents of the eluent of 50%ethanol were isolated and purificated by macroporous resin,silica gel column chromatography and semi preparative liquid chromatography.The structure of chemical constituents were identified on the basis of its MS、¹H-NMR、¹³C-NMR、2D-NMR.（3）Prediction of the main targets of iridiod in Scrophulariae against cerebral ischemiaTo construct the 3D chemical structures of the 35 compounds from the TCMSP database and reported in the literature by chemdraw software.To upload the structures to the pharmmapper database to predict the protein targets.To annotate the information of targets with the aid of Mas 3.0 biological molecular function software.To construct the ingredients-targets-pathways network by the Cytoscope software.Comparing the network with the mechanism of cerebral ischemia to illustrate the mechanism of iridoid glucosides.Results（1）Optimization of the hot reflux extraction of iridoid glycoside in Radix Scrophulariae by response surface methodThe optimum extraction condition is that we choose anhydrous methanol as solvent,liquid ratio is 1:15,extraction temperature is 73℃,extraction time is 1.2h.（2）The separation and identification of chemical constituents of Radix Scrophulariae7 constituents were obtained by using separation methods and modern chromatographic techniques,and identified as:Cinnamic Acid（1）,6’-O-cinnamoylharpagide（2）,8-O-E-p-methoxycinnamoylharpagide（3）,Harpagoside（4）,Harpagide（5）,Geniposide（6）,Acubin（7）.And constituents3 was first obtained from Radix Scrophulariae.（3）Prediction of the main targets of iridiod in Scrophulariae against cerebral ischemiaThe results of prediction of targets indicate that active ingredients involve 115targets.Ras-related C3 botulinum toxin substrate 1,Cell division control protein 42homolog,Signal transducer and activator of transcription 1-alpha/beta,Caspase-3,Tyrosine-protein kinase JAK2,Transforming growth factor beta-2,Tyrosine-protein kinase ABL1,3-phosphoinositide-dependent protein kinase 1,Apoptotic protease-activating factor 1 were high-frequency proteins.The results of annotation and analysis of targets indicate that 64 signal pathways were involved by the iridoid.14 of them are about disease,5 of them are about endocrine system,7 of them are about immune system,9 of them are about signal transduction,23 of them are about metabolism.The anti-cerebral ischemia activity of Scorphulariae may be related to its ability to relieve acidosis,inflammation,oxidative stress and apoptosis caused bu cerebral ischemia.Conclusion（1）The fit degree and credibility of the model are good,and the extraction process can improve the extraction rate of iridoid.（2）8-O-E-p-methoxycinnamoylharpagide was first obtained from Radix Scrophulariae.（3）The research reflects the features of tradition Chinese medicine in multi-components,multi-targets and multi-pathways,and provides a scientific basis for elucidation of the anti-cerebral ischemia mechanism of Radix Scrophulariae.