| Isoquinolines is a kind of compounds which contain isoquinoline or four hydrogen isoquinoline as basic units.Huge nμMber of these compounds are complex structures and mainly distributed in about 27 families of plant.Studies found that some isoquinoline compounds could inhibit vascular smooth musclecontraction in response to KCl,but sensitivity were obviously different.As far as the current research is concerned,isoquinoline compounds usually contain 1 to 3 isoquinoline units,thus we speculate that isoquinoline unit is the active center,but the structure-activity relationship of action of isoquinohnes on smooth muscle relaxation is unclear.In this study,we selected 20 isoquinolines,including seven isoquinolines of the Chinese herb,Embryo Loti,to study the structure-activity relationship of action of isoquinolines on smooth muscle relaxation in response to KCl,and then filtered out the strong and irreversible alkaloids as target to find out target protein,so it could help us elucidate the molecular mechanism of isoquinolines and provide the basis for reasonable drug molecule design.1.Isolation,purification and identification of the isoquinolines of Embryo LotiMediμM pressure colμMn preparation and reversed phase high-performance liquid chromatography(RP-HPLC)were used to isolate,purify alkaloids from Embryo Loti total alkaloids,and they were identified by 1H NMR and 13C NMR.Seven isoquinolines were identified,including nuciferine,pronuciferine,liensinine,isoliensinine,neferine,armepavine and neoliensinine.Using these two kinds of chromatographic separation methods obtained the pure alkaloid composition,and were shortened the time of separation.The separation method was also relatively stable and mature.2.Isoquinolines for relaxation effect in mice smooth muscle of the gutThe second-order branches of mesenteric artery were cleaned of adipose and mounted on a wire myograph.we selected 20 isoquinolines,including differentnμMber of isoquinoline unit,to measure the action of isoquinolines on smooth muscle relaxation in response to KCl,and calculate the IC50 alue,to study the structure-activity relationship of isoquinoline unit nμMber on smooth muscle relaxation.The results found that 20 different concentrations of isoquinolines could relax on gut smooth muscle of mice,but the effect is different.After isoquinoline unit reached number two,which forces showed no(or weak)reversible,indicating the number of the isoquinoline unit is closely related to the affinity of the compounds.3.Mechanism of Isoquinolines on gut smooth muscle of mice relaxation3.1 The effect of Isoquinolines on myosin regulatory light chain(MRLC)phosphorylationTo explore the mechanism of isoquinolines on gut smooth muscle of mice relaxation,whether related to the inhibition of MRLC phosphorylation.In this paper,we selected the strongest pesticide effect of Tetrandrine and the stronger effect of irreversible reaction Neoliensinine as the research object from 20 isoquinolines.With depolarization by 124 mM KCl,they were acting on gut smooth muscle of mice,and then extracting proteins.Western blot analyses were performed for measurements of myosin regulatory light chain phosphorylation.We found that Tet and Neo could inhibit MRLC phosphorylation.3.2 Molecular mechanism of Isoquinolines on gut smooth muscle of mice relaxationIn order to elucidate the molecular mechanism of Isoquinolines,we used the latest photochemical method to find a target protein with this kind of compounds Neo was selected as the main object to identify specific Neo-binding protein from a complex mixture of macromolecules by using a new biological molecules TFPA-PEG3-Biotin as a direct capture tool.Compounds of Biotin could be combined with Neutr Avidin Agarose Resin affinity resin.Then we used SDS-polyacrylamide gel electrophoresis to identify differentially expressed proteins.Its molecular weight was between 35 and 40 kda range.Eventually the catalytic subunit of protein phosphatase(PP1C)was identified by LC-MS/MS. |
