Studies On Active FXa Inhibitors Of The Root Of Achyranthes Bidentata

Thrombosis is the important reason of high incidence and mortality rates of cardiovascular disease,and anticoagulants is widely used for the prevention and treatment of venous and arterial thromboembolism diseases.The traditionary anticoagulants mainly for unfractionated heparin(UFH),low-molecular-weight heparin(LMWH)and warfarin,but there are many restrictions so that the clinical use of them had been limited strongly.There is a need for new anticoagulants that safety,effectiveness and convenient.Currently,the most development potential anticoagulant is the small-molecule,oral,direct Factor Xa inhibitor,and there have been multiple synthesis monomer into different clinical test periods.Drugs for invigorating blood circulation and eliminating stasis of traditional chinese medicine are used for the prevention and treatment of stagnation of blood in clinic at long-term,and the curative effect of anti coagulation is certain.This essay attempts to find natural FXa inhibitors from the drugs for invigorating blood circulation and eliminating stasis,and to provide the lead compounds for the development of small-molecule,oral Factor Xa inhibitor afterward.This essay establishes the methods of testing FXa activity in vitro with the chromogenic substrates and continuous rate methods.Based on this to use Xarelto as positive control,preliminary screening the ethanol-extraction content of 26 commonly used activating blood circulation to dissipate blood stasis plants.The results indicate that the inhibition to FXa of Herba Artemisiae Anomalae,Semen Persicae,Herba Lycopi,Radix Curcumae,Rhizoma Chuanxiong,Radix Salviae Miltiorrhizae and Semen and Rsdix Achyranthis Bidentatae are more than 40%under a final reaction concentration of 166 μg/mL,and demonstrate that there are direct FXa inhibitors in plants.Separating three active compounds from the root of ethyl acetate part of Achyranthis Bidentatae with active tracer combined with and several kinds of chromatographic methods(inculding silica gel column chromatography,gel column chromatography,reversed phase column chromatography,preparation of liquid phase)and identificating structures of these three compounds through the physical and chemical properties,NMR spectrum,mass spectrum and so on.They are dibutyl phthalate,phthalic acid 2-(α-ethyl)hexyl and oleanolic acid-3-O-β-D-(6’-methyl)-pyran-glucuronidase.And their inhibition rate to FXa is 23.3%,36.5%,84.1%,respectively under a final reaction concentration of 166 μg/mL.Further Using the chromatography such as macroporous resin,reversed-phase column chromatography,preparative high performance liquid and so on to separate the normal butanol part of achyranthes,we get five compounds which identify with the method of physicochemical property,NMR spectroscopy and mass spectrum as rhapontisterone,3-O-(β-D-glucose)-oleanolic acid-28-O-(β-D-glucose),ginsenoside R0,oleanolic acid 3-O-P-D-glucuronopyranoside and oleanolic acid-28-O-β-D-mannose.And their inhibition rate to FXa is 13.6%,50.0%,2.1%,86.4%,31.9%under a final reaction concentration of 166 μg/mL.The IC50 of the three compounds oleanolic acid-3-O-0-D-(6’-methyl)-pyran-glucuronidase,3-0-((3-D-glucose)-oleanolic acid-28-(β-D-glucose)and oleanolic acid 3-O-β-D-)-glucuronopyranoside is 15.2 μmol/L,171.1 μmol/L,and 95.4 μmol/L,respectively according to the results of determining.It is found that the inhibition is dropping if the 28-bit of oleanolic acid has substitution.On the contrary,the inhibition is significantly enhanced after the uronic acid esterification of 3-bit of oleanolic acid.Further study the enzymatic kinetics and selective of the compound oleanolic acid-3-O-p-D-(6’-methyl)-pyran-glucuronidase as a inhititor of FXa.It is found that this compound is competitive inhibitor of FXa and its Ki is 4.071 ± 0.5 pmol/L.It has a good selectivity to FXa so that its inhibitory rate of FXa is higher than other serine protease in human.This essay was the first to show some plant-medicines for activating blood circulation to dissipate blood stasis also have inhibition ingredients to FXa,and provide reference for reasonable utilization of drugs for invigorating blood circulation and eliminating stasis in clinical application.And discover triterpenoid saponin of oleanane type have direct inhibition to FXa.Among the total,the inhibitive effect of oleanolic acid-3-O-β-D-(6’-methyl)-pyran-glucuronidase is strongest,as well as the study of the enzymatic kinetics and selective,provide the basis for the research and development of this class of compounds.