Study On The Antithrombotic Effect Of Ligustrazine Derivative F3

Cardiovascular and cerebrovascular diseases are the general term of cardiovascular and cerebrovascular diseases,and the number one killer of human health in the world,including hyperlipidemia,stroke,cerebral infarction,blood viscosity,hypertension and atherosclerosis.Pathological thrombosis leads to vascular occlusion,ischemic stroke,myocardial infarction and other cardiovascular diseases.Platelet activation is the key factor of thrombosis.Platelets are non-nucleated blood cells formed by the cytoplasm of marrow megakaryocytes,which play an important role in physiological hemostasis,maintenance of vascular integrity,and some pathological processes,such as atherosclerosis,thrombosis,unstable angina,inflammatory reaction,Alzheimer's disease and tumor metastasis.Under normal physiological conditions,the healthy blood system releases inhibitory signals and platelets is leaving in a resting state.When blood vessels are damaged,blood flow is changed or stimulated by chemical substances,platelets are activated,resulting in the aggregation,deformation,adhesion and release of granular contents.Anti-platelet drugs are widely used in the primary and secondary prevention of thrombotic cardio-cerebrovascular diseases.However,there are individual differences and hemorrhage in anti-platelet drugs.Ligustrazine is an effective component of traditional Chinese medicine,ligustrong,which has a variety of pharmacological effects,such as protecting vascular endothelium,anti-platelet and anti-oxidative stress,and is widely used in clinical occlusive cardiovascular and cerebrovascular diseases.Ligustrazine with pharmacological activities,however,is not strong,fast metabolism and low bioavailability.Therefore,according to the splicing principle in pharmaceutical chemistry,the structure of 3,5,6-trimethylpyrazinic acid and the structure of cinnamic acid were combined to design a new type of ligustrate phenolic.Objective:To evaluate the antithrombotic effect of ligustrazine derivative F3.Methods:1.Effects of F3 on platelet aggregation in vitro:Turbidimetric method was used to determine the platelet aggregation.Platelet-rich plasma were extracted from rat and pretreated with F3(the final concentration was 6.25?M,12.5 ?M,25 ?M,50?M and 100?M).The platelet aggregation was induced by ADP,collagen,AA and thrombin respectly and the aggregation rate was observed.2.Effect of F3 on platelet aggregation in vivo:Wistar rats were randomly divided into control group(0.5%CMC-Na),and F3 groups(50 mg/kg,25 mg/kg and 6.25 mg/kg),and ligustrazine group(TMP,200 mg/kg).The blood sample was selected and the platelet-rich plasma were extracted after the gavage administration.The platelet aggregation was induced by ADP,collagen,AA and thrombin respectly and the aggregation rate was observed.3.The effects of F3 on tail bleeding time in mice:Mice were divided in F3 groups(87.5 mg/kg,175 mg/kg and 350 mg/kg),clopidogrel group(30 mg/kg)and 0.5%CMC-Na group.Two hours after gavage administration,the tail of mouse was cut 2-3 mm and then was put into 37 0C normal saline,the bleeding time was observed.4.Effect of F3 on common carotid artery thrombosis in rats:Wistar rats were randomly divided into control group(0.5%CMC-Na),F3 groups and ligustrazine group.Thrombogenesis in common carotid artery was induced by electric stimulation with 1.5 mA and 7 min.The obstruction time was measured.5.The effect of F3 on pulmonary embolism induced by AA in mice:Mice were randomly divided into control group,F3 group(350 mg/kg,175 mg/kg and 87.5 mg/kg),and ligustrazine group(400 mg/kg).Two hours after administration by gavage,arachidonic acid(20 mg/kg,0.02 ml/10g)was injected through vena caudalis,and the number of death within 3 minutes was counted.6.Effect of F3 on arteriovenous bypass thrombosis in rats:Rats were randomly divided into control group(0.5%CMC-Na),ligustrazine group(200 mg/kg),and F3(50 mg/kg,25 mg/kg,and 6.25 mg/kg)groups.Two hours after administration by gavage,the arteriovenous bypass circulation was established.After 15 min,the weight of arteriovenous bypass thrombosis was weighed.7.Preliminary metabolic studies of F3 in vivo:The carotid sinus blood sample was slected after gavage administration.F3 metabolism was analysed by high performance liquid chromatography.Results:1.Effects of F3 on platelet aggregation in vitro:The results showed that F3 had no significantly inhibitory effect on platelet aggregation induced by ADP,collagen,thrombin and AA in vitro.2.Effect of F3 on platelet aggregation in vivo:The result showed that F3 could significantly inhibit the platelet aggregation induced by ADP,AA and collagen.However,F3 had no effect on the platelet aggregation induced by thrombin.3.The effects of F3 on tail bleeding time in mice:F3(175 mg/kg and 350 mg/kg)could obviously prolong the bleeding time.4.Effect of F3 on common carotid artery thrombosis in rats:At the dose of 50 mg/kg,F3 could significantly prolong the obstruction time.This result suggests F3 has inhibitory effect on thrombosis formation.5.The effect of F3 on pulmonary embolism induced by arachidonic acid in mice:The result showed that F3 at dose of 175mg/kg and 350mg/kg could significantly reduced the mortality of mice and the survival rate was significantly improved.6.Effect of F3 on arteriovenous bypass thrombosis in rats:The weight of arteriovenous bypass thrombosis in rats was significantly reduced at the dose of 50mg/kg of F3.7.Preliminary metabolic studies of F3 in vivo:The ligustrazine was not found in the blood sample.Conclusion:F3 could inhibit platelet aggregation in vivo,prolong tail bleeding time in mice,and inhibit thrombosis.These results suggest that F3 has antithrombotic effects.