| Objective:To explore the effective substance of anti-platelet aggregation on ethyl acetate extract,and lay the foundation for the development of antiplatelet drugs.Methods:Kunming mice were used as the subjects,we used improved Karber’s method to calculation LD50 and 95%confidence-limit,and record the clinical manifestations and weight changes.Used the slice method to determine clotting time in mice,and calculate the half effective concentration.SD rats were used as the subjects,and the blood transfusion components were analyzed by HPLC-DAD-ESI-MS method to determine the chemical structure of serum.Used classical silica gel column chromatography,and combined TLC with liquid chromatography to purify the active extract,used turbidimetric method to verify the antiplatelet aggregation of the purified product.Results:The LD500 value of ethyl acetate extract was 2.4194 g/kg,which was equivalent to 201.617 g crude drug/kg,95%confidence limit was 2.1050 to2.7801 g/kg,and the body weight of survival mice close to the blank mice in14 days.The results of the determination of coagulation time in mice showed that there was significant difference between the high dose group and the middle dose group to blank group,there was no significant difference in low dose group.There was significant difference between the high dose group and the aspirin group,and its effect was better than that of aspirin;the results showed that EC50 was 25.2 to 32.2 mg/kg,which equivalent to 2.10 to 2.68 g crude drug/Kg and the treatment index was 84.3.The 6 components were analyzed by HPLC-DAD-ESI-MS method,one of which was the in vivo conversion component of Procumbenoside L,and 5 were the original constituents,which were 6-hydroxy justicidin B,6-hydroxy justicidin A,6-hydroxy justicidin C,justicidin B,taiwanin E methyl ether.The results of turbidimetric method show the antiplatelet aggregation of the purified was stronger than the ethyl acetate extract.Conclusion:The toxicity of ethyl acetate was lower in general,and it is clinically safe.The results showed that ethyl acetate extract had good antiplatelet aggregation in vivo,and it was effective substances of Justicia.The results of serum pharmacological studies provide the basis for the study on the pharmacological of antiplatelet aggregation.The antiplatelet aggregation of the purified product is stronger than that the ethyl acetate extract,which indicates that the purification process is basically feasible and can lay the foundation for the development of antiplatelet aggregation drug preparation. |
