Both course of thrombopoiesis and thromboembolus of clinicopathologic lead to thrombosis disease, which is characterized by high rate of incidence, relapse, death and casualty. Many serious diseases in clinical include deep venous thrombosis, coronary heart disease, myocardial infarction, stroke and pulmonary embolism etc. belong to thrombosis disease. With an up ward tendency it will become the first burden in the year of 2020 and more attention has been focused on it. It is recognized by the world as one of tough problems of medicine and sociology.The dried flower of safflower(Carthamus tinctorius L.) is a traditional medicine in the treatment of cardiovascular and cerebrovascular diseases. Safflower injection is unilateral injection through water extraction and alcohol precipitation. It has been used in clinical for about 30years, mainly effective for disease prevention and treatment such as occlusive cerebrovascular disease, coronary heart disease and vasculitis and so on. With the purpose of reasonably wide range applications and international market competition, the Pharmacology material basis of need further research by using modern science and technology to achieve effective substance and clarify its mechanism. But the current domestic and international research on safflower injection mainly in clinical observation and pharmacological research, the material basis of its efficacy has not been reported.On the material basis research for efficacy on Chinese medicine, a very important aspect is that the effect of component selection methods should reflect characteristics of traditional Chinese medicines including the overall effect, multi-target and multi-component synergistic. Platelet cells in solid-phase extraction combine HPLC is a screening biologically active compounds method. It screens biologically active compounds from Complex system by the specific binding of the cells with biological activity and effective compounds. This screening method is suitable for the separation and analysis of active ingredient of Chinese medicine with many targets characteristics.The study will be split the overall chemical of safflower into five sub-groups of chemical substances by colμmn chromatography, and screen effective chemical group by pharmacological test. Then screen compounds effective on platelet by the methods of Platelet cells in solid-phase extraction combine HPLC, and then separates and analyses those compounds. Finally, evaluate the pharmacological effects of those compounds combination with platelet by pharmacological experimental. In this paper, safflower injection was studied gradually in-depth by the strategy of hierarchical, systematic and progressive optimization.This paper is divided into the following parts.1. Split the overall chemical safflower injection into five sub-groups of chemical substances by different column materials. The pharmacological test of ADP induced platelet aggregation in vitro showed that Group 1 and 5 with stronger inhibition of platelet aggregation than Group 3,4, but Group 2 has a strong role in promoting platelet aggregation.2. Screen the composition effects on platelet from safflower extract, the active chemical substance group 2, group 5 of safflower injection by the tests of platelet cells in solid-phase extraction combine HPLC. Ten compounds were screened from Safflower Extract, and identify two compounds, including HSYA and SYB. Three compounds were screened from group 2, and identify one compound, which is HSYA. Thirteen compounds were screened from group 5, and identify one compound, which is 6-hydroxy-kaempferol 3-O-glucoside.3. Obtain hydroxyl safflower yellow A, safflower yellow B and 6-hydroxy-kaempferol 3-O-glucoside that combine with platelet. We found the compounds in two yellow ribbons and HSYA are the same Rf value in the process of purification HSYA. The two compounds were identified by UV, IR, MS, NMR, CD and other spectroscopy. The result shows that the two compounds are not enantiomers mutually; however, the structure of the two compounds is quite similar to each other. They are each other isomers. 4. Evaluate antithrombotic effects of the compounds that combine with platelet by Pharmacological tests of rat platelet aggregation induced by ADP and the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT). Pharmacological results show that compound I, SYB and 6-hydroxy-kaempferol 3-O-glucoside was a platelet aggregation inhibitor and they also exhibited prolong activated partial thromboplastin time of rat plasma and prothrombin time. Those indicate that compoundsâ… , SYB and 6-hydroxy-kaempferol 3-O-glucoside have inhibitory effect on endogenous and exogenous coagulation system. Compoundsâ…¡showed strong positive impacts on platelet aggregation by induced ADP and it exhibited prolong prothrombin time and shorten activated partial thromboplastin time of rat plasma. Those indicate that compoundsâ…¡has inhibitory effect on exogenous coagulation system, but has positive impacts on endogenous coagulation system.By the preliminary study, using solid phase extraction of platelet cells screened thirteen effect components from safflower injection, identified four kinds of those compounds, and confirmed the anticoagulant effect by in vitro pharmacological experiments. |