| Objective:1.By comparing the acute toxicity of different extracts from Coreopsis Tinctoria Nutt on mice,and combined with the HPLC fingerprint and multiple linear regression to analyze the element which plays the most important role in causing the death of mice.2.By measuring 5 extracts impact on the clotting time and bleeding time in mice to determin whether Coreopsis Tinctoria Nutt has anticoagulant effect,and screening the better anticoagulant effect extract.and to provide the safety data for improving the extraction technology.3.By studying the effect of ETE of Coreopsis Tinctoria Nutt on Acute Thrombosis model in rat to further determine its effect and possible mechanism of anticoagulant and the prevention of thrombosis.4.By conducting the Ussing Chamber model to study absorption features of chlorogenic acid,Flavanomarein,Marein,3,5-dicaffeoylquinic acid of Extracts in different intestinal segment of rat.5.Through in situ single pass intestinal perfusion model to test the absorption features of chlorogenic acid,Flavanomarein,Marein,3,5-dicaffeoylquinic acid of Extracts in small intestine.Methods:1.To measure the maximum dose and maximal tolerance dose（MTD）of all the extracts,to measure the median lethal dose(LD50)by Bliss,and to record the death and weight changes;to measure the fingerprints of the extracts by HPLC,and to determine the element which mostly induced the death of mice by analyzing the absorption peak of the extracts by HPLC fingerprint with multiple linear regression.2.Chose Kunming mice as the research object,and based on the previous test of pesticide effectiveness to determine the drug dosage,dosing 7 days continuiously,at the last time after 30 min,test the bleeding time by tail cutting method,and test the clotting time by the glass method.3.Chose Sprague Dawley rats as the research object,with aspirin as the positive control drug,divide the ETE of extracts into high,medium and low three dose groups,continuously given 21 days drug,and at the last time after 1h to set up the acute thrombosis model,meanwhile to determin the TXB2,6-Keto-PG,APTT,PT,TT,FIB and thrombus weight,make thrombus pathological,study the impact of ETE of Coreopsis Tinctoria Nutt on mice acute thrombosis.4.Design high,medium and low dose concentration according to thrombosis prevention experiment result.By adopting“Quantitative Analysis Multi-components and with Single Market（QAMS）”to test four major ingredients’accumulative absorption volume Q,absorption rate constant Ka and apparent permeability coefficient Papp in Coreopsis tinctoria Nutt extractive in different intestinal segment in rat through Ussing Chamber model by HPLC.5.Design high,medium and low dose concentration according to thrombosis prevention experiment result.By adopting“Quantitative Analysis Multi-components By Single Market（QAMS）”to test four major ingredients’Ka and Pepp in rat through situ single pass intestinal perfusion model by HPLC.Results:1.The extracts include aqueous extract by spray drying（SD）,aqueous extract by vacuum drying（VD）process,ethanol extract（ETE）,ethyl acetateextracted component,（AC）and the ethyl acetateextracted residuum（AR）.Among those extracts,the maximum dose of SD and AR is 36g/kg,the MTD of the VD is 26g/kg,the LD50（95%confidence limits）of ETE and AC are 19.565（17.558-21.734）g/kg and16.414（13.987-34.725）g/kg,respectively;Under the high dose situation,3,5-dicaffeoylquinic acid properly is the component which mostly contributes to the death of mice.2.The 5 extracts can prolong the coagulation time（P<0.01）and bleeding time（P<0.01）,and ETE shows the best.3.the hige and medium dose of ETE can prolong APTT（P<0.01）,the high,medium and low dose of ETE can prolong PT（P<0.01）,the high,medium and low dose of ETE can decrese the TXB2 content（P<0.01）,and increase the6-Keto-PG content,the high and medium dose of ETE decrease the thrombus weight（P<0.01）,the low dose of ETE decrease the thrombus weight（P<0.05）.4.The Papp of chorogenic acid and flavanomarein in duodenum,jejunum,ileum and colon shows at1.0×10-610×10-6cm·s-1.The Papppp of marein in duodenum and jejunum is lower than1.0×10-6,and with the number of 1.0×10-610×10-6cm·s-1 in ileum and colon.The Papp of 3,5-O-dicaffeoylquinic acid in duodenum is smaller than1.0×10-6,while shows1.0×10-610×10-6cm·s-1 in jejunum,ileum and colon.5.The Pepp of chorogenic acid,flavanomarein,marein and 3,5-O-dicaffeoylquinic acid in small intestine show at0.03×10-42×10-44 cm·s-1.Conclusion:1.Under the high dose situation,the ETE and AC will lead the death,andthe 3,5-dicaffeoylquinic acid properly is the component which mostly contributes to the death of mice.2.The 5 extracts show the antithrombin activity and ETE presents the best.3.The ETE shows thrombosis prevention effectiveness and can prolong the APTT,PT,meanwhile can decrese the thrombus weight,and may impact the coagulation function by restraining the TXB2 and promoting the 6-Keto-PG.4.14chemical materials of ETE were tested and identified,and 10 of them can be absorbed by the intestinal,The four chemical components of the extractive can all be absorbed by the intestinal canal of rat which all conforms to zero order absorption and the effect of absorption in ileum present the best.5.14 chemical materials of ETE were tested and identified,and the peak area of 10 tested differently before and after the intestinal perfusion;the absorotion of the four chemical components of the extractive are influenced by the concentration and category as the medium permeability compounds. |
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