Fundamental Research Of The Interaction Of Medicine,Aptamer And Protein

In this passage, microdialysis coupled with high pressure liquid chromatograph(MD-HPLC) and capillary electrophoresis(CE) are applied to study the interaction of drug molecule, thrombin and 29 nt human thrombin apatamer. There are four parts in the thesis.(1) In the dialysis experiment, speed of perfusate, incubating time, temperature is optimized. Interaction between drug molecules in Salvia miltiorrhiza and bovine thrombin are characterized by MD-HPLC. The determination of total affinity between drug molecule,including salvianic acid A sodium(SAS), protocatechuic acid(PA), ferulic acid(FA) in Salvia miltiorrhiza and bovine thrombin is respectively 460, 912, 5481 M-1.(2) A capillary zone electrophoresis method for determination of drug molecules in Salvia miltiorrhiza, B-Thr and Apt29 is established. The determination of total binding affinity is: PA to B-Thr is 3.39×104M-1and FA to B-Thr is1.05×104M-1. SAS has no binding. Compared to MD-HPLC, CE has advantages such as lower cost, faster analysis,and higher sensitivity. Apt29 can combine with heparin binding site on B-Thr.Determination of interaction between Apt29 and drug molecules in Salvia miltiorrhiza, is first studied. The determination of total binding affinity is: PA to Apt29 is 1.32×104 M-1 and FA to B-Thr is 1.48×104 M-1. SAS is no binding. Apt29 can bind to B-Thr specifically.Apt29 can bind to B-Thr comparatitively. In the combination study, PA occupies heparin binding site, while FA occupies other sites.(3) A capillary zone electrophoresis method for determination of Loureirin(LA),Loureirin B(LB), Loureirin C(LC), pterostilbene(PTS), resveratrol(RES) and7’,4-dihydroxylflavone(DHF), homoisoflavanones(HIF) are established. PH, KCl concentration, separate voltage and temperature are optimized. Interactions between these drug molecules, Apt29 and thrombin are studied by CZE. The determination of total binding affinity is: LA to B-Thr is 4.73×104 M-1 and PTS to B-Thr is 1.63×105 M-1. There is no binding between other five molecules and B-Thr. The combining interaction between flavoniods and B-Thr is related to the molecule structure and groups. In this passage, it is the first time to study the interaction between drug molecules in Dragon’s blood(LA, LB,LC, RES, HIF) and Apt29. The total bindings of LA, LB and DHF to Apt 29 arerespectively 1.16×105 M-1、2.26×105 M-1、2.26×104 M-1, while nk value of LC, RES to Apt29 can’t be detected. The distinctions in the molecule structure will lead to interaction differences. The binding study between PTS and H-Thr, B-Thr are performed by affinity capillary electrophoresis(ACE). The binding constant is obtained according to mobility migration. Thrombin origin affects the interaction between molecule in Dragon’s blood and thrombin. In CZE and ACE research, bind constants between PTS to B-Thr are in the same order.(4) A non-aqueous capillary electrophoresis method(NACE) for determination of gambogic acid(GA) in gamboge was established based on the optimized conditions. The effect of compositions, concentration, p H, additives including methanol, acetonitrile andβ-CD, applied voltage and temperature is thoroughly investigated. Quantification of GA in gamboge from different places including Vietnam, Thailand, Burma, India was1.67±0.04~472.40±7.75 mg/g.