| Effective drugs are the main way to prevent and treat human disease. The establishment of the method for screening active ingredient has been the forefront research questions in the filed of medicine, pharmacy, chemistry and others. The major purpose of this paper is to establish orientated immobilized β2-adrenergic receptor (β2-AR) affinity chromatography to screening the pharmaceutically active ingredient, and apply it to screen the active ingredient and mechanism study; Using zebrafish as the animal model to study the angiogenic effect of Isopropyl-3-(3,4-dihydroxyphenyl)-2hydroxypropanoate. The paper includes four chapters, the main contribution of the author was showed as follows:1. Set β2-AR as an example, we established the method for screening the active ingredient of ephedra which was based on the oriented immobilized β2-AR affinity chromatography. Gene recombination method was used to get the histidine-tagged receptor. Using the high density imidazolyl in his-tags in the fusion protein, β2-AR was immobilized onto the surface of macroporous silica gel by diazonium salt reaction. The results showed that the protein bonding content was200.17±7.47pmol/g and the active ingredient of ephedra was ephedrine. Compared with the traditional random immobilization method, the oriented method can effectively reduce the loss of active binding sites in the process of immobilization, uniform orientation and conformation of the protein. This method is expected to provide a new way for the efficient screening of active ingredients of traditional Chinese medicine.2. Stationary phase containing the immobilised receptor was used to investigate the interaction between β2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β2-AR. At37℃, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02)×103M for pseudoephedrine, with the binding sites of (8.92±0.06)×10-4M. Thermodynamic studies showed that the binding of the two compounds to β2-AR was a spontaneous reaction with exothermal processes. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.3.In this chapter, based on the advantages of the new drug screening animal model zebrafish in the cardiovascular system, using the blood vessels fluorescent transgenic zebrafish TG (VEGFR2:GFP) to study angiogenic effect of the Isopropyl-3-(3,4-dihydroxyphenyl)-2hydroxypropanoate. The vascular injury model was made by using PTK787(growth factor tyrosine kinase inhibitor agent). Results showed thatDanshen isopropyl whose concentation was ranged from0.01to1.0μM can significantly resistant the vascular injury induced by PTK787, the relative production rate of the blood vessels were32.0±0.6%,41.1±5.6%,52.4±0.7%, respectively. The results showed that Isopropyl-3-(3,4-dihydroxyphenyl)-2hydroxypropanoate have the significant vascular antagonism for PTK787induced injury and in a dose-dependent manner. |
PDF Full Text Request