| Respiratory system disease is one of the major chronic non-infectious diseases with the characters of high morbidity,drug dependence,and recurrent episodes.New technologies and methods are urgently needed to develop highly effective and innovative drugs to achieve effective prevention and treatment of such diseases.It has become one of the emerging difficulties and hot issues in the field of medicine and health.Traditional Chinese medicine?TCM?is one of the major sources for defending respiratory diseases.The bioactive compounds screening from TCMs becomes a bottleneck problem in the research and development of innovative Chinese medicines and one of the emerging difficulties and hot issues in the field of medicine and health.Sanzi Yangqin Decoction?SYD?is a widely used recipe and usually prescribed for fighting against the respiratory system diseases.Focusing on the unclear anti-asthmatic active compounds and targets,we established a new chromatographic method based on the immobilized?2-adrenoceptor??2-AR?and applied it to bioactive compounds screening in SYD in this thesis.This study is expected to achieve rapid analysis of the anti-asthma compounds and solve the bottleneck problem of a long research cycle and low accuracy from TCMs.The method is believed to provide a reference for the bioactive compounds screening from other TCMs and play a positive role in innovative drug development.The thesis is divided into four chapters,and among which the main contributions are as follows:1.A one-step immobilization method for?2-AR was established.Taking?2-AR as a probe,O6-alkylguanine DNA alkyltransferase?hAGT?was fused at the C terminus of the receptor.The fusion receptor was induced and expressed in E.coli BL21?DE3?.According to the specific bio-orthogonal reaction between hAGT and O6-benzylguanine derivatives modified gel,?2-AR was captured and immobilized by a one-step method from the cell lysate through covalent bond.Scanning electron microscopy?SEM?,immuno-scanning electron microscopy?Immuno-SEM?,high-performance liquid chromatography coupled with mass spectroscopy?HPLC-MS?and other techniques were used for morphological,functional and micro-environmental characterization of the immobilized?2-AR.The result illustrated that?2-AR can be oriented immobilized on the gel surface as a monolayer with high specificity,high activity,and high stability.It can provide new method for the rapid preparation of receptor chromatography with high activity.2.Taking salbutamol,methyoxynamine,and terbutaline as probes,we used frontal analysis and perturbation peak method to investigate the binding interaction of the drugs and the immobilized?2-AR.The results were also verified by molecular docking.The data showed that the three drugs had one type of common binding sites on the immobilized?2-AR.Isothems of the three drugs were best fitted with Langmuir model.The binding constants of the three drugs were 1.63×104L/mol,3.03×104L/mol,and 0.76×104L/mol.Hydrogen bond and Van der Waal's force were the main driving forces of the interaction.The exposed amino acids were Ser 203,Ser 204,Ser 207 and Asp 113.It is proved that immobilized?2-AR still preserves the ability to recognize its specific ligands.The established receptor chromatography can be applied in studying the binding interaction between the receptor and the ligand,which provides a new chromatographic method for targeted bioactive compounds screening.3.We used the established,immobilized receptor-based,chromatographic model to screen the anti-asthma compounds from SYD that targets to?2-AR.The pharmacological effect of the screened compounds was evaluated by the tension changes of the trachea ring in vitro.Molecular docking was used for validating the pharmacological effect.The result showed that rosmarinic acid and sinapine thiocyanate were the specific bioactive compounds targeted?2-AR in SYD.The two compounds exert the pharmacological effect through a concentration-dependent manner when the trachea was pre-treated with high potassium.And the concentration ranges were tested to be 10-9-10-44 mol/L and 10-12-10-77 mol/L,respectively.While when the trachea was pre-treated with ICI 118551,a specific antagonist of?2-AR,the relaxation effect was inhibited.It proved that the antitussive and anti-asthmatic effects of the two compounds were exerted through specific identifying and binding with?2-AR.Molecular docking revealed that the two compounds mainly interacted with the Ser 203,Ser 204,Ser207,Tyr 316 and Asn 312 of?2-AR.Our method is proved to be a powerful alternative in targeted screening and evaluating the bioactive compounds for anti-asthma effects. |
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