Chromatographic Screening And Evaluation Of Active Components Targeting Two G Protein Coupled Receptors From Siraitia Grosvenorii

As the product of long-term clinical practice in the field of medicine and health in China,traditional Chinese medicine（TCM）has the advantages of high safety,exact efficacy,and low cost,and has become an important source of innovative drug research and development.However,TCM and its prescription are complex in composition,flexible in compatibility,and have the characteristics of multi-target,multi-pathway,and multi-component synergism,which challenging the research and development of TCM.The core of solving the above challenges is how to screen targeted active components from TCM,explore the material basis of TCM,clarify their mechanisms and reveal the scientific connotation.Based on the random immobilization method for theβ₂-adrenergic receptor（β₂-AR）in previous work,we established a method for immobilizingβ₂-AR and M₃-muscarinic acetylcholine receptor（M₃R）with high activity,screened and evaluated the anti-asthmatic components in Luo-Han-Guo.This work will provide a basis for the promotion and application of receptor chromatography in screening the targeted active components from TCM.It has significance for the research on the effective material basis of TCM and drug discovery and development.The full text is divided into five chapters.The author’s main contributions are as follows:1.The establishment of the oriental immobilization method forβ₂-AR and M₃R.On the basis of random immobilization in previous work,to improve the activity and stability of the immobilized receptors,we immobilized the haloalkane dehalogenase（Halo）-taggedβ₂-AR and Halo-tagged M₃R receptor on the surface of amino microspheres by a one-step reaction to prepare two types of receptor chromatographic stationary phase through the specific dehalogenation reaction between Halo-tag and its substrate haloalkanes.The morphology of the immobilizedβ₂-AR and M₃R was performed by Scanning electron microscope.The element composition was characterized by Energy dispersive spectrometer and X-ray photoelectron spectroscopy.The specificity and stability of the immobilizedβ₂-AR were characterized by the retention behaviors of terbutaline,methoxyphenamine,and bambuterol on theβ₂-AR column.The specificity and stability of immobilized M₃R were characterized by the retention behaviors of atropine,darifenacin,pilocarpine on the M₃R column.The results indicated that compared with the random immobilization method,the established one-step immobilization method can significantly improve the ligand recognition activity and stability of the receptor.The immobilized receptors can recognize their specific ligands according to the differences in the retention times,which provides a method for screening the active components in TCM.2.The orientally immobilizedβ₂-AR and M₃R can be used to evaluate ligand-receptor interaction.Injection amount-dependent analysis and non-linear chromatography were used to study the interaction between ligands and immobilizedβ₂-AR and M₃R for evaluating the binding strength of the ligands and receptors.Molecular docking was applied to explore the mechanism of the bindings.The results showed that the association constants of terbutaline,methoxyphenamine,and bambuterol withβ₂-AR were 1.51×10⁴,1.23×10⁴,and 1.93×10⁴L/mol by injection amount dependent analysis,and 1.08?10⁴,0.34?10⁴,and 7.85?10⁴ L/mol by nonlinear chromatography,respectively.The association constants of atropine,darifenacin,pilocarpine with M₃R were measured by the two methods were 0.28×10⁵,1.92×10⁵,1.19×10⁵L/mol,and 3.49?10⁴,28.70?10⁴,5.24?10⁴ L/mol,respectively.The measured association constants were consistent with the data reported in the literature,and they followed the order of bambuterol>terbutaline>methoxyphenamine.The main amino acid residues ofβ₂-AR that interact with bambuterol,terbutaline,and methoxyphenamine were Asn 312;Ser 203;Phe193,Asn 312,etc.The main amino acid residues of M₃R that interact with atropine,darifenacin,and pilocarpine were Asn 152;Trp 192;Ala 235,Asn 507,etc.Hydrogen bonds and hydrophobic interactions are the main forces between the ligands and the two receptors.The work provides a method for evaluating the binding strength and mechanism of the interactions between drugs and receptors.It is expected to provide a reference for the rapid evaluation of other drug-receptor interactions.3.The rapid screening and evaluation method for anti-asthmatic components in Luo-Han-Guo targetingβ₂-AR and M₃R.We screened Luo-Han-Guo extract by affinity chromatography with immobilizedβ₂-AR and M₃R,collected the retained components,and identified the structures of the components by HPLC-MS/MS.The interactions between the retained components andβ₂-AR and M₃R were studied and evaluated by injection amount-dependent analysis and nonlinear chromatography.The binding mechanism was explored by molecular docking and tracheal experiment in vitro.The results showed that11-O-mogroside V was the active component that targets M₃R and Mogroside V was the active component that acting on bothβ₂-AR and M₃R.The association constants of Mogroside V withβ₂-AR were 8.21×10⁴ L/mol and 1.60×10⁴ L/mol by injection amount dependent analysis and nonlinear chromatography,respectively.The association constants of Mogroside V with M₃R were 2.10×10⁴ L/mol and 2.00×10⁴ L/mol by injection amount dependent analysis and nonlinear chromatography respectively.Molecular docking showed that Thr 110 and Thr 195 ofβ₂-AR and Ala 237 and Leu198 of M₃R were the main amino acid residues that interact with Mogroside V.This study provides a method for rapid screening the targeted components forβ₂-AR and M₃R in traditional Chinese medicine.It also provides a methodological reference for the rapid screening and evaluation of the targeted components for other receptors from traditional Chinese medicine.4.To clear that mogroside V has a significant anti-asthmatic effect.The anti-asthmatic effect of mogroside V was evaluated by determining the content of inflammatory cytokines and observing the morphological changes in lung tissue in the ovalbumin（OVA）‐induced allergic asthma mice model.Mass spectrometry with multiple reaction monitoring was used to study the pharmacokinetics of mogroside V in rats.The results showed that Luo-Han-Guo extract,mogrosideⅤ,11-O-mogrosideⅤ,compound middle,and high doses can reduce IL-4,IL-5,IL-13,IL-17,Ig E,OVA Ig E,and Ig G₁ levels,up-regulate IFN-γlevel,regulate the balance of Th1 and Th2,control the airway inflammation in asthma model mice,reduce the inflammatory infiltration in lung tissues,and improve the morphology of lung tissues in asthmatic mice.The effect of the high-dose group was the most obvious one.The level of inflammatory cytokines was no statistically different compared with the normal group,and the morphology of lung tissue was similar to the normal group.Mogroside V reached the C_maxat 45 minutes and showed fast absorption and slow elimination in rats,indicating that mogroside V has good druggability.The above results provide a basis for clarifying the anti-asthmatic components in Luo-Han-Guo and have positive significance for the mogroside V-based research and development of innovative drugs from traditional Chinese medicine.