Research On The In Vivo Process Of Ziziphi Spinosae Semen Extract

RATIONALES: Ziziphi Spinosae Semen(ZSS),was first documented in the Divine Husbandman's Classic of the Materia Medica,which has the effect of nourishing the heart and the liver,calming the nerves,soothing the sweat.ZSS has a variety of pharmacological effects such as sedative hypnosis,anxiolytic,antidepressant,anti-inflammatory and anti-convulsion.In the natural drug chemical separation mode,some of the pharmacodynamic substances for the improvement of sleep by the ZSS have been clarified.However,using natural medicinal chemistry as a means,the quality control model of reference to chemicals does not match the overall function of traditional Chinese medicine(TCM),ignoring the complexity of TCM and the integrity of the body,and separating the correlation between chemical composition and in vivo processes.In most cases,the active ingredient of TCM needs to be absorbed into the blood,and the active compounds can be produced by metabolic to exert the overall pharmacodynamic effect.Therefore,the selection of active ingredients should be more accurate and comprehensive in connection with the analysis of drug efficacy and metabolism.It is unclear which components of the ZSS are absorbed into the body,and which components are absorbed into the body and coordinate with the target of the body.ZSS is the first choice for treatment of insomnia in clinical practice.It is taken in the form of decoction.In summary,the ZSS extract was used as the research objects,and its composition were studied based on in vivo process.The results not only reveal the chemical basis of ZSS treatment for insomnia,but also have important significance for quality control evaluation.OBJECTIVE: Comprehensively characterize the chemical constituents in the insomnia model rats after oral administration of ZSS extract,and identify the main prototype components and metabolites.Explore the metabolism of different types of chemical components.METHOD&CONTENT:1.Preparation and quality evaluation of ZSS extractAccording to the research strategy of standard decoction,the preparation process and quality evaluation of ZSS extract were studied.The method of crushing,extraction time and drying method of ZSS were investigated by using the content of paste and the content of spinosin,jujuboside A and jujuboside B.According to the pharmacopoeia method,the indicator components of ZSS slices and ZSS extract were determined,and the transfer rate and extract rate of were investigated.The multi-dimensional fingerprints of HPLC-UV/ELSD were established to evaluate the consistency of the ZSS extract.A LC-MS/MS method for the simultaneous determination of nine indicator components of ZSS extract was established.2.Deep characterization of in vivo chemical constituents in ZSS extract based on UPLC Q Orbitrap MSIn order to solve the bottleneck problem of the reference substance lacking in vivo components and its metabolites,the new strategy for analyzing the metabolic processes of the multi-components of ZSS was explored.Established in vitro chemical composition research method of ZSS extract based on UPLC Q Orbitrap MS.PCPA-induced rat insomnia model was prepared to evaluate the pharmacodynamic effect of ZSS.Optimize sample preparation,chromatographic conditions,mass spectrometry conditions,and data acquisition to obtain high resolution mass and multistage mass spectral data.The characteristic diagnostic ion technology was used to identify the target compound and obtain its parental information.On this basis,the non-targeted prototype and metabolite analysis methods were established.The complex components and metabolism of the insomnia model rats were studied.3.Pharmacokinetics study of six main components in ZSS extractUPLC Q Orbitrap MS technique was used to establish a method for the determination of six main chemical components of ZSS extract with palmatine hydrochloride,daidzein and astragalosides ? and as internal standards.The method was applied to the pharmacokinetics of normal and PCPA-induced insomnia rats treated with ZSS extract.RESULTS:1.ZSS was mechanically pulverized,and boiled twice with water for 120 min to prepare the ZSS extract,and the extract was prepared as a lyophilized powder.The fingerprints of ZSS extract have extremely high similarity,both above 0.9.The ointment rate of 10 batches of ZSS extract was 19.28~21.96%,and the transfer rate of index components was 64.10~90.11%,which met the standard.The method for determining the content of nine components by LC-MS/MS was sensitive and specific.2.Molecular prediction based on the accurate molecular weight of the compound,and combined with the mass spectrometry cleavage of different types of compounds and fragment ions,a total of 41 compounds in ZSS were identified,including 19 flavonoids,11 saponins,9 alkaloids and 2 other types of compounds.The high dose group of ZSS(30g/kg)can significantly increase the 5-HT in the serum of model rats.Based on the in vivo composition analysis strategy,30 prototype components and 26 metabolites were identified from the urine of insomnia model rats;and 14 prototypes and 17 metabolites in the blood were identified by extracting ion currents.The main metabolic reactions in PCPA-induced insomnia rats include oxidation,reduction,hydrolysis,sulfation,glucuronidation and methylation.3.The UPLC Q Orbitrap MS method for determining the content of six components in rat plasma has small matrix interference and high sensitivity.Compared with normal rats,the plasma exposure of spinosin and 6'''-feruloylspirnosin was reduced,and the plasma clearance of both was significantly increased.In addition,the Tmax of jujuboside A and jujuboside B in the model group was significantly decreased,and the T1/2 of jujuboside A was obviously increased.Compared with the normal group,the Cmax,AUC0-t and CL had a weak trend,but there was no significant difference.The PCPA insomnia model significantly affected the absorption and in vivo exposure of six chemical components.CONCLUSION: In this paper,the method of quality evaluation of ZSS extract was established,which provided a stable and controllable material basis for the study of in vivo process.On the basis of the corresponding relationship between in vitro and in vivo chemical composition groups,the targeted chemical composition characterization strategy of LC-HR/MS was studied,which provided a useful reference for the deep characterization of the target components in the complex metabolic system of TCM.The metabolites in serum and urine were systematically characterized,and the chemical composition of ZSS under the condition of pathological model was preliminarily expounded.The pharmacokinetics characteristics of three kinds of representative components in ZSS were clarified,which laid a foundation for further study on the quality markers of ZSS and provided a scientific basis for the construction of scientific and reasonable quality standard of ZSS..