Study On Quality Standard Of Zishen Yutai Pill And Its Mechanism In The Treatment Of Recurrent Spontaneous Abortion

Objectives:Based on the fingerprint of index chemical components and qualitative and quantitative comprehensive analysis and control,the overall quality standard of Zishen Yutai pill was established and chemically characterized;Then,based on the main components of Zishen Yutai pill and relying on network pharmacology and molecular docking technology,the molecular mechanism of Zishen Yutai pill in the treatment of recurrent spontaneous abortion is predicted;Then human chorionic trophoblast cells（HTR-8/SVneo cells）were used to screen and verify the material basis of potential efficacy,so as to lay a foundation for clarifying the mechanism of Zishen Yutai pill in the treatment of recurrent spontaneous abortion.Methods:1.Characterization of chemical constituents of Zishen Yutai pill based on UHPLC/Q-TOF-MS/MSUHPLC/Q-TOF-MS/MS technology is adopted,and Waters aquity BEH C₁₈（2.1mm×100mm,1.7μm）is a chromatographic column;Gradient elution was carried out with acetonitrile-0.05%formic acid water as mobile phase;Flow rate:0.3 m L/min;Column temperature 37℃,injection volume 2μL.Using ESI source and positive and negative ion mode,the atomization gas is high-purity nitrogen,capillary voltage is 3.5 k V,collision energy is 15 V,and the atomization gas pressure is 45psi（1psi=6894.8Pa）;Dry gas temperature 350℃;Dry gas flow rate:11 L/min;Scanning range m/z 100-1200.2.Establishment of HPLC characteristic fingerprint and determination of multi components in Zishen Yutai pillBy HPLC,Agilent ZORBAX SB-C₁₈（4.6×250mm,5μm）is a chromatographic column;Gradient elution was carried out with acetonitrile-0.1%phosphoric acid water as mobile phase;Flow rate:1.0 m L/min;Column temperature 40℃,injection volume 10μL;The detection wavelength was 254nm and the acquisition time was 60min.Taking sweroside as the reference peak,11 batches of Zishen Yutai pill samples were evaluated and analyzed by using the similarity evaluation system of TCM（2012A version）,the characteristic fingerprint of Zishen Yutai pill was established,and the contents of sweroside,loganin and isochlorogenic acid C were determined at the same time.3.Network pharmacological analysis of Zishen Yutai pill in the treatment of recurrent spontaneous abortionFirstly,Swiss ADME and previous research reports were used to predict the potential active components of absorption,distribution,metabolism and excretion（ADME）of Zishen Yutai pill.Secondly,the potential targets of Zishen Yutai pill were collected through TCMSP and Swiss Target Prediction database;Use Gene Cards,Dis Ge NET and OMIM databases to collect disease-related targets,so as to obtain the intersection targets of disease and drugs.Apply them to the STRING platform to build PPI network,so as to select the key targets of the effective active components of Zishen Yutai pill in the treatment of recurrent spontaneous abortion.Thirdly,Omic Share platform and DAVID database were used for GO biological process enrichment analysis and KEGG signal pathway analysis.Finally,the affinity of key compounds to key targets in the pathway was preliminarily evaluated by molecular docking.Firstly,according to previous research reports and Swiss ADME platform,potential active ingredients meeting the characteristics of oral bioavailability（OB）and drug like（DL）were screened.Then,the action targets of potential active ingredients were collected through TCMSP and Swiss Target Prediction database.Secondly,Gene Cards,Dis Ge NET and OMIM databases are used to collect disease-related targets,so as to obtain the intersection targets of disease and drugs.It is applied to the STRING platform to construct the target protein interaction（PPI）to select the key targets of potential active components of Zishen Yutai pill in the treatment of recurrent spontaneous abortion.Thirdly,Omic Share platform and DAVID database were used for gene ontology（GO）biological process and Kyoto Encyclopedia of Genes and Genomes（KEGG）signal pathway enrichment analysis.Finally,the affinity of key compounds to key targets in the pathway was preliminarily evaluated by molecular docking.4.Preliminary screening of pharmacodynamic substances of Zishen Yutai pill in the treatment of recurrent spontaneous abortionHTR-8/SVneo cells were cultured in vitro,and the injury model was established by LPS induction.At the same time,the main chemical components of Zishen Yutai pill were added for cell intervention.CCK-8 kit was used to screen the suitable dosage concentration and detect the effect of the main chemical components of Zishen Yutai Pill on the proliferation of HTR-8/SVneo injury model.Results:1.According to the retention time of chromatographic peak and the fragmentation law of mass spectrum,combined with the relevant information of compound database and reference substance,39 chemical components of Zishen Yutai pill were identified.According to its chemical structure,it can be divided into organic acids and their derivatives,terpenes,alkaloids,anthraquinones,flavonoids and so on.2.The characteristic fingerprint of Zishen Yutai pill was established,and 9 common peaks were calibrated.Through the comparison of the reference materials,five common peaks were identified,which were 5-hydroxymethylfurfural（peak 2）,loganic acid（peak 5）,sweroside（peak 6）,loganin（peak 7）and isochlorogenic acid C（peak 9）.The similarity between each batch of Zishen Yutai pill samples and the reference fingerprint was greater than 0.900.The linear relationship among sweroside,loganin and isochlorogenic acid C was good（r>0.999）.The mass fractions of the above three components in 13 batches of Zishen Yutai pill powder were 592.62-816.19,208.02-289.26 and 69.10-281.14μg/g.3.After screening 30 potential active components and 650 action targets of Zishen Yutai pill,167 targets for recurrent abortion were obtained after intersection with 1078 disease targets.Through PPI protein interaction analysis and network topology parameters,29 key targets such as PIK3CA,STAT3,PIK3R1,MAPK1 and TP53 were finally screened.The enrichment pathways of key targets mainly include PI3K-AKT signal pathway,thyroid hormone signal pathway,estrogen signal pathway and MAPK signal pathway.PI3K-AKT signal pathway is the signal pathway with the most enrichment targets,involving 12 targets and 21 active components.4.The CCK-8 experiment results showe that 20μM quercetin,cauloside A and emodin could significantly inhibit the activity of HTR-8/SVneo cells（P<0.01）,while quercitrin,atractylenolide III and tetrahydroxystilbene glucoside showed certain proliferation promoting effects（P<0.05）.The results of CCK-8 test showed that compared with LPS group,the cell viability of LPS+verbascoside group,LPS+quercetin group,LPS+emodin group and LPS+rubiadin group was inhibited（P<0.01）,while the cell viability of LPS+tetrahydroxystilbene glucoside group,LPS+pinoresinol diglucoside group,LPS+ginsenoside Rb1 group and LPS+atractylenolide III group was significantly increased（P<0.01）.Conclusion:1.The main components of Zishen Yutai pill include 14 organic acids and their derivatives、13 terpenoids、3 alkaloids、3 anthraquinones and 2 flavonoids.2.The characteristic fingerprint of Zishen Yutai pill was established.Combined with the method of simultaneous determination of multiple components,9 common peaks were calibrated and 5 common peaks were identified.The similarity of 11 batches of Zishen Yutai pill samples was not less than 0.900,and the samples of 13 batches of Zishen Yutai pill contained 592.62～816.19μg/g of sweroside,208.02～289.26μg/g of loganin and69.10～281.14μg/g of isochlorogenic acid C.3.The analysis of network pharmacology method shows that PI3K-AKT signal pathway may be the key pathway of Zishen Yutai pill in the treatment of recurrent abortion,and EGFR may be the key target.4.Tetrahydroxystilbene glucoside、pinoresinol diglucoside、ginsenoside Rb1 and atractylenolide III have protective effects on HTR-8/SVneo cells induced by LPS,which lays an experimental foundation for the subsequent validation of pharmacodynamic substances in in vivo model.