Study On The Compatibility Mechanism Of Anemarrhena Asphodeloides And Fritillariae Cirrhosae Based On Process In Vivo And Metabonomics

The compatibility of Anemarrhena asphodeloides(ZM)and Fritillariae cirrhosae(CBM)has a long history of application in China.They are mostly used for lung heat cough,lung dryness cough,and also for cough with water deficiency and fire excess.It was first collected in the famous ancient traditional Chinese medicine prescriptions such as Jingyue Quanshu and Jijiu Xianfang.At present,the research on the compatibility of the two herbs is limited to the study of pharmacological effects and preliminary pharmacokinetics,and there are no reports on the changes of chemical components in vivo and in vitro and the mechanism of anti asthma after the compatibility of them.The material basis and pharmacological mechanism of the compatibility of ZM and CBM need to be systematically studied.Objective: Using UPLC-QTOF-MS technology,focusing on the analysis of in vitro and in vivo chemical components,we aimed to study the changes of in vitro chemical components after compatibility and the mutual influence of the absorption of various compatible components in the normal rat model.Then,combined with network pharmacology,metabonomics,pharmacodynamics research methods,this paper makes an overall study on the anti asthma mechanism of ZM and CBM.The compatibility mechanism of drug pair was systematically studied from in vitro and in vivo chemical composition,efficacy and mechanism.Finally,combined with metabolic profile,network pharmacology,molecular docking technologies,the potential anti asthma mechanism of peimine was studied.Methods:1.The metabolic fingerprints of ZM and CBM were established by UPLC-QTOF-MS technology,and the chemical components of ZM and CBM were identified and analyzed by metabolic fingerprints.Combined with plant metabolomics technology and quality background deduction method,the contents of chemical components of single decoction of ZM,CBM and combined decoction of ZM-CBM drug pair were analyzed and compared,and the changes of chemical components of ZM and CBM were studied.2.UPLC-QTOF-MS technique was used to establish the identification and analysis method of chemical components of ZM and CBM absorbed in normal rat plasma.Further,Peak View software was used to extract the peak area of absorbed components,combined with multivariate statistical analysis technology,the peak areas of components absorbed in blood before and after the compatibility ZM or CBM were compared and analyzed to find out the different components and content change trend before and after the compatibility.3.Firstly,the active components and targets of ZM and CBM were screenned through the database.By establishing the “component-diseasetarget network” of ZM and CBM,the possible anti-asthma mechanism of ZM and CBM was explained.The compatibility mechanism was explored by comparing the changes of anti-asthma mechanism before and after compatibility.4.A rat asthma model sensitized by ovalbumin was established.Pharmacodynamic indexes were used to investigate the anti asthmatic effect of ZM,CBM and ZM-CBM drug pair extract.Combined with UPLC-QTOF-MS technology,the metabolomics of plasma samples was studied to investigate the differences of plasma metabolic profiles between model rats and control rats,as well as the changes of plasma profiles of model rats after administration of ZM,CBM and ZM-CBM drug pair.The pathway analysis of different metabolites was carried out to evaluate the effectiveness of drug pair compatibility from the perspectives of metabolomics and pharmacodynamics,and to explore the relevant mechanism of drug pair compatibility in the treatment of asthma.5.Using UPLC-QTOF-MS technology,combined with software and molecular network analysis,the metabolic profiles of peimine in different liver microsomes(human liver microsomes,dog liver microsomes,rat liver microsomes and mouse liver microsomes)were studied.Secondly,the potential anti-asthmatic mechanism of peimine and its metabolites and the changes of efficacy of peimine after metabolism were analyzed by network pharmacology.Finally,molecular docking was used to study the interaction between peimine and its metabolites and the core targets of asthma.Results: 1.A total of 138 chemical components were identified based on UPLC-QTOF-MS technology,including 67 components in CBM and 71 components in ZM.By multivariate statistical analysis and quality background deduction method,55 different components with significant changes before and after compatibility were selected,including 21 belonging to ZM and 34 belonging to CBM.Based on heat map analysis,the content of 14 components in ZM increased after compatibility,such as diosgenin,timosaponin A Ⅲ,gitogenin,sarsapogenin,neomangiferi,quercetin,while the content of 5 components decreased after compatibility,such as mangiferin and timosaponin J.The contents of 34 components of CBM decreased,such as imperialine,peimine,isoverticine,peimisine,adenine.The results showed that the compatibility of ZM and CBM changed the dissolution of many components.2.Based on UPLC-QTOF-MS technology,85 absorbed components were identified,including 44 components in CBM and 41 components in ZM.Peak area of absorbed components were extracted by Peak View software.Through multivariate statistical analysis,10 components with significant changes before and after compatibility were selected,including 4 components of ZM and 6 components of CBM.The study on the overall difference of body components before and after the compatibility of ZM and CBM showed that the compatibility could significantly reduce the plasma contents of timosaponin A3,anemarsaponin B＿qt,timosaponin A1,mangiferin in ZM and peimine,verticine N-oxide,imperial N-oxide and pingbeinine B in CBM.It also significantly increased the plasma contents of isoverticine and zhebeinone in CBM.After deducting the effect of dissolution change in vitro,the compatibility promoted the absorption of components of CBM and inhibited the absorption of components of ZM.3.There are 16anti-asthma components,164 anti-asthma targets and 20 regulatory pathwaysin ZM;CBM has 12 anti-asthma components,78 anti-asthma targets and 19 regulatory pathways.The results of PPI network showed that ZM-CBM drug pair played an anti asthmatic effect by core targets such as HIF1 A,CASP3,Jun,TNF,EGFR,VEGFA,STAT3,PTGS2,MMP9 and Src through a variety of active components.It involved multiple pathways,mainly including calcium signaling pathway,Th17 cell differentiation pathway,PI3K-Akt signaling pathway,c GMP-PKG signaling pathway and arachidonic acid metabolism pathway.The results showed that there were 16 common pathways between ZM and drug pair,7 common pathways between CBM and drug pair,and 3 unique pathways only for drug pair.It showed that the anti asthmatic effect of ZM-CBM drug pairwas the synergistic effect of ZM and CBM,and the mechanism was more similar to that of ZM.At the same time,a new action pathway was produced after compatibility.4.The results of anti asthmatic efficacy evaluation showed that ZM,CBM and ZM-CBM drug pair had obvious anti asthmatic effects.They could effectively reduce the airway resistance of asthmatic model rats,significantly improve the pathological changes of asthmatic model rats.Among them,ZM-CBM drug pair had synergistic anti asthmatic effects.The results of plasma metabonomics showed that there were 56 metabolites and 4 metabolic pathways in ZM treatment group.There were 52 metabolites and 4 metabolic pathways in CBM treatment group.There were 62 metabolites and 5 metabolic pathways after adjustment of drug pair.After adjustment of drug pair,the number of metabolites and metabolic pathways was higher than that of single use.Metabolic pathway analysis showed that,α-linolenic acid metabolism,linolenic acid metabolism,unsaturated fat biosynthesis,glycerol phospholipid metabolism and arachidonic acid metabolism are the main regulatory pathways of ZM-CBM drug pair against asthma.The results of correlation analysis between plasma metabolomics and network pharmacology showed that the regulation of arachidonic acid metabolism are the main mechanisms of the compatibility of ZM and CBM to enhance anti asthma.5.The results of in vitro liver microsomes experiment showed that a total of 19 one-phase metabolites were detected(17 in human liver microsomes,11 in dog liver microsomes,13 in rat liver microsomes and 9 in mouse liver microsomes).Peimine was metabolized in multiple ways,mainly including deoxygenation,oxidation,dehydrogenation,ketogenesis,dehydration,demethylation and methylation metabolism.The results of PCA and cluster analysis showed that the metabolic profile of human liver microsomes was significantly different from that of the other three species.Compared with mice and dogs,the metabolic characteristics of rats were closer to that of humans.The results of network pharmacology showed that a total of 266 component targets were obtained.The targets regulated by metabolite were 190 more than that by peimine.298 asthma targets were obtained by databases,and 49 common targets were obtained after intersection.The results of PPI network showed that peimineand its metabolitesacted synergistically on core targets such as ADRB2,SLC6A4,HSP90AA1,CHRNA7,ADRA1 B,CHRM1,SLC6A3 and SLC6A2.Cholinergic synaptic pathway,smooth muscle contraction pathway and PI3 K Akt signal pathway were the main metabolic pathways of peimine.Molecular docking results showed that there was a strong interaction between peimine and its metabolites and ADRB2,and hydrogen bond was the main form of interaction.Conclusion: Through the study of chemical composition in vivo and in vitro,efficacy and anti-asthma mechanism,the material basis and anti-asthma mechanism of compatibility and synergistic effect of ZM-CBM were clarified.By the perspective of chemical component dissolution,it was found that the material basis of its synergistic effect was related to the overall superposition of the content changes of various effective components.By the perspective of blood components in vivo,the material basis of the compatibility of ZM and CBM was that the compatibility promoted the absorption of main prototype components in CBM in vivo.Pharmacodynamic studies showed that ZM,CBM and ZM-CBM drug pair all improved the symptoms of asthmatic model rats to a certain extent,among which the drug pair had the most obvious effect.Network pharmacology and plasma metabolomics studies showed that ZM,CBM and ZM-CBM drug pair improved the metabolic disorder of asthma model rats to a certain extent,and their intervention effects were related to linolenic acid metabolism,arachidonic acid metabolism α-Linolenic acid metabolism and glutathione metabolism.Among them,the drug pair had the most effect on regulating differential metabolites and metabolic pathways,indicating that the combination of ZM and CBM has better effect than that of the same dose of ZM and CBM alone,which confirms the rationality and advantage of the compatibility of ZM and CBM.This study has clarified the action mechanism and scientific connotation of ZM-CBM and provided valuable reference examples and methodological reference for the systematic research of traditional Chinese medicine.