Anxiolytic Mechanism Study Of Gardenia Japonica Based On Metabolomics And Network Pharmacology

Objective:Anxiety disorder is a mental disease threatening human's life.Gardenia jasminoides Ellis(GJE)is a natural drug with anxiolytic effect,but its anxiolytic mechanism has not been fully elucidated,which limits the development of its application in new drugs.Therefore,aim of this study is to verify the anxiolytic effect of GJE,and then to explore the endogenous metabolites in anxious rats as well as the callback of metabolites under intervention of GJE basing on the non-targeted metabonomics technology of liquid chromatography-mass spectrometry,and to construct the metabolic regulatory pathway of GJE,and to expound the mechanism of anxiolytic effect of GJE from the metabolic level;In addition,network pharmacology was used to explore the mechanism of anxiolytic effect of GJE in the aspect of the potential constituents,targets and pathways,and to clarify the mechanism of anxiolytic effect of GJE from the molecular level.Methods:1.The male SD rats were randomly divided into control group,model group,positive drug(diazepam group,intraperitoneal injection 1.38 mg/(kg · d)for once),GJE low-dose group(intragastric administration of 53 mg/(kg·d)for seven days)and GJE high-dose group(intragastric administration of 90 mg/(kg·d)for seven days).Diazepam was the positive drug.The anxiolytic effect of GJE was verified by elevated plus maze test.2.Based on the non-target metabolomics technology of Ultra Performance Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry(UPLC-Q-TOF/MS),the plasma of rats in control group,model group,diazepam group,GJE low-dose group and GJE high-dose group were collected after the Elevated plus maze test.The differential metabolites in the plasma beteween model group compared and control group were found by multivariate statistical analysis and univariate statistical analysis,and then the relative content in medication administration groups of the above-mentioned metabolites was analyzed.At the same time,the pathway enrichment of differential metabolites was analyzed to elucidate the anxiolytic mechanism of GJE.3.The anxiolytic chemical constituents of GJE was analyzed based on UPLC-Q-TOF/MS and Masslynx software:an Waters Acquity UPLC BEH C18(100 mm × 2.1 mm,1.7 ?m)was performed for gradient elution with the mobile phase of 0.1%formic acid solution(A)and 0.1%formate acetonitrile solution(B),the optimum elution condition is:0?14 min,2%B;14.0?16.5 min,2%-60%B;16.5?18.0 min,60%-98%B;18?20.0 min,98%B,the injection volume was 5 ?l,the flow rate was 0.3 ml/min,the column temperature was 40?.The electrospray ionization(ESI)was used for sample MS data in negative ion mode,the collection range was m/z 50-1200.Chemical constituents was identificated and matched based on Masslynx software and literature report.4.PharmMapper,TCMSP and other databases were used to predict and screen the targets of GJE;OMIM,DrugBank and other databases were combined to search the anxiety targets;The potential anxiolytic targets of GJE were obtained by cross analysis.The protein-protein interaction network was constructed based on String,Cytoscape software was used for topological analysis to find the potential core targets of GJE in the treatment of anxiety disorder.Then GO and KEGG biological functions of the core targets were analyzed by String,the possible pharmacodynamic constituents,and the targets and core pathways of GJE of anxiolytic effect were searched.Results:1.The results of Elevated plus maze test showed that compared with the model group,53 mg/kg for seven days and 90 mg/kg GJE by gavage and 1.38 mg/kg diazepam by single intraperitoneal injection could increase the percentage of time and times of rats entering the open arm(P<0.05),and did not increase or reduce the total times.2.The results of non-targeted metabonomics of rat plasma based on UPLC-Q-TOF/MS showed that there were significant changes in thirty-two metabolites in model rats,including citric acid,arachidonic acid,glycine,indoxylsulfuric acid,urea,palmitic acid,stearic acid and some lipids,mainly involved in lipid metabolism,amino acid metabolism,energy metabolism.Fifteen of them were recalled by GJE low-dose and eight of them were recalled by GJE high-dose,including urea,glycine,citric acid and some lipids,it is suggested that GJE can play an anxiolytic role in metabolism by regulating tricarboxylic acid cycle,phospholipid metabolism and glycine metabolism and other metabolic reactions.3.Twenty-three constituents in the GJE were identified by UPLC-Q-TOF/MS combined with Masslynx software in negative ion mode,mainly including iridoids,crocins and monoterpenes.4.Based on network pharmacology,we found that twenty-one potential active constituents in GJE may act on forty-six targets related to anxiety,such as SLC6A3,CNR1,MAOB,MAO A,GABA,ERBB2,involving catecholamine binding,regulation of amine transport,neuroactive ligand-receptor interaction,5-hydroxytryptaminergic synaptic pathway,cholinergic synapse,etc,which may by affecting the release of 5-HT,DA and other neurotransmitters,intervening in oxidative stress,alleviating neuron damage and other ways to play the anxiolytic effect.Conclusion:The results showed that GJE by gavage for seven days continuously(53 mg/kg,90 mg/kg)had anxiolytic effect on SD male rats;Metabonomics results show that GJE can interfere with tricarboxylic acid cycle,phospholipid metabolism and glycine metabolism and other metabolisms;Network pharmacology results show that the iridoids and crocins in GJE can affect multiple pathways through multiple targets to coordinate anxiety.This study laid a foundation for the study of elucidating pharmacodynamic mechanism and the development and utilization of GJE.