Screening The Effective Components Of Suanzaoren Decoction On The Treatment Of Chronic Restraint Stress Induced Anxiety-Like Mice

RATIONALES:Suanzaoren Decoction（SZRD）,listed in“Synopsis of the golden chamber”compiled by Zhong Jing Zhang of the Han Dynasty,was a classical Chinese medicine prescription.It was composed of Ziziphi Spinosae Semen,Poria,Anemarrhenae Rhizoma,Chuanxiong Rhizoma and Glycyrrhizae Radix et Rhizoma.The classical application of SZRD was nourishing blood,calming the mind,clearing heat and eliminating irritability.The original intention of its formulation was to alleviate the sleep disorders caused by people’s displacement and anxiety.At present,there are many commercially available preparations of SZRD such as tablets,granules,concentrated granules,which were usually used for treating deficiency,disturbance,sleepless,palpitation,night sweat,dizziness and vertigo.Clinical studies have reported that SZRD and its additive formula had been used to treat anxiety.Anxiety referred to excessive and unrealistic worry and is inconsistent with the actual situation and interferes with daily functioning.Anxiety patients often exhibited sleep disorders due to continuous tension,fear and anxiety.Epidemiologic surveys show that 40%of anxious patients present with insomnia first,and 72%have insomnia after or at the same time as an anxiety disorder.It has become an urgent problem to be solved that find the alternative drugs with safe and effective.The chemical composition of traditional Chinese prescription was complex,but also difficult to understand the effect of TCM compounds on the body and then change its biochemical reaction.It was well known that TCM exerted its therapeutic effect through influencing the physiological function via the chemical components.Taking SZRD as example,in vitro and in vivo studies combined with network pharmacology were utilized in the present study.OBJECTIVE:To clarify the effective components of SZRD in improving anxiety and insomnia,and to explore the key pathway and target of SZRD in treating CRS anxiety model.METHOD&CONTENT:1.Study on the chemical material basis of SZRD.A method for characterization and analysis of chemical constituents of SZRD was established based on UPLC-Q-TOF-MS/MS.A quality evaluation method of SZRD combined with HPLC-DAD-ELSD fingerprint and multi-component determination was establish.The raw materials from different origin and different sources were collected to prepare 10 batches of SZRD,and different dosage forms of SZRD from mainland China,Hong Kong,Taiwan and Japan were collected.The established method was used to evaluate the fingerprint similarity and determine the content of 13 components of 10 batches of SZRD and 10 batches of commercially available products with different dosage forms.2.Pharmacodynamic study on SZRD treating anxiety.The chronic restraint stress induced anxiety model was used to evaluate the effectiveness of SZRD on the prevention and treatment of anxiety from the whole animal level.With diazepam as positive control,SZRD（7,14 g/kg）was used to intervene CRS model mice.Behavioral evaluation was performed by elevated cross maze test,pentobarbital induced sleep test,open field test,horizontal cross wire test,etc.The levels of neurotransmitters such as serotonin,norepinephrine and gamma-aminobutyric acid in hippocampus and corticosterone and corticotropin-releasing hormone in serum were determined.The histopathological analysis of hippocampal tissue was performed.3.To identify the potential effective components from SZRD.A method of serum metabolomics based on UPLC-Q-TOF-MS/MS was established using the chinmedomics strategy.Multivariate statistical analysis was used,including supervised partial least squares discriminant analysis and student t-test,to screen the difference metabolites between the blank group and the model group,and unsupervised principal component analysis and relative distance method were used for metabolic profile analysis.KEGG pathway was analyzed by the relative abundance of CRS biomarkers.At the same time,serum chemical analysis was carried out to identify the prototype components into the blood.Spearman correlation analysis was used to analyze the correlation between serum transitional components and biomarkers,and the absolute correlation coefficient was greater than 0.55as the indicator to screen the potential pharmacodynamic components.4.Using network pharmacology to verify pharmacodynamic ingredients and predict targets.SWISS target prediction,TCMIP,TCMSP and Genecards databases were used to predict the components,anxiety and insomnia targets of Suanzaoren Decoction,and STRING database was used to calculate the interaction strength of proteins at intersection targets.KEGG pathway enrichment was performed on the selected key targets using DAVID database,and a"prescription-components-targets-pathways"network was established after the key pathways were focused,and the key targets were screened by combining with the key pathways focused by metabolomics,so as to provide a potential way for Suanzaoren Decoction to improve anxiety and insomnia.RESULTS:The average yield of 10 batches of SZRD’s dry extract was 21.57%.Five and four common peaks were calibrated in HPLC-DAD fingerprint at 282 nm and 335 nm,with their similarity ranged from 0.907 to 0.999,from 0.926 to 0.999,respectively.Four common peaks were calibrated in HPLC-ELSD fingerprint,and the similarity ranged from0.946 to 0.999.All the calibration curves showed good linearity with correlation coefficients（r）no less than 0.9959,the average recovery was 93.69%,and the average RSD was 2.04%.The content of neomangiferin,coclaurine,mangiferin,isomangiferin,liquiritin,spinosin,ferulic acid,6′′′-feruloylspinosin,isoliquiritin,timosaponin BII,jujuboside A,jujuboside B and glycyrrhizic acid in 10 SZRD commercial products in different dosage forms were 0.30-3.38,0.02-0.20,0.27-6.04,0.33-1.38,1.54-12.02,0.16-0.87,0.16-1.27,0.04-0.35,0.25-3.12,3.66-20.92,0.36-0.98,0.32-0.89,4.90-36.37 mg/g,respectively.2.SZRD possessed anxiolytic effect at 7 g/kg by increasing the percentages of entries open arms entries and time spent in open arms,and improving hippocampus 5-HT,GABA and NE levels,as well as decreasing serum CORT and CRH level.SZRD also exerted sedative effect at 14 g/kg by prolonging sleeping time and decreasing sleep latency with no muscle relaxation effect.A total of 110 compounds of SZRD were identified and 20 of them were absorbed in blood.21 serum biomarkers were identified after intervention of SZRD,which were involved into arachidonic acid metabolism,tryptophan metabolism,sphingolipid metabolism and linoleic acid metabolism.3.By analyzing the correlation of the identified biomarkers and blood components,11effective components closely associated with arachidonic acid metabolism related to IL-1β,IL-6,TNF and tryptophan metabolism related to HTR_1A,HTR_2A,HTR_2C,HTR_3A,MAO_A,MAO_B,SLC6A4 were extracted.4.The network pharmacological results showed that 435 potential targets related to effective components of Suanzaoren Decoction and 397 potential targets related to anxiety and insomnia,and 67 common targets were obtained.32 targets with strong protein-protein interaction（degree greater than average）were selected for pathway enrichment,and the first51 key pathways such as TNF signaling pathway and tryptophan biosynthesis were selected.The“prescription-serum immigrant components-targets-pathways”network was constructed,including 11 components,32 targets and 51 pathways.Molecular docking results showed that the binding energy of the 8 components with 4 proteins(TNF,HTR_1A,MAO_B,SLC6A4)were all less than-6 mmol/kcal,and the binding energy of Jujubogenin sarsaponin with 4 proteins（except TNF and sarsaponin）were also less than-6 mmol/kcal.CONCLUSION:The results indicated that the new approach was applicable in the routine analysis and quality control of SZRD products.The study might provide a basis for quality control of SZRD,and further pharmacokinetic study of SZRD in vivo.The current study proved that the integration of chinmedomics and network pharmacology,was a powerful approach to investigate the effective components and therapeutic mechanisms of SZRD,and provided a solid basis for quality marker（Q-marker）of SZRD.