The Establishment And Application Of Targeted Phenolomics Methods

Objective:Through the previous experimental results and related literature research,we found that anti-anxiety effect is a kind of biological activity shared by natural small molecule phenols(NSMPs),and phenolic hydroxyl is an essential group.In the tyrosine and phenylalanine metabolic pathways,there is a kind of metabolite,endogenous small molecule phenols(ESMPs),which is produced by the neurotransmitters dopamine and noradrenaline.NSMPs and ESMPs are very similar in structure.We propose the hypothesis that there is a close relationship between the changes of esmps and neurodegenerative diseases,and the specific changes of esmps can indicate neurodegenerative diseases;nsmps plays a therapeutic role in neurodegenerative diseases by regulating the metabolism of ESMPs.The aim of this study is to establish the analysis method of targeted phenolomics,and then to establish the Alzheimer’s disease and anxiety model respectively,to investigate the specific changes of ESMPs and the mechanism of eugenol and ferulic acid regulating ESMPs to play the role of anti-anxiety and treatment of Alzheimer’s disease.Methods:1:A quantitative analysis method of plasma ESMPs was established based on UPLC-QqQ-MS/MS.Firstly,the appropriate ion source and scanning mode were selected,and the mass spectrum parameters such as ion channel,collision energy and cone voltage were optimized.Secondly,the chromatographic conditions were comprehensively investigated to optimize the chromatographic parameters such as chromatographic column,mobile phase type,additive type and volume fraction,elution gradient,flow rate and column temperature.Finally,the specific property,standard curve,quantitative limit and detection limit,precision,stability and accuracy were investigated to determine the analytical method of liquid chromatography mass spectrometry which can quantify ESMPs;2:Morris water maze test was used to evaluate the scopolamine induced AD model,and then the specific changes of ESMPs in AD model were investigated based on targeted phenolomics,and then the potential ESMPs that can characterize AD were screened;at the same time,the effect of ferulic acid on AD and the mechanism of regulating ESMPs on AD were investigated;3:The anxiety-like behavior model was established by using the elevated plus maze experiment,and then the specific changes of ESMPs in the anxiety model were investigated based on the targeted phenolomics,and then the ESMPs that can potentially represent anxiety disorders were screened;at the same time,the anxiolytic effect of eugenol and the anxiolytic mechanism by regulating ESMPs were investigated;4:RT-PCR and ELISA were used to investigate the gene and protein expression of ESMPs related metabolizing enzymes and the regulation of eugenol in anxiety model.Results:1:A method of UPLC-QqQ-MS/MS was established for the analysis of target phenolomics.The results of the methodology meet the requirements of guiding principles for validation of biological sample analysis methods and Chinese Pharmacopoeia(2015).The method is sensitive and reliable,and can be used for the quantitative detection of plasma ESMPs;2:The contents of THCM and HPAA in plasma of AD model mice induced by scopolamine were up-regulated,while the contents of HPPT,HBA,PHCA,SLA and DHBA were down regulated.Among them,SLA and DHBA are the key ESMPs,which can specifically characterize the pathogenesis of AD.Ferulic acid can reduce six kinds of ESMPs with abnormal metabolism to normal level,which indicates that ferulic acid has a better effect on AD from the metabolic level of ESMPs,and ferulic acid may play a role in the treatment of AD by regulating the level of ESMPs;3:The plasma levels of PHCA and DOPEGAL were up-regulated,HDPL,HPAT,HBA,DHPP,HMBH,HVA and DHPG were down regulated.Among them,PHCA,DHPG and HVA screening are the key ESMPs,which can specifically characterize the onset of anxiety.Eugenol has anxiolytic activity in a dose-dependent manner.Eugenol can reduce six kinds of ESMPs,such as PHCA,back to normal level,and improve the metabolism disorder of ESMPs in anxiety rats;4:In the anxiety model group,SIRT1,MAOA and COMT enzyme content and gene expression were significantly up-regulated,ALDH enzyme content was significantly down regulated,gene expression was significantly up regulated,AR enzyme content and gene expression were significantly down regulated;eugenol could effectively inhibit the up regulation of SIRT1,MAOA and COMT enzyme content and gene expression in the anxiety model group,callback ALDH enzyme expression and gene expression,and up regulate ar enzyme expression.Eugenol may play an anxiolytic role by regulating SIRT1-MAOA signal pathway and affecting the expression of related metabolic enzymes and genes of ESMPsConclusion:The content of ESMPs in Alzheimer’s disease and anxiety disorder changed specifically;eugenol and ferulic acid played the role of anti-anxiety and treatment of Alzheimer’s disease by regulating ESMPs.