Study On The Pharmacokinetics And Metabolomics Of Aβ-induced AD Model Rat Of Valeriana Amuremsis

As the most common neurodegenerative disease,the incidence rate of Alzheimer’s disease(AD)has been increasing in recent years,which has brought great burden on the mental and economic of the patients.Therefore,the research and development of new antiAD drugs are paid attention in the medical field.Valeriana amurensis,a perennial herb,has been taxonomically placed in the genus Valeriana which belongs to the family of Valerianaceae.In previously,the chemistry and pharmacodynamics of were systematically studied,which led the treatment of V.amurensis on AD was discovered for the first time.And the effective fraction of V.amurensis was determined as 50%ethanol eluted fraction from macroporous resin chromatography.Further studies showed that monoepoxy lignans and bisepoxy lignans were the neuroprotective basis of V.amurensis,and the later was more effective than the former.On the basis of previous work,In this study,we would continue carrying out study on V.amurensis as following:1.Simultaneous analysis 9 lignans from V.amurensis by using of UPLC-MS/MS.Established a quantitative analysis on eight bisepoxy lignans and a monoepoxy lignan from V.amurensi simultaneous by using of ultra high performance liquid chromatography with tandem mass spectrometry(UPLC-MS/MS).Using CSH Fluoro-Phenyl(150 mm×2.1 mm,1.7 μm)column was used,acetonitrile-water as mobile phase with the flow rate at 0.2 mL/min.ESI source,negative scan,multiple reaction monitoring mode(MRM),the injection source voltage:-4500 V,ion source temperature:550℃,Atomizing gas(Gas1):55 psi,heated gas(Gas2):55 psi,and air curtain air:10 psi as the MS conditions.As a result,9 constituents showed good linear relationship at the measurement concentrations,instrument precision RSD<4.91%and the average recoveries ranged from 95.91 to 102.04%.The analyzed method had been used for quantitative analysising 9 analytes from 10 batches of Valeriana amurensis.The level of 9 analytes in Valeriana amurensis were decreasing with storage time extending and the level of 9 analytes from wild V.amurensis were higher than cultivated.The analyte of(-)-massoniresinol 3α-O-β-D-glucopyranoside was not discovered in cultivated V.amurensis,while the level of analyte(+)-pinoresinol-4,4’-di-O-β-D-glucopyranoside(PDG)were the highest in Valeriana amurensis.2.V.amurensis extract on plasma pharmacokinetics in rats.According to the pre experimental results,a total of 9 lignan constituents in the plasma of rats can be detected 4.Build a method to determine the content of(+)-8-hydroxypinoresinol4’-O-β-D-glucopyranoside(HG),prinsepiol-4-O-β-D-glucopyranoside(PG),(+)-pinoresinol-,4’-di-Oβ-D-glucopyranoside(PDG),and(-)-massoniresinol 3α-O-β-D-glucopyranoside(MG)in rat plasma after administration V.amurensis extraction.Analyte and ethyl 4-hydroxybenzoate(IS)were used Waters ACQUITY UPLC HSS T3(100 mm×2.1 mm,1.7μm)column to separate,acetonitrile-water gradient,flow rate of 0.3 mL/min,and analysis time 10 min.ESI source in negative ion mode scan MRM.Linear correlations coefficient of 4 analytes are R>0.99,day and intra-day precision less than 13.85%accuracy between-2.20%-12.30%in rat plasma extraction recovery was more than 81.10%.The method of pharmacokinetic studies on HG,PG,PDG,MG have been established for oral administration in rats.Their peak plasma concentration were 27.33,33.54,9.85,24.95 ng/mL,while Tmax is between 0.58-0.67h,T1/2 is between 2.13-3.93 h.3.Study on the distribution of V.amurensis extract in rat tissues.Investigation four lignan constituents on HG,PG,PDG,MG distribution in the rat tissues and the drug concentration variety of time dynamic law in organization.Select 0.5,1,1.5,2 and 2.5 h five time points after oral administration in rats,and select heart,liver,spleen,lung,kidney,brain tissue.Using UPLC-MS/MS analysis method identification of the four main organs lignan ingredients change in V.amurensis extraction in rats in vivo.The results showed that after administration,4 components in the liver,spleen,lung tissue concentrations were higher,and in the renal tissue of low concentration,the concentration in the heart is in after intragastric administration of 2 h began to increase and is subsequently reduced,and four components which can pass through the blood brain barrier.4.Effect on V.amurensis extraction and lignan constituents on CYP450 enzyme activity.Evaluate the effect on V.amurensis extract and nine lignans on human liver microsomes,six kinds of isoforms CYP2A6,CYP2B6,CYP2C9,CYP2C19,CYP2D6,CYP3A4 activity.Coumarin,butylamino propiophenone,tolbutamide,omeprazole,dextromethorphan,and testosterone were used as CYP2A6,CYP2B6,CYP2C9,CYP2C19,CYP2D6,CYP3A4 probe substrate,and 7-hydroxycoumarin,bupropion hydroxybutyrate,4-hydroxy tolbutamide,5hydroxy-omeprazole which are the specific metabolite of demethylated and hydroxylated were used as an activity analysis indicators,and establish Cocktail probe substrate human liver microsomes in vitro model.The results showed that extraction of V.amurensis had different degrees of inhibition on four subtypes:CYP2B6,CYP2C9,CYP2D6 and CYP3 A4 enzymes.The IC50 values were 87.49,1.73,68.29,2.80 μg/mL.In 9 lignans components,MG has moderate inhibition on CYP2A6 with IC50 value 8.51μM;DG has moderate inhibition on CYP2D6 with IC50 value 8.73μM;MDG had moderate inhibition on CYP2B6 and CYP2C9 with IC50 values 5.41μM and 8.20μM,respectively.Thus this indicate that drug interactions in clinical applications should be paid attention to V.amurensis extraction,and predict that other ingredients can also inhibit CYP450 enzymes in V.amurensis extraction,in addition to the nine lignans in this study.5.Plasma Metabolomics study on effective part of V.amurensis in rat models of Alzheimer disease.Discuss the change of endogenous metabolites in the AD model rats after giving V.amurensis extraction and the intervention of metabolic pathways in AD model rats.In this chapter,AD model was established by Aβ1-42 bilaterally injected into hippocampal of rats.After drug intervention,observe the effect on V.amurensis extraction on rat hippocampal morphology by pathological observation.The technology of ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)method were used for the detection combined with Principle component analysis(PCA),orthogonal partial least squares to latent structures discriminate analysis(OPLS-DA),56 metabolite ions were considered as potential biomarkers in plasma,which include the positive ion mode 48,the negative ion mode 8.These biomarkers included glycerol,sphingomyelin,fatty acids,diglycerides and triglycerides,and metabolic pathways involved in phospholipid metabolism,fatty acid metabolism.The results showed that after high dose group therapy,plasma metabolite showed significant pullback trend,V.amurensis middle-dose and low-dose group have a callback trend to the sham group,and inference,the initial inference drawn:the therapeutic effect of V.amurensis extract on Alzheimer’s disease may play a role through the influence of glycerol phospholipid metabolism,sphingomyelin and fatty acid metabolism of pathway.In summary,the medicine of V.amurensis extract pharmacokinetic and metabolic studies of systematic study,for V.amurensis in the treatment of Alzheimer’s drug development provide the previous research and theoretical basis.