Qualitative Analysis And In Vivo Metabolic Profile Study Of Lagotis Brachystachys Maxim Based On UPLC-Q-TOF-MS Technology

Objection:To establish HPLC fingerprints of 17 batches of Lagotis brachystachys,compare the chromatograms of different producing areas,and analyze the total extract of Lagotis brachystachys in negative ion mode combined with UPLC-Q-TOF-MS technology The purpose of the research is to provide a reference for the quality control of the medicinal materials of Lagotis brachystachys;UPLC-Q-TOF-MS technology was used to identify the absorbed components and some metabolites of the total extract of Lagotis brachystachys after oral administration in negative ion mode in rats.To provide a theoretical basis for probing the medicinal material basis of Lagotis brachystachys;to study the effects of plantamajoside,tricin,and chrysoeriol on the inflammation model induced by sodium urate(MSU)in RAW264.7 macrophages.Methods:(1)Using InertSustain C18 chromatographic column(4.6mm x 250mm,5μm),the mobile phase is acetonitrile-0.1%formic acid water gradient elution;flow rate 1mL·min-1;column temperature 30℃;detection wavelength is 256mm,The injection volume is 10μL.The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2012 Edition)" was used to calculate the similarity,and the SPSS statistical software(version 21.0)was used to cluster the data and compare the HPLC chromatograms of different origins.(2)Aglient ZORBAX Eclipse Plus C18 column(2.1x100mm,1.8μm);the mobile phase is 0.1%formic acid water(A)-acetonitrile(B solution).Negative particle mode:gradient elution 0～48min,10%～35%B;48～55min,35%～95%B.The flow rate is 0.25 mL/min,and the injection volume is 2 μL.In the negative ion mode,the PeakView software is used to fit the elemental composition of the mass spectrum information,combined with the comparison of the reference substance,the mass spectrum parameters and retention time of the compounds in the literature,etc.,to analyze the structure information of the compounds in Lagotis brachystachys.(3)Rats were orally administered at a dose of 12g/kg of crude drug,and blood,urine,and feces were collected at different time points within 24 hours after administration.UPLC-Q-TOF-MS technology was used to identify these collected biological samples and analyze the short-ear rabbit ear grassland components and some metabolites.(4)Monosodium urate crystals were used to stimulate mouse RAW264.7 macrophages to establish an inflammation model.The SRB method was used to detect the effects of plantamajoside,tricin,chrysoeriol,and colchicine on the viability of RAW264.7 cells.After modeling,the tumor necrosis factor-α(TNF-α)was detected,and it was detected by Western-Blot.Protein expression of TOLL-like receptors(TLR4,TLR2),Caspase-1(Caspase-1),NOD-like receptor protein 3(NLRP3),and nuclear transcription factor-kB(NF-κB)Level.Results:(1)Established the HPLC fingerprint of Lagotis brachystachys,identified 20 common peaks,and identified 10 chemical components,namely chrysoeriol,psyllium,tricin,avidin,plantamajoside,Verbascoside,Luteolin,Luteolin,8-epi-loganic acid,Triticein 7-O-β-D-glucopyranoside.The similarity between the fingerprints of 17 batches of Lagotis brachystachys and the control map was 0.644～0.977,and the cluster analysis could be divided into 5 categories.(2)Through the analysis of UPLC-ESI-MS mass spectrometry data by PeakView software,combined with literature data,a total of 72 components are identified,including 35 flavonoids,19 phenethyl alcohol glycosides,iridoid glycosides and saponins 9 each.(3)A total of 37 prototype compounds were identified from rat blood,urine,and feces,including 14 flavonoids,15 phenethanol glycosides,and 8 saponins.Using luteolin,tricin and plantamajoside as targets,a total of 43 metabolites were identified from rat blood,urine,and feces,including 23 metabolites of luteolin and 14 metabolites of tricin.There are 6 metabolites of plantamajoside.(4)Through cell experiments,it was found that the low and high dose of plantamajoside group,the low and high dose group of tricinin,the low and high dose group of coylicin significantly reduced the level of TNF-α;the low and high dose of plantamajoside group,The low and high-dose groups of tricin,the low and high-dose coylidin groups significantly reduced the TLR4 protein level,the low and high-dose coylidin groups significantly reduced the TLR2 protein level,the low and high-dose psyllium group,the high-dose coylidin,and the high dose of tricin Caspase-1 protein levels were significantly reduced in the high-dose psyllium group,the low-dose and high-dose coylidin group,and the high-dose coylidin group,the high-dose coylivine group,and the high-dose coylidin group.The NF-κB protein level was significantly reduced in the low-dose and high-dose coylidin group.Conclusion:The results of this study provided the differences in the 20 common peaks of 17 different batches of Lagomorpha brevisiae,which provided a certain reference for the follow-up quality control research of Lagotis brachystachys.The qualitative analysis of the chemical components of the total extract of Lagotis brachystachys provides a reference for elucidating the material basis of its medicinal effect and a theoretical basis for its quality control.This article also reported for the first time the study of the in vivo metabolism of Lagotis brachystachys extract,which provides a certain reference and basis for the pharmacokinetics and mechanism of pharmacodynamics of Lagotis brachystachys.Through cell experiments on the RAW264.7 macrophage inflammation model induced by sodium urate crystals,the anti-inflammatory activities of plantamaj oside,tricin and chrysoeriol and their mechanism of action have been explored.The three compounds belong to Lagotis brachystachysthe prototype components absorbed by the extract in rats provide a further theoretical basis for the material basis of its medicinal effect and medicinal action mechanism of Lagotis brachystachys.