Investigation On Therapeutic Basis Matter Of Trollius Ledebourii And Metabolites Identificaion Of Osthole Using Ultra-high-performance Liquid Chromatography Coupled To Quadrupole Time-of-flight Mass Spectrometry

Trollius ledebourii,also known as Tropaeolum majus and Ludilian,is the dried flowers of Trollius chinensis Bunge.and Trollius ledebourii Reichb.in the buttercup family and was widely distributed in Inner Mongolia and the Hebei province in northern China.Trollius ledebourii is a type of traditional Chinese medicine?TCM?that is commonly found in tea with multiple functions such as the treatment of tonsillitis,periostitis and conjunctivitis.It has a long history for treatments of aphtha,throat swelling,toothache,eye illness and damp-heat,which were initially recorded in Gang Mu Shi Yi.Trollius ledebourii has been more involved in Mongolian medicine and is often used as a type of tea for heat-clearing and detoxifying in the populus.Five different ephedrine formulations of Trollius ledebourii prescription preparations were included in the Chinese Pharmacopoeia?2015?,and orientin was set as the index component for determination.Modern pharmacological studies have shown that Trollius ledebourii possesses bioactive activities,including anti-inflammation,antioxidant,antimicrobial,antivirus and anticancer properties.The chemical constituents of Trollius ledebourii are flavonoids,phenolic acids and alkaloids.The flavonoids are the biologically active fractions.Previous studies have proved that orientin,vitexin and2''-O-?-L-galactopyranosylorientin were the major constituents with high contents and strong bioactivities in Trollius ledebourii.In this study,a rapid and sensitive method ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry?UHPLC-Q-TOF-MS/MS?was developed for the identification of the multi-constituents in Trollius ledebourii and metabolites in vitro and in vivo in rats after oral administration of orientin and vitexin,respectively.A total of 37chemical constituents in Trollius ledebourii extract were unambiguously or tentatively identified,and 12 metabolites of orientin and 23 metabolites of vitexinweredetectedprimarily.High-performanceliquid chromatography-quadrupoleiontraptandemmassspectrometry?HPLC-QTRAP-MS/MS?was applied for the simultaneous determination of11 compounds,which were either with high contents or strong bioactivities in Trollius ledebourii.The results might provide the basis for quality control analysis of Trollius ledebourii.Osthole?OST?,a natural simple coumarin,is the active component in many popular medical plants and fruits,including Cnidium monnieri?L.?Cuss.,Peucedanum ostruthium and Citrus aurantium.In recent years,pharmacological studies revealed that OST has diverse effects on anticancer,anti-apoptosis and neuroprotection.Currently,OST has attracted tremendous attention due to its insect control application on crop planting,and it has become a novel plant-based pesticide at home and abroad with the advantage of environmental protection and small toxicity.A total of 72 metabolites of OST were identified in vitro and in vivo by a novel SWATH acquisition method based on UHPLC-Q-TOF-MS/MS technology.The proposed bio-transformation routes of OST provide scientific and reliable support for safety and effect in clinical application.Part one Qualitative analysis of chemical composition of Trolliusledebourii using UHPLC-Q-TOF-MS/MSObjective:To establish a rapid qualitative method using UHPLC-Q-TOF-MS/MS for identification of constituents in Trollius ledebourii.Methods:Analysis was performed on a Poroshell 120 EC-C₁₈ column?100 mm×2.1 mm,2.7?m?eluted with 0.1%formic acid-water?A?and acetonitrile?B?in a gradient program.The mass spectrometer was operated in the positive mode with electrospray ionization?ESI?sources.Results:The results indicated that 37 compounds in the extract from Trollius ledebourii had been identified based on exact masses,retention times,chromatographic behaviors and characteristic MS/MS ions.They were 17flavonoid glycosides,6 flavones,3 flavonols,1 dihydroflavone,8 phenolic acids,1 amide and 1 triterpene.In addition,pectolinarin,naringenin,diosmetin,paeonol,ferulic acid,caffeic acid,protocatechuic acid and isorhamnetin were identified from Trollius ledebourii for the first time.Conclusions:The clear fragment patterns of the main compositions also contributed to its metabolites identification in the later subsequent.UHPLC-Q-TOF-MS/MS used for analysis of Trollius ledebourii extract will shed some new lights on the quality control of traditional Chinese herbs.Part two Quantification of 11 chemical compositions in Trolliusledebourii by HPLC-QTRAP-MS/MSObjective:To develop an HPLC-QTRAP-MS/MS method for the determination of(2''-O-?-L-galactopyranosylorientin,orientin,vanillic acid,vitexin,hyperoside,ferulic acid,luteolin,quercetin,apigenin,naringenin,and acacetin in Trollius ledebourii.Methods:Analysis was performed on a Diamonsil C₁₈ column?150mm×4.6 mm,5?m?eluted with 0.1%formic acid-water?A?and acetonitrile?B?in a gradient program.The flow rate was 0.8 mL/min,the injection volume was 10?L,and the column temperature was 30oC.The multiple-reaction monitoring scanning?MRM?was employed for the quantification with switching electrospray ion source polarity in negative mode.The ion spray voltage was set at-4500 V and the turbo spray temperature was maintained at650oC.Results:The regression equations showing linear relationships between peak areas and contents of each compound were obtained?r²>0.9987?.The average recoveries of the compounds ranged from 98.15%to 101.6%and the precision in terms of RSD was in the range from 0.28%to 1.95%.Conclusions:The method is rapid,simple and sensitive.It could be used as a quantitative determination method for the eleven components including flavonoids and phenolic acids in Trollius ledebourii simultaneous.Part three Metabolites identificaion of two bioactive constituents inTrollius ledebourii in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometryObjective:To develop a rapid method based on UHPLC-Q-TOF-MS/MS to separate and characterize metabolites in rat liver microsomes?RLMs?,rat plasma,rat bile,rat urine and rat feces after oral administration of orientin and vitexin,respectively.Methods:RLMs was applied as metabolic reaction model in vitro.Orientin and vitexin were incubated for phase?and phase?bio-transformations.Each test was set up blank group,control group and sample group with three parallel incubations.The rat plasma,bile,urine and feces samples were collected after intragastric administration of orientin and vitexin in rats,respectively.All of collected samples were analyzed by UHPLC-Q-TOF-MS/MS.Samples were separated on a Poroshell 120 EC-C₁₈ column?100 mm×2.1mm,2.7?m?,coupled with a C₁₈ pre-column?Security Guard^??.The column temperature was kept at 40oC.It was found that the peak shapes and responses of analytes were becoming better with the mobile phase for a mixture 0.1%formic acid and acetonitrile with an optimized linear gradient elution.The high solution mass spectra were obtained in the positive ESI mode.IDA criteria were provided for data acquisition.The identification of possible metabolites and the summary of metabolic pathways of orientin and vitexin were conducted by chromatographic retention times,MS spectra,MS/MS spectra and MetabolitePilot^TM 1.5 software.Results:Through the above analysis strategies,a total of 12 metabolites of orientin and 23 metabolites of vitexin were elucidated and identified,respectively.Conclusions:The metabolic pathways were summarized systematically.Oxidation,methylation,acetylation,reduction,loss of C₆H₁₀O₅ and glucuronide conjugation were the major biotransformation routes of both of them in rats.Part four SWATH acquisition mode for metabolites identification ofosthole in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometryObjective:Toestablishanovelstragetybasedon UHPLC-Q-TOF-MS/MS coupled with SWATH acquisition method for rapid metabolites identification of osthole.Methods:UHPLC-Q-TOF-MS/MS and MetabolitePilot^TM2.0 software with PCVG filtering were applied to observe and filter probable metabolites of OST firstly.The high resolution mass data were acquired by data-independent acquisition mode?DIA?,i.e.,SWATH,which could improved the hit rate of low-level and trace metabolites.A novel data processing method?KPIs?was employed for rapid hunting and identification more accurate metabolites as an assistant tool.Then,the most possible metabolites in RLMs,plasma,bile,urine and feces were identified by comparing with reference standards and analyze their MS and MS/MS spectra.Results:A total of 72 metabolites of OST were identified in vitro and in vivo,including 39 metabolites in RLMs,20 metabolites in rat plasma,32metabolites in rat bile,32 metabolites in rat urine and 37 metabolites in rat feces samples.Conclusions:OST possessed high metabolic activity in RLMs and in rats.The most amount and varieties of metabolites were detected in rat RLMs and feces.The characteristic metabolic bio-transformation routes of phase?were oxydrolysis,demethylation,3,4-epoxide and aldehylation.Glucuronide conjugation,S-cysteine conjugation,sulfate conjugation,phosphorylation and N-Acetylcysteine conjugation reactions were main metabolic pathways of phase?.This proposed bio-transformation routes of OST provide scientific and reliable support for full understanding of the metabolism of coumarins.