| Multiple species from the Panax genus have exhibited tonifying effects to human health,of which Panax ginseng C.A.Meyer?PG?,P.quinquefolius L.?PQ?,and P.notoginseng?Burk.?F.H.Chen?PN?,are the most reputable thereof.The roots,stems,leaves,and flower buds of three Panax species,can be used as the raw drug materials or as the sources of healthcare products,which are receiving wide consumption.The results of phytochemistry studies showed that,the aboveground and underground parts of three species are brimful with ginsenosides,which are the main bioactive ingredients.Compared with the root part,researches in respect of the flower buds of these three species,are extremely rare at present.Three flower buds of Panax species have distinguishing features in appearance,however,it is impossible to differentiate which raw materials are used,owing to the destruction of appearance and microscopic features when the extracts are prepared from the raw materials.While they have saponins in abundance,nevertheless few studies currently are available regarding the saponin composition of three flower buds of Panax species,especially the divergence between them.The establishment of"Identification Markers"of flower buds of P.ginseng?PGF?,P.quinquefolius?PQF?and P.notoginseng?PNF?separately,by systematic analysis,is conducive to the accurate identification of Panax-derived TCMs.As a core research task for the General Program of National Natural Science Foundation of China?“Monomethod Characterization of Structure Analogs-Based and Multi-technology Integrated New Mode of TCM Quality Standard Investigation”?and National Key R&D Plan-TCM Modernization Research Project?“Identification of Active Ingredients of Ginseng and Research of Group-Effect Relationship”?,this thesis,using PGF,PQF and PNF as the research objects,integrated two high-resolution LC/MS platforms,to establish the off-line three-dimensional liquid chromatography/quadrupole-Orbitrap mass spectrometry?3D-LC/Q-Orbitrap-MS?and ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry?UHPLC/IM-QTOF-MS?approaches,aiming to explicate the material basis systematically,to discover novel saponin structures,and to search for the different components among the three flower buds via a comprehensive metabolome differential analysis,with markers suitable for the accurate identification.Off-line three-dimensional liquid chromatography was established,for the first time,integrated with high-resolution mass spectrometry enhanced data dependent acquisition?DDA?technology,to separate,characterize and identify the saponins in the PGF,PQF and PNF as much as possible,and as a result,a total of 1754 ginsenosides were identified or tentatively characterized from three flower samples,among which 553 saponins were identified from PGF,596 saponins from PQF,and 605 saponins from PNF,respectively,realizing the in-depth characterization of complex saponins.Moreover,a chromatographic separation system with powerful separation profit based on strong anion exchange chromatography-hydrophilic interaction chromatography-reversed phase ultra-high performance liquid chromatography?SAXIEC-HILIC-RPUHPLC?was established to achieve classification and in-depth characterization of acidic and neutral saponins,by screening extensively chromatographic column;An ion exchange chromatography system using a Pheno Sphere SAX column with methanol/10 m M ammonium acetate-0.1%formic acid as the mobile phase,which is capable of separating neutral saponins from acidic saponins;while using Water XBridge Amide column as HILIC and acetonitrile/0.1%formic acid as mobile phase to achieve saponin separation according to polarity difference?from low to high sugar content?.Separation of saponins in consideration of distribution difference by reversed phase ultra-high performance liquid chromatography using a Waters BEH Shield RP18 column and acetonitrile/0.1%formic acid as mobile phase,was achieved.The establishment of an improved DDA acquisition method,in view of Q-Orbitrap high-resolution mass spectrometer,which contained a list of the precursor ions,fulfilled sensitive detection of target saponins in segmented samples and automatic collection of unknown mass component fragment information simultaneously.On the basis of an in-house ginsenoside database?recording 499 known ginsenosides,corresponding to 169different mass numbers?,the negative ion mode[M–H]–mass-to-charge ratio was calculated,in addition,the integer and decimal parts were double filtered by 1000 times?mass loss:fixed variation range±10 m Da?,as a result,106,102 and 94 target masses of PGF,PQF and PNF,from the negative full scan data,which were screened to construct precursor ion list respectively.A data collection method including the list of precursor ions,Full MS/dd-MS2?with"if idle pick others"and dynamic exclusion function turned on?was established on UHPLC/Q-Orbitrap-MS,36 segmented samples of three kinds of flower buds separated and prepared were analyzed at the same time by IEC-HILIC.It is shown that the establishment of 3D-LC-MS method has excellent precision?RSD<5%?and reproducibility?RSD<10%?,by means of the methodological verification.Reference compounds comparison?74 samples?,identification and derivation of saponin structure derived from processes of high-resolution MS data element composition analysis,cleavage path analysis,in-house database retrieval and comparison of retention behavior.Consequently,553,596,and 605 saponins,in this study,were identified or tentatively characterized from PGF,PQF and PNF,containing a large number of structurally novel saponins..Compared with the traditional one-dimensional liquid chromatography-mass spectrometry method,it is able to increase the number of identifiable components by approximately 6 times through three-dimensional liquid chromatography-mass spectrometry,indicating that it has great advantage in the research of Chinese medicine material basis systems.On the one hand,this thesis,on the basis of systematic material basis research,established an untargeted metabolomics method,which was founded on UHPLC/IM-QTOF-MS,characterized and compared the differences of saponins among PGF,PQF and PNF by mass sample analysis,and meanwhile,identified 32 potential difference components,and constructed"Identification Markers"of three kinds flower buds.On the other hand,in this paper,a method for characterizing ginsenosides was established,which relied on optimizing key MS parameters?Capillary Voltage:–1.0 k V;Cone Voltage:20 V;Collision Energy Ramp:80–120e V?,reversed-phase chromatography?Column:Waters BEH Shield RP18;mobile phase:acetonitrile-0.1%formic acid/water-0.1%formic acid?and negative ion model data-independent HDMSE,achieved on the Waters VionTMIMS-QTOF high resolution LC-MS instrument.Here,a untargeted metabolomics analysis workflow was demonstrated:1)Metabolomics profile analysis of 42 batches of PGF,PQF and PNF samples was performed by UHPLC/IM-QTOF-HDMSE;2)UNIFI?data correction?and Progenesis QI software?Deconvolution:Peak Alignment and Peak Extraction?are used to automatically process multiple batches of data,which generated a list of metabolic characteristics including t R,m/z,and normalized peak area and collision cross section-CCS;3)Filtering the obtained data according to the"80%rule"and"30%variation"principles;4)Using SIMCA-P software to further perform multivariate statistical analysis to reveal the potential difference components;5)Verifying and identifying the structure of the differential saponin through Target MS/MS.Furthermore,OPLS-DA modeling analysis was performed on 1506 ions after filtering,and VIP threshold 3.0 was set to discover and identify 32 potential differential components.Among them,ginsenoside Rb3?M1?,Ra1?M2?,m-Rc/m-Rb2/m-Rb3 isomer?M3?,Ra1/Ra2 isomer?M4?,Rb1?M5?,Ra3 isomer?M6?,which are the most important components to distinguish PQF/PGF/PNF:1)The content of Rb1 in PGF is medium,but the other 5 markers are scant;2)The contents of Rb3,m-Rc/m-Rb2/m-Rb3 isomers and Rb1 are moderate in PQF,differently,the other 3 markers is exiguous;3)Ra1,Ra1/Ra2 isomers?M4?and Ra3 isomers?M6?are rich in PQF,with medium content of the other 3 markers.In addition,these ginsenoside markers in the next work,which need to be isolated by target and confirmed by NMR.It is capable of achieving accurate identification for the flower buds of three congeneric Panax species,by the above research results.Three-dimensional liquid chromatography-mass spectrometry was applied,to research on material basis of TCM system,and the metabolomics technology based on ion-mobility high resolution mass spectrometry was used in the quality control of TCM in this report.The off-line three-dimensional liquid chromatography-high resolution mass spectrometry technique,which was able to achieve targeted and untargeted characterization at the same time,combining ion exchange chromatography,hydrophilic interaction chromatography and reversed phase chromatography,capable of realizing the deep characterization of acid and neutral active components in TCM.Untargeted metabolomics is an effective approach for accurate identification of TCM.This paper provides a methodological reference for the interpretation of material basis and precise quality control of TCM system. |
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