Investigations On The Systematic Chemical Basis Elucidation Of Panax Ginseng,Red Ginseng And The Processing-induced Holistic Chemical Transformations

White ginseng（WG）is the dried root of the Panax ginseng C.A.Mey,while Red ginseng（RG）is a product obtained by steaming and drying fresh ginseng.Many studies have shown that the transformation of ginsenosides caused by heating treatment is significantly related to the improvement of biological activity.Systematic interpreting of the chemical compositions of WG and RG,elucidation of the overall chemical transformation mediated by steaming,and establishment of the characteristic saponin markers,are considered to be important to achieve the precise quality control of WG and RG,optimize the processing parameters,reasonably guide clinical medication,and promote the development of ginseng.In this paper,multiple techniques,such as systematic phytochemical separation,multi-dimensional chromatography-high resolution mass spectrometry,nontargeted metabolomics,and mass spectrometry imaging（MSI）are used to isolate and prepare saponin compounds from WG,and then deeply characterize and identify the saponin compositions of WG and RG,holistic depiction of steaming-mediated chemical transformation to construct the characteristic markers for both WG and RG.In this thesis,a variety of column chromatography（D101 macroporous adsorption resin,medium-pressure C18 column）,preparative and semi-preparative high performance liquid chromatography,were used to systematically separate and purify ginsenosides from the root of P.ginseng.We could isolate and identify 42 ginsenoside compounds（1–42）,including a new oleanolic acid-type ginsenoside（1）and an intra-species firstly isolated compound（2,notoginsenoside FP₁）.The structure of the new ginsenoside was established by high-resolution mass spectrometry,and one-dimensional and two-dimensional NMR analyses,as oleanolic acid 3-O-[β-D-glucopyranosyl-（1→2）-β-D-glucopyranosyl]-28-O-β-D-glucopy-ranosyl-（1→6）-β-D-glucopyranoside,named ginsenoside R_O1（1）.The other 40 known saponins were identified as:ginsenoside Rg₁（3）,notoginsenoside R₄（4）,ginsenoside Ra₂（5）,-Rb₁（6）,-Rb₂（7）,-Rd（8）,notoginsenoside Rt（9）,ginsenoside Rc（10）,-Ra₁（11）,20-O-glucosyl-ginsenoside Rf（12）,malonylginsenoside Rb₁（13）,malonylginsenoside Rb₂（14）,ginsenoside Re₂（15）,-Re₃（16）,malonylfloralginsenoside Rd₅（17）,malonylginsenoside Rc（18）,ginsenoside Rg₃（19）,ginsenoside Re（20）,20（R）-ginsenoside Rh₂（21）,20（S）-ginsenoside Rh₂（22）,ginsenoside Rf（23）,20（S）-protopanaxatriol（24）,ginsenoside F₃（25）,-Rk₁（26）,-F₅（27）,-Rg₂（28）,-Rb₃（29）,20（R）-ginsenoside Rh₁（30）,compound K（31）,ginsenoside F₁（32）,-F₂（33）,-R_O（34）,notoginsenoside R₁（35）,chikusetsusaponin IVa（36）,20（S）-ginsenoside Rh₁（37）,ginsenoside Rg₅（38）,-Rk₃（39）,notoginsenoside Fe（40）,-R₂（41）and ginsenoside Ra₃（42）.The phytochemical separation work,in this thesis,further enriched the diversity of saponin compounds in P.ginseng,and provided reference compounds for WG and RG in the material basis research and difference analysis based on LC/MS.An approach appliable to the systematic multi-component characterization of traditional Chinese medicine（TCM）was established,based on offline two-dimensional liquid chromatography/ion mobility quadrupole time-of-flight high-resolution mass spectrometry（2D-LC/IM-QTOF-MS）,enabling dimension-enhanced four-dimensional separation.This technique,in combination with the in-house ginsenoside database-driven standardized procedures of intelligent peak annotation by the UNIFI platform,as a result,we could separate and characterize a total of 323 ginsenosides（including 286 from WG and 306 from RG）,and 125 thereof may have not been isolated from the Panax genus.First,the key parameters of chromatography-mass spectrometry（stationary phase,mobile phase,column temperature,elution gradient;ion source parameters like capillary voltage,cone voltage,and ramp collision energy）were optimized in turn through single-factor experiments,and an offline 2D-LC system configured with an XBridge Amide column of hydrophilic interaction chromatography（HILIC）and a reversed-phase HSS T3 column was established.Orthogonality of the system,determined by the asterisk equations,was 0.76,and the effective peak capacity was 976.This 2D-LC/MS method was verified to be stable and reproducible.The Vion?IMS-QTOF hybrid high-resolution mass spectrometer was used,and the application of data independent high-resolution MS^E（HDMS^E）for data collection in the negative ESI mode could enable four-dimensional separation of the WG and RG extracts and provide five-dimensional structure-related information(t _R-HILIC,t _R-RP,CCS,MS¹,MS²)of each saponin component.Compared with MS^E,HDMS^Ecan better separate ginsenosides and directly provide CCS information.On the basis of systematic searching of the literature on phytochemical investigation of ginsenosides,an"in-house ginsenoside database"was established,which recorded 504 known ginsenoside structures（name,molecular formula,and structure）.This database was imported into the UNIFI^TMsoftware,together with determined structural information of 58 saponin reference compounds.An efficient metabolite identification procedure,based on automatic annotation of the HDMS^Edata by UNIFI was created（including analysis method setting,data collection,data correction,data processing and identification,reconfirmation of the identification results）.By the prediction of small molecules fragmentation,experimental MS¹/MS²data and the theoretical MS information were matched,which greatly reduced the analysis time and presented reproducible identification results.The experiments in this chapter added one-dimensional chromatographic separation（HILIC）and ion mobility（IM）separation,which greatly increased the peak capacity.The CCS values measured by ion mobility provide more one-dimensional information orthogonal to mass spectrometry for molecular structure identification,especially in the discrimination of isomers.Finally,286 ginsenosides could be identified from WG,and 306 from RG,with a total of 323 ginsenosides（including 119 PPD type,87 PPT type,21 OA type,17 malonylated,and 76 others）.This laid the foundation for the difference analysis of WG and RG.In addition,the established offline2D-LC/IM-QTOF-HDMS^Emethod could also serve as a magnifying glass to initially explore the differences between WG and RG.In this thesis,a nontargeted metabolomics approach,based on ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry（UHPLC/IM-QTOF-MS）,was developed,which was utilized to analyze the overall chemical transformation of P.ginseng steamed by different time points.Comparing the metabolome of WG and RG obtained by both self-made and commercially purchased,led to the identification of 18 potential differential components,based on which the characteristic markers of WG and RG were discovered.And MSI was applied to determine the tissue distribution of these differential markers.We established a nontargeted metabolomics analysis procedure,by the integration of IM-QTOF-MS,UNIFI,and Progenesis QI software:1)negative-mode HDMS^Emetabolic feature collection;2)multi-batch HDMS^Edata correction and data deconvolution（peak alignment,peak extraction,and normalization）;3)metabolic feature list reprocessing by 80%rule and 30%variation rule;4)to discover the differential metabolites based on principal component analysis（PCA）,orthogonal partial least squares difference analysis（OPLS-DA）,and VIP values（variable importance in projection）;5)identification of the potential markers.The transformation degree of the chemical components of P.ginseng under different processing time（2 h,3 h,and 4 h）was firstly studied according to the above procedure,and the time-dependent dynamic transformation process was found through the PCA score plot.Differential analysis was performed on WG and RG self-made in the laboratory,and when the VIP threshold was set to2.5,22 differential ions were identified.In a similar manner,comparing the commercial WG with RG,25 differential ions were identified.Among them,m-Rb₁（isotopic peak m/z1195.6070）,ginsenoside Ro,M6（PPD+3Glc+2Xyl+Mal）,m-Rc（isotopic peak m/z1165.5967）,m-Rb₂（isotopic peak m/z 1165.5970）are the Identification Markers for WG;ginsenoside Rb₂,ginsenoside Rd,and ginsenoside Rg₃are the Identification Markers for RG.On this basis,further MSI analysis was performed on the masses of 5 markers(M18:ginsenoside Rg₅（isomer）;M15:ginsenoside Rg₃（isomer）;M4:ginsenoside Ro（isomer）;M12:Ginsenoside Rd（isomer）;M10:m-Rc（isomer）among the 18 potentially processed related markers was performed.The specific distribution of five ginsenosides markers in the root tissue of WG and RG that higher content of ginsenosides Rg₅and Rg₃can be seen in the cork and phloem,and ginsenoside Ro,m-Rc（isomer）have higher content in the cambium.In addition,MSI can visually show the difference in the content of different markers in different groups of samples.The contents of M18 and M15 in RG were higher than those of WG,while the contents of M4,M12,and M10,in WG were higher than those in RG.These trends were basically consistent with the results obtained by nontargeted metabolomics.It is worth noting that the change trend of metabolites reflected by MSI is the total intensity of all isomers that can be ionized.If these isomers have different change trends,the results presented by MSI may be different from those obtained by LC-MS.In this thesis,for the first time,an offline 2D-LC/IM-QTOF-HDMS^E-based dimension-enhanced metabolites characterization technique was established to support four-dimensional separation of the mixed samples,and the in-depth characterization of saponins both in WG and RG was realized.Nontargeted metabolomics analysis procedure,based on UHPLC/IM-QTOF-MS,UNIFI and Progenesis QI,was created,and further applied to depict and identify the relevant processing markers for P.ginseng as a whole.MSI was first used to study the tissue distribution of processing markers.These results will play a positive role to achieve the precise quality control of WG and RG,to rationally guide the clinical medication,and to promote the development of the ginseng industry.Meanwhile,these studies provide a methodological reference for the researches of the systematic material basis and the processing mechanism of TCM.