Application Of LC-MS In The Discovery Of Q-Marker Of Glycyrrhiza Uralensis And Paeonia Lactiflora With Different Processing Degrees And Metabolic Transformation Of Ginsenosides By Gut Microbiota

The combination of liquid chromatography(LC)technology with mass spectrometry(MS)detection(LC-MS)is increasingly used in drug analysis at different stages of discovery and development:drug discovery,product characterization,metabolic studies(in vitro and in vivo),and identification of impurities and metabolites.In this paper,the LC-MS technology was applied to the discovery of Q-marker of Glycyrrhiza uralensis and Paeonia lactiflora with different processing degrees,and metabolic transformation of ginsenosides by gut microbiota.In the first and second parts,metabonomics method was applied to search for the chemical markers of prepared slices of G.uralensis with different honey processing degrees and P.lactiflora with different wine processing degrees.Coupled with metabolomics analytical methods,ultra-performance liquid chromatography hyphenated with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF/MS)and ultra-performance liquid chromatography hyphenated with triple quadrupole tandem mass spectrometry(UPLC-TQ-MS/MS)were applied to generate global chemical profiles of the raw material of G.uralensis and P.lactiflora and their prepared slices with different processing degrees.According to the UNIFI theoretical database combined with the library of standard samples,a total of 57 chemical components and 11 chemical components were identified from G.uralensis and P.lactiflora,respectively.Among them,37 and 56 compounds were respectively identified in positive and negative ion modes for G.uralensis while 7 and 10 compounds for P.lactiflora.Unsupervised principal component analysis(PCA)showed that the chemical ingredients differ a lot from the processing degrees.Supervised orthogonal partial least squares discriminant analysis(OPLS-DA)was applied to differentiate the moderate processing group and the raw group and partial least squares discriminant analysis(PLS-DA)was applied to differentiate the less processing group,the moderate processing group and the excessive processing group.The results showed that licoricesaponin E2,licoricesaponin G2 and glycyrrhizic acid of G.uralensis and paeoniflorin,benzoylpaeoniflorin of P.lactiflora have obvious quantitative difference.Additionally,it was found that ethyl gallate only existed in wine-process P.lactiflora through the analysis of the data set.Finally,reference standards of G.uralensis and P.lactiflora were quantified by UPLC-TQ-MS/MS,respectively.It was found that the contents of glycyrrhizic acid and paeoniflorin were the highest when the processing was moderate and their contents decreased when the processing was excessive,which could be used as the chemical markers of G.uralensis and P.lactiflora with different processing degrees,and help supervise the processing degrees of the prepared slices.Ethyl gallate only exists in P.lactiflora,and its content increased with the processing time,which can be used as a chemical marker before and after processing of P.lactiflora.The findings provide scientific evidence for quality control of evaluation of processing degree of G.uralensis with honey processing,and P.lactiflora with wine processing.In the third part,the similarities and differences of protopanaxadiol(PPD)-type ginsenosides Rb1,Rb2,Rb3 and Rc metabolism in human gut microbiota were studied by UPLC-Q-TOF/MS.A total of 15 metabolites including 13 deglycosylated metabolites and two oxidative metabolites were identified.They are ginsenosides Rd,Rg3,F2,Compound K,PPD,Compound O,Compound Y,Compound Mx,Compound Mc-1,Compound Mc,gypenosides XVII,LXXV,IX,and Compound K Oxide,PPD Oxide.On the basis of the formation rates of the metabolites detected and the metabolic pathways consulted in previous study,the metabolic pathways of PPD-type ginsenosides were speculated:Rb1/Rb2/Rb3/Rc→Rd→F2→Compound K→PPD,which is mainly the hydrolysis reaction of selective elimination of glycosylation.In addition,ginsenoside Rd is a metabolite produced by the hydrolysis of the outer sugar at C-20 position of the parent compounds by gut microbiota.And by monitoring the peak area of parent compounds and their metabolites,it was found that Rd was generated the fastest in the incubation solution of the four parent compounds.At the same time,the content of Rbl decreased the fastest and the content of Rd was produced the fastest in the incubation solution of Rb1.It is suggested that the hydrolysis rate of the terminal glycosidic bond at C-20 position by gut microbiota was faster,in which the terminal glucosidic bond was hydrolyzed the fastest.The terminal glycosidic bond at C-20 position is presumed to have high enzyme catalytic efficiency,and the enzyme catalytic efficiency of its terminal glucosidic bond is the highest.Therefore,the different substituents at C-20 position could affect the stability of PPD ginsenoside in gut microbiota.The result provides scientific evidence for the determination of the pharmacodynamic basis of PPD-type Ginsenosides by gut microbiota.