Studies On Therapeutic Material Basis And Metabonomics Of Zhi-zi-da-huang Decoction Against Liver Injury

Zhi-Zi-Da-Huang decoction(ZZDHD),a classic Traditional Chinese medicine(TCM)formula recorded in "Jin-Kui-Yao-Lue"(Synopsisof Golden Chamber)for the treatment of alcoholic jaundice,is composed of four crude drugs:Gardenia jasminoides Ellis(Zhi-Zi),Rheum palmatum L.(Da-Huang),Citrus aurantium L.(Zhi-Shi)and Sojae Semen Praeparatum(Dan-Dou-Chi).It has been widely used in China for thousands of years to treat liver diseases including acute or chronic hepatitis,alcoholic hepatitis cholestasis and jaundice,etc.In the present thesis,ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS)and ultra-high performance liquid chromatography coupled with tandem mass spectrometry(UHPLC-MS/MS)techniques were developed to systematically investigate the inherent chemical constituents,the absorbed constituents and metabolites of ZZDHD in rat plasma,the pharmacokinetics of eight active ingredients of ZZDHD in rat plasma,the metabolism of ZZDHD in rat urine,and the metabolomics of ZZDHD in the serum,urine and liver of liver injury ratsA sensitive and reliable UHPLC-Q-TOF-MS method was established to separate and identify the inherent chemical constituents of ZZDHD,and the absorbed components and metabolites in rat plasma after oral administration of ZZDHD.Moreover,the differences of the absorbed components and metabolites in healthy,cholestatic liver injury(CLI)and alcoholic liver injury(ALI)rats were also compared.With the optimized conditions,a total of 82 compounds were identified or tentatively characterized.Of the 82 compounds,21 compounds were identified by comparing the retention time and MS data with reference standards,the others were characterized by analyzing MS data and retrieving the reference literature.In addition,31 compounds were identified from Gardenia jasminoides Ellis,10 compounds were identified from Rheum palmatum L.,33 compounds were identified from Citrus aurantium L.,and 8 compounds were identified from Sojae Semen Praeparatum.Results indicated that iridoid glycosides,anthraquinones,flavonoids,coumarins,glycosides of crocetin,monoterpenoids,tannins,organic acids and limonins were major chemical constituents in ZZDHD.By comparing the retention time,high resolution mass data of blank plasma and dosed plasma,43 constituents,including 21 prototype compounds and 22 metabolites were identified or tentatively characterized in healthy rats,and 39 constituents,including 21 prototype compounds and 18 metabolites were identified or tentatively characterized in CLI and ALI rats.Results indicated that glucuronidation and sulfation were the main metabolic pathways of iridoid glycosides and anthraquinones,glucuronidation was the main metabolic pathways of flavanone-related compounds.To compare the pharmacokinetic behaviors of active constituents between healthy and liver injury rats after oral administration of ZZDHD,a selective,sensitive and reliable UFLC-MS/MS method was developed for the simultaneous determination of two iridoid glycosides(geniposide and genipin gentiobioside),two anthraquinones(rhein and emodin)and four flavonoidglycosides(isonaringin,naringin,hesperidin and neohesperidin),the major active ingredients of ZZDHD,in rat plasma.Paeoniflorin was used as internal standard(IS).After liquid-liquid extraction with ethyl acetate-isopropanol(1:1,v/v),separation was achieved on a Shim-pack XR-ODS C18 column(75 mm × 3.0 mm,2.2 μm)using gradient elution with a mobile phase consisting of water(containing 0.1%formic acid)and acetonitrile at a flow rate of 0.4 mL·min-1.Detection was performed on 4000 QTRAP mass spectrometry equipped with turbo ion spray source in the negative ionization and multiple reaction monitoring(MRM)mode.The intra-and inter-day precisions(as relative standard deviation)were less than 11.4%,and accuracy(as relative error)was within±10.0%.The calibration curves were linear over a range of 4-2000 ng·mL-1,0.5-250 ng·mL-1,2-1000 ng·mL-1,0.1-50 ng·mL-1,1-500 ng·mL-1,2-1000 ng·mL-1,1-500 ng·mL-1 and 2-1000 ng·mL-1 for geniposide,genipin gentiobioside,rhein,emodin,isonaringin,naringin,hesperidin and neohesperidin,respectively,and the lower limits of quantification(LLOQ)were 4,0.5,2,0.1,1,2,1,2 ng-mL-1.The extraction recoveries of the analytes and IS from rat plasma were all more than 86.0%.The method was fully validated and applied to compare the pharmacokinetic profiles of the analytes in normal,CLI,and ALI rats after oral administration of ZZDHD.Results showed that there were remarkable differences in pharmacokinetic properties of the analytes between normal and CLI and ALI group.The established UHPLC-Q-TOF-MS method was applied to study the metabolism of ZZDHD in rat urine,and compare the differences between healthy,CLI and ALI rats.By comparing the retention time and high resolution mass data of blank urine and dosed urine,a total of 59 constituents,including 39 prototype compounds and 22 metabolites,were identified or tentatively characterized,including 11 iridoids,7 anthraquinones,33 flavonoids,5 monoterpenoids and 3 coumarins in healthy,CLI and ALI rats.No other prototype compounds and metabolites were found.The metabolomics approach based on UHPLC-Q-TOF-MS has been developed to characterize the global serum,urine and liver metabolic profiles associated with CLI and investigate the curative mechanisms of ZZDHD.The analytical data were processed via principle component analysis(PCA),and score plot and loading plot were obtained.A total of 17 potential biomarkers were screened out in rat serum,including 4 acylcarnitines(linoleyl carnitine,palmitoyl carnitine,elaidic carnitine and stearoyl carnitine),8 LPCs[LPC(18:3),LPC(16:1),LPC(20:4),LPC(22:6),LPC(18:2),LPC(22:5),LPC(20:3)and LPC(18:1)],2 bile acids(glycocholic acid and cholic acid),tryptophan,creatine and sphingosine-1-phosphate.7 potential biomarkers were screened out in rat urine,including 2 amino acids(phenylalanine and isoleucine),2 bile acids(glycocholic acid and cholic acid),5-methylcytosine,kynurenic acid and creatine.7 potential biomarkers were screened out in rat liver,including 2 amino acids(prolyl-arginine and methionyl-arginine),2 bile acids(glycocholic acid and cholic acid),and 3 sphingosines(C16 sphinganine,phytosphingosine and sphinganine).It mainly involved in fatty acid transportation and metabolism,phospholipid catabolism,amino acid metabolism,cholesterol metabolism and nucleotide metabolism.PCA score piots of analytical data indicated the general differences among different groups and the influence of ZZDHD on CLI,and ZZDHD could definitely inhibit the CLI development and reverse some pathological biomarkers.The metabolomics approach based on UHPLC-Q-TOF-MS has been developed to characterize the global serum,urine and liver metabolic profiles associated with ALI and investigate the curative mechanisms of ZZDHD.The analytical data were processed via principle component analysis(PCA),and score plot and loading plot were obtained.A total of 6 potential biomarkers were screened out in rat serum,including 4 phosphatides[glycerophosphocholine,LPC(18:2),LPE(16:0)and LPC(20:2)],tryptophan and linoleic acid.7 potential biomarkers were screened out in rat urine,including 2 nucleotides(cytosine and 5-methylcytosine),4 amino acids(phenylalanine,tyrosyl-proline,n-butyrylglycine and hydroxyprolyl-valine),and kynurenic acid.6 potential biomarkers were screened out in rat liver,including 2 nucleotides(xanthine and hypoxanthine),2 phosphatides[LPC(20:4)and LPE(16:0)],L-carnitine and arginyl-glycine.It mainly involved in fatty acid transportation and metabolism,phospholipid catabolism,amino acid metabolism,and nucleotide metabolism.PCA score piots of analytical data indicated the general differences among different groups and the influence of ZZDHD on ALI,and ZZDHD could definitely inhibit the ALI development and reverse some pathological biomarkers.With the direction of theory and clinical practice of TCM,utilizing the knowledge of Chinese material medica science,analytical chemistry,pharmacokinetics,metabonomics and computer technology,the chemical constituents,the absorbed constituents and metabolites in rat plasma,and the metabolism of ZZDHD in rat urine were studied,the pharmacokinetic behaviors of active constituents of ZZDHD in healthy and liver injury rats were also compared.Meanwhile,the action mechanism of ZZDHD against liver injury was revealed based on metabonomics theory.This research provided essential data for clinical use,research and development of ZZDHD.