| Pyrexia usually is a systemic pathological process that can lead to metabolic disorders.Metabonomics as a powerful tool not only can reveal the pathological mechanisms,but also can give insight into the progression of pyrexia from another angle.Because of the characteristics of Chinese patent medicine including multiple components and multi-target actions,the study of therapeutic basis of Chinese medicine formula becomes more and more important for evaluating Chinese medicines on a scientific basis and promoting the modernization of Chinese materia medica.In the present study,using a combined method of metabonomics and pharmacokinetics based on high-pressure liquid chromatography-mass spectrometry,we investigated the therapeutic basis of Gegenqinlian Decoction(GQLD)on pyrexia in rat model of pyrexia.An ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry(UHPLC-FT-ICR-MS)metabonomic approach was employed for the first time to investigate the plasma biochemical characteristics of pyrexia induced by three methods and to reveal subtle metabolic changes under the condition of pyrexia so as to explore its mechanism.Partial least squares-discriminant analysis(PLS-DA)was used to investigate the differences between the pyrexia groups and the matched control groups.Fifty-two endogenous metabolites were identified and putatively identified as potential biomarkers primarily associated with phospholipid metabolism,sphingolipid metabolism,fatty acid oxidation metabolism,fatty acid amides metabolism and amino acid metabolism,and related to bile acid biosynthesis and glycerolipid catabolism.LysoPC(14:0),LysoPC(18:3),LysoPC(20:4),LysoPC(16:0),phytosphingosine,Cer(d18:0/12:0),N-[(4E,8E)-1,3-dihydroxyoctadeca-4,8-dien-2-yl]hexadecanamide,oleamide,fatty acid amide C22:1,tryptophan,acetylcarnitine,palmitoylcarnitine and stearoylcarnitine were considered as common potential biomarkers of pyrexia rats induced by three methods.Sphinganine and glycocholic acid were unique potential biomarkers for TCMP group.LysoPC(18:2),LysoPC(20:3)and fatty acid amide C22:2 were unique potential biomarkers for YP group.LysoPC(20:5),LysoPC(22:6),LysoPC(22:5),LysoPE(0:0/18:0),sphingosine 1-phosphate,linoleoyl ethanolamide,cis-5-tetradecenoylcarnitine,tetradecanoylcarnitine,trans-hexadec-2-enoyl carnitine,linoleyl carnitine and elaidic carnitine were unique potential biomarkers for DNP group.An UPLC-MS metabonomic method was developed for metabolites profile analysis of plasma from pyrexia model rats,which was the premise and reference of drawing up a reasonable dose regimen.The pyrexia groups were the traditional Chinese medicine pyrexia group,yeast pyrexia group and 2,4-dinitrophenol pyrexia group,respectively.In view of the different effective time of each pyrexia rat model and the simplicity and reproducibility of the model.Therefore,pyrexia rat model induced by yeast was used to study on the therapeutic pyrexia of GQLD.An UHPLC-FT-ICR-MS method was employed to investigate the major constituents in GQLD.Accurate mass measurements for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds.In total,134 compounds in GQLD were identified or tentatively characterized mainly including flavonoids,alkaloids and triterpenoids.These results could provide a basis for quality control and further in vivo research of GQLD.An UHPLC-FT-ICR-MS method was established to profile the metabolic fate of GQLD in rat plasma,urine,bile and feces.A total of 174 compounds mainly including flavonoids,alkaloids and triterpenoids in rat plasma,urine,bile and feces after oral administration of GQLD were identified or tentatively characterized by comparison with retention times,accurate mass within 5 ppm error and their characteristic MS fragmentation ions.Among compounds in rats,107 compounds are prototypes and 67 compounds are metabolites.Results demonstrated that metabolic pathways of GQLD in rats included methylation,demethylation,hydrogenation,hydrolysis,hydroxylation,methoxylation,sulfate conjugation and glucuronide conjugation.This study lays the foundation for the study of therapeutic basis of GQLD.Pyrexia rats were treated with different medicines,an UHPLC-FT-ICR-MS metabonomic approach was employed to investigate the plasma biochemical characteristics using PCA and PLS-DA.Itraconazole,aspirin,and GQLD had obvious preventive effects on pyrexia.The main biomarkers of the itraconazole group,aspirin group,and GQLD group were slightly different.GQLD can make most of the markers tend to be normal.It may be that the synergetic actions of multi-ingredients in GQLD.A rapid,sensitive,selective and accurate UPLC-MS/MS method was developed and fully validated for simultaneous determination of puerarin,daidzein,baicalin,wogonoside,wogonin,berberine,palmatine,jateorhizine,coptisine,isoliquiritin,liquiritigenin,glycyrrhizic acid and glycyrrhetinic acid in rat plasma after oral administration of GQLD.The liquid-liquid extraction method with ethyl acetate/butyl alcohol(2:1,v/v)was used to prepare the plasma samples.The simple,sensitive,specific,and handy method met the requirements of biological sample analysis,and the method was successfully applied to a comparative pharmacokinetic study of GQLD in normal and pyrexia rats.In conclusion,there were statistically significant differences in the pharmacokinetic parameters of AUC(0-t)and CL values for puerarin,Tmax value for baicalin,wognoside,coptisine,isoliquiritin and liquiritigenin,Cmax value for daidzein,palmatine and jatrorrhizine,t1/2 value for jatrorrhizine observed between normal and pyrexia model rats.These significant differences in the pharmacokinetic behaviors may result from pathological state of pyrexia.It demonstrated that the absorption and metabolism of the main components in GQLD were influenced under the pyrexia pathological condition.These results could provide some beneficial guidance for GQLD in the clinical application. |
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