| Hyperuricemia is a disease caused by metabolic disorder of purine,and the prevalence has been increasing.It does harm to people’s health and relates colsely with a variety of diseases such as gout,coronary heart disease and myocardial infarction,and has became a part of the metabolic syndrome.Cortex Fraxini is a traditional Chinese medicine and has the effect of drying dampness,astringent,improving eyesight,etc.Researches of home and abroad proves that Cortex Fraxini has the efficacy of anti-inflammatory and lowering uric acid in blood,and it can be used for the treatment of hyperuricemia.However,the mechanism of treatment is still not clear.The ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry(UHPLC-FT-ICR-MS)method was used to identify the main chemical components in Cortex Fraxini and the metabolic profile in rats after administration,clarifying the efficacy material basis of Cortex Fraxini.Based on UPLC-MS/MS,the multi-component pharmacokinetic study of Cortex Fraxini was conducted,and the differences of pharmacokinetic behavior in hyperuricemic model and normal rats was compared,confirming the pharmacological effects of chemical components absorbed into blood.Based on 1H-NMR and LC-MS/MS technology,the metabonomics analysis platform was established to illustrate the whole metabolism characteristics and abnormal metabolic pathways in hyperuricemic rats.The changes of endogenous metabolites was also monitored before and after the treatment of Cortex Fraxini to find the deeper relationship between these factors and correlations.This research illustrated the treatment mechanism for hyperuricemia used by Cortex Fraxini,and laid the foundation for the further research and development of Cortex Fraxini.1.Characterization of chemical constitutes in Cortex Fraxini and the study of its reduction effect on uric acidThe powder of Cortex Fraxini was extracted in boiled water under refluxing.The filtrate was precipitated with 2.5-fold volume of ethanol(90%,v/v)and D101 macroporous resin column was used to purify the extracting solution of Cortex Fraxini.The elutions were collected and finally freeze-dried to give the purified powder of extract of Cortex Fraxini.UHPLC-FT-ICR-MS method was established for the separation and characterization of the constituents of Cortex Fraxini.LC separation was conducted on a C18 column(150 mm × 2.1 mm,1.8 μm).The mobile phase consists of water and methanol,and the mass detector was operated in positive and negative model.According to the results,33 chemical compounds were characterized,and coumarins,phenylethanoid glycosides,iridoid glycosides,phenylpropanoids and lignans were the main constituents of Cortex Fraxini.Coumarins were the major components characterized,and eight compounds were accurately identified by comparison with reference compounds.The rats used for hyperuricemic model construction were fed with a high-purine diet:Oteracil potassium 100 mg/kg/d,adenine 200 mg/kg/d and yeast extract 10 g/kg/d.Rats in the prevention group were given Cortex Fraxini extract at a dose of 0.5 g/kg/d at the same time.All rats were given their treatments for four weeks.The plasma levels of uric acid(UA)were determined using commercial kits.After four weeks gavage of a high-purine diet,the UA level of were increased markedly compared with normal group,indicating the occurrence of hyperuricemia.After the forth week,the hyperuricemic model rats were given Cortex Fraxini extract(0.5 g/kg/d)and allopurinol tablets(50 mg/kg/d),respectively.After four weeks of treatment with Cortex Fraxini extract and allopurinol,the UA level decreased significantly,suggesting that the Cortex Fraxini could be used to treat hyperuricemia.Rats in prevention group were given high-purine diet and Cortex Fraxini extract at the same time,and the UA levels had no significant change,indicating the Cortex Fraxini extract had the preventive effect of hyperuricemia.2.Comparative pharmacokinetic study of the main components of Cortex Fraxini after oral administration in normal and hyperuricemic ratsAn efficient and rapid ultra-performance liquid chromatography mass spectrometry(UPLC-MS)method was developed and validated for simultaneous quantitation of six coumarins(aesculin,fraxin,aesculetin,fraxetin,sopoletin and 7-hydroxycoumarin)in normal and hyperuricemic rats plasma after oral administration of Cortex Fraxini.The method could successfully be applied for pharmacokinetics studies.Hymecromone was used as internal standard and the plasma was precipitated with acetonitrile before analysis.LC separation was performed on a Universil XB C18 column(150 mm × 2.1 mm,1.8 μm)with a flow rate at 0.2 ml/min.The mobile phase contained 0.1%formic acid in water and methanol.The electrospray ionization(ESI)source was operated in positive and the detection of the analytes was in the multiple reaction monitoring mode(MRM).The intra-day and inter-day precision were less than 12.7%,and the accuracy was within ± 10.4%.The calibration curves were linear over a range of 10-2000 ng/ml for aesculin,5-1000 ng/ml for fraxin,aesculetin and fraxetin,2.5-500 ng/ml for sopoletin,and 1-200 ng/ml for 7-hydroxycoumarin.The lower limits of quantification(LLOQ)were 10 ng/ml for aesculin,5 ng/ml for fraxin,aesculetin and fraxetin,2.5 ng/ml for sopoletin,and 1 ng/ml for 7-hydroxycoumarin.The extraction recoveries of the six analytes were all greater than 85.5%.After the Cortex Fraxini extract was administrated to rats at a dose of 1.7 g/kg body weight,the pharmacokinetic behavior of six coumarins in normal and hyperuricemic rats plasma was determined.Results showed that for some of analytes,the pharmacokinetic parameters(AUC0-t,AUC0-∞,Cmax,Tmax and CL)were significantly different between normal and hyperuricemic rats.The different pharmacokinetic parameters might result from the renal impairment or the change of metabolic enzymes in the pathological state.3.1H-NMR and MS based metabolomics study of the therapeutic effect of Cortex Fraxini on hyperuricemic ratsMetabolomics based on 1H-NMR and MS was used to study the therapeutic effect of Cortex Fraxini on hyperuricemic rats.Subsequently,metabolomics analysis was conducted using samples of plasma,kidney and urine from the rats in control group,hyperuricemic model group,Cortex Fraxini treatment group,allopurinol treatment group and Cortex Fraxini prevention group.Orthogonal partial least squares-discriminant analysis(OPLS-DA)combined with principal component analysis(PCA)were used to detect potential biomarkers.A total of 26 biomarkers were identified as being primarily involved in amino acid metabolism,lipid metabolism,purine metabolism,amino acid metabolism and carbohydrate metabolism,and hyperuricemia can disturb the balance of many of these metabolic pathways in vivo.The variations in biomarkers revealed the therapeutic mechanism of Cortex Fraxini,and a number of these are not only significant for early diagnosis but also for predicting hyperuricemia.The levels of acetoacetate and 3-hydroxybutyrate in the Cortex Fraxini treatment group were reduced markedly and returned to normal levels,and the GSH level increased significantly compared with the model group.The level of creatinine,aspartate,leucine,phenylalanine,tryptophan and ornithine,all recovered after Cortex Fraxini treatment.In contrast,not many significant changes were observed in the above biomarkers after treatment with allopurinol.4.Metabolic profile of aesculin and Cortex Fraxini in ratsA reliable and sensitive UHPLC-FT-ICR-MS method was developed for systematical screen and identification of the metabolic profile in rats after oral administration of aesculin and Cortex Fraxini.After oral administration of esculin(100 mg/kg)or Cortex Fraxini(1.7 g/kg)for rats,plasma,urine,feces and bile samples were collected for 24 h to screen metabolites.The chromatographic separation was performed on a Universil XB C18 column(150 mm × 2.1 mm,1.8 μm)and eluted by a gradient program.The mobile phase contained water and methanol,mass spectra were performed with electrospray ionization(ESI)source operated in positive ion mode.As a result,a total of 19 metabolites,including 10 phase Ⅰmetabolites and 9 phase Ⅱ metabolites were found and identified after administrated with aesculin.The Cortex Fraxini underwent complex biotransformation process in vivo.A total of 80 constituents,including 28 prototype compounds and 52 metabolites were identified.Five types of prototype structure were identified,including coumarins,iridoid glycosides,phenylethanoid glycosides,lignans and phenylpropanoids,and 20 phase Ⅰ metabolites and 32 phase Ⅱ metabolites were observed.The metabolites were mainly coumarin derivatives.Results showed that metabolic pathways included hydrolysis,dehydrogenation,hydroxylation,methylation and dehydrogenation in phase Ⅰ metabolites,and glucuronidation,sulfation and glycine conjugation in phase Ⅱ metabolites. |
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