The Study On Hypolipidemic Therapeutical Components And Metabonomics Of Green Tea

In the present study,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 of green tea in rat plasma,the pharmacokinetics of seven active ingredients of green tea in rat plasma and the metabolomics of green tea in the serum and urine of hyperlipemia rats.A sensitive and reliable UHPLC-Q-TOF-MS method was established to separate and identify the inherent chemical constituents of green tea.With the optimized conditions,a total of 62 compounds were identified or tentatively characterized.Of the 62 compounds,20 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.Among them,21 compounds were identified as flavonols,20 compounds were identified as flavones,2 compounds were identified as alkaloids,16 compounds were identified as Phenolic acid relatives,1 compounds were identified as organic ester and 1 compounds were identified as amino acid.Results indicated that catechins,flavones and alkaloids were major chemical constituents in green tea.A sensitive and reliable UHPLC-Q-TOF-MS method was established to separate and identify the absorbed components and metabolites in rat plasma after oral administration of green tea extract.By comparing the retention time,high resolution mass data of blank plasma and dosed plasma,28 constituents,including 14 prototype compounds and 14 metabolites were identified or tentatively characterized in rats.Results indicated that glucuronidation and sulfation were the main metabolic pathways of catechins.To study the pharmacokinetic behaviors of active constituents in rats after oral administration of green tea extract,a selective,sensitive and reliable UFLC-MS/MS method was developed for the simultaneous determination of seven catechins(catechin epicatechin,gallocatechin,epigallocatechin,epicatechin-3-gallate,gallocatechin-3-gallate and epigallocatechin-3-gallate.ethyl gallate was used as internal standard(IS).After liquid-liquid extraction with ethyl acetate-isopropanol(1:1,v/v),separation was achieved on a Venusil MP C18 column(100 mm × 2.1 mm,2.2 ?m)using gradient elution with a mobile phase consisting of water(containing 0.1%formic acid),acetonitrile and methanol 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 andmultiple reaction monitoring(MRM)mode.The intra-and inter-day precisions(as relative standard deviation)were less than 14.3%,and accuracy(as relative error)was between-8.8%and 7.5%.The calibration curves were linear over a range of 8-8000 ng/mL,8-8000 ng/mL,30-30000 ng/mL,3-3000 ng/mL,10-10000 ng/mL,5-5000 ng/mL and 15-15000 ng/mL for catechin epicatechin,gallocatechin,epigallocatechin,epicatechin-3-gallate,gallocatechin-3-gallate and epigallocatechin-3-gallate,respectively,and the lower limits of quantification(LLOQ)were 8,8,30,3,10,5 and 15 ng/mL.The extraction recoveries of the analytes and IS from rat plasma were all more than 72.0%.The method was fully validated andapplied to the pharmacokinetic profiles of the analytes rats after oral administration of green tea extract.Results showed that that the seven catechins have linear pharmacokinetic characteristics in rats.The metabolomics approach based on UHPLC-Q-TOF-MS has been developed to characterize the global serum and urine metabolic profiles associated with hyperlipidaemia and investigate the curative mechanisms of green tea.The analytical data were processed via principle component analysis(PCA),and score plot and loading plot were obtained.A total of 14 potential biomarkers were screened out in rat serum,including 2 acylcarnitines(propionyl carnitine,palmitoyl carnitine),2 amino acids(valine,lysine),6 LPCs[LPC(15:0),LPC(20:5),LPC(22:6),LPC(18:2),LPC(18:3),LPC(17:0)],1 bile acids(deoxycholic acid)and 3 sphingolipids(sphinganine,dihydrosphingosine,phytosphingosine).6 potential biomarkers were screened out in rat urine,including taurine,gluconic acid,allantoin,uric acid,aminoadipic acid,p-cresol,cholic acid,p-cresol sulfate.It mainly involved in fatty acid transportation and metabolism,phospholipid catabolism,amino acid metabolism,cholesterol metabolism,sphingolipids metabolism and carbohydrate metabolism.PCA score plots of analytical data indicated the general differences among different groups and the influence of green tea extract on hyperlipidemia,and green tea extract could definitely inhibit the hyperlipidemia 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,metabonomicsand computer technology,the chemical constituents,the absorbed constituents and metabolites in rat plasma and the pharmacokinetic behaviors of active constituents of green tea were also studied.Meanwhile,the hypolipidemic action mechanism of green tea was revealed based on metabonomics theory.This research provided essential data for research and development of green tea.