| Based on liquid chromatography-mass spectrometry technology,Guan-Xin-Shu-Tong capsule(GXSTC)was selected as the objective of the study.Comprehensive profiling of ingredients in Guan-Xin-Shu-Tong capsule,prototype components and their metabolites screening in rat plasma,pharmacokinetic study of active ingredients and metabolimics study on hyperlipidemia and acute blood stasis rats after oral administration of Guan-Xin-Shu-Tong capsule were achieved and deeply investigated.1.A comprehensive analytical method using quadrupole-time-of-flight mass spectrometry(Q-TOF/MS)was developed for the first time to investigate the compounds in vitro and active components in healthy and heart-disease-modeled rats after oral administration of GXSTC,based on the multiple data processing methods including extracted ion chromatography,mass defect filtering and fragment filtering approach.In vitro,74 compounds were identified in GXSTC,among them,18 compounds were unambiguously identified by comparing the retention time and MS/MS data as standards,and the other 56 compounds were tentatively characterized by the accurate quasi-molecular ion mass and MS/MS fragmentation pattern.Among 74 compounds,22 compouns were derived from Choerospondias axillaris,48 compounds were derived from Salvia miltiorrhiza Bunge,8 compounds were from Syzigium aromaticum and 1 alkaloid was from Tabaschir.The results showed that flavonoids,organic acids,propionic acid,diterpenoids,coumarins,alkaloids and steroids were the main compounds of GXSTC.In vivo,24 prototype components and 20 metabolites originated from GXSTC were detected among healthy group,hyperlipidemia group and acute blood stasis group rats after oral administration of GXSTC.24 prototype components showed no differences between the normal group and the pathological model groups,including 9 diterpenoids,8 phenolic acids,5 organic acids,1 alkaloid and 1 flavonoid.But 20 metabolites were different in each group,including 12 phenolic acid related metabolites,7 diterpenoid related metabolites and 2 flavonoids related metabolites.The developed method for comprehensive analysis and identification of GXSTC in vitro and in vivo was proved to be rapid and accurate,which may contribute to the further quality control and pharmacology study.2.A validated and sensitive analytical method using the ultra fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS)was reported for the first time to simultaneously determine and investigate the pharmacokinetics of five phenolic acids including gallic acid,danshensu,protocatechuic acid,rosmarinic acid and chlorogenic acid,and four diterpenoids including dihydrotanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA in rats.The established method was successfully applied to compare the pharmacokinetics of nine compounds in normal and modeled rats with acute blood stasis(ABS)after oral administration of GXSTC.After liquid-liquid extraction with ethyl acetate from blood,the separation of analytes and internal standard(simvastatin and sulfamethoxazole)were achieved by Shim-pack XR-ODS C18 column(75 mm × 3.0 mm,2.2μm)and mobile phase was composed of acetonitrile and water(containing 0.1%formic acid)under gradient elution conditions,with an electrospray ionization source in both positive and negative ion modes with multiple reaction monitoring mode.The limit of quantification of all the analytes was 0.025-1.250 ng/ml.Intra-day and inter-day precision were less than 10.9%and the mean extraction recoveries of analytes and internal standards from rat plasma were all more than 75%.Compared with the normal group,the pharmacokinetic parameters for AUC0→t,AUC0-∞ and Cmax associated with gallic acid,rosmarinic acid,cryptotanshinone and tanshinone IIA increased significantly(P<0.05)after oral administration of ABS rats.In addition,the t1/2 of tanshinones significantly prolonged and appeared double peak curve.The Tmax of dihydrotanshinone obviously increased.3.To acquire the changed metabolism pathways and search for the potential biomarkers,a metabonomics approach of GXSTC against hyperlipidemia was developed based on LC-MS/MS.Multivariate statistical analysis revealed obvious differentiation between the normal group,hyperlipidemia group,positive group and GXSTC treated group in both positive and negative ion modes.A total of 16 metabolites were identified in plasma as potential biomarkers,including phenylalanine,metyrosine,glutamate,L-threonine,cortisol,cytosine,galactose,2-oxoadipic acid,gluconic acid,indoleacetaldehyde,5-(2-Hydroxyethyl)-4-methylthiazole,2-keto-glutaramic acid,uric acid,LPE(16:0)and sphingosine.Meanwhile,19 metabolites were screened out in urine samples,including phenylacetylglycine,tryptophan,tyrosine,cysteine,tryptophanol,L-glutamic acid-5-phosphate,theophylline,nicotinic acid,creatinine,3-cresotinic acid,indoleacetaldehyde,alpha hydroxy hippuric acid,carnitine acid,taurine,glucose,uric acid,a-ketoglutaric acid oxime,2-phosphoglycerate acid and glycerate acid.They mainly involved taurine metabolism,fatty acid metabolism,amino acid metabolism,cholesterol metabolism,nucleotide metabolism and a series of metabolic processes.The results showed that such a metabonomics approach could not only provide a systematic view of hyperlipidemia-related metabolism,but also reveal the mechanism of therapeutic effect of GXSTC in treating hyperlipidemia.4.To acquire the changed metabolism pathways and search for the potential biomarkers,a metabonomics approach of GXSTC against acute blood stasis was developed based on LC-MS/MS.Multivariate statistical analysis revealed obvious differentiation between the natural group,acute blood stasis group,positive group and GXSTC treated group in both positive and negative ion modes.A total of 16 metabolites were were identified as potential biomarkers in plasma samples,including ornithine,L-serine,tryptophan,L-arginine,N-methyl hydantoin,creatine,L-acetylcarnitine,2-deoxy keto acid,uric acid,tyramine,phytosphingosine,LPC(18:2),LPC(16:0),docosahexaenoic acid,arachidonic acid and linoleic acid.A total of 14 metabolites were identified as potential biomarkers in urine samples,including beta glutamic acid,ornithine,L-allo-threonine,stachyose,fructose,sorbitol,pyruvic acid,creatine,iodohippurate,benzonic acid,acetic acid,guanidine-succinic acid,succinate and spermidine.It mainly involved energy metabolism(fatty acid oxidation pathway and glucose metabolism),amino acid metabolism(arginine and proline metabolism,arginine and ornithine metabolism,tyrosine metabolism,phenylalanine metabolism,metabolism of glutamate,glycine,serine and threonine metabolism and tryptophan metabolism),purine metabolism,sphingolipid metabolism,glycerol phospholipid metabolism,arachidonic acid metabolism and a series of metabolic processes.The metabonomics approach could provide a systematic view of acute blood stasis-related metabolism,and reveal the mechanism of therapeutic effect of GXSTC in treating acute blood stasis. |
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