Study On The In Vivo Process Of Constituents From Ilex Pubescens And Its Intervention On Stasis Model Rats According To Metabolomics

Clinical applications of traditional Chinese medicine(TCM)have drawn more and more attention,which provided a new thought for treating thrombotic diseases in those with contraindication and high-risk populations.Ilex pubescens Hook.et Arn(Aquifoliaceae),belonging to Ilex plants,mainly contains triterpenoids,iridoids,lignans,organic acids,etc.The dry root of I.pubescens natively calling Mao-Dong-Qing(MDQ)has the functions of clearing heat and detoxifying,activating blood circulation and removing blood stasis.Pharmacological investigations demonstrated that I.pubescens could treat cardiovascular and arterial thrombosis diseases.Some previous researches were difficult to interpret overall antithrombotic characteristics and in vivo mechanisms of I.pubescens.In this study,the chemical substances from I.pubescens and the in vivo action process of constituents were analyzed.The intervention of different parts of I.pubescens on stasis model rats according to metabolomics was also researched.Our purpose was to clarify the anti-thrombosis mechanisms arising from I.pubescens,which was characterized by multi-components and multi-pathways synergy.1.Qualitative and quantitative studies on the components of I.pubescens(1)Qualitative studies on the components of I.pubescens by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MS)UPLC-Q/TOF-MS technology was used to quickly and accurately identify various constituents of I.pubescens.Chemical profiles of I.pubescens were analyzed in both positive and negative ion modes.In the collected TICs,lignans and phenolic acids were mainly distributed in 0?10 min as well as triterpenoids mainly concentrating in 6?20 min.A total of 74 compounds in I.pubescens were identified in use of combining methods EIC and MDF.These identified components included 45 triterpenoids,4 lignans,3 iridoids,2 flavonoids and 20 other compounds of phenolic acids,amino acids and fatty acids.The components of I.pubescens were detected in plasma after administration based on the established method.As a result,a total of 45 chemical components were detected including 29 triterpenoids,16 other compounds including iridoids,lignans and fatty acids.(2)Simultaneously determination of 9 triterpenoids from I.pubescens by UPLC-Q/TOF-MSAccording to the previous studies,triterpenoids are the main active ingredients.Therefore,monitoring the content of triterpenoids was an important indicator controlling the quality of I.pubescens materials.A rapid and practical strategy based on UPLC-Q/TOF-MS had been established for simultaneously determination of nine triterpenoid compounds including ilexoside O,ilexsaponin B3,pedunculoside,ilexsaponin B2,ilexsaponin A1,ilexsaponin B1,ilexgenin A,calenduloside E and ilexpubson in I.pubescens.The content of nine triterpenoids in twelve batches of root,root bark and heartwood were analyzed.The content of ilexgenin A?ilexsaponin A1 and ilexsaponin B3 were generally more than that of the other six triterpenoids.Among them,ilexgenin A and ilexsaponin A1 were supreme content in I.pubescens.The concentrations of these nine triterpenoids in the root bark of I.pubescens were significantly higher than those in root and heartwood.2.In vivo metabolic transformation of triterpenoids from I.pubescensIlexgenin A(IA)and ilexsaponin A1(IA1)with supreme content were chosen as the marker compounds for the pharmacokinetic studies.The blood samples were collected via the oculi chorioideae vein according to the time schedule after orally administeration.The pharmacokinetics of IA and IA1 in vivo as well as the time-effect relationship of the metabolites were researched in order to provide reference for further study on the metabolic mechanism of triterpenoids from I.pubescens.(1)Pharmacokinetics of total triterpenoid saponins(IPTS)and the monomers(IA and IA1)from I.pubescensUPLC-Q/TOF-MS method for simultaneous determination of IA and IA1 in rat plasma was established by optimizing the liquid phase,the values of collision energy,characteristic ions and declustering potential,etc.In the present study,we not only investigated the pharmacokinetics of IA1 and IA in rats plasma,but also compared the pharmacokinetic profiles of the active components in rats after consumptions of IPTS versus pure compounds in negative ion mode.And the pharmacokinetics differences between the two analytes were compared after handling by Drug And Statistics(DAS)software.The two analytes showed good linearity for 0.38-1040 ng/mL(IA)and 0.13-1060 ng/mL(IA1).Meanwhile,the specificities,matrix effects,precisions,accuracies and stabilities of the two components were validated in accordance with the guidance for the bioanalytical methods validation.The results showed that the absorbtion of IA1 and IA was relatively fast,while the time to peak of IA transformed from IA1 was delayed at 5.67-6.33 h for Tmax.Under the developed analytical conditions,the obtained values of main pharmacokinetic parameters(Cmax and AUC0-t)indicated that the pure compounds were more efficient than the IPTS in IA1 and IA absorption.(2)Metabolites of IA and IA1The metabolites of IA and IA at 0,4,8,12,24 and 48 h in plasma after intragastric administration were identified by Metabolite PilotTM 1.5 and Peak View? 2.0 softwares.According to the retention time and MSMS information of the candidate metabolites,21 metabolites from IA and 32 metabolites from IA1 were identified.A variety of metabolite types have been concluded such as hydroxylation,oxidation,sulfate conjugation,glucuronidation,deglycosylation,methylation,demethylation,N-Acetylcysteine conjugation,S-Cysteine conjugation,etc.At 0-8 h,the most metabolites were identified.The number of the identified metabolites at 8-24 h and 24-48 h was relatively reduced.There were specific differences in metabolic types in different periods.At 8-24 h,M13 was presumed to be generated by the sulfate conjugation,while the metabolites M16 and M19 were detected at 24-48 h were possibly formed by the S-Cysteine conjugation and N-Acetylcysteine conjugation,respectively.3.The therapeutic effect of I.pubescens extract on blood stasis model rats according to pharmacodynamics and metabolomicsIn this study,an acute blood stasis model was established by adrenaline combined with ice bath.The antithrombotic effect of I.pubescens extract(IP)was evaluated and the non-targeted metabolomics was applied to discover the potential pathological mechanism.The anti-thrombotic efficacy among IP,total triterpenoid saponins(IPTS)and non-saponins fraction(IPNT)of I.pubescens were compared on the basis of the critical indexes.UPLC-Q/TOF-MS based on serum metabolomics was employed to find differential metabolites and metabolic pathways associated with blood stasis to elucidate the mechanism of antithrombotic activity of IP,which provided a reference to understand the multi-targets pharmacological actions for activating blood circulation and removing blood stasis arising from I.pubescens.(1)Antithrombotic effect of I.pubescens extractBiochemical indexes of hemorheology and coagulation function including platelet aggregration rate(PAR),whole blood viscosity(WBV),activated partial thromboplastin time(APTT),thrombin time(TT),prothrombin time(PT),fibrinogen concentration,thromboxane B2(TXB2),6-keto-prostaglandin F1?(6-keto-PGF1?),endothelin(ET)and endothelial nitric oxide synthase(eNOS)were detected for evaluating the effects of IP on the model rats.As a result,the antithrombotic effect of IP could be explained by the improvement of vascular endothelium damage,anticoagulation and inhibition of platelet aggregation.Furthermore,the effects of different fractions(IP,IPTS and IPNT)of I.pubescens on hemorheology,coagulation function and vascular endothelial function were investigated.The results showed that IP had the most significant effect followed by IPTS,and IPNT did not show a regulatory sign.(2)The therapeutic effect of IP on blood stasis model rats according to serum metabolomicsSerum metabolomics based on UPLC-Q/TOF-MS method was firstly applied to the overall profiling of serum metabolites in blood stasis rats.With pattern recognition analysis as principal component analysis(PCA)and orthogonal projections to latent structures discriminant analysis(OPLS-DA)by SIMCA software,a clear separation between model group and control group was achieved.The pathway analysis of the potential biomarkers was performed with MetPA.Among eighteen identified metabolites,eleven differential biomarkers reflecting the therapeutic effects of IP on blood stasis syndrome have been elaborated for the significant change.Metabolic pathway analysis showed that the key metabolic pathways associated with blood stasis were arachidonic acid metabolism and glycerophospholipid metabolism.(3)The therapeutic effect of IP,IPTS and IPNT on blood stasis model rats according to lipidomicsGlycerophospholipid metabolism was mainly potential lipid pathway for IP to exert efficacy.On the basis of discovery,CH2Cl2-CH3OH extraction method was adopted to enrich the lipids in serum.Lipidomics profiling was applied to investigate the therapeutic effects of different fractions(IP,IPTS and IPNT)of I.pubescens.The studies had found that sphingolipid metabolism and glycerolipid metabolism were as well as important targeting lipid pathways besides glycerophospholipid metabolism.The degree to regulate the differential markers as LPC(16:0),LPC(18:2),LPC(18:0),LPC(18:1)and CE(20:4)by different fractions of I.pubescens were compared.As a result,IP had the most significant effect followed by the IPTS,and the IPNT did not show a regulatory sign.By the researches mentioned above,1)the UPLC-Q/TOF-MS method for simultaneously analyzing the in vivo and in vitro chemical constituents of I.pubescens was established.2)the pharmacokinetic characteristics and metabolic regularity of typical triterpenoids in I.pubescens were elucidated.3)the antithrombotic mechanism of I.pubescens could be explained by regulating arachidonic acid metabolism and lipids metabolism integrated with improving vascular endothelial damage and inhibiting platelet aggregation according to pharmacodynamics and metabolomics.4)triterpenoids were potential pharmacodynamic substances from I.pubescens.The anti-thrombosis mechanisms arising from I.pubescens,which was characterized by multi-components and multi-pathways synergy,were clarified.It provides basic references for the further research of I.pubescens.