The Study On Metabolomics And Pharmacokinetics Of Shexiang Baoxin Pill

Traditional Chinese Medicine (TCM) formulae are multi-herbal medicines, which consistof principal, subordinate, adjuvent and guide drugs, with characteristic of multi-components,multi-targets and multi-effect pathways. TCMs are the centralized reflection of spirits ofintegrity and syndrome differentiation. TCMs have been widely used in clinic for2000yearsand many of them obtained satisfied therapeutic effects for coronary heart disease (CHD). Asthe complex pathology of CHD, TCMs' treatment has more significant advantages than thosesingle-targeted medicines. Those bioactive compounds in TCMs can regulate differentpathways simultaneously and play the synergistic effect, thus inhibit the development of CHD.On the other side, the bioactive components have low affinity with their targets which causethe low toxicity of TCMs lead to the long administration of TCMs.Cardiovascular diseases (CVDs) is one of the largest killers in the world, of whichcoronary heart disease (CHD) is the No. one. As the complexity mechanism of CHDdevelopment, the effect of single target medicine was not significant. The mortality of CHD isstill not decreased. Nowadays, there are some efficient TCMs for the prevention and treatmentof CHD in clinic, including SuXiaoJiuXin Pill, Danshen Pill, Danshen Tablet and ShexiangBaoxin Pill (SBP). The remedy effectiveness of these TCMs have been confirmed andverified through a long time clinical application. But there are some defects which still existin TCMs such as unknown components, unclear mechanism and difficulty in quality controletc. So it is urgent to carry out depth study on TCMs using modern technology to reveal themechanism, control the quality, produce rational praeparatum and carry forward the spirit ofour traditional medical system.In this dissertation, the mechanism of SBP was investigated by using metabolomicmethod, and the absorption, metabolism and pharmacokinetic character of main bioactivecomponents in SBP were also discussed. Through metabolomic study, we identified someacute myocardial infarct (AMI) related pathways in metabolite level, established themetabolite network in the development of AMI, demonstrated the reverse effect of SBP inAMI model rats, revealed the mechanism of SBP in molecular level. Metabolomic studydemonstrated that SBP can inhibit the development of AMI through regulatingmulti-pathways. On the other side, this study made up a deficiency in the pharmacokinetic field. We analyzed the different absorption ability of main bioactive compouds in SBP in aCaco-2model, discussed their main metabolism enzemys and species differences. The studywill profit the investigation of their changes in vivo. We also studied the pharmacokinetics ofginsenosides and bufadienolides in SBP in vivo. The study demonstrated the efficiency andrationality of TCMs' compatibility.In the study of metabolomics, we firstly investigated the prevention effect of SBP in AMImodel. To identify the biomarkers in early period of AMI in rat serum, a metabolomicapproach using reversed-phase liquid chromatography/quadrupole time-of-flight massspectrometry (LC-Q-TOF-MS) was developed. By combining with partial least squaresdiscriminant analysis (PLS-DA),14biomarkers in rat serum were identified. Therse identifiedbiomarker are mainly related to inflammation, hypertrophy and oxidative injury process. It isdemonstratd that inflammation, hypertrophy and oxidative injury are the most relatedpathological changes in early period of AMI. Through analysis of the network of theseidentified biomarkers, hypertrophy pathway showed seriously disturpted. These biomarkersnot only supplied a systematic view of the progression of AMI in early period but alsoprovided the theoretical basis for the prevention or treatment of AMI. SBP showed satisfiedprevention effects in AMI rat. The effects of SBP were not only showed in scoring plot, whichdemonstrated the state of serum in SBP group was near sham group, but also in the results ofbiomarkers' regulation. SBP could reverse4biomarkers to normal level includingcorticosterone, aldosterone, cortisol and epinephrine. These reversed biomarkers mainlyinclude in the process of myocardial hypertrophy. So it demonstrated that the pretreatment ofSBP could inhibit the pathway of myocardial hypertrophy, thus receiving protective effects onAMI.On the other side, we also investigated the treatment effect of SBP using metabolomicmethod in rat plasma and urine with14days administration SBP (14mg/kg/d) after AMImodel finished. Urine and serum in3,7,15d were collected and used for biochemical andmetabolomic analysis. The serum enemzy determination and infarction areaanalysisdemonstrated that the AMI model was successful and SBP definitely had thetherapeutic effects. In the serum metabolomic study,15biomarkers in rat plasma wereidentified by combining with PLS-DA. The regulated biomarkers are mainly related to theenergy metabolism, oxidative stress, hypertrophy and inflammation pathways. SBP coulddefinitely inhibit the AMI development and reverse some pathological biomarkers including deoxyinosine, glycerate, cytidine, LTA4and deoxycorticosterone to normal level. As LTA4isthe metabolite of arachidonic acid, and cytidine and deoxycorticosterone are both myocardialhypertrophy related factors, it suggested that SBP can inhibit inflammation and cardiachypertrophy, thus protecting the myocardium and achieving the aim of treatment. To obtainthe comprehensive acknowledgement about SBP' therapeutic mechanism, the urinemetabolomic study was also done. In the study of urine metabolomic study,16biomarkerswere identified, and8of them were related to energy metabolism pathway. Through theestablishment of metabolites network, it exhibited that citric acid cycle related network ofenergy metabolism was acutely perturbed. It could be seen that SBP could reverse the urinestate of the AMI rat to normal through scoreing plot. SBP also could regulate some identifiedbiomarkers to the normal level such as creatine, uridine, glutamate, oxalosuccinic acid andnicotinamide mononucleotide. As these5biomarkers are all related to energy metabolism, itdemonstrated that the therapeutic effects of SBP are mainly through regulating disturpedenergy metabolism pathway. Through the urine and serum metabolomic analysis in AMI rats,we deeply discussed AMI AMI related mechanism and revealed the therapeutic mechanism ofSBP, which is mainly throuh inhibiting inflammation, myocardial hypertrophy and regulatingthe dysfunction of energy metabolism.In pharmacokinetics study of SBP, vivo and vitro method were used to study thecharacters of metabolism and kinetics of main bioactive components. In vitro experiment, aCaco-2absorption model was used to evaluate the transport ability of borneol, muscone,cinnamic acid, cholic acid, ginsenoside Rb1, ginsenoside Re and bufalin (main bioactivecompounds in SBP) in intestine and found the enhance effect of borneol and muscone on theabsorptive transport of ginsenosides. Then we used human and rat liver microsome to analyzethe metabolism enzemys and species difference of these bioactive components. The resultsdemonstrated that the metabolism enzemys of borneol, isoborneol and bile acid are uridinediphosphate－glucuronosyltransferases (UGTs), and the cytochrome p-450(CYPs) are themain metabolism enzemys of ginsenosides and bufadienolides. Species differences existed inthe metabolism pathways of bufalin and bile acid. On the other side, the absorptionenhancement ability of borneol was conformed through pharmacokinetics study in vivo. Thepharmacokinetics characters of ginsenoside Rb1, Rc, Re and Rg1were firstly comparedbetween SBP and SBP without borneol exist. The study demonstrated that theconcentration-time curves in SBP were definitely different by comparing with those in SBP without borneol exist. The ginsenosides' concentration in blood could be highly elevated withthe borneol existed simultaneously. To further confirm the enhancement effect of borneol inginsenosides' absorption, the pharmacokinetics parameters after oral administration of singleginsenoside compound or single ginsenoside combined with borneol was studied. The resultdemonstrated that borneol can definitely enhance the ability of ginsenosides' absorption andevaluate their blood concentration. The pharmacokinemics study of bufadienolides in SBP invivo exhibited that the the bufadienolides have smaller Tmax and higher Cmax after oraladministration of Venenum Bufonis than those after oral administration of SBP. Though theCmax was lower after SBP administration, the areas of concentration-time curves were notsignificantly changed. It is demonstrated that the pharmacokinetic characters of buadienolideswere significantly changed after SBP compatibility. The study results facilitated themechanism study of efficacy enhancing and toxicity reducing of SBP.