Studies Of Pharmacokinetics Of Dahuang Mudan Decoction And Metabonomic On IBD Rats

Dahuang mudan decoction(DMD),originally described in "Synopsis of Golden Chamber" in Eastern Han Dynasty,is a famous Traditional Chinese Medicine(TCM)formula composed of Rhubarb,Moutan bark,Prunus persica,Wax gourd kernel(seeds of Benincasa hispida)and Mirabilite(a mineral,Na2SO4·10H2O).DMD has been widely utilized for more than thousand years to treat intestinal carbuncle in China,thus far new pharmacological logical effects of DMD were continuously found,such as appendicitis,inflammatory bowel disease,pelvic inflammatory disease and acute pancreatitis.Inflammatory bowel disease(IBD)is a violation of the digestive tract and accompanied by abnormal immune systemic disease,its pathogenesis features characterized by repeated attacks,which makes it difficult to cure.In recent years,the incidence of IBD is on the rise globally.With the changes of lifestyle and dietary habits of Chinese,the incidence of IBD is on the rise in China,which make it increasingly important to reveal the pathogenesis and the mechanism of drug treatment of IBD.So far,there is no study which can systematically elucidating the mechanism of DMD in the treatment on IBD.DMD contains many chemical constituents,but not all of them are active ingredients,in this case of unclear pharmacodynamic material basis,the research on the mechanism of action of DMD will be limited.At present,some effective methods such as active site study,fingerprint study,serum pharmacokinetics,pharmacokinetics and metabolomics have been established in the basic research on pharmacodynamics of formulas.Therefore,this paper aims to reveal the pharmacodynamic material basis of DMD and investigate the mechanism of its treatment of IBD.In this study,investigations of pharmacokinetics and metabolomics on the pharmacodynamic material basis of DMD and its effect on IBD were carried out.1.Firstly,a rapid and sensitive ultra-high performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry(UPLC-Q/TOF-MS/MS)was developed for the identification of chemical constituents in the DMD.Based on the accurate mass measurement,the reference substances,the retention times and the mass spectra fragmental patterns,63 compounds were identified or tentatively characterized in DMD.The study preliminarily illuminated the major chemical constituents of DMD.Moreover,27 prototype constituents were determined in rat's plasma after oral administration of extract of DMD.2.Then the plasma concentrations of five absorted bioactive constituents were simultaneously quantified and further applied into a pharmacokinetic study.The quantitative analysis methods of emodin,aloe-emodin,rhein,paeoniflorin and amygdalin in rat plasma were established and applied to the pharmacokinetic study.The results show that the five compounds have good linear relationships between the determination of concentration range(r>0.997).The developed method was validated for sufficient specifificity,precision,accuracy and sensitivity.The extraction rate and the matrix effect of the analyses were all ranging from 84.79%-100.38%and 86.3%-112.2%,respectively.And the intra-day precisions were all less than 12.09%,and the inter-day precisions were all less than 6.61%.Which showed that the established method is simple feasible,accurate and reliable and has good reproducibility and would be useful for the pharmacokinetic study of five compounds of DMD.3.Establisment of IBD model and the DMD's therapeutic effect evaluation.32 male SD rats were randomly divided into four groups with eight rats in each group.And each group was arranged as follows:control group,model group,DMD group and Mesalazine group.The IBD rats were established with 2,4,6-trinitrobenzenesulfonic acid(Trinitrobenzene sulfonic acid,TNBS)/ethanol(90 mg/kg).To evaluate the intervened effect of DMD,the general ethology,blood routine,serum inflammatory cytokine levels and the intestinal histopathological changes of all rats were detected and observed.The results showed that,in Model group,disease activity index(DAI),colon injury,while blood cell counts(WBC),lymphocyte counts(Lymph),granulocyte counts(Gran)and red blood cell counts(RBC)were significantly higher than those in the control group(P<0.05).Meanwhile,the content of inflammatory cytokines including IL-6 and TNF-a,in vivo,were increased compared with control group(P<0.05).Above results were in accordance with the evaluation indexs of IBD.However,after intervention with DMD,the blood routine indexes and the inflammatory cytokines in vivo,tended to be normal,and the histopathological changes of intestine were improved,which indicated that DMD has a good therapeutic effect on alleviating IBD symptom,regulating immunoreation and releasing the intestinal injury.4.Investigation of the therapeutic mechanism of DMD on IBD rats by using metabolomic approach.Serum and urinary metabolomic approachs based on UPLC-Q/TOF-MS/MS were performed to investigate the changes in the endogenous metabolites in order to evaluate the integral efficacy of DMD on IBD model rats.Principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminate analysis(OPLS-DA)were used to find the potential metablic markers and to screen the targeted metabolic pathways,which may be related to therapeutic effects of DMD on IBD rats.The results showed that 8 potential metabolic markers were screened from serum,which maybe related to the mechanisms of DMD on IBD model rats.Meanwhile,the results showed that 6 potential metabolic markers were screened from urine.Compared with the control group,the abnormal contents of the potential metabolic markers in IBD model rats were tended to be normal level after the intervention of DMD,and involved in Biosynthesis of phenylalanine,tyrosine and tryptophan,phenylalanine metabolism,glycine,serine and threonine metabolism,arginine and proline metabolism,sphingolipid metabolism,cysteine and methionine metabolism,valine,leucine and isoleucine biosynthesis and caffeine metabolism.etc.Based on UPLC-Q/TOF-MS/MS,the study has clarified the main chemical basis of DMD and revealed the pharmacokinetic characteristics of the five main components absorbed into rats.It was found that DMD had a good therapeutic effect on TNBS/ethanol-induced IBD rats,and the mechanism of its treatment of IBD was revealed from the perspective of metabolomics.