Metabolic Profiling,Pharmacokinetics Combined With Quality Control Research Of Xiaokeyinshui Extracts Combination

Xiaokeyinshui extract combination（XEC）was developed from Xiaokeyinshui formula,which was recorded in Bencaogangmu.XEC was the mixture of Coptidis Rhizoma extracts,Liriopes Radix extracts,Momordica Charantia extracts and Cassiae Semen extracts.Our previous research clarified the chemical composition of four herbal extracts,the pharmacological action and the underlying mechanism of XEC.However,the therapeutic material basis and quality markers of XEC were still unclear.Thus,this thesis was focused on the investigation of metabolic profiles of XEC,which was helpful to further clarify the therapeutic material basis and quality markers of XEC.Based on this,the systematic quality control methods of XEC were established.The results of this research were as follows.1.Identification of prototypes and metabolites of XEC in vivo.A UPLC-QE-HRMS method was developed to characterize the ingredients of XEC in plasma,urine,feces and bile samples of rats.After administration of XEC,132 and 152 ingredients were identified in normal rats and T₂DM rats,respectively.Various structural compounds were found,including alkaloids,triterpenoid saponins,anthraquinones and naphthopyrones.Berberine,aruantio-obtusin,coptisine,epiberberine and palmatine were the representative components of XEC.The main metabolic pathways of XEC included demethyleneization,demethylation,hydroxylation,hydrolysis,reduction,methylation,sulfation and glucuronidation.Besides,3 prototypes（demethyleneberberine,dedimethylberberine and berberrubine）and 14 phase II metabolites（i.e.glucuronide conjugates desmethylenorrubrofusarin,glucuronide conjugates torachrysone,and sulfonic acid conjugated demethylaruantio-obtusin）were only found in T₂DM rats.2.Tissue distribution of XEC.The xenobiotics of XEC in heart,liver,kidney,lung,spleen and ileum of T₂DM rats were characterized by the same UPLC-QE-HRMS method.A total of 87 ingredients were identified in these tissues.Prototypes were distributed widely in all tissues,while metabolites were mainly distributed in the liver,kidney and ileum.Furthermore,a rapid UPLC-MS/MS method was developed to determine the content of berberine,coptisine,epiberberine,palmatine,magnoflorine and aruantio-obtusin in liver.The T_max of berberine,palmatine,epiberberine and aurantio-obtusin occurred at 1 h after administration,while the T_max of coptisine and magnoflorine was located at 2 h.3.Pharmacokinetics（PK）of XEC.A rapid UPLC-MS method was established for the determination of berberine,coptisine,palmatine,epiberberine,berberrubine,magnoflorine and aurantio-obtusin of XEC in plasma.Compared with the Xiaokeyinshui extract combination-normal rats group（XEC-NC）and Coptidis rhizoma extracts-type 2 diabetes mellitus group（CRE-T₂DM）,the T_max values of berberine,palmatine,coptisine,epiberberine and magnoflorine increased,while the C_max values of them decreased in Xiaokeyinshui extract combination-type 2 diabetes mellitus group（XEC-T₂DM）.Besides,the C_max of berberrubine and AUC_0-tof all alkaloids increased in XEC-T₂DM group.As for the aurantio-obtusin,there was no significant difference of the PK parameters between XEC-NC group and XEC-T₂DM group.The results indicated that the diabetes status and the compatibility of XEC could decrease the absorption rate and increase the total uptake of berberine,coptisine,palmatine,magnoflorine and epiberberine,while not for aurantio-obtusin.4.Quality control of XEC.The results of metabolic profiles,tissue distribution and PK research unclosed the pharmacodynamic substances and quality markers of XEC.Based on this,thin layer chromatography（TLC）methods were developed for the identification of huang lian,bitter melon and jue ming zi in XEC.The fingerprints of 10batches of XEC were then illustrated by HPLC-UVD and HPLC-ELSD methods.There were 11 and 22 common peaks in HPLC-UVD and HPLC-ELSD fingerprints,respectively.the similarities of the fingerprints of XEC were all higher than 0.90.Besides,by a HPLC-UVD quantitative method,the average contents of berberine,coptisine,palmatine,epiberberine and aurantio-obtusin in 10 batches XEC were determined as 7.08%,2.45%,1.26%,0.71%and 0.20%,respectively.And the content of total polysaccharides in XEC was 31.91%by a UV quantitative method.In this study,the results of metabolic profile,tissue distribution and pharmacokinetics research confirmed the pharmacodynamics materials and quality markers of XEC.Various analytical techniques were used in the development of qualitative and quantitative methods,which laid a foundation for the research of XEC,as well as provided a successful example for the quality control research of traditional Chinese medicine.