New Methods And Strategies For Multi-component Pharmacokinetics Of Traditional Chinese Medicine

Multi-component pharmacokinetics of Traditional Chinese Medicine(TCM)is important for clarifying its theory and mechanism.Due to the complexity and diversity of TCM components,and their complex and uncertain interactions with biological system,there are many bottleneck problems for the multi-component pharmacokinetics of TCM,such as(1)the complex chemical composition and diverse metabolic reaction make it difficult to annotate their structures in vivo;(2)endogenous interferences from matrix,and lack of authentic standards bring difficulties to the establishment of qualitative and quantitative methods;(3)the unclear pharmacodynamic index and active components hamper the evaluation of pharmacokinetic results.Therefore it is necessary to apply advanced techniques to establish the fast,accurate,comprehensive qualitation methods and the sensitive,fast,accurate,wide dynamic range quantitation method for multiple components derived from TCM.It is also important to establish new strategies from a deeper and broader perspective,and to support the multi-component pharmacokinetic study of TCM.In this thesis,aimed at some bottlenecks in the multi-component pharmacokinetics of TCM,Epimedii Wushanensis Folium(EWH),active fraction of Xiao-Xu-Ming decoction(AF-XXMD),and Er-xian decoction(EXD)were subjects to explore new techniques and strategies for the multi-component pharmacokinetics of TCM.The first part of this thesis proposed a systematic and logical strategy for the pharmacokinetic marker selection based on the comprehensive metabolic pathways to solve the bottleneck of the multi-component pharmacokinetics of TCM without authentic standards.The strategy first employed high-performance liquid chromatography coupled with high resolution and multiple stage mass spectrometry(HPLC-HRMSn)and mass spectral trees similarity filter technique(MTSF)to identify metabolites derived from five main prenylflavonoid monomers,and to deduce the metabolic pathways of five prenylflavonoids from metabolite identification results.A total of 14 mainly exposed compounds including prototypes and glucuronide conjugates were selected as potential pharmacokinetic markers.The pharmacokinetic profiles of 14 major compounds were monitored in unhydrolyzed plasma after oral administration of 5 prenylflavonoid monomers by a high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method;Due to the lack of authentic standards for glucuronide conjugated metabolites,enzyme hydrolysis was used to transform 14 major compounds to 10 potential markers with authentic standards and metabolic relationships.The HPLC-MS/MS method was then established for the quantitation of 10 potential markers in hydrolyzed plasma.For chromatographic separation,a quaternary gradient separation effect was achieved using the binary liquid pumps through the optimization of the composition and mixing time of mobile phases A and B,and the EpiA-C/icariin cross-talk and icaritin matrix effect were eliminated;For mass spectrometric detection,the segmented multiple reaction monitoring mode was used to shorten the duty cycle and increase the sensitivity of the analytes.The established method was well validated and applied to determine the pharmacokinetic parameters of 10 potential marker compounds in plasma after oral administration of five prenylflavonoid monomers and EWH.A Kendall’s tau-b rank correlation analysis of the dynamic concentration changes of the detected compounds with time in the unhydrolyzed and hydrolyzed plasma was performed.One of our most important findings was that glucuronide conjugates of icaritin were the main circulating metabolites of prenylflavonoids after administration.Furthermore,total icaritin content accurately reflected both systemic exposure levels and dynamic changes of prenylflavonoids in rats.Icaritin,characterized by high abundance and a strong correlation with prenylflavonoid levels(r>0.5),could be used as the marker compound for the pharmacokinetics of prenylflavonoids in EWH extract.In addition,the pharmacokinetic properties of icaritin were incorporated into an integrated mathematical model to predict the exposure level of EWH in rats.The predicted and experimental AUC(area under the curve)values showed strong agreement,further supporting the hypothesis that icaritin in hydrolyzed plasma can be used as a pharmacokinetic marker for the dynamic exposure levels of prenylflavonoids of EWH.The second part of this thesis studied the multi-component pharmacokinetics of AF-XXMD in focal cerebral ischemic rats.A total of 68 comounds in AF-XXMD were analyzed and identified by an HPLC-HRMSn method and MTSF technique.In addition,21 abundant compounds were first identified from plasma and brain,and selected as effective candidates based on pharmacological activity prediction by reverse molecular docking.A high resolution and rapid liquid chromatography tandem mass spectrometry(RRLC-MS/MS)was established to determine these effective candidates in plasma and brain after oral administration of AF-XXMD.In sample preparation,ascorbic acid was added into plasma to prevent the oxidation of baicalin and baicalein,and the lipid processing solid phase extraction cartridge was used to selectively remove the phospholipid compounds,which caused the matrix effect of glycyrrhizic acid in plasma and brain.For chromatographic separation,RRLC can effectively separate three pairs of isomers.For mass spectrometric detection,the segmented positive/negative ionization switching mode and trace addition of ammonium formate to mobile phases were employed to enable good mass response.The established method was well validated according to FDA guidance on bioanalytical method validation.Focal cerebral ischemia rats modeled by the permanent occlusion of bilateral common carotid arteries method were used in the present study.Plasma and cerebral pharmacokinetic differences of these effective compounds were compared between control and focal cerebral ischemic rats.Results indicated that the pharmacokinetic behaviors of AF-XXMD were found to significantly change in focal cerebral ischemic rats including the blood-brain permeability,cereral dynamic changes,brain exposure,and brain tissue distribution.In cerebral ischemic rats,flavonoids and chromones were identified as the predominant form in plasma,whereas chromones and alkaloids were identified as the major form in brain.The efficacy of AF-XXMD against cerebral ischemia is relevant to the synergistic effects of these compounds in targeting different receptors and pathways.The third part of this thesis proposed a feasible strategy to explore the pharmacodynamic basis of TCM from holistic view of disease treatment.EXD was used as subject investigated.Compound identification,metabolomics,and bioinformatics techniques were integrated to investigate the active material basis including exogenous compounds identified in TCM,endogenous metabolites disrupted by TCM,and relationships between these compounds and pharmacology or action mechanism.A new strategy was first developed to discover and identify unknown compounds of TCM.The strategy guarantees the fast discovery of candidate structural information and provides efficient structure clues for identification including four steps in sequence:(1)to establish an HPLC-HRMSn method,discover potential compounds,and obtain sub-structure information by the MTSF technique,based on high resolution mass(HRMS)and multiple-stage mass spectrometric(MSn)data;(2)to classify potential compounds into known chemical classes by discriminant analysis(DA)on the basis of retention time(RT)and HRMS data;(3)to hit the candidate structural information of compounds by intersection sub-structure between MTSF and DA;(4)to annotate and confirm candidate structures by isotope intensity distribution data.This strategy showed the high irrelevant ion exclusion efficiency over 41%while the structural information of 553 potential compounds were obtained and 71 compounds were identified.Based on 71 chemical components as templated compounds,a total of 163 compounds(including 13 prototypes)were identified by MTSF technique in rat plasma,urine,feces,and bile after oral administration of EXD.A high-throughput UHPLC-MS/MS method was developed for profiling both ketolic and phenolic sex steroids in biological samples.A total of 20 sex steroids were quantified in a single run within 28 min with 500 μL serum samples.The human serum,prepared through liquid-liquid extraction and subsequently derivatized using Girard P offline,was automatically injected twice under the automated injection program.For the first injection,Girard P-derivatized ketolic sex steroids were loaded onto the column,and subsequently,the second injection were online derivatized by dansyl chloride in the injector needle.The dansyl-labeled phenolic sex steroids were then loaded onto the column.The diverter valve worked in coordination with the injection program to import the derivatized sex steroids and remove excess derivatization reagents.The two types of derivatives were individually analyzed in a step-by-step manner.In method validation,the linear range was 2-400 pg/mL with the correlation coefficient higher than 0.988,and the lower limit of quantitation was 2-4 pg/mL.In addition,all analytes’ precision relative standard deviation were less than 20%with accuracy 80-120%.The method validation results indicated that this method could meet the requirements of quantifying sex steroids in biological samples.The effect of EXD on ovariectomy(OVR)rats was investigated at different time points.Four groups were set including a shamoperation,an OVR model,an OVR model treated with EXD,and an OVR model treated with nylestriol.Body weight,blood lipid index(total cholesterol,high density lipoprotein,low density lipoprotein,and triglycerides),and liver oil red staining were used to evaluate the model and EXD effect at different time points.At the same time,a lipidomics study was performed using sex steroid,lipid,and ceramide analysis platforms and statistical analysis to find biomarkers for the effect OVR pathology,EXD treatment and time lapse on plasma and liver metabolites.The mechanism of EXD regulatory effect on OVR-induced lipid disorder at different OVR stages was systematically clarified based on the lipid synthesis and metabolism pathways.Results indicated that EXD could effectively regulate the disorder of sex steroids and lipids of OVR.Of the investigated time points,4 week was noted as the most significant in terms of the biomarker numbers,including 15 glycerides,10 phospholipids,4 sex steroids in plasma and 8 glycerides,10 phospholipids in liver.In addition,the regulatory effect of EXD was closely associated with time in terms of the number,subclass,and repeated potential biomarkers at different OVR stages.Finally,reverse molecular docking of EXD-derived compounds in vivo was used to predict the active components and action mechanism of EXD regulation.Predicted active compounds were then used to bridge the gap between endogenous metabolites and chemical compounds in EXD.The regulation of EXD on the triglyceride biosynthesis pathway was further validated by the Western-blot and ELISA methods,further revealing the action mechanism of EXD.