The Identification Of Constituents And Metabolites In Lishukang Capsule And The Effects Of Lishukang Capsule On The Pharmacokinetics Of Olsalazine

Lishukang capsule?LSK?is an anti-hypoxia Tibetan medicine prescription.It is composed of Gymnadenia conopsea?L.?R.Br,Dracocephalum tanguticum Maxim,Rhododendron anthopogonoides Maxim,Rhodiola kirilowii?Regel?Maxim,Phellodendron amurense Rupr and Glycyrrhiza uralensis Fisch.Some constitutions of individual herbs in LSK were reported previously.However,the active constitutions of LSK remain unclear.UHPLC-HRMS was used for the analytical characterization of traditional Chinese medicine?TCM?prescriptions widely.Complexity of chemical components in TCM prescriptions made data processing difficult and time-consuming.Up to now,a rapid and widespread data analysis pattern concentrated on identifying both phytochemical components in vitro and their metabolites in vivo of TCM prescriptions has rarely been reported.TCM or probiotics combined with 5-aminosalicylic acid?5-ASA?agents were two major therapies to improve the inflammatory bowel disease?IBD?treatment effect and reduce clinical relapse rate.Pharmacological advantages of drugs combined administration have been explored in many preclinical and clinical studies,but the effects of TCM or probiotics on the pharmacokinetics of 5-ASA agents had rarely been reported.In this study,a new MS-network analysis pattern concentrated on mass spectral data processing of TCM prescriptions had been developed based on UHPLC-Q-TOF-MS to identify constituents and metabolites from LSK.A pharmacokinetic study foucsed on evaluateing the effects of LSK?an example of TCM?and YJK?Lactobacillus acidophilus,an example of probiotics?on OLZ?an example of 5-ASA agents?had been carried out in UC rats based on UHPLC-QQQ-MS.The study was consisted of two chapters.Chapter 1:A new MS-network analysis pattern for rapid identification of constituents and metabolites from LSK based on UHPLC-Q-TOF-MSObjective:To develope a rapid method for TCM prescriptions mass spectral data processing based on UHPLC-Q-TOF-MS.Using the method to identify the constituents and metabolites of LSK.Methods:The chromatographic separation was performed on a Waters ACQUITY UPLC BEH T3 column.Methanol-acetonitrile?1:1,v/v?-aqueous with 0.1%formic acid were selected as the mobile phase.Mass spectrometry detection was used information dependent acquisition?IDA?mode under both positive and negative ion mode.A new MS-network analysis pattern integrated the intrinsic structural correlation of phytochemical components with their MS properties was established in the date processing.The MS-network analysis pattern was applied to identify the constituents and metabolites of LSK.Results:A series of MS-Networks including flavones and flavone glycosides,alkaloids,phenolic acids,saponins and benzylester glucosides were preliminary established by MS-network analysis pattern.278 compounds including 9 potential novel compounds in LSK were unambiguously assigned or tentatively identified.57 prototype constituents and metabolites of LSK in rat plasma were identified.The possible metabolic pathways of metabolites were investigated by MS-network analysis pattern further.Conclusion:The MS-network analysis pattern could give an integral solution in the qualitative analysis of TCM prescriptions and set up a bridge between phytochemical compounds and its metabolites.The investigations for LSK also provided a basis for its further study.Chapter 2:A pharmacokinetic study on evaluating the effects of LSK and YJK on OLZ in UC rats based on UHPLC-QQQ-MSObjective:To develope and validate a method for the determination of olsalazine?OLZ?and its main metabolite 5-aminosalicylic acid?5-ASA?and N-acetyl-5-aminosalicylic acid?N-AC-5-ASA?in rat plasma based on UHPLC-QQQ-MS.Applying the method to evaluate the effects of LSK and YJK on the pharmacokinetics of OLZ.Methods:Acetoxyacetic acid was used as internal standard?IS?.Protein precipitation process was used to extract OLZ,5-ASA,N-AC-5-ASA and IS from plasma.The chromatographic separation was achieved with a mobile phase consisting of 0.2%formic acid,10 mM ammonium acetate in acetonitrile/water?50:50,v/v?and 0.2%formic acid,10 mM ammonium acetate in acetonitrile/water?95:5,v/v?by using gradient elution on a Waters ACQUITY UPLC BEH Amide column.Mass spectrometry operated under the multiple reaction-monitoring?MRM?mode using the electrospray ionization technique.After treated different groups of TNBS-induced colitis rats with LSK?at a dose of 2 g/kg/d?and YJK?at a dose of 1×10⁷ CFU/d?for 21 days,pharmacokinetic studies of OLZ,5-ASA and N-AC-5-ASA was carried out at single oral dose of 100 mg/kg OLZ.Results:The method had only 6 min run time.The achieved LLOQ of OLZ,5-ASA and N-AC-5-ASA was 10 ng/mL.Three analytes had an excellent linearity in the range of 10–1000 ng/mL?R²>0.99?.The intra-and inter-day precisions and accuracies were within±14%.OLZ,5-ASA and N-AC-5-ASA were stable during different conditions.The The pharmacokinetic results showed that LSK had the tendency to reduce the C_maxax of OLZ,but there was no significant difference compared with the control group?p=0.088?,and YJK had a tendency to increase OLZ t_1/2,but there was no significant difference compared with the control group?p=0.061?.YJK had no effect on the exposure of 5-ASA and N-AC-5-ASA in blood,the AUC_0-t of 5-ASA and N-AC-5-ASA in the YJK group were basically similar to the control group?5-ASA,P>0.839;N-AC-5-ASA,P>0.898?.Although LSK had a tendency to reduce the exposure of 5-ASA and N-AC-5-ASA in blood,there were no significant difference in the AUC_0-t of 5-ASA and N-AC-5-ASA between LSK group and control group?5-ASA,P>0.225;N-AC-5-ASA,P>0.176?.The T_max of 5-ASA and N-AC-5-ASA in LSK group was significantly delayed 2 h compared with the control group?5-ASA,P>0.011;N-AC-5-ASA,P>0.011?.Conclusion:In this experiment,LSK and YJK did not increase the exposure of OLZ,5-ASA and N-AC-5-ASA in blood,and had no effect on their pharmacokinetics.So there was no risk of their combined administration for IBD treatment.The developed LC-MS method can be used to investigate the pharmacokinetic effects of other TCM or probiotics on other 5-ASA agents.