The Metabolism And Pharmacokinetics Of Capilliposide B And Capilliposide C In Lysimachia Capillipes Hemsl Extract

Lysimachia capillipes Hemsl (Primulaceae), a folk medicinal plant, is used as a remedy for the treatment of colds and rheumatoid arthritis. A series of triterpenoid saponins and flavonoids have been isolated and elucidated, among which capillipo-side B (LC-B) and capilliposide C (LC-C) are the major components. In previous work, capilliposide, total saponins of L. capillipes Hemsl extract (LCE), could effectively inhibit tumor growth in animal models. In addition, in vitro study of LC-B and LC-C indicated significant cytotoxic activities against various human cell lines. However, there is little knowledge about absorption, distribution, metabolism and excretion (ADME) of LC-B and LC-C in vivo. Therefore, to provide scientific evidence for further development of LC-B and LC-C as remedies, this study will investigate the metabolism and pharmacokinetics of LC-B and LC-C in vitro and in vivo.1. The pharmacokinetic study of LC-B and LC-C in ratA reliable LC-MS/MS method was developed and validated for simultaneous determination of LC-B and LC-C in rat plasma. Rat plasma and whole blood samples were pretreated with dichlorvos, an esterase inhibitor, preventing the hydrolysis of analytes in biological samples. LC-A, the potential hydrolytic metabolite of LC-B and LC-C, was characterized by LC-QTRAP-MS/MS. According to the result of PK study of LC-B and LC-C in rat, the system exposure of LC-B and LC-C was very low after oral administration of LCE, indicating the bioavailability of LC-B and LC-C in rat was poor.2. The biotransformation of LC-C in vitro by rat intestinal microflora and the biological activity evaluation of its metabolite poolLC-C was anaerobically incubated with pooled rat intestinal microflora.92.7%LC-C was biotransformed in rat intestinal microflora. Human cancer cell lines HepG2, PC-3and A549were treated with metabolites pool of LC-C, respectively, which followed by cell viability assays. The metabolites were characterized using UHPLC-QTOF-MS/MS. As a result, significant cytoxicity was observed for the metabolites pool, from which six metabolites were identified.3. The preparation and bioactive evaluation of LC-ALC-A, esterolysis product of LC-B and LC-C, was prepared by sodium hydroxide, and was elucidated by mass spectrometry and nuclear magnetic resonance. The purity of LC-A was98.2%determined by HPLC-ELSD. In vitro cytotoxicity study, LC-A significantly inhibited the proliferation of human cancer HepG2cells. The IC50was17.46±1.55μg/mL. 4. The metabolism of LC-A, LC-B and LC-C in vitro and in vivoThe metabolic stability of LC-A, LC-B and LC-C was performed by rat liver microsomes and human liver microsomes in vitro, respectively. As a result, these compounds were stable in either rat liver microsomes or human liver microsomes, which suggested that CYP450s was not the major enzyme involved in their metabolism. An UHPLC-QTRAP-MS/MS method was employed to investigate the in vivo metabolism of LC-B and LC-C after oral administration of LCE in mice for10days. Consequently, twenty one metabolites were characterized in plasma, urine and feces. The deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-B and LC-C in mice. In addition, some phase I metabolites were also detected in feces.5. Tissue distribution of LC-A, LC-B and LC-C after intravenous administration ofLC-B and oral administration of LCE in miceA LC-MS/MS method was developed and validated for simultaneous determination of LC-A, LC-B and LC-C in tissues. LC-B was widely distributed throughout the whole body except for muscle and LC-A was distributed in liver and intestine after intravenous administration. LCE was orally administrated to mice continuously for10days. Both of the LC-B and LC-C were distributed in various tissues. LC-A was mainly found in stomach and intestine.6. The excretion of pure LC-B following intravenous administrationWe developed and validated a LC-MS/MS method to simultaneously determined LC-A, LC-B and LC-C in rat urine and feces. The SPE procedure with urine and feces samples directly acidified with1%formic acid was optimized, which allowed LC-B to be reproducibility retained on SPE sorbent. To overcome the nonspecific binding in urine assays,3mM CHAPS, as anti-adsorptive agent, was added to urine. The total urinary and fecal accumulative ratio of LC-B and LC-A in rat was9.69%, which indicated that LC-B may be excreted as other metabolic forms.