Studies On The Pharmacokinetics Of Antitumor Constituents From Cudrania Tricuspidata

Cudrania tricuspidata (Carr.) Bur. belongs to the Moraceae family and is widely distributed over China, Korea, and Japan. Its roots are applied in clinic for the treatment of digestive apparatus tumor, especially gastric carcinoma, and are also used as Chinese folk medicine "Chuan-po-shi" together with the roots of C. cochinchinensis (Lour) against gonorrhea, rheumatism, jaundice, boils, scabies, bruising, and dysmenorrhea. In early 1980s, Zhemu syrups was made from it’s root extract for the treatment of gastrointestinal cancer. But its active ingredients were not clear and there were no qualitative and quantitative standards for the quality control. Our preliminary research showed that the extract of C. tricuspidata had significant anti-tumor activity, and synergistic effect is showed when flavonoids from Cudrania tricuspidata combined with chemotherapeutics. Investigations on the bioactive constituents indicated that most of the isoprenylated xanthones possessed significant cytotoxicity. To the best of our knowledge, there is no report on the pharmacokinetics of isoprenylated xanthones. Therefore, the present study focuses on the pharmacokinetics of antitumor constituents in order to promote the research and development of C. tricuspidata.1. Isolation and identification of antitumor constituents from C. tricuspidata.Repeated column chromatography and semi-prepared HPLC of the ethanol extract of C. tricuspidata afforded five isoprenylated xanthones, cudratricusxanthone B (1), macluraxanthone B (2), cudratricusxanthone D (3), cudratricusxanthone N (4), and cudratricusxanthone O (5). Compounds 4 and 5 are new compounds, whose structures were identified by spectroscopic method. Compound 4 was screened for cytotoxicity against six kinds of human tumor cell lines, including gastric carcinoma (BGC-823), lung adenocarcinoma (A549), melanoma (K111), vascular endothelial (ECV-304), colon carcinoma (LS174T), and erythromyeloblastoid leukemia (K562). It showed significant inhibitory effects on the six kinds of cell lines with IC50 values of 2.51-11.72μg/mL.2. Pharmacokinetic study of the antitumor constituents of C. tricuspidata in rats.Cudratricusxanthone B (CXB) and cudratricusxanthone N (CXN) with significant antitumor effects and high content were selected as the representative bioactive constituents from C. tricuspidata for the pharmacokinetic study. (1) Pharmacokinetic study of CXB in ratsAn HPLC-ESI-MS/MS method for the quantification of CXB in rat plasma was developed and validated. The method involved liquid-liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C18 column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the cudraxanthone H (I.S.). The method was sensitive with an LLOQ at 1.0 ng/ml for CXB using 100μl rat plasma. The standard curves were linear over the concentration range of 1-500 ng/ml for CXB in rat plasma. The intra-and inter-batch accuracy for CXB at four concentrations was between 100%±15%, the RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/ml using 100μl of plasma. The results indicate that it is suitable for the analysis of CXB in biological samples.The method was successfully applied to the pharmacokinetic study of CXB in rats. After an intravenous administration of CXB 5.0 mg/kg to male Sprague-Dawley (SD) rats, the concention-time profiles were fitted a three-compartmental model. The results showed that CXB distributed quickly and eliminated slowly after, and AUC0-t was 566.0±76.4 ng h/mL.(2) Pharmacokinetic study of CXN in ratsAn HPLC-ESI-MS/MS method for the quantification of CXN in rat plasma was developed and validated. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 395.0/339.1 for CXN and m/z 381.6/269.2 for the cudraxanthone H (I.S.). The other conditions were the same as the method for the quantification of CXB. The method was sensitive with an LLOQ at 1.0 ng/ml for CXN using 50μl rat plasma. The standard curves were linear over the concentration range of 1-500 ng/ml for CXN in rat plasma. The intra-and inter-batch accuracy for CXN at four concentrations was between 100%±15%, the RSDs were less than 9.85%. The lower limit of quantification for CXN was 1.0 ng/ml using 100μl of plasma. The results indicate that it is suitable for the analysis of CXN in biological samples.The method was successfully applied to the pharmacokinetic study of CXN in rats. After an intravenous administration of CXN 2.0 mg/kg to male SD rats, the concention-time profiles were fitted a three-compartment model. CXN was distributed quickly and eliminated slowly, and AUC0-twas 354.6±106.7 ng h/mL. After an oral administration of CXN 40 mg/kg, the concention-time profiles could not be described by the classical compartmental models. The absolute bioavailability was only 5.92%.3. Preliminary study on the mechanism of absorption of antitumor constituents of C. tricuspidata.Cudratricusxanthone N (CXN) was selected as the representative bioactive constituent from C. tricuspidata for the study on the mechanism of absorption.Caco-2 cell was used to study on the mechanism of absorption of CXN, the effects of time, pH, temperature, concentration of donor solutions and P-glycoprotein inhibitor on the uptake of CXN were investigated. The determination of CXN was performed by HPLC-UV. The uptake of CXN increased almost linearly when the concentration of CXN varied from 6.25-50μg/mL. Passive diffusion played a major role in the uptake of CXN. The uptake of CXN is positively correlated to uptake time and temperature. The P-glycoprotein inhibitor Verapamil had no effect on the uptake of CXN, while cyclosporine A decreased the uptake of CXN on the condition of high concentration of donor solutions of CXN.