| Cirsium japonicum DC., a member of the family Compositae, is a wild perennial herb found in many areas of China, Korea and Japan. It is listed in the Chinese pharmacopoeias and has been used in Chinese medicine as an antihemorrhagic, antihypertensive and prevent epistaxis, metrorrhagia. Phytochemical investigation of Cirsium japonicum DC. proves that many constituents such as flavonoids, sterols, volatile oils, long-chain alcohols, and furans are contained in this medicine. Pharmacological studies indicated that the flavonoids components have the function of antihemorrhagic and antitumor. To our knowledge, there are several articles about the phytochemical investigation and pharmacological research of Cirsium japonicum DC., but there is no report yet of pharmacokinetic study of Cirsium japonicum DC. Therefore, an intensive investigation on absorption, distribution, excretion and in vitro metabolism of main bioactive components of Cirsium japonicum DC. was necessary. A sensitive LC-MS/MS method was established for simultaneous determination of pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and apigenin in rat plasma after oral administration Cirsium japonicum DC. extract. The pharmacokinetic parameters were obtained; An HPLC-MS/MS method was established for simultaneous determination of pectolinarin, linarin, pectolinarigenin in rat urine, bile, feces and tissues after oral administration Cirsium japonicum DC. extract. This method was successfully apply to the excretion and distribution study; An UHPLC-Q-TOF-MS/MS method was established for in vitro metabolism of pectolinarin, linarin and pectolinarigenin.Part one LC-MS/MS determination and pharmacokinetic study of seven flavonoids in rat plasma.Objective: To develope an HPLC-MS/MS method for simultaneousdetermination of pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and apigenin in rat plasma.Methods: Chromatographic separation was performed on a Diamonsil C18 column(150 mm × 4.6 mm, 5 μm), The mobile phase was consisted of methanol(A) and 0.1% formic acid aqueous solution(B) using a gradient elution of 35%-65% A at 0-1.5 min, 65% A at 1.5-6 min, 65%-95% A at 6-8 min, The flow rate was set at 0.8 m L/min. The detection was performed on a tandem mass system equipped with a turbo ion spray interface in positive and negative modes simultaneously. The turbo spray temperature was kept at 650℃. Twelve rats were divided into two groups randomly, Blood samples were collected from the fossa orbitalis vein at 0, 2, 5, 10, 15, 25, 60, 90, 120, 180 min for group A and 0, 5, 10, 25, 60, 90, 180, 360, 720, 1140, 1800 min for group B after a single oral administration. Plasma samples were pretreated by a single-step protein precipitation with methanol, and sulfamethoxazole was used as internal standard(IS).Results: Good linearity was obtained for seven analytes. The correlation coefficient for every calibration curve was higher than 0.9954. Analytes were rapidly absorbed, achieving Cmax at 5 min except for apigenin(at 360 min), the Cmax values for pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin, acacetin and apigenin were 876.77±97.34 ng/m L, 86.79±1.70 ng/m L, 6.13±0.12 ng/m L, 32.85±2.50 ng/m L, 37.2±2.04 ng/m L, 19.02±1.29 ng/m L and 148.26±20.63 ng/m L, respectively. A double-peak phenomenon of apigenin is presented, and this phenomenon may be relevant to entero-hepatic recirculation.Conclusion: A sensitive HPLC-MS/MS method was first established for simultaneously determination of seven flavonoids in rat plasma after oral administration of Cirsium japonicum DC. extract. The results showed that Concentrations of pectolinarin, linarin, pectolinarigenin, hispidulin, diosmetin and acacetin reached peak value rapidly and tapered after oral administration.Part two LC-MS/MS determination and excretion study of three flavonoids in rat urine, bile and feces.Objective: To develope an HPLC-MS/MS method for simultaneous determination and excretion study of pectolinarin, linarin and pectolinarigenin in rat urine, bile and feces.Methods: Chromatographic separation was performed on a Diamonsil C18 column(150 mm × 4.6 mm, 5 μm), The mobile phase was consisted of methanol(A) and 0.1% formic acid aqueous solution(B) using a gradient elution of 35%-90% A at 0-1.5 min, 90%-95% A at 1.5-4.5 min, 95% A at 4.5-6.5 min, 35%A at 6.6 min. The flow rate was set at 0.8 m L/min. The mass spectrometer was operated in the positive ESI mode with multiple reaction monitoring(MRM) at unit resolution. The turbo spray temperature was kept at 650℃. After administration, the urine and feces samples were collected at 0-4, 4-8, 8-12, 12-24, 24-36, 36-48 h. Bile samples of each rats were collected at 0-2, 2-4, 4-6, 6-8, 8-12, 12-24 and 24-36 h. All samples were pretreated before determination.Results: The excretion data of pectolinarin in urine, bile and feces indicated that about 0.07%, 0.99% and 14.96% was excreted after administration of 24 h, accounted for 94.52%, 95.27% and 99.14% of total excretion. The excretion data of linarin in urine, bile and feces indicated that about 0.047%, 0.36% and 10.50% was excreted after administration of 24 h, accounted for 97.53%, 95.13% and 99.66% of total excretion. The excretion data of pectolinarigenin in urine and feces indicated that about 2.21% and 25.85% was excreted after administration of 36 h, accounted for 98.17% and 99.98% of total excretion. Pectolinarigenin had not been detected in bile samples.Conclusion: An accurate and reliable method was first applied to the determination of three flavonoids in rat urine, bile and feces after oral administration of Cirsium japonicum DC. extract. Results showed that the three flavonoids were excreted mainly by the fecal route, and pectolinarigenin was not excreted via bile.japonicum DC. extract Part three Tissue distribution study of pectolinarin, linarin and pectolinarigenin in rats after oral administration of CirsiumObjective: To develope an HPLC-MS/MS method for simultaneous determination and distribution study of pectolinarin, linarin and pectolinarigenin in rat tissues.Methods: Chromatographic separation was performed on a Diamonsil C18 column(150 mm × 4.6 mm, 5 μm), The mobile phase was consisted of methanol(A) and 0.1% formic acid aqueous solution(B) using a gradient elution of 35%-90% A at 0-1.5 min, 90%-95% A at 1.5-4.5 min, 95% A at 4.5-6.5 min, 35%A at 6.6 min. The flow rate was set at 0.8 m L/min. The mass spectrometer was operated in the positive ESI mode with multiple reaction monitoring(MRM) at unit resolution. Twenty rats were divided into four groups randomly. These four groups of rats were euthanized by decapitation at 5 min, 10 min, 25 min and 45 min after oral administration of 5 m L/kg Cirsium japonicum DC. extract, respectively. Tissues including heart, liver, spleen, lung, kidney and brain were pretreated before determination.Results: The highest concentration levels of pectolinarin and linarin were detected in the spleen(960.76±130.72 ng/g for pectolinarin at 5 min, 350.76±62.44 ng/g for linarin at 5 min). While, the highest concentration levels of pectolinarigenin was obeserved in the kidney(581.94±82.70 ng/g at 10 min). These three flavonoids could be detected in tissues, and the contents were significantly decreased after administration of 45 min.Conclusion: An HPLC-MS/MS method was first established for simultaneously determination of pectolinarin, linarin and pectolinarigenin in rat tissues after oral oral administration of Cirsium japonicum DC. extract. The abundant blood-supply organs had a high content of these three flavonoids. The three flavonoids could cross the blood-brain barrier and eliminate rapidly after oral administration. Pectolinarin, linarin and pectolinarigenin has no long-term accumulate in the rat tissues.Part four In vitro metabolism of pectolinarin, linarin and pectolinargenin from Cirsium japonicum DC.Objective: To establishe A UHPLC-Q-TOF-MS/MS method for phase I and phase metabolism study of pectolinarin, linarin and pectolinarigenin in Ⅱrat liver microsomes.Methods: Incubation system of phase I: The incubation mixture was conducted in a 0.1 mol/L K2HPO4 buffer(p H=7.4) consisting of 1.3 mmol/L β-NADP, 3.3 mmol/L glucose-6-phosphate, 1.0 U/m L glucose-6-phosphate dehydrogenase, 3.3 mmol/L Mg Cl2 and rat liver microsomes(1.0 mg/m L); Incubation system of phase Ⅱ: Reaction mixtures contained 1 mg/m L rat liver microsomes, 2 mmol/L UDPGA, 8 mmol/L Mg Cl2 and 25 μg/m L alamethicin in 50 m M Tris-HCl buffer(p H=7.4). Reactions were initiated after the addition of analytes. The samples were detected in IDA modes by UHPLC-Q-TOF-MS/MS. Peakview 1.2 and Metabolite Pilot 1.5 were applied to the analysis of metabolite.Results: One phase I metabolites(M1) was detected for pectolinarin; one phase I metabolites(M2) was detected for linarin; five phase I metabolites(M3, M4, M5, M6, M7) was detected for pectolinarigenin. The phase Ⅱmetabolites of three flavonoids had not been detected.Conclusion: High resolution techniques were first applied to in vitro metabolism study of pectolinarin, linarin and pectolinarigenin. The result of experiment shows that these three flavonoids are easy to loss-CH2 from parent nucleus and no glucuronidation metabolites were found in the three samples. |
PDF Full Text Request