The Quality Control Of Radix Angelicae Dahuricae And The Metabolism Of Cnidilin In Vitro And Vivo

Radix Angelicae Dahuricae, dried radix of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. and Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss.) Shan et Yuan, belongs to Umbelliferae family. It have been frequently used for common acesodyne and cough, especially for headache and toothache for over 2000 years. Coumarins are the main effective components responsible for their activities, such as antihistamine, promotion of lipometabolism, spasmolysis, antibacterial, anticancer, excitation of motor and respiratory center, inhibition of arachidonic acid-induced platelet aggregation, etc. In this artical, we have simultaneously made a quantitation of eight coumarins in Radix Angelicae Dahuricae by micellar electrokinetic capillary chromatography, in order to represent the integral quality of Radix Angelicae Dahuricae extensively.The metabolism of medicine is a very important part in the pharmaceutical research. Studying the metabolism of medicine is useful to acknowledge the effect, side effect and toxicity of medicine, and can provide the suitable dosage and manner. We have studied the metabolism and excretion of cnidilin, which is a kind of coumarin, a main effective component in Radix Angelicae Dahuricae. And the results are helpful for us to start the further study of the transformation pathways and excretion pharmacokinetics of Radix Angelicae Dahuricae.Part One Simultaneous quantitation of eight coumarins in Radix Angelicae Dahuricae by micellar electrokinetic capillary chromatographyObjective: To establish the method to simultaneously determine Scopoletin, Xanthotoxol, Xanthotoxin, Bergapten, Oxypeucedanin, Imperatorin, Cnidilin and Isoimperatorin by micellar electrokinetic capillary chromatography.Methods: Separations were carried out using fused-silica capillary (Beckman Technologies, USA) of 60 cm total length (50 cm effective length)×75μm i.d. The running buffer was composed of 10 mmol/L borax, 40 mmol/L sodium dodecyl sulfate (SDS) and 20% acetonitrile. The applied voltage was 22 kV and the temperature was 20℃. The detection wavelength was 214 nm.Results: The 8 coumarins were separated successfully within 20 min. A good linearity (r≥0.9971) was found in the range of 1.25~20.04, 1.78~28.53, 0.44~7.01, 0.67~10.74, 7.62~121.86, 3.94~63.08, 2.89~46.21, 3.02~48.35 ng/mL for the 8 components, respectively. The recoveries were between 92.7%~105.6%, which were meet the requirement of the analysis.Conclusion: With this method, 8 coumarins were simultaneous determined. The operation of the method is simple, quick, accurate and can be used as a new method for the quality control of Radix Angelicae Dahuricae.Part Two The metabolism of cnidilin in vitroObjective: In order to find the metabolites, the method of the microbial transformation and the rat intestinal flora were used for studying the metabolism of cnidilin in vitro. And the metabolites were obtained by the preparative.Methods: 1. The microbial transformation: The metabolites were discovered by comparing the HPLC-PDA chromatograms of 20 fungal strains samples with the corresponding blanks, and were prepared by the fungal which could transform the cnidilin the most completely. The metabolites were identified by 1H-NMR and ESI-MS, and the chemical structures of them were confirmed. 2. The rat intestinal flora: cnidilin was anaerobically incubated with rat stool in vitro to find the metabolites.Results: 1. The microbial transformation: 4 metabolites were found by the fungal AS 3.970. Two of them were prepared and identified by 1H-NMR and ESI-MS, which are 3",8-methoxy-isoimperatorin and 5"-hydroxyl-8-methoxy-isoimperatorin. 2. The rat intestinal flora: there was no metabolite found in the incubation sample solution.Conclusion: The microbial transformation and the intestinal flora could be used for the metabolism of coumarins in vitro, which was helpful to study the transformation pathways of Radix Angelicae Dahuricae.Part Three Determination of cnidilin and its two metabolites in rat plasma by high performance liquid chromatography- electrospray ionization tandem mass spectrometryObjective: To establish a method for the simultaneous determination of cnidilin and its two metabolites in rat plasma by HPLC coupled with electrospray ionization tandem mass spectrometry after the orally administrating of cnidilin.Methods: After the plasma was added with methanol used as protein precipitant, which was collected from the rat retro-orbital vein, the samples were analyzed with gradient elution consisted of 0.5‰aqueous formic acid and methanol (containing 0.5‰formic acid) in the multiple-reaction monitoring mode. Pimpinellin was used as internal standardResults: Cnidilin and its two metabolites were separated successfully within 8 min. A good linearity (r≥0.9975) was found in the range of 25~3200,3.91~500 ng/mL for the 3 analytes, respectively. The recoveries of low, medium and high concentration of quality control samples were between 92.7%~105.6%, which were meeting the requirement of the analysis of the biological sample.Conclusion: The operation of the method is accurate, quick and sensitive and can be used as a new method for simultaneous determination of medicine and its metabolites in rat plasma. With this method, not only the pathway of the biological transformation could be studied, but also the pharmacokinetic parameter of the medicine could be obtained, which was helpful to the study of the metabolism of Radix Angelicae Dahuricae in vivo.Part Four The excretion study of cnidilinObjective: To investigate bile and stool pharmacokinetics of cnidilin in rats. And the metabolites of cnidilin were determined in bile and stool, which was helpful for the metabolism of cnidilin in vivo.Methods: After the bile and stool were collected after oral administration of cnidilin, the analytes were separated on a reverse phase C18 column with gradient elution consisted of 0.5‰aqueous formic acid and methanol (containing 0.5‰formic acid) in the multiple-reaction monitoring mode. Pimpinellin was used as internal standard (IS) to determine cnidilin and its two metabolites.Results: Cnidilin in bile was completely excreted in 22 h. Main excretive amount of loganin was above 80% in 0~6 h, and the drug recovery in bile was about 1.95%; while in stool, cnidilin was completely excreted in 36 h, main excretive amount was about 95% in 0~24 h, and the drug recovery in stool was about 1.48%.Conclusion: In this study, a high sensitive and selectivity LC-MS method for on-line qualitative analysis of cnidilin and its two metabolites in bile and stool of rats has been developed. The biochemical samples were easily treated and the analysis time was short. The method was helpful for the study of excretion of Radix Angelicae Dahuricae in rats.