Studies On The Method For Quality Control And Pharmacokinetics Of Ixeris Sonchifolia

Ixeris sonchifolia （Bge.） Hance, commonly named Kudiezi in China, belongs to the plant family Compositae. It is one of the important folk medicines in Traditional Chinese Medicine （TCM） and has been widely used in China for many years. I. sonchifolia possesses various activities, in the modern clinical practices, which has been used to treat coronary heart disease, stroke, myocardial infarction and cerebrovascular diseases. In this paper the chemical constituents, antibacterial activity and quality control method, as well as pharmacokinetics on a few active constitutes of I. sonchifolia were studied.The chemical constituents of I. sonchifolia were systematically studied and 15 compounds were isolated and purified by silica gel, polyamide, sephadex LH-20, ODS and PHPLC column chromatography. Utilizing chemical and spectroscopic methods （UV, IR, NMR, MS）, the structures of 14 compounds were fully characterized, including 8 flavonoids, 4 sesquiterpene lactones,β-sitosterol and daucosterol.The method for HPLC-fingerprint analysis of I. sonchifolia was established. The different sources of samples were analyzed and 25 co-possessing peaks were selected as the fingerprint peaks of I. sonchifolia. The similarity was calculated by cosine, and based on the result of similarity analysis, the evaluation criterions for quality control of the herb were established. Furthermore, the main constituents of I. sonchifolia were identified by comparing their retention behaviors with standard solutions.RP-HPLC method was developed for the determination of adenosine in I. sonchifolia. The extraction process of adenosine was optimized by orthogonal design. The result showed that the optimal extracting condition was water consumed fifty times the amount of material, and one time for thirty minutes. Good linearity was observed over the range of 1.02～30.6μg、mL^-1 for adenosine, and the average recovery was 98.9% （RSD=1.8%）. HPLC-DAD method was developed for simultaneous determination of chlorogenic acid, caffeic acid and three flavonoids. Good linearities were observed for the five compounds and the recoveries varied from 94.1% to 100.7% （RSD<5.0%）. The method for simultaneous quantitative analysis of 6 flavonoids in I. sonchifolia was performed by using HPLC-MS technique. The samples were analyzed by using negative electrospray ionization MS in selected ion monitoring mode. The linear calibration curves were plotted with satisfied relative coefficients, the recoveries were in the range of 95.1%～100.7% （RSD<4.0%）. HPLC-DAD method was developed for simultaneous determination of 4 sesquiterpene lactones in I. sonchifolia, which showed good linearities for each marker and the recoveries were in the range of 96.4%～100.9% with RSD less than 3.0%.The central composite design-response surface methodology was adopted to optimize the extraction process of sesquiterpene lactones in I. sonchifolia. The contents of 4 sesquiterpene lactones were selected as assessment indices. Three factors were studied in this experiment, including the concentration of solvent, duration of extraction and quantity of solvent. The result showed that the optimal extracting condition was 70% methanol consumed twenty times the amount of material, and one time of ultrasonic extraction for 40 min.A quantitative analysis method was established for simultaneous quantification of 5 main chemical constituents in Kudiezi Injection, a major preparation of I. sonchifolia, and was applied for the determination of 4 batches of Kudiezi Injection.A rapid and sensitive LC-ESI-MS method was developed for the determination of luteolin-7-O-β-D-glucoside in rat plasma using liquid-liquid extraction and isoquercitrin as internal standard. The selected ions for luteolin-7-O-β-D-glucoside and isoquercitrin were m/z 448.95 and m/z 464.95, respectively. Good linearity was observed over the range of 20.0-2000 ng·mL^-1 with a lower limit of quantification of 20.0 ng·mL^-1. The intra-day and inter-day precisions at low, medium and high concentrations were found to be less than 6.5% and 10.4% relative standard deviation. The accuracy, expressed as relative error, was within±4.5%. The extraction recovery was no less than 84.5%. The validated method has been applied to the pharmacokinetic study of luteolin-7-O-β-D-glucoside in rat plasma after intravenous administration of Kudiezi Injection. The mean plasma concentration-time curves was plotted, and the main pharmacokinetic parameters were T_1/2, 0.70 h; K_e,1.05 h^-1; and AUC_0-∞,113.9 ng·h·mL^-1, respectively.A simple and reliable LC-ESI-MS method was developed for simultaneous determination of luteolin-7-O-gentiobiside and luteolin-7-O-β-D-glucuronide in rat plasma. After deproteinized by acetonitrile, the plasma samples were analyzed with puerarin as internal standard. The detection utilized selected ion monitoring at m/z 609.00 for luteolin-7-O-gentiobiside, m/z 460.95 for luteolin-7-O-β-D-glucuronide and m/z 415.10 for puerarin, respectively. The assay showed good linearities over the range of 20.0～2000 ng·mL^-1 for luteolin-7-O-gentiobiside and 0.08～20μg·mL^-1 for luteolin-7-O-β-D-glucuronide. The lower limits of quantification were 20.0 ng·mL^-1 and 0.08μg·mL^-1 , respectively. The method was shown to be reproducible and reliable with intra-day precisions below 7.2% and 5.4%, inter-day precisions below 8.7% and 8.4% and accuracies within±8.3% and±6.4%, respectively. The extraction recoveries for the two analytes and the internal standard were found to be above 76.6%. The LC-MS method has been applied to the pharmacokinetic studies after the rats were administered Kudiezi Injection, the solution of luteolin-7-O-gentiobiside and the solution of luteolin-7-O-β-D-glucuronide via intravenous route, respectively. Under the two administration conditions, the main pharmacokinetic parameters for luteolin-7-O-gentiobiside were compared as follows: T_1/2, 0.68 h and 0.58 h; K_e, 1.21 h^-1 and 1.44 h^-1; AUC_0-∞, 165.5 ng·h·mL^-1 and 163.2 ng·h·mL^-1, respectively. The main pharmacokinetic parameters for luteolin-7-O-β-D-glucuronide were compared as follows: T_1/2, 0.48 h and 0.41 h; K_e, 1.59 h^-1 and 2.03 h^-1, AUC_0-∞, 0.962μg·h·mL^-1 and 0.955μg·h·mL^-1, respectively.The antibacterial activities of 15 compounds were investigated in vitro. The results manifested that most compounds had strong antibacterial activities against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, but only a few compounds showed conspicuous antibacterial activities against Candida albicans.Under the direction of theory and clinical practice of TCM, utilizing the knowledge of Chinese material medica science, analytical chemistry, pharmacology, pharmacokinetics and chemometrics, the chemical constituents and quality assessment methods of I. sonchifolia were studied. The pharmacokinetics of three active components in rat plasma were also investigated to some extend. This research provided an exploration for the modernization of TCM.