| Part 1. Determination of aristolochic acid and study on its pharmacokinetices in mice by HPLCNephrotoxicity caused by traditional Chinese medicines containing Aristolochic Acid has extensively become one of the focuses in the traditional Chinese medicine circles both in China and in the foreign countries since the publication of "Letter to Industry-FDA Concerned About Botanical Products, Including Dietary Supplement, Containing Aristolochic Acid " on May 16th 2000" and "Letter to Health Care Professionals-FDA Concerned About Botanical Products, Including Dietary Supplement, Containing Aristolochic Acid " on May 31st 2000 by FDA. But the Chinese medical circles' knowledge of aristolochic acid (AA) is still resting on the level of 30 years ago. It is really stagnation. "Safety first, efficiency second" is the general principle for the international examination and approval of medicines. In order to clarify the pharmacokinetic characteristic of aristolochic acid, the pharmacokinetic research of Aristolochic acid in Caulis Aristolochiae Manshuriensis was carried out. The drug was given orally and the concentration of aristolochic acid in mice plasma determine by HPLC. Objective: To establish a reversed-phase HPLC method for the study on pharmacokinetics of aristolochic acid in the extract solution from Caulis Aristolochiae Manshuriensis and Guanxinsuhe capsule in mice after single oral administration.Methods: Mice were divided into different groups randomly, six mice each group and were given orally at the dose of 4mg · kg-1 AA. Collect blood samples from eye after given drug at the prearranged time (5,10,15,20,30,45,60,90,120,150,180,240min), and then they were drawn into tubes containing heparin sodium respectively. The plasma samples were separated by centrifugation at 3000 rpm for 10min, add three-times volume of methanol to deposit protein, centrifugat, transferr the upper liquid, filterr, inject 20μL of each sample into the HPLC. DiamonsilTM C18 column was used as an analytical column (250mm×4.6mm, 5μm). The mobile phase consisted of 72 volume of methanol, 27 volume of water and 1 volume of acetic acid at the flow rate of 1.0ml · min-1. The UV detection was set at 315nm and the experiment was carried out at the room temperature of 20℃. The sensitivity was set at 0.5AUFS. Record the peak area of aristolochic acid and calculat concentration of aristolochic acid in plasma. Cope the original dates with the 3P97 and NDST pharmacokinetics software. Make the best compartment model according to the least AIC and the biggest corrlation coefficient obtained from real concentration. Results: The calibration curve of aristolochic acid was linear in the range from 0.1098 to 3.66μg·mL-1 (r=0.9998, n=7). Thelowest determination concentration of aristolochic acid was 0.0183μg·mL-1. The recoveries and relative standard deviations of aristolochic acid were 99.34%(0.80%), 97.23%(1.13%)and 97.14% ( 0.97%) when it was in high, middle and low concentration solutions respectively. The relative standard deviations of within-day and between-day precisions for the method were all less than 4.9%. After single oral administration of the solution containing Caulis Aristolochiae Manshuriensis to mice, the mean plasma concentration-time course was found to fit a one-compartment model. The main pharmacokinetic parameters of aristolochic acid were as follows: T1/2(ka), T1/2(ke), T(max) , AUC, Cmax were 3.94min, 16.29min, 10.65min, 104.88(μg·mL-1)·min, 2.84μg·mL-1. Conclusion: It is a sensitive,specific and simple method to determine concentration of aristolochic acid in plasma and its pharamacokinetic parameters and characteristics was obtained. Key words: HPLC,CAULIS ARISTOLOCHIAE MANSHURIENSIS,aristolochic acid,plasma concentration,pharmacokinetics... |
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