| Background:Crocetin, a brick red orthogonal crystalline compound with polyunsaturated conjugated olefine acid structure, which falls into the category of carotenoid, is one of the major effective components in saffron crocus and it was extracted from cape jasmine which belongs to gamene family. Crocetin exerts many pharmacologiacl effects on nervous, cardiovascular, respiratory and urinary system. As per our previous study, it was shown that crocetin was mainly metabolized by CYP3A,1A2 enzyme. But there was no in-depth study into whether crocetin have effects on the activity of CYP450 enzyme and will its interaction with other drugs impact the metabolism of crocetin. Therefore, it is necessary to study the effects of crocetin on CYP450 enzyme so as to avoid the interaction when crocetion was co-administrated with other drugs. The model of human 、rat microsomes and rat in vivo metabolism were established to explore the effects of crocetin on two sub-enzymes which were involved in the metabolism.Objective:The aim of this research was to investigate the effects of crocetin on the activity of CYP3A、1A2 enzyme through the model of human、rat microsomes in vitro and rat in vivo metabolism experiment, so as to further extend the research on crocetin and to provide theoretical references for the rational administration of crocetin clinically. Method:1. The HPLC method was established to determine the concentration of MDZ、 PHE in human microsomes and rat microsomes.2. The HPLC method was established to determine the concentration of MDZ in rat plasma.3. The respective optimum reaction condition for MDZ and PHE in human and rat liver microsomes were determined by investigating three different factors which were incubation time、the concentration of protein and the concentration of substrate.4. The effects of crocetin on the activity of CYP3A4、CYP1A2 sub-enzyme in human and rat liver microsomes was researched.5. By intraperitoneally injecting different dosages of crocetin which was followed by tail vein injection of same dosage of MDZ, the effect of crocetin on the pharmacokinetics of MDZ mediated by CYP3A in rats was explored.Results:1. The HPLC method was established to determine the concentration of MDZ, PHE in rat microsomes and the concentration of MDZ, PHE in human microsomes.2. The HPLC method was established to determine the concentration of MDZ in rat plasma.3. MDZ was incubated in rat liver microsomes at 37 ℃ and 120r/min. Within the period 1-40min, the elimination rate of MDZ was increased with the increase of time. Thus, the optimum incubation time was 40min. Within the concentration of protein ranged from 0.05-0.3mg/ml, the elimination rate of MDZ was increased with the increase of the concentration of protein and the optimum protein concentration was defined at 0.3mg/ml. When the substrate concentration ranged from 2.5-1 OμM, the metabolism rate was increased most significantly with the increase of substrate concentration, so the optimum substrate concentration was 10μM. PHE was incubated in rat liver microsomes under 37 ℃ and 120r/min circumstances. Within the period 5-40min, PHE was eliminated linearly. When the incubation time was more than 40 minutes, the elimination rate of PHE was flatlined. Thus, the optimum incubation time was 40min. Within the range 0.1-0.4mg/ml, the elimination rate of PHE was increased with the increase of the concentration of protein and the optimum protein concentration was defined at 0.4mg/ml. When the substrate concentration ranged from 5-80μM, the metabolism rate was increased most significantly with the increase of substrate concentration, so the optimum substrate concentration was 80μM.4. When the concentration of crocetin ranged from 0.5 to 80μM, there were significant differences (P<0.05) in rat liver microsomes in terms of crocetin effect on the activity of CYP3A enzyme, the average metabolic rate of MDZ were decreased by 15.55%,35.27%,48.77%,59.74%,61.72%,67.88%,71.11%(P<0.05) respectively. When the concentration of crocetin ranged from 0.5 to 80(iM, there were significant differences (P<0.05) in rat liver microsomes in terms of crocetin effect on the activity of CYP1A2 enzyme, the average metabolic rate of PHE were decreased by 10.17%, 21.38%、33.43%、52.09%、63.01%、77.32%、81.24%(P<0.05) respectively. By SPSS software, the IC50 values of crocetin to CYP3A and CYP1A2 in rat liver microsomes were caculated to be 6.814 μM,9.157μM respectively.5. MDZ was incubated in human liver microsomes at 37 ℃ and 120r/min. Within the period 1-30min, MDZ was eliminated linearly. When the incubation time was more than 30 minutes, the elimination rate of MDZ was flatlined. Thus, the optimum incubation time was 30min. Within the range 0.05-0.2mg/ml, the elimination rate of MDZ was increased with the increase of the concentration of protein and the optimum protein concentration was defined at 0.2mg/ml. When the substrate concentration ranged from 2.5-1 OμM, the metabolism rate was increased most significantly with the increase of substrate concentration. So the optimum substrate concentration was 10μM. PHE was incubated in human liver microsomes under 37℃ and 120r/min circumstances. Within the period 5-40min, the elimination rate of PHE was increased with the increase of time. Thus, the optimum incubation time was 40min. Within the range 0.1-0.4mg/ml, PHE was eliminated linearly and the optimum protein concentration was defined at 0.4mg/ml. When the substrate concentration ranged from 5-80μM, the metabolism rate was increased most significantly with the increase of substrate concentration. So the optimum substrate concentration was 80μM.6. When the concentration of crocetin ranged from 0.5 to 80μM, there were significant differences (P<0.05) in human liver microsomes in terms of crocetin effect on the activity of CYP3A4 enzyme. The average metabolic rate of MDZ were decreased by 25.36%,32.37%,38.91%,45.96%,51.74%,59.24% 67.72%(P<0.05)respectively, when the concentration of crocetin was within the range which was mentioned above. When the concentration of crocetin ranged from 0.5 to 80μM, there were significant differences (P<0.05) in human liver microsomes in terms of crocetin effect on the activity of CYP1A2 enzyme. The average metabolic rate of PHE were decreased by 13.48%、30.55%、33.72%、42.67%、48.01%、56.62%、 67.43%(P<0.05) respectively, when the concentration of crocetin was within the range which was mentioned above. By SPSS software, the IC50 values of crocetin to CYP3A4 and CYP1A2 in human liver microsomes were caculated to be 13.271μM、 19.225μM respectively.7. Major pharmacokinetic parameters of four MDZ groups:control group: AUC(o-t)(361.209mg·Lmin-1), AUC(0-∞)(403.953mg·L·min-1), MRT(0-t) (37.78 min), MRT(0-∞)(62.362min), t1/2z(42.193 min), CLz(0.07 L·min-1·kg-1), Vz(1.135 L·kg-1); low dose group:AUC(o-t)(454.895mg·L·min-1), AUC(0-∞)(499.209mg·L·min-1), MRT(0-t) (49.004 min), MRT(0-∞)(75.421 min), t1/2z(52.806 min), CLz(0.024 L·min-1·kg-1), Vz(0.785 L·kg-1); medium dose group:AUC(o-t)(636.417mg·L·min-1), AUC(0-∞)(704.421mg·L·min-1), MRT(0-t) (57.212 min), MRT(0-∞)(92.241 min), ti/2z(64.404 min), CLz(0.017L·min-1·kg-1), Vz(0.553 L·kg-1); high dose group: AUC(0-t)(1050.388mg·L·min-1),AUC(0-∞)(1177.015mg·L·min-1), MRT(0-t) (64.681 min), MRT(0-∞)(107.915 min), t1/2z(77.145 min), CLz(0.006L·min-1·kg-1), Vz(0.316L·kg-1). After being administered three different doses of crocetin, the AUC of MDZ was evidently higher than the control group which was only injected with MDZ, which indicated that crocetin exerts significant impact on the plasma concentration of MDZ. And the higher the concentration of crocetin was, the stronger the influence was.Conclusion:1. The HPLC method established for the determination of MDZ, PHE concentra-tion in rat and human microsomes and MDZ, PHE concentration in rat, human microsomes was sensitive and reliable.2. The HPLC method established for the determination of MDZ concentration in rat plasma was sensitive and reliable.3. Crocetin excerts medium inhibition on both activitys of CYP1A2 and CYP3A sub-enzyme in rat liver microsomes.4. Crocetin excerts weak inhibition on CYP1A2 and CYP3A4 sub-enzyme in human microsomes.5. In rats, after being administered three different doses of crocetin, the AUC of MDZ was evidently higher than the control group which was only injected with MDZ, which indicated that crocetin exerts significant impact on the plasma concentration of MDZ. And the higher the concentration of crocetin was, the stronger the influence was. |
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