[关键词]
[摘要]
目的 研究茵陈活性成分滨蒿内酯在BeWo细胞模型上的跨胎盘摄取转运特性。方法 建立滨蒿内酯及内标物质双氯芬酸钠在细胞裂解液、Hank's平衡盐溶液(HBSS)中的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法,并进行了方法学验证;MTT法考察滨蒿内酯(1、5、20、50、100、200、400、800、1 000 μ mol/L )作用4 h对BeWo细胞活性的影响;应用建立的UPLC-MS/MS法考察给药时间、药物浓度、温度、pH值、转运蛋白抑制剂对BeWo细胞摄取滨蒿内酯的影响;以BeWo单层细胞为体外模型,应用Transwell孔板,考察给药时间、药物浓度、转运蛋白抑制剂对滨蒿内酯的跨胎盘转运的影响。结果 建立的UPLC-MS/MS分析方法专属性强,灵敏度高,重现性好,提取回收率、基质效应和样品稳定性等均符合生物样品药物含量的分析测定要求。滨蒿内酯在浓度为1~800 μmol/L时对BeWo细胞无明显毒性作用。滨蒿内酯在BeWo细胞上的摄取随时间和浓度变化呈线性增加,在所选取的浓度范围内无饱和趋势,不符合米氏方程;滨蒿内酯在BeWo细胞上的摄取量在4℃和37℃条件无显著性差异,表明摄取转运过程不受温度的影响;与生理条件下(pH 7.4)比较,在酸性条件下(pH 5.0或6.5),摄取量略有降低,无显著性差异;碱性条件下(pH 8.6),摄取量显著减少(P<0.001);滨蒿内酯在细胞膜刷状缘侧的摄取不受各类膜转运蛋白底物或抑制剂的影响。滨蒿内酯在BeWo细胞上的2个方向的转运速率无显著差异;二者表观渗透系数(Papp)值极为接近,药物外排率(Pratio)约为1;在考察的浓度范围(5、50、100、200、400 μmol/L)内,滨蒿内酯在母-胎和胎-母2个方向上转运均呈线性增加,且无饱和趋势;滨蒿内酯在BeWo单层细胞的转运不受各类膜转运蛋白底物或抑制剂的影响。结论 滨蒿内酯在BeWo细胞上的摄取及跨膜转运机制为非载体蛋白介导的被动转运。
[Key word]
[Abstract]
Objective To explore the transplacental uptake and transport properties of scoparone, the active ingredient of Artemisiae Scopariae Herba, in BeWo cell model.Methods A method for the determination of scoparolide and diclofenac sodium in cell lysate and Hank's balanced salt solution (HBSS) by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was established and validated; MTT method was used to determine the effect of scoparolide (1, 5, 20, 50, 100, 200, 400, 800 and 1 000) μmol/L on activity of BeWo cells after 4 h treatment. The effects of administration time, drug concentration, temperature, pH and transporter inhibitor on the uptake of scoparolide by BeWo cells were investigated by UPLC-MS/MS. The Transwell plate was used to investigate the effects of administration time, drug concentration and transporter inhibitor on the transplacental transport of scoparolide. Results The established UPLC-MS/MS method has strong specificity, high sensitivity and good reproducibility. The extraction recovery, matrix effect and sample stability all meet the requirements for the determination of drug content in biological samples. When the concentration of scoparolide was 1 - 800 μmol/L, there was no obvious toxic effect on BeWo cells. The uptake of scoparolide in BeWo cells increased linearly with time and concentration, and there was no saturation trend in the selected concentration range, which did not conform to Michaelis Menten equation. There was no significant difference in the uptake of scoparolide in BeWo cells at 4 ℃ and 37 ℃, indicating that the uptake and transport of scoparolide were not affected by temperature; Compared with physiological conditions (pH 7.4), the intake decreased slightly under acid condition (pH 5.0 or 6.5), and there was no significant difference. Under the alkaline condition (pH 8.6), the uptake was significantly reduced (P < 0.001). The uptake of scoparolide on the brush edge of cell membrane was not affected by various membrane transporter substrates or inhibitors. There was no significant difference in the transport rate of scoparolide between the two directions in BeWo cells; The apparent permeability coefficient (Papp) of the two drugs were very close, and the drug efflux rate (pratio) was about 1; In the investigated concentration range (5, 50, 100, 200, 400 μmol/L), the transport of scoparolide increased linearly in both maternal fetal and fetal maternal directions, and there was no saturation trend. The transport of scoparolide in BeWo monolayer cells was not affected by various membrane transporter substrates or inhibitors. Conclusions The uptake and transmembrane transport of scoparone in BeWo cells is probably passive diffusion.
[中图分类号]
R945
[基金项目]
江苏省研究生创新实践项目(SJCX20-0557)