目的 研究茵陳活性成分濱蒿內(nèi)酯在BeWo細(xì)胞模型上的跨胎盤攝取轉(zhuǎn)運(yùn)特性。方法 建立濱蒿內(nèi)酯及內(nèi)標(biāo)物質(zhì)雙氯芬酸鈉在細(xì)胞裂解液、Hank's平衡鹽溶液（HBSS）中的超高效液相色譜-串聯(lián)質(zhì)譜（UPLC-MS/MS）分析方法，并進(jìn)行了方法學(xué)驗(yàn)證；MTT法考察濱蒿內(nèi)酯（1、5、20、50、100、200、400、800、1 000 μ mol/L ）作用4 h對BeWo細(xì)胞活性的影響；應(yīng)用建立的UPLC-MS/MS法考察給藥時(shí)間、藥物濃度、溫度、pH值、轉(zhuǎn)運(yùn)蛋白抑制劑對BeWo細(xì)胞攝取濱蒿內(nèi)酯的影響；以BeWo單層細(xì)胞為體外模型，應(yīng)用Transwell孔板，考察給藥時(shí)間、藥物濃度、轉(zhuǎn)運(yùn)蛋白抑制劑對濱蒿內(nèi)酯的跨胎盤轉(zhuǎn)運(yùn)的影響。結(jié)果 建立的UPLC-MS/MS分析方法專屬性強(qiáng)，靈敏度高，重現(xiàn)性好，提取回收率、基質(zhì)效應(yīng)和樣品穩(wěn)定性等均符合生物樣品藥物含量的分析測定要求。濱蒿內(nèi)酯在濃度為1～800 μmol/L時(shí)對BeWo細(xì)胞無明顯毒性作用。濱蒿內(nèi)酯在BeWo細(xì)胞上的攝取隨時(shí)間和濃度變化呈線性增加，在所選取的濃度范圍內(nèi)無飽和趨勢，不符合米氏方程；濱蒿內(nèi)酯在BeWo細(xì)胞上的攝取量在4℃和37℃條件無顯著性差異，表明攝取轉(zhuǎn)運(yùn)過程不受溫度的影響；與生理?xiàng)l件下（pH 7.4）比較，在酸性條件下（pH 5.0或6.5），攝取量略有降低，無顯著性差異；堿性條件下（pH 8.6），攝取量顯著減少（P<0.001）；濱蒿內(nèi)酯在細(xì)胞膜刷狀緣側(cè)的攝取不受各類膜轉(zhuǎn)運(yùn)蛋白底物或抑制劑的影響。濱蒿內(nèi)酯在BeWo細(xì)胞上的2個(gè)方向的轉(zhuǎn)運(yùn)速率無顯著差異；二者表觀滲透系數(shù)（P_app）值極為接近，藥物外排率（P_ratio）約為1；在考察的濃度范圍（5、50、100、200、400 μmol/L）內(nèi)，濱蒿內(nèi)酯在母-胎和胎-母2個(gè)方向上轉(zhuǎn)運(yùn)均呈線性增加，且無飽和趨勢；濱蒿內(nèi)酯在BeWo單層細(xì)胞的轉(zhuǎn)運(yùn)不受各類膜轉(zhuǎn)運(yùn)蛋白底物或抑制劑的影響。結(jié)論 濱蒿內(nèi)酯在BeWo細(xì)胞上的攝取及跨膜轉(zhuǎn)運(yùn)機(jī)制為非載體蛋白介導(dǎo)的被動(dòng)轉(zhuǎn)運(yùn)。

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 (P_app) of the two drugs were very close, and the drug efflux rate (p_ratio) 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.

R945

江蘇省研究生創(chuàng)新實(shí)踐項(xiàng)目（SJCX20-0557）