目的 建立测定细胞中东莨菪内酯(scopoletin,Sco)含量的分析方法,考察包载Sco的聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇(soluplus)胶束给药系统(Sco-Ms)在Caco-2细胞上的摄取和代谢,初步探究Sco-Ms促进Sco口服吸收的机制。方法 采用液相色谱-串联质谱(LC-MS/MS)法,结合酶水解法,测定细胞样品中Sco原药及代谢物含量,计算Sco的细胞摄取率和代谢率;并利用多种抑制剂考察Sco-Ms的入胞途径。结果 Sco在5~1 000 ng·mL-1内线性良好;各时间点Sco-Ms在Caco-2细胞上的摄取率均显著高于Sco;Sco-Ms,显著降低了Sco在细胞中的代谢率;Sco-Ms主要通过网格蛋白介导的內吞作用和胞饮作用进入细胞。结论 提高细胞对Sco的摄取;同时降低Sco的代谢率,是Sco-Ms促进Sco口服吸收的重要机制。
Abstract
OBJECTIVE To develop an analytical method for the quantification of scopoletin (Sco) and investigate the cellular uptake and metabolism of polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol (soluplus)-based Sco micelles (Sco-Ms) in Caco-2 cells, as well as exploring the possible mechanisms involved in the oral absorption of Sco-Ms.METHODS Combined with enzymatic hydrolysis for pretreatment, a liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-MS/MS) method was developed for the quantification of Sco and its corresponding metabolite. Then, cellular uptake efficiency and metabolic rate of Sco were calculated.RESULTS This method was proven to be linear over the concentration range of 5-1 000 ng·mL-1. Cellular uptake of Sco-Ms increased significantly compared with that of free Sco at various time points. Meanwhile, Sco-Ms inhibited the enzymatic degradation of Sco. Sco-Ms were primarily internalized into enterocytes via macropinocytosis and clathrin-dependent pathways.CONCLUSION Enhanced cellular uptake and decreased metabolic rate are pivotal mechanisms by which Sco-Ms promotes oral absorption of Sco.
关键词
东莨菪内酯 /
聚乙烯己内酰胺-聚乙酸乙烯酯-聚乙二醇 /
胶束 /
Caco-2细胞 /
液相色谱-串联质谱
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Key words
scopoletin /
soluplus /
micelle /
Caco-2 cells /
LC-MS/MS
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中图分类号：
R944
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参考文献
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脚注
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基金
成都医学院四川养老与老年健康协同创新中心项目资助(YLZBZ1808);成都医学院科研基金课题资助(CYZ17-07);宁波市科技创新团队资助(2015C110027)
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