独活中6种香豆素类成分在Caco-2细胞单层模型中的吸收转运研究

Absorption and transport of 6 coumarins isolated from the roots of Angelica pubescens f.biserrata in human Caco-2 cell monolayer model

目的:研究独活中6种香豆素类成分(伞形花内酯、甲氧基欧芹素、二氢欧山芹素、二氢欧山芹醇乙酯、当归醇-A和当归醇-B)在人源结肠腺癌细胞系Caco-2细胞单层模型的吸收和转运。方法:采用Caco-2细胞单层模型研究6种香豆素类化合物由绒毛面(AP侧)到基底面(BL侧)和从BL侧到AP侧两个方向的转运过程,采用高效液相色谱法测定其在AP和BL侧的含量,计算表现渗透系数(apparent permeability coefficient,P_(app)),并与阳性对照药普萘洛尔和阿替洛尔进行比较。采用ATP耗竭剂碘乙酰胺和MRP2抑制剂MK571添加法探讨当归醇-B的转运机制。结果:6种香豆素类成分皆以被动扩散为主要方式被Caco-2细胞吸收、转运。伞形花内酯、甲氧基欧芹素、二氢欧山芹素、二氢欧山芹醇乙酯、当归醇-A和当归醇-B由AP侧到BL侧的P_(app)分别为(2.679±0.263)×10^(-5)、(1.306±0.324)×10^(-5)、(0.595±0.086)×10^(-6)、(0.293±0.041)×10^(-6)、(1.532±0.444)×10^(-5)和(1.413±0.243)×10^(-5)cm/s;由BL侧到AP侧的P_(app)分别为(3.381±0.410)×10^(-5)、(0.898±0.134)×10^(-5)、(0.510±0.183)×10^(-6)、(0.222±0.025)×10^(-6)、(1.203±0.280)×10^(-5)和(0.754±0.092)×10^(-5)cm/s。在本实验条件下,易吸收的阳性对照药普萘洛尔由AP侧到BL侧的P_(app)值为2.18×10^(-5)cm/s,不易吸收的阳性对照药阿替洛尔由AP侧到BL侧的P_(app)值为2.77×10^(-7)cm/s。上述6个香豆素类化合物中,伞形花内酯、甲氧基欧芹素、当归醇-A和当归醇-B的P_(app)值与普萘洛尔的P_(app)值在一个数量级;二氢欧山芹素和二氢欧山芹醇乙酯的P_(app)值在易吸收和不易吸收的阳性对照药之间。以EBSS代替HBSS作为转运介质,在碘乙酰胺和MK571存在下,当归醇-B从AP侧到BL侧和从BL侧到AP侧转运的P_(app)值与HBSS为转运介质的对照组相比皆没有统计学意义上的变化。回收率实验中,伞形花内酯的平均总回收率为(83.31±3.52)%、当归醇-A的平均总回收率为(77.39±7.38)%;甲氧基欧芹素、二氢欧山芹素和当归醇-B的平均总回收率在50%～65%之间,二氢欧山芹醇乙酯的平均总回收率低于10%。甲氧基欧芹素和二氢欧山芹素在Caco-2细胞中的蓄积率分别为(36.15±5.87)%和(53.90±4.39)%。结论:6种香豆素皆以被动扩散为主要方式被Caco-2细胞单层吸收和转运;伞形花内酯、甲氧基欧芹素、当归醇-A和当归醇-B是良好吸收的化合物,二氢欧山芹素和二氢欧山芹醇乙酯的吸收性属中等。在Caco-2细胞中,甲氧基欧芹素和二氢欧山芹素有蓄积,二氢欧山芹醇乙酯具有代谢不稳定性。当归醇-B的转运不受转运介质EBSS(pH 6.5)的影响,碘乙酰胺或MK571的存在亦不影响当归醇-B在两个方向的转运。

Objective： To study the absorption and transepithelial transport of six coumarins （umbelliferone, osthole, columbianadin, columbianetin acetate, angelol-A and angelol-B, isolated from the roots of Angelica pubescens f. biserrata） in the human Caco-2 cell monolayer model. Methods： The in vitro cultured human colon carcinoma cell line, Caco-2 cell monolayer model, was applied to study the absorption and transport of the six coumarins from apical （AP） to basolateral （BL） side and from BL to AP side. The six coumarins were measured by reversed-phase high-performance liquid chromatography （HPLC） coupled with ultraviolet absorption detector. Transport parameters and apparent permeability coefficients （Papp） were calculated and compared with those of propranolol as a control substance of high permeability and atenolol as a control substance of poor permeability. The transport mechanism of angeloI-B was assayed by using iodoacetamide as a reference standard to inhibit ATP-dependent transport and MK571 as a well-known inhibitor of MRP2. Results： The absorption and transport of six coumarins were passive diffusion as the dominating process. The Papp values of umbelliferone, osthole, columbianadin, columbianetin acetate, angeloI-A and angeloI-B from AP to BL side were （2. 679±0.263）×10^-5, （1.306±0.324）×10^-5, （0.595±0.086） ×10^-6, （2.930±0.410） ×10^-6, （1. 532±0. 444） ×10^-5 and （1. 413±0. 243） ×10^-5 cm/s, and from BL to AP side were （3. 381±0. 410） ×10^-5, （0. 898±0. 134） ×10^-5, （0. 510±0. 183） ×10^-6, （0. 222±0. 025）×10^-6, （1. 203±0. 280） ×10^4 and （0. 754±0. 092） ×10^-5 cm/s, respectively. In this assay, the Papp value of propranolol was 2.18×10^-5 cm/s and the Papp value of atenolol was 2.77 ×10^-7 cm/s. Among the 6 coumarins, the Papp values of umbelliferone, osthole, angelol-A and angelol-B from AP to BL side were identical with that of propranotol, and columbianadin and columbianetin acetate lied between propranolol and atenolol. When replaced the HBSS with EBSS, and iodoacetamide or MK-591 were used in the experiment, the Papp of angelol-B had no statistical difference as compared with the control group. In the mean total recoveries, umbelliferone was （83.31±3.52） %, angeloI-A was （77.39±7.38） %, osthole, cotumbianadin and angeloI-B were between 50% to 65 %, and columbianetin acetate was lower than 10%. The accumulation rates of osthole and columbianadin in theCaco-2 cells were （36.15±5.87） % and （53.90±4.39） %, respectively. Conclusion： The absorption and transport of umbelliferone, osthole, columbianadin, columbianetin acetate, angelol-A and angeloI-B are passive diffusion as the dominating process in Caco-2 cell monolayer model. Umbelliferone, osthole, angeloI-A and angeloI-B are estimated to be highly absorbed compounds, and columbianadin and columbianetin acetate are estimated to be moderately absorbed compounds. In the Caco- 2 cells, osthol and columbianadin appear to accumulate, and columbianetin acetate may be metabolited. The absorption and transport of angeloI-B are not influenced by the change of pH and the presence of iodoacetamide or MK571.