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Journal of Chinese Integrative Medicine ›› 2008, Vol. 6 ›› Issue (4): 392-398.doi: 10.3736/jcim20080413

• Original Experimental Research • Previous Articles     Next Articles

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

Xiu-wei Yang1(), Qing-mei Guo2, Ying Wang1   

  1. 1. Department of Natural Medicines,State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University, Beijing 100083, China
    2. College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province 250014, China
  • Received:2007-08-02 Online:2008-04-20 Published:2008-04-15
  • Contact: YANG Xiu-wei E-mail:xwyang@bjmu.edu.cn

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 angelol-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, angelol-A and angelol-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 -5 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 propranolol, 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) %, angelol-A was (77.39±7.38) %, osthole, columbianadin and angelol-B were between 50% to 65%, and columbianetin acetate was lower than 10%. The accumulation rates of osthole and columbianadin in the Caco-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 angelol-B are passive diffusion as the dominating process in Caco-2 cell monolayer model. Umbelliferone, osthole, angelol-A and angelol-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 angelol-B are not influenced by the change of pH and the presence of iodoacetamide or MK571.

Key words: Caco-2 cell, coumarins, intestinal absorption, apparent permeability coefficient

CLC Number: 

  • R285.5

Figure 1

Chemical structures of 6 coumarins 1: Umbelliferone; 2: Osthole; 3: Columbianadin; 4: Columbianetin acetate; 5: Angelol-A; 6: Angelol-B."

Figure 2

Speed-rate of 6 coumarins transported from AP side to BL side in the Caco-2 cell monolayer model 1: Umbelliferone; 2: Osthole; 3: Columbianadin; 4: Columbianetin acetate; 5: Angelol-A; 6: Angelol-B. Coumarins were incubated with Caco-2 cells for 90 min. The values presented M±SD (n=3)."

Figure 3

Speed-rate of 6 coumarins transported from BL side to AP side in the Caco-2 cell monolayer model 1: Umbelliferone; 2: Osthole; 3: Columbianadin; 4: Columbianetin acetate; 5: Angelol-A; 6: Angelol-B. Coumarins were incubated with Caco-2 cells for 90 min. The values presented M±SD (n=3)."

Table 1

Papp values of 6 coumarins from bidirections at different concentrations ($\bar{x}$±s)"

Compounds Papp AP→BL
(×10-5 cm/s)
Papp BL→AP
(×10-5 cm/s)
Papp AP→BL/PappBL→AP AT (%) TRR (%)
Umbelliferone
25 (μmol/L) 2.470±0.278 2.980±0.433 0.83 3.31±0.17 83.58±6.35
50 (μmol/L) 2.638±0.148 3.640±0.297 0.72 2.32±0.49 81.66±1.22
100 (μmol/L) 2.929±0.125 3.517±0.158 0.83 1.01±0.11 84.68±0.82
Average 2.679±0.263 3.381±0.410 0.79 2.21±1.06 83.31±3.52
Osthole
25 (μmol/L) 1.553±0.331 0.975±0.125 1.59 30.05±0.01 52.30±0.01
50 (μmol/L) 1.385±0.116 0.898±0.122 1.54 36.64±0.41 55.95±3.06
100 (μmol/L) 0.955±0.237 0.819±0.152 1.17 41.75±1.02 55.99±0.38
Average 1.306±0.324 0.898±0.134 1.46 36.15±5.87 54.75±2.75
Columbianadin
25 (μmol/L) 0.630±0.107 0.719±0.078 0.88 56.89±2.53 59.27±4.65
50 (μmol/L) 0.497±0.045 0.425±0.029 1.17 55.96±2.09 53.58±3.19
100 (μmol/L) 0.657±0.016 0.384±0.058 1.71 48.86±2.89 46.57±4.12
Average 0.595±0.086 0.510±0.183 1.17 53.90±4.39 53.14±6.52
Columbianetin acetate
25 (μmol/L) 0.253±0.026 0.173±0.048 1.46 6.51±0.53 8.39±0.12
50 (μmol/L) 0.343±0.021 0.252±0.075 1.36 2.39±0.29 10.25±0.12
100 (μmol/L) 0.284±0.011 0.241±0.020 1.18 5.11±2.15 8.57±0.81
Average 0.293±0.041 0.222±0.025 1.33 4.92±2.13 9.07±0.98
Angelol-A
25 (μmol/L) 1.976±0.065 1.577±0.177 1.25 5.93±0.59 86.89±0.69
50 (μmol/L) 1.639±0.088 0.968±0.030 1.69 3.74±0.36 71.06±2.61
100 (μmol/L) 0.980±0.092 1.065±0.018 0.92 2.37±0.11 74.21±0.63
Average 1.532±0.444 1.203±0.280 1.27 4.01±1.79 77.39±7.38
Angelol-B
25 (μmol/L) 1.496±0.128 0.656±0.042 2.28 3.11±0.70 74.34±7.34
50 (μmol/L) 1.604±0.036 0.837±0.042 1.92 2.28±0.59 67.10±1.70
100 (μmol/L) 1.140±0.279 0.769±0.060 1.48 1.83±0.06 50.25±8.07
Average 1.413±0.243 0.754±0.092 1.87 2.41±0.73 63.94±12.08

Figure 4

Time kinetics curve of angelol-B transported from AP side to BL side in the Caco-2 cell monolayer model The final concentration of angelol-B was 50 μmol/L. The values presented M±SD (n=3)."

Figure 5

The Papp values of angelol-B presented with EBSS, iodoacetamide or MK571 in Caco-2 cell monolayer model The values presented M±SD (n=3)."

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