Fig 1.
Chemical structures of hordenine (A) and its metabolite hordenine sulfate (B).
Fig 2.
Extracted ion chromatograms of standard solution containing 500 nM hordenine and hordenine sulfate diluted in MeOH/H2O (1+19, v/v) (A) and in cell culture matrix (B). Both qualifier and quantifier MRM transitions are shown. The matrix signal (intensity: 1,52×105 cps) is resulting from cell culture medium.
Fig 3.
Relative transepithelial/transendothelial electrical resistance (TEER) and electrical capacitance (cCL,) of Caco-2 cells (A1/A2) and primary porcine brain capillary endothelial cells (PBCEC, B1/B2) incubated with 1 μM hordenine or solvent (neg, 0.1% DMSO) for 48 h (n = 3 × 3).
The data are normalized to the initial values at the beginning of the transport studies and represented as means ± standard deviations. Significant differences (p < 0.05) between the test substances and the respective negative control were calculated in the Student’s t-test and marked with asterisks.
Table 1.
Permeability coefficients of Lucifer Yellow (LY) after 48 h preincubation with hordenine (1 μM) and solvent (0.1% DMSO) (n = 3 × 2).
Fig 4.
Passive transfer (A) and active transport (B) of hordenine through the Caco-2 monolayer over the course of 48 h. The passive transfer of 1 μM hordenine from apical (ap) to basolateral (ba) compartment was determined (n = 3 × 3). Hordenine and hordenine sulfate (abbreviated as sulfate) were quantified using HPLC-MS/MS and normalized to the initial amount (0.76 nmol) on the apical side. For active transport experiments equimolar concentrations of 200 nM hordenine were applied in the apical and basolateral compartment (n = 3 × 3). Data are presented as means ± standard deviations. Statistically significant differences between concentration in the two compartments are marked by asterisks (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).
Fig 5.
Passive transfer (A) and active transport (B) of hordenine through the PBCEC monolayer. The passive transfer of hordenine from apical (ap) to basolateral (ba) compartment was determined after apical application of 1 μM hordenine (n = 3 × 3). Hordenine was quantified using HPLC-MS/MS and normalized to the initial amount (0.76 nmol) on the apical side. The active transport experiments based on the recovery of 200 nM hordenine in the apical and basolateral compartment after incubation of equimolar concentrations in both compartments (n = 3 × 3). Data are presented as means ± standard deviations.
Table 2.
Permeability coefficients of hordenine calculated in the Caco-2 and BBB model in comparison to test compounds with their literature reference.
Permeation of hordenine from apical to basolateral compartment of the Caco-2 or PBCEC monolayer with apical application of 1 μM hordenine (passive transfer study, n = 3×3).
Fig 6.
Schematic illustration of transport and metabolism of hordenine through the intestinal barrier (A) and the blood-brain barrier (B).