Active Hydrophilic Components of the Medicinal Herb Salvia miltiorrhiza (Danshen) Potently Inhibit Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8)

Abstract

Many active components of herbal products are small organic anions, and organic anion transporters were previously demonstrated to be a potential site of drug-drug interactions. In this study, we assessed the inhibitory effects of six hydrophilic components of the herbal medicine Danshen, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and tanshinol, on the function of the murine organic anion transporters, mOat1 and mOat3. All of Danshen components significantly inhibited mOat1- and mOat3-mediated substrate uptake (P < 0.001) with lithospermic acid (LSA), protocatechuic acid, rosmarinic acid (RMA), and salvianolic acid A (SAA) producing virtually complete inhibition under test conditions. Kinetic analysis demonstrated that LSA, RMA, and SAA were competitive inhibitors. As such, K(i) values were estimated as 14.9 ± 4.9 μM for LSA, 5.5 ± 2.2 μM for RMA, and 4.9 ± 2.2 μM for SAA on mOat1-mediated transport, and as 31.1 ± 7.0 μM for LSA, 4.3 ± 0.2 μM for RMA, and 21.3 ± 7.7 μM for SAA on mOat3-mediated transport. These data suggest that herb-drug interactions may occur in vivo on the human orthologs of these transporters in situations of polypharmacy involving Danshen and clinical therapeutics known to be organic anion transporter substrates.

Figure 1

Chemical structures of six active hydrophilic Danshen components. MW: molecular weight.

Figure 2

Inhibition profile of mOat1 and mOat3. (a) Inhibition of mOat1-mediated uptake of [³H]PAH (5 μM) by LSA, PCA, RMA, SAA, SAB, TSL, and probenecid (1000 μM) was measured in the CHO-mOat1 cells (10 min). Background PAH accumulation was measured in CHO-FRT cells in the absence of inhibitor and is shown to provide a clear gauge of the low background in the experimental system. (b) Inhibition of mOat3-mediated uptake of [³H]ES (1 μM) by LSA, PCA, RMA, SAA, SAB, TSL, and probenecid (1000 μM) was measured in the CHO-mOat3 cells (10 min). Background ES accumulation was measured in CHO-FRT cells in the absence of inhibitor and is shown to provide a clear gauge of the low background in the experimental system. Values are mean ± S.D. of triplicate values. ***denotes P < 0.001 as determined by one-way ANOVA followed by Dunnett's t-test.

Figure 3

Michaelis-Menten kinetics of PAH transport in CHO-mOat1 cells. In order to calculate K _i values for mOat1, the K _m value for PAH needed to be determined in the CHO-mOat1 cell system. Uptake of [³H]PAH was measured for 2 min at room temperature in CHO-mOat1 (closed triangles) and CHO-FRT (open squares) cells in order to construct a saturation curve. The corrected curve (closed circles) was obtained by subtracting the nonspecific background uptake as measured in the CHO-FRT cells from CHO-mOat1 accumulation to allow analysis of mOat1-mediated activity. Experiments were repeated three times in triplicate, and Michaelis constant (K _m) values were calculated by nonlinear regression using the Michaelis-Menten model. The K _m for PAH on mOat1 was estimated as 13.0 ± 3.3 μM (mean ± S.E.M.). Graph shown is from a representative experiment with values plotted as mean ± S.D. (n = 3).

Figure 4

K _i determination for LSA, RMA, and SAA in the CHO-mOat1 cell line. Two minute uptake of [³H]PAH (5 μM) in CHO-mOat1 cells was measured in the presence of increasing concentrations (10⁻⁷ to 5 × 10⁻⁴M) of LSA, RMA, and SAA. Data were corrected for nonspecific background measured in the CHO-FRT cells prior to kinetic analysis. K _i values were determined with non-linear regression and the “one-site competition” model using GraphPad Prism software. Experiments were repeated three times in triplicate with the mean K _i ± S.E.M. reported in Table 2. Graphs shown are from representative experiments with values plotted as mean ± S.D. (n = 3).

Figure 5

K _i determination for LSA, RMA, and SAA in the CHO-mOat3 cell line. One minute uptake of [³H]ES (1 μM) in CHO-mOat3 cells was measured in the presence of increasing concentrations (10⁻⁷ to 4 × 10⁻⁴M) of LSA, RMA, and SAA. Data were corrected for nonspecific background measured in the CHO-FRT cells prior to kinetic analysis. K _i values were determined with non-linear regression and the “one-site competition” model using GraphPad Prism software. Experiments were repeated three times in triplicate with the mean K _i ± S.E.M. reported in Table 2. Graphs shown are from representative experiments with values plotted as mean ± S.D. (n = 3).