Interactions of 172 plant extracts with human organic anion transporter 1 (SLC22A6) and 3 (SLC22A8): a study on herb-drug interactions

Drugs and reagents

Probenecid, furosemide, warfarin and poly-D-Lysine were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 6-Carboxyfluorescein (6-CF) was obtained from Aladdin (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco (Gaithersburg, MD, USA). Hygromycin B was purchased from Solarbio (Beijing, China). BCA protein assay kit was purchased from Cwbio (Beijing, China). Methanol, n-hexane, dichloromethane, and n-butanol were purchased from J&K Chemical (Beijing, China). All other chemicals, unless indicated, were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Plant extracts

A total of 37 medicinal and economic plants were collected by staff members from the New York Botanical Garden (Mr. Daniel Atha) and the National Herbarium of the Republic of Georgia (Dr. Manana Khutsishvili) during 2006 at various sites in the Republic of Georgia, Armenia, Azerbaijan, and the United States. Voucher specimens have been prepared and deposited in the National Herbarium of Georgia and the New York Botanical Garden. Each sample was identified by the staff of these herbaria using standard botanical methods. A list of the taxa investigated in the current study and their relative information are presented in Table S1. The detailed preparation method of plant extracts is shown in Fig. S1. Briefly, methanol extracts were prepared from either the entire plant or discrete parts of the plant. Each methanol extract was further fractionated to provide four fractions, namely n-hexane (H), dichloromethane (D), n-butanol (B) and aqueous (A). As a result, a total of 172 plant extracts were prepared, and maintained at −20 °C until use.

Cloning and expression of human OAT1 and OAT3

HEK293 cells (bought from Invitrogen, Carlsbad, CA, USA) stably expressing OAT1 and OAT3 were established using the Flp-In expression system (Invitrogen) according to the manufacturer’s protocol as described in a previous publication (Chen et al., 2009). In brief, the cDNAs of OAT1 (GenBank accession number NM_004790) and OAT3 (GenBank accession number NM_004254) were obtained from OriGene (Rockville, MD, USA), and sub-cloned into pcDNA5/FRT (Invitrogen). They were co-transfected with pOG44 into Flp-in-293 cells. Stable cells (HEK-OAT1 and HEK-OAT3) were selected in the presence of hygromycin B (75 μg∕ml). Cells were routinely grown in DMEM containing 10% FBS, 1% streptomycin/penicillin and hygromycin B (75 μg∕ml) in a humidified incubator at 37 °C and 5˘% CO₂. The mRNAs of OAT1 and OAT3 in three cell lines were quantified by quantitative real-time PCR. The method and primer sequences are shown in Table S2.

Uptake assay

Cell uptake assay was performed as previously described with some modifications (Hsueh et al., 2016). Cells were seeded at a density of 7 × 10⁴ cells per well in poly-D-lysine-coated 96-well culture plates. Transport assays were performed 16-h post seeding in preheated uptake buffer (135 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 0.8 mM MgSO₄, 28 mM glucose, and 13 mM Hepes, pH 7.2). 6-CF was used as the fluorescent substrate for both hOAT1 and hOAT3 according to a previous study (Duan et al., 2012). The 6-CF alone or 6-CF with different inhibitors (probenecid or plant extracts) was incubated for 4 min in HEK-OAT1 cells and 10 min in HEK-OAT3 cells. Uptake was terminated by adding ice-cold phosphate-buffered saline (PBS), and quickly washed three times with ice-cold PBS. The cells in each well were lysed with 100 μl of 20 mM Tris–HCl containing 0.2% TritonX-100. An aliquot of 50 μl lysate was used to determine fluorescence using a Tecan Infinite M200 (Mannedorf, Switzerland) with excitation and emission wavelengths at 485 and 528 nm, respectively. The protein concentration of the cell lysate was determined using a BCA Protein Assay Kit (Cwbio, Beijing, China). Uptake values were standardized against protein content, and were measured in triplicate.

Uptake kinetics were characterized by measuring the uptake of increasing concentration of 6-CF (0–500 μM) in cells stably expressing hOAT1 (4 min) and hOAT3 (10 min). Michaelis constants were obtained by fitting to the Michaelis–Menten equation V = V_maxS∕(K_m + S), where V_max is the maximum transport rate, S is the substrate concentration and K_m is the substrate concentration reaching half-maximal uptake rate (Graphpad Prism version 5, San Diego, CA, USA).

The fluorescence of each extract in the presence or absence of 6-CF was measured to rule out detection error. Probenecid was loaded into four corners of each 96-well plate, and used as a positive control. The stock solutions of plant extracts were prepared using DMSO with a final concentration of 1 mg/ml. The initial concentration for each extract was set at 5 μg∕ml. Every well contained the same concentration of DMSO (0.5%) to eliminate the potential effect of solvent. Uptake of 6-CF (4 μM) was quantified for 4 min for OAT1 and 10 min for OAT3 in the presence or absence of plant extracts with a final concentration of 5 μg∕ml. Before the initial screening, the reproducibility of uptake experiments was evaluated. The uptake of 6-CF (4 μM) in HEK-OAT1 and HEK-OAT3 cells were measured in the absence and presence of probenecid (100 μM) in three plates with four repeated wells per uptake assay in each plate. The results demonstrated that the inhibitory effect of probenecid on OAT-mediated 6-CF uptake was reproducible (Fig. S3). Due to the large number of samples, for the initial screening study, each uptake experimental sample was measured in triplicates in the same assay. For those plant extracts showing more than 50% inhibition, the uptake experiments were repeated twice to validate the results.

Those plant extracts showing more than 50% inhibition of 6-CF uptake were selected to carry out concentration-dependent inhibition experiments. To determine IC₅₀ (50% inhibitory concentration) values, three-fold serial dilutions of the tested extracts (0–5 μg∕ml) and probenecid (0–200 μM) were prepared in triplicate in a 96-well plate and mixed with an equal volume of 6-CF (4 μM). In the experiment, we found that DMSO at concentrations up to 2% had little effect on OAT-mediated 6-CF uptake (Fig. S4). Therefore, the control group contained 0.25% DMSO for the concentration-dependent inhibition experiments. After the uptake was terminated at 4 min and 10 min for OAT1 and OAT3 respectively, intracellular fluorescence was determined as above. IC₅₀ values were estimated by non-linear regression analysis, using the following equation: $Y=100∕\left(\left(1+X\stackrel{ˆ}{}HillSlope\right)∕\left({\mathrm{IC}}_{50}\stackrel{ˆ}{}HillSlope\right)\right)$; where X is the logarithm of inhibitor concentration; Y is the normalized response; and HillSlope is the Hill slope factor (Graphpad Prism Version 5; Graphpad, San Diego, CA, USA).

Pharmacokinetic studies

Pharmacokinetic study of furosemide was performed according to a previous publication (Ma et al., 2014). Male Sprague–Dawley (SD) rats (200–220 g) were obtained from Vital River Laboratories (Beijing, China), and housed in the certified animal facility at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences (CAMS, Tianjin, China). All of the experimental protocols and animal handling were performed with the approval of the Animal Use Committee at the CAMS (No. 1202). Rats were acclimated for 1 week in a temperature controlled room (23 ± 2 °C), with a relative humidity of 55 ± 10%. Food and water were supplied ad libitum. The rats were fasted for 12-h before the experiment. For the interaction by Juncus effusus extract, a dose of 100 mg/kg of this extract (dissolved in 40% PEG400) was administered to the rats by either oral gavage or tail vein injection at 5 min before administration of 10 mg/kg FS (dissolved in saline) intravenously through the tail vein. For the control group, a same volume of 40% PEG400 was administered to the rats at 5 min before the injection of a same volume of saline. About 700 μl blood was collected into heparinized tubes via the orbital venous sinus at 2, 5, 10, 15, 30, 60, 90, and 120 min. Plasma sample (250 μl) was pipetted into a microcentrifuge tube, and 1 ml acetonitrile (containing 40 μg∕ml warfarin as internal standard) was added. The mixture was vortexed and centrifuged at 20,000 rpm for 15 min. The supernatant was dried in vacuum and then was reconstituted in 200 μL mobile phase for HPLC injection.

Quantification of furosemide by HPLC-UV

The concentrations of furosemide were quantified using a modified HPLC method (Jankowski, Skorek-Jankowska & Lamparczyk, 1997; Ma et al., 2014). Chromatographic analysis was performed on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a G1311C Quaternary pump, G1329B autosampler (0.1–100 μL), G1316A column oven (273–333 K), and G1315D Diode Array Detector (190–950 nm). The analytical column was a ZORBAX SB-C18 columm (4.6 × 150 mm, 5 µm; Agilent, Santa Clara, CA, USA) with a precolumn. The mobile phase was a mixture of acetonitrile and distilled water (69:31, v/v) with 0.3 M sodium acetate buffer (pH 5.0). The column temperature is 30 °C with a flow rate of 1 ml/min. The injection volume was 10 μl, and the detection wavelength was set at 280 nm.

Data analysis

For the pharmacokinetics, five rats per group were used. Data with error bars represent means ± S.D. Pharmacokinetic parameters were calculated using a non-compartmental analysis by using PKSolver (Zhang et al., 2010). Data were analyzed with two-tailed unpaired Student’s t-test. The p value for statistical significance was set to be <0.05.

Characterization of OAT1- and OAT3-overexpressing cells

HEK293 cells stably expressing OAT1 and OAT3 (HEK-OAT1 and HEK-OAT3) were validated by both mRNA expression and uptake function. As shown in Fig. S2, the mRNA expressions of OAT1 and OAT3 were almost undetectable in HEK-EV cells, whereas they are markedly increased in HEK-OAT1 and HEK-OAT3 cells, respectively. Both HEK-OAT1 and HEK-OAT3 cells exhibited a time- and concentration-dependent increase in the uptake of 6-CF (Fig. 1A). The estimated K_m values for the uptake of 6-CF by hOAT1 and hOAT3 were 10.86 ± 0.64 μM and 112.9 ± 14.24 μM, respectively. The V_max values of 6-CF uptake for HEK-OAT1 and HEK-OAT3 cells were 168.5 ± 2.27 and 71.89 ± 3.42 pmol/mg protein/min, respectively. Probenecid is a classic inhibitor for OAT1 and OAT3. The estimated IC₅₀ value of probenecid was 29.42 μM for OAT1, and 8.662 μM for OAT3 (Fig. 1B). Fluorescence images of cells treated with 6-CF in the presence or absence of probenecid were taken using a fluorescence microscope (Nikon E100; Nikon, Tokyo, Japan). As shown in Fig. 1C, no detectable fluorescence was observed in HEK-EV cells treated with 6-CF. Both HEK-OAT1 and HEK-OAT3 cells treated with 6-CF showed a strong fluorescence, which was almost completely abolished by co-incubating probenecid. Therefore, HEK-OAT1 and HEK-OAT3 cells were successfully established and characterized by functional assay.

Effects of 172 plant extracts on OAT1- and OAT3-mediated 6-CF transport

Among the 172 plant extracts investigated in the initial screening study, 59 extracts (9H, 24D, 18B, and 10A) significantly inhibited the 6-CF uptake in HEK-OAT1 cells, whereas nine extracts (2H, 2B and 5A) significantly increased the OAT1-mediated uptake of 6-CF (Fig. 2 and Table 1). Similarly, 55 extracts (8H, 28D, 16B, and 3A) significantly inhibited the OAT3-mediated uptake of 6-CF, whereas seven extracts (2B and 5A) increased the 6-CF uptake in HEK-OAT3 cells.

Three hexane extracts (Achillea biebersteinii, Chaerophyllum bulbosum, Symphytum asperum), 17 dichloromethane extracts (Anchusa azurea, Camphorosma lessingii, Chaerophyllum bulbosum, Echium russicum, Elaeagnus orientalis, Glycyrrhiza glabra, Hypericum scabrum, Juncus effusus, Juniperus oblonga (RT), Juniperus oblonga (LF + FR), Melandrium album, Polygonum hydropiper, Primula macrocalyx, Solanum dulcamara (ST + RT), Stachys lavandulifolia, Symphytum asperum, Zygophyllum fabago), 12 n-butanol extracts (Anchusa azurea, Artocarpus altilis, Chaerophyllum bulbosum, Echium russicum, Glycyrrhiza glabra, Hypericum scabrum, Juniperus oblonga (LF + FR), Mentha longifolia, Scutellaria orientalis, Symphytum asperum, Thymus kotschyanus, Zygophyllum fabago), and one aqueous extract (Thymus kotschyanus) showed inhibitory effects on both OAT1 and OAT3.

IC₅₀ values of plant extracts on OAT1- and OAT3-mediated 6-CF transport

As shown in Table 1, 18 extracts for OAT1 and 25 extracts for OAT3 showed more than 50% inhibition. These 43 extracts were selected for further concentration-dependent inhibition studies. Some extracts did not show a good concentration-dependent inhibition on 6-CF uptake and thus were excluded from further investigation (Fig. S5). Three extracts (Camphorosma lessingii (D), Geranium tuberosum (B), Polygonum hydropiper (D)) were strong inhibitors for OAT1 with IC₅₀ values being <5 μg∕ml (2.441, 2.966, and 1.878 μg∕ml, respectively) (Fig. 3A). Fourteen extracts (Anchusa azurea (A and B), Astracantha microcephala (D), Chaerophyllum bulbosum (H), Echium russicum (B), Glycyrrhiza glabra (D), Juncus effusus (D), Juniperus oblonga (LF + FR) (B and D), Mentha longifolia (B), Polygonum hydropiper (D), Primula macrocalyx (D), Thymus kotschyanus (B), Symphytum asperum (B)) were strong inhibitors of OAT3 with IC₅₀ values being <5 μg∕ml (Fig. 3B).

Interaction of Juncus effusus (D) with FS in rats

The mean plasma concentration–time profiles of FS in the presence and absence of Juncus effusus (D) in rats is shown in Fig. 4. Pharmacokinetic parameters of FS were summarized in Table 2. Results indicated that pretreatment with Juncus effusus (D) significantly altered the pharmacokinetics of FS in rats. The AUC_0–t values of FS were increased by 80% and 55% after oral and intravenous coadministration with Juncus effusus (D), respectively. Therefore, concomitant use of Juncus effusus (D) with substrates of OAT1 and OAT3 should require close monitoring for potential HDIs.