**1. Introduction**

Oral drug administration is a preferred route, offering the advantages of convenience and safety. Many drug interactions with foods and other drugs occur via alteration of drug absorption. There are absorptive transporters, such as organic anion transporting polypeptide (OATP) and secretory transporters, including *<sup>P</sup>*-glycoprotein (*P*-gp), associated with drug absorption. To improve drug therapy, it is necessary to investigate possible interactions mediated by transporters that could alter systemic exposure of drugs.

P-gp, belonging to the ATP binding cassette superfamily, is an ATP-dependent efflux protein that excretes drugs out of cells [1–3]. P-gp is an important factor limiting the absorption of drugs and plays a key role in drug distribution and resistance [3,4]. For example, P-gp overexpression induced by a hypoxic environment in many cancers decreases the effects of chemotherapy [5,6]. Furthermore, drug–drug interactions may occur when substrates of P-gp (e.g., cimetidine, digoxin, doxorubicin, fexofenadine, and vinblastine) are coadministered with inhibitors of P-gp (e.g., atorvastatin, ketoconazole and quinidine) or inducers of P-gp (e.g., rifampin and clotrimazole) [7,8]. The OATP family is also an important transporter for drug disposition. The OATP members of the solute carrier (SLC) family, contributes to the uptake of substrates, including endogenous compounds

and drugs [9,10]. Drug–drug interactions and food–drug interactions mediated by these two active transporters—P-gp and OATP—have been reported. In addition, a study on medication use patterns revealed that 50% of 2590 study participants had taken at least one prescription drug during the week prior to the study, and 16% of them had taken one or more herbals/supplements [11,12]. Given that St. John's wort was found to increase *P*-gp expression [13], it is necessary to evaluate the e ffects of herbal supplements on these transporters. Despite the widespread use of herbal drugs in combination with drugs, there has been little research on the interactions between drugs and herbal medicines.

This study investigated the e ffects of*Rumex acetosa (R. acetosa)* extract on *P*-gp and OATP1A2 in vitro and on fexofenadine absorption in vivo. *R. acetosa*, used in folk remedies for skin diseases, has been singled out as a natural herbal medicine for its potential to be used in combination with fexofenadine [14]. *R. acetosa* is widely distributed in eastern Asia and decoction of this plant has been used for the treatment of several health disorders such as fever, gastro-intestinal problems, inflammatory diseases. It is belonging in the Polygonaceae family, known to produce many biologic metabolites [15]. Particularly, *R. acetosa* is rich in anthraquinones and flavonoids that have anti-inflammatory and antiproliferative effects [16,17]. Emodin, a major anthraquinone component of *R. acetosa* extract, is reported that has the potential for *P*-gp mediated drug interaction [18] and has various pharmacological e ffects, such as antidiabetic [19] and anticancer activities [20].

Fexofenadine, a selective histamine H1 receptor antagonist, is widely used for seasonal allergic rhinitis and chronic idiopathic urticarial treatment [21]. There is no evidence for cardiotoxicity associated with fexofenadine, the active metabolite of terfenadine, even though terfenadine is not used anymore due to the risk of cardiac arrhythmia. Fexofenadine was selected as a model drug that is a marker substrate of *P*-gp [22] and OATP1A2 [23]. Fexofenadine is considered a good model drug, because only around 5% of its dose is metabolized and most of the dose is excreted into urine (11%) and feces (80%) as the unchanged form [24,25], which means that metabolism can be excluded in interpreting the pharmacokinetics of fexofenadine.

To date, there have been many drug interaction studies involving *P*-gp or OATP. However, there have been few studies concerning drug interactions with herbal medicines involving both *P*-gp and OATP1A2. Furthermore, it has been reported that the emodin acts on *P*-gp as an inducer [26] or an inhibitor [18]. Our results clarify the inhibitory e ffect of emodin on the *P*-gp through in vitro and in vivo study. In addition, our findings include the fact that *R. acetosa* extract could a ffect drug absorption via intervention in the OATP-mediated influx and the aqueous solubility. These results indicate that the effects of herbal medicines such as plant extracts, on drug absorption must be considered in terms of not only e fflux through *P*-gp, but also OATP-mediated influx and the aqueous solubility.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Fexofenadine hydrochloride and emodin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethyl sulfoxide (DMSO), terfenadine, verapamil, Dulbecco's modified Eagle's medium (DMEM) with high glucose, MEM non-essential amino acid solution (NEAA) and glutamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and water were purchased from Fisher Scientific Korea (Seoul, Korea). Emodin, emodin-8- *O*-β-<sup>d</sup>-glucoside, chrysophanol, chrysophanol-8- *O*-β-<sup>d</sup>-glucoside, physcion and physcion-8- *O*-β-<sup>d</sup>-glucoside isolated from *R. acetosa* were obtained from the pharmacognosy laboratory of the College of Pharmacy at Gyeongsang National University (Jinju, Korea) [27]. Fetal bovine serum (FBS), N-2-hydroxyethylpiperazine–N-2-ethanesulfonic acid (HEPES) and Hanks' balanced salt solution (HBSS) were purchased from Corning (Manassas, VA, USA). Penicillin–streptomycin, Opti-MEM and 0.25% ( *w*/*v)* trypsin–EDTA were purchased from Gibco (Carlsbad, CA, USA). Phosphate bu ffered saline (PBS) was purchased from Welgene (Gyeongsan, Korea). An MDR assay kit (fluorometric) was purchased from Abcam (Cambridge, UK).

#### *2.2. R. acetosa Extract*

The *R. acetosa* extract was prepared by previously reported procedure [27]. Briefly, the dried whole part of *R. acetosa* was extracted with 70% ethanol. The extraction was performed by the Soxhlet extractor for 3 h at 80 ◦C. The extract was filtered and lyophilized.

The total phenol content and total flavonoid content of *R. acetosa* extract were 74.5 mg GAE (gallic acid equivalent)/g of dry weight and 180.3 μg QAE (quercetin equivalent)/g of dry weight, respectively. The contents of anthraquinones in *R. acetosa* extract were determined by HPLC. The contents of emodin, emodin-8-*O*-β-<sup>d</sup>-glucoside, chrysophanol, chrysophanol-8-*O*-β-<sup>d</sup>-glucoside, physcion and physcion-8-*O*-β-<sup>d</sup>-glucoside in *R. acetosa* extract were 0.94 ± 0.15%, 1.29 ± 0.06%, 0.68 ± 0.09%, 0.77 ± 0.12%, 0.17 ± 0.02% and 0.41% ± 0.05% (*w*/*w*), respectively. The values were expressed as mean ± standard deviation.
