**3. Results**

#### *3.1. Confirmation of ABCC11-Mediated Transport Activity*

Prior to screening the ABCC11-inhibitory activities of natural products, we verified the transport assay system used in the present study. Immunoblotting with the anti-ABCC11 antibody confirmed the expression of ABCC11 protein as a matured *N*-linked glycoprotein in the plasma membrane vesicles prepared from the ABCC11-expressing cells (Figure 1a). No detectable expression of ABCC11 was observed in the control vesicles. We then measured the ATP-dependent DHEA-S transport into the ABCC11-expressing plasma membrane vesicles (Figure 1b). The DHEA-S transport activities of the ABCC11 vesicles were remarkably higher than those of the mock vesicles, which was enough for the quantitative evaluation of ABCC11-mediated DHEA-S transport activity in subsequent processes.

**Figure 1.** Expression and function of ABCC11. (**a**) Immunoblot detection of ABCC11 protein in the plasma membrane vesicles using an anti-ABCC11 antibody. Mock means plasma membrane vesicles that were prepared from control cells transfected with an empty pcDNA3.1/hyg(−) vector. Arrowhead: matured ABCC11 as an *N*-linked glycosylated protein. Na+/K<sup>+</sup>-ATPase (a plasma membrane protein) was used for a loading control. (**b**) [1,2,6,7-3H(N)]-dehydroepiandrosterone sulfate (DHEA-S) transport activities. Plasma membrane vesicles were incubated with or without ATP for 5 min. In this assay, all incubation mixtures contained 1% dimethyl sulfoxide (DMSO). Data are expressed as the mean ± SD; *n* = 3. Statistical analyses for significant differences were performed using Bartlett's test, followed by a parametric Tukey–Kramer multiple-comparison test. Different letters indicate significant differences between groups (*p* < 0.05).

#### *3.2. Screening the ABCC11-Inhibitory Activities of Plant Extracts*

For the ABCC11-inhibitory properties of natural products, we focused on plants commonly found in the human diet including citruses, tea leaves, soybeans, and miso, a traditional grain-based fermented food in Japan [16]. Each sample was extracted with water and then dialyzed, and the resulting outer layer was lyophilized and reconstituted in water at 10 mg/mL. The 34 obtained concentrates (final concentration at 100 ppm) were used for screening the ABCC11-inhibitory activity (Figure 2). Since the extract of soybean (*Glycine max*) showed the highest inhibitory activities (approximately 70% inhibition) and soybean is a common crop consumed globally, we further explored the ingredients therein responsible for the ABCC11-inhibitory activity.

**Figure 2.** Screening of inhibitory effects of various plant extracts on the transport activity of ABCC11. Inhibitory effect of each plant extract on the ABCC11-mediated [1,2,6,7-3H(N)]-DHEA-S transport activity was investigated by the vesicle transport assay. Plasma membrane vesicles (0.375 mg/mL in the reaction mixture) were incubated with the extract (100 ppm) in the presence of 50 μM [1,2,6,7-3H(N)]-DHEA-S for 5 min; 1% water was used for the vehicle control. Data are expressed as % of vehicle control, and they represent averages of two independent experiments.

#### *3.3. Fractionation and Isolation of Glycine Max (Soybean) Extract by Chromatographic Separations*

To determine the ABCC11-inhibitory ingredients in the water extract of soybeans, further fractionation was conducted with liquid chromatographic separations in a total of three steps (Figure 3). First, the water extract was separated with a preparative MPLC system to yield 12 fractions (Fr.#1-12) (Figure 4a). The ABCC11-inhibitory activities of these 12 fractions were measured at 100 ppm (Figure 4b). Fr.#1-9 showed no significant effect, whereas Fr.#10-12 significantly inhibited the ABCC11-mediated DHEA-S transport. Secondly, since Fr.#11 exhibited the highest activity, we next further separated it with a similar preparative MPLC to give a total of six subfractions (Fr.#11-1 to Fr.#11-6), as described

in Materials and Methods (Section 2.7). Monitoring the MPLC effluent at 254 nm showed that, among the six subfractions, the main compounds were collected in Fr.#11-5, which had the highest ABCC11-inhibitory activity (approximately 22% of inhibition at 50 ppm) among the six subfractions.

**Figure 3.** Separation scheme used to fractionate ABCC11 inhibitors in the soybean extract. In each separation step, the fraction with the highest ABCC11-inhibitory activity is colored in red. DP: dominant peak (details are described in Materials and Methods).

**Figure 4.** ABCC11-inhibitory activities for each fraction of soybean extract from the first separation step with preparative medium-pressure liquid chromatography (MPLC). (**a**) A preparative MPLC chromatogram for separating the water extract of soybeans. The chromatogram was recorded at 265 nm. Red line indicates linear gradients of solvent B (0.2% formic acid in acetonitrile). (**b**) ABCC11-inhibitory activity profile of each fraction (100 ppm) obtained from the first separation process. The effects on ABCC11-mediated [1,2,6,7-3H(N)]-DHEA-S transport activity were investigated by the vesicle transport assay; 1% methanol was used for the vehicle control. Data are expressed as % of vehicle and the mean ± SD; *n* = 3. \*\*, *p* < 0.01 vs. control (Dunnett's test).

Thirdly, to isolate the substances responsible for the ABCC11 inhibition, Fr.#11-5 was further subjected to recycling HPLC, which was repeated to afford components from peak #11-5-1 and peak #11-5-2 (denoted as Fr.#11-5-1 and Fr.#11-5-2, respectively; Figure 5a). All the wastes of this process were collected and further processed as Fr.#11-5-3. All three subfractions showed ABCC11-inhibitory activities at 20 ppm, and Fr.#11-5-2 was the most active (Figure 5b) and therefore the object of further analysis. Of note, the re-chromatography of Fr.#11-5-2 followed by LC-Q-TOF-MS and LC-DAD analyses suggested that this subfraction was mainly composed of a single substance that should be responsible for the ABCC11-inhibitory activity (Figure 5c). Indeed, a full LC-Q-TOF-MS scan of Fr.#11-5-2 revealed a constituent with a retention time of 5.83 min. Ions were detected in the positive ion mode at *m*/*z* 271.0616 and 293.0428, which corresponded to the [M+H]<sup>+</sup> and [M+Na]<sup>+</sup> of the constituent, respectively (Figure 5d).

**Figure 5.** Isolation of an ABCC11-inhibitory ingredient by means of recycling preparative HPLC. (**a**) Recycling preparative HPLC chromatograms for the separation of fractions Fr.#11-5-1 and Fr.#11-5-2. The upper chromatogram was recorded with a refractive index detector, and the lower one was recorded with a diode array and multiple-wavelength detector at 254 nm. After separation under the recycling mode (0–120 min), the mode was changed; Fr.#11-5-1 (123–126 min) and Fr.#11-5-2 (160–176 min) were collected, and all the wastes were collected and further processed as Fr.#11-5-3. R, recycled peaks for Fr.#11-5-1; r, recycled peaks for Fr.#11-5-2. (**b**) ABCC11-inhibitory activities of each subfraction (20 ppm) in terms of ABCC11-mediated [1,2,6,7-3H(N)]-DHEA-S transport activity measured by the vesicle transport assay; 1% DMSO was used for the vehicle control. Data are expressed as % of vehicle and the mean ± SD; *n* = 3. \*\*, *p* < 0.01 vs. control (Dunnett's test). (**c**) Purity verification of the isolated ingredient in Fr.#11-5-2 by spectrometric analyses. Left: UV chromatograms recorded at 265 nm. Right: LC-quadrupole time-of-flight-MS (LC-Q-TOF-MS) base peak chromatograms, excluding peaks derived from the plasticizing materials and injected solvent. †, a specific peak in Fr.#11-5-2 with a retention time of 5.83 min. (**d**) Full scan mass spectrum obtained in the positive ion mode of this peak (indicated by † in **c**) at 5.83 min. The inset is the magnified view for ions at *m*/*z* 271.0616 and 293.0428, which corresponded to the [M + H]+ and [M + Na]+ of the target constituent, respectively.

#### *3.4. Structural Characterization of the Putative ABCC11 Inhibitor Derived from Soybeans*

We next conducted a series of spectrometric analyses (Figure 6) to obtain structural information about the candidate active ingredient, which was almost completely isolated from the soybean extract into Fr.#11-5-2. Based on accurate mass information from the LC-Q-TOF-MS analysis (Figure 5d), the elemental composition of the target analyte was determined as C15 H10 O5 (Δ−5.51 and Δ−2.84 ppm from [M+H]<sup>+</sup> and [M+Na]<sup>+</sup>, respectively). The three major soy isoflavones are genistein, daidzein, and glycitein, with the respective formulas (monoisotopic mass) of C15 H10 O5 (270.0528), C15 H10 O4 (254.0579), and C16 H10 O4 (284.0685) [17]. Additionally, isoflavones exhibit an intense UV absorption between 240 and 280 nm associated with their benzoyl system, and the target analyte showed a similar spectrometric feature. Therefore, we hypothesized that the active ingredient would be genistein (Figure 6a). This hypothesis was tested by spectroscopic analyses, which demonstrated that the Fr.#11-5-2 and authentic genistein were identical in their retention time (Figure 6b), accurate mass of parent ion and the ratios of adduct ions (Figure 6c), photoabsorption spectrum (Figure 6d), and MS/MS spectrum (Figure 6e). Hence, the isolated substance should be genistein.

**Figure 6.** Chemical characterization of an ABCC11 inhibitory activity-guided fraction from soybean extract. Fraction (Fr.) #11-5-2 (upper panels) and authentic genistein (lower panels) were analyzed by a high-performance liquid chromatography instrument coupled with a diode array and multiple wavelength detector (DAD) and Q-TOF-MS system. (**a**) Chemical structure of genistein. (**b**) Extracted ion chromatograms (EICs) with a single peak at *m*/*z* 271.0621 in the positive ESI spectrum. (**c**) MS spectrums with a retention time of 5.83 min for the parent ion. (**d**) DAD spectrums. (**e**) Information on the fragment ions derived from MS/MS analyses.

#### *3.5. Identification of the Active Ingredient as Genistein*

To check whether genistein was indeed responsible for inhibiting the ABCC11 function, we examined the e ffects of genistein and the other two major soy isoflavones (daidzein and glycitein), as well as their metabolites (genistein 7-β-D-glucuronide 4'-sulfate, daidzein 7-β-D-glucuronide 4'-sulfate, and equol) on the ABCC11 function (Figure 7a,b). As expected, genistein inhibited ABCC11; its ABCC11-inhibitory activity was the highest among the tested compounds at 100 μM. The further examination of its concentration-dependent inhibitory e ffects revealed an IC50 of 61.5 μM (Figure 7c). If the Fr.#11-5-2 of soybean extract only contained genistein, 20 ppm of this subfraction corresponded to approximately 74 μM of genistein. The detected ABCC11-inhibitory effect of Fr.#11-5-2 at 20 ppm was approximately 40% (Figure 5b), and this is consistent with the measured concentration-dependent effects of genistein (Figure 7c). After combining these results and the determined structural characters (Figure 6), we concluded that the active ingredient in the Fr.#11-5-2 was indeed genistein.

**Figure 7.** Effects of soybean flavonoids and their metabolites on the transport activity of ABCC11. (**a**) Chemical structures. Gein-7G-4'S, genistein 7-β-D-glucuronide 4'-sulfate; dein-7G-4'S, daidzein 7-β-D-glucuronide 4'-sulfate. (**b**) Inhibitory effects of each flavonoid (100 μM) on ABCC11-mediated [1,2,6,7-3H(N)]-DHEA-S transport. (**c**) Concentration-dependent inhibition of ABCC11-mediated DHEA-S transport by genistein. Data are expressed as % of vehicle and the mean ± SD; *n* = 3–6. \*\*, *p* < 0.01 vs. control (Dunnett's test).

Daidzein and glycitein only exhibited weak and minimal ABCC11-inhibitory activity, respectively. (*S*)-equol, which is a daidzein-derived metabolite produced by the intestinal bacterial flora in human intestines [18], showed a stronger effect than daidzein (Figure 7b). Moreover, compared with the non-conjugated forms, the glucuronide-sulfate diconjugates of genistein and daidzein showed lower inhibitory activities, suggesting that the polyfunctionalization-mediated structural enlargement might affect the interaction between the soy isoflavones and ABCC11 protein.

#### *3.6. Investigation of ABCC11-Inhibitory Activities of Other Dietary Flavonoids*

Finally, we investigated the effects of other dietary flavonoids of interest on the ABCC11 function. The chemical structures of the selected compounds are shown in Figure A1. As shown in Table 2, at 100 μM (the same concentration used in Figure 7b), 13 of the flavonoids lowered the ABCC11-mediated DHEA-S transport to less than 30% of that of the control. Among them, luteolin, nobiletin, myricetin, quercetagetin, isoliquiritigenin, and phloretin powerfully inhibited the transport activity of ABCC11. Additionally, hardly any ABCC11-inhibitory activity was observed for (+)-catechin, (−)-epicatechin, (−)-epigallocatechin, and (+)-gallocatechin in this study, but their galloylated forms inhibited ABCC11, thus suggesting that the gallic acid esterified with catechins would be an important chemical structure for an interaction with ABCC11. These results provide a framework for the further investigation of naturally derived ABCC11 inhibitors.


**Table 2.** ABCC11-inhibitory activities of dietary flavonoids.

Inhibitory effects of each food ingredient (100 μM) on ABCC11-mediated [1,2,6,7-3H(N)]-DHEA-S transport activity were investigated by using plasma membrane vesicles (0.5 mg/mL in the reaction mixture) prepared form ABCC11-expressing or control adenovirus-infected 293A cells. Additionally, major green tea catechins, based on a previous study [19], were tested in this study. Data are expressed as % of vehicle and the mean ± SD; *n* = 3. \*, Values were calculated under 0; †, one-sample *t*-test (vs. vehicle control as 100%); NS, not significantly different from control (*p* > 0.05).
