Characterization of Natural Aryl Hydrocarbon Receptor Agonists from Cassia Seed and Rosemary

Many recent studies have suggested that activation of the aryl hydrocarbon receptor (AhR) reduces immune responses, thus suppressing allergies and autoimmune diseases. In our continuing study on natural AhR agonists in foods, we examined the influence of 37 health food materials on the AhR using a reporter gene assay, and found that aqueous ethanol extracts of cassia seed and rosemary had particularly high AhR activity. To characterize the AhR-activating substances in these samples, the chemical constituents of the respective extracts were identified. From an active ethyl acetate fraction of the cassia seed extract, eight aromatic compounds were isolated. Among these compounds, aurantio-obtusin, an anthraquinone, elicited marked AhR activation. Chromatographic separation of an active ethyl acetate fraction of the rosemary extract gave nine compounds. Among these compounds, cirsimaritin induced AhR activity at 10–102 μM, and nepitrin and homoplantagenin, which are flavone glucosides, showed marked AhR activation at 10–103 μM.


Introduction
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that is present in mammalian cells and tissues. The AhR has also been referred to as dioxin receptor because it binds environmental pollutants (e.g., dioxins) and is involved in biotoxicity linked to xenobiotic AhR ligand exposure in animals, including cancer, reproductive impairment, and immunological impairment [1][2][3]. Although studies have identified numerous xenobiotic ligands for the AhR, such as dioxins, the essential functions of the AhR are largely unknown; therefore, the AhR is still regarded as an orphan receptor.
Functional elucidation of AhR activation by non-toxic ligands such as food constituents has been reported in recent years [4][5][6]. The AhR has been identified as a target of several signaling pathways that cross-talk with its own regulatory pathway, such as proteasomal degradation, redox-sensitive transcription factors, and mitogen-activated protein kinases (MAPKs) [7,8]. Several studies have also found that the AhR plays an important role in immune system function [9][10][11][12]. For example, activation of the AhR is associated with various effects on dendritic cells (DCs) and regulatory T cells and has been shown to mediate the Th1/Th2 cell balance. These cells play a major role in the development of food allergies, an increasing health problem in both humans and animals. Despite existing knowledge regarding the risk factors of and cellular mechanisms underlying food allergies, no approved treatments are yet available. Activation of the AhR by dioxin-like compounds has been shown to suppress allergic sensitization by reducing the absolute number of precursor and effector T cells, preserving CD4 + CD25 + Foxp 3+ T reg cells, and affecting DCs and their interactions with effector T cells. Additionally, tranilast, an anti-allergy drug, has been shown to cause significant upregulation of microRNA (miR)-302 by activation of the AhR [13]. Thus, dietary ligands of the AhR may have anti-inflammatory, anti-allergy, anti-cancer, and immunoregulatory effects. However, while although the role of the AhR in the response to environmental toxins is widely accepted, its broader role in adapting the response to natural ligands is limited. Therefore, it is necessary to characterize various natural AhR ligands.
In the current study, we sought to further characterize AhR agonists present in foods. We examined the AhR activities of 37 health food materials using an in vitro reporter gene assay called the chemicalactivated luciferase gene expression (CALUX) assay [14][15][16]. Active sample extracts were subsequently fractionated, and chromatography was performed to characterize the fractions containing AhR activity and associated individual constituents.

Identification and AhR Activity of Constituents
To characterize the active components in sample 5 (cassia seed extract), the extract was first partitioned with organic solvent for separation into n-hexane-, ethyl acetate-, and water-soluble fractions. As shown in Figure 2a, AhR activity was present only in the ethyl acetate extract, which was separated by chromatography over Sephadex LH-20 with ethanol to afford 10 fractions (Frs. 1-10).
influence of this glycosidic feature on the activity of the related anthraquinones was similar to our previous findings that the AhR activity of isoflavones tended to be weakened by glycosidation [4]. It is notable that the presence of a hydroxyl group at C-8 on the anthraquinone skeleton is necessary for AhR activation. Additionally, aurantio-obtusin (4), which was the most active compound, had a hydroxyl group at C-7 and C-9, which may also contribute to AhR activation. However, to discuss the structure-activity relationships in anthraquinones, additional data from more compounds are required. The results of the present study revealed that AhR activation by the cassia seed extract is associated with anthraquinones and that aurantio-obtusin (4) may be an important natural AhR agonist.
As mentioned earlier, AhR activation tends to be weakened by glycosidation of the parent AhR ligand. This tendency has been observed even for flavonoid ligands [4]. In the present study, nepitrin (15) and homoplantagenin (16), which are flavone glucosides, were found to have noticeable AhR activity. Some compounds characterized as potential AhR agonist candidates in the current study have been reported to have various biological functions beneficial to human health. For example, lipolytic, antilipogenic, and antiproliferative activities have been identified as biological properties of cirsimaritin (14) [17], and nepitrin (15) has been reported to have anti-inflammatory and gastroprotective activity [18,19]. Recently, several studies have reported that activation of AhR may be involved in various immune responses as described above; therefore, natural AhR ligands are expected to have beneficial regulatory roles in humans, mediating anti-allergy and anti-cancer effects. Further studies on AhR-activating ingredients derived from natural foods may clarify both the physiological significance of the AhR and the benefits derived from food constituents. The reversed-phase (RP) HPLC conditions were as follows: column, L-column ODS (5 μm, 150 × 2.1 mm i.d.) (Chemicals Evaluation and Research Institute, Tokyo, Japan); mobile phase, 5% acetic acid (solvent A) and acetonitrile (solvent B) (0-30 min, 0%-50% B in A; 30-35 min, 50%-85% B in A; 35-40 min, 85%-85% B in A); injection volume, 2 μL; column temperature, 40 °C; flow rate, 0.3 mL/min; and detection, 200-400 nm. TLC was performed on Silica Gel 60 F 254 plates (Merck, Darmstadt, Germany), and the spots were visualized under a UV lamp (254 nm). Column chromatography was conducted using Sephadex LH-20 (GE Healthcare, Little Chalfont, England), MCI Gel CHP-20P (75-150 μm) (Mitsubishi Chemical Co., Tokyo, Japan), YMC GEL ODS-AQ (AQ12S50) (YMC Co., Ltd., Kyoto, Japan), and Silica Gel 60 (Nacalai Tesque, Kyoto, Japan) columns.

Samples and Reagents
The reagents used in the present study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Nacalai Tesque, and 37 health food materials, as shown in Table 1, were obtained from Uchida Wakanyaku Ltd. (Tokyo, Japan), Tochimoto Tenkaido Ltd. (Osaka, Japan), and Nagaoka Perfumery Ltd. (Osaka, Japan). The species were identified by the Herbarium of the College of Pharmaceutical Sciences, Matsuyama University, where the voucher specimens were deposited. All other chemicals were of analytical reagent grade.

Extraction
The health food samples were prepared as follows: The materials (1 g) were homogenized in aqueous ethanol [ethanol/water (4:1)] (30 mL) for 10 min and filtered. The filtrates were concentrated under reduced pressure and freeze-dried. Fr. 4 (180 mg) was subjected to column chromatography over silica gel 60 (ϕ 2.0 × 20 cm) with chloroform/methanol (9:1) to give obtusin 2-O-glucoside (5) (4.1 mg). These known compounds were identified by direct comparison with valid standards or by comparison of their spectral data with those reported in the literature [20,21].

Estimation of AhR Ligand Activity
The extracts and compounds were dissolved in DMSO and evaluated for AhR-binding activity using a luciferase assay (CALUX assay). The CALUX assay for AhR ligand activity was conducted as follows. Mouse hepatoma H1L1 cells (ca. 1.5 × 10 5 cells/well) were cultured in 96-well culture plates, and the samples were dissolved in DMSO and then added at final concentrations of 1-10 2 μg/mL (or μM in compound)] in three steps in fractions. The final DMSO concentration was 1% in the cell culture medium. The plates were incubated at 37 °C in 5% CO 2 for 24 h for optimal expression of luciferase activity. After incubation, cell viability was confirmed using a microscope. Subsequently, the medium was removed and the cells were lysed. After addition of luciferin as the substrate, luciferase activity was determined using a luminometer (Centro LB960; Berthold, Bad Wildbad, Germany) and recorded as RLUs. The values represent the mean ± SD of at least two or three independent determinations for each experiment. Statistical significance was analysed using the Student's t test.

Conclusions
In this study, we examined the effects of 37 health food materials on AhR activity using a reporter gene assay and found that cassia seed and rosemary extracts elicited notable AhR activation. To characterize the AhR-activating substances within these extracts, the respective extracts were subjected to fractionation followed by estimation of AhR activity. Eight compounds were isolated and identified from the active fractions of the cassia seed extract. Among them, aurantio-obtusin (4), an anthraquinone, was characterized as an effective AhR-activating ligand. In rosemary, nine compounds were isolated from the active extract. Nepitrin (15) and homoplantagenin (16), which are flavone glucosides, showed marked AhR-binding activity.