Arctigenin from Fructus Arctii (Seed of Burdock) Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) and ovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.


Introduction
Arctium lappa (burdock) is a biennial plant of the Arctium genus in the Asteraceae family. Its root is widely used as a dietary ingredient, and its seed (called Fructus Arctii) is used as a traditional herbal medicine in oriental countries such as China, Japan, and Korea. In particular, F. Arctii has been used for the treatment of sore throat, urticaria, and furuncle. The anti-inflammatory and antioxidant properties of F. Arctii are supported by its ethnopharmacological use for the treatment of inflammation, wherein it inhibits proinflammatory factors such as nuclear factor kappa B (NF-B), inducible nitric oxide synthase, and oxidative stress [1,2].
Intestinal epithelial cells regulate the influx of nutrients and the efflux of xenobiotics against substances entering the gut intestinal tract [3]. The tight junction (TJ) proteins expressed in the cells play a key role in intestinal barrier function by protecting against toxic substances, allergens, and macromolecules derived from food [4]. However, dysfunction or destruction of TJ may cause inflammatory diseases such as inflammatory bowel disease, irritable bowel syndrome, leaky gut syndrome, and food allergy [5][6][7][8]. Recently, various components derived from food and plants have been known to enhance intestinal barrier function. For example, theaflavins in black tea could enhance intestinal barrier function by increasing the expression of TJ-related proteins (occludin, claudin-1, and zonula occludens-1) through the activation of AMP-activated protein kinase in Caco-2 cells [9].
The Caco-2 cells, which are derived from colon carcinoma, spontaneously undergo the process of differentiation that leads to the formation of monolayers. The cell monolayers have been used as an in vitro model that mimics the human intestinal epithelium [10,11]. In this study, we established an in vitro system to evaluate the effect of foodand plant-derived extracts including F. Arctii on intestinal barrier function in Caco-2 cell monolayers. In particular, the effect of F. Arctii was focused on intestinal barrier function, and the permeability was measured using ovalbumin (OVA) as one of substrates absorbed through paracellular diffusion. Fructus Arctii (400 g) was pulverized to powder, and then the extract from the powder was obtained by reflux extraction in 95% ethanol (4 L) for 3 h twice. The ethanol extract was concentrated under vacuum in a rotary evaporator and then freeze-dried for 3 days (84 g, yield 21.1%).

Cell
Culture. The Caco-2 cells were cultured at 37 ∘ C in humidified air containing 5% CO 2 . The cells were maintained in a 100 mm dish with DMEM containing 1000 mg/L of glucose and supplemented with 10% FBS, 1% NEAA, 100 U/mL of penicillin, and 100 g/mL of streptomycin. The cells were seeded at a density of 2 × 10 5 cells/mL on a 12-transwell plate (Costar, Corning, NY, USA) and allowed to grow for 3 weeks for the experiments.

Measurement of Transepithelial Electrical
Resistance. The integrity of the Caco-2 cell monolayers was checked by measuring the transepithelial electrical resistance (TEER) by using a Millicell-ERS device (Millipore, Bedford, MA, USA). The monolayers of Caco-2 cells (300-500 Ω cm −2 ) were preincubated with HBSS for 30 min at 37 ∘ C in a CO 2 incubator to stabilize the cell monolayers. The cell monolayers were treated with each sample and bile salts for 60 min, and OVA was added for 3 h at 37 ∘ C. The presence of bile salts weakens intestinal barrier function [12]. However, the bile salts actually exist in gastrointestinal tract in vivo, and the addition of bile salts in experimental in vitro system can mimic in vivo system. The TEER value was measured, and OVA influx was also detected by ELISA.

ELISA.
Coating anti-OVA primary antibodies (ab17290) and horseradish peroxidase-(HRP-) conjugated anti-OVA . Each solvent (chloroform, ethyl acetate, butanol, and water) was sequentially added in FAE solution, and then each fraction was sequentially partitioned with hexane, chloroform, ethyl acetate, butanol, and water ( Figure 1). All solvent fractions were evaporated until dry. The detailed method and yields of solvent fractions are shown in Table 1.

Determination of Total Phenolic Content.
Measurement of total phenolic content (TPC) was based on the method described by Shin et al. [13]. TPC was calculated using tannic acid (Sigma-Aldrich) as a standard.

HPLC Analysis.
High-performance liquid chromatography (HPLC) analysis was carried out with a Jasco PU-2080 plus liquid chromatography system (JASCO, Tokyo, Japan) equipped with multiwavelength detector Jasco UV-2075 plus (JASCO, Tokyo, Japan). Samples were injected to 1 g/mL by autoinjector AS-2057 plus (JASCO, Tokyo, Japan), and ODS C18 column (250 × 4.6 mm) was used to separate compounds at 40 ∘ C in column adapter CO-2060 plus (JASCO, Tokyo, Japan). Solvents were used mixture of acetonitrile (A) and water (B) in gradient mode (eluent A: 20 to 40% in 35 min), and the flow rate was 1 mL/min. UV wavelength for detection was 280 nm.  Cary, NC, USA). Differences between the experimental data were assessed by 1-way analysis of variance (ANOVA), followed by Duncan's multiple-range test; a value of < 0.05 was considered significant.  (Figure 2(a)). In contrast, the other eight natural material extracts had no effect on the TEER value. Furthermore, the FAE dramatically enhanced the TEER value in a dosedependent manner (Figure 2(b)). Next, we examined whether the FAE-induced enhancement of intestinal barrier function could suppress allergen permeation via paracellular diffusion pathway. The results showed that FAE treatment reduced OVA permeation across Caco-2 cell monolayers by enhancing the intestinal barrier function (Figures 2(c) and 2(d)).

Effects of Natural Material Extracts on Intestinal Barrier Function in Monolayers of
These results indicated that F. Arctii could enhance intestinal barrier function, preventing allergens and toxic and xenobiotic substrates from entering the intestine.

Effects of FAE Fractions on TEER and OVA Flux in Caco-2 Cell Monolayers.
To investigate the active components of FAE, FAE was fractionated using different polar solvents such as hexane, chloroform, ethyl acetate, butanol, and water. Among the five fractions extracted from FAE, chloroform and ethyl acetate fractions significantly increased TEER value in Caco-2 monolayers and also inhibited the OVA permeation across Caco-2 cell monolayers ( Figure 3). Although TEER values were significantly increased by hexane and butanol fractions, the increases were similar to the level of TEER value in initial control (normal condition). Therefore, we thought that the hexane and butanol fractions recovered TEER values reduced by bile salts in Caco-2 cell monolayers. These results demonstrated that the FAE-induced enhancement of intestinal barrier function was owing to compounds in chloroform and ethyl acetate fractions. Thus, we investigated TPC of the fractions to determine the properties of these components.  compounds. Therefore, we suggest that the active components of chloroform and ethyl acetate fractions might be nonpolar polyphenols.

Effects of Single Component (Arctiin and Arctigenin) on TEER and OVA Flux in Caco-2 Cell
Monolayers. Next, we analyzed the components in chloroform and ethyl acetate fractions using HPLC. The results showed four peaks (P1, P2, P3, and P4), which included chloroform and ethyl acetate fractions (Figures 4(a)-4(b)). The two main peaks, P2 and P4, were associated with arctiin and arctigenin, respectively. We then investigated the effects of arctiin and arctigenin on intestinal barrier function and OVA permeation across Caco-2 cell monolayers. Arctigenin, not arctiin, enhanced the TEER value and inhibited OVA permeation (Figures 4(c) and 4(d)). Moreover, the TEER increased and the OVA flux

Discussion
Arctigenin and arctiin are lignans found in many plants of the Asteraceae family, and their physiological functions have been identified by researchers. Typical functions of arctiin include protective [14], anti-inflammatory [15], and antidiabetic [16] effects, whereas those of arctigenin were anti-inflammatory [17], anti-cancer [18], and neuroprotective [19] effects. In the present study, we showed that arctigenin on TEER values were measured in the cell monolayers, and amount of permeated OVA was also detected in the basolateral side. Each value is presented as mean ± SD ( = 3). Bars are significantly different from the control at * * < 0.01. Data were analyzed using ANOVA followed by Duncan's multiple-range test. from F. Arctii enhanced the intestinal barrier function in human intestinal epithelial Caco-2 cells, but arctiin has no such effect. Both arctigenin and arctiin were well-known active components of F. Arctii. Furthermore, arctiin is the glycoside of arctigenin. Nevertheless, they have different physiological and pharmacological functions and mechanisms. For example, arctigenin effectively inhibited intestinal inflammation (body weight loss, proinflammatory cytokines, and crypt destruction) by suppressing the activation of mitogen-activated protein kinases and NF-B in dextran sulfate sodium-induced colitis model [20]. However, arctiin did not significantly ameliorate dextran sulfate sodiuminduced symptoms such as body weight loss, inflammatory index, colon length, and myeloperoxidase activity. Our results also showed different functions of arctigenin and arctiin on intestinal barrier function. Therefore, although arctigenin is an aglycon of arctiin, the physiological effects of arctigenin differ with arctiin.
Our result showed that arctigenin from F. Arctii enhanced intestinal barrier function. However, we suggest that other components also may have the potential on enhancement of intestinal barrier function. For example, chlorogenic acid decreased intestinal permeability through increasing intestinal expression of tight junction proteins such as occludin and zonula occludens-1 in weaned rats challenged with lipopolysaccharide [22]. Therefore, we will investigate the effects of other components from F. Arctii on intestinal barrier function in further study.

Conclusions
In summary, F. Arctii enhanced intestinal barrier function in human intestinal epithelial Caco-2 cells. Active component of the extract was arctigenin. We expect that the reinforcing effect of arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases. To identify active components in Fructus Arctii extract, HPLC analysis was carried out with a Jasco PU-2080 plus liquid chromatography system equipped with multiwavelength detector. Ethyl acetate and chloroform fractions were injected (1 g/mL each) by autoinjector, and an ODS C18 column (250 × 4.6 mm) was used to separate compounds at 40 ∘ C in column adapter. Solvents used were a mixture of acetonitrile (eluent A) and water (eluent B) in gradient mode (eluent A: 20 to 40% in 35 min), and the flow rate was 1 mL/min. UV wavelength for detection was 280 nm. (c-f) The effects of arctiin and arctigenin on TEER and OVA permeability were examined in Caco-2 cells. Each value is presented as mean ± SD ( = 3). Bars are significantly different from the control at * < 0.05 and * * < 0.01. Data were analyzed using ANOVA followed by Duncan's multiple-range test.