Note Effects of Soybean Isoflavones on the Release of Chemical

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Effects of Soybean Isoflavones on the Release of Chemical Mediators from Rat Peritoneal Exudate Cells by Allergic Reaction in Vitro

1 2 3 4* Mikako Takasugi , Kazuko Shimada , Koji Yamada and Hirofumi Arai

1Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kyushu Sangyo University, 2-3-1 Matsukadai, Higashi-ku, Fukuoka 813-8503, Japan 2Department of Nutrition, Faculty of Nursing and Nutrition, Yamaguchi Prefectural University, 3-2-1 Sakurabatake, Yamaguchi 753-8502, Japan 3Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki Higashi-ku, Fukuoka 812-8581, Japan 4Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, 165 Koencho, Kitami 090- 8507, Japan

Received December 9, 2013 ; Accepted February 18, 2014

Soybean isoflavones are expected to reduce the risks of various diseases. In this study, anti-allergic effects of soybean isoflavones such as genistein and daidzein, and equol, a metabolite of daidzein, were investigated by

measuring the chemical mediators, leukotriene B4 (LTB4) and histamine, released from rat peritoneal exudate cells

(PEC) in vitro. Genistein and equol significantly suppressed the release of LTB4 from PEC stimulated by calcium ionophore without cytotoxicity, whereas the inhibitory effect of daidzein was weak. In contrast, they had no effect on the release of histamine from calcium ionophore-stimulated PEC. These data suggest that the soybean isoflavones and metabolite may contribute to allergy symptom relief by inhibiting leukotriene production, but not histamine release.

Keywords: soybean, isoflavone, equol, leukotriene, allergy

Introduction promotes the hydrolysis of arachidonic acids from phospholipids

The number of patients with allergic diseases shows an of the cell membrane by phospholipase A2. Leukotrienes (LTs) are increasing trend in Japan. It is known that mast cells play a key produced by lipoxygenase (LOX) reactions with arachidonic acids role in immediate-type hypersensitivity such as food allergies and and are then released into the extracellular space. Meanwhile, the hay fever. The cell signaling in mast cells is triggered by the cross- cell signaling also leads to histamine release from mast cells by linking of high-affinity IgE receptors (FcεRI) via IgE–antigen degranulation. LT and histamine are the chemical mediators that complexes on the cell surface, which induces protein facilitate mucus secretion, smooth muscle contraction, and phosphorylation and Ca2+ influx (Siraganian, 2003). This in turn leukocyte chemotaxis, which cause immediate-type

Abbreviations BSA; bovine serum albumin, FcεRI; high-affinity IgE receptors, HPLC; high-performance liquid chromatography, LOX; lipoxygenases, LT; leukotriene, PEC; peritoneal exudate cells

*To whom correspondence should be addressed. E-mail: [email protected] 726 M. Takasugi et al.

hypersensitivity (Amin, 2012; Galli et al., 2008). To date, anti- LTB4 release assay The induction of chemical mediators allergy drugs such as LT receptor antagonists, 5-LOX inhibitors release from PEC and their determination were performed by (Scow et al., 2007), and histamine H1 receptor antagonists have Matsuo’s method with some modifications (Matsuo et al., 1996). been generally used for symptomatic treatment. In recent years, it PEC were re-suspended in 50 µL of Tyrode buffer (2.0 × 106 cells/ has been expected that food components could improve allergy mL) containing 0.1% BSA, 0.9 mmol/L CaCl2, and various symptoms without side effects. concentrations of isoflavones dissolved in ethanol, and stimulated Isoflavones are a class of flavonoids that are abundant in with 5 µmol/L calcium ionophore (A23187) at 37℃ for 20 min. legumes, generally as glycosides. Genistein and daidzein (Fig. 1) The stimulation was terminated by the addition of 50 µL of are the major soybean isoflavone aglycones generated because of acetonitrile:methanol (30:25, v/v) containing 250 ng of intestinal digestion of their glycosides, genistin and daidzin, prostaglandin B2 as an internal standard. The cell lysate was respectively. Furthermore, a part of daidzein is enzymatically centrifuged at 10,000 × g for 10 min, and the supernatant was metabolized to equol (Fig. 1) by intestinal bacteria. It has been subjected to high-performance liquid chromatography (HPLC) on suggested that genistein, daidzein, and equol are remarkable an ODS-A column (150 × 6.0 mm I.D.; YMC, Kyoto, Japan) at functional food components that may reduce the risks of diseases room temperature. The samples were then eluted with 5 mmol/L such as osteoporosis, cancer, and cardiovascular diseases (Ishimi et ammonium acetate aqueous solution:acetonitrile:methanol: (9:6:5, al., 1999; Mathey et al., 2007; Ren et al., 2001; Yamakoshi et al., v/v/v) at a flow rate of 1.0 mL/min and LTB4 was monitored at 2000). Genistein, daidzein, and equol are known to behave as 280 nm. phytoestrogens and antagonists by binding to estrogen receptors Histamine release assay PEC were re-suspended in 2 mL of and G protein-coupled receptor 30 (Setchell et al., 2002; Kuiper et Tyrode buffer (1.0 × 106 cells/mL) containing 0.1% BSA, al., 1998; Thomas and Dong, 2006). Anti-inflammatory or anti- 0.9 mmol/L CaCl2, and 100 µmol/L isoflavones, and stimulated allergy effects of soybean isoflavones were reported by some with 5 µmol/L A23187 at 37℃ for 20 min. The stimulation was research groups using allergic murine models (Bao et al., 2011; terminated by cooling at 4℃ for 15 min. The cell suspension was Masilamani et al., 2011). Genistein and daidzein also regulated centrifuged at 300 × g for 10 min, and the histamine content of the mucosal immune responses; and the effect of genistein was supernatant was determined by fluorescence photometry as follows stronger than that of daidzein (Wei et al., 2012). Our research (Shore et al., 1959). One milliliter of the supernatant was mixed group has reported that various polyphenols in foods can suppress with 1 mL of Tyrode buffer, 0.75 g of NaCl, 0.5 mL of 5 mol/L the release of chemical mediators from rat peritoneal exudate cells NaOH, and 5 mL of n-butanol:chloroform (3:2, v/v) for 2 min. (PEC) containing mast cells, rat basophilic leukemia cells After centrifugation at 270 × g for 5 min, 4 mL of the organic (RBL−2H3), and human basophilic cells (KU812) in vitro (Matsuo solvent layer was recovered and mixed with 1.5 mL of n-heptane et al., 1996; 1997; Tachibana et al., 2000). These papers imply that and 1.5 mL of 0.1 mol/L HCl for 2 min. After centrifugation at soybean isoflavones have the potential to modulate inflammation 270 × g for 5 min, 1 mL of the HCl layer was recovered and mixed or the allergic system. However, the anti-allergic effects of soybean with 0.15 mL of 1 mol/L NaOH and 0.1 mL of 0.2% isoflavones have not been sufficiently addressed. In the present o-phthalaldehyde. After incubation at room temperature for 5 min, study, we examined the effects of genistein, daidzein, and equol on the reaction was terminated by the addition of 0.14 mL of 0.25 mol/ the release of LTB4 and histamine from rat PEC in vitro. L H2SO4, and fluorescence intensity was then measured with excitation at 360 nm and emission at 450 nm. The percentage of Materials and Methods histamine release was calculated as follows: histamine release (%) Materials Genistein and daidzein were purchased from Funakoshi (Tokyo, Japan). (±) Equol was obtained from Extrasynthese (Genay, France). Preparation of PEC Rat PEC were prepared according to the method provided by Matsuo et al. (1996). Tyrode buffer (20 mL/ rat) containing 0.1% bovine serum albumin (BSA) was injected into the peritoneal cavity of 8-week-old male Sprague−Dawley rats (Kyudo, Saga, Japan), followed by gentle massaging of the abdomen for 2 min. After abdominal incision, Tyrode buffer containing PEC was collected and centrifuged at 200 × g for 10 min at 4℃. The precipitated cells were suspended in a modified ammonium chloride buffer (150 mmol/L NH4Cl, 10 mmol/L

KHCO3, 10 mmol/L EDTA−2Na, pH 7.4) and incubated for 5 min at 4℃ to remove contaminating erythrocytes. Fig. 1. Structures of genistein, daidzein, and equol. Soybean Isoflavones Suppress Leukotriene B4 Release from Immune Cells 727

Fig. 2. Effect of genistein, daidzein, and equol on LTB release from rat peritoneal exudate cells (PEC). 4 _ Genistein (A), daidzein (B), or equol (C) were added to PEC at 1 100 μmol/L and then LTB4 release was induced by calcium a-d ionophore (A23187). LTB4 was determined by HPLC with UV detection. Each value represents mean ± SE (n = 3). Values not sharing a common letter are significantly different atp < 0.01. N.D.: not detected.

= (test − negative control)/(total − negative control) × 100. The and daidzein exist as aglycones of genistin and daidzin at 0 _ 15 mg supernatant from the unstimulated cells was used as the negative and 3 _ 23 mg in 100 g of dry soybean, respectively (Tsukamoto et control, while the supernatant from the treatment with 5% Triton-X al., 1995). When soybean isoflavone glycosides are ingested, they 100 was used as total. are hydrolyzed to aglycones and glucose by b−glucosidase in the Statistical analysis Data are expressed as mean ± standard intestine. It has also been reported that the ingestion of 60 g of error. The statistical significance of differences was analyzed by baked soybean powder increased the blood plasma level of the Tukey-Kramer test. Differences with p values of less than 0.01 daidzein and genistein to about 1.6 µmol/L and 2.4 µmol/L in 2 h, were considered statistically significant. respectively (Watanabe et al., 1998). Moreover, a fraction of daidzein can be metabolized to equol by enterobacterial enzymes Results and Discussion in the intestine (Setchell et al., 1984 and 2002; Karr et al., 1997). Figure 2 shows the effect of genistein (A), daidzein (B), and Setchell et al. (2002) reported that an oral dose of 25 mg of equol equol (C) on the release of LTB4 from calcium ionophore- resulted in approximately 1.3 µmol/L of equal in the plasma in 6 h. stimulated rat PEC. LTB4 is an appropriate index of LTs released Our results suggest that equol, rather than daidzein and genistein, 6 from PEC (Matsuo et al., 1996). PEC (1.0 × 10 cells) released may act as a LTB4 production inhibitor at the physiological about 7 ng of LTB4 in the absence of isoflavones. It has been concentration, although animal experiments need to be performed reported that genistein inhibited LTB4 and LTC4 production from to confirm the effective concentrations in vivo. Further studies on calcium ionophore (A23187)-stimulated bovine peripheral blood the influences of their metabolite, especially conjugates (Thomas et mononuclear cells and macrophage-like cells, respectively (Atluru al., 2001; Qiu et al., 2005), after absorption on the inhibitory and Gudapaty, 1993; Glaser et al., 1993). In our experimental activities against LTB4 production are also required. condition, the LTB4 release was significantly suppressed to less We have shown that flavonoids such as epigallocatechin gallate than 50% by 100 µmol/L genistein, although the effect was not and myricetin can inhibit histamine release from rat PEC (Matsuo dose-dependent at the concentration of 1 _ 100 µmol/L. Daidzein et al., 1996; Yamada et al., 1999). Several research groups have slightly decreased LTB4 release at 100 µmol/L. Equol significantly reported that eriodictyol, luteolin, and quercetin inhibited mast cell inhibited LTB4 release in a dose-dependent manner at the degranulation (Yoo et al., 2012; Kimata et al., 2000; Matsuda et concentration of 1 _ 100 µmol/L. These isoflavones had no al., 2002). Herein, we investigated the effect of genistein, daidzein, cytotoxicity at 100 µmol/L for 20 min (data not shown). We had and equol on histamine release, another chemical mediator, from previously shown a structure−activity relationship of flavonoids calcium ionophore-stimulated rat PEC. As shown in Fig. 3, such as quercetin and luteolin for the inhibition of LTB4 production 100 µmol/L of genistein, daidzein, and equol did not suppress in PEC, which suggested that the C4-carbonyl group has an histamine release from PEC by calcium ionophore stimulation. For important role in the activities (Yamada et al., 1999). The present this reason, genistein and equol may inhibit only LOX reaction data are not sufficient to reveal the structure−activity relationships generating LTs, while they may not suppress the degranulation of isoflavones as a LTB4 production inhibitor. Most of genistein resulting histamine release. It is known that PEC includes not only 728 M. Takasugi et al.

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