Inhibitory effects of adlay bran (Coix lachryma-jobi L. var. ma-yuen Stapf) on chemical mediator release and cytokine production in rat basophilic leukemia cells

Journal of Ethnopharmacology 141 (2012) 119–127

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Inhibitory effects of adlay bran (Coix lachryma-jobi L. var. ma-yuen Stapf) on chemical mediator release and cytokine production in rat basophilic leukemia cells Hong-Jhang Chen, Yi-Chen Lo, Wenchang Chiang ∗ Graduate Institute of Food Science and Technology, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan

a r t i c l e

i n f o

Article history: Received 12 October 2011 Received in revised form 3 February 2012 Accepted 6 February 2012 Available online 14 February 2012 Keywords: Allergic response Adlay bran Degranulation Histamine Cytokines

a b s t r a c t Ethnopharmacological relevance: Adlay (Job’s tears, Coix lachryma-jobi L. var. ma-yuen Stapf) has long been used in China to treat rheumatism. Aim of the study: We investigated the anti-allergic effects of adlay bran on rat basophilic leukemia (RBL)2H3 cells. Materials and methods: To evaluate the anti-allergic effects of adlay bran, the release of histamines and cytokines were measured using ELISA. To explore the mechanism of these effects, the protein expression levels were determined using western blotting. Results: A 40.8 ␮g/mL concentration of the ethyl acetate fraction of the ethanolic extracts of adlay bran (ABE-EtOAc) effectively inhibited mast cell degranulation. The 40–100% EtOAc/Hex subfractions of ABEEtOAc inhibited histamine release with an IC50 of 71–87 ␮g/mL. Moreover, the ABE-EtOAc subfractions suppressed the secretion of interleukin (IL)-4, IL-6 and tumor necrosis factor-␣ in the RBL-2H3 cells, indicating that adlay bran can inhibit cytokine secretion in the late phase of the allergic reaction. In addition, adlay bran reduced the intracellular production of reactive oxygen species, inhibited the phosphorylation of Akt and decreased the expression of protein kinase C. Furthermore, six phenolic acids and one flavone were isolated. Of these compounds, luteolin showed the most potent inhibitory activity (IC50 = 1.5 ␮g/mL). Conclusion: Adlay bran extract reduced the release of histamines and cytokines and suppressed the production of Akt. These combined effects influenced the signal transduction in RBL-2H3 cells, thereby revealing the mechanisms of the anti-allergic effects of adlay. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Allergy is a type of immune dysfunction and is a serious health problem worldwide. Along with new industrial developments and changes in lifestyles, the morbidity of allergic diseases is

Abbreviations: ABE, the ethanolic extracts of adlay bran; ABE-BuOH, the 1butanol soluble fraction from the ethanolic extracts of adlay bran; ABE-EtOAc, the ethyl acetate fraction from the ethanolic extracts of adlay bran; ABE-Hex, the nhexane-soluble fraction from the ethanolic extracts of adlay bran; ABE-H2 O, the water-soluble fraction from the ethanolic extracts of adlay bran; DCFH-DA, 2 ,7 dichlorodihydrofluorescin diacetate; DMSO, dimethyl sulfoxide; Fc␧RI, Fc receptor for IgE; IL, interleukin; MAPK, mitogen-activated protein kinase; OVA, ovalbumin; RBL, rat basophilic leukemia; ROS, reactive oxygen speciesl TNF-␣tumor necrosis factor-alpha. ∗ Corresponding author at: 1, Roosevelt Road, Sec. 4, Taipei 10617, Taiwan. Tel.: +886 2 3366 4115; fax: +886 2 2363 8673. E-mail address: [email protected] (W. Chiang). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.02.009

increasing, and the average age of allergy sufferers is trending downward (Abdel-Raheem et al., 2005). Adopting dietary changes to regulate the immune system has been a popular means of reducing allergic symptoms and avoiding the unnecessary use of drugs (Paul et al., 2006). Adlay (Job’s tears, Coix lachryma-jobi L. var. ma-yuen Stapf) is an annual plant that has long been used in China to treat warts, chapped skin, rheumatism, and neuralgia. Adlay has been reported to have various immunomodulatory activities, such as anti-complementary, anti-inflammatory and anti-allergic effects (Takahashi et al., 1986; Otsuka et al., 1988; Seo et al., 2000; Hsu et al., 2003; Chen et al., 2010). Moreover, the consumption of adlay extracts can increase the activities of cytotoxic T cells and natural killer (NK) cells (Hidaka et al., 1992). The methanolic extract of adlay seeds inhibits NO and O2 −• production by activated macrophages (Seo et al., 2000). Benzoxazinoid compounds isolated from adlay roots show anti-histamine release activity in rat peritoneal mast cells (Otsuka et al., 1988). We have shown

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that a diet of 20% dehulled adlay can suppress the production of IgE against an ovalbumin (OVA) antigen, modulate the Th1/Th2 immune responses via reduced IL-5 secretion, and increase IL-2 production (Hsu et al., 2003). Accordingly, adlay is an effective treatment for allergic symptoms. Allergic symptoms can be caused by both unbalanced cytokines and the abnormal degranulation of mast cells. The effects of adlay on the cytokine secretion and signal transduction in mast cells remain unclear. Murine peritoneal mast cells have been widely used as a single peritoneal lavage. Nevertheless, it is difficult to establish primary cultures of mast cells for routine work, due to the limited amounts of mast cells obtained from murine tissues. Mast cells are present in all normal tissues and are believed to play crucial roles in tissue homeostasis, wound healing, and host defense, particularly against bacterial infection. The chronic activation of mast cells contributes to the pathophysiology of many allergic diseases through the synthesis and release of numerous proinflammatory mediators and cytokines (Bradding et al., 2006). In the present work, RBL-2H3 (rat basophilic leukemia) cells were used because of the high affinity of their Fc receptor for IgE (Fc␧RI) and their release of proinflammatory mediators, such as histamine, interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-␣) (Bischoff, 2007). Upon degranulation in mast cells, ␤-hexosaminidase is released along with histamine. Therefore, this enzyme is used as a marker for mast cell degranulation (Mastuda et al., 2002; Matsuda et al., 2004; Tewtrakul and Itharat, 2006; Choi et al., 2007). Furthermore, it has been suggested that intracellular reactive oxygen species (ROS) are generated by mast cells following incubation with calcium ionophore A23187 (Wolfreys and Oliveira, 1997). Interestingly, Gushchin et al. (1990) demonstrated that intracellular ROS stimulated histamine release and triggered cytokine production. Therefore, the production of ROS was evaluated (Nakano et al., 2005). In this study, we demonstrated that several subfractions of ethanolic extract adlay can modulate the immune response through the inhibition of cytokine secretion and mast cell degranulation in RBL-2H3 cells. 2. Materials and methods 2.1. Materials Bovine serum albumin (BSA), calcium ionophore (A23187), 2 ,7 -dichlorodihydrofluorescin diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT), and other chemicals were obtained from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) and a trypsin–EDTA solution were obtained from Biological Industries (Haemek, Israel). Antibodies for immunoblotting, such as anti-Akt1 and -PKC, were purchased from Santa Cruz Biotechnology (San Diego, CA). Other antibodies, such as anti-phospho-ERK (1/2), -phospho-Akt (Ser473), -phospho-p38, phospho-JNK, and -␤-actin, were from Cell Signaling (Danvers, MA). 2.2. Preparation of ethanolic extracts and various fractions from the bran of adlay seeds The ethanolic extracts of adlay bran were derived according to the procedure described by Lee et al. (2008) with several modifications. Adlay bran powder (20 kg) was extracted with 200 L of ethanol three times (4–5 days each time) for two weeks at room temperature. To minimize the ethanol consumption during the adlay bran ethanol extraction, we prolonged the extraction time. The plant residue was removed by filtration, and the

ethanolic extracts were combined and concentrated under reduced pressure using a rotary vacuum evaporator. The ethanolic extracts of adlay bran were termed “ABE.” The dry ABE extract (1000 g) was suspended in 10 L of H2 O followed by several partitioning processes with various extraction solvents, including n-hexane, ethyl acetate, 1-butanol, and water. The resulting fractions were ABE-Hex (the n-hexane-soluble fraction), ABE-EtOAc (the ethyl acetatesoluble fraction), ABE-BuOH (the 1-butanol-soluble fraction), and ABE-H2 O (the water-soluble fraction), respectively. The ABE-EtOAc (395 g) was coated with 395 g of silica gel (230–400 mesh) and subjected to column chromatography on silica gel (230–400 mesh) with successive elutions using a Hex/EtOAc and EtOAc/MeOH gradient solvent system. The subfractions with the same TLC patterns were combined into one fraction; therefore, 9 fractions were obtained: A (0–20% EtOAc/Hex), B (20–25% EtOAc/Hex), C (25–30% EtOAc/Hex), D (30–40% EtOAc/Hex), E (40–60% EtOAc/Hex), F (60–80% EtOAc/Hex), G (80–90% EtOAc/Hex), H (90–100% EtOAc/Hex), and I (100% MeOH). A simplified version of the process used is shown in Fig. 1. The activities of the various subfractions were tested using the methods described above. The activities of subfractions ABE-EtOAc-E and F were combined and further purified using column chromatography (silica gel eluted with 60–80% EtOAc/Hex) and semi-preparative HPLC on a LiChrosorb Si-60 column at 2 mL/min using 20–60% CH2 Cl2 /EtOAc, yielding syringaldehyde, vanillic acid, ferulic acid, protocatechuic acid, caffeic acid, p-coumaric acid and luteolin. Each compound was identified using NMR and MS, and TLC and HPLC were employed to analyze the purity of each compound by comparing it with samples of known purities of 90–95%.

2.3. Spectral identification of purified compounds The identities of all of the compounds were determined with the use of spectroscopic methods, including 1-D NMR and MS spectral analysis. Syringaldehyde: EIMS, m/z 182 [M]+ ; 1 H NMR (400 MHz, acetone-d6 ) ı 3.92 (s, 6H, OCH3 ), 7.24 (s, 2H, H-2, H-6), 9.80 (s, 1H, CHO). The data were in agreement with the reported literature (Kim et al., 2003). Vanillic acid: EIMS, m/z 152 [M]+ ; 1 H NMR (400 MHz, acetone-d6 ) ı 3.91 (s, 3H, OCH3 ), 6.91 (d, J = 8.2 Hz, 1H, H-5), 7.56 (d, J = 1.5 Hz, 1H, H-2), 7.60 (dd, J = 8.2, 1.5 Hz, 1H, H-6). The data were in agreement with the reported literature (Harrison et al., 1995). Ferulic acid: EIMS, m/z 194 [M]+ ; 1 H NMR (400 MHz, acetone-d6 ) ı 3.93 (s, 3H, OCH3 ), 6.28 (d, J = 16.4 Hz, 1H, H-8), 6.91 (d, J = 8.0 Hz, 1H, H-5), 7.03 (dd, J = 8.0, 2.0 Hz, 1H, H-6), 7.09 (d, J = 2.0 Hz, 1H, H2), 7.69 (d, J = 16.4 Hz, 1H, H-7). The data were in agreement with the reported literature (Kim and Kim, 2000). Protocatechuic acid: EIMS, m/z 154 [M]+ ; 1 H NMR (400 MHz, acetone-d6 ) ı 6.89 (d, J = 8.2 Hz, 1H, H-5), 7.47 (dd, J = 8.2, 1.0 Hz, 1H, H-6), 7.50 (d, J = 1.0 Hz, 1H, H-2). The data were in agreement with the reported literature (Hopper and Mahadevan, 1997). Caffeic acid: EIMS, m/z 180 [M]+ ; 1 H NMR (400 MHz, acetoned6 ) ı 6.26 (d, J = 16.0 Hz, 1H, H-8), 6.86 (d, J = 8.4 Hz, 1H, H-5), 7.05 (dd, J = 8.4, 2.0 Hz, 1H, H-6), 7.16 (d, J = 2.0 Hz, 1H, H-2), 7.53 (d, J = 16.0 Hz, 1H, H-7). The data were in agreement with the reported literature (Bolzani et al., 1991). p-Coumaric acid: EIMS, m/z 164 [M]+ ; 1 H NMR (400 MHz, acetone-d6 ) ı 6.33 (d, J = 15.9 Hz, 1H, H-8), 6.88 (d, J = 8.0 Hz, 2H, H-3, H-5), 7.54 (d, J = 8.0 Hz, 2H, H-2, H-6), 7.60 (d, J = 15.9 Hz, 1H, H-7), 8.90 (brs, 1H, OH). The data were in agreement with the reported literature (Iwagawa et al., 1984). Luteolin: EIMS, m/z 286 [M]+ ; 1 H NMR (400 MHz, CDCl3 ) ı 6.24 (d, J = 2.0 Hz, 1H, H-6), 6.52 (d, J = 2.0 Hz, 1H, H-8), 6.57 (s, 1H, H3), 6.90 (d, J = 8.4 Hz, 1H, H-5 ), 7.45 (d, J = 2.4 Hz, 1H, H-2 ), 7.48

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Fig. 1. Procedure for extracting, partitioning, and fractionating the anti-allergic fractions from adlay bran.

(dd, J = 8.4, 2.4 Hz, 1H, H-6 ), The data were in agreement with the reported literature (Peters et al., 1986). 2.4. Cell culture The RBL-2H3 cells were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan) and were cultured with DMEM containing 15% heat-inactivated FBS, 100 U/mL penicillin–streptomycin, and 2% glutamine in a 5% CO2 incubator at 37 ◦ C. The cells were detached with a trypsin–EDTA solution. After washing, the cells were resuspended in fresh medium and used for subsequent experiments. 2.5. Assay of cell viability Cell viability was analyzed with the MTT assay, as previously described (Mosmann, 1983). Briefly, the RBL-2H3 cells were seeded at 105 cells/well in 96-well plates. After being washed with Siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2 , 25 mM PIPES, and 40 mM NaOH, pH 7.4), the cells were pretreated with various test samples and 1 ␮M A23187 for 12 h. The medium was removed, the filtered MTT solution was added to each well (2.5 mg MTT/well), and the plates were incubated at 37 ◦ C for 2 h. Any unreacted dye was removed after 2 h. The insoluble MTT formazan crystals were dissolved in DMSO at room temperature for 15 min. The cell viability was determined using a microplate reader at 570 nm (Molecular Devices, CA). The cell viability of the various test groups was calculated using the following equation: (absorbance of the test group/absorbance of the control) × 100. All of the test samples discussed above were dissolved in DMSO. The final DMSO concentration was <0.1%, and 0.1% DMSO was used as a control.

2.6. Assay of ˇ-hexosaminidase release The ␤-hexosaminidase release assay was performed according to the method described by Matsuda et al. (2004) with several modifications. Briefly, the RBL-2H3 cells were seeded into 24-well plates at 2 × 105 cells/well and incubated at 37 ◦ C overnight. The cells were washed with an incubation buffer consisting of Siraganian buffer supplemented with 5.6 mM glucose, 1 mM CaCl2 , and 0.1% BSA and were incubated in 160 ␮L of the incubation buffer for 10 min at 37 ◦ C. Subsequently, 20 ␮L of each test sample solution was added to each well and incubated for 10 min followed by the addition of 20 ␮L of calcium ionophore (A23187, final concentration of 1 ␮M) and a 10-min incubation at 37 ◦ C to stimulate degranulation in the cells. Quercetin served as a positive control (Kawai et al., 2007). The reaction was stopped by cooling on ice for 10 min. The cellular supernatant (50 ␮L) was transferred into 96-well plates and incubated with 50 ␮L of the substrate (1 mM p-nitrophenyl-Nacetyl-␤-d-glucosaminide) in 0.1 M citrate buffer (pH 4.5) at 37 ◦ C for 1 h. The reaction was stopped by adding 200 ␮L of a stop solution (0.1 N Na2 CO3 /NaHCO3 , pH 10.0). The absorbance at 405 nm was measured with a microplate reader (Molecular Devices, CA). The percentage of ␤-hexosaminidase release was calculated as follows: (the supernatant OD value of the stimulated cells – the supernatant OD value of the unstimulated cells) × 100/(the OD value of the total cell lysate – the supernatant OD value of the unstimulated cells). The total cell lysates were obtained using 1% Triton X-100. The IC50 values were determined graphically.

2.7. Assay of histamine release The histamine release assay protocol was similar to that described above for A23187-stimulated degranulation. Briefly,

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Table 1 Anti-allergic effects of various fractions and subfractions of adlay bran on cell viability and ␤-hexosaminidase release in A23187-stimulated RBL-2H3 cells. Inhibition of ␤-hexosaminidase releasea (%) 25 ␮g/mL

50 ␮g/mL

IC50 (␮g/mL)

Cell viabilityb (%)

100 ␮g/mL

ABE-Hex ABE-EtOAc ABE-BuOH ABE-H2 O

11.2 17.8 3.7 −4.9

± ± ± ±

4.2* 3.8* 2.3* 6.1*

25.9 74.8 5.1 1.9

± ± ± ±

1.5* 8.3* 4.8* 1.0*

55.3 92.6 7.8 5.2

± ± ± ±

04.8* 04.7* 02.3* 04.3*

91.1 40.8 >100 >100

96.3 ± 0.9 100.4 ± 3.3 93.7 ± 0.7 98.7 ± 7.4

ABE-EtOAc subfractions A B C D E F G H I

10.0 2.1 2.5 5.0 −1.1 −8.9 −1.5 −2.1 −9.2

± ± ± ± ± ± ± ± ±

5.6* 3.6* 1.4* 7.8* 3.7* 7.0* 12.8* 10.6* 8.0*

7.3 7.7 2.8 −0.5 9.9 35.7 45.0 64.8 −9.7

± ± ± ± ± ± ± ± ±

12.2* 10.1* 3.8* 7.1* 8.8* 8.1* 13.5* 13.7* 12.4*

9.1 2.8 2.3 0.4 76.5 85.9 88.9 93.0 8.1

± ± ± ± ± ± ± ± ±

05.0* 01.9* 09.5* 03.4* 06.7* 04.0* 03.3* 06.3* 12.8*

>100 >100 >100 >100 68.8 56.5 63.2 70.7 >100

117.5 ± 6.3 114.4 ± 3.3 119.8 ± 4.7 106.1 ± 7.1 97.4 ± 1.4 100.9 ± 1.3 91.5 ± 5.3 91.8 ± 2.0 98.3 ± 2.6

Quercetin

5 ␮M 34.9 ± 0.2*

9.0

103.7 ± 1.1

a b *

10 ␮M 58.2 ± 3.5*

20 ␮M 74.9 ± 0.8*

Each value represents the mean ± SD of three experiments. The concentrations of the adlay bran extracts and the quercetin used in the cell viability assay were 100 ␮g/mL and 20 ␮M, respectively. Denote significant differences from the control, p < 0.05.

RBL-2H3 cells were treated with various test samples and stimulated with 1 ␮M A23187. The supernatants (100 ␮L) were collected 20 min after the stimulation and were assayed for histamine release using an ELISA kit (IBL, Hamburg). 2.8. Measurement of IL-4, IL-6 and TNF-˛ The inhibitory effects on the A23187/PMA-stimulated release of cytokines in RBL-2H3 cells were evaluated similarly to the A23187stimulated degranulation described above. Briefly, RBL-2H3 cells were treated with various test samples and stimulated with 1 ␮M A23187 and 50 nM PMA for 5 h. The concentrations of IL-4, IL-6 and TNF-␣ in the cellular supernatants were determined using a commercial ELISA kit (eBioscience, San Diego, CA). 2.9. Western blotting The cells were washed twice with ice-cold PBS and then lysed in RIPA lysis buffer (20 mM Tris–HCl, 2 mM EDTA, 500 ␮M sodium orthovanadate, 10 ␮g/mL aprotinin, 10 mM NaF, 1% Triton X-100, and 0.1% SDS, pH 7.4) on ice for 20 min. The lysates were clarified by centrifugation at 10,000 × g for 30 min at 4 ◦ C. The protein contents of the supernatants were measured with the Bio-Rad Protein Assay Kit. Equal amounts of the samples were subjected to SDSPAGE using 8% running gels. The proteins were transferred onto a PVDF membrane, which was blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight at 4 ◦ C with anti-p38, -phospho-ERK, -JNK, -PKC, -Akt or -␤-actin antibodies. After hybridization with the primary antibodies, the membrane was incubated with an HRP-labeled secondary antibody for 2 h. The final detection was performed with enhanced chemiluminescence (ECL) western blotting reagents (Amersham Pharmacia Biotech). 2.10. Measurement of intracellular ROS production The intracellular production of ROS was monitored as described by Nakano et al. (2005). DCFH-DA is a nonpolar compound that is converted into a membrane-impermeable nonfluorescent polar derivative (DCFH) by cellular esterase following its incorporation into cells. The trapped DCFH is rapidly oxidized to fluorescent 2 ,7 -dichlorofluorescein (DCF) by intracellular peroxides, such as hydrogen peroxide. In this study, RBL-2H3 cells were seeded into 6-well plates at 106 cells/well and incubated with the test samples

for 20 min at 37 ◦ C. Subsequently, the cells were washed with the incubation buffer and incubated with 5 ␮M DCFH-DA for 30 min at 37 ◦ C. Next, 1 ␮M A23187 was added, and the cells were incubated at 37 ◦ C for 10 min before being harvested. The cells were washed, resuspended in ice-cold PBS, and analyzed using a FACSCalibur (Becton-Dickinson; excitation and emission at 488 and 530 nm, respectively) equipped with CellQuest for 10,000 cells per test.

2.11. Statistical analysis The results are expressed as the mean ± standard deviation (SD) of three experiments. The differences between specific means were analyzed with a one-way analysis of variance (ANOVA) using SPSS software, ver. 11.0 (SPSS, Chicago, IL). The group means were compared using a one-way ANOVA followed by Dunnett’s test. Probability (p) values less than 0.05 were considered statistically significant.

3. Results 3.1. Effects of ABE fractions on ˇ-hexosaminidase release and cell viability Using release of ␤-hexosaminidase by cell degranulation as an indicator of anti-allergic activity in cells, we studied the effects of various fractions of ABE on degranulation in A23187-stimulated RBL-2H3 cells. First, we confirmed there were no cytotoxicities in any of the test samples (Table 1). The ABE-EtOAc fractions exhibited better inhibit degranulation than the lower-polarity fraction (ABE-Hex) or the higher-polarity fractions (ABE-BuOH and ABEH2 O). In addition, the ␤-hexosaminidase release assay revealed that the ABE-EtOAc fraction effectively suppressed the degranulation in a concentration-dependent manner (IC50 = 40.8 ␮g/mL). Thus, the ABE-EtOAc fraction was separated into nine subfractions (A–I) according to the chromatographic results. The higher-polarity subfractions (40–100% EtOAc/Hex) of ABE-EtOAc-E, -F, -G, and -H produced a significant inhibition of cell degranulation at a concentration of 100 ␮g/mL, but the other subfractions did not. These data indicate that major anti-allergic compounds may exist in the 40–100% EtOAc/Hex subfractions of the ABE-EtOAc fraction.

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Therefore, these four subfractions (ABE-EtOAc-E, -F, -G, and -H) were subjected to further investigation.

3.2. Effects of ABE-EtOAc subfractions on histamine release We identified four effective subfractions of ABE-EtOAc that inhibited cell degranulation. Next, we examined the effects of these subfractions on the secretion of histamine in mast cells because histamine is the primary mediator of the allergic response. The addition of these subfractions at 50 ␮g/mL significantly reduced the level of histamine release in A23187-stimulated RBL-2H3 cells (Table 2). The ABE-EtOAc-F subfraction (100 ␮g/mL) displayed the strongest inhibition of histamine release (∼70%) compared with the other subfractions. These results imply that these subfractions have the potential to inhibit allergic responses in cells.

3.3. Effects of the ABE-EtOAc subfractions on cytokine secretion IL-4, IL-6 and TNF-␣ are inflammatory mediators that are synthesized from cell membrane phospholipids in activated cells and are involved in the allergic reaction. Mast cells secrete IL-4, the primary allergic cytokine. In our results, the basal levels of IL4 secreted by unstimulated RBL-2H3 cells were below 2 pg/mL. In contrast, higher levels of IL-4 (221 pg/mL) were observed in activated cells stimulated with A23187 and PMA for 5 h. Fig. 2A shows the inhibitory effects of the ABE-EtOAc subfractions on IL-4 secretion. When the cells were cultured with 25 or 50 ␮g/mL of the subfractions, only modest inhibitions of IL-4 secretion were observed. However, higher concentrations (100 ␮g/mL) of certain subfractions produced significant decreases in IL-4 secretion: reductions of ∼97% and ∼93% occurred in the G and H subfractions, respectively. These profound effects of the ABE-EtOAc subfractions on IL-4 secretion were comparable to those observed in the quercetin-treated cells. IL-6 is the major inflammatory cytokine released from mast cells in the late phase of the allergic response. As shown in Fig. 2B, the effective ABE-EtOAc subfractions significantly suppressed the secretion of IL-6. At a concentration of 100 ␮g/mL, the subfractions inhibited IL-6 secretion to the following degrees: ABE-EtOAcF (49%) > ABE-EtOAc-E (48%) > ABE-EtOAc-H (30%) > ABE-EtOAc-G (24%). Interestingly, ABE-EtOAc-E and -F produced greater inhibitions of IL-6 secretion than did the other subfractions. The results indicate that the mechanisms of regulating IL-4 and IL-6 secretion may differ between subfractions E and F and the other subfractions. TNF-␣ is a potent inflammatory cytokine involved in many pathophysiological conditions, including allergy. The ABE-EtOAc subfractions significantly suppressed the secretion of TNF-␣ in the A23187/PMA-stimulated RBL-2H3 cells (Fig. 2C). The ABE-EtOAcE subfraction caused the weakest inhibition of TNF-␣ secretion. Unlike the effects of the other subfractions, the inhibition by the ABE-EtOAc-E subfraction was not concentration-dependent

3.4. Effects of the ABE-EtOAc subfractions on intracellular ROS generation Intracellular ROS have been implicated in mast cell degranulation. An ROS-dependent pathway has been shown to mediate the activation of PKC in mast cells (Matsui et al., 2000). Accordingly, we observed that the expression of PKC was markedly reduced compared with the basal levels in cells pretreated with 70 ␮g/mL of the ABE-EtOAc subfractions (Fig. 3A). To measure the generation of intracellular ROS in the A23187-stimulated RBL-2H3 cells, we assayed the production of H2 O2 using flow cytometry. Fig. 3B shows that A23187 stimulated intracellular ROS generation in the

Fig. 2. Inhibitory effects of the various subfractions of ABE-EtOAc on the secretion of (A) IL-4, (B) IL-6, and (C) TNF-␣ in A23187-stimulated RBL-2H3 cells. Each column represents the mean ± SD of three experiments. *p < 0.05 was considered to represent a statistically significant difference from the control.

RBL-2H3 cells (solid line). All of the ABE-EtOAc subfractions effectively decreased the generation of ROS (bold line). 3.5. Effects of the ABE-EtOAc subfractions on the MAP kinase cascades Three major mitogen-activated protein kinases (MAPKs), including ERKs, JNKs, and p38, are activated in A23187-stimulated RBL-2H3 cells. As shown in Fig. 4, the RBL-2H3 cells treated with 1 ␮M A23187 alone showed dramatic inductions of the phosphorylation of Akt and ERK1/2 but not JNK or p38 (lane 2). The phosphorylation of ERK1/2 was suppressed by the ABE-EtOAc subfractions at a concentration of 70 ␮g/mL (lanes 3–6). There were no obvious effects on the phosphorylation levels of the JNK and p38 MAP kinases when the cells were treated with various subfractions of ABE-EtOAc. However, Akt phosphorylation was suppressed by the ABE-EtOAc subfractions. Notably,

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Table 2 Effects of the ABE-EtOAc subfractions on histamine release from A23187-stimulated RBL-2H3 cells. Inhibition of histamine releasea,b (%)

ABE-EtOAc-E ABE-EtOAc-F ABE-EtOAc-G ABE-EtOAc-H Quercetin

IC50 (␮g/mL)

25 ␮g/mL

50 ␮g/mL

100 ␮g/mL

36.7 ± 8.9 ± 0.7 ± 13.9 ± 5 ␮M 32.6 ±

40.2 ± 41.5 ± 27.2 ± 31.0 ± 10 ␮M 51.8 ±

60.4 ± 69.7 ± 58.0 ± 59.8 ± 20 ␮M 66.5 ±

4.5* 1.8 0.6 1.9* 1.4*

10.6* 03.0* 00.7* 04.0* 4.8*

4.5* 1.7* 4.5* 1.6*

71.3 71.2 87.1 83.2

4.9*

11.5

Each value represents the mean ± SD of three experiments. b The histamine level was 126.2 ± 4.5 ng/mL in the non-stimulated group and 408.3 ± 5.1 ng/mL in the A23187-stimulated group. The detection limit for histamine is 0.14 pg/mL. * Denote significant differences from the control, p < 0.05. a

Fig. 3. Effects of the subfractions of ABE-EtOAc on protein kinase C (PKC) activation and reactive oxygen species (ROS) production in A23187-stimulated RBL-2H3 cells. (A) The expression of PKC was analyzed by western blotting. The cells were untreated (lane 1), treated with 1 ␮M A23187 alone for 20 min (lane 2), or treated with 70 ␮g/mL of either ABE-EtOAc-E (lane 3), ABE-EtOAc-F (lane 4), ABE-EtOAc-G (lane 5), or ABE-EtOAc-H (lane 6) and co-incubated with 1 ␮M A23187 for 20 min. (B) The production of ROS was analyzed using flow cytometry. FL1-H indicates DCFH-DA fluorescence, which signifies the production of ROS. Filled gray peaks, cells induced without 1 ␮M A23187 as the blank. Solid peaks, cells induced with 1 ␮M A23187 for 20 min as the control. Bold peaks, cells treated with 70 ␮g/mL of various subfractions of ABE-EtOAc and subsequently induced with 1 ␮M A23187 for 20 min.

H.-J. Chen et al. / Journal of Ethnopharmacology 141 (2012) 119–127

Fig. 4. Effects of the subfractions of ABE-EtOAc on the signaling pathways in A23187-stimulated RBL-2H3 cells. The cells were treated without (lane 1) or with (lane 2) 1 ␮M A23187 for 20 min or were treated with 70 ␮g/mL of ABE-EtOAc-E (lane 3), ABE-EtOAc-F (lane 4), ABE-EtOAc-G (lane 5), or ABE-EtOAc-H (lane 6) and co-incubated with 1 ␮M A23187 for 20 min.

ABE-EtOAc-E and -F displayed stronger suppressive effects on Akt phosphorylation than on ERK phosphorylation (lanes 3 and 4, respectively). ABE-EtOAc-H also suppressed the phosphorylation of Akt (lane 6). The different effects of the various subfractions of ABE-EtOAc on multiple signaling pathways provide additional evidence of the existence of at least two anti-allergic compounds in adlay bran. 3.6. Effects of the active constituents isolated from the ABE-EtOAc subfractions on ˇ-hexosaminidase release A bioassay-guided separation of ABE-EtOAc produced six phenolic acids (syringaldehyde, vanillic acid, ferulic acid, protocatechuic acid, caffeic acid, and p-coumaric acid) and one flavone (luteolin). These compounds were identified by their spectroscopic data (1-D NMR and MS) and were compared with published data. The anti-allergic activities of various phenolic compounds, as estimated from their effects on degranulation in A23187-stimulated RBL-2H3 cells, are shown in Table 3. All of these compounds, except caffeic acid and ferulic acid, exhibited inhibitory activity against mast cell degranulation. Luteolin produced the most potent inhibitory activity of all of the constituents of ABE-EtOAc (IC50 = 1.5 ␮g/mL). The other phenolic compounds showed the following weak levels of degranulation inhibition: p-coumaric acid (22.9%) > vanillic acid (16%) > syringaldehyde (10.2%) > protocatechuic acid (7.3%) at a concentration of 200 ␮g/mL. 4. Discussion In our preliminary experiments, the IC50 values of anti␤-hexosaminidase release were similar between the ethanolic extracts of adlay hull, testa and bran at concentrations of 118.3, 105.3 and 104.5 ␮g/mL, respectively (data not shown). The antiallergic compounds in the roots of adlay have been isolated (Otsuka et al., 1988). However, the other compounds in the edible parts of adlay have not been identified. Previously, we found that the compounds from the ethyl acetate fractions of adlay testa, which were less polar than other compounds, exhibited anti-allergic activity (Chen et al., 2010). These results indicate that adlay contains a variety of anti-allergic compounds. Therefore, in the present study, we chose to isolate compounds from different parts of the adlay seed. The seeds were divided into hull, testa, and dehulled adlay by

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gentle blowing with an electric fan. The dehulled adlay, an edible part of the adlay seed, was separated into bran and polished adlay. Mast cell secretory granules contain ␤-hexosaminidase, which is released in amounts that exhibit a quantitative relationship with the histamine that is released following the immunologic stimulation of the cells (Schwartz et al., 1979). To characterize the effects of various fractions of adlay bran, we used the ␤-hexosaminidase release assay as a high throughput method to screen the adlay samples. Ca2+ ionophores, such as A23187, are believed to bypass the receptor-mediated events by directly conveying Ca2+ across the plasma membrane to activate PKC (Baumgartner et al., 1994). A23187 has been shown to induce the release of cytokine receptors independently by increasing the intracellular Ca2+ level (Choi et al., 2007). Therefore, we chose 1 ␮M A23187 as the stimulating agent to induce a 30–60% level of ␤-hexosaminidase release. Quercetin, a common flavanol that is rich in plants, functions as an anti-allergic agent (Kawai et al., 2007). Table 1 shows that 10 ␮M quercetin markedly decreased ␤-hexosaminidase release (58%, p < 0.05), validating this throughput model for the analysis of anti-allergic effects. In this study, adlay bran significantly inhibited the allergic response in RBL-2H3 cells. Furthermore, the ABE-EtOAc-E, -F, -G, and -H subfractions suppressed histamine release in A23187stmulated RBL-2H3 cells, with IC50 values of 71.2–87.1 ␮g/mL (Table 1). Histamine release was inhibited 60–70% by ABE-EtOAc subfractions at a concentration of 100 ␮g/mL. We have previously shown that a 20% dehulled adlay diet suppresses the production of IgE against OVA antigen and allergic cytokines (Hsu et al., 2003). Our present results are consistent with these earlier observations and further confirm the contribution of adlay bran to both adaptive and innate immunity. Mast cells are a potential source of inflammatory cytokines, such as IL-4, IL-6, and TNF-␣, and they play a key role in allergic diseases (Bischoff, 2007). A single A23187 stimulation may not be sufficient to activate the allergic response; a secondary signal is critical for immune activation. Therefore, the A23187/PMA co-activated mast cells were able to produce remarkable increases in the secretion of IL-4, IL-6 and TNF-␣. The IL-4-induced B-cell class switching to IgE is the most important cytokine target in the anti-allergic pathway. The subfractions of ABE-EtOAc significantly suppressed the inflammatory cytokines, especially the secretion of IL-4 (Fig. 2A). These results agree with our previous data, which showed that the dehulled adlay diet decreased the IgE production in OVAsensitized mice, implying that the anti-allergic compounds in adlay bran can suppress the allergic response via the inhibition of IL-4 in mast cells. RBL-2H3 cells do not store TNF-␣ in secretory granules, instead releasing it through a Golgi-dependent mechanism, which is tightly regulated by Ca2+ and PKC (unlike constitutive secretion) (Baumgartner et al., 1994). In the A23187/PMA-stimulated RBL-2H3 cells examined in the present study, the ABE-EtOAc subfractions reduced the secretion of TNF-␣ (Fig. 2C). The reductions in the expression of PKC and the phosphorylation of ERK caused by these subfractions suggest that a release of anti-TNF-␣ antibodies occurred. Our results mirror the effects of azelastine, an anti-allergic drug that works via the same mechanism of decreasing PKC expression (Abdel-Raheem et al., 2005). Although the inhibition of IL-6 secretion by the ABE-EtOAc subfractions was not as strong as that of TNF-␣ secretion (Fig. 2B), the significant decreases in IL-6 secretion caused by the ABE-EtOAc subfractions indicate that adlay bran suppresses not only Th2 cytokines but also inflammatory cytokines. The various compounds found in adlay bran may use different mechanisms to suppress the allergic response. Two major anti-allergic compounds that have been isolated from the rhizomes of Dioscorea membranacea shows different anti-allergic effects. Dioscorealide B, which is isolated form Dioscorea membranacea, inhibits the degranulation of mast cells in the early phase of the

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Table 3 Effects of the various compounds isolated from the ABE-EtOAc subfractions on ␤-hexosaminidase release from A23187-stimulated RBL-2H3 cells. Inhibition (%)a

Caffeic acid Ferulic acid p-Coumaric acid Protocatechuic acid Syringaldehyde Vanillic acid Luteolin a *

IC50 (␮g/mL)

25 ␮g/mL

50 ␮g/mL

100 ␮g/mL

200 ␮g/mL

2.1 ± 10.1 0.7 ± 2.6 6.5 ± 0.7 0.9 ± 0.2 2.9 ± 2.4 4.9 ± 6.2 0.5 ␮g/mL 10.8 ± 1.4*

2.8 ± 5.3 0.5 ± 5.9 16.5 ± 1.6* 1.2 ± 5.0 6.3 ± 8.8 4.4 ± 2.8 1 ␮g/mL 31.4 ± 2.7*

0.6 ± 3.4 3.9 ± 4.6 17.9 ± 6.9* 4.9 ± 5.5 5.8 ± 2.9 9.2 ± 5.2 1.5 ␮g/mL 51.9 ± 5.2*

2.7 ± 0.3 5.5 ± 6.5 22.9 ± 2.8* 7.3 ± 2.1* 10.2 ± 9.0* 16.0 ± 3.8* 2 ␮g/mL 72.4 ± 3.7*

>200 >200 >200 >200 >200 >200 1.5

Each value represents the mean ± SD of three experiments. Denote significant differences from the control, p < 0.05.

allergic response, but dioscorealide A suppresses cytokine secretion in the late phase (Tewtrakul and Itharat, 2006). Similarly, we identified two highly potent subfractions of ABE-EtOAc; ABE-EtOAc-F produced a better inhibition of degranulation in mast cells, but ABE-EtOAc-E suppressed the secretion of cytokines. The decrease in Akt phosphorylation in ABE-EtOAc-H-treated cells implies that the anti-allergic effects of ABE-EtOAc are mediated through the inhibition of cytokine secretion in the late phase of the allergic response. However, adlay bran need to be further separated into their constituent components. The p38 and MAPK pathways are the major targets of asthma medications, and the level of PKC expression is positively correlated to the release of cytokines (Gilfillan and Tkaczyk, 2006). The phosphorylation of Akt and ERK provides the main stimulus for mast cell degranulation in the early phase of the allergic response. The anti-degranulation effects of the ABE-EtOAc subfractions were mediated through the suppression of Akt and ERK phosphorylation (Fig. 3). Several studies have reported similar effects from various anti-allergic compounds. Sinomenine, which was isolated from Sinomenium acutum, suppresses the Akt and MAPK pathways to decrease IL-4 and TNF-␣ secretion (Huang et al., 2008). Ginsenodise Rb1 from Panax ginseng has anti-allergic effects that are evidenced by a decrease in IL-4 secretion through the MAPK pathway (Liao et al., 2006). However, we did not observe significant changes in the expression levels of JNK and p38. These conflicting results concerning the activation of the MAPK pathway may be due to different types of stimulation required by different cell types or their different responses to stimulation. Nevertheless, these MAPK-activated mediators, which include anti-histamines, IL-4 antagonists and anti-inflammatory compounds, are useful targets in current allergic therapy (Walsh, 2005). Furthermore, PKC signaling results in the generation of ROS in A23187-stimulated RBL-2H3 cells, which indicates that ROS participate in positive feedback to cause the phosphorylation of FAK, which, in turn, induces histamine release and activates NK-␬B to enhance cytokine production in mast cells (Matsui et al., 2000). In our study, the ABE-EtOAc subfractions suppressed PKC expression and inhibited ROS production. These results suggest the existence of an alternative mechanism to reduce cytokine release in response to immune stimulation. Taken together, these data on the effects of adlay bran on the modulation of signal transduction in RBL-2H3 cells provide strong evidence of the anti-allergic effects of this plant. Cereals have been shown to have antioxidant properties that are attributed to their phenolic compounds (Baublis et al., 2000). Many studies have reported that certain cereals may have effects on allergic symptoms. For example, ethanolic extracts of pigmented black rice show anti-IL-1, -IL-6 and -TNF-␣ activities in A23187stimulated RBL-2H3 cells (Choi et al., 2007). Extracts of buckwheat grain inhibit histamine release and the transcription of IL-4 in RBL-2H3 cells (Kim et al., 2003). The active compounds that have been isolated from various cereals include phenolic compounds,

flavones and phytosterols. Our findings indicate that adlay bran possesses anti-allergic properties. The compounds extracted from adlay bran include caffeic acid, ferulic acid, p-coumaric acid, protocatechuic acid, syringaldehyde, vanillic acid and luteolin. Our previous research revealed the benefits of the different flavonoids that are abundant in adlay bran (Chen et al., 2011). It has been reported that flavonoids have anti-allergic effects; for example, luteolin exerts such effects via the suppression of ERK phosphorylation (Mastuda et al., 2002). The IC50 value (5.2 ␮M) of luteolin found in the present work is similar to that reported in the literature (Mastuda et al., 2002). Although phenolic compounds have been reported to possess antioxidant activity, we found that phenolic compounds have a limited effect on degranulation. Also, p-Coumaric acid has been reported to exert a weak inhibitory effect on degranulation in RBL-2H3 cells (Matsuda et al., 2004). There have been few reports of the anti-allergic effects of the other phenolic compounds isolated from ABE-EtOAc fractions of A23187-stimulated RBL-2H3 cells. Our results indicate that these phenolic compounds may not be the main anti-allergic active components in adlay bran (Table 3). However, it has been reported that there is no correlation between anti-allergic and anti-oxidative effects (Yamada and Tachibana, 2000). In addition, the complement activation pathway may serve as a central regulator of allergic disorders. The anti-complementary polysaccharides isolated from adlay may inhibit the C3a- and C5a-activated degranulation of mast cells, indicating their anti-allergic properties (Takahashi et al., 1986). Therefore, the flavonoids and polysaccharides found in adlay bran may produce anti-allergic effects via the MAPK and anticomplementary pathways. In summary, we found that ABE suppressed the production of histamines and inflammatory cytokines in sensitive RBL-2H3 cells and identified the subfractions of ABE-EtOAc that were active in this process. Additionally, we isolated several compounds that displayed mast cell degranulation inhibition activity. In conclusion, ABE positively modulates the immune system and alleviates allergic symptoms. Therefore, the inhibitory effects of ABE on allergic responses may provide an additional strategy for the treatment of allergic diseases.

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