Anti-inflammatory effects and chemical study of a flavonoid-enriched fraction from adlay bran

Food Chemistry 126 (2011) 1741–1748

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Anti-inflammatory effects and chemical study of a flavonoid-enriched fraction from adlay bran Hong-Jhang Chen a, Cheng-Pei Chung a,1, Wenchang Chiang a,⇑, Yun-Lian Lin b,c,⇑ a

Graduate Institute of Food Science and Technology, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan National Research Institute of Chinese Medicine, Taipei, Taiwan c School of Pharmacy, National Taiwan University, Taipei 100, Taiwan b

a r t i c l e

i n f o

Article history: Received 29 June 2010 Received in revised form 25 November 2010 Accepted 14 December 2010 Available online 19 December 2010 Keywords: Anti-inflammation Adlay bran Flavonoids New aurone derivative LC/MS

a b s t r a c t Anti-inflammation-guided fractionation and purification were used to evaluate the bioactivity and components of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) bran. Results showed that the fraction with high phenolic and flavonoid contents from the ethanol extracts of adlay bran suppressed LPS-stimulated IL-6 and TNF-a secretions in a concentration-dependent manner in RAW 264.7 cells and murine peritoneal macrophages. Fifteen compounds, including a novel aurone derivative, two chromones, one dihydrochalcone, one chalcone, four flavanones, five flavones and one isoflavone, were isolated from the active fraction. The structure of the new compound was elucidated by spectroscopic methods, including 1D and 2D NMR and MS. All of the isolates are reported for the first time from adlay except naringenin. LC/MS was also provided as an analytical platform. Our results suggest that flavonoids in adlay bran, partially at least, contribute to its anti-inflammatory effect. Thus, adlay bran may be beneficial to the health of consumers. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Chronic inflammation contributes to the pathogenesis of a number of diseases that affect a significant part of the human population. Nitric oxide (NO) is an inorganic free radical that is implicated in pathological processes such as chronic and acute inflammation (Salerno, Sorrenti, Di Giacomo, Romeo, & Siracusa, 2002). NO is produced by the oxidation of L-arginine, catalysed by NO synthase (NOS). In the NOS family, inducible (i)NOS, in particular, is involved in the pathological overproduction of NO, and can be expressed in response to proinflammatory agents such as tumour necrosis factor (TNF)-a and lipopolysaccharide (LPS) in

Abbreviations: ABE, the ethanol extracts of adlay bran; ABE-BuOH, the n-butanol soluble fraction from the ethanol extract of adlay bran; ABE-EtOAc, the ethylacetate fraction from the ethanol extract of adlay bran; ABE-Hex, the n-hexane soluble fraction from the ethanol extract of adlay bran; ABE-H2O, the water-soluble fraction from the ethanol extract of adlay bran; CE, catchin equivalents; DMSO, dimethyl sulfoxide; GAE, gallic acid equivalents; IL, interleukin; LC/MS, liquid chromatography/mass spectrometry; TIC, total ion current; TNF-a, tumour necrosis factor-alpha. ⇑ Corresponding authors. Addresses: 1, Roosevelt Road, Sec. 4, Taipei 106, Taiwan. Tel.: +886 2 3366 4115; fax: +886 2 2363 8673 (W. Chiang), 155-1, Li-Nung St., Sec. 2, Taipei 112, Taiwan. Tel.: +886 2 2820 1999x6531; fax: +886 2 2826 4266 (Y.-L. Lin). E-mail addresses: [email protected] (H.-J. Chen), [email protected] (W. Chiang), [email protected] (Y.-L. Lin). 1 Address: 155-1, Li-Nung St., Sec. 2, Taipei 112, Taiwan. Tel.: +886 2 2820 1999x6521; fax: +886 2 2826 4266. 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.074

various cell types, including macrophages. Furthermore, activated macrophages cause inflammation, which is mediated by proinflammatory mediators, including TNF-a, interleukin (IL)-6, and NO (Libby, 2006). There is growing evidence that systemic inflammation is associated with increased risks of chronic diseases, such as inflammatory bowel disease and cancer (Atreya & Neurath, 2005). Therefore, the application of dietary components, aside from anti-inflammatory drugs, has become a focus of interest. Diets for preventing chronic diseases associated with inflammation were recently proposed as a therapeutic strategy (O’Keefe, Gheewala, & O’Keefe, 2008). Adlay (Job’s tears, Coix lachryma-jobi L. var. ma-yuen Stapf) is an annual crop, which has long been used in Chinese medicine to treat warts, chapped skin, rheumatism, and neuralgia (Lu, Li, & Dong, 2008). It was reported to have various activities, such as anticancer (Lee, Lin, Cheng, Chiang, & Kuo, 2008), anti-inflammatory (Seo et al., 2000), and antiallergic effects (Chen, Shih, Hsu, & Chiang, 2010). Various constituents were isolated from adlay, such as anti-inflammatory benzoxazinoids from the roots (Otsuka, Hirai, Nagao, & Yamasaki, 1988), phenolic compounds with antiallergic properties from the testa (Chen et al., 2010), cytotoxic lactams from the bran (Lee et al., 2008), and antioxidative lignans from the hulls (Kuo et al., 2002). However, anti-inflammatory and related components of adlay bran, the edible part of adlay, are still unclear. Therefore, in this work, we investigated the anti-inflammatory effect of adlay bran with in vitro and ex vivo cell cultures, and undertook a chemical study of the bioactive fraction. Meanwhile, liquid chromatographic

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(LC)/mass spectrometric (MS) analyses were also developed as a quality-control platform. 2. Materials and methods 2.1. Materials and general methods Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lipopolysaccharide (LPS), and other chemicals were obtained from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (Grand Island, NY, USA). Foetal bovine serum (FBS), red blood cell (RBC) lysis solution, and antibiotic–antimycotic were obtained from Biological Industries (Haemek, Israel). Silica gel (230–400 mesh), Sephadex™ LH-20 (Amersham Biosciences, Uppsala, Sweden), and a semi-preparative Si column (LiChrosorb Si-60, Merck, Darmstadt, Germany) were used for column chromatography. Solvents (analytical grade) were purchased from Merck. Infrared (IR) spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer (Thermo Electron, Akron, OH, USA). Optical rotation was measured on a Jasco P-2000 polarimeter (Hachioji, Tokyo, Japan). Ultraviolet (UV) spectra were measured on a Hitachi U-3310 spectrophotometer (Hitachi, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were run in CD3OD on a Varian unity INOVA-500 or VNMRS 600 (Varian, Palo Alto, CA, USA), using standard pulse sequences. Mass spectra (ESIMS) were recorded on a Finnigan MAT LCQ ion trap mass spectrometer system (Thermoquest, San Jose, CA, USA). 2.2. Determination of the total phenolic content The total phenolic content was determined according to the Folin–Ciocalteu method (Moyo, Ndhlala, Finnie, & Van Staden, 2010). Briefly, 200 ll of each sample (2000 lg/ml) were separately mixed with 2 ml of distiled water, 1 ml of Folin–Ciocalteu reagent, and 5 ml 20% sodium carbonate. After incubation in the dark for 20 min at room temperature, the absorbance was measured at 735 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The amount of Folin–Ciocalteu reagent was substituted by the same amount of distiled water in the blank. Gallic acid (0, 200, 400, 600, 800, and 1000 lg/ml) was used to make a calibration curve. The total phenolic content was expressed as mg gallic acid equivalents (GAE)/g of extract. 2.3. Determination of the total flavonoid content The AlCl3 method was used to determine the total flavonoid content (Chang, Yang, Wen, & Chern, 2002). Briefly, 0.5 ml of each sample (2000 lg/ml) was separately mixed with 0.1 ml of 10% aluminium chloride, 0.1 ml of 1 M potassium acetate, and 2.8 ml of distiled water. After incubation at room temperature for 30 min, the absorbance was measured at 415 nm with a microplate reader (molecular devices). The amount of 10% aluminium chloride was substituted by the same amount of distiled water in the blank. Catechin (0, 100, 200, 400, 600, and 800 lg/ml) was used to make a calibration curve. The total flavonoid content was expressed as mg catechin equivalents (CE)/g of extract. 2.4. Preparation of the ethanol extract and various fractions from the bran of adlay seeds and isolation and identification of chemical constituents Adlay seeds were collected from Taichung, Taiwan, in 2008, and divided into the hull, testa, and dehulled adlay by gentle blowing with an electric fan. The dehulled adlay, the edible part of adlay

seed, was separated into the bran and polished adlay. Adlay bran powder (2 kg) was extracted three times with 20 l of ethanol at room temperature for 2 weeks. The ethanol extracts were combined and concentrated under reduced pressure in a rotary vacuum evaporator. The dry ethanol extract (ABE, 365 g) was suspended in 4 l of 90% MeOH in H2O, followed by sequential partitioning with hexane, ethyl acetate (EtOAc), and n-butanol (BuOH), to give hexane-soluble (ABE-Hex, 155 g), ethyl-acetate-soluble (ABE-EtOAc, 145 g), n-butanol-soluble (ABE-BuOH, 12 g), and water-soluble fractions (ABE-H2O, 47 g). ABE-EtOAc was then subjected to silica gel (230–400 mesh) column chromatography with successive elution by a Hex/EtOAc gradient and MeOH solvent system. Subfractions with the same thin-layer chromatography (TLC) patterns were combined into one fraction; thus 9 fractions were obtained: A (0% to 20% EtOAc/Hex), B (20% to 25% EtOAc/Hex), C (25% to 30% EtOAc/Hex), D (30% to 40% EtOAc/Hex), E (40% to 60% EtOAc/Hex), F (60% to 80% EtOAc/Hex), G (80% to 90% EtOAc/Hex), H (90% to 100% EtOAc/Hex), and I (100% MeOH). The activities of various subfractions were tested in the system described below, and the bioactive subfractions, ABE-EtOAc-E–I were combined (28.3 g) and further purified over a Sephadex LH-20 column (eluted with MeOH) and semi-preparative high-performance liquid chromatography (HPLC) on a LiChrosorb Si-60 column, using a mobile phase of acetone: EtOAc: Hex of 2:1:5 at a flow rate of 1.5 ml/min to yield 1 (1.2 mg), 2 (3.0 mg), 3 (6.6 mg), 4 (14.6 mg), 5 (30.5 mg), 6 (3.5 mg), 7 (3.6 mg), 8 (1.3 mg), 9 (5.2 mg), 10 (4.1 mg), 11 (6.4 mg), 12 (2.9 mg), 13 (2.3 mg), 14 (10.6 mg) and 15 (2.1 mg). 20 ,6-Dihydroxy-40 -methoxydihydroauronol (1). Amorphous powder. ESIMS: m/z 289 [M+H]+; ½a2 5D = +13.3° (c 0.3, CH3OH); CD (c 0.035, MeOH) 287.7 (+0.58), 238.9 (1.32), 212.2 (1.75), 207.1 (2.40); IR mmax 3334, 1623, 1461, 1374, 1255, 1148 cm1; UV (MeOH)(log e) kmax 282 (3.78), 286 (3.79) nm; 1H NMR (CD3OD, 500 MHz): d 3.51 (1H, dd, J = 14.5, 7.5, H-9a), 3.53 (1H, dd, J = 14.5, 6.0, H-9b), 3.73 (3H, s, 40 -OCH3), 4.21 (1H, br d, J = 6.6, H-7), 5.46 (1H, m, H-8), 6.29 (1H, d, J = 2.4, H-1), 6.37 (1H, d, J = 2.4, H-30 ), 6.43 (1H, dd, J = 8.4, 2.4, H-50 ), 6.48 (1H, dd, J = 8.4, 2.4, H-5), 7.16 (1H, d, J = 8.4, H-60 ), 7.28 (1H, d, J = 8.4, H-4); 13C NMR (CD3OD, 125 MHz): d 40.9 (t, C-9), 55.9 (q, OCH3), 67.5 (d, C-7), 80.1 (d, C-8), 97.5 (d, C-1), 104.0 (d, C-30 ), 107.3 (d, C-60 ), 110.7 (d, C-5), 112.9 (s, C-3), 120.9 (s, C-10 ), 126.0 (d, C-50 ), 133.2 (d, C-4), 158.0 (s, C-2), 160.8 (s, C-6), 162.0 (s, C-20 ), 162.5 (s, C40 ); key H-H COSY correlations: H-9/H-8/H-7; key HMBC correlations: H-7/C-2 & C-8, H-4/C-2 & C-6, H-8/C-2 & C-7, H-30 /C-20 & C-40 , H-30 /C-20 & C-40 , H-50 & 40 -OCH3/C-40 , H-5/C-3, H-60 /C-20 & C40 ; key NOESY correlations: H-9/H-7, H-8 & H-60 , 40 -OCH3/H-30 & H-50 , H-4/H-5. 5,7-Dihydroxychromone (2). ESIMS: m/z 177 [M-H], 1H NMR (CD3OD, 500 MHz): d 6.18 (1H, d, J = 6.0, H-3), 6.19 (1H, d, J = 1.5, H-6), 6.32 (1H, d, J = 1.5, H-8), 7.97 (1H, d, J = 6.0, H-2). 5-Hydroxy-7-methoxychromone (3). ESIMS: m/z 193 [M+H]+; 1H NMR (CD3OD, 500 MHz): d 3.68 (3H, s, OCH3), 6.18 (1H, d, J = 6.0, H-3). 6.20 (1H, d, J = 2.0, H-6), 6.33 (1H, d, J = 2.0, H-8), 7.96 (1H, d, J = 6.0, H-2). Davidigenin (4). ESIMS: m/z 257 [M-H]; 1H NMR (CD3OD, 500 MHz): d 2.90 (2H, t, J = 7.5, H-b), 3.17 (2H, t, J = 7.5, H-a), 6.24 (1H, d, J = 2.0, H-30 ), 6.33 (1H, dd, J = 9.0, 2.0, H-50 ), 6.68 (2H, d, J = 8.5, H-3, 5), 7.05 (2H, d, J = 8.5, H-2, 6), 7.70 (1H, d, J = 9,0, H-60 ). Isoliquiritigenin (5). ESIMS: m/z 255 [M-H]; 1H NMR (CD3OD, 500 MHz): d 6.28 (1H, d, J = 2.0, H-30 ), 6.41 (1H, dd, J = 8.5, 2.5, H50 ), 6.84 (2H, d, J = 8.5, H-3, 5), 7.61 (1H, d, J = 15.5, H-a), 7.62 (2H, d, J = 8.5, H-2, 6), 7.79 (1H, d, J = 15.5, H-b), 7.97 (1H, d, J = 8.5, H-6’). Naringenin (6). ESIMS: m/z 271 [M-H]; 1H NMR (CD3OD, 500 MHz): d 2.69 (1H, dd, J = 17.0, 3.0, H-3a), 3.11 (1H, dd,

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J = 17.0, 13.0, H-3b), 5.34 (1H, dd, J = 13.0, 3.0, H-2), 5.88 (1H, d, J = 2.0, H-8), 5.89 (1H, d, J = 2.0, H-6), 6.81 (2H, d, J = 8.5, H-30 , 50 ), 7.31 (2H, d, J = 8.5, H-20 , 60 ). Homoeriodictyol (7). ESIMS: m/z 301 [M-H], 1H NMR (CD3OD, 500 MHz): d 2.70 (1H, dd, J = 17.5, 3.0, H-3a), 3.13 (1H, dd, J = 17.5, 13.0, H-3b), 3.88 (3H, s, OCH3), 5.34 (1H, dd, J = 13.0, 3.0, H-2), 5.88 (1H, d, J = 2.5, H-6), 5.90 (1H, d, J = 2.5, H-8), 6.82 (1H, d, J = 9.0, H-50 ), 6.92 (1H, dd, J = 9.0, 2.0, H-60 ), 7.07 (1H, d, J = 2,0, H-20 ). Hesperetin (8). ESIMS: m/z 301 [M-H]; 1H NMR (CD3OD, 500 MHz): d 2.72 (1H, dd, J = 17.4, 3.0, H-3a), 3.04 (1H, dd, J = 17.4, 12.0, H-3b), 3.88 (3H, s, OCH3), 5.32 (1H, dd, J = 12.0, 3.0, H-2), 5.88 (1H, d, J = 2.0, H-6), 5.91 (1H, d, J = 2.0, H-8), 6.93 (1H, dd, J = 9.0, 2.0, H-60 ), 6.94 (1H, d, J = 9.0, H-50 ), 6.95 (1H, d, J = 2,0, H-20 ). Liquiritigenin (9). ESIMS: m/z 255 [M-H]; 1H NMR (CD3OD, 600 MHz): d 2.70 (1H, dd, J = 17.0, 3.0, H-3a), 3.05 (1H, dd, J = 17.0, 12.5, H-3b), 5.37 (1H, dd, J = 12.5, 3.0, H-5), 6.34 (1H, d, J = 2.0, H-8), 6.48 (1H, dd, J = 8.8, 2.0, H-6), 6.81 (2H, d, J = 8.4, H30 ,50 ), 7.32 (1H, d, J = 8.4, H-2), 7.72 (1H, d, J = 8.4, H-5). Chrysoeriol (10). ESIMS: m/z 299 [M-H], 1H NMR (acetone-d6, 500 MHz): d 3.98 (3H, s, OCH3), 6.24 (1H, d, J = 2.0, H-6), 6.58 (1H, d, J = 2.0, H-8), 6.68 (1H, s, H-3), 6.99 (1H, d, J = 8.5, H-50 ), 7.59 (1H, dd, J = 8.5, 1.5, H-60 ), 7.62 (1H, d, J = 1.5 , H-20 ), 8.57 (1H, br s, OH), 9.76 (1H, br s, OH), 13.00 (1H, br s, OH). 3,40 ,5,7-Tetramethoxyflavone (11). ESIMS: m/z 365 [M+Na]+; 1H NMR (CD3OD, 600 MHz): d 3.89, 3.92, 3.95, and 4.03 (3H each, s, OCH3), 6.62 (1H, d, J = 2.0, H-6), 6.68 (1H, d, J = 2.0, H-8), 7.10 (2H, d, J = 9.0, H-30 , 50 ), 7.97 (2H, d, J = 9.0, H-20 , 60 ). 3,30 ,40 ,5,7-Pentamethoxyflavone (12). ESIMS: m/z 395 [M+Na]+; 1 H NMR (CD3OD, 600 MHz): d 3.92, 3.93, 3.94, 3.95, and 3.96 (3H each, s, OCH3), 6.66 (1H, d, J = 2.0, H-6), 6.69 (1H, d, J = 2.0, H-8), 7.13 (1H, d, J = 9.0, H-50 ), 7.55 (1H, d, J = 2.0, H-20 ), 7.66 (1H, dd, J = 9.0, 2.0 H-60 ). Tangeretin (13). ESIMS: m/z 395 [M+Na]+, 1H NMR (CD3OD, 600 MHz): d 3.88, 3.89, 3.92, 4.02, and 4.10 (3H each, s, OCH3), 6.67 (1H, s, H-3), 7.11 (2H, d, J = 9.0, H-30 , 50 ). 7.98 (2H, d, J = 9.0, H-20 , 60 ). 3,30 ,40 ,5,6,7,8-Heptamethoxyflavone (14). ESIMS: m/z 455 [M+Na]+; 1H NMR (CD3OD, 600 MHz): d 3.82, 3.90, 3.91, 3.92, 3.93, 4.00, and 4.09 (3H each, s, OCH3), 7.14 (1H, d, J = 9.0, H-50 ), 7.76 (1H, d, J = 2.4, H-20 ), 7.85 (1H, dd, J = 9.0, 2.4, H-60 ). Formononetin (15). ESIMS: m/z 267 [M-H], 1H NMR (CD3OD, 500 MHz): d 3.82 (3H, s, OCH3), 6.83 (1H, d, J = 2.0, H-8), 6.92 (1H, dd, J = 9.0, 2.0, H-6), 6.97 (2H, d, J = 9.0, H-30 , 50 ), 7.46 (2H, d, J = 9.0, H-20 , 60 ), 8.04 (1H, d, J = 9.0, H-5), 8.14 (1H, s, H-2). 2.5. Cell culture Mouse macrophage cells of the RAW264.7 cell line were obtained from the Bioresource Collection and Research Centre (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan) and were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml of penicillin–streptomycin, and 2% glutamine in a 5% CO2 incubator at 37 °C. Cells were detached by washing and scraping. After washing, cells were resuspended in fresh medium and used for subsequent experiments. 2.6. Preparation of peritoneal macrophages BALB/c mice (6–7 weeks old, 19–25 g) were purchased from LASCO (BioLASCO, Taipei, Taiwan). Mice were kept in an airconditioned, pathogen-free room at a temperature of 23 ± 2 °C on a regulated 12-h light/dark cycle. They were housed individually in stainless-steel wire cages and fed a commercial diet (Lab Rodent Chow 5001, Ralston Purina, St. Louis, MO, USA). Animal care and

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handling conformed to accepted guidelines, and were approved by the Institutional Animal Care and Use Committee of National Taiwan University. Murine peritoneal macrophages were collected from the peritoneal cavity of normal BALB/c mice (Otsuka et al., 1988). Briefly, peritoneal fluid was collected by an intraperitoneal (i.p.) injection of 10 ml of ice-cold DMEM. After washing with RBC lysis buffer, peritoneal macrophages were centrifuged and grown in DMEM supplemented with 10% FBS and the antibiotic–antimycotic. Cells were seeded at 106 cells/well and allowed to adhere for 2 h at 37 °C in 5% CO2. After non-adherent cells were removed by washing with PBS, adherent cells were used for subsequent experiments. 2.7. Assay of cell viability Cell viabilities of RAW264.7 cells and murine peritoneal macrophages were determined by an MTT assay, as previously reported (Chen et al., 2010). The cell viability of various test groups was determined by the equation: (absorbance of the test group/absorbance of the control)  100%. All test samples mentioned above were dissolved in DMSO, and the final concentration of DMSO was <0.1%; 0.1% DMSO was used as the control group. 2.8. Assay of NO release An NO release assay was carried out according to the method described by Matsuda, Morikawa, Ando, Toguchida, and Yoshikawa (2003). Briefly, RAW264.7 cells were seeded at 105 cells/well in a 96-well plate. After washing with PBS, cells were pretreated with various test samples and 1 lg/ml of LPS for 24 h. The supernatant (100 ll) was mixed with the same volume of Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in water) and incubated for 15 min in the dark. The absorbance of the water-soluble purplish-red product was read at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The amount of NO was calculated by a calibration curve established with 0.15–100 lM NaNO2. 2.9. Measurement of IL-6 and TNF-a The inhibitory effects on LPS-stimulated release of cytokines in RAW264.7 cells and murine peritoneal macrophages were evaluated, as described above. Briefly, RAW264.7 cells and murine peritoneal macrophages were treated with various test samples and stimulated with 1 or 10 lg/ml of LPS for 24 and 48 h. The concentrations of IL-6 and TNF-a in the supernatants were determined by commercially available ELISA kits (eBioscience, San Diego, CA, USA). 2.10. HPLC/MS analysis HPLC analyses were carried out using a Finnigan MAT HPLC system (Thermo Electron, Akron, OH, USA), which was equipped with an analytical InertsilÒ ODS-3 column (4.6  250 mm  5 lm) and a guard column at a constant flow rate of 0.3 ml/min. The mobile phases A (0.01% formic acid in double-distiled water, pH 3.30) and B (acetonitrile: methanol 1:1, v/v) were used according to a programmed protocol: 0–10 min, 30–40% B; 10–20 min, 40–60% B; 20–27 min, 60–75% B; 27–35 min, 75–78% B; 35–40 min, 78–85% B; 40–50 min, 85–95% B; and 50–70 min, 95% B. The UV detector was operated at a wavelength of 254 nm. This system was coupled with a Finnigan MAT LCQ ion trap mass spectrometer system (Finnigan MAT, San Jose, CA, USA) which was operated in the electrospray ionisation (ESI) mode. An aliquot of the bioactive fraction (20 ll) was directly introduced into the column through

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the autosampler (Finnigan MAT AS3000), where nitrogen was used as the nebulising and drying gas. The operating parameters used were as follows: a gas temperature of 250 °C, a spray needle voltage of 5 kV, a nebulizer pressure of 60 psi, and auxiliary gas of 30 psi. An ion trap, containing helium damping gas, was introduced, following the manufacturer’s protocols. The mass spectra were acquired in an m/z range of 100–1000 with 5 microscans and a maximum ion injection time of 200 ms. 2.11. Statistical analysis Results are expressed as the means ± standard deviation (SD) of three experiments. Differences between specific means were analysed by one-way analysis of variance (ANOVA), using the SPSS system vers. 11.0 (SPSS, Chicago, IL, USA). Group means were compared using one-way ANOVA, followed by Dunnett’s test. Probability values (p) of <0.05 were considered statistically significant. 3. Results and discussion 3.1. Effects of subfractions on NO release and cell viability NO formation and cytokine secretions suggested a high correlation with various pathophysiological processes, including inflammation (Chen et al., 2001). The suppressive effect of the ABEEtOAc on the production of NO was evaluated in LPS-stimulated RAW264.7 cells. As shown in Fig. 1A, LPS significantly increased NO production compared to the unstimulated control, and subfractions ABE-EtOAc-E–I (40–100% EtOAc/Hex) presented significant inhibition of NO production at the concentration of 25 lg/ml, but the other subfractions did not. These data indicate that major anti-inflammatory components possibly exist in the 40–100% EtOAc/Hex subfractions of the ABE-EtOAc. For the cytotoxicity assay, cell viability showed no significant difference between the control and ABE-EtOAc-E–I subfractions (12.5–50 lg/ml) (Fig. 1B). Therefore, these five subfractions were subjected to further investigation. 3.2. Effects of subfractions of the ABE-EtOAc on the secretion of cytokines IL-6 and TNF-a are well-known inflammatory mediators that modulate acute and chronic inflammatory conditions (Libby, 2006). Macrophage cell lines, such as murine RAW264.7 and human THP-1 cells, have been used as rapid in vitro screening methods to study anti-inflammatory agents (Singh, Tabibian, Venugopal, Devaraj, & Jialal, 2005). In the present study, RAW264.7 cells and murine peritoneal macrophages were used for screening. As shown in Fig. 2, ABE-EtOAc subfractions (50 lg/ ml) significantly inhibited IL-6 and TNF-a production in both LPS-stimulated RAW264.7 cells and peritoneal macrophages. Inhibition of IL-6 secretion occurred in the order ABE-EtOAc-F (89%) > ABE-EtOAc-I (70%) > ABE-EtOAc-G (69%) > ABE-EtOAc-E (65%) > ABE-EtOAc-H (64%) at the concentration of 25 lg/ml in LPS-stimulated RAW264.7 cells (Fig. 2A). Similar results were obtained for IL-6 secretion in a concentration-dependent manner in LPS-stimulated peritoneal macrophages (Fig. 2C). Although only modest inhibition of TNF-a secretion was observed at the concentrations of 12.5 and 25 lg/ml, high concentrations (50 lg/ml) of each ABE-EtOAc subfraction effectively diminished TNF-a production in LPS-stimulated RAW264.7 cells by up to 48% (Fig. 2B). The generation of TNF-a was also concentration-dependently decreased in LPS-stimulated murine peritoneal macrophages (Fig. 2D). Meanwhile, pretreatment of RAW264.7 cells and primary peritoneal macrophages with 12.5, 25, and 50 lg/ml subfractions

Fig. 1. Effects of ABE-EtOAc subfractions of the ethanol extract on (A) nitrite production and (B) cell viability in LPS-stimulated RAW264.7 cells. Each column represents the mean ± SD of three experiments. RAW264.7 cells were stimulated by 1 lg/ml of lipopolysaccharide (LPS) for 24 h.

of the ABE-EtOAc for 24 h had no direct cytotoxicity according to an MTT assay (data not shown). Recent studies have shown that components from cereal sources, such as buckwheat (Ishii et al., 2008), may have anti-inflammatory activity and offer nutritional benefits against inflammation. Therefore, dietary components and nutritional supplements have also been proposed to prevent certain inflammatory diseases (O’Keefe et al., 2008). In this study, we showed that adlay bran possesses anti-inflammatory activity. More in vitro and in vivo studies on the beneficial components and action mechanisms of the ABE-EtOAc or its subfractions are warranted. 3.3. Phenolic and flavonoid compositions Certain levels of phenolic contents and flavonoids have been shown to exhibit an antioxidant capacity and have redox properties, e.g. reducing agents, hydrogen donors, and reactive oxidative species scavengers (Robards, Prenzler, Tucker, Swatsitang, & Glover, 1999). The total phenolic contents of the ABE-EtOAc subfractions are presented in Table 1, expressed in milligrammes of GAE per gramme of extract. The total phenolic contents of different subfractions ranged from 6.6 ± 0.8 to 19.1 ± 0.7 mg GAE/g of extract. The bioactive subfractions (ABE-EtOAc E–I) were significantly higher than the others. The flavonoid contents of the subfractions (at 3.5 ± 1.2 to 13.7 ± 1.8 mg CE/g of extracts) revealed

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Fig. 2. Effects of ABE-EtOAc subfractions on proinflammatory cytokine production from LPS-stimulated RAW264.7 cells and peritoneal macrophages. Values are expressed as the means ± SD of three independent experiments, each with triplicates and statistically analysed using Student’s t-test. ⁄p < 0.05, indicates a significant difference from the control. RAW264.7 cells were stimulated by 1 lg/ml of LPS for 24 h of incubation. Murine peritoneal macrophages were stimulated by 10 lg/ml of LPS for 48 h of incubation.

a trend similar to that of the total phenolic contents. Concentrations of flavonoids in the bioactive subfraction (8.2 ± 2.4 to 13.7 ± 1.8 mg CE/g of extracts) were much higher than those in the other subfractions. It was reported that flavonoids act on enzymes and pathways involved in anti-inflammatory processes (Matsuda et al., 2003). Our results indicated that the higher flavonoid contents in ABEEtOAc-E–I subfractions produced higher anti-inflammatory effects in the NO assay (Fig. 1). Similar results were obtained for the suppression of cytokine release (Fig. 2). Moreover, ratios of the total flavonoid/total phenolic contents in the bioactive ABE-EtOAc Table 1 Phenol and flavonoid contents of ABE-EtOAc subfractions of the ethanol extract of adlay bran. Subfraction

Total phenolsa

Total flavonoidsb

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

7.4 ± 1.1 6.6 ± 0.8 10.1 ± 0.9 13.0 ± 2.1 16.2 ± 1.0 17.8 ± 8.1 19.1 ± 0.7 15.0 ± 1.7 13.6 ± 1.0

3.5 ± 1.2 3.6 ± 2.1 4.9 ± 0.2 7.0 ± 1.8 10.1 ± 0.6 13.7 ± 1.8 13.6 ± 2.9 9.9 ± 3.3 8.2 ± 2.4

a The total phenol content of extracts is shown as mg gallic acid equivalent/g extract. b The total flavonoid content of extracts is shown as mg catechin equivalents/g extract.

subfractions ranged from 0.60 to 0.77. The results suggest that flavonoids may act as major and important anti-inflammatory components in the active fractions. 3.4. Identification of isolated compounds from the ABE-EtOAc and their effects on NO production The ABE was successively partitioned with Hex, EtOAc, and nBuOH to yield Hex-, EtOAc-, n-BuOH-, and H2O-soluble fractions. The EtOAc fraction showed an anti-inflammatory effect, when subjected to column chromatography over silica gel. Bioactive subfractions E–I were combined and then subjected to further chromatography, using Sephadex LH-20 and semi-preparative silica gel columns, to afford 15 isolates (Fig. 3). Among them, compound 1 was identified as a new compound. The remaining compounds were: two chromones, 5,7-dihydroxy-chromone (2) (Simon, Chulia, Kaouadji, & Delage, 1994) and 5-hydroxy-7-methoxy-chromone (3) (Vasconcelos, Silva, & Cavaleiro, 1998); one dihydrochalcone, davidigenin (4) (Severi et al., 1998); one chalcone, isoliquiritigenin (5) (Kong, Zhang, Pan, Tan, & Cheng, 2000); four flavanones, naringenin (6) (Panadda & Larry, 2001); homoeriodictyol (7) (Garo, Maillard, Antus, Mavi, & Hostettmann, 1996), hesperetin (8) (Lee, Choi, Park, Park, & Kim, 2004), and liquitiritigenin (9) (Kong et al., 2000); five flavones of chrysoeriol (10) (Bentamene et al., 2008), 3,40 ,5,7-tetramethoxyflavone (11) (Dong et al., 1999), 3,30 ,40 ,5,7-pentamethoxy-flavone (12) (Joseph-Nathan, Abramo-Bruno, & Torres, 1981), tangeretin (13)

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4' OCH3

HO 5

1

O 7

4

HO H

9 1'

H

8

RO

O

6

4 OH 2

HO 4'

OH O

OH

α

OH O

2 R=H 3 R=CH3

1

β 1'

4

1

4 R2 OR3

OH

HO

HO

O

R1 OH O

HO

3

6

OCH3 OH

4'

1'

6 R1=OH, R2=R3=H 7 R1=OH, R2=OCH3, R3=H 8 R1=R2=OH, R3=CH3 9 R1=R2= R3=H

5

O

O OH O 10

R4 OCH3

R3 H3CO R2 H3CO

O

O

HO

O

R1 O

11 R1=OCH3, R2=R3=R4=H 12 R1=R4=OCH3, R2=R3=H 13 R1=R4=H, R2=R3=OCH3 14 R1= R2=R3=R4=OCH3

OCH3

15

Fig. 3. Structures of the isolated compounds.

(Li, Lo, & Ho, 2006), and 3,5,6,7,8,30 ,40 -heptamethoxyflavone (14) (Li et al., 2006); one isoflavone, formononetin (15) (Schliemann, Ammer, & Strack, 2008). Each known compound was identified by NMR and MS and comparison with authentic samples or with published data. Compound 1 was obtained as a colourless amorphous powder. The ESIMS spectrum of 1 showed a quasi-molecular ion peak at m/z 289 [M+H]+. The UV spectrum displayed absorption peaks at 282 and 286 nm. The IR spectrum suggested the presence of hydroxy (3334 and 1148 cm1) and aromatic moieties (1623 cm1). 1H and 13C NMR spectra showed three ABX-type signals at dH 3.51 (dd, J = 14.5, 7.5), 3.53 (dd, J = 14.5, 6.0), and 5.46 (m), dC 40.9 (t) and 80.1 (d); dH 6.29 (d, J = 2.4), 6.48 (d, J = 8.4, 2.4) and 7.28 (d, J = 8.4), dC 97.5 (d), 110.7 (d) and 133.2 (d); dH 6.37 (d, J = 2.4), 6.43 (dd, J = 8.4, 2.4), and 7.16 (d, J = 8.4), dC 126.0 (d), 104.0 (d), and 107.3 (d), a methoxy group (dH 3.73; dC (55.9 (q)), and an oxymethine proton [dH 4.21 (br d, J = 6.6); dC (67.5 (d)]. The H–H COSY spectrum showed a spin–spin coupling partner sequence H-9/H-8/ H-7. The connectivities of the A-ring and C-ring, and C-ring and Bring were determined by key HMBC correlations at H-7/C-2 and C8, H-4 & H-8/C-2, H-30 /C-20 & C-40 , H-30 /C-20 & C-40 , H-5 & 40 -OCH3/ C-40 , H-5/C-3 (Fig. 4). Further structural confirmation was obtained from the NOESY spectrum, where correlations were seen of H-9 with H-7, H-8 and H-60 , and of methoxyl with H-30 and H-50 (Fig. 4). From the above results, the structure of the compound was assigned as 20 ,6-dihydroxy-40 -methoxydihydroauronol. The isolated compounds 1, 3, 5, 11, 12, 13 and 14 were tested for LPS-stimulated NO production in RAW264.7 at 12.5, 25 and 50 lM, respectively (Fig. 5). Results showed that compounds 5, 13 and 14 significantly inhibited NO production, but compound 14 revealed significant cytotoxicity at all of the above concentrations (data not shown). Various flavonoids and polymethoxyflavones were reported to have a broad spectrum of biological activity, including cytotoxicity (Beutler et al., 1998; Li et al., 2009), inducing apoptosis in adipocytes (Sergeev, Li, Ho, Rawson, & Dushenkov, 2009), anti-inflammation (Li et al., 2009; Matsuda et al., 2003). As Table 1 shows,

OCH3 HO

O HO H

Fig. 4. Key HMBC (

H

OH

) and NOESY (M) correlations of compound 1.

Fig. 5. Effect of isolated compounds on NO production from LPS-stimulated RAW264.7 cells. Values are expressed as the means ± SD of three independent experiments, each with triplicates and statistically analysed using Student’s t-test. ⁄ p < 0.05, indicates a significant difference from the control. RAW264.7 cells were stimulated by 1 lg/ml of LPS for 24 h of incubation.

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high total phenolic content and total flavonoids were found in ABEEtOAc subfractions E–I; flavonoids were the major components in the subfractions and some of the isolated compounds showed significant anti-inflammatory effect (Fig. 5). Our results suggest that flavonoids in adlay bran partially, at least, contributed to its antiinflammatory effect. This is the first report of polymethoxyflavones being isolated from a cereal, and their various bioactivities mean that adlay can be used as a functional food. 3.5. LC/MS identified major flavonoids in the ABE-EtOAc Fifteen isolated compounds were analysed by LC/MS. In Fig. 6, the UV chromatogram and total ion current (TIC) of the LC/MS

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for the bioactive fraction are shown. Peaks were identified as 5,7dihydroxychromone (tR = 30.15 min, [M-H] m/z, 177), naringenin (tR = 35.77 min, [M-H] m/z, 271), homoeriodictyol (tR = 36.07 min, [M-H] m/z, 301), hesperetin (tR = 36.69 min, [M-H] m/z, 301), davidigenin (tR = 37.73 min, [M-H] m/z, 257), chrysoeriol (tR = 38.40 min, [M-H] m/z, 299), isoliquiritigenin (tR = 39.13 min, [M-H] m/z, 255), and formononetin (tR = 40.65 min, [M-H] m/z, 267), in a negative ion chromatogram of LC/MS (Fig. 6A). As shown in Fig. 6B, 20 ,6-dihydroxy-40 -methoxydihydroauronol (tR = 31.61 min, [M+H]+ m/z, 289), 5-hydroxy-7-methoxychromone (tR = 37.17 min, [M+H]+ m/z, 193), naringenin (tR = 35.60 min, [M+H]+ m/z, 273), 3,5,6,7,8,30 ,40 -heptamethoxyflavone (tR = 40.62 min, [M+H]+ m/z, 433), 3,40 ,5,7-tetramethoxyflavone (tR = 44.44 min, [M+Na]+ m/z, 365), liquiritigenin (tR = 48.08 min, [M+Na]+ m/z, 433), 3,30 ,40 ,5,7,-pentamethoxyflavone (tR = 40.65 min, [M+Na]+ m/z, 395), and tangeretin (tR = 50.63 min, [M+H]+ m/z, 395), were analysed in the positive ion mode. Therefore, LC/MS analysis can be used as a quality-control platform. 4. Conclusions LPS-stimulated production of inflammatory mediators, in both RAW 264.7 cells and murine peritoneal macrophages, was used as a screening platform. iNOS and cytokine productions of IL-6 and TNF-a were targeted molecules, to examine the anti-inflammatory effects of adlay bran extract and the bioactive subfractions. The present results showed that the higher-polarity subfractions of the ABE-EtOAc potently inhibited NO synthesis (Fig. 1A). LPS stimulated both IL-6 and TNF-a secretions in RAW264.7 cells and peritoneal macrophages (Fig. 2) with no direct cytotoxicity at the treated concentrations. High levels of total phenolic contents and flavonoids in the anti-inflammatory subfractions were determined. The ratio of total flavonoids and phenolic contents in the bioactive ABE-EtOAc subfractions ranged from 0.60 to 0.77. Using column chromatography, 15 compounds were isolated and identified from the active fraction. Among them, 13 compounds were elucidated as flavonoids. In addition to naringenin (6), all of the other compounds are reported as being isolated from adlay bran for the first time, and compound 1 was identified as a novel compound. Compounds 1, 3, 5, 11, 12, 13 and 14 were tested for inhibition of NO production in RAW264.7 cells. Among them, 5, 13 and 14 significantly inhibited NO production at 50 lM (Fig. 5). These results showed that flavonoids were the major components of the ABEEtOAc responsible for the anti-inflammatory activities. An LC/MS analysis, to determine the flavonoids, was developed in this study. In a negative ion chromatogram of LC/MS (Fig. 6A), eight flavonoids were detected even though those components had very close retention times in HPLC. Isomers, such as homoeriodictyol (7) and hesperetin (8), with same mass spectrum (m/z = 301), could be successfully separated by HPLC. Furthermore, the polymethoxyflavonoes were detected by positive ion chromatograms (Fig. 6B). The results showed that the LC/MS analysis was useful for quality control of flavonoids. This is the first investigation of the anti-inflammation activity and chemical components of adlay bran. Our results suggest that flavonoids in adlay bran contributed, at least partially, to its antiinflammatory effect. Thus, adlay bran may be a source of natural inflammatory inhibitors, and may be beneficial to the health of consumers.

Fig. 6. HPLC and LC/MS chromatography (A) total negative ion currents and (B) total positive ion currents of flavonoid-enriched fractions by HPLC/MS. (1) 20 ,6dihydroxy-40 -methoxydihydroauronol, (2) 5,7-dihydroxychromone, (3) 5-hydroxy7-methoxychromone, (4) davidigenin, (5) isoliquiritigenin, (6) naringenin, (7) homoeriodictyol, (8) hesperetin, (9) liquiritigenin, (10) chrysoeriol, (11) 3,5,7,40 tetramethoxyflavone, (12) 3,5,7,30 ,40 -pentamethoxyflavone, (13) tangeretin, (14) 3,5,6,7,8,30 ,40 -heptamethoxyflavone, and (15) formononetin.

Acknowledgements The authors gratefully acknowledge the financial support from the National Science Council, Taiwan. We also thank Dr. Ming Jaw Don, a Research Fellow of the National Research Institute of

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