Arch. Pharm. Res. DOI 10.1007/s12272-014-0387-4
RESEARCH ARTICLE
Lignan and flavonoids from the stems of Zea mays and their antiinflammatory and neuroprotective activities Ye-Jin Jung • Ji-Hae Park • Jin-Gyeong Cho • Kyeong-Hwa Seo • Dong-Sung Lee • Youn-Chul Kim • Hee-Cheol Kang • Myoung-Chong Song Nam-In Baek
•
Received: 8 December 2013 / Accepted: 2 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract The stems of Zea mays L., otherwise known as cornstalks, were extracted with 80 % aqueous MeOH, and the concentrated extract was successively partitioned with ethyl acetate (EtOAc), normal butanol, and water. From the EtOAc fraction, a new lignan along with three known flavonoids, tricin (1), salcolin A (2), and salcolin B (3), were isolated. The chemical structure of the lignan was determined to be tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-one (4) through spectroscopic data analyses including NMR, MS, and IR. All compounds were isolated for the first time from this plant. The isolated compounds were evaluated for their inhibitory activity against NO production in Lipopolysaccharideinduced RAW 264.7 cells and their protective activity in glutamate-induced cell death in HT22 cells. The compounds 1, 2 and 4 showed anti-inflammatory effects with IC50 values of 2.63, 14.65, and 18.91 lM, respectively, as well as neuroprotective effects with EC50 values of 25.14, 47.44, and [80 lM, respectively.
Y.-J. Jung J.-H. Park J.-G. Cho K.-H. Seo N.-I. Baek (&) Graduate School of Biotechnology and Oriental Medicinal Materials and Processing, Kyung Hee University, Yongin 446-701, Republic of Korea e-mail: [email protected] D.-S. Lee Y.-C. Kim College of Pharmacy, Wonkwang University, Iksan 570-749, Republic of Korea H.-C. Kang R&D Center, GFC Co., Ltd, Suwon 443-813, Republic of Korea M.-C. Song Intelligent Synthetic Biology Center, KAIST, Daejeon 305-701, Republic of Korea
Keywords Anti-inflammation Cornstalk HT22 Neuroprotection RAW 264.7 Tetrahydro-4,6-bis(4hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1one Zea mays
Introduction Zea mays L. (Gramineae), or maize, is a widely cultivated crop in most tropical regions of the world. In 2012, 860 million metric tons of corn was produced in the world (World Agricultural Production 2013), and 83,210 and 73,612 metric tons of corn were produced in the Republic of Korea in 2012 and 2011, respectively (Agricultural Production [Wheat, Maize], 2012). About 110,000 metric tons of agricultural waste due to cornstalks were generated in the Republic of Korea in 2011 (Park et al. 2011; Choi et al. 2012). The total amount of cornstalks generated in the world is expected to be more than 12 billion metric tons. Most parts of the maize plant are extensively used in several varieties of food products, livestock and poultry feed, and cosmetic materials. In addition, their antioxidant and anti-inflammatory activities, as well as their role in the inhibition of melanogenesis have also been reported (Wang et al. 2012; Ebrahimzadeh et al. 2008; Kim et al. 2009). Chemical compounds isolated from corn silk (the style of maize) include terpenoids, steroids, saccharides, cerebrosides and flavonoid glycosides such as chrysoeriol 7-O-bglucopyranoside and chrysoeriol-6-C-b-L-boivinopyranoside (Suzuki et al. 2003; Ren and Ding, 2007; Liu et al. 2011). Phenolic acids, flavonoids and steroids have been isolated from corn leaves (Liu et al. 2012), feruloylated saccharides and hydroxycinnamic acid derivatives have been isolated from corn bran (Allerdings et al. 2006; Kim
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et al. 2009), and flavonoids have been isolated from corn bract (Wang et al. 2010; Zhang et al. 2011). However, there have been no studies on the stems of Z. mays L. (cornstalks), with the exception of an analysis on the cellulose in cornstalks (Gramera and Whistler 1963). Thus, this study was initiated to investigate the bioactive compounds in cornstalk. This study reports the isolation of a flavonoid, tricin (1), two flavonolignans, salcolin A (2) and salcolin B (3), and a new lignan, neo-olivilic acid (4), as well as their anti-inflammatory and neuroprotective properties. These compounds were isolated for the first time from the stems of Z. mays L. and showed an inhibitory effect on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophage cells and a protective effect on glutamate-induced cell death in HT22 cells.
Materials and methods
DMSO, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from SigmaAldrich Co. Ltd (St. Louis, MO, USA). 96-Well tissue culture plates and other tissue culture dishes were purchased from Falcon (Becton–Dickinson, Oxnard, CA, USA). All other chemicals and reagents were of analytical grade and were obtained from Sigma-Aldrich, unless indicated otherwise.
Plant materials The dried stems of Z. mays L. were supplied by GFC Co., Ltd, Suwon, Korea in June 2011 and were identified by Professor Dae-Keun Kim, Woosuk University, Jeonju, Korea. A voucher specimen (KHU2011-0620) is reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
General experimental procedures Extraction and isolation The silica gel (SiO2), octadecyl SiO2 (ODS), Waters ODS, and Sephadex LH-20 resin used for column chromatography (c.c.) were Kiesel gel 60 (63–200 lm, Merck, Darmstadt, Germany), Lichroprep RP-18 (40–60 lm, ˚ (55–105 lm, Waters, Milford, Merck), Prep C18 125 A MA, USA), and SephadexTM LH-20 (GE Healthcare BioSciences, Piscataway, NJ, USA), respectively. Flash c.c. was carried out using the SNAPÒ Cartridge KP-Sil (Biotage, Uppsala, Sweden). The thin layer chromatography (TLC) analysis was carried out using a Kiesel gel 60 F254 and a RP-18 F254S plates (Merck), and detection was performed by a UV lamp (Spectroline Model ENF-240 C/F, Spectronics Corporation, Westbury, NY, USA) and 10 % H2SO4 solution by spraying and heating. NMR spectra were recorded on a 400 MHz FT-NMR spectrometer (Varian Inova AS 400, Palo Alto, CA, USA), and deutrium solvents for NMR were purchased from Merck Co. Ltd Melting points were measured using a Fisher-John’s Melting Point Apparatus (Fisher Scientific, Miami, FL, USA) with a microscope. Optical rotations were measured from a JASCO P-1010 digital polarimeter (Tokyo, Japan) and IR spectra were obtained from a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). FAB-MS was recorded on a JEOL JMS-700 spectrometer (Tokyo, Japan), EI-MS was recorded on a JEOL JMSAX 505-WA spectrometer (Tokyo, Japan), and ESI–MS was recorded on a Finnigan LCQ Advantage Spectrometer (Thermo Scientific, Waltham, MD, USA). Roswell Park Memorial Institute (RPMI) medium 1640, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). LPS,
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The dried and chopped stems of Z. mays (8 kg) were extracted with 80 % aqueous MeOH (80 L 9 3) at room temperature for 24 h. The concentrated MeOH extracts (2 kg) were suspended in H2O (0.8 L) and then extracted with ethyl acetate (EtOAc, 0.8 L 9 4). Immediately following, H2O (0.6 L) was added to the H2O fraction and then extracted with n-butanol (n-BuOH, 1.0 L 9 4). The extracts were concentrated to produce the residues of the EtOAc fraction (CSE, 102 g), the n-BuOH fraction (CSB, 83 g), and the H2O fraction (CSW, 1.8 kg), respectively. The EtOAc fraction was applied to a SiO2 c.c. (12.5 cm 9 12 cm) and eluted with n-hexane–EtOAc (2:1 ? 1:1, 75 L of each), CHCl3–MeOH (10:1, 20 L), and CHCl3–MeOH–H2O (10:3:1, 8 L). The eluting solutions were monitored by TLC to produce 18 fractions (CSE-1–CSE-18). CSE-15 (1.59 g, elution volume/total volume [Ve/Vt] 0.89–0.90) was subjected to the SiO2 c.c. (4.5 9 13 cm) eluted with CHCl3–MeOH (30:1 ? 15:1, 4.9 L of each) resulting in 16 fractions (CSE-15-1 to CSE15-16). CSE-15-6 (233 mg, Ve/Vt 0.07-0.10) was subjected to an ODS c.c. (3 9 7 cm) eluted with MeOH-H2O (2:1, 1.3 L) to produce eight fractions (CSE-15-6-1 to CSE-15-6-8). CSE-15-6-4 (56 mg, Ve/Vt 0.09–0.28) was applied to the ODS c.c. (2.5 9 6 cm) eluted with MeOHH2O (3:2, 0.9 L) yielding four fractions (CSE-15-6-4-1 to CSE-15-6-4-4). CSE-15-6-4-2 (52 mg, Ve/Vt 0.11–0.48) was applied to a Sephadex LH-20 c.c. (1.5 9 35 cm) eluted with MeOH-H2O (4:1, 0.3 L) yielding seven fractions (CSE-15-6-4-2-1 to CSE-15-6-4-2-7) along with purified compound 1 (CSE-15-6-4-2-5, 4 mg, Ve/Vt 0.460.60, TLC [ODS F254S] Rf 0.40, MeOH–H2O = 4:1). CSE-
Lignan and flavonoids from the stems of Zea mays
15-9 (218 mg, Ve/Vt 0.12–0.16) was applied to the Sephadex LH-20 c.c. (1.5 9 65 cm) eluted with MeOHH2O (4:1, 0.6 L) to produce 16 fractions (CSE-15-9-1 to CSE-15-9-16). CSE-15-9-6 (21 mg, Ve/Vt 0.25-0.31) was applied to the ODS c.c. (1 9 5 cm) eluted with MeOHH2O (2:1, 0.13 L), yielding ten fractions (CSE-15-9-6-1 to CSE-15-9-6-10) along with purified compound 2 (CSE-159-6-3, 4 mg, Ve/Vt 0.09–0.13, TLC [ODS F254S] Rf 0.47, MeOH-H2O = 3:1). CSE-16 (9.74 g, Ve/Vt 0.90–0.93) was applied to the SiO2 flash c.c. (SNAP cartridge KP-Sil, 100 g) washed with EtOAc and then was eluted with CHCl3–MeOH (30:1 ? 20:1 ? 12:1, 3.9 L of each) and EtOH–MeOH (4:1, 1.5 L), resulting in eight fractions (CSE-16-1 to CSE-16-8). CSE-16-5 (3.56 g, Ve/Vt 0.48–0.61) was subjected to the SiO2 c.c. (4 9 12 cm) eluted with CH2Cl2–MeOH (25:1, 1.8 L) to give five fractions (CSE-16-5-1 to CSE-16-5-5), and CSE-16-5-2 (2.15 g, Ve/Vt 0.02–0.08) was applied to the Sephadex LH-20 c.c. (2.5 9 55 cm) eluted with MeOH–H2O (4:1, 1.2 L) to produce 14 fractions (CSE-16-5-2-1 to CSE-16-5-2-14). CSE16-5-2-8 (384 mg, Ve/Vt 0.24–0.32) was subjected to the Waters ODS c.c. (3.5 9 9 cm) eluted with MeOH-H2O (3:2 ? 2:1 ? 3:1, 0.8 L of each) to yield 16 fractions (CSE16-5-2-8-1 to CSE-16-5-2-8-16). CSE-16-5-2-8-5 (25 mg, Ve/Vt 0.07–0.10) was applied to the SiO2 c.c. (2.5 9 12 cm) eluted with CH2Cl2–MeOH (40:1 ? 20:1 ? 10:1, 0.4 L of each), yielding 11 fractions (CSE-16-5-2-8-5-1 to CSE-16-52-8-5-11) along with purified compound 3 (CSE-16-5-2-8-58, 4 mg, Ve/Vt 0.36-0.39, TLC [ODS F254S] Rf 0.51, MeOH– H2O = 3:1). CSE-10 (3.33 g, Ve/Vt 0.32–0.37) was subjected to the SiO2 c.c. (4 9 12 cm) eluted with n-hexaneCHCl3–MeOH (15:1:1, 3.2 L), resulting in four fractions (CSE-10-1 to CSE-10-4). CSE-10-3 (2.20 g, Ve/Vt 0.28–0.87) was applied to the Waters ODS c.c. (4 9 10 cm) eluted with MeOH-H2O (1:1 ? 4:1 ? 5:1, 4.0 L of each), yielding 15 fractions (CSE-10-3-1 to CSE-10-3-15), and CSE10-3-4 (54 mg, Ve/Vt 0.02–0.04) was also subjected to the Waters ODS c.c. (2.5 9 8 cm) eluted with MeOH–H2O (2:3, 0.5 L) to give 11 fractions (CSE-10-3-4-1 to CSE-10-3-4-11). CSE-10-3-4-8 (27 mg, Ve/Vt 0.29–0.50) was applied to the SiO2 c.c. (2 9 12 cm) eluted with n-hexane–EtOAc (3:2, 6.9 L) to yield six fractions (CSE-10-3-4-8-1 to CSE-10-3-48-6) along with purified compound 4 (CSE-10-3-4-8-3, 10 mg, Ve/Vt 0.44-0.71, TLC [ODS F254S] Rf 0.60, MeOH– H2O = 2:1).
Tricin (1) Pale yellow powder (CH3OH); negative FAB-MS m/z 329 [M-H]-; IR (CaF2 plate, m) 3,413, 2,941, 2,842, 1,664, 1,611 cm-1; 1H-NMR (400 MHz, pyridine-d5, dH) 7.42
(2H, s, H-20 , 60 ), 6.98 (1H, s, H-3), 6.85 (1H, d, J = 2.0 Hz, H-8), 6.74 (1H, d, J = 2.0 Hz, H-6), 3.87 (6H, s, 30 , 50 -OCH3); 13C-NMR (100 MHz, pyridine-d5, dC) 182.74 (C-4), 165.98 (C-7), 164.72 (C-2), 163.24 (C-5), 158.65 (C-9), 149.48 (C-30 , 50 ), 142.64 (C-40 ), 121.58 (C-10 ), 105.48 (C-20 , 60 ), 105.08 (C-10), 104.56 (C-3), 100.06 (C-6), 95.08 (C-8), 56.65 (C-30 , 50 -OCH3). Salcolin A (2) Yellow amorphous powder (CH3OH); ½a24 D ? 2.6° (c = 0.10, CH3OH); negative ESI–MS m/z 525 [M-H]-; IR (CaF2 plate, m) 3,369, 2,938, 1,652, 1,611, 1,588, 1,512, 1,495, 1,356, 1,263, 1,159, 1,124, 841 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 7.20 (2H, s, H-20 , 60 ), 7.02 (1H, d, J = 1.6 Hz, H-200 ), 6.87 (1H, dd, J = 8.0, 1.6 Hz, H-600 ), 6.74 (1H, d, J = 8.0 Hz, H-500 ), 6.67 (1H, s, H-3), 6.44 (1H, br s, H-8), 6.18 (1H, br s, H-6), 5.00 (1H, d, J = 6.4 Hz, H-700 ), 4.28 (1H, ddd, J = 6.4, 3.6, 3.2 Hz, H-800 ), 3.93 (6H, s, 30 , 50 -OCH3), 3.83 (3H, s, 300 -OCH3), 3.80 (1H, dd, J = 12.0, 3.6 Hz, H-900 a), 3.40 (1H, dd, J = 12.0, 3.2 Hz, H-900 b); 13C-NMR (100 MHz, CD3OD, dC) 183.72 (C-4), 166.62 (C-7), 165.05 (C-2), 163.21 (C5), 159.43 (C-9), 154.68 (C-30 , 50 ), 148.75 (C-300 ), 147.20 (C-400 ), 140.97 (C-40 ), 133.51 (C-100 ), 127.95 (C-10 ), 120.84 (C-600 ), 115.83 (C-500 ), 111.72 (C-200 ), 105.87 (C-3), 105.37 (C-10), 105.02 (C-20 , 60 ), 100.39 (C-6), 95.29 (C-8), 88.87 (C-800 ), 74.45 (C-700 ), 62.04 (C-900 ), 56.93 (C-30 , 50 -OCH3), 56.37 (C-300 -OCH3). Salcolin B (3) Yellow amorphous powder (CH3OH); ½a24 D - 5.0° (c = 0.10, CH3OH); negative ESI–MS m/z 525 [M-H]-; IR (CaF2 plate, m) 3,364, 2,933, 1,649, 1,607, 1,590, 1,495, 1,457, 1,358, 1,244, 1,166, 1,121, 832 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 7.20 (2H, s, H-20 , 60 ), 6.98 (1H, d, J = 1.6 Hz, H-200 ), 6.81 (1H, dd, J = 8.0, 1.6 Hz, H-600 ), 6.72 (1H, d, J = 8.0 Hz, H-500 ), 6.68 (1H, s, H-3), 6.44 (1H, br s, H-8), 6.18 (1H, br s, H-6), 4.90 (1H, d, J = 5.6 Hz, H-700 ), 4.44 (1H, ddd, J = 5.6, 5.2, 3.2 Hz, H-800 ), 3.94 (1H, dd, J = 12.0, 5.2 Hz, H-900 a), 3.90 (6H, s, 30 , 50 -OCH3), 3.82 (3H, s, 300 -OCH3), 3.67 (1H, dd, J = 12.0, 3.2 Hz, H-900 b); 13C-NMR (100 MHz, CD3OD, dC) 183.72 (C-4), 166.62 (C-7), 165.13 (C-2), 163.20 (C5), 159.42 (C-9), 154.82 (C-30 , 50 ), 148.64 (C-300 ), 146.96 (C-400 ), 140.54 (C-40 ), 133.79 (C-100 ), 127.79 (C-10 ), 120.85 (C-600 ), 115.65 (C-500 ), 111.55 (C-200 ), 105.78 (C-3), 105.35 (C-10), 105.02 (C-20 , 60 ), 100.37 (C-6), 95.29 (C-8), 87.50 (C-800 ), 74.23 (C-700 ), 61.93 (C-900 ), 56.89 (C-30 , 50 -OCH3), 56.36 (C-300 -OCH3).
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Tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3Hfuro[3,4-c]furan-1-one (4) ½a24 D
Pale brown amorphous powder (CH3OH); - 5.4° (c = 0.50, CH3OH); high resolution-EI-MS m/z 372.1201 [M]? (calcd. 372.1209, 2.3 ppm); IR (CaF2 plate, m) 3,407, 2,928, 2,843, 1,759, 1,602, 1,517, 1,462, 1,370, 1,274, 1,195, 1,032, 845 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 6.95 (1H, d, J = 2.0 Hz, H-20 ), 6.93 (1H, d, J = 2.0 Hz, H-2), 6.83 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.82 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.80 (1H, d, J = 8.0 Hz, H-5), 6.77 (1H, d, J = 8.4 Hz, H-50 ), 5.38 (1H, d, J = 4.0 Hz, H-7), 5.22 (1H, d, J = 4.0 Hz, H-70 ), 4.28 (1H, dd, J = 9.2, 6.8 Hz, H-9a), 4.01 (1H, dd, J = 9.2, 4.8 Hz, H-9b), 3.85 (3H, s, 30 -OCH3), 3.84 (3H, s, 3-OCH3), 3.65 (1H, dd, J = 9.6, 4.0 Hz, H-80 ), 3.29 (1H, m, H-8); 13C-NMR (100 MHz, CD3OD, dC) 179.67 (C-90 ), 149.36 (C-3), 149.16 (C-30 ), 148.19 (C-4), 147.45 (C-40 ), 133.20 (C-10 ), 132.41 (C-1), 119.78 (C-6), 119.50 (C-60 ), 116.43 (C-50 ), 116.16 (C-5), 110.66 (C-2), 110.60 (C-20 ), 87.15 (C-7), 85.07 (C-70 ), 73.79 (C-9), 56.50 (C-3-OCH3), 56.46 (C-30 OCH3), 54.44 (C-80 ), 50.99 (C-8). RAW 264.7 cell culture RAW 264.7 cells, a murine macrophage-like cell line, were cultured in DMEM medium supplemented with penicillin G (100 units/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), and 10 % heat-inactivated FBS in a humidified atmosphere with 5 % CO2 and 95 % air at 37 °C. Measurement of nitrite (NO production) NO production was determined by measuring the amount of NO2- (nitrite) in the cell culture. NO2- levels were determined by the method based on the Griess reaction, as previously described (Lee et al. 2004; Chun et al. 2012). To test the inhibitory effect of the compounds on NO production, RAW 264.7 cells were incubated in 96-well cell culture plates (2 9 104 cells/well) for 1 h, pretreated with various concentrations of test compounds (1–4; 10, 20, 40 lM) for 12 h, and then stimulated with 1 lg/mL LPS for an additional 18 h. Subsequently, an aliquot of each cell culture supernatant (100 lL) was mixed with 100 lL of Griess reagent (0.1 % [w/v] N-[1-naphathyl]ethylenediamine and 1 % [w/v] sulfanilamide in 2 % [v/v] phosphoric acid) in a new 96-well plate for 10 min at room temperature. The absorbance was measured at 550 nm and the NO2- concentration was determined by comparison to the standard curve generated by the sodium nitrite (NaNO2) concentration. Butein (10 lM), a flavonoid with inhibitory effects on NO production, was used as a positive control.
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HT22 cell culture Mouse hippocampal HT22 cells, a subclone of the HT4 hippocampal cell line, were obtained from Prof. InheeMook (Seoul National University, Seoul, Korea). The cells were maintained at 5 9 105 cells/mL in DMEM medium supplemented with penicillin G (100 Units/mL), streptomycin (100 mg/mL), L-glutamine (5 mM), and 10 % heatinactivated FBS in a humidified atmosphere with 5 % CO2 and 95 % air at 37 °C. Cytoprotective activity assay For the assessment of cytoprotective activity, the HT22 cells were cultured in 96-well plates (105 cells/mL) for 24 h and pretreated with either compounds (1–2, 4) or the positive control trolox for 4 h. Subsequently, the cells were exposed to glutamate (5 mM) for 12 h. Individual compounds were tested at concentrations of 10, 20, 40, and 80 lM and each experiment was performed in triplicate. Cell viability was evaluated using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, as previously described (Shrestha et al. 2012). Briefly, cells were incubated with 0.5 mg/mL MTT for 4 h at 37 °C, the medium was discarded, acidic isopropanol (0.04 N HCl) was added, and after incubating for 30 min, absorbance was measured at 590 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The resulting half maximal effective concentration (EC50) was expressed as the percentage of viability versus control. Trolox (50 lM) was used as a positive control. Statistical analysis Data values were expressed as the means ± SDs of three independent experiments. To compare the data from each group and treatment concentration, one-way analysis of variance (ANOVA) was used followed by the NewmanKeuls post hoc test. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software, Inc., San Diego, CA, USA). Results and discussion The stems of Z. mays were extracted in 80 % MeOH, and concentrated extracts were successively partitioned using EtOAc, n-BuOH, and H2O. Among the fractions, the EtOAc fraction was used for the isolation of metabolites through column chromatography to produce a flavonoid, two flavonolignans, and a lignan. A known flavonoid and two known flavonolignans were respectively identified as 40 ,5,7-trihydroxy-30 ,50 -dimethoxyflavone, tricin (1), tricin-40 -O-(threo-b-guaiacyl glyceryl) ether,
Lignan and flavonoids from the stems of Zea mays
salcolin A (2), and tricin-40 -O-(erythro-b-guaiacyl glyceryl) ether, salcolin B (3) based on the interpretation of spectroscopic data including NMR, MS, and IR, as well were confirmed by comparison of the data with those reported in literatures (Bouaziz et al. 2002; Jiao et al. 2007; Huang et al. 2010). All of them were isolated for the first time from cornstalks (Fig. 1). Compound 4 was isolated as a pale brown amorphous powder and showed an orange-brown color on a TLC plate by spraying with 10 % H2SO4 and then heating. The molecular weight was determined to be 372 from the molecular ion peak [M]? m/z 372 in the EI/MS spectrum, and the molecular formula was determined to be C20H20O7 from the peak [M]? m/z 372.1201 (calcd. 372.1209 for C20H20O7) in the HR-EI/MS. The IR absorbance bands of hydroxyl (3,407 cm-1), lactone ring (1,759 cm-1) and the aromatic groups (1,602 cm-1) were observed. In the 1HNMR spectrum, six olefin methine proton signals at dH 6.95 (1H, d, J = 2.0 Hz), 6.93 (1H, d, J = 2.0 Hz), 6.83 (1H, dd, J = 8.4, 2.0 Hz), 6.82 (1H, dd, J = 8.0, 2.0 Hz), 6.80 (1H, d, J = 8.0 Hz), and 6.77 (1H, d, J = 8.4 Hz) in the lower magnetic region indicated the presence of two 1,2,4-trisubstituted benzene ring moieties. In the oxygenated region, two oxygenated methine proton signals at dH 5.38 (1H, d, J = 4.0 Hz) and 5.22 (1H, d, J = 4.0 Hz) were observed and an oxygenated methylene proton signal was separately detected at dH 4.28 (1H, dd, J = 9.2, 6.8 Hz) and 4.01 (1H, dd, J = 9.2, 4.8 Hz), which showed germinal coupling with each other. In addition, two methoxy proton signals at dH 3.85 (3H, s) and 3.84 (3H, s) and two methine proton signals at dH 3.65 (1H, dd, J = 9.6, 4.0 Hz) and 3.29 (1H, m) were observed. From the 1H-NMR data, compound 4 was expected to be a furano lignan. The 13C-NMR spectrum showed 20 carbon signals including two methoxy carbon signals (dC 56.50, 56.46), confirming that compound 4 was a lignan. In the low magnetic field, a lactone carbonyl carbon signal (dC 179.67), four oxygenated olefin quaternary carbon signals (dC 149.36, 149.16, 148.19, 147.45), two olefin quaternary carbon signals (dC 133.20, 132.41), and six olefin methine carbon signals (dC 119.78, 119.50, 116.43, 116.16, 110.66, 110.60) were observed, predicting the presence of two benzene ring moieties. In the oxygenated region, two oxygenated methine carbon signals (dC 87.15, 85.07) and one oxygenated methylene carbon signal (dC 73.79) were detected, and two methine carbon signals (dC 54.44, 50.99) were observed in the high magnetic region. As a result, the structure of compound 4 was confirmed to be a lignan with a lactone moiety. In the gCOSY spectrum (Fig. 2), the oxygenated methylene proton signals (dH 4.28, H-9a; 4.01, H-9b) correlated with the methine proton signal (dH 3.29, H-8), which had a correlation with the oxygenated methine proton signal (dH 6.38, H-7), and the methine proton signal (dH 3.65, H-80 ). The H-80 signal showed a cross peak with
the oxygenated methine proton signal (dH 6.22, H-70 ). According to the above gCOSY data, compound 4 was considered to have a tetrahydrofuran unit in the molecular. In the gHMBC spectrum (Fig. 2), the H-7 signal correlated with the olefin quaternary carbon (dC 132.41, C-1), the olefin methine carbons (dC 119.78, C-6; 110.66, C-2), and the oxygenated methylene carbon (dC 73.79, C-9) signals, and the H-70 signal correlated with the lactone carbonyl carbon (dC 179.67, C-90 ), olefin quaternary carbon (dC 133.20, C-10 ), and the olefin methine carbon (dC 119.50, C-60 ; 110.60, C-20 ) signals. Both the H-9a and H-9b signals showed cross peaks with the oxygenated methine carbon (dC 87.15, C-7) and the methine carbon (dC 54.44, C-80 ) signals, and the H-80 signal showed correlation with the C-90 . As a result, this compound was identified to be a lignan, in which two guaiacyl moieties were combined at the C-7 and C-70 of tetrahydrofuran and tetrahydrofuran linked with five-membered lactone ring at C-8 and C-80 , respectively. The olefin methine proton signals (dH 6.95, H-20 ; 6.83, H-60 ) correlated with the oxygenated olefin quaternary carbon (dC 147.45, C-40 ) and the olefin methine proton signals (dH 6.93, H-2; 6.82, H-6) correlated with the oxygenated olefin quaternary carbon (dC 148.19, C-4), suggesting the position of C-4 and C-40 . The olefin methine proton signals (dH 6.80, H-5 and 6.77, H-50 ) showed cross peaks with the oxygenated olefin quaternary carbon (dC 149.36, C-3 and 149.16, C-30 ), suggesting that the positions of the oxygenated quaternary carbons position were C-3 and C-30 , respectively. From the correlation of the two methoxy proton signals (dH 3.85, 30 -OCH3; 3.84, 3-OCH3) with C-30 and the oxygenated olefin methine carbon (dC 149.36, C-3) signals, respectively, the two methoxy group were determined to be at C-30 and C-3, respectively. To determine the stereo structure of compound 4, the NOSEY experiment was accomplished. In the NOESY spectrum (Fig. 2), the H-7 signal showed cross peaks with H-8, and the H-70 signal showed a correlation with H-80 . There were no cross peaks between H-8 and H-80 , between H-7 and H-80 , between H-7 and H-70 , and between H-70 and H-8. Therefore, the arrangements of the four substituents on the tetrahydrofuran ring are cis–trans–cis configuration. Finally, based on the interpretation of the spectroscopic data and in comparison with those in the literatures (Lavie et al. 1974; Eklund and Sjo¨holm 2003), compound 4 was identified to be tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-one, which has been once synthesized by Eklund and Sjo¨holm (2003). It was isolated for the first time in nature. The compounds 1, 2, and 4 isolated from the Z. mays stems were evaluated for an inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells. Various concentrations of the compounds (10–40 lM) were used to treat the LPS-stimulated macrophages cells. A well-known
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Y.-J. Jung et al. Fig. 1 Chemical structures of compounds 1–4 isolated from the stems of Z. mays L.
Fig. 2 Key gHMBC, gCOSY, and NOESY correlations of compound 4
active chalcone, butein (10 lM), was used as a positive control. Figure 3 shows that three compounds inhibited LPS-induced NO production. The IC50 values of 1, 2, and 4 were determined to be 2.63, 14.65, and 18.91 lM, respectively (Table 1). Compound 1 showed similar inhibitory effects at all concentrations, while the inhibitory effects of 2 and 4 were dose-dependent. 40 lM of compounds 2 and 4 showed almost same activity as 10 lM of butein. Several previous studies have also reported that some flavonoids and flavonolignans including tricin (1), salcolin A (2), and salcolin B (3) have shown inhibitory effects on NO production in mouse peritoneal macrophages (Mohanlal et al. 2011) and on NO and PGE2 production in RAW 264.7 cells (Moscatelli et al. 2006). Jeong et al.
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Fig. 3 Anti-inflammatory effects of compounds 1, 2 and 4 on NO production in RAW 264.7 cells. Cells were pre-treated for 12 h with the indicated concentrations of the compounds, and stimulated 18 h with LPS (1 lg/mL). Each bar represents the mean ± S.D. of three independent experiments, *p \ 0.05. Butein (10 lM) was used as a positive control
Lignan and flavonoids from the stems of Zea mays Table 1 Inhibitory effects of compounds 1, 2 and 4 from the stems of Zea mays L. on NO production in RAW 264.7 cells and the protective effects against cell death in HT22 cells Compound
RAW 264.7 (IC50)a
HT22 (EC50)b
Butein
5.78
–
Trolox
–
15.8
1 2
2.63 14.65*
25.14* 47.44*
4
18.91*
[80
a
IC50 value of each compound was defined as the concentration (lM) that caused 50 % inhibition of NO production in LPS-induced RAW 264.7 cells. *p \ 0.05 compared to the group treated with LPS b EC50 value of each compound was defined as the concentration (lM) that caused 50 % protection effect of cell death in glutamatedinduced HT22 cells. *p \ 0.05 compared to the group treated with glutamate
4 as well as compounds 1-3 can be useful as potential antiinflammatory agents. The continuing study of these compounds as therapeutic materials against some diseases caused by inflammation will be very valuable. Compounds 1, 2 and 4 were also evaluated for their effects on glutamate-induced cell death in HT-22 cells. Compound 1 showed considerable cell viability (93.2 %, data not shown) and a protective effect (90.5 %) against glutamate-induced cell death at concentrations at 80 lM while trolox, the positive control, showed 81.8 % protection effect at 50 lM (Fig. 4). Compounds 1 and 2 showed a dose-dependent effect, and their EC50 values were determined to be 25.14 and 47.44 lM, respectively (Table 1). Previously, compound 1 was reported to have protective effects in PC12 cells against Ab-induced toxicity with an ED50 of 20.3 lM (Na et al. 2010). These results suggest that compounds 1 and 2 can be useful as potential neuroprotective agents. The continuing study of these compounds as therapeutic materials with neuroprotective activity will be very valuable. In conclusion, this study was initiated to search for new active compounds from the stems of Z. mays L. One new lignan and three known flavonoids were isolated through repeated column chromatography such as SiO2, ODS, and Sephadex LH-20, and identified on the basis of spectroscopic data analysis of NMR, IR, UV and MS. Compounds 1–2 and 4 showed inhibitory activity on NO production in LPS-stimulated RAW 264.7 cells and compounds 1 and 2 showed protective activity in glutamate induced oxidative injury in HT22 cells. Therefore, these results show the possibility for the biomedical use of these compounds and the extracts as anti-inflammatory or neuroprotective agents. Further studies are needed to determine the mechanisms and structure–activity relationships involved in the effects of each isolated compound. Acknowledgments This work was supported by the Next Generation Bio-Green 21 Program (PJ009574) from the Rural Development Administration, Republic of Korea.
References
Fig. 4 The neuroprotective effects of compounds 1, 2 and 4 on glutamate-induced cell death in HT22 cells. The exposure of HT22 cells to 5 mM glutamate for 12 h increased reactive oxygen species production. Each bar represents the mean ± S.D. of three independent experiments, *p \ 0.05. Trolox (50 lM) was used as a positive control
(2011) reported the inhibitory activity of compound 3 and another flavonolignan from the aerial parts of Oryza sativa L. on NO production. These results suggest that compound
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RESEARCH ARTICLE
Lignan and flavonoids from the stems of Zea mays and their antiinflammatory and neuroprotective activities Ye-Jin Jung • Ji-Hae Park • Jin-Gyeong Cho • Kyeong-Hwa Seo • Dong-Sung Lee • Youn-Chul Kim • Hee-Cheol Kang • Myoung-Chong Song Nam-In Baek
•
Received: 8 December 2013 / Accepted: 2 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract The stems of Zea mays L., otherwise known as cornstalks, were extracted with 80 % aqueous MeOH, and the concentrated extract was successively partitioned with ethyl acetate (EtOAc), normal butanol, and water. From the EtOAc fraction, a new lignan along with three known flavonoids, tricin (1), salcolin A (2), and salcolin B (3), were isolated. The chemical structure of the lignan was determined to be tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-one (4) through spectroscopic data analyses including NMR, MS, and IR. All compounds were isolated for the first time from this plant. The isolated compounds were evaluated for their inhibitory activity against NO production in Lipopolysaccharideinduced RAW 264.7 cells and their protective activity in glutamate-induced cell death in HT22 cells. The compounds 1, 2 and 4 showed anti-inflammatory effects with IC50 values of 2.63, 14.65, and 18.91 lM, respectively, as well as neuroprotective effects with EC50 values of 25.14, 47.44, and [80 lM, respectively.
Y.-J. Jung J.-H. Park J.-G. Cho K.-H. Seo N.-I. Baek (&) Graduate School of Biotechnology and Oriental Medicinal Materials and Processing, Kyung Hee University, Yongin 446-701, Republic of Korea e-mail: [email protected] D.-S. Lee Y.-C. Kim College of Pharmacy, Wonkwang University, Iksan 570-749, Republic of Korea H.-C. Kang R&D Center, GFC Co., Ltd, Suwon 443-813, Republic of Korea M.-C. Song Intelligent Synthetic Biology Center, KAIST, Daejeon 305-701, Republic of Korea
Keywords Anti-inflammation Cornstalk HT22 Neuroprotection RAW 264.7 Tetrahydro-4,6-bis(4hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1one Zea mays
Introduction Zea mays L. (Gramineae), or maize, is a widely cultivated crop in most tropical regions of the world. In 2012, 860 million metric tons of corn was produced in the world (World Agricultural Production 2013), and 83,210 and 73,612 metric tons of corn were produced in the Republic of Korea in 2012 and 2011, respectively (Agricultural Production [Wheat, Maize], 2012). About 110,000 metric tons of agricultural waste due to cornstalks were generated in the Republic of Korea in 2011 (Park et al. 2011; Choi et al. 2012). The total amount of cornstalks generated in the world is expected to be more than 12 billion metric tons. Most parts of the maize plant are extensively used in several varieties of food products, livestock and poultry feed, and cosmetic materials. In addition, their antioxidant and anti-inflammatory activities, as well as their role in the inhibition of melanogenesis have also been reported (Wang et al. 2012; Ebrahimzadeh et al. 2008; Kim et al. 2009). Chemical compounds isolated from corn silk (the style of maize) include terpenoids, steroids, saccharides, cerebrosides and flavonoid glycosides such as chrysoeriol 7-O-bglucopyranoside and chrysoeriol-6-C-b-L-boivinopyranoside (Suzuki et al. 2003; Ren and Ding, 2007; Liu et al. 2011). Phenolic acids, flavonoids and steroids have been isolated from corn leaves (Liu et al. 2012), feruloylated saccharides and hydroxycinnamic acid derivatives have been isolated from corn bran (Allerdings et al. 2006; Kim
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et al. 2009), and flavonoids have been isolated from corn bract (Wang et al. 2010; Zhang et al. 2011). However, there have been no studies on the stems of Z. mays L. (cornstalks), with the exception of an analysis on the cellulose in cornstalks (Gramera and Whistler 1963). Thus, this study was initiated to investigate the bioactive compounds in cornstalk. This study reports the isolation of a flavonoid, tricin (1), two flavonolignans, salcolin A (2) and salcolin B (3), and a new lignan, neo-olivilic acid (4), as well as their anti-inflammatory and neuroprotective properties. These compounds were isolated for the first time from the stems of Z. mays L. and showed an inhibitory effect on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophage cells and a protective effect on glutamate-induced cell death in HT22 cells.
Materials and methods
DMSO, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from SigmaAldrich Co. Ltd (St. Louis, MO, USA). 96-Well tissue culture plates and other tissue culture dishes were purchased from Falcon (Becton–Dickinson, Oxnard, CA, USA). All other chemicals and reagents were of analytical grade and were obtained from Sigma-Aldrich, unless indicated otherwise.
Plant materials The dried stems of Z. mays L. were supplied by GFC Co., Ltd, Suwon, Korea in June 2011 and were identified by Professor Dae-Keun Kim, Woosuk University, Jeonju, Korea. A voucher specimen (KHU2011-0620) is reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
General experimental procedures Extraction and isolation The silica gel (SiO2), octadecyl SiO2 (ODS), Waters ODS, and Sephadex LH-20 resin used for column chromatography (c.c.) were Kiesel gel 60 (63–200 lm, Merck, Darmstadt, Germany), Lichroprep RP-18 (40–60 lm, ˚ (55–105 lm, Waters, Milford, Merck), Prep C18 125 A MA, USA), and SephadexTM LH-20 (GE Healthcare BioSciences, Piscataway, NJ, USA), respectively. Flash c.c. was carried out using the SNAPÒ Cartridge KP-Sil (Biotage, Uppsala, Sweden). The thin layer chromatography (TLC) analysis was carried out using a Kiesel gel 60 F254 and a RP-18 F254S plates (Merck), and detection was performed by a UV lamp (Spectroline Model ENF-240 C/F, Spectronics Corporation, Westbury, NY, USA) and 10 % H2SO4 solution by spraying and heating. NMR spectra were recorded on a 400 MHz FT-NMR spectrometer (Varian Inova AS 400, Palo Alto, CA, USA), and deutrium solvents for NMR were purchased from Merck Co. Ltd Melting points were measured using a Fisher-John’s Melting Point Apparatus (Fisher Scientific, Miami, FL, USA) with a microscope. Optical rotations were measured from a JASCO P-1010 digital polarimeter (Tokyo, Japan) and IR spectra were obtained from a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). FAB-MS was recorded on a JEOL JMS-700 spectrometer (Tokyo, Japan), EI-MS was recorded on a JEOL JMSAX 505-WA spectrometer (Tokyo, Japan), and ESI–MS was recorded on a Finnigan LCQ Advantage Spectrometer (Thermo Scientific, Waltham, MD, USA). Roswell Park Memorial Institute (RPMI) medium 1640, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). LPS,
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The dried and chopped stems of Z. mays (8 kg) were extracted with 80 % aqueous MeOH (80 L 9 3) at room temperature for 24 h. The concentrated MeOH extracts (2 kg) were suspended in H2O (0.8 L) and then extracted with ethyl acetate (EtOAc, 0.8 L 9 4). Immediately following, H2O (0.6 L) was added to the H2O fraction and then extracted with n-butanol (n-BuOH, 1.0 L 9 4). The extracts were concentrated to produce the residues of the EtOAc fraction (CSE, 102 g), the n-BuOH fraction (CSB, 83 g), and the H2O fraction (CSW, 1.8 kg), respectively. The EtOAc fraction was applied to a SiO2 c.c. (12.5 cm 9 12 cm) and eluted with n-hexane–EtOAc (2:1 ? 1:1, 75 L of each), CHCl3–MeOH (10:1, 20 L), and CHCl3–MeOH–H2O (10:3:1, 8 L). The eluting solutions were monitored by TLC to produce 18 fractions (CSE-1–CSE-18). CSE-15 (1.59 g, elution volume/total volume [Ve/Vt] 0.89–0.90) was subjected to the SiO2 c.c. (4.5 9 13 cm) eluted with CHCl3–MeOH (30:1 ? 15:1, 4.9 L of each) resulting in 16 fractions (CSE-15-1 to CSE15-16). CSE-15-6 (233 mg, Ve/Vt 0.07-0.10) was subjected to an ODS c.c. (3 9 7 cm) eluted with MeOH-H2O (2:1, 1.3 L) to produce eight fractions (CSE-15-6-1 to CSE-15-6-8). CSE-15-6-4 (56 mg, Ve/Vt 0.09–0.28) was applied to the ODS c.c. (2.5 9 6 cm) eluted with MeOHH2O (3:2, 0.9 L) yielding four fractions (CSE-15-6-4-1 to CSE-15-6-4-4). CSE-15-6-4-2 (52 mg, Ve/Vt 0.11–0.48) was applied to a Sephadex LH-20 c.c. (1.5 9 35 cm) eluted with MeOH-H2O (4:1, 0.3 L) yielding seven fractions (CSE-15-6-4-2-1 to CSE-15-6-4-2-7) along with purified compound 1 (CSE-15-6-4-2-5, 4 mg, Ve/Vt 0.460.60, TLC [ODS F254S] Rf 0.40, MeOH–H2O = 4:1). CSE-
Lignan and flavonoids from the stems of Zea mays
15-9 (218 mg, Ve/Vt 0.12–0.16) was applied to the Sephadex LH-20 c.c. (1.5 9 65 cm) eluted with MeOHH2O (4:1, 0.6 L) to produce 16 fractions (CSE-15-9-1 to CSE-15-9-16). CSE-15-9-6 (21 mg, Ve/Vt 0.25-0.31) was applied to the ODS c.c. (1 9 5 cm) eluted with MeOHH2O (2:1, 0.13 L), yielding ten fractions (CSE-15-9-6-1 to CSE-15-9-6-10) along with purified compound 2 (CSE-159-6-3, 4 mg, Ve/Vt 0.09–0.13, TLC [ODS F254S] Rf 0.47, MeOH-H2O = 3:1). CSE-16 (9.74 g, Ve/Vt 0.90–0.93) was applied to the SiO2 flash c.c. (SNAP cartridge KP-Sil, 100 g) washed with EtOAc and then was eluted with CHCl3–MeOH (30:1 ? 20:1 ? 12:1, 3.9 L of each) and EtOH–MeOH (4:1, 1.5 L), resulting in eight fractions (CSE-16-1 to CSE-16-8). CSE-16-5 (3.56 g, Ve/Vt 0.48–0.61) was subjected to the SiO2 c.c. (4 9 12 cm) eluted with CH2Cl2–MeOH (25:1, 1.8 L) to give five fractions (CSE-16-5-1 to CSE-16-5-5), and CSE-16-5-2 (2.15 g, Ve/Vt 0.02–0.08) was applied to the Sephadex LH-20 c.c. (2.5 9 55 cm) eluted with MeOH–H2O (4:1, 1.2 L) to produce 14 fractions (CSE-16-5-2-1 to CSE-16-5-2-14). CSE16-5-2-8 (384 mg, Ve/Vt 0.24–0.32) was subjected to the Waters ODS c.c. (3.5 9 9 cm) eluted with MeOH-H2O (3:2 ? 2:1 ? 3:1, 0.8 L of each) to yield 16 fractions (CSE16-5-2-8-1 to CSE-16-5-2-8-16). CSE-16-5-2-8-5 (25 mg, Ve/Vt 0.07–0.10) was applied to the SiO2 c.c. (2.5 9 12 cm) eluted with CH2Cl2–MeOH (40:1 ? 20:1 ? 10:1, 0.4 L of each), yielding 11 fractions (CSE-16-5-2-8-5-1 to CSE-16-52-8-5-11) along with purified compound 3 (CSE-16-5-2-8-58, 4 mg, Ve/Vt 0.36-0.39, TLC [ODS F254S] Rf 0.51, MeOH– H2O = 3:1). CSE-10 (3.33 g, Ve/Vt 0.32–0.37) was subjected to the SiO2 c.c. (4 9 12 cm) eluted with n-hexaneCHCl3–MeOH (15:1:1, 3.2 L), resulting in four fractions (CSE-10-1 to CSE-10-4). CSE-10-3 (2.20 g, Ve/Vt 0.28–0.87) was applied to the Waters ODS c.c. (4 9 10 cm) eluted with MeOH-H2O (1:1 ? 4:1 ? 5:1, 4.0 L of each), yielding 15 fractions (CSE-10-3-1 to CSE-10-3-15), and CSE10-3-4 (54 mg, Ve/Vt 0.02–0.04) was also subjected to the Waters ODS c.c. (2.5 9 8 cm) eluted with MeOH–H2O (2:3, 0.5 L) to give 11 fractions (CSE-10-3-4-1 to CSE-10-3-4-11). CSE-10-3-4-8 (27 mg, Ve/Vt 0.29–0.50) was applied to the SiO2 c.c. (2 9 12 cm) eluted with n-hexane–EtOAc (3:2, 6.9 L) to yield six fractions (CSE-10-3-4-8-1 to CSE-10-3-48-6) along with purified compound 4 (CSE-10-3-4-8-3, 10 mg, Ve/Vt 0.44-0.71, TLC [ODS F254S] Rf 0.60, MeOH– H2O = 2:1).
Tricin (1) Pale yellow powder (CH3OH); negative FAB-MS m/z 329 [M-H]-; IR (CaF2 plate, m) 3,413, 2,941, 2,842, 1,664, 1,611 cm-1; 1H-NMR (400 MHz, pyridine-d5, dH) 7.42
(2H, s, H-20 , 60 ), 6.98 (1H, s, H-3), 6.85 (1H, d, J = 2.0 Hz, H-8), 6.74 (1H, d, J = 2.0 Hz, H-6), 3.87 (6H, s, 30 , 50 -OCH3); 13C-NMR (100 MHz, pyridine-d5, dC) 182.74 (C-4), 165.98 (C-7), 164.72 (C-2), 163.24 (C-5), 158.65 (C-9), 149.48 (C-30 , 50 ), 142.64 (C-40 ), 121.58 (C-10 ), 105.48 (C-20 , 60 ), 105.08 (C-10), 104.56 (C-3), 100.06 (C-6), 95.08 (C-8), 56.65 (C-30 , 50 -OCH3). Salcolin A (2) Yellow amorphous powder (CH3OH); ½a24 D ? 2.6° (c = 0.10, CH3OH); negative ESI–MS m/z 525 [M-H]-; IR (CaF2 plate, m) 3,369, 2,938, 1,652, 1,611, 1,588, 1,512, 1,495, 1,356, 1,263, 1,159, 1,124, 841 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 7.20 (2H, s, H-20 , 60 ), 7.02 (1H, d, J = 1.6 Hz, H-200 ), 6.87 (1H, dd, J = 8.0, 1.6 Hz, H-600 ), 6.74 (1H, d, J = 8.0 Hz, H-500 ), 6.67 (1H, s, H-3), 6.44 (1H, br s, H-8), 6.18 (1H, br s, H-6), 5.00 (1H, d, J = 6.4 Hz, H-700 ), 4.28 (1H, ddd, J = 6.4, 3.6, 3.2 Hz, H-800 ), 3.93 (6H, s, 30 , 50 -OCH3), 3.83 (3H, s, 300 -OCH3), 3.80 (1H, dd, J = 12.0, 3.6 Hz, H-900 a), 3.40 (1H, dd, J = 12.0, 3.2 Hz, H-900 b); 13C-NMR (100 MHz, CD3OD, dC) 183.72 (C-4), 166.62 (C-7), 165.05 (C-2), 163.21 (C5), 159.43 (C-9), 154.68 (C-30 , 50 ), 148.75 (C-300 ), 147.20 (C-400 ), 140.97 (C-40 ), 133.51 (C-100 ), 127.95 (C-10 ), 120.84 (C-600 ), 115.83 (C-500 ), 111.72 (C-200 ), 105.87 (C-3), 105.37 (C-10), 105.02 (C-20 , 60 ), 100.39 (C-6), 95.29 (C-8), 88.87 (C-800 ), 74.45 (C-700 ), 62.04 (C-900 ), 56.93 (C-30 , 50 -OCH3), 56.37 (C-300 -OCH3). Salcolin B (3) Yellow amorphous powder (CH3OH); ½a24 D - 5.0° (c = 0.10, CH3OH); negative ESI–MS m/z 525 [M-H]-; IR (CaF2 plate, m) 3,364, 2,933, 1,649, 1,607, 1,590, 1,495, 1,457, 1,358, 1,244, 1,166, 1,121, 832 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 7.20 (2H, s, H-20 , 60 ), 6.98 (1H, d, J = 1.6 Hz, H-200 ), 6.81 (1H, dd, J = 8.0, 1.6 Hz, H-600 ), 6.72 (1H, d, J = 8.0 Hz, H-500 ), 6.68 (1H, s, H-3), 6.44 (1H, br s, H-8), 6.18 (1H, br s, H-6), 4.90 (1H, d, J = 5.6 Hz, H-700 ), 4.44 (1H, ddd, J = 5.6, 5.2, 3.2 Hz, H-800 ), 3.94 (1H, dd, J = 12.0, 5.2 Hz, H-900 a), 3.90 (6H, s, 30 , 50 -OCH3), 3.82 (3H, s, 300 -OCH3), 3.67 (1H, dd, J = 12.0, 3.2 Hz, H-900 b); 13C-NMR (100 MHz, CD3OD, dC) 183.72 (C-4), 166.62 (C-7), 165.13 (C-2), 163.20 (C5), 159.42 (C-9), 154.82 (C-30 , 50 ), 148.64 (C-300 ), 146.96 (C-400 ), 140.54 (C-40 ), 133.79 (C-100 ), 127.79 (C-10 ), 120.85 (C-600 ), 115.65 (C-500 ), 111.55 (C-200 ), 105.78 (C-3), 105.35 (C-10), 105.02 (C-20 , 60 ), 100.37 (C-6), 95.29 (C-8), 87.50 (C-800 ), 74.23 (C-700 ), 61.93 (C-900 ), 56.89 (C-30 , 50 -OCH3), 56.36 (C-300 -OCH3).
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Y.-J. Jung et al.
Tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3Hfuro[3,4-c]furan-1-one (4) ½a24 D
Pale brown amorphous powder (CH3OH); - 5.4° (c = 0.50, CH3OH); high resolution-EI-MS m/z 372.1201 [M]? (calcd. 372.1209, 2.3 ppm); IR (CaF2 plate, m) 3,407, 2,928, 2,843, 1,759, 1,602, 1,517, 1,462, 1,370, 1,274, 1,195, 1,032, 845 cm-1; 1H-NMR (400 MHz, CD3OD, dH) 6.95 (1H, d, J = 2.0 Hz, H-20 ), 6.93 (1H, d, J = 2.0 Hz, H-2), 6.83 (1H, dd, J = 8.4, 2.0 Hz, H-60 ), 6.82 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.80 (1H, d, J = 8.0 Hz, H-5), 6.77 (1H, d, J = 8.4 Hz, H-50 ), 5.38 (1H, d, J = 4.0 Hz, H-7), 5.22 (1H, d, J = 4.0 Hz, H-70 ), 4.28 (1H, dd, J = 9.2, 6.8 Hz, H-9a), 4.01 (1H, dd, J = 9.2, 4.8 Hz, H-9b), 3.85 (3H, s, 30 -OCH3), 3.84 (3H, s, 3-OCH3), 3.65 (1H, dd, J = 9.6, 4.0 Hz, H-80 ), 3.29 (1H, m, H-8); 13C-NMR (100 MHz, CD3OD, dC) 179.67 (C-90 ), 149.36 (C-3), 149.16 (C-30 ), 148.19 (C-4), 147.45 (C-40 ), 133.20 (C-10 ), 132.41 (C-1), 119.78 (C-6), 119.50 (C-60 ), 116.43 (C-50 ), 116.16 (C-5), 110.66 (C-2), 110.60 (C-20 ), 87.15 (C-7), 85.07 (C-70 ), 73.79 (C-9), 56.50 (C-3-OCH3), 56.46 (C-30 OCH3), 54.44 (C-80 ), 50.99 (C-8). RAW 264.7 cell culture RAW 264.7 cells, a murine macrophage-like cell line, were cultured in DMEM medium supplemented with penicillin G (100 units/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), and 10 % heat-inactivated FBS in a humidified atmosphere with 5 % CO2 and 95 % air at 37 °C. Measurement of nitrite (NO production) NO production was determined by measuring the amount of NO2- (nitrite) in the cell culture. NO2- levels were determined by the method based on the Griess reaction, as previously described (Lee et al. 2004; Chun et al. 2012). To test the inhibitory effect of the compounds on NO production, RAW 264.7 cells were incubated in 96-well cell culture plates (2 9 104 cells/well) for 1 h, pretreated with various concentrations of test compounds (1–4; 10, 20, 40 lM) for 12 h, and then stimulated with 1 lg/mL LPS for an additional 18 h. Subsequently, an aliquot of each cell culture supernatant (100 lL) was mixed with 100 lL of Griess reagent (0.1 % [w/v] N-[1-naphathyl]ethylenediamine and 1 % [w/v] sulfanilamide in 2 % [v/v] phosphoric acid) in a new 96-well plate for 10 min at room temperature. The absorbance was measured at 550 nm and the NO2- concentration was determined by comparison to the standard curve generated by the sodium nitrite (NaNO2) concentration. Butein (10 lM), a flavonoid with inhibitory effects on NO production, was used as a positive control.
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HT22 cell culture Mouse hippocampal HT22 cells, a subclone of the HT4 hippocampal cell line, were obtained from Prof. InheeMook (Seoul National University, Seoul, Korea). The cells were maintained at 5 9 105 cells/mL in DMEM medium supplemented with penicillin G (100 Units/mL), streptomycin (100 mg/mL), L-glutamine (5 mM), and 10 % heatinactivated FBS in a humidified atmosphere with 5 % CO2 and 95 % air at 37 °C. Cytoprotective activity assay For the assessment of cytoprotective activity, the HT22 cells were cultured in 96-well plates (105 cells/mL) for 24 h and pretreated with either compounds (1–2, 4) or the positive control trolox for 4 h. Subsequently, the cells were exposed to glutamate (5 mM) for 12 h. Individual compounds were tested at concentrations of 10, 20, 40, and 80 lM and each experiment was performed in triplicate. Cell viability was evaluated using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, as previously described (Shrestha et al. 2012). Briefly, cells were incubated with 0.5 mg/mL MTT for 4 h at 37 °C, the medium was discarded, acidic isopropanol (0.04 N HCl) was added, and after incubating for 30 min, absorbance was measured at 590 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The resulting half maximal effective concentration (EC50) was expressed as the percentage of viability versus control. Trolox (50 lM) was used as a positive control. Statistical analysis Data values were expressed as the means ± SDs of three independent experiments. To compare the data from each group and treatment concentration, one-way analysis of variance (ANOVA) was used followed by the NewmanKeuls post hoc test. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software, Inc., San Diego, CA, USA). Results and discussion The stems of Z. mays were extracted in 80 % MeOH, and concentrated extracts were successively partitioned using EtOAc, n-BuOH, and H2O. Among the fractions, the EtOAc fraction was used for the isolation of metabolites through column chromatography to produce a flavonoid, two flavonolignans, and a lignan. A known flavonoid and two known flavonolignans were respectively identified as 40 ,5,7-trihydroxy-30 ,50 -dimethoxyflavone, tricin (1), tricin-40 -O-(threo-b-guaiacyl glyceryl) ether,
Lignan and flavonoids from the stems of Zea mays
salcolin A (2), and tricin-40 -O-(erythro-b-guaiacyl glyceryl) ether, salcolin B (3) based on the interpretation of spectroscopic data including NMR, MS, and IR, as well were confirmed by comparison of the data with those reported in literatures (Bouaziz et al. 2002; Jiao et al. 2007; Huang et al. 2010). All of them were isolated for the first time from cornstalks (Fig. 1). Compound 4 was isolated as a pale brown amorphous powder and showed an orange-brown color on a TLC plate by spraying with 10 % H2SO4 and then heating. The molecular weight was determined to be 372 from the molecular ion peak [M]? m/z 372 in the EI/MS spectrum, and the molecular formula was determined to be C20H20O7 from the peak [M]? m/z 372.1201 (calcd. 372.1209 for C20H20O7) in the HR-EI/MS. The IR absorbance bands of hydroxyl (3,407 cm-1), lactone ring (1,759 cm-1) and the aromatic groups (1,602 cm-1) were observed. In the 1HNMR spectrum, six olefin methine proton signals at dH 6.95 (1H, d, J = 2.0 Hz), 6.93 (1H, d, J = 2.0 Hz), 6.83 (1H, dd, J = 8.4, 2.0 Hz), 6.82 (1H, dd, J = 8.0, 2.0 Hz), 6.80 (1H, d, J = 8.0 Hz), and 6.77 (1H, d, J = 8.4 Hz) in the lower magnetic region indicated the presence of two 1,2,4-trisubstituted benzene ring moieties. In the oxygenated region, two oxygenated methine proton signals at dH 5.38 (1H, d, J = 4.0 Hz) and 5.22 (1H, d, J = 4.0 Hz) were observed and an oxygenated methylene proton signal was separately detected at dH 4.28 (1H, dd, J = 9.2, 6.8 Hz) and 4.01 (1H, dd, J = 9.2, 4.8 Hz), which showed germinal coupling with each other. In addition, two methoxy proton signals at dH 3.85 (3H, s) and 3.84 (3H, s) and two methine proton signals at dH 3.65 (1H, dd, J = 9.6, 4.0 Hz) and 3.29 (1H, m) were observed. From the 1H-NMR data, compound 4 was expected to be a furano lignan. The 13C-NMR spectrum showed 20 carbon signals including two methoxy carbon signals (dC 56.50, 56.46), confirming that compound 4 was a lignan. In the low magnetic field, a lactone carbonyl carbon signal (dC 179.67), four oxygenated olefin quaternary carbon signals (dC 149.36, 149.16, 148.19, 147.45), two olefin quaternary carbon signals (dC 133.20, 132.41), and six olefin methine carbon signals (dC 119.78, 119.50, 116.43, 116.16, 110.66, 110.60) were observed, predicting the presence of two benzene ring moieties. In the oxygenated region, two oxygenated methine carbon signals (dC 87.15, 85.07) and one oxygenated methylene carbon signal (dC 73.79) were detected, and two methine carbon signals (dC 54.44, 50.99) were observed in the high magnetic region. As a result, the structure of compound 4 was confirmed to be a lignan with a lactone moiety. In the gCOSY spectrum (Fig. 2), the oxygenated methylene proton signals (dH 4.28, H-9a; 4.01, H-9b) correlated with the methine proton signal (dH 3.29, H-8), which had a correlation with the oxygenated methine proton signal (dH 6.38, H-7), and the methine proton signal (dH 3.65, H-80 ). The H-80 signal showed a cross peak with
the oxygenated methine proton signal (dH 6.22, H-70 ). According to the above gCOSY data, compound 4 was considered to have a tetrahydrofuran unit in the molecular. In the gHMBC spectrum (Fig. 2), the H-7 signal correlated with the olefin quaternary carbon (dC 132.41, C-1), the olefin methine carbons (dC 119.78, C-6; 110.66, C-2), and the oxygenated methylene carbon (dC 73.79, C-9) signals, and the H-70 signal correlated with the lactone carbonyl carbon (dC 179.67, C-90 ), olefin quaternary carbon (dC 133.20, C-10 ), and the olefin methine carbon (dC 119.50, C-60 ; 110.60, C-20 ) signals. Both the H-9a and H-9b signals showed cross peaks with the oxygenated methine carbon (dC 87.15, C-7) and the methine carbon (dC 54.44, C-80 ) signals, and the H-80 signal showed correlation with the C-90 . As a result, this compound was identified to be a lignan, in which two guaiacyl moieties were combined at the C-7 and C-70 of tetrahydrofuran and tetrahydrofuran linked with five-membered lactone ring at C-8 and C-80 , respectively. The olefin methine proton signals (dH 6.95, H-20 ; 6.83, H-60 ) correlated with the oxygenated olefin quaternary carbon (dC 147.45, C-40 ) and the olefin methine proton signals (dH 6.93, H-2; 6.82, H-6) correlated with the oxygenated olefin quaternary carbon (dC 148.19, C-4), suggesting the position of C-4 and C-40 . The olefin methine proton signals (dH 6.80, H-5 and 6.77, H-50 ) showed cross peaks with the oxygenated olefin quaternary carbon (dC 149.36, C-3 and 149.16, C-30 ), suggesting that the positions of the oxygenated quaternary carbons position were C-3 and C-30 , respectively. From the correlation of the two methoxy proton signals (dH 3.85, 30 -OCH3; 3.84, 3-OCH3) with C-30 and the oxygenated olefin methine carbon (dC 149.36, C-3) signals, respectively, the two methoxy group were determined to be at C-30 and C-3, respectively. To determine the stereo structure of compound 4, the NOSEY experiment was accomplished. In the NOESY spectrum (Fig. 2), the H-7 signal showed cross peaks with H-8, and the H-70 signal showed a correlation with H-80 . There were no cross peaks between H-8 and H-80 , between H-7 and H-80 , between H-7 and H-70 , and between H-70 and H-8. Therefore, the arrangements of the four substituents on the tetrahydrofuran ring are cis–trans–cis configuration. Finally, based on the interpretation of the spectroscopic data and in comparison with those in the literatures (Lavie et al. 1974; Eklund and Sjo¨holm 2003), compound 4 was identified to be tetrahydro-4,6-bis(4-hydroxy-3-methoxyphenyl)-1H,3H-furo[3,4-c]furan-1-one, which has been once synthesized by Eklund and Sjo¨holm (2003). It was isolated for the first time in nature. The compounds 1, 2, and 4 isolated from the Z. mays stems were evaluated for an inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells. Various concentrations of the compounds (10–40 lM) were used to treat the LPS-stimulated macrophages cells. A well-known
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Y.-J. Jung et al. Fig. 1 Chemical structures of compounds 1–4 isolated from the stems of Z. mays L.
Fig. 2 Key gHMBC, gCOSY, and NOESY correlations of compound 4
active chalcone, butein (10 lM), was used as a positive control. Figure 3 shows that three compounds inhibited LPS-induced NO production. The IC50 values of 1, 2, and 4 were determined to be 2.63, 14.65, and 18.91 lM, respectively (Table 1). Compound 1 showed similar inhibitory effects at all concentrations, while the inhibitory effects of 2 and 4 were dose-dependent. 40 lM of compounds 2 and 4 showed almost same activity as 10 lM of butein. Several previous studies have also reported that some flavonoids and flavonolignans including tricin (1), salcolin A (2), and salcolin B (3) have shown inhibitory effects on NO production in mouse peritoneal macrophages (Mohanlal et al. 2011) and on NO and PGE2 production in RAW 264.7 cells (Moscatelli et al. 2006). Jeong et al.
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Fig. 3 Anti-inflammatory effects of compounds 1, 2 and 4 on NO production in RAW 264.7 cells. Cells were pre-treated for 12 h with the indicated concentrations of the compounds, and stimulated 18 h with LPS (1 lg/mL). Each bar represents the mean ± S.D. of three independent experiments, *p \ 0.05. Butein (10 lM) was used as a positive control
Lignan and flavonoids from the stems of Zea mays Table 1 Inhibitory effects of compounds 1, 2 and 4 from the stems of Zea mays L. on NO production in RAW 264.7 cells and the protective effects against cell death in HT22 cells Compound
RAW 264.7 (IC50)a
HT22 (EC50)b
Butein
5.78
–
Trolox
–
15.8
1 2
2.63 14.65*
25.14* 47.44*
4
18.91*
[80
a
IC50 value of each compound was defined as the concentration (lM) that caused 50 % inhibition of NO production in LPS-induced RAW 264.7 cells. *p \ 0.05 compared to the group treated with LPS b EC50 value of each compound was defined as the concentration (lM) that caused 50 % protection effect of cell death in glutamatedinduced HT22 cells. *p \ 0.05 compared to the group treated with glutamate
4 as well as compounds 1-3 can be useful as potential antiinflammatory agents. The continuing study of these compounds as therapeutic materials against some diseases caused by inflammation will be very valuable. Compounds 1, 2 and 4 were also evaluated for their effects on glutamate-induced cell death in HT-22 cells. Compound 1 showed considerable cell viability (93.2 %, data not shown) and a protective effect (90.5 %) against glutamate-induced cell death at concentrations at 80 lM while trolox, the positive control, showed 81.8 % protection effect at 50 lM (Fig. 4). Compounds 1 and 2 showed a dose-dependent effect, and their EC50 values were determined to be 25.14 and 47.44 lM, respectively (Table 1). Previously, compound 1 was reported to have protective effects in PC12 cells against Ab-induced toxicity with an ED50 of 20.3 lM (Na et al. 2010). These results suggest that compounds 1 and 2 can be useful as potential neuroprotective agents. The continuing study of these compounds as therapeutic materials with neuroprotective activity will be very valuable. In conclusion, this study was initiated to search for new active compounds from the stems of Z. mays L. One new lignan and three known flavonoids were isolated through repeated column chromatography such as SiO2, ODS, and Sephadex LH-20, and identified on the basis of spectroscopic data analysis of NMR, IR, UV and MS. Compounds 1–2 and 4 showed inhibitory activity on NO production in LPS-stimulated RAW 264.7 cells and compounds 1 and 2 showed protective activity in glutamate induced oxidative injury in HT22 cells. Therefore, these results show the possibility for the biomedical use of these compounds and the extracts as anti-inflammatory or neuroprotective agents. Further studies are needed to determine the mechanisms and structure–activity relationships involved in the effects of each isolated compound. Acknowledgments This work was supported by the Next Generation Bio-Green 21 Program (PJ009574) from the Rural Development Administration, Republic of Korea.
References
Fig. 4 The neuroprotective effects of compounds 1, 2 and 4 on glutamate-induced cell death in HT22 cells. The exposure of HT22 cells to 5 mM glutamate for 12 h increased reactive oxygen species production. Each bar represents the mean ± S.D. of three independent experiments, *p \ 0.05. Trolox (50 lM) was used as a positive control
(2011) reported the inhibitory activity of compound 3 and another flavonolignan from the aerial parts of Oryza sativa L. on NO production. These results suggest that compound
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