Article pubs.acs.org/JAFC
Hydrolyzed Methylhesperidin Induces Antioxidant Enzyme Expression via the Nrf2−ARE Pathway in Normal Human Epidermal Keratinocytes Tetsuya Kuwano, Manabu Watanabe, Daiji Kagawa, and Takatoshi Murase* Biological Science Research, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan ABSTRACT: Methylhesperidin (MHES) is a mixture of methylated derivatives of the citrus flavonoid hesperidin and is used as a food or pharmaceutical additive. Dietary MHES could be hydrolyzed by gut microflora to give aglycons. Therefore, we prepared hydrolyzed methylhesperidin (h-MHES) and assessed its pharmacological activity in human epidermal keratinocytes. h-MHES promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and the expression of cytoprotective genes (e.g., heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC)). h-MHES also increased intracellular glutathione levels and reduced UVB-induced reactive oxygen species. Moreover, h-MHES increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and a p38 MAPK inhibitor significantly attenuated h-MHES-induced HO-1 and GCLC expression. Furthermore, when we purified the components of h-MHES, we identified two methoxy-chalcones as novel Nrf2 activators. Our study demonstrates that h-MHES can induce cytoprotective gene expression and reduce oxidative stress via the Nrf2−ARE pathway in keratinocytes, suggesting that MHES may contribute to the suppression of UVB-induced skin damage in vivo. KEYWORDS: aglycon, keratinocyte, methylhesperidin, Nrf2, UVB
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INTRODUCTION Oxidative stress is defined as an imbalance between the production of free radicals and reactive metabolites, so-called oxidants, and their elimination by protective mechanisms, referred to as antioxidant systems.1 As the boundary between the body and the environment, the skin is chronically exposed to both endogenous and environmental pro-oxidant agents, leading to the generation of reactive oxygen species (ROS).2 ROS that are generated by aging, UV light, and cigarette smoke damage cells and organs; therefore, antioxidant systems are important to maintain cellular homeostasis. The skin antioxidant system consists of a network of enzymatic and nonenzymatic antioxidants.3 Among the enzymatic antioxidants, glutathione peroxidase, catalase, and superoxide dismutase play pivotal roles.4 Cellular nonenzymatic antioxidants include α-tocopherol, ascorbic acid, and glutathione.4 Nuclear factor erythroid 2-related factor 2 (Nrf2, also known as NFE2L2) functions as a master regulator of these antioxidant systems for cytoprotection in response to oxidative and electrophilic stresses.5 Under stress-free conditions, Nrf2 is degraded via the ubiquitin−proteasome pathway in a Kelch-like ECH-associated protein 1 (Keap1)-dependent manner. When the thiol group of Keap1 senses oxidative stress, Keap1 releases Nrf2. Nrf2 is then stabilized, translocates into nuclei, and binds to the antioxidant response element (ARE) site of various antioxidant genes.6 Thus, the Nrf2−ARE pathway regulates the expression of antioxidant genes, including heme oxygenase-1 (HO-1 also known as HMOX1), NAD(P)H:quinine oxidoreductase 1 (NQO1), glutathione peroxidase (GPX), glutamate cysteine ligase modifier subunit (GCLM), glutamate cysteine ligase catalytic subunit (GCLC), and thioredoxin (TXN).7 The Nrf2−ARE pathway thus contributes to cytoprotection against oxidative stress. Numerous studies have shown that Nrf2 © 2015 American Chemical Society
protects many cell types and organs from various toxic insults and disease pathogenesis (carcinogens, electrophiles, ROS, inflammation, UV light, and cigarette smoke).8 Methylhesperidin (MHES) is a mixture of methylated derivatives of hesperidin, a citrus flavonoid that is used as a food or pharmaceutical additive. MHES consists of several flavanones and chalcones that differ in terms of the numbers and positions of their methyl groups.9,10 Flavanones and chalcones show a wide range of pharmacological effects, including anti-inflammatory, antibacterial, and anticancer activities,11−14 which suggests that MHES would similarly have a range of pharmacological effects. However, to date, there have been no reports about the pharmacological activity of MHES. Dietary hesperidin is hydrolyzed by the β-glucosidase of microflora before it is absorbed into the small intestine to give hesperetin, the aglycon form of hesperidin.15 Here, we hydrolyzed MHES and assessed the pharmacological activity of hydrolyzed methylhesperidin (h-MHES) in human keratinocytes, with the goal of identifying dietary agents that can protect the skin against oxidative stress.
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MATERIALS AND METHODS
Materials. Preparation of h-MHES by Acid Hydrolysis. MHES was obtained from Alps Pharmaceutical Industry Co. Ltd. (Gifu, Japan). To a stirred solution of MHES (80 g) in 50% ethanol (4 L), 12 M HCl (240 mL) was added at room temperature, and the mixture was refluxed for 16 h. The mixture was then cooled to room temperature and neutralized with 4 M sodium hydroxide (690 mL). The mixture Received: Revised: Accepted: Published: 7937
April 21, 2015 August 20, 2015 August 27, 2015 August 27, 2015 DOI: 10.1021/acs.jafc.5b01992 J. Agric. Food Chem. 2015, 63, 7937−7944
Article
Journal of Agricultural and Food Chemistry was subsequently evaporated, and the residue was extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give h-MHES (37 g) as a yellow solid. The product was dissolved in dimethyl sulfoxide (DMSO) prior to use in cell experiments. Analysis of h-MHES by Means of High-Performance Liquid Chromatography (HPLC). HPLC analysis was performed by using an Agilent 1220 Infinity LC (Agilent Technologies, Santa Clara, CA, USA) with UV detection (254 nm) and an InertSustain C18 column (⌀ 3.0 × 50 mm, GL Science, Tokyo, Japan). The temperature of the column oven was set at 40 °C. The mobile phase consisted of 0.1% formic acid in water (solution A) and acetonitrile (solution B); the run conditions were as follows: isocratic elution, 70% A/30% B, 0−1.5 min; linear gradient from 70% A/30% B to 30% A/70% B, 1.5−5.5 min; linear gradient from 30% A/70% B to 0% A/100% B, 5.5−6.0 min. The flow rate was 1.0 mL/min. Isolation of Flavanones (AG1, AG2) and Chalcones (AG3, AG4). Thin-layer chromatography (TLC) was performed by using TLC silica gel 60 F254 (Merck, Darmstadt, Germany); spots were visualized by using UV light and treatment with phosphomolybdic acid stain followed by heating. Flash column chromatography was performed by using Hi-Flash column silica gel (Yamazen, Osaka, Japan). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 spectrometer with Cryoprobe (Bruker, Newark, DE, USA). Chemical shifts (δ) are reported in parts per million based on the resonance of tetramethylsilane (0 ppm for 1H NMR in CDCl3) or the respective solvent (1H NMR, 2.49 ppm in DMSO-d6; 13C NMR, 77.0 ppm in CDCl3 or 39.7 ppm in DMSO-d6) as the internal standard. The h-MHES (3.32 g) was fractionated by using flash column chromatography and eluted with hexane/ethyl acetate/methanol. Ten fractions (F1−F10) were collected. 5,7-Dihydroxy-3′,4′-dimethoxyflavanone (AG1) (865 mg) was obtained as a white solid by recrystallizing the F2 fraction from hexane/ethyl acetate: 1H NMR (DMSO-d6) δ 12.13 (1H, s), 10.80 (1H, s), 7.11 (1H, d, J = 1.9 Hz), 7.01 (1H, dd, J = 8.3, 1.9 Hz), 6.95 (1H, d, J = 8.3 Hz), 5.88 (1H, d, J = 2.2 Hz), 5.87 (1H, d, J = 2.2 Hz), 5.46 (1H, dd, J = 12.9, 3.0 Hz), 3.75 (3H, s), 3.74 (3H, s), 3.32 (1H, d, J = 17.1, 12.9 Hz), 2.68 (1H, dd, J = 17.1, 3.0 Hz); 13C NMR (DMSOd6) δ 196.3, 166.6, 163.5, 162.8, 149.0, 148.7, 130.9, 119.3, 111.4, 110.5, 101.7, 95.8, 95.0, 78.5, 55.5, 42.1. 7-Hydroxy-5,3′,4′-trimethoxyflavanone (AG2) (609 mg) was obtained as a pale yellow solid from the F7 and F8 fractions: 1H NMR (DMSO-d6) δ 10.53 (1H, s), 7.07 (1H, d, J = 1.9 Hz), 6.98 (1H, dd, J = 8.4, 1.9 Hz), 6.94 (1H, d, J = 8.4 Hz), 6.04 (1H, d, J = 2.1 Hz), 5.95 (1H, d, J = 2.1 Hz), 5.35 (1H, dd, J = 12.7, 2.8 Hz), 3.75 (3H, s), 3.73 (3H, s), 3.71 (3H, s), 3.03 (1H, d, J = 16.5, 12.7 Hz), 2.52 (1H, dd, J = 16.5, 2.8 Hz); 13C NMR (DMSO-d6) δ 188.1, 164.7, 164.6, 162.6, 149.3, 149.1, 131.8, 119.4, 111.9, 110.8, 104.8, 96.0, 93.6, 78.6, 56.0, 55.9, 45.3. 4′-Hydroxy-3,4,2′,6′-tetramethoxychalcone (AG3) (156 mg) was obtained as a pale yellow solid from the F5 fraction: 1H NMR (DMSO-d6) δ 9.82 (1H, s), 7.25 (1H, d, J = 2.0 Hz), 7.15 (1H, dd, J = 8.4, 2.0 Hz), 7.10 (1H, d, J = 16.0 Hz), 6.93 (1H, d, J = 8.4 Hz), 6.84 (1H, d, J = 16.0 Hz), 6.10 (2H, s), 3.77 (3H, s), 3.76 (3H, s), 3.62 (6H, s); 13C NMR (DMSO-d6) δ 194.1, 160.4, 158.4, 151.3, 149.4, 144.4, 127.7, 127.6, 123.3, 111.9, 110.9, 110.2, 92.4, 55.99, 55.95, 55.89. 4′,6′-Dihydroxy-3,4,2′-trimethoxychalcone (AG4) (112 mg) was obtained as an orange solid from the F3 fraction: 1H NMR (DMSOd6) δ 13.72 (1H, s), 10.58 (1H, s), 7.69 (1H, d, J = 15.6 Hz), 7.60 (1H, d, J = 15.6 Hz), 7.26 (2H, m), 7.00 (1H, d, J = 8.8 Hz), 5.99 (1H, d, J = 2.1 Hz), 5.89 (1H, s), 3.85 (3H, s), 3.81 (3H, s), 3.79 (3H, s); 13C NMR (DMSO-d6) δ 192.2, 166.4, 165.1, 162.9, 151.4, 149.3, 142.9, 128.1, 125.6, 123.1, 112.1, 111.1, 105.6, 96.2, 56.4, 56.0, 55.9. Synthesis of 2′,4′,6′-Trihydroxy-3,4-dimethoxychalcone (AG5). To a stirred solution of 2′,4′,6′-trihydroxyacetophenone monohydrate (2.01 g) in tetrahydrofuran (50 mL), diisopropylethylamine (18.8 mL) and chloromethyl methyl ether (3.28 mL) were added at 0 °C; the
mixture was then stirred at room temperature for 9.5 h. After the solution was cooled to 0 °C, water (10 mL) was added. Then the mixture was extracted with ethyl acetate. The organic layer was washed with 0.5 M HCl and brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by using flash chromatography (hexane/ethyl acetate) to give 2′-hydroxy4′,6′-bis-methoxymethoxyacetophenone (1.71 g, 62%) as a white solid: 1 H NMR (CDCl3) δ 13.71 (1H, s), 6.24 (1H, d, J = 2.5 Hz), 6.22 (1H, d, J = 2.5 Hz), 5.23 (2H, s), 5.15 (2H, s), 3.50 (3H, s), 3.45 (3H, s), 2.64 (3H, s); 13C NMR (CDCl3) δ 203.2, 166.8, 163.4, 160.3, 106.9, 97.1, 94.4, 94.0, 56.7, 56.5, 33.1. To a stirred solution of 2′-hydroxy-4′,6′-bis-methoxymethoxyacetophenone (1.12 g) in methanol (20 mL), veratraldehyde (728 mg) and potassium hydroxide (2.85 g) in water (2 mL) were added at 0°C, and the mixture was stirred at room temperature for 55 h. Then, the mixture was neutralized with 2 M sodium hydroxide (30 mL) and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by using flash column chromatography (hexane/ethyl acetate) to give 6′hydroxy-3,4-dimethoxy-2′,4′-bis-methoxymethoxychalcone (1.13 g, 64%) as a yellow solid: 1H NMR (DMSO-d6) δ 12.15 (1H, s), 7.49 (1H, d, J = 15.7 Hz), 7.45 (1H, d, J = 15.7 Hz), 7.28 (1H, d, J = 1.9 Hz), 7.24 (1H, dd, J = 8.4, 1.9 Hz), 6.99 (1H, d, J = 8.4 Hz), 6.26 (1H, d, J = 2.3 Hz), 6.23 (1H, d, J = 2.3 Hz), 5.24 (2H, s), 5.20 (2H, s), 3.785 (3H, s), 3.783 (3H, s), 3.375 (3H, s), 3.368 (3H, s); 13C NMR (DMSO-d6) δ 192.8, 162.0, 161.2, 157.8, 151.1, 148.9, 143.6, 127.4, 125.6, 123.1, 111.6, 110.3, 109.1, 96.6, 94.53, 94.45, 93.8, 56.2, 55.9, 55.6, 55.4. To a stirred solution of 6′-hydroxy-3,4-dimethoxy-2′,4′-bis-methoxymethoxycalcone (1.13 g) in methanol (60 mL), 1M HCl (30 ml) was added at room temperature, and refluxed for 50 min. After the solution was cooled to room temperature, the mixture was diluted with water (60 mL), and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by using flash chromatography (hexane/EtOAc) to give 2′,4′,6′-trihydroxy-3,4-dimethoxychalcone (AG5) (585 mg, 66%) as an orange solid: 1H NMR (DMSO-d6) δ 12.53 (2H, s), 10.40 (1H, s), 8.01 (1H, d, J = 15.6 Hz), 7.65 (1H, d, J = 15.6 Hz), 7.25−7.23 (2H, m), 7.01 (1H, d, J = 8.9 Hz), 5.82 (2H, s), 3.792 (3H, s), 3.788 (3H, s); 13C NMR (DMSO-d6) δ 191.6, 164.8, 164.4, 150.9, 148.9, 142.1, 127.8, 125.0, 122.6, 111.7, 110.6, 104.2, 94.8, 55.6, 55.4. Cell Culture. Human epidermal keratinocytes (Cascade Biologics, Portland, OR, USA) were grown in EpiLife medium containing 0.06 mM Ca2+, human keratinocyte growth supplement (0.4% bovine pituitary extract, 10 μg/mL insulin, 0.67 μg/mL hydrocortisone, and 0.1 ng/mL human epidermal growth factor), and an antibiotic mixture (50 μg/mL gentamicin and 50 ng/mL amphotericin B) (Cascade Biologics). HEK293 cells (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Both cell types were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air until 80% confluence and then used for experiments. Microarray Analysis. Total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). cRNA synthesis and labeling reactions were performed using a Low Input Quick Amp Labeling Kit (Agilent Technology) according to the manufacturer’s protocol. Array hybridization was performed using a Whole Human Genome microarray 4 × 44K ver.2 (Agilent Technology). The hybridized array slide was scanned with a SureScan Microarray Scanner (Agilent Technology). The microarray data were normalized by using GeneSpring GX 11.5 software (Agilent Technology). The cutoff value was set at 0.5−2.0 for the ratio (>2.0, up-regulation; 2.0 or
