Ergothioneine As an Anti-Oxidative/Anti-Inflammatory Component in Several Edible Mushrooms
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Food Sci. Technol. Res., 17 (2), 103–110, 2011 Ergothioneine as an Anti-Oxidative/Anti-Inflammatory Component in Several Edible Mushrooms 1* 2 3 4 2 2 Tomomi ito , Manami Kato , Hironobu tsuchida , Etsuko harada , Toshio niwa and Toshihiko osawa 1 Hokkaido University of Education Asahikawa Campus, 9-Hokumoncho, Asahikawa 070-8621, Japan 2 Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa, Nagoya 464-8601, Japan 3 Aichi Mizuho College, 86-1, Haiwa, Hiratobashi-cho, Toyota, Aichi 470-0394, Japan 4 Iwade Research Institute of Mycology, Tsu, Mie 514-0012, Japan Received May 8, 2010; Accepted December 1, 2010 We measured the anti-oxidative and anti-inflammatory activities of hot water extracts prepared from 11 species of mushrooms. Anti-oxidative activity was evaluated using the oxygen radical absorbance capacity method, and anti-inflammatory activity was examined by measuring the inhibition of lysine chloramine formation by hypochlorous acid. Hot water extracts of Grifola gargal (G. gargal) showed the strongest anti-oxidative activity and inhibitory effects on lysine chloramines. Hot water extracts of G. gargal were evaluated by HPLC and divided into 8 fractions. The most active fraction among these 8 frac- tions was further purified by preparative HPLC. The active component isolated by HPLC was identified as ergothioneine (EGT) using spectral analysis. On HPLC analysis, the EGT content in G. gargal was the highest among the 11 species of mushrooms. We also examined the protective role of EGT by examining the inflammatory response of adipocyte cells induced by tumor necrosis factor-α. Keywords: mushroom, ergothioneine, oxygen radical absorbance capacity, anti-inflammation, 3T3-L1, interleukin 6 Introduction Mushrooms have been used as traditional medicines It has been speculated that antioxidants act as scavengers since ancient times, and the results of recent studies have in- of reactive oxygen species (ROS), and play an important role dicated that they have significant pharmacological properties in the prevention of aging and diseases, such as atherosclero- (Lindequist et al., 2005). The best known of these is the im- sis, diabetes, cancer and cirrhosis (Halliwell and Gutteridge, munomodulatory effect of polysaccharides belonging to the 1984). β-glucans (Moradali et al., 2007). In addition, they have anti- It is also known that arteriosclerosis is related to the oxi- tumor (Mizuno et al., 1990) and anti-inflammatory (Dore et dation of the low-density lipoprotein (LDL), excess platelet al., 2007) activities. aggregation, and induction of adhesion molecules. It has Although studies have focused on the antioxidant proper- been noted that arteriosclerosis is closely related to inflam- ties of mushrooms using several methods, little information mation, as 3-chloro-tyrosine produced during the inflamma- is available about the oxygen radical absorbance capacity tion process was detected in the blood or tissue of arterio- (ORAC). ORAC assay directly measures the antioxidant ac- sclerosis patients (Hazen and Heinecke, 1997). In addition, tivities against the peroxyl radical. We have measured ORAC hypochlorous acid (HOCl), produced by neutrophils through values in various edible mushrooms. Recently the anti-in- an inflammatory response, reportedly forms lysine chlora- flammatory effects of mushrooms have been reported (Zhang mine that reacts with the amino-group of the lysine residue et al., 2002). We also evaluated the anti-inflammatory effects in proteins, finally producing 3-chloro-tyrosine by reacting of the mushrooms using a novel in vitro method that evalu- with tyrosine (Hazen et al., 1998; Ishitsuka et al., 2007). ates the chlorination of lysine induced by HOCl. Here, we measured the anti-oxidative and anti-inflam- *To whom correspondence should be addressed. matory activities of hot water extracts prepared from various E-mail: [email protected] mushrooms and attempted to isolate the active component 104 t. ito et al. from these mushrooms. Preparation of hot water extracts from mushrooms Each dried mushroom sample (10 g) was mixed with 200 Materials and Methods mL of hot water and was then heated for 30 min in a boiling Materials Eleven species of mushrooms were obtained water bath with occasional stirring for effective extraction. from the Iwade Research Institute of Mycology, in dried After centrifugation using an angle rotor (TA-9BH; TOMY powder form. The complete names of these mushrooms are SEIKO, Tokyo, Japan) at 10,000 rpm for 10 min, superna- listed in Table 1. Disodium fluorescein (FL), 2,2'-azobis(2- tants were obtained by filtration and the precipitates were amidinopropane)-dihydrochloride (AAPH), Trolox, sodium twice extracted with 200 mL of hot water as described previ- hypochlorite (NaOCl), DMSO, HCl, dexamethasone (DEX) ously. The combined supernatant solutions were evaporated and 3-isobutyl-1-methylxanthine (MIX) were purchased in vacuo to 100 mL. Extracts (10 mL) were directly used for from Wako Pure Chemical Industries (Osaka, Japan). Benzo- the quantitative analysis of EGT. Another extract (90 mL) ylglycyllysine (BGL) was a product of the Peptide Institute, was freeze-dried, dissolved in distilled water (10 mg/mL) Inc. (Osaka, Japan). l-(+)-Ergothioneine (EGT) and insulin and stored at −20℃ until use. Extract yields were expressed were obtained from Sigma (St. Louis, MO). as percentages on a dry weight basis (Table 1). Cell culture 3T3-L1 cells were obtained from the Japa- Anti-oxidative activity assay The anti-oxidative activity nese Collection of Research Bioresources (Osaka, Japan). of the hot water extracts from the mushrooms was examined Cells were grown in high-glucose Dulbecco’s Modified Ea- by ORAC. The ORAC value for each sample was evaluated gle’s Medium (DMEM; Gibco) supplemented with 10% fetal according to methods similar to that of Dávalos et al. (2004). bovine serum (FBS), 100 µg/mL penicillin, and 100 units/ Reactions were carried out in 75 mM phosphate buffer (pH mL streptomycin in a 5% CO2-containing atmosphere. DEX 7.4). The FL (150 µL; 125 nM final concentration) and sam- and MIX were dissolved for cell treatment in DMSO at 1 ple solutions (25 µL) were placed in a black 96-well plate. mM and 250 mM, respectively. Insulin was dissolved in 0.02 Mixtures were preincubated for 10 min at 37℃, and AAPH N HCl at 2 mM and sterile filtered (0.22 µm). Tumor necro- solution (25 µL; 18.75 mM final concentration) was added. sis factor-α (TNF-α) was purchased from R&D Systems, Inc. The fluoroskan ascent (Thermo Electron Co., Waltham, MA) (Minneapolis, MN), and was dissolved for cell treatment in was programmed to record fluorescence every 5 min for phosphate-buffered saline (PBS) containing 0.1% bovine 60 min (Ex. 485, Em. 538 nm). The ORAC activity of the serum albumin (BSA) at 10 µg/mL. l-EGT was dissolved for samples was calculated from the differences in the areas un- cell treatment in PBS. der the FL quenching curves of Trolox, blank and samples, and was expressed as µM of Trolox equivalence per mg/mL (ORAC value). Table 1. Mushroom species and yield of hot water extracts from several mushrooms. Structural determination of chlorinated BGL BGL (20 mM final concentration) was mixed with NaOCl (60 mM Yield of extract Mushroom species HOCl final concentration) and then incubated at 37℃ for 15 (g/100g of dry mushroom) min. Concentrations of HOCl were spectrophotometrically -1 -1 Grifola gargal 46.5 determined using ε292 = 350 M cm (Morris, 1966). After Grifola frondosa 48.2 incubation, the reaction was purified by reverse phase HPLC. Pleurotus ostreatus 34.6 Chromatography was carried out using a Develosil ODS Pleurotus eringii 49.1 HG-5 column (i.d. 20 × 250 mm, Nomura Chemicals, Aichi, Lentinula edodes 35.7 Japan) with gradient elution using solvent A (water contain- Pholiota nameko 46.6 ing 0.05% acetic acid) and solvent B (methanol containing Agrocybe cylindracea 39.7 0.05% acetic acid): 0-5 (3% B), 5-40 (3-100% B), 40-50 (100% B), 50-51 (100-3% B), 51-60 min (3% B) (condition Leucopaxillus giganteus 46.8 1). Flow rate was 6 mL/min with UV detection (245 nm). Agaricus blazei 46.5 The novel peak (retention time 38 min) was concentrated and Tricholoma sp. 67.5 dried. The product (6.9 mg) was analyzed by NMR using a Cyttaria espinosae 47.8 Bruker ARX400 spectrometer (400 MHz; Karlsruhe, Ger- Each dried mushroom sample (10 g) was extracted 3 times in hot many) and liquid chromatography-mass spectrometry (LC- water (200 mL) for 30 min. Extracts were evaporated and freeze- MS) (Micromass VG Platform II, Micro-mass, Manchester, dried. Extract yields are expressed as percentages based on dry United Kingdom) in negative electrospray ionization mode. weight. The LC-MS was carried out using a Develosil ODS HG-5 Anti-Inflammation of Ergothioneine 105 column (i.d. 4.6 × 250 mm) with gradient elution (condition ed with water containing 0.1% triethylamine at a flow rate 1) at 0.8 mL/min. 0.8 mL/min (Dubost et al., 2006). EGT was quantified by N-chloro-benzoylglycyllysine (N-ClBGL) MS m/z: (ESP- monitoring the absorbance at 260 nm, and comparison with a − 1 ) 342.1, 344.1 (M-H) ; H-NMR (400 MHz, DMSO-d6): δΗ standard curve obtained using authentic l-EGT. 8.74 (dd, 1H, J = 6.0, 12.0 Hz, H-6), 8.20 (d, 1H, J = 7.6 Hz, Cell culture and differentiation 3T3-L1 cells were H-3), 7.92 (brs, 1H, H-5''), 7.88 (d, 2H, J = 6.8 Hz, H-2'), 7.52 subcultured every 3 or 4 days at approximately 90% conflu- (d, 1H, J = 7.2 Hz, H-4'), 7.46 (d, 2H, J = 14.4 Hz, H-3'), 4.22 ence. Cells were grown in 24-well plates (0.5 mL/well) and (m, 1H, H-2), 3.98 (dd, 1H, J = 6.0, 16.4 Hz, H-5a), 3.87 (dd, reached confluence in 3 days.