Ergothioneine As an Anti-Oxidative/Anti-Inflammatory Component in Several Edible Mushrooms

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 fractions 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. At this point (day 0), cells 1H, J = 6.0, 16.4 Hz, H-5b), 2.73 (m, 2H, H-4''), 1.72 (m, were switched to differentiation medium (DMEM, 10% FBS, 2H, H-1''), 1.56 (m, 2H, H-3''), 1.35 (m, 2H, H-2''); 13C-NMR 1 µM DEX, 0.25 mM MIX, and 2 µM insulin) and incu-

(100 MHz, DMSO-d6): δC 173.6 (C-1), 169.2 (C-4), 166.6 bated. After 3 days (day 3), medium was replaced with 2 µM (C-7), 134.2 (C-1'), 131.5 (C-4'), 128.4 (C-3'), 127.5 (C-2'), insulin-containing DMEM, and the medium was refreshed 51.8(C-2), 42.5 (C-5), 38.6 (C-4''), 30.6 (C-1''), 26.6 (C-3''), with insulin medium 2 times more every second day (Rof- 22.4 (C-2''). fey et al., 2006). After a 9-day post-induction, media were Anti-inflammatory activity assay Anti-inflammatory ac- replaced with l-EGT (0-5 mM)-containing DMEM. Treated tivity was determined based on the inhibition of lysine resi- cells were incubated for 2 h with the media. Cells were then due chlorination by HOCl. BGL (1 mM final concentration) washed twice with cold PBS and treated with TNF-α (10 ng/ was mixed with the hot water extracts of mushrooms (0.2 mL). After a 20 h incubation, culture medium was collected mg/mL final concentration) and was then incubated with Na- for measuring Interleukin 6 (IL-6). Cell viability was as- OCl (2 mM HOCl, final concentration) in phosphate buffer sessed using MTT assay, as described previously (Roffey et (pH 7.4) at 37℃ for 1 h. After incubation, the reaction was al., 2006). terminated by cooling with ice. The N-ClBGL produced from Enzyme-linked immunosorbent assay (ELISA) for IL-6 the reaction mixtures was analyzed by reverse phase HPLC Culture medium was diluted with PBS (× 5) and the sample using a Develosil ODS HG-5 column (i.d. 4.6 × 250 mm) (100 µL) was used for IL-6 assay. IL-6 secretion by treated with a gradient elution using solvent A (water containing 3T3-L1 adipocytes was measured using a DuoSet ELISA 0.05% acetic acid) and solvent B (methanol containing 0.05% Mouse IL-6 immunoassay kit (R&D Systems, MN) accord- acetic acid): 0-5 (3% B), 5-30 (3-100% B), 30-40 (100% B), ing to the supplier’s protocol. 40-41 (100-3% B), 41-50 min (3% B) with UV (245 nm) de- Statistical analysis All data are expressed as means ± tection. The flow rate was 0.8 mL/min. The inhibition rate (%) standard deviation (S.D.) and each value represents a mini- was calculated as follows: mum of three (n = 3-4) replicate experiments. The differ- 100 × [ N-ClBGL (Control) − N-ClBGL (Sample) ] / [ N- ences between values were compared by two-way unpaired t ClBGL (Control) ] test. HPLC separation of Grifola gargal (G. gargal) extracts Freeze-dried hot water extract from G. gargal was re- Results and Discussion dissolved in water and separated by HPLC using a Develosil Yield of hot water extracts from mushrooms Dried ODS HG-5 column (i.d. 8 × 250 mm) with a gradient elution mushroom samples of 11 species were extracted in hot wa- using solvent A (water containing 0.01% trifluoroacetic acid ter and freeze-dried. The yields of hot water extracts are ) and solvent B (methanol containing 0.01% trifluoroacetic presented in Table 1, and ranged from 34.6 to 67.5 g/100 g acid): 0-5 (0% B), 5-15 (0-100% B), 15-25 (100% B), 25-26 dry weight. Thus, about half of the materials from the mush- (100-0% B), 26-35 min (0% B) with UV (260 nm) detec- rooms were solubilized in hot water. tion. The flow rate was 2.0 mL/min. The most active frac- ORAC assay Table 2 shows the results of ORAC for tion (Fraction 3; Table3) was further purified by preparative the hot water extracts from the mushrooms. Extracts from G. HPLC as described above. A total of 1.7 mg of compound gargal produced the highest ORAC values. ORAC assay has was isolated. provided significant information regarding the antioxidant Quantification of EGT by HPLC Extracts diluted (× 10) capacity of various biological samples from pure compounds with water were pre-treated with the Sep Pac (Waters Corp., (Wang et al., 1997) to complex matrices (Cao et al., 1996), Milford, CT). The diluted samples (0.4 mL) were applied to such as tea and vegetables. It is known that mushrooms have Sep Pac (C18, 360 mg, 0.7 mL) and were eluted with water anti-oxidant activity (Mau et al., 2002). Tsai et al. (2006) (1.2 mL). Samples (20 µL) were applied to two Develosil measured the anti-oxidant activity of hot water extracts from ODS HG-5 columns (i.d. 4.6 mm × 250 mm) and were elut- Agrocybe cylindacea, and we found ORAC values that indi- 106 T. Ito et al.

Table 2. Anti-oxidative and anti-inflammatory activities of mushroom extracts in hot water.

a Inhibitory effect on protein Mushroom species ORAC value (μM TE/mg/mL) a,b chlorination by HOCl (%)

Grifola gargal 200.2 ± 44.8 38.7 ± 8.9 Grifola frondosa 171.3 ± 32.7 24.2 ± 11.8 Pleurotus ostreatus 107.0 ± 26.8 28.8 ± 9.7 Pleurotus eringii 45.5 ± 17.9 19.5 ± 12.5 Lentinula edodes 81.5 ± 9.6 33.5 ± 12.3 Pholiota nameko 75.8 ± 3.6 26.4 ± 11.6 Agrocybe cylindracea 194.3 ± 33.7 35.8 ± 11.3 Leucopaxillus giganteus 160.2 ± 32.8 33.6 ± 10.4 Agaricus blazei 180.4 ± 25.3 32.0 ± 11.5 Tricholoma sp. 178.8 ± 16.4 29.5 ± 12.8 Cyttaria espinosae 127.2 ± 2.9 22.6 ± 3.7

Assays for anti-oxidative and anti-inflammatory activities were as described inMaterials and Methods. a Values are expressed as means ± S.D. of triplicate measurements. b N-ClBGL produced from the reaction of HOCl and BGL was analyzed by HPLC. The inhibition rate was calculated from the peak area. cated similarly high anti-oxidant activity (Table 2). Puttaraju et al. (2006) compared the anti-oxidant properties of water and methanolic extracts from 23 species of mushrooms. They used various assays for anti-oxidant activity, such as the conjugated diene method, reducing power, scavenging ability for 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals and chelating ability for ferrous and cupric ions. However, evaluation on the basis of ORAC has not yet been reported. We examined the antioxidant activity of mushrooms based on ORAC, and the results showed that all the mushroom samples have anti-oxidant activity (Table 2). Inhibition of protein chlorination by HOCl The heme enzyme myeloperoxidase, synthesized and secreted by neutrophils and monocytic/macrophage cells, is an important endogenous source of oxidants. It uses hydrogen peroxide

(H2O2) generated by the phagocyte NADPH oxidase and chloride ions to produce the potent cytotoxin, HOCl (Furt- müller et al., 2000). HOCl is a strong oxidant generated by human neutrophils, and has the potential to cause significant tissue damage. HOCl is able to chlorinate lysine residues in proteins and produce chloramines (Panzenboeck et al., 1997). We used BGL as a model of lysine residues and found Fig. 1. HPLC chromatogram of reaction mixture of BGL that BGL is chlorinated by the reaction with HOCl. HPLC with HOCl (A). Chemical structures of bezoylglycyllysine analysis showed that the reaction of HOCl and BGL resulted and N-chlorobenzoylglycyllysine (B). in the formation of a major product (Fig. 1A). We purified Reaction mixtures of HOCl (4 mM) and BGL (2 mM) were incubat- the peak sample and performed structural analysis using LC- ed at 37℃ for 1 h. HPLC used a Develosil ODS HG-5 column (i.d. 1 13 4.6 mm × 250 mm) with a gradient elution using solvent A (water MS, H-NMR and C-NMR measurements. LC-MS analysis containing 0.05% acetic acid) and solvent B (methanol containing showed a molecular ion at m/z 342.1 and 344.1. The rela- 0.05% acetic acid): 0-5 (3% B), 5-40 (3-100% B), 40-50 (100% B), tive intensity of 342.1:344.1 was 3:1. This suggested the 50-51 (100-3% B), 51-60 min (3% B) eluted with 0.8 mL/min. Anti-Inflammation of Ergothioneine 107 presence of a chlorine. The product was finally indentified cidate the active components in G. gargal, the extract was as N-ClBGL by LC-MS, 1H-NMR and 13C-NMR, as shown separated into 8 fractions by HPLC. The 8 fractions were re- Fig. 1B (Kato et al., 2006). Using N-ClBGL formation in the dissolved at the same concentration (1 mg/mL) and assayed BGL/HOCl system, we then screened the activities of the for ORAC and inhibition of protein chlorination by HOCl mushroom extracts for scavenging and/or reducing the chlo- (Table 3). F3 had the highest anti-oxidative activity and anti- rinating intermediates in vitro. Table 2 shows the inhibitory inflammatory activity. Therefore, we purified F3 by prepara- effects on protein chlorination by HOCl of mushroom ex- tive HPLC. The active compound in F3 was identified as tracts in hot water. The G. gargal extract also had the highest EGT (Ergothioneine, 2-mercaptohistidine trimethylbetaine) inhibitory effects on the chlorination of lysine by HOCl. (Fig. 2) by co-injection into HPLC and 1H-NMR (Kimura, et Elevated levels of 3-chloro-tyrosine, a specific end prod- al., 2005). EGT was isolated as a component from Lyophyl- uct of the reaction between HOCl and tyrosine residues in lum connatum by Kimura et al. (2005). The IC50 (inhibitory proteins, have been detected in atherosclerotic tissue (Hazen concentration) of l-EGT on the inhibitory effect of protein and Heinecke, 1997). This strongly suggests that oxidative chlorination by HOCl was 325 µM. protein reactions involving HOCl are related to the chronic EGT content in several mushrooms We then compared inflammatory disorder. HOCl is produced by neutrophils in the EGT levels in several mushrooms by HPLC (Table 4). vivo and first forms lysine chloramines from the reaction Among 11 species of mushrooms, G. gargal contained the with the lysine residue in protein, before producing 3-chloro- highest levels of EGT. These results showed that EGT is one tyrosine by reacting with tyrosine (Hazen et al., 1998). of the active components in G. gargal. Dubost et al. (2006) Therefore, we measured the lysine chloramines produced reported that the EGT contents of Pleurotus eringii, Grifola by HOCl and confirmed’ anti-inflammatory activity of the frondosa, Pleurotus ostreatus and Lentinus edodes were 1.72, mushrooms. 1.84, 2.01 and 2.09 mg/g dw, respectively. We believe that Recently, the relationship between the oxidative stress and inflammation has been investigated (Ishitsuka et al., 2007). We examined the anti-oxidative and anti-inflammatory activities of hot water extracts from various mushrooms. G. gargal had the strongest anti-oxidative and anti-inflammatory activities in vitro (Table 2). We then attempted to isolate the active components from G. gargal. Isolation and identification of anti-oxidative and anti- Fig. 2. Chemical structure of Ergothioneine. inflammatory components from G. gargal extracts To elu-

Table 3. Anti-oxidative and anti-inflammatory activities of fraction separated fromG. gargal extracts.

Fraction separated from G. gargal Inhibitory effect on protein ORAC value (μM TE/mg/mL) extracts (Retention time, yield (mg) a ) chlorination by HOCl (%) b

F1 ( 2.2- 4.4 min, 2.0) 33.3 37.8 F2 ( 4.4- 5.5 min, 11.5) 42.5 31.6 F3 ( 5.5- 6.3 min, 0.5) 1179.1 100.0 F4 ( 6.3- 8.9 min, 1.8) 162.9 32.7 F5 ( 8.9-14.8 min, 3.4) 404.6 68.0 F6 (14.8-17.0 min, 2.9) 373.7 75.8 F7 (17.0-21.5 min, 1.2) 165.7 72.2 F8 (21.5-25.0 min, 1.7) 67.3 9.9

Hot water extracts from G. gargal were separated by HPLC using a Develosil ODS HG-5 column (i.d. 8 mm × 250 mm) with a gradient elution using solvent A (water containing 0.01% trifluoroacetic acid) and solvent B (methanol containing 0.01% trifluoroacetic acid ) : 0-5 (0% B), 5-15 (0-100% B), 15-25 (100% B), 25-26 (100-0% B), 26-35 min (0% B) with UV (260 nm) detection. Flow rate was 2.0 mL/min. The eluate was separated into 8 fractions. a Yield was defined as dried fraction eluted, when the G. Gargal extract (about 19.1 mg) was injected into column. Assays were performed as described in Table 2, except that the final concentration was 0.4 mg/mL. 108 T. Ito et al.

Table 4. Ergothioneine contents in several mushrooms.

Ergothioneine Mushroom species a (mg/g of dry mushroom)

Grifola gargal 2.04 ± 0.20 Grifola frondosa 0.67 ± 0.06 Pleurotus ostreatus 1.98 ± 0.04 Pleurotus eringii 1.41 ± 0.12 Lentinula edodes 0.40 ± 0.03 Pholiota nameko 0.46 ± 0.11 Agrocybe cylindracea 1.29 ± 0.07 Leucopaxillus giganteus 1.70 ± 0.14 Agaricus blazei 0.00 ± 0.00 Tricholoma sp. 0.91 ± 0.14 Cyttaria espinosae 0.00 ± 0.00 Fig. 3. Effects of ergothioneine on TNF-α-induced IL-6 secretion by 3T3-L1 cells. EGT was analyzed by HPLC. Conditions were as described in Cells were pretreated with l-EGT for 2 h, and were then treated Materials and Methods. with TNF-α (10 ng/mL) for 20 h. IL-6 in the culture media was ana- a Values are expressed as means ± S.D. of triplicate measure- lyzed by ELISA (n = 3). ments.

this discrepancy in EGT contents is due to differences in the in the 3T3-L1 cells. The increasing concentrations of l-EGT strains, analytical methods and extract methods. Agaricus decreased IL-6 release in the 3T3-L1 cells, and 1.0 mM, 2.5 blazei and Cyttaria espinosae contained little EGT, but they mM and 5.0 mM l-EGT decreased IL-6 to approximately showed anti-oxidative and anti-inflammatory activity. We 79%, 65% and 48%, respectively. Cell viability remained at presumed that they had active components other than EGT. > 90% after all of the above treatments, as assessed by MTT Indeed, fractions other than F3 had inhibitory activity (Table assay (data not shown). 3). Recently, Laurenza et al. (2008) used a C2C12 cell Effects of EGT on IL-6 secretion in TNF-α-induced 3T3- model on free fatty acid-induced lipotoxicity to examine the L1 cell culture The main biological functions of EGT protective role of EGT, and found that EGT was able to exert include reduction of oxidative stress in the kidney and liver anti-inflammatory effects by inhibiting IL-6 modulation. We of rats treated with ferric nitrilotriacetic acid (Deiana et al., found that l-EGT suppressed TNF-α-induced IL-6 release in 2004), and modulating the potential toxicity of N-acetyl cys- adipocytes. Thus, EGT would regulate IL-6, which is known teine/H2O2 in neuronal cells (Aruoma et al., 1999). The func- to induce insulin resistance in adipocytes. tional ability to scavenge singlet oxygen, hydroxyl radicals EGT is biosynthesized only by mycobacteria and fungi and peroxyl radicals (Akanmu et al., 1991; Aruoma et al., (Melville et al., 1956); no EGT synthesis has been detected 1997) has already been reported. Colognato et al. (2006) and in higher plants or animal species (Melville et al., 1957).

Rahman et al. (2003) reported that EGT inhibited both H2O2- Hence, in humans, EGT is only absorbed through diet (Mel- induced cell death and the TNF-α-mediated activation of NF- ville and Eich, 1956). Moreover, EGT has recently attracted κB. attention because of its identification as the key biogenic Fontana et al. (2007) reported that visceral fat is an substrate of the organic cation transporter OCTN1 (gene important site for IL-6 secretion, and provides a potential symbol: SLC22A4) (Gründemann et al., 2005). mechanistic link between visceral fat and systemic inflam- In conclusion, we established the anti-inflammatory activ- mation in individuals with abdominal obesity. Based on this ity of edible mushrooms by measuring the inhibitory effects information, we examined the inhibitory effects of l-EGT on on lysine chloramine formation induced by HOCl. Hot water the IL-6 secretion by TNF-α-induced 3T3-L1 cells. Increased extracts of G. gargal showed the strongest anti-oxidative and IL-6 release was observed in 3T3-L1 cells following 20 h anti-inflammatory activity among the 11 species of mush- of exposure to TNF-α, as compared with untreated (control) rooms. The main active component in G. gargal was identi- cells (Fig. 3). Pre-treatment of 3T3-L1 cells for 2 h with l- fied as EGT. We also applied the active compound to TNF- EGT significantly inhibited the TNF-α-induced IL-6 release α-stimulated murine adipocytes, and we found that EGT had Anti-Inflammation of Ergothioneine 109 anti-inflammatory activity on adipocytes. Therefore, EGT- covery of the ergothioneine transporter. Proc. Natl. Acad. Sci. containing edible mushrooms may have beneficial effects on USA, 102, 5256-5261. human health. Halliwell, B. and Gutteridge, J.M.C. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J., 219, Acknowledgements The authors are grateful to Daicel Chemical 1-14. 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