�-Hydroxyergothioneine, a New Ergothioneine Derivative

from the Mushroom Lyophyllum connatum, and Its Protective

Activity against Carbon Tetrachloride-Induced Injury

in Primary Culture Hepatocytes

Chieko KIMURA,1 Manabu NUKINA,1;2;y Kiharu IGARASHI,1;2 and Yoko SUGAWARA3

1Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences,

Iwate University (Yamagata University), 1-23 Wakaba-cho, Tsuruoka, Yamagata 997-8555, Japan2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University,

1-23 Wakaba-cho, Tsuruoka, Yamagata 997-8555, Japan3School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan

Received September 21, 2004; Accepted November 10, 2004

A new ergothioneine derivative named �-hydroxyer-

gothioneine was isolated from the mushroom Lyophyl-

lum connatum. Ergothioneine,N-hydroxy-N0,N0-dimeth-

ylurea, and connatin (N-hydroxy-N0,N0-dimethyl-

citrulline) were also isolated. All the compounds dis-

played the ability to scavenge free radicals, based on a

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scaveng-

ing assay. Structural determination, including the

absolute stereochemistry of �-hydroxyergothioneine,

was achieved by spectroscopic analysis and X-ray

crystallography. The radical scavenging activity of �-

hydroxyergothioneine was almost the same as that of

ergothioneine. �-Hydroxyergothioneine showed the

greatest protective activity against carbon tetrachlo-

ride-induced injury in primary culture hepatocytes.

Key words: ergothioneine; Lyophyllum connatum; radi-

cal scavenger; 1,1-diphenyl-2-picrylhydra-

zyl (DPPH); hepatocytoprotective effect

Antioxidants are known to play an important role inthe prevention of diseases induced by free radicals andof oxidative deterioration of food materials.1) A partic-ularly good source of antioxidants is plant material, andto a lesser extent material of microbial origin.2–6)

Since the Tohoku area is rich in mushroom species,we are interested in searching for potential antioxidantspresent in these organisms. We have screened for DPPHradical scavenging activities7) (Table 1) and have foundthat Lyophyllum connatum (osiroisimeji in Japanese) isone of the active species.

In the present study we isolated the following fourDPPH-active constituents from Lyophyllum connatum:ergothioneine8,9) (ERT) (2), N-hydroxy-N0,N0-dimeth-

ylurea10) (3), connatin10) (4), and a new ERT derivativenamed �-hydroxyergothioneine (HERT) (1) (Fig. 1).ERT is a well-known natural antioxidant that isbiosynthesized only by mycobacterium and fungi.11)

Interestingly, ERT can accumulate in the red blood cellsand liver of the rat,12) as well as in those tissues of otheranimals13) and humans.14) Plants can take up ERT fromsoil microorganisms, and animals through their dietarysources.15) Interestingly, ERT acts as a scavenger ofsinglet oxygen,16) hydroxyl,17) and peroxyl radicals.18)

N-Hydroxy-N0,N0-dimethylurea and connatin, bothdisplaying characteristic violet color reactions withferric chloride, have been isolated as components fromLyophyllum connatum in Germany.10)

We report here the isolation and structural determi-nation, including absolute configurations, of HERT, andthe isolation and identification of three other com-pounds. The radical scavenging activity of these com-pounds is reported. The effect of HERT and ERT on ratprimary culture hepatocytes upon CCl4-induced injury isalso reported.

Materials and Methods

General. NMR spectral data were recorded on a JNM-EX 400FT-NMR spectrometer. Chemical shifts wereexpressed as � values using DSS as an internal standard.Mass spectra in positive ESI were recorded on a JEOLMS700 instrument. Polyethylene glycol 300 (PEG300)was used as an internal standard for high-resolutionESIMS analysis. HREIMS, EIMS, and FABMS wererecorded on a JEOL JMS-SX102A instrument. Perfluo-roalkyl phosphazine (Ultramark 1621) was used as aninternal standard for FABMS analysis. UV spectral data

y To whom correspondence should be addressed. Fax:+81-235-28-2812; E-mail: mnukina@tds1.tr.yamagata-u.ac.jp

Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl; HERT, �-hydroxyergothioneine; ERT, ergothioneine; ESIMS, electrospray ionization mass

spectrometry

Biosci. Biotechnol. Biochem., 69 (2), 357–363, 2005

were recorded on a Jasco UVIDEC-510. IR spectra weremeasured as a KBr disc with a Jasco A-202 spectrom-eter. HPLC was carried out using an LC-8A pumpconnected to a Shimadzu SPD-10A UV–VIS detector.Column chromatography was performed with SephadexLH-20, Sephadex G-10 (Amersham Biosciences,Uppsala, Sweden), and SiO2 (70–230 mesh, Merck,Darmstadt, Germany). Analytical thin-layer chromatog-raphy (TLC) was carried out with Silica gel 60 F254aluminium plates (Merck). DPPH was from TokyoKasei Kogyo (Tokyo, Japan). L-(+)-Ergothioneine innersalt was from Sigma Chemical (Poole, U.K.).

Extraction and preliminary separation. We haveobtained fruit bodies of Lyophyllum connatum severaltimes from the Tohoku area, but the amounts have notbeen great in the past few years. Fortunately we knowthat the same mushroom is obtainable in quantity inHokkaido (Japan). Therefore, for extraction of the activeprinciples of the mushroom we have used fruit bodiessupplied from there. The fruit bodies were collected inHokkaido late in September 2003. The mushroom(2.0 kg, fresh weight) was extracted with acetone for3 days, then filtered and concentrated under reducedpressure. The residue was extracted with ethyl acetate inorder to remove lipophilic materials, and the aqueouslayer was then freeze-dried. The freeze-dried material(38 g total) was re-dissolved in water (4��10 g in50ml) and applied to an Amberlite IR120B (Hþ form)ion exchange column (�5� 28 cm). After the columnwas washed with water (1-liter), the bound materialwas eluted with pyridine–H2O (ratio 1:10) (500ml).The pyridine–H2O eluate (7.24 g) was freeze-dried andseparated by column chromatography on SiO2 (3:6�33 cm, SiO2 60, 63–200 mm) and eluted with EtOH(0.3-liter), EtOH–H2O (ratio 95:5) (4.0-liter) and H2O(0.3-liter). The eluate was collected in 300ml aliquots,separated into 15 fractions.

Isolation of HERT (1). 1 was obtained from Fr. 8–10(fractions eluting between 2.1–3.0-liter) of the SiO2

column chromatography. These fractions were concen-trated under reduced pressure and freeze-dried. Theresidue (576mg) was purified by Sephadex G-10column chromatography (1:6� 104 cm, H2O). Fractionseluting between 120–130ml were freeze-dried. Theresidue (109.3mg) was recrystallized from EtOH–H2Oto afford 1 (57.3mg).

Isolation of ERT (2). 2 was obtained from Fr. 15 (thefraction eluting between 4.1–4.4-liter) of the SiO2

column chromatography. This fraction was concentratedunder reduced pressure and freeze-dried. The residue(1.6 g) was purified by Sephadex G-10 column chroma-tography (each 0.8 g, 3:2� 103 cm, H2O). Fractionseluting between 0.4–0.5-liter were freeze-dried. Theresidue (7.5mg) was purified by HPLC (Develosil C30-UG-5, �10� 250mm, H2O, UV:210, 2ml/min), andfreeze-dried to give 2 (0.7mg) as a colorless powder.

Isolation of N-hydroxy-N0,N0-dimethylurea (3). A

Table 1. List of Mushrooms Examined and Their Activity Rates by

the DPPH Method

Family Scientific Name Activity Rate

Agaricaceae Agaricus arvensis �Phaeolepiota aurea �

Amanitaceae Amanita citrina var. citrina þAmanita vaginata var. vaginata þAmanita virgineoides þAmanita virosa þþ

Bolbitiaceae Agrocybe praecox �Boletaceae Tylopilus neofelleus �Clavariaceae Clavulinopsis fusiformis �

Clavulinopsis pulchra �Coprinaceae Psathyrella candolliana þCortinariaceae Cortinarius pseudosalor �

Descolea flavoannulata þGymnopilus aeruginosus þ

Ganodermataceae Elfvingia applanata þþGeoglossaceae Trichoglossum hirsutum f. hirsutum �Gomphidiaceae Gomphidius maculatus �Hygrophoraceae Hygrocybe acutoconica f. japonica �

Hygrocybe coccinea þHygrocybe conica þ

Hymenochaetaceae Fuscoporia obliqua þþLeotiaceae Leotia lubrica f. lubrica þ

Neobulgaria pura �Morchellaceae Morchella esculenta var. esculenta þþPhallaceae Phallus impudicus þþPluteaceae Pluteus atricapillus þPolyporaceae Coriolus hirsutus þ

Coriolus versicolor �Daedaleopsis purpurea þFomes fomentarius þþLaetiporus sulphureus var. miniatus þþLenzites betulina �Polyporellus badius �

Rhodophyllaceae Rhodophyllus clypeatus þRhodophyllus crassipes þRhodophyllus quadratus �

Russulaceae Lactarius chrysorrheus þLactarius hatudake þþLactarius piperatus þþRussula compacta þRussula emetica �Russula foetens þRussula senecis �

Sarcoscyphaceae Wynnea gigantea �Strophariaceae Naematoloma fasciculare þþ

Naematoloma sublateritum þþPholiota lubrica þPholiota lenta þStropharia rugosoannulata �

Tricholomataceae Armillariella mella �Laccaria bicolor þLaccaria laccata þLaccaria vinaceoavellanea �Lampteromyces japonicus �Lyophyllum connatum þþLyophyllum decastes �Marasmius maximus �Mycena amygdalina �Mycena crocata �Mycena epipterygia �Mycena haematopoda þOudemansiella platyphylla �Oudemansiella radicata þPleurocybella porrigens �Tricholoma flavovirens þTricholoma ustale þ

Xylariaceae Daldinia concentrica �

These mushroom specimens were kindly supplied by members of the

Tohoku Branch of the Mycological Society of Japan.

For preliminary evaluation of the presence of radical scavengers in the

mushrooms, fresh mushroom specimens were extracted by acetone and an

appropriate portion of the acetone solution were tested for the scavenging

DPPH radical. The activity rate was roughly estimated by color change in

the DPPH solution: yellow (þþ, full reaction), dark yellow (þ, partial

reaction), or violet (�, no reaction).

358 C. KIMURA et al.

portion of the water eluate from Amberlite IR120Bchromatography was freeze-dried. The residue (3.3 g)was separated by Sephadex LH-20 column chromatog-raphy (�2:2� 142 cm, MeOH). Active fractions werecombined, concentrated under reduced pressure, andpurified by preparative HPLC (Develosil C30-UG-5,�10:0� 250mm, H2O, UV:210, 2ml/min). The peak ata retention time of 17min was collected and freeze-driedto give 3 (2.3mg) as a light pink powder.

Isolation of connatin (4). 4 was obtained from aseparate lot of Lyophyllum connatum that was collectedin Hokkaido in 2002. The mushroom (3.6 kg, freshweight) was extracted with acetone and separated usingAmberlite IR120B in the way described above. A portionof the residue (1.1 g) was separated by SiO2 columnchromatography (�2:2� 20 cm, SiO2 60, 63–200 mm)using EtOH (100ml) and EtOH–H2O (ratio 95:5)(500ml). Fractions eluting between 250–275ml werecombined, concentrated under reduced pressure, andfreeze-dried. The residues (35.1mg) were further puri-fied by preparative HPLC (Develosil C30-UG-5, 10:0�250mm, H2O, UV:210, 2ml/min) to give 4 (13.3mg).

HERT (1). Colorless prisms (EtOH–H2O). mp 195–200 �C (dec.). ½��D þ35� (c 0.27, H2O). UV �max (H2O)nm ("): 250 (9200); +NaOH: 235; +HCl: 250;+NaOH+HCl+NaOH: 235. IR �max (KBr) cm�1:3400, 3200, 3100, 2860, 1620 (–COO�), 1490, 1355,1065. HRMS (positive ESI) m=z: 268.0723 [M + Na]þ

(calcd. for C9H15N3O3SNa: 268.0732), m=z: 246.0920[M + H]þ (calcd. for C9H16N3O3S: 246.0912). The

1Hand 13C NMR data in D2O are shown in Table 1.

ERT (2). Colorless powder. mp 229 �C. ½��D þ99�

(c 0.27, H2O). NMR �H (D2O): 6.80 (1H, s, H50), 3.89(1H, dd, J ¼ 4, 12Hz, H2), 3.28 (9H, s, þNMe3), �3:22(2H, m, H3). NMR �C (D2O): 173.0 (C1), 158.8 (C20),126.7 (C40), 118.2 (C50), 80.0 (C2), 55.0 (þNMe3), 25.7(C3).

N-hydroxy-N0,N0-dimethylurea (3). light pink powder.mp 101–103 �C. MS (positive FAB) m=z: 105 [M +H]þ. NMR �H (D2O): 2.78 (s). NMR �C (D2O): 165.4(C=O), 38.4 (NMe2).

Connatin (4). Colorless powder. mp 189–190 �C(dec.). ½��D þ5� (c 0.27, H2O). UV �max (H2O) nm:210. IR �max (KBr) cm�1: 3435, 1630, 1590, 1405. MS(positive FAB) m=z: 220 [M + H]þ. NMR �H (D2O):3.78 (1H, t, J ¼ 6Hz, H1), 3.29 (2H, t, J ¼ 7Hz, H5),2.95 (6H, s, H7), 1.94 (2H, m, H3), 1.75 (2H, s, H4).NMR �C (D2O): 177.5 (C1), 168.9 (C6), 57.5 (C2), 55.4(C5), 40.4 (C7), 30.6 (C3), 24.8 (C4).

X-ray analysis of HERT (1). Intensity data(�14 � h � 14, �17 � k � 17, �17 � l � 17) werecollected using a crystal, 0:6� 0:5� 0:4mm3, obtainedfrom an EtOH–H2O solution, by Rigaku MercuryCCD-system with MoK� (� ¼ 0:7107 �A) up to �max ¼32:5�. Crystal data were: C9H15O3N3S.H2O, formulaweight 263.32, orthorhombic, space group P212121,a ¼ 9:773ð1Þ, b ¼ 11:292ð2Þ, c ¼ 11:583ð2Þ �A, V ¼1278:3ð3Þ �A3, Z ¼ 4, and Dx ¼ 1:386Mgm�3. Datareduction and numerical absorption correction(�ðMoK�Þ ¼ 0:261mm�1) were performed using Crys-talClear19) with Rint ¼ 0:032. The structure was solvedby the direct method using SHELXS, and refined bySHELXL.20) The atomic positions of one crystal watermolecule and all hydrogen atoms were located fromdifference maps. The absolute configuration wasconfirmed by Flack parameter21) x ¼ 0:03ð6Þ. The finalR value was 0.037 for 4517 observed reflections(jFoj > 4ðFoÞ). Crystallographic data (excluding struc-ture factors) for the structure in this paper have beendeposited with the Cambridge Crystallographic DataCenter as supplementary publication no. CCDC 251136.Copies of the data can be obtained, free of charge, onapplication to CCDC, 12 Union Road, CambridgeCB2 1EZ, U.K., (fax: +44-1223-336033, or by e-mail:deposit@ccdc.cam.ac.uk).

Measurement of DPPH radical scavenging activity.The DPPH radical scavenging activity of each com-pound was measured according to the method of Suda,22)

with slight modifications, and expressed as a Troloxequivalent. Each compound in 50% EtOH was added toa 1:1:1 mixture of 400 mM DPPH in EtOH, 0.2 M MES(2-morpholinoethaesulphonic acid) buffer (pH 6.0), and50% EtOH. The solution was mixed using a vortex

Fig. 1. Structure of Compounds 1–4.

�-Hydroxyergothioneine from Lyophyllum connatum 359

mixer (10 s pulse) and the absorbance at 520 nm wasmeasured after 20min. A standard curve was preparedby repeating the same experiment using Trolox in EtOHinstead of the sample. The Trolox equivalent of thesample was calculated using the standard curve forTrolox.

Cell experiment. Hepatocytes were prepared fromWistar strain rat (250 g) by the collagenase perfusionmethod (Matsuda et al., 2002).23) A 12 — well tissueculture plate containing fresh William’s medium sup-plemented with 10% newborn bovine serum (NBS),insulin (10�4 M), and dexamethasone (10�4 M) wasinoculated with cell suspension (3� 105 cells/ml inWilliam’s medium containing 10% NBS and 30 mg/mlkanamycin). The culture was precultured for 4 h at 37 �Cunder a 5% CO2 atmosphere. The medium was thenexchanged and the culture continued for a further 24 hunder the same conditions. The medium was exchangedwith fresh medium containing either 500 ml DMSO per100ml (Con: control group without sample and +ERTor +HERT group), or medium containing both 500 mlDMSO and 300 ml CCl4 per 100ml (for the +CCl4,+ERT+CCl4, and +HERT+CCl4 groups). A 15 mlaliquot of the medium containing 4.4 mM of ERT or3.8 mM of HERT was added to the well of the +ERT or+HERT group, and the +ERT+CCl4 or +HERT+CCl4group. The final concentrations of ERT and HERT in themedium in each well were 66 nM and 57 nM respectively.For the wells containing the Con and +CCl4 groups,medium without sample was added. After 90minincubation, the medium was collected and aspartic acidaminotransferase (AST) and alanine aminotransferaseactivities (ALT) were measured.24) The final concen-tration of CCl4 in the medium in each well was settled at30mM. AST and ALT activities were expressed ininternational units (IU: mmol/min/l) in the medium. Inthis paper we also express AST and ALT activities as

hepatocyte AST and ALT activities, since the enzyme isreleased into the medium from hepatocyte cells.

Results

Structural determination of HERT (1)HERT (1) was obtained as colorless prisms; mp 185–

195 �C (dec.). The molecular formula C9H15N3O3S ofthe new compound was deduced from an [M + H]þ

peak at 246.0920 [M + H]þ (calcd. for C9H16N3O3S:246.0912) in the positive HR-ESIMS data together withthe NMR data (Table 2) and X-ray crystallographicanalysis.

The EIMS of 1 did not show a molecular ion peak.Prominent peaks observed by HR-EIMS of 1 are theseions of m=z 183.0833 ([M–CO2–H2O]

þ, calcd. forC8H13N3S: 183.0830), m=z 169.0676 ([M–CO2–OH–CH3]

þ, calcd. for C7H11N3S: 169.0674), m=z 154.0419([M–CO2–OH–(CH3)2]

þ, calcd. for C6H8N3S:154.0439), m=z 128.0076 ([M–CH2–CO2–N(CH3)3]

þ,calcd. for C4H4N2OS: 128.0044) and m=z 58.0666([N(CH3)3–H]

þ, calcd. for C3H8N: 58.0657). Thesefragment ions indicate the presence of the trimethyl-ammonium group and a heterocyclic fragment. Some ofthe possible fragmentations of 1 in EIMS are shownin Fig. 2.

Table 2. NMR Spectral Data of HERT (1) (in D2O, DSS as an

internal standard, 400MHz)

Position�H

(JHz)�C

1JðC,HÞ(Hz)

HMBC

(H to C)

1 — 171.8

2 4.00 (1H, d, J ¼ 10Hz) 83.2 151 1, 3, -þNMe33 5.27 (1H, d, J ¼ 10Hz) 66.8 147 2, 40

-þNMe3 3.39 (9H, s) 56.4 146

20 — 159.9

40 — 131.0

50 6.92 (1H, s) 118.2 199 20, 40

Fig. 2. HR-EIMS of 1.

360 C. KIMURA et al.

In the UV (H2O) spectrum of 1, the maximalabsorption band (250 nm, " 9,200) was hypsochromi-cally shifted to 235 nm by the addition of base (1NNaOH). This characteristic behavior is reminiscent of a2-thioimidazol ring, such as that of ergothioneine.

The 1H NMR and 13C NMR spectral data of 1 areshown in Table 2. The 1HNMR spectrum showed asharp singlet (�H 3.39, 9H, s), two protons coupled toeach other (�H 4.00 and �H 5.27, each 1H, d, J ¼ 10Hz)and an aromatic proton (�H 6.92, 1H, s). In the 13C NMRspectrum of 1, seven peaks were observed. The DEPTspectra of 1 showed methyl carbons (�C 56.4), threemethine carbons (�C 66.8, �C 83.2, and �C 118.2), andthree quaternary carbons (�C 131.0, �C 159.9, and �C171.8). The 1H and 13C NMR spectra indicate resonancetypical of a trimethylammonium group at �H 3.39 (9H, s)and �C 56.4. The signal at �C 171.8 can be assigned to acarboxylate carbon, the presence of which was support-ed by the IR (KBr) absorption band at 1620 cm�1

(–COO�). The coupling constant (J ¼ 10Hz) betweenH2 and H3 indicated the anti-perpendicular arrangementof these protons. In the HMBC, the crosspeaks betweenH2 to C1 and methyl carbons of the þNMe3 indicated abetaine moiety at C2. In the HMBC, there werecrosspeaks between H2 to C3, between H3 to C2 andC40, and between H50 to C40 and C20. The couplingconstant of 1JðC,HÞ of C50 is 199Hz, indicating thepresence of a heterocyclic ring in 1. These 1H NMR and13C NMR spectral data of 1 revealed good correlationwith those of ERT, except for the signals of H3 and C3.The chemical shifts at H3 (�H 5.27) and C3 (�C 66.8) of1 were in good agreement with those of (2S,3S)-�-hydroxyhistidine (at �H 5.42 and �C 65.0).25) Based onthese spectral data, the new compound 1 was assigned tobe �-hydroxyergothioneine.

To confirm the absolute configuration of 1, X-raycrystallographic analysis was performed. An ORTEPdrawing26) of the molecule is shown in Fig. 3. Themolecular structure deduced from the HR-EIMS and 1Hand 13C NMR spectral data was confirmed. Moreover,the absolute configuration was determined to be (2S,3S).The thioimidazole ring is a thione form, not a thiol form,

as is the case with L-ergothioneine.27) The crystals of 1were monohydrate. The crystal water molecule washydrogen bonded to the sulfur atom of the thioimidazolering and the oxygen atom of the carboxyl group as aproton donor and to N10 of the thioimidazole ring as aproton acceptor. There were no significant differences inbond lengths and angles of 1 and ERT, except for thebond length of C2–C3 (1.555(2) �A), which was a littlelonger than the corresponding bond distance of ERT(1.526(4) �A). The conformations of the two compoundswere very similar with the nitrogen atom N4 trans to thethioimidazole ring in both cases. The oxygen atom of thehydroxyl group was nearly trans to C1 ( (C1–C2–C3–O10) = �166:2ð1Þ�) and gauche to N30 ( (N30–C40–C3–O10) = �63:5ð2Þ�).Accordingly, 1 was determined to be (2S,3S)-�-

hydroxyergothioneine.

Identification of ERT (2), N-hydroxy-N0,N0-dimeth-ylurea (3) and connatin (4)ERT (2) was identified by direct comparison with the

authentic sample on the basis of the melting point, thespecific rotation, and the NMR data.N-Hydroxy-N0,N0-dimethylurea (3) and connatin (4)

were identified on the basis of melting point, FABMS,and the NMR data.10,28) In addition, compound 3 wasprepared by a previously reported method for directcomparison.28) The synthetic product and the naturalsample were found to be identical.

DPPH radical scavenging activityAs shown in Table 3, both HERT and ERT have

radical scavenging activities almost equivalent to that ofTrolox, which is known as a water-soluble vitamin Eanalog. Connatin and N-hydroxy-N0,N0-dimethylureawere weaker than HERT or ERT under these exper-imental conditions.

Prevention of hepatocyte injuryAs shown in Fig. 4, AST and ALT activities in

hepatocytes cultured with either ERT or HERT did notdiffer from the control group. AST and ALT activities inhepatocytes cultured with carbon tetrachloride signifi-

Fig. 3. ORTEP Drawing26) of 1.

Table 3. The DPPH Radical Scavenging Activities of HERT, ERT,

N-Hydroxy-N0,N0-Dimethylurea, and Connatin

CompoundsTrolox equivalent

(mmol Trolox eq/mmol sample)

HERT 0.94

ERT 0.94

N-Hydroxy-N0,N0-dimethylurea 0.14

Connatin 0.18

Each compound was added to the DPPH solution (see ‘‘Materials and

Methods’’), and after 20min the decrease in the absorbance at 520 nm was

measured. Then the Trolox equivalent was deduced from the standard curve

obtained by Trolox. Radical scavenging activity is shown as Trolox

equivalent/mmol sample.

�-Hydroxyergothioneine from Lyophyllum connatum 361

cantly increased when compared to the control (Congroup). Intriguingly, the addition of ERT and HERT tothe hepatocytes cultured in the presence of carbontetrachloride significantly relieved the increase in ASTand ALT activities induced by carbon tetrachloride (e.g.,compare the +CCl4 group with the +ERT+CCl4 and+HERT+CCl4 groups). Furthermore, the reduction inhepatocytotoxicity was significantly greater for HERTthan for ERT.

Discussion

ERT was first isolated from ergot by Tanret in 1909,8)

and is now recognized to be an important antioxidant ofbiological origin.29) ERT has been detected in mamma-lian tissues including the liver, the kidney, the eye,seminal fluid, and erythrocytes.30) These tissues accu-mulate ERT from dietary sources. ERT is known to beproduced by mycobacterium and fungi, including mush-rooms. This paper is the first report of the occurrence ofERT in the mushroom Lyophyllum connatum.There are very few natural products having a histidine

betaine moiety. Two such examples include clithio-neine31) and sclerothionine,32) which have 20-substitutedthioimidazol groups. HERT, which has a hydroxyl groupat the � position of the histidine betaine moiety, is thefirst example of this class of naturally occurringcompounds. Since ERT is biosynthesized from histidinevia hercynine,33) HERT can be generated from hydroxy-histidine.25) Examination of the occurrence of HERT inother fungal species and mushrooms is interesting froma chemotaxonomical viewpoint. Since the DPPH radicalscavenging activity of HERT and ERT was the same, thehydroxyl group of HERT appears to have little effect on

the ability to scavenge free radicals under theseexperimental conditions.

Neither ERT nor HERT displayed apparent cytotox-icity at the concentrations used in our experiments, sincethe AST and ALT activities of the Con, ERT, and HERTgroups were almost identical, but ERT and HERT gavehepatocytes significant protection against the toxiceffects of carbon tetrachloride. Cytochromes P450 inthe hepatocyte microsome are known to generate �CCl3as a reactive radical species from carbon tetrachloride.34)

Therefore these results indicate that ERT and HERTprovide the liver with protection against radical-inducedhepatocytotoxicity. Since HERT is more effective thanERT in protecting hepatocytes against the toxic effectsof carbon tetrachloride, the additional hydroxyl group ofHERT appears to have a beneficial effect under theexperimental conditions used in this study. Since theantioxidative activities of ERT and HERT expressed asTrolox equivalent were almost equal, the higher uptakeof HERT with the hydroxyl group by cells might beconcerned with the stronger protective effect of HERTthan of ERT. Although a protective effect of ERTagainst liver injury induced by ethionine in rats has beenreported previously,35) this is the first report of ERTprotecting against liver hepatocytotoxity induced byradical species such as �CCl3. Our results indicate thatERT and HERT might also be effective in suppressingoxidative stress involving lipid peroxidation induced byradical species in vivo. The protective effect of these twocompounds using cultured hepatocytes showed HERT tobe significantly better than ERT. It will be interesting tocompare the protective effects of HERT and ERTagainst oxidation in vivo to determine the pronouncedeffect of the additional hydroxyl group present in HERT.

Fig. 4. Effect of ERT and HERT on CCl4-Induced Hepatocyte Injury.

Values are mean � s.e. of four wells in a microplate. Data for each of the 6 groups were statistically analyzed by Duncan’s multiple range test

after a one-way analysis of variance (ANOVA).36) Values with different letters are significantly different at p < 0:05. AST (A) and ALT (B)

activities were measured in the medium, which contained AST and ALT released from rat primary culture hepatocytes cultured in the absence of

CCl4 (Con, +ERT, and +HERT groups), and in the presence of CCl4 with or without sample (+CCl4, +ERT+CCl4, and +HERT+CCl4groups). Activities are shown in international units (IUs) per medium. The precise method for activity measurement is described in the text.

362 C. KIMURA et al.

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