Isolation and characterization of a human intestinal bacterium, Eubacterium sp. ARC-2, capable of demethylating arctigenin, in the essential metabolic process to enterolactone

Article abstract of DOI:10.1248/bpb.30.904

Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage

Full text of DOI:10.1248/bpb.30.904

9
04
Biol. Pharm. Bull. 30(5) 904—911 (2007)
Vol. 30, No. 5
Isolation and Characterization of a Human Intestinal Bacterium,
Eubacterium sp. ARC-2, Capable of Demethylating Arctigenin, in the
Essential Metabolic Process to Enterolactone
a
a
a
b
c
Jong-Sik JIN, Yu-Feng ZHAO, Norio NAKAMURA, Teruaki AKAO, Nobuko KAKIUCHI, and
Masao HATTORI*,
a
a
b
Institute of Natural Medicine, University of Toyama; 2630 Sugitani, Toyama 930–0194, Japan: Faculty of
c
Pharmaceutical Sciences, University of Toyama; 2630 Sugitani, Toyama 930–0194, Japan: and Graduate School of
Natural Science and Technology, Kanazawa University; Kakumacho, Kanazawa 920–1192, Japan.
Received November 15, 2006; accepted January 31, 2007; published online February 13, 2007
Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized
to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes in-
clude deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intes-
tinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)buty-
rolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eu-
bacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By
incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of
arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed
by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-
1,4-butanediol by demethylation.
Key words human intestinal bacteria; plant lignan; arctigenin; Eubacterium; demethylation
Phytoestrogens are estrogen-like compounds found in
In the present paper, we report the isolation and characteri-
many edible plants and are generally categorized as zation of a bacterial strain, Eubacterium (E.) sp. ARC-2, ca-
isoflavones and lignans according to their chemical struc- pable of demethylating in lignans. The structural requirement
1,2)
tures. Experimental and epidemiological studies that phy- of demethylation catalized by this bacterium was quite differ-
13)
toestrogens have potential health benefits in hormone-de- ent from that previously isolated by Wang et al.
pendent diseases such as breast and prostate cancers, have There are many studies of microbial O-demethylation
led to intense interest in their transformation and absorp- systems. Some reports showed O-demethylation by oxyge-
3
—5)
tion.
nase or by tetrahydrofolate (THF)-dependent methyltrans-
1
5—21)
Transformation and absorption of the plant lignans have ferase.
The importance of THF and ATP in the anaero-
6,7)
22)
been studied
END) were first isolated from urine of humans and animal effects of THF and ATP on demethylation of arctigenin by E.
species in 1980. ENL and END are usually termed mam- sp. ARC-2, a strictly anaerobic bacterium.
since enterolactone (ENL) and enterodiol bic O-demethylation was reported. Here, we investigated
(
malian lignans because they possess phenolic hydroxyl
Moreover, we examined the biotransformation of (ꢀ)-seco-
groups only in the meta position of aromatic rings unlike isolariciresinol and (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-
8)
plant lignans. ENL and END are transformed from the 3-(3,4-dimethoxybenzyl)-1,4-butanediol [(ꢁ)-secoisolarici-
plant lignans by bacteria in the intestinal tract and absorbed resinol 4ꢂ-O-methyl ether] by E. sp. ARC-2.
into the body. They exhibit biological ability such as estro-
9
—11)
genic and antiestrogenic acivities
as well as antioxida- MATERIALS AND METHODS
12)
tive activity.
The transformations of plant lignans like secoisolari-
General An anaerobic incubator, EAN-140 (Tabai Co.,
ciresinol diglucoside, pinoresinol diglucoside, arctiin, and Osaka, Japan), was used for incubation of fecal suspensions
matairesinol, to ENL and END by intestinal bacteria include and intestinal bacteria. Optical rotations were measured in
a series of reaction such as deglucosylation, ring cleavage, MeOH solutions with a DIP-360 automatic polarimeter
13)
demethylation, dehydroxylation, and oxidation. Wang et al.
(Jasco Co., Tokyo, Japan), UV spectra with a UV-2200
isolated two bacteria responsible for transforming secoisolar- UV–VIS recording spectrophotometer (Shimadzu Co.,
iciresinol diglucoside to ENL and END. Peptostreptococcus Kyoto, Japan), melting points with a Yanagimoto micro hot-
sp. strain SDG-1, one of these bacterial strains, capable of stage melting point apparatus (Yanagimoto Co., Tokyo,
demethylation, however, did not transform tetra-O-methyl- Japan), and electron impact mass spectra (EI-MS) with a
secoisolariciresinol. This observation suggested that an ad- JMS-GC mate mass spectrometer at an ionization voltage
1
13
jacent hydroxyl group to the methoxy group was necessary of 70 eV (JEOL Co., Akishima, Japan). H- and C-NMR,
1
1
for demethylation by P. sp. SDG-1.
H– H-correlated spectroscopy (COSY), heteronuclear mul-
14)
On the other hand, Xie et al. reported that arctigenin was tiple quantum coherence (HMQC), heteronuclear multiple
transformed to ENL, indicating the presence of bacteria ca- bond coherence (HMBC), and nuclear Overhauser effect
pable of demethylating vicinal dimethoxy groups to yield spectroscopy (NOESY) experiment were taken on Varian
1
13
free hydroxyl group(s).
UNITY 500 ( H: 500 MHz, C: 125 MHz) and Varian Gem-
∗
To whom correspondence should be addressed. e-mail: saibo421@inm.u-toyama.ac.jp
© 2007 Pharmaceutical Society of Japan

May 2007
905
1
13
23
ini 300 ( H: 300 MHz, C: 75 MHz). High performance liq-
(ꢀ)-Dihydroxyenterodiol: Amorphorus powder. [a]D
uid chromatography (HPLC) was performed on a CCPM-II ꢀ19.0° (cꢄ0.11, MeOH). This compound was identified by
Tosoh, Tokyo, Japan) equipped with a Tosoh UV-8020 spec- comparing the H- and C-NMR spectra with that pub-
trometer and a Shimadzu C-R8A chromatopac (Shimadzu, lished.
1
13
(
13)
Kyoto, Japan) under the following conditions: column, TSK-
(ꢁ)-Dihydroxyenterodiol [(2R,3R)-2,3-Bis(dihydroxyben-
2
3
gel ODS-80Ts (Tosoh Co., Tokyo, Japan, 4.6 mmꢃ150 mm); zyl)-1,4-butanediol]: Amorphorus powder. [a] ꢁ5.7° (cꢄ
D
mobile phase, H O (solvent system A) and CH CN (solvent 0.15, MeOH). This compound was identified by comparing
2
3
1
13
27)
system B) in a gradient mode (B from 25 to 26.2% in the H- and C-NMR spectra with that published.
0 min); flow rate, 1.0 ml/min; detection, UV 280 nm; tem- Isolation of an Human Intestinal Bacterial Strain Ca-
perature, room temperature. Thin-layer chromatography pable of Demethylating 8 to Yield 1 A bacterial suspen-
5
(
(
TLC) was carried out on silica gel pre-coated 60 F254 plates sion from fresh feces of a healthy volunteer was repeatedly
0.25 mm, Merck Co., Darmstadt, Germany) and spots were cultured in 2 ml of GAM broth containing 0.5 mM arctigenin
detected under a UV lamp or exposing I vapor. Silica gel as a substrate at 37 °C in an anaerobic incubator. A portion of
2
BW-820 MH (Fuji silysia, Aichi, Japan) was used for column the culture, possessing metabolic activity, was seeded on
chromatography. The DNA sequences were aligned using GAM agar plates and anaerobically incubated for 24 h at
‘
DNASIS’ version 3.0 (Hitachi Software Engineering Co., 37 °C. Colonies were picked up and screened for their activ-
Tokyo, Japan). ity of transforming 8 to 1. Such a procedure was repeated
Plant Materials The seeds of Arctium lappa L. were until a single strain having demethylation acitivity was iso-
purchased from Tochimoto Tenkaido Co. (Osaka, Japan). lated. Characterization of a bacterial strain was carried out
Chemicals and Media General anaerobic medium according to the methods of Mitsuoka and the Bergey’s
25)
26)
(GAM) broth was purchased from Nissui Co. (Tokyo, Japan). manual.
Arctigenin (8) was isolated from the seeds of A. lappa as fol-
Sequencing the Bacterial 16S rRNA Gene The bac-
lows: The softened and grinded seeds (1 kg) were extracted terium was incubated at 37 °C in an anaerobic incubator for
two times by immersing in MeOH (5 l) overnight. The com- 2 d and collected by centrifugation at 10000ꢃg for 10 min.
bined solutions were concentrated to 450 ml under vacuum, Total DNAs were extracted with a DNeasy Plant Mini Kit
to which 50 ml of water was added. The mixture was ex- (Qiagen, Germany) following the manufacturer’s protocol.
tracted with 300 ml of hexane and the residual solution was The 16S rRNA gene was amplified by polymerase chain re-
hydrolyzed with 2 N HCl at 80 °C for 2 h. The mixture was action (PCR) with two forward and reverse primer sets based
extracted with ethyl acetate and the ethyl acetate-soluble on those of various strains from the database: Bac 1F
fraction was evaporated to yield a residue (46.2 g), which (AGAGTTTGATCCTGGCTCAG) and Bac 1R (CCGTAT-
was chromatographed on a silica gel column (8 cmꢃ36 cm) TACCGCGGCTGCTG); and Bac 3F (TAACTACGTGCC-
eluting with CHCl –MeOH (100 : 1→20 : 1). Then, (ꢁ)- AGCAGCCG) and Bac 3R (CCCGGGAACGTATTCAC-
3
14)
arctigenin (15.0 g) [mp 103—104 °C, lit.
mp 100.5— CG). Amplification proceeded in a reaction mixture contain-
2
3
14)
23
D
1
ꢁ
01.5 °C; [a] ꢁ20.3° (cꢄ0.054, MeOH), lit.
[a]
ing 1 U of KOD-Plus DNA polymerase (Toyobo, Osaka,
D
2
3)
25.8° (MeOH), lit. [a] ꢁ27.5° (MeOH)] was obtained Japan), 1ꢃPCR buffer mix, 0.8 mM dNTP mix (0.2 mM
D
by crystallization from MeOH–ether. Secoisolariciresinol each), 1 mM MgSO , 0.3 mM of each primer, and 100 ng of
4
diglucoside (SDG) was provided by Suntory Co. (Osaka, template DNA. The PCR program was as follows: 94 °C for
Japan) and purified by repeated column chromatography in 2 min, 30 cycles of 94 °C for 14 s, 55 °C for 30 s, 68 °C for
our laboratory. (ꢀ)-Secoisolariciresinol was obtained as fol- 45 s, and finally 68 °C for 5 min. The PCR products were pu-
lows: SDG (96 mg) was dissolved in acetate buffer (pH 4.5) rified using a QIA Quick PCR Purification Kit (Qiagen, Ger-
and incubated with 100 mg of naringinase at 37 °C overnight. many), and directly sequenced using a Bigdye Terminator
The mixture was extracted with ethyl acetate and the ethyl Cycle Sequencing Kit (Applied Biosystems, Foster city, CA,
acetate soluble fraction was evaporated to yield a residue, U.S.A.) wth primers (Bac 1F, Bac 1R, Bac 3F, and Bac 3R)
which was chromatographed on a silica gel column eluting and an ABI PRISM 310 (Applied Biosystems, Foster city,
with CHCl –MeOH (20 : 1→10 : 1). Then, (ꢀ)-secoisolari- CA, U.S.A.).
3
ciresinol (43 mg) was obtained by crystallization from ethyl
Incubation of Arctigenin (8) with E. sp. ARC-2 and
acetate. (ꢁ)-Secoisolariciresinol 4ꢂ-O-methyl ether, (ꢀ)-di- Isolation of Metabolites (ꢁ)-Arctigenin (1 g in 10 ml
hydroxyenterodiol and (ꢁ)-dihydroxyenterodiol were ob- MeOH) and a bacterial suspension (50 ml) of E. sp. ARC-2
24)
tained by modified methods of Makela et al. from arcti- were added to 1 l of GAM broth and incubated at 37 °C in an
genin, (ꢀ)-secoisolariciresinol and (ꢁ)-secoisolariciresinol anaerobic incubator for 62 h. The reaction mixture was then
4
ꢂ-O-methyl ether, respectively.
extracted three times with ethyl acetate. The organic layer
(
ꢀ)-Secoisolariciresinol: Colorless prism. mp 102— was evaporated under reduced pressure to give a residue
2
3
1
04 °C. [a] ꢀ41° (cꢄ0.2, MeOH). This compound was (1.726 g). The residue was applied to a column of silica gel,
identified by comparing the H- and C-NMR spectra with which was eluted with a solvent system, CHCl –MeOH
D
1
13
3
13)
that published.
2R,3R)-2-(4-Hydroxy-3-methoxybenzyl)-3-(3,4-di- Fractions B—D were chromatographed on a silica gel col-
methoxybenzyl)-1,4-butanediol [(ꢁ)-Secoisolariciresinol 4ꢂ- umn (CHCl : MeOHꢄ100 : 1), followed by HPLC (22 or
(50 : 1) to give fractions A—E. Fraction E gave 1 (63 mg).
(
3
2
3
O-Methyl Ether]: Colorless prisms. mp 100—101 °C. [a]
14.2° (cꢄ0.077, MeOH). This compound was identified (14.6 mg), 7 (8.7 mg) from fraction B; compound 6 (20 mg)
by comparing the H- and C-NMR spectra with that pub- from fraction C; compounds 2 (5.4 mg), 3 (2.7 mg), 4
lished.
24% of CHCN in H O) to yield compounds 5 (16.3 mg), 6
D
3 2
ꢁ
1
13
29)
(63.5 mg) from fraction D. An original compound of arcti-

9
06
Vol. 30, No. 5
genin (121 mg) was recovered from the reaction mixture.
ration of ethyl acetate in vacuo, the residue was dissolved in
(
2R,3R)-2,3-Bis(3,4-dihydroxy)butyrolactone [(ꢁ)-Dihy- 0.3 ml of MeOH. The MeOH solution was filtered through a
2
3
droxyenterolactone] (1): Colorless oil. [a] ꢁ25.9° (cꢄ 0.2 mm membrane filter, and a 10 ml portion was injected to a
D
27)
25
D
0
.159, MeOH) [lit. [a] ꢁ14° (MeOH)]. This compound column for HPLC analysis. Concentrations of arctigenin (8)
1
was identified by comparing the H-NMR spectrum with that and its metabolites were calculated from the calibration
27)
published.
curves of the respective authentic samples.
(
2R,3R)-2-(4-Hydroxy-3-methoxybenzyl)-3-(3,4-dihy-
Incubation of Compounds 5, 6, and 7 with E. sp. ARC-
A 100 ml portion of precultured E. sp. ARC-2 was inocu-
2
3
droxybenzyl)butyrolactone (2): Amorphorus powder. [a]
2
D
27)
25
D
ꢁ
22.8° (cꢄ0.114, MeOH) [lit. [a] ꢁ20° (MeOH)]. This lated to 2 ml of GAM broth containing 0.5 mM arctigenin (for
1
compound was identified by comparing the H-NMR spec- induction of methyltransferase) and anaerobically incubated
27)
trum with that published.
for 24 h. After confirming of complete transformation to di-
(
2R,3R)-2-(3,4-Dihydroxybenzyl)-3-(4-hydroxy-3-me- hydroxyenterolactone (1) by HPLC, compounds 5, 6 and 7
1
thoxybenzyl)butyrolactone (3): Amorphorus powder. H- were separately added to a bacterial suspension and incu-
NMR (CDCl , 500 MHz): d 2.45—2.64 (4H, m, H-2, 3, 7ꢂ), bated anaerobically for 1 h. A 100 ml aliquot was then taken
3
2
.85 (2H, d, Jꢄ6.5 Hz, H-7ꢅ), 3.83 (3H, s, –OCH ), 3.88 (1H, out and analyzed as described above.
3
dd, Jꢄ8, 8.8 Hz, H -4), 4.12 (1H, dd, Jꢄ7, 8.8 Hz, H -4),
Preparation of Cell-Free Extracts E. sp. ARC-2 was
a
b
6.45 (1H, d, Jꢄ2 Hz, H-2ꢂ), 6.51—6.53 (2H, m, H-6ꢅ, 6ꢂ), cultured in 500 ml of GAM broth containing 0.1 mM arcti-
6.60 (1H, d, Jꢄ1.5 Hz, H-2ꢅ), 6.75 (1H, d, Jꢄ7.5 Hz, H-5ꢅ), genin (inducer) under anaerobic conditions at 37 °C for 11 h.
6.80 (1H, d, Jꢄ8 Hz, H-5ꢂ) (the numbering of protons is indi- The cells were harvested and then suspended in 100 mM
cated in Fig. 3). UV l
(MeOH) nm (e): 221 (12200), 281 phosphated buffer (pH 7.3). The cell suspensions were soni-
max
ꢀ
(
6100). EI-MS (rel. int.) m/z: 123 (73), 137 (100), 344 [M]
cated by 10 sonic bursts of 30 s each (Branson Sonifier 250,
Branson Ultrasonics Corporation, Danbury, CT, U.S.A.) on
2
3
(
48). [a] ꢁ24.0° (cꢄ0.075, MeOH).
D
(
2R,3R)-2-(3,4-Dihydroxybenzyl)-3-(3-hydroxy-4-me- ice, and then centrifuged at 100000ꢃg for 60 min (Ultracen-
1
thoxybenzyl)butyrolactone (4): Amorphorus powder. H- trifuge Beckman Optima XL-70, Beckman Instruments,
NMR (CD OD, 500 MHz): d 2.33—2.59 (4H, m, H-2, 3, 7ꢂ), Fullerton, CA, U.S.A.) at 4 °C. The supernatants were filtered
3
2
.74 (1H, dd, Jꢄ7, 14.5 Hz, H -7ꢅ), 2.81 (1H, dd, Jꢄ5.5, with 0.45 mm microfilter and then used as cell-free extracts.
a
14.5 Hz, H -7ꢅ), 3.78 (3H, s, –OCH ), 3.84 (1H, t, Jꢄ8.5 Hz,
Measurement of Demethylation Activity The cell-free
b
3
H -4), 4.01 (1H, dd, Jꢄ7.5, 8.5 Hz, H -4), 6.47 (2H, dd, Jꢄ2, extracts containing 0.6 mM arctigenin, 4 mM THF, and 4 mM
a
b
8
Hz, H-6ꢅ, 6ꢂ), 6.53 (1H, d, Jꢄ2 Hz, H-2ꢂ), 6.64 (1H, d, ATP were anaerobically incubated at 37 °C for 24 h. A 100 ml
Jꢄ2 Hz, H-2ꢅ), 6.69 (1H, d, Jꢄ8 Hz, H-5ꢅ), 6.77 (1H, d, aliquot was taked out and extracted with ethyl acetate. After
Jꢄ8 Hz, H-5ꢂ) (the numbering of protons is indicated in Fig. evaporation of the ethyl acetate in vacuo, the residue was dis-
3
). UV l
(MeOH) nm (e): 220 (12200), 281 (5900). EI- solved in 0.3 ml of MeOH. The MeOH solution was filtered
max
ꢀ
23
D
MS (rel. int.) m/z: 123 (68), 137 (100), 344 [M] (49). [a]
ꢁ
through a 0.2 mm membrane filter, and a 10 ml portion was
32.5° (cꢄ0.52, MeOH).
injected to a column for HPLC analysis. Concentrations of
(
2R,3R)-2,3-Bis(4-hydroxy-3-methoxybenzyl)butyrolac- arctigenin (8) and its metabolites were calculated from the
2
3
tone (5): Amorphorus powder. [a] ꢁ23.6° (cꢄ0.057, calibration curves of the respective authentic samples.
D
23)
MeOH) [lit. [a] ꢁ50.0° (MeOH)]. This compound was
Incubation of (ꢀ)-Secoisolariciresinol and (ꢁ)-Secoiso-
D
1
identified by comparing the H-NMR spectrum with that lariciresinol 4ꢂ-O-Methyl Ether with E. sp. ARC-2
A
23)
published.
100 ml portion of precultured E. sp. ARC-2 was inoculated to
2R,3R)-2-(3,4-Dihydroxybenzyl)-3-(3,4-dimethoxyben- 2 ml of GAM broth containing 0.3 mM (ꢀ)-secoisolari-
(
2
3
zyl)butyrolactone (6): Amorphorus powder. [a] ꢁ40.3° ciresinol or (ꢁ)-secoisolariciresinol 4ꢂ-O-methyl ether and
D
14)
25
D
(
cꢄ0.197, MeOH) [lit. [a] ꢁ42.8° (MeOH)]. This com- anaerobically incubated for 72 h. A 100 ml aliquot was taken
1
pound was identified by comparing the H-NMR spectrum out and extracted with ethyl acetate. After evaporation of the
14)
with that published.
ethyl acetate in vacuo, the residue was dissolved in 0.3 ml of
(
2R,3R)-2-(4-Hydroxy-3-methoxybenzyl)-3-(3-hydroxy-4- MeOH. The MeOH solution was filtered through a 0.2 mm
1
methoxybenzyl)butyrolactone (7): Amorphorus powder. H- membrane filter, and a 10 ml portion was injected to a col-
NMR (CDCl , 500 MHz): d 2.43—2.62 (4H, m, H-2, 3, 7ꢂ), umn for chiral HPLC analysis under the following condi-
3
2
.92 (2H, m, H-7ꢅ), 3.82—3.86 (7H, m, H -4, –OCH ꢃ2), tions: column, chiral CD-Ph (Shiseido, Tokyo, Japan,
a 3
4
.09 (1H, dd, Jꢄ7, 9 Hz, H -4), 6.47 (1H, dd, Jꢄ2.3, 8 Hz, 4.6 mmꢃ250 mm); mobile phase, 0.1% TFA (solvent system
b
H-6ꢂ), 6.58 (1H, d, Jꢄ2.3, H-2ꢂ), 6.63 (1H, dd, Jꢄ2, 8 Hz, A) and CH CN (solvent system B) in a gradient mode (B
3
H-6ꢅ), 6.67 (1H, d, Jꢄ2, H-2ꢅ), 6.72 (1H, d, Jꢄ8 Hz, H-5ꢂ), from 25 to 31% for 18 min, B from 31 to 42% from 18 to
6
.83 (1H, d, Jꢄ8 Hz, H-5ꢅ) (the numbering of protons is indi- 40 min); flow rate, 0.5 ml/min; detection, UV 280 nm; tem-
cated in Fig. 3). UV l
(MeOH) nm (e): 223 (12900), 281 perature, 30 °C. The retention times of (ꢀ)-dihydroxyentero-
max
ꢀ
23
D
(
6300). EI-MS (rel. int.) m/z: 137 (100), 358 [M] (34). [a]
diol, (ꢁ)-dihydroxyenterodiol, (ꢀ)-secoisolariciresinol and
(ꢁ)-secoisolariciresinol 4ꢂ-O-methyl ether were 14.3, 15.1,
ꢁ31.0° (cꢄ0.087, MeOH).
Time Course for the Transformation of Arctigenin (8) 23.8 and 31.5 min, respectively.
by E. sp. Strain ARC-2 GAM broth (3 ml) containing
arctigenin (a final concentration of 0.6 mM) was incubated RESULTS
with a bacterial suspension of E. sp. ARC-2 (150 ml) at 37 °C
under anaerobic conditions. A 100 ml aliquot was taked out at
Isolation of a Bacterial Strain Capable of Transforming
intervals of 1 h and extracted with ethyl acetate. After evapo- (ꢁ)-Arctigenin (8) to (ꢁ)-2,3-Bis(3,4-dihydroxybenzyl)bu-

May 2007
907
tyrolactone (Dihydroxyenterolactone) (1) from a Human Table 1. Biochemical Characteristics of E. sp. ARC-2 and E. limosum
Fecal Suspension Through repeated culture of a bacterial
a)
Characterisitic
E. sp. ARC-2
E. limosum
mixture of human feces in GAM broth containing 0.5 mM
arctigenin (8), and screening of colonies on GAM agar plates
for demethylation activity, a bacterial strain (strain ARC-2)
capable of transforming 8 to 1 was isolated. Strain ARC-2
was strictly anaerobic, Gram-positive, and regular rods
Gram stain
Aerobic growth
Motility
Esculin hydrolysis
Indole production
Starch hydrolysis
Nitrate reduction
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ
(Table 1). Based on these characteristics, strain ARC-2 was
ꢁꢀ
ꢁ
considered to belong to the genus Eubacterium. The strain
was not able to produce indole and nitrite, and hydrolyzed es-
culin but not starch. In addition, it produced butyric acid
from glucose predominantly and fermented erythritol, fruc-
tose, glucose, mannitol, and ribose (Table 2). Therefore, the
strain was concluded to be an Eubacterium sp. with charac-
teristics similar to those of E. limosum (Genbank accession
no. M59120), and was named E. sp. ARC-2 (EF413640).
Furthermore, the 16S rRNA gene sequence had 99% similar-
ity to that of E. limosum (Fig. 1).
26)
a) Data of “Bergey’s Manual of Systematic Bacteriology.” Symbols: ꢀ, positive;
ꢁ
, negative.
Table 2. Comparative Fermentation Reactions of E. sp. ARC-2 and E.
limosum (ATCC8486)
a)
Carbohydrate
E. sp. ARC-2
E. limosum
Amygdalin
Arabinose
Cellobinose
Dextrin
Erythritol
Esculin
Fructose
Glucose
Glycerol
Glycogen
Inositol
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
ꢀ
ꢀ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
w
ꢁ
ND
ꢀ w
ꢁ
ꢀ
ꢀ
ꢁ w
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ w
ꢀ w
ꢁ w
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ w
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
d
Transformation of (ꢁ)-Arctigenin (8) by E. sp. ARC-2
After anaerobic incubation of arctigenin (8) with E. sp.
ARC-2 for 12 h, seven metabolites together with compound 1
were detected in the culture by HPLC analysis (Fig. 2). For
isolating the metabolites, a large amount of arctigenin (8)
was cultured with E. sp. ARC-2 under the similar conditions.
The culture was extracted with ethyl acetate and the extract
was subject to silica gel column chromatography and HPLC.
Seven metabolites (1—7) (Fig. 3) were isolated and the
1
13
Inulin
Lactose
structures of these metabolites were determined by H-, H-
NMR (Table 3), 2D-NMR and MS spectroscopy.
Maltose
Mannitol
Mannose
Melbiose
Melezitose
Raffinose
Rhamnose
Ribose
Salicin
Sorbitol
Starch
Sucrose
Trehalose
Xylose
Compounds 5—7 and 2—4 showed molecular ion peaks at
ꢀ
ꢀ
m/z 358 [M] and 344 [M] , respectively, in the ESI-MS
spectra. The formers were 14 mass units less than the molec-
ular ion of (ꢁ)-arctigenin (8), suggesting that 5—7 were di-
methyl derivatives of dihyroxyenterolactone, while the latters
were 28 mass units less, suggesting that 2—4 were mono-
methyl derivatives of dihydroxyenterolactone. This was con-
firmed by the presence of only two or one signal(s) for O-
methyl proton(s) in comparison with three siglet signals of
1
arctigenin in the H-NMR spectra.
For determining the location of methoxy groups of com-
pounds 5—7, heteronuclear multiple-bond coherence
2
5)
a) Data of “A Color Atlas of Anaerobic Bacteria,” “Bergey’s Manual of System-
(HMBC) experiments were undertaken to see HMBC corre-
26)
atic Bacteriology.” Symbols: ꢀ, positive (pH below 5.5); ꢁ, negative (pH upper 5.9);
lations between signals of methoxy protons and aromatic car- w, weak reaction (pH 5.5—5.9); d, 40—60% of strains positive; ND, no data.
Fig. 1. Dendrogram of Phylogenic Affiliation of E. sp. ARC-2
A 16S rRNA gene sequence-based phylogenic tree shows relationship between newly isolated E. sp. ARC-2 (EF413640) and other closely related species. The sequence of E. sp.
ARC-2 was compared to those from Genbank database with ClustalW program.

9
08
Vol. 30, No. 5
13
Table 3.
C-NMR Spectral Data of Compounds 1—8
a,c)
a,d)
b,d)
a,c)
b,d)
b,d)
b,d)
a,c)
C
1
2
3
4
5
6
7
8
1
2
3
4
181.7
47.7
42.6
181.4
47.9
42.4
179.1
46.5
41.1
181.7
47.7
42.5
178.6
46.6
41.0
179.9
46.5
40.8
178.5
46.7
41.0
181.5
47.7
42.4
72.8
72.8
71.5
72.8
71.3
71.7
71.3
72.9
1
2
3
4
5
6
7
1
2
3
4
5
6
7
ꢅ
130.8
117.5
146.3
145.1
116.4
121.9
35.1
131.5
116.8
146.3
144.9
116.4
121.0
38.6
130.5
113.8
148.8
146.2
116.0
122.9
35.2
131.3
116.7
146.2
144.8
116.3
120.9
38.7
130.1
116.0
143.6
142.7
115.1
121.6
34.2
129.7
111.0
146.5
144.2
114.4
121.3
38.3
130.7
117.4
146.3
145.1
116.4
121.9
35.1
132.7
116.7
147.4
147.6
112.8
121.0
38.4
129.4
111.3
146.5
144.4
114.0
121.9
34.6
129.6
110.8
146.4
144.2
114.3
121.2
38.4
129.8
116.1
144.0
142.9
115.2
121.5
34.0
130.5
111.7
148.9
147.7
111.3
120.7
38.0
129.4
111.5
146.5
144.4
114.2
122.0
34.6
131.0
114.6
145.5
145.2
110.6
120.0
38.0
130.7
113.8
149.0
146.4
116.1
123.0
35.4
132.8
113.5
150.4
149.1
113.0
122.0
38.8
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
–
–
Me
Me
56.3
55.9
56.4
55.8
55.9
55.8
55.9
55.9
56.0
56.4
56.3ꢃ2
Measured in a) CD OD and b) CDCl . NMR data performed on spectrometers of c) 125 MHz and d) 75 MHz.
3
3
Fig. 2. HPLC Profile of Metabolites of Arctigenin by E. sp. ARC-2
A 100 ml portion of precultured E. sp. ARC-2 was inoculated to 2 ml of GAM broth containing 0.5 mM arctigenin, and anaerobically incubated for 12 h. A 100 ml aliquot was
then taken out and analyzed under the following conditions: Column, TSK-gel ODS-80Ts (4.6 mm i.d.ꢃ150 mm, Tosoh); solvent, CH CN 25→26.2% (50 min); detector, UV at
3
280 nm; flow rate, 1 ml/min; temp., room temperature.
carbons at C-3ꢂ and C-4ꢂ, and compound 5 showed correla-
tions between signals of methoxy protons and carbons at C-
3
5
ꢅ and C-3ꢂ. Therefore, the structures of compounds 6 and
were concluded to be 3ꢅ-desmethylarctigenin, and 4ꢂ-
desmethylarctigenin, respectively.
In addition, compound 2 was identified as dihydroxyen-
terolactone 3ꢅ-O-methyl ether on the basis of the correlations
between signals of methoxy protons and a carbon at C-3ꢅ in
the HMBC experiment and a correlation between signals of
methoxy protons and an H-2ꢅ proton in the NOESY experi-
ment. As compound 3 showed HMBC correlations between
signals of methoxy protons and a carbon at C-3ꢂ, the struc-
ture was determined to be dihydroxyenterolactone 3ꢂ-O-
methyl ether. Compound 4 was concluded to be dihydroxyen-
terolactone 4ꢂ-O-methyl ether on the basis of a NOESY cor-
relation between signals of methoxy protons and an H-5ꢂ
proton.
Fig. 3. Arctigenin and Its Metabolites by E. sp. ARC-2
bons. Compound 7 showed HMBC correlations between sig-
The metabolic processes were deduced by time course ex-
nals of methoxy protons and signals of carbons at C-3ꢅ and periments and structures of isolated metabolites. A stepwise
C-4ꢂ. The structure of compound 7 was consequently deter- demethylation from arctigenin to dihydroxyenterolactone
mined to be 3ꢂ-desmethylarctigenin. Compound 6 showed was proceeded by incubation with E. sp. ARC-2 (Fig. 4).
HMBC correlations between signals of methoxy protons and Compounds 5, 6 and 7 were independently incubated with E.

May 2007
909
Fig. 4. Time Course of Transformation of (ꢁ)-Arctigenin (8) to (ꢁ)-Dihydroxyenterolactone (1) by E. sp. ARC-2
Fig. 5. Possible Metabolic Processes of Arctigenin by E. sp. ARC-2
sp. ARC-2 for confirming of transformation pathways to ARC-2 cells.
compounds 2, 3 and 4. The HPLC analysis of an incubation
Substrate Specificity of E. sp. ARC-2 The substrate
mixture showed that compound 5 was demethylated to yield specificity of this bacterium was assayed using (ꢀ)-secoiso-
compounds 2 and 3, compound 6 was transformed to 3 and lariciresinol and (ꢁ)-secoisolariciresinol 4ꢂ-O-methyl ether
4
, compound 7 to 2 and 4, respectively (data not shown). (Fig. 6). All methoxy groups of two compounds were elimi-
Based on these findings, we drew metabolic pathways of nated to yield (ꢀ)-dihydroxyenterodiol and (ꢁ)-dihydroxy-
demethylation of arctigenin (Fig. 5). Arctigenin (8) was enterodiol, respectively, by incubation with E. sp. ARC-2.
transformated by E. sp. ARC-2 to yield a number of metabo-
lites, but the main pathway seems to be 8→7→4→1 on the DISCUSSION
basis of the time course experiments of the respective
metabolites and the structural consideration.
Biotransformation of lignans by intestinal bacteria has
Effects of Tetrahydrofolate and ATP on Demethylation been studied for a long time. Elimination of glucose,
by a Cell-Free Extract of E. sp. ARC-2 When arctigenin methoxy, and hydroxy groups is essential steps to yield END
14)
was incubated with a cell-free extract of E. sp. ARC-2, only or ENL from precursors such as arctiin, pinoresinol di-
27)
13)
3
6.4% for mono-desmethylarctigenins and 6.0% for di- glucoside, and secoisolariciresinol diglucoside. In the
desmethylarctigenins were produced and the rest was recov- course of studies on the metabolic activation of herbal medi-
ered as the strarting material (Tabel 4). However, addition of cines, we isolated some bacterial strains involved in the me-
ATP (4 mM) increased the percentage of demethylated prod- tabolism of lignans, and investigated their characteristics and
ucts, which accounted for 39.2% for mono-desmethylarcti- metabolites.
genins and 9.6% for di-desmethylarctgenins. Addition of
THF (4 mM) also increased the demethylated products up to teria capable of demethylation. Since E. sp. ARC-2 was simi-
5.0% and 23.4% for mono-desmethylarctigenins, and di- lar to E. limosum in characteristics and 16S rRNA gene se-
We isolated a bacterial strain, E. sp. ARC-2 as one of bac-
4
desmethylarctigenins, respectively. Moreover, addition of quence, we anaerobically incubated arctigenin (8) with either
both ATP and THF appreciably increased in the percentage E. limosum (ATCC 8486) or E. sp. ARC-2 and obtained the
of the demethylated products, which accounted for 39.5%, same metabolite dihydroxyenterolactone. However, the shape
40.7% and 7.2% for mono-, di-, and tri-desmethylarcti- and viscosity of colony in BL agar plates was different from
genins, respectively. The demethylation by the cell-free ex- each other. Especially, the high viscosity of E. limosum was
tract seems to proceed via 8→7→4→1 as that by E. sp. apparent in contrast with that of E. sp. ARC-2.

9
10
Vol. 30, No. 5
Table 4. Effects of Tetrahydrofolate (THF) and ATP on Demethylation by a Cell-Free Extract (CFE) of E. sp. ARC-2
Recovered
Dimethyl der.
Monomethyl der.
Free form
8
7
6
5
4
3
2
1
3
6.4
6.0
0.8
CFE
57.4
51.2
29.0
12.6
0.2
0
22.0
24.8
28.3
27.5
9.8
4.6
5.4
5.3
4.2
3.8
1.4
2.7
4.3
7.0
3
9.2
9.6
CFEꢀATP
CFEꢀTHF
9.0
5.4
1.5
4
5.0
23.4
2.7
2.6
7.2
11.4
16.4
29.6
3
9.5
40.7
4.1
CFEꢀATPꢀTHF
7.8
Relative concentration (%).
22)
transferase.
For investigating substrate specificity, (ꢀ)-secoisolari-
ciresinol and (ꢁ)-secoisolariciresinol 4ꢂ-O-methyl ether were
anaerobically incubated with E. sp. ARC-2. Both of the com-
pounds were tranformed to dihydroxyenterodiol, indicating
that demethylation by E. sp. ARC-2 is taken place regardless
of their (ꢀ)- or (ꢁ)-forms. Moreover, dihydroxyenterolac-
tone was obtained by anaerobic incubation of arctiin with E.
sp. ARC-2 in GAM broth at 37 °C (data not shown). Vanillic
acid, isovanillic acid and 3,4-dimethoxybenzoic acid were
transformed to protocatechuic acid by incubation with this
bacterium. Syringic acid and 3,4,5-trimethoxybenzoic acid
were transformed to gallic acid, too (data not shown). E. sp.
ARC-2 showed low substrate specificity compared with P. sp.
SDG-1, which requires a 4-hydroxy-3-methoxy phenyl ring
Fig. 6. Biotranformation of (ꢀ)-Secoisolariciresinol and (ꢁ)-Secoisolari-
ciresinol 4ꢂ-O-Methyl Ether by E. sp. ARC-2
13)
system as a substrate. The former is more preferable for
modification of naturally occurring methylated compounds to
We isolated seven demethylated metabolites (1—7) of 8 by free hydroxyl forms. As regards microbial demethylation, it
incubation with E. sp. ARC-2 although the production of was recently reported that E. limosum had metabolic activity
these metabolites seems to vary depending on the bacteri- capable of O-demethylation of biochanin A, formononetin,
28)
al constitutions in the gastro-intestinal flora of humans. and glycitein.
Through the transformation experiments and the time course Since dehydroxylation in the metabolic processes of lig-
of intermediate and final metabolite formations, the meta- nans to enterolactone or enterodiol takes place only in the
bolic pathway was speculated as shown in Fig. 5. In the pres- presence of vicinal two hydroxy groups, the accumulation of
ent study, 8 was demonstrated to transform through the major metabolic intermediates having free 3,4-dihydroxy phenyl
processes of 8→7→4→1 by E. sp. ARC-2, although it was residues catalyzed by intestinal bacteria such as E. sp. ARC-
reported to proceed through a processes of 8→6 by a bacter- 2, may be preferable for the formation of phytoestrogenic
14)
ial mixture of human feces. This difference of metabolic substances.
processes between a single bacterium and bacterial flora is
important to understand the actual event in the intestinal
Acknowledgements Part of this study was financially
tract. Furthermore, when we added 8 to E. sp. ARC-2 al- supported by the aid of Ministry of Education, Culture,
ready proliferated in GAM broth, the major process was Sports, Science and Technology (MEXT) as the 21st Century
8
→(6, 7)→4→1 (data not shown). This means the pattern of of Center of Excellence Program and the Sasakawa Scientific
biotransformation by intestinal bacteria may be altered due to Research Grant from The Japan Science Society.
the number of bacteria or the substrates present in the intes-
tinal environment.
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essary for demethylating activity of E. sp. ARC-2, suggesting
that demethylation is catalyzed by THF-dependent methyl-
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