Further studies on a human intestinal bacterium Ruminococcus sp. END-1 for transformation of plant lignans to mammalian lignans

Article abstract of DOI:10.1021/jf900902p

A human intestinal bacterium Ruminococcus (R.) sp. END-1 capable of oxidizing (-)-enterodiol to (-)-enterolactone, enantioselectively, was further investigated from the perspective of transformation of plant lignans to mammalian lignans; A cell-free extra

Full text of DOI:10.1021/jf900902p

J. Agric. Food Chem. 2009, 57, 7537–7542 7537
DOI:10.1021/jf900902p
Further Studies on a Human Intestinal Bacterium
Ruminococcus sp. END-1 for Transformation of Plant Lignans
to Mammalian Lignans
†,‡
IN
,†
*
J
ONG-SIK
J
AND
MASAO
HATTORI
†Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan, and
‡Benno Laboratory, Center for Intellectual Property Strategies, RIKEN, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan
A human intestinal bacterium Ruminococcus (R.) sp. END-1 capable of oxidizing (-)-enterodiol to
(-)-enterolactone, enantioselectively, was further investigated from the perspective of transfor-
mation of plant lignans to mammalian lignans; A cell-free extract of the bacterium transformed
(-)-enterodiol to (-)-enterolactone through an intermediate, enterolactol. The bacterium showed not
only oxidation but also demethylation and deglucosylation activities for plant lignans. Arctiin and
secoisolariciresinol diglucoside were converted to (-)-dihydroxyenterolactone and (þ)-dihydroxyen-
terodiol, respectively. Moreover, by coincubation with Eggerthella sp. SDG-2, the bacterium
transformed arctiin and secoisolariciresinol diglucoside to (-)-enterolactone and (þ)-enterodiol,
respectively.
KEYWORDS: Mammalian lignan; human intestinal bacteria; enterolactol; enantioselective oxidation
INTRODUCTION
and strain ARC-1 (EF413639), which were isolated from human
feces. Eg. sp. SDG-2 transformed (þ)-dihydroxyenterodiol and
(-)-dihydroxyenterolactone to (þ)-enterodiol and (-)-enterolac-
tone, respectively, but left out (-)-dihydroxyenterodiol and
(þ)-dihydroxyenterolactone. Conversely, strain ARC-1 trans-
formed (-)-dihydroxyenterodiol and (þ)-dihydroxyenterolactone
to (-)-enterodiol and (þ)-enterolactone, respectively, but left out
(þ)-dihydroxyenterodiol and (-)-dihydroxyenterolactone.
Ruminococcus (R.) sp. END-1 (EF451052) and strain END-2
(EF451053) are human intestinal bacteria capable of oxidizing
enterodiol to enterolactone (20). The former transformed
(-)-enterodiol to (-)-enterolactone, selectively. The latter oxi-
dized (þ)-enterodiol to (þ)-enterolactone.
Enterodiol and enterolactone are metabolites of several plant
lignans such as pinoresinol diglucoside, secoisolariciresinol diglu-
coside, sesamin, and arctiin by gut microflora (1-4). Since
metabolites of these lignans are categorized as phytoestrogens,
their biological activities have been extensively studied. They
showed estrogenic and antiestrogenic activities (5-7) as well as
antioxidant activity (8). Moreover, it is reported that enterolac-
tone inhibits the growth of prostate cancer cell lines in vitro (9,10)
and mammary carcinomas in vivo (11), indicating the possible
association with epidemiological studies that phytoestrogens
have potential health benefits in hormone-dependent diseases
such as breast, colon, and prostate cancers (12-14).
The metabolic processes of precursors to enterodiol and
enterolactone by intestinal bacteria include deglucosylation,
demethylenation, ring cleavage, demethylation, dehydroxylation,
and oxidation of diols to γ-butyrolactones. The bacteria respon-
sible for these processes have been reported. (1, 2, 15-18).
However, both compounds occur as enantiomeric, mirror image
forms in nature (Figure 1). Enterolactone, isolated from incuba-
tion of arctiin and pinoresinol diglucoside with intestinal bacteria,
was a (-)-form, while that isolated from incubation of secoiso-
lariciresinol diglucoside was a (þ)-form. Interconversion between
the respective enantiomers did not occur during bacterial meta-
bolism (19). Therefore, metabolic specificity must be considered
between enantiomers as well as their biological activities.
In this article, we report additional properties of R. sp. END-1
in the metabolism of plant lignans, as regards kinetics in trans-
formation, substrate specificity, and coculture with another
bacterium, Eg. sp. SDG-2.
MATERIALS AND METHODS
General. An anaerobic incubator, model EAN-140 (Tabai Co., Osaka,
Japan), was used for the incubation of fecal suspensions and intestinal
bacteria. Optical rotations were measured in MeOH solutions with a DIP-
360 automatic polarimeter (Jasco Co., Tokyo, Japan). 1H- and 13C NMR
spectra were measured with Varian Unity 500 (1H, 500 MHz; 13C, 125
MHz) and Varian Gemini 300 (1H, 300 MHz; 13C, 75 MHz). Thin-layer
chromatography (TLC) was carried out on silica gel precoated 60 F254
plates (0.25 mm, Merck Co., Darmstadt, Germany), and spots were
detected under a UV lamp or exposing I2 vapor. Silica gel BW-820 MH
(Fuji Silysia, Aichi, Japan) was used for column chromatography.
Chemicals and Media. General anaerobic medium (GAM) was
purchased from Nissui Co. (Tokyo, Japan). (-)-Arctigenin, secoisolar-
iciresinol diglucoside, (þ)-secoisolariciresinol, (þ)-dihydroxyenterodiol,
We recently reported enantioselective dehydroxylation by
Eggerthella (Eg.) sp. SDG-2 (Genbank accession no. EF413638)
*Corresponding author. Tel: þ81-76-434-7630. Fax: þ81-76-434-
5060. E-mail: saibo421@inm.u-toyama.ac.jp.
© 2009 American Chemical Society
Published on Web 07/24/2009
pubs.acs.org/JAFC

7538 J. Agric. Food Chem., Vol. 57, No. 16, 2009
Jin and Hattori
system A) and CH3CN (solvent system B) in gradient modes [(-)-
enterodiol, (-)-enterolactone, and enterolactol analysis, B from 20 to
50% for 30 min, (-)-hydroxyenterolactone analysis, B from 10 to 35% in
50 min]; flow rate, 1.0 mL/min; detection, UV at 280 nm; temperature,
30 °C. High-purity nitrogen was used as dry gas at a flow rate of 10 L/min,
with a temperature of 360 °C. Helium was used as nebulizer at 50 psi. The
ESI interface and mass spectrometric parameter were optimized to obtain
maximum sensitivity.
Bacterial Growth and Kinetics in Biotransformation with R. sp.
END-1. GAM broth (4 mL) containing (-)-enterodiol (a final concen-
tration of 0.25 mM) was incubated with a bacterial suspension of R. sp.
END-1 (400 μL) at 37 °C under anaerobic conditions. A 100 μL aliquot
was taken out at 3 h intervals and extracted three times with 200 μL of ethyl
acetate. After evaporation of ethyl acetate in vacuo, the residue was
dissolved in 0.3 mL of MeOH. The MeOH solution was filtered through a
0.45 μm membrane filter, and a 10 μL portion was injected into a column
for HPLC analysis. HPLC was performed on a CCPM-II (Tosoh, Tokyo,
Japan) equipped with a Tosoh UV-8020 spectrometer and a Shimadzu
C-R8A chromatopac (Shimadzu, Kyoto, Japan) under the following
conditions: column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan,
4.6 mm ꢀ 150 mm); mobile phase, 0.1% TFA (solvent system A) and
CH3CN (solvent system B) in a gradient mode (B from 30 to 50% in
20 min); flow rate, 1.0 mL/min; detection, UV 280 nm; temperature, room
temperature. Concentrations of (-)-enterodiol and (-)-enterolactone
were calculated from calibration curves of the respective authentic
samples. Optical density at 540 nm was measured with a UV-2200 UV-
vis recording spectrophotometer (Shimadzu Co., Kyoto, Japan).
Preparation of a Cell-Free Extract. R. sp. END-1 was cultured
under anaerobic conditions at 37 °C for 12 h in GAM broth containing
0.2 mM (-)-enterodiol (inducer), harvested, and then suspended in
100 mM phosphate buffer (pH 7.3). The cell suspensions were sonicated
by 10 sonic bursts of 30 s each (Branson Sonifier 250, Branson Ultrasonics
Corporation, Danbury, CT, USA) on ice and then centrifuged at 100,000g
for 60 min (Ultracentrifuge Beckman Optima XL-70, Beckman Instru-
ments, Fullerton, CA, USA) at 4 °C. The supernatants were filtered with a
0.45 μm microfilter and then used as cell-free extracts.
Figure 1. Enantiomers of enterodiol, enterolactone, dihydroxyenterodiol,
and dihydroxyenterolactone.
(-)-dihydroxyenterodiol, (þ)-dihydroxyenterolactone, (-)-dihydroxyen-
terolactone, (þ)-enterodiol, (-)-enterodiol, (þ)-enterolactone, and
(-)-enterolactone were prepared by the same methods of Jin et al. (21).
Enterolactol was prepared by a modified method of Xia et al. (22).
Enterolactol [(3R,4R)-3,4-bis(3-hydroxybenzyl)-tetrahydrofuran-2-ol]:
amorphous powder. EI-MS m/z: 300 [M]þ. 1H NMR (CD3OD, 300
MHz): δ 2.10, 2.55 (m, H-3, 4, 70, 700), 3.52 (t, J = 8.0 Hz, Ha-5), 3.70
(t, J = 8.5 Hz, Ha-5), 3.83 (t, J = 8.5 Hz, Hb-5), 3.99 (t, J = 8.0 Hz, Hb-5),
5.12 (m, H-2), 6.64, 7.07 (m, H-10, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500, 600). 13C
NMR (CD3OD, 75 MHz): δ 34.9, 39.6, 40.0, 44.1, 47.5, 53.0, 54.8, 72.5,
73.1, 99.8, 104.2, 113.7, 113.8, 113.9, 114.0, 116.3, 116.3, 116.7, 116.8,
120.7, 120.8, 121.1, 121.2, 130.1, 130.2, 142.5, 143.0, 143.2, 143.6, 158.1,
158.2, 158.2, 158.3.
(-)-Secoisolariciresinol was prepared from pinoresinol diglucoside as
follows: a bacterial suspension (50 mL) of strain PUE (23) and Eg. sp.
SDG-2 was inoculated to 500 mL of GAM broth containing pinoresinol
diglucoside (100 mg) and incubated at 37 °C in an anaerobic incubator for
180 h. The reaction mixture was then extracted three times with 500 mL of
ethyl acetate. The organic layer was evaporated under reduced pressure to
yield a residue. The residue was applied to a column of silica gel (45 g),
which was eluted with a solvent system of CHCl3-MeOH (20:1). Then,
fractions including (-)-secoisolariciresinol were concentrated under re-
duced pressure and applied to a column of Sephadex LH-20, which was
eluted with a solvent system of H2O-MeOH (1:1) to yield (-)-secoisolar-
iciresinol (36.8 mg). The compound was identified by comparing the
1H- and 13C NMR spectra with those published (1). (þ)-Desdimethylpi-
noresinol was prepared from pinoresinol diglucoside as follows: a bacterial
suspension (2 mL) of Eubacterium (E.) sp. ARC-2 was inoculated to
200 mL of GAM broth containing 50 mg of pinoresinol diglucoside and
incubated at 37 °C in an anaerobic incubator for 132 h. The reaction
mixture was then extracted three times with 300 mL of ethyl acetate. The
organic layer was evaporated under reduced pressure to yield a residue.
The residue was applied to a column of silica gel (62 g), which was eluted
with a solvent system of CHCl3-MeOH (15:1). Then, fractions including
(þ)-desdimethylpinoresinol were concentrated under reduced pressure
and applied to a column of Sephadex LH-20, which was eluted with a
solvent system of H2O-MeOH (1:1) to yield (þ)-desdimethylpinoresinol
(8 mg). (þ)-Desdimethylpinoresinol: amorphous powder. [R]2D3 58.0 (c =
Incubation of R. sp. END-1 with Substrates. Each substrate
(0.1 mM) was anaerobically incubated with R. sp. END-1 for 120 h at
37 °C, and then a 100 μL aliquot was taken out and extracted three times
with 200 μL of ethyl acetate. After evaporation of ethyl acetate in vacuo,
the residue was dissolved in 0.3 mL of MeOH. The MeOH solution was
filtered through a 0.45 μm membrane filter, and a 10 μL portion was
injected onto a column for LC/MS analysis. The conversion of substrates
was confirmed by TLC.
Coincubation of R. sp. END-1 and Eg. sp. SDG-2. Each substrate
(0.1 mM) was anaerobically incubated with R. sp. END-1 and Eg. sp.
SDG-2 for 120 h at 37 °C, and then a 100 μL aliquot was taken out and
treated as described above.
RESULTS
Bacterial Growth and Biotransformation of (-)-enterodiol to
(-)-enterolactone by R. sp. END-1. Figure 2 shows bacterial
growth and time course of (-)-enterolactone formation by
R. sp. END-1 in GAM broth containing 0.25 mM (-)-enterodiol.
The bacterium grew maximal within 18 h under anaerobic
conditions monitored by turbidity at 540 nm. The metabolic
activity of oxidizing (-)-enterodiol to (-)-enterolactone was
observed accompanied by bacterial growth. (-)-Enterodiol
was decreased in amount from 9 h, while (-)-enterolactone was
gradually increased. The maximal concentration of (-)-entero-
lactone was obtained at 30 h. A metabolic intermediate was
detected during the cultivation but not measured quantitatively
because of a small amount (data not shown).
1
0.729, MeOH). H NMR (CD3OD, 300 MHz): δ 3.07 (2H, m, H-8, 80),
3.78 (2H, dd, J = 3.5, 9.0 Hz, Ha-9, 90), 4.19 (2H, dd, J = 7.0, 9.0 Hz, Hb-9,
90), 4.62 (2H, d, J = 4.5 Hz, H-7, 70), 6.68 (2H, dd, J = 2.5, 8.0 Hz, H-6, 60),
6.73 (2H, d, J = 8.0 Hz, H-5, 50), 6.80 (2H, d, J = 2.5 Hz, H-2, 20). 13C
NMR (CD3OD, 75 MHz): δ 55.2 (C-8, 80), 72.5 (C-9, 90), 87.3 (C-7, 70),
114.3 (C-2, 20), 116.1 (C-5, 50), 118.7 (C-6, 60)þ, 133.6 (C-1, 10), 145.8 (C-3,
30), 146.2 (C-4, 40). EI-MS m/z: 331 [M þ H] .
LC/MS Analysis. HPLC/MS was performed on an Agilent 1100
system (Agilent Technologies, Waldbronn, Germany) with a photodiode
array detector, an Agilent 1100 series binary pump, an Esquire 3000plus
mass spectrometer (Bruker Daltonic GmbH, Bremen, Germany) coupled
with an ESI interface, and an ion trap mass analyzer, under the following
conditions: column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan,
4.6 mm ꢀ 150 mm); mobile phase, 0.1% trifluoroacetic acid (TFA, solvent
Identification of an Intermediate in the Transformation of
(-)-enterodiol by R. sp. END-1. Since a little amount of the
metabolic intermediate was detected, we obtained a relatively
large amount of it for structural elucidation by incubation of
(-)-enterodiol with a cell-free extract of R. sp. END-1. The same

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J. Agric. Food Chem., Vol. 57, No. 16, 2009 7539
with retention times of 36.9 and 36.5 min, respectively (Figure 4).
The former was identical with that obtained by incubation of
(-)-dihydroxyenterolactone with Eg. sp. SDG-2.
Demethylation Activity of R. sp. END-1. The demethylation
activity of R. sp. END-1 was demonstrated by incubation of
either (-)-arctigenin or (-)-secoisolariciresinol, which resulted
in the formation of (-)-dihydroxyenterolactone. Moreover,
(þ)-secoisolariciresinol was transformed to (þ)-dihydroxyenter-
odiol by the bacterium (Figure 5).
β-Glucosidase Activity of R. sp. END-1. R. sp. END-1 showed
β-glucosidaseactivity for some plant lignans. Pinoresinol digluco-
side and secoisolariciresinol diglucoside were converted to
(þ)-desdimethylpinoresinol and (þ)-dihydroxyenterodiol, re-
spectively, by elimination of a glucose moiety, in which the latter
product was further demethylated (Figure 5).
Figure 2. Transformation kinetics of (-)-enterodiol to (-)-enterolactone
by R. sp. END-1.
Transformation of Plant Lignans by Coincubation of R. sp.
END-1 and Eg. sp. SDG-2. When pinoresinol diglucoside was
anaerobically incubated with both R. sp. END-1 and Eg. sp.
SDG-2 at 37 °C, (-)-enterolactone was detected in an ethyl
acetate extract from the bacterial suspension by analytical LC/
MS with a chiral column. In the conversion of pinoresinol
diglucoside to (-)-enterolactone, R. sp. END-1 participated in
deglucosylation, demethylation, and oxidation, while Eg. sp.
SDG-2 participated in ring cleavage and dehydroxylation
(Figure 6). In the same way, R. sp. END-1 was responsible
for deglucosylation and demethylation of secoisolariciresinol
diglucoside and Eg. sp. SDG-2 for dehydroxylation to pro-
duce (þ)-enterodiol. Moreover, arctiin was also converted to
(-)-enterolactone by co-operation of both bacteria.
DISCUSSION
The oxidative lactonization of dibenzylbutane-1,4-diols is
essential in the formation of enterolactone from plant lignans
such as pinoresinol diglucoside, secoisolariciresinol diglucoside,
and sesamin by intestinal flora. Some bacteria capable of oxidiz-
ing enterodiol to enterolactone have been reported; Clavel et al.
isolated strain ED-Mt61/PYG-s6, which was classified into
Lactonifactor longoviformis as an enterolactone-producing bac-
terium from human feces (16, 18). We also isolated two bacterial
species, R. sp. END-1 and strain END-2, capable of enantiose-
lectively oxidizing enterodiol to enterolactone from human feces.
R. sp. END-1 enantioselectively converted diols such as (-)-
dihydroxyenterodiol, (-)-hydroxyenterodiol, and (-)-enterodiol
to the corresponding γ-lactones, indicating that the oxidation
proceeds regardless of the presence or absence of a para hydro-
xyl group in the phenyl rings. 40- Hydroxyenterolactone and
400- hydroxyenterolactone were detected during the transforma-
tion of pinoresinol diglucoside (1), indicating that oxidation takes
place both sides at C-1 and C-4 in the butane-1,4-diol moiety.
When (-)-enterodiol was incubated with R. sp. END-1, enter-
olactol was detected as an intermediate; however, we could not
analyze it quantitatively because of a faint amount of formation
during the incubation. Although this compound was newly identi-
fied in the present experiment as an intermediate of the transfor-
mation of precursor lignans to enterolactone, a similar compound,
(-)-lactol, was already reported in plant metabolic processes.
Secoisolariciresinol dehydrogenase from Forsythia intermedia
converted (-)-secoisolariciresinol to (-)-matairesinol via (-)-
lactol (22). The secoisolariciresinol dehydrogenase was enantiose-
lective in the oxidation of secoisolariciresinol. A similar dehydro-
genase has not been isolated from R. sp. END-1 yet. However,
because cell-free extract of the strain converted to (-)-enterodiol to
enterolactol and (-)-enterolactone, further experiments will make
clear the enzymatic transformation.
Figure 3. Transformation of (-)-enterodiol by cell-free extract of R. sp.
END-1 and ESI-MS spectra of enterolactol. (A) LC/MS total ion chromato-
gram profile of the transformation of (-)-enterodiol to (-)-enterolactone
by cell-free extract of R. sp. END-1. (B) LC/MS total ion chromatogram
profile of synthesized enterolactol. (C) Possible metabolic pathway of
(-)-enterodiol by R. sp. END-1.
metabolite was detected by LC/MS analysis (Figure 3A). The
metabolite had a quasi-molecular ion peak at m/z 323 [M þ Na]þ
in the MS spectrum, indicating 2 mass units higher than that of
enterolactone and 2 mass units lower than that of enterodiol,
suggesting a reduction product of enterolactone. We synthesized
it from (-)-enterolactone by reduction with LiEt3BH and
analyzed it under the same conditions. The synthesized com-
pound showed the retention time and mass fragment patterns
(Figure 3B) same as those of an isolated metabolite in bacterial
transformation.
Substrate Specificity of R. sp. END-1 in Oxidation Activity.
(-)-Dihydroxyenterodiol was transformed to (-)-dihydroxyen-
terolactone through oxidative lactonization by anaerobic
incubation with R. sp. END-1 (data not shown). However, (þ)-
dihydroxyenterodiol, an enantiomer of (-)-dihydroxyenterodiol,
was not transformed by this bacterium, indicating the strain has
enantioselectivity to a substrate in oxidation. For studying
selectivity in oxidation of other dibenzylbutanediols, R. sp.
END-1 was incubated with (-)-hydroxyenterodiol (0.1 mM),
and we obtained two isomers of 40- and 400- hydroxyenterolactone

7540 J. Agric. Food Chem., Vol. 57, No. 16, 2009
Jin and Hattori
Figure 4. Transformation of (-)-hydroxyenterodiol by R. sp. END-1 and ESI-MS spectra of two metabolites. (A) HPLC elution profile of the transformation of
(-)-hydroxyenterodiol to two types of hydroxyenterolactone. (B) HPLC elution profile of 40- hydroxyenterolactone isolated from incubation of Eg. sp. SDG-2
with (-)-dihydroxyenterolactone. (C) Possible metabolic pathway of (-)-hydroxyenterodiol by R. sp. END-1.
R. sp. END-1 takes part in not only oxidation but also
demethylation and deglucosylation of plant lignans, in which
methoxy groups of (-)-arctigenin and both (þ)- and (-)-secoi-
solariciresinols were converted to free hydroxyl groups without
any enantioselectivity. Since demethylation and deglucosylation
are essential metabolic processes in the transformation of pre-
cursor lignans to enterodiol and enterolactone, several secoiso-
lariciresinol diglucoside-deglycosylating and secoisolariciresinol-
demethylating bacteria were reported (2, 16, 24). Wang et al.
reported the Peptostreptococcus (P.) sp. strain SDG-,1 which was
isolated from a human fecal suspension as a bacterium capable of
demethylating. P. sp. SDG-1 demethylated (þ)-secoisolariciresi-
nol to (þ)-dihydroxyenterodiol. However, this strain could not
convert dimethylsecoisolariciresinol, i.e., only methoxy groups
having a vicinal hydroxyl group could be eliminated by P. sp.
SDG-1. R. sp. END-1 showed low substrate specificity compared
with that of P. sp. SDG-1. These bacteria having demethylation
activity may contribute to a variety of metabolic processes
converting some lignans to phytoestrogens in the gastrointestinal
tract. Moreover, the bacteria with diverse metabolic activities
such as R. sp. END-1 bring complicated processes into meta-
bolism. The metabolic intermediates or final metabolites can be
absorbed and show several biological activities.
Eg. sp. SDG-2 had ring cleavage activity as well as dehydro-
xylation like Eg. lenta (AF292375) and showed 99% similarity
with the latter in the 16S rRNA gene sequencing analysis (21,25).
Hence, pinoresinol diglucoside was converted to (-)-enterolac-
tone by coincubation of only two bacteria, R. sp. END-1 and

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J. Agric. Food Chem., Vol. 57, No. 16, 2009 7541
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Figure 6. Transformation of plant lignans by coincubation of R. sp. END-1
and Eg. sp. SDG-2.
Eg. sp. SDG-2. In order to produce enterolactone from pinor-
esinol diglucoside, five metabolic processes are concerned; R. sp.
END-1 participates in deglucosylation, demethylation, and oxi-
dation, while Eg. sp. SDG-2 participates in ring cleavage and
dehydroxylation activity. The bacterial cooperation leads to the
formation ofenterolactone from pinoresinol diglucoside. Because
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