The C-glucosyl bond of puerarin was cleaved hydrolytically by a human intestinal bacterium strain PUE to yield its aglycone daidzein and an intact glucose

Article abstract of DOI:10.1248/cpb.59.23

C-Glycosides are usually resistant against acidic hydrolysis and enzymatic treatments because C-1 of the sugar moiety is directly attached to the aglycone by C-C bonding. Nevertheless, a human intestinal bacterium, strain PUE, can cleave the C-glucosyl bo

Full text of DOI:10.1248/cpb.59.23

January 2011
Regular Article
Chem. Pharm. Bull. 59(1) 23—27 (2011)
23
The C-Glucosyl Bond of Puerarin Was Cleaved Hydrolytically by a
Human Intestinal Bacterium Strain PUE to Yield Its Aglycone
Daidzein and an Intact Glucose
a,b
a
a
a
a
Kenichi NAKAMURA, Tomohiro NISHIHATA, Jong-Sik JIN, Chao-Mei MA, Katsuko KOMATSU,
b
,a
Makoto IWASHIMA, and Masao HATTORI*
a
b
Institute of Natural Medicine, University of Toyama; 2630 Sugitani, Toyama 930–0194, Japan: and Faculty of
Pharmaceutical Sciences, Suzuka University of Medical Science; 3500–3 Minamitamagaki, Suzuka, Mie 513–8670, Japan.
Received May 28, 2010; accepted October 8, 2010; published online October 15, 2010
C-Glycosides are usually resistant against acidic hydrolysis and enzymatic treatments because C-1 of the
sugar moiety is directly attached to the aglycone by C–C bonding. Nevertheless, a human intestinal bacterium,
1)
strain PUE, can cleave the C-glucosyl bond of puerarin to yield its aglycone daidzein. To clarify the mechanism
of the cleaving reaction, we tried to identify the structure of the metabolite derived from the sugar moiety of
puerarin. To detect it easily, deuterium labeled puerarin, [6ꢀ,6ꢀ-D ]puerarin, was prepared in 7 steps. Sugars con-
2
tained in a metabolite mixture from [6ꢀ,6ꢀ-D ]puerarin was analyzed by an HPLC-electrospray ionization (ESI)-
2
MS method after treatment of sugars with 1-phenyl-3-methyl-5-pyrazolone (PMP). Since deuterium labeled glu-
cose was detected in the metabolite mixture of [6ꢀ,6ꢀ-D ]puerarin, we concluded that puerarin was metabolized
2
to daidzein and an intact glucose by strain PUE. As C-1 of the sugar was hydroxylated instead of hydrogenating,
C-glucosyl bond-cleaving reaction is not reduction but hydrolysis. This is the first report of revealing the reaction
manner and the exact products of C-glucosyl bond-cleaving reaction.
Key words C-glucoside; puerarin; human intestinal bacterium
Many types of glycosides are widely distributed as major
constituents in medicinal plants. However, due to their hy-
drophilic properties, they are poorly absorbed from gastro in-
testinal tract into body fluid, but their aglycones formed by
intestinal bacterial hydrolysis are readily absorbed. There-
fore, intestinal bacteria play an important role in increasing
absorption and efficacy of drugs including glycosides. In na-
ture, there is a special type of glycoside, C-glycosides, such
as those of flavone, isoflavone, chromone, xanthone, an-
Fig. 1. Two Possible Processes for Cleaving C-Glucosyl Bond of Puerarin
throne, and gallic acid. Since C-1 of the sugar ring is directly
connected to the aglycone by C–C bonding, C-glycosides are Results
usually resistant against acidic and enzymatic hydrolysis in Synthesis of [6ꢀ,6ꢀ-D ]puerarin (8) Since puerarin (1)
2
contrast with the corresponding O-glycosides. Thus, with the has only one primary alcohol at C-6 of the sugar moiety, we
prospect of improving the stability, various C-glycoside ana- targeted it for labeling with deuterium. First, 1 was treated
logues mimicking bioactive but labile O-glycosides have with benzaldehyde and zinc chloride to give 2. Other hy-
2)
been prepared for developing the new drugs. In addition, a droxyl groups of 2 were acetylated with acetic anhydride in
lot of publications describing in detail about O-glycosidase pyridine to obtain 3. Benzylidene acetal of 3 was hydrolyzed
appeared, but that of C-glycosidase did not.
under an acidic condition to afford 4. The primary hydroxyl
On the other aspects, in the last two decades, C-glucosyl of 4 was oxidized to carboxylic acid with a Jones reagent to
bond-cleaving reactions by human intestinal bacteria were re- give 5. The acetyl groups of 5 were removed by alkali to give
1
,3—11)
ported.
Despite of the growing importance of C-glyco- 6, then, the carboxyl group was esterified to yield 7 by a
sides in the field of pharmaceuticals, characteristic features usual method. We failed the reduction of 7 to the correspon-
of C-glucosyl bond-cleaving enzymes involved in its metabo- ding alcohol with LiAlD due to decomposition, but 8 was
4
lism were still remain to be unclear.
Recently, we isolated a new bacterial species named strain mixture containing NaBD4. In this reaction, the addition of
prepared from 7 by slow addition of CD OD into the reaction
3
12)
PUE that transforms a C-glucoside puerarin to its aglycone CH OH instead of CD OD gave a mixture of 8 and [6ꢀ-H,6ꢀ-
3
3
1)
daidzein. The aglycone moiety is normally detected easily D]puerarin (ca. 1 : 1 ratio), because hydrogen atoms origi-
because of UV active, however, the liberated sugar segment nated from CH OH were also incorporated into the reduction
3
1
from C-glucoside has not been yet identified. The structural products by any chance. In the H-NMR spectrum of 8 ob-
elucidation of the liberated sugar moiety could yield a valu- tained with the CD OD condition, the signals for H-6ꢀ disap-
3
able clue in revealing the reaction mechanism (Fig. 1). In the peared and in the high-resolution electrospray ionization
present paper, we describe the structural elucidation of the mass spectrometric (HR-ESI-MS) spectrum, the [MꢁH] ion
sugar moiety after cleavage of a C-glucosyl bond of puerarin appeared at m/z 417.1154 (Calcd for C H D O : 417.1149).
2
1
17
2
9
by anaerobic incubation with a human intestinal bacterium, These results confirmed that the product was puerarin labeled
strain PUE, and a cell-free extract. with two deuterium at C-6ꢀ.
∗
To whom correspondence should be addressed. e-mail: iwashima@suzuka-u.ac.jp
© 2011 Pharmaceutical Society of Japan

2
4
Vol. 59, No. 1
Fig. 2. LC-ESI-MS Spectra of PMP-Labeled Sugars
(a) Standard PMP-glucose, (b) PMP-labeled sugar from the metabolite of puerarin,
(
c) PMP-labeled sugar from the metabolite of [6ꢀ,6ꢀ-D ]puerarin.
2
Reagents: (a) PhCHO, ZnCl , (b) Ac O, pyridine, (c) 80% AcOH, 50 °C, (d) CrO ,
2
2
3
H SO , H O, acetone, (e) K CO , MeOH, (f) MeOH, H SO , (g) (i) NaBD , THF,
2
4
2
2
3
2
4
4
6
0 °C, (ii) slow addition of CD OD.
3
Chart 1
HPLC-ESI-MS Analysis of a [6ꢀ,6ꢀ-D ]Puerarin
2
Metabolite Mixture In general, it is hard to analyze vari-
ous sugars by an HPLC-ESI-MS method because of the lack
of good chromatographic separation and the difficulty in ion-
ization of sugars. To resolve these problems, many kinds of
13)
Fig. 3. Conversion of [6ꢀ,6ꢀ-D ]Puerarin to Daidzein and [6,6-D ]Glucose
2 2
by a CFE of Strain PUE
precolumn derivatization methods have been reported. Of
these, we adopted a method of PMP-labeling using 1-phenyl-
14)
3
-methyl-5-pyrazolone (PMP). A cell-free-system instead
of living bacterial system was selected since the bacteria may results suggested that puerarin was metabolized to daidzein
consume liberated sugars quickly as a carbon source, and and an intact glucose by a human intestinal bacterium, strain
identification of the sugars seems to be difficult. A pilot PUE (Fig. 3).
study indicated that a cell-free extract (CFE) of strain PUE
Time Course of Conversion of Puerarin to Daidzein
required for manganese ion on cleaving the C-glucosyl bond and Glucose To confirm further the fact that the metabolite
of puerarin, as observed in the case of mangiferin C-glucosyl derived from the sugar moiety of puerarin was D-glucose, an
11)
cleaving enzyme. Puerarin or [6ꢀ,6ꢀ-D ]puerarin was added enzymatic method using a Glucose (GO) assay kit (SIGMA,
2
to a CFE in the presence of MnCl , and the reaction mixture U.S.A.) was performed to the CFE. The key enzyme of the
2
was anaerobically incubated at 37 °C for 6 h. The mixture kit was glucose oxidase which specifically oxidized b-D-glu-
was filtered through a 10 kDa membrane, and a filtrate in- cose to gluconic acid and hydrogen peroxide. Generated hy-
cluding low molecular weight compounds was treated with a drogen peroxide reacts with o-dianisidine in the presence of
1
5)
PMP reagent before analyzing by HPLC-ESI-MS.
peroxidase to form a colored product. L-Glucose, on the
16)
Figure 2 shows the LC-MS spectra of (a) standard PMP- contrary, does not react with glucose oxidase at all. A cell-
glucose, (b) PMP-labeled sugar from the metabolite of puer- free-system instead of living bacterial system was selected
arin, and (c) PMP-labeled sugar from the metabolite of for the same reason as pointed out above. In the preparation
[
6ꢀ,6ꢀ-D ]puerarin.
of the CFE, strain PUE was cultured in general anaerobic
2
Standard PMP-glucose was eluted at 22.8 min and the medium (GAM) broth containing puerarin to induce the
[MꢂH] ion appeared at m/z 511.4. We could find a peak of puerarin metabolizing enzyme. The collected bacterial cells
the identical retention time and mass spectrum as PMP-glu- were washed with buffer before sonication, but quite a little
cose on (b), indicating the presence of glucose in the metabo- amount of daidzein was remained in CFE regrettably because
lite mixture of puerarin. On (c), a peak with the same reten- of its hydrophobicity. To the CFE of strain PUE were added
tion time of PMP-glucose was also observed, however, the puerarin and MnCl , and the mixture was anaerobically incu-
2
[MꢂH] ion appeared at m/z 513.3. The pseudo molecular ion bated at 37 °C (Fig. 4). Almost whole puerarin was con-
for this PMP-labeled sugar was detected two mass units sumed before 8 h incubation, with the increase of daidzein
higher than that of PMP-glucose, which could be concluded and glucose. No increase of glucose was observed in the
that the sugar was originated from [6ꢀ,6ꢀ-D ]puerarin. These CFE containing neither puerarin nor MnCl . These observa-
2
2

January 2011
25
Chemicals Strain PUE was isolated from human feces as previously de-
1)
scribed. Puerarin was isolated from the roots of Pueraria lobata (WILLD.)
OHWI (Tochimoto Tenkaido Co., Osaka, Japan). GAM broth was purchased
from Nissui Seiyaku Co. (Tokyo, Japan). 1-Phenyl-3-methyl-5-pyrazolone
(
PMP) was obtained from TCI (Tokyo, Japan). CD OD (99.8%) and NaBD4
3
(99%) were purchased from Cambridge Isotope Laboratories, Inc. (U.S.A.).
A glucose (GO) assay kit was obtained from Sigma (U.S.A.).
Synthesis of Deuterium-Labeled Puerarin. 4ꢀ,6ꢀ-O-Benzylidenepuer-
arin (2) 1 (433 mg, 1.04 mmol) and zinc chloride (140 mg, 1.03 mmol)
were dissolved in benzaldehyde (3 ml) and the mixture was stirred for 3 h at
room temperature. The reaction mixture was poured into water, and the pre-
cipitate was washed with water and hexane. The precipitate was purified by
silica gel column chromatography [CHCl –MeOH (15 : 1)] to yield 2
3
1
(
3
368 mg, 70%) as a white solid. H-NMR (CD OD) d: 3.64 (1H, m, H-5ꢀ),
Fig. 4. Time Course of Conversion of Puerarin (ꢀ) to Daidzein (ꢁ) and
Glucose (ꢂ) by a Cell-Free Extract of Strain PUE
3
.73 (1H, dd, Jꢃ9.0, 9.5 Hz, H-4ꢀ), 3.78 (1H, dd, Jꢃ8.2, 9.5 Hz, H-3ꢀ), 3.83
(1H, br t, Jꢃ10.4 Hz, 6ꢀa), 4.28 (1H, dd, Jꢃ5.0, 10.4 Hz, H-6ꢀb), 4.39 (1H,
0
.3 mM puerarin and 1 mM MnCl were added to the CFE of strain PUE. To the glu-
2
dd, Jꢃ8.2, 10.0 Hz, H-2ꢀ), 5.20 (1H, d, Jꢃ10.0 Hz, H-1ꢀ), 5.65 (1H, s, Ph-
CH), 6.86 (2H, d, Jꢃ8.7 Hz, H-3ꢄ, 5ꢄ), 7.01 (1H, d, Jꢃ8.9 Hz, H-6), 7.33—
7.38 (3H, m, three protons of Ph), 7.38 (2H, d, Jꢃ8.7 Hz, H-2ꢄ, 6ꢄ), 7.52—
cose of CFE (ꢃ) were added neither puerarin nor MnCl . A little amount of daidzein at
2
0
h is trace back to CFE preparation. The enzyme is induced only when strain PUE was
cultured with puerarin.
7
.56 (2H, m, two protons of Ph), 8.07 (1H, d, Jꢃ8.9 Hz, H-5), 8.21 (1H, s,
1
3
H-2). C-NMR d: 69.8 (C-6ꢀ), 72.8 (C-5ꢀ), 73.2 (C-2ꢀ), 76.0 (C-1ꢀ), 76.7
tions combined with the spectral data of obtaining sugar
(C-3ꢀ), 82.6 (C-4ꢀ), 103.0 (Ph-CH), 112.8 (C-8), 116.3 (three carbon signals
product indicated the absolute configuration of the glucose of 3ꢄ, 5ꢄ, 6), 118.3 (C-4a), 124.1 (C-1ꢄ), 125.7 (C-3), 127.6, 129.1, 129.9
liberated from puerarin to be confirmed as D-form.
and 139.2 (six carbon signals of Ph), 128.3 (C-5), 131.4 (two carbon signals
of C-2ꢄ, 6ꢄ), 154.4 (C-2), 158.7 (two carbon signals of C-4ꢄ, 8a), 163.2 (C-
ꢂ
7
), 178.2 (C-4). ESI-MS (positive) m/z: 505.4 [MꢂH] .
Discussion
2
ꢀ,3ꢀ,4ꢁ,7-Tetra-O-acetyl-4ꢀ,6ꢀ-O-benzylidenepuerarin (3) Acetic an-
The C–C bonds of C-glucosides are difficult to disconnect
hydride (1 ml) was added dropwise to a mixture of 2 (151 mg, 0.30 mmol) in
by chemical and biological methods. However, a human in- pyridine (2 ml) and stirred for 2 h at room temperature. The reaction mixture
was poured into water and extracted three times with ethyl acetate. Com-
bined organic layer was washed with 1 M HCl and brine, dried over magne-
sium sulfate, and concentrated under reduced pressure. The residue was pu-
testinal bacterium, strain PUE, can cleave the C-glucosyl
1)
bond of puerarin. The reaction mechanism is unclear, and
the structure of the metabolite derived from a sugar moiety
of puerarin is not determined. For the purpose of elucidating
rified by silica gel column chromatography [hexane–acetone (8 : 3)] to yield
1
3
(170 mg, 85%) as a slightly yellow solid. According to the H-NMR spec-
the sugar metabolite, we synthesized [6ꢀ,6ꢀ-D ]puerarin. trum, inseparable two conformational isomers (ca. 3 : 4 ratio) were existed in
2
the product, because free rotation of the C-glucosyl bond was restricted due
After [6ꢀ,6ꢀ-D ]puerarin was incubated with a cell-free ex-
tract of strain PUE, sugars contained in the metabolite mix-
ture was prelabeled with PMP and analyzed by HPLC-ESI-
MS. As a result, deuterium-labeled glucose was detected in dd, Jꢃ9.8, 10.3 Hz, H-6ꢀa), 3.99 (1H, m, H-5ꢀ), 4.04 (1H, t, Jꢃ9.4 Hz, H-
2
to the steric hindrance between a phenolic hydroxyl group at C-7 and a
1
17)
bulky sugar moiety at C-8. H-NMR data of one of the isomer (A)
(
DMSO-d ) d: 1.65—2.44 (12H, four singlet signals of acetates), 3.77 (1H,
6
the metabolite mixture of [6ꢀ,6ꢀ-D ]puerarin. We conse- 4ꢀ), 4.26 (1H, dd, Jꢃ4.8, 10.3 Hz, H-6ꢀb), 5.22 (1H, d, Jꢃ10.0 Hz, H-1ꢀ),
2
5
7
.54 (1H, dd, Jꢃ9.2, 9.4 Hz, H-3ꢀ), 5.67 (1H, s, Ph-CH), 5.77 (1H, m, H-2ꢀ),
.22 (2H, d, Jꢃ8.6 Hz, H-3ꢄ, 5ꢄ), 7.30 (1H, d, Jꢃ8.8 Hz, H-6), 7.35—7.42
quently concluded that puerarin was converted to daidzein
and intact glucose by strain PUE. Furthermore, liberation of
glucose was also confirmed by an enzymatic glucose detec-
tion method using glucose oxidase. The reason why strain signals of acetates), 68.1 (C-6ꢀ), 70.2 (C-5ꢀ), 70.3 (C-2ꢀ), 71.8 (C-1ꢀ), 72.7
PUE cleaves the C-glucosyl bond of puerarin may be as- (C-3ꢀ), 78.3 (C-4ꢀ), 101.0 (Ph-CH), 118.2 (C-8), 121.5 (C-6), 122.3 (two
cribed that this intestinal bacterium had ability to utilize lib-
erated glucose as an energy source by cleaving C-glucosyls.
A C-glucosyl bond-cleaving reaction is not hydrogenolysis
(5H, m, five protons of Ph), 7.64 (2H, d, Jꢃ8.6 Hz, H-2ꢄ, 6ꢄ), 8.16 (1H, d,
13
Jꢃ8.8 Hz, H-5), 8.70 (1H, s, H-2). C-NMR d: 20.3—21.5 (four carbon
carbon signals of C-3ꢄ, 5ꢄ), 122.4 (C-4a), 123.7 (C-3), 126.6, 128.6, 129.5
and 137.6 (six carbon signals of Ph), 127.7 (C-5), 129.2 (C-1ꢄ), 130.4 (two
carbon signals of C-2ꢄ, 6ꢄ), 150.8 (C-4ꢄ), 153.4 (C-7), 154.8 (C-2), 155.9 (C-
1
8a), 169.0—170.2 (four carbon signals of acetates), 175.0 (C-4). H-NMR
but a new type of hydrolysis, because C-1 of the sugar moi- data of another isomer (B) (DMSO-d ) d: 1.65—2.44 (12H, four singlet sig-
6
nals of acetates), 3.67 (1H, dd, Jꢃ9.7, 10.3 Hz, H-6ꢀa), 3.91 (1H, ddd,
Jꢃ5.1, 9.6, 9.7 Hz, H-5ꢀ), 3.98 (1H, m, H-4ꢀ), 4.23 (1H, dd, Jꢃ5.1, 10.3 Hz,
H-6ꢀb), 5.46 (1H, m, H-3ꢀ), 5.52 (2H, m, H-1ꢀ, 2ꢀ), 5.78 (1H, s, Ph-CH),
ety was hydroxylated. 1-Deoxyglucose possibly produced in
the other reductively-cleaving mechanism (hydrogenolysis)
did not detect in the metabolite mixture, thus means that the
reaction with strain PUE does not include any reduction
7
.20 (2H, d, Jꢃ8.6 Hz, H-3ꢄ, 5ꢄ), 7.32 (1H, d, Jꢃ8.8 Hz, H-6), 7.35—7.42
(
5H, m, five protons of Ph), 7.63 (2H, d, Jꢃ8.6 Hz, H-2ꢄ, 6ꢄ), 8.18 (1H, d,
1
3
process. Until now, purification and characterization of the Jꢃ8.8 Hz, H-5), 8.62 (1H, s, H-2). C-NMR d: 20.3—21.5 (four carbon
signals of acetates), 67.9 (C-6ꢀ), 70.5 (C-5ꢀ), 71.1 (C-1ꢀ), 71.3 (C-2ꢀ), 72.8
C-glucosyl bond-cleaving enzyme of puerarin has not been
(C-3ꢀ), 78.1 (C-4ꢀ), 100.8 (Ph-CH) 118.4 (C-8), 121.7 (C-4a), 122.2 (two
accomplished. Further study has to provide the new findings
of novel glycoside hydrolase family catalyzing the hydrolysis
of C-glycosides.
carbon signals of C-3ꢄ, 5ꢄ), 122.9 (C-6), 123.6 (C-3), 126.6, 128.6, 129.5
and 137.6 (six carbon signals of Ph), 127.6 (C-5), 129.2 (C-1ꢄ), 130.6 (two
carbon signals of C-2ꢄ, 6ꢄ), 150.8 (C-4ꢄ), 154.3 (two carbon signals of C-7,
8
a), 154.9 (C-2), 169.0—170.2 (four carbon signals of acetates), 175.0 (C-
ꢂ
Experimental
4). ESI-MS (positive) m/z: 673.4 [MꢂH] .
General An anaerobic incubator, EAN-140 (Tabai Co., Osaka, Japan),
was used for incubation with a bacterium, strain PUE or a cell-free extract
2ꢀ,3ꢀ,4ꢁ,7-Tetra-O-acetylpuerarin (4) 3 (326 mg, 0.485 mmol) was dis-
solved in 80% acetic acid (6 ml) and the mixture was stirred for 3 h at 50 °C.
1
13
1
(
6
CFE). H- and C-NMR were taken on Varinan NMR system 600 ( H, After cooling, water was added to the reaction mixture and the mixture was
13
00 MHz; C, 125 MHz). Chemical shifts are given on a d (ppm) scale with extracted three times with ethyl acetate. Combined organic layer was washed
1 13
either CH OH ( H, 3.31 ppm; C, 49.0 ppm) for CD OD or dimethyl sulfox-
with brine, dried over magnesium sulfate, and concentrated under reduced
3
3
1
13
ide (DMSO) ( H, 2.49 ppm; C, 39.7 ppm) for DMSO-d6 as the internal pressure. The residue was purified by silica gel column chromatography
standard. HR-ESI-MS spectrum was taken with a micrOTOF-Q (Bruker
Daltonics Inc., Billerica, U.S.A.). UV spectra were measured by a UV-2200
UV–VIS recording spectrophotometer (Shimadzu Co., Kyoto, Japan).
[CHCl –MeOH (50 : 1)] to yield 4 (230 mg, 81%) as a white solid. Two con-
3
formational isomers (ca. 1 : 1 ratio) were existed because of the steric inter-
1
17)
action between an acetyl group at C-7 and a bulky sugar. H-NMR data of

2
6
Vol. 59, No. 1
ꢂ
one of the isomer (A) (DMSO-d ) d: 1.61—2.43 (12H, four singlet signals
m/z: 431.2 [MꢂH] .
Daidzein-8-C-glucuronic Acid Methyl Ester (7) A catalytic amount of
6
of acetates), 3.48 (1H, m, H-6ꢀa), 3.57 (2H, m, H-4ꢀ, 5ꢀ), 3.74 (1H, m, H-
6
ꢀb), 4.95 (1H, d, Jꢃ10.1 Hz, H-1ꢀ), 5.17 (1H, m, H-3ꢀ), 5.60 (1H, m, H-2ꢀ), H SO was added to a solution of 6 (140 mg, 0.326 mmol) in MeOH (5 ml)
2 4
7
.20 (2H, d, Jꢃ8.4 Hz, H-3ꢄ, 5ꢄ), 7.30 (1H, d, Jꢃ8.7 Hz, H-6), 7.64 (2H, d, and the mixture was stirred overnight at room temperature. Water was added
1
3
Jꢃ8.4 Hz, H-2ꢄ, 6ꢄ), 8.15 (1H, d, Jꢃ8.7 Hz, H-5), 8.69 (1H, s, H-2). C-
NMR d: 20.3—21.2 (four carbon signals of acetates), 61.3 (C-6ꢀ), 68.4 (C-
to the mixture, which was then extracted three times with ethyl acetate.
Combined organic layer was washed with brine, dried over magnesium sul-
fate, and then concentrated under reduced pressure. The residue was purified
by silica gel column chromatography [CHCl –MeOH–H O (10 : 1.5 : 0.1)] to
5
6
ꢀ), 70.3 (C-2ꢀ), 71.3 (C-1ꢀ), 76.7 (C-3ꢀ), 81.4 (C-4ꢀ), 118.9 (C-8), 121.4 (C-
), 122.2 (two carbon signals of C-3ꢄ, 5ꢄ), 122.4 (C-4a), 123.4 (C-3), 127.3
3
2
1
(C-5), 129.3 (C-1ꢄ), 130.3 (two carbon signals of C-2ꢄ, 6ꢄ), 150.7 (C-4ꢄ),
yield 7 (79 mg, 55%) as a slightly yellow solid. H-NMR (CD OD) d: 3.54
3
153.3 (C-7), 155.0 (C-2), 155.9 (C-8a), 168.8—170.3 (four carbon signals
(1H, t, Jꢃ9.0 Hz, H-3ꢀ), 3.76 (1H, dd, Jꢃ9.0, 9.7 Hz, H-4ꢀ), 3.77 (3H, s,
COO-Me), 4.00 (1H, d, Jꢃ9.7 Hz, H-5ꢀ), 4.28 (1H, m, H-2ꢀ), 5.12 (1H, d,
Jꢃ9.8 Hz, H-1ꢀ), 6.84 (2H, dd, Jꢃ2.2, 8.7 Hz, H-3ꢄ, 5ꢄ), 6.99 (1H, d,
Jꢃ8.9 Hz, H-6), 7.37 (2H, dd, Jꢃ2.2, 8.7 Hz, H-2ꢄ, 6ꢄ), 8.06 (1H, d,
1
of acetates), 174.9 (C-4). H-NMR data of another isomer (B) (DMSO-d )
6
d: 1.61—2.43 (12H, four singlet signals of acetates), 3.48—3.51 (2H, m, H-
4
ꢀ, 6ꢀa), 3.62 (1H, m, H-5ꢀ), 3.67 (1H, m, H-6ꢀb), 5.10 (1H, dd, Jꢃ9.3,
1
3
9
.5 Hz, H-3ꢀ), 5.22 (1H, d, Jꢃ10.0 Hz, H-1ꢀ), 5.40 (1H, dd, Jꢃ9.5, 10.0 Hz, Jꢃ8.9 Hz, H-5), 8.19 (1H, s, H-2). C-NMR d: 52.8 (COO-Me), 72.3 (C-
H-2ꢀ), 7.20 (2H, d, Jꢃ8.5 Hz, H-3ꢄ, 5ꢄ), 7.30 (1H, d, Jꢃ8.7 Hz, H-6), 7.63
2ꢀ), 73.3 (C-4ꢀ), 75.9 (C-1ꢀ), 79.4 (C-3ꢀ), 81.2 (C-5ꢀ), 112.3 (C-8), 116.2
(three carbon signals of C-3ꢄ, 5ꢄ, 6), 118.5 (C-4a), 124.1 (C-1ꢄ), 125.6 (C-3),
128.4 (C-5), 131.4 (two carbon signals of C-2ꢄ, 6ꢄ), 154.5 (C-2), 158.7 (two
carbon signals of C-4ꢄ, 8a), 163.2 (C-7), 171.4 (C-6ꢀ), 178.2 (C-4). ESI-MS
(
2H, d, Jꢃ8.5 Hz, H-2ꢄ, 6ꢄ), 8.15 (1H, d, Jꢃ8.7 Hz, H-5), 8.63 (1H, s, H-2).
1
3
C-NMR d: 20.3—21.2 (four carbon signals of acetates), 60.7 (C-6ꢀ), 67.8
(
(
C-5ꢀ), 70.6 (C-1ꢀ), 71.1 (C-2ꢀ), 77.0 (C-3ꢀ), 81.6 (C-4ꢀ), 119.0 (C-8), 121.6
C-4a), 122.2 (two carbon signals of C-3ꢄ, 5ꢄ), 122.9 (C-6), 123.4 (C-3),
ꢂ
(positive) m/z: 445.3 [MꢂH] .
1
4
27.2 (C-5), 129.3 (C-1ꢄ), 130.5 (two carbon signals of C-2ꢄ, 6ꢄ), 150.7 (C-
ꢄ), 154.3 (two carbon signals of C-7, 8a), 155.0 (C-2), 168.8—170.4 (four
[6ꢀ,6ꢀ-D ]Puerarin (8) NaBD (16 mg, 0.38 mmol) was added to a solu-
tion of 7 (34 mg, 0.077 mmol) in tetrahydrofuran (THF) (1 ml) and the mix-
2
4
carbon signals of acetates), 175.0 (C-4). ESI-MS (positive) m/z: 585.5
ture was stirred for 30 min at 60 °C. CD OD (400 ml) was slowly added to
3
ꢂ
[
MꢂH] .
ꢁ,7-Di-O-acetyldaidzein-8-C-(2,3-di-O-acetyl) Glucuronic Acid (5)
A Jones reagent (2.17 mmol CrO , 180 ml H SO , 900 ml H O) was added to
the reaction mixture and the mixture was stirred for 1 h at 60 °C. After cool-
ing, the reaction was quenched with 0.1 M HCl (5 ml). The mixture was ex-
tracted three times with BuOH and combined organic layer was concentrated
under reduced pressure. The residue was purified by silica gel column chro-
matography [CHCl –MeOH–H O (10 : 1.5 : 0.1)] to yield 8 (10 mg, 31%) as
4
3
2
4
2
a solution of 4 (440 mg, 0.753 mmol) in acetone (5 ml). The mixture was
stirred for 2 h at room temperature, and then quenched by addition of 2-
propanol at 0 °C. Water was added to the reaction mixture and the mixture
was extracted three times with ethyl acetate. Combined organic layer was
3
2
1
a slightly yellow solid. H-NMR (CD OD) d: 3.47 (1H, m, H-5ꢀ), 3.52—
3
3.54 (2H, m, H-3ꢀ, 4ꢀ), 4.12 (1H, m, H-2ꢀ), 5.10 (1H, d, Jꢃ10.0 Hz, H-1ꢀ),
6.85 (2H, d, Jꢃ8.7 Hz, H-3ꢄ, 5ꢄ), 6.99 (1H, d, Jꢃ8.9 Hz, H-6), 7.38 (2H, d,
washed with water, a 1% NaHCO solution and brine. The organic layer was
3
13
dried over magnesium sulfate, and then concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
Jꢃ8.7 Hz, H-2ꢄ, 6ꢄ), 8.06 (1H, d, Jꢃ8.9 Hz, H-5), 8.19 (1H, s, H-2). C-
NMR d: 62.1 (C-6ꢀ), 71.7 (C-4ꢀ), 73.0 (C-2ꢀ), 75.6 (C-1ꢀ), 80.0 (C-3ꢀ), 82.7
(C-5ꢀ), 113.2 (C-8), 116.2 (three carbon signals of C-3ꢄ, 5ꢄ, C-6), 118.4 (C-
4a), 124.2 (C-1ꢄ), 125.6 (C-3), 128.1 (C-5), 131.4 (two carbon signals of C-
[
CHCl –MeOH (50 : 1)] to afford 5 (220 mg, 49%) as a white solid. The
3
products consisted of inseparable two conformational isomers, similar to
1
17)
compounds 3 and 4. H-NMR data of one of the isomer (A) (DMSO-d6) 2ꢄ, 6ꢄ), 154.5 (C-2), 158.7 (two carbon signals of C-4ꢄ, C-8a), 163.2 (C-7),
ꢁ
d: 1.62—2.43 (12H, four singlet signals of acetates), 3.82 (1H, m, H-4ꢀ),
178.3 (C-4). HR-ESI-MS (negative) m/z 417.1154 [MꢁH] (Calcd for
4
.07 (1H, d, Jꢃ9.7 Hz, H-5ꢀ), 5.05 (1H, d, Jꢃ10.0 Hz, H-1ꢀ), 5.23 (1H, dd, C H D O : 417.1149).
2
1
17
2
9
Jꢃ9.1, 9.5 Hz, H-3ꢀ), 5.65 (1H, dd, Jꢃ9.5, 10.0 Hz, H-2ꢀ), 7.20 (2H, d,
Preparation of a Cell-Free Extract Strain PUE was cultured under
Jꢃ8.7 Hz, H-3ꢄ, 5ꢄ), 7.30 (1H, d, Jꢃ8.7 Hz, H-6), 7.62 (2H, d, Jꢃ8.7 Hz, H- anaerobic conditions at 37 °C for 12 h in 1 l of GAM broth containing 0.3
1
3
2
2
7
ꢄ, 6ꢄ), 8.16 (1H, d, Jꢃ8.7 Hz, H-5), 8.72 (1H, s, H-2). C-NMR d: 20.3—
1.3 (four carbon signals of acetates), 69.9 (two carbon signals of C-2ꢀ, 4ꢀ),
1.5 (C-1ꢀ), 76.1 (C-3ꢀ), 79.0 (C-5ꢀ), 118.6 (C-8), 121.5 (C-6), 122.2 (two
mM puerarin. The bacterial cells were collected by centrifugation, washed
twice with 50 mM potassium phosphate buffer (pH 7.5), and then suspended
in 25 ml of same buffer. Cells were disrupted by sonication on ice and cen-
trifuged at 100000ꢅg for 60 min at 4 °C to obtain a supernatant as a cell-
carbon signals of C-3ꢄ, 5ꢄ), 122.4 (C-4a), 123.7 (C-3), 127.6 (C-5), 129.3
(C-1ꢄ), 130.4 (two carbon signals of C-2ꢄ, 6ꢄ), 150.8 (C-4ꢄ), 153.5 (C-7), free extract.
1
54.9 (C-2), 155.9 (C-8a), 168.9—170.3 (four carbon signals of acetates),
Preparation of a Metabolite Mixture of Puerarin or [6ꢀ,6ꢀ-D ]Puer-
2
1
170.0 (C-6ꢀ), 174.9 (C-4). H-NMR data of another isomer (B) (DMSO-d ) arin A mixture of a cell-free extract (1 ml) , MnCl (1 mM) and puerarin or
6
2
d: 1.62—2.43 (12H, four singlet signals of acetates), 3.79 (1H, dd, Jꢃ9.5,
[6ꢀ,6ꢀ-D ]puerarin (each 0.5 mM) was anaerobically incubated for 6 h at
.7 Hz, H-4ꢀ), 4.04 (1H, d, Jꢃ9.7 Hz, H-5ꢀ), 5.16 (1H, dd, Jꢃ9.1, 9.5 Hz, H- 37 °C. The reaction mixture was filtrated through Centriplus YM-10 (Milli-
2
9
3
7
ꢀ), 5.36 (1H, d, Jꢃ10.0 Hz, H-1ꢀ), 5.41 (1H, dd, Jꢃ9.1, 10.0 Hz, H-2ꢀ), pore, U.S.A.) with a 10 K molecular weight cut off by centrifugation at
.20 (2H, d, Jꢃ8.7 Hz, H-3ꢄ, 5ꢄ), 7.29 (1H, d, Jꢃ8.8 Hz, H-6), 7.62 (2H, d,
3000ꢅg for 30 min at 4 °C. The low molecular weight filtrate was labeled
using a PMP reagent described follows.
1
3
Jꢃ8.7 Hz, H-2ꢄ,6ꢄ), 8.16 (1H, d, Jꢃ8.8 Hz, H-5), 8.62 (1H, s, H-2). C-
NMR d: 20.3—21.3 (four carbon signals of acetates), 69.9 (C-4ꢀ), 70.7 (C-
PMP Labeling Standard PMP-glucose was prepared as previously re-
14)
1
ꢀ), 70.8 (C-2ꢀ), 76.0 (C-3ꢀ), 79.6 (C-5ꢀ), 118.6 (C-8), 121.7 (C-4a), 122.2
ported. PMP labeling of liberated sugars were as follows: A 0.5 M PMP
(
1
(
two carbon signals of C-3ꢄ, 5ꢄ), 123.1 (C-6), 123.5 (C-3), 127.4 (C-5),
29.3 (C-1ꢄ), 130.5 (two carbon signals of C-2ꢄ, 6ꢄ), 150.8 (C-4ꢄ), 154.3 were added to a solution (50 ml) of low molecular weight metabolites, and
two carbon signals of C-7, 8a), 154.9 (C-2), 168.9—170.3 (four carbon sig-
solution (100 ml) in MeOH and 0.3 M aqueous sodium hydroxide (100 ml)
the mixture was incubated for 30 min at 70 °C. After cooling, the mixture
nals of acetate), 170.0 (C-6ꢀ), 175.0 (C-4). ESI-MS (positive) m/z: 599.4
was neutralized with 0.1 M HCl, diluted with water (400 ml), and washed
ꢂ
[MꢂH] .
three times with CHCl , then aqueous layer was concentrated. The residue
3
Daidzein-8-C-glucuronic Acid (6) K CO (177 mg, 1.28 mmol) was
was dissolved in MeOH (500 ml), and an aliquot was injected into a column
of the HPLC-ESI-MS system.
2
3
added to a solution of 5 (156 mg, 0.261 mmol) in MeOH (5 ml) and the mix-
ture was stirred for 2 h at room temperature. After acidified with 1 M HCl,
the reaction mixture was extracted three times with BuOH, and then com-
bined organic layer was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography [CHCl –MeOH–H O
HPLC-ESI-MS Analysis HPLC was performed on an Agilent 1100
system (Agilent Technologies, Germany) with a photodiode array detector
and Agilent 1100 series binary pump, and an Esquire 3000 plus mass spec-
trometer (Bruker Daltonik GmbH, Germany) coupled with an ESI interface
and an ion trap mass analyzer. Conditions for analyzing PMP-sugars are as
3
2
1
(
8 : 3 : 0.5)] to yield 6 (60 mg, 53%) as a slightly yellow solid. H-NMR
(
9
CD OD) d: 3.55 (1H, dd, Jꢃ8.8, 9.1 Hz, H-3ꢀ), 3.73 (1H, dd, Jꢃ9.1, follows: column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan, 4.6 mmꢅ
3
.7 Hz, H-4ꢀ), 3.97 (1H, d, Jꢃ9.7 Hz, H-5ꢀ), 4.22 (1H, m, H-2ꢀ), 5.13 (1H, d,
150 mm); mobile phase, 22% acetonitrile in 0.1% acetic acid for 30 min on
Jꢃ10.0 Hz, H-1ꢀ), 6.85 (2H, dd, Jꢃ2.0, 8.7 Hz, H-3ꢄ, 5ꢄ), 7.00 (1H, d, isocratic mode; flow rate, 1.0 ml/min; temperature, 30 °C; monitoring, TIC
Jꢃ9.0 Hz, H-6), 7.38 (2H, dd, Jꢃ2.0, 8.7 Hz, H-2ꢄ, 6ꢄ), 8.07 (1H, d,
(total ion chromatogram) and EIC (extracted ion chromatogram at 511.4 or
513.3) in the positive mode by ESI-MS.
Time Course A CFE (20 ml) containing MnCl2 (1 mM) and puerarin
(0.3 mM) was anaerobically incubated at 37 °C. The amount of puerain,
daidzein, and glucose in the CFE were measured at 0, 1, 2, 3, 4, 5, 6, 8, 10,
and 12 h.
1
3
Jꢃ9.0 Hz, H-5), 8.21 (1H, s, H-2). C-NMR d: 72.5 (C-2ꢀ), 73.4 (C-4ꢀ),
5.8 (C-1ꢀ), 79.5 (C-3ꢀ), 81.0 (C-5ꢀ), 112.5 (C-8), 116.2 (three carbon sig-
nals of C-3ꢄ, 5ꢄ, 6), 118.5 (C-4a), 124.2 (C-1ꢄ), 125.6 (C-3), 128.3 (C-5),
31.4 (two carbon signals of C-2ꢄ, 6ꢄ), 154.5 (C-2), 158.7 (two carbon sig-
nals of C-4ꢄ, 8a), 163.2 (C-7), 172.9 (C-6ꢀ), 178.3 (C-4). ESI-MS (positive)
7
1

January 2011
27
Quantitative Analysis of Puerarin and Daidzein Puerarin and
daidzein were extracted twice with BuOH (200 ml) from the reaction mixture
istry, 28, 1289—1290 (1989).
6) Che Q., Akao T., Hattori M., Kobashi K., Namba T., Chem. Pharm.
Bull., 39, 704—708 (1991).
7) Che Q., Akao T., Hattori M., Tsuda Y., Namba T., Kobashi K., Chem.
Pharm. Bull., 39, 757—760 (1991).
8) Meselhy M. R., Kadota S., Hattori M., Namba T., J. Nat. Prod., 56,
39—45 (1993).
9) Li Y., Meselhy M. R., Wang L., Ma C., Nakamura N., Hattori M.,
Chem. Pharm. Bull., 48, 1239—1241 (2000).
10) Sanugul K., Akao T., Li Y., Kakiuchi N., Nakamura N., Hattori M.,
Biol. Pharm. Bull., 28, 1672—1678 (2005).
(
100 ml) mentioned above. The extract was evaporated to dryness, dissolved
in 50% MeOH, and analyzed by HPLC. Concentrations of puerarin and
daidzein were calculated from the calibration curves of the respective au-
thentic samples. HPLC conditions were as follows: recorder, C-R6A Chro-
matopac; pump, LC-6A; system controller, SCL-6B; monitor, SPD-6A; in-
jector, SIL-9A; column, TSK-gel ODS-80Ts (Tosoh Co., Tokyo, Japan,
4
5
.6ꢅ150 mm); flow rate, 1 ml/min; detection, 250 nm; solvent system, 15—
0% acetonitrile in 0.1% trifluoroacetic acid in a linear gradient for 20 min.
Quantitative Analysis of Glucose An aliquot of the reaction mixture
(
250 ml) was heated for 5 min at 90 °C. After centrifugation at 6000ꢅg, the
11) Sanugul K., Akao T., Nakamura N., Hattori M., Biol. Pharm. Bull., 28,
2035—2039 (2005).
12) Soai K., Oyamada H., Takase M., Ookawa A., Bull. Chem. Soc. Jpn.,
amount of glucose in the supernatant was measured with a Glucose (GO)
assay kit.
5
7, 1948—1953 (1984).
References and Notes
13) Lamari F. N., Kuhn R., Karamanos N. K., J. Chrom. B, 793, 15—36
(2003).
14) Honda S., Akao E., Suzuki S., Okuda M., Kakehi K., Nakamura J.,
Anal. Biochem., 180, 351—357 (1989).
1)
2)
3)
4)
5)
Jin J. S., Nishihata T., Kakiuchi N., Hattori M., Biol. Pharm. Bull., 31,
621—1625 (2008).
Yang G., Schmieg J., Tsuji M., Franck R. W., Angew. Chem. Int. Ed.,
3, 3818—3822 (2004).
Hattori M., Kanda T., Shu Y., Akao T., Kobashi K., Namba T., Chem.
Pharm. Bull., 36, 4462—4466 (1988).
Hattori M., Shu Y., El-Sedawy A., Namba T., Kobashi K., Tomimori
T., J. Nat. Prod., 51, 874—878 (1988).
Hattori M., Shu Y., Tomimori T., Kobashi K., Namba T., Phytochem-
1
4
15) Bergmeyer H. U., “Methods of Enzymatic Analysis,” 2nd English Edi-
tion, New York, Academic Press, 1974, pp. 1205—1212.
16) Pazur J. H., Methods Enzymol., 9, 82—87 (1966).
1
13
17) H- and C-NMR were taken as a conformational mixture. The as-
signments of them were performed by gCOSY, gHSQC, gHMBC and
gHSQC-TOCSY analysis.