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100 : 4) to afford compounds 2 (105.8 mg) and 3 (97.2 mg).
5.30 Hz, Hb-7ꢁ), 3.85 (1H, t, Jꢆ8.94, Ha-4), 4.05 (1H, dd, Jꢆ8.94, 7.24 Hz,
Hb-4), 6.37 (1H, dd, Jꢆ7.95, 2.18 Hz, H-6ꢂ), 6.50 (1H, d, Jꢆ2.18 Hz, H-2ꢂ),
6.63—6.66 (4H, m, H-2ꢁ, H-4ꢁ, H-6ꢁ, H-5ꢂ), 7.10 (1H, t, Jꢆ7.95 Hz, H-5ꢁ).
13C-NMR: Table 1. CD (MeOH): De281 ꢀ0.51, De230 ꢀ3.13 (dm3 molꢀ1
cmꢀ1).
Arctiin (1, 748 mg) and 140 ml of an HIB mixture were added to 1400 ml
of GAM broth and the mixture was anaerobically incubated at 37 °C. At in-
tervals, 100 ml reaction mixture was removed and extracted three times with
n-BuOH (saturated with H2O, containing 0.1% acetic acid). The n-BuOH so-
lutions were combined and evaporated to give an H2O suspension. The H2O
suspension was chromatographed on a Diaion HP-20 column eluted with
H2O, 50% aqueous MeOH, and MeOH. The 50% aqueous MeOH and
MeOH fractions were combined and applied to a Sephadex LH-20 column
eluted with MeOH–H2O (6 : 4). Of the 18 fractions collected, fractions 4—
10 were subjected to silica gel column chromatography eluted with
CHCl3–MeOH (100 : 0Æ100 : 10) and ODS column chromatography eluted
with 50% aqueous MeOH to afford compounds 4 (69.7 mg) and 5 (5.2 mg).
Fractions 11—16 were chromatographed on a silica gel column eluted with
CHCl3 : MeOH (100 : 2Æ100 : 5) and then a Sephadex LH-20 column to
give compounds 6 (79.3 mg) and 7 (48 mg).
Compound 7 [(ꢀ)-Enterolactone]: Amorphous powder. [a]D25 ꢀ46.0°
MeOH
(cꢆ0.15, MeOH). UV l
(e): 218 (10000), 275 (3800), 281 sh (3500)
max
nm. IR (KBr) nmax: 3394 (–OH), 1747 (g-lactone CO) cmꢀ1. EI-MS m/z:
298 [M]ꢃ. H-NMR (CDCl3, 400 MHz): d 2.50 (2H, m, Ha-7ꢂ, H-3), 2.60
1
(2H, m, Hb-7ꢂ, H-2), 2.88 (1H, dd, Jꢆ14.01, 6.77 Hz, Ha-7ꢁ), 2.96 (1H, dd,
Jꢆ14.01, 5.30 Hz, Hb-7ꢁ), 3.90 (1H, dd, Jꢆ9.18, 7.52 Hz, Ha-4), 4.12 (1H,
dd, Jꢆ9.18, 7.00 Hz, Hb-4), 6.47 (1H, t, Jꢆ1.90 Hz, H-2ꢂ), 6.58 (1H, br d,
Jꢆ7.76 Hz, H-6ꢂ), 6.61 (1H, t, Jꢆ1.9 Hz, H-2ꢁ), 6.69—6.75 (3H, m, H-4ꢁ,
6ꢁ, 4ꢂ), 7.13 (1H, t, Jꢆ7.90 Hz, H-5ꢁ), 7.16 (1H, t, Jꢆ7.90 Hz, H-5ꢂ). 13C-
NMR: Table 1. CD (MeOH): De280 ꢀ0.26, De220 ꢀ3.60 (dm3 molꢀ1 cmꢀ1).
Time Course for the Transformation of Arctiin (1) by HIB Sixty mi-
croliters of 100 mM arctiin (1) in MeOH and 600 ml of an HIB mixture were
added to 6 ml of GAM broth, and the mixture was incubated at 37 °C under
anaerobic conditions. A 100 ml aliquot was removed at intervals and ex-
tracted with n-BuOH (saturated with H2O, containing 0.1% acetic acid,
100 mlꢄ3). After evaporation of n-BuOH in vacuo, the residue was dis-
solved in 0.5 ml of MeOH. The MeOH solution was diluted with water to a
volume of 1 ml and filtered through a 0.2 mm membrane filter, and a 5 ml
portion was injected on a column for HPLC analysis. Metabolites were well
separated and detected under the conditions mentioned above. Concentra-
tions of arctiin (1) and its metabolites were calculated according to the cali-
bration curves of the respective authentic samples.
Effects of Arctiin (1) and Its Metabolites on the Growth of Human
Breast Cancer MCF-7 Cells Human breast cancer MCF-7 cells were pur-
chased from the Institute of Physical and Chemical Research (Wako, Japan).
Medium A: Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Life
Technologies) supplemented with 5% fetal bovine serum (FBS, Gibco BRL,
Life Technologies), penicillin (100 units/ml), and streptomycin (100 mg/ml)
(Gibco BRL, Life Technologies). Medium B: Phenol red-free DMEM
(Gibco BRL, Life Technologies, New York, U.S.A.) supplemented with 10%
heat-inactivated charcoal/dextran-treated human serum.25) MCF-7 cells were
maintained in medium A and subcultured for 3 or 4 d. The cells were col-
lected by trypsinization (0.25% trypsin, Nacalai Tesque, Kyoto, Japan) and
suspended in medium B (5000 cells/100 ml), then seeded in a 96-well culture
plate (100 ml/well). After 24 h culture, the medium was changed with 90 ml
of fresh medium (medium B) and 10 ml of a sample solution. After continu-
ous culture for 4 d, the proliferation of cells was assessed using an MTT
assay.26) Estradiol, arctiin (1), and its metabolites were dissolved in dimethyl
sulfoxide (DMSO) to a concentration of 10ꢀ2 M and diluted with medium B
before use.
Incubation of Arctiin (1) with an RIB Mixture A 0.5 ml portion of
the RIB mixture and 50 ml of 100 mM arctiin (1) in MeOH were added to
5 ml of GAM broth and anaerobically incubated at 37 °C for 1 week. The re-
action mixture was removed at intervals and extracted with n-BuOH (satu-
rated with H2O, containing 0.1% acetic acid). The n-BuOH solution was
checked by TLC, as described above.
Compound 2 [(ꢀ)-Arctigenin]: Colorless prisms (MeOH). mp 100.5—
MeOH
101.5 °C. [a]D25 ꢀ25.8° (cꢆ0.20, MeOH). UV l
(e): 231 (8800), 281
max
(4000) nm. IR (KBr) nmax: 3424 (OH), 1762 (g-lactone CO) cmꢀ1. EI-MS
m/z: 372 [M]ꢃ. H-NMR (CDCl3, 400 MHz): d 2.45—2.66 (4H, m, H-2, 3,
1
7ꢂ), 2.92 (2H, m, H-7ꢁ), 3.81 (3H, s, –OCH3), 3.82 (3H, s, –OCH3), 3.85
(3H, s, –OCH3), 3.88 (1H, dd, Jꢆ9.18, 7.28 Hz, Ha-4), 4.13 (1H, dd,
Jꢆ9.18, 7.24 Hz, Hb-4), 6.46 (1H, d, Jꢆ1.94 Hz, H-2ꢂ), 6.55 (1H, dd,
Jꢆ8.19, 1.94 Hz, H-6ꢂ), 6.61 (1H, dd, Jꢆ7.99, 1.94 Hz, H-6ꢁ), 6.64 (1H, d,
Jꢆ1.94 Hz, H-2ꢁ), 6.75 (1H, d, Jꢆ8.19 Hz, H-5ꢂ), 6.83 (1H, d, Jꢆ7.99 Hz,
H-5ꢁ). 13C-NMR: Table 1. CD (MeOH): De282 ꢀ0.102, De233 ꢀ3.06
(dm3 molꢀ1 cmꢀ1).
Compound 3 [(2R,3R)-2-(3ꢁ,4ꢁ-Dihydroxybenzyl)-3-(3ꢂ,4ꢂ-dimethoxyben-
zyl)butyrolactone]: Amorphous powder. [a]D25 ꢀ42.8° (cꢆ0.13, MeOH).
MeOH
UV l
(e): 228 (12000), 281 (5800) nm. IR (KBr) nmax: 3421 (–OH),
max
1751 (g-lactone CO) cmꢀ1. EI-MS m/z: 358 [M]ꢃ. 1H-NMR (CDCl3,
400 MHz): d 2.48—2.64 (4H, m, H-2, 3, 7ꢂ), 2.85 (2H, d, Jꢆ5.88 Hz, H-7ꢁ),
3.81 (3H, s, –OCH3), 3.85 (3H, s, –OCH3), 3.88 (1H, dd, Jꢆ8.94, 7.24 Hz,
Ha-4), 4.14 (1H, dd, Jꢆ8.94, 7.00 Hz, Hb-4), 6.48 (1H, d, Jꢆ1.94 Hz, H-2ꢂ),
6.51 (1H, dd, Jꢆ8.19, 1.94 Hz, H-6ꢁ), 6.57 (1H, dd, Jꢆ8.19, 1.94 Hz, H-6ꢂ),
6.63 (1H, d, Jꢆ1.94 Hz, H-2ꢁ), 6.75 (1H, d, Jꢆ8.19 Hz, H-5ꢁ), 6.76 (1H, d,
Jꢆ8.19 Hz, H-5ꢁ). 13C-NMR: Table 1. CD (MeOH): De282 ꢀ0.65, De233
ꢀ3.08 (dm3 molꢀ1 cmꢀ1).
Compound 4 [(2R,3R)-2-(3ꢁ-Hydroxybenzyl)-3-(3ꢂ,4ꢂ-dimethoxybenzyl)-
butyrolactone]: Amorphous powder. [a]D25 ꢀ51.1° (cꢆ0.12, MeOH). UV
Effects of Compound 4 and (ꢀ)-Enterolactone (7) on Estradiol-Medi-
ated Proliferation of MCF-7 Cells Tamoxifen was dissolved in DMSO to
give a concentration of 10ꢀ2 M and the solution was diluted with medium B
before use. Either tamoxifen (final concentration 1 mM), compound 6 (final
concentration 1 or 10 mM), or (ꢀ)-enterolactone (7) (final concentration 1 or
10 mM) was added to the culture containing 10ꢀ10 M estradiol as described
above.
MeOH
max
l
(e): 224 (11000), 276 (5000) nm. IR (KBr) nmax: 3309 (–OH), 1747
(g-lactone CO) cmꢀ1. EI-MS m/z: 342 [M]ꢃ. 1H-NMR (CDCl3, 400 MHz): d
2.47—2.51 (2H, m, H-3, Ha-7ꢂ), 2.59—2.62 (2H, m, H-2, Hb-7ꢂ), 2.89 (1H,
dd, Jꢆ14.01, 6.77 Hz, Ha-7ꢁ), 2.98 (1H, dd, Jꢆ14.01, 5.30 Hz, Hb-7ꢁ), 3.82
(3H, s, –OCH3), 3.85 (3H, s, –OCH3), 3.88 (1H, dd, Jꢆ9.18, 7.72 Hz, Ha-4),
4.14 (1H, dd, Jꢆ9.18, 7.24 Hz, Hb-4), 6.48 (1H, d, Jꢆ2.18 Hz, H-2ꢂ), 6.56
(1H, dd, Jꢆ7.99, 1.94 Hz, H-6ꢂ), 6.61 (1H, t, Jꢆ2.18 Hz, H-2ꢁ), 6.68 (1H,
br d, H-6ꢁ), 6.71 (1H, dd, H-4ꢁ), 6.76 (1H, d, Jꢆ7.99 Hz, H-5ꢂ), 7.14 (1H, t,
Jꢆ7.72 Hz, H-5ꢁ). 13C-NMR: Table 1. CD (MeOH): De280 ꢀ0.46, De231
ꢀ3.11 (dm3 molꢀ1 cmꢀ1).
References
1) Setchell K. D. R., Lawson A. M., Mitchell F. L., Adlercreutz H., Kirk
D. N., Axelson M., Nature (London), 287, 740—742 (1980).
2) Adlercreutz H., Fotsis T., Heikkinen R., Dwyer J. T., Woods M.,
Goldin B. R., Gorbach S. L., Lancet, 11, 1295—1298 (1982).
3) Pietinen P., Stumpf K., Mannisto S., Kataja V., Uusitupa M., Adler-
creutz H., Cancer Epidemiol. Biomarkers Prevention, 10, 339—344
(2001).
4) Axelson M., Sjovall J., Gustafsson B. E., Setchell K. D. R., Nature
(London), 298, 659—660 (1982).
5) Borriello S. P., Setchell K. D. R., Axelson M., Lawson A. M., J. Appl.
Bacteriol., 58, 37—43 (1985).
6) Welshons W. V., Murphy C. S., Koch R., Calaf G., Jordan V. C., Breast
Cancer Res. Treat., 10, 169—175 (1987).
7) Mousavi Y., Adlercreutz H., J. Steroid Biochem. Molec. Boil., 41,
615—619 (1992).
8) Lin X., Switzer B. R., Demark-Wahnefried W., Anticancer Res., 21,
3995—3999 (2001).
9) Hirose M., Yamaguchi T., Lin C., Kimoto N., Futakuchi M., Kono T.,
Nishibe S., Shirai T., Cancer Lett., 155, 79—88 (2000).
10) Nose M., Fujimoto T., Nishibe S., Ogihara Y., Planta Med., 58, 520—
Compound 5 [(2R,3R)-2-(3ꢁ-Hydroxybenzyl)-3-(3ꢂ-hydroxy-4ꢂ-methoxy-
benzyl)butyrolactone]: Amorphous powder. [a]D25 ꢀ30.7° (cꢆ0.10, MeOH).
MeOH
max
UV l
(e): 220 (10000), 279 (4100) nm. IR (KBr) nmax: 3450 (–OH),
1747 (g-lactone CO) cmꢀ1. EI-MS m/z: 328 [M]ꢃ. 1H-NMR (CDCl3,
400 MHz): d 2.43—2.48 (2H, m, H-3, Ha-7ꢂ), 2.56—2.59 (2H, m, H-2, Hb-
7ꢂ), 2.90 (1H, dd, Jꢆ13.77, 6.77 Hz, Ha-7ꢁ), 2.97 (1H, dd, Jꢆ13.77,
5.30 Hz, Hb-7ꢁ), 3.85 (1H, dd, Jꢆ8.94, 7.72 Hz, Ha-4), 3.87 (3H, s, –OCH3),
4.10 (1H, dd, Jꢆ8.94, 7.24 Hz, Hb-4), 6.50 (1H, dd, Jꢆ7.95, 2.18 Hz, H-6ꢂ),
6.59 (1H, d, Jꢆ2.18 Hz, H-2ꢂ), 6.61 (1H, t, Jꢆ2.0 Hz, H-2ꢁ), 6.72 (1H, m,
H-6ꢁ), 6.74 (1H, m, H-4ꢁ), 6.74 (1H, d, Jꢆ7.95 Hz, H-5ꢂ), 7.17 (1H, t,
Jꢆ7.72 Hz, H-5ꢁ). 13C-NMR: Table 1. CD (MeOH): De279 ꢀ0.46, De229
ꢀ3.10 (dm3 molꢀ1 cmꢀ1).
Compound 6 [(2R,3R)-2-(3ꢁ-Hydroxybenzyl)-3-(3ꢂ,4ꢂ-dihydroxybenzyl)-
butyrolactone]: Amorphous powder. [a]D25 ꢀ36.7° (cꢆ0.12, MeOH). UV
MeOH
l
(e): 220 (12000), 281 (4500) nm. IR (KBr) nmax: 3332 (–OH), 1751
max
(g-lactone CO) cmꢀ1. EI-MS m/z: 314 [M]ꢃ. 1H-NMR (CD3OD, 400 MHz):
d 2.35 (1H, m, Ha-7ꢂ), 2.47 (1H, m, H-3), 2.51 (1H, m, Hb-7ꢂ), 2.65 (1H, m,
H-2), 2.83 (1H, dd, Jꢆ14.01, 7.0 Hz, Ha-7ꢁ), 2.92 (1H, dd, Jꢆ14.01,