M. Ishibashi et al.
FULL PAPERS
specimens (6–484) were deposited in our laboratory.
purified by ODS-HPLC (the same column; eluted with 66% aqueous
MeOH; flow rate, 2 mLminꢀ1) to give compound 12 (2.3 mg, tR =22 min).
Fraction 5f was purified by ODS-HPLC (the same column; eluted with
65% aqueous MeOH; flow rate, 2 mLminꢀ1) to give compounds 8 and
10 (6.3 and 6.6 mg, tR =12 and 18 min, respectively).
Cell cultures: HCT116 and DLD1 cells were purchased from ATCC;
SW480 cells were derived from the Institute of Development, Aging, and
Cancer, Tohoku University; STF/293 cells were a generous gift from
Prof. Jeremy Nathans (Johns Hopkins Medical School). STF/293, 293T,
SW480, and HCT116 cells were cultured in Dulbeccoꢁs modified eagle
medium (Wako) with 10% FBS. DLD1 cells were cultured in RPMI-
1640 medium (Wako) with 10% FBS. Cultures were maintained in a hu-
midified incubator at 378C in 5% CO2/95% air.
Eleutherinoside
B (1): Reddish brown powder, FAB-MS m/z 615
[M+H]+; HRFABMS m/z 615.2336, calcd for C28H39O15 [M+H]+,
615.2289; IR (ATR):n˜max =3326, 2932, 1611, 1241, 901, and 808 cmꢀ1; UV
(MeOH) lmax nm (log e) 340 (4.7), 326 (4.7), 311 (4.7), and 234 (4.8);
½aꢁ2D4 =ꢀ2.7 (c=1.0, MeOH/H2O 1:1); 1H and 13C NMR data in Table 2.
Transfection and luciferase assay: Transient transfection was performed
using Lipofectamine 2000 (Invitrogen, USA). Briefly, 1ꢂ105 cells (293T
or SW480) were split into 24-well plates. After 24 h, cells were transfect-
ed with 1 mg of the luciferase reporter constructs (SuperTopflash or Su-
perFopflash, a generous gift from Prof. Randall T. Moon, University of
Washington) and 0.05 mg of pRL-CMV plasmid (Promega, USA) for nor-
Eleutherinoside
C (2): Reddish brown powder, FAB-MS m/z 599
[M+H]+, 637 (M+ K)+; HRFABMS m/z 637.1887, calcd for C28H38O14K
[M+K]+, 637.1899; IR (ATR): n˜max =3365, 1610, 1361, 1058, 836, and
751 cmꢀ1; UV (MeOH) lmax nm (log e) 337 (4.0), 324 (4.0), and 236 (4.9);
½aꢁ2D4 =ꢀ5.0 (c=0.25, MeOH/H2O 1:1); 1H and 13C NMR data in Table 2.
malization. At 3 h posttransfection, compounds were added with
a
Eleutherinoside
D (3): Reddish brown powder, FAB-MS m/z 571
[M+H]+, 609 [M+K]+; HRFABMS m/z 609.1575, calcd for C26H34O14K
[M+K]+, 609.1586; IR (ATR): n˜max =3651, 1659, 1198, and 751 cmꢀ1; UV
(MeOH) lmax nm (log e) 334 (4.6), 319 (4.6), 306 (4.6), and 234 (4.9);
medium containing FBS. Of note, 293T cells were treated with com-
pounds in a FBS-containing medium combined with 15 mm of LiCl. Cells
incubated for 24 h were lysed in Passive lysis buffer (Promega,
50 mLwellꢀ1) and luciferase activity was measured with a Dual-Glo Luci-
ferase Assay System (Promega). For stable reporter cells, STF/293 cells
(3ꢂ104) were split into 96-well plates and 24 h later treated with com-
pounds combined with 15 mm LiCl. After incubation for 24 h, cells were
lysed with CCLR (cell culture lysis reagent; 20 mLwellꢀ1, Promega) and
luciferase activity was measured with a Luciferase Assay System (Prome-
ga). Assays were performed at least in triplicate.
½aꢁ2D4 =ꢀ10.9 (c=0.2, MeOH/H2O 3:2); H and 13C NMR data in Table 2.
1
Eleutherinoside E (4): Yellow powder, FAB-MS m/z 571 [M+H]+, 609
[M+K]+; HRFABMS m/z 609.1566, calcd for C26H34O14K [M+K]+,
609.1586; IR (ATR): n˜max3308, 1604, 1198, and 841 cmꢀ1; UV (MeOH)
lmax nm (log e) 366 (3.5), 339 (3.9), and 228 (4.7); ½aꢁ2D4 =ꢀ26.0 (c=0.50
MeOH/H2O 1:1); 1H and 13C NMR data in Table 2.
Enzymatic hydrolysis of eleutherinosides A–E: Eleutherinoside A (5,
9.0 mg) in HOAc/NaOAc buffer (pH 4.3, 4.5 mL) was treated with narin-
ginase (45.0 mg, Wako) at 378C for 23 h. After cooling to room tempera-
ture, water was added to the reaction mixture, and the mixture was parti-
tioned with EtOAc. The aglycon in the EtOAc layer was purified by
preparative TLC eluted with hexane/EtOAc 2:3. The sugar in the aque-
ous layer was purified by passage through an ODS sep-pak column. Eleu-
therinoside B (1, 6.4 mg), eleutherinoside C (2, 5.7 mg), eleutherinoside
D (3, 1.0 mg), and eleutherinoside E (4, 1.0 mg) were treated in the same
way as 1 to yield aglycons and sugars of each compound. The aglycon of
2 (2.1 mg) was found to be identical with isoeleutherin (9) by a compari-
son of NMR and ½aꢁD data. The aglycon of 3 (0.3 mg) was identical with
12 by a comparison of NMR and ½aꢁD data with literature data.
Assay of cell viability (FMCA assay):[23] SW480, HCT116, DLD1, and
293T cells (6ꢂ103) were split into 96-well plates and incubated for 24 h.
Cells were treated with compounds and incubated for 24 h. They were
treated with fluorescein diacetate (Wako) in PBS buffer (10 mgmLꢀ1),
and after 1 h of incubation, fluorescence was detected. Assays were per-
formed at least in triplicate.
Activity-guided extraction and isolation: The MeOH extract (10.3 g) of
bulbs of E. palmifolia was dissolved in 10% aqueous MeOH, and parti-
tioned successively with EtOAc (100 mLꢂ2) and nBuOH (100 mLꢂ2) to
give three layers (EtOAc layer, 2.5 g, 91% inhibition of TCF/b-catenin
transcription; nBuOH layer, 3.9 g, 65% inhibition; water layer, 56% in-
hibition at 5 mgmLꢀ1, respectively). The BuOH layer was then separated
by chromatography on a silica gel PSQ100B column (f=28ꢂ500 mm) to
give fraction 1a (72 mg, eluted with 1100 mL of CHCl3/MeOH 9:1, 4.3%
inhibition at 2.5 mgmLꢀ1), 1b (215 mg, eluted with 1100 mL of CHCl3/
MeOH 8.5:1.5, 5.9% inhibition at 2.5 mgmLꢀ1), 1c (177 mg, eluted with
400 mL of CHCl3/MeOH 8:2, 67% inhibition at 2.5 mgmLꢀ1), 1d (842 mg,
eluted with 600 mL of CHCl3/MeOH 8:2, 64% inhibition at 2.5 mgmLꢀ1),
and 1e (2.3 g, eluted with 4300 mL of MeOH, 12% inhibition at
2.5 mgmLꢀ1). Fraction 1c was suspended in CHCl3 and the precipitate
was collected to give eleutherinoside A (5, 40 mg). Fraction 1d was puri-
fied by ODS-HPLC (Mightysil RP18 column, f=10ꢂ250 mm; eluted
with 47% aqueous MeOH; flow rate, 2 mLminꢀ1) to give eleutherinoside
E (4, 2.5 mg, tR =7 min), compound 6 (91.2 mg, tR =15 min), eleutherino-
side D (3, 4.5 mg, tR =18 min), eleutherinoside B (1, 26.2 mg, tR =20 min),
eleutherinoside C (2, 33.7 mg, tR =23 min) and compound 7 (104.8 mg,
Comparison of optical data of 12 and ventilong C: CD (MeOH) for 12:
lmax nm (De) 261 (ꢀ0.35), 301 (0.42), 393 (ꢀ0.12); CD (MeOH) for venti-
long C:[13] lmax nm (De) 280 (ꢀ0.17), 320 (0.09), 408 (ꢀ0.15). Optical rota-
tion: 12, ½aꢁ2D4 =+41.5 (c=1.00, CHCl3); ventilong C,[13] ½aꢁ2D2 =+144 (c=
0.16, CHCl3).
HPLC analysis of sugar components in eleutherinosides B–E: The sugars
obtained by hydrolysis of eleutherinosides B–E (0.5 mg of each) were dis-
solved in pyridine (1 mL) and treated with l-cysteine methyl ester hydro-
chloride (2 mg) at 608C for 1 h. The resulting thiazolidine derivatives
were analyzed by HPLC. HPLC conditions: detection: UV at 210 nm;
column: Inertsil NH2 (f=4.6ꢂ250 mm); eluent: 95–75% CH3CN/H2O
for 60 min, linear gradient; flow rate: 1 mLminꢀ1. Each retention time
was identical with that of the authentic sample. Methyl-2-(d-gluco-penta-
hydroxypentyl)thiazolidine-4(R)-carboxylate: tR =23.4 and 29.4 min;
methyl-2-(l-glucopentahydroxypentyl)thiazolidine-4(R)-carboxylate: tR =
24.6 and 28.8 min.
Isolation of cellular extracts: SW480 cells (5ꢂ105) were seeded into
60 mm dishes, and after 24 h of incubation were treated with compound 9
for 24 h. Cells were then collected by scraping and whole cell protein was
obtained by lysing the cells in ice-cold lysis buffer containing 20 mm Tris-
HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100, 1% Sodium deoxycho-
late, 0.1% SDS, 0.1 mm NaF, and 1% protease inhibitor cocktail (Nakar-
ai tesque Inc.) for 20 min. The lysates were centrifuged at 13000 rpm,
48C for 30 min and supernatants were stored at ꢀ808C before use.
tR =33 min). The EtOAc layer was then separated on
a silica gel
PSQ100B column (f=27ꢂ490 mm) to give fraction 5b (219 mg, eluted
with 2000 mL of Hexane/EtOAc 8.5:1.5, 10.4% inhibition at
2.5 mgmLꢀ1), 5c, and 5d (458 and 133 mg, eluted with 200 mL of hexane/
EtOAc 7.5:2.5 , 94.7% inhibition at 2.5 mgmLꢀ1), 5e, 5f (48 and 44 mg,
eluted with 200 mL of hexane/EtOAc 7:3, 99.5% and 74.3% inhibition,
respectively), and 5g (compound 11; 43 mg, eluted with 200 mL of
hexane/EtOAc 7:3, 94.6% inhibition), 5 h, 5i, and 5j (155, 357, and
570 mg, eluted with 1100 mL of hexane/EtOAc 5:5, EtOAc/acetone 5:5,
and MeOH; 56.1%, 18.4%, and 21.9% inhibition at 2.5 mgmLꢀ1, respec-
tively). Fraction 5c was purified by ODS-HPLC (YMC pack Pro C18
column, f=10ꢂ250 mm; eluted with 70% aqueous MeOH; flow rate,
2 mLminꢀ1) to give compounds 15, 13, and 14 (2.6, 14.4, and 74.0 mg,
tR =12, 19 and 22 min, respectively). Fraction 5d was purified by ODS-
HPLC (the same column; eluted with 65% aqueous MeOH; flow rate,
2 mLminꢀ1) to give compound 9 (42.4 mg, tR =28 min). Fraction 5e was
Isolation of cytoplasmic and nuclear proteins: SW480 cells were treated
in the same way as the cellular extract. Cells were then tripsinized and
cytoplasmic and nuclear proteins were obtained using NE-PER nuclear
and cytoplasmic extraction reagents (Pierce) following the manufactur-
erꢁs protocol, and the proteins were stored at ꢀ808C.
546
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2009, 4, 540 – 547