6
A. Fujiwara et al. / Phytochemistry xxx (2015) xxx–xxx
reaction mixture was concentrated in vacuo, subjected to HPLC
purification using an Inertsil ODS-EP column (u6 ꢀ 250 mm,
4.8. Assessment of anti-iCSC-10A activity
5
lm, GL Sciences), and eluted by a linear gradient from H2O–AcOH
iCSCL-10A cells were generated and maintained as described
previously (Nishi et al., 2014a). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin. Cell prolifera-
tion and cytotoxicity were evaluated using the Cell Counting Kit-
8 (CCK-8) (Dojindo Molecular Technologies, Kumamoto, Japan).
For the tumor sphere formation assay, cells were seeded in 96-well
ultra-low-attachment surface plates (Corning) at a density of
5 ꢀ 103 cells/well and cultured in serum-free DMEM-Ham’s F12
nutrient mixture (1:1, v/v) supplemented with 5 mg/mL insulin,
0.5 mg/mL hydrocortisone, 2% B27, and epidermal growth factor
(20 ng/mL). Cell viability was evaluated using the CellTiter-GloTM
Luminescent Cell Viability Assay (Promega, Madison, WI, USA)
according to the manufacturer’s instructions. IC50 values were
determined by fitting the data to a sigmoidal dose–response curve
using ImageJ software (NIH, Bethesda, MD, USA).
(100:0.1, v/v) to CH3CN within 20 min, followed by elution in
CH3CN at 1 mL minꢁ1. Peracetylated eucommicin A (2) was recov-
ered as a single peak at 20.0 min. 1H-NMR (CD3OD, 400 MHz) dH
2.0–2.1 (4 ꢀ 3H, overlapped, s, 4q-OAc, 40q-OAc, 5q-OAc, 50q-OAc),
0
2.2 (2 ꢀ 3H, 2s, 1q-OAc, 1q -OAc), 2.2 (4 ꢀ 3H, overlapped,
s, 3-OAc, 30-OAc, 4-OAc, 40-OAc), 2.03, 2.06 (2 ꢀ 1H, m, H-6q
a
,
,
2
H-6q0a), 2.42, 2.48 (2 ꢀ 1H, dd, 2J = 15.9, J2q
a,3q = 3.5, H-2q
a
H-20q
a
), 2.60 (2 ꢀ 1H, m, H-6qb, H-60qb), 2.70 (2 ꢀ 1H, m,
3
3
3
4
H-2qb, H-20qb), 3.87 (1H, ddd, J8,8 = 10, J7,8 (or J7 ,8 ) = 6.3, J7 ,8
0
0
0
0
4
3
3
0
0
0
(or J7,8 ) = 1.0, H-8 or H-8 ), 3.92 (1H, ddd, J8,8 = 10, J7,8 (or
3
3
4
4
0
0
0
0
0
J7 ,8 ) = 7.6, J7 ,8 (or J7,8 ) = 1.0, H-8 or H-8 ), 4.27 (1H, ddd,
3
3
4
4
0
0
0
0
0
0
J7,7 = 10, J7,8 (or J7 ,8 ) = 6.3, J7 ,8 (or J7,8 ) = 1.0, H-8 or H-8 ),
3
3
3
4
4
0
0
0
0
0
4.31 (1H, ddd, J8,8 = 10, J7,8 (or J7 ,8 ) = 7.6, J7 ,8 (or J7,8 ) = 1.0,
H-8 or H-80), 5.08, 5.09 (2 ꢀ 1H, dd, 9.9, 4.3, H-4q, H-40q), 5.51,
5.56 (2 ꢀ 1H, m, H-3q, H-30q), 5.54 (2H, m, H-5q, H-50q), 8.86, 8.88
(2 ꢀ 1H, dd, 8.4, 2.0), 6.91, 6.95 (2 ꢀ 1H, d, 2.0, H-2, H-20), 6.97,
6.98 (2 ꢀ 1H, d, 8.4, H-5, H-50); 13C-NMR (CD3OD, 400 MHz) dC
4.9. LC–MS/MS detection of eucommicin A (1)
20.4–20.5 (3-O(CO)CH3, 30-O(CO)CH3, 4-O(CO)CH3, 40-O(CO)CH3),
20.9–21.1 (4q-O(CO)CH3, 40q-O(CO)CH3, 5-O(CO)CH3, 50q-O(CO)
Eucommicin A (1) extracted from freshly-harvested leaves of E.
ulmoides was detected by LC–MS/MS after each purification step.
Each sample was separated using a Capcell Pak C18 MGIII column
(2.0 mm i.d. ꢀ 250 mm, Shiseido, Tokyo, Japan) and eluted by vary-
ing the relative concentrations of solvents A (H2O–AcOH (100:0.1,
v/v)) and B (MeOH–AcOH (100:0.1, v/v)) in a linear gradient from
0% B to 50% B over a 10-min period, followed by a linear gradient
from 50% B to 100% B over an 8-min period. The flow rate was
0.2 mL minꢁ1. The mass spectrometer was operated in the multiple
reaction monitoring (MRM) mode. The ion source (Turbo V ion
source) was operated in the negative ESI mode. The source param-
eters were set as follows: curtain gas, 40 psi; temperature, 500 °C;
spray gas (GS1), 50 psi; dry gas (GS2), 80 psi; and ion spray voltage,
ꢁ4500 V. The m/z 707 ? 191 transition was monitored (collision
energy, ꢁ56 V; collision cell exit potential, ꢁ2.0 V; declustering
potential, ꢁ110 V). Both quadrupoles were set at unit resolution.
CH3), 21.4–21.5 (1q-O(CO)CH3, 10q-O(CO)CH3), 32.8, 33.0 (C-2q,
C-20q), 37.9, 38.0 (C-6q, C-60q), 44.2, 44.3 (C-8, C-80), 46.0, 46.1
(C-7, C-70), 68.7, 69.0 (C-5q, C-50q), 73.1, 73.5 (C-4q, C-40q), 80.8,
80.9 (C-1q, C-10q), 124 (overlapped, C-2, C-20, C-5, C-50), 127.2,
127.4 (C-6, C-60), 138.3, 138.4 (C-1, C-10), 142.3 (overlapped, C-3,
C-30), 143.3, 143.4 (C-4, C-40), 169.8–169.9 (overlapped, 3-O(CO)
CH3, 30-O(CO)CH3, 4-O(CO)CH3, 40-O(CO)CH3), 171.6, 171.7 (1q-O
(CO)CH3, 10q-O(CO)CH3), 171.8–171.9 (overlapped, 4q-O(CO)CH3,
40q-O(CO)CH3, 5-O(CO)CH3, 50q-O(CO)CH3), 172.6, 172.9 (C-9,
C-90), 173.8, 173.9 (C-7q, C-70q).
4.7. Compound 2 (3,30,4,40-tetramethoxy-b-truxinic acid dimethyl
ester)
Dried eucommicin A (1) (1.2 mg) was dissolved in MeOH
100 lL, added to excess ethereal diazomethane solution, and incu-
Acknowledgements
bated for 1 h. After concentration to dryness, the methylated
eucommicin A was treated with 0.1 M NaOMe in MeOH at ambient
temperature for 1 h. The reaction mixture was acidified with 1 M
AcOH and partitioned between H2O and EtOAc. The organic layer
was dried (Na2SO4) and concentrated in vacuo, giving compound
2 as a white solid. EI-MS m/z (rel. int.): 144 (1.9), 222 (100,
[M/2]+), 300 (2.4), 351 (0.47, [M-3 ꢀ OMe]+), 382 (0.53, [M-
2 ꢀ OMe]+), 413 (1.2, [M-OMe]+), 444 (0.35, M+). 1H-NMR (CD3OD,
400 MHz) dH 3.60 (6H, s, CH3-12, CH3-120), 3.75 (12H, s, CH3-10,
CH3-100, CH3-11, CH3-110), 3.91 (2H, detected as a doublet-like
The authors would like to thank Noriko Ikawa and Yukiko
Watanabe for their technical assistance with the cytotoxicity and
tumor sphere formation assays. This work was supported in part
by the Creation of Innovation Centers for Advanced Interdisci-
plinary Research Areas Program, a grant-in-aid from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan
(to A. R. and M. N.), and the Innovation Promoting Program of Ibar-
aki University (to Y. S.).
References
3
3
4
multiplet, ddd, J8,8 = 11, J7,8 = 7, J7 ,8 = 1.0, H-8, H-80), 4.24 (2H,
0
3
3
detected as
a doublet-like multiplet, ddd, J7,7 = 11, J7,8 = 7,
Carmignani, M., Volpe, A.R., Monache, D.F., Botta, B., Espinal, R., de Bonnevaux, S.C.,
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4
4
J7,8 = 1.0, H-7, H-70), 6.45 (2H, d, J2,6 = 2.0, H-2, H-20), 6.67 (2H,
0
3
4
3
dd, J5,6 = 8.2, J2,6 = 2.0, H-6, H-60), 6.77 (2H, d, J5,6 = 8.2, H-5, H-
50); 1H-NMR (acetone-d6, 400 MHz) dH 3.63 (6H, s, CH3-12, CH3-
120), 3.70 (6H, s, CH3-10, CH3-100), 3.70 (6H, s, CH3-11, CH3-110),
3
0
3.92 (2H, detected as a doublet-like multiplet, ddd, J8,8 = 11,
J7,8 = 3J7 ,8 = 7.0, J7 ,8 = 4J7,8 = 1.0, H-8, H-80), 4.25 (2H, detected
3
4
0
0
0
0
3
3
0
0
as
a
doublet-like multiplet, ddd, J7,7 = 11, J7,8 = 3J7 ,8 = 7,
J7,8 = 4J7 ,8 = 1.0, H-7, H-70), 6.60 (2H, d, J2,6 = 2.0, H-2, H-20), 6.69
4
4
0
0
3
4
3
(2H, dd, J5,6 = 8.2, J2,6 = 2.0, H-6, H-60), 6.74 (2H, d, J5,6 = 8.2, H-
5, H-50); 13C-NMR (CD3OD, 400 MHz) dC 43.9 (C-8, C-80), 45.6 (C-
7, C-70), 52.0 (C-12, C-120), 56.0 (C-10, C-100), 56.0 (C-11, C-110),
112.2 (C-5, C-50), 113.4 (C-2, C-20), 120.9 (C-6, C-60), 132.7 (C-1,
C-10), 148.9 (C-3, C-30), 149.9 (C-4, C-40), 173.6 (C-9, C-90 [
a]20D
0 (c 0.05, MeOH).
Please cite this article in press as: Fujiwara, A., et al. Eucommicin A, a b-truxinate lignan from Eucommia ulmoides, is a selective inhibitor of cancer stem