J Nat Med
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20), 116.5 (C-50), 120.3 (C-60), 127.5 (C-10), 147.5 (C-30),
148.6 (C-40), 163.6 (C-9), 166.0 (C-5), 169.3 (C-7), 172.4
(C-100), 192.7 (C-4). HR-FAB-MS: 599.3569 ([M?H]?,
C35H51O8?; calcd. 599.3584).
595.1604). HR-ESI-MS: 593.1349 [M-H]-, C34H25O10
;
calcd. 593.1448).
Hydrolysis of compounds 1–3 and identification
of the hydrolysis products
(2R,3R)-3-O-Docosanoyltaxifolin (=(2R,3R)-2-(3,4-di-
hydroxyphenyl)-5,7-dihydroxy-4-oxochroman-3-yl
do-
cosanoate; 2). Amorphous solid. UV (MeOH): 232 (4.06),
284 (4.04), 297 (4.03), 332sh (3.75). CD (MeOH) ([h]):
327 (?5156), 292 (-28125). IR (KBr): 3435 (OH), 1737
(ester C=O), 1643 (C=O). 1H-NMR (CDCl3): dH 0.87 (3H,
t, J = 6.5 Hz, H-2200), 1.25 (34H, br s, H-400–H-2100), 1.57
(2H, br q, J = 7.0 Hz, H-300), 2.33 (1H, dt, J = 15.5,
7.0 Hz, H-200), 2.40 (1H, dt, J = 15.5, 7.5 Hz, H-200), 5.64
(1H, d, J = 12.0 Hz, H-2), 6.34 (1H, d, J = 2.0 Hz, H-8),
6.43 (1H, d, J = 12.0 Hz, H-3), 6.47 (1H, d, J = 2.0 Hz,
H-6), 7.23 (1H, dd, J = 7.5, 2.0 Hz, H-60), 7.29 (1H, d,
J = 7.5 Hz, H-50), 7.65 (1H, d, J = 2.0 Hz, H-20); 13C-
NMR (CDCl3): dC 14.3 (C-2200), 22.9 (C-2100), 25.3 (C-300),
29–30 (C-400–1900), 32.1 (C-2000), 34.1 (C-200), 72.7 (C-3),
82.1 (C-2), 96.6 (C-8), 97.8 (C-6), 101.8 (C-10), 116.2 (C-
20), 116.5 (C-50), 120.3 (C-60), 127.5 (C-10), 147.5 (C-30),
148.6 (C-40), 163.6 (C-9), 165.0 (C-5), 169.3 (C-7), 172.4
(C-100), 192.7 (C-4). HR-FAB-MS: 627.3887 ([M?H]?,
C37H55O8?; calcd. 627.3897).
Compounds 1, 2, and 3 (each ca 5 mg) were hydrolyzed
by 1 N HCl/MeOH under reflux for 1 h. The reaction
solutions were poured into water and extracted with
EtOAc. The EtOAc solutions were washed with sat.
NaHCO3 aq. and water, successively, and were
evaporated in vacuo. MeOH (0.2 ml) was added to the
dried products and filtered to give MeOH-soluble polar
parts. The residual parts were resolved with EtOAc to
give nonpolar parts. The three polar parts showed the
same spots on TLC (silica gel, CHCl3:MeOH = 6:1) and
the same peaks on HPLC (Mightysil RP-18 GP). The
polar parts were purified by HPLC to yield a pure com-
pound (2.5 mg) that was measured via ESI-MS and NMR
and identified as taxifolin. The three nonpolar parts from
comp. 1, 2, and 3 were analyzed via ESI-MS to yield
pseudmolecular ion, m/z 349.3069 [M ? Na]?, m/
z 377.3382 [M ? Na]?, and m/z 405.3700 [M ? Na]?,
respectively, which were C21H42O2 for methyl eicosano-
ate, C23H46O2 for methyl docosanoate, and C25H52O2 for
methyl tetracosanoate.
(2R,3R)-3-O-tetracosanoyltaxifolin (= (2R,3R)-2-(3,4-
dihydroxyphenyl)-5,7-dihydroxy-4-oxochroman-3-yl tetra-
cosanoate; 3). Amorphous solid. UV (MeOH): 232 (4.06),
284 (4.04), 297 (4.03), 332sh (3.75). CD (MeOH) ([h]):
326 (?2321), 293 (-18545). IR (KBr): 3401 (OH), 1736
Cell lines and culture conditions
1
(ester C = O), 1643 (C = O). H-NMR (CDCl3): dH 0.87
Human lung and colorectal cancer cell lines, A549 and HT-
29, were obtained from ATCC (Lockville, MD, USA).
A549 and HT-29 cells were maintained in Dulbecco’s
modified Eagle’s medium (D-MEM) (D6046, Sigma) and
D-MEM/F-12 medium (D8062, Sigma) with 10 % heat-
inactivated fetal bovine serum (FBS) and 5 mg/ml of
gentamicin, respectively, at 37 °C in a humidified atmo-
sphere containing 5 % CO2.
(3H, t, J = 7.0 Hz, H-2400), 1.25 (38H, br s, H-400–H-2300),
1.57 (2H, br q, J = 7.0 Hz, H-300), 2.33 (1H, dt, J = 15.5,
7.0 Hz, H-200), 2.40 (1H, dt, J = 15.5, 7.5 Hz, H-200), 5.64
(1H, d, J = 12.0 Hz, H-2), 6.34 (1H, d, J = 2.0 Hz, H-8),
6.43 (1H, d, J = 12.0 Hz, H-3), 6.47 (1H, d, J = 2.0 Hz,
H-6), 7.23 (1H, dd, J = 8.0, 2.5 Hz, H-60), 7.29 (1H, d,
J = 8.0 Hz, H-50), 7.65 (1H, d, J = 2.5 Hz, H-20); 13C-
NMR (CDCl3): dC 14.3 (C-2400), 22.9 (C-2300), 25.3 (C-300),
29–30 (C-400–2100), 32.1 (C-2200), 34.2 (C-200), 72.7 (C-3),
82.1 (C-2), 96.6 (C-8), 97.8 (C-6), 101.8 (C-10), 116.2 (C-
20), 116.5 (C-50), 120.3 (C-60), 127.5 (C-10), 147.5 (C-30),
148.6 (C-40), 163.6 (C-9), 165.0 (C-5), 169.3 (C-7), 172.4
(C-100), 192.7 (C-4). HR-FAB-MS: 655.4229 ([M?H]?,
C39H59O8?; calcd. 655.4210).
Growth-inhibition assay
A 190-ll volume of an exponentially growing cell sus-
pension (1 9 104 cells/1.9 ml) was seeded in a 96-well
microtiter plate, and 10 ll of each drug at various con-
centrations was then added 24 h after the seeding of tumor
cells. Drugs were initially dissolved in DMSO and diluted
to each concentration with culture medium. After incuba-
tion for 96 h at 37 °C, 10 ll of TetraColor ONE (Seika-
gaku Biobusiness Co., Tokyo, Japan) was added to each
well, and the plates were incubated for a further hour at
37 °C. After incubation, the optical density was measured
at 450 nm with a microplate reader (SPECTRA max
PLUS, Molecular Devices, CA, USA), and the concentra-
tion causing 50 % inhibition of cell proliferation (IC50) was
6-Methyl-7,40,700-tri-O-methylamentoflavone
(=5-hy-
droxy-2-{3-[5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4-
oxo-4H-chromen-8-yl]-4-methoxyphenyl}-7-methoxy-6-
methyl-4H-chromen-4-one; 4). Pale yellow powder.
[a]2D3 = –5.2° (c = 0.04, CH3CN). IR (KBr): 3320 (OH),
1655 (C=O), 1605 (arom.). UV (CH3CN): 215 (51900),
272 (39000), 323 (35700). CD (CH3CN) ([h]): 325
(-1657), 305 (?308). NMR spectra: see Table 1. HR-
FAB-MS: 595.1626 ([M?H]?, C34H27O10
?
;
calcd.
123