8
70 J ournal of Natural Products, 1998, Vol. 61, No. 7
Lee et al.
for the structure-activity relationships were purchased
carcinoma), HCT-15 (human colon adenocarcinoma),
and SKOV-3 (human ovary adenocarcinoma) were grown
in RPMI-1640 medium supplemented with 10% heat-
inactivated fetal bovine serum plus 1% antibiotics.
CCD-18Co (human normal colon) and HT-1197 (human
bladder carcinoma) were grown in DMEM medium with
the same supplements. Cells were cultured in T-flasks
in CO2 incubator supplied with 5% CO2 and 95% humid
from Sigma Chemical Co.
Extr a ction a n d Isola tion . The fresh sarcotestas (3
kg) were extracted three times with CHCl3 in an
ultrasonic apparatus for 3 h and, upon removal of the
solvent in vacuo, yielded the CHCl3 extract (80 g).
Separation of PI-PLCγ1 inhibitory principles from the
CHCl3 extract was carried out by bioassay-guided
separation; it was fractionated by column (6 × 65 cm)
chromatography over Si gel (800 g, 230-400 mesh)
using CHCl3-MeOH mixtures with increasing polarity
4
air at 37 °C. Cells were maintained 5-10 × 10 cells/
mL and subcultured 2-3 times per week; 0.05% trypsin-
EDTA was used for dissociating monolayer cells. Cell
4
suspension (1-8 × 10 cells/well) of log phase was added
(100:1f2:1) to give six fractions, of which fractions 2
to each well of a 96-well plate to determine the human
cancer cell growth inhibition of compounds and incu-
bated in a CO2 incubator at 37 °C. After day 1,
compounds were treated, and cells were cultured for two
additional days. The final DMSO concentration was
adjusted to 0.5% in all samples. Each experiment was
performed in triplicate. Viable cells were counted by
and 4 showed potent activity. Fraction 2 (34 g) was
subjected to column chromatography over Si gel (5.2 ×
4
0 cm, 230-400 mesh, 350 g, n-hexanes-EtOAc 10:1)
and Sephadex LH-20 (3.4 × 49 cm, n-hexane-CH2Cl2-
MeOH 5:5:1) to give four subfractions. A semiprepara-
tive HPLC (YMC-Pack C18, 4 µm, 10 × 250 mm,
CH3CN-MeOH 10:1, 254 nm, 2 mL/min) was used for
further purification of subfraction 2, which led to the
isolation of compounds 1 (52 mg, tR 28.28 min), 2 (45
mg, tR 46.25 min), 3 (15 mg, tR ) 30.72 min), and 4 (13
mg, tR 52.72 min). Semipreparative HPLC was carried
out twice over Phenomenex C18 (5 µm, 10 × 250 mm,
3
1
the SRB method.
Cell growth inhibition index was
determined as IC50; the drug concentration result-
ing in 50% growth inhibition was compared to the un-
treated control. Adriamycin was used as a positive
control.
Id en tifica tion of Com p ou n d s 1-10. The struc-
tures of compounds 1-10 (Figure 1) were identified,
respectively, as cardanols {3-[8′ (Z)-pentadecenyl]phenol
2
54 nm, 2 mL/min, solvent a MeOH-OHAc 99:1; solvent
b MeOH-AcOH-H2O 99:1:10) for subfraction 3, to
provide compounds 5 (16 mg, b; tR 113.10 min), 6 (18
mg, a; tR 30.99 min), and 7 (15 mg, b; tR 104.14 min).
From fraction 4 (20 g), compounds 8 (200 mg, tR 102.32
min), 9 (50 mg, tR 85.00 min), and 10 (13 mg) were
separated using vacuum column chromatography (7 ×
(1), 3-[10′ (Z)-heptadecenyl]phenol (2), 3-tridecyl phenol
(3), and 3-pentadecyl phenol (4)}; phenolic acids {6-[8′
(Z)-pentadecenyl]salicylic acid (5), 6-[10′ (Z)-heptade-
cenyl]salicylic acid (6), and 6-tridecyl phenol (7)}; and
bilobols {5-[8′ (Z)-pentadecenyl]resorcinol (8), 5-[10′ (Z)-
heptadecenyl]resorcinol (9), and 5-tridecyl resorcinol
2
0 cm, 270 g, 15 µm, n-hexane-Me2CO 10:1f3:1),
Sephadex LH-20 (3.4 × 49 cm, n-hexane-CH2Cl2-
MeOH 5:5:1), and semipreparative HPLC (Phenomenex
HC 12 8 O, 56 : µ1 m) . , 10 × 250 cm, 254 nm, 2 mL/min, MeOH-
1
13
(
10)} by IR, H NMR, C NMR, and MS. These
structures were confirmed by comparison with those of
1
5,19,32-33
literature data.
The positions of double bonds
in compounds 1, 2, 5, 6, 8, and 9 were determined from
In Vitr o P I-P LCγ1 Assa y. The PI-PLCγ1 assay was
performed by the method of Rhee et al.30 In brief, the
enzyme solution was dissolved in 50 mM HEPES/NaOH
product ions by collisionally activated dissociation of [M
+
-
H + 2Li] precursor ions, which were generated by
+
33
FABMS using a Li containing matrix.
(
pH 7.0), 3 mM CaCl2, and 1 mM EGTA, and a reaction
P r ep a r a tion of Com p ou n d s 1, 5, a n d 8 Der iva -
tives. Cardanol C15:1 (1) 3 mg and bilobol C15:1 (8) 5
mg were acetylated by Ac2O in pyridine at room tem-
perature, and yielded O-acetyl 3-[8′ (Z)-pentadecenyl]-
phenol (cardanol C15:1 acetate, 1a ) 2 mg and 1, 3-O-
diacetyl 5-[8′ (Z)-pentadecenyl]resorcinol (bilobol C15:1
diacetate, 8a ) 3.5 mg. Phenolic acid C15:1 (5) 10 mg was
esterified by CH2N2 in Et2O in an ice bath, to give
methyl 6-[8′ (Z)-pentadecenyl]salicylate (phenolic acid
C15:1 methyl ester, 5a ) 12 mg. Phenolic acid C 15:1
methyl ester (5a ) 5 mg was acetylated by the method
described previously, and methyl O-acetyl 6-[8′ (Z)-pen-
tadecenyl]salicylate (methyl phenolic acid C15:1 acetate,
3
mixture contained 0.02 µCi [ H-inositol] PI, 10 nM PI
and 0.1%-SDC. The enzyme reaction was initiated by
,
adding enzyme at 37 °C and terminated after 10 min
by adding 1 mL of CHCl3-MeOH-concentrated HCl
(50:50:0.3, by volume) and 0.3 mL of 1 N HCl containing
3
mM EGTA. Samples were vortexed and centrifuged
at 2000 rpm for 10 min, and 0.5 mL of the aqueous
phase was placed in a scintillation vial, and the radio-
3
activity of [ H-inositol] IP was counted. Each inhibitor
was tested in triplicate at each point. Amentoflavone
was used as a positive control. The inhibitory modes
of action were kinetically analyzed by changing the con-
centration of the PI (2-50 µM) in the presence of various
concentrations of the inhibitors (0, 2, and 10 µg/mL).
5
b) 3.5 mg was obtained.
Ack n ow led gm en t. This work was supported by the
Cell Lin es a n d Cu ltu r e Med ia . Cell lines (A-549,
MCF-7, HCT-15, SKOV-3, CCD-18Co, and HT-1197)
were obtained from the Korean Cell Line Bank, Seoul
National University, Seoul, Korea. Media (DMEM and
RPMI-1640), and antibiotics (penicillin-streptomycin)
were purchased from Gibco-BRL (Grand Island, NY).
Fetal bovine serum was obtained from Hyclon (Logan,
UT).
Korea Science and Engineering Foundation (KOSEF)
through the Research Center for New Drug Develop-
ment (RCNDD) at Seoul National University.
Refer en ces a n d Notes
(
1) Nishizuka, Y. Science 1992, 258, 607-614.
2) Berridge, M. J . Nature 1993, 361, 315-325.
(
(3) Suh, P. G.; Ryu, S. H.; Moon, K. H.; Suh, H. W.; Rhee, S. G.
Proc. Natl. Acad. Sci. (USA) 1988, 85, 5419-5423.
4) Suh, P. G.; Ryu, S. H.; Moon, K. H.; Suh, H. W.; Rhee, S. G. Cell
Gr ow th In h ibition Assa y of Ca n cer Cell. A549
(
(human lung carcinoma), MCF-7 (human breast adeno-
1988, 54, 161-169.