A cytotoxic and apoptosis-inducing sesquiterpenoid isolated from the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk)

Article abstract of DOI:10.1248/cpb.56.1168

Repeated silica gel and octadecyl silica gel (ODS) column chromatography of the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk) led to the isolation of a new sesquiterpenoid, 3-((S)-2-methylbutyryloxy)-costu-1(10),4(5)- dien-12,6α-olide (2), along with two previously reported sesquiterpenoids: 8α-angeloyloxy-3β,4β-epoxy-6βH,7αH,8βH-guaia- 1(10),11(13)-dien-12,6α-olide (1, carlaolide B) and 3β,4β-epoxy- 8α-isobutyryloxy-6βH,7αH,8βH-guaia-1(10),11(13)-dien-12, 6α-olide (3, carlaolide A). The structure of compound 2 was elucidated by spectroscopic data analysis, including one dimensional (1D) and two dimensional (2D) nuclear magnetic resonance (NMR) experiments. Of the isolates, compound 2 exhibited potent cytotoxicity against human cervix adenocarcinoma cells and induced apoptosis.

Full text of DOI:10.1248/cpb.56.1168

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Notes
Chem. Pharm. Bull. 56(8) 1168—1172 (2008)
Vol. 56, No. 8
A Cytotoxic and Apoptosis-Inducing Sesquiterpenoid Isolated from the
Aerial Parts of Artemisia princeps PAMPANINI (Sajabalssuk)
Myun-Ho BANG,a Min-Woo HAN,b Myoung-Chong SONG,b Jin-Gyeong CHO,b Hae-Gon CHUNG,c
d
e
f
b
,b
*
Tae-Sook JEONG, Kyung-Tae LEE, Myung-Sook CHOI, Se-Young KIM, and Nam-In BAEK
a The Skin Biotechnology Center, Kyung Hee University; Suwon 443–766, Korea: b The Graduate School of Biotechnology
& Plant Metabolism Research Center, Kyung Hee University; Suwon 446–701, Korea: c Ganghwa Agricultural R&D
Center; Incheon 417–833, Korea: d National Research Laboratory of Lipid Metabolism & Atherosclerosis, Korea Research
Institute of Bioscience and Biotechnology; Daejeon 305–333, Korea: e Department of Biochemistry, College of Pharmacy,
f
Kyung Hee University; Seoul 130–701, Korea: and Department of Food Science and Nutrition, Kyungpook National
University; Daegu 702–701, Korea. Received March 4, 2008; accepted May 9, 2008; published online May 23, 2008
Repeated silica gel and octadecyl silica gel (ODS) column chromatography of the aerial parts of Artemisia
princeps PAMPANINI (Sajabalssuk) led to the isolation of a new sesquiterpenoid, 3-((S)-2-methylbutyryloxy)-costu-
1(10),4(5)-dien-12,6a-olide (2), along with two previously reported sesquiterpenoids: 8a-angeloyloxy-3b,4b-
epoxy-6bH,7aH,8bH-guaia-1(10),11(13)-dien-12,6a-olide (1, carlaolide B) and 3b,4b-epoxy-8a-isobutyryloxy-
6bH,7aH,8bH-guaia-1(10),11(13)-dien-12,6a-olide (3, carlaolide A). The structure of compound 2 was eluci-
dated by spectroscopic data analysis, including one dimensional (1D) and two dimensional (2D) nuclear magnetic
resonance (NMR) experiments. Of the isolates, compound 2 exhibited potent cytotoxicity against human cervix
adenocarcinoma cells and induced apoptosis.
Key words Artemisia princeps; carlaolide A; carlaolide B; 3-((S)-2-methylbutyryloxy)-costu-1(10),4(5)-dien-12,6a-olide; cy-
totoxicity; apoptosis
More than 200 Artemisia species have been described loxy-6bH,7aH,8bH-guaia-1(10),11(13)-dien-12,6a-olide
worldwide, with 38 identified in Korea; many species have (carlaolide A), respectively, through the comparison of their
yet to be undescribed.1) Knowledge of these undescribed spectroscopic data with those in the existing literature8,9)
species is important, not only to enable their conservation, (Fig. 1).
but also for the discovery of novel compounds which may be
Compound 2, which was obtained as a yellowish, amor-
effective in treating diseases. Artemisia has traditionally been phous powder from chloroform (CHCl3), exhibited ab-
used in medicine as an antiphlogistic, an anodyne and a car- sorbance bands of ester (1763 cmꢀ1) and olefine (1670 cmꢀ1)
diotonic drug.2) The chemical constituents of the genus in the infra red spectrometer (IR) spectrum. The molecular
Artemisia have been studied by a number of researchers who ion peak [M]ꢁ at m/z 332 in the electron ionization mass
have isolated terpenoids, sesquiterpenoids, lignans, phenyl- spectrometer (EI-MS) spectrum and m/z 332.2006 in the high
propanoids.3—5) As of yet, however, There has been very little resolution (HR) EI-MS spectrum as a mass number of
research focusing on Artemisia princeps PAMPANINI (Saja- C20H28O4 (calcd mass numberꢂ332.1987) were obtained.
balssuk).
The proton-NMR (1H-NMR) spectrum showed two exometh-
“Sajabalssuk” is a variety of Artemisia princeps, an annual ylene signals [dH 6.25 (H-13a) and dH 5.51 (H-13b)], two
herb that grows in the wild in Ganghwa Island, Korea. Saja- olefine methine signals [dH 4.89 (H-1) and dH 4.86 (H-5)],
balssuk has been used as an antiphlogistic, a cardiotonic two oxygenated methine signals [dH 5.18 (H-3) and dH 4.57
drug, and to reduce bleeding, regulate menses and cure men- (H-6)], two methine signals [dH 2.57 (H-7) and dH 2.39 (H-
strual disorders, and alleviate pain. The constituents of Saja- 2ꢃ)], three methylene signals exhibiting germinal couplings
balssuk include flavonoids, such as eupatilin, jaceosidin, api- (J2) [dH 2.48 and dH 2.25 (H-2); dH 2.10 and dH 1.62 (H-8);
genin and eupafolin6) and steroids, such as b-sitosterol, er- dH 2.42 and dH 2.41 (H-9)], one methylene signal [dH 1.65
gosterol peroxide, daucosterol and stigmasterol.7) This paper
details the isolation and identification of two previously
known and one new sesquiterpenoids from the aerial parts of
Artemisia princeps PAMPANINI (Sajabalssuk), and evaluates
their cytotoxic and apoptosis-inducing activities. The ethyl
acetate (EtOAc) layer of the 80% methanol extract was de-
veloped using silica gel thin layer chromatography (TLC).
After spraying a 10% H2SO4 solution and heating the TLC
plate, the spots showed purple coloration, indicating the pres-
ence of sesquiterpenoids in the fraction. Repeated silica gel
and octadecyl silica gel (ODS) column chromatography of
the EtOAc layer yielded three sesquiterpenoids, compounds
1—3.
Fig. 1. Chemical Structures of Sesquiterpenoids Isolated from the Aerial
Parts of Artemisia princeps PAMPANINI (Sajabalssuk)
Compounds 1 and 3 were identified as 8a-angeloyloxy-
The two-way arrows indicate the correlations between proton and proton signals in
the NOESY spectrum, and one-way arrows indicate the long-range correlations be-
tween proton and carbon signals in the gHMBC spectrum.
3b ,4b -epoxy-6b H,7a H,8b H-guaia-1(10),11(13)-dien-
12,6a-olide (carlaolide B) and 3b,4b-epoxy-8a-isobutyry-
∗ To whom correspondence should be addressed. e-mail: nibaek@khu.ac.kr.
© 2008 Pharmaceutical Society of Japan

August 2008
1169
(H-3ꢃ)], and two allylic singlet methyl signals [dH 1.69 (H- methyl ester from compound 2 was 8.59 min. The stereo-
15) and dH 1.44 (H-14)], a doublet methyl signal at dH 1.14 chemistry of chiral carbon (C-2ꢃ) for 2-methylbutanoic acid
(H-5ꢃ), and a triplet methyl signal at dH 0.90 (H-4ꢃ). In the from compound 2 was therefore determined to be an S-con-
13C-NMR spectrum, a total of twenty signals were observed. figuration. Finally, the structure of compound 2 was con-
In the sp2 carbon region we observed two carbonyl carbon cluded to be 3-((S)-2-methylbutyryloxy)-costu-1(10),4(5)-
signals [dC 175.4 (C-1ꢃ), dC 170.0 (C-12)], three olefine qua- dien-12,6a-olide (Fig. 1). This is the first report addressing
ternary carbon signals [dC 139.4 (C-4), dC 138.8 (C-10), and the isolation of compound 2 from a natural source.
dC 138.6 (C-11)], two olefine methine carbon signals [dC
Guaianolide sesquiterpenoids have been reported to have
125.5 (C-5) and dC 124.2 (C-1)], and one exomethylene car- cytotoxic effects on various cancer cell lines.17,18) We previ-
bon signal [dC 120.0 (C-13)]. In the sp3 carbon region, sig- ously reported that costunolide sesquiterpenoids are potent
nals were observed for two oxygenated-methine carbons [dC cytotoxins19) and induce apoptosis in human cancer cells via
81.0 (C-6) and dC 78.6 (C-3)], two methine carbons [dC 50.0 depletion of intracellular thiols.20) Accordingly, in this study
(C-7) and dC 41.3 (C-2ꢃ)], four methylene carbons [dC 41.0 the three isolated sesquiterpenoids were evaluated for their
(C-8), dC 32.2 (C-2), dC 28.3 (C-9), and dC 26.8 (C-3ꢃ)] and cytotoxic and apoptosis-inducing abilities.
four methyl carbons [dC 16.7 (C-5ꢃ), dC 16.4 (C-14), dC 12.7
The cytotoxic activity of the three isolated sesquiter-
(C-15), and dC 11.7 (C-4ꢃ)]. We concluded, therefore, that penoids in human cervical adenocarcinoma (HeLa), human
compound 2 is a germacrane-type sesquiterpenoid with two leukemia (U937) and human lung adenocarcinoma (A549)
olefins—one between C-1 and C-10 and another between C-4 cell lines was examined using a colorimetric 3-(4,5-di-
and C-5—an exomethylene on C-1, and a 2-methylbutyry- methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
loxy group on C-3. Determination of the final structure of assay. Each compound showed significant cytotoxicity
compound 2, including the location of the functional group, against all cancer cells (Table 1). Cytotoxicity most likely oc-
was accomplished using gradient heteronuclear multiple- curs at level of replication as opposed to an effect on the
bond connectivity (gHMBC). The exomethylene signals at biosynthesis of DNA precursors.21) The active chemicals may
dH 6.25 (H-13a) and dH 5.51 (H-13b) were correlated with penetrate the cell membrane to the DNA and react with nu-
methine carbons at dC 50.0 (C-7) and dC 81.0 (C-6). Two cleophiles, especially the cystein sulfhydryl groups of en-
methyl signals, H-15 (dH 1.69) and H-14 (dH 1.44), corre- zymes, through a Michael-type addition.22) Variance in cyto-
lated with two olefine quaternary carbons, C-4 (dC 139.4) toxicity may therefore largely be explained by differences in
and C-10 (dC 138.8). These observations revealed the cos- electronics, lipophilicity and molecular geometry.23) The cy-
tunolide skeleton. The oxygenated methine proton signal at totoxicity of compounds 1—3 was chemically mediated by
dH 5.18 (H-3) correlated with the carbonyl carbon of 2- an a,b-unsaturated carbonyl structure—e.g. an a-methylene-
methylbutyryloxy at dC 175.5 (C-1ꢃ), indicating that the 2- g-lactone—and organic acid moieties—angeloyl, methylbu-
methylbutyryloxy group attaches to C-3 through an ester tyroyl and isobutyroyl—which increase the lipophilicity of
bond. The relative stereochemistry for chiral carbons such as the molecule. Compared to other compounds, compound 2
C-3, C-6 and C-7, and the configurations of double bonds has more double bonds in the ring structure and showed rela-
were determined using a NOE experiment. The H-3 proton tively higher activity. The level of activity exhibited by com-
signal at dH 5.18 showed a NOE effect with an olefin proton pound 2 is almost identical to the well known anti-cancer
signal at dH 4.86 (H-5) and H-5 with a methine proton signal compound, cisplatin. The double bonds increase the electron
at dH 2.57 (H-7); this indicates that the stereostructure of the cloud of the molecules and can, in addition, influence the
double bond between C-4 and C-5 was E. Both H-5 and H-7 molecular form with conformations. The presence of an
showed the same configuration. The NOESY spectrum ex- epoxide group in compounds 1 and 3 will also increase the
hibited correlations between H-6 (dH 4.57) and an equatorial electron cloud; however, these structures appeared to have
proton signal at dH 1.62 (H-8b), and between H-7 and an only a minor effect on activity.23) Therefore, we conclude that
axial proton signal at dH 2.11 (H-9a), which indicates they the double bond has more of an influence on activity level
have identical configurations. The cross peaks between H-9a than the epoxide group.
and an olefin proton signal at dH 4.89 (H-1), and between an
Because apoptosis-inducing compounds are potential anti-
allylic methyl proton signal at dH 1.69 (H-15) and an axial tumor agents, we tested the ability of compound 2 to induce
proton signal at dH 2.48 (H-2b) verify the stereochemistry of apoptosis in HeLa cells. Apoptotic cells have several typical
a double bond between C-1 and C-10 as E. The absolute
stereochemistry of compound 2 was confirmed by a compari- Table 1. The Cytotoxicity of Sesquiterpenoids Isolated from Aerial Parts
of Artemisia princeps PAMPANINI (Sajabalssuk) against Human Cancer Cell
Lines
son of specific rotation ([a]D ꢁ133.2) and the chemical
shifts and the coupling patterns of C-3 (dH 5.18, dd, Jꢂ10.4,
5.2 Hz; dC 78.6), C-6 (dH 4.57, dd, Jꢂ9.2, 9.6 Hz; dC 81.0),
and C-7 (dH 2.57, m; dC 50.0) in the NMR spectra with those
IC50 (mg/ml)
Compound
HeLa
U937
A549
of 3S,6S,7R-3b-hydroxygermacra-1(10)E,4E,11(13)-trien-
12,6a-olide [3b-hydroxycostunolide, hanphyllin; [a]D
ꢁ155.210); ꢁ103.811)] and other relevant components.12—15)
The configuration of C-2ꢃ was subsequently determined
using GC analysis with a chiral column.16) The retention
times of (S)-2-methylbutanoic acid methyl ester and (R)-2-
methylbutanoic acid methyl ester appeared at 8.66 min and
1
2
3
22.8
15.5
22.9
15.7
17.2
16.0
22.6
12.5
43.5
22.1
40.5
24.9
Cisplatin
IC50 is defined as the concentration that results in a 50% decrease in the number of
cells compared to that of the control cultures in the absence of compound. The data are
9.27 min, respectively, while that of 2-methylbutanoic acid representative examples for tests performed in triplicate.

1170
Vol. 56, No. 8
Fig. 3. Activation of Caspase-3 during Compound 2-Induced Apoptosis
After treatment with 2.5 and 15 mg/ml compound 2, the cleavage of procaspase-3
was examined by Western blot analysis.
the involvement of caspase activation in compound 2-in-
duced apoptosis in HeLa cells. Western blotting was per-
formed to analyze caspase activation. Treatment of cells with
compound 2 (2.5, 10 mg/ml) for 24 h increased the cleavage
form of procaspase-3 (Fig. 3). These results indicate that
compound 2 is a potent inducer of apoptosis in HeLa cells
via caspase-3 activation. Although further structure–activity
relationship studies are necessary, a sesquiterpenoid such as
compound 2, has the potential to be a powerful anti-tumor
agent. Moreover, Artemisia princeps PAMPANINI (Sajabalssuk),
which is frequently ingested in Korea in both traditional
medication and as an ingredient when cooking, might prove
useful for the development of anti-cancer functional foods or
medicine.
Experimental
General Experimental Procedures Melting points were determined on
a Fisher–John apparatus and uncorrected. Optical rotation was measured on
a JASCO P-1010 digital polarimeter (Jasco, Tokyo, Japan). The IR spectrum
was run on a Perkin Elmer spectrum One FT-IR spectrometer (Perkin Elmer,
Norwalk, U.S.A.). EI-MS was recorded on a JEOL JMS 700 (JEOL, Tokyo,
Japan). 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were taken
on a Varian Unity Inova AS 400 FT-NMR spectrometer (Varian, California,
U.S.A.). Isotopic analysis was carried out using a liquid scintillation counter
(1450 Microbeta Trilux Wallac Cy, Turku, Finland).
Plant Materials Artemisia princeps PAMPANINI (Sajabalssuk) was har-
vested at Ganghwa in 2003, and was provided by Ganghwa Agricultural
R&D Center, Incheon, Korea, and identified by Prof. Jae-Ho Pyee of
Dankook University, Seoul, Korea. The Sajabalssuk was stored in the shade
for two years and then used for the experiments. A voucher specimen
(KHU-NPCL-051020) was lodged at the Laboratory of Natural Products
Chemistry, Kyung Hee University, Suwon, Korea.
Fig. 2. Accumulation of Cells with Sub-G1 DNA Content (A) and DNA
Fragmentation (B) in Compound 2-Treated HeLa Cells
Chemicals Methanol-d4 and CDCl3 as the internal standard was pur-
chased from Sigma (St.Louis, MO, U.S.A.). RPMI Medium 1640, Dul-
becco’s Modified Eagle Medium (GIBCO BRL, Life Technologies Inc., NY,
U.S.A.) and Penicillin-Streptomycin were purchased from GIBCO (Grandis-
land, NY, U.S.A.). Fetal bovine serum (FBS) was obtained from Hyclone
(Logan, UT, U.S.A.). MTT, RNase, leupeptin, aprotinin, phenylmethyl-
sulfonylfluoride (PMSF), 4ꢃ,6-diamidino-2-phenylindole-dihydrochloride
(DAPI) and propidium iodide (PI) were purchased from Sigma Chemical
Co. (St. Louis, MO, U.S.A.). Antibodies for caspase-3 and b-actin were pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
HeLa cells were treated with compound 2 at the indicated concentrations for 24 h,
and then induction of apoptosis was estimated. (A) Accumulation of cells with sub-G1
DNA content. Cell distribution according to DNA content was measured by PI incorpo-
ration and apoptotic DNA contents (sub-G1 phase) analyzed by flow cytometry. (B) The
extent (%) of DNA fragmentation was determined by a fluorometric method using
DAPI. Data presented are the meansꢅS.D. of results from three independent experi-
ments. Cisplatin was compared as a positive control. ∗ pꢆ0.05 vs. control group; signif-
icance between treated groups were determined using the Student–Newman–Keuls
method.
features, such as accumulation of cells with sub-G1 DNA
content, DNA fragmentation into nucleosomal fragments and
the appearance of nuclear condensation.24,25) The number of
HeLa cells in the sub-G1 fraction increased in a concentra-
tion-dependent manner (Fig. 2A). Compared to the control
cells (0.64%), treatment with compound 2 (15 mg/ml) for
24 h resulted in 41.44% sub-G1 ratio (Fig. 2A). In addition,
compound 2 also induced DNA fragmentation in a concen-
tration-dependent manner in the HeLa cells (Fig. 2B). Be-
cause it has been suggested that cytosolic aspartate-specific
proteases, called caspases, are responsible for the intentional
Extraction and Isolation The dried aerial parts of Artemisia princeps
PAMPANINI (Sajabalssuk) (4 kg) were extracted at room temperature with 80%
methanol (MeOH, 22 l). The extract was partitioned with water (4 l), EtOAc
(4 lꢄ2) and normal butanol (n-BuOH, 4 lꢄ2), successively, and concen-
trated in vacuo to obtain the EtOAc (47 g, SJBE), n-BuOH (107 g, SJBB),
and H2O extracts (45 g, SJBW). The EtOAc extract was applied to a silica
gel (70—230 mesh) column (8ꢄ25 cm) for chromatography (c.c), and
eluted with n-hexane : EtOAc (7 : 1→5 : 1→3 : 1→1 : 1, each 5 l) with moni-
toring by TLC to produce 20 fractions (SJBE-1 to SJBE-20). SJBE-11
(Ve/Vtꢂ0.55, elution volume/total volume, 664 mg) was applied to the silica
gel c.c. (4ꢄ5 cm) and eluted with n-hexane : EtOAc (5 : 1, 3 l), which re-
sulted in 19 fractions (SJBE-11-1 to SJBE-11-19). The SJBE-11-5 (Ve/Vtꢂ
0.26, 190 mg) fraction was subjected to ODS c.c. (2ꢄ5 cm) and eluted with
disassembly of a cell into apoptotic bodies,26) we examined MeOH : H2O (2 : 1, 1 l) to produce purified compound 1 (SJBE-11-5-5,

August 2008
1171
Flow-Cytometric Cell Analysis by PI Staining The cell cycle distribu-
tion has been previously described.28) The cells were collected by centrifuga-
tion at 2000 rpm for 4 min. The cell pellets were then resuspended in 1 ml of
phosphate-buffered saline (PBS), fixed in 70% ice-cold ethanol, and kept in
a freezer overnight. The fixed cells were centrifuged, washed once in PBS
and resuspended in PI staining solution containing 50 mg/ml of propidium
iodide and 100 mg/ml of RNase A. The cell suspension, which was shielded
from light, was incubated for 30 min at 37 °C and analyzed using the FACS
(Becton-Dickinson, Heidelberg, Germany). A total of 10000 events were ac-
quired for analysis using CellQuest software.
Ve/Vtꢂ0.71, ODS TLC Rfꢂ0.5 in MeOH : H2Oꢂ5 : 1, 19 mg). The SJBE-
12 (Ve/Vtꢂ0.6, 1.26 g) fraction was applied to a silica gel c.c. (4ꢄ10 cm)
and eluted with n-hexane : EtOAc (5 : 1, 3 l) to give 10 fractions (SJBE-12-1
to SJBE-12-10), and the SJBE-12-4 (Ve/Vtꢂ0.4, 401 mg) fraction was ap-
plied to an ODS c.c. (2ꢄ4 cm) and eluted with MeOH : H2O (2 : 1, 1.5 l) to
yield compound 2 (SJBE-12-4-10, Ve/Vtꢂ0.9, ODS TLC Rfꢂ0.4 in
MeOH : H2Oꢂ7 : 1, 17 mg). The SJBE-12-7 (Ve/Vtꢂ0.7, 100 mg) fraction
was eluted with MeOH : H2O (5 : 1, 2 l) from an ODS c.c. (2ꢄ4 cm) to pro-
duce compound 3 (SJBE-12-7-7, Ve/Vtꢂ0.7, Silica gel TLC Rfꢂ0.5 in n-
hexane : EtOAcꢂ3 : 1, 38 mg).
DAPI Assay DNA fragmentation was quantitated, as previously re-
ported.26) In brief, cells were lysed in a solution containing 5 mM Tris–HCl
(pH 7.4), 1 mM EDTA, and 0.5% (w/v) Triton X-100 for 20 min on ice. The
lysate and supernatant obtained after centrifugation at 27000ꢄg for 20 min
were sonicated for 40 s, and the level of DNA in each fraction was measured
with a fluorometric method using DAPI. The amount of fragmented DNA
was calculated as the ratio of the amount of DNA in the supernatant to that
in the lysate.
8a-Angeloyloxy-3b,4b-epoxy-6bH,7aH,8bH-guaia-1(10),11(13)-dien-
12,6a-olide (1, Carlaolide B): Yellowish powder (MeOH); mp 180—181 °C;
[a]D ꢀ11.3 (cꢂ0.52, CHCl3); EI-MS m/z: 344 [M]ꢁ; IR n (KBr) cmꢀ1
:
1765, 1735, 1668, 1625.
3-((S)-2-Methylbutyryloxy)-costu-1(10),4(5)-dien-12,6a-olide (2): Yel-
lowish powder (MeOH); mp 103—105 °C; [a]D ꢁ133.2 (cꢂ1.03, MeOH);
EI-MS m/z: 332 [M]ꢁ, 217, 189, 175, 150, 123, 109, 53; HR-EI-MS m/z
332.1996 [M]ꢁ (Calcd for C20H28O4ꢂ332.1987); IR n (KBr) cmꢀ1: 1763,
Western Blot Analysis HeLa cells (2.5ꢄ107) were harvested and
washed twice with ice-cold PBS. Cell pellets were then lysed in an ice-cold
cell extraction buffer [50 mM HEPES, pH 7.0, 250 mM NaCl, 5 mM EDTA,
0.1% NP-40, 0.1 mM PMSF, 0.5 mM dithiothreitol (DTT), 5 mM NaF, 0.5 mM
Na orthovanadate) containing 5 mg/ml each of leupeptin and aprotinin and
incubated for 30 min at 4 °C. Cell debris was removed by microcentrifuga-
tion (10000 g, 5 min), followed by quick freezing of the supernatants. Pro-
tein concentration was determined by a Bio-Rad protein assay reagent. Cel-
lular proteins (50 mg) were electroblotted onto nitrocellulose membrane fol-
lowing separation on a 10—15% SDS-polyacrylamide gel electrophoresis.
The immunoblot was incubated overnight with blocking solution (5% skim
milk in TBS containing 0.1% Tween 20) at 4 °C, and then incubated for 4 h
with a 1 : 1000 dilution of anti-caspase-3 antibody. Blots were washed three
times with TTBS (Tris-buffered saline, 0.1% Tween 20), and then incubated
with a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary an-
tibody for 1 h at room temperature, washed again three times with TTBS,
and then developed by enhanced chemiluminescence (Amersham Life Sci-
ence, Arlington Heights, IL, U.S.A.).
1
1670, 1450, 1140; H-NMR (400 MHz, CDCl3) dH: 6.25 (1H, d, Jꢂ3.2 Hz,
H-13a), 5.51 (1H, d, Jꢂ3.2 Hz, H-13b), 5.18 (1H, dd, Jꢂ10.4, 5.2 Hz, H-3),
4.89 (1H, br d, Jꢂ9.6 Hz, H-1), 4.86 (1H, d, Jꢂ9.6 Hz, H-5), 4.57 (1H, dd,
Jꢂ9.2, 9.6 Hz, H-6), 2.57 (1H, m, H-7), 2.48 (1H, m, H-2a), 2.42 (1H, m,
H-9b), 2.39 (1H, qt, Jꢂ7.2, 6.4 Hz, H-2ꢃ), 2.25 (1H, m, H-2b), 2.11 (1H, m,
H-9a ), 2.10 (1H, m, H-8a), 1.69 (3H, br s, H-15), 1.65 (2H, qd, Jꢂ7.2,
6.4 Hz, H-3ꢃ), 1.62 (1H, m, H-8b), 1.44 (3H, br s, H-14), 1.14 (3H, d,
Jꢂ7.2 Hz, H-5ꢃ), 0.90 (3H, d, Jꢂ7.2 Hz, H-4ꢃ); 13C-NMR (100 MHz,
CDCl3) dC: 175.5 (C-1ꢃ), 170.0 (C-12), 139.4 (C-4), 138.8 (C-10), 138.6 (C-
11), 125.5 (C-5), 124.2 (C-1), 120.0 (C-13), 81.0 (C-6), 78.6 (C-3), 50.0 (C-
7), 41.3 (C-2ꢃ), 41.0 (C-9), 32.2 (C-2), 28.3 (C-8), 26.8 (C-3ꢃ), 16.7 (C-5ꢃ),
16.4 (C-14), 12.7 (C-15), 11.7 (C-4ꢃ).
3b,4b-Epoxy-8a-isobutyryloxy-6bH,7aH,8bH-guaia-1(10),11(13)-dien-
12,6a-olide (3, Carlaolide): White powder (MeOH); mp 183—191 °C; [a]D
ꢀ16.5 (cꢂ0.1, CHCl3); EI-MS m/z: 332 [M]ꢁ, 314; IR n (KBr) cmꢀ1: 1760,
1735, 1670, 1625.
Alkaline-Hydrolysis of Compound 2 Followed by Methylesterification
and GC Analysis Compound 2 (3 mg) was dissolved in a 10% KOH solu-
tion (3 ml, H2O–MeOHꢂ1 : 10) and refluxed for 2 h, at which time TLC
(CHCl3–MeOHꢂ7 : 1) indicated that the reaction was complete. After the
reaction mixture was neutralized with cation exchange resin (DOWEX
50WX4-400, Aldrich Inc., Milwaukee, WI, U.S.A.) and filtrated, the solution
was concentrated under reduced pressure to dryness. The concentrate was
dissolved in MeOH (1 ml) and esterified by adding diazomethane in ether
(ca. 1 ml). Gas chromatography was used to analyze the solution. A Shi-
madzu gas chromatograph Model GC-14B (Japan), equipped with an on-
column injection system and FID was used. The column used was an Alltech
Chiraldex G-Ta capillary column (30 mꢄ0.25 mm i.d., film thickness
0.125 mm; Alltech Associates Inc., Deerfield, IL, U.S.A.). The operating
conditions were as follows: carrier gas flow (N2), 1.5 ml/min with split; H2,
60 kPa; air, 50 kPa; make-up gas (N2), 29 ml/min; injector, 200 °C; detector,
220 °C. The oven temperature was maintained at 40 °C for 4 min, then in-
creased to 80 °C at the rate of 2 °C/min, and subsequently maintained at
80 °C for 4 min. 0.5 ml of sample was injected directly into the inject port.
Both the (S)-(ꢁ)-2-methylbutanoic acid (2 mg, Sigma-Aldrich Korea, Yon-
gin, Korea) and (ꢅ)-2-methylbutanoic acid (2 mg, Sigma-Aldrich Korea,
Yongin, Korea) were esterified and analyzed using the same procedure as de-
scribed previously. The retention times of (S)-2-methylbutanoic acid methyl
ester and (R)-2-methylbutanoic acid methyl ester were at 8.66 min and
9.27 min, respectively.
Statistical Analysis Data presented are the meansꢅS.D. from three in-
dependent experiments. The Student–Newman–Keuls method was used to
compare treated groups and controls. ∗ pꢆ0.05 indicates statistical signifi-
cance.
Acknowledgements This study was supported by the Ganghwa R&D
Center of ‘Evaluation of Biological activity and Pharmacological efficacy
for plants indigenous to Ganghaw (Incheon)’ and by a grant from the Korean
Science & Technology Foundation through the Plant Metabolism Research
Center, Kyung Hee University.
References
1) Tang W., Eisenbrand G., “Chinese Drugs of Plant Origin,” Springer
Verlag., Berlin, 1992, pp. 159—183.
2) Cho Y. H., Chiang M. H., Kor. J. Intl. Agric., 13, 313—320 (2001).
3) Brown G. D., Lian G. Y., Sy L. K., Phytochemistry, 64, 303—323
(2003).
4) Maroco J. A., Sanz-Chervra J. F., Garcia-Lliso V., Valles-Xirau J., Phy-
tochemistry, 44, 1133—1137 (1997).
5) Park J. C., Young H. S., Yu Y. B., Lee J. H., Kor. J. Pharmacogn., 25,
70—72 (1994).
6) Ryu S. N., Kang S. S., Kim J. S., Ku B. I., Korean J. Crop Sci., 49,
452—456 (2004).
7) Bang M. H., Kim D. H., Yoo J. S., Lee, D. Y., Song M. C., Yang H. J.,
Jeong T. S., Lee K. T., Choi M. S., Chung H. G., Baek N. I., J. Korea
Soc. Appl. Biol. Chem., 48, 418—420 (2005).
8) Rustaiyan A., Sedaghat S., Acta Horticulturae, 667, 65—69 (2005).
9) Abbas H., Nazak A., Farahnaz K. S., Abdolhossein R., DARU, 11,
171—174 (2003).
10) Tarasov N. D., Abdullaev N. D., Kasymov Sh. Z., Sidyakin G. P.,
Yagudaev M. R., Khim. Prirodn. Soedin., 1, 62—65 (1978).
11) Asakawa Y., Toyota M., Takemoto T., Phytochemistry, 20, 257—261
(1981).
Cell Culture Human cervical adenocarcinoma HeLa, human histocytic
lymphoma U937 and human lung adenocarcinoma A549 cell lines were pur-
chased from the Korean cell line bank (KCLB, Seoul, Korea) and cultured in
RPMI 1640 media supplemented with 10% heat-inactivated FBS, penicillin
(100 units/ml), and streptomycin sulfate (100 mg/ml). Cells were maintained
at 37 °C in an atmosphere of 5% CO2 in air.
Cell Viability Assay Cytotoxicity was assessed by the MTT assay.27)
Briefly, cells were seeded at 1ꢄ105/ml in each well containing 100 ml of the
RPMI medium supplemented with 10% FBS in a 96-well plate. After 12 h,
various concentrations of tested samples were added. After 48 h, 50 ml of
MTT (5 mg/ml stock solution, in PBS) was added per well and the plates
were incubated for an additional 4 h. The medium was discarded and the for-
mazan blue, which formed in the cells, was dissolved with 100 ml DMSO.
The optical density was measured at 540 nm using an Enzyme-Linked Im-
muno Sorbent Assay (ELISA) system version 2 (Amersham Pharmacia
Biotech).
12) Sigstad E. E., Catalan C. A. N., Gutierrez S. B., Diaz J. G., Goedken V.
L., Herz W., Phytochemistry, 30, 1933—1940 (1991).
13) Irwin M. A., Geissman T. A., Phytochemistry, 12, 875—878 (1973).
14) Fang F., Sang S., Chen K. Y., Gosslau A., Ho C.-T., Rosen R. T., Food
Chem., 93, 497—501 (2005).

1172
Vol. 56, No. 8
15) Bohlmann F., Ang W., Trinks C., Jakupovic J., Huneck S., Phytochem- 22) Abeysekera B. F., Abramowski Z., Towers G. H. N., Biochem. System.
istry, 24, 1009—1015 (1985).
Ecol., 13, 365—369 (1985).
16) Kwon D. Y., Yang H.-J., Yoon S. H., Food Sci. Biotechnol., 8, 139—
141 (1999).
23) Scotti M. T., Fernandes M. B., Ferreira M. J. P., Emerenciano V. P.,
Bioorg. Med. Chem., 15, 2927—2934 (2007).
17) Ma J. Y., Wang Z. T., Xu L. S., Xu G. J. A., Phytochemistry, 50, 113—
114 (1999).
18) Fatima H., Otto S., Jorg, H., J. Nat. Prod., 65, 523—526 (2002).
19) Lee M. G., Lee K. T., Chi S. G., Park J. H., Biol. Pharm. Bull., 24,
303—306 (2001).
24) Wyllie A. H., Nature (London), 284, 555—556 (1980).
25) Ormerod M. G., Collins M. K., Rodrigues-Tarduchy G., Robertson D.,
J. Immunol. Methods, 153, 57—65 (1992).
26) Cervia D., Martini D., Garcia-Gil M., Di Giuseppe G., Guella G., Dini
F., Bagnoli P., Apoptosis, 11, 829—843 (2006).
20) Choi J. H., Ha J., Park J. H., Lee J. Y., Lee Y. S., Park H. J., Choi J. W., 27) Park H. J., Kwon S. H., Han Y. N., Choi J. W., Miyamoto K., Lee S.
Masuda Y., Nakaya K., Lee K. T., Jpn. J. Cancer Res., 93, 1327—
1333 (2002).
21) Woynarowski J. M., Konopa J., Mol. Pharmacol., 19, 97—102 (1981).
H., Lee K. T., Arch. Pharm. Res., 24, 342—348 (2001).
28) Choi J. H., Seo B. R., Seo S. H., Lee K. T., Park J. H., Park H. J., Choi
J. W., Itoh Y., Miyamoto K., Arch. Pharm. Res., 25, 80—84 (2002).