Phytomedicine 17 (2010) 935939

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Phytomedicine
journal homepage: www.elsevier.de/phymed

Short communication

1 S-1 -Acetoxyeugenol acetate: A new chemotherapeutic natural compound

against MCF-7 human breast cancer cells
Noor Hasima a, , Lionel In Lian Aun a , Mohamad Nurul Azmi b , Ahmad Nazif Aziz e ,
Eswary Thirthagiri c , Halijah Ibrahim d , Khalijah Awang b
a
Dept. of Genetics & Molecular Biology, (ISB), Faculty of Science, University Malaya, 50603, Kuala Lumpur, Wilayah Persekututan, Malaysia
b
Centre for Natural Product Research and Drug Discovery (CENAR), Dept. of Chemistry, Faculty of Science, University Malaya, 50603, Kuala Lumpur, Malaysia
c
Cancer Research Initiative Foundation, Sime Darby Medical Centre, Outpatient Centre, 47500, Subang Jaya, Selangor, Malaysia
d
Dept. of Ecology and Biodiversity, (ISB), Faculty of Science, University Malaya, 50603, Kuala Lumpur, Malaysia
e
Dept. of Chemical Science, Faculty of Science and Technology, University Malaysia Terengganu, Mengabang Telipot, 21030, Kuala Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Keywords: Medicinal plants containing active natural compounds have been used as an alternative treatment for
1 S-1 -Acetoxychavicol acetate cancer patients in many parts of the world especially in Asia (Itharat et al. 2004). In this report, we describe
1 S-1 -Acetoxyeugenol acetate the cytotoxic and apoptotic properties of 1 S-1 -acetoxyeugenol acetate (AEA), an analogue of 1 S-1 -
Alpinia conchigera
acetoxychavicol acetate (ACA), isolated from the Malaysian ethno-medicinal plant Alpinia conchigera
Apoptosis
Griff (Zingiberaceae) on human breast cancer cells. Data from MTT cell viability assays indicated that
Cytotoxicity
Breast cancer AEA induced both time- and dose-dependant cytotoxicity with an IC50 value of 14.0 M within 36 h of
treatment on MCF-7 cells, but not in HMEC normal control cells. Both annexin V-FITC/PI ow cytometric
analysis and DNA fragmentation assays conrmed that AEA induced cell death via apoptosis. AEA was
also found to induce cell cycle arrest in MCF-7 cells at the G0 /G1 phase with no adverse cell cycle arrest
effects on HMEC normal control cells. It was concluded that AEA isolated from the Malaysian tropical
ginger represents a potential chemotherapeutic agent against human breast cancer cells with higher
cytotoxicity potency than its analogue, ACA.
2010 Elsevier GmbH. All rights reserved.

Introduction cells (Campbell et al. 2007), human T cell lymphoma (Ichikawa

Alpinia conchigera, also known locally as lengkuas ranting,
lengkuas kecil, lengkuas padang, lengkuas getting or chengke-
nam (Janssen and Scheffer 1985) is a herbaceous perennial, 25
feet tall, found in eastern Bengal and southwards to Peninsular
Malaysia and Sumatera (Burkill 1966). It is used as a condiment
in the northern states of Peninsular Malaysia and occasionally in
traditional medicine in the east coast to treat fungal infections.
In Thailand, the rhizomes are used in traditional Thai medicine to
relieve the gastro-intestinal disorders and in the preparation of Thai
food dishes (Matsuda et al. 2005).
Traditional medicine from various plant types containing active
natural compounds such as chalcones (Hsu et al. 2006), xanthoan-
gelol (Tabata et al. 2005) and licochalcone-A (Fu et al. 2004) have
all been reported as potential drugs for the treatment of cancer.
In recent years, the pro-apoptotic effects of 1 S-1 -acetoxychavicol
acetate (ACA) from the Thai ginger isolate, Languas galanga and
Alpinia galanga, have been documented in human breast carcinoma
Corresponding author. Tel.: +603 79675921; fax: +603 79675908.
E-mail addresses: hasima@um.edu.my, lionelin 81@yahoo.com (N. Hasima).
et al. 2005) and in the inhibition of tumor-promoter-induced
EpsteinBarr virus (Kondo et al. 1993). Even though previous stud-
ies have shown that ACA isolates from ginger exhibited anti-tumor
properties against a wide variety of cancers, there have been no
reports thus far on the cytotoxic and apoptotic effects of the closely
related 1 S-1 -aceotxyeugenol acetate (AEA) (Fig. 1) on human
breast cancer cells.
This study describes the purication, characterization and bio-
logical activity of six natural ACA analogs isolated from the
rhizomes of the Malaysian wild-ginger, Alpinia conchigera (Zingib-
eraceae). We report herein the phytochemical data as well as our
preliminary cytotoxic and apoptotic effects of AEA (Fig. 2) on MCF-7
human breast cancer cells.
Materials and methods
Plant material
Rhizomes of Alpinia conchigera Griff. were collected from Jeli
province of Kelantan, East-coast of Peninsular Malaysia. The sample
was identied by Prof. Dr. Halijah Ibrahim from the Institute of
Biological Science, Faculty of Science, University Malaya. A voucher

0944-7113/$ see front matter 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2010.03.011
936 N. Hasima et al. / Phytomedicine 17 (2010) 935939

Fig. 1. Phenylpropanoids isolated from Alpinia conchigera (Zingiberaceae).

specimen (KL5049) was deposited in the Herbarium of Chemistry
Department, Faculty of Science, University Malaya.
Reagents
RPMI-1640, MEGM, fetal bovine serum (FBS) and antibiotics
were purchased from Lonza Inc. (USA). MTT reagent, Annexin V-
FITC/PI apoptosis detection kit, propidium iodide (PI), paclitaxel,
RNase A and Suicide TrackTM DNA Ladder Isolation Kit were pur-
chased from EMD Chemicals Inc. (Calbiochem, San Diego, CA, USA).
Extraction and isolation natural compounds
Air-dried and powdered rhizomes of Alpinia conchigera (2.1 kg)
were extracted with dichloromethane at room temperature (72 h).
The solvent was evaporated in vacuo to give dichloromethane
extract. The extract was subjected to column chromatography (CC)
on silica gel (Merck Kiesegel 60) with stepwise gradient of hexane-
ethyl acetate. Fractions were collected separately and concentrated
in vacuo at 40 C. Fractions with similar proles in TLC were pooled
together to obtain six subfractions which were then subjected to
further chromatographic analysis which yielded six compounds
(16). The structures of compounds 16 were determined based
on comparison of the 1 H and 13 C NMR data with those reported
in the literatures (Lee and Ando 2001; Ando et al. 2005; Janssen
and Scheffer 1985; Yang and Eilerman 1999; Barik et al. 1987;
Mitsui et al. 1976). All spectral data were obtained on the following
instruments; IR on the PerkinElmer FT-IR spectrometer RX1, UV
on a Shimadzu UV-160A UV-Visible Recording Spectrophotome-
ter, NMR on a JEOL (Japan Electronic Optics Laboratory Co. Ltd.,
Tokyo, Japan) JNM-LA400 FT-NMR spectrometer system (400 MHz)
and MS on a Shimadzu GC-MS spectrometer (HP 6890 Series Mass
Selective Detector and HP 6890 Series GC System).
LCMS analysis
LCMS analysis was done using Shimadzu LCMS-IT-TOF
(Columbia, MD, USA) with a binary pump, an automatic injector and

Fig. 2. Cell cycle distribution of MCF-7 and HMEC cells using ow cytometry after staining with propidium iodide (PI). (A) MCF-7 human breast cancer cells before and after
AEA treatment for 12 and 24 h. (B) HMEC normal breast cell controls were treated with AEA for 12 and 24 h followed by cell cycle analysis. I: Sub-G1 ; II: G0 /G1 ; III: S; IV:
G2 /M. All experiments are a representative of 20,000 cells and the percentage of cells in all cell cycle phases are indicated.
 N. Hasima et al. / Phytomedicine 17 (2010) 935939 937

Table 1
MTT cytotoxic effects of ACA analogs from Alpinia conchigera on human breast cancer cells (MCF-7) and human normal breast cells (HMEC).

Cell lines Compound n Treatment (h) IC50 value (M) Viability (% cells)a

HMEC Paclitaxel (positive control) 3 36 n/a 95.2

1 S-1 -Acetoxychavicol acetate (ACA), 1 3 36 n/a 79.4
1 S-1 -Acetoxyeugenol acetate (AEA), 2 3 36 n/a 86.8

MCF-7 Paclitaxel (positive control) 3 36 15.0 16.2

1 S-1 -Acetoxychavicol acetate (ACA), 1 3 36 20.0 0.0
1 S-1 -Acetoxyeugenol acetate (AEA), 2 3 36 14.0 0.0
1 -Hydroxychavicol acetate, 3 3 36 n/a 70.1
trans-p-Coumaryl diacetate, 4 3 36 n/a 72.7
p-Hydroxycinnamyl acetate, 5 3 36 n/a 73.9
p-Hydroxycinnamaldehyde, 6 3 36 n/a 70.6
a
Percentage of cell viability after 36 h incubation upon maximum treatment with 80.0 M of each compound.

a photodiode array detector (SPD-M20A). Separations were car-
ried out on a Waters Xbridge C18 column (50 2.1 mm, 2.5 m).
A binary gradient solvent system of double-distilled water (elu-
ent A)acetonitrile (eluent B) was used as follows: 100% A and
0% B (0.0 min), 0% A and 100% B (6.0 min), 100% A and 0% B
(8.510.0 min). Flow-rate at 0.5 ml/min was used and absorbance
was detected at 254 nm. All tested solutions were ltered through
Whatman 13 mm, 0.2-m nylon membrane syringe lters before
use.
annexin V anti-coagulant (200.0 g/mg) was added to AEA treated
MCF-7 and HMEC cells. All samples were centrifuged and re-
suspended in cold binding buffer and PI (30.0 g/ml). Detection
of signals from a 2.0 104 cells were obtained using the BD
FACSCaliburTM ow cytometer (Becton Dickenson, Mountain View,
CA, USA) and CellQuest Pro IVD software (Becton Dickenson, Moun-
tain View, CA, USA).
DNA fragmentation assay

Cell lines and cultivation conditions
Two human cell lines were used in this study, comprising
of human breast adenocarcinoma (MCF-7) (CARIF, Malaysia) and
human mammary epithelial cells (HMEC) (Lonza, USA), which was
used as a normal cell control. MCF-7 cells were cultured as mono-
layers in RPMI-1640, supplemented with 10.0% (v/v) FBS, 100 U/ml
penicillin and 100.0 g/ml streptomycin, while HMEC cells were
cultured in MEGM. All cultures were maintained at 37 C in 5.0%
CO2 and 95.0% air.
Cells were treated with AEA for 12 and 24 h before harvesting
and total DNA was extracted from both untreated and treated cells
using the Suicide TrackTM DNA Ladder Isolation Kit according to
the manufacturers protocol. Extracted DNA was analysed on a 1.0%
(w/v) agarose gel electrophoresis and stained with ethidium bro-
mide. Fragmentation of DNA was observed under UV illumination
and visualized using a gel documentation system (Alpha Inotech,
USA).
Statistical analysis

MTT cell viability assay
Cytotoxic effects of all ACA analogues on MCF-7 and HMEC
cells were determined by measuring the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye metabolism. All
ACA analogues were dissolved in DMSO to a nal concentration
of 10.0 mM. Briey, 2.0 104 cells were treated in triplicates with
each ACA analogue and paclitaxel. MTT reagent (5.0 mg/ml) was
added and cells were incubated in the dark at 37 C. DMSO was
added to dissolve purple formazon precipitates and a microtiter
plate reader (Tecan Sunrise , Switzerland) was used to detect
absorbance/reference at 570/650 nm.
Cell cycle analysis
Cells were seeded treated with AEA for 12 and 24 h before har-
vesting. Fixation was done with 70% (v/v) ethanol and incubated
at 20 C overnight. Fixed cells were washed with PBS and stained
in 1.0 ml of PBS containing 50.0 g/ml PI and 0.1 mg/ml RNase A.
Fluorescence from a population of 2.0 104 cells were detected
using the BD FACSCaliburTM ow cytometer (Becton Dickenson,
Mountain View, CA, USA) and CellQuest Pro (IVD) software (Becton
Dickenson, Mountain View, CA, USA).
Annexin V-FITC/PI analysis
Detection of apoptosis was conducted using the Annexin
V-FITC/PI apoptosis detection kit according to manufacturers pro-
tocol. Briey, media binding reagent containing FITC-conjugated
All cell proliferation and ow cytometry experiments were car-
ried out in triplicates. Data from all experiments were presented
as mean values with standard deviation (SD). One-way ANOVA
was used to determine the statistical signicance of results with
p 0.05.
Results
Isolation and purication of phenylpropanoids from Alpinia
conchigera
The main focus of this study was to chemically characterize AEA
and other ACA natural analogs from Alpinia conchigera, followed
by implicating their degree of cytotoxicity and apoptotic effects
in the treatment of human breast cancer. Isolation and purica-
tion of the compounds from dichloromethane (CH2 Cl2 ) extract
yielded six puried compounds: 1 S-1 -acetoxychavicol acetate, 1
(1.62 g, 23.1%), 1 S-1 -acetoxyeugenol acetate, 2 (6.0 mg, 0.08%),
1 -hydroxychavicol acetate, 3 (56.8 mg, 0.81%), trans-p-coumaryl
diacetate, 4 (153.8 mg, 2.20%), p-hydroxycinnamyl acetate, 5
(3.0 mg, 0.04%) and p-hydroxycinnamaldehyde, 6 (77.7 mg, 1.11%).
The chemical structures of all six phenylpropanoids were found to
correspond with previous reported references and are illustrated
in Fig. 1.
AEA inhibits the proliferation of MCF-7
The MTT cell viability assay was used to examine the effects
of ACA analogues on the proliferation of MCF-7 and HMEC cells.
938 N. Hasima et al. / Phytomedicine 17 (2010) 935939

Fig. 3. AEA potentiates apoptosis mediated cell death in MCF-7 human breast cancer cells. (A) Detection of apoptosis using ow cytometry after annexin V-FITC/propidium
iodide (PI) staining. MCF-7 cells were treated with AEA at IC50 concentrations for 12 and 24 h. Viable cells are in the lower left quadrant, early apoptotic cells are in the
lower right quadrant, late apoptotic cells are in the upper right quadrant and non-viable necrotic cells are in the upper left quadrant. Dot plots are a representative of 20,000
cells from a single replicate. (B) Bar graph representing mean values from independent triplicate experiments. (C) Conrmation of apoptosis mediated cell death through
observation of DNA laddering using the DNA fragmentation assay. MCF-7 cells were treated with AEA for 12 and 24 h followed by analysis of extracted DNA on 1.0% (w/v)
agarose gel electrophoresis. M: 100 bp DNA size marker.

As shown in Table 1, only ACA and AEA out of the six analogs
tested were successful in inhibiting the growth of MCF-7 cells with
IC50 values of 20.0 and 14.0 M respectively. Cytotoxicity induced
by both ACA and AEA were found to be dose and time depen-
dant with 100% inhibition achieved after 36 h of treatment. The
anti-proliferative effects of both ACA and AEA were found to be
minimal when tested on HMEC normal human breast cells control.
Solvent controls showed that the viability of cells were insigni-
cantly affected (data not shown), indicating that cytotoxicity was
not induced by the presence of DMSO, which has been shown to
be cytotoxic at high concentrations (Violante et al. 2002). AEA also
displayed comparable levels of cytotoxicity with paclitaxel which
was used as a positive control drug. Our preliminary results sup-
port the potential development of AEA as an anti-proliferative drug.
Because the cytotoxicity effects ACA have been previously reported,
we focused our attention to AEA, where all consecutive experi-
ments were carried out based on IC50 values obtained from our
present MTT data.
AEA induces apoptosis mediated cell death in MCF-7 cells
Lastly, we investigated whether AEA was inducing cell death
via apoptosis or necrosis in MCF-7 cells using DNA fragmentation
assay and double uorescence staining of annexin V-FITC/PI ow
cytometry assay. As shown in Fig. 3A, the population of cells indi-
cated a shift from viable cells to early and late stage apoptosis,
followed by secondary necrosis after 12 and 24 h AEA treatment.
The percentage of both early and late apoptotic cells increased from
2.84% to 36.13% after 24 h AEA exposure (Fig. 3B). The occurrence of
apoptosis mediated cell death was conrmed through the activa-
tion endonucleases-mediated nucleosome excision leading to the
observation of DNA laddering of about 180200 bp. Fig. 3C demon-
strated partial and complete fragmentation of MCF-7 genomic DNA
after 12 and 24 h of AEA treatment respectively, which represents
one of the major hallmarks of apoptosis.
Discussion

AEA induces cell cycle arrest in MCF-7 cells
We next evaluated the effects of AEA on cell cycle proles using
ow cytometry PI-based staining. As shown in Fig. 2A, cultivation of
MCF-7 cells with AEA signicantly increased the population of sub-
G1 phase from 1.62% to 52.71% after 24 h incubation. The increase in
the sub-G1 population was consistent with a reduction in the G0 /G1
phase from 79.07% to 37.34% after 24 h. This indicated a potential
cell cycle arrest during the G0 /G1 phase as there were no signicant
changes in both S and G2 /M phases even after 24 h of AEA treatment.
Cell cycle proles were consistent for HMEC normal human breast
cell controls after 12 and 24 h exposure to AEA (Fig. 2B). Therefore,
we concluded that AEA induces G0 /G1 arrest in MCF-7 cells, and did
not cause any signicant changes to normal HMEC cells.
Cells undergoing apoptosis can be observed via multiple
hallmarks such as the appearance of a sub-diploid DNA peak
during PI-based cell cycle analysis, the staining of exposed phos-
phatidylserine on the cell surface which is demonstrated during
annexin V-FITC/PI ow cytometry, and the consistent laddering
of genomic DNA (Renehan et al. 2001; Majno and Joris 1995).
All observations reported in this study strongly suggest that AEA
induces both anti-proliferative and apoptotic effects on MCF-7 with
no cytotoxic effects on HMEC normal human breast cells. Varia-
tions in cell cycle distribution after treatment with AEA, indicated
by changes in PI intensity of stained DNA suggest that AEA inhibits
cell cycle progression effects at the G0 /G1 phase while unaffecting
HMEC normal cells. The effect of AEA in terms of cell cycle arrest
in this study has been consistent with past reports on ACA, where
 N. Hasima et al. / Phytomedicine 17 (2010) 935939
it strongly increased the population of myeloma cells in the G0 G1
phase and the sub-G1 phase (Ito et al. 2005). Our current cell cycle
studies in response to AEA treatments thus necessitates the need
for additional molecular signalling studies on specic cyclin reg-
ulatory proteins, particularly CDK4 and CDK6 which are involved
in G1 progression, and also various CDK inhibitors which may be
up-regulated following AEA treatment.
Previous reports have shown that ACA from different plant
species inhibits cellular carcinogenesis through various means such
as the mitochondrial- and fas-mediated pathway (Ito et al. 2004),
the inhibition of iNOS gene (Ohata et al. 1998), the induction of
TRAIL (Ito et al. 2005) and the suppression of NF-B pathway and
its regulated gene products (Ichikawa et al. 2005). Since NF-B acts
as a transcription factor that regulates the transcription of genes
corresponding to immune responses, proliferation, inammation,
anti-/pro-apoptotic genes and cell cycle regulators (Beinke and Ley
2004), it is likely that both ACA and AEA shares similar apoptotic
mechanics by primarily targeting the NF-B pathway, which could
then trigger a cascade of rippling effects on other downstream
apoptotic pathways.
Considering that only ACA and AEA (out of all six phenyl-
propanoid compounds) were successful in manifesting its cytotoxic
effects, a few observations were noted. On the basis of
structureactivity relationship studies, the contributions of 2 -3
terminal double bond of ACA and AEA is highly responsi-
ble for activity. Evidently, analogue 4, 5 and 6 which were
devoid of the 2 3 terminal double bond did not show sig-
nicant cytotoxic effect on MCF-7 cells. In addition, the para
substitution of the acetoxy and 1 -acetoxypropenyl group at the
benzene ring of ACA and AEA were found to be essential for
biological activity. Murakami et al reported that both acetoxy
group in ACA and AEA were necessary in cellular permeability
properties because the analogue without the 1 -acetyl group (1 S-
1 -hydroxychavicol acetate, 3) resulted in the elimination of its
cytotoxicity.
Previous studies using esterase inhibitors also found that the
acetoxyl groups in ACA were subjected to acetate elimination
through hydrolyzation by intracellular esterase activity in order
to maintain its retention within the cell (Murakami et al. 2000)
thus resulting in an intracellular modied ACA candidate structure
which targets specic downstream molecules. To date, little has
been known about the chemical properties of AEA in relation to its
biological implications in comparison to ACA. We are currently in
the process of elucidating the molecular mechanism by which AEA
modulates its effects through global gene expression and proteins
expression studies, as well as evaluating the synergistic effects of
AEA as a chemosensitizer when used in combination with other
anti-cancer drugs.
Overall, this study provides evidence of 1 S-1 -acetoxyeugenol
acetate (AEA) isolated from the Malaysian Alpinia conchigera Griff.,
of possessing anti-cancer properties through the induction of cell
cycle arrest and apoptosis in MCF-7 human breast carcinoma. Our
current ndings also conclude that AEA induces comparable, if
not, better cytotoxic effects with paclitaxel, and is a signicantly
more potent anti-cancer compound in comparison to its analogue,
ACA.
Acknowledgements
This study was supported by the University of Malaya Post-
graduate Research Grant (PPP) (PS127-2008A), the Ministry of
939
Higher Education (MOHE) through the Fundamental Research
Grant Scheme (FRGS) (FP062-2006A) and the Centre for Natural
Product Research and Drug Discovery (CENAR) Grant (4911274).
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