PHYTOTHERAPY RESEARCH

Phytother. Res. 22, 695–698 (2008)

Published online 18 March 2008
CANCER in Wiley InterScience EFFECTS OF CURCUMA XANTHORRHIZA
CHEMOPROTECTIVE 695
(www.interscience.wiley.com) DOI: 10.1002/ptr.2418

Cancer Chemoprotective Effects

of Curcuma xanthorrhiza

Jae Hee Park1, Kwang Kyun Park1,2,3, Mi Jeong Kim1,3, Jae Kwan Hwang4, Sun Kyu Park1,3
and Won Yoon Chung1,2,3,*
1
Department of Oral Biology, Yonsei University College of Dentistry, Seoul, 120-752, Korea
2
Oral Cancer Research Center, Yonsei University College of Dentistry, Seoul, 120-752, Korea
3
Brain Korea 21 Project, Yonsei University College of Dentistry, Seoul, 120-752, Korea
4
Department of Biotechnology and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea

Curcuma xanthorrhiza Roxb. (Zingiberaceae) is a medicinal plant widely spread in South East Asia. In
particular, it is commonly used not only for food and medicinal purposes in Indonesia, but also for the topical
treatment of acne and skin inflammations as Thai traditional medicine. It was found that the methanol extract
of C. xanthorrhiza inhibited significantly 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagene-
sis of Salmonella typhimurium TA98 and TA100 in the presence of S9, and the mutagenesis induced by H2O2
and tert-butylhydroperoxide in S. typhimurium TA102, respectively. In addition, 12-O-tetradecanolyphorbol-
13-acetate(TPA)-induced mouse ear edema was markedly inhibited by pretreatment with C. xanthorrhiza
extract. C. xanthorrhiza extract dose-dependently reduced ODC expression in mouse skin with TPA-induced
acute inflammation. Furthermore, repeated treatment with 0.1% C. xanthorrhiza extract reduced the average
number of tumors per mouse and the percentage of tumor-bearing mice in a multistage mouse skin carcinogenesis
induced by DMBA and TPA. These results demonstrate that the methanol extract of C. xanthorrhiza possesses
cancer chemopreventive potential. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Curcuma xanthorrhiza; antimutagenicity; antiinflammatory activity; multistage skin carcinogenesis; chemoprevention.

1985), antiinflammatory activity in murine models

INTRODUCTION
Cancer chemoprevention is defined as the administra-
tion of agents to prevent the induction of, or to inhibit
or delay the progression of cancer (Sporn and Newton,
1979). A chemopreventive agent may also inhibit or
reverse carcinogenesis at a premalignant stage (Kelloff
et al., 1994). All stages of carcinogenesis, termed initia-
tion, promotion and progression, can be targeted for
chemopreventive intervention (Gupta and Mukhtar,
2002). Chemoprevention is recognized as an important
approach to control malignancy and recent studies have
focused on the search for desirable chemopreventive
agents. Natural products, particularly dietary substances,
have played an important role in creating new chemo-
preventive agents (Surh, 2003).
Curcuma xanthorrhiza Roxb. (Zingiberaceae), com-
monly known as temu lawak or Javanese turmeric, has
been traditionally used in Indonesia and Thailand for
food and medicinal purposes to treat stomach diseases,
liver disorders, constipation, rheumatic diseases, haem-
orrhoids and skin inflammation (Yasni et al., 1994; Lin
et al., 1995). It has been reported that C. xanthorrhiza
and its active compounds confer antitumor activity
against Sarcoma 180 ascites in mice (Itokawa et al.,
* Correspondence to: Dr Won Yoon Chung, Department of Oral
Biology, Yonsei University College of Dentistry, Shinchon-Dong 134,
Seodaemoon-Ku, Seoul 120-752, Korea.
E-mail: wychung@yumc.yonsei.ac.kr
Contract/grant sponsor: Korea Ministry of Health and Welfare; contract/
grant number: 02-PJ1-PG10-20802-0006.
Copyright © 2008 John Wiley & Sons, Ltd.
Copyright © 2008 John Wiley & Sons, Ltd.
treated with ethyl phenylpropiolate, carrageenan or
acetic acid (Claseson et al., 1996; Lee et al., 2002) and
antibacterial activity (Hentschel et al., 1996; Hwang
et al., 2000; Rukayadi et al., 2006).
In the present study, to assess the chemoprotective
potential of the methanol extract of C. xanthorrhiza,
its antimutagenic activity on carcinogen-induced bac-
terial mutagenesis and inhibitory effects on a 12-O-
tetradecanolyphorbol-13-acetate(TPA)-induced acute
inflammation and multistage skin carcinogenesis in mice
were investigated. Furthermore, its effect on TPA-
induced activation of ornithine decarboxylase (ODC),
a biochemical hallmark of tumor promotion (O’Brien
et al., 1975), was also explored.
MATERIALS AND METHODS
Preparation of the plant extract. The dried rhizomes
(100 g) of C. xanthorrhiza purchased at the market of
Yokjakarta in Indonesia (Voucher specimen No.
YS98015) were pulverized and extracted with 75% MeOH
(w/v; 400 mL) three times. The extract was filtered and
concentrated with a rotary evaporator under reduced
pressure. The concentrated extract was then freeze-dried
to remove solvent completely. The dried extract was
placed in a −20 °C deep freezer until analysis.
Animals. Female ICR mice (5–6 weeks of age) were
supplied from the Dae-Han Biolink Experimental
Animal Center (Daejeon, Korea). The animals were
Received
Phytother. 13 November
Res. 22, 2006
695–698 (2008)
Accepted 28DOI:
September 2007
10.1002/ptr
696 J. H. PARK ET AL.
fed with standard pellet chow and water was supplied
ad libitum. All the animals were maintained at 22 ±
2 °C and relative humidity 50–55% under 12 h light and
dark cycle. Animal studies were performed after the
experimental protocols approved by the Institutional
Animal Ethics Committee, Yonsei University College
of Dentistry, Seoul, Korea.
Chemicals. Tert-butylhydroperoxide (t-BOOH), NADP+,
glucose 6-phosphate, hydrogen peroxide (H2O2), 7,12-
dimethylbenz[a]anthracene (DMBA), TPA, dimethylsul-
foxide (DMSO) were purchased from Sigma Chemical
Co. (St Louis, MO). The S9 extract from the livers of
Arochlor 1254-induced male Sprague-Dawley rats was
obtained from ICN Pharmaceuticals, Inc. (Aurora, OH,
USA). All other chemicals and solvents used were com-
mercial products of analytical grade.
Ames/Salmonella mutagenicity assay. The bacterial
mutagenicity assays were conducted with Salmonella
typhimurium TA98, TA100 and TA102 by using the
liquid pre-incubation method developed by Maron and
Ames (1983). Briefly, S. typhimurium TA98, TA100 and
TA102 were grown in Oxoid nutrient broth medium,
respectively. Each 11 h culture (100 μL) of S. typhimurium
TA98 and 100 was added to the mixture (600 μL)
containing DMBA (200 nmol/plate) and the indicated
dose of C. xanthorrhiza extract with S9 mixture. An
11 h culture of S. typhimurium TA102 was added to the
mixture containing H2O2 (200 μg/plate) or t-BOOH
(100 μg/plate) in the presence or absence of a given C.
xanthorrhiza extract. After pre-incubation for 30 min
at 37 °C in a rotary shaker, the mixture was transferred
to 2 mL top agar containing 0.5 mM histidine–biotin
and poured onto minimal glucose plates. The plates
were incubated for 48 h at 37 °C and the number of
His+ revertant colonies was counted.
TPA-induced mouse ear edema. The right ear of
each female ICR mice (6 weeks of age, six per group)
were topically treated with the indicated dose of C.
xanthorrhiza extract in 50 μL vehicle (DMSO–acetone:
50–50, v/v) 30 min prior to the application of 5 nmol
TPA in 50 μL vehicle. Their left ears were treated with
vehicle alone. The control group received vehicle only
on both ears. Four hours later, the mice were killed
by cervical dislocation and the ears were cut off. The
right and left ear punches were taken from each mouse
with a 6 mm cork borer. Edema was measured as the
difference between the weight of the right ear punch
and that of the left. The increases in mass of the ear
punches were directly proportional to the degree of
inflammation.
ODC expression. Female ICR mice (6 weeks of age,
three per group) were topically treated on their shaven
back with the indicated doses of extract in 0.2 mL vehi-
cle (DMSO–acetone: 15–85, v/v) 30 min prior to the
application of 10 nmol TPA in 0.2 mL vehicle. Control
mice were treated with vehicle alone. 4 h later, the mice
were killed and their dorsal skins were excised for
isolation of protein. After the fat was removed on ice,
the remaining skin tissues were immediately pulverized
in liquid nitrogen. The pulverized skins were homogen-
ized with a Polytron tissue homogenizer and lysed in
ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM
Copyright © 2008 John Wiley & Sons, Ltd.
NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM
sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM
Na3VO4, 1 μg/mL leupeptin) containing protease inhibi-
tors for 1 h. The lysates were centrifuged at 14 000 rpm
for 30 min at 4 °C. The supernatants were collected
and the protein concentration was determined by BCA
protein assay. The aliquot of total protein containing
30 μg protein was boiled in sodium dodesylsulfate (SDS)
sample buffer for 5 min and resolved in 12% SDS-
PAGE gel. After electrophoresis, the proteins in gel
were transferred to a polyvinylidene fluoride (PVDF)
membrane (Amersham, Arlington Heights, IL). The
blots were blocked with 5% non-fat dry milk in phos-
phate buffered saline containing 0.1% Tween-20 (PBST)
for 2 h at room temperature and then washed in PBST
buffer. The membranes were incubated with a 1:1000
dilution of primary antibodies for ODC (Santa Cruz
Biotechnology) for 4 h at 4 °C. Blots were washed three
times with PBST at 5 min intervals followed by incuba-
tion with a 1:5000 dilution of horseradish peroxidase-
conjugated secondary antibody for 1 h and again washed
in PBST three times. The transferred proteins were
visualized with an enhanced chemiluminescence detec-
tion kit (Amersham Pharmacia Biotech, Inc.).
Two-stage mouse skin carcinogenesis. The dorsal region
of 6-week-old female ICR mice (30 per group) was
shaved with an electric clipper. To evaluate the anti-
promoting activity of C. xanthorrhiza extract, the mice
were treated topically with a single dose of 0.2 μmol
DMBA dissolved in 0.2 mL acetone. One week later,
the DMBA-initiated mice were treated topically with
7.5 nmol TPA in 0.2 mL vehicle three times weekly
for 19 weeks. 0.1% C. xanthorrhiza extract dissolved
in 0.2 mL vehicle (DMSO–acetone: 15–85, v/v) was
topically applied 30 min before each TPA treatment.
The control mice were pretreated with vehicle alone.
Tumors of more than at least 1 mm in diameter were
counted and recorded biweekly, and the results
were expressed as the average number of tumors
per mouse (tumor multiplicity) and the percentage
of tumor-bearing mice (tumor incidence).
Statistical analysis. Data were expressed as the mean
± standard error (SE) of at least three independent
experiments. The statistical significance of differences
was determined with the Student’s t-test. Values of
p < 0.05 were considered to be statistically significant.
RESULTS AND DISCUSSION
Carcinogenesis is a multistep process, exemplified by
the initiation, promotion and progression (Slaga et al.,
1980; Hursting et al., 1999). Tumor initiation is an
irreversible event that begins when DNA in a cell or
population of cells is damaged by exposure to exogen-
ous or endogenous carcinogens. If this damage is not
repaired, it can lead to genetic mutations, resulting in
an alteration in responsiveness of the mutated cells
to their microenvironment. Tumor promotion is char-
acterized by selective clonal expansion of the initiated
cells through alterations in the expression of critical
genes whose products regulate proliferation, inflamma-
tion and differentiation. Benign papillomas are produced
Phytother. Res. 22, 695–698 (2008)
DOI: 10.1002/ptr
 CANCER CHEMOPROTECTIVE EFFECTS OF CURCUMA XANTHORRHIZA 697

in the tumor promotion stage, which is reversible at

least in early stages and requires repeated and pro-
longed applications of a promoting agent. Progression
is also an irreversible process. During tumor progres-
sion, preneoplasic cells develop into malignant tumors
due to additional genetic alterations and progressive
genomic instability, accompanied ultimately by inva-
sion and metastasis. Based on the underlying mecha-
nism by which they exert protective effects in a specific
stage of multistep carcinogenesis, chemopreventive
agents are classified into two major categories, block-
ing agents and suppressing agents (Wattenberg, 1981).
Blocking agents inhibit tumor initiation by blocking
genetic mutation by chemical carcinogens and/or endo-
genous carcinogens. Suppressing agents are considered
to inhibit malignant expression of initiated cells, in
either the promotion or the progression stage. Most of
them demonstrate antioxidative, antiinflammatory and
antiproliferative activity.
In the present study, it was found that the methanol
extract of C. xanthorrhiza inhibited significantly DMBA- Figure 1. Inhibitory effect of the methanol extract of C.
induced bacterial mutagenesis in S. typhimurium TA98 xanthorrhiza on TPA-induced mouse ear edema (A) and ODC
and TA100 in the presence of S9 (Table 1) and the expression (B). (A) The right ear of each female ICR mouse was
topically treated with the indicated doses of C. xanthorrhiza
mutagenesis induced by H2O2 and t-BOOH in S. extract in 50 μL vehicle (DMSO–acetone: 50–50, v/v) 30 min prior
typhimurium TA102 (Table 2), respectively. As shown to the application of 5 nmol TPA in 50 μL vehicle. Four hours
in Fig. 1A, TPA-induced mouse ear edema was inhibited later, ear punches 6 mm in diameter were taken from each
markedly by treatment with C. xanthorrhiza extract at mouse. Edema was measured as the difference between the
weight of the right ear punch and that of the left. Data are
5.0 mg. expressed as mean ± SE obtained from six mice/group. Experi-
Tumor promotion of skin carcinogenesis induced by ments were repeated twice independently. * Significantly dif-
TPA in mice is closely related to inflammatory response ferent from the TPA alone treated group (p < 0.05). (B) Each
such as development of edema, hyperplasia, induction female ICR mouse skin was topically treated with C. xanthorrhiza
of proinflammatory cytokine interleukin-1α and en- extract in 0.2 mL vehicle (DMSO–acetone: 15– 85, v/v) 30 min
prior to the application of 10 nmol TPA in 0.2 mL vehicle. 4 h
hanced release of reactive oxygen species (ROS), and after the TPA treatment, protein expression was measured by
induction of epidermal ornithine decarboxylase (ODC) western blot analysis as described in Materials and Methods.
The western blot shown is representative of results obtained
from three animals.

Table 1. Antimutagenic activity of C. xanthorrhiza extract on

DMBA-induced mutagenesis in S. typhimurium TA98 and TA100

His+ revertant (% inhibition) (Murakami et al., 2000). ODC, a rate-limiting enzyme

Extract in the synthesis of polyamines that play pivotal roles in
(μg/plate) TA98 TA100 cell growth and proliferation, has been recognized as a
well-known biomarker of tumor promotion. Elevated
0 441 ± 9 814 ± 8
0.5 338 ± 45 (23)a 650 ± 34 (20)a
levels of ODC are consistently detected in transformed
1.0 246 ± 8 (44)a 479 ± 41 (41)a cell lines, virtually in all-animal tumors and in certain
2.5 159 ± 6 (64)b 306 ± 59 (62)b tissues predisposed to tumorigenesis. Transgenic mice
overexpressing ODC in hair follicle keratinocytes were
Each data represent the mean ± SE from triplicate plates. shown to be much more sensitive to DMBA-induced
a
p < 0.05, b p < 0.001, Significantly different from the plates carcinogenesis than littermate controls, and did not
treated with DMBA alone. require treatment with a tumor promoter to develop
tumors. These results suggest that ODC overexpression
is a sufficient condition for tumor promotion in this
Table 2. Antimutagenic activity of C. xanthorrhiza extract on model (O’Brien et al., 1997). Therefore, suppressing
oxidant-induced mutagenesis in S. typhimurium TA102 ODC activation during the carcinogenic progression has
been recognized as a commonly acceptable approach
H2O2 t-BOOH
for the effective inhibition of tumor promotion.
+
Extract His revertant Extract His+ revertant The present study showed that the methanol extract
(μg/plate) (% inhibition) (μg/plate) (% inhibition) of C. xanthorrhiza dose-dependently reduced ODC
expression in TPA-treated mouse skin. Treatment with
0 423 ± 8 0 897 ± 16 C. xanthorrhiza extract alone did not affect ODC
0.5 369 ± 16 (13)a 1.0 731 ± 38 (19)a
expression (Fig. 2B). Furthermore, to examine the
1.0 254 ± 20 (40)a 10 372 ± 43 (59)b
2.5 88 ± 13 (80)b 50 31 ± 19 (97)b
antitumor-promoting activity, the inhibitory effect of
C. xanthorrhiza extract on TPA-induced tumor forma-
Each data represent the mean ± SE from triplicate plates. tion in DMBA-initiated mouse skin was investigated.
a
p < 0.05, b p < 0.001, Significantly different from the plates All the DMBA-initiated mice treated with 7.5 nmol
treated with H2O2 or t-BOOH alone, respectively. TPA for 19 weeks after initiation by DMBA developed
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 695–698 (2008)
DOI: 10.1002/ptr
698 J. H. PARK ET AL.

Figure 2. Inhibitory effect of C. xanthorrhiza extract on mouse skin tumor promotion. DMBA-initiated female ICR mice received
vehicle (DMSO–acetone: 15–85, v/v) or C. xanthorrhiza extract 30 min prior to each topical application of 7.5 nmol TPA three times
weekly for 19 weeks, as described in Materials and Methods. Control animals received vehicle alone and did not produce papillomas.
Tumors of at least 1 mm in diameter were counted and recorded biweekly, and the results were expressed as the average number
of tumors per mouse (tumor multiplicity) and the percentage of tumor-bearing mice (tumor incidence).

an average of 15.5 ± 2.3 skin tumors per mouse (tumor
multiplicity). The repeated pretreatment with 0.1%
C. xanthorrhiza extract reduced tumor multiplicity
to 10.9 ± 2.1 (p < 0.05). In addition, topical application
of C. xanthorrhiza extract lowered the percentage of
tumor-bearing mice (tumor incidence) to 87% at the
termination of the experiments, as shown in Fig. 2.
In conclusion, the methanol extract of C. xanthorrhiza
may inhibit tumor formation by blocking carcinogen-
induced mutagenesis, TPA-induced inflammation and
TPA-induced expression of ODC protein. These
results demonstrate that the methanol extract of C.
xanthorrhiza possess cancer chemopreventive potential.
Acknowledgement
This study was supported by a grant of the Korea Health 21 R&D
Project, Ministry of Health & Welfare (02-PJ1-PG10-20802-0006),
and by the Korea Research Foundation Grant, funded by the Gov-
ernment of the Republic of Korea (KRF-2055-005-J05902).

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Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 695–698 (2008)
DOI: 10.1002/ptr