The American Journal of Chinese Medicine, Vol. 32, No.

2, 281290
2004 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine

Antimicrobial Activity and Cytotoxicity of

the Essential Oil of Curcuma zedoaria

Eric Y.C. Lai,* Charng-Cherng Chyau, Jeng-Leun Mau, Chien-Chou Chen,

Yi-Jui Lai, Ching-Fang Shih, Long-Liu Lin

*Departmentsof Nursing and Food Science and Nutrition

Hungkuang University, Taichung 433-2, Taiwan
Department of Food Science, National Chung-Hsing University

Taichung 402-27, Taiwan

Department of Applied Chemistry, National Chiayi University

Chiayi 60083, Taiwan

Abstract: The chemical compositions of the essential oil of Curcuma zedoaria (Berg.) Rosc.
were analyzed by gas chromatography-mass spectrometry (GC-MS) and showed a high
content of epicurzerenone and curdione representing 46.6% and 13.7% of the total oil,
respectively. The essential oil was evaluated for potential antimicrobial activity against
Staphylococcus aureus, Escherichia coli, Pseudomonasa aeruginosa, Vibrio parahaemolyticus,
Salmonella typhimurium and Bacillus cereus. V. parahaemolyticus was sensitive to the
presence of the essential oil, while the most resistant strain appeared to be E. coli. Based on 3-
(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay, nitroblue
tetrazolium (NBT) reduction and cell morphology, the essential oil of C. zedoaria could inhibit
the proliferation of human promyelocytic leukemia HL-60 cells. These results suggest that the
essential oil has the antimicrobial activity against some of Gram- positive and negative
pathogenic microorganisms and the components of the extract lead to the apoptosis of human
cancer cell line.

Keywords: Curcuma zedoaria; Essential Oil; Antimicrobial Activity; Vibrio parahaemolyticus;

Cytotoxicity.

Introduction

Antimicrobial activity has been reported for the essential oils from several sources, such as
Origanum scabrum and Origanum microphyllum (Aligiannis et al., 2001), Micromeria
cristata subsp. Phrygia (Tabanca et al., 2001), and Calycotome villosa (Loy et al., 2001).

Correspondence to: Dr. Long-Liu Lin, Department of Applied Chemistry, National Chiayi University, 300 University
Road, Chiayi 60083, Taiwan. Fax: (+886) 5-271-7901, E-mail: llin@mail.ncyu.edu.tw

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The use of antimicrobial compounds from natural vegetation could have a great impact in
preserving food storage from contamination, and in controlling plant and human diseases of
microbial origin (Balandrin et al., 1985; Conner, 1993). Besides, the essential oils derived
from many plants are known to possess other biological effects, such as antifungal, insecticidal
and cytotoxic activities (Thompson, 1989; Konstantopoulou et al., 1992; Apisariyakul
et al., 1995; Gonzalez et al., 1997; Rahman et al., 2000; Islam et al., 2001).
Curcuma zedoaria (Berg.) Rosc. (Zingiberaceae), the so-called Er-Jyur in Chinese, has
long been used as a folk medicine. Traditionally, the dried rhizome of C. zedoaria is selected
to process drinks or to be extracted as medicine. It has been reported that the boiling water
extracts of C. zedoaria had a moderate antimutagenic activity against benzo[a]pyrene (Lee
and Lin, 1988). Many sesquiterpenes have been isolated from the aqueous acetone extracts
of C. zedoaria rhizome and the major compounds, such as furanodiene, germacrone and
curcumin, were found to have potent protective effect on D-galactosamine/lipopolysaccharide-
induced liver injury in mice (Matsuda et al., 1998). A crude ethanolic extract of C. zedoaria
has also shown the inhibitory activity against OVCAR-3 cells and the three identified
curcuminoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, are responsible
for this activity (Syu et al., 1998). In this investigation, we demonstrate the antimicrobial
and cytotoxic activities of the essential oil of C. zedoaria against bacteria and HL-60 cell
line, respectively.

Materials and Methods

Chemicals, Plant Material, Bacterial Strains and Cell Culture

Nutrient broth and Bacto agar for bacterial culture were purchased from Difco Laboratories
(Detroit, MI, USA). Dulbeccos Modified Eagles Medium (DMEM) and trypsin was acquired
from Invitrogen Corporation (San Diego, CA, USA). Penicillin-streptomycin solution,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), nitroblue tetrazolium
(NBT) and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical
Co. (Saint Louis, MO, USA). All other chemicals were commercial products of analytical
grade or molecular biological grade.
The dried rhizome of C. zedoaria imported from Sichuan Providence, the Peoples
Republic of China, was purchased from a Chinese medicine store in Changhwa, Taiwan.
The sample was ground (particle size, 20 mesh) in a comminuted mill (Ultra Centrifugal
Mill Type ZM1, Retsch GmbH & Co., Haan, Germany). The ground sample was then stored
in a dark glass at 20C until use.
The following strains of bacteria purchased from Culture Collection and Research Center
(CCRC), Hsin-Chu, Taiwan, were used as test organisms for antimicrobial assay:
Staphylococcus aureus (ATCC 6538P), Escherichia coli (ATCC 23815), Pseudomonas
aeruginosa (ATCC 31156), Vibrio parahaemolyticus (ATCC 17802), Salmonella
typhimurium (ATCC 14028) and Bacillus cereus (ATCC 11778). Bacteria were grown either
in nutrient broth or in nutrient agar and incubated at 37C for S. aureus, E. coli, P. aeruginosa,
V. parahaemolyticus and S. typhimurium, or at 30C for B. cereus.

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 THE ESSENTIAL OIL OF CURCUMA ZEDOARIA 283

The human promyelocytic leukemia HL-60 cell line was also obtained from CCRC and
grown in DMEM supplemented with 5% fetal calf serum, 2 mM glutamine, 100 units
penicillin/ml and 100 g streptomycin/ml.

Isolation of Essential Oil

Simultaneous steam distillation and solvent extraction was conducted from a modified Likens-
Nickerson apparatus (extraction time, 2 hours) on 100 g of ground sample. Fifty milliliters
of a solvent mixture of n-pentane and diethyl ether (1:1, v/v) was applied in the extraction.
The resulting extract was dehydrated over Na2SO4 anhydrous and filtered with Whatman
no. 1 filter paper. After the solvent was distilled off by Vigreux column (i.d. 1.5 100 cm),
the extracted oil was weighed and stored at 20C until the chemical analyses. The
essential oil was filtered through a nylon filter (pore size, 0.22 mm) before antimicrobial and
cytotoxic assays.

Chemical Characterization

Chemical analyses were performed by GC-MSD (Hewlett-packard 6890 GC and 5973A

MSD, EI, 70 eV) with a CP-Wax 52CB column (60 m 0.25 mm i.d. fused silica capillary
column, film thickness = 0.25 m; Chrompack, Middelburg, The Netherlands) and He as
the carrier gas (flow rate, 1 ml/min). GC temperature program was set at 40C as the initial
temperature and at 220C as the final temperature with an increased speed of 3C/minute.
GC injector temperature and GC-MSD interface temperature were 250 and 265C,
respectively. Identification of components was performed on the basis of linear retention
indices, mass spectra from HP Wiley MS Chemstation libraries (6th edition, G1034, Rev.
C.00.00, Palo Alto, CA, USA) and literature (Jennings and Shibamoto, 1980). The relative
amount of individual components of the oil was expressed as percent peak area relative to
total peak area.

Antimicrobial Assay

The selected microorganisms were incubated for 24 hours under favorable conditions for
proliferation. The cultivated cultures were diluted with 1% peptone water to give a final
concentration in the test peri dish of ~103 colony forming unit (CFU)/plate. The essential
oil of C. zedoaria was added to the sterilized nutrient broth to reach final concentrations of
100, 200, 300, 400 and 500 ppm, respectively. The same medium without the addition
of the essential oil was used as a control. The poured plates were then incubated at
37C or 30C and counted after 24 hours for the bacterial number. The inhibition is defined
as follow: (original CFU the essential oil-treated CFU)/original CFU 100%.

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Cytotoxic Assays

The MTT assay is a well-known quantitative colorimetric method to assess the cytotoxicity
of a chemical on cultured cells (Denizot and Lang, 1986). For the assay, HL-60 cells were
trypsinized in a solution of 0.25% trypsin and seeded into 96-well microplates at a density
of 3.0 104 cells/0.1 ml/well. After the cells were grown for 24 hours to a subconfluent
state, 100 l of the culture medium containing various concentrations of the essential oil was
added to each well to give a final concentration of 250, 500, 1000, 2500 and 5000 ppm,
respectively, and the cultivation was continued for 48 hours in a CO2 incubator. After
incubation, 50 l of DMEM containing 2 mg MTT/ml was added to each well, and the
microplates were cultivated for an additional 4 hours. The cells were harvested by
centrifugation (1200 g, 10 minutes), and dissolved in dimethyl sulfoxide (DMSO). The
formation of formazan was assessed using an ELISA reader (Hyperion Inc., Miami, FL,
USA) exactly as described by the manufacturer. MTT reduction (%) is defined as follows:
(A540nm-experiment A650nm-experiment)/(A540nm-control A650nm-control) 100%.
NBT reducing activity was determined by the method of Matsuhisa and Mori (1995)
with a slight modification. Briefly, 500 l of the treated cells were harvested by centrifugation,
washed once with 500 l of phosphate-buffered saline (PBS), and resuspended in 200 l of
a solution containing 2 mg NBT/ml and 100 g PMA/ml. The resulting materials were
incubated at 37C for 30 minutes in a CO2 incubator. Afterwards, 10 l of the sample was
withdrawn and placed on a hemocytometer for cell counting. NBT reduction (%) is defined
as follows: formazon-forming cells/total cells 100%.
The cells treated with the essential oil were fixed and stained with Lius stain according
to the procedure of Chang et al. (1993) and examined at high magnification (400). Apoptoxic
cells were identified on the basis of cell-membrane damage and the loss of granules.

Statistical Analysis

Results are expressed as mean SE. Data were analyzed by ANOVA for repeated measures
with p < 0.05 considered statistically significant.

Results

Chemical Composition of the Essential Oil

The identification of the essential oil of C. zedoaria by GC-MS is presented in Fig. 1. Totally,
13 compounds were identified in this study. The major components are epicurzerenone
(46.64%), curdione (13.66%) and 5-isopropylidene-3,8-dimethyl-1(5H)-azulenone (9.15%).
Some other minor components include 1,8-cineole (1.36%), camphor (1.46%), -elemene
(1.90%), -terpineol (1.45%), -curcumene (0.70%), curcumol (1.89%) and isocucumenol
(1.84%). C. zedoaria is known to contain high amounts of sesquiterpenes and monoterpenes
(Tang and Eisenbrand, 1992); however, less than 3% of the terpenes was identified in the
essential oil. Together with a previous report (Ma et al., 1995), it is worth to mention that the
major ketones represent approximately 71% of the essential oil.

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 THE ESSENTIAL OIL OF CURCUMA ZEDOARIA 285

Figure 1. Total ion chromatogram of the essential oil of C. zedoaria. Peaks denote: 1, -pinene; 2, -penene;
3, 1,8-cineole; 4, camphor; 5, -elemene; 6, -terpineol; 7, -curcumene; 8, -elemenone; 9, epicurcumenone;
10, curdione; 11, curcumol; 12, isocurcumenol; 13 to 15, unknown compounds and 16, 5-isopropylidene-3,8-
dimethyl-1(5H)-azulenone.

Antimicrobial Activity

Six different bacteria strains were used to study the antimicrobial activity of the essential oil.
The selected microorganisms include two Gram-positive (S. aureus and B. cereus) and three
Gram-negative (E. coli, P. aeruginosa, V. parahaemolyticus and S. typhimurium) bacteria. As
shown in Table 1, V. parahaemolyticus was the most sensitive pathogen to the essential oil
with a percentage inhibition of 99.9 9.2 at dosage of 500 ppm, followed by S. aureus,
B. cereus, S. typhimurium and P. aeruginosa (p < 0.05). However, only 50.6 3.6% inhibition
for E. coli was observed at the dosage of 500 ppm (Table 1). Consistently, E. coli was reported
as the most resistant characteristic in the evaluation of antimicrobial activity of Garcinia
atroviridis Grif. Ex T. Anders extracts against four bacteria strains (Mackeen et al., 2000).

Table 1. Antimicrobial Activity of the Essential Oil of C. zedoria

Microorganism Inhibition (%)*
100 ppm 200 ppm 300 ppm 400 ppm 500 ppm
S. aureus 38.5 2.3 63.1 5.6 82.7 7.1 91.4 6.9 98.5 7.3
E. coli 9.1 1.6 38.5 6.1 35.5 2.3 57.4 5.3 50.6 3.6
P. aeruginosa 5.6 1.9 19.7 3.7 53.9 3.8 58.1 4.6 69.5 5.9
V. parahaemolyticus 81.5 8.2 98.5 8.1 99.2 7.4 99.8 5.6 99.9 9.2
S. typhimurium 31.2 5.9 43.1 5.8 54.6 4.5 67.2 5.8 74.3 5.2
B. cereus 19.3 3.4 58.9 6.8 67.8 6.9 92.3 7.9 93.8 6.7
*Data presented were the means of three independent tests (p < 0.05).

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Cytotoxicity

MTT assay quantifies mitochondrial activity by measuring the formation of a dark blue
formazan product formed by the reduction of the tetrazolium ring of MTT. The reduction of
MTT is thought to mainly occur in the mitochondria through the action of succinate
dehydrogenase, therefore providing a measure of mitochondrial function (Slater et al., 1963).
As shown in Fig. 2, a significant decrease in MTT reducing activity was observed in human
promyelocytic leukemia HL-60 cell line treated with 500 ppm of the essential oil, and less
than 20% of the activity was retained when the concentration increased to 1000 ppm,
indicating that the essential oil of C. zedoria is toxic to the HL-60 cells.

Figure 2. Effects of the essential oil on MTT (A) and NBT (B) reducing activities of HL-60 cells. Cells were
seeded in a microplate and treated with the essential oil as described in the Materials and Methods section. Data
presented were the means of three independent experiments. The control activity is normalized to 100%. The
differences between the control and the essential oil-treated sample were significant (p < 0.05).

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Differentiation effects of the essential oil were also evaluated in HL-60 cells by analysis
of its NBT reducing activity, which is a typical marker for the differentiation of myeloid
leukemia cells (Theodore, 1990). As shown in Fig. 2, the essential oil had a differentiation-
repressing effect to the tested cell line. Treatment with the essential oil at a dosage of 250
ppm caused a complete loss of NBT reducing activity in the cell line and more than 90% of
the cells showing morphologic mal-maturation with the disappearance of cytoplasmic granules
(Fig. 3B). In the presence of a higher dosage of the essential oil, the breakdown of cell
membrane and the release of cytoplasmic components were observed (Figs. 3C and D).
These results further suggested that the essential oil of C. zedoria inhibits efficiently the
monocytic differentiation of human promyelocytic leukemia HL-60 cell line.

Figure 3. Morphological changes of human promyelocytic leukemia HL-60 cell line treated with the essential oil.
A, untreated cells; B, cells treated with the essential oil at a dosage of 250 ppm; C, cells treated with the essential
oil at a dosage of 500 ppm; and D, cells treated with the essential oil at a dosage of 1000 ppm.

Discussion

The phytochemical studies of C. zedoria essential oil revealed the presence of phenolic
compounds. Essential oils rich in phenolic compounds are reported to possess high levels of
antimicrobial activity (Sivropoulou et al., 1996; Panizi et al., 1993), and the inhibition is
related to the concentration of phenols (Cruz et al., 2001). Since the phenolic components
represent less than 1% of the C. zedoria essential oil, it is possible that other compounds
should have an inhibitory effect against the strains tested. Recently, camphor, identified as
one of the major constituents of Cistus salviifolius essential oil, has been shown to be active
against Gram-positive bacteria (Demetzos et al., 2002). Likely, this compound may have a
similar contribution to the antimicrobial activity of the essential oil of C. zedoria.

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It has been reported that curcumin from C. zedoria plays an important role in the inhibitory
effect to OVCAR-3 cells (Syu et al., 1998). Several works demonstrated that this phenolic
compound possesses antioxidant, free radical scavenger, anti-inflammatory and anti-
carcinogenic activities (Srimal and Dhawan, 1973; Sharm, 1976; Ruby et al., 1995). Since
curcumin passes easily through the plasma membranes into the cytosol (Oetari et al., 1996),
the presence of this compound will cause phosphatidylserine exposure, and increase plasma
permeability (Jaruga et al., 1998). Although curcumin is one of the main components of the
ethanolic extract of C. zedoaria (Syu et al., 1998), this compound is non-volatile and could
not be found in the essential oil of C. zedoaria. Therefore, it remains to be investigated
whether other terpenes in the essential oil of C. zedoaria may confer cytotoxic activity
against leukemia cell line as has been demonstrated in Gorgonian Isis hippuris (Sheu et al.,
2000) and Atractylodes ovata (Wang et al., 2002).
The previous studies showed that the main compounds with biological activities in the
extracts of C. zedoaria are sesquiterpenes (Matsuda et al. 1998; Syu et al., 1998). In our
study, however, terpenes represented less than 3% of the essential oil. Although terpenes
in the essential oil might play an important role for antimicrobial, antioxidant and cytotoxic
activities, other components, especially epicurzerenone, curdione and 5-isopropylidene-3,8-
dimethyl-1(5H)-azulenone, could also be involved in the action. Studies are presently
underway to measure the major constituents alone or in combination with ionizing radiation
on tumor growing in animals. The resulting information will contribute to our better
understanding of anti-carcinogenic activity of the natural constituents from C. zedoaria.

Acknowledgments

This research work was supported by a grant (HKHSC 89-04) from the Scientific Council of
Hungkuang University, Taiwan.

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