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

*
Departments of 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

281
282 E.Y.C. LAI et al.

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.
 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 Na 2SO4 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
3
concentration in the test peri dish of ~10 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%.
284 E.Y.C. LAI et al.

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
4
3.0 10 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 CO 2 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:
(A A )/(A A ) 100%.
540nm-experiment 650nm-experiment 540nm-control
650nm-control
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 CO 2 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.
 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).
286 E.Y.C. LAI et al.

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).
 THE ESSENTIAL OIL OF CURCUMA ZEDOARIA 287

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.
288 E.Y.C. LAI et al.

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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