Anticancer and Antimicrobial Activities of Zerumbone from the Rhizomes of Zingiber zerumbut
Ahmad B. Abdul,
Siddig I. Abdelwahab,
Adel S. Al-Zubairi,
Manal M. Elhassan
Syam M. Murali
The aim of this study is to evaluate the anticancer
and antimicrobial activities of zerumbone (ZER) from the rhizomes of Zingiber
zerumbut. ZER is a crystalline sesquiterpene from the wild ginger,
Z. zerumbet. This bioactive component has its unique structure,
with a cross-conjugated ketone in an 11-membered ring, as well as remarkable
biological activity. Thus, this compound has been isolated from the fresh
rhizomes of Z. zerumbet using steam distillation and evaluated
for its antimicrobial and anticancer activities. The antimicrobial effects
were examined using disc diffusion method and group of microorganism,
namely known as Methicilin resistant Staphylococcus aureus,
Pseudomonas aeruginosa, Salmonella choleraesuis, Bacillus
subtilis, Candida albicans, Aspergillus ochraceaus
and Sacchoromyces cerevisiae. However, MTT assay was performed
to determine the anti-cancer properties of zerumbone on human cervical
cancer cells (HeLa) compared to cisplatin as positive control. Zerumbone
has shown a dose dependent (p<0.05) anti-bacterial effect on S.
choleraesuis, while no antifungal activity were observed. Zerumbone
was also able to exert an antiproliferative effect towards cervical cancer
cell line (HeLa) in time-dependent manner (p<0.05) (24, 48 and 72 h).
It could be concluded that, zerumbone with its unique chemical structure
and versatile pharmacological activities might be a potential primer to
develop new curative agents for possible various ailments.
Zingiber zerumbet (L.) Sm., known as lempoyang among the
Malays, is a member of the family Zingiberaceae and used in the traditional
medicine as a cure for swelling, sores, loss of appetite, worm infestation
in children (Somchit and Nur-Shakirah, 2003). This plant has shown anti-tumor
(Sakinah et al., 2007), anti-inflammatory (Murakami et al.,
2002) and suppressant of cyclooxygenase-2 properties (Tanaka et al.,
2001). Zerumbone is a crystalline monocyclic sesquiterpene derived from this
plant. This bioactive component has its unique structure, with cross-conjugated
ketone in an 11-membered ring, as well as remarkable biological activities.
It has been reported that zerumbone constitute about 37% of Z. zerumbet
(Matthes et al., 1980; Sakinah et al., 2007). Moreover,
this compound showed a potential candidate for the development of anticancer
treatment. Therefore, screening of this compound the biologically is needed.
The objective of this research is to investigate antimicrobial and cytotoxic
properties of zerumbone.
MATERIALS AND METHODS
Zerumbone and sample preparation: ZER was isolated using steam
distillation method (Kitayama et al., 1999). Briefly, the fresh
plant of Z. zerumbet was sliced and placed in flask and heated
using Mentel heater. This flask was connected with a special glass ware
(Dienstag), to collect the volatile essential oil of the boiled plant
material. The collected volatile oil was crystallized spontaneously using
circulating cool water during the extraction procedure. To obtain a pure
material of zerumbone, recrystallization was performed using hexane for
three times followed with thin layer chromatography to check the purity.
Then the crystals of zerumbone were obtained and kept at -4°C for
further biological activities.
Cell culture and MTT cytotoxicity assay: Human Cervical cancer
cell line (HeLa) was obtained from American Type Culture Collection (ATCC),
Maryland, USA. ATCC protocol recommended that RPMI 1640 can used for HeLa
cell cells which was purchased from Culture Labs (Australia). The disposable
items (Tissue culture flask, Filter system, 96-well plates) were purchased
from NUNCTM, Denmark. Trypsin, EDTA, Feotal Calf Serum, Amphotericin
B and Penicillin Streptomycin were ordered from FlowLab (Australia). The
MTT (Microtetrazolium) powder was purchased from Amresco and the DMSO
(Dimethylsulphoxide) was purchased from Sigma Aldrich. MTT assay reading
was performed (n = 3) using ELISA plate reader (Universal Micro plate
Antimicrobial assay: Minimum inhibitory concentration method was
utilized to test the antimicrobial properties of zerumbone against fungi
and bacteria compared to standard antimicrobial agents using disc diffusion
method. The diameter of the clear zone was measured in millimeter, A group
of microorganism was utilized, namely known as MRSA = Methicilin resistant
Staphylococcus arureus; PA = Pseudomonas aeruginos,
SC = Salmonella choleraesuis, BS = Bacillus subtilis, CA
= Candida albicans, AO = Aspergillus ochraceaus and
SCE = Sacchoromyces cerevisiae. The bacterial and fungal
stock cultures were maintained on nutrient agar (Oxoid, UK) and potato
dextrose agar (Oxoid, UK) slants, respectively. For the purpose of antimicrobial
assay, the microorganisms were cultured in nutrient broth for bacteria
and potato dextrose broth for fungi at 30°C overnight. The concentrations
of the cultures were adjusted turbidometrically at wavelength of 600 nm
to 500,000-1000,000 colony forming units (CFU) per mL.
Statistical analysis: Data was presented as Mean±SD as
a measure of descriptive statistics. Dependency of antimicrobial treatment
of zerumbone on dose was tested statistically using Pearson Correlation
Coefficient. A p-value of <0.05 was considered statistically significant.
All statistical analysis was performed SPSS statistical package software
version 16.0 (USA).
Cytotoxicity assay: As shown in Fig. 1, ZER was
able to exert the antiproliferative effects towards cervical cancer cell
line, HeLa, tested in time-dependent manner (p<0.05) (24, 48 and 72
h). The IC50 value which is the concentration required for
50% growth inhibition of zerumbone towards HeLa cell viability is 20.30±1.2
μM mL-1. Comparatively, cisplatin, a commercial drug with
anti-neoplastic activity was used as a positive control in this study.
Cisplatin revealed an inhibitory effect on HeLa cells with an IC50 value
of 5.45±0.44 μM mL-1.
Antimicrobial activity of zerumbone: Zerumbone was tested for
its ability to inhibit the growth of certain bacteria and fungi using
disc diffusion method. Table 1 showed the results of
the antimicrobial experiment. Whereby, the compound did not show any antifungal
properties in C. albicans, A. ochraceaus and S. cerevisiae
compared to the standard drug, Nystatin. However, zerumbone has shown
a dose dependent (p<0.05) anti-bacterial effect on S. choleraesuis.
In the anti-bacterial screening test, streptomycin was functioned as standard
drug (positive control).
||Effects of zerumbone on cell viability of HeLa cell
lines. Treatment of zerumbone on HeLa cell lines significantly decreases
the number of viable cells with IC50 values of 20.30±1.2
μM mL-1 (A 24 h, B 48 h and C 72 h). Y-axis is the
percentage of inhibition; X-axis is the concentration of zerumbone
||Antimicrobial activity of zerumbone on selected bacteria
and fungi based on disc diffusion method (n = 3)
Natural products as alternative form of health care and the development
of microbial resistance to available antibiotics have led researchers
to investigate the antimicrobial activity of medicinal plants (Marjorie,
1999). These medicinal plants have also studied for their anticancer properties
(Gordaliza, 2007). One of these potential biologically active plants is
Z. zerumbet which shown a versatile pharmacological properties
such as anti-atherosclerosis (Eguchi et al., 2007), anti-inflammatory,
insulin-like grow factor-1 and induced Waf-1 gene expression, glutathione
S-transferase activity and heat shock protein. Zerumbone was also found
to exert induction of differentiation and cytoprotective activity (Rodriguez
et al., 1997). Thus, the present study was proposed to investigate
the antimicrobial activity of zerumbone using gram-positive and negative
bacteria and fungi. In addition, the anticancer properties of this compound
were also evaluated using human cervical cancer cell line (HeLa). The
IC50 value which is the concentration required for 50% growth
inhibition of zerumbone towards HeLa cell viability is 20.30± 1.2
μM mL-1. This value could be accepted among the recommended
clinical range of new anti-cancer drugs. Comparatively, cisplatin, a commercial
drug with anti-neoplastic activity was used as a positive control in this
study. Cisplatin is used widely in the treatment of ovarian, bladder,
cervical and testicular cancer (Teni and Maria, 2004; Janson et al.,
2008). The results of this study are supported with the previous data
showed that zerumbone has antiproliferative effects towards human hepatic,
leukemic (Xian et al., 2007) and colonic adenocarcinoma
cells (Sakinah et al., 2007). Zerumbone is bioactive crystalline
sesquiterpene and has unique structure, with a cross-conjugated ketone
in an 11-membered ring (Kitayama et al., 2003). This unique structure
could be used as a primer to develop new antimicrobial agents that have
the ability to overcome the current microbial resistance. Obtained results
of this research paper revealed that zerumbone showed no anti-fungal effects
against C. albicans, A. ochraceaus and S. cerevisiae.
However; this compound has shown a dose dependent anti-bacterial effect
on S. choleraesuis but not on methicillin resistant S.
aureus, P. aeruginosa, S. choleraesuis and B. subtilis.
Moreover, it has been reported that this compound did not show any antibacterial
activity against B. subtilis but its derivative has shown antibacterial
agents that inhibit histidine protein kinase YycG of B. subtilis
(Yamamoto et al., 2001; Kitayama et al., 2007). Moreover,
it further suggest the potentiation of ZER as a preface to identify
new synthesized antimicrobial agents.
In conclusion, zerumbone has shown remarkable anti-proliferative properties
against human cervical cancer cells and antibacterial effects against
S. choleraesui. Moreover, these versatile biological activities
might be a corner stone to obtain new anticancer and antimicrobial agents
The authors wish to express sincere appreciation to the University of
Putra Malaysia for the financial support of this investigation and to
the Laboratory of Natural Products, Institute of Bioscience, UPM for providing
technical guidelines for phytochemical analysis [RUGS 91143].
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