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In Vitro Activity of Xanthorrhizol Isolated from

the Rhizome of Javanese Turmeric ( Curcuma
xanthorrhiza Roxb.) Against Candida albicans
Biofilms

Article in Phytotherapy Research July 2013

DOI: 10.1002/ptr.4834 Source: PubMed

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 PHYTOTHERAPY RESEARCH
Phytother. Res. 27: 10611066 (2013)
Published online 12 September 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.4834

In Vitro Activity of Xanthorrhizol Isolated from

the Rhizome of Javanese Turmeric (Curcuma
xanthorrhiza Roxb.) Against Candida albicans
Biolms

Yaya Rukayadi1* and Jae-Kwan Hwang2

1
Department of Food Science, Faculty of Food Science and Technology, and Laboratory of Natural Products (LHS), Institute of
Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia
2
Department of Biotechnology, Yonsei University, Seoul 120-749, Korea

The purpose of this study was to investigate the activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. on
Candida albicans biolms at adherent, intermediate, and mature phase of growth. C. albicans biolms were formed in
at-bottom 96-well microtiter plates. The biolms of C. albicans at different phases of development were exposed to
xanthorrhizol at different concentrations (0.5 mg/mL - 256 mg/mL) for 24 h. The metabolic activity of cells within the
biolms was quantied using the XTT reduction assay. Sessile minimum inhibitory concentrations (SMICs) were
determined at 50% and 80% reduction in the biolm OD490 compared to the control wells. The SMIC50 and SMIC80
of xanthorrhizol against 18 C. albicans biolms were 4 16 mg/mL and 8 32 mg/mL, respectively. The results
demonstrated that the activity of xanthorrhizol in reducing C. albicans biolms OD490 was dependent on the
concentration and the phase of growth of biolm. Xanthorrhizol at concentration of 8 mg/mL completely reduced in
biolm referring to XTT-colorimetric readings at adherent phase, whereas 32 mg/mL of xanthorrhizol reduced
87.95% and 67.48 % of biolm referring to XTT-colorimetric readings at intermediate and mature phases, respectively.
Xanthorrhizol displayed potent activity against C. albicans biolms in vitro and therefore might have potential
therapeutic implication for biolm-associated candidal infections. Copyright 2012 John Wiley & Sons, Ltd.

Keywords: amphotericin B; biolm; Candida albicans; in vitro; xanthorrhizol.

metabolites may have antifungal activity, and the active

INTRODUCTION
Candida species are emerging as major agents of nosoco-
mial infections. Indeed, the majority of manifestations
of candidiasis at both mucosal and systemic sites are asso-
ciated in one way or another with biolms on inert or
biological surfaces (Douglas, 2003; Ramage et al., 2006).
Moreover, manifestations of infections associated with the
formation of Candida biolms, most notably C. albicans,
include those occurring on devices such as indwelling intra-
vascular catheters (Jabra-Rizk et al., 2004; Chandra et al.,
2001). Indwelling intravascular catheters represent a risk
factor that is associated with nosocomial C. albicans
infections (Crump and Collignon, 2000). Furthermore, for-
mation of C. albicans biolms has important clinical reper-
cussions because of their increased resistance to antifungal
therapy and the ability of cells within biolms to withstand
host immune defense (Douglas, 2003; Ramage et al., 2006).
The development of resistance in known fungal
pathogens and the emergence of new fungal pathogens
intrinsically resistant to currently available antibiotics
demonstrate the urgent importance of identifying novel
antifungal agents (Ficker et al., 2003). Plant secondary
* Correspondence to: Yaya Rukayadi, Department of Food Science, Faculty
of Food Science and Technology, and Laboratory of Natural Products (LHS),
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor Darul Ehsan, Malaysia.
E-mail: yrukayadi@food.upm.edu.my
Copyright 2012 John Wiley & Sons, Ltd.
molecules could be models for synthetic antifungal
agents (Rocha et al., 2004). According to Beuscher et al.,
traditional medicinal plants can provide a reservoir of
pharmacologically active substances or drugs for fungal
treatment (1994). The antifungal effect of essentials oils
of many plants and their specic anticandidal activity is
also well established (Arras and Usai, 2001; Chami et al.,
2004). Xanthorrhizol (Fig. 1), a natural sesquiterpenoid
isolated from the rhizome of Java turmeric (Curcuma
xanthorrhiza Roxb.), is widely used in medical and
dental practice due to its potent bactericidal, antioxidant,
anti-inammatory, anticancer, and antifungal activities
(Hwang et al., 2000a,2000b; Kim et al., 2004; Lim et al.,
2005). We have previously reported the antifungal activity
of xanthorrhizol against planktonic fungal cells such as
Candida species, Malassezia species, and opportunistic
lamentous fungi (Rukayadi et al., 2006; Rukayadi and
Hwang, 2007a,2007b). Xanthorrhizol also has ability to
prevent and remove Streptococcus mutans biolms
(Rukayadi and Hwang, 2006a,2006b). Moreover,
xanthorrhizol exhibits potent activity against non-Candida
albicans Candida (C. glabrata, C. guilliermondii, and
C. parapsilosis) biolms in vitro (Rukayadi et al., 2011).
However, to our knowledge, the activity of xanthorrhizol
against sessile cells in C. albicans biolms has never been
demonstrated. Our main objective in this study was to
evaluate the activity of xanthorrhizol against C. albicans
biolms so that potential therapeutic implications for
biolm-associated candidal infections could be dened.
Received 13 March 2012
Revised 09 July 2012
Accepted 03 August 2012
1062 Y. RUKAYADI AND J.-K. HWANG
14
4 at 37 C (2008). After incubation time, the medium was
5 thoroughly washing the biolm three times in sterile PBS.
3 The microtiter plates were drained in an inverted position
6 2
12
1
7 9 11
15 8 10 13
Figure 1. Structure of xanthorrhizol.
MATERIALS AND METHODS
Candida albicans isolates. Two reference Candida
albicans used in this study were obtained from the
American Type Culture Collection (ATCC 10231;
Rockville, MD, USA) and the Korean Culture Center of
Microorganisms (KCCM 50062; Seoul, Korea). Sixteen
clinical C. albicans isolates were obtained from body uids,
blood, urine, and genital of patients (The Research
Institute of Bacterial Resistance, College of Medicine,
Yonsei University, Korea). The species were identied
by conventional methods or using Vitex YBC cards
(bioMeriex, Marcy IEtoile, France), according to the
manufacturers instruction and stored at 70 C.
Growing candidal cultures and preparation of inocula. C.
albicans strains were cultured on Sabouraud dextrose agar
plates (SDA) (Difco, Spark, MD, USA) for 48 h at 37 C.
20 ml of Sabouraud dextrose broth (SDB) (Difco, Spark,
MD, USA) medium in a 150-mL ask was used for propa-
gation of candidal cells as described previously by Pierce
et al. (2008). Flasks containing SDB medium were
inoculated with a loopful of cells from the stock cultures
on SDA and incubated overnight in an orbital shaker
(180 rpm) at 30 C. Cells were harvested from the over-
night-grown liquid cultures by centrifugation (3000 g) for
5 min at 4 C. The pellets were washed twice in sterile
phosphate-buffered saline (PBS; 10 mmol/L, pH 7.4). The
washed pellets were suspended in approximately 20 mL
of RPMI 1640 without bicarbonate supplemented with
L-glutamine (WelGENE, Daegu, Korea) and buffered
with 165 mmol/l morpholinepropanesulfonic acid (MOPS;
Sigma, Steinheim, Germany) to pH 7. Cell suspensions
were diluted in the same medium and counted using a
hemocytometer and a bright eld microscope with a  40
objective to get a nal concentration of 1  106 cells/mL,
which is designated as the standardized inocula (Pierce
et al., 2008).
In vitro biolm formation. C. albicans biolms were
formed in the wells of commercially available presterilized,
polystyrene, at-bottomed, 96-well microtiter plates as
described previously by Bachman et al. (2002) and Pierce
et al. (2008) with a slight modication. Briey, biolms
were formed by pipetting 100 mL of the standardized
inoculum into selected wells of the microtiter plates
and covered the entire microtiter plate with its original
lid, then sealed with paralm. The plates were placed
Copyright 2012 John Wiley & Sons, Ltd.
inside incubator and incubated statically for 4 h (adherent
phase), 24 h (intermediate phase), and 72 h (mature phase)
discarded and nonadherent cells were removed by
by blotting with paper towels to remove any residual.
Biolms were ready to be processed for anticandidal
susceptibility testing assays (Rukayadi et al., 2011; Pierce
et al., 2008).
Preparation of anticandidal agents. Xanthorrhizol (Fig. 1)
was isolated in its pure form from the ethylacetate fraction
of the methanol extract of Java turmeric or temulawak
(C. xanthorrhiza Roxb.) according to the method of
Hwang et al. (2000b). The isolated xanthorrhizol was
dissolved in 10% dimethylsulfoxide to obtain 10 mg/mL
stock solutions. Amphotericin B, purchased from Sigma
(St. Louis, MO, USA), was dissolved in sterile-distilled
water to form a 512 mg/mL stock solution. Xanthorrhizol
and amphotericin B were diluted in RPMI 1640 without
bicarbonate supplemented with L-glutamine and buffered
with MOPS for nal concentrations of 256 mg/mL
and 64 mg/mL, respectively, which is designated as the
high working concentration of anticandidal agent
(Pierce et al., 2008).
In vitro biolm treatment. A 200 mL of the high working
concentration of anticandidal agent was added to the
corresponding wells on column 1 of each microtiter plate
containing C. albicans biolms. A 100 mL of RPMI 1640
without bicarbonate supplemented with L-glutamine and
buffered with MOPS was added to each well in columns
211. Wells in column 12 were remained empty. Anticandi-
dal agent (100 mL) from the wells of column 1 were
removed and added to the adjacent wells in column 2,
which already contain 100 mL of medium, mixed gently,
and repeated moving right until the wells of column 10,
after which the nal 100 mL volume from the wells of
column 10 after mixing was discarded. Wells in column 11
were the positive control (untreated biolm or biolm
not exposed to anticandidal agent). Wells in column 12
were negative control. The plates were covered with their
lids, sealed with paralm, and incubated for 24 h at 37 C
(Pierce et al., 2008). The experiments were performed
three times with four replicate wells for each experiment.
Biolm quantication. The anticandidal activity of
xanthorrhizol and amphotericin B on C. albicans biolms
was monitored using the XTT [2,3 - bis (2-methoxy-
4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]
reduction assay, adapted from a previous report
(Bachman et al., 2002; Ramage et al., 2001; Hawser et al.,
1998). Briey, XTT (Sigma, Steinheim, Germany) was
prepared in a saturated solution at 0.5 g/L in Ringers lac-
tate. The solution was lter-sterilized through a 0.22-mm-
pore-size lter (MILLEXWGV, Carrighwohill, Ireland),
aliquoted, and stored at 70 C. Prior to each assay, an ali-
quot ofstock XTT was thawed, and menadione (Sigma,
Steinheim, Germany; 10 mmol/L prepared in acetone)
was added to a nal concentration of 1 mmol/L. A 100-ml-
aliquot of the XTT-menadione solution was then added
to each prewashed biolm as well as to negative control
wells for the measurement of background XTT reduction
levels. The plates were then incubated in the dark for
23 h at 37 C. A colorimetric change in the XTT
Phytother. Res. 27: 10611066 (2013)
 ANTI-CANDIDA BIOFILM ACTIVITY OF XANTHORRHIZOL 1063

reduction assay, which directly correlates with the meta- Table 1. Results of anticandidal susceptibility testing of biolms
bolic activity of the biolm, was then measured in a micro- formed by C. albicans type strain ATCC 10231, KCCM 50062,
titer plate reader (VERSAMAX, Sunnyvale, CA, USA) at and 16 clinical C. albicans isolates against amphotericin B and
490 nm. Anticandidal activities for each isolates were xanthorrhizol for 24 h (intermediate growth) biolms.
expressed as percentage of the mean optical density at
490 nm wavelength (OD490) determined by XTT reduc- Amphotericin B (mg/mL) Xanthorrhizol (mg/mL)
tion assay of anticandidal-treated biolms compared to Candida isolate SMIC50 SMIC80 SMIC50 SMIC80
untreated biolms, which was considered to be 100%
(in this case values for column 11). Sessile minimum ATCC 10231 1 2 8 16
inhibitory concentrations (SMICs) were determined for KCCM 50062 0.5 1 4 8
each C. albicans isolate: SMIC50 and SMIC80 are the Clinical No. 1 2 4 16 32
anticandidal concentrations at which a 50% or 80% Clinical No. 2 1 2 8 16
decrease in biolm OD490 was detected in comparison Clinical No. 3 1 2 4 8
with the control biolms formed by the same C. albicans Clinical No. 4 0.5 1 4 8
isolate in the absence of anticandidal agent (Pierce et al., Clinical No. 5 0.5 1 4 8
2008). The experiments were performed three times with Clinical No. 6 2 4 16 32
four replicate wells for each experiment. Clinical No. 7 2 4 16 32
Clinical No. 8 1 2 16 32
Growth inhibition curves. To test the fungicidal activity of Clinical No. 9 0.5 1 4 8
xanthorrhizol against C. albicans biolm, the biolms at Clinical No. 10 1 2 8 16
different phase of growth were prepared and treated with Clinical No. 11 1 2 8 16
increasing concentrations of anticandidal as described Clinical No. 12 0.5 1 4 8
above. Treated biolms were washed with sterile PBS Clinical No. 13 2 4 16 32
two times to remove nonadherent cells and xanthorrhizol. Clinical No. 14 0.5 1 4 8
Biolms from the bottom of the wells were scraped by a Clinical No. 15 1 2 8 16
metal loop. Biolms were resuspended in 100 mL of MOPS Clinical No. 16 1 2 8 16
buffered RPMI 1640 medium and serially diluted. An
appropriate volume (100, 40, or 20 mL, depending on the
dilution and the concentration of xanthorrhizol), was The activity of xanthorrhizol in reduction C. albicans
spread onto SDA plates and incubated at 37 C for 48 h biolms was dependent on the concentration and the
or more (until the colonies were seen on the plates) to phase of growth of biolm. At adherent phase (4 h), a
determine the numbers CFU/mL. The experiments sharp reduction in the biolm OD490 as assessed by the
were performed three times with four replicate wells for XTT reduction assay was demonstrated when C. albicans
each experiment. biolms were exposed to amphotericin B and xanthorrhizol
at concentrations of 0.5 mg/mL and 2 mg/mL, respectively.
Statistical analysis. Statistical analysis was performed The metabolic activity of cells within the biolms of
with Students t test. Analyses were performed using C. albicans at adherent phase was completely abolished
Prism version 3.00 for Windows (GraphPad Software, by amphotericin B and xanthorrhizol at concentrations
San Diego, CA, USA). P values of <0.05 were consid- of 4 mg/mL and 8 mg/mL, respectively (Fig. 2A).
ered statistically signicant. Further experiments show that treatment with 2 mg/mL
of amphotericin B and 8 mg/mL of xanthorrhizol resulted
signicant reduction in the biolm OD490 of C. albicans
at intermediate phase (up to 50% reduction, P = 0.016) of
RESULTS growth. Moreover, treatment with 4 mg/mL of amphoteri-
cin B and 32 mg/mL of xanthorrhizol resulted signicant
SMIC50 and SMIC80 of amphotericin B and xanthorrhizol reduction in the biolm OD490 of C. albicans up to 85%
(P = 0.001) (Fig. 2B). Amphotericin B and xanthorrhizol
C. albicans biolms at intermediate phase of growth achieved the reduction in biolm OD490 of C. albicans at
(24 h old) were used for susceptibility test of amphotericin mature phase of growth by 90% at high concentration of
B and xanthorrhizol. The results of the susceptibility test of 64 mg/mL and 128 mg/mL (P = 0.001), respectively (Fig. 2C).
C. albicans biolms to amphotericin B and xanthorrhizol
are summarized in Table 1. All C. albicans biolms were Growth inhibition curves
susceptible to amphotericin B and xanthorrhizol, with
SMIC50s ranging from 0.5 2 mg/mL and 4 16 mg/mL, The colony counts of viable cells in biolms after treatment
respectively. SMIC80 of amphotericin B and xanthorrhizol with xanthorrhizol are presented in Fig. 3. The mean
against C. albicans biolms were 1 4 mg/mL and colony cell count determined in control (untreated)
8 32 mg/mL, respectively. These results indicate that C. albicans biolm at adherent (4 h), intermediate (24h)
50% or 80% of C. albicans biolms can be reduced in the and mature phase (72) of growth was 4.5  106 CFU/mL,
biolm OD490 by specic concentrations of xanthorrhizol. 1.8  108 CFU/mL and 3.6  109 CFU/mL, respectively.
The biolms of C. albicans at the adherent phase of growth
In vitro activity of amphotericin B and xanthorrhizol during were completely killed by xanthorrhizol at concentration
the different phases of C. albicans biolm development of 16 mg/mL. These results indicated that C. albicans
biolms at adherent phase of growth can be eradicated
The results of in vitro activity of amphotericin B and by xanthorrhizol at concentration of 16 mg/mL
xanthorrhizol during the different phases of C. albicans (Fig. 3). Xanthorrhizol attained the maximum signicantly
biolms development of growth are presented in Fig. 2. decrease in colony count reaching >3 log units at
Copyright 2012 John Wiley & Sons, Ltd. Phytother. Res. 27: 10611066 (2013)
1064 Y. RUKAYADI AND J.-K. HWANG

Figure 2. Activity of different amphotericin B and xanthorrhizol concentrations against biofilms of 18 C. albicans isolates at adherent phase (4 h) (A),
intermediate phase (24 h) (B), and mature phase (72 h) (C) growth. Values for the XTT reduction assay are expressed as the average percentage of
OD490 of the wells containing xanthorrhizol-treated biofilms compared to that of the control wells (untreated wells, considered to be 100%). Results
are from three times experiments performed with four replicate wells.

concentration of 32 mg/ml against C. albicans biolm
at intermediate phase of growth (Signicantly different
from the control, P = 0.01). The killing activity (fungicidal)
of xanthorrhizol against C. albicans biolm at mature
phase of growth was >3 log units at concentration of
32 mg/mL (Signicantly different from the control,
P = 0.026). However, the results showed that xanthorrhizol
failed to reach fungicidal activity in intermediate phase
and mature phase growth of C. albicans at concentration
of 256 mg/mL (Fig. 3).
DISCUSSION
Fungal biolm-associated infections show uniform
resistance to a wide spectrum of the currently available
conventional antifungal agents. This includes resistance to
the new triazoles, which generally regarded to be fungi-
static with extended activity against many azole-resistant
organisms (Jabra-Rizk et al., 2004). Therefore, fungal
biolm-associated infections are difcult to treat, which
emphasizes the need to develop antifungal drugs showing
activity against biolm-associated organisms. In addition,
Copyright 2012 John Wiley & Sons, Ltd.
theurapeutics used against mycoses are often toxic and
may result in dangerous drugdrug interactions (Shin and
Pyun, 2004). Research of new and effective natural
products, showing anticandidal activity against biolm cells
with low cytotoxicity, is likely to signicantly impact
the treatment, as well as the management, of biolm-
associated fungal infection (Chami et al., 2005). Some
essential oils have been shown to possess antifungal effects
(Agarwal et al., 2008). However, use of the whole essential
oil does not allow the determination of the active agent
because of the complexity of its components. Moreover,
it is possible that the minor components of essential oils
may exhibit some toxic or adverse effects in vivo (Chami
et al., 2005). Xanthorrhizol, a natural single compound that
is nontoxic to humans, can be isolated from the rhizome of
Java turmeric (C. xanthorrhiza Roxb.) and might be a good
candidate to prevent and kill C. albicans biolms.
There are few reports concerning the susceptibility of
C. albicans biolms to a natural single compound isolated
from medicinal plants. He et al. (2007) found that eugenol,
the major phenolic component of clove essential oil,
displayed activity against C. albicans cells in biolms
in vitro, where the SMIC50 was 500 mg/mL. Our ndings
show that xanthorrhizol had in vitro activity against
Phytother. Res. 27: 10611066 (2013)
 ANTI-CANDIDA BIOFILM ACTIVITY OF XANTHORRHIZOL
Figure 3. Growth inhibition curves of adherent phase (4 h) (filled
rhombus), intermediate phase (24 h) (filled squares), and mature phase
(72 h) (filled triangles) growth of C. albicans biofilms treated with
xanthorrhizol at concentrations 0.5 256 mg/mL. Values are
expressed as the average percentage of log10 cfu/ml of sessile cells
in the wells containing xanthorrhizol-treated biofilms compared to that
of the control wells (untreated wells, considered to be 100%). Results
are from three times experiments performed with four replicate
wells.*Significantly different from the control treatment (P < 0.05).
C. albicans cells within biolms, where the SMIC50 was
16 mg/mL. This result indicates that the anti-biolm activ-
ity of xanthorrhizol against C. albicans biolms was much
stronger than that of eugenol. Even though the SMIC50
of xanthorrhizol against C. albicans biolm was higher than
that of ampothericin B (Table 1), the SMIC50 of xanthor-
rhizol against C. albicans biolms was much lower than
that of uconazole (>1024 mg/mL) (Pierce et al., 2008;
Ramage et al., 2001).
Although complete reduction in the biolm OD490 at
intermediate phase and mature phase of growth were not
achieved by treatment with xanthorrhizol, the experiment
showed a >80% reduction in the biolm OD490 with
32 mg/mL and 128 mg/mL xanthorrhizol, respectively
(Fig. 2B and 2C). These concentrations were much lower
than the concentration of eugenol required for reduce up
to 80% sessile cells within C. albicans biolms (2000 mg/L
of eugenol) (He et al., 2007).
Because of the intrinsic recalcitrance of microbial
biolms and the difculty in eradicating cells once a biolm
has been established (intermediate phase and mature
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In this experiment, amphotericin B seemed to be more
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C. albicans biolms but normally at those concentrations
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Acknowledgment
This work was supported by the Yonsei Biomolecule Research Initiative of
the Brain Korea 21 Project.
Conict of Interest
The authors have declared that there is no conict of interest.
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