Open Journal of Medical Microbiology, 2011, 1, 7-11
doi:10.4236/ojmm.2011.11002 Published Online December 2011 (
Copyright © 2011 SciRes. OJMM
Combination Effect of Miconazole with Polygodial
against Candida albicans
Isao Kubo, Sang Hwa Lee, Kuniyoshi Shimizu
Department of Enviro n mental Science, Policy and Management, University of California, Berkeley, USA
Received November 4, 2011; revised November 11, 2011; accepted December 5, 2011
The combination effect of miconazole with polygodial against Candida albicans was investigated by an in
vitro checkerboard method. Isobolograms, fractional inhibitory concentration (FIC), and fractional fungicidal
concentration (FFC) indices were used for evaluating the interaction between compounds combined. The
combination of miconazole with polygodial exhibited strong synergism on both fungistatic and fungicidal
action against this opportunistic pathogen.
Keywords: Polygodial, Miconazole, Candida albicans, Combination, Synergism
1. Introduction
Opportunistic fungal infection in humans has become a
serious and increasingly common problem due to the ad-
vent of broad spectrum antibiotics. This has been further
exacerbated with the use of corticosteroids and immu-
nosuppressive drugs. Secondary fungal infection in HIV
infected patients is also a particularly difficult treatment
problem [1-3]. Only a relatively a few number of drugs
are available for the treatment of systemic fungal dis-
eases. Of these, many have major weaknesses in spectra,
potency, safety, and pharmacokinetic properties [4]. Mi-
conazole (1), an imidazole derivative, has broad activity
against most strains of yeasts, dermatophytes, and As-
pergillus spp. By oral or intravenous administration, it is
effective against systemic candidiasis, but its antifungal
efficacy is relatively weak and toxicity is high, compared
to fluconazole, itraconazole, and ketoconazole. Therefore,
miconazole is mainly used topically for yeast and other
localized fungal infections [5-7]. In general, miconazole
is fungistatic rather than fungicidal. Hence, studies are
needed to enhance the total biological activity of mi-
conazole by combining it with “other substances”, possi-
bly converting it from fungistatic to fungicidal.
In our continuing search for antifungal agents from non-
microbial sources, many plant secondary metabolites have
been characterized as active principles. Among them, po-
lygodial (2) (see Figure 1 for structures) isolated from the
sprouts of Polygonum hydropiper (Polygonaceae) (re-
classified as Persicaria hydropiper) [8], used as a food
Cl H
Figure 1. Structure of miconazole (1) and polygodial (2).
spice in Japan, was noted to possess potent fungicidal
activity against yeast-like fungi Candida albicans, Crypto-
coccus neoformans, Saccharomyces cerevisiae, and also
filamentous fungi, including Trichophyton mentagrophytes ,
T. rubu rum , and Penicillium marneffei [9]. Subsequently,
polygodial was found to enhance the antifungal activity
of antibiotics such as actinomycin D and rifampicin [10].
The combination of antifungal drugs with phytochemi-
cals may be one way to address to the urgent need for
effective and selective antifungal therapeutics [11]. In
order to gain new insights into combination effects of two
or more antifungal compounds on a molecular basis, the
study of miconazole in combination with polygodial against
C. albicans was performed as a model using an in vitro
checkerboard method [12].
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2. Methods
The test strain of Candida albicans ATCC 18804 used
for the experiment was purchased from American Type
Culture Collection (Rockville, MD). The procedures used
for antifungal assay were the same as previously descri-
bed [9]. Polygodial was available from our previous work
[4,13]. Miconazole was purchased from Sigma Chemical
Co. (St. Louis, MO). The FIC index was based on the
MICs of the combined compounds. The FIC is calculated
as (MICa combination/MICa alone) + (MICb combina-
tion/MICb alone), where a and b are the two compounds
used. The FFC index was based on their MFCs. The cal-
culation method was the same with FIC. The FIC or FFC
presented are significant values obtained from the checker-
board matrix. Best values are reported, with the exception
of antagonistic activity for which the worst values are
reported. The values of FIC and FFC indices were used
to define the interaction of combined compounds: syner-
gistic (X < 0.5), additive (1 < X > 0.5), indifferent (4 < X
> 1), or antagonistic (X > 4) [9,13,14].
3. Results and Discussion
Antifungal activity of miconazole and polygodial was
tested against C. albicans and the results are listed in
Table 1 [13,14]. Polygodial exhibited potent fungicidal
activity nearly comparable to amphotericin B. Based on a
time-kill curve experiment, polygodial showed strong and
fast fungicidal activity against C. albicans under growing
conditions and this activity was strongly increased at non-
growing conditions. This result is agreeable with previ-
ous reports [13,14]. The difference in the minimum inhi-
bitory concentration (MIC) and minimum fungicidal con-
centration (MFC) of polygodial is no more than 2-fold,
indicating no residual fungistatic activity. In a time-kill
curve experiment, polygodial showed strong and fast fun-
gicidal activity against C. albicans under growing condi-
tions [15]. In contrast to polygodial, the difference in MIC
and MFC of miconazole is 8-fold. Combination effects of
miconazole with polygodial were investigated by com-
paring their MICs and MFCs against C. albicans using a
checkerboard method [12]. The MIC and MFC of both
compounds were used as standards for evaluating their
combination effects.
Table 1. Antifungal activity (μg/mL) of miconazole, poly-
godial and amphotericin B against C. albicans.
Compounds Tested MIC (MFC)
Miconazole 6.25 (50)
Polygodial 3.13 (6.25)
Amphotericin B 1.56 (3.13)
In combination with miconazole, polygodial exhibited
a potent synergistic effect on antifungal action as shown
in Figure 2. Namely, when 0.2 µg/mL of miconazole was
combined with 0.313 µg/mL of polygodial, the growth of
C. albicans was completely inhibited. The FIC was cal-
culated as 0.16 [18]. The combination of polygodial and
miconazole demonstrated strong synergism based on their
FFC of 0.19. More importantly, the MFC of miconazole
was decreased from 50 µg/mL to 3.13 µg/mL when it
was combined with 0.78 µg/mL of polygodial. It appears
that polygodial exhibits strong synergistic effect to both
FIC 0.16
Polygodial (g/ml)
0.0 0.5 1.0 1.5 2.0 2.5
Miconazole (g/ml)
Figure 2. Resulting isobologram of the MICs (a) and MFCs
(b) obtained with combinations of miconazole and poly-
godial against C. albicans. The fractional inhibitory concen-
tration (FIC) or fractional fungicidal concentration (FFC)
index was calculated with the MICs or MFCs of the com-
bined compounds that exhibited the best antifungal combi-
nation effect. Data were obtained by the checkerboard
broth dilution technique at 30˚C [13,18].
Copyright © 2011 SciRes. OJMM
the fungistatic and the fungicidal actions of miconazole.
It appears that the antifungal action of miconazole can be
converted to fungicidal when it is combined with small
amounts of polygodial. In general, antimicrobial therapy
is dependent on both drugs’ growth inhibition and the
host’s immune system. However, systemic fungal disease
occurs commonly in patients with seriously impaired im-
mune systems, so fungicidal properties of antifungal agents
are considered to be very important [16]. Since azole
antifungal agents, including miconazole are only fung-
istatic [7], the combination of miconazole and polygodial
could be useful. Miconazole and polygodial were found
to have a particularly strong synergistic effect on fungi-
cidal action against C. albicans. Hence, this combination
was further investigated by their time-kill curve against
this yeast-like pathogen [17]. For the inoculum of 5 × 106
colony forming units (CFU)/mL, 6.25 µg/mL of polygo-
dial completely killed the initial inoculum within 12 h,
whereas a concentration of 3.13 µg/mL did not show
fungicidal activity as shown in Figure 3. Miconazole alone
did not exhibit any lethal activity at the concentration of
25 µg/mL within 48 h. However, when it was combined
with 3.13 µg/mL (equivalent to 1/2 MFC) of polygodial,
the complete lethal action of miconazole was observed
within 24 h. Subsequently, the concentration of micona-
zole could be further reduced to 0.78 µg/mL when the
assay was extended to 48 h.
The fungicidal mechanism of polygodial is associated
with its specific dialdehyde structure. It does not act by a
single defined process but rather, has multiple functions
by which it exerts the potent fungicidal action [13]. How-
ever, its fungicidal activity primarily comes from its abi-
lity to act as a nonionic surface-active agent (surfactant),
disrupting the lipid-protein interface. For example, poly-
godial is known to induce leakage by disrupting the mem-
brane surface [10,13]. The binding of polygodial to cell
surface alone is unlikely to explain its entire antifungal
mechanism, but may be large part of it. The surfactant con-
cept may also explain in part the permeability of foreign
molecules. In combination with polygodial, more mico-
nazole may enter the cells through pores derived from
membrane damage by polygodial [10]. Once inside the
cells, miconazole is known to inhibit 14-lanosterol de-
methylase, a microsomal cytochrome P450-dependent en-
zyme, causing ergosterol depletion [18]. The subsequent
replacement of ergosterol with lanosterol results in al-
terations to the plasma membrane, particularly its per-
meability. The similar potentiation mechanism of actin-
omycin D by this sesquiterpene was previously described
[10,19]. It seems that polygodial facilitates the transmem-
brane transport of miconazole (foreign compound) into
the cells [14,20].
Polygodial is known to inhibit the plasma membrane
Time (min)
0 12243648
Log CFU/mL
Figure 3. Effect of miconazole and polygodial on the growth
of C. albicans. Symbols indicate the concentration of poly-
godial and miconazole: Drug free (); poylgodial 3.13 μg/mL
(); miconazole 25 μg/mL (); 3.13 μg/mL (), 0.78 μg/mL
() of miconazole in combination with polygodial 3.13
H+-ATPase by disrupting and disorganizing the hydrogen
bonds at the lipid bilayer-protein interface [13]. The pre-
vious report suggests that the plasma membrane H+-ATPase
is also a site of antifungal action for miconazole [21].
This ATPase is mainly involved intercellular pH regula-
tion and hence, evolves into potential target for rational
drug design [22]. However, the inhibition of the glucose-
induced medium acidification around MFC of polygodial
was 25%, which was rather weak. The high cell density
such as 108 cells/mL was needed for the acidification
assay as compared with MIC and MFC assay performed
by 106 cells/mL. Therefore, the fungicidal potency of
polygodial was thought to be weakened by the high cell
density. The inhibition of plasma membrane H+-ATPase
alone could not explain the fungicidal action of poly-
godial. The data obtained are consistent with an effect on
the lipid bilayer-protein interface rather than a direct in-
teraction of H+-ATPase. All of these are agreeable with
the previous report that the primary active site of poly-
godial is at the membrane [19]. In addition to H+-ATPase,
other plasma membrane proteins may also be disrupted
by polygodial. For example, the resistance mechanism of
C. albicans is known to involve changes in cellular ef-
flux mechanisms [23], the fungal equivalent to the mi-
conazole resistant efflux pumps. Polygodial may disrupt
the efflux pump (membrane protein), similar mechanisms
described for the H+-ATPase. The drug efflux pumps are
based on energy-dependent efflux and both miconazole
and polygodial are common inhibitors of the plasma mem-
brane H+-ATPase (P-type) and the mitochondrial ATPase
(F-type) [24].
Copyright © 2011 SciRes. OJMM
In addition to polygodial, anethole also acts synergis-
tically with several antifungal agents. For example, ane-
thole was reported to enhance the fungicidal activity of
polygodial 128-fold against C. albicans in combination
with a sublethal concentration of anethole [17]. On the
other hand, anethole exhibited a strong synergistic effect
on fungistatic action of an azole antifungal agent, mico-
nazole, against this opportunistic fungus but a marginal
synergistic effect on fungicidal action. Thus, C. albicans
cells appeared to adapt to this combination stress, even-
tually recovering and growing normally. Interestingly, a
structurally similar phenyl propanoid, eugenol character-
ized from the same source, did not show this synergistic
activity at all, although it was found to exhibit fungicidal
activity against S. cerevisiae and C. albicans with each
MFC of 800 μg/mL.
Anethole was previously described to exhibit in vivo
synergistic effect on the fungicidal activity of the anethole/-
polygodial-containing compound against C. albicans,
supporting its present clinical application [25]. On the
basis of the result obtained with polygodial, the study was
further extended to see if the enhancing activity to mi-
conazole is specific to only polygodial’s dialdehyde struc-
tural feature. In order to facilitate it, a structurally simple
primary aliphatic alcohol, decanol was selected since
amphipathic primary aliphatic alcohols exhibit antifungal
activity against C. albicans. Among these alcohols, de-
canol was noted to exhibit the most potent fungicidal
activity with an MFC of 50 µg/mL against C. albicans.
Hence, miconazole was combined with decanol to see if
the same combination effect can also be observed against
C. albicans. The combination of miconazole and decanol
synergistically retarded the growth of C. albicans, but
had only marginal synergism on their fungicidal action.
Thus, C. albicans cells appeared to adapt to this combi-
nation stress, eventually recovering and growing normally.
To reveal the different combination effect between po-
lygodial and decanol, the leakage of 260 nm absorbing
materials and K+ ion from C. albicans cells was checked.
Polygodial induced leakage of 260 nm absorbing materi-
als, similar to those described for combination against S.
cerevisiae [10]. In contrast to polygodial, decanol did not
induce a leak of 260 nm absorbing materials although it
did induce a leak of K+ ion. The leakage of K+ ion by
decanol was observed within 30 min treatment accompa-
nied by significant loss of cell viability, suggesting that
decanol quickly affects the plasma membrane of C. al-
bicans cells forming rather smaller size pores than poly-
godial. The size formed may not be large enough to fa-
cilitate the transmembrane transport of miconazole into
the cells.
In conclusion, the combination effect of miconazole
with polygodial is truly synergistic, as shown by the va-
rious concentrations less than half-MIC or MFC of the
combined counterpart. It seems that the combination of
miconazole and polygodial targets the extracytoplasmic
region, and thus does not need to enter the cell, thereby
avoiding most cellular pump-based resistance mecha-
nisms. The combination strategy of antifungal drugs and
phytochemicals for the purpose of effectively controlling
systemic fungal pathogens is promising.
4. Acknowledgements
The authors are grateful to Dr. M. Lewin for his critical
reading of the manuscript, and Dr. M. Himejima and Dr.
K. Fujita for performing antifungal assay at earlier stage
of the work.
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