Alkylphenol activity against candida spp. and microsporum canis: A focus on the antifungal activity of thymol, eugenol and o-methyl derivatives

Article abstract of DOI:10.3390/molecules16086422

In recent years there has been an increasing search for new antifungal compounds due to the side effects of conventional antifungal drugs and fungal resistance. The aims of this study were to test in vitro the activity of thymol, eugenol, estragole and anethole and some O-methyl-derivatives (methylthymol and methyleugenol) against Candida spp. and Microsporum canis. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). The minimum fungicidal concentrations (MFC) for both Candida spp. and M. canis were found by subculturing each fungal suspension on potato dextrose agar. Thymol, methylthymol, eugenol, methyleugenol, anethole, estragole and griseofulvin respectively, presented the following MIC values against M. canis: 4.8-9.7; 78-150; 39; 78-150; 78-150; 19-39 μg/mL and 0.006-2.5 μg/mL. The MFC values for all compounds ranged from 9.7 to 31 μg/mL. Concerning Candida spp, thymol, methylthymol, eugenol, methyleugenol, anethole, estragole and amphotericin, respectively, showed the following MIC values: 39; 620-1250; 150-620; 310-620; 620; 620-1250 and 0.25-2.0 μg/mL. The MFC values varied from 78 to 2500 μg/mL. All tested compounds thus showed in vitro antifungal activity against Candida spp. and M. canis. Therefore, further studies should be carried out to confirm the usefulness of these alkylphenols in vivo.

Full text of DOI:10.3390/molecules16086422

Molecules 2011, 16, 6422-6431; doi:10.3390/molecules16086422
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Alkylphenol Activity against Candida spp. and Microsporum
canis: A Focus on the Antifungal Activity of Thymol, Eugenol
and O-Methyl Derivatives
1
,
2
3
4
Raquel O.S. Fontenelle *, Selene M. Morais , Erika H.S. Brito , Raimunda S.N. Brilhante ,
4
5
5
6
Rossana A. Cordeiro , Ynayara C. Lima , Nilce V.G.P.S. Brasil , André J. Monteiro
4
4,7
José J.C. Sidrim and Marcos F.G. Rocha
1
Centre of the Agricultural Sciences and Biological, Acaraú Valley State University, 62040-370,
Sobral, CE, Brazil
2
3
4
Department of Chemistry, State University of Ceará, 60740-000, Fortaleza, CE, Brazil;
E-Mail: selene@uece.br
Department of the Veterinary, Faculty of Veterinary Medicine, Superior Institute of Applied
Theology, 62050-100, Sobral, CE, Brazil; E-Mail: sallesbrito@yahoo.com.br
Department of Pathology and Legal Medicine, Faculty of Medicine, Medical Mycology Specialized
Center, Federal University of Ceará, 60441-750, Fortaleza, CE, Brazil; E-Mails: brilhante@ufc.br
(
R.S.N.B.); ross_aguiar@yahoo.com.br (R.A.C.); sidrim@ufc.br (J.J.C.S.); mfgrocha@gmail.com
M.F.G.R.)
(
5
Department of Organic and Inorganic Chemistry, Federal University of Ceará, 60455-760,
Fortaleza, CE, Brazil; E-Mails: yna.colares@gmail.com (Y.C.L.)
Nilcegramosapompeu@yahoo.com.br (N.V.G.)
6
7
Department of Statistics and Applied Mathematics, Federal University of Ceará, 60455-760,
Fortaleza, CE, Brazil; E-Mail: jalles@ufc.br
Postgraduate Program in Veterinary Science, State University of Ceará, 60740-000, Fortaleza, CE,
Brazil
*
Author to whom correspondence should be addressed; E-Mail: raquelbios@yahoo.com.br;
Tel.: +55-81-8104-0745. Fax: +55-85-3495-8692.
Received: 11 May 2011; in revised form: 15 July 2011 / Accepted: 26 July 2011 /
Published: 29 July 2011
Abstract: In recent years there has been an increasing search for new antifungal
compounds due to the side effects of conventional antifungal drugs and fungal resistance.

Molecules 2011, 16
The aims of this study were to test in vitro the activity of thymol, eugenol, estragole and
6423
anethole and some O-methyl-derivatives (methylthymol and methyleugenol) against
Candida spp. and Microsporum canis. The broth microdilution method was used to
determine the minimum inhibitory concentration (MIC). The minimum fungicidal
concentrations (MFC) for both Candida spp. and M. canis were found by subculturing each
fungal suspension on potato dextrose agar. Thymol, methylthymol, eugenol, methyl-
eugenol, anethole, estragole and griseofulvin respectively, presented the following MIC
values against M. canis: 4.8–9.7; 78–150; 39; 78–150; 78–150; 19–39 µg/mL and
0
.006–2.5 μg/mL. The MFC values for all compounds ranged from 9.7 to 31 µg/mL.
Concerning Candida spp, thymol, methylthymol, eugenol, methyleugenol, anethole,
estragole and amphotericin, respectively, showed the following MIC values: 39; 620–1250;
1
50–620; 310–620; 620; 620–1250 and 0.25–2.0 μg/mL. The MFC values varied from 78
to 2500 µg/mL. All tested compounds thus showed in vitro antifungal activity against
Candida spp. and M. canis. Therefore, further studies should be carried out to confirm the
usefulness of these alkylphenols in vivo.
Keywords: thymol; eugenol; estragole; methyl-derivatives; dermathophytes; Candida
1
. Introduction
Dermatophytosis is the most important superficial mycoses in both humans and animals. This
clinical condition is caused by a group of related filamentous fungi of the genera Epidermophyton,
Microsporum and Trichophyton [1]. The conventional treatment of fungal diseases is limited compared
to antibiotic therapy for bacterial infections [2]. Treatment of dermatophyte infection involves
primarily oral and/or topical formulations of azoles or allylamines, particularly itraconazole and
terbinafine [3]. More recently, the increasing problems of antifungal drug resistance of dermatophytic
fungi have been described [4].
The various forms of candidiasis are the most frequent causes of fungal infection in humans. A
progressive increase in the frequency of candidemia has been observed, particularly among patients
receiving antibiotics, immunosuppressive therapy or parenteral nutrition, as well as among patients
exposed to invasive medical procedures [5]. Antifungal resistance in Candida spp strains has been
observed in both humans and animals [5-7]. The resistance of Candida spp to currently available
antifungal drugs represents
prophylactic strategies.
a
major challenge for future empirical therapeutic and
Therefore, numerous essential oils have been tested for both their in vitro and in vivo antimycotic
activity. These oils have great potential as antifungal agents for use alone or together with standard
antifungal therapies [2]. Essential oils that contain alkylphenols such as thymol, eugenol, estragole,
and anethole have demonstrated antifungal properties [8-12].
Our previous studies with essential oils from species of Crotons [10] and Lippia sidoides [8] have
shown their antifungal activity against Candida spp. and M. canis. In the GC/MS analysis of these oils,
the main constituent found in the essential oil of L. sidoides was thymol, while in the essential oil of

Molecules 2011, 16
6424
Croton nepetaefolius this was methyleugenol and for Croton zenhtneri its was estragole/anethole.
Based on these studies, the present research was carried out to evaluate the antifungal activity of
alkylphenols (thymol, eugenol, estragole and anethole) and O-methyl-derivatives of thymol and
eugenol (Figure 1) against Candida spp. and M. canis in vitro. The O-methyl-derivatives were
obtained by reaction of the phenolic group with dimethyl sulfate in alkaline medium [13].
Figure 1. Chemical structures of antifungal alkylphenols.
2
. Results and Discussion
The screening results for the antifungal activity of the alkylphenols (thymol, eugenol, estragole and
anethole) and thymol/eugenol methyl-derivatives against M. canis and Candida spp. are shown in
Table 1, while Table 2 shows the MICs and MFCs of thymol, methylthymol, eugenol, methyleugenol,
anethol and estragole against M. canis. A statistically significant difference was observed when
comparing MICs (p = 0.0256) and MFCs (p = 0.0256) for thymol to MICs (p = 0.0231) and MFCs
(
p = 0.0231) for eugenol against M. canis strains.
Table 2 also shows the MICs and MFCs of thymol, methylthymol, eugenol, methyleugenol,
anethole and estragole against Candida spp. There was statistical significance for the MIC
(
p = 0.0196) and MFC (p = 0.0196) of thymol in relation to the other alkylphenols.
Many essential oils exhibit antioxidant and antimicrobial activities. Phenols, such as thymol,
carvacrol, and eugenol, are among the most active natural antioxidants and antimicrobials found in
essential oils. However, due to their poor water solubility and the requirement for high concentrations
to reach a therapeutic effect, the efficiency of these compounds in treatment is limited [14].

Molecules 2011, 16
6425
Table 1. Screening of antifungal activity of alkylphenols against M. canis and Candida
spp. by the agar-well diffusion method.
Growth Inhibition Zones (mm)
Alkylphenols (10,000 µg/mL)
Controls
thymol methyl- eugenol methyl-
anethole
estragole
griseofulvin amphotericin B
Strains
thymol
eugenol
(1000 µg/mL)
(5 µg/mL)
M. canis
CEMM 01-3-188
CEMM 01-5-190
35
40
22
20
26
30
20
20
16
20
23
20
55
47
-
-
C. albicans
CEMM 01-3-075
CEMM 01-3-069
18
17
18
13
12
8
10
7
11
9
11
8
-
-
14
10
Essential oils rich in alkylphenols frequently present antimicrobial activity [2]. This has been
corroborated by several other studies on the effect of essential oils of Croton species and Lippia
sidoides, which are rich in thymol, methyl-eugenol, estragole and anethole, and were shown to have
antifungal properties against Candida spp. and M. canis [8,10]. In addition, the antimicrobial activity
of thymol was demonstrated against oral pathogens [9]; the inhibitory effects of anethole and eugenol
were evaluated based on the growth and toxin production of Aspergillus parasiticus [14].
Eugenol was shown to be effective against fungi isolated from onychomycosis [15]; a fungistatic
dodecanol combined with a sublethal amount of anethole showed fungicidal activity against
S. cerevisiae [16] and the anti-Candida effects of estragole in combination with ketoconazole or
amphotericin B was demonstrated [17].
This work demonstrates the great potential of alkylphenols as antifungal agents against Candida
spp. and Microsporum canis, as observed by the relevant MIC and MFC levels of thymol, anethole,
estragole and methyl-derivatives (methylthymol and methyleugenol). Although all the tested
compounds showed MIC and MFC values, which support their antimicrobial activity against fungi of
medical interest, thymol stood out for its potent effect in vitro on both the dermatophyte M. canis and
the Candida spp.
Corroborating these results, Botelho et al. [9] demonstrated that thymol has potent antimicrobial
activity against Candida albicans, with minimum inhibitory concentrations ranging from 625 to
1
0,000 µg/mL. More recently, Guo et al. [18] showed the antifungal activity of thymol against clinical
isolates of fluconazole-sensitive and -resistant Candida albicans strains. In addition, Gayoso et al. [15]
showed that eugenol is a strong antifungal agent against yeast (C. albicans, C. tropicalis, C. krusei)
and dermathophytes (T. rubrum and T. mentagrophytes), suggesting this alkylphenol is a promising
compound for the development of antifungal drugs. It was also reported that anethole shows
synergistic effects on the antifungal activities of phytochemicals against Saccharomyces cerevisiae and
C. albicans [16].

Molecules 2011, 16
6426
Table 2. Minimum inhibitory and fungicidal concentrations of alkylphenols against M. Canis and Candida spp. evaluated
by the broth microdilution method.
Alkylphenols (µg/mL)
eugenol methyl-eugenol
Controls (μg/mL)
Strains
thymol
methyl-thymol
MIC MFC
1250 (4) 2500 (4) 620 (3)
50 (1)
1250 (1) 2500 (1) 310 (2)
anethole
estragole
MIC MFC
1250 (4) 1250 (3) 2500 (3)
amphotericin B
griseofulvin
MIC
MFC
MIC
MFC
1250 (3)
310 (1)
620 (2)
MIC
MFC
1250 (3)
620 (1)
620 (2)
MIC
MFC
MIC
0.5 (2)
0.25 (2)
1.0 (1)
0.5 (1)
0.5
MIC
-
C. albicans (n = 4)
C. tropicalis (n = 2)
39 (4)a
39 (2)
150
78 (4)
620 (3)
310 (1)
310 (2)
620 (4)
1
620 (1)
1250 (1)
1250 (2)
78 (2)
310
620 (2)
620
1250 (2) 620 (2)
-
-
-
6
20 (1)
1250 (1)
2500
C. krusei (n = 1)
ATCC 22019
1250
620
620
1250
1250
620
620
1250
1250
1250
1250
620
620
1250
1250
C. parapsiloses
150
310
1250
2500
620
2.0
-
(
n = 1)
ATCC 6528
M. canis (n = 6)
9.7 (3)
19 (3)
150 (4)
78 (2)
310 (4)
150 (2)
39 (6)
78 (6)
150 (2)
78 (4)
310 (2) 150 (3)
150 (4) 78 (3)
310 (3)
150 (3)
39 (4)
19 (2)
78 (4)
39 (2)
0.25 (2)
4
.8 (3)
9.7 (3)
0.125 (3)
0
.006 (1)
a
Represents the number of strains of a species for the MIC and MFC indicated.

Molecules 2011, 16
6427
3. Experimental
3.1. General
Estragole, anethole and eugenol were purchased from Sigma Chemical Co. (St. Louis, MO, USA)
and thymol was supplied by Vetec (Duque de Caxias, RJ, Brazil). The methyl-derivatives were
synthesized as previously described by Furniss et al. [13]. Briefly, eugenol or thymol (0.05 mol) was
suspended in a cold solution of NaOH (13.3 g, 0.33 mol) in water (81.25 mL) with vigorous stirring.
Dimethyl sulfate (14.4 g, 0.12 mol) was added in one portion, and the mixture was shaken vigorously
for 20 minutes, while the temperature was maintained below 35 °C by external cooling. A second
portion of dimethyl sulfate (14.4 g, 0.12 mol) was added and the temperature was allowed to rise to
4
0–45 °C. At the end of the reaction, the reaction mixture was transferred to a separatory funnel. The
organic layer, containing the methyl-derivatives, was washed with distilled water and dried with
anhydrous sodium sulfate. The solvent was then evaporated to furnish the corresponding methyl
derivatives. The chloroform was eliminated in a rotary evaporator to obtain pure methyl derivatives,
1
13
which were analyzed by H-, C-NMR and mass spectroscopy [19]. The presence of the absorption
1
3
band in 55.2 δ of the OCH
3
group in the C-NMR and the corresponding peak at 3.87 δ in the
1
H-NMR spectrum of O-methylthymol confirmed the methylation of thymol. In the spectra of
1
O-methyl-eugenol, the mass spectra confirmed the molecular mass at m/z 178, in the H-NMR
1
3
spectrum two peaks at 3.88 δ and 3.90 δ appeared, while in the C-NMR there were also two peaks for
the methoxyl groups at 55.7 δ and 55.9 δ. The spectral data of the methyl derivatives of thymol and
eugenol are listed below.
1
3
O-Methylthymol. C-NMR (δ, CDCl
3
): 155.6 (C1), 136.3 (C2), 126.8 (C3), 121.1 (C4), 134 (C5),
1
1
6
7
11.4 (C6), 25.4 (C7), 22.8 (C8), 22.8 (C9), 21.3 (C10), 55.2 (OCH
.4, CH 8/CH 9), 2.39 (s, CH 7), 3.34 (h, 6.4, H7), 3.87 (s, OCH
.17 (d, 5.6, H4).
3
). H-NMR (δ, CDCl
3
): 1.26 (d,
3
3
3
3
), 6.73 (s, H6), 6.81 (d, 5.6, H3),
O-Methyleugenol. MS-EI m/z (Int. %): 178 (100), 163 (45), 151 (15), 147 (50), 131 (14), 115 (26), 103
1
3
(
78), 91 (84), 77 (39), 65 (38), 51 (22). C-NMR (δ, CDCl
3
): 147.3 (C1), 148.8 (C2), 111.2 (C3),
1
32.6 (C4), 120.4 (C5), 111.8 (C6), 39.7 (C7), 137.8 (C8), 115.6 (C9), 95.9 (C10), 95.7 (C11).
1
3 3 3
H-NMR (δ, CDCl ): 3.36 (d, 2.5, H7), 3.88 (OCH ), 3.90 (OCH ), 5.09 (dd, 9.3; 2.9, H9), 5.98 (m,
H8), 6.76 (d, 4.7, H-5), 6.83 (m, H3, H6).
.2. Fungal strains
The strains were obtained from the fungus collection of the Specialized Medical Mycology
3
Center—CEMM (Federal University of Ceará, Brazil), where they were maintained in saline (0.9%
NaCl), at 28 °C. At the time of the analysis, an aliquot of each suspension was taken and inoculated on
potato dextrose agar (DIFCO, Detroit, MI, USA), and then incubated at 28 °C for 2–10 days. A total of
six strains of M. canis and six Candida spp. (four C. albicans and two C. tropicalis) were included
in this study. Both M. canis and Candida spp. strains were isolated from dogs. In addition,
C. parapsilosis (ATCC 22 019) and C. krusei (ATCC 6528) strains were used as a quality controls.

Molecules 2011, 16
.3. Agar-well diffusion method
6428
3
The inocula preparation for the agar-well diffusion method was carried out as previously described
8,10,20]. The screening of the antifungal activity of the alkylphenols and methyl derivatives of
[
thymol and eugenol was performed against Candida spp and M. canis strains by the agar-well
diffusion method, as previous reported [8,10,20]. In brief, Petri dishes (15 cm in diameter) were
prepared with potato dextrose agar (DIFCO) and then wells (6 mm in diameter) were cut from the
agar. Each fungal suspension was inoculated on the surface of the agar and 0.100 mL of each
suspension was placed into the wells. After incubation at 28 °C, for 3–5 days for Candida spp. and
5–8 days for M. canis, all the dishes were examined for growth-inhibition zones and the diameters of
these zones were measured in millimeters. Each experiment was repeated at least twice. The
alkylphenols and methyl-derivatives were dissolved in DMSO to obtain a test concentration of
1
0,000 µg/mL. griseofulvin (1000 µg/mL; Sigma Chemical Co.) and amphotericin B (5 µg/mL; Sigma
Chemical Co.) were prepared in distilled water and tested as positive controls for M. canis and
Candida spp., respectively.
3
.4. Broth microdilution method
For the broth microdilution method, the inocula preparation was carried out as previously described
[
6-8,10]. The minimum inhibitory concentration (MIC) for Candida spp. was determined by the broth
microdilution method, in accordance with the Clinical and Laboratory Standards Institute—CLSI
formerly NCCLS; M27-A2) [21]. The broth microdilution assay for M. canis was performed as
(
described by Jessup et al. [22], Fernandez-Torres et al. [23], and Brilhante et al. [24], based on the
M38-A document [25]. The minimum fungicidal concentrations (MFC) for both Candida spp. and
M. canis were found according Fontenelle et al. [8].
The microdilution assay was performed in 96-well microdilution plates and the alkylphenols were
tested in concentrations ranging from 4 to 5,000 µg/mL. Growth and sterile control wells were
included in each experiment. The microplates were incubated at 37 °C and read visually after two days
for Candida spp. and five days for M. canis, as previously reported [6,7,10,20,23,25]. All isolates were
run in duplicate. The MIC was defined as the lowest alkylphenol concentration that caused 100%
inhibition of visible fungal growth. The results were read visually as recommended by CLSI. The
MFC was determined by subculturing 100 µL of solution from subsequent MIC well on potato
dextrose agar at 28 °C. The MFCs were determined as the lowest concentration resulting in no growth
on the subculture after two days for Candida spp. and five days for M. canis [6-8,10].
3
.5. Statistical analysis
The MICs and MFCs of the aromatic compounds and methyl derivatives were analyzed by the
Wilcoxon and Friedman tests. The result was considered significant at p < 0.05. The followings
comparisons were carried out: thymol vs. methyl-thymol; eugenol vs. methyl-eugenol; and differences
among the isolated alkylphenols.

Molecules 2011, 16
. Conclusions
Our results demonstrate that alkylphenols showed in vitro antifungal activity against Candida spp.
6429
4
and M. canis. Since thymol and eugenol are skin irritants, further studies should be carried out to
establish whether or not methyl derivatives of thymol and eugenol possess better pharmacokinetics and
safety characteristics than their parent compounds for in vivo usefulness.
Acknowledgements
We acknowledge the financial support of FUNCAP (Ceará State Research Foundation) and CNPq
(
National Counsel for Technological and Scientific Development; Brazil, Proc. CNPq: 307402/2010-0;
Proc. CNPq: 562296/2010-7).
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