Journal of Applied Microbiology 2004, 97, 12891296

doi:10.1111/j.1365-2672.2004.02417.x

Antifungal effects of herbal essential oils alone and in

combination with ketoconazole against Trichophyton spp.
S. Shin and S. Lim
College of Pharmacy, Duksung Womens University, Dobongku, Seoul, South Korea
2003/0470: received 4 June 2003, revised 6 July 2004 and accepted 7 July 2004

ABSTRACT
S . S H I N A N D S . L I M . 2004.

Aims: To determine the effects of herbal essential oils on Trichophyton spp. growth and to evaluate the effects of
Pelargonium graveolens oil and its main components citronellol and geraniol combined with ketoconazole against
Trichophyton spp.
Methods and Results: Growth inhibition of six Trichophyton spp. by herbal essential oils was accessed and the
combined effects of P. graveolens oil and its main components citronellol and geraniol were evaluated using a
checkerboard microtitre assay against T. schoenleinii, T. erinacei and T. soudanense. The essential oil fraction of
P. graveolens and its main components, geraniol and citronellol, exhibited strong synergism with ketoconazole
against T. schoenleinii and T. soudanense, with fractional inhibitory concentration (FIC) indices in the range of
018038.
Conclusions: The antifungal effects of ketoconazole against Trichophyton spp. are enhanced significantly by
administering it in combination with the essential oil fraction of P. graveolens or its main components, because of
strong synergism, especially against T. soudanense and T. schoenleinii.
Significance and Impact of the Study: The combination of ketoconazole and the essential oil fraction from
P. graveolens or its main components for treatment of infections caused by Trichophyton species may reduce the
minimum effective dose of ketoconazole, and thus minimize the side-effects of ketoconazole.
Keywords: citronellol, essential oil, geraniol, ketoconazole, Pelargonium graveolens, synergism, Trichophyton.

INTRODUCTION
Natural antifungal agents are in great needed because
currently used therapeutics have toxic side-effects, may
interact with other drugs, and become ineffective as a result
of the rapid development of fungal resistance (Gundidza
1993; Hammer et al. 1998, 1999; Shahi et al. 1999).
Trichophyton is a fungal species that causes superficial
mycoses commonly known as tinea infections in various
areas in humans and other animals. Ketoconazole is one of
the commonly used antifungal drugs administered orally for
the treatment of both superficial and deep infections caused
by Trichophyton. However, the unpleasant side-effects of
Correspondence to: Seungwon Shin, College of Pharmacy, Duksung Womens
University, Ssangmoondong 419, Dobongku, Seoul, 132-714, South Korea
(e-mail: swshin@duksung.ac.kr).

2004 The Society for Applied Microbiology

this drug include nausea, abdominal pain and itching, and its
toxicity limits its therapeutic use in many cases. Moreover,
the therapeutic response may be slow, and thus inappropriate for treatment of patients with severe or rapidly
progressive mycoses. In addition, the efficacy of ketoconazole is poor in immunosuppressed patients and in the
treatment of meningitis.
Essential oils are one of the most promising groups of
natural compounds for the development of safer antifungal agents. However, their poor absorption from the
human intestine and relatively mild antifungal activity
compared with commercial, synthetic antifungal drugs
may ultimately limit their clinical application in the
treatment of systemic fungal infections (Garg and Siddiqui 1992; Adam et al. 1998; Bidlack et al. 2000; Shin and
Kang 2001).

1290 S . S H I N A N D S . L I M

Many essential oils are useful for treatment of tinea and

their in vitro and in vivo antifungal effects have been
evaluated (Hammer et al. 2000; Inouye et al. 2001; Harris
2002). In general, their antifungal activity is mild compared
with commonly used antibiotics. Many essential oils are only
fungistatic and high concentrations are needed for fungicidal
activity (Cassella et al. 2002). To enhance the efficacy of
essential oils, the combined use of different oils has been
evaluated recently for potential synergistic effects. The
combination of essential oils with synthetic antifungals will
probably result in a more effective therapy though (Yoon
et al. 1994; Suresh et al. 1997; Giordani et al. 2001; Shin
and Kang 2003). However, synergistic effects between
essential oils and common antifungal drugs against Trichophyton have not yet been reported until now.
In order to develop a broad-spectrum natural antiTrichophyton agent, we evaluated the activities of essential
oils, used for the treatment of fungal infections in aromatherapy and complementary medicine, against six species of
Trichophyton fungi by broth dilution and disc diffusion tests.
In addition, we evaluated the synergistic effects of the
essential oils from P. graveolens, a potent oil containing the
highly active and stable primary alcohols citronellol and
geraniol, and ketoconazole, a commonly used therapeutic for
Trichophyton infections, against Trichophyton spp.

M A T E R I A LS A N D M E T H O D S
Preparation of samples for testing
anti-Trichophyton activities
The essential oils from Cedrus atlantica (wood), Citrus
bergamia (fruit), Cymbopogon citratus (leaf), Eukalyptus
golbulus (leaf), Juniperus communis (fruit), Lavandula angustifolia (flower), Melaleuca alternifolia (leaf), Pelargonium
graveolens (leaf), Pogestemon patchouli (leaf), Rosmarinus
officinalis (herb), Styrax tonkinensis (wood) and Thymus
vulgaris (herb) were extracted by steam distillation. The
compositions of the essential oils were analysed by gas
chromatography-mass spectrometry (GC-MS) on a
Hewlett-Packard 6890 GC and Hewlett-Packard 5973
MSD apparatus using an HP-5 capillary column (Shin
2003). Citronellol, geraniol, benzoic acid, thymol, 1,8-cineol
and ketoconazole were purchased from Sigma Chemical Co.
(St Louis, MO, USA).
Fungal strains
Six Trichophyton spp. were obtained from the Korean
Culture Center of Microorganisms (KCCM). Trichophyton
erinacei KCCM 60411, T. mentagrophytes KCCM 11950,
T. rubrum ATCC 6345, T. tonsurans KCCM 11866,
T. schoenleinii KCCM 60477 and T. soudanense KCCM

60448 were cultured in yeast and malt extract broth (YM;

Difco 0712) for 48 h at 25
C. The turbidity of the cell
suspension was measured at 600 nm and adjusted with
medium to match the 05 McFarland standard [105106
colony-forming units (CFU) ml)1].
Determination of MIC
Essential oil samples were serially diluted with YM broth to
obtain solutions with 02564 mg ml)1 essential oil; 10 ll
Tween 80 was added to each solution. After shaking, 100-ll
aliquots of the essential oil solutions were added to wells of 96well microtitre plates. A 100-ll suspension of each of the six
Trichophyton species was adjusted to 104105 CFU ml)1, and
then added to individual wells and cultivated at 25
C. The
MIC was defined as the lowest concentration that completely
inhibited visible fungal growth after 72 h. Each organism was
also cultured with a blank solution containing Tween 80 at
concentrations equivalent to those in the test solutions to
certify that these vehicles did not affect fungal growth.
Disc diffusion assay
Fungal broth culture aliquots were added to Sabouraud
s
dextrose agar medium and uniformly distributed. Sterile
paper discs (8 mm; Advantec, Dublin, CA, USA) were
impregnated with 50 ll of 2550% (12525 mg per disc)
ethanol solutions of each agent, and after alcohol evaporation
the discs were placed on the culture plates. The width of the
zone of inhibition (mm) was measured from the boundary of
the disc after cultivation at 25
C for 72 h. Values are given
as mean and S.D. of tests performed in triplicate.
Checkerboard microtitre test
Ten serial twofold dilutions of geraniol, citronellol, the
essential oil fraction from P. graveolens, and ketoconazole
were prepared using the same solvents as in the MIC tests.
Fifty-micolitre aliquots of each P. graveolens oil dilution
were added to the wells of a 96-well plate in a vertical
orientation and 10-ll aliquots of each ketoconazole dilution
were added in a horizontal orientation so that the plate
contained various concentration combinations of the two
compounds. Following this, each well was inoculated with
100 ll (ca 5 104 CFU per well) of one of three Trichophyton (T. erinacei, T. soudanense or T. schoenleinii) fungal
suspensions and cultivated at 25
C. Fractional inhibitory
concentration (FIC) was calculated by dividing the MIC of
the combination of geraniol and ketoconazole by the MIC of
geraniol or ketoconazole alone. The FIC index, obtained by
adding both FIC, was interpreted as indicating a synergistic
effect when it was 05, as additive or indifferent when it
was >05 and 20, and as antagonistic when it was >20

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

1291

HERB OILS AGAINST TRICHOPHYTON SPP.

fungal sensitivity did not differ remarkably in the broth

dilution and agar methods. However, the MIC and MFC of
the essential oils tested varied significantly.
Among the oil fractions tested, C. citratus and E. golbulus
were the most potent inhibitors of all fungi tested, with MIC
of <0125025 mg ml)1 and MFC of <01251 mg ml)1.
As expected, T. vulgaris and its main component thymol had
an especially high MIC value ranging between 025 and
1 mg ml)1. Thymol was more active than the total oil
fraction of T. vulgaris. The oil of P. graveolens as well as its
main components, citronellol and geraniol, also strongly
inhibited these fungi, with MIC of 0252 mg ml)1. The
oils of P. patchouli, which are widely used as therapeutics for
various microbial infections, exhibited unexpectedly moderate or relatively poor activity in this study.

(White et al. 1996). Similar checkerboard experiments were

also performed to test the combined effect of the essential oil
fraction from P. graveolens or citronellol and ketoconazole.
An isobologram was constructed from the checkerboard data
to depict the synergism between P. graveolens oil and
ketoconazole against Trichophyton spp. A checkerboard
experiment was also performed to determine the effect of
combining citronellol or geraniol with ketoconazole.
Statistical treatment of the results
The analysis of data was performed with the SPSS
program version 110 (SPSS Inc., Chicago, IL, USA).
Analysis of variance tests were conducted using the
general one-way ANOVA with post hoc comparison of mean
values by LSD.

Growth inhibition of Trichophyton spp. on

Sabourauds agar plates

RESULTS

The results of the disc diffusion tests (Table 2) also indicate

that these oils are significantly potent against Trichophyton
spp. Consistent with the MIC assay results, the oils of
C. citrates, L. angustifolia, P. graveolens, R. officinalis and
T. vulagris exhibited strong inhibition of >39 mm in most of
the experimental concentrations of 25 and 125 mg per disc.
The results from tests with T. vulagris and its main
component thymol, and P. graveolens and its main components citronellol and geraniol were similar, at 125 mg per
disc. However, not all the fungal inhibition results on

MIC of the herbal oils and their main components

We used the main components (benzoic acid, 1,8-cineol,
thymol, citronellol and geraniol) of the essential oils in
S. tonkinensis, E. golbulus, T. vulgaris and P. graveolens,
respectively, as determined by GC-MS analysis of the oils
(Shin 2003). MIC assay results of the tested oils are shown
in Table 1.
Most of the oils evaluated exhibited significant inhibitory
activity against all six of the Trichophyton species tested. The

Table 1 Minimum inhibitory concentration (MIC)* and minimum fungicidal concentration (MFC) of herbal oils against Trichophyton spp.
Fungi
T. erinacei

T. mentagrophytes

T. rubrum

T. schoenleinii

T. soudanense

T. tonsurans

Essential oil

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

C. atlantica
C. bergamia
C. citratus
E. globulus
J. communis
L. angustifolia
M. alternifolia
P. graveolens
P. patchouli
R. officinalis
S. tonkinensis
T. vulgaris
Benzoic acid
1,8-Cineol
Citronellol
Geraniol
Thymol

2
4
025
025
05
05
05
05
8
4
1
05
<0125
025
05
05
025

4
16
05
1
4
4
3,12
2
>32
16
2
1
0125
2
4
2
05

1
2
<0125
025
4
2
1
05
>32
8
2
1
025
<0125
1
05
05

2
4
<0125
05
8
4
>32
1
>32
16
4
1
05
025
2
1
1

1
1
<0125
<0125
1
05
1
05
2
8
2
1
025
05
2
1
05

2
4
0125
05
4
4
>32
05
8
8
2
1
<025
05
4
1
1

1
2
<0125
025
2
2
2
05
2
4
1
05
<025
025
1
05
05

2
4
0125
05
4
4
>32
05
8
8
2
1
<025
05
4
1
1

025
05
<0125
025
05
025
8
025
05
05
05
05
025
025
05
025
05

05
2
<0125
05
2
05
16
05
>32
1
1
1
05
05
1
05
1

05
1
<0125
<0125
2
1
1
05
8
8
2
1
025
05
2
05
05

1
2
0125
0125
4
2
2
1
>32
16
4
2
05
1
4
1
1

*Values are given as mean values (mg ml)1) from the triplicate experiments.
2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

1292 S . S H I N A N D S . L I M

Table 2 Growth inhibition (mm)* of Trichophyton spp. on Sabouraud
s agar plates

Fungi
Essential oil
C. atlantica
I
II
C. bergamia
I
II
C. citratus
I
II
E. globules
I
II
III
J. communis
I
II
L. angustifolia
I
II
M. alternifolia
I
II
P. graveolens
I
II
III
P. patchouli
I
II
R. officinalis
I
II
S. tonkinensis
I
II
III
T. vulgaris
I
II
III
Benzoic acid
III
1,8-cineol
I
II
III
Citronellol
III
Geraniol
III

T. erinacei

T. mentagrophytes

T. rubrum

T. schoenleinii

73 076f
33 076c

92 029e
53 029c

10 000a
07 029a

73 058e
43 115c

80 050f
50 200e

60 100c
28 029b

87 076e
60 100d

33 058b
25 150a

>390
>390

>390
>390

>390
>390

>390
>390

T. soudanense
87 058d
58 076c
143 153
78 029
>390
>390

T. tonsurans
72 076d
38 029b
33 058
23 058a
>390
>390

250 153
180 000
00 000a

210 208
130 153
00 000a

150 200
90 200e
00 000a

98 058
62 115d
00 000a

>390
>390
00 000a

100 029f
58 076c
00 000a

28 029b
17 029b

58 029c
27 058b

38 029c
110 058

65 132
33 029b

52 029b
28 029

47 058
22 029a

>390
>390
130 058
48 029e
>390
>390
40 050
53 104e
33 058c
>390
>390
150 153
87 058f
00 000a
>390
>390
45 087

>390
>390

>390
>390

>390
>390

87 058e
53 029c

93 058e
67 115e

160 115f
83 058

>390
>390
68 104d

>390
>390
43 104d

>390
>390
105 132

30 000b
27 029b

33 153c
23 058b

>390
>390
160 050
110 058
00 000a
>390
>390
78 104

330 115
200 029
170 029
130 050
00 000a
>390
>390
47 058d

70 050e
60 000d
>390
>390
170 029f
110 029
00 000a
>390
>390
108 126

>390
>390
233 153
117 029e
>390
>390
87 126
47 058b
45 078b
>390
>390

>390
>390
52 029c
35 087b
>390
>390
95 050f
73 058d
57 058c
310 058
170 000

190 200
162 076
00 000a

290 153
190 100
00 000a

>390
>390
113 064e

>390
>390
82 104e

13 029a

25 050b

57 058d

28 029b

63 058c

58 029c

95 050
15 050a
00 000a

20 000a
12 076a
00 000a

27 058b
18 029a
00 000a

77 153e
37 058b
00 000a

12 029a
00 000a
00 000a

30 000
18 029a
0 000a

42 029d

57 058c

38 029c

45 050c

65 087

85 050e

40 000d

62 076c

37 029c

47 058c

88 029d

93 058f

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

HERB OILS AGAINST TRICHOPHYTON SPP.

1293

Table 2 (Contd)
Fungi
Essential oil

T. erinacei

T. mentagrophytes

T. rubrum

T. schoenleinii

T. soudanense

T. tonsurans

Thymol
III

88 076f

73 058d

93 058e

93 058

63 153c

58 029c

I, 25 mg per disc; II, 125 mg per disc; III, 125 mg per disc.
*The width (mm) of growth inhibition of fungi was measured from the boundary of discs.
The values are the mean and S.D. from the triplicate experiments.
Data in each column followed by the same letters are not significantly different at the 5% level.

which was highly active against all five Trichophyton spp.

and contained, relatively stable primary monoterpene
alcohols (citronellol and geraniol) as main components. As
shown in Table 3, the MIC of ketoconazole combined with
P. graveolens oil against T. erinacei, T. schoenleinii and
T. soudanense were remarkably decreased, with FIC indices
ranging between 018 and 056. Moreover, fungal susceptibility to ketoconazole was enormously improved by combination with the oil or its main components. In an experiment
with T. erinacei and T. soudanense, the FIC of ketoconazole
when combined with citronellol or geraniol was 006 and
013. FIC indices indicate the strongest synergism between
P. graveolens oil and ketoconazole against T. soundanense,
with an FIC index of 018. Similar results were obtained by
the combination of ketoconazole with geraniol or citronellol,
with an FIC index of 018. The isobologram based on the
checkerboard titre assay results against T. schoenleinii

Sabourauds agar plates were consistent with results from the

MIC assays. For example, the radius of inhibition by
E. globulus oil and its main component 1,8-cineol was smaller
than expected based on its MIC. J. communis and P. patchouli
oils were relatively ineffective against all the fungi tested,
with inhibition zones between 28 and 73 mm even at 25 mg
per disc on Sabourauds dextrose agar plate. The widths of
the fungal inhibition zones were generally dose dependent.
Combined effects of P. graveolens oil and its main
components and ketoconazole
To identify synergism of the essential oils with and
ketoconazole to enhance the efficacy of antifungal drugs,
checkerboard assays were performed and used to construct
an isobologram. Among the 12 essential oil fractions used in
the MIC and disc diffusion tests, we chose P. graveolens oil,

Table 3 Fractional inhibitory concentration (FIC) and FIC indices

T. erinacei
Sample
P. graveolens oilketoconazole
P. graveolens oil (mg ml)1)
Ketoconazole (lg ml)1)
CitronellolKetoconazole
Citronellol (mg ml)1)
Ketoconazole (lg ml)1)
GeraniolKetoconazole
Geraniol (mg ml)1)
Ketoconazole (lg ml)1)

T. schoneleinii

T. soudanense

MICo

MICc

FIC

FICI

MICo

MICc

FIC

FICI

MICo

MICc

FIC

FICI

05
16

025
1

050
006

056

05
8

0125
05

025
006

031

025
16

0031
1

012
006

018

05
16

025
1

050
006

056

1
8

025
1

025
013

038

05
16

0062
1

012
006

018

05
16

025
1

050
006

056

05
8

0125
1

025
013

038

025
16

0031
1

012
006

018

MICo, MIC of one sample alone; MICc, MIC of one sample of the most effective combination.
FIC of oil

MIC of oil in combination with ketoconazole

MIC of oil alone

FIC of ketoconazole

MIC of ketoconazole in combination with oil

MIC of ketoconazole alone

FICI FIC of oil FIC of ketoconazole

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

1294 S . S H I N A N D S . L I M

Ketoconazole (mg ml1)

(a)

(b)

(c)

0008

0008

0008

0006

0006

0006

0004

0004

0004

0002

0002

0002

0000

0000

0000

02

00

04

06

00

P. graveolens oil (mg ml1)

02

04

06

08

10

00

Citronellol (mg ml1)

02

04

06

Geraniol (mg ml1)

Fig. 1 Isobolograms revealing the synergistic and additive effect of Pelargonium graveolens oil (a), citronellol (b) and geraniol (c) with ketoconazole on
Trichophyton schoenleinii growth inhibition

Ketoconazole (mg ml1)

(a)

(b)

(c)

0016

0016

0016

0012

0012

0012

0008

0008

0008

0004

0004

0004

0000

0000

0000
00

03
01
02
P. graveolens oil (mg ml1)

00

02
04
Citronellol (mg ml1)

06

00

01
02
Geraniol (mg ml1)

03

Fig. 2 Isobolograms revealing the synergistic action of Pelargonium graveolens oil (a), citronellol (b) and geraniol (c) in combination with ketoconazole
on Trichophyton soudanense growth inhibition

(Fig. 1) also indicates synergism between the combined

compounds by the obviously deviating curves to the left.
The patterns of the curves for P. graveolens oil and geraniol
activity were highly similar. The isobologram against
T. soudanense confirms these results. The curves depicted
in Fig. 2 also show strong synergism from the combination
of ketoconazole with P. graveolens oil, citronellol or geraniol
by the distinctly left-deviating curves (Davidson and Parish
1989). Thus, geraniol is responsible for a large part of the
activity of the P. graveolens oil fraction (Nidiry 1998).
DISCUSSION
With the goal of developing a new highly active antifungal
therapeutic combination of herbal essential oils and
synthetic antifungal agents, we began by comparing the
antifungal properties of 12 essential oils and some of their

main components against six species of Trichophyton that

commonly cause tinea infections. These filamentous fungi
act as dermatophytes in humans and cattle by digesting
keratin. Tinea is typically treated by topical or systemic
administration of polyene or azole antibiotics, including
ketoconazole, which are efficacious but problematic, as
previously mentioned (Sugar et al. 1987; Von Moltke et al.
1996; Giordani et al. 2001; McMichael 2001). The in vivo
and in vitro anti-Trichophyton activity of herbal essential oils
has focused mainly on T. rubrum and T. mentagrophytes
(Kishore et al. 1993; Wannissorn et al. 1996; Adam et al.
1998; Shahi et al. 1999; Inouye et al. 2001; Cassella et al.
2002; Cimanga et al. 2002; Giamperi et al. 2002; Harris
2002; Patra et al. 2002). However, the efficacy of herbal oils
and their main components against the broad-spectrum
Trichophyton spp., and especially the combined efficacy of
these oils with ketoconazole, have not previously been

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

HERB OILS AGAINST TRICHOPHYTON SPP.

evaluated extensively. We did not compare our MIC and

disc diffusion test data with data of previous reports because
essential oil composition is variable and dependent on many
factors such as plant chemotype, and culture, harvesting and
storage conditions. Therefore, results from unrelated studies
may differ because of these uncontrolled variables rather
than anti-fungal activity (Lis-Balchin et al. 1996; Pandey
et al. 1996; Kaul et al. 1997, 1998; Cosentino et al. 1999;
Rao et al. 2001; Faleiro et al. 2003). Based on our MIC and
disc diffusion test results, we selected P. graveolens oil and
its main components, the relatively stable monoterpene
primary alcohols, citronellol (1720%) and geraniol (589%),
to investigate in combination with ketoconazole. The oils
from C. citrates, L. angustifolia, R. officinalis and T. vulgaris
also exhibited strong inhibition against all the fungi tested in
this study. Alcohols could be clinically practical antifungal
agents because of their stability, especially in different pH
conditions, while the clinical utility of phenolic compounds
such as eugenol or thymol may be restricted in higher pH
conditions.
The synergistic effects of P. graveolens oil in combination
with ketoconazole against Trichophyton spp. had not previously been investigated. Of the Trichophyton species tested,
little variation in susceptibility to the oils alone was
observed. We selected three relatively rare pathogenic
Trichophyton species to investigate the synergistic effects of
the oil and the main oil components and ketoconazole.
However, the FIC results and the FIC indices differed. As
shown in Table 3, the combination of P. graveolens oil and
its components with ketoconazole exhibited especially strong
synergistic inhibition against T. soudanense and T. schoenleinii. In the same experiment, inhibition by the combination
was additive against T. erinacei. We cannot currently explain
the mechanistic reason for these differences, as the antifungal mechanisms of essential oils have not yet been clarified.
However, the inhibitory mechanism may be associated with
the fungal cell wall composition or construction, upon
consideration of the active mechanism of ketoconazole
(Godwin and Michniak 1999; Scholar and Pratt 2000).
In conclusion, we suggest the combination of P. graveolens
oil and ketoconazole for the treatment of Trichophyton
species, especially T. soudanense and T. schoenleinii, which
originated in Africa and have spread to other parts of the
world in recent decades (Nzelibe and Gugnani 1984; Kalter
and Hay 1988; Ruebben and Krause 1996; Lamb and
Rademaker 2001) may reduce the efficacious dose of
ketoconazole and thus minimize the side-effects of ketoconazole. In addition, the therapeutic use of essential oils may
also provide a solution for the rapid development of fungal
resistance and drugdrug interactions that are problematic
with the currently available common antifungal therapeutics. Further in vivo experiments are necessary to assess the
potential for therapeutic application.

1295

ACKNOWLEDGEMENTS
This study was supported by a grant from Korea Science
and Engineering Foundation (KOSEF, no. R04-2002-00000058-0). The authors acknowledge the support.
REFERENCES
Adam, K., Sivropoulou, A., Kokkini, S., Lanaras, T. and Arsenakis,
M. (1998) Antifungal activities of Origanum vulgare subsp. hirtum,
Mentha spicata, Lavandula angustifolia, and Salvia fruticosa essential
oils against human pathogenic fungi. Journal of Agricultural and Food
Chemistry 46, 17391745.
Bidlack, W.R., Omaye, S.T., Meskin, M.S. and Topham, D. (2000)
Phytochemicals as Bioactive Agents, pp. 106110. Lancaster, UK:
Technomic Publishing Company.
Cassella, S., Cassella, J. and Smith, I. (2002) Synergistic antifungal
activity of tea tree (Melaleuca alternifolia) and lavender (Lavandula
angustifolia) essential oil against dermatophyte infection. Internatinal
Journal of Aromatheraphy 12, 215.
Cimanga, K., Apers, S., de Bruyne, T., Van Miert, S., Hermans, N.,
Totte, J., Pieters, L., Vlietinck, A.J. et al. (2002) Chemical
composition and antifungal activity of essential oils of some aromatic
medicinal plants growing in the Democratic Republic of Congo.
Journal of Essential Oil Research 14, 382387.
Cosentino, S., Tuberoso, C.I.G., Pisano, B., Satta, M., Mascia, V.,
Arzedi, E. and Palmas, F. (1999) In-vitro antimicrobial activity and
chemical composition of Sardinian Thymus essential oils. Letters in
Applied Microbiology 29, 130135.
Davidson, P.M. and Parish, M.E. (1989) Methods for testing the
efficacy of food antimicrobials. Food Technology 43, 148155.
Faleiro, M.L., Miguel, M.G., Ladeiro, F., Venancio, F., Tavares, R.,
Brito, J.C., Figueiredo, A.C., Barroso, J.G. et al. (2003) Antimicrobial activity of essential oils isolated from Portuguese endemic
species of Thymus. Letters in Applied Microbiology 29, 130135.
Garg, S.C. and Siddiqui, N. (1992) Antifungal activity of some
essential oil isolates. Pharmazie 47, 467.
Giamperi, L., Fraternale, D. and Ricci, D. (2002) The in vitro action of
essential oils on different organisms. Journal of Essential Oil Research
14, 312318.
Giordani, R., Trebaux, J., Masi, M. and Regli, P. (2001) Enhanced
antifungal activity of ketoconazole by Euphorbia characias latex
against Candida albicans. Journal of Ethnopharmacology 78, 15.
Godwin, D.A. and Michniak, B.B. (1999) Influence of drug lipophilicity on terpenes as transdermal penetration enhancers. Drug
Development and Industrial Pharmacy 25, 905915.
Gundidza, M. (1993) Antimicrobial of essential oil from Schinus molle.
Central African Journal of Medicine 39, 231234.
Hammer, K.A., Carson, C.F. and Riley, T.V. (1998) In-vitro activity of
essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea
tree oil products, against Candida spp. Journal of Antimicrobial
Chemotheraphy 42, 591595.
Hammer, K.A., Carson, C.F. and Riley, T.V. (1999) Antimicrobial
activity of essential oils and other plant extracts. Journal of Applied
Microbiology 86, 985990.
Hammer, K., Carson, C.F. and Riley, T.V. (2000) In vitro activities of
ketoconazole, econazole, miconazole, and Melaleuca alternifolia (tea

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x

1296 S . S H I N A N D S . L I M

tree) oil against Malassezia species. Antimicrobial Agents and

Chemotherapy 44, 467469.
Harris, R. (2002) Progress with superficial mycoses using essential oils.
Internatinal Journal of Aromatheraphy 12, 8391.
Inouye, S., Uchida, K. and Yamaguchi, H. (2001) In-vitro and in-vivo
anti-Trichophyton activity of essential oils by vapor contact. Mycoses
44, 99107.
Kalter, D.C. and Hay, R.J. (1988) Onychomycosis due to Trichophyton
soudanense. Clinical and Experimental Dermatology 13, 221227.
Kaul, P.N., Rao, B.R., Bhattacharya, A.K., Mallavarapu, G.R. and
Ramesh, S.I. (1997) Changes in chemical composition of rose
scented geranium (Pelargonium sp.) oil during storage. Journal of
Essential Oil Research 9, 115117.
Kaul, P.N., Rao, B.R., Bhattacharya, A.K. and Mallavarapu, G.R. (1998)
Aberrations in the composition of the herb oil of rose scented geranium
(Pelargonium species). Journal of Essential Oil Research 10, 439441.
Kishore, N., Mishra, A.K. and Chansouria, J.P.N. (1993) Fungitoxicity of essential oils against dermatophytes. Mycoses 36, 211215.
Lamb, S.R. and Rademaker, M. (2001) Tinea due to Trichophyton
violaceum and Trichophyton soudanense in Hamilton, New Zealand.
The Australasian Journal of Dermatology 42, 260263.
Lis-Balchin, M., Deans, S.G. and Hart, S. (1996) Bioactive geranium
oils from different commercial sources. Journal of Essential Oil
Research 8, 281290.
McMichael, A.J. (2001) Effective management of tinea capitis in the
pediatric population. Journal of New Developments in Clinical
Medicine 19, 93104.
Nidiry, E.S. (1998) Structure-fungitoxicity relationships of the monoterpenoids of the essential oils of peppermint (Mentha piperita) and
scented geranium (Pelargonium graveolens). Journal of Essential Oil
Research 10, 628632.
Nzelibe, F.K. and Gugnani, H.C. (1984) Use of locally available plant
materials for mycological media. Mycosen 27, 519526.
Pandey, M.C., Sharma, J.R. and Dikshit, A. (1996) Antifungal evaluation
of the essential oil of Cymbopogon pendulus (Nees ex Steud.) Wats. cv.
Praman. Flavour and Fragrance Journal 11, 257260.
Patra, M., Shahi, S.K., Midgely, G. and Dikshit, A. (2002) Utilization
of essential oil as natural antifungal against nail-infective fungi.
Flavour and Fragrance Journal 17, 9194.
Rao, B.R., Sastry, K.P., Battattacharya, A. and Kaul, P. (2001) Yield
and chemical composition of rose-scented geranium (Pelargonium

species) oil at different time of harvesting. Journal of Essential Oil

Research 10, 456459.
Ruebben, A. and Krause, H. (1996) Tinea superficialis capitis due to
Trichophyton soudanense in African Immigrants. Mycoses 39, 397398.
Scholar, E.M. and Pratt, W.B. (2000) The Antimicrobial Drugs, pp. 348.
Oxford, UK: Oxford University Press.
Shahi, S.K., Shukla, A.C., Bajaj, A.K., Medgely, G. and Dikshit, A.
(1999) Broad spectrum antimycotic drug for the control of fungal
infection in human beings. Current Science 76, 836839.
Shin, S. (2003) Anti-Aspergillus activities of plant essential oils and
their combination effects with ketoconazole or amphotericin B.
Archives of Pharmacal Researches 26, 389393.
Shin, S. and Kang, C.A. (2001) Studies on composition and antifungal
activities of essential oils from cultivars of Brassica juncea L. Korean
Journal of Pharmacognosy 32, 140144.
Shin, S. and Kang, C.A. (2003) Antifungal activity of the essential oil
of Agastache rugosa Kuntze and its synergism with ketoconazole.
Letters in Applied Microbiology 36, 111115.
Sugar, A.M., Alsip, S.G., Galgiani, J.N., Graybill, J.R., Dismukes,
W.E., Claud, G.A., Craven, P.C. and Stevens, D.A. (1987)
Pharmacology and toxicity of high-dose ketoconazole. Antimicrobial
Agents and Chemotherapy 31, 18741878.
Suresh, B., Sriram, S., Dhanaraj, S.A., Elango, K. and Chinnaswamy,
K. (1997) Anticandidal activity of Santolina chamaecyparissus volatile
oil. Journal of Ethnopharmacology 55, 151159.
Von Moltke, L.L., Greenblatt, D.J., Harmatz, J.S., Duan, S.X.,
Harrel, L.M., Crotreau-Bibbo, M.M., Pritchard, G.A., Wright, C.E.
et al. (1996) Triazolam biotransformation by human liver microsomes in vitro: effects of metabolic inhibitors and clinical conformation of a predicted interaction with ketoconazole. Journal of
Pharmacology and Experimental Therapeutics 276, 370379.
Wannissorn, B., Jarikasem, S. and Soontorntanasart, T. (1996)
Antifungal activity of lemon grass oil and lemon grass oil cream.
Phytotherapy Research 10, 551554.
White, R.L., Burgess, D.S., Manduru, M. and Bosso, J.A. (1996)
Comparison of three diffenent in vitro methods of detecting synergy:
time-kill, checkerboard, and E test. Antimicrobial Agents and
Chemotherapy 40, 19141918.
Yoon, S.Y., Eo, S.K., Lee, D.K. and Han, S.S. (1994) Antimicrobial
activity of Ganoderma lucidum extract alone and in combination with
some antibiotics. Archives of Pharmacal Research 6, 438442.

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 12891296, doi:10.1111/j.1365-2672.2004.02417.x