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Received on 22 September 2007; revised 28 January 2008.
Address for correspondence: Dr. Cristiane de Bona da Silva. Universidade
Federal de Santa Maria, Centro de Ciências da Saúde, Curso de Farmácia,
Departamento de Farmácia Industrial, Av. Roraima, 1000, prédio 26,
Santa Maria/RS. Zip code: 97105-900. Brazil. Phone: + 55 55 3220-
8452; Fax: + 55 55 3220-9371. E-mail: csbona@smail.ufsm.br. This
research was supported by CNPq.
The Brazilian Journal of Infectious Diseases 2008;12(1):63-66.
© 2008 by The Brazilian Journal of Infectious Diseases and Contexto
Publishing. All rights reserved.
Antifungal Activity of the Lemongrass Oil and Citral Against Candida spp.
Cristiane de Bona da Silva
1,2
, Sílvia S. Guterres
1
, Vanessa Weisheimer
1
and Elfrides E.S.Schapoval
1
1
Programa de Pós-Graduação em Ciências Farmacêuticas Pos-Graduation program in Pharmaceuthical Sciences, Pharmaceutical School,
Federal University of Rio Grande do Sul, Porto Alegre, RS;
2
CPharmaceutical School, Department of Pharmaceutical Industry, Federal
University of Santa Maria; Santa Maria, RS, Brazil
Superficial mycoses of the skin are among the most common dermatological infections, and causative organisms
include dermatophytic, yeasts, and non-dermatophytic filamentous fungi. The treatment is limited, for many
reasons, and new drugs are necessary. Numerous essential oils have been tested for both in vitro and in vivo
antifungal activity and some pose much potential as antifungal agents. By using disk diffusion assay, we evaluated
the antifungal activity of lemongrass oil and citral against yeasts of Candida species (Candida albicans, C. glabrata,
C. krusei, C. parapsilosis and C. tropicalis). This study showed that lemongrass oil and citral have a potent in vitro
activity against Candida spp.
Key-Words: lemongrass oil, citral, antifungal activity, Candida spp.
Cutaneous fungal infections are common diseases in
humans, and can also be caused by dermatophytic fungi and
some yeasts. Superficial candidiasis is a common infection of
the skin, oral cavity and esophagus, and vagina, although
most infections occur in patients debilitated or who are
immunocompromised. Candida albicans is responsible for
many of these infections, but occasionally other members
of the genus are associated, and generally infect the skin,
nails, or mucous membranes [1-4]. In vulvovaginal
candidiasis, C. albicans affects 85-95% of women, and C.
glabrata affects 10-20% [5]; C. tropicalis and C. krusei can
also be associated with these infections [5,6]. Moreover, the
specie C. parapsilosis is related to onychomicoses caused
by Candida [6].
The conventional treatment of fungal disease is limited,
and part of the reason is due to the limited spectrum of the
currently antifungal drugs, and the expensive treatment,
particularly due to the need of prolonged therapy. Thus, new
drugs and alternative therapies are necessary, including
natural products.
Essential oils of aromatic plants species are used in
industries for the production of soaps, perfumes and toiletries.
Many of them are also used in traditional medicine for various
purposes. Investigations concerning the evaluation of the
biological activities of essential oils of some medicinal plants
have revealed that some of them exhibited antibacterial,
antifungal and insecticidal properties [7]. Because of the
antimicrobial properties showed by essential oils, the
aromatherapy has been used for treatment of serious skin
diseases, in special, superficial mycoses [8].
Cymbopogon citratus (DC) Stapf (Gramineae) is an herb
worldwide known as lemongrass. The tea made from its leaves
is popularly used in Brazil as antispasmodic, analgesic, anti-
inflammatory, antipyretic, diuretic and sedative [9]. The volatile
oil obtained from fresh leaves of this plant is widely used by
the perfumes and cosmetics industries [10].
Lemongrass oil is characterized for monoterpenes
compounds, and citral is the major component, present at
levels of, approximately, 65-85%. Citral (3,7-dimethyl-2,6-
octadienal) is the name given to a natural mixture of two
isomeric acyclic monoterpene aldehydes: geranial (trans-citral,
citral A) and neral (cis-citral, citral B) (Figure 1). In addition to
citral, the lemongrass oil consists of small quantities of
geraniol, geranylacetate and monoterpene olefins, such as
myrcene [10].
Figure 1. Chemical structure of the citral.
Studies on the antimicrobial, especially antibacterial and
antifungal, activity of lemongrass oil and its components were
reported [11-15]. The antiviral activity of this oil against Herpes
simplex virus-1 was also demonstrated [16].
In this work, in order to estimate the possibility of using
lemongrass oil as an antifungal agent for skin diseases
(cutaneous candidiasis and dermatomycosis), the antifungal
activity of this oil and its main component, citral, against
Candida species was studied.
Material and Methods
Lemongrass Oil and Citral
The lemongrass oil (Cymbopogon citratus) was
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BJID 2008; 12 (February)
commercially obtained (Destilaria Maripá, Brazil) and the citral
(assigned purity of 95%) was supplied by Sigma-Aldrich
(Darmstadt, Germany). The percentage of citral (neral and
geranial) in the lemongrass oil was 76%, assayed by gas
chromatography.
Antifungal Assay
The antifungal activity of the lemongrass oil and citral
against yeasts was determined using the disk diffusion method
[13]. Eight strains of Candida species were used: Candida
albicans ATCC 10231, Candida albicans ATCC 18804,
Candida albicans CI-I (clinical isolate), Candida albicans
CI-II, Candida krusei ATCC 6258, Candida glabrata ATCC
2001, Candida tropicalis ATCC 750 and Candida parapsilosis
ATCC 22019. The cultures of Candida spp. were cultivated
on Sabouraud dextrose 4% agar (SDA) at 25ºC for 48 hours.
Suspensions of yeasts were prepared in saline solution of
25% ± 2% turbidity, obtained at 580 nm, using a suitable
spectrophotometer (Analyser Model 800, São Paulo, Brazil).
Seeded agar plates were prepared by pouring 20 mL of SDA
into each sterile plate. After solidification of medium, each
plate was overlaid with 5 mL of SDA, which was previously
inoculated with 1% (v/v) of the suspensions of yeasts.
Lemongrass oil and citral were applied on filter paper disks
(2.0, 4.0 and 8.0 μL/disk) of 6 mm in diameter. Nystatin solution
(0.3 mg/mL) was used as antifungal reference substance (20.0
μL/disk). These disks were placed on the surface of seeded
agar plates (one disk for plate). All plates were incubated at
35ºC ± 2ºC for 24 hours. The inhibition zone was determined
by measuring the diameter of the clear zone around each disk
with a paquimeter (Starret®, Itu, Brazil). Six disks were used
for each sample.
Results
In this work, the antifungal activity against other Candida
species was also studied, and the results obtained showed the
important antifungal activity of lemongrass oil and citral. Candida
albicans is an important pathogen of human infections;
moreover, other species can be associated with some infections.
Different yeasts were tested and the antifungal activity of
lemongrass oil and its major component (citral) was evaluated
against Candida spp. Results from the antifungal activity are
summarized in Tables 1 and 2. They show that the lemongrass
oil and the citral exhibited action against all Candida spp.,
especially C. albicans species, with a partial inhibition when
a volume of 8.0 μL of each sample was employed (diameter
inhibition zone higher than 40 mm). On the other hand, C.
krusei was the more resistant specie, showing a diameter
inhibition zone minor than 20 mm using 8.0 μL of each sample.
In the present work, the citral concentration in lemongrass oil
Antifungal Activity of Lemongrass and Citral
Table 2. Diameter of inhibition zone of the citral against some Candida species (n = 6)
Table 1. Diameter of inhibition zone of the lemongrass oil against some Candida species (n = 6)
Test strain
Diameter of inhibition zone (mm)
Lemongrass oil (
μμ
μμ
μL) Nystatin solution
(0.3 mg/mL, 20.0
μμ
μμ
μL)
2.0 4.0 8.0
C. albicans ATCC 10231 18.6 27.4 > 40 18.8
C. albicans ATCC 18804 13.3 22.5 > 35 18.8
C. albicans CI-I 12.6 27.3 > 40 17.8
C. albicans CI-II 14.4 27.3 > 50 17.9
C. glabrata ATCC 2001 19.3 25.1 > 30 17.3
C. krusei ATCC 6258 12.3 14.4 19.6 15.4
C. parapsilosis ATCC 22019 8.9 18.3 28.6 13.5
C. tropicalis ATCC 750 12.8 19.2 29.5 15.6
Test strain
Diameter of inhibition zone (mm)
Reference citral (
μμ
μμ
μL) Nystatin solution
(0.3 mg/mL, 20.0
μμ
μμ
μL)
2.0 4.0 8.0
C. albicans ATCC 10231 14.6 27.8 > 40 18.6
C. albicans ATCC 18804 12.5 20.6 > 35 18.8
C. albicans CI-I 14.8 20.3 > 40 17.8
C. albicans CI-II 14.5 26.1 > 35 18.5
C. glabrata ATCC 2001 18.8 22.5 > 30 17.3
C. krusei ATCC 6258 12.3 14.3 19.7 15.1
C. parapsilosis ATCC 22019 9.0 18.1 27.5 14.0
C. tropicalis ATCC 750 12.4 22.2 32.6 18.1
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65
was 76%, and the citral quantity expected in 2, 4 and 8 μL of
this oil was, approximately, 1.35, 2.70 and 5.41 mg, respectively.
Discussion
Numerous essential oils have been tested for in vivo and
in vitro antimycotic activity and some demonstrated to be
potential antifungal agents. Their mechanism of action appears
to be predominantly on the fungal cell membrane, disrupting
its structure causing leakage and cell death; blocking the
membrane synthesis; inhibition of the spore germination,
fungal proliferation and cellular respiration [8]. Because of
high volatility and lipophilicity of the essential oils, they are
readily attached to penetrate into the cell membrane to exert
their biological effect [17].
The antifungal activity of lemongrass oil has been tested
against some species that induced human infections, including
dermatophyte species and C. albicans [11,12,18,19]. Onawunmi
(1989) reported that the MIC value obtained for citral against
C. albicans was 0.05% (v/v), and this value was similar to
those obtained in studies with lemongrass oil [11]. In addition,
the lemongrass oil (100 μg/mL) and citral (25-200 μg/mL)
inhibited the mycelial growth of C. albicans, suggesting the
potential value of lemongrass oil for the treatment of cutaneous
candidiasis [19]. Additionally, pre-clinical studies were
conducted with ointments containing essential oils, including
lemongrass oil. The animals were infected with dermatophyte
fungi (T. rubrum and M. gypseum), and the results showed
the efficacy of this preparation [20].
The susceptibility of Candida spp. to antifungal agents
is not uniform, and several resistance mechanisms have been
related (changes in the cell wall or plasma membrane,
mutations, sequestration of the antifungal in organelle-like
vacuoles, or chromosomal changes). Candida krusei is
resistant to some triazoles [3], and these observations can
explain the minor efficacy showed by lemongrass oil and citral.
Nystatin is used as a reference substance, and in comparison
with that drug, the samples of the oil and citral showed higher
activity. In addition, using volumes of 4.0 and 8.0 μL of the
lemongrass oil and the reference citral it was observed
asymmetric diameter inhibition zones and lower growing around
these inhibition zones. These observations can be explained
by the evaporation of the essential oil and by different vapor
concentration into the agar layer. In a previous work carried out
in our laboratory, a total inhibition of the growing yeast was
observed using 20.0 μL of the lemongrass oil [13].
The antifungal activity presented by lemongrass oil and
citral were similar, and corroborates with literature, which
indicated significant association between the effect and the
presence of citral in lemongrass oil [11,12,21]. Literature points
that citral acts as a fungicidal agent because it is able to form
a charge transfer complex with an electron donor of fungal
cells, resulting in fungal death [22].
These results demonstrated the potentiality of using the
lemongrass oil instead of the citral in pharmaceuticals
preparations, because of its similar antifungal activity, the
minor cost and toxicity. Citral is a widely used fragrance and
flavor material with a strong lemon-like odor. It is reported to
be sensitizer and irritant [23,24]. Opdyke (1976) reported that
three aldehydes (cinnamic aldehyde, phenylacetaldehyde and
citral) were shown to be sensitizers in a human maximization
test; however, essential oils that contain significant amounts
of each material did not induce sensitization [25].
It was observed very good effectiveness and broad-
spectrum activity against Candida species. In our researches,
topic formulations containing this oil were developed and
evaluated [26], and subsequent clinical studies are necessary.
Besides, in vivo and in vitro studies have been conducted to
assess skin irritation.
Acknowledgements
The authors are grateful to Professor Maria L. Scroferneker
of the Instituto de Ciências Básicas da Saúde/UFRGS for
providing the species. This research was supported by CNPq.
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