Revista Brasileira de Farmacognosia

Brazilian Journal of Pharmacognosy

20(5): 734-741, Out./Nov. 2010 Received 20 Oct 2009; Accepted 4 Dec 2009; Available online 24 Sep 2010.

In vitro antifungal activity and cytotoxic effect of essential oils and

extracts of medicinal and aromatic plants against Candida krusei and
Article

Aspergillus fumigatus

Julieth Correa-Royero,1,3 Verónica Tangarife,1,3 Camilo Durán, 2,3 Elena Stashenko,2,3

Ana Mesa-Arango*,1,3

1
Department of Microbiology and Parasitology, School of Medicine Carrera 51D Nº 62-29, University of Antioquia,
Medellin, Colombia,
2
Research Centre for Biomolecules, CIBIMOL, Industrial University of Santander, Bucaramanga, Colombia,
3
Research Centre of Excellence, Cra. 27 Calle 9 Campus UIS, Edificio C 45, Complejo Piloto Agroindustrial
CENIVAM, Bucaramanga, Colombia.

RESUMO: “Atividade antifúngica in vitro e os efeitos citotóxicos de óleos essenciais

e extratos de plantas medicinais e aromáticas contra Candida krusei e Aspergillus
fumigatus” As plantas são geralmente utilizadas na medicina tradicional como agentes
antimicrobianos e seus óleos essenciais e extratos foram conhecidos por possuir atividade
antifúngica. O objetivo deste estudo foi avaliar in vitro a atividade de 32 óleos essenciais
e 29 extratos contra Candida krusei e Aspergillus fumigatus, bem como o efeito citotóxico
em células Vero. A curva do tempo-morte e a interação entre antifúngicos e Chenopodium
ambrosioidese do extrato de Myrcia cucullata mostraram atividade antifúngica contra C.
krusei (geometric means of the minimal inhibitory concentration [GM-MIC] 7,82 e 31,25 µg/
mL, respectivamente). Lippia citriodora foi ativa contra C. krusei e A. fumigatus (GM-CIM =
99,21 µg/mL e 62,5 µg/mL, respectivamente). Os testes de tempo-morte feitos com óleo de C.
ambrosioides mostraram atividade fungicida em 4x MIC. A interação do óleo C. ambrosioides
com itraconazol e anfotericina B foi testada pela técnica de xadrez. Nenhuma interação
foi detectada pela combinação do óleo C. ambrosioides com anfotericina B e itraconazol
(intervalo fractional inhibitory index [FICI] = 1,03-1,06 e 1,03-1,00, respectivamente). Os
ensaios de citotoxicidade para todas as amostras foram realizadas com MTT. Apenas os óleos
Hedyosmun sp. e L. dulcis foram citotóxicos.

Unitermos: Candida krusei, Aspergillus fumigatus, óleos essenciais, extratos, curva do tempo-
morte, técnica de xadrez.

ABSTRACT: The plants are usually used in traditional medicine as antimicrobial agents and
their essential oils and extracts have been known to possess antifungal activity. The aim of this
study was to evaluate the activity of 32 essential oils and 29 extracts in vitro against C. krusei
and A. fumigatus as well as the cytotoxic effect on Vero cells. Time-kill curve and interaction
between antifungal and the most active sample against C. krusei, was also evaluated. The oils
from C. ambrosioides and the extract of M. cucullata showed antifungal activity against C. krusei
(GM-MIC 7.82 and 31.25 µg/mL, respectively). L. citriodora was active against C. krusei and A.
fumigates (GM-MIC = 99.21 µg/mL and 62.5 µg/mL respectively). Time-kill assays done with
C. ambrosioides oil showed fungicidal activity at 4x MIC. The interaction of C. ambrosioides
oil with itraconazole and amphotericin B was tested following the chequerboard technique.
No interaction was detected for the combination of C. ambrosioides oil with amphotericin B
and itraconazole (FICI range = 1.03-1.06 and 1.03-1.00, respectively). Cytotoxicity assays for
all samples were carried out with MTT. Only the oil from Hedyosmun sp. and L. dulcis were
cytotoxic.

Keywords: Candida krusei, Aspergillus fumigatus, essential oil, extract, checkerboard, time-kill
curve.

734 *E-mail: [email protected], Tel. +57 4 219 60 59, Fax: +57 4 219 60 66. ISSN 0102-695X
DOI: 10.1590/S0102-695X2010005000021
In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei

INTRODUCTION MATERIALS AND METHODS

Candida spp. and Aspergillus spp. are responsible Plant material

for the majority (80 to 90%) of fungal infections (Espinel-
Ingroff et al., 2005). Candida species produce infections Stems, leaves, flowers and roots of 57 medicinal
ranging from non-life-threatening mucocutaneous and aromatic plants were collected in different regions of
illnesses to invasive processes that may involve any organ. Colombia as part of a survey conducted by CENIVAM,
C. albicans is the predominant species causing infection; a Research Centre devoted the study of aromatic plants
however infections due to other species such as C. and medicinal in Colombia. The taxonomic identification
krusei, associated with intrinsic resistance to fluconazole, of the botanical samples was performed by Dr. Jose Luis
decreased susceptibility to flucytosine and amphotericin Fernandez at the National Herbarium from Colombia,
B, and resistance to echinocandins, are on the increase Institute of Natural Sciences, Faculty of Sciences,
(Capoor et al., 2005, Cantón et al., 2008, Pfaller et al., Universidad Nacional de Colombia, Bogota, where
2008). Additionally, C. krusei has the ability to adhere to voucher specimens were deposited.
a host surface or a prosthesis leading to the formation of The plants selected belong to the following
biofilms which further facilitate adhesion and infection genera: Salvia (S. aratocensis subsp. suratensis 516326,
(Parahitiyawa et al., 2006). S. rubriflora Kunth 521064, S. amethystina J.E. Smith
Aspergillosis encompasses a broad spectrum of 521071, S. bogotensis Benth 521063, S. rubriflora Epling
diseases caused by members of the genus Aspergillus, 521107, S. melaleuca subsp. totensis 521038, S. melaleuca
the most frequently responsible species are A. flavus subsp. melaleuca 521076, S. bogotensis 521074), Lippia
and A. fumigatus. The last specie is one of the major (L. citriodora 484334, L. dulcis 512079, L. origanoides
causes of contamination of intrahospital environments, 512075, L. citriodora 480749, L. schlimii Turcz 521078),
infecting patients by inhalation, direct inoculation through Lepechinia (L. salviifolia Kunth subsp. salviifolia
surgical tools, mechanical ventilation, air conditioners 521070, L. conferta 521068), Hyptis (H. suaveolens
or filters (Haiduven, 2008). Additionally, the emergence (L) Poit 512267, H. perbullata 521095), Ocimum (O.
of resistance of this fungus to itraconazole and elevated campechianum 512263, O. tenuiflorum L. 516925),
minimum inhibitory concentrations (MIC) to voriconazole, Croton (Croton spp. 519596, C. leptostachyus 519598),
posaconazole, and the experimental azole, ravuconazole Lantana (L. camara 520293, L. fucata Lindl. 521031).
has appeared. A. fumigatus has also displayed variable Additionally, the following plants were also selected,
susceptibility to amphotericin B (Snelders et al., 2008, Swinglea glutinosa 521530, Pimienta racemosa 512227,
Qiao et al., 2008). Siparum conica 519971, Nectandra acutifolia 520339,
The development of fungal drug resistance, Calycolpus moritzianus 517006, Artemisia vulgaris L.
drug-related toxicity, significant drug interactions and 517002, Turnera aff. diffusa Willd. ex Schult. 516293,
insufficient bioavailability of the conventional antifungal Rosmarinus officinalis 531011, Hedyosmum scaberrium
drugs, has encouraged the search for new alternatives Standl. 517321, Satureja brownei (Sw.) Briqn 519796,
among natural products (Cavaleiro et al., 2006). Plants Aloysia tripilla 517189, Chenopodium ambrosioides L.
are usually used in traditional medicine as antimicrobial 519592, Morella parvifolia (Benth) 517726, Schinus molle
agents and their essential oils and extracts have been L. 517747, Calyptranthes sp. 517736, Bursera graveolens
known to possess antibacterial and antifungal proprieties (Kunth) Triana & Planch. 517753, Cymbopogon citratus
(Cowan, 1999; Tempone et al., 2008). Previous reports 531013, Aristolochia anguicida 512207, Morinda royoc
have suggested that several essential oils and extracts show L. 512222, Eugenia cf. uniflora 512226, Stachytarpheta
important antifungal activity against yeasts, dermatophytes mutabilis (Jacq) Vahl 512281, Calea glomerata 512231,
and Aspergillus isolates, and have therapeutic potential, Myrcia cucullata 512228, Tagetes lucida 512074,
mainly against fungal diseases involving mucosal, Hedyosmum racemosum 517005, Psychotria poeppigiana
cutaneous and respiratory tract infections (Cavaleiro et al., müll Arg. 512218, Phyllanthus acuminatus 512239, Eriope
2006; Tempone et al., 2008; Tavares et al., 2008). crassipes Benth 512260, Guatteria metensis 512205,
The aim of this study was to evaluate the activity Aristolochia ringens 512238, Coccocypselum hirsutum
against C. krusei and A. fumigatus, in vitro, as well as the Bartl 519995, Alternanthera lanceolata 519981, Baccharis
cytotoxic effect of essential oils and extracts of Colombian prunifolia 521077, and Sigesbeckia agrestis 521062.
plants. Additionally, the combined effects of itraconazole
and amphotericin B, and the pharmacodynamics with Extracts and essential oils extraction
the most active samples were evaluated against C. krusei
by the chequerboard method and the time-kill curves, Essential oils (32 oils) and extracts (29) were
respectively evaluated. The essential oils were extracted from dried
stems, leaves, flowers and roots (300 g) by microwave-
assisted hydrodistillation (30 min, 250 mL water), using

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 Julieth Correa-Royero, Verónica Tangarife, Camilo Durán, Elena Stashenko, Ana Mesa-Arango

a Clevenger-type distillation apparatus and a Dean-Stark Time-kill assay

distillation trap in a domestic microwave oven (Kendo
MO-124, 2.5 GHz, 800 W), as described (Stashenko et The in vitro pharmacodynamics of C.
al., 2004). The extracts were obtained from 40 g of dried ambrosioides L. oil with C. krusei ATCC 6258 was
leaves of each plant, macerated with 200 mL ethanol and performed as described by Klepser et al. (1998). One
left in suspension for seven days at 28 °C. The mixture hundred microliters of an initial inoculum ranging from
was filtered and concentrated using a Buchi rotavapor. 1-5 x 105 CFU/mL was seeded onto flat-bottomed 96-
Stock solutions of 20 mg/mL for oils and 40 mg/mL well microtitration plates and 100 µL of each sample, at
for the extracts, were prepared in DMSO for subsequent concentrations of 0.5, 1, 2 and 4x MIC, were added. The
bioassays. samples were incubated at 35 °C with agitation. At 0, 4, 8,
12, and 24 h, 10 µL were removed from each test solution
Antifungal activity assays for three serial dilutions (1:10). Volumes of 10 µL were
then spread onto potato dextrose agar and incubated at
The antifungal activity of the oils and extracts 37 °C for 24 h to determine the number of CFU/mL. The
was evaluated following the Clinical and Laboratory limit of detection was 100 CFU/mL. Time-kill curves with
Standards Institute M38-A (CLSI M38-A, 2002) protocol itraconazole and amphotericin B served as fungistatic and
for filamentous fungi, and the standard method proposed by fungicide controls, respectively. Experiments were carried
the Antifungal Susceptibility Testing Subcommittee of the out in duplicate in two separate experiments. Time-kill
European Committee on Antibiotic Susceptibility Testing curves were constructed by plotting of mean±standard
(AFST-EUCAST) for yeasts (Cuenca-Estrella et al., 2003). deviation (SD) of colony count (log10 CFU/mL) as a
C. krusei (ATCC 6258) and A. fumigatus (ATCC 204305) function of time (hours) with the statistical package Prisma
were used to evaluate antifungal activity. Briefly, duplicate (GraphPad Software, Inc., USA, 2007). Fungicidal activity
100 µL samples of five serial dilutions of the essential oils was defined as ≥3 - log10 (≥99.9%) reduction in CFU/mL
and extracts were dispensed into 96-well microtitration from the starting inoculum (0.5- 2.5 x 105 CFU/mL).
plates (Becton Dickinson, New Jersey, USA) at final
concentrations between 31.25-500 µg/mL. Amphotericin Cytotoxicity assay
B and itraconazole (Sigma-Aldrich, Co, MO, USA) were
used as positive controls at final concentrations of 0.031- Cercopithecus aethiops African green monkey
16 µg/mL. Tween 80 was included at a final concentration kidney cells (Vero cell line ATCC CCL-81) were used.
of 0.001% (v/v) to enhance oil solubility. One hundred The cells were grown in Eagle's minimum essential
microlitres of the fungal inoculum of 1-5 x 105 CFU/mL medium (MEM) supplemented with 10% of fetal bovine
and 0.8 x 104-1x105 CFU/mL for yeast and filamentous serum (FBS), 100 units/mL of penicillin, 100 mg/mL of
fungi, respectively, were added. For the AFST-EUCAST streptomycin, 20 mg/mL of L-glutamine, 0.14% NaHCO3,
method, the MIC was determined after 24 h of incubation and 1% each of nonessential amino acids and a vitamin
at 35 °C and defined as the lowest concentration that solution. The cytotoxicity of the essential oils and
resulted in 90% reduction of growth. For the CLSI M38-A their components was examined in vitro using an MTT
method, the MIC were determined after 48 h of incubation (dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
at 35 °C, and defined as the lowest essential oil and extract (Sigma, New Jersey, USA) assay as described (Betancur-
dilution that resulted in total inhibition of visible growth. Galvis et al., 2002). Briefly, cells were plated at 1.4 x 105
Essential oils and extracts were considered active when cells/mL in 96-well flat-bottomed plates, and incubated at
they presented MIC values ≤500 µg/mL. The MIC values 37 ºC in a humidified incubator with 5% CO2. After 24
were expressed as geometric means (GM-MIC) of tests h of incubation, each diluted oil or extract was added to
performed in duplicate in the three different assays against the appropriate wells, and the plates were incubated for
each of the fungi species. an additional 48 h at 37 ºC. Supernatants were removed
from the wells, and 28 μL (2 mg/mL) of an MTT solution
Interaction of essential oils and drugs in MEM, supplemented with 10% FBS, were added to
each well. Plates were incubated for 1.5 h at 37 ºC, and
Assays were performed in flat-bottomed 96-well then 130 μL of DMSO were then added to dissolve the
microtitration plates using the chequerboard method (Vitale purple formazan crystals that were produced. The plates
et al. 2005). C. krusei was used at a final concentration were placed on a shaker for 25 min, and absorbance was
of 0.5-2.5 x 105 CFU/mL. The final concentrations of the read at 550 nm on a multiwell spectrophotometer (Titertek
itraconazole and amphotericin B ranged from 0.004 to 2 μg/ Uniskan). The minimal dilution of the essential oils that
mL and the essential oil of C. ambrosioides L.from 0.488 induced 50% growth inhibition of the cells was expressed
to 31.25 μg/mL. The fractional inhibitory index (FICI) was as IC50. The IC50 values for each compound were obtained
calculated and the interpretation was determined as follow: by linear regression analysis of the dose-response curves
≤0.5 Synergistic effect; >0.5 but <4, no interaction; and ≥4 generated from the absorbance data with the statistical
antagonistic effect (Vitale et al. 2005).
Rev. Bras. Farmacogn.
736 Braz. J. Pharmacogn.
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In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei

package R (Development Core Team, Vienna, Austria, the classification of biological activity used by Holetz et
2008). IC50 values were expressed as the Mean±SD of al. (2002), the extract of M. cucullata (GM-MIC = 31.25
two independent experiments done in quadruplicate. μg/mL) and the oil of C. ambrosioides L. (GM-MIC =
7.82 μg/mL) displayed strong activity against C. krusei.
RESULTS AND DISCUSSION The activity of both samples was similar or higher than
fluconazole when it was evaluated with ten clinical isolate
MIC of 23 active samples (GM≤500 µg/mL), of C. krusei (GM-MIC = 32 and 64 μg/mL) (Espinel-
tested against C. krusei and A. fumigatus and the respective Ingroff et al., 2005). Additionally, MIC value obtained
IC50 values are presented in Table 1. Six essential oils, and with C. ambrosioides oil was lower than the reported
three extracts showed activity only against C. krusei (GM- by Vasquez et al. (2000) for Melaleuca alternifolia oil
MIC range 7.82 to 500 µg/mL), whereas four essential oils (tea tree oil) with an isolate of C. krusei (8-16 μg/mL).
and two extracts were active only against A. fumigatus M. alternifolia oral solution has been used as an natural
(GM-MIC range 125 to 500 µg/mL). Seven oils and one topical antiseptic, furthermore it has showed efficacy for
extract showed a major spectrum of activity because they treatment of fluconazole-resistant oropharyngeal Candida
were active against both strains. There is no agreement on infections in AIDS patients (Jandourek et al., 1998) and for
the level of acceptance for plant products when compared treatment of vaginal candidiasis (Hammer et al., 1998)
with standards; therefore, some authors consider only In Colombia, the leaves, roots and flowers of C.
activity comparable to antibiotics, while others consider ambrosioides have been approved as anthelmintic and
even higher values (Hennebelle et al., 2008). According to antidiarrhea (Fonnegra & Jimenez, 2007). In the present

Table 1. Geometric Means of Minimal Inhibitory Concentration (GM-MIC) and Inhibitory Concentration 50 (IC50) of essential oils
and extracts with antifungal activity.
GM-MIC (µg/mL) IC50±SD (µg/mL)
Samples
C. krusei A. fumigatus Vero Cell line R2
T. lucida (512074)* >500 250 ≥200 NA
P. racemosa (512227) * >500 250 ≥200 NA
N. acutifolia (520339)* 250 500 57.5±9.3 0.88
L. citriodora (484334) * 222.7 99.21 37.9±4.3 0.89
L. dulcis (512079) * >500 125 ≤25 NA
L. origanoides (512075) * 250 125 60.4±11.2 0.83
L. citriodora (480749) * 99.21 62.5 116.9±6.6 0.97
R. officinalis (531011) * 500 >500 53.6±11.4 0.80
H. racemosum-fruits (517005) * 396.85 >500 32.0±5.6 0.76
Hedyosmun sp. (517005) * 396.85 250 28.1±1.6 0.97
T. aff. diffusa Willd. Ex Schult. (516293)* 99.21 500 52.2±5.2 0.93
Hedyosmum scaberrium Standl. (517321)* >500 500 ≤25 NA
A. triphylla (517189)* 250 99,21 ≥200 NA
C. ambrosioides L. (519592)* 7.82 >500 ≥200 NA
B. graveolens (Kunth) Triana & Planch. (517753)* 500 >500 28.0±4.8 1.00
T. lucida (512074)* 500 >500 31.9±6.4 0.70
C. citratus (531013)* 314.98 >500 ≥200 NA
M. royoc L. (512222)** 250 250 ≥200 NA
E. cf. uniflora (512226) ** 250 >500 117.4±11.9 0.92
M. cucullata (512228)** 31.25 >500 ≥200 NA
S. amethystina J.E. Smith (521071) ** >500 500 ≥200 NA
L. fucata Lindl. (521031)** 500 >500 ≤25 NA
H. perbullata (521095) ** >500 250 ≥200 NA
Amphotericin B 0.630 1.260 - -
Itraconazole 0.125 0.157 - -
*Essential oil; ** Extract; R : linear regression coefficient; NA, Not applicable; The GM-MIC of actives essential oil and extract are indicated
2

in bold.

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 Julieth Correa-Royero, Verónica Tangarife, Camilo Durán, Elena Stashenko, Ana Mesa-Arango

study, the essential oil of one plant of the same species, result from the action of these terpenes. The only extract
was not active against A. fumigatus (GM-MIC>500 μg/ active against both strains (GM-MIC of 250 µg/mL) was
mL). However, the oil of the same plant evaluated with that of M. royoc L. Extracts from other species such as M.
the poisoned food technique completely inhibited mycelial elliptica and M. angustifolia displayed different biological
growth of this filamentous fungi at 100 µg/mL (Kumar et activities, including antifungal (Xiang et al., 2008; Ali et
al., 2007). This difference in antifungal activity between al., 2000). Nevertheless, to our knowledge, the antifungal
essential oils from plants of the same species can be explained activity of M. royoc L has not been reported.
by the chemotype (chemical composition), geographical The criteria of cytotoxic activity for the crude
region of plant collection, age of plant, extraction method extracts, as established by the American National Cancer
of oils, method used to assess the antifungal activity or Institute (USA), is an IC50<30 µg/mL (Suffness & Pezzuto,
by the intra or inter species differences (Samaranayake 1990). According to this criterion, the essential oils of
et al., 1995; Cruz et al., 2007; Maksimovic et al., 2008). Hedyosmun spp. and L. dulcis, were the only cytotoxic
Sharma & Tripathi (2008) have shown that the antifungal oils. This finding is very important given the need to find
activity of essential oils is better evaluated in liquid than in new antimycotic compounds with low-toxic effects.
solid media, since in the latter, the hydrophobic nature of To obtain oils and extracts active against C.
the majority of essential oil components hamper uniform krusei and A. fumigatus is important because C. krusei
diffusion of these substances through the agar medium (de has been recognized as a potentially multidrug-resistant
Cerqueira et al., 2007). We used the standard microdilution pathogen and A. fumigatus is the main cause of invasive
methods M38-A and AFST-EUCAST for the evaluation of aspergillosis in immunocompromised patients; moreover,
antifungal susceptibility of filamentous fungi and yeasts, only a limited number of drugs are available (Brock
respectively. These methods make it possible to reduce et al., 2008, Pfaller et al., 2008). Additionally, both C.
the amount of oil used, to simultaneously evaluate several krusei and A. fumigatus, have the ability to form biofilms
samples, and reproducibility in the results. in biomedical devices that come in contact with skin,
Chemical analyses of the oil of C. ambrosioides mucosal or inert surfaces (Quindós et al, 2009; Seidler et
were previously carried out in our laboratory. The main al., 2008). Previous studies have reported the reduction
component of the oil is the terpene ascaridole (31.0%) of C. albicans biofilms by essential oils (Agarwal et
(unpublished data). The antifungal activity of this terpene al., 2008). Therefore, based on this information and our
has been previously demonstrated against Aspergillus results, a possible application of these natural products
flavus, A. glaucus, A. ochraceous, Colletotrichum could reside in the control of biofilm formation.
gloeosporioides, A. niger, C. musae and Fusarium The time-kill dynamic process is used for the
oxysporum (Jardim et al., 2008). It is possible that anti-C. evaluation of new antimicrobial agents. This makes
krusei activity obtained in this study could be explained by it possible to determine if the agent has a fungistatic or
a higher concentration of ascaridole. fungicidal effect, and if the killing process is concentration
H. perbullata is the extract that showed the best or time-dependent (Pfaller et al., 2004). Analysis of the
activity against A. fumigatus (GM-MIC = 250 µg/mL). time-kill curves of oil from C. ambrosioides L. with C.
This species is native of the Chicamocha canyon (Boyacá krusei did not have a significant effect at 0.5 x and 1 x MIC,
and Santander, Colombia), and is known as “Mastranto whereas a fungicidal effect (CFU /mL >3 log units) was
azul del Chicamocha” (Fernandez-Alonso, 2006). This is detected at 4 x MIC, after 4 h (Figure 1a). This behavior
an aromatic plant whose antimicrobial activity as extract or was comparable to the effect of amphotericin B (Figure
essential oil has not been reported. However, essential oils 1b). Fungicidal activity is clinically more important than
of other species such as H. suaveolens and H. ovalifolia fungistatic activity, particularly in HIV patients, because
have antibacterial, anti-C. albicans and anti-dermatophyte prophylactic use of fungistatic drugs has been associated
activity (Asekun et al., 1999; Souza et al., 2003). Extracts with an increased frequency of innate or acquired drug
(1) and oils (7) were active against both C. krusei and A. resistance in clinical isolates (Monk & Goffeau, 2008).
fumigatus; L citriodora was the most active (GM-MIC At 1 x MIC there was a reduction in growth of UFC/mL
= 99.21 and 62.5 µg/mL, respectively). The antifungal until approximately 4 h, with renewed growth after 8 h of
activity of essential oils of various species of the Lippia incubation. At 24 h the colony values were close to those of
genus have been reported but not of L. citriodora (Oliveira the growth controls. This analysis was made in comparison
et al., 2007; Hennebelle et al., 2008). Chemical analyses of with the time-kill curve obtained with amphotericin B and
oil from C. ambrosioides have also been carried out in our itraconazole (Figure 1b and Figure 1c).
laboratory: the main components were geranial (18.9%) Potential synergy of essential oils with antibiotics
and neral (15.6%) (unpublished data). Both compounds has previously been considered with the aim of increasing
were also the major compounds of the essential oil of the rate of fungal killing, shorten the duration of therapy,
Thymus pannonicus (41.42 and 29.61%, respectively) to avoid the emergence of drug resistance, expand the spectrum
show anti-C. albicans activity (Maksimovic et al., 2008); of activity, and decrease drug-related toxicity by allowing
consequently, the anti-mycotic activity of L. citriodora can lower doses of antifungal agents to be administered (Shin

Rev. Bras. Farmacogn.

738 Braz. J. Pharmacogn.
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In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei

& Lim, 2004). Unfortunately, in this study an indifferent more hydrophobic than other species. This physicochemical
effect was found when oil of C. ambrosioides L. was characteristic could at least partly explain the susceptibility
combined with itraconazole or amphotericin B (FICI range of this yeast to the oils due to the hydrophobic nature of
= 1.03-1.06 and 1.03-1.00, respectively). the majority of the components of the essential oil (de
The studies carried out in our laboratory have Cerqueira et al., 2007).
shown that C. krusei is more susceptible to essential In conclusion, the essential oil of L. citriodora
oils than C. parapsilosis (Mesa-Arango et al., 2007). was the sample with the broadest spectrum of activity.
Samaranayake et al. (1995) demonstrated that C. krusei is Moreover the essential oil of C. ambrosioides and

(a) (b)
10 10
Log 10 UFC/mL

8 8

Log 10 UFC/mL
6 6

4 4

2 2

0 0
0 6 12 18 24 0 4 8 12 16 20 24
Time (hours) Time (hours)

(c)
10
Growth Control
8 0.5 MIC
Log 10 UFC/mL

1 MIC
6 2 MIC
4 MIC
4

0
0 6 12 18 24
Time (hours)

Figure 1. Time-kill plots of (a) C. ambrosioides L. oil, (b) amphotericin B and (c) itraconazole against C. krusei ATCC 6258.
● Growth control; ■, 0.5 x MIC; ▲, 1 x MIC; ▼, 2 x MIC; ♦, 4 x MIC.

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