Anti-Candida Activity of Essential Oils from Eucalyptus species. A Preliminary Study
Janaina Priscila Barbosa*, Thaís Rossini de Oliveira, Daniele de Godoy Penteado Bragado Puppin, Anderson Laerte Teixeira, Giovana Cláudia Boni, Simone Nataly Busato de Feiria and José Francisco Höfling
Department of Oral Diagnosis, State University of Campinas, Brazil
Submission: January 05, 2017; Published: April 19, 2018
*Corresponding author: Janaina Priscila Barbosa, Department of Oral Diagnosis, University of Campinas, Brazil, Tel/Fax: +55(19)2106-5322;
Email: [email protected]
How to cite this article: Janaina Priscila Barbosa, Thaís Rossini de Oliveira, Daniele de Godoy Penteado Bragado Puppin, Anderson Laerte Teixeira, et al. Anti-
Candida Activity of Essential Oils from Eucalyptus species. A Preliminary Study. Adv Dent & Oral Health. 2018; 8(2): 555740. DOI: 10.19080/ADOH.2018.08.555740
Members of the genus Eucalyptus belonging to the Myrtaceae family originate in Australia but have been naturalized on most continents. They are popularly used as an anesthetic, expectorant, anti-inflammatory and antiseptic, because they present antibacterial, antifungal, anti-inflammatory and analgesic properties through their essential oils. Faced with the increase resistance of microorganisms to antimicrobials has increased the studies of medicinal plants as an alternative agent for treatments and prevention of infections. Candidiasis is an infection caused by Candida spp. Some strains are already reported as resistant to traditional therapies. Therefore, the present study evaluated the antifungal activity of two species of Eucalyptus spp. through the microdilution method in broth determining the MIC (Minimum Inhibitory Concentration) and determining the CFM (Minimum Fungicidal Concentration). The essential oils of Eucalyptus citriodora and Eucalyptus globulus are biologically active against Candida spp. tested, exhibiting promising antifungal properties and can be used in combination with other traditional antimitotics.
The use of medicinal plants is an age-old practice where man sought use natural resources to improve their health. The beginning of the use of plants as a resource for the treatment of diseases was empirical, with a view that plants could be an alternative against the cure of diseases .
This practice has grown significantly throughout the world in recent years. Some factors favored the increase of its use, such as the option of natural therapies and a greater interest as an alternative of anti-microorganisms. The erroneous belief that natural products are superior to conventional drugs without causing harm to health, associated to the no satisfaction with pharmaceuticals results and self-medication , has led researches to work in the scientific search and validation of medicinal plants in the development of new medicinal products or therapeutic combinations due to the resistance of antimicrobial drugs already in use [3,4]. Antimicrobial resistance is reported as one of the greatest threats to human health . This alarming growth of multi resistant bacterial infections due to the excessive use of antimicrobials spectrum has been a factor that has favored the dissemination, mainly of microorganisms of the genus Candida, increasing their chances of causing diseases .
The incidence of Candida strains spp. resistance to antifungal treatment has increased in the last decades, being a concern for professionals of health . The most commonly used antifungal
for the treatment of candidoses belong to the azoles family (fluconazole, ketoconazole and itraconazole), and polyenes (nystatin and amphotericin B). Excessive use of these drugs is one of the factors that may favor the development of resistant strains to antifungals . Candidiasis is a most commonly diseases caused by Candida albicans species. These infections are of broad spectrum and affect mainly immunodepressed patients, HIV-positive, cancer patients, patients who make excessive use of antibiotics and those who use immunosupressors therapies as in the case of transplants .
C. albicans is a pleomorphic fungus, presenting in the yeast form, or as filamentous with the production of hyphae. Such morphological transition capacity is known as polymorphism due to the formation of germ tubes and the consequent development of this form is considered a virulence factor of this specie, which also has the capacity of chlamydospores formation. The two forms are related to the infectious process, and the yeast form has a greater ability to human dissemination while the hyphae have the ability to invade tissues of the host phagocytic cells [9,10].
Resistance to antifungals treatment has becoming a serious public health problem, which justifies the search for new alternatives with antifungal potential , and through numerous plant efficiency and reliability as new drugs .
The members of the genus Eucalyptus, belonging to the
Myrtaceae family is originated from Australia, but has been
found on most continents. The Eucalyptus is popularly used as
anesthetic, expectorant, anti-inflammatory and antiseptic .
They are known in medicine as antibacterial, antifungal and having
inflammatoy activities, beside analgesic properties through their
essential oils. Essential oils from members of the genus Eucalyptus
are also used in the pharmaceutical and cosmetics industries due
to their biological activities .
In view of the increase in antimicrobial resistance cases,
studies looking for new drugs to treat infections having medicinal
plants as coadjuvants or a promising source for new treatment
has been an alternative of research. The objective of this study
was to preliminary evaluate the antifungal activity of Eucalyptus
spp essential oils against Candida yeasts using the microdilution
method (MIC), minimal inhibitory concentration in broth M27-A3
, and the determination of the MFC (Minimum Fungicidal
Samples of the essential oils were tested for their activity
on Candida spp strains determining the Minimum Inhibitory
Concentration (MIC) following the recommendations of the
M27-A3 protocol .
Inoculum Adjustment: It was prepared in saline solution,
equivalent to 5.0 x 106 CFU/mL in spectrophotometer. The
inoculum was standardized at 2.5 x 103 by serial dilution. In a
sterile microplate, 100 μl of RPMI was distributed in all wells,
the first column added 100μl of the essential oil at the initial
concentration, and then the serial microdilution of the essential
oils was carried out. After the dilution process was added 100μl of
the adjusted inoculum, giving the following groups:
a) Positive control group: culture medium and inoculum,
for observation of yeast growth)
b) Negative control group 1: culture medium only, for
observation possible contamination
c) Negative control group 2: culture medium, essential oil
and diluent, the purpose of observing possible contaminations
in the microdilution)
d) Negative control group 3: culture medium and diluent,
for the purpose to observe possible toxic effects of the diluent
on the yeast
e) Test group: Eucalyptus citriodora essential oil
(Concentration initial 16 mg/mL), or Eucalyptus globulus
essential oil (16mg/ml initial concentration) or antifungal
treatment commercial Fluconazole (initial concentration
64μg/ml) or Amphotericin B (Initial concentration 16 μg/ml).
The plates were incubated for 24 hours at 37°C in an aerobiose
oven, and visual reading was performed . The tests were
performed in triplicate.
The determination of the Minimum Fungicidal Concentration
(MFC) was made in Sabouraud Dextrose Agar plates - SDA (KASVI).
The 96 wells were homogenized by pipetting, and an aliquot of
10μl was transferred to the Petri dish. After incubation at 37°C for
48h, the lowest fungicidal concentration was established. MFC was
determined as the lowest concentration of essential oils, which
does not allow the growth of any fungal colony in solid culture
through visual reading to confirm the fungicidal and fungistatic
effects of the essential oils tested . The assays were performed
The essential oils were tested against Candida reference
strains in order to determine their inhibitory effect through broth
microdilution technique. Both oils tested showed inhibitory
activity on the planktonic cells of Candida spp (Table 1).
Inhibitory activities of up to 8 mg/mL were detected, and
inhibitory concentrations ranged from 0.5 mg/ml and 0.125 mg/
ml for Eucalyptus citriodora essential oil and 8 mg/ml to 1 mg/ml
for essential oil of Eucalyptus globulus.
Fluconazole and amphotericin B antifungal agents were also
tested against Candida spp. in order to determine the minimum
inhibitory concentration through broth microdilution technique.
Fluconazole showed lower inhibitory activity when compared to
the antifungal Amphotericin B, which demonstrated inhibitory
activity at concentrations between 0.25 μg/ml and 1 μg/ml (Table
After determining the MIC values of the essential oils, an
aliquot of the susceptibility assay was used to determine the
Minimum Fungicidal Concentration (MFC) against the strains
of Candida spp. The essential oil of E. citriodora demonstrated fungicidal activity, varying its effect at concentrations between
0.25 mg/mL and 0.5mg/mL. The E. globulus essential oil showed
fungicidal activity at concentrations of 8 mg/mL and 1 mg/ml
In recent years, treatment against candidias is remains a
concern due to toxicity and the development of resistance to
commercial antifungal agents . The secondary metabolism
of plants promotes the production of active compounds that are
involved in their defense system. These metabolites are extracted
in different ways and different parts of the plant, but mainly make
up the essential oils and extracts produced with different vehicles.
The genus Eucalyptus was cultivated and exploited on a large
scale for many years. Several species of the genus are used in
folk medicine as an antiseptic and for infections treatments of
the upper respiratory tract. The essential oils extracted from the
leaf of this plant exhibit various biological effects as antibacterial,
antifungal and antioxidant activity. The antimicrobial, analgesic
and anti-inflammatory properties of E. citriodora and E. globulus
species have been reported .
In the present study, the data presented through the
microdilution method  showed antifungal activity of the oils
tested against planktonic cells of Candida spp. For E. citriodora
essential oil, MIC concentrations ranged from 0.5mg/mL to
0.125mg/mL among the strains tested. E. globulus oil showed MIC
from 2mg/mL to 1 mg/mL for the strains tested, except for the
Candida rugosa strain IZ 12, which had its inhibited growth only at
8 mg/mL concentration (Table 1).
The minimal fungicidal concentration (MFC) of E. citriodora
essential oil in the Candida strains tested was between 0.25
mg/ml and 0.5 mg / ml. The E. globulus essential oil had MFC
concentrations between 1 mg/mL and 8mg/mL (Table 1).
These data corroborate with the literature indicating
that Eucalyptus species show antimicrobial activity by the
presence of bioactive compounds in their essential oil (Luqman
et al) . According to Tyagi et al.  E. globulus oil showed
antimicrobial activity against B. subtillis and S. aureus. E. globulus
oil also showed activity against yeast cells, inhibiting the growth
of Candida albicans and Saccharomyces cerevisiae. Antibacterial
activity against the species Fusobacterium nucleatum and
Porphyromonas gingivalis were also observed by Harkat-Madouri
et al. . In a study by Dhakad et al. , E. globulus exhibited
antibacterial and antifungal activity. E. citriodora essential oil has
also demonstrated antifungal activity against filamentous fungi
species . Luqman et al.  also reported the antimicrobial
activity of E. citriodora against Candida albicans, Aspergillus
flavus and Aspergillus niger, and against the bacterial strains
of Escherichia coli and Mycobacterium smegmatis. In addition,
oil from E. citriodora exhibited antifungal activity against the
dermatophytes Microsporum canis, Trichophyton rubrum and
Trichophyton mentagrophytes .
All these data, when analyzed together, show that Eucalyptus
spp species are potentially antibacterial and antifungal species,
exhibiting action against these microorganisms in different ways.
Different methodologies of extraction from different parts of the
plants can exhibit different bio components with various capacity
of microorganism’s inhibition suggesting that more researches to
look for understand such phenomena must be taken in account.
Since there are several reports in the literature indicating
the antimicrobial action of these plant species, the phenomena
of microbial resistance to commercial antifungal, and a smaller
possibility to synthesizing new drugs, together with the increase of
infections by these microorganisms, justify more extensive studies
in the attempt to evaluate the antifungal potential of essential oils
using lower concentrations in the synergistic combination with
conventional drugs in an attempt to decrease or avoid side effects
of these drugs, potentializing their antifungal activity, should be
carried out as part of our future researchs, added of citoxicity and
anti-cancer tests of such bio-activity plant components, including
other parts of these plants.
This research refers to a preliminary study about antifungal
potential of the Eucalyptus spp. essential oils against the Candida
genus. New investigations as to evaluate the effect of the essential
oils of the biofilm of Candida albicans; to evaluate the effect of
essential oils on Candida albicans biofilm through Scanning
Electron Microscopy; to evaluate the possible cytotoxic effect of
essential oils on epithelial cells of the HaCat strain and also to
evaluate the possible cytotoxic effect of essential oils “in vivo”
in Galleria mellonella larvae are being carried out in an attempt
to amplify the knowledge about this plants and its antifungal