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Journal of Medicinal Plants Research Vol. 5(17), pp. 4147-4156, 9 September, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Chemical composition, antioxidant and antifungal
potential of Melaleuca alternifolia (tea tree) and
Eucalyptus globulus essential oils against oral
Candida species
Emira Noumi1*, Mejdi Snoussi2, Hafedh Hajlaoui1, Najla Trabelsi3, Riadh Ksouri3,
Eulogio Valentin4 and Amina Bakhrouf1
1 Department of Microbiology, Faculty of Pharmacy, Monastir, University of Monastir, Laboratory Analysis,
Treatment and Recovery of environmental pollutants and Products, Tunisia.
2Laboratory Waste Water Treatment, and Research Center for Water Technology (CERT), Technopole de Borj-Cédria,
BP 273 – Soliman, Tunisia.
3Laboratory of plant adaptation to abiotic stresses, Biotechnology Centre, Technopole Borj-Cédria (CBBC), BP 901,
2050 Hammam-Lif, Tunisia.
4Department of Microbiology and Ecology, Faculty of Pharmacy, University of Valencia, Burjassot, Valencia, Spain.
Accepted 28 February 2011
Eucalyptus globulus and Melaleuca alternifolia essential oils has been of interest to researchers
because they are traditionally used for the treatment of fungal infections and especially candidiasis.
The chemical composition of hydrodistilled essential oils were analyzed by gas chromatography-mass
spectrometry (GC-MS), and their antifungal activity was tested against 32 Candida strains including 15
species. The antioxidant activities (DPPH, reducing power, and superoxide anion radical-scavenging
activity) were also investigated. Tea tree essential oil was particularly rich on terpinen-4-ol (40.44%),
gamma terpinene (19.54%) and 1,8-cineole (95.61%) and alpha-pinene (1.5%) for the E. globulus oil. E.
globulus oil was more efficient and had the best antifungal effect on oral Candida albicans and Candida
glabrata strains comparing to the results obtained with Amphotericin B. Even at low concentrations,
these oils drastically impair the maximum yield and growth rate of both C. albicans and C. glabrata on
YPD medium. The Tea Tree essential oil displayed the highest DPPH scavenging ability with the lowest
IC50 value (IC50, 12.5 µg/ml), the greater reducing power and bleaching of β-carotene (EC50, 24 µgml-1 and
IC50, 42 µgml-1, respectively) as compared to E. globulus oil and BHT. These findings support the
interest of E. globulus and M. alternifolia essential oils as an efficient oral hygiene tool (anti-Candida
spp.) and as a source of antioxidant compounds.
Key words: Candida, Melaleuca alternifolia, Eucalyptus globulus, gas chromatography-mass spectrometry,
antioxidant, antifungal activities.
INTRODUCTION
Medicinal plants have been used as a source of remedies
since ancient times and the ancient Egyptians were
familiar with many medicinal herbs and were aware of
their usefulness in treatment of various diseases (Abu-
Shanab et al., 2004). In Tunisia, many plant extracts and
*Corresponding author. E-mail: emira_noumi@yahoo.fr. Tel: +
216 73 466244. Fax: + 216 73 461830.
essential oils have been shown to exert biological activity
in vitro and in vivo, which justified research on traditional
medicine focused on the characterization of antimicrobial
activity of these plants (Snoussi et al., 2008; Hajlaoui et
al., 2008, 2009, 2010; Noumi et al., 2010a,b).
The oil of Melaleuca alternifolia contains ~100
components, which are mostly monoterpenes,
sesquiterpenes and related alcohols. The essential oil
obtained by steam distillation from the leaves have long
been used in aboriginal traditional medicine of Australia
4148 J. Med. Plant. Res.
as remedies for wounds and cutaneous infections, to
treat many pathological conditions such as empyema,
ringworm, paronychia, tonsillitis, stomatitis and vaginal
infections (Humphrey, 1930; Penfold and Morrison,
1937). Tea tree oil has been used medicinally in
Australia, with uses relating primarily to its antimicrobial
(Carson and Riley, 1993; Carson et al., 2002, Mondello et
al., 2003), anti-inflammatory and antifungal especially
anticandidal properties (Hammer et al., 1998, 2000). Tea
tree oil efficiency was confirmed in the treatment of
dandruff (Satchell et al., 2002) and oral candidiasis
(Jandourek et al., 1998; Hammer et al., 2004). Data from
an animal model also indicate that it may be effective in
the treatment of vaginal candidiasis (Hammer et al.,
2003).
The genus Eucalyptus, (family: Myrtaceae) is native to
Australian region. The genus Eucalyptus comprises well-
known plants of over 600 species of trees. Eucalyptus
globulus is increasingly used in traditional medicine for
various medical implications such as antibacterial, anti-
inflammatory, and antipyretic effects. The plant is popular
for this, it is cultivated in subtropical and Mediterranean
regions more than other species. The essential oil of
leaves of Eucalyptus species has been the object of
several studies antibacterial, antioxidant,
antihyperglycemic and antifungal activity (Derwich et al.,
2009).
The aim of this study was to compare the antifungal
activities of the essential oils of E. globulus and M.
alternifolia against a range of Candida species
associated with oral disorders, evaluating minimal
inhibitory and minimal fungicidal concentrations, and
kinetic parameters in an attempt to contribute to the use
of these as alternative products for microbial control and
as a natural source of antioxidant components.
MATERIALS AND METHODS
Plant material and essential oil
M. alternifolia (tea tree) essential oil (leaves) was purchased from
Arkomédika (Laboratories Pharmaceutiques, BP 28-06511 Carros,
France). E. globulus commercialized essential oil was kindly
provided by the “Laboratoire de Pharmacognosie, Monastir
(Tunisia).
Gas chromatography-mass spectrometry (GC-MS) analysis
conditions
The analysis of the essential oil was performed using a Hewlett
Packard 5890 II GC, equipped with a HP-5 MS capillary column (30
m· 0.25 mm i.d., 0.25 µm) and a HP 5972 mass selective detector.
For GC–MS detection an electron ionization system with ionization
energy of 70 eV was used. Helium was the carrier gas, at a flow
rate of 1 ml/min. Injector and MS transfer line temperatures were
set at 220 and 290°C, respectively. Column temperature was
initially kept at 50°C for 3 min, then gradually increased to 150°C at
a 3° C/min rate, held for 10 min and finally raised to 250°C at
10°C/min. Diluted samples (1/100 in acetone, v/v) of 1 µl were
injected manually and in the splitless mode. The components were
identified based on the comparison of their relative retention time
and mass spectra with those of standards, NBS75K library data of
the GC–MS system and literature data (Adams, 2001). The results
were also confirmed by the comparison of the compounds elution
order with their relative retention indices on non-polar phases
reported in the literature (Adams, 2001).
Antioxidant activities
1,1-Diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion
radical-scavenging activity
The effect of the tested essential oil on DPPH degradation was
estimated according to the method described by Hajlaoui et al.
(2010). The essential oil was diluted in pure methanol at different
concentrations, and then 2 ml were added to 0.5 ml of a 0.2 mmol/L
DPPH methanolic solution. The mixture was shaken vigorously and
left standing at room temperature for 30 mn. The absorbance of the
resulting solution was then measured at 517 nm measured after 30
min. The antiradical activity (three replicates per treatment) was
expressed as IC50 (µg/ml), the antiradical dose required to cause a
50% inhibition. A lower IC50 value corresponds to a higher
antioxidant activity of essential oil. The ability to scavenge the
DPPH radical was calculated using the following equation:
DPPH scavenging effect (%) = [(A0 -A1) x 100]/A0 (1)
Where A0 is the absorbance of the control at 30 min, and A1 is the
absorbance of the sample at 30 min. Superoxide anion scavenging
activity was assessed using the method described by Trabelsi et al.
(2010). The reaction mixture contained 0.2 ml of essential oil has
different concentration, 0.2 ml of 60 mM PMS stock solution, 0.2 ml
of 677 mM NADH and 0.2 ml of 144 mM NBT, all in phosphate
buffer (0.1 mol/l, pH 7.4). After incubation at ambient temperature
for 5 min, the absorbance was read at 560 nm against a blank.
Evaluating the antioxidant activity was based on IC50. The IC50
index value was defined as the amount of antioxidant necessary to
reduce the generation of superoxide radical anions by 50%. The
IC50 values (three replicates per treatment) were expressed as µg/
ml.
As for DPPH., a lower IC50 value corresponds to a higher
antioxidant activity of plant extract. The inhibition percentage of
superoxide anion generation was calculated using the following
formula:
Superoxide quenching (%) = [(A0 -A1) x 100]/A0
Where A0 and A1 have the same meaning as in Equation (1).
Reducing power
The ability of the extracts to reduce Fe3+ was assayed by the
method of Oyaizu (1986). Briefly, 1 ml of each essential oil were
mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of
1% K3Fe(CN)6. After incubation at 50°C for 25 mn, 2.5 ml of 10%
trichloroacetic acid was added and the mixture was centrifuged at
650 x g for 10 min. Finally, 2.5 ml of the upper layer was mixed with
2.5 ml of distilled water and 0.5 ml of 0.1% aqueous FeCl3. The
absorbance was measured at 700 nm.
The mean of absorbance values were plotted against
concentration and a linear regression analysis was carried out.
Increased absorbance of the reaction mixture indicated increased
reducing power. EC50 value (mg/ml) is the effective concentration at
which the absorbance was 0.5 for reducing power. Ascorbic acid
was used as positive control.
β
ββ
β-Carotene-linoleic acid model system (β
ββ
β-CLAMS)
The β-CLAMS method by the peroxides generated during the
oxidation of linoleic acid at elevated temperature. In this study the
β-CLAMS was modified for the 96-well micro-plate reader according
the protocol described by Koleva et al. (2002). In brief, the β-
carotene was dissolved in 2 ml of CHCl3, to which 20 mg of linoleic
acid and 200 mg of tween 40 were added. CHCl3 was removed
using rotary evaporator. Oxygenated water (100 ml) was added,
and the flask was shaken vigorously until all material dissolved.
This test mixture was prepared fresh and using immediately. To
each well, 250 µl of the reagent mixture and 35 µl sample or
standard solution were added. The plate was incubated at 45°C.
Readings were taken at 490 nm using visible/UV microplate kinetics
reader (EL x 808, Bio-Tek instruments). Readings of all samples
were performed immediately (t = 0 mn) and after 120 mn of
incubation. The antioxidant activity (AA) of the extracts was
evaluated in term of β-carotene blanching using the following
formula:
AA (%) = [(A0-A1)/A0]*100
Where A0 is the absorbance of the control at 0 min, and A1 is the
absorbance of the sample at 120 mn. The results are expressed as
IC50 values (µg/ml). All samples were prepared and analyzed in
triplicate.
Antifungal activity
A total of 32 Candida strains including 15 species (Candida
albicans, Candida dubliniensis, Candida glabrata, Candida
parapsilosis, Candida krusei, Candida famata, Candida kefyr,
Candida sake, Candida holmii, Candida lusitaniae, Candida
intermedia, Candida atlantica, Candida maritima, Pichia
guillermondii and Pichia jardinii) were used in this study. Clinical
isolates were taken from the oral cavity of patients by using a
swabbing method.
A sterile cotton swab (Nippon Menbo, Tokyo, Japan) was
immediately cultured into Sabouraud Chloramphenicol agar (Bio-
rad, France) to obtain isolated colonies. All isolates were incubated
at 30°C for 24 to 48 h and yeast-like colonies were isolated and
identified by the ID 32C (bio-Mérieux, Marcy-l’Étoile, France)
assimilation kit. The ATCC Candida species were used as
reference strains.
Disc diffusion method
The anti-Candida spp. activity was achieved by the agar-well
diffusion method according the protocol described by Hajlaoui et al.
(2010).
All Candida strains were inoculated into Sabouraud
chloramphenicol agar and incubated for 18 h at 37°C. The yeast
cultures were harvested and than suspended in sterile saline (0.8%
NaCl) and the cell density was adjusted to 107 cells/ml (OD540= 0.5).
For the antifungal activity of the plants oils used in this study, three
sterile 6 mm paper discs (Whatman paper N°3), impregnated with
30 mg of essential oil (10 µl/disc) were placed on the inoculated
surface. Plates were then incubated at 37°C for 18 to 24 h. The
ATCC strains were used as a quality control strains. The diameter
of the zones of inhibition around each disc were examined after 24
h, measured and recorded as the mean diameter (mm) of complete
growth-inhibition. As a positive control, 10 µg of amphotericin B
(Fungizone, BioBasic INC) was used.
Tests were done in triplicate and results given as mean average
(Table 2).
Noumi et al. 4149
Microdilution method for the determination of the (minimal
inhibition concentration) MIC and ( minimal fungicidal
concentration) MFC
The MIC and the MFC values were determined for all Candida
strains according the protocol described by Hajlaoui et al. (2010).
The inoculums of the yeast strains were prepared from 12 h
Sabouraud dextrose broth cultures and suspensions were adjusted
to an optical density of 0.5 at 540 nm. The 96-well plates were
prepared by dispensing into each well 95 µl of nutrient broth (1%
NaCl) and 5 µl of the inoculum. A 100 µl aliquot from the stock
solutions of each plants extract was added into the first wells. Then,
100 µl from the serial dilutions were transferred into eleven
consecutive wells. The last well containing 195 µl of nutrient broth
(1% NaCl) without essential oil and 5 µl of the inoculum on each
strip was used as the negative control. The final volume in each
well was 200 µl. The plates were incubated at 37°C for 24 h. The
plants extract tested in this study was screened two times against
each strain. The MIC was defined as the lowest concentration of
the compounds to inhibit the growth of the microorganisms. The
MFC values were interpreted as the highest dilution (lowest
concentration) of the sample, which showed clear fluid with no
development of turbidity and without visible growth. All tests were
performed in triplicate.
Effect of the essential oils on the kinetic growth of Candida
strains on YPD broth
The effect of M. alternifolia and E. globulus essential oils on the
kinetic growth of C. albicans (15B) and C. glabrata (15T) strains was
tested. Cultures were grown on YPD broth for 18 to 24 h at 37°C.
The enrichment cultures were used to inoculate the sterile glass
bottles containing 30 ml of new YPD broth and the initial OD600 was
adjusted at 1 (107 to 108 cfu/ml). These bottles were then prepared
at different concentration with the two tested essential oils (1/2 MIC,
MIC and MFC) and incubated on rotatory shaker (150 rpm) at 37°C.
At regular time intervals, fungal growth was evaluated by measuring
absorbance at 600 nm using the spectrophotometer after 0, 1, 2, 3,
6, 9, 12 and 24 h of incubation. All values were conducted in
triplicate and average values were calculated using the SPSS 13.0
statistics package for Windows. Morphology of the fungal cells was
observed under a binocular light microscope.
RESULTS AND DISCUSSION
Essential oil composition
Thirty five components were identified in the essential oil
of Tea tree and only thirteen in the essential oil of E.
globulus. The main compounds of the oils are given in
Table 1, where the components were listed according to
their elution on the Innowax column. Tea tree essential
oils was particularly rich on: terpinen-4-ol (40.44%), γ-
terpinene (19.54%), α-terpinene (7.69%), 1,8-cineole
(5.20%), para-cymene (4.74%), α-terpineol (3.31%), α-
terpinolene (3,09%), α-pinene (2.67%), alloaroma-
dendrene (1.47%), ∆-Cadinene (1.47%), ledene (1.20%)
α-thujene (0.90%), myrcene (0.75%), β-pinene (0.73%),
aromadendrene (0.52%).
Our results are in accordance with previous works
dealing about the chemical composition of both M.
alternifolia and E. globulus essential oils. In fact, six
4150 J. Med. Plant. Res.
Table 1. The main components identified in the essential oils of M. alternifolia and E. globulus used in this study.
Compounds identified *(KI) HP-5 Percentage Identification
M. alternifolia essential oil (Total identified components 97.19%)
terpinen-4-ol 1186 40.44 MS, KI
γ-terpinene 1062 19.54 MS, KI
α-terpinene 1019 7.69 MS, KI
1,8-cineole 1033 5.20 MS, KI
para-cymene 1026 4.74 MS, KI
α-terpineol 1195 3.31 MS, KI
α-terpinolene 1089 3.09 MS, KI
α-pinene 935 2.67 MS, KI
E. globulus essential oil (Total identified components 99.19%)
1-8,cineole 1040 95.61 MS, KI
α-pinene 935 1.50 MS, KI
myrcene 991 0.53 MS, KI
β-pinene 979 0.40 MS, KI
α-terpineol 1192 0.28 MS, KI
*: (KI) HP-5: Kovats index.
chemotypes of M. alternifolia essential oil have been
described including terpinen-4-ol, terpinolene chémotype
and four 1-8 cineole chemotypes (Williams, 1998; Homer
et al., 2000). Tea tree oil (TTO) is composed of terpene
hydro-carbons, mainly mono-terpenes, sesquiterpenes,
and their associated alcohols. Early reports on the
composition of TTO described 12, 21, and 48 compo-
nents (Carson et al., 2006). In addition, the seminal work
done by Brophy and collaborators examined over 800
TTO samples by GC and GC/MS and reported
approximately 1010 components and their range of
concentrations as follow: terpinen-4-ol (40.1%), gamma
terpinene (23.0%), alpha terpinene (10.4%), 1,8-cineole
(5.1%), terpinolene (3.1%), para-cymene (2.9%), alpha-
pinene (2,6%), alpha terpineol (2.4%), aromadendrene
(1.5%), delta Cadinene (1.3%), limonene (1%), sabinene
(0.2%), globulol (0.2%) and viridoflorol (0.1%).
1,8-cineole (95.61%) and alpha-pinene (1.5%) were the
main components of E. globulus essential oil tested in the
present work. In fact, multiple studies have been reported
on the chemical composition of the essential oils of
Eucalyptus species belonging to different regions in the
world. The chemical compositions of the leaf oils of
Eucalyptus from various parts of the world have been
reported and the 1.8-Cineole was identified as the major
component in from samples growing in Taiwan, Uruguay,
Algeria, Burundi, Congo, Mozambique, Greece, Australia,
Tunisia, Italy, Nigeria, Turkey and Morocco (Boland et al.,
1991; Dethier et al., 1994; Derwich et al., 2009).
Antifungal activity
Early data on the susceptibility of fungi to tea tree and
E. globulus essential oils were largely limited to Candida
albicans, which was a commonly chosen model test
organism. We investigated in the present study the
antifungal activity of M. alternifolia and E. globulus
essential oils against several Candida species including
those isolated from Tunisian patients suffering from oral
candidiasis. Antifungal effects are reported as inhibition
zones using the disc diffusion method and in vitro activity
as MIC and MFC values (Table 3).
The two plant essential oils showed significant
antifungal activity against all Candida strains tested.
Overall, the best antifungal activity was against C.
albicans ATCC 90028 for M. alternifolia (19.33 mm) and
against C. glabrata ATCC 90030 for E. globulus oil (22.33
mm). Essential oil of E. globulus was more efficient and
had the best antifungal effect for oral C. albicans strain
(15B) (IZ= 19.33 mm) comparing to the results obtained
with Amphotericin B (IZ= 11 mm) and also for C. glabrata
ATCC 90030 strain (IZ= 22.33 mm) comparing to
Amphotericin B results (IZ= 14.33 mm). Table 3
summarizes the MIC and MFC of the two plants essential
oils. The lowest values of MIC were seen against two C.
glabrata isolates with E. globulus oil (strains 15T and
ATCC 90030; MIC: 0.078 mg/ml), followed by 0.156
mg/ml for C. albicans isolates (strains 15BB and ATCC
90028). The MFC values were similar for all Candida
tested strains (10 mg/ml). As to the he standard
antifungal drug used in this work, Amphotericin B was
more active against all oral and reference Candida
strains (MIC range: 0.012 to 0.39 mg/ml; MFC range:
0.195 to 1.562 mg/mg) comparing the two essential oils.
The medicinal properties of tea tree oil were first
reported by Penfold in the 1920s. Contemporary data
clearly show that the broad-spectrum activity of TTO
Noumi et al. 4151
Table 2. Antifungal activity of M. alternifolia and E. globulus oils against Candida strains.
Strains
Inhibition zone in diameter (mm ± SD). MIC and MFC (mg/ml)
M. alternifolia E. globulus AmB
IZ MIC MFC IZ MIC MFC IZ MIC MFC
C. albicans
ATCC 90028 19.33±0.57 0.312 >10 17.66±0.57 0.156 10 11 0.097 0.781
ATCC 2091 21±1 0.0097 >10 20±0 0.0097 5 14.66±0.57 0.024 0.781
17 12.66±0.57 0.0097 >10 19.66±0.57 0.0097 5 10.66±1.52 0.097 0.781
H8 12±0 0.0097 10 17±1 0.039 2.5 11.66±0.57 0.097 0.195
H5 14.66±0.57 0.0195 10 12±0 0.156 2.5 9.66±0.57 0.39 0.195
15B 15.33±0.57 0.625 10 19.33±0.57 0.156 10 11±1 0.012 0.781
H3 15.66±0.57 1.25 10 21.66±0.57 0.156 5 9.66±0.57 0.012 0.097
16 16.33±0.57 0.0195 >10 24.33±0.57 0.0097 10 11.66±0.57 0.024 0.04
4 18.66±0.57 0.0097 10 18.33±0.57 0.0097 10 11±1 0.006 0.048
I2 19.66±0.57 5 10 26.33±1.15 0.0097 1.25 11.33±0.57 0.012 0.024
14 19±1 0.0097 10 20.66±0.57 0.0097 5 11±1 0.048 0.781
H2 20.33±0.57 0.156 >10 24.33±0.57 0.0195 5 10.33±0.57 0.097 0.195
65 21.33±0.57 0.0097 10 17.66±0.57 0.0097 5 10.66±1.15 0.097 1.562
11 21.66±1.154 0.0097 5 21±1 0.0097 10 7± 0 0.048 1.562
21 24.33±0.57 0.0097 >10 16±1 0.0097 10 11±1 0.097 0.195
7 24±0 0.0097 10 17.66±0.57 0.0097 10 9.66±0.57 0.048 0.39
10 25.66±0.57 0.0097 >10 19.33±0.57 2.5 10 11± 0 0.048 1.562
C. parapsilosis
I3 14.66±0.57 5 >10 15.33±0.57 0.0195 10 11±1 0.097 0.195
C. kefyr
CECT 1017 19.33±1.15 0.0097 5 19.33±0.57 0.0097 5 10.66±1.15 0.195 0.39
35 25.33±0.57 0.0097 10 20.66±0.57 0.0195 10 9.66±0.57 0.39 1.562
C. glabrata
ATCC 90030 14.33±0.57 0.625 10 22.33±0.57 0.078 10 14.33 ± 0.57 0.195 1.562
15T 12±0 0.0097 10 12.66±1.15 0.0097 10 10.66 ± 0.57 0.195 0.39
I1 11.66±0.57 0.0195 2.5 16.33±0.57 0.0097 >10 10.33±1.15 0.39 0.195
Others
C. dubliniensis CECT 11455 15±0 0.0097 5 20.33±0.57 0.0097 5 11.33±0.57 0.012 0.195
C. lusitaniae CECT 1145 15.33±0.57 0.0097 >10 18.66±1.15 0.0097 2.5 12.33±1.15 0.097 0.195
4152 J. Med. Plant. Res.
Table 2. Contd.
C. sake CECT 1044 16.33±0.57 0.0097 10 17.33±1.15 0.097 2.5 12±1 0.097 0.39
Pichia jadinii CECT 1060 17.66±0.57 0.0097 10 16.33±0.57 0.0097 5 11.66±1.15 0.006 0.195
C. famata CECT 11957 20.66±0.57 0.0097 >10 21.33±0.57 0.0097 5 12.33±0.57 0.195 0.39
C. intermedia CECT 11869 20±0 0.0097 >10 19±0 0.0097 5 12±0 0.012 0.195
Pichia guilliermondii CECT 1456 20±0 0.0097 10 16.33±1.15 0.0097 5 12.33±0.57 0.097 0.195
C. atlantica CECT 1435 21±1 0.0097 10 20.66±0.57 0.0097 5 11±0 0.195 0.39
C. maritima CECT 1435 24.66±0.57 0.0097 10 23.66±0.57 0.0097 2.5 12.66±1.15 0.097 0.195
Table 3. Antioxidant activities of M. alternifolia and E. globulus essential oils compared to BHT ones: DPPH, superoxide
radicals and β-Carotene bleaching test. Reducing power was expressed as EC50 values (µg/ml).
Essential oils BHT
M. alternifolia E. globulus
DPPH IC50 (µg.ml-1) 12.5 57 11.5
O2.- IC50 (µg.ml-1) 26.6 14 1.5
RP EC50 (µg.ml-1) 24 48 75
β-carotenes IC50 (µg.ml-1) 42 48 75
DPPH radical–scavenging activity is expressed as IC50 values (µg/ml); RP: reducing power was expressed as EC50 values (µg/ml);
β-Carotenes bleaching test is expressed as IC50 values (µg.ml-1); O2.-: Superoxide anion radical-scavenging activity is expressed as
IC50 values (µg/ml).
includes antibacterial, antifungal, antiviral, and
antiprotozoal activities. Of all these properties,
antimicrobial activity has received the most
attention. For this, TTO is employed for its
antimicrobial property and is incorporated as the
active ingredient in many tropical formulations
used to treat cutaneous infections. In fact, our
results agree with previous works dealing about
the high susceptibility of a wide range of yeasts,
dermatophytes, and other filamentous fungi
(Carson et al., 2006).
The antifungal activity of TTO is due to its
lypophilic nature, which facilitates skin
penetration. In this context, it has been clinically
evaluated for the treatment of several superficial
fungal infections, including onychomycosis (Syed
et al., 1999), tinea (Tong et al., 1992) and
refractory oral candidiasis (Jandourek et al.,
1998). In 1998, Hammer and colleagues tested in
vitro the antifungal activity of 24 essential oils
against fourteen Candida spp. isolates and
founded that E. globulus essential oil inhibit the
growth of C. albicans ATCC 10231 at MIC=1%
(v,v) and from 0.12 to 0.5% for all Candida
species tested. In 2005, Tampieri and colleagues
founded that 1,8-cineole (81.4%) and limonene
(7.01%) were the main components of E. globulus
essential oil and that these two components have
the same fungistatic activity at >1000 and 1000
ppm respectively. The highest antifungal activity
was observed in three active principles including
(trans-cinnamaldehyde, 1-decanol and β-
phellandrene) with MIC= 50 ppm even after 48 h
or 7 days of application. In the same year (2005),
Devkatte and colleagues studied the in vitro
efficacities of 38 plant essential oils against four
isolates of C. albicans. Twenty three of them
caused a 1-30 mm zone of inhibition (ZOI),
seventeen oils caused a 10-20 mm ZOI and six
showed a 1-9 mm ZOI. The E. globulus oil caused 6.3 to
10 mm ZOI and tea tree caused 11 to 24 mm ZOI. Seven
oils were found to be the most effective with MICs values
ranging from 0.03 to 0.15% concentration and tea tree
cause fungicidal effect at 0.25% concentration
comparatively to 1.5 - 2.5% for E. globulus oil.
Growth kinetics of C. albicans (15B) and C. glabrata
(15T) on YPD medium in the presence of increasing
concentrations of E. globulus and M. alternifolia essential
oils (Figure 1) showed that, even at low concentrations,
these oils drastically impair the maximum yield and
growth rate of both fungi. In fact, as can be shown in
Figure 1, a concentration as low as 0.078 mg/ml
(Eucalyptus oil) and 0.3125 mg/ml (tea tree) inhibits the
growth of both C. albicans and C. glabrata strains. At
high concentrations (MFC): 10 mg/ml (respectively for
Eucalyptus and tea tree plant oils), the growth of C.
albicans and C. glabrata strains was inhibited signalling
the fungicidal effect of these oils within the first hours of
the experiment. A comparison of the curves obtained with
different concentrations of E. globulus and M. alternifolia
oils confirms the highest efficiency of the second plant oil
on these two Candida strains. Both investigated C.
albicans and C. glabrata strains were susceptible to tea
tree plant oil at MIC values of 0.625 µg/ml, respectively.
All of the untreated Candida cells were round or oval in
shape and their number was significantly reduced
depending on the concentration of tea tree oil added
(Figure 2).
In fact, tea tree oil and components appear to affect
membrane properties and integrity in a manner
consistent with other lypophilic, membrane-active agents
such as the terpenes, thymol (Shapiro and Guggenheim,
1995) and geraniol (Hisajima et al., 2008).
Mondello et al. (2003) showed that TTO inhibited the
growth of all isolates tested inclusive those resistant to
fluconazole and Itraconazole and that the MICs values
ranged from 0.15 to 0.5%. MIC90s were 0.25 and 0.5% for
azole-susceptible and –resistant C. albicans strains
respectively, 0.125% for C. krusei and C. glabrata, and
0.06% for C. neoformans and C. parapsilosis. All azole-
resistant isolates of C. albicans were killed within 30 mn
by 1% TTO and within 60 mn by 0.25% TTO at pH 7. At
pH 5, the decrease in viable count was less rapid,
nonetheless, a 100% killing within 30 mn by TTO 1% was
achieved.
Also, TTO inhibits the formation of germ tubes, or
mycelial conversion, in C. albicans. Hammer and
colleagues have shown that germ tube formation was
completely inhibited in the presence of 0.25 and 0.125%
TTO. Recently, we reported that M. alternifolia essential
oil has an antimycelial activity against C. albicans isolates
higher than E. globulus essential oil. In fact, only 1/2 MIC
(0.312 mg/ml) of M. alternifolia was able to inhibit totally
mycelium in C. albicans isolate while 2 MIC (0.312
mg/ml) of the second essential oil was necessary to
inhibit germ tube formation in the same strain (Noumi et
Noumi et al. 4153
al., 2010a).
Antioxidant activities
Table 3 illustrates scavenging of the DPPH radical by M.
alternifolia and E. globulus essential oils. The scavenging
effect of essential oil and standard (BHT) on the DPPH
radical expressed as IC50 values were 12.5 µg/ml for M.
alternifolia oil and 11.5 µg/ml for BHT. The results
obtained with the PMS-NADH-NBT system demonstrated
that the inhibiting capacities of superoxide were very
interesting for E. globulus essential oil (IC50=14 µg/ml)
comparing to the results obtained for M. alternifolia oil
(IC50=26.6 µg/ml), but these results are inferior as
compared to BHT value obtained with the same test
(IC50=1.5 µg/ml).
Another reaction pathway in electron donation is the
reduction of an oxidized antioxidant molecule to
regenerate the ‘‘active” reduced antioxidant. As showed
in Table 2, the reducing power of M. alternifolia essential
oil, expressed as CE50, was clearly more important than
the reducing power of E. globulus (24 and 48 µg/ml,
respectively) and that of positive control BHT (75 µg/ml).
The results obtained with β-carotene bleaching test
demonstrate that the two essential oils have
approximately the same IC50 values (42 and 48 µg/ml
respectively for M. alternifolia and E. globulus essential
oils). These results are clearly more important than
positive control BHT (IC50=75 µg/ml). In 2010, Mishra and
colleagues tested the phytochemical analysis and
antioxidant activities of the essential oil extracted from
eucalyptus leaves. These authors founded that the free
radical scavenging activity of the different concentrations
of the leaf oil (10, 20, 40, 60 and 80% (v/v) in DMSO) of
E. globulus increased in a concentration dependent
fashion. In DPPH method, the oil in 80% (v/v)
concentration exhibited 79.55 ± 0.82%. In nitric oxide
radical scavenging assay method, it was found that 80%
(v/v) concentration exhibited 81.54 ± 0.94% inhibition.
Conclusion
Our results showed that tea tree essential oil exhibited
important antioxidant activities comparatively to the
Eucalyptus essential oil. In addition, there was a little
inter-species variation in susceptibility and all Candida
spp. tested were uniformly susceptible. Although
essential oil values where high when compared with
those of Amphotericin B, but these results were in
interest as we were dealing with an essential oil and not a
pure product. The present study together with previous
analysis supports the antibacterial properties of M.
alternifolia and E. globulus essential oils and suggests
them as antibacterial additives. Additional clinical trials of
these oils have to be performed if they are to be used for
4154 J. Med. Plant. Res.
Figure 1. Growth kinetics of C. albicans (15B) and C. glabrata (15T) on YPD medium in absence (C ♦) and presence of respectively ½ MIC (■), MIC (▲) and MFC (x) (mg ml-1)
of E. globulus (A) and M. alternifolia (B) essential oils. The essential oils were added to each experimental culture in zero time. Data represent the mean value of three measures
of the optical density at 600 nm.
Noumi et al. 4155
Figure 2. Microscopic examination showing the effect of E. globulus essential oil on the growth
of C. albicans (strain 15B) tested at ½ MIC (1), MIC (2) and MFC (3) concentrations
(Magnification 400×). The first photo (C) represents C. albicans (strain 15B) growing without
essential oil in YPD after 24 h at 37°C.
medicinal purposes.
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