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Article
Antifungal Activity of Oleuropein against
Candida albicans—The In Vitro Study
Nataša Zori´c 1, *, Nevenka Kopjar 2, Ivan Bobnjari´c 3, Igor Horvat 3, Siniša Tomi´c 1
and Ivan Kosalec 3
1Agency for Medicinal Products and Medical Devices of Croatia (HALMED), Ksaverska cesta 4,
HR-10000 Zagreb, Croatia; sinisa.tomic@halmed.hr
2Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000 Zagreb, Croatia;
nkopjar@imi.hr
3Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39,
HR-10000 Zagreb, Croatia; ivan.bobnjaric@gmail.com (I.B.); igor.horvat@gmail.com (I.H.);
ikosalec@pharma.hr (I.K.)
*Correspondence: natasa.zoric@halmed.hr; Tel.: +385-1-4884-225
Academic Editors: Daniela Barlocco and Fiorella Meneghetti
Received: 1 November 2016; Accepted: 24 November 2016; Published: 28 November 2016
Abstract:
In the present study we investigated activity of oleuropein, a complex phenol present
in large quantities in olive tree products, against opportunistic fungal pathogen Candida albicans.
Oleuropein was found to have
in vitro
antifungal activity with a minimal inhibitory concentration
(MIC) value of 12.5 mg
·
mL
−1
. Morphological changes in the nuclei after staining with fluorescent
DNA-binding dyes revealed that apoptosis was a primary mode of cell death in the analyzed samples
treated with subinhibitory concentrations of oleuropein. Our results suggest that this antifungal agent
targets virulence factors essential for establishment of the fungal infection. We noticed that oleuropein
modulates morphogenetic conversion and inhibits filamentation of C. albicans. The hydrophobicity
assay showed that oleuropein in sub-MIC values has significantly decreased, in both aerobic and
anaerobic conditions, the cellular surface hydrophobicity (CSH) of C. albicans, a factor associated with
adhesion to epithelial cells. It was also demonstrated that the tested compound inhibits the activity of
SAPs, cellular enzymes secreted by C. albicans, which are reported to be related to the pathogenicity
of the fungi. Additionally, we detected that oleuropein causes a reduction in total sterol content in the
membrane of C. albicans cells, which might be involved in the mechanism of its antifungal activity.
Keywords: oleuropein; antifungal activity; Candida albicans; virulence factors
1. Introduction
Candida species are commensal organisms that normally colonize mucosal surfaces of healthy
individuals and, under conditions of host weakness, can become opportunistic pathogens.
Among Candida species, Candida albicans is the predominant cause of invasive fungal infections;
however, in recent years, a growing incidence of infections caused by non-albicans species has been
observed [
1
]. An increase in serious human infections in immunocomprised patients caused by
fungi and a progression of drug resistance to conventional therapeutics triggered a need for more
effective treatment.
Several studies have reported that olive leaf extract and its constitutes, particularly oleuropein
and hydroxytyrosol, have health benefits, including antioxidant and antimicrobial properties [2–5].
Oleuropein was found to inhibit the growth of Staphylococcus aureus,Bacillus subtilis,
and Psudomonas solanecearum [
6
]. It was shown that it also inhibits germination and sporulation
of Bacillus megaterium [
7
], and an outgrowth of germinating spores of Bacillus cereus [
8
]. The activity
Molecules 2016,21, 1631; doi:10.3390/molecules21121631 www.mdpi.com/journal/molecules
Molecules 2016,21, 1631 2 of 9
of oleuropein was investigated
in vitro
against Mycoplasma hominis,M. fermentas,M. pneumoniae,
and M. pirum. Oleuropein inhibited mycoplasmas at concentrations from 20 to 320 mg
·
L
−1
[
9
].
Considering that molecules that derive from natural sources have considerable antifungal properties
and can be a promising source for the development of new anti-candidal therapy [
10
], we performed
in vitro
tests to examine the effect of this phenolic compound on C. albicans, one of the most important
opportunistic fungal pathogen, and to investigate its possible mechanism of action.
2. Results and Discussion
Antifungal susceptibility testing was used to estimate the drop of viability up to 90% in
comparison to the control (untreated cells). Our findings showed that minimal inhibitory concentration
(MIC) of oleuropein against C. albicans was 12.5 mg·mL−1.
A quantitative fluorescent-dye exclusion assay revealed (Table 1) that oleuropein significantly
(p< 0.05, Pearson chi-square test) reduced cell viability compared to the negative control at certain
applied concentrations (12.5 mg·mL−1; 1.25 mg·mL−1; 0.195 mg·mL−1).
Table 1.
Results of the quantitative fluorescent assay for simultaneous identification of apoptotic
and necrotic cells due to the loss of membrane integrity in Candida albicans ATCC 10231 treated with
oleuropein in vitro for 18 h.
Sample Viable Cells (%) Non-Viable Cells
ΣApoptosis (%) Necrosis (%)
OLP116.3 ±2.1 83.7 ±2.1 OLP2,OLP3,NC
39.3
±
11.2
OLP2,OLP3,NC,PC
44.3
±
10.2
OLP2,OLP3,NC,PC
OLP261.3 ±6.0 38.7 ±6.0 OLP3,NC,PC 29.0 ±4.6 OLP3,NC,PC 9.7 ±1.5 NC
OLP376.0 ±6.6 24.0 ±6.6 NC,PC 14.7 ±5.5 NC,PC 9.3 ±1.5 NC
PC 11.7 ±3.2 88.3 ±3.2 NC 78.7 ±0.6 NC 9.7 ±3.8 NC
NC 97.3 ±0.6 2.7 ±0.6 1.0 ±1.0 1.7 ±1.1
Three hundred cells per sample per each experimental point were analyzed. Mean values
±
SD are shown.
OLP—concentration of oleuropein (OLP
1
—12.5 mg
·
mL
−1
, OLP
2
—1.25 mg
·
mL
−1
, OLP
3
—0.195 mg
·
mL
−1
);
PC—positive control; NC—negative control (RPMI). Statistical significance of data was evaluated using a
χ2
test. The level of statistical significance was set at p< 0.05. The abbreviations next to the means indicate from
which groups the relevant group differs with statistical significance.
Analysis of C. albicans cells following 18 h
in vitro
exposure to oleuropein indicated a cytotoxic
effect of oleuropein that was concentration-dependent. Further, intergroup comparison of viable vs.
dead cells using the Pearson chi-square test revealed statistically significant differences (
p< 0.05
)
between tested concentrations. In the samples treated with concentrations, 1.25 mg
·
mL
−1
and
0.195 mg
·
mL
−1
of oleuropein apoptosis was a predominant type of cell death. Differentiation between
viable and dead cells after treatment with the test agent and the altered morphology of the nuclear
chromatin visualized by fluorescence microscopy is shown in Figure 1.
Molecules 2016, 21, 1631 2 of 8
that molecules that derive from natural sources have considerable antifungal properties and can be a
promising source for the development of new anti-candidal therapy [10], we performed in vitro tests
to examine the effect of this phenolic compound on C. albicans, one of the most important opportunistic
fungal pathogen, and to investigate its possible mechanism of action.
2. Results and Discussion
Antifungal susceptibility testing was used to estimate the drop of viability up to 90% in
comparison to the control (untreated cells). Our findings showed that minimal inhibitory concentration
(MIC) of oleuropein against C. albicans was 12.5 mg·mL−1.
A quantitative fluorescent-dye exclusion assay revealed (Table 1) that oleuropein significantly
(p < 0.05, Pearson chi-square test) reduced cell viability compared to the negative control at certain
applied concentrations (12.5 mg·mL−1; 1.25 mg·mL−1; 0.195 mg·mL−1).
Table 1. Results of the quantitative fluorescent assay for simultaneous identification of apoptotic and
necrotic cells due to the loss of membrane integrity in Candida albicans ATCC 10231 treated with
oleuropein in vitro for 18 h.
Sample Viable Cells (%) Non-Viable Cells
ΣApoptosis (%) Necrosis (%)
OLP1 16.3 ± 2.1 83.7 ± 2.1 OLP2,OLP3,NC 39.3 ± 11.2 OLP2,OLP3,NC,PC 44.3 ± 10.2 OLP2,OLP3,NC,PC
OLP2 61.3 ± 6.0 38.7 ± 6.0 OLP3,NC,PC 29.0 ± 4.6 OLP3,NC,PC 9.7 ± 1.5 NC
OLP3 76.0 ± 6.6 24.0 ± 6.6 NC,PC 14.7 ± 5.5 NC,PC 9.3 ± 1.5 NC
PC 11.7 ± 3.2 88.3 ± 3.2 NC 78.7 ± 0.6 NC 9.7 ± 3.8 NC
NC 97.3 ± 0.6 2.7 ± 0.6 1.0 ± 1.0 1.7 ± 1.1
Three hundred cells per sample per each experimental point were analyzed. Mean values ± SD are
shown. OLP—concentration of oleuropein (OLP1—12.5 mg·mL−1, OLP2—1.25 mg·mL−1, OLP3—0.195
mg·mL−1); PC—positive control; NC—negative control (RPMI). Statistical significance of data was
evaluated using a χ2 test. The level of statistical significance was set at p < 0.05. The abbreviations
next to the means indicate from which groups the relevant group differs with statistical significance.
Analysis of C. albicans cells following 18 h in vitro exposure to oleuropein indicated a cytotoxic
effect of oleuropein that was concentration-dependent. Further, intergroup comparison of viable vs.
dead cells using the Pearson chi-square test revealed statistically significant differences (p < 0.05) between
tested concentrations. In the samples treated with concentrations, 1.25 mg·mL−1 and 0.195 mg·mL−1 of
oleuropein apoptosis was a predominant type of cell death. Differentiation between viable and dead
cells after treatment with the test agent and the altered morphology of the nuclear chromatin
visualized by fluorescence microscopy is shown in Figure 1.
Figure 1. Appearance of C. albicans blastospores treated with oleuropein following staining with
ethidium bromide and acridine orange according to the fluorescent-dye exclusion method: viable
normal blastospores excluded ethidium bromide, and their nuclei were bright green with an intact
structure. Non-viable cells had orange to red colored chromatin with organized structure. Apoptotic
cells were bright green with highly condensed or fragmented nuclei.
Figure 1.
Appearance of C. albicans blastospores treated with oleuropein following staining
with ethidium bromide and acridine orange according to the fluorescent-dye exclusion method:
viable normal blastospores excluded ethidium bromide, and their nuclei were bright green with
an intact structure. Non-viable cells had orange to red colored chromatin with organized structure.
Apoptotic cells were bright green with highly condensed or fragmented nuclei.
Molecules 2016,21, 1631 3 of 9
In order to understand mode of action of antifungal agent, it is necessary to investigate its effect
on virulence factors, which are essential for development of infection in the host. It was believed
in the past that yeasts passively participate in the process of pathogenesis and the establishment of
fungal infection. An immunocomprised host was considered the only mechanism responsible for
the establishment of opportunistic infection. Today, it is known that yeasts actively participate in the
pathophysiology of the disease using mechanisms called virulence factors [
10
]. The main advantages
of targeting virulence are a higher number of potential targets for novel antifungal therapeutics,
the preservation of host microbioma, and weaker selective pressure for the development of antibiotic
resistance [11].
C. albicans is a polymorphic fungus and is able to undergo reversible morphological transition
between yeast and filamentous forms. It has been reported that the growth of hyphae promotes
virulence and plays an important function in tissue invasion and resistance to phagocytosis [
12
].
This morphological change occurs in response to external stimuli, including nutrient availability,
high temperature, pH, and the presence of host macrophages [13].
We evaluated the inhibitory effect of oleuropein under hyphal-inducing conditions. For the
test, we used complex media containing 10% serum, which is the most potent inducer of the hyphal
morphological state [13].
After incubation at 35
◦
C for 3 h, statistically significant (p< 0.05) inhibition of morphological
transition of C. albicans cells to filamentous form was observed for samples treated with subinhibitory
concentrations (10 mg
·
mL
−1
, 5 mg
·
mL
−1
, and 1 mg
·
mL
−1
) of oleuropein in comparison to the negative
control. The results presented in Figure 2show the modulation of the morphogenetic conversion of
C. albicans under the influence of oleuropein.
Molecules 2016, 21, 1631 3 of 8
In order to understand mode of action of antifungal agent, it is necessary to investigate its effect
on virulence factors, which are essential for development of infection in the host. It was believed in the
past that yeasts passively participate in the process of pathogenesis and the establishment of fungal
infection. An immunocomprised host was considered the only mechanism responsible for the
establishment of opportunistic infection. Today, it is known that yeasts actively participate in the
pathophysiology of the disease using mechanisms called virulence factors [10]. The main advantages
of targeting virulence are a higher number of potential targets for novel antifungal therapeutics, the
preservation of host microbioma, and weaker selective pressure for the development of antibiotic
resistance [11].
C. albicans is a polymorphic fungus and is able to undergo reversible morphological transition
between yeast and filamentous forms. It has been reported that the growth of hyphae promotes
virulence and plays an important function in tissue invasion and resistance to phagocytosis [12].
This morphological change occurs in response to external stimuli, including nutrient availability,
high temperature, pH, and the presence of host macrophages [13].
We evaluated the inhibitory effect of oleuropein under hyphal-inducing conditions. For the test,
we used complex media containing 10% serum, which is the most potent inducer of the hyphal
morphological state [13].
After incubation at 35 °C for 3 h, statistically significant (p < 0.05) inhibition of morphological
transition of C. albicans cells to filamentous form was observed for samples treated with subinhibitory
concentrations (10 mg·mL−1, 5 mg·mL−1, and 1 mg·mL−1) of oleuropein in comparison to the negative
control. The results presented in Figure 2 show the modulation of the morphogenetic conversion of
C. albicans under the influence of oleuropein.
Figure 2. Effect of different concentrations of oleuropein (OLP) on germ-tube formation in C. albicans;
NC—intact cells. The data are shown as means ± SD from three independent experiments (* p < 0.05
in comparison to NC).
Important virulence factors that facilitate pathogenicity of Candida are host recognition, which
enables the pathogen to bind to the host cells and proteins, and adherence to host surfaces. Additionally,
Candida species can adhere to the surfaces of medical devices and form biofilms. The initial attachment of
Candida cells is mediated by non-specific factors (hydrophobicity and electrostatic forces) and
promoted by specific adhesins on the surface of fungal cells [14].
Studies have suggested that cell surface hydrophobicity is involved in adherence to epithelial
cells and is associated with pathogenic potential of the yeasts [15].
The hidrophobicity assay in our study showed that oleuropein below the MIC value induced a
statistically significant change in CSH levels (p < 0.05), in comparison to the control for C. albicans,
after 24 h of incubation aerobically at 25 °C by decreasing hydrophobicity from 34.05% ± 1.85% to
13.55% ± 2.46% at a concentration of 97.6 μg·mL−1 and from 34.05% ± 1.85% to 14.92% ± 1.99% at a
concentration of 48.83 μg·mL−1 of oleuropein (Figure 3a). It was noted that oleuropein has a stronger
influence on the decrease of CSH levels when samples are incubated 24 h at 25 °C under 10% CO2
conditions (Figure 3b). A statistically significant decrease in hydrophobicity under anaerobic conditions
in comparison to the control was observed at an oleuropein concentration of 97.6 μg·mL−1 (from
Figure 2.
Effect of different concentrations of oleuropein (OLP) on germ-tube formation in C. albicans;
NC—intact cells. The data are shown as means
±
SD from three independent experiments (* p< 0.05 in
comparison to NC).
Important virulence factors that facilitate pathogenicity of Candida are host recognition,
which enables the pathogen to bind to the host cells and proteins, and adherence to host surfaces.
Additionally, Candida species can adhere to the surfaces of medical devices and form biofilms.
The initial attachment of Candida cells is mediated by non-specific factors (hydrophobicity and
electrostatic forces) and promoted by specific adhesins on the surface of fungal cells [14].
Studies have suggested that cell surface hydrophobicity is involved in adherence to epithelial
cells and is associated with pathogenic potential of the yeasts [15].
The hidrophobicity assay in our study showed that oleuropein below the MIC value induced a
statistically significant change in CSH levels (p< 0.05), in comparison to the control for C. albicans,
after 24 h of incubation aerobically at 25
◦
C by decreasing hydrophobicity from 34.05%
±
1.85% to
13.55%
±
2.46% at a concentration of 97.6
µ
g
·
mL
−1
and from 34.05%
±
1.85% to 14.92%
±
1.99%
at a concentration of 48.83
µ
g
·
mL
−1
of oleuropein (Figure 3a). It was noted that oleuropein has a
stronger influence on the decrease of CSH levels when samples are incubated 24 h at 25
◦
C under 10%
Molecules 2016,21, 1631 4 of 9
CO
2
conditions (Figure 3b). A statistically significant decrease in hydrophobicity under anaerobic
conditions in comparison to the control was observed at an oleuropein concentration of 97.6
µ
g
·
mL
−1
(from 34.05%
±
1.85% to 7.44%
±
2.54%) and a concentration of 48.83
µ
g
·
mL
−1
(from 34.05%
±
1.85%
to 4.78% ±1.33%).
Molecules 2016, 21, 1631 4 of 8
34.05% ± 1.85% to 7.44% ± 2.54%) and a concentration of 48.83 μg·mL−1 (from 34.05% ± 1.85% to
4.78% ± 1.33%).
(a) (b)
Figure 3. Effect of different concentrations of oleuropein (OLP) on modulation of cellular surface
hydrophobicity in (a) aerobic conditions; and (b) anaerobic conditions; NC-intact cells. The data are
shown as means ± SD from three independent experiments (* p < 0.05 in comparison to NC).
Most therapies for fungal infections target the ergosterol biosynthesis pathway or its end
product ergosterol. This membrane sterole is unique to fungi, and is necessary for growth and the
normal membrane function of fungal cells. The primary mechanism of action by which, for example,
commonly used azole antifungal drugs inhibit yeast cell growth is through disruption of the normal
sterol biosynthetic pathway, leading to a reduction in ergosterol biosynthesis [16].
We tested the effect of oleuropein on the membrane of C. albicans cells using an ergosterol
synthesis assay. Figure 4 shows the modulation of ergosterol biosynthesis at different concentrations
of oleuropein.
Figure 4. Modulation of ergosterol content at different concentration of oleuropein (OLP);
PC-amphotericin 1 μg·mL−1; NC-intact cells. Results represent the mean of three experiments ± SD
(p < 0.05 in comparison to NC).
Analysis showed that, at subinhibitory concentrations, our phenolic component had altered sterol
content and subsequently affected the cell membrane. Further, our date indicated that the compound
decreased ergosterol content in a dose-dependent fashion. Intergroup comparisons revealed statistically
significant differences (p < 0.05) between tested concentrations. At the highest concentration
(1.25 mg·mL−1), oleuropein caused a 28% reduction in total sterol content.
Additional Candida factors important in adherence, tissue penetration, invasion, and destruction
of host tissue are extracellular hydrolytic enzymes [17]. The most significant hydrolytic enzymes
involved in the virulence of C. albicans are SAP proteins [18]. Their relationship with the virulence has
been demonstrated by the degradation of cellular substrates such as proteins related to immunological
and structural defenses of the host [19]. The present study revealed that the cultivation of C. albicans
Figure 3.
Effect of different concentrations of oleuropein (OLP) on modulation of cellular surface
hydrophobicity in (
a
) aerobic conditions; and (
b
) anaerobic conditions; NC-intact cells. The data are
shown as means ±SD from three independent experiments (* p< 0.05 in comparison to NC).
Most therapies for fungal infections target the ergosterol biosynthesis pathway or its end product
ergosterol. This membrane sterole is unique to fungi, and is necessary for growth and the normal
membrane function of fungal cells. The primary mechanism of action by which, for example, commonly
used azole antifungal drugs inhibit yeast cell growth is through disruption of the normal sterol
biosynthetic pathway, leading to a reduction in ergosterol biosynthesis [16].
We tested the effect of oleuropein on the membrane of C. albicans cells using an ergosterol
synthesis assay. Figure 4shows the modulation of ergosterol biosynthesis at different concentrations
of oleuropein.
Molecules 2016, 21, 1631 4 of 8
34.05% ± 1.85% to 7.44% ± 2.54%) and a concentration of 48.83 μg·mL−1 (from 34.05% ± 1.85% to
4.78% ± 1.33%).
(a) (b)
Figure 3. Effect of different concentrations of oleuropein (OLP) on modulation of cellular surface
hydrophobicity in (a) aerobic conditions; and (b) anaerobic conditions; NC-intact cells. The data are
shown as means ± SD from three independent experiments (* p < 0.05 in comparison to NC).
Most therapies for fungal infections target the ergosterol biosynthesis pathway or its end
product ergosterol. This membrane sterole is unique to fungi, and is necessary for growth and the
normal membrane function of fungal cells. The primary mechanism of action by which, for example,
commonly used azole antifungal drugs inhibit yeast cell growth is through disruption of the normal
sterol biosynthetic pathway, leading to a reduction in ergosterol biosynthesis [16].
We tested the effect of oleuropein on the membrane of C. albicans cells using an ergosterol
synthesis assay. Figure 4 shows the modulation of ergosterol biosynthesis at different concentrations
of oleuropein.
Figure 4. Modulation of ergosterol content at different concentration of oleuropein (OLP);
PC-amphotericin 1 μg·mL−1; NC-intact cells. Results represent the mean of three experiments ± SD
(p < 0.05 in comparison to NC).
Analysis showed that, at subinhibitory concentrations, our phenolic component had altered sterol
content and subsequently affected the cell membrane. Further, our date indicated that the compound
decreased ergosterol content in a dose-dependent fashion. Intergroup comparisons revealed statistically
significant differences (p < 0.05) between tested concentrations. At the highest concentration
(1.25 mg·mL−1), oleuropein caused a 28% reduction in total sterol content.
Additional Candida factors important in adherence, tissue penetration, invasion, and destruction
of host tissue are extracellular hydrolytic enzymes [17]. The most significant hydrolytic enzymes
involved in the virulence of C. albicans are SAP proteins [18]. Their relationship with the virulence has
been demonstrated by the degradation of cellular substrates such as proteins related to immunological
and structural defenses of the host [19]. The present study revealed that the cultivation of C. albicans
Figure 4.
Modulation of ergosterol content at different concentration of oleuropein (OLP);
PC-amphotericin 1
µ
g
·
mL
−1
; NC-intact cells. Results represent the mean of three
experiments ±SD
(p< 0.05 in comparison to NC).
Analysis showed that, at subinhibitory concentrations, our phenolic component had altered
sterol content and subsequently affected the cell membrane. Further, our date indicated that the
compound decreased ergosterol content in a dose-dependent fashion. Intergroup comparisons revealed
statistically significant differences (p< 0.05) between tested concentrations. At the highest concentration
(1.25 mg·mL−1), oleuropein caused a 28% reduction in total sterol content.
Additional Candida factors important in adherence, tissue penetration, invasion, and destruction
of host tissue are extracellular hydrolytic enzymes [
17
]. The most significant hydrolytic enzymes
Molecules 2016,21, 1631 5 of 9
involved in the virulence of C. albicans are SAP proteins [
18
]. Their relationship with the virulence has
been demonstrated by the degradation of cellular substrates such as proteins related to immunological
and structural defenses of the host [
19
]. The present study revealed that the cultivation of C. albicans
in the presence of oleuropein caused a statistically significant (p< 0.05) inhibition of SAP activity
(Figure 5).
Molecules 2016, 21, 1631 5 of 8
in the presence of oleuropein caused a statistically significant (p < 0.05) inhibition of SAP activity
(Figure 5).
Figure 5. SAP activity of C. albicans treated with different concentrations of oleuropein OLP
(1/4 MIC—3.12 mg·mL−1, 1/2 MIC—6.25 mg·mL−1, MIC—12.5 mg·mL−1); PC—pepstatin 0.1·mg·mL−1.
Results represent the mean of three experiments ± SD (* p < 0.05 in comparison to PC).
It should be noted that the effect of oleuropein on the inhibition of SAP activity was
dose-dependent. Oleuropein caused approximately 13% of inhibition at a concentration of 3.12 mg·mL−1
(1/4 MIC), 20% at a concentration of 6.25 mg·mL−1 (1/2 MIC), and 30% at a concentration of 12.5 mg·mL−1
(MIC). Results from the present study suggest that oleuropein inhibition of SAP activity might be
related to decreased pathogenicity of C. albicans.
In conclusion, the findings of our study show that oleuropein has promising in vitro activity
against C. albicans. This antifungal agent targets virulence factors essential for the establishment of
opportunistic infection. Additional studies are necessary to further investigate the mechanisms of
action of oleuropein and the possible development of a new antifungal therapeutic.
3. Materials and Methods
3.1. Materials
Candida albicans strains from the stock culture collection of the Department of Microbiology,
Faculty of Pharmacy and Biochemistry, University of Zagreb, were used for all tests performed in
this study. Oleuropein (Extrasynthese, Genay, France) was dissolved in a pH 7.4 phosphate buffer to
prepare stock solution at a concentration of 50 mg·mL−1. All other chemicals and reagents, unless
otherwise specified, were purchased from Sigma (St. Louis, MO, USA).
3.2. Methods
3.2.1. Antimicrobial Susceptibility Testing
The minimum inhibitory concentration (expressed as MIC 90%) of oleuropein against C. albicans
ATCC 10231 was assessed using the EUCAST Def. 7.3 procedure [20]. Serial broth microdilution of
oleuropein in RPMI 1640% + 2% glucose (w/v) from 25 mg·mL−1 to 12.21 μg·mL−1 was performed in a
sterile flat-bottom 96 well microtiter plate inoculated with 100 μL of C. albicans suspension adjusted
to cell density of 0.5 McFarland units (Nephelometer, bioMerioux, France). The plate was incubated
aerobically for 24 h at 35 °C. After incubation, 10 μL samples from each dilution were transferred to
Sabouraud 2% (w/v) glucose agar and further incubated for 48 h at 35 °C. Control wells contained
100 μL of cell suspension and oleuropein solvent.
3.2.2. Identification of Apoptotic and Necrotic Cells Due to Loss of Membrane Integrity
Differentiation between viable and dead cells of C. albicans ATCC 10231 was determined using the
fluorescent dye exclusion method [21]. One hundred microliters of inoculum suspension (1.5 McFarland
units) were mixed with 900 μL of RPMI 1640 (with 2% of glucose) containing oleuropein in concentrations
Figure 5.
SAP activity of C. albicans treated with different concentrations of oleuropein OLP
(1/4 MIC—3.12 mg
·
mL
−1
, 1/2 MIC—6.25 mg
·
mL
−1
, MIC—12.5 mg
·
mL
−1
); PC—pepstatin
0.1
·
mg
·
mL
−1
. Results represent the mean of three experiments
±
SD (* p< 0.05 in comparison to PC).
It should be noted that the effect of oleuropein on the inhibition of SAP activity was
dose-dependent. Oleuropein caused approximately 13% of inhibition at a concentration of
3.12 mg
·
mL
−1
(1/4 MIC), 20% at a concentration of 6.25 mg
·
mL
−1
(1/2 MIC), and 30% at a
concentration of 12.5 mg
·
mL
−1
(MIC). Results from the present study suggest that oleuropein inhibition
of SAP activity might be related to decreased pathogenicity of C. albicans.
In conclusion, the findings of our study show that oleuropein has promising
in vitro
activity
against C. albicans. This antifungal agent targets virulence factors essential for the establishment of
opportunistic infection. Additional studies are necessary to further investigate the mechanisms of
action of oleuropein and the possible development of a new antifungal therapeutic.
3. Materials and Methods
3.1. Materials
Candida albicans strains from the stock culture collection of the Department of Microbiology,
Faculty of Pharmacy and Biochemistry, University of Zagreb, were used for all tests performed in this
study. Oleuropein (Extrasynthese, Genay, France) was dissolved in a pH 7.4 phosphate buffer to prepare
stock solution at a concentration of 50 mg
·
mL
−1
. All other chemicals and reagents, unless otherwise
specified, were purchased from Sigma (St. Louis, MO, USA).
3.2. Methods
3.2.1. Antimicrobial Susceptibility Testing
The minimum inhibitory concentration (expressed as MIC 90%) of oleuropein against C. albicans
ATCC 10231 was assessed using the EUCAST Def. 7.3 procedure [
20
]. Serial broth microdilution of
oleuropein in RPMI 1640% + 2% glucose (w/v) from 25 mg
·
mL
−1
to 12.21
µ
g
·
mL
−1
was performed in
a sterile flat-bottom 96 well microtiter plate inoculated with 100
µ
L of C. albicans suspension adjusted
to cell density of 0.5 McFarland units (Nephelometer, bioMerioux, France). The plate was incubated
aerobically for 24 h at 35
◦
C. After incubation, 10
µ
L samples from each dilution were transferred to
Sabouraud 2% (w/v) glucose agar and further incubated for 48 h at 35
◦
C. Control wells contained
100 µL of cell suspension and oleuropein solvent.
Molecules 2016,21, 1631 6 of 9
3.2.2. Identification of Apoptotic and Necrotic Cells Due to Loss of Membrane Integrity
Differentiation between viable and dead cells of C. albicans ATCC 10231 was determined
using the fluorescent dye exclusion method [
21
]. One hundred microliters of inoculum suspension
(1.5 McFarland units) were mixed with 900
µ
L of RPMI 1640 (with 2% of glucose) containing oleuropein
in concentrations of 12.5 mg
·
mL
−1
, 1.25 mg
·
mL
−1
, and 0.195 mg
·
mL
−1
. Amphotericin (1
µ
g
·
mL
−1
)
served as a positive control. The samples were incubated at 35
◦
C for 3 h. DNA-binding dyes (ethidium
bromide and acridine orange) were added to the samples at a final concentration of 100
µ
g/mL (1:1;
v/v), and samples were analyzed. Three classes of cells were observed: viable, apoptotic, and
necrotic cells.
3.2.3. Inhibition of Germ-Tube Formation
The test organism C. albicans ATCC 10231 was cultured on Sabouraud 2% (w/v) glucose agar
(Merck, Darmstadt, Germany) for 24 h at 37
◦
C, aerobically. Inoculum suspension (0.5 McFarland units)
for the assay was prepared from fresh culture in physiological saline. The analysis was performed
according to the method of Zuzarte and colleagues [
22
] with slight modifications. Briefly, test tubes
contained 100
µ
L of inoculum suspension and 900
µ
L of 10% (v/v) fetal bovine serum (FBS) with
10 mg
·
mL
−1
, 5 mg
·
mL
−1
, and 1 mg
·
mL
−1
oleuropein. The negative control contained no oleuropein.
The samples were incubated at 35
◦
C for 3 h. Three hundred cells were counted in a Neubauer chamber
using phase-contrast microscopy and the number of yeast cells with germ-tubes versus non-germinated
cells were calculated.
3.2.4. Modulation of Cellular Surface Hydrophobicity Levels
Modulation of cellular surface hydrophobicity (CSH) levels were assessed according to the method
reported by Ishida et al. [
23
]. Inoculum suspension was prepared from fresh cultures of C. albicans
ATCC 10231 in a pH 7.4 phosphate buffer with optical density of 0.5
±
0.05 at 620 nm. Yeasts were
treated with 97.6
µ
g
·
mL
−1
, 48.83
µ
g
·
mL
−1
, and 24.41
µ
g
·
mL
−1
of oleuropein and incubated at 25
◦
C
for 24 h. Additionally, incubation was also carried out at 25
◦
C for 24 h under 10% CO
2
conditions.
Following incubation, 1.2 mL of treated yeast suspension was added to 0.6 mL of xylene, mixed by
vortexing for 30 s, and left for 10 min at room temperature until the 2 phases separated. The aqueous
phase of the sample was measured in a spectrophotometric 96-well plate reader (iEMS Reader,
Labsystem, Finland) at 620 nm and hydrophobicity was calculated using the following equation:
(Acontrol −Atest)×100/Acontrol (1)
where Acontrol is the optical density before treatment, and Atest is the optical density after treatment.
3.2.5. Modulation of Membrane Ergosterol Content
The inhibition of ergosterol synthesis was determined according to the method of
Arthington-Skaggs et al. [
24
] in inoculums prepared from fresh cultures of C. albicans ATCC
10231 treated with different concentrations of oleuropein (24.41
µ
g
·
mL
−1
, 195.31
µ
g
·
mL
−1
,
and 1250
µ
g
·
mL
−1
). The samples were incubated at 37
◦
C for 18 h on an orbital shaker (170 rpm)
aerobically. The sample treated with amphotericin served as a positive control (1
µ
g
·
mL
−1
). Following
incubation, the cells were harvested by centrifugation (2700
×
g, 5 min), and the weight of the cell
pellet was determined. Three milliliters of freshly prepared alcoholic potassium hydroxide solution
(25% m/v) was added to each pellet and vortexed vigorously for 1 min. Obtained cell suspensions
were transferred to borosilicate glass tubes and incubated for one hour at 85
◦
C in a water bath and
then allowed to cool. The sterol extraction was enabled by the addition of water:n-heptane mixture
(1:3 v/v) followed by vortexing for 3 min. The produced heptane layer was transferred to a new
borosilicate glass tube with a screw cap and stored (
−
20
◦
C). Prior to scanning, 0.6 mL of sterol
extract was diluted in 100% ethanol (1:5) and scanned between 240 and 300 nm at 5 nm (Varian Cary
Molecules 2016,21, 1631 7 of 9
1 UV-Vis spectrophotometer, Agilent, Santa Clara, CA, USA). The ergosterol content was calculated as
a percentage of the wet weight of the cell using the following equations:
%ergosterol + %24(28) DHE = [(A281.5/290) ×F]/cell mass (2)
%24(28) DHE = [(A230/518) ×F]/cell mass (3)
%ergosterol = [%ergosterol + %24(28) DHE] −%24(28) DHE (4)
where F is the factor of sample dilution in ethanol (1:5), and 290 and 518 are the E values (in percentages
per centimeter) determined for crystalline ergosterol and 24(28) DHE, respectively.
3.2.6. Inhibition of Secreted Aspartyl Proteinase Activity
The influence of oleuropein on the secreted aspartyl proteinases (SAP) activity of C. albicans
was evaluated according to the method described by Yordanov et al. [
25
]. Fresh culture of vaginal
clinical isolate of C. albicans MFBF 11103 from the stock culture collection of the Department of
Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, was used for the test.
The secretion of aspartyl proteinases was induced by adding C. albicans suspension (0.5 McFarland
units) to a BSA-Remold liquid medium (2% (m/v) glucose, 0.1% (m/v) KH
2
PO
4
, 0.5% (m/v) MgSO
4
,
0.7% (m/v) YNB, and 1% (m/v) BSA (without (NH
4
)
2
SO
4
and amino acids)). The samples were
cultivated at 27
◦
C for 7 days on orbital shaker (150 rpm) aerobically. Following cultivation, cells were
removed, and filtrated culture supernatant was used to evaluate the influence of oleuropein on the
enzyme activity. Test tubes contained 0.2 mol
·
L
−1
sodium citrate–HCl buffer, 1% BSA in the same
buffer, culture supernatant, and oleuropein in different concentrations (12.5 mg
·
mL
−1
, 6.25 mg
·
mL
−1
,
and 3.12 mg
·
mL
−1
). The sample treated with protease inhibitor pepstatin A dissolved in 5% DMSO
served as a positive control. Test tubes with mixtures were incubated at 37
◦
C for 60 min (T
60
), and the
reaction was terminated with 20% trichloroacetic acid. An additional control was prepared by adding
20% trichloroacetic acid to all ingredients simultaneously (T
0
). Following centrifugation at 3000
×
g
for 30 min, 160
µ
L of clear supernatant was mixed with 40
µ
L of dye reagent concentrate Coomassie
Brilant Blue G-250, and the optical density (OD) at 595 nm was measured. Protease activity was
calculated as the difference in the OD: T60 −T0.
4. Statistical Analysis
All experiments were performed as triplicates at three independent occasions. Presented values
are the mean and standard deviation.
Statistical significance of the data was evaluated using a one-way ANOVA with Dunnett’s post
test and an X2test. The level of statistical significance was set at p< 0.05.
Author Contributions:
N.Z. is responsible for design of the study and for participation in all aspects of the
work, including the analysis and interpretation of data and the drafting of the manuscript; N.K. participated in
a fluorescent dye exclusion assay including the analysis and interpretation of acquired data; I.B. participated
in inhibition of the ergosterol synthesis assay; I.H. participated in the cellular surface hydrophobicity assay;
S.T. participated in the design of the study and helped to draft the manuscript; I.K. conceived of the study and
participated in its design and coordination.
Conflicts of Interest: The authors declare no conflict of interest.
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