Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism with fluconazole and nystatin against Candida spp.

Article abstract of DOI:10.1016/j.diagmicrobio.2016.08.002

We tested the antifungal potential of caffeic acid and 8 of its derivative esters against Candidaalbicans ATCC 90028 and 9 clinical isolatesand carried out a synergism assay with fluconazole and nystatin. Propyl caffeate (C3) showed the best antifungal ac

Full text of DOI:10.1016/j.diagmicrobio.2016.08.002

Synthesis, antifungal activity of caffeic acid derivative esters, and their
synergism with fluconazole and nystatin against Candida spp.
Jana ´ı na de C a´ ssia Orlandi Sardi, Fernanda Patr ´ı cia Gullo, Irlan Almeida
Freires, Nayla de Souza Pitangui, Maicon Petr oˆ nio Segalla, Ana Marisa
Fusco-Almeida, Pedro Luiz Rosalen, Lu ´ı s Oct a´ vio Regasini, Maria Jos e´
Soares Mendes-Giannini
PII:
DOI:
Reference:
S0732-8893(16)30239-5
doi: 10.1016/j.diagmicrobio.2016.08.002
DMB 14156
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date:
Revised date:
Accepted date:
7 March 2016
11 July 2016
5 August 2016
Please cite this article as: de C ´a ssia Orlandi Sardi Jana ´ı na, Gullo Fernanda Patr ´ı cia,
Freires Irlan Almeida, de Souza Pitangui Nayla, Segalla Maicon Petr oˆ nio, Fusco-Almeida
Ana Marisa, Rosalen Pedro Luiz, Regasini Lu ´ı s Oct ´a vio, Mendes-Giannini Maria Jos ´e
Soares, Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism
with fluconazole and nystatin against Candida spp., Diagnostic Microbiology and Infectious
Disease (2016), doi: 10.1016/j.diagmicrobio.2016.08.002
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ACCEPTED MANUSCRIPT
Synthesis, antifungal activity of caffeic acid derivative esters, and their
synergism with fluconazole and nystatin against Candida spp.
1
,3,#
1,#
3
Janaína de Cássia Orlandi Sardi , Fernanda Patrícia Gullo , Irlan Almeida Freires ,
1
2
1
Nayla de Souza Pitangui Maicon Petrônio Segalla , Ana Marisa Fusco-Almeida , Pedro
3
2,4
1,*
Luiz Rosalen , Luís Octávio Regasini , Maria José Soares Mendes-Giannini
1
.
Department of Clinical Analysis, Laboratory of Clinical Mycology, Faculty of
Pharmaceutical Sciences, UNESP – Univ Estadual Paulista, Araraquara, SP, 14801-902
Brazil.
2
Institute of Chemistry, Department of Biochemistry and Chemical Technology, UNESP –
Univ Estadual Paulista, Araraquara, SP 14800-060, Brazil
3
Department of Physiological Sciences, Piracicaba Dental School, University of Campinas,
Piracicaba, SP, 13414-90, Brazil
4
Institute of Biosciences, Letters and Exact Sciences, Department of Chemistry and
Environmental Sciences, São José do Rio Preto, SP 15054-000, Brazil
#
These authors contributed equally to this work.
Correspondence to:
*
Dr. Maria José Soares Mendes Giannini, Faculty of Pharmaceutical Sciences of
Department of Clinical Analysis, Laboratory of Clinical Mycology, Univ Paulista
UNESP), R. Expedicionários do Brasil, 1621, CEP. 14801-902, Araraquara, São Paulo,
(
Brazil
Tel: +55 – 16 – 3301 - 5716
e-mail: gianninimj@gmail.com / janasardi@gmail.com
Key words: Synergism, Candida albicans, Caffeic acid, Antifungal
1

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Abstract
We tested the antifungal potential of caffeic acid and eight of its derivative esters against C.
albicans ATCC 90028 and nine clinical isolates, and carried out a synergism assay with
fluconazole and nystatin. Propyl caffeate (C3) showed the best antifungal activity against
the tested strains. When in combination, C3 markedly reduced the MIC of fluconazole and
nystatin with synergistic effect up to 64-fold. Finally, C3 showed a high IC50 value and
Selective Index (SI) against oral keratinocytes, demonstrating low toxicity against this cell
type and selectivity for yeast cells. Further research should confirm its antifungal potential
for development of combined therapy to treat C. albicans infections.
Introduction
Oral candidiasis is one of the most common opportunistic infections afflicting
humans, with Candida albicans as the major causative agent of this disease (Garcia-Cuesta
et al., 2014). The complexity of interactions between Candida and other microorganisms in
the host, mainly bacteria, suggest that several mechanisms are involved in yeast fitness to
the oral cavity. Some studies have shown that Candida spp. can co-aggregate with bacteria
in dental plaque. This feature may be an important factor for the onset of oral candidiasis as
well as fungal colonization of carious cavities and periodontal pockets (Sardi et al., 2012;
Thurnheer et al., 2015). The presence of yeasts in subgingival regions may contribute to the
pathogenesis of periodontal disease or increase the chance of candidemia, especially in
cases of immunosuppression (Hannula et al., 2001; Reynaud et al., 2001; Al Mubarak et
al., 2013). In addition, it has been well documented that systemic diseases such as diabetes
and AIDS; physiological conditions such as pregnancy, infancy or old age; nutritional
factors; treatment with broad-spectrum antibiotics; use of immunosuppressive drugs and
corticosteroids; xerostomia, and use of dentures, may predispose the individual to develop
candidiasis (Soll, 2002; Tekeli et al., 2004; Manfredi et al., 2006).
The current therapy with antifungals has serious drawbacks, in particular due to
toxic effects to human cells and adverse effects (Epstein et al., 2002; Gabler et al., 2008).
As the drugs used to treat candidiasis are not always specific and properly prescribed
(targeting the causative agent of infection), there has been a significant increase in
resistance of Candida spp. to traditional antifungal drugs. The increasing microbial
resistance rates may also be a result of long-term drug exposure or selection of strains with
2

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intrinsic resistance mechanisms (Ying et al., 2013; Fernandez-Ruiz et al., 2015; Liao et al.,
2
015; Seifi et al., 2015; Freitas et al., 2015). Therefore, the development of novel strategies
to minimize the toxic effects of current antifungals and improve their effectiveness, has
been strongly encouraged.
Natural products have continued to be a rich source of new drugs with clinically
significant biological targets. Over the past 34 years, 49% of FDA-approved
chemotherapeutic drugs were either natural products or directly derived therefrom
(Newman & Cragg, 2016). There is a great interest of the pharmaceutical industry in the
discovery of new molecules of natural origin or even their combination with existing drugs,
in order to improve efficacy, potency, safety, tolerability, and decrease production costs,
side effects and selection of resistant strains (Svetaz et al., 2016). A number of studies in
the literature have established the value of combined antifungal therapy against resistant
strains, in particular standard drugs with naturally-occurring agents (Pippi et al., 2015; Han
et al., 2016).
Caffeic acid (3,4-dihydroxycinnamic acid) is an important phenolic compound
commonly found in plants, foods and propolis samples, particularly in the form of caffeic
acid phenethyl ester (CAPE) (Paracatu et al., 2014; Rzepecka-Stojko et al., 2015). It is
better known for its pharmacological properties, including antimicrobial, antioxidant, anti-
inflammatory and anticancer (Balachandran et al., 2012; Kuo et al., 2015). Nevertheless,
modifications of the caffeic acid structure into esters or amides, for instance, may generate
novel analog molecules with enhanced and desired biological activity (Touaibia et al.,
2
011), particularly as antimicrobials (Fu et al., 2010).
Herein, we investigated the antifungal potential of caffeic acid and eight of its
derivative esters against C. albicans ATCC 90028 and nine oral clinical isolates. The most
active molecule, propyl caffeate (C3), was selected for a synergism assay with fluconazole
and nystatin against the C. albicans strains, and tested for its toxicity on oral keratinocytes
(NOK cells).
Material and Methods
Synthesis of Esters. Caffeic acid (0.2 mM) solution and corresponding alcohols (20 mM)
º
were prepared at 5 C with a solution of N, N′-dicyclohexylcarbodiimide (DCC, 1.0 mM) in
p-dioxane (3.0 mL). After the solution was stirred for 48 h, the solvent was removed under
reduced pressure. The residue was partitioned three-times with EtOAc and filtered. The
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filtrate was serially washed with saturated aqueous citric acid solution (three times),
saturated aqueous NaHCO (three-times), water (two-times), dried over MgSO , and
3
4
evaporated under reduced pressure. The crude products were purified over a silica gel
column using an isocratic system of CHCl –MeOH (98:2). The modifications made in
3
caffeic acid molecule are shown in Table 1.
Microorganisms. Candida albicans ATCC 90028 strain and nine highly virulent clinical
isolates of C. albicans obtained from the oral cavity of patients with diabetics and
periodontitis (Sardi et al., 2012), were used in this study. This study was approved by the
research ethics committee at Piracicaba Dental School, University of Campinas, Piracicaba,
SP, Brazil (protocol no. 062/2008).
Determination of Antifungal Activity. The Minimum Inhibitory Concentration (MIC) of
caffeic acid and its eight derivatives against C. albicans ATCC 90028 was determined
using 96-well microplates based on the protocol M27-A3 of the CLSI (2008), with
modifications. The esters that showed the lowest MIC values against C. albicans ATCC
9
0028 (C3, C4 and C6) were then tested against nine clinical isolates of C. albicans. The
synthetic compounds of caffeic acid were diluted in DMSO and tested in concentrations
ranging from 250 µg/mL to 0.48 µg/mL (Scorzoni et al., 2007). The inoculum was
3
prepared (λ 530 nm, Abs 0.08-0.1) and diluted to 2.5 x 10 CFU/mL. The plates were
o
incubated at 35 C for 24 h. The MIC was determined as the lowest concentration of the
1
00
compound inhibiting visible fungal growth as indicated by 0.1% resazurin (Sigma-Aldrich,
St. Louis, MO, USA). Aliquots from the wells corresponding to the MIC and higher
®
concentrations were sub-cultured on Sabouraud Dextrose Agar (Difco , Detroit, MI, USA)
for determination of the Minimum Fungicidal Concentration (MFC). The MFC was defined
as the lowest concentration of the compound causing no visible growth on the agar plate.
Combinatorial Antifungal Activity (Synergism Assay). The ester which showed the best
activity against C. albicans strains (C3) was combined with conventional antifungals
commonly used for the treatment of candidiasis, fluconazole and nystatin. Their
combinatorial antifungal activity was determined through the checkerboard method using
9
6-well microplates (Dai et al., 2015). A mathematical calculation was used to generate the
fractional inhibitory concentration index (FICI), as follows: FICI = (MIC compound 1 in
combination / MIC compound 1 alone) + (MIC compound 2 in combination / MIC
compound 2 alone). The combinations were classified as synergistic (FICI ≤ 0.5), additive
4

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(0.5 < FICI ≤ 1.0), indifferent (1.0 < FICI < 4.0) and antagonistic (FICI ≥ 4.0) (Soares et al.,
2
014).
Cytotoxic Effects on Oral Keratinocytes. The most active ester derivative (C3) as well as
fluconazole and nystatin were tested for their cytotoxicity against keratinocytes from the
oral mucosa of humans (NOK cells) provided by the Department of Medicine at Harvard
Medical School (Dr. Karl Munger). NOK cells were maintained in culture medium for
keratinocytes without fetal bovine serum (Gibco, Life Technologies) at 36.5ºC and 5% CO2.
4
Cells were seeded onto a 96-well plate at a density of 5 x 10 cells/well for 24 h. Then the
cells were exposed to the treatments (C3, Fluconazole, Nystatin, and their combinations)
for 24 h. Cell viability was determined by adding aliquots of 10µL of MTT solution (5
mg/mL) (Sigma-Aldrich, St. Louis, MO) to each well. The plates were incubated at 37ºC
for 4 h to allow for visualization of precipitated formazan crystals. Aliquots of 100 µL of
isopropyl alcohol were added per well and absorbance was read using a spectrophotometer
at 560 nm (Mosmann et al., 1983). Hydrogen peroxide (10%) was used as a positive
control for cytotoxicity and untreated cells were considered as the negative control. Based
on this cell viability assay, we next determined the half maximal inhibitory concentration
(
IC ) for the esters, standard-drugs and their combinations. The IC50 was defined as the
50
effective concentration of the compound able to inhibit 50% of the NOK cells. The
establishment of this concentration was essential to calculate the selectivity index (SI)
(
IC50/MIC ratio), which was used as an indicator of potential toxicity against normal cells
lines at the effective therapeutic concentration for each strain. Hence, the selectivity index
(SI) indicated the relationship between drug toxicity against C. albicans and the host cells
(Gullo et al., 2012; Mora-Navarro et al., 2016).
Statistical Analysis. All assays were performed in triplicate of independent experiments.
The data were analyzed on Graphpad Prism 5.0 (San Diego, CA, USA) by one-way
analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, with a
significance level of 5%.
Results
Structure-Activity Relationship of Caffeic Acid Derivatives. A screening for antifungal
activity of caffeic acid and eight derivative esters against C. albicans was carried out.
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These esters differ by the number of constituent carbons in the molecule, as shown in Table
1
. Caffeic acid and all of its synthesized derivatives showed a fungicidal activity against C.
albicans ATCC 90028 and presented a structure-activity relationship. As seen in Table 2,
the caffeic acid molecule (C0) without structural change and the derived C1 showed MIC
and MFC values of 125 µg/mL. The derivatives C2, C5, C7 and C8 showed MIC and MFC
values of 31.25 µg/mL. C3 and C4 showed strong fungicidal activity against C. albicans
ATCC 90028, with MIC and MFC values of 15.62 µg/mL, together with C6 which showed
MIC and MFC equal to 7.81 µg/mL. Thus, the compounds C3, C4 and C6 – which were
better than their originating caffeic acid molecule – were further tested against nine oral
clinical isolates of C. albicans. As shown in Table 3, the compound C3 demonstrated the
best activity when compared with the other caffeic acid derivatives (C4 and C6), with MIC
and MFC values ranging between 7.81 and 62.5 µg/mL. The MIC and MFC of C4 and C6
ranged between 31.25-125 µg/mL and 7.81-≥250 µg/mL, respectively.
Combinatorial Antifungal Activity with Standard Drugs. Given the strong antifungal
activity of C3 against C. albicans strains, we next investigated its combinatorial activity
with the standard drugs fluconazole and nystatin. A synergistic activity was observed for
the combination between C3 and fluconazole against most strains, with FICI values
ranging from 0.06 to 0.5. Indifferent activity was observed against two clinical isolates,
with FICI values close to 2.0. When comparing the MIC values of C3 and fluconazole
alone and combined, we observed a decrease in their MIC and potentiation of fluconazole
of 2 to 64-fold (Table 4). The combination of C3 with nystatin was advantageous
(synergistic) against three out of the ten strains when compared with the MIC values of the
compounds tested alone, with potentiation of nystatin by 8- to 64-fold. Additive activity
was observed against four clinical isolates and C. albicans ATCC 90028 strain, with FICI
values ranging from 0.53 to 1.0. The additive activity of the combination showed
potentiation of nystatin between 32- and 64-fold.
Toxic Effects on Oral Keratinocytes. A viability assay was carried out to evaluate the
toxicity of C3 and standard drugs alone and in combination, against NOK cells. The
antifungal compound C3 showed low toxicity at the tested concentrations, with a high IC50
value of 420 µg/mL. Likewise, fluconazole and nystatin showed high IC50 values of 320
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µg/mL and 400 µg/L, respectively. When combined, both C3 and the antifungals had their
SI value increased, indicating selectivity for yeasts rather than NOK cells (Table 5).
Figure 1 shows the percentage of cell viability of NOK cells treated with the
antifungal compounds at different concentrations. The data showed that treatment with C3,
fluconazole and nystatin maintained over 80% cell viability at all tested concentrations.
Hydrogen peroxide, used as a positive control, markedly affected cell viability, with a
significant difference when compared to C3 and standard drugs (P < 0.05).
Discussion
Opportunistic infections caused by Candida spp. have still been considered a
recurrent health issue with high burden worldwide (Rodriguez-Tudela et al., 2015; Denning
&
Gugnani, 2015). Thus, novel therapeutic approaches are much needed to treat Candida
infections, including the use of naturally-occurring agents. In this study, we demonstrate
the antifungal potential of caffeic acid derivatives against C. albicans and their successful
synergism with antifungals commercially available.
Caffeic acid is commonly found in fruits (Balachandran et al., 2012) and propolis
samples (Freires et al., 2016) used in daily life products and folk medicine. We showed that
caffeic acid molecule has fungicidal activity against C. albicans ATCC 90028.
Nevertheless, the structural modifications performed in this study rendered the caffeic acid
molecule much more effective in terms of fungicidal activity, showing a structure-activity
relationship. The addition of 2 to 8 carbon atoms in the caffeic acid molecule increased its
anti-Candida activity. Other studies have also demonstrated the relationship between the
number of carbons and the antifungal activity of the molecule (Nihei et al., 2003; Soares et
al., 2014). Among the synthesized compounds, C3 showed better activity when compared
with other caffeic acid derivatives (C4 and C6), showing lower minimum inhibitory and
fungicidal concentrations against most strains. Balachandran et al. (2012) isolated methyl
caffeate from Solanum torvum plant and showed antibacterial activity against Gram-
negative and Gram-positive bacteria, in addition to antifungal activity against C. albicans
and Apergillus flavus. These findings confirm the antimicrobial potential of caffeic acid
derivatives.
Of note, the clinical isolates used in this study showed lower susceptibility to
nystatin and fluconazole when compared with the reference strain of C. albicans ATCC
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9
0028. This could be due to acquisition of resistance mechanisms as the isolates belonged
to diabetic patients. A recent study showed that this group of patients is prone to develop
Candida infections in cases of poor glycemic control (Zomorodian et al., 2016).
Microbial resistance has raised concern over the last decades as the investment in
antibiotic discovery is declining considerably over time when compared to high-priced
drugs such as chemotherapeutics (Bax & Green, 2015). To make it worse, failure in
prescribing the appropriate drug, misuse, and long hospital stay have led to the emergence
of azole-resistant isolates, particularly in cases of invasive infections (Liao et al., 2015;
Sanguinetti, Posteraro & Lass-Flörl, 2015). This opens avenues for the development of
combined antifungal therapy, in which drugs with different mechanisms of action (or not)
are combined to enhance their antifungal potency and avoid selection of resistant strains.
Here, we demonstrated the successful combination of propyl caffeate (C3) with fluconazole
and nystatin against C. albicans strains. A study performed by Lee & Han (2005)
demonstrated the synergistic effect of the combination between amphotericin B and
berberine (alkaloid) against C. albicans. These authors also tested this combination in mice
with candidemia and observed that the combined treatment prolonged the lives of mice in
2
2 days compared to those treated with amphotericin B alone. Other studies have reported
the successful combination of essential oils with conventional drugs, including nystatin,
fluzonazole and micafugin (Aprotosoaie et al., 2008; Rosato et al., 2009; Rodrigues et al.,
2
012; Stringaro et al., 2014).
With the purpose of future clinical use, we also investigated the effects of C3
against oral keratinocytes and compared them with those of the standard drugs. NOK cells
were chosen for this study as they constitute the main epithelial cell type lining the oral
mucosa, which would be highly exposed in case of administration of an oral suspension or
a solution for oral candidiasis. Overall, low toxicity was found for C3, fluconazole and
nystatin, with IC values higher than their MIC/MFC. These agents also showed a high SI
5
0
value, which means that if the index is greater than 10.0 the compound exhibits selectivity
for killing yeasts rather than the host’s cells. The combination between C3 and fluconazole
led to a considerable increase in their SIs, highlighting their selectivity for yeast cells.
Collectively, our findings indicate that the propyl ester modification of caffeic acid
molecule (C3) has strong fungicidal activity and potentiated the effects of fluconazole and
nystatin against C. albicans strains, with little effects against oral keratinocytes. Further
8

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studies should focus on the effects of these combinations against Candida biofilms and
establish C3 mechanism(s) of action.
The authors declare no conflict of interest.
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Figure 1
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Table 1. Nomenclature, molecular formulas and the chemical structures of the caffeic acid
derivative esters tested in this study.
Code
C0
Nomenclature Molecular Formula
Chemical Structure
Caffeic acid
C H O
9 8 4
C1
Methyl caffeate
C
10
H
8
O
4
C2
C3
C4
Ethyl caffeate
Propyl caffeate
Butyl caffeate
C
11
12
13
H
12
H
14
H
16
O
4
O
4
O
4
C
C
C5
C6
Pentyl caffeate
Hexyl caffeate
C
C
14
H
18
O
4
H
20
O
4
15
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C7
C8
Heptyl caffeate
C
16
H
22
O
4
Octyl caffeate
C
H
24
O
4
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Table 2. Antifungal activity of caffeic acid derivative esters against Candida albicans
ATCC 90028.
Caffeic Acids
Candida albicans ATCC 90028
Derivates
MIC (µg/mL)
125
MFC (µg/mL)
125
C0
C1
125
125
C2
C3
31.25
15.62
15.62
31.25
7.81
31.25
15.62
15.62
31.25
7.81
C4
C5
C6
C7
31.25
31.25
4.0
31.25
31.25
4.0
C8
Nystatin
Fluconazole
0.5
0.5
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Table 3. Antifungal activity of caffeic acid derivative esters (C3, C4 and C6) against nine oral clinical
isolates of Candida albicans. MIC/MFC values are expressed in µg/mL.
C. albicans
clinical isolates
Ca#22
C3
C4
C6
Fluconazole
Nystatin
MIC MFC
MIC MFC
31.25 31.25
MIC MFC
MIC MFC
MIC MFC
7.81
7.81
7.81
125
125
7.81
125
125
8.0
8.0
8.0
2.0
0.5
8.0
8.0
8.0
4.0
8.0
8.0
8.0
2.0
0.5
8.0
8.0
8.0
4.0
8.0
8.0
8.0
4.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
4.0
8.0
8.0
8.0
8.0
8.0
Ca#25
31.25 31.25 62.50 62.50
15.62 15.62 31.25 31.25
Ca#45
Ca#50
31.25 31.25
31.25 31.25 62.50 62.50 >250 >250
7.81 7.81 31.25 31.25 7.81 7.81
31.25 31.25 62.50 62.50 >250 >250
125
125
>250 >250
Ca#61
Ca#62
Ca#63
Ca#105
Ca#124
31.25 31.25
62.50 62.50
125
125
125
125
>250 >250
>250 >250
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Table 4. Antifungal effects of propyl caffeate (C3) combined with fluconazole and nystatin
against Candida albicans ATCC 90028 and nine oral clinical isolates. MIC values are
expressed in µg/mL.
FCZ + C3
C3
NYST + C3
FICI
C. albicans strains
FCZ
MIC)
0.25
Effect /Potentiation
FCZ
NYST
(MIC)
0.125
0.125
4.0
C3
Effect /Potentiation
NYST
FICI
(
(MIC)
0.12
(MIC)
ATCC 90028
Ca#22
0.50
2.01
0.06
0.13
0.37
0.50
2.01
0.18
0.07
0.12
Synergistic /2X
Indifferent/64X
Synergistic/16X
Synergistic/8X
Synergistic/4X
Synergistic/2X
Indifferent/64X
Synergistic/8X
Synergistic/16X
Siyergistic/ 8X
7.81
0.53
2.01
1.50
1.06
0.24
1.01
2.01
0.51
1.03
0.28
Additive/32X
0.125
0.5
15.62
0.12
15.62
31.25
15.62
3.90
Indifferent/64X
Indifferent/2X
Indifferent/16X
Synergistic/8X
Indifferent/64X
Indifferent/64X
Additive /64X
Indifferent/32X
Synergistic/32X
Ca#25
Ca#45
1.0
0.12
0.5
Ca#50
0.5
3.90
0.5
Ca#61
0.25
0.125
1.0
0.12
0.125
0.125
0.125
0.25
31.25
15.62
15.62
31.25
15.62
Ca#62
15.62
1.95
Ca#63
Ca#105
Ca#124
0.5
0.48
0.5
0.12
0.25
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Table 5. IC50 value and Selective Index (SI) of propyl caffeate (C3), fluconazole (FCZ)
and nystatin (NYST) showing the toxicity of the compounds against yeasts in relationship
to human oral keratinocytes (NOK cells).
Compound
C3
IC (µg/mL)
SI (alone)
SI (combination)
5
0
420
320
400
13.5
40
26.9 (FCZ) / 13.5 (NYST)
FCZ
320
100
NYST
50
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Highlights


Caffeic acid and eight of its derivative esters were tested for their activity against Candida spp.
Propyl caffeate (C3) was the most active compound against reference strain and clinical
isolates.



When combined, C3 markedly reduced the MIC of fluconazole and nystatin up to 64-fold.
C3 showed low toxicity against oral keratinocytes and high selectivity for yeast cells.
Further research should focus on its combined therapy to treat C. albicans-related infections.
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