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Article
In Vitro and In Vivo Anti-Candida spp. Activity of
Plant-Derived Products
Reginaldo dos Santos Pedroso 1,2,3,†, Brenda Lorena Balbino 1, †, Géssica Andrade 1,
Maria Cecilia Pereira Sacardo Dias 1, Tavane Aparecida Alvarenga 1,
Rita Cássia Nascimento Pedroso 1, Letícia Pereira Pimenta 1, Rodrigo Lucarini 1,
Patrícia Mendonça Pauletti 1, Ana Helena Januário 1, Marco Túlio Menezes Carvalho 4,
Mayker Lazaro Dantas Miranda 5and Regina Helena Pires 1, *
1Universidade de Franca, Franca 14404-600, SP, Brazil; rpedroso@ufu.br (R.d.S.P.);
brendalorenabalbino@hotmail.com (B.L.B.); gessicaandrade16123@gmail.com (G.A.);
ceciliasacardo@outlook.com (M.C.P.S.D.); tavanealvarenga@gmail.com (T.A.A.);
ritinha-pedroso@hotmail.com (R.C.N.P.); leticia_pimenta94@hotmail.com (L.P.P.);
rodrigolucarini@hotmail.com (R.L.); patricia.pauletti@unifran.edu.br (P.M.P.);
ana.januario@unifran.edu.br (A.H.J.)
2Escola Técnica de Saúde, Universidade Federal de Uberlândia, Uberlândia 38400-902, MG, Brazil
3Programa de Pós-graduação em Ciências da Saúde, Universidade Federal de Uberlândia,
Uberlândia 38400-902, MG, Brazil
4Universidade Estadual de Minas Gerais, Passos 37902-407, MG, Brazil; marcotulioibc@outlook.com
5Instituto Federal do Triângulo Mineiro, Campus Uberlândia Centro, Uberlândia 38.064-300, MG, Brazil;
maykermiranda@iftm.edu.br
*Correspondence: regina.pires@unifran.edu.br
†Both authors contributed equally to this work.
Received: 15 September 2019; Accepted: 8 November 2019; Published: 11 November 2019
Abstract:
Candidiasis therapy, especially for candidiasis caused by Candida non-albicans species,
is limited by the relatively reduced number of antifungal drugs and the emergence of antifungal
tolerance. This study evaluates the anticandidal activity of 41 plant-derived products against Candida
species, in both planktonic and biofilm cells. This study also evaluates the toxicity and the therapeutic
action of the most active compounds by using the Caenorhabditis elegans–Candida model. The planktonic
cells were cultured with various concentrations of the tested agents. The Cupressus sempervirens,
Citrus limon, and Litsea cubeba essential oils as well as gallic acid were the most active anticandidal
compounds. Candida cell re-growth after treatment with these agents for 48 h demonstrated that
the L. cubeba essential oil and gallic acid displayed fungistatic activity, whereas the C. limon and C.
sempervirens essential oils exhibited fungicidal activity. The C. sempervirens essential oil was not toxic
and increased the survival of C. elegans worms infected with C. glabrata or C. orthopsilosis. All the
plant-derived products assayed at 250
µ
g/mL affected C. krusei biofilms. The tested plant-derived
products proved to be potential therapeutic agents against Candida, especially Candida non-albicans
species, and should be considered when developing new anticandidal agents.
Keywords: plant-derived products; Candida;C. elegans; anticandidal agents
1. Introduction
Infections caused by yeasts belonging to the genus Candida affect especially immunocompromised
individuals, children, elderly, individuals hospitalized in Intensive Care Units (ICU), and users of
invasive devices [
1
]. Vulvovaginitis caused by Candida and Candida-associated stomatitis also represent
important infections in the field of Public Health [2,3].
Plants 2019,8, 494; doi:10.3390/plants8110494 www.mdpi.com/journal/plants
Plants 2019,8, 494 2 of 17
Factors such as the use of immunosuppressive drugs, broad-spectrum antibiotics, and antifungal
agents for prophylaxis have increased the number of patients that are susceptible to opportunistic
diseases, including candidiasis, particularly candidiasis caused by non-albicans Candida (NAC) species
such as C. glabrata,C. krusei,C. parapsilosis,C. tropicalis, and, more recently, C. auris [4].
Depending on the microenvironment’s nutritional content, micro-organisms, including Candida,
can grow in the planktonic or the biofilm form. The biofilm is represented by aggregated, organized,
and functional micro-organisms embedded in an exopolymeric matrix, which allows irreversible
adhesion to biotic or abiotic surfaces [
5
]. Microbial biofilms are the main cause of hospital infections
and the source of many recurrent and persistent diseases [
4
,
5
]. Furthermore, NAC species leading
to infections, including species that may be resistant to more than one class of antifungal agents,
have contributed to increasing the intrinsic or acquired resistance of Candida isolates to antifungal
drugs [4,5].
The search for alternatives for the primary or complementary therapy of infections caused by
Candida has been constant. In this context, plant-derived products allow the discovery of new agents
with potential application in the clinical setting and in the development of drugs for systemic and/or
topical use [6].
Plants have several secondary metabolites that display antimicrobial activity. For instance,
plant essential oils (EOs) consist of various naturally associated compounds among which terpenes
(monoterpenes and sesquiterpenes), aromatic compounds (aldehyde, alcohol, phenol, and methoxy
derivative), and terpenoids (isoprenoids) predominate [
7
]. In the case of yeast-like fungi, terpenes
have been reported to inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, cell
growth and signaling modulators, apoptosis initiators, and cell cycle arrest inducers [8].
In addition, essential oils may be rich in phenolic compounds, which present antioxidant properties
due to their ability to act as hydrogen donors, reducing agents, singlet oxygen depleters, and metal
chelators [
7
,
9
,
10
]. Thus, the activity of essential oils is related to the composition, functional groups,
and synergistic interactions between the components [
9
], not to mention that the harvesting period
also determines the concentrations of the main components in the plant oil [10].
Approximately half of the drugs that were approved for use between 1940 and 2006 were derived
from natural products [
11
], which exhibited antimicrobial, anticancer, antidiabetic, and antidepressant
actions, among others [
10
]. This highlights that plants are a potential source of bioactive molecules.
However, there is limited knowledge about the activity of plant-derived products against NAC species,
mainly against C. glabrata and C. krusei, which are less susceptible or resistant to fluconazole, the most
widespread antifungal for prophylactic and/or therapeutic use [4].
The development of novel antifungal drugs requires both safety and toxicity assessment. Currently,
in vivo
studies have increasingly employed the so-called alternative models, which replace animals
such as mice and rats with other animals, such as the Danio rerio fish, the Galeria mellonella larvae,
and the Caenorhabditis elegans nematode. These alternative models are suitable to investigate acute or
systemic toxicity, pharmacokinetics, and infections. C. elegans offers advantages that include its easy
handling in the laboratory, reasonable cost, and known genome, which enables toxic compounds to be
screened and their antifungal activity against Candida yeasts to be evaluated [12].
In this study, the antifungal action of plant-derived products against Candida species growing both
as planktonic and biofilm cells has been investigated. In addition, the toxicity and the effectiveness of
the most active compounds have been studied by using a C. elegans–Candida infection model.
2. Results
Forty-one plant-derived products were tested against six Candida species (Table 1). Among the
tested essential oils (EOs, 30 samples), the Cupressus sempervirens (cypress) EO presented the best result;
it acted against all the Candida species. The minimum inhibitory concentration (MIC) values were
250, 250, 62.5, 31.25, 62.5, and 31.25
µ
g/mL against C. albicans,C. tropicalis,C. krusei,C. glabrata,C.
parapsilosis, and C. orthopsilosis, respectively (Table 1).
Plants 2019,8, 494 3 of 17
Table 1. Minimal Inhibitory Concentration (µg/mL) values of essential oils obtained against Candida species.
Essential Oils Candida albicans
SC 5314
Candida tropicalis
ATCC 13803
Candida krusei
ATCC 6258
Candida glabrata
ATCC 2001
Candida parapsilosis
ATCC 22019
Candida orthopsilosis
ATCC 96141
Betula pendula Roth. >2000 >2000 >2000 >2000 >2000 >2000
Cananga odorata >2000 >2000 >2000 >2000 2000 >2000
Cedrus atlantica >2000 >2000 >2000 >2000 >2000 >2000
Cinnamomum zeylanicum (leaves) 500 500 500 500 500 500
Citrus aurantium (Petitgrain) >2000 >2000 >2000 >2000 >2000 >2000
Citrus limon (L.) Burm 500 250 500 250 500 500
Citrus nobilis (peels) 2000 >2000 >2000 2000 >2000 >2000
Citrus reticulata (peels) 2000 1000 250 1000 1000 250
Citrus reticulata Blanco (dry leaves) >2000 >2000 >2000 2000 2000 2000
Citrus reticulata Blanco (peels) 1000 2000 500 1000 1000 1000
Citrus reticulata var. cravo (peels) >2000 >2000 >2000 2000 >2000 2000
Citrus reticulata Blanco (fresh leaves) 2000 2000 >2000 >2000 >2000 >2000
Citrus sinensis L (peels) >2000 >2000 >2000 >2000 >2000 >2000
Cupressus sempervirens (leaves) 250 250 62.5 31.25 62.5 31.25
Cymbopogon citratus (DC) Stapf 2000 >2000 2000 2000 >2000 >2000
Cymbopogon martinii 2000 2000 2000 2000 2000 2000
Cymbopogon nardus 2000 2000 2000 2000 2000 2000
Eucalyptus globulus >2000 >2000 1000 2000 >2000 >2000
Eugenia caryophyllus >2000 >2000 2000 2000 2000 2000
Litsea cubeba 500 1000 62.5 250 500 250
Melaleuca alternifolia >2000 >2000 2000 >2000 >2000 >2000
Mentha arvensis L. 2000 2000 2000 2000 2000 2000
Mentha piperita L. >2000 >2000 >2000 >2000 >2000 >2000
Origanum vulgare L. 500 500 1000 500 1000 1000
Pelargonium graveolens >2000 >2000 2000 2000 2000 >2000
Piper aduncun L. (leaves) >2000 >2000 >2000 >2000 >2000 >2000
Piper aduncun L. (inflorescences) >2000 >2000 >2000 2000 >2000 2000
Piper aduncun L. (branches) 2000 >2000 >2000 2000 2000 2000
Psidium cattleyanum (dry leaves) >2000 >2000 >2000 >2000 >2000 >2000
Rosmarinus officinalis >2000 >2000 >2000 >2000 >2000 >2000
Plants 2019,8, 494 4 of 17
The Citrus limon EO provided MIC values of 250
µ
g/L against C. tropicalis and C. glabrata (Table 1).
The Litsea cubeba EO yielded MIC values of 62.5, 250, and 250
µ
g/mL against C. krusei, C. glabrata, and
C. orthopsilosis, respectively (Table 1). Finally, the Citrus reticulata EO afforded MIC values of 250
µ
g/mL
against both C. krusei and C. orthopsilosis (Table 1). The first three products were tested against Candida
in the following assays.
According to the adopted criteria for antimicrobial activity, none of the five plant extracts tested
here (Table 2) were active against the evaluated Candida strains, their MIC values were higher than
2000 µg/mL.
Table 2. Minimal inhibitory concentrations (MIC) of plant extracts against Candida species.
Plant Extracts
Candida Species (MIC µg/mL)
C. albicans
ATCC 5314
C. tropicalis
ATCC 13803
C. krusei
ATCC 6258
C. glabrata
ATCC 2001
C.
parapsilosis
ATCC 22019
C.
orthopsilosis
ATCC 96141
Anacardium occidentale
(ethanolic extract) >2000 >2000 >2000 >2000 >2000 >2000
Anacardium othonianum
(ethanolic extract) >2000 >2000 >2000 >2000 >2000 >2000
Curcuma longa
(ethanolic extract) >2000 >2000 >2000 >2000 >2000 >2000
Curcuma longa L.
(aqueous extract) >2000 >2000 >2000 >2000 >2000 >2000
Vochysia divergens stem
(ethanolic extract) >2000 -* >2000 >2000 >2000 -*
-*: They were not evaluated.
Among the seven plant-derived compounds tested herein (Table 3), gallic acid showed the greatest
activity; its MIC values were 125, 31.25, 250, and 250
µ
g/mL against C. krusei,C. glabrata,C. parapsilosis,
and C. orthopsilosis, respectively, so this acid was selected for further studies. Against the C. parapsilosis
ATCC 22019 and the C. krusei ATCC 6258 reference strains, amphotericin B (AMB) gave MIC values of
0.25 and 1.00 µg/mL, respectively.
Table 3.
Minimal inhibitory concentration (MIC) of plant-derived compounds against Candida species.
Compounds
Candida Species/MIC (µg/mL)
C. albicans
SC5314
C. tropicalis
ATCC 13803
C. krusei
ATCC 6258
C. glabrata
ATCC 2001
C.
parapsilosis
ATCC 22019
C.
orthopsilosis
ATCC 96141
Alpha-bisabolol
>2000 >2000 2000 >2000 >2000 >2000
Benzoic acid >2000 >2000 >2000 >2000 >2000 >2000
Caffeic acid >2000 >2000 1000 500 >2000 500
Ferulic acid >2000 >2000 >2000 >2000 >2000 >2000
Gallic acid 500 1000 125 31.25 250 250
Menthol >2000 >2000 >2000 >2000 >2000 >2000
Salicylic acid >2000 >2000 >2000 >2000 >2000 >2000
The EOs were extracted from Cupressus sempervirens and Citrus limon leaves and Litsea cubeba fruits
in 0.65%, 1.5%, and 1.0% yield, respectively. GC-MS and GC-FID analyses helped to identify 13, 9, and
11 chemical constituents in the EOs extracted from Cupressus sempervirens (total 99.1%) and Citrus limon
(total 98.1%) leaves and Litsea cubeba fruits (total of 96.2%), respectively. Sabinene, terpinen-4-ol, citral,
limonene, neral, and geraniol were the major compounds in these EOs. Table 4lists all the identified
compounds, retention indexes, and relative area percentages (% RA).
Plants 2019,8, 494 5 of 17
Table 4.
Chemical composition of essential oils (EOs) from Cupressus sempervirens and Citrus limon
leaves and Litsea cubeba fruits.
Compounds % RA
RI C. sempervirens C. limon L. cubeba
α-Pinene 934 8.0 - 2.0
Sabinene 969 20.3 - 1.3
β-Pinene 974 - - 2.0
Myrcene 991 6.0 - 1.3
δ-2-Carene 1001 4.0 - -
p-Cymene 1023 5.0 - -
Limonene 1024 3.9 53.4 37.0
cis-Limonene oxide 1129 - 2.0 -
trans-Limonene oxide 1133 - 7.0 -
γ-Terpinene 1054 4.0 - -
Citronelol 1150 - 3.8 -
Linalool 1095 - 1.9 4.0
Terpinen-4-ol 1177 15.4 - -
α-Terpineol 1186 2.4 - 2.3
Neral 1238 5.0 11.0 31.4
Citral 1249 20.0 - 12.0
Geraniol 1268 - 9.0 1.2
Nerol 1363 - 6.0 -
Geraniol acetate 1384 - 4.0 -
β-Caryophyllene 1415 - - 1.7
δ-Cadinene 1522 3.0 - -
Cedrol 1598 2.1 - -
Total 99.1 98.1 96.2
RI: Retention index; % RA: relative area percentage.
The Litsea cubeba EO was tested against C. krusei, and gallic acid was assayed against C. glabrata
and C. krusei. The tested agents were fungistatic at all the concentrations (Figure 1A–C). The Citrus
limon EO at 0.5
×
MIC (125
µ
g/mL) or 1
×
MIC (250
µ
g/mL) exerted fungicidal action against C. tropicalis
(Figure 1D) after 4 h. The Citrus limon EO at 2
×
MIC (500
µ
g/mL) had a fungicidal effect on C. tropicalis
(Figure 1D) and C. glabrata (Figure 1E) after 2 h. A fungicidal effect emerged after exposure of C.
orthopsilosis (Figure 1F) to the C. sempervirens EO at 0.5
×
MIC (15.6
µ
g/mL), 1
×
MIC (31.25
µ
g/mL), and
2
×
MIC (62.5
µ
g/mL) for 8, 6, and 4 h, respectively. This same EO at 0.5
×
MIC exhibited fungistatic
activity against C. glabrata after 8 h. This EO, at 1
×
MIC or 2
×
MIC, displayed a fungicidal effect
against C. glabrata after 12 h (Figure 1G).
Table 5depicts the minimal biofilm-inhibiting concentration (MBIC) and the minimal
biofilm-eradicating concentration (MBEC) obtained with the Litsea cubeba,Citrus limon, and Cupressus
sempervirens EOs and gallic acid. The Cupressus sempervirens EO gave the best antibiofilm activity; the
MBIC and MBEC values ranged between 62.5 and 1000
µ
g/mL against all the Candida species (Table 5).
The lowest MBIC and MBEC values were achieved against Candida krusei at 62.5 and 250
µ
g/mL,
respectively (Table 5).
Plants 2019,8, 494 6 of 17
Plants 2019, 8, x FOR PEER REVIEW 6 of 18
A B
C D
E F
G
Figure 1.
Kill assays for plant-derived products against Candida species. The concentrations 0.5
×
MIC, 1
×
MIC, and 2
×
MIC correspond to: (
A
)Litsea cubeba
×
C. krusei: 31.25, 62.5, and 125
µ
g/mL;
(
B
) Gallic acid
×
C.glabrata 31.25, 62.5, and 125
µ
g/mL; (
C
) Gallic acid
×
C. krusei: 62.5, 125, and
250
µ
g/mL; (
D
)Citrus limon
×
C. tropicalis: 125, 250, and 500
µ
g/mL; (
E
)Citrus limon
×
C. glabrata: 125,
250, and 500
µ
g/mL; (
F
)Cupressus sempervirens
×
C. orthopsilosis: 15.62, 31.25, and 62.5
µ
g/mL; (
G
)
Cupressus sempervirens
×
C. glabrata: 15.62, 31.25, and 62.5
µ
g/mL. AMB: 4
µ
g/mL amphotericin B and
Untreated: Candida species’ growth without plant-derived products. The results are expressed as the
mean colony-forming units (CFU)/mL ±standard deviation from three independent experiments.
Plants 2019,8, 494 7 of 17
Table 5. Activity of the essential oils and gallic acid on biofilm formation and against preformed Candida species biofilms.
Compounds
Candida Species (µg/mL)
C. albicans
SC 5314
C. glabrata
ATCC 2001
C. tropicalis
ATCC 13803
C. krusei
ATCC 6258
C. parapsilosis
ATCC 22019
C. orthopsilosis
ATCC 96141
MBIC * MBEC ** MBIC * MBEC ** MBIC * MBEC ** MBIC * MBEC ** MBIC * MBEC ** MBIC * MBEC **
Citrus limon (L.) Burm 2000 1000 1000 1000 2000 1000 125 250 2000 1000 1000 1000
Cupressus sempervirens 1000 1000 250 1000 500 1000 62.5 250 500 1000 125 1000
Gallic acid >2000 >2000 >2000 >2000 >2000 >2000 250 500 500 >2000 >2000 >2000
Litsea cubeba 2000 1000 2000 1000 2000 1000 250 1000 1000 1000 2000 1000
* MBIC: minimum biofilm formation inhibiting concentration (capable of reducing ≥90% optical density (OD) compared to the control free of chemical substances); ** MBEC: minimum
biofilm-eradicating concentration (capable of reducing ≥90% OD compared to the control free of chemical substances).
Plants 2019,8, 494 8 of 17
The antifungal activity findings obtained in the
in vitro
assays were confirmed by using an
in vivo
infection model, namely the Caenorhabditis elegans–Candida infection assay, which is regarded
as an infection model to study Candida-associated infections. Initially, the toxicity of the selected
plant-derived products was evaluated by testing approximately 15–20 late-L4 larvae in each well
of a 96-well microplate, exposed to concentrations of the selected products of 0.5
×
MIC,
1×MIC
,
and 2 ×MIC
at 25
◦
C for 24 h. The Litsea cubeba and Cupressus sempervirens EOs at concentrations
between 31.25 and 125
µ
g/mL as well as gallic acid at concentrations between 15.62 and 250
µ
g/mL
were not toxic (p>0.05) against C. elegans as compared to untreated larvae (data not shown). In turn,
the Citrus limon EO at 0.5
×
MIC (125
µ
g/mL) was not toxic (p>0.05), but this same EO at 1
×
MIC
(250
µ
g/mL) and 2
×
MIC (500
µ
g/mL) was significantly toxic (p<0.05 and p<0.0001, respectively)
(data not shown).
The C. glabrata-infected larvae were treated with the Cupressus sempervirens (Figure 2A) and Citrus
limon (Figure 2B) EOs and gallic acid (Figure 2C) at 25
◦
C for four days. Only in the presence of
Cupressus sempervirens was a higher frequency of viable larvae maintained (Figure 2A). In contrast,
the larvae infected with C. krusei, and treated with the Litsea cubeba EO (Figure 2D) at concentrations
between 31.25 and 125
µ
g/mL or gallic acid (Figure 2E) at concentrations between 62.5 and 250
µ
g/mL,
were not cured of candidiasis.
The Citrus limon EO at concentrations between 125 and 500
µ
g/mL was used to treat larvae
infected with C. tropicalis (Figure 2F). After 24 h, a small percentage of dead larvae (5–10%) was
detected. However, after 48 h, 35% and 55% of the larvae died at EO concentrations of 125
µ
g/mL and
250–500
µ
g/mL, respectively. At the end of four days, only 40% and 10–15% of the larvae survived,
respectively, at the same concentrations.
Lastly, worms infected with C. orthopsilosis were treated with the Cupressus sempervirens EO
(Figure 2G). Exposure to this EO (15.62 to 62.5
µ
g/mL) increased the survival of C. elegans worms
infected with C. orthopsilosis as compared to the treated control. At four days postinfection, 80–85% of
the infected and treated larvae survived.
Plants 2019,8, 494 9 of 17
Plants 2019, 8, x FOR PEER REVIEW 10 of 18
Figure 2. Survival curves of the responses to the tested compound concentrations from the Caenorhabditis
elegans–Candida infected model. Nematode survival diminished at 0.5 × MIC, 1 × MIC, and 2 × MIC for all the
compounds, except for the Cupressus sempervirens EO. A—C. elegans infected with C. glabrata and treated with
C. sempervirens EO, B—C. elegans infected with C. glabrata and treated with C. limon EO, C—C. elegans infected
with C. glabrata and treated with gallic acid, D—C. elegans infected with C. krusei and treated with L. cubeba EO,
E—C. elegans infected with C. krusei and treated with gallic acid, F—C. elegans infected with C. tropicalis and
treated with C. limon, G—C. elegans infected with C. orthopsilosis and treated with C. sempervirens. The untreated
control group is represented by the green lines; the infected control, the fungicidal control drug (amphotericin
B), and the different concentrations of the tested compounds are represented by symbols. The results were
obtained from three independent experiments with at least three replicates.
C. elegans infected with C. glabrata treated with C. limon C. elegans infected with C. glabrata treated with C. sempervirens
C. elegans infected with C. glabrata treated with gallic ac id C. elegans infected with C. krusei treated with L. cubeba
C. elegans infected with C. tropicalis treated with C. limon C. elegans infected with C. krusei treated with gallic acid
C. elegans infected with C. orthopsilosis treated with C. sempervirens
Figure 2.
Survival curves of the responses to the tested compound concentrations from the Caenorhabditis
elegans–Candida infected model. Nematode survival diminished at 0.5
×
MIC, 1
×
MIC, and 2
×
MIC for
all the compounds, except for the Cupressus sempervirens EO. (
A
)—C. elegans infected with C. glabrata
and treated with C. sempervirens EO, (
B
)—C. elegans infected with C. glabrata and treated with C. limon
EO, (
C
)—C. elegans infected with C. glabrata and treated with gallic acid, (
D
)—C. elegans infected with
C. krusei and treated with L. cubeba EO, (
E
)—C. elegans infected with C. krusei and treated with gallic
acid, (
F
)—C. elegans infected with C. tropicalis and treated with C. limon, (
G
)—C. elegans infected with C.
orthopsilosis and treated with C. sempervirens. The untreated control group is represented by the green
lines; the infected control, the fungicidal control drug (amphotericin B), and the different concentrations
of the tested compounds are represented by symbols. The results were obtained from three independent
experiments with at least three replicates.
Plants 2019,8, 494 10 of 17
3. Discussion
In Brazil, plant-derived products have gained importance because of the publication of Resolution
971 (3 May 2006) [
13
] and Act 5813 (22 June 2006) [
14
], which regulate the National Policy on Integrative
and Complementary Practices and the National Policy on Medicinal and Phytotherapeutic Plants,
respectively. These regulations introduced the use of medicinal plants and phytotherapeutic drugs
into the Unified Health System (SUS) and aimed to ensure safe access of the Brazilian population
to these medications as well as their rational application, promoting the sustainable use of the
national biodiversity.
In this scenario, this study evaluated the antifungal potential of plant-derived products (essential
oils, Brazilian native plant extracts, and plant constituents) that have been employed as antimicrobials
in folk medicine. Among the EOs assessed herein, the Cupressus sempervirens, Citrus limon, and Litsea
cubeba EOs are noteworthy. According to literature data, the EO extracted from Cupressus sempervirens
leaves exhibits similar chemical composition to the composition identified here, albeit with different
percentages. For instance, Selim et al. [
15
] reported that
α
-pinene (48.6%),
δ
-3-carene (22.1%), limonene
(4.6%), and
α
-terpinolene (4.5%) are the main constituents of Cupressus sempervirens studied in Saudi
Arabia, whereas Ibrahim et al. [
16
] described
α
-pinene (21.15%), terpinen-4-ol (6.98%), allo-ocimene
(24.00%), and
α
-cedrol (23.68%) as the main components of Egyptian Cupressus sempervirens. As
for Cupressus sempervirens growing in Brazil, we determined some of these substances at lower
concentrations as well as the compounds sabinene (20.3%) and citral (20.0%), which were reported at
higher concentrations in the EO extracted from Cupressus sempervirens leaves for the first time (Table 4).
The Cupressus sempervirens anticandidal activity against C. albicans has been demonstrated, but it
has not been defined as fungistatic or fungicidal [
17
,
18
]. Here, we showed the greater susceptibility of
C. glabrata and C. orthopsilosis to the Cupressus sempervirens EO (Table 1). These Candida species have
been cited as causing a significant increase in Candida infections in the last few years [
19
]. Additionally,
C. glabrata is the NAC species that has been the most commonly isolated from the environment and by
health practitioners in a Brazilian Tertiary Hospital [
20
], and C. orthopsilosis has been identified as the
prevalent organism among yeasts isolated from the hydraulic system of a hemodialysis facility [21].
Exposure to the Cupressus sempervirens EO completely inhibited C. orthopsilosis (Figure 1F) and
C. glabrata (Figure 1G) cells. The fungicidal action of this EO could be partly attributed to its
major constituents such as sabinene, citral, and terpinen-4-ol, which have been reported to display
antimicrobial effects [
22
–
24
]. Moreover, at 2 x MIC, this EO provided the same effect as 4
µ
g/mL AMB
against C. orthopsilosis (Figure 1F), which is the best antifungal drug concentration with fungicidal
action that has been described in in vivo studies [25].
The C. elegans larvae infected with C. glabrata (Figure 2A) or C. orthopsilosis (Figure 2G) and treated
with the Cupressus sempervirens EO at concentrations between 15.62 and 62.5
µ
g/mL had significantly
(p<0.05) higher survival as compared to the infected larvae control four days postinfection. This
suggested that this EO might be a valuable antifungal agent against Candida infections. Besides
that, this EO showed antibiofilm activity (MBIC or MBEC) against all the tested Candida non-albicans
species (Table 5), which pointed out that it could be an adjuvant in the treatment of biofilm-associated
Candida-non-albicans infections.
The Citrus limon EO exhibited anticandidal activity (MIC) against all the assayed strains (Table 1).
At concentrations between 125 and 500
µ
g/mL and between 250 and 500
µ
g/mL, this EO displayed a
fungicidal effect against C. tropicalis ATCC 13803 (Figure 1D) and C. glabrata ATCC 2001 (Figure 1E),
respectively. The antifungal mechanism of the Citrus limon EO is associated with its main component,
limonene [
26
], which was detected at a similar concentration to the concentration reported by Campelo
et al. [
27
]. Limonene damages the C. albicans cell wall/membrane, thereby modifying cellular adhesion
and plasticity, pH, and ionic content [
26
]. In addition, such damage causes oxidative stress and
consequent DNA damage, resulting in cell cycle modulation and apoptosis as demonstrated by Thakre
et al. [
28
]. The high concentration of limonene in the EO could contribute to its nematocidal effect [
29
].
Plants 2019,8, 494 11 of 17
The Litsea cubeba EO is known to possess diverse biological properties, among which the
antimicrobial action is worthy of note [
30
]. Here, this EO afforded the best activity against C. krusei
ATCC 6258, with MIC and MBIC values of 62.5 and 250
µ
g/mL, respectively. Its anti-Candida
effect can be justified by the presence of chemical constituents such as limonene, citral, neral,
terpinen-4-ol, and geraniol (Table 4), which have previously been described to present anti-Candida
activity [
26
,
31
]. Moreover, in agreement with a previous study [
32
], we confirmed the Litsea cubeba EO
nematocidal activity.
Gallic acid had great inhibitory action against C. glabrata and C. krusei (Table 1), but better
antibiofilm activity against C. krusei (Table 5). Previous studies have shown antifungal activity for gallic
acid against C. albicans and filamentous fungi [
33
–
36
]; however, its activity against Candida biofilms
has been poorly investigated. This compound can inhibit biofilm formation [
34
], as confirmed by the
C. krusei antibiofilm result (MBIC) recorded here. Gallic acid toxicity to C. elegans larvae has been
reported at concentrations starting from 120 µg/mL [36].
The fungistatic and fungicidal actions of the EOs make them promising alternatives to treat
superficial candidiasis; that is, to treat oral candidiasis and denture stomatitis by topical administration,
since they can be included in mouth rinses or toothpastes [
37
]. Interestingly, we detected that all the
assayed plant-derived products at 250
µ
g/mL had an effect on C. krusei biofilms (Table 5), suggesting that
these products might be valuable antifungal agents in the therapy against C. krusei biofilm-associated
infections. This organism is an important pathogenic Candida species that is frequently refractory
to conventional antimicrobial agents and has been isolated from patients with oral candidiasis [
38
].
Further studies using an
in vivo
biofilm-associated animal model (e.g., a rat model of acute dermal
toxicity) are necessary to confirm that the EOs might be useful to treat candidal biofilm-associated
infections, especially topical infections.
4. Materials and Methods
4.1. Essential Oils
Citrus reticulata (peel), Citrus reticulata Blanco (peel and fresh and dry leaves), Citrus reticulata var.
cravo (peel), Cupressus sempervirens (leaves), Citrus limon (L.) Burm (leaves), and Litsea cubeba (fruits)
were harvested in Rio Verde (17
◦
99.4
0
63.2
0
’ S and 51
◦
05.2
0
44.6
0
’ W), GO, Brazil, on January 2nd, 2017, at
09:00. Voucher specimens (#CR-25, #CRB-25
0
, #CRC-25
0
’, #CS556, #CL89, and #LC2800) were deposited
in the herbarium at the Instituto Federal Goiano (IF-GOIANO), in Rio Verde. Briefly, distilled water
(500 mL) was added to the plant material (100 g) and transferred to a Clevenger-type apparatus. The
essential oil (EO) was collected, and the remaining water was eliminated with anhydrous sodium
sulfate, which was followed by filtration. This method was used in triplicate, and the obtained EOs
were kept under refrigeration (4
◦
C). The mean quantities of the EOs (w/w) were obtained on the basis
of the plant material weight and data from three experiments.
Cananga odorata,Cedrus atlantica,Citrus aurantium (Petitgrain), Citrus sinensis L. (peel), Cymbopogon
martinii,Cymbopogon nardus,Eucalyptus globulus,Eugenia caryophyllus,Melaleuca alternifolia,Mentha
arvensis,Mentha piperita L., Origanum vulgare L., Pelargonium graveolens,Piper aduncum L., and Rosmarinus
officinalis EOs were purchased from FERQUIMA
®
(Vargem Grande Paulista, SP, Brazil). Betula pendula
Roth., Citrus nobilis (peel), and Psidium cattleyanum (fresh leaves) EOs were acquired from LASZLO
®
(Belo Horizonte, MG, Brazil). Cinnamomum zeylanicum and Cymbopogon citratus (DC) Stapf EOs were
obtained from AROMATERÁPICA®(Sorocaba, SP, Brazil).
4.2. Plant Extracts
Anacardium occidentale L. and Anacardium othonianum cashew nuts were obtained from a local
market in Franca (Oct/2013) and from Montes Claros de Goias (Mar/2017). Voucher specimens (SPFR
16040 and HJ3793) were deposited in the Biology Department Herbarium in the Plant Systematics
Laboratory of the Faculty of Philosophy, Sciences and Letters of Ribeir
ã
o Preto, University of S
ã
o Paulo,
Plants 2019,8, 494 12 of 17
Brazil (Herbarium SPFR) and in the Herbarium Jataiense Germano Guarim Neto of the Goi
á
s Federal
University, Brazil (Herbarium HJ). The air-dried powdered A. occidentale and A. othonianum nuts were
extracted with ethanol.
Vochysia divergens was collected in the Pantanal area, in the State of Mato Grosso (16
◦
35
0
22” S and
56
◦
47
0
83” W) in January 2017. A voucher specimen UFMT 39559 was deposited in the Herbarium of
the Mato Grosso Federal University (UFMT), Brazil (Herbarium UFMT). The V. divergens stem barks
were powdered and exhaustively extracted by maceration at room temperature; ethanol was employed.
After filtration, the solvent was removed under reduced pressure to yield the ethanolic extract.
The Curcuma longa L. extracts were provided by Dr. Marco T
ú
lio Menezes Carvalho, from the
State University of Minas Gerais, MG, Brazil. The dried rhizomes were obtained from a local market
(September 2017) in Passos, MG (20
◦
43
0
13
0
’ S and 46
◦
36
0
36
0
’ W), and the powdered material was stored
in the dark. The extracts were obtained by maceration of the powdered curcuma rhizomes, at room
temperature; boiling water or ethanol/water (50:50, v/v) was employed.
4.3. Plant-Derived Compounds
The plant-derived compounds gallic acid, caffeic acid, ferulic acid, benzoic acid, salicylic acid,
menthol, and alpha-bisabolol were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4.4. Candida Species
Reference strains of six Candida species, including C. albicans SC 5314, C. glabrata ATCC 2001,
C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. tropicalis ATCC 13803, and C. orthopsilosis ATCC
96141 were used in this study. The strains were maintained at
−
70
◦
C in sterile distilled water plus
50% glycerol and subcultured in Sabouraud dextrose agar (SDA, Difco, Detroit, MI) and CHROMagar
Candida medium (Becton Dickinson and Company, Sparks, MD) at 37
◦
C for 24 h to ensure purity
and viability.
4.5. Minimum Inhibitory Concentration Determination
The
in vitro
antifungal susceptibility assays of all the natural products were performed by the
broth microdilution method according to the adapted protocol M27-S4 from the Clinical and Laboratory
Standards Institute [
39
]. Sterile microtiter plates (Corning Inc., NY, USA) were used. The inoculum
size was 2.5
×
10
3
cells/mL. The final concentration of amphotericin B (AMB) and the tested products
ranged from 0.03 to 16
µ
g/mL and from 3.90 to 2.000
µ
g/mL, respectively. AMB and all the natural
products were solubilized in DMSO (2%) and diluted in Roswell Park Memorial Institute (RPMI 1640,
Sigma-Aldrich, St. Louis, MO, USA) medium added with 0.2% glucose. The C. parapsilosis ATCC
22019 and C. krusei ATCC 6258 strains and AMB were included as quality control [
39
]. The minimum
inhibitory concentration (MIC) was determined with the fluorometric indicator resazurin at 0.01%
(w/v) [
40
]. MIC was defined as the lowest antifungal/product concentration that maintained a blue
hue. The AMB breakpoint adopted herein was
≤
1
µ
g/mL, whereas AMB >1
µ
g/mL was considered as
resistant [
41
]. The wells where micro-organism growth occurred displayed the pink color. Plant-derived
products were considered active when MIC was <100
µ
g/mL, moderately active when MIC ranged from
100 up to 500
µ
g/mL, and weakly active when MIC was >500 and <1000
µ
g/mL. Above 1000
µ
g/mL,
the products were considered inactive [42]. All the tests were conducted in triplicate.
4.6. Identification of the Chemical Composition of the EOs
Gas chromatography-flame ionization detection and gas chromatography–mass spectrometry
analyses were accomplished with Shimadzu QP2010 Plus and GCMS2010 Plus (Shimadzu Corporation,
Kyoto, Japan) systems. The GC-MS and GC-FID conditions and the identification of the chemical
constituents of the EOs were carried out in agreement with the methodology proposed by Santos et
al. [43].
Plants 2019,8, 494 13 of 17
4.7. Time-Kill Curves
All the compounds and natural products were evaluated at 0.5
×
MIC, 1
×
MIC, and 2
×
MIC
for each more susceptible Candida strain, at predetermined incubation time points (0, 2, 4, 6, 8, 12,
24, and 48 h). AMB was used at 4
µ
g/mL [
25
]. The Candida strains were subcultured on Sabouraud
Dextrose Agar (SDA) plates at 35
◦
C for 24 h, suspended in 5 mL of RPMI 1640, and adjusted to a
0.5 McFarland turbidity standard (1 to 5
×
10
6
cells/mL) with a nephelometer (ATB 1550, BioM
é
rieux,
France). Next, the micro-organism suspension was diluted in RPMI 1640, to give a new suspension
containing 2.5–5.0
×
10
4
cells/mL, and the plant product was added at an appropriate concentration.
The latter suspension was incubated at 35
◦
C for 48 h, and 100
µ
L aliquots were removed at each time
point. Tenfold serial dilutions were performed, and 10
µ
L aliquots were plated on SDA plates and
incubated at 35
◦
C for 24 h. The mean colony-forming unit (CFU) value was converted to the respective
log CFU/mL value. The data were plotted as log CFU/mL against time point. A fungicidal activity was
considered to exist when micro-organism growth decreased by at least 3 log
10
CFU/mL as compared
to the initial inoculum, to result in a reduction of 99.99% of CFU/mL. In turn, a fungistatic activity
was considered to exist when micro-organism growth was less than 99.9% or <3 log
10
in CFU/mL as
compared to the initial inoculum [22,44]. The experiments were conducted in triplicate.
4.8. Evaluation of the Effects on Biofilm
Two groups of tests were completed to evaluate the activity of the tested plant products
against Candida biofilms: i) inhibition of biofilm formation was assessed to determine the minimal
biofilm-inhibiting concentration (MBIC) and ii) effect on the preformed biofilm was analyzed
to determine the minimal biofilm-eradicating concentration (MBEC). Three EOs, namely the
Litsea cubeba,Citrus limon, and Cupressus sempervirens EOs, and one plant-derived compound
(gallic acid) were selected after MIC determination. A sterile 96-well flat-bottom microtiter
plate (Corning) was used. Inhibition/eradication of biofilm formation by the Candida strains
was assayed according to a previously described methodology [
45
]. The EOs and gallic acid
(concentration range from 1.95 to 2000
µ
g/mL) were dissolved in DMSO and two-fold diluted
in RPMI 1640 medium at 35
◦
C for 48 h. Biofilm viability was measured by the tetrazolium salt
(sodium 3
0
- [1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) hydrated benzene
sulfonic acid, XTT) reduction assay, by adding 100
µ
L of XTT-menadione to each well. Wells
containing culture medium/biofilms/XTT/menadione (positive control) and wells containing culture
medium/XTT/menadione (negative control) were included. The optical density (OD) was read on a
microtiter plate reader (Asys - Eugendorf, Salzburg, Austria) at a wavelength of 492 nm [
45
]. The
effective concentration of the EO/chemical compound capable of reducing
≥
90% OD as compared to
the control free of a chemical substance (100% of survivors) was considered as the MBIC or MBEC.
Each experimental condition was tested in triplicate, and the arithmetic mean of the results was used
to present the results.
4.9. In Vivo Toxicity to Caenorhabditis elegans
Acute toxicity was measured after exposure of Caenorhabditis elegans to the tested plant product for
24 h. The AU37 [glp-4(bn2) I; sek-1(km4) X] mutant strain was kindly provided by the S
ã
o Paulo State
University, Dr. J
ú
lio de Mesquita Filho, Instituto de Ci
ê
ncia e Tecnologia, Departamento de Bioci
ê
ncias
e Diagn
ó
stico Bucal, S
ã
o Jos
é
dos Campos, SP, Brazil. Nematodes grew on nematode growth medium
(NGM) agar plates, seeded with Escherichia coli OP50 and incubated at 16
◦
C. They were synchronized
by treatment with sodium hypochlorite, transferred, and incubated in NGM without E. coli OP50.
The worms were then washed with NaCl 50 mM. Around 20 worms were added to the wells of
96-well microplates containing culture broth (60% 50mM NaCl, 40% BHI (brain heart infusion broth),
cholesterol (10
µ
g/mL), kanamycin (90
µ
g/mL), and ampicillin (200
µ
g/mL)). Selected plant-derived
products were added to each well at 0.5
×
MIC, 1
×
MIC, or 2
×
MIC; AMB was added at 1.0
µ
g/mL. The
Plants 2019,8, 494 14 of 17
96-well microplates were maintained at 25
◦
C, and individual worm survival was assessed after 24 h,
Nematodes were considered dead when they were rod-shaped and did not respond to touching [
46
,
47
].
Two independent experiments were carried out for each treatment.
4.10. Infection Assay of Caenorhabditis elegans–Candida Species
C. elegans AU37 fed with E. coli OP50 were maintained at 16
◦
C and synchronized as described
above. Candida species were grown on BHI-agar, and worms in stage L4 were added to the plates
containing each Candida species. Next, the plates with Candida and worms were incubated at 25
◦
C
for 2 h. Then, the worms were washed with 50 mM NaCl, and C. elegans suspension was adjusted
to contain 15–20 L4 larvae per well in a 96-well microplate [
46
]. In each well, the plant product was
added at 0.5
×
MIC, 1
×
MIC, or 2
×
MIC concentrations. The plates were incubated at 25
◦
C for
four days. Worms not treated with a plant-derived product and worms infected with Candida species
served as controls. Worm survival was expressed as a percentage of worm viability at day zero. Three
independent experiments with at least three replicates were performed.
5. Statistical Analysis
Statistical analyses were done with the Program GraphPad Prism 5.0 (GraphPad Software Inc., San
Diego, CA, USA). The survival curve of C. elegans was plotted by using the Kaplan–Meier method, and
the survival differences were analyzed by log-rank (Mantel–Cox). A value of p<0.05 was considered
statistically significant.
Author Contributions:
Conception or design: R.d.S.P., P.M.P., A.H.J., M.L.D.M., R.H.P.; Experimental work
and data analysis: R.d.S.P., B.L.B., M.C.P.S.D., M.L.D.M., T.A.A., R.C.N.P., L.P.P., R.H.P.; Contributed with
reagents/materials/analysis tools: R.L., G.A., M.T.M.C., M.L.D.M., P.M.P., A.H.J., R.H.P.; Manuscript writing and
final approval of the version to be published: R.S.P., B.L.B., P.M.P., A.H.J., M.L.D.M., R.H.P.
Funding:
This work was supported by the State of S
ã
o Paulo Research Foundation – FAPESP (grant # 2018/02333-0;
#2018/20828-7 and #2017/26517-0) and by Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior
(CAPES, Finance Code 001).
Acknowledgments:
The authors are grateful to Fabiano Guimar
ã
es Silva and Marcos Antonio Soares, who
furnished the A. othonianum and V. divergens plant materials, respectively; to Erika do Amaral for the botanical
classification of C. limon, C. reticulata, C. reticulata Blanco, C. reticulata var. cravo, C. sempervirens, and L. cubeba;
and to Liliana Scorzoni for donating the C. elegans AU37 and E. coli OP50 strains. The authors also acknowledge
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Conflicts of Interest: The authors declare no conflict of interest.
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