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In Vitro and In Vivo Anti-Candida spp. Activity of Plant-Derived Products



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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; (R.d.S.P.); (B.L.B.); (G.A.); (M.C.P.S.D.); (T.A.A.); (R.C.N.P.); (L.P.P.); (R.L.); (P.M.P.); (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;
5Instituto Federal do Triângulo Mineiro, Campus Uberlândia Centro, Uberlândia 38.064-300, MG, Brazil;
Both authors contributed equally to this work.
Received: 15 September 2019; Accepted: 8 November 2019; Published: 11 November 2019
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
g/mL aected 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 aect especially immunocompromised
individuals, children, elderly, individuals hospitalized in Intensive Care Units (ICU), and users of
invasive devices [
]. 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
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 [
]. Microbial biofilms are the main cause of hospital infections
and the source of many recurrent and persistent diseases [
]. 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 [
]. 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 [
]. Thus, the activity of essential oils is related to the composition, functional groups,
and synergistic interactions between the components [
], 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 [
], which exhibited antimicrobial, anticancer, antidiabetic, and antidepressant
actions, among others [
]. 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 oers 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 eectiveness 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
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 ocinalis >2000 >2000 >2000 >2000 >2000 >2000
Plants 2019,8, 494 4 of 17
The Citrus limon EO provided MIC values of 250
g/L against C. tropicalis and C. glabrata (Table 1).
The Litsea cubeba EO yielded MIC values of 62.5, 250, and 250
g/mL against C. krusei, C. glabrata, and
C. orthopsilosis, respectively (Table 1). Finally, the Citrus reticulata EO aorded MIC values of 250
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
ATCC 22019
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
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.
Candida Species/MIC (µg/mL)
C. albicans
C. tropicalis
ATCC 13803
C. krusei
ATCC 6258
C. glabrata
ATCC 2001
ATCC 22019
ATCC 96141
>2000 >2000 2000 >2000 >2000 >2000
Benzoic acid >2000 >2000 >2000 >2000 >2000 >2000
Caeic 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).
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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
g/mL) or 1
MIC (250
g/mL) exerted fungicidal action against C. tropicalis
(Figure 1D) after 4 h. The Citrus limon EO at 2
MIC (500
g/mL) had a fungicidal eect on C. tropicalis
(Figure 1D) and C. glabrata (Figure 1E) after 2 h. A fungicidal eect emerged after exposure of C.
orthopsilosis (Figure 1F) to the C. sempervirens EO at 0.5
MIC (15.6
g/mL), 1
MIC (31.25
g/mL), and
MIC (62.5
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 eect
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
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
respectively (Table 5).
Plants 2019,8, 494 6 of 17
Plants 2019, 8, x FOR PEER REVIEW 6 of 18
Figure 1.
Kill assays for plant-derived products against Candida species. The concentrations 0.5
MIC, 1
MIC, and 2
MIC correspond to: (
)Litsea cubeba
C. krusei: 31.25, 62.5, and 125
) Gallic acid
C.glabrata 31.25, 62.5, and 125
g/mL; (
) Gallic acid
C. krusei: 62.5, 125, and
g/mL; (
)Citrus limon
C. tropicalis: 125, 250, and 500
g/mL; (
)Citrus limon
C. glabrata: 125,
250, and 500
g/mL; (
)Cupressus sempervirens
C. orthopsilosis: 15.62, 31.25, and 62.5
g/mL; (
Cupressus sempervirens
C. glabrata: 15.62, 31.25, and 62.5
g/mL. AMB: 4
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.
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
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
and 2 ×MIC
at 25
C for 24 h. The Litsea cubeba and Cupressus sempervirens EOs at concentrations
between 31.25 and 125
g/mL as well as gallic acid at concentrations between 15.62 and 250
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
g/mL) was not toxic (p>0.05), but this same EO at 1
g/mL) and 2
MIC (500
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
g/mL or gallic acid (Figure 2E) at concentrations between 62.5 and 250
were not cured of candidiasis.
The Citrus limon EO at concentrations between 125 and 500
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
g/mL and
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
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. AC. elegans infected with C. glabrata and treated with
C. sempervirens EO, BC. elegans infected with C. glabrata and treated with C. limon EO, CC. elegans infected
with C. glabrata and treated with gallic acid, DC. 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, GC. 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. (
)—C. elegans infected with C. glabrata
and treated with C. sempervirens EO, (
)—C. elegans infected with C. glabrata and treated with C. limon
EO, (
)—C. elegans infected with C. glabrata and treated with gallic acid, (
)—C. elegans infected with
C. krusei and treated with L. cubeba EO, (
)—C. elegans infected with C. krusei and treated with gallic
acid, (
)—C. elegans infected with C. tropicalis and treated with C. limon, (
)—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 dierent 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) [
] and Act 5813 (22 June 2006) [
], 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 dierent
percentages. For instance, Selim et al. [
] reported that
-pinene (48.6%),
-3-carene (22.1%), limonene
(4.6%), and
-terpinolene (4.5%) are the main constituents of Cupressus sempervirens studied in Saudi
Arabia, whereas Ibrahim et al. [
] described
-pinene (21.15%), terpinen-4-ol (6.98%), allo-ocimene
(24.00%), and
-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 [
]. 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 [
]. 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 [
], 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 eects [
]. Moreover, at 2 x MIC, this EO provided the same eect as 4
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
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
g/mL and between 250 and 500
g/mL, this EO displayed a
fungicidal eect 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 [
], which was detected at a similar concentration to the concentration reported by Campelo
et al. [
]. Limonene damages the C. albicans cell wall/membrane, thereby modifying cellular adhesion
and plasticity, pH, and ionic content [
]. In addition, such damage causes oxidative stress and
consequent DNA damage, resulting in cell cycle modulation and apoptosis as demonstrated by Thakre
et al. [
]. The high concentration of limonene in the EO could contribute to its nematocidal eect [
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 [
]. Here, this EO aorded the best activity against C. krusei
ATCC 6258, with MIC and MBIC values of 62.5 and 250
g/mL, respectively. Its anti-Candida
eect 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 [
]. Moreover, in agreement with a previous study [
], 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 [
]; however, its activity against Candida biofilms
has been poorly investigated. This compound can inhibit biofilm formation [
], 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 [
]. Interestingly, we detected that all the
assayed plant-derived products at 250
g/mL had an eect 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 [
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
’ S and 51
’ W), GO, Brazil, on January 2nd, 2017, at
09:00. Voucher specimens (#CR-25, #CRB-25
, #CRC-25
’, #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
ocinalis 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
o Preto, University of S
o Paulo,
Plants 2019,8, 494 12 of 17
Brazil (Herbarium SPFR) and in the Herbarium Jataiense Germano Guarim Neto of the Goi
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
22” S and
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
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
’ S and 46
’ 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, caeic 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
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
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 [
]. Sterile microtiter plates (Corning Inc., NY, USA) were used. The inoculum
size was 2.5
cells/mL. The final concentration of amphotericin B (AMB) and the tested products
ranged from 0.03 to 16
g/mL and from 3.90 to 2.000
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 [
]. The minimum
inhibitory concentration (MIC) was determined with the fluorometric indicator resazurin at 0.01%
(w/v) [
]. MIC was defined as the lowest antifungal/product concentration that maintained a blue
hue. The AMB breakpoint adopted herein was
g/mL, whereas AMB >1
g/mL was considered as
resistant [
]. The wells where micro-organism growth occurred displayed the pink color. Plant-derived
products were considered active when MIC was <100
g/mL, moderately active when MIC ranged from
100 up to 500
g/mL, and weakly active when MIC was >500 and <1000
g/mL. Above 1000
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
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
g/mL [
]. 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
cells/mL) with a nephelometer (ATB 1550, BioM
France). Next, the micro-organism suspension was diluted in RPMI 1640, to give a new suspension
containing 2.5–5.0
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
L aliquots were removed at each time
point. Tenfold serial dilutions were performed, and 10
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
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
in CFU/mL as
compared to the initial inoculum [22,44]. The experiments were conducted in triplicate.
4.8. Evaluation of the Eects 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) eect 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 [
]. The EOs and gallic acid
(concentration range from 1.95 to 2000
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
- [1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) hydrated benzene
sulfonic acid, XTT) reduction assay, by adding 100
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 [
]. The
eective 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
o Paulo State
University, Dr. J
lio de Mesquita Filho, Instituto de Ci
ncia e Tecnologia, Departamento de Bioci
e Diagn
stico Bucal, S
o Jos
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
g/mL), kanamycin (90
g/mL), and ampicillin (200
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
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 [
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
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 [
]. 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 dierences 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.
This work was supported by the State of S
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).
The authors are grateful to Fabiano Guimar
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.
Pappas, P.G.; Lionakis, M.S.; Arendrup, M.C.; Ostrosky-Zeichner, L.; Kullberg, B.J. Invasive candidiasis. Nat.
Rev. Dis. Primers. 2018,4, 18026. [CrossRef] [PubMed]
Kong, E.F.; Kuchar
, S.; Van Dijck, P.; Peters, B.M.; Shirtli, M.E.; Jabra-Rizk, M.A. Clinical implications
of oral candidiasis: Host tissue damage and disseminated bacterial disease. Infect. Immun.
,83, 604–613.
[CrossRef] [PubMed]
Sobel, J.D.; Sobel, R. Current treatment options for vulvovaginal candidiasis caused by azole-resistant Candida
species. Expert Opin. Pharmacother. 2018,19, 971–977. [CrossRef] [PubMed]
Friedman, D.Z.P.; Schwartz, I.S. Emerging fungal infections: New patients, new patterns, and new pathogens.
J. Fungi 2019,5, E67. [CrossRef] [PubMed]
5. Fanning, S.; Mitchell, A.P. Fungal biofilms. PLoS Pathog. 2012,8, 1002585. [CrossRef] [PubMed]
Asong, J.A.; Amoo, S.O.; McGaw, L.J.; Nkadimeng, S.M.; Aremu, A.O.; Otang-Mbeng, W. Antimicrobial
activity, antioxidant potential, cytotoxicity and phytochemical profiling of four plants locally used against
skin diseases. Plants 2019,8, 350. [CrossRef] [PubMed]
Serra, E.; Hidalgo-Bastida, L.; Verran, J.; Williams, D.; Malic, S. Antifungal activity of commercial essential
oils and biocides against Candida albicans.Pathogens 2018,7, 15. [CrossRef] [PubMed]
Plants 2019,8, 494 15 of 17
Zore, G.B.; Thakre, A.D.; Jadhav, S.; Karuppayil, S.M. Terpenoids inhibit Candida albicans growth by aecting
membrane integrity and arrest of cell cycle. Phytomedicine 2011,18, 1181–1190. [CrossRef] [PubMed]
Nazzaro, F.; Fratianni, F.; Coppola, R.; De Feo, V. Essential oils and antifungal activity. Pharmaceuticals
10, 86. [CrossRef] [PubMed]
Ghasemzadeh, A.; Ghasemzadeh, N. Flavonoids and phenolic acids: Role and biochemical activity in plants
and humans. J. Med. Plants Res. 2011,5, 6697–6703. [CrossRef]
Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod.
79, 629–661. [CrossRef] [PubMed]
Doke, S.K.; Dhawale, S.C. Alternatives to animal testing: A review. Saudi Pharm. J.
,23, 223–229.
[CrossRef] [PubMed]
Brazil Ministry of Health, Minister’s Oce. Ordinance No. 971, May 3th, 2006: Approves the National Policy for
Integrative and Complementary Practices (Pol
tica Nacional de Pr
ticas Integrativas e Complementares [PNPIC]) in
the Single Health System (Sistema
nico de Sa
de—SUS); Ministry of Health: Brasilia, Brasil, 2006. Available
online: (accessed on 9 July 2019).
Republic Presidence, Deputy Chief of Legal Aairs. Ordinance No. 5813, 22 July 2006: Approves the National
Policy of Medicinal Plants and Herbal Medicines and Other Measures; Oce of the Presidential Sta: Brasilia,
Brasil, 2006. Available online:
(accessed on 9 July 2019).
Selim, S.A.; Adam, M.E.; Hassan, S.M.; Albalawi, A.R. Chemical composition, antimicrobial and antibiofilm
activity of the essential oil and methanol extract of the Mediterranean cypress (Cupressus sempervirens L.).
BMC Complement Altern. Med. 2014,14, 179. [CrossRef] [PubMed]
Ibrahim, N.A.; El-Seedi, H.R.; Mohammed, M.M.D. Constituents and biological activity of the chloroform
extract and essential oil of Cupressus sempervirens L. Chem. Nat. Compd. 2009,45, 309–313. [CrossRef]
Afsharzadeh, M.; Naderinasab, M.; Tayarani Najaran, Z.; Barzin, M.; Emami, S.A.
In vitro
activities of some Iranian conifers. Iran J. Pharm. Res. 2013,12, 63–74. [PubMed]
Boukhris, M.; Regane, G.; Yangui, T.; Sayadi, S.; Bouazizc, M. Chemical composition and biological potential
of essential oil from Tunisian Cupressus sempervirens L. J. Arid Land Stud. 2012,22, 329–332.
Turner, A.S.; Butler, G. The Candida pathogenic species complex. Cold Spring Harb. Perspect. Med.
4, a019778. [CrossRef] [PubMed]
Savastano, C.; de Oliveira Silva, E.; Gonçalves, L.L.; Nery, J.M.; Silva, N.C.; Dias, A.L. Candida glabrata among
Candida spp. from environmental health practitioners of a Brazilian Hospital. Braz. J. Microbiol.
367–372. [CrossRef] [PubMed]
Pires, R.H.; Santos, J.M.; Zaia, J.E.; Martins, C.H.; Mendes-Giannini, M.J. Candida parapsilosis complex
water isolates from a haemodialysis unit: Biofilm production and
in vitro
evaluation of the use of clinical
antifungals. Mem. Inst. Oswaldo Cruz 2011,106, 646–654. [CrossRef] [PubMed]
Leite, M.C.A.; Bezerra, A.P.B.; Sousa, J.P.; Guerra, F.Q.S.; Lima, E.O. Evaluation of antifungal activity
and mechanism of action of citral against Candida albicans.Evid. Based Complement Alternat. Med.
2014, 378280. [CrossRef] [PubMed]
Araujo, F.M.; Passos, M.G.V.M.; Lima, E.O.; Roque, N.F.; Guedes, M.L.S.; Souza-Neta, L.C.; Cruz, F.G.;
Martins, D. Composition and antimicrobial activity of essential oils from Poiretia bahiana C. Müller
(Papilionoideae-Leguminosae). J. Braz. Chem. Soc. 2009,20, 1805–1810. [CrossRef]
Hammer, K.A.; Carson, C.F.; Riley, T.V. Antifungal activity of the components of Melaleuca alternifolia (tea
tree) oil. J. Appl. Microbiol. 2003,95, 853–860. [CrossRef] [PubMed]
Andes, D.; Safdar, N.; Marchillo, K.; Conklin, R. Pharmacokinetic-pharmacodynamic comparison of
amphotericin B (AMB) and two lipid-associated AMB preparations, liposomal AMB and AMB lipid complex,
in murine candidiasis models. Antimicrob. Agents Chemother. 2006,50, 674–684. [CrossRef] [PubMed]
Prabajati, R.; Hernawan, I.; Hendarti, H.T. Eects of Citrus limon essential oil (Citrus limon L.) on
cytomorphometric changes of Candida albicans.Dent. J. 2017,50, 43–48. [CrossRef]
27. Campelo, L.M.L.; Sá, C.G.; Feitosa, C.M.; Sousa, G.F.; Freitas, R.M. Chemical constituents and toxicological
studies of the essential oil extracted from Citrus limon Burn (Rutaceae). Rev. Bras. Pl. Med.
,15, 708–716.
Plants 2019,8, 494 16 of 17
Thakre, A.; Zore, G.; Kodgire, S.; Kazi, R.; Mulange, S.; Patil, R.; Shelar, A.; Santhakumari, B.; Kulkarni, M.;
Kharat, K.; et al. Limonene inhibits Candida albicans growth by inducing apoptosis. Med. Mycol.
565–578. [PubMed]
Abdel-Rahman, F.H.; Alaniz, N.M.; Saleh, M.A. Nematicidal activity of terpenoids. J. Env. Sci. Health B.
48, 16–22. [CrossRef] [PubMed]
Zhang, W.; Hu, J.F.; Lv, W.W.; Zhao, Q.C.; Shi, G.B. Antibacterial, antifungal and cytotoxic isoquinoline
alkaloids from Litsea cubeba.Molecules 2012,17, 12950–12960. [CrossRef] [PubMed]
Kandimalla, R.; Kalita, S.; Choudhury, B.; Dash, S.; Kalita, K.; Kotoky, J. Chemical composition and
anti-candidiasis mediated wound healing property of Cymbopogon nardus essential oil on chronic diabetic
wounds. Front. Pharmacol. 2016,7, 198. [CrossRef] [PubMed]
Kamle, M.; Mahato, D.K.; Lee, K.E.; Bajpai, V.K.; Gajurel, P.R.; Gu, K.S.; Kumar, P. Ethnopharmacological
properties and medicinal uses of Litsea cubeba.Plants 2019,8, 150. [CrossRef] [PubMed]
Li, Z.-J.; Liu, M.; Dawuti, G.; Dou, Q.; Ma, Y.; Liu, H.G.; Aibai, S. Antifungal activity of gallic acid
in vitro
and in vivo. Phytother. Res. 2017,31, 1039–1045. [CrossRef] [PubMed]
Teodoro, G.R.; Gontijo, A.V.L.; Salvador, M.J.; Tanaka, M.H.; Brighenti, F.L.; Delbem, A.C.B.; Delbem,
Koga-Ito, C.Y. Eects of acetone fraction from Buchenavia tomentosa aqueous extract and gallic acid on Candida
albicans biofilms and virulence factors. Front. Microbiol. 2018,9, 647. [CrossRef] [PubMed]
mara, C.R.S.; Shi, Q.; Pedersen, M.; Zbasnik, R.; Nickerson, K.W.; Schlegel, V. Histone acetylation increases
in response to ferulic, gallic, and sinapic acids acting synergistically
in vitro
to inhibit Candida albicans
yeast-to-hyphae transition. Phytother. Res. 2019,33, 319–326. [CrossRef] [PubMed]
Singulani, J.L.; Scorzoni, L.; Gomes, P.C.; Nazar
, A.C.; Polaquini, C.R.; Regasini, L.O.; Fusco-Almeida, A.M.;
Mendes-Giannini, M.J.S. Activity of gallic acid and its ester derivatives in Caenorhabditis elegans and zebrafish
(Danio rerio) models. Future Med. Chem. 2019,9, 1863–1872. [CrossRef] [PubMed]
Haas, A.N.; Wagner, T.P.; Muniz, F.W.; Fiorini, T.; Cavagni, J.; Celeste, R.K. Essential oils-containing
mouthwashes for gingivitis and plaque: Meta-analyses and meta-regression. J. Dent.
,55, 7–15.
[CrossRef] [PubMed]
Hu, L.; He, C.; Zhao, C.; Chen, X.; Hua, H.; Yan, Z. Characterization of oral candidiasis and the Candida
species profile in patients with oral mucosal diseases. Microb. Pathog.
,134, 103575. [CrossRef] [PubMed]
Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility
Testing of Yeasts; Fourth Informational Supplement, Document M27-S4; CLSI: Wayne, PA, USA, 2012.
Far, F.E.; Al-Obaidi, M.M.J.; Desa, M.N.M. Ecacy of modified Leeming-Notman media in a resazurin
microtiter assay in the evaluation of in-vitro activity of fluconazole against Malassezia furfur ATCC 14521.
J. Mycol. Med. 2018,28, 486–491. [CrossRef] [PubMed]
Alastruey-Izquierdo, A.; Melhem, M.S.C.; Bonfietti, L.X.; Rodriguez-Tudela, J.L. Susceptibility test for fungi:
Clinical and laboratorial correlations in medical mycology. Rev. Inst. Med. Trop. S. Paulo
,57, 57–64.
[CrossRef] [PubMed]
Holetz, F.B.; Pessini, G.L.; Sanches, N.R.; Cortez, D.A.; Nakamura, C.V.; Filho, B.P. Screening of some plants
used in the Brazilian folk medicine for the treatment of infectious diseases. Mem. Inst. Oswaldo Cruz
1027–1031. [CrossRef] [PubMed]
Santos, L.S.; Alves, C.C.F.; Estevam, E.B.B.; Martins, C.H.G.; Silva, T.S.; Esperandim, V.R.; Miranda, M.L.D.
Chemical composition,
in vitro
trypanocidal and antibacterial activities of the essential oil from the dried
leaves of Eugenia dysenterica DC from Brazil. J. Essent. Oil Bear. Plants 2019,22, 347–355. [CrossRef]
n, E.; Pem
n, J.; Viudes, A.; Quind
s, G.; Gobernado, M.; Espinel-Ingro, A. Minimum fungicidal
concentrations of amphotericin B for bloodstream Candida species. Diagn. Microbiol. Infect. Dis.
203–206. [CrossRef]
Pierce, C.G.; Uppuluri, P.; Tristan, A.R.; Wormley, F.L., Jr.; Mowat, E.; Ramage, G.; Lopez-Ribot, J.L. A simple
and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to
antifungal susceptibility testing. Nat. Prot. 2008,3, 1494–1500. [CrossRef]
Plants 2019,8, 494 17 of 17
Scorzoni, L.; de Lucas, M.P.; Mesa-Arango, A.C.; Fusco-Almeida, A.M.; Lozano, E.; Cuenca-Estrella, M.;
Mendes-Giannini, M.J.; Zaragoza, O. Antifungal ecacy during Candida krusei infection in non-conventional
models correlates with the yeast
in vitro
susceptibility profile. PLoS ONE
,8, e60047. [CrossRef]
Tampakakis, E.; Okoli, I.; Mylonakis, E.A. C. elegans-based, whole animal,
in vivo
screen for the identification
of antifungal compounds. Nat. Protoc. 2008,3, 1925–1931. [CrossRef] [PubMed]
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... Thus, given the antifungal drug resistance patterns making treatment of candidiasis difficult, it is essential to search for new antifungals against these opportunistic human pathogens responsible for frequent nosocomial infections. In this context, plant-derived products including EOs have demonstrated promising results in vitro and in vivo against several Candida species and thus, could be considered useful in the development of novel anticandidal drugs [34,35]. ...
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In this study, 10 essential oils (EOs), from nine plants (Cinnamomum camphora, Curcuma longa, Citrus aurantium, Morinda citrifolia, Petroselinum crispum, Plectranthus amboinicus, Pittosporum senacia, Syzygium coriaceum, and Syzygium samarangense) were assessed for their antimicrobial, antiaging and antiproliferative properties. While only S. coriaceum, P. amboinicus (MIC: 0.50 mg/mL) and M. citrifolia (MIC: 2 mg/mL) EOs showed activity against Cutibacterium acnes, all EOs except S. samarangense EO demonstrated activity against Mycobacterium smegmatis (MIC: 0.125–0.50 mg/mL). The EOs were either fungistatic or fungicidal against one or both tested Candida species with minimum inhibitory/fungicidal concentrations of 0.016–32 mg/mL. The EOs also inhibited one or both key enzymes involved in skin aging, elastase and collagenase (IC50: 89.22–459.2 µg/mL; 0.17–0.18 mg/mL, respectively). Turmerone, previously identified in the C. longa EO, showed the highest binding affinity with the enzymes (binding energy: −5.11 and −6.64 kcal/mol). Only C. aurantium leaf, C. longa, P. amboinicus, P. senacia, S. coriaceum, and S. samarangense EOs were cytotoxic to the human malignant melanoma cells, UCT-MEL1 (IC50: 88.91–277.25 µg/mL). All the EOs, except M. citrifolia EO, were also cytotoxic to the human keratinocytes non-tumorigenic cells, HaCat (IC50: 33.73–250.90 µg/mL). Altogether, some interesting therapeutic properties of the EOs of pharmacological/cosmeceutical interests were observed, which warrants further investigations.
... The solution is not only the search for new antimicrobials but also the exploration of the synergistic interactions of already existing drugs with natural compounds such as plant products. Essential oils (EOs) show antibiotic, antifungal, insecticidal, and antiviral activities and are obtained from various plants through different techniques including fermentation, enfleurage, extraction, and steam distillation [1,2]. Their mechanism of action has been extensively reported in the literature showing that also minor components in EOs can play an important role in the antimicrobial, anti-inflammation, and anti-oxidant activity either alone or in combination with commercial agents, even if limited knowledge exists regarding their activity against biofilms and also host-cell cytotoxicity [3]. ...
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The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.
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This study aimed to screen for essential oils with antibiofilm effect on Candida albicans . The antifungal effect of 15 essential oils was evaluated on C. albicans planktonic cells, and the most active essential oils were tested for anti-biofilm property. Toxicity to Vero cells was also assessed. Thymus vulgaris and Allium sativum essential oils showed higher fungistatic effects on C. albicans MYA-2876 and C. albicans ATCC 18804. Both essential oils also showed an anti-biofilm effect. Thymus vulgaris and Allium sativum essential oils showed low and moderate cytotoxicity, respectively. The results obtained in this study open promising possibilities for the elaboration of mouthwashes and topical formulations to improve the conventional treatment of oral candidiasis.
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Bis(2-carboxyphenyl) succinate (disalicylic acid; DSA) is composed of two salicylic acids connected by a succinyl linker. Here, we propose its use as a new, synthetic plant-protection agent. DSA was shown to control Pectobacterium brasiliense, an emerging soft-rot pathogen of potato and ornamental crops, at minimal inhibitory concentrations (MIC) lower than those of salicylic acid. Our computational-docking analysis predicted that DSA would inhibit the quorum-sensing (QS) synthase of P. brasiliense ExpI more strongly than SA would. In fact, applying DSA to P. brasiliense inhibited its biofilm formation, secretion of plant cell wall-degrading enzymes, motility and production of acyl–homoserine lactones (AHL) and, subsequently, impaired its virulence. DSA also inhibited the production of AHL by a QS-negative Escherichia coli strain (DH5α) that had been transformed with P. brasiliense AHL synthase, as demonstrated by the biosensors Chromobacterium violaceaum CV026 and E. coli pSB401. Inhibition of the QS machinery appears to be one of the mechanisms by which DSA inhibits specific virulence determinants. A new route is proposed for the synthesis of DSA, which holds greater potential for use as an anti-virulence agent than its precursor SA. Based on these findings, DSA is an excellent candidate for repurposing for new applications.
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Objectives: Candida albicans is resistant to commercial antifungal agents. Therefore, it is desirable to use material derived from natural sources as an antifungal agent. Essential oil from Citrus limon peel is able to inhibit the growth of C. albicans in vitro. The purpose of this study was to determine the most effective concentration of essential oil from C. limon peel with regards to the inhibition of C. albicans cyto-morphometric changes and biofilm formation in vivo. Methods: Male Wistar rats weighing 200-300 g were inoculated with C. albicans for 48 h and then given a single dose of oral methylprednisolone as an immunosuppressant. Essential oil from C. limon peel, in a gel form and at three different concentrations (0.39%, 0.78% and 1.56%), was applied twice a day for 2 days. The rats were killed after 48 h and then palatal mucosa tissues were prepared and examined with a scanning electron microscope (SEM) with regards to C. albicans, cyto-morphometric changes and biofilm formation. Results: Essential oil from C. limon peel at a concentration of 1.56% showed the strongest ability to inhibit C. albicans growth when compared to 0.78% and 0.39%. At a concentration of 1.56%, essential oil from C. limon peel disrupted cyto-morphometric changes; cells that were neither in intact nor colonised were evident, the filaments around the cells were smooth, the layer of biofilm had disappeared and there was no evidence of hyphae formation. Conclusion: The effect of essential oil from C. limon peel on cyto-morphometric changes and biofilm formation was concentration-dependent. Essential oil from C. limon peel at a dose of 1.56% showed the strongest ability to inhibit cyto-morphometric changes and biofilm formation. These findings demonstrate that essential oil of C. limon peel is a potential antifungal candidate for the treatment of candidiasis.
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Peppers (Capsicum sp.) are special to produce spices due to their fruit's color and active principles, which bestow aroma and flavor. This study aimed to analyze phenolic compounds found in an ethanolic extract from Capsicum chinense unripe fruit (var. bode pepper) (henceforth, EE-BP) by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) and at investigating their in vitro leishmanial, antifungal, and cytotoxic potential. Several phenolic compounds were analyzed in EE-BP, such as gallic acid, protocatechuic acid, gentisic acid, caffeic acid, vanillic acid, p-coumaric acid, ferulic acid, kaempferol-3-O-robinobiosideo, naringenin, capsaicin, and dihydrocapsaicin. EE-BP exhibited activity against promastigote forms of Leishmania amazonensis (IC50 = 23.82 µg/mL) and was highly active against Candida glabrata (MIC = 50 µg/mL), C. parapsilosis (MIC = 62.5 µg/mL), C. krusei (MIC = 100 µg/mL) and C. tropicalis (MIC = 125 µg/mL). Besides, EE-BP revealed high toxicity against Artemia salina (LC50 = 70.5 µg/mL). Results showed, for the first time, that EE-BP has antiparasitic, antifungal, and cytotoxic potential, which can be better investigated by further preclinical and clinical (in vivo) studies.
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Although orthodox medications are available for skin diseases, expensive dermatological services have necessitated the use of medicinal plants as a cheaper alternative. This study evaluated the pharmacological and phytochemical profiles of four medicinal plants (Drimia sanguinea, Elephantorrhiza elephantina, Helichrysum paronychioides, and Senecio longiflorus) used for treating skin diseases. Petroleum ether and 50% methanol extracts of the plants were screened for antimicrobial activity against six microbes: Bacillus cereus, Shigella flexneri, Candida glabrata, Candida krusei, Trichophyton rubrum and Trichophyton tonsurans using the micro-dilution technique. Antioxidant activity was conducted using 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging and β-carotene linoleic acid models. Cytotoxicity was determined against African green monkey Vero kidney cells based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Spectrophotometric and Gas Chromatography-Mass Spectrometry (GC-MS) methods were used to evaluate the phytochemical constituents. All the extracts demonstrated varying degrees of antimicrobial potencies. Shigella flexneri, Candida glabrata, Trichophyton rubrum and Trichophyton tonsurans were most susceptible at 0.10 mg/mL. In the DPPH test, EC50 values ranged from approximately 6–93 µg/mL and 65%–85% antioxidant activity in the β-carotene linoleic acid antioxidant activity model. The phenolic and flavonoid contents ranged from 3.5–64 mg GAE/g and 1.25–28 mg CE/g DW, respectively. The LC50 values of the cytotoxicity assay ranged from 0.015–5622 µg/mL. GC-MS analysis revealed a rich pool (94–198) of bioactive compounds including dotriacontane, benzothiazole, heptacosane, bumetrizole, phthalic acid, stigmasterol, hexanoic acid and eicosanoic acid, which were common to the four plants. The current findings provide some degree of scientific evidence supporting the use of these four plants in folk medicine. However, the plants with high cytotoxicity need to be used with caution.
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: The landscape of clinical mycology is constantly changing. New therapies for malignant and autoimmune diseases have led to new risk factors for unusual mycoses. Invasive candidiasis is increasingly caused by non-albicans Candida spp., including C. auris, a multidrug-resistant yeast with the potential for nosocomial transmission that has rapidly spread globally. The use of mould-active antifungal prophylaxis in patients with cancer or transplantation has decreased the incidence of invasive fungal disease, but shifted the balance of mould disease in these patients to those from non-fumigatus Aspergillus species, Mucorales, and Scedosporium/Lomentospora spp. The agricultural application of triazole pesticides has driven an emergence of azole-resistant A. fumigatus in environmental and clinical isolates. The widespread use of topical antifungals with corticosteroids in India has resulted in Trichophyton mentagrophytes causing recalcitrant dermatophytosis. New dimorphic fungal pathogens have emerged, including Emergomyces, which cause disseminated mycoses globally, primarily in HIV infected patients, and Blastomyces helicus and B. percursus, causes of atypical blastomycosis in western parts of North America and in Africa, respectively. In North America, regions of geographic risk for coccidioidomycosis, histoplasmosis, and blastomycosis have expanded, possibly related to climate change. In Brazil, zoonotic sporotrichosis caused by Sporothrix brasiliensis has emerged as an important disease of felines and people.
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Natural products are a very important source of bio-molecules for drug to treat infectious diseases. The essential oil of Eugenia dysenterica DC. dried leaves (Ed-EO), of the Myrtaceae family, was analyzed by gas chromatography with flame ionization detector (GC/FID) and by mass spectrometry (GC/MS) in order to identify their volatile components. A total of 24 compounds were identified from Ed-EO. The major compounds found in Ed-EO were limonene (16.0 %), caryophyllene oxide (15.0 %), citral (9.0 %), trans-caryophyllene (8.0 %), and 1,8-cineole (7.3 %). The in vitro biological activities of Ed-EO were investigated against trypomastigote forms of Trypanosoma cruzi and bacteria of the genus Streptococcus. Our results demonstrated that Ed-EO, tested against T. cruzi, affected trypomastigote growth in a dose-dependent manner. The IC 50 of Ed-EO was 9.5 μg/mL, while the IC 50 of benznidazole (positive control) was 9.8 μg/mL, revealing remarkable trypanocidal action. The minimum inhibitory concentrations (MICs) were determined by the broth dilution method in 96-well microplates. The Ed-EO displayed moderate antibacterial activity against Streptococcus mitis (MIC = 250 μg/mL), S. sanguinis (MIC = 200 μg/mL), S. sobrinus (MIC = 400 μg/mL), and S. salivarius (MIC = 400 μg/mL), and strong activity against S. mutans (MIC = 31.2 μg/mL). These results suggest that the oil of E. dysenterica dried leaves could be tested in future studies for the treatment of Chagas disease and dental caries.
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The genus Litsea is predominant in tropical and subtropical regions of India, China, Taiwan, and Japan. The plant possesses medicinal properties and has been traditionally used for curing various gastro-intestinal ailments (e.g., diarrhea, stomachache, indigestion, and gastroenteritis) along with diabetes, edema, cold, arthritis, asthma, and traumatic injury. Besides its medicinal properties, Litsea is known for its essential oil, which has protective action against several bacteria, possesses antioxidant and antiparasitic properties, exerts acute and genetic toxicity as well as cytotoxicity, and can even prevent several cancers. Here we summarize the ethnopharmacological properties, essentials oil, medicinal uses, and health benefits of an indigenous plant of northeast India, emphasizing the profound research to uplift the core and immense potential present in the conventional medicine of the country. This review is intended to provide insights into the gaps in our knowledge that need immediate focus on in-situ conservation strategies of Litsea due to its non-domesticated and dioecious nature, which may be the most viable approach and intense research for the long-term benefits of society and local peoples.
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A promising anti-Candida activity of Buchenavia tomentosa extracts was recently described. In the present work, experiments were carried out to determine the fraction with higher antifungal activity from a B. tomentosa extract. Acetone fraction (AF) was obtained from the aqueous extract from dried leaves (5 min/100°C) and it was the most effective one. Gallic acid (GA) was identified by electrospray ionization mass spectrometry (ESI–MS) and also chosen to perform antifungal tests due to its promising activity on Candida albicans. Minimal inhibitory and fungicidal concentrations (MIC and MFC) were determined by broth microdilution technique. The effect on virulence factors of C. albicans was evaluated, and the cytotoxicity was determined. MIC50 and MIC90 values were both equal to 0.625 mg ml-1 for AF and 2.5 and 5 mg ml-1, respectively, for GA. AF and GA showed ability to inhibit C. albicans adherence and to disrupt 48 h-biofilm. AF and GA were effective in reducing the formation of hyphae of C. albicans SC5314. AF and GA decreased adherence of C. albicans to oral epithelial cells. AF and GA showed slight to moderate toxicity to Vero cells. This result suggests further studies for topic use of these compounds. AF, which contains a combination of several molecules, presented greater potential of antimicrobial activity than GA, with lower values of MIC and lower cytoxicity.
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Management of oral candidosis, most frequently caused by Candida albicans, is limited due to the relatively low number of antifungal drugs and the emergence of antifungal tolerance. In this study, the antifungal activity of a range of commercial essential oils, two terpenes, chlorhexidine and triclosan was evaluated against C. albicans in planktonic and biofilm form. In addition, cytotoxicity of the most promising compounds was assessed using murine fibroblasts and expressed as half maximal inhibitory concentrations (IC50). Antifungal activity was determined using a broth microdilution assay. The minimum inhibitory concentration (MIC) was established against planktonic cells cultured in a range of concentrations of the test agents. The minimal biofilm eradication concentration (MBEC) was determined by measuring re-growth of cells after pre-formed biofilm was treated for 24 h with the test agents. All tested commercial essential oils demonstrated anticandidal activity (MICs from 0.06% (v/v) to 0.4% (v/v)) against planktonic cultures, with a noticeable increase in resistance exhibited by biofilms (MBECs > 1.5% (v/v)). The IC50s of the commercial essential oils were lower than the MICs, while a one hour application of chlorhexidine was not cytotoxic at concentrations lower than the MIC. In conclusion, the tested commercial essential oils exhibit potential as therapeutic agents against C. albicans, although host cell cytotoxicity is a consideration when developing these new treatments.
Novel treatments are needed to prevent candidiasis/candidemia infection due to the emergence of Candida species resistant to current antifungals. Considering the yeast‐to‐hyphae switch is a critical factor to Candida albicans virulence, phenols common in plant sources have been reported to demonstrating their ability to prevent dimorphism. Therefore, phenols present in many agricultural waste stress (ferulic (FA) and gallic (GA) acid) were initially screened in isolation for their yeast‐to‐hyphae inhibitory properties at times 3, 6, and 24 hr. Both FA and GA inhibited 50% of hyphae formation inhibitory concentration (IC50) but at a concentration of 8.0 ± 0.09 and 90.6 ± 1.05 mM, respectively, at 24 hr. However, the inhibitory effect of FA increased by 1.9–2.6 fold when combined with different GA concentrations. GA and FA values decreased even lower when sinapic acid (SA) was added as a third component. As evidenced by concave isobolograms and combination indexes less than 1, both GA:F A and GA:FA:SA combinations acted synergistically to inhibit 50% hyphae formation at 24 hr. Lastly, acetylation of histone H3 lysine 56 acetylation (H3K56) was higher in response to the triple phenolic cocktail (using the IC50 24 hr inhibitory concentration level) comparable with the nontreated samples, indicating that the phenols inhibited hyphal growth in part by targeting H3K56 acetylation.
Introduction: Clinicians are increasingly challenged by patients with refractory vulvovaginal candidiasis (VVC) caused by azole-resistant Candida species. Fluconazole resistant C.albicans is a growing and perplexing problem following years of indiscriminate drug prescription and unnecessary drug exposure and for which there are few therapeutic alternatives. Regrettably, although the azole class of drugs has expanded, new classes of antifungal drugs have not been forthcoming, limiting effective treatment options in patients with azole resistant Candida vaginitis. Areas covered: This review covers published data on epidemiology, pathophysiology and treatment options for women with azole-resistant refractory VVC. Expert opinion: Fluconazole resistant C.albicans adds to the challenge of azole resistant non-albicans Candida spp. Both issues follow years of indiscriminate drug prescription and unnecessary fluconazole exposure. Although an understanding of azole resistance in yeast has been established, this knowledge has not translated into useful therapeutic advantage. Treatment options for such women with refractory symptoms are extremely limited. New therapeutic options and strategies are urgently needed to meet this challenge of azole drug resistance.
Invasive candidiasis is an important health-care-associated fungal infection that can be caused by several Candida spp.; the most common species is Candida albicans, but the prevalence of these organisms varies considerably depending on geographical location. The spectrum of disease of invasive candidiasis ranges from minimally symptomatic candidaemia to fulminant sepsis with an associated mortality exceeding 70%. Candida spp. are common commensal organisms in the skin and gut microbiota, and disruptions in the cutaneous and gastrointestinal barriers (for example, owing to gastrointestinal perforation) promote invasive disease. A deeper understanding of specific Candida spp. virulence factors, host immune response and host susceptibility at the genetic level has led to key insights into the development of early intervention strategies and vaccine candidates. The early diagnosis of invasive candidiasis is challenging but key to the effective management, and the development of rapid molecular diagnostics could improve the ability to intervene rapidly and potentially reduce mortality. First-line drugs, including echinocandins and azoles, are effective, but the emergence of antifungal resistance, especially among Candida glabrata, is a matter of concern and underscores the need to administer antifungal medications in a judicious manner, avoiding overuse when possible. A newly described pathogen, Candida auris, is an emerging multidrug-resistant organism that poses a global threat.
Background: Malassezia furfur is lipodependent yeast like fungus that causes superficial mycoses such as pityriasis versicolor and dandruff. Nevertheless, there are no standard reference methods to perform susceptibility test of Malassezia species yet. Aims: Therefore, in this study, we evaluated the optimized culture medium for growth of this lipophilic yeast using modified leeming-Notman agar and colorimetric resazurin microtiter assay to assess antimycotic activity of fluconazole against M. furfur. Results: The result showed that these assays were more adjustable for M. furfur with reliable and reproducible MIC end-point, by confirming antimycotic activity of fluconazole with MIC of 2μg/ml. Conclusion: We conclude that this method is considered as the rapid and effective susceptibility testing of M. furfur with fluconazole antifungal activity.