Journal of Ethnopharmacology 295 (2022) 115403

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Bioactive compounds with antifungal activity against pathogens isolated

from pregnant woman: Gallesia integrifolia (garlic wood) is a promising
treatment for vulvovaginal candidiasis
Amanda Nascimento Vasques de Souza a, c, Maria Graciela Iecher Faria b, e,
Cristiane Edna da Rocha a, Gisele Strieder Philippsen g, Gabriela Catuzo Canonico Silva b,
Gustavo Ratti da Silva a, d, Rodrigo Sadao Inumaro f, José Eduardo Gonçalves f,
Zilda Cristiani Gazim b, e, Samantha Wietzikoski a, Francislaine Aparecida dos Reis Lívero a, c, d, e,
Flavio Augusto Vicente Seixas h, Evellyn Claudia Wietzikoski Lovato a, c, *
a
Laboratory of Preclinical Research of Natural Products, Paranaense University, Umuarama, PR, Brazil
b
Laboratory of Chemistry of Natural Products, Paranaense University, Umuarama, PR, Brazil
c
Postgraduate Program in Medicinal Plants and Phytotherapics in Basic Attention, Paranaense University, Umuarama, PR, Brazil
d
Postgraduate Program in Animal Sciences with Emphasis on Bioactive Products, Paranaense University, Umuarama, PR, Brazil
e
Postgraduate Program in Biotechnology Applied to Agriculture, Paranaense University, Umuarama, PR, Brazil
f
Postgraduate Program in Clean Technologies and Cesumar Institute of Science, Technology and Innovation, Cesumar University, Maringá, PR, Brazil
g
Universidade Federal do Paraná, Jandaia do Sul, PR, Brazil
h
Department of Technology, Universidade Estadual de Maringá, Umuarama, PR, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: The leaves, bark, and roots of Gallesia integrifolia are consumed in folk medicine
Candida albicans through infusion, decoction, and topical preparation by crushing because of its pharmacological properties in
Candida glabrata several peripheral system disorders, including microbial infections. The presence of various molecules in
Gallesia integrifolia
different parts of the plant likely confers this species’ fungicidal action, but scientific evidence is lacking. Vul­
3,5-dithiahexanol-5,5-dioxide
vovaginal candidiasis mainly affects women of reproductive age. When left untreated, it can cause pregnancy
Methionine
Ethyl ester complications. Currently available antifungals often cause undesirable side effects. New alternative therapeutic
Phytol strategies based on medicinal plants have been proposed.
Aim: To investigate the antifungal activity of G. integrifolia against vulvovaginal candidiasis secretion in pregnant
women.
Materials and methods: Antifungal activity was determined by the minimum inhibitory concentration (MIC),
determined by broth microdilution method using Candida spp (NEWP1210), C. albicans (CCCD-CC001),
C. tropicalis (CCCD-CC002) standard and clinical isolates from pregnant women with vulvovaginal candidiasis.
Nystatin and fluconazole were used as positive controls. The chemical composition of essential oils that were
extracted from leaves, flowers, and fruits of G. integrifolia was determined by gas chromatography coupled to
mass spectrometry. Reverse docking was used to suggest a possible target in Candida. Conventional docking was
used to identify the most probable compound that inhibits fungal growth.
Results: A total of 24 compounds were identified, accounting for ~99% of volatile constituents in the essential
oils. Leaves of G. integrifolia contained 3,5-dithiahexanol-5,5-dioxide (40.93%), flowers contained methionine
ethyl ester (46.78%), and fruits contained 2,8-dithianonane (54.01%) as the most abundant compounds. The
MICs of essential oils of leaves, flowers, and fruits of G. integrifolia against standard strains of Candida spp,

Abbreviations: Å, angstrom; ANOVA, analysis of variance; C. albicans, Candida albicans; C. tropicalis, Candida tropicalis; C. glabrata, Candida glabrata; C. albicans LSS,
C. albicans enzyme lanosterol synthase; CLSI, Clinical and Laboratory Standards Institute; G. integrifolia, Gallesia integrifolia; GC-MS, gas chromatography-mass
spectrometry; MIC, minimum inhibitory concentration; MFC, minimum fungicidal concentration; MW, molecular weight; LAN, lanosterol substrate; LSS, enzyme
lanosterol synthase; SEA, Similarity ensemble approach; SD, standard deviation; UNIPAR, University of Paraná.
* Corresponding author. Laboratório de Pesquisa Pré-clínica de Produtos Naturais, Universidade Paranaense, Praça Mascarenhas de Moraes, 4282, Caixa Postal
224, Umuarama, PR, CEP 87502-210, Brazil.
E-mail address: evellyn@prof.unipar.br (E.C. Wietzikoski Lovato).

https://doi.org/10.1016/j.jep.2022.115403
Received 8 April 2022; Received in revised form 20 May 2022; Accepted 22 May 2022
Available online 25 May 2022
0378-8741/© 2022 Elsevier B.V. All rights reserved.
A.N.V. de Souza et al. Journal of Ethnopharmacology 295 (2022) 115403

C. albicans, and C. tropicalis ranged from 13.01 to 625.00 μg/mL. The essential oil of flowers more effectively
inhibited Candida spp. Essential oils of leaves and flowers were similar to fluconazole against C. albicans.
Essential oils of flowers and fruits were similar to fluconazole against C. tropocalis. In Candida yeast species that
were isolated from vaginal secretion samples from pregnant patients, the MICs of leaves and flowers ranged from
52.08 to 5000.00 μg/mL. The essential oil of leaves (277.77 μg/mL) was the most active against C. albicans. No
significant differences were found between the essential oils of leaves and flowers against C. glabrata. Docking
simulations suggested that phytol in leaves and flowers was responsible for the antimicrobial effect.
Conclusion: The present results suggest the potential therapeutic use of G. integrifolia, especially its leaves and
flowers, against Candida and vulvovaginal candidiasis.

1. Introduction β-bisabolene, β-sesquiphellandrene, zingiberene, and α-bergamotene

Vulvovaginal candidiasis is a common clinical condition that is
characterized by itch, redness, inflammation, and vulvovaginal
discharge that are caused by Candida species (Willems et al., 2020). The
onset of sexual activity is an important risk factor for infection. Ac­
cording to epidemiological data, approximately 50% of women aged up
to 25 years have had at least one episode of vulvovaginal candidiasis
(Makanjuola et al., 2018). Furthermore, 70–75% of women of repro­
ductive age have at least one episode of vulvovaginal candidiasis
throughout life (Zeng et al., 2018). Of these women, 10% have recurrent
infection, representing approximately 138 million women worldwide
(Powell and Nyirjesy, 2014; Van Riel et al., 2021).
Candida albicans has been considered the main etiological agent of
vulvovaginitis, but the prevalence of non-albicans Candida species, such
as C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. dubliniensis, and
C. glabrata has emerged globally, increasing scientific interest in these
species (Makanjuola et al., 2018; Anh et al., 2021). Furthermore, many
of these species have intrinsic resistance to antifungal treatment,
resulting in treatment failure (Silva et al., 2019; Salazar et al., 2020).
Therefore, new antifungal drugs with novel targets and different
mechanisms of action need to be developed (Berkow and Lockhart,
2017; Colombo et al., 2017; Di Mambro et al., 2019).
Medicinal plants are a rich source of medicines that have been used
for centuries to treat various human ailments. Researchers have inves­
tigated medicinal plants to provide scientific evidence and justification
for the use of herbal medicine preparations and identify new promising
species and bioactive components that are effective against various
pathogens (Jamshidi-Kia et al., 2018). Medicinal plants play an impor­
tant role in the development of drugs that act in novel ways or overcome
microorganisms’ mechanisms of resistance (Anand et al., 2019; Nwonu
et al., 2019; Akash, 2021).
Gallesia integrifolia (Spreng.) Harms, Phytolaccaceae, is a native and
endemic plant of Brazil that is widely distributed in the Amazon, Caa­
tinga, Cerrado, and Atlantic rain forest (Arunachalam et al., 2016;
Forzza et al., 2012). It is commonly known as “pau-d’alho” or “garlic
plant” because of the strong odor of garlic that is peculiar to all parts of
the plant (Lima et al., 2010). In traditional medicine, its leaves and bark
are popularly used as a remedy for flu, cough, pneumonia, worms,
gonorrhea, prostate tumors, and rheumatism (Lorenzi, 2002). Tea from
the leaves and stem bark is used to treat ulcers (Grandtner and Chevr­
ette, 2013). Boiled leaves and stem bark are also used to treat intestinal
worms and respiratory and lymphatic diseases. Fresh crushed leaves are
used topically to treat abscesses, otitis, and gonorrhea (Balbach, 1993).
The infusion of roots is used to treat rheumatism and ulcers in some
Brazilian communities, and the essential oil is used to treat gonorrhea
(Barbosa et al., 1999; Muñoz et al., 2000; Bieski et al., 2012). An eth­
nopharmacological study by Bueno et al. (2019) found that G. integrifolia
has other therapeutic indications, such as back pain, bronchitis, rheu­
matism, and constipation. The phytochemical characterization of this
species indicates a predominance of organosulfur compounds with the
main presence of 2,8-dithianonane, dimethyl trisulfide, and lenthionine
(Bortolucci et al., 2021a). Other compounds have also been identified,
such as α-santalene, phytol, bis disulfide bis, methyl disulfide methyl,
(Arunachalam et al., 2017). Preclinical studies of G. integrifolia have
reported insecticidal (Bortolucci et al., 2021b), antibacterial (Bussmann
et al., 2010; Arunachalam et al., 2016), antitumor, antimicrobial, anti­
oxidant (Barbosa et al., 1999; Anwar et al., 2008; Bussmann et al.,
2010), fungicidal (Raimundo et al., 2018), gastroprotective (Aru­
nachalam et al., 2017), antinociceptive (Silva Júnior et al., 2013), and
anticytotoxic (Bortolucci et al., 2021b) effects.
The present study investigated the antifungal activity of
G. integrifolia against vulvovaginal candidiasis discharge in pregnant
women.
2. Materials and methods
2.1. Patient recruitment
Before collecting clinical samples, the study received approval from
the Ethics Committee on Research Involving Human Beings of UNIPAR
(CAEE no. 39975620.2.0000.0109). The participants provided both
verbal and written consent for vaginal secretion collection for research
purposes. Pregnant women (18–42 years old) who were receiving pre­
natal care at the Mother Paranaense Regional Outpatient Clinic,
Umuarama, Paraná, Brazil, with symptoms of vulvovaginal candidiasis
(at least two symptoms, including itch, irritation, foul odor, discharge,
painful urination, or pain during intercourse) and who had a diagnosis of
recurrent urinary tract infection (two or more episodes in the last 6
months or three or more episodes in the last year), with use of antibiotic
therapy, considering that the is a risk factor for candidiasis, were
included in the study (Chiavari-Frederico et al., 2020). Women who
were not pregnant, who were asymptomatic for candidiasis, who pre­
sented structural genetic abnormalities in the genital or urinary system,
who had genetic or drug-induced immune deficiency, who had neuro­
logical impairment that affects urinary tract function, who had a ma­
lignant disease of the pelvis, who had undergone pelvic radiotherapy
treatment, and who were unable to provide vaginal specimens were
excluded from the study (Tsega and Mekonnen, 2019).
2.2. Collection of clinical samples and microorganism identification
A total of 10 vaginal secretion samples from pregnant patients were
collected through a specular examination using swabs that contained
Aimes medium with activated charcoal (Transystem™, Copan Italia,
Brescia, Italy). The samples were obtained from patients who were
receiving prenatal care at the Mother Paranaense Regional Outpatient
Clinic, Umuarama, Paraná, Brazil, from May to August 2021. Immedi­
ately after collection, the samples were kept in Stuart transport medium
(Olen®) until sowing time, which was performed on the same day in
Agar Sabouraud medium (Kasvi®). For positive cultures, a fresh slide
was prepared to check for yeast growth, confirm micromorphology in
cornmeal Agar medium (Himedia®), and cultivate yeast growth in
Chromogenic Candida Agar culture medium (Laborclin®) that allows
the isolation and differentiation of most Candida species.

2
A.N.V. de Souza et al.
2.3. Clinical inoculum
To prepare the clinical inoculum, yeast cells from 24 h of culture at
35 ◦ C on Sabouraud Agar were suspended in sterile saline (0.9% NaCl).
The concentration was adjusted to 90% ± 2% transmittance in a spec­
trophotometer (Spectra Max Plus) at 530 nm, compatible with 1.0–5.0
× 106 CFU/mL. From this suspension, two new dilutions were made. The
first 1:50 dilution was made in sterile saline solution, and the second
1:20 dilution was made in Mueller Hinton Broth (Kasvi®) medium.
2.4. Plant material and essential oil extraction
The leaves, flowers, and fruits of Gallesia integrifolia were collected in
Umuarama, Paraná, Brazil (S23◦ 46′ 16′′ , W53◦ 19′ 38′′ ). An exsiccate was
deposited in the Herbarium of the State University of West Paraná
(UNIOESTE), Cascavel, Paraná, Brazil (no. 1716), and was registered in
the National System for Management of Genetic Heritage and Associated
Traditional Knowledge (SisGen, no. A7B8825). Leaves and flowers were
collected in December 2018, and fruits were collected in May and June
2019. Fresh leaves, flowers, and fruits of G. integrifolia were fragmented
with water (1:10) in an industrial blender for 5 min and immediately
submitted to hydrodistillation in a modified Clevenger apparatus for 3 h
(Raimundo et al., 2017). At the end of distillation, essential oil was
removed from the apparatus, transferred to amber vials, and stored at
4 ◦ C until use (Brasil, 2010). The plant name was checked on April 08,
2022 at http://www.theplantlist.org and found to be approved.
2.5. Gas chromatography-mass spectrometry
The chemical identification of compounds in leaves, flowers, and
fruits of Gallesia integrifolia essential oils was performed using gas
chromatography coupled to mass spectrometry (GC-MS; Agilent 7890B-
5977A MSD). The capillary column was an HP-5MS UI 5% (30 m × 0.25
mm × 0.25 μm), with an initial temperature of 60 ◦ C that was main­
tained for 3 min, followed by heating to 300 ◦ C at 5 ◦ C/min for 10 min
and then heating to 310 ◦ C at 10 ◦ C/min, which was maintained for 10
min. Helium was used as the carrier gas at a linear speed of 1 mL/min.
The injector temperature was 300 ◦ C. The injection volume was 1 μL.
The injection occurred in split mode (20:1). The transfer line was kept at
280 ◦ C. The ionization source and quadrupole temperatures were 230 ◦ C
and 150 ◦ C, respectively. Mass spectrometry was performed at a scan
range of 30–550 m/z with a solvent delay of 3 min. The compounds were
identified by comparing them to mass spectra in NIST 11.0 libraries and
comparing their retention indices (RIs) that were obtained by a standard
homologous series C7–C28 (Adams, 2017).
2.6. Preparation and selection of essential oils to assess antimicrobial
activity
The leaves, flowers, and fruits G. integrifolia essential oils were
weighed and diluted in water using 2% Tween 80 as the solubilizing
agent. The stock concentration was 10,000 μg/mL, and the working
range on the microplate was 9.7–5000 μg/mL.
Preliminarily, screening of the antifungal activity of leaves, flowers,
and fruits of G. integrifolia essential oils against standard strains was
performed with Candida tropicalis CCCD-CC002 (CEFAR BF–C 468
strain), Candida albicans CCCD-CC001 (CEFAR BF–H 709 strain), and
Candida spp (NEWPROVE1210) to select essential oils with the best
activity. Essential oils that were extracted from leaves and flowers of
G. integrifolia were selected for antifungal evaluation against clinical
yeast that was isolated from vaginal secretion samples from pregnant
patients.
2.7. Determination of MIC and minimum fungicidal concentration
Susceptibility tests were performed according to document M27-A3
3
Journal of Ethnopharmacology 295 (2022) 115403
(CLSI, 2008) with modifications for natural products. The tests were
performed in 96-well sterile plastic microplates that were arranged in
eight series from A to H, each with 12 wells numbered 1 to 12. Each row
corresponded to a yeast strain and received 50 μL of the corresponding
inoculum. In the wells of columns 2 to 11, aliquots of Mueller Hinton
Broth medium (50 μL) were added, and 100 μL was added in column 12.
The oils (50 μL) were added in columns 1 and 2 of the microplates. With
a multichannel pipette, a serial dilution (1:2) was performed from the
second column. The following controls were included: negative control
(Mueller Hinton broth only), microorganism control (culture medium
plus inoculum, without the addition of antifungal or oil), and thinner
control (Tween 80 and inoculum). As positive controls, fluconazole (64
μg/mL) and nystatin (64 μg/mL) were used. The standard strains
Candida tropicalis CCCD-CC002 (CEFAR BF–C 468 strain) and Candida
albicans CCCD-CC001 (CEFAR BF–H 709 strain) were used as reference
yeasts. The finished microplates were incubated in an oven at 35 ◦ C/24
h. Readings were then performed, and the MIC was determined. The MIC
was considered the lowest concentration at which the essential oils
inhibited 100% of yeast growth. Readings were performed with the
addition of 10 μL of 1.0% 2,3,5-triphenyltetrazolium chloride developer
(Reatec®) to each well followed by incubation of the microplates at
35 ◦ C for 20 min (Beloti et al., 1999).
After the readings, 2 μL of each well was cultivated in Mueller Hinton
agar culture medium and incubated at 35 ◦ C/24 h to determine the
minimum fungicidal concentration (MFC) of the essential oils against
the microorganisms. All tests were performed in triplicate.
2.8. Bioinformatics
Three-dimensional structures of the compounds were obtained from
the PubChem database or drawn by the Marvin program (chemaxon.
com) and later analyzed by the SEA Predictions server (Keiser et al.,
2007) to identify possible metabolic targets that are present in the fungi
by reverse docking. The results indicated that the enzyme lanosterol
synthase (LSS E.C.: 5.4.99.7) is a possible target, which motivated
modeling of the LSS structure from C. albicans (C. albicans LSS; UniProt
ID: q04782) in the Apo form using the AlphaFold2 server (Jumper et al.,
2021). The human homologous enzyme that was crystallized in the
presence of lanosterol substrate (LAN) was used as a template (Protein
Data Bank ID: 1w6k; Thoma et al., 2004) for geometric docking of the
LAN ligand in C. albicans LSS using the Coot program (Emsley et al.,
2010).
To correct possible stereochemical clashes, the C. albicans LSS-LAN
complex was submitted to structure minimization by molecular dy­
namics using the NAMD2 program (Phillips et al., 2005). The structure
of the C. albicans LSS-LAN complex was virtually immersed in a periodic
box whose edges were at least 15 Å away from the outermost surface of
the protein, and a sufficient amount of sodium counterions was added to
neutralize system charges. The Charmm36 force field (Mackerell et al.,
2004) was used for the protein, waters, and salts. The ligand force field
was generated by the SwissParam server (Zoete et al., 2011) in the same
format. Minimization occurred in stages. In the first stage, the system
was subjected to 20,000 steps of minimization by a conjugate gradient,
in which the ligand atoms were fixed in space to preserve crystallo­
graphic structure information. In the second stage, all atoms of the
system were subjected to a new 10,000-step conjugate gradient, and the
structure that emerged from this stage was used in conventional docking
simulations.
The docking simulations were performed using two software pro­
grams. The AutoDock Vina program used the standard search and
ranking algorithm, with search box dimensions at 20, 20, and 20
centered on LAN. The Gold program used CHEMPLP as a score function,
200% efficiency, and a search radius of 15 Å centered on the LAN ligand.
The simulations were performed in quadruplicate by each program, and
mean relative scores were calculated according to the protocol of da
Silva et al. (2021), with the LAN score as a cutoff.
A.N.V. de Souza et al. Journal of Ethnopharmacology 295 (2022) 115403

2.9. Statistical analysis
Differences between groups were assessed using analysis of variance
(ANOVA), followed by Tukey’s post hoc test. Values of p < 0.05 were
considered statistically significant. The results are expressed as mean ±
standard deviation (SD). The statistical analyses were performed using
Statistica 13.3 software.
3. Results
3.1. Microbiological identification of vaginal secretion samples from
pregnant patients
Table 1 shows that of the 10 clinical samples that were isolated from
vaginal secretions from pregnant women, five were positive for two
strains of Candida spp, identified as Candida albicans (30%) and Candida
glabrata (20%).
3.2. Chemical composition of G. integrifolia essential oil
against Candida spp, C. albicans, and C. tropicalis standard strains ranged
from 13.01 to 625.00 μg/mL (Table 3). The one-way ANOVA indicated a
significant difference in activity against Candida spp (F1,3 = 2542.23, p
< 0.001), C. albicans (F1,3 = 1133.48, p < 0.001), and C. tropicalis (F1,3 =
4.61, p < 0.05) between the essential oils. Tukey’s post hoc test showed
that the essential oil that was extracted from flowers of G. integrifolia
(MIC = 16.27 μg/mL) was the most active against the Candida spp strain
compared with leaves (MIC = 156.25 μg/mL), fruits (MIC = 625 μg/
mL), and the positive control fluconazole (MIC = 53.33 μg/mL).
C. albicans was more susceptible to essential oils that were extracted
from leaves (MIC = 32.55 μg/mL) and flowers (MIC = 13.01 μg/mL)
compared with fruits (p < 0.05). No significant difference was found
between essential oils from leaves and flowers of G. integrifolia and the
positive control fluconazole (p > 0.05). C. tropicalis was significantly
inhibited (p < 0.05) by essential oils that were extracted from flowers
(16.27 μg/mL) and fruits (52.08 μg/mL). Considering the preliminary
results of the antifungal activity of G. integrifolia essential oils, we
evaluated their antifungal efficacy in clinical samples that were isolated
from pregnant patients.

Table 2 shows the gas chromatography profile and probable chem­ 3.4. Antifungal activity
ical composition of the leaves, flowers, and fruits of G. integrifolia
essential oils. Essential oil that was extracted from leaves of G. integ­ Table 4 shows individual MICs and MFCs for each clinical yeast
rifolia contained 3,5-dithiahexanol-5,5-dioxide (40.93%, C4H10O3S2, isolate. The MIC for leaves ranged from 52.08 to 5000.00 μg/mL. The
MW = 170.3) as the most abundant compound, followed by 1,3,5-tri­ MIC for flowers ranged from 208.33 to 1666.66 μg/mL. The MBCs for
thiane (14.25%, C3H6S3, MW = 138.3) and N-ethyl-1,3-dithioi­ clinical samples were >5000.00 μg/ml.
soindole (13.03%, C10H13NS2, MW = 211.4). Essential oil that was Table 5 shows the in vitro antifungal activity of G. integrifolia essential
extracted from flowers of G. integrifolia contained methionine ethyl oils that were extracted from leaves and flowers against Candida yeast
ester (46.78%, C7H15NO2S, MW = 177.27) as the most abundant com­ species that were isolated from vaginal secretion samples from pregnant
pound, followed by methyl p-tolyl sulfide (17.64%, C8H10S, MW = patients. The MIC of G. integrifolia essential oils (leaves and flowers)
138.23) and N-ethyl-1,3-dithioisoindole (13.24%, C10H13NS2, MW = against C. albicans and C. glabrata ranged from 277.77 to 2604.16 μg/
211.4). Essential oil that was extracted from fruits of G. integrifolia mL. The one-way ANOVA indicated a significant difference between the
contained 2,8-dithianonane (54.01%, C7H16S2, MW = 164.3) as the essential oils against C. albicans (F1,3 = 8.48, p < 0.001) and C. glabrata
most abundant compound, followed by dimethyl trisulfide (16.22%, (F1,3 = 4.62, p < 0.01). Tukey’s post hoc test showed that the essential oil
C2H6S3, MW = 126.3) and lenthionine (14.87%, C2H4S5, MW = of leaves (277.77 μg/mL) was the most active compared with flowers
188.4). (659.72 μg/mL). With regard to inhibiting C. glabrata, Tukey’s post hoc
did not indicate significant differences between the essential oils of
3.3. Screening of antifungal activity of G. integrifolia essential oils against leaves and flowers (p > 0.05).
standard yeast strains
3.5. Identification of target structure, modeling, and docking
The MIC of G. integrifolia essential oils of leaves, flowers, and fruits
The structures of the compounds in the library were submitted to the
Table 1 SEA (Similarity ensemble approach) Predictions server, which verifies
Microbiological identification of yeast isolated from vaginal secretion samples the similarity of a given compound with a database of ligands of known
from pregnant patients. targets. If similarity between the query and ligand of a given protein is
Sample Initial Cornmeal Chromogenic Final found, then the query likely also binds to this target. The phytol com­
culture agar Candida agar identification pound was found to have similarity to a ligand of LSS (MaxTC: 0.38; e-
1 yeast C. albicans green C. albicans value: 5.063 × 10− 24), a target that is also present in Candida albicans.
positive To test whether phytol or another component of the essential oil is
2 yeast C. albicans green C. albicans able to bind LSS, the structure of the C. albicans LSS enzyme was
positive
3 yeast – – –
modeled in the presence of LAN. The AlphaFold2 server generates only
negative Apo-shaped structures. Therefore, the LAN ligand was later inserted by
4 yeast C. glabrata lilac/mauve C. glabrata geometric docking. This caused stereochemical clashes that need to be
positive corrected by minimizing the complex (Fig. 1).
5 yeast – – –
The simulation programs were unable to perform docking of the low-
negative
6 yeast – – – MW compounds that are listed in Table 2. Therefore, only relative mean
negative scores of docked compounds are shown in Fig. 2.
7 yeast – – – The results of the docking simulations suggest that the ligand phytol
negative (PubChem CID: 5280435) is the component of the essential oil of Gal­
8 yeast
lesia integrifolia that most likely binds the C. albicans LSS enzyme, fol­
– – –
negative
9 yeast C. glabrata lilac/mauve C. glabrata lowed by the ligands S-phenyl-p-toluene-thiosulfonate and 5-methyl-2-
positive phenylindole. The intermolecular interactions that phytol makes with
10 yeast C. albicans green C. albicans the C. albicans LSS enzyme are very similar to interactions that are made
positive
by the lanosterol substrate, highlighting the charge-dipole interaction
(− ) = absent. with the Asp449 residue (Fig. 3).

4
A.N.V. de Souza et al. Journal of Ethnopharmacology 295 (2022) 115403

Table 2
Chemical composition of Gallesia integrifolia essential oils extracted from leaves, flowers, and fruits.
Peak Compoundsa RIbcalc Relative area (%) for essential oils Identification method

Leaves Flowers Fruits

1 dimethyl disulfide 808 t 3.45 0.91 a, b, c

2 2,4-dithiapentane 810 4.16 t t a, b, c
3 2,3,5-trithiahexane 885 3.79 2.29 t a, b, c
4 1,2,4-trithiolane 973 1.04 1.81 0.76 a, b, c
5 dimethyl trisulfide 1101 1.13 t 16.22 a, b, c
6 1,3,5-trithiane 1137 14.25 t t a, b, c
7 methyl p-tolyl sulfide 1138 – 17.64 – a, b, c
8 1,2,4,5-tetrathiane 1262 t t 5.81 a, b, c
9 n.i. 1344 – 0.56 – a, b, c
10 dimethyl tetrasulfide 1367 5.59 t – a, b, c
11 2,8-dithianonane 1478 t t 54.01 a, b, c
12 3,6-dithiaoctan-1-ol 1506 t – 0.63 a, b, c
13 n.i. 1559 – – 0.53 a, b, c
14 3,6-dioxa-8-mercaptooctane-1-ol 1618 1.88 – t a, b, c
15 3,5-dithiahexanol-5,5-dioxide 1634 40.93 6.14 t a, b, c
16 methionine ethyl ester 1716 5.29 46.78 t a, b, c
17 3,6-dithiaoctan-1,8-diol 1718 t 0.62 t a, b, c
18 Lenthionine 1761 0.66 0.56 14.87 a, b, c
19 ethanol, 2-(octylthio) 1780 0.66 t t a, b, c
20 Hexathiepane 1779 t – 5.48 a, b, c
21 N-ethyl-1,3-dithioisoindole 1847 13.03 13.24 t a, b, c
22 5-methyl-2-phenylindole 1914 t t 0.58 a, b, c
23 Phytol 1916 4.00 3.04 – a, b, c
24 propyl-undecenoate 2194 2.23 0.53 – a, b, c
25 3-([(methylthio)methy]sulfonyl)-1-phenyl-1-propanone 2321 0.54 – t a, b, c
26 S-phenyl-p-toluene-thiosulfonate 2325 t 3.10 t a, b, c
27 n.i. 2328 0.55 – – a, b, c
Total Identification 99.18 99.20 99.27

a: Compounds listed in order of elution in HP-5MS column; b: Identification based on retention index (RI) using a homologous series of n-alkane C7–C28 on Agilent HP-
5MS column; c: Identification based on comparison of mass spectra using with NIST 11.0 library. Relative area (%): percentage of area occupied by the compounds in
the chromatogram. n.i. = not identified. t = trace. (− ) = absent.

Table 3
Antifungal activity of Gallesia integrifolia essential oils extracted from leaves, flowers, and fruits against standard strains of different Candida species.
Minimum inhibitory concentration (μg/mL)

Microorganism Source Commercial drugs (control) Gallesia integrifólia essential oils

Nystatin Fluconazole Leaves Flowers Fruits

Candida spp NEWPROVE1210 >64 53.33 ± 18.47 156.25 ± 0.001* 16.27 ± 5.64*, + 625.00 ± 0.001*
Candida albicans (CEFAR BF–H 709) CCCD-CC001 >64 34.66 ± 28.09# 32.55 ± 11.27# 13.01 ± 5.64# 625.00 ± 0.001
Candida tropicalis (CEFAR BF–C 468) CCCD-CC002 >64 32.00 ± 0.001++ 117.18 ± 67.65 16.27 ± 5.64++ 52.08 ± 22.55++

The data are expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance, followed by Tukey (*p < 0.01 compared with
Fluconazole; +p < 0.01 compared with leaves and fruits; #p < 0.01 compared with fruits; ++p < 0.01 compared with leaves).

Table 4
Antifungal activity of Gallesia integrifolia essential oils extracted from leaves and flowers against Candida yeast species isolated from vaginal secretion samples from
pregnant patients and standard strains, expressed as minimal inhibitory concentration (MIC; μg/mL) and minimum fungicidal concentration (MFC).
Sample Commercial drugs (control) Gallesia integrifólia

Nystatin Fluconazole Leaves Flowers

MIC (μg/ MFC (μg/ MIC (μg/mL) MFC (μg/ MIC (μg/mL) MFC (μg/ MIC (μg/mL) MFC (μg/
mL) mL) mL) mL) mL)

1 >64 >64 >64 >64 52.08 ± 22.55 >5000 208.33 ± 90.21 >5000
2 >64 >64 >64 >64 156.25 ± 0.001 >5000 520.83 ± 180.42 >5000
4 >64 >64 >64 >64 5000.00 ± >5000 1666.66 ± >5000
0.001 721.68
9 >64 >64 >64 >64 208.33 ± 90.21 >5000 833.33 ± 360.84 >5000
10 >64 >64 >64 >64 625.00 ± 0.001 >5000 1250.00 ± 0.001 >5000
Candida albicans (CEFAR BF–H >64 >64 42.66 ± >64 91.14 ± 59.67 >5000 32.55 ± 11.27 >5000
709) 18.47
Candida tropicalis (CEFAR BF–C >64 >64 42.66 ± >64 32.70 ± 11.41 >5000 19.53 ± 0.001 >5000
468) 18.47

The data are expressed as mean ± SD.

5
A.N.V. de Souza et al. Journal of Ethnopharmacology 295 (2022) 115403

Table 5 4. Discussion
Antifungal activity of Gallesia integrifolia essential oils extracted from leaves and
flowers against yeast species isolated from vaginal secretion samples from Candida spp genus yeasts are opportunistic pathogenic fungi that are
pregnant patients, expressed as minimal inhibitory concentration (MIC). part of the human microbiome. Under certain conditions, such as
Minimum inhibitory concentration (μg/mL) immunosuppression, changes in vaginal pH, antibiotic therapy, hospital
Microorganism n Commercial drugs Gallesia integrifólia admission, urinary catheter use, chronic diseases, and pregnancy,
vaginal secretion (control) among others (Dadar et al., 2018), the presence of these fungi may
Nystatin Fluconazole Leaves Flowers
progress to systemic infection (Polke et al., 2015; Romo, and Kumamoto,
2020). The main species of Candida infections, Candida albicans, is found
Candida albicans 3 277.77 ± 659.72 ±
in several anatomical sites, such as the oropharynx and genitourinary
>64 >64
264.53* 473.78
Candida glabrata 2 >64 >64 2604.16 ± 1250.00 ± and gastrointestinal tracts. This species is the most pathogenic, multi­
2625.12 684.65 resistant, and responsible for the majority of invasive candidiasis cases
(Anh et al., 2021).
The data are expressed as mean ± standard deviation (SD). Data were analyzed
by one-way analysis of variance, followed by Tukey (*p < 0.01 compared with An estimated 40% of pregnant women worldwide may have vulvo­
Flowers). vaginal candidiasis, with C. albicans accounting for ~90% of cases,
followed by C. glabrata accounting for ~12% of cases (Soares, and
Pereira, 2018; Yadav and Prakash, 2016; Alo et al., 2012; Alli et al.,
2011). Corroborating these statistics, the present study found that 50%
of the clinical samples were contaminated by yeast, and C. albicans was
the most frequent (30%), followed by C. glabrata (20%).
High levels of hormones (e.g., estrogen and progesterone) facilitate
the adhesion and multiplication of yeast that predispose pregnant
women to primary candidiasis and recurrence. When left untreated,
yeast infection can contribute to the development of gestational com­
plications (Freitas et al., 2020; Gómez-Rodríguez et al., 2019). However,
unlike other types of vaginitis, no consensus has been reached in the
literature that Candida colonization during the gestational period is
associated with a higher risk of premature birth, despite emerging evi­
dence and systematic reviews that report that the eradication of candi­
diasis in pregnancy, even asymptomatic cases, can reduce the risk of
premature birth, late miscarriage, and other complications (Hay and
Czeizel, 2007; Schuster et al., 2020).
The treatment and control of fungal infection with antifungal agents,
such as clotrimazole, fluconazole, itraconazole, and nystatin, signifi­
cantly lowers rates of premature birth and other complications, but not
all of these standard treatments are available in public healthcare sys­
tems, thereby limiting therapy and resulting in recurrent episodes, long
treatment times, and therapeutic failure (Ghaddar et al., 2020; Mathe­
son and Mazza, 2017; Brandolt et al., 2017).
Fig. 1. Ribbon model of C. albicans LSS (green) bound to lanosterol substrate Antifungal resistance was also observed in the clinical samples in the
(space-filling) used in the docking simulations. (For interpretation of the ref­
present study, in which 100% of the isolated samples did not respond to
erences to colour in this figure legend, the reader is referred to the Web version
the positive controls nystatin and fluconazole (>64 μg ml− 1; suscepti­
of this article.)
bility analysis performed according to the CLSI M27-A2 protocol). New
therapeutic agents that are effective and less expensive and have fewer
adverse effects need to be developed.
G. integrifolia has antioxidant and antifungal properties (Bortolucci
et al., 2021a; Raimundo et al., 2021; Spada et al., 2019). The medicinal
characteristics of its leaves, flowers, fruits, and bark have been studied.
The present study evaluated the antifungal activity of essential oils from
different parts of G. integrifolia against vulvovaginal candidiasis in
samples of vaginal secretions from pregnant women. Essential oils with
organosulfur compounds have broad-spectrum antimicrobial activity in
vitro (Silva et al., 2018) against medical, food, and agricultural patho­
gens (Bortolucci et al., 2021b; Raimundo et al., 2018).
The results of the present study supported our hypothesis that
fungicidal activity was related to specific compounds in essential oils
that were extracted from different parts of the plant. The chromato­
graphic analysis of G. integrifolia essential oils indicated that 3,5-dithia­
hexanol-5,5-dioxide was a major component in leaves (40.93%),
methionine ethyl ester was the major component in flowers (46.78%),
and 2,8-dithianonane was the major component in fruits (54.01%).
Fig. 2. Relative mean scores of compounds found in Gallesia integrifolia Corroborating our findings, Raimundo et al. (2021) reported that the
essential oil. most prominent essential oil compounds were 2,8-dithianonane (52.6%)
in fruits, 3,5-dithiahexanol-5,5-dioxide (38.9%) in leaves, and methio­
nine ethyl ester (45.3%) in flowers. Other major compounds were also
found, including dimethyl trisulfide (15.3%) and lenthionine (14.7%) in

6
A.N.V. de Souza et al.
Fig. 3. Two-dimensional diagram of the map of interactions that the ligands phytol (A) and lanosterol (B) make with the C. albicans LSS enzyme, with van der Waals
contacts represented in green and the charge-dipole interaction shown as dotted lines. The figure was generated by the PoseView program (Stierand and Rarey,
2010). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
fruits, 1,3,5-trithiane (13.7%) and N-ethyl-1,3-dithioisoindole (12.6%)
in leaves, and methyl p-tolyl sulfide (17.1%) and N-ethyl-1,3-dithioi­
soindole (13.4%) in flowers.
Against the standard strains, our results showed that the essential oil
that was extracted from flowers more effectively inhibited Candida spp.
Leaves and flowers were similar to fluconazole against C. albicans, and
flowers and fruits were similar to fluconazole against C. tropocalis. These
results encouraged us to further evaluate essential oils that were
extracted from leaves and flowers in clinical samples that were collected
from pregnant women who presented symptoms of vulvovaginal
candidiasis. In Candida yeast species that were isolated from vaginal
secretion samples from pregnant patients, the MICs of leaves and flowers
ranged from 52.08 to 5000.00 μg/mL, and leaves (277.77 μg/mL) were
the most active against C. albicans.
Bioinformatics analyses may provide clues into the ways in which
compounds from essential oils of G. integrifolia can affect metabolism of
the microorganism. Reverse docking is a tool that identifies the most
likely targets based on the similarity of a given compound at query to a
database of ligands from their respective enzymes. Thus, it is possible to
associate the query molecule with a particular metabolic target, based
on similarity to ligands of this target. The enzyme LSS is part of the third
module of the ergosterol biosynthesis pathway (Roessner et al., 1993)
and catalyzes the cyclization of (S)-2,3 oxidosqualene to lanosterol, a
reaction that forms the sterol core that is essential for the cell wall
structure in fungi (Kelly et al., 1990). The CYP51 enzyme also partici­
pates in this process and is a target of all azole antifungals (Zonios and
Bennett, 2008). These findings demonstrate the efficiency of inhibiting
enzymes that are involved in ergosterol biosynthesis to control fungal
infections.
The reverse docking experiments suggested that the phytol com­
pound that was present in essential oils of G. integrifolia could bind to
LSS. Thus, conventional docking with the modeled enzyme of C. albicans
showed that, among all compounds that were present in the essential oil,
phytol was the one that would most likely bind, although it was not the
component with the highest concentration in the samples. However,
7
Journal of Ethnopharmacology 295 (2022) 115403
docking simulations only suggest a possible interaction mechanism be­
tween a protein and ligand, and the score that is provided by this
interaction should be regarded as related to probability and not be
confused with binding affinity (Kato-Schwartz et al., 2020).
Thus, experimental validation is very important to confirm pre­
dictions of simulations. Table 2 shows that the highest concentrations of
phytol in the G. integrifolia essential oils were from leaves (4.00%) and
flowers (3.04%), vegetal parts that confer the greatest antifungal effects
(i.e., lower MICs) against Candida. This hypothesis is supported by the
fact that phytol has already been reported to have antifungal actions
against bacteria and fungi (Ghaneian et al., 2015), including Candida
spp (Lima et al., 2020). However, the mechanism of action of this
compound has not been thoroughly studied. Altogether, this evidence
reinforces the proposition that phytol inhibits the enzyme LSS and ul­
timately affects the synthesis of ergosterol that is necessary for fungus
survival.
Finally, the present study found that G. integrifolia exerted antifungal
activity against standard strains and yeast species of Candida that were
isolated from cultures of vaginal secretions from pregnant women,
suggesting its potential medicinal use as a therapeutic agent against
Candida, the primary cause of vulvovaginal candidiasis. The limitations
of this in vitro study are that preclinical and clinical research was not
conducted. We also did not evaluate toxicity of the essential oils in
pregnant women. Such toxicological evaluations will be critical for
determining the safety and efficacy of this plant. Further high-
throughput screening in animal studies and clinical trials are needed
to confirm that specific G. integrifolia metabolites may be potential
drugs.
5. Conclusion
Bioactive compounds in leaves, flowers, and fruits of G. integrifolia
essential oils presented antifungal activity against standard strains and
Candida yeast species that were isolated from cultures of vaginal se­
cretions from pregnancy women. Leaves were more efficient against
A.N.V. de Souza et al.
C. albicans, and flowers were more efficient against C. glabrata. The re­
sults suggest that the compound phytol is responsible for this beneficial
effect of G. integrifolia against Candida, the etiological agent of vulvo­
vaginal candidiasis.
Financial Support
The authors are grateful to Universidade Paranaense, Graduate
Program in Medicinal Plants and Phytotherapeutics in Basic Attention,
and Graduate Program in Animal Science with Emphasis on Bioactive
Products for financial support. This work was supported by grants from
Paranaense University (UNIPAR; no. 124718) and Fundação Araucária
(no. PIB2021101000002 and 87/2021).
CRediT authorship contribution statement
Amanda Nascimento Vasques de Souza: Methodology, Investiga­
tion, Data curation. Maria Graciela Iecher Faria: Methodology,
Investigation, Data curation. Cristiane Edna da Rocha: Methodology,
Investigation, Data curation. Gisele Strieder Philippsen: Methodology,
Investigation, Data curation. Gabriela Catuzo Canonico Silva: Meth­
odology, Investigation, Data curation. Gustavo Ratti da Silva: Meth­
odology, Investigation, Data curation. Rodrigo Sadao Inumaro:
Methodology, Investigation, Data curation. José Eduardo Gonçalves:
Methodology, Investigation, Data curation. Zilda Cristiani Gazim:
Methodology, Investigation, Data curation. Samantha Wietzikoski:
Methodology, Investigation, Data curation. Francislaine Aparecida
dos Reis Lívero: Methodology, Investigation, Data curation, Writing –
original draft, Supervision, Writing – review & editing. Flavio Augusto
Vicente Seixas: Methodology, Investigation, Data curation, Writing –
original draft. Evellyn Claudia Wietzikoski Lovato: Conceptualiza­
tion, Funding acquisition, Project administration, Methodology, Inves­
tigation, Data curation, Writing – original draft, Supervision, Writing –
review & editing.
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Beatriz Pascoali Cunha, Camile Fernanda Squi­
satti, Gabriel Henrique do Prado Gonçalves, Heloisa Tonial, Joana de
Angelis Ponte e Silva, and Renata Carolina Della Riva for their assistance
with the experiments and UNIPAR and Fundação Araucaria for
providing the research grant and fellowships that enabled this study.
References
Adams, R.P., 2017. Identification of Essential Oil Components by Gas Chromatography/
massspectrometry, fourth one ed. Allured, Carol Stream, USA, p. 804.
Akash, M.S., 2021. A review on antibacterial properties of natural plants. Int. J. Adv. Res.
Biol. Sci. 8 (2) https://doi.org/10.22192/ijarbs.2021.08.02.002, 140-14.
Alli, J.A.O., Okonko, I.O., Odu, N.N., Kolade, A.F., Nwanze, J.C., 2011. Detection and
prevalence of Candida isolates among patients in Ibadan, Southwestern Nigeria.
J. Microbiol. Biotechnol. Res. 1, 176–184.
Alo, M.N., Anyim, C., Onyebuchi, A.K., Okonkwo, E.C., 2012. Prevalence of
asymptomatic infection of candidiasis and vaginal trichomoniasis among pregnant
women in Abakaliki, South-Eastern Nigeria. J. Nat. Sci. Res. 2, 87–91.
Anand, U., Jacobo-Herrera, N., Altemimi, A., Lakhssassi, N., 2019. A comprehensive
review on medicinal plants as antimicrobial therapeutics: potential avenues of
biocompatible drug discovery. Metabolites 9 (11), 258. https://doi.org/10.3390/
metabo9110258.
Anh, D.N., Hung, D.N., Tien, T.V., Dinh, V.N., Son, V.T., Luong, N.V., Van, N.T.,
Quynh, N.T.N., Van Tuan, N., Tuan, L.Q., Bac, N.D., Luc, N.K., Anh, L.T., Trung, D.
M., 2021. Prevalence, species distribution and antifungal susceptibility of Candida
albicans causing vaginal discharge among symptomatic non-pregnant women of
reproductive age at a tertiary care hospital. Vietnam. BMC Infect Dis. 21 (1), 523.
https://doi.org/10.1186/s12879-021-06192-7.
Anwar, A., Burkholz, T., Scherer, C., Abbas, M., Lehr, C.M., Diederich, M., Jacob, C.,
2008. Naturally occurring reactive sulfur species, their activity against Caco-2 cells,
8
Journal of Ethnopharmacology 295 (2022) 115403
and possible modes of biochemical action. J. Sulphur Chem. 29 (3–4), 251–268.
https://doi.org/10.1080/17415990802195623.
Arunachalam, K., Ascêncio, S.D., Soares, I.M., Aguiar, R.W.S., Silva, L.I., Oliveira, R.G.,
Balogun, S.O., Martins, D.T.O., 2016. Gallesia integrifolia (Spreng.) Harms: in vitro
and in vivo antibacterial activities and mode of action. J. Ethnopharmacol. 184,
128–137. https://doi.org/10.1016/j.jep.2016.03.005.
Arunachalam, K., Balogun, S.O., Pavan, E., de Almeida, G.V.B., de Oliveira, R.G.,
Wagner, T., Cechinel Filho, V., de Oliveira Martins, D.T., 2017. Chemical
characterization, toxicology and mechanism of gastric antiulcer action of essential
oil from Gallesia integrifolia (Spreng.) Harms in the in vitro and in vivo experimental
models. Biomed. Pharmacother. 94, 292–306. https://doi.org/10.1016/j.
biopha.2017.07.064.
Balbach, A., 1993. As Plantas Curam [The Plants that Heal]. Missionary Publishing
house, São Paulo, Brazil.
Barbosa, L.C.A., Demuner, A.J., Teixeira, R.R., Madruga, M.S., 1999. Chemical
constituents of the bark of Gallesia gorazema. Fitoterapia 70, 152–156. https://doi.
org/10.1016/s0367-326x(99)00014-3.
Beloti, V., Barros, M.A., Freitas, J.C., Nero, L.A., Souza, J.A., Santana, E.H.W., Franco, B.
D.G.M., 1999. Frequency of 2,3,5-triphenyltetrazolium chloride (TTC) non-reducing
bacteria in pasteurized milk. Revista de Microbiologia [online] 30 (2), 137–140.
https://doi.org/10.1590/S0001-37141999000200009.
Berkow, E.L., Lockhart, S.R., 2017. Fluconazole resistance in Candida species: a current
perspective. Infect. Drug Resist. 10, 237–245. https://doi.org/10.2147/IDR.
S118892.
Bieski, I.G.C., Santos, F.R., de Oliveira, R.M., Espinosa, M.M., Macedo, M.,
Albuquerque, U.P., de Oliveira Martins, D.T., 2012. Ethnopharmacology of
medicinal plants of the Pantanal region (Mato Grosso, Brazil). Evid.-Based
complement. Alternative Med., 272749 https://doi.org/10.1155/2012/272749,
2012.
Bortolucci, W.C., Raimundo, K.F., Fernandez, C.M.M., Calhelha, R.C., Ferreira, I.C.F.R.,
Barros, L., Gonçalves, J.E., Linde, G.A., Colauto, N.B., Gazim, Z.C., 2021a.
Cytotoxicity and anti-inflammatory activities of Gallesia integrifolia (Phytolaccaceae)
fruit essential oil. Nat. Prod. Res. 18, 1–6. https://doi.org/10.1080/14786419.2021.
Bortolucci, W.C., Oliveira, H.L.M., Oliva, L.R., Gonçalves, J.E., Piau Júnior, R.,
Fernandez, C.M.M., Colauto, N.B., Linde, G.A., Gazim, Z.C., 2021b. Crude extract of
the tropical tree Gallesia integrifolia (Phytolaccaceae) for the control of Aedes aegypti
(Diptera: Culicidae) larvae. Rev. Biol. Trop. 69 (1), 153–169. https://doi.org/
10.15517/rbt.v69i1.41225.
Brandolt, T.M., Klafke, G.B., Gonçalves, C.V., Bitencourt, L.R., de Martinez, A.M.B.,
Mendes, J.F., Meireles, M.C.A., Xavier, M.O., 2017. Prevalência de Candida spp. em
amostras cérvico-vaginais e a suscetibilidade in vitro de isolados. Braz. J. Microbiol.
48 (1), 145–150. https://doi.org/10.1016/j.bjm.2016.09.006.
Brasil, 2010. Farmacopeia Brasileira. Agência Nacional de Vigilância Sanitária, 5a ed.
Anvisa, Brasília, p. 546.
Bueno, N.R., Campos, E.P., Silva, M.S., Rezende, K.S., Lima, B.B.M., 2019. Levantamento
etnofarmacológico e farmacológico de plantas medicinais comercializadas em
Rondonópolis (MT). Rev. Biodiversidade. 18, 2–20.
Bussmann, R.W., Glenn, A., Sharon, D., 2010. Antibacterial activity of medicinal plants
of Northern Peru – can traditional applications provide leads for modern science?
Indian J. Tradit. Knowl. 9, 742–753. http://nopr.niscair.res.in/handle/123456
789/10330.
Chiavari-Frederico, M.O., Barbosa, L.N., Carvalho dos Santos, I., Ratti da Silva, G.,
Fernandes de Castro, A., de Campos Bortolucci, W., Barboza, L.N., Campos, C.F.A.A.,
Gonçalves, J.E., Menetrier, J.V., Jacomassi, E., Gazim, Z.C., Wietzikoski, S., Dos Reis
Lívero, F.A., Wietzikoski Lovato, E.C., 2020. Antimicrobial activity of Asteraceae
species against bacterial pathogens isolated from postmenopausal women. PLoS One
15 (1), e0227023. https://doi.org/10.1371/journal.pone.0227023.
Clinical and Laboratory Standards Institute (CLSI), 2008. Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard, third ed.
Clinical and Laboratory Standards Institute, Wayne, PA. CLSI M27-A3.
Colombo, A.L., Almeida Júnior, J.N., Guinea, J., 2017. Emerging multidrug-resistant
Candida species. Curr. Opin. Infect. Dis. 30 (6), 528–538. https://doi.org/10.1097/
QCO.0000000000000411.
da Silva, T.B.V., Castilho, P.A., Sá-Nakanishi, A.B., Seixas, F.A.V., Dias, M.I., Barros, L.,
Ferreira, I.C.F.R., Bracht, A., Peralta, R.M., 2021. The inhibitory action of purple tea
on in vivo starch digestion compared to other Camellia sinensis teas. Food Res. Int.
150 (Pt A), 110781 https://doi.org/10.1016/j.foodres.2021.110781.
Dadar, M., Tiwari, R., Karthik, K., Chakraborty, S., Shahali, Y., Dhama, K., 2018. Candida
albicans - biology, molecular characterization, pathogenicity, and advances in
diagnosis and control - an update. Microb. Pathog. 117, 128–138. https://doi.org/
10.1016/j.micpath.2018.02.028.
Di Mambro, T., Guerriero, I., Aurisicchio, L., Magnani, M., Marra, E., 2019. The Yin and
Yang of current antifungal therapeutic strategies: how can we harness our natural
defenses? Front. Pharmacol. 10, 80. https://doi.org/10.3389/fphar.2019.00080.
Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., 2010. Features and development of
Coot. Acta Crystallogr. D66, 486–501. https://doi.org/10.1107/
S0907444910007493.
Forzza, R.C., Baumgratz, J.F.A., Bicudo, C.E.M., Canhos, D.A.L., Carvalho, A.A.,
Coelho, M.A.N., Costa, A.F., Costa, D.P., Hopkins, M.G., Leitman, P.M., Lohmann, L.
G., Lughadha, E.N., Maia, L.C., Martinelli, G., Menezes, M., Morim, M.P., Peixoto, A.
L., Pirani, J.R., Prado, J., Queiroz, L.P., Souza, S., Souza, V.C., Stehmann, J.R.,
Sylvestre, L.S., Walter, B.M.T., Zappi, D.C., 2012. New Brazilian floristic list
highlights conservation challenges. Bioscience 62 (1), 39–45. https://doi.org/
10.1525/bio.2012.62.1.8.
Freitas, L.F.Q., Maia, L.R.S., Deus, M.R.A.R., Oliveira, S.R., Peres, A.L., 2020. Frequency
of Microorganisms in Vaginal Discharges of High-Risk Pregnant Women from a
A.N.V. de Souza et al.
Hospital in Caruaru, Pernambuco, Brazil, vol. 56. Jornal Brasileiro de Patologia e
Medicina Laboratorial, e2062020. https://doi.org/10.5935/1676-2444.20200048
[online].
Ghaddar, N., Anastasiadis, E., Halimeh, R., Ghaddar, A., Dhar, R., AlFouzan, W.,
Yusef, H., El Chaar, M., 2020. Prevalence and antifungal susceptibility of Candida
albicans causing vaginal discharge among pregnant women in Lebanon. BMC Infect.
Dis. 20 (1), 32. https://doi.org/10.1186/s12879-019-4736-2.
Ghaneian, M.T., Ehrampoush, M.H., Jebali, A., Hekmatimoghaddam, S., Mahmoudi, M.,
2015. Antimicrobial activity, toxicity and stability of phytol as a novel surface
disinfectant. Environ. Health Eng. Manag. J. 2 (1), 13–16. https://ssrn.com/abstract
=2610015.
Gómez-Rodríguez, L.D.C., Campo-Urbina, M.L., Ortega-Ariza, N., Bettin-Martínez, A.,
Parody-Muñoz, A., 2019. Prevalence of potentially pathogenic microbiological
agents in vaginal exudates of asymptomatic pregnant women, Barranquilla,
Colombia, 2014-2015. Rev. Colomb. Obstet. Ginecolog. 70 (1), 49–56. https://doi.
org/10.18597/rcog.3183.
Grandtner, M.M., Chevrette, J., 2013. Dictionary of Trees, vol. 2. Elsevier, South
America.
Hay, P., Czeizel, A.E., 2007. Asymptomatic trichomonas and candida colonization and
pregnancy outcome. Best Pract. Res. Clin. Obstet. Gynaecol. 21 (3), 403–409.
https://doi.org/10.1016/j.bpobgyn.2007.02.002.
Jamshidi-Kia, F., Lorigooini, Z., Amini-Khoei, H., 2018. Medicinal plants: past history
and future perspective. J. Herbmed. Pharmacol. 7 (1), 1–7. https://doi.org/
10.15171/jhp.2018.01.
Jumper, J., Evans, R., Pritzel, A., et al., 2021. Highly accurate protein structure
prediction with AlphaFold. Nature 596, 583–589. https://doi.org/10.1038/s41586-
021-03819-2.
Kato-Schwartz, C.G., de Sá-Nakanishi, A.B., Guidi, A.C., Gonçalves, G.A., Bueno, F.G.,
Zani, B.P.M., Mello, J.C.P., Bueno, P.S.A., Seixas, F.A.V., Bracht, A., Peralta, R.M.,
2020. Carbohydrate digestive enzymes are inhibited by Poincianella pluviosa stem
bark extract: relevance on type 2 diabetes treatment. Clin. Phytosci. 6 (31) https://
doi.org/10.1186/s40816-020-00177-w.
Keiser, M., Roth, B., Armbruster, B., Ernsberger, P., Irwin, J.J., Shoichet, B.K., 2007.
Relating protein pharmacology by ligand chemistry. Nat. Biotechnol. 25, 197–206.
https://doi.org/10.1038/nbt1284.
Kelly, R., Miller, S.M., Lai, M.H., Kirsch, D.R., 1990. Cloning and characterization of the
2,3-oxidosqualene cyclase-coding gene of Candida albicans. Gene 87 (2), 177–183.
https://doi.org/10.1016/0378-1119(90)90299-7.
Lima, I.L., Longui, E.L., Andrade, I.M., Garcia, J.N., Zanatto, A.C.S., Morais, E.,
Florsheim, S.M.B., 2010. Efeito da procedência em algumas propriedades da madeira
de Gallesia integrifolia (Spreng.). Harms. Rev. Inst. Flor. 22 (1), 61–69.
Lima, T.L.C., Souza, L.B.F.C., Tavares-Pessoa, L.C.S., Santos-Silva, A.M.d., Cavalcante, R.
S., Araújo-Júnior, R.F.d., Cornélio, A.M., Fernandes-Pedrosa, M.F., Chaves, G.M.,
Silva-Júnior, A.A.d., 2020. Phytol-loaded solid lipid nanoparticles as a novel
anticandidal nanobiotechnological approach. Pharmaceutics 12 (871). https://doi.
org/10.3390/pharmaceutics12090871.
Lorenzi, H., 2002. Árvores brasileiras: Manual de identificação e cultivo de plantas
arbóreas nativas do Brasil, fourth ed. Instituto Plantarum de Estudos da Flora Ltda,
São Paulo.
Mackerell Jr., A.D., Feig, M., Brooks III, C.L., 2004. Extending the treatment of backbone
energetics in protein force fields: limitations of gas-phase quantum mechanics in
reproducing protein conformational distributions in molecular dynamics
simulations. J. Comput. Chem. 25, 1400–1415. https://doi.org/10.1002/jcc.20065.
Makanjuola, O., Bongomin, F., Fayemiwo, S.A., 2018. An update on the roles of non-
albicans Candida species in vulvovaginitis. J Fungi (Basel). 4 (4), 121. https://doi.
org/10.3390/jof4040121.
Matheson, A., Mazza, D., 2017. Recurrent vulvovaginal candidiasis: a review of guideline
recommendations. Aust. N. Z. J. Obstet. Gynaecol. 57 (2), 139–145. https://doi.org/
10.1111/ajo.12592.
Muñoz, V., Sauvain, M., Bourdy, G., Arrázola, S., Callapa, J., Ruiz, G., Choque, J.,
Deharo, E., 2000. A search for natural bioactive compounds in Bolivia through a
multidisciplinary approach. Part III. Evaluation of the antimalarial activity of plants
used by Alteños Indians. J. Ethnopharmacol. 71 (1–2), 123–131. https://doi.org/
10.1016/s0378-8741(99)00191-9.
Nwonu, C., Ilesanmi, O., Agbedahunsi, J., Nwonu, P., 2019. Natural products as veritable
source of novel drugs and medicines: a review. Int. J. Herb. Med. 7 (1), 50–54.
Phillips, J.C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C.,
Skeel, R.D., Kalé, L., Schulten, K., 2005. Scalable molecular dynamics with NAMD.
J. Comput. Chem. 26, 1781–1802. https://doi.org/10.1002/jcc.20289.
Polke, M., Hube, B., Jacobsen, I.D., 2015. Candida survival strategies. Adv. Appl.
Microbiol. 91, 139–235. https://doi.org/10.1016/bs.aambs.2014.12.002.
Powell, A.M., Nyirjesy, P., 2014. Recurrent vulvovaginitis. Best Pract. Res. Clin. Obstet.
Gynaecol. 28 (7), 967–976. https://doi.org/10.1016/j.bpobgyn.2014.07.006.
Journal of Ethnopharmacology 295 (2022) 115403
Raimundo, K.F., de Campos Bortolucci, W., Silva, E.S., Pereira, A.F.B., Sakai, O.A.,
Piau, R., Goncalves, J.E., Linde, G.A., Gazim, Z.C., 2017. Chemical composition of
garlic wood (Gallesia integrifolia) (Phytolaccaceae) volatile compounds and their
activity on cattle tick. Aust. J. Crop. Sci. 11 (8), 1058–1067. https://doi.org/
10.3316/informit.170372647220443.
Raimundo, K.F., Bortolucci, W.C., Glamočlija, J., Soković, M., Gonçalves, J.E., Linde, G.
A., Colauto, N.B., Gazim, Z.C., 2018. Antifungal activity of Gallesia integrifolia fruit
essential oil. Braz. J. Microbiol. 49 (Suppl. 1), 229–235. https://doi.org/10.1016/j.
bjm.2018.03.006. Suppl 1.
Raimundo, K.F., Bortolucci, W.C., Rahal, I.L., Oliveira, H.L.M., Piau Júnior, R., Campo, C.
F.A.A., Gonçalves, J.E., Linde, G.A., Colauto, N.B., Gazim, Z.C., 2021. Insecticidal
activity of Gallesia integrifolia (Phytolaccaceae) essential oil. Bol. Latinoam. Caribe
Plant. Med. Aromat. 20 (1), 38–50. https://doi.org/10.37360/blacpma.21.20.1.3.
Roessner, C.A., Min, C., Hardin, S.H., Harris-Haller, L.W., McCollum, J.C., Scott, A.I.,
1993. Sequence of the Candida albicans erg7 gene. Gene 127 (1), 149–150. https://
doi.org/10.1016/0378-1119(93)90631-C.
Romo, J.A., Kumamoto, C.A., 2020. On commensalism of Candida. J. Fungi (Basel,
Switzerland) 6 (1), 16. https://doi.org/10.3390/jof6010016.
Salazar, S.B., Simões, R.S., Pedro, N.A., Pinheiro, M.J., Carvalho, M.F.N.N., Mira, N.P.,
2020. An Overview on conventional and non-conventional therapeutic approaches
for the treatment of candidiasis and underlying resistance mechanisms in clinical
strains. J Fungi (Basel). 6 (1), 23. https://doi.org/10.3390/jof6010023.
Schuster, H.J., Jonghe, B.A., Limpens, J., Budding, A.E., Painter, R.C., 2020.
Asymptomatic vaginal Candida colonization and adverse pregnancy outcomes
including preterm birth: a systematic review and meta-analysis. Am. J. Obstetr.
Gynecol. MFM 2 (3), 100163. https://doi.org/10.1016/j.ajogmf.2020.100163.
Silva, J.P., Nascimento, S.C., Okabe, D.H., Pinto, A.C., Oliveira, F.R., Paixão, T.P.,
Siqueira, M.L.S., Baetas, A.C., Andrade, M.A., 2018. Antimicrobial and anticancer
potential of Petiveria alliacea L. (Herb to “tame the master”): a review. Pharmacogn
12, 85–93. https://doi.org/10.4103/phrev.phrev_50_17.
Silva, L.N., de Mello, T.P., de Souza Ramos, L., Branquinha, M.H., Dos Santos, A.L.S.,
2019. New and promising chemotherapeutics for emerging infections involving
drug-resistant non-albicans Candida species. Curr. Top. Med. Chem. 19 (28),
2527–2553. https://doi.org/10.2174/1568026619666191025152412.
Silva Júnior, A.J., de Campos-Buzzi, F., Romanos, M.T., Wagner, T.M., Guimarães, A.F.,
Filho, V.C., Batista, R., 2013. Chemical composition and antinociceptive, anti-
inflammatory and antiviral activities of Gallesia gorazema (Phytolaccaceae), a
potential candidate for novel anti-herpetic phytomedicines. J. Ethnopharmacol. 150
(2), 595–600. https://doi.org/10.1016/j.jep.2013.09.005.
Soares, F.M., Pereira, R.M., 2018. Abordagem atual da candidíase vulvovaginal no
período gravídico. Revista Bahiana de Saúde Pública. 42 (1), 199–207. https://doi.
org/10.22278/2318-2660.2018.v42.n1.a2600.
Spada, G., Uesugi, G., Silva, R.B., Silva, M.R., 2019. Qualidade de mudas de Pau-d’alho
sob diferentes doses e frequências de aplicação de nutrientes. Colloquium Agrariae
15 (2), 121–132. https://doi.org/10.5747/ca.2019.v15.n2.a291.
Stierand, K., Rarey, M., 2010. Drawing the PDB: protein− Ligand complexes in two
dimensions. ACS Med. Chem. Lett. 1 (9), 540–545. https://doi.org/10.1021/
ml100164p.
Thoma, R., Schulz-Gasch, T., D’Arcy, B., et al., 2004. Insight into steroid scaffold
formation from the structure of human oxidosqualene cyclase. Nature 432, 118–122.
https://doi.org/10.1038/nature02993.
Tsega, A., Mekonnen, F., 2019. Prevalence, risk factors and antifungal susceptibility
pattern of Candida species among pregnant women at Debre Markos Referral
Hospital, Northwest Ethiopia. BMC Pregnancy Childbirth 19 (1), 527. https://doi.
org/10.1186/s12884-019-2494-1.
Van Riel, S.J.J.M., Lardenoije, C.M.J.G., Oudhuis, G.J., Cremers, & N.A.J., 2021.
Treating (recurrent) vulvovaginal candidiasis with medical-grade honey-concepts
and practical considerations. J Fungi (Basel). 7 (8), 664. https://doi.org/10.3390/
jof7080664.
Willems, H.M.E., Ahmed, S.S., Liu, J., Xu, Z., Peters, B.M., 2020. Vulvovaginal
candidiasis: a current understanding and burning questions. J Fungi (Basel). 6 (1),
27. https://doi.org/10.3390/jof6010027.
Yadav, K., Prakash, S., 2016. Prevalence of vulvovaginal candidiasis in pregnancy. Glob.
J. Med. Med. Sci. 4 (1), 108–116.
Zeng, X., Zhang, Y., Zhang, T., Xue, Y., Xu, H., An, R., 2018. Risk factors of vulvovaginal
candidiasis among women of reproductive age in Xi’an: a cross-sectional study.
BioMed Res. Int., 9703754 https://doi.org/10.1155/2018/9703754, 2018.
Zoete, V., Cuendet, M.A., Grosdidier, A., Michielin, O., 2011. SwissParam: a fast force
field generation tool for small organic molecules. J. Comput. Chem. 32, 2359–2368.
https://doi.org/10.1002/jcc.21816.
Zonios, D.I., Bennett, J.E., 2008. Update on azole antifungals. Semin. Respir. Crit. Care
Med. 29 (2), 198–210. https://doi.org/10.1055/s-2008-1063858.