Journal of Ethnopharmacology 301 (2023) 115856

Contents lists available at ScienceDirect

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

Antifungal Annona muricata L. (soursop) extract targets the cell envelope of

multi-drug resistant Candida albicans
Lara M. Campos a, Ari S.O. Lemos a, Irley O.M. Diniz a, Lucas A. Carvalho a, Thiago P. Silva b,
Paula R.B. Dib c, Eugênio D. Hottz c, Luciana M. Chedier d, Rossana C.N. Melo b, Rodrigo
L. Fabri a, *
a
Bioactive Natural Products Laboratory, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
b
Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
c
Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
d
Plant Chemistry Laboratory, Department of Botany, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Juiz de Fora, MG, Brazil

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

Keywords: Etnopharmacological relevance: Annona muricata L. (soursop) is traditionally used in the treatment of inflam­
Antifungal activity matory diseases, cancer, and infections caused by fungi. The therapeutic activity explored by its medicinal use is
Annona muricata generally associated with its phytoconstituents, such as acetogenins and alkaloids. However, its potential anti­
Candida albicans
fungal bioactivity as well as its mechanism of action remains to be established.
Chemical diversity
Fungal cells
Aim of the study: To evaluate the antifungal activity of the ethanolic extract of A. muricata leaves against
Cell viability multidrug-resistant Candida albicans (ATCC® 10231).
Material and methods: Phytoconstituents were detected by UFLC-QTOF-MS. The minimum inhibitory concen­
tration was determined, followed by the determination of the minimum fungicidal concentration. For planktonic
cells, the growth curve and cell density were evaluated. Studies to understand the mechanism of action on the
cell envelope involved crystal violet permeability, membrane extravasation, sorbitol protection, exogenous
ergosterol binding assay, metabolic activity, and cell viability. Furthermore, mitochondrial membrane potential
was assessed.
Results: Our analyses demonstrated a significant inhibitory effect of A. muricata, with the ability to reduce fungal
growth by 58% and cell density by 65%. The extract affected both the fungal plasma membrane and cell wall
integrity, with significant reduction of the cell viability. Depolarization of the fungal mitochondrial membrane
was observed after treatment with A. muricata. Rutin, xi-anomuricine, kaempferol-3O-rutinoside, nornuciferine,
xylopine, atherosperminine, caffeic acid, asimilobine, s-norcorydine, loliolide, annohexocin, annomuricin,
annopentocin, and sucrose were identified as extract bioactive components.
Conclusions: Our findings show that the A. muricata extract is a source of chemical diversity, which acts as a
potential antifungal agent with promising application to the therapy of infections caused by C. albicans.

1. Introduction The restricted number of antifungal drugs available challenges the

treatment of invasive pathologies. Currently, only four different classes
Candida albicans is a microorganism of clinical importance in inva­ of these drugs are used to treat numerous fungus-associated diseases:
sive human diseases and generates high healthcare costs worldwide. polyenes, azoles, nucleoside analogues, and echinocandins (Gintjee
These infections are considered emerging, especially in the immuno­ et al., 2020). Fungal pathogens are increasing their multidrug resistance
compromised population (Chen et al., 2020). However, current thera­ properties and evolving virulence factors, which is particularly prob­
peutic choices for the treatment of invasive fungal infections are limited lematic in clinical settings (Jamiu et al., 2021). Alternative therapies
and are associated with high toxicity (Van Daele et al., 2019). such as natural products have been studied and directed towards

* Corresponding author. Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz
de Fora, Minas Gerais CEP 36036-900, Brazil.
E-mail address: rodrigo.fabri@ufjf.br (R.L. Fabri).

https://doi.org/10.1016/j.jep.2022.115856
Received 23 August 2022; Received in revised form 3 October 2022; Accepted 18 October 2022
Available online 22 October 2022
0378-8741/© 2022 Elsevier B.V. All rights reserved.
L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

understanding mechanisms of action, epidemiology, antifungal resis­ voltage of the ion source 40 V, a capillary voltage 4500 V, and a capillary
tance, and pathogenesis. Such models have become relevant due to the temperature of 220 ◦ C. The full scan mass acquisition was performed by
high incidence of fungal infections caused by Candida species and their scanning from 100 up to 1000 m/z range.
increasing resistance to conventional treatments (Newman, 2017).
Annona muricata L. is a tropical plant fruit species that belongs to the 2.4. Fungal strain
Annonaceae family and is commonly known as soursop. In India and
tropical America and Africa, A. muricata is extensively used as an eth­ The studies were performed using the C. albicans species (ATCC
nomedicine against a variety of human ailments and diseases, especially 10231), characterized for having resistance to itraconazole, vor­
cancer, diabetes, rheumatism and parasitic infections (Moghadamtousi iconazole, anidulafungin and fluconazole (ATCC, 2017). Professor
et al., 2015), in addition to other uses (Moghadamtousi et al., 2015; Marcelo Gonzaga de Freitas Araújo from the Universidade Federal de
Quílez et al., 2018; Coria-Téllez et al., 2018; Gavamukulya et al., 2017). São João del Rey, campus Centro Oeste Dona Lindu, kindly provided this
Extracts and phytoconstituents of A. muricata are considered to have microorganism for the study. The strain was cultured overnight at 35 ◦ C
multiple beneficial effects with potential antimicrobial, in RPMI-1640 culture media before each experiment. All experiments
anti-inflammatory, anti-protozoal, antioxidant, insecticidal, and larvi­ and following analyzes were done in triplicates.
cidal activities (Coria-Téllez et al., 2018; Gavamukulya et al., 2017; Kim
et al., 2020; Quílez et al., 2018). These activities are associated with 2.5. Minimum inhibitory concentration (MIC)
compounds found in fruits and leaves, mainly acetogenins, phenolic
compounds, alkaloids, and terpenes (Leite et al., 2020). Cytotoxic effects MIC was determinated by assay using the protocol described by CLSI
for tumor cells, hepatoprotective and hypoglycemic activities also are (CLSI modified, 2017). The tests were performed using RPMI 1640
reported for A. muricata natural compounds (Mishra et al., 2013; Ade­ media. Yeasts were grown overnight at 35 ◦ C on Sabouraud Dextrose
wole and Ojewole, 2009; Kim et al., 2020). Agar (ASD). In 96-well microplates, successive microdilutions of 1000 to
Few previous studies have shown the fungicidal property activity of 7.8 μg/mL were performed. For this preparation, stock solutions of 2.5
extracts and fractions of A. muricata against Candida species, including mg/mL in Dimethyl sulfoxide (DMSO) 1% were used. In microplates that
C. albicans strains, in vitro (Pai et al., 2016; Cesar et al., 2021). However, already contained 100 μL of RPMI 1640, 80 μL of this stock solution was
the mechanism of action of A. muricata extracts against C. albicans, added. The final volume of 200 μL was completed by adding 20 μL of
especially multidrug-resistant strains, remains to be established. Here, inoculum (3 × 105 CFU/mL), based on the standard 1.0 McFarland scale.
we investigate the antifungal activity and mechanisms of action of The plates were incubated at 35 ◦ C for 24 h. A growth control (RPMI
ethanolic extract of A. muricata (AME) against a multidrug-resistant 1640 + extract) were simultaneously performed. Nystatin (10–0.16
Candida albicans strain. μg/mL), fluconazole (10,000–39 μg/ml), and itraconazole (400–3.2
μg/mL) were used as positive controls. DMSO solvent control was also
2. Material and methods performed. The MIC was calculated by the lowest dilution that shows a
complete inhibition of the strain on which the tests were performed. All
2.1. Plant material tests were performed in duplicate.

The leaves of A. muricata L. were collected in March 2018, in the city 2.6. Minimum fungicidal concentration (MFC)
of Recreio, Minas Gerais, Brazil, at the following coordinates 19◦52′
30,8′′ S; 43◦58′ 31,7′′ with license number A032F41-23 SISGEN/ Aliquots of 10 μL were taken from the plate samples in the wells
BRAZIL. Dr. Vinícius Antônio de Oliveira Dittrich, from the Botany without visible growth in the MIC assay. The removed aliquots were
Department of the Federal University of Juiz de Fora (UFJF), made the streaked onto ASD plates, which were then incubated at 35 ◦ C for a
identification of the plant. Voucher specimens (CESJ 46006) of the plant period of 24 h. All tests were done in triplicate. The MFC is reported as
parts were deposited in the Leopoldo Krieger Herbarium at UFJF. the lowest concentration of the extract capable of causing yeast death
(Lemos et al., 2020).
2.2. Preparation of the extract
2.7. Fungal killing assay
The leaves from A. muricata L. were dried in an oven with forced air
circulation at 40 ◦ C and pulverized to obtein a dry plant material (6.5g). AME was tested to determine the growth curve for C. albicans.
A static maceration was performed with ethanol as a liquid phase, at Freshly grown fungal strains were inoculated into tubes containing
room temperature to extract the material. The extract was concentrated RPMI 1640 media supplemented with AME at 35 ◦ C. Absorbance was
by reduced pressure evaporation, obtaining the ethanolic extract of measured at 560 nm at 0, 4, 8, 12, 24, 30, 36, 48, 60 and 72 h. Graphs of
A. muricata (AME) 6.6% yield. turbidity versus incubation time were plotted. Growth rate curves were
studied to evaluate signs of fungicidal effect of AME. Nystatin was used
2.3. Ultra-fast liquid chromatography-Quadrupole time of flight mass as positive control. Fungal strains inoculated in RPMI 1640 served as the
spectrometry (UFLC-QTOF-MS) analysis growth control. The procedure was performed in triplicate (Lemos et al.,
2020).
AME (5 mg) was analyzed by ultra-fast liquid chromatography
coupled with mass spectrometry in the positive ion mode using a Shi­ 2.8. Fungal cell density
madzu UFLC (Nexera model) and a Bruker mass spectrometer (QTOF
Compact model) with an electrospray ionization source. The mobile Fungal counts were performed in cytocentrifugation separation
phase employed was water acidified with formic acid, pH = 3 (phase A), based on Silva et al. (2014), using a marker that binds to DNA double
and methanol (phase B), the injection flow rate was 0.4 mL/min with a strand and emits a blue fluorescence, called 4’,6-dia­
running time of 12 min. The column used was a Kinetex 2.6 μm, C18- midino-2-phenylindole (DAPI; Joux and Lebaron, 2000). Strains of
100A, 100 mm × 3.0 mm. The chromatographic run began with 40% C. albicans were inoculated in RPMI 1640 media tubes containing AME
of phase B, increasing to 70% of B at 8.20 min and 95% of B at 9.70 min. (MIC value), then incubated at 35 ◦ C for 24 h. The microorganisms that
Subsequently, the mobile phase was returned to 40% B at 10.20 min, to were inoculated in RPMI 1640 were used as growth control and the
re-equilibrate the column, following up to 11.50 min, the running ended positive controls were incubated with nystatin (MIC value). Samples
in 12 min. The ionization conditions were set as follows: electrospray were stained with DAPI at a final concentration of 5 μg/mL, fixed with

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L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

4% formaldehyde and prepared in a cytocentrifuge (Shandon cytospin 4, triplicate. Cells were washed with Hank’s Buffered Salf Solution HBSS
Thermo Electron) at 452g for 10 min at high acceleration. A fluores­ (1500 rpm for 5 min) and incubated with 1.25 μg/mL of propidium
cence microscope (Olympus BX-60) was used to count the yeast cells on iodide (PI). The incubation time was 30 min. The effect of treatments on
the slides in 10 random fields at 1000× magnification using immersion the cell envelope was evaluated using the FACSCanto II flow cytometer
oil. For fungal quantification the following formula was used: with the acquisition of 10,000 events. In the analysis of the data through
(nxa)/(Vxa), in which n = number of cells counted, a = area of the grid the software FlowJo®, the percentage of positive cells for the IP was
counted, and V = sample volume x volume counted/total volume. An­ determined.
alyzes were performed in triplicates.
2.9.6. Cellular metabolic activity
2.9. Cell envelope studies Yeast viability was assessed by intracellular metabolic activity by the
LIVE/DEAD Yeast Viability kit (Molecular Probes), which consists of two
2.9.1. Measurement of permeability with crystal violet fluorescent markers, FUN-1 and Calcofluor White (CW). C. albicans
Crystal violet (CV) assay was performed as described by Campos strain was inoculated into RPMI 1640 media tubes containing AME (MIC
et al. (2018) for the evaluation of membrane permeability changes. value) and incubated for 24 h at 35 ◦ C. For growth and positive controls,
Thereafter, freshly grown fungal lines in RPMI 1640 media were inoc­ fungi were inoculated in RPMI 1640 or nystatin (MIC value), respec­
ulated into tubes supplemented with AME and nystatin (MIC value) at tively. All the treatments were performed in triplicate. Yeast suspensions
35 ◦ C temperature, then incubated for 4 h. The fungal cell suspensions (1 mL of each sample, n = 3) were stained with 1 μL of FUN 1 and 5 μL of
were centrifuged at 1000g/10 min and the pellet was resuspended in 10 CW. After incubation at 35 ◦ C for 30 min in the dark, slide samples were
μg/mL crystal violet solution and subsequently incubated for 10 min at prepared by cytocentrifugation (Shandoncytospin 4, ThermoElectron) at
35 ◦ C. A spectrophotometer (Multiskan Go, Thermo Scientific, Waltham, 452g for 10 min (SILVA et al., 2014). The resulting slides were analyzed
MA, USA) with a wavelength of 570 nm was used to measure the optical by fluorescence microscopy (BX-60, Olympus) and.yeast cell counts
density (OD) of the supernatant obtained from the centrifugation of the were done by using both the U-MWU2 fluorescent filter (CW) and the
samples (1000 g/15 min). For the growth control, the OD of the su­ U-MWB filter (FUN-1). The proportion of viable active/non-viable
pernatant of the normal group of untreated cells was used. The OD value inactive yeasts were determined by differential cell counting in 10
of the crystal violet solution was taken as 100%. Analyzes were per­ random fields.
formed in triplicates. The following formula was used to express the
percentage of crystal violet uptake: (OD value of the sample/OD value of 2.9.7. Mitochondrial membrane potential (MMP)
the CV solution) × 100 = percentage violet crystal uptake. MMP was determined using tetramethylrhodamine (TMRE), a probe
used to label active mitochondria (Tian et al., 2017). The fungal cell
2.9.2. Nucleotide leakage suspension (3 × 106 CFU/mL) was treated with AME or nystatin (both
The methodology was performed according to Campos et al. (2018). with concentrations relative to the MIC value) for 4 h. To control the
C. albicans was incubated in RPMI 1640 media at 35 ◦ C for 24 h. Sub­ experiment, the standards carbonyl cyanide 4-(trifluoromethoxy)phe­
sequently, the culture was centrifuged at 1000 rpm for 5 min, washed nylhydrazone - FCCP (10 μM) and oligomycin A (15 μg/mL) were used in
and resuspended in 10 mM Phosphate-buffered saline (PBS) solution cultures for 10 min before incubation with TMRE. Cultures incubated
(pH 7.4), reaching a final density of 3 × 106 CFU/mL. The microor­ with buffer alone were used as treated and untreated growth controls.
ganisms were incubated with AME and nystatin (MIC value) at different After exposure, cells were centrifuged at 1500 rpm for 5 min, washed
times (0, 1, 2, 3, and 4 h). As a control, we used cells incubated with 10 with HBSS buffer and incubated with 20 μM TMRE at 35 ◦ C for 30 min.
mM PBS (pH 7.4). Supernatants obtained from the suspensions were After labeling, the material was again centrifuged and resuspended in
centrifuged at 3000 rpm for 15 min and analyzed at 260 nm. Only at 4 h, HBSS buffer. The average fluorescence intensity (MFI) of the suspen­
total proteins were quantified by the Lowry method (1951). The pro­ sions were quantitatively analyzed in a FACSCanto II flow cytometer
cedures were performed in triplicate. (Becton & Dickinson) with 10,000 acquired events. The assays were
carried out in triplicate and the FlowJo® program was used for the
2.9.3. Ergosterol effect assay analyses.
The MIC of AME was determined according to the methodology of
Khan et al. (2013) and Leite et al. (2020). In a 96-well microplate, 2.10. Statistical analysis
containing ergosterol-enriched RPMI 1640 media (400 μg/mL), serial
microdilution was performed. A dilution of the AME stock solution was The statistical analysis used to compare the positive control and
performed at concentrations ranging from 16.000 to 25 μg/mL. For the treated groups (AME and nystatin) was performed by ANOVA followed
positive control, nystatin (100–0.78 μg/mL) was used. After 24 h of by the Bonferroni test (P < 0.05), using Prism 6.0.1 software (GraphPad
incubation at 35 ◦ C, the MIC values were determined. All tests were software, San Diego, CA, United States).
performed in duplicate.
3. Results
2.9.4. Sorbitol protection assay
To evaluate the osmoprotection of sorbitol, a model described by 3.1. Chemical analysis of AME by UFLC-QTOF-MS
Frost et al. (1995) and Khan et al. (2013). In a sterile 96-well microplate
containing RPMI 1640 media enriched with 0.8 M sorbitol, serial The chromatographic profile obtained by UFLC-QTOF-MS was ac­
microdilution was performed. The stock solution of AME was diluted at quired and 14 compounds were identified as described in Table 1.
concentrations ranging from 16000 to 125 μg/mL. The determination of
the MIC values was done after incubation for 24 h at 35 ◦ C. All tests were 3.2. Minimal inhibitory concentration (MIC) and minimum fungicidal
performed in duplicate. concentration (MFC)

2.9.5. Cell viability The minimum inhibitory concentration (MIC) value for AME was
Plasma membrane integrity was assessed by the method described by 1000 μg/mL. In this concentration, AME showed fungistatic effect
Lee and Kim (2016). The fungal cell suspension (3 × 106 CFU/mL) was against C. albicans. The MIC values obtained for fluconazole, itracona­
treated with AME or nystatin, with a final concentration equivalent to zole, and nystatin were 10000, 200, and 2.5 μg/mL, respectively. These
MIC, for 4 h and incubated at 35 ◦ C. The treatments were performed in results evidenced a resistance by C. albicans for fluconazole and

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L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

Table 1
Compounds identified in the ethanolic extract from A. muricata leaves (AME) by UFLC-QTOF-MS in ESI (+).
Compound Compound Names Chemical Calculated Mass Error Rt Main fragments (m/z) Reference
Classes formulae mass found (ppm) (Min)

Saccharide Sucrose C12H22O11 342.1162 342.1068 − 27.68 1.1 381.0700 (M + K+); Cardozo et al., 2012
203.0461 (M-Glic + Na+)
Flavonoid Rutin C27H30O16 610.15346 610.1409 − 20.58 2.1 611.1482 (M + H); Matsushige et al., 2012a
303.0424 (M-Rha-Glic)
Alkaloid xi-Anomuricine C19H23NO4 329.1628 329.1539 − 27.03 2.4 330.1612 (M + H) Leboeuf et al., (1981)
Flavonoid Kaempferol-3O- C27H30O15 594.1585 594.1461 − 20.95 2.7 595.1534 (M + H); Nawwar et al., 2012
rutinoside 449.0961 (M-Rha)
Alkaloid Nornuciferine C18H19NO2 281.14166 281.1338 − 27.95 3.0 282.1411 (M + H) Hasrat et al., 1997a; Hasrat
et al., 1997b;
Alkaloid Xylopine C18H17NO3 295.1209 295.1129 − 27.14 3.4 296.1202 (M + H) Fofana et al., 2011
Alkaloid Atherosperminine C20H23NO2 309.1729 309.1648 − 26.45 4.2 310.1721 (M + H); Leboeuf et al., (1981)
223.1723 (M–HCN–4CH3)
Phenolic acid Caffeic acid C9H8O4 180.0422 180.0518 52.87 4.4 203.0410 (M + Na); Nam et al., 2017
181.0591 (M + H)
Alkaloid Asimilobine C17H17NO2 267.1260 267.1046 − 80.11 4.8 306.0678 (M + K+) Hasrat et al., 1997a; Hasrat
et al., 1997b;
Alkaloid S- Norcorydine C19H21NO4 327.1471 327.1388 − 25.49 5.7 328.1461 (M + H) Matsushige et al., 2012b
Benzofuran Loliolide C11H16O3 196.1100 196.1103 +1,47 8.2 197.11 76 (M + H) Cárdenas et al., 2021
Acetogenin Annohexocin C35H64O9 628.4553 628.4555 +0.30 18.1 629.4628 (M + H) Zeng et al., 1995
Acetogenin Annomuricin A,C, or C35H64O8 612.4604 612.4603 − 0,16 18.6 613.4676 (M + H) Jaramillo et al., 2000; Kim
E et al. (1998); Wu et al., 1995.
Acetogenin Annopentocin A, B, C35H64O8 612.4604 612.4604 0.00 18.8 613.4677 (M + H) Zeng et al., 1996
or C

Rt - Retention time.

itraconazole, and sensitivity for nystatin. nystatin treatments in comparison with growth control along 72 h (P
˂0.05; Fig. 1B). Fungi treated with AME reduced their growth around
3.3. Fungal killing assay 58% when compared to the growth control (Fig. 1B). Indeed, both
treatments, AME and nystatin, extended the lag phase (between 4 and
The fungal growth kinetics of C. albicans was acquired through op­ 12h) and induced a decrease in the growth cycle curve (in log phase)
tical density (600 nm) for 72 h (Fig. 1A). Analyses of area under the when compared to the growth control (Fig. 1A).
curve showed a significant reduction of fungal growth in AME and

Fig. 1. Fungal growth of C. albicans cultures

treated withethanolic extract from
A. muricata leaves (AME) and nystatin
(NYS). . (A) Growth curves of treated and
non-treated (growth control, CG) groups
acquired by optical density. (B) Area under
the curve analyses of growth curves. (C) Cell
density of the growth control and treated
groups at 24 h. Fungal cells were stained
with DAPI and counted under fluorescence
microscopy to assess cell density. The letters
indicate a statistically significant difference
in relation to growth control (GC) (a) and
nystatin (NYS) (b) (ANOVA followed by
Bonferroni, P < 0.05). The experiments were
performed in triplicate and the data repre­
sent the mean ± SD.

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L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

3.4. Fungal cell density Table 2

Quantification of total proteins after membrane leakage in fungal
Using fluorescence microscopy, we investigated the effect of AME on cells treated with ethanolic extract from A. muricata leaves (AME)
cell density (Fig. 1C). Quantitative analyses showed that yeast cell and nystatin.
density was significantly reduced in 65% and 63% for AME and nystatin Samples Total proteins (mg/mL)
treatments, respectively (P ˂0.05). AME 1.39a,b ± 0.08
Nystatin 1.03a ± 0.1
Growth control 0.9 ± 0.05
3.5. Measurement of permeability with crystal violet
The letters indicate a statistically significant difference in relation
Our data indicate a growth in fungal cell permeability evidenced by to growth control (a) and nystatin (b) (ANOVA followed by Bon­
the increase in CV uptake (Fig. 2A). After treatment with AME the CV ferroni, P < 0.05). All experiments were performed in triplicate,
and the data represent the mean ± SD.
uptake increased in 3.6-fold when compared to the growth control (P
˂0.05).
Table 3
3.6. Nucleotide leakage In vitro antifungal activity of ethanolic extract from A. muricata leaves (AME)
against C. albicans in the presence and absence of sorbitol and exogen ergosterol
The effect on cell permeability can be evaluated by measuring the for the microtiter dilution broth assay.
release of an intracellular component (nucleotides) into the medium Samples MIC (μg/ MIC with sorbitol (μg/ MIC with ergosterol (μg/
from cells washed in buffer. The release of nucleotides from C. albicans mL) mL) mL)

was highest when these cells were treated with AME and nystatin AME 1000 8000 8000
already in the first hours of treatment (Fig. 2B). Upon treatment with Nystatin 2.5 – 20
AME, a 6-fold increase was obtained when compared to control (P
˂0.05). Analyses of area under the curve showed a significant an
3.8. Cell viability
increased extravasation of extracellular fungal components in AME and
nystatin treatments in comparison with growth control along 4 h (P
The ability to damage the cell envelope was assessed by PI after
˂0.05; Fig. 2C). At the end of the 4 h of treatment with AME and
treatment with AME. A change in cell profile was clearly observed when
nystatin, we can also observe a greater release of proteins, indicating
compared to control, when analyzing the parameters of size and gran­
extravasation to the extracellular medium (Table 2).
ularity (Fig. 3A and B). The fluorescence intensity was mainly increased
when C. albicans was treated with AME. Fig. 3C shows a change in
3.7. Ergosterol and sorbitol protection assays fluorescence intensity in the histograms after treatment of the fungal
cells with AME. 38% and 14.5% of the cells were damaged and inter­
These assays evaluated the ability of AME to disrupt the fungal cell nalized PI for AME and nystatin, respectively (Fig. 3D) (P ˂0.05). AME
wall and membrane, which form the cell envelope. An increase in the significantly increased the amount of unviable fungal cells when
MIC value was observed in both assays (Table 3), indicating that AME compared to control and nystatin treated group (Fig. 3E) (P ˂0.05).
has a possible activity on the structures that make up the cell envelope.

Fig. 2. Effect of ethanolic extract from

A. muricata leaves (AME) and nystatin (NYS)
on C. albicans crystal violet uptake (A) and
nucleotide leakage (B). In (A) is shown the
graph of the mean proportion of crystal vi­
olet uptake by the yeast cells, which deno­
tated cell membrane permeability. (B)
Shows the kinetics of nucleotide leakage
obtained by optical density along 4 h of
control growth (CG) cultures and nystatin
(NYS) and AME treated cultures. (C) Area
under the curve analyses of kinetics of
nucleotide leakage. The letters indicate a
statistically significant difference in relation
to growth control (GC) (a) and nystatin
(NYS) (b) (ANOVA followed by Bonferroni,
P < 0.05). All experiments were performed
in triplicate, and the data represent the
mean ± SD. (For interpretation of the ref­
erences to colour in this figure legend, the
reader is referred to the Web version of this
article.)

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L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

Fig. 3. Activity of ethanolic extract from A. muricata leaves (AME) and nystatin (NYS) on free yeast cells stained with propidium iodide (PI) and analyzed by flow
cytometry. (A and B) FSC x SSC dot plot graph; (C and D) histogram representing fluorescence intensity; (E) graph representing non-viable cells, where there was IP
internalization. The letters indicate a statistically significant difference in relation to growth control (GC) (a) and nystatin (NYS) (b) (ANOVA followed by Bonferroni,
P < 0.05). All experiments were performed in triplicate, and the data represent the mean ± SD.

3.9. Cellular metabolic activity control (23% and 45%, respectively) (Fig. 4B) (P ˂0.05). After Fun-1
staining yeast cells with a full metabolic activity were seen as bright
Metabolic activity analyses was performed using the LIVE/DEAD green cells with CIVS stained in red (Fig. 4C and 4Ci, arrowheads).
Yeast Viability kit®. By CW staining visualization by fluorescence mi­ Quantitative analyses under a fluorescence microscope showed that
croscopy (Fig. 4A) we noticed that AME and nystatin significantly treatment with nystatin and MFM led to a significant reduction of the
reduced the number of yeast cells with intact cell wall when compared to metabolic activity of yeast cells (Fig. 4D).

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L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

Fig. 4. Metabolic activity of C. albicans in plank­

tonic cultures. In (A) observe a representative
image of AME-treated yeast cells fluorescent
marked with calcofluor white (CW), which
stained intact cell walls. In (B) graph shows the
number of yeasts with intact cell wall. In (C)
observe FUN-1 -stained yeasts cells treated with
AME Note in detail (Ci) green active viable cells
exhibiting intravesicular structures stained in red
(arrowhead), while cells with low or no activity
show diffuse yellow to green staining. (D) Shows
the proportion of active cells. The letters indicate
a statistically significant difference in relation to
growth control (GC) (a) and nystatin (NYS) (b)
(ANOVA followed by Bonferroni, P < 0.05). All
experiments were performed in triplicate, and the
data represent the mean ± SD. (For interpretation
of the references to colour in this figure legend,
the reader is referred to the Web version of this
article.)

3.10. Mitochondrial membrane potential (MMP) concentration capable of preventing the growth of a microorganism in
subcultures free of antimicrobial substances, which predicts the death
TMRE allows the labeling of active mitochondria. AME treatment and fungicidal action of the sample (Belanger and Hancock, 2021). Our
alters the fungal cell dispersion profile when compared to control results showed that AME has promising antifungal activity with fungi­
(Fig. 5A and B) Cytometer analyses of the cells provide different fluo­ static effect at MIC value.
rescence intensity data, which treatment with the extract was able to We analyzed the behavior of C. albicans in the presence of AME and
decrease this intensity compared to the control (Fig. 5C). Both AME and nystatin on MIC values. Monitoring fungal growth and death on the
nystatin caused a depolarization in the mitochondrial membrane, action of compounds are often used to understand antimicrobial action
leading to inactivation of mitochondria, where AME and nystatin over time (Foerster et al., 2016). As nystatin, AME showed a potential to
significantly reduced by 79% and 81% the fluorescence intensity when delay the adaptation phase by interfering with the initiation of cell di­
compared to control (Fig. 5D) (P ˂0.05). vision, repairing some molecular damage, or changing the synthesis of
cellular components necessary for its normal growth (Freire et al., 2017;
4. Discussion Lemos et al., 2018; Toledo et al., 2020). In the log phase, characterized
by accentuated growth and high metabolic activity, a decrease in this
There are several mechanisms that lead C. albicans to "escape" the stage was detected as a response to the extract treatment, indicating an
action of conventional antifungal drugs, such as overexpression of efflux interference with cell integrity, unlike nystatin, which maintained
pumps and alteration of the cell membrane and biofilm formation, growth (Campos et al., 2018). A reduction in cell density was clearly
which are the most dominant resistance strategies (Hans et al., 2019). associated with the decrease in the growth curve of C. albicans after
Thus, the focus of this work was to study possible mechanisms of action treatment with AME (Fig. 1C).
of the ethanolic extract from A. muricata leaves (AME), targeting the AME was effective in injuring the fungal cell envelope (Fig. 2). The
treatment for infections caused by this species and the possibility of increase in membrane permeability was observed when there was a
reducing these virulence factors. greater uptake of crystal violet after incubation with AME, which was
Initially, the antifungal activity was evaluated by determining the superior to the treatment with nystatin. Hydrophobic crystal violet ex­
MIC, which corresponds to the lowest effective concentration of the hibits low penetration in normal cells, however, in cells with damage to
sample that inhibits visible fungal growth after an incubation period. the cell envelope there is accumulation. Therefore, cellular uptake of
The minimum fungicidal concentration corresponds to that crystal violet is easily increased when the envelope is defective, but is

7
L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

Fig. 5. Action of ethanolic extract from A. muricata leaves (AME) and nystatin (NYS) on the mitochondrial membrane potential of C. albicans. (A and B) FSC x SSC dot
plot graph; (C) histogram representing fluorescence intensity; (D) Graph depicting the fluorescence intensity of cells treated with NYS, AME, FCCP and oligomicin A
after TMRE labeling. The letters indicate a statistically significant difference in relation to growth control (a), NYS (b), AME (c), and FCCP (d) (ANOVA followed by
Bonferroni, P < 0.05). All experiments were performed in triplicate, and the data represent the mean ± SD.

not detected when intact (Baena-Santillán et al., 2021). The leakage of containing exogenous ergosterol, providing an impediment to binding to
cytoplasmic materials indicates damage to the fungal cell envelope (Lee membrane ergosterol (Pippi et al., 2019). An increase in the MIC value
and Kim, 2016; Zorić et al., 2017), being confirmed and superior when of AME was observed in the presence of exogenous ergosterol, thus
compared to nystatin after treatment with AME. In addition to the suggesting the ability of compounds present in AME to bind to fungal
nucleotide permeability, protein release into the extracellular environ­ ergosterol.
ment can also serve as a marker of membrane damage (Niu et al., 2020), Membrane integrity analysis was evaluated by flow cytometry using
as demonstrated in the AME-treated groups. propidium iodide (PI), a reagent that binds to double-stranded DNA,
Other results have also shown that AME has activity on the fungal which is not internalized in cells with intact plasma membranes.
cell envelope (cell wall and plasma membrane). The use of antifungal Therefore, fungal cells with damaged membranes are able to internalize
agents can cause damage to the fungal cell wall, but in the presence of PI (Bouarab et al., 2021). A significant increase in cell damage was
osmoprotectants, such as sorbitol, this damage is minimized (Essid et al., detected in cells treated with AME, in accord with other tests performed
2017). Substances with an antifungal effect act on essential components for the cell envelope, which indicated a more effective action of the
of the cell or fungal wall and cause cell lysis in the absence of osmo­ extract compared to nystatin. This effect is related to damage to the
protectant. However, if a stabilizer is present in the medium, the cells fungal cell wall, as well as the plasma membrane, which would explain
will continue to grow (Frost et al., 1995). When sorbitol is used, there is the greater internalization of PI.
an increase in the MIC value, revealing the action of AME in targeting In order to confirm the effect of AME on the cell envelope, the
this substance, which is relevant for cell wall synthesis. Some antifungals metabolic activity assay was performed using specific markers.. FUN-1
exhibit membrane-damaging effects by targeting ergosterol, which is a labels the cytoplasm of all yeasts in yellow-green. In cells with an
structural lipid essential for many aspects of cell physiology. Damage to intact plasma membrane and normal metabolic function, FUN-1 fluo­
this structure can form transmembrane pores that lead to a change in rescence is converted and marks intracellular vacuoles in orange-red.
membrane permeability and consequent disruption (Lone et al., 2020). Calcofluor White M2R detects the chitin of the cell wall with blue
Antifungals, as nystatin, whose action target is binding to membrane fluorescence, regardless of the metabolic state (Campos et al., 2021;
ergosterol suffer a shift in their activity when there is a culture medium Ingham et al., 2012).

8
L.M. Campos et al. Journal of Ethnopharmacology 301 (2023) 115856

Core mitochondrial functionality is the ability to pump protons diseases, elucidating the mechanism of action in the fight against
across the inner membrane to create a gradient with high potential Candida albicans and opening perspectives for the expansion of new
energy, the mitochondrial membrane potential (Tian et al., 2017). There therapies, such as the development of formulations applicable in anti­
are substances capable of altering mitochondrial polarization (Saka­ fungal treatment, including the fight against virulence factors, such as
muru et al., 2016). One of the mechanisms is the uncoupling of protons, biofilms.
which allows the flow of protons through the mitochondrial membrane
independent of adenosine triphosphate (ATP) synthesis, which was Author’s contributions
generated by the use of FCCP. Another mechanism is the blocking of
mitochondrial ATP formation by the action of oligomycin A. Its mech­ R.L.F., E.D.H., T.P.S., and R.C.N.M. conceived and designed the ex­
anism of action is related to the binding to the proton channel in the Fo periments; L.M.C., A.S.O.L., I.O.M.D., L.A.C., and P.R.D. performed the
portion of ATP synthase. As a result, oligomycin A blocks mitochondrial experiments; L.M.C., T.P.S., and R.L.F. analyzed the data; R.L.F., E.D.H.,
ATP synthesis, proton translocation and oxygen binding (Hearne et al., and R.C.N.M. contributed to the reagents/materials; R.L.F., L.M.C., and
2020; Lippe et al., 2019). TMRE is used to quantify changes in MMP in T.P.S. wrote the paper. All authors have read, edited, and agreed to the
living cells, by flow cytometry, by labeling active mitochondria. TMRE is published version of the manuscript.
a red/orange dye capable of permeating into cells. This substance is
positively charged, which causes it to readily accumulate in active Funding
mitochondria, due to its relative negative charge. Depolarized or inac­
tive mitochondria have reduced membrane potential and do not This work was supported by grants from the Conselho Nacional de
sequester the TMRE. Our results from fungal cells treated with extract Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and the
and nystatin indicated a reduced membrane potential by depolarized Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Grant
membranes relative to untreated cells. These findings indicate that there Number: APQ-01357-21 to Rodrigo L. Fabri). Scholarships were pro­
was no binding of TMRE at the mitochondrial membrane, and conse­ vided by the Federal University of Juiz de Fora (UFJF/Brazil and the
quently, a reduction in the mean fluorescence intensity was observed, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES,
demonstrating the mitochondrial depolarization induced by the treat­ Brazil).
ments. Therefore, AME has shown a high modulating effect on anti­
fungal activity, highlighting changes in both virulence and the cell
CRediT authorship contribution statement
envelope of C. albicans.
For a better understanding of plant drugs, it is important to perform
Lara M. Campos: Methodology, Formal analysis, Methodology,
phytochemical profiling with a maximum number of markers available
Formal analysis, Conceptualization, Writing - original draft. Ari. S.O.
for an extract. Species of the genus Annona, are known for the presence
Lemos: Methodolgy, Formal analysis. Irley O.M. Diniz: Methodology.
of acetogenins and alkaloids, which have antifungal, antibacterial and
Thiago P. Silva: Formal analysis, Supervision, Writing – review &
cytotoxicity activities previously reported in the literature (Coria-Téllez
editing. Paula R.B. Dib: Methodology. Eugênio D. Hottz: Methodol­
et al., 2018; Quílez et al., 2018; Nugraha et al., 2019; Pinto et al., 2017).
ogy, Resources. Luciana M. Chedier: Formal analysis, Writiong - review
To identify these and other chemical constituents, which could be
& editing. Rossana C.N. Melo: Resources, Supervision, Writing – re­
related to antifungal activity, we investigated the chemical profile of
view & editing. Rodrigo L. Fabri: Resources, Formal analysis, Funding
AME by UFLC-QTOF-MS analysis. Fourteen compounds were identified
acquisition, Writing - original draft, Writing – review & editing.
in AME: rutin, xi-anomuricine, kaempferol-3O-rutinoside, nornucifer­
ine, xylopine, atherosperminine, caffeic acid, asimilobine, s-norcor­
ydine, loliolide, annohexocin, annomuricin, annopentocin and sucrose, Declaration of competing interest
and some of them have well-known antifungal activity (Quílez et al.,
2018). The flavonoids identified have recognized antifungal activity The authors declare that they have no known competing financial
(Han, 2009; Rocha et al., 2019; Tatsimo et al., 2012), such as does interests or personal relationships that could have appeared to influence
xylopine, a benzylisoquinoline alkaloid (Villar et al., 1986) and caffeic the work reported in this paper.
acid (Rossatto et al., 2021; Sardi et al., 2016).
This is the first record of activity against Candida albicans for the Data availability
chemical composition of the extract under study. Thus, based on these
literature data, the chromatographic analysis of AME suggests that the The data that has been used is confidential.
afore mentioned compounds may be responsible for the antifungal ac­
tivity against multi-drug resistant C. albicans found for A. muricata Acknowledgments
extract, and, could be acting in synergism with one another present in
the extract. The authors are grateful to Dr. Vinícius Antônio de Oliveira Dittrich
from the Department of Botany/Federal University of Juiz de Fora for
5. Conclusion the botanical identification of the species and to Delfino Antônio Cam­
pos from the Department of Biochemistry/Federal University of Juiz de
The chemical characterization of the ethanolic extract from Annona Fora for technical support.
muricata leaves (AME) revealed the presence of alkaloids, flavonoids,
acetogenins, phenolic acids, benzofurans, and disaccharides. AME
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