4.3 7.

Article

Antimicrobial, Antivirulence, and

Antiparasitic Potential of Capsicum
chinense Jacq. Extracts and Their
Isolated Compound Capsaicin

Ralciane de Paula Menezes , Meliza Arantes de Souza Bessa , Camila de Paula Siqueira,
Samuel Cota Teixeira, Eloisa Amália Vieira Ferro, Mário Machado Martins, Luis Carlos Scalon Cunha
and Carlos Henrique Gomes Martins

Special Issue
Candida spp. Biofilm: Oral and Systemic Implications
Edited by
Prof. Dr. Cláudia Helena Silva-Lovato, Dr. Adriana Barbosa Ribeiro and
Prof. Dr. Helena Freitas Oliveira Paranhos

https://doi.org/10.3390/antibiotics11091154
 antibiotics
Article
Antimicrobial, Antivirulence, and Antiparasitic Potential of
Capsicum chinense Jacq. Extracts and Their Isolated
Compound Capsaicin
Ralciane de Paula Menezes 1,2 , Meliza Arantes de Souza Bessa 2 , Camila de Paula Siqueira 2 ,
Samuel Cota Teixeira 3 , Eloisa Amália Vieira Ferro 3 , Mário Machado Martins 4 , Luis Carlos Scalon Cunha 5
and Carlos Henrique Gomes Martins 2, *

1 Technical School of Health, Federal University of Uberlândia, Uberlândia 38400-732, MG, Brazil
2 Laboratory of Antimicrobial Testing, Federal University of Uberlândia, Uberlândia 38400-732, MG, Brazil
3 Laboratory of Immunophysiology of Reproduction, Institute of Biomedical Sciences,
Federal University of Uberlândia, Uberlândia 38400-732, MG, Brazil
4 Institute of Biotechnology, Federal University of Uberlândia, Uberlândia 38400-902, MG, Brazil
5 Chemistry Department, Federal Institute of Triângulo Mineiro, Uberaba 38064-790, MG, Brazil
* Correspondence: [email protected]

Abstract: Bacterial, fungal, and parasitic infections increase morbimortality rates and hospital costs.
Citation: Menezes, R.d.P.; Bessa, This study aimed to assess the antimicrobial and antiparasitic activities of the crude extract from the
M.A.d.S.; Siqueira, C.d.P.; Teixeira, seeds and peel of the pepper Capsicum chinense Jacq. and of the isolated compound capsaicin and to
S.C.; Ferro, E.A.V.; Martins, M.M.; evaluate their ability to inhibit biofilm formation, eradicate biofilm, and reduce hemolysin production
Cunha, L.C.S.; Martins, C.H.G. by Candida species. The crude ethanolic and hexane extracts were obtained by maceration at room
Antimicrobial, Antivirulence, and temperature, and their chemical compositions were analyzed by liquid chromatography coupled to
Antiparasitic Potential of Capsicum mass spectrometry (LC–MS). The antimicrobial activity of the samples was evaluated by determining
chinense Jacq. Extracts and Their the minimum inhibitory concentration. Inhibition of biofilm formation and biofilm eradication by the
Isolated Compound Capsaicin.
samples were evaluated based on biomass and cell viability. Reduction of Candida spp. hemolytic
Antibiotics 2022, 11, 1154.
activity by the samples was determined on sheep blood agar plates. The antiparasitic action of the
https://doi.org/10.3390/
samples was evaluated by determining their ability to inhibit Toxoplasma gondii intracellular prolifera-
antibiotics11091154
tion. LC–MS-ESI analyses helped to identify organic and phenolic acids, flavonoids, capsaicinoids,
Academic Editors: Cláudia and fatty acids in the ethanolic extracts, as well as capsaicinoids and fatty acids in the hexane extracts.
Helena Silva-Lovato, Adriana Antifungal action was more evident against C. glabrata and C. tropicalis. The samples inhibited biofilm
Barbosa Ribeiro, Helena Freitas
formation and eradicated the biofilm formed by C. tropicalis more effectively. Sub-inhibitory concen-
Oliveira Paranhos and María
trations of the samples significantly reduced the C. glabrata and C. tropicalis hemolytic activity. The
Auxiliadora Dea-Ayuela
samples only altered host cell viability when tested at higher concentrations; however, at non-toxic
Received: 30 June 2022 concentrations, they reduced T. gondii growth. In association with gold standard drugs used to treat
Accepted: 18 August 2022 toxoplasmosis, capsaicin improved their antiparasitic activity. These results are unprecedented and
Published: 26 August 2022 encouraging, indicating the Capsicum chinense Jacq. peel and seed extracts and capsaicin display
Publisher’s Note: MDPI stays neutral antifungal and antiparasitic activities.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: antimicrobial activity; biofilm; Candida spp.; capsaicin; Capsicum chinense Jacq.; Toxoplasma
iations. gondii; virulence

Copyright: © 2022 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland.
Health care-related infections (HAI) caused by multidrug-resistant bacteria are increas-
This article is an open access article
distributed under the terms and
ingly common in hospitals worldwide, and ESKAPEEc (Enterococcus faecium, Staphylococcus
conditions of the Creative Commons
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli,
Attribution (CC BY) license (https:// and Enterobacter spp.) has been the most isolated species [1]. HAIs caused by fungal species,
creativecommons.org/licenses/by/ especially Candida spp., have also increased, significantly impacting morbimortality rates
4.0/). and hospital costs [2].

Antibiotics 2022, 11, 1154. https://doi.org/10.3390/antibiotics11091154 https://www.mdpi.com/journal/antibiotics

Antibiotics 2022, 11, 1154 2 of 22

Candida species can produce virulence factors (e.g., hydrolytic enzymes and biofilms)
that help initiate and maintain the infectious process [3]. Hydrolytic enzymes degrade host
tissues (which facilitates the onset of infection) and help the pathogen obtain nutrients to
multiply and propagate [4]. Microorganisms can form biofilms, one of their main defense
mechanisms—biofilms make microorganism removal from biotic and abiotic surfaces
difficult. They prevent contact between the matrix’s antifungal agents and microbial
cells [5]. Production of these virulence factors, associated with indiscriminate use of
antimicrobials, has raised the number of infections by resistant fungal isolates, thereby
limiting therapeutic options and increasing mortality rates [3].
Parasitic infections are a major public health concern because they impact morbidity
rates. Among such infections, toxoplasmosis, a foodborne zoonotic infection caused by Tox-
oplasma gondii [6], stands out. It has severe clinical manifestations in immunocompromised
patients, fetuses, and newborns [6,7]. Regarding treatment, sulfadiazine combined with
pyrimethamine (SDZ + PYR) is the first choice; however, although this option suppresses
active infection, it does not cure latent infection [8]. In addition, these classic drugs are
associated with serious maternal and infant side effects and cases of treatment failure,
suggesting parasite resistance to SDZ + PYR [9]. In the face of these issues, several studies
have reported the antimicrobial, antivirulence, and antiparasitic effects of many natural
compounds, and promising results have been published recently [5,10].
The genus Capsicum has 35 known pepper species; Capsicum chinense is one of the
most common species in Brazil and Central America [11]. The chemical composition of
peppers belonging to this genus includes phytochemical compounds such as flavonoids,
carotenoids, vitamins C, D, and E, and capsainoids, whose medicinal activity has been
proven [11,12]. Capsainoids underlie the characteristic pungency of the genus. Capsaicin is
the most abundant molecule and has relevant anti-inflammatory and antitumor actions,
not to mention that it helps to control cholesterol and obesity [13]. Nevertheless, its antimi-
crobial [14], antiparasitic [15], and antivirulence [16] activities remain little investigated.
In this study, we aim to evaluate the antimicrobial and antiparasitic activities of the
crude extracts from the seeds and peel of the pepper Capsicum chinense Jacq. and of the
isolated substance capsaicin and to investigate their ability to inhibit biofilm formation,
eradicate biofilm, and reduce hemolysin production by Candida species.

2. Results
2.1. MIC, MBC, and MFC Determination
The samples PPHE (Pepper Peel Hexane Extract), PPEE (Pepper Peel Ethanolic Extract),
PSHE (Pepper Seed Hexane Extract), PSEE (Pepper Seed Ethanolic Extract), and CPS
(capsaicin) did not show antimicrobial action against the investigated bacteria within the
evaluated concentration range (0.0115 and 400 µg/mL). However, they displayed antifungal
activity, especially against C. glabrata (ATCC 2001) and C. tropicalis (CI). The seed extracts
and capsaicin were the most active (Table 1).

2.2. Inhibition of Biofilm Formation

The samples inhibited biofilm formation by C. tropicalis (CI) better than the other
isolates evaluated. Capsaicin and seed extracts were the most effective (MICB50 = 93.75 and
187.5 µg/mL, respectively; Figure 1A,B,E). PSHE at 375 µg/mL inhibited biofilm formation
by C. tropicalis (CI). As for the reduction in the cell viability of the C. tropicalis (CI) biofilm
by at least 50%, IC50 values varied between 55.01 and 288.5 µg/mL; PSEE provided the
lowest value. Finally, 93.75 µg/mL PSEE considerably reduced the number of viable cells
in the biofilm (Figure 1).
Antibiotics 2022, 11, 1154 3 of 22

Table 1. Minimum inhibitory concentration and minimum fungicide concentration of the hexane and
ethanolic extracts from Capsicum chinense Jacq. peel and seeds, capsaicin, and amphotericin B against
Candida spp.

Isolates Minimum Inhibitory Concentration/Minimum Fungicide Concentration (µg/mL)

CPS 1 PPHE 2 PPEE 3 PSHE 4 PSEE 5 Amphotericin B

MIC 6 MFC 7 MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC

C. albicans—ATCC 90028 1500 1500 1500 1500 3000 3000 3000 3000 1500 1500 0.5 0.5

C. albicans—CI 8 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 0.25 0.25

C. parapsilosis—ATCC
- - 3000 - - - - - 1500 1500 0.5 0.5
22019

C. parapsilosis—CI - - 3000 - - - - - 3000 3000 0.125 0.125

C. tropicalis—ATCC 13803 - - 3000 3000 3000 - 3000 - 3000 - 0.5 0.5

C. tropicalis—CI 187.5 187.5 187.5 750 750 750 187.5 375 93.75 93.75 0.125 0.125

C. glabrata—ATCC2001 187.5 187.5 1500 1500 3000 3000 375 375 187.5 187.5 0.25 0.25

C. glabrata—CI 1500 1500 1500 1500 3000 3000 1500 1500 750 1500 0.25 0.25

C. krusei—ATCC 6258 1500 1500 1500 750 3000 3000 750 1500 3000 3000 1.0 1.0

C. krusei—CI 1500 1500 750 750 750 750 187.5 1500 1500 1500 0.06 0.06

Note: 1 Capsaicin; 2 Pepper Peel Hexane Extract; 3 Pepper Peel Ethanolic Extract; 4 Pepper Seed Hexane Extract;
5Pepper Seed Ethanolic Extract; 6 Minimum Inhibitory Concentration; 7 Minimum Fungicide Concentration; 8
Clinical Isolate, -: >3000 µg/mL was considered inactive.

Regarding C. glabrata ATCC 2001, PSEE and PSHE gave the best MICB50 values: 375 and 750
µg/mL, respectively (Figure 2A,B). Biomass inhibition was high from 750 µg/mL PSEE (Fig-
ure 2A). PSEE and PSHE also afforded the best IC50 values: 107.1 and 751.9 µg/mL, respectively.
Biofilm cell viability inhibition was high from 187.5 µg/mL PSEE (Figure 2A).

2.3. Biofilm Eradication

Figure 3 shows how the ability of the extracts and capsaicin to eradicate the C. glabrata
(ATCC 2001) and C. tropicalis (CI) biofilms varied. The samples effectively reduced the cell
viability of the preformed C. tropicalis (CI) biofilm. The best samples were CPS and PSEE,
which gave MBEC50 of 187.5 µg/mL and provided 80% biofilm eradication at the highest
concentrations (3000 µg/mL) (Figure 3B). Except for PPHE, all the samples at the highest
tested concentrations destroyed over 80% of the preformed C. tropicalis (CI) biofilm.
Concerning the preformed C. glabrata (ATCC 2001) biofilm, its cell viability decreased
significantly when the samples were tested at concentrations above 1500 µg/mL (Figure 3A).

2.4. Hemolysin Inhibition

C. glabrata (ATCC 2001) exposure to 1⁄2 MIC of PSHE, PSEE, and PPHE significantly
reduced the hemolytic activity (p ≤ 0.05) compared to the control. Treatment with PSEE
inhibited this activity by 48.6% (Table 2). As for C. tropicalis CI, except for PPEE, all the
samples significantly reduced the hemolytic activity. Capsaicin notably resulted in 39.6%
inhibition (p < 0.0001) (Table 2).

2.5. Antiparasitic Action

2.5.1. Cytotoxicity Assay on Host Cells
BeWo cells lost viability after treatment with high PSHE and CPS doses for 24 h. The
minimal dose that elicited the toxic effect was 512 µg/mL for PSHE and 256 µg/mL for
CPS as measured by viability loss (Figure 4B,E). PPEE, PPHE, and PSEE did not alter cell
viability at any tested concentration (Figure 4A,C,D). BeWo cells treated with 1.2% DMSO
did not lose cell viability (Figure 4). The half Cytotoxic Concentration (CC50) against BeWo
cells was 144.33 µg/mL for CPS, while for the active extracts, the CC50 was not determined.
Antibiotics 2022, 11, 1154 4 of 22

Figure 1. Inhibition of C. tropicalis CI biofilm by the hexane and ethanolic extracts from Capsicum
chinense Jacq. peel and seeds. (A–D), capsaicin (E), and amphotericin B (F). The blue line refers to the
curve of the OD values of biomass in relation to the concentration of the tested sample. The black line
refers to the curve of the OD values of biofilm metabolic activity in relation to the concentration of
the tested sample. MICB50 values refer to the concentration of the sample that was able to inhibit
biofilm formation by at least 50% in relation to the biomass [17]. The IC50 values indicate the
sample concentration that was able to inhibit biofilm metabolic activity by half compared to the
control group [18]. The low, moderate, and high classification refers to biofilm formation inhibition
in biomass, where high inhibition corresponds to OD < 0.44, moderate inhibition corresponds to
0.44 < OD < 1.17, and low inhibition corresponds to OD > 1.17. This stratification was based on the
classification of Candida spp. regarding biofilm formation as proposed by Zambrano et al. [19].
Antibiotics 2022, 11, 1154 5 of 22

Figure 2. Inhibition of C. glabrata ATCC2001 biofilm by the hexane and ethanolic extracts from
Capsicum chinense Jacq. peel and seeds (A–D), capsaicin (E), and amphotericin B (F). The blue line
refers to the curve of the OD values of biomass in relation to the concentration of the tested sample.
The black line refers to the curve of the OD values of biofilm metabolic activity in relation to the
concentration of the tested sample. MICB50 values refer to the sample concentration that inhibited
biofilm formation by at least 50% in relation to the biomass [17]. The IC50 values indicate the
concentration of the sample that was able to inhibit biofilm metabolic activity by half compared to the
control group [18]. The low, moderate, and high classification refers to biofilm formation inhibition
in biomass, where high inhibition corresponds to OD < 0.44, moderate inhibition corresponds to
0.44 < OD < 1.17, and low inhibition corresponds to OD > 1.17. This stratification was based on the
classification of Candida spp. regarding biofilm formation as proposed by Zambrano et al. [19].
 μ
Antibiotics 2022, 11, 1154
μ 6 of 22

Figure 3. Percentage of viable cell eradication from the preformed C. glabrata (ATCC2001) (A) and
C. tropicalis (CI) (B) biofilms at different concentrations of the hexane and ethanolic extracts from
the Capsicum chinense Jacq. peel and seeds, capsaicin, and amphotericin B (C). The MBEC50 values
(Minimum Biofilm Eradication Concentration) refer to the sample concentration that was able to
reduce the cell viability of the preformed biofilm by at least 50%.
Antibiotics 2022, 11, 1154 7 of 22

Table 2. Hemolytic index and percentage of hemolytic activity inhibition for C. glabrata (ATCC2001)
and C. tropicalis (CI) after exposure to 1⁄2 MIC of capsaicin, hexane and ethanolic extracts from Capsicum
chinense Jacq. peel and seeds, and amphotericin B.

C. glabrata (ATCC 2001)

Control CPS 1 PPHE 2 PPEE 3 PSHE 4 PSEE 5 Amphotericin B
Average Hi 0.35 0.47 0.5 0.47 0.48 0.52 0.42
% inhibition - 34.3% 42.8% 34.3% 37.1% 48.6% 20%
p valor 0.13 0.002 * 0.71 0.03 * 0.006 * >0.99
C. tropicalis (CI 6 )
Control CPS PPHE PPEE PSHE PSEE Amphotericin B
Average Hi 0.53 0.74 0.70 0.61 0.67 0.69 0.70
% inhibition - 39.6% 32.1% 15.1% 26.4% 30.2% 32.1%
p valor <0.0001 * 0.0002 * 0.99 0.0067 * 0.0012 * 0.0004 *
Note: 1 Capsaicin; 2 Pepper Peel Hexane Extract; 3 Pepper Peel Ethanolic Extract; 4 Pepper Seed Hexane Extract;
5Pepper Seed Ethanolic Extract; 6 Clinical Isolate; * p value statistically significant value.

Figure 4. Host cell viability. For 24 h, BeWo cells were treated in twofold serial dilution (ranging from
4 to 512 µg/mL)μof pepper peel ethanolic extract (PPEE) (A), pepper peel hexane extract (PPHE) (B),
pepper seed ethanolic extract (PSEE) (C), pepper seed hexane extract (PSHE) (D), and capsaicin (CPS)
(E), Cells incubated with culture medium alone (negative control; black column) were considered as
100% viability. Data are expressed as means ± standard deviation. Significant differences detected by
the Kruskal–Wallis test and Dunn’s multiple comparison post-test are labeled (statistically significant
when p < 0.05). ** p < 0.01, **** p < 0.0001.

2.5.2. PPEE, PPHE, and PSEE impaired T. gondii Intracellular Proliferation in BeWo cells
For 24 h, T. gondii-infected BeWo cells were treated
μ with non-toxic concentrations
μ
μ
in twofold serial dilutions of PPEE, PPHE, or PSEE (4 to 512 µg/mL), PSHE (4 to 256
µg/mL),β or CPS (4 to 128 µg/mL). T. gondii intracellular proliferation was quantified by
μ
μ μ μ

μ
Antibiotics 2022, 11, 1154 8 of 22

measuring the β-galactosidase activity of viable parasites. All the tested samples inhibited
parasite proliferation, as follows: PPEE (256 and 512 µg/mL) (Figure 5A), PPHE (256 and
512 µg/mL) (Figure 5C), PSEE (512 µg/mL), and CPS (64 and 128 µg/mL) (Figure 5E).
PSHE was not able to control parasite growth (Figure 5B). The SDZ + PYR treatment
reduced parasite proliferation by about 50% compared to the untreated group (Figure 5).
The Half Inhibitory Concentration (IC50) against T. gondii tachyzoites was 42.12 µg/mL
for CPS, with a selective index (SI) of 3.43. The IC50 and SI for the active extracts were
not determined.

Figure 5. T. gondii intracellular proliferation. For 24 h, T. gondii-infected BeWo cells were treated
with non-toxic concentrations in twofold serial dilutions, as follows: PPEE (A), PPHE (B), and PSEE
(C), (all at concentrations ranging from 4 to 512 μµg/mL), PSHE (4 to 256 μ µg/mL) (D), and CPS (4 to
μ
128 µg/mL) (E), Significant differences detected by the Kruskal–Wallis test and Dunn’s multiple
comparison post-test are labeled (statistically significant when p < 0.05). * Comparison between
infected/untreated cells and infected/treated cells. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.0001.

2.5.3. CPS Potentiates the action of SDZ + PYR to Control Parasite Growth
μ
For 24 h, intracellular T. gondii tachyzoites were allowed to grow in BeWo cells in
the presence of SDZ + PYR (200 +μ 8 µg/mL, respectively) alone or in combination with
μ
different CPS concentrations (4 to 128 µg/mL). Association of SDZ + PYR with CPS (4 to
128 µg/mL) significantly reduced parasite proliferation μcompared to CPS alone (Figure 6).
In addition, only the combination of SDZ + PYR with 64 µg/mL CPS inhibited T. gondii
growth significantly more effectively than SDZ + PYR alone and CPS alone (Figure 6).
Antibiotics 2022, 11, 1154 9 of 22

Figure 6. The effects of the association of CPS and SDZ + PYR to control intracellular parasite
proliferation. Infected BeWo cells were treated with CPS (4 to 128 µg/mL) alone or in the presence of
SDZ + PYR (200 + 8 µg/mL, respectively) for 24 h. Next, μ T. gondii proliferation was quantified by
measuring β-galactosidase activity. Significant differences detected by the Kruskal–Wallis test and
μ
Dunn’s multiple comparison post-test are labeled (statistically significant when p < 0.05). * Compari-
β son between CPS alone with CPS plus (SDZ + PYR). $ Comparison to both CPS alone and SDZ + PYR.
$ p < 0.05, *** p < 0.001, **** p < 0.0001.

2.6. Analysis of the Chemical Profile of C. chinense Jacq. Extracts by LC-ESI-MS

The samples evaluated in the biological assays (PPHE, PPEE, PSHE, and PSEE) were
analyzed by LC-MS-ESI to identify their chemical composition. Table 3 lists the chemical
constituents present in the C. chinense Jacq. extracts.
Organic and phenolic acids, flavonoids, capsaicinoids, and fatty acids were the main
constituents of the ethanolic extracts from peel and seeds. As for the hexane extracts, they
contained capsaicinoids and fatty acids as major constituents.
Antibiotics 2022, 11, 1154 10 of 22

Table 3. Compounds identified in PPEE, PPHE, PSEE and PSHE by LC-MS in negative mode.

Rt Exact Molecular
N. [M-H]– Error (ppm) Fragmentos MS2 Tentative Identity References
(Min) Mass Formula
10 eV: 181, 163, 119, 101,
1 0.75 181.0740 181.0745 −2.7 C6 H14 O6 Sorbitol 1 [20]
89, 71, 59
10 eV: 173, 158, 127, 109,
2 0.81 191.0563 191.0561 1.04 C7 H12 O6 Quinic acid 1,2 [20,21]
93, 85
3 0.96 128.0354 128.0353 0.78 10 eV: 128, 112, 99, 88 C5 H7 NO3 Pyroglutamic acid 1,2 [20]
4 1.10 117.0194 117.0193 0.85 10 eV: 99, 73 C4 H6 O4 Succinic acid 1,2 [20,21]
5 1.18 292.1429 292.1435 −2.0 10 eV: 202, 130 C12 H22 NO7 Fructosyl-leucine/isoleucine 1 [22]
6 1.37 169.0143 169.0142 0.59 10 eV: 125 C7 H6 O5 Gallic acid 1,2 [20,21]
7 1.59 164.0724 164.0723 0.60 10 eV: 147, 103, 72 C9 H11 NO2 Phenylalanine 1,2 [20]
3,4-dihydroxybenzoic acid
8 2.27 153.0194 153.0193 0.65 10 eV: 109 C7 H6 O4 [20,23]
(Protocatechuic acid) 1,2
9 2.52 117.0561 117.0557 3.41 10 eV: 99, 87, 71 C5 H10 O3 Hydroxyisovaleric acid 1,2 [20]
Dihydroxybenzoic acid methyl
10 2.73 329.0877 329.0878 −0.30 10 eV: 269, 209, 167 C14 H18 O9 [21]
ether-O-hexoside 1
11 2.92 181.0509 181.0506 1.65 10 eV: 163, 135, 119 C9 H10 O4 Hydroxyphenyllactic acid 1 [20,24]
12 3.33 137.0246 137.0244 1.45 10 eV: 123, 93, 65 C7 H6 O3 Hydroxybenzoic acid 1,2 [20,25]
13 3.66 131.0713 131.0714 −0.76 10 eV: 131, 99, 59 C6 H12 O3 Hydroxycaproic acid I 1,2 [24]
14 4.03 210.0774 210.0772 0.95 20 eV: 163, 124, 94 C10 H13 NO4 Methoxytyrosine 1 [26]
15 4.34 131.0713 131.0714 −0.76 10 eV: 131, 99, 85, 69 C6 H12 O3 Hydroxycaproic acid II 1,2 [27]
10 eV: 165, 147, 119, 103,
16 4.80 165.0559 165.0557 1.21 C9 H10 O3 3-phenyllactic acid 1,2 [28,29]
91, 73
2-hydroxycinnamic acid
17 4.91 163.0402 163.0401 0.61 10 eV: 147, 119, 103 C9 H8 O3 [23]
(p-coumaric acid) 1
20 eV: 563, 503, 473, 443, Apigenin-6-glucoside-8-arabinoside
18 5.03 563.1409 563.1406 0.53 C26 H28 O14 [23]
425, 383, 353 (Isoshaftoside) 1,2
Luteolin-6-C-glucoside or
19 5.27 447.0931 447.0933 −0.44 20 eV: 357, 327, 285 C21 H20 O11 Luteolin-8-C-glucoside [23]
(Iso)orientin 1
20 5.24 193.0505 193.0506 −0.51 10 ev: 178, 149, 134 C10 H10 O4 Ferulic acid 1 [30]
Apigenin-8-C-glucoside
21 5.46 431.0983 431.0984 −0.23 10 eV: 431, 342, 311, 183 C21 H20 O10 [23,31]
(Vitexin) 1
22 6.32 187.0974 187.0976 −1.06 5 eV: 187, 125, 97 C9 H16 O4 Azelaic acid 1,2,3,4 [32]
20 eV: 343, 300, 301, 271,
23 6.37 447.0933 447.0933 0.0 C21 H20 O11 Quercetin 3-O-rhamnoside 1 [20,21]
227, 179, 151, 109
20 eV: 357, 315, 314, 299,
24 7.01 461.1089 461.1089 0.0 C22 H22 O11 Isorhamnetin 3-O-rhamnoside 1 [33]
295, 271, 199, 151
20 eV: 291, 211, 171, 137, 1,2
25 8.34 327.2179 327.2177 0.61 C18 H32 O5 Trihydroxyoctadecdienoic acid I [22]
85
10 eV: 273, 201, 171, 137, 1,2
26 8.49 327.2175 327.2177 −0.61 C18 H32 O5 Trihydroxyoctadecdienoic acid II [22]
85
20 eV: 329, 311, 293, 275,
Hydroxyoctadecanedioic acid I or
27 8.75 329.2333 329.2333 0.0 229, 211 201, 183, 171, C18 H34 O5 [21–23]
Trihydroxyoctadecenoic acid I 1,2
139, 127
20 eV: 329, 311, 293, 275,
Hydroxy-octadecanedioic acid II or
28 8.85 329.2332 329.2333 −0.30 229, 211, 201, 183, 171, C18 H34 O5 [21–23]
Trihydroxy-octadecenoic acid II 1,2
139
Antibiotics 2022, 11, 1154 11 of 22

Table 3. Cont.

Rt Exact Molecular
N. [M-H]– Error (ppm) Fragmentos MS2 Tentative Identity References
(Min) Mass Formula
20 eV: 329, 311, 293, 275,
Hydroxyoctadecanedioic acid III or
29 8.97 329.2334 329.2333 0.30 229, 211, 201, 183, 171, C18 H34 O5 [21–23]
Trihydroxyoctadecenoic acid III 1,2
139
30 9.05 287.2231 287.2228 1.00 20 eV: 287, 269, 241, 211 C16 H32 O4 1 [22]
Dihydroxy-hexadecanoic acid
31 9.12 304.1916 304.1918 −0.65 10 eV: 289, 168, 116 C18 H27 NO3 Capsaicin 1,2,3,4 [34]
20 eV: 329, 311, 293, 275, Hydroxyoctadecanedioic acid IV or
32 9.19 329.2335 329.2333 0.60 C18 H34 O5 [22,23]
229, 201, 171, 139 Trihydroxyoctadecenoic acid IV 1,2
10 eV: 309, 291, 265, 209,
33 9.45 309.2065 309.2071 −1.9 C18 H30 O4 Oxoepoxyoctadecenoic acid I 1,2 [35]
185, 171, 149, 113
34 9.65 306.2072 306.2075 −0.97 20 eV: 247, 170 C18 H29 NO3 Dihydrocapsaicin 1,2,3,4 [20,36]
20 eV: 309, 291, 273, 249,
35 9.76 309.2072 309.2071 0.32 C18 H30 O4 Oxoepoxyoctadecenoic acid II 1,2 [37]
201, 185, 171, 155, 137
20 eV: 293, 275, 249, 201, Dihydroxy-octadecadienoic acid or
36 9.91 311.2230 311.2228 0.64 C18 H32 O4 [38]
185, 171, 155, 139 Linoleic acid hydroperoxide 1,2,3,4
20 eV: 293, 275, 249, 201, Dihydroxyoctadecadienoic acid or
37 10.18 311.2229 311.2228 0.32 C18 H32 O4 [38]
185, 171, 155, 139 Linoleic acid hydroperoxide 1,2,3,4
Hydroxyoctadecatrienoic acid I or
38 293.2124 293.2122 0.68 20 eV: 275, 235, 171, 121 C18 H30 O3 [22,38]
10.56 Oxooctadeca-dienoic acid I 1,2
39 291.1968 291.1966 0.68 20 eV: 291, 273, 185, 121 C18 H28 O3 Oxooctadecatrienoic acid 3 [20]
Hydroxyoctadecatrienoic acid II or
40 293.2119 293.2122 −1.0 20 eV: 275, 235, 171, 121 C18 H30 O3 [22,38]
10.85 Oxooctadeca-dienoic acid II 1,2,3,4
20 eV: 277, 259, 233, 195,
41 295.2278 295.2279 −0.33 C18 H32 O3 Hydroxyoctadecadienoic acid I 1,2 [22]
171, 151, 123
20 eV: 293, 275, 249, 221,
42 293.2119 293.2122 −1.0 C18 H30 O3 Oxooctadecadienoic acid III 1,2,3,4 [22,38]
10.90 197, 185, 149, 125
43 295.2277 295.2279 −0.67 20 eV: 277, 235, 183, 171 C18 H32 O3 Hydroxyoctadecadienoic acid II 1,2,4 [22]
44 11.86 277.2170 277.2173 −1.0 20 eV: 277, 233 C18 H30 O2 Octadecatrienoic acid 3,4 [21]
20 eV: 279, 254, 218, 185, Octadecadienoic acid 2,3,4
45 12.23 279.2331 279.2330 0.35 C18 H32 O2 [21]
151, 171, 211 (Linoleic acid)
Hexadecanoic acid 3,4
46 12.58 255.2334 255.2330 −1.56 25 eV: 255, 237, 201 C16 H32 O2 [21]
(Palmitic acid)

Note: Rt = Retention time. 1 Pepper Peel Ethanolic Extract; 2 Pepper Seed Ethanolic Extract; 3 Pepper Peel Hexane Extract; 4 Pepper seed hexane extract.
Antibiotics 2022, 11, 1154 12 of 22

3. Discussion
There are no reports on the antimicrobial action of extracts and compounds isolated
from C. chinense Jacq. However, some studies have already demonstrated the antibacterial
potential of molecules isolated from other peppers belonging to the genus Capsicum against
clinical isolates of Streptococcus pyogenes (MIC values between 64 and 128 µg/mL) [16] and S.
aureus (MIC = 1.2 µg/mL) [14] and standard strains of Porphyromonas gingivalis ATCC 33277
(MIC = 16 mg/mL), Enterococcus faecalis ATCC 6057 (MIC = 25 µg/mL), Escherichia coli ATCC
25922 (MIC = 5 µg/mL) and Klebsiella pneumoniae ATCC 29665 (MIC = 0.6 µg/mL) [14].
However, the samples investigated herein do not inhibit bacterial growth in the tested
concentration range. The fact that we did not detect antimicrobial action of the extracts
and capsaicin against bacterial species, unlike what was reported by other authors, can
be explained by the fact that all bacterial isolates evaluated were multiresistant. Perhaps,
the way in which the tested compounds prevent bacterial growth is inhibited by some
resistance mechanism that the bacteria present.
The antifungal action of extracts and molecules isolated from peppers belonging to the
genus Capsicum, including capsaicin, has been little reported in the literature. Most studies
have been carried out with phytopathogenic fungi, such as Aspergillus parasiticus [39] and
Penicillium expansum [40]. Ozçelik et al. [41] evaluated the antimicrobial action of several
Capsicum spp. components against standard C. albicans ATCC 10231 and C. parapsilosis
ATCC 22019 strains and found MIC values lower than 16 µg/mL. The discrepancy between
these results and the results of the present study might be related to the different types of
extracts analyzed in each study and to the origin of the plant material.
However, we have found that PSEE, PSHE, PPHE, and capsaicin present MIC values
lower than 200 µg/mL against C. glabrata and C. tropicalis, which, according to Holetz
et al. [42], indicates that these samples have antifungal action. Furthermore, capsaicin
exerts a fungicidal action based on the MIC and MFC values. Nevertheless, according
to Dorantes et al. [43], the capsaicin mechanism of action remains unknown, but this
compound is believed to lyse the cell wall and consequently kill cells.
According to Pappas et al. [2] and Colombo et al. [44], C. glabrata and C. tropicalis are the
most frequent non-albicans species in HAIs in North America, Latin America, and Asia. In
addition, C. glabrata and C. tropicalis isolates present increased fluconazole resistance [45,46],
which limits the therapeutic options available for treating invasive candidiasis. Therefore,
discovering molecules from natural compounds with promising antifungal action may lead
to a therapeutic strategy in the future.
Candida spp. produce virulence factors during the infectious process, contributing
to worsening patient prognosis [3,5]. In recent years, there has been greater interest in
investigating the attributes that contribute to the pathogenicity of this genus and finding
ways to inhibit or reduce virulence factor production [5,46,47]. Among virulence factors,
biofilm formation plays a crucial role in Candida spp. resistance to antifungal agents [5,48].
Thus, developing and using compounds that inhibit biofilm formation should be evaluated
as an important therapeutic strategy when treating invasive candidiasis [46].
The results of this study are good, especially concerning the C. tropicalis clinical isolate—some of
the samples tested here can inhibit biofilm formation by 50% even at sub-inhibitory concentrations.
Regarding the preformed biofilm, the concentrations needed for reducing biofilm viability by 50%
or more are higher than MICB50, which should be expected. As the fungal biofilm matures, its
architecture becomes more resistant, making it difficult for compounds with antifungal action to
penetrate fungal cells [48]. No literature study has evaluated the ability of extracts from pepper
belonging to the genus Capsicum and of capsaicin to inhibit biofilm formation or eradicate preformed
Candida spp. biofilms, so this is the first report in this sense. However, in studies carried out with other
extracts and molecules, expressive inhibition of the biofilm formed by C. tropicalis clinical isolates has
only been possible when concentrations equal to or greater than the MIC were employed [49].
Likewise, at sub-inhibitory concentrations, most samples investigated herein can
significantly reduce the C. glabrata and C. tropicalis hemolytic activity, indicating the an-
tivirulence potential of C. chinense Jacq. Nevertheless, no literature study has evaluated
Antibiotics 2022, 11, 1154 13 of 22

the anti-enzymatic action of extracts and molecules from Capsicum spp. against Candida
species, so this is a pioneering study in this sense.
Iron absorption from the lysis of red blood cells may be related to Candida spp. re-
sistance to fluconazole [50]. In this context, crude extracts and molecules isolated from
natural compounds that have relevant antivirulence action can be evaluated as possible
adjuvants in the treatment of invasive infections to reduce the pathogen’s virulence and,
consequently, optimize the action of antifungals at lower concentrations.
Despite the relevance of exoenzymes for Candida spp. virulence, the action of natural
compounds in producing these enzymes remains poorly studied, mainly in relation to
hemolysin. Most literature has evaluated compounds’ interference in phospholipase and
proteinase production [51].
To assess the anti-Toxoplasma activity of the investigated samples, we used human
trophoblastic cells (BeWo cells), a well-established in vitro experimental model widely
used for studying human congenital toxoplasmosis [10]. PPEE, PPHE, PSEE, and CPS can
efficiently control T. gondii intracellular proliferation in BeWo cells at concentrations that
are not toxic to host cells. This highlights the selective potential of the tested compounds
against parasites. Additionally, the CPS antiparasitic activity can be potentialized when it
is combined with the classical treatment (SDZ + PYR) against congenital toxoplasmosis.
Piperaceae extracts have distinct pharmacological properties, especially against par-
asites [52] and tumor cell lines [53]. However, few studies have reported their anti-T.
gondii activity. Corroborating with our study, Leesombun et al. [54] assessed the effects
of ethanolic extracts from Thai piperaceae plants Piper betle, P. nigrum, and P. sarmentosum
against infection with T. gondii by using in vitro and in vivo models. They demonstrated
that P. betle is more effective than the other extracts in controlling the parasitic infection
in HFF cells and mice [54]. Similarly, the water and ethanol extract from P. nigrum and
Capsicum frutescens can reduce the number of T. gondii tachyzoites in the peritoneal fluid of
infected mice [55].
Although numerous studies have shown the therapeutic potential of members be-
longing to the family Piperaceae, most of them have been limited to crude extracts. On
the other hand, for the first time, our study has demonstrated the anti-T. gondii action of
extracts from C. chinense Jacq. seeds and peel. We revealed the antiparasitic action of the
isolated compound capsaicin using a model of congenital toxoplasmosis. Thus, C. chinense
Jacq. can be an alternative source of compounds for treating congenital toxoplasmosis, as
highlighted using capsaicin, which controlled the T. gondii growth rate with low toxicity
effects on the host cells.
With respect to the chemical composition of extracts from C. chinense Jacq. peel and
seeds, LC-ESI-MS analysis, allowed us to identify several classes of metabolites such as
organic and phenolic acids, flavonoids, capsaicinoids, and fatty acids. Particularly, organic
acids, phenolic acids, and flavonoids occurred only in the ethanolic extracts of C. chinense
Jacq. peel and seeds. Compounds of these classes of metabolites are known in the Capsicum
genus, and some of them have already been identified in C. chinense fruit [30,56,57].
The organic acids identified in the extracts from C. chinense Jacq. peel and seeds
include quinic (2), pyroglutamic (3), succinic (4), hydroxyisovaleric (9), hydroxycaproic
(13), and 3-phenyllactic (16) acids. Among these acids, quinic (2) and succinic (4) acids
have already been determined in the fruit and different parts of the C. chinense fruit. We
have not found other organic acids such as citric, malic, and fumaric acids reported in C.
chinense fruits [56,57].
The phenolics protocatechuic (8), p-coumaric (17), and ferulic (20) acids identified here
have already been reported in the ethanolic extract from C. chinense fruits [30]. Genistic,
caffeic, and vanillic phenolic acids found in C. chinense by Santos et al. [30] were not
observed in this study.
Concerning flavonoids, we have identified isoshaftoside (18) in the peel and seed
extracts and (iso)orientin (19), vitexin (21), quercetin 3-O-rhamnoside (23), and isorhamnetin
3-O-rhamnoside (24) in the peel ethanolic extract. Surprisingly, all these compounds are
Antibiotics 2022, 11, 1154 14 of 22

glycoside flavones. Similar results were observed in the study by Materska and Perucka [58]
with the Capsicum annuum pericarp and fruit, from which several flavonoid glycosides
were isolated, including quercetin 3-O-rhamnoside (23) identified in C. chinense peel.
Some studies with peppers have shown that qualitative and quantitative variations
between their constituents may occur [56,57,59]. Some factors such as genetics, cultivar type,
maturation stages, irrigation, environmental conditions, soil type, seasonality, extraction
processes, and analytical methods can promote these variations [59].
Phenolic acids and flavonoids are related to several biological activities; however, their
antifungal actions have been highlighted [60]. Plant extracts rich in phenolic compounds
and isolated compounds such as gallic acid (6), protocatechuic acid (8), p-coumaric acid
(17), ferulic acid (20), and hydroxybenzoic acid (12) exert activity against various Candida
species [60,61]. In particular, flavonoid aglycones may also contribute to the activity of
ethanolic extracts because many of these compounds have antifungal effects alone or in
synergistic combination with conventional medicines [62]. Some studies have also shown
that flavonoid glycosides alone or in combination are potent anti-T. gondii agents [63].
Capsaicinoids are another group of metabolites that can be found in C. chinense extracts.
Here, we have detected capsaicin (31) and dihydrocapsaicin (34). These two compounds
have already been found to be the major capsaicinoids in the fruit of C. chinense and other
peppers such as Capsicum annuum and Capsicum baccatum [56].
Capsaicinoids, particularly capsaicin, have been linked to several biological properties,
including antibacterial and antifungal effects [13,30,64] and antiparasitic activity [65]. Based
on literature data and the results obtained herein for the capsaicin standard, this molecule,
together with dihydrocapsaicin (34), may contribute to the activities we have observed for
the extracts.
Still concerning the chemical composition of C. chinense peel and seeds, they contain
several fatty acids that have great antifungal potential, including activity against Candida
spp. [66,67]. Moreover, the antiparasitic potential of fatty acids has also been evidenced [68].
Therefore, apart from capsaicin, other chemical constituents identified in the extracts may
have exerted some effect during the assays.
The chemical composition of C. chinense Jacq. peel and seeds agree with the chemical
composition of other species belonging to the genus Capsicum and other works on C.
chinense. The classes of metabolites and some identified constituents have already been
shown to be active in the biological assays, justifying the promising results found in
this study.

4. Materials and Methods

4.1. Obtaining the Extract and Isolated Substances from the Pepper C. chinense Jacq.
About 2 kg of C. chinense Jacq. pepper fruit was collected on 24 February 2020 in
the rural area of the municipality of Paranaiguara, Goiás, Brazil. Coordinates: Latitude
18◦ 87′ 82” South, Longitude: 50◦ 67′ 37” West. Prof. Dr. Jimi Naoki Nakajima kindly
identified the material, and a testimonial was deposited at the Uberlandense Herbarium of
the Federal University of Uberlândia (UFU) under registration number HUFU 80347. This
project was submitted to the National System for the Management of Genetic Heritage and
Associated Traditional Knowledge (SISGEN) and is registered under No. A9D4A2D.
After the collection and identification steps, the peel and seeds were separated and
placed in a circulating air oven at 35 ◦ C for 7 days for drying. Then, the plant material was
ground in a knife mill, and the ethanolic and hexane extracts were obtained by maceration
at room temperature, as described by Silva et al. [69]. Briefly, in Erlenmeyer flasks, the
material from the peel (0.55 kg) and seeds (0.16 kg) was initially extracted with hexane P.A.
(400 mL) at room temperature for 48 h. This procedure was repeated four times. The final
volume of hexane that was used in the procedure (1.6 L) was filtered and removed in a
rotary evaporator under reduced pressure at 40 ◦ C to give the pepper peel hexane extract
(PPHE) and pepper seed hexane extract (PSHE). Subsequently, the remaining plant material
was extracted from the peel and seeds with ethanol P.A. (400 mL). The same steps performed
Antibiotics 2022, 11, 1154 15 of 22

for the extraction with hexane were followed, leading to the pepper peel ethanolic extract
(PPEE) and pepper seed ethanolic extract (PSEE). The capsaicin used in the study was
obtained commercially (Sigma-Aldrich, Darmstadt, Germany) with purity ≥95%.

4.2. Analysis by High-Performance Liquid Chromatography Coupled to Mass Spectrometry

The C. chinense Jacq. extracts were analyzed by LC-MS on a liquid chromatograph
(Agilent, model Infinity 1260) coupled to a high-resolution mass spectrometer QTOF
(Quadrupole Time of Flight—Agilent, model 6520 B) with electrospray ionization source
(ESI). The chromatographic conditions were Agilent Zorbax C18 column (2.1 mm × 50 mm,
1.8 µm) and ultrapure water with formic acid (0.1% v/v) (mobile phase A) and methanol
(mobile phase B). A volume of 1.0 µL of the sample (2 mg mL−1 ) was injected into the
chromatograph. The gradient elution system consisted of 10% B (0 min), 98% B (0–10 min),
and 98% B (10–17 min) at a flow rate of 0.6 mL min−1 . The ionization parameters were
nebulizer pressure of 58 psi and drying gas at 8 L min–1 at a temperature of 220 ◦ C; energy
of 4.5 kV was applied to the capillary. The analysis was performed in the negative mode
[M-H]− under high resolution (MS).
The molecular formula was proposed for each compound according to a list sug-
gested by the MassHunter Workstation Qualitative Analysis Software Agilent® (Version
10.0) following the smallest difference between the experimental mass and the exact mass,
error in ppm, unsaturation equivalence, and nitrogen rule.. The molecular ions’ sequential
mass spectrometry (MS2 ) was performed at different collision energies. The chemical
composition of the extract was proposed by comparing the obtained mass spectra of the
fragments and the mass obtained under high resolution with other works in the literature,
Metlin library [20], and PubChem database [24,26–28,35–37]. The standard capsaicin
(Sigma-Aldrich, Darmstadt, Germany) with purity ≥95% in methanol (300 µg mL−1 ) was
also analyzed under the same conditions to confirm its presence in the extracts.

4.3. Microorganisms
Microbiological assays were performed with standard strains from the American
Type Culture Collection (ATCC) and clinical isolates (CI) of multidrug-resistant bacteria
and Candida species from previous research and maintained at the Antimicrobial Assay
Laboratory (LEA-UFU). The assayed microorganisms were Enterococcus faecalis (ATCC1299
and CI), Klebsiella pneumoniae (ATCC13883 and CI), Pseudomonas aeruginosa (48 1997a EPM
and CI), Staphylococcus aureus (ATCC BAA 44 and CI), Staphylococcus epidermidis (ATCC
14990 and CI), Candida albicans (ATCC 90028 and CI), Candida glabrata (ATCC 2001 and CI),
Candida krusei (ATCC 6258 and CI), Candida parapsilosis (ATCC 22019 and CI), and Candida
tropicalis (ATCC 13803 and CI). All the isolates belong to the LEA-UFU culture collection
and are kept under cryopreservation at −20 ◦ C.

4.4. Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal/Fungicide Concentration (MBC/MFC)
The broth microdilution technique determined the antimicrobial activity of the extracts
PPEE, PPHE, PSEE, and PSHE and capsaicin (CPS). This was determined by the broth microdi-
lution technique, as proposed by the Clinical and Laboratory Standards Institute in documents
M07-A9 and M27 for assays with bacterial and fungal isolates, respectively [70,71]. MIC,
defined as the lowest concentration of the compound capable of inhibiting microorganism
growth, was determined.
The final concentrations of the tested samples varied between 0.0115 and 400 µg/mL
and 0.98 and 3000 µg/mL in the bacterial and fungal assays, respectively. The antimicrobials
amphotericin B (0.031 to 16 µg/mL) and tetracycline (0.0115 to 5.9 µg/mL) and the isolates
C. krusei ATCC 6258, C. parapsilosis ATCC 22019, E. coli ATCC 25922, and S. aureus ATCC
25923 were used as test controls. The plates were incubated at 37 ◦ C for 24 h and read
after 30 µL of 0.02% aqueous resazurin solution was added to observe microbial growth.
The development of a blue and pink color indicated the absence and presence of growth,
Antibiotics 2022, 11, 1154 16 of 22

respectively. MIC was the lowest concentration that maintained the blue color in the
supernatant medium [72].
Then, 10 µL of the inoculum was removed from each well before resazurin was
added to determine MBC and MFC, defined as the lowest concentration of the test sample
without any microbial growth. The removed sample was plated with Muller–Hinton agar
(bacteria) and Sabouraud Dextrose agar-ASD (yeasts). The presence or absence of growth
was observed after incubation at 37 ◦ C for 24 h. The tests were performed in triplicate in
independent experiments.

4.5. Antivirulence Action Evaluation

Only the isolates that showed promising MIC results according to the classification
proposed by Holetz et al. [42] were selected. For plant extracts, MIC values lower than
100 µg/mL, between 100 and 500 µg/mL, from 500 to 1000 µg/mL, and higher than
1000 µg/mL correspond to good antimicrobial activity, moderate antimicrobial activity,
weak antimicrobial activity, and inactivity, respectively. Thus, only the samples and isolates
for which MIC was lower than 500 µg/mL were selected.

4.5.1. Candida spp. Antibiofilm Assay

The ability of PPEE, PPHE, PSEE, PSHE, and CPS to inhibit biofilm formation and
eradicate preformed biofilm was evaluated against C. glabrata (ATCC2001) and C. tropicalis
(CI) based on biomass and cell viability.
The tests were performed in 96-well plates, and sample preparation and assay were
performed according to the broth microdilution methodology proposed by CLSI [71].
The fungal inoculum was prepared according to Pierce et al. [73]. The final concen-
tration was adjusted to 1 × 106 cel/mL. Then, aliquots of this suspension were added to
96-well plates containing the samples diluted in RPMI-1640 with 2% glucose and buffered
with MOPS ([N-morpholino] propane sulfonic acid) to obtain a final concentration ranging
from 0.98 to 3000 µg/mL. The plates were incubated at 37 ◦ C for 24 h for biofilm formation
and adhesion.
Plates intended for biomass evaluation were processed according to Marcos-Zambrano
et al. [19] with modifications. Briefly, after incubation, well contents were gently aspirated,
and the plates were washed three times with phosphate-buffered saline (PBS, pH: 7.2)
to remove non-adhered cells. Then, the plates were fixed with methanol for 15 min and
stained with 0.1% crystal violet solution for 20 min, and the solution was removed after
submerging the plates in a container with distilled water. Finally, the adhered crystal was
solubilized by adding 33% acetic acid for 30 min, and the absorbance of each well was
determined after the plates were read in a spectrophotometer at a wavelength of 595 nm.
Thus, it was possible to determine the minimum inhibitory concentration of the biofilm
(MICB50 ), defined as the lowest concentration of the sample capable of inhibiting biofilm
formation by at least 50% [17].
Plates reserved for evaluating biofilm cell viability were processed according to Pierce
et al. [73] and Oliveira et al. [46] with modifications. Then, after incubation, well contents
were gently aspirated, and the wells were washed with PBS three times (to remove non-
adhered cells). Next, 50 µL of menadione and 2,3-bis (2-methoxy- 4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide (MTT) with a final concentration of 0.5 mg/mL were added,
and the plates were incubated at 37 ◦ C for 6 h. After that, the formazan product was
solubilized by adding 100 µL of dimethylsulfoxide (DMSO) for 10 min, and 80 µL from
each well was transferred to another plate and read in a spectrophotometer at a wavelength
of 490 nm. Thus, it was possible to calculate the concentration capable of inhibiting the cell
viability of the biofilm by 50% (IC50 ) [18].
Then, 100 µL of the isolate suspension with a concentration of 1 × 106 CFU/mL was
added to the wells of the plates and incubated at 37 ◦ C for 24 h to assess the ability of the
samples to eradicate the preformed biofilm. Then, the non-adhered cells were removed by
washing the wells with PBS three times, and aliquots of the samples diluted in RPMI 1640-
Antibiotics 2022, 11, 1154 17 of 22

MOPS were added to obtain a final concentration between 0.98 and 3000 µg/mL. The plates
were incubated again at 37 ◦ C for 24 h, and cell biomass and viability were determined as
described above. Thus, it was possible to determine the concentration capable of eradicating
at least 50% of viable cells from the preformed biofilm (MBEC50 ) [74].
Amphotericin B (final concentration between 0.031 and 16 µg/mL) was used as a test
control. Tests were performed in triplicate in independent experiments.

4.5.2. Hemolysin Production Inhibition

The ability of the samples to inhibit or reduce Candida spp. hemolytic activity at a sub-
inhibitory concentration (1⁄2 MIC) was determined as proposed by El-Houssaini et al. [47]
and Brondani et al. [75] with modifications. Briefly, from a 24-h culture of the isolate, a
suspension was formed in tubes containing PBS with turbidity equivalent to tube 0.5 on the
McFarland scale. Then, 500-µL aliquots of this suspension were added to tubes containing
500 µL of RPMI-1640-MOPS plus the test sample so that the final concentrations of the
compounds in the tubes were equivalent to the 1⁄2 MIC values and that the final amount of
fungal cells was 1 × 106 cells/mL in each tube. The material was incubated at 37 ◦ C for 24 h.
After this exposure, the tubes were centrifuged at 3000 rpm for 10 min, the supernatant
was discarded, and the pellet was washed with PBS and centrifuged again under the same
conditions. The procedure was repeated two more times, and the pellet was resuspended
in PBS.
Then, 5 µL of this suspension was deposited in equidistant points of Petri dishes
containing ASD plus 7% defibrinated sheep blood and incubated at 37 ◦ C for 48 h. The
hemolytic activity was evidenced by the presence of a hemolysis halo around the colony.
The hemolytic index (Hi) was determined by the ratio between the colony diameter (dc)
and the hemolysis halo plus colony diameter (dcp). The results were classified as negative
(Hi = 1), moderate (0.63 < Hi < 1), and severe (Hi ≤ 0.63) [76]. Amphotericin B was used as
a test control.
The assays were performed in triplicate at two different times. The suspension con-
taining only the RPMI1640-MOPS isolate and broth was used as a positive control, and
amphotericin B was used as a control. The test results were expressed as a percentage of
inhibition of hemolytic activity according to El-Houssaini et al. [47]. Data normality was ver-
ified using the Shapiro–Wilk test. The significance of hemolysis inhibition was determined
through analysis by the ANOVA One Way and Kruskal–Wallis test. GraphPad Prisma
software, version 8.2, was used, and p-values lower than 0.05 were considered significant.

4.6. Antiparasitic Effects

4.6.1. Cell Culture and Parasite Maintenance
Human trophoblast cells (BeWo lineage) were commercially purchased from the
American Type Culture Collection (ATCC, Manassas, VA, USA). They were maintained in
RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin,
and 10% heat-inactivated fetal bovine serum (FBS) in a humidified incubator at 37 ◦ C
and in 5% CO2 . T. gondii tachyzoites (highly virulent RH strain, 2F1 clone) constitutively
expressing the β-galactosidase gene were cultured as previously described [77].

4.6.2. Host Cell Viability

PPEE, PPHE, PSEE, PSHE, and CPS were solubilized in DMSO and diluted in sup-
plemented RPMI 1640 medium to form a stock solution of 640 µg/mL as previously
published [10]. Briefly, BeWo cells (3 × 104 cells/200µL/well) were seeded in 96-well
microplates and treated or not with different concentrations of the tested samples (ranging
from 4 to 512 µg/mL; twofold serial dilutions) at 37 ◦ C and in 5% CO2 for 24 h. Cells
were also incubated with 1.2% DMSO (concentration used in the highest treatment dose:
512 µg/mL). We referred to published data to establish the work concentration of sulfa-
diazine (SDZ) and pyrimethamine (PYR) (200 + 8 µg/mL). The chosen doses have been
shown as non-toxic for BeWo cells [78]. BeWo cells were incubated with 5 mg/mL MTT
Antibiotics 2022, 11, 1154 18 of 22

reagent at 37 ◦ C in 5% CO2 for 3 h, which was followed by addition of 10% SDS and 0.01 M
HCl (37 ◦ C, 5% CO2 , 18 h,) [79]. Absorbance (570 nm) was measured with a multi-well scan-
ning spectrophotometer. Cell viability was expressed in percentages, with the absorbance
of cells incubated with culture medium only considered as 100% viability (Viability %).
Dose-response inhibition curves (log (inhibitor) vs. normalized response—variable slope)
were obtained using GraphPad Prism Software version 9.3.0.

4.6.3. T. gondii Intracellular Proliferation Assay by β-galactosidase Activity

BeWo cells (3 × 104 cells/200 µL/well) were seeded in 96-well microplates. After
adhesion, the cells were infected with a highly virulent T. gondii strain (RH strain, 2F1
clone) at a multiplicity of infection (MOI) of 3:1 (ratio of parasites per cell) in RPMI 1640
medium containing 2% FBS at 37 ◦ C in 5% CO2 . After 3 h of invasion, the medium was
discarded, and the cells were rinsed with culture medium and incubated at 37 ◦ C and in
5% CO2 for 24 h with non-toxic concentrations in twofold serial dilutions, as follows: PPEE,
PPHE, and PSEE (4 to 512 µg/mL), PSHE (4 to 256 µg/mL), and CPS (4 to 128 µg/mL).
Based on the literature, the present study used the standard treatment with SDZ + PYR
(200 + 8 µg/mL, respectively) for comparison with the treatments [78]. The number of
tachyzoites was calculated compared to a standard curve produced with free tachyzoites. T.
gondii-infected BeWo cells incubated with a culture medium in the absence of any treatment
were used as negative treatment control (non-inhibited parasite growth) [10,79]. Dose-
response inhibition curves (Log (inhibitor) vs. normalized response—Variable slope) were
obtained using GraphPad Prism Software version 9.3.0. In addition, the selectivity indexes
(SI) were calculated based on the CC50 BeWo cells/IC50 T. gondii ratio.
Finally, we assessed whether the anti-T. gondii action of CPS could be potentialized in
the presence of SDZ + PYR. Briefly, BeWo cells (3 × 104 cells/200 µL/well) were seeded
in 96-well microplates and infected with T. gondii tachyzoites (3:1) in RPMI 1640 medium
containing 2% CO2 . After 3 h, non-invaded parasites were removed by washing with a
culture medium. The cells were treated with CPS (4 to 128 µg/mL) alone or in the presence
of a certain concentration of SDZ + PYR (200 + 8 µg/mL, respectively) at 37 ◦ C and in
5% CO2 for 24 h. As controls, the cells were incubated with a culture medium only or
with SDZ + PYR alone. Finally, T. gondii intracellular proliferation was calculated by the
β-galactosidase assay, as mentioned above.

5. Conclusions
The results of this study are unprecedented and encouraging. We have shown that the
extracts from C. chinense Jacq. and capsaicin display antifungal action. In addition, they
can significantly inhibit the production of virulence factors (such as biofilm formation and
hemolytic activity) that are important for the onset and maintenance of invasive candidiasis
by C. glabrata and C. tropicalis thereby indicating their antivirulence potential. Furthermore,
we have demonstrated the antiparasitic potential of capsaicin at concentrations that are
not toxic to host cells, which attests to the selectivity of this compound toward Candida
spp. and T. gondii. However, studies involving microscopy and molecular assays are
needed to elucidate capsaicin’s antifungal and antivirulence action and understand the
pathways through which this molecule exerts this effect. In vivo studies for evaluating the
toxicity and behavior of capsaicin in the fight against pathogens in the host should also be
carried out to confirm the results presented here so that in the future, this compound can
be considered a possibility for treating fungal and parasitic infections.

Author Contributions: R.d.P.M.: Conceptualization; Data curation; Formal analysis; Funding acquisition;
Investigation; Methodology; Project administration; Resources; Validation; Visualization; Writing—original
draft; Writing—review & editing. M.A.d.S.B.: Formal analysis; Investigation; Methodology; Writing—review
& editing. C.d.P.S.: Methodology; Writing—review. S.C.T.: Methodology; Writing—original draft; Writing-
review & editing. E.A.V.F.: Writing—original draft; Writing—review & editing. M.M.M.: Methodology;
Writing—review. L.C.S.C.: Methodology; Writing—original draft; Writing—review & editing. C.H.G.M.:
Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project
Antibiotics 2022, 11, 1154 19 of 22

administration; Resources; Supervision; Validation; Visualization; Writing—review & editing. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received financial support of the Programa de Pós Graduação em Imunologia
e Parasitologia aplicadas and Pró Reitoria de Pesquisa e Pós Graduação of the Federal University
of Uberlândia.
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Supe-
rior (CAPES)—Financing Code 001.
Conflicts of Interest: The authors declare no conflict of interest.

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