Available via license: CC BY 4.0
Content may be subject to copyright.
Citation: Viturino, J.J.F.; Camilo, C.J.;
de Carvalho, N.K.G.; Nascimento,
J.B.d.; da Silva, M.I.; Pereira da Silva,
M.; Castro, J.W.G.; Souza, G.G.d.O.;
Rodrigues, F.F.G.; da Costa, J.G.M.
Chemical Composition, Antibacterial
and Antibiotic-Modifying Activity of
Croton grewioides Baill Essential Oil.
Future Pharmacol. 2024,4, 731–742.
https://doi.org/10.3390/
futurepharmacol4040039
Academic Editor: Juei-Tang Cheng
Received: 19 August 2024
Revised: 23 September 2024
Accepted: 8 October 2024
Published: 18 October 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Chemical Composition, Antibacterial and Antibiotic-Modifying
Activity of Croton grewioides Baill Essential Oil
JoséJonas Ferreira Viturino 1,2 , Cicera Janaine Camilo 1, Natália Kelly Gomes de Carvalho 2,
Joice Barbosa do Nascimento 1,2 , Maria Inacio da Silva 1,2, Mariana Pereira da Silva 2,
JoséWalber Gonçalves Castro 1,2, Geane Gabriele de Oliveira Souza 1,2, Fabíola Fernandes Galvão Rodrigues 1,2
and JoséGalberto Martins da Costa 1, 2, *
1Programa de Pós-Graduação em Química Biológica, Departamento de Química Biológica,
Universidade Regional do Cariri, Rua Coronel Antônio Luíz—Pimenta, Crato 63105-00, CE, Brazil;
jjonasferreira5@gmail.com (J.J.F.V.); janainecamilo@hotmail.com (C.J.C.);
joicebarbosa.bio@gmail.com (J.B.d.N.); maria.i.silva@urca.br (M.I.d.S.); josewalber.castro@urca.br (J.W.G.C.);
geane.souza@urca.br (G.G.d.O.S.); fabiolafer@gmail.com (F.F.G.R.)
2
Laboratório de Pesquisa de Produtos Naturais, Departamento de Química Biológica, Universidade Regional
do Cariri, Rua Coronel Antônio Luíz—Pimenta, Crato 63105-010, CE, Brazil;
nataliakellygc@gmail.com (N.K.G.d.C.); mariana.pereira@urca.br (M.P.d.S.)
*Correspondence: galberto.martins@gmail.com
Abstract: Background: Croton grewioides Baill., a species native to the Caatinga, popularly known as
canela de cunhã, is used in traditional medicine to treat gastrointestinal diseases such as diarrhea,
gastritis and stomach ulcers. The combination of essential oils with antibiotics reveals several
beneficial effects associated with the increased efficacy of these drugs against pathogenic agents.
Through this perspective, this study aimed to identify the chemical composition of the essential oil of
C. grewioides (OECG) and evaluate its antibacterial and antibiotic-modifying activities against standard
and multiresistant bacteria. Methods: To analyze the compounds present in the oil, the techniques
of gas chromatography coupled with mass spectrometry (GC/MS) and gas chromatography with a
flame ionization detector (GC/FID) were used. In the bacteriological tests, the Minimum Inhibitory
Concentration (MIC) was obtained by the broth microdilution technique. The modulating effect of
the essential oil was determined by calculating the subinhibitory concentration, followed by a serial
microdilution of the antibiotics. The MIC reduction factor (CRF) was calculated, and its data were
expressed as a percentage. Results: The analysis of the chemical composition identified the presence
of the major compound estragole with a relative abundance of 50.34%. The MIC values obtained
demonstrated efficacy in K. pneumoniae isolated from urine with MIC values of 512
µ
g/mL. OECG
potentiated the effects of all antibiotics tested on the strains S. aureus ATCC 29213, K. pneumoniae
Carbapnemase, E. coli ATCC 25922 and S. aureus ATCC 29213 with their CRF of 97.65%, 99.80%, 99.85%
and 99.88%, respectively. Conclusions: Thus, the essential oil of C. grewioides presents synergistic
effects when combined with the antibiotics tested, in addition to acting in the fight against bacterial
resistance to antibiotics.
Keywords: antibacterial activity; Croton grewioides Baill; essential oil; synergistic effect
1. Introduction
The discovery of antibiotics led to a reduction in mortality rates caused by bacterial
infections in hospital settings. However, the widespread and uncontrolled use of these
drugs has promoted the emergence of resistant microorganisms [
1
], making research into
natural products a promising alternative for combating microbial resistance [2].
In Brazil, the native fauna and flora have been used since before colonization by
native peoples to treat illnesses, accumulating enormous knowledge of medicinal flora [
3
].
Despite having one of the greatest biodiversities in the world, research on native products of
Future Pharmacol. 2024,4, 731–742. https://doi.org/10.3390/futurepharmacol4040039 https://www.mdpi.com/journal/futurepharmacol
Future Pharmacol. 2024,4732
Brazilian flora is relatively underdeveloped [
4
]. Among the Brazilian biomes, the Caatinga
encompasses a large number of plant species with therapeutic potential, but few studies
have focused on the area of chemistry. Therefore, research on species from the Caatinga is
still in its infancy, and their use is reserved, in most cases, for use by traditional and local
communities [5].
The species Croton grewioides Baill. (Euphorbiaceae), commonly known as canela de
cunhã, is widely distributed throughout the Nordeste region of Brazil and is characteristic
of the Caatinga biome [
6
]. Its use in traditional medicine is through teas and infusions
of fresh leaves, commonly used in the treatment of gastrointestinal disorders such as
diarrhea, gastritis and in the treatment of stomach ulcers, in addition to being used as
a sedative agent, relieving pain symptoms and having a calming effect [
7
–
11
]. Its anti-
inflammatory, hepatoprotective and insecticidal effect is also reported, used by small and
medium-sized farmers for pest control [12]. These reported activities are likely associated
with the chemical constituents anethole and estragole, which are present in the essential oil
of the leaves of C. grewioides.
Several studies report that the association of essential oils with antibiotics brings with
it beneficial effects in their use, such as increasing the efficacy of these drugs, which leads
to a reduction in the pathogenicity of the tested strains [
13
–
15
]. Thus, given the high
prevalence of antibiotic-resistant microorganisms and the limited microbiological studies
on C. grewioides, this study aimed to identify the chemical composition of the essential
oil of C. grewioides and evaluate its antibacterial and modifying activities of beta-lactam
antibiotics (amoxicillin and penicillin) and aminoglycosides (gentamicin and amikacin)
against standard and multidrug-resistant bacteria.
2. Materials and Methods
2.1. Collection and Obtaining of Essential Oil
Aerial parts of C. grewioides were collected in the municipality of Crato, Ceará-Brazil,
between August and September 2022 in the morning. A specimen was deposited in the
Herbarium Caririense Dárdano de Andrade Lima of the Regional University of Cariri,
where it was assigned registration number 1619. The essential oil was obtained by hy-
drodistillation (Clevenger type apparatus, Spalabor Instruments Inc., Presidente Prudente,
SP, Brazil), where the sample of the dry plant material (2 kg) was crushed and subjected to
distillation for 4 h. The obtained oil was dried over Na2SO4, yielding 0.20% (w/w).
2.2. GC-MS Analysis
GC-MS was performed with a Shimadzu GC-MS QP2010 series (Shimadzu Scien-
tific Instruments Inc., Columbia, MD, USA). The compounds present in the essential
oil of C. grewioides were separated on a capillary column SH-Rtx-5 (30 m
×
0.25 mm
I.D.
×
0.25 mm film). The analysis time was 30 min and followed the following tempera-
ture program: 80–180
◦
C at 4
◦
C/min, then to 246
◦
C at 6.6
◦
C/min, closing with 10 min
at 280
◦
C, at 3.4
◦
C/min. Helium was used as a carrier gas; the injector temperature was
220
◦
C; the flow rate was 1.5 mL/min; and the split ratio was 1:100. The MS detection
conditions were as follows: interface temperature, 230
◦
C and íon source, 200
◦
C; Ionization
mode, EI; electron energy, 70 eV; mass range, 40–350 m/z. Compounds were identified by
the use of online NIST-library spectra and literature MS data. The results were expressed
as mean area (%) ±standard deviation (n= 3).
2.3. GC Analysis
GC analysis was performed with a Shimadzu GC-2010 Plus equipped with FID (Shi-
madzu Scientific Instruments Inc., Columbia, MD, USA). Compounds were separated on
30 m
×
0.25 mm i.d.
×
0.25 mm film SH-Rtx-5 capillary column. The analysis was per-
formed in 30 min, according to the following temperature program: 80–180
◦
C at 4
◦
C/min,
then to 246
◦
C at 6.6
◦
C/min, closing with 10 min at 280
◦
C, at 3.4
◦
C/min. A split injection
was conducted with a split ratio of 1:15; flow rate: 1.5 mL/min; injection volume: 1
µ
L of
Future Pharmacol. 2024,4733
500 ppm solution prepared with dichloromethane; and helium was used as carrier gas. The
linear retention index was obtained by injecting a mixture of C8–C40 linear hydrocarbons
under the same conditions as the samples, as described by Van Den Dool and Kratz (1963).
The identity of the compounds was confirmed by comparing their retention indices and
mass spectra with those taken from the literature [16].
2.4. Antibacterial Activity
2.4.1. Determination of Minimum Inhibitory Concentration (MIC)
To determine the antibacterial activity, the broth microdilution method containing
the Brain Heart Infusion Broth (BHI) culture medium was used based on the CLSI docu-
ment [
17
]. The following strains were used: Escherichia coli ATCC 25922, Staphylococcus
aureus ATCC 29213, Klebsiella pneumoniae Clinical Urine Isolate (ICU), Escherichia coli 06,
Staphylococcus aureus 10, Klebsiella pneumoniae Carbapnemase (KPC) and Klebsiella pneumo-
niae Betalactamase (BLSE).
The essential oil of C. grewioides (OECG) was prepared at a concentration of 1024
µ
g/mL
diluted in sterile water and DMSO. In the assay, serial microdilutions of 100
µ
L of OECG
were performed where the control group contained only the inoculum. The tests were
conducted in triplicate and incubated in the microbiological incubator at 37 º C for 24 h.
The readings were performed by the colorimetric method using 25
µ
L of 0.01% sodium
resazurin [17].
2.4.2. Antibiotic Modulation Tests
The antibiotics used to evaluate the OECG modulating activity were amoxicillin,
penicillin, gentamicin and amikacin. The methodology for performing the test was adapted
from Coutinho et al. [
18
]. Based on the calculation of subinhibitory concentrations (MIC/8),
a serial microdilution of the respective antibiotics was performed. The negative control
contained only OECG and the inoculum, and the positive control contained the antibiotics
and the bacterial inoculum. The tests were performed in triplicate, where the plates were
placed in an incubator at 37
◦
C for 24 h, and then the bacterial growth was evaluated by
the colorimetric method with sodium resazurin (0.01%).
2.4.3. Determination of the MIC Reduction Factor
To determine the MIC Reduction Factor (FRC) expressed as a percentage, the value
of X was calculated using the formula below. To perform this calculation, a simple rule of
three was used, which obtained the following conclusions: X is equal to the values of the
test MIC (which contains the essential oil together with the antibiotic) multiplied by the
value of 100 divided by the values of the control MIC (antibiotics and bacteria). Thus, the
values obtained in the CRF calculation represent the percentage of reduction in the MIC
values in the presence of the essential oil.
FRC = 100 −X
X = CIM test ×100/CIM control
FCR = 100 −(CIM test ×100/CIM control)
2.4.4. Statistical Analysis
The tests were analyzed by two-way ANOVA followed by the Bonferroni test using
GranphPad Prism 10.0 software (accessed on 10 June 2024). Results expressed at p< 0.05
were considered statistically significant.
3. Results and Discussion
3.1. Composition of C. grewioides Essential Oil
The evaluation of the chemical composition of the essential oil extracted from the aerial
parts of C. grewioides revealed the presence of 2.92% of monoterpenoid compounds, 5.45%
Future Pharmacol. 2024,4734
of oxygenated monoterpenes, 4.40% of sesquiterpenes, 0.42% of oxygenated sesquiterpenes,
69.73% of phenylpropanoids and 10.99% of ethers. The major compound identified in
the chemical analysis was estragole with 50.34%, followed by the compounds methyl
eugenol with 19.39% and anisole with 10.99%. In total, 18 compounds were identified by
chromatography analysis (Figures 1and 2), whose percentages are described in Table 1.
In a study conducted by Santos et al. [
19
] that evaluated the chemical composition of
essential oils from three chemotypes of C. grewioides extracted from branches, in the first
chemotype, the major component was (E)-anethole with 70.5%. In the second chemotype,
the major component was eugenol with 84.2%. Finally, in the third chemotype, (Z)-methyl
with 53.4% was presented. In another study carried out by Silva et al. [
17
], the composition
of the essential oil of C. grewioides extracted from branches revealed (E)-anethole as the
major component with 47.8%. Comparing these studies with the components identified in
this research, the absence of the compound (E)-anethole in the chromatographic analyses
is demonstrated, which can be explained by environmental factors such as collection site,
collection period, part of the plant used, vegetative state of the plant, genetic factors, among
others [20–22].
Future Pharmacol. 2024, 4, FOR PEER REVIEW 4
3. Results and Discussion
3.1. Composition of C. grewioides Essential Oil
The evaluation of the chemical composition of the essential oil extracted from the
aerial parts of C. grewioides revealed the presence of 2.92% of monoterpenoid compounds,
5.45% of oxygenated monoterpenes, 4.40% of sesquiterpenes, 0.42% of oxygenated ses-
quiterpenes, 69.73% of phenylpropanoids and 10.99% of ethers. The major compound
identified in the chemical analysis was estragole with 50.34%, followed by the compounds
methyl eugenol with 19.39% and anisole with 10.99%. In total, 18 compounds were iden-
tified by chromatography analysis (Figures 1 and 2), whose percentages are described in
Tabl e 1.
Figure 1. Chromatogram GC/MS of Croton grewioides essential oil.
Figure 2. Chromatogram GC/FID of Croton grewioides essential oil.
Figure 1. Chromatogram GC/MS of Croton grewioides essential oil.
Future Pharmacol. 2024, 4, FOR PEER REVIEW 4
3. Results and Discussion
3.1. Composition of C. grewioides Essential Oil
The evaluation of the chemical composition of the essential oil extracted from the
aerial parts of C. grewioides revealed the presence of 2.92% of monoterpenoid compounds,
5.45% of oxygenated monoterpenes, 4.40% of sesquiterpenes, 0.42% of oxygenated ses-
quiterpenes, 69.73% of phenylpropanoids and 10.99% of ethers. The major compound
identified in the chemical analysis was estragole with 50.34%, followed by the compounds
methyl eugenol with 19.39% and anisole with 10.99%. In total, 18 compounds were iden-
tified by chromatography analysis (Figures 1 and 2), whose percentages are described in
Tabl e 1.
Figure 1. Chromatogram GC/MS of Croton grewioides essential oil.
Figure 2. Chromatogram GC/FID of Croton grewioides essential oil.
Figure 2. Chromatogram GC/FID of Croton grewioides essential oil.
Future Pharmacol. 2024,4735
Table 1. Compounds identified (GC/MS) in the essential oil of the aerial parts of C. grewioides.
No. Compounds Mean ±Standard
Deviation (%)
RT
(min) IR 1IR 2
1α-pinene 0.766 ±0.092 3.973 1.070 1.050
2 camphene 0.413 ±0.050 4.141 1.090 1.097
3 sabinene 0.250 ±0.127 4.380 1.111 1.123
4 2-β-pinene 0.243 ±0.125 4.455 1.116 1.119
5β-myrcene 0.400 ±0.173 4.530 1.122 1.142
6 1,8-cineole 4.650 ±0.412 5.016 1.156 1.164
7β-ocimene 1.336 ±0.219 5.156 1.165 1.145
8 camphor 1.143 ±0.164 6.094 1.223 1.243
9 isoborneol 0.146 ±0.020 6.345 1.236 1.255
10 estragole 49.61 ±0.688 6.539 1.246 1.226
11 anisole 11.36 ±0.352 7.250 1.283 1.264
12 methyl eugenol 19.05 ±0.653 8.108 1.339 1.359
13 β-elemene 1.286 ±0.111 8.318 1.355 1.375
14 trans-caryophyllene 0.826 ±0.020 8.645 1.380 1.400
15 isoledene 0.290 ±0.069 8.800 1.391 1.373
16 methyl-trans-isoeugenol 6.340 ±0.485 8.943 1.403 1.422
17 caryophyllene oxide 1.750 ±0.243 10.357 1.553 1.572
18 α-selinene 0.287 ±0.090 11.326 1.628 1.648
Results expressed as the average area of each compound (%)
±
DP (n= 3). RT: Retention time (min); IR
1
: Linear
retention index obtained; IR 2: Linear retention index obtained from the literature.
Studies evaluating the chemical composition of the essential oil extracted from the
leaves of C. grewioides, reported by Costa et al. [
23
] and Siqueira et al. [
24
] reveal the presence
of estragole with 76.8% and 45.95%, respectively, as the major components present in its oil.
As in the studies conducted by Leite et al. [
25
] and Andrade et al. [
8
], its percentage present
in the essential oil is 78% and 84.7%, whose estragole compound and its percentages are
close to those found in this study.
The variation in the chemical composition of essential oils is likely a result of the plant’s
adaptive mechanisms, which utilize secondary metabolites to defend against predators
and to adapt to variations in both biotic and abiotic environmental conditions [26].
3.2. Antibacterial Activity
The antibacterial activity of C. grewioides essential oil tested on standard strains demon-
strated significant results against the K. pneumoniae (ICU) strain with an MIC value of
512
µ
g/mL. In multidrug-resistant strains, the oil presented better results on the K. pneu-
moniae (KPC) and K. pneumoniae (BLSE) strains, with MIC values of 512 and 256
µ
g/mL,
respectively. The other MIC values of the strains used in this study are described in Table 2.
In the strains tested, there was no inhibition of bacterial growth even at a concentration
of 1024
µ
g/mL of OECG. This lack of inhibition was due to the fact that the strains tested
present biochemical mechanisms related to bacterial resistance, which implies a high degree
of pathogenicity, since the strains tested in this study are involved in various infections in
hospitalized patients, mainly due to urinary tract infections, as is the case with strains of
E. coli and K. pneumoniae [27].
Studies such as those by Rodrigues and collaborators [
28
], which demonstrate the
antibacterial potential of the C. grewioides essential oil against standard strains of Pseu-
domonas aeruginosa ATCC 15442 and S. aureus ATCC 12692, obtained minimum inhibitory
Future Pharmacol. 2024,4736
dose (MID) results of 1 and 0.5 mg/L of air in S. aureus. These findings corroborate the
results obtained in this research, suggesting that compounds present in the essential oil,
such as estragole and anethole, which are commonly reported in chemical studies, are
directly related to the antibacterial effects observed in the tests.
Table 2. Minimum Inhibitory Concentration (MIC) values of OECG against standard and multiresis-
tant bacteria.
Organisms MIC (µg/mL)
Multidrug-resistant strains
SA 10 ≥1024
EC 06 ≥1024
KPC 512
KP BLSE 256
Standard strains
EC 25922 ≥1024
SA 29213 ≥1024
KP ICU 512
SA 10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse; KP
ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; and EC 25922—E. coli ATCC 25922.
3.3. Modulating Activity
The beta-lactam antibiotics used in this study were amoxicillin and penicillin, which
have a mechanism of action associated with the inhibition of bacterial cell wall synthesis,
leading to a destabilization of the cell wall’s macrostructure and leakage of intracellular
fluid due to membrane lysis. The aminoglycoside antibiotics were amikacin and gentamicin,
which act by inhibiting the protein synthesis mechanism, specifically in the 30S subunit of
bacterial ribosomes [29].
From the results obtained in the modulation tests, OECG enhanced the effects of
amoxicillin on strains of S. aureus 10, K. pneumoniae (ICU) and S. aureus ATCC 29213, with
their respective MIC Reduction Factors (FRC) of 85%, 84.16% and 97.65%. As for penicillin,
it presented results on strains of S. aureus 10 and S. aureus ATCC 29213, with its most
potent effects on K. pneumoniae (KPC) with an FRC of 99.95%, E. coli 06 with 99.80% and
K. pneumoniae (ICU) with 98.02%. The remaining results are detailed in Table 3.
When associated with gentamicin, OECG demonstrated promising results in both
standard and multidrug-resistant strains, except for K. pneumoniae (KPC), which presented
a negative FRC, indicating an antagonistic effect. Of these results, the standard strains
obtained an FRC of 99.85% in E. coli ATCC 25922, 99.80% in S. aureus ATCC 29,213 and
99.75% in K. pneumoniae (ICU). As for amikacin, OECG was more effective against S. aureus
ATCC 29,213 with an FRC of 99.88%.
Table 3. Minimum inhibitory concentrations (
µ
g/mL) of antibiotics in the absence and presence of
C. grewioides essential oil and their MIC reduction factors (FRC).
Substance Organisms MIC of Antibiotics (µg/mL)
Amoxicillin Penicillin Gentamicin Amikacin
Control SA 10 40 8 8 40
EC 06 40 256 8 40
KP BLSE 256 1024 1024 32
KPC 645 1024 812 512
KP ICU 101 406 203 161
SA 29213 256 256 128 256
EC 25922 256 512 203 128
Future Pharmacol. 2024,4737
Table 3. Cont.
Substance Organisms MIC of Antibiotics (µg/mL)
Amoxicillin Penicillin Gentamicin Amikacin
OECG test SA 10 6 0.5 0.5 2
EC 06 161 0.5 0.5 3
KP BLSE 256 1024 256 161
KPC 1024 0.5 1024 512
KP ICU 16 8 0.5 4
SA 29213 6 40 0.25 0.30
EC 25922 203 512 0.30 20
FRC SA 10 85% 93.75% 93.75% 95%
EC 06 −302.5% 99.80% 93.75% 92.5%
KP BLSE 0% 0% 75% −403.12%
KPC −58.76% 99.95% −26.10% 0%
KP ICU 84.16% 98.02% 99.75% 97.51%
SA 29213 97.65% 84.37% 99.80% 99.88%
EC 25922 20.7% 0% 99.85% 84.37%
SA 10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli ATCC 25922;
OECG—C. grewioides essential oil; FRC—MIC reduction factor.
In the graph shown in Figure 3, the modulating effect of OECG associated with the
antibiotic amoxicillin against the strains used in this study and a synergistic effect were
observed in the multiresistant strain S. aureus 10, and in all standard strains tested. In
the other strains tested, an antagonistic effect was observed in S. aureus 10, E. coli 06,
K. pneumoniae (BLSE), K. pneumoniae (KPC).
Future Pharmacol. 2024, 4, FOR PEER REVIEW 7
Table 3. Minimum inhibitory concentrations (µg/mL) of antibiotics in the absence and presence of
C. grewioides essential oil and their MIC reduction factors (FRC).
Substance Organisms MIC of Antibiotics (µg/mL)
Amoxicillin Penicillin Gentamicin Amikacin
Control SA 10 40 8 8 40
EC 06 40 256 8 40
KP BLSE 256 1024 1024 32
KPC 645 1024 812 512
KP ICU 101 406 203 161
SA 29213 256 256 128 256
EC 25922 256 512 203 128
OECG test SA 10 6 0.5 0.5 2
EC 06 161 0.5 0.5 3
KP BLSE 256 1024 256 161
KPC 1024 0.5 1024 512
KP ICU 16 8 0.5 4
SA 29213 6 40 0.25 0.30
EC 25922 203 512 0.30 20
FRC SA 10 85% 93.75% 93.75% 95%
EC 06 −302.5% 99.80% 93.75% 92.5%
KP BLSE 0% 0% 75% −403.12%
KPC −58.76% 99.95% −26.10% 0%
KP ICU 84.16% 98.02% 99.75% 97.51%
SA 29213 97.65% 84.37% 99.80% 99.88%
EC 25922 20.7% 0% 99.85% 84.37%
SA 10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922; OECG—C. grewioides essential oil; FRC—MIC reduction factor.
In the graph shown in Figure 3, the modulating effect of OECG associated with the
antibiotic amoxicillin against the strains used in this study and a synergistic effect were
observed in the multiresistant strain S. aureus 10, and in all standard strains tested. In the
other strains tested, an antagonistic effect was observed in S. aureus 10, E. coli 06, K. pneu-
moniae (BLSE), K. pneumoniae (KPC).
Figure 3. Modulating potential of OECG on the antibiotic activity of amoxicillin against strains SA 10—
S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli
ATCC 25922. Source: author. Two-way ANOVA followed by Bonferroni post-test, using GraphPad
Prism 10.0 software. **** p< 0.0001.
OECG also showed synergistic effects in association with penicillin. Figure 4demon-
strates the synergistic effects in all strains tested, with the exception of K. pneumoniae
(BLSE) and E. coli ATCC 29213, which showed an equivalence of means. One of the main
Future Pharmacol. 2024,4738
mechanisms of bacterial resistance to beta-lactam antibiotics is the production of the beta-
lactamase enzyme that hydrolyzes the beta-lactam ring of these antibiotics, inhibiting
their action [
30
]. The lack of effects of OECG associated with penicillin against K. pneu-
moniae (BLSE) may be related to its resistance mechanism of the type of producer of the
beta-lactamase enzyme that acts to inhibit the action of these drugs [31].
Future Pharmacol. 2024, 4, FOR PEER REVIEW 8
Figure 3. Modulating potential of OECG on the antibiotic activity of amoxicillin against strains SA
10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922. Source: author. Two-way ANOVA followed by Bonferroni post-test, us-
ing GraphPad Prism 10.0 software. **** p < 0.0001.
OECG also showed synergistic effects in association with penicillin. Figure 4 demon-
strates the synergistic effects in all strains tested, with the exception of K. pneumoniae
(BLSE) and E. coli ATCC 29213, which showed an equivalence of means. One of the main
mechanisms of bacterial resistance to beta-lactam antibiotics is the production of the beta-
lactamase enzyme that hydrolyzes the beta-lactam ring of these antibiotics, inhibiting
their action [30]. The lack of effects of OECG associated with penicillin against K. pneu-
moniae (BLSE) may be related to its resistance mechanism of the type of producer of the
beta-lactamase enzyme that acts to inhibit the action of these drugs [31].
Figure 4. Modulating potential of OECG on the antibiotic activity of penicillin against strains SA 10-
S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli
ATCC 25922. Source: author. ns—not significant. Two-way ANOVA followed by Bonferroni post-
test, using GraphPad Prism 10.0 software. **** p < 0.0001.
Carbapinemase-producing bacteria exhibit a broad resistance mechanism against
several beta-lactam antibiotics, acting in the hydrolysis of drugs such as penicillin, ceph-
alosporins, monobactams and carbapenems [32,33]. In this study, OECG potentiated the
effect of penicillin, providing evidence that it may be involved in increasing membrane
permeability, in which the entry of the drug into the cell is facilitated, in addition to acting
at the genetic level, altering its resistance mechanism, since the drug is able to enter the
cell, overcoming enzymatic barriers and inhibiting peptidoglycan synthesis, leading to the
rupture and extravasation of intracellular content [34].
Amoxicillin, which also acts to inhibit cell wall synthesis, showed synergism in stand-
ard strains and multiresistant S. aureus 10. The mechanism is similar to that of penicillin.
Furthermore, more effective inhibition by the drug was expected in standard strains, since
these do not have as many resistance mechanisms as multiresistant strains.
In corroboration with these data, Medeiros et al. [35] evaluated the effects of the C.
grewioides essential oil on the modulation of the antibiotics norfloxacin and tetracycline
against Staphylococcus aureus (SA-1199B) with the efflux pump resistance mechanism, and
they demonstrated that in the absence of the oil, the antibiotic presents an inhibitory con-
centration of 64 µg/mL for norfloxacin and 32 µg/mL for tetracycline. In the presence of
the oil, these values dropped to 16 µg/mL for norfloxacin and 0.5 µg/mL for tetracycline,
Figure 4. Modulating potential of OECG on the antibiotic activity of penicillin against strains SA 10-
S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli
ATCC 25922. Source: author. ns—not significant. Two-way ANOVA followed by Bonferroni post-test,
using GraphPad Prism 10.0 software. **** p< 0.0001.
Carbapinemase-producing bacteria exhibit a broad resistance mechanism against several
beta-lactam antibiotics, acting in the hydrolysis of drugs such as penicillin, cephalosporins,
monobactams and carbapenems [
32
,
33
]. In this study, OECG potentiated the effect of
penicillin, providing evidence that it may be involved in increasing membrane permeability,
in which the entry of the drug into the cell is facilitated, in addition to acting at the genetic
level, altering its resistance mechanism, since the drug is able to enter the cell, overcoming
enzymatic barriers and inhibiting peptidoglycan synthesis, leading to the rupture and
extravasation of intracellular content [34].
Amoxicillin, which also acts to inhibit cell wall synthesis, showed synergism in stan-
dard strains and multiresistant S. aureus 10. The mechanism is similar to that of penicillin.
Furthermore, more effective inhibition by the drug was expected in standard strains, since
these do not have as many resistance mechanisms as multiresistant strains.
In corroboration with these data, Medeiros et al. [
35
] evaluated the effects of the
C. grewioides essential oil on the modulation of the antibiotics norfloxacin and tetracycline
against Staphylococcus aureus (SA-1199B) with the efflux pump resistance mechanism, and
they demonstrated that in the absence of the oil, the antibiotic presents an inhibitory
concentration of 64
µ
g/mL for norfloxacin and 32
µ
g/mL for tetracycline. In the presence
of the oil, these values dropped to 16
µ
g/mL for norfloxacin and 0.5
µ
g/mL for tetracycline,
enhancing the effect of the antibiotic, even in the presence of the overexpressed gene for
the efflux pump.
The effect of OECG against aminoglycoside antibiotics showed that amikacin
(Figure 5) presented synergistic effects in all strains tested except K. pneumoniae (BLSE),
which presented an antagonistic effect, and K. pneumoniae (KPC), which demonstrated
equivalence to the control. As for gentamicin (Figure 6), synergistic effects were observed
in all strains tested, except for K. pneumoniae (KPC), which exhibited an antagonistic effect.
Future Pharmacol. 2024,4739
Future Pharmacol. 2024, 4, FOR PEER REVIEW 9
enhancing the effect of the antibiotic, even in the presence of the overexpressed gene for
the efflux pump.
The effect of OECG against aminoglycoside antibiotics showed that amikacin (Figure
5) presented synergistic effects in all strains tested except K. pneumoniae (BLSE), which
presented an antagonistic effect, and K. pneumoniae (KPC), which demonstrated equiva-
lence to the control. As for gentamicin (Figure 6), synergistic effects were observed in all
strains tested, except for K. pneumoniae (KPC), which exhibited an antagonistic effect.
Figure 5. Modulating potential of OECG on the antibiotic activity of amikacin against strains SA
10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922. Source: author. ns—not significant. Two-way ANOVA followed by Bon-
ferroni post-test, using GraphPad Prism 10.0 software. **** p < 0.0001.
Figure 6. Modulating potential of OECG on the antibiotic activity of gentamicin against strains SA
10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922. Source: author. Two-way ANOVA followed by Bonferroni post-test, us-
ing GraphPad Prism 10.0 software. **** p < 0.0001.
Protein synthesis inhibitors that act on bacterial ribosomes in the 30S and 50S subu-
nits are mainly classified as aminoglycosides, tetracyclines, and aphenicols, among others.
These antibacterial drugs have greater selectivity precisely because they target compo-
nents of the bacterial ribosome that are distinct from those of eukaryotic ribosomes [29].
Figure 5. Modulating potential of OECG on the antibiotic activity of amikacin against strains SA 10—
S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli
ATCC 25922. Source: author. ns—not significant. Two-way ANOVA followed by Bonferroni post-test,
using GraphPad Prism 10.0 software. **** p< 0.0001.
Future Pharmacol. 2024, 4, FOR PEER REVIEW 9
enhancing the effect of the antibiotic, even in the presence of the overexpressed gene for
the efflux pump.
The effect of OECG against aminoglycoside antibiotics showed that amikacin (Figure
5) presented synergistic effects in all strains tested except K. pneumoniae (BLSE), which
presented an antagonistic effect, and K. pneumoniae (KPC), which demonstrated equiva-
lence to the control. As for gentamicin (Figure 6), synergistic effects were observed in all
strains tested, except for K. pneumoniae (KPC), which exhibited an antagonistic effect.
Figure 5. Modulating potential of OECG on the antibiotic activity of amikacin against strains SA
10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922. Source: author. ns—not significant. Two-way ANOVA followed by Bon-
ferroni post-test, using GraphPad Prism 10.0 software. **** p < 0.0001.
Figure 6. Modulating potential of OECG on the antibiotic activity of gentamicin against strains SA
10—S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Car-
bapenemáse; KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC
25922—E. coli ATCC 25922. Source: author. Two-way ANOVA followed by Bonferroni post-test, us-
ing GraphPad Prism 10.0 software. **** p < 0.0001.
Protein synthesis inhibitors that act on bacterial ribosomes in the 30S and 50S subu-
nits are mainly classified as aminoglycosides, tetracyclines, and aphenicols, among others.
These antibacterial drugs have greater selectivity precisely because they target compo-
nents of the bacterial ribosome that are distinct from those of eukaryotic ribosomes [29].
Figure 6. Modulating potential of OECG on the antibiotic activity of gentamicin against strains SA 10—
S. aureus 10; EC 06—E. coli 06; KP BLSE—K. pneumoniae BLSE; KPC—K. pneumoniae Carbapenemáse;
KP ICU—K. pneumoniae clinical urine isolate; SA 29213—S. aureus ATCC 29213; EC 25922—E. coli
ATCC 25922. Source: author. Two-way ANOVA followed by Bonferroni post-test, using GraphPad
Prism 10.0 software. **** p< 0.0001.
Protein synthesis inhibitors that act on bacterial ribosomes in the 30S and 50S subunits
are mainly classified as aminoglycosides, tetracyclines, and aphenicols, among others.
These antibacterial drugs have greater selectivity precisely because they target components
of the bacterial ribosome that are distinct from those of eukaryotic ribosomes [
29
]. For
amikacin, promising results were observed in both standard and multidrug-resistant strains,
with synergistic effects reported in all standard strains as well as in the multidrug-resistant
E. coli 06 and S. aureus 10.
Essential oils, due to their hydrophobic nature, interfere in essential biochemical
processes for the cell when they come into contact with bacterial membranes, such as the
respiratory chain and energy production, which leads to the deregulation of membrane
permeability, facilitating the entry of antibiotic drugs, in addition to interfering in the efflux
pump resistance mechanism, since this requires the binding of the adenosine triphosphate
(ATP) molecule for its functioning in the “expulsion” of toxic substances [28,36].
Future Pharmacol. 2024,4740
Linked to the hydrophobic character of essential oils, their effects on the antibacterial
and modulating activity reported in this research may be related to the various compounds
of the terpene class present in their composition that have an intrinsic relationship to this
activity [37].
4. Conclusions
The data obtained in this research allow us to conclude that OECG, when associated
with the tested antibiotics, increased its efficacy in the main strains of clinical relevance,
with the most promising effects observed in E. coli 06, K. pneumoniae (KPC), S. aureus
ATCC 29213 and E. coli ATCC 25922. Thus, OECG has the potential to be used in future
antimicrobial therapies, acting as a modulating agent in the fight against bacterial resistance
to antibiotics. However, further research on its action on the permeability of the bacterial
membrane, genetic alterations of its resistance mechanism, in addition to analyses of
isolated compounds are necessary to verify whether the efficacy of the essential oil is linked
to the major compound and thus able to use it in future antibacterial treatments.
Author Contributions: Research, writing, formatting, methodology, investigation, J.J.F.V.; methodol-
ogy, investigation, C.J.C.; formal analysis, data curation, N.K.G.d.C.; formal analysis, data curation,
writing, J.B.d.N.; methodology, investigation, M.I.d.S.; methodology, investigation, M.P.d.S.; formal
analysis, data curation, J.W.G.C.; methodology, investigation, G.G.d.O.S.; methodology, investigation,
validation, writing, F.F.G.R.; conceptualization, methodology, supervision, writing, J.G.M.d.C. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors upon request.
Acknowledgments: This work was carried out at the Laboratório de Pesquisa de Produtos Naturais
(LPPN) of the Departamento de Química Biológica at the Universidade Regional do Cariri (URCA),
with support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos
e Projetos (FINEP), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico
(FUNCAP), Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) and Instituto Nacional de
Ciência e Tecnologia—Alimentos (INCT-ALIM).
Conflicts of Interest: The authors declare no conflicts of interest.
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