Food Bioscience 50 (2022) 102128

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Evaluation of antibacterial and toxicological activities of essential oil of

Ocimum gratissimum L. and its major constituent eugenol
Julio Cesar Silva a, Raimundo Luiz Silva Pereira a, Thiago Sampaio de Freitas a,
Janaína Esmeraldo Rocha a, Nair Silva Macedo a, Carla de Fatima Alves Nonato a,
Marina Leite Linhares b, Daniely Sampaio Arruda Tavares c, Francisco Assis Bezerra da Cunha a,
Henrique Douglas Melo Coutinho a, *, Sidney Gonçalo de Lima d,
Francisco Nascimento Pereira-Junior e, Francisco Paulo Araújo Maia f, Ivo Cavalcante Pita Neto f,
Fabiola Fernandes Galvão Rodrigues a, George Joaquim Garcia Santos a
a
Department of Biological Chemistry, Regional University of Cariri, R. Cel. Antonio Luis 1161, 63105000, Crato, CE, Brazil
b
University Center Doutor Leão Sampaio, Av. Leão Sampaio 400, 63040000, Juazeiro do Norte, CE, Brazil
c
Research and Development Center for Medicines, Federal University of Ceara, R. Coronel Nunes de Melo 1000, 60430275, Fortaleza, CE, Brazil
d
Chemistry Department, Federal University of Piaui, Campus Universitário Ministro Petrônio Portella, 64049550, Teresina, PI, Brazil
e
Center for Agricultural and Biodiversity Sciences - CCAB, Federal University of Cariri, Crato, Ceará, Brazil
f
CECAPE - College of Dentistry, Juazeiro do Norte, CE, 63024-015, Brazil

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

Keywords: The present study aimed to comparatively analyze the antibacterial activity, combined with antibiotics, and
Antibacterial agents toxicological activity of the essential oil of O. gratissimum L. and its major compound, eugenol. The phyto­
Drosophila melanogaster chemical analysis of the essential oil was performed by gas chromatography coupled with mass spectrometry.
Eugenol
The minimum inhibitory concentration and combined antibiotic activity were performed by microdilution.
Ocimum gratisismumum L. essential oils
Drosophila melanogaster was the model organism chosen to evaluate the toxicological response of substances. The
chromatographic assay showed eugenol as the major compound of the essential oil of O. gratissimum L. The
microbiological assays showed that in terms of antibacterial activity, both products showed similar activity, quite
significant in Gram-negatives, such as those of strains of Pseudomonas aeruginosa. Similar results were verified in
the modulatory activity, allowing the verification that the bioactivity performed by the essential oil of
O. gratissimum L. is mainly influenced by its major constituent. Although the EC50 of the compounds varied
subtly, the essential oil was less toxic compared to eugenol. The information obtained in the present study
demonstrates that the essential oil of O. gratissimum L. is a promising antibacterial agent and better tolerated than
its majority compound alone, in the model organism D. melanogaster.

1. Introduction number of deaths (HE et al., 2020). Research for new drugs is essential,
given the obsolescence of many drugs and their common side effects (Al
Medicinal plants have always been used in traditional medicine Zuhairi et al., 2020). Thus, studies with essential oils, as well as the
practices since the prehistoric period, becoming a centuries-old habit screening of phytochemicals and isolated compounds, have grown in
linked to many cultures (Badke et al.., 2011). The usefulness offered by recent years, due to the increase in bacterial resistance to antibiotics.
medicinal plants in the treatment of diseases is associated with their (Achmit et al., 2021; Kuhn et al., 2019).
secondary metabolites and their biological effects, which have been Essential oils are volatile compounds of different combinations and
studied in recent years (Rimawi et al., 2020). abundant in flowers, leaves and bark of different species (SWAMY et al.,
The discovery of new drugs for the treatment of bacterial infections 2020), In addition, they can contain different concentrations of sub­
has ensured the extension of human life expectancy, minimizing the stances, such as monoterpenes, sesquiterpenes and phenylpropanoids,

* Corresponding author.
E-mail address: hdmcoutinho@gmail.com (H.D. Melo Coutinho).

https://doi.org/10.1016/j.fbio.2022.102128
Received 1 August 2022; Received in revised form 11 October 2022; Accepted 17 October 2022
Available online 25 October 2022
2212-4292/© 2022 Elsevier Ltd. All rights reserved.
J.C. Silva et al. Food Bioscience 50 (2022) 102128

applicable in the industry, pharmacy and food (Kucukbay et al., 2014). 2.4. Preparation of the essential oil of Ocimum gratissimum L. And
In this context, the Lamiaceae family has gained prominence due to eugenol
its medicinal and cosmetic properties. It is estimated that this family has
a total of 200 genera and more than 6900 species distributed throughout In a test tube 10 mg of essential oil and 500 μL of DMSO were added.
the world (Mohammadhosseini, 2017). Among these species, Ocimum This solution was transferred to another tube and diluted in 9265 mL of
gratissimum L. is an aromatic herb that has been introduced extensively sterile distilled water, resulting in a solution with a final concentration
in tropical and subtropical regions, originating in the African continent of 1,024 μg/mL that was used in all tests. The same process was repeated
and subspontaneously in Brazil, mainly in the Northeast region (Sharma for eugenol.
et al., 2011).
Borges et al. (2012) distinguished eugenol as the major constituent of 2.5. Determination of the minimum inhibitory concentration (MIC)
Ocimum gratissimum L. This phenolic, aromatic and lipophilic compound
showed substantial biological activities, including antibacterial effects The bacterial strains Escherichia coli ATCC 25922, Escherichia coli 06,
(Zhang et al., 2018). The widespread use of medicinal plants and their Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 9027,
chemical components can generate toxic responses (Cunha et al., 2015). Pseudomonas aeruginosa 24, Staphylococcus aureus ATCC 25923, Staph­
Therefore, the Drosophila melanogaster (fruit fly) model is important in ylococcus aureus 10 and Streptococcus mutans ATCC 0446 were seeded in
the evaluation of the pharmacological and toxicological effects offered Petri dishes containing HIA culture medium and incubated at 37 ◦ C for
by metabolites derived from medicinal plants (Zemolin et al., 2014). 24 h.
Thus, this study characterized the chemical profile of the essential oil Then, a sample of each culture was dragged and sterile dilution in
of Ocimum gratissimum L., evaluating its antibacterial effect and its ac­ test tubes containing saline, in triplicate. After this procedure, the
tivity associated with antibiotics. A similar analysis was performed for turbidity was adjusted according to the McFarland 0.5 scale, by which
eugenol alone, comparing its effect with those presented for the essential the intensity of bacterial multiplication in liquid culture medium is
oil. In addition, both substances were analyzed for their effects on determined, established by an increase in the particles that oppose the
inducing mortality or interfering with the negative geotaxis mechanism passage of light, causing opacity in the medium, which will increase as
of fruit flies. the bacteria grow, and the analysis is performed against a standard scale,
which ranges from 0.5 to 10. A 100 μL aliquot of each bacterial inoculum
2. Materials and methods (referring to 10% of the total solution) was transferred to a tube con­
taining 900 μL of a 10% Brain and Heart Infusion Broth (BHI) solution.
2.1. Botanical material Each well of a 96-well microdilution plate was filled with 100 μL of
the solution formed, and then the essential oil was microdiluted in a 1:1
Two hundred and 10 g (210 g) of Ocimum gratissimum L. leaves were ratio, the concentrations ranged from 512 μg/mL to 8 μg/mL. A well
collected in July 2020 at the Crato Municipal Nursery, located at the with no essential oil added was used as a positive control of bacterial
Chico Mendes Institute for Biodiversity Conservation – ICMBio, ac­ growth. The same process performed for the essential oil was repeated
cording to coordinates 7◦ 14′ 27.7′′ S 39◦ 25′ 01.4′′ W. The leaves were with eugenol. The reading of the Minimum Inhibitory Concentration
collected at 12:00 p.m., eugenol peak time, as stipulated by Borges and (MIC) was performed by colorimetric method after 24 h of incubation at
collaborators (2012). An excicata of the specimen was deposited at the 37 ◦ C in a bacteriological oven. Bacterial growth was analyzed by adding
Herbarium Dardano de Andrade de Lima – HCDAL of the Regional 20 μL of resazurin (0.4 mg/ml) to each well. After the addition of
University of Cariri - URCA, Crato/CE, under number 14.389. resazurin, the plates were incubated for 1 h at room temperature and
after this period the colorimetric variation was observed, since there was
no bacterial growth in the wells that remained blue, and there was
2.2. Essential oil extraction and obtaining the eugenol
bacterial growth in the wells that changed from blue to pink coloring
(Gallucci et al., 2009.). The tests were performed in triplicate.
The extraction of the essential oil of Ocimum gratissimum L. was
carried out by the hydrodistillation method, using the Clevenger-type
2.5.1. Evaluation of modulating antibiotic activity
apparatus. The leaves were crushed, placed in a 5.0 L glass flask and
To evaluate the activity associated with antibiotics, was used the
placed in contact with 2.5 L of distilled water for subsequent boiling for
method described by Coutinho and collaborators et al. (2010). The
2 h. The oil was separated by adding sodium sulfate (Na2SO4) anhydrous
substances (chlorpromazine, eugenol and essential oil) were tested in
and preserved in refrigeration (− 4 ◦ C) until its use. Eugenol (Sigma-
their sub-inhibitory concentrations (MIC/8). Each substance was diluted
Aldrich) was kindly provided by Dr. Francisco Assis Bezerra da Cunha
in tubes containing 900 μL of BHI 10%, where 100 μL of the bacterial
from the Semi-Arid Bioprospecting and Alternative Methods Laboratory
inoculum of the strains Escherichia coli 06, Pseudomonas aeruginosa 24
- LABSEMA, Regional University of Cariri (URCA), Crato/CE.
and Staphylococcus aureus 10 were added separately. 100 μL of the ob­
tained concentration were placed in each well of the microdilution plate.
2.3. Chemical analysis of the essential oil of Ocimum gratissimum L Microdilutions were performed with antibiotics amikacin, ampicillin,
norfloxacin and penicillin and the enzymatic inhibitor sulbactam
Analyzes in CG/MS were performed on the chromatograph (GCMS- (associated with Ampicillin), all of them with an initial concentration of
QP2010 SE, AOC-5000 auto injector from SHIMADZU), following the 1,024 μL/mL. The last well of each plate was used as a growth control.
following analysis conditions: Injector 250 ◦ C; initial oven temperature The plates were incubated in an oven at 37 ◦ C for 24 h and the minimum
of 50 ◦ C, with a first heating ramp of 5 ◦ C/min to 180 ◦ C, remaining for inhibitory concentrations of each tested substance were determined by
4 min; a second ramp of 10 ◦ C/min to 260 ◦ C, remaining for 10 min; Split colorimetric method, through the addition of resazurin (Gallucci et al.,
ratio of 1:100; interface temperature: 250 ◦ C and 250 ◦ C source; mass 2009). The tests were performed in triplicate.
range from 50 to 400 Da; ionization by electron impacts, 70 eV. For
component chromatography, a DB5-MS (Agilent) column, 30 m × 0.25 2.6. Toxicity assay
mm with an inner film thickness of 0.25 μm and a stationary phase of
diphenyl dimethylpolysiloxane and helium as carrier gas was used. The 2.6.1. Breeding and stocking of Drosophila melanogaster
identification was carried out by comparing the mass spectra with those Drosophila melanogaster (Harwich strain) was obtained from the
of the Nist08 libraries and comparison with the base records of the National Species Stock Center, Bowling Green, OH. The flies were grown
literature. according to methodology described by Cunha et al. (2015) in 340 mL

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J.C. Silva et al. Food Bioscience 50 (2022) 102128

glass containers and grown in medium containing: 83% corn mass, 4% 3. Results
sugar, 4% lyophilized milk, 4% soybean, 4% wheat or oat bran and 1%
salt). When cooking the mixture, 1 g of Nipagin (Methylparaben) was 3.1. Chemical profile of Ocimum gratissimum L. essential oil
added. After the mixture cooled in the growth flasks, 1 mL of a solution
containing Saccharomyces cerevisiae was added. The yield of the essential oil of Ocimum gratisismum (EOOg) was
The flies were grown in BOD type incubator at a temperature of 25 ◦ C 0.39%. Phytochemical characterization, performed by mass chroma­
± 1 ◦ C, with light-dark cycles 12:12 h and a relative humidity of 60%. tography, identified the presence of 10 compounds, as can be seen in
Fig. 1.
2.6.2. Mortality assay When analyzing the constituents individually, it is observed that
Mortality tests were performed in accordance with the methodology eugenol (63.25%), cineole (10.83%), beta-silinene (8.43%) and trans-
of Cunha et al. (2015). Adult flies, male and female, were placed in 130 caryophyllene (6.85%) are the major components (Table 1).
mL containers (6 cm high and 6.5 cm in diameter). The pots were
divided into control and test groups. One milliliter (1 mL) of 20% su­ 3.2. Minimum inhibitory concentration (MIC)
crose solution in distilled water was placed at the bottom of all control
pots. The test groups consisted of pots that contained eugenol or The values obtained for the MICs of each substance were considered
essential oil of Ocimum gratissimum L. The fumigation methodology was clinically significant when less than 1,000 μg/mL (Houghton et al.,
adopted, where the compounds are added on a filter paper in the tube 2007). The Minimum Inhibitory Concentration (MIC) obtained in the
cap and thus are volatilized and absorbed. The concentrations of 1, 5 and tests with eugenol (EUG) demonstrated its action against the strains:
10 μL/mL of eugenol and essential oil of O. gratissimum were used, with a Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 9027,
counting time of 1, 2, 3, 6, 9, 12, 24 and 48 h. The temperature of 25 ◦ C Pseudomonas aeruginosa 24, Staphylococcus aureus ATCC 25923 and
± 1 ◦ C was maintained throughout the test, as well as the relative hu­ Streptococcus mutans ATCC 0446 (Table 2a).
midity of 60% in a BOD type incubator with a light-dark cycles 12: 12 h. Following the analysis of antibacterial activity, the EOOg showed
The tests were performed in sextuplicate, with each pot containing a results similar to those obtained by the EUG, with clinically significant
total of 20 flies. responses evidenced against the same strains tested (Table 2b).

2.6.3. Negative geotaxis assay 3.3. Evaluation of the associated activity of eugenol and essential oil of
Damage to the locomotor system was determined by the negative Ocimum gratissimum L
geotaxis test, as described by Cunha et al. (2015). Live flies, after
exposure to eugenol or essential oil of O. gratissimum at predetermined An evaluation of the associated activity of EUG and EOOg with an­
times, were taken to the bottom of the pots. After 1 min, the flies that tibiotics was also carried out. In addition, the effect of the combination
reached 4 cm in height in the containers were counted and this pro­ of chlorpromazine (CPMZ), associated with the antibiotics of interest,
cedure was repeated after the 1-min interval. and sulbactam (SULB) in combination with ampicillin was also tested.
The results obtained in this analysis were distributed in sections, orga­
2.7. Statistical analysis nized according to pairs of antibiotics, which act on the external and
internal portion of the bacterial cell.
The antibacterial assays were performed in triplicates and the results Fig. 2a and b represent the results of the combination of the com­
were expressed as the geometric mean. Statistical hypothesis analysis pounds mentioned above, against the Escherichia coli 06 strain. A
was performed for the antibacterial assays using a Two-Way ANOVA possible synergistic effect was evidenced in the association between
followed by Bonferroni’s post hoc test, using the GraphPad Prism 7.0 CPMZ + norfloxacin, and EUG + norfloxacin. The same could be seen
software. To analyze the toxicity data, a two-way ANOVA followed by a with amikacin, when associated with EUG and EOOg. On the other hand,
Tukey’s multiple comparisons test were performed. No statistical dif­ CPMZ when associated with amikacin, caused an increase in MIC,
ferences using the same concentration as a function of time were resulting in a possible antagonistic phenomenon (Fig. 2a).
observed. It was observed that the combination of sulbactam and ampicillin
caused a reduction in MIC, from 1,024 μg/mL to 128 μg/mL, possibly
potentiating the activity of ampicillin against the strain tested (Fig. 2b).
Fig. 2c and d shows a combined activity of the antibiotics nor­
floxacin, amikacin, penicillin and ampicillin, for the bacteria

Fig. 1. Chromatogram CG-MS of the essential oil of Ocimum gratissimum L. (1) 1,8-cineole-eucalytol; (2) Cis-ocimene; (3) 1-alpha-terpineol; (4) Eugenol; (5) Beta-
element; (6) Trans-caryophyllene; (7) Alpha-humulene; (8) Germacrene-D; (9) Beta-selinene; (10) Alpha-selinene.

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J.C. Silva et al. Food Bioscience 50 (2022) 102128

Table 1
Relative composition of Ocimum gratissimum L. essential oil.
Nº RT Similarity (%) Constituent Kovats Index Molecular Formula Absolute Area Relative Area

1 9.525 95 1,8-cineole eucalytol 991 C10H18O 648767 10,83%

2 9.574 93 Cis-ocimene 1050 C10H16 112333 1,88%
3 14.428 93 l-alpha-terpineol 1189 C10H18O 50805 0,85%
4 18.955 97 Eugenol 1356 C10H12O2 3787946 63,25%
5 20.077 91 Beta-elemene 1375 C15H24 50833 0,85%
6 20.959 96 Trans-caryophyllene 1419 C15H24 410342 6,85%
7 21.921 94 Alpha-humulene 1438 C15H24 60036 1,00%
8 22.585 96 Germacrene-D 1480 C15H24 182146 3,04%
9 22.810 95 Beta-selinene 1485 C15H24 504974 8,43%
10 22.985 94 Alpha-selinene 1494 C15H24 180493 3,01%

*RT – Retention Time.

of EUG and EOOg. For both, concentrations of 1, 5 and 10 μL/mL were

Table 2
used. EOOg showed a EC50 equal to 2,677 μL/mL after 6 h of exposure.
Minimum inhibitory concentration (MIC) of eugenol (a) and essential oil of
The concentration of 1 μL/mL did not differ significantly when
Ocimum gratissimum L. (b) against bacterial strains.
compared to the control. The concentration of 5 μL/mL showed greater
STRAINS MIC (μg/mL) toxicity after 48 h of contact. After 6 h of exposure to a concentration of
(a) EUGENOL Escherichia coli ATCC 25922 1024 10 μL/mL of EOOg, it started to increase significantly, demonstrating the
Escherichia coli 06 1024 toxicity at this concentration. (Fig. 3a).
Klebsiella pneumoniae ATCC 4352 512
There was variation in the mortality pattern according to the EUG
Pseudomonas aeruginosa ATCC 9027 406.37
Pseudomonas aeruginosa 24 322.54 concentration used, which presented EC50 equivalent to 2,681 μL/mL,
Staphylococcus aureus ATCC 25923 203.19 after 6 h of exposure. Concentrations of 1 and 5 μL/mL showed a sig­
Staphylococcus aureus 10 1024 nificant degree of mortality after 48 and 24 h of exposure, respectively.
Streptococcus mutans ATCC 0446 322.54 On the other hand, the concentration of 10 μL/mL differed from the
(b) ESSENTIAL OIL Escherichia coli ATCC 25922 1024
Escherichia coli 06 1024
control after 6 h, being more toxic in relation to the others (Fig. 3b).
Klebsiella pneumoniae ATCC 4352 645.08 Thus, EOOg was less offensive when compared to EUG.
Pseudomonas aeruginosa ATCC 9027 812.75 The concentration of 1 μL/mL of EOOg was not significant in the
Pseudomonas aeruginosa 24 512 impairment of D. melanogaster geotaxy. Fact that differs from that
Staphylococcus aureus ATCC 25923 80.63
visualized for the concentration of 5 μL/mL, which caused an interfer­
Staphylococcus aureus 10 1024
Streptococcus mutans ATCC 0446 812.75 ence in the flight ability from 12 h of the beginning of the tests. The
concentration of 10 μL/mL of EOOg caused the negative geotaxis of the
fruit flies to be compromised right after the first 3 h of testing, with a
Pseudomonas auruginosa 24. Chlorpromazine continued to intensify the substantial degree of impairment (Fig. 4a).
action of norfloxacin, as well as with Escherichia coli 06. This possible The eugenol also affected the negative geotaxis of D. melanogaster.
synergistic effect was also evidenced in the association CPMZ + ami­ Fig. 4b shows how much each concentration varied compared to the
kacin and EUG + amikacin. However, the EUG + norfloxacin combi­ control. In this case, it is verified that the exposure to 1 μL/mL of the
nation was possibly antagonistic, with a corresponding increase in the compound causes impairment of the mobility of the flies after 48 h of
inhibitory concentration of the antibiotic against the strain tested. This exposure, while the concentration of 5 μL/mL causes the same effect
information can be seen in Fig. 2c. after 12 h of contact. The previous exposure to 10 μL/mL of EUG was
Sulbactam continued to intensify the action of ampicillin, by even more toxic, in view of the negative geotaxis impairment even in the
reducing the MIC from 512 μg/mL to 4 μg/mL, on the other hand, there first counting time.
was a possible antagonistic process in the association of this antibiotic
with the EUG, given the increase in MIC from 512 μg/mL to 812.7 μg/mL 4. Discussion
(Fig. 2d).
Fig. 2e shows a possible antagonism in the combination of nor­ Antibiotic resistance has become a serious public health problem in
floxacin + CPMZ and norfloxacin + EUG against Staphylococcus recent years. Some publications try to alert to the need to create research
aureus10. This antagonism was repeated for amikacin + CPMZ and priorities, aiming to find new antibacterial molecules, because of the
amikacin + EUG. However, there was an intensification of amikacin restricted therapeutics and the lack of new chemical structures, with
activity, when combined with essential oil, similarly to what happened efficient mechanisms of action (Carneiro et al., 2014; Duval; Grare,
in the Escherichia coli 06 strain. Demoré, 2019). Research such as the one carried out by Saraiva et al.
Interestingly, EUG and EOOg caused a possible synergistic effect (2021), who compared the intrinsic and antibacterial effect of Laurus
(reduction of MIC from 1,024 μg/mL to 161.3 μg/mL and 128 μg/mL, nobilis essential oil and its major compound 1,8-cineole is important to
respectively) when combined with penicillin against Staphylococcus delimit the activity of essential oils and isolated compounds, thus
aureus 10 (Fig. 2f) which it did not happen with the strains Escherichia demonstrating which of these are more effective.
coli 06 and Pseudomonas aeruginosa 24. In view of the Gram-positivity of The main bacteria associated with the development of resistance
Staphylococcus aureus, it is assumed that this effect for penicillin is not mechanisms are Gram-negative, including Escherichia coli, Klebsiella
prominent in Gram-negatives. The combination of sulbactam + ampi­ pneumoni, Pseudomonas aeruginosa (Duval; Grare, Demoré, 2019), given
cillin and EOOg + ampicillin also potentiated the control effect, the the scenario presented above and in addition to the fact that since the
latter contrary to what was seen in the other strains. production of new antibiotics is practically discontinued nowadays,
studies like ours are essential, as they collaborate with the scientific
dissemination of antibacterial substances, comparing them and pre­
3.4. Toxicological activity senting which one is the most expressive in terms of biological activity
and tolerability in model organisms.
The Drosophila melanogaster model was chosen to assess the toxicity

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J.C. Silva et al. Food Bioscience 50 (2022) 102128

Fig. 2. Evaluation of antibiotics modifying activity (Amikacin, Ampicillin, Norfloxacin and Penicillin) by EUG and EOOg against bacterial strains (Escherichia coli 06,
Pseudomonas aeruginosa 24 and Staphylococcus aureus 10).

Fig. 3. Effect of Essential oil of Ocimum gratissimum L. (a) and eugenol (b) on the mortality test of Drosophila melanogaster at different concentrations and time.

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J.C. Silva et al. Food Bioscience 50 (2022) 102128

Fig. 4. Effect of the Essential oil of Ocimum gratissimum L. (a) and eugenol (b) on the negative geotaxis behavior of Drosophila melanogaster in different concentrations
and time.

Pessoa et al. (2015) chemically characterized the essential oil of carried out with Ocimum basilicum L. showed that the association of its
O. gratissimum L., distinguishing the following compounds: eugenol essential oil, rich in eugenol, with Neomycin and with Amikacin gen­
(67.30–74.99%), β -selinene (8.18–8.27%), β-ylangene (4 0.97 to erates an intensifying effect, represented by the reduction of the MIC of
5.09%), viridiflorene (3.38–3.47%), 1,8-cineole (3.07–3.11%) and these antibiotics against Staphylococcus aureus (NUNES et al., 2014). The
y-himachalene (2.00–2.62%). The relative composition of EOOg pre­ same was verified in this study for the EOOg and even for the EUG.
sented in this study was similar to that presented by Pessoa et al. (2015), Aguiar et al. (2015) studied the direct modulatory effect of the
given what was presented for eugenol (63.25%) and β-selinene (8.43%). essential oil of O. gratissimum L. in association with the aminoglycosides
Catherine et al. (2012) mentioned the antibacterial effect of eugenol amikacin and gentamicin. The significant results obtained in tests
against Escherichia coli and Staphylococcus aureus bacteria. Its activity involving the bacteria Escherichia coli, Pseudomonas aeruginosa and
through the microtiter methodology was considerable, demonstrated Staphylococcus aureus were antagonistic. In this study, we present the
through the following minimum inhibitory concentrations: Bacillus ce­ possibility of synergism for amikacin (against Escherichia coli and
reus (0.125 μg/mL), Escherichia coli (0.125 μg/mL), Helicobacter pylori Staphylococcus aureus), norfloxacin (against Pseudomonas aeruginosa),
(0.0312 μg/mL), Staphylococcus aureus (0.25 μg/mL) and Streptococcus ampicillin and penicillin (against Staphylococcus aureus).
pyogenes (0.5 μg/mL). A slightly higher but also significant MIC was Synergistic processes occur through the inhibition of the sequence of
obtained for Pseudomonas aeruginosa (8 μg/mL) (Jeyakumar & Law­ a common biochemical pathway, as well as enzymes that protect mi­
rence, 2021). croorganisms, due to the combination of active agents in the cell wall or
Aguiar et al. (2015) examined the antibacterial action of EOOg their association with components that act in other pathways, which
against the standard and resistant strains of Escherichia coli, Pseudomonas facilitates penetration into the cell (Santiesteban-López, Palou, &
aeruginosa and Staphylococcus aureus. In this one, only the MIC for the López-Malo, 2007).
standard strain of Staphylococcus aureus was significant, which corrob­ The factors that cause antagonism in drug combinations are still
orates this study. In our study, EOOg was equally effective against poorly understood. However, it is known that the association of bacte­
standard strains of Klebsiella pneumoniae, standard and resistant strains riostatic and bactericidal agents, the combined use of compounds that
of Pseudomonas aeruginosa, standard strains of Staphylococcus aureus and compete for the same target in a microorganism and the molecular in­
Streptococcus mutans. Joshi (2013) also demonstrated that the essential teractions between different active components, are the main reasons for
oil of Ocimum gratissimum L. has considerable activity against Escherichia the development of antagonistic processes (Goñi et al., 2009).
coli and Klebsiella pneumoniae. Eugenol acts by creating deformations in the bacterial membrane,
In the present study, we demonstrated that the antibacterial activity impairing its integrity and morphology, which causes an overflow of the
of the essential oil of Ocimum gratissimum L. and the isolated eugenol are intracellular content and inactivation of the microorganism (Debao
equivalent, that is, both compounds showed a response against the same et al., 2019). In addition, there is also a predisposition of the EUG to
strains, namely: Klebsiella pneumoniae ATCC 4352, Pseudomonas aerugi­ bacterial genomic DNA, responsible for the aggregation of DNA mole­
nosa ATCC 9027, Pseudomonas aeruginosa 24, Staphylococcus aureus cules. Due to its lipophilic character, typical of monoterpenes, there is
ATCC 25923 and Streptococcus mutans ATCC 0446. The best activity was interaction with polysaccharides, fatty acids and lipids of the bacterial
demonstrated for the bacterium Pseudomonas aeruginosa, since the sub­ cell membrane, thus causing a disturbance in the permeable barrier
stances showed activity against both standard and resistant strains. This (Limaverde et al., 2017).
result is quite significant since the aforementioned bacterium (Gram- A similar mechanism can be seen in the use of essential oils, capable
negative) is a relevant opportunistic pathogen, directly associated with of compromising the integrity of the bacterial membrane and the
nosocomial infections (Tuon, Gortz, & Rocha, 2012). conformation of its cell wall, facilitating the influx of drugs (Matias
EUG causes a disturbance in the lipid fraction of the bacterial et al., 2011). The hydrophobic compounds in these oils, such as eugenol,
membrane, causing a change in permeability, which facilitates the can adhere both to the plasma membrane and to phospholipids and
interaction of the compound with intracellular portions (Jeyakumar & membrane proteins, thus promoting the assimilation of antibiotics (Luz
Lawrence, 2021). The mechanism of action of EOOg is related to its et al., 2014).
majority composition, with the prevalence of eugenol, as described by The conformation of the cellular envelope of microorganisms in­
Joshi (2013). duces variations in the effectiveness of chemical components against
The evaluation of the combined activity was performed for the an­ bacteria. The thick layers of peptidoglycans in Gram-positive bacteria
tibiotics amikacin, ampicillin, norfloxacin and penicillin, analyzing the ensure greater sensitivity to bioactive components, which can interact
effect of the interaction of EUG and EOOg with these drugs. Studies with the bacterial envelope in different ways and degrees (Requena,

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J.C. Silva et al. Food Bioscience 50 (2022) 102128

Vargas, & Chiralt, 2019). This phenomenon justifies the action of EOOg Ocimum gratissimum L. The antibacterial activity of the essential oil of
by potentiating the action of amikacin and penicillin against Staphylo­ O. gratissimum L. and eugenol was similar and promising against both
coccus aureus 10 and EUG by intensifying the action of penicillin against standard and resistant strains of Pseudomonas aeruginosa, and the com­
the same strain in this study. bined activity with antibiotics was also significant and similar for both
Chlorpromazine, in non-antibacterial concentrations, has been used substances. The evaluation of toxicological activity revealed that the
as an agent capable of inhibiting efflux pumps, Coutinho et al. (2010) essential oil of O. gratissimum L. is better tolerated by the model or­
discussed the MIC-lowering effect of chlorpromazine, noting its com­ ganism Drosophila melanogaster, which, comparatively, may collaborate
bined effect with amikacin, kanamycin and tobramycin, in addition to with greater effectiveness in the possibility of administration combined
describing an antagonistic effect for gentamicin and neomycin, with with antibiotics. More studies are recommended, using other toxicity
data referring to tests with Escherichia coli. The combination of SULB models.
with ampicillin considerably broadens the spectrum of action of the
latter, against Klebsiella pneumoniae and Staphylococcus aureus, for Informed consent
example. Specific tests should be performed with both compounds,
against strains that express active efflux proteins and the beta-lactamase Informed consent was obtained from all individual participants
enzyme, since the results presented in our study are not sufficient to included in the study.
infer the inhibition of possible resistance processes.
According to Zhang et al. (2016), oxygenated monoterpenes are Ethical statement
agents of considerable toxicity against Drosophila melanogaster, which
can be represented by the LC50 variation of 0.015 and 0.02 μl/L. This This article does not contain any studies with animals performed by
data agrees with that presented for the EUG in this study, taking into any of the authors.
account the EC50 value equivalent to 2,681 μL/mL. This is associated
with the specific characteristics of EUG, such as the presence and posi­
Authorship contribution declaration
tion of its hydroxyl group, as well as the characteristic positioning of its
double bonds, which can influence differently the degree of mortality of
Júlio César Silva: Methodology, writing and validation. Raimundo
fruit flies, as seen for α-pinene and β-pinene (Xie et al., 2014; Zhang
Luiz Silva Pereira: Methodology. Thiago Sampaio de Freitas: Software.
et al., 2016).
Janaína Esmeraldo Rocha: Validation. Nair Silva Macedo: Methodology.
The toxicological response of essential oils is related to the additive
Carla de Fatima Alves Nonato: Methodology. Marina Leite Linhares:
and synergistic effects of different compounds (Essoung et al., 2020).
Formal analysis. Daniely Sampaio Arruda Tavares: Formal analysis.
Tests carried out with Ocimum tenuiflorum showed its fumigant capacity
Francisco Assis Bezerra da Cunha: Resources, Supervision. Henrique
against S. oryzae, by inhibiting the enzyme acetylcholinesterase (AChE).
Douglas Melo Coutinho: Conceptualization, Supervision. Sidney Gon­
This action is represented by the LC50 value of 479 μL/L (Bhavya,
çalo de Lima: Resources, Supervision. Francisco Nascimento Pereira
Chandu, & Devi, 2018).
Júnior: Writing-review and editing. Francisco Paulo Araujo Maia:
A similar effect was observed for EOOg in this study, whose mortality
Conceptualization, Supervision. Fabiola Fernandes Galvão Rodrigues:
was similar to that of EUG, which is justified, as the literature describes
Conceptualization, Supervision. George Joaquim Garcia Santos:
EUG as one of the main compounds of Ocimum gratissimum L. In
Conceptualization, Supervision.
particular, the compounds present in essential oils confer characteristics
volatiles, which facilitate their entry into the intracellular environment,
the influx of terpenes, for example, can cause the formation of free Declaration of competing interest
radicals, leading to death (Hua et al., 2018).
The toxicological effect on the negative geotaxis of Drosophila mel­ The authors declare that they have no known competing financial
anogaster was prominent for EUG and EOOg. As a survival strategy in interests or personal relationships that could have appeared to influence
adverse situations, fruit flies increase the activity levels of the enzyme the work reported in this paper.
acetylcholinesterase (AChE), hydrolyzing acetylcholine and preventing
it from accumulating under unfavorable environmental conditions or Data availability
stress (Hu et al., 2019). Thus, the EUG and EOOg acted by inhibiting the
action of AChE, allowing the stress of the flies to impair of their loco­ Data will be made available on request.
motor system.
The results of toxicological activity demonstrate that O. gratissimum Acknowledgments
L. essential oil is slightly more tolerated in the model organism
D. melanogaster than eugenol, as seen in negative geotaxis and mortality This study was funded by the Fundação Cearense de Apoio ao
assays. Previous studies did not mention the toxicity of EOOg using the Desenvolvimento Científico e Tecnológico – FUNCAP; Conselho Nacio­
chosen model, so the results obtained are also unprecedented for the nal de Desenvolvimento Científico e Tecnológico – CNPq.
essential oil. Toxicity tests are important, as the practice of using natural
products and their inputs can be unsafe when overused (Duarte et al.,
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