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European Journal of Integrative Medicine xxx (2014) xxx.e1–xxx.e6

Original article

Antibacterial and modifying-antibiotic activities of the essential oils of

Ocimum gratissimum L. and Plectranthus amboinicus L.
José J.S. Aguiar, Cicera P.B. Sousa, Mariana K.A. Araruna, Maria K.N. Silva, Aline C. Portelo,
Jeferson C. Lopes, Victória R.A. Carvalho, Fernando G. Figueredo, Vanessa C.N. Bitu,
Henrique D.M. Coutinho ∗ , Thiago Adolfo Sobreira Miranda, Edinardo F.F. Matias
Faculdade Leão Sampaio Unidade Saúde, Juazeiro do Norte, Ceará, Brazil
Received 12 August 2014; received in revised form 9 October 2014; accepted 13 October 2014

Abstract
Introduction: Developing resistance to antimicrobial agents is increasingly observed for many microorganisms is increasingly becoming a problem
worldwide. The aim of the present work was to evaluate the antibacterial and antibiotic-modifying activity of essential oils of Ocimum gratissimum
and Plectranthus amboinicus (Lamiaceae), alone and combined.
Methods: Standard and multiresistant bacterial strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were utilized,
and antibiotics of the aminoglycoside class were tested, using the microdilution technique.
Results: The antibacterial effect of the O. gratissimum L. and P. amboinicus L. oils alone and combined have a minimum inhibitory concen-
tration (MIC) ≥ 1024 ␮g/mL, except against E. coli ATCC10536, which showed a MIC = 128 ␮g/mL and against S. aureus ATCC25923 with
MIC = 512 ␮g/mL. The drug-modifying effect of the essential oil of O. gratissimum L. resulted in an antagonism, reducing the effect of antibiotics,
against all bacterial strains assayed. However, the essential oil of P. amboinicus L. showed a synergistic effect, potentiating the antibiotic activity of
these drugs against the bacterial strains assayed. When the mixture of the O. gratissimum and P. amboinicus oils was combined with the antibiotic,
a synergistic effect was observed.
Conclusions: The data obtained are promising, but further studies are needed to isolate the active compounds and to conduct pharmacological
tests in vivo, making it possible to develop new therapeutic alternatives for the treatment of diseases caused by multiresistant microorganisms.
© 2014 Elsevier GmbH. All rights reserved.

Keywords: Ocimum gratissimum; Plectranthus amboinicus; Antibiotic-modifying activity; Antibacterial; Essential oils

Introduction humans and animals. The genus Staphylococcus is commonly

Bacteria are prokaryotic microorganisms that reproduce by
binary fission and characterized by the presence of filamentous
double-strand circular DNA. They are differentiated by their cell
wall, which is used to classify whether they are Gram-positive
or Gram-negative [1]. These microscopic organisms are usually
found on the surface of the skin, mucosae and intestinal tract of
∗
Corresponding author at: Laboratório de Microbiologia e Biologia Molecu-
lar, Av. Cel Antonio Luiz, 1161, CEP: 63105-000, Crato, CE, Brazil.
Tel.: +55 88831021212; fax: +55 8831021291.
E-mail address: hdmcoutinho@gmail.com (H.D.M. Coutinho).
distributed in nature and occurs in the normal microbiota [2]. The
bacteria are usually found as single cells, doublets, or chains and
are classified as important human pathogens, causing diseases
with a fatal prognosis [3].
The bacterial species Escherichia coli is classified as a
microorganism capable of causing severe infections and is asso-
ciated with a variety of diseases, included sepsis, urinary tract
infection (UTI), meningitis and gastroenteritis. On the other
hand, Pseudomonas aeruginosa is a Gram-negative bacillus
with polar flagella, which confer motility, where it causes vari-
ous cutaneous infections, in cases where there are severe burns
and also for example, in endocarditis in immunocompromised
patients [1].

http://dx.doi.org/10.1016/j.eujim.2014.10.005
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Please cite this article in press as: Aguiar JJS, et al. Antibacterial and modifying-antibiotic activities of the essential oils of Ocimum gratissimum
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The ability to develop resistance to antimicrobial agents is
a characteristic observed among microorganisms in general.
However, bacteria are able to develop different mechanisms of
resistance that are genetically coded. Resistant genes can be
acquired through DNA mutation and transfer [4].
With the increase in incidence of resistance to antibiotics,
substances derived from plants may provide a potential alterna-
tive [5,6], given that natural products of plant origin can alter
the action of antimicrobial agents, either by enhancing or reduc-
ing the activity of the drugs [7]. Over recent years, many plants
have been evaluated not only for their antibacterial effects but
also for their antibiotic-modifying activity [8]. Medicinal plants
are therapeutic resources used by traditional cultures for vari-
ous illnesses and conditions. They have pharmacological effects
which have aroused the interest of scientists, with the purpose of
developing studies directed at developing traditional medicines
[9].
For example, accordingly, the family Lamiaceae has a variety
of species commonly utilized in the cosmetic and pharmaceuti-
cal industry [10]. One of these species is Ocimum gratissimum
Lamiaceae, a hardy perennial plant, commonly found all over
Brazil and popularly known as alfavaca. It is utilized in the
treatment of illnesses that affect the airways, and is character-
ized by its high percentage of eugenol [11]. Pharmacological
assays have shown antioxidant, anticonvulsant, anesthetic,
dental analgesic, fungicidal [12], and antimicrobial activities
[13].
Plectranthus amboinicus L. is another representative of the
family Lamiaceae with aromatic characteristics, readily found in
Brazil [14]. In the Northeast of Brazil, the Chapada do Araripe,
the local community utilizes “hortelã gorda,” as it is popularly
called, in a home remedy syrup for treating respiratory ailments
[15]. This species has a scientific synonymy: Coleus amboinicus
Lour or Coleus aromaticus Benth [16] and is routinely used in
culinary services, and as a phytotherapeutic product and as pri-
mary matter in the pharmaceutical industry [10]. Its constituents
identified by chemical and phytochemical assays, include ger-
macrene, thymol and carvacrol, have excellent antimicrobial
[17], and anti-inflammatory [15] activities.
The aim of the present study was to evaluate the antibacterial
and antibiotic-modifying activity of essential oils of O. gratis-
simum Lamiaceae and P. amboinicus Lamiaceae, alone and
combined against standard and multiresistant bacterial strains.
Materials and methods
Bacteriological material
The strains bacterial utilized in the present study were strains
of S. aureus (SA-ATCC25923 and SA358), E. coli (EC-ATCC
10536 and EC 27) and P. aeruginosa (PA-ATCC15442 and
PA03) with the resistance profile identified in Table 4. All strains
were maintained on slants with heart infusion agar (HIA, Difco
Laboratories LTDA). Before the assay, the cells were grown for
24 h at 37 ◦ C in brain heart infusion broth (BHI, Difco Labora-
tories LTDA).
Please cite this article in press as: Aguiar JJS, et al. Antibacterial and modifying-antibiotic activities of the essential oils of Ocimum gratissimum
L. and Plectranthus amboinicus L. Eur J Integr Med (2014), http://dx.doi.org/10.1016/j.eujim.2014.10.005
Plant material
The leaves of O. gratissimum L. and P. amboinicus L. were
collected around 08:00 h, in the garden of medicinal plants
of Universidade Regional do Cariri – URCA, located in the
municipality of Crato-Ceará, Brazil, geographic coordinates of
7◦ 13 46 S, 39◦ 24 32 W. A dried specimen was deposited in
the Herbario Caririense Dardano de Andrade Lima, of Univer-
sidade Regional do Cariri – URCA, under Nos. 3978 and 26433,
respectively.
Extraction of oils and preparation of solutions and
antibacterial tests
The leaves of O. gratissimum L. and P. amboinicus L. were
triturated, each made into 400 g amounts, placed separately in
glass flasks, submersed in distilled water and boiled for 2 h in a
Clevenger type oil extractor [18]. The water/oil mixture obtained
of each plant was separated, treated with anhydrous sodium sul-
fate and filtered. The oils were prepared at a concentration of
10 mg/mL, dissolved in DMSO (dimethyl sulfoxide), and then
diluted with distilled water to a concentration of 1024 ␮g/mL
(10% of DMSO) [7].
Phytochemical characterization
The identification of the classes of secondary metabolites
in the essential oil of O. gratissimum L. and P. amboinicus
L. was done using a ShimadzuQP2010 series GC/MS system
equipped with an Rtx-5MS capillary column (30 m × 0.25 mm
i.d., 0.25 ␮m film thickness); helium was the carrier gas at
1.5 mL/min, flow rate was 0.8 mL/min, using split mode. The
injector and detector temperatures were 250 and 200 ◦ C, respec-
tively. The column temperature was programmed from 35 to
180 ◦ C at 4 ◦ C/min and then 180 to 250 ◦ C at 10 ◦ C/min. The
mass spectrometer was operated at an ionization energy of 70 eV.
The identification of individual components was based on their
fragmentations in the NIST 8mass spectral library and through
comparison with the literature data [19].
Antibacterial activity tests
MIC (minimum inhibitory concentration) was determined
using a microdilution assay in broth medium [20], with an inocu-
lum of 100 ␮L of each strain in 96-well microtiter plates. The cell
suspension was in BHI broth and had a starting concentration of
105 CFU/mL, with 2-fold serial dilutions. Solutions of samples
and of their respective combinations (100 ␮L) were added to
each well. The final concentrations varied from 8 to 512 ␮g/mL.
The controls were with the standard antibiotics amikacin and
gentamicin, whose final concentrations varied 2500–2.4 ␮g/mL.
The plates were incubated at 35 ◦ C for 24 h. Afterwards, viable
cells were stained with resazurin and the color change was deter-
mined [21]. The MICs were recorded as the lowest concentration
needed to inhibit growth.
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Table 1 Table 2
Origin of bacterial strains and antibiotic resistance profile. Chemical composition (%) of essential oil of Ocimum gratissimum L.
Bacterial strain Origin Resistance Component RT (min)a (%)
profile
␤-Pinene 3.58 1.59
Staphylococcus aureus SA358 Surgical Oxa, Gen, ␤-Ocimene 4.09 4.47
wound Tob, Ami, p-Cineol 4.15 15.17
Can, Neo, ␤-Linalol 4.85 0.37
Para, But, Sis, ␣-Terpineol 6.15 0.73
Net Eugenol 8.42 65.26
Staphylococcus aureus ATCC 25923 ␤-Elemene 8.90 0.66
Escherichia coli EC27 Surgical Ast, Ax, Amp, (E)-Caryophyllene 9.37 3.04
wound Ami, Amox, ␣-Humulene 9.82 0.47
Ca, Cfc, Cf, Germacrene-D 10.17 1.91
Caz, Cip, Clo, ␤-Selinene 10.26 4.40
Im, Can, Szt, ␥-Gurjunene 10.37 1.57
Tet, Tob
Total 99.64
Escherichia coli ATCC 10536
Pseudomonas aeruginosa PA03 Cathetertip Cpm, Ctz, Im, a Retention time.
Cip, Ptz, Lev,
Mer, Ami
Pseudomonas aeruginosa ATCC 15442 et al. [23] reports that variation of yield of oil essential and
concentration of constituents can be caused by various environ-
Ast, aztreonan; Ax, amoxacillin; Amp, ampicillin; Ami, amikacin; Amox,
amoxillin, Ca, cefadroxil; Cfc, cefaclor; Cf, cephalothin; Caz, ceftazidime;
mental factors.
Cip, ciproflaxacin; Clo, chloramphenicol; Im, imipenem; Can, kanamycin; Szt, Table 3 shows the MIC of samples tested against standard
sulfamethotrim, Tet, tetracycline; Tob, tobramycin; Oxa, oxacillin; Gen, genta- and multiresistant strains of E. coli, S. aureus and P. aeruginosa.
micin; Neo, neomycin; Para, paramomycin; But, butirosin; Sis, sisomicin; Net, Comparatively, the essential oils separate and combined showed
Netilmicin; (–), absence of resistance or non-significant resistance. the same MIC with the exception of EOPA and its combina-
tion with EOOG against EC-ATCC 10536, which produced
Determination of antibiotic resistance-modifying activity a better antibacterial activity with MIC of 128 ␮g/However,
when EOOG was tested against SA-ATCC 25923, the MIC was
Antibiotic-modifying activity was determined by the method 512 ␮g/mL.
described by Coutinho et al. [7], where the solutions of essential Figs. 1 and 2 show the results of evaluating the modulatory
oils and their combinations were tested at sub-inhibitory concen- activity of oils when combined with amikacin and gentami-
trations (MIC/8). A volume of 100 ␮L of the solution containing cin. The results demonstrated that the combination of EOPA
10% BHI, inoculum and sample was added to each well in the with aminoglycosides had a synergistic effect against all strains
alphabetic direction of the plate. Next, 100 ␮L of the antibiotic tested with significance (p < 0.001). The combinations of EOOG
solution were added to the first well and serially diluted on 2- with aminoglycosides demonstrated antagonism, with excep-
fold scale on all wells, excepted the last one. The concentrations tion of the combination with gentamicin against PA03 and with
of aminoglycosides varied gradually from 2500 to 2.44 ␮g/mL. amikacin against EC 27, however without significance. But the
The plates were incubated at 35 ◦ C for 24 h, then the viable cells simultaneous combination of EOPA and EOOG with the antibi-
were determined by resazurin staining. otics revealed that the antagonistic effect with EOOG prevailed
over the effect of EOPA, causing interference capable of lower-
Statistical analysis ing its synergistic effect.

The results of tests were obtained in triplicate and expressed Table 3

as geometric mean. Statistical analysis was carried out using Chemical composition (%) of essential oil of P. amboinicus L.
ANOVA followed by Bonferroni post-tests, utilizing GraphPad Component RT (min)a (%)
Prism 6.0 [22], and p < 0.05 was considered significant. Copaene 8.73 8.03
α-Amorphene 8.90 4.84
Results (E)-Caryophyllene 9.37 18.91
␣-Humulene 9.82 1.74
α-Curcumene 10.05 0.89
The yield of essential oil from O. gratissimum L. was 0.45%, Germacrene-D 10.17 38.60
compatible with the finding of Vieira et al. [18], The chromato- ␤-Bisabolene 10.38 4.04
graphic analysis showed that the major constituent was eugenol ␦-Cadinene 10.64 3.16
(65%)(Table 1), these findings are in accordance with the study trans-Nerolidol 11.03 6.29
Caryophyllene oxide 11.55 2.13
by Sartoratto et al. [12]. On the other hand, P. amboinicus L.
Zingiberene 15.70 2.49
yielded 0.15% essential oil, which 38.60% was germacrene-D
as the major secondary metabolite (Table 2), a higher percentage Total 91.12
than that described by Bandeira et al. [17]. However, Nogueira a Retention time.

Please cite this article in press as: Aguiar JJS, et al. Antibacterial and modifying-antibiotic activities of the essential oils of Ocimum gratissimum
L. and Plectranthus amboinicus L. Eur J Integr Med (2014), http://dx.doi.org/10.1016/j.eujim.2014.10.005
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Fig. 1. Comparative analysis of essential oils alone and combined with amikacin.

Fig. 2. Comparative analysis of essential oils alone and combined with gentamicin.

Discussion [13,29]. Germacrene-D is the major terpene in the essential oils

Natural products from several plants have demonstrated an
antibacterial potential against several bacteria using different
methods, gaseous or direct contact as Porella arboris-vitae,
Cymbopogon citratus and Croton zehntneri [24–26].
The synergistic activity observed can be due to terpenoids
present in EOPA that are synthesized by plants in response to
microbial infections [27,28], where they are able to alter the
cell wall or disrupt the cell membrane facilitating drug uptake
in study, with proven antimicrobial activity [30].
Harvey et al. [31] reported that aminoglycosides are used
in the treatment of enteric infections caused by Gram-negative
bacteria and against sepsis and that their indiscriminate use is
one of the causes of bacterial resistance. They also noted that
high or frequent doses of aminoglycosides can cause irreversible
adverse toxicity, such as ototoxic and/or nephrotoxic reactions.
Accordingly, the combination of aminoglycosides with EOPA
can be a natural medicine alternative against multi-resistant

Table 4
Minimum inhibitory concentration (MIC) of essential oils of Ocimum gratissimum and Plectranthus amboinicus (␮g/mL).
Essential oil EC27 EC-ATCC 10536 SA358 SA-ATCC 25923 PA03 PA-ATCC 15442

EOOG ≥1024 ≥1024 ≥1024 512 ≥1024 ≥1024

EOPA ≥1024 128 ≥1024 ≥1024 ≥1024 ≥1024
EOOG + EOPA ≥1024 128 ≥1024 ≥1024 ≥1024 ≥1024

EOOG, essential oil of Ocimum gratissimum; EOOG, essential oil of Plectranthus amboinicus; EC, Escherichia coli; SA, Staphylococcus aureus; PA, Pseudomonas
aeruginosa.

Please cite this article in press as: Aguiar JJS, et al. Antibacterial and modifying-antibiotic activities of the essential oils of Ocimum gratissimum
L. and Plectranthus amboinicus L. Eur J Integr Med (2014), http://dx.doi.org/10.1016/j.eujim.2014.10.005
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bacteria, since it lowers the necessary dose at which the amino-
glycoside is therapeutically effective, thereby diminishing the
toxicity of antibiotics.
The evident antagonistic action observed with EOOG can be
explained by the chelating property of secondary metabolites
such as eugenol in the essential oil in study, capable of causing
mutual chelation. This effect possibly accounts for the reduc-
tion in the activity of antibiotics in the presence of EOOG [32].
However, these findings are at odds with the results of Escobar
[33] who described the major constituent eugenol as a phenyl-
propanoid compound with substantial antibacterial activity.
The observations of Oliveira et al. [34] indicated the inter-
ference of essential oils with antibiotics, but their study
demonstrated an antagonistic effect in some combinations of
essential oils and aminoglycosides. This characteristic of essen-
tial oils varies depends on the type of antibiotic, of essential oil
tested in combination and on the type of bacterial strain assayed.
Conclusions
On the basis of the results obtained, we conclude that EOPA
when combined with aminoglycosides has a synergistic effect
in antibiotic toxicity toward resistant bacterial strains, where
EOPA is thereby a possible source of product natural with bac-
terial resistance-modifying activity. However, the combination
of antibiotics with EOOG resulted in an antagonistic effect,
decreasing the antimicrobial activity of aminoglycosides. Fur-
ther studies are needed to isolate the active compounds and
develop new therapeutic alternatives for the treatment of diseases
caused by multiresistant microorganisms.
Authors’ contribution
JJSA, CPBS and MKAA performed the antimicrobial assays;
MKNS, ACP, JCP and VRAC performed the modulatory assays;
FGF and VCNB performed the GC–MS analysis; HDMC and
EFFM supervised the work and wrote the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors are thankful for the support and cooperation
from the Faculdades Leão Sampaio FALS-CE, URCA/LPPN/
LMBM/LFQM Universidade Regional do Cariri-CE/
Laboratory of Regional Natural Products Research/Laboratory
of Microbiology and Molecular Biology. Dr. A. Leyva helped
with English translation and editing of the manuscript.
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