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Revista Brasileira de Ciências Farmacêuticas
Brazilian Journal of Pharmaceutical Sciences
vol. 43, n. 1, jan./mar., 2007
Inhibitory effect of ββ
ββ
β-pinene, αα
αα
α-pinene and eugenol on the growth of
potential infectious endocarditis causing Gram-positive bacteria
Aristides Medeiros Leite1, Edeltrudes de Oliveira Lima2 , Evandro Leite de Souza3, Margareth de
Fátima Formiga Melo Diniz1, Vinícius Nogueira Trajano2, Isac Almeida de Medeiros1
1Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, João Pessoa, 2Laboratório de Micologia,
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal da Paraíba,
João Pessoa, 3Departamento de Nutrição, Centro de Ciências da Saúde, Universidade Federal da Paraíba,
João Pessoa
This study was led with the purpose of evaluating the effectiveness
of eugenol, β-pinene and α-pinene in inhibiting the growth of
potential infectious endocarditis causing gram-positive bacteria.
The phytochemicals Minimum Inhibitory Concentration-MIC was
determined by solid medium diffusion procedure, while the
interference of the MIC values on the bacterial cell viability was
performed by viable cells count. Staphylococcus aureus, S.
epidermidis, Streptococcus pneumoniae and S. pyogenes strains were
used as test microorganisms. The assayed phytochemicals showed
effectiveness in inhibiting all assayed bacteria strains presenting
MIC values between 2.5 and 40 μL/mL. Eugenol showed the lowest
MIC values which were between 2.5 and 5 μL/mL for the most
bacteria strains. MIC values found to the phytochemicals were
able to inhibit the cell viability of S. aureus providing a total
elimination of the bacteria inoculum in a maximum time of 24 hours
of exposure. These data showed the interesting antibacterial
property of the assayed phytochemicals and support their possible
and rational use in the antimicrobial therapy.
Uniterms
• Endocarditis
• Gram positive bacteria
• Phytochemicals
• Antibacterial activity
*Correspondence:
E.L. Souza
Rua Radialista Antônio Assunção de
Jesus, 680/102,
58052-230 - João Pessoa - PB, Brasil
E-mail: evandroleitesouza@ccs.ufpb.br
INTRODUCTION
Infectious endocarditis (IE) is an infectious disease
attacking mainly the heart valves endocardium. Most ca-
ses of IE has been related to infections caused by
Streptococcus and Staphylococcus genus, although
bacteria inserted in the HACEK group (Haemophillus
parainfluenzae, H. aphrophilus, H. paraphrophilus, H.
influenzae, Actinobacillus actinomycetemcomitans,
Cardiobaccterium hominis, Eikenella corrodens,
Kingela kingae and K. denitrificans) have presented an
emerging importance as IE causing agents (Baddour et
al., 2005; Bashore et al., 2006). Fungi can act as IE
etiological agents in immunosuppressed people and/or in
catheter and valve prosthesis users (Niwa et al., 2003;
Watanabe et al., 2003; Anguera et al., 2005).
A. M. Leite, E. O. Lima, E. L. Souza, M. F. F. M. Diniz, V. N. Trajano, I. A. Medeiros122
Even regarding the advances in the clinical therapy the
IE still presents a high mortality rate (20% to 30% of the
cases). It has been related to the development of antibiotic
resistance by IE causing agents, mainly when regarded the
Staphylococcus genus because the appearance of methicillin-
resistant Staphylococcus aureus (MRSA) (Nakatani et al.,
2003; Ishiwada et al., 2005). Some researchers have
reported that the rising of MRSA strains has been responsible
for an increase in the action of Staphylococcus genus as IE
causing agent. On the other hand, the participation of
Streptococcus genus in IE etiology has decreased (Ako et al.,
2003; Pigrau et al., 2005; Ferreiros et al., 2006).
Currently there has been an increasing interest in
studying the biological properties of plant and derivatives
in order to discover alternative biologically active
compounds (Seidil, 2000; Araújo et al., 2004; Lima et al.,
2005). Plant products have received a renewed interest in
their use as alternative source of antimicrobial
compounds because the uncontrolled use of the
antimicrobial chemotherapy (Dorman, Deans, 2000). The
successful history of microbial chemocontrol lies in the
continuous rising of microbial strains with resistance to
the classical antibiotics (Notermans, Hoogenboon-
Verdegaal, 1992). Researches regarding the antimicrobial
activity of phytochemicals have been yet little emphasized
being the most studies addressed to evaluate the
antimicrobial effect of essential oils and extracts (Nasci-
mento et al., 2000; Souza et al., 2005).
This study aimed to evaluate the effectiveness of
eugenol, β-pinene and α-pinene in inhibiting the growth of
Gram-positive bacteria known as potential IE etiological
agents.
MATERIAL AND METHODS
Phytochemicals
Eugenol, α-pinene and β-pinene were supplied by
Department of Pharmaceutical Sciences, State University
of Ponta Grossa, Paraná, Brazil. The phytochemicals were
tested at concentrations ranging from 160 to 1.25 μL/mL,
and the solutions were prepared prior to the antimicrobial
assays according to Souza et al. (2007). Molecular
structures of the phytochemicals are shown in Figure 1.
Bacteria strains
Staphylococcus aureus ATCC 13150, S. aureus ATCC
6538, S. aureus ATCC 25932, S. aureus ATCC LB 126,
S. epidermidis SSI 1, S. epidermidis ATCC 12228,
Streptococcus pyogenes ATCC 19615, Streptococcus
pyogenes ATCC 8668 and S. pneumoniae ATCC 11773 were
used as test microorganisms. These strains were supplied by
Laboratory of Clinical Bacteriology, Department of
Pharmaceutical Sciences, Health Sciences Center, Federal
University of Paraíba, Brazil. Stock cultures were maintained
on nutrient agar slants at 4 °C. Inocula used in antimicrobial
assays were obtained from overnight cultures grown on
nutrient agar slants at 37 °C and diluted in sterile saline
solution (0.85 % w/v) to have a final concentration of 106
colony forming unity (cfu)/mL (adjusted according to the
turbidity of 0.5 McFarland scale tube).
Determination of the Minimum Inhibitory
Concentration
Microplate bioassay was used to determine the
Minimum inhibitory concentration - MIC of the assayed
phytochemicals. For this was used a microplate with 96
wells (flatted bottom) and cap. The 96-well microplates
were prepared dispensing into each well 100 μL of doubled
strength nutrient broth inoculated with 1 mL of the
bacterium inoculum prior the assay (1:9 v/v). 100 μL of the
phytochemical solution with their respective concentration
was transferred into seven consecutive wells. Final volume
in each well was 200 μL. The solution having the highest
concentration was added into the first well and the lowest
concentration was added into the penultimate well. The last
well contained 200 μL of nutrient broth inoculated with the
bacterium inoculum was used as positive control (strain
viability). The microplate was asseptically sealed, followed
by mixing on plate shaker (300 rpm) for 30 s, and incubated
at 28 – 30 °C/48 h (Viljoen et al., 2003; Sahin et al., 2004).
Antibacterial activity was detected using a colorimetric
method by adding 200 μL of resauzurin staining (0.1 %)
aqueous solution in each well at the end of the incubation
period. MIC was defined as the lowest phytochemical
concentration able to inhibit the bacteria growth, as
FIGURE 1 - Molecular structures of phytochemicals used
in the antimicrobial assays.
O
OH
CH
3
α-pinene β-pineneeugenol
Inhibitory effect of β-pinene, α-pinene and eugenol 123
indicated by resauzurin staining (bacteria died cells are not
able to change the staining color by visual observation –
blue to red) (Palomino et al., 2002; Burt, Reinders, 2003).
Kill time study
Kill time study was carried out with MIC values
previously found in the microplate bioassay. For this was
used the viable cells count method. 5mL of double strength
nutrient broth was inoculated with 1mL of the bacterium
suspension (approximately 106 cfu/mL). After that, 4mL of
the phytochemical solution, with concentration adjusted to
provide a phytochemical final concentration similar to the
MIC previously determined, was added to the system and
followed by shaking for 30 s using Vortex. The system was
incubated at 37 °C. At different time intervals (1, 2, 4, 8,
12 and 24 hs) of exposure, 1mL of the suspension was
serially diluted (10-1 – 10-5) in sterile peptone water
(0.1% w/v) and inoculated on nutrient agar Petri dishes for
24 h at 37 °C (Viljoen et al., 2003; Souza et al., 2007). The
mean number of colonies (cfu/mL) was counted and
compared with that found in the control assay in which the
essential oil solution was replaced by sterile distilled water.
The results were expressed in log of cfu/mL.
Statistical analysis
Statistical analysis was performed to determine
significant differences (P<0.05) by Tukey test in the
bacteria kill time assays. For this was used Sigma stat 2.03
computer program.
RESULTS AND DISCUSSION
MIC values found for eugenol, α-pinene and β-pinene
are shown in Table 1. The assayed phytochemicals presented
interesting antimicrobial potential noted by low MIC values.
MIC values oscillated between 2.5 (eugenol x S. pneumoniae
ATCC 11773) to 40 μL/mL (β-pinene x S. epidermidis
ATCC 12228). All assayed bacteria strains were sensitive to
the phytochemicals. On the other hand, some strains
presented resistant behavior to the standard antibiotics,
mainly to gentamicin. Eugenol provided the most intense
antibacterial effect showing MIC values between 2.5 to
5 μL/mL for the most bacteria strains. S. aureus presented
as the most resistant bacterium with MIC values between 10
and 20 μL/mL for the tested phytochemicals. For the other
bacteria species were found MIC values oscillating between
2.5 to 5 μL/mL for most interactions.
TABLE I - MIC of some phytochemicals on potential infectious endocarditis causing gram-positive bacteria
Bacteria Phytochemicals (μL/mL) Penicillin Gentamicin
Eugenol β-pinene α-pinene (10 μg/mL) (10 μg/mL)
S. aureus 10 20 20 S R
ATCC 13150
S. aureus 10 20 20 S S
ATCC 6538
S. aureus 10 20 20 S S
ATCC 25923
S. aureus 10 20 10 S S
LB 126
S. epidermidis 520 5 SS
SSI 1
S. epidermidis 540 5 SS
ATCC 12228
S. pyogenes 520 5 SR
ATCC 19615
S. pyogenes 520 10 SR
ATCC 8668
S. pneumoniae 2.5 20 5 R S
ATCC 11773
S: sensitive; R: resistant
A. M. Leite, E. O. Lima, E. L. Souza, M. F. F. M. Diniz, V. N. Trajano, I. A. Medeiros124
Figure 2, 3 and 4 show the effect of eugenol,
β-pinene and α-pinene MIC values on the S. aureus
ATCC 6538 cell viability. S. aureus was chosen to be
included in the kill time assays because it is known as the
main etiological agent of IE acquired at the community or
in nosocomial environment (Niwa et al., 2003; Ishiwada
et al., 2005). Phytochemicals MIC values were able to
cause a significant (p<0.05) inhibition of S. aureus cell
viability providing a bactericidal effect in a maximum
time of 24 hours of exposure. Most intense inhibitory
effect was showed by β-pinene and eugenol causing a total
elimination of the initial bacterial inoculum after 8 hours
of exposure. β-Pinene and eugenol presented a
bacteriostatic effect until 2 hours of exposure and from
this time on it was established a progressive decrease in
the bacteria cell count. α-pinene provided a bacteriostatic
effect until 4 hours of exposure being established its cidal
effect after 24 hours of exposure.
Some researches have found an antimicrobial
effectiveness in phytochemicals, although these data still
are considered as incipient when compared to the
availability of researches regarding the antimicrobial
properties of plant extracts and essential oils (Vasquez et
al., 2001; Nostro et al., 2004). Gayoso et al. (2004) and
Lima et al. (2005) found antimicrobial effectiveness of
pinenes against moulds and pathogen yeasts providing a
cidal effect between 2 and 5 hours of exposure. Hao et al.
(1998) reported antimicrobial activity of eugenol on Gram
positive bacteria, including S. aureus strains.
Phytochemicals are small organic biomolecules
generally hydrophobic and designated as naturally
occurring antibiotics. Cytoplasm membrane coagulation,
breakdown of protons motive force, breakdown of electron
flux and active transport unbalance are some events
responsible for providing the antimicrobial property of
phytochemicals (Sikkema, 1995; Carlson et al., 2002).
These biological events have been believed for no occurring
separately being some of them activated as consequence of
other ones (Sikkema, 1995; Burt, 2004).
Our data show the intense antimicrobial potential
of eugenol, β-pinene and α-pinene which were able to
inhibit significantly the growth and cell viability of
potential infectious endocarditis causing gram-positive
bacteria. These results support the recognizing of
phytochemicals as alternative antimicrobial compounds
to be used in pharmaceutical formulations used in the
antibacterial therapy. Further researches are needed to
FIGURE 3 - Effect of β-pinene (20 μL/mL) on the S. aureus
ATCC 6538 viable cells number.
0
2
4
6
8
10
12481224
β-pinene
control
hours
Log cfu/mL
FIGURE 2 - Effect of eugenol (10 μL/mL) on the S. aureus
ATCC 6538 viable cells number.
0
2
4
6
8
10
1 2 4 8 12 24
eugenol
control
hours
Log cfu/mL
FIGURE 4 - Effect of α-pinene (20 μL/mL) on the S.
aureus ATCC 6538 viable cells number.
0
2
4
6
8
10
12481224
α-pinene
control
hours
Log cfu/mL
Inhibitory effect of β-pinene, α-pinene and eugenol 125
evaluate their antimicrobial effectiveness against
pathogen microorganisms able to act as etiological
agents of different infections diseases, as well as
regarding their toxicological and pharmacological
aspects.
RESUMO
Efeito inibitório de eugenol, β-pineno e α-pineno
sobre o crescimento de bactérias Gram-positivas
potencialmente causadoras de endocardite infecciosa
Este estudo foi conduzido com a proposta de avaliar a
efetividade de eugenol, β-pineno e α-pineno em inibir o
crescimento de cepas de bactérias Gram-positivas poten-
cialmente causadoras de endocardite infecciosa. A Con-
centração Inibitória Mínima-CIM dos fitoconstituintes foi
determinada através do método de difusão em meio sóli-
do, enquanto a interferência da CIM sobre a viabilidade
celular bacteriana foi avaliada através da contagem de
células viáveis. Cepas de Staphylococcus aureus, S.
epidermidis, Streptococcus pneumoniae e S. pyogenes fo-
ram utilizadas como microrganismos teste nos ensaios
antimicrobianos. Os fitoconstituintes ensaiados mostra-
ram efetividade em inibir todas as cepas bacterianas uti-
lizadas como microrganismos testes apresentando valores
de CIM entre 2.5 e 40 μL/mL. Eugenol apresentou os
menores valores de CIM, os quais estiveram entre 2,5 e
5 μL/mL para a maioria das cepas bacterianas. Os valo-
res de CIM encontrados para os fitoconstituintes foram
capazes de inibir a viabilidade celular de S. aureus cau-
sando uma total eliminação do inóculo bacteriano em um
tempo máximo de 24 horas de exposição. Estes dados
mostram o intenso potencial antibacteriano dos
fitoconstituintes ensaiados e suportam sua possível e ra-
cional aplicação na terapia antimicrobiana.
UNITERMOS: Endocardite. Bactérias Gram-positivas.
Fitoconstituintes. Atividade antibacteriana.
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Recebido para publicação em 09 de agosto de 2006.
Aceito para publicação em 28 de fevereiro de 2007.
