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Antibacterial and modifying-antibiotic activities of the essential oils of Ocimum gratissimum L. and Plectranthus amboinicus L.
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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 concentration (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 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 Molecular, Av. Cel Antonio Luiz, 1161, CEP: 63105-000, Crato, CE, Brazil. Tel.: +55 88831021212; fax: +55 8831021291. E-mail address: [email protected] (H.D.M. Coutinho).
humans and animals. The genus Staphylococcus is commonly 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 associated 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 various cutaneous infections, in cases where there are severe burns and also for example, in endocarditis in immunocompromised patients [1].
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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 alternative [5,6], given that natural products of plant origin can alter the action of antimicrobial agents, either by enhancing or reducing 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 various 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 pharmaceutical 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 characterized 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 primary matter in the pharmaceutical industry [10]. Its constituents identified by chemical and phytochemical assays, include germacrene, 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. gratissimum 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 Laboratories LTDA).
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 Universidade 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 sulfate 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, respectively. 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 inoculum 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 determined [21]. The MICs were recorded as the lowest concentration needed to inhibit growth.
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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J.J.S. Aguiar et al. / European Journal of Integrative Medicine xxx (2014) xxx.e1–xxx.e6 Table 1 Origin of bacterial strains and antibiotic resistance profile.
Table 2 Chemical composition (%) of essential oil of Ocimum gratissimum L.
Bacterial strain
Origin
Resistance profile
Staphylococcus aureus SA358
Surgical wound
Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net
Surgical wound
Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Can, Szt, Tet, Tob
Staphylococcus aureus ATCC 25923 Escherichia coli EC27
Escherichia coli ATCC 10536 Pseudomonas aeruginosa PA03
Cathetertip
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Cpm, Ctz, Im, Cip, Ptz, Lev, Mer, Ami
Pseudomonas aeruginosa ATCC 15442 Ast, aztreonan; Ax, amoxacillin; Amp, ampicillin; Ami, amikacin; Amox, amoxillin, Ca, cefadroxil; Cfc, cefaclor; Cf, cephalothin; Caz, ceftazidime; Cip, ciproflaxacin; Clo, chloramphenicol; Im, imipenem; Can, kanamycin; Szt, sulfamethotrim, Tet, tetracycline; Tob, tobramycin; Oxa, oxacillin; Gen, gentamicin; Neo, neomycin; Para, paramomycin; But, butirosin; Sis, sisomicin; Net, Netilmicin; (–), absence of resistance or non-significant resistance.
Determination of antibiotic resistance-modifying activity Antibiotic-modifying activity was determined by the method described by Coutinho et al. [7], where the solutions of essential oils and their combinations were tested at sub-inhibitory concentrations (MIC/8). A volume of 100 L of the solution containing 10% BHI, inoculum and sample was added to each well in the alphabetic direction of the plate. Next, 100 L of the antibiotic solution were added to the first well and serially diluted on 2fold scale on all wells, excepted the last one. The concentrations of aminoglycosides varied gradually from 2500 to 2.44 g/mL. The plates were incubated at 35 ◦ C for 24 h, then the viable cells were determined by resazurin staining. Statistical analysis The results of tests were obtained in triplicate and expressed as geometric mean. Statistical analysis was carried out using ANOVA followed by Bonferroni post-tests, utilizing GraphPad Prism 6.0 [22], and p < 0.05 was considered significant. Results The yield of essential oil from O. gratissimum L. was 0.45%, compatible with the finding of Vieira et al. [18], The chromatographic analysis showed that the major constituent was eugenol (65%)(Table 1), these findings are in accordance with the study by Sartoratto et al. [12]. On the other hand, P. amboinicus L. yielded 0.15% essential oil, which 38.60% was germacrene-D as the major secondary metabolite (Table 2), a higher percentage than that described by Bandeira et al. [17]. However, Nogueira
Component
RT (min)a
(%)
-Pinene -Ocimene p-Cineol -Linalol ␣-Terpineol Eugenol -Elemene (E)-Caryophyllene ␣-Humulene Germacrene-D -Selinene ␥-Gurjunene
3.58 4.09 4.15 4.85 6.15 8.42 8.90 9.37 9.82 10.17 10.26 10.37
1.59 4.47 15.17 0.37 0.73 65.26 0.66 3.04 0.47 1.91 4.40 1.57
Total a
99.64
Retention time.
et al. [23] reports that variation of yield of oil essential and concentration of constituents can be caused by various environmental factors. Table 3 shows the MIC of samples tested against standard and multiresistant strains of E. coli, S. aureus and P. aeruginosa. Comparatively, the essential oils separate and combined showed the same MIC with the exception of EOPA and its combination with EOOG against EC-ATCC 10536, which produced a better antibacterial activity with MIC of 128 g/However, when EOOG was tested against SA-ATCC 25923, the MIC was 512 g/mL. Figs. 1 and 2 show the results of evaluating the modulatory activity of oils when combined with amikacin and gentamicin. The results demonstrated that the combination of EOPA with aminoglycosides had a synergistic effect against all strains tested with significance (p < 0.001). The combinations of EOOG with aminoglycosides demonstrated antagonism, with exception of the combination with gentamicin against PA03 and with amikacin against EC 27, however without significance. But the simultaneous combination of EOPA and EOOG with the antibiotics revealed that the antagonistic effect with EOOG prevailed over the effect of EOPA, causing interference capable of lowering its synergistic effect. Table 3 Chemical composition (%) of essential oil of P. amboinicus L. Component
RT (min)a
(%)
Copaene α-Amorphene (E)-Caryophyllene ␣-Humulene α-Curcumene Germacrene-D -Bisabolene ␦-Cadinene trans-Nerolidol Caryophyllene oxide Zingiberene
8.73 8.90 9.37 9.82 10.05 10.17 10.38 10.64 11.03 11.55 15.70
8.03 4.84 18.91 1.74 0.89 38.60 4.04 3.16 6.29 2.13 2.49
Total a
91.12
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 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
[13,29]. Germacrene-D is the major terpene in the essential oils 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 EOPA EOOG + EOPA
≥1024 ≥1024 ≥1024
≥1024 128 128
≥1024 ≥1024 ≥1024
512 ≥1024 ≥1024
≥1024 ≥1024 ≥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.
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bacteria, since it lowers the necessary dose at which the aminoglycoside 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 reduction 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 phenylpropanoid compound with substantial antibacterial activity. The observations of Oliveira et al. [34] indicated the interference of essential oils with antibiotics, but their study demonstrated an antagonistic effect in some combinations of essential oils and aminoglycosides. This characteristic of essential oils varies depends on the type of antibiotic, of essential oil tested in combination and on the type of bacterial strain assayed.
[3] [4]
[5]
[6]
[7]
[8] [9]
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 bacterial resistance-modifying activity. However, the combination of antibiotics with EOOG resulted in an antagonistic effect, decreasing the antimicrobial activity of aminoglycosides. Further studies are needed to isolate the active compounds and develop new therapeutic alternatives for the treatment of diseases caused by multiresistant microorganisms.
[10]
[11]
[12]
[13]
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.
[14]
[15] [16]
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.
[17]
[18]
[19]
[20]
[21]
References [1] Murray PR. Microbiologia médica. 4a ed. Rio de Janeiro: Guanabara Koogan; 2004. [2] Coutinho HDM, Costa JGM, Lima EO, Falcão-Silva VS, SiqueiraJr JP. Herbal therapy associated with antibiotic therapy: potentiation
[22]
xxx.e5
of the antibiotic activity against methicillin-resistant Staphylococcus aureus by Turnera ulmifolia L. BMC Complement Altern Med 2009;9: 1–13. Vermelho AB, Bastos MCF, Branquinha MHS. Bacteriologia Geral. 1a ed. Rio de Janeiro: Guanabara Koogan; 2007. Carneiro JCO (MSc thesis) Padrão de consumo de antibacterianos em uma UTI geral: correlac¸ão com a resistência bacteriana. Universidade de Brasília; 2006. Oliveira FQ, Gobira B, Guimarães C, Batista J, Barreto M, Souza M. Espécies vegetais indicadas na odontologia. Rev Bras Farmacogn 2007;17:466–76. Silva JG, Souza IA, Higino JS, Siqueira-Junior JP, Pereira JV, Pereira MSV. Atividade antimicrobiana do extrato de Anacardium occidentale Linn em amostras multiresistentes de Staphylococcus aureus. Rev Bras Farmacogn 2007;17:572–7. Coutinho HDM, Costa JGM, Siqueira JR, Lima JPEO. In vitro antistaphylococcal activity of Hyptis martiusii Benth against methicillin resistant Staphylococcus aureus-MRSA strains. Braz J Pharmacogn 2008;18:670–5. Simon G. Anti-staphylococcal plant natural products. Nat Prod Rep 2004;21:263–77. Alvim NATI, Ferreira MA, Cabral IE, Filho AJA. O uso de plantas medicinais como recurso terapêutico: das influências da formac¸ão profissional às implicac¸ões éticas e legais de sua aplicabilidade como extenc¸ão da prática de cuidar realizada pela Enfermeira. Rev Latino Am Enfermagem 2006;14:3–10. Sousa VC, Lorenzi H. Botânica sistemática Guia ilustrado para identificac¸ão das familias de angiospermas da flora brasileira. 3a ed. Nova Odessa: Instituto Plantarum; 2005. Pereira RS, Sumita TC, Furlan MR, Jorge AOC, Ueno M. Atividade antibacteriana de óleos essenciais em cepas isoladas de infecc¸ão urinária. Rev Saúde Publica 2004;38:326–8. Sartoratto A, Machado ALM, Delarmelina C, Filgueira GM, Duarte MCT, Rehder VLG. Composition and antimicrobial activity of essential oils fron aromatic plants used brazil. Braz J Microbiol 2004;35: 275–80. Matias EFF (MSc thesis) Avaliac¸ão da atividade antibacteriana e moduladora da resistência bacteriana à aminoglicosídeos de extratos polares e apolares de Croton campestris A. (velame), Ocimum gratissimum L. (alfavaca) e Cordia verbanacea DC. (erva-baleeira). Crato, CE: Universidade Regional do Cariri – URCA; 2010. Vieira RF, Graye RJ, Paton A, Simon JE. Uso de marcadores químicos no estudo da diversidade genética de ocimum gratissimum L. Rev Bras Farmacogn 2002;12:126–9. Matos FJA. Farmácias vivas. 3a ed. Fortaleza: Ed. UFC; 1998. Nogueira RJMC, Junior JFS. Resistencia estomática, tensão de água no xilema e teor de clorofilia em genótipos de gravioleira. Sci Agric 2001;58:491–5. Bandeira JM, Barbosa FF, Rodrigues ICS, Bacarin MA, Peters JÁ, Braga EJB. Composic¸ão do óleo essencial de quatro espécie do gênero Plectranthus. Rev Bras Plantas Med 2011;13:157–64. Silva MGF (BSc thesis) Atividade antioxidante e antimicrobiana in vitro de óleos essenciais e extratos hidroalcóolicos de manjerona (Origanum majorana L.) e manjericão (Ocimum basilicum L.). Pato Branco: Universidade Tecnológica Federal do Paraná – UTFPA; 2011. Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. Carol Stream: Allured Publishing Corporation; 2001. p. 455. NCCLS – National Committee For Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 6th ed. Wayne: NIH; 2003. Salvat AA, Antonnacci L, Fortunato RH, Suarez EY. Screening of some plants from northern Argentina for their antimicrobial activity. Lett Appl Microbiol 2001;32:293–7. Matias EFF, Alves EF, Santos BS, Souza CES, Ferreira JVA, Lavor AKLS, et al. Biological activities and chemical characterization of Cordia verbenacea DC. as tool to validate the ethnobiological usage. Evid Based Complement Altern Med 2013;2013:1–7.
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
+Model EUJIM-367; No. of Pages 6 xxx.e6
ARTICLE IN PRESS J.J.S. Aguiar et al. / European Journal of Integrative Medicine xxx (2014) xxx.e1–xxx.e6
[23] Nogueira MA, Diaz G, Sakumo L. Caracterizac¸ão química e atividade biológica do óleo essencial de Lippia alba cultivada no Paraná. Rev Ciênc Farm Básica Apl 2007;28:273–8. [24] Tyagi AK, Bukvicki D, Gottardi D, Veljic M, Guerzoni ME, Malik A, et al. Antimicrobial potential and chemical characterization of Serbian liverwort (Porella arboris-vitae): SEM and TEM observations. Evid Based Complement Altern Med 2013;2013:1–7. [25] Tyagi AK, Gottardi D, Malik A, Guerzoni ME. Chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil. LWT – Food Sci Technol 2014;57:731–7. [26] Rodrigues FFG, Costa JGM, Coutinho HDM. Synergy effects of the antibiotics gentamicin and the essential oil of Croton zehntneri. Phytomedicine 2009;16:1052–5. [27] Dixon RA, Dey PM, Lamb CJ. Phytoalexins: enzymology and molecular biology. Adv Enzymol Relat Areas Mol Biol 1983;55:1–69. [28] Ho KY, Tsai CC, Huang JS, Chen CP, Lin TC, Lin CC. Antimicrobial activity of tannin components from Vaccinium vitisidaea L. J Pharm Pharmacol 2001;53:187–91.
[29] Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564–82. [30] Iacobellis NS, Cantore PL, Capasso F, Senatore F. Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils. J Agric Food Chem 2005;53:57–61. [31] Harvey RA, Champe PC, Fisher BD. Microbiologia ilustrada. 2a ed. São Paulo: Editora Artmed; 2008. [32] Behling EB, Sendão MC, Francescato HDC, Antunes LMG, Bianchi MLP. Flavonoid quercetin: general aspects and biological actions. Alimentos e Nutric¸ão 2004;15:285–92. [33] Escobar LRG. Eugenol: propriedades farmacológicas y toxicológicas. Ventajas y desvantajas de su uso. Rev Cubana Estomatol 2002;39: 139–56. [34] Oliveira RAG, Lima EOL, Vieira WL, Freire KRL, Trajano VN, Lima IO, et al. Estudo da interferência de óleos essenciais sobre a atividade de alguns antibióticos usados na clínica. Rev Bras Farmacogn 2006;16: 77–82.
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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 concentration (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 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 Molecular, Av. Cel Antonio Luiz, 1161, CEP: 63105-000, Crato, CE, Brazil. Tel.: +55 88831021212; fax: +55 8831021291. E-mail address: [email protected] (H.D.M. Coutinho).
humans and animals. The genus Staphylococcus is commonly 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 associated 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 various 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 1876-3820/© 2014 Elsevier GmbH. All rights reserved.
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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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 alternative [5,6], given that natural products of plant origin can alter the action of antimicrobial agents, either by enhancing or reducing 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 various 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 pharmaceutical 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 characterized 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 primary matter in the pharmaceutical industry [10]. Its constituents identified by chemical and phytochemical assays, include germacrene, 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. gratissimum 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 Laboratories LTDA).
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 Universidade 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 sulfate 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, respectively. 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 inoculum 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 determined [21]. The MICs were recorded as the lowest concentration needed to inhibit growth.
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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Table 2 Chemical composition (%) of essential oil of Ocimum gratissimum L.
Bacterial strain
Origin
Resistance profile
Staphylococcus aureus SA358
Surgical wound
Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net
Surgical wound
Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Can, Szt, Tet, Tob
Staphylococcus aureus ATCC 25923 Escherichia coli EC27
Escherichia coli ATCC 10536 Pseudomonas aeruginosa PA03
Cathetertip
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Cpm, Ctz, Im, Cip, Ptz, Lev, Mer, Ami
Pseudomonas aeruginosa ATCC 15442 Ast, aztreonan; Ax, amoxacillin; Amp, ampicillin; Ami, amikacin; Amox, amoxillin, Ca, cefadroxil; Cfc, cefaclor; Cf, cephalothin; Caz, ceftazidime; Cip, ciproflaxacin; Clo, chloramphenicol; Im, imipenem; Can, kanamycin; Szt, sulfamethotrim, Tet, tetracycline; Tob, tobramycin; Oxa, oxacillin; Gen, gentamicin; Neo, neomycin; Para, paramomycin; But, butirosin; Sis, sisomicin; Net, Netilmicin; (–), absence of resistance or non-significant resistance.
Determination of antibiotic resistance-modifying activity Antibiotic-modifying activity was determined by the method described by Coutinho et al. [7], where the solutions of essential oils and their combinations were tested at sub-inhibitory concentrations (MIC/8). A volume of 100 L of the solution containing 10% BHI, inoculum and sample was added to each well in the alphabetic direction of the plate. Next, 100 L of the antibiotic solution were added to the first well and serially diluted on 2fold scale on all wells, excepted the last one. The concentrations of aminoglycosides varied gradually from 2500 to 2.44 g/mL. The plates were incubated at 35 ◦ C for 24 h, then the viable cells were determined by resazurin staining. Statistical analysis The results of tests were obtained in triplicate and expressed as geometric mean. Statistical analysis was carried out using ANOVA followed by Bonferroni post-tests, utilizing GraphPad Prism 6.0 [22], and p < 0.05 was considered significant. Results The yield of essential oil from O. gratissimum L. was 0.45%, compatible with the finding of Vieira et al. [18], The chromatographic analysis showed that the major constituent was eugenol (65%)(Table 1), these findings are in accordance with the study by Sartoratto et al. [12]. On the other hand, P. amboinicus L. yielded 0.15% essential oil, which 38.60% was germacrene-D as the major secondary metabolite (Table 2), a higher percentage than that described by Bandeira et al. [17]. However, Nogueira
Component
RT (min)a
(%)
-Pinene -Ocimene p-Cineol -Linalol ␣-Terpineol Eugenol -Elemene (E)-Caryophyllene ␣-Humulene Germacrene-D -Selinene ␥-Gurjunene
3.58 4.09 4.15 4.85 6.15 8.42 8.90 9.37 9.82 10.17 10.26 10.37
1.59 4.47 15.17 0.37 0.73 65.26 0.66 3.04 0.47 1.91 4.40 1.57
Total a
99.64
Retention time.
et al. [23] reports that variation of yield of oil essential and concentration of constituents can be caused by various environmental factors. Table 3 shows the MIC of samples tested against standard and multiresistant strains of E. coli, S. aureus and P. aeruginosa. Comparatively, the essential oils separate and combined showed the same MIC with the exception of EOPA and its combination with EOOG against EC-ATCC 10536, which produced a better antibacterial activity with MIC of 128 g/However, when EOOG was tested against SA-ATCC 25923, the MIC was 512 g/mL. Figs. 1 and 2 show the results of evaluating the modulatory activity of oils when combined with amikacin and gentamicin. The results demonstrated that the combination of EOPA with aminoglycosides had a synergistic effect against all strains tested with significance (p < 0.001). The combinations of EOOG with aminoglycosides demonstrated antagonism, with exception of the combination with gentamicin against PA03 and with amikacin against EC 27, however without significance. But the simultaneous combination of EOPA and EOOG with the antibiotics revealed that the antagonistic effect with EOOG prevailed over the effect of EOPA, causing interference capable of lowering its synergistic effect. Table 3 Chemical composition (%) of essential oil of P. amboinicus L. Component
RT (min)a
(%)
Copaene α-Amorphene (E)-Caryophyllene ␣-Humulene α-Curcumene Germacrene-D -Bisabolene ␦-Cadinene trans-Nerolidol Caryophyllene oxide Zingiberene
8.73 8.90 9.37 9.82 10.05 10.17 10.38 10.64 11.03 11.55 15.70
8.03 4.84 18.91 1.74 0.89 38.60 4.04 3.16 6.29 2.13 2.49
Total a
91.12
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 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
[13,29]. Germacrene-D is the major terpene in the essential oils 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 EOPA EOOG + EOPA
≥1024 ≥1024 ≥1024
≥1024 128 128
≥1024 ≥1024 ≥1024
512 ≥1024 ≥1024
≥1024 ≥1024 ≥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.
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bacteria, since it lowers the necessary dose at which the aminoglycoside 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 reduction 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 phenylpropanoid compound with substantial antibacterial activity. The observations of Oliveira et al. [34] indicated the interference of essential oils with antibiotics, but their study demonstrated an antagonistic effect in some combinations of essential oils and aminoglycosides. This characteristic of essential oils varies depends on the type of antibiotic, of essential oil tested in combination and on the type of bacterial strain assayed.
[3] [4]
[5]
[6]
[7]
[8] [9]
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 bacterial resistance-modifying activity. However, the combination of antibiotics with EOOG resulted in an antagonistic effect, decreasing the antimicrobial activity of aminoglycosides. Further studies are needed to isolate the active compounds and develop new therapeutic alternatives for the treatment of diseases caused by multiresistant microorganisms.
[10]
[11]
[12]
[13]
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.
[14]
[15] [16]
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.
[17]
[18]
[19]
[20]
[21]
References [1] Murray PR. Microbiologia médica. 4a ed. Rio de Janeiro: Guanabara Koogan; 2004. [2] Coutinho HDM, Costa JGM, Lima EO, Falcão-Silva VS, SiqueiraJr JP. Herbal therapy associated with antibiotic therapy: potentiation
[22]
xxx.e5
of the antibiotic activity against methicillin-resistant Staphylococcus aureus by Turnera ulmifolia L. BMC Complement Altern Med 2009;9: 1–13. Vermelho AB, Bastos MCF, Branquinha MHS. Bacteriologia Geral. 1a ed. Rio de Janeiro: Guanabara Koogan; 2007. Carneiro JCO (MSc thesis) Padrão de consumo de antibacterianos em uma UTI geral: correlac¸ão com a resistência bacteriana. Universidade de Brasília; 2006. Oliveira FQ, Gobira B, Guimarães C, Batista J, Barreto M, Souza M. Espécies vegetais indicadas na odontologia. Rev Bras Farmacogn 2007;17:466–76. Silva JG, Souza IA, Higino JS, Siqueira-Junior JP, Pereira JV, Pereira MSV. Atividade antimicrobiana do extrato de Anacardium occidentale Linn em amostras multiresistentes de Staphylococcus aureus. Rev Bras Farmacogn 2007;17:572–7. Coutinho HDM, Costa JGM, Siqueira JR, Lima JPEO. In vitro antistaphylococcal activity of Hyptis martiusii Benth against methicillin resistant Staphylococcus aureus-MRSA strains. Braz J Pharmacogn 2008;18:670–5. Simon G. Anti-staphylococcal plant natural products. Nat Prod Rep 2004;21:263–77. Alvim NATI, Ferreira MA, Cabral IE, Filho AJA. O uso de plantas medicinais como recurso terapêutico: das influências da formac¸ão profissional às implicac¸ões éticas e legais de sua aplicabilidade como extenc¸ão da prática de cuidar realizada pela Enfermeira. Rev Latino Am Enfermagem 2006;14:3–10. Sousa VC, Lorenzi H. Botânica sistemática Guia ilustrado para identificac¸ão das familias de angiospermas da flora brasileira. 3a ed. Nova Odessa: Instituto Plantarum; 2005. Pereira RS, Sumita TC, Furlan MR, Jorge AOC, Ueno M. Atividade antibacteriana de óleos essenciais em cepas isoladas de infecc¸ão urinária. Rev Saúde Publica 2004;38:326–8. Sartoratto A, Machado ALM, Delarmelina C, Filgueira GM, Duarte MCT, Rehder VLG. Composition and antimicrobial activity of essential oils fron aromatic plants used brazil. Braz J Microbiol 2004;35: 275–80. Matias EFF (MSc thesis) Avaliac¸ão da atividade antibacteriana e moduladora da resistência bacteriana à aminoglicosídeos de extratos polares e apolares de Croton campestris A. (velame), Ocimum gratissimum L. (alfavaca) e Cordia verbanacea DC. (erva-baleeira). Crato, CE: Universidade Regional do Cariri – URCA; 2010. Vieira RF, Graye RJ, Paton A, Simon JE. Uso de marcadores químicos no estudo da diversidade genética de ocimum gratissimum L. Rev Bras Farmacogn 2002;12:126–9. Matos FJA. Farmácias vivas. 3a ed. Fortaleza: Ed. UFC; 1998. Nogueira RJMC, Junior JFS. Resistencia estomática, tensão de água no xilema e teor de clorofilia em genótipos de gravioleira. Sci Agric 2001;58:491–5. Bandeira JM, Barbosa FF, Rodrigues ICS, Bacarin MA, Peters JÁ, Braga EJB. Composic¸ão do óleo essencial de quatro espécie do gênero Plectranthus. Rev Bras Plantas Med 2011;13:157–64. Silva MGF (BSc thesis) Atividade antioxidante e antimicrobiana in vitro de óleos essenciais e extratos hidroalcóolicos de manjerona (Origanum majorana L.) e manjericão (Ocimum basilicum L.). Pato Branco: Universidade Tecnológica Federal do Paraná – UTFPA; 2011. Adams RP. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. Carol Stream: Allured Publishing Corporation; 2001. p. 455. NCCLS – National Committee For Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 6th ed. Wayne: NIH; 2003. Salvat AA, Antonnacci L, Fortunato RH, Suarez EY. Screening of some plants from northern Argentina for their antimicrobial activity. Lett Appl Microbiol 2001;32:293–7. Matias EFF, Alves EF, Santos BS, Souza CES, Ferreira JVA, Lavor AKLS, et al. Biological activities and chemical characterization of Cordia verbenacea DC. as tool to validate the ethnobiological usage. Evid Based Complement Altern Med 2013;2013:1–7.
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
+Model EUJIM-367; No. of Pages 6 xxx.e6
ARTICLE IN PRESS J.J.S. Aguiar et al. / European Journal of Integrative Medicine xxx (2014) xxx.e1–xxx.e6
[23] Nogueira MA, Diaz G, Sakumo L. Caracterizac¸ão química e atividade biológica do óleo essencial de Lippia alba cultivada no Paraná. Rev Ciênc Farm Básica Apl 2007;28:273–8. [24] Tyagi AK, Bukvicki D, Gottardi D, Veljic M, Guerzoni ME, Malik A, et al. Antimicrobial potential and chemical characterization of Serbian liverwort (Porella arboris-vitae): SEM and TEM observations. Evid Based Complement Altern Med 2013;2013:1–7. [25] Tyagi AK, Gottardi D, Malik A, Guerzoni ME. Chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil. LWT – Food Sci Technol 2014;57:731–7. [26] Rodrigues FFG, Costa JGM, Coutinho HDM. Synergy effects of the antibiotics gentamicin and the essential oil of Croton zehntneri. Phytomedicine 2009;16:1052–5. [27] Dixon RA, Dey PM, Lamb CJ. Phytoalexins: enzymology and molecular biology. Adv Enzymol Relat Areas Mol Biol 1983;55:1–69. [28] Ho KY, Tsai CC, Huang JS, Chen CP, Lin TC, Lin CC. Antimicrobial activity of tannin components from Vaccinium vitisidaea L. J Pharm Pharmacol 2001;53:187–91.
[29] Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564–82. [30] Iacobellis NS, Cantore PL, Capasso F, Senatore F. Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils. J Agric Food Chem 2005;53:57–61. [31] Harvey RA, Champe PC, Fisher BD. Microbiologia ilustrada. 2a ed. São Paulo: Editora Artmed; 2008. [32] Behling EB, Sendão MC, Francescato HDC, Antunes LMG, Bianchi MLP. Flavonoid quercetin: general aspects and biological actions. Alimentos e Nutric¸ão 2004;15:285–92. [33] Escobar LRG. Eugenol: propriedades farmacológicas y toxicológicas. Ventajas y desvantajas de su uso. Rev Cubana Estomatol 2002;39: 139–56. [34] Oliveira RAG, Lima EOL, Vieira WL, Freire KRL, Trajano VN, Lima IO, et al. Estudo da interferência de óleos essenciais sobre a atividade de alguns antibióticos usados na clínica. Rev Bras Farmacogn 2006;16: 77–82.
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