http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(5): 560–565 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.853810
ORIGINAL ARTICLE
Chemical composition and evaluation of modulatory of the antibiotic activity from extract and essential oil of Myracrodruon urundeuva
1
Laboratory of Microbiology and Molecular Biology and 2Laboratory of Research in Natural Products, University of the Region of Cariri, Crato (CE), Brazil Abstract
Keywords
Context: The combination of antibiotics with natural products has demonstrated promising synergistic effects in several therapeutic studies. Objective: The aim of this study was to determine the effect of a combination of an ethanol extract of Myracrodruon urundeuva Fr. All. (Anacardiaceae) (aroeira plant) and its essential oil with six antimicrobial drugs against multiresistant strains of Staphylococcus aureus and Escherichia coli from clinical isolates. Materials and methods: After identification of the chemical components by GC-MS, the antibacterial activity of the natural products and antibiotics was assessed by determining the minimal inhibitory concentration (MIC) using the microdilution method and concentrations ranging 8–512 mg/mL and 0.0012–2.5 mg/mL, respectively. Assays were performed to test for a possible synergistic action between the plant products and the antimicrobials, using the extract and the oil at a sub-inhibitory concentration (128 mg/mL) and antibiotic at concentrations varying between 8 and 512 mg/mL. Results: The GC-MS analysis identified the main compound as d-carene (80.41%). The MIC of the natural products was 41024 mg/mL, except against S. aureus ATCC25923. Only the combinations of the natural products with gentamicin, amikacin and clindamycin were effective against S. aureus 358, enhancing the antibiotic activity by reducing the MIC. Conclusions: The extract from aroeira showed a higher antibacterial activity and the oil was more effective in potentiating the activity of conventional antibiotics.
Aminoglycosides, aroeira, clindamycin, Escherichia coli, essential oil, Staphylococcus aureus, synergism
Introduction In the last decades, scientists have searched for new compounds with activity against various infections and low toxicity to patients. This concern has been due to the increase in bacterial resistance to antibiotics along with increased incidence of side effects associated with commonly used drugs and the clinical importance given to infections particularly in immunocompromised patients (Cars et al., 2011). Staphylococcus aureus is widespread in nature and belongs to the normal microbiota of the skin and mucosa of animals, including birds. Some strains of Staphylococcus are frequently recognized as etiological agents of opportunistic infections in various animals and humans (Coutinho et al., 2009). S. aureus is one of the most common etiological agents of purulent infections (including, furuncles, carbuncles, abscess, myocarditis, endocarditis, meningitis, pneumonia
Correspondence: Saulo Relison Tintino, Centre of Biological Sciences and Health, University of the Region of Cariri, Crato (CE), Rua Cel. Antonio Luis 1161, Pimenta, 63105-000, Brazil. Tel: þ55(88)31021212. Fax: þ55(88) 31021291. E-mail: [email protected]
History Received 17 April 2013 Revised 20 September 2013 Accepted 6 October 2013 Published online 19 November 2013
and bacterial arthritis) (Matias et al., 2010a). Escherichia coli is one of the main pathogens that cause infectious diseases in humans. These bacteria produce enterotoxins, which have been studied extensively for their properties and role in diarrheal disease. The activity of cytotoxins and their role in human infections have been identified (Matias et al., 2010a), especially with regard to urinary tract infections (Matias et al., 2010b). Resistance to antibiotics is a growing concern in the treatment of many bacterial diseases (Cars et al., 2011). Antimicrobial resistance increases morbidity and mortality in patients with infections, while costs to health institutions are greatly increased (Coutinho et al., 2005). In view of this situation, there is an increased need to develop new drugs with superior antibacterial properties in combating infections (Michelin et al., 2005). Natural products from plants may be able to change or modulate the action of antibiotics, enhancing or reducing their activity (Coutinho et al., 2008). In recent years, many plants have been evaluated not only for their direct antibacterial action but also as modifiers of antibiotic activity (Coutinho et al., 2010).
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Fernando G. Figueredo1, Bruno F. F. Lucena1, Saulo R. Tintino1, Edinardo F. F. Matias1, Nadghia F. Leite1, Jacqueline C. Andrade1, Lavouisier F. B. Nogueira1, Edson C. Morais1, Jose´ G. M. Costa2, Henrique D. M. Coutinho1, and Fabiola F. G. Rodrigues2
Antimicrobial activity of Myracrodruon urundeuva
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DOI: 10.3109/13880209.2013.853810
The use of natural products, mainly the chemical components of plants with antimicrobial properties, have contributed to significant therapeutic outcomes (Albuquerque & Hanazaki, 2006; Oliveira et al., 2007). Prior knowledge of the classes of chemical compounds present in the plant of interest is necessary to determine the levels of active principles. Once the presence of certain secondary metabolites has been established, a biological study is carried out (Figueredo et al., 2013). Myracrodruon urundeuva Fr. All. (Anacardiaceae) is popularly known as ‘‘aroeira,’’ ‘‘aroeira-do-sertao’’ or ‘‘urundeuva.’’ It is a deciduous species that is heliophytic and selectively xerophytic (Lorenzi & Matos, 2002). It is a large tree that can measure more than 20 feet high. Its majestic appearance stands out in the hinterlands flora and can be found frequently on hill slopes (Lorenzi & Matos, 2002). This plant is often used for its medicinal properties such as antiseptic, antidiarrheal and wound healing (Duarte et al., 2009). Pharmacological assays have demonstrated its antimicrobial (Lima et al., 2006; Sa´ et al., 2009), antiinflammatory (Albuquerque et al., 2007) and antitumor (Duarte et al., 2009) activities. Leaves and stem bark are indicated for the treatment of inflammatory, painful and ulcerative conditions (Albuquerque et al., 2007). The objective of this study was to determine the antibacterial activity of the ethanol extract and essential oil of leaves of M. urundeuva as a modifying agent of resistance to conventional antibiotics in standard and multiresistant strains of E. coli and S. aureus and to identify the main secondary metabolites of these natural products.
Material and methods Bacterial material The bacterial strains used were E. coli (EC ATCC10536 and EC27) and S. aureus (SA ATCC25923 and SA 358), and their resistance profile is given in Table 1. All strains were maintained on heart infusion agar (HIA, Difco Laboratories Ltd.). Before the tests, the strains were grown for 18 h at 37 C in brain heart infusion broth (BHI, Difco Laboratories Ltd.). Leaves of M. urundeuva were collected at Penaforte, Ceara, Brazil, in September 2011 at 8a.m. The plant material was identified by Dr. Maria Arlene Pessoa da Silva, and a voucher specimen was deposited in the respective herbal collections in the Herbario Caririense Dardano de AndradeLima (HCDAL) of the Department of Biological Sciences (URCA), registration no. 1547.
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Preparation of ethanol extracts of M. urundeuva The ethanol extract was prepared using 144 g of leaves, which were sliced and then packed in a container with sufficient solvent to submerge the plant material. After 72 h, the extract was filtered and concentrated in a rotary vacuum evaporator (model Q-344B, Quimis, Brazil) and using a hot-water bath (model Q-214 M2, Quimis, Brazil) (Brasileiro et al., 2006). Obtaining essential oil The essential oil was obtained by hydrodistillation in a Clevenger-type device modified by Gottlieb (1960). Fresh leaves of M. urundeuva were placed in a 5-L flask, together with 3 L of distilled water and heated for 2 h. Afterwards, the water/oil mixture obtained was separated, and the essential oil of M. urunduiva (EOMU) was treated with anhydrous sodium sulfate, filtered, and kept under refrigeration until the time of analysis. Phytochemical prospecting The phytochemical tests to detect the presence of heterosides, tannins, flavonoids, steroids, triterpenes, coumarins, quinones, organic acids and alkaloids were performed according to the method described by Matos (1997). The tests were based on visual observation of the change in color or formation of precipitate after the addition of specific reagents. Chemical analysis of the essential oil Oil analysis was performed using a Shimadzu GC MS – QP2010 series (GC/MS system): Rtx-5MS capillary column (30 m 0.25 mm, 0.25 mm film thickness); helium carrier gas at 1.5 mL/min; injector temperature of 250 C; detector temperature of 290 C. The oven temperature was programmed from 60 C (isothermal for 1.5 min), with an increase of 5 C/min to 180 C, then 10 C/min to 280 C, ending with a 10 min isothermal period at 280 C. The split ratio was 1:200 and injection volume was 1 mL, using a 1:200 dilution of essential oil in CHCl3. Solvent cut time ¼ 2.5 min. This method had a run time of 30 min, and the mass spectrometer was operated at 70 eV ionization energy. Identification of individual components was based on their mass spectral fragmentation according to the NIST 08 mass spectral library and retention indices, and comparison with published data. The individual components were identified by matching their mass spectra based on an impact energy of 70 eV with the database using the library constructed by the spectrometer (Wiley 229) and two other computers using retention indices
Table 1. Origin of the bacterial strains and profile of resistance to antibiotics. Bacteria Escherichia coli 27 Escherichia coli ATCC10536 Staphylococcus aureus 358 Staphylococcus aureus ATCC25923
Origin
Profile of resistance
Surgical wound ATCC Surgical wound ATCC
Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Can, Szt, Tet, Tob – Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net –
Ast: Aztreonam; Ax: Amoxicillin; Amp: Ampicillin; Ami: Amikacin; Amox: Amoxicillin; Ca: Cefadroxil; Cfc: Cefaclor; Cf: Cefalotin; Caz: Ceftazidime; Cip: Ciprofloxacin; Chlo: Chloramphenicol; Im: Imipenem; Kan: Kanamycin; Szt: Sulfametim; Tet: Tetracycline; Tob: Tobramycin; Oxa: Oxacillin; Gen: Gentamicin; Neo: Neomycin; Para: Paramomycin; But: Butirosin; Sis: Sisomicin; Net: Netilmicin; (—): No resistance or resistance without relevance. ATCC: American Type Culture Collection.
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as a preselection (Alencar et al., 1984, 1990), as well as by visual comparison of the fragmentation pattern with that reported in the literature (Adams, 2001; Stenhagen et al., 1974).
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Antibacterial test (MIC) and modulation of antibiotic activity MIC (minimal inhibitory concentration) was determined with a microdilution assay (NCCLS, 2003) utilizing an inoculum of 100 mL of each strain, suspended in BHI broth up to a final concentration of 105 CFU/mL in 96-well microtiter plates, using two-fold serial dilutions. MIC was defined as the lowest concentration at which no growth was observed. Each well received 100 mL of each extract solution. The final concentrations of the extracts varied from 8 to 512 mg/mL. MIC for the antibiotics was determined in BHI by the microdilution assay utilizing suspensions of 105 CFU/mL and a drug concentration range of 0.0012–2.5 mg/mL (two-fold serial dilutions). To evaluate the ethanol extract and oil as modulators of antibiotic activity, the MIC of the antibiotics from the classes aminoglycosides, lincosamides and b-lactams were evaluated in the presence and absence of the plant samples in sterile microplates. Antibiotics were evaluated at concentrations ranging from 0.5 to 512 mg/mL. All antibiotics tested were obtained from Sigma. The plant extracts were mixed with BHI broth at subinhibitory concentrations MIC/8 or concentration eightfold lower than MIC. In the modulation test, antibiotic solutions were prepared by adding sterile distilled water at a double concentration (1024 mg/mL) in relation to the initial concentration in a volume of 100 mL and serially diluted 1:1 in 10% BHI broth, with or without plant extract. The mixture of antibiotic and plant extract (100 mL) was added to each well containing 100 mL of bacterial suspension diluted (1:10). The same controls used in the evaluation of MIC for the extracts were also used for the modulation assay (Coutinho et al., 2008). The plates were incubated at 35 C for 24 h, and bacterial growth was assessed by the use of resazurin. Antibacterial assays were performed in triplicate and results were expressed as average of replicates.
Results and discussion The crude extract and essential oil evaluated in this study, after the entire preparation procedure, showed yields of 4.5 and 0.6%, respectively. A pilot study was conducted using only DMSO, but no antibacterial or modulatory activity was observed, indicating a nontoxic effect. Phytochemical prospecting of extracts showed the presence of several classes of secondary metabolites exhibiting a wide variety of biological activities such as antimicrobial (Matias et al., 2010a; Figueredo et al., 2013), antioxidant (Barreiros & David, 2006), and antitumor and antiophidic (Okuda et al., 1989). Table 2 shows the presence of various potentially bioactive compounds in the extracts evaluated, namely tannin pyrogallates, anthocyanins, anthocyanidins, flavones, flavonols, xanthones, chalcones, aurones, flavononols, leucoanthocyanidins, catechins and flavonones.
Pharm Biol, 2014; 52(5): 560–565
Table 2. Phytochemical prospecting of the ethanol extract of Myracrodruon urundeuva. Metabolites Extracts EEMU
1 –
2 þ
3 –
4 þ
5 þ
6 þ
7 þ
8 þ
9 þ
10 þ
11 þ
12 þ
13 þ
14 þ
1: phenols; 2: tannin pyrogallates; 3: tannin phlobaphenes; 4: anthocyanins; 5: anthocyanidins; 6: flavones; 7: flavonols; 8: xanthones; 9: chalcones; 10: aurones; 11: flavonols; 12: leucoanthocyanidins; 13: catechins; 14: flavonones; 15: alkaloids; (þ): presence; (): absence. EEMU: Ethanol extract of Myracrodruon urundeuva.
Table 3. Chemical constituents of the essential oil from the leaves of Myracrodruon urundeuva Allema˜o. Components a-Pinene b-Myrcene d-Carene o-Cymene a-Limonene Terpinolene Artemiseole 2-caren-4-ol p-Cymen-8-ol 2-Pinen-4-ona a-Bergamotene Caryophyllene oxide Total
Tr (min)a
IKb
(%)
3.56 4.22 4.63 4.84 4.92 6.04 7.12 8.05 8.21 11.56 11.88 18.28
939 987 1006 1023 1031 1051 1088 1111 1160 1189 1420 1497
1.90 0.62 80.41 1.09 1.89 0.43 0.44 0.52 0.61 0.77 1.95 1.81 92.44
Tr: retention time; IK: index of Kovats
Studies of antimicrobial activities of the extracts of M. urundeuva, as well as its chemical composition, were reported by Ju´nior et al. (2009), who demonstrated antibacterial activity of this plant against pathogenic bacterial strains using the disk diffusion method. Monteiro et al. (2005) observed a high level of tannins and flavonoids in extracts of the leaves and bark of this species, which were also present in our sample. In another study, Silva et al. (2012) found saponins in their leaf extract. Nobre-Ju´nior et al. (2009) demonstrated the neuroprotective effect of chalcones isolated from this plant. The presence of alkaloids in the leaves of M. urundeuva has also been reported (Silva et al., 2010). The use of essential oils in the search for new antibacterial compounds has led to promising results in many studies (Fontenelle et al., 2007; Rodrigues et al., 2010; Reis et al., 2011). Essential oils consist of a complex mixture of various bioactive components, so it is necessary to identify which compound is responsible for antimicrobial activity. The study of the chemical composition of the essential oil of M. urundeuva (EOMU) was performed by gas chromatography coupled to mass spectrometry (CG/EM), and the components were identified by comparison of their mass spectra and retention time (Rt) with data in the literature (Adams, 2001). The Kovats retention indices (KI) were determined using a homologous series of n-alkanes chromatographed under the same conditions as the samples. Thus, it was possible to identify 92.44% of the constituents, with the majority being d-carene (80.41%), a-bergamotene (1.95%), a-pinene (1.90), a-limonene (1.89) and caryophyllene oxide (1.81%) (Table 3).
Antimicrobial activity of Myracrodruon urundeuva
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DOI: 10.3109/13880209.2013.853810
The results found in the chemical analysis of the essential oil are consistent with previous studies which showed that most of the constituents of M. urundeuva are generally of the monoterpene class and that d-carene is the most encountered (Montanari, 2010). Table 4 shows the MIC values of the ethanol extract and essential oil tested against standard and multiresistant strains of E. coli and S. aureus. Comparatively, the samples of EEMU and EOMU showed the same MIC, with the exception of EEMU against SA-ATCC 25923, which showed better antibacterial activity with a MIC of 512 mg/mL. Natural products derived from medicinal plants have been used as a source of new bioactive compounds especially for the treatment of infectious diseases. Therefore, it is truly possible to use plants as a source of new bioactive compounds effective against bacteria resistant to conventional antibiotics (Buttler & Buss, 2006). These products of plant or animal origin can alter the effect of antibiotics, either increasing or decreasing their activity and may thus be termed modifiers of antibiotic activity (Coutinho et al., 2008; Rodrigues et al., 2009). Some of the classes of secondary metabolites identified in this study have been characterized as modifiers of antibiotic activity, such as terpenes (Nicolson et al., 1999), tannins (Figueredo et al., 2013; Matias et al., 2010a), flavonoids and derivatives (Sato et al., 2004). Table 5 shows the influence of the plant samples on the activity of the aminoglycosides and clindamcycin, demonstrating a synergistic effect, with a decrease in MIC against the Gram-positive strains. The most representative effect was seen with the combination of 128 mg/mL (MIC/8) EOMU with the antibiotics in the culture medium, showing a potentiation of amikacin combined with EOMU against SA 358 with a decrease in MIC from 64 to 8 mg/mL.
Table 4. Minimal inhibitory concentration (MIC) of the ethanol extract and essential oil of Myracrodruom urundeuva (mg/mL). Extracts and antimicrobial by natural products
EC27
EEMU EOMU
1024 1024
EC-ATCC10536 SA358 SA-ATCC25923 1024 1024
1024 1024
512 1024
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
Table 5. Minimum inhibitory concentration (MIC) of aminoglycosides and clindamycin in the presence and absence of ethanol extract and essential oil of Myracrodrum urunduiva at a concentration of MIC/8 (128 mg/mL).
Antibiotics Gentamicin Amikacin Clindamycin
563
The synergistic activity observed might have been due to the presence of secondary metabolites present in the extracts and oil, such as tannins and flavonoids, which are synthesized by plants in response to microbial infection (Dixon et al., 1983; Ho et al., 2001), where they are capable of disrupting the cell wall or plasma membrane facilitating drug uptake (Figueredo et al., 2013; Matias et al., 2010a; Tsuchiya et al., 1996). Due to uptake by normal cells, host toxicity is common to all aminoglycosides except streptomycin. Nephrotoxicity, ototoxicity and neuromuscular blockade are the most important toxic effects of aminoglycosides (Oliveira, 2006; Vallejo et al., 2001). The adverse effects of clindamycin consist mainly of gastrointestinal disorders that can manifest as severe diarrhea and pseudomembranous colitis (Katzung, 2005). The combination of natural products with aminoglycosides and clindamycin may be an alternative to minimize the undesirable effects of these antibiotics, when used for the treatment of Gram-positive infections since the combination leads to a synergistic effect, thereby significantly reducing the MIC of these drugs and allowing the use of lower doses for therapeutic success. The Gram-positive strain was more sensitive to the combination of antibiotics with plant substance compared with the Gram-negative strains. These results agree with those found in the literature (Veras, 2011), showing that Gramnegative bacteria are more resistant to the action of natural products, such as extracts and essential oils, because the outer membrane present in these bacteria forms a complex envelope, protecting them against the action of these antimicrobials (Holley & Pattel, 2005; Oladimeji et al., 2004). Besides, these bacteria possess other resistance mechanisms such as efflux pump, enzymes that cleave the beta-lactam ring (b-lactamases), and change in penicillin-binding protein, among others (Bush, 2002; Enright et al., 2002). However, our results are at odds with Deans and Ritchie (1987) who pointed out that the antibacterial action of essential oils is independent of Gram reaction (Dorman & Deans, 2000). Table 6 shows the influence of the plant samples on the activity of the b-lactam, demonstrating no synergistic effect. All b-lactam antibiotics inhibit bacterial growth by interfering with penicillin-binding protein, where the transpeptidation reaction is inhibited, blocking the synthesis of peptidoglycan and leading to cell death (Katzung, 2005). Many of these antibiotics penetrate Gram-negative bacteria through protein channels present in their outer membrane. Table 6. Minimum inhibitory concentration (MIC) of beta-lactam in the presence and absence of ethanol extract and essential oil of Myracrodrum urundeuva at a concentration of CIM/8 (128 mg/mL).
EC 27
SA 358
EC 27
SA 358
MIC combined
MIC combined
MIC combined
MIC combined
MIC
EEMU
EOMU
MIC
EEMU
EOMU
Antibiotics
CIM
EEMU
EOMU
CIM
EEMU
EOMU
64 128 1024
64 128 1024
64 128 1024
8 64 16
4 16 4
2 8 8
Benzetacil Ampicillin Oxacillin
1024 1024 1024
1024 1024 1024
1024 1024 1024
512 128 0.5
512 128 0.5
512 128 0.5
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
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Through these channels, the drug can reach its receptor in the cell wall and exert its antibacterial action (Nikaido, 1994). However, this activity of b-lactam alone or in combination with natural products against this strain was not observed in the present study, which can be related to resistance mechanisms and the presence of b-lactamase and other factors already mentioned (Bush, 2002; Enright et al., 2002). We observed b-lactam activity in the Gram-positive strains, but there was no modification of antibiotic activity in combinations with natural products.
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Conclusion The results of this study indicate that the ethanol extract and essential oil of M. urundeuva are an alternative source of natural products with antibacterial action, since they possess compounds with antibacterial activity recognized as tannins, flavonoids and terpenes, besides indicating their possible use in combination with conventional antibiotics against Grampositive bacterial strains. We conclude that the ethanol extract of aroeira has greater antibacterial activity, and that the oil is more effective in enhancing antibiotic activity against the S. aureus 358 strain.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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DOI: 10.3109/13880209.2013.853810
Oladimeji FA, Orafidiya LO, Okeke IN. (2004). Physical properties and antimicrobial activities of leaf essential oils of Lippia multiflora Moldenke. Int J Arom 14:162–8. Oliveira JFP, Cipullo JP, Burdmann EA. (2006). Nefrotoxicidade dos aminoglicosidios. Braz J Cardio Surger 21:444–52. Oliveira RAG, Lima EO, Souza EL, et al. (2007). Interference of Plectranthus amboinicus (Lour.). Spreng essential oil on the anti-Candida activity of some clinically used antifungals. Rev Bras de Farmacog 17:186–90. Reis FB, Souza VM, Thomaz MRS, et al. (2011). Use of Carnobacterium maltaromaticum cultures and hydroalcoholic extract of Lippia sidoides Cham. against Listeria monocytogenes in fish model systems. Int J Food Microbiol 146:228–34. Rodrigues FFG, Costa JGM, Coutinho HDM. (2009). Synergy effects of the antibiotics gentamicin and the essential oil of Croton zehtneri. Phytomedicine 16:1052–5. Rodrigues FFG, Costa JGM, Coutinho HDM. (2010). Enhancement of the antibiotic activity of gentamicin by volatile compounds of Zanthoxylum articulatum. Indian J Med Res 131:833–5. Sa´ RS, Argolo ACC, Napolea˜o TH, et al. (2009). Antioxidant, Fusarium growth inhibition and Nasutitermes cornigerrepellent activities of secondary metabolites from Myracrodruon urundeuva heartwood. Int Biodeter Biodegrad Barking 63:470–7.
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Sato Y, Shibata H, Arakaki N, Higuti T. (2004). 6,7-Dihydroxyflavone dramatically intensifies the susceptibility to S-lactam antibiotics in methicillin-resistant and sensitive Staphylococcus aureus. Antimicrob Agents Chemother 48:1357–60. Silva NLA, Miranda FAA, Conceic¸a˜o GM. (2010). Triagem fitoquı´mica de plantas de cerrado, da a´rea de protec¸a˜o ambiental municipal do Inhamum, Caxias, Maranha˜o. Scient Plen 6:1–17. Stenhagen E, Abrahamson S, Mclafferty FW. (1974). Registry of Mass Spectra Data Base. Washington DC: Government Printing Office. Tsuchiya H, Sato M, Miyazaki T, et al. (1996). Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus. J Ethnopharmacol 50: 27–34. Vallejo JC, Silva MN, Oliveira JAA, et al. (2001). Deteccao precoce de ototoxicidade usando emissoes otoacusticas produtivas de distorcao. Rev Bras Otorrinolari 6:845–51. Veras HNH. (2011). Caracterizac¸a˜o quı´mica e avaliac¸a˜o da atividade antimicrobiana e antiinflamato´ria to´pica do o´leo essencial de Lippia sidoides CHAM. (VERBENACEAE). Dissertac¸a˜o (Mestrado) – Programa de Po´s-graduac¸a˜o em Bioprospecc¸a˜o Molecular da Universidade Regional do Cariri-URCA.
ORIGINAL ARTICLE
Chemical composition and evaluation of modulatory of the antibiotic activity from extract and essential oil of Myracrodruon urundeuva
1
Laboratory of Microbiology and Molecular Biology and 2Laboratory of Research in Natural Products, University of the Region of Cariri, Crato (CE), Brazil Abstract
Keywords
Context: The combination of antibiotics with natural products has demonstrated promising synergistic effects in several therapeutic studies. Objective: The aim of this study was to determine the effect of a combination of an ethanol extract of Myracrodruon urundeuva Fr. All. (Anacardiaceae) (aroeira plant) and its essential oil with six antimicrobial drugs against multiresistant strains of Staphylococcus aureus and Escherichia coli from clinical isolates. Materials and methods: After identification of the chemical components by GC-MS, the antibacterial activity of the natural products and antibiotics was assessed by determining the minimal inhibitory concentration (MIC) using the microdilution method and concentrations ranging 8–512 mg/mL and 0.0012–2.5 mg/mL, respectively. Assays were performed to test for a possible synergistic action between the plant products and the antimicrobials, using the extract and the oil at a sub-inhibitory concentration (128 mg/mL) and antibiotic at concentrations varying between 8 and 512 mg/mL. Results: The GC-MS analysis identified the main compound as d-carene (80.41%). The MIC of the natural products was 41024 mg/mL, except against S. aureus ATCC25923. Only the combinations of the natural products with gentamicin, amikacin and clindamycin were effective against S. aureus 358, enhancing the antibiotic activity by reducing the MIC. Conclusions: The extract from aroeira showed a higher antibacterial activity and the oil was more effective in potentiating the activity of conventional antibiotics.
Aminoglycosides, aroeira, clindamycin, Escherichia coli, essential oil, Staphylococcus aureus, synergism
Introduction In the last decades, scientists have searched for new compounds with activity against various infections and low toxicity to patients. This concern has been due to the increase in bacterial resistance to antibiotics along with increased incidence of side effects associated with commonly used drugs and the clinical importance given to infections particularly in immunocompromised patients (Cars et al., 2011). Staphylococcus aureus is widespread in nature and belongs to the normal microbiota of the skin and mucosa of animals, including birds. Some strains of Staphylococcus are frequently recognized as etiological agents of opportunistic infections in various animals and humans (Coutinho et al., 2009). S. aureus is one of the most common etiological agents of purulent infections (including, furuncles, carbuncles, abscess, myocarditis, endocarditis, meningitis, pneumonia
Correspondence: Saulo Relison Tintino, Centre of Biological Sciences and Health, University of the Region of Cariri, Crato (CE), Rua Cel. Antonio Luis 1161, Pimenta, 63105-000, Brazil. Tel: þ55(88)31021212. Fax: þ55(88) 31021291. E-mail: [email protected]
History Received 17 April 2013 Revised 20 September 2013 Accepted 6 October 2013 Published online 19 November 2013
and bacterial arthritis) (Matias et al., 2010a). Escherichia coli is one of the main pathogens that cause infectious diseases in humans. These bacteria produce enterotoxins, which have been studied extensively for their properties and role in diarrheal disease. The activity of cytotoxins and their role in human infections have been identified (Matias et al., 2010a), especially with regard to urinary tract infections (Matias et al., 2010b). Resistance to antibiotics is a growing concern in the treatment of many bacterial diseases (Cars et al., 2011). Antimicrobial resistance increases morbidity and mortality in patients with infections, while costs to health institutions are greatly increased (Coutinho et al., 2005). In view of this situation, there is an increased need to develop new drugs with superior antibacterial properties in combating infections (Michelin et al., 2005). Natural products from plants may be able to change or modulate the action of antibiotics, enhancing or reducing their activity (Coutinho et al., 2008). In recent years, many plants have been evaluated not only for their direct antibacterial action but also as modifiers of antibiotic activity (Coutinho et al., 2010).
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Fernando G. Figueredo1, Bruno F. F. Lucena1, Saulo R. Tintino1, Edinardo F. F. Matias1, Nadghia F. Leite1, Jacqueline C. Andrade1, Lavouisier F. B. Nogueira1, Edson C. Morais1, Jose´ G. M. Costa2, Henrique D. M. Coutinho1, and Fabiola F. G. Rodrigues2
Antimicrobial activity of Myracrodruon urundeuva
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DOI: 10.3109/13880209.2013.853810
The use of natural products, mainly the chemical components of plants with antimicrobial properties, have contributed to significant therapeutic outcomes (Albuquerque & Hanazaki, 2006; Oliveira et al., 2007). Prior knowledge of the classes of chemical compounds present in the plant of interest is necessary to determine the levels of active principles. Once the presence of certain secondary metabolites has been established, a biological study is carried out (Figueredo et al., 2013). Myracrodruon urundeuva Fr. All. (Anacardiaceae) is popularly known as ‘‘aroeira,’’ ‘‘aroeira-do-sertao’’ or ‘‘urundeuva.’’ It is a deciduous species that is heliophytic and selectively xerophytic (Lorenzi & Matos, 2002). It is a large tree that can measure more than 20 feet high. Its majestic appearance stands out in the hinterlands flora and can be found frequently on hill slopes (Lorenzi & Matos, 2002). This plant is often used for its medicinal properties such as antiseptic, antidiarrheal and wound healing (Duarte et al., 2009). Pharmacological assays have demonstrated its antimicrobial (Lima et al., 2006; Sa´ et al., 2009), antiinflammatory (Albuquerque et al., 2007) and antitumor (Duarte et al., 2009) activities. Leaves and stem bark are indicated for the treatment of inflammatory, painful and ulcerative conditions (Albuquerque et al., 2007). The objective of this study was to determine the antibacterial activity of the ethanol extract and essential oil of leaves of M. urundeuva as a modifying agent of resistance to conventional antibiotics in standard and multiresistant strains of E. coli and S. aureus and to identify the main secondary metabolites of these natural products.
Material and methods Bacterial material The bacterial strains used were E. coli (EC ATCC10536 and EC27) and S. aureus (SA ATCC25923 and SA 358), and their resistance profile is given in Table 1. All strains were maintained on heart infusion agar (HIA, Difco Laboratories Ltd.). Before the tests, the strains were grown for 18 h at 37 C in brain heart infusion broth (BHI, Difco Laboratories Ltd.). Leaves of M. urundeuva were collected at Penaforte, Ceara, Brazil, in September 2011 at 8a.m. The plant material was identified by Dr. Maria Arlene Pessoa da Silva, and a voucher specimen was deposited in the respective herbal collections in the Herbario Caririense Dardano de AndradeLima (HCDAL) of the Department of Biological Sciences (URCA), registration no. 1547.
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Preparation of ethanol extracts of M. urundeuva The ethanol extract was prepared using 144 g of leaves, which were sliced and then packed in a container with sufficient solvent to submerge the plant material. After 72 h, the extract was filtered and concentrated in a rotary vacuum evaporator (model Q-344B, Quimis, Brazil) and using a hot-water bath (model Q-214 M2, Quimis, Brazil) (Brasileiro et al., 2006). Obtaining essential oil The essential oil was obtained by hydrodistillation in a Clevenger-type device modified by Gottlieb (1960). Fresh leaves of M. urundeuva were placed in a 5-L flask, together with 3 L of distilled water and heated for 2 h. Afterwards, the water/oil mixture obtained was separated, and the essential oil of M. urunduiva (EOMU) was treated with anhydrous sodium sulfate, filtered, and kept under refrigeration until the time of analysis. Phytochemical prospecting The phytochemical tests to detect the presence of heterosides, tannins, flavonoids, steroids, triterpenes, coumarins, quinones, organic acids and alkaloids were performed according to the method described by Matos (1997). The tests were based on visual observation of the change in color or formation of precipitate after the addition of specific reagents. Chemical analysis of the essential oil Oil analysis was performed using a Shimadzu GC MS – QP2010 series (GC/MS system): Rtx-5MS capillary column (30 m 0.25 mm, 0.25 mm film thickness); helium carrier gas at 1.5 mL/min; injector temperature of 250 C; detector temperature of 290 C. The oven temperature was programmed from 60 C (isothermal for 1.5 min), with an increase of 5 C/min to 180 C, then 10 C/min to 280 C, ending with a 10 min isothermal period at 280 C. The split ratio was 1:200 and injection volume was 1 mL, using a 1:200 dilution of essential oil in CHCl3. Solvent cut time ¼ 2.5 min. This method had a run time of 30 min, and the mass spectrometer was operated at 70 eV ionization energy. Identification of individual components was based on their mass spectral fragmentation according to the NIST 08 mass spectral library and retention indices, and comparison with published data. The individual components were identified by matching their mass spectra based on an impact energy of 70 eV with the database using the library constructed by the spectrometer (Wiley 229) and two other computers using retention indices
Table 1. Origin of the bacterial strains and profile of resistance to antibiotics. Bacteria Escherichia coli 27 Escherichia coli ATCC10536 Staphylococcus aureus 358 Staphylococcus aureus ATCC25923
Origin
Profile of resistance
Surgical wound ATCC Surgical wound ATCC
Ast, Ax, Amp, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Im, Can, Szt, Tet, Tob – Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net –
Ast: Aztreonam; Ax: Amoxicillin; Amp: Ampicillin; Ami: Amikacin; Amox: Amoxicillin; Ca: Cefadroxil; Cfc: Cefaclor; Cf: Cefalotin; Caz: Ceftazidime; Cip: Ciprofloxacin; Chlo: Chloramphenicol; Im: Imipenem; Kan: Kanamycin; Szt: Sulfametim; Tet: Tetracycline; Tob: Tobramycin; Oxa: Oxacillin; Gen: Gentamicin; Neo: Neomycin; Para: Paramomycin; But: Butirosin; Sis: Sisomicin; Net: Netilmicin; (—): No resistance or resistance without relevance. ATCC: American Type Culture Collection.
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as a preselection (Alencar et al., 1984, 1990), as well as by visual comparison of the fragmentation pattern with that reported in the literature (Adams, 2001; Stenhagen et al., 1974).
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Antibacterial test (MIC) and modulation of antibiotic activity MIC (minimal inhibitory concentration) was determined with a microdilution assay (NCCLS, 2003) utilizing an inoculum of 100 mL of each strain, suspended in BHI broth up to a final concentration of 105 CFU/mL in 96-well microtiter plates, using two-fold serial dilutions. MIC was defined as the lowest concentration at which no growth was observed. Each well received 100 mL of each extract solution. The final concentrations of the extracts varied from 8 to 512 mg/mL. MIC for the antibiotics was determined in BHI by the microdilution assay utilizing suspensions of 105 CFU/mL and a drug concentration range of 0.0012–2.5 mg/mL (two-fold serial dilutions). To evaluate the ethanol extract and oil as modulators of antibiotic activity, the MIC of the antibiotics from the classes aminoglycosides, lincosamides and b-lactams were evaluated in the presence and absence of the plant samples in sterile microplates. Antibiotics were evaluated at concentrations ranging from 0.5 to 512 mg/mL. All antibiotics tested were obtained from Sigma. The plant extracts were mixed with BHI broth at subinhibitory concentrations MIC/8 or concentration eightfold lower than MIC. In the modulation test, antibiotic solutions were prepared by adding sterile distilled water at a double concentration (1024 mg/mL) in relation to the initial concentration in a volume of 100 mL and serially diluted 1:1 in 10% BHI broth, with or without plant extract. The mixture of antibiotic and plant extract (100 mL) was added to each well containing 100 mL of bacterial suspension diluted (1:10). The same controls used in the evaluation of MIC for the extracts were also used for the modulation assay (Coutinho et al., 2008). The plates were incubated at 35 C for 24 h, and bacterial growth was assessed by the use of resazurin. Antibacterial assays were performed in triplicate and results were expressed as average of replicates.
Results and discussion The crude extract and essential oil evaluated in this study, after the entire preparation procedure, showed yields of 4.5 and 0.6%, respectively. A pilot study was conducted using only DMSO, but no antibacterial or modulatory activity was observed, indicating a nontoxic effect. Phytochemical prospecting of extracts showed the presence of several classes of secondary metabolites exhibiting a wide variety of biological activities such as antimicrobial (Matias et al., 2010a; Figueredo et al., 2013), antioxidant (Barreiros & David, 2006), and antitumor and antiophidic (Okuda et al., 1989). Table 2 shows the presence of various potentially bioactive compounds in the extracts evaluated, namely tannin pyrogallates, anthocyanins, anthocyanidins, flavones, flavonols, xanthones, chalcones, aurones, flavononols, leucoanthocyanidins, catechins and flavonones.
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Table 2. Phytochemical prospecting of the ethanol extract of Myracrodruon urundeuva. Metabolites Extracts EEMU
1 –
2 þ
3 –
4 þ
5 þ
6 þ
7 þ
8 þ
9 þ
10 þ
11 þ
12 þ
13 þ
14 þ
1: phenols; 2: tannin pyrogallates; 3: tannin phlobaphenes; 4: anthocyanins; 5: anthocyanidins; 6: flavones; 7: flavonols; 8: xanthones; 9: chalcones; 10: aurones; 11: flavonols; 12: leucoanthocyanidins; 13: catechins; 14: flavonones; 15: alkaloids; (þ): presence; (): absence. EEMU: Ethanol extract of Myracrodruon urundeuva.
Table 3. Chemical constituents of the essential oil from the leaves of Myracrodruon urundeuva Allema˜o. Components a-Pinene b-Myrcene d-Carene o-Cymene a-Limonene Terpinolene Artemiseole 2-caren-4-ol p-Cymen-8-ol 2-Pinen-4-ona a-Bergamotene Caryophyllene oxide Total
Tr (min)a
IKb
(%)
3.56 4.22 4.63 4.84 4.92 6.04 7.12 8.05 8.21 11.56 11.88 18.28
939 987 1006 1023 1031 1051 1088 1111 1160 1189 1420 1497
1.90 0.62 80.41 1.09 1.89 0.43 0.44 0.52 0.61 0.77 1.95 1.81 92.44
Tr: retention time; IK: index of Kovats
Studies of antimicrobial activities of the extracts of M. urundeuva, as well as its chemical composition, were reported by Ju´nior et al. (2009), who demonstrated antibacterial activity of this plant against pathogenic bacterial strains using the disk diffusion method. Monteiro et al. (2005) observed a high level of tannins and flavonoids in extracts of the leaves and bark of this species, which were also present in our sample. In another study, Silva et al. (2012) found saponins in their leaf extract. Nobre-Ju´nior et al. (2009) demonstrated the neuroprotective effect of chalcones isolated from this plant. The presence of alkaloids in the leaves of M. urundeuva has also been reported (Silva et al., 2010). The use of essential oils in the search for new antibacterial compounds has led to promising results in many studies (Fontenelle et al., 2007; Rodrigues et al., 2010; Reis et al., 2011). Essential oils consist of a complex mixture of various bioactive components, so it is necessary to identify which compound is responsible for antimicrobial activity. The study of the chemical composition of the essential oil of M. urundeuva (EOMU) was performed by gas chromatography coupled to mass spectrometry (CG/EM), and the components were identified by comparison of their mass spectra and retention time (Rt) with data in the literature (Adams, 2001). The Kovats retention indices (KI) were determined using a homologous series of n-alkanes chromatographed under the same conditions as the samples. Thus, it was possible to identify 92.44% of the constituents, with the majority being d-carene (80.41%), a-bergamotene (1.95%), a-pinene (1.90), a-limonene (1.89) and caryophyllene oxide (1.81%) (Table 3).
Antimicrobial activity of Myracrodruon urundeuva
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DOI: 10.3109/13880209.2013.853810
The results found in the chemical analysis of the essential oil are consistent with previous studies which showed that most of the constituents of M. urundeuva are generally of the monoterpene class and that d-carene is the most encountered (Montanari, 2010). Table 4 shows the MIC values of the ethanol extract and essential oil tested against standard and multiresistant strains of E. coli and S. aureus. Comparatively, the samples of EEMU and EOMU showed the same MIC, with the exception of EEMU against SA-ATCC 25923, which showed better antibacterial activity with a MIC of 512 mg/mL. Natural products derived from medicinal plants have been used as a source of new bioactive compounds especially for the treatment of infectious diseases. Therefore, it is truly possible to use plants as a source of new bioactive compounds effective against bacteria resistant to conventional antibiotics (Buttler & Buss, 2006). These products of plant or animal origin can alter the effect of antibiotics, either increasing or decreasing their activity and may thus be termed modifiers of antibiotic activity (Coutinho et al., 2008; Rodrigues et al., 2009). Some of the classes of secondary metabolites identified in this study have been characterized as modifiers of antibiotic activity, such as terpenes (Nicolson et al., 1999), tannins (Figueredo et al., 2013; Matias et al., 2010a), flavonoids and derivatives (Sato et al., 2004). Table 5 shows the influence of the plant samples on the activity of the aminoglycosides and clindamcycin, demonstrating a synergistic effect, with a decrease in MIC against the Gram-positive strains. The most representative effect was seen with the combination of 128 mg/mL (MIC/8) EOMU with the antibiotics in the culture medium, showing a potentiation of amikacin combined with EOMU against SA 358 with a decrease in MIC from 64 to 8 mg/mL.
Table 4. Minimal inhibitory concentration (MIC) of the ethanol extract and essential oil of Myracrodruom urundeuva (mg/mL). Extracts and antimicrobial by natural products
EC27
EEMU EOMU
1024 1024
EC-ATCC10536 SA358 SA-ATCC25923 1024 1024
1024 1024
512 1024
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
Table 5. Minimum inhibitory concentration (MIC) of aminoglycosides and clindamycin in the presence and absence of ethanol extract and essential oil of Myracrodrum urunduiva at a concentration of MIC/8 (128 mg/mL).
Antibiotics Gentamicin Amikacin Clindamycin
563
The synergistic activity observed might have been due to the presence of secondary metabolites present in the extracts and oil, such as tannins and flavonoids, which are synthesized by plants in response to microbial infection (Dixon et al., 1983; Ho et al., 2001), where they are capable of disrupting the cell wall or plasma membrane facilitating drug uptake (Figueredo et al., 2013; Matias et al., 2010a; Tsuchiya et al., 1996). Due to uptake by normal cells, host toxicity is common to all aminoglycosides except streptomycin. Nephrotoxicity, ototoxicity and neuromuscular blockade are the most important toxic effects of aminoglycosides (Oliveira, 2006; Vallejo et al., 2001). The adverse effects of clindamycin consist mainly of gastrointestinal disorders that can manifest as severe diarrhea and pseudomembranous colitis (Katzung, 2005). The combination of natural products with aminoglycosides and clindamycin may be an alternative to minimize the undesirable effects of these antibiotics, when used for the treatment of Gram-positive infections since the combination leads to a synergistic effect, thereby significantly reducing the MIC of these drugs and allowing the use of lower doses for therapeutic success. The Gram-positive strain was more sensitive to the combination of antibiotics with plant substance compared with the Gram-negative strains. These results agree with those found in the literature (Veras, 2011), showing that Gramnegative bacteria are more resistant to the action of natural products, such as extracts and essential oils, because the outer membrane present in these bacteria forms a complex envelope, protecting them against the action of these antimicrobials (Holley & Pattel, 2005; Oladimeji et al., 2004). Besides, these bacteria possess other resistance mechanisms such as efflux pump, enzymes that cleave the beta-lactam ring (b-lactamases), and change in penicillin-binding protein, among others (Bush, 2002; Enright et al., 2002). However, our results are at odds with Deans and Ritchie (1987) who pointed out that the antibacterial action of essential oils is independent of Gram reaction (Dorman & Deans, 2000). Table 6 shows the influence of the plant samples on the activity of the b-lactam, demonstrating no synergistic effect. All b-lactam antibiotics inhibit bacterial growth by interfering with penicillin-binding protein, where the transpeptidation reaction is inhibited, blocking the synthesis of peptidoglycan and leading to cell death (Katzung, 2005). Many of these antibiotics penetrate Gram-negative bacteria through protein channels present in their outer membrane. Table 6. Minimum inhibitory concentration (MIC) of beta-lactam in the presence and absence of ethanol extract and essential oil of Myracrodrum urundeuva at a concentration of CIM/8 (128 mg/mL).
EC 27
SA 358
EC 27
SA 358
MIC combined
MIC combined
MIC combined
MIC combined
MIC
EEMU
EOMU
MIC
EEMU
EOMU
Antibiotics
CIM
EEMU
EOMU
CIM
EEMU
EOMU
64 128 1024
64 128 1024
64 128 1024
8 64 16
4 16 4
2 8 8
Benzetacil Ampicillin Oxacillin
1024 1024 1024
1024 1024 1024
1024 1024 1024
512 128 0.5
512 128 0.5
512 128 0.5
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
EEMU: ethanol extract of M. urundeuva; EOMU: essential oil of M. urundeuva; EC: E. coli; SA: S. aureus.
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Through these channels, the drug can reach its receptor in the cell wall and exert its antibacterial action (Nikaido, 1994). However, this activity of b-lactam alone or in combination with natural products against this strain was not observed in the present study, which can be related to resistance mechanisms and the presence of b-lactamase and other factors already mentioned (Bush, 2002; Enright et al., 2002). We observed b-lactam activity in the Gram-positive strains, but there was no modification of antibiotic activity in combinations with natural products.
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Conclusion The results of this study indicate that the ethanol extract and essential oil of M. urundeuva are an alternative source of natural products with antibacterial action, since they possess compounds with antibacterial activity recognized as tannins, flavonoids and terpenes, besides indicating their possible use in combination with conventional antibiotics against Grampositive bacterial strains. We conclude that the ethanol extract of aroeira has greater antibacterial activity, and that the oil is more effective in enhancing antibiotic activity against the S. aureus 358 strain.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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