Evidence-Based Complementary and Alternative Medicine

Volume 2014 (2014), Article ID 786586, 7 pages

http://dx.doi.org/10.1155/2014/786586

Research Article

Phytochemistry and Preliminary Assessment of the Antibacterial Activity of Chloroform Extract of Amburana cearensis (Allemão) A.C. Sm. against Klebsiella pneumoniae Carbapenemase-Producing Strains

Mirivaldo Barros Sá,¹ Maria Taciana Ralph,¹ Danielle Cristina Oliveira Nascimento,¹ Clécio Souza Ramos,² Isvânia Maria Serafin Barbosa,³ Fabrício Bezerra Sá,⁴ and J. V. Lima-Filho1,5

¹Departamento de Biologia, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-900 Recife, PE, Brazil

²Departamento de Ciências Moleculares, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-900 Recife, PE, Brazil

³Departamento de Biofísica, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-900 Recife, PE, Brazil

⁴Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-900 Recife, PE, Brazil

⁵Laboratório de Microbiologia e Imunologia, Departamento de Biologia, Universidade Federal Rural de Pernambuco, R. Dom Manoel de Medeiros s/n, Campus Dois Irmãos, 52171-900 Recife, PE, Brazil

Received 24 November 2013; Revised 7 January 2014; Accepted 10 January 2014; Published 17 March 2014

Academic Editor: Ulysses Paulino Albuquerque

Copyright © 2014 Mirivaldo Barros Sá et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The chloroform extract of the stem bark of Amburana cearensis was chemically characterized and tested for antibacterial activity.The extract was analyzed by gas chromatography and mass spectrometry. The main compounds identified were 4-methoxy-3-methylphenol (76.7%), triciclene (3.9%), α-pinene (1.0%), β-pinene (2.2%), and 4-hydroxybenzoic acid (3.1%). Preliminary antibacterial tests were carried out against species of distinct morphophysiological characteristics: Escherichia coli, Salmonella enterica Serotype Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentration (MIC) was determinate in 96-well microplates for the chloroform extract and an analogue of themain compound identified, which was purchased commercially.We have shown that plant’s extract was only inhibitory (but not bactericidal) at the maximum concentration of 6900 μg/mL against Pseudomonas aeruginosa and Bacillus cereus. Conversely, the analogue 2-methoxy-4-methylphenol produced MICs ranging from215 to 431 μg/mL against all bacterial species.New antibacterial assays conducted with such chemical compound against Klebsiella pneumoniae carbapenemase-producing strains have shown similarMICresults and minimumbactericidal concentration (MBC) of 431 μg/mL.We conclude that A. cearensis is a good source of methoxy-methylphenol compounds,which could be screened for antibacterial activity againstmultiresistant bacteria fromdifferent species

1. Introduction

Amburana cearensis (Allemão) A.C. Sm. (Fabaceae) is a native plant from Brazilian semiarid region widely used in folk medicine to treat nervous disorders, headaches, asthma, sinusitis, bronchitis, flu, and rheumatic pain, with the stem bark and seeds commonly being consumed as tea or infusion preparations [1–3]. The stem bark is rich in coumarin (1,2-benzopyrone), whose pharmacological properties include anti-inflammatory, antinociceptive, and bronchodilator effects [4–7]. Indeed, four new compounds (p-hydroxybenzoic acid, aiapin, and two stereoisomers of o-coumaric acid glycoside) were recently identified, showing plants’ repertory of potential bioactive substances [8]. However, although A. cearensis has been broadly used in treatment of respiratory infections, only a few studies report antimicrobial activity in plants’ extracts [9, 10], and, therefore, its potential as a source of new antimicrobials was not fully investigated.

The spread of multidrug resistance among bacteria from various sources [11–16] has made natural product researchers increase their effort on screening plant extracts for compounds with broad spectrum of antimicrobial activity. For example, Tragia involucrata L., Citrus acida (Roxb. Hook.f.), and Aegle marmelos (L.) Correa ex Roxb. showed wide inhibitory action against several multidrug-resistant human pathogens, particularly, Burkholderia pseudomallei and Staphylococcus aureus, which was related to the high contents of phenolic or polyphenolic compounds in methanol extracts [17, 18]. Likewise, recent antibacterial assays with stem bark ethanol extracts of A. cearensis have shown growth inhibition of a broad range of pathogens of veterinary interest [10]. In particular, hospital-acquired infections caused by Klebsiella pneumoniae (Enterobacteriaceae) have increased in recent years due to emergence of carbapenemase- producing strains [19, 20]. These bacteria are capable of hydrolyzing carbapenems, penicillins, cephalosporins, and aztreonam [21], and, therefore, the search for new therapeutics is forthcoming.

Here, a preliminary assessment of the antibacterial activity of A. cearensis chloroform extracts against human clinical isolates of K. pneumoniae carbapenemase-producing strains was conducted. Moreover, the plants’ extract was chemically characterized and an analogue of the major compound identified was also tested against bacteria. Although the chemical composition of plant extracts varies under influence of seasonal and climatic conditions [22], in the present study, we report A. cearensis as a new source of methoxy-methylphenol compounds with antibacterial activity. These are phenol derivatives with potential applications as antiseptics and biocides. These data are discussed in light of previous studies.

2. Material and Methods

2.1. Collection and Botanical Identification

The stem bark of A. cearensis was collected in the municipality of Salgueiro, Pernambuco, Brazil (latitude 08°04′27′′ south and longitude 39°07′09′′ west). The plant’s identification was made by comparing the aerial parts with exsiccate samples (voucher number 46090) deposited in the collection of Vasconcelos Sobrinho Herbarium, Department of Biology, Federal Rural University of Pernambuco, under the care of Dr. Suzene Izidio da Silva.

2.2. Plant Extract

Fresh plant material was collected and dried in oven at a temperature range of 45°C to 50°C for 48 h. The dried material was ground in a blade mill to obtain a fine homogeneous powder. This material was weighed and extracted by maceration using chloroform (P.A.) as solvent extractor in the ratio of 1 : 3 (w : v). The resulting mixture remained for 48 hours under agitation every two hours. The extract was filtered and concentrated in a rotary evaporator under reduced pressure at a temperature of 45°C for complete solvent removal. Stock solutions were prepared with extracts using dimethyl sulfoxide (DMSO) as solvent (100 mg/mL), which were kept in a refrigerator at −20°C until use [23].

2.3. Analysis by Gas Chromatography Coupled to Mass Spectrometry (GS/MS)

The plant extract was analyzed by GS/MS using a Varian 431-GC chromatograph coupled to a Varian 220-MS mass spectrometer, equipped with a J & W Scientific DB5 fused silica capillary column (). The temperature of the injector and detector was set at 260°C with the furnace temperature programmed in a range of 60–240°C at 3°C/min. The mass spectra were obtained with a 70 eV electron impact, 0.84 scan/sec m/z 40–550. The carrier gas used was helium at a flow rate of 1 mL/min. A stock solution of 2 mg/mL was prepared and 1.0 L was injected for analyses. The identification of the constituents was carried out by comparison with previously reported values of retention indices, obtained by coinjection of oil samples and C₁₁–C₂₄ linear hydrocarbons and calculated using the van Den Dool and Kratz equation [24], by direct comparison of the spectra with spectra stored in libraries of equipment (NIST21 and NIST107) as well as with the spectra and retention times of authentic compounds reported previously in the literature for comparison. Subsequently, the MS acquired for each component was matched with those stored in the NIST21, NIST107 mass spectral library of the GC-MS system and with other published mass spectral data [25].

2.4. Microorganism and Growth Conditions

Preliminary assessment of antibacterial activity was conducted with bacteria of distinct morphophysiological characteristics, such as Escherichia coli (facultative anaerobe, gram-negative, nonencapsulated, extracellular bacteria, Enterobacteriaceae), Salmonella enterica Serotype Typhimurium (facultative anaerobe, gram-negative, nonencapsulated, intracellular bacteria, Enterobacteriaceae), Pseudomonas aeruginosa (aerobe, gram-negative, extracellular bacteria, non-Enterobacteriaceae), Staphylococcus aureus (facultative anaerobe, gram-positive, non-spore-forming, extracellular bacteria), Listeria monocytogenes (facultative anaerobe, gram-positive, non-spore-forming, intracellular bacteria),and Bacillus cereus (aerobe, gram-positive, spore-forming, extracellular bacteria) belonging to the collection of the Laboratory of Microbiology and Immunology of Federal Rural University of Pernambuco (UFRPE). Klebsiella pneumoniae carbapenem-resistant strains (KPC) were obtained from human clinical cases and kindly provided by Dr. Marcia Moraes (University of Pernambuco). Single colonies from fresh cultures were streaked in tubes containing Brain Heart Infusion Agar after growth (37°C/18–24 h) and kept at 8°C until use.

2.5. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The MIC of the plant extract was determined by the microdilution method [34] in 96-well plates at concentrations ranging from 6.7 to 6900 µg/mL. In addition, the analogue 2-methoxy-4-methylphenol of the main compound identified in plant extracts (4-methoxy-3-methylphenol) was purchased commercially (Sigma) and also investigated. The density of the bacterial suspension was adjusted to approximately 10⁵ CFU/mL, and the results were read after an incubation period of 24 hours at 37°C. The MIC was the lowest concentration causing inhibition of visible growth. In this case, aliquots of 0.1 mL were transferred to plates containing Mueller-Hinton agar. The minimum bactericidal concentration (MBC) was considered the lowest concentration that resulted in no growth after incubation for 24 h at 37°C. All assays were performed in duplicate.

3. Results and Discussion

The Brazilian floras are worldwide known as a source of biologically active compounds with biodegradable properties, and, therefore, screening of plant extracts is often a first step procedure for isolation of phytochemicals [23]. However, studies that correlate antibacterial activity of extracts to the presence of specific compounds are rare. For instance, ethanol extracts of the stem bark of A. cearensis were reported to be inhibitory against Staphylococcus epidermidis, S. aureus, Klebsiella spp., Salmonella spp., Enterobacter aerogenes, Streptococcus pyogenes, Proteus mirabilis, Pseudomonas aeruginosa, and Shigella flexneri, but no chemical characterization was performed [10]. Here, we report the phytochemistry and preliminary assessment of A. cearensis chloroform extracts as a source of novel antimicrobials against multiresistant Klebsiella strains.

The GC/MS data showed a major peak at the retention time of 7.8 min with relative concentration of 76.7% (Figure 1). The interpretation of the mass spectra in comparison with spectra reported in the literature led to identification of the benzenoid 4-methoxy-3-methylphenol. Other compounds included tricyclene (3.9%), -pinene (1.0%), -pinene (2.2%), and 4-hydroxybenzoic acid (3.1%). The complete list of compounds identified in plants’ extract is reported in Table 1. Several compounds have been identified in the bark of A. cearensis, such as trans -3,4-methyl dimetoxicinamato, cis-3,4-dimethoxy-methyl cinnamate, 3-methoxy-4-hydroxy-methyl cinnamate, 4-hydroxy-methyl benzoate, 3,4-dihydroxy methyl benzoate, 3-hydroxy-4-methoxy methyl benzoate, catechol, guaiacol, a-ethoxy-p-cresol, benzenemethanol 4-hydroxy, 4-methoxy-methylphenol, 2,3-dihydrobenzofuran, and anthraquinone (chrysophanol), with predominance of coumarins being often reported [35].

Table 1: Chemical composition of the chloroform extract of the stem bark of Amburana cearensis.

Compoundsa
Relative area (%)
RIb
RIc

Tricyclene
3.9
919
921

α-Pinene
1.0
931
932

β-Pinene
2.2
974
974

6,10-Dodecatrien-1-ol, 3,7,11-trimethyl
2.3d
—
—

4-Methoxy-3-methylphenol
76.7d
—
—

Terpinolene
0.3
1087
1088

1,3,8-p-Menthatriene
0.5
1107
1108

4-Hydroxybenzoic acid
3.1d
—
—

Longifolene
1.7
1405
1407

cis-Muurolo-4(14),5-diene
3.2
1462
1465

Total
94.9
—
—

Compounds are listed in order of their elution from a DB-5 column; bRI = retention indices relative to C₇–C₃₀  -alkanes; cRI = retention indices from the literature. dIdentified by direct comparison of the spectra with spectra stored in libraries of equipment as well as with the spectra and retention times of authentic compounds reported previously in the literature for comparison.

Figure 1: Chromatogram of chloroform extract of the stem bark of A. cearensis.

The initial screening for antibacterial activity has shown that plants’ chloroform extract was only inhibitory against P. aeruginosa and B. cereus at the highest concentration of 6900 µg/mL (Table 2). P. aeruginosa is responsible for different etiological processes in immunocompetent and immunocompromised patients in hospitals, whereas B. cereus is an opportunistic pathogen [36, 37]. On the other hand, the analogue compound 2-methoxy-4-methylphenol showed broad spectrum activity against all bacteria tested (Table 3). Indeed, the MIC ranged from 215 to 431 µg/mL against K. pneumoniae carbapenemase-producing strains being also bactericidal at 431 µg/mL (Table 3). The carbapenems imipenem, meropenem, and ertapenem are often used as last therapeutic choices against gram-negative multidrug-resistant bacteria [38]. Moreover, these bacteria usually show resistance to aminoglycosides and fluoroquinolones due to the presence of gene gnr and [39]. Thus, the risk of nosocomial infections is higher for patients in hospital’s intensive care units [40, 41].

Table 2: Antibacterial activity of the chloroformextract of A. cearensis and the analogue 2-methoxy-4-methylphenol.

Bacterial species
Chloroform extract
2-Methoxy-4-methylphenol
Ciprofloxacin

MIC*
MBC
MIC
MBC
MIC
MBC

µg/ mL

Salmonella enterica Typhimurium
—
—
215
431
<6.7
<6.7

Escherichia coli
—
—
215
431
<6.7
<6.7

Pseudomonas aeruginosa
>6900
—
431
>6900
<6.7
<6.7

Bacillus cereus
>6900
—
431
3450
<6.7
107

Listeria monocytogenes
—
—
215
862
<6.7
<6.7

Staphylococcus aureus
—
—
215
862
<6.7
<6.7

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

Table 3: Antibacterial activity of 2-methoxy-4-methylphenol against Klebsiella pneumoniae carbapenemase-producing strains.

K. pneumonia strains
2-Methoxy-4-methylphenol
Ciprofloxacin

MIC*
MBC
MIC
MBC

(µg/mL)

KPC 201
215
431
<6.7
<6.7

KPC 199
215
431
<6.7
<6.7

KPC +
215
431
<6.7
215

KPC 278
215
431
<6.7
431

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration.

According to PubChem Compound Database, 4-methoxy-3-methylphenol is also known as 14786-82-4, AG-D-93185, NSC168522, AC1L6RKC, SureCN263095, and 4-methoxy-3-methylphenol [42]. Although anticancer assays were previously carried out with the compound, it was reported to be inactive against tumor model L1210 leukemia in mice [42]. While we were not aware of previous antimicrobial studies conducted with 4-methoxy-3-methylphenol, we hypothesize that the action mechanism of the molecule against bacteria is possibly due to 3-methylphenol compound (m-cresol) well known as an oxidizing agent [43]. Yet, m-cresol is a methyl derivative of phenol that has been used as precursor of amylmetacresol present in commercial antiseptic formulation.

The array of chemical compounds in A. cearensis extracts varies despite the type of solvent used (Table 4). Accordingly, we have shown that chloroform extracts of A. cearensis are good sources of methoxy-methylphenol compounds with antibacterial activity against several bacterial species and clinical isolates of multidrug-resistant K. pneumonia. Thus, the plants’ chloroform extract could be exploited as a source of antiseptics or biocides against drug-resistant bacteria from different species.

Table 4: Phytochemicals reported in extracts of A. cearensis.

Part used
Solvent
Secondary metabolites
Antibacterial activity
Reference

Stem bark
Ethanol
Not reported
Staphylococcus epidermidis
[9]

Stem bark
Ethanol
Not reported
Cocci strains, Enterobacteria, non-fermenting bacteria
[10]

Stem bark and leaves
Ethanol
Anthocyanins, anthocyanidins, flavones, chalcones, aurones, leucoanthocyanidins
Not reported
[11, 13]

Seeds
Butanol and hydroethanol
Flavonoids, proanthocyanidins, anthocyanins, carotenoids
Not reported
[26]

Aerial parts and xylopodium
Ethanol
Protocatechuic acid, vanillic acid, coumarin, amburoside
Not reported
[27]

Resin
Methanol and chloroform
Chalcone, 2′,4,4′-trihydroxy chalcone (isoliquiritigenin a) (1), 2′,4′, dihydroxy-3′,4′-methoxychalcone (2), 7,8,3′,4′-tetramethoxy isoflavone, 2′,4,4′-trihydroxy (isoliquiritigenin) (1);

2′,4′,dihydroxy-3′,4′-methoxy (2),7,8,3′,4′-tetramethoxy isoflavone
Not reported
[28]

Stem bark
Ethanol
Coumarin and phenolic compounds (isokaempferide and amburoside)
Not reported
[29]

Wood powder
Hydroethanol
1-Dodecanol; 2-ethyl-hexane acid; dihydrocoumarin; coumarin (1,2- benzopyrone)
Not reported
[30]

Stem bark
Ethanol
Isoflavonoid (afromorsin)
Not reported
[31]

Stem bark
Hexane and chloroform
Cumarina Coumarin (1,2 - benzopirona) (1,2-benzopyrone)
Not reported
[32]

Trunk bark
Ethanol
Coumarin and vanillic acid
Not reported
[33]

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors thank Dr. Suzene Izidio da Silva for botanical identification and Dr. Marcia Moraes for providing the Klebsiella strains used in the present study.

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