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

Authors: Mirivaldo Barros Sá; Maria Taciana Ralph; Danielle Cristina Oliveira Nascimento; Clécio Souza Ramos; Isvânia Maria Serafin Barbosa; Fabrício Bezerra Sá; J. V. Lima-Filho
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

The chloroform extract of the stem bark ofAmburana cearensiswas 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 entericaSerotype Typhimurium,Pseudomonas aeruginosa,Staphylococcus aureus,Listeria monocytogenes, andBacillus 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 againstPseudomonas aeruginosaandBacillus 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 againstKlebsiella pneumoniaecarbapenemase-producing strains have shown similarMICresults and minimumbactericidal concentration (MBC) of 431 μg/mL.We conclude thatA. cearensisis a good source of methoxy-methylphenol compounds,which could be screened for antibacterial activity againstmultiresistant bacteria fromdifferent species


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## Body

## 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, althoughA. cearensishas 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 involucrataL.,Citrus acida (Roxb. Hook.f.), andAegle marmelos(L.) Correa ex Roxb. showed wide inhibitory action against several multidrug-resistant human pathogens, particularly,Burkholderia pseudomallei andStaphylococcus 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 ofA. cearensis have shown growth inhibition of a broad range of pathogens of veterinary interest [10]. In particular, hospital-acquired infections caused byKlebsiella 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 ofA. cearensis chloroform extracts against human clinical isolates ofK. 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 reportA. 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 ofA. 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 (30
m
×
0.25
mm
×
0.25
mm). 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/secm/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 μL 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 asEscherichia 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),andBacillus 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.


## 2.1. Collection and Botanical Identification

The stem bark ofA. 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 (30
m
×
0.25
mm
×
0.25
mm). 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/secm/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 μL 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 asEscherichia 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),andBacillus 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 ofA. cearensis were reported to be inhibitory againstStaphylococcus epidermidis, S. aureus, Klebsiella spp.,Salmonella spp.,Enterobacter aerogenes, Streptococcus pyogenes, Proteus mirabilis, Pseudomonas aeruginosa, andShigella flexneri,but no chemical characterization was performed [10]. Here, we report the phytochemistry and preliminary assessment ofA. cearensischloroform extracts as a source of novel antimicrobials against multiresistantKlebsiella strains.The GC/MS data showed a major peak at the retention time of 7.8 min with relative concentration of 76.7% (Figure1). 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%), α-pinene (1.0%), β-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 ofA. 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 ofAmburana 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
—
—
aCompounds are listed in order of their elution from a DB-5 column; bRI = retention indices relative to C7–C30  n-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 ofA. cearensis.The initial screening for antibacterial activity has shown that plants’ chloroform extract was only inhibitory againstP. aeruginosa andB. 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, whereasB. 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 againstK. 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 genegnr and b
l
a
kpc [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 ofA. 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 entericaTyphimurium
—
—
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 againstKlebsiella 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 inA. cearensis extracts varies despite the type of solvent used (Table 4). Accordingly, we have shown that chloroform extracts ofA. cearensis are good sources of methoxy-methylphenol compounds with antibacterial activity against several bacterial species and clinical isolates of multidrug-resistantK. 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 ofA. 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]



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*Source: 786586-2014-03-17.xml*