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Antibacterial activity of compounds isolated from Caesalpinia coriaria (Jacq) Willd against important bacteria in public health
Microbial Pathogenesis 136 (2019) 103660
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Antibacterial activity of compounds isolated from Caesalpinia coriaria (Jacq) Willd against important bacteria in public health
T
Agustín Olmedo-Juáreza, Tania Isabel Briones-Roblesb, Adrian Zaragoza-Bastidac,**, Alejando Zamilpad, Deyanira Ojeda-Ramírezc, Pedro Mendoza de Givesa, Jaime Olivares-Péreze, Nallely Rivero-Perezc,* a
Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID SAI-INIFAP), Carretera Federal Cuernavaca-Cuautla No. 8534 / Col. Progreso, C.P. 62550, Jiutepec, Morelos, A.P. 206-CIVAC, Mexico b Universidad Politécnica de Morelos. Boulevard Cuauhnáhuac #566, Col. Lomas del Texcal, Jiutepec, Morelos, CP 62550, Mexico c Área Académica de Medicina Veterinaria y Zootecnia, Instituto de Ciencias Agropecuaria, Universidad Autónoma del Estado de Hidalgo, Av. Universidad Km 1, Ex-Hda. de Aquetzalpa, 43600, Tulancingo, Hgo, Mexico d Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social, Argentina No. 1. Col. Centro, CP 62790, Xochitepec, Morelos, Mexico e Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Guerrero, Guerrero, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Caesalpinia coriaria (Jacq) Willd Methyl gallate Gallic acid Antibacterial activity
Antimicrobial resistance has been increasing in recent years and is most frequently found in pathogenic microorganisms resistant or multiresistant to drugs. The secondary metabolites of plants have been evaluated as alternatives for control and treatment of these microorganisms. The aim of this study was to isolate and identify the secondary metabolites with antibacterial activity from Caesalpinia coriaria (Jacq) Willd fruit. Hydroalcoholic extract (CCHA), was subjected to a bipartition with ethyl acetate giving two fractions an aqueous (Aq-F) and an organic (EtOAc-F). The isolation of bioactive fraction (EtOAc-F) allowed obtain two important compounds, methyl gallate (1) and gallic acid (2). These compounds were identified by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). The CCHA, both fractions and the isolated compounds were evaluated in vitro to determine their Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Listeria monocytogenes and Staphylococcus aureus. Gallic acid (2) showed the lowest MIC on S. typhi, (0.156 mg/mL), L. monocytogenes and S. aureus (1.25 mg/mL), while methyl gallate (1) had the best inhibitory effect against E. coli and P. aeruginosa (1.25 mg/mL). On the other hand, methyl gallate (1) showed the best MBC on P. aeruginosa (2.50 mg/mL), and gallic acid (2) had the lowest MBC on P. aeruginosa and L. monocytogenes. In conclusion, methyl gallate (1) and gallic acid (2) are the compounds responsible for the antibacterial activity of Caesalpinia coriaria fruit.
1. Introduction In February 2017, the World Health Organization (WHO) published a list of bacteria that show resistance or multiresistance to antibiotics (carbapenems, vancomycin, methicillin, fluoroquinolones, clarithromycin and cephalosporins) and that assigned levels of priority depending on the degree of resistance and potential danger to public health (critical, high and medium). Bacteria from the WHO list includes Pseudomonas aeruginosa, Escherichia coli (critical priority), Staphylococcus aureus, Salmonella spp. (high priority), Shigella spp. and Streptococcus pneumoniae (medium priority) [1]. Antibiotic resistance in bacteria can develop due to mechanisms *
such as de novo mutations, acquisition of genetic material from other microorganisms, transformation, transduction or metabolic adaptations to the drug, among other mechanisms [2,3]. This reduces the effectiveness of drugs and increases the possibility of spreading microorganisms to other people or animals, directly threatening public health [1]. Because of the increase in resistant pathogenic microorganisms, new alternatives to treatment and control, such as bacteriocins, phagotherapy and secondary metabolites from plants (phytochemicals) with antibacterial activity, have been evaluated [4–6]. Caesalpinia coriaria (Jacq) Willd, known as cascalote in Mexico, is a tree native to tropical America and the West Indies. It is used in Mexican traditional medicine due to its anti-inflammatory, analgesic,
Corresponding author. Corresponding author. E-mail addresses: [email protected] (A. Zaragoza-Bastida), [email protected] (N. Rivero-Perez).
**
https://doi.org/10.1016/j.micpath.2019.103660 Received 1 December 2018; Received in revised form 5 August 2019; Accepted 6 August 2019 Available online 06 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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ATCC9027; Salmonella typhi, ATCC14028), accordance with the CLSI guidelines [13].
healing, hemostatic, antidiarrheal, antiproliferative, hypercholesterolemic, antiarthritic, antiacne, hepatoprotective, anticancer and antimicrobial properties [7,8]. With respect to the antimicrobial properties, some studies indicate that the aqueous extract of the fruit and the alcoholic extract of the leaves of C. coriaria possess an antimicrobial effect against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Xanthomonas pathovars [9,10]. However, C. coriaria's active antibacterial compounds have not yet been identified. The aim of this study was to isolate and identify secondary metabolites with antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhi and Staphylococcus aureus from the fruit of Caesalpinia coriaria (Jacq) Willd.
2.4.1. Minimal inhibitory concentration (MIC) The MIC was determined using the microdilution method described by Mothana et al.,2009 and Kaewpiboon et al., 2012 [14,15]. The concentrations evaluated were 100, 50, 25, 12.50, 6.25, 3.12, 1.56, and 0.78 mg/mL for CCHA, Aq-F and EtOAc-F and 10.0, 5.0, 2.50, 1.12, 0.62, 0.31, 0.15 and 0.07 mg/mL for the pure compounds. EtOAc-F, C1F15 and C1F17 were suspended in 4% methanol in nutrient broth, while the rest were dissolved in nutrient broth. Next, 100 μL duplicates of each concentration of extract, fraction or compound and 10 μL of a bacterial suspension, adjusted to 0.5 McFarland standard (150 × 106 cells/mL) in nutrient broth (DIFCO ®) solution, were added to 96-well plates. The plates were incubated at 37 °C for 24 h at 70 rpm. Kanamycin (AppliChem 4K10421) and Tetracycline (aMReSCO 1135B29) (64–0.5 μg/mL) were used as the positive controls, while nutrient broth and 4% methanol in nutrient broth were used as the negative controls. The MIC was determined using a colorimetric method with tetrazolium salts [16]. After incubation, 20 μL of p-iodonitrotetrazolium solution [0.04% (w/v)] was added to each well, and the plates were incubated at 37 °C for 30 min at 70 rpm. Bacterial viability was confirmed by the observation of pink coloration after the addition of the piodonitrotetrazolium salt. The MIC of each sample was the lowest concentration that inhibited visible bacterial growth [14,15].
2. Materials and methods 2.1. Collection and identification of plant The fruit of Caesalpinia coriaria was harvested from Palmar Grande located in the Amatepec Municipality, in the State of Mexico, Mexico (18°23′24.8″N, 100°17′03.5″W), between March and April of 2016. An herbarium sample was deposited at the Herbarium of the Centro de Investigación en Biodiversidad y Conservación of the Universidad Autónoma de Morelos (CIByC, UAEM) with the code number 35274. The fresh fruit was washed and then dried at room temperature in the dark. 2.2. Preparation of hydroalcoholic extract
2.4.2. Minimal bactericidal concentration (MBC) After incubation and previous addition of p-iodonitrotetrazolium, 5 μL from each well was inoculated in Mueller-Hinton agar and incubated at 37 °C for 24 h. The MBC was the lowest concentration of antimicrobial agent that kills > 99.9% of the initial bacterial population and when no visible growth of the bacteria was observed on the plates [16].
The dried Caesalpinia coriaria fruit (1000 g) was macerated using an aqueous methanol solution (70%, 1:10 ratio, w/v) at room temperature for 24 h to obtain an extract. The extract was filtered using Whatman filter paper (Whatman ® 4). After filtration the solvent was eliminated using a rotary evaporator (Büchi R-300, Switzerland) to obtain a semisolid extract, which was finally freeze-dried and stored at −4 °C until the phytochemical analysis and antimicrobial evaluation.
2.4.3. Statistical analysis The MIC and MBC results were normalized using log10 and were analyzed by a completely randomized design through ANOVA using the general linear model (GLM). Differences among means were assessed by Tukey's multiple comparison statistical analysis at the p = 0.05 level of significance using SAS program, version 9.0 [17].
2.3. Identification of major compounds The identification of major compounds from C. coriaria fruits was performed trough a chemical fractionation of a hydroalcoholic extract (CCHA), which was previously reported by García-Hernández et al., 2019 [11]. The bipartition of CCHA with ethyl acetate allowed obtained an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F) [12]. Then two important compounds from this last fraction (methyl gallate, 1 and gallic acid, 2) were isolated. In the present study, the CCHA, AqF, EtOAc-F and compounds (1 and 2) were utilized in the antibacterial assays. The chemical analysis (HPLC and NMR) in the extract, fractions and compounds from C. coriaria fruits was carried out based according to García-Hernández et al., 2019 [11]. Gallic acid and methyl gallate were identified by comparison of the retention times and UV spectra with the reference standards (Rt = 7.08 and 9.3 min, respectively) (Sigma-Aldrich, St Louis Mo, USA).
3. Results 3.1. Identification of major compounds The CCHA, Aq-F and EtOAc-F analysis resulted in 122.0, 93.04.0 and 19.76 g/kg of dry matter, respectively. On the other hand, the chromatographic analysis in the CCHA, Aq-F and EtOAc-F revealed the presence of gallic acid derivates (Fig. 1 ACD). The chromatographic purification of EtOAc-F allowed us to obtain two pure compounds: methyl gallate (1) and gallic acid (2). The chromatographic analysis by HPLC of 1 and 2 revealed the presence of methyl gallate and gallic acid, respectively (Fig. 2). To identify the structure of these compounds, structural elucidation was performed by NMR spectra data analysis (1H, 13C) and were compared with the data described in the literature. According to the chemical shift displacements of 1H and 13C NMR, C1F15 corresponds to methyl gallate (compound 1) and C1F17 to gallic acid (compound 2) (Fig. 3).
2.3.1. NMR analysis Spectroscopic data of 1H and 13C NMR of C1F15, C1F17 were analyzed in a Bruker Avance III HD 500 MHz NMR Spectrometer. CD3OD was used as the solvent. 2.4. Antibacterial assay
3.2. Antibacterial assay
To determinate the antibacterial activity, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for extract, fractions and metabolites from Caesalpinia cariaria against two Gram-positive bacteria (Listeria monocytogenes, ATTCC19113; Staphylococcus aureus, ATCC6538) and three Gram-negative bacteria (Escherichia coli, ATCC35218; Pseudomonas aeruginosa,
3.2.1. Minimal inhibitory concentration (MIC) The broth microdilution method was used to determine the minimal inhibitory concentration for CCHA, Aq-F, EtOAc-F, methyl gallate (1) and gallic acid (2). Table 1 shows that CCHA inhibits the growth of 2
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Fig. 1. HPLC chromatogram of A) hydroalcoholic extract CCHA, B) aqueous fraction (Aq-F) and C) organic fraction (EtOAc-F) at λ = 280 nm.
4. Discussion
Gram-positive and Gram-negative bacteria and that EtOAc-F was more active than Aq-F. In addition, there are some differences between the activity of methyl gallate (1) and gallic acid (2). Methyl gallate (1) was the most active treatment against E. coli (MIC = 1.25 mg/mL, P = 0.0001). In contrast, gallic acid (2) was statistically (P = 0.0001) more active than methyl gallate (1) against S. typhi (MIC = 0.15 and 0.62 mg/mL, respectively). Moreover, the MIC values for methyl gallate (1) and gallic acid (2) against P. aeruginosa were equal (1.25 mg/mL, P = 0.0001), but it was statically lower than the MIC of CCHA, Aq-F and EtOAc-F. Finally, Gram-positive bacteria L. monocytogenes and S. aureus were more sensitive to EtOAc-F (MIC = 0.78 and 0.39 mg/mL, respectively). These results were statistically lowest (P = 0.0001) than the MIC values obtained for CCHA, Aq-F, methyl gallate (1) and gallic acid (2).
Currently, drug resistance reported in human and animal pathogens is an important problem in public health, and the use of plants with antimicrobial activity is a potential source of new antibiotics that offer therapeutic benefits and affordable treatments [18,19]. The Fabaceae family has 745 genera and more than 19,500 species of legumes [20], including Caesalpinia species, which have astringent, antiseptic, antimicrobial, anti-inflammatory and chemopreventive or antineoplastic effects [8]. Caesalpinia coriaria (cascalote) possesses some pharmacological properties such as anti-inflammatory, antiproliferative and antimicrobial effects attributed to the presence of dodecadien-1-ol, n-hexadecanoic, tridecanoic and octadecatrienoic acids, 3-cyclopentylpropionamide, phytol, 2-ethyl-9,12,15-octadecatrienoate, squalene, tannins, flavonoids, glycosides, stigmasterol, ethyl gallate, gallic acid and vitamin E in the plant [7,8,21]. Despite reports about the antibacterial activity of this plant, the chemical components responsible of these pharmacological effects have not been identified. Table 1 shows the MIC obtained for CCHA, Aq-F and EtOAc-F. The hydroalcoholic extract of C. coriaria fruit (CCHA) obtained in this study has a slightly antibacterial effect against all tested bacteria (MIC = 25.0 to 3.12 mg/mL). On the other hand, the bipartition procedure increased the antibacterial effect. In fact, EtOAc-F was the most active fraction (E. coli = 1.56, P. aeruginosa = 6.25, S. typhi = 3.12, L. monocytogenes 0.78, S. aureus = 0.39 mg/mL). EtOAc-F was sixteen fold more active
3.2.2. Minimum bactericidal concentration (MBC) Minimum bactericidal concentration (MBC) was determined by the lowest concentration of compound at which no colony-forming units (CFUs) were detected on solid media. The MBC values corresponding to extract, fractions and isolated compounds are shown in Table 2. Gallic acid (2) was the most effective compound (P = 0.0001) against all tested bacteria. This compound killed 99.9% of L. monocytogenes and P. aeruginosa at 5 mg/mL and 99.9% of E. coli, S. typhi, S. aureus at 10 mg/ mL. On the other hand, methyl gallate (1) only showed bactericidal activity against P. aeruginosa at 2.5 mg/mL (P = 0.0001). 3
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Fig. 2. HPLC chromatogram of A) organic fraction (EtOAc-F), B) C1F15 and C) C1F17.
between both compounds [24]. On the other hand, Ekaprasada et al. (2015) obtained a MIC of 7.5 mg/mL against S. aureus and E. coli using the disk diffusion susceptibility method with methyl gallate isolated from the leaves of Toona sureni as can see our results are three and six time better that the obtained in the Ekaprasada et al., experiment [25]. Ahmed et al. (2017) obtained an MIC of 0.1 and 0.05 mg/mL of the same compounds obtained from Glochidion superbum leaves on methicillin-resistant S. aureus isolated from blood and pus [26]. In this study gallic acid (2) showed MIC values of 1.25 mg/mL for P. aeruginosa, 2.5 mg/mL for E. coli, 1.25 mg/mL for S. aureus, 1.25 mg/mL for L. monocytogenes and 0.15 mg/mL for S. typhi. Akiyama et al., in 2001, determined that 8 mg/mL was the MIC to the gallic acid when was evaluated on S. aureus, this concentration is greater than the obtained in the present experiment, but the strains used in our experiment were ATCC and Akiyama et al., evaluated strains isolated from furuncle lesions and impetigo [27]. Borges et al. (2013) reported the MIC for gallic acid (Sigma Aldrich) on different bacterial genera including P. aeruginosa (0.50 mg/mL), E. coli (1.50 mg/mL), S. aureus (1.75 mg/mL) and L. monocytogenes (2.0 mg/mL). Our results are not totally comparable with those
against E. coli; more active against P. aeruginosa, fourteen fold more active against L. monocytogenes and fifteen fold more active against S. aureus than CCHA against the same bacteria. These results suggested to us that the antibacterial compounds from C. coriaria fruit were in the organic fraction. Chromatographic purification of EtOAc-F allows us identified methyl gallate (1) and gallic acid (2) as major components. Methyl gallate (1) and gallic acid (2) are phenolic compounds with antioxidant, anti-inflammatory, antibacterial, antifungal, and antiviral activity. These pharmacological actions have been associated with the capacity of these compounds to form ionic and peptide bonds, thus modulating enzymatic activity, ion channels, transporters and transcription factors [20,22]. Furthermore, gallic acid shows a bacteriostatic effect against methicillin-resistant Staphylococcus aureus, E. coli, P. aeruginosa and S. typhi [19,23]. The MIC values for methyl gallate (1) obtained in this study (Table 1) against S. typhi, S. aureus and E. coli (0.62, 2.5 and 1.25 mg/ mL, respectively). Choi et al. (2009) reported MIC of 0.125–0.5 mg/mL against nalidixic acid-resistant Salmonella species and 0.125 mg/mL for E. coli [24]. These results are different because Choi et al., mixed methyl gallate with nalidixic acid and described a synergic activity 4
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Table 2 Minimum Bactericidal Concentration (mg/mL) of extract, fractions and compounds obtained from Caesalpinia coriaria (Jacq) Willd fruits for E. coli, P. aeruginosa, S. typhi, L. monocytogenes and S. aureus. Evaluated compound
Bacteria E. coli
CCHA Aq-F EtOAc-F Methyl gallate (1) Gallic acid (2) Tetracycline* Kanamycin* Nutrient broth Nutrient broth + 4% methanol
CCHA Aq-F EtOAc-F Methyl gallate (1) Gallic acid (2) Tetracycline* Kanamycin* Nutrient broth Nutrient broth + 4% methanol
Bacteria P. aeruginosa
S. typhi
L. monocytogenes
S. aureus
25.00 g 3.12f 1.56 d 1.25c
12.50 d 25.00e 6.25c 1.25 b
3.12e 3.12e 3.12e 0.62 d
3.12f 1.56e 0.78c 5.00 g
6.25 g 3.12f 0.39c 2.50e
2.50e 8.00 b 4.00a NA NA
1.25 b 32.00a 32.00a NA NA
0.15c 4.00a 8.00 b NA NA
1.25 d 4.00 b 1.00a NA NA
1.25 d 8.00 b 4.00a NA NA
100.0 25.0f 6.2e 2.5c 5.0 d 32.0 b 16.0a NA NA
g
S. typhi e
25.0 100.0f 12.5 d NA 10.0c 32.0 b 16.0a NA NA
L. monocytogenes d
25.0 50.0e 12.5c NA 5.0 b 16.0a 16.0a NA NA
S. aureus 25.0 d 100.0e 12.5c NA 10.0 b 16.0a 16.0a NA NA
0.39 mg/mL) is lowest than the observed with methyl gallate (5 and 2.5 mg/mL) and gallic acid (1.25 mg/mL for both) for L. monocytogenes and S. aureus. This effect could be due to synergism between compounds contained in EtOAc-F, which being separated by the bipartition, loses or decrease their activity. There are studies that indicate than the secondary metabolites, can act synergistically or antagonist depending on the target organism [30]; this effect was observed in the present study, since for Gram-positive a synergistic effect was observed and for Gram-negative the effect was antagonic. The results of MIC analysis are not a viable option to fully evaluate the effectiveness of a drug since the pharmacokinetic and pharmacological properties of the drugs must also be considered [31]. Because viable cells may still be present in each well, the minimum bactericidal concentration (MBC) is evaluated to analyze both results and to determine whether the drug (extract) has a bacteriostatic or bactericidal effect [32]. In the present study gallic acid (2) showed the best MBC, 5 mg/mL for L. monocytogenes and P. aeruginosa and 10 mg/mL for E. coli, S. aureus, S. typhi. Borges et al. (2013) reported similar MBC values for gallic acid (Sigma-Aldrich) against E. coli (5.0 mg/mL), L. monocytogenes (5.5 mg/mL) and S. aureus (5.25 mg/mL) [18]. However, in that experiment, the mechanism of action of gallic acid was facilitated with the inclusion of DMSO in the bacterial inoculum. Fu et al., in 2016 reported 0.63 mg/mL as the MBC to gallic acid over S. aureus and 5 mg/ mL against E. coli, S. typhimurium and P. aeruginosa; the differences observed between both experiments could be associated with the strains used and the methods employed for extract and concentrate the secondary compounds [28]. On the other hand, methyl gallate (1) only showed bactericidal activity against P. aeruginosa (2.5 mg/mL). With respect to the mechanism of action of these compounds on bacteria, methyl gallate and gallic acid can produce hyperpolarization of the cell membrane [19,33]. The negative charge on the surface of the bacterial cell wall (zeta potential) is associated with lipopolysaccharides (LPS) or phospholipids in Gram-negative bacteria and with peptidoglycan or lipoteichoic acid in Gram-positive bacteria. This charge is affected by electrostatic changes caused by external agents that alter the permeability of the bacterial cell surface and cause their death [34,35]. Furthermore, gallic acid induces a loss of Ca2+ through the cell membrane of E. coli [36].
Table 1 Minimal Inhibitory Concentration (mg/mL) of extract, fractions and compounds obtained from Caesalpinia coriaria (Jacq) Willd fruit for E. coli, P. aeruginosa, S. typhi, L. monocytogenes and S. aureus.
E. coli
50.0 50.0e 25.0 d NA 10.0c 16.0 b 4.0a NA NA
P. aeruginosa
NA = No activity, CCHA = Hydroalcoholic extract of Caesalpinia coriaria fruits Aq-F = aqueous fraction, EtOAc-F = organic fraction, *Concentration in μg/ mL, Different literals in the column show significant differences (P ≤ 0.05) among the compounds evaluated.
Fig. 3. Chemical structure of methyl gallate (1) and gallic acid (2).
Evaluated compound
e
NA = No activity, CCHA = Hydroalcoholic extract of Caesalpinia coriaria fruits, Aq-F = aqueous fraction, F-AcEtO = organic fraction. *Concentration in μg/ mL, Different literals in the column show significant differences (P ≤ 0.05) among the compounds evaluated.
reported by Borges et al. (2013), who used DMSO (dimethyl sulfoxide) to modify the permeability of the cytoplasmic membrane during the time that the bacteria were incubated with gallic acid (30 min) [19]. In contrast, in our study the incubation time was without DMSO. In the present study was found a better antibacterial effect of gallic acid obtained from C. coriaria fruit against S. aureus and L. monocytogenes, even in absence of DMSO. Others studies found that gallic acid showed antibacterial activity and inhibition against S. aureus (0.63 mg/mL), E. coli, S. typhimurium and P. aeruginosa (2.5 mg/mL); these concentration are lowest than obtained in our study against S. aureus, similar for E. coli, highest on P. aeruginosa and S. typhi, the disagreements can be explained by the different microbial strains employed and extraction and concentrations processes of extract used in each experiment [28]. In 2016 Lima et al., reported a MIC for gallic acid against S. aureus, E. coli and P. aeruginosa resistant to drugs above 1024 μg/mL, results that match with our results, except for S. typhi (150 μg/mL); these authors indicated that gallic acid have synergistic effect with Norfloxacin and Gentamicin over S. aureus [29]. As can see in Table 1, the MIC obtained with EtOAc-F (0.78 y
5. Conclusion Despite reports about the antibacterial activity of Caesalpinia coriaria (Jacq) Willd, the chemical components responsible of this 5
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pharmacological effect had not been identified until now. In the present experiment were evaluated hidroalcoholic extract (CCHA), aqueous fraction (Aq-F), organic fraction (EtOAc-F), methyl gallate (1) and gallic acid (2) obtained from Caesalpinia coriaria fruits against E. coli, P. aeruginosa, L. monocytogenes, S. typhi and S. aureus. Methyl gallate (1) and gallic acid (2) isolated from EtOAc-F are the compounds associated with the antibacterial activity of Caesalpinia coriaria (Jacq) Willd fruits. These compounds could be a pharmacological alternative for the treatment of diseases caused for E. coli, P. aeruginosa, L. monocytogenes, S. typhi and S. aureus, microorganism that are on the WHO list, with critical and high priority; because have generated resistance or multiresistance to drugs, a global public health problem.
[13]
[14]
[15]
[16]
[17] [18]
Conflicts of interest
[19]
All authors declare that they have no competing interests. [20]
Funding Part of this SIGI:8215734475).
[21]
work
was
supported
by
INIFAP
(Project
[22]
Acknowledgements
[23]
This study formed part of the thesis work of Miss Tania Isabel Briones-Robles to obtain a degree in Biotechnology Engineering (at the Universidad Politécnica del Estado de Morelos, at Jiutepec, Morelos, Mexico).
[24]
[25]
[26]
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Grundhöfer, et al., Biosynthesis and subcellular distribution of hydrolyzable tannins, Phytochemistry 57 (6) (2001) 915–927 https://doi.org/10.1016/S00319422(01)00099-1. A. Chanwitheesuk, et al., Antimicrobial gallic acid from Caesalpinia mimosoides Lamk, Food Chem. 100 (3) (2007) 1044–1048 https://doi.org/10.1016/j. foodchem.2005.11.008. J.G. Choi, et al., Antibacterial activity of methyl gallate isolated from Galla Rhois or carvacrol combined with nalidixic acid against nalidixic acid resistant bacteria, Molecules 14 (5) (2009) 1773–1780 https://doi.org/10.3390/molecules14051773. M.T. Ekaprasada, et al., Antibacterial Activity of methyl gallate isolated from the leaves of Toona sureni, Int. J. Adv. Sci. Eng. Inf. Technol. 5 (4) (2015) 280–282 https://doi.org/10.18517/ijaseit.5.4.537. M.D. Ahmed, et al., Antimicrobial activity of methyl gallate isolated from the leaves of Glochidion superbum against hospital isolates of methicillin resistant Staphylococcus aureus, Nat. Prod. 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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Antibacterial activity of compounds isolated from Caesalpinia coriaria (Jacq) Willd against important bacteria in public health
T
Agustín Olmedo-Juáreza, Tania Isabel Briones-Roblesb, Adrian Zaragoza-Bastidac,**, Alejando Zamilpad, Deyanira Ojeda-Ramírezc, Pedro Mendoza de Givesa, Jaime Olivares-Péreze, Nallely Rivero-Perezc,* a
Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID SAI-INIFAP), Carretera Federal Cuernavaca-Cuautla No. 8534 / Col. Progreso, C.P. 62550, Jiutepec, Morelos, A.P. 206-CIVAC, Mexico b Universidad Politécnica de Morelos. Boulevard Cuauhnáhuac #566, Col. Lomas del Texcal, Jiutepec, Morelos, CP 62550, Mexico c Área Académica de Medicina Veterinaria y Zootecnia, Instituto de Ciencias Agropecuaria, Universidad Autónoma del Estado de Hidalgo, Av. Universidad Km 1, Ex-Hda. de Aquetzalpa, 43600, Tulancingo, Hgo, Mexico d Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social, Argentina No. 1. Col. Centro, CP 62790, Xochitepec, Morelos, Mexico e Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Guerrero, Guerrero, Mexico
A R T I C LE I N FO
A B S T R A C T
Keywords: Caesalpinia coriaria (Jacq) Willd Methyl gallate Gallic acid Antibacterial activity
Antimicrobial resistance has been increasing in recent years and is most frequently found in pathogenic microorganisms resistant or multiresistant to drugs. The secondary metabolites of plants have been evaluated as alternatives for control and treatment of these microorganisms. The aim of this study was to isolate and identify the secondary metabolites with antibacterial activity from Caesalpinia coriaria (Jacq) Willd fruit. Hydroalcoholic extract (CCHA), was subjected to a bipartition with ethyl acetate giving two fractions an aqueous (Aq-F) and an organic (EtOAc-F). The isolation of bioactive fraction (EtOAc-F) allowed obtain two important compounds, methyl gallate (1) and gallic acid (2). These compounds were identified by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). The CCHA, both fractions and the isolated compounds were evaluated in vitro to determine their Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Listeria monocytogenes and Staphylococcus aureus. Gallic acid (2) showed the lowest MIC on S. typhi, (0.156 mg/mL), L. monocytogenes and S. aureus (1.25 mg/mL), while methyl gallate (1) had the best inhibitory effect against E. coli and P. aeruginosa (1.25 mg/mL). On the other hand, methyl gallate (1) showed the best MBC on P. aeruginosa (2.50 mg/mL), and gallic acid (2) had the lowest MBC on P. aeruginosa and L. monocytogenes. In conclusion, methyl gallate (1) and gallic acid (2) are the compounds responsible for the antibacterial activity of Caesalpinia coriaria fruit.
1. Introduction In February 2017, the World Health Organization (WHO) published a list of bacteria that show resistance or multiresistance to antibiotics (carbapenems, vancomycin, methicillin, fluoroquinolones, clarithromycin and cephalosporins) and that assigned levels of priority depending on the degree of resistance and potential danger to public health (critical, high and medium). Bacteria from the WHO list includes Pseudomonas aeruginosa, Escherichia coli (critical priority), Staphylococcus aureus, Salmonella spp. (high priority), Shigella spp. and Streptococcus pneumoniae (medium priority) [1]. Antibiotic resistance in bacteria can develop due to mechanisms *
such as de novo mutations, acquisition of genetic material from other microorganisms, transformation, transduction or metabolic adaptations to the drug, among other mechanisms [2,3]. This reduces the effectiveness of drugs and increases the possibility of spreading microorganisms to other people or animals, directly threatening public health [1]. Because of the increase in resistant pathogenic microorganisms, new alternatives to treatment and control, such as bacteriocins, phagotherapy and secondary metabolites from plants (phytochemicals) with antibacterial activity, have been evaluated [4–6]. Caesalpinia coriaria (Jacq) Willd, known as cascalote in Mexico, is a tree native to tropical America and the West Indies. It is used in Mexican traditional medicine due to its anti-inflammatory, analgesic,
Corresponding author. Corresponding author. E-mail addresses: [email protected] (A. Zaragoza-Bastida), [email protected] (N. Rivero-Perez).
**
https://doi.org/10.1016/j.micpath.2019.103660 Received 1 December 2018; Received in revised form 5 August 2019; Accepted 6 August 2019 Available online 06 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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ATCC9027; Salmonella typhi, ATCC14028), accordance with the CLSI guidelines [13].
healing, hemostatic, antidiarrheal, antiproliferative, hypercholesterolemic, antiarthritic, antiacne, hepatoprotective, anticancer and antimicrobial properties [7,8]. With respect to the antimicrobial properties, some studies indicate that the aqueous extract of the fruit and the alcoholic extract of the leaves of C. coriaria possess an antimicrobial effect against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Xanthomonas pathovars [9,10]. However, C. coriaria's active antibacterial compounds have not yet been identified. The aim of this study was to isolate and identify secondary metabolites with antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhi and Staphylococcus aureus from the fruit of Caesalpinia coriaria (Jacq) Willd.
2.4.1. Minimal inhibitory concentration (MIC) The MIC was determined using the microdilution method described by Mothana et al.,2009 and Kaewpiboon et al., 2012 [14,15]. The concentrations evaluated were 100, 50, 25, 12.50, 6.25, 3.12, 1.56, and 0.78 mg/mL for CCHA, Aq-F and EtOAc-F and 10.0, 5.0, 2.50, 1.12, 0.62, 0.31, 0.15 and 0.07 mg/mL for the pure compounds. EtOAc-F, C1F15 and C1F17 were suspended in 4% methanol in nutrient broth, while the rest were dissolved in nutrient broth. Next, 100 μL duplicates of each concentration of extract, fraction or compound and 10 μL of a bacterial suspension, adjusted to 0.5 McFarland standard (150 × 106 cells/mL) in nutrient broth (DIFCO ®) solution, were added to 96-well plates. The plates were incubated at 37 °C for 24 h at 70 rpm. Kanamycin (AppliChem 4K10421) and Tetracycline (aMReSCO 1135B29) (64–0.5 μg/mL) were used as the positive controls, while nutrient broth and 4% methanol in nutrient broth were used as the negative controls. The MIC was determined using a colorimetric method with tetrazolium salts [16]. After incubation, 20 μL of p-iodonitrotetrazolium solution [0.04% (w/v)] was added to each well, and the plates were incubated at 37 °C for 30 min at 70 rpm. Bacterial viability was confirmed by the observation of pink coloration after the addition of the piodonitrotetrazolium salt. The MIC of each sample was the lowest concentration that inhibited visible bacterial growth [14,15].
2. Materials and methods 2.1. Collection and identification of plant The fruit of Caesalpinia coriaria was harvested from Palmar Grande located in the Amatepec Municipality, in the State of Mexico, Mexico (18°23′24.8″N, 100°17′03.5″W), between March and April of 2016. An herbarium sample was deposited at the Herbarium of the Centro de Investigación en Biodiversidad y Conservación of the Universidad Autónoma de Morelos (CIByC, UAEM) with the code number 35274. The fresh fruit was washed and then dried at room temperature in the dark. 2.2. Preparation of hydroalcoholic extract
2.4.2. Minimal bactericidal concentration (MBC) After incubation and previous addition of p-iodonitrotetrazolium, 5 μL from each well was inoculated in Mueller-Hinton agar and incubated at 37 °C for 24 h. The MBC was the lowest concentration of antimicrobial agent that kills > 99.9% of the initial bacterial population and when no visible growth of the bacteria was observed on the plates [16].
The dried Caesalpinia coriaria fruit (1000 g) was macerated using an aqueous methanol solution (70%, 1:10 ratio, w/v) at room temperature for 24 h to obtain an extract. The extract was filtered using Whatman filter paper (Whatman ® 4). After filtration the solvent was eliminated using a rotary evaporator (Büchi R-300, Switzerland) to obtain a semisolid extract, which was finally freeze-dried and stored at −4 °C until the phytochemical analysis and antimicrobial evaluation.
2.4.3. Statistical analysis The MIC and MBC results were normalized using log10 and were analyzed by a completely randomized design through ANOVA using the general linear model (GLM). Differences among means were assessed by Tukey's multiple comparison statistical analysis at the p = 0.05 level of significance using SAS program, version 9.0 [17].
2.3. Identification of major compounds The identification of major compounds from C. coriaria fruits was performed trough a chemical fractionation of a hydroalcoholic extract (CCHA), which was previously reported by García-Hernández et al., 2019 [11]. The bipartition of CCHA with ethyl acetate allowed obtained an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F) [12]. Then two important compounds from this last fraction (methyl gallate, 1 and gallic acid, 2) were isolated. In the present study, the CCHA, AqF, EtOAc-F and compounds (1 and 2) were utilized in the antibacterial assays. The chemical analysis (HPLC and NMR) in the extract, fractions and compounds from C. coriaria fruits was carried out based according to García-Hernández et al., 2019 [11]. Gallic acid and methyl gallate were identified by comparison of the retention times and UV spectra with the reference standards (Rt = 7.08 and 9.3 min, respectively) (Sigma-Aldrich, St Louis Mo, USA).
3. Results 3.1. Identification of major compounds The CCHA, Aq-F and EtOAc-F analysis resulted in 122.0, 93.04.0 and 19.76 g/kg of dry matter, respectively. On the other hand, the chromatographic analysis in the CCHA, Aq-F and EtOAc-F revealed the presence of gallic acid derivates (Fig. 1 ACD). The chromatographic purification of EtOAc-F allowed us to obtain two pure compounds: methyl gallate (1) and gallic acid (2). The chromatographic analysis by HPLC of 1 and 2 revealed the presence of methyl gallate and gallic acid, respectively (Fig. 2). To identify the structure of these compounds, structural elucidation was performed by NMR spectra data analysis (1H, 13C) and were compared with the data described in the literature. According to the chemical shift displacements of 1H and 13C NMR, C1F15 corresponds to methyl gallate (compound 1) and C1F17 to gallic acid (compound 2) (Fig. 3).
2.3.1. NMR analysis Spectroscopic data of 1H and 13C NMR of C1F15, C1F17 were analyzed in a Bruker Avance III HD 500 MHz NMR Spectrometer. CD3OD was used as the solvent. 2.4. Antibacterial assay
3.2. Antibacterial assay
To determinate the antibacterial activity, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for extract, fractions and metabolites from Caesalpinia cariaria against two Gram-positive bacteria (Listeria monocytogenes, ATTCC19113; Staphylococcus aureus, ATCC6538) and three Gram-negative bacteria (Escherichia coli, ATCC35218; Pseudomonas aeruginosa,
3.2.1. Minimal inhibitory concentration (MIC) The broth microdilution method was used to determine the minimal inhibitory concentration for CCHA, Aq-F, EtOAc-F, methyl gallate (1) and gallic acid (2). Table 1 shows that CCHA inhibits the growth of 2
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Fig. 1. HPLC chromatogram of A) hydroalcoholic extract CCHA, B) aqueous fraction (Aq-F) and C) organic fraction (EtOAc-F) at λ = 280 nm.
4. Discussion
Gram-positive and Gram-negative bacteria and that EtOAc-F was more active than Aq-F. In addition, there are some differences between the activity of methyl gallate (1) and gallic acid (2). Methyl gallate (1) was the most active treatment against E. coli (MIC = 1.25 mg/mL, P = 0.0001). In contrast, gallic acid (2) was statistically (P = 0.0001) more active than methyl gallate (1) against S. typhi (MIC = 0.15 and 0.62 mg/mL, respectively). Moreover, the MIC values for methyl gallate (1) and gallic acid (2) against P. aeruginosa were equal (1.25 mg/mL, P = 0.0001), but it was statically lower than the MIC of CCHA, Aq-F and EtOAc-F. Finally, Gram-positive bacteria L. monocytogenes and S. aureus were more sensitive to EtOAc-F (MIC = 0.78 and 0.39 mg/mL, respectively). These results were statistically lowest (P = 0.0001) than the MIC values obtained for CCHA, Aq-F, methyl gallate (1) and gallic acid (2).
Currently, drug resistance reported in human and animal pathogens is an important problem in public health, and the use of plants with antimicrobial activity is a potential source of new antibiotics that offer therapeutic benefits and affordable treatments [18,19]. The Fabaceae family has 745 genera and more than 19,500 species of legumes [20], including Caesalpinia species, which have astringent, antiseptic, antimicrobial, anti-inflammatory and chemopreventive or antineoplastic effects [8]. Caesalpinia coriaria (cascalote) possesses some pharmacological properties such as anti-inflammatory, antiproliferative and antimicrobial effects attributed to the presence of dodecadien-1-ol, n-hexadecanoic, tridecanoic and octadecatrienoic acids, 3-cyclopentylpropionamide, phytol, 2-ethyl-9,12,15-octadecatrienoate, squalene, tannins, flavonoids, glycosides, stigmasterol, ethyl gallate, gallic acid and vitamin E in the plant [7,8,21]. Despite reports about the antibacterial activity of this plant, the chemical components responsible of these pharmacological effects have not been identified. Table 1 shows the MIC obtained for CCHA, Aq-F and EtOAc-F. The hydroalcoholic extract of C. coriaria fruit (CCHA) obtained in this study has a slightly antibacterial effect against all tested bacteria (MIC = 25.0 to 3.12 mg/mL). On the other hand, the bipartition procedure increased the antibacterial effect. In fact, EtOAc-F was the most active fraction (E. coli = 1.56, P. aeruginosa = 6.25, S. typhi = 3.12, L. monocytogenes 0.78, S. aureus = 0.39 mg/mL). EtOAc-F was sixteen fold more active
3.2.2. Minimum bactericidal concentration (MBC) Minimum bactericidal concentration (MBC) was determined by the lowest concentration of compound at which no colony-forming units (CFUs) were detected on solid media. The MBC values corresponding to extract, fractions and isolated compounds are shown in Table 2. Gallic acid (2) was the most effective compound (P = 0.0001) against all tested bacteria. This compound killed 99.9% of L. monocytogenes and P. aeruginosa at 5 mg/mL and 99.9% of E. coli, S. typhi, S. aureus at 10 mg/ mL. On the other hand, methyl gallate (1) only showed bactericidal activity against P. aeruginosa at 2.5 mg/mL (P = 0.0001). 3
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Fig. 2. HPLC chromatogram of A) organic fraction (EtOAc-F), B) C1F15 and C) C1F17.
between both compounds [24]. On the other hand, Ekaprasada et al. (2015) obtained a MIC of 7.5 mg/mL against S. aureus and E. coli using the disk diffusion susceptibility method with methyl gallate isolated from the leaves of Toona sureni as can see our results are three and six time better that the obtained in the Ekaprasada et al., experiment [25]. Ahmed et al. (2017) obtained an MIC of 0.1 and 0.05 mg/mL of the same compounds obtained from Glochidion superbum leaves on methicillin-resistant S. aureus isolated from blood and pus [26]. In this study gallic acid (2) showed MIC values of 1.25 mg/mL for P. aeruginosa, 2.5 mg/mL for E. coli, 1.25 mg/mL for S. aureus, 1.25 mg/mL for L. monocytogenes and 0.15 mg/mL for S. typhi. Akiyama et al., in 2001, determined that 8 mg/mL was the MIC to the gallic acid when was evaluated on S. aureus, this concentration is greater than the obtained in the present experiment, but the strains used in our experiment were ATCC and Akiyama et al., evaluated strains isolated from furuncle lesions and impetigo [27]. Borges et al. (2013) reported the MIC for gallic acid (Sigma Aldrich) on different bacterial genera including P. aeruginosa (0.50 mg/mL), E. coli (1.50 mg/mL), S. aureus (1.75 mg/mL) and L. monocytogenes (2.0 mg/mL). Our results are not totally comparable with those
against E. coli; more active against P. aeruginosa, fourteen fold more active against L. monocytogenes and fifteen fold more active against S. aureus than CCHA against the same bacteria. These results suggested to us that the antibacterial compounds from C. coriaria fruit were in the organic fraction. Chromatographic purification of EtOAc-F allows us identified methyl gallate (1) and gallic acid (2) as major components. Methyl gallate (1) and gallic acid (2) are phenolic compounds with antioxidant, anti-inflammatory, antibacterial, antifungal, and antiviral activity. These pharmacological actions have been associated with the capacity of these compounds to form ionic and peptide bonds, thus modulating enzymatic activity, ion channels, transporters and transcription factors [20,22]. Furthermore, gallic acid shows a bacteriostatic effect against methicillin-resistant Staphylococcus aureus, E. coli, P. aeruginosa and S. typhi [19,23]. The MIC values for methyl gallate (1) obtained in this study (Table 1) against S. typhi, S. aureus and E. coli (0.62, 2.5 and 1.25 mg/ mL, respectively). Choi et al. (2009) reported MIC of 0.125–0.5 mg/mL against nalidixic acid-resistant Salmonella species and 0.125 mg/mL for E. coli [24]. These results are different because Choi et al., mixed methyl gallate with nalidixic acid and described a synergic activity 4
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Table 2 Minimum Bactericidal Concentration (mg/mL) of extract, fractions and compounds obtained from Caesalpinia coriaria (Jacq) Willd fruits for E. coli, P. aeruginosa, S. typhi, L. monocytogenes and S. aureus. Evaluated compound
Bacteria E. coli
CCHA Aq-F EtOAc-F Methyl gallate (1) Gallic acid (2) Tetracycline* Kanamycin* Nutrient broth Nutrient broth + 4% methanol
CCHA Aq-F EtOAc-F Methyl gallate (1) Gallic acid (2) Tetracycline* Kanamycin* Nutrient broth Nutrient broth + 4% methanol
Bacteria P. aeruginosa
S. typhi
L. monocytogenes
S. aureus
25.00 g 3.12f 1.56 d 1.25c
12.50 d 25.00e 6.25c 1.25 b
3.12e 3.12e 3.12e 0.62 d
3.12f 1.56e 0.78c 5.00 g
6.25 g 3.12f 0.39c 2.50e
2.50e 8.00 b 4.00a NA NA
1.25 b 32.00a 32.00a NA NA
0.15c 4.00a 8.00 b NA NA
1.25 d 4.00 b 1.00a NA NA
1.25 d 8.00 b 4.00a NA NA
100.0 25.0f 6.2e 2.5c 5.0 d 32.0 b 16.0a NA NA
g
S. typhi e
25.0 100.0f 12.5 d NA 10.0c 32.0 b 16.0a NA NA
L. monocytogenes d
25.0 50.0e 12.5c NA 5.0 b 16.0a 16.0a NA NA
S. aureus 25.0 d 100.0e 12.5c NA 10.0 b 16.0a 16.0a NA NA
0.39 mg/mL) is lowest than the observed with methyl gallate (5 and 2.5 mg/mL) and gallic acid (1.25 mg/mL for both) for L. monocytogenes and S. aureus. This effect could be due to synergism between compounds contained in EtOAc-F, which being separated by the bipartition, loses or decrease their activity. There are studies that indicate than the secondary metabolites, can act synergistically or antagonist depending on the target organism [30]; this effect was observed in the present study, since for Gram-positive a synergistic effect was observed and for Gram-negative the effect was antagonic. The results of MIC analysis are not a viable option to fully evaluate the effectiveness of a drug since the pharmacokinetic and pharmacological properties of the drugs must also be considered [31]. Because viable cells may still be present in each well, the minimum bactericidal concentration (MBC) is evaluated to analyze both results and to determine whether the drug (extract) has a bacteriostatic or bactericidal effect [32]. In the present study gallic acid (2) showed the best MBC, 5 mg/mL for L. monocytogenes and P. aeruginosa and 10 mg/mL for E. coli, S. aureus, S. typhi. Borges et al. (2013) reported similar MBC values for gallic acid (Sigma-Aldrich) against E. coli (5.0 mg/mL), L. monocytogenes (5.5 mg/mL) and S. aureus (5.25 mg/mL) [18]. However, in that experiment, the mechanism of action of gallic acid was facilitated with the inclusion of DMSO in the bacterial inoculum. Fu et al., in 2016 reported 0.63 mg/mL as the MBC to gallic acid over S. aureus and 5 mg/ mL against E. coli, S. typhimurium and P. aeruginosa; the differences observed between both experiments could be associated with the strains used and the methods employed for extract and concentrate the secondary compounds [28]. On the other hand, methyl gallate (1) only showed bactericidal activity against P. aeruginosa (2.5 mg/mL). With respect to the mechanism of action of these compounds on bacteria, methyl gallate and gallic acid can produce hyperpolarization of the cell membrane [19,33]. The negative charge on the surface of the bacterial cell wall (zeta potential) is associated with lipopolysaccharides (LPS) or phospholipids in Gram-negative bacteria and with peptidoglycan or lipoteichoic acid in Gram-positive bacteria. This charge is affected by electrostatic changes caused by external agents that alter the permeability of the bacterial cell surface and cause their death [34,35]. Furthermore, gallic acid induces a loss of Ca2+ through the cell membrane of E. coli [36].
Table 1 Minimal Inhibitory Concentration (mg/mL) of extract, fractions and compounds obtained from Caesalpinia coriaria (Jacq) Willd fruit for E. coli, P. aeruginosa, S. typhi, L. monocytogenes and S. aureus.
E. coli
50.0 50.0e 25.0 d NA 10.0c 16.0 b 4.0a NA NA
P. aeruginosa
NA = No activity, CCHA = Hydroalcoholic extract of Caesalpinia coriaria fruits Aq-F = aqueous fraction, EtOAc-F = organic fraction, *Concentration in μg/ mL, Different literals in the column show significant differences (P ≤ 0.05) among the compounds evaluated.
Fig. 3. Chemical structure of methyl gallate (1) and gallic acid (2).
Evaluated compound
e
NA = No activity, CCHA = Hydroalcoholic extract of Caesalpinia coriaria fruits, Aq-F = aqueous fraction, F-AcEtO = organic fraction. *Concentration in μg/ mL, Different literals in the column show significant differences (P ≤ 0.05) among the compounds evaluated.
reported by Borges et al. (2013), who used DMSO (dimethyl sulfoxide) to modify the permeability of the cytoplasmic membrane during the time that the bacteria were incubated with gallic acid (30 min) [19]. In contrast, in our study the incubation time was without DMSO. In the present study was found a better antibacterial effect of gallic acid obtained from C. coriaria fruit against S. aureus and L. monocytogenes, even in absence of DMSO. Others studies found that gallic acid showed antibacterial activity and inhibition against S. aureus (0.63 mg/mL), E. coli, S. typhimurium and P. aeruginosa (2.5 mg/mL); these concentration are lowest than obtained in our study against S. aureus, similar for E. coli, highest on P. aeruginosa and S. typhi, the disagreements can be explained by the different microbial strains employed and extraction and concentrations processes of extract used in each experiment [28]. In 2016 Lima et al., reported a MIC for gallic acid against S. aureus, E. coli and P. aeruginosa resistant to drugs above 1024 μg/mL, results that match with our results, except for S. typhi (150 μg/mL); these authors indicated that gallic acid have synergistic effect with Norfloxacin and Gentamicin over S. aureus [29]. As can see in Table 1, the MIC obtained with EtOAc-F (0.78 y
5. Conclusion Despite reports about the antibacterial activity of Caesalpinia coriaria (Jacq) Willd, the chemical components responsible of this 5
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pharmacological effect had not been identified until now. In the present experiment were evaluated hidroalcoholic extract (CCHA), aqueous fraction (Aq-F), organic fraction (EtOAc-F), methyl gallate (1) and gallic acid (2) obtained from Caesalpinia coriaria fruits against E. coli, P. aeruginosa, L. monocytogenes, S. typhi and S. aureus. Methyl gallate (1) and gallic acid (2) isolated from EtOAc-F are the compounds associated with the antibacterial activity of Caesalpinia coriaria (Jacq) Willd fruits. These compounds could be a pharmacological alternative for the treatment of diseases caused for E. coli, P. aeruginosa, L. monocytogenes, S. typhi and S. aureus, microorganism that are on the WHO list, with critical and high priority; because have generated resistance or multiresistance to drugs, a global public health problem.
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Conflicts of interest
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All authors declare that they have no competing interests. [20]
Funding Part of this SIGI:8215734475).
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work
was
supported
by
INIFAP
(Project
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Acknowledgements
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This study formed part of the thesis work of Miss Tania Isabel Briones-Robles to obtain a degree in Biotechnology Engineering (at the Universidad Politécnica del Estado de Morelos, at Jiutepec, Morelos, Mexico).
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