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Antimicrobial Annona muricata L. (soursop) extract targets the cell membranes of Gram-positive and Gram-negative bacteria
Industrial Crops & Products 107 (2017) 332–340
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Antimicrobial Annona muricata L. (soursop) extract targets the cell membranes of Gram-positive and Gram-negative bacteria
MARK
Nícolas de C.C. Pintoa, Lara M. Camposa, Anna Carolina S. Evangelistaa, Ari S.O. Lemosa, Thiago P. Silvab, Rossana C.N. Melob, Caroline C. de Lourençoc, Marcos J. Salvadorc, ⁎ Ana Carolina M. Apolôniod, Elita Scioa, Rodrigo L. Fabria, a
Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Brazil Laboratory of Cell Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil c Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, São Paulo, Brazil d Laboratory of Bacterial Physiology and Molecular Genetics, Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Annona muricata Antibacterial activity Membrane permeability Alkaloids Plant extract
Annona muricata has become an interesting subject in the search for new therapeutic agents. We investigated the bacterial mode of action of the methanolic extract of A. muricata leaves (AML). AML extract was tested against several bacteria strains by broth microdilution susceptibility method. The bacterial killing assay, bacterial abundance and membrane viability analysis were made using fluorescent probes. The nucleotide leakage and outer membrane (OM) permeability assays were used to verify membrane destabilization. The biochemical reaction profile was carried out on a VITEK®2 system. For UPLC-ESI–MS/MS Analysis an Acquity UPLC system was used. AML was active against both Gram-negative and Gram-positive bacteria, showing greater activity against S. aureus, S. typhimurium and E. faecalis. AML exhibited rapid time dependent kinetics of bacterial killing. DAPI staining revealed that AML inhibited the bacterial growth, while the LIVE/DEAD BacLight analysis showed that AML induced an increase in dead cells. AML increased nucleotide leakage and was also capable of increasing the OM permeability in the tested bacteria. Differences between the stressed clones and controls observed in the biochemical characterization were not enough to modify the strain identity. UPLC-ESI–MS/MS analysis revealed the presence of the alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline. Our findings demonstrate, for the first time, a broad spectrum of antibacterial activity for AML and identify that bacterial membranes (both plasma and outer membranes) are primary targets of this extract. Based on these observations, AML has a good potential for the design of novel antimicrobial agents.
1. Introduction The emergence and spread of microbial resistance is growing each day, thereby necessitating the development of new antimicrobials (Malik et al., 2017). Hospital-acquired infections are becoming a growing concern (WHO, 2015). Based on this, studies have shown that natural products appear to be resources for valuable resistance breaking molecules (Newman and Cragg, 2012). Plant derived compounds are of particular interest, based on their already proven antimicrobial activity (Jamkhande et al., 2016; Mocan et al., 2015; Mocan et al., 2016; Vlase et al., 2014). In this context, Annona muricata (Fig. 1) has become an interest subject in the search for new therapeutic agents. This species belongs to
the Annonaceae family, which consists of 29 genera and 390 species with tropical and subtropical distribution worldwide (Joly, 1979). It is popular known as soursop or prickly custard apple, and the fruits are edible and commonly used to make ice cream, teas and other drinks (Degnon et al., 2013). Several parts of this plant are traditionally used to treat cancer, diabetes, hypertension, fever, gastrointestinal disorders, and parasitic infections (Adjanohou, 1996). Various pharmacological studies have confirmed the traditional uses of Annona muricata (Adewole and Caxton-Martins, 2006; N’gouemo et al., 1997). In particular, the antimicrobial properties of Annona muricata leaves (AML) have attracted attention due to their potential to also treat viral infections (Florence et al., 2014), hyperlipidemia (Adewole and Ojewole, 2008) and neglected human diseases,
⁎ Corresponding author at: Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais CEP 36036 900, Brazil. E-mail addresses: [email protected], [email protected] (R.L. Fabri).
http://dx.doi.org/10.1016/j.indcrop.2017.05.054 Received 19 December 2016; Received in revised form 12 May 2017; Accepted 28 May 2017 Available online 09 June 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.4. Serial dilution assay for determination of the minimal inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) of AML was determined by microdilution techniques in Mueller Hinton broth (MHB) as described by CLSI (2010). Bacteria were cultured overnight at 37 °C for 24 h in MHA. Sample stock solution was two-fold diluted from 5000 to 2.5 μg mL−1 (final volume = 80 μL) and a final dimethyl sulfoxide (DMSO) concentration ≤1%. Then, 100 μL of MHB and 20 μL of 108 CFU mL−1 (according to McFarland turbidity standards) of standardized bacterial suspensions were inoculated onto microplates and the test was performed in a volume of 200 μL. Plates were incubated at 37 °C for 24 h. The same tests were performed simultaneously for growth control (MHB + bacteria + AML vehicle) and sterility control (MHB + AML vehicle) as well as the positive control with Chloramphenicol (500–0.24 μg mL−1). The MIC values were calculated as the highest dilution showing complete inhibition of the tested strain. Analyses were performed in triplicate. Extracts with MIC values ≤100 μg mL−1 were considered significantly active (Kuete, 2010).
Fig. 1. Annona muricata plant with focus on the Annona muricata leaves (AML).
including leishmaniasis and schistosomiasis (Jaramillo et al., 2000), which still account for a large proportion of morbidity and mortality worldwide. Although the antimicrobial properties of the soursop have previously been reported (Takahashi et al., 2006), its mechanisms of action are not understood. In the present study, we address the mechanism of antibacterial action of A. muricata leaf (AML) extract. Different approaches were used to verify if compounds present in AML are capable of inducing membrane destabilization, which may contribute to a better understanding of their mechanism of action. In addition, the identification of alkaloids that could be related with this bioactivity was also attempted.
2.5. Minimum bactericidal concentration (MBC) The MBC of AML was determined by a modification of the method of Spencer and Spencer (2004). Samples (10 μL) were taken from plates with no visible growth in the MIC assay and inoculated on freshly prepared MHA plates, and later incubated at 37 °C for 24 h. The MBC was taken as the concentration of the extract that did not show any growth on a new set of agar plates. 2.6. Bacterial growth curve
2. Material and methods
AML was tested to determine the time-kill kinetics for E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 strains. Freshly grown bacterial strains containing approximately 108 CFU mL−1 in saline were inoculated in tubes of MHB supplemented with different concentrations of AML (MIC, 0.5MIC and 0.25MIC values) at 37 °C and the optical density at 600 nm (OD 600nm) was recorded at 2, 4, 6, 8 and 24 h. Graphs of turbidity versus incubation time were plotted. These growth rate curves were studied for any signs of bactericidal effects of AML. Chloramphenicol (MIC values) was used as a positive control. Bacterial strains inoculated in MHB with AML vehicle served as control of bacterial growth (CBG). The experiment was carried out in triplicate (Babii et al., 2016).
2.1. Plant material Annona muricata L. leaves (AML) were collected in Recreio, Minas Gerais State, Brazil, in March, 2011. The plant was identified by a botanical specialist (Dr. Vinícius Antônio de Oliveira Dittrich, Department of Botany, Federal University of Juiz de Fora, Brazil). A voucher specimen (CESJ 46006) was deposited at the Leopoldo Krieger Herbarium at the Federal University of Juiz de Fora.
2.2. Preparation of the extract
2.7. Bacterial abundance
After being oven dried at 40 °C for two days, 6.5 g of the powdered leaves of the plant were extracted by static maceration with methanol (5 × 200 mL) for five days at room temperature. The solvent was evaporated using a rotatory evaporator at 40 °C to produce the methanolic leaf extract in 6.6% yield (AML). The extract was kept in tightly stoppered bottles under refrigeration until used for the biological testing and chemical analysis.
Bacteria enumeration was performed in citocentrifugation preparations as before (Silva et al., 2014a). Bacterial strains of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 in saline were inoculated in tubes of MHB containing AML (MIC value) and incubated at 37 °C for 24 h. Bacterial strains inoculated in MHB with AML vehicle served as negative controls and positive controls were incubated with Chloramphenicol (MIC values). Samples were diluted 10 times (1 mL) in saline, fixed with particle-free 37% formaldehyde (0.2 μm filtered) to a final concentration of 2%, stained with DAPI (final concentration 0.01 μg mL−1) and placed in mega funnels (Shandon Mega Funnel, Thermo, UK) for immediate centrifugation in a cytocentrifuge (Shandon Cytospin 4, Thermo, United Kingdom), at 452g at high acceleration for 10 min. Acceleration and speed were established by the procedures for medical microbiology provided by the Cytospin manufactureŕs manual. Analyses were performed on a fluorescence microscope (BX-60, Olympus, Melville, NY, USA) and U-MWU2 filter (330–385 nm excitation wavelengths). Bacteria numbers were determined by counting 20 random fields at 1000× magnification using an ocular grid. The final count was determined by multiplying by the dilution factor (10×).
2.3. Microbial strains The sample was evaluated against Gram-positive microorganisms: Bacillus cereus ATCC 14579, Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 6538 as well as Gram-negative microorganisms: Enterobacter aerogenes ATCC 13048, Enterobacter cloacae ATCC 23355, Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica serovar Typhimurium ATCC 13311, Salmonella enterica serovar Choleraesuis ATCC 10708 and Shigella dysenteriae ATCC 13313. The strains were cultured overnight at 37 °C in Mueller Hinton agar (MHA) before each experiment.
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MA, USA) using a UHPLC column (2.1 × 50 mm, 1.7 μm particle size) at a temperature of 30 °C. A gradient of (A) deionized purified water with 1% formic acid and (B) methanol (Tedia®, Brazil) starting with 20% B and ramping to 100% B at 5 min, holding to 5.50 min, then returning to initial conditions and re-equilibrating at 7 min. Detection in positive ion modes was achieved on an Acquity TQD mass spectrometer (Micromass Waters®, Milford, MA, USA) with capillary voltage3000 V, Cone–30 V, source temperature 150 °C; desolvation temperature 350 °C. In the identification of the detected compounds, the chromatographic retention time (co-injection of the standards method) and the MS/MS data and spectrums of the standard compounds were compared with retention time and spectrums of the extract for confirmation, using analytical standards in the same chromatographic conditions. Moreover, previously the ion of interest was subjected to MS/MS analysis to make sure that the m/z corresponded to the same compound as the standard by direct insertion mass spectrometry with electrospray ionization in positive ion mode (ESI–MS/MS). The analyses were carried out at least in triplicate.
2.8. Bacteria viability For evaluation of bacteria viability, bacterial samples (diluted 10 times in saline) were stained with the LIVE/DEAD BacLight kit (Molecular Probes) which enables differentiation between bacteria with intact and damaged plasma membranes (Boulos et al., 1999; Freese et al., 2006). This kit contains a mixture of fluorescent stains (SYTO 9 and propidium iodide) that differ both in their spectral characteristics and their ability to penetrate healthy bacterial cell membranes. SYTO 9 stains viable cells whereas propidium iodide stains non-viable cells. Bacterial strains of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 in saline were inoculated in tubes of MHB containing AML (MIC value) incubated at 37 °C for 24 h. Bacterial strains inoculated in MHB with AML vehicle or incubated with chloramphenicol (MIC values) served as negative and positive controls, respectively. Bacteria were stained by adding 1 mL of each sample to 3 μL of BacLight. In the dark, samples were placed in megafunnels (Shandon Mega funnel, Thermo, UK) for immediate centrifugation in a cytocentrifuge as described above. Samples were prepared by using regular slides without any coating. Analyses were performed on a fluorescence microscope (BX-60, Olympus, Melville, NY, USA) using U-MWB filter (450–480 nm excitation wavelengths) which allows simultaneous visualization of both markers. Bacteria were directly counted in 10 random fields using an ocular grid at 1,000× magnification and the average percentage of live/dead bacteria was established for each slide sampled.
2.13. Statistical analysis Data were presented as median and 95% confidence interval. Each data point represented mean ± SD from at least two independent experiments performed in triplicate. Statistical differences between the treatments and the control were evaluated by ANOVA test followed by Bonferroni (p > 0.05).
2.9. Nucleotide leakage 3. Results The experiment was performed according to Tang et al. (2008) with some modifications. The overnight culture of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 at 37 °C was washed and resuspended in 10 mM PBS (pH 7.4), reaching a final density of about 108 CFU ml−1. Strains were incubated with AML (MIC value) for different times (2, 4, 6 and 8 h); strains incubated with 10 mM PBS (pH 7.4) were used as control. Following incubation, the cell suspensions were centrifuged at 10,000 g for 10 min and the supernatants were then diluted appropriately. The optical density at 260 nm was then recorded at room temperature. The experiment was carried out in triplicate.
3.1. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) AML showed a broad spectrum of antibacterial activity, with clear effectiveness against both Gram-negative and Gram-positive bacteria. However, the three bacteria species most susceptible to AML according to MIC values were E. faecalis, S. typhimurium and S. aureus, in this order (Table 1). We found that AML was bactericidal for S. typhimurium, S. aureus and P. aeruginosa (MBC/MIC = 1) and bacteriostatic for the others bacteria tested (MBC/MIC ≠ 1). Effectiveness does not necessarily mean mortality for bacterial cell. For E. faecalis, whose MIC value was lower (39 μg mL−1), the activity was bacteriostatic; while for P. aeruginosa, whose MIC was 16 times higher (625 μg mL−1), the activity was bactericidal. Chloramphenicol’s activity was bacteriostatic for both bacteria. The AML extract was more effective against E. faecalis, S.
2.10. Outer membrane permeability Outer membrane (OM) permeability was determined according to the method described by Hao et al. (2009), with some modifications. The strain used was S. typhimurium ATCC 13311. Briefly, overnight cultures (5 × 108 CFU mL−1) were inoculated into MHB containing AML alone and with the hydrophobic antibiotic erythromycin at concentrations ranging from 0.1 to 20 μg mL−1 with or without AML (MIC value). The media were then added to sterilized 96-well microplates and incubated at 37 °C for 24 h. The bacterial growth was measured at 450 nm using a spectrophotometer.
Table 1 Antibacterial activity of the methanolic extract Annona muricata leaves (AML). AML
Chloramphenicol
MICa
MBCa
MBC/MIC
MICa
625 39 156 625
1250 78 156 2500
2 2 1 4
3.13 6.25 12.5 25.0
1250 625 625
5000 2500 625
4 4 1
12.5 3.13 25.0
625
5000
8
3.13
78
78
1
12.5
313
1250
4
25.0
2.11. Study of biochemical reactions Bacillus cereus ATCC 14579 Enterococcus faecalis ATCC 19433 Staphylococcus aureus ATCC 6538 Enterobacter aerogenes ATCC 13048 Enterobacter cloacae ATCC 23355 Escherichia coli ATCC 10536 Pseudomonas aeruginosa ATCC 9027 Salmonella Choleraesuis ATCC 10708 Salmonella Typhimurium ATCC 13311 Shigella dysenteriae ATCC 13313
The clone (AML stressed clone, chloramphenicol stressed clone and control clone) from E. faecalis, S. aureus and S. typhimurium reference strains were subjected to the study of biochemical reactions. The tests were carried out on VITEK®2 system. The VITEK®2 GP ID Card (identification of Gram-positive bacteria) and GN ID Card (identification of fermenting and non-fermenting Gram-negative bacilli), which offer rapid and accurate identification of a broad range of clinically significant bacterial pathogens, were use for biochemicals. 2.12. UHPLC-ESI–MS/MS analysis AML was analyzed on an Acquity UHPLC system (Waters®, Milford,
a
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Values in μg mL−1.
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Fig. 2. Activity kinetics of methanolic extract of Annona muricata leaves (AML) against S. aureus (A); S. typhimurium (B) and E. faecalis (C). Choramphenicol (CHL) was used as positive control, and bacteria inoculated in MHB with AML vehicle was used as bacteria growth control (CBG). The experiments were carried out triplicate, and data represent the mean ± SD.
3.4. Nucleotide leakage and outer membrane permeability
typhimurium and S. aureus, based upon their MIC values and, as such, these bacteria were chosen as targets to further investigate the AML extract’s activity.
As shown in (Fig. 4), AML increased at 19%, 41% and 30% the nucleotide release in S. aureus, S. typhimurium and E. faecalis respectively, compared to the controls. The efflux of nucleotides from the intracellular compartment was more significant for Gram-negative than Gram-positive bacteria. Fig. 5 clearly shows synergism between AML and erythromycin suggesting that AML was able to increase the OM permeability in the tested bacteria.
3.2. Bacterial killing assay The AML vehicle does not affect bacterial growth as the shape of the bacterial growth curve was typical for all species tested. However, all tested bacteria showed a dose-dependent growth decrease when exposed to AML leaf extract (Fig. 2), with bacteria death by lysis induced at the MIC concentration. For S. aureus, all concentrations inhibited the growth cycle curve when compared to the control treated with vehicle (Fig. 2A). For S. typhimurium (Fig. 2B) AML extended the lag phase by two hours, and then induced the bacteria death by lysis at the MIC value. For E. faecalis (Fig. 2C), it was observed that the bacterial life cycle was forced into the death phase at the 6th hour of treatment. At other concentrations, the bacterial growth was increased, but this growth was lower than the vehicle treatment.
3.5. Study of biochemical reaction Clones from three tested strains exhibited some alteration in biochemical reactions after antimicrobial stress when compared with the original, unstressed control. For S. aureus the L-lactate alkalinization (ILATk) and α-glucosidase (AGLU) reactions were converted from negative to positive in the AML clone, and the pyrrolidonyl arylamidase (PyrA) and bacitracin-tests (BACI) were converted from positive to negative in the chloramphenicol clone. For S. typhimurium the gammaglutamyl transpeptidase (GGT), urease (URE) and ILATk reactions were converted from negative to positive in the AML clone and tyrosine arylamidase (TyrA) and GGT were converted from negative to positive in the chloramphenicol clone. Finally, in E. faecalis, leucine arylamidase (LeuA) and alanine arilamidase (AlaA) were converted from a positive to a negative reaction in the AML clone, while only LeuA was altered by the chloramphenicol clone. If differences between the stressed clones and controls were observed in the biochemical characterization, it was not enough to modify the identity of the strain identification. The percentage of trust was 98%, 97% and 98% for Chloramphenicol and 99%, 93% and 97% for AML clones, respectively for S. aureus, S. typhimurium and E. faecalis.
3.3. Bacterial abundance and viability Fluorescence microscopy after DAPI staining showed that AML inhibited the bacterial abundance compared to the control group, reducing the bacterial growth by 28%, 26% and 40% for S. aureus, S. typhimurium and E. faecalis, respectively (Fig. 3A). AML treatment caused a decrease in cell density and an increase in cell death. The cytocentrifuge preparations of the florescent viability probe test (Live/Dead® BacLight) enabled a clear imaging of live (green)/dead (red) bacteria in both AML-treated and control cultures (Fig. 3C). Quantitative results showed that AML (MIC value for 24 h) for S. aureus, S. typhimurium and E. faecalis induced an increase in the percentage of dead cells (34%, 34% and 24%, respectively) compared to the vehicle (Fig. 3B). Chloramphenicol showed 8.5%, 4.5% and 1% of dead cells, respectively.
3.6. UPLC-ESI–MS/MS analysis The chromatografic profile of the AML was acquired and the compounds identified are described in Table 2. The chemical structure of each compound is shown in Fig. 6. 335
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Fig. 3. Effect of methanolic extract of Annona muricata leaves (AML) on bacterial growth of S. aureus, S. typhimurium and E. faecalis. Cultured bacteria were stained with DAPI or LIVE/ DEAD® BacLight™ and counted under fluorescence microscopy for evaluation of cell density (A) and viability (B), respectively. AML treatment induced both decrease of cell density (A) and increase of cell death (B) compared to control groups treated with AML vehicle. In (C), representative images of treated and non-treated bacteria after BacLight staining. Note that viable/live (green) and non-viable/dead (red) bacteria are clearly observed and dead cells increases with the AML treatment. Letters indicate statistically difference from controls treated with AML vehicle for S. aureus (a), S. typhimurium (b), E. faecali (c) (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD of bacteria counted. Scale bar: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
time-dependent kinetics showed AML was more effective than chloramphenicol, which is a bacteriostatic drug. Following the standard method for the determination of bactericidal effects, i.e. bacterial killing assay, we performed two additional approaches using fluorescent probes to investigate the effect of the plant extract on bacterial growth/death in treated cultures (AML at MIC value and controls, for 24 h). First, bacterial abundance was quantified by fluorescence microscopy after DAPI staining. AML treatment induced a decrease in cell density and an increase in cell death, which corroborated the bacterial killing assay results. This cell reduction may be related to both potential effects of AML in inhibiting bacterial growth and/or inducing death and cell lysis. Secondly, we investigated bacterial death using a florescent viability probe (Live/Dead® BacLight), which enables direct identification of loss of plasma membrane integrity. Cells with damaged membranes are unable to maintain an electrochemical potential and are considered as dead, so that individual imaging of bacteria is helpful to recognize bacterial viability and their physiological functions at a single-cell level (Joux and Lebaron, 2000). The increase of dead cells induced by AML was significant, which is in accordance with previous studies that demonstrated the bactericidal activity after plant-extract treatments
Although the antibacterial potential of A. muricata has been previously reported, this study shows for the first time that the AML extract has a broad spectrum of antibacterial activity, against both Gram-negative and Gram-positive bacteria, as shown by the MIC/MBC assay. A time-dependent bactericidal effect takes place when the concentration of the antibacterial drug exceeds the MIC for the microorganism, while a concentration-dependent bactericidal effect occurs when an antibiotic has a high concentration at the binding site to eliminate the microorganism (Anantharaman et al., 2010). Our timekill kinetic analyses showed that AML exhibited rapid time dependent kinetics of bacterial killing for both Gram negative and Gram positive bacteria, mainly at MIC value. Thus, AML may potentially contain infections with greater rapidity, compared to antibacterial agents that exhibit slow killing kinetics. Comparing the activity of the AML with chloramphenicol at MIC concentration, we observed a similar action in the early stages, but a better inhibition after the bacterial lag phase. Although these events occurred for all three of the bacteria species studied, it is important to note that even for E. faecalis, for which the AML extract demonstrated bacteriostatic activity (Table 1), the rapid
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Fig. 4. Mode of action of methanolic extract of Annona muricata leaves (AML). Effect of AML on the amount of the total nucleotide released from S. aureus (Sa), S. typhimurium (St) and E. faecalis (Ef). Letters indicate statistically difference from controls treated with AML vehicle for S. aureus (a), S. typhimurium (b), E. faecali (c) (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD.
Our results show that AML is effective against both Gram-negative and Gram-positive bacteria, but it’s antimicrobial activity is organism dependent. Furthermore, the results suggest that the inhibition of bacterial growth and the induction of cell death by AML may both be related to the same mechanism (membrane destabilization). Several models for studying the mode of action of antibacterial drugs have been described in the literature. In this context, nucleotide leakage and OM permeability assays are associated with damages to bacterial membranes, so that these models were chosen to clarify how AML is able to inhibit bacterial proliferation (Hao et al., 2009). As DNA and RNA are released after membrane disruption, these nucleotides were quantified by monitoring the absorbance at 260 nm. AML probably induced the disruption of the cytoplasmic membrane after 2 h as the nucleotide release began to increase after this time, especially in Gram-negative bacteria.
through viability analyses using Baclight (Abdullah et al., 2014; You et al., 2013). Noteworthy, this viability analyses is unable to detect alterations prior to cell lysis. This means that AML treatment was able to elicit changes at the cellular level, which are indicative of cell death, i.e., loss of plasma membrane integrity. Additionally, as the treatment with chloramphenicol, a well-known bacteriostatic drug (Bernatová et al., 2013), showed very low proportion of dead cells, our viability results reinforce the potential bactericidal effect of AML for the three bacteria tested. Abundance analyses and cell viability evaluation corroborate to MBC/MIC for the identification of antimicrobial activity, which indicates that AML also has a potential bacteriostatic effect on E. faecalis, as the increase in the proportion of dead cells (24%) is smaller than the reduction of the total bacterial count (40%). Therefore, the microscopic approaches used in the present study were very important to confirm and reinforce perspectives about the mode of action of AML.
Fig. 5. Mode of action of methanolic extract of Annona muricata leaves (AML). Outer membrane permeability of S. typhimurium induced by AML in association with erytromicin. aStatistically different from ERY + AML treated. (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD.
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Table 2 Active compounds identified in the methanolic extract Annona muricata leaves (AML) by UHPLC–MS/MS analysis. Compound
Retention time standards (min)a
Retention time (min)a
[M+H]+ (m/z)
MS-MS íons (m/z) and relative abundance (%), in positive íon mode
Anonaine Asimilobine Liriodenine Nornuciferine Xylopine Reticuline Coripalmine
2.98 2.29 3.43 2.50 3.00 1.69 2.27
2.99 2.32 3.41 2.51 3.09 1.71 2.25
266 268 276 282 296 330 342
266→ 268→ 276→ 282→ 296→ 330→ 342→
a
266 268 276 282 296 299 342
(18), 249 (100), 234 (8), 219 (28), 191 (46) (10), 251 (100), 236 (15), 219 (51), 191 (80) (100) (100) (100) (30), 192 (100), 175 (12), 151 (7), 143 (16), 137 (22) (100)
UHPLC-ESI–MS/MS analysis using analytical standards in the same chromatographic for identification of the compounds detected in AML.
pores, solubilization of membrane proteins, and changes in the structure/function of the phospholipid bilayer of the membrane (Chen and Cooper, 2002). On the basis of the results of the bacterial membrane permeability studies, it can be stated that the antibacterial action of AML is induced, at least in part, by changes in the permeability of the cell wall and to the bacterial cytoplasmic membrane. It is well accept that antibiotics induce bacterial biochemical-physiological alterations (Dwyer et al., 2014; Lobritz et al., 2015; Santos et al., 2011). Changed phenotypic profiling has been utilized to discover of the mechanism of action of antimicrobial drugs (Athamneh et al., 2014). Our results corroborate this idea. The differences observed in stressed and control clones for the three species studied indicate the negative expression of enzymes involved in secondary metabolism and positive expression (induction) of enzymes involved with energetic metabolism. These data are consistent with those found in other tests. Poor or inadequate energetic metabolism interferes directly in the logarithmic phase of bacterial growth leading to a bacteriostatic action. Furthermore, even in part, the energy metabolism is related to the cytoplasmic membrane. Therefore, interferences in the membrane could induce cell lysis. For a better global positioning of any herbal drug, it is important to establish a chemical profile based upon the maximum available number of markers for an extract (Fabri et al., 2013). For this reason, the chromatographic profile of AML was acquired. Plants from the genus Annona, Annonaceae family, are well known by the presence of acetogenins, which have been reported as potent antimicrobial and cytotoxic compounds. Almost 30 acetogenins were already isolated from this species (Pandey and Barve, 2011). To identify other chemical constituents which could be possibly related to antimicrobial activity, we investigated the alkaloid profile of AML by UHPLC-ESI–MS/MS
The bacterial cell wall is a rigid structure that covers the cytoplasmic membrane and gives structural support to bacteria. In Grampositive bacteria, the cell wall is thick consisting of several layers of peptidoglycan and surface glycopolymers such as teichoic acids. In Gram-negative bacteria, the cell wall is relatively thin and is composed of a single layer of peptidoglycan, lipoproteins, lipopolysaccharides, and the OM, which is important as a molecular barrier that prevents the loss of intracellular proteins, and reduces the access of hydrolytic enzymes and some drugs, particularly hydrophobic antibiotics (SilvaJúnior et al., 2014). The influence of AML on S. typhimurium OM permeability was determined by a synergistic growth inhibition assay in association with the hydrophobic antibiotic erythromycin. The OM of the Gram-negative cell wall is known to be an effective permeability barrier against hydrophobic antibacterial agents, including erythromycin. This antibiotic does not infiltrate effectively in the intact Gram-negative OM, however it can permeate the OM of the certain OM-defective mutants, as well as the OM damaged by chelators or polycations (Viljanen et al., 1990). Therefore, erythromycin was chosen as a probe to detect any possible increase in OM permeability induced by AML. The synergism between AML and erythromycin strongly indicated that AML was able to disrupt OM permeability. Co-administration of two or more antibiotics is a common medical practice because it results in synergistic effects, broadening the spectrum of action or reducing bacterial resistance and toxicity (Silva-Júnior et al., 2014). In these assays, it is not possible to clarify how the compounds present in AML interact with the cytoplasmic membrane components and exactly how it elicits the leakage of nucleotides. A number of mechanisms for this interaction have been proposed to explain the action of antibacterial drugs on the cytoplasmic membrane, with emphasis on formation of
Fig. 6. Chemical structures of the compounds identified in the methanolic extract of Annona muricata leaves (AML) by UHPLC-ESI–MS/MS analysis.
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(−)- Anonaine induces DNA damage and inhibits growth and migration of human lung carcinoma H1299 cells. J. Agric. Food Chem. 59, 2284–2290. Costa, E.V., Cruz, P.E.O., Lourenço, C.C., Moraes, V.R.S., Nogueira, P.C.L., Salvador, M.J., 2013. Antioxidant and antimicrobial activities of aporphinoids and other alkaloids from the bark of Annona salzmannii A. DC. (Annonaceae). Nat. Prod. Res. 27, 1002–1006. Degnon, R.G., Adjou, E.S., Noudogbessi, J.P., Metome, G., Boko, F., Dahouenon-Ahoussi, E., Mohamed, S., Sohounhloue, D.K.C., 2013. Investigation on nutritional potential of soursop (Annona muricata L.) from Benin for its use as food supplement against protein-energy deficiency. Int. J. Biosci. 3, 135–144. Dwyer, D.J., Belenky, P.A., Yang, J.H., Cody MacDonald, I., Martell, J.D., Takahashi, N., Chan, C.T.Y., Lobritz, M.A., Braff, D., Schwarz, E.G., Ye, J.D., Pati, M., Vercruysse, M., Ralifo, P.S., Allison, K.R., Khalil, A.S., Ting, A.Y., Walker, G.C., Collins, J.J., 2014. 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Biological activities of leaves of ethnomedicinal plant, Borassus flabellifer Linn. (Palmyra palm): an antibacterial, antifungal and antioxidant evaluation. Bull. Facul. Pharm. 54, 59–66. Jaramillo, M.C., Arango, G.J., Gonzalez, M.C., Robledo, S.M., Velez, I.D., 2000. Cytoxicity and antileishmanial activity of Annona muricata pericarp. Fitoterapia 71, 183–186. Joly, A.B., 1979. Botânica, introdução à taxonomia vegetal, third ed. Companhia Nacional, São Paulo. Joux, F., Lebaron, P., 2000. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect. 2, 1523–1535. Kuete, V., 2010. Potential of Cameroonian plants and derived products against microbial infections: a review. Planta Med. 76, 1479–1491. Likhitwitayawuid, K., Angerhofer, C.K., Chai, H., Pezzuto, J.M., Cordell, G.A., Ruangrungsi, N., 1993. Cytotoxic and antimalarial alkaloids from the tubers of Stephania pierrei. J. Nal. Prod. 56, 1468–1478. Malik, T.A., Kamili, A.N., Chishti, M.Z., Ahad, S., Tantry, M.A., Hussain, P.R., Johri, R.K., 2017. Breaking the resistance of Escherichia coli: antimicrobial activity of Berberis lycium Royle. Microb. Pathog. 102, 12–20. Marti, G., Eparvier, V., Morleo, B., Le Ven, J., Apel, C., Bodo, B., Amand, S., Dumontet, V., Lozach, O., Meijer, L., Guéritte, F., Litaudon, M., 2013. Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/Cyclin B and DYRK1A. Molecules 18, 3018–3027. Mocan, A., Vlase, L., Raita, O., Hanganu, D., Păltinean, R., Dezsi, S., Gheldiu, A.M.,
analysis, using analytical standards in the same chromatographic conditions. The literature reports the wide occurrence of benzylisoquinoline alkaloids in Annonacaea spp. and they may be considered as good chemical and biological markers for this family (González-Esquinca et al., 2014). Bioactivities are described for many of them (Costa et al., 2013; Kuete, 2010; Vendramin et al., 2013; Silva et al., 2014b). The cytotoxic and antimicrobial activities of anonaine (Chen et al., 2011; Kuete, 2010; Paulo et al., 1992; Villar et al., 1987), asimilobine (Costa et al., 2013; Woo et al., 1995), liriodenine (Hsieh et al., 2005; Hufford et al., 2011; Woo et al., 1997), nornuciferine (Marti et al., 2013), xylopine (Florence et al., 2014; Hai-Bo et al., 2013; Likhitwitayawuid et al., 1993) and reticuline (Orhan et al., 2007; Suresh et al., 2012; Tsai et al., 1989) are well known. Chen et al. (2011) reported that anonaine induces DNA damage, activation of caspase 3, 7, 8 and 9, and poly ADP ribose polymerase cleavage, although more profound information regarding the mechanism of action of other compounds identified in this study are not well described yet. Thus, based on these literature data, the chromatographic analysis of AML suggests that the aforementioned compounds may be responsible for the antimicrobial activity found for A. muricata extract, and, could be acting in synergism with one another and/or acetogenins present in the extract. 5. Conclusion This study reported, for the first time, that the antimicrobial activity of A. muricata presents a broad spectrum of action, and that bacterial membranes (both plasma and outer membranes) are primary targets of the bioactive compounds. The alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline may be, at least in part, responsible for the bioactivities reported in this study. In addition, our results corroborate to the ethnopharmacological uses of this specie. It is also noteworthy to mention that A. muricata presents a remarkable phytomedicinal potential, and it is commonly used as food, which encourages further studies to evaluate whether the ingestion of this plant may be useful to treat or prevent bacterial infections, as several antimicrobial compounds were already identified in this species. From the results obtained with the extract of the plant evaluated here, we could expect its possible application, in the future, associated with other antimicrobials, such as erythromycin, in order to avoid antibacterial resistance. However, until then, more studies are necessary. Conflict of interest The authors declare no competing financial interest. Acknowledgments The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), to the Federal University of Juiz de Fora (UFJF)/Brazil, to CAPES, CNPq, FAPESP and FAEPEX-UNICAMP for financial support, and also to Dr. Vinícius Antônio de Oliveira Dittrich from the Department of Botany/Federal University of Juiz de Fora for the botanical identification of the species. References Abdullah, S., Gobilik, J., Chong, K.P., 2014. In vitro antimicrobial mode of action of Cynodon dactylon (L.) Pers. solid phase extract (SPE) against selected pathogens. In: International Conference on Civil, Biological and Environmental Engineering. CBEE, Istanbul, Turkey. pp. 66–72. Adewole, O.S., Caxton-Martins, E.A., 2006. Morphological changes and hypoglycemic effects of Annona muricata Linn. (Annonaceae) leaf aqueous extraction pancreaticcells of streptozotocin-treated diabetic rats. Afr. J. Biomed. Res. 9, 173–187. Adewole, O.S., Ojewole, J.A.O., 2008. Protective effects of Annona muricata (Linn) (Annonaceae) leaf aqueous extract on serum lipid profiles and oxidative stress in hepatocytes of streptozotocin-treated diabetic rats. Afr. J. Tradit. Complement. Altern. Med. 6, 30–41. Adjanohou, E., 1996. Organization of African Unity, Scientific, Technical, and Research
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Suresh, H.M., Shivakumar, B., Shivakumar, S.I., 2012. Cytotoxicity of aporphine alkaloids from the roots of Annona reticulata on human cancer cell lines. Int. J. Plant. Res. 2, 57–60. Takahashi, J.A., Pereira, C.R., Pimenta, L.P., Boaventura, M.A., Silva, L.G., 2006. Antibacterial activity of eight Brazilian Annonaceae plants. Nat. Prod. Res. 20, 21–26. Tang, Y.L., Shi, Y.H., Zhao, W., Hao, G., Le, G.W., 2008. Insertion mode of a novel anionic antimicrobial peptide MDpep5 (Val-Glu-Ser-Trp-Val) from Chinese traditional edible larvae of housefly and its effect on surface potential of bacterial membrane. J. Pharm. Biomed. Anal. 48, 1187–1194. Tsai, I.L., Liou, Y.F., Lu, S.T., 1989. Screening of isoquinoline alkaloids and their derivatives for antibacterial and antifungal activities. Gaoxiong Yi Xue Ke Xue Za Zhi. 5, 132–145. Vendramin, M.E., Costa, E.V., Santos, E.P., Pinheiro, M.L.B., Barison, A., Campos, F.R., 2013. Chemical constituents from the leaves of Annona rugulosa (Annonaceae). Biochem. Syst. Ecol. 49, 152–155. Viljanen, P., Matsunaga, H., Kimura, Y., Vaara, M., 1990. The outer membrane permeability-increasing action of deacylpolymyxins. J. Antibiot. 44, 517–523. Villar, A., Mares, M., Rios, J.L., Canton, E., Gobernado, M., 1987. Antimicrobial activity of benzylisoquinoline alkaloids. Pharmazie 42, 248–250. Vlase, L., Mocan, A., Hanganu, D., Benedec, D., Gheldiu, A.M., Crișan, G., 2014. Comparative study of polyphenolic content, antioxidant and antimicrobial activity of four Galium species (Rubiaceae). Dig. J. Nanomater. Biostruct. 9 (3), 1085–1094. World Health Organization, 2015. Antimicrobial resistance: draft global action plan on antimicrobial resistance, in: T.W.H. ASSEMBLY (Ed.), Provisional Agenda Item 15.1 Ed, World Health Organization, Geneva. http://apps.who.int/gb/ebwha/pdf_files/ WHA68/A68_20-en.pdf (Acessed 18.12.16). Woo, M.H., Cho, K.Y., Zhang, Y., Zeng, L., Gu, Z.M., Mclaughlin, J.L., 1995. Asimilobin and cis- and trans-murisolinones, novel bioactive annonaceous acetogenins from the seeds of Asimina triloba. J. Nat. Prod. 58, 1533–1542. Woo, S.H., Reynolds, M.C., Sun, N.J., Cassady, J.M., Snapka, R.M., 1997. Inhibition of topoisomerase II by liriodenine. Biochem. Pharmacol. 54, 467–473. You, Y.O., Choi, N.Y., Kang, S.Y., Kim, K.J., 2013. Antibacterial activity of Rhus javanica against methicillin-resistant Staphylococcus aureus. Evid. Based Complement. Alternat. Med. 1–8.
Oprean, R., Crișan, G., 2015. Comparative studies on antioxidant activity and polyphenolic content of Lycium barbarum L. and Lycium chinense Mill Leaves. Pak. J. Pharm. Sci. 28, 1511–1515. Mocan, A., Zengin, G., Uysal, A., Gunes, E., Mollica, A., Degirmenci, N.S., Alpsoy, L., Aktumsek, A., 2016. Biological and chemical insights of Morina persica L.: a source of bioactive compounds with multifunctional properties. J. Funct. Foods 25, 94–109. N’gouemo, P., Koudogbo, B., Tchivounda, P., Akono-Nguema, C., Etoua, M.M., 1997. Effects of ethanol extract of Annona muricata on pentylenetetrazol-induced convulsive seizures in mice. Phytother. Res. 11, 243–245. Newman, D.J., Cragg, G.M., 2012. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 75, 311–335. Orhan, I., Özçelik, B., Karaoğlu, T., Şener, B., 2007. Antiviral and antimicrobial profiles of selected isoquinoline alkaloids from Fumaria and Corydalis species. J. Biosci. 62, 19–26. Pandey, N., Barve, D., 2011. Phytochemical and pharmacological review on Annona squamosa Linn. Int. J. Res. Pharm. Biol. Sci. 2, 1404–1412. Paulo, M.Q., Barbosa-Filho, J.M., Lima, E.O., Maia, R.F., Barbosa, R.C., Kaplan, M.A., 1992. Antimicrobial activity of benzylisoquinoline alkaloids from Annona salzmanii D.C. J. Ethnopharmacol. 36, 39–41. Santos, K.V., Carvalho, M.A.R., Martins, W.A., Andrade, H.M., Veloso, L.C., Santos, S.G., Apolônio, A.C.M., Diniz, C.G., Nicoli, J.R., Farias, L.M., 2011. Phenotypic changes in laboratory derived ertapenem resistant Escherichia coli strain. J. Chemother. 23, 135–139. Silva, F.M., Souza, A.D., Koolen, H.H., Barison, A., Vendramin, M.E., Costa, E.V., Ferreira, A.G., Pinheiro, M.L., 2014a. Phytochemical study of the alkaloidal fractions of Unonopsis duckei R. E. Fr. guided by electrospray ionization ion-trap tandem mass spectrometry. Phytochem. Anal. 25, 45–49. Silva, T.P., Noyma, N.P., Duque, T.L., Gamalier, J.P., Vidal, L.O., Lobão, L.M., ChiariniGarcia, H., Roland, F., Melo, R.C., 2014b. Visualizing aquatic bacteria by light and transmission electron microscopy. A Van Leeuw. 105, 1–14. Silva-Júnior, I.F., Oliveira, R.G., Soares, I.M., Alvim, T.C., Ascêncio, S.D., Martins, D.T.O., 2014. Evaluation of acute toxicity, antibacterial activity, and mode of action of the hydroethanolic extract of Piper umbellatum L. J. Ethnopharmacol. 151, 137–143. Spencer, A.L.R., Spencer, J.F.T., 2004. Public Health Microbiology: Methods and Protocols, first ed. Human Press Inc., New Jersey.
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Antimicrobial Annona muricata L. (soursop) extract targets the cell membranes of Gram-positive and Gram-negative bacteria
MARK
Nícolas de C.C. Pintoa, Lara M. Camposa, Anna Carolina S. Evangelistaa, Ari S.O. Lemosa, Thiago P. Silvab, Rossana C.N. Melob, Caroline C. de Lourençoc, Marcos J. Salvadorc, ⁎ Ana Carolina M. Apolôniod, Elita Scioa, Rodrigo L. Fabria, a
Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Brazil Laboratory of Cell Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil c Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, São Paulo, Brazil d Laboratory of Bacterial Physiology and Molecular Genetics, Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Annona muricata Antibacterial activity Membrane permeability Alkaloids Plant extract
Annona muricata has become an interesting subject in the search for new therapeutic agents. We investigated the bacterial mode of action of the methanolic extract of A. muricata leaves (AML). AML extract was tested against several bacteria strains by broth microdilution susceptibility method. The bacterial killing assay, bacterial abundance and membrane viability analysis were made using fluorescent probes. The nucleotide leakage and outer membrane (OM) permeability assays were used to verify membrane destabilization. The biochemical reaction profile was carried out on a VITEK®2 system. For UPLC-ESI–MS/MS Analysis an Acquity UPLC system was used. AML was active against both Gram-negative and Gram-positive bacteria, showing greater activity against S. aureus, S. typhimurium and E. faecalis. AML exhibited rapid time dependent kinetics of bacterial killing. DAPI staining revealed that AML inhibited the bacterial growth, while the LIVE/DEAD BacLight analysis showed that AML induced an increase in dead cells. AML increased nucleotide leakage and was also capable of increasing the OM permeability in the tested bacteria. Differences between the stressed clones and controls observed in the biochemical characterization were not enough to modify the strain identity. UPLC-ESI–MS/MS analysis revealed the presence of the alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline. Our findings demonstrate, for the first time, a broad spectrum of antibacterial activity for AML and identify that bacterial membranes (both plasma and outer membranes) are primary targets of this extract. Based on these observations, AML has a good potential for the design of novel antimicrobial agents.
1. Introduction The emergence and spread of microbial resistance is growing each day, thereby necessitating the development of new antimicrobials (Malik et al., 2017). Hospital-acquired infections are becoming a growing concern (WHO, 2015). Based on this, studies have shown that natural products appear to be resources for valuable resistance breaking molecules (Newman and Cragg, 2012). Plant derived compounds are of particular interest, based on their already proven antimicrobial activity (Jamkhande et al., 2016; Mocan et al., 2015; Mocan et al., 2016; Vlase et al., 2014). In this context, Annona muricata (Fig. 1) has become an interest subject in the search for new therapeutic agents. This species belongs to
the Annonaceae family, which consists of 29 genera and 390 species with tropical and subtropical distribution worldwide (Joly, 1979). It is popular known as soursop or prickly custard apple, and the fruits are edible and commonly used to make ice cream, teas and other drinks (Degnon et al., 2013). Several parts of this plant are traditionally used to treat cancer, diabetes, hypertension, fever, gastrointestinal disorders, and parasitic infections (Adjanohou, 1996). Various pharmacological studies have confirmed the traditional uses of Annona muricata (Adewole and Caxton-Martins, 2006; N’gouemo et al., 1997). In particular, the antimicrobial properties of Annona muricata leaves (AML) have attracted attention due to their potential to also treat viral infections (Florence et al., 2014), hyperlipidemia (Adewole and Ojewole, 2008) and neglected human diseases,
⁎ Corresponding author at: Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais CEP 36036 900, Brazil. E-mail addresses: [email protected], [email protected] (R.L. Fabri).
http://dx.doi.org/10.1016/j.indcrop.2017.05.054 Received 19 December 2016; Received in revised form 12 May 2017; Accepted 28 May 2017 Available online 09 June 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.4. Serial dilution assay for determination of the minimal inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) of AML was determined by microdilution techniques in Mueller Hinton broth (MHB) as described by CLSI (2010). Bacteria were cultured overnight at 37 °C for 24 h in MHA. Sample stock solution was two-fold diluted from 5000 to 2.5 μg mL−1 (final volume = 80 μL) and a final dimethyl sulfoxide (DMSO) concentration ≤1%. Then, 100 μL of MHB and 20 μL of 108 CFU mL−1 (according to McFarland turbidity standards) of standardized bacterial suspensions were inoculated onto microplates and the test was performed in a volume of 200 μL. Plates were incubated at 37 °C for 24 h. The same tests were performed simultaneously for growth control (MHB + bacteria + AML vehicle) and sterility control (MHB + AML vehicle) as well as the positive control with Chloramphenicol (500–0.24 μg mL−1). The MIC values were calculated as the highest dilution showing complete inhibition of the tested strain. Analyses were performed in triplicate. Extracts with MIC values ≤100 μg mL−1 were considered significantly active (Kuete, 2010).
Fig. 1. Annona muricata plant with focus on the Annona muricata leaves (AML).
including leishmaniasis and schistosomiasis (Jaramillo et al., 2000), which still account for a large proportion of morbidity and mortality worldwide. Although the antimicrobial properties of the soursop have previously been reported (Takahashi et al., 2006), its mechanisms of action are not understood. In the present study, we address the mechanism of antibacterial action of A. muricata leaf (AML) extract. Different approaches were used to verify if compounds present in AML are capable of inducing membrane destabilization, which may contribute to a better understanding of their mechanism of action. In addition, the identification of alkaloids that could be related with this bioactivity was also attempted.
2.5. Minimum bactericidal concentration (MBC) The MBC of AML was determined by a modification of the method of Spencer and Spencer (2004). Samples (10 μL) were taken from plates with no visible growth in the MIC assay and inoculated on freshly prepared MHA plates, and later incubated at 37 °C for 24 h. The MBC was taken as the concentration of the extract that did not show any growth on a new set of agar plates. 2.6. Bacterial growth curve
2. Material and methods
AML was tested to determine the time-kill kinetics for E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 strains. Freshly grown bacterial strains containing approximately 108 CFU mL−1 in saline were inoculated in tubes of MHB supplemented with different concentrations of AML (MIC, 0.5MIC and 0.25MIC values) at 37 °C and the optical density at 600 nm (OD 600nm) was recorded at 2, 4, 6, 8 and 24 h. Graphs of turbidity versus incubation time were plotted. These growth rate curves were studied for any signs of bactericidal effects of AML. Chloramphenicol (MIC values) was used as a positive control. Bacterial strains inoculated in MHB with AML vehicle served as control of bacterial growth (CBG). The experiment was carried out in triplicate (Babii et al., 2016).
2.1. Plant material Annona muricata L. leaves (AML) were collected in Recreio, Minas Gerais State, Brazil, in March, 2011. The plant was identified by a botanical specialist (Dr. Vinícius Antônio de Oliveira Dittrich, Department of Botany, Federal University of Juiz de Fora, Brazil). A voucher specimen (CESJ 46006) was deposited at the Leopoldo Krieger Herbarium at the Federal University of Juiz de Fora.
2.2. Preparation of the extract
2.7. Bacterial abundance
After being oven dried at 40 °C for two days, 6.5 g of the powdered leaves of the plant were extracted by static maceration with methanol (5 × 200 mL) for five days at room temperature. The solvent was evaporated using a rotatory evaporator at 40 °C to produce the methanolic leaf extract in 6.6% yield (AML). The extract was kept in tightly stoppered bottles under refrigeration until used for the biological testing and chemical analysis.
Bacteria enumeration was performed in citocentrifugation preparations as before (Silva et al., 2014a). Bacterial strains of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 in saline were inoculated in tubes of MHB containing AML (MIC value) and incubated at 37 °C for 24 h. Bacterial strains inoculated in MHB with AML vehicle served as negative controls and positive controls were incubated with Chloramphenicol (MIC values). Samples were diluted 10 times (1 mL) in saline, fixed with particle-free 37% formaldehyde (0.2 μm filtered) to a final concentration of 2%, stained with DAPI (final concentration 0.01 μg mL−1) and placed in mega funnels (Shandon Mega Funnel, Thermo, UK) for immediate centrifugation in a cytocentrifuge (Shandon Cytospin 4, Thermo, United Kingdom), at 452g at high acceleration for 10 min. Acceleration and speed were established by the procedures for medical microbiology provided by the Cytospin manufactureŕs manual. Analyses were performed on a fluorescence microscope (BX-60, Olympus, Melville, NY, USA) and U-MWU2 filter (330–385 nm excitation wavelengths). Bacteria numbers were determined by counting 20 random fields at 1000× magnification using an ocular grid. The final count was determined by multiplying by the dilution factor (10×).
2.3. Microbial strains The sample was evaluated against Gram-positive microorganisms: Bacillus cereus ATCC 14579, Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 6538 as well as Gram-negative microorganisms: Enterobacter aerogenes ATCC 13048, Enterobacter cloacae ATCC 23355, Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica serovar Typhimurium ATCC 13311, Salmonella enterica serovar Choleraesuis ATCC 10708 and Shigella dysenteriae ATCC 13313. The strains were cultured overnight at 37 °C in Mueller Hinton agar (MHA) before each experiment.
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MA, USA) using a UHPLC column (2.1 × 50 mm, 1.7 μm particle size) at a temperature of 30 °C. A gradient of (A) deionized purified water with 1% formic acid and (B) methanol (Tedia®, Brazil) starting with 20% B and ramping to 100% B at 5 min, holding to 5.50 min, then returning to initial conditions and re-equilibrating at 7 min. Detection in positive ion modes was achieved on an Acquity TQD mass spectrometer (Micromass Waters®, Milford, MA, USA) with capillary voltage3000 V, Cone–30 V, source temperature 150 °C; desolvation temperature 350 °C. In the identification of the detected compounds, the chromatographic retention time (co-injection of the standards method) and the MS/MS data and spectrums of the standard compounds were compared with retention time and spectrums of the extract for confirmation, using analytical standards in the same chromatographic conditions. Moreover, previously the ion of interest was subjected to MS/MS analysis to make sure that the m/z corresponded to the same compound as the standard by direct insertion mass spectrometry with electrospray ionization in positive ion mode (ESI–MS/MS). The analyses were carried out at least in triplicate.
2.8. Bacteria viability For evaluation of bacteria viability, bacterial samples (diluted 10 times in saline) were stained with the LIVE/DEAD BacLight kit (Molecular Probes) which enables differentiation between bacteria with intact and damaged plasma membranes (Boulos et al., 1999; Freese et al., 2006). This kit contains a mixture of fluorescent stains (SYTO 9 and propidium iodide) that differ both in their spectral characteristics and their ability to penetrate healthy bacterial cell membranes. SYTO 9 stains viable cells whereas propidium iodide stains non-viable cells. Bacterial strains of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 in saline were inoculated in tubes of MHB containing AML (MIC value) incubated at 37 °C for 24 h. Bacterial strains inoculated in MHB with AML vehicle or incubated with chloramphenicol (MIC values) served as negative and positive controls, respectively. Bacteria were stained by adding 1 mL of each sample to 3 μL of BacLight. In the dark, samples were placed in megafunnels (Shandon Mega funnel, Thermo, UK) for immediate centrifugation in a cytocentrifuge as described above. Samples were prepared by using regular slides without any coating. Analyses were performed on a fluorescence microscope (BX-60, Olympus, Melville, NY, USA) using U-MWB filter (450–480 nm excitation wavelengths) which allows simultaneous visualization of both markers. Bacteria were directly counted in 10 random fields using an ocular grid at 1,000× magnification and the average percentage of live/dead bacteria was established for each slide sampled.
2.13. Statistical analysis Data were presented as median and 95% confidence interval. Each data point represented mean ± SD from at least two independent experiments performed in triplicate. Statistical differences between the treatments and the control were evaluated by ANOVA test followed by Bonferroni (p > 0.05).
2.9. Nucleotide leakage 3. Results The experiment was performed according to Tang et al. (2008) with some modifications. The overnight culture of E. faecalis ATCC 19433, S. aureus ATCC 6538 and S. typhimurium ATCC 13311 at 37 °C was washed and resuspended in 10 mM PBS (pH 7.4), reaching a final density of about 108 CFU ml−1. Strains were incubated with AML (MIC value) for different times (2, 4, 6 and 8 h); strains incubated with 10 mM PBS (pH 7.4) were used as control. Following incubation, the cell suspensions were centrifuged at 10,000 g for 10 min and the supernatants were then diluted appropriately. The optical density at 260 nm was then recorded at room temperature. The experiment was carried out in triplicate.
3.1. Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) AML showed a broad spectrum of antibacterial activity, with clear effectiveness against both Gram-negative and Gram-positive bacteria. However, the three bacteria species most susceptible to AML according to MIC values were E. faecalis, S. typhimurium and S. aureus, in this order (Table 1). We found that AML was bactericidal for S. typhimurium, S. aureus and P. aeruginosa (MBC/MIC = 1) and bacteriostatic for the others bacteria tested (MBC/MIC ≠ 1). Effectiveness does not necessarily mean mortality for bacterial cell. For E. faecalis, whose MIC value was lower (39 μg mL−1), the activity was bacteriostatic; while for P. aeruginosa, whose MIC was 16 times higher (625 μg mL−1), the activity was bactericidal. Chloramphenicol’s activity was bacteriostatic for both bacteria. The AML extract was more effective against E. faecalis, S.
2.10. Outer membrane permeability Outer membrane (OM) permeability was determined according to the method described by Hao et al. (2009), with some modifications. The strain used was S. typhimurium ATCC 13311. Briefly, overnight cultures (5 × 108 CFU mL−1) were inoculated into MHB containing AML alone and with the hydrophobic antibiotic erythromycin at concentrations ranging from 0.1 to 20 μg mL−1 with or without AML (MIC value). The media were then added to sterilized 96-well microplates and incubated at 37 °C for 24 h. The bacterial growth was measured at 450 nm using a spectrophotometer.
Table 1 Antibacterial activity of the methanolic extract Annona muricata leaves (AML). AML
Chloramphenicol
MICa
MBCa
MBC/MIC
MICa
625 39 156 625
1250 78 156 2500
2 2 1 4
3.13 6.25 12.5 25.0
1250 625 625
5000 2500 625
4 4 1
12.5 3.13 25.0
625
5000
8
3.13
78
78
1
12.5
313
1250
4
25.0
2.11. Study of biochemical reactions Bacillus cereus ATCC 14579 Enterococcus faecalis ATCC 19433 Staphylococcus aureus ATCC 6538 Enterobacter aerogenes ATCC 13048 Enterobacter cloacae ATCC 23355 Escherichia coli ATCC 10536 Pseudomonas aeruginosa ATCC 9027 Salmonella Choleraesuis ATCC 10708 Salmonella Typhimurium ATCC 13311 Shigella dysenteriae ATCC 13313
The clone (AML stressed clone, chloramphenicol stressed clone and control clone) from E. faecalis, S. aureus and S. typhimurium reference strains were subjected to the study of biochemical reactions. The tests were carried out on VITEK®2 system. The VITEK®2 GP ID Card (identification of Gram-positive bacteria) and GN ID Card (identification of fermenting and non-fermenting Gram-negative bacilli), which offer rapid and accurate identification of a broad range of clinically significant bacterial pathogens, were use for biochemicals. 2.12. UHPLC-ESI–MS/MS analysis AML was analyzed on an Acquity UHPLC system (Waters®, Milford,
a
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Fig. 2. Activity kinetics of methanolic extract of Annona muricata leaves (AML) against S. aureus (A); S. typhimurium (B) and E. faecalis (C). Choramphenicol (CHL) was used as positive control, and bacteria inoculated in MHB with AML vehicle was used as bacteria growth control (CBG). The experiments were carried out triplicate, and data represent the mean ± SD.
3.4. Nucleotide leakage and outer membrane permeability
typhimurium and S. aureus, based upon their MIC values and, as such, these bacteria were chosen as targets to further investigate the AML extract’s activity.
As shown in (Fig. 4), AML increased at 19%, 41% and 30% the nucleotide release in S. aureus, S. typhimurium and E. faecalis respectively, compared to the controls. The efflux of nucleotides from the intracellular compartment was more significant for Gram-negative than Gram-positive bacteria. Fig. 5 clearly shows synergism between AML and erythromycin suggesting that AML was able to increase the OM permeability in the tested bacteria.
3.2. Bacterial killing assay The AML vehicle does not affect bacterial growth as the shape of the bacterial growth curve was typical for all species tested. However, all tested bacteria showed a dose-dependent growth decrease when exposed to AML leaf extract (Fig. 2), with bacteria death by lysis induced at the MIC concentration. For S. aureus, all concentrations inhibited the growth cycle curve when compared to the control treated with vehicle (Fig. 2A). For S. typhimurium (Fig. 2B) AML extended the lag phase by two hours, and then induced the bacteria death by lysis at the MIC value. For E. faecalis (Fig. 2C), it was observed that the bacterial life cycle was forced into the death phase at the 6th hour of treatment. At other concentrations, the bacterial growth was increased, but this growth was lower than the vehicle treatment.
3.5. Study of biochemical reaction Clones from three tested strains exhibited some alteration in biochemical reactions after antimicrobial stress when compared with the original, unstressed control. For S. aureus the L-lactate alkalinization (ILATk) and α-glucosidase (AGLU) reactions were converted from negative to positive in the AML clone, and the pyrrolidonyl arylamidase (PyrA) and bacitracin-tests (BACI) were converted from positive to negative in the chloramphenicol clone. For S. typhimurium the gammaglutamyl transpeptidase (GGT), urease (URE) and ILATk reactions were converted from negative to positive in the AML clone and tyrosine arylamidase (TyrA) and GGT were converted from negative to positive in the chloramphenicol clone. Finally, in E. faecalis, leucine arylamidase (LeuA) and alanine arilamidase (AlaA) were converted from a positive to a negative reaction in the AML clone, while only LeuA was altered by the chloramphenicol clone. If differences between the stressed clones and controls were observed in the biochemical characterization, it was not enough to modify the identity of the strain identification. The percentage of trust was 98%, 97% and 98% for Chloramphenicol and 99%, 93% and 97% for AML clones, respectively for S. aureus, S. typhimurium and E. faecalis.
3.3. Bacterial abundance and viability Fluorescence microscopy after DAPI staining showed that AML inhibited the bacterial abundance compared to the control group, reducing the bacterial growth by 28%, 26% and 40% for S. aureus, S. typhimurium and E. faecalis, respectively (Fig. 3A). AML treatment caused a decrease in cell density and an increase in cell death. The cytocentrifuge preparations of the florescent viability probe test (Live/Dead® BacLight) enabled a clear imaging of live (green)/dead (red) bacteria in both AML-treated and control cultures (Fig. 3C). Quantitative results showed that AML (MIC value for 24 h) for S. aureus, S. typhimurium and E. faecalis induced an increase in the percentage of dead cells (34%, 34% and 24%, respectively) compared to the vehicle (Fig. 3B). Chloramphenicol showed 8.5%, 4.5% and 1% of dead cells, respectively.
3.6. UPLC-ESI–MS/MS analysis The chromatografic profile of the AML was acquired and the compounds identified are described in Table 2. The chemical structure of each compound is shown in Fig. 6. 335
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Fig. 3. Effect of methanolic extract of Annona muricata leaves (AML) on bacterial growth of S. aureus, S. typhimurium and E. faecalis. Cultured bacteria were stained with DAPI or LIVE/ DEAD® BacLight™ and counted under fluorescence microscopy for evaluation of cell density (A) and viability (B), respectively. AML treatment induced both decrease of cell density (A) and increase of cell death (B) compared to control groups treated with AML vehicle. In (C), representative images of treated and non-treated bacteria after BacLight staining. Note that viable/live (green) and non-viable/dead (red) bacteria are clearly observed and dead cells increases with the AML treatment. Letters indicate statistically difference from controls treated with AML vehicle for S. aureus (a), S. typhimurium (b), E. faecali (c) (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD of bacteria counted. Scale bar: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
time-dependent kinetics showed AML was more effective than chloramphenicol, which is a bacteriostatic drug. Following the standard method for the determination of bactericidal effects, i.e. bacterial killing assay, we performed two additional approaches using fluorescent probes to investigate the effect of the plant extract on bacterial growth/death in treated cultures (AML at MIC value and controls, for 24 h). First, bacterial abundance was quantified by fluorescence microscopy after DAPI staining. AML treatment induced a decrease in cell density and an increase in cell death, which corroborated the bacterial killing assay results. This cell reduction may be related to both potential effects of AML in inhibiting bacterial growth and/or inducing death and cell lysis. Secondly, we investigated bacterial death using a florescent viability probe (Live/Dead® BacLight), which enables direct identification of loss of plasma membrane integrity. Cells with damaged membranes are unable to maintain an electrochemical potential and are considered as dead, so that individual imaging of bacteria is helpful to recognize bacterial viability and their physiological functions at a single-cell level (Joux and Lebaron, 2000). The increase of dead cells induced by AML was significant, which is in accordance with previous studies that demonstrated the bactericidal activity after plant-extract treatments
Although the antibacterial potential of A. muricata has been previously reported, this study shows for the first time that the AML extract has a broad spectrum of antibacterial activity, against both Gram-negative and Gram-positive bacteria, as shown by the MIC/MBC assay. A time-dependent bactericidal effect takes place when the concentration of the antibacterial drug exceeds the MIC for the microorganism, while a concentration-dependent bactericidal effect occurs when an antibiotic has a high concentration at the binding site to eliminate the microorganism (Anantharaman et al., 2010). Our timekill kinetic analyses showed that AML exhibited rapid time dependent kinetics of bacterial killing for both Gram negative and Gram positive bacteria, mainly at MIC value. Thus, AML may potentially contain infections with greater rapidity, compared to antibacterial agents that exhibit slow killing kinetics. Comparing the activity of the AML with chloramphenicol at MIC concentration, we observed a similar action in the early stages, but a better inhibition after the bacterial lag phase. Although these events occurred for all three of the bacteria species studied, it is important to note that even for E. faecalis, for which the AML extract demonstrated bacteriostatic activity (Table 1), the rapid
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Fig. 4. Mode of action of methanolic extract of Annona muricata leaves (AML). Effect of AML on the amount of the total nucleotide released from S. aureus (Sa), S. typhimurium (St) and E. faecalis (Ef). Letters indicate statistically difference from controls treated with AML vehicle for S. aureus (a), S. typhimurium (b), E. faecali (c) (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD.
Our results show that AML is effective against both Gram-negative and Gram-positive bacteria, but it’s antimicrobial activity is organism dependent. Furthermore, the results suggest that the inhibition of bacterial growth and the induction of cell death by AML may both be related to the same mechanism (membrane destabilization). Several models for studying the mode of action of antibacterial drugs have been described in the literature. In this context, nucleotide leakage and OM permeability assays are associated with damages to bacterial membranes, so that these models were chosen to clarify how AML is able to inhibit bacterial proliferation (Hao et al., 2009). As DNA and RNA are released after membrane disruption, these nucleotides were quantified by monitoring the absorbance at 260 nm. AML probably induced the disruption of the cytoplasmic membrane after 2 h as the nucleotide release began to increase after this time, especially in Gram-negative bacteria.
through viability analyses using Baclight (Abdullah et al., 2014; You et al., 2013). Noteworthy, this viability analyses is unable to detect alterations prior to cell lysis. This means that AML treatment was able to elicit changes at the cellular level, which are indicative of cell death, i.e., loss of plasma membrane integrity. Additionally, as the treatment with chloramphenicol, a well-known bacteriostatic drug (Bernatová et al., 2013), showed very low proportion of dead cells, our viability results reinforce the potential bactericidal effect of AML for the three bacteria tested. Abundance analyses and cell viability evaluation corroborate to MBC/MIC for the identification of antimicrobial activity, which indicates that AML also has a potential bacteriostatic effect on E. faecalis, as the increase in the proportion of dead cells (24%) is smaller than the reduction of the total bacterial count (40%). Therefore, the microscopic approaches used in the present study were very important to confirm and reinforce perspectives about the mode of action of AML.
Fig. 5. Mode of action of methanolic extract of Annona muricata leaves (AML). Outer membrane permeability of S. typhimurium induced by AML in association with erytromicin. aStatistically different from ERY + AML treated. (ANOVA followed by Bonferroni, P < 0.05). All experiments were carried out in triplicate, and data represent the mean ± SD.
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Table 2 Active compounds identified in the methanolic extract Annona muricata leaves (AML) by UHPLC–MS/MS analysis. Compound
Retention time standards (min)a
Retention time (min)a
[M+H]+ (m/z)
MS-MS íons (m/z) and relative abundance (%), in positive íon mode
Anonaine Asimilobine Liriodenine Nornuciferine Xylopine Reticuline Coripalmine
2.98 2.29 3.43 2.50 3.00 1.69 2.27
2.99 2.32 3.41 2.51 3.09 1.71 2.25
266 268 276 282 296 330 342
266→ 268→ 276→ 282→ 296→ 330→ 342→
a
266 268 276 282 296 299 342
(18), 249 (100), 234 (8), 219 (28), 191 (46) (10), 251 (100), 236 (15), 219 (51), 191 (80) (100) (100) (100) (30), 192 (100), 175 (12), 151 (7), 143 (16), 137 (22) (100)
UHPLC-ESI–MS/MS analysis using analytical standards in the same chromatographic for identification of the compounds detected in AML.
pores, solubilization of membrane proteins, and changes in the structure/function of the phospholipid bilayer of the membrane (Chen and Cooper, 2002). On the basis of the results of the bacterial membrane permeability studies, it can be stated that the antibacterial action of AML is induced, at least in part, by changes in the permeability of the cell wall and to the bacterial cytoplasmic membrane. It is well accept that antibiotics induce bacterial biochemical-physiological alterations (Dwyer et al., 2014; Lobritz et al., 2015; Santos et al., 2011). Changed phenotypic profiling has been utilized to discover of the mechanism of action of antimicrobial drugs (Athamneh et al., 2014). Our results corroborate this idea. The differences observed in stressed and control clones for the three species studied indicate the negative expression of enzymes involved in secondary metabolism and positive expression (induction) of enzymes involved with energetic metabolism. These data are consistent with those found in other tests. Poor or inadequate energetic metabolism interferes directly in the logarithmic phase of bacterial growth leading to a bacteriostatic action. Furthermore, even in part, the energy metabolism is related to the cytoplasmic membrane. Therefore, interferences in the membrane could induce cell lysis. For a better global positioning of any herbal drug, it is important to establish a chemical profile based upon the maximum available number of markers for an extract (Fabri et al., 2013). For this reason, the chromatographic profile of AML was acquired. Plants from the genus Annona, Annonaceae family, are well known by the presence of acetogenins, which have been reported as potent antimicrobial and cytotoxic compounds. Almost 30 acetogenins were already isolated from this species (Pandey and Barve, 2011). To identify other chemical constituents which could be possibly related to antimicrobial activity, we investigated the alkaloid profile of AML by UHPLC-ESI–MS/MS
The bacterial cell wall is a rigid structure that covers the cytoplasmic membrane and gives structural support to bacteria. In Grampositive bacteria, the cell wall is thick consisting of several layers of peptidoglycan and surface glycopolymers such as teichoic acids. In Gram-negative bacteria, the cell wall is relatively thin and is composed of a single layer of peptidoglycan, lipoproteins, lipopolysaccharides, and the OM, which is important as a molecular barrier that prevents the loss of intracellular proteins, and reduces the access of hydrolytic enzymes and some drugs, particularly hydrophobic antibiotics (SilvaJúnior et al., 2014). The influence of AML on S. typhimurium OM permeability was determined by a synergistic growth inhibition assay in association with the hydrophobic antibiotic erythromycin. The OM of the Gram-negative cell wall is known to be an effective permeability barrier against hydrophobic antibacterial agents, including erythromycin. This antibiotic does not infiltrate effectively in the intact Gram-negative OM, however it can permeate the OM of the certain OM-defective mutants, as well as the OM damaged by chelators or polycations (Viljanen et al., 1990). Therefore, erythromycin was chosen as a probe to detect any possible increase in OM permeability induced by AML. The synergism between AML and erythromycin strongly indicated that AML was able to disrupt OM permeability. Co-administration of two or more antibiotics is a common medical practice because it results in synergistic effects, broadening the spectrum of action or reducing bacterial resistance and toxicity (Silva-Júnior et al., 2014). In these assays, it is not possible to clarify how the compounds present in AML interact with the cytoplasmic membrane components and exactly how it elicits the leakage of nucleotides. A number of mechanisms for this interaction have been proposed to explain the action of antibacterial drugs on the cytoplasmic membrane, with emphasis on formation of
Fig. 6. Chemical structures of the compounds identified in the methanolic extract of Annona muricata leaves (AML) by UHPLC-ESI–MS/MS analysis.
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analysis, using analytical standards in the same chromatographic conditions. The literature reports the wide occurrence of benzylisoquinoline alkaloids in Annonacaea spp. and they may be considered as good chemical and biological markers for this family (González-Esquinca et al., 2014). Bioactivities are described for many of them (Costa et al., 2013; Kuete, 2010; Vendramin et al., 2013; Silva et al., 2014b). The cytotoxic and antimicrobial activities of anonaine (Chen et al., 2011; Kuete, 2010; Paulo et al., 1992; Villar et al., 1987), asimilobine (Costa et al., 2013; Woo et al., 1995), liriodenine (Hsieh et al., 2005; Hufford et al., 2011; Woo et al., 1997), nornuciferine (Marti et al., 2013), xylopine (Florence et al., 2014; Hai-Bo et al., 2013; Likhitwitayawuid et al., 1993) and reticuline (Orhan et al., 2007; Suresh et al., 2012; Tsai et al., 1989) are well known. Chen et al. (2011) reported that anonaine induces DNA damage, activation of caspase 3, 7, 8 and 9, and poly ADP ribose polymerase cleavage, although more profound information regarding the mechanism of action of other compounds identified in this study are not well described yet. Thus, based on these literature data, the chromatographic analysis of AML suggests that the aforementioned compounds may be responsible for the antimicrobial activity found for A. muricata extract, and, could be acting in synergism with one another and/or acetogenins present in the extract. 5. Conclusion This study reported, for the first time, that the antimicrobial activity of A. muricata presents a broad spectrum of action, and that bacterial membranes (both plasma and outer membranes) are primary targets of the bioactive compounds. The alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline may be, at least in part, responsible for the bioactivities reported in this study. In addition, our results corroborate to the ethnopharmacological uses of this specie. It is also noteworthy to mention that A. muricata presents a remarkable phytomedicinal potential, and it is commonly used as food, which encourages further studies to evaluate whether the ingestion of this plant may be useful to treat or prevent bacterial infections, as several antimicrobial compounds were already identified in this species. From the results obtained with the extract of the plant evaluated here, we could expect its possible application, in the future, associated with other antimicrobials, such as erythromycin, in order to avoid antibacterial resistance. However, until then, more studies are necessary. Conflict of interest The authors declare no competing financial interest. Acknowledgments The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), to the Federal University of Juiz de Fora (UFJF)/Brazil, to CAPES, CNPq, FAPESP and FAEPEX-UNICAMP for financial support, and also to Dr. Vinícius Antônio de Oliveira Dittrich from the Department of Botany/Federal University of Juiz de Fora for the botanical identification of the species. References Abdullah, S., Gobilik, J., Chong, K.P., 2014. In vitro antimicrobial mode of action of Cynodon dactylon (L.) Pers. solid phase extract (SPE) against selected pathogens. In: International Conference on Civil, Biological and Environmental Engineering. CBEE, Istanbul, Turkey. pp. 66–72. Adewole, O.S., Caxton-Martins, E.A., 2006. Morphological changes and hypoglycemic effects of Annona muricata Linn. (Annonaceae) leaf aqueous extraction pancreaticcells of streptozotocin-treated diabetic rats. Afr. J. Biomed. Res. 9, 173–187. Adewole, O.S., Ojewole, J.A.O., 2008. Protective effects of Annona muricata (Linn) (Annonaceae) leaf aqueous extract on serum lipid profiles and oxidative stress in hepatocytes of streptozotocin-treated diabetic rats. Afr. J. Tradit. Complement. Altern. Med. 6, 30–41. Adjanohou, E., 1996. Organization of African Unity, Scientific, Technical, and Research
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