|Year : 2011 | Volume
| Issue : 1 | Page : 1-7
Absence of the antibacterial activity of the crude extracts and compounds isolated from M. rubiginosa against extended-spectrum β-lactamase producing enterobacteria
Geisiany M de Queiroz1, Maria Gorete M de Souza1, Tatiane C de Carvalho1, Luciana A Casemiro1, Wilson R Cunha2, Carlos H. G. Martins1
1 Laboratory of Research in Applied Microbiology, University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600 Franca-SP, Brazil
2 Nucleus of Exact Sciences and of the Earth, University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600 Franca-SP, Brazil
|Date of Web Publication||15-Jul-2011|
Carlos H. G. Martins
Laboratório de Pesquisa em Microbiologia Aplicada, Universidade de Franca, Av. Dr. Armando Salles Oliveira, 201. CEP: 14404-600, Franca, SP
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The present study evaluates the antibacterial activity (Minimum Inhibitory Concentration- MIC, Minimum Bactericidal Concentration- MBC) and the Fractional Inhibitory Concentration (FIC) of crude extracts and compounds isolated from Miconia rubiginosa against ten clinical bacterial isolates and one standard bacterial strain Expanded-Spectrum β-lactamase (ESBL)-producing enterobacteria. Materials and Methods: The crude extracts were obtained by maceration and the compounds isolated were purified through different chromatography techniques, and then structures were identified for physical methods of organic analysis. The evaluation of the antibacterial activity for the microdiluition in broth technique. Results: The MIC and MBC result for the ethanolic extract was 2.0 mg/mL. For the dichloromethane extract and ursolic acid and oleanolic acid, MIC was >2.0 mg/mL, but no bactericidal activity (MBC) was observed. The FIC values achieved with the combinatioin of the crude extracts with clavulanic acid were not significantly different. Conclusion: The ethanolic extract from Miconia rubiginosa exhibits better antibacterial activity, but the two isolated compounds are not active against the tested bacterial isolates. The combination of the crude extracts with clavulanic acid does not lead to synergism, and there are no statistical differences between the two crude extracts in this sense.
Keywords: Antibacterial activity, Beta-lactamase, Miconia rubiginosa, oleanolic acid, ursolic acid
|How to cite this article:|
de Queiroz GM, de Souza MM, de Carvalho TC, Casemiro LA, Cunha WR, Martins CH. Absence of the antibacterial activity of the crude extracts and compounds isolated from M. rubiginosa against extended-spectrum β-lactamase producing enterobacteria. J Pharm Negative Results 2011;2:1-7
|How to cite this URL:|
de Queiroz GM, de Souza MM, de Carvalho TC, Casemiro LA, Cunha WR, Martins CH. Absence of the antibacterial activity of the crude extracts and compounds isolated from M. rubiginosa against extended-spectrum β-lactamase producing enterobacteria. J Pharm Negative Results [serial online] 2011 [cited 2019 Sep 22];2:1-7. Available from: http://www.pnrjournal.com/text.asp?2011/2/1/1/82982
| Introduction|| |
Resistance mechanisms are part of the natural evolution of bacteria. They occur when the bacterial cells are exposed to inadequate antimicrobial agents, which destroy the sensitive strains whilst the resistant ones survive. This process is known as selective pressure. Bacteria develop natural defense mechanisms, so that they can grow and multiply the fastest possible. Such mechanisms include mutations or acquisition of resistance genes from other cells, as in the case of plasmidial conjugation. ,
Enterobacteria are Gram-negative, nonspore-producing, facultative anaerobic bacilli that ferment glucose. They belong to the Enterobacteriaceae family and have great biological importance due to their pathogenicity and the appearance of bacterial isolates that are multiresistant to the antimicrobial agents currently used in therapeutics. 
The expanded-spectrum β-lactamase enzymes (ESBL) are capable of inactivating penicillins, cephalosporins, and other related antimicrobial agents that hydrolyze the β-lactam ring. However, ESBL are usually inhibited by compounds such as clavulanic acid, sulbactan, and tazobactan.,, It has been demonstrated tha the combination of β-lactam antibiotics with inhibitors of β-lactamases displays in vitro activity against a range of ESBL-producing pathogenic enterobacteria. Most of the inhibitors of β-lactamases do not present significant intrinsic antibacterial activity, so they act as suicidal inhibitors by competing with β-lactam antibiotics in the active range of β-lactamases. This culminates with an irreversible interaction between the enzyme and the inhibitors.
ESBL-producing enterobacteria have been more frequently isolated from clinical samples originated from hospitalized patients; however, they can also be found in samples of community origin. These bacterial isolates can appear sporadically, thereby bearing no relation to epidemic nor leading to nosocomial outbreaks. The treatment of patients infected with ESBL-producing bacterial strains is limited to the prescription of a few broad-spectrum drugs, which are bound to fail in the presence of microorganisms that produce multiple β-lactamases.
Plants are considered true, complex biochemical laboratories that synthesize the natural active lead drugs, among several other substances. In fact, natural products are involved in the development of 44% of all the new pharmaceuticals. In some areas involving the treatment of diseases such as cancer and infectious disorders, around 60% of the prescribed drugs are of natural origin. 
The Melastomataceae is one of the most important families of the neotropical flora, with about 4200 to 5000 species belonging to 11 tribes and 185 genera. , Among the genera found in Brazil, the Miconia stands out and is widely distributed along Tropical America. 
From a chemical viewpoint, some studies have been accomplished with species belonging to the genus Miconia. Significant results concerning antimicrobial, ,, analgesic,  and trypanocidal activity  have been achieved with the quinones, cumarins, and triterpenes isolated from these plants.
Starting with a comparative study the authors demonstrated that the ethanolic and dichloromethane crude extracts from M. rubiginosa exhibit antimicrobial activity.  The methanolic extract from the wood of M. racemosa displays inhibitory activity against two strains of Plasmodium falciparum resistant to chloroquine and quinine,  and still observed that the ethanolic extract from the leaves of M. ciliata presents sedative activity. 
The substances isolated from M. rubiginosa, namely ursolic acid (UA) (3b-hydroxy-urs-12-en-28-oic acid) and its isomer oleanolic acid (OA) (3b-hydroxy-olea-12-en-28-oic acid), are tripterpenes widely distributed in medicinal herbs in the form of free acid, aglycone, or saponin triterpenoid.  Their antimicrobial activities have been demonstrated,  they inhibit the growth of Staphylococcus aureus, Gram-negative bacteria, and Mycobacterium tuberculosis;  they display antiviral activity against the Human Immunodeficiency Virus (HIV);  and they serve as antibacterial agents against oral microorganisms  and resistant Enterococcus spp. and Staphylococcus aureus strains. 
Therefore, it is mandatory that the effects of extracts and substances isolated from plants are investigated, so that new approaches for the treatment of patients with infectious diseases caused by ESBL-producing enterobacteria can be found. The present work aimed to evaluate the antibacterial activity of the ethanolic and dichloromethane crude extracts and compounds isolated from M. rubiginosa. The synergistic interaction between the crude extracts from M. rubiginosa and clavulanic acid against ESBL-producing enterobacteria was also examined.
| Materials and Methods|| |
Ten clinical bacterial isolates provided by a private laboratory of clinical analyses located in none the state of São Paulo were employed. These isolates had different origins [Table 1], and their use was approved by the Research Ethics Committee of the University of Franca, registered under protocol number 080/07. A standard strain of Klebsiella pneumoniae (ATCC-700603), acquired from the "American Type Culture Collection", maintained at -20°C in a freezer in the Laboratory of Research in Applied Microbiology (LaPeMA) of the University of Franca, was also utilized.
The biochemical identification of the enterobacterial species was accomplished by the commercial system "BBL Crystal Identification Systems Enteric/Nonfermenters ID Kit" (Becton and Dinckinson, Lincoln Park, USA), according to the manufacturer's instructions. The screening test for ESBL production by the bacterial isolates was performed by the method of approach of disks as previously described. 
The species M. rubiginosa was collected in the area located at Rodovia Tancredo Neves (Franca/SP-Claraval/MG), in the northeast of the state of São Paulo, with approximate coordinates of 20°30' S and 40°20' WG, altitudes between 810 and 870 m, in an area comprising 19.66 hectares of cerrado. Identification of the plant was accomplished by Dra. Ângela Borges Martins, at the Institute of Biology of the University of Campinas (UNICAMP). The species was deposited in the Herbarium of this same Institute (UEC 10830). To obtain the ethanolic and dichloromethane crude extracts from M. rubiginosa, the aerial parts (4 kg) of the plant were dried and stabilized in a stove with circulating air (Marconi®, Piracicaba, São Paulo, Brazil) at a temperature of 40°C. Soon afterwards, they were triturated in knive-mill (Marconi®), until the powder form was achieved. The resulting powder was submitted to maceration with dichloromethane and then ethanol/water (96:4 v/v) for seven days, at 25°C, for production of the crude extract. The whole material resulting from the maceration process was filtered and concentrated under reduced pressure at a temperature of 60°C by means of a rotary evaporator (Marconi®), until all the solvent was eliminated. For purification and isolation of the ursolic and oleanolic acids, the obtained dichloromethane extract was submitted to several chromatographic processes (Vacuum-Liquid Chromatography, VLC and High Performance Liquid Cromatography, HPLC), and after your structures were identified for physical methods of organic analysis, as previously described. 
Determination of the Minimum Inhibitory Concentration (MIC) by the broth microdilution technique in 96-well microplates (Techno Plastic Products® , Trasadingen, Switzerland) as established by NCCLS  was employed for evaluation of the antibacterial activity of the crude extracts and the isolated compounds diluted in dimethyl sulfoxide (DMSO).
Control cultures were also accomplished, namely a culture presenting bacterial growth, a sterile culture of the broth Müeller-Hinton (Difco, Sparks, MD, USA), a solvent control using DMSO at concentrations varying from 1% to 5%, and the positive control Imipenen (Merck Sharp and Dohme, São Paulo, Brazil) at concentrations ranging between 0.0001 mg/mL and 0.0295 mg/mL. All these controls were accomplished for analysis validation. The crude extracts and compounds isolated from M. rubiginosa were investigated at concentrations between 0.3 mg/mL and 2.0 mg/mL. The microplates were covered and incubated at 37°C for 24 hours. After the incubation period, 15 μL of a 0.02% resazurin (Sigma-Aldrich® , New York, USA) sterile aqueous solution was added to each well for analysis of the results. This developing medium facilitates verification of the presence of microbial growth; a blue coloration indicates absence of microbial growth, whereas the red color is an indication of the growth of viable cells.
For determination of the Minimum Bactericidal Concentration (MBC), before the revelation process with the aid of pipette multicanal each well of the microplates was subplate in agar Müeller Hinton prepared previously. Determination of the Fractional Inhibitory Concentration (FIC) was accomplished in 96-well microplates (Techno Plastic Products® ), and results were interpreted as previously described,  using the following calculations:
- FIC of A= MIC of A in combination/MIC of A alone
- FIC of B= MIC of B in combination/MIC of B alone
- FIC of combination= FIC of A+ FIC of B
Interpretation of the results: FIC> 0.5 to 4 Indifferent/ FIC> 4 Antagonism / FIC≤ 0.5 Synergysm
| Results|| |
Results from the identification of species belonging to the genera Escherichia sp and Klebsiella sp demonstrated that of the 10 researched bacterial isolates, 3 belonged to the species Escherichia coli, 6 to the species Klebsiella oxytoca, and 1 to the species K. pneumoniae. For all the bacterial isolates, the screening test confirmed the production of ESBL enzymes, with formation of a "Ghost Zone" in the approach of disks. At the end of the whole process of extract stabilization and maceration, a total of 206.6 g ethanolic extract and 202.8 g dichloromethane extract were obtained from M. rubiginosa. The chemical structures of the compounds isolated from M. rubiginosa are represented in [Figure 1].
The MIC and MBC values found for the ethanolic extract were to 2.0 mg/mL, whilst for the dichloromethane extract MIC was superior to 2.0 mg/mL. The isolated compounds ursolic and oleanolic acids gave MIC and MBC values higher than 2.0 mg/mL. As for the Imipenen (Merck Sharp and Dohme) control, MIC values varied from <0.001 mg/mL to 0.001 mg/mL, whereas for DMSO the results were higher than 5%. The results obtained for the controls confirm the precision of the experiments. For determination of the FIC values, the lowest crude extract and clavulinic acid concentrations capable of inhibiting bacterial growth were employed, namely 2.0 mg/mL in the case of the ethanolic extract, between 3.6 and 8.0 mg/mL for the dichloromethane extract (carried out in a separate study), and 0.3 mg/mL for clavulanic acid. Interpretation of the results from the FIC calculations demonstrated that the combination of the crude extracts with the synthetic inhibitor of β-lactamases, clavulanic acid, did not give rise to synergysm, this results are represented in [Table 2] and [Table 3]. FIC determination was not accomplished for the compounds isolated from M. rubiginosa ursolic acid and oleanoic acid.
|Table 2: Results found for the ethanolic extract (A) and for the clavulanic acid (B) alone and in combination (A/B) against microorganisms valued (mg/mL)|
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|Table 3: Results found for the dichloromethane extract (A) and for the clavulanic acid (B) alone and in combination (A/B) against microorganisms valued (mg/mL).|
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| Discussion and Conclusions|| |
Over the last 20 years, countless β-lactamic drugs have been developed for the reason that they act against the hydrolytic action of the ESBL enzymes. However, the indiscriminate use of such drugs has favored the selection of ESBL-producing bacteria that are resistant to these pharmaceuticals. 
Previous studies have reported that the crude extract and the compounds isolated from M. rubiginosa display satisfactory antimicrobial activity. ,, A comparative study of four screening techniques, in order to evaluate their efficacy, in this study, the ethanolic and dichloromethane crude extracts from the aerial parts of M. rubiginosa where shown to exhibit activity against five microorganisms, namely E. faecalis, K. rhizophila, E. coli, P. aeruginosa, and S. choleraesuis, but they were not active against S. aureus. The antimicrobial activity of the abovementioned crude extracts was analyzed by the microdilution broth method for determination of MIC values. Results for both extracts varied between 250 μg/mL and >400 μg/mL for the tested microorganisms. 
The antimicrobial activity of compounds isolated from plants of the Brazilian cerrado, including ursolic and oleanolic acids, against seven oral microorganisms, namely S. sanguinis, S. mutans, S. sobrinus, S. mitis, S. salivarus, C. albicans, and E. faecalis. The antimicrobial activity of the two acids was investigated by the broth microdilution method for determination of MIC values, which ranged between 30 μg/mL and 90 μg/mL in the case of the tested microorganisms, for both substances. 
The evaluated the activity of the ursolic and oleanolic acids obtained from Salvia officinalis against bacterial isolates presenting two different resistance forms. Two isolates were Vancomycin-Resistant Enterococcus (VRE) belonging to E. faecalis and E. faecium, while one isolate consisted of Methicillin-Resistant Staphylococcus (MRS) belonging to S. aureus. Three Gram-negative bacterial isolates were also analyzed, namely E. coli, P. aeruginosa, and S. marcescens. MIC values obtained from analysis of the antimicrobial activity by the broth microdilution method varied from 4 μg/mL to 16 μg/mL for ursolic acid, and from 8 μg/mL to 16 μg/mL for oleanolic acid, in the case of the VRE and MRS isolates. As for the Gram-negative isolates, both acids furnished MIC values >128 μg/mL. The latter results were explained in terms of the outer lipoprotein membrane present in these bacteria. 
In the literature there are reports on the combination of plant extracts with synthetic antibiotics, with a view to investigating possible synergistic effects. The test the simultaneous use of the alcoholic extract from Brazilian and Bulgarian propolis with some synthetic antibiotics commonly prescribed against Salmonella Typhi.  To this end, the Pour Plate methodology after serial incubations was utilized, and significant synergism was detected. Also, the synergistic effect between eight different crude extracts and synthetic antibiotics employed against Staphylococcus aureus, using the methodology of Kirby and Bauer were assessed. Results demonstrating synergism and antagonism were achieved. 
Comparing literature reports with ours, it can be noticed that not only the crude extracts but also the compounds isolated from the species M. rubiginosa display good antibacterial activity, ,,, even against resistant isolates.  However, the aforementioned literature studies did not test the effects of the crude extracts and the isolated compounds from M. rubiginosa against ESBL-producing bacteria, which may explain the results from our present research. Besides being producers of ESBL, the bacteria studied here are Gram-negative; that is, they have an outer membrane. In other words, the bacterial isolates tested here exhibit a form of resistance that challenges the currently employed therapeutics, since they present pronounced resistance to state-of-the-art antimicrobial agents, such as the synthetic third-generation antimicrobial cephalosporins.
Finally, of the investigated M. rubiginosa extracts, the ethanolic crude extract was the one that furnished the best results. Therefore, the activity of this extract should not be related to the presence of the ursolic and oleanolic acids, once high concentrations of these triterpenes are present in the dichloromethane extract. Hence, the activity of the ethanolic extract should be due to the presence of more polar substances that possibly act together and potentialize the action of this crude extract.
The proposed combination of the crude extracts from M. rubiginosa with clavulanic acid aimed to reveal a possible synergistic effect between the extracts and this β-lactamase inhibitor. However, when the calculations proposed were applied, it could be seen that in the present case the combination was indifferent for all the bacterial isolates. Moreover, to the best of our knowledge, there are no studies in the literature evaluating the effect of such combinations on ESBL-producing enterobacteria, so no comparisons with results from other works could be made. In conclusion, we would like to highlight the importance of more comprehensive studies that will detect the actual mechanisms underlying the action of crude extracts and compounds isolated from medicinal plants on β-lactamases, which should allow for new discoveries about their antibacterial effect.
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[Table 1], [Table 2], [Table 3]