Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Evaluation of the Interaction between the Poincianella pyramidalis (Tul.) LP Queiroz Extract and Antimicrobials Using Biological and Analytical Models

  • Thiago P. Chaves ,

    Contributed equally to this work with: Thiago P. Chaves, Felipe Hugo A. Fernandes, Jocimar S. Santos, Ana Cláudia D. Medeiros

    Affiliations Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil, Department of Natural Sciences, Universidade Federal do Piauí, Bom Jesus, Piauí, Brazil

  • Felipe Hugo A. Fernandes ,

    Contributed equally to this work with: Thiago P. Chaves, Felipe Hugo A. Fernandes, Jocimar S. Santos, Ana Cláudia D. Medeiros

    Affiliations Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil, School of Pharmaceutical Sciences, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil

  • Cleildo P. Santana ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Jocimar S. Santos ,

    Contributed equally to this work with: Thiago P. Chaves, Felipe Hugo A. Fernandes, Jocimar S. Santos, Ana Cláudia D. Medeiros

    Affiliation Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Francinalva D. Medeiros ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Délcio C. Felismino ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Vanda L. Santos ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of Pharmacology, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Raïssa Mayer R. Catão ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of Research in Microbiology, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

  • Henrique Douglas M. Coutinho ,

    ‡ These authors also contributed equally to this work.

    Affiliation Laboratory of Microbiology and Molecular Biology, Universidade Regional do Cariri, Crato, CE, Brazil

  • Ana Cláudia D. Medeiros

    Contributed equally to this work with: Thiago P. Chaves, Felipe Hugo A. Fernandes, Jocimar S. Santos, Ana Cláudia D. Medeiros

    Affiliation Laboratory of drug assay and development, Universidade Estadual da Paraíba, Campina Grande, Paraíba, Brazil

Evaluation of the Interaction between the Poincianella pyramidalis (Tul.) LP Queiroz Extract and Antimicrobials Using Biological and Analytical Models

  • Thiago P. Chaves, 
  • Felipe Hugo A. Fernandes, 
  • Cleildo P. Santana, 
  • Jocimar S. Santos, 
  • Francinalva D. Medeiros, 
  • Délcio C. Felismino, 
  • Vanda L. Santos, 
  • Raïssa Mayer R. Catão, 
  • Henrique Douglas M. Coutinho, 
  • Ana Cláudia D. Medeiros


Poincianella pyramidalis (Tul.) LP Queiroz (Fabaceae) is an endemic tree of northeastern Brazil, occurring mainly in the Caatinga. Its medicinal use is widespread and is an important therapeutic option against diarrhea, dysentery, and respiratory and urinary infections, among other diseases. In this study we determined the chemical marker and evaluated the interaction between P. pyramidalis extract and a commercial antimicrobial through the use of biological and analytical models. To obtain the extract, an ethanol-water mixture (50:50 v/v) was used as solvent. It was nebulized in a spray dryer using colloidal silicon dioxide as a drying adjuvant. The extract (ENPp) was subjected to HPLC analysis to verify the presence of certain secondary metabolites. The Minimum Inhibitory Concentration (MIC) of the extract against Gram-negative bacteria was determined by broth microdilution and the MIC of synthetic antimicrobial drugs in the presence and absence of the extract. The antioxidant activity of ENPp was evaluated by the DPPH method. The compatibility between the antimicrobial and the extract was evaluated by thermal analysis (TG/DTA). The acute toxicity of the extract was evaluated in vivo in rodents. The results indicate significant additive action of the extract on synthetic antibiotics, considerable antioxidant activity and absence of toxicity. This extract shows high potential for the development of formulations for antimicrobial therapy when used with a vegetable-active ingredient.


The emergence of antibiotics was one of the greatest advances in modern medicine. These substances play a key role in the successful treatment of infections that used to take patients’ lives, and they also help increase life expectancy. However, the widespread and indiscriminate use of antibiotics has contributed to the emergence of resistant pathogens, including multidrug-resistant strains [1,2]. This problem has been aggravated in recent decades and has recently been recognized as one of the greatest threats to human health [3,4].

A particular concern is the case of the multiresistant Gram-negative bacteria. These microorganisms, which are intrinsically resistant to different antibiotics, have an outer membrane of low permeability that restricts access of the antimicrobial agents to their targets inside the cell, and this concern, together with the resistance mechanisms of the acquired-like efflux pump, enzymatic degradation, and change in drug target site, protect the bacteria against the deleterious effects of these agents [5]. A major threat to the global level caused by these bacteria are nosocomial infections because the treatment of patients in critical condition in an intensive care unit (ICU) or the treatment of other immunosuppressed patients becomes more complex if the condition is associated with increased morbidity and mortality [6,7].

The problem of increased antimicrobial resistance becomes even more menacing when the delay in the discovery and development of new antibiotics is taken into account. The number of such drugs is still quite limited, which endangers the essential role played by antibiotics in current medical practices [8].

The aforementioned problems urgently require new therapeutic strategies. Of special importance is the search for new drugs derived from biological sources in which molecules, predominantly secondary metabolites, contribute to their development [9]. Another approach to improving the efficacy of existing antimicrobials and suppressing the emergence of multidrug-resistant strains involves the use of products that potentiate the activity of these substances [1012]. These products can improve the effectiveness of the antibiotic in eliminating or delaying the emergence of antibiotic resistance [13].

Plant extracts are known to have antimicrobial properties and may play an important role in therapeutic treatments. For this reason, a growing number of studies in different countries have been conducted to demonstrate the effectiveness of these extracts [1416]. Besides the direct antimicrobial activity, plant species have been tested as potential adjuvants by modifying the microbial resistance [17,18].

Combinations of antimicrobial drugs and natural products of vegetable origin, in which these products act as adjuvants, constitute a promising approach for the treatment of infections. The natural products would replace at least a part of the synthetic substances in the formulations and would eventually reduce the undesirable effects of these substances in the human body [19].

Poincianella pyramidalis (Tul.) L.P. Queiroz (Fabaceae) is an arboreal species with wide distribution in the Brazilian semiarid region. Until recently, this species was known as Caesalpinia pyramidalis Tul., But due to a taxonomic update, it came to be called P. pyramidalis [20]. Its parts, especially its bark, leaves, and flowers, are used in traditional medicine for the treatment of several diseases such as influenza, cough, diarrhea, dysentery, respiratory infections, urinary infections, and inflammation in general [2127]. Among the biological activities of P. pyramidalis described in the literature, we can highlight the antibacterial [28,29], antifungal [30], antioxidant [31], gastroprotective [32], anti-inflammatory, antinociceptive [33] and antihelminthic [34] activities.

This work is aimed to investigate the interaction between P. pyramidalis extract and antimicrobial drugs through the use of biological and analytical models.

Material and Methods

Plant material

Bark of P. pyramidalis were collected on the farm “Farinha”, municipality of Pocinhos, PB, Brazil (7°07´54.53´´S e 36°07´14.51´´O), in January 2014. A voucher specimen (CSTR 5036) was deposited in the herbarium of the Center for Health and Rural Technology at Federal University of Campina Grande.

Preparation of extract

The plant materials were dried in an air circulation oven at 40°C. Subsequently it was ground in a knife mill with a particle size of 10 mesh. The hydroalcoholic extract obtained by extraction was assisted by ultrasound at 40°C for 60 min, using ethanol-water mixture (50:50 v/v) as solvent. Out below has been subjected to spray drying in a Mini Spray Dryer Labmaq PS-1, with onset temperature 120°C, air flow of 40 L min-1, drying air flow rate 3 ml min-1. The nebulized extract (ENPp) was dried with adjuvant using colloidal silicon dioxide (Aerosil 200®) at 20% on dry weight basis.

Chemical assays

Determination of total polyphenols.

The total polyphenol content of plant extracts was measured by Folin-Ciocalteu method [35]. The extracts were dissolved in distilled water to obtain a final concentration 200 μg mL-1. From each solution, a 1 mL aliquot was added to 1 mL of 1 mol L-1 Folin-Ciocalteu reagent (Sigma-Aldrich). This mixture remained undisturbed for 2 min before the addition of 2 mL of 20% (w/v) Na2CO3 solution and left undisturbed for 10 min. Thereafter the reading was performed Spectrophotometer Shimadzu, at 757 nm. The calibration curve was obtained with a stock solution of gallic acid (Sigma-Aldrich) (1000 μg mL-1), from which dilutions were made at concentrations between 1 and 40 μg mL-1.

Determination of total flavonoids.

The total flavonoids were determined by the AlCl3 method [35]. The extracts were diluted with methanol at 1000 μg mL-1. To the 5 ml of each test solution was added the same volume of 2% (w/v) AlCl3 solution in methanol. This mixture remained undisturbed for 10 min before the UV spectrophotometric reading at 415 nm wavelength. The total flavonoids were determined by the calibration curve using quercetin (Sigma-Aldrich) as standard at concentrations between 2 and 30 μg mL-1.

Determination of condensed tannins.

The content of condensed tannins was verified through the method described by Makkar and Becker [36] wherein 0.25 ml of the sample was added to 1.5 mL vanillin (Sigma-Aldrich) dissolved in methanol (4% w/v) and subsequently in 0.75 mL of concentrated HCl (37%). After the HCl addition, the tube content was shaken in water bath at 30°C for 3–4 seconds before being read on a spectrophotometer at a 500 nm wavelength. Catechin (Sigma-Aldrich) was used as standard at concentrations between 10 and 100 μg mL-1.

Determination of saponins.

The quantification of total saponins followed the method described by Makkar et al.[37]. First, 250 μL of an 8% vanillin solution in ethanol was added to a 250 μL extract solution in 80% methanol; then 2.5 mL of 72% sulfuric acid were added. The tubes were incubated at 60°C in a water bath for 10 minutes and then transferred to an ice bath to rest for 4 minutes. The absorbance reading at 544 nm was performed against a blank consisting of the vanillin solution, 80% methanol and sulfuric acid. The calibration curve was obtained from a disogenin (Sigma-Aldrich) solution at concentrations between 100 and 500 μg mL-1.

Determination of major chemical compound.

We used a liquid chromatograph Ultra Efficiency (UPLC), Shimadzu, equipped with two pumps model LC-20AD, autosampler SIL-20-AHT, oven column CTO-20A, detector with variable wavelength UV/Vis, model SPD-20A, controller CBM-20A, automatic computerized integrator with software LC Solution®. The stationary phase was composed of a column Gemini—NX C18 (250 x 4.60 mm, 5 μm). The mobile phase consisted of an isocratic mixture of acetic acid 0.1%: metanol (90:10, v/v). Analyses were performed under controlled temperature (30°C), using a flow 1 mL min-1 and injection volume of 20μL. All samples were amended with 0.45μm syringe filters diameter.

Antioxidant activity

The antioxidant activity of ENPp was originally assessed by the ability of the antioxidant substances present in the sample to capture the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl). The tests were conducted using the method described by Dhar et al. [38], with adaptations. Initially, the DPPH solution was prepared at 0.200 mM in ethanol. 500 μL of this solution was added to 500 μL of diluted extract in ethanol in concentrations ranging between 50 and 3.125 μg mL-1. The mixture remained at rest in the dark at room temperature for 30 minutes before the absorbance was read in a spectrophotometer at UV 517 nm. Gallic acid and quercetin were used as standards. The ability to scavenge DPPH radicals was calculated by the following equation: (1) where, ABScontrol is the absorbance of the DPPH radical + ethanol; ABSsample It is the absorbance of the DPPH radical + extract or standard.

The inhibitory concentration (IC50) and effective concentration (EC50) were estimated as described by Kroyer [39] and Prakash et al. [40]. The IC50 was determined by plotting the DPPH elimination ability against the logarithm of the concentration of the sample, while the EC50 was calculated using the following equation: (2)

Microbiological assays

Were used standard strains American Type Culture Collection (ATCC) and clinical isolates of Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae whose phenotypic profile is described in Table 1. The strains were maintained on slants of tubes with Mueller-Hinton Agar, and, before testing, cultured at 37°C for 24 hours, plates with the same culture medium.

Table 1. Bacterial strains used and their phenotypic profile of antimicrobial resistance.

Determination of Minimum Inhibitory Concentration (MIC).

The minimum inhibitory concentration (MIC) was determined by a microdilution method in 96-well plates using Mueller-Hinton broth [41]. Colonies of microorganisms were suspended in a 0.9% saline solution, and by a spectrophotometric method at 625 nm, the suspension adjusted to a final concentration of 5 x 106 CFU mL-1. Serial dilutions of the extract in the range of 1000 to 2.4 μg mL-1 and antibiotics in the range of 2500 to 2.4 μg mL-1 were performed. Dimethyl sulfoxide (DMSO) 10% was included as a negative control. The plates were incubated at 37 ± 1°C for 24 hours. Bacterial growth was indicated by addition of 20 μL of 0.01% aqueous resazurin solution (Sigma-Aldrich) with incubation at 37 ± 1°C for 2 h. MIC values were identified as the lowest concentration in which no bacterial growth is visible. The assays were performed in triplicate.

Modulation of antimicrobial resistance.

Evaluation of extracts as modulators of antibiotic resistance was performed according to Coutinho et al.[42]. The MIC of the antibiotic was determined in presence and absence of sub-inhibitory concentrations (MIC/8) of EESb. Plates were incubated as described above and each assay was performed in triplicate.

Evaluation of acute toxicity

The study was carried out in strict accordance with the Standard Operating Procedures (Laboratory of Pharmacology at the State University of Paraiba, Campina Grande, Brazil) approved by veterinarian, which monitored frequently the animals by physical condition assessments of the health. All efforts were exerted in order to reduce the suffering of experimental animals. The protocol was approved by the Ethics Committee on Animal Use of Faculty of Medical Sciences of Campina Grande (No: 5618092015). Disease-free albino Wistar rats (Rattus norvegicus) (6–8 weeks age and 200–220 g weight) were used for this study. The animal house were obtained of Federal University of Paraíba, João Pessoa, Paraíba, Brazil. The animals were housed in rat standard plastic cages (n = 6) with stainless steel coverlids and wood shavings. All rats underwent a period of at least 7 days of acclimatization prior to the procedure, being socialized with contact including humans. The animals were handling with care to minimize stress. The researchers confirm that the laboratory had a protocol in place for the use of humane endpoints in cases where animals became severely ill or moribund during the experiment, but no had death or behavioral changes in animals. They remained in polypropylene boxes, in single sex groups, at room temperature (22°C ± 3°C) and humidity (50% ± 20%) and 12 hrs light/dark cycles. The animals received standard laboratory pellets and water ad libitum both for the adaptation period (7 days) and during the trial, except the period of 12 hours prior to the experiment in which the access to food was restricted. Throughout the experiments, all of the animals received humane care according to the ‘‘Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences [43].

The animals were divided in groups of 6 males and 6 females, which received, orally, the dose of extract to 2000 mg kg-1. A control group was treated with saline, the same used to resuspend the extract. Immediately after administration of the extract, the animals were evaluated behaviorally carefully during the first 4 hours, as recommended by the handshake protocol and evaluation of clinical signs of OECD [44] and daily for 14 days after administration. They were also observed the consumption of water and feed. Animals were sacrificed under anesthesia with ketamine/xylazine (0.5 mL of 100 mg mL-1 ketamine combine with 0.05 mL of 20 mg mL-1 xylazine) at a dosage of 0.55 mL/ 100g body weight (b.w.). After sacrifice was carried out weighing and macroscopic analysis of the viscera (liver, kidneys, spleen, lungs and heart).

Statistical analysis

The results of the microbiological tests were expressed as a geometric mean. It was applied to a two-way analysis of the variance, followed by the Bonferroni post-test, and was applied to toxicity testing through an analysis of the variance with Tukey's post-test using GraphPad Prism 5.0 software.

Thermal analysis

The thermoanalytical profiles were obtained using a simultaneous TG-DTA analyzer, model DTG-50 (Shimadzu). Samples (5.0 ± 0.2 mg) were accommodated in a platinum crucible, and subjected to a heating program from 30 to 900°C, at 10°C min-1, in inert nitrogen atmosphere (50 mL min-1). The samples consisted of antibiotics and extract analyzed separately and in binary mixtures, the proportion 1:1.

The DTA module is calibrated with indium standard (mp = 156.6°C). And the calibration of TG module, was used a standard calcium oxalate monohydrate. Curves were analyzed in the TA60 software, version 2.21.

Results and Discussion

Chemical assays

The secondary metabolite content is shown in Table 2. The content of the total number of polyphenols and tannins is high compared to the total flavonoids. Although the method above is a quantitative method, it fails to predict the composition of each individual compound as well as the possible quantification of nonphenolic compounds [45].

Table 2. Content of secondary metabolites present in P. pyramidalis extract obtained spectroscopy in the visible region.

Other studies carried out using the phytochemical with P. pyramidalis extracts revealed the presence of secondary metabolites such as saponins, ursolic acid, sitosterol, Cinnamic derivatives, flavonoids, quercetin, proanthocyanidins, catechin, gallic acid, and ellagic acid [29,46,47].

Identification of the major chemical compound using liquid chromatography was performed, and, based on the retention time parameter (TR), was compared with the TR values for the analyzed standards of gallic acid, catechin, quercetin, rutin, and kaempferol, with the RT values of peaks observed in the EEPp. The results indicated the presence of gallic acid, which can be used as a chemical marker of P. pyramidais (Fig 1). In a study conducted by Santana et al. [48] with the ethanol extract of the plant, the researchers found the presence of rutin, which was absent in this study. This difference may be related to parameters such as the place and time of collection and the stage of plant development, among other factors [35].

Fig 1. Chromatograms of nebulized extract of P. pyramidalis showing its chemical marker.

Antioxidant activity

The search for substances that might reduce the risk of developing chronic diseases caused by oxidative stress [49] is increasing. This risk includes several pathological and toxicological processes such as aging, transformation, cell killing, cancer induction, autoimmune diseases, and heart diseases, among others [50,51]. Among the most promising substances in the study are those of vegetable origin, such as polyphenols, flavonoids, alkaloids, terpenoids, carotenoids, etc. [5254].

The scavenging capacity of the free radicals of P. pyramidalis extract was compared through its ability to eliminate the DPPH radical. The data from these tests are shown in Table 3, expressed as a percentage of the inhibition of DPPH, IC50, and EC50. When looking at the percentage of the inhibition of DPPH, we found that the extract showed a high inhibition rate, with no statistically significant difference between it and gallic acid. On the other hand, the IC50 and EC50 extract was significantly higher than the standards. Its inhibition rate may be related to the fact that the extract is a complex mixture of substances as the patterns are pure substances.

Table 3. Antioxidant activity of the extract and standards towards DPPH.

There are references in the literature that mention that P.pyramidalis has a good number of phenolic compounds [46]. These substances are very important components of plant extracts and contribute directly to the elimination of radicals because of their hydroxyl groups [55,56]. Gallic acid, found in P. pyramidalis extract, is one of these compounds, and there are reports in the literature of its antioxidant properties [5759].

Antimicrobial and modulatory activities

As for antimicrobial activity, the ENPp had no clinically significant effect on any of the studied strains. Dall'Agnol et al. [60] and Rios and Recio [61] reported that plant extracts are considered inactive with MIC > 1 000 μg mL-1; they are low-activity holders with CIM between 500 and 1000 μg mL-1 and show moderate activity with MIC values between 100 and 500 μg mL- 1 and good activity when MIC is ≤ 100 μg mL-1. The latter activity levels have good potential for the determination and purification of active compounds. The absence of antimicrobial activity presented here is consistent with the study conducted by Silva et al. [62], who detected the inactivity of the ethanol extract of P. pyramidalis on standard strains and clinical isolates of E. coli, P. aeruginosa, and K. pneumoniae.

Gallic acid, a chemical marker of ENPp, was also subjected to microbial susceptibility testing and showed no significant activity on strains tested in this study (MIC > 1000 μg mL-1). This result corroborates the study by Chanwitheesuk et al. [63], in which the gallic acid also presented MIC > 1000 μg mL-1 on several bacterial strains, Gram positive, Gram negative, and fungal. A similar result was observable in the study of Jayaraman et al. [64] in tests with standard and multidrug resistant strains of P. aeruginosa. Borges et al. [65], in trials with E. coli, S. aureus, and Listeria monocytogenes, obtained MIC values > 1000 μg mL-1. However, against P. aeruginosa, the gallic acid showed MIC = 500 μg mL-1. Other studies report MIC values for gallic acid that can be considered active. Sanchez-Maldonado et al. [66] obtained MIC = 490 μg mL-1 against E. coli. Vaquero et al. [67], using the agar diffusion method, obtained inhibition halos against standard strains of E. coli (ATCC 35218 and ATCC 25922), with gallic acid concentrations ranging between 200 and 1000 μg mL-1. The discrepancy between the findings of this and other studies may be related to the method used to determine the antimicrobial activity, as well as the phenotypic profile of the strains used.

The resistance presented by Gram-negative bacteria is possibly related to the presence of the outer membrane of the bacterial wall, which forms a semipermeable barrier composed mainly of phospholipids, lipopolysaccharides, and proteins. This barrier hinders the passage of antimicrobial drugs and is linked to the high intrinsic resistance of these bacteria [68,69]. Beyond this barrier, these microorganisms may have other mechanisms that prevent the antimicrobial from reaching the target. It is able to detect the change in the composition of the outer membrane, eliminating porins, induction efflux pumps, and enzymatic degradation of the antimicrobial [70,71].

In modulatory activity, assays have calculated a subinhibitory concentration (MIC / 8) of the extract, which are associat with antibiotics. Because the extract did not prevent efficacy, CIM considered for this calculation was the highest concentration tested and has, therefore, a subinhibitory concentration of 125 μg mL-1. In Fig 2A, 2B, 2C and 2D, we can see that, after combination with both the extract and gallic acid, there was a significant increase in the gentamicin activity on the E. coli strains 401, 534, and 613. Against E. coli 523, chloramphenicol reduced MIC in both associations, while gentamicin, only in association with the extract. Moreover, the association with gallic acid caused a significant increase in nitrofurantoin (E. coli 401), ampicillin (E. coli 523), and norfloxacin (E. coli 534 e 613). In assays with P. aeruginosa strains (Fig 2E, 2F e 2G), we found the additive effect of the extract and gallic acid in the action of gentamicin on all strains tested, ceftriaxone (P. aeruginosa 106 e 117), ciprofloxacin, and cefepime (P. aeruginosa 117). The extract also led to a significant reduction of ciprofloxacin, the MIC for P. aeruginosa 106, while the opposite effect resulted in gallic acid. Also, on this same strain, the association with gallic acid increased MIC of cephalothin. In assays with K. pneumoniae (Fig 2H), the antibiotic whose activity has intensified in both associations, was gentamicin. However, there was a significant increase in CIM clindamycin when it was added to the extract.

Fig 2. Modulatory activity of ENPp against of Gram negative strains.

A—E. coli 401; B—E. coli 523, C—E. coli 613; D = E. coli 534; E—P. aeruginosa 106; F—P. aeruginosa 117; G—P. aeruginosa 208. H—K. pneumoniae 110. ***—statistically significant with P value < 0.001; ns–not statistically significant value with P > 0.05. CFL = Cephalothin, NIT = Nitrofurantoin, CF = Ceftriaxone, GEN = Gentamicin, CLI = Clindamycin, AMP = Ampicillin, CLO = Chloramphenicol, NOR = Norfloxacin, CIP = Ciprofloxacin, CPM = Cefepime.

The best results in the interactions with the extract or gallic acid were observed in assays with gentamicin, whose CIM on all strains was reduced. This antimicrobial belongs to the class of aminoglycoside, which in turn is the class to which the most commonly used anti-infectious agents in clinical practice belong [72]. They are able to interact with different portions of rRNA, causing deleterious effects on mRNA translation process polypeptide causing inhibition of protein synthesis or production of defective proteins [7274]. The main mechanisms of resistance to these antimicrobial agents include the enzymatic degradation (N-acetylation, adenylation, or O-phosphorylation), reduction in the intracellular concentration of the antibiotic by changes in membrane permeability and transport by active efflux; changes in the target 30S ribosomal subunit; and changes in the antimicrobial binding site [75,76].

A particularly important feature presented by the aminoglycosides is the ability to act synergistically with other drugs [76]. This feature is particularly important in the search for new therapeutic alternatives because of the emergence of resistant strains and toxicity caused by these drugs [77]. In the literature, there are studies that demonstrate the resulting synergism of the associations between β-lactams and aminoglycosides [78,79] and between aminoglycosides and natural products [8082].

The combination of plant extracts and antibiotics is well documented in the literature. In many cases, this association led to an additive effect, resulting in improved antimicrobial activity against various multidrug-resistant strains [8388].

The reduction in the MIC of gentamicin after association with ENPp, or its chemical marker, may be related to the promotion of drug entry into the bacterial cell. Gallic acid acts on cell membranes, leading to irreversible changes in its characteristics relating to intra- and extracellular permeability. The chemical-physical properties it is altered by changes in the hydrophobicity and decreased negative charge of the surface. It favors the occurrence of breaks or the formation of pores, with consequent loss of essential intracellular components for the bacterial life [65].

The ENPp as various types of plant extracts and phytochemicals presents a wide variety of compounds; therefore, other mechanisms may be related to the additive effects of the combination of antibiotics and ENPp. A wide variety of compounds present in the extract can act on different targets [89] and may be in one or multiple targets at once. Among the mechanisms by which the extracts can interfere with microbial growth is the inactivation of enzymes, transport and receptor proteins, and DNA/RNA, besides acting in the suppression of bacterial resistance mechanisms [9092]. Moreover, the said plurality of compounds and their mechanisms of action provide a low risk for increasing resistance because they afford the greatest difficulties for microbial adaptation [93].

Acute toxicity

During the 14 days following the statement of administration, there was no death among the animals, making it impossible to calculate the LD50. There was also no change in the behavior of the animals. When the consumption of water and feed and the average weight of the organs of animals were evaluated, there was no statistically significant difference between the group treated with the extract and the control group (P <0.05).

Thermal analysis

Differential thermal analysis (DTA).

The DTA curve of ENPp shows three processes of the sample phase transitions. The first, endothermic at 53.36°C, is possibly related to loss of water, solvent (ethanol), or volatile compounds in the sample. The following two peaks are exothermic of crystallization: the second at 348.30°C and the third at 425.17°C (Table 4).

In the DTA curve of norfloxacin, the first peak of the endothermic nature at 179.69°C was observed. The second peak endotherm occurred at 223.84°C, which corresponds to the delay of the drug fusion process that occurs between 220.00 and 221.00°C [94,95]. In the binary mixture of antibiotic + extract, we observed that the first endothermic peak occurred at a temperature of 179.36°C, which is characteristic of the drug. The second peak was in 219.51°C, which corresponds to the anticipation of the melting process of the drug. From the third, occurring in 428.63°C, the thermal sample decomposition process begins. In the mixture, there was a change in the heat peaks in advance of the drug melting temperature and suppression of peaks observed in the extract. These changes are possibly due to interactions between the extract and antibiotic (Table 4/Fig 3).

Fig 3. DTA curves of nebulized extract of P. pyramidalis, antimicrobials and their binary mixtures.

(A) Norfloxacin, (B) Ampicillin, (C) cephalothin, (D) Ciprofloxacin (E) Clindamycin, (F) Nitrofurantoin, (G) Ceftriaxone, (H) Gentamicin, (I) Cefepime, (J) Chloramphenicol.

Four events were recorded in the DTA curve of ampicillin. The first, which were endothermic, recorded in temperatures of 63.52°C, related to the dehydration sample; the second event was in 218.30°C, which probably corresponds to the delayed melting process, with accompanying decomposition, of one of its anhydrous forms [95,96]; the third, at 363.24°C, was the beginning of the decomposition of ampicillin. In the curve produced with the mixture, we observed that the first two endothermic peaks of the drug were maintained at the same temperature but with less reaction heat involved in the endothermic processes. The removal of the exothermic peaks of the drug and the extract in the binary mixture curve is an indication of a strong chemical interaction between the components of the sample (Table 4/Fig 3B).

The DTA curve of cephalothin showed an endothermic peak at 167.27°C, probably related to the sample fusion process between 160.0 and 160.5°C [95], and an exotherm at 212.63°C, which corresponds to their decomposition processes [97]. With the mixture produced from the extract, a glass transition was observed at a temperature of 151.33°C and an exothermic peak at 211.66°C, both associated with the degradation of the mixture (Table 4/Fig 3C).

In the DTA curve of ciprofloxacin, two events occur, the first, of an endothermic nature, was observed at 144.45°C, corresponding to the loss of the acetylene group (C2H2) of the drug [98]. The second, at 318.40°C, corresponds to the melting process of the drug, which occurs between 318.0 and 320.0°C [95,99]. In the curve of the mixture, four endothermic peaks were observed. The first, at 134.90°C, resulted from the sample moisture loss and the drug group C2H2[98]. The second, in 296.64°C, is probably related to the anticipation of the drug fusion process. The two endotherms, at 405.53 and 430.63°C, demonstrate the sample decomposition process. These differences point to an interaction between the substances in the mixture (Table 4/Fig 3D).

Five events were observed in the DTA curve clindamycin, the first, of endothermic nature, which occur at temperatures of 109.00°C, concern the dehydration of the sample. The event at 148.97°C, probably related to the delayed drug fusion process, should occur in the temperature range of 141 to 143°C [95]. From the peak observed at 170.18°C, the process of drug decomposition begins. Moreover, from the curve of the mixture, only endothermic events occurred, the first at a temperature of 101.86°C, referring to the loss of moisture in the mixture. The second, at 147.23°C was probably due to the retardation of the drug fusion process. And the third, of 204.84°C, was also attributed to the delay in the onset of decomposition in the mixture. The exothermic peaks present in the drug and the mixture were removed, indicating a possible incompatibility between the drug and the nebulized extract of P. pyramidalis (Table 4/Fig 3E).

In the DTA curve of nitrofurantoin, there was an endothermic event at 272.07°C only. The absence of previous events indicates that the sample was completely free from humidity. The first observed event is related to the early decomposition of the drug, which should take place in temperatures above 270°C [95]. There was still a last endothermic peak at 307.84°C, ending the process of decomposition. In the mixture two exothermic events were observed. The first, at 263.06°C, related to the anticipation of the decomposition process of nitrofurantoin. And the other, in 423.83°C, corresponded to the extract degradation. These events indicate that there should probably be a strong incompatibility between the drug and the substances present in the extract (Table 4/Fig 3F).

The DTA curve of ceftriaxone showed a endothermic event at 80.90°C, attributed to the dehydration process of the sample. The second, at 148.22°C, was probably responsible for initiating the melting/decomposition process of the drug, given that ceftriaxone melts with decomposition when heated to over 155°C [95,100]. The last exothermic peak, at 270.27°C, corresponds to the continuation of the drug decomposition process [101]. The DTA curve of the binary mixture ceftriaxone + extract showed two endothermic events and three exothermic. The first endotherm, of 154.40 and 85.12°C, corresponds to the first two peaks observed in the drug, as does the first exotherm, which occurred at 273.23°C, though with a delay. The second exothermic peak, at temperature 356.14°C, is probably the first peak decomposition extract that was anticipated. And, the last peak exotherm, at 546.50°C, corresponds to the lagging end of the extract decomposition process (Table 4/Fig 3G).

The gentamicin curve showed three endothermic peaks. The first of the sample, to 75.05°C, is related to water loss. The second and third peaks occurred at 252.84°C and 299.71°C. According to the Merck Index [95], the melting point of gentamicin sulfate is between 218 and 237°C. Aquino et al. [102] reported that gentamicin sulphate, the raw material, is characterized by a range of endothermic peaks related to the fusion of the different isoforms. In the curve of the binary mixture, three endothermic peaks were observed, all three of which correspond to the drug. The first peak occurred at 67.93°C, while the second and third occurred at 252.46 and 299.15°C, respectively. The temperatures of the last two peaks came very close to the peaks of the drug (Table 4/Fig 3H).

The DTA curve of Cefepime presented three endothermic peaks and an exothermic. The first two occurred at 50.13°C and 117.38°C and are related to the sample moisture loss. The third, 182.04°C in the drug, is linked to the fusion process that occurs at close to 182°C [103]. The last exothermic peak, at 645.78°C, is associated with the drug degradation process. In Cefepime + extract mixture, the first three peaks of the drug were maintained, occurring at 69.05, 108.11, and 186.27°C, respectively. With respect to the last peak, there was a delay in the melting point of the drug (Table 4/Fig 3I).

Chloramphenicol has two exothermic events at 184.87 and 279.09°C, probably linked to the melting and decomposition of the drug process. These results contrast with those found by Macedo [104], in which the fusion process of Chloramphenicol was observed in the 155.2°C exothermic event, characteristic of the drug decomposition that occurred at 244.1 and 257.8°C (Table 4/Fig 3J).

Thermogravimetry (TG).

In the TG curves of the samples, their thermal decomposition processes were observed. The first, when it occurs at temperatures up to 100°C, refers to the loss of moisture from the sample, which, in the case of the nebulized extract of P. pyramidalis, was 6.36%. The second step is assigned to the main stage of decomposition. It indicates the beginning of the degradation process, which for this extract occurred at a temperature of 208.99°C, with a mass loss of 14.67% (Table 5/ Fig 4). During this step, many chemical chains were broken, probably caused by carbon dioxide, other gases, and novel compounds, which join to form more stable compounds. They subsequently decompose at higher temperatures. From the second stage, there is a gradual mass loss, corresponding to the whole thermal decomposition of the sample.

Fig 4. TG curves of nebulized extract of P. pyramidalis, antimicrobials and their binary mixtures.

(A) Norfloxacin, (B) Ampicillin, (C) Cephalothin, (D) Ciprofloxacin (E) Clindamycin, (F) Nitrofurantoin, (G) Ceftriaxone, (H) Gentamicin, (I) Cefepime, (J) Chloramphenicol.

Table 5. TG Data relating to the stages of decomposition of nebulized extract, antibiotic and their binary mixtures.

At the end of the decomposition process, which generally occurs above 400°C, a mineral residue is present. This residue corresponds to the ash content of the sample, which in the case of P. pyramidalis extract was 58.84%. It occurred at a temperature range from 429.47 to 370.02°C. With this residue was the EEPp high, caused by the extract being dried using a 20% colloidal silicon dioxide, an amorphous silica that only degrades at temperatures above 1600°C [105].

The drugs cephalothin, ceftriaxone, ciprofloxacin, clindamycin, and cefepime showed a three-step thermal decomposition, and the degradation stages for these samples began in 206.36 (CFL), 263.84 (CFO), 130.16 (CIP), 207.55 (CLI), and 189.01°C (CPM). The antimicrobials norfloxacin, ampicillin, and nitrofurantoin had four steps. The first step starts at temperatures of 206.36 (NOR), 209.52 (AMP), 236.47 (NIT), and 244,38°C (GEN). Chloramphenicol has five stages of decomposition, the first one starting to 194.03°C (Table 5/ Fig 4).

The TG curves of the binary mixtures showed that the mixtures produced with all the drug extracts anticipated the degradation process, except for cefepime. However, when comparing the curves of gentamicin, with its respective binary mixture, we observed that the difference between the temperatures at which the sample began decomposition processes is very small.


The results of this study indicate that the extract of the P. pyramidalis bark has significant antioxidant action on the radical DPPH. The extract showed no significant antimicrobial activity against multirresistant strains; however. When it was combined with certain synthetic antibiotics. Its MIC was significantly reduced. The TG/DTA curves of the antimicrobials and their binary mixtures with the extract indicate a possible physicochemical interaction between the extract and the antibiotic. Whose mixture with the extract showed no additive effect in microbiological assays. This interaction can be confirmed by additional techniques such as X-ray diffraction. and FT-IR.

The nebulized extract of P. pyramidalis has significant potential for use as an adjuvant component formulation for use in antimicrobial therapy and is a promising alternative in combating multidrug-resistant bacteria.

Author Contributions

Conceived and designed the experiments: ACDM TPC. Performed the experiments: TPC FHAF FDM CPS JSS VLS. Analyzed the data: TPC FDM VLS HDMC RMRC. Contributed reagents/materials/analysis tools: ACDM DCF. Wrote the paper: TPC ACDM FDM.


  1. 1. Kumar A. Schweizer HP. Bacterial resistance to antibiotics: Active efflux and reduced uptake. Adv Drug Deliv Rev. 2005; 57: 1486–1513. pmid:15939505
  2. 2. Pogue JM. Kaye KS. Cohen DA. Marchaim D. Appropriate Antimicrobial Therapy in the Era of Multidrug Resistant Human Pathogens. Clin Microbiol Infect. 2015; 21: 302–312. pmid:25743999
  3. 3. Davies. S. C. Annual Report of the Chief Medical Officer. Volume Two. 2011. Infections and the rise of antimicrobial resistance. Department of Health. London. 2013.
  4. 4. World Economic Forum. Global Risks 2014. Available:
  5. 5. Poole K. Multidrug resistance in Gram-negative bacteria. 2001; 4: 500–508. pmid:11587924
  6. 6. Vincent JL. Rello J. Marshall J. Silva E. Anzueto A. Martin CD. et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA. 2009; 302: 2323–2329.
  7. 7. Karaiskos I. Giamarellou H. Multidrug-resistant and extensively drug-resistant Gram-negative pathogens: current and emerging therapeutic approaches. Expert Opin Pharmacother. 2014; 10: 1351–1370.
  8. 8. Rossolini GM. Arena F. Pecile P. Pollini S. Update on the antibiotic resistance crisis. Curr Opin Pharmacol. 2014;18: 56–60. pmid:25254623
  9. 9. Taylor PW. Alternative natural sources for a new generation of antibacterial agents. Int J Antimicrob Agents. 2013; 42: 195–201. pmid:23796893
  10. 10. Kalana L. Wrigh GD. Antibiotic adjuvants: multicomponent anti-infective strategies. Expert Rev Mol Med. 2011; e5. pmid:21342612
  11. 11. Bernal P. Molina-Santiago C. Daddaoua A. Llamas MA. Antibiotic adjuvants: identification and clinical use. Microb Biotechnol. 2013; 6: 445–449. pmid:23445397
  12. 12. Gill EE. Franco OL. Hancock REW. Antibiotic Adjuvants: Diverse Strategies for Controlling Drug-Resistant Pathogens. Chem Biol Drug Des. 2015; 85: 56–78. pmid:25393203
  13. 13. Farha MA. Brown ED. Discovery of antibiotic adjuvants. Nat Biotechnol. 2013; 31: 120–122. pmid:23392510
  14. 14. Mabona U. Viljoen A. Shikanga E. Marston A. Van Vuuren S. Antimicrobial activity of southern African medicinal plants with dermatological relevance: From an ethnopharmacological screening approach. to combination studies and the isolation of a bioactive compound. J Ethnopharmacol. 2013; 148: 45–55. pmid:23545456
  15. 15. Madikizela B. Aderogba MA. Finnie JF. Van Staden J. Isolation and characterization of antimicrobial compounds from Terminalia phanerophlebia Engl. & Diels leaf extracts. J Ethnopharmacol. 2014; 156: 228–234. pmid:25218320
  16. 16. Vieira DRP. Amaral FMM. Maciel MCG. Nascimento FRF. Libério SA. Rodrigues VP. Plant species used in dental diseases: Ethnopharmacology aspects and antimicrobial activity evaluation. J Ethnopharmacol. 2014; 155: 1441–1449. pmid:25046828
  17. 17. Gibbons S. Anti-staphylococcal plant natural products. Nat Prod Rep. 2004; 21: 263–277. pmid:15042149
  18. 18. Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspects Med. 2006; 27: 1–93. pmid:16105678
  19. 19. Hemaiswarya S. Kruthiventi AK. Doble M. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine. 2008; 15: 639–652. pmid:18599280
  20. 20. Queiroz LP. New combinations in Libidibia (DC.) Schltd. and Poincianella Britton & Rose (Leguminosae. Caesalpinioideae). Neodiversity. 2010;5:11–12.
  21. 21. Silva ACO. Albuquerque UP. Woody medicinal plants of the Caatinga in the state of Pernambuco (Northeast Brazil). Acta bot bras. 2005; 19: 17–26.
  22. 22. Agra MF. Baracho GS. Nurit K. Basílio IJLD. Coelho VPM. Medicinal and poisonous diversity of the flora of “Cariri Paraibano”. Brazil. J Ethnopharmacol. 2007; 111: 383–395. pmid:17236731
  23. 23. Albuquerque UP. Medeiros PM. Almeida ALS. Monteiro JM. Lins Neto EMF. Melo JG. et al. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: A quantitative approach. J Ethnopharmacol. 2007; 114: 325–354. 2007. pmid:17900836
  24. 24. Lucena RFP. Albuquerque UP. Monteiro JM. Almeida CFCBR. Florentino ATN. Ferraz JSF. Useful Plants of the Semi-Arid Northeastern Region of Brazil–A Look at their Conservation and Sustainable Use. Environ Monit Assess. 2007; 125: 281–290. pmid:17219240
  25. 25. Lucena RFP. Nascimento VT. Araújo EL. Albuquerque UP. Local Uses of Native Plants in an Area of Caatinga Vegetation (Pernambuco. NE Brazil). Ethnobotany Research & Applications 2008; 6: 003–013. 2008.
  26. 26. Cartaxo SL. Souza MMA. Albuquerque UP. Medicinal plants with bioprospecting potential used in semi-arid northeastern Brazil. J Ethnopharmacol. 2010; 131: 326–342. pmid:20621178
  27. 27. Silva FS. Ramos MA. Hanazaki N. Albuquerque UP. Dynamics of traditional knowledge of medicinal plants in a rural community in the Brazilian semi-arid region. Braz J Pharmacogn. 2011; 21: 382–391.
  28. 28. Lima MRF. Luna JS. Santos AF. Andrade MCC. Santana AEG. Genet JP. et al. Anti-bacterial activity of some Brazilian medicinal plants. J. Ethnopharmacol. 2006; 105: 137–147. pmid:16356672
  29. 29. Saraiva AM. Saraiva CL. Gonçalves AM. Soares RR. Mendes FO. Cordeiro RP. et al. Antimicrobial activity and bioautographic study of antistaphylococcal components from Caesalpinia pyramidalis Tull. Braz. J. Pharm. Sci. 2012; 48: 147–154.
  30. 30. Cruz MCS. Santos PO. Barbosa JRAM. Melo DLFM. Alviano CS. Antoniolli AR. et al. Antifungal activity of Brazilian medicinal plants involved in popular treatment of mycoses. J Ethnopharmacol. 2007; 111: 409–412. pmid:17234376
  31. 31. Alviano WS. Alviano DS. Diniz CG. Antoniolli AR. Alviano C. Farias LM. et al. In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine. Arch. Oral Biol. 2008; 53: 545–552. pmid:18243157
  32. 32. Ribeiro AR. Diniz PF. Estevam CS. Pinheiro M. Albuquerque RLC Jr. Thomazzi S. Gastroprotective activity of the ethanol extract from the inner bark of Caesalpinia pyramidalis in rats. J Ethnopharmacol. 2013; 147: 383–388. pmid:23506986
  33. 33. Santos AC. Ailane MPR. Passos FCA. Camargo EA. Estevam CS. Santos MRV. et al. Antinociceptive and anti-inflammatory effects of Caesalpinia pyramidalis in rodents. Braz. J. Pharmacogn. 2011; 21: 1077–1083.
  34. 34. Borges-dos-Santos RR. Santos JLL. Farouk Z. David JM. David JP. Lima JWM. Biological Effect of Leaf Aqueous Extract of Caesalpinia pyramidalis in Goats Naturally Infected with Gastrointestinal Nematodes. Evid. Based. Complementary Altern. Med. 2012.
  35. 35. Chaves TP. Santana CP. Véras G. Brandão DO. Felismino DC. Medeiros ACD. et al. Seasonal variation in the production of secondary metabolites and antimicrobial activity of two plant species used in Brazilian traditional medicine. Afr J Biotechnol. 2013; 12: 847–853.
  36. 36. Makkar HPS. Becker K. Vanillin-HCl method for condensed tannins: Effect of organic solvents used for extraction of tannins. J Chem Ecol. 1993; 4: 613–621.
  37. 37. Makkar HPS. Siddhuraju P. Becker K. Plant Secondary Metabolities. Totowa. New Jersey: Humana Press; 2007.
  38. 38. Dhar P. Bajpai PK. Tayade AB. Chaurasia OB. Srivastava RB. Singh SB. Chemical composition and antioxidant capacities of phytococktail extracts from trans-Himalayan cold desert. BMC Complement Altern Med 2013; 13: 259. pmid:24098968
  39. 39. Kroyer GT. Red clover extract as antioxidant active and functional food ingredient. Innov Food Sci Emerg Technol. 2004; 5: 101–105.
  40. 40. Prakash D. Upadhyay G. Singh BN. Singh HB. Antioxidant and free radical-scavenging activities of seeds and agri-wastes of some varieties of soybean (Glycine max). Food Chem. 2007. 104: 783–790.
  41. 41. CLSI–Clinical and Laboratory Standards Institute. 2012. Performance Standards for Antimicrobial Susceptibility Testing. Twenty Second Informational Supplement. ninth ed. Document M100–S22. Pensilvania. USA: NIH.
  42. 42. Coutinho HDM. Costa JGM. Lima EO. Falcão-Silva VS. Siqueira-Júnior JP. Herbal therapy associated with antibiotic therapy: Potentiation of the antibiotic activity against methicillin-resistant Staphylococcus aureus by Turnera ulmifolia L. BMC Complement Altern Med. 2009; 9: 13–17. pmid:19426487
  43. 43. OECD, OECD Guidance Document on Acute Oral Toxicity Testing, OECD, Paris, France, 2000.
  44. 44. Garber J. Barbee R. Bielitzki J. Clayton L. Donovan J. et al. Guide for the care and use of laboratory animals. Washington DC: National Academic Press. 2010. 220 p.
  45. 45. Robya MHH. Sarhan MA. Selima KAH. Khalel KI. Evaluation of antioxidant activity. total phenols and phenolic compounds in thyme (Thymus vulgaris L.). sage (Salvia officinalis L.). and marjoram (Origanum majorana L.) extracts. Ind Crops Prod. 2013; 43: 827–831.
  46. 46. Monteiro JM. Lins Neto EMF. Amorim ELC. Strattmann RC. Araújo EL. Albuquerque UP. Tannin concentration in three simpatric medicinal plants from caatinga vegetation. Rev Árvore. 2005; 29: 999–1005.
  47. 47. Santos CA. Passos AMPR. Andrade FC. Camargo EA. Estevam CS. Santos MRV. Thomazzi SM. Antinociceptive and anti-inflammatory effects of Caesalpinia pyramidalis in rodents. Braz J Pharmacogn. 2011; 21: 1077–1083
  48. 48. Santana DG. Santos CA. Santos ADC. Nogueira PCL. Thomazzi SM. Estevam CH. et al. Beneficial effects of the ethanol extract of Caesalpinia pyramidalis on the inflammatory response and abdominal hyperalgesia in rats with acute pancreatitis. J Ethnopharmacol. 2012; 142: 445–455. pmid:22626927
  49. 49. Aruoma OI. Free radicals. oxidative stress. and antioxidants in human health and disease. J Am Oil Chem Soc. 1998. 75: 199–212.
  50. 50. Halliwell B. Murcia MA. Chirico S. Aruoma OI. Free radicals and antioxidants in food and in vivo: What they do and how they work. Crit Rev Food Sci Nutr. 1995; 35: 7–20. pmid:7748482
  51. 51. Zwart LL. Meerman JHN. Commandeur JNM. Vermeulen NPE. Biomarkers of free radical damage: Applications in experimental animals and in humans. Free Radic Biol Med. 1996; 26: 202–26. pmid:9890655
  52. 52. Procházková D. Boušová I. Wilhelmová N. Antioxidant and prooxidant properties of flavonoids. Fitoterapia. 2011; 82: 513–523. pmid:21277359
  53. 53. González-Burgos E. Gómez-Serranillos MP. Terpene Compounds in Nature: A Review of Their Potential Antioxidant Activity. Curr Med Chem. 2012;19: 5319–5341. pmid:22963623
  54. 54. Choi DY. Lee YJ. Hong JT. Lee HJ. Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer's disease. Brain Res Bull. 2012; 87: 144–153. pmid:22155297
  55. 55. Hatano T. Edamatsu R. Mori A. Fujita Y. Yasuhara E. Effect of interaction of tannins with co-existing substances. VI. Effects of tannins and related polyphenols on superoxide anion radical and on DPPH radical. Chem Pharm Bull. 1989; 37: 2016–2021.
  56. 56. Duh PD. Tu YY. Yen GC. Antioxidant Activity of Water Extract of Harng Jyur (Chrysanthemum morifolium Ramat). Food Sci Technol. 1999; 32: 269–277.
  57. 57. Strlič M. Radovič T. Kolar J. Pihlar B. Anti- and Prooxidative Properties of Gallic Acid in Fenton-Type Systems. J. Agric. Food Chem. 2002; 50: 6313–6317. pmid:12381109
  58. 58. Yen GC. Duh PD. Tsai HL. Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid. Food Chem. 2002; 79: 307–313.
  59. 59. Yilmaz Y. Toledo RT. Major Flavonoids in Grape Seeds and Skins: Antioxidant Capacity of Catechin. Epicatechin. and Gallic Acid. J. Agric. Food Chem. 2004; 52: 255–260. pmid:14733505
  60. 60. Dall'Agnol R. Ferraz A. Bernardi AP. Albring DC. Sarmento L. Lamb L. et al. Antimicrobial activity of some Hypericum species. Phytomedicine. 2003; 10: 511–516. pmid:13678236
  61. 61. Ríos JL. Recio MC. Medicinal plants and antimicrobial activity. J Ethnopharmacol. 2005; 100: 80–84. pmid:15964727
  62. 62. Silva CHTP. Peixoto Sobrinho TJP. Saraiva AM. Pisciottano MNC. Amorim ELC. Phytochemical profile and antibacterial activity of bark and leaves of Caesalpinia pyramidalis Tul. and Sapium glandulosum (L.) Morong. J Med Plants Res. 2012; 6: 4766–4771.
  63. 63. Chanwitheesuk A. Teerawutgulrag A. Kilburn JD. Rakariyatham N. Antibacterial activity of Gallic acid from Caesalpinia mimosoides Lamk. Food Chem. 2007;100:1044–48.
  64. 64. Jayaraman P. Sakharkar MK. Lim CS. Tang TH. Sakharkar KR. Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro. Int. J. Biol. Sci. 2010; 6: 556–68. pmid:20941374
  65. 65. Borges A. Ferreira C. Saavedra MJ Simões M. Antibacterial Activity and Mode of Action of Ferulic and Gallic Acids Against Pathogenic Bacteria. Microb Drug Resist. 2013; 19: 256–65. pmid:23480526
  66. 66. Sánchez-Maldonado AF. Schieber A. Ganzle MG. Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria. J. Appl. Microbiol. 2011; 111: 1176–1184. pmid:21895894
  67. 67. Vaquero MJR. Alberto MR. Manca de Nadra MC. Antibacterial effect of phenolic compounds from different wines. Food Control. 2007; 18: 93–101.
  68. 68. Hancock REW. The bacterial outer membrane as a drug barrier. Trends Microbiol. 1997; 5: 37–42. pmid:9025234
  69. 69. Savage PB. Multidrug-resistant bacteria: overcoming antibiotic permeability barriers of gram-negative bacteria. Ann Med. 2001; 33: 167–171. pmid:11370769
  70. 70. Denyer SP. Maillard JY. Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria. J Appl Microbiol. 2002; 92 Suppl: 35S–45S. pmid:12000611
  71. 71. Denyer SP. Stewart GSAB. Mechanism of action of disinfectants. Int Biodeterior Biodegradation. 1998; 41: 261–268.
  72. 72. Kotra L.P. Haddad J. and Mobashery S. Aminoglycoside: perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicrob Agents Chemother. 2000; 44: 3249–3256. pmid:11083623
  73. 73. Noller HF. Ribosomal RNA and translation. Annu Rev Biochem. 1991; 60:191–227.
  74. 74. Dunkle JA. Vinal K. Desai PM. Zelinskaya N. Savic M. West DM. et al. Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA. Proc Natl Acad Sci U S A. 2014; 111: 6275–6280. pmid:24717845
  75. 75. Mingeot-Leclercq MP. Glupczynski Y. Tulkens PM. Aminoglycosides: Activity and Resistance. Antimicrob Agents Chemother. 1999; 43: 727–737. pmid:10103173
  76. 76. Shakil S. Khan R. Zarrilli R. Khan AU. Aminoglycosides versus bacteria–a description of the action. resistance mechanism. and nosocomial battleground. J Biomed Sci. 2008; 15: 5–14. pmid:17657587
  77. 77. Avent M. L. Rogers B. A.. Cheng A. C. and Paterson D. L. Current use of aminoglycosides: indications. pharmacokinetics and monitoring for toxicity. Int Med J. 2011; 41: 441–449. pmid:21309997
  78. 78. Giamarellou H. Zissis NP. Tagari G. Bouzos J. In vitro synergistic activities of aminoglycosides and new beta-lactams against multiresistant Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1984; 25:534–536. pmid:6428310
  79. 79. Wu YL. Scott EM. Po AL. Tariq VN. 1999. Ability of azlocillin and tobramicyn in combination to delay or prevent resistance development in Pseudomonas aeruginosa. J Antimicrob Chemoter. 44:389–392. pmid:10511408
  80. 80. Sousa EO. Rodrigues FFG. Campos AR. Lima SG. Costa JGM. Chemical composition and synergistic interaction between aminoglycosides antibiotics and essential oil of Lantana montevidensis Briq. Nat Prod Res. 2013; 27: 942–945. pmid:22475025
  81. 81. Barreto HM. Lima IS. Coelho KMRN. Osório LR. Mourão RA. Santos BHC. et al. Effect of Lippia origanoides H.B.K. essential oil in the resistance to aminoglycosides in methicillin resistant Staphylococcus aureus. Eur J Integr Med. 2014; 6: 560–564.
  82. 82. Coutinho HDM. Rodrigues FFG. Nascimento EMM. Costa JGM. Falcão-Silva VS. Siqueira-Júnior JP. Synergism of Gentamicin and Norfloxacin with the Volatile Compounds of Lippia microphylla Cham. (Verbenaceae). J Essent Oil Res. 2011; 23: 24–28.
  83. 83. Aqil F. Ahmad I. Owais M. Evaluation of anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and synergy of some bioactive plant extracts. Biotechnol J. 2006; 1: 1093–1102. pmid:17004300
  84. 84. Coutinho HDM. Costa JGM. Falcão-Silva VS. Siqueira-Júnior JP. Lima EO. Effect of Momordica charantia L. in the resistance to aminoglycosides in the methicillin-resistant Staphylococcus aureus. Comp Immunol Microbiol Infect Dis. 2010a; 33: 467–471. pmid:19732954
  85. 85. Coutinho HDM. Costa JG. Lima EO. Falcão-Silva VS. Siqueira-Júnior JP. Potentiation of antibiotic activity by Eugenia uniflora and Eugenia jambolanum. J Med Food. 2010b; 13: 1024–1026. pmid:20482280
  86. 86. Bitu VCN. Fecundo HTF. Costa JGM. Coutinho HDM. Rodrigues FFG. Santana NM. et al. Chemical composition of the essential oil of Lippia gracilis Schauer leaves and its potential as modulator of bacterial resistance. Nat Prod Res. 2014; 28: 399–402. pmid:24479735
  87. 87. Figueiredo FG. Lucena BFF. Matias EFF. Tintino SR. Leite NF. Andrade JC. et al. Chemical composition and evaluation of modulatory of the antibiotic activity from extract and essential oil of Myracrodruon urundeuva. Pharm Biol. 2014; 52: 560–565. pmid:24251788
  88. 88. Tintino SR. Souza CES. Guedes GMM. Costa JI. Duarte FM. Chaves MCO. et al. Modulatory antimicrobial activity of Piper arboretum extracts. Acta Bot Croat. 2014; 73: 281–289.
  89. 89. Juven BJ. Kanner J. Schved F. Weisslowicz H. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J Appl Bacteriol. 1994; 76: 626–631. pmid:8027009
  90. 90. Imming P. Sinning C. Meyer A. Drugs. their targets and the nature and number of drug targets. Drug Discov. 2006; 5: 821–834. pmid:17016423
  91. 91. Schmidt B. Ribnicky DM. Poulev A. Logendra S. Cefalu WT. Raskin I. A natural history of botanical therapeutics. Metabolism. 2008; 57 (Suppl 1): S3–S9. pmid:18555851
  92. 92. Wagner H. Ulrich-Merzenich G. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009; 16: 97–110. pmid:19211237
  93. 93. Matias EEF. Alves EF. Santos BS. Souza CES. Ferreira JVA. Lavor AKLS. et al. Biological activities and chemical characterization of Cordia verbenacea DC as tool to validate the ethnobiological usage. Evid. Based Complement. Alternat. Med. 2013. 1–7.
  94. 94. Sustar B. Bukovec N. Bukovec P. Polymorphism and stability of Norfloxacin. (1-ethyl-6-fluoro-1.4-dihydro-4-oxo-7- (1-piperazinil)-3-quinolinocarboxylic acid. J Therm Anal. 1993;40: 475–48.
  95. 95. O’Neil MJ. Heckelman PE. Dobbelaar PH. Roman KJ. Kenny CM. Karaffa LS. The Merck Index. 15th ed.; RSC Publishing. 2013.
  96. 96. Baraldi C. Tinti A. Ottanic S. Gamberinia MC. Characterization of polymorphic ampicillin forms. J Pharm Biomed Anal. 2014; 100: 329–340. pmid:25194347
  97. 97. Otsuka M. Kaneniwa N. Effect of grinding on the crystallinity and chemical stability in the solid state of cephalothin sodium. Int J Pharm. 1990; 62: 65–73.
  98. 98. Sadeek SA. El-Shwiniy WH. Zordok WA. El-Didamony AM. Spectroscopic. structure and antimicrobial activity of new Y(III) and Zr(IV) ciprofloxacin. Spectrochim Acta A Mol Biomol Spectrosc. 2011; 78: 854–867. pmid:21208824
  99. 99. Dorofeev VL. Arzamastsev AP. Veselova OM. Melting point determination for the analysis of drugs of the fluoroquinolone group. Pharmaceutical Chemistry Journal. 2004; 38: 333–335.
  100. 100. Owens HM. Dash AK. Ceftriaxone sodium: comprehensive profile. Profiles Drug Subst Excip Relat Methodol. 2003; 30: 21–57. pmid:22469539
  101. 101. Masoud MS. Ali AE. Elasala GS. Synthesis. spectral. computational and thermal analysis studies of metalloceftriaxone antibiotic. J Mol Struct. 2015; 1084: 259–273.
  102. 102. Aquino RP. Auriemma G. Mencherini T. Russo P. Porta A. Adami R. et al. Design and production of gentamicin/dextrans microparticles by supercritical assisted atomisation for the treatment of wound bacterial infections. Int J Pharm. 2013; 440: 188–194. pmid:22917746
  103. 103. Ferdous S. Sultan Z. Bashar T. Rahman A. Islam S. In vitro and In vivo Studies of Drug-Drug Interaction between Metformin and Cefepime. Pharm Anal Acta. 2015; 6:348.
  104. 104. Macedo RO. Aragão CFS. Nascimento TG. Macêdo AMC. Applications of Thermogravimetry in the quality control of chloramphenicol tablets. J Therm Anal Calorim. 1999; 56: 1323–1327.
  105. 105. Flörke OW. Graetsch HA. Brunk F. Benda L. Paschen S. Bergna HE. et al. Silica. Ullmann's Encyclopedia of Industrial Chemistry. Wiley Online Library. 2008.