Evaluation of the potential modulator of bacterial resistance , acute toxicity and chemical composition of Schinopsis brasiliensis Engl .

Universidade Federal do Piauí, Departamento de Ciências da Natureza, BR 135, km 03, Planalto Horizonte, Bom Jesus PI, Brazil. Laboratório de Desenvolvimento e Ensaios de Medicamentos (LABDEM) – Universidade Estadual da Paraíba, Rua das Baraúnas, 351 Bairro Universitário, Campina Grande, Paraíba, Brazil. Laboratório de Pesquisa em Microbiologia, Centro de Ciências Biológicas e da Saúde, Universidade Estadual da Paraíba, R. Baraúnas, 351 Bairro Universitário, Campina Grande, Paraíba, Brazil. Laboratório de Farmacologia, Centro de Ciências Biológicas e da Saúde, Universidade Estadual da Paraíba, R. Baraúnas, 351 Bairro Universitário, Campina Grande, Paraíba, Brazil.


INTRODUCTION
Brazil is known throughout the world for its biodiversity and complex cultural heterogeneity, which was influenced by indigenous, European, and African cultures in the colonial period (Elisabetsky and Wannmacher, 1993; *Corresponding author.E-mail: anaclaudia@uepb.edu.br.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Albuquerque et al., 2007).The direct relationship between the population and the local flora has generated a rich system of knowledge about its therapeutic properties.This ethnopharmacological knowledge is a great starting place for researchers in search of new drugs (Fabricant and Farnsworth, 2001;Balunas and Kinghorn, 2005).
In recent years, the use of plant extracts for therapeutic purposes has become a growing research interest (Oliveira and Petrovick, 2010).These extracts consist of a complex of plant metabolites whose therapeutic effectiveness is related to the phytochemical interaction wherein molecules perform additional tasks.Thus, it is not surprising that within plant mixtures, secondary metabolites may be more biologically active than the individual components (Lila and Raskin, 2005;Wagner, 2011).Among the Brazilian medicinal plants, Schinopsis brasiliensis Engl.(Anacardiaceae), a thorny tree popularly known as braúna, baraúna, braúna-parda and braúna-do-sertão can be highlighted (Lorenzi, 1992).It is one of the most important medicinal plants for communities located in the Caatinga in Northeastern Brazil where people use its bark in various mixtures designed to treat flu symptoms, colds, diarrhea, dysentery, fractures, general inflammations, general infections and sexual impotence (Albuquerque et al., 2005(Albuquerque et al., , 2007(Albuquerque et al., , 2011;;Silva and Albuquerque, 2005;Albuquerque and Oliveira, 2007;Almeida et al., 2006Almeida et al., , 2010)).
Despite the importance and widespread use of S. brasiliensis in folk medicine, there are few experimental studies that seek to evaluate its pharmacological and phytochemical properties.The presence of a considerable concentration of phenolic compounds such as tannins and flavonoids as well as antioxidant and antimicrobial properties has been reported (Saraiva et al., 2011(Saraiva et al., , 2013)).A new alkyl phenol, methyl 6-eicosanyl-2hydroxy-4-methoxybenzoate and an unusual steroid 5a,8a-epidioxyergosta-6,22-dien-3-b-ol have been isolated from this plant (Cardoso et al., 2005).Because of its current high use value for the Caatinga communities and its potential to be framed in various categories of ethnobotanical use, S. brasiliensisis the Brazilian Institute for the Environment's (IBAMA) list of species was threatened with extinction (Lucena et al., 2007).Knowing the phytochemical and pharmacological properties of S. brasiliensis may, in addition to finding new bioactive molecules, provide subsidies for the conservation and sustainable use of this species.Thus, this study aims to evaluate the phytochemical properties, modulating activity of the bacterial resistance, and the acute toxicity of the extract of S. brasiliensis.

Plant material and ethanolic extraction
The bark of five different specimens of S. brasiliensis Engl, (Anacardiaceae), were collected in the semiarid region of the Paraíba state, with a voucher specimen prepared and deposited in the herbarium Professor Jayme Coelho de Morais (Herbarium Code EAN), Federal University of Paraíba, under the number EAN-14049.The plant material was dried in a circulating air oven at 40±1°C and crushed into slicer with a particle size of 10 mesh.The powdered plant particles (100 g) were extracted exhaustively with 96% ethanol by percolation, and subsequently the concentration conducted on a rotary evaporator.

Drugs
Penicillin, ampicillin, erythromycin, amoxicillin, levofloxacin, cephalothin and gentamicin were acquired from a local pharmacy.All drugs were dissolved in sterile 0.9% saline solution.

Determination of total polyphenols
The total polyphenol content of plant extracts was measured by Folin-Ciocalteu method (Chandra and Mejia, 2004).The extract were dissolved in distilled water to obtain a final concentration of 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 method described by Meda et al. (2005).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 Ultraviolet (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 (1993) 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 hydrochloric acid (HCl) (37%).After the HCl addition, the tube content was shaken in water bath at 30°C for 3 to 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. (2007).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 min, and then transferred to an ice bath to rest for 4 min.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 .

Microbiological assays
It were used clinical isolates Staphylococcus aureus resistant to ampicillin, ciprofloxacin, cephalexin, erythromycin, penicillin and amoxicillin and Escherichia coli resistant to amoxicillin, cephalothin, levofloxacin, chloramphenicol, tetracycline and gentamicin.The minimum inhibitory concentration (MIC) was determined by a microdilution method in 96-well plates using Mueller-Hinton broth (CLSI, 2012).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 10 6 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 h.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.Evaluation of extracts as modulators of antibiotic resistance was performed according to Coutinho et al. (2010).The MIC of the antibiotic was determined in presence and absence of sub-inhibitory concentrations (MIC/8) of EESb.Plates were incubated as described earlier and each assay was performed in triplicate.

Acute toxicity
Wistar rats (131 to 258 g) were used in this research.They were maintained under standardized conditions of light (light-dark cycle of 12 h), relative humidity (60% ± 15%) and temperature (25 ± 2°C) with free access to food and water.The animals were acclimatized to laboratory conditions before the commencement of the experiment.All experiments were approved by the Ethics Committee on Animal Research CCTR/UFCG under register no.196/2014.The animals were kept in groups of five per cage.Prior to testing, the animals were fasted for 12 hours.The extract was administered by gavage at a concentration of 2000 mg kg -1 , and the animals were maintained under the aforementioned conditions, being observed within 30 min andat 1, 2 and 4 h after the extract administration to observe the emergence of toxic signs (Almeida et al., 1999).At 24, 48 and 72h after the administration of the extract, deaths were recorded and the consumption of water and food were measured and the animal was weighed.A control group was treated with a saline solution, the same solution used to dilute the extract.After the period of observation, the surviving animals were dissected, including the resection and weighing of organs (liver, kidneys, heart, lungs and spleen), which were also examined macroscopically.

Statistical analysis
The microbiological test results were expressed as geometric means.Two-way analysis of variance followed by Bonferroni posttest was applied.For acute toxicity test, the results were expressed as the mean ± standard deviation (SD) and subjected to an analysis of variance (ANOVA) followed by Dunnett's post-test or a Tukey-Kramer test.The confidence limit was 95% (p < 0.05).The statistical software used was GraphPad Prism-5 ® , version 2007.

Phytochemical tests
The total content of the major groups of secondary metabolites present in the extract of S. brasiliensis are shown in Table 1.The values were expressed as gallic acid equivalents (GAE) per gram of the crude extract to phenolic compounds, quercetin equivalents (QE) for flavonoids, catechin equivalent (CE) for the condensed tannin and disogenin equivalent (DE) to saponins.

Microbiological assays
The EESb showed efficacy against four strains tested with the lowest MIC values acquired on S. aureus strains, SA ATCC 25923 and SA16 strains, sensitive to 7.8 and 15.6 µg ml -1 respectively.However, over the strains of E. coli, EESb showed no significant activity (MIC > 1000 µg ml -1 ).In the assays of modulation of resistance, the EESb showed the best results on strains of E. coli, significantly reducing (<0.001) the MIC of all antibiotics tested.The most pronounced decrease was of cephalothin, whose MIC was reduced from 49.6 to 12.4 μg ml -1 .For S. aureus, the combination of extract that provided significant results (p < 0.001) was with erythromycin; the MIC decreased from 1000.0 to 793.7 μg ml -1 (Figures 1  and 2).

Acute toxicity
In the acute toxicity test, the rats used showed no behavioral changes after EESb administration at 2000 mg Kg -1 v.o.Feed intake and water remained stable in the control group and the group treated with the extract.However, when macroscopically analyzing the viscera of the animals in the group treated with the extract compared with the control group, the results point to possible hepatotoxic and nephrotoxic action of EESb, considering the weights of the kidneys and liver were  significantly decreased (P < 0.05).Such toxic effect can be proven only by histopathology.During the observation of the animals after the administration of the extracts, there were no deaths; it was not possible to determine the mean lethal dose (LD 50 ).

Phytochemical tests
The concentrations of some substances in the methanol extract of EESb has already been measured by Saraiva et al. (2011), who found a higher concentration of polyphenols (825.65±40.99mg of tannic acid equivalent per gram of extract) and lower flavonoid concentration (11.29±0.94mg of rutin equivalent per gram of extract).This difference may be related to the solvent used in the production of extracts; while the aforementioned authors used methanol, this study used ethanol.Other aspects that may also have influenced the difference in the concentration of secondary metabolites in both studies are the collection site of plant material and intrinsic factors such as the stage of plant development and tissues differentiation (Chaves et al., 2013;Mirdehghan and Rahemi, 2007;Ncube et al., 2011).Rios and Recio (2005), suggest that in order to have clinical relevance, the MIC of crude extracts must not be higher than 1000 µg ml -1

Microbiological assays
. Considering this parameter, the EESb showed efficacy against both strains of S. aureus.Extracts of S. brasiliensis showed the antimicrobial activity reported in the literature.Chaves et al. (2011) when testing the hydroalcoholic extract, Saraiva et al. (2011) the methanol extract and Saraiva et al. (2013) different proportions of different fractions of methanolic extract showed efficacy against gram positive, gram negative and fungal strains through an agar diffusion test.However, the impossibility of measuring the amount of extract spread on the agar affects the ease of using this method to accurately determine the MIC (Hadacek and Greger, 2000).
The use of plant extracts associated with other natural or synthetic antibiotics is well documented in the literature.This association has shown that some plant extracts seem to work synergistically with the antibiotic used, resulting in better antimicrobial activity against several multidrug-resistant strains (Sato et al., 2004;Aqil et al., 2006;Coutinho et al., 2009aCoutinho et al., ,b, 2010;;Ncube et al., 2012;Figueiredo et al., 2013).
The mechanisms related to the synergistic effects of combining antibiotics and extracts relate primarily to factors such as the multiplicity of targets on which the mixture of bioactive compounds can act, including enzymes, receptors, ion channels, transport proteins, Chaves et al. 847 DNA/RNA, antibodies and many others, as well as the suppression of bacterial resistance mechanisms for compounds present in the extract (Imming et al., 2006;Schmidt et al., 2008;Wagner and Ulrich-Merzenich, 2009).Both the antimicrobial activity and the synergistic effect may be related to the richness of S. brasiliensis regarding polyphenolic compounds and saponins since some of these compounds have already been cited as exhibiting such activities (Cowan, 1999;Gibbons, 2005Gibbons, , 2008)).Tannins can prejudice the metabolism of bacteria, inhibiting enzymes, oxidative phosphorylation and the electron transport system and neutralizing microbial adhesins and proteins in the cell envelope (Cowan, 1999;Scalbert, 1991).In turn, flavonoids and saponins can destabilize bacterial membranes, breaking the barrier function and forming pores respectively; both of which cause intramembranous leakage of materials (Ikigai et al., 1993;Francis et al., 2002;Cushnie and Lamb, 2005;Sung and Lee, 2008).

Acute toxicity
The low toxicity of extracts of S. brasiliensis suggested in this assay has been demonstrated by studies on plants of the same family.For example, Ayoka et al. (2006) evaluated extracts of Spondias mombin L., and Konan et al. (2007) assessed a hydroalcoholic extract of Anacardium occidentale L. and observed the absence of toxic symptoms or the death of animals.It is suggested that further tests should be performed to confirm the toxicological profile of the plant

Conclusion
The tests performed demonstrated that the extract obtained from the bark of S. brasiliensis is an effective antimicrobial, and with the capacity to modulate bacterial resistance activity and may be considered to possess low toxicity.This study represents the first report of these biological activities on the quantitative phytochemistry of ethanol extract of S. brasiliensis, and the results that justify the ethnopharmacological use of this plant in Brazilian traditional medicine against certain types of infections.However, further studies are needed in order to understand the mechanism of antimicrobial and modulating action and the toxicological profile of the plant.

Table 1 .
Total content of phytochemical constituents of the ethanolic extract of S. brasiliensis.