Development of a rich fraction in phenolic compounds with high antioxidant and antimicrobial activity in Amburana cearensis seeds extracts

1 Natural Products Research Laboratory, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil. 2 Faculty of Medicine, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil. 3 General and Applied Microbiology Laboratory, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil. 4 Faculty of Medicine, Natural Products Research Laboratory, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte), Federal University of Tocantins, Palmas, Tocantins 77020-210, Brazil. 5 Department of Biotechnology, Biodiversity and Biotechnology Post Graduate Program of the Amazon (Bionorte) Gurupi, Federal University of Tocantins, Tocantins 77404-970, Brazil.


INTRODUCTION
Amburana cearensis, known as umburana or cumaru is a leguminous plant of Fabaceae family, used in perfumery and for pharmaceutical purposes; it can be observed in practically all of America, from Peru to Argentina (Canuto and Silveira, 2006).Many parts of this plant are used in traditional medicine, stem bark and leaves contain phenolic compounds with anti-inflammatory and antioxidant effects (Leal et al., 2003), they are also useful in conducting several physiological disorders such as diabetes mellitus, hypertension, vascular fragility and improvement of the health of gastrointestinal tract (Scalbert et al., 2005).The seeds are used for stomach and liver diseases (Leal et al., 2008).Besides, some peptides from A. cearensis have antifungal (dos Santos et al., 2010) and antimicrobial activity (Sá et al., 2011).
The antimicrobial activity of phenolics and flavonoids are also well documented (Erdemoglu et al., 2007;Xia et al., 2011).The phenolics compounds can affect the growth and metabolism of bacteria, activating or inhibiting the microbial growth according to their constitution and concentration (Alberto et al., 2006;Nazzaro et al., 2009).
The need to find new antimicrobial substances against microorganisms presents a challenge in the treatment of infectious disease (Gonçalves and Santana, 2010).
Currently, many bacterial strains are resistant to almost all antimicrobials.The search for antibacterial properties of plant extracts has been encouraged and intensified, substances derived from plants constitute approximately 25% of medically prescribed agents in industrialized countries (Sá et al., 2011).
Several studies have demonstrated the presence of secondary metabolites and antimicrobial activity in leaves and stem bark of the A. cearensis, however, the seeds do not present similar results (Lima et al., 2013), this is already expected because the main constituents of most seeds are carbohydrates, lipids and proteins, followed by other constituents in smaller amounts such as vitamins, minerals and water (Vaclavik and Christian, 2006).Furthermore, the solubility of secondary compounds depends on the type of solvent used, degree of polymerization, as well as interaction with other food constituents and formation of insoluble complexes (Naczk and Shahidi, 2004), indicating the need for further studies with this part of the plant.
Due to the widespread use of A. cearensis for medicinal purposes and the scarcity of phytochemical reports of its seeds in the literature, it has become essential to carry out its study, to discover the possible response for the therapeutic properties of A. cearensis.The aim of this study was to determine the antioxidant capacity and antimicrobial activity of A. cearensis extracts against bacteria strains.

MATERIALS AND METHODS
This study was done in Natural Products Research Laboratory of Aguiar et al. 649 Federal University of Tocantins (UFT), Palmas Campus, Tocantins, Brazil.

Plant
The seeds of A. cearensis were obtained from popular commerce in the city of Palmas, Tocantins, Brazil, and their authenticity recognized by EMBRAPA Herbarium, voucher (CPAP 5948).The seeds were dried in a stove at 45°C and ground using knives crusher .The ground seeds were stored in bottles and kept at room temperature and covered from light and humidity (Al-Marby et al., 2016).

Seed extract preparation
To prepare the extracts, 15 g of powdered seeds were extracted for 4 h using the Soxhlet apparatus (Mabiki et al., 2013).First, an extraction was performed with 80% ethanol at the boiling temperature of the solvent, resulting in the crude extract (CEA).
Subsequently, another extraction was carried out using different solvents, n-hexane, methanol, 80% ethanol and water, from the lowest to the highest polarity to extract the maximum seed components.
Each solvent was used after being dried in a stove at 40°C for 24 h.The solvents were removed on a rotary evaporator at -600 mm Hg (Fisaton 804) at 45°C.They were dried in the exhaust hood and stored in an amber bottle and kept at 4°C.The hexanic, methanol, 80% ethanol, and water extracts were respectively named as, hexanic extract (HEA), methanol extract (MEA), ethanol extract (EEA), and aqueous extrac (AEA).

Phytochemical screening
To elucidate which compounds were mainly present in CEA extract, previous phytochemical analysis was achieved according to previously described methods (Matos, 2009), based on coloration/precipitation tests.This analysis was complemented by thin layer chromatography (TLC).The test was fulfilled using benzene-ethyl acetate-formic acid-methanol (60/30/10/5, v/v/v/v) as the mobile phase.
Then, dried plates have been sprayed with a 1% solution of 2aminoethyl diphenylborinate in methanol and spray with a 0.5% ethanolic solution of polyethylene glycol (NEU/PEG reagent).Next, the plates were visualized using a darkroom viewing cabinet (SOLAB®, model SL 204) UV light at 365 nm.The retention factor (Rf) values were calculated measuring the distance reached by the extract divided by the distance of the mobile phase and compared with Rf of standards rutin, quercetin, and morin.

Determination of phenolic compounds and total flavonoid content
Total phenolic content was quantified using the Folin-Ciocalteu method as described by Amorim et al. (2008) and determined by interpolation of the absorbance of the samples and the calibration curve constructed with standard, gallic acid in methanol (y = 1.3631x + 0.0213, adjusted r 2 = 0.962).The result was exposed as mg gallic acid equivalents (GAE) per gram of A. cearensis extract *Corresponding author.E-mail: alineaires13@uft.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 (mg GAE/g).
Total flavonoid content was performed using the method described by Soares et al. (2014) with few modifications and determined by using the absorbance of the samples against a calibration curve (y = 1.3336x + 0.0078, adjusted r 2 = 0.9999) constructed with the rutin standard and expressed as milligrams of rutin equivalents (RE) per gram of dry extract (mg RE/g).

Evaluation of antioxidant activity
The antioxidant activity was performed by the 1,1-diphenyl-2picrylhydrazyl (DPPH) (Sigma-Aldrich®) assay, described by Peixoto-Sobrinho et al. (2011).Standards were used as rutin and ascorbic acid (Sigma-Aldrich®).The percentage scavenging values were calculated following the equation: where AA is the antioxidant activity, AS is the absorbance of the sample, Ablank is the absorbance of the blank, and A0 is the absorbance of DPPH without sample.
The IC50 value calculated indicates the concentration of a sample required to decrease the absorbance at 517 nm by 50%.The IC50 was expressed in μg/mL.

HPLC analysis
The A. cearensis extracts were analyzed by high-performance liquid chromatography (HPLC) at Scientific Instrumentation Laboratory (LABIC) UFT-Palmas, Tocantins, Brazil.The HPLC system (Shimadzu, Tokyo, Japan) consisted of a chromatograph (LC-10 Avp series) with a pump (LC-10 AD), a degasser (DGU-14A) to pump the mobile phase, rheodyne manual injector (20 μL loop) and class integrator (LC-10A), a UV-Vis (SPD -10A) detector and a column oven (CTO 10A).The extracts and standards were prepared with methanol and filtered with Millipore membrane (0.22 μm pore size).The separation was achieved by a gradient system, using a reverse-phase Phenomenex Luna 5 mm C18 (2) (250 4.6 mm 2 ) column with direct-connect C18 Phenomenex Security Guard Cartridges (4 3.0 mm 2 ) filled with similar material as the main column.Mobile Phase A was 0.1% phosphoric acid in Milli-Q water and mobile phase B was 0.1% phosphoric acid in Milli-Q water/acetonitrile/methanol (54:35:11).Flow rate: 1 ml/min, temperature: 22 1C.UV detection was done at 280 nm.The standards used were gallic acid, rutin, ellagic acid, naringin, myricetin, morin, quercetin, naringenin, and kaempferol (Sigma®).The compounds were expressed in micrograms per milligram of extract (μg/mg) by correlating the area of the analyte with the calibration curve of standards built in concentrations of 4.5 to 18 μg/mL.

Antimicrobial activity in broth
The minimum inhibitory concentration was determined by the microplate dilution technique (96 holes) according to the methodology described according to the M7-A6 standard of the Manual 38 Clinical and Laboratory Standards Institute (CLSI, 2006), the most recommended method for this determination (Benfatti et al., 2010).The microplate wells were filled with 100 μL of Muller-Hinton broth medium (MHB-Micro Med), then 100 μL of extract solutions were added and a serial dilution was performed: 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μg/mL.In addition, 20 μL of the microorganism suspensions were inoculated into each well of the microplates.As a positive control, chloramphenicol was used at the same dilutions as extracts.Control of the culture medium, bacterial growth control and negative control (solvents) were also performed.The microplates were incubated in an incubator at 37°C for 24 h, all tests were performed in triplicate.
The MIC was expressed as the lowest concentration that inhibited growth, which was judged by the well.The turbidity in each well was then measured at 450 nm by using a spectrophotometer (GT -722G).The MIC values equal to or less than 1000 μg/mL were considered active (Kuete, 2010).

Statistical analysis
The experiment was totally randomized, with three replicates per treatment.The ANOVA test was obtained using the ASSISTAT 7.6 beta program.

Phytochemical screening
Many studies have demonstrated the profuse presence of coumarin, flavonoids and tannins in stem bark and leaves of A. cearensis as responsible for pharmacological activities of this species according to the effects observed in tests pure substances (Marinho et al., 2004;Canuto, 2006); however, these phenolic compounds have not yet been well documented in seeds, this can occur because secondary metabolites link competitively for some solvents during the extraction process (Naczk and Shahidi, 2004).In this work, the sequential extraction process eliminated this interference, favoring the extraction of phenolic compounds.A similar extraction methodology was performed by Zhu et al. (2010) in a study with Portulaca oleracea L, obtaining such results.Seed extraction from A. cearensis provided 1.315 g (8.73%) hexane extract, 3.877 g (25.91%) methanolic extract, 1.009 g (6.66%) hydroethanolic extract and 0.876 g (5.33%) aqueous extract.
Table 1 shows the total phenol, flavonoids and the evaluation of total antioxidant activity of A. cearensis seed extracts.The EEA extract presented higher phenolic  and flavonoid content, this high antioxidant activity is probably due to the type of extraction performed, since the EEA is an extract practically free of interferents as fatty compounds, moreover, this extract was the only one which presented rutin in HPLC analysis (Figure 3).Values below the IC 50 reflect high antioxidant activity.EEA extract values were very close to the ascorbic acid and rutin patterns (Figure 2).

HPLC analysis
The fingerprinting of A. cearensis extracts obtained by HPLC is as shown in Figure 3 2.
Rutin is a powerful free radical scavenger that has a significant therapeutic potential against cancer; morin possess antibacterial activity (Pereira et al., 2015).Catechin is considered antifungal (Anand and Rai, 2016) and has shown activity against many bacteria (Shahid et al., 2016).
The HPLC data made evident that the A. cearensis seeds extracts obtained are plentiful in phenolic compounds of pharmacological importance and antioxidant significance.Different phenolic compounds appear in the different extracts, demonstrating the extraction method used was effective to elucidate the potential stored in A. cearensis seeds.EEA extract has the highest number of phenolic compounds (Table 2) corroborating with results of DPPH and TLC tests.The not identified peaks shown in the chromatograms (Figure 3) can be phytocomponents with important biological activity and different potential.Hence, more research with A. cearensis seeds could improve the pharmacological knowledge about this plant.

Antibacterial assay
The bioassays with extracts of A. cearensis showed that EEA and AEA extratcs had activity against E. coli, P. aeruginosa, S. flexneri, L. monocytogenes and S. aureus, with MIC values ranging from 250 to 1000 µg/mL.CEA, HEA and MEA extracts had no significant effect against these microorganisms (Table 3).There is no report of antibacterial activity in A. cearensis seeds extracts prior to this work.Santos et al. (2010) have shown activity of peptide from A. cearensis seeds only against phytopathogenic fungi and yeasts and Lima et al. (2013) showed no antibacterial activity against strains of S. aureus, E. coli and P. aeruginosa with their A. cearensis extract.Catechin and naringin is present in EEA extract (Table 2), it is known that these phenolic compounds act against microorganisms (Anand and Rai, 2016), then it explains the antimicrobial activity of this extract.The standard antibiotic for this assay, chloramphenicol,
. The retention time of samples and the standards made possible the identification of various phenol compounds, such as: gallic acid (time 16.5 min), catechin (time 23.7 min), rutin (time 40.6 min), ellagic acid (time 41.5 min), naringin (time 43.4 min), myricetin (time 46.8 min), and morin (time 49.5 min).The substances detected in extracts are shown in Table

Table 1 .
Total phenolic content, total flavonoids content and antioxidant activity (DPPH IC50) of seed extracts obtained by sequential extraction.

Table 2 .
Phenol compounds identified by HPLC in A. cearensis seeds extracts.