Evaluation of the antimicrobial and antioxidant activity of 7-hydroxy-4 ’ , 6-dimethoxy-isoflavone and essential oil from Myroxylon peruiferum L . f

This study evaluated the antibacterial, antifungal, and antioxidant effect of 7-hydroxy-4’,6dimethoxy-isoflavone and essential oil of Myroxylon peruiferum. The compound was isolated and its structure elucidated by NMR. The chemical composition of essential oil determined by gC-MS analysis. To evaluation of antimicrobial activity, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) were performed. In addition to analysis of antioxidant activity, DPPH radical scavenging tests, iron chelating assay (FIC), antioxidant reducing power assay (FRAP) and β-carotene bleaching assay (BCB) were performed. For the essential oil were identified 24 organized compounds having as main constituents; Germacrene D (17.2%), α-pinene (14.8%) and E-caryophyllene (10.8%). The results showed that isoflavone (2000 to 156 μg/mL) and essential oil (5.0 to 1.25%) present antibacterial and antifungal activity against Gram-positive bacteria and filamentous fungi. The isoflavone and the essential oil also presented antioxidant activity in all the tests, mainly on inhibition of the oxidation of β-carotene test concentrations ranging from 60 to 100%. In conclusion, isoflavone and essential oil from M. peruiferum present an antimicrobial alternative against gram-positive bacteria, especially of the genus Staphylococcus and dermatophyte fungi of the genus Trichophyton, as well as a natural compound antioxidant.


INTRODUCTION
Plants have been used for medicinal purposes since the beginning of human civilization for prevention, treatment and cure of several diseases (De Carvalho et al. 2013).The evolution of scientific knowledge has intensified studies with medicinal plants relating their chemical composition to its effects (Cavalcante et al. 2013).In fact, several studies have investigated the presence of chemical compounds with biological potential in plant extracts and essential oils, commonly in plants used in folk medicine by communities (guimarães et al. 2014, Dhifi et al. 2016, Jantapan et al. 2017).
The emergence of the resistant microorganisms is one of the principal public health problems of this century, increasing exponentially of the numbers of infections caused by microorganisms no longer susceptible to the common medicines used to treat them (Prestinaci et al. 2015).Studies of novel products of plant origin with antimicrobial activity have increasing interest, since emergence of resistant bacteria and fungi to conventional antimicrobials agents, aggravating this problem of global public health (Silva et al. 2012).Thus, extracts or compounds isolated from plant species used in folk medicine can be promising sources for the investigation of new antimicrobial agents (Al-Fatimi et al. 2007).
Bacteria that are usually involved in hospitalacquired infections include gram-negative and gram-positive, being Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli the main agents (Khan et al. 2015, Horan et al. 2008).On the other hand, although the epidemiology of fungal diseases has changed over the past few decades, Candida species continue to be the main fungal pathogens responsible for most cases of severe fungal disease, such as candidiasis (Bongomin et al. 2017).While dermatophytes, such as Trichophyton rubrum, remain the most common fungal pathogens causing superficial fungal infections, especially onychomycosis, tinea pedis and tinea cruris (Silva-Rocha et al. 2017, Foster et al. 2004, Drakensjö and Chryssanthou 2011, Simonnet et al. 2011).
Myroxylon peruiferum belongs to the Fabaceae family, popularly known as "cabriúva" or "bálsamo".It is a deciduous plant, heliophytic, having about 10 to 20 meters of height.Their leaves are composed of pinnacles, occurring almost throughout the country.Their wood is suitable for furnitures, claddings and civil constructions because of its high resistance to rot (lorenzi 1998).
Regarding popular knowledge, lorenzi and Matos (2002) report the use of leaves, fruits and resin of M. peruiferum to for fighting wounds and ulcers.According to lorenzi and Matos (2008), leaves, fruits and resin can be used for treating asthma, rheumatism, phlegm, external wounds, headache, torticollis and tuberculosis.França et al. (2008), in an extensive consultation on medicinal literature, states that M. toluifera resin is used to treat coughing caused by bronchitis.Santos et al. (1988) described that M. balsamum (l.) Harms, popularly known as "Bálsamo-to-tolú", is an effective antiseptic of the urinary tract.
Thus, the aim of this study was evaluated the antibacterial and antifungal activities on microorganism clinically relevant and the antioxidant activities of an isoflavone and essential oil isolated from the leaves of M. peruiferum.Moreover, the isolation of the isoflavone and the chemical profile of the essential oil was described.
For Minimum Bactericidal Concentration (MBC), 10 μL of wells that having no visible microbial growth were inoculated onto Petri dishes with TSA medium.MBC was considered as the lower concentration capable of completely inhibiting microbial growth in plaques.

MINIMUM INHIBITORY CONCeNTRATION (MIC) AND MINIMUM FUNgICIDAl CONCeNTRATION (MFC)
The minimum inhibitory concentration was determined by the method described by Clinical and laboratory Standards Institute-ClSI (ClSI M27-A2 2008), while the minimum fungicidal concentration (CFM) was according to Fontenelle et al. (2007).The compound and the essential oil were prepared in DMSO and mineral oil, respectively.100 μL of sterile RPMI 1640 was inoculated into each well of the microdilution plate followed by 100 μL of sample added to the first microwell column and serially diluted until column 11; the concentration range was of 5 to 0.009 mg/ mL.Subsequently, 100 μL of the inoculum was added to all wells making a final volume of 200 μL per well.The MIC was defined by the smallest test concentration capable of inhibiting visually fungal growth.The MFC was defined by the lowest concentration which resulted in non-growth after 2 days for Candida spp.and 5 days for T. rubrum spp.after transferring 100 μL solution from wells without turbidity to agar dextrose 28 (Fontenelle et al. 2007(Fontenelle et al. , 2008)).
ANTIOXIDANT ACTIVITY

Determination of DPPH radical scavenging activity
The ability of the compound and the essential oil to scavenge the radicals DPPH was measured with reference to Duan et al. (2006).Test samples consisted of a methanolic solution containing 0.16 mM DPPH added to different concentrations of the isoflavone (6.25 to 400 μg/mL) and the essential oil (2.5 to 7.8%).The control was DPPH solution without sample, and the blank consisted of sample without DPPH solution.Samples, controls and blanks were incubated in the dark at 25°C for 30 minutes and the optical density was measured at 517 nm using a microplate reader (Biochrom Asys

Iron chelating activity (FIC)
The iron ion chelating activity assay was performed with reference to Wang et al. (2009).The samples were prepared using deionized water to obtain concentrations equal to previous test where 2 mM iron chloride (FeCl 2 ) and 5 mM ferrozine were added.Blank and control were prepared with distilled water replacing ferrozine and samples, respectively.Samples were incubated at 25°C for 10 minutes and the optical density was measured at 562 nm using a microplate reader (Biochrom Asys UVM 340).eDTA (ethylenediamine tetraacetic acid) was used as a positive control.The FIC activity was calculated by the following equation:

Iron reduction method (FRAP)
The antioxidant capacity of iron reduction of the sample was determined according to the method described by ganesan et al. (2008).Initially, the isofl avone at concentrations ranging from 400 to 6.25 μg/mL and the essential oil from 2.5 to 7.8% were mixed with 0.2 M sodium phosphate buff er (pH 6.6) and 1% potassium ferricyanide.Samples were incubated at 50°C for 20 minutes.After cooling, 10% trichloroacetic acid was added.An aliquot was mixed with 0.1% iron hydrochloride (FeCl 3 ) and distilled water.After 10 minutes, the optical density of this mixed was measured at 700 nm using a microplate reader (Biochrom Asys UVM 340).Butylhydroxyanisole (BHA) was used as a positive control.Higher the optical density, greater the compound's ability to reduce iron.

Oxidation inhibition with β-carotene and linoleic acid (BCB)
The ability of the compound and the essential oil to inhibit the oxidation of β-carotene and linoleic acid was determined by a combination of the methods described by Chew et al. ( 2008) and Dhongade and Chandewar (2013).In brief, the reagent for this assay consists of 3 mL of 0.1 mg/mL β-carotene dissolved in chloroform, 40 mg linoleic acid and 400 mg Tween 40.The chloroform was evaporated using a rotary evaporator and then ultra-pure water (Milli-Q) was added to the solution.The samples were adjusted to the same concentrations as before.
The optical density of the solutions was initially measured at 470 nm using a microplate reader (Biochrom Asys UVM 340), then again after 3 hours of incubation at 50°C.Butylhydroxyanisole (BHA) was used as a positive control for this assay.
The antioxidant activity was calculated by the following equation:  I.The results of gas chromatography coupled to mass spectrometry (Cg-MS) showed the identification and quantification of 24 compounds organized by elution order in column DB-5 corresponding to 85.50% of the chemical composition of the oil, having the germacrene D (17.2%), α-pinene (14.8%) and E-caryophyllene (10.8%) as the main.The main components (e) and (Z)-nerolidol, α-bisabolol and (E, E)-farnesol were reported by Wanner et al. (2010).
The study of the chemical composition of M. peruiferum showed a predominantly presence of nerolidol and a variety of flavonoids being reported for the first time in the literature (Maranduba et al. 1979).Through the chemical analysis of the essential oil of bark and of the essential oil of trunk of M. peruiferum several phenolic substances were found.Furthermore, in its resinous fraction were found monoterpenoids, sesquiterpenoids, phenylpropanoids, alcohols and phenylpropanoids derivatives (Akisue 1971, 1972a, b, Oliveira et al. 1978, Maranduba et al. 1979).Variations in the chemical composition of essential oils, secondary metabolism products of plant, occur due to changes in abiotic factors as the geographic location of the plant, season and even time of day when the plants are exposed (Dudareva et al. 2004, gobbo-Neto andlopes 2007).
The results of the antibacterial activity (Table II The isoflavone isolated from the ethyl acetate extract, the most predominant compound of the species, shows a proven antibiotic activity against Helicobacter pylori (Ohsaki et al. 1999).The ethanolic extract as well as the hexanic and chloroform fractions of the shell of M. balsamum showed activities against resistant bacteria, especially S. aureus resistant to methicillin and P. aeruginosa isolated from hospital infections, revealing their medicinal use.The bioassay with the extract identified the isoliquiritigenin calzone, an antimicrobial compound that has potential against S. aureus, S. epidermidis and S. haemolyticus (Machado et al. 2005).
Staphylococcus were more susceptible to the action of isoflavone and EOMP than Gram-negative bacteria (Table II).The difference in the susceptibly bacterial probably occurred due to structure of the cell wall in gram-negative bacteria, composed of an external membrane, constituting a second lipid bilayer (Vaara 1992, Arunachalam et al. 2016).The intrinsic resistance of gram-negative bacteria to many different drugs is largely attributed to the architecture of the cell envelope and multidrug efflux pumps.The outer membrane and the efflux machinery work together to reduce the intracellular concentration of various metabolites, essential oil and antibiotics so that the bacterium is able to resist the action of a range of structurally diverse compounds (Blair et al. 2015).
The results in the antifungal assays showed MIC and MFC values for the isoflavone of 156 and 312 μg/mL against T. rubrum LABMIC 0207; 312 and 625 μg/mL against T. rubrum LABMIC 0208; 625 and 1250 μg/mL against T. rubrum lABMIC 0209; and 1250 and 2500 μg/mL against T. rubrum lABMIC 0210, respectively.The eOMP showed MIC and MFC on T. rubrum lABMIC 0207, T. rubrum lABMIC 0208 and T. rubrum lABMIC 0209 at concentrations ranging from 5000 to 1250 μg/mL (Table II).It was observed that the EOMP did not present inhibition effectiveness against the yeast strains tested.
The isoflavonoids acquired considerable importance for exhibiting diverse biological activities like antioxidant, antifungal, bactericidal, anti-inflammatory, estrogenic and contraceptive (Jang et al. 2003).Furthermore, essential oils have been described for their antibacterial and antifungal properties, whose use may represent an advance against resistance mechanisms that inactivate standard antifungal (Castro 2010, Saad et al. 2010, Tempone et al. 2008).Thus, the discovery of natural products with antibacterial and antifungal activity may represent a new therapy through the production of phytotherapics (Khan et al. 2012, Mendes 2011, Silva et al. 2009) (Hummelova et al. 2014).
The formononetine (7-hydroxy, 4'methoxyisoflavone) has several pharmacological properties such as antimicrobial, healing potential, antilipid effect, antioxidant, hyperlipidemic, cardioprotective activity, antitumor, antidiabetic and neuroprotective activity (Vishnuvathan et al. 2016).Certain authors consider the lipophilicity of the constituents of essential oils as a property that explains the antimicrobial activity, which allows bacterial inhibition through the permeability and depolarization of the cytoplasmic membrane.With the permeability of membrane, increase the proton evasion of cells and the modification of electrical potential of the membrane, indications of membrane damage and consequently cell death (Cowan et al. 2011, Rúa et al. 2011).
Regarding antioxidant activity, in the DPPH scavenging assay (Figures 2a and 3a), the isoflavone and the eOMP showed antioxidant activities at all concentrations tested with percentages of approximately 60%.The ascorbic acid (positive control) presented significantly higher values than the samples in all concentrations tested, with percentage values of 70-100%.The ability to bind to metal ions, specifically to the iron ion (Figures 2b and 3b), the compound showed dose- dependent chelating action with percentages of 10-20% in the concentrations of 6.25 to 400 μg/mL, while the eOMP showed a percentage of 40-100% in concentrations of 0.078 to 2.5%.The positive control (EDTA) showed values significantly from 10 to 100% when compared to compound and essential oil.The samples were also tested for their ability to reduce Fe 3+ to Fe 2+ .The results obtained showed that the optical density remained stable at the concentrations of isoflavone; but the optical density increased in accordance with the concentrations for the eOMP when compared to the positive control (BHA) (Figures 2c and 3c).
Finally, it was evaluated the ability of the samples to inhibit β-carotene oxidation.Both the essential oil and the isoflavone showed a high oxidizing activity in up to 60 hours of storage varying from 80 to 100% and inhibiting β-carotene oxidation in values close to these found in the positive control (BHA) (Figures 2d and 3d).

CONCLUSIONS
In this study, we have isolated the compound 7-hydroxy-4', 6-dimethoxy-isoflavone and described the chemical composition of essential oil of M. peruiferum.Furthermore, the isoflavone and essential oil exhibits antifungal e antibacterial, mainly on gram-positive bacteria and dermatophytes fungi.
Moreover, in the antioxidant assays the samples showed a potential antioxidant action in different trial.Thus, the essential oil and the isoflavone isolated of M. peruiferum can be considered as potential antimicrobial agents against infections and contaminations caused by bacteria and fungus.In addition, they can be used against oxidative stress present in infections.
An Acad Bras Cienc (2019) 91(2) e20180204 5 | 13 UVM 340).Ascorbic acid was used as a positive control.The percentage of the DPPH radical scavenging eff ect was calculated by the following equation(Duan et al. 2006): assays, the percent values obtained in the DPPH, FIC and BCB assay for each of the concentrations tested were converted to absolute values, submitted to angular transformation and compared by Student's t-test for independent values.The optical density values obtained in the FRAP test were also analyzed using Student's t-test for independent values.Values of p < 0.05 were considered statistically signifi cant.spectra showed 17 carbons signals, being two signals of methoxy groups.The isolated compound of the species was identified as 7-hydroxy-4',6-dimethoxy-isoflavone whose RMN 1 H, RMN 13 C spectral data are according to literature values (Jha et al. 1980).This compound was isolated by Jha et al. (1980), from the desoxybenzoin obtained by condensation of phenols with derivatives of arylacetic acids or aliphatic acids.Fernandes et al. (2009) isolated the isoflavone from suspension cell cultures and callus of Dipteryx odorata, popularly known as afrormosin or castanin.The chemical structure of the isoflavone is shown in Figure 1.The essential oil yield was 2.0% (w/w) dry weight.The chemical composition of the volatile constituents of the essential oil of M. peruiferum (eOMP) and their respective percentages are set out in Table
showed that formononetine has antifungal effects for Candida albicans Y0109 and C. albicans SC5314 with MIC values of 8 μg/mL.Li et al. (2012) isolated isoflavone from tobacco roots and demonstrated antibacterial effect (over 90% inhibition) against Proteus vulgaris, Staphylococcus aureus, Escherichia coli and Bacillus subtilis.The eOMP showed MIC and MFC of 1.25 and 2.5 µl/ml for C. albicans URM-6543 and C. tropicalis URM-6741, 1.25 μL/mL for MIC and MFC for C. glabrata URM-6393 and 0.625 and 1.25 µl/ml for C. krusei URM-6391 and C. parapsilosis URM-6557 (Costa et al. 2017).Several studies have evaluated the antibacterial and antifungal activity and reported different types of action mechanisms owing to large variety in the chemical composition of essential oils(Burt 2004, Di Pasqua et al. 2010, greay and Hammer 2011, Qiu et al. 2011).

Figure 3 -
Figure 3 -Antioxidant activity of the essential oil from Myroxylon peruiferum l.f.Test DPPH (a), FRAP (b), FIC (c) and BCB (d).White column: sample; Gray column: positive control.*Statistically significant in relation to the positive control, when p < 0.05.

TABLE II Antibacterial and antifungal activities of the isoflavone and essential oil from Myroxylon peruiferum L.f.
lABMIC-Microbiology laboratory.ATCC-American Type Culture Collection.N.I -No inhibitory activity.
) showed that isoflavone was able to inhibit the bacterial growth of Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228 with MIC values of 2000 μg/mL for both