A New Prenylisoflavone from the Antifungal Extract of Leaves of Vatairea guianensis Aubl

A new compound, 5,7,3’-trihydroxy-4’-methoxy-8-prenylisoflavone, was isolated from the leaves of Vatairea guianensis Aubl. (Fabaceae), together with two known isoflavones lupiwighteone and 5,7,4’-trihydroxy-3’-methoxy-8-prenylisoflavone. All isolated compounds were characterized based on infrared (IR), UV, H and C nuclear magnetic resonance (NMR), including 2D NMR analyses and high resolution mass spectrometry. The ethanolic extract from V. guianensis leaves displayed activity against Candida dubliniensis, C. albicans and C. krusei. However, the EtOAc fraction from that extract exhibited more significant activity than the ethanolic extract, showing antifungal activity for all fungi species investigated. The major compound 5,7,3’-trihydroxy4’-methoxy-8-prenylisoflavone isolated from that EtOAc fraction was also active against C. parapsilosis and C. dubliniensis.


Introduction
Many plants from Brazilian biomes such as the Cerrado, Atlantic Forest and Amazon Forest have been used as natural medicines by local communities for treating tropical diseases such as fungal and bacterial infections. 1,2The population makes use of these traditional medicines through medical prescriptions, self-medication, home remedies and other means. 3Among them Vatairea guianensis Aubl.(Fabaceae), a plant native to the Amazon, popularly known as "fava de impingem" (ringworm bean) is used in folk medicine for treating mycoses. 4,5he genus Candida has at least 15 distinct species that cause human disease, such as C. kefyr, C. tropicalis, C. lusitaniae, C. dubliniensis, C. guilliermondii and C. rugosa, but the five most common pathogens are: C. glabrata, C. tropicalis, C. parapsilosis, C. krusei and the most common and well-studied of the disease-causing species in that genus is C. albicans. 6Candidiasis may present in forms ranging from superficial skin lesions 7 to disseminated infections and in recent decades, there has been a substantial increase in the occurrence of invasive fungal infections (IFIs) caused by C. albicans, 8 which can naturally colonize the skin, genital, and/or intestinal mucosa in up to 70% of healthy individuals 9 and is the most common bloodstream pathogen in the USA, responsible for 50% of the cases. 6n invasive candidiasis, echinocandin plays a central role in the treatment, but there has recently been an increased incidence of resistant strains in both nosocomial and in community fungal infections. 10Despite the large number of antifungal medicines available on the market, aside from their side effects, drug resistance is an obstacle in treating.An important resistant phenotype in Candida is azole resistance and of greatest concern is the evolution of multidrug-resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. 11iven the increasing occurrence of fungal infections in humans along with increasing resistance to available medicines, it is important to identify new antifungal compounds that will enable development of new medicines. 12][15][16] The present paper reports the isolation and structural characterization by spectroscopic and spectrometric methods of a new compound, named 5,7,3'-trihydroxy-4'-methoxy-8-prenylisoflavone (3), along with other two known isoflavones lupiwighteone (1) and 5,7,4'-trihydroxy-3'-methoxy-8-prenylisoflavone (2) (Figure 1).The ethanolic extract, hexanic, EtOAc and MeOH/H 2 O fractions and compounds 1 and 3 were evaluated for their antifungal activities against certain pathogenic fungi but, due to insufficient quantities, the antifungal testing of compound 2 was not carried out.

General experimental procedures
The melting point (mp) was determined on a Quimis Q340S23 melting point analyzer.UV spectra were obtained from HPLC equipped with DAD Prominence SPDM-20A (Shimadzu, Tokyo, Japan).Infrared (IR) spectra were recorded on a Shimadzu Corporation IR Prestige 21 spectrometer (Tokyo, Japan) with KBr pellets.Nuclear magnetic resonance (NMR) spectra, including 1D and 2D experiments (see Supplementary Information), were recorded on a Varian Mercury-300 spectrometer (Palo Alto, CA, USA), operating at 300 MHz at 1 H and 75 MHz at 13 C, using acetone-d 6 , CD 3 OD or a mixture (CDCl 3 and a few drops CD 3 OD) as deuterosolvents (0.6 mL).Mass spectrometry (MS) analysis was performed on a XEVO G2-SQ-TOF mass spectrometer (Waters Corp., Milford, MA, USA) equipped with a lockspray source where an internal reference compound (leucine-enkephalin) was introduced simultaneously with the analyte for accurate mass measurements.Electrospray mass spectra data were recorded in a positive ionization mode for a mass range from m/z 50 to 1000 with a scan time of 0.1 s.The source temperature was set to 150 °C with a cone gas flow of 20 L h -1 .The desolvation gas flow was set to 600 L h -1 at a temperature of 250 °C.The capillary was set at 3.5 kV with cone voltage at 20 V. MassLynx software (Waters, Milford, MA, USA) was used for system control and data acquisition.High-performance liquid chromatography (HPLC) was carried out in a semi-preparative LC-8A Shimadzu system with a SPD-10AV Shimadzu UV detector (Tokyo, Japan), using a Phenomenex Gemini C18 column (250 × 10 mm, 5 μm), isocratic system of 50% acetonitrile-water and a flow rate of 4.7 mL min -1 .Detection was performed at 254 and 282 nm.All solvents were filtered through a 0.45 mm nylon membrane filter prior to analysis.Open column chromatography was run using silica gel 60 (70-230 Mesh, Macherey-Nagel, Düren, Germany).Thin layer chromatography (TLC) was performed on precoated silica gel aluminium sheets (Macherey-Nagel, Düren, Germany) by detection with a spraying reagent (vanillin/sulfuric acid/EtOH solution) followed by heating at 100 °C and with NP-PEG reagent (diphenylborinic acid aminoethyl ester-polyethylene glycol) for flavonoid detection.

Plant material
Leaves of V. guianensis were collected in November 2010 in the city of Belém, state of Pará, Brazil.Identification was performed by Manoel R. Cordeiro from Embrapa Amazônia Oriental, Pará, Brazil, and a voucher specimen (IAN -187050) has been deposited in the herbarium at Embrapa Amazônia Oriental.

In vitro antifungal activity
The minimal inhibitory concentrations (MICs) were determined by broth microdilution methods based on the Clinical and Laboratory Standards Institute (CLSI) reference protocol M38-A2. 17All the test strains were subcultured on Sabouraud dextrose agar (SDA) (Sigma-Aldrich, Saint Louis, MO, USA), incubated for 24-72 hours at temperature 30 °C, and their inocula were prepared according to procedures reported by Daboit et al. 18 That involved scraping across the surface of the fungal colonies with a sterile pipette and sterile saline solution (0.85%), containing 0.05% Tween 40.After standing for 3-5 min, at room temperature for deposition of larger particles, the concentration of spores in the supernatant was adjusted spectrophotometrically (530 nm) to a per cent transmission in the 80-82 range for Candida albicans (INGOS 40175), Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 22019) and Candida dubliniensis (CBS 7987).These suspensions were diluted to 1:50 in a synthetic RPMI-1640 medium buffered with morpholinopropanisulfonic acid (MOPS; Sigma ® , Saint Louis, MO, USA).
The extracts, fractions and pure compounds were primarily dissolved in dimethyl sulfoxide (DMSO) and diluted in sodium bicarbonate-free RPMI 1640 medium (Sigma ® , Saint Louis, MO, USA) buffered with 165 mmol L -1 MOPS, pH 7.0, and supplemented with 4 mmol L -1 L-glutamine.The final DMSO concentration was maintained as less than 1%.Concentrations ranged H and 13 C NMR data were recorded at 300 and 75 MHz, respectively.Multiplicity and coupling constants (J in Hz) are in parentheses.Number of hydrogen atoms bound to carbon atoms deduced by comparative analysis of 1 H and DEPT 13 C NMR and HETCOR spectra; b1 H- 13 C HMBC correlations are from the carbon atoms specified to the protons indicated.from 1024 to 0.125 μg mL -1 for extracts and fractions; for pure compounds they ranged from 256 to 0.125 μg mL -1 , and the assay was carried out as reported previously. 19he MIC was determined as the lowest concentration of the crude extract, fractions and the pure compound that inhibited the complete growth of the organisms.
Minimum fungicidal concentrations (MFCs) were established following the incubation time determined for the MIC.Thirty μL from each well with complete growth inhibition were inoculated onto SDA plates and incubated at 30 °C for up to 72 h.The MFC was defined as the lowest concentration of the extracts, fractions and pure compounds, in which there was either no growth or a growth up to seven colonies, which corresponds to a 99.9% kill; 20 fluconazole was used as the standard antibiotic.All experiments were conducted in triplicate.

Results and Discussion
Compound 1 was identified as lupiwighteone by comparing its spectral data with those reported in literature. 21,22This substance is being reported for the first time for the genus Vatairea and presents important antiinflammatory activity according to Paoletti et al., 23 as well as being considered a very potent phytoestrogen. 24ompound 2 was identified as 5,7,4'-trihydroxy-3'-methoxy-8-prenylisoflavone.This substance has been reported only once for the plant Wyethia mollis; 25 however, the data from HRESITOF-MS, 1 H and 13 C NMR are being reported for the first time in this paper.
Compound 3 was obtained as a pale yellow solid, with the molecular formula C 21 H 20 O 6 , based on the [M + H] + peak at m/z 369.1352 (calcd.for [M + H] + , C 21 H 20 O 6 + H + , 369.1338) in the HRESITOF-MS, and confirmed by 1 H and 13 C NMR experiments (Table 1).The 1 H NMR signal at d H-2 7.85 and 13 C NMR signals at d C-2 152.9, d C-3 122.7 and d C-4 180.9, were typical of isoflavones. 26Additionally, the 1 H NMR spectrum exhibited signals in the aromatic region at d H 6.84 (1H, d, J 8.4 Hz), 6.93 (1H, dd, J 1.9 and 8.4 Hz) and 6.99 (1H, d, J 1.9 Hz), which indicated an ABX spin system of a 1,3,4-trisubstituted phenyl group, as well as one singlet at d H 6.24 assigned to a pentasubstituted benzene ring.The signal singlet at d H 3.83 indicated the presence of an OMe group connected to an aromatic ring.All couplings were confirmed through analysis of the 1 H-1 H correlation spectroscopy (COSY) spectrum.Besides the signals related to C-ring carbons, the 13 -9).The location of the OH and OMe groups at C-3' and C-4' of the B-ring, respectively, was sustained by combining the HMBC cross-peaks from H-6' (d H 6.93) and OMe (d H 3.83) to C-4' (d C 147.2) with the nuclear Overhauser effect (NOE) observed in the NOE difference spectra, which revealed spatial interactions between H-5' and OMe-4'.Therefore, 3 was characterized as 5,7,3'-trihydroxy-4'-methoxy-8-prenylisoflavone.This compound is a new natural product.
In evaluating the inhibition of fungal growth by V. guianensis, the extracts that presented MICs ≤ 800 μg mL -1 and the pure compounds with MICs ≤ 250 μg mL -1 were defined as active, these values being based on the work of Stein et al. 27 The results of the MICs obtained in this study are shown in Table 2.
The most significant result of the minimum inhibitory concentration for the EtOH extract from leaves was 32 μg mL -1 for C. dubliniensis.For the strains of fungi C. albicans and C. krusei the EtOH extract was also active showing MIC of 128 μg mL -1 , although it was inactive for C. parapsilosis (MIC > 1024 μg mL -1 ) (Table 2).

Conclusions
Phytochemical investigation of the antifungal fraction (EtOAc) from V. guianensis leaves resulted in the isolation of a new isoflavone (3) besides other two known isoflavones.These compounds are in agreement with the typical chemical profile of plants of the Vatairea genus and Fabaceae family.The results of evaluating the antifungal activity of V. guianensis indicate the potential of this species, but it is necessary to expand the studies to provide scientific support to popular use of this plant in the treatment of skin infections caused by fungi.

Figure 1 .
Figure 1.Chemical structures of isoflavones 1-3 isolated from the leaves of V. guianensis.

Table 1 .
13e 1 H and13C NMR chemical shifts (d Η in ppm) and HMBC correlations of compound 3 in CDCl 3 and a few drops of CD 3 OD a ')] was confirmed as attached at C-8 by the 3 J C,H correlations in the HMBC experiments (Table 1) between the signals at d H 7.85 (H-2) and 3.35 (H-1'') with the signal at d C 155.2 (C

Table 2 .
Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of crude extract, fractions and constituents isolated from V. guianensis against pathogenic microorganisms New Prenylisoflavone from the Antifungal Extract of Leaves of Vatairea guianensis Aubl.
−: Not detected in the assay conditions.A J. Braz.Chem.Soc.1136