New Isoflavones from the Leaves of Vatairea guianensis Aublé

Das folhas de Vatairea guianensis Aublé foram isoladas quatro isoflavonas identificadas como, 5,3’,-diidroxi-4’-metoxi-2”,2”-dimetilpirano-(5”,6”:8,7)-isoflavona (1), 5,7-diidroxi-3’,4’metilenodioxi-8-prenil-isoflavona (2) e 5,3’-diidroxi-4’-metoxi-7-O-β-glicopiranosídeo-8-prenilisoflavona (3) e derrona (4), juntamente com cinco triterpenos identificados em mistura de lupeol, α-amirina, β-amirina, germanicol e ácido betulínico. As substâncias 1-3 são novos produtos naturais, porém 1 e 2 já foram citados como produtos de síntese. No entanto, todas essas substâncias são relatadas pela primeira vez para essa espécie. Suas estruturas químicas foram elucidadas com base nos seus dados de ressonância magnética nuclear (RMN) 1D e 2D e por espectrometria de massas de alta resolução. O extrato etanólico das folhas e os compostos 1-3 foram avaliados quanto ao seu potencial sequestrador do radical DPPH• (2,2-difenil-1-picril-hidrazila) e os resultados mostram que o extrato apresentou alta atividade (CI50 = 6,2 ± 0,4 μg mL ), enquanto as substâncias testadas apresentaram baixo poder antioxidante (CI50 ≥ 29,5 ± 2,5 μg mL) quando comparadas com TROLOX (CI50 = 4,5 ± 0,4 μg mL ).


Introduction
The genus Vatairea (Fabaceae) includes seven species distributed in Brazil, the Guianas and the Atlantic coastal regions of Central America and Mexico. 1 The species Vatairea guianensis Aublé is a plant native to the Amazon, known as "fava bolacha" or "fava de impingem". 2Its fruits, bark from the stems, roots and leaves are commonly used for treating surface micoses. 3,4Few scientific references citing biological activities for this species have been found in the literature.Fruits are the only part of the plant that has been studied in terms of biological potential, presenting in vitro action against Leishmania amazonensis, 5 antimicrobial 6 and topical healing activity. 7Among the components reported for the plant are antraquinones chrysophanol, physcion and emodin, antrones 9-anthronechrysophanol acid, 9-anthronephyscion and 10-anthronephyscion, triterpenes oleanolic acid and dihydromacaerinic acid lactone. 4,8These reports of phytochemical studies are restricted to fruits, heartwood and bark of the stems.The leaves of V. guianensis were chosen for this study in an effort to identify their chemical composition and assess their potential for scavenging radical DPPH • .Four isoflavones were isolated (1-4) (Figure 1) together with a mixture of known triterpenes lupeol, α-amyrin, β-amyrin, germanicol and betulinic acid.The ethanolic extract of the leaves and isoflavones 1-3 were evaluated for their potential to scavenge DPPH • .

Experimental
General UV spectra were obtained from a liquid chromatograph (LC) equipped with diode array detection (DAD) Prominence SPDM-20A (Shimadzu, Tokyo, Japan).Nuclear magnetic resonance (NMR) spectra including 1D and 2D experiments were recorded on a Varian Mercury-300 spectrometer, operating at 300 MHz at 1 H and 75 MHz at 13 C, using CDCl 3 or CD 3 OD as solvent (0.6 mL).Mass spectral analyses were performed on UltrOTOF-Q (Bruker Daltonics, Billerica, MA, USA) in the cationized ion region.The heated capillary and voltage were maintained at 250 o C and 3 kV, respectively.A 20 V cone energy for ion extraction and mass spectrometry data were acquired at positive and negative modes for all compounds.High performance liquid chromatography (HPLC) was carried out in a semi-preparative LC-8A Shimadzu system with 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 35% acetonitrile-water, and a flow rate 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, Maherey-Nagel).Thin layer chromatography (TLC) was performed on precoated silica gel aluminium sheets (Maherey-Nagel) by detection with a spraying reagent (vanillin/sulfuric acid/EtOH solution) followed by heating at 100 °C and with reagent NP-PEG (diphenylborinic acid aminoethylester-polyethylene glycol) for flavonoid detection.

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

DPPH assays
A stock solution of DPPH • radical (0.5 mmol L -1 ) in methanol was prepared.The solution was diluted in methanol (60 µmol L -1 approx.)measuring an initial absorbance of 0.62 ± 0.02 in 517 nm at room temperature.The reaction mixture was composed by 1950 µL of DPPH • solution and 50 µL of the samples diluted in different methanol portions.The final concentrations were of 10.0, 7.5, 5.0 and 2.5 µg mL -1 for the crude extract, 75.0, 50.0, 25.0 and 12.5 µg mL -1 for substances 2 and 3 and 8.0, 6.0, 4.0 and 2.0 µg mL -1 for the Trolox standard.For each sample a methanol blank was also measured.The absorbance was measured at 517 nm during 60 min.All experiments were conducted in triplicate and Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) was used as the standard antioxidant.The radical scavenging activity of each sample was calculated by the DPPH • inhibition percentage (%I DPPH ) according to equation 1, where A and B are the blank and sample absorbance values in the end reaction. 9) The concentration of antioxidant required for 50% scavenging of DPPH • radicals (IC 50 ) was determined by linear regression using Windows/Excel.All analyses were performed in triplicate.The data were expressed as means ± standard deviation (SD) and analyzed using GraphPad (version 5.0).One-way analysis of variance (ANOVA) and Tukey multiple comparisons were performed in order to test any significant differences between the means.Differences between means at the 5% level were considered significant., 367,11816); 1 H and 13 C NMR spectral data: see Tables 1 and 2.  13 C NMR spectral data: see Tables 1 and 2. , 531.18664); 1 H and 13 C NMR spectral data: see Tables 1 and 2.

Results and Discussion
The hexane fraction from the ethanol extract of V. guianensis leaves was fractionated as indicated in the Experimental section leading to the isolation of new isoflavones 1-3, together with known compounds derrone (4), lupeol, α-amirin, β-amirin, germanicol and betulin acid, which were identified by comparison of their spectral data with those reported in literature. 10,11ompound 1 was obtained as an amorphous pale green solid, with the molecular formula C 21 H 18 O 6, based on the [M+H] + peak at m/z 367.11901 (calc.for C 21 H 19 O 6 + , 367.11816) in the HRESITOF-MS, and confirmed by 1 H and 13 C NMR experiments (Tables 1 and 2, respectively).The 1 H NMR signals at δ H 7.87 (H-2) and 13 C NMR signal at δ C 152.5 (C-2), 123.7 (C-3) and 180.8 (C-4), were typical of isoflavones. 12Additionally, the 1 H NMR spectrum exhibited signals in the aromatic region at δ H 6.91 (d, 1H, J 8.1 Hz), 7.03 (brd, 1H, J 8.1 Hz) and 7.06 (brs, 1H), which indicated an ABX spin system of a 1,3,4-trisubstituted phenyl group, as well as one singlet at δ H 6.28 assigned to a pentasubstituted benzene ring.The signals observed at δ H 5.58 and 6.67 (d, 1H each, J 9.9 Hz) and 1.47 (s, 6H) revealed a 2,2-dimethylchromene ring attached to an aromatic ring, whereas the singlets at δ H 12.95 and 3.91 indicated the presence of a hydroxyl chelated to carbonyl and one OMe group connected to an aromatic ring, respectively.All couplings were confirmed through analysis of the 1 H-1 H COSY spectrum.Besides the signals related to C-ring carbons, the 13 C NMR spectrum of 1 exhibited another 17 signals attributed to eighteen carbons with aid of the HETCOR and HMBC (Table 2) experiments.The 2,2-dimethylchromene ring attached to A-ring at C-7 and C-8 was deduced by 3 J C,H correlations from H-2 (δ H 7.87) and H-4'' (δ H 6.67) to C-8a (δ C 152.1) observed in the HMBC spectrum (Table 2), as well as 2 J C,H correlation between H-6 (δ H 6.28) and OH-5 (δ H 12.95) with C-5 (δ C 162.2).The location of the OMe and OH groups at C-4' and C-3' of the aromatic B-ring, respectively, was supported by the 3 J C,H correlations from H-6' (δ H 7.03) and OMe-4' (δ H 3.91) to oxidized aromatic carbon C-4' (δ C 146.8) and confirmed by the NOE effect observed in the NOE difference experiment, which revealed spatial interactions between H-5' (δ H 6.91) and OMe-4' (δ H 3.91).
The crude extract of leaves showed strong radical scavenging capacity, with inhibition percent of 77.2 at 22.0% at concentrations of 10.0 at 2.5 µg mL -1 , respectively, and IC 50 value of 6.2 ± 0.4 µg mL -1 (Trolox, IC 50 = 4.4 ± 0.1 µg mL -1 ).The results obtained from isolated isoflavones 1-3 showed that all tested samples were capable of scavenging free radical DPPH • , however, compound 1 was weakly active with an inhibition percentage below 50.0% at 100 µg mL -1 .Compounds 2 and 3 presented greater inhibition effects, with inhibition percentage above 50.0%during 60 min of reaction at 25 and 50 µg mL -1 concentrations, respectively.However, compounds 2 and 3 were about 6 and 14 times less active than Trolox with IC 50 values of 29.5 ± 2.5 µg mL -1 and 64.3 ± 2.6 µg mL -1 , respectively.The free radical scavenging activity of flavonoids and other phenols is mostly due to their aromatic hydroxyl groups, which afford greater stability to the phenolic radical as soon as it is formed, after one hydrogen radical donation to DPPH • . 19Although compounds 1-3 possess two phenolic hydroxyl groups each, the isoflavonoid 2 was more effective in promoting DPPH • reduction, when compared to 1 and 3. Early studies showed that the presence of a substituent (sugar residues) in position C-7 of A-ring in the flavonoids reduces the sequestering activity of the radicals, since the flavonoid structure loses its coplanarity due to the presence of voluminous groups, 20,21 and methylation in hydroxyl group in the para-position decreased DPPH • scavenging activity. 22On the other hand, the methylenedioxy function contributes to stabilization of the phenoxy radical. 23The fact that much higher values of IC 50 were found for the isolated compounds when compared to Trolox suggests that the significant anti-radical activity exhibited by the ethanolic extract of V. guianensis leaves may be attributed to the effect of synergism in the substances present in this extract.

Conclusions
Phytochemical investigation from the leaves of Vatairea guianensis (Fabaceae) resulted in the isolation of compounds belonging to the isoflavone and triterpene classes, and it should be noted that the isoflavone class is being cited for the first time for this species.The evaluation of antioxidant activity, based on the method of sequestering radical DPPH • demonstrated that the ethanolic extract of V. guianensis leaves presents a high power for scavenging radical DPPH • , similar to the TROLOX standard, while the isoflavones tested present low anti-radical reaction, which suggests that the greatest antioxidant potential of the extract may be associated with synergism among its components.

Figure 1 .
Figure 1.Structures of the isoflavones isolated from V. guianensis.