Dihydroflavonols from the leaves of Derris urucu ( Leguminosae ) : Structural Elucidation and DPPH Radical-Scavenging Activity

Derris urucu é uma planta da Amazônia com propriedades inseticida e ictiotóxica. Estudos com esta espécie reportam a presença de flavonóides, principalmente rotenóides, bem como de estilbenos. A partir do extrato etanólico das folhas de Derris urucu (Leguminosae), três novos diidroflavonóides, denominados urucuol A (1), B (2) e C (3) e o diidroflavonol isotirumalina (4), foram isolados e identificados. As estruturas destes compostos foram elucidadas por uma extensiva análise espectroscópica de RMN uni e bidimensional, UV, IV e dados de EM, além de comparação com dados da literatura. Os compostos isolados (1-4) foram avaliados quanto ao seu potencial sequestrador do radical DPPH e apresentaram baixo poder antioxidante quando comparados ao antioxidante comercial trans-resveratrol.


Introduction
Amazonian ecosystems are rich in plants with insecticide and piscicide properties, and those belonging to the Derris genus are the most used. 1 These plants, in the Amazon area, are called "timbó".3][4][5] In addition to rotenoids, others minor flavonoids, such as flavanones, isoflavanones and chalcones, together with stilbenes, have been also described from the roots of Derris urucu. 6Flavonoids belong to a group of naturally occurring compounds with a number of biological activities, such as antibacterial, antimutagenic, cytotoxic and anticarcinogenic, [7][8][9][10] together with antioxidant activity, which is one of the most studied. 11,12Antioxidant activity arises from the ability of flavonoids to scavenge free radicals and thus eliminate reactive oxygen species. 13,146][17] Therefore, the antioxidant activity of these natural compounds is related to a number of different mechanisms such as free radical scavenging, hydrogen donation, singlet oxygen quenching, metal ion chelation, and acting as a substrate for radicals such as superoxide and hydroxide. 18Because oxidative stress is known to cause many diseases, scientists have become more interested in natural sources to fight it, looking for active components from plants in recent years.Vol. 20, No. 6, 2009   Nevertheless, some phenolic compounds increase oxidative stress and toxicity because of their prooxidant properties. 19he balance between antioxidant or prooxidant properties can be determined by the scavenger capacity of radical oxygen or nitrogen species using spectrophotometric methods, such as DPPH, which has been applied to the phenolic compounds commonly present in natural products.The spectrophotometric technique employs the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH • ), which shows a characteristic UV-Vis spectrum with a maximum of absorbance close to 517 nm in methanol.The addition of an antioxidant compound results in a decrease of absorbance proportional to the concentration and antioxidant activity of the compound. 202][23][24][25][26] It is easy to perform, highly reproducible and comparable with other methods such as ABTS, reduction of superoxide anion and inhibition of lipid peroxidation. 27,28n particular, DPPH • free radical has been used to assess the ability of phenolic compounds to transfer labile H atoms to radicals. 29Total H atom donating capacities are evaluated in the EC 50 index, defined as the concentration needed to reduce 50% of DPPH • free radical.
Thus, in this work we have investigated leaves of D. urucu for the first time, searching for compounds with potential antioxidant activity, resulting in the isolation of three new dihydroflavonols named urucuol A (1), B (2) and C (3), as well as dihydroflavonol isotirumalin (4) (Figure 1), which were evaluated for the ability of DPPH • radical-scavenging.Our purpose in this work was also contribute to a better understanding of the mechanistic features of antioxidant processes of the dihydroflavonols isolated from Derris urucu.

Results and Discussion
The dried leaves of Derris urucu were extracted with EtOH.The ethanolic extract was fractionated by silica gel column chromatography affording six fractions.Chromatographic separation of the EtOAc-soluble fraction by semi-preparative HPLC led to the purification of pure substances 1-4 (Figure 1).
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, 36 so the dihydroxylated dihydroflavanols 1 and 4 were more effective in promoting DPPH reduction, compared with the monohydroxylated 2 and the unhydroxylated 3, the least active.
Dihydroflavonols show a different behavior compared with flavones or flavonols.The methylation in the hydroxyl group at the para-position decreased DPPH scavenging activity.8][39] An additional methylation in the meta-position significantly decreased DPPH scavenging capacity.Based on all the information cited, it is possible to justify the lesser antioxidant activity of the compounds tested compared to the resveratrol, whose structure presents trihydroxylation at the para-and meta-positions and an all conjugated system.

Experimental
General IR spectra were obtained in a Bomen MB-102 spectrophotometer, using the thin solid film method.UV spectra were obtained from a Shimadzu Prominence 20A LC equipped with DAD.NMR spectra, including 1 H-1 H COSY, HETCOR, HMBC experiments, were recorded on a Varian Mercury-300 spectrometer, operating at 300 MHz for 1 H and 75 MHz for 13 C, using d-chloroform as solvent and internal standard.Mass spectral analyses were performed at low resolution on a Quattro-LC instrument (Micromass, Manchester, UK) provided with an ESI ion source and a triple quadrupole mass analyzer.High resolution analyses were performed on UltrOTOF-Q (Brucker, Daltonics, Billerica MA, USA) only in the cationized ion region.After a systematic investigation, the heated capillary and the voltage were maintained at 250 °C and 3 kV, respectively.A 20 V (cone energy) was applied for the ion extraction and the mass spectrometry data were acquired in the positive mode for all compounds.HPLC was carried out in a preparative LC-8A Shimadzu system with SPD-10AV Shimadzu UV detector (Tokyo, Japan); using a Phenomenex Gemini C18 column (250 mm × 10 mm, 5μm), an isocratic system of water/acetonitrile (46:54) and a flow rate of 4.7 mL per min.Detection was performed at 270 and 320 nm.All solvents were filtered through a 0.45 mm membrane filter prior to use.Absorbance measurements were recorded on a Spectrum UV SP-220 spectrophotometer.

Plant material
The leaves of Derris urucu were collected in January 2006, in the forest reserve of EMBRAPA-Amazônia Oriental in Belém, Pará State, Brazil.A voucher specimen (IAN 179599) was deposited at the herbarium of this institution.

DPPH assays
A methanolic solution (25 mg L −1 ) of the radical DPPH • was prepared daily and protected from light.Absorbance was recorded to check the stability of the radical throughout the time of analysis.The effect of phenolic compounds on the DPPH • absorbance was estimated by using the procedure described in the literature. 20Different sample concentrations dissolved in methanol were added to DPPH • methanolic solutions.Absorbance at 517 nm was recorded at different time intervals until the reaction reached an equilibrium.The initial absorbance was close to 1.100-1.150 in all cases.The blank reference cuvette contained methanol.All measurements were performed in duplicate.Six different concentrations of each phenolic compound studied were assayed in order to check the linearity of response and to establish the antioxidant activity values in an adequate linear range.All phenolic compounds were properly dissolved in methanol.

Data analysis
Reaction kinetics of phenols with DPPH • were registered for each antioxidant concentration tested.From these plots, the percentage of DPPH • remaining at the steady state (DPPH • rem) was determined as %DPPH • rem = (A f / A 0 ) × 100.A 0 and A f correspond to the absorbances at 517 nm of the radical at the beginning and at the steady state, respectively.Time at steady state was used in order to ensure that reaction did not progress further.Concentrations of the phenolic compounds in the reaction medium were plotted against the percentages of the remaining DPPH • at the end of the reaction in order to obtain the EC 50 index, defined as the amount of antioxidant needed to decrease the initial DPPH • concentration by 50%.Analysis of variance and linear correlations tests were performed using the BIOSTAT® version software package.

Urucuol A (1)
Pale yellow powder; IR (thin solid film) n max /cm  13 C NMR spectral data: see Tables 1 and 2.

Figure 2 .
Figure 2. Dose-response curve for radical scavenging activity of the dihydroflavonols 1-4 isolated from leaves of the Derris urucu by DPPH method at different concentrations.trans-resveratrol (RESV) was used as reference compound.

Figure S3 .
Figure S3.ESI mass spectrum of the compound 1 isolated from leaves of Derris urucu.

Figure S10 .
Figure S10.ESI mass spectrum of the compound 2 isolated from leaves of Derris urucu.

Figure S15 .
Figure S15.ESI mass spectrum of the compound 3 isolated from leaves of Derris urucu.

Figure S22 .
Figure S22.ESI mass spectrum of the compound 4 isolated from leaves of Derris urucu.

Table 1 .
1H NMR Chemical Shifts (d H in ppm) and Coupling Constants (J in Hz) of Compounds 1-4 in CDCl 3 a 1 H NMR data were recorded at 300 MHz.b Multiplicity and coupling constant (J, Hz) are in parenthesis.