Identification and Antioxidant Activity of Several Flavonoids of Inga Edulis Leaves

Um extrato metanol-água das folhas de Inga edulis foi fracionado para identificar os compostos polifenólicos. Os compostos identificados foram o acido gálico, a catequina, a epicatequina, a miricetina-3-ramnopiranosídeo, a quercetina-3-glucopiranosídeo e a quercetina3-ramnopiranosídeo. A capacidade antioxidante do extrato e dos polifenóis puros foi medida pelo teste ORAC e comparada com o teor em fenólicos totais (TP). O extrato bruto seco apresentou valores de ORAC (11.16 mmol TE per g) e TP (496.5 mg GAE per g) muito altos. Os compostos identificados foram responsáveis, respectivamente, por 9.53 % e 12.10 % dos valores ORAC e de TP do extrato de folhas de Inga edulis.


Introduction
Inga edulis Mart.(Leguminosae) is a tree widespread in the tropical secondary forest of the Amazonian Region and is known by the population for its sweet fruits.I. edulis leaves have been used in the folk medicine as antiinflammatory and anti-diarrheic products. 13][4][5] They can be divided into various classes on the basis of their molecular structure. 2,6 PLC coupled with a UV-Vis diode array detector (DAD) has been a method of choice for the separation and quantification of polyphenols. 7,8However, the complete analysis of the absolute structure and configuration of a polyphenol requires techniques, such as 1 H and 13 C NMR spectroscopy, mass spectrometry or X-ray diffraction. 2everal phenolic compounds have already been identified in different species of the genus Inga.Gallic and ellagic acids were isolated from the leaves of I. verna 9 and three flavonoids (5,7,3´,4´-tetrahydroxy-3-methoxyflavone; 6,3´,4´-trihydroxyaurone and 5,7,4´-trihydroxy-6,8-dimethylflavonone) were isolated from the roots of I. edulis. 10Methanol-water extracts from Inga leaves have shown particularly high values, of both antioxidant capacity and polyphenolic content. 1,9 veral methods can be used to measure the antioxidant capacity of plant polyphenolic extracts.Among these, the Oxygen Radical Antioxidant Capacity (ORAC) assay is considered as a reference method. 11It measures the antioxidant inhibition of peroxyl radical induced oxidations by H atom transfer and thus reflects the classical radical chain breaking antioxidant activity. 11,12he Folin-Ciocalteu assay, which is widely used to quantify the Total Phenolic (TP) content, can be considered as another antioxidant method since the mechanism involved is an electron transfer aiming at neutralizing an oxidant species. 11he aim of this work was to fractionate the methanolwater extract of I. edulis leaves, in order to identify some of its major polyphenolic compounds and to evaluate their contribution to the antioxidant activity of the extract by using both the ORAC and the Folin-Ciocalteu assays.

Results and Discussion
Solid phase extraction (SPE) cartridge fractionation of the crude extract allowed separating four fractions.Table 1 presents the distribution of the antioxidant activity (ORAC and Folin-Ciocalteu values) among the four obtained fractions.Fractions 2 (eluted with 20% MeOH) and 3 (eluted with 60% MeOH) are clearly enriched in antioxidants, since the crude extract presented a content of 496.5 mg GAE per g of dry extract, while the fractions 2 and 3 showed 753.3 and 721.9 mg GAE per g of dry fraction, respectively.The particularly high values obtained for the crude extract in terms of both ORAC (11.16 ± 1.03 mmol Trolox Equivalent per g dry crude extract -mmol TE per g DCE) and TP assays (496.5 ± 75.3 mg gallic acid equivalents -GAE per g DCE) confirm the literature data for the Inga species. 1,9 he results obtained with the two methods to evaluate the antioxidant capacity were highly correlated, when expressed per gram of dry fraction (DF).Such correlations between the TP and ORAC assays have already been observed for many other plant extracts. 1,11Interestingly enough, the antioxidant capacity (TP and ORAC) of fractions 2 and 3 were similar when expressed per gram of DF (Table 1), indicating that the compounds present in these fractions contribute in a similar manner to the total antioxidant capacity of the extract.
Figure 1A presents a typical HPLC chromatogram of the crude methanol-water extract of I. edulis leaves.Detection was monitored at a range from 200 to 600 nm, but the selected chromatogram was recorded at 270 nm (all phenolics) and 370 nm (flavonols). 7,8 igure 1A shows that all flavonols have retention time higher than 19 min, whereas almost all other phenolics elute earlier.Figures 1B and 1C show the HPLC chromatograms corresponding to the fractions 2 and 3 and reveal that fraction 3 contains the flavonolic compounds and the epicatechin, whereas fraction 2 is almost totally free of flavonols.Semi-preparative HPLC of fractions 2 and 3 led to the isolation of the two major flavonoids (retention times (RT) of 16.6 and 22.2 min in the analytical HPLC, Figure 1A).4][15][16] Additional polyphenols were also identified, either by comparison of retention time and UV-Vis spectra with pure standards and co-elution HPLC tests after mixing of standards with the crude extract.These compounds are gallic acid (1) (RT of 5.4 min in Figure 1A), catechin (2) (RT of 13.8 min), quercetin-3-O-α-L-glucopyranoside (5) (RT of 23.2 min) and quercetin-3-O-α-L-rhamnopyranoside (6) (RT of 25.2 min).All identified compounds (Figure 2) were quantified in the DCE and their concentrations are given in Table 2 with their respective ORAC and TP values.Among these compounds, epicatechin, myricetin-3-O-α-L-rhamnopyranoside and catechin are the major phenolics and they presented, respectively, 25.43 ± 0.14, 16.51 ± 0.20 and 9.67 ± 0.80 mg per g DCE.ORAC values of myricetin-3-rhamnopyranoside and quercetin-3glucopyranoside were presented for the first time and the ORAC values of the other compounds are in accordance with literature data. 17,18 he theoretical contributions of these polyphenols to the total antioxidant capacity (ORAC or TP value) of the DCE were calculated on the basis of their concentration determined by HPLC analysis (Table 2).The sum of their theoretical contribution resulted in 1.06 mmol TE per g DCE for ORAC assay and 60.09 mg GAE per g DCE for TP content, which correspond, respectively, to 9.53 % and 12.10 % of the value measured in the crude extract (Table 1).A standard mixture of these identified polyphenols was prepared in the same proportion as in the DCE.This solution presented 1.53 mmol TE per g DCE for ORAC assay (13.7 %) and 55.10 mg GAE per g DCE for TP content (11.09 %).
In conclusion, the results confirmed the high antioxidant capacity of methanol-water extract from Inga edulis leaves.The additive contribution of the six compounds identified to the total antioxidant capacity represented more than 10 % of the total phenolics and ORAC value.Further studies are thus needed to complete the chemical characterization of the phenolics present in I. edulis leaves which contributes to the antioxidant activity.The potential role of the phenolic from I. edulis leaves in the anti-inflammatory properties and other biological activities may be the subject of other investigations.

Plant material
Inga edulis Mart was taxonomically identified by a pharmacognosy expert of the Botanical Museum "Emilio Goeldi" (Belem, State of Para, Brazil) comparing with a voucher specimen deposited in this herbarium (MG 0153192).The leaves of the plant to be studied were collected in Belem city.The leaf samples were carried to the laboratory directly after harvest and were cut in small pieces (49.9 ± 0.09 % of dry matter (DM)).They were then lyophilised for 48 h, milled until reaching a granulometry below 1 mm (94.5 ± 0.15 % of DM), and stored at -20 °C under N 2 atmosphere.

Extraction of the phenolic compounds
Lyophilised leaf samples were diluted in a methanolwater solution (50:50, v:v) in proportion of 1 g per 20 mL.The mixtures were agitated and then left for 1 hour in the dark at room temperature.The suspensions were filtered through a Whatman n o 1 paper under vacuum and concentrated in a rotary evaporator in order to eliminate the organic solvent.The aqueous solutions obtained were then cooled and lyophilised for 48 h (97.0 ± 0.35 % of DM).The dry crude extracts (DCE) were stored at -20 °C under N 2 atmosphere until further processing or analysis.1.5 mg DCE were dissolved in one milliliter acidified water (1 % formic acid) and poured on a SPE C 18 column cartridge (Strata 55 µm, 70 A, 100 mg per mL, Phenomenex, Torrance, CA).The polyphenolic compounds were eluted using increasing proportions of methanol in acidified water (1 % formic acid).Four fractions were separated.Fractions 1, 2, 3 and 4 were eluted with 0 % (0.26 mg obtained from 1.5 mg DCE), 20 % (0.39 mg), 60 % (0.77 mg) and 100 % (0.01 mg) methanol, respectively.Fractions 2 and 3 were further used for the isolation of specific phenolic compounds.The procedure was done in triplicate, and these results were expressed as a mean of this triplicate.Cartridges of 5g per 20 mL had been used for attainment of bigger amount for isolation in semipreparative HPLC.All chemical solvents used were HPLC grade purchased from Merck (Darmstadt, Germany) and the polyphenol standards were from Sigma Chemical Co (St. Louis, MO) and Extrasynthese (Genay, France).

Analytical HPLC
The HPLC system employed was a Shimadzu system series LC-10Avp (Tokyo, Japan) equipped with a Class VP chromathography data station software, autosampler SIL-10AF, column oven CTO-10AS, and diode array detection system SPD-M20A to monitor all wavelengths in the range from 200 to 600 nm, with a scan rate of 1 nm.For the column, Gemini C 18 (5 µm, 250 × 4.6 mm i.d.) joined with a Gemini C 18 guard column (3.0 × 4.0 mm i.d.) (Phenomenex, Torrance, CA), was used at 30 °C.Gradient elution was performed with 1 % formic acid in ultra-pure water (solvent A) and acetonitrile (solvent B), delivered at a flow rate of 1.0 mL per min as follows: 8-35% B in 35 min, 35-100% B in 5 min and 100% B during 5 min.The gradient elution was followed by a 10 min post-run at initial conditions for equilibration of the column.All solvents were filtered through a 0.45 µm membrane filter prior to analysis.The injection volume for the extract was 20 µL.

Semi-preparative HPLC and identification
Phase reverse semi-preparative HPLC was conducted on a Varian HPLC/UV-Vis system (Las Vegas, NV) fitted with a Gemini C 18 column (5 µm, 250 × 10 mm).In order to have enough mass to the RMN experiments (minimum of 10 mg of each compound), a repetitive chromatographic procedure was applied, as follows: thirty mg of fraction 2 or 3 diluted in 500 µL of an acetonitrile:acidified water (1% formic acid) (80:20, v:v) solution were injected and the elution process was performed in an isocratic way with the injection solution at a flow rate of 4.7 mL per min.In case of co-elution, the collected peaks were evaporated to dryness, redissolved in 500 µL of injection mixture containing 15% acetonitrile instead of 20%, and submitted to a second isocratic HPLC run with 15% acetonitrile.The isolated compounds were evaporated to dryness and respective mass were dissolved in 0.6 mL of methanol-d 4 .The 1 H and 13 C NMR spectra were obtained using a Varian Mercury 300 NMR spectrometer (Las Vegas, NV) recorded at 300 and 75 MHz using the own solvent as internal standard.
Activity of Several Flavonoids of Inga Edulis Leaves J. Braz.Chem.Soc.

Table 2 .
Concentration of polyphenols identified in the dry crude extract (DCE) of Inga edulis leaves and ORAC and TP values from standard compounds and theoretical contribution to DCE