Antioxidant capacity of extracts and isolated compounds from Stryphnodendron obovatum Benth

The extract from stem bark of Stryphnodendron obovatum Benth. was chromatographed on a Sephadex® LH-20 column, and yielded nine compounds: gallic acid (GA), p-hydroxybenzoic acid (PHB), gallocatechin (GC), epigallocatechin (EPG), 4’-O-methylgallocatechin (MGC), epigallocatechin(4b→8)-epigallocatechin (EPEP), epigallocatechin-(4b→8)-gallocatechin (EPGC), robinetinidol(4a→8)-gallocatechin (ROGC) and robinetinidol-(4b→8)-epigallocatechin (ROEP). Evaluation of the antioxidant capacity in vitro by the methods of DPPH free radical (IC50; mg/mL) and reduction of the phosphomolybdenum complex (RAC) gave the following results, respectively: crude extract 4.52 and 0.8242; ethyl-acetate fraction 4.04 and 0.9537; aqueous fraction 5.58 and 0.9275. The crude extract and ethyl-acetate fraction were shown to possess an antioxidant capacity comparable to that of vitamin C (4.93 and 1.0). The values obtained by the DPPH free-radical method for the isolated compounds were IC50 (μM): GA=8.89; PHB=10.12; GC=16.46; EPG=13.20; MGC=21.00; EPEP=6.89; EPGC=4.91; ROGC=7.78 and ROEP=6.20. Vitamin C and trolox showed 30.11 and 30.10, respectively. Dimers showed greater activity in scavenging free radicals, possibly related to the number of hydroxyls. However, compounds without a hydroxyl at position 5 of the A-ring (5-deoxy-proanthocyanidins) did not change the antioxidant activity of the DPPH free radical, as evaluated here for the first time. Among the monomers, there appeared to be a direct relationship in scavenging of free radicals because of the stereochemistry of the compounds. The presence of a methyl radical on the B-ring significantly reduced the scavenging of free radicals of gallocatechin. All compounds showed greater scavenging of radicals than vitamin C and trolox, and these two compounds showed no significant difference from each other.


INTRODUCTION
Stryphnodendron obovatum Benth. is a native species of the Brazilian cerrado (savanna), and belongs to the family Leguminosae (Cronquist, 1988).It is a perennial shrub about 3-4 meters in height.Popularly known as "barbatimão", this plant has long been used in the treatment of several diseases and particularly in cases of skin ulcerations, principally as a wound healant and antiseptic.The chemical constituents of members of the genus Stryphnodendron evaluated to date include, in the bark as well as in the leaves or beanlike seeds: sapogenins (Tursch et al., 1963(Tursch et al., , 1966)), galactomannans (Reicher et al., 1993), and tannins (Mello, Petereit, Nahrstedt, 1996a, 1996b, 1999).
The objective of the present study was to evaluate the antioxidant capacity of extracts and isolated condensed tannins from the stem bark of S. obovatum.

Antioxidant activity in vitro
The ethyl-acetate fraction, the aqueous fraction, the crude extract and the isolated compounds were analyzed by the method of scavenging of the DPPH free radical (Amarowicz et al., 2004), and the extracts were evaluated by the method of reduction of the phosphomolybdenum complex, to demonstrate the total antioxidant capacity (May, 1999).
The method of scavenging of the DPPH (2,2-diphenyl-1-picrilhydrazil) radical was read in a spectrophotometer at 517 nm, and the results were expressed as IC 50 (quantity of antioxidant necessary to reduce by 50% the initial free-radical DPPH concentration).The calculations were done by linear regression.
The results from the phosphomolybdenum-complex reduction method were expressed as RAC (relative antioxidant capacity).Absorbance was read at 695 nm.The antioxidant capacity of the extracts was expressed in relation to ascorbic acid, used as a standard, with the reference antioxidant activity considered as 1 (100%).

Statistical analysis
The results are presented as mean ± standard deviation of the mean.The experiments were performed in triplicate.The statistical analysis was done by nonparametric Anova, Tukey's method, with the program GraphPad Prism 3.0 ® .

RESULTS AND DISCUSSION
The crude extract (CE), ethyl-acetate (EtOAc) and aqueous (AQ) fractions from the stem bark of S. obovatum were evaluated for their free-radical scavenging capacity by means of the stable free-radical DPPH, and compared to the standard vitamin C (Figure 1).
The CE and EtOAc extracts of S. obovatum showed significant antioxidant capacity compared to vitamin C (P<0.005).The EtOAc fraction had a greater capacity to reduce the DPPH radical than did the CE and AQ.This probably occurred because the tannin content in this fraction [55.01±0.36%(CV=0.65%)]was higher than in the CE [36.58±0.35%(CV=0.98%]or AQ [29.54±0.23%CV=0.58%)] fractions.In certain species, such as Vitis vinifera, the extract that demonstrated the highest DPPH radical-scavenging activity, was also the richest in polyphenols (condensed tannins) (Guendez et al., 2005).In Geranium niveum (IC 50 7.3 mg) the methanolic extract showed higher antiradical activity by DPPH than dimeric (geranin A) or trimeric (geranin D) compounds (Maldonado et al., 2005), and in Prunus domestica the semipurified fraction containing proanthocyanidin oligomer (IC 50 2.1 mM) displayed more potent activity than chlorogenic acid, epicatechin and ascorbic acid (Kimura et al., 2008).These findings showed that the extracts gave better results than did monomeric, dimeric or trimeric condensed tannins.
The results of the evaluation of the total antioxidant capacity by the method of reduction of the phosphomolybdenum complex are shown in Figure 2.
Figure 2 shows the values found for the antioxidant capacity of the crude extract, the ethyl-acetate fraction, and the aqueous fraction in relation to vitamin C (200 mg mL), referenced as 1.0.Vitamin C is recognized for its antioxidant power, which is assigned the value of unity (May, 1999), and is frequently used as a reference in this test (Prietto, Pineda, Aguilar, 1999).The ethyl-acetate fraction showed the highest potential to reduce the phosphomolybdenum complex (RAC=0.9537±0.067;RSD%=7.02),followed by the aqueous fraction (RAC=0.9275±0.0498;RSD%=5.37) and the crude extract (RAC=0.8242±0.0445;RSD%=5.39).Statistical analysis indicated a significant difference between vitamin C (1.0) compared to the crude extract (P < 0.005), demonstrating that CE has no capacity to reduce the phosphomolybdenum complex.
Obviously it is not possible to compare the different antioxidant methods, but the EtOAc fraction did show a compatible antioxidant capacity in the two methods evaluated, whereas the AQ fraction differed in respect to the CE.
It is evident that the interaction of a potential antioxidant with the DPPH radical depends on its structural conformation.Certain compounds react very rapidly with the DPPH radical, reducing a number of DPPH molecules corresponding to the number of available hydroxyl groups (Antolovich et al., 2002).
The phosphomolybdenum complex method is based on the reducing potential of the hydroxyl group of the 6-hydroxychroman ring, which is shared by all flavan-3ols (condensed tannins) and flavonoids.Thus, this method employs the reduction of Mo(VI) to Mo(V) by the sample analyte (Prieto, Pineda, Aguilar, 1999).
Therefore, it is expected that the free-radical scavenging activity of flavonoids, tannins and other phenols is mostly due to their aromatic hydroxyl groups, which confer great stability on the phenolic radical as soon as it is formed, after donation of one hydrogen radical to DPPH.According to our results, one can conclude that the ethyl-acetate fraction, in both methods, showed the greatest capacity to scavenge free radicals.As can be observed, the chemical evaluation of the ethyl-acetate fraction indicated the presence of a large quantity of poly-phenolic compounds (condensed tannins, see chemical considerations below).
These data demonstrate unequivocally that dimeric condensed tannins show significantly different free-radical scavenging activity from the monomers of flavan-3-ols (gallocatechin and epigallocatechin).There was a significant difference between gallocatechin and epigallocatechin, which may be related to the stereochemistry of the compounds.Note the presence of a methoxyl group at position 4' of gallocatechin, which reduced its free-radical scavenging activity compared to the non-substituted compound.There was a significant difference between dimers with a hydroxyl group at position 5 (prodelphinidins) and those without a hydroxyl group at this position (prorobinetinidins).Similarly to the monomers, the dimers showed the same characteristic in relation to the stereochemistry.There was a statistical difference among all the dimers; however, the differences were smaller between EPEP and ROEP, and between EPEP and ROGC, whereas for all other combinations the differences were greater (data not shown).One can deduce that in substances with relative stereochemistry of type 2,3-cis, 3,4-trans and 2,3-trans, 3,4-cis in the "upper" unit, since the "lower" unit shows the 2,3-cis configuration, the statistical differences are smaller, showing some similarity in antioxidant activity; and this similarity is therefore related to the stereochemistry of the substances.There was no significant difference between vitamin C and trolox.
This confirmed the observation of van Acker et al. (1996) that compounds with groups of the pyrogallol type (trihydroxylated) possess greater activity than compounds with groups of the catechol type (dihydroxylated).
However, this is the first report to compare antioxidant activity between proanthocyanidins with or without a hydroxyl group at A ring position 5.For this evaluation there are no currently available parameters for comparison, because 5-deoxyproanthocyanidins have been isolated from only a few plant species, including: Stryphnodendron spp., Acacia mearnsii, A. mollissima and Schinopsis spp.Nahrstedt, 1996aNahrstedt, , 1996bNahrstedt, and 1999)).A similar situation was obtained with 4'-O-methylgallocatechin, which has been isolated from only Panda oleosa and Stryphnodendron spp.(Garcia et al., 1993;Mello, Petereit, Nahrstedt, 1996a).
The EtOAc fraction obtained from the CE of stem bark of Stryphnodendron obovatum Benth.was chromatographed on a Sephadex LH-20 column, and fractions 6, 7, 8 and 9 were further purified by high-speed countercurrent chromatography (HSCCC) to obtain compounds 1-5.Fraction 10 resulted after HSCCC and derivation by acetylation in compounds 6-9.The structural identification of all biflavonoid prodelphinidins and prorobinetinidins (6-9) was established by the physical properties [ 1 H NMR, circular dichroism (CD), and DCI-mass spectrometry] of the corresponding peracetate derivatives.
Compound 7a showed similarities to compound 6a, differing only in the coupling constant of the protons of the F heterocyclic ring (H-2 and H-3), with a value of J 2,3 =9.9 Hz.Thus, a relative 2,3-trans configuration for the "lower" unit was confirmed.The mass spectrum showed the same pattern of fragmentation, with the pseudo-molecular ion (positive mode) [M+18] + m/z 1133 for compounds 6a and 7a.The 4β linkage of the flavanyl substituent and hence the 4R absolute configuration of 7a was confirmed by a positive Cotton effect in the CD spectrum at 220-240 nm (Botha, Ferreira, Roux, 1978a;Barret et al., 1979;Botha et al., 1981a).These compounds were previously isolated and characterized in Stryphnodendron adstringens (Mart.)Coville (Mello, Petereit, Nahrstedt, 1996a, 1996b, 1999).
Peracetate compounds 8a and 9a showed similarities with the corresponding prodelphinidins, except for the Aring region and the analyses of the chemical shifts of the heterocyclic C-ring protons.Therefore, these compounds were identified as robinetinidol-(4a→8)-gallocatechin (2 mg; 0.0006% of the CE) and robinetinidol-(4b→8)epigallocatechin (22.5mg; 0.0068% of the CE) through NMR (1D and 2D), DCI-MS, and CD with authentic samples and in comparison with literature.The 1 H NMR data for compounds 8a and 9a are seen in Tables I and II.
The peracetate derivative (8a) of compound 8 (Figure 6) produced a [M+18] + peak at m/z 1074 in the DCI-mass spectrum.The 1 H NMR spectrum of 8a had the characteristic spin system of an all-trans dimeric prorobinetinidin, as evidenced by the coupling constants of the heterocyclic protons (J 2,3(C) =9.6 Hz; J 3,4(C) =9.8 Hz; J 2,3(F) =8.6 Hz), an aromatic AMX spin system and two two-proton singlets at d 6.97 and 6.65, indicating the presence of pyrogalloltype B-and E-rings, respectively.Determination of the "upper" 5-deoxyflavan-3-ol unit was facilitated by the long-range coupling between H-5(A) (d 6.89) and H-4(C)   The dominance of one rotamer (Fletcher et al., 1977), the chemical shift of H-3(C) (d 5.79; t, SJ=19.5 Hz), the chemical shift of the B-ring protons and the high-amplitude negative Cotton effect in the 220-240 nm region of the CD spectrum of 8a, all confirmed a (4b→8) linkage and, thus, a 4S absolute configuration.Consequently, 8 was characterized as robinetinidol-(4b→8)-gallocatechin, previously reported as a prominent metabolite from Acacia mearnsii (Drewes et al., 1966b;Botha, Ferreira, Roux, 1978b;Botha et al., 1981b;Viviers et al., 1983) and also present in S. adstringens (Mello, Petereit, Nahrstedt, 1996a, 1996b, 1999) and in S. polyphyllum Mart.(Lopes, 2003).This is the first time that this compound has been reported for S. obovatum, which gives a preliminary indication of a close chemotaxonomic relationship between the species.
The 1 H NMR spectrum of compound 9a showed the aromatic substitution pattern and also the 5-deoxyproanthocyanidin character, as evident from an AMX spin system (d 6.26-6.86), a residual D-ring proton at d 6.66 and two two-proton singlets at d 6.78 and 7.12.As in the prodelphinidin compounds, the signals were assigned to the equivalent E-and B-ring protons, respectively, with the aid of a 1 H-1 H COSY experiment with the 2-H(C) and 2-H(F) resonances as reference signals.The "upper" 5-deoxyflavan-3-ol unit was determined as for compound 8a, by the long-range coupling between H-5(A) (d 6.86) and H-4(C) (d 4.47) ('W'-coupling) from a 2D NMR ( 1 H-1 H COSY).The coupling constants of the C-ring protons (J 2,3 =7.8 Hz; J 3,4 =5.4 Hz) confirmed the 2,3-trans-3,4-cis relative configuration, whereas the 2,3-cis configuration of the "lower" unit was evident from the small coupling constants of the heterocyclic protons (J 2,3 <2.0 Hz).The chemical shift and the splitting pattern of proton 3(C) are useful for the differentiation of dimers with 2,3-trans-3,4-cis and 2,3-trans-3,4-trans configuration, only in the case of methoxyacetylated 5-deoxyproanthocyanidins (Fletcher et al., 1977).The chemical shift of the H-6(A) (d 6.44), H-8(A) (d 6.26) and H-2(F) (d 4.42), and the clear dominance of one rotamer indicate the (4→8) interflavanyl linkage.The chemical shift of the proton 3(C) between d 5.27-5.42 was similar to those of methoxyacetylated biflavonoids of corresponding configuration, and suggest a 2,3-trans-3,4cis stereochemistry.The 2R,3S,4R absolute configuration was determined from the high-amplitude positive Cotton effect in the 220-240 nm region of the CD spectrum of 9a.Compound 9 was determined as robinetinidol-(4b→8)epigallocatechin (Figure 7), which was previously isolated from S. adstringens (Mello, Petereit, Nahrstedt, 1996a, 1996b, 1999); this is the first time that the compound, has been reported for S. obovatum.