The genus Caesalpinia comprises ca. 100 species, distributed widely in tropical and subtropical regions [1] . About 17 species of the genus are widespread in China, and 14 species of the genus have long been used in Chinese traditional medicine for the treatment of rheumatism and inflammatory diseases [2] . Caesalpinia ferrea Mart. is a species belonging to Leguminosae family commonly known in Brazil as “jucá” or “pau-ferro”. It occurs in Brazil from the Northeast Region to the State of Rio de Janeiro and it is widely utilized in folk medicine due to its several therapeutic properties such as anti-inflammatory, analgesic, antimicrobial and antipyretic [3] . From an ethanol extract of Caesalpinia ferrea four phenolic compounds were isolated and identified by spectral data: organic acid 1 and ester 2 (from green beans), biflavonoid 3 and phytoalexin 4 (from stem). Additionally, from this same extract, other constituents (two triterpenes, two steroids, two acid and fatty alcohol) were detected only by GC/MS. The structures of phenolic compounds were elucidated based on spectral studies, especially 1D and 2D NMR experiments.

Compound 1 was isolated as a white amorphous solid from EtOH extract of the green beans of C. ferrea by Sephadex LH-20 column (MeOH). The IR spectrum showed bands at 3600 - 2500 cm⁻¹ (broadband indicative of the OH group of carboxylic acid), 1689 cm⁻¹ (carboxyl group) and 1610, 1542 and 1448 cm⁻¹ (aromatic ring). ¹H NMR spectrum of 1 exhibited one only signal at δ_H 7.06 (s, 2 H) which indicates a tetra substituted aromatic ring containing two equivalent hydrogens. The ¹³C NMR spectrum exhibited five resonances, however the signals at δ_C 145.52 and 110.53 with high relative intensities, were assigned to two carbon atoms each. Therefore, there appeared to be seven carbons in 1. All the signals in the ¹³C NMR spectrum were in the region of sp² carbons (included those in δ_C 170.52 assigned to carbonyl carbon of a conjugated acid carboxylic), and the comparison with the ¹³C NMR DEPT spectrum showed four signals to five non-hydrogenate carbons and one signal to two methine carbons. The analysis of these spectral data (Table 1) and comparison with spectral data of literature [4] [5] identified 1 as 3,4,5-trimethoxybenzoic acid, commonly known as gallic acid (Figure 1).

Compound 2 was obtained as a yellow amorphous solid. The EtOH extract of the green beans was partitioned between hexane and EtOAc and fraction hexane was subjected to cc Si gel to yield 2. ¹H NMR of 2 exhibited one only signal at δ_H 7.45 (s, 2 H) which indicates a substituted aromatic ring containing two equivalent hydrogens as in 1. The ¹³C NMR of 2 exhibited seven sp² carbons signals, and the comparison with the DEPT ¹³C NMR spectrum showed one signal to methine (δ_C 110.25) and six signals to non-hydrogenated carbons (Table 1). The only record in the HMQC experiment on 2 correlated the carbon signal at δ_C 110.25 with the singlet of the two aromatic hydrogens at δ_H 7.45 (2 H), while the HMBC experiment correlated these two hydrogens with carbons signals at δ_C 107.61; 112.37; 139.71; 148.17 and 159.20. Thus, the structure of 2 was determined on the basis of 2D-NMR spectroscopy and by comparison with spectral data of literature [6] to be a derivative ester dimer from gallic acid, the 4,4',5,5',6,6'-hexahidroxydifenic-2,6,6'-dilactone, known as ellagic acid (Figure 1).

Compound 3 was obtained as a white amorphous solid from EtOH extract of the stem of C. ferrea by Sephadex LH-20 column (MeOH). The IR spectrum showed bands at 3100 cm⁻¹ (broad band typical of one or more OH groups), 1618 cm⁻¹ (chelated carbonyl or conjugated double bond), 1571, 1498 and 1450 cm⁻¹ (aromatic ring), 1233, 1161 cm⁻¹ (C-O/C-C bounds) and 820 cm⁻¹ (=C-H bounds). The ¹³C NMR spectrum (Table 2) showed 28 signals, all as sp² carbons, including two to carbon carbonyl atoms (δ_C 183.96 and 184.24). The comparison with the DEPT 135 NMR spectrum revealed 18 quaternary and 10 methines carbons. However, the methine carbon signals at δ_C 129.46 and 116.94 with high relative intensities, each correlated to two carbon atoms, then the spectra indicated the presence of 30 carbon atoms, consistent with the compound being a biflavonoide with molecular formula C₃₀H₁₈O₁₀ in accordance with the peaks at m/z 539 [M + H]⁺ and 537 [M – H]⁻ in the HR-TOF-MS spectra obtained using ESI ionization. In the ¹H NMR spectrum, an AA'BB' benzenoid spin system was inferred from the signals at δ_H 7.58 (d, 2 H, J = 8.7 Hz) and 6.67 (d, 2 H, J = 8.7 Hz) in accordance with the signals of methine carbons in δ_C 129.46 (2CH) and 116.94 (2CH). The ¹H NMR spectrum also exhibited two singlet of one hydrogen each at δ_H 6.61 and 6.59, characteristic of flavone units (hydrogens attached to the C-3 in the flavonoid skeleton); signals at δ_H 8.07 (1 H, d, J = 2.1 Hz), 7.89 (1 H, dd, J = 8.6, 2.1 Hz) and 7.10 (1 H, d, J = 8.6 Hz) revealed an AMX coupling system in the 3'''-4'''-bisubstituted B_I ring of 3 indicating that C-3''' was the position of linkage of the two flavonoids units [7] ; two meta-coupled hydrogens signals in A_I ring appeared at δ_H 6.17 (1 H, d, J = 1.8 Hz) and 6.29 (1 H, d, J = 1.8 Hz). Thus, the signals of the hydrogen atoms of the flavonoid unit I, were: δ_H 6.61 (s, H-3''), 6.17 (d, H-6''), 6.29 (d, H-8''), 8.07 (d, H-2'''), 7.89 (dd, H-6''') and 7.10 (d, H-5'''). Further, one hydrogen signal appeared at δ_H 6.32 (1H, s) which was attributed to the hydrogen H-6 (A_II ring) assuming that C-8 was the position of linkage of the two flavonoid units. Thus, the signals of the hydrogen atoms of the flavonoid unit II, were: δ_H 6.59 (s, H-3), 6.32 (s, H-6), 7.58 (d, H-2'/H-6') and 6.67 (d, H-3'/H-5'). All the chemical shifts of carbons connected with hydrogens were confirmed using the HSQC experiment (Table 2). The HMBC spectrum showed that H-6 (δ_H 6.32) and H-2''' (8.07) were correlated with resonances at δ_C 106.91 (C-8) and that H-5''' (7.10) was correlated with the resonance at 122.59 (C-3'''). These correlations were important to confirm that the linkage between both flavonoid units occurred by B_I and

A_II rings, and indicated 3 as a biflavonoid (Figure 1) with a C-8-C-3''' interflavonoid linkage corresponding to the amentoflavone series [7] [8] . The ¹H- and ¹³C NMR signal assignments (Table 3) were achieved by combination of ¹H-H COSY, HSQC and HMBC spectral data, and comparison with literature values [9] . To our knowledge, this is the first report of isolation of 3 from Caesalpinia ferrea.

Compound 4 was obtained as a yellow amorphous solid from EtOH extract of the stem of C. ferrea by Sephadex LH-20 column (MeOH). The IR spectrum of 4 showed intense broadband at 3204 (OH groups), 1584, 1509 and 1460 (aromatic ring), 1146, 986, 963 (C-O/C-C bounds) and 827 cm⁻¹ (=C-H bounds). Analysis of the NMR spectral data showed that 4 was composed of two substituted phenyl rings connected by one double bond. The two olefinic hydrogens [δ_H 6.79 (1 H, d, J = 16.0 Hz) and 6.95 (1 H, d, J = 16.0 Hz)] showed a ³J_HH = 16.0 Hz indicating a trans-configuration. Further, examination of the ¹H NMR spectrum indicated the presence of one symmetrically trisubstituted aromatic ring [δ_H 6.15 (1 H, t, J = 2.1 Hz) and 6.44 (2 H, d, J = 2.1 Hz)], and one 1,4-disubstituted aromatic ring [δ_H 6.76 (2 H, d, J = 8.6 Hz) and 7.34 [2 H, d, J = 8.6 Hz)]. The ¹³C NMR spectrum displayed ten signals, six methine and four quaternary carbon atoms; chemical shift suggested that tree carbons were oxygenated (δ_C 159.80 (2C) and 158.51 (C)), and thus consistent with the structure of trans-3,5,4'- trihydroxyestilbene (4), as supported by the HMBC spectrum through the correlations of H-2/H-6 (δ_H 6.44) with C-3/C-5 (δ_C 159.80), H-2'/H-6' (δ_H 7.34) and H-3'/H-5' (δ_H 6.76) with C-4' (δ_C 158.51). Table 3 gives the ¹H and ¹³C NMR chemical shift assignments of 4 and have been confirmed by DEPT, ¹H-¹H COSY, HSQC and HMBC experiments. In addition, spectral data of compound 4 were compared with spectral data of literature [10] . To our knowledge, this is the first report of isolation of 4, known as resveratrol (Figure 1), from Caesalpinia ferrea.

This work demonstrated a practical application of spectroscopic techniques in the identification of natural products. Thus, using high-resolution mass spectrometry, and especially the two-dimensional NMR spectroscopy, two previously unpublished components (3 and 4) were characterized from the species Caesalpinia ferrea, which are of potential importance to human health as antioxidant and also in food.

The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) e Fundação Cearense de Apoio ao Desenvol- vimento Científico e Tecnológico (FUNCAP) for the scholarship and for financial support.