Chemical Constituents from Caesalpinia férrea : Identification and 1 H and 13 C Resonance Assignment

In a phytochemical investigation of Caesalpinia ferrea (Leguminosae), four aromatic compounds (1-4) have been isolated and identified. Their structures have been assigned based on data provided by spectroscopic techniques, including 2D NMR experiments. Compounds 3 and 4 are being reported for the first time for Cesalpina ferrea.


Introduction
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.

Results and Discussion
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). 1 H 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 13 C 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 13 C NMR spectrum were in the region of sp 2 carbons (included those in δ C 170.52 assigned to carbonyl carbon of a conjugated acid carboxylic), and the comparison with the 13 C 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. 1 H 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 13 C NMR of 2 exhibited seven sp 2 carbons signals, and the comparison with the DEPT 13 C 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).

General
IR spectrum were recorded on a Perkin-Elmer model Spectrum 100 FTIR spectrophotometer using KBr disks.NMR data were performed on Bruker DPX 300 and DRX 500 spectrometers, with TMS as internal standard.Mass spectra were determined on Shimadzu QP 5050A spectrometer, and HR-ESI-MS were acquired using a Q-TOF mass spectrometer.Column chromatography (CC) was conducted using silica gel 60 (0.040 -0.0063 mm; 230 -400 mesh, Merck), and TLC was performed on precoated silica gel polyester sheets (kieselgel 60 F 254 , 020 mm, Merck).All compounds were detected by spraying with vanlin/perchloric acid/EtOH solution followed by heating at 100˚C.

Plant Material
The pods and stems of C. ferrea were collected at Acarape County, State of Ceará, Brazil.A voucher sample is deposited in the Herbarium Prisco Bezerra of the Departamento de Biologia, Universidade Federal do Ceará.

Conclusion
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.

Table 1 .
1H and 13 C spectral data for compounds 1 and 2 in CD 3 OD.

Table 2 .
1H and 13 C spectral data for compound 3 in CD 3 OD.