Abstracts

SHORT REPORT

Others flavonoids from Ouratea hexasperma (Ochnaceae)

Juliana F. de S. Daniel^I; Mário Geraldo de Carvalho^I,* * e-mail: mgeraldo@ufrrj.br FAPESP helped in meeting the publication costs of this article. ; Renata da S. Cardoso^I; Maria de F. Agra^II; Marcos N. Eberlin^III

^IDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, Br 465 Km. 7, 23851-970 Seropédica - RJ, Brazil

^IICentro de Ciências da Saúde, LTF, Universidade Federal da Paraíba, 58051-970 João Pessoa -PB, Brazil

^IIIInstituto de Química, Universidade Estadual de Campinas, 13083-970 Campinas - SP, Brazil

ABSTRACT

Chromatographic fractionation of the extracts from the leaves of Ouratea hexasperma (Ochnaceae) afforded a new flavone dimer 5-hydroxy-7-methoxyflavone-(4'-O-8")-5",4"' -dihydroxy-7"-methoxyflavone besides known agathisflavone, 7"-methylagathisflavone, epicatechin, 6-C-glycopyranosyl-luteolin and 3-O-glycopyranosyl-quercetin. The IR, NMR and mass spectra data analyses were used to establish the structures of these compounds.

Keywords:Ouratea hexasperma, Ochnaceae, flavone dimer, agathisflavone, lanaraflavone

RESUMO

O fracionamento cromatográfico dos extratos das folhas de Ouratea hexasperma (Ochnaceae) forneceu, além do novo biflavonóide 5-hidróxi-7-metoxiflavona-(4'-O-8")-5",4"'-diidróxi-7" -metoxiflavona, a agathisflavona, 7"-metilagathisflavona, epicatequina, 6-C-glicopiranosil-luteolina e a 3-O-glicopiranosil-quercetina já registradas na literatura. As estruturas desses compostos foram estabelecidas através da análise dos dados espectrométricos de IV, EM e RMN.

Introduction

The genus Ouratea (Ochnaceae) comprises 300 tropical species occurring mainly in South America,¹ and has been reported to be used in folk medicine for the treatment of rheumatic and gastric distress. In previous reports we described the phytochemical study of species from the Ochnaceae including Luxemburgia^2-4 and Ouratea^5-8 genera. We have also isolated biisoflavonoids⁹ and a flavone dimer from Ouratea hexasperma collected in Amazon cerrado.¹⁰ In further investigations, we have also detected cytotoxic and antitumour activities^11,12 and DNA topoisomerase inhibition¹² by biflavonoids from those species. Besides the biflavonoids as phytochemical constituents, it was found other farmacological activities of Ouratea species.^13-15 The present paper describes the isolation of a new biflavonoid, its acetyl and pentamethyl derivatives, besides the first identification of known flavonoids, agathisflavone, epicatechin, 6-C-glycopyranosyl-luteolin and 3-O-glycopyranosyl-quercetin in O. hexasperma. The significant amount of 7"-methylagathisflavone from the leaves of Ouratea hexasperma collected in Amazon cerrado¹⁰ was confirmed in this species of Mata Atlântica. The spectral data, including 1D and 2D NMR experiments of the natural substances and of the derivatives were used to establish the structures and the unambiguous ¹H and ¹³C NMR assignments.

Experimental

General procedures

Melting points are uncorrected. IR spectra were recorded on a Perkin-Elmer 1605 FT-IT spectrophotometer using KBr discs or NaCl film. ¹H (200.0 MHz) and ¹³C (50.3 MHz) NMR spectra recorded on a Bruker AC 200 spectrometer using D₃CSOCD₃, D₃CCOCD₃ or CDCl₃ and TMS as internal standard. HRESIMS were obtained with a VG 7070E-HF spectrometer using (methanol:H₂O, Ar as CAD), CE 20 eV for MS and 45 eV for MS-MS) in negative mode for 1 and (methanol:H₂O) + formic acid in positive mode for 1a. HPLC analysis was performed using a LC-6AD Shimadzu isocratic pump. Analyses were carried out in 5 mm Betasil C₁₈ preparative column 250 mm x 20 mm, using a mixture of methanol/water (6:4) as mobile phase, the eluted fraction at 3 mL min^-1 (1.0 mL injection volume) yielded a glycoside mixture. Column chromatography with silica gel (Merck and Aldrich 0.05-0.20 mm); TLC: silica gel H and G (Merck and Aldrich) were used to analyze the fractions collected from column chromatography (CC) with visualization by UV (254 and 366 nm), AlCl₃-EtOH (1%) or exposure to iodine vapor.

Plant material

The leaves of Ouratea hexasperma St.-Hil (Ochnaceae) were collected in João Pessoa, Paraíba State, Brazil, in October 2002. Voucher specimen (Nº JPB-21438) is deposited at the Herbarium Prof. Lauro Pires Xavier Universidade Federal da Paraíba, João Pessoa-PB, Brazil.

Extraction and isolation

Air dried leaves of O. hexasperma (585.0 g) were extracted exhaustively with CH₂Cl₂ and MeOH at room temperature. The solvents were removed under vacuum to yield residues LD (14.52 g) and LM (133.0 g). The LD residue (13.5 g) was filtered on a silica gel column using CH₂Cl₂, EtOAc and MeOH. The fraction eluted with CH₂Cl₂ (LDD, 7.5 g) was analyzed by TLC, IR and ¹H NMR and a mixture of aliphatic alcohols was identified. The fraction eluted with EtOAc (LDA, 4.0 g) was crystallized from MeOH to afford 7,7^"-O-dimethyllanaraflavone (1, mp 325-328 ºC, 98.2 mg). The methanolic extract (LM, 133.8 g) was partitioned with hexane:diethyl ether 1:1, EtOAc and MeOH. The hexane-ether residue (LMH, 4.53 g) was dissolved in acetone and filtered on a sephadex LH-20 column, eluted with MeOH, to give 7^'-O-methylagathisflavone (2, 217.0 mg), agathisflavone (3, 13.3 mg) and epicatechin (30.0 mg). The EtOAc residue (LMA, 52.2 g) was chromatographed on a silica gel column, using mixtures of chloroform and methanol, increasing the polarity to 100% methanol, to give 90 fractions. Fractions 28-31 yielded 7'-O-methylagathisflavone (2, 1.2 g). Fraction 53-70 (400.0 mg) were chromatographed on a silica gel column and eluted on a C₁₈ HPLC preparative column (using a ShimadzuLC-6AD isocratic pump) to yield a mixture of 6-C-glycopyranosyl-luteolin and 3-O-glycopyranosyl-quercetin (30.0 mg).

7,7"-O-dimethyllanaraflavone, (1), C₃₂H₂₂O₁₀

Yellow crystals, mp 325-328ºC (methanol). HRESIMS/MS: 693.1554 [M^+• + 1, 70% (C₃₈H₂₂O₁₃ + H⁺)], 443 (55), 401 (40), 361 (80) and 301 (95), (Scheme 1). ¹H and ¹³C NMR: (Table 1).

7,7"-O-dimethyl-5,5",4"-triacetyllanaraflavone, (1a)

7,7^"-O-dimethyllanaroflavone (1, 33.0 mg) was dissolved in pyridine (2.0 mL) and Ac₂O (2 mL). the solution was allowed to stand for 24h at room temperature. The usual work-up afforded a residue which was crystallized from MeOH to give the triacetyl derivative 1a (mp 125-130ºC, 20.6 mg). ¹H and ¹³C NMR: (Table 1).

Lanaraflavone-pentamethyl ether, (1b)

Compound 1 (20.0 mg), Me₂SO₄ (0.19 mL), dry Me₂CO (2.0 mL) and K₂CO₃ (0.2 g) were heated under reflux for 2 h. After removal of the inorganic salts with H₂O, the solution was dried over Na₂SO₄ and concentrated to dryness. The crude residue was subjected to silica gel column, eluting with CHCl₃ to give the penta-O-methyl derivative 1b (mp 265 ºC, 12.0 mg). H¹ NMR data:¹⁶, ¹³C NMR: (Table 1).

Results and Discussion

7"-O-methylagathisflavone (2), agathisflavone (3), epicatechin and the mixture of 6-C-glycopyranosyl- luteolin and 3-O-glycopyranosyl-quercetin were identified by ¹H and ¹³C NMR spectral analysis of the natural substances and comparison of the data with values described in the literature.^10,17-19 The ¹H - {¹H}-noe spectra resulting from irradiation of H-3, 3", 6" (in 2, 2a and 3) showed nOe of H-2',6', 2'",6'", HO-5" and H₃CO-7", in the respective structures; from irradiation of the methoxy groups (in 2, 2a) were detected NOE in the signals of H-6" (2 and 2a), H-3',5' and H-3'",5'" (2a). This information and the absence of NOE in the signals of H₃CO- and of HO-5 in the spectra from irradiation of H-8 (in 2) confirmed the structures of these biflavonoids.

The IR spectrum of 1 exhibited absorption bands at max 3420 cm^-1 (OH), 1658 cm^-1 (conjugated carbonyl) and 1600, 1500 and 1440 cm^-1 (aromatic ring). The peak at m/z 566 (2%, M^•+) observed in the LREIMS and the ¹H and ¹³C (BBD and DEPT) NMR spectral analysis was used to make the expanded molecular formula (C=O)₂ C₁₅(CH)₁₃(OMe)₂(OH) ₃O₃ = C₃₂H₂₂O₁₀. This formula is consistent with a biflavonyl ether skeleton with one free hydroxyl group (d_H 10.39), two H-bonded hydroxy groups (d_H 12.83 and 12.88), two methoxy groups (d_H 3.83 and 3.86, s, 3H), four doublets at d_H 8.06, 7.59, 7.17 and 6.74, corresponding to two AA'BB' systems in two para-substituted aromatic rings, three singlets at d_H 6.94, 6.85 and 6.73 and two doublets (2.0 Hz) at 6.35 and 6.76. The d_CH at 104.8, 104.2, 98.1, 96.4 and 92.8 are compatible with C-3, -3', -6, -8 and -6"of 1. Comparison of the carbon chemical shifts of 1 with those of 7-methyllanaraflavone registered in the literature⁸ revealed the shielding of the methine carbons CH-6 (98.1), CH-8 (92.8), CH-6" (96.4) through a g effect of the carbon atom in the O-methyl group at C-7 and C-7". The absence of additional signal of CH near 93.0 ppm indicated that C-8 is involved in the connection of the flavonoid moieties. The ¹H{¹H}-nOe difference spectra of 1 with irradiation at 3.86 showed nOe at d_H 6.73(s) and irradiation at 3.83 showed NOE at 6.76(d, 2 Hz) and 6.35 (d, 2.0 Hz) (Table 2). The LRESIMS-MS (negative mode) of ion with m/z 565 (40%) yields peaks at 550(60%), 283(100%) and 255(50%) that are in agreement with dimethoxylanaraflavone. These information along with the absence of a nOe on the doublets for H-3',5" and 3"', 5"' confirmed the location of the methoxy group at 7 and 7" and also the 4'-O-8 connection. The homonuclear 2D ¹Hx¹H-COSY was used to make the chemical shift assignment of 1 (Table 1). The analysis of 2D ¹Hx¹H-COSY and ¹H{¹H}-nOe difference spectra (Table 2) together with the peaks at m/z (%) 693.1554 [M^+• + 1, 70% (C₃₈H₂₂O₁₃ + H⁺)], 443 (55), 401 (40), 361 (80) and 301 (95) in the HRESIMS (positive mode, Scheme-1) of the acetyl derivative (1a) corroborates the dimethoxylanaraflavone structure. Irradiation at 3.89 (H₃CO-7) yields an NOE at d_H 6.59(H-6) and 6.84 (H-8) and irradiation at 3.93 (H₃CO-7") afforded NOE at 6.78 (H-6"). The nOe observed at the chemical shifts of H-3"',5"', H-6" and H-6 with irradiation at H₃CCO (in 1a) confirmed the location of three hydroxy groups at 4"', 5" and 5 in the natural biflavone 1. Additional Noe observed at the doublet of H-2',6'and of H-2'",6'" by irradiation at H-3 and H-3" (Table 2) and the cross peaks in the ¹Hx¹H-COSY of 1a were used to make the complete hydrogen chemical shifts assignments (Table 1). The LRESIMS-MS (positive mode) spectra of the ion with m/z 693 allowed us to confirm the three acetyl group with peaks at m/z (%): 651 (100), 609 (90) and 567 (10). The same analysis was made with ions (set of species: fragment of 1a, formic acid, methanol and water) at m/z 443, 401, 361 and 301 to identify the peaks of 1a with m/z 383, 326, 283 and 255 (Scheme 1). Treatment of 1 with dimethylsulfate gave the penta-O-methyl derivative (1b, mp 265 ºC) with the same ¹H NMR chemical shifts registered in the literature¹⁶. Thus, it was possible to confirm the structure of the new lanaraflavone derivative (1) and to make the complete carbon-13 chemical shift assignment of the pentamethyl derivative 1b, which has not been previously reported (Table 1).

Acknowledgements

The authors are grateful to CNPq, CAPES and FAPERJ for scholarships and financial support.

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Received: March 2, 2004

Published on the web: March 4, 2005

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