Diploflavone, a new flavonoid from Diplotropis ferruginea Benth. (Fabaceae)

SHORT REPORT

Diploflavone, a new flavonoid from Diplotropis ferruginea Benth. (Fabaceae)

Jackson Roberto G. S. Almeida^{I, II}; José Maria Barbosa-Filho^{*, I}; Analúcia G. S. Cabral^I; Maria de Fátima Agra^I; Emidio V. Leitão da-Cunha^{I, III}; Marcelo S. da Silva^I; Silene C. do Nascimento^IV; Raimundo Braz-Filho^V

^ILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa - PB, Brazil

^IIUniversidade Federal do Vale do São Francisco, CP 252, 56306-410 Petrolina - PE, Brazil

^IIIDepartamento de Farmácia, CCBS, Universidade Estadual da Paraíba, 58100-000 Campina Grande - PB, Brazil

^IVDepartamento de Antibióticos, Universidade Federal de Pernambuco, 50740-521 Recife - PE, Brazil

^VSetor de Química de Produtos Naturais- LCQUI-CCT - Universidade Estadual do Norte Fluminense, 28015-620 Campos, Rio de Janeiro - RJ, Brazil

ABSTRACT

The chemical examination of Diplotropis ferruginea Benth. resulted in the isolation of a new 3-methoxyflavone, 3-methoxy-6-O-prenyl-6",6"-dimethylchromene-(7,8,2",3")-flavone, to which was given the trivial name diploflavone (1); as well as the known 3,6-dimethoxy-6",6"-dimethylchromene-(7,8,2",3")-flavone (2). The structure of the new compound was established by spectral analyses. Cytotoxic activity of the isolated compounds was tested against the cells NCI-H292 (lung carcinoma), HEp-2 (larynx carcinoma) and KB (oral epidermoid carcinoma). The cells HEp-2 were the most affected by the substances tested.

Keywords:Diplotropis ferruginea, Fabaceae, flavonoids, cytotoxicity

RESUMO

A análise química de Diplotropis ferruginea Benth. resultou no isolamento da 3-metoxiflavona, 3-metoxi-6-O-prenil-6",6"-dimetilcromeno-(7,8,2",3")-flavona, à qual foi dado o nome trivial de diploflavona (1), bem como da 3,6-dimetoxi-6",6"-dimetilcromeno-(7,8,2",3")-flavona (2). A estrutura do novo composto foi estabelecida por análises espectrais. A atividade citotóxica dos compostos isolados foi testada contra células NCI-H292 (carcinoma de pulmão), HEp-2 (carcinoma de laringe) e KB (carcinoma epidermóide oral). As células HEp-2 foram as mais afetadas pelas substâncias testadas.

Introduction

The Fabaceae have a cosmopolitan distribution, consisting of ca 700 genera and more than 17000 species.¹ The genus, Diplotropis consists of approximately 22 species, including, Diplotropis ferruginea Benth. Investigations of only two species have been reported in the literature: the isolation of quinolizidine alkaloids from Diplotropis martiusii,² and flavonoids from Diplotropis purpurea.³

Diplotropis ferruginea is a tree native to Northeastern Brazil, where it is popularly known as "sucupira". It is used in folk medicine for the treatment of rheumatism, arthritis and diabetes.⁴ Recently, a chemical investigation of this species resulted in the isolation of lupeol, ethyl 2-hydroxy-4-methoxy-6-propyl benzoate⁵ and of the flavonoid 3,4,5,8-tetramethoxy-6,7,2",3"-furanoflavan.⁶ Spasmolytic activity was reported for the crude EtOH extract of this plant.⁷

This paper describes the isolation of two more flavonoids, whose structures were established by spectroscopic techniques, mainly EIMS and 1D and 2D NMR.

Experimental

General experimental procedures

Melting points were determined on a REICHERT, model R3279 "Kofler" apparatus, and are uncorrected. IR spectra were obtained in KBr on a BOMEM model MB 100 spectrophotometer. ¹H and ¹³C NMR spectra were run on a Jeol Eclipse+ 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C, using CDCl₃ as solvent (approximately 10 mg of sample were dissolved in 0.5 mL of solvent and transferred into a 5 mm NMR tube) and solvent signals were used as internal reference for the chemical shifts d_H 7.26 (CHCl₃) and d_C 77.00 (CDCl₃). The one-dimensional (1D) ¹H and ¹³C NMR spectra were acquired under standard conditions (5 mm multinuclear probe). The two-dimensional (2D) experiments were acquired and processed with the Delta software provided by Jeol. Standard pulse sequences were used for all experiments. ¹H-¹H-COSY spectra were obtained with X-points 512/Y-points 256, X-resolution 11.7 Hz/Y-resolution 23.4 Hz, X-acquisition time 85.4 ms/Y-acquisition time 42.7 ms, pulse 90º, relaxation delay 1.5 s, zerofill: 4. For homonuclear 2D ¹H-¹H-NOESY experiments were used mixing time 0.5 s, X-points 512/Y-points 256, X-resolution 11. 7Hz/Y-resolution 23.4 Hz, X-acquisition time 85.4 ms/Y-acquisition time 42.7 ms, pulse 90º relaxation delay 1.5 s, zerofill: 4. Two-dimensional inverse hydrogen detected heteronuclear shift correlation ¹H-¹³C-HMQC-¹J_CH spectra were obtained with ¹J_CH = 140 Hz, X-points 1024/Y-points 128, X-resolution 5.86 Hz/Y-resolution 196 Hz, X-pulse 90º/Y-pulse 90º, X-acquisition time 0.17 s/Y-acquisition time 5.09 ms, pulse 90º, relaxation delay 2.0 s, gradient 1/3 1 ms square, zerofill: 4. Two-dimensional inverse hydrogen detected heteronuclear long-range correlation ¹H-¹³C-HMBC-ⁿJ_CH (n=2 and 3) experiments were carried out by using ⁿJ_CH = J constant 140 Hz/J long range 8 Hz, X-points 1024/Y-points 128, X-resolution 5.86 Hz/Y-resolution 196 Hz, X-pulse 90º/Y-pulse 90º, X-acquisition time 0.17 s/Y-acquisition time 5.09 ms, pulse 90º, relaxation delay 2.0 s, gradient 1/3 1 ms square, zerofill: 4. EIMS were measured at 70 eV on a GC/MS System Shimadzu QP-5050.

Plant material

The stem bark of Diplotropis ferruginea was collected in the municipality of Caraúbas, State of Rio Grande do Norte, Northeastern Brazil in May 2002. Botanic material was identified by Prof. Maria de Fátima Agra, of the Laboratório de Tecnologia Farmacêutica. A voucher specimen (AGRA & D. ALMEIDA 5559) is deposited at the Herbario Prof. Lauro Pires Xavier (JPB), of the Universidade Federal da Paraíba.

Extraction and isolation

The dried and powdered stem bark of D. ferruginea (3 kg) was exhaustively extracted with 95% EtOH at room temperature. The extract was concentrated under vacuum yielding 95 g of the crude product. This was suspended in a MeOH:H₂O (3:7 v/v) mixture and partitioned with hexane, CHCl₃ and EtOAc. The hexane fraction was then subjected to silica gel column chromatography and eluted with hexane, CHCl₃ and MeOH in an increasing polarity gradient to give 152 fractions. The fractions were monitored by TLC and classified into 25 groups. Fraction 97-102 was purified by preparative TLC over silica gel using CHCl₃:MeOH (9:1) to afford flavonoid 1 (61 mg) and the fraction 89-96 was purified in the same way using hexane:EtOAc (2:1) to afford flavonoid 2 (123 mg).

Biological assay

The cytotoxic activity assays were based on the methylazoetetrazolium (MTT) method or the 3-(4,5-dimethylazol-2-yl)-3,5-diphenyltetrazolium bromide method.⁸ For the evaluation of cytotoxity the cellular strain HEp2 (larynx carcinoma) NCIH-292 (lung carcinoma) KB (mouth carcinoma)⁹ with proven viability were used. The cells were grown in MEM- Minimal Essential Medium¹⁰ with 10% bovine fetal serum containing 1% antibiotics solution (penicillin 1000 UI mL^-1 + streptomycin 250 mg mL^-1) and 1% glutamine (200 µM). A cellular suspension of 5x10⁴ cells mL^-1 was used and distributed in plates of 96 wells. The test samples of 0.15 mL were added into each well. The plates were incubated for 72 h at 37 ºC in a humid atmosphere enriched with 5% CO₂. After incubation 15 mL MTT in phosphate buffered saline (BPS) solution at (5 mg mL^-1) was added into each well. After 2h the culture medium was removed and 100 µL of DMSO were added in each well for quantitation of blue formazan. The readings were performed with the aid of a Multskan ELX 800 cell reader (Bio-Tec Instruments – USA) at 540 nm.

3-Methoxy-6-O-prenyl-6",6"-dimethylchromene -(7,8,2",3")-flavone or diploflavone, (1)

It was obtained as amorphous powder, mp 163-165 ºC. IR (KBr) n_max/cm^-1: 3062, 2971, 2847, 1620, 1404, 1379, 1300, 1100. EI-MS: m/z (%): 418 (8, [M⁺]), 364 (19), 349 (100, [M⁺ - prenyl]), 335 (38) (Calc. for C₂₆H₂₆O₅). ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz) (Table 1).

3,6-Dimethoxy-6",6"-dimethylchromene-(7,8,2",3") -flavone, (2)

It was obtained as amorphous powder, mp 203-204 ºC. IR (KBr) n_max/cm^-1: 2995, 2844, 1615, 1402, 1382, 1300, 1100. EI-MS: m/z (%): 364 (63, [M⁺]), 349 (100, [M⁺ - CH₃]), 319 (4) (Calc. for C₂₂H₂₀O₅). ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz).

Results and Discussion

Flavonoid 1 was obtained as a colorless amorphous solid. Its molecular formula was deduced as C₂₆H₂₆O₅ (14 degrees of unsaturation), supported by the occurrence of the molecular ion at m/z 418 in the MS, in combination with ¹H and ¹³C-APT-NMR spectral data. The IR spectrum showed absorptions at 1620 cm^-1, attributed to an a-b unsaturated carbonyl group; 3062 cm^-1 attributed to unsaturated C-H and absorptions in the region 1379-1404 cm^-1, suggesting the presence of a gem-dimethyl group. ¹H NMR of 1 showed signals at d_H 8.07 (2H, br, d J = 7.7 Hz) and 7.56-7.46 (3H, m) which indicates the possibility of a mono-substituted ring B in a flavonoid. The presence of a 2,2-dimethylchromene moiety was indicated by the characteristic signals of its two vinyl hydrogens forming an AB system¹¹ at d_H 5.74 (1H, d, J = 9.9 Hz) and 6.87 (1H, d, J = 9.9 Hz) and a signal at d_H 1.54 (6H, s) attributed to the two methyl groups. A signal at d_H 1.79 (6H, s) was also observed and signals at d_H 4.68 (1H, d, J = 6.2 Hz) and 5.53 (1H, t, J = 6.2 Hz), suggesting the presence of a prenyl group in the molecule. This suggestion is confirmed by the ¹³C-APT NMR spectra which shows signals at d_C 18.25 and 25.72, for 2 methyl carbons and a methylene carbon at d_C 66.29. The chemical shift of the methylene carbon in the ¹³C NMR indicates that the prenyl group is bound to an oxygen atom. The HMBC experiment showed the location of the O-prenyl group at C-6, due to the ³J_CH correlation between the signal at d_H 4.68 (prenyl's methylene hydrogens) with the signal at d_C 146.06 (C-6). The analysis of all the spectral data for 1 led to the elucidation of its structure as 3-methoxy-6-O-prenyl-6",6"-dimethylchromene-(7,8,2",3")-flavone. This substance is described here for the first time and was given the trivial name diploflavone.

Flavonoid 2 was isolated as a colorless amorphous solid. Its molecular formula deduced as C₂₂H₂₀O₅ (13 degrees of unsaturation), was confirmed by the molecular ion at m/z 364 in the MS in combination with ¹H-NMR (1D and 2D ¹H-¹H-COSY) and ¹³C-APT-NMR spectral data. IR and ¹H and ¹³C-NMR spectra showed the similarity with substance 1. The only difference between the two substances was the absence of the prenyl moiety in 2, having a methoxy in the same position. The presence of the methoxy group was indicated by the signal at d_H 3.97 (3H, s). The substance was thus characterized as the flavonoid 3,6-dimethoxy-6",6"-dimethylchromene-(7,8,2",3")-flavone (2), previously isolated from Bowdichia virgilioides and the NMR data are in accordance with the literature. ¹²

The 2D experiments HMQC and HMBC were used to confirm the ¹H and ¹³C chemical shifts of 1 (Table 1) and 2.¹²

The cytotoxic activity of cells NCI-H292 and KB were not affected by flavonoids 1 and 2, however, cells HEp-2 were affected by diploflavone (1). At the concentration of 10 µg mL^-1 it showed an inhibition of proliferation of 41% (Table 2).

Acknowledgements

The authors are grateful to the Instituto do Milênio do Semi-Árido (IMSEAR/CNPq), CAPES and FAPERJ by financial support. Sincere thanks are also due to NAPRALERT.

Received: January 13, 2005

Published on the web: October 31, 2005

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  13 Jan 2005