Bioactive flavone dimers from Ouratea multiflora (Ochnaceae)

Dímeros flavônicos bioativos de Ouratea multiflora (Ochnaceae)

Abstracts

O fracionamento cromatográfico do extrato orgânico das folhas de Ouratea multiflora forneceu os flavonóides diméricos, heveaflavona, 7<FONT FACE=Symbol>¢¢</FONT>,4<FONT FACE=Symbol>¢¢¢</FONT>-dimetilamentoflavona, podocarpusflavona-A e amentoflavona. Suas estruturas foram elucidadas com base nos dados espectrais, incluindo experimentos bidimensionais de RMN, das substâncias naturais. A atividade antibiótica de todos os isolados foi avaliada, usando-se as bacterias Gram-positivas Staphylococcus aureus and Bacillus subtilis. Teste de citotoxicidade nas linhagens de linfoma de ratos (L5178) e KB também foram conduzidos para avaliar os extratos e os flavonóides isolados. a triagem biológica para a avaliação de atividade antioxidante e inibidora de acetil colinesterase foram conduzidas pela técnica da bioautografia com DPPH e teste pelo teste de Ellman respectivamente.

Ochnaceae; Ouratea multiflora; biflavonoids; antibacterial activity

Chromatographic fractionation of the organic extract from leaves of Ouratea multiflora afforded the flavone dimers heveaflavone, amentoflavone-7<FONT FACE=Symbol>¢¢</FONT>,4<FONT FACE=Symbol>¢¢¢</FONT>-dimethyl eter, podocarpusflavone-A and amentoflavone. Their structures were elucidated from spectral data, including 2D-NMR experiments of the natural substances. Biological activities of all isolates were evaluated, using antimicrobial assay against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, cytotoxicity assay against mouse lymphoma (L5178) and KB cell lines, TLC screening for acetylcholinesterase inhibitors and antioxidant activity measured by DPPH test.

Ochnaceae; Ouratea multiflora; biflavonoids; antibacterial activity

ARTIGO

Bioactive flavone dimers from Ouratea multiflora (Ochnaceae)

Dímeros flavônicos bioativos de Ouratea multiflora (Ochnaceae)

Carlos Alberto Carbonezi^I; Lidilhone Hamerski^I; A. A. Leslie Gunatilaka^II; Alberto Cavalheiro^I; Ian Castro-Gamboa^I; Dulce Helena Siqueira Silva^I; Maysa Furlan^I; Maria Claudia Marx Young^III; Marcia Nasser Lopes^I; Vanderlan da Silva BolzaniI, ^*

^IInstituto de Química, Universidade Estadual Paulista, UNESP, CP 355, 14801-970, Araraquara, SP, Brazil

^IIArid Land Research Institute, University of Arizona, Tucson, AZ 85706-6800, USA

^IIISeção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, 01061-970, São Paulo, SP, Brazil

ABSTRACT

Chromatographic fractionation of the organic extract from leaves of Ouratea multiflora afforded the flavone dimers heveaflavone, amentoflavone-7¢¢,4¢¢¢-dimethyl eter, podocarpusflavone-A and amentoflavone. Their structures were elucidated from spectral data, including 2D-NMR experiments of the natural substances. Biological activities of all isolates were evaluated, using antimicrobial assay against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, cytotoxicity assay against mouse lymphoma (L5178) and KB cell lines, TLC screening for acetylcholinesterase inhibitors and antioxidant activity measured by DPPH test.

Keywords: Ochnaceae, Ouratea multiflora, biflavonoids, antibacterial activity.

RESUMO

O fracionamento cromatográfico do extrato orgânico das folhas de Ouratea multiflora forneceu os flavonóides diméricos, heveaflavona, 7¢¢,4¢¢¢-dimetilamentoflavona, podocarpusflavona-A e amentoflavona. Suas estruturas foram elucidadas com base nos dados espectrais, incluindo experimentos bidimensionais de RMN, das substâncias naturais. A atividade antibiótica de todos os isolados foi avaliada, usando-se as bacterias Gram-positivas Staphylococcus aureus and Bacillus subtilis. Teste de citotoxicidade nas linhagens de linfoma de ratos (L5178) e KB também foram conduzidos para avaliar os extratos e os flavonóides isolados. a triagem biológica para a avaliação de atividade antioxidante e inibidora de acetil colinesterase foram conduzidas pela técnica da bioautografia com DPPH e teste pelo teste de Ellman respectivamente.

Unitermos: Ochnaceae, Ouratea multiflora, biflavonoids, antibacterial activity.

INTRODUCTION

Species of the Ochnaceae family are distributed in tropical and subtropical zones throughout the World (Hegnauer, 1969; Carvalho et al., 2000) This family is characterized by the presence of flavonoids and biflavonoids and terpenoids as main secondary metabolites (Oliveira et al., 2002; Estevam et al 2005). Several members of the Ouratea genus are employed for the extraction of edible oil, and are used as medicinal plants (Moreira et al., 1994; Agra et al., 2007). As part of our research program on the bioactive constituents of Atlantic Forest plant species, we have investigated Ouratea multiflora collected in Juréia Reserve, São Paulo State. As result, four flavonoid dimers: heveaflavone (Geiger, 1986) amentoflavone-7¢¢,4¢¢¢-dimethyl-ether (Geiger, 1986), podocarpusflavone-A (Geiger, 1986; Markham et al., 1987) and amentoflavone (Geiger, 1986; Markham et al., 1987; Felício et al., 2001), were isolated from the ethanolic extract of the leaves. Additionally, the biological activity of the isolates was also evaluated.

MATERIAL AND METHODS

General procedure

¹H and ¹³C NMR, inverse heteronuclear HMQC and HMBC as well as COSY experiments were performed on a Varian Inova 500 MHz instrument operating at 500 MHz for hydrogen, and 125 MHz for carbon, respectively. Pyridine-d₅ or DMSO-d₆ were used as solvents and TMS as internal standard. TLC was performed on precoated aluminum sheets (silica F₂₅₄, 0.25 mm, Merck, Parmtadt, Germany) with detection provided by UV light (254 and 366 nm) and by spraying with anisaldeyde reagent followed by heating (120 ºC). Silica gel (230-400 mesh ASTM, particle size 0.040-0.063 µM, Merck) or Sephaex LH-20 (Pharmacia) were used in the CC fractionations.

Plant material

Ouratea multiflora was collected at the Botanical reserve of Juréia, São Paulo, Brazil and a voucher was placed at the Botanical Institute, São Paulo State, Brazil.

Extraction and isolation

Dried powdered leaves of Ouratea multiflora (118.0 g) were extracted by maceration using ethanol at room temperature. The extract was filtered and the solvent was removed under vacuum. The crude extract (3.44 g) was dissolved in n-BuOH. Subsequent addition of water afforded two phases which were evaporated to dryness under reduced pressure. The n-BuOH fraction (2.10 g) was solubilized with methanol/water (8:2 v/v) and partitioned with n-hexane, chloroform and ethyl acetate, successively. The ethyl acetate phase after concentration of the solvent (980.0 mg), was chromatographed on a Sephadex LH-20 column with a gradient elution of MeOH/H₂O. The separation was monitored by TLC, and eluted fractions exhibiting similar appearances were combined, yielding 20 fractions. Fractions 7 and 8 showed a solid precipitation. The solid was washed with cold methanol to afford heveaflavone (Geiger, 1986) (1) (21.2 mg). Fraction 13 was chromatographed on silica gel yielding amentoflavone 7", 4"'-dimethyl ether (2) (10.8 mg). Fraction 15 gave a powder after precipitation. This powder was washed with cold methanol to afford podocarpusflavone-A 3 (Markam et al., 1987) (8.9 mg). Fraction 17 was chromatographed on silica gel using CHCl₃:MeOH (4:6) yielding amentoflavone 4 (Geiger, 1986; Markham et al., 1987) (15.0 mg).

Antimicrobial assay

Sterile filter paper disks were impregnated with 20 mg of samples using DMSO as carrier solvent. The impregnated disks were then placed on agar plates previously inoculated with Staphylococcus aureus (ATCC 2592) and Bacillus subtilis (DSM 2105). Solvent controls were incubated at 37 ºC for each organism, and after incubate time of 24 h at 37 ºC, antimicrobial activity was recorded as clear zones (in mm) of inhibition surrounding the disk. The sample was considered active when the inhibition surrounding the disk was greater than 7 mm.

Cytotoxicity assay

Antiproliferative activity was examined against two cell lines and was determined through an MTT assay as described earlier (Edrada et al., 1996).

TLC screening for acetylcholinesterase inhibitors

The protocol adopted for this in vitro assay is described by Ellman and co-workers (Ellman et al., 1961).

Determination of the radical-scavenging activity

The determination of the antioxidant activity was tested according to a protocol described elsewhere (Cardoso et al., 2004; So; Lewis, 2002).

RESULTS AND DISCUSSION

The chromatographic fractionation of the ethanol extract from the leaves of O. multiflora afforded heveaflavone (1), amentoflavone-7¢¢,4¢¢¢-dimethyl-ether (2), podocarpusflavone-A (3), and amentoflavone (4).

The ¹³C NMR spectrum of compound 1 showed 31 signals, which were attributed to twelve sp² CH, including signals at d_CH 114.1 and 127.6, each representing two carbon atoms, three sp³ carbons (d_CH3 55.1, 55.5 and 55.9), sixteen sp² quaternary carbons and two carbonyl groups (d_C 181.6 and 182.2) (Table 2). The ¹H NMR spectrum showed signals for two chelated hydroxyls at d_H 13.19 and 12.93 (OH-5 and OH-5"), three aromatic methoxyl groups, a 1,3,4-trisubstituted aromatic ring (B ring) and a 1,4-disubstituted (B' ring). The ¹H NMR spectra [1D and 2D (¹H-¹H-COSY)] showed a para-substituted aromatic ring at d_H 7.55 (d, J 9.0 Hz, H-2"', H-6"'), d_H 6.82 (d, J 9.0 Hz, H-3"', H-5"'), two meta aromatic hydrogens at d_H 6.23 (d, J 2.5 Hz, H-6), 6.49 (d, J 2.5 Hz, H-8), commonly observed in a flavone nucleous. The structure of 1 was established on the basisi of HMBC spectra, which showed heteronuclear long-range couplings of quaternary carbons C-7'' (d_C 161.2), C-7 (d_C 164.8) and C-4"' (d_C 162.4) with the hydrogens of methoxyl groups at d_H 3.72 (OMe-7''), 3.77 (OMe-7), and 3.81 (OMe-4"'), respectively, and by comparison of their spectral data with those reported for heveaflavone (Geiger, 1986).

Compound 2 also showed spectral features of a flavonoid dimer derivative (Table 2). The ¹H MNR spectrum (Table 1) of 2 showed the presence of to chelated hydroxyl and at d_H 3.83 and d_H 3.75 two methoxyl groups. The ¹H-¹H-COSY spectrum of compound 2 showed a set of doublets at d_H 7.66 (d, J 9.5 Hz, H-2"', H-6"') and d_H 6.91 (d, J 9.5 Hz, H-3"', H-5"') of an AA'BB' system, besides the signals at d_H 7.98 (d, J 2.5 Hz, H-2'), 7.14 (d, J 8.5, H-5') and 8.01 (dd, J 8.5; 2.5, H-6') attributed to a 1,3,4-trisubstituted aromatic ring. Furthermore, the ¹H-¹H-COSY spectrum showed signals with a meta coupling pattern at d_H 6.17 (d, J 2.5 Hz, H-6) and 6.45 (d, J 2.5 Hz, H-8) corresponding to an A ring of a flavone. The HMBC spectrum of 2 revealed correlations of the hydrogen-bonded OH-5 (d_H 12.94) with C-5 (d_C 161.4), C-6 (d_C 98.8) and C-10 (d_C 103.7), while OH-5" (d_H 13.20) correlated with C-5" (d_C 161.4), C-6" (d_C 95.6) and C-10" (d_C 104.2). From the long range coupling of H-6 (d_H 6.17) and H-3 (d_H 6.82) with C-10 (d_C 103.7); H-3"' and H-5"' (d_H 6.91) with C-1"' (d_C 122.9); and H-3" (d_H 6.92) with C-10" (d_C 104.2) thus, the structure of 2 was established to be a dimer. A literature search confirmed the structure of 2 as 7",4"'-dimethoxy-amentoflavone (Geiger, 1986).

¹H and ¹³C NMR spectra of 3 showed signals at d_H 13.05, d_C 182.2, d_H 12.95, and d_C 181.8 of two hydrogen of chelated hydroxyl groups, and an O-methyl group at d_H 3.74 (d_C 55.5). A detailed analysis of cross peaks from the ¹H-¹H COSY spectrum showed two doublets for a mono-oxygenated para disubstituted system at d_H 7.65 (d, J 9.0 Hz, H-2"', H-6"') and 6.91 (d, J 9.0 Hz, H-3"', H-5"'), one trioxygenated tetra substituted ring with alternating oxy-substituents at d_H 6.17 (d, J 1.5 Hz, H-6) and d_H 6.45 (d, J 1.5 Hz, H-8), and one mono-oxygenated trisubstituted system at d_H 7.98 (d, J 2.5 Hz, H-2'), 7.15 (d, J 8.5 Hz, H-5') and 8.00 (dd, J 8.5; 2.5 Hz, H-6'). Additional 1D and 2D NMR data indicated a pentasubstituted aromatic ring with a 5,7-dioxygenation pattern for the A' ring of a flavone (d_H 6.41, H-6") and two singlets (d_H 6.81 (H-3), d_H 6.86 (H-3'') of both flavones units in compound 3. Analysis of HMBC spectra revealed characteristic correlations which defined the positions of aromatic rings and their substitution patterns. Correlations of H-3 (d 6.81) with C-2 (d 163.8), C-4 (d 181.7), C-10 (d 103.7), C-1' (d 121.1) and H-3" (d 6.86) with C-2" (d 163.2), C-4" (d 182.2), C-10" (d 103.7), C-1"' (d 123.0) established the linkages between rings C/B and C"/B", respectively. Moreover, the correlation between H-2' (d 7.98) and C-8" (d 104.0) showed the linkage between C-3'and C-8". The ROESY correlation observed between methoxyl group at d_H 3.74 and the aromatic protons at d_H 6.91 (d, J 9.0 Hz, H-3"', H-5"') justify the location of the methoxyl group at C-4"' of the B' aromatic ring. These data are in agreement with those published for podocarpusflavone-A (Geiger, 1986; Markham, et al., 1987).

¹H NMR spectrum of 4 showed two signals at d_H 13.09 and 12.96 due to chelated phenolic hydroxyls, and two doublets at d_H 7.56 (J 9.0 Hz, H-2"', H-6"'), and 6.70 (J 9.0 Hz, H-3"', H-5"') assigned to an AA'BB' aromatic system. Signals of a trisubstituted aromatic ring containing one oxygenated carbon were observed at d_H 8.00 (d, J 2.5 Hz, H-2'), 7.14 (d, J 9.0 Hz, H-5'), and 7.99 (dd, J 9.0; 2.5 Hz H-6'), along with doublets of a tetrasubstituted aromatic ring containing three oxygenated carbons at d_H 6.18 (J 2.5 Hz, H-6) and 6.45 (J 2.5 Hz, H-8). Analysis of the HMBC spectra evidenced correlations of the hydroxyl group with hydrogen bonded by cross peak at d_H 13.09 (OH-5) with C-5" (d 161.1), C-6" (d 98.7), and C-10" (d 103.6) and d_H 12.96 (OH-5") with C-5 (d 162.1), C-6 (d 98.9) and C-10 (d 103.7), which are in agreement to those of amentoflavone (Geiger, 1986; Markham et al., 1987).

Previous studies on ¹³C NMR data for amenthoflavone derivative dimers indicated a close relationship between substituent effects and chemical shift for the inter-flavonoid linkage: a) amenthoflavones; I-3' (+ 6 ppm), II-8'' (+ 10 ppm), b) dihydroamenthoflavone; I-3'(+ 4 ppm), II-8" (+ 9 ppm), which can be diagnostic values in the recognition of new biflavones in the amenthoflavone series (Markhan, et al., 1987; Felício, et al., 2001). The values of ¹³C NMR spectra of 1-4 (Table 2) followed logically the average found to the series reported and indicated that O-methylation induced shifts that are in the same direction as those reported for monoflavonoids (Markham, 1978).

The biflavonoids were assayed for cytotoxicity toward mouse lymphoma (L5178) and melanoma cancer cell line (KB) (Edrada et al., 1996). None of the compounds were active toward the cell line, however, they showed weak activity against the Gram-positive bacteria S. aureus and B. subitilis at the concentrations of 0.5 and 10 µg/mL (Table 3). The reference antibiotic, streptomycin sulfate, inhibited the growth of all bacterial species tested in this study at 5 µg/mL (zone inhibition = 30-35 mm). Compounds 1-4 also were submitted to preliminary TLC screening for selecting potential acetylcholinesterae (AchE) inhibitors (Ellman et al., 1961), in which none inhibited the enzyme at 0.1 and 1.0 µM concentrations. The antioxidant activities of biflavones 1-4 were tested toward DPPH radical (Table 3) (Cardoso et al. 2004). Compound 4 showed moderate scavenging activity (IC₅₀ 18.5 µg/mL) in this test (So; Lewis, 2002), while 1-3 showed weak scavenging activity which confirms the dependence of antioxidant activity with the number of free aromatic hydroxyl groups of tested compounds.

ACKNOWLEDGMENT

This work was funded by grants of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) as part of the Biota-FAPESP - The Biodiversity Virtual Institute Program (www.biotasp.org.br), grant n° 03/02176-7 awarded to V. da S. Bolzani, Principal Investigator. V. da S. B., M.F. and C.A.C acknowledge CNPq and CAPES for researcher and Ph.D. fellowships. The authors wish to thank Arid Land Research Institute University of Arizona (Tucson, USA) by complementation of the fellowship awarded to C.A.C .

Received 05/14/07. Accepted 07/28/07

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Publication Dates

• Publication in this collection
  11 Oct 2007
• Date of issue
  Sept 2007

History

• Received
  14 May 2007
• Accepted
  28 July 2007