New flavonoids and other constituents from Ouratea hexasperma (Ochnaceae)

SHORT REPORT

New flavonoids and other constituents from Ouratea hexasperma (Ochnaceae)

Mário G. de Carvalho^I,*; Luciano R. Suzart^I; Lorena C. Cavatti^I; Maria Auxiliadora C. Kaplan^II

^IDepartamento de Química, Universidade Federal Rural do Rio de Janeiro, Br 465 Km 7- Seropédica, 23890-000 Rio de Janeiro-RJ, Brazil

^IINúcleo de Pesquisa em Produtos Naturais Bloco H, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Ilha do Fundão, Rio de Janeiro-RJ, Brazil

ABSTRACT

Chromatographical fractionation of the methanol extract from the branches of Ouratea hexasperma (Ochnaceae) afforded two new flavonoids, 7-O-β-D-glucopyranosyl-6-(3-methylbut-2-enyl)-5,4'-dihydroxyflavanonol (6-β,β-dimethylallylaromadendrin-7-O-β-D-glucoside) and 7-O-β-D-glucopyranosyl-6-(3-methylbut-2-enyl)-3,5,4'-trihydroxyflavone (6-β,β-dimethylallylkaempferol-7-O-β-D-glucoside) besides a mixture of sitosterol and stigmasterol,lupeol, sitosterol-3-O-β-D-glycopyranoside and 2,4-dihydroxyphenylacetic acid. The structures were established by analysis of IR, ESI-MS, and NMR spectra of the natural substances, and those of the dimethyl ether and peracetyl-derivatives of the new flavone, as well as comparison with literature values.

Keywords: Ochnaceae, Ouratea hexasperma, glucopyranosyl-prenylflavonoids, prenylflavanonol glucoside, prenylflavone glucoside, 2,4-dihydroxyphenylacetic acid

RESUMO

O estudo fitoquímico do extrato metanólico de galhos de Ouratea hexasperma (Ochnaceae) forneceu dois novos flavonóides, 7-O-β-D-glicopiranosil-6-(3-metilbut-2-enil)-5,4'-diidroxiflavanonol (6-β,β-dimetilalil-7-O-β-D-glucopiranosil-aromadendrina) e 7-O-β-D-glicopiranosil-6-(3-metilbut-2-enil)-3,5,4'-triidroxiflavona (6-β,β-dimetilalil-7-O-β-D-glucopiranosil-kaemperol) além de uma mistura de sitosterol e estigmasterol, lupeol, 3-O-β-D-glicopiranosil-sitosterol e o ácido 2,4-diidroxifenilacético. As estruturas dessas substâncias foram estabelecidas através de análise dos espectros de IV, EM-IES e RMN das substâncias naturais e dos derivados peracetílico e éter dimetílico da nova flavona, além de comparação com dados da literatura.

Introduction

Previous phytochemical and pharmacological investigations on Ouratea species (Ochnaceae) have shown the presence of terpenoids, isoflavonoids, flavonoid glycosides, and more frequently biflavones which are considered as chemical markers forthis genus.¹ DNA topoisomerase inhibition, cytotoxic and antitumoral activities of biflavonoids^2-4 have been described as well as other pharmacological activities of extracts from Ouratea species.^5-7 So far, the phytochemical study of O. hexasperma identified biisoflavanones hexaspermone A, hexaspermone B and hexaspermone C, biflavones (6 6")-bigenkanine, 7,7"-dimethyl-lanaraflavone, 7"-methyl-agathisflavone and agathisflavone, as well as the mono flavonoids 5,7,4'-trimethoxy-isoflavone, epicatechin, 6-C-glycopyranosyl-luteolin and 3-O-glycopyranosyl-quercetin.^8-11 The present paper reports the results of an additional phytochemical study of the extract from the branches of O. hexasperma, describing the identification of two new prenylflavonoid glucosides, including some known compounds.

Results and discussion

The chromatographic fractionation of the methanol extract from O. hexasperma branches afforded sitosterol and stigmasterol as an inseparable mixture, lupeol, sitosterol-3-O-β-D-glucopyranoside, 2,4-dihydroxyphenylacetic acid (3) and two new flavonoids, 7-O-β-D-glucopyranosyl-6-γ,γ-dimethylallyl-5,4'-dihydroxyflavanonol (1) and 7-O-β-D-glucopyranosyl-6-γ,γ-dimethylallyl-3,5,4'-trihydroxyflavone (2) (Figure 1).

The known sterols, β-sitosterol glucoside, its acetyl derivative, and lupeol were identified by spectral data analysis and comparison with literature values.^11,12

The flavanonol glucoside 1 was identified from its NMR and mass spectra. The aglycone moiety was deduced to be γ,γ-dimethylallylflavanonol by the presence of the fragment m/z 355.1227 (C₂₀H₁₉O₆) in the HR-EISMS (Figure 2), which was supported by the ¹H NMR spectral data as follows: two doublets at δ 5.11 and 4.55 (J 11.0 Hz) of H-2 and H-3; two doublets (J 8.0 Hz, 2H) of an AA'BB' system (H-2',6' and 3',5', ring B), and two singlets at δ 6.22 (H-8) and 12.04 (HO-5) characteristic of a flavanonol structure. The presence of γ,γ-dimethylallyl group was identified by two signals of three-protons at δ 1.75 and 1.61 (-CMe₂), multiplets at δ 3.31, 3.14 (CH₂-CH=C<), and the signal at δ 5.17 (t, 7.0 Hz, CH₂-CH=C<). The ¹³C NMR chemical shifts of the prenyl group (C-1'"-5'"), the δ_CH at 94.2 (CH-8) and δ_C at 199.7 (C-4) and 109.9 (C-6) detected in the ¹³C NMR (BBD and DEPT) and HMQC spectra (Table 1) were compatible with this group being located at C-6 of flavononol (Figure 1). According to the values of J_H-2,H-3 11Hz, δ_CH-2 83.2 and δ_CH-3 71.8, the aryl and hydroxyl substituents at C-2 and C-3 are equatorially oriented (2,3-trans), with the same stereochemistry proposed for xeractinol.¹³ The sugar moiety was identified from five additional resonances of oxymethyne carbons including a signal at δ_CH 100.2 (HC-1"), and of a CH₂ at 60.7 (H₂C- 6") (Table 1), corresponding to a O-β-D-glucopyranosyl moiety.¹⁴ The analysis of ¹³C NMR (BBD and DEPT) and HMQC spectra, and data comparison of 1 (Table 1) with those of prenylflavanonol glycosides^14,15 corroborated the structure of a 6-prenylflavanonol glycoside. The signals observed in the HMBC spectra such as ⁿJ_CH of C-7 and C-9 with H-8, C-6 with H-8, 1'" and 2'", the chemical shift of CH-8 (94.2), and the nOe signal of H-1" [ 4.92 (d, J 8.0 Hz)] detected by irradiation at 6.22 (s, H-8) using the NOEDIFF technique, assigned the location of γ,γ-dimethylallyl and O-glucopyranosyl groups at the 6 and 7 positions, respectively (Figure 1). The peaks detected in the HR-ESI mass spectra (MS) at m/z: 517.1747 (M-H, 1a), 355.1227 (M-162, 1b), 327.1268 (1c), and 219.0696 [M - 162 (C₆H₁₀O₅) – 136 (C₈H₈O₂)] (Figure 2) and peaks detected by MS/MS (see experimental) confirmed the structure of the new flavonoid (1) as 7-O-β-D-glucopyranosyl-6-γ,γ-dimethylallyl-5,4'-dihydroxyflavanonol (Table 1).

The structure of 2 was identified by NMR and mass spectra. The δ_H 8.08 (d, J 8.0 Hz), 6.95 (d, J 8.0 Hz) of an AA'BB' system (H-2',6' and 3',5', ring B), two singlets at δ_H 12.04 (HO-5) and 6.89 (H-8) in the ¹H NMR spectrum,and the chemical shifts of C-4 (176.1) and CH-8 (93.2) detected in the ¹³C NMR were in agreement with a kaempferol structure with a substituent at position 6. Further examination of the ¹H, ¹³C NMR (BBD and DEPT) and HMQC spectra, and comparison with the spectral data of 1 and of 8-γ,γ-dimethylallylkaemperol glucosides¹⁴ allowed the identification of the γ,γ-dimethylallyl (C-1'"-5'") side chain and the O-β-D-glucopyranosyl (C-1"-6") moiety (Table 1). The structure of the prenylkaempferol glycoside was supported by peaks detected in the HR-ESI mass spectra of 2 with m/z 515.1550 (M-H, 2c), 353.1049 (M-162, 2d) and 219.0696 [M–162 (C₆H₁₀O₅) –134 (C₈H₆O₂)] (Figure 2), besides peaks detected by MS/MS spectra (see Experimental). The signals of nOe at δ_H 6.89 (H-8) and δ_H 4.90 (H-1"), detected by irradiation at 4.90 (H-1"), and at 6.89 (H-8), respectively, assigned location of the allyl group at C-6, and the O-glucopyranosyl unit at C-7 in the flavone nucleous (Figure 1, Table 1). The analyses of ¹H and ¹³C NMR spectra of themethoxyl derivative 2a, and the acetyl derivative 2b (see Experimental and Table 1) were used as additional information to confirm the structure of 2. nOe signals detected in the NOEDIFF spectrum of 2a, at δ_H 5.1 (H-1"), 8.05 (H-2',6'), and 7.18 (H-3',5'δ) by irradiation at δ_H 6.90 (H-8), 3.85 (MeO-3) and at 3.87 (MeO-4'), respectively, confirmed the location of two O-methyl groups at 3 and 4', and the O-glucopyranosyl group at position 7 (see Experimental). Comparison of the above data with those of 8-γ,γ-dimethylallylkaemperol glycosides¹⁵ established the structure of 2 as the new flavonol glycoside, 6-γ,γ-dimethylallylkaempferol-7-O-β-D-glucopyranoside.

Related compounds have been previously isolated from other plant species, such as 7-O-glycopyranosyl-6-prenyl-3,5,3',4'-tetrahydroxyflavanone, the only glycoside of this type isolated from Ochnaceae (Ochna integerrima),¹⁵ 7-O-glycopyranosyl-6-(3-hydroxy-3-methyl-butyl-3,5,4'-trihydroxyflavanone isolated from Phellodendron chinese (Rutaceae),¹⁶ and the diphyllosides identified in Epimedium diphyllum (Berberidaceae).¹⁴

The structure of the dihydroxy-phenylacetic acid 3 was based on the IR ν_max /cm^-1: 3437-2160 (O-H), 1695(C=O)], and NMR spectra. Its ¹H NMR (1D and 2D) in MeO-d₄ displayed three signals of an aromatic ABC system, and a singlet at δ 3.4 (2H), whereas the ¹³C NMR (BBD and DEPT) in MeO-d₄ showed signals at δ 130.2 (CH), 104.9 (CH), 104.1(CH), and 43.7 (CH₂), besides five quaternary carbon including the carboxylic acid signal (δ_C 176.9). These data were compatible with the molecular formula C₈H₈O₄, further supported by mass spectra (m/z 168, M^+.). Comparison of the NMR data of 3 with those of 2,5-dihydroxyphenyl- and 3,4-dihydroxyphenyl-acetic acids recorded in CDCl₃ + DMSO-d₆,¹⁷ 2,4-dihydroxyphenyl acetic acid recorded in D₂O,¹⁸ as well as using spectral prediction software, led us to propose the structure of 3 as2,4-dihydroxyphenyl acetic acid, what hydrogen and ¹³Cchemical shift are found in the literature but in different solvent (D₂O).¹⁸ This compound has been previously isolated from Nigella damascena seeds (Pamiculaceae)¹⁹ and Nigella damascena seeds.²⁰

Experimental

General procedures

Melting points have not been corrected. IR spectra were recorded on a Perkin-Elmer 1605 FT-IT spectrophotometer. ¹H (200.0 MHz) and ¹³C (50.3 MHz) NMR spectra were recorded on a Brüker AC 200 spectrometer using DMSO-d₆, CD₃OD or CDCl₃ with TMS as internal standard. HRESI mass spectra were obtained with a Brüker Daltonics UltrOTOF-Q, Billerica, MA, spectrometer using (H₂O, Ar as CAD), CE 20 eV for MS and 45 eV for MS/MS in negative mode. Electron Ionization Mass Spectra (LREI-MS) of 3 was taken using a gas chromatograph coupled to a mass spectrometer (GC-MS) on a Varian Saturn 2000 using an ion trap at 70 eV. The Advanced Chemistry Development (ACD/Labs) software V8. 14 for Solaris 1994-2007 ACD/Labs was used for prediction of ¹H and ¹³C NMR chemical shifts of 3. Column chromatography with silica gel (Vetec and Aldrich 0.05-0.20 mm) and Sephadex LH-20 (Sigma, USA); silica gel F254 G (Vetec) was used for preparative TLC; aluminum backed (Sorbent) silica gel plates w/UV254 were used for analytical TLC, with visualization underUV (254 and 366 nm), withAlCl₃:EtOH (1%), Lieberman-Burchard and/or Godin reagents, or exposure to iodine vapor.

Plant material

The branches 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

The dried and powdered branches of O. hexasperma (410.0 g) were extracted with methanol at room temperature. The solvent was removed under vacuum furnishing a residue (44.0 g). The crude methanol residue (30.0 g) was filtered on silica gel with CH₂Cl₂ and EtOAc, yielding, after removal of the organic solvent, two residues named OHMC (196.0 mg) and OHMAc (15.5 g). The residue OHMC was fractionated by CC on silica gel eluted with a mixture of CH₂Cl₂:EtOAc with increasing polarity to EtOAc (100%), and the resulting fractions grouped according to their TLC profile. Fractions 2-10 yielded a hydrocarbon mixture, Fr_15-25 furnished a mixture of steroids, and Fr_27-40 yielded lupeol (300 mg). Fraction OHMAc (10.0 g) was fractionated on a silica gel column, eluted with CH₂Cl₂:MeOH with increasing polarity to 100% MeOH. Thirthy sub fractions were collected, and assembled according to their composition, as checked by TLC. The sub fractions 2-4 yielded a mixture of sitosterol and stigmasterol (25.0 mg); fractions 6-7 furnished a residue (mp 308-310 ºC, 40.0 mg) insoluble in chloroform, that was identified as sitosterol glycoside, from the formation of its acetyl derivative (Ac₂O:Pyridine). Fractions 8-9 were purified by preparative TLC using CH₂Cl₂:MeOH (8:2) to yield compound 3 (gum, 5.0 mg). Fr_11-15 was submitted to Sephadex LH-20 column employing methanol as eluent. First pure fractions were grouped and crystallized from ethyl acetate to give 1 (mp 172-173 ºC, 90.0 mg), whereas the last fractions yielded a mixture of 1 and 2. Fr_20-25 was submitted to Sephadex LH-20 CC using methanol as eluent, to furnish a mixture of 1 and 2. Last chromatographic fractions yielded 2 as anyellow solid(mp 179-180 ºC, 80.0 mg). The flavone 2 was treated with diazomethane to yield 2a (gum, 12 mg), and with Ac₂O/pyridine (1:1) to form the peracetyl derivative 2b (gum, 20 mg; Table 1).

7-O-β-D-glucopyranosyl-6-(3-methylbut-2-enyl)-5,4'-dihydroxyflavanonol (1)

mp 172-173ºC (EtOAc). ¹H NMR (200 MHz) and ¹³C (50.3 MHz) DMSO-d₆, Table 1; ESI-MS [H₂O] m/z (ion, %): 607.2093 (M–H + 5×H₂O, 10), 553.1509 (M – H + 2×H₂O, 75), 517.1747 (1a, M – H, 100), 515.1508 (M–H – H₂, 90), 355.1227 (1b, 55), 327.1268 (1c, 9) 255.2360 (219 + H₂O, 10); MS/MS: {553}: 515.1628 (45), 355.1240 (100); {517}: 517.1739 (30), 515.1623 (45), 355.1235 (100), 327.1268 (15), 219.0696 (20); {515}: 355.1235 (100), 327.1268 (20), Figure 1.

7-O-β-D-glucopyranosyl-6-(3-methylbut-2-enyl)-3,5,4'-trihydroxyflavone (2)

mp 179-180 ºC (amorphous powder); ¹H NMR (200 MHz) and ¹³C NMR (50.3 MHz) DMSO-d₆Table 1; ESI-HRMS [H₂O] m/z (ion, %): 605.1894 (M–H + 5×H₂O, 10), 551.1331 (M–H + 2×H₂O, 10), 515.1550 (2c, M – H, 100;), 399.1319 (219 + [162] + H₂O, 15), 353.1049 (2d, 2), 255.2344 (219 + H₂O, 10); MS/MS: {515}: 353.1073 (100); {353}: (219.0594, 100), Figure 1.

7-O-β-D-glucopyranosyl-6-(3-methylbut-2-enyl)-3,4'-dimethoxy-5-hydroxyflavo-ne (2a)

A methanol solution of 2 (12 mg) was treated with ethereal diazomethane, to yield the dimethyl ether 2a (12 mg, gum): ¹H NMR (200 MHz, DMSO-d₆) δ 13.0 (s, HO), 8.05 (d, J 8.0 Hz, H-2',6'), 7.18 (d, J 8.0 Hz, H-3',5'), 6.9 (s, H-8), 5.1 (d, J 9 Hz, H-1"), 5.2 (m, H-2'"), 4.1 (brs, H-6"), 3.1-3.7 (m, H-2",3",4",5" and H-1'"), 3.85 (s, MeO-3), 3.87 (s, MeO-4'), 1.8 (s, H-5'", 3H), 1.6 (s, H-4'", 3H); ¹H{¹H}-nOe by NOEDIFF experiments: H-8 (δ 6.9){H-1"(5.1)}, MeO-3 (3.85){H-2',6'(8.05)}, MeO-4' (3.87){H-3',5'(7.18)}.

Peracetyl derivative of 2 (2b)

A mass of 25 mg of 2 was dissolved in a mixture of Ac₂O:pyridine (1:1) and the solution was allowed to stand for 24 h at room temperature. The usual work-up yielded 2b (20 mg, gum): ¹H (200 MHz, CDCl₃) and ¹³C NMR (50.3 MHz, CDCl₃), Table 1.

2,4-dihydroxyphenylacetic acid ( 3 )

Gum; IR (KBr) ν_max/cm^-1): 3437-2160(OH), 2922, 1695 (C=O), 1604, 1558, 1452 and 1293; ¹H NMR (200 MHz, MeO-d₄) δ 6.85 (d, J 8.0 Hz, H-6), 6.28 (d, J 2.4 Hz, H-3), 6.20 (dd, J 8.0, 2.4 Hz, H-5) and 3.4 (s, 2H, H-7); ¹³C NMR (50.3 MHz, DMSO-d₆) δ 176.9 (C-8), 158.9 (C-4), 156.7 (C-2), 130.2 (CH-6), 115.4 (C-1), 104.1 (CH-3), 104.9 (CH-5), 43.7 (CH₂-7). LRMS: m/z (%): 168 (M^+., 2%), and 150 (M^+. – H₂O, 100%).

Acknowledgments

The authors are grateful to CNPq, FAPERJ and CAPES for scholarships and financial support, as well as to T.M.S. da Silva and M. de F. Agra for providing the plant material and N. P. Lopes of Faculdade de Farmácia-USP-Ribeirão Preto for the HR-ESIMS.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br, as PDF file.

Received: July 30, 2007

Web Release Date: August 20, 2008

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Publication Dates

• Publication in this collection
  03 Oct 2008
• Date of issue
  2008

History

• Received
  30 July 2007
• Accepted
  20 Aug 2008