New Biflavonoid and Other Constituents from Luxemburgia nobilis (EICHL)

Abstracts

Chromatographic fractionation of the organic extracts from the leaves and branches of Luxemburgia nobilis (Ochnaceae) afforded sitosterol, sitosterol-3-O-betaD-glucopyranoside, friedelin, friedelinol, a mixture of triterpenes lupeol, alpha-amyrin and beta-amyrin, rutin, epicatechin, a mixture of two chalcones, 2,4,3',4'-tetrahydroxychalcone and 2,4,4'-trihydroxychalcone, two known biflavones, amentoflavone and robustaflavone along with a new biflavonoid, 5,7,4'-trihydroxyflavone-(3'-O-4'")-5",7"-dihydroxyflavanone. The structures were established from spectral data, including 2D-NMR experiments of the natural substances and of the acetyl and methyl ether derivatives of the new biflavone.

Luxemburgia nobilis; Ochnaceae; flavonoids; steroids; triterpenes

O fracionamento cromatográfico dos extratos orgânicos das folhas e galhos de Luxemburgia nobilis (Ochnaceae) forneceu o sitosterol, sitosterol-3-O-betaD-glicopiranosil, friedelina, friedelinol, a mistura dos triterpenos lupeol, alfa-amirina e beta-amirina, rutina, epicatequina, uma mistura de duas chalconas, isoliquiritigenina e 3'-hidróxiisoliquiritigenina, duas biflavonas conhecidas, amentoflavona e robustaflavona além de uma biflavona nova, 5,7,4'-triidróxiflavona-(3'-O-4"')-5",7" -diidróxiflavanona. As estruturas foram definidas através dos dados espectrométricos incluindo experimentos bidimensionais de RMN das substâncias naturais e dos derivados metilados e acetilados da biflavona nova.

Short Report

New Biflavonoid and Other Constituents from Luxemburgia nobilis (EICHL)

Márcia C. C. de Oliveira^a, Mário G. de Carvalho ^a*, Cleber J. da Silva^a and Alceni A. Werle^b

^aDepartamento de Química ¾ ICE, Universidade Federal Rural do Rio de Janeiro, 23851-970, Seropédica - RJ, Brazil

^bDepartamento de Química ¾ ICEB, Universidade Federal de Ouro Preto, 35400-000, Ouro Preto - MG, Brazil

O fracionamento cromatográfico dos extratos orgânicos das folhas e galhos de Luxemburgia nobilis (Ochnaceae) forneceu o sitosterol, sitosterol-3-O-bD-glicopiranosil, friedelina, friedelinol, a mistura dos triterpenos lupeol, a-amirina e b-amirina, rutina, epicatequina, uma mistura de duas chalconas, isoliquiritigenina e 3'-hidróxiisoliquiritigenina, duas biflavonas conhecidas, amentoflavona e robustaflavona além de uma biflavona nova, 5,7,4'-triidróxiflavona-(3'-O-4"')-5",7" -diidróxiflavanona. As estruturas foram definidas através dos dados espectrométricos incluindo experimentos bidimensionais de RMN das substâncias naturais e dos derivados metilados e acetilados da biflavona nova.

Chromatographic fractionation of the organic extracts from the leaves and branches of Luxemburgia nobilis (Ochnaceae) afforded sitosterol, sitosterol-3-O-bD-glucopyranoside, friedelin, friedelinol, a mixture of triterpenes lupeol, a-amyrin and b-amyrin, rutin, epicatechin, a mixture of two chalcones, 2,4,3',4'-tetrahydroxychalcone and 2,4,4'-trihydroxychalcone, two known biflavones, amentoflavone and robustaflavone along with a new biflavonoid, 5,7,4'-trihydroxyflavone-(3'-O-4'")-5",7"-dihydroxyflavanone. The structures were established from spectral data, including 2D-NMR experiments of the natural substances and of the acetyl and methyl ether derivatives of the new biflavone.

Keywords:Luxemburgia nobilis, Ochnaceae, flavonoids, steroids, triterpenes

Introduction

The Ochnaceae family has been characterized as a major source of biflavonoids and up to now it has been best represented by Ouratea,^1-5Ochna^6-9 and Lophira^10-12 genera. In a previous report, we described the inhibition of murine tumor growth, antiproliferative effects and activation of apoptosis on Erlich tumor cells by flavones isolated from Ouratea hexasperma¹³ and from Ouratea semisserrata.¹⁴ There is only one record of a phytochemical work on a Luxemburgia genus where we described the isolation and identification of steroids, fatty acids, betulinic acid, the diterpene epimanoyl oxid, atranorin and two new triglycerides.¹⁵

In this paper, we report the structure determination of a new biflavonoid, 2",3"-dihydroochnaflavone, two known biflavones, amentoflavone and robustaflavone, the flavonoids rutin, epicatechin, and two chalcones, along with fatty acids, sitosterol, 3-O-b-D-glucopyranosylsitosterol and five pentacyclic triterpenes isolated from the branches and leaves of L. nobilis.

Results and Discussion

The chromatographic fractionation of the methanol extract from the branches and also of the hexane, ethyl acetate and methanol extracts from the leaves of L. nobilis afforded hexadecanoic, eicosanoic and tetraeicosanoic acids, a new biflavonoid, 2",3"-dihydroochnaflavone (1); two known biflavones, amentoflavone (2) and robustaflavone (3); epicatechin (4); two chalcones, isoliquiritigenin (5) and 3'-hydroxyisoliquiritigenin (6); rutin (7); sitosterol (8); sitosterol 3-O-b-D-glucopyranoside (9); friedelin (10); friedelinol (11) and a mixture of lupeol (12), a-amyrin (13) and b-amyrin (14).

The ¹³C NMR spectrum of compound 1 shows 28 signals including two signals at d_CH 128.80 and 116.30, each representing two carbon atoms, eight sp² CH, two sp³ carbons (d_CH 78.57 and d_CH2 42.38), fourteen sp² quaternary carbons (4xC and 10xC-O) and two carbonyl groups (d_C 182.22 and 196.48). The ¹H NMR spectrum shows two signals at d 11.99 and 12.71 indicating the presence of two chelated hydroxyls, which were confirmed by the IR spectrum which exhibits a broad OH absorption band at 3495 cm^-1 and also a chelated carbonyl absorption at 1646 cm^-1. The NMR spectrum shows ten aromatic hydrogen signals including two sets of meta-coupled doublets (¹H,¹H-COSY) at d 6.11 and 6.37 (2.0 Hz) and d 5.81 and 5.82 (2.1 Hz) which belong to the H-6 and H-8 atoms of two flavonoid moieties. These data are in agreement with a flavonoid dimeric structure. The molecular formula C₃₀H₂₀O₁₀, which was obtained by HREIMS m/z [M⁺,30] 540.10565 (calc. 540.10050 for C₃₀H₂₀O₁₀) confirms the latter observation. The presence of a singlet at d 6.62 (one hydrogen) and three double doublets at d 5.39 (16.6, and 12.7 Hz), 3.11 (16.6, 12.7 Hz) and 2.66 (16.6, 6.0 Hz) led us to propose a flavone and flavanone unit for the dimer. The data above imply that carbons 6 and 8 of each unit are not involved in the interflavonoid linkage. Ring B of the flavone unit was identified by three hydrogen signals at d 7.06 (d, 8.7 Hz), 7.62 (d, 2.0 Hz) and 7.80 (dd, 8.7 and 2,0 Hz) corresponding to H-5', H-2'and H-6'of this moiety. Furthermore, the ¹H NMR spectrum also shows a set of AA'BB' doublets (J 7.8 Hz, 2H each) at d 7.36 and 6.83 which were assigned to H-2"',6"' and H-3"',5"' of the flavanone moiety, respectively. The cross peaks observed in the ¹³C,¹H-COSY-ⁿJ _CH (n = 2 and 3, HMBC) spectra of 1 show heteronuclear long-range couplings of C-1'with H-5' and of C-1"' with H-3"',5"' which confirm rings B of both flavone and flavanone, respectively. These observations and comparison of the UV absorption maxima (288 and 332 nm) and NMR data with those of the biflavonoid 2,3-dihydroochnaflavone, isolated from Ochna obtusata,⁶ revealed these to be identical compounds. The differences between the chemical shift of the AA'BB' hydrogen in 1 [d 7.36 and 6.83 (d, 7.8 Hz, 2H each)] and the values for the same set for the 2,3-dihydroochnaflavone reported in the literature⁶ [d 8.03 and 7.08 (d, 9.0 Hz, 2H)] led to propose the 2",3"-dihydroochnaflavone structure for 1. The treatment of 1 with diazomethane yielded 1a with three methoxy groups and two chelated hydroxyls. The results obtained from NOEDIFF-NMR experiments on this derivative, performed with irradiation at the methoxy groups did not reveal any signal enhancements at the doublet at d 7.62 (d, 2.0 Hz, H-2') and at 7.36 (d, 7.80 Hz, H-3"',5"') but did show nOe at the doublets at d 7.06 (H-5'), 6.11 (H-6), 6.37 (H-8) 5.81(H-6") and 5.82 (H-8"). These observations further confirm the C-3'-O-C-4'" connection between the flavone and flavanone moieties. The comparison of the ¹³C NMR spectral data of 1 with those of 2,3-dihydroochnaflavone⁶ along with the analysis of the ¹³C,¹H-COSY, ⁿJ_CH (n = 1, HMQC, Table 1; n = 2 and 3, HMBC) allowed to define the structure of 1 as the new biflavonoid 4',5,7-trihydroxyflavone-(3'-O-4"')-5",7"-dihydroxyflavanone or 2",3"-dihydroochnaflavone. The ¹H and ¹³C-NMR data of 1b were used to confirm the proposed structure.

Compounds 2, 3 and 4 were characterized as amentoflavone, epicatechin and robustaflavone, respectively, with the help of 1D and 2D ¹H and ¹³C NMRanalysis of the natural substances and comparison with literature data.^16-20

Scheme

The molecular formulas of 5 and 6 were determined to be C₁₅H₁₂O₄ and C₁₅H₁₂O₅ from the low-resolution mass spectrum, which showed peaks at m/z 256 (5) and 272 (6), in combination with the ¹H and ¹³C-NMR spectra (HBBD and DEPT). The 1D and 2D ¹H (¹H,¹H-COSY and NOESY) and ¹³C-NMR (HMQC and HMBC) spectra of the mixture of 5 and 6 were analyzed and compared with those of isoliquiritigenin (5) reported in the literature²¹. The remaining hydrogen and carbon-13 signals observed in the 1D and 2D NMR spectra along with the peak with m/z 172 in the mass spectrum were used to assign the additional structure in the mixture as the chalcone 2,4,3',4'-tetrahydroxychalcone (6) registered in the literature.²²

Compound 7 was characterized as rutin by 1D and 2D ¹H and ¹³C NMR spectral analysis of the natural substances and comparison with literature data.²⁰ The treatment of 7 with diazomethane followed by treatment with Ac₂O and pyridine yielded 7a, with three methoxyl and seven acethyl groups. The results obtained from NOEDIFF-NMR experiments on this derivative performed with irradiation at the methoxyl groups did not reveal signal enhancements of hydrogens bound to anomeric carbons but showed nOe at the doublets at d 6.19 (H-6), 6.39 (H-8), 7.52-7.55 (H-2') and 6.84 (H-5'). These observations further confirm the C-3-O-glycosyl moiety in the flavone and allowed to identify 7 as rutin.^22,23

The known natural steroid 8, its glycoside 9 and the terpenoids 10-14 were identified by analysis of their spectral data including the acetyl derivative 9a and comparison with literature values, mainly ¹³C NMR chemical shifts described for sitosterol (8),^24,25 sitosterol-3O-b-D-glycopiranoside (9)²⁶ and the mixture of lupeol, a-amyrin and b-amyrin (12-14), friedelin (10) and friedelinol (11) .^27,28

Experimental

General procedure

Mp's are uncorrected. NMRspectra in CD₃OD(1, 2 ,3) or CDCl₃ (1a) were recorded on Bruker spectrometers (200 and 500 MHz for ¹H and 50.3 and 125 MHz for ¹³C, respectively) and on a Varian Unity 400 (400 MHz for ¹H and 100 MHz for ¹³C) spectrometer using TMS as internal standard. eims: direct inlet at 70 eV on a VG auto Spec-300 spectrometer; CC: silica gel (Merck and Aldrich 0.05-0.20 mm); TLC: silica gel H or G (Merck and Aldrich) was used to analyse the fractions collected from CC with visualization by UV (254 and 366) and exposure to iodine vapor; UV: recorded in MeOH with a DMS 80 Varian spectrophotometer; IR spectra were recorded on KBr disks on a Perkin-Elmer 1420 spectrophotometer.

Plant material

Luxemburgia nobilis (Ochnaceae) was collected in morro de São Sebastião, Ouro Preto, Minas Gerais, Brazil and authenticated by botanist Jorge L. Silva. A voucher specimen (N^o 6737) is deposited at the Herbário José Badini of the Instituto de Ciências Exatas e Biológicas of the Universidade Federal de Ouro Preto, Minas Gerais state, Brazil.

Extraction and isolation

Dried and powdered leaves and branches were successively extracted by maceration using organic solvents at room temperature. The solvents were removed under vacuum to yield residues from hexane (LNLH, 2.0 g), ethyl acetate (LNLA, 17.7 g) and methanol (LNLM, 20 g) from the leaves and hexane (LNBH, 3.85 g) and methanol (LNBM, 20.0 g) from the branches of L. nobilis. The LNLH residue was fractionated on a silica gel column (A) using hexane, CH₂Cl₂ and methanol increasing the polarity to 100% methanol. The A-1/4, A-6/9 and A-31/35 fractions were crystallized and yielded hexadecanoic acid (mp 68 °C, 200.0 mg, acetone), a mixture of tetraeicosanoic and eicosanoic acids (130.0 mg, acetone) and sitosterol (8, 97.0 mg, hexane). The LNLA residue was chromatographed on a silica gel column (B) using CH₂Cl₂/MeOH increasing the polarity to 100% MeOH. The B-1/48 fractions were fractionated on a flash column of silica gel using CHCl₃ and yielded friedelin (10, mp 300 ^oC, 107.0 mg). fractions B-49/54 and B-55/64 were filtered on silica gel and sephadex columns using CHCl₃/MeOH (9:1) and afforded biflavone 2 (88.80 mg) and biflavone 1 (130.0 mg), respectively. The LNLM residue was fractionated on a silica gel column (C) using ethyl acetate increasing the polarity to 100% methanol. Fractions C-10/15 were filtered on a sephadex column and purified by preparative TLC using CHCl₃/MeOH and yielded triterpenes friedelinol (11, mp 301 ^oC, 45 mg) and friedelin (10, 53 mg); fractions C-26/30 were dissolved in methanol and after addition of CHCl₃ afforded a precipitate corresponding to the biflavone 3 (gum, 50.0 mg). fractions C-32-39 yielded 1 (mp 220 ^oC, 295.0 mg) after precipitation from acetone. The work up of residue LNBH has been previously described.¹⁵ Finally, LNBM residue was subjected to column chromatography (D) on silica gel using ethyl acetate/methanol increasing the polarity to 100% methanol. Fraction D-2 was purified with a silica gel column and preparative TLC using CHCl₃/MeOH (9:1) to yield a mixture of triterpenes lupeol (12), b-amyrin (13) and a-amyrin (14) (80.0 mg) besides epicatechin (4, oil, 30.0 mg). fractions D-8/12 were filtered on a silica gel column using CH₂Cl₂/MeOH (7:3) affording epicatechin (4, 200 mg). fractions D-18/20 yielded a residue identified as 3O-bD-glucopyranosylsitosterol (9, mp 300 ^oC, 35.0 mg). Filtration on sephadex column of fractions D-33/35 yielded two fractions which were recrystallized from EtOAc:MeOH (9:1) and further purified by preparative TLC affording the same glycoside 9 (85.00 mg) and a mixture of chalcones 5 and 6. Compound 7 (1.00 g), known as rutin, was obtained from filtration of D-36/63 with sephadex using MeOH as solvent.

4',5,7-trihydroxyflavone-(3'-O-4"')-5",7"-dihydroxy flavanone (1): mp 220 ºC (EtOAc). UV: l_max^MeOH /nm (log e): 288 (3,29), 332 (3,42) nm. [a]_D: +7.0 (Me₂CO, c 0.6), IR n_max /cm^-1: 3433, 3096, 1773, 1693, 1646, 1617, 1507, 1473, 1428, 1371, 1337, 1266, 1193, 1130, 1077, 1030, 902, 841(KBr). ¹H NMR (500 MHz, methanol-d₄) and ¹³C NMR (125 MHz, methanol-d₄), Table-1; EI-MS (70 ev), m/z (%) [M^+., 540 (13)], 389 (6), 314 (5), 286 (5), 272 (11), 212 (7), 179 (5), 166 (11), 152 (29), 137 (16), 126 (100), 110 (26), 97 (20), 81 (23), 69 (47), 57 (34); HREIMS m/z [M^+.] 540.10565 (calcd 540.10050 for C₃₀H₂₀O₁₀).

4',7-dimethoxy-5-hydroxyflavone-(3'-O-4"')-7" -methoxy-5"-hydroxyflavanone (1a),trimethyl ether of 1: Prepared by treating a methanol solution of 1 (20 mg) with ethereal diazomethane. After evaporation of the solvent, the residue was dissolved in acetone and purified by CC on silica gel. A fraction eluted with acetone yielded 1a (20 mg): mp 186 °C (AcOEt). UV: l_max^MeOH/nm (log e): 210 (3.60), 270 (3.20), 380 (3.2), 330 (3.06). IR n_max /cm^-1: 3443, 3076, 2935, 2840, 1643, 1612, 15606, 1440, 1378, 1266, 1115, 1160 893 (KBr); ¹H (400 MHz, D₃CCOCD₃); ¹³C (50.3 MHz, CDCl₃) NMR, Table-1. ¹H-NMR-NOEDIFF in CDCl₃.

Peracetyl derivative of 1 (1b): The peracetate of 1 (1b), was prepared with Ac₂O, pyridine and DMAP at room temperature for 24 h and was isolated as colorless needles from acetone: mp 230 °C; IR n_max /cm^¾1 1772, 1694, 1646 (KBr); ¹H NMR (200 MHz, CDCl₃): d 7,64 (dd, 1H, J 8.5, 2.0 Hz, H-6'), 7.45 (d, 1H, J 2.0 Hz, H-2'), 7.44 (d, 2H, J 8.8 Hz, H-2'", 6'"), 7.30 (d, 1H, J 8.5, H-5'), 7.27 (d, 1H, J 2.2 Hz, H-8), 7.05 (d, 2H, J 8.8 Hz, H-3'", 5'"), 6.82 (d, 1H, J 2.2 Hz, H-6), 6.78 (d, 1H, J 2.2 Hz, H-8"), 6.52 ( d, 1H, J 2.2 Hz, H-6"), 6.51 (s, 1H, H-3), 5.48 (dd, J 13.08, 2.8 Hz, H-2"), 3.05 (dd, 1H, J 16.7, 13.08, H-3" ax), 2.78 (dd, 1H, J 16.7, 2.8, H-3" eq), 2.20, 2.32, 2.38, 2.39 and 2.40 (s, 3H each, OAc-5,7,4',5",7"); ¹³C NMR (50 MHz, CDCl₃): d 167.90 (C-2), 108.87 (C-3), 176.17 (C-4), 155.87 (C-5), 113.85 (C-6), 160.97 (C-7), 109.11 (C-8), 153.96 (C-9), 111.50 (C-10), 133.56 (C-1'), 118.17 (C-2'), 148.80 (C-3'), 144.56 (C-4'), 124.66 (C-5'), 122.29 (C-6'), 78.97 (C-2"), 44.96 (C-3"), 188.99 (C-4"), 156.84 (C-5"), 110.55 (C-6"), 163.11 (C-7"), 109.05 (C-8"), 150.11 (C-9"), 114.50 (C-10"), 130.07 (C-1"'), 128.06 (C-2'", 6"'), 118.54 (C-3"', 5"'), 151.16 (C-4"'), 168-169,5 (O-COCH₃), 20,7-21,8 (O-COCH₃).

Acknowledgements

The authors are grateful to Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for scholarships and financial support. We thank Dr. E. R. Silveira, Universidade Federal do CE, Fortaleza, Ceará, Brazil, for [a]_D readings and NMR spectra (500 MHz for ¹H and 125 MHz for ¹³C).

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Received: November 1, 2000

Published on the web: December 12, 2001

Publication Dates

• Publication in this collection
  20 June 2002
• Date of issue
  Feb 2002

History

• Received
  01 Nov 2000