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Megastimanes and ergostane type steroid from leaves Cratylia mollis (Leguminosae)

Abstracts

From the methanolic extract of the leaves of Cratylia mollis were obtained by chromatographic techniques (3S,5S,6S,9R)-3,6-dihydroxy-5,6-dihydro- β-ionol (1), and a new bis-norisoprenoid named (4S*, 6S*)-4-but-1E-enyl-4,6-dihydroxy-3,5,5-trimethyl-cyclohex-2-enone (2) as well as 5a,8a-epidioxyergosta-6,22-dien-3-β-ol. The structures of the pure compounds were elucidated based on MS, ¹H and 13C NMR spectroscopic data analyses.

megastimanes; Leguminosae; Cratylia mollis


Do extrato metanólico das folhas de Cratylia mollis, foram isolados através de técnicas cromatográficas, (3S,5S,6S,9R)-3,6-di-hidróxi-5,6-diidro- β-ionol (1) e um novo nor-isoprenóide identificado como (4S*, 6S*)-4-but-1E-enil-4,6-diidróxi-3,5,5-trimetil-ciclo-hex-2-enona (2) além do 5a,8a-epidioxiergosta-6,22-dien-3-β-ol. As estruturas foram elucidadas por meio da análise dos dados de EM, IV, RMN ¹H e 13C.


SHORT REPORT

Megastimanes and ergostane type steroid from leaves Cratylia mollis (Leguminosae)

Luciano S. LimaI; Marcos V. B. LimaII; Juceni P. DavidII; Ana M. GiuliettiIII; Luciano P. de QueirozIII; Jorge M. DavidI, * * e-mail: jmdavid@ufba.br

IInstituto de Química, Universidade Federal da Bahia, 40170-290 Salvador-BA, Brazil

IIFaculdade de Farmácia, Universidade Federal da Bahia, 40170-290 Salvador-BA, Brazil

IIIDepartamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, 44031-460 Feira de Santana-BA, Brazil

ABSTRACT

From the methanolic extract of the leaves of Cratylia mollis were obtained by chromatographic techniques (3S,5S,6S,9R)-3,6-dihydroxy-5,6-dihydro- β-ionol (1), and a new bis-norisoprenoid named (4S*, 6S*)-4-but-1E-enyl-4,6-dihydroxy-3,5,5-trimethyl-cyclohex-2-enone (2) as well as 5a,8a-epidioxyergosta-6,22-dien-3-β-ol. The structures of the pure compounds were elucidated based on MS, 1H and 13C NMR spectroscopic data analyses.

Keywords: megastimanes, Leguminosae, Cratylia mollis

RESUMO

Do extrato metanólico das folhas de Cratylia mollis, foram isolados através de técnicas cromatográficas, (3S,5S,6S,9R)-3,6-di-hidróxi-5,6-diidro- β-ionol (1) e um novo nor-isoprenóide identificado como (4S*, 6S*)-4-but-1E-enil-4,6-diidróxi-3,5,5-trimetil-ciclo-hex-2-enona (2) além do 5a,8a-epidioxiergosta-6,22-dien-3-β-ol. As estruturas foram elucidadas por meio da análise dos dados de EM, IV, RMN 1H e 13C.

Introduction

Cratylia is one of 670 genera belonging to Leguminosae family,1 it is included in the Phaseolae tribe (subtribe Diocleinae)2 and this position is maintained until the most recent classifications.3 This genus comprises only five species, C. argentea (Desvaux) O. Kuntze, C. bahiensis L. P. de Queiroz, C. hypargyrea Martius ex Benth., C. intermedia (Hassler) L. P. de Queiroz and C. mollis Martius ex Benth. From chemical point of view the subtribe genera can be characterized by the presence of the non-proteic aminoacids, especially canavanine.4 However, there are no data of the occurrence of this aminoacid in Cratylia species. On the other hand, other chemical characteristic of Cratylia is the presence of lectins in their seeds which shows great similarity with the lectins isolated from seeds of other species of same tribe.5

Cratilya mollis is a legume shrub native to the Northeast semi-arid region of Brazil, especially in "caatinga". This species is popularly known as "camaratuba" or "camaratu" and is highly resistant to desiccation. The leaves have been an alternative source of nutrition for cattle, being recommended to be employed by locals as forage to improve cattle's nutrition, especially during the dry seasons, contributing to regional development of the semi-arid.6 However, in spite of studies the about of the biological activities of this and related species7 to date there are no phytochemical studies regarding C. mollis.

In the present work it is described the phytochemical study of leaves of Cratylia mollis led to isolation of two bis-norisoprenoids (1 and 2) besides the 5a,8a-epidioxyergosta-6,22-dien-3-b-ol (3).

Results and Discussion

The Cl3-norisoprenoid (1) is known as 3,6-dihydroxy-5,6-dihydro-β-ionol. It was identified by analysis of ESIMS, IR, optical rotation, 1H and 13C NMR and comparison with data previously published in the literature.8 Moreover, HMQC, HMBC and COSY spectra permitted to attribute unequivocally all the NMR signals. The 13C NMR data of 1 are compatible with that previously described for (3S,5R,6S,9R)-3,6-dihydroxy-5,6-dihydro- β-ionol obtained from Apollonias barbujana (Lauraceae)8 except for C-1. However, the HMQC and HMBC of 1 permitted to attribute the peak at δ 16.4 to C-13 by the observed correlations of one doublet at δ 0.79 and this carbon in the HMQC and C-4 (δ 39.9) and C-6 (δ 78.1) observed in the HMBC experiment. This findings permitted to determinate an axial position of CH3-13 instead of equatorial as previously proposed and, concluded compound 1 is (3S,5S,6S,9R)-3,6-dihydroxy-5,6-dihydro- β-ionol. Thus, it is a must a revision of 13C NMR data and structure of the megastimane isolated from A. barbujana.

The 13C NMR spectra (including DEPT experiments) of 2 showed signals were assigned to four methyls, four methines, one methylene and four non-hydrogenated carbons. These findings besides the protoned molecular ion [M-H]+ observed at m/z 223.1330 in the negative HRESIMS permitted to propose molecular formula C13H20O3 (requires 223.1334) which was indicative of a C13-norisoprenoid derivative. The signals at d 198.5, 126.7 and 163.4 observed in the 13C NMR spectra, and the singlet at d 5.95 in the 1H NMR spectra were indicative of presence of a α ,β -unsaturated ketone group. These findings together with the additional chemical shifts indicated the structural similarity of 2 and the known megastigmane glycosides 4 and 5.9 The HMQC spectrum was elucidative once it permitted to identify the correlations of closer hydrogen signals with respective carbon resonances. However, the analysis of HMBC spectral data was conclusive to confirm the structure of compound 2. Briefly, the correlations of H-4 (δ 5.95), H-7 (δ 5.79) and the C-6 (d 78.9) and, H-10 (δ 1.22) and C-9 (δ 49.6) and C-8 (δ 135.6) were indicative the E-butenyl group is attached at C-6 (Table 1).

The relative configuration of compound 2 was determinate by phase-sensitive NOESY, once the spatial interactions of H-7 and H-2 were indicative the butenyl group and H-2 were in same face (Figure 2). The proposed relative stereochemistry was also confirmed by analysis of coupling constant of derivative 2a, obtained as main product of reduction of 2 by NaBH4/MeOH. The 1H NMR spectrum of 2a showed the peak of H-3 as a double doublet at d 3.4 (J 12.9 6.5 Hz) revealing pseudodiaxial coupling of H-3 and H-2, which allowed to confirm the relative configuration of compound 2.


The APCI-MS, 1H NMR and 13C NMR spectra of compound 3 and comparison with data previously describe in literature10 permitted to identify this steroid. However correlations observed in COSY, HMQC and HMBC experiments indicated the values of 13C NMR data previously established for C-6 and C-7 must be changed.

This is the first occurrence of the megastimane 4-but-1-enyl-4,6-dihydroxy-3,5,5-trimethyl-cyclohex-2-enone (2). Compound 1 was previously isolated from Apollonias barbujana (Lauraceae)8 but this is the first time it is being reported in Leguminosae family. Compound 3 was previously isolated from fungus Lactarium volemus, Schinopsis brasiliensis and Typha latifolia.10 However the detailed analysis of correlations observed in HMQC and HMBC experiments permitted to attribute unequivocally the C-13 NMR data for this steroid.

Experimental

General procedures

1H (300 MHz); 13C NMR and DEPT (75 MHz) experiments were carried out in a Varian mod. Gemini 2000. HMQC, NOESY and HMBC were run on a Varian INOVA 500: chemical shifts were recorded in d (ppm) from the solvent peak relative to TMS; APCI and ESIMS were obtained on Shimadzu LCMS-2010; HRESIMS was recorded on Bruker micrOTOF II, IR spectra were taken on a Varian mod. 640-IR spectrophotometer and optical rotations were measured with a Perkin Elmer polarimeter mod. 341.

Column chromatography was carried out on silica gel 60 (Akros 0.04-0.073 mm) and, silica gel TLC plates were used to monitor the chromatographic fractionment employing iodine fumes, Libermann-Bouchard spray reagent, and UV light (254/366 nm).

Plant material

Botanical material of C. mollis was collected at Jacobina, Bahia State, a region where "caatinga" vegetation is prevalent. A voucher is deposited at Herbarium of Universidade Estadual de Feira de Santana under number LP5119.

Extraction and isolation

The powdered leaves (4.1 Kg) were repeatedly extracted with MeOH at room temperature. The leaf crude extract was immediately partitioned with CHCl3/MeOH:H2O (6:4), and after the evaporation of CHCl3 under vacuum, the extract (73.2 g) obtained was partitioned with hexane/MeOH:H2O (9:1). The hydromethanolic partition phase (28.89 g) was submitted to CC using Silica gel as adsorvent and eluted with mixtures CHCl3:MeOH with gradient of polarity (95:5 → 3:2). The fractions (1.88 g) eluted with CHCl3:MeOH (9:1) were jointed and submitted to another CC on silica gel which was eluted with mixtures of CHCl3:MeOH (95:5 and 9:1). The fractions (88.8 mg) eluted with the system CHCl3:MeOH (95:5) were subjected to PTLC and developed with a mixture of CHCl3:MeOH:HOAc (90:9:1). This procedure permitted to obtain compound 1 (15.0 mg). The fractions (175 mg) eluted with CHCl3:MeOH (9:1) were rechromatographed on Sephadex LH-20 column with Hex:CH2Cl2 (2:8) as eluent to yield compound 2 (25.0 mg).

The hexane partition phase (42.9 g) was submitted to a CC on Si gel 60 with mixtures of Hex:EtOAc. The fractions (4.5 g) eluted with Hex:EtOAc (95:5) were further submitted to a flash CC on silica gel eluted with CHCl3:MeOH (98:2) affording compound 3 (7.2 mg).

(1S,4S,6S)-1-(3-Hydroxy-but-1E-enyl)-2,2,6-trimethyl-cyclohexane-1,4-diol or (3S,5S,6S,9R)-3,6-Dihydroxy-5,6-dihydro-β-ionol (1)

Colorless syrup. [α]D20 -7.0° (c 0.48, MeOH), 1H and 13C NMR: Table 1.

(4S*, 6S*)-4-But-1E-enyl-4,6-dihydroxy-3,5,5-trimethyl-cyclohex-2-enone (2)

Oil. [a]D20 + 56.0° (c 1.47, MeOH) ESIMS (m/z) 223 [M-H]-, and 255 [M-H+MeOH], IR νmax/cm -1:3200-3600 (OH), 1713 (C=O), 1656 (C=C), 1H and 13C NMR: Table 1.

Reaction of reduction of compound 2

Compound 2 (5 mg) was dissolved em MeOH (2.0 mL) and added a suspension containing NaBH4 in MeOH. The mixture was stirred at room temperature during 30 min. After the solvent was evaporated the residue was dissolved in CHCl3 and 2a (3.5 mg) was obtained.

Acknowledgments

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), IMSEAR (CNPq/MCT), FAPESB and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship support and grants.

Supplementary Information

Supplementary information for compounds 1-3 is available free of charge at http://jbcs.sbq.org.br, as a PDF file.

References

1. Joly, A. B.; Botânica- Introdução à Taxonomia Vegetal, Cia Editora Nacional: São Paulo, Brasil, 1998.

2. Bentham, G.; Commentationes de Leguminosarum Generibus, Sollingeri, J. B., ed., Vienna, 1837.

3. Hutchinson, J.; The Genera of Flowering Plants, vol. 1, Claredon Press: Oxford, 1964; Baudet, J. C.; Bull. Jard. Bot. Nat. Belg. 1978, 48, 183; Lackey, J. A. In Advances in Legume Systematics, part 1, Polhill, R. M.; Raven, P. H., eds., Kew Royal Botanic Gardens: England, 1981.

4. Turner, B. L.; Harborne, J. B.; Phytochemistry 1967, 6, 863.

5. Moreira, R. A.; Barros A. C. H.; Oliveira, J. T. A.; Braz. Arch. Biol. Technol. 1985, 28, 172.

6. Paiva, P. M. G.; Oliva; M. L. V.; Fritz, H.; Coelho, L. C. B. B.; Sampaio, C. A. M.; Phytochemistry 2006, 67, 545.

7. Andrade, C. A. S.; Correia, M. T. S.; Coelho, L. C. B. B.; Nascimento, S. C.; Santos-Magalhães, N. S.; Int. J. Pharm. 2004, 278, 435; Maciel, E. V. M.; Araujo-Filho, V. S.; Nakazawa, M.; Gomes, Y. M.; Coelho, L. C. B. B.; Correia, M. T. S.; Biologicals 2004, 32, 57; Paiva, P. M. G.; Souza, A. F.; Oliva, M. L. V.; Kennedy, J. F.; Cavalcanti, M. S. M.; Coelho, L. C. B. B.; Sampaio, C. A. M.; Bioresour. Technol. 2003, 88, 75; Barreiros, A. L. B. S.; David, J. P.; Queiroz, L. P. de; David, J. M.; Phytochemistry 2000, 55, 805.

8. Wirth, J.; Guo, W.; Baumes, R.; Gunata, Z.; J. Agric. Food Chem. 2001, 49, 2917; Perez, C.; Trujillo, J.; Almonacid, L. N.; Trujillo, J.; Navarro E.; Alonso, S. J.; J. Nat. Prod. 1996, 59, 69.

9. Kanchanapoom, T.; Chumsri, P.; Kasai, R.; Otsuka, H.; Yamasaki, K.; Phytochemistry 2003, 63, 985.

10. Yue, J-M.; Chen, S-M.; Lin, Z-W.; Phytochemistry 2001, 56, 801; Cardoso, M. P.; David, J. M.; David, J. P.; Nat. Prod. Res. 2005, 19, 431; Greca, M. D.; Mangoni, L.; Molinaro, A.; Monaco, P.; Previtera, L.; Gazz. Chim. Ital. 1990, 120, 391.

Received: November 25, 2008

Web Release Date: September 28, 2009

Supplementary information


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  • 1. Joly, A. B.; Botânica- Introdução à Taxonomia Vegetal, Cia Editora Nacional: São Paulo, Brasil, 1998.
  • 2. Bentham, G.; Commentationes de Leguminosarum Generibus, Sollingeri, J. B., ed., Vienna, 1837.
  • 3. Hutchinson, J.; The Genera of Flowering Plants, vol. 1, Claredon Press: Oxford, 1964;
  • Baudet, J. C.; Bull. Jard. Bot. Nat. Belg 1978, 48, 183;
  • Lackey, J. A. In Advances in Legume Systematics, part 1, Polhill, R. M.; Raven, P. H., eds., Kew Royal Botanic Gardens: England, 1981.
  • 4. Turner, B. L.; Harborne, J. B.; Phytochemistry 1967, 6, 863.
  • 5. Moreira, R. A.; Barros A. C. H.; Oliveira, J. T. A.; Braz. Arch. Biol. Technol 1985, 28, 172.
  • 6. Paiva, P. M. G.; Oliva; M. L. V.; Fritz, H.; Coelho, L. C. B. B.; Sampaio, C. A. M.; Phytochemistry 2006, 67, 545.
  • 7. Andrade, C. A. S.; Correia, M. T. S.; Coelho, L. C. B. B.; Nascimento, S. C.; Santos-Magalhães, N. S.; Int. J. Pharm. 2004, 278, 435;
  • Maciel, E. V. M.; Araujo-Filho, V. S.; Nakazawa, M.; Gomes, Y. M.; Coelho, L. C. B. B.; Correia, M. T. S.; Biologicals 2004, 32, 57;
  • Paiva, P. M. G.; Souza, A. F.; Oliva, M. L. V.; Kennedy, J. F.; Cavalcanti, M. S. M.; Coelho, L. C. B. B.; Sampaio, C. A. M.; Bioresour. Technol. 2003, 88, 75;
  • Barreiros, A. L. B. S.; David, J. P.; Queiroz, L. P. de; David, J. M.; Phytochemistry 2000, 55, 805.
  • 8. Wirth, J.; Guo, W.; Baumes, R.; Gunata, Z.; J. Agric. Food Chem. 2001, 49, 2917;
  • Perez, C.; Trujillo, J.; Almonacid, L. N.; Trujillo, J.; Navarro E.; Alonso, S. J.; J. Nat. Prod. 1996, 59, 69.
  • 9. Kanchanapoom, T.; Chumsri, P.; Kasai, R.; Otsuka, H.; Yamasaki, K.; Phytochemistry 2003, 63, 985.
  • 10. Yue, J-M.; Chen, S-M.; Lin, Z-W.; Phytochemistry 2001, 56, 801;
  • Cardoso, M. P.; David, J. M.; David, J. P.; Nat. Prod. Res. 2005, 19, 431;
  • Greca, M. D.; Mangoni, L.; Molinaro, A.; Monaco, P.; Previtera, L.; Gazz. Chim. Ital. 1990, 120, 391.
  • *
    e-mail:
  • Publication Dates

    • Publication in this collection
      14 Oct 2011
    • Date of issue
      2009

    History

    • Accepted
      28 Sept 2009
    • Received
      25 Nov 2008
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