Gram-scale isolation of isobrucein B and neosergeolide from Picrolemma sprucei Hook. f.

Isolamento de isobruceina B e neosergeolida de Picrolemma sprucei Hook. f. em escala-grama

NOTAS E COMUNICAÇÕES

Gram-scale isolation of isobrucein B and neosergeolide from Picrolemma sprucei Hook. f.

Isolamento de isobruceina B e neosergeolida de Picrolemma sprucei Hook. f. em escala-grama

Ellen Cristina Costa da Silva^I; Rodrigo César das Neves Amorim^II; Wanderli Pedro Tadei^III; Adrian Martin Pohlit^IV

^ICurso de Pós-graduação em Química de Produtos Naturais, Instituto de Ciências Exatas, Universidade Federal do Amazonas (UFAM), Avenida General Rodrigo Otávio Jordão Ramos, 3000, Aleixo Campus Universitário, 69.077-000, Manaus, Amazonas, Brazil

^IICurso de Pós-graduação em Biotecnologia, Universidade Federal do Amazonas (UFAM), Avenida General Rodrigo Otávio Jordão Ramos, 3000, Aleixo Campus Universitário, 69.077-000, Manaus, Amazonas, Brazil

^IIICoordenação de Pesquisas em Ciências da Saúde (CPCS), Instituto Nacional de Pesquisas da Amazônia (INPA), Avenida André Araújo, 2936, Petrópolis, CEP 69083-000, Manaus, Amazonas, Brazil

^IVCoordenação de Pesquisas em Produtos Naturais (CPPN), Instituto Nacional de Pesquisas da Amazônia (INPA), Avenida André Araújo, 2936, Petrópolis, CEP 69083-000, Manaus. Telefone / FAX [phone]. E-mail: [e-mail]

ABSTRACT

Quassinoids neosergeolide and isobrucein B, obtained from Picrolemma sprucei, have proven in vitro antitumor, antimalarial, anthelminthic, cytotoxic, insecticide and leishmanicidal activities. There is interest in the in vivo pharmacological study of these natural compounds and their semi-synthetic derivatives, however, the quantities obtained in previous extraction processes have been shown to be a limiting factor for continuation of these studies. Herein, we describe a method for obtaining grams of these quassinoids whose purification relies only on recrystallization.

Keywords: Caferana, Simaroubaceae, Quassinoid, Recrystallization.

RESUMO

Os quassinóides neosergeolida e isobruceína B, obtidos de Picrolemma sprucei, possuem atividades antitumoral, antimalárica, anti-helmíntica, citotóxica, inseticida e anti-leishmania comprovadas em estudos in vitro. Há interesse no estudo farmacológico in vivo dessas substâncias naturais e de seus derivados semi-sintéticos, porém a quantidade obtida nos processos de extração tem se mostrado um fator limitante à continuação desses estudos. No presente trabalho, descrevemos um método para obtenção de gramas desses quassinóides cuja purificação depende apenas de cristalização fracionada.

Palavras-chave: Caferana, Simaroubaceae, Quassinóide, Recristalização.

Isobrucein B (1) (Kupchan et al., 1975) and neosergeolide (2) (Schpector et al., 1994) are bioactive secondary metabolites belonging to the class of highly oxygenated terpenoids known as quassinoids, a class which is exclusively found in the Simaroubaceae plant family (Figure 1). Their isolation on small scales has been described previously from the stems and roots of Picrolemma sprucei (synonym P. pseudocoffea Ducke) (Moretti et al., 1982), one of several plant species known in the Brazilian Amazon as "caferana" (Silva et al., 1977). Other species known as caferana are Tachia guyanensis Aubl. (Gentianaceae) and Quiina rhytidopus Tul. (Quiinaceae) (Corrêa, 1926; Silva et al., 1977). Compound 1 was also obtained from the leaf extract of P. sprucei (Polonsky et al., 1984). The antileukemic, antifeedant, antimalarial (Fandeur et al., 1985; Andrade-Neto et al., 2007), leishmanicidal and anthelmintic (Nunomura et al., 2006) activities of these quassinoids have been reviewed recently (Amorim and Pohlit, 2006) and a study on the cytotoxic, larvicidal, antimalarial and hemolytic activity of 1 and 2 and two semi-synthetic derivatives has been recently published (Silva et al., 2009)

Despite more than 25 years of interest on the part of chemists and pharmacologists in quassinoids from P. sprucei, methods for obtaining them are still both resource and time-consuming. Moreover, non-sustainable extraction directly from the Amazon forest is the available method for obtaining P. sprucei at present (Amorim and Pohlit, 2006). This fact limits the potential usefulness of this Amazonian medicinal plant and the quassinoids obtained from it. Studies are underway to develop sustainable production methods for this plant and its tissues (Barros and Pohlit, unpublished work). In this report, we describe a method for obtaining both 1 and 2 on gram-scales without the need for preparative chromatographic methods and using fractional recrystallization in the purification step.

Collection was performed in Silves Municipality, in Amazonas State, Brazil, in April, 2003. Voucher specimens have been previously deposited at the UFAM Herbarium (Silva 5729 & 5730). Identification was performed by Dr. Wayt Thomas as Picrolemma sprucei Hook. f.(Wayt Thomas, personal comm.). Roots and stems were cut into small pieces while fresh and allowed to dry in the shade and were then ground.

In a pilot-scale, stainless-steel Soxhlet extractor, powdered stems and roots were degreased by continuous extraction with hexanes. The solvent was evaporated from the marc which was then exhaustively extracted in the same apparatus with distilled hot water. The water extracts were combined and concentrated using rotatory evaporation in vacuo with a 50-55ºC bath. The resulting concentrated water extract was re-suspended in 700 mL of water and continually extracted with chloroform. The three concentrated chloroform extracts (totaling 42.8 g, after total evaporation) were each separately dissolved in a minimum of hot water and acetone (2:1, v/v) (Silva, 2006). Crystallization occurred upon cooling of the resulting solutions. The first chloroform extract (CF1) yielded three crops of crystals (PPT1.1, PPT1.2 and PPT1.3) and a filtrate. The second and third chloroform extracts (CF2 and CF3) yielded one crop each (PPT2 and PPT3, respectively). After recrystallization, PPT1.1, PPT1.2, PPT2 and PPT3 yielded a total of 1.75 g (0.027 %) of pure 2. Recrystallizations of PPT1.3 yielded 1.17 g (0.018 %) of pure 1 (Scheme 1). Identification was performed by comparison of physical and spectral data with literature data (Moretti et al., 1982; Vieira et al., 2000). The above isolation and purification procedures were monitored by analyzing extracts, fractions and substances by normal-phase thin-layer chromatography (TLC) using ether : isopropyl alcohol (9:1) as mobile-phase. TLC plates were conveniently developed by contact with iodine vapor and illumination with UV light (254nm). The R_F values for 1 and 2 were 0.6 and 0.4, respectively under these conditions. The melting points (m.p.) obtained for 1 and 2 were 258-260.1°C and 202-204.1°C, respectively.

Moretti et al. (1982) used degreasing and exhaustive, serial hot water infusions which are a lengthy and tedious procedure, especially on a pilot scale. In our adaptation, the use of continuous liquid-solid extraction for these tandem steps provided quassinoid-rich, partially concentrated water extracts. Whereas in Moretti et al. (1982) the chloroform extract was chromatographed to yield 1 and 2 on milligram scales, our method obviates chromatography altogether. The key recrystallization step removes polar, water soluble substances present in the chloroform extract and provides for differential recrystallization of 1 and 2.

This method should aid researchers interested in obtaining 1 and 2 for pharmacological studies. Cold and room temperature extraction with non-chlorinated solvents, followed by recrystallization, are under study to further develop multi-gram isolation of 1 and 2.

ACKNOWLEDGEMENTS

This work was supported by the Brazilian National Research Council (CNPq) through several grants [PPG-7 557106/2005-2, PNOPG 520354/1999-0 and 550260/2001-3] as well as the Bioamazonia-Basa-Fepad Contract. The authors thank CBA (Centro de Biotecnologia da Amazônia) for NMR spectra and E.C.C.S. and R.C.N.A. recognize scholarships received from CAPES and CNPq (142115/06-1), respectively.

LITERATURE CITED

Andrade-Neto, V.F.; Pohlit, A.M.; Pinto, A.C.S.; Silva, E.C.C.; Nogueira, K.L.; Melo, M.R.S.; Henrique, M.C.; Amorim, R.C.N.; Silva, L.F.R.; Costa, M.R.F.; Nunomura, R.C.S.; Nunomura, S.M.; Alecrim, W.D.; Alecrim, M.G.; Chaves, F.C.M.; Vieira, P.P.R. 2007. in vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants. Memórias do Instituto Oswaldo Cruz, 102: 359-365.

Amorim, R.C.N.; Pohlit, A.M. 2006. Picrolemma sprucei Hook. f.: Uso tradicional, princípios ativos e seus derivados semi-sintéticos, exploração comercial e econômica. Fitos, 2: 19-26.

Corrêa, M.P. 1926. Dicionário de Plantas Úteis do Brasil e das Exóticas Cultivadas do Brasil e das Exóticas Cultivadas. Imprensa Nacional, Rio de Janeiro, RJ, Brasil, 747pp.

Fandeur, T.; Moretti, C.; Polonsky, J. 1985. in vitro and in vivo assessment of the antimalarial activity of sergeolide. PlantaMedica, 51:20-23.

Kupchan, S.M.; Britton, R.W.; Lacadie, J.A.; Ziegler, M.F.; Sigel, C.W. 1975. The isolation and structural elucidation of bruceantin and bruceantinol, new potent antileukemic quassinoids from Brucea antidysenterica. Journal of Organic Chemistry, 40: 648-654.

Moretti, C.; Polonsky, J.; Vuilhorgne, M.; Prange, T. 1982. Isolation and structure of sergeolide, a potent cytotoxic quassinoid from Picrolemma pseudocoffea. Tetrahedron Letters, 23: 647-650.

Nunomura, R.C.S., Silva, E.C.C., Oliveira, D.F., Garcia, A.M., Boeloni, J.N., Nunomura, S.M., Pohlit, A.M. 2006. in vitro studies of the anthelmintic activity of Picrolemma sprucei Hook. f. (Simaroubaceae). Acta Amazonica, 36: 327-330.

Polonsky, J.; Bhatnagar, S.; Moretti, C. 1984. 15-deacetyl-sergeolide, a potent antileukemic quassinoid from Picrolemma pseudocoffea. Journal of Natural Prodructs, 47: 994-996.

Silva, E.C.C. 2006. Isolamento, transformação química e atividade biológica in vitro dos quassinóides neosergeolida e isobruceína B. Dissertação de Mestrado, Universidade Federal do Amazonas, Manaus, Amazonas. 191pp.

Silva, E.C.C.; Cavalcanti, B.C.; Amorim, R.C.N.; Lucena, J.F.; Quadros, D.S.; Tadei, W.P.; Montenegro, R.C.; Costa-Lotufo, L.V.; Pessoa, C.; Moraes, M.O.; Nunomura, R.C.S.; Nunomura, S.M.; Melo, M.R.S.; Andrade-Neto, V.F.; Silva, L.F.R.; Vieira, P.P.R.; Pohlit, A.M. 2009. Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives) isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae). Memórias do Instituto Oswaldo Cruz, 104(1), in press.

Silva, M.F.; Lisbôa, P.L.B., Lisbôa, R.C.L. 1926. Nomes vulgares de plantas amazônicas. INPA, Belém, PA, Brasil, 222pp.

Schpector, J.Z.; Castellano, E.E.; Fho, E.R.; Vieira, I.J.C. 1994. A new quassinoid isolated from Piclolemma pseudocoffea. Acta Crystallographica C, 50: 794-797.

Vieira, I.J.C.; Rodrigues-Filho, E.; Fernandes, J.B.; da Silva, M.F.G.F.; Vieira, P.C. 2000. Complete ¹H and ¹³C chemical shift assignments of a new C₂₂-quassinoid isolated from Picrolemma sprucei Hook by NMR spectroscopy. Magnetic Resonance in Chemistry, 38: 805-808.

Recebido em 12/04/2008

Aceito em 22/08/2008

Publication Dates

• Publication in this collection
  27 May 2009
• Date of issue
  Mar 2009

History

• Accepted
  22 Aug 2008
• Received
  12 Apr 2008