Indexación: ScieloThe lichen Pseudocyphellaria nudata is a species endemic to southern South América. From the lichen tallus, methyl orsellinate, 2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde, methyl-evernate, tenuiorin, hopan-6ß,22-diol and hopan-6α,76,22-triol were isolated and identified as the main lichen constituents. This is the first report of the occurrence of 2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde in lichens.http://www.scielo.cl/scielo.php?script=sci_arttext&pid=s0717-97072008000300017&nrm=is

Cuellar, M.

Espinoza, L.

Quilhot, W.

Rubio, C.

Soto, C.

English

Repositorio Institucional Académico (RIA) de la Universidad Andrés Bello

J. Chil. Chem. Soc., 53, Nº 3 (2008)1624PHENOLICS, DEPSIDES AND TRITERPENES FROM THE CHILEAN LICHEN PSEUDOCYPHELLARIA NUDATA (ZAHLBR.) D.J. GALLOWAYM. CUELLAR1*, W.  QUILHOT1, C. RUBIO1, C. SOTO1,  L. ESPINOZA2, and H. CARRASCO31Departamento de Ciencias  Químicas y Recursos Naturales, Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña Nº 1093, Valparaíso, Chile.2Departamento de Química, Universidad Técnica Federico Santa María, Av. España Nº 1680, Valparaíso, Chile.3 Departamento de Ciencias Químicas, Universidad Andrés Bello, Campus Viña del Mar,  Los Fresnos Nº 52, Viña del Mar, Chile.(Received: January 23, 2008 - Accepted: April 28, 2008)ABSTRACTThe lichen  Pseudocyphellaria nudata  is a  species endemic to southern South America. From the lichen tallus, methyl orsellinate, 2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde, methyl-evernate, tenuiorin, hopan-6ß,22-diol and hopan-6α,7ß,22-triol were isolated and identified as the main lichen constituents. This is the first report of the occurrence of  2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde in lichens.Keywords: Pseudocyphellaria nudata, lichen, monophenols, depsides, triterpenes.INTRODUCTIONIn the cool temperate rainforests of southern South America, lichen species belonging to genus Pseudocyphellaria are common1. The genus Pseudocyphellaria is represented in Chile by 54 species occurring from latitude 23ºS to 56ºS. The mains constituents of the species have been identified by thin layer chromatography1,2. Pseudocyphellaria nudata (Zahlbr.) D.J. Galloway (Lobariaceae, lichenized Ascomycota) is endemic to southern South America. It is a robust, coriaceous lichen, with the cyanobacteria Nostoc as photobiont. The genus Pseudocyphellaria is characterized by a wide chemical diversity, including substances from the major secondary metabolism pathways. The acetate polymalonate pathway produces depsides, depsidones, and usnic acid; from the shikimic acid pathway, terphenylquinones and principally pulvinic acid derivatives are produced while the mevalonic acid pathway led to sterols and terpenoids3. The occurrence of triterpenoids, especially the hopane derivatives, produced also by cyanobacteria and other organisms4, have attracted considerable interest in the organic geochemistry as indicators of sources of sedimentary organic matter and biomarkers of mature petroleum-containing layers 5,6,7. According to Galloway1, “the identity of the compounds appearing on thin layer chromatograms remain to be definitively characterized, and that provides an exciting opportunity for future work”. The aim of this work was the identification of the main constituents of a Chilean collection of P. nudata by spectroscopic means.EXPERIMENTALGeneral experimental proceduresThe 1H and 13C NMR spectra were recorded in a Bruker AVANCE spectrometer model 400 operating at 400 MHz for 1H and 100 MHz for 13C, using CDCl3 and DMSO-d6 as solvents. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. Melting points were determined on a Stuart-Scientific SMP3 apparatus. Optical rotation were measured with a sodium lamp (λ= 589 nm, D line) on a Perkin Elmer 241 digital polarimeter equipped with 1 dm cells. Column chromatography (CC) used silica gel Merck 60 G (0,032-0,063 nm), the fractions were monitored by thin-layer chromatography (TLC) used chromatoplates silica gel Merck 60 F254. The spots were visualized under UV light (254/365 nm) and developed using H2SO4 spray reagent 1. Plant material.Pseudocyphellaria nudata (Zahlbr.) D.J. Galloway (118 g) was collected from bark of Nothofagus spp.  at 1000-1100 m altitude  in Conguillio National Park (39º,39`S; 71º,43`W), Chile, in November 2005. The species was determined by W. Quilhot. Voucher specimens are deposited in the Lichen Herbarium (UV), Facultad de Farmacia, Universidad de Valparaíso Extraction and isolation: The air dried lichen sample was ground, and extracted successively with chloroform and acetone (2 L), each time 72 h at room temperature. The solvents were removed under reduced pressure to give a chloroform (6.08 g) and acetone (4.50 g) extracts.The chloroform extract was chromatographed on silica gel using a mixture of dichloromethane and ethyl-acetate with increasing polarity (49:1 and 1:49), collecting 8 ml in each fraction (260 fractions). The fractions were combined based on TLC and 1H-NMR monitoring. Fractions 5-8 were combined and reduced to dryness. Recrystallisation in Et2O:MeOH (1:1) afforded an impure precipitate which was removed from the solution, and further concentration led to the isolation of a pure precipitated compound, identified as methyl evernate (1) (5.8 mg), colorless crystalline powde, mp 137-138ºC. 1H-NMR (CDCl3): 11.57 (1H, s, OH-2’); 11.35 (1H, s, OH-2); 6.71 (1H, d, J = 2.3 Hz, H-3’); 6.60 (1H, d, J = 2.3 Hz, H-5’); 6.38 (1H, d, J = 2.7 Hz, H-5); 6.37 (1H, d, J = 2.7 Hz, H-3); 3.98 (3H, s, CO2Me); 3.83 (3H, s, OMe); 2.62 (3H, s, H-8); 2.58 (3H, s, H-8’). 13C-NMR (CDCl3): 171.7 (C-7’); 169.7 (C-7); 166.5 (C-4’); 164.8 (C-4); 164.4 (C-2); 153.9 (C-2’); 143.4 (C-6, C-6’); 116.6 (C-5’); 111.9 (C-5); 110.4 (C-1’); 108.8 (C-3’); 104.3 (C-1); 98.9 (C-3); 55.4 (OMe); 52.3 (CO2Me); 24.6 (C-8); 24.2 (C-8’) in accordance with literature8.Fractions 16-38 afforded tenuiorin (2) (1.61 g), white crystalline powder, mp 180-182 ºC. 1H-NMR (CDCl3): 11.61 (1H, s, OH-2’’); 11.34 (1H, s, OH-2); 11.15 (1H, s, OH-2’); 6.78 (1H, d, J = 1.5 Hz, H-3’); 6.73 (1H, d, J = 1.5 Hz, H-3’’); 6.69 (1H, d, J = 1.5 Hz, H-5’); 6.61 (1H, d, J = 1.5 Hz, H-5’’); 6.38 (2H, s, H-3 + H-5); 3.99 (3H, s, CO2Me); 3.84 (3H, s, OMe); 2.70 (3H, s, H-8’); 2.64 (3H, s, H-8); 2.59 (3H, s, H-8’’). 13C-NMR (CDCl3): 171.7 (C-7’’); 169.6 (C-7)*; 169.3 (C-7’)*; 166.6 (C-4’)#; 165.2 (C-4’’)#; 164.9 (C-4); 164.4 (C-2); 154.8 (C-2’)†; 153.6 (C-2’’) †; 143.8 (C-6’); 143.6 (C-6’’); 143.4 (C-6); 117.2 (C-5’); 116.4 (C-5’’); 112.0 (C-5); 110.7 (C-1’’); 109.6 (C-1’); 109.2 (C-3’); 108.7 (C-3’’); 104.2 (C-1); 98.9 (C-3); 55.4 (OMe); 52.4 (CO2Me); 24.6 (C-8); 24.5 (C-8’); 24.2 (C-8’’).†, *, # assignment may be interchanged, in accordance with literature8.From fractions 42-50 a residue was obtained which was recrystallized in Et2O:MeOH (1:1) to afford methyl orsellinate (3) (16 mg), colorless crystalline powder, mp 142-143 ºC.1H-NMR (CDCl3): 11.78 (1H, s, OH-2); 6.28 (1H, d, J = 2.1 Hz, H-5); 6.23 (1H, d, J = 2.1 Hz, H-3); 5.63 (1H, bs, OH-4); 3.92 (3H, s, CO2Me); 2.48 (3H, s, Me-8). 13C-NMR (CDCl3): 172.1 (C-7); 165.3 (C-4); 160.3 (C-2); 144.0 (C-6); 111.4 (C-5); 105.6 (C-1); 101.3 (C-3); 51.9 (CO2Me); 24.2 (C-8) in accordance with literature8.Fractions 78-92 yielded the compound hopan-7ß,22-diol (4) (13.1 mg), colorless crystalline powder, mp 230-231 ºC, [α]D25 = +26.3º (c 0.54 CHCl3). 1H-NMR (CDCl3): 3.89 (1H, dd, J = 5.0, 10.9 Hz, H-7); 2.23 (1H, m, H-21); 1.21 (3H, s, Me-29); 1.17 (3H, s, Me-30); 1.04 (3H, s, Me-28); 0.98 (3H, s, Me-27); 0.86 (3H, s, Me-23); 0.80 (3H, s, Me-25); 0.79 (3H, s, Me-24); 0.77 (3H, s, Me-28). 13C-NMR (CDCl3): 73.8 (C-22); 73.5 (C-7); 53.5 (C-17); 53.2 (C-5); 51.1 (C-21); 50.5 (C-9); 50.1 (C-13); 47.7 (C-8); 44.1 (C-18); 43.4 (C-14); 41.8 (C-3), 41.4 (C-19); 40.2 (C-1); 38.4 (C-15); 37.4 (C-10); 33.2 (C-23); 33.0 (C-4); 30.8 (C-30); 29.6 (C-6); 28.8 (C-29); 26.4 (C-20); 24.1 (C-12); 22.3 (C-16); 21.5 (C-24); 20.7 (C-11); 18.6 (C-2); 17.7 (C-27); 16.1 (C-28); 15.5 (C-25); 11.2 (C-26) in accordance with literature8.Fractions 148-187 yielded the compound hopan-6α,7ß,22-triol (5) (1.46 g), colorless crystalline powder, mp 226-227 ºC, [α]D25 = +46.9º (c 0.62 Py). 1H-NMR (CDCl3): 3.72 (1H, dd, J = 8.6, 10.7 Hz, H-6); 3.57 (1H, d, J = 8.6 Hz, H-7); 2.23 (1H, m, H-21); 1.20 (3H, s, Me-29); 1.18 (3H, s, Me-30); 1.15 (3H, s, Me-23); 1.05 (6H, s, Me-26 + Me-27); 1.00 (3H, s, Me-24); 0.88 (3H, s, Me-25); 0.77 (3H, s, Me-28). 13C-NMR (CDCl3): 79.4 (C-7); 73.8 (C-22); 73.3 (C-6); 56.8 (C-5); 53.5 (C-17); 51.0 (C-21); 49.9 (C-13); 49.6 (C-9); 47.3 (C-14); 44.0 (C-18); 43.6 (C-19); 43.5 (C-8); 41.4 (C-3); 40.3 (C-1, C-15); 38.8 (C-10); 36.3 (C-23); 33.6 (C-4); 30.8 (C-30); 28.8 (C-29); 26.4 (C-20); 23.9 e-mail: mauricio.cuellar@uv.clJ. Chil. Chem. Soc., 53, Nº 3 (2008)1625(C-12); 22.2 (C-16); 22.0 (C-24); 20.7 (C-11); 18.5 (C-2); 17.7 (C-27); 16.7 (C-25, C-26); 16.1(C-28) in accordance with literature8. The acetone extract was chromatographed on silica gel using a mixture of dichloromethane and ethyl-acetate with increasing polarity (48:2 and 2:48), collecting 8 ml in each fraction (230 fractions). Fractions 8-14 afforded tenuiorin (3) (248 mg) and the fractions 120-131 yielded compound hopan-6α,7ß,22-triol (5) (284 mg), both compound previously found in the chloroform extract. Fractions 165-183 were combined and reduced to dryness. Recrystallisation in Et2O:MeOH (1:1) afforded an impure precipitate which was removed from the solution, and further concentration led to isolation of a pure precipitated compound, identified as 2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde (6) (20 mg), colorless crystalline powder, mp 155-156 ºC. 1H- NMR (DMSO-d6): 10.42 (1H, s, H-7); 7.06 (1H, s, H-5); 3.89 (3H, s, OMe); 2.47 (3H, s, H-9); 2.17 (3H, s, H-8). 13C-NMR (DMSO-d6): 186.9 (C-7); 162.6 (C-2); 151.0 (C-4); 137.6 (C-6); 114.5 (C-1); 113.2 (C-3); 112.9 (C-5); 21.7 (C-9); 9.7 (C-8) in accordance with literature8. Fig. 1.- Metabolites isolated from P. nudata.RESULTS AND DISCUSSIONIn the family Lobariaceae the most complex chemistry is found in Pseudocyphellaria; it has the most richly diverse chemistry of any genus, with several compounds synthesized via the shikimic acid pathway8,9,10 in addition to contributions from other biosynthetic pathway. The genus is also especially rich in triterpenoids from several different series (hopane, stictane secostictane, lupane and fernene series), exhibiting an evolution in chemical complexity which reflect both geological age and phylogenetic relationships11,12.The compounds isolated from P. nudata were previously reported from several species of the genus. Since, with the exception of the triterpene hopane-6α,7β,22-triol (5), other compounds informed in P. nudata by means of TLC, including stictic, constictic acid, cryptostic  and constictic acids, methyl gyrophorate1, were not detected, probably  because the low concentrations of the compounds in the thalli.Tenuiorin (2) and methyl orsellinate (3) are currently found in species of Pseudocyphellaria and in other lichen genus3.The phenolic 2-methoxy-3,6-dimethyl-4-hydroxybenzaldehyde (6), however, is reported for the first time from a  lichen. It was isolated from the fungus Aspergillus silvaticus13, where additionally were reported metabolites related to 3-methylorsellinate; also methyl β-orcinolcarboxylate was isolated from Stereocaulon alpinum14.Hopanoids are pentacyclic triterpenes occurring predominantly in bacteria15. It has been suggested that the compounds are of great importance as cell membrane stabilizers in bacteria16. Hopanoids were discovered in nitrogen-fixing bacteria17,18, and also occurs in soil bacteria as Bradyrhizobium root nodules in legume plants19.The hopanoid hopan-7ß,22-diol (4) has been isolated from P. impressa and P crocata4,9, and hopan-6α,7ß,22-triol (5) from P. crocata and Nephroma laevigatum10. Hopanoids are very common in lichens with green algae and/or cyanobacteria as primary or secondary photobionts20.ACKNOWLEGMENTSThis research was supported by the grants DIPUV 21/2005 and DIPUV 22/2005 from Dirección de Investigación, Universidad de Valparaíso. Our thanks to Corporación Nacional Forestal (IX Región) for logistic assistance in the field work.REFERENCES1. D. J. Galloway, Biblioth. Lichenol. 46, 1, (1992). 2. J.W. Bjerke, D.J. Galloway, A. Elvebbak, W. Quilhot, Crypt. Mycol. 24, 59, (2003).3. C. Culberson, W.L. Culberson, A. Johnson Am. Bryol. Lichenol. Soc., Missouri Botanical Garden, St. Louis, 1977.4. J.D. Connolly, R.A. Hill Dictionary of Terpenoids Chapman & Hall, 1991.5. S. Sakata, J.M Hayes, A.R. Mc Taggart, R.A Evans, K.J. Leckrone, R.K. Togasaki, Geochim. Cosmochim. Acta, 61, 5379, (1997).6. S. Hanish, D. Ariztegui, W. Püttmann, Org. Geochem. 34, 1223, (2003).7. J.S. Sinninghe Damsté, I.C. Rijpstra, S. Schouten, J. Fuerst, M.S.M. Jetten , M.. Strous, Org. Geochem. 35, 561, (2004).8. S. Huneck, I. Yoshimura, Identification of Lichen Substances, Springer-Verlag, Berlin-Heildelberg-New York, 1996.9. J.A. Garbarino, W. Quilhot, M. Piovano, C. Rubio, Bol. Soc. Chil. Quím. 36, 229, (1991).10. M. Piovano, M.C. Chamy, J.A. Garbarino.  Bol. So. Chil. Quím. 46, 1, (2001).11. D.J. Galloway, J. Linn. Soc. Bot. 96, 45, (1988).12. D.J. Galloway, Symbiosis, 11, 327, (1991).13. N. Kawahara, K. Nozawa, S. Nakajima, S.I. Udagawa, K.I. Kawai, Chem. Pharm. Bull. 36, 398, (1988).14. J. P. Hylands, K. Ingolfsdottir, Phytochemistry, 24, 127, (1985).15. G. Ourisson, M. Rohmer, K. Poralla, Annu. Rev. Microbiol. 41, 301, (1987).16. K.  Poralla, E. Kannenberg, A. Blume, FEBS Lett. 113, 107, (1980).17. A.N. Berry, R.A. Moreau, A.D. Jones, Plant Physiol. 95, 111, (1991).18. C. Vilchezc, P. LLopiz, P. Neunlist, K. Poralla, M. Rohmer,  Microbiol. 140, 2749, (1994).19. E.L. Kannenberg, M. Perzl, Th. Härtner, Microbiol. Lett. 127, 255, (1995).20. B. Büdel, C. Scheidegger, in Lichen Biology, Th.H. Nash III ed. Cambridge University Press, Cambridge, 1996; pp. 37-64.

Phenolics, depsides and triterpenes from the chilean lichen pseudocyphellaria nudata (zahlbr.) D.J. Galloway

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Phenolics, depsides and triterpenes from the chilean lichen pseudocyphellaria nudata (zahlbr.) D.J. Galloway

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