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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.51 no.4 Concepción Dec. 2006 


J. Chil. Chi. Soc., 51, N°.4 (2006), p.1057-1060





1Departamento de Agroindustrias. Facultad Ingeniería Agrícola, Universidad de Concepción-Chillán. 2Departamento de Botánica. Facultad de Ciencias Naturales y Oceanográficas. Universidad de Concepción, Casilla 160-Concepción-Chile. Fax: 56-41-221569, Email: 3Departamento Ciencias Básicas, Facultad de Ciencias. Universidad del Bío-Bío, Chillán-Chile. 4Lehrbereich Biotechnologie der Universität, Paul-Ehrlich-Straße 23, D-67663 Kaiserslautern, Germany. 5Division of Organic Chemistry 2, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.


We reported seven compounds isolated from mycelial cultures of Chilean Basidiomycetes. The compound structures were elucidated by spectroscopic methods. Two polyacetylenes were isolated from Gymnopilus spectabilis: hepta-4,6-diyn-3-ol(1) and 7-chloro-hepta-4,6-diyn-3 ol (2). We isolated the aromatic compounds p-anisaldehyde (3) and 3-chloro-p-anisaldehyde (4) from Hypholoma capnoides. H. fasciculare produced the compounds (3), (4),3,5-dichloro-4-metoxi-benzyl alcohol (5) and the sesquiterpen naematolon (6). Finally, H. sublateritiumproduced (5) and another sesquiterpen, marasmal (7). The compounds (1), (2), (5), and (7) showed biological activity against the tested model microorganism.

Keywords: biological activity, basidiomycetes, Chilean fungi


Among the sources of bioactive metabolites, less intensively investigated organisms like the higher fungi seem highly promising in terms of new structures with interesting biological activities. In recent decades, interesting compounds of different biogenetic origins have been isolated from Basidiomycetes and were found to have antibacterial, antifungal, phytotoxic, nematicidal, cytostatic, antiviral, and other pharmacological activities. Basidiomycetes inhabit most climate zones, from arctic to tropical rain forest. Fruiting bodies can be collected from substrates like leaves, wood, dung, or soil.

Mycelial cultures are usually derived from spores or tissues plugs, which are allowed to germinate and grow on a solid medium consisting of yeast extract, malt extract, and glucose. Our search for novel biologically active metabolites from Chilean basidiomycetes led to previous reports of 4 new bioactive compounds, the Himanimides, from Serpula himantioides (1) and a new antifungal and cytotoxic triterpenoid, Favolon-B, isolated from fermentations of Mycena sp. (2).In the present study, we report the results obtained from submerged cultures of Gymnopilus spectabilis, Hypholoma capnoides,H. fasciculare, and H. sublateritium.


Producing organisms and fermentation

Fruiting bodies of G. spectabilis, H. capnoides,H. fasciculare, and H. sublateritium were collected in a forest close to Concepción (36º 47' SL, 73º 7'WL),Chile. Mycelial cultures were produced from spore prints of fruiting bodies. These were grown in YMG medium composed of 0.4% yeast extract, 1% malt extract, 0.4% glucose, and 1.5% agar with a pH of 5.5.

Fermentations were carried out in YMG medium at 24ºC in a 20 liter fermentor (Braun Biostat U) with aeration (4.0 liter/air/minute) and agitation (120 rpm); 200 ml of a well-grown culture (10 days approx.) were used as inoculum. At the end of the carbon source, the fermentation were stopped and the liquid culture was separated from the mycelium by filtration. (G. spectabilis = 6 days; H. capnoides = 14 days;H. fasciculare = 12 days and H. sublateritium = 10 days).


The compounds were extracted from the culture filtrate by absorption onto Mitsubishi DIAION HP-21 resin. The resin was washed with methanol and acetone, yielding crude extracts. These extracts were evaporated at reduced pressure and applied onto a column containing silica gel (Merck 60, 0.063-0.2 um; column 3 x 30 cm). Elution was carried out with hexane, ethyl-acetate, and methanol. Final purification was achieved by preparative TLC (Merck, Silica gel 60 F254) and preparative HPLC (Jasco model PU-980 with a diode array detector; column: Macherey and Nagel, 250 x 21.2 mm containing Nucleosil C18 (7 mm) flow rate: 5 ml/min). Active compounds were isolated by bioassay-guided fractionation.

Biological assays

Antimicrobial activity was determined in the serial dilution assays or the plate diffusion assays as described by (3).The extracts of crude and pure substances to be tested were dissolved in the most effective solvent (100 µl/ml). Each solution (10 µl) was pipetted onto a sterile antibiotic filter disc and placed onto appropriate growth medium for the respective test organism. The crude extracts and pure compounds were tested against bacteria such as Bacillus brevis,B. subtilis, Streptococcus pyogenes, and Staphylococcus aureus and the following fungi: Mucor miehei,Nematospora coryli, Paecilomyces variotii and Penicillium notatum.

Spectroscopic Data

To elucidate the structures, conventional methods such as Mass, UV, IR, 1H-RMN, and 13C-RMN spectra were used. Mass spectra were registered with a Hewlett Packard 5890 Series II, the UV spectra were obtained with a Perkin Elmer l16, and the IR spectra with a Bruker IFS 48. 1H NMR were recorded at 250 MHz and 13C NMR at 65 MHz on Bruker spectrometers; chemical shifts (ppm) are related to (CH3)4Si as internal references.

hepta-4,6-diyn-3-ol (1): 1H NMR (CDCl3) d (ppm): 4.32(t, 1H), 3.45(bs, 1H), 2.19(s,1H), 1.76-1.65(m,2H), 0.97(t,3H). 13C-RMN(CDCl3) d (ppm): 76.89(C-5), 68.67(C-7), 68.18(C-6), 67.24(C-4), 63.48(C-3), 30.22(C-2), 9.1(C-1). MS: m/z(int.rel) 107(3), 90(23), 86(16), 79(100), 77(23), 74(18), 68(15), 62(18), 51(29).

7-chloro-hepta-4,6-diyn-3-ol (2): 1H NMR (CDCl3) d (ppm): 4.32(t, 1H), 3.45(bs, 1H), 2.19(s,1H), 1.76-1.65(m,2H), 0.97(t,3H). 13C NMR (CDCl3) d (ppm): 102 (C-7), 79 (C-6), 90 (C-5), 91(C-4), 53 (C-3), 25(C-2), 17 (C-1). MS: m/z (int. rel) 143(1), 142(1), 143(5), 125(1), 116(2), 115(32), 114(5), 113(100), 107(24), 96(5), 85(34), 77(24), 61(18), 50(29).

p-anisaldehyde(3):IR: nmax 3077, 2969, 1698, 1601, 1578, 1511, 1461, 1427, 1394, 1302, 1261, 1216, 1151, 1109, 855, 894, 767, 645. 1H NMR (CDCl3) d (ppm): 9.86 (s, 1H), 7.82 (d, 1.7 Hz, 2H), 6.98 (d, 1.9 Hz, 2H), 3.86(s, 3H). 13C NMR(CDCl3) d (ppm): 190.70(s), 164.63(s), 131.93(d), 129.97(s), 114.33(d), 55.53(q). MS: m/z (int. rel) 138(0.3), 137(2.5), 136(66), 135(69), 107(15), 92(14), 77(23), 65(9), 63(14), 51(10).

3-chloro-p-anisaldehyde (4): 1H NMR (CDCl3) d (ppm) 3.96 (s, 3H), 7.02 (d, J = 8.5 Hz, 1H), 7.75 (dd, J = 1.9, 8.5 Hz, 1H), 7.87 (d, J = 1.9 Hz, 1H), 9.82 (s, 1H). 13C NMR (CDCl3) d (ppm): 56.26(q), 111.44 (d), 123.43 (s), 130.00 (s), 130.29 (d), 130.93(d), 159.54 (s), 189.42 (d). MS: m/z (int. rel) 169(100), 154(1), 141(12), 126(18), 111(11), 99(20), 77(21), 63(31), 50(10).

3,5 dichloro-4 methoxybenzyl alcohol(5): 1H NMR (CDCl3) d (ppm): 4.5 (s, 3H), 7.5 (s, 2H), 2.7 (s, 2H).13C NMR (CDCl3) d (ppm): 43.5(t),78.9(q), 128(d), 130(d), 152 (s), 156 (s), 157.2(s). MS.:m/z (int.rel)206 (100), 171 (94), 141(56), 128(25), 111(25), 108 (30), 101(20), 99(73), 77(37).

Naematolon(6): 1H-NMR(CDCl3) d (ppm): 2.24 (dd, J = 11.3, 10 Hz, 1H), 4.76 (dd, J = 11.3, 5.7 Hz, 1H), 6.26 (dq, J = 9 , 1.5 Hz, 1H), 5.83 (dd, J = 9, 0.5 Hz, 1H), 3.24 (dddd, J = 10, 10, 5.4, 0.8 Hz, 1H), 1.16 (s, 3H), 1.36 (s, 3H), 1.79 (dd, J = 1.4, 0.5 Hz, 1H), 5.77 (d, J = 0.8 Hz, 1H), 5.83 (br s, 1H), 2.16 (s), 3.64(d, J = 5.7 Hz, 1H).

Marasmal (7):IR: nmax : 3465, 3264, 1754, 1687, 655, 1632, 1370, 1365. 1H-NMR(CDCl3) d (ppm): 9.36(s, CHO), 7.15 (br d, H-7), 5.31(br s, 1H), 4.34 (br dd, J = 2.8, 2.8 Hz, 1H), 3.69 (m, 1H), 3.51 (br dd, J = 2.8, 2.8 Hz, 1H), 2.43( br ddd, J = 19.4, 7.0, 3.5, 1.5 Hz, 1H), 2.12( br ddd, J =12.8, 3.0, 2.0 Hz, 1H), 1.95(ddd, J = 14.8, 2.8, 2.8 Hz, 1H),1.87(dd, J = 12.8,3.5 Hz, 1H), 1.13(s, 3H), 0.99(s, 3H). 13C NMR (CDCl3) d (ppm): 195.2(d), 178.9 (s)156.2 (d), 140.4 (s), 101.5 (d), 77.9(d), 70.8(d), 54.6(s), 47.7(d), 38.2(s), 37.3(d), 30.4(q), 28.9(t), 25.8(q), 23.0(t). MS: m/z (int. rel) 260(M+ - H2O, 15), 250(20), 234(9), 232(35), 206(23), 188(25), 157(38), 151(25), 143(36), 135(46), 133(37), 119(27), 117(33), 107(27), 69(44), 67(20), 65(22), 55(29), 53(27).


All reported compounds are known and were isolated from the filtrated liquid (see Figure 1) Two polyacetylenes were isolated from G. spectabilis: hepta-4,6-diyn-3-ol(1) and 7-chloro-hepta-4,6-diyn-3 ol (2).These compounds are probably derived by stepwise desaturation of saturated fatty acids using crepenynic acid as key intermediaries in the biosynthesis of many polyacetylenes in fungi (4).The strong biological activity of compounds (1) and (2) can be traced back to their reactive triple bonds (5). The antimicrobial activities of the pure compounds are presented in Table 1.

H. capnoides produced p-anisaldehyde (3) and 3-chloro-p-anisaldehyde (4); H. fasciculare (3), (4) 3,5-dichloro-4-metoxi-benzyl alcohol (5) and naematolon (6) and H. sublateritium(5) and marasmal (7).

The known compounds (3), (4) and (5) have been isolated from mycelial cultures and from natural substrates of several common-wood and forest-litter degrading fungi, e.g. Pleurotus pulmonaris, Bjerkandera adusta, and Pholiota squarrosa (6,7). These compounds showed negative activity against the bacteria and fungi tested. Compounds (6) and (7) are fungal sesquiterpens formed via the humulane-protoilludane biosynthesis pathway. The caryophyllane derivative naematolon (6) has also been isolated from fermentations of several Hypholoma (Naematoloma) species and Panus strains (8). Naematolon showed weak antibacterial and antifungal activity. On the other hand, marasmal, possessing the marasmane skeleton, has previously been reported from liquid cultures of the fungi Marasmius oreades (9). This compound was preferentially active toward the fungi tested as compared to bacteria.



This work has been completed thanks to Fondecyt-Chile Nº.1040445, International Foundation for Science (Grant Nº F/3972-1),Dirección de Investigación, Universidad de Concepción and DIUBB, a financial grant from the Universidad del Bío- Bío.



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