J. Chil. Chem. Soc., 50, N 1 (2005)

STUDIES ON CHILEAN FUNGI III. FREE AND BOUND STEROLS FROM MYCENA CHLORINELLA* (BASIDIOMYCETES)

MARISA PIOVANO¹, MARCO CLERICUZIO², SILVIA TABASSO², MARÍA CRISTINA CHAMY¹, JUAN A. GARBARINO¹, GIOVANNI VIDARI³ and PAOLA VITA-FINZI³.

¹Departamento de Química, Universidad Técnica Federico Santa María, Casilla 110-V, Valparaíso, Chile; e-mail: marisa.piovano@usm.cl.
²Dipartimento di Chimica Generale ed Organica Applicata, Università di Torino, Via P. Giuria 7, 10125 Torino, Italy.
³Dipartimento di Chimica Organica, Università di Pavia, Via Taramelli 10, 27100 Pavia, Italy.

ABSTRACT

A mixture of oleic, linoleic and a-linolenic esters (1b-d, 2b-d) of ergosterol (1a) and episterol (2a) was isolated from the fruiting bodies of Mycena chlorinella. Ergosterol and its endoperoxide (3) were also found as free sterols. In addition, relatively large amounts of the nucleoside adenosine (4) were isolated.

Keywords: Mycena chlorinella, basidiomycete, fungus, sterols, fatty acid, sterol esters, adenosine.

INTRODUCTION

Mycena, Basidiomycetes of the Tricholomataceae family (1), constitutes a saprophytic, non-symbiotic, cosmopolitan genus of fungi. It comprises more than 500 described species which grow on decomposing plant material such as rotten wood, leaves, conifer needles, etc. No Mycena species have any nourishing value, while M. pura and its allies are classified as poisonous, presumably due to the presence of muscarine and epi-muscarine (2). This genus appears to be a very promising source of new bioactive secondary metabolites: from pure cultures of several Mycena species, a number of different molecular structures have been isolated, such as polyacetylenes from M. viridimarginata (3), a fulvene sesquiterpene from M. leaiana (4) and the diterpene citricolic acid from the pathogenous M. citricolor (5).

Noteworthy for their biological activities are oudemansines and strobilurines, metabolites of mixed biosynthethic origin (shikimic-acetate), isolated from pure cultures of many Mycena species such as M. galopoda, M. atromarginata, M. rosella, M. vitilis, etc. (6). These molecules are potent antibiotics and fungicides, acting on the bc1 segment of the respiratory chain (7).

Much less work has been done on the fruiting bodies collected in nature; the diterpene tintinnadiol, from fruiting bodies of M. tintinnabulum, is one of the few examples (8).

In Chile, a minimum of 37 species are recognized, distributed from the IV (Coquimbo) Region to the X (Lake) Region (9). No chemical studies on them have been published.

This paper deals with a first partial characterization of the chemical contents of M. chlorinella (Lange) Singer, a small fungus growing abundantly on fallen Pinus needles in Central Chile. It has probably been introduced from North America together with Pinus radiata plantations (10).

EXPERIMENTAL

Plant material

The fungal material was collected in June 2003 near Lake Peñuelas, Valparaiso, V Region (39 09' S, 71 30' W), and identified by one of us (M. Clericuzio). Voucher specimens are deposited in the Herbarium at the Universidad Técnica Federico Santa María, Valparaiso, Chile.

Apparatus

The GC-MS apparatus was a Hewlett-Packard 5970 B mass spectrometer equipped with a HP 5890 gas-chromatograph.

The HPLC-MS apparatus was an ion-trap Finnigan LCQ mass spectrometer system (APCI ionization mode).

The NMR spectra were recorded on a Bruker Avance 400 MHz instrument.

General Procedure.

Crushed fresh fruiting bodies (3000 g) were extracted first with CH₂Cl₂, and then with MeOH.

The nonpolar extract (8,1 g) was chromatographed on a silica Merck gel GF 254 column and eluted with toluene and then with toluene-EtOAc mixtures (5:1 to 1:1). The order of elution was: esterified ergosterol and episterol, free ergosterol and ergosterol peroxide.

LC-MS (APCI)

657 (M + H)⁺ of linolenyl ergosterol (1b);

659 (M + H)⁺ of linoleyl ergosterol (1c) and linolenyl episterol (2b);

661 (M + H)⁺ of oleyl ergosterol (1d) and linoleyl episterol (2c);

663 (M + H)⁺ of oleyl episterol (2d).

Methanolysis of esters.

25 mg of the mixture were dissolved in 1.0 ml CH₂Cl₂, and 0.25 ml of 20% solution of MeONa in MeOH was added. The reaction was stirred at 30 C for 20 min, after which time the reaction was quenched with dilute HCl and then extracted with Et₂O. The resulting crude material was subjected to CC (silica gel) using hexane-CH₂Cl₂ mixtures to yield a mixture of methyl oleate, methyl linoleate and methyl linolenate (eventually identified by LC-MS), and a mixture of ergosterol (1a) and episterol (2a) which could not be separated.

Oxidation of ergosterol (1a) to its endoperoxide (3)

15 mg of the mixture (1a + 2a) were dissolved in n-PrOH (2.0 mL), and a catalytic amount of methylene blue was added. Air was bubbled through the solution, with stirring, for 24 h, under irradiation with sunlight. After this time, tlc showed that all the ergosterol had been oxidized to its endoperoxide. The solution was dried under vacuum and loaded on a silica gel column and eluted with hexane-EtOAc (9:1 to 3:1), yielding episterol (2a) in pure form.

Episterol (2a) (7 mg), amorphous solid, MS/EI) m/z (% rel. abund.): 398 (M⁺, 6) 383 (11), 314 (16), 271 (100). ¹³C-NMR (100 MHz, CDCl₃) d: 37.2 (C1), 29.7 (C2), 71.2 (C3), 38.1 (C4), 49.6 (C5), 39.7 (C6) 117.6 (C7), 139.7 (C8), 40.4 (C9), 34.3 (C10), 21.7 (C11), 31.6 (C12), 43.5 (C13). 55.1 (C14), 23.1 (C15), 28.0 (C16), 56.1 (C17), 12.0 (C18), 13.2 (C19), 36.3 (C20), 19.0 (C21), 34.7 (C22), 31.2 (C23), 157.0 (C24), 33.9 (C25), 22.1 (C26), 22.0 (C27), 106.1 (C28).

The methanol extract (6,6 g) was suspended in H₂O (25 ml) and extracted twice with 25 ml of 2-butanone. The organic phase was dried with Na₂SO₄ and then evaporated under vacuum, to yield 3.5 g of subextract. The material was chromatographed on RP-18 silica gel with H₂O-CH₃CN (2:1) as eluent. The first fraction (350 mg) was further purified by means of Sephadex LH-20 molecular exclusion chromatography eluting with MeOH, yielding adenosine.

Adenosine (4) (28 mg) glassy solid; MS (ESI): 268.2 (M + H)⁺; 290.2 (M + Na)⁺. ¹³C-NMR (100 MHz, MeOH-d₄) d: 139.4 (C2), 142.0 (C4), 150.0 (C5), 153.5 (C6), 130.8 (C8), 91.2 (C10), 88.2 (C11), 75.5 (C12), 72.7 (C13), 63.5 (C14).

From the CH₂Cl₂ extract of M. chlorinella, an oily nonpolar substance was isolated, having Rf 0.55 in hexane­CH₂Cl₂ (1:1), moderately UV-active, giving a strongly purple coloured spot on tlc when developed with sulfoanisaldehyde. NMR analysis of this material showed that it was actually a mixture of sterols esterified with different fatty acids. Further separation of this mixture was not possible by standard liquid chromatography.

This mixture was characterized by HPLC-MS (see Experimental), where it was eluted as a single peak, showing a range of molecular weights from 656 to 662, the peak corresponding to 660 being the most prominent one.

In order to further characterize this sample, we performed a transesterification with freshly prepared MeONa. This procedure afforded a mixture of fatty acid methyl esters and two free sterols, which was submitted to CC.

The fatty acid methyl esters were identified by GC-MS as those of oleic, linoleic and a-linolenic acids, in approximately equal proportions, with an additional trace of palmitic acid.

By comparison with authentic samples, one of the two free sterols was identified as ergosterol, the commonest sterol in fungi.

The identity of the second sterol remained unclear, and it was not possible to completely separate it from ergosterol. To achieve this separation, we took advantage of the easy oxidability of the conjugated 5-6,7-8 double bonds of ergosterol by singlet oxygen. After complete oxidation of ergosterol to the endoperoxide, direct phase CC allowed us to obtain the second sterol in pure form. After inspection of its ¹H and ¹³C-NMR spectra, and the ei-MS spectrum (mw = 398), it was identified as episterol (ergosta­7,24 (28)-dien-3b-ol), a less common sterol that is believed to be an intermediate in the biosynthesis of ergosterol (11).

The presence of fatty acid esters of ergosterol and episterol has been already reported from fruiting bodies of various Basidiomycetes (12).

The most likely functions of sterol fatty acid esters are as storage compounds and as contributors to the regulation of the sterol concentrations, and also as membrane constituents (13). Interestingly enough, ergosterol was also found in the free alcohol form (together with its endoperoxide), while episterol was only found as fatty acid esters.

The methanol extract showed the presence of several metabolites, one of which was identified as adenosine. Adenosine, a nucleoside, is a many-sided drug, with predominantly cardiovascular effects (14). In fungi, it was postulated to be involved in nutrition, reproduction and morphogenesis, and in dimorphism (15).

1. Singer, R. The Agaricales in modern taxonomy. Koetz Scientific, Koenigstein . Germany, 1-981. (1986).

2. Stadelmann, R.J., Müller, E., Eugster, C.H. Helv. Chim. Acta 59, 2432-2436. (1976).

3. Jente, R., Bosold, F., Bäuerle, Anke, T. Phytochemistry 24, 553- 559. (1985).

4. Hartig, U., Anke, T., Scherer, A., Steglich, W. Phytochemistry 29, 3942-3944. (1990).

5. Ayer, W., Dufresne, C. Bull. Soc. Chim. Belges 95, 699-706. (1986).

6. Bäuerle, J., Anke, T. Planta Med. 39, 195-196. (1980).

7. Daferner, M., Anke, T., Hellwing, V. Steglich, W. J. Antibiot 51, 816-822. (1998).

8. Engler, M., Anke, T., Sterner, O. Phytochemistry 49, 2591-2593. (1998).

9. Barrera, M.E. Catálogo de la colección de Hongos de R. Singer. Publicación ocasional 40. Dirección de Bibliotecas, Archivos y Museos. Museo Nacional. 1-45. (1984).

10. Maas Geesteranus, R.A. In Mycenas of the Northern Hemisphere. Edita KNAW, Amsterdam, 1-493. (1992).

11. Griffiths, K.M., Bacic, A., Howlett, B.J. Phytochemistry, 62, 147- 153 (2003).

12. Rösecke, J., Köning, W.A. Phytochemistry , 54, 603-610. (2000).

13. Elliot, C.G. Sterols in fungi: their functions in growth and reproduction. In advances in Microbial Physiology 15. Rose and Tempest (Eds.). Academic Press. 121-173. (1977).

14. Galduf, J., Monte, E., Escrivá, J., Romá, E., García, J. Farm. Hosp. 20, 279-288. (1995).

15. Domodon, D., He, W., De Kimpe, N., Höfte, M., Poppe, J. Phytochemistry 65, 181-187. (2004).

ACKNOWLEDGMENTS

This work was funded by DGIP, UTFSM (Project # 13.03.01). We also thank Fundación Andes, Convenio C-13672.

^*For Part II See Studies on Chilean Fungi II. "Structural Characterization of Metabolites from Pholiota spumosa (Basidiomycete). Clericuzio et al. (2004). Croat. Chem. Acta, 77 (2004) 605-611.