Extraction and analysis of indole derivatives from fungal biomass

Jochen Gartz

(Received 2 September 1993/Accepted 29 September 1993)

The occurance and extraction of indole derivatives in six species from four genra of higher fungi were investigated. By using pure method for extraction of the mushrooms analysis revealed the highest concentractions of psilocybin and baeocystin. The psilo cin content of the species was higher by using aqueous solutions of alcohols than with methanol alone but was an artificial phenomenon caused by enzymatic destruction of psilocybin. The extraction with dilute acetic acid yielded better results than with t he water containing alcohols.

In the last 15 years many papers have been published about the occurance and determination of psychotropic tryptamine derivatives like psilocybin, psilocin and baeocystin in fungi (GARTZ 1992, 1993).

Various extraction procedures of these substances from mushrooms have been ised mainly with methanol as solvent (BEUG and BIGWOOD 1982, GARTZ 1987, SOTTOLANO and LURIE 1983).

In 1985 an aqueous-organic extraction method with acetic acid for these compounds was described (CASALE 1985). Recently, Czech analysts have used aqueous solutions of methanol and ethanol (pure or in presence of potassium-nitrate) for extraction of the indole derivatives in Psilocybe bohemica SEBEK (KYSILKA and WURST 1990, WURST et al 1992). They claimed that it was possible to have found more psilocin with aqueous ethanol extraction than with pure methanol and that a dissimilar extraction of the alka loids by using both new systems could be achieved. In this work the extraction procedures of psilocybin, psilocin and baeocystin from varoius mushroom species including P. bohemica SEBEK were studied by using methanol and the recommended mixtures of sol vents (CASALE 1985, KYSILKA and Wurst 1990, WURST et al. 1992), respectively.

Materials and methods

Fungal material: Cultivated mushrooms: Psilocybe semilanceata (FR.) KUMM from horse manure compost (GARTZ 1991); Psilocybe cubensis (EARLE) SINGER grown on cow dung/rice grain mixture (GARTZ 1989a); P. bohemica from rice grain/water (GARTZ and MUELLE R 1989; Gymnopilus purpuratus (COOKE and MASS.) SINGER from rice grain/saw dust medium (GARTZ and MUELLER 1990, GARTZ 1991).

Naturally grown mushrooms: Panaeolus cyanescens (BK. & BK.) SACC. (leg. Oahu, Hawaii 13.11.88); Inocybe aeruginascens BABOS (leg. Potsdam 20.05.1987); P. bohemica SEBEK (leg. near Sazava, Czech Republic 15.11.89).

All basidiocarps were dried at room temperature. Possible present residual water was removed from the mushrooms by freeze-drying. Voucher speciments of each species have been deposited in the herbarium of the Univeristy of Leipzig (LZ).

Extraction: (Samples (0.01 - 0.1 g) of dried ground mushrooms were extracted with 5 to 20 ml of methanol for 0.5 to 12 hours by using a magnetic stirrer at room temperature. Under equal conditions the mixtures with aqueous acetic acid (CASALE 1985) and a queous ethanol (psilocin) and methanol (psilocybin) (KYSILKA and WURST 1990, WURST et al. 1992) were used for extraction of the same batch of mushrooms. In the cases with aqueous alcohols as solvent a different extraction time for psilocybin (10 min) and psilocin (160 min) was performed (KYSILKA and WURST 1990). By using of dilute acetic acid the solution was placed in a boiling water bath for 10 min after extraction and anaysis and was then analysed 10 min after extraction and analysis and was then anal ysed again (CASALE 1985).

The filtration and analysis of the indole derivatives by using HPLC and TLC were described elsewhere (GARTZ 1987, SEMERDZIEVA et al. 1986, WURST et al. 1992). An analysis of the extracts for enzymes of the phosphatase type was also carried out (WEBER a nd HORITA 1963).

Results

In this investigation the extraction of psilocin, psilocybin and baeocystin with pure methanol was not completely after 30 min in all species and even 6 hours in analysis of P. cubensis and G. purpuratus. But the full extraction of the alkaloids from al l mushrooms was reached after 12 hours. After this time no traces of indole derivatives could be detected after subsequent extraction of the fungal material with aqueous solutions of ethanol/methanol or acetic acid as well as with chloroform for psilocin.

Baeocystin as incompletely methylated counterpart and possible precursor of psilocybin (GARTZ 1989a) was found in all species by using methanol but in some cases only in very small amounts (Table 1).

The psilocybin and psilocin content was in the same order of magnitude as that found earlier (GARTZ 1992, 1993). This substance seems to be a phosphoric acid ester like psilocybin and baeocystin. Similar concentrations of psilocin were detected in the extracts of P. cubensis and G. purpuratus by using an aqueous solution of acetic acid versus pure methanol (Table 2).

By using the new solvent mixtures containing ethanol and methanol for extraction it was found that more psilocin could be detected in extracts of every species but always smaller amounts of psilocybin than with pure methanol (Table 3).

Additionally, a high activity of enzymes of the phosphatase type could be detected in these aqueous solutions from all species. In contrast to these results only the extracts of P. cubensis and P. cyanescens showed a significant enzymatic activity b y using acetic acid as solvent. In these cases psilocybin was completely dephosphorylated to psilocin by heating the acid extracts and no baeocystin could be detected in P. cyanescens.

Discussion

It is well known that an extraction procedure with methanol needs much time (up to 12 hours) at room temperature (BEUG and BIGWOOD 1982, GARTZ 1987, SEMERDZIEVA et al. 1986) or one hour at 45 C (SCOTTOLANO and LURIE 1983) for complete extrraction.

In our investigations psilocin could be found in high concentrations as well as psilocybin after simple extraction with methanol from various species (GARTZ 1987, 1989c, 1991). When undertaking quantitave analysis of levels of indole derivatives after biotransformation of tryptamine and similar compounds in fruiting mycelia of P. cubensis the highest concentrations of psilocin in every mushroom for example could be detected by using methanol (GARTZ 1989a, b). By using aqueous methanol and ethanol as so lvent for analysis of P. bohemica the Czech analysts have not always analyzed the same batch of mushrooms during their comparative study of extraction methods (KYSILKA, pers. communication 1989).

We generally found variations from one mushroom to another in every species even within P. bohemica from a single location (GARTZ and MUELLER 1989) and also in controlled cultures (GARTZ 1991). Additionally, the high activity of enzymes of the phosphat ase type in the aqueous solutions of alcohols was already described in aqueous mycelial extracts of P. cubensis and other psilocybin containing mushrooms many years ago (BOCKS 1968, GARTZ 1993, WEBER and HORITA 1963). These enzymes were also extracted wit h the water containing solvents and caused a partial dephosphorylation of psilocybin to psilocin (Tables 1 and 3). By using these aqueous soluions it was also observed that in some cases bluish mixtures have been resulted after extraction as a sign of par tial oxydation of psilocin (BOCKS 1968, GARTZ 1989a, WEBER and HORITA 1963). It is also interesting that most of the baeocystin was destroyed during the extraction procedure with water containing alcohols (Tables 1 and 3).

CASALE (1985) described the rapid formaion of psilocin after complete dephosphorylation of psilocybin by heating the dilute acetic acid extract. It is now quite clear that the decomposition under these conditions is an enzymatic reaction and was not ca used by the acid alone. For example the phosphoric acid ester psilocybin, baeocystin and aeruginascin in these acidic extracts from I. aeruginacens were stable during heating in contrast to the behaviour of the same alkaloids in solutions of P. cubensis a nd P. cyanescens. It seems that active enzymes of the phosphatase type could be extracted with aqueous acetic acid only in these two species in contrast to water containing alcohols as extraction method. In the past attempts at the sparation of psilocybin and psilocin simply using mixtures of organic solvents and water were also unsatisfactory (THOMSON 1980).

This investigation shows that the high percentage of psilocin detected in P. bohemica (KYSILKA and WURST 1990, WURST et al. 1992) and not found earlier (GARTZ and MUELLER 1989) was an artificial phenomenon casued by enzymatic destrucion of psilocybin w hich is common in different species by using water containing organic solvents. Extraction with pure methanol is the safest method to obtain the genuine indole derivatives from mushroom species of various genera.

Acknowledgements

The author thanks the following persons: G. DREWITZ, J. ALLEN, G.K. MUELLER and M. SEMERDZIEVA who geneously supplied herbarium material and/or valuable information.

Refernces

BEUG, M.W. and BIGWOOD, J., 1982. Psilocybin and psilocin levels in twenty species from several genera of wild mushrooms in the Pacific Northwest, U.S.A. J. Ethnopharm, 5, 271-289.

BOCKS, S.M., 1968. The metabolism and psilocin and psilocybin by fungal enzymes. Biochem. J., 106, 12-13.

CASALE, J.F., 1985. An aqueous-organic extraction method for the isolation and identification of psilocin from hallucinogenic mushrooms. J. Forensic Sci., 30, 247-250.

GARTZ, J., 1985. Zur Isolierung des Baeocystins aus den Fruchtkoerpern einer Psilocybe-Art. Pharmazie, 40, 274.

GARTZ, J., 1987. Variation deer Indolalkaloide von Psilocybe cubensis durch unterschiedliche Kultivierungsbedingungen. Beitraege z. Kenntnis d. Pilze Mitteleuropas, 3, 275-281.

GARTZ. J., 1989a. Biotransformation of tryptamine derivatives in mycelial cultures of Psilocybe. J. Basic Microbiol., 29, 347-352.

GARTZ, J., 1989b. Biotransformation of tryptamine in fruiting mycelia of psilocybe cubensis. Planta Med., 55, 249-250.

GARTZ, J., 1989c. Occurence of psilocybin, psilocin and baeocystin in Gymnopilus purpuratus. Persoonia, 14, 19-22.

GARTZ, J. and MUELLER, G.K., 1990. Analysis and cultivation of fruit bodies and mycelia of Psilocybe bohemica. Biochem. Physiol. Pflanzen, 184, 337-341.

GARTZ, J. und MUELLER, G.K., 1990. Versuche zur Kultur von Gymnopilus purpuratus, Purpurflaemmling. Myk. Mitt. blatt (Halle), 33, 29-30.

GARTZ, J., 1991. Further investigations on psychoactive mushrooms of the genera Psilocybe, Gymnopilus and Conocybe. Ann. Mus. civ. Rovereto (Italy), Sez. sc. nat., 7, 265-274.

GARTZ, J., 1992. New aspects of the occurance chemistry and cultivaion of European hallucinogenic mushrroms. Ann. Mus. civ. Rovereto (Italy), Sez. sc. nat., 8, 107-124.

GARTZ, J., 1993. Narrenschwaemme. Psychotrope Pilze in Europa in Europa. Herausforderung an Forschung und Wertsystem. Editions Heuwinkel. Genf/Neuallschwill.

KYSILKA, R. and WURST, M., 1990. A novel extraction procedure for psilocybin and psilocin determination in mushroom samples. Planta Med., 56, 327-328.

SEMERDZIEVA, M. and WURST, M., KOZA, T. und GARTZ, J., 1986. Psilocybin in Fruchtkoerpern von Inocybe aeruginascens. Planta Med., 47, 83-85.

SOTTOLANO, S.M. and LURIE, I.S., 1983. The quantitation of psilocybin in hallucinogenic mushrooms using high performance liquid chromatography. J. Forensic Sci., 28, 931-935.

THOMSON, B.M. 1980. Analysis of psilocybin and psilocin in mushroom extracts by reversed-phase high performance liquid chromatography. J. Forensic Sci., 25, 779-785.

WEBER, L.J. and HORITA, A., 1963. Oxydation of 4 and 5-hydroxy-indole derivatives by mammalian cytochrome oxydase. Life Sciences 1, 44-49.

WURST, M., KYSILKA, R., and KOZA, T., 1992. Analysis and islolation of indole alkaloids of fungi by high-performance liquid chromatography. J. Chromatogr., 593, 201-208.