249 Biotransformation of Tryptamine in Fruiting Mycelia of Psilocybe cubensis

Jochen Gartz!

1 Institute of Biotechnology, Academy of Sciences of the GDR, PermoserstraBe 15, GDR-7050 Leipzig, German Democratic Republic

Received: March 13, 1988

Extraction and analysis Abstract The extraction procedure and the analysis ofthe in- dole alkaloids by using HPLC and TLC were described in the previous Mycelial cultures ofPsilocybe cubensis, with papers (3, 8-10). The presence or absence of tryptamine was de- the ability to form psilocybin and psilocin de-novo, also hy- monstrated by TLC as described by Stijve et al. (11). droxylated and methylated fed tryptamine to give psilocin in up to 3,3 % dry mass of the obtained fruit bodies. By using Results and Discussion HPLCand TLC, it was found that these mushrooms contain only a small amount of psilocybin (0.01 ~0.2 % dry mass). The cow dung-rice mixture actually produced The values of psilocin are the highest described in any the first flush of mushrooms earlier than the cultivations on rye mushrooms. (with casing) (2) and rice (3), respectively. They yielded an aver- age of 3 g dry mass per 109 substratum.

Under the same culture conditions, the fruc- Introduction tification times, the yields, and sizes of mushrooms as well as the blueing feature (3)were equal when the growth media also Psilocybe cubensis (Earle) Sing. is a subtropi- contained high concentrations of tryptamine. Initial experi- cal mushroom and contains the indole alkaloid psilocybin and ments without the addition of tryptamine were performed to only small amounts of its dephosphorylated counterpart psilo- determine the content ofpsilocybin and psilocin in comparison cin (1~4). Variations in these metabolites have been well de- with experiments using other culture conditions and/or media monstrated by investigations of fruit bodies cultivated under (2,3). controlled conditions on a rye-grain medium (2) and rice sub- stratum (3), respectively. The levels of psilocybin and psilocin varied from one flush to the next, but generally were much the same The study of psilocybin biosynthesis in sub- as those in the other experiments (2, 3) (Table 1). Consistently merged culture of P. cubensis showed that radioactive tryp- low levels ofpsilocin were found in the mushrooms without the tamine functioned as a better precursor than tryptophan (5~ 7). addition oftryptamine to the substratum. Additionally, psilocin It was found that not less than 22.4 % of the psilocybin formed generally was absent in the first flush as was also observed in was derived from the labelled precursor tryptamine (5). The earlier investigations (2, 3). Table 1 shows that the fed tryp- level of psilocin was generally zero in the mycelial tissue from tamine gives high values of psilocin in each flush from the cul- these experiments (5~ 7). tures.

In the present paper, the biotransformation of fed tryptamine in fruiting mycelia of P. cubensis is described. Table 1 Variation of psilocybin and psilocin levels in P. cubensis as a function of flush number from the cultivations with (a) and without (b) addition of tryp- Materials and Methods tamine (25 mM concentration). Cultivation of Psilocybe cubensis Flush Psilocin Psilocybin No. (% dry mass) (% dry mass) A dried cow dung/rice-grain mixture (2: 1) with a b a b twice the amount of water was used to obtain fast fructifications without casing of a strain (3) of P. cubensis. A 25 mM concentration of tryp- 1 2.l 0.01 0.55 0.48 tamine (as hydrochloride) was added to this medium. Cultivations with- 2 3.3 0.01 0.02 0.51 out the addition of any tryptamine were also tested. The methods of cul- 3 2.8 0.02 0.20 3.l 0.07 0.46 tivations were described in (3). 4 0.09 5 2.9 0.l5 0.l3 0.61 The first sporocarps were produced by cultures of P. cub ens is in 3 to 4 weeks. The cultures continued to produce mushrooms in five flushes. Each flush was harvested as soon as the sporocarps were mature. The mushrooms were immediately freeze-dried, sealed in plas- These psilocin levels are uncommonly high tic, and stored at -10°C until analysis. (from 2.1 to 3.3 %) since values reported for psilocin in dried mushrooms are always below 1 % (1~4, 12, 13). =

250 Planta Medica 55 (1989) Jochen Gartz

Inocybe aeruginascens Babos contains only References traces of psilocin but high amounts of the incompletely methy- lated psilocybin (baeocystin) (9). In contrast to the initial exper- 1 Heim, R., Hofmann, A. (1958) Compt. Rend. 247, 557. iments without an addition of tryptamine, the mushrooms gen- 2 Bigwood, J., Beug, M.W. (1982) J. Ethnopharm. 5, 287. Gartz, J. (1987) Beitrage zur Kenntnis der Pilze Mitteleuropas 3, 275. erally contained only small amounts of psilocybin. The tryp- Badham, E. (1984)]. Ethnopharm. 10,249. tamine level was always zero in each mushroom. In this case no 5 Agurell, 5., Blomkvist, 5.,Catalfomo, P.(1966) Acta Pharm. Suecica tryptamine was additionally found in the methanolic extract of 3,37. the vegetative mycelia from the substratum. 6 Agurell, S., Nilsson, J. L. G. (1968) Acta Chern. Scand. 22, 1210. 7 Agurell, 5., Nilsson, J. L. G. (1968) Tetrahedron Lett. 1063. In a previous report, Gartz (3) was unable to 8 Gartz, J. (1985) Pharmazie 40, 134. 9 Gartz, J. (1987) Planta Med. 53, 539. detect baeocystin in P. cubensis. But Repke et al. (14) reported 10 Semerdzieva, M., Wurst, M., Koza, T., Gartz, J. (1986) Planta Med. traces of baeocystin in other strains of P. cubensis about 10 52,83. years ago. They suggested that many non-specific enzyme sys- 11 Stijve, T., Hischenhuber, c., Ashley, D. (1984) Z. Mykol. 50, 361. tems exist in fungi which have the ability to oxidize exogenously 12 Beug, M.W., Bigwood, J. (1982)J. Ethnopharm. 5, 271. added compounds, as well as normal, obligatory intermediates 13 Ohenoja, E., Jokiranta, J., Makinen, T., Kaikkonen, A., Airaksinen, M.M. (1987)J. Nat. Prod. 50, 741. (14). 14 Repke, D. B., Leslie, D. T., Guzman, G. (1977) Lloydia 40,566. The results in Table 1 show that the enzyme systems in P. cubensis have a high hydroxylation and methyla- tion capacity to convert added tryptamine to psilocin. It is possi- ble that a reduced amount of phosphate in the culture media decreased the biosynthesis of psilocybin from psilocin in the mycelia.

P. cubensis also failed to produce detectable amounts ofbaeocystin under these culture conditions.

Acknowledgements

The author thanks the following persons: G. Dre- witz, T. Stijve, G. K. Muller, and M. Gey who generously supplied valu- able information.