A Novel Extraction Procedure for Psilocbin and Psilocin Determination in Mushroom Samples

A Novel Extraction Procedure for Psilocbin and Psilocin Determination in Mushroom Samples
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  A Novel Extraction Procedure for Psilocyhin and Psilocin Determi- nation in Mushroom Samples Roman Kysilka 2 and Milan Wurst' Institute of Microbiology, Czechoslovak Academy of Sciences Videns-ka 1083, 142 20 Prague 4, Czechoslovakia 2 Address for correspondence Received: October 3, 1989 Results and Discussion The composition of the extraction agent has a fundamental influence on the yield of extraction. For test- ing we used aqueous solutions of methanol and ethanol, in which the tested substances are soluble (2). The depen- dence of psilocybin and psilocin yield on the alcohol con- centration (pure or in presence of potassium nitrate) is illus- trated in Fig. 1. It follows from this that an optimal extrac- tion agent for psilocybin is quite unsuitable for psilocin. 327 Since 1958, when psilocybin was disco- vered as an active principle of hallucinogenic mushrooms of the genus Psilocybe (1), many papers have been published about the determination of psychotropic indole derivatives. High-performance liquid chromatography is now almost exclusively used for the quantitative determination of these substances (2, 3, 4, 5, 6, 7). A key step in the analytical procedure is the extraction of the compounds from the biological material. However, meagre attention has been paid to the extraction procedure. Hofmann et al. (2) found that psilocybin and psilocin are well soluble in methanol, and have sub- sequently used solely methanol for extraction. Other au- thors then used this solvent with or without modifications (4,6, 7, 9, 10, 11, 12, 13).Psilocybin and psilocin were found in con-centrations up to 1.0% and 0—0.16%, respectively, in fruit bodies of Psilocybe bohemica Sebek extracted with me- thanol. In this work we optimized the extraction conditions for psilocybin and psilocin from fruit bodies ofPsilocybe mushrooms and studied the influence of extrac- tion agent composition, its quantity and extraction time on the yield of these compounds. High-performance liquidchromatographic method was used for quantification of psilocybin and psilocin. Materials and MethodsExtraction The dried fruit bodies of mushroom Psilocybe bohemica were cut and then perfectly homogenized in a glass mor-tar. The extraction was performed in 20 ml vials at a constant sam- ple weight of 10mg. It was carried out on a reciprocal shaker with a 15mm amplitude and a frequency of 1.2 Hz. The crude extract was filtered through 1 mm PTFE filter before injection into the HPLC apparatus. HPLC analysis Chromatographic analysis was performed on LC- 3B liquid chromatograph (Perkin-Elmer, USA). Column (250 x4mm ID.) was packed with Silasorb SPH C18 7.5 m (Lachema, Czechoslovakia). Mobile phase consists of methanol, water and acetic acid 10 : 90: 1 (for psilocybin), or 35 : 65: 1 (for psilocin). A spectrophotometric and electrochemical detection was used for quantification of both substances (14).  of alcohol Fig. 1 Dependence of the determined amount of psilocybin and psilocin onthe composition of the extracting agent (ã methanol/water/saturated KNO3; O methanol/water; A ethanol/water). We assume that the satisfactory values with methanol obtained by spiking (9) can be attributed to the ex- tracellular supply of the spiked substances whereas in realsamples they are intracellular. A statistically significant dependence of the extraction yield on the amount of solvent was not found in range of 0.05—0.9 mllmg for both compounds. We also tested the influence of extraction time (0—lOh) on the yield of both substances. The time de- pendence may be well described by the equation (1). C = a.(1_et) (1) in both cases. C is the determined concentration in time oft, a and b are constants (the limit of a is the actual concentra-tion of substance in the sample, his the rate constant of theextraction). The calculated values for psilocybin and psilo- cinwere a = 1.159, b = —41.429 and a = 0.190, b = —2.580, respectively. The extraction of psilocin thus appears to be much slower than that of psilocybin. On the basis of these results, we devised animproved procedure for extraction of psilocybin and psilo- cm from samples of mushroom fruit bodies. 0 20 40 6080 100 Letters    D  o  w  n   l  o  a   d  e   d   b  y  :   Y  a   l  e   U  n   i  v  e  r  s   i   t  y   L   i   b  r  a  r  y .   C  o  p  y  r   i  g   h   t  e   d  m  a   t  e  r   i  a   l .  328 Planta Med. 56(1990) Letters standard deviation for 15 parallel measurementsT criterion for significance test (15), critical value is 2.765 (alpha = 0.010) Hofmann, A., Helm, R., Brack, A., Kobel, H. (1958) Experientia 14, 107— 109. 2 White, P. C. (1979) J. Chromatogr. 169,453—456. Christiansen, A. L., Rasmussen, K. E. (1982) J. Chromatogr. 244, 357—364. ' Christiansen, A. L., Rasmussen, K. E. (1983) J. Chromatogr. 270, 293 —299. Vanhaelen-Fastre, R., Vanhaelen, M. (1984) J. Chromatogr. 312, 467—472. 6 Wurst, M., Semerdzieva, M., Vokoun, J. (1984) J. Chromatogr. 286, 229—235. Kysilka, R., Wurst, M., Pacakova, V., Stulik, K., Haskovec L. (1985) J. Chromatogr. 320, 414—420. 8 Hofmann, A., Helm, R., Brack, A., Kobel, H., Frey, A., Ott, H., Petrzilka, Th., Troxier, F., (1959) HeIv. Chim. Acta 42, 1557—1572.Beug, M. W., Bigwood, J. (1981) J. Chromatogr. 207, 379—385. 10 Christiansen, A. L., Rasmussen, K. E., Tonnesen, F. (1981) J. Chro- matogr. 210, 163—167. Stijve, T., Kuyper, Th. W. (1985) Planta Med. 385—387. 12 Semerdzieva, M., Nerud, F. (1973) Ces. Mykologie 27, 42—47. Perkal, M., Blackman, G. L., Ottrey, A. L., Turner, L. K. (1980) J. Chromatogr. 196, 180—184. 14 Kysilka, R., Wurst, M. (1989) J. Chromatogr. 464,434—437. 15 Miller, J. C., Miller, J. N. (1988)Analyst 113, 1351—1356. We thank Mrs. Marta Semerdzieva, Department of Experimental Mycology of our institute, for providing us with the biological material. References silocybin: 10mg of finely powdered sam- ple is extracted for 10 minutes in 0.50 ml of 75% methanol(saturated with potassium nitrate). The extract is then cen- trifuged, filtered through a membrane filter, and analyzed by HPLC. Psilocin: An analogous procedure is used for extraction, the difference being in the composition of the extraction agent (75% water/ethanol) and time (160 mm). The results obtained with the new extrac-tion procedure were compared with the conventional ex- traction in methanol [e.g. (11)]; the results are summarizedin Table 1. The mean yields are significantly different evenon a high confidence level. Only 76% psilocybin and about8 % psilocin were found with the original extraction proce-dure in comparison with the new procedure. Table 1 procedure bohem/ca(Poricko,Comparison of the results of conventional and optimized in the determination of psilocybin and psilocin middle Bohemia, 1983). in extraction Psilocybe methodconventional optimized T6 psilocybin average 0.9321.223 S.D. 0.1490.129 5.76 psilocin average 0.041 0.448 S. D. 0.0 19 0.05529.75 The results pose a question if the absence or the very low concentrations of psilocin determined in the presence of substantially higher values of psilocybin may not result from an incorrect analytical procedure. Accord- ing to our results, the contents ofpsilocybin and psilocin are comparable in Psilocybe bohemica, in contrast to current literature data. Acknowledgements    D  o  w  n   l  o  a   d  e   d   b  y  :   Y  a   l  e   U  n   i  v  e  r  s   i   t  y   L   i   b  r  a  r  y .   C  o  p  y  r   i  g   h   t  e   d  m  a   t  e  r   i  a   l .
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