PL43326B1 - - Google Patents

Description

The invention relates to a method for the preparation of psychotropic substances psilocybin and psilocin from certain species of mushrooms. The method according to the invention consists in the fact that from Psilocybe mexicana Heim or from artificially grown mushroom material obtained from cultures or biological variants or mutants of this fungus by inoculating natural or artificial cultures and maturing these cultures in light or in the dark at a constant temperature between 18 and 27 ° C, the active substances are extracted according to known methods and then purified, separated from each other and possibly freed from the halogen. Moreover, it was found that psilocybin and psy- locin can be obtained pure according to with the above-mentioned tap method, starting from the mushroom material *) The patent owner stated that the inventor was Roger Cailleux. of natural origin of the genera Psilocybe, Stropharia, Conocybe, Panaeolus, Amanita, or Russula, or of artificially grown mushroom material obtained from the cultures of individual species of fungi or their biological variants or mutants. It is already known (R. Heim, Cr Hebd.Seances Acad. Sci. 242, 965 (1956)) that several species of mushrooms of natural origin from Mexico, Eastern Siberia, Kamchatka and other countries of Asia and America are used for ritual purposes or as stimulants or stunning agents. They have a specific effect on the body, and above all on the human psyche. These so-called hallucination mushrooms are of the following genera: Psylocybe, Stropharia, Panaeolus, Conocybe, Amanita, Russula. Hitherto, however, it has not been possible to isolate the active substances from fungi of natural origin or from artificially grown fungi. It was also impossible to start from fungi (with hallucinatory properties) of natural origin in the laboratory to grow species in which active substances would be formed in sufficient quantity to obtain these bodies on a preparative scale. The invention relates to a method by which it is possible to to obtain hitherto unknown active substances of hallucinating mushroom species, ^ first of all Psiilocybe mexicana Heim, Psilocybe caerulescens Murrill var. Mazatecorum Heim, P. caerulescens Murrill var. nigripes Heim, P. Zapotecorum Heim, P. semperviva Heim and Cailleux, P. Aztecorum Heim, Stropharia cubensis Earle. These active substances can be obtained pure according to known methods, either directly from mushroom material of natural origin or from mushroom material grown from cultures derived from natural or artificial substrates, the cultures of these cultures being carried out according to such methods. Generally, the procedure is as follows: for cultivation on natural substrates, compost is made of fermented wheat straw, a mixture of leaves and stems of corn, and a bit of wild grass, which is rinsed with plenty of running water, ground in a clay bowl and autoclaved. The compost is inoculated with the mycelium of the original cultures and matured for about two weeks at 24-27 ° C, while the cultures are covered with sterile sand and left at 18-27 ° C in glass boxes under daylight, while maintaining a constant humidity . After four to five weeks, fruit appears: harvesting takes place within one to two months of the onset of spore formation. However, since obtaining more starting material in this way is troublesome, other methods of breeding have been developed. that mushrooms in vitro set on nutrient-rich substrates, instead of mycelium with fruits, in large quantities, almost exclusively active fungus and partially sclerosis, and on substrates poor in nutrient known fruitfuls. For example, for nutrient solution on an agar medium (with 1.5% agar content), the optimum pericarp production at the dry matter concentration of the sweet extract is from 0.2 to 0.7%, while the optimum for mycelium and sclerotia production is it lies at a concentration of 4-10% depending on the species of fungus. While daylight is necessary for the formation of the pericarp, it has been found, in contrast, that when the cultures mature in the dark, there is mainly abundant sclerotia or mycelium formation. The best yields of active mushroom material (mycelium and sclerotia) are obtained when the surface cultures are placed on a nutrient solution of sweet extract (beer wort or commercial preparation of malt extract) with a dry substance content of 4-10% and when these the cultures are allowed to mature in the dark at a constant temperature between 22 ° and 26 ° C. For good growth, 0.2% agar should be added to the nutrient solution. In this almost liquid medium the fungus has a sufficient support to quickly create a closed mycelium cover. The addition of Fe11 salt is necessary, and it turns out that the addition of zinc, potassium, calcium and magnesium salts, containing nitrate, phosphate and This method of breeding represents a great technical advance, as it allows in a shorter time and with less labor compared to the cultivation on natural substrates, obtaining more than ten times greater yield of active starting material. The active mushroom material (fruit, sclerosis or mycelia) is carefully dried in a stream of air or in a vacuum at a temperature of 20-40 ° C, finely ground and thoroughly extracted with water, a lower aliphatic alcohol or a mixture of water and organic solvent. mixing with water. Extractions are carried out at room temperature. The extracts are evaporated in a low-temperature vacuum, the residue is degreased with petroleum ether and extracted with acetone or a chloroform-alcohol mixture to free it from inactive accompanying substances. The remaining ballast substances are separated by dissolving the residue in as little water as possible and reducing it again with anhydrous ethanol or acetone. The filtrate is evaporated under vacuum at low temperature. Further purification is carried out by chromatography in a column with a powder - 2 - cellulosic. Elution is carried out with water-saturated butanol or other water-immiscible alcohol. The collected fractions are tested with Keller's reagent (iron chloride in acetic acid and concentrated sulfuric acid) for the content of active substances. The fractions showing a positive color reaction are pooled and, if necessary, separated again on a chromatographic column filled with cellulose powder. The faster moving zone is eluted from the chromatogram, which gives a pure blue color in the Keller reaction, from which the active ingredient called "psilocin" is obtained, while the slower moving zone yields a second, quantitatively predominant active ingredient "Psilocybin", which gives a violet color reaction. The active substances obtained from the column already in a fairly pure form contain chlorine and therefore do not crystallize. It is only by chemical treatment that the halogen can be removed, thereby obtaining crystallizable compounds. To this end, the aqueous solution of the active bodies is treated with silver carbon and the permeate is freed of the excess silver ions with the aid of hydrogen sulphide. The permeate is then concentrated in a vacuum at low temperature, the active substances crystallizing from the concentrated solution. For analytical purposes, psilocybin is added can be recrystallized again from methanol or from water. Fine, snow-white needles are obtained from water, and from methanol - colorless hexagonal plates or prisms, containing methanol, which melt decomposed at 195-200 ° C. The obtained compound is dissolved in 120 parts by volume of boiling methanol or in 20 parts by volume of boiling water: in higher alcohols or other organic solvents it is very sparingly or completely insoluble. For analysis, pure substances are dried in vacuum at 100 ° C, with a weight loss of 10.4%. The elemental analysis results correspond to the sum formula Cs2Hl704N2P (molecular weight 284.2). Psilocybin is an amphoteric compound, optically inactive, and dissolves readily to form salts in dilute aqueous solutions of mineral acids and alkalis. A solution of psilocybin in 80% aqueous ethanol is slightly acidic (pH = 5.2). The ultraviolet spectrum in methanol solution shows peaks at 222, 267 and 290 µl. For analysis, the psilocins are purified by rechromatography on a column packed with cellulose powder with saturated butanol water or by treatment with acidic sodium carbonate in the solution. and shaking with ether or other organic solvent. The elemental analysis results are in accordance with the formula C 2 H 16 NO 2. Psilocin crystallizes from methanol or acetone: it is moderately soluble in water and readily soluble in dilute acid. Melting point 173-176 ° C (decomposition) Psilocin is characterized by an ultraviolet spectrum (in methanol solution) with maxima at 222, 260, 267, 283 and 293 mp, and a Keller color reaction, The exertion of psilocybin gives a pure blue color. When injected subcutaneously or orally, 2-8 mg of psilocybin produces an externally manifested sense of blogostasis, accompanied by a lack of excitability and apathy. At higher doses, apart from the vegetative symptoms, some perceptible external changes occur. The discussed compounds can be used to study mental disorders and psychoses of various origins and can be used as an effective aid in the psychotherapeutic treatment of the mentally ill. Example 1. Psilocybin and psilocin from Psilocybin mexicana Heim of natural origin For cultivation on natural substrate, compost is made from fermented wheat straw. The compost is washed with plenty of running water, ground in an earthenware bowl, autoclaved, then inoculated with mycelium from the original cultures and matured for approximately 2 weeks at 24-27 ° C. After this time, the cultures are covered with sterile sand and left in glass boxes under daylight at a temperature of 21-22 ° C. After 4 to 5 weeks they appear fruitfully: harvesting takes place within one to two months after the spores began to form. - 3 - Ripe fruitfuls of Psylocybe mexicana Heim are then carefully dried in a stream of air at 30 ° C. 54 g of the dried mushrooms are finely ground and shaken once with 600 ml and three times with 300 ml of methanol. within 1/2 hour. The extracts are purified and evaporated to dryness in a vacuum. Remaining 12 g. The residue after extraction with methanol is rubbed four times with 250 ml of petroleum ether and more than once with 100 ml of chloroform with the addition of 10% alcohol to remove fats. The still undissolved residue is dissolved in 10 ml of water and 100 ml of absolute alcohol are slowly added, while the solution is enriched with active ingredients. These operations are repeated 2 or 3 more times. The decanted solutions are vacuum evaporated to dryness, redissolved in methanol and 20 g of cellulose powder with 5% water added are added. The methanol is evaporated again in a vacuum and the cellulose powder, loaded with active ingredient, is introduced into a column filled with 100 g of powdered cellulose with 5% water added (the column is previously purified by washing with butanol saturated with water). Elution is carried out with butanol saturated with water and fractions of 20 ml are collected. After evaporation of the individual fractions, the residue is tested for the content of active substances by means of a color Keller reaction. To this end, 0.25 mg of a sample from the evaporation residue is treated with 2 ml of Keller's reagent. The fractions with a positive color reaction are pooled. Dissolve the amorphous powder in 20 ml of water and shake it with 0.5 g of silver carbonate. After filtering, the silver ions are removed from the solution with hydrogen sulphide and the solution then carefully thickens. From the concentrated solution, psilocybin crystallizes in the form of colorless, fine needles (yield 200 mg). Psilocybin is obtained from the pericarp only in trace amounts. is obtained in an analytically pure state after recrystallization from methanol or water. It is dissolved in 120 parts by weight of boiling methanol or in 20 parts by weight of water at the boiling point. Colorless prisms are obtained from methanol, which melt at a temperature of 195-220 ° C with simultaneous decomposition. The given values correspond, according to the analysis, to the sum formula CiaH ^ Oa ^ P ^ The ultraviolet spectrum obtained in methanol shows the following maxima: 222 m [ i. 267 µm. i 290 µm. The new active ingredient is amphoteric in nature. It dissolves in both dilute aqueous alkali solutions as well as aqueous acid solutions. A solution of psilocybin in an 80% aqueous alcohol solution reacts acidically (pH-5.2). Example II. Preparation of Psilocybin and Psilocin from Stropharia cubensis Earle of Natural Origin. Stropharia cubensis Earle fruit harvested in Mexico are air-dried in the shade at room temperature. 24.2 g of the dried pericarp is finely ground and shaken at room temperature once with 300 ml and three times with 150 ml of methanol for 1/2 hour. The extracts are combined and evaporated to dry under vacuum. For degreasing, the residue (6 g) is rubbed four times with 125 ml of petroleum ether and then three times with 50 ml of chloroform containing 10% ethanol. The still undissolved, about 5 g., Are dissolved in 5 ml. Of water, and other accompanying substances are precipitated from this solution by slowly treating with 50 ml. Absolute ethanol, the solution enriched with active substances. Purification in the same way is repeated two or three more times. The decanted solutions are combined and evaporated to dryness in a vacuum. The residue is taken up in methanol and 10 g of cellulose powder with 5% water are added. The methanol is evaporated off in a vacuum and the cellulose powder containing the active substance is placed on a column filled with 50 g of cellulose powder containing 5% water (previously the column is washed with saturated butanol). water). Elution is carried out with water-saturated butanol and 10 ml fractions are collected. The individual fractions are evaporated under high vacuum at a bath temperature of not more than 50 ° C, and the active substance content is tested with glacial acetic acid containing ferric chloride and concentrated sulfuric acid. To this end, samples of the residue of 8.85 rtig are treated with 2 cm3 of the reagent Kel- - 4 -lera. The active fractions give a purple (psilocybin) or pure blue (psilocin) color in the Keller reaction. Fractions yielding a positive color reaction of the same color are combined together. The amorphous powder obtained as a residue after evaporation of the above fractions dissolves in 2 ml of water and shaken it with 0.25 g of silver carbonate. After filtering, the excess silver ions are removed with hydrogen sulfide. Psilocybin crystallizes from the concentrated solution under conservation conditions in the form of colorless fine needles. 58 g of crystalline psilocybin are obtained, with a yield of 0.24% in relation to the dry pericarp, and traces of psilocin. Example III. Obtaining psilocybin and psilocin from pure cultures of Psilocybe sem- perviva Heim et Cailleux. a. Preparation of the vaccine: Pure spore cultures of the Mexican hat mushrooms Psilocybe semperviva Heim et Cailleux are fed to the broth and agar culture. For this purpose, the spores falling from the gills of the ripe pericarp are placed on a sterile tray, placed on the beer wort with agar and matured. The material for inoculation is obtained from the primary cultures prepared in this way: The mycelium is scraped with a sterile water supply so as to form a suspension containing fine mycelium clots. This suspension is inoculated into Erlenmeyer flasks which contain porcelain pot and fillings in the shape of a saddle. The experiments are best carried out in 300 ml Erlenmeyer flasks containing 50 g of a saddle-shaped filling (size 1 cm, weight about 0.9 g) and 80 ml of nutrient solution, consisting of a beer wort of the content of about 4% of dry substance and 0.2% of agar. The culture matures at 24 ° C, after two weeks a tight cover of mycelium forms on the surface. The whole culture is shaken for 30 minutes on a rotary shaker, the saddle-like filling with its sharp edges grinding the mycelium. In the process, a fine suspension of mycelium filaments is produced. The vaccine thus obtained is sufficient to inoculate 50 liters of medium. b. Production of culture: Fresh, light beer wort without the addition of hops is diluted with water to the content of 8% dry substance / One liter of this solution contains: FeSO4. 7H 2 O 0.00417 g ZnSC 4 • 7H 2 O 0.00172 g Ca (NO 3) 2 1.0 g KI 12 PO 4 0.0624 g MgSO 4. 7H 2 O 0.0624 g KCl 0.0312 g Agar-Agar 2.0 g This medium is dispensed in 1 liter aliquots into penicillin flasks and autoclaved for 25 minutes at 108 ° C. After cooling, the contents of the flasks are inoculated with 2 ml of a suspension, P. semperviva Heim et Cailleux. The resulting cultures mature in the dark at 24-26 ° C. A mycelial cover is produced as described under Ilia. c. Isolation of mushroom material: After seven weeks, the mature culture is passed through gauze, the mushroom material is squeezed and dried in a vacuum at 30 ° C. of one gray 100 penicillin flasks, containing 100 liters of nutrient after 49 weeks 2540 g of dry material, ie 25.4 g per liter of nutrient set. The culture effluent, which also contains some active substances, is processed in the same way as the mushroom material described below. d. Preparation of active substances: 306 g of the dried mushroom material is thoroughly pulverized and shaken three times with chloroform in the amount of 500 ml each, and then three times with chloroform containing 10% ethanol in the amount of 500 ml. Inactive accompanying substances are deposited in the process. to the solution in an amount of 2.8 g. The pre-extracted mushroom material is leached exhaustively once with 3 liters and three times with 1.5 liters of methanol. The combined extracts are evaporated to dryness under reduced pressure, leaving a 17.5 g light brown precipitate. To separate from the fatty impurities, this precipitate is dissolved in 17.5 ml of water and the slurry shaken once with 500 ml and twice with 250 ml of petroleum ether. The solution in petroleum ether contains 0.75 g of inactive accompanying substances: The residual aqueous solution is concentrated in a vacuum to about 25 ml and mixed with vigorous stirring. 250 ml of absolute ethanol. The solution containing the active substances is separated by decantation from the sticky precipitate which is thereby formed. The precipitate is redissolved in a small amount of water and treated with ten times the amount of absolute ethyl alcohol. This sludge treatment by loss is repeated twice more. The solutions are combined and evaporated in a vacuum to dryness. 11.7 g of a residue is obtained, which contains the total amount of active substances. For chromatography in a cellulose column, the residue is dissolved in as little as possible of 50% methanol and the resulting solution is thoroughly mixed with 40 g of cellulose powder and dried in a vacuum. . The cellulose powder, thus loaded with the substance, is introduced into a cellulose column, the filling of which was prepared by the desliming of S50 g of cellulose powder with butanol saturated with water. The chromatogram is developed with butanol saturated with water, the 100 ml fractions are separated and evaporated under high vacuum at the bath temperature not exceeding 50 ° C. The intermediate fractions (3.35 g) which give a positive reaction to the color with Keller's reagent are purified once more by chromatography on cellulose powder. When developing the chromatogram with water-saturated butanol, the fractions of 50 ml are separated and, after evaporation in high vacuum at a bath temperature of 50 ° C or less, tests the color Keller reaction, in the following division: Fraction no. Residue Color reaction Keller mg 1 - 7 8 - 16 17 - 20 21 - 30 31 - 43 1270 87 79 1053 758 negative pure blue negative violet negative The residue of fractions 8-16 (87 mg) provides 45 mg of pure psilocin after silver carbonate treatment as described in example II. Fractions 21-30 (1053 mg) yield 765 mg of pure psilocybin after treatment with silver carbonate. In the same manner, the active ingredients are obtained from the feed after culture. For this purpose, the percolate is concentrated in a vacuum to about 1/10 of its volume. The accompanying substances are precipitated with ten times the volume of methanol, then filtered off, and the solution is evaporated to dryness under a vacuum and the residue is extracted three times with ten times the amount of methanol. The residue after evaporation of the methanol extracts is further processed as above. From 12 liters of culture feed, 80 mg of psilocybin and 6 mg of psilycin are obtained. Overall yield: 845 mg psilocybin and 51 mg psilocin, corresponding to 0.28% or 0.017% of dry mushroom material. Example IV. Obtaining psilocybin and psilocin from pure cultures of Psilocybe mexicana Heim. This example gives a detailed description of the in vitro cultivation of Psilocybe mexicana Heim mushrooms to produce mycelium and sclerotia, as well as the preparation of pure active substances. Fresh, hop-free wort. The beer is diluted with water to the content of 4.0-4.5% of dry substance. For each liter of this solution is added: FeSO4-7H2O 0.00417 g ZnSO4-7H2O 0.00172 g agar-agar 2.0 g This medium is poured in 500 ml portions into 1.6 liter Fernbach flasks and sterilized in an autoclave for 25 minutes at 108 ° C. After cooling, the contents of the flasks were inoculated with 2 ml of the Psilocybe mexicana Heim mushroom suspension. The vaccine is obtained (by placing the spores of pure cultures of the mentioned cap fungi on the beer wort and agar. Spores falling off the gills of the ripe pericarp, are placed on the broth and agar medium and matured. From the thus produced primary cultures, the vaccine can be obtained. as follows: the mycelium, with a sterile water supply, scrape off a sharp spatula so that a suspension of fine mycelium filaments is formed This vaccine is inoculated into a culture medium placed in Erlenmeyer flasks (in which the brine-like porcelain fillers are placed). Experiments with Erlenmeyer flasks containing 50 g of saliva-like filling shapes (size - 1 cm, weight about 0.9 g) and 80 ml of medium, consisting of a beer wort with a value of about 4% of dry substance and 0, ^% - 6 -'agar. In the maturation period at 24 ° C, after 2 weeks it forms for the mycelium is tightly covered. The entire culture is rotated for 30 minutes on an agitator, the saddle shapes grinding the mycelium so that a fine suspension of mycelium filaments is formed. The vaccine obtained in this way is sufficient to inoculate 25 liters of nutrient medium. The inoculated cultures mature in the dark at a temperature of 24-26 ° C. The already described mycelium cover is formed, obliterating numerous sclerotia, which generally reach a size of about 1 cm (in individual cases they can be much larger). In order to separate the mycelium and sclerotia, the pure cultures are filtered through gauze, pressed and dried in an oven at 35-40 ° C. After 62 days of harvesting, 1149 g of dry material (sclerotia and mycelium) are obtained from one gray 135 Fernbadh flasks, containing 67 liters of nutrient solution, which corresponds to 17.14 g per liter of nutrient solution. for example, 612 g of dried material, consisting of starch and mycelium, are extracted and extracted 3 times with chlorophore in the amount of 1 liter and 3 times with chloroform with the addition of 10% ethanol in the amount of 1 liter. 5.6 g of inactive companion bodies are transferred to the extract. The pre-extracted mushroom material is then leached thoroughly with methanol (once for 6 liters and 3 times for 3 liters). The combined methanol extracts are evaporated to dryness under reduced pressure and 35 g of a light brown residue is obtained. This residue is poured with 35 ml of water to remove fatty contamination and shaken once with 1 liter and twice for 0 . 5 libra of petroleum ether. The petroleum ether now contains 1.5 g of inactive accompanying substances. The remaining aqueous solution is previously concentrated in a vacuum to about 50 ml and then 500 ml of anhydrous ethanol are added with vigorous stirring. The active substance solution is separated by decantation from the sticky precipitate formed. . The precipitate is once more dissolved in a little water and re-treated with ten times the amount of absolute alcohol. These lose operations are repeated 2 more times. The solutions are combined and evaporated in a vacuum to dryness. A solid residue of 23.4 g is obtained, which contains the total amount of the active substances. For chromatography on a cellulose column, the cadmium is dissolved in as little as possible 50% methanol. and the solution is thoroughly mixed with 80 g of cellulose powder, after which the material thus obtained is dried in a vacuum. The cellulose powder, thus loaded, is loaded onto a cellulose column, the packing of which was prepared by desludging 700 g of cellulose powder with butanol saturated with water. The chromatogram is developed with butanol saturated with water, and fractions of 200 ml are separated, which are evaporated under high vacuum at a bath temperature not exceeding 50 ° C. The following division is obtained: Fraction No. 1-14 15-34 35-40 Table 1 Residue S 2.170 6.660 3.540 12.370 Keller color reaction negative positive - negative The remainder of about 11 g is retained on the column. Fractions 15-34, which provide a total of 6.660 g of residue, contain a certain amount of active substances. For further purification, they are again chilled & graphed as described below. 6.6 g of the residue is dissolved in as little methanol as possible, the resulting solution is saturated with 20 g of cellulose powder, dried and then charged to a column filled with 600 g of cellulose powder treated as above. When developing the chromatogram with water-saturated butanol, fractions of 100 ml are separated, which, after evaporation in a high vacuum, are tested for a color reaction. The following division is obtained: Table 2 Fraction No. 1- 8 9-17 18-21 22-31 32-45 Residue mg 2.950 180 155 912 1.510 Keller color reaction negative pure blue negative violet negative "" "Fraction residue 9 -17 (180 mg) provides after treatment with silver carbonate as described in example 1, 90 mg of pure "psilocin. Fractions 22-31 (table 2) provide, after treatment with silver carbonate described in example I, 619 mg of pure" psilocin ". Psilocybin having the properties described in Example 1. The active substances were obtained from the culture filtrate in the same manner as described above for sclerotia. To this end, the percolation of the cultures is concentrated in vacuo to about 1/10 of its volume, infused with ten times the volume of methanol, percolated and the residue extracted three times with ten times the amount of methanol. The residue after evaporation of the methanol extracts is worked up further as described above. 10 liters of culture feed yield, for example, 220 mg of psilocybin and 12 mg of psilocin. Preparation of psilocybin and psy-lynin from pure cultures of Stropharia cubensis Earle. The nutrient is prepared as follows: fresh, light beer wort without the addition of hops diluted in running water to 6% of the dry substance content. To each liter of this solution is added: FeSO4-7H20 0.0021 g ZnSO4.7H20 0.0009 g Ca / NO3 / 2 1.0 g KH2P04 0.0624 g MgSO4.7H20 0.0624 g KCl 0.0312 g Agar- 2.0 g agar. This medium is sterilized as in example III and inoculated with 2 ml of Stopharia cubensis Earle mushroom suspension per liter of medium. The preparation of this vaccine in suspension is carried out as in example III. After 52 days of dark aging at 24 ° C., from a charge of 20 liters, 452 g of dry mushroom material, ie 22.6 g per liter of medium, are obtained. The preparation of the active ingredients from the mushroom material is carried out as described in Example III. Yield: 1083 mg of pure crystalline psilocybin and 45 mg of psilocin, corresponding to a yield of 0.24% or 0.010% of dry mushroom material. Example VI. Preparation of psilocybin and psilocin from Psilocybe semperviva Heim et Cailleux of natural origin. Mature fruit bearing fruit of Psilocybe semperviva Heim et Cailleux obtained by artificial cultivation on a natural nutrient (compost) is carefully dried in a stream of air at a temperature of 30 ° C. 32 g of the dried pericarp is finely ground and shaken at room temperature for 1/2 hour, once with 300 ml of methanol and three times with 150 ml of methanol. The combined extracts are evaporated to dryness in a vacuum. For degreasing, the residue (3.3 g) is rubbed four times with 125 ml of petroleum ether and three times with chloroform, which contains 10% ethanol in an amount of 50 ml each. A residue of 6.5 g is obtained, which is dissolved. It hides in 6 ml of water. This solution is treated slowly with 60 ml of absolute ethanol in order to precipitate the remaining accompanying substances, the solution being enriched with the active substances. Cleansing in this way is repeated two more times. The solutions are decanted, blended together and evaporated to dryness in a vacuum. The residue is dissolved in methanol and chromatographed on cellulose powder as described in Example II. The active bodies thus obtained are freed from halogen by treatment with silver carbonate. After recrystallization, 160 mg of crystalline psilocybin and 32 mg of psilocin are obtained, corresponding to a content of 0.5% or 0.1% of the dried mushroom material. Example VII. Preparation of psilocybin from Psilocybe caerulescens Murril var. Mazatecorum Heim of natural origin. Mature fruit bearing fruit of Psilocybe caerusce lens Murrill var. Mazatecorum Heim obtained by artificial cultivation on natural nutrient medium (compost) is carefully dried, then the dried mushroom material (7.3 g) is finely ground and extracted thoroughly with methanol as described in Example 2. The extracts are combined and evaporated in a vacuum to dryness. The residue obtained as in Example II is further processed to obtain the active ingredients. 14.6 mg of psilocybin are obtained (relative to dried mushroom material). Psilocin is not obtained from a processed mushroom sample. Example VIII. Obtaining psilocybin from pure cultures of Psilocybe carulescens Murrill var. Mazatecorum Heim. * The breeding environment is prepared by diluting the fresh beer wort with running water without the addition of hops to a dry substance content of 4.0%. For each liter of this solution is added: corn steep 10.0 g FeSO4-7H20 0.00834 g Ca / OH / 2 0.20 g K2HP04 0.20 g NH4ÓH 0.25 g Agar-agar 2.0 g - 8 - This medium is sterilized as described in Example III, and inoculated with a mycelial suspension of pure culture of Psilocibe caerulescens Murrill var. Heim ma- terum from Meklik, and the produced cultures are matured in the dark at a temperature of 26 ° C. After 48 days, 20.4 g of dry mushroom material were collected per liter of solution. The separation and purification of the active substances from this material was carried out as described in Example III. Single Landing Yield; from 10 liters of nutrient solution: 449 mg of psiocybin, corresponding to 0.23% of the dried mushroom material. No psilocin was found in this load. Example IX. Obtaining psyiocybin from Psilccibe Zapotecorum Heim of natural origin. Fruitful fruitful Psilccibe Zapotecorum Heim harvested and picked in Mexico in the area called "pays chatino" is carefully dried (remaining 42.4 g), finely ground and shaken with methanol as indicated in Example 2 The combined methanol extracts are evaporated to dryness under vacuum. The residue is further worked up as indicated in Example 2. 212 mg of pure psyiocybin, corresponding to 0.5%, is obtained. Psilocin is not obtained from the processed mushroom material. X. Obtaining psyiocybin from pure cultures of Psilócibe Zapotecorum Heim. Fresh, pale beer wort without the addition of hops is diluted in running water to 4.5% dry substance. For each liter of this solution is added: * FeSO4 corn steep NH4OH K2HPO4 Agar-agar pH of the solution: 5.5 This culture medium is sterilized as described in example III and inoculated with a mycelial suspension of a pure culture which obtained from the spores of a mature pericarp of Psilocibe Zapotecorum Heim from "pays chatamo", Mexico. After 57 days of dark maturation at 24 ° C., a charge of 25 liters is obtained, 430 g of dried mushroom material, ie 17.2 g per liter of nutrient solution. The preparation of the active ingredients from this material is carried out according to the method of Example III. Yield: 903 mg of pure psyiocybin, which corresponds to a content of 0.21% in relation to the dry mushroom material. Psilocins aiie are obtained. «Example XI. Obtaining psyiocybin and psilocin from Psilócibe Aztecorum Heim of natural origin. Ripe fruitful Psilócibe Aztecorum Heim harvested in Mexico in the Aztec country (On Popo-catepetl at an altitude of 3300-3500 m above sea level) is carefully dried, it is ground finely and thoroughly extracted with methanol as described in example II. The extracts are combined and evaporated in a vacuum to dryness. The residue is worked up in order to isolate the active ingredients as set out in Example 2. 570 mg of pure psyiocybin and 47 mg of psilocin are obtained, corresponding to a content of 0.2% or 0.017% of the dry mushroom material (285 g). Example XII. Obtaining psyiocybin and psilocin from pure cultures of Psilócibe Aztecrum Heim. The composition of the nutrient medium is: 0.00834 g 10.0 0.30 0.30 2.0 gg era malt extract - FeS047H20 ZnOS4-7H, C Ca / NOjj / a KH2P04 MgSO4-7H20 KC1 Agar-agar 100 g 0.00417 g 0.00172 g 1.0 g 0.25 g 0.25 g J 0.125 g 2.0 g running water to 1000 ml The medium is autoclaved during 25 minutes at 108 ° C. 1 liter is inoculated with 2 ml of Psilócibe Aztecorum Heim mushroom suspension. The preparation of this suspension vaccine is carried out as described in the example of Ilia. Cultures mature in the dark for 45 days at 24 ° C and harvested as described in example Hic. * The yield from 10 liters of nutrient medium is 86.5 g of dry mycelium. The finely ground mycelium is shaken at room temperature for 1/2 hour with 1 liter of 80% ethanol. The filtration residue is extracted * three more times in the same manner. The residue obtained by evaporating the combined extracts (8.9 g) is leached successively twice with 100 ml of petroleum ether, twice with 80 ml of chloroform and twice with 50 ml of acetone in order to separate from fatty substances and inactive ballast substances. . This gives 5.6 g of a water-soluble powder which is dissolved in 6 ml of water. The solution is slowly mixed with 80 ml of acetone while stirring, the precipitate is separated off, which then dissolves in a little water and is precipitated again. ten times the amount of acetone. This sludge treatment is repeated two more times with some tin substances passing into the acetone-water extract. The extracts are evaporated in a vacuum and the residue (2.8 g) is purified and separated by chromatography on a cellulose column. After chromatography twice, 225 mg of crystalline psilocybin and 15 mg of psilocin are obtained, corresponding to a yield of 0.26 % or 0.017% of dry mushroom material. Example XIII. Preparation of the culture. The nutrient solution is obtained as follows: The fresh wort (pale without hops) is diluted with running water to a dry substance content of 4.0 to 4.5%. To one liter of this solution is added: FeSO4.7H20 0.00209 g ZnSO4-7H20 0.00086 g Ca / NO3 / 2 0.25 g KH2P04 0.0625 g MgSO4 0.0625 g KC1 0.031 g Agar-agar 2.0 g This medium is aged and inoculated as in example IV. After 48 days of maturation at 24 ° C, it achieves a yield of 55 liters from one charge, a yield of 924 g of sclerotia and mycelium (dry matter), which is 16.6 g per liter of nutrient solution. The preparation of active bodies from this material is carried out as described in example IV. Yield: 0.018% psilocin and 0.12% psilocybin based on dry mushroom material. Example XIV. Obtaining the culture. The nutrient solution is obtained as follows: Fresh beer wort (light, hop-free) is diluted in running water to a dry substance content of 4.0-4.5%. For each liter of this solution is added: FeSO4'7H20 0.00209 g ZnS04.7H20 0.00086 g Ca / NOa / a 1.0 g Agar-agar 2.0 g The growth medium is sterilized, inoculated and matured as in example IV . After 48 days, 20.5 g of dry mycelium and sclerotia are collected per liter of nutrient solution, from which the active substance is isolated as in Example IV. Yield: 0.011% psilocin and 0.14% psilocybin on dried mushroom material. Example XV. Obtaining the culture. The nutrient solution is obtained as follows: Fresh beer wort (light, hop-free) is diluted in running water to a dry substance content of 4.0-4.5%. To each liter of this solution is added: FeSO4.7H2O 0.00209 g ZnSO4.7H2O 0.00086 g KH2PO4 0.25 g Agar-agar 2.0 g. Further treatment is performed as described in example IV. After 48 days, the yield of dried sclerosis and mycelium amounts to 15.9 g per liter of nutrient solution. The active ingredients are isolated as described in Example IV. Yield: 0.02% psilocin and 0.11% psilocybin on dried mushroom material. Example XVI. Obtaining the culture. The nutrient solution is obtained as follows: Fresh beer wort (light, hop-free * diluted in running water to a dry substance content of 4.0-4.5%. To each liter of this solution is added: FeSO4.7H20 0.00209 g ZnSO4.7H2O 0.00086 g corn steep 20.0 g Agar-agar 2.0 g. The further treatment is carried out as described in Example 4. The yield of dried clusters and mycelium after 48 days is 29.3 g per liter of nutrient solution. The isolation of the active bodies is carried out as described in example IV. Yield: 0.012% psidocin and 0.22% psilocybin in relation to the dry mushroom material. Example XVII. Culture production. g FeSO4-7H20 0.00417 g ZnSO4.7H20 0.00172 g running water 1000 ml The medium is sterilized in an autoclave at 108 ° C for 25 minutes. 1 liter of the medium is inoculated with 10 ml of Psilocibe mexicana mushroom suspension, the preparation of the vaccine in suspension is as described in p December IV. After inoculation, the fungus multiplies? on this medium at 24 ° C. In doing so, mycelium spheres similar to sago are formed, as is known in the propagation of lower fungi. After 30 days, the mycelium is separated by drainage. 7.0 g of dry mycelium per liter of nutrient solution are obtained. 430 g of dried, finely ground mycelium is shaken at room temperature for 1/9 hour with 4.5 liters of 80% ethanol. After filtering off the residue, it is extracted in the same way three more times. The residue from the evaporation of the combined extracts (41 g) is leached successively twice with 500 ml of petroleum ether, twice with 400 ml of chloroform and twice with 200 ml of acetone to separate the ballast substances. There is 25 g of a water-soluble powder which is dissolved in 25 ml of water. 250 ml of acetone are slowly added to the solution with good stirring, and the precipitate, separated from the liquid, is dissolved again in a small amount of water and re-precipitated with ten times the amount of acetone. These operations with the acetone-insoluble precipitate are repeated twice more, with all active substances passing into the water-acetone flow. These extracts are evaporated with good stirring and the residue (9.8 g) is separated further by chromatography on a cellulose column as described in Example IV. After chromatography twice, 32 mg of crystalline psilocin and 225 mg of psilocin are obtained, corresponding to yield 0.007 or 0.05% on dry mycelium. PL

Claims (11)

1. Claims 1. A method for obtaining pure psilocybin and psilocin from certain species of fungi, characterized in that the mushroom material of natural origin of the genus Psilocybe, Stropharia, Painaeolus, Conocybe, Amaróta or Russula, or with artificial ¬ the non-propagated mushroom material obtained from the cultures of these fungi or their biological variants and mutants by inoculation of artificial medium and maturation of these cultures in daylight or in the dark at a constant temperature between 18-27 ° C, the active substances are extracted by known methods, psilocybin and psilocybin (purifies, separates one substance from another and frees from halogen. 2. The method according to claim Psilocybe mexicana Heim is used as a fungus of the genus Psdlocybe. 3. The method according to p. The mushroom of the genus Psilocybe the mushroom Psilocybe semperviva Heim et Cadlleux is used. 4. The method according to p. The mushroom of the genus Psilocybe, Psilocybe caerulescens Munill var. Mazatecomm Heim. 5. The method according to p. The mushroom of the genus Psdlocibe is Psilocybe Zapotecorum Heim. 6. The method according to p. The mushroom of the genus Psilocybe the mushroom Psilocybe Aztecorum Heim is used. 7. The method according to p. The mushroom of claim 1, wherein the Stropharia cubensis Earle mushroom is used as the mushroom of the genus Stropharia. 8. The method according to p. A method according to any of the preceding claims, characterized in that the fungi are propagated on the natural substrate and the cultures are allowed to mature in daylight at a constant temperature between 18-27 ° C. 9. The method according to p. 1 to 7, characterized in that the starting material is mycelium and sclerotia, and the fungi are grown in glass on artificial nutrients from sweet extract with a dry matter concentration of 4-10% depending on the type of fungus, and the nutrients are added to 0.0004-0.0010 g / l of gall ion and 2 g / l of agar and the cultures are allowed to mature in the dark at a constant temperature of 22 to 26 ° C. 10. The method according to p. The process according to any of the claims 1-7, characterized in that the degreased mushroom material is extracted with water or with an organic solvent which is miscible with water, and the non-active accompanying substances are separated from the aqueous solution by fractionated precipitation with an organic solvent which is miscible with water. water, and the active substances contained in the filtrate are separated by absorption on cellulose powder and eluted with butanol saturated with water. 11. The method according to p. A method according to any one of claims 1-7, characterized in that psilocybin and psilocybin are freed from the halogen before or after separating them from the halogen in a known manner by means of silver carbonate. SANDOZ A. G. Zastepca: dr Andrzej Au, patent attorney 930. RSW "Press", Kielce PL