BE575588A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



  Process for obtaining psilocybin and psilocin.



   The present invention relates to a process for obtaining compounds endowed with psychotropic properties, hitherto unknown, psilocybin and psilocin, from mushrooms. The applicant has found a process for obtaining these compounds, a process according to which the active bodies of the substance are extracted by known methods.
Psilocybe mexicana Heim or artificially developed cryptogamic material obtained from cultures or biological variants or mutants of this fungus by inoculation of natural or artificial nutrient media and incubation of such cultures in daylight or darkness , at a constant temperature between 18 and 27, these active substances are purified, they are separated from one another and optionally the halogen is eliminated.

        

 <Desc / Clms Page number 2>

 



   The Applicant has also found that starting with fungal materials of natural origin of the species Psilocybe, Stropharia, Conocybe, Panaeolus. Amanita or Russula or artificially developed cryptogamic materials, obtained from cultures of these kinds of fungi or from their biological variants or mutants, psilocybin and psilocin were obtained, according to the procedure indicated above, at the pure state.



   We already knew (R. Heim, C.r.hebd. Sessions Acad. Sci.



  242, 965 (1956) that certain species of natural mushrooms from Mexico, Eastern Siberia, Kamtschatka and other parts of Asia and America were used by the Aborigines for ritual purposes or as stimulants or narcotics, because the absorption of these fungi produces very specific effects on the body and especially on the mind of man. These mushrooms, known as hallucinogenic mushrooms, include the following species: Psilocybe, Stropharia, Panaoelus, Conocybe, Amanita, Russula.



   But so far we had not succeeded in isolating the active bodies from samples of naturally occurring fungi or artificially cultivated fungal material, and neither had it been possible, starting from natural fungi, to cultivating in the laboratory the species endowed with hallucinogenic properties, under conditions such that the active starting material is twisted in sufficient quantities to allow the technical extraction of the active substances.



   The Applicant has found a process according to which one obtains in the pure state, by known methods, the active bodies hitherto unknown of species of hallucinogenic fungi, preferably of Psilocybe mexicana Heim, of Psilocybe caerulescens Nurrill var. . Mazatecorum Heim, from Psilocybe caerulescens Myrrill var.

   nigripes Heim, Psilocybe Zapotecorum Heim, Psilocybe semperviva Heim and Cailleux, Psilocybe Aztecorum Heim, Stropharia cubensis Earle, either directly from fungal material of natural origin, or from cultured fungal material from

 <Desc / Clms Page number 3>

 nant from cultures grown on natural or artificial substrates according to cultivation methods which make it possible to obtain quantities of active starting material sufficient for the industrial extraction of the active substances.



   The process is preferably carried out as follows: for cultivation on a natural substrate, a compost is prepared consisting of fermented wheat straw, a mixture of leaves and stalks of corn or of wild herbs, it is washed thoroughly with running water, distributed in earthen capsules and sterilized in an autoclave. The compost is seeded with mycelium from primary cultures, incubated at 24-27 for about 2 weeks, the cultures are covered with sterile sand and left on their own in glass boxes in daylight. , at a temperature of 18 to 27, keeping the humidity constant.

   Fruiting bodies appear after 4 to 5 weeks; the harvest is carried out from time to time for 1 to 2 months, when the formation of the spores begins.



   As it is however rather complicated to obtain, by this method, of rather large quantities of starting material, other methods of cultures were developed. The Applicant has thus found that by operating in vitro on substrates rich in nutrients, the fungi formed, instead of mycelium with fruiting bodies, almost exclusively active mycelium and partially sclerotia, but on the other hand formed on poor substrates. in nutritive matter, the known fruiting bodies.

   For example, on nutrient agar (containing 1.5% agar-agar), optimum fruiting occurs at a dry substance concentration of malt extract of 0.2-0.7%; on the other hand, the optimum formation of mycelium and sclerotia takes place at concentrations of 4 to 10% depending on the species of fungus.



   While daylight is essential for the formation of fruiting bodies, the applicant has found that, on the other hand, the formation of sclerotia or mycelium was more abundant.

 <Desc / Clms Page number 4>

 when incubating cultures in the dark. The best yields of active cryptogamic materials (mycelium and sclerotia) are obtained by carrying out surface cultures with nutrient solutions containing malt extract (beer wort or commercial preparation of malt extract) with a content of 4 to 10% dry extract and incubating these cultures in the dark at a constant temperature between 22 and 26.

   To obtain good growth, 0.2% agar-agar must be added to the nutrient solution: in this still almost liquid medium the fungus finds just enough support to quickly form a tightly closed layer of mycelium.



  It is necessary to add divalent iron salts; it is also very advantageous to add zinc salts of potassium, calcium and magnesium, nitric, phosphoric and sulfuric ions as well as yeast extract or "Cornsteep Solids" (aqueous bath concentrate of maceration of corn).



   This cultivation process represents a great technical progress because it makes it possible to obtain, more quickly and with less labor, a yield of active raw material more than tenfold, compared to cultivation on natural substrate.



   The active cryptogamic material (fruiting bodies, sclerotia or mycelium) is carefully dried at 20-40 ° C, in a stream of air or under reduced pressure, very finely ground and extracted at room temperature until exhausted. with water, a lower aliphatic alcohol or a mixture of water and an organic solvent miscible with water. The extracts are evaporated under reduced pressure at low temperature, the residue is degreased with petroleum ether and, in order to remove the inactive impurities therefrom, it is extracted with acetone or with a mixture of chloroform and alcohol. The other inert charges are removed by dissolving the residue in a minimum of water and repeated precipitation with absolute ethanol or acetone; the filtrate is evaporated at low temperature under reduced pressure.



   The purification is continued by passage chromatography on cellulose powder; onelute with water-saturated butanol or

 <Desc / Clms Page number 5>

 with another water immiscible alcohol. In the fractions collected, the presence of active substances is detected with Keller's reagent (glacial acetic acid containing ferric chloride and concentrated sulfuric acid). The fractions giving a positive color reaction are pooled and, if necessary, they are again subjected to chromatographic separation on a column of cellulose powder.

   From the chromatogram a more rapidly migrating zone is eluted which gives an active substance, named psilocin, characterized by a pure blue color reaction with Keller's reagent, while a slower moving zone gives a second active substance, quantitatively predominant, psilocybin which gives a purple color reaction.



   The active substances obtained in a fairly pure state after chromatography contain halogen and, in this state, they do not crystallize: it is only by chemical treatment that one can remove the halogen and obtain compounds. crystallized. To this end, an aqueous solution of the active substances is treated with silver carbonate, the excess silver ions are removed from the filtrate with the aid of hydrogen sulphide and the mixture is concentrated at low temperature under pressure. reduced: the substances then crystallize in the concentrated solution.



   For analysis, the psilocyanin is recrystallized once again in methanol or in water. After recrystallization from water, it is in the form of fine snow-white needles; after recrystallization from methanol, it is obtained in the form of colorless hexagonal plates or prisms containing methanol. These crystals melt at 195-200 with decomposition. The compound dissolves in 120 parts of boiling methanol or in 20 parts of boiling water; in higher alcohols or in other organic solvents it is very hardly soluble or insoluble.



   For analysis it is dried at 1000 under high vacuum, which produces a weight reduction of 10.4%. The results of the analysis

 <Desc / Clms Page number 6>

 elemental correspond to the crude formula C12H17O4N2P (molecular weight: 284.2). Psilocybin is an optically inactive amphoteric compound which readily dissolves, giving rise to salts, in dilute aqueous mineral acids and dilute aqueous alkalies.



  The solution of psilocybin in 80% aqueous ethanol has a weakly acidic reaction (PH 5.2). In methanolic solution, it gives an ultraviolet spectrum exhibiting maxima at 222, 267 and 290 m.



   For the analysis, the psilocin is purified by a new chromatography on a column of cellulose powder with butanol saturated with water, or by treatment with sodium bicarbonate in aqueous solution and extraction with ether or an or- ganic. The results of the elemental analysis correspond to the crude formula C12H16NO2. Psilocin crystallizes from methanol or acetone; it is a compound sparingly soluble in water, easily soluble in dilute acid. Its melting point is 173-176 (with decomposition).



   Psilocin is characterized on the one hand by its ultraviolet spectrum (in methanolic solution) which has maxima at 222, 260. 267, 283 and 293 m / u and on the other hand by the pure blue color that it gives with the Keller's reagent, unlike psilocybin.



   When injected subcutaneously or given orally 2 to 8 mg, psilocybin causes a pronounced euphoria in humans which is followed by sluggishness and a sensation of apathy ... in higher doses, in addition to vegetative phenomena, certain alterations in perception occur. These compounds are to be used for the study of mental illnesses and psychoses of different origins and they can be applied to psychopaths as useful auxiliaries in psychotherapeutic treatment.



   The following examples illustrate the present invention without in any way limiting its scope.

 <Desc / Clms Page number 7>

 



  EXAMPLE 1.-
Extraction of psilocybin and psilocin from Psilocybe mexicana Heim of natural origin.



   In order to carry out cultivation on a natural substrate, compost is prepared from fermented wheat straw, washed with plenty of running water, distributed in earthen capsules and sterilized in an autoclave. . The compost is seeded with mycelium from primary cultures, incubated at 24-27 for about 2 weeks, the cultures are covered with sterile sand and left on their own in glass boxes in daylight. , at a temperature of 21-22. After 4 to 5 weeks the fruiting bodies appear; when spore formation begins, harvest is carried out from time to time for 1 to 2 months.



  Then, in a current of air at 30, carefully dry the mature fruiting bodies of Psilocybe mexicana Heim. 54 g of the dried mushrooms are very finely ground and shaken once with 600 cc and 3 with 300 cc of methanol for half an hour each time. The extracts are combined and evaporated to dryness under reduced pressure. This gives a residue of 12 g.



   In order to degrease it, this residue is kneaded 4 times with 250 cm3 of petroleum ether, then another 3 times with 100 cm3 of chloroform supplemented with 10% alcohol. The still undissolved residue (about 10 g) is dissolved in 10 cm3 of water and 100 cm3 of absolute alcohol is slowly added, which results in an enrichment of the solution with active substance. This operation is repeated 2 to 3 times.



  The decanted solutions are concentrated to dryness under reduced pressure, the residue is dissolved again in methanol and 20 g of cellulose powder supplemented with 5% water are added. The methanol is evaporated off under reduced pressure and the cellulose powder retaining the active substance is introduced onto a column of 100 g of cellulose powder + 5% water (the column was washed beforehand with butanol saturated with water). Eluted with water-saturated butanol and 20 cm 3 fractions collected. The evaporation residues of the various fractions are subjected to the Keller color reaction to

 <Desc / Clms Page number 8>

 detect the presence of active bodies. To this end, 2 cm3 of Keller's reagent is added to test portions of 0.25 -ce of evaporation residue.



   The fractions are combined giving a positive reaction.



  The amorphous powder is dissolved in 20 cm3 of water and shaken with 0.5 g of silver carbonate. After filtration, the silver ions are removed from the solution with dihydrogen sulfide and then carefully concentrated. In the concentrated solution, the psilocybin crystallizes in fine colorless needles (amount obtained: 200 mg).



   From the fruit bodies, only traces of psilocin are obtained.



   By further recrystallization from methanol or from water, psilocybin is obtained in the analytically pure state.



  It dissolves in 120 parts of boiling methanol or in 20 parts of boiling water. From the methanolic solution, colorless prisms are obtained, melting at 195-220 with decomposition.



   The results of the analysis correspond to the crude formula C12 H17O4N2P. The ultraviolet spectrum, taken in methanol, shows the following maxima: 222 m / u, 267 m / u and 290 m / u.



   The new active body has an amphoteric character. It dissolves with formation of salts in both dilute aqueous alkalis and aqueous acids. The solution of psilocybin in 80% aqueous alcohol has an acidic reaction (pH 5.2).



  EXAMPLE 2. -
Extraction of psilocybin and psilocin from Stropharia cubensis Earle of natural origin.



   Carefully, the spore-bearing apparatuses of Stropharia cubensis Earle collected in Mexico are dried in air, in shade and at room temperature. 24.2 g of dried spore-bearing apparatus are very finely ground and, at room temperature, they are shaken once with 300 cm3 and 3 times with 150 cm3 of methanol each time, for half an hour each. times. The extracts are combined and evaporated to dryness under reduced pressure. To degrease it,

 <Desc / Clms Page number 9>

 the residue (6 g) is kneaded 4 times with 125 cm3 of petroleum ether each time and a further 3 times with 5C cm3 of chloroform containing 10% ethanol each time.

   The not yet dissolved residue (about 5 g) is dissolved in 5 cm of water and, by slowly adding to this solution 50 cm3 of ethanol, other impurities are precipitated, which produces an enrichment of the solution in active substances. . The purification is repeated 2 to 3 more times in the same way. The decanted solutions are combined and concentrated to dryness under reduced pressure.



  The residue is taken up in methanol and 10 g of cellulose powder supplemented with 5% water are added. The methanol is evaporated off under reduced pressure and the cellulose powder, to which the active substance adheres, is introduced onto a column of 50 g of cellulose powder supplemented with 5% water; beforehand, the column was washed with water-saturated butanol. Elution is carried out with water-saturated butanol and 10 cc fractions are collected. The isolated fractions are evaporated under high vacuum at a bath temperature of not more than 50, and the presence of active substances is examined with glacial acetic acid containing ferric chloride and concentrated sulfuric acid. To this end, 2 cm3 of Keller's reagent is added to samples of 0.25 mg of residue.

   The active fractions are characterized by the appearance of a purple color (psilocybin) or a pure blue color (psilocin).



   The fractions giving a positive colored reaction of the same shade are pooled. The amorphous powder is dissolved in the previous evaporation residues in 2 cm3 of water and shaken with 0.25 g of silver carbonate. After filtration, the excess silver ions are removed by means of hydrogen sulphide. In the solution, after careful concentration, the psilocybin crystallizes into fine, colorless needles. 58 mg of crystallized psilocybin are obtained, ie a yield of 0.24%, based on the dried fruiting bodies, and a trace of psilocin.



    EXAMPLE 3.-
Extraction of psilocybin and psilocin from pure cultures of Psilocybe semperviva Heim and Cailleux.

 <Desc / Clms Page number 10>

 a) Preparation of the seed body:
Pure cultures of basidiospores of the Mexican fungus Psilocybe semperviva Heim et Cailleux are coated on a beer-agar-agar medium. For this purpose, the spores falling from the lamellae of a mature fruiting body are collected on a sterile support, these spores are placed on a medium of beer wort and agar-agar and incubated. With the primary cultures thus obtained, the seed material is prepared in sufficient quantity as follows:
Under sterile tap water, the mycelium is scraped with a rough spatula so as to obtain a suspension of fine mycelium flakes.

   With this suspension are inoculated Erlenmeyer flasks containing a nutrient solution and porcelain stool-shaped fillers, such as those ordinarily used to fill distillation columns. Best results are obtained with 300 cm Erlenmeyer flasks containing 50 g of stool-shaped filler (size: 1 cm; weight: approx. 0.9 g) and 80 cm3 of nutrient solution consisting of 4% beer wort about dry extract and 0.2% agar-agar.



   The culture is incubated at a temperature of 24; after two weeks a compact layer of mycelium has formed on the surface. The entire culture is shaken for 30 minutes on the rotary shaker. With their sharp edges the filling bodies crush the mycelium: the result is a fine suspension of mycelium flakes. The seed body thus obtained is sufficient to inoculate 50 liters of nutrient solution. b) Preparation of the culture.



   Fresh, clear beer wort, not containing added hops, is diluted with tap water to bring the dry extract content to 8%. To each liter of this solution we add:

 <Desc / Clms Page number 11>

 
0.00417 g of FeSO4.7H20 0.00172 g of Znso4.7H20
1.0 g of Ca (NO3) 2 0.0624 g of KH2PO4
0.0624 g of MgSO4.7H20 0.0312 g of KC1
2.0 g of agar
This nutrient solution is introduced in portions of one liter each time into vials of suitable size, similar to penicillin vials, and sterilized by autoclaving at 108 for 25 minutes. After cooling, the flasks are inoculated with 2 cm3 of a suspension of Psilocybe semperviva Heim and Cailleux each time.

   The cultures obtained are incubated at 24-26 in the dark. The mycelium layer described in Example 3 under a) is formed. c) Isolation of the cryptogamic material.



   After six weeks the mature culture is filtered through gauze, the fungal material is squeezed and dried at reduced pressure. With 100 bottles of penicillin containing 100 liters of nutrient solution, we obtain, by harvesting after 49 days, 2540 g of dry matter, i.e. 25.4 g per liter of nutrient solution involved. The culture filtrate, which also contains certain quantities of active substances, is treated by the same process as that which is described in the following paragraph for the cryptogamic material. d) Extraction of active bodies.



   306 g of dried cryptogamic material are sprayed very finely, shaken 3 times with 500 cm 3 of chloroform each time and 3 more times with 500 cm 3 of chloroform containing 10% ethanol each time. 2.8 g of inactive impurities thus go into solution. The cryptogamic material previously extracted is exhausted, once with 3 liters and 3 times with 1.5 liters of methanol each time.



  The extracts are combined and evaporated to dryness under reduced pressure.

 <Desc / Clms Page number 12>

 



  This gives a light brown residue of 17.5 G. To remove the lipoid impurities, this residue is taken up in 17.5 cm3 of water and the suspension is shaken once with 500 cm3 and twice with 250 cm3 each time. petroleum ether. The solution in petroleum ether contains 0.75 g of inactive impurities. The remaining aqueous solution was concentrated under reduced pressure to about 25 cm3 and, with vigorous stirring, 250 cm3 of absolute ethanol was slowly added dropwise. From the sticky precipitate which forms, the solution containing the active substances is separated by decantation. The precipitate is dissolved once more in a little water and treated with a tenfold amount of absolute ethanol. With the residue, this purification is repeated twice more by precipitation.

   The solutions are combined and evaporated to dryness under reduced pressure. A solid residue of 11.7 g is obtained which contains all of the active substances. rour chromatography on the cellulose column, the residue is dissolved in the minimum of 50% methanol, the solution is mixed well with 40 g of cellulose powder and the material is dried under reduced pressure. The cellulose powder thus loaded with the substance is introduced onto a cellulose column prepared by suspending 350 g of cellulose powder in butanol saturated with water. It is developed by the passage process with butanol saturated with water: fractions of 100 cm3 each are thus separated and evaporated under high vacuum at a bath temperature not exceeding 50.

   Average fractions (3.35 g) give a positive Keller reaction. To complete the purification, they are chromatographed once again by the same process on cellulose powder.



   By development with water-saturated butanol, fractions of 50 cm 3 each are obtained which are evaporated under high vacuum at a bath temperature of at most 50, and which are then examined by the color Keller reaction. We obtain the following distribution:

 <Desc / Clms Page number 13>

 
 EMI13.1
 
<tb> Fraction <SEP> Residue <SEP> in <SEP> Colored <SEP> reaction <SEP> of
<tb>
<tb> N <SEP> mg <SEP> Keller.
<tb>
<tb>
<tb>
<tb>



  1 <SEP> - <SEP> 7 <SEP> 1270 <SEP> negative
<tb>
<tb>
<tb>
<tb> 8 <SEP> - <SEP> 16 <SEP> 87 <SEP> blue <SEP> pure
<tb>
<tb> 17 <SEP> - <SEP> 20 <SEP> 79 <SEP> negative
<tb>
<tb>
<tb>
<tb> 21- <SEP> 30 <SEP> 1053 <SEP> purple
<tb>
<tb>
<tb> 31 <SEP> - <SEP> 43 <SEP> 758 <SEP> negative
<tb>
 
After the treatment with silver carbonate carried out as described in Example 2, the residue of fractions 8 to 16 (87 mg) gives 45 mg of pure psilocin. Fractions 21 to 30 (1053 mg) provide, after treatment with silver carbonate, 765 mg of pure psilocybin.



   From the culture filtrate the active bodies are extracted by the same process. To this end, the filtrate is concentrated under reduced pressure to about 1/10 of its volume. It is precipitated with a ten-fold volume of methanol, the impurities which have precipitated are removed by filtration, the solution is evaporated to dryness under reduced pressure and the residue is extracted three times with a ten-fold quantity of methanol. The treatment of the evaporation residue of the methanolic extracts is continued as indicated above.



  From 12 liters of culture filtrate, 80 mg of psilocybin and 6 mg of psilocin are obtained. Total quantities obtained 845 mg of psilocybin and 51 mg of psilocin, or respectively contents of 0.28% and 0.17% with respect to the dried cryptogamic material.



    EXAMPLE 4.-
Extraction of psilocybin and psilocin from pure cultures of Psilocybe mexicana Heim.



   In this example there will be found an exact description, on the one hand, of the cultivation of the fungus Psilocybe mexicana Heim in vitro for the production of mycelium and sclerotia, on the other hand, of the isolation of the pure active substances.

 <Desc / Clms Page number 14>

 



   Fresh, clear beer wort, not added with hops, is diluted with tap water until the dry extract leader is 4.0-4.5%. To each liter of this solution we add:
0.00417 g of FeSO4.7H2O
0.00172 g of ZnSO4.7H2O
2.0 g of agar
This nutrient solution is introduced at a rate of 500 cm3 per vial into Fernbach flasks with a capacity of 1.6 liters each and sterilized by autoclaving at 108 for 25 minutes.



  After cooling, the flasks are inoculated with 2 cm3 of a suspension of the fungus Psilocybe mexicana Heim each time. The seed body is prepared by depositing pure cultures of basidiospores of the mentioned fungus on a medium of beer wort and agar-agar. The spores which fall from the lamellae of a mature fruiting body are collected on a sterile support, deposited on agar-agar-beer wort and incubated. From the primary cultures which are formed in this way, the seed body can be prepared as follows: under sterile tap water, the mycelium is scraped with a rough spatula so as to obtain a suspension of fine flakes of mycelium.

   With this suspension, Erlenmeyer flasks are inoculated which contain a nutrient solution and porcelain fillers in the form of this stool.



  The best results are obtained with 300 cm3 Erlenmeyer flasks containing 50 g of saddle-shaped filler (size: 1 cm; weight = approximately 0.9 g) and 80 cm3 of nutrient solution consisting of beer wort. about 4% dry extract and 0.2% agar-agar. By incubation at 24, a compact surface layer of mycelium forms after two weeks. The entire culture is stirred for 30 minutes on a rotary shaker. The stool-shaped fillers then crush the mycelium, resulting in a fine suspension of mycelium flakes.



  The seed body thus obtained is sufficient to seed 25 liters of nutrient solution.

 <Desc / Clms Page number 15>

 



   The inoculated cultures are incubated at 24-26 in the dark. It forms the layer of mycelium already described with very many sclerotia reaching in general a dimension of up to about 1 cm (some can become much larger. To separate the mycelium and the sclerotia, the mature cultures are filtered through through gauze, press and dry at 35-40 in an oven.



  If we started from 134 flasks of Fernbach containing 67 liters of nutrient solution, we obtain, by harvesting after 62 days, 1149 g of dry matter (sclerotia and mycelium) that is to say 17, 14 g per liter of nutrient solution used.



   In order to isolate the active substances, 612 g of dried sclerotia and mycelium are sprayed very finely, for example, it is extracted 3 times beforehand with 1 liter of chloroform each time. then three more times each time with 1 liter of chloroform supplemented with 10 ethanol. 5.6 g of inactive impurities then pass into the preliminary extract. The previously extracted cryptogamic material is then used up, once with 6 liters, then another 3 times with 3 liters of methanol each time. The methanolic extracts are combined and evaporated to dryness under reduced pressure, thereby obtaining 35 g of a light brown residue. In order to remove the lipoid impurities therefrom, this residue is suspended in 35 cm3 of water and it is shaken once with 1 liter and twice with 0.5 liter of petroleum ether each time.

   Petroleum ether contains 1.5 g of inactive impurities. The remaining aqueous solution is first concentrated under reduced pressure to about 50 cm3, after which 500 cm3 of ethanol is added with vigorous stirring. From the sticky precipitate which is then formed, the solution containing the active substances is separated by decantation. The precipitate is dissolved again in a little water and treated with a tenfold quantity of absolute alcohol. This precipitation operation is repeated twice with the residue.



  The solutions are combined and concentrated to dryness under reduced pressure. A solid residue of 23.4 g is obtained which contains all the active substances.

 <Desc / Clms Page number 16>

 



   For the chromatography on a cellulose column, the residue is dissolved in the minimum of 50% methanol, the solution is mixed well with 80 g of cellulose powder and the material is dried under reduced pressure. The cellulose powder thus loaded with the substance is introduced onto a cellulose column which gold, has prepared by suspending 700 g of cellulose powder in butanol saturated with water. Developed with water-saturated butanol by the pass method and separated fractions of 200 cm3 each. They are concentrated under high vacuum at a bath temperature not exceeding 50. We then obtain the following distribution: TABLE 1.
 EMI16.1
 
<tb>



  Fraction <SEP> hesidu <SEP> in <SEP> g <SEP> reaction <SEP> colored <SEP> of
<tb>
<tb>
<tb> n <SEP> Keller.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  1 <SEP> - <SEP> 14 <SEP> 2,170 <SEP> negative
<tb>
<tb>
<tb>
<tb> 15 <SEP> - <SEP> 34 <SEP> 6,660 <SEP> positive
<tb>
<tb>
<tb>
<tb>
<tb> 35 <SEP> - <SEP> 40 <SEP> 3,540 <SEP> negative
<tb>
<tb>
<tb>
<tb>
<tb> 12,370
<tb>
 the remainder, ie approximately il g, is retained in the column.



   Fractions 15 to 34, which provide a residue of 6.660 g in total, close the totality of the active substances. In order to complete the purification they are chromatographed again as described below. The residue of 6.66 g is dissolved in the minimum of methanol and with the solution 20 g of cellulose powder are impregnated. After drying, this powder is introduced onto a column of 600 g of cellulose powder previously treated as indicated above. By developing with water-saturated butanol, fractions of 100 cm3 each are collected. After evaporating them under high vacuum, they are subjected to the color reaction.

   We then obtain the following distribution:

 <Desc / Clms Page number 17>

    TABLE 2.
 EMI17.1
 
<tb> Fraction <SEP> residue <SEP> in <SEP> traction <SEP> colored <SEP> of
<tb>
<tb> n <SEP> mg <SEP> Keller.
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>



  1 <SEP> - <SEP> 8 <SEP> 2.950 <SEP> negative
<tb>
<tb>
<tb> 9 <SEP> - <SEP> 17 <SEP> 180 <SEP> blue <SEP> pure
<tb>
<tb>
<tb>
<tb> 18 <SEP> - <SEP> 21 <SEP> 155 <SEP> negative
<tb>
<tb>
<tb> 22 <SEP> - <SEP> 31 <SEP> 912 <SEP> purple '
<tb>
<tb>
<tb>
<tb> 32 <SEP> - <SEP> 45 <SEP> 1.510 <SEP> negative
<tb>
 
After treatment with silver carbonate as described in Example 1, the residue from fractions 9 to 17 (180 mg) provides 90 mg of pure psilocin.



   After the treatment with silver carbonate as described in Example 1, fractions 22 to 31 (Table 2) give 619 mg of pure psilocybin having the properties described in Example 1. The active substances are extracted. culture filtrate by the same process as that previously described for sclerotia.



  To this end, the culture filtrate is concentrated under reduced pressure to about 1/10 of its volume. It is precipitated with a ten-fold volume of methanol, filtered and the residue is extracted 3 times with a ten-fold quantity of methanol each time. one continues the treatment of the evaporation residue of the methanol extracts in the manner previously described. From 10 liters of culture filtrate, for example, 220 mg of psilocybin and 12 mg of psilocin are obtained.



  EXAMPLE 5.-
Extraction of psilocybin and psilocin from pure cultures of Stropharia cubensis Earle.



   A nutrient solution is prepared as follows: fresh and clear beer wort, not added with hops, is diluted with tap water to a dry extract content of 6%. To each liter of this solution we add:

 <Desc / Clms Page number 18>

 0.0021 g of FeSO4.7H2O 0.0009 g of ZnSO4.7H2O
 EMI18.1
 1.0 g of Ca (PF03) 2 0.0624 g of KR2P04 0.0621 g of MgS04.rIHZO
0.0312 g of KC1
2.0 g of agar
This nutrient solution is sterilized as in Example 3 and inoculated by adding, per liter, 2 cm3 of a suspension of the fungus Stropharia cubensis arle from Cambodia. This sowing suspension is prepared as indicated in Example 3 under a).

   After 52 days of incubation in the dark at 24, we obtain,
 EMI18.2
 if we started with 20 liters, 452 s of cryto material, a: i; ue dried, that is to say 226, g per liter. The active bodies are isolated from the cryptogenic material by proceeding as indicated in Example 3. Quantities obtained = 1.033 mg of crystal psilocybin.
 EMI18.3
 pure and 45 ...: 16 of psilocin, i.e. J, 2 ,, and O, 010 / respectively, relative to the cryptogenic substance SCII (e.



  EXEiPLw- 6.- Lxtraczion of psilocybin and psilocin from PSilocybe se # ervib2 Reia and Cailleux of natural origin.



   The mature fruiting bodies of Psilocybe semperviva Hein and Cailleux obtained by artificial cultivation on natural nutrient media (compost) are carefully dried at 30 by a current of air. 32 g of dried fruiting bodies are ground very finely and shaken at room temperature once with 300 cm3 and three times with 150 cm3 of methanol each time for 1/2 hour. Extracts are combined and evaporated to dryness under reduced pressure. To degrease it, knead the residue (3.3 g)
 EMI18.4
 four times each time with 125 cc of petroleum eïlC1 and three times with each time 50 cm3 of chloroform containing 10; 6 ethanol. A residue of 6.5 g is obtained.

   This residue is dissolved in 6 cm3 of water and, to the solution, 60 cm3 of absolute ethanol is slowly added.

 <Desc / Clms Page number 19>

 to precipitate other impurities and thus enrich the solution in active substances. The purification is repeated two more times in the same way. The solutions are decanted, combined and evaporated to dryness under reduced pressure. The residue is taken up in metha-
 EMI19.1
 no and it is chromatographed on COûD1e cellulose powder described in Example 2. By treatment with silver carbonate the halogen is removed from the active substances thus obtained. After recrystallization,
 EMI19.2
 obtains' 1 Lf c4c-- s 0 h cristllisc and 32 s of ilccie, ie a content of 0.5J and 01 / d respectively with respect to the dried cryptogamic material.



  EXAMPLE 7.-
Extraction of psilocybin from Psilocybe caerulescens
 EMI19.3
 ¯Iurrill var. Mazatecorusi Breast of natural origin.



  Ripe fruiting bodies of Psilocvbe caerule: cens Aurrill var. azatecorus ei obtained by artificial cultivation on natural nutrient media (compost), after which the dried fungal material is very finely ground (7.3 g)
 EMI19.4
 and it is exhausted with methanol thus cu-lil has been described in 1-lexein-ile 2. The extracts are combined and evaporated to dryness under reduced pressure. The treatment of the residue is continued as indicated in Example 2 turn to reach the active bodies. 14.6 m ± of pure psilocybin is obtained, ie a content of 02% of psilocybin ();:; I 'r, nwprt at lz 7! 1;, d :: dried cryptogamic i.ère). From the treated mushroom sample no psilocin is obtained.



  EXAMPLE 8. -
Extraction of psilocybin from pure cultures of Psilo-
 EMI19.5
 cybe caerulescens 1> Iurrill var. î-lazatecorusi Heils.



   The culture medium is prepared by diluting clear, fresh beer wort, not added with hops, with tap water to bring the dry extract content to 4.0%. ± each liter of this solution is added:

 <Desc / Clms Page number 20>

   10, it g of nCornsteep Solids "0.00834 of ReSO4.7H2O 0.20 g of Ca (OH) 2,
0.20 g of KH2PO4 0.25 g of NH4OH
2.0 g of agar-agar pH of the solution:

   5.4
This nutrient solution is sterilized as in Example 3, it is ensenece with the suspension of mycelium of a pure culture of Psilocybe caerulescens urrill var. Mazatecorum Heim from the lexicon and the resulting cultures are incubated in 26 to 3 darkness. After 48 days, 20.4 g of dried cryptogamous material are collected per liter of solution. The isolation and purification of the active bodies from this material are carried out according to the indications given in Example 3. From 10 liters of nutrient solution, one obtains: 449 mg of psilocybin, ie a content of 0, 22% based on the dried cryptogamic material. Psilocin is not found in the treated material.



  EXAMPLE 9. -
Extraction of psilocybin from Psilocybe Zapotecorum Heim of natural origin.



   Carefully dry (residue 42.4 g) the mature fruiting bodies of Psilocybe Zapotecorum Heim which have been collected and sorted in Chatino country (Mexico), are very finely ground and shaken with methanol as indicated. to Example 2. The methanolic extracts are combined and evaporated to dryness under reduced pressure. The treatment of the residue is continued as indicated in Example 2. 212 mg of pure psilocybin are obtained, ie a content of 0.5%. From the treated cryptogamic material, no psilocin is obtained.



  EXAMPLE 10. -
Extraction of psilocybin from pure cultures of Psilocybe Zapotecorum Hein.

 <Desc / Clms Page number 21>

 



   Clear and fresh beer wort, without the addition of hops, is diluted with tap water to bring the dry extract content to 4.5%. To each liter of this solution is added:
0.00834 g of FeSO4
10.0 g of "Cornsteep Solids" 0.30 g of NH4OH
0.30 g of KH2PO4
2.0 g of agar-agar pH of the solution: 5.5.



   This culture medium is sterilized as described in Example 3 and is inoculated with the suspension of -mycelium of a pure culture obtained from spores of mature fruiting bodies of Psilocybe Zapotecorum Heim from Chatino country. from Mexico. After 57 days of incubation in the dark at 24, 430 g of dried cryptogamic material, that is to say 17.2 g per liter, is obtained from 25 liters. To isolate the active bodies of this material, one proceeds according to the indications given in Example 3. Quantity obtained: 903 mg of pure psilocybin, ie a content of 0.21% with respect to the dried cryptogamic material. No Psilocin.



  EXAMPLE 11. -
Extraction of psilocybin and psilocin from Psilocybe Aztecorum Heim of natural origin.



   The mature fruiting bodies of Psilocybe Aztecorum Heim collected in Mexicue in the Aztec region (on the Popocatépelt at an altitude of 3,300 to 3,500 m above sea level) are carefully dried. They are very finely ground and thoroughly extracted with ethanol as described in Example 2. The extracts are combined and concentrated to dryness under reduced pressure.



  The treatment of the residue is continued as indicated in Example 2 to obtain the active substances. 570 mg of pure psilocybin and 47 mg of psilocin are obtained, ie contents of 0.2; and 0.017%, respectively, based on the dried cryptogamic material (285 g).

 <Desc / Clms Page number 22>

 



  EXAMPLE 12. -
Extraction of psilocybin and psilocin. from pure cultures of Psilocybe Aztecorum Heim.



   The nutrient solution is prepared as follows:
100 g of malt extract 0.00417 g of FeSO4.7H20
0.00172 g of ZnSO4.7H20
1.0 g of Ca (NO3) 2
0.25 g of 0.25 g MgSO4.7H20 of cm3
0.125 g of KCl 210 g of agar-agar and the whole is spread with tap water up to 1000 cm3.



   The nutrient solution is autoclaved at 108 for 25 minutes. 1 liter of this nutrient solution is inoculated with 244 of a suspension of the fungus Psilocybe Aztecorum Heim.



  This seeding suspension is prepared according to the indications given in Example 3 under a). The cultures are incubated in the dark for 45 to 24 days and harvested as described in Example 3 under c). Amount obtained from 10 liters of nutritive solution: 86.5 g of dried mycelium. The very finely ground mycelium is shaken with 1 liter of 80% aqueous ethanol for 1/2 hour at room temperature. After removing the residue by filtration, this residue is extracted three more times in the same way. The extracts are combined and evaporated.

   To remove the lipoid impurities and inert fillers from the evaporation residue (8.9 g), extraction is carried out successively twice with 100 cm3 of petroleum ether, twice with 80 cm3 of chloroform and twice with 50 cm3 of 'acetone. 5.6 g of a water-soluble powder remain, which are dissolved in 6 cm3 of water. With good stirring, 60 cm3 of acetone is slowly added to the solution, the precipitate formed is separated off, it is redissolved in a little water and it is precipitated once again with a tenfold quantity of acetone.

   We repeat again

 <Desc / Clms Page number 23>

 twice this purification by precipitation on the part insoluble in acetone, after which all of the active substances are passed into the aqueous-acetone extracts. The extracts are evaporated off under reduced pressure and the purification of the residue is completed.



  (2.8 g) by chromatography on a column of cellulose powder as indicated in Example 3.



   After two chromatographies, 225 mg of crystallized psilocybin and 15 mg of psilocin are obtained, ie yields of 0.26% and of 0.017, b, relative to the dried cryptogamic material.



  EXAMPLE 13.-
Preparation of a culture.



   A nutrient solution is prepared as follows:
Fresh beer wort (clear and free of hops) is diluted with tap water to bring the dry extract content to 4.0 - 4.5% To each liter of this solution is added: 0.00209 g of FeSO4.7H20 0.00086 g of ZnSO4.7H20
0.25 g of Ca (NO3) 2 0.0625 g of KH2PO4
0.0625 g of MgSO4 0.31 g of KC1
2.0 g of agar
This nutrient solution is sterilized as in Example 4 and sown. After 48 days of incubation at 24, 924 g of sclerotia and mycelium (dry extract) are obtained from 55 liters, c ' i.e. 16.6 g per liter.

   The isolation of the active substance can be carried out as described in Example 4. Yields: psilocin 0.018 and psilocybin 0.12 based on the dried cryptogamic material.



  EXAMPLE 14 Repair of a Culture.



   A nutrient solution is prepared as follows:
Fresh beer wort (clear and free of hoppers) is diluted with tap water to increase the extract content.

 <Desc / Clms Page number 24>

 dry at 4.0 - 4.5% To each liter of this solution is added:
 EMI24.1
 0.00209 g of eSO. T3z0
0.00086 g of ZnSO4 7H20
1.0 g of Ca (N03) 2-
2.0 g agar
The nutrient solution is sterilized as in Example 4, seeded and then incubated. After 48 days, 20.5 g of dried sclerotia and mycelium are harvested per liter of nutrient solution, from which the active bodies are isolated as in Example 4. Quantities obtained: psilocin 0.011% and psilocybin relative to the dried cryptogamic material.
 EMI24.2
 



  MM, * eLZ-25, -
Preparation of a culture:
A nutrient solution is prepared as follows:
Fresh beer wort (clear and free from hoppers) is diluted with tap water to bring the dry extract content to 4.0 - 4.5%. To each liter of this solution is added:
0.00209 g of FeSO4.7H20
0.00086 g of ZnSO47H20
 EMI24.3
 OY25 g of KH2P0,
2.0 g of agar
The treatment is continued as described in Example 4. After 48 days the production, per liter of nutrient solution, of dried sclerotia and mycelium is 15.9 g. To isolate the active bodies, the procedure is as in Example 4. Yields: psilocin 0.02% and psilocybin 0.11% of the dried cryptogamic material.



  EXAMPLE 16.-
Preparation of a culture.



   A nutrient solution is prepared as follows:
Fresh beer wort (clear and free of hops) is diluted with tap water to bring the dry extract content to 4.0 - 4.5. To each liter of this solution is added:

 <Desc / Clms Page number 25>

 
0.00209 g of FeSO4.7H20 0.00036 g of ZnS04.7H20
20.0 g of Cornsteep Solids
2.0 g of agar-agar.



   The treatment is continued as indicated in Example 4.



  After 48 days, 29.8 g of dried mycelium and sclerotia are obtained per liter of nutrient solution.



   The active bodies are isolated as indicated in Example 4.



  Yields: psilocin 0.012; and psilocybin 0.22% of the dried cryptogamic material.



  EXAMPLE 17.-
Preparation of a culture.



   A nutrient solution is prepared as follows:
45 g malt extract
0.00417 g of FeSO4.7H20
0.00172 g of ZnSO4.7H20
1000 Cm3 of city water.



   The nutrient solution is autoclaved at 108 for 25 minutes. 1 liter of this solution is inoculated with 10 cm3 of a suspension of the fungus Psilocybe mexicana. The preparation of this seeding suspension is carried out according to the indications given in Example 4. After seeding, the fungus is cultivated in this nutrient solution, by the mobile immersion process, at 24. Tapioca-like spheres of celium form, as is known in the submerged culture of lower fungi. After 30 days the mycelium is filtered off and 7.0 g of dried mycelium are obtained per liter of nutrient solution.



   430 g of finely ground dry mycelium are shaken for half an hour at room temperature with 4.5 liters of 80% aqueous etanel. After separation of the residue by filtration, this residue is extracted 3 more times in the same way. The extracts are combined, then evaporated. To remove the inert charges from the residue (41 g), extraction is carried out successively with 2 times 500 cm3 of petroleum ether,

 <Desc / Clms Page number 26>

 2 times 400 cm3 of chloroform and 2 times 200 cm3 of acetone. There remains 25 g of a water soluble powder which is dissolved in 25 cm3 of water.

   While stirring well, slowly add 250 cm3 of acetone, then the liquid is separated and precipitated and the latter is redissolved in a little water. Thereafter, it is precipitated again with a ten-fold amount of acetone. This operation is repeated twice more on the part insoluble in acetone: this treatment has the effect of causing all of the active substances to pass into the aqueous-acetone extracts. These extracts are evaporated with good stirring and the residue (9.8 g) is chromatographed on a cellulose column, as indicated in Example 4.



   After further chromatography, 32 mg of crystallized psilocin and 225 mg of psilocybin are obtained, ie respective yields of 0.007 and 0.05% relative to the dry mycelium.


    

Claims (1)

ABSTRACT. ------------ The present invention comprises in particular: 1) A process for obtaining psilocybin and psilocin, compounds hitherto unknown, endowed with psychotropic properties, from fungi, process according to which the active bodies are extracted, by known methods, from cryptogami materials - ques of natural origin of the Psilocybe, Stropharia, Panaeolus, Conocybe, Amanita or r.ussula species, or of artificially developed cryptogamic materials, obtained from cultures of these fungi or from their biological variants or mutants , by inoculating natural or artificial nutrient media and incubating these cultures in daylight or in the dark, at a constant temperature between 18 and 27, these active substances are purified, they are separated from one another and optionally the halogen is removed. 2. - Modes of execution of the process specified under 1, having the following peculiarities taken separately or according to the various possible combinations: a) the bodies are extracted by known methods <Desc / Clms Page number 27> active ingredients of Psilocybe mexicana Heim or of artificially developed cryptogamic materials, obtained from pure cultures or from biological variants or mutants of this fungus, by inoculation of natural or artificial nutrient media and incubation of these cultures in daylight or in the dark, at a constant temperature between 18 and 27, the active substances are purified, they are separated from one another and optionally the halogen is eliminated; b) the fungus Stropharia cubensis Earle is used as starting material; c) the fungus Psilocybe semperviva Heim et Cailleux is used as starting material; d) the fungus Psilocybe caerulescens Murrill var is used as starting material. Mazatecorum Heim; e) the fungus Psilocybe Zapotecorum Heim is used as starting material; f) the fungus Psilocybe Aztecorum Heim is used as starting material; g) to obtain an active fungal material (fruiting bodies), the fungi are grown on a natural substrate and the cultures are incubated in daylight at a temperature between 18 and 27; h) to obtain an active cryptogamic material (mycelium and sclerotia) in large enough quantity, the fungi are cultivated in vitro on artificial nutrient media based on malt extracts having a dry extract concentration of 4 to 10% depending on the es - species of fungus, 0.0004 to 0.0010 g of ferrous ions per liter and 2 per liter of agar-agar are added to the nutrient solution and the cultures are incubated in the dark at a constant temperature between 22 and 26; i) the defatted cryptogamic material is subjected to extraction with water or with an organic solvent miscible with water, the inactive impurities are removed from the aqueous solution of the <Desc / Clms Page number 28> extraction residue by fractional precipitation with an organic solvent miscible with water and the active substances contained in the filtrate are separated from each other by absorption on cellulose powder and elution with saturated butanol. 'water; j) the halogen is removed from the active substances obtained which contain it, before or after having separated them, by treatment with silver carbonate according to known methods.