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Offlineranonar
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Registered: 07/27/06
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Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz * 2
    #7680529 - 11/26/07 05:39 AM (13 years, 6 months ago)

OK, I spent Sunday night browsing through the shroomery archives. and I found this remark:

"Hm. How come nothing ever comes of this talk, even though it happens every few years? Just like the tryptamine HCL talk."

http://www.shroomery.org/forums/showflat.php/Number/7049809#7049809

Then I read more shroomery posts about Gottlieb's Psilocybin Producers Guide, Workmans' comments that liquid cultures are difficult to test for bacterial contamination, other ppl's comments that liquid mycelium is not potent, the synthetic agar recipe which is supposed to remedy that (it isn't BTW) etc etc.

And then I realized that all those problems are already solved. They were solved in the nineteen fifties already but the idiot who first wrote the "psilocybin producer's guide" made a few big mistakes in the translation. And THAT is why the liquid broth method does not work: in liquids the potency of the mycelium peaks and then goes down. The "Psilocybin Producers Guide" method really can work but only when the mycelium is harvested at exactly the right moment - which is difficult to catch.

A solution to this problem is to use soft agar cultures. For some reason the production peak of the alkaloids is much smoother, one can just wait until the mycelium covers the surface of the agar and then harvest. Using soft agar also solves the contamination problem of liquid cultures since contaminations only appear at the surface and are easily visible. There are many more advantages of using this method (like producing some 150 grams of very clean, no-need-to-be-cleaned-from-seeds-or-vermiculite MexA sclerotia per liter of malt agar medium, which means that almost half of the dry agar ingredients are converted into dry mycelial mass and no expensive grasseeds are needed anymore. Or how about growing thick mats of super potent azurescens or PanCyan mycelium).

The honor of the re-discovery of the benefits of the Brack&Kobel method (the entire method is quoted at the end of this post) should go to dr. Jochen Gartz, who published about it (under pseudonym and in German) in 2003 in Arno Adelaars book Alles Ueber Psilos (=everything about mushrooms). He also compared different mycelia for potency. Not surprising is that azurescens and PanCyan are the winners. These findings are confirmed by Brack&Kobel original patents, which point at another woodlover (Psilocybe caerulescens, copies of the original culture are now in the hands of Workman) as prime psilocybin producer.

Anyway, here is the write-up of the method I made earlier, plus an attempt to write the history of it. Enjoy!

An excellent method to produce psilocybin in large amounts on an agar substrate was discovered in 1958 by drs. Arthur Brack and Hans Kobel. That method was patented, both in Europe and in the US, by the Swiss Pharmaceutical company Sandoz (currently operating under the name Novartis International AG). The American patent was filed at May 11, 1965 and co-signed by Albert Hofmann (who first isolated psilocin and psilocybin), Roger Heim (who collected the Psilocybe mexicana spores which were used for the first cultivated psilocybian cultures) and Roger Cailleux (who collaborated with Heim to grow the first psilocybian mushrooms in the laboratory). In 1976 this Brack&Kobel method was translated and pseudonymously published by ‘Adam Gottlieb’ with the title The Psilocybin Producers Guide - How to produce 5000 doses of organic psilocybin in a small room every week. Ronin Publishing reprinted the book in 1997, re-edited by Bob Harris (author of Growing Wild Mushrooms), under the title Psilocybin Production. The text is also published on-line at http://diseyes.lycaeum.org/teo/psilprod.gui and http://www.erowid.org/plants/mushrooms/mushrooms_cultivation20.shtml

When reading the Gottlieb text one gets the impression that the author has heard something about the Brack&Kobel method but doesn’t know the rights of it. The latter is quite literally so because ‘Gottlieb’ nor Sebastian Orfali (responsible for the 1997 Ronin edition) mention about the patent or even the original publishers: both editions of the brochure are presented as if it were an original publication.

Soft agar cultures

A more severe shortcoming of the Gottlieb version is that the author completely missed the essential components of the Brack&Kobel method. As a result of that nobody will ever be able to produce the promised ‘5000 doses of organic psilocybin’ with it. The essential components of the Brack&Kobel method, the ones which made the method original and patentable, were the following:

One does not need to go through the fruiting process of the mushrooms to get psilocybin; a pure mycelial surface culture is sufficient;
a mycelium of optimum potency can be produced from beerwort, i.e the raw material which is used for beer brewing. Expensive additives or complicated recipes are unneeded. The mycelium can produce both sclerotia (‘Philosopher Stones’ in modern lingo) and mushrooms on this substrate, depending on the concentration of beerwort in the agar;
the efficiency of the technique can be greatly improved by adjusting the concentration of the agar agar powder.

It is especially the third observation which makes the patent so useful. Brack and Kobel did not use a solid agar medium like the ones which are described in other cultivation techniques. Nor did they use a liquid medium like the Gottlieb book suggests. They used something in between: a soft agar culture. The beauty of a soft agar medium is that it ensures that the growing mycelium doesn’t sink to the bottom of the cultivation container where it can not breathe and stops growing.

Problems of liquid cultures

Lack of oxygen is a major problem in liquid cultures. Paul Stamets devotes a full chapter on liquid cultures in his cultivation bible Growing Gourmet and Medicinal Mushrooms. In there mycelia are grown out on petri dishes of solid agar, fragmented in an Eberbach blender, incubated in Erlenmeyer flasks and aerated by use of a magnetic stirbar and a stirplate which is put at 100-200 rpm. All this use of expensive equipment and the extremely contamination-sensitive practice of culture stirring becomes obsolete when one only adds some agar gelling agent to the medium, so that the mycelium stays at the surface. But not too much agar: the harder the surface of the substrate the thinner the mycelium layer which will form and the lower its mass: this is the result of the mycelium being unable to penetrate the agar.

A concentration of 2 promille of agar, about one tenth of the concentration which is used for a solid medium, supports a biological efficiency which is over two and a half times that of PF Substrate. And even though the mycelial tissue contains only half as much alkaloid as the mushrooms may do, high yields (approximately twenty dried grams per liter of medium!) along with easy and rapid cultivation of mycelial tissue more than compensate for the lower psilocybin content. The higher viscosity of a soft agar medium compared to a liquid one provides in another desirable quality as well: the conversion of nutrients into alkaloids proceeds at a much more constant rate. In liquid cultures the mycelium needs to be harvested at the moment that the alkaloid production peaks. After that the mycelium begins to break down the alkaloids and may use them up in a matter of days. In August 1963, the researchers Catalfomo and Tyler pointed out that, in their experiments with Psilocybe cubensis mycelium, that moment was at exactly seven days after inoculation. However, that moment is difficult to recognize in liquid cultures, especially when they are constantly stirred to provide them of oxygen. Catalfomo and Tyler had to monitor the acidity of the medium and the growth speed of the mycelium to find it. In contrast, by using a soft agar culture one can just wait until the mycelium has covered the surface of the substrate.

Psilocybe azurescens tops the charts

In 1999 the German mycologist dr Jochen Gartz dedicated a chapter on the soft agar cultivation method accompanied with three beautiful photo’s of Psilocybe cubensis fruiting on that medium in an erlenmeyer flask in his book Narrenschwämme.

Experiments with other mushrooms and the same method were published in August 2003 in Arno Adelaars’ German book Alles über Psilos. Handbuch der Zauberpilze.

Arno Adelaars’book

The researcher(s?) published their chapter pseudonymous (‘by F. Spitzkegulus & B. Paramycelius’) under the title ‘Psilocybin-Pilzkultur ganzjährig zu Hause’. They used sixty grams of malt sugar per litre and found mycelia of Panaeolus cyanescens (also known as Copelandia and ‘blue meanie’) and Psilocybe azurescens (the ‘flying saucer mushroom’) to be one and a half to double the potency of Psilocybe cubensis mycelium. The mycelia of the indigenous psilocybian mushrooms of Europe were not as potent: only Psilocybe cyanescens mycelium was about the same as that of Psilocybe cubensis but mycelia of Panaeolus subbalteatus and Psilocybe semilanceata contained only half as much psilocybin.

the original Brack&Kobel method:

OBTAINING PSILOCYBIN AND PSILOCIN FROM FUNGAL MATERIAL

United States Patent Office
3,183,172

Patented May 11, 1965

Roger Heim, Paris, France, Albert Hofmann, Botimingen, Basel-Land, Arthur Brack, Riehen, near Basel, and Hans Kobel, Basel, Switzerland, and Roger Cailleux, Pavillons-sous-Bois, France, assignors to Sandoz Ltd., Basel, Switzerland, a Swiss firm

No Drawing. Filed Feb. 16, 1959, Ser. No. 793,234
Claims priority, application Switzerland, Feb. 21, 1958,
56,143; July 30, 1958, 62,393
21 ClaIms. (Cl. 195-80)

The present invention relates to psilocybin and psilocin and to the preparation thereof from the so-called hallucinogenic fungi: Psilocybe, Stropharia, Panaeolus, Conocybe, Amanita and Russula. It has heretofore been impossible to isolate the active substance from samples of the natural fungi or from artificially cultured fungal material; nor has it been possible to culture hallucinogenically active species starting from natural fungi-under conditions that would produce active material in amounts sufficient for obtaining the active substance on a preparative scale.
A primary object of the present invention is the embodiment of an industrially feasible process whereby the hitherto unknown active principles of hallucinogenic fungal species, notably Psilocybe mexicana Heim,
Psilocybe caerulescens Murrill var. nigripes Heim, Psilocybe zapotecorum Heim, Psilocybe semperviva Heim and Cailleux, Psilocybe aztecorum Heim and Stropharia cubensis Earle. are obtained free from halogen.
This object is realized according to the present invention by extracting the active principals either directly from the fungal material of natural origin or from cultures of the fungi or of biological variants or mutants thereof which have been grown on natural or artificial substrates by culture techniques which make it possible to obtain sufficient amounts of active material to permit the isolation of the latter on a preparative scale. Briefly stated, the cultures are incubated in daylight or in the dark at a constant temperature between 18 and 27 centigrade and, after purification, the active substance is separated and freed from halogen, where present.
The process, more specifically, may be carried out as follows: For a culture on a natural substrate, a compost consisting of fermented wheat straw and a mixture of corn leaves and corn stems or stalks of wild grasses is washed well under running water, poured into earthenware dishes and sterilized in an autoclave. The compost is inoculated with mycelium from primary cultures and incubated for approx. two weeks at 24-27 centigrade. The cultures are then covered with sterile sand and left undisturbed in a glass box in the daylight at a temperature of 18-27 centigrade, the moisture content being kept constant.
The fruit bodies appear after 4 to 5 weeks. They are harvested from time to time for a period of 1 or 2 months as soon as the formation of the spores has started.
In developing an alternative process which is better adapted for large scale operation, it was found quite unexpectedly that, grown in vitro on substrates rich in nutritive material, the fungi produce active mycelium and sclerotia in large quantities and only a very small number of fruit bodies; on substrates poor in nutrients, however, the familiar fruit bodies are produced. On an agar medium containing 1.5% by weight of agar, a concentration of 0.2 to 0.7% by weight of dry substance of malt extract is the optimum for the formation of fruit bodies, whereas concentrations of 4 to 10% by weight, depending upon the particular fungal species employed, are optimal for the formation of mycelium and sclerotia.
While daylight is indispensable for the formation of fruit bodies, it has been found that sclerotia and mycelium are formed in greater profusion if the cultures are incubated in the dark. The largest yields of active fungal material (mycelium and sclerotia) are obtained by preparing surface cultures with culture media of malt extract (beerwort or commercial preparations of malt extracts) containing 4 to 10% by weight of dry substance, and by incubation of these cultures in the dark at a constant temperature between 22 and 26 centigrade. Addition of 0.2% by weight of agar to the culture medium enhances good growth. This medium is just sufficiently firm-though it is still almost liquid-to permit the fungus to grow quickly, forming a well-knit mycelium layer. Addition of ferrous salts is very desirable; addition of zinc, potassium, calcium and magnesium salts, of nitrate, phosphate and sulphate ions as well as of yeast extract or of corn steep solids are also found to be very advantageous.
This process of cultivation is a great technical improvement, as it gives a high yield of active starting material; the yield is actually ten times greater than that obtained from a culture on natural substrates and the artificial method takes less time and involves less work.
The active fungal material (fruit bodies, sclerotia and mycelium) is carefully dried in an air current or under reduced pressure at 20-40 centigrade, finely ground and thoroughly extracted with a lower aliphatic alcohol or with a mixture of water and a water-miscible organic solvent at room temperature (20 to 30 centigrade). The extracts are concentrated under reduced pressure at low temperature. The residue is defatted with petroleum ether and extracted with acetone or chloroform-alcohol to remove inactive accompanying material. Other ballast material is separated off by dissolving the residue in as little water as possible and repeated precipitation with absolute ethanol or acetone; the filtrate is concentrated under reduced pressure at low temperature.
Further purification is advantageously carried out by chromatography on cellulose powder in a through-flow process; elution is performed with water saturated butanol or another alcohol not miscible with water. The fractions collected are tested for their content of active substance by means of the Keller reagent (glacial acetic acid containing iron chloride and concentrated sulphuric acid). The fractions showing a positive colour reaction are combined and, if necessary, chromatographed again on a column of cellulose powder. From the through-flow chromatogram a rather rapidly travelling zone is eluted. This yields a product containing an active substance, "psilocin," characterized by a clear blue Keller reaction, while from a zone travelling more slowly, a product containing a second active substance, "psilocybin," is obtained in larger amounts.
The active substances are obtained in a fairly pure state from the column but contain halogen and do not crystallize out as psilocybin and psilocin. The halogen may be removed only by chemical treatment, crystalline compounds being thereby obtained. For this purpose, an aqueous solution of the active material is treated with silver carbonate or silver oxide. Excess silver ions are removed with hydrogen sulphide and the remaining solution is concentrated under reduced pressure at low temperature, the substances crystallizing out from the concentrated solution.
For analysis, psilocybin is recrystallized from methanol or water. Recrystallization from water yields very fine white needles; from methanol colourless hexagonal plates or prisms are produced; these contain methanol and melt at 195-200 centigrade (with decomposition). The compound dissolves in 120 parts by weight of boiling methanol or in 20 parts by weight of boiling water; it dissolves difficulty in higher alcohols and other organic solvents. The crystals are dried in a high vacuum at 100 centigrade, a decrease in weight of 10.4% taking place. The results of elementary analysis give the empirical formula C12H17O4N2P (molecular weight 284.2). Psilocybin is an amphoteric compound. It is optically inactive and readily soluble in dilute aqueous mineral acids and in dilute aqueous alkalis with which it forms salts. A solution of psilocybin in 80% (by weight) aqueous ethanol has a faintly acid reaction (pH 5.2). The UV-spectrum in a methanolic solution shows maxima at 222, 267 and 290 µm.
For analysis, the psilocin is purified by another chromatographic operation on a column of cellulose powder, using water-saturated butanol or by treatment with potassium bi-carbonate in an aqueous solution and extraction with ether or an organic solvent. The results of elementary analysis give the empirical formula C12H16NO2. Psilocin crystallizes from methanol or acetone; it is moderately soluble in water but dissolves readily in dilute acid. M.P. 173-176 centigrade. (with decomposition). Psilocybin is the phosphoric acid ester of psilocin 4-hydroxy-dimethyl tryptamine (Experientia, vol. 14, 1958, pages 397-399).
Psilocin is characterized by an UV-spectrum in a methanolic solution with maxima at 222, 260, 267, 283 and 293 µm and by the Keller colour reaction which shows -unlike psilocybin- a clear blue colour.
The products of the present invention are psychotropically active and find use in therapy as tranquillisers. Subcutaneous injection or oral administration of 2 to 8 mg. of psilocybin produces a pronounced euphoric mood accompanied by a lack of spontaneity and a feeling of indifference. When administered in higher dosage, there occur changes in perception together with autonomic symptoms.
Both psilocin and psilocybin are useful for research into mental disease and psychoses. They are also useful as an aid to psychotherapy in mental patients (tranquillisation, anxiety repressing, etc.).
In the following illustrative examples, parts by weight bear the same relationship to parts by volume, as do grams to millilitres. Percentages are by weight. Temperatures are in degrees centigrade.

EXAMPLE 1
Psilocybin and psilocin from fruit bodies of Psilocybe mexicana Heim obtained by artificial cultivation.
For cultivation on a natural substrate a compost of fermented wheat straw is prepared, washed thoroughly with running water, poured into earthenware pots and sterilized in the autoclave. The compost is inoculated with mycelium from primary cultures of Psilocybe mexicana Heim and incubated for approx. two weeks at 24-27 centigrade. The cultures are then covered with sterile sand and left in a glass case at a temperature of 21-22 centigrade in the daylight. After 4 to 5 weeks the fruit bodies appear when the spores are beginning to form, the fruit bodies are gathered from time to time over a period of 1 to 2 months. The ripe fruitbodies of Psilocybe mexicana Heim are then, carefully dried in an air current at 30 centigrade. 54 parts by weight of the dried fungi are finely powdered and extracted once with 600 parts by volume and three times with 300 parts by volume of methanol for 30 minutes each time. The extracts are combined and evaporated to dryness in vacuo. Residue: 12 parts by weight.
To defat the methanol residue, it is rubbed four times with 250 parts by volume of petroleum ether and three times with 100 parts by volume of chloroform containing 10% of ethanol. The undissolved residue of approximately 10 parts by weight is dissolved in 10 parts by volume of water and the solution is mixed gradually with 100 parts by volume of absolute ethanol. The amount of active substance in the solution is thereby increased. This operation is repeated two or three times. The solutions are decanted off and evaporated to dryness under reduced pressure; the residue is again dissolved in methanol and the solution treated with 20 parts by weight of cellulose powder containing 5% of water. The methanol is evaporated off under reduced pressure and the cellulose powder bearing the active material is poured onto a column of 100 parts by weight of cellulose powder containing 5% of water, the column having previously been washed with water-saturated butanol. The column is eluted with water-saturated butanol and fractions of 20 parts by volume are collected.
The evaporation residues from the individual fractions are tested for their content of active material by means of the Keller colour reaction. For this purpose, 2 millilitres of Keller reagent are added to samples of 0.25 milligram of evaporation residue.
The fractions showing a positive colour reaction are combined. The amorphous powder is dissolved in 20 parts by volume of water and shaken with 0.5 parts by weight of silver carbonate. The solution is filtered and desilverized with hydrogen sulphide and then carefully concentrated. Psilocybin crystallizes from the concentrated solution in the form of fine colourless needles (yield 200 parts by weight).
Psilocin is obtained only in trace amounts from the fruit bodies. Psilocybin is obtained in an analytically pure state by a further recrystallization from methanol or water. It dissolves boiling methanol or in water at the boiling point.
Colourless prisms are obtained from methanol, which melt at 195-220 centigrade (with decomposition).
The results of elementary analysis give the empirical formula C12H17O4N2P. The UV-spectrum in a methanolic solution shows the following maxima: 222 µm (micrometer), 267 µm and 290 µm.
The new substance is amphoteric. It dissolves to form salts in diluted aqueous alkalis as well as in aqueous acids. A solution of psilocybin in 80% aqueous alcohol has an acid reaction, (pH 5.2).

EXAMPLE 2
Obtaining psilocybin and psilocin from Stropharia cubensis Earle obtained by artificial cultivation

The fruit bodies of Stropharia cubensis Earle collected in Mexico are carefully dried at room temperature in a shady place in the air. 24.2 parts by weight of dried fruit bodies are thoroughly ground and extracted once with 300 parts by volume and then three times with 150 parts by volume of methanol, each time at room temperature for 30 minutes. The extracts are combined and evaporated to dryness under reduced pressure. The residue (6 parts by weight) is defatted by rubbing well four times with 125 parts by volume of petroleum ether and three more so times with 50 parts by volume of chloroform each time, the chloroform containing 10% of ethanol. The undissolved residue (59 parts by weight) is dissolved in 5 parts by volume of water. From this solution other products are precipitated by slowly adding 50 parts by volume of absolute ethanol so that the active substance accumulates in the solution. The purification is repeated two or three times in the same manner. The decanted solutions are combined and evaporated to dryness in vacuo. The residue is taken up in methanol and treated with 10 parts by weight of cellulose powder containing 5% of water. The methanol is evaporated off under reduced pressure and the cellulose powder bearing the active substance is poured onto a column of 50 parts by weight of cellulose powder containing 5% of water, the column having previously been washed with water-saturated butanol. After extraction with water-saturated butanol, fractions of 10 parts by volume are collected. The individual fractions are evaporated in a high vacuum at a maximum bath temperature of 50 centigrade and are tested for their content of active material by means of glacial acetic acid containing ferric chloride and concentrated sulphuric acid. For this purpose, 2 millilitres of Keller reagent are added to samples of 0.25 milligram of the residue. The active fractions are characterized by a violet (psilocybin) or a clear blue (psilocin) Keller reaction. The fractions showing a positive colour reaction of the same shade are combined. The amorphous powder of the above evaporation residues is dissolved in 2 parts by volume of water and extracted with 0.25 parts by weight of silver carbonate. After filtering, the excess of silver ions is removed with hydrogen sulphide. Upon carefully concentrating the solution, the psilocybin crystallizes as thin colourless needles.

EXAMPLE 3
Obtaining psilocybin and psilocin from pure cultures of Psilocybe semperviva Heim and Cailleux

(a) PREPARING THE INOCULATION MATERIAL

Pure cultures of the basidiospores of the Mexican fungus Psilocybe semperviva Heim and Cailleux are grown on beerwort-agar. For this purpose, the spores falling from the lamellae of a ripe fruit body are collected on sterile material, placed on beerwort-agar and incubated. From the primary cultures so produced the inoculation material is prepared in the following manner in sufficient amount:
The mycelium is scraped off with a rough spatula, a suspension of fine mycelium flakes being formed. This suspension is used to inoculate flasks containing a culture medium and saddle-shaped porcelain filters such as are used for filling distillation columns.
The best results are obtained with 300 millilitre flasks containing about 50 saddle-shaped filters (size 1 cm., weight approx. 0.9 gram) and 80 millilitres of a culture medium consisting of beerwort containing approx. 4% of dry substance and 0.2% of agar. The culture is incubated at a temperature of 24 centigrade. After 2 weeks a compact mycelium layer has formed on the surface. The whole culture is shaken for 30 minutes on the rotating shaking machine, the sharp edges of the saddle-shaped filters grinding the mycelium so that a fine suspension of mycelium flakes is formed. The inoculation material so obtained is sufficient to inoculate 50 litres of culture medium.

(b) PREPARATION OF CULTURE
Fresh and clear beerwort, without hops, is diluted with tap water to a content of 8% of dry substance. To each litre of this solution there are added:

Ingredient Amount (grams)
FeSO4 ° 7H20 0.00417
ZnSO4 ° 7H2O 0.00172
Ca(NO3)2 1.0
KH2PO4 0.0624
MgSO4 ° 7H2O 0.0624
Kcl 0.0312
Agar-agar 2.0

This culture medium is poured into penicillin flasks and the latter are then sterilized in the autoclave at 108 centigrade for 25 minutes. On cooling, the flasks are each inoculated with 2 millilitres of a suspension of Psilocybe semperviva Heim and Cailleux. The cultures so obtained are incubated in the dark at 24-26 centigrade, the mycelium layer described under 3(a) thus being formed.

(c) ISOLATION OF THE FUNGAL MATERIAL

After 7 weeks the ripe culture is filtered through a gauze tissue, the fungal material is squeezed out and dried in vacuo at 30 centigrade. 2540 grams of dry material, i.e. 25.4 grams per litre of culture medium are obtained after 49 days from a batch of 100 penicillin flasks containing 100 litres of culture medium. The culture filtrate containing active material is worked up according to the procedure described in the following paragraph for the fungal material.

(d) OBTAINING THE ACTIVE MATERIAL
306 parts by weight of the dried fungal material are finely powdered and shaken 3 times with 500 parts by volume of chloroform each time and 3 more times with 500 parts by volume of chloroform containing 10% of ethanol. 2.8 parts by weight of inactive accompanying substance are thereby dissolved. The pre-extracted fungal material is thoroughly extracted once with 3000 parts by volume and 3 times with 1500 parts by volume of methanol each time. The combined extracts are evaporated to dryness under reduced pressure, a clear brown residue of 17.5 parts by weight remaining. In order to remove fatty impurities from this residue, it is taken up in 17.5 parts by weight of water and the suspension is extracted once with 500 parts by volume and twice with 250 parts by volume of petroleum ether each time. The petroleum ether solution contains 0.75 parts by weight of inactive accompanying material. The residual aqueous solution is concentrated under reduced pressure to about 25 parts by volume and is then treated with 250 parts by volume of absolute ethanol, while being vigorously stirred. From the sticky precipitate so produced, the solution containing active material is separated by decantation. The precipitate is redissolved in a little water and treated with a tenfold quantity of absolute ethanol. This purification by precipitation is repeated twice with the residue. The solutions are combined and evaporated to dryness in vacuo. There remains a solid residue of 11.7 parts by weight containing the whole amount of active material. For chromatography on a column of cellulose, the residue is dissolved in as little of 50% methanol as possible.
The solution is mixed well with 40 parts by weight of cellulose powder and the material is dried under reduced pressure. The cellulose powder bearing the active substance is poured onto a column of cellulose prepared by suspending 350 parts by weight of cellulose powder in water-saturated butanol. The column is developed in a through-flow process with water-saturated butanol, fractions of 100 parts by volume each being thereby separated and then concentrated under a high vacuum at a bath temperature not exceeding 50 centigrade. The intermediate fractions (3.35 parts by weight) show a positive Keller colour reaction and are chromatographed again in the same way for further purification.
Fractions of 50 parts by volume each are obtained by developing with water-saturated butanol and, after evaporation in a high vacuum at a bath temperature not exceeding 50 centigrade, are tested by means of the Keller colour reaction.
The fractions obtained are as follows:

Fraction No. Residue parts by weight Keller colour reaction
1-7 1.27 Negative
8-16 0.087 Clear blue
17-20 0.079 Negative
21-30 1.053 Violet
31-43 0.758 Negative

Upon treatment with silver carbonate, the residue of fractions 8-16 yields 0.045 part by weight of pure psilocin as described in Example 2. Fractions 21-30 yield 0.765 parts by weight of pure psilocybin after treatment with silver carbonate.
The active material is obtained from the culture filtrate by the same process. For this purpose, the filtrate is concentrated in vacuo to approx. 1/10th of its volume. It is then precipitated with a tenfold volume of methanol and filtered off from the precipitated accompanying material. The solution is evaporated to dryness in vacuo and the residue is extracted 3 times with a tenfold amount of methanol. The evaporation residue of the methanol extracts is worked up as described above. 80 milligrams of psilocybin and 6 milligrams of psilocin are obtained from each 12 litres of culture filtrate.

EXAMPLE 4
Obtaining psilocybin and psilocin from pure cultures of Psilocybe mexicana Heim

In this example a detailed description is given of the cultivation of Psilocybe mexicana Heim in vitro for the production of mycelium and sclerotia as well as of the method of obtaining the pure active material.
Fresh and clear beerwort, without hops, is diluted with tap water to a content of 4.0-4.5% of dry substance. To this solution there are added:

Ingredient Amount (grams)
FeSO4 ° 7H20 0.00417
ZnSO4 ° 7H2O 0.00172
Agar-agar 2.0

500 millilitre portions of this culture medium are poured into 1.6 litre Fernbach flasks and the latter are then sterilized in the autoclave at 108 centigrade for 25 minutes. On cooling, the flasks are each inoculated with 2 millilitres of a suspension of the fungus Psilocybe mexicana Heim. The inoculation material is prepared by growing pure cultures of basidiospores of the said fungus on beerwort agar. The spores falling from the lamellae of a ripe fruit body are collected on sterile material, placed on beerwort-agar and incubated. From the primary cultures so produced the inoculation material may be prepared in the following manner:
The mycelium is scraped off with a rough spatula under a stream of sterile tap water. In this way a suspension of fine mycelium flakes is obtained. This suspension is used to inoculate Erlenmeyer flasks containing a culture medium and saddle-shaped porcelain filters. The best results are obtained with 300 millilitre Erlenmeyer flasks containing a 50 gram saddle-shaped filter (size 1 cm., weight approx. 0.9 gram) and 80 millilitres of a culture medium consisting of beerwort containing approx. 4% of dry substance and 2% of agar. The culture is incubated at a temperature of 24 centigrade; after 2 weeks a compact mycelium layer has formed on the surface. The whole culture is shaken for 30 minutes on the rotating shaking machine, the saddle-shaped filters grinding the mycelium so that a fine suspension of mycelium flakes is formed. The inoculation material so obtained is sufficient to inoculate 25 litres of culture medium. The inoculated cultures are incubated at 24-26 centigrade in the dark. A mycelium layer bearing many sclerotia is formed, the sclerotia in general reaching a size of 1 cm. (some of them may grow even larger). In order to separate the mycelium and the sclerotia, the ripe cultures are filtered through a gauze tissue, squeezed out and dried in a drying oven at 35-40 centigrade. 1149 grams of dry material (sclerotia and mycelium), i.e. 17.14 grams per litre of culture medium are obtained after 62 days from a batch of 134 Fernbach flasks containing 67 litres of nutrient solution.
The active material can be obtained as follows: 612 parts by weight of dry substance consisting of dried sclerotia and mycelium are finely pulverized and pre-extracted three times with 1000 parts by volume portions of chloroform and three times with 1000 parts by volume portions of chloroform containing 10% of ethanol. 5.6 parts by weight of inactive accompanying material are thus pre extracted. The pre-extracted fungal material is then extracted thoroughly once with 6000 parts by volume and three more times with 3000 parts by volume of methanol. The combined methanol extracts are evaporated to dryness under reduced pressure to give 35 parts by weight of a clear brown residue. In order to remove fatty impurities from this residue, it is washed in 35 parts by volume of water and shaken once with 1000 parts by volume and twice with 500 parts by volume of petroleum ether. The petroleum ether contains 1.5 parts by weight of inactive accompanying substance. The remaining aqueous solution is first concentrated under reduced pressure to approx. 50 parts by volume and is then treated with 500 parts by volume of absolute ethanol with vigorous stirring. From the sticky precipitate so produced, the solution containing active material is separated by decantation. The precipitate is redissolved in a little water and treated with a ten-fold amount of absolute alcohol. This purification by precipitation is repeated twice with the residue. The solutions are combined and evaporated to dryness under reduced pressure. There remains a solid residue of 23.4 parts by weight containing the whole amount of active material. For chromatography on a column of cellulose, the residue is dissolved in as little of 50% methanol as possible.
The solution is mixed well with 80 parts by weight of cellulose powder and the material is dried under reduced pressure. The cellulose powder bearing the active substance is poured onto a column of cellulose prepared by suspending 700 parts by weight of cellulose powder in water-saturated butanol. The column is developed in a through-flow process with water-saturated butanol, fractions of 200 parts by volume being thereby separated and then concentrated under a high vacuum at a bath temperature not exceeding 50 centigrade.
The fractions obtained are as follows:

TABLE 1
Fraction No. Residue parts by weight Keller colour reaction
1-14 2.170 Negative
15-34 6.650 Positive
35-40 3.540 Negative

Approx. 11 parts by weight remain in the column.
Fractions 15 to 34 yield a residue of 6.660 parts by weight and contain the whole amount of active substance. They are chromatographed again as described below for further purification. The residue of 6.660 parts by weight is dissolved in as little methanol as possible and is then used to impregnate 20 parts by weight of cellulose powder. After drying, the cellulose powder is poured onto a column of 600 parts by weight of cellulose powder, which has been treated as described above. Fractions of 100 parts by volume each are obtained by developing with water-saturated butanol and, after evaporation in a high vacuum, are tested by means of a colour reaction. The fractions obtained are as follows:

TABLE 2
Fraction No. Residue parts by weight Keller colour reaction
1-8 2.950 Negative
9-17 0.180 Clear Blue
18-21 0.155 Negative
22-31 0.912 Violet
32-45 1.510 Negative

The residue from fractions 9—17 yields 0.090 part by weight of pure psilocin after treatment with silver carbonate as described in Example 1. After treatment with silver carbonate as described in Example 1, fractions 22—31 (Table 2) yield 0.619 parts by weight of pure psilocybin having the properties described in Example 1.

The active material is obtained from the culture filtrate by the same process as described above for the sclerotia. For this purpose the culture filtrate is concentrated under reduced pressure to approx. 1/10 of its volume. After precipitation with a tenfold volume of methanol and subsequent filtration, the residue is extracted three times with a tenfold amount of methanol. The evaporation residue of the methanol extracts is worked up as described above. From every 10 litres of culture filtrate 220 milligrams of psilocybin and 12 milligrams of psilocin are obtained

EXAMPLE 5

Psilocybin and psilocin from pure cultures of Stropharia cubensis Earle

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of 6% of dry substance. To each litre of this solution there are added:

Ingredient Amount (grams)
FeSO4 ° 7H20 0.0021
ZnSO4 ° 7H2O 0.0009
Ca(NO3)2 1.0
KH2PO4 0.0624
MgSO4 ° 7H2O 0.0624
Kcl 0.0312
Agar-agar 2.0

This culture medium is sterilized as described in Example 3 and inoculated with 2 millilitres of a suspension of the fungus Stropharia cubensis Earle (from Kambodscha) per litre of nutrient solution. The inoculation material is prepared as described in Example 3a. After incubation for 52 days at 24 centigrade in the dark, there are obtained 452 grams of dried fungal material, i.e. 22.6 grams per litre from a batch of 20 litres. The active substance is obtained from the fungal material in the manner described in Example 3. Yield: 1083 milligrams of pure crystalline psilocybin and 45 milligrams of psilocin.

EXAMPLE 6

Psilocybin and psilocin from fruit bodies of Psilocybe semperviva Heim and Cailleux

Ripe fruit bodies of the fungus Psilocybe semperviva Heim and Cailleux obtained from artificial cultures on a natural medium are carefully dried in an air current at 30 centigrade. 32 parts by weight of the dried fruit bodies are finely ground and shaken for 30 minutes at room temperature once with 300 parts by volume and three times with 150 parts by volume of methanol each time. The combined extracts are evaporated to dryness under reduced pressure. To remove the fat, the residue (8.3 parts by weight) is rubbed 4 times with 125 parts by volume of petroleum ether each time and three times with 50 parts by volume of chloroform containing 10% of ethanol. The residual 6.5 parts by weight are dissolved in 6 parts by volume of water and, in order to precipitate other substances, the solution is slowly treated with 60 parts by volume of absolute ethanol; the amount of active substance in the solution is thereby increased. The purification is repeated twice in the same manner. The solutions are decanted, combined and evaporated to dryness under reduced pressure. The residue is taken up in methanol and chromatographed on cellulose powder as described in Example 2. From the active material so obtained, the halogen is removed by treatment with silver carbonate. After recrystallization there are obtained 0.160 parts by weight of crystalline psilocybin and 0.032 parts by weight of psilocin.

EXAMPLE 7

Psilocybin from fruit bodies of Psilocybe caerulescens Murrill var. mazatecorum Heim of natural origin

Ripe fruit bodies of the fungus Psilocybe caerulescens Murrill var. mazatecorum Heim obtained from artificial cultures on a natural medium are carefully dried. The dried fungal material (7.3 parts by weight) is finely ground and extracted thoroughly with methanol as described in Example 2. The extracts are combined and evaporated to dryness under reduced pressure. The residue is worked up as described in Example 2. There is obtained 0.0146 part of pure psilocybin.

EXAMPLE 8

Psilocybin from pure cultures of Psilocybe caerulescens Murrill var. mazatecorum Heim

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of 4% of dry substance. To each litre of this solution there are added:
Ingredient Amount (grams)
Cornsteep solids 10.0
FeSO4 ° 7H20 0.00824
Ca(OH)2 0.20
K2HPO4 0.20
NH4 OH 0.25
Agar-agar 2.0
pH of the solution 5.4.

This culture medium is sterilized as described in Example 3 and inoculated with a mycelium suspension prepared from a pure culture of the fungus Psilocybe caerulescens Murrill var. mazatecorum Heim (from Mexico). The cultures so obtained are incubated at 26 centigrade in the dark. After 48 days there are obtained 20.4 grams of dried fungal material per litre of nutrient solution.
The active substance is isolated and purified as described in Example 3. A batch of 10 litres of culture medium yields 449 milligrams of psilocybin.

EXAMPLE 9

Psilocybin from fruit bodies of Psilocybe zapotecorum Heim of natural origin

Ripe fruit bodies of the fungus Psilocybe zapotecorum Heim (collected in the "pays chatino" in Mexico) are carefully dried (residue 42.4 grams), finely ground and extracted with methanol as described in Example 2. The combined methanol extracts are evaporated to dryness under reduced pressure and the residue is worked up as described in Example 2. There are obtained 212 mg. of pure psilocybin.

EXAMPLE 10

Psilocybin from pure cultures of Psilocybe zapotecorum Heim

Fresh and clear beerwort, without hops, is diluted with tap water to a content of dry substance of 4.5%. To each litre of this solution there are added:
Ingredient Amount (grams)
Cornsteep solids 10.0
FeSO4 0.00834
K2HPO 0.30
NH4 OH 0.30
Agar-agar 2.0

pH of the solution 5.5.

This culture medium is sterilized as described in Example 3 and inoculated with a mycelium suspension of a pure culture obtained from spores of ripe fruit bodies of the fungus Psilocybe zapotecorum Heim (from the "pays chatino" in Mexico). After incubation for 57 days at 24 centigrade in the dark, there are obtained 430 grams of dried fungal material, i.e. 17.2 grams per litre from a batch of 25 litres. The active substance is obtained by the process described in Example 3. Yield: 903 milligrams of pure psilocybin.

EXAMPLE 11

Psilocybin and psilocin from Psilocybe aztecorum Heim of natural origin

Ripe fruit bodies of the fungus Psilocybe aztecorum Heim (collected in Mexico in the region of the Aztecs on Mt. Popocatepetl at an altitude of 10,800 to 11,500 feet) are carefully dried, finely ground and extracted thoroughly with methanol as described in Example 2. The extracts are combined and evaporated to dryness under reduced pressure. The residue is worked up as described in Example 2. There are obtained 570 milligrams of pure psilocybin and 47 milligrams of psilocin.
EXAMPLE 12

Psilocybin and psilocin from pure cultures of Psilocybe aztecorum Heim

A culture medium is prepared as follows:

Ingredient Amount (grams)
Malt extract 100
FeSO4 ° 7H20 0.00417
ZnSO4 ° 7H2O 0.00172
Ca(NO3)2 1.0
KH2PO4 0.25
MgSO4 ° 7H2O 0.25
Kcl 0.125
Agar-agar 2.0
Tapwater up to 1000 milliliters

This culture medium is sterilized in an autoclave at 108 centigrade for 25 minutes. 1 litre of the nutrient solution is inoculated with 2 millilitres of a suspension of the fungus Psilocybe aztecorum Heim. The inoculation material is prepared as described in Example 3a. The cultures are incubated for 45 days at 24 centigrade in the dark and then harvested as described in Example 3c. Yield from 10 litres of culture medium: 86.5 grams of dried mycelium.
86.5 parts by weight of finely ground mycelium is shaken out at room temperature for 30 minutes with 1000 parts by volume of 80% aqueous ethanol. After filtration, the residue is extracted three more times in the same way. To remove the fatty accompanying products and inactive ballast material, the evaporation residue of the combined extracts is successively extracted twice with 100 parts by volume of petroleum ether, twice with 80 parts by volume of chloroform and twice with 50 parts by volume of acetone. There remain 5.6 parts by weight of water-soluble powder, which are dissolved in 6 parts by volume of water. 60 parts by volume of acetone are slowly added, while stirring vigorously. The precipitate thus obtained is separated, dissolved once more in a little water and precipitated again with a tenfold quantity of acetone. This purification by precipitation is repeated twice with the share not soluble in acetone, the whole amount of active material passing over into the aqueous acetone extracts. The extracts are evaporated in vacuo and the residue (2.8 parts by weight, is further purified by chromatography on a column of cellulose powder as described in Example 3.

After double chromatography, there are obtained 0.225 parts by weight of crystalline psilocybin and 0.015 parts by weight of psilocin.

Example 13

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of dry material of 4.0 to 4.5%. To each litre of this solution there are added:

Ingredient Amount (grams)
ZnSO4 ° 7H2O 0.0086
FeSO4 ° 7H20 0.00209
Ca(NO3)2 0.25
KH2PO4 0.0625
MgSO4 0.0625
Kcl 0.031
Agar-agar 2.0

This culture medium is sterilized and inoculated as described in Example 4. After incubation at 24 centigrade for 48 days, there is obtained a yield of 924 grams of sclerotia and mycelium (dried material), i.e. 16.6 grams per litre, from a batch of 55 litres. The active material -psilocin and psilocybin- may be obtained therefrom as described in Example 4.

Example 14

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of dry material of 4.0 to 4.5%. To each litre of this solution there are added:

Ingredient Amount (grams)
FeSO4° 7H20 0.00209
ZnSO4 ° 7H2O 0.00086
Ca(NO3)2 1.0
Agar-agar 2.0

This culture medium is sterilized, inoculated and incubated as described in Example 4. After 48 days there are obtained 20.5 grams of dried sclerotia and mycelium per litre of culture medium. The active material -psilocin and psilocybin- is isolated as described in Example 4.

Example 15

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of dry material of 4.0 to 4.5%. To each litre of this solution there are added:

Ingredient Amount (grams)
FeSO4° 7H20 0.00209
ZnSO4 ° 7H2O 0.00086
KH2PO4 0.25
Agar-agar 2.0

This culture medium is worked up as described in Example 4. After 48 days there is obtained a yield of 15.9 grams of dried sclerotia and mycelium per litre of nutrient solution. The active material -psilocin and psilocybin- is isolated as described in Example 4.

Example 16

A culture medium is prepared as follows: Fresh and clear beerwort, without hops, is diluted with tap water to a content of dry material of 4.0 to 4.5%. To each litre of this solution there are added:

Ingredient Amount (grams)
FeSO4° 7H20 0.00209
ZnSO4 ° 7H2O 0.00086
“Cornsteep solids” 20.0
Agar-agar 2.0


This culture medium is sterilized, inoculated and incubated as described in Example 4. After 48 days there is obtained a yield of 29.8 grams of dried sclerotia and mycelium per litre of nutrient solution. The active material -psilocin and psilocybin-is isolated as described in Example 4.
Example 17

A culture medium is prepared as follows:

Ingredient Amount (grams)
FeSO4° 7H20 0.00417
ZnSO4 ° 7H2O 0.00172
Malt extract 45
Agar-agar 2.0
Tap water up to 1000 millilitres.

This culture medium is sterilized at 108 centigrade for 25 minutes in an autoclave. 1 litre of the nutrient solution is inoculated with 10 millilitres of a suspension of the fungus Psilocybe mexicana. The inoculation material is prepared as described in Example 4. After inoculation, the fungus is cultured in this nutrient solution at 24 centigrade using an agitated submersion procedure. There are formed sago-like mycelium balls. After 30 days the mycelium is separated by filtration and yields 7.0 grams of dry mycelium per litre of culture medium.
430 parts by weight of dried and finely ground mycelium are shaken for 30 minutes with 4500 parts by volume of 80% aqueous ethanol at room temperature. After filtering off the residue, it is extracted three more times in the same manner. To remove ballast material, the evaporation residue of the combined extracts (41 parts by weight) is successively extracted twice with 500 parts by volume of petroleum ether, twice with 400 parts by volume of chloroform and two more times with 200 parts by volume of acetone. There remain 25 parts by weight of a water-soluble powder, which are dissolved in 25 parts by volume of water. 250 parts by volume of acetone are slowly added, while stirring vigorously. The liquid is then separated from the precipitate, the latter is redissolved in a little water and precipitated again with a tenfold quantity of acetone. This operation is repeated twice with the part not soluble in acetone, all the active material passing over into the aqueous acetone extracts. The extracts are evaporated with vigorous stirring and the residue (9.8 parts by weight) is further purified by chromatography on a cellulose column as described in Example 4.
After chromatographing twice, there are obtained 0.032 parts by weight of crystalline psilocin and 0.225 parts by weight of psilocybin. While in the preceding illustrative examples methanol alone is exemplified as the extracting agent, other aliphatic alcohols such as ethanol, propanol and isopropanol can also be used in like manner and with equal success.

What is claimed is:
A process for obtaining the psychotropically active compounds psilocybin and psilocin, which comprises extracting the active principles from fungal material of one of the species Psilocybe mexicana Heim, Stropharia cubensis Earle, Psilocybe semperviva Heim and Cailleux, Psilocybe caerulescens Murrill var. mazatecorum Heim, Psilocybe zapotecorum Heim, Psilocybe aztecorum Heim and Psilocybe caerulescens Murrill var. nigripes Heim, by means of the extractant action on said material of a water-miscible organic solvent for such active principles said solvent being selected from the group consisting of water, lower aliphatic alcohol, and a mixture of water and lower aliphatic alcohol, severally isolating the active principles from the resulting extract, and treating the so-obtained material with a silver ions-yielding halogen-acceptor and thus freeing the material of halogen, whereby the compounds psilocybin and psilocin and obtained; the compound psilocybin having the empirical formula C12H17O4N2P being recrystallizable from water as fine white needles and from methanol as colorless hexagonal crystals containing crystallization-methanol and melting at 195-200 centigrade with decomposition, being optically inactive, amphoteric and soluble in dilute aqueous mineral acids and in dilute aqueous alkalis, and being characterized in methanolic solution by a UV-spectrum shown maxima at 222, 267 and 290 µm and the compound psilocin having the empirical formula C12H18NO2, melting at 173-176 centigrade with decomposition, and being characterized by a clear blue Keller color reaction and in methanolic solution by a UV-spectrum showing maxima at 222, 260, 267, 283 and 293 µm.
a process according to claim 1, wherein the fungal material being extracted is natural fungal material.
a process according to claim 2, wherein the extractant is a lower aliphatic alcohol.
a process according to claim 3, wherein the natural fungal material is Psilocybe mexicana Heim.
a process according to claim 3, wherein the natural fungal material is Stropharia cubensis Earle.
a process according to claim 3, wherein the natural fungal material is Psilocybe semperviva Heim and Cailleux.
a process according to claim 3, wherein the natural fungal material is Psilocybe caerulescens Murril var. mazatecorum Heim.
a process according to claim 3, wherein the natural fungal material is Psilocybe zapotecorum Heim.
a process according to claim 3, wherein the natural fungal material is Psilocybe aztecorum Heim.
a process according to claim 3, wherein the fungal material is in the form of fruit bodies which have been cultured on a natural substrate and the cultures have been incubated at a constant temperature between 18 and 27 centigrade.
a process according to claim 1, wherein the fungal material being extracted is artificially cultured material obtained from cultures of a fungus selected from the group consisting of Psilocybe mexicana Heim, Stropharia cubensis Earle, Psilocybe semperviva Heim and Cailleux, Psilocybe caerulescens Murrill var. mazatecorum Heim, Psilocybe zapotecorum Heim, Psilocybe aztecorum Heim and Psilocybe caerulescens Murrill var. nigripes Heim and biological mutants and variants thereof by inoculating a substrate with said fungus and incubating the cultures at a constant temperature between 18 and 27 centigrade.
a process according to claim 11, wherein the extractant is a lower aliphatic alcohol.
a process according to claim 12, wherein the fungus is Psilocybe mexicana Heim.
a process according to claim 12, wherein the fungus is Stropharia cubensis Earle.
a process according to claim 12, wherein the fungus is Psilocybe semperviva Heim and Cailleux.
a process according to claim 12, wherein the fungus is Psilocybe caerulescens Murrill var. mazatecorum Heim.
a process according to claim 12, wherein the fungus is Psilocybe zapotecorum Heim.
a process according to claim 12, wherein the fungus is Psilocybe aztecorum Heim.
a process according to claim 12, wherein the fungus is cultured in vitro on an artificial substrate of a 4 to 10% concentration of dry malt extract, 0.0004 to 0.0010 gram of iron (II) ions and 2 grams of agar-agar being added per litre of nutrient solution employed, and the culture being incubated in the dark
a process according to claim I, wherein the halogen-acceptor is silver oxide.
a process according to claim I, wherein the halogen-acceptor is silver carbonate.


References Cited by the Examiner

UNITED STATES PATENTS

1,632,312 6/27 Raeth 195-80
2,422,230 6/47 Foster et al. 195-80
2,825,734 3/58 Specter 260-319
2,850,518 9/58 Gaertner 260-319
2,850,520 9/58 Merian 260-319
2,955,073 10/60 De Beer 167-65
2,997,422 8/61 Tedeschi 167-65

OTHER REFERENCES
Wilkins: Annals Appl. Biol., vol. 31, No. 4, 1944, pp. 261-270.
Balenovic: Archiv. Kem, vol. 27, pp. 15-20 (1955) (obtained thin C.A., vol. 49, 1955, p. 14907!).
Wasson: Mycologia, vol.. 50, 1958, pp. 147-148. Experientia, vol. 14, 1958, pp. 397-399.
Heim et al.: Experientia, 14, pp. 107-109, 1958.

A. LOUIS MONACELL, Primary Examiner.
MORRIS 0. WOLK, NICHOLOS S. RIZZO, Examiners.


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Offlineelcharrosays
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: ranonar]
    #7680552 - 11/26/07 06:13 AM (13 years, 6 months ago)

hahaha congratulations on quite an extensive and formidable first post


--------------------


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Offlineranonar
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: elcharrosays]
    #7680608 - 11/26/07 06:56 AM (13 years, 6 months ago)

"congratulations on quite an extensive and formidable first post"

thanks. But perhaps I should not have posted it in the Mushroom cultivation forum - there are so many posts here that this method disappears from view in a few hours.

But IMHO it deserves to be known better. After all, in 2008 the Brack&Kobel method is exactly 50 years old. That needs to be celebrated - using soft agar is the greatest unknown method ever hidden in plain view.


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Offlineranonar
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: ranonar]
    #7680636 - 11/26/07 07:28 AM (13 years, 6 months ago)

Another big plus of the Back&Koble soft agar method is the possibility of making standardized extracts, i.e. it does not really matter anymore if the mycelium is potent or weak.

The first step to a standardized extract is to get the pure psilocybin crystals. The Brack&Kobel method makes it look like that it is extremely time consuming to get to them, but thanks to our friend Sasha Shulgin we know now that it can be done alot simpler:

A most simple extraction method for psilocybin by using a Soxhlet extractor apparatus
was published on March 5th 2003 by the medicinal chemist dr. Alexander Shulgin at the
website http://www.cognitiveliberty.org/shulgin/adsarchive/extraction.htm: “a Soxhlet extraction of 12
grams of powdered Psilocybe cubensis, using 95% ethanol. When the 60mL of extract cooled to room
temperature, many small transparent, colorless crystals had formed on the bottom of the container”.

So that's it. You only need to replace the soxhlet with something cheaper. How about a simple reflux setup in a pasta sauce jar?

The crystals which precipitate on the bottom can be redissolved in water. Please use lab grade, pure demineralised water here (not drinking water).

According to the Kobel&Brack patent the solubility of psilocybin in pure (distilled) water of 20 centigrade (68 degrees Fahrenheit) is 8.3 milligram per milliliter. Put some of the crystals which precipitated from the ethanol, put them in a vial with pure water, close the vial. Place vial in boiling water, so thet the crystals in the vial rdissolve. Take the vial out, wait until the crystals come out again and the vial it is at 20 centigrade (roomtemperature). If you want to be absolutely sure you can measure the acidity, the pH should be 5.3

Now you *know* that the water contains exactly 8.3mg of psilocybin per ml. No matter how weak or potent
the mycelium starting material ever was!

To doublecheck the potency of the extract you can use a color reagent such as DMCA (relatively easy to get at a pharmacy, just ask them, it is a color reagent for microscopy purposes not a dangerous precursor to something evil).

A good method to determine the psilocybin concentration was published in 1984: ‘Occurence of 5-hydroxylated indole
derivatives in Paneolina foenescii (Fries) Kuehner from various origin.’ authors T. Stijve,
C. Hischenhuber, D. Ashley. Source: Z. Mycol. 50: 361 (1984).

The paper reported a color reagent with a sensitivity for psilocybin of 25 nanograms. The color reagent was 4-
dimethylamino cinnamaldehyde (DMCA) (Fluka no 39421) which was used as a solution
of 0,5 g in 10 ml fuming concentrated hydrochloric acid, mixed with 50 ml methanol. It
produced a violet color upon contact with psilocybin on cellulose.


The above mentioned 8.3mg is 332,000 times as much as 25ng. So if the authors of that article are right then
a milliliter of psilocybin saturated water can be mixed in a litre of pure water, 1ml of this
dilution can be mixed in 100ml of pure water, a milliliter of that can be diluted with two
more milliliter of pure water and a ml of that final 1:332,000 dilution would still produce a
violet color when it was placed on a cellulose surface, allowed to dry and DMCA was
sprayed on it!


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: ranonar]
    #7680652 - 11/26/07 07:46 AM (13 years, 6 months ago)

Very interesting info all around, thank you for the posts! And welcome to the site, I hope you will choose to stick around.

This post may be more suited for the Advanced Mycology forum, although you will likely get more general attention in the cultivation forum here.


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Offlineranonar
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: urayasan]
    #7680774 - 11/26/07 09:24 AM (13 years, 6 months ago)

I almost forgot something. Brack&Kobel as well as Gartz used Penicillin bottles to cultivate sclerotia on soft agar, for a photo of a penicillin bottle see for instance

http://www.shroomtalk.com/forum/index.php?act=attach&type=post&id=998

The benefit of penicillin bottles is a big surface combined with a small opening. The small opening is needed to reduce the contamination risk. But since Rush Wayne published his peroxide method we know that it is possible to handle agar cultures in open air as long as some 10 ml of 3 percent hydrogen peroxide is mixed in the agar medium.

When we do that in the Kobel&Brack agar formula it yields a possibility to use tupperware containers to grow mycelial sheets or sclerotia instead of expensive penicillin bottles.

A complete write-up of this idea was published in Entheogen Review under the name "Moksha method". I recommend to pick one up:

http://www.entheogenreview.com


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: ranonar]
    #7680813 - 11/26/07 09:45 AM (13 years, 6 months ago)

Oops I meant 10ml of peroxide per liter of medium. Also for peroxide it makes a big difference if you use a soft agar culture or a liquid one. In a liquid culture the peroxidase enzymes of the mycelium can travel freely through the medium: the peroxide is gone in a few days and the culture recontaminates (in the mean time most of the mycelium was under the waterline, where it was unable to breathe, and most likely became the victim of the peroxide). But in soft agar the peroxide continues to protect the medium because the peroxidase enymes can't go very far. And the mycelium stays on the surface, fully able to breathe and to defend itself.

Result: you can just add 60 grams of malt, 2 grams of yeast extract (Entheogen Review recommends British Marmite)and 2 grams of agar to a liter of cold water, heat until it boils, add 10ml of 3 percent peroxide and pour everything in a clean tupperware container. Wait until everything has cooled down and add a wedge of mycelium (or a clean sclerotium or a piece of colonized uncontaminated spawn). Now click the tupperware lid on (which should have a hole in it which is covered with some tyvek for aeratium), allow the culture to grow for a few weeks at roomtemperature and harvest the mycelial sheet or the sclerotia. Wash the agar jelly off by using water in which a gram of vitamin c is dissolved (prevents premature blueing) and you end up with a shitload of the most beautiful sclerotia you have ever seen.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz [Re: ranonar]
    #15143037 - 09/27/11 07:20 PM (9 years, 8 months ago)

Greetings I have always been a fan of this thread and am currently working on a project to compile all relevant data to this research. If such a thing already exists please feel free to point me in that direction. It is in my belief this technique if continuously developed within this community could greatly benefit affordable psychedelic research for the medical community.

It is astounding to see how far from brf cakes people have taken things... That being said I do wish to contribute an interesting document that I ran across today in an unexpected source. Contained within LSD-25 & Tryptamine Synthesis by Otto Snow is an interesting article that is rarely associated with psilocybin - Increasing Psilocybin & Psilocin Content of Cultivated Carpophores Using Tryptamine Not the only article of this nature, however I never see it referenced so I would like to compare notes.

I am also curious with the emerging of the d-limonene cactus tek is this feasible with mushrooms? Can anyone point me to scientific document regarding citrus and psilocybin/psilocin absorption? I see a lot of posts suggesting it but I was curious if this could also be a potential ally using the d-limonene for the extract? Please forgive me as I am still new at chemistry but am attempting to compile all of my research.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: ranonar]
    #15143430 - 09/27/11 08:35 PM (9 years, 8 months ago)

This thread was moved from Mushroom Cultivation.

Reason:
This is off topic in cultivation.  I must have missed it four years ago.
RR


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: RogerRabbit]
    #15143829 - 09/27/11 09:49 PM (9 years, 8 months ago)

:smile: I would love to see your input on this topic yours was one skilled cultivator's name I did not see tossed into this hat while doing this research.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: RogerRabbit]
    #15540514 - 12/19/11 05:46 PM (9 years, 5 months ago)

Quote:

RogerRabbit said:
Reason:
This is off topic in cultivation.  I must have missed it four years ago.
RR



not sure why this was moved? is it just since it talks about extraction? the key point is growing on SOFT agar, the talk of extraction just shows it is active. I think this could be very useful for growing LCs and being able to visually see contaminants, it also seems to generate a lot more myc so could be used to inoculate a lot more jars. Probably a lot more than a GLC jar would.

Quote:

ranonar said:This suspension is used to inoculate flasks containing a culture medium and saddle-shaped porcelain filters such as are used for filling distillation columns...

The whole culture is shaken for 30 minutes on the rotating shaking machine, the sharp edges of the saddle-shaped filters grinding the mycelium so that a fine suspension of mycelium flakes is formed. The inoculation material so obtained is sufficient to inoculate 50 litres of culture medium.



These are often called "Raschig rings"
http://en.wikipedia.org/wiki/Raschig_ring
They try and increase surface area inside distillation columns so the vapours have a large area to condense on


I used to distil, and modern hobby distillers use stainless steel scrubbers, which have massive surface area per unit volume.


I wonder how well these would work with soft agar, it might be too tightly knit, but they could colonise like PF jars. It might be hard to get it off, but perhaps it could be extracted and boiled and the spent myc might come off.

Raschig rings can be made out of many things, like drinking straws cut up, but they may not take the heat of an autoclave. Though the guy who started this thread has grown on soft agar just treated with H2O2 using wedges. Some have used glass marbles.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: blackout]
    #15544928 - 12/20/11 02:11 PM (9 years, 5 months ago)

:awethumb: Awesome post! Glad this got dug up again, very cool info!


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: dwpineal]
    #26189245 - 09/15/19 09:10 AM (1 year, 8 months ago)

This sounds amazing, being so new to this, I do not understand the lack of wider spread interest?


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OfflineThePurpleWiggle
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: ThePurpleWiggle]
    #26191052 - 09/16/19 08:24 AM (1 year, 8 months ago)

Hello? Anybody there?


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: ThePurpleWiggle]
    #26191673 - 09/16/19 02:24 PM (1 year, 8 months ago)

Quote:

ThePurpleWiggle said:
Hello? Anybody there?



yo dont do that. Its been less than 15 hours, dont bump your own thread yet.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: jbgtaa]
    #26191763 - 09/16/19 03:02 PM (1 year, 8 months ago)

Quote:

jbgtaa said:
Quote:

ThePurpleWiggle said:
Hello? Anybody there?



yo dont do that. Its been less than 15 hours, dont bump your own thread yet.




This isnt his thread its an 11 year old post with the last comment being 7+ years ago of course nobody is here :rofl2:


--------------------
Bods Easy AF Oat Prep Tek
https://www.shroomery.org/forums/showflat.php/Number/24126032

Pastywhytes No Pour Agar Tek (PastyPlates)
https://www.shroomery.org/forums/showflat.php/Number/19208976

Shaperdreamings Shoebox Assembly Tek
https://www.shroomery.org/forums/showflat.php/Number/26009662



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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: ThePurpleWiggle]
    #26191898 - 09/16/19 04:05 PM (1 year, 8 months ago)

Quote:

ThePurpleWiggle said:
This sounds amazing, being so new to this, I do not understand the lack of wider spread interest?



Neither do I, if I grew a lot I would certainly be putting a lot of effort into growing sclerotia in soft agar. Who knows, maybe some commercial growers do and just keep quiet about it.

It seems in the US people selling shrooms need to be selling actual shrooms, as I guess many customers are suspect of anything else.

Growers would be wary of growing out myc as you can not easily tell if it has contaminated. Growing out myc to ingest has pretty much always been frowned upon in most forums I looked at.


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: blackout]
    #26192483 - 09/16/19 08:10 PM (1 year, 8 months ago)

I apologise for my lack of etiquette. I have never been an active member of any forum, noted.

Good point on myc being frowned upon for consumption, though it being grown of soft agar, while a thicker biomass than hard agar provides, would likely still allow for fairly accurate view of contams being a mostly 2d plane, and anything growing must start from the top and penetrate downward.

Here is another more appealingly titled thread I put in the cultivation section should any readers be interested https://www.shroomery.org/forums/showflat.php/Number/26191459/fpart/1/vc/1


--------------------
All's good :smile:


Edited by ThePurpleWiggle (09/16/19 08:46 PM)


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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: ThePurpleWiggle]
    #26192729 - 09/16/19 10:40 PM (1 year, 8 months ago)

Hi all :smile:
I have never been an active member here or of the DMT nexus, occasional long time lurker. These threads referenced can be found at the bottom of the post.

I not long ago I found an old post of an older tek that had been long forgotten and thought to be bunk due to a reportedly terrible translation.

https://www.shroomery.org/forums/showflat.php/Number/7680529

The Brack and Kobel method of psilocybin production.

The claim is that mycelium in some mushrooms (maybe not pan cyans, as one member showed a law enforcement study of pan cyans that tested no psilocybin prior to pinning, though consuming pre-fruited mycelium of Cubes anecdotally produces effect) is active, and it's speed of growth makes it a viable option for rapid actives production. The mistranslation is generally interpreted as myc grown in LC.
However, LC myc catabolises it's actives very quickly at exactly the one week mark and requires close and difficult (complex, apparatus for testing) monitering to be harvested in the small optimal window. The other common mis-interpretation is to grow on hard agar. This results in myc that is unable to penetrate the surface, thereby providing too thin a layer of myc, low yields.

The solution posed is that agar mixed to 1/10th the dilution of hard agar allows for mycelial penetration. and one ought to harvest once the myc has carpeted the surface.

Reportedly effectively done by Brack and Kobel whilst working for sandoz in the 50s, also effectively used to grow sclerotia and fruit from this substrate. The sclerotia is extremely clean and easily harvested, and the agar is rightly priced if using it at 1/10th regular dilution.

The corrected translation of the tek recieved very little interest, admittedly the title and body of text is offputtingly dense in the old post.

I am curious to see if this is a viable tek for producing actives at 6x the speed. (So much more in the fruit, I realise, still I see potential benefit here, grow bulk then and extract to another medium if eating heaps of myc is unappealing, the tek also mentions an extraction tek (I know fast degradation, make some agar jellies with it or mix it with some other protective medium, add flavours too :smile: )

I tried to revive the old thread to little avail. Then I tried making a post in the cultivation thread, mixed responses. A lot of ill will toward the concept. A good point was made by footpath; "Just about everyone knocks what they don't try and don't even take time to read anything legitimate about. They mainly just proliferate speculative rumors from the populous. Ultimately, I am 99% certain that 99% of the people here Do Not have the means to produce anything beyond anecdotal evidence. And that, most of the time, they don't even bother to create that."

Now, please, forgive my arrogance for making three posts on the same topic and necroing an 11 year old thread about an abandoned tek from the 50s. I am curious, and see potential benefit to the community at large, perhaps the advanced crowd will be more likely to share in my curiosity.

I will also post this to the DMT nexus.

I am a noob. I know nothing. I am in process of growing my first PF jars. I have no experience with agar. I am a student on government assistance who has just been kicked out of home and am shortly to live in a tent in the backyard of my law abiding Aunt. My pf jars have had to be moved to another location 3 hours from my Aunts. There will be renovations there in a months time for a month and a half. My jars will have to be spawned to completely autonomous bulk monotubs on my first grow.

I am an avid reader and have a lot of curiosity, that is just about all that is going for me as a cultivator.

If anyone feels any curiosity toward such a beautiful tek, a week, a little agar and some curiosity is the minimum requirements for some results. I believe that others may have favourable conditions, supplies and skills for such experimentation earlier than I will be able to.

Have a go if you will and tell us all how you go :smile:

Look at this, promising anecdotal reports regarding growing strong, active azurescens mycelial mats on agar. Easier than sclerotia even and better substrate for sclerotia too.
https://www.shroomery.or...hp/Cat/0/Number/7789758

Spent cake tea, effective -
https://www.shroomery.or...umber/14257992#14257992

Eating cakes and mushroom stem bases on surface of cakes -
https://www.shroomery.or...umber/14992441#14992441

Mycelium extraction teks -
https://www.shroomery.or...hp/Number/539138#539138

https://www.shroomery.or.../Number/4387553#4387553

General cultivation shroomery thread -
https://www.shroomery.or...r/26191459/fpart/1/vc/1

Advanced cultivation thread -
https://www.shroomery.or...lat.php/Number/26192695

Law enforcement study on what stage of Pan cyan myc contains actives, with pan cyans they found that it isn't until pinning, note, I've heard reports of other species that contain notable actives prior to pinning and this is the only source I found to say this about pan cyan, we trust cops right haha-
http://www.fanaticus.com/forensic.htm

The old post on the supposedly correctly translated Brack and Kobel method -
https://www.shroomery.or...wflat.php/Number/7680529


--------------------
All's good :smile:


Edited by ThePurpleWiggle (09/16/19 11:21 PM)


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Offlinejbgtaa
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Re: Brack&Kobel, the Psilocybin Producers Guide and Jochen Gartz (moved) [Re: DnDRnD]
    #26192868 - 09/16/19 11:55 PM (1 year, 8 months ago)

Quote:

DnDRnD said:
Quote:

jbgtaa said:
Quote:

ThePurpleWiggle said:
Hello? Anybody there?



yo dont do that. Its been less than 15 hours, dont bump your own thread yet.




This isnt his thread its an 11 year old post with the last comment being 7+ years ago of course nobody is here :rofl2:




I didnt even realize :rofl2:

If ths thread is eleven years old, and with this content... jesus christ why even revive it...


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