Psilocybin Extraction Study

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the following is for informational use only. we do not condone misuse of this information in any way. with that said heres some juicy info thanks to my friend D who referred me to it. "long, but interesting" ..check it out...for informational purposes ony, that is


United States Patent Office
3,183,172
Patented May 11, 1965

OBTAINING PSILOCYBIN AND PSILOCIN FROM FUNGAL MATERIAL
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 m~.

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 m~ 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 I 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
mjs, 267 m~s and 290 m~.
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(N03)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 sub stance 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
12.370
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:

1. 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 mj~ 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 m~.

2. a process according to claim 1, wherein the fungal material being
extracted is natural fungal material.

3. a process according to claim 2, wherein the extractant is a lower
aliphatic alcohol.

4. a process according to claim 3, wherein the natural fungal material is
Psilocybe mexicana Heim.

5. a process according to claim 3, wherein the natural fungal material is
Stropharia cubensis Earle.

6. a process according to claim 3, wherein the natural fungal material is
Psilocybe semperviva Heim and Cailleux.

7. a process according to claim 3, wherein the natural fungal material is
Psilocybe caerulescens Murril var. mazatecorum Heim.

8. a process according to claim 3, wherein the natural fungal material is
Psilocybe zapotecorum Heim.

9. a process according to claim 3, wherein the natural fungal material is
Psilocybe aztecorum Heim.

10. 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.

11. 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.

12. a process according to claim 11, wherein the extractant is a lower
aliphatic alcohol.

13. a process according to claim 12, wherein the fungus is Psilocybe
mexicana Heim.

14. a process according to claim 12, wherein the fungus is Stropharia
cubensis Earle.

15. a process according to claim 12, wherein the fungus is Psilocybe
semperviva Heim and Cailleux.

16. a process according to claim 12, wherein the fungus is Psilocybe
caerulescens Murrill var. mazatecorum Heim.

17. a process according to claim 12, wherein the fungus is Psilocybe
zapotecorum Heim.

18. a process according to claim 12, wherein the fungus is Psilocybe
aztecorum Heim.

19. 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

20. a process according to claim I, wherein the halogen-acceptor is silver oxide.

21. a process according to claim I, wherein the halogen-acceptor is silver carbonate.

[Soliver1]

Woof - heavy stuff, way above m capabilities, but interesting to know that someone's doing it right!



soliver

[sleepwalker]

hmmog.thats a long post lol.good ifo man.i didnt read it all i dont do extracts but hey thats good ifo being pushed.keep it up.

[Hippie3]

is there going to be a quiz ?

[thepope]

Wow, I read it and I am intrigued by the implications of a large scale liquid culture,
a huge vat liquid culture like a 55 gallon drum or maybe start smaller: a few 5 gallon carboys, Foaf has a couple sitting around and plans to experiment with this later on this year :-)

From 100 liters of this LC, these guys filtered out MORE THAN 2.5 KG of mycelium, doesn't this contain just as much Psilocybin as the basidiocarp?

Thanks for the find!

[thepope]

Lol, I didn't realize that this thread was a year old

And upon doing the math, they only got one good dose from each five liters of LC, that is totally not worth the trouble, and this process wouldn't be feasible

Unless one used UV or mutagens to create a high alkaloid producing strain, such as was done in order to create a decent yielding claviceps

A psilocybe that produced actives at 1g/L like the high yielding claviceps,
would be a very worthwile deal though

[cap]

Originally Posted by thepope

Wow, I read it and I am intrigued by the implications of a large scale liquid culture,
a huge vat liquid culture like a 55 gallon drum or maybe start smaller: a few 5 gallon carboys, Foaf has a couple sitting around and plans to experiment with this later on this year :-)
From 100 liters of this LC, these guys filtered out MORE THAN 2.5 KG of mycelium, doesn't this contain just as much Psilocybin as the basidiocarp?
Thanks for the find!

it will NEVER contain as much magic as the basidiocarp.
not with UV radiation, not even with Dr. Albert Hofmann himself holding your hand..
i posted this at a time when someone on the forums
was trying to grow cubes>extract mycelium for goodies.

if workin with cubes, grow them out (fruit) then extract.
in the case of sclerotia, you have an increasing amount
of psilo. building up as time goes on and stones form in vitro

[aumbrellaforainydays]

this is interesting stuff. i'll bet their not making much money off that patent, being its illegal and all.

so they tested:
Psilocybe mexicana
Stropharia cubensis
Psilocybe semperviva
Psilocybe caerulescens
Psilocybe zapotecorum
Psilocybe aztecorum

Stropharia cubensis, looks like they did use cubes. they shoulda went north and found some woodlovers to play around with.