Studies in the hallucinogenic principles of Amanita muscaria, L.
by A V Subbaratnam
A THESIS Submitted to the Graduate Faculty I n partial fulfillment of the requirements for the degree
of Doctor of Philosophy in Chemistry at Montana State College
Montana State University
© Copyright by A V Subbaratnam (1961)
Abstract:
Both fresh and dry mushrooms, Amanita muscaria, brave been examined chemically and
physiologically. The mushrooms were subjected to elaborate fractionations with comparative
pharmacological evaluation, to locate the hallucinogen 'pilzatropine' which has been elusive for over a
century now. The hallucinogenic principles have been located definitively in a water soluble portion of
the mushroom, freed from extraneous materials. Similar water concentrates from four successive
batches of mushroom have been found to have LSD-like properties and devoid of muscarinic effects.
Minute quantity of a potent crystalline substance, designated 'Z', m.p. I35-38°G (dec), C15H30015K4
(provisional) has been isolated from the water concentrate. Attempts are being made to characterize 'Z',
with the micro quantity available. Some indolic components are being separated from mother liquors.
Constituents occur in very small amounts in the mushroom. Isolation procedures have been evolved to
minute detail and for easy repetition to obtain constituents in sufficient quantity from large batches of
mushroom.
Potassium gluconate and a fixed oil consisting of esters of oleic acid have been isolated for the first
time from A. muscaria. Mannitol, fumaric acid and some minor constituents like crystalline substances,
m.p. 172-74 °, m.p. I60-65 °, and a β-glycoside, m.p. 106 °C (dec) have been found in the mushroom.
. - STUDIES Ifif THE HALLUCINOGENIC
PRINCIPLES OF AMANITA MUSGARI A, L.
by
A. V. SUBBARATNAM
A THESIS
Submitted to the Graduate Faculty
In
p a rtia l
fu lfillm e n t o f the requirements
for the degree o f
Doctor o f Philosophy in Chemistry
at
Montana S tate College
Dean, Graduate D ivision
Bozeman, Montana
August, I 961
Laboratory, POONA-8 , In d ia .
He was associated with P ro f. R. 0 . Shah from
February 1950 to July 1956 a t the N. 0 . L.
H is services were lent on deputation by the C . S . I .R. to the A li­
garh Muslim U n iv e rsity from the end o f July 1956 for a period o f three
years to help the U n iversity in the development of researches on indigen­
ous drugs, used In Unani M edicine.
A pril
He stayed a t the U n iv e rsity t i l l
1958 as a Senior Research Fellow .
On May 2, 1958 he s a ile d for
U. S . A. for higher studies on leave o f absence.
He has published some papers and taken some p a te n ts .
lis te d under Appendix I .
These are
He was a member of the In s titu tio n o f Chemists,
India; Indian Science Congress Association; Northwest S c ie n tific Associ­
ation (U. S . A . ) .
He has recently been awarded a grant by the Asia
Foundation to defray the cost o f membership in the American Chemical
Society.
He married Srimathi . Shakuntala in September 1949.
daughters, Sumati (10) and Hema ( 4 ) .
They have two
-A ACKNOWLEDGEMENTS
I
express my deep sense o f g ra titu d e to D r. W illiam B. Cook for
having afforded me an opportunity to v i s i t Iil. S . A. fo r higher studies.
H is guidance and advice during my graduate studies and research from
June 1958 to August 1961 have been in valuable.
My h e a r tfe lt thanks are due to D r. Kenneth J . Goerlng and
D r. Graeme L . Baker for t h e ir personal in te r e s t, h o s p ita lity and p ro fessi.onal. help a t al l tim es.
I wish to thank the follow ing:
1.
Members of my Graduate Committee for t h e ir helpful c ritic is m
and encouragement.
2.
S ta ff p f the Chemistry Department, fellow graduate students
and various friends a t Montana S tate College for t h e ir ready assistance
with equipment, e tc .
.3 .
Medical Sciences Research Foundation for fin an cia l support
through a Research Fellowship during my tenure as a graduate student.
4,
,P r o f. R.' C. Shah (Dy. D ir e c to r), P ro f. G. I . Finch, F .R .S .
(then D ir e c to r), P ro f. K. Venkataraman (present D ire c to r) of National
Chemical Laboratory, POONA-8, In dia and P ro f. M. S . Thacker, D ire c to rGeneral, G .S .I.R ., India for granting me leave of absence fo r studies
abroad.
The C . S . I .R .,a u th o ritie s have been kind enough to accept my re sig ­
nation from A pril 24, 196.1 ( a f t e r my continuous association fo r nearly
17 years) absolving me of a ll
legal l i a b i l i t i e s in order to enable me to
—15—
get my Ph. D. degree.
5.
D r. Salimuzzaman S id d iq u i, F. R. S
(D ire c to r, Pakistan G. S .
I . R .) fo r in it ia t in g me into researches on natural products and for his
sustained high hopes on my progress ever since.
6.
D r. Edward Pelican and the team of Pharmacologists for th e ir
valuable biolo gical data,
7.
Varian Associates fo r N. M. R. spectra
8.
M/S Ohas, P fiz e r for a sample of pure potassium gluconate.
9.
Granger Research Laboratories, Washington, D. G ., and the
Mycologjcal S ociety, C a lifo r n ia , for the c o lle c tio n of mushrooms,
AmanIt a musearia,
10,
My w ife , S rim a th i. Shakuntala for her s a c rific e s and partaking
a ll. p riv a tio n s c h e e rfu lly during the la s t ten years of our married l i f e ,
in a struggle fo r my higher education, which seemed hopeless for so long.
F in a lly , I dedicate th is th esis in fond memory o f three of my best
frie n d s , who inspired me with confidence during t h e ir short live s and
died t r a g ic a lly young:
( I a te ) P ro f. A. N. Ratnagir i swaran; ( la t e ) D r, K.
Ramachandran and ( I a te ) D r. S , RajagopaI an.
—Smm
TABLE OF CONTENTS
L I STT OF TABLES * * * * * * * # * * @ @ * * * # * * # * * * # * » @ » * * * * # * » * * » # # # * # ^ * @ * * * @ 0
Page
6
LIST OF FIGURES AND ILLUSTRATIONS ......................... .............. ......................
IO
LIST OF CHARTS (FLOW SHEETS) ..........................................
9
I
ABSTRACT **e 6 **»##*#»**e#^ *#**»*#****#*#*####**##*##»*##*#4#»*$*«
13
I NTRODLiGTI ON # # * * * * # * * # * # * * * * * # * * * * * 0***#**#»»##**#*#»#*»*#####*#
1.4
L ite ra tu re Review ..................
On Musoar i ne .......................................... .............. ^
.............
Hallucinogenic Factors .................................................
Other Gonstituents o f Amanita Muscaria ......................................
14
15
20
25
DlSCUSSI ON AND SUGGESTI ONS FOR FURTHER RESEARCH
30
EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35
P a rt J_ : Examination of dry mushroom (255 g) ................. .
I ) A lkaloidal constituents ........................................................
11) SoxhLet e x tra c tio n ...................................................................
M i i E xtractions a t room temperature ................... ..
C ry s ta llin e substance, X r 1 m.p. 172-74°O
...........
Water concentrate, A ,M. t 6 (%. 10), with
L S D - Iik e a c t iv it y
..................................... ..
A. M. 20, Xy 2Q .............. ......................... ........................ .
36
36
36
40
40
P art JM. : Examination o f fresh mushroom (6 .5 Kg) . . . . . . . .
Examination of various frac tio n s .............................
A. M. 21 , X jT , 21 ...................................................... ................
43
45
48
41
43
P a rt TTQ Fresh mushrooms (200 lb s .) ............. ..........................
50
A)
. Methanol ic Extracts .................................................
, 50
B) . Water E xtracts ........................... .................................
87
a) The acetone soluble fra c tio n ( T j T .12 ) . . . . . . . . . .
53
from A ). Methanol I c extra c ts
■ ___
A. M. 19/20 1A1, IL L . I 21, o il from JJX. I 2 . . . .
56
b) The 7 methanol ex tra c ts ("ITT. I 22)
___ from TTT.! 2 ................................................ ......................
62
11 I .Xf » fumaric .aei d ... ....................... ............. ..
68
The ether insoluble fra c tio n 1F 1 (TIT. 1222) o f the
concentrate from 70# methanol ............... .
72
Examination of the acetone insoluble fra c tio n ,
III .II
75
Mannitol from Amanita muscaria ..................... .
. SI
C ry s ta llin e product, m.p. 106° G (dec) (j3-gl ucosi d e ).
83
-7 .
Page
P a rt TT: Fresh mushrooms (4 6 .5 Kg) ...........; .............................................. 8-7
E xtractions a t Abbott Laboratory es ............................................ ... 89' .
Potassium gIuconate from Amanita musearia ........................... ..
94 ,
oi l fra c tio n s , Ab. A. I l l (TV.2121) , Ab. A. TV
(T V .2 12 2 ), and Ab. A. V (TV. 2 123) ......................................... 99
G ry s ta llin e product, m. p. 160-65°© ' (dec) ...............................
Aqueous concentrate 1C1, T V .3 with LSD -like
physiological properties .............................................................. /(.6#
C rystal I ine substance 1Z ' , .................................................. ..
m.p* 135-38°© (d e c )., (0,
5K4 , p ro v is io n a l)
(LSD-1i ke mater ia l ) ................... .................. .....................................108
PWARMACOLOGI GAL DATA;................... .................. ..
. P roperties of acetyl choline .......................
D e ta ils o f biolo gical te s ts ......................................
Fractions from P art ^ .................................................. ..
A . M. 20, ]K1 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fractions from P art TT; biolo gical data ............... ..
Comparative potencies of frac tio n s from P art TT
Fractions from P art i l l ; biolo gical data . . . . . . . . . . .
Comparative potencies o f fractio n s from P art 11 I . . .
B iological a c t iv it y of TV. I . TV.2I I and T V . 3 ............
B iological data on o il frac tio n s from P art JJZ . . . . . .
C ry s ta llin e product 1Z' (LS D -like substance) . . . . . . .
Potassium gluconate a n d p -g Iucoside ................... ..............
D r. Pe 11can s comments
SOMMARY
%
.
V\%
116
I 18
122
123
124
125
127
128
129
130
130
134
APPEMDIX
. . O . . . . . . . . . . . . . o . . . . . . . . . I 36-440
L I ST OF REFEREMGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . o . . . . . . . . . . . . . 14 1
$
ft W
-
.
I-1
Lv
' W'Sr'*
'
LIST QF TABLES
I.
Low y ie ld o f muscarine from Amanita muscarla ...................
T T , Fractions from P art T
111 .
TV.
.
VT♦
Fractions from P a rt TT
......................... ............................ ..
...............................
' Page
16
116
(21
Fractions from P art TT: ............................................ .................. ..
biolo gical data
| 22
Comparative potencies o f frac tio n s from P art TT ...........
123
Fractions from P art T l I .......... . . ' ......................................... ..
123
VTT.
F racti ons from P art TTTi ................... ............................ ..............
biolo gical data
| 24
Vl 11 .
Comparati ve potencies of fractio n s from Part T l T ...........
I 25
TT.
Fractions from P art TV ................................................................
| 26
T.
B iological a c t iv it y o f fra c tio n s ,
__
Ab. A. (TV. I ) , Ab. B-R (JV..2I I ) and Ab. G (H /.3 ) . . . . . .
| 27
B iological data on o il
128
XT.
)
frac tio n s from P art TV
........
—
LIST 0F GMARTS
. (Flow sheets elu c id a tin g fra c tio n a tio n s In the Experim ental«)
Chart I .............................................................................................................
Page
37
Chart 2 ...................................................... .................... .............................. ..
39
Chart 3 ......................... ... ..................... .......................................................
44
Chart 4 ................................................ ...................... ........................... ...
46
Chart 5 ............. ........................................................................... ..
49
Chart 6 ....................... .................................................................................. ..
51
Chart I
54
.............. ............................................................................... ..
Chart 7a ..................... ................ .......................... .......................... ..............
52
Chart 8 ............... .................................................... .............
55
Chart 9 ........... ...................................................................................
63
Chart IO ....................................................................... ...................... ............
73
Chart 11 .............................................. ...................... ........................... ..
76
Chart 12 .......................................... ..............................................................
77
Chart 13 ....................... ............................................................... ..................
82
Chart 14 .........^ ............ .............. .................... .................. ..
88
Chart 15 ............. ........................................... .........................................
90
Chart 16 ................................................ .......................................... ............ .
91
Chart 17 ...................................................... .................... ..............................
93
Chart 18 ..........................................................................................................
100
Chart 19 ..........................................................................................................
104
Chart 20 ........................... ......................................................... ................
107
-1 0 LIST OF FIGURES AND ILLUSTRATIONS
Figure
1. In fra re d Spectrum (Nujol m ull) o f A. M. 20,
........... ......
2\
2.
In fra re d Spectrum (Nujol m ull) of A. M. 21,
3.
In fr a red Spectrum (c a p illa r y film ) o f A. M. 19/20 .............
A /I , IL L , 121 I
57
4.
In fra re d Spectrum (c a p illa r y film ) o f A. M, 19/20 A /2 ,
TiT. 1 2 1 2 ......... ................................... .................... ...................
57
5.
In fra re d Spectrum (c a p illa r y film ) of acids from
A .M . 19/20, A /I (TIT. 1211) .................... ............... ..................
58
In fra re d Spectrum ( cap! I I ary film ) o f acids from
A .M . 19/20, A/2 (I I I .1 2 1 2 )..............................................................
58
In fra re d Spectrum (c a p illa r y f? Im) o f Methyl Esters ■
o f Acids from A .M . 19/20 A/I ( I I 1.1 21 I) ...................
59
In fra re d Spectrum (c a p iI I ary f i Im) of Methyl Esters
o f Acids from A .M . 19/20 A/2 ( I I I .1212) .................................
59
9.
In fra re d Spectrum (Nujol
m ull) o f tran s -A c o n itic Acid
65
10.
In fra re d Spectrum (Nujol
m ull) o f c i s-A co n itic Acid . . . . . . .
65
11.
In fra re d Spectrum (Nujol
m ull) of Fumaric Acid .......................
66
12.
In fra re d Spectrum (Nujol
I 96-97°G (dec)
m ull) of TTT.Xj , m.p. .......................
66
13.
In fra re d
Spectrum (c a p illa r y film ) of Ca (Chart 13) ..............
74
14.
In fra re d Spectrum (c a p illa r y film ) of ether-so lu b le
fra c tio n of 1F 1 ( I Li . I 222) ..............................................................
74
15.
In fra re d Spectrum (Nujol m ull) o f Mannitol
................................
78
16.
In fra re d Spectrum (Nujol m ull) o f Mannitol from
mushroom ..................... ..............................................................................
78
In fra re d Spectrum (Nujol m ull) o f B-glucoside, m.p. 106°G
(dec) from mushroom
..................................... ..
86
In fra re d Spectrum (Nujol m ull) o f Trehalose . . . . . . . . . . . . . . .
86
.6 .
7.
8.
17.
18.
...............
Page
47
.....
47
-I I Figure
19. In fra re d Spectrum (Nujol m ull) o f potassium
g I ucon ate from mushroom ................................................ ................
20.
21.
22.
23.
Page
95
In fra re d Spectrum (Nujol mu I I ) of potassium
gluconate prepared from g Iucono-deI a t - 1actone ............... ..
95
In fra re d Spectrum (Nujol m ull) of potassium
gluconate (Ohas. P fiz e r ) ............................. ..
96
In fra re d Spectrum (Nujol m ull) o f potassium
gluconate (Matheson, Qoleman & B e ll) .................
:96
In fra re d Spectrum (c a p illa r y film ) o f o i l , Ab. A.
I I I . I V .21 21 .......................................... .............. ..............................
I'd i'
In fra re d Spectrum (c a p illa r y film ) o f o i l , Ab. A.
H > TT .2122 .........................................................................................
I OI
25.
In fra re d Spectrum (c a p illa r y film ) of o i l , Ab. A.
V., TV.2I 23 .................................................. '........................................
. ,
102 '
27.
In fra re d Spectrum (c a p illa r y film ) of A. M.'IS 0 /1 ,
TT.5-01 .......................................................... ................ ......................
24.
61
I
28*
In fra re d Spectrum (c a p illa r y film ) o f A. M. 1/20 Ej
29.
In fra re d Spectrum (Nujol H u ll) o f c r y s ta llin e product
1Z 1, m, p . 135-38°0 (dec) from water concentrate . . . . . . .
109 '
In fra re d Spectrum (FlouroIube) of c r y s ta llin e produce
1Z ', m. p . 135-38° (dec) from water concentrate . . . . . . . .
I b9
30.
31.
....
61
Thin sheet chromatogram of a c o n itic acid
(tra n s — and ci s™
67
32.
Thin sheet chromatogram of TTT.X| and fumaric acid
69
33.
Thin sheet chromatogram of Tl I .X. , fumaric acid
and maleic acid ........................... ..v . . . . . . . . . . . . . . . . . . . . . . .
34.
Paper chromatogram o f glucose from acid hydrolysate of
j3-g l ucosi de, m. p. 106°G (dec) with ... samples o f
glucose and xylose .....................................................................
!
71
84
-Ar-
■
""
“ I
35.
36.
37.
2
~
■;
Page
Hallucinogens o f vegetable o rig in ................................................
23
Thin sheet chromatogram o f mannitol from A. muscaria
p a ra lle l with a sample o f mannitol ...............................................
Thin sheet chromatogram o f potassium gluconate from
A. muscaria p a ra lle l with samples o f potassium gluconate.
80
98
y;:
—13—
ABSTRACT
Both fresh and dry mushrooms. Amanita muscarfa, hrave been examined
chem ically and p h y s io lo g ic a lly . The mushrooms were subjected to elabor­
ate fra c tio n a tio n s with comparative pharmacological ev alu a tio n , to locate
the hallucinogen ' p i Iza tro p in e ' which has been elusive fo r over a century
now. The hallucinogenic p rin c ip le s have been located d e f in it iv e ly in a
water soluble portion of the mushroom, freed from extraneous m a te ria ls .
S im ila r water concentrates from four successive batches o f mushroom have
been found to have LS D -like properties and devoid of muscarinic e ffe c ts .
Minute q u an tity o f a potent c r y s ta llin e substance, designated 1Z ' , m.p.
I35-38°G (d e c ), C15H30O15K4 (p ro v is io n a l) has been isolated from the
water concentrate. Attempts are being made to ch aracterize 1Z ' , with
the micro qu an tity a v a ila b le . Some in d o lic components are being separated
from mother I i quors.
Constituents occur in very small amounts in the mushroom. Is o la ­
tio n procedures have been evolved to minute d e ta il and fo r easy re p e titio n
to obtain constituents in s u ffic ie n t q u an tity from large batches of mush­
room.
Potassium gluconate and a fixed o il consisting o f esters of o le ic
acid have been isolated fo r the f i r s t time from A. muscaria. M annitol,
fumaric acid and some minor constituents lik e c r y s ta llin e substances,
m.p. 172 -74 *0 , m.p. I60-65°C , and a p - g l ycoside, m.p. 106*0 (dec) have
been found in the mushroom.
-.3
x
-1 4 . I NTRODUCTI ON
Innumerable In v e s tig a to rs , a ll over the world, bo tan ists, chemists,
explorers, mycologists, path o lo g ists, pharmacologists, physiologists and
1
p ra c titio n e rs o f psycho-therapy have examined Amanita Muscaria , |_. , ( F r .)
.02
00
S. F. Gray , (L . ex F r .) Quel . , from time to tim e, with d iffe r e n t
view p o in ts .
As a r e s u lt, an overwhelming mass of lit e r a t u r e , a t times
mingled with seemingly contradictory observations, has accumulated around
th is mushroom.
Natural Iy , one wonders why th is p a rtic u la r mushroom,
common Iy known as f ly agaric a ttra c te d so much a tte n tio n .
L ite ra tu re Revfew
Strange beauty is ascribed to Amanita muscaria:
"crowned by a
v iv id red cap dotted with white spots, i t was depicted by Walt Disney in
the Dance o f the Mushrooms with Tschaikowsky’ s Nutcracker S uite mus?c In
25
F a n ta s ia ."
Before A. 0 . 1256, A lb ert Magnus^ noted th a t the fres h ly cut
mushroom placed in a dish of m ilk or water k ille d f li e s (Musca).
f li e s were said to have died by ingesting mushroom ju ic e .
Linnaeus6^
reported th a t the mushroom was advocated for k ill in g bed bugs.
the a b il it y of the mushroom to k i l l
The
However,
f li e s has never been confirmed
sc'ienti fic a l I y.
Wassons®^ have dwelt a t length on the possible o rig in of the
a p p e lla tio n ' f I y 1 to the mushroom.
They believe th a t the word ' f l y 1
(o r bug) was meant o r ig in a lly to describe the temporary demented con­
d itio n produced by the fungus; d erivation from th e doubtful t o x ic ity of
-1 5 6 f A. muscaria towards f lie s appears +6 be fa r fetched.
Explorers and tr a v e lle r s who toured S ib e ria e a rly in the 18th
century described v iv id ly the use o f A. muscaria by Siberian trib e s in
r itu a ls and orgy.
recent a r tic le s
These reports 4 6 ,7 9 ,8 3,8 5 Jiave been recounted in
*
*
f
( c f . Appendix 2 for a b r ie f d e s c rip tio n ).
I t has also been stated th a t the mushroom was responsible for th e .s o c a lle d Berserk fury o f the Viking period.
Fabing^ has quoted r e fe r ­
ences a t length in his a r t i c le rlOh Going Beserki
a Neurochemical Inquiry
In support o f the hypothesis th a t the furious rage of the Berserks in.
the heyday of Viking c u ltu re , a thousand years ago, was brought about
by A* muscaria.
T y le r
82
contradicts the above view c itin g reference to
QQ
review o f lit e r a t u r e pn th is to p ic by Thorsen .
A. Drew o f the Department o f Neurology a t the U n iv e rsity of
Michigan has described the e ffe c ts o f A., muscaria on a middle-aged
tavern keeper who ate the mushrooms accidently in October 1955.
p riv a te communication quoted by Fabing
25
This
is reproduced in Appendix 3 .
I t must, however, be cautioned th a t in modern times there has not been
any authentic case o f d e fin itiv e value to prove s c ie n t if ic a lly th a t
h allu c in a tio n s re s u lt from eating A. muscaria.
Another point to be
noted is th a t instances o f death re s u ltin g from A. muscaria have been
ra re , and few.
On Muscarine
Studies in the is o la tio n of the a c tiv e p rin c ip le s o f A., muscaria
began nearly 150 years ago.
Al I the in vestig ations centered around mqs-
Table I
Low y ie ld of muscarine from Amanita muscaria
Wt. o f Fresh Mushroom Processed
Authors
Y ie ld
King, H .50
25.5 Kg
I 24 mg. muscarine a u rLchIoride
Kog I , F . e t a 151
1250 Kg
I 37 mg. c r y s ta llin e reineckate
of muscarine.
Kogl, F. e t a 153
E
:
r. '
• -■
1250 Kg
540 mg . crude.;muscari ne
c h lo rite , admixed with
choline c h lo rid e '.*
1136 Kg
710 mg. crude muscarine
ch i or Id e .*
Y."
L
•'
260 mg. pure muscarine
c h lo rid e .
BaIenovIG, K. e t a I
3
1
Eugster, 0 . H .20' 2 *
and Waser-
r- .
I 24 Kg
^These figures have been computed by me from the o rig in a l a r t ic le s .
to have not recorded th e ir fin a l y ie ld s .
The authors re fe rred
-1 7 car I ne, the main a lk a lo id .
The Is o la tio n o f muscarine c a lls fo r a lo t
o f experience, patience and perseverance.
on an average 0.002# muscarine.
About 500 Kg. o f mushrooms w ill be re­
quired to Is o la te one gram o f muscarine.
when r e la t iv e ly pure.
The fresh mushrooms contain
Muscarine crystal Iiz e s only
During the p u rific a tio n and enrichment followed
through b iolo gical e v alu a tio n , the loss in muscarine ranges about 30#.
Table I gives an idea o f the poor yei Ids o f muscarine obtained by four
groups o f representative in v e s tig a to rs , from King®® in 1922 to present
day.
Al I these in vestig ations were c a rrie d out in c o llab o ratio n with
pharmacologists.
B iological evalu atio n , stepwise, o f each fra c tio n was
found to be essential to locate the p a rtic u la r fra c tio n rich in muscar­
ine*
Physiological properties o f muscarine, lik e stopping an isolated
fro g ’ s heart in d ia s to le were used.
These bioassays were helpful in recording a c t iv it y of various
fractio n s from the mushroom in terms o f muscarinic units and so fo r th .
Yet the bioassays also caused a lo t o f confusion.
choline and acetyl choline besides muscarine.
A. musoaria contains
Bioassays could not e a s ily
distinguish between muscarine and a c e ty lc h o lin e .
An is o la tio n scheme
based mainly on bioassay could not possibly lead td 1 pure muscarine.
Kogl e t a I
53
re a liz e d t h is , q u ite la te in th e I r in ve s tig atio n s .
They reported, in 1957, the follow ing a c t iv it ie s on fro g 's h e a rt.
Q
Muscarine: - 2 .3 x 10
Acetyl choline:
units/m g.
8
- 10 x 10 units/m g.
-
Eugster and Waser
18-
made use o f bioassays, but a t the same time
developed s p e c ific p a r titio n chromatographic techniques to lo ca lis e mus­
c a rin e .
T h e ir In vestig ations were extensive, processing 2600 Kg. of the
QQ
mushrooms c o n tin u a lly from 1953 onwards.
Wilkinson
cred ited the above
authors with success in obtaining the f i r s t pure muscarine c h lo rid e .
Balenovic e t a l^ found th a t countei—current e x tra c tio n was not
s u ita b le for the separation o f reineckates o f quarternary bases from
A. muscaria.
By switching to chromatography with ion-exchange resins
and c e llu lo s e columns, Balenovic and Stefanae^ could is o la te muscarine
pure.
Keehl e t al
57
also obtained pure muscarine chlo ride by prelim inary
I
frac tio n a tio n s on resins and p u rific a tio n by chromatography. Eugster and
M ulIer^Z USed ion-exchange resins and column chromatography fo r the iso­
la tio n o f muscarine from Inocybe p a to u illa rd i (y ie ld , 0 .0 3 7 # ), Inocybe
fa s tiq ia ta ( y ie ld . 0.01#) and Inocybe umbrlna ( y ie ld , 0 .0 0 3 # ), during
th e ir search fo r b e tte r sources o f muscarine.
The stru c tu re o f muscarine b a ffle d a ll the in v e s tig a to rs , t i l l
very recent years.
S tructural studies resulted In varying degradation
products in d iffe r e n t la b o ra to rie s .
nostic reports were erroneous.
Functional group analyses and diag­
Chemists considered the problem was ih -
triguiing because o f -stereo s p e c ific ity o f natural muscarine.
By p a r titio n chromatography, muscarine was a t la s t obtained pure
in m illigram q u an tity but confirmed as a sin g le e n tity in d iffe r e n t lab­
o ra to rie s .
Immediately a f t e r , the s tru c tu re o f muscarine was elucidated .
-1 9 with the use of in fra re d spetitroscopy and X-ray analysis.;
The various
stereoisomers have been synthesized and evaluated pharm acologically.
;
x
■ •
'v
'
Al I the above work leading to stru c tu re e lu c id a tio n , syhthesis and
physiological properties o f muscarine and re la ted products has been the
subject o f three recent and e x c e lle n t review a r tic le s with extensive
bibliographyi
I.
The Hi story and Chemis try o f Muscarine.
Q uart. R ev., XV (No. 2 ),
15 3 -7 1 (1 9 6 1 ).
by S . W ilk inson,8®
(87 main references, covering
the period, .1811 - I9 6 0 ).
'
V 2.
0.
Breve revue d*ensemble sur Ia chimie de Ia Muscarine,
by
H . IiE u g s te r,^ (22 references from 1869-1960); Revue de Mycologie,
XXIV (No. 5 ) , 369-85 (1959)
3.
The Story of Muscarine, by K. Bowden and 6 . A. Mogey8 .
J . Pharm. Pharmacol. ,
10. 145-56 ( 1958) (55 references from 1811-1957).
A chronological h isto ry o f muscarine from 181 I to 1924 can be found
in the f i r s t a r t i c le of Kogl e t al^*
' v
in 1931 on A. muscaria.
A. muscaria in terested not only the chemists for so long.
I t be­
came an a c tiv e source o f experimentation fo r pharmacologists from 1869,
with the work of Schmiedeberg and Koppe.
75 '
19
Dixon, in 1907 was struck by•
the s im ila r ity of the e ffe c ts o f muscarine reproducing some o f the r e - •
sponses, due to the Vagus stim ulation of the parasympathetic system.
He
advanced a hypothesis th a t the vagus nerve lib erated a m uscarine-like
substance which acted as a chemical tra n s m itte r of nerve Impulses.
DlXpn's
hypothesis met with universal skepticism; he was discouraged from follow ing
-2 0 th fs promising f ie ld of in v e s tig a tio n .
Dixon's hypothesis was proved to
be s u b s ta n tia lly c o rrect by Hunt and T a v e a u ,^ and by D a le ^ il *
th e ir studies on acetyl c h o lin e .
in
Muscarine became a foundation stone of
modern pharmacology.
Hal Iuci nogenic factors
Muscarine engulfed lik e an octopus the chemical and pharmacolog­
ical study o f A. muscaria.
As a re s u lt the o rig in a l
impetus for research
on th is mushroom, to find out the a c tiv e p rin cip le s responsible for the
psychotropic action was lo s t.
Schmiedeberg^® became curious with the observation o f Petri® ^In a
d is s e rta tio n in 1880 th a t there was a lim itin g fa c to r in the dose of mus­
carine required to cause cardiac a r r e s t.
P e tri mentioned th a t when the
dose of muscarine exceeded the lim it , the heart was accelerated instead
o f being stopped.
P e tri used in his experiments a commercial sample of
muscarine prepared from A. muscaria by Gehe and Go. In Dresden.
berg investigated an id en tic a l sample of commercial muscarine.
Schmiede
He con­
cluded th a t the product contained in addition to muscarine and choline,
a s ig n ific a n t quantity o f a base, s im ila r to atropine in antagonizing the
a c tiv ity of muscarine.
U nfortunately, i t was not ascertained i f ( I ) th is
base was a new a lk a lo id which occurred in A. muscaria in some cases
(2 ) th is was an a r t i f a c t formed from other basic compounds during the
preparation of muscarine or (3 ) th is was an accidental ad u ltera tio n with
one o f the known a lk a lo id s of Solanaceae.
Ten years la te r in 1891, K o b e rt^ '^ 6 reported th a t f ly agaric
-2 1 contained besides choline and muscarine, a new a c tiv e p rin c ip le , s im ila r
to the unknown base o f Schmiedeberg.
Kobert named th is new compound
"pi I zatropine" or a lte r n a tiv e ly ''muscari di ne".
He reported th a t pi Iz a -
tropinb d ila te d the constricted p u p il, accelerated the slowed heart and
was f in a lly excreted unchanged In u rin e .
In 1903,' Harmsem^ could not fin d any a tro p in e -lik e substance in
the crude muscarine Isolated by him from A. muscaria co llected in Bavaria,
as determined by pharmacological tdsts on the frog heart and eye of the
c a t.
Harmsen emphasized th a t his experiments should not ru le out the
p o s s ib ility o f pi Izatro p in e occurring in the mushroom.
He considered i t
as probable th a t developmental and environmental factors might influence
the occurrence o f pi Izatropine in A. muscaria .
Hondaz*"3 , in 1911, isolated from A. muscari a two bases,
and p -
myketosine in very small q u a n tity , not s u ffic ie n t to allow determination
o f th e ir physical and chemical p ro p ertie s .
Tes,ts on frogs and rabbits
indicated th a t only in r e la t iv e ly large doses,,both o ( - and J3- myketosine
exhibited hypnosis.
Honda re fra in e d from speculating on the re la tio n s h ip
o f these compounds to pi I zatro pin e.
In 1953, Wieland and M o tz e l^ re-examined the fungus, AmanIta
mappa.
They concluded th a t the base from Amanita mappa which was pre­
viously named "mappin' was id en tical with bufptenine.
an example of coincidence in nomenclature.
c a lle d toadstoo ls.
Here again was
Mushrooms are also sometimes
Bufotenine was f i r s t isolated from the secretion of
the parotid gland o f the common European toad, Bufo vulg aris Laur.
70
-2 2 In the same communication WIeIand and Motzel reported to have
detected the presence o f bufotenine In Amanita panther!na and in small
q u a n titie s in Amanita muscaria by paper chromatography.
In 1955, Lewls®^ reported the is o la tio n of ^ -hyoscyamine from
Aman It a panther Ina ( y ie ld , 1.8 mg/Kg) and from Aman It a muscaria ( y ie ld ,
1.1 mg/Kg).
Lewis thought .th at Robert's pi Izatropine might have been
^-hyoscyamine.
He cautioned th a t the usual adm inistration of atropine
fo r treatm ent of mushroom poisoning in general as inadvisable.
Curiously enough, ju s t a year before the report of Lewis, in 1954,
K w a s n i e w s k i ^ examined 10 Kg. o f fresh A. muscaria.
did not d ila te pupil of human eye.
The basic fra c tio n
He concluded th a t no a tro p in e -lik e
substance was present in the mushroom in ve s tig ate d .
The presence of an a tro p in e -lik e p rin c ip le in the panther fungus.
Amanita panth er!na was reported by In o k o ^ towards the end o f la s t
century.
Fiske
28
■■
examined some poisonous plants o f New Jersey, and
reported th a t A. muscaria contained the a lk a lo id s , muscarine, choline
and a tro p in e .
Brady and T y le r
9
examined Amanita pantherina re c e n tly .
They could not detect hyoscyamine or bufotenine by paper chromatography.
But in Panaeolus campanulatus,
95
T y le r detected serotonin by paper
chromatography.
E u g s te r^ has been working on A. muscaria p e rs is te n tly from 1953.
He reports th a t though 2600 Kg. o f the mushrooms were processed in his
la b o ra to rie s , hyoscyamine and bufotenine were not found.
Iished in vestig ations with M u lle r,
23
I n h i s unpub-
he detected an indole with a spectrum
-2 3 -
Z
t rf
K
R
I
[
—
H A rt
©.
V z
CHrl CHa N
J
A=RrHj SEROTON I M
M U S C A R I N E"
R=R=CHj j GU F=OT E N I N
_
Me
oM e,
MESCALINE
x
Me
0 ^ H 11
T e T R f ) H y D R 0 C . A N N ft B I N O L
Q^H
O H ^ H 2 N -C H j
P s iL o c y Ek I n
l .SD
- 25
Figure 35
Hallucinogens of vegetable o rig in
-24~
p ra c tic al Iy s im ila r to th a t of 'p s ilo c y b in 1.41
However, the indole from
A. muscaria which was suspected to be a 4-hydroxy d e riv a tiv e of indole
was so la b ile th a t i t could not be is o la te d .
I t appeared to have chemi­
cal properties d iffe r e n t from l P s iIo c y b in ' .
The research team headed by Or. A. Hofmann a t the Sandoz Labora­
to r ie s , Basle, Sw itzerland have in recent years been screening in to x ic a t-- Ing mushrooms of Mexican o rig in chem ically and pharm acologically.
They
have been fortunate to have P ro f. Roger Heim, an able mycologist as an
a c tiv e co lla b o ra to r fo r the id e n tific a tio n and c o lle c tio n o f various
species of PsI Iocybe, Stropharia , Gonocybe, e t c .^
From PsiIocybe
mexicana. Heim, the above team isolated an a c tiv e psychotropic p rin c ip le ,
'p s ilo c y b in '.
Psilocybin has been proved to have the stru c tu re of phos­
phoric acid e s ter o f 4-hydroxy-dimethyl tryptam ine,4 *
wide in te re s t adding to the speculation^4 ' ^
This has aroused
th a t most o f the known
hallucinogenic m aterials o f vegetable o rig in ( F ig .35) excepting t e t r a ­
hydrocannabinol are Indol i e .
Pharmacological evaluation of various
81
substituted indoles has also been c a rrie d o u t.
I t is of in te re s t in th is connection to re fe r to the 'o l o l Iu quI'
problem which has been the subject o f research for some years in th is
laboratory by Gook and Keel and.4^
They Isolated from the seeds of
Rivea corymbosa, a glucoside to which they advanced a provisional stru c­
tu re on the basis o f dehydrogenation studies on the agIycone and possible
bIo genetic re la tio n s h ip s to other natural products.
They found th a t the
seeds contained minute qu antity of a lk a lo id s which could not be is o la te d .
%
“ 25“
Hofmann and Tscherter
42
using th in sheet chromatography have since
then succeeded in is o la tin g m icro -q u an tities of three c r y s ta llin e indole
a lk a lo id s id e n tifie d as lysergic acid d e riv a tiv e s from the seeds of
Rivea corymbose ( L .) Hall ie r FM I us.
They report th a t the same a lk a lo id s
were also found in Ipomoea t r ic o lo r , Cav.
Just as the e a rly erroneous formula o f Kogl e t al"5* for muscarine
is quoted in many recent books on Medicinal chemistry. Biochemistry and
Pharmacology, s im ila r ly statements have been made to the e ffe c t th a t
muscarine is responsible fo r the psychotropic action o f A. muscarla.
Even Bowden and Mogey
8
in t h e ir recent painstaking review a r t i c le , quote
"the tru e base (muscarine) is almost c e rta in to be a valuable h e u ris tic
tool - i t has fo r instance already given c le a r confirm ation
2
of the
presence o f a tro p in e -lik e a c tio n s ."
E u g s te r^ has c la r if ie d th is issue.
He states th a t muscarine has
no action on the central nervous impulses, while the In to xicatio n s of the
-I-,
23
mushroom are due to e x c ita tio n of the CNS to a g re a t e x te n t.
Eugster
concludes n o s ta lg ic a lly th a t the problem o f muscarine has been solved
but the o rig in a l purpose o f research on the hallucinogens o f A. muscarla
has been forgotten and remains u n f u lf ille d .
shared by T y le r
82
This conclusion is equally
1
th a t "the fin a l chapter of the story of the ambiguous
a lk a lo id , pi I z a tro p ine, remains to be w ritte n ."
Other Constituents o f AmanIt a muscarla
A)
Choline and acetyl choline have been encountered by many in v e s tig a to rs .
In t h e ir e a rly work on the colouring m atter of the mushrooms, Kogl and
-2 6 Erxleben"^ isolated an orange yellow pigment, muscarufin, C25H |e^g> m.p.
275.5° (y ie ld ;
850 mg. from 500 Kg. of A. muscaria) ,
and assigned to
i t the follow ing stru ctu re:
H ooc
Eugster
23
Ch = C h - C h = C h - C o oh
reports th a t te s ts c a rrie d out in his laboratory in dicate th a t
the red colour of A. muscaria is due to a t least 4 compounds o f d iffe re n t
shades of colo ur.
No one a fte r Kogl has so fa r succeeded in is o la tin g
the colouring m atter(s ) in a c r y s ta llin e s ta te .
Eugster
23
casts doubt
on the terphenyl quinone stru ctu re of Kogl for muscarufin , on the basis
of absorption sp e c tra .
In a very recent prelim inary communication, Kogl, Salemink and
S chuller54 reported the is o la tio n of a new a lk a lo id , muscaridin (y ie ld :
300 mg. of muscaridin a u ric h lo rid e , m.p. I 29-131°C (uncorrected) from
1035 Kg. of A. muscaria) .
Muscaridin is reported to be the quarternary
trim ethyl ammonium s a lt of 6-ami no-2 ,3-dihydroxy-hexane:
CH3- ^ H 2-CH2-CH2-C-C-CH3
CH3
OH Ah
C l"
B)
L ite ra tu re on other minor constituents in , and varied properties
of A. muscaria col Iected through Chemical Abstracts from 1907 on­
wards by the author o f th is th esis:
V
~27~
Buschmann
I2
found in A. muscaria, stored in methanol, one day
a fte r gathering. Xanthine and, in greater amount hypoxanthine.
Clark and Kantor^^ processed A. muscaria according to the method
o f Marmsen^ fo r the is o la tio n o f muscarine and s im ila r substances.
The
re s u ltin g waxy, sem i-crystal I Ine products were in jected in lymph sacs of
fro g s .
Al I the frogs died in a few minutes with the classical symptoms
o f muscarine in to x ic a tio n .
58
Kung
reported th a t from extracts of A. muscaria, choline, pu tresc in e , herzynine (h is tid in e betaine) could be is o la te d .
31
Gerber
studied the coagulating properties in 86 species o f B asidomycetes, on m ilk , f a in t ly a c id u la ted .
He found th a t Amanita ph alIoides
coagulated boiled or fresh m ilk a t 40-55°> a ll other Amanitas required
2.5 hours fo r coagulation of m ilk .
Ford^® studied the d is trib u tio n o f poisons in various Amanitas.
He reported th a t A. muscaria contained muscarine, an a g g lu tin in and a
hemolysin.
an hour.
The agglutin was a glucoside, re s is ta n t to b o ilin g for h@ilf
For the is o la tio n of the glucoside, the fungi were extracted
with water and f ilt e r e d through a berkefeld f i l t e r .
To the f i l t r a t e
evaporated to a small bulk, ethanol was added in excess.
The re su ltin g
p re c ip ita te was freed from protein by uranyI acetate in a lk a lin e s o lu tio n .
From the p ro te in -fre e s o lu tio n , the glucoside was p re c ip ita te d by basic
lead a c e ta te .
bicarbonate.
powers.
The p re c ip ita te was washed with water and decomposed by
The re s u ltin g dark brown f i l t r a t e possessed agglu tin atin g
A fte r hydrolysis.w ith a c id , i t reduced F eh lIn g i S s o lu tio n .
It
-2 8 gave no colour reactions for pentoses.
Ford reported th a t I t was the
f i r s t a g g lu tin in to be isolated by chemical means; and the only glyco­
side known to have blood-agglutinating powers.
Radais and .S a rto ry ^ studied immun.ity o f rabbits toward mushrooms.
Juice expressed from the caps o f a large q u an tity o f mushrooms, preserved
with essence o f mustard was in jected In tra p e rito n e a lly in to ra b b its .
minimum lethal dose was 8 c .c .
The
Death occurred in 24 hours, preceded by
g a s tro in te s tin a l disturbance and p a ra ly s is .
Continued small doses re ­
sulted in immunity, reaching a peak a t the end o f 4 months.
But a sus­
pension of in je c tio n fo r one month resulted in loss o f resistance and the
animals died.from 8 c .c , doses,
Zel I ner®® found th a t the substance he had Isolated from A. muscarla
e a rlier® ^ was not pure e rg o s te ro l, but a m ixture o f two substances.
The
m ixture yielded a new product, ©26^40® ’ HgO, m. p . 159°, and a cerebros id e , m. p. 133°.
to s te ro l (y ie ld ;
Among the minor c o n stitu en ts. King
50
obtained a phy­
89 mg. from 25.5 Kg. o f A. muscaria) , m. p. I58-59°C,
whose analyses did not agree with GgG^gO, but with the formula,
®26^44®•
King concluded th a t I t must be e rg o s te ro l.
KOI, leucine, mannitol and fumaric a c id .
o f leucine e a r lie r in the mushroom.
Ee also obtained
L u d g w i noted the presence
In an e a r lie r communication, Z e lin e r
mentioned th a t A* muscaria contained propionic and fumaric acids, mykose
(treh alo s e ) and other u n id e n tifie d minor constitu en ts.
Herrmann
40
has given data on the odour and other c h a ra c te ris tic s
fo r id e n tific a tio n of about 40 species o f mushrooms.
He reported th a t
89
-29the odour o f mushrooms was due to ethereal o i ls , re sin s , terpenes, e tc .
which need chemical in v e s tig a tio n .
Bard and ZeIJner^ examined the polysaccharides o f various fungi
including A. muscaria.
Glucose (1 .7 6 $ ), mannitol (6 .0 2 $ ), mycetlde
(7 .4 0 $ ) and viscosin (5 .4 3 $ ) were found in A. muscaria.
The la s t two
components were c o llo id a l and contained pentosans (0 .8 6 $ ) and methyl
pentosans (0 .6 2 $ ).
Lapicque and N a tta n -L a rrie r
60
reported th a t the ju ic e
o f A. muscaria acted in the same manner on nervous and on muscular e x c ita ­
b ility .
Ohretien and Leblois*^ studied the to x ic ity of various mushrooms
to dogs.
A. muscaria dried a t 38° Was found to be non-toxic.
Rabb and R en z^ reported th a t A. muscaria causes no hemolysis
while some other Amanita species do.
B inet e t a l 6 reported th a t the
e x tra c t o f -A. muscaria produced a marked contraction o f the bronchioles
of the perfused isolated dog lung.
Atropine antagonized th is a c tio n .
The to x ic substance was found to be in the cap o f the mushroom,
e s p e cia lly in the c u tic le and not in the fo o ts ta lk .
S im ila r resu lts were
obtained by thq same authors^ with experiments on guinea p ig .
A. muscaria grown in Uruguay
was found to contain muscarine.
Amanita muscarina, P e rs ., growing w ild in Russia4"^ was found to contain
ascorbic acid:
dry mushroom.
A. muscaria.
11 mg. per lOOg. of fresh mushroom and 4 mg* per IOOg. of
Scandinavian workers detected d ihydroxy glutamic acid in
-3 0 DISCUSSI ON AND SUGGESTIONS FOR FURTtER RESEARCH
Important observations can be made as a re s u lt o f experience
gained in the present work and a review o f lite r a tu r e on Aman It a muscarI a .
The a b il it y of mushrooms to k i l l
f li e s (o r insects) or repel them
needs to be v e r if ie d .
The hallucinogenic p rin c ip le (s ) in the mushroom is /a r e not e a s ily
metabolised.
To somehow account for the ambiguous ' p i Iz a tro p In e *, in vestigators
-V'.
during the la s t hundred years and more looked for an atro p in e-typ e a LkaI o i d, or an indole lik e bufotenin.
,
C lim atic and environmental factors have been mooted to account for
the v a ria b le presence/absence of pi I z a tro p ine.
Constituents are present in minute amounts in the mushroom.
D efin­
it i v e work demands processing o f large q u a n titie s of mushroom, about
2000 K g ..a t a tim e .
V.
Constituents are la b ile .
is o la tio n .
Routine techniques w ill not s u ffic e for
S p e c ific dependable methods as have been evolved In th is
laboratory are necessary to follow each c o n s titu e n t.
Various pharmacological syndromes are present th a t are not always
e a s ily d istin g u is h a b le .
B iological data needs careful
in te rp re ta tio n .
The present in ve s tig atio n on A. muscarla was undertaken with a
sing le aim:
to c la r if y the elusive * p iI z a tro p ine' problem.
Unlike other
in v e s tig a to rs , the search was not re s tric te d to a mere hypothetical
a lk a lo id .
In vestig atio n s have been c a rrie d out in c o llab o ratio n with an
—3 1~
able team of pharmacologists.
Both dry and fresh mushrooms co lle cte d by
authentic agencies (Granger Research Laboratories, Washington, D. G ., and
Mycologieal S ociety, C a lifo r n ia ) have been examined s y ste m a tic a lly .
The
various fr b c tio n a tion and is o la tio n procedures are presented f u lly under
four parts o f the Experimental with illu s tr a tio n s ;
'
'
'
was subjected to pharmacological ev alu a tio n .
Each fnain frac tio n
j ' . i
'■
B iological data proved h e lp fu l, but no undue re lia n c e was placed
on them.
Changes were made in is o la tio n techniques progressively.
f
■
.
Elaborate anc| elegant fra c tio n a tio n s were c a rrie d opt In t u it iv e ly with
.
.
,
4 batches o f pishroom.
As a .r e s u lt , i t has been established th a t the
u ltim ate water soluble fra c tio n of the mushroom freed from a ll possible
lip id s ,, proteins and other organic and Inorganic constituents has LSD•
"
'
■
.'0;
lik e p ro p e rtie s . This water concentrate has been cleanly separated from
^
"r
'N
the antagonistic m uscarine-like.product.
F
An extremeIy hygroscopic but r e la t iv e ly pure c r y s ta llin e substance
........... %
■
■
has f in a lly been Isolated In a very small quantity (yield s 307 mg. from
__
•<
40.5 KG mushrooms. P art _TV o f Experim ental) from the potent water con­
c e n tra te .
The c r y s ta llin e substance designated iZ l , m.p. 135-3^°G (dec)
■ *e:v
:
has been found to have LSD -Iike e ffe c ts .
-
From the mother liquors and
remaining w ater-concentrate freed from inorganic s a lts , lik e KGI-, some
In d o li c . components are being separated through th in sheet chromatography.
The work is laborious and time.consuming.
I t is lik e ly to involve well
over 500 chromatographic plates and necessary zeal in developing s p e c ific
'
:
'
:
:-V :
techniques to locate each individual, component in q pure s ta te .
-ji
.
■
v;
—32*"
Whether ' p i Iz a tr o p ime' is concentrated in the above water concen­
tr a t e or whether the hallucinogenic a c t iv it y of the mushroom is due to a
combined drug syndrome w ill have to. be proved by u ltim ate human experi­
ments.
The point being stressed is th is :
there is a p o s s ib ility th a t
the psychic e ffe c ts o f the mushroom may not reside in one p a rtic u la r
substance lik e the hypothetical
the fa ilu r e o f a ll
' p i Iz a tr o p in e '.
in vestig ato rs in the p a s t.
This may account for
For example, the water
concentrate is o la te d , a fte r so much s tru g g le, with d e fin ite LSD-Iike
e ffe c ts may contain besides c r y s ta llin e 1Z 1 a host o f other substances,.
The presence in minute q u a n titie s o f these other substances may accelerate
or diminish psychic e ffe c ts with v a ria tio n in dosage of the water-concen­
tra te .
The hallucinogenic p rin c ip le s need not necessarily belong to the
category of a lk a lo id s , indoles or gIucosides.
They need not even be
organic or organo -inorganic substances. Even an Inorganic component
93
lik e potassium thiocyanate,
i f present, can co ntrib ute to the psychosis
o f th e mushroom to some e x te n t,
Curiously enough, the mushroom has been found to contain, in the
present in v e s tig a tio n , a number o f potassium s a lts :
KGI (considerable
q u a n tity ); potassium gluconate (which has been isolated fo r the f i r s t
time from A. muscaria); c r y s ta llin e ' Z ' , e 15^30®j
(p ro v is io n a l);
A. H . 20 and A* M. 21, c r y s ta llin e substances isolated from s im ila r
water-concentrates (P arts T and TT of Experim ental) with LSD-I ike e ffe c ts .
Why does potassium occur so frequently?
Mas potassium any lin k
with "sympathotonia ", a term coined by our team of pharmacologists to
-3 3 express 'hyperpyrexia-m ydriasis' drug syndrome, c h a ra c te ris tic of LSDIfk e m aterials?
tro p in e ' problem.
Speculation w ill on Iy add to the confusion o f ' p i I zaExperimentation alone can t e l l .
■ Experiments are underway to determine as much of the stru ctu re o f
c r y s ta llin e 'Z ' as possible by micromethods.
With the m icro-quantity
a v a ila b le and hindered by it s hygroscopic nature, 'Z ' cannot possibly
be worked through c la s s ic al approach.
Results o f WIR spectra of 'Z ' with
in te rp re ta tio n are being eagerly awaited.
A . muscaria has not so fa r been reported to contain fixed o i ls .
Considerable qu antity o f an o il consisting mainl.y of esters o f o le ic acid
has been is o lated and fra c tio n a te d .
fractio n s h,bs been c a rrie d o u t.
P relim inary examination of the o il
The o i l s are being screened for in s e c ti­
cidal and insect repel I ant p ro p e rtie s .
D etailed examination of the o i l s
i
wi I I have to be c a rrie d o u t.
Various minor constituents have been encountered in the present
In vestig ations on A. muscaria.
Mannitol and fumaric acid detected by
in vestig ato rs in the past were not apparently characterized wel l .
the present work these have been id e n tifie d s a t is f a c t o r ily .
In
Potassium
gluconate is a new minor constituent Iso lated and characterized In the
curren t work.
In vestig atio n s on A. muscaria have been extensive.
The chances
o f obtaining other d e fin itiv e constituents l i ke the jB-glucoside, m. p.
106°G (d e c ), (C hart 13, P a rtjT T ) and the c r y s ta llin e substances, m. p.
172-74°G (C hart 2, P art T) and m. p. 160-650C (dec) (Chart 18, Part TT)
7
-34"
In s u ffic ie n t amounts w ill be meagre and d i f f i c u l t .
tie s should not be overlooked.
Yet these p o s s ib ili­
The various fractio n s indicated in the
experimental as reserve m aterial w ill also have to be examined c a re fu lly .
Arrangements w i11 have to be made to process a t least about 2000 Kg
o f mushrooms.
C ry s ta llin e lZ f and various other constituents indicated so
fa r can be Isolated in s u ffic ie n t amount fo r stru c tu ra l e lu c id a tio n , i f
the procedures o u tlin ed are followed f a it h f u l ly .
The re s t of the potent
water concentrate should be worked up as Indicated with the help o f th in
sheet chromatography.
Comparative pharmacological evaluation may aid in
locating other potent constituents which w ill have to be tackled in ­
genuously.
One point needs to be borne in mind.
L ite ra tu re indicates th a t
only the Siberian tr ib e s practiced the mushroom orgy with A. Muscaria.
I t is possible th a t the mushrooms grown in th a t p a rtic u la r area alone
may be hallucino genic.
One needs to v e r ify by actual human consumption,
with a ll precautions and proper s c ie n t if ic recording, the presence or
absence o f psychotropic a c t iv it y in A. muscaria col Iected from various
parts o f the world.
Studies in the v a ria tio n , both q u a lita tiv e and
q u a n tita tiv e , o f hallucinogenic p rin c ip le (s ) due to changes in clim ate
and soil o f f e r wide scope fo r research,
-3 ^EXPERIMENTAL
The experimental data Is presented under four parts fo r the sake of
convenience, interspersed with flow-sheets (c h a rts ) and fig u re s , often
with cross references to maintain c o n tin u ity and c l a r i t y .
Negative re ­
s u lts and fra c tio n a tio n s e tc . th a t u ltim a te ly are o f no immediate value
have been e ith e r deleted or a t best merely indicated wherever deemed
necessary.
No fr a c tio n ^ however in s ig n ific a n t i t might have appeared from
time to tim e , was ever discarded or b e lit t le d t i l l
the end, and was only
j
set aside, on the basis o f pharmacological data, for fu tu re work,
To prevent the formation o f a r te fa c ts , high temperatures and undue
exposure to a i r , strong acids and bases were avoided,
As fa r as possible
a ll the work was c a rrie d out a t or near-about room tem perature.
An in e rt
nitrogen atmosphere was maintained whenever considered expedient.
That
the experimental procedures did not produce chemical a lte r a tio n of plant
m aterials present in the fresh p lan t and th a t the te s t m aterials contained
no b a cterial pyrogens was confirmed by the pharmacologists by the "clean­
ness" of biolo gical re s u lts they obtained.
Al I the m elting points are corrected unless otherwise s ta te d ,
Microanalyses were c a rrie d out by Berkeley A nalytical Laboratory, Univer­
s ity o f C a lifo r n ia , Berkeley, C a lifo r n ia , and G e IIe r Laboratories,
Bardonia. New York.
p u rity were used,
Solvents and chemicals of a n a ly tic a l reagent grade
Abbreviations lik e ether fo r diethyl ether and S kelly
for Skellysolve eF 1, a petroleum fra c tio n with a b o ilin g point range of
30-60° have been used in d e s crip tio n .
• -3 6 PART r
EXAMINATION OF DRY MUSHROOM (255g)
P r e lf mi nary experiments
i)
Al kale? dal constituents
a) Prol I Ius flu id / “ether ( 2 5 ), chloroform ( 8 ) , ethanol ( 2 . 5 ) and
ammonia ( I ) _ / e x tra c tio n 39 of the powdered mushroom ( 20g) gave a basic
fra c tio n (4 0 .8 mgn, 0.204^) designated as A. M V l (code no. T . l ) for
biolo gical e v alu a tio n .
This fra c tio n , however, did not give any apprecia­
ble p re c ip ita te with a lk a lo id a l reagents (M ayer's, Dragendorff ' s ) .
b) To detect the presence o f any a lk a lo id a l complex, samples of
dry mushroom were (A) extracted with a c id ifie d methanol and (B) mixed
.
with s u ffic ie n t aq. (5$) NagOOg to make a uniform ly wetted pulp and then
extracted with chloroform.
i
Examination, o f the above fo r lib e ra te d a lk a lo id s gave no p o sitive
Indication^
I i ) SoxhIe t e x tra c tio n (Ghart I )
The powdered drug (25g> was extracted in succession with Skel Iy ,
e th e r, chloroform and methanol in a soxhlet e x tra c to r (ISO m l),
a) The residue from Skel Iy was a lig h t brown o il with c h aracteris­
t i c odour o f the o rig in a l dry mushroom ( I .8051g ) »
I t was dissolved in
ether (30 c . c . ) and shaken out in a separating funnel tw ice with d ilu te
NOI (2#, 10 c . c , , '5 c . c . ) and d ilu te a c e tic acid (2 $ , 10 c . c . , 5 c .c .)
f
and washed with Water (5 c . c . , 5 c . c . ) .
washings did not contain any a lk a lo id .
The aqueous a c id ic solutions and
AMANITA MUSCARIA
Dry powder - 25 g.
Extn. (soxh let) with Skellysolve 1F 1
4Marc
Soxhlet in succession
with e th e r, CHC13 and
CHtOH
V
Ether (0.2720)
CHCL3 (0.2084)
CH3OH (6.4180)
Total
6.8984g. residue
Ske Iy E xt.
Solvent removed
neutral
frn .
0.3390g
A.M.7
(1 .7 )
V
Res due
Light Brown Oil (1.8051g)
----------^---------------- 1 - 2204*
basic fr n . acidic frn
n e g lig ib le (0.3230g)
AJM .3
d .3 )
neutral frn .
(0.4970g)
A.M. 7
(1 .7 )
CHART I
basic frn .
(0 .0 3 5 g .)
A.M. 2
(1 .2 )
acidic frn .
(0 .0423g .)
A.M. 4
(T .4 )
-vl
I
-38 —
The ethereal
layers were extracted with d ilu te NagOO^ (@#, 10 c . c . ,
10 c .c ^ ) and washed with w ater.
The carbonate and washings did not ex­
t r a c t any s ig n ific a n t amount o f a c id ic constitu en ts.
The ethereal solu­
tio n was then extracted with d ilu te KOM (2#, 10 c . c . , 5 c . c . ) and washed
with w ater.
The remaining ether solu tion was dried over anhydrous NagSO^
and yielded on removal o f solvent a neutral fra c tio n A. M, 7 (code no. T .7
0 .3 3 9 0 g ).
The aqueous a lk a lin e e x tra c t was cooled, a c id ifie d and extracted
with e th e r .
The ether layers were washed with water, dried over NagSO^
and evaporated.
The re s u ltin g a c id ic fra c tio n (0.323g) was labeled
A. M. 3 (code No. T .3 ) fo r biolo gical te s ts .
b) From the ether e x tra c tiv e was obtained a yellowish brown semis o lid (O.2720g, 1,088$) which was almost insoluble in d ilu te HgSO^ and
gave negative te s ts for a lk a lo id s .
e) The residue from chloroform (0.2084g, 0.8336$) was dark brown
with a c h a ra c te ris tic odour.
A small acid soluble fra c tio n o f the
chloroform e x tra c t was found to contain no a lk a lo id s .
d) The dark brown residue from methanol (6 ,4 l8 g , 25.672$) gave
in s ig n ific a n t p re c ip ita te s with a Ik a I o i dal reagents.
)
Microscopic manipulation by means o f solvents and mixed solvents,
of each residue s e p arately, did not y ie ld any c r y s ta llin e product.
Hence
a ll o f them were worked up together and separated in to a neutral fra c tio n
A. M. 7 (T .7 , 0,497g), basic fra c tio n , A. M. 2 (T .2 , 0.035g) and an a c id ic
fra c tio n , A. M. 4 (T .4 , 0„0423g) fo r biolo gical te s ts .
AMANITA MUSCARI A
Dry Powder - 50g - (I5 0 g )
Ether + MeOH (9 5 :5 )
-------------------------1
Ethereal E xtracts
4
Marc
IeOH e x t.
r
I
4
MeOH e x ts .
»
Ma *c
jW ater extn.
Water e x ts.
acetone
I
^ fra c tio n a tI on____
AjlM. 10
(1 . 10)
soluble
A.M. 9
I
-V
Neutral
middle
y — ^
Ej
E2
A.M.5 A.M.6
(1 .5 ) (1 .6 )
Insoluble
A.M. 8
(1. 8)
(1 i 9)
X l . I m.p. 172-4*C
CHART 2
basic
A.M.2
4acidic
A.M .4
(1 . 2)
(1 .4 )
%
I
Iii)
E x tractio n s a t room temperature (Chart 2)
a ) The powdered mushroom (50g) was macerated and percolated a t
room temperature with e th e r, containing 5% methanol, over a period of
two weeks.
On removal o f solvent from the e x tra c ts , a th ic k syrup was
obtained consisting mainly o f neutral m aterial with n e g lig ib le amounts
o f a c id ic and basic fra c tio n s .
The neutral product on r e frig e ra tio n yielded a pale yellow top
fra c tio n , A. M. 5 (T .5 , 0.7074g) and a brown heavy wax (l.6 5 7 3 g ) a t the
bottom.
The wax on t r it u r a t io n with acetone in the cold yielded a lig h t
brown p r e c ip ita te , A. M. 6 (T.6, I.IS I3 g ) which was hygroscopic.
In a
c a p illa ry i t changed in colour a t 193*0, softened from 218*0 and melted
between 234-38*0 (d e c .).
The defatted mushroom was next extracted in lots with methanol in
a Waring blender.
The f i l t r a t e s on removal o f solvent under reduced
pressure yielded a th ic k syrup.
This Was t r itu r a te d (magnetic s t ir r e r )
repeated Iy with fresh acetone and c e n trifu g e d .
The c o lle c tiv e p re c ip i­
ta te s , A. M. 8 (T.8, Q.95g) were submitted fo r biolo gical te s ts .
The acetone solu tion (supernatants) yielded a residue, A. M. 9 .
A major p a rt o f i t (G.5258g) was sent fo r biological te s ts and the
remainder worked up with solvents.
F in a lly from methanol, colourless
needles X | , (6 2 .8 mg), m.p. 17 2-74*0. were obtained.
Found: G, 39.42# and H, 7.64#; NI. W. (? ) - Insoluble in t camphor and
(B erkeley)
no absorption in the u .v . (200-400 mjp)
—4 1—
The marc l e f t a f t e r methanol extractio n s was then repeated Iy ex­
tra c te d with d is t ille d water (Waring blen dor).
were concentrated under reduced pressure.
The aqueous f il t r a t e s
The aqueous concentrate,
A. M. IO (12 c . c , , T. 10) had an intense fecal odour, was f Iourescent in.
in u .v . and gave a p o s itiv e K e lle r 's te s t (in d ic a tiv e o f in d o les ).
0 f the various frac tio n s A, M. I to A. M. IO (%%I to T . 10) sent
fo r biolo gical e v alu atio n , A. M. 10 (1 /3 of the concentrate, 4 c .c . con­
sumed fo r biolo gical e xp eriemtns) was reported to e x h ib it hallucinogenic
a c t iv it y In te s t anim als.
Attempts a t is o la tio n o f c r y s ta llin e products
through various solvents (w ith the aid of microscope) lik e aq. methanol,
acetone, dioxane e tc . from A. M. 10 were not f r u i t f u l , even though some
elusive c r y s ta llin e m aterials could be observed under the microscope.
The concentrate was run through a c e llu lo s e col unin prepared with
n-butanol and eluted with moist n-butanol.
Dark coloured solutions
were obtained f i r s t , followed by yellow e lu an ts.
run on a t ig h t ly packed c e llu lo s e column.
The dark solution was
This time i t was held as the
top zone and butanol e lu tio n yielded more of the yellow e lu an ts.
The
dark zone was eluted with w ater.
The yellow butanol eluants were freed of solvent a t room tempera**
tu re ( l ^ ) ♦
Descending paper chromatography of the residue with A) meth­
anol ( 2 ) , n-butanol ( I ) , benzene ( I ) and water ( I ) and B) ethyl acetate
( 4 ) , pyridine (2 ) and water (4 ) indicated the separation of a t least two
components f Iourescent in u .v .
At one stage, a micro quantity o f c r y s ta llin e product (u .v . maxi ma) fa 258 m y ,
98 mp) was obtained.
-4 2 The dark coloured solution was evaporated in vacuum and the r e s i­
due in minimum q u an tity o f water was shaken up with dioxane.
The gummy
mass which separated out was again taken up with water and subjected to
repeated e x tra c tio n with dioxane.
The to ta l dioxane solutions were re frig e ra te d overnight and cen­
tr ifu g e d .
A small dark brown viscous m aterial separated o u t.
c le a r golden yellow supernatants, solvent was removed.
From the
But c r y s t a lliz a ­
tio ns from various solvents were o f no a v a il.
b) Al I the remainder o f the dry powdered mushroom a v a ila b le for
in vestig atio n (150 g .) was worked up as above and a tte n tio n focused
mainly on the u ltim ate water e x tr a c t.
The fin a l aqueous s o lu tio n , con­
centrated to about 120 c .c . (corresponding to A. M- ND) was absorbed on
a t ig h t ly packed c e llu lo s e column, prepared with moist n-bu tan ol.
column was c a re fu lly pushed out and cut into three zones:
The
I } top-black
2) middle-brown and 3) bo tto m -ligh t brown.
Each zone was extracted in succession with n-butanol, dioxane
and w ater.
The butanol, dioxane and water e x tra c tiv e s o f each zone were
worked up s e p a ra te ly .
The residue from each fra c tio n was subjected to
c r y s ta lliz a tio n by a ll known methods, but in vain.
Each o f them was chromatographed on c e llu lo s e , chromosorb e tc .
Eluants were co lle cte d in fractio n s ( a r b i t r a r i l y , on the basis of
visual colour and in te n s ity of f Iourescence in u .v .) and worked up.
The re su lts were negative y ie ld in g no c r y s ta llin e substances.
-4 3 FInaI Iy , when a ll the above frac tio n a tio n s proved f r u it le s s , they
were a ll co lle cte d to g e th e r, freed o f solvents, dissolved in water,
tre a te d -with s ilv e r carbonate and f il t e r e d .
The f i l t r a t e s were d e s iIv e r -
ed with MgS, f ilt e r e d and c o n c e n t r a t e d Qn r e fr ig e ra tio n , a b it t e r
sem i-crystal 11ne product was obtained.
On i n i t i a l c r y s ta lliz a tio n from
methanol and then th r ic e crystal I i zed from w ater, a potassium s a lt ,
A. M. 20, X | '2 0 * long colourless needles (y ie ld 606 mg) m. p . in d e fin ite
ranging between 200°-230°C (dec) was obtained and most of i t (426 mg)
subjected to b iolo gical te s ts .
PART TT
EXAMINATION OF FRESM MUSHROOM
E a r lie r experiments indicated th a t the a c tiv e p rin c ip le s were
present main Iy in the water soluble portion of the mushrooms.
mushrooms contain on the average, about 95£ o f w ater,
The fresh
Hence arrangements
were made for the c o lle c tio n of fresh mushrooms which were asked to be
dropped in to cans o f methanol immediately on picking.
The f i r s t shipment was received on Feb. 17, 1959 evening.
The
mushrooms strain ed through cheese-cloth folds (Chart 3) weighed 6,5 kg
(dry w t.«s488g).
They were crushed in porcelain ware and th e pulp was
extracted with methanol a t room temperature by maceration, percolation
and shaking In lots (mechanical s h a k er).
A fte r s ix extraction s of each
lot. with fresh s o lv e n t, the marc was pressed out of adhering solvent and
s im ila r ly extracted with d is t ille d water (5 extraction s each b a tc h ).
Fresh Mushroom (P a rt IT )
(in methanol)
S t r a lned
T
jvieOH
" '
*
Whole Mushrooms
crushed and
e x t. with MeOH
Added on
MeOH
Exts .
(I7 0 g .)
Soluble
0 1 .5 )
water exts
Marc
(2 l3 g )
CONCENTRATE
MeOH
+ acetone
Insoluble
Less Soluble
<H.3>
CHART 3
More Soluble
(TT.2)
-4 4
Acetone
water exts
■
-4 5 The to ta l methanol Caq) e x tra c tiv e s were freed o f solvent a t room
temperature.
The dark brown residue C170 g) was t r itu r a te d In batches
repeatedly with acetone (magnetic s t i r r e r ) .
The acetone insoluble mass
on drying in vacuum was a semi-sol id m a te ria l, A. M. 14 (T T .4 , 150 g)
soluble in water (pH 5 .0 ) .
A brown, viscous o i l , A. M. 15 (7 T .5 ) , 18.95 g) m iscible with
water (pH 3 .6 5 ) was obtained on removal of solvent from the acetone f i l ­
tr a te s .
The water e x tracts o f the marc were evaporated a t room temperature.
In s p ite o f repeated f i l t r a t io n s , there was always some mushroom pulp
.
.
;
c a rrie d through a t each stage of e x tra c tio n . Then by successive r e f r ig ­
e ratio n and f i l t r a t i o n a ll the "muck" (71 g) was removed.
The aqueous
concentrate, A. M. I I (TT. 1, 34 g on dry weight basis,. pH 4 ,0 ) corres­
ponding to A. M. 10 (T . 10) was trea te d with a mixture o f methanol and
acetone (2 :1 , 4 e x tra c tio n s , magnetic s t i r r e r ) .
The residue from the
r e la tiv e ly more soluble fra c tio n , A. M.. 12 (TT.2, 20.412 g) and also the
residue obtained on removal of solvent in vacuum from the less soluble
p o rtio n , A. M. 13 (J_!_.3, 11.03 g, pH 3 .9 ) along with A. M. I I (11 .1 ),
A. M. 14 (TT.4) and A. M. 15 (TT.5) were sent for biological te s tin g .
Examin a tion o f the various fractio n s
■ r. 5
The residue from A. M. 13 was macerated with methanol (about a
month, in the r e fr ig e r a to r , (Chart 4 ) . . The methanol, insoluble portion
:s
Was t r itu r a t e d exhaustively (magnetic s t i r r e r ) in succession with moist
butanol and dioxane.
The fin a l
insoluble residue was taken up with water.
A. M. 13 (TT.3)
MeOH_______
'F
MeOH Solution
if
Insoluble
n-BuOH
Insoluble
BuOH Solution
D ioxane
r™
Insoluble
3
Dioxane Soluble
i
Ox
I
Water
Aqueous Solution
Ag2CO3 , f i l t e r , d esilver (H2S)
f i I t e r , evaporate (N2)
Concentrate
(Very small qu antity o f c ry s ta ls , A. M. 2I(X__ 9 I )
(IR spectrum d iffe r e n t from A.M. 20 (X-y- 20 )
—
CHART 4
47
Figure I
In fra re d Spectrum ( NujoI m ull) of A. M. 20, Xy 2o
W AVELEN G TH
IN
M ICRO N!
IN
KAYSER6
W im m
W AVENUMBER
Figure 2
In fra re d Spectrum ( NuJoI m ull) of A. M. 21, XI I .21
-4 8 tre a te d with s ilv e r carb onate* f il t e r e d , and d e s iIvered through HgS.
From the f i l t r a t e s a f t e r p a tie n t working a small q u an tity o f a c ry s ta l­
lin e product A. M. 21
) was obtained which had in fra re d spectrum
d iffe r e n t from th a t o f A. M. 20 (X > isolated e a r lie r from the sample
I -»20
■o f dry mushroom.
A. M. 12 ( I T . 2} and A. M. 14 (T T .4 ) were worked up as descri bed
for A. M. 13 ( T F .3 ) .
Each one of the corresponding fra c tio n s , soluble
in methanol, n-butanol and dioxane was examined c lo s e ly .
Although traces
o f c ry s ta ls (under microscope) could be discerned occasionally no c ry s ta l­
lin e product could be is o la te d .
A. M. 15 (T T .5 ), dissolved in e th e r, was chromatographed on a
column of alumina prepared with Skel I y .
did not contain any product.
S k e lly eluants (fra c tio n s 1-5)
E lu tion with a mixture of e th e r and S,kel1y
(9; I ) (fra c tio n s 6 - I0 ) yielded an o i l , A, M.. 15 c /I (T T .5 -0 I ) consisting
mainly o f esters (from IR spectrum. F ig . 2 7 ).
E lu tion w ith a mixture
of ether ( I ) and methanol (3 ) (fra c tio n s 11-14) yielded a sm aller quantity
of another o i l , A. M. 15 c /2 (T T .5 -0 2 ).
* S ilv e r carbonate was used fo r p u r ific a tio n . Hofman^* reported th a t
s ilv e r carbonate aided in the is o la tio n o f ’ p s ilo c y b in 1.
Fresh Mushroom (A p r!I -June, 1959)
200 lb s , (preserved in methanol)
(P a rt 111)
S t r a ined
MeOH (a q .)
Solutions
Mushrooms
Pulped and exhausted with
MeOH a t room temperature
Added
Methanol Ic
^ E xtracts
TTTJ
r
Water Extracts
TTT .2
CHART 5
E xtraction with water
----------- 1
Final Marc
dry weight 3952 g.
I
49-
------------------ 1
Marc
-SOPART 111
FRESH MUSHROOMS - 200 Ib s .
E a rly In March 1959 a shipment of 8 cans (25 lbs* of mushroom in
each) o f fresh mushroom preserved in methanol was received.
lik e mud, d i r t and straw in the cans were separated.
Im purities ■■
The aqueous methan­
ol i c d ia lysate (about 15 gallons) was f ilt e r e d through cheese-cToth
(Chart 5) and then f i l t e i —paper.
The mushrooms were ground (in the s ta in ­
less steel meat grinder o f Animal Husbandry Department, Campus) on
March 24, 1959 to a fin e pulp.
The pulpy mass was extracted with methanol
a t room temperature by maceration and percolation during a: period of three
months (A p ril-J u n e 1959) in improvised conical-shaped s ta in le s s steel
perco lato rs.
The extracted mushrooms were pressed, f ilt e r e d (basket
c e n trifu g e ) and then extracted exhaustively with d is t ille d w ater.
A .) Methanolic E xtracts
Solvent was removed from the methanolic extracts' (about 85 g a l­
lons) under reduced pressure a t room temperature, y ie ld in g a th ic k syrup
(1675 g ) .
A number.of prelim inary experiments were c a rrie d out with
the concentrate.
Following Is a ty p ic a l
fra c tio n a tio n c a rrie d out with
a portion o f the concentrate, (Chart 6 ) , In these prelim inary t r i a l s ,
i ) The concentrate (80 g) was mixed with crushed ice and repeated Iy ex­
trac te d with e th e r.
The concentrate from ether solution was freed o f a
small, qu an tity o f gummy mass by judicious addition of Skel I y ,
tr a te s were chromatographed on a column of alumina.
The f i l ­
E lu tion with a m ix- .
tu re o f S k e lly and e th e r, increasing the ether content o f the mixture
grad u ally, resulted in the separation of three o il fractions^
Methanol Concentrate
T iT . I
80 g. (A . M. 1/20)
crushed ice
ether extraction
I
Ether insoluble
aq. solution
Ether solution
Skel Iy added
dropwise into
batches. F ilte re d
crushed ice
n-butanol
e xtractio n
FTeci p I t a t e s
gummy
InsoTubTe
residue
contained
Kc I e tc .
F iItr a te s
Chromatographed
on alumina
eluants
Skel Iy +
ether
n-outanol
extracts
solvent
removed under
f
■reduced pressure
CONCENTRATE
Three main frn s.
r e f r ig e r a t ion
I f ^2 ' and Et o ils
addition of
n-butanol
v
Waxy
Butanol
Crystals?
decantates
I cel Iulose
I c o lumn
E Iuants
(Solvent c r y s ta lliz a tio n .
Treatment with s ilv e r carbonate e tc .)
CHART 6
Total Concentrate
( 15 9 0 g .), m _ .l, A.M. 19/20
S tirre d with
acetone.
20 extractions a t
room temperature
Acetone
Insoluble
n r .ii
Acetone
Solution
TTT.I2
i
Ul
M
CHART 7a
-5 3 CA. M. 1/20 EI , E2 and E3 ) .
The ether insoluble fra c tio n from the o rig in a l methanolic concen­
t r a t e was mixed with additional crushed ice and extracted with n-butanol.
The butanol insoluble residue was found to contain inorganic s a lts
(KOI e t c .)
The butanol e x tra c t was concentrated a t reduced pressure.
Experi­
ments with various solvent systems were t r ie d with the concentrate.
c r y s ta llin e m aterial was is o la te d .
No
R e frig e ra tio n for considerable time
and treatm ent with increasing q u a n titie s o f n-butanol resulted in the
separation of a waxy m a te ria l.
This m a te ria l, sem i-crystal lin e under the
microscope, yielded no d e fin ite product.
The butanol decantates were
chromatographed on c e llu lo s e , and the various eluant (n -b u tan o l) fractions
examined.
C ry s ta lliz a tio n could not be induced through solvents.
T re a t­
ment with s ilv e r carbonate, d e s ilve rin g and fu rth e r work fa ile d to y ie ld
a pure substance,
I i ) The fin a l scheme o f fra c tio n a tio n o f the methanol Ic concentrate is
o u tlined under Chart 7 , and follow ing C harts,
The concentrate ( I590g) was s tir r e d in batches with acetone
continuously (about 20 extractio n s o f each, with fresh solvent each tim e,
using magnetic s t i r r e r ) , thus separating i t in to two main frac tio n s :
th a t
soluble in acetone and th a t insoluble in acetone.
a) The acetone soluble fra c tio n ( I l 1.1 2 ) on removal o f solvent was
obtained as a th ic k concentrate (730 g) soluble fre e ly in ether and less
in Skel I y ,
I t was p a rtitio n e d in lo ts , between equal amounts o f S kelly
and 70# methanol (a q ).
Acetone Solution 111.12
Solvent removed under
reduced pressure, Ng
Concentrate (730 g .)
P a rtitio n between Skel Iy
and 10% aq. MeOH
n!'
Skel Iy layers
aq. MeOH Solution
TTT-122
Solvent removed
=Yeed of solvent under
reduced pressure
V
Concentrates
A.M. 19/20 1A1 260 g.
n r. I 21
Shaken out with ether
Ethereal Solution
Solvent removed
T fT . 1221
CHART 7
Ether Insoluble Portion
I
A. M. 19/20 1F1
(aq. concentrate, I55g.
140 cc. pH 2 .5 )
IT T . I 222
Ul
A. M. 19/20 'A' (240g.) I I I .121
Alumina Chromatography
(23 x 5.5 cm.)
___
A. M. 19/20 A /2.T7T. I 21 2
Thick, brownish o il ( I 24g)
(s k e lIy eluants)
Sap. equivalen t= 245.8
Neut. equivalent of acids
(on hydrolysis) = 290.3
A. M. 19/20 A/ Ij Il I .121 I
Light yellowish o il (5 8 g .)
(s k e lI y e l wants)
Sap. equivalent = 235.1
Newt, equivalent o f acids
(on hydrolysis) = 298.4
Vl
Vl
I
IR spectra o f o rig in a l 1A1, Fraction I ,
acids from I and Methyl esters of acids;
Fraction 2, acids from 2 and Methyl
esters of acids.
Gas Chromatogram of Methyl esters of
acids from I and 2,
CHART 8
—
56 —
The Skel Iy solution on removal o f solvent (Mg) yielded an o il
( 118 . 121. 260 g ) .
1A1
i t was chromatographed on a column o f alumina
(23 x 5 .5 cm) prepared in Skel I y .
The f i r s t eluants with S k e lly (.1500 c . c . )
y ie ld ed , on removal of solvent (Chart 8 ) , a lig h t yellow ish o il (58 g,
I 11.1211. A. NI. 19 /2 0 A / I ) .
Subsequent S k e lly eluants (3 li t e r s ) yielded
a viscous brown o il ( I 24 g, Il j . I 21 2. A. NI. 19/20 A /2 ).
Further eluants
(fra c tio n s 3-16) with Skel Iy , mixture o f S k e lly and ether (w ith gradual
increase o f e t h e r ) , e th e r, m ixture o f ether and methanol and u ltim a te ly
methanol and water did not give any d e fin ite product.
The o rig in a l o il
( ' A ' , 111.121) and the two o il
frac tio n s (A. NI.
19/20 A /I = VVi .1 21 I and A. M. 19/20 A/2 = I I l .1212) obtained on chroma­
tographic separation were a ll submitted fo r biological te s ts .
At one
stage, the pharmacologists reported th a t fra c tio n I I I . 1212 was potent,
ha I Iucinogenical Iy while the o th ers, Y l 1,121 and 111, 1211 exhibited
in s ig n ific a n t e f f e c t s .
The o ils were examined as i t was f e l t th a t there might be a lip o id
soluble hallucinogen with the fa tty o ils acting as c a r r ie r s .
No such
product could be isolated through the usual methods o f solvent fra c tio n a t i on.
The sap o n ificatio n equivalents o f the o ils
77
(weight o f fa t
s a p o n ifia b le by one gram - equivalent of KCfcl) were very clo se, as well
as th e n e u tra liz a tio n equivalents^? o f t h e ir fa tty acids.
-5 7 -
F igure 3
In fra re d Spectrum (c a p illa r y film ) of A. M. 19/20 A / I , I I I . I 21 I
2000
1900
1800
W AVENUM BER
IN
KAYSER S
F igure 4
In fr a r e d Spectrum ( c a p i l l a r y f i l m ) o f A. M. 19/20 A /2 , I I I .1212
58-
F igure 5
In fr ar ed Spectrum ( c a p i l l a r y f i l m) o f acids from
A. M . 19/20, A/I ( H i . I 21 I)
W AVENUMBER
IN
K AYSER S
Figure 6
In fr a r e d Spectrum ( c a p i l l a r y f i l m ) o f acid s from
A. M. 19/20, A/2 (T T T .I2 I2 )
-5 9 -
W AVENUM BER
IN
K AYSER S
F i gure I
I n f r a r e d Spectrum ( c a p i l l a r y f i l m ) o f Methyl E s t e r s
o f A c i ds from A. M. 19/20 A/I ( I I I . 1211)
W AVELEN G TH
IN
M IC R O N S
W AVENUMBER
Figure 8
In fr a r e d Spectrum ( c a p i l l a r y f i l m ) o f Methyl E ste rs
o f Acids from A. M. 19/20 A/2 (I I I .1212)
-60Fraction
Sapn. equivalent
Neutral I zation
Equivalent
TTT. 121 I
235.1
298.4
T IT . 1212
245.8
290.3
For the determination of sap o n ificatio n e q u ivalen t,
77
weighed quan­
t i t y of o il was r e f I taxed on steam-bath with an excess o f standard KOM I n
ethylene g ly c o l.- The amount o f a lk a li consumed was estimated by backt it r a t io n with standard a c id .
A blank was run under the same conditions.
For c o lle c tin g the f a tty acids, o il was saponified with KOM (in
methanol or ethylene g ly c o l) and the saponified product freed of neutral
fra c tio n by e x tra c tio n with Skel Iy , ether and ethyl acetate and then
a c id ifie d with cold d ilu te HO I .
The lib e ra te d acids were taken up in
e th e r, careful Iy rinsed with water, dried over anhydrous sodium sulphate
and evaporated under n itro gen.
The acids were dried over p a ra ffin shav­
ings in H2804. in vacuum desicator for IR spectra, determination of neu­
t r a liz a t io n equivalents6^ and for the preparation of methyl esters using
ethereal dI azomethane.
No serious attempts were made to is o la te the
neutral fra c tio n , suspected to be g ly c e ro l.
The in fra red spectra o f the o i ls , the acids and th e I r esters from
both the frac tio n s I lT .1211 and I I I .1212 are shown in F ig . 3 -8 .
chromatograms o f the methyl esters were id e n tic a l.
Gas
The in dication was
th a t from both frac tio n s they were p r in c ip a lly esters o f o le ic acids.
Small q u a n titie s o f other acids saturated and unsaturated appeared to be
present.
—6 1—
1500
W AVENUMBER
1400
1300
1200
IN K AYSER S
UOQ
1000
Figure 27
In fra re d Spectrum (c a p illa r y film ) of A. M. 15 C / I , 7 T .5 -0 I
W A V E LE N G T H
W AVENUMBER
Figure 28
In fr a r e d Spectrum ( c a p i l l a r y f i l m ) o f A. M. I /20 E|
-6 2 The IR spectra o f o ils obtained s im ila r ly from e a r lie r samples
o f mushroom vi; * A. M. 15 c /l (TT.S-OI ) , A. M. I5 c / 2 (T T .5 -0 2 ) and A. M.
I / 20, E IyE2 and
were very s im ila r to those o f M t .1211 and 111. 1212.
b) The 7Q# methanol e xtracts ( I T l . I 22) were concentrated under
reduced pressure.
The concentrate was shaken out repeated Iy with diethyl
ether to remove any o il
(which being very sparingly soluble in Skel Iy
might have escaped in to the methanol layer during p a r t it io n ) .
o f e th e r, an o il
On removal
1E 1 ( I I I . 1221) was obtained which was suspended in a
large amount o f S k e lly and chromatographed on alumina (3 .5 x 29 cm)
(C hart 9 ) .
Eluant
Remarks
1.
S k e lly
No residue
2.
S k e lly (9 0 ) Ether (10)
No residue
3.
S k e IIy (70) Ether (30) 400 c .e .
Oi I
4.
Skel Iy (50) Ether (50) ;l l i t e r
"O )
S k e lly (20) Ether (80) 700 c„c.
.\0)
S k e lly (10) Ether (90) 500 c .c .
--O')'
Ether
300 c .c .
Sem i-crystal 11ne r e s idue
200 c .c .
■
200 c .c .
L i t t l e r e s idue
9.
Ether (90) MeOH (10)
• - -..O,,
Ether (80) MeOH (20 )
10.
Ether (60) MeOM (40)
200 c .c .
L i t t l e residue
II.
Ether (4 0 ) MeOH (60)
600 c .c .
Very I i t t l e r e s idue
12.
Ether (20) MeQH (80)
200 c .c .
No res I due.
5.
6.
7.
8.
v ’-1
*
L i t t l e residue
L i t t l e r e s idue
L i t t l e r e s idue
L i t t l e residue
A. M. 19/20 ' E ' , I I 1 . 1221
I
II
(S k e lIy + Ether 1: 1)
C ry s ta ls , m.p. 193°C (dec)
(Fumaric acid?)
I L l- x I
CHART 9
v
v
Ml
IV
(Ether + MeOH eluants)
O
I (O il)
CSkeIIy + Ether 7 :3 )
Alumina Chromatography (29 x 3.5 cm.)
Crystal 11ne Products
(Micro)
-64 13.
Ether C10) MeOH (90) 400 e .c .
Very l i t t l e residue
14.
Methanol
Very l i t t l e residue
15.
Water
I lite r
L i t t l e residue
Solvent was removed fromchromatographic fra c tio n number 4 ,
/S k e lly (50) and ether ( 5 0 ) / .
The residue on re frig e ra tio n yielded a
small q u an tity o f a c r y s ta llin e product (100 mg).
On c r y s ta lliz a tio n
from water i t came out as needles, m. p. 196-97*0 ( d e c .) .
/p a p illa r y
introduced a t 100*0; m elting point depended on ra te o f h e a tin g ./
About
h a lf o f the m aterial (44 mg). I I I .Xj was used for biolo gical te s ts .
was soluble in water and methanol and very sparingly so in e th e r.
ium fusion indicated the absence o f nitorgen.
with Fol i n 's , ^
Kel I e r 's , 4 *
It
Sod­
I t gave negative te s ts
and ninhydrin reagents.
An aqueous solu­
tio n was a c id ic (pH 2 .8 -3 .0 ) and decolorized bromine-water and KMnO^.
The n e u tra liz a tio n equivalent o f the product, c a rrie d out as
below was found to be 5 7 .2 2 .
The substance (2 2 .8 mg) was taken up In warm, n e u tra l, 50# ethanol
in a stoppered fla s k , cooled and t it r a t e d against standard NaOH
(37.80 c .c . o f 0.01054 1N1 a lk a li was consumed) using phenolphthalein
I ndicator
-6 5 -
F lgure 9
In fra re d Spectrum (Nujol m ull) of tran s -A c o n itic Acid
Figure IO
In fra re d Spectrum (Nujol m ull) of c is -A c o n itic Acid
- 66“
F ig u re 11
In fr a r e d Spectrum (N u jo l m u ll) o f Fum arIc A cid
W AVELEN G TH
IN
M ICRO N!
F ig u re 12
In fra re d Spectrum (N ujo l m u ll) o f I I I . X |. m .p. I96-97°C (dec)
-6 7 -
Sol vent Front
F igure 31
Thin sheet chromatogram o f:
I ) trans-aconi t i c acid
2 ) TTT.X| and 3) c is -a c o n itic acid
-6 8 —
The m elting p o in t ,,9 6 - 9 7 ° 0 (d e c )., unsaturation, and n e u tra liz a ­
tio n e q u iva len t, 57.22 (Al. E . may be such as 1, 1/2 or 1/3 o f molecular
weight) indicated t h a t . i t could be tra n s -a c o n itic a c i d , m . p .
I 94-95°G (N. E. = 58 .0 3 , M. W. = 174.11).
191°,
Eowever, in fra re d spectra o f
samples o f tra n s -a c o n itic acid (General Bio-chem icals, Ohio) and e is a c o n itlc acid (N u tritio n a l Biochemicals G orporation, Ohio) d iffe re d
considerably from the spectrum o f the mushroom product, jT T .X j > m. p.
l96-97°0 (d e c ).
(F ig . 9 -1 2 ).
Ascending th in sheet chromatography
confirmed the n o n -id e n tity .
Aqueous solutions of trans-aconitic acid,.TTT.X| and cis-aconitic acid
were spotted p a ra lle l on a s ili c a sh eet.
The s ilic a sheet was kept for
three hours in a glass chamber containing 200 ml of the solvent system;
Ethyl acetate ( 5 0 ), P yridine (30) and water (2 0 ).
o u t, and allowed to dry a t rotimi tem perature.
a fres h ly mixed reagent;
94
The sheet was taken
I t was then sprayed with
P yridine (7 ) and a c etic anhydride ( 3 ) .
sprayed sheet was dried in a current o f hot a i r .
The
Both e ls - and tra n s -
a c o n itic acids exhibited brown spots (on a white background) while the
c r y s ta llin e product M l .X| did not.
Fumaric acid has been reported to be present in Amanita muscaria
by e a r lie r in v e s tig a to rs , though apparently not character!zed.®®'®®
The m elting point o f fumaric acid has been v a ried ly reported:
77
200°C j
34
287° (293^5)°G , sublimes a t 20OOG, b .p . 290°G ; 300-2° (2 8 6 -7 , 282-4)°G
in a sealed tube, sublimes above 200° in open vessels*^; 287°(closed
c a p illa r y , rapid h e a tin g ), sublimes a t 200°G . ^
Fumaric acid being d i-
carboxylic (O4H4O4 , M. W. I 16.07) has the same n e u tra liz a tio n equivalen t,
-6 9 -
Solvent fro n t
- —© --------------------------------- O
I
2
Figure 22
Thin sheet chromatogram o f:
2) fumaric acid
I) H I .Xj
and
-7 0 , 58.03 as a e o n ltic acid (CfiH6Og, M. W. 174.11), a tric a rb o x y lic a cid .
Thin sheet chromatography o f 111 .X| p a ra lle l with a sample of fumQric acid (N u tritio n a l Biochemicals Corporation, Ohio) indicated id e n tity .
A s ilic a sheet with samples spotted was developed for I hour and 40
minutes In the solvent systemi n-butanol ( 10) , formic acid ( 2$ and
to
water (1 5 ). Bromophenol blue (0*04#. in 95# ethanol and a few drops o f
d ilu te NaOH fo r adjusting to a d e fin ite blue colour, pH 6 .7 ) was used as
spraying reagent.
ta in e d .
Id en tic a l yellow spots on a blue background were ob­
(F ig . 3 2 ).
The c r y s ta llin e product 11j .X^ was re a d ily soluble In w ater,
Fumaric acid is only sparingly soluble in water, 0 .7 g/100 ml a t 250C
and 9 .8 g/100 ml a t 100* 0 .3 4
Even in a closed c a p illa r y , TTT.X| melted
a t 195*0 in p a ra lle l experiments with fumaric a c id .
M aleic a c id , m. p.
130.5*0 is e a s ily soluble in w ater, 7 8.8 g/100 ml a t 25®C and 392.6 g/100
ml a t 9 7 . 5* 0 .34
Samples of fumarie a c id , TTT.X | , maleic acid prepared by gentle
warming with water o f maleic anhydride (Eastman Kodak) and also an old
sample of maleic acid (Eastman Kodak) were chromatographed on a s ilic a
sheet, fo r two hours, in the same solvent system: n-butanol ( 10) , formic
10
acid (2 ) and water ( 1 5 ). Bromophenol spray
gave id en tic a l yellow spots
fo r fumaric acid and 111 .X [ . while the yellow spots o f maleic acids were
lower.
The old sample o f maleic acid gave a tin y yeI low zone a t the same
level o f fumaric acid in d icatin g the presence o f some fumaric acid in i t .
(F ig . 3 3 ).
The chromatogragram o f aconit i c acids (Fig 31) gave Id en tical
-7 1 Sol vent Front
___ «a_______
o
-O-
O
3
2
F i gure 33
Thin sheet chromatogram o f:
I) Fumaric acid;
2) I I I .X |; 3) M aleic acid from maleic anhydride;
4) M aleic acid
-7 2 brown spots fo r c is - and trans-isom ers.
Of course, in the tran s -a c id
there was a lower brown spot in d icatin g the presence o f some other con­
s titu e n t or im pu rity.
The in fra red spectra of fumaric acid and 111.Xi were very close.
However, i t appears th a t while T
l f consi st s mainly o f fumaric acid,
there is some im purity present as fu rth e r evinced by elemental analysis
below:
Found: G, 41.65#; H, 4.64#; M. W. 136
CGeIle r )
Fumaric acid (G4H4O4 , M. W. 116 .0 7 ) requires:
A c o n itic acid (C6HgOg, M. W. 174.11)
G, 41.39#;
M, 3.47#
Pharmacologists reported th a t 11 |'.X| produced only hyperpyrexia
and no other o b je c tiv e symptoms, lik e m ydriasis.
Ho more m aterial is
a v a ila b le fo r fu rth e r p u rific a tio n and preparation of d e riv a tiv e s lik e
methyl e s te rs .
Micro qu an tity o f c r y s ta llin e products separated from the other
chromatographic fra c tio n s , but fu rth e r work on these was Shelved,
The ether insoluble fra c tio n o f the concentrate from 70# methanol
(C hart 7 ).
1F 1 ( 111 .1 222) was taken up with water (140 c . c . , 155 g,
pH 2 .5 ) and r e frig e ra te d .
the usual methods.
No c r y s ta llin e product could be isolated through
I t was passed through a DEAE ( d iethyl-am inoethyl) c e l­
lulose column (Chart 10) and eluted with n-butanol, a r b it r a r i ly col I e c t i ng
various fra c tio n s .
stance.
None of them yielded d ire c tly any c r y s ta llin e sub­
The to ta l concentrates were then taken up with absolute ethanol
and fra c tio n a l Iy p re c ip ita te d with e th e r.
Gummy insoluble m aterials which
A. M. 19/20 'F ' ( 111. 1222)
DEAE-ceI Iulose
n-BuOH eluted
Y
E Iuants
Freed of solvent under
reduced pressure, Ng
Y
Concentrate
Taken up with absolute
EtOH and fractionated
with ether
♦
Y
Soluble
Insoluble
C rystals
m.p. I06°C
CHART IO
74
Figure 13
In fra re d Spectrum (c a p illa r y film ) of Ca (c h a rt 13)
Figure 14
In fra re d Spectrum (c a p illa r y film ) of
e th e r-s o lu b le fra c tio n of 1F 1 ( I I I . 1222)
separated did not y ie ld any c r y s ta llin e si/bstance on fu rth e r m anipulation.
The soluble fra c tio n was found to have an In fra red spectrum
(c a p illa r y film ) id en tic a l With the ether soluble fra c tio n
(Chart 15)
from the butanol concentrate o f the to ta l acetone insoluble frac tio n
(V IT . 11) separated from the main methanol concentrate of the mushrooms.
(Charts 7 , 11, 12 and 13).
Pharmacological reports indicated s im ila r
muscarinic a c t iv it y concentrated in both fractio n s 0F 1 (TTT.1 222)
and 1C1 (TTT. I I I ) .
By p e rs is ten t working a very small q u an tity o f a c r y s ta llin e pro­
duct, m. p . I06°C (dec) was co lle cte d from the a lc o h o l-eth er soluble
portion id e n tic a l with th e same product obtained.from
(Chart 13) des­
cribed la te r on.
Examination o f the acetone-?nsoluble fra c tio n I I I . I I ( Charts 7 & I I )
This was extracted exhaustively with n-butanol in batches
(mechanical shaker).
The butanol insoluble pitchy brown semi-sol Id
yielded a fra c tio n soluble in absolute methanol.
The concentrate 1D1
( ITl . I 12. 250g) was soluble in water (pH 4 .7 ) and consisted mostly of
inorganic m a te ria l, KGl being predominant.
Pharmacologists reported
th a t th is fra c tio n did not appear to be hallucinogenic*
I t was set aside.
From the butanol so lu tio n s, solvent was removed under reduced
pressure (Mg) and the concentrate 1G1 (TTT. I l l ,
160.5 g, pH 3 .4 ) on
re frig e ra tio n over a period o f two months yielded a sem i-crystal lin e
deposit which was f ilt e r e d (Chart 12).
The c r y s ta llin e mass was washed
(to remove the adhering colouring m atter and im p u rities) with methanol
Acetone In s o lu b le F ra c tio n ( 111 . 11)
n-BuOH
I-
'K
Residue
BuOH Solution
Solvent removed under
reduced pressure, Ng
A. M. 19/20 'C '
(160.5 g ., pH 3 .4 )
( IL L .im
Absolute MeOH
Absolute MeOH
solution
Solvent removed
A. M. 19/20 'D' (250 g .)
(TTT. 112)
CHART I l
Residue
I
Ox
I
A. M. 19/20 'C ' (160.5 g . , pH 3 .4 )
(TFT. M l)
R e frIg e r a tion, f i I t r a t i on, e tc .
C rystals (0.927 g .)
m.p. I69-70°C (from
absolute EtOH)
(DL-M a n n ito l)
77k
tra te s
Solvent removed - residue taken up with
moist BuOH, run through DEAE-celIulose
(22 x 2.5 c m .). Eluted with n-BuOH
E Iu a n ts
C ellulose (42 x 6 cm.)
E Iu a n ts
S o lv e n t removed under
reduced p re s s u re , N2
ir
Res due
CHART 12
-~j
i
-7 8 W AVELEN G TH
IN
M IC R O N S
Figure 15
In fra re d Spectrum (Nujol m ull) of Mannitol
1600
1500
W AVENUM BER
1400
1300
1200
IN K AYSER S
Figure 16
In fra re d Spectrum (Nujol m ull) o f Mannitol from mushroom
-7 9 and fra c tio n a l Iy crystal I i zed from hot absolute ethanol, when i t came out
as colourless needles, m. p. I69-70°O ( y ie ld , 0.927 g ) .
te s ts with ninhydrin, F o lio ’ s , M olisch,
71
J t gave negative
K e lle r 's and Liebermann reagents.
I t gave a p o s itiv e te s t with periodic acid in dicating
v ic in a l hydroxyls.
I t did not show any depression in m.p. on admixture with a sample of
m annitol, m. p. 16 6-67*0.
From elemental analysis and m elting point i t
could reasonably be id e n tifie d to be D L r M a n n i t o I (o r possibly a mixture
o f D-M annitol, m. p. 166* and DL-MannitoI, m. p. 1 70*).
-
The In frared
\
spectra o f the substance and Dririannitpl sample\;were almost id en tical
(F ig . 1 5 -16 ).
There were minor differences in .th e fin g e r p r in t region.
78
Thin sheet chromatography
confirmed the id e n tity with m annitol.
Aqueous solutions of the c r y s ta llin e product p a ra lle l with a sample of
mannitol (Eastman Kodak) were spotted on a s ilic a sheet.
The s ilic a
sheet was kept fo r 55 minutes in a glass chamber containing the solvent
system:
Ethyl acetate (5 0 ), Pyridine (3 0 ) and Water (2 0 ).
sheet was taken out and dried a t room tem perature.
with 50# H2SO4 and dried In oven.
The s ilic a
I t was then sprayed
Id en tic a l dark zones were observed
with both the products (F ig . 3 6 ).
Found:
(G eM er)
0 , 39.10#;
H, 7.56#
Mannitol requires: 0 , 39.56#;
(G6H14O6 , M. W.. 182.17)
H, 7.74#
Thin sheet chromatography is fa s te r than the c u rre n tly known paper
chromatographic methods, fo r the Id e n tific a tio n of carbohydrates.
Both
the techniques Involve equally the same length of tim e In the search for
-8 0 Sol vent Front
I
2
Figure 36
Thin sheet chromatogram o f:
1) Mannitol (sample from Eastman Kodak)
2) Mannitol isolated from A. hnuscaria
—8 1—
s u ita b le solvent systems and spray-reagents fo r id e n tific a tio n .
Paper chromatography was c a rrie d out e a r lie r to id e n tify the
mannitol iso lated from A. muscaria. with a known sample o f m annitol,
From IO to 14 hours, were needed fo r development using the solvent
system:
n-butanol ( 6) , pyridine (4 ) and water ( 3 ) .
n itr a te was used as a spraying reagent.
91
*
92
Ammoniacal s ilv e r
I f the sprayed paper is
dried fa s t in a curren t o f hot a i r , the background burns dark brown,
making I t d i f f i c u l t to see c le a rly the re s u ltin g id en tic a l chocolate
coloured spots o f m annitol.
This d i f f i c u lt y can be avoided by allowing
the paper, a f t e r spraying with ammonIacaI s ilv e r n it r a t e , to dry f i r s t
a t room temperature fo r a few hours and then exposing fo r a few minutes
to hot a i r .
The f i l t r a t e s
l e f t a f t e r removal of mannitol were freed o f solvent,
taken up In minimum qu an tity o f moist butanol and passed through a DEAE
(d ieth ylam ino eth yI) c e llu lo s e column (22 x 2.5 cm).
The butanol eluants
were then passed through a c e llu lo s e column (42 x 6 cm) and eluted again
with n-b u ta n o l.
Various a r b it r a r i ly c o lle c te d fractio n s o f eluant were
subjected to a number of prelim inary experiments in the hope o f is o la tin g
some c r y s ta llin e substance but without success» Then the to ta l residue
was subjected to elaborate fra c tio n a tio n s as indicated,under Chart 13.
The In fra red spectrum o f C^, the ether soluble fra c tio n in the
above scheme (F ig . 13) was found to be id en tic a l with th a t o f the ether
soluble portion of 1F 1 (T 7 T .I2 2 2 ) (F ig . 14) as were the pharmacological
reports o f muscarinic a c t iv it y concentrated in these two fra c tio n s , as
Resi due
(From 1C1 through Chromatography)
Exhaustive and successive ethereal
p re c ip ita tio n in batches
4
R recipIt a t e
Residue
MeOH
MeOH
S olution
I
F iIt r a t e s
THF
Dry Ether
--------T-------------------------I
THF
Freeip it a t e
Fi tra te s
Solution
I
(m .p. I 06°C)
THF
Solvent removed (charcoal)
v
C rystals
|________ I
____
Residue
THF solution
(pH 3 .8 )
MeOH
Ether
Resi due
Acetone c r y s ta lliz a tio n
Crystals m.p. 105*
m.p. 106* (from
acetone + MeOH)
IR Spectra
Ether Me0H Solutlon
Ether
Solvent
removed
Resi due
MeOH t Ether
Solvent removed
cr 4
(pH 4 .6 )
CHART 13
THF = te tra h y d ro fu ra n
00
1
-8 3 a l ready noted.
The maximum amount of the c r y s ta llin e product m. p, IOS0G
(dec) was obtained from
C haracterizatio n of the crystal Iin e product m. p. IOS0C ( dec?.
The to ta l crude crystal I iz a te (O.S285g) was c ry s ta lliz e d from a
m ixture of methanol and acetone and f in a l ly from hot absolute ethanol.
On very slow c r y s ta lliz a tio n during a period of nearly three months,
colourless needles ( y ie ld , 292 mg), m. p. I06°C (dec) were obtained.
Sodium fusion indicated the absence o f nitrogen and halogen.
Molisoh and Red Tetrazolium
27
Tests with
fo r reducing sugars were negative.
Test
with periodic acid was p o s itiv e .
Mo residue was l e f t a f t e r ig n itio n and emission spectrum indicated
absence of inorganic contaminants.
A fte r acid hydrolysis (20 mg, refluxed with 5 e .c . o f 5# HGl bn
steam bath fo r 2 ,5 hours) i t gave a p o s itiv e te s t with Red T e tra z o l(urn.
27
' ! a
Besides, the tim e taken to develop the pink colour
when tested ,with Red
Tetrazolium along with known sugars indicated the lik e lih o o d of glucose
being released on hydrolysis.
The hydrolysate was n e u tralized with
, concentrated and
chromatographed on Whatman paper p a ra lle l with standard glucose.
The
solvent system consisted o f Pyridine ( 4 ) , n-butanol ( 6 ) and water ( 3 ) .
Spraying with G D -I^ indicated id e n tity with glucose.
streaking due to the MaGl present.
There was a l i t t l e
The hydrolysate was desalted by pass­
ing through M. B, 3 resin (mixed bed) and then chromatographed as above.
Glucose and no other sugar was observed to be present when sprayed with
GD-I (F ig . 3 4 ).
-8 4 Solvent Front
2
3
Figure 34
Paper chromatogram o f:
I) Glucose sample; 2) acid hydrolysate of
Jg-glucoside, m.p. I06°C (dec) from A. muscaria
3) Xylose sample
"85—
In another experiment, the c r y s ta llin e product was incubated (42°C,
6 hours) with emuIs in and chromatographed, p a ra lle l with standard glucose.
Spraying with GD-I indicated id en tic a l Rf value and no other components„ This
indicated th a t the c r y s ta llin e substance m.p. 106°0 could be a B~glucoside.
A cetylation of the g Iucostde with a c etic anhydride and pyridine was attem pt­
ed tw ice but no c r y s ta llin e acetyl d e riv a tiv e could be c o lle c te d .
ia l was l e f t fo r the Is o la tio n and id e n tific a tio n of the aglycone.
No mater­
The
substance had a s lig h t tendency to sublime in vacuum a t I QQ0G CI mm pres­
sure) but in an open c a p illa ry i t decomposed only above it s melting p o in t.
Found;
(B erkeley) C, 40.73#; U , 6.80#; M. W. 350
(S e lle r )
0 , 44.08#; H, 7.51#; Loss in weight 6.95#
The d iffe re n c e in a n a ly tic a l values, for the same substance, is prob­
ably due to varying degree of hydration of sample analyzed in the above two
la b o ra to rie s .
a g lucoside.
known.
Al I the a v a ila b le evidence indicates th a t the substance is
This can be s e ttle d only a f t e r the nature of the aglycone is
In the meanwhile one may feel th a t the substance may be a disac- ■
charide monohydrate, G |2^22« ^2° (M. W. 360) wi l l analyze for C, 40,00# and
M, 6.60#, in close agreement with a n a ly tic a l values obtained from Berkeley
A nalytical Laboratory fo r the substance, g Iucoside, m.p. 106*8 isolated
from Ai muscaria .
Trehalose, © | 2^22^ I I *1 ZHgO, m. p. 96.5-97 »5°G is the closest known
disaccharide with non-reducing end.
a t times in young mushrooms.
Trehalose Is also reported to be found
However, the in frared spectra o f a sample of
trehalose and the c r y s ta llin e substance, m. p. 106*0 (dec) from the mush­
room d if fe r considerably (F ig . 17, 18) in s p ite o f unequal in te n s ity of
absorption spectra.
-86 -
Figure 17
In fra re d Spectrum (Nujol m ull) of j3 -g lucoside,
m. p . 106°C (dec) from mushroom
W AVELEN G TH
IN
M IC R O N S
12
1900
1800
1700
1600
1500
W AVENUM BER
1400
1300
1200
IN KAYSERS
1100
KS
Figure 18
In fra re d Spectrum (Nujol m ull) of Trehalose
13
14 15 16 ,
-8 7 B•
Water E xtracts
The pressed marc ( l e f t a f t e r e x tra c tio n with methanol) was e x tra c t
ed with d is t ille d water a t room temperature by maceration, percolatio n,
shaking in batches (mechanical shaker), f i l t r a t i o n
e tc .
This was
(July-August 19 5 9 ).
in a basket centrifuge
a very tedious process during a period o f two months
The f i l t r a t e s were concentrated a t room temperature
(a ir-b lo w in g , vacuum d i s t i lla t io n e t c ) .
The concentrate (fecal odour) was chromatographed on a c e llu lo s e
column (42 x 6 cm) (Chart 14) and eluted with moist butanol .
The butanol
eluants on removal of solvent under reduced pressure yielded a golden
yellow syrup (1 .5 g ) , containing m icro-crystal Iin e m a te ria l, f Iourescent
In u .v . and giving a p o s itiv e te s t with K e lle r 's reagent fo r Indoles.
With small q u a n titie s of the concentrate a t a tim e, various micro experi­
ments were t r ie d (chromatography, solvent fra c tio n a tio n , e t c ) , but the
major p a rt was reserved fo r future work.
A ttention was focused on the
next lo t of mushrooms, described under P art TV.
A fte r butanol e lu tio n , the column was washed with d is t ille d w ater,
f ilt e r e d ( c e lit e ) and concentrated.
I t was found to contain a good deal
o f inorganic components.
The fin a l dry marc, a fte r water e x tra c tio n , weighed 3952 g.
PART TV
As a re s u lt o f experience gained in working up the i n i t i a l batches
of fresh mushrooms I t was considered desirable to process the mushrooms
more e f f ic ie n t ly in a pharmaceutical p ilo t p lan t and obtain the concen­
tra te s fo r in v e s tig a tio n .
Accordingly, s ix cans of fresh mushroom In
W ater E x tr a c ts
C o n ce n tra te d a t room te m p e ra tu re
>f
C o n c e n tra te ( fe c a l o d o r)
Chromatographed on C e llu lo s e Column
(4 2 x 6 cm .)
Residue on Column
------- *
BuOH e lu a n ts
k
I
W ater ( c e l i t e )
F ilt r a t io n , e tc .
Freed o f s o lv e n t under
reduced p re s s u re , Ng
Y
W ater E x tr a c ts
I
C o n ce n tra te
Y
C o n c e n tra te (1 .5 g . )
( C r y s ta ls , ln d o l lc , K e lle r 's T e s t e t c . )
CHART 14
methanol received in May I960 were processed a t the Abbott Laboratories,
North Chicago, I l l i n o i s , in June 1960 under the d ire c t supervision of
D r. Gook.
The extractio n s were based upon those adopted e a r lie r .
One s ig n ific a n t change was th a t the i n i t i a l aqueous methanolic
extracts were c o lle cte d separately and worked up, distinguishing them
\
from subsequent dry (absolute) methanolic e x tra c tiv e s .
The mushrooms and methanol together weighed 98,6 Kg.
•
Solvent
was drained through c lo th ( 5 8 . 1 Kg) and the whole mushrooms (40.5 Kg)
were pulped in a meat grinder (Chart 15).
F ilt r a t io n through a stone­
ware f i l t e r under vacuum was not s a tis fa c to ry .
f i l t r a t e was co lle cte d by th is method.
Only four gallons of
Then, the pulpy mushrooms were
run in batches in a basket c en trifu g e (4 Kg. a t a tim e t i l l
was reduced to about I .2 Kg).
the weight
About 20-25 gal Ions of f i l t r a t e s was
co llected by th is method.
The to ta l aqueous methanol Ic e xtracts (pH 6 .0 0 , containing approxi­
mately 6Q% water) were freed of solvent below 25°C under reduced pressure
in a L « T . V. (low temperature vacuum) s t i l l .
The concentrate 'A 1
(TV. I ) was obtained as a th ic k , dark brown syrup (3628 g, pH 5 .1 ) .
The residual pulp (1 1 .4 Kg) was a ir -d r ie d a t 35°G overnight to
5 .9 Kg (corresponding to 3.604 Kg dry weight) and was extracted with
absolute methanol (9 9 .6 #) in batches (Chart 16). Each batch was e x tr a c t, I
ed with about 12 gallons o f methanol by the follow ing successive operations
using lig h tn in g s t ir r e r fo r c irc u la tio n o f solvent;
Fresh Mushroom (Par+ TV)
(Preserved in MeOH; received in May, I960
E xtractions a t Abbot Laboratories in June, i960)
Drained through cloth
I
I
MeOH (aq)
Extracts
(58.1 kg)
Pulped in meat-grinder
and f ilt e r e d through
stone-ware f i l t e r and
basket centrifuge
Added on
'I'
F ilt r a t e s
(25 g a l)
V
06-
Sol vents
removed
below 25°C
in L .T .V .
S till
Mushroom (4 0 .5 k g .)
Residual pulp
11.4 kg.
a ir -d r ie d a t
35°C ., overnight
Concentrate 1A1
Dark brown th ic k syrup
( 3628g. j pH 5 .1 )
V
5 .9 kg.
in. I
CHART 15
i
A ir-D rie d Mushroom Pulp
(5 .9 kg.
3.604 kg. dry weight)
E xtraction with
MeOH (99.6%) in batches
I 2 gal Ions each time
s t ir r in g
c l rc u la tin g
standing with MeOH, e tc .
f?I te r
Marc
F iltr a te s
Basket C e n trifuge
Marc
F iIt r a t e s
Added on
Water e xtractio n
S t ir r in g , f i l t e r , etc
Final Marc
Solvent removed
in L .T .V . S ti 11
below 25°C
Concentrate 1B1
Lig h t Brown syrup
(2947 g . , pH 7 .5 )
Water Extracts
L .T .V . Sti 11
d i s t i I led
'V
Aqueous Concentrate 1C1
(3288 g. 33 g. s o lid s ) T V .3
(CHART 16)
-9 2 1.
S + irrin g fo r h a lf an hour, standing for 1/2 hour and
filte r in g .
2.
( F ilt r a t e s c o n tain ed '-/4 .7 # water)
S tir r in g for I hour, c irc u la tin g for 2 hours and
f I Ite rin g (water content of fI Itr a te s
3.
I .4#)
C irc u la tin g fo r 2 hours and f il t e r in g (water content
-■0.6#) (Chart 16).
The extracted mushroom pulp was centrifuged when about 3 lit e r s
o f f i l t r a t e s were c o lle c te d .
K g).
The mare weighed 4 .5 Kg (dry weight 2.656
I t was extracted with deionized water by s t ir r in g for about an hour
a t a time and f i l t e r i n g .
still.
The f i l t r a t e s were concentrated in the L . T . V.
The concentrate (4 .2 Kg) was processed in th is labo ratory.
was found to contain mushroom pulp and was th e re fo re r e f ilt e r e d .
It
This
f i l t r a t e weighed 3288 g. corresponding to 33 g . dry weight, 1C 1, T V .3.
The combined absolute methanolic extracts on d i s t i lla t io n in
L 1JT. V. s t i l l
pH 7 . 5 ) .
below 25°G gave a lig h t brown syrup 'B*(% V.2, 2947 g,
On keeping for a few days In the r e fr ig e r a to r , i t deposited a
waxy mass embedded with crystal lin e substance and was worked up as out­
lined under Shart 17.
S h a ra cte rization of the crystal Iin e product
The to ta l c r y s ta llin e product, m. p . I8 3 °0 , recrystal 11 zed from
aqueous methanol e t c ., ( illu s t r a t e d in Chart 17) was f in a lly re c ry s ta l11 zed by taking up in minimum q u an tity o f water, adding absolute ethanol
dropwise to f a in t tu r b id ity and s e ttin g asid e.
Thus repeated frac tio n a l
c r y s ta lliz a tio n s yielded a to ta l o f 606.6 mg., colourless needles, m. p.
186°S (dec) ( F ir s t crop o f c ry s ta ls , 0 .4 1 20g; Second crop, 0 .1 926g and
C oncentrate 1B 1 IV .2
Cooled, small quantity
of semi-crystal lin e sub­
stance embedded in waxy
m aterial separated.
Decanted
4
T h ick, waxy
lower portion
Clear; top
Supernatant
C h ille d
Decanted
I
Wax
I
Decantate
Washed with benzene,
e th e r, acetone, e tc .
Organic
Washings
Evaporate i n
va< cuum
3"
Resi due
Crystal I i zed (tw ice
from water + MeOH
(charcoal)
V
Colourless, c r y s ta llin e
needles, m.p. I83°C (very
low y ie ld )
Concentrate
Abs. MeOH
Cooled (3 days)
F iIte rp d
F i lt r a t e
"""I'
C ry s ta llin e Product
m.p. 175-80°C
Washed with e th er,
acetone, e tc .
S o l v e n t B
Washings
/ S
C ry s ta llin e
product
m.p. 175-80°C
Evaporated
ri n vaccuum
Concentrate B-I
H .2 I
CHART 17
vO
W
I
C ry s ta l / I i zed from
/ water +
/
MeOH
Colorless needles
(0.3107 g .) m.p.
183° C
-9 4 th e la s t crop about 2 mg).
Aqueous solution of the c ry s ta ls was nearly neutral ({5H 7.00 with
close-range pH p a p e r).
and halogen.
Fusion with sodium indicated absence of nitrogen
Tests with Mol Isch, Fol in 's , FeGI3 , K e lle r 's and L Ieb er-
mann's reagents were negative.
I t gave a p o sitiv e te s t with periodic
acid but acetyl a t I on was not successful .
I t gave negative te s ts with
Red T e tra z o li urn both before and a f t e r treatm ent with a c id .
Paper chroma­
tography of acid hydrolysate p a ra lle l with known sugars was not d e fin itiv e
When heated on a platinum s p atu la. I t burned lik e sugars.
flame was c h a ra c te ris tic o f potassium s a lts .
The small amount of residue
on the spatula gave a basic aqueous solution (pH 1 0 .0 0 ),
trum indicated the presence
The
Emission spec­
o f only potassium in moderate concentration.
From elemental analyses, m. p. e t c ., the substance was suspected
to be potassium gluconate.
Hence a sample o f potassium gluconate was
prepared from gluoono d e lta lactone
(N u tritio n a l Biochemicals Corpora­
tio n , Ohio) and t h e ir in frared spectra appeared to be very s im ila r
(F ig . 19, 2 0 ).
Found:
C, 5 0 .3 3 % ', H, 4.81#
(B erkeley)
(G e IIe r) G.> 30.70#; H, 4.91#; K, 16.59#; 0, 47.72#; M. W. 107,
C, 30.99#; H, 4.57#; K, 16.54#; 0, 47.97#; M. W. J>07
.
'
>
Potassium Gluconate, GgHjiOy K (M.W. 234) requires:
0 , 30.77#; H, 4.70#; K, 16.66#; 0 , 47.86#
The preparation o f a pure s a lt of gluconic acid presents consider­
able problems*.
The in fra re d spectra o f samples of potassium gluconate
obtained from.Chas, P fiz e r (99# pure) and Matheson Coleman & Bell (98#)
95-
Figure I 9
In fra re d Spectrum ( Nuj o I m ull) o f potassium gluconate from mushroom
MO------ MO------ 700------ 600
W AVENUM BER
IN
KAYSERS
F igure 20
In fra re d Spectrum (Nujol m ull) of
potassium gluconate prepared from g lu c o n o -d e lta -lactone
-9 6 W AVELEN G TH
IN
M ICRO N!
Figure 21
In fra re d Spectrum (Nujol m ull) of
potassium gluconate (Chas. P fiz e r )
Figure 22
In fra re d Spectrum (Nujol m ull) of
potassium gluconate (Matheson, Coleman & B e ll)
are shown in F ig . 21, 22.
A thorough search o f lite r a tu r e revealed th a t
the m elting point o f potassium gluconate has been recorded by oryl-y one
in v e s tig a to r ,^ in In d ia , as 185°, Keenan and W eisberg^ measured optical
properties o f some s a lts of gluconic acid fo r id e n tific a tio n .
Th eir data
fo r potassium gluconate agrees well for the substance iso lated from the '
> *,
mushroom.
1.
1.
irre g u la r grains and rods.
2.
The indices o f re fra c tio n ;
n^ =
.523, n,
P
I .548
Hg =■ I-.565, a ll + 0 .003.
3.
E x tin c tio n p a ra lle l
For microscopical id e n tific a tio n , the c r y s ta llin e m aterial from
the mushroom was immersed in a liq u id with index I .524.
consisted o f irre g u la r grains and rods.
The c rystals
A sing le crystal was located.
On ro ta tin g the stage, the e x tin c tio n was p a ra lle l with the disappearance
o f crystal boundary.
On another s lid e the substance was immersed in a
liq u id with index I .564.
Rotating the stage, a fu ll c ir c le , caused no
disappearance o f the crystal boundary.
Id e n tity with potassium gluconate was confirmed fu rth e r by th in
sheet chromatography on s ili c a sheet.
Aqueous solutions of samples of
potassium gluconate (Chas. P fiz e r and Matheson, Coleman & B e ll) were
spotted p a ra lle l with a solution of the c r y s ta llin e product from the
mushroom.
The s i l i c a sheet was kept for two hours in a glass chamber
containing the solvent system;
, water ( 1 5 ) .
n-butanol ( 10) , formic acid ( 2 ) and
The sheet was taken out and dried a t room temperature.
On
-9 8 -
2
uj
-------------------------------------0 ------
O
Sol vent Front
________
F igure 37
Thin sheet chromatogram o f: Potassium gluconate
I ) Sample from Matheson; 2) Isolated from
A. muscaria; 3) Sample from Chas. P fiz e r
■99spraying with 50£ HLSO. and drying in oven, id en tical dark zones, on a
white background, were exhibited by a ll the th re 6 substances (F ig . 37,).
The concentrate B-I ( W . 2 I ) from the main, deoamtate ( from the
absolute methanolic e x tra c t,
=
TV.2) a fte r removal of the above -
c r y s ta llin e product was fractio n ated through e xtractio n with ether
(containing a l i t t l e S k e lly to aid in c la r if ic a t io n ) in to an insoluble
(TV. 2 11) and an ethereal solution (TV.2 12) as outlined in
fra c tio n , B-R
Ghart 18.
1
From the ethereal solu tion on removal o f solvent was obtained an
o i l , corresponding to the o il
e a r lie r work.
1A1,
TTT. 121 (Charts 7 , 8 ) , obtained in
I t was taken up in S k e lly and chromatographed on alumina
(3 0 .x 5.5 cm) .
(Chart 18)
E Iuant
Ii te r
Remarks
1.
S k e lIy l
2.
S k e lIy 1.5 I ite rs
3.
S k e lIy l
4.
Sk (80) Ether (20) 400 c .c .
5.
Sk (60) Ether (40) I l i t e r
Very l i t t l e r e s idue
6.
Sk (20) Ether (80) I l i t e r
A l i t t l e more residue
7.
E ther, 4 lit e r s
8.
Ether (60) MeOH (4 0 ) 500 c .c .
Very l i t t l e residue
9.
Ether (20) MeOH (80) 500 c .c .
Very l i t t l e residue
I it e r
10. MeQH, 4 lit e r s
The in fra re d spectra o f the
25.985g - ye I low o iI
(Ab. A - Ill = I V .2121)
'54.6224g - reddish brown o il
(Ab. A-IV = 11.2122),
I 2.6726g - brown o il
(Ab. A. V = JV .2I23)
Very l i t t l e residue
Very I i t t l e r e s idue
Small q u an tity o f th in film
fractio n s were id en tic a l with
I'
150879
C oncentrate B -I
IV .21
I Exhausted with ether
_________ I (+ a l i t t l e S kelly for c l a r i f i c a t i on)
InsolubIe
Middle
Ethereal Solution
B -^11.211 (2 5 2 .3g .)
(T 7.2I 2)
S kelly
washed with e th e r,
Chi lie d
benzene, ether
Decanted
'F
v
ResI due
Sol vent washings
Ethereal
Slimy
Solution
P re c ip ita te
Dissolved in water (m in.)
Extn with CHC13 + MeOH
( 1 : 1 ) , 15 extns, 100 ml each
Solvent removed
'f
Residue
"
"
y
Evaporated in
vacuum
i>
Concentrate. Taken up
in S k e lly and chroma­
tographed on alumina
(30 x 5.5 cm.)
Skel Iy eluted:
'V
S k e l I y e l uted:
Resi due
1. yellow o il - 25.98g (T7.2I2I)A b .A .T T T
Crystal I i zed from
2 . reddish brown o il - 54.62g. (T V .2 122)
water + MeOH (charcoal)
Ab.A. T V
and then abs. EtOH
3 . brown o il - 12.67 g (T V .2 123) Ab.A.V
colorless p la te s , m.p.
IR Spectra id en tical w /o iI fra c tio n 1A1
160-165°C. (d e c ).
A.M. 19 /20 A/I and A /2 from e a r lie r working (200
(micro q u an tity)
lbs. lo t) Ether + MeQH Eluted: Micro
q u an titie s of some semi­
crystal 11ne products.
CHART 18
i
100
CHCI , + MeOH
s o lution
-IO Im ic r o n
W AVENUMBER
IN
;
• i
i
1300
1200
K AYSER S
Figure 23
In fra re d Spectrum (c a p illa ry film ) of o i l , Ab. A . I I V. IV .2121
W AVELEN G TH
IN
M ICRO NS
Figure 24
In fra re d Spectrum (c a p illa ry film ) of o i l , Ab. A. I V , I V.2122
-1 0 2 -
W AVELEN G TH
W AVENUMBER
IN
KAY S E R S
F i gure 25
In fra re d Spectrum (c a p illa r y film ) of o i l , Ab. A. V , IV .2123
-1 0 3 M. 19 /2 0 , 1A1 A / I , and A/2 obtained e a r lie r from fresh mushroom, 200
IsbW-^Chapt 8 ) .
Pharmacological evaluation of these oi ls Indicated them
to be devoid o f any noticeable hallucinogenic p ro p ertie s .
Experiments are underway to determine whether these o ils have any
in s e c tic id a l and/or Insect repel I ant p ro p e rtie s .
The ether Insoluble fra c tio n B-R ( I T , 21 I ) was also reported to
have no hallucinogenic a c t iv it y .
By fra c tio n a tio n s , as illu s tr a te d in
Chart 18, a m icro-quantity of c r y s ta llin e product, m. p. 160-65°C (dec)
was separated but fu rth e r experiments on th is fra c tio n were postponed.
Examination o f aqueous methanolic concentrate (TV. I )
As the hallucinogenic p rin c ip le s were mainly concentrated in the
water soluble e x tra c tiv e o f the mushrooms, the mushrooms were so process,ed
’
(a t the Abbott Laboratories) as already explained in the beginning of
P a r tJ V
to c o lle c t the i n i t i a l aqueous methanol i c concentrate £60#
water content) separately and examine i t .
U n fortunately, pharmacological
reports on th is fra c tio n was fa r from encouraging.
I t was worked up as
illu s tr a te d under Chart 19 and the various fractio n s reserved for future
e x p lo ra tio n ,
A tten tion was mainly focused on the fin a l water e x tra c tiv e
from the fIjiarc1,
Aqueous concentrate from the marc 1G1 TV.3
The aqueous concentrate (3288 g. corresponding to onIy 33 g.
of to ta l s o lid s ) was again the most a c tiv e hallucinogenic fra c tio n in
th is whole working.
Innumerable experiments were c a rrie d out with th is p a rtic u la r
Aqueous Methanolic Concentrate
'A ', T f J , (3628g. pH 5 .1 )
E xtraction
wi th
Ether
"I
I
Pale Yellow Ethereal
S o lu tio n . Washed, dried e tc .
Solvent removed
Ether insoluble
W .I2
I
Fractionation
with acetone
( s t ir r in g e tc .)
Concentrate, A. E .
TV. 11
Moi st
n-butanol
Butanol Solution
Insoluble
Solvent removed
Red. pressure
v
Concentrate
TT- 122
CHART 19
I
Concentrate
A.A. T V .121
i
104
•V
Acetone
Solution
Insoluble
V
*■(
-1 0 5 fra c tio n .
Various solvent systems were t r ie d with chromatographic
screening (column, paper, th in -s h e e t e t c .)
F in a lly the follow ing proce­
dure was found to be best suited and adopted.
(Chart 2 0 ).
Most of the
water was removed by evaporation a t room temperature under nitrogen a t ­
mosphere in batches.
The syrupy concentrate was re frig e ra te d for a few
days when an inorganic deposit consisting mostly of KCI s e ttle d down.
The decantate was divided in to small portions and then added to an array
of flasks containing absolute e th a n o l.
A lc o h o lic ,f ilt r a t e from each on
microscopic manipulation with solvents indicated presence of c ry s ta llin e
m a te ria l, pale yellow in colour with c h a ra c te ris tic fecal odour, f Iourescent in u . v . and generally responding to K e lle r 's te s t for indoles.
The alcoho lic f i l t r a t e s , co lle cte d together (pH 6 .0 ) were evapor­
ated a t room temperature (R in c o ).
The residue (about 2.5 g) was
induced to c r y s t a lliz e by successive solution in minimum qu antity of
methanol, and treatm ent with a judicious mixture of ethyl acetate and
acetone.
The e n tire q u an tity of c r y s ta llin e m aterial (246.8 mg) obtained
thus (over a period of two months) was almost colourless, excepting for
a fa in t yellowish tin g e .
I t melted in d e fin ite ly between 130-35°G (dec)
(c a p illa ry introduced a t I 00°G ).
burned with a smoky flame.
On heating over a platinum spatula i t
Its aqueous solution (pH 6 . I - 6 .2 ) had a
tin g lin g ta s te and was f Iourescent In u .v .
and ^ 3 1 0 mp was recorded fo r the substance.
Absorption maxima a t />\260mp
Test for nitrogen on
sodium fusion was very do ubtful, but i t gave p o s itiv e te s ts with ninhydrin
and K elle r 's .
-106
T h in sheet chromatography ( s ili c a p late s ) with various solvent
systems, (alo n g ,w ith samples of psylocybin, p s ilo e in , e t c .) indicated
the separation of two to three components f Iourescent in u .v .
A fte r repeated c r y s ta lliz a tio n from absolute methanol, colourless
crys ta ls (104.8 mg) were obtained (weakly f Iourescent in u .v . and not
showing any other component on th in sheet chromatography).
Unfortunate­
ly , about 1/3 o f the pure c r y s ta llin e product (34.7 mg) sent fo r biologic
a I evaluation was lo s t.
Through p e rs is ten t c r y s ta lliz a tio n o f mother liquors and also
subjecting the various p re c ip ita te s from absolute ethane I obtained
e a r lie r to the same treatm ent ( i . e . , dissolving in minimum quantity of
w ater, adding in batches to absolute ethanol in a series o f containers,
and working up the ethanolic f il t r a t e s a f t e r removal of solvent with
methanol, ethyl acetate and acetone), three more crops o f the same
c r y s ta llin e m aterial (to ta l of 202.6 mg) were collected and the various
mother liquors e tc . c a re fu lly preserved in a r e fr ig e r a to r .
For biolo gical te s ts , 12 mg dissolved in I c .e . o f water were
sent (in a sealed ampoule) again.
As expected i t was reported to be
a c tiv e and devoid o f muscarinic e f f e c t s .
The fin a l pure c y rs ta lI Ine product (to ta l y ie ld : 307.4 mg) was
extremely hygroscopic.
I t did not e x h ib it any sharp m. p. and melted
between 135-38°O (dec) (m.p. recorded a f t e r repeated t r i a l s , c a p illa ry
being introduced a t IOO0O and examined under microscope Immediately
a fte r m elting showing no re sid u e ).
On ig n itio n over a platinum spatula.
Aqueous Concentrate
' C , I I . 3 (3288g. fi It r a t e
33g. dry weight)
Most of the water removed
by evaporation, d is t illa t io n ( I mm)
e tc . a t room temp. (N2) in batches
CONCENTRATE
R e fr igerated
Decanted
Freeip ita te s
Inorganic
(K cI)
(C rystal I i zed
from water)
*
decantates
I Added in small batches
to EtOH in a series of
^ containers
*
F iItr a te s
I
P re c ip ita te s
I
Concentrates
mother
I i quors
CHART 20
S
'Z*
o
--108I t burned lik e an organic substance.but gave a purple flame ch a rac te ris ­
t i c o f potassium.
was basic.
The residue l e f t was very l i t t l e whose aqueous- solution
Emission spectrum indicated only the presence o f potassium.
Found:
0 , 30,17#; H, 4.83#; K, 25.23# and M. W. 615; (fii, absent)
(Gel I e r)
c I5 h3 0 °I5 k4
(M.W. 606.79)
requires:
1
.
G, 29.7#; H, 4.98#; K, 25.77#
The c r y s ta llin e substance, designated as 1Z ' , does not appear to
contain any sugar moiety in the molecule.
Tests with Molisch and Red
T e tra z o ii urn were negative both before and a f t e r treatm ent with acid and
emu I s in .
These experiments were c a rrie d out p a ra lle l with the |) - g Iuco-
s id e , m. p . 106°G (dec) isolated from the mushroom.
The in fra re d spectra (Nujolm ulI and f Io u ro lwbe) o f the c ry s ta llin e
substance are shown in F ig . 29^30.
A sample of the substance (23.4mg)
has been sent to the Vari an Associates, Palo Al to , C a lifo rn ia for NMR
spectrum and in te rp re ta tio n .
The fin a l mother liquors preserved in the r e fr ig e r a to r , have
again been subjected to intensive chromatographic fra c tio n a tio n s .
Attempts are being made to is o la te any fu rth e r c r y s ta llin e products,
/
'
■
probably in d o lic in nature.
Column chromatography was not successful.
Thrn sheet chromato­
graphy on s ili c a sheet, for one hour, in the solvent system:
Ethyl
acetate (5 0 ), Pyridine (30) and water (20) resulted in thd separation
o f three d is tin c t zones a ll showing bluish green f Iourescence in u . v.
-1 0 9 -
B H
Iw
■
I
4500
4000
rr
;1|
T
m
:
:
I
I!
:
•\
::
■
m
M ICRO NS
:'
Ht
&00
IN
II!2rX
m
eH
W AVELENG TH
H i3000
3500
it
m M
m
I! I
-
m
2000
ill1800
1900
1700
1600
1500
W AVENUM BER
ii
1400
1300
1200
IN K AYSER S
'iB
1100
1000
■
I ;;;;
900
I:I
H
I .
800
Figure 29
In fra re d Spectrum CNujoI m ull) of c r y s ta llin e product, 1Z 1,
m. p . 135-38°C (dec) from water concentrate
Figure 30
In fra re d Spectrum (FIouroIube) of c r y s ta llin e product ' Z ' ,
m. p . 135-38° (dec) from water concentrate
600
Hence, the to ta l mother liquors are being worked up by th is technique.
This may involve more than 500 p la te s .
s e p a ra te ly .
Each zone w ill have to be eluted
Concentrate from each should again be sent fo r biological
te s ts , to fin d out whether any o f these have LSD-like p ro p ertie s .
The eluants from lowest zone on rechromatography on alumina sheet
in the same solvent system gives fu rth e r separatio n.
Besides the zones
e x h ib itin g bluish green f Iouresence in u . v , , there is a d is tin c t yellow
component.
Working on the above lin e s , combined with fra c tio n a l c r y s t a lliz a ­
tio n may u ltim a te ly lead to some p o s itiv e re s u lts .
ADDENDA
a)
1Z 1 was sent to 'B erkeley' fo r elemental analysis to check
the values obtained e a r lie r from 'Gel I e r 1t
Found: 0 , 30.3#; H, 4.6#
(Berkeley)
Equivalent Weight (from potentiom etric t it r a t io n curve) = 218.
Apparently three free carboxyl groups (COOH) are present In 'Z '
besides four COOK.
b) N. M. R. data from Varian Associates, Palo A lto , C a lifo rn ia ,
on 'Z 1: - 6 la b ile hydrogens, 8 methyl groups and one methyl keto group
In the mol ecu I e .
x
c) P relim inary experiments in dicate th a t the o il
frac tio n s Iso­
lated from A. muscaria are devoid o f oral t o x ic ity towards insects.
PHARMACOLOGICAL DATA
At the o u ts e t, i t was planned to submit each fra c tio n , system­
a t ic a lly , fo r biolo gical e valu atio n .
B iological screening has been of
assistance in the present in ve s tig atio n in the separation of a potent
water soluble fra c tio n .
This fra c tio n has been found by our team of .
pharmacologists to have LSD -like properties and free from muscarinic
'
v
e ffe c ts .
D r. Edward Pelican (Acting Head, Department o f Pharmacology and
Experimental Therapeutics, Boston U n iv e rsity College of Medicine) and
his associates c arried out the biological assays.
Concise data from
t h e ir unpublished re su lts and p riv a te communications are presented along.
with relevan t information from standard tre a tis e s on Pharmacology,
I I 32 "
'
fo r coherence.
Human experiments were e v id en tly ru le d out a t these stages of in ­
v e s tig a tio n .
Some reasonable biolo gical c r it e r i a had, perfo rce, to be
used, with te s ts c a rrie d out on dumb anim als.
Hence the primary in te re s t
of the pharmacologists was to measure the r e la tiv e a b il it ie s o f various
m a te ria ls , isolated by us from the mushroom, to produce e ffe c ts in animals
s im ila r to those observed by the adm inistration of LSD and muscarine re ­
s p e c tiv e ly .
LSD (Lysergic acid diethyl amide) is used in p s y c h ia tric therapy
and research because o f it s unique central a c tio n s .
32
In extreme Iy
minute doses (less than Gh5 microgram per Kg. o r a lly ) i t is reported to
produce highly s p e c ific psychic a lte r a tio n s .
Most impressive among these
- 1I 2—
are disturbances o f visual perception.
Visual h a llu c in a tio n s are v iv id ly
coloured and ra p id ly change.
Pelican e t al have termed the drug syndrome c h a ra c te ris tic of LSD
in animal species, lik e ra b b its , as "SympathotomI a ".
This means product­
ion o f hyperpyrexia ( fever) and mydriasis (p u p illa ry d ila t a t io n ) .
Con-
t r a r i l y , "Parasympathontoic" e ffe c ts , lik e miosis (p u p illa ry c o n s tric tio n ),
excessive s a liv a tio n , excessive Iaerim ation are associated with muscarinelik e products.
The term "muscarinic e ffe c ts " is widely used in physiology and '
:
pharmacology while describing the ro le o f endogenous acetyl choline in
n e u ro -effecto r transmission o f parasympathetic impulses.
Muscarine was
once thought to be the substance which transm itted parasympathetic im­
pulses.
"While the sympathetic system is known to be a c tiv e in a ll
parts of the body during conditions o f stress, fig h t and
fe a r, the parasympathetic system is a c tiv e in a restin g
person, e . g. asleep a f t e r a meal . Impulses passing down
the vagus nerve ( I ) slow the pulse ( 2) quicken the movements
o f the in te s tin e s (3 ) increase the secretions in to the a l i ­
mentary t r a c t and (4 ) c o n s tric t the bronchioles so th a t
breathing may be noisy. The e ffe c ts of. acetyl chol ine are
s im ila r to those ju s t described, but i t has other e ffe c ts
as w e ll. D ale*6""T8 showed th a t i t acted in p a rt lik e mus­
carine and in p a rt lik e n ic o tin e . The muscarinic actions
are the same as the parasympathetic a c tio n s . The muscarinic
actions o f acetyl choline are on the smooth muscle while the
n ic o tin ic action is on the s tria te d muscle.
P ro p e rties o f acetyI choline
I.
(a ) A smalI dose causes a fa I I in blood pressure due to
vasodiIa t a t ion.
(b ) A larger dose causes v a so d ilata tio n and slows the
heart in a d d itio n .
/.
- I IJ (c ) I f atropine is given, both these e ffe c ts are abolished.
(d ) | f now a large dose o f acetyl choline is given, i t w ill
cause a ris e o f blood pressure due to the release of
adrenaline from the-suprarenal gland and to stim ulatio n
o f the c e lls o f the postgag I io nic fib re s of the sympa­
th e tic gang lia. The c e lls send impulses to the blood
vessels and c o n s tric t them. This is the n ico tin e actio n ,
fo r nico tine has the same e f f e c t .
2.
I t increases In te s tin a l movements and s e cretio n s.
3.
I t c o n s tric ts the pupil and the bronchioles. ■
4.
I t causes contraction of the skeletal muscle e a s ily
o b ta inabIe in the fro g .
5.
I t stim ulates the receptors in c a ro tid body, and the
impulses set up cause increased r e s p ir a tio n ." !I
D e ta ils o f biological te s ts
Al I te s ts were performed on male albino, rabbits weighing about 2 Kg.
The ambient temperatures were'between 75° and SG0F .
Light' in te n s itie s
varies between 5 1 and 20 foot candles in the experimental area.
Depending on the s o lu b ility c h a ra c te ris tic s of the m aterials
screened, they were prepared fo r Intravenous adm inistration e ith e r by
dissolving in s t e r i le , pyrogen-free 0.9# KIaGl solution or by suspension
in s t e r i le , pyrogen-free po lyvinyIp r r o l i done s o lu tio n .
Relevant data concerning the weight of dry solids in each sample
and the fresh mushroom weight corresponding to each fra c tio n and also
s o lu b ilitie s and other c h a ra c te ris tic s were supplied to the pharmacolo­
g is ts .
- I 14Wi +h the above data each m aterial was screened f i r s t .
observations were made.
Control
B iological assays were then c a rrie d o u t.
Three
to s ix male albino rabbits were studied a t each dose le v e l, with negative
and p o sitiv e c o n tro ls ,
A group of animals which received no treatment
formed the negative control and a group which received only the menstruum
in which the te s t m aterial was administered served as p o s itiv e c o n tro l.
At frequent in te rv a ls before and a f t e r adm inistration o f each
sample, pupil diameter was measured with a m illim e te r scale and rectal
temperature determined with a c lin ic a l thermometer.
made of the general behaviour of the anim als.
Observations were
Frequency o f occurence of
excessive s a liv a tio n , Iacrim atio n , the dose a t which death occured e tc .
were recorded.
These observations were continued for a t least four hours
a fte r dosing.
For each m a te ria l, for a ll dose levels and for a ll animals a t each
dose le v e l, the mean maximum increase or decrease in pupil diameter was
plotted against the logarithm of the corresponding dose.
For each mater­
i a l , the mean maximum change in body temperature for a ll animals a t a
given dose le v e l, less the mean temperature change in the p o s itiv e control
group was p lo tte d against the logarithm of the corresponding dose.
S tra ig h t lines best f i t t i n g the data were determined by the method of
least squares, or the method of averages.
From the computed (by In te r ­
p o la tio n ) d o se-effect-curves, e ffe c tiv e doses and units were defined ■ ,
thus:
Doses:
I ) The dose o f m aterial
in mg (o f dry s o lid ) per Kg. of body
weight required to increase the pupil diameter I mm (m ydriatic
e ffe c t) or decrease the pupil diameter I mm (m io tic e f f e c t ) . ■
2 ) The dose o f m aterial
in mg (o f dry s o lid ) per Kg. of body
weight required to increase the body temperature l.5 °G .
Units:
I ) U nit m ydriatic a c t iv it y was defined as the a c t iv it y o f a m a te ria l, I mg. o f which per Kg. of body weight caused p u p illa ry
- I
d i Ia ta tio n of I mm.
2) U n it m iotic a c t iv it y was possessed, by d e fin itio n , by a
m aterial which in dose o f I mg/Kg caused p u p illa ry c o n s tric tio n
of I mm.
3) U n it hyperpyrexia a c t iv it y was defined as the a c t iv it y .o f a
m aterial which in a dose of I mg/Kg caused Increase in rectal
temperature of I .5°G . ;
in a male albino ra b b it of about 2 Kg. under the conditions of te s ts . .
I
-1 1 6 -
Tab'l.e TF
FRACTIONS FROM PART T
R e f.
Dry
Mushroom
Weight
A.H. I
(T . I )
P t. I ; basic
f r n . (Pro I1Iiu s f lu id )
20g.
A.M.2
(1 .2 )
Charts 1,2;
basic frn.
A.M.3
(1 .3 )
Chart I ;
a c id ic f r n .
from Skel Iy
(SoxhIet)
A.M.4
(1 .4 )
Charts I ,2;
a c id ic f r n .
A.M.5
(1 .5 )
A.M.6
(1 -6 )
F rn .
.
Amt.
i soI ated
4 0 .8mg.
Amt.
for
B .T .*
9 mg
75g
35 mg
(appx)
25 g
32.3mg
32mg-
75g
5Omg
(appx)
3 8 .4mg
Chart 2;
,neutral fr n .
50g
707.4mg.
Chart 2;
Neutral frn .
insoluble in
acetone
50g
A.M.7
(1 .7 )
Chart I ;
Neutra I f r n .
from Soxhlet
e x tr a c ti ve
^g
836mg.
3 9 .9 1mg
A.M.8
(1 -8 )
Chart 2;
Acetone in soIubIe f r n .
from the
methanolic
e x tra c t!v e
o f defatted
mushroom
50g
95mg
46.40mg
in 3 ml
of water
33.3 mg
44.53mg
•V
Dose*
Object!ve
Symptoms
-
-
-
-
I 73mg/Kg
-
■5.2mg/Kg
none
-
none
I . 1813mg. 23.02mg
I
9 .2mg/Kg
3.5mg/Kg
none
At I Omg/
Kg le v e l,
hypoaetiv ity fIa c c id ity ,
s Iow-labored
breathing
-1 1 7 -
Frn
-
Dry
Mushroom
Weight
TSb1Le TT (continued)
Amf.
Amt.
i sofor
Iated
B .T .*
Ajei .9
(j_.9)
Chart 2;
Aeetone
Bbluble
fra c tio n
from the
methanol Ic
e x tra c t!v e
o f defatted
mushroom
50g
Ig (appx.)
A.M . IQ
( T . I0 )
Chart 2 pp.
Final water
e x tra c t o f
marco
50g
I 2mI
(cone.)
( 0 . 2g
to ta l
sol i ds)
52.58mg'
4 ml-
Dose *
O b je c tive
Symptoms
;■
I .53
mg/Kg
D iIated
pup!Is
sal I v a tI on,
rapid re sp ira tio n a t
higher dose
levels
0.019ml Hypoactiv ity ,
per Kg fI aceId ity
slow labour- ,
ed. b re a th -.
I ng) )>art j a I .
p a ra ly s is ,
miosis s i de
position &
convuIsions,
a t 0.1 mg/Kg
Death a fte r
3 .8 hrs.
*B . T . indicates B iological Tests
*Dose stands fo r amount required to ra is e body temperature to IwS0G
Fractions A .M .I, A.M. 2, A.M. 4 and A.M- 6 were not te s te d .
;
For the dry mushroom i t s e i f a dose o f 20mg/Kg was reported to be
required to ra is e body temperature by l.5 ° C . S a liv a tio n and miosis
were then noticed.
\
Fractions A.M. 10, A.M. 9 and A.M. 8 , in o rder, were potent in
producing hyperpyrexia.
- I 18,2) C ry s ta llin e substance, A. M. 2 0 ,Xj 2o (Potassium s a lt from water
e x tra c t o f 'marc1 from '150 g. of dry mushroom):
Q uantity Iso lated :
606 mg.
Q uantity sent to D r. Pelican:
426 mg.
Symptoms
H yp e ra c tiv ity
ED-50
■ mg/Kg
. 22.4
Hyperpyrexia
2.9
Rapid Respiration
9 .6
Tremors
22.4
Ataxia o f G a it
22.4
Dose required to ra is e body temperature to I .5°C:
5 .2 mg/Kg
Dose required to ra is e body temperature, to 3 . 0°'C:
123 mg/Kg
(e x trap o lated )
The follow ing experiments were c a rrie d out a t the campus,
Bozeman, on male mice with the kind cooperation of. the S ta ff o f Veterln
ary Research In s t it u t e , on January 16, 1959, 3:30-4:00 P. M.
Mouse
Marked
(I)
( 2)
(3 )
(4 )
(5 )
( 6)
(7 )
Dose of A. M., 20 in
physioloqicaI s a lin e
O.8 lmg/0 .l ml, intravenous
Q.9I mg/0.1 ml, intravenous
0.41 mg/0.1 ml, intravenous
0.41 mg/0.1 ml, intravenous
0 .20 mg/0 . I ml, intravenous
0 .20 mg/0.1 ml, intravenous
2 . 1 mg/I m l, vIn trap er it o n ia I
Observations on
behaviour unti I
Jan. 22, 1959.
normal
normal
norma I
norma I
normal
norma I
normal
The mice were fed re g u la rly (w ith mice food borrowed from D r, B. L.
Johnson's Laboratory) and given water with the dropping b o ttle on the
/
cage p e rio d ic a lly .
No changes were observed u n til January 22, 1959
evening.
At 5 p.m ., on January 22, 1959, 10 mg. (approxim ately) o f the
aqueous mother liq u o rs .o f A. M. 20 were dissolved in the water b o ttle . .
The mice were normal u n til 24th evening.
At 11:30, on the night of the
24th, mouse (5 ) was found sluggish, but showed re flu x a c tio n . When poked
/
.
■. ' .
with a sp I !in te r, the muscles twiitched. I t died about 12:15, noon of 25th.
About 1:30, mice ( 6 ) and (7 ) showed signs of approaching death.
By 3:15
In the afternoon, excepting mice (2 ) and (3 0 , a I I the other fiv e were
dead.
'The survivors were observed re g u la rly .
/
They, both, were active
:
and moved about normally in the cage, u n til 11:00 a.m. o f 26th.
I
noon, about 12:15, they died, a f t e r sh iverin g .
By
D r. Wi I Iiam Hi. S ip p e l, Physician, Bozeman, administered me 3 .2 mg.
o f zA; vM. 20 in 2 ml. of normal physiological sa lin e intram uscularly a t
4:52 p.m ., Jan. 17 th , 1959.
D r. Sippel took careful notes, fo r nearly
three hours, o f my body temperature and pupil s iz e .
No appreciable
changes were detected.
At about 10:30 p.m. in the n ig h t, I f e l t fe v e ris h .
/
tu re appeared to ra is e slowly fo r about 2 hours.
to normal by e a rly dawn of 18th.
no h a llu c in a tio n s .
The tempera-
I t gradual Iy receded
My sleep was disturbed.
I experienced
My only feeling s were uneasiness and a n x ie ty .
On
-r l 20-
+he 18th t was tre a te d with p e n le iI l i n as a prophylactic a t the Student
Health C enter.
The above experiments, on looking back, appear to be crude.
They
r e fle c t my impetuousness owing to the small quantity of dry mushroom,
255g., a v a ila b le for in vestig atio n s fo r nearly nine months (June 1958 March 1959) and the delay in obtaining pharmacological re su lts from Dr.
P e lica n .
3.
Fractions from P art TT (Chart 3) is o la te d from fresh mushroom, 6.5 Kg,
corresponding to 488 g (rounded to 506 g.) on dry weight basis:
IU , H
Tables
&I.
The weights recorded are approximate as the mushrooms examined were
dropped by the c o lle c to rs in to methanol immediately a fte r p icking.
Accur­
ate weight of fresh and dry mushrooms, with standard d e v ia tio n s , w ill have
to be ascertained in future work.
However, other in vestig ato rs have re ­
corded th a t 100 g. o f fresh mushroom on drying yielded 5 to 6 g. of the
dried materi a I .
- !2 1 -
Table I 11
Amt.
i soIated
Fm .
a .m :
II
■34 g_
(H .D
A. M. 12
( H - 2)
■20.412 g
A. M. 13
(H -3 )
11.03 g
A . M . 14
C H -4 )
A.. M. 15
C H -5)
Fracti on
A. M.
II
Fractions from P art ■
- H
Sample
Total
'Wt. of Fresh
fo r
:Solids in
Mushroom
B .T .
Equi valent
Samp Ie
to Sample
2 .4 g I n
IOmI
water
4.555 g in
t'h 20 ml
water
2 .4 g
460 g
Wt. of Dry
Mushroom
Equi valent
to Sample
35 g
4.555 g
I I 2 g ■'
I .45 Kg
6.9675 g
in 25 ml
water
6.9675 g
318 g
4.14 Kg
150 g
37.0795 g
37.0795 g
123 g
I .6 Kg
18.95 g
6.9948 g
6.9948 g'
184.g ‘
2 .4 Kg
Dose required to ra is e body temperature to ,!.S0O
0.162 mg/Kg
A. M. 12
18.2
mg/Kg
A. M. 13
4 ,2 3
mg/Kg
A. M. 14
21 .4
mg/Kg
A. M. 15
6.6
mg/Kg
- I 22Tab I e l V
Fractions from P art TT;
Symptoms
Hypoactiv ity .
A.M. I I
(IE * D
. 6 .3
biological data
ED-50 mg/Kq
A.M. 12 A.M, 13 A.M: 14
C II.2 )
(H *3 )
< E *4 ),
A.M. 15
( I I . 5)
-
32.4
59,6
16.2
Hyperpyrexia
0.05
10.5
3 .6
16.6
4.1
Rapid Respiration
5 .6
45.7
15.8
144.5
12.0
141 .3
117.5
’ 43.6
Convulsions
120.2
Death (24 h r s ,)
31 .6
Death ( 4 h r s ))
89.1
-
4 6,8
-
■
-
43.6
128.8
I 17.5
I I .2
44.7
I I .2
-
-
-
x
—
Sal I v a tI on
5 .2
45.7
Lacrimation
17.8
-
Slowing of Respir­
ation
17.0
-
Prone Position
89.1
83.1
4 4 .8
-
16.2
Loss of R ig h ting
Reflex
38,0
1T
104.7
117.5
-
Miosis
16.6
57.5
66.1
87.1
32.4
Vasodi I afatbf.on
16.6
—
-
-
Pupi I I ary
Di I a t a t i on
' Ataxia of g a it
-
37.2
53.7
-1 2 3 The Comparative potencies of the fiv e fractions in producing hyper­
pyrexia, mydriasis and miosis are given in Table Y .
Table V
Comparative potencies o f fractio n s from P art
Hyperpyrexic
potency
(u n its /q )
Fraction
TT
Mydri ati'c
potency
(u n its /q )
M io tic
potency
(u n its /q )
A. M. 11
20820
33330
833
A. M. 12
58
95
... 17
A. M. 13
301
2105
16
A. M. 14
47
O
A. M. 15
152
83
" 16
f
36
Table Vl
F ra c ti on
Amt.
i soI ated
F r a c tions from P art 111
Total
Sample .
for
Solids
In
B.T.
Sample
Wt. o f Fresh Mush­
room Equivalent to
Samp Ie
6 .2 lbs.
A.M. 19/20 'A'
CTTlM 21)
260 g
8.5. ig
8 . Sg Coi I )
A.M. 19/20 A/I
C T T T .I2 II)
58 g
10.1 g
IO J g ( I Ight
ye I I ow oi I )
3 3 .I lbs.
A.M. 19/20 A/2
(T T T .I2 I2 )
124 g
13.1 g
13.1 g (brown
oi I )
20
A.M* 19/20 'G'
(T T T M H )
I 60 .5 9 22.3 g
6.516 g
A.M. 19/20 ’ 0 '
.CTTT- 1:i 27)
250 g
2 3 .D g
4,267 g ■
A.M. 19/20 'F '
CTTT. 1222)
155 g
23.5 g
6,017 g
Ib s ,
26.4 lbs.
88 lbs.
28.8 lbs.
T a b le Vl I
Fractions from P art I I I ;
Symptoms ■
•‘
' ' e"
HypoaGtivity
(IL L . I 21)
2.09
(IL L . 1211)
biological data
—
----•- ~
ED-50 ma/Kd
•
( I n Vl 21 2).
.(Tl I . 111) T m t t t i 2) ~ ( I I I . 1222) .
200
I .84
1700
50
^ 3-4 0 0 0
—
-
—
0.24
Gonvu Is io n s .
>400
-
-
0.95
Death
> 400
275
316
. I .54
2.17
I .28
Hyperpyrexia
Rapid Respiration
i IOO
I .28
0 .4
200
250
I .28
I .2
-
I .23
I..29
Slowing of Respira tio n
-
-
-
0.24
Prone position
-
-
-
I .15
275
316
0.97
I .27
I .23
Loss o f Righting R eflex
>400
. 1.2
-
Miosis
-
-
w
0.08
I .28
0.52
Vasodi Iavic:.
-
-
-
2 .8
16.98
2.87
316
200
0 .9
I .64
I .20
Ataxia o f g a it
Mydriasis
>400
.3430-
32 to 100
76
'
- I 24-
224
- I 25—
The c r y s ta llin e substance,
TTT.Xj, m.p. I96-97°G . (dec) (ftimanlc)
acid?) was reported to produce only hyperpyrexia and not mydriasis or
m iosis.
The comparative potencies o f the various fra c tio n s , in units per
gram o f dry s o lid are shown in Table V I11.
Table V ll l
Gomparative potencies of frac tio n s from P art j'lT
Hyperpyrexic
potency
(u n its /g )
M aterial
A.M. 19/20 'A'
(IL L . I 21)
A.M. 19/20 A /I
TTTT. 1211)
'
A.M. 19/20 A/2
C Q I - I 212)
A.M. 19/20 1G1
(T rr-IH )
Mydria tic
potency
(u n lts /g )
M io tic
potency
(u n lts /g )
8 .3
54
0
0.6
59
0
18
^ 17
0
0*
0
3510
■v1
.
A.M. 19/20 1D1
(3 1 1 . 112 )
A.M. 19/20 1F 1
( I I I . i 222 )
I I l.X i (fum arie acid?)
_ 378
14
0*
103
0
■ 2445
0
^Fractions 111,111 and jJJ_.l222 exhibited only hypothermia
.0
-
Table I Y
L
——
Fractions from P art I V
F racti on
Reference
Amount
i so lated
Samp Ie
fo r B .T.
Total
Wt. o f Fresh
Solids in
Mushroom
Sample
Equivalent to Sample
Chart IS, Aq.
Methanol Concentrate
3628 g
69.59 g
47.73 g
777:g
Ab1 1B-R1
11.211
Chart 18
Ether insoluble
from absolute
methanol concentrate
252,3 g
71.18 g
36.64 g
I I .42 Kg
AL 1G1
U .3
Charts 16, 20
Water concentrate
o f the *marc1
3288 g
59.84 g
0.8486 g
Ab..'A1 H I
T V .2 I2 I
Chart 18
yellow o il
on chromatography
25,98 g
2.92 g
- -1 ;
Ab.A T?
12*2122
Chart 18
reddish brown o iI
on chromatography
54.62 g
3.272 g
AL A T
H .2 I2 3
Chart 18
brown o iI on
chromatography .
12,67 g
2.048 g
.
7a>\ Kg
2.92 g
4 .5 5 .Kg
I
3.272 g
2.43 Kg
2,048 g
6.55 Kg
126-
A bJA1
iv . I
-!■27Tab le X
B iological a c t iv it ie s o f fra c tio n s ,
Ab. A C H - 1> > Ab. B-R (TY.2I I ) and Ab . 0 ( W * 3 )
Symptoms
Ab. A.
IV . I
ED-50 ma/Kq
Ab. B-R
J V .2 II
Ab/G
1 1 .3
Hypomoti I It y
I .48
I .50.
0.39.
A taxia of G a it
2.15
I ,65
-
Loss o f R ighting Reflex
2.32
2.19
-
Prone Position
2.15
*•*
Miosis
I .03
I .38
S a liv a tio n
I .04
I .02
LacrImation
I .04
^ 2.40
Hyperpyrexia
I .01
2.40
Slowing o f Respiration
I .01
I .63
Convulsions
2 .32
2.40
Death
2 .32
2.25
Op Isthotonos
'2.32
2,37
Proptosis
2.15
-
Hypotonia
""
W
I .53
!
I .71
Mydriasis_
0.40
more than
0.65 less
than 0,90
—
I .5
Opisthotonos: Tetan ic spasm in which the body or trunk is arched, dorsa l l y concave, with the head and po sterior bent both toward one another.
Proptosis:
Protrusion or prolapse e s p e c ia lly of the ey eb all*
- I 28—
Table 2<L
B iological data on o il
Symptoms
frac tio n s from P art JJf
Correspondi hq doses .In mg/Kg
Ab. A. 111
Ab. A. V
Ab* A. TT
T T .2 I 22
T ? . 21 23~
I r . 2i 2i
Hypomoti I It y
10, 100, 316
562
5 6 .2 , 100,
316, 562
Mydriasis
(2 m.m. max.)
10, 100, 316
(2 i T i . m . max.)
5 6 .2 , 100,
316, 562
-
Ataxia o f Gsait
100,316,562
Prone Position
316, '562
Laboured R espiration
.31562
316,. 562
316, 562
316; 562
316, 562
Death
316, 562
562
Hypothermia
( I .3 *0 )
a t 562
—
(1 .5 mm. m in .T
a t 3 .1 6 , I0.Q>
100. 00,
. 316, 562» '
;
562
' -
316, 562
Loss of Righting Reflex
Hyperpyrexia
562
316, 562
none
( I . 6°G )at
316 (0.4°G )
a t 5 6.2 CO;9°C)
’
a t 562
( 0 .5 - 0 .8°G)
a t 10 & 100
( 0 . 2oG) a t
' 3/16 & 100
;•
^Mydriasis Increased with dose In the case o f o i l , T v .2123
Maximum d ila t io n , I .0 mm a t I ,0 mg/Kg
Maximum d ila t io n , 3 .0 mm a t 3.16 mg/Kg
Maximum d ila t io n , 4 ,0 mm a t 10,. 100 and 316 mg/Kg
( O J 6G) a t
10 (0.5°G9
a t 316
C©„85°G)
a t 562
-1 2 9 © ry s ta ll I ne product, lZ t , m. p. I35-38°G (d e c ), CCj
5K^,
p ro v is io n a l), isolated from the water concentrate, Ab. ' 0 ' . , TV .3 ; Total qu an tity Isolated :
307.4 mg.
Samples sent fo r biolo gical te s ts :
a) 34.7 mg were lo s t,
b) 12 mg I n l m l, o f d is t iI Ied water.
Reports o f te s t a t two dose I eveIs
Low level dose;
1,0 mg/Kg.
a) M ydriasis resulted 30 minutes a f t e r adm inistration and p e rs is t­
ed thence fo r 30 minutes.
Pupil d ila tio n was,-2 mm above normal.
b) Three hours a f t e r a d m in istratio n , there was a temperature raise
o f 0.9°G .
Hyperpyrexia lasted less than an hour.
High level dose:
6.25 mg/Kg
a) M ydriasis was Immediate.
I t persisted for 30 minutes.
Pupil d ila tio n was i n i t i a l l y 1.5 mm above normal, increasing to 2 mm.
Ia t e r .
b) T h ir ty minutes a f t e r a d m in istratio n , temperature rose to
0 .7 * 0 .
Hyperpyrexia persisted for 30 minutes.
c) F ifte e n minutes a f t e r a d m in istratio n , there was rapid resp ira ­
tio n p e rs is tin g for 2 hours.
d) There were no other symptoms.
-1 30-
Qther samp Ies on which Pharmacoloqioal -reports are expected soon:
1)
Potassium gluconate; m. p. 186*0 (dec) (P a rt T T , Chart 17)
Q uantity isolated :
606.6 mg.
Sample sent fo r b iolo gical te s ts :
IN
mg.
2) B -g l ucosI de, m. p. 106*0 (dec) (P a rt TFT. Chart 13)
Q uantity isolated :
292 mg
Sample sent fo r biolo gical te s ts :
3 0 .7 mg
B r. Pel lean's Comments
Hyperpyrexia, per Se, is not necessarily accompanied by or necessar­
i l y produces mydriasis or m iosis.
For example, i l l .Xj (fum arlc acid?)
produced hyperpyrexia but neith er mydriasis nor m iosis.
I f , then, a
m aterial produces mydriasis accompanied by hyperpyrexia i t is suggestive .
of the m aterials containing a sing le substance capable o f producing a
unique drug syndrome ( o f . nSympathotonia" o f LSD).
From bid-assays, the most e f f ic ie n t e x tra c tio n o f the material
with LSD-Mke e ffe c t appears to have been made by the method which
yielded fra c tio n A. M. I I . , ( I T <■I ) .
The most e f f ic ie n t separation of
the muscarin e - lIke m aterial from the m aterial with LS D -like a c t iv it y was
made by the procedures which yielded fra c tio n s , A. M. 19/20 1G1, IT i'. I I I
and A. NI* 19 /20 1F 1, 111 .1222.
These procedures also gave the highest
y ie ld s o f m uscarine-lIke m a te r ia l.
The lethal dose o f Schmiedebergt S muscarine, a f t e r intravenous
adm inistration to rabbits was apparently of the order o f 6-10 mg/Kg.
The median lethal dose o f A. M. 19/20 1G1, TFT. I l l ,
the most potent
-1 3 1 muscarin e - like m aterial
Fraser
in th is series was 5 .9 mg/Kg o f dry s o lid .
found th a t pure muscarine produced tra n s ie n t e ffe c ts on the
blood pressure and the uterus o f anesthetized ra b b it a f t e r intravenous
adm inistration of doses ranging from 5-10 micrograms/Kg.
19/20 1Q1, I I I . I l l
M aterial A. M.
produced prolonged e ffe c ts on the pupil a fte r in tr a ­
venous adm instration o f doses o f 285 micrograms/Kg of dry s o lid .
I t is
obvious th a t *G ', 111 . 1I I compares favourably with samples o f muscarine
isolated from natural sources.
That a sin g le m aterial exists In the fra c tio n s , with musearineIik e action is suggested by the fa c t th a t a ll of the m aterials tested
which produced miosis also produced excessive s a liv a tio n .
In co ntrast,
i t can be said th a t n e ith er hyperpyrexia nor the pro stratio n which may
accompany i t w ill produce s a liv a tio n .
Excessive s a liv a tio n was not,
fo r instance, observed a f t e r the adm inistration o f I I I .Xj Cfumaric acid)
in doses s u ffic ie n t to produce fe ver.
S p e c ific pharmacodynamic studies were not performed to id e n tify
the mechanism o f action of the m aterial
in the mushroom responsible for
the production o f hyperpyrexia-m ydriasis drug syndrome.
Data obtained -
during the course o f the bio-assays suggest th a t the m aterial is LS D -like,
ra th e r than a tro p in e -1ik e .
Fever produced by LSD is produced a t doses
fa r lower than the lethal dose; fever produced by a tro p in e -1Ike agents
occurs a ft e r n e a r-le th a l doses.
The dose of A. M. 11, T T .I required to
e levate body temperature I .S0C was 0.16 mg/Kg.
o f A. M. I I was 26.7 mg/Kg.
The median lethal dose
R atio o f the two.doses is o f the order to
-13 2be expected o f LSD-IIke m aterials rath er than a tro p in e -lik e m a te ria ls .
Although precise data are not a v a ila b le , review of the lite r a tu r e
shows th a t, under the conditions o f our experiments, LSD i t s e l f might be
expected to produce hyperthermia a f t e r doses of the order o f 25-50 m icrograms/Kg.
According-to our d e fin itio n , pure LSD has a potency of the
order o f 20,000-40,000 u n its /g of dry m a te ria l.
The LSD -like material
present in the p lan t e x tracts studied is of the same order o f potency as '
LSD i t s e l f .
Computation o f the r a tio :
M y d ria tic potency/mio tic potency, from
biological data yie ld s a value which measures the degree to which LSDlik e m aterial was separated from the m uscarine-like m aterial in each
samp I e .
In regard to the o il
fra c tio n s , the most e f f ic ie n t separation of
LSD -like m aterial from m uscarine-like m aterial was made In fra c tio n ,
A. M. 19/20 A /2, 111. 1212, which unfortunately gave a low y ie ld .
though n e ith er A. M. 19/20 A, TjT. I 21 and A. M . 19/20 A /1 ,
A l­
TTT. 121 I
produced m iosis, TTT.121 produced hypothermia a t the highest dose level
studied, 400 mg/Kg. 111. 121 I a t the highest dose level studied, 300 hng/Kg.
produced an i n i t i a l hypothermia followed by hyperthermia.
Hence, we
believe these two o i ls , I I l .121 and M l .1211 to be less well separated
from m uscarine-like substance than IlT . 1212.
Our conclusions are based on such assumptions th a t the separation
procedures did not produce chemical a lte r a tio n of m aterials present in
the fresh p lan t and th a t the te s t m aterials coiitaiirte'd hotb a c te rfalinpyrow,
gens.
The "Cleanness" o f the .results obtained in d ic a te s /th a t fgw,
-1 3 3 I f any, b io lo g ic a lly a c tiv e contaminants were present in the samples.
SUMMARY:
Bio-assays in d icate th a t the mushroom probably contained two,
separable, b io lo g ic a lly a c tiv e substances.
e ffe c ts in ra b b its :
One substance had LSD-IIke
the other had muscarime-1iRe e f f e c t s .
In the mater
i a Is in which they were present in the highest concentrations, the sub­
stances had potencies comparable to those o f LSD and muscarine, respect­
iv e ly .
- I 34—
SUMMARY
1.
The mushroom. Amanita muscarla. both fresh and dry, has been examined
chem ically and p h y s io lo g ic a lly .
2.
The u ltim ate water-concentrate o f the ’ marc' has been . found on re­
peated experim entation, to have LSD -like p ro p ertie s , d ila tin g the pupil
and producing hyperpyrexia.
3.
From the water concentrate, a potent c r y s ta llin e m aterial with LSD-
lik e a c t iv it y has. been iso lated in small q u an tity and designated 1Z 1,
Z, m.p. I35-380(S> (d e c ), C| ^H^qOj^
-
is being studied with the help o f NMR
spectroscopy.
4 ,. A fixed o il has been isolated and fra c tio n a te d .
o f esters o f o le ic a c id .
I t consists mainly .
The o ils are being screened fo r insect repel­
len t and in s e c tic id a l p ro p ertie s .
5.
M annitol, fumaric a c id , and some c r y s ta llin e substances, m, p . 172-
74 *6 , m.p. 160-65*0, and a p-glyeo side, m.p. 106*0 (dec) have been found
in the mushroom.
Potassium gluconate has been isolated fo r the f i r s t
tim e from the mushroom among the minor constitu en ts.
6.
Is o la tio n and fra c tio n a tio n procedures have been worked out in d e t a il,
p erfectin g them for easy re p e titio n in fu tu re work.
Jndolic components have been located in the water concentrate and
are being subjected, to th in sheet chromatographic separation.
7.
The experimental has been presented under four parts with il lu s t r a ­
tio n s .
The subject o f 'pi Iz a tro p In e ', the elusive hallucinogen-, has been
discussed and fo r future research some valuable suggestions have been
made.
-1 3 5 -
'
8.
Pharmacological data has been included.
9.
A prelim inary communication on the above work was presented a t the
I . U. P. A. C. Symposium on the chemistry of Natural Products, Austral ia ,
• I960.
—136APPEND I X
I
L i s t o f P u b li c a tio n s o f A. V. Subbaratnam
1.
Oarotehoid pigments o f pyrethrum flow ers.
Subbaratnam and Pi I la y , J. S c i. Ind.: Res. 6B (1 9 4 7 ), 100.
2.
A IkaIdI dal constituents of GI o r ibsa sugerba Lin n ,
Subbaratnam, J. ,Sei . In dustrie R es., I IB (1952), 446.
3.
Dntersuehungen uber die In h a lts s to ffe der Q loriosa superba Linn.
Subbaratnam/.LilePharmazle , 8 (1 9 5 3 ), 1041
4.
Studies in the a Ik a lo idal constituents o f G loriosa supbera Linn.
, Subbaratnam, J . S e i. in d u s tr. R es., 13B' ( 19 5 4 ), 670.
5.
Ghemical Examination o f the Bark o f Tabernaemontana fllchotoma, Roxb.
Subbaratnam, Current Science,, In d ia , 25, 66 ( 1954)
6.
Chemical Examination of Gloriosa superba Linn.
Subbaratnam, B u ll, of the N at. In s t. S c i. (In d ia ) Nd. 4 (1 9 55 ), 27.
7.
Chemical Examination o f the Bark of Tabernaemontana heyneana Wal I .
Subbaratnam, J . S c i. In d . Res. I4B (1 9 55 ), 424.
8.
Studies on the I r r i t a n t Factor o f Pyrethrum Flowers.
Subbaratnam and S id d iq u I, J. Sci » In d . Res. I SB ( 19 5 6 ), 243.
9.
Chemical Examination o f the Seeds o f Alanqium Iamarcki?., Thwaites:
P a rt I - A lkaloidal Constituents o f the Seed Kernels.
Subbaratnam and S id d lq u i, J . S c i. Ind, Res. ,1SB (1 9 5 6 ), 432.
- rr.
10.
-
'
Chemical Examination of the Seeds of E ryth rin a lndica Lam.
Subbaratnam, J . S c i. In d . Res, I SB (1 9 56 ), 210.
II.
Studies In the EflalIucinogenie P rin c ip le s o f Amanita muscarja.
Cook and Subbaratnam, Abstract 50 o f papers presented a t
• .
1, 0 .P .A .0 . Symposium on the Chemistry of Natural Products, I960,
A u s tra lia .
12.
Chemical Examination o f the Roots o f Coceu I us Ieaeba D. G.
Cook and Subbaratnam, Abstract 136, I .U »P.AeGe Symposium, I960,
A u s tra li a .
13.
S tructure o f G lo rio s ine (to be published).
-1 3 7 Patemts and Mon-teehnlcal Notes
1.
Manufacture o f m osquito-repelIa n t compositions.
M itr a ,. Subbaratnam and S id d iq u i, Indian Patent No. 34410 (1946).
Non-teehnical note, J . S c i. ] nd ustr. Res. _5 (1 9 4 6 ), 28,
2.
Improvements in or re la tin g to manufacture of concentrated extracts
o f pyrethrum.
,
S id d iq u i, Subbaratnam and BagchI, Indian P a t. No. 35338 (1946).
Non-technical note, J . S e t. I ndustr. R es., _6 (1 9 4 7 ), 284.
3.
Improvements in or r e la tin g to preventing the d e te rio ra tio n of
p y re th rins and preserving the b iolo gical s t a b ili t y o f pyrethrum
preparations.
S id d iq u i, Subbaratnam and Sharma, Indian p a t. No. 35567 (1946),
4.
Subbaratnam,.A. V. e t a l . , Proc. 34th Indian Science Congress
A bstracts, Sec. 4 , 1947, 22.
-1 5 8 APPENDIX 2
74
From nDrugs and th e M ind" by R obert S . de Ropp, 7 > pp, 278-80,
S t. M a rtin 's Press, New York, 1957.
"Moving now to a very d iffe r e n t p a rt o f the world, the ioy
■ plains bounded on the north by the Siberian Sea and on the
east by the Bering Sea, we fin d a curious "passport to happi­
ness" in use among the tr ib e s who In h ab it these Inclement re ­
gions. The crimson f ly a g a ric . Amanita muscaria, is known
to most people as a decorative fungus which figures in count­
less illu s tr a tio n s o f f a ir y s to rie s along with dwarfs In red
caps, mottled fawns, elv e s , and other assorted flo r a and
fauna. The p rim itiv e peoples o f northeastern A sia, the
Tungus, Yakuts, Ghukches, Koryaks, and Kamaehadales, put
th is fungus to a very d iffe r e n t use. I t is c a lle d muchamor
and is eaten as an in to x ic a n t during the long w inters when the
sun scarcely ris e s above the horizon and to ta l boredom is an
ever present th r e a t, tinder these circumstances any form of
in e b ria tio n may be considered b e tte r than none and the Koryak,
whose tastes are not re fin e d , prefers the f ly agaric to any
other agent and w ill even give a whole reindeer fo r a single
dried mushroom, as the fungus i t s e l f does not grow so fa r
n o rth . The f l y agaric presents.other advantages which appeal
to the t h r i f t y , fo r the a c tiv e p rin c ip le is excreted unchanged
in the urine and can thus be-used again. Thus an agaric orgy
among the Koryaks is s ta rte d by the women, who chew the dried
fungus and r o ll the chewed substance in to sausages which are
then swallowed by the men. As.the party warms up the p a r t i­
cipants grow liv e ly . Some shout and sing, some hold conversa­
tio ns with imaginary beings, some r e la te with d e lig h t th a t
they have made vast fortunes, some leap to and fro across the
room, imagining every tin y obstacle in t h e ir path to be so
high th a t they must jump to get over i t . Then, when the
i n i t i a l j o l l i t y has somewhat worn o f f , there are shouts of
"pass the po t", and one o f the women, enters with a t i n can
into which a ll present u rin ate with enthusiasm. The can is
then passed around and each partakes o f the s t i l l warm urin e,
gaiety is restored to the p a rty and the leaping and singing
recommence,
This is a form o f social gathering which might not appeal to
an asethete but., as was mentioned above, the Koryak Is not
re fin e d . Besides, the precious agarics are very expensive
and the winters are long, so th e re is every inducement to
prolong the orgy. C e rta in ly as a euphoriant the f l y agaric
- 139“
leaves much to be desired and excessive indulgence in the drug
leads to an attack of raving madness. The h a llu c in a tio n s be­
come d estructive and dangerous, ending in acts of violence o r,
in some cases, s e lf-m u tila tio n . The a c tiv e p rin c ip le in f ly
agaric which produces these h a llu c in a tio n s appears not to have
been is o la te d . The c h ie f to x ic substance in th is fungus is
an a lk a lo id c a lle d muscarine which, in s u ffic ie n t doses, stops
the beating o f the h e a rt. But whatever the substance is th a t
affords such joy to Koryaks, Tungus, Ghekches, Yakuts, -et
c e te ra , i t c e rta in ly cannot be muscarine."
—| 40APPENDIX 3
From the a r t i c le "On Going Berserk:
a Neurochemical Inquiry" by
Howard D. FabIng^5 , The S c ie n tific Monthly (Am. Asscn. Adv. S c i . ) , 8 5 .
232-37 (1 9 5 6 ).
V
"Drew re p o rts , the p a tie n t, a middle-aged tavern keeper,
picked some w ild mushrooms and ate them a t 10:00 o ’ clock
one might In October 1955, These mushrooms were la te r
id e n tifie d by the botany department as Amanita muscaria.
Two hours a fte r ingestion the p a tie n t had an explosive onset
of d iarrh ea, profuse sweating, excessive s a liv a tio n and
v e rtig o . He f e ll asleep and wakened a t 2:00 A.M- completely
d iso rie n te d , ir r a tio n a l and v io le n t. When he was admitted
to U n iv e rsity H o s p ita l, Ann Arbor, M ic h ., his nail beds were
dusky and he appeared cyanotic; blood pressure was 150/90,
pulse 120, re s p ira tio n 24 per minute, and his temperature
9 8 *4 *. He did not react to deep pain s tim u la tio n , but re ­
sponded to p in p ric k , tie was disoriented In a ll three spheres.
Somnolence a lte rn a te d with periods o f excitem ent. He thought
th a t he was in hell and id e n tifie d the In tern e , nurses, and
attending physicians as C h ris t, Satan, God, or angels. Nur­
sing notes on admission in d icate th a t he was threshing about
, in bed, ta lk in g constantly and ir r a t io n a lly ,
As the day wore on, the content of his h a llu c in a to ry and
i I Iusional output remained almost e n tir e ly r e lig io u s , He
constantly m lsldem tified a t a l l resident physician as
C h ris t. He kept re fe rrin g to nurses and other attendants
as God or angels. He f e l t th a t he was in the Garden of
Eden, and then in h e ll . As evening came he cleared up
m entally, lo st his motor excitem ent, and f e l t relaxed.
Al I laboratory te s ts were w ithin normal lim its . He appear­
ed to be recovered on the follow ing morning and was d is - .
charged."
-1 4 1 LIST QF REFERENCES
• I.
A lt , L . L . , Anal . Chem.,, 27, 749-51 (1 9 5 5 ).
2.
Ambache, N ., and Less in , A. W*, J. Physiol . , 127 , 449 (1955);
Cf: C. A. 49, 9163a (1 9 5 5 ).
3.
Bal enovic," K.,, C erar, D ., Gaspert, B ., and Gal I Jan, T . , Arhiv
Za Kemiju,, 27, 107-16 (1 9 55 ).
4.
Balenovic, K ., and Stefanac, Z . , Chem. and In d ., 23 (1 9 5 6 ).
5.
Bard, L . , and Z e lln e r , J ., Monatsh., 44, 9-17 ( 1923); through
0 . A. 1 7 , 290S9 (1 9 2 3 ).
6.
Bi n et, L *, Marek, J . , and B u rste in, M. , Compt. rend. soe. b io l. , I 29.
.272-73 (1 9 38 ).
7.
Bi n e t, L . , Marek, J . , arid B u rste in, M ., Ann. p h ysio l, physioco ohlm.
b i o l . , 1 4 , 645 (1938); through 0 . A. 33, 7365 (1 9 3 9 ).
8.
Bowden, K ., and Mogey, G. A ., J. Pharm, Pharmacol. , J_0, 145-56 (1 9 58 ).
9.
Brady, L* R ., and T y le r , V. E . , J r . , J. Am. Pharm. Assn., (S c !. Ed.)
XLVIII (No. 7 ) . 417-19 (1 9 5 9 ).
- ‘ ■
i
:
Buch, M. L
Montgomery, R ., and P o rte r, W. L . , A nali Chem., 24,
489-91 (1952)
,
10.
11.
12.
Burn, J. M ., "Lecture Notes on Pharmacology", B lackw ell, Oxford (1 9 5 3 ).
(/
BuschmanriflE ., Pharm. P o s t., 45, S53-4; through C .A ., 6_, 24852 (1 9 12 ).
13.
Chretien and L e b lo is, Rev. path©I . comp. hyg. gen., 28, 131 (1928);
B u ll, soc. hyg. a lim e n t., Jj5, 253-54 (1928); through G .A ., 22,
46692 (1 9 2 8 ).
14*
C la rk, E . O ., and Kantor, J. L . , Mycologia , 3 , 175-88; through
G.A ., 6 , I ISO2 (1 9 1 2 ).
15 .
-----
Cook, W. B i , and Subb'aratnam, A. V ., Abstract 50, |J,.I3.P.A.C.
Symposium on the Chemistry o f Natural Products, A u s tra lia , I960.
16.
D ale, H. E ., J . Pharmacol, and Exper. Therap. , 6_, 147-90 (1914)
17.
Dale, H. H ., Feldberg, W., and Vogt, M ., J. P hysio l. , 8 6 , 353-80 (1936)
18.
D ale, H. H ., Science, 90, 393 (1 9 3 9 ).
-1 42,r
19.
Dixon, W. E ., Med. Mag., J_6 , 454-57 (1 9 0 7 ).
20.
Eugs+er, 0 . M ., and Waser, P. G ., Experien+ia, 10, 298 (1 9 5 4 ).
21.
Eugster, C -M * , MGlv . Ghim. A c ta ., 39, 1002-23 (1 9 5 6 ).
22.
Eugster, 0 . M*, and M u lle r, G ., Melv . Ghim. A c ta ., 42, 1189 (1 9 59 ).
23.
Eugster, C. M ., Revue de Mycologie, XXIV (No. 5 ) , 369-85 (1959).
24.
Fabing, E. B i, and Hawkins, J. R ., Science,123, 886 (1 9 5 6 ).
25.
Fading, H. D ,, S c i. Monthly (Am. Assn. Adv.
26.
Fabing, H. D ., Harper’ s Magazine, 214 (No. 1284), 50 (1 9 5 7 ),
27.
F ie s e r, L . F . , "Experiments in Organic Ghemis try " , T h ird , Revised
E d ., D. G. Heath, Boston, 1957.
28.
Fiske, J . G *, N. J . Agr. E xpt. S ta . B u ll ., 261, 3-30 (1932),.
2$,
Fernando Rosa-Mato, AnaJes assoc, quim. Farm. Uruguay, 44 TNo, 3 ) ,
9-15 (1941); through C, A. 36, 4542® (1 9 42 ).
30.
Ford, W* W., J. Pharmacol., J_, 275-87? through G. A. £ , 2314 (1 9 10 ).
31.
Gerber, G *, Gompt. rend. soc. b l o t ., 67, 612-4; through G. A», 4 ,
16285, (1 9 1 0 ).
32.
Goodman, L . S ., and Gilman, A ., "The Pharmacological Basis of
Therapeutics", Macml I Ian, New York, 1956.
33.
Gordon,
T . , Thornburg, W*, and Werum, L. N«, Anal 4 Ghem., 28,
849-55 (1 9 5 6 ).
34.
Handbook of Qhemistry and Physics, 39th E d ., Chemical Rubber, Ohio,
• (1 9 5 8 ).
35.
Harmsen, E», Arch, exp. P athol, Pharmakol. , Naunyn-Schmledeberg’ s ,
SO, 361 (1 9 0 3 ).
36.
H eilbron, I an, E d -ln -c h le f, "D ictionary of Organic Compounds",
Vol . TT., Oxford U n iv e rsity Press, New York, 1953,
37.
Heim, R ., "Les actions nerveuses provoquees par less champignons",
Science, e t Nature, 29, 1-8 (S e p t-O c t., 1958).3
*
8
38.
Henry, T . A ., "The P lan t A lkaloids” , IV E d ., pp. 659, B la k is to n ,
P h ila d e lp h ia , 1949.
S c i . ) , 83, 232-37
(1 9 56 ).
—143—
39'.
Henry, T . A.> in " A lle n 's Commercial Organic A nalysis", Vol . V I I ,
pp♦ 6, B Ia k is+on, P h ila d e lp h ia , 1929.
40*
Herman, E , , Pharm. Zentral hal I e, B6, 555-59 (1915); through G. A.
J 0 , I7677 .(19 1 6 ).
41.
Hofmann, A ., Frey, A ., O t t , H i , , P e t r i z l lk a , T h ., and T ro xIe r , F *,
E xperienti a , 14-,- 397-99 (1958) .
42.
Hofmann, A *, and T s c h e rter, H*,, E xperie n tia , _l_6, 414 (I9 6 0 ) .
43*
Honda, J * , Arch.* exp. P a th o l. Pharmakol * , Naunyn-Schml edeberg’ s , .
65, 454 (1 9 1 1 ),
44.
Hunt and Tavpau, B r i t . Med. J . , TT, 1788'(1906),.
45.
Inoko, Y * , M i t t , med, Fac. Q n iv ., Tokyo, J_, 313 (188799); through
Pharm. j . , 50, 1069 (1891)*
46.
Joehelsen, W., "R eligion and Myths o f the Koryak" In Jessup North
P a c ific Expedition V I, Am. Museum Natural H is to ry , V o l„ 10,
pp., I -382, New York, 1956.
47.
Keel and, W. E . , Ph. D. Thesis, Montana S tate C ollege, Bozeman,
Montana, August 1960. ■
48..
Keenan, G., L * , and Welsberg, M* S«, J. Phy. Chem., 23 , 791-94 (1 9 29 ),
49.
Kezel I , T . A ., and Dzhaparidze, L » I . , B u ll. Acad. S c i, Georgian
S .S .S .R .,
99.3-96 (1944); through G.A. £1. 3172 e (1 9 47 ).
50.
King, H ., J . Ghem. Soc., 121 . 1743-53
(1 9 2 2 ).
51.
Kogl, F . , Duisberg, H ., and Erxleben,
H ., Ann., 489,156-92
52.
Kogl, F ., and Erxleben, H ., Ann., 479. I I -26 (1 9 30 ).
53.
Kogl., F . , Salemink, 0 , A ., Schoutten, M ., and Jel I i nek,
T ra v . OhIm.., 76, 109-27 (1 9 57 ).
54.
Kogl, F . , Salemlnk, G, A ., and S c h u lle r, P. L . , Rec. T ra v „ Ghlm., 79,
278-81/ (I9 6 0 ) .
55.
Kobert, R ., S t. P etersb. med. Woehensehr., No. 51 and 52 (1891);
through Jahresber. Pharm., 27, 815 (1:892).5
*
6
56.
Kobert, R ., S ltz b e r , Dorpat* N a tu rf. V e rs ,, 9_, 535 < 1892);
through Ghem, Z e n tr ., £3 ( I I ) , 929 (1 8 9 2 ),”’
(1931).
F», Ree.
'
-1 4 4 57.
Kuehl, A. F*> J r . , L ebel, N ., and R ic h te r, J . W..# J . Am. Ghem. Soc.,
77, 6663-65, (1956?.
58.
Kung, A ., Z» P hysio l. Ghem., 9^, 241-50; through C> A* 8 , 35754 (1914)
59.
Kwasniiwski, V ,, Deut. Apoth-Ztg. V er. Suddent.
1177-9 (1 9 5 4 ).
60.
Laplcque, M i, and N a tta n -L a rrier,.'M»> Oompt. rend. sec. b i o l. , 96,
934-35 (1 9 2 7 ).
61.
Leach, M ., E d ., "Standard D ictio n ary o f F o lk lo re , Mythology and
Legend", pp* 26, Funk and Vsfagnal Is , New York, 1949.
'6 2 .
Apoth* Z t g * ,. 94,
Lewis, B-*, S'* A frican Med. J ., 29.» 262-3 (1 9 55 ).
63.
Linnaeus, 0.., Skansa Resa, pp* 430, Stockholm, 1751*
64.
Ludwig, Jahresber* Ghem,, 516 (1 8 6 2 ).
65.
Magnus, A ., "Be Vegetabi 11 bus"; through Ramsbottom, J . , "Mushrooms
and to a d sto o ls", Gol I in s , London, 1953*
66.
Merck Index, S ixth E d .» Merck & G o., Rahway, N. J ., 1955*
.
67.
N ie d e rl, J . B *, and N id e rl, V ., "Organic Q u a n tita tiv e MicroTAnsalysis",
Second E d ,, John W iley, New York, 1948.
68.
Nilakantum, S* V ., J . Scl , T e c h ., In d ia , 2 , 3 9 -5 1 (.1936).
-69.
P e t r i,
"B eitrage Zur Lehre Von den Hemmungsapparaten des Herzens",
Berne, 188©; through' .Sehmiedeberg, 0 .
70.
Phi s a lix , G ., and B ertrand, © ., Gompt. rend, sec. b i o l , , 54, 932
(1 9 0 2 ),
71.
Pigman, W .,E d ,, "The Garbohydrates", Academic Press, New York, 1957.
72.
Raab, H. A ., and Renz, J . , Z* P h y s io l. Ghem,, 216, 224-28 (1933);
through B. A ., 27, 3236? ( ! 9 3 3 ) .
73.
Radais and S a rto ry , Gompt. re n d ., 151. 156-8; through G. A. 4 , 3255
(1 9 1 0 ).
74.
de Rppp,,R. S ., Drugs and the Mind, pp 278-80, S t. M a rtin ’ s Press,
New York, 1957.7
5
75.
Schmiedeberg, 0 . , and Kdppe, R ., "Das Muscarin", Vogel, L e ip zig , 1869.
/
g
—145—
76.
Schmiedeberg, G.> Arch. exp. P a th o l. Pharmakol., Naynyn-Sehmiedeb e rg 's , h4, 376 (1 8 8 1 ).
77.
S h rIner, R'. L .# and Fuson, R. © ., " Id e n tific a tio n of Organic
Compounds", Second E d ., John W iley, New York, 1940.
78.
S ta h l, E ., Schroter, G ,, K ra ft, G ., and Renz, R ., PharmazIe , JJ_,
633-37 (1 9 5 6 ).
79.
Von S tra h Ienberg, P. J . , "An H is to ric - Georgraphieal Description o f
the North and Eastern P art o f Europe and A sia", English Transla­
t io n , London, 1736.
80.
Thorsen , P ., F r ie s ia ., 3 P 331 ( 1948)
81.
T ro x le r, F . , Seeman, F . , and Hofmann, A ., Helv . .Qhim. Acta, XLh .
2073-2103 (1 9 5 9 ).
82.
T y le r , V . E ., J r . , Am. J . Pharm., 130. 264-69 (1 9 5 8 ).
83.
Vanderl Ip , S ., "In search of a Siberian Klondike", pp. 212, 1903.
84.
V Irtanen, A. I . , and E t t a la , T . , Acta. Chem. Scand., _M_, 182-84 (1957)
85.
Wasson^ V. P ., and Wasson, R. G ., "Mushrooms, Russia and H is to ry ",
pp. 190-214, Vol J_, Pantheon Books, New York, 1957.
86.
Wasson, R. G ., "The Hallucinogenic Mushrooms of Mexico", T r . New
York. Acad. Sc?., 2I_, 325-36 (1 9 5 9 ).
87.
Welland, T . , and M o tze l, W., Ann., 581 * 10-16 (1 9 5 3 ).
88.
W ilkinson, S ., Q uart. Rev., XV (No. 2 ) , 153-171,(1961),
89.
Zel In e r, J . , Monatsh, 25 , 537 ( 1904)
26, 727 (1905)
through Chern. Z e n t., 77 (T T ). 615 (1906)
90.
Zel In e r, J . , Monatsh, 3 2 , 133-42; through G. A ., 3 2266
91.
Block, R. J . , Durrum, E . L . , and Zwelg, G ., "A Manual o f Paper:
Chromatography and- Paper E lectrophoresis", Academic Press,
New York, 1955.
92 .
Q
(1911),
• ■
Lederer, E . , and Lederer, M ., "Chromatography", 2nd E d ., E ls e v ie r,
New York, 1957.
—
146 -
93.
B a rn e tt, H. J . M
Jackson, I . V, , and Spaulding, W. B .,
J . Am. Med. Assoc., 147, 1554-58 ( 1951) .
94.
©odin, P ., ©hem. and Imd., 1424 (1 9 5 4 ).
95.
T y le r , V. E ., Science, 128. 718 (1 9 5 8 ).
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