[Agr.
Biol.
Chem.,
Vol.
29,
No.
The
Part I.
10,
p.
949•`955,
House
Extraction
1965]
Fly Attractants
in Mushrooms
and Activities of the Attractive
Components
in Amanita muscaria (L) Fr.
By
Toshio
Laboratory
MUTO
of
.
The
Pesticide
Tokyo
and
Chemistry,
University
Several
active
Amanita
solvent
one
concentrates
muscaria
extraction
fraction
of
the
referred
It
was
on
long
belonging
to
and
to
known
fruiting
the
et
chemical
acids
and
acid
and
been
rather
ble
to
the
ples
the
attracting
by
of
anticipation,
various
agents
methods
adsorbent
and
extracting
finally
several
with
a
novel
of
known
house
steam
fly
attracting
distillation
body.
Among
colorless
crystals,
exhibited
a
ness
the
exhaustive
active
m.p.
marked
property
and
concentrates,
22•`23•Ž,
with
attractiveness
was
isolated
as
to
colorless
chromatographycaly
a
house
crystals
proved
the
the
with
a
purity.
muscaria
species
and
from
the
using
the
fruiting
solvents,
substances
fly
and
established
a
at-
fly
is
ex
in
cap
in
15cm.
process
of
September
of
near
the
the
Tokyo
concentrates;
one
comparatively
strong
attractive
1) T. Takemoto and M. Nakajima, Yakugaku-Zassi, 84,
1183(1964).
2) T. Takemoto and M. Nakajima, ibid., 84, 1186 (1964).
3) T. Takemoto and M. Nakajima, ibid., 84, 1230 (1964).
4) T. Takemoto and M. Nakajima, ibid., 84. 1232 (1964).
bodies
were
off-white
the
in
Life
a
left
of
parts
the
up
the
from
pink
10•`
to
about
mushroom
the
at
Research
the
warts,
stalk
October
University
end
the
of
region
Institute
of
of
Education
in
Prefecture.
harvest,
within
as
mushroom
by
period
early
Nagano
After
This
bodies
a
Plateau
Sugadaira,
lost
and
in
known
used
mountaneous
with
fruiting
to
mentioned
the
and
northern
and
Amanita
above
characterized
harvested
a same
easily
extraction.
spotted
The
precipition
the
property
the
using
possessing
of
of
selected
from
diameter,
were
active
Fr.
and
colored
of
obtainable
mushrooms
Japan
Preservation
advantages
attracting
native
and
authors
material
15cm
body
ion
house
of
With
extracted
(L)
of
RESULTS
Potency
mushroom
the
starting
princi
mushrooms.
Mushroom
Attracting
abundantly,
attracted
AND
the
of
remarka
house
the
Fly
Considering
had
mushrooms
active
of
House
species
we
marked
have
the
amino
are
some
a
Harvest
toxic
tricholomic
another
flies
the
EXPERIMENTAL
death.
acidic
of
the
of
such
we
components
changers,
them
bodies
potency
tractive
in
house
for
and
elucidated
However,
that
existence
responsible
this
as
acid.1•`4)
fruiting
supposed
isolated
names
interested
as
gather
to
mushrooms,
the
phenomenon
long
Tricholomataceae
structures
ibotenic
the
fruiting
mushrooms
poisoned
successfully
proposed
the
isolated
flies
some
get
those
of
1965
through
homogeneity
house
families
al.
in
was
9,
Agriculture
Tokyo
females.
of
and
components
their
that
for
obtained
juice
D3
mature
bodies
Amanitaceae
Takemoto
as
proved
especially
the
were
filtered
to
chromatographycally
flies,
Fr.
of
Education,
June
responsible
(L.)
SUGAWARA
Faculty
of
Received
of
Ryozo
the
few
at
attracting
weeks
ambient
potency
when
the
conditions;
was
fruiting
even
950
Toshio
preserved
in
a refrigerator
disappeared
a
within
process
consisting
bodies
a
cloth
and
placing
found
in
to
be
the
original
loss
of
six
covering
a refrigerator
quite
the
was
storage.
stored
was
detected
Hence
used
for
tion
pH
of
0•Ž
no
of
extraction
of
room
width,
controlled
in
at
at
along
the
Three
26•‹
cedure,
at
to
side
a
height
hundreds
of
after
one
and
L.),
left
Petri
more
dishes,
were
2cm
20
placed
dishes
an
to
30ml
of
the
shelf
the
the
in
the
usually
while
supply
the
with
centre
potencies
pared
had
that
flies
for
of
based
fallen
that
number
of
(16
hours)
samples
on
of
of
and
the
visited
the
visiting
be
placed
the
drowned
flies
the
in
which
drowned
during
number
of
the
far
ex
basins
flies,
counted
both
over
to
about
in
to
7.
distilled
the
distilled
procedure
to
was
distillate
500ml
bath
the
pH
intermittently
added
to
adjusted
the
to
original
repeated
exhibited
to
concentration,
was
was
a
The
water
while
After
above
debris.
a
being
water
solution
as
through
concentrated
flask
done
ranges:
prepared
filtered
in
pH
10.
stable
60•`70•Ž
the
at
was
some
was
the
within
above
and
pressure
back
again
and
remove
filtrate
solution
of
30min.
juice
7
at
decreases
specified
pH
12
condition,
work
filtered
to
at
and
the
the
does
reflux
beyond
2
the
juice
1 to
the
markedly
reduced
and
in
proved
filtered
extraction
Celite
flask
ap
Components
from
below
the
brown
This
com
flies
of
500ml
attractive
mutually
layer
adjusted
water
and
the
test
the
retained
but
pH
of
the
Active
under
in
adjusted
under
after
diluted
obtaining
ranging
the
was
of
illumina
a
9,
maintained
time,
and
The
counts
Although
the
may
be
were
of
is
to
at
dark
to
dishes
under
milk
basins
variable
period
were
water,
samples
Petri
room.
the
the
but
the
cotton
the
having
ceeded
suitable
powdered
into
really
a
the
period.
30cm
of
of
of
in
pH
potency
3000ml
with
volume
than
wicked
solutions
the
and
enrichment
stability
throughout
diameter,
test
at
Hence
alternatively
The
overnight
tion,
the
room
in
aqueous
number
simultaneously.
experiment.
example,
step.
of
temperature;
7
pro
mutual
Juice.
drop
10min.
room
that
potency
from
test
suitably
of
for
in
preliminary
not
the
days
the
Filtered
room
(Musca
4
in
9cm
same
longer
to
so
Five
some
before
and
on
interval
tested
left
deep
containing
according
than
longer
shelf
flies
released
yield
Fractionation
potency
this
for
grade
tested
accurate
before
were
the
Primary
A
potencies,
extraction,
to
attracting
were
shelf.
of
simplicity
an
require
concentrates
proximate
entrance.
the
the
step
of
with
latter
extracting
attracting
that
their
the
the
Since
samples
of
concentra
of
reasons
the
flies
time
simultaneously
guide
impossible
or
according
the
floor
each,
house
were
or
the
from
over
of
the
watts
53cm
aged
30min.
containing
from
thousand
emergence,
width
from
20
evenly
to
domestica
wall
a
a
the
of
simultaneously
height,
humidity,
with
62cm
lights,
in
relative
shelf
of
illuminate
245cm
in
by
expression
evaluation
a
325cm
as
reproducibility.
the
the
in
the
of
Therefore,
employed
considered
of
period
with
components
components
good
juice
Potency
of
60%
a
height
left
Attracting
and
and
with
a
Fly
dimensions
depth
fluoresent
fixed
as
House
the
equipped
30cm
at
of
with
294cm
was
active
and
number
longer
experiments.
solutions
Procedure
A
the
a
correlation
active
was
was
active
and
after
good
performed
after
principles.
Assay
the
unitary
apparent
filtered
a
of
count
was
even
the
of
time
drowned
showed
retention
potency;
potency
period
found
toluene
about
SUGAWARA
fruiting
the
for
Ryozo
filtering
with
at
effective
attracting
months'
thus
adjusting
7•`8,
potency
the
at
and
However,
harvest,
cotton,
juice
the
crushing
after
of
filtered
-2•Ž,
months.
of
immediately
with
the
at
three
MUTO
twice
no
the
pH
volume.
more
until
detectable
attrac
tiveness.
The
distilled
extracted
the
The
a
with
with
organic
water,
portions
were
400ml
hexane
layers
dried
were
over
combined
combined,
anhydrous
and
three
times.
washed
Na2SO4
and
7
The
House
Fly
Attractants
distilled. The residual pale yellow oil was
dissolved in a small quantity of hexane and
charged on a column of alumina prepared by
pouring a hexane slurry of 50g material into
a glass tube, 4cm in diameter.
The column
was developed
with hexane
followed by
methanol.
The hexane eluate (300ml) and
methanol eluate (100ml) on evaporation
of
the solvents yielded 440mg of colorless oil
and 150mg of yellowish viscous liquid.
Both
residues exhibited house fly attracting potencies
when tested with the above specified pro
cedure.
Thus they were referred to as the
fractions A and B respectively.
The residual solution after the above steam
distillations still retained a remarkable
house
fly attracting potency.
After a variety of
solvents at several pH conditions had been
examined regarding the efficiency of extracting
the residual components,
nearly all the re
maining potency was found to be removed by
exhaustive extraction with chloroform followed
by butanol at pH 2. Thus the residual solu
tion, 500ml in volume, was adjusted to pH 2
and repeatedly shaken with 50ml portion of
chloroform.
The chloroform
layers were
separated, combined (ca. 500ml), washed with
water, dried over Na2SO4 and evaporated
under reduced pressure.
The viscous brown
residue was dissolved in a small volume of
hexane and charged to a column prepared by
pouring a hexane slurry of 50g alumina into
a glass tube, 4cm in diameter, and allowing
to settle.
The column was eluted in succes
sion with 500ml of hexane, 500ml of benzene,
1000ml of 1% methanol-benzene,
500ml of
5% methanol-benzene
and finally 100ml of
methanol. The hexane eluate on evaporation
of the solvent yielded 75mg of colorless vis
cous oil; 1% methanol-benzene
eluate, 946mg
of pale yellow viscous residue;
and 5%
methanol-benzene eluate, 113mg of pale yellow
viscous residue.
These concentrates
which
exhibited house fly attracting potencies in the
above described methods were referred to as
C, D and E respectively.
Some residual
in
Mushrooms.
potency
Part
I
remaining
extraction
in
with
removed
with
the
butanol;
to
the
after
was
the
filtrate
house
after
the
completely
flies
the
became
extraction
butanol.
Thin-Layer
Chromatography
Homogeneity
Since
of
the
the
primarily
a
an
the
thin-layer
and
to
Silica
Gel
carrier.
adsorbing
ones,
of
was
water
paste
and
applied
over
thickness
of
0.25mm.
by
and
samples
heating
lengths
in
an
oven,
were
a
glass
105•Ž
9 to
11cm
with
in
oven
with
for
with
2%
acetate-benzene
a glass
20%
PHOTO. 1. Thin-Layer
Chromatograms
D and Crystalline
D3.
The left plate was
the right plate, with
being spotted
from
two crystallizations
an
to
were
spotted
either
in
with
plates
plates
plates
ethyl
an
the
homogeneous
The
10%
as
of
mixed
to
developed
sprayed
Co.
grams
clean
The
or
from
Merck
throughly
at
cooled.
methanol-benzene
to
employing
G.
material
further
checked
Kagakukikai
Twenty-five
distilled
most
among
was
being
Yamato
of
30min.
flies
it
by
50ml
activated
the
the
house
homogeneity
made
adsorbent
test
exhibited
chromatography
apparatus
Co.
D
separated
while
Checking
D.
attractiveness
purified
with
for
Fraction
fraction
remarkable
a
filtrate
chloroform
with
indifferent
951
jar,
sulfuric
dried
acid
of a Crude
developed with 2% methanol-benzene;
10% ethyl acetate-benzene,
each plate
left to right with crude D, D3 after
and D3 after three crystallizations.
952
Toshio
and
again
shown
heated
in
typically
spot
brown
spots
the
three
of
of
0.14•`0.16,
the
and
D3,
0.48•`0.53
in
The
from
RF
values
in
2%
Purification
While
the
checked
of
with
purified
the
gel
(a
into
allowed
The
crude
a
a
1%
methanol-benzene
rated
of
and
The
spot
D3
of
the
D1
and
the
solvent,
only,
D2 spots.
eluate
and
and
5ml
more
D3,
slightly
six
FIG. 1.
by
those
50ml
the
the
frac
fractions
giving
fractions
of
end
the
solution
substance;
spectrum
75.51%;
in
2920,
2848,
1655,
1460,
1413,
1378,
LR. Spectrum of Crystalline D3 (liquid film).
in
ethyl
less
hexane,
and
characteristic
ultraviolet
region
measured
in
concentration 1mg/ml
absorption
measured
3000,
a
0,
somewhat
when
the
for
(1.79%
no
the
absorption
a
11.54%;
11.31%;
and
water;
3480,
H,
chloroform,
ether,
at
molecu
method,
[ƒ¿]D=•}0
in
the
white
H,
ethanol
maximum
an
by
apparent
the Rast's
75.56%;
and
in
flies,
obtained
calculated
solube
methanol,
hexane
infrared
C,
731.12;
soluble
except
a
an
with
C,
benzene
in
absorption
about
W.
very
acetate,
benzene
of
the
M.
house
far
properties:
difference),
13.13%;
soluble
so
characterized
analysis,
C46H820b:
exhibited
a chromatographically
was
(by
chloroform);
in
Crystal
to
fraction
with
formula,
evapo
than
passage
finally
The
cut
was
in
followed
D2
to
chromatography.
nothing
of
D2
the
dissolved
after
elementary
with
the
separated
potencies
a
m.p.
22•`23•Ž;
determined
12.95%
charged
colorless
which
physico-chemical
0,
developed
fraction
thin-layer
giving
consisting
containing
both
residue
and
was
following
693;
dissolved
attractive
crystals
diameter,
12cm.
of
concentrates
D3,
of
of
was
benzene
and
appeared
in
of
procedure
homogeneity,
platelets;
lar weight
benzene,
height
(670mg)
Each
to the
fraction
1000ml
tions
the
a
column
50ml.
subjected
4cm
to
of
The
fractions
and
tube,
settle
volume
column.
Chemi
in
several
less
form
proved
50g
crystallizations
313mg
Properties
above
component
in
further
Mallinckrodt
suspended
fraction
the
was
repeated
and
residue.
22•`23•Ž.
the
the
or
the
thin-layer
D
chromatography;
of
glass
D
small
gel
was
to
described
being
combined,
waxy
D3
more
eluates
on
yield
m.p.
Among
D
of
fraction
product
Co.)
and
in
silica
Works
Fraction
above
the
with
silica
the
only
were
a colorless
Physico-Chemical
line
acetate-benzene,
homogeneity
chromatography,
poured
Some
0.30•`0.33
spot
give
ether-methanol
through
Further
cal
waxy
platelets,
respectively.
of
residue
coloring
0.55•`0.61
ethyl
D3
to
coloring
the
SUGAWARA
giving
the
evaporated
rapidly
0.08•`0.10,
10%
As
concentrate
Ryozo
slowly
with
and
and
and
min.
D
the
and
D2
0.37•`0.43
for30
crude
spots:
D1,
methanol-benzene
and
115•Ž
1,
gave
violet
of
at
Photo.
MUTO
maxima
as
1743,
1238,
liquid
1720
1167,
in a
film
at
(shoulder),
1118,
1091,
The
House
FIG. 2.
1017,
940
NMR
and
721cm-1
spectrum
was
measured
thyl
in
silane
House
as
Fly
CCl4
an
NMR
Spectrum
a 60mc.
including
2
whitish
with
which
in
Potencies
of
water
filtered
Amanita
juice
muscaria
tracted
active
less
attracting
flies
gathered
active
of
(L.)
the
Fr.
juice,
solutions
tained
during
crystalline
be
Ex.
of
of
distilled
the
distance
about
emergence
released.
500
of
ten
Within
To
were
60cm
from
days
one
containing
on
a
trace
the
a
batch
5 hours,
had
159
which
on
Petri-dish,
placed
room
in
having
been
flies
a
which
at
previously
females
major
the
started
in
a
to
the
flies
as
the
in
crystalline
a hexane
60cm
the
the
solution
the
were
After
females
the
flies
males)
was
food.
gradu
24
again
210
a
dish,
from
food
basin.
of
and
on
15 cm
a
in
of
(452
an
shelf.
22•`23•Ž)
giving
chromatography
over
the
well
in
the
and
of
the
as
gathered
water
proportion
abdomen
on
deep
dropped
distance
and
Then
milk
had
12
room
wicked
30mg
swarming
drowned
Ex.
started
(138
at
whitish
in
flies
3cm
distilled
attracted
662
30ml
been
of
flies
ally
the
out
hatch
hours.
cotton
of
D3
(m.p.
on
the
thin-layer
100ml
The
shelf
the
taken
the
placed
containing
had
thin-layer
the
only
in
to
in
10
of
dish,
flies
powdered
were
all
Petri
preparation
single
spot
This
died
died
started
for
supply
nearly
a
(m.p.
20mg
even
while
house
with
flask
D3
hexane.
carrying
and
males)
male)
released
filled
water
diameter,
dropped
one
feeding
examples
containing
placed
in
before
a
21
sample,
had
were
dishes
a
food,
flies
eggs
and
test
which
Erlenmyer
After
the
with
thousand
batch
without
ob
diameter,
1ml
left
and
with
was
before
as
the
and
One
days
extraction
the
2.
a
Petri
original
of
of
female
from
concentrates
by
in
another
water
flies
the
dropped
the
several
used.
in
dissolved
and
of distilled
them
of
water
sample
Petri-dish
flies;
Of
revealed
was
30ml
of
contaminated
9cm
and
or
containing
preparation
as
deep
a
fraction,
ex
below.
chromatography
of
D3
D,
2cm
the
of
basin.
Ex.
of
more
house
and
course
still
amount
as
dishes
some
the
Crystalline
20•`21•Ž)
to
drowned.
described
1.
well
Petri
and
filtered
bodies
exhibited
the
and
will
as
potencies
to
basins
the
fruiting
concentrates
concentrates
into
the
portion
ovipositor
basin
(one
953
swollen
projecting
two
Extracted
a major
the
I
D3 (CC14).
abdomen
those
tetrame
Part
of Crystalline
and,
Components
The
Mushrooms.
Fig.
standard.
Attracting
in
1):
solution
internal
Attractants
in
(Fig.
represented
Fly
hours,
including
with
swollen,
were
found
basin.
3.
About
500
emergence
room.
5
The
flies
from
days
before
Petri
a batch
dishes
were
having
released
containing
954
4mg,
Toshio
2mg
and
1mg
of the
same
MUTO
and
crystalline
Ryozo
SUGAWARA
Amanitaceae,
preparation
of D3 dropped
to 30ml
water
were placed
alternatively
with
three
Petri
dishes containing
distilled water at an interval
of 40cm.
After 16 hours, 26 (18 females and
8 males), 22 (16 females and 6 males) and 12
aiming
(8 females and 4 males) flies died in the dishes
containing
4mg, 2mg and 1mg of the sample,
respectively,
while 3 (1 male and 2 females),
6 (1 male and 5 females)
and 3 (4 females)
died in the water basins.
After 10 days of the above experiment,
500
flies from the same batch, thus having passed
15 days after the commencement
of emergence
were released in the room and the house fly
attracting
potency
of the same sample
was
similarly
tested. Within 16 hours 27 (25 females
and 2 males), 11 (7 females and 4 males) and
12 (4 females and 8 males) flies drowned
in
the basins containing
300, 30 and 3 mcg per
dish respectively,
while only one fly was found
in each of the three water basins.
A comparison
of these two similar
experi
ments carried
out at the interval
of 10 days
as well as other
experiments
suggested
the
sensitivity
of house flies to D3 increases along
with the adult days after emergence.
Ex. 4. A sample of A, somewhat
purified
by a vacuum
distillation
but not yet suf
ficiently
pure for establishing
a strict identity,
was added to 30ml of distilled
water in Petri
dishes, 9cm in diameter,
at rates of 10mg,
1mg and 0.1mg
per dish.
The Petri dishes
were placed
on the shelf alternatively
with
those containing
distilled
water.
After
16
hours, the numbers
of drowned
flies were 111,
27 and 9 in the basins of 10mg,
1mg, and
0.1mg respectively,
while the numbers
of the
drowned
flies in the water
basins were 9, 9
and 2.
period
DISCUSSION
Anticipating
any principles
the long known phenomenon
for
flies
gather
to the fruiting bodies of the mushrooms
belonging
to the families
Tricholomaceae
and
the
ponents.
of
material
a
essentially
bodies
obtainable
in
liquid
and
after
satisfactory
Of
the
ated
for
retaining
active
through
the
exhaustive
referred
to
as
of
I. R.
and
istence
straight
and
free
fatty
well
suggested
a
the
of
ex
a
long,
carbonyl
method
neither
were
group
rather
times
employed
so
accurate
in
nor
satisfied
results
many
the
weight,
residue
test
reproduced
peated
form
problem,
molecular
acid,
is
we
in
structure
group.
the
experiment
and
component,
isolated
another
esterified
hydroxyl
sensitive,
fraction
chemical
spectra
an
Admitting
this
is
chain
a
be
analysis,
NMR
of
be quite
distillation
a
the
elementary
im
to
potency.
of
could
component
of
fruiting
portions
found
the
Although
this
data
process
the
extraction,
D3,
crystals.
at
limited
concentrates
steps
solvent
very
solid
was
several
a
disclosed
separating
harvest
first
fly
a valuable
in
above
consisting
into
mediately
house
mushrooms,
the
com
were
the
the
year,
experiments
supposed
difficulties
preserving
potency
of
the
some
in
starting
out
of
Although
tracting
of
carried
isolation
experienced
so
with
the
of
similar
tests
during
the
course
re
of
extraction.
Seemingly,
D3
and
the
other
with
advance
some
4•`5
increased
to
or
of
for
in
drowned
the
the
the
of
increasing
more,
the
large
drowned
and
proportion
showing
of
increased
might
the
those
at
high
suggested
which
flies
males;
even
found
it was
flies
the
those
than
females
were
regard,
of
the
the
sensitive
house
maturity
and
sensitivity
in
abdomen
to
appeared
particularly
more
flies,
sensitivity
with
Still
ovipositors
In
this
it
apparent
females
swollen
projecting
frequency.
to
food.
were
flies
increased
emergence
females,
eggs
house
life;
their
their
the
suggested
bearing
whitish,
adult
after
with
of
of
concentrates
their
along
desire
flies
sensitivity
active
days
proportion
the
responsible
that house
we
at
egg
be
due
bearing
females.
The
results
of
this
experiment
clearly
proved
The
House
Fly
Attractants
the long known
ability
of the mushroom
to
attract house flies is attributable
to some active
components
present
in the fruiting
body.
Since the fractions other than D3, for example,
A, C, D2 and E, though
minor components,
are supposed
to have not so inferior
levels of
activity and relations with D, in structure,
we
now intend further
purification
of these frac
tions as well as the elucidation
of the chemical
structure of D3.
in
Mushrooms.
Part
I
Acknowledgement.
Dr.
H.
Ando
of Science,
for
for
their
Mr.
Tokyo
kindest
harvesting
was
of
K.
are
very
Sato
the
of
University
instructions
the
investigation
grant-in-aid
We
and
the
955
Ministry
to
Faculty
of Education
and
mushrooms.
partly
grateful
the
Also,
suppoted
of
cooperation
by
Education.
this
the