Occurrence of Cholest-4-en-3-one in Red Alga, Meristotheca papulosa


Kanazawa, A; Yoshioka, M

Nippon Suisan Gakkaishi 37(5): 397-403

1971


Bulletin
of
the
Japanese
Society
of
Scientific
Fisheries
Vol.
37,
No.
5,
1971
397
Occurrence
of
Cholest-4-en-3-one
in
Red
Alga,
Meristotheca
papulosa
Akio
KANAZAWA
and
Mitsuki
YOSHIOKA*
(Recieved
November
2,
1970)
Hitherto,
little
is
known
about
the
occurrence
of
4
4
-3-keto-steroids
in
the
plant
kingdom.
In
this
study,
an
attempt
to
clarify
the
occurrence
of
keto-steroid
in
the
red
alga,
Meristotheca
papulosa
was
carried
out.
The
algal
keto-steroid
isolated
by
a
column
chromatography
and
thin-layer
chromato-
graphy
gave
m.p.
75
-
79°C,
MW
(mass
spectrum):
384,
p-phenylenediamine
reaction:
positive,
2,4-dinitrophenylhydrazine
reaction
:
positive.
Ultraviolet
absorption
spectral
and
mass
spectral
analyses
suggested
the
structure
of
4
4
-3-keto-steroid.
This
algal
steroid
was
identified
as
cholest-4-en-3-one
by
a
thin-layer,
paper,
and
gas-liquid
chromatography.
Moreover,
it
was
ascertained
that
the
labeled
cholest-4-en-3-one
was
formed
as
a
metabolite
of
cholesterol-
1.4C
by
algal
tissues.
The
cholest-4-en-3-one,
an
intermediate
in
the
bioconversion
of
cholesterol
to
steriod
hormones,
has
not
yet
been
shown
in
animals
and
plants.
However,
in
the
case
of
alga,
it
is
suggested
that
cholesterol
is
oxidized
to
cholest-4-en-3-one
for
lack
of
steroid
C20-C221yase
in
the
red
alga.
It
has
been
known
that
4
4
-3-keto-steroids
occur
widely
in
the
animal
kingdom
and
play
an
important
function
as
a
sexual
and
adrenocortical
hormones.
However,
little
is
known
about
the
occurrence
of
it
in
the
plant
kingdom.
Cholesterol
peculiar
to
animals
has
been
found
from
red
algae"',
brown
algae'',
and
green
algae.
Therefore,
if
V-
hydroxysteroid
dehydrogenase
(EC
1.1.1.51)
and
steroid
4-isomerase
(EC
5.3.3.1)
exist
in
algae,
the
formation
of
4
4
-3-keto-steroid
from
cholesterol
may
occur
as
well
as
in
animals.
This
paper
describes
the
results
of
our
search
for
4
4
-3-keto-steroids
in
an
edible
red
alga.
Material
and
Methods
Material.
Meristotheca
papulosa,
which
is
an
edible
red
alga
available
in
com-
mercial
quantities
in
Japan
was
used
in
this
experiment.
The
material
was
collected
in
May
1969,
at
the
south
coast
of
Kagoshima,
and
dried
in
dark.
Isolation
of
keto-steroid.
The
procedure
for
isolating
keto-steriod
from
red
alga
is
shown
in
Fig.
1.
The
dried
material
(1.6
kg)
was
pulverized
and
extracted
twice
with
7
volumes
of
dichloromethane.
The
organic
phase
was
washed
with
volume/20
water,
and
then
evaporated
to
dryness
in
vacuo
at
a
bath
temperature
of
40°C.
The
residue
(12
g)
was
*
Laboratory
of
Fisheries
Chemistry,
Faculty
of
Fisheries,
Kagoshima
Univ.,
Kagoshima,
Japan.
(Itigle
i*"
.1Elakt:
fiE9Mic4-9kM*%)
398
Meristotheca
papulosa
(1.6
kg)
+7
volumes
CH2Cl2
extracted
Extract
Residue
+CH2C12
Extract
Residue
washed
with
H2O
+Na2SO4
(anhydrous)
filtered
Extract
evaporated
under
N2
at
40°C
CH2C12
extract
(12
g)
Silica
gel
column
chromatography
Eluent:
Hexane-acetone
Steroid
fraction
(4
g)
Alumina
column
chromatography
Eluent:
Hexane-benzene
Na2SO4
Keto-steroid
fraction
Thin-layer
chromatography
Solvent:
Benzene-acetone
(4:
1)
Keto-steroid
(Rf
0.45-0.75)
Thin-layer
chromatography
Solvent:
Heptane-ethylacetate
(4:
1)
Sterol
fraction
I
Cholesterol
I
(500
mg)
Rf
0.40
keto-steroid
Rf
0.47
keto-steroid
crystallized
from
ethanol
and
methanol
Unknown
compound
Cholest-4-en-3-one
I
(2.5
mg)
Fig.
1.
Procedure
for
isolating
keto-steriod
from
Meristotheca
papulosa.
initially
chromatographed
on
a
column
of
silica
gel
(Kieselgel
G,
Merck)
with
hexane,
hexane-acetone
(100:1),
hexane-acetone
(50:1),
hexane-acetone
(20:1),
and
hexane-
acetone
(10:1)
as
an
eluent.
When
the
steroid
fraction
gained
(4
g)
was
rechromato-
graphed
on
a
column
of
alumina
(grade
II,
Merck)
with
hexane,
hexane-benzene
(5:1),
hexane-benzene
(3:1),
hexane-benzene
(1:1),
and
hexane-benzene
(1:2)
as
an
eluent,
it
was
divided
into
a
sterol
fraction
and
a
keto-steroid
fraction.
The
sterol
fraction
gave
500
mg
of
cholesterol
after
crystallization.
4
4
-3-Keto-steroid
fraction
was
further
sub-
jected
to
a
thin-layer
chromatography
using
two
solvent
systems:
benzene-acetone
(4:1)
and
heptane-ethylacetate
(4:1).
The
keto-steroid
on
the
chromatograms
was
located
under
UV-light
(253
mac),
and
scraped
off
the
plate
by
spatura,
and
then
eluted
with
dichloro-
methane-methanol
(9:1)
from
the
absorbent.
In
the
latter
solvent
system,
4
4
-3-keto-
steroid
was
divided
into
Rf
0.40
and
0.47
keto-steroids.
Rf
0.40
keto-steroid,
after
recrystallizations
from
ethanol
and
methanol,
gave
2.5
mg
of
crystals.
Rf
0.47
keto-steroid
remained
as
a
non-crystals.
399
Thin-layer
chromatography
(TLC),
paper
chromatography
(PC)
and
gas-liquid
chroma-
tography
(GLC).
For
the
purpose
of
identifying
the
4
4
-3-keto-steroid,
TLC,
PC
and
GLC
were
used.
In
TLC,
chromatoplate
(20
x
20
cm)
coated
with
Kieselgel
G—Kieselgel
GF
a
.
(5:1)
to
a
thickness
of
0.5
mm
was
used.
PC
was
carried
out
by
a
descending
method
using
Toyo
filter
paper
No.
51.
GLC
was
performed
with
a
Shimadzu
model
GC-3AF
chromatographic
unit,
equipped
with
flame
ionization
detector.
The
column
packings
used
were
1.5%
SE-30,
1.0
%
QF-1,
and
1.5
%
OV-17.
Spectra
analyses.
Ultraviolet
absorption
spectrum
was
taken
with
a
Shimadzu
multipurpose
spectrophotometer
MPS-50
L.
Mass
spectrum
was
measured
on
a
Hitachi
RMU-6D
instrument
(chamber
voltage,
70
eV).
Incubation
of
alga
with
cholesterol-"C.
Cholesterol-4-
14
C
(33.5
mCi/mM)
was
pur-
chased
from
Daiichi
Pure
Chemicals
Co.,
Ltd.
(Japan).
The
purity
of
this
precursor
was
ascertained
by
TLC
and
GLC.
Cholest-4-en-3-one
was
obtained
from
Mann
Research
Laboratories
(U.S.A.).
Meristotheca
papulosa
collected
in
May
1970,
at
the
Hanase-point
in
Kagoshima
was
immediately
kept
in
the
cold
sea
water
and
carried
to
the
laboratory.
The
alga
was
thoroughly
washed
with
the
sterilized
sea
water
and
the
extraneous
matter
was
re-
moved.
Two
hundred
and
fifty
g
of
Meristotheca
papulosa
was
chopped
finely
and
incubated
with
0.5
pCi
of
cholesterol-4-
14
C
(dissolved
in
0.1
ml
of
ethanol)
in
250
ml
of
the
vitamin-free
ASP-6
medium'
)
containing
cofactor
(1
mg
of
nicotinamide
adenine
dinucleotide
phosphate)
and
antibiotics
(480,000
units
of
penicillin
G
and
10
mg
of
aureomysin)
at
25°C
for
40
hours.
At
the
end
of
16
and
24
hours
incubation,
cofactor
and
antibiotics
were
added
to
the
medium.
Isolation
and
identification
of
cholest-4-en-3-one-
14
C
from
the
incubated
alga.
After
the
incubation,
the
medium
and
incubated
laver
were
extracted
twice
with
7
volumes
of
dichloromethane,
respectively.
Two
dichloromethane
extracts
were
combined,
and
to
this,
3
mg
of
authentic
cholest-4-en-3-one
was
added
as
a
carrier
for
radioactive
steroid
produced.
The
extracts
were
evaporated
to
dryness
in
vacuo
below
40°C.
The
residue
was
subjected
to
a
silica
gel
column
chromatography,
alumina
column
chromatography,
and
thin-layer
chromatography
by
the
same
manner
as
mentioned
above
(Fig.
1.).
The
conclusive
identification
of
radioactive
steroid
produced
was
based
on
the
con-
stant
specific
activities
of
the
crystals.
Measure
of
radioactivity.
The
radioactivities
of
the
materials
were
measured
by
a
Beckman
liquid
scintillation
counter,
model
LS-150.
The
toluene
solution
of
PPO
(0.6
%)
and
POPOP
(0.02
%)
was
used
as
a
scintillator.
The
efficiency
of
counting
of
radioactive
materials
was
approximately
65
%.
Rf
Meristotheca
steroid
Cholest-4-en-
3-one
Solvents
Benzene-acetone
(4:1)
0.48
Chloroform-methanol
(97:3)
0.58
Hexane-acetone
(2:1)
0.67
Heptane-ether
(4:1)
0.13
Heptane-ethylacetate
(4:1)
0.42
Acetic
acid-H
2
0
(84:16)
0.61
n-Propanol-methanol-H20
(15:82:3)
0.44
Methanol-H
2
0
(95:5)
0.26
TLC
0.48
0.57
0.67
0.13
0.42
PC
0.60
0.44
0.25
SE-30
QF-1
OV-17
2.43
9.41
2.34
2.43
9.35
2.34
Relative
retention
time*
Meristotheca
steroid
Cholest-4-en-
3-one
Column
packings**
400
Results
Identification
of
cholest-4-en-3-one
in
Meristotheca
papulosa.
The
algal
steroid
isolated
from
Meristotheca
papulosa
gave
m.p.
75-79°C,
MW
(mass
spectrum):
384,
:
241
mp,
p-phenylenediamine
reaction
:
positive,
2,4-dinitrophenylhydrazine
reaction:
positive.
The
chromatographic
mobilities
of
algal
steroid
investigated
on
a
TLC,
PC
and
GLC
are
shown
in
Tables
1
and
2.
Table
1.
TLC
and
PC
analyses
of
algal
steroid
and
authentic
cholest-4-en-3-one.
Table
2.
GLC
analysis
of
algal
steroid
and
authentic
cholest-4-en-3-one.
*
Relative
to
cholestane
**
SE-30:
Column
2.0
m
x
4
mm
I.D.,
1.5%
SE-30
on
60-80
mesh
Chromosorb
W;
Column
temperature
224°C;
Nitrogen
1.0
kg/cm'
QF-1:
Column
2.0
m
x
4
mm
I.D.,
1.0%
QF-1
on
60-80
mesh
Chromosorb
W;
Column
temperature
205°C;
Nitrogen
1.0
kg/cm'
OV-17:
Column
3.0
m
x
4
mm
I.D.,
1.5
%
OV-17
on
80-100mesh
Shimalite
W;
Column
temperature
240°C;
Nitrogen
1.0
kg/cm'
The
algal
steroid
revealed
the
same
mobilities
as
authentic
cholest-4-en-3-one
in
a
TLC
and
PC
using
the
various
solvent
systems.
The
gas-chromatographic
retention
time
of
this
substance
was
identical
with
that
of
cholest-4-en-3-one
on
three
different
columns.
The
mass
spectrum
of
algal
steroid
showed
prominant
peaks
at
m/e
384
(M+),
369
(M+-C1-1
3
),
342
(M
+
-CH
2
C0),
and
124.
The
peaks
at
m/e
124
and
342
are
charac-
401
teristic
of
11
4
-3-keto-steroids
9,10)
.
On
the
basis
of
the
above
data,
the
algal
steroid
was
assingned
the
structure
of
cholest-4-en-3-one.
Biosynthesis
of
cholest-4-en-3-one
by
Meristotheca
papulosa.
After
the
incubation
of
Meristotheca
papulosa
with
cholesterol-
14
C,
the
cholest-4-en-3-one-
14
C
was
isolated
from
incubates.
The
radioactivities
in
various
purification
steps
of
cholest-4-en-3-one
are
shown
in
Table
3.
Table
3.
Radioactivities
in
the
purification
steps
of
cholest-4-en-3-one
from
the
alga
incubated
with
cholesterol-4-
14
C.
Purification
steps
Radioactivity
(dpm)
Dichloromethane
extract
Silica
gel
column
chromatography
Alumina
column
chromatography*
Thin-layer
chromatography
Benzene-acetone
(4:1)
Thin-layer
chromatography
Heptane-ethylacetate
(4:1)
465,000
321,000
3,680
2,550
1,910
The
radioactivity
of
the
sterol
fraction
(precursor)
eluted
from
an
alumina
column
chromatography
was
257,000
dpm.
The
radioactivities
of
sterol
fraction
and
keto-steroid
fraction
separated
by
an
alumina
column
chromatography
were
257,000
dpm
and
3,680
dpm,
respectively.
Fur-
ther,
during
the
chromatographic
purification
steps
by
TLC,
the
cholest-4-en-3-one
showed
a
significant
radioactivities,
successively.
A
percentage
of
conversion
of
cholesterol-
14
C
to
cholest-4-en-3-one-
14
C
by
the
algal
tissues
was
0.41
%
of
the
total
radioactivity
recovered
from
the
dichloromethane
extracts.
Finally,
to
the
chromatographically
purified
cholest-4-en-3-one-
14
C,
about
5
mg
of
authentic
cholest-4-en-3-one
was
added,
and
recrystallized
several
times
from
the
different
solvent
systems.
As
shown in
Table
4,
the
specific
activities
of
the
crystals
were
found
to
be
constant
in
the
last
three
crystallizations.
Table
4.
Recrystallization
of
cholest-4-en-3-one-
14
C
from
the
alga
incubated
with
cholesterol-4-
14
C.
Crystallization
1
st
2
nd
3
rd
Solvent
systems
Acetone-water
Methanol
Ethanol
Specific
activity
(dpm/mg)
211
225
212
Discussion
The
keto-steroid
isolated
from
the
red
alga,
Meristotheca
papulosa
was
identified
as
cholest-4-en-3-one
by
melting
point,
molecular
weight,
TLC,
PC,
GLC,
ultraviolet
ab-
402
sorption
spectrum,
and
mass
spectrum.
Furthermore,
it
was
demonstrated
that
cholest-
4-en-3-one
was
formed
from
the
labeled
precursor
cholesterol,
in
algal
tissues,
The
cholest-4-en-3-one
which
is
presumed
to
be
an
intermediate
in
the
bioconver-
sion
of
cholesterol
to
steroid
hormones
has
not
yet
been
found
in
animals
and
plants.
In
the
case
of
animals,
as
shown
in
(I),
it
has
been
generally
suggested
that
cholesterol
CH3
C=0
/\1
2
\
(I)
Steroid
C2
0
-C22
lyase
30-Hydroxysteroid
dehydrogenase
Steroid
bisomerase
HO/\/N/
H0/\/\/
Cholesterol
Pregnenolone
CH3
C=0
\1
2
\
I
Progesterone
\/\
\/\
3A-Hydroxysteroid
dehydrogenase
—›
Steroid
4-isomerase
HO/\/\/
Cholesterol
01\,\/
Cholest-4-en-3-one
was
oxidized
by
313-hydroxysteroid
dehydrogenase
and
steroid
LI-isomerase
after
the
cleavage
of
its
side-chain
by
enzyme
cutting
between
C
20
and
C22
(steroid
C20—C22
lyase).
On
the
other
hand,
in
the
case
of
alga,
it
is
suggested
that
cholesterol
may
be
oxidized
to
cholest-4-en-3-one
for
lack
of
steroid
C20
C22
lyase
in
it,
as
shown
in
(II).
Recently,
the
oxidation
of
cholesterol
to
cholest-4-en-3-one
has
been
demonstrated
by
a
few
micro-
organisms"
").
It
seems
reasonable
to
suppose
that
either
red
alga,
belonging
to
the
lowest
class
in
plant,
does
not
possess
steroid
C2O
C22
lyase,
or
the
rate
of
cleavage
of
side-
chain
may
be
so
slow,
as
well
as
in
a
few
microoranisrns.
In
any
case,
the
occurrence
of
cholest-4-en-3-one
in
the
marine
red
alga
is
an
interesting
subject
to
biosynthetic
study
of
steroid.
403
Acknowledgements
The
authors
are
indebted
to
Dr.
K.
KASHIWADA
and
to
Mr.
S.
TESHIMA,
University
.of
Kagoshima,
for
their
kind
advices.
Mass
spectrometric
analysis
was
performed
by
the
member
of
the
InStitute
of
Applied
Microbiology,
University
of
Tokyo,
to
whom
-
the
authors
wish
to
express
their
thanks.
References
1)
K.
TSUDA,
S.
AKAGI,
and
Y.
KISHIDA:
Science,
126,
927-928
(1957).
2)
K.
TSUDA,
S.
AKAGI,
and
Y.
KISHIDA:
Chem.
Pharm.
Bull.
(Tokyo),
6,
101-104
(1958).
3)
K.
TSUDA,
S.
AKAGI,
Y.
KISHIDA,
R.
HAYATSU,
and
K.
SAKAI:
ibid.,
6,
724-727
(1958).
4)
A.
SArro
and
D.
R.
LDLER:
Can.
J.
Biochem.,
44,
1195-1199
(1966).
5)
G.
F.
GIBBONS,
L.
J.
GOAD,
and
T.
W.
GOODWIN:
Phytochern.,
6,
677-683
(1967).
6)
D.
R.
IDLER,
A.
SAITO,
and
P.
WISEMAN:
Steroids,
11,
465-473
(1968).
7)
N.
IKEKAWA,
N.
MORISAKI,
K.
TSUDA,
and
T.
YOSHIDA:
ibid.,
12,
41-48
(1968).
8)
L.
PROVASOLI,
J. J.
A.
MCLAUGHLIN,
and
M.
R.
DROOP:
Arch.
Mikrobiol.,
25,
392-428
(1957).
9)
H.
BUDZIKIEWICZ,
C.
DJIERASSI,
and
D.
H.
WILLIAMS:
Structure
Elucidation
of
Natural
Products
by
Mass
Spectrometry,
Vol.
2,
89
pp.,
Holden
Day,
San-Francisco
(1964).
10)
R.
H.
SHAPIRO,
J.
M.
WILSON,
and
C.
DJIERASSI:
Steroids,
1,
1-6
(1963).
11)
W.
CHARNEY
and
H.
L.
HERZOG:
Microbial
Transformations
of
Steroids,
217-218
pp.,
Academic
Press,
New
York
and
London
(1967).
12)
H.
Itzuick
and
A.
NArro:
Microbial
Transformation
of
Steroids
and
Alkaloids,
217
pp.,
Univ.
of
Tokyo
Press,
Tokyo
and
Univ.
Park
Press,
Pennsylvania
(1967).
:13)
H.
bzw(A,
M.
In3A,
S.
TESHIMA,
and
Y.
MINEMURA:
Z.
,41Ig.
Mikrobiol.,
9,
443
148
(1969).