Steroid, triterpenoid, and flavonoid constituents of Euphorbia pulcherrima Villd. leaves


Khafagy, S.; Nazmi, N.; Salam, N.A.; Eldin, A.S.

Acta Pharmaceutica Jugoslavica 30(2): 103-107

1980


Light petroleum, ether, and alcoholic extracts of Euphorbia pulcherrima Willd. leaves, yielded 3 teriterpenes, 2 steroids and 3 flavonoids. By using UV, IR, NMR, and MS spectral methods as well as available standard sample comparison, the compounds were found to be germanicol acetate (yield 0.25%), germanicol (0,025%), brein (0.0054%), (3-sitosterol (0.042%), (3-sito steryl 3-D-glucoside (0.01 %), kaempferol 3-0-glucoside (0.013%), kaempferol 3-0 -rutinoside (0.11%) and rutin (0.012%). The first two compounds exhibited moulting activity when assayed on S podoptera littoralis Bosid. (cotton leaf worm).

Acta
Pharm.
Jugosl.
30
(1980)
103-107
Steroid,
triterpenoid,
and
flavonoid
constituents
of
Euphorbia
pulcherrima
Willd.
leaves
SAAD
KHAFAGY
NAWAL
NAZMI
NABIL
ABDEL
SALAM
AHMED
SEIF
ELDIN
Faculty
of
Pharmacy,
University
of
Alexandria,
Egypt
Received
February
4,
1980
Light
petroleum,
ether,
and
alcoholic
extracts
of
Euphorbia
pulcherrima
Willd.
leaves,
yielded
3
triterpenes,
2
steroids
and
3
flavonoids.
By
using
UV,
IR,
NMR,
and
MS
spectral
methods
as
well
as
available
standard
sample
compari-
son,
the
compounds
were
found
to
be
germa-
nicol
acetate
(yield
0.25%),
germanicol
(0,025%),
brein
(0.0054%),
0-sitosterol
(0.042%),
13-sitoste-
ryl
3-D-glucoside
(0.01%),
kaempferol
3-0-glu-
coside
(0.013%),
kaempferol
3-0-rutinoside
(0.11%)
and
rutin
(0.012%).
The
first
two
com-
pounds
exhibited
moulting
activity
when
as-
sayed
on
Spodoptera
littoralis
Bosid.
(cotton
leaf
worm).
Euphorbia
pulcherrima
Willd.,
»Christmas
flowere,
is
an
ornamental
plant
widely
cultivated
in
Egypt
(1,
2,
3).
It
was
reported
that
all
parts
of
the
plant
are
poisonous
and
that
the
bracts
have
insecticidal
properties
(1,
4,
5).
The
literature
encompasses
scattered
chemical
studies,
carried
out
abroad,
on
the
latex,
stems,
bracts
and
flowers
(2,
6,
7,
8).
However,
no
pu-
blications
could
be
found
dealing
with
the
chemistry
of
the
leaves,
although
it
has
been
reported
(4)
that
a
small
child
died
after
eating
a
single
leaf.
This
study
of
the
leaves
was
undertaken
as
a
result
of
a
preliminary
examination
of
the
light
petroleum
extract
which
showed
to
be
just
as
potent
as
the
insect-moulting
hormones,
when
assayed
on
Spodoptera
littoralis
Bosid.
(cotton
leaf
worm).
EXPERIMENTAL
Plant
material
The
plant
was
collected
from
cultivated
areas
in
several
public
and
spe-
cial
gardens
in
Alexandria.
It
was
identified
as
Euphorbia
pulcherrima
Willd.
by
the
late
professor
Dr.
V.
Tackholm.
Isolation
procedure
2.5
kg
of
the
powdered
leaves
were
successively
extracted,
at
room
tem-
perature,
with
light
petroleum
(50-70
°C),
ether,
and
ethanol
(95%).
103
S.
Khafagy
et
al.:
Steroid,
triterpenoid
and
flavonoid
constituents
of
Euphorbia
pulcherrima
Willd.
leaves,
Acta
Pharm.
Jugosl.
30
(1980)
103-107.
Light
petroleum
extract
Refrigeration
of
the
light
petroleum
extract
(110
g)
furnished
a
yellowish-
-white
deposit
that
was
repeatedly
crystallised
from
methanol
to
give
com-
pound
DA«
m.p.
279-80
°C,
(yield
0.25%).
The
light
petroleum
mother
liquor
was
saponified
with
20%
alcoholic
potash.
Part
of
the
unsaponifiable
matter
(17
g)
was
chromatographed
on
an
alumina
column
(Merck,
400
g)
and
eluted,
in
succession,
with
benzene
(fractions
1-58,
200
ml
each),
then
with
1%
methanol
in
benzene
(fractions
59-71).
Separate
crystallisation
of
material
from
fractions
(27-58),
(60-65)
and
(69-71),
respectively,
from
methanol,
gave
the
following
products:
compound
»B«,
m.p.
172-4
°C
(yield
0.275%),
5-sitosterol
(yield
0.042%),
and
compound
»D«,
m.p.
222-4
°C
(yield
0.0054%).
Ethereal
extract
The
ethereal
extract
gave,
on
concentration,
a
greenish
deposit
which
was
repeatedly
crystallised
from
ethanol
to
give
fi-sitosteryl
3-D-glucoside,
m.p.
284-6
°C
(yield
0.01%).
Alcoholic
extract
The
extract
was
freed
from
inorganic
material
and
the
solvent
was
eva-
porated
to
give
294
g
of
a
dark
brown
residue;
35
g
of
this
residue
were
chromatographed
on
a
silica
gel
column
(Chemapol,
700
g).
The
column
was
eluted
with
EtOAc
(fractions
1-29,
200
ml
each)
then
with
5%
methanol
in
EtOAc
(fractions
30-75).
Separate
crystallization
of
material
from
fractions
(30-45)
and
(63-75),
respectively,
from
methanol
gave
2
flavonoids
iden-
tified
as
kaempferol
3-0-glucoside,
m.p.
229-31
0
C
(yield
0.013%)
and
rutin,
m.p.
183-5
°C
(yield
0.012%)
whereas
crystallization
of
fractions
(56-61)
from
distilled
water
gave
a
flavonoid
»G«,
m.p.
176-8
°C
(yield
0.11%).
Characterisation
and
identification
of
the
isolated
compounds:
a-Sitosterol,
13-sitosteryl
3-D-glucoside,
kaempferol
3-0-glucoside
and
rutin
were
identified
by
direct
comparison
of
mp's,
mmp's,
with
reference
sam-
ples
TL
chromatograms,
IR
and
UV
spectra.
Compound
»A«:
m.p.
278-80
°C
(MeOH),
yield
0.25%.
Gives
a
bright
red
colour
in
Liebermann's
and
Salkowski's
tests.
IR
(KCl
cm
-
')
:
1718
(C
=
0
of
an
ester),
1640
(C
=
C),
1450,
1385,
1375
(gem-dimethyl),
1242
(C-O-C
of
ace-
tate),
840
and
820
(cyclic
compound).
Ms,
m/e:
W
468,
453
(M
+
(M+
acetic),
393,
218,
207,
205,
204,
203,
190,
189,
177.
NMR
CC1
4
):
singlets
at
0.7,
0.78,
0.88,
0.95
and
1.04
(24
H,
8
x
CH
),
1.4
10
x
C
H2)
,
(8
ppm
60
MHO,
1.94
(3H,
OCOCH
3
),
multiplet
at
4.05
(1
proton
nex
t
3
to
acetyl
group),
CH
3
),
408
multiplet
at
4.82
(H,
vinylic
proton).
1.6
(20
H,
Compound
0.13«:
m.p.
172-4
°C
(Me0H),
yield
0.025%.
Gives
a
bright
red
colour
in
Liebermann's
and
Salkowski's
tests.
IR
(KCI
cm
-
'):
3350
(OH),
1640
(C
=
C),
1450,
1380,
1370
(gem-dimethyl).
Ms,
m/e:
M+
426.
411
(1‘
11+
CH
3
),
408
(M+
H
2
O),
218,
207,
205,
204,
203,
190,
189,
177.
NMR
(8
PPM
60
MH
z
,
CC1
4
):
0.64,
0.7,
0.85,
0.9
and
1.02
(24
H,
8
x
CH
3
),
1.18,
1.7
(
20
H
,
10
x
CH
2
)
and
a
multiplet
at
4.8
(H,
v
i
ny
li
c
proton).
Compound
»B«
was
detected
in
the
light
petroleum
extract
before
saponification,
but
it
was
iso
-
lated
afterwards
from
the
unsaponifiabl
e
matter.
Acetylation
of
compound
»B«:
gave
crystals
m.p.
279-80
°C,
that
were
found
to
be
identical
with
com-
104
S.
Khafagy
el
al.:
Steroid,
triterpenoid
and
flavonoid
constituents
of
Euphorbia
pulcherrima
leaves,
Acta
Pltartn.
Jugosl.
30
(1980)
103-107.
pound
»A«.
The
signals
in
the
NM'R
spectra
of
compounds
»A«
and
»B«
between
0.64
and
1.04,
attributed
to
8
overlapping
methyl
groups,
as
well
as
the
band
characteristic
of
gem-dimethyl
groups
in
the
IR,
were
good
evi-
dence
for
a
pentacyclic
triterpenoid
constitution.
The
molecular
ion
appear-
ing
in
the
Ms
of
compound
»B«
at
m/e
426,
indicated
a
monohydroxy
triter-
penoid
skeleton
(C
30
H
50
0).
The
Ms
showed
the
diagnostically
important
peaks
that
characterise
the
oleanene
series
and
particularly
the
A-18
group
(9).
Moreover,
the
multiplet
in
the
NMR
at
8
4.8
and
the
band
in
the
IR
at
1640
cm
-
'
confirmed
the
presence
of
a
nuclear
double
bond.
In
the
Ms,
cleavage
of
ring
C
yielded
the
ion
m/e
207
(23%),
enclosing
ring
A
and
B,
characteristic
for
3-hydroxy
pentacyclic
triterpenes.
This
ion
was
accompa-
nied
by
the
ion
m/e
218
(85%)
comprising
rings
D
and
E.
A
conclusive
proof
of
the
structure
was
obtained
from
the
highly
pronounced
signal
at
m/e
177
(73%)
characteristic
for
0
-18
oleanenes.
By
comparing
the
above
findings
with
those
published
(10)
for
A-18
oleanene
compounds,
material
»B«
was
found
to
be
identical
with
germani-
col*.
The
Ms
of
compound
»A«
showed_
a
molecular
ion
at
m/e
468,
followed
by
peaks
at
m/e
453
(25%),
408
(4%)
and
393
(8%)
due
to
loss
of
methyl,
acetyl
and
combination
of
both.
Saponification
of
material
»A«
yielded
crystals,
m.p.
172-4
°C,
identical
with
compound
»B«.
Accordingly
compound
»A«
is
ger-
manicol
acetate.
Compound
»Do:
m.p.
220-1
°C
(MeOH),
yield
0.0054%.
Gives
positive
Liebermann's
and
Salkowski's
tests;
acetyl
deriv.,
m.p.
198-9
°C
(MeOH).
IR
(KBr
3340-3420
(OH),
1640-1660
(C
=
C),
1470,
1410,
1385,
1360
(gem-dimethyl),
1040
(-C-0),
1000,
975,
870,
850
and
830.
Ms,
m/e:
MI
-
442,
427
(NI+
-
CH
3
),
424
(M+
-
H
2
0),
409
(M'
-
CH
3
+
H
2
0),
406
(M+
-
2H
2
0),
391,
301,
273,
271,
257,
255,
234
(100%),
219,
218,
216,
207,
205,
191,
189.
The
colour
tests,
as
well
as
the
bands
in
the
IR
at
1385,
1360
and
1660-
-1640
cm',
indicated
an
unsaturated
triterpenoid
structure.
The
Ms
showed
a
mol.
ion
at
m/e
442,
pointing
to
a
dihydroxy
triterpene,
the
spectrum
showed
in
addition
the
diagnostically
important
peaks,
that
characterise
A-12
pentacyclic
triterpenes,
resulting
from
a
retro-Diels-Alder
fragmentation
(9).
The
appearance
of
the
highly
pronounced
peak
m/e
191
(84%)
in
comparison
with
that
at
m/e
203
(12%)
indicated
that
compound
»Do«
is
closely
related
to
the
ursene
series
(11).
The
strong
band
in
the
IR
at
3340-3420
cm
-1
as
well
as
the
ease
of
acetylation
and
formation
of
a
crystalline
derivative
confirm
that
the
oxygen
atoms
make
part
of
two
hydroxy
groups,
which
are
either
primary
and/or
equatorially
oriented
secondary
alcohols.
By
analogy
to
all
other
triterpenes
of
the
tx-amyrin
series,
one
of
the
oxygens
might
be
attached
at
C-3.
The
position
of
the
other
is
suggested
by
the
Ms
spectrum.
The
high
intensity
base
peak
at
m/e
234
(100%)
giving
rise
to
peaks
at
m/e
219,
218
and
203,
as
a
result
of
sequential
loss
of
hydroxy
and
methyl
groups,
shows
that
this
hydroxy
group
is
present
either
in
the
form
of
a
hydroxyme-
thylene
group
at
C-29
or
30
in
ring
E,
or
at
C-17
in
ring
D,
or
as
separate
hydroxy
and
methyl
groups.
The
absence
of
a
mass
peak
at
M+
-
31
chara-
cteristic
for
a
loss
of
-CH
2
OH
excludes
the
possibility
of
the
occurence
of
the
*
An
authentic
sample
of
germanicol
was
not
available
for
direct
comparison.
105
S.
Khafagy
et
al.:
Steroid,
triterpenoid
and
flavonoid
constituents
of
Euphorbia
pulcherrima
leaves,
Acta
Pharm.
Jugosl.
30
(1980)
103-107.
-CH
3
and
-OH
as
-CH
2
OH.
Accordingly,
OH
and
CH
3
should
occupy
different
locations.
The
low
intensity
ion
peak
m/e
203
(12%)
confirms
that
the
C47
substituent
is
a
methyl
group.
Therefore,
the
second
hydroxyl
might
be
at
C-16,
21
or
22.
Referring
to
literature,
one
may
suggest
that
compound
DM(
might
be
similar
to
brein
(12)
(3,21-dihydroxy
ursan-12-ene),
m.p.
216-7
°C,
acetyl
deriv.
m.p.
196
°C.
Compound
»G«:
m.p.
176-8
°C
(H
2
0),
yield
0.11%.
This
compound
is
a
flavonoid
glycoside;
gave
a
green
colour
with
FeCl
3
T.S.
and
magenta
red
with
Mg/HC1.
Under
UV
light
it
appears
as
a
purple
spot
changing
to
yellow
when
treated
with
ammonia
vapours.
UV
max.
(in
Me0H):
270,
300,
370
nm;
UV
shifts
after
addition
of
NaOMe:
280,
330,
405;
AlC1
3
:
280,
305,
350,
400;
alCl
3
/HCI:
280,
305,
350,
400;
Na
OAC:
280,
310,
390;
Na
OAC/H
3
B0
3
:
270,
305,
357.
Acid
hydrolysis
yielded
glucose,
rhamnose
and
an
aglycone
identi-
fied
as
kaempferol
by
direct
comparison
with
a
reference
sample.
The
UV
of
both
aglycone
and
glycoside
indicated
that
the
sugar
is
linked
at
C-3.
NMR
(6
ppm,
DMSO-d
6
):
1
(S,
3H,
CH
3
);
6.6,
6.4
(2
d,
2H,
J
=
2.511,,
H-8,
14-6);
8.2,
7.03
(2d,
2H,
J
=
8.5
H
z
,
H-2.6,
H-3.5).
The
2
signals
attributed
to
C-1
sugar
protons
(5.8
5.5
and
4.5
ppm)
suggested
that
it
is
a
diglycoside
which
was
confirmed
by
acid
hydrolysis.
Moreover,
that
appearance
of
a
strong
signal
at
6
1.0
for
3
protons
confirmed
that
the
sugar
part
is
rhamnose.
It
was
con-
cluded
that
the
sugar
is
a
3-0
rutinoside
from
the
chemical
shift
in
the
NMR
observed
for
C-1
and
C-CH
3
signals.
Therefore,
compound
»G«
is
kaempferol
3-0
rutinoside.
Moulting
activity
of
germanicol
and
its
acetate
(compounds
DA«
&
»B«):
Topical
application**
of
germanicol
and
its
acetate
(in
hexane)
on
the
dorsum
of
the
thorax
of
the
6th
instar
larvae
of
Spodoptera
(cotton-leaf
worm)
showed
that
22
and
51%
respectively,
pupae
failed
to
.
develop
into
adult
form.
The
eff
ec
ti
ve
d
oses
were
found
to
be
3µg
of
germa-
nicol
and
1µg
of
germanicol
acetate.
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1.
J.
M.
Watt,
M.
G.
Breyer-Brandwifk,
The
Medicinal
and
Poisonous
Plants
of
South
and
Eastern
Africa,
E.
S.
Livingstone
Ltd.,
2nd
ed.,
London
1962,
1393.
2.
G.
Ponsinet,
G.
Ourisson,
Phytochemistry
7
(1968)
89.
3.
A.
K.
Bedevian,
Illustrated
Polyglottic
Dictionary
of
Plant
Names,
Argus
and
Papazian
Presses,
1936,
270.
4.
E.
Claus,
V.
Tyler,
L.
Bray,
Pharmacognosy,
6th
ed.,
Lea
&
Febiger,
Philadelphia
1940,
459.
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L.
J.
Cassarett
(ed.),
T
ox
i
co
l
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the
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of
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Macmillan
Publishing
Comp.,
New
York
1975,
593.
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H.
N.
Khastgir,
B.
P.
Pradhan,
J.
Indian
Chem.
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(1967)
159.
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A.
Xorge,
J.
G.
D.
Domingues,
M.
Ma.
De
Lourdes,
G.
M.
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R.
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1184.
8.
J.
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2673.
9.
H.
Budzikiewicz,
J.
M.
Wilson,
C.
Djerassi,
J.
Amer.
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(1963)
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10.
Yamaguchi,
Spectral
Data
of
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Products,
Vol.
1,
Elsevier
Publishing
Comp.,
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10
*
11.
J.
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C.
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12.
I.
Heilbron
et
al.,
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of
Organic
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6th
ed.,
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and
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Ltd.,
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vol.
1,
295.
De
**
The
insect-moulting
test
was
kindly
p
er
f
ormed
by
Prof.
Dr.
F.
El
-Gayyar,
Plant
Protection
pt.,
Faculty
of
Agriculture,
Univ.
of
Alexandria.
106
S.
Khafagy
et
al.:
Steroid,
triterpenoid
and
flavonoid
constituents
of
Euphorbia
pulcherrima
Wild.
leaves,
Acta
Pharm.
Jugosl.
30
(1980)
103-107.
IZVOD
Steroidi,
triterpenoidi
i
flavonoidi
u
ligdu
Euphorbia
pulcherrima
WilId.
SAAD
KHAFAGY,
NAWAL
NAZM
I
\
NIIDEL
SALAM
i
AHMED
SELF
ELDIN
Eterski,
petroleterski
i
alkoholni
ekstrakti
li§da
Euphorbia
pulcherrima
Willd.
sadr2e
3
terpena,
2
steroida
i
3
flavonoida.
Primjenom
spektroskopskih
metoda
(UV,
IR,
NMR
i
MS)
utvrclene
su
strukture
sljededih
spojeva:
germa-
nikol-acetat
(sadrIaj
0.25%),
germanikol
(0.025%),
brein
(0.0054%)
0-sitosterol
(0.042%),
0-sitosteril-3-D-glukozid
(0.01%),
kemferol-3-O-glukozid
(0.013%),
kern-
ferol-3-O-rutinozid
(0.11%)
i
rutin
(0.012%).
Ispitivanja
germanikola
i
germa-
nikol
acetata
pokazala
su
da
obje
supstancije
djeluju
na
mitarenje
Spodoptera
littoralis.
Farmaceutski
fakultet,
University
of
Alexandria,
Egipat
107