Preliminary studies on the anti-schistosomal effect of Ammi majus L.


Abdulla, W.A.; Kadry, H.; Mahran, S.G.; el-Raziky, E.H., el-Nakib, S.

Egyptian Journal of Bilharziasis 4(1): 19-26

1978


Ammi majus L., a plant belonging to the family Umbellifereae was found to possess a certain antischistosomal effect in mice infected with S. mansoni. This was proved by applying two techniques, the worm burden and the Oogram change. Bergapten, a pure furocoumarin naturally present in Ammi majus was found to have certain schistosomicidal effect.

2
Egypt,
J.
Bilh.,
4,
No.
1,
19-26
(1977)
..f.
,;
-
,..:_______,
..
1
,
c
Preliminary
Studies
on
the
Anti-Schistosomal
Effect
of
Amini
majus
L.
W.A.,
Abdul
la,
*
H.,
Kadry**
S.
G.,
Mihr
an*,
El
-Raziky***,
E.H.
and
S.
El-Nalcib,*
*
C.I.D.
-
Research
- 74 -
ind
,
Control
Laboratories,
.
Cairo;
:`-'E?ypt
;'''"
Fdeulty
of
Plormacy.
Tanta
University
and
"''''
Faculty
of
Medicine,
Cairo
University.
i
A
ltNtr
m.t.tus
L.,
a
plant
belonging
to
tile
family
Umbellifereae
was
tifoand
to
possess
a
certain
antischistosomal
effect
in
mice
infected
with
S.
In:mold.
This
was
proved
by
applying
two
teehinques,
the
worm
burden
and
the
Oogram
change.
Bergapten,
a
pure
furocoumarin
naturally
present
in
Ammi
nz.ijus
was
found
to
have
certain
schistosomicidal
effect.
In
the
control
of
schistosomiasis,
an
endemic
disease
which
affects
the
majority
of
the
Egyptian
population
some
of
the
indigenous
plants
have
been
inc-
luded
in
our
program
to
be
screened
for
antibilharzial
activity.
Ammi
majus
L.,
a
herbaceous
indigenous
annual
plant
belonging
to
the
family
Umbelliferae
was
screened.
Bergapten
which
is
one
of
the
important
furocumarins
present
in.the
Ammi
majus
L.
has
been
also
screened
for
its
'antibilharzial
activity.
The
screening
method
of
the
antibilharzial
effect
in
mice
infected
with
S.
mansoni
depend
on
the
reduction
of
the,
worm
burden
and
the
Oogram
change.
Material
Samples
Ammi
majus
fruits
Petroleum
ether
extract
of
Ammi
majus.
-
The
residue
of
the
petroleum
ether
extract.
Water
decoction
of
Ammi
majus.
Equipment
'Soxhlet
apparatus.
Desaga
equipment
for
TLC.
iRota.evaporator.
Crude
Bergapten.
Pure
crystalline
Bergapten.
Tartar
emetic.
20
w
.A
,
ANDULLA
,
et
al.
Experimental
Preparation
of
extracts
a.
Petroleum
ether
extract
Amrni
majus
fruits
(400
g)
were
extracted
with
petroleum
ether
(40-60°)
in
a
continuous
extraction
apparatus
till
exhaustion
(2
ml
evaporated
to
dryn-
ess
left
residue).
b.
Petroleum
ether
residue
The
petroleum
ether
extract
was
concentrated
and
then
kept
in
the
refri-
gerator.
On
cooling,
a
greyish
-white
residue
was
formed,
fi
ltered
and
washed
with
petroleum
ether
till
the
filtrate
was
nearly
colourless.
The
residue
was
dried
at
60%
for
3
hr
and
weighed.
The
weight
of
the
dried
residue
was
7.5
g
i.e.,
1.87
%
of
the
drug.
2.
Ether,
chloroform
and
alcohol
96
%
extracts
These
extracts
were
prepared
in
the
same
way
as
the
forementioned
extract.
Each
time
the
drug
was
removed
from
the
soxhlet,
dried
a
and
then
repacked.
3.
The
water
-decoction
Ammi
majus
fruits
were
extracted
with
boiling
water
(5%),
for
30
min
and
fi
ltered.
The
fi
ltrate
was
kept
in
the
refrigerator.
4.
Bergapten
The
crude
Bergapten*
was
purified
by
several
crystallisation
from
boil-
ing
alcohol
95%,
m.p.
of
the
pure
crystalline
Bergapten
189-190°.
Analysis
a
:
Investigation
of
extracts
by
TLC
The
formentioned
samples
were
applied
to
silica
gel
G
plates
0.25
mm
thick,
previously
activated
by
heating
at
105°
for
30
min.
The
best
separation
was
achieved
by
the
solvent
system,
ethyl
acetate:
toluene:
water
(50.75:50)..
After
running,
the
plates
were
dried
at
room
temperature
and
the
spots
were
best
seen
and
localized
under
u.v.
(350
m)
as
illustrated
in
Fig.
1.
'By,
examining
the
chromatoplate
of
the
different
samples
one
can
conc-
lude
the
following
:
1.
All
extracts
except
the
water
decoction
showed
2
to
4
main
spots,
(a,
b,
c,
d)
three
of
which
of
a
greenish
-yellow
fl
uorescence
and
one
of
which
is
identical
in
R
f
value
and
colour
as
that
of
Bergapten.
*
kindly
supplied
by
Memphis
Firm.
EGYPT.
J.
BILE
;
4,
No.
1
(1977)
ANTI-SCHISTOSOMAL
EFFECT
OF
d.IIkII
MAJUS
L.
21
Sil
ica
gel
G
Ethyl
Acetate:Toluene:Water
50
75
50
d
O
C7
c
9Y
2
3
4
5
6
7
1.
chloroform
extract
2.
Petroleum
ether
extract
3.
Petroleum
ether
residue
4.
Bergatpen
5.
Water
decoction
6.
Alcohol
extract
7.
Ether
extract;
g.y.
greenish
yellow
fl
uorescence
a
b,
c,
g.y.
fluorescent
spots.
2.
The
petroleum
ether
and
ether
extracts
showed
nearly
the
same
no.
of
t
spots
(7-9)
3.
The
water
decoction
showed
one
main
spot
greenish
-yellow
in
flur-
escence
and
corresponding
in
R
E
value
and
colour
to
that
of
Bergapten.
4.
The
pure
Bergapten
showed
one
greenish
-yellow
fl
uorescent
spot.
b.
Chemical
tests
for
comnarins
The
presence
of
flurocoumarins
(Coumarins)
in
all
samples
especially
that
of
petroleum
ether,
was
confirmed
by
the
usual
coumarin
reactions(•).
All
sampls
gave
positive
tests.
Anti-schistosomal
Screening
1.
Antischistosomal
investigations
of
the
extracts
The
investigations
were
carried
out
to
determine
the
antibilharzial
effect
of
the
different
extractive
of
Ammi-majus
on
experimentally
infected
albino
mice.
EGYPT.
J.
BILE.
,
4,
No:
1
(1977)
22
.A
,
ABDULLA
,
et
aV.
'The
samples
used
in
this
investigation
were
as
follows:
1.
The
petroleum
ether
residue
of
the
extract.
2.
water
decoction.
3.
Bergapten.
The
study
of
the
anti-schistosoma/
activity
of
these
samples
depends
on
the
reduction
of
the
worm
-burden
and
the
Oogram
change.
Albion
mice
each
weighing
from
15-20
were
infected
with
1C0
cercaria
of
Sanansoni
per
animal
adopting
the
method
of
Moore
et
al.
(
2
).
The
animals
were
then
divided
into
4
groups:
The
1st
group:
infected
but
non
-treated
(control)
The
2
nd
group:
infected
and
treated
with
5%
Ammi
majus
decoction
400.
mg
kg/day
per
as
during
6
successive
days.
The
3
rd
group:
infected
and
treated
with
petroleum
ether
residue
15
mg/kg
day
orally
in
6
successive
days.
The
4
th
group:
infected
and
treated
with
tartar
-emetic
15
mg/kg/day
].P.
during
6
successive
days.
Methods
1.
Wection
of
mice
The
infection
was
done
by
adopting
the
method
of
Moore
et
al.
(
2
)
100
cercaria
of
S.
mansoni
were
injected
p.c.
in
each
mice.
2.
Perfusion
technique
The
recovery
of
the
adult
worms
was
achieved
by
using
the
method
of
Duval
and
Dewitt(
3
).
3.
Oogram
technique
The
evaluation
of
the
schistosomicidal
effect
was
ascertained
by
the
Oogram
technique
after
Pellegrino
and
Paria(
4
).
*
A
suspension
of
the
residue
in
a
concentration
of
0.15%
was
prepared
using
glycerin,.
Tween
80
(5%)
and
water
to
the
required
volume.
EGYPT.
J:
BILH.,.
4;
No:
1
(1977)
ANTI-SCHISTOSOMAL
EFFDCT
OF
1UIMI
MAJUS
L.
23
The
screening
of
the
schistosomicidil
effect
using
infected
albino
mice,
'usually
employs
the
following
technique
adopted
by
Lambert
(
s
),
Pellegino
:and
Katz
(
6
).
These
techniques
depend
on
the
following
Criteria:
1.
Increase
of
the
survival
time
of
treated
mice.
2.
Reduction
of
the
eggs
from
Faeces
after
treatment.
3.
Hepatic
shift
of
schistosomes.
.4.
Reduction
of
the
number
of
worms
-burden
recovered
from
treated
mice
group
as
compared
with
the
untreated
one.
.5.
Change
in
the
number
and
character
of
eggs
in
the
intestine
(00grain)
provids
a
simple,
sinsitive
and
reliable
criteria.
The
presnt
investigation
depend
on
the
last
two
criteria:
Raduction
of
the
worm
-burden
(Schubert)(
7
).
Oogram
technique
by
Pellegrino
(
4
).
TABLE
1.
The
anit-schistosomal
effect
of
Amin/
mains
on
infected
mice
Group
.Control
Ammi
InCljliS
decoction
Dose
mg/kg
Oogram
change
No.
of
worm
burden/mouse
400
0/20
7.2
2.7
5.7
0
Total
21.3
Worm
-burden
reduction
wa%
0.0%
6/10
2.4
Petroleum
ether
residue
Tartar
-emetic
15
7/10
15
10/10
6.0
2.6
0.7
3.0
0.1
1.4
0.9
1.2
5.9
10.3
5.1
72.3
49.3
'76.0
11
Preliminary
Investigation
of
the
antischistosomal
effect
of
Bergapten
Albino
mice
(50),
each
weighing
from
15-20
g
were
infected
with
100
cercarea
of
ganansoni
per
animal
adopting
the
method
of
Moore
et
al
e).
The
animals
were
then
divided
into
5
groups,
each
of
10
mice.
The
first
group:
infected
but
non
treated
(control).
The
second,
third
and
fourth
infected'
and
treated
with
Bergapten
suspension*,
10,15,20
mg/kg,
day
during
,
.6
susaessive
days.
The
fifth:
infected
and
treated
with
tartar
emetic
15
mg/kg/day
I.P
for
6
sucessive
days:'
ZGYPT
.
.
J.
4,
No.
1
(1977)
24
.A
,
ABDULLA
,
et
al:
TABLE
2.
The
anti-schistosoma/
effect
of
Bergapten
on
the
infected
albino
mite.
No.
of
worm
burden/mouse
Group
Dose
nighkg
Oogram
change
Warm
-burden
reduction%
0
0
c
0
+
T
Control
6;10
7.5
4.6
1.7
15.5
0.0
Bergapten
10
6/10
4.4
2.3
0.4
7.5
51.6
Bergapten
15
7/10
1.0
0.3
2.0
5.3
65.8
Bergapten
20
9/10
3.2
0.8
0.6
5.2
66.4
Tartar
emetic
15
10/10
1.4
0.5
0.3
2.5
83.8
*
A
suspension
of
Bergapten
in
the
concentrations
of
0
,
1
to
0
.
2
%
was
prepar
ed
usin
g.
Glycerin,
Tween
80
(5%)
and
water.
to
the
required
volume.
Results
and
Discussion
FrOm
the
above
data,
Table
1,
it
is
obvious
that
the
samples
under
investiga.
tion
posess
a:
certain
anti-schistosomal
effect
if
given
orally
to
mice
in
a
dose
of
15
mg/kg/day
in
case
of
the
petroleum
other
residue
(furococumarin)
and
orally
in
a
dose
of
400
mg/kg/day
in
case
of
Ammi
niajus
decoction.
Although,
the
Oogram
change
in
case
of
the
decoction
is
not
significant,
the
worm
-burden
reduction
is
comparatively
high
if
compared
with
that
of
the
petroleum
other
residue
and
standared
Tartar
-emetic.
The
petroleum
ether
residue,
however,
shows
an
Ougram
change
of
7/10.
On
the
other
hand,
promising
anti-schistosomal
results
were
obtained
by
using
Bergapten,
a
substance
belonging
to
the
furocoumarin
group.
From
Table
2,
it
is
clear
that
by
increasing
the
dose
of
Bergapten,
an
increase
in
the
effect
was
observed.
A
dose
of
20
mg/kg
produces
a
significant
Oogram
change
of
9/10
and
a
worm
-burden
reduction
of
66.4
%.
The
schistosomicidal
effect
of
Ammi
majus
L.
might
be
therefore,
attributed
io
the
presence
of
certain
constituents,
the
furocoumarins.
Their
presence
to
all
samples
was
confirmed
by
chemical
and
chromatographic
analysis..
IWYPT.
J.
BILII.
4,
Na.
1•
(1977)
ANTI-SCHISTOSOMAL
EPFTICT
OF
AMMI
MAJUS
L.
2,5
Summary
Ammi
majus
L.
in
the
form
of
a
water
decoction
or
a
petroleum
ether'
extract,
was
found
to
have
a
certain
antischistosomal
effect
in
mice.
The
worm
burden
reduction
and
the
Oogram
change,
caused
by
the
decoction
was
72%
and
6/10
respectively.
In
case
of
the
petroleum
ether
extract,
the
Oogram
change
and
the
worm
-burden
reduction
was
7/10
and
49
-
respectively.
On
the
other
hand,
Bergapten,
a
furocoumarin,
in
the
dosage
of
20
mg/kg,
proved
to
have
a
schistosomicidal
effect,
represented
by
9/10
Oogram
change.
and
66%
worm
burden.
References
1.
Feigl,
F.,
"Spot
Tests
in
Organic
Analysis",
6'l
edition,
Flaevier
Publishing
Co._
New
York,
N.Y.,
P.
584
(1960).
2.
Moore,D.V.,
Yolles,
T.K.
and
Melenecy,
H.E.,
J.
Parasitol.,35,
156
(1949).
3.
Duval,
R.H.
and
Dewitt,
W.B.,
J.
Trop.
Med.
Hyg.
16,
483
(1967).
4.
Pellegrino,
J.
and
Faria,
J.,
Am.
J.
Trop.
Med.
Ilyg,
14,
363
(1965).
5.
Lambert,
C.R.,
Trans.
Roy.
soc
Trop.
Med.
Hyg.
61,
559
(1967).
6.
Pellegrino,
,J.
and
Katz,
N.,
Ann.
N.Y.
Acad.
Sci.
160,
429
(1969).
7.
Schubert
M.,
Am.
J.
Trop.
Med.
28,
121
(1948).
(Received
24/2/1976)c.
MGM'.
J.
BILE.,
4,
No.
.1
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ABDULLA;
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