Pakistan J. Med. Res.
Vol. 42 No.3, 2003
Al-Jabre
S*,
Al-Akloby
OM*,
Al-Qurashi
AR**,
Akhtar
N**,
Al-Dossary
A*,
Randhawa
MA***
Department
of
*Dermatology,
**
Microbiology
and
***Pharmacology,
College
of
Medicine,
King
Faisal
University,
Dammam,
Kingdom
of
Saudi
Arabia
SUMMARY
Nigella sativa (N. Sativa) seed called as ‘Habbah Al-Sauda’ in Arabic, is commonly used as a natural remedy for many ailments over 2000 years and frequently added to bread and prickles as a flavouring agent. Recently, many active principles have been isolated from N. Sativa including thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellidine, nigellimine-N-oxide and alpha-hedrin. Besides, many other pharmacological effects, activity of N. sativa oil, ether extract and its active principle, thymohydroquinone, has been reported in the literature against a number of bacteria (including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and yeast (Candida albicans). Therefore, we thought that N. sativa or some of its active principles might have useful action against Aspergillus as well, which commonly causes opportunistic infection in immune deficient individuals. We studied the growth of Aspergillus niger in dermasel agar containing 0.25, 0.5, 1 & 2 mg/ml of thymoquinone; drmasel agar with selective supplement and dermasel agar alone. There was 0, 25, 77.1 and 100% inhibition of A. niger with 0.25, 0.5, 1 & 2 mg/ml of thymoquinone on day 7, giving MIC as 2 mg/ml. We hope our study will promote further investigations to determine usefulness of thymoquinone or N. sativa in the treatment and prevention of Aspergillosis.
INTRODUCTION
Nigella sativa, one of the Ranunculaceae, commonly grows in the Middle East, Eastern Europe and Middle Asia. In Arab countries it is commonly known as ‘Habba Al-Sauda’ or ‘Habba Al-Baraka’. N. Sativa seed is frequently added to bread and prickles as a flavouring agent and used as a natural remedy for many ailments over 2000 years 1, 2.
Recently
many
active
principles
have
been
isolated
from
N.
sativa,
including
thymoquinone,
thymohydroquinone,
dithymoquinone,
thymol,
carvacrol,
nigellicine,
nigellimine-N-
oxide,
nigellidine
and
alpha-hedrin
3,
4,
5,
6,
7
&
8.
Over
the
last
few
decades,
a
number
of
pharmaco-therapeutic
effects
of
various
extracts
of
N.
sativa
and
some
of
its
active
principles
have
been
investigated,
using
modern
scientific
techniques,
and
recently
reviewed
by
Randhawa
and
Al-Ghamdy
9.
Anti-bacterial
effect
of
the
phenolic
fraction
of
N.
sativa
oil
was
first
reported
by
Topozada
et
al10.
Thymohydroquinone
was
later
isolated
4
and
found
to
have
high
activity
against
gram-positive
microorganisms.
Later,
the
diethyl-ether
extract
of
N.
sativa
was
reported
to
inhibit
Staphylococcus
aureus,
Pseudomonas
aeruginosa
and
Escherichia
coli.
It
also
showed
synergistic
effect
with
streptomycin
&
gentamycin
and
additive
effect
with
spectinomycin,
erythromycin,
tobramycin,
doxycycline,
chloramphenicol,
nalidixic
acid,
ampicillin,
lincomycin
and
co-trimoxazole.
In
addition,
the
extract
also
inhibited
a
pathogenic
yeast,
Candida
albicans11.
Recently,
crude
extracts
of
N.
sativa
were
reported
to
have
a
promising
effect
on
multi-resistant
organisms
including
gram-positive
and
gram-
negative
bacteria12.
In
view
of
the
wide
spectrum
of
anti-microbial
activity
of
N.
sativa,
we
thought
it
might
have
useful
activity
against
some
fungi,
like
Aspergillus,
besides
Candida
albicans.
Aspergilli
comprise
a
group
of
rapidly
growing
hyaline
moulds,
that
are
widely
distributed
in
the
environment
and
are
common
contaminants
of
clinical
laboratories.
They
commonly
cause
opportunistic
infection
in
humans.
Aspergillosis
may
present
as
well
defined
clinical
syndromes
involving
a
variety
of
sites
and
organ
systems:
pulmonary,
disseminated,
central
nervous
system,
cutaneous,
endocardial
and
nasoorbital.
It
almost
exclusively
occurs
in
patients
who
are
immuno-suppressed
or
neutropenic,
particularly
in
transplant
recipients
and
those
with
leukemias
or
lymphomas.
Aspergillosis
is
also
reported
in
corticosteroid
use,
intravenous
drug
abuse
and
later
stages
of
AIDS.
Of
the
some
700
Aspergillus
species
described,
only
19
have
been
cited
as
causing
human
infections;
of
these
only
4
are
recovered
with
any
frequency
in
hospitalized
patients:
Aspergillus
fumigatus,
A.
flavus,
A.
niger
and
A.
terreus
13.
MATERIALS
AND
METHODS
Strains
of
A.
niger
were
isolated
from
contamination
in
petri-dishes
being
used
for
some
culture
sensitivity
tests
in
the
Department
of
Microbiology,
College
of
Medicine,
King
Faisal
University,
Dammam.
The
specimens
were
sub-cultured
on
Dermasel
agar
(Oxoid)
and
incubated
at
30o
C
for
7
days.
The
growth
was
identified
as
A.
niger
by
microscopy
after
staining
with
lactophenol
cotton
blue.
Thymoquinone
was
dissolved
in
small
amount
of
sterile
distilled
water
and
then
mixed
with
sufficient
presterilized
dermasel
agar
to
obtain
2
mg/ml
of
thymoquinone.
Serial
dilutions
were
then
made
to
get
thymoquinone
1,
0.5
and
0.25
mg/ml.
The
isolates
of
A.
niger
were
subcultured
on
three
sets
of
culture
media.
Set
1:
dermasel
agar
only,
Set
2:
dermasel
agar
with
dermasel
selective
supplement
(containing
cycloheximide
200
mg/500
ml
and
chloramphenicol
25
mg/500
ml)
and
Set
3:
dermasel
agar
with
0.25,
0.5,
1
&
2
mg/ml
of
thymoquinone.
Four
petri-plates
were
inoculated
in
each
set.
A
mycelial
disc,
5
mm
in
diameter,
cut
from
the
periphery
of
5-7
day
old
cultures
of
A.
niger
in
dermasel
agar
was
aseptically
inoculated
onto
different
sets
of
media.
The
inoculated
plates
were
incubated
at
30o
C.
The
cultures
were
examined
on
the
3rd,
5th
and
7th
day
and
results
interpreted
by
visual
impression
as
well
as
by
measurement
of
the
mean
diameter
of
the
growth
of
Aspergillus14.
The
percentage
inhibition
of
A.
niger
with
different
concentrations
of
thymoquinone
was
then
calculated
by
taking
its
growth
in
non-drug
dermasel
agar
as
100%.
The
growth
of
A.
niger
in
dermasel
agar
with
the
selective
supplement
was
taken
as
active
control.
RESULTS
There
was
0,
25,
77.1
and
100%
inhibition
of
A.
niger
with
0.25,
0.5,
1
&
2
mg/ml
of
thymoquinone
on
day
7,
giving
MIC
as
2
mg/ml
(table
1),
while
with
the
selective
supplement
the
inhibition
was
only
32.7
%.
A.
niger
covered
the
entire
area
of
perti-plates
(9
cm
diameter,
taken
as
100%)
when
inoculated
in
dermasel
agar
alone
on
day
3
to
7
.
Table
1:Percentage
inhibition
of
the
growth
of
Aspergillus
niger
with
different
concentrations
of
thymoquinone
on
Day
3,
5
and
7.
|
Thymoquinone
mg/ml |
%
inhibition
of
A.
niger
growth |
||
|
Day
3 |
Day
5 |
Day
7 |
|
|
0.
25 |
25 |
Nil |
Nil |
|
0.
5
|
76.8 |
55.4 |
25 |
|
1 |
100 |
91.8 |
77.1 |
|
2 |
100 |
100 |
100 |
The
mean
diameter
of
the
growth
of
A.
niger
in
dermasel
agar
containing
various
concentrations
of
thymoquinone
and
dermasel
agar
with
selective
supplement
is
given
in
table
2a
&
2b,
respectively.
Table
2:
Growth
of
Aspergillus
niger
in
dermasel
agar
containing
different
concentrations
of
thymoquinone
and
in
dermasel
agar
with
selective
supplement.
Dermasel
agar
with
thymoquinone
|
Concentrations mg/ml |
Mean
diameter
+
SD
(cm)
of
A.
niger
growth
(n
=
4) |
||
|
Day
3 |
Day
5 |
Day
7 |
|
|
0.25 |
+++(*) |
++++(**)
|
++++ |
|
0.5 |
2.09
+
0.2 |
4.01
+
0.49 |
+++
|
|
1 |
Nil |
0.74
+
0.43 |
2.06
+
0.8 |
|
2 |
Nil |
Nil |
Nil |
Dermasel
agar
with
selective
supplement
|
Mean
diameter
+
SD
(cm)
of
A.
niger
growth
(n
=
4) |
||
|
Day
3 |
Day
5 |
Day
7 |
|
2.05
+
0.11 |
4.35
+
0.39 |
6.06
+
0.11 |
*
Growth
of
A.
niger
covering
¾
of
the
petri-plate
**
Growth
of
A.
niger
completely
covering
the
petrti-
plate
(9
cm),
taken
as
100%.
DISCUSSION
N.
Sativa
has
been
used
for
many
ailments
in
ancient
Greek,
Indian
and
Arabic
medicines.
For
example,
Ibne-Sina
recommended
the
use
of
N.
sativa
for
the
treatment
of
various
microbial
infections
of
skin
(warts,
collar-stud
abscess
and
other
abscesses,
chronic
fungal
infections
such
as
ring
worm),
eye
and
gastrointestinal
tract
as
well
as
for
the
expulsion
of
worms
from
the
intestines15.
In
the
present
study
we
observed
a
dose
related
anti-fungal
effect
of
thymoquinone,
an
active
principal
of
N.
sativa;
0.25,
0.5,
0.1
and
0.2
%
thymoquinone
inhibited
0,
25,
77.1
and
100
%
of
A.
niger
with
0.25,
0.5,
1
&
2
mg/ml
of
thymoquinone
on
day
7.
Earlier,
25-400
micrograms
of
diethyl-ether
extract
per
disc
of
N.
sativa
seed
has
been
reported
to
inhibit
a
concentration
dependent
growth
of
Gram-positive
and
Gram-negative
bacteria11.
Moreover,
inhibition
in
the
growth
of
coliforms,
staphylococci
and
yeasts/moulds
in
the
processed
cheese
spread
by
0.1
-
0.3%
hexane-extracted
N.
Sativa
oil
has
also
been
reported
16.
There
was
some
growth
of
A.
niger
in
dermasel
agar
with
selective
supplement
on
day
3,
5
&
7
as
shown
in
table
2b.
Which
shows
that
the
selective
supplement
(containing
cycloheximide
200
mg/500
ml
and
chloramphenicol
25
mg/500
ml)
failed
to
completely
inhibit
the
growth
of
A.
niger.
We
hope
our
study
will
promote
further
investigations
to
determine
usefulness
of
N.
sativa
in
the
treatment
and
prevention
of
Aspergillosis
and
possibly
other
local
and
systemic
fungal
infections
in
immuno-compromised
patients.
CONCLUSION
Thymoquinone,
an
active
principle
of
Nigella
sativa,
effectively
inhibited
the
growth
of
Aspergillus
niger
(MIC
2mg/ml).
ACKNOWLEDGMENT
We
gratefully
acknowledge
Dr.
Mastour
S.
Al-Ghamdi,
Vice
Dean
(Academic
Affairs),
College
of
Medicine,
King
Faisal
University,
Dammam,
Saudi
Arabia,
for
the
provision
of
thymoquinone
base
for
our
study.
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