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www.LJMR.com.ly5365-ISSN: 2312 Res an J MedyLib
Vol. 8, No. 1: 2014 59
Studies of the antimicrobial activity of Black Seed Oil from Nigella
Sativaon Staphylococcus aureus and Escherichia coli
Najib M. Sufya
1*
, Rehab R. Walli
2
, Fatima M. Wali
1
, Marwa S. Alareiba
1
and Basma M. Doro
1
1
Departments of Microbiology and Immunology and
2
Biochemistry and Clinical Biochemsitry, Faculty
of Pharmacy, University of Tripoli, Tripoli, Libya
*Correspondance to najibsufa@yahoo.com
Abstract: It is well known that various plants (whole or some parts) are of definite
and useful use for human benefit and well fare. One of these benefits is the ability of
many plant seeds, fruits and different parts of exerting antimicrobial activity. The aim
of theses uses is the treatment of various infectious diseases. This would be clearly
understood that such plant may display a role in inactivating the underlying causes of
diseases. These are to include various bacteria and may some of fungi if not extended
to include viruses. Methods: In this study, commercially available black seed oil was
collected from the public market in Libya. The latter was reconstituted with sterile
deionised water to various concentrations and then tested for the antimicrobial activity
using both Gram positive Staphylococcus aureus (ATCC 6538) and Gram negative
Escherichia coli (ATCC C600). Antimicrobial activity evaluations were performed by
the cub-cut agar method and the killing curve (survival curve) method. A further
investigation was to understand the mechanism of action of black seed oil and how it
inactivates bacterial cells was carried out. All antimicrobial investigation studies were
referenced with the efficacy of 5% (v/v) phenol. Results: A distinctive zone of
inhibition was detected at various concentrations of the black seed oil. This was
clearly demonstrated against both S. aureus and E. coli. Survival curve experiments
have demonstrated that S. aureus was more susceptible than E. coli. This was clearly
illustrated in the reduction of log of survivors. This was displayed by 1.7 log kill in
the case of S. aureus whilst 0.9 log of kill for E. coli. Further spectrophotometric
experiments displayed a detection of bacterial organelles over a wave length at A
350nm
.
This absorbance was followed by the significant reduction in the concentration of
theses organelles. Conclusion: The black seed oil has shown an antimicrobial activity
that able to inactivate both S. aureus and E. coli. Such activity would be elucidated in
its ability to target bacterial cell envelope causing its damage. This might result in
bacterial lysis as a result of losing bacterial components and/or target bacterial
organelles. Black seed oil, therefore, in addition to its role as a part of food
constituents would be of a great benefit for the treatment of various infectious
diseases that are of bacterial origin
Introduction
Many of antimicrobials that are used
today are related in terms of natural
structure. In many cases, chemically
synthesized drugs have obtained their
modulated structure from nature and/or
modified from herbal sources. With
better techniques and knowledge, syn-
thesized compounds from natural
plants will most likely also lead to
better results in the field of antimic-
robial therapy and the control of
infectious diseases (1, 2).
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Vol. 8, No. 1: 2014 60
Plants are rich in a wide variety of
phytochemical products, such as
tannins, terpenoids, alkaloid sand
flavonoids, which have been found in
vitro to have antimicrobial properties
(3).
Black seed is an annual herbaceous
plant that is believed to be indigenous
to the Mediterranean region but has
been cultivated into other parts of the
world including Arab dessert, Northern
Africa and some parts of Asia. Black
seed originates from the common
fennel flower plant (Nigella sativa) of
the buttercup (Ranunculaceae) family.
Nigella sativa and its black seed are
known by other names, varying
between places. Some named it black
caraway others named it black cumin
(Kalonji) or even coriander seeds.
Nevertheless, this is Nigella sativa,
which has been known and used from
ancient times (3-5) in curing diseases.
The Prophet Muhammad (Peace be
Upon Him) said in his divine wisdom
speech about the Black seed "Use this
Black seed; it has a cure for every
disease except death" (SahihBukhari).
Black seed unquestionably has a
positive and stabilizing effect on the
human body. This would result in
supporting the human health and
welfare that may go beyond curing
simple disorders such as skin diseases
and allergies. It would extend to
support the immune system that
intuitively helps the body in curing and
eliminating the underlying cause of
many diseases (6, 7), purify the blood,
protect against irritants and support
bodies during recovery (2, 4, 5). Many
studies have displayed the antimic-
robial activity of Black seed oil against
variety of microorganisms. Such effect
was displayed against E. coli,
Salmonellaspp, Shigella spp. and some
strains of Vibrios bacteria.
This was also extended to inhibit the
proliferation of some fungal growth (8-
10). Several studies displayed that the
black seed oil contains over 100
valuable nutrients. It contains 21%
protein, 38% carbohydrates and 35%
plant fats and oils. Among these
constituents are the Thymoquinone,
Nigellone, and some fixed oils. Other
ingredients are to include linoleic acid,
oleic acid, calcium, potassium, iron,
zinc, magnesium, selenium, vitamin A,
vitamin B, vitamin B
2
, Niacin, and
vitamin C (11, 12). These constituents,
in addition to others phenolics and
flavonoids display a number of
medical uses. For examples: treating
urinary and respiratory tract infections,
relieving cough and digestive problems
and possible activity against
methicillin resistant Staphylococcus
aureus (13-15). Its effect extended to
display antipa-rasitic action and insect
repellent activities, as well as relieve
vomiting and diarrhea, and flatulent
colic, and to help counteract and
relieve from the griping action (10, 16,
17).
To the best of our knowledge, no
previous studies were reported about
the antimicrobial activity of black seed
oil in Libya at least. Thus this study
was aimed to evaluate the antimic-
robial activity of the commercially
black seed oil and understanding its
possible effect on challenged bacteria.
Materials and methods
Chemicals and Preparations: Black
seed oil at different concentrations
(v/v) of 10%, 30%, 50%, 70% and
90% were prepared into phosphate
buffer saline (PBS) (0.01 M) where the
pH is adjusted to 7.0 and kept at cold
room. Solubility was performed first
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Vol. 8, No. 1: 2014 61
into 0.02% DMSO (18) then volume
completed by PBS to desired concentr-
ation. Nutrient broth and Nutrient agar
media used to grow bacterial strains.
All dehydrated media were obtained
from BDH (Poole, U.K.). All reagents
were of the highest grade of purity
(Aristar
®
) of the BDH suppliers.
Microorganism and Culture Maint-
enance: Staphylococcus aureus
(ATCC 6538) and Escherichia coli
(ATCC C600) strains were maintained
in long term preserve system. This was
prepared into the lab using 10% (v/v)
glycerol in nutrient broth media. The
stock was dispensed into 1 ml aliquots
and stored at - 80
o
C till required. Fresh
cultures were prepared from frozen
stocks prior to every experimental
procedure. This was performed by
streaking, using sterile loop, freshly
prepared nutrient agar plates which
were then incubated at 37° C for 18hrs,
from which a colony was used to run
the experiments.
Antimicrobial Susceptibility Exper-
iments: All antimicrobial susceptibility
experiments were conducted by
transferring 5ml of the tested strains
grown at late exponential phases (O.D.
of A
470nm
A0.9 and 1.2, respectively)
for S. aureus and E. coli strains. This
will give bacterial suspension of 1x10
8
cfu per ml. Absorbance was measured
using the spectrophotometer (PU 8675
Vis spectrophotometer), Philips,
Germany. The Black seed oil was
added, to the bacterial suspension, to
give final concentrations (v/v) of 10%,
30%, 50%, 70% and 90%. A sample of
volume 1 ml was taken every 1 hr
intervals up to 6 hrs and then at 24 hrs
where the serial dilutions were perfor-
med and the viable count calculated
using the spread plate technique.
Viable count was performed by
transferring 100 µl of each sample
taken onto predried nutrient agar plates
where then incubated for 24 hrs and
counted for viable cfu per ml. Data
were expressed as the log of survivors
against exposure time. These were
used to set up the survival curve (dose
response curve). All experiments were
performed in triplicates and the mean
was used throughout this study.
Experiments were also designed to
understand the effect of the Black seed
oil on which part of the bacterial cell.
This was performed by collecting
samples of the exposed populations
every 1hr up to 6 hrs and then at 24 hrs
to measure the absorbance at the wave
length 350 nm. Data were expressed as
the absorbance reading A
350nm
against
the exposure time. This will indicate,
based on the absorbance reading,
which part of the bacterial cell was
targeted by the Black seed oil. In
preliminary evaluation of the anti-
microbial activity of the Black seed oil,
cup cut agar method was performed. A
5% (v/v) phenol was used and data
were compared to that of Black seed
oil.
Results
Antimicrobial Susceptibility Experiments
The cup-cut agar method was used as a
preliminary step for the qualitative
evaluation of the antimicrobial activity
of the Black seed oil at the concentr-
ations of 10%, 30%, 50% and 70%
(v/v). In the case of E. coli, activity
was not clearly observed. Black seed
oil concentrations were, therefore,
elevated to 90%, whereas a clear zone
of inhibition on E. coli was then
determined. On the other hand,
inactivating activity of black seed oil
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Vol. 8, No. 1: 2014 62
was clearly observed at 10%, 30%,
50% and 70% (v/v) on S. aureus. The
zone of inhibition was related to the
extract concentration (Table 1).
It should be mentioned that the S.
aureus showed a remarkable susceptib-
ility over that shown by the E. coli.
This was clearly noticed on the diam-
eter of the zone of inhibition. In gener-
al both bacteria were susceptible to the
effect of the Black seed oil at different
concentrations, where the E. coli
showed susceptibility only at the 90%
concentration (Table 1).
Table 1: Activity of the Black seed oil on S.
aureus and E .coli expressed as zone of
inhibition in mm.
* 5% phenol showed Zone of inhibition (mm) = 26
** 5% phenol showed Zone of inhibition (mm) = 25
All experiments performed in triplicate and repeated
three times, where the mean used to plot the data
As the Black seed oil has proved to
display a significant antimicrobial
activity, it is very important therefore
to reference this activity to one of the
most potent agents. In this context the
phenol 5% (v/v) was the reference
antimicrobial agent used (Table 1). In
the case of S. aureus, the zone of
inhibition of 70% oil represented about
1.5 fold of the phenol one. This was
38mm for the extract and 26mm for the
phenol. The zone of inhibition for
50%, 30% and 10% concentrations
were 25 mm, 24 mm and 18 mm,
respectively. In the case of E. coli, its
susceptibility was also observed. This
was 13 mm for the oil (90%) compared
to that of the 5% phenol (25 mm). This
represents about half fold of the phenol
activity (Table 1). No activity was
detected for the concentrations 10, 30,
50 and 70%.
This study was also designed to evalu-
ate the antimicrobial activity of Black
seed oil quantitatively by determine the
remaining of survival cells after expos-
ure to different concentrations over
period of times. Results in Figures 1
and 2 showed antibacterial activity of
the Black seed oil 70% and 90%
towards S. aureus and E. coli,
respectively.
Figure 1: Effect of Black seed oil on S.
aureus. ●control, ○ 70% black seed oil.
In the case of S. aureus, the level of
bacterial kill was clearly demonstrated.
This was about 1.7 log reduction
compared to the control over 24 hrs
exposure (Fig. 1). The level of
reduction in the cfu per ml was from
1.76 x 10
9
to 4.6 x 10
7
cfu per ml. This
displayed that about 97% of the
population were killed. Similarly, E.
coli populations were also susceptible
but with lesser extent. This was
demonstrated in the rate of bacterial
survivals over 24 hrs exposure to 90%
of the agent concentration. The redu-
ction in the E. coli survivals was meas-
ured by about 0.9 log reductions (Fig.
2).
Bacteria
Concentration (v/v)
10
%
30
%
50
%
70
%
90%
Zone of Inhibition in mm
S. aureus*
18
24
25
38
Not
applied
E. coli**
No
No
No
No
13
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Vol. 8, No. 1: 2014 63
This indicated that about 80% of the
population was killed and 20%
survived. It is therefore clear that the S.
aureus population is more susceptible
to the effect of the black seed oil than
that of the E. coli one. It is very
important to notice that the bacterial
population used to investigate the
antibacterial activity of the Black seed
oil was collected specifically at the late
exponential phase for both E. coli and
S. aureus (Figs. 3 and 4). This was
intended to avoid both the exponential
phase that characterized by its
susceptibility to the effect of
antimicrobials and the stationary phase
where the bacteria are already started
to die. This will impact that the
obtained results was mostly reflect the
action of the oil minimizing the
incidence of biological and physio-
logical variations of the bacteria.
Further investigation in this study was
aimed to understand the possible antib-
acterial action of the Black seed oil on
the bacterial cell. An experiment of
measuring the absorbance of chall-
enged bacterial populations at low
wave length ranged at A
350nm
was
designed. This was aimed to measure
the absorbance of bacterial structure
when small bacterial organelles such as
RNA, DNA and ribosomes are
suspended and released into the
medium.
Figure 2: Effect of Black seed oil on E. coli.
●control, ▫ 90% black seed oil.
In such instances, if reading was
detected at such low wave lengths, this
would therefore indicate the possibility
of antibacterial agents has an effect on
the bacterial envelope and bacterial
organelles were released and absor-
bance was detected.
Figure 3: Growth curve of S. aureus.
Figure 4: Growth curve of E. coli
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Vol. 8, No. 1: 2014 64
In this study, detections of light
absorbance at A
350nm
were succeeded.
This would intuitively displayed that
black seed oil managed to distort and
interfere with the cell envelop for both
S. aureus and E. coli to various
extents. All detections were explained
in Figure 5 over 24 hrs exposure. All
samples were collected alongside with
the viable count sampling for colony
forming unit measurements. Figure 5
has showed that the absorbance was
relatively high at the first 3 hrs
exposure compared to those at next
exposures up to 6hrs whilst, on the
other hand, it was higher than that at
24 hrs exposure. The degree of varia-
tion was higher in the case of S. aureus
than that of the E. coli by about 10
folds (Figure 5). This would clearly
display that Gram positive bacteria are
more susceptible to be inactivated by
black seed oil, which would be
explained in the variation in their cell
envelope structure. These data are in
consistence with the data collected
above (Figs. 1, 2).
Figure 5: Optical density measurements for
challenged S. aureus(○) and E. coli(▫) populations
to70% and 90% black seed oil respectively at A
350nm
over 24hrs exposure
Discussion
When comparing the activity of the
Black seed oil to the most powerful
antimicrobial and biocide, data shown
that the oil potency was variable based
on the type of bacteria. This was about
1.5 and 0.5 folds for the S. aureus and
E. coli respectively, of that of phenol.
This would indicate that such potency
could be the reason behind its
acceptability for community and public
use and, in treating various cases of
bacterial infections. Similarly, the
ability of the black seed oil for
observed inactivation of the population
over 24 hrs of exposure is another
evidence of its antibacterial activity.
This was clearly demonstrated in the
survival curve method. The survivors
fractions recovered on the plates were
2% and 20 % for both S. aureus and E.
coli respectively. In this respect, the
phenol 5% v/v had eradicated the
whole population upon exposure.
This was demonstrated at zero time of
exposure where the plates showed no
recovered colonies (data not shown).
Such inactivating activity of the Black
seed oil was demonstrated by dramatic
decrease in the number of survivors. In
such instances, the black seed oil
would clearly play a very important
role in inactivating bacterial populat-
ions. This would be elucidated in
understanding its mechanism of action.
The authors suggest that the oil was
able to interfere with bacterial envel-
ope function. Doing so will lead to the
inability of bacterial cell to control
molecule movements which might lead
to bacterial lysis and burst. This would
eventually lead to cell death.
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Vol. 8, No. 1: 2014 65
In this context, preliminary inform-
ation about understanding the possible
mechanism of action of the black seed
oil was obtained. This was achieved
by conducting an experiment of
measuring the absorbance of challen-
ged bacterial populations at low wave
length (A
250 - 350nm
). This will detect
the bacterial cell organelles if they are
released into the medium (15, 19-21).
Findings of optical density measure-
ments for challenged S. aureus and E.
coli populations to 70% and 90%
black seed oil respectively at A
350nm
over 24hrs exposure had confirmed
these findings where detection of abso-
rbance A
350nm
was achieved. Such
readings would laid the principle of
bacterial cell envelope is the primary
target for black seed oil. In one hand,
S. aureus was more susceptible with
very low absorbance reading at 24hrs
exposure. On the other hand, E. coli
susceptibility was significantly less
with a higher absorbance (10 folds) at
24 hrs exposure. In both cases, non
treated control samples showed no
absorbance detections at A
350nm
confirming these findings. In this
manner, the bacterial physiology of the
cell envelope of Gram negative that
differ from that of Gram positive
bacteria in having the layer of outer
membrane would contribute for such
variation in susceptibility (21) and
therefore in absorbance detection. In
details, the outer membrane layer of
the Gram negative bacteria would
retard the access of the black seeds
molecules (considering molecule size)
to a certain extent. This would result in
delaying the onset of bacterial kill and
therefore the magnitude of organelles
detection (22-24). Conversely, the
absence of the outer membrane in the
case of Gram positive bacteria
eliminates such factor (25, 26) where
the black seed oil molecules found
their way to interfere with bacterial
wall and therefore enable early
detection of the cell organelles.
Interestingly, further reduction in the
reading of absorbance A
350nm
was
significantly detected over the expos-
ure time. Such dramatic decrease for
both bacteria, considering the magnit-
ude in reduction, would indeed, extend
the postulation for the ability of black
seed oil to further target the bacterial
organelles in its action. This would be
expected either directly by inactivating
such organelles or by indirectly throu-
gh the role of bacterial lysozymes
(autolysin enzymes) (27-29). Bacterial
lysozymes are normally produced
when bacteria are exposed to stress
agent and the recovery is a futile
attempt (30, 31). Therefore the black
seed oil would further have the ability
in activating the release of such
enzymes explaining the ultimate and
rapid inactivation of bacteria.
Previous studies postulated that Black
seed oil might have an effect in the
prevention and treatment of severe
bacterial infections, especially those
that are difficult to treat and/or are
antibiotic resistant. They postulated
their mechanism of action on S. aureus
and E. coli is to disintegrate the cell
envelope releasing associated bacterial
material from the cells to the
surrounding medium. Such effect
might be attributed to a number of the
components of the black seed oil (32)
that are related to either the disruptive
ability of the bacterial envelope and/or
inducing the release of some autolytic
enzymes as postulated above. Such
postulations would deserve further
investigation and implying that the
interference with the bacterial
permeability barrier is not the sole
mechanism of action. In this context, a
possible identification of the oil const-
ituents and performing molecular inve-
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Vol. 8, No. 1: 2014 66
stigation is strongly suggested.
According to the present study, it can
be concluded that Black seed oil had
proven to show significant antiba-
cterial activity against various bacterial
types and through different inhibitory
mechanisms. This confirms the
satisfactory use of the Black seed oil at
least help in treating various
underlying causes of different
infectious diseases.
Acknowledgments: The authors would
like to thank all people who supported
and helped in performing this research.
We would like to thank: research
assistants Asma-Ejhany and Najat
Magerhy for their help and support.
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