Potential of Black Seed Oil (Nigella Sativa) as Immunomodulator in
Mice (Mus Musculus) Infected with Salmonella Typhimurium
Eka Yunita Wulandari
1
, Dwi Krihariyani
2
and Suliati
2
1
Departement of Immunology, Postgraduate School Universitas Airlangga, Surabaya, Indonesia
2
Study program of Medical Laboratory Technology, Health Polytechnic Surabaya, Surabaya, Indonesia
Keywords: Black Seed Oil, Immunomodulator, Salmonella typhimurium.
Abstract: Typhoid fever is a systemic disease mainly caused by Salmonella typhi that spreads systemically into blood
circulation and leads to bacteria accumulation in the intestine. Black seed oil (Nigella sativa) contains a
chemical substance called thymoquinone acting as immunomodulator that can enhance immune system to
prevent bacteria colonization in the intestine. This study was to determine the potential of black seed oil as
immunomodulator to mice infected with Salmonella typhimurium. This study is an experimental research
using completely randomized design (CRD) with 4 treatments and 6 replications. Animals used were 2-
month-old male mice, weighing 25 - 30 g totaling 24. Treatments in this study included K- (normal mice),
K+ (mice infected with Salmonella typhimurium i.p), P1 (mice orally administered with black seed oil for 7
days and infected with Salmonella typhimurium i.p), and P2 (mice orally administered with black seed oil
for 14 days and infected with Salmonella typhimurium i.p). 7 days after infection, mice intestines were
collected and total number of bacteria were counted. Data were analyzed using One-Way ANOVA and Post
Hoct test. The result shows ANOVA p-value = 0.006 and p-value of Post Hoc = 0.004, proving that black
seed oil has potential as immunomodulator in mice infected with Salmonella typhimurium.
1 INTRODUCTION
Salmonella sp is gram-negative bacteria with rod
shape from Enterobacter famili and pathogenic for
animals and human. Salmonella sp cause enteritis,
sistemical infection, and enterical fever in human.
There are four important serotypes of Salmonella
which mainly cause enterical fever in human, such
as Salmonella typhi, Salmonella paratyphi A,
Salmonella paratyphi B, and Salmonella paratyphi C
(Jawetz et.al, 2013).
Salmonella typhimurium is usually used in
laboratory research as model of typhoid fever in
human because there are differences of genetic
marker expressed at Salmonella typhi and
Salmonella typhimurium resulting in inability of
Salmonella typhi to infect mice compared to how
Salmonella typhi cause typhoid fever in human
(Mathur et.al, 2013).
Typhoid fever or known as “tifoid, tipes, or
typhus” is a systemical acute infection in human
gastrointestinal tract caused by Salmonella enterica,
especially Salmonella typhi (Paputungan et.al,
2016).
WHO (World Health Organization) predicted
that incidence rate of typhoid worldwide increase in
average number of 800 cases per 100.000 people
every year. One of the reasons of high incidence rate
of typhoid fever in Indonesia is because of bad
sanitation which causes Salmonella sp can spread
easily (Depkes, 2013; Paputungan et.al, 2016).
Antibiotics use is one of the way to overcome
typhoid fever. Although antibiotics had had good
contribution to help fight against infections
worldwide, the fact is infection is still one of the
cause of human death. It is because bacteria has
ability to protect themselves against antibacterial
drugs, gradually leading to resistence (Soleha, 2015;
Umiana, 2015).
Many cases of antibiotics resistance and use of
drugs with specific side effects make a shift in
society. Using principal of Back to Nature, people
think to utilize natural plants as drug. Furthermore,
society now thinks about early prevention against
infection, for example by consuming supplements
made of natural plants. Substances in those natural
plants which are able to enhance body immune is
Wulandari, E., Krihariyani, D. and Suliati, .
Potential of Black Seed Oil (Nigella Sativa) as Immunomodulator in Mice (Mus Musculus) Infected with Salmonella Typhimurium.
DOI: 10.5220/0007543904070411
In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 407-411
ISBN: 978-989-758-348-3
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
407
called immunomodulator (Mardiana, 2012; Sulisti
et.al, 2014).
Black seed (Nigella sativa) or known as
Habbatussauda is spices coming from middle east
that had been used as medicine. In hadist of Bukhari
Muslim, Rasulullah SAW encourages us to use
black seed that can cure all diseases, except Al-Sam /
death (Putra, 2011).
Black seed become more familiar nowadays
because there are many thibbun nabawi-based
medication which is referred to Al-Qur’an and As-
Sunnah. Some of Islamic references believe that
black seed is one of herbs used based on sunnah.
People now can use black seed easier because they
are produced in many practical package such as
capsule, oil, or powder. Black seed are empirically
used to enhance immunity, stamina, cure diabetes,
kolesterol, cancer, athma, inflamation, stroke, ulcer,
and many other infections.
There have not been many studies about using of
black seed oil (Nigella sativa) products to be
immunomodulator against typhoid fever. Because of
that reason, researcher intended to do experiment to
know potential of black seed oil (Nigella sativa) as
immunomodulator to mice (Mus musculus) infected
with Salmonella typhimurium.
2 MATERIALS AND METHOD
2.1 Research Design
The study used true experiment method with Posttest
Only Control Group as research design. This study
was aimed to know potential of black seed oil
(Nigella sativa) as immunomodulator to mice (Mus
musculus) infected with Salmonella typhimurium.
Researcher will compare mice acting as positive and
negative controls to mice which were given
treatments. There were 4 treatments and 6
replications in each treatment.
2.2 Place and Time
This research took place in Microbiology and
Zoology Laboratory Faculty of Math and Science,
University of Jember starting from May until July
2017.
2.3 Materials
2.3.1 Animal Test
Animal used were male mice of Balb/c strain with
age ±2 months weighing 25 – 30 gram, totaling 24
and were divided into four groups such as :
1. K (-) : without any treatment
2. K (+) : Salmonella typhimurium 10
8
infection i.p
3. P1 : Black seed oil for 7 days and
Salmonella typhimurium 10
8
infection
i.p
4. P2 : Black seed oil for 14 days and
Salmonella typhimurium 10
8
infection
i.p
2.3.2 Black Seed Oil
Black seed oil used during research was bought from
Jalan Nyamplungan Ampel, Surabaya.
2.3.3 Bacteria
Salmonella typhimurium ATCC (American Type
Culture Collection) 14028 was obtained from Unit
Layanan Pengujian/ULP (Assesment Service Unit)
Faculty of Pharmacy, Universitas Airlangga
Surabaya.
2.3.4 Bacterial media
Bacterial media used in this study were Salmonella
Shigella Agar (SSA) and Luria Bertani Broth.
2.4 Procedure
Mice were acclimatized in cages for 7 days and
given standardized foods and drinks. All laboratory
glasswares were sterilized using autoclave at
temperature of 121
o
C for 15 minutes. Bacterial
suspension was made based on infection dose of 10
8
.
Black seed oil was administered orally as much
as 0,2 ml for 7 days to mice in group P1 and 14 days
to mice in group P2. Mice in group K (+), P1, and
P2 were infected with Salmonella typhimurium via
intraperitonal.
Confirmation test was performed on the third day
after infection and performed through following
steps : First, 0.1 ml blood was collected from mice
via intraorbital. Blood then was placed in microtube
filled with 0.3 ml EDTA. Blood from microtube was
poured inside reaction tube filled with 5 ml Luria
Bertani Broth. The mixture then was incubated at
temperature of 37
o
C for 24 hours. During incubation
period, the mixture was shaken at 120 rpm. The
ICPS 2018 - 2nd International Conference Postgraduate School
408
mixture was inoculated on petri dish using pour
plate method and SSA. Sample was homogenized
and incubated at temperature of 37
o
C for 24 hours.
Seven days after Salmonella typhimurium
infection, all mice were knocked out with cloroform,
belly was opened and intestine was collected.
Intestine was cleaned, weighed, mashed using
mortar, diluted in certain volume of saline to create
dilution of 10
-1
and serial dilutions were performed.
Using vortex, dilution of intestine was homogenized.
The dilution was cultured on SSA media using pour
plate method. After incubation period for 24 hours at
temperature of 35
o
C, number of colony was counted
using colony counter.
3 RESULTS AND DATA
ANALYSIS
Figure 1 : Salmonella typhimurium on SSA media
Table 1: Calculation of Salmonella typhimurium colony in
mice (Mus musculus) intestine of K (-) group.
Treatment
Average number of
colony
(CFU/gram)
K (-)
0
K (+)
TNTC
P1
6.2 x 10
8
P2
4.9 x 10
8
TNTC : Too Numerous To Count
CFU : Colony Forming Unit
Data were further analyzed using post hoc test
and showed sig. 0,004 < 0,05, suggesting that there
was significant difference between group P1 (7 days
consumption of black seed oil) and group P2 (14
days consumption of black seed oil).
Table 2 : Normality test using Kolomogrov Smirnov
One-Sample Kolmogorov-Smirnov Test
data
N
17
Normal Parameters
a
Mean
5.61E7
Std.
Deviation
1.276E7
Absolute
.238
Most Extreme Differences
Positive
.238
Negative
-.144
Kolmogorov-Smirnov Z
.981
Asymp. Sig. (2-tailed)
.291
a. Tes distribution is Normal
Normality test using Kolomogrov Smirnov
showed that data of colony number of Salmonella
typhimurium in mice intestine had normal
distribution.
Table 3 : Homogenity test
Levene Statistic
df1
df2
Sig.
5.900
2
14
.014
Homogenity test using showed that data of
colony number of Salmonella typhimurium in mice
intestine were homogenous with p value of 0.014.
Table 4 : Two-way ANOVA
ANOVA
Number of Colony
Sum of
Squares
df
Mean
Square
F
Sig.
Between
Groups
1.346E15
2
6.728E1
4
7.47
7
.006
Within
Groups
1.260E15
14
8.999E1
3
Total
2.605E15
16
Data were further analyzed using One-Way
ANOVA parametric test. From One-Way ANOVA
parametric test, sig.0,006 < 0,05 was obtained,
meaning that there was difference of Salmonella
typhimurium colony number between P1 (7 days
consumption of black seed oil) and P2 (14 days
consumption of black seed oil).
Table 5 : Post Hoc Test
(I)
gro
up
(J)
gro
up
Mean
Differ
ence
(I-J)
Std.
Error
Si
g.
95%
Confidence
Interval
Lower
Bound
Upp
er
Bou
nd
P1
P2
1.888
E7
5.47
7E6
.0
04
713665
3.12
3063
0013
Potential of Black Seed Oil (Nigella Sativa) as Immunomodulator in Mice (Mus Musculus) Infected with Salmonella Typhimurium
409
.55
P2
P1
-
1.888
E7
5.47
7E6
.0
04
-
3.06E7
-
7136
653.
12
Data were further analyzed using post hoc test
and showed sig. 0,004 < 0,05, suggesting that there
was significant difference between group P1 (7 days
consumption of black seed oil) and group P2 (14
days consumption of black seed oil).
4 DISCUSSIONS
Positive control (not given black seed oil but
infected with Salmonella typhimurium) showed the
highest colony number of Salmonella typhimurium
among all groups. This is occurred because without
any intervention from herbs, Salmonella
typhimurium could penetrate easily to intestinal
epithels as the first process of entering mice body.
Salmonella typhimurium in positive control
group penetrated epithelial intestine easily, causing
bacterial antigens were trapped by Peyer’s patches.
As the result, local immune response of intestine
was triggered. Antigen-presenting dendritic cells
(DCs) then delivered signal, allowing signal to be
recognized by other parts of intestine without
interfering reading process of antigens coming from
commensal intestinal bacteria or other pathogens. B
cells and memory cells were activated by antigen-
presenting cells at Peyer’s patches. These activated
cells migrated toward exact location of antigen
target, associated with multiplication of intestinal
immune response. During infection, endotoxin of
Salmonella (LPS) triggered macrophage and other
phagocyte cells to produce and release various
cytokines such as IL-1, TNF-α, and IL-6 (Broz et.al,
2012).
Compared to positive control, number of
Salmonella typhimurium colony of mice orally
administered black seed oil for 7 days (group P1)
and mice orally administered black seed oil for 14
days (group P2) decreased. It was due to the
existence of an active substituent in black seed oil
known as thymoquinone. Thymoquinone is believed
to serve as immunomodulator. Ability of
thymoquinone to be immunmodulator is closely
related to its function to enhance immunity in
intestine as early barrier to Salmonella typhimurium
exposure. Thymoquinone in black seed oil helps
improving the release of digestive enzymes so that
nutritions which are needed to fight pathogens can
be absorbed better. Thymoquinone also takes part in
decreasing number of coliforms in mice ceccum
which is known as stable environment for pathogens
and commensal bacteria (Erener et.al, 2010).
Thymoquinone in black seed oil stimulated T
lymphoctye during the process of bacteria
elimination by intestinal macrophage. T cells
proliferated inside thymus to become cells involved
in expression of specific T cell reseptors. Those
specific T cell reseptors were derived into CD4+ or
CD8+. CD4+ and CD8+ cells are cell surface
proteins which determine the main subpopulation of
T cells, CD4 cells (T helper) and CD8 cells (T
cytotoxic). Differentiated T cells then migrated to
infection site and give systemic protection (Salem
et.al, 2011; Ahmad, 2013).
The study showed that consuming black seed oil
for 14 days were more effective to prevent
Salmonella typhimurium to grow in mice intestine
than black seed oil for 7 days. It was relevant with
former research by Musthoza (2017) that the longer
period herbs were consumed, better immunity would
be formed. This was because herbs take more time
to establish and improve whole body system by
repairing damaged cells and organs.
5 CONCLUSIONS
The black seed oil is proven to have potential to be
immunomodulator in mice infected with Salmonella
typhimurium.
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Potential of Black Seed Oil (Nigella Sativa) as Immunomodulator in Mice (Mus Musculus) Infected with Salmonella Typhimurium
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