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Advances in Environmental Biology, 5(2): 483-490, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Effect of Black Cumin (Nigella Sativa) Seed Oil on Gastric Tissue in Experimental
Colitis
1
3
Emekli-Alturfan E, 1Yarat A., 1Tunali-Akbay T, 1Isik F, 1Yenidogan G, 2Sener G, 2Sehirli O,
Pisiriciler R, 3Ak E, 4Altintas A
1
Marmara University, Faculty of Dentistry, Department of Biochemistry, İstanbul/Turkey
Marmara University, Faculty of Pharmacy, Department of Pharmacology, Istanbul / Turkey
3
Marmara University, Faculty of Dentistry, Department of Histology& Embryology, İstanbul/Turkey
4
Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, Eskisehir/Turkey
2
Emekli-Alturfan E, Yarat A., Tunali-Akbay T, Isik F, Yenidogan G,
Pisiriciler R, Ak E, Altintas A: Effect of Black Cumin (Nigella Sativa)
Tissue in Experimental Colitis
Sener G, Sehirli O,
Seed Oil on Gastric
ABSTRACT
The black cumin (Nigella sativa) (NS), the member of Ranunculacea family, is a plant widely used
as a spice in our country. Its seed oil contains 21% protein, 35 % carbohydrate, 35-38 % lipid. The
aims of this study were to investigate if the proinflammatory cytokines generated in trinitrobenzene
sulphonic acid (TNBS)-induced colitis affect the gastric tissue and to determine the effects of orally
administered NS seed oil (ORIGO ‘100 % naturel Black Cumin Seed Oil’, 2.5 ml/kg; orally) on the
gastric tissue. Rats were grouped as control (n=5), NS treated control (n=5), colitis (n=6) and NS
treated colitis (n=7). NS was given 5 minutes later than the induction of colitis and the treatment was
continued for 3 days. Three days after the induction of colitis, all rats were decapitated and gastric
tissues were removed and homogenized. Total sialic acid (SA), glutathione (GSH), malondialdehyde
(MDA) levels; catalase (CAT), superoxide dismutase (SOD) activities were measured in the homogenates.
Gastric tissue samples were also examined cytologically. TNF-a, IL-1b and IL-6 and LDH levels were
determined in blood samples. In the NS treated control group SA levels were significantly decreased
when compared with the control group. In the colitis group increased plasma proinflammatory cytokines
and decreased tissue CAT, MDA and SA levels suggest that activation of immun system protects the
gastric tissues. On the other hand in the NS treated colitis group, significantly increased gastric tissue
CAT activity compared with the colitis group indicate that thymoquinone content of black cumin has
SOD like activity. Therefore, black cumin seed oil may have protective effects on gastric tissue.
Key words: Black Cumin (Nigella sativa), gastric tissue, colitis, sialic acid, superoxide dismutase,
catalase, malondialdehyde
Introduction
Ulcerative colitis (UC) is characterized by
oxidative and nitrosative stress, leukocyte
infiltration and upregulation of proinflammatory
cytokines. Inflammatory bowel disease (IBD)
consists of Crohn's disease (CD) and UC where
genetic, immunologic and environmental factors
are involved in the initiation and perpetuation of
these chronic intestinal diseases [1,2].
The bowel disease in UC is traditionally
considered to be confined to the large intestine.
Corresponding Author:
Ebru Emekli-Alturfan, Marmara University Faculty of Dentistry Department of Biochemistry
Nisantası, 34365, Istanbul, Turkey.
E-mail : [email protected],
Tel: 90 212 233 66 27
Fax: 90 212 246 52 47
Adv. Environ. Biol., 5(2): 483-490, 2011
However, a number of investigators have reported
that diffuse gastritis is a common finding in
patients with UC [3-6]. Berrebi et al. [7]
suggested that the gut inflammatory reaction in
patients with UC is not restricted to the large
intestine. However the characteristics of the
inflammatory infiltrate in UC-associated gastritis
are not known [8].
Plant remedies have become increasingly
popular and are often preferred to synthetically
derived pharmaceuticals. Nigella sativa Linn. (NS)
(a dicotyledon of the Ranunculaceae family),
commonly known as black seed or black cumin,
is an annual plant that has been traditionally
used for culinary and medicinal purposes as a
natural remedy for a number of illnesses and
conditions that include asthma, hypertensin,
diabetes, inflammation, cough, bronchitis, headache,
eczema, fever, dizziness and influenza. NS seed
oil contains more than 30 of a fixed oil and
0.4-0.45 w/w of a volatile oil. In volatile oil
analysis many components has been characterized,
but the major ones were thymoquinone
(27.8%–57.0%), ρ-cymene (7.1%–15.5%),
carvacrol(5.8%–11.6%), t-anethole (0.25%–2.3%), 4terpineol(2.0%–6.6%) and longifoline (1.0%–8.0%).
The oil of NS is so beneficial due to its content
of over 100 components such as aromatic oils,
trace elements, vitamins and enzymes. It contains
58% of essential fatty acids including omega 6
and omega 3. These are necessary for the
forming of prostaglandin E1 that balances and
strengthens the immune system giving it the
power to prevent infections and allergies and
control chronic illnesses [9-11].
Of the several animal models of intestinal
inflammation, the well-characterized haptene
reagent TNBS-induced colitis resembles human
UC in its various histological features including
infiltration of colonic mucosa by neutrophils and
macrophages and increased production of
inflammatory mediators including T helper type 1
profile of cytokines [12].
The aims of this study were to investigate,
if the inflammation markers formed in
trinitrobenzene sulphonic acid (TNBS)-induced
colitis affect the gastric tissue and to determine
the possible protective effects of orally
administered NS seed oil on the gastric tissue.
Materials and Methods
484
(65–70%) were kept constant. The animals were
fed a standard pellet and food was withdrawn
overnight before colitis induction. Access to water
was allowed ad libitum. Experiments were
approved by the Marmara University School of
Medicine Animal Care and Use Committee.
Rats were grouped as control (n=5), NS
treated control (n=5), colitis (n=6) and NS treated
colitis group (n=7).
2.2. Nigella Sativa Oil Fatty Acid Analyses:
A fatty acid analysis of NS (BCO, Origo,
“100 % natural black cumin oil”, Gaziantep,
Turkey) was performed at Anadolu University,
School of Pharmacy, Department of
Pharmacognosy, Eskisehir, Turkey by gas
chromatography technique.
2.3. Induction of Colitis and Ns Administration:
Animals were fasted for 18 hours before the
induction of colitis. Under light ether anesthesia,
a polyethylene catheter (PE-60) was inserted into
the colon with its tip positioned 8 cm from the
anus. To induce colitis, a single solution of 1
mL of a 30 mg/mL TNBS solution, dissolved in
40% ethanol in saline was instilled. The rats in
the control group were subjected to the same
procedure with the exception that an equal
volume of isotonic saline was substituted for
TNBS. NS or saline (2.5 mL kg-1) were given
orally 5 minutes later than the induction of
colitis and the treatment was continued for the
following 3 days. On the fourth day of colitis
induction, rats were decapitated and trunk blood
was collected for the assessment of the proinflammatory cytokines, TNF-α, IL-1β, and IL-6
and LDH levels. Distal 8 cm of the colon
obtained from each animal were initially examined
for recording macroscopic damage scores. Gastric
tissues were removed and and then stored at
!20NC until the determination of malondialdehyde
(MDA; an index of lipid peroxidation), glutathione
(GSH; a key antioxidant), superoxide dismutase
(SOD: an antioxidant enzyme), catalase (CAT: an
antioxidant enzyme), sialic acid (SA; one of the
markers of membrane integrity) and total protein
(TP) levels in the gastric tissue.
2.4. Biochemical Analyses:
2.4.1. Serum Cytokine Levels and Ldh Activities:
2.1. Animals:
Both sexes of Wistar albino rats (200–250 g)
were kept in a light- and temperature-controlled
room with 12:12-hour light–dark cycles, where the
temperature (22 ± 0.5 0C) and relative humidity
Serum
LDH
was
determined
spectrophotometrically using an automated analyzer
[13]. Serum levels TNF-α, IL-1β, and IL-6 were
quantified using enzyme-linked immunosorbent
assay (ELISA) kits according to the
Adv. Environ. Biol., 5(2): 483-490, 2011
manufacturer’s instructions and guidelines
(Biosource International, Camarillo, CA). These
particular assay kits were selected because of
their high degree of sensitivity, specificity, interand intraassay precision, and small amount of
plasma sample required conducting the assay.
485
resulted in the formation of a red colored
substance extracted in cyclohexanone. Its
absorbance at 549 nm was read and the result
expressed in mg SA per gram of protein .
TP level was determined by the method of
Lowry [19], using bovine serum albumin as a
standard, reading absorbance at 500 nm.
2.4.2. Gastric Tissue Mda Levels:
2.5. Cytological Examinations:
The MDA levels were measured by the
method of Ledwozyw for products of lipid
peroxidation [14]. Results were expressed as nmol
MDA per mg protein.
Gastric tissue samples were smeared over a
glass microscope slide and fixed with air. Then
they were stained with Giemsa stain [20] and
microscopically examined (x100).
2.4.3. Gastric Tissue Gsh Levels:
2.6. Statistics:
GSH
was
determined
by
the
spectrephotometric method using Ellman’s reagent
[15] and the results were expressed as mg GSH
per gram of protein.
2.4.4. Gastric Tissue Sod Activity:
SOD activity in the gastric tissue samples
was measured according to the previously
described method [16]. Briefly, measurements were
performed in cuvettes containing 2.8 mL 50 mM
potassium phosphate (pH = 7.8) with 0.1 mM
EDTA, 0.2 mM riboflavin in 10 mM potassium
phosphate (pH 7.5), 0.1 mL of 6 mM odianisidin and tissue extract. Cuvettes with all
their components were illuminated with 20-W
Slylvania Grow Lux fluorescent tubes that were
placed 5 cm above and to one side of cuvettes
maintaining a temperature of 37oC. Absorbance
was measured at 460 nm and the result
expressed as SOD U per mg of protein.
Statistical analysis was carried out using
Instat statistical package (GraphPad Software, San
Diego, CA). All data are expressed as mean
values ± SD. Following the assurance of normal
distribution of data, groups of data were
compared with Mann Whitney test. Values of
p<0.05 were regarded as significant.
3. Results:
The groups were checked for the differences
in weight at the beginning and at the end of the
experiment, but none were found.
3.1. Nigella Sativa
Analyses:
Oil Fatty Acid Composition
Table 1 shows the fatty
NS oil. According to the
linoleic (C18:2n-6) and oleic
the major fatty acids in NS
acid composition of
fatty acid analysis
(C18:1n-9) acids are
oil.
2.4.5. Gastric Tissue Cat Activity:
3.2. Blood Parameters:
CAT activity was measured by the Aebi
method [17]. The principle of this method was
based on the hydrolyzation of H 2 O 2 and
decreasing absorbance at 240 nm. The conversion
of H2O2 into H2O and 1/2 O2 in 1 min under
standard condition was considered to be the
enzyme reaction velocity. Result were expressed
as CAT U per mg of protein.
2.4.6. Total Sa and Tp Levels:
To determine total SA levels, the gastric
tissue homogenate was first incubated at 80 °C
for 1 h in diluted sulfuric acid in order to
liberate bound SA and then Warren's method was
applied [18]. This method consisted of oxidizing
SA with periodate, terminated by the addition of
arsenite and then adding thiobarbituric acid. This
Serum TNF-α, IL-1β, IL-6 and LDH levels
significantly increased in colitis group when
compared with the control group. NS significantly
decreased these parameters in the colitis group
(Table 2).
3.3. Gastric Tissue Parameters:
Gastric tissue SA, catalase, MDA, SOD and
GSH levels are given in Table 3. In the NS
treated control group SA levels significantly
decreased when compared with the control group.
In the colitis group gastric tissue CAT, MDA
and SA levels decreased significantly compared
with the control group. NS treatment in the
colitis group significantly increased gastric tissue
CAT activity when compared with the colitis
Adv. Environ. Biol., 5(2): 483-490, 2011
group.
3.4. Cytological Examinations:
Gastric tissue samples obtained from the
colitis group revealed epitheloid cell groups that
confirmed colitis. In the NS oil given colitis
group, epitheloid cells were degenerated and they
486
disapperared. Moreover macrophages were
observed in the same group. Increased necrotic
material and bacteria in the colitis group were
found to be decreased in the NS administered
colitis group (Figure 1-3).
When the gastric tissues of NS given control
group examined cytologically no difference was
observed.
Fig. 1: Big round parietal cells with eosinophilic cytoplasm are observed in the Control group. Original
magnification x400, May Grunwald Stain.
Fig. 2: Epitheloid cell groups are observed in the Colitis group. Original magnification x400, May
Grunwald Stain.
Adv. Environ. Biol., 5(2): 483-490, 2011
487
Fig. 3: Gastric cells, fatty cells and macrophages (arrow) are observed in Nigella sativa treated colitis
group. Original magnification x400, May Grunwald Stain.
Table 1: Nigella sativa oil fatty acid composition
Content
Myristic acid (14:0)
Palmitic acid (16:0)
Palmitoleic acid (16:1)
Margaric acid (17:0)
Stearic acid (18:0)
Oleic acid (18:1)
Linoleic acid (18:2)
Linolenic acid (18:3)
Arachidonic acid (20:0)
11-eicosenoic acid (20:1)
Behenic acid(22:0)
Lignoseric acid (24:0)
Saturated
Unsaturated
Unsaturated/Saturated
(%)
0.1
7.1
trace
trace
3.2
27.7
59.2
0.5
trace
0.2
0.6
trace
11.6
87.6
7.6
Table 2: Serum TNF-α, IL-1β, IL-6 levels and LDH activities of the groups
Control
Control+NS
Colitis
Colit+NS
TNF-α (pg/ml)
6.15 ± 2.57
6.01 ± 2.17
44.23 ± 21.63 ***
18.28 ± 7.72 ++
IL-1β (pg/ml)
10.3 ± 4.1
11.3 ± 5.5
60.4 ± 14.7 ***
27.7 ± 12.5 *, +++
IL-6 (pg/ml)
22.3
8.3
20.8
7.3
65.4
32.5 **
33.3
15.5 +
LDH (U/L)
1065
368
939
324
2919
412 ***
1800
508 ++
Values are expressed as mean ± standard deviation. P values< 0.05 are considered statistically significant.
* p<0.05, ** p<0.01, *** p<0.001 compared with the control group
+ p<0.05, ++ p<0.01, +++ p<0.001 compared with the colitis group
TNF-α : tumor necrosis factor, IL1β-: Interleukin 1-beta, IL-6: Interleukin 6, LDH: lactate dehydrogenase. NS: Nigella sativa
Table 3: Sailic acid, Glutathione, Malondialdehyde levels and Superoxide dismutase, and Catalase activities of the groups
Control
Control+NS
Colitis
Colitis+NS
SA mg/g protein
17±2.24
10.64±2.27*
9.9±5.3*
15.71±4.03
GSH mg/g protein
0.6±0.11
0.79±0.13
0.49±0.09
0.5±0.15
SOD U/mg protein
1±0.21
1.2±0.29
1.03±0.61
0.56±0.19
MDA nmol/mg protein 2.19±0.34
2.4±0.25
1.74±0.27*
1.97±0.59
CAT U/mg protein
518.8±38.40
465.8±50.41
373.0±73.39*
472.1±28.29**
Values are expressed as mean ± standard deviation. P values< 0.05 are considered statistically significant.
*p<0.05 compared with the control group, ** p<0.05 compared with the colitis group.
SA: Sialic acid, GSH: Glutathione, SOD: Superoxide diamutase, MDA: Malondialdehyde, CAT: Catalase, NS: Nigella sativa
Adv. Environ. Biol., 5(2): 483-490, 2011
Discussion:
Excessive production of pro-inflammatory
mediators such as TNF-α, IL-1β, IL-6, IL-8,
leukotriene B4 and platelet activating factor, and
the presence of highly activated inflammatory
cells such as neutrophils, monocytes and
macrophages are common characteristic of
ulcerative colitis [7,11,21]. Accordingly in the
present study, serum TNF-α, IL-1β, and IL-6
increased in TNBS induced colitis group. This
inflammatory status has been reversed by NS oil.
Moreover increased serum LDH activity of the
colitis group decreased with NS oil administration
which shows the improved tissue damage by NS
oil.
There is extensive experimental literature
supporting the concept that during gastrointestinal
inflammation, a complex interaction occurs
between immune cells, epithelial and mesenchymal
cells, and the neurones of both the intrinsic and
extrinsic innervation of the gut [21,22].
Importantly, these events are not restricted to the
inflamed region [21,23,24]. Therefore we
investigated the gastric tissue oxidant-antioxidant
parameters and SA levels in experimental colitis.
In the present study gastric MDA levels and
CAT activity
decreased in the colitis group
compared with the control group.
Lipid
peroxidation is a free radical-mediated process
which leads to structural modification of complex
lipid protein components such as biomembranes
and lipoproteins [25]. This oxidative deterioration
of polyunsaturated lipids is often associated with
cellular malfunction. The products of lipid
peroxidation are capable of interacting with DNA
and cause oxidative damage [26]. SOD plays a
to hydrogen
key role in catalyzing O2•!
peroxides and oxygen.
CAT catalyzes the
reduction of hydrogen peroxide to molecular
oxygen and water. Thus, this enzyme helps to
protect the tissues from highly reactive hydroxyl
radicals (•OH), derived from H2O2. Since H2O2
can undergo Fenton reaction in the presence of
transition metal ions such as Fe2+ and Cu2+ and
can also react with O2•! in the Haber-Weiss
reaction to produce hydroxyl radical (•OH), the
reduced CAT activity might result in increased
formation of (•OH), which is a potent free radical
[27].
In the present study the reduced lipid
peroxidation in the gastric tissue might be a
consequence of reduction of hydrogen peroxide to
molecular oxygen and water by CAT to protect
the tissue from highly reactive hydroxyl radicals
(•OH), derived from H2O2. This in turn might
have led to the depletion of CAT. NS treatment
in the colitis group significantly increased CAT
488
activity in the gastric tissue when compared with
the colitis group which may be due to the SOD
like activity of thymoquinone content of black
cumin since thymoquinone has been reported to
act as SOD-like substances to scavenge O2.produced by xanthine/xanthine oxidase system
[28,29]. Therefore, NS may have protective
effects on gastric tissue.
The other parameter investigated in the
present study was SA in gastric tissue.
The
gastrointestinal tract is covered by a protective
mucus gel layer. The turnover of this gel layer
is essential for hydration, mechanical protection,
the physical removal of contaminants and
provision of suitable environment for the renewal
of other defensive molecules that are incorporated
into mucus [30]. The key component of the
mucus is predominantly mucin glycoprotein that
is synthesized and secreted by mucous neck cells
in the stomach and goblet cells in the intestine
[31]. Mucin is made up of fucose, galactose, Nacetylglucosamine, N-acetylgalactosamine and SA.
The SA usually occupy the terminal position of
the oligosaccharide chain of the glycoconjugate
and these terminal residues have a significant
influence on the mucus charge, mucus rheology
and mucus degradation. In this way they
contribute to the high viscosity of the mucus
lining and protect the endothelial layers of the
intestine and stomach. Desialylation of mucus
may lead to degradation of the mucus [32].
Sialate aldolase is a sialidase localized in the
cytosol of mammalian cells or secreted by
bacteria into the medium.
The role of this
enzyme is the degradation of SA in mammalian
cells therefore it increases cellular levels of SA.
However the role of these enzymes in
inflammation is a largely unknown field deserving
more attention [33]. In the present study SA
levels decreased in the colitis group which may
be a consequence of the inflammatory response
formed due to TNBS induced colitis. On the
other hand, NS treatment in the colitis group
decreased SA levels significantly when compared
with the control group.
Studies investigating the gastric tissue in
ulcerative colitis are limited in the literature. Hu
et al [22] hypothesised that inflammation of the
colon results in sensitisation of extrinsic afferent
nerve fibres leading not only to hyperalgesia but
also to hyperactive neuronal reflex pathways
affecting gastric motor function. They reported on
the presence and pathogenesis of disturbances in
gastric motility in rats with experimental distal
colitis. Berrebi et al. [7] investigated cytokine
and chemokine receptor expression in the gastric
mucosa and rectal mucosa of a group of
Adv. Environ. Biol., 5(2): 483-490, 2011
pediatric patients with UC. They found the
gastric mucosa macroscopically normal in all of
the patients with UC who were examined.
However chronic gastritis was diagnosed
histologically in 13 of 14 patients, who were
without evidence of Helicobacter pylori infection.
Most of these patients were judged to have mild,
diffuse mononuclear cell infiltrates of the gastric
mucosa by the evaluating gastrointestinal
pathologists. The inflammatory infiltrate in the
gastric biopsy specimens consisted mainly of T
and B cells. In the present study, gastric tissue
samples obtained from the colitis group revealed
epitheloid cell groups that confirmed colitis. In
the NS oil given colitis group, epitheloid cells
were degenerated and mostly disapperared.
Macrophages were observed in the same group
showing strengthened defense system of the
gastric tissue. Moreover increased necrotic material
and bacteria in the colitis group were found to
be decreased in the NS
administered colitis
group.
The use of natural anti-inflammatory products
provides an attractive and relatively non-toxic
alternative to modulate inflammatory disorders.
Consequently NS may have an important role in
modulating the inflammatory response of the
gastric tissue in colitis.
489
7.
8.
9.
10.
11.
12.
13.
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