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483 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. 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