The investigation of silymarin effect on colon ulcer induced acetic acid in mice Balb/C

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1 Available online at Annals of Biological Research, 2012, 3 (7): ( ISSN CODEN (USA): ABRNBW The investigation of silymarin effect on colon ulcer induced acetic acid in mice Balb/C Vahid Hemayatkhah Jahromi 1*, Farshid Kafilzadeh 1, Habibollah Johari 2 1 Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran 2 Department of Biology, Darab Branch, Islamic Azad University, Darab, Iran ABSTRACT Colon ulcer is an inflammatory chronic colon disease and free radicals play an important role in its pathogenesis. Free radicals increase the severity of the disease by decreasing the antioxidants. Silymarin is a flavonoid component that its extracted of Silybum marianum. Silymarin contains antioxidants and inhibits the inflammatory reactions. The aim of this research, is the investigation of anti- inflammatory effect of silymarin in the treatment of colon ulcer induced acetic acid in mice Balb/C spicies.in this study, 32 mice are divided into 4 groups (n=8).these groups include:control group with no colitis report,the Sham group with colitis report that are not treated and the groups with colitis report that received 10 and 20 mg/kg B.W. of silymarin orally.the treatment groups received silymarin daily, for 1 week after having the disease. The drugs are fed to the mice per oral using gaster tubes. To induce colitis, 1 ml of acetic acid (%4) is injected into the rectum. In the one week after induction of colitis, the animals sacrificed and the damages of the colon investigated in Morris and Murthy methods.the results of this study shown acetic acid causes severe inflammation and damages of the colon. Also, silymarin is effective in improving of the inflammation and repair of the colon ulcer induced acetic acid. So, making use of silymarin can be considered as a suggestive way for the colitis treatment. Key words: Mice, acetic acid, colon ulcer, silymarin INTRODUCTION Colon ulcer is a chronic disease that people affects with different ages and inflammatory agents and free radicals have an important role in its creation [1,2]. Inflammation of white blood cells and macrophages to the intestinal mucosal tissue is the symptoms of this disease [3]. Activated white blood cells produce free radicals in the intestinal mucosal tissues, that cause lipid peroxidation, the increasing of permeability of blood vessels, the increasing of neutrophils enterance and expanding of inflammation in the intestinal mucosal tissue. Release of inflammatory mediators and enzymes are caused intestinal damage, injury, bleeding and diarrhea. Also, free radicals are reduce anti-oxidant agents and thus are increase the severity of the disease [4]. The controlling of inflammation and reducing of the adverse effects of drugs is important goals in colitis ulcerative. The best way to treatment is usage of drugs like corticosteroids and aminosalysilates, but these drugs have severe side effects [5]. So, for disease control and prevention are conducted extensive studies of natural healing techniques including the use of anti-oxidants substances [6,7]. Anti-oxidants materials capable to prevent of releasing of free radicals and cells and tissues damage in the body [8]. 3691

2 Silymarin is a flavonoid compound that is extracted from Silybum marianum [9]. This plant has anti-oxidant and anti-inflammatory properties [10]. Silymarin is as well as cell membrane stabilizers, and is enhancing cellular glutathione concentration. Glutathione responsible for detoxification of free radicals in the body [11,12]. Traditionally, this herb has been used to increasing of milk secretion and depression of liver and kidney congestion [13]. Silymarin is very similar to steroid hormones and these hormones are effective via protein production in the increasing of gene expression rate [9]. Anticancer effects of silymarin have been studied on skin cells, colon, ovary and nervous system cancers [4]. Silymarin prevents of lipoproxidation and damage to cell membranes [14]. So, with regard to cases of heavy use of silymarin, in this study, its effect has been investigated on reduction of intestinal mucosal inflammation and tissue repair induced by acetic acid in mice that resembles is similar to human intestinal ulcers [15]. MATERIALS AND METHODS This is an experimental study. In this study, all the work ethics of laboratory animals has been considered. Of 32 mice was used of Balb / C race, weighing approximately 36 grams and aging days prepared from Serum and Vaccine Research Center, Shiraz. Then, animals were trasferrd to animal house in Islamic Azad University, Jahrom, and lighting - darkness conditions (12 hours lighting and 12 hours darkness ) were maintained. Mice fed the standard diet (Pellet). Animals were divided into four groups (n=8) into the following: group1: control and intact mice without causing disease group2: Sham mice have with intestinal ulcer(no treated with silymarin) group3: mice with colitis + 10 mg/kg silymarin that was given orally and group4: mice with colitis +20 mg/kg silymarin that was used orally. Animals in groups 3 and 4 received silymarin daily, for 1 week after causing disease. silymarin was given as gavage. To create colitis (ulcer) was used acetic acid (%4) as intrarectal [14]. So how do, the mice were kept for 24 hrours in the fasting state and only water was used. For colitis, animals anesthetized briefly with ether and a catheter into the rectum and 1 ml acetic acid (%4) injected into the rectum.. One week after the diagnosis of colitis, for each mouse,was removed 5 cm section of large intestine. Then, colon cut to length and was claened with cold saline and was weighting. Part of the colon tissue samples taken for histological studies and was established in Bowen, s fixative. Colon macroscopic damage were scoring to the Morris method [16] according to the following features from 0 to 5: No damage (0), localized damage without ulcers (1), linear ulcers without severe inflammation (2), linear ulcers with inflammation at one point (3), inflammation or sores in two places or more (4) and inflammation and ulcers in two places or more, or a large ulcer and inflammation over 1 centimeter (5). For microscopic studies, samples of colon dehydrate after being fixed in alcohol and were placed in paraffin and sections 5 microns were provided by microtome. Then, sections stained with hematoxylin - eosin. Degree of injury of krypt microscopic and inflammation severity was evaluated to the Murthy method [17]: the normal krypt (0), the loss of a third krypt (1), the loss of two thirds krypt (2) total loss of krypt and healthy epithelium (3) and total loss of epitheliumt and krypt (4). Data analysis, was done with software SPSS and ANOVA and was plotted necessary histograms. RESULTS Macroscopic studies Acetic acid (%4) injected into the rectum causes colon ulcer in animals (figure 1). Colon macroscopic damage score showed significant difference (P<0.05) in the group with colitis compared with control group (histogram1). Macroscopic damage in different groups showed the highest rate of injuries related to non-treated colitis group with silymarin. In treatment groups (10 and 20 mg/kg body weight), macroscopic damage were decreased significantly (P<0.05) (histogram 1). Tissue damage and inflammation of the colon from the microscopic view Vast damage of Intestinal epithelial cells and severe inflammation of white blood cells observed in the colitis group compared with control group that these changes are characteristic of intestinal ulcer disease (figures 2,3,4 and 5 and 3692

3 histogram 2). Comparison of microscopic inflammation score and degree of tissue damage in different groups showed that the highest score is treated to the colitis group (histogram 2). Silymarin (in the values of 10 and 20 mg/kg B.W.) reduced significantly (P<0.05) microscopic intestinal damage and inflammatory response compared with colitis group (histogram 2) figure1- The image of macroscopic of colon. Inflammation associated with bleeding is seen (2 and 3) clearly. 1 control group. 2 colitis group without treating. 3 cutting of intestine length in colitis group without treating. 4 cutting of colon length in the treatment group (10 mg/kg silymarin). 5 cutting of colon length in the treatment group (20 mg/kg silymarin) histogram1- The effect of silymarin on the macroscopic intestinal damage in colitis group induced by acetic acid *significant level (P<0.05 ) histogram 2 The effect of silymarin on the microscopic intestinal damage in colitis group induced by acetic acid. * significant level (P<0.05) 3693

4 Figure3- photomicrograph of colon in colitis group 1-the loss of epithelial cells 2-the loss of krypt 3-inflamation of white blood cells staining:hematoxilin-eosin magnification: 400x Figure2- photomicrograph of colon in colitis group In this picture, vast damage is observed in intestine mucosal tissue. submucosa -B mucosa -A magnification: 400x Staining:hematoxilin-eosin figure5- photomicrograph of colon in control group. staining: hematoxilin-eosin magnification: 400x figure4- photomicrograph of colon in colitis group treated with silymarin. staining: hematoxilin-eosin magnification: 400x DISCUSSION AND CONCLUSION Inflammatory bowel diseases, is the most common gastrointestinal diseases.colon ulcer (colitis ulcerative) is a chronic inflammatory bowel disease that inflammatory agents and free radicals have an important role in its creation [1]. Inflammation of white blood cells and macrophages to the intestinal mucosal tissues are the obvious symptoms of the disease [4]. By activated neutrophils are produced in intestinal mucosal tissues, free radicals including ion peroxide, hydrogen peroxide and hydroxyl radicals. These factors lead to lipid peroxidation, the increased permeability of blood vessels,the increasing of neutrophils enterance and inflammation development in the mucosal tissue [18]. Currently the best method of treatment is using anti-inflammatory drugs such as corticosteroids and aminosalysilates but these drugs have severe side effects [3]. So, for disease control and prevention are conducted extensive studies of natural healing techniques particularly the use of anti oxidants [5]. In the present study, the effect of antiinflammation of silymarin have been investigated in the treatment of intestinal mucosa ulcer. Recent study results showed that acetic acid causes loss of epithelial cells and intestinal krypt that is considered a microscopic signs of disease. Also, macroscopic view is seen the inflammation and ulcers in the colon wall that is another signs of disease [5]. Study results indicate that silymarin is effective in the elimination of intestinal inflammation and tissue damage. Possibly, anti-oxidant and anti-inflammatory properties of silymarin compounds have been pouring heals. Silymarin is a flavonoid compound that comes from Silybum marianum and its anti-oxidant properties is very high. These properties have been proven in laboratory animals. Anti-oxidant compounds can neutralize free radicals and prevent cell damage caused by free radicals [9,5,19]. 3694

5 Studies show silymarin inhibits nuclear transcription factors in the expression of genes involved in inflammatory processes and cancer. Research suggests that silymarin, its activities, does via TNF-α [16]. These effects are induced through the inhibition of phosphorylation and dysfunction tumor necrosis factor - alpha, which is a preinflammatory agent [20]. Tumor necrosis factor - alpha is an inflammatory agent with a significant role in the creation of inflammatory bowel disease. Increased serum levels of this factor is confirming in patients with intestinal ulcers (colitis) the same subject [21,22]. Research results conducted on the anti-inflammatory mechanism of action suggests that silymarin is able to inhibit lipopolysaccharides activating macrophages. Induction of preinflammatory proteins is carried out by these polysaccharides [22]. Besides, free radicals, prostaglandins and interleukins are effective in creating colitis ulcerative [23]. Prostaglandin-E2 as an agent chemiotaxis, are caused neutrophil accumulation in the mucosa and the formation of the active types of oxygen and oxidative damage in cells and acid arashidonic production of cell membrane phospholipids [24]. Arashidonic acid is converted by the cyclooxygenase enzymes to various prostaglandins. Cyclooxygenase is made during inflammation by chemical stimulators in the inflammated tissues [20]. Studies show, silymarin compounds (silybin, silydynein and silychristin) can be prevented the formation of prostaglandins [25]. Inhibition of prostaglandins formation is related to inhibition of the cyclooxygenas enzymes[19]. So, as colitis ulcerative as is discussed a chronic illness and also, these disease has frequent return periods, using silymarin can be effective in preventing the disease back. So,we can conclude that silymarin could have a effective role in elimination of bowel inflammation. Also, treatment with silymarin can not side effects of chemical drugs.terefore, it is suggested to used as a way to treatment and prevention of disease. Howeve, it is better to do further research works in molecular and enzymes levele. Acknowledgments The authors are indebted to the participants of Islamic Azad University, Jahrom branch. We wish to thank Saberi M. for their technical assistance. REFERENCES [1] SB. Hanauer, N. Engl. J. Med., 1996, 334: [2] SB. Hanauer, Inflamm. Bowel. Dis., 2006, 12: 3-9. [3] J. Wang, YX. Fu, Immunol. Rev,. 2005, 204: [4] L. Radko, W. Cybulski, J. Pre-Clin. Clin Res., 2007, 1(1): [5] KA. Head, JS. Jurenka, Altern. Med. Rev., 2003, 8: [6] MA. Gassull, Aliment. Pharmacol., 2004, 20: [7] BJ. Geerling, A. Badart, RW. Stockbrugger, RJ. Beumer, Eur. J. Clin. Nutr., 2000, 54(6): [8] J. Pravda, World J. Gastroenterol., 2005, 28: [9] N. Dixit, S. Baboota, K. Kohli, S. Ahmad, Indian J. Pharmacol., 2007, 39(4): [10] SK. Manna, A. Mukhopadhyay, BB. Aggarwal, J. Immunol., (12): [11] A. Feher, I. Lang, K. Nekam, Biochem. Sci., 1990, 15: [12] M. Laforge, Pharmacol., 1990, 39: [13] DA. Dermar, The Review of Natural Product, St. Louis, 2001, Lst ed. Facts and Comparisions, [14] RC. Parish, PL. Doering, Vet. Hum. Toxical., 1986, 28: [15] AR. Jurjus, NN. Khoury, J. Pharmacol._Toxicol. Methods., 2004, 50: [16] GP. Morris, PL. Beck, MS. Herridge, JL. Wallact, Gastroenterology., 1989, 96: [17] SN. Murthy, HS. Cooper, H. Shim, RS. Shah, SA. Ibrahim, DJ. Sedergran, Dig. Dis. Sci., 1993, 38(9): [18] DC. Alarcon, M. Martin, E. Maruenda, J. Pharm. Pharmacol., 1992, 44: [19] R. Saller, R. Meier, R. Brignoli, Drug., : [20] JS. Kang, YJ. Jeon, SK. Park, KH. Yang, HM. Kim, Biochem. Pharmacol., 2004, 67(1): [21] O. Brostrom, Med. Clin. North Am., 1990, 74: [22] S. Toovey, E. Hudson, WF. Hendry, Gut,. 1981, 22: [23] AG. Dalgleish, K. O Byrne, Cancer Treat. Res., 2006, 130:1-38. [24] YZ. Almallah, SW. Ewen, A. El-Tahir, NA. Mowat, PW. Brunt, TS. Sinclair, SD. Heys, O. Eremin, J. Clin. Immunol., 2002, 20(1): [25] I. Durak, R. Cetin, E. Devrim, J. Erguder, Life Sciennces., 2005, 76: