Antioxidant mediated antiulcer effect of Eupatorium triplinerve Vahl against acetic acid induced ulcerative colitis in mice

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Antioxidant mediated antiulcer effect of Eupatorium triplinerve Vahl against acetic acid induced ulcerative colitis in mice Manigandan Krishnan , Richard L. Jayaraj , Jayasekar Megala , Namasivayam Elangovan ∗ Department of Biotechnology, Periyar University, Salem 636011, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 20 October 2013 Accepted 12 December 2013 Available online xxx Keywords: Antiulcer Antioxidant Eupatorium triplinerve Myeloperoxidase activity Lipid peroxidation AgNORs

a b s t r a c t Purpose: Ulcerative colitis (UC) is associated with tainted neutrophil infiltration, deregulated proinflammatory mediators, characterized by severe oxidative stress of the intestine. In the present study, an effort was made to evaluate the effect of methanolic fractions of Eupatorium triplinerve (E. triplinerve) (ET) on acetic acid induced ulcerative colitis in male adult mice. Methods: Colitis in mice was induced with 3.0% acetic acid (v/v) in saline via rectal route. Pre-intervention with E. triplinerve extract (100 mg and 200 mg kg−1 body weight, oral) and reference drug ranitidine (50 mg kg−1 body weight used as reference, oral) 4 days before induction of colitis and was extended up to 8 days. Results: The phase of inflammation before E. triplinerve extract pre-treatment significantly showed attenuated macroscopic damage, argyrophilic nuclear organization regions (AgNORs) count and histological changes. Similarly, extract also effectively detracts the activity of both Myeloperoxidase (MPO) and Malondialdehyde (MDA) levels by enhancing the cellular antioxidant enzyme levels (Glutathione-s-transferase [GST], Glutathione peroxidise [GPx] and Catalase [CAT]) at the site of ulceration. Conclusions: The E. triplinerve based therapy resolved that some constituents in extract have an antiulcer effect against UC at colon specific area through its inviolable radical scavenging activity. © 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction Inflammatory bowel disease (IBD) is a chronic revenant immunological ailment of gastrointestinal tract associated with austere ulceration, relapse bleeding and diarrhoea [1,2]. Crohns disease and ulcerative colitis (UC) are the two sorts of IBD which eventually lead to mucosal damage and disruption of epithelial cells in large intestine. Hence, the etiology of UC remains vague, considered being multi-factorial such as diet, environment and genetic factors [3,4]. Subject with prolonged (or) chronic ulceration or inflammation at colon site may have the chance of incurring colorectal cancer [5]. Quite a few reports avowed that an overexpression of deregulated immunological mediators (Reactive oxygen species [ROS], neutrophils, macrophages, mast cells and lymphocytes) acts as an intense contribution in pathological events of UC [6–8]. Under physiological conditions, an equilibrate state was maintained between oxidants and antioxidants, but it was

∗ Corresponding author. Room No. 216 A, Department of Biotechnology, Periyar University, Salem 636011, Tamilnadu, India. Tel.: +919789097142. E-mail addresses: [email protected] (M. Krishnan), [email protected] (R.L. Jayaraj), [email protected] (J. Megala), [email protected], [email protected] (N. Elangovan).

impaired under morbid events [9]. Oxidative stress mediated by onset of free radicals through neutrophil activation renders the antioxidant system to be inadequate in epithelial cell, which can lead to cell damage and lipid peroxidation [10,11]. Due to complex pathogenesis of UC, the therapy to potentially reduce the severity or to prevent the incident rate was not yet defined. The present available therapies for UC involve treatment with antiinflammatory agents (glucocorticosteroids and 5-aminosalicylic acid), immunomodulatory agents (azathioprine, mercaptopurines and cyclosporine), but they will provoke adverse side effects [12]. Hence, plant-based remedies will navigate to find out the unknown bioactive compounds to combat several disorders [13]. Eupatorium triplinerve Vahl, an ornamental erect perennial herb with aromatic leaves belongs to the family of Asteraceae. The whole plant is used as folk medicine for the treatment of various diseases (colic, stomach pain, edema, depurative and snake bite). The biochemical constituent of E. triplinerve mainly consists of coumarin family substituted by methyl and hydroxyl side chains. Similarly, it is also reported to possess anticancer [14], antifungal, antibacterial [15], hepatoprotective [16], and neuroprotective activities [17]. Several rodent models have been established to find the effective drug that mimics the inflammatory mediator in ulcerative colitis [18,19]. However, the protective action against ulcerative condition was not yet defined so far in E. triplinerve. In this study, we

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investigated the antiulcer effect of E. triplinerve extract on acetic acid-induced colitis model in mice. 2. Materials and methods 2.1. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 5,5 dithiobis (2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), 1-chloro-2, 4-dinitrobenzene (CDNB), O-dianisidine dihydrochloride, sodium azide, trichloroacetic acid (TCA), hydrogen peroxide (H2 O2 ), ethylene diamine tetra acetic acid (EDTA), hemotoxylin, eosin and ranitidine were purchased from Sigma Chemical Co., St. Louis, MO, USA. All other chemicals are of analytical grade.

2.3.3. Induction and treatment schedule Animals were randomly divided into five groups (n = 6). Group I served as normal animals which received oral administration of saline (0.9% w/v) for 7 days. Group II animals on 4th day received intrarectal induction of 0.2 mL of 3.0% acetic acid (v/v) and allowed it for 30 seconds then 0.5 mL of saline (0.9%) was injected in order to withdraw the prior solution from the colon. Group III animals were pre-treated with standard ranitidine (50 mg/kg., p.o.) for 7 days and 0.2 mL of 3.0% acetic acid induced intrarectally on 4th day. Groups IV and V were pre-treated with E. triplinerve extract (100 mg and 200 mg/kg., p.o., based on acute toxicity study [16]) for 7 days and acetic acid was induced as per group III. Treatment in all groups was continued till 8th day. The animals were euthanized on 8th day; colon of each mouse was removed and weighed. The tissues of all groups of animal were used for analyzing damage scores, histological patterns and biochemical parameters.

2.2. Preparation of plant extract Healthy plants of E. triplinerve were collected from State Forestry Research Institute, Chennai, TamilNadu and deposited in the herbarium of Department of Biotechnology, Periyar University, Salem (PU/BT/Eupatorium triplinerve Vahl/voucher specimen No.: 012/2010). The plant leaf was washed thoroughly with running tap water and shade dried for 2–3 weeks to get consistent weight. The dried plants were grounded to coarse powder and extracted using methanol (100%, w/v) in soxhlet apparatus for 6 h. The extract was concentrated by using rotary evaporator at 40–50 ◦ C under reduced pressure. 2.3. Methods 2.3.1. Preliminary plant antioxidant screening 2.3.1.1. DPPH radical scavenging assay. 1,1-Diphenyl-2picrylhydrazyl (DPPH) radical was assayed by the method of Liyana-Pathiranan and Shahidi [20]. The reaction mixture containing 0.135 mM DPPH in methanol followed by 1.0 mL of plant extract (20–100 ␮g/mL) in methanol. The mixture was left in dark at room temperature for 30 min. The absorbance was measured at 517 nm using quercetin as standard in UVspectrophotometer (Systronics-2203, India). The DPPH percentage inhibition was calculated by the following equation: DPPH radical (%) = [(Abscontrol − Abssample )/Abscontrol ] × 100. 2.3.1.2. Ferric Reducing Antioxidant Property Assay (FRAP). FRAP was assayed as described by the method of Oyaizu Yen and Duh [21]. The reaction mixture contains different concentrations of plant extracts (20–100 ␮g/mL) in 0.2 M phosphate buffer (pH 6.6) and 1.0% ferrocyanate. The temperature of the mixture was raised to 50 ◦ C for 20 min and 2.5 mL of 10% TCA was added after centrifugation at 3000 g for 10 min. To the supernatant, equal volume of distilled water and 1.0% ferric cyanide was added. The absorbance was measured at 700 nm using quercetin as standard. The intensity of the absorbance determines the antioxidant activity of plant extract. 2.3.2. Experimental design Adult male Swiss albino mice (6–8 weeks old, weight varying from 22–26 g) were housed in polycarbonate cages at Central Animal Facility, Periyar University, Salem. They were kept in room with a 12-hour day-night cycle, temperature of 25 ± 3 ◦ C and humidity of 45–65%. They were fed with commercial mice feed and water ad libitum. The experiment was conducted after obtaining institutional animal ethical committee clearance (1085/ac/07/PUIAEC/2012/11).

2.3.4. Determination of E. triplinerve on colonic damage The severity of ulcer area on colon tissues was assessed according to the scaling grade of Morris et al. [22]. Absence of macroscopic damage: score 0, localized hyperaemia with devoid of ulcers: score 1, elongated ulcers without inflammation: score 2, ulcers with inflammation at one site: score 3, more than one site of inflammation with ulceration: score 4, extension of more than 1.0 cm length of ulcers with erosions in colonic region: score 5, respectively. 2.4. Biochemical analysis 2.4.1. Assessment of myeloperoxidase (MPO) levels Myeloperoxidase, an index of neutrophil infiltration, was assayed according to the modified method of Goldblum et al. [23]. The reaction mixtures contained 0.1 mL of tissue homogenate, 0.59 M O-dianisidine dihydrochloride (w/v) in 50 mM phosphate buffer (pH 6.0) and 0.0005% H2 O2 (v/v). The change in absorbance was read at 460 nm for one minute by using spectrophotometer. One unit of MPO activity is defined as the change in absorbance per min by 1.0 at room temperature. It was calculated by using the formula MPO activity (U/g) = X/weight of tissue taken, where X = 10 × change in absorbance/min/volume of supernatant taken. 2.4.2. Evaluation of lipid peroxidation and antioxidant enzyme contents The level of lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels by the method of Ohkawa et al. [24]. The amount of MDA was expressed as nmol/g of wet colonic tissues by using molar extinction 1.56 × 105 M1 cm−1 . The levels of glutathione peroxidase (GPx-E.C: 1.11.1.9) were measured according to Rotruck et al. [25]. The enzyme activity was measured at 420 nm and the values are expressed as n moles of GSH oxidized/min/mL of enzyme. Glutathione-S-transferase (GST-E.C: 2.5.1.18) was performed as described by Habig et al. [26]. The specific activity was expressed as micromole of CDNB-GSH conjugates formed/min/mg of protein using 9.6 as molar extinction co-efficient. The protein content was measured by the method of Lowry et al. [27]. Catalase (CAT-E.C: 1.11.1.6) was quantified by the method of Luck [28] and the enzyme activity was calculated and expressed in ␮moles of H2 O2 consumed/units/mg of protein. 2.5. Histopathological studies A portion of the colon tissue samples was fixed in 10% neutral buffered formalin solution. The fixed tissues were dehydrated in graded ethanol, hot infiltrated and embedded in paraffin wax. The embedded blocks were sectioned in microtome at 6.0 ␮m size. The sectioned tissues were placed on slides followed by deparaffinization, rehydration and then stained with hemotoxylin and eosin. The

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slides were then visualized under light microscope (Magnus MLXi, Olympus). 2.6. AgNOR staining and quantification Evaluation of silver nitrate stained nuclear organization regions in colonic tissues was performed by the method of Rosana et al. [29]. Paraffin-embedded sections (6.0 ␮m) were deparaffinised with xylene for 30 minutes, dehydrated with graded ethanol for 10 minutes time interval. After distilled water washing, slides were immersed with one volume of staining solution (50% silver nitrate, 2.0% gelatine and 1.0% formic acid) in dark for 60 minutes and then rinsed in deionised water. The stained sections were dehydrated with ethanol, xylene cleared and mounted with DPX. The appearance of brown or black dots within the nucleus or outside the nucleolus upon silver-staining was examined under light microscope (Magnus MLXi, Olympus) and the counted data were expressed as number of AgNORs/nucleus. 2.7. Statistical analysis Statistical analysis was conducted using one-way ANOVA with Dunnett’s test for multiple comparisons using SPSS version 16.0. The values are expressed as mean ± SEM of n observations, where ‘n’ represents the number of mice (n = 6 animals for each group). P < 0.05 was considered statistically significant. 3. Results In the present investigation, DPPH radical scavenging activity of E. triplinerve extracts with varying concentrations (50–200 ␮g/mL) were compared with standard (quercetin) as shown in Fig. 1A. The maximum inhibitory concentration (IC50 ) of E. triplinerve plant extract was observed to be 83.89 ␮g when compared to quercetin standard (51.28 ␮g). Similarly, FRAP scavenging property of E. triplinerve extracts was in the range of 0.604–1.0 Abs.

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The absorbance range of extracts was significantly lower than that of standard (0.0681–1.6 Abs) as shown in Fig. 1B. Hence, the above in vitro result predicts the radical scavenging property of E. triplinerve. 3.1. E. triplinerve attenuated acetic acid-induced colitis All the animals survived during the entire course of experiment. Intrarectal instillation of acetic acid (3.0%) vividly decreased the body weight and macroscopic appearances (haemorrhagic ulceration, hyperemia and mucosal edema) in all groups, whereas ranitidine and E. triplinerve treated groups showed recovery of macroscopic appearances (Fig. 2). The clinical status of macroscopic score, colon weight/length ratio and colon length was found to be significantly (P < 0.001) elevated in acetic acid-induced group as compared to normal group. Treatment with rantidine for 7 days showed mild improvement in colonic weight/length ratio whereas in E. triplinerve extract pre-treated groups, the above-mentioned factors were significantly reduced (P < 0.05) as compared to acetic acid group (Table 1). 3.2. Effect of E. triplinerve on antioxidant and stress marker enzymes Fig. 3 renders the levels of MPO and MDA in colonic tissues of experimental animals. The activities of these enzyme levels were significantly (P < 0.05) elevated in colonic tissue of acetic acid instilled mice as compared to control mice. E. triplinerve extract (100 mg and 200 mg/kg, p.o.) treatment considerably (P < 0.05) reduced the MPO and MDA activities of colonic damage when compared to acetic acid-induced animals. Hence, extract at the concentration of 200 mg/kg was found to be more potent to that

A DPPH % inhibition

100

Quercetin EC50=51.28µ g

80

Eupatorium triplinerve EC50=83.89µ g

60 40 20 0 0

50

100

150

200

250

Concentration in µ g/ml

B Absorbance at 700nm

1.5

Quercetin r 2=0.912

1.0

Eupatorium triplinerve r2= 0.901

0.5

0.0 0

50

100

150

200

250

Concentration inµ µg/ml Fig. 1. A. DPPH radical scavenging activities of E. triplinerve. B. Ferric reducing power of E. triplinerve methanol subfractions compared with standard quercetin.

Fig. 2. Effect of E. triplinerve extracts on change in body weight induced by acetic acid in mice. Treatment with ET extract for 7 days showed acceptable recovery in body weight when compared with other groups while acetic acid-induced group exhibits significant decline in body weight than that of control group (A). Macroscopic appearance of acetic-induced colon tissues in mice. No macroscopic damage in control group. Mucosal hyperemia mediated hemorrhagic ulceration with stiffened colonic walls in 3.0% acetic acid group. Ranitidine group: mucosal edema exists and ulcers can be seen. ET treated groups: macroscopic damages decreased (B).

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Fig. 3. Effect of methanolic fractions of E. triplinerve on (A) myeloperoxidase (MPO), (B) malondialdehyde (MDA) levels in acetic acid-induced colitis. Both the enzyme levels were increased after AA administration. Dose-dependent treatment with ET atrophied these parameters. Values are expressed in mean ± SEM (one-way ANOVA followed by Dunnett’s test). a P < 0.05 compared to control group; b P < 0.05 compared to AA group and # non-significant vs control group.

Fig. 4. Effect of E. triplinerve extracts on (A) catalase (CAT), (B) GPx, (C) GST enzyme levels and (D) AgNOR precipiations per nucleus in colonic tissue homogenate of experimental colitis groups. Values are expressed in mean ± SEM (one-way ANOVA followed by Dunnett’s test). a P < 0.05 compared to control group; b P < 0.05 compared to AA groups; and # non-significant compared to AA group.

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Table 1 Effect of E. triplinerve on wet weight of colonic segment, diarrhoea from mice on induced colitis. Groups

n

Colon length (cm)

Group I: control Group II: acetic acid (3.0%) Group III: ranitidine 50 mg/kg b.wt. Group IV: E.T 100 mg/kg, b.wt. Group V: E.T 200 mg/kg, b.wt.

6 6 6 6 6

10.2 5.3 7.0 6.5 6.6

± ± ± ± ±

0.2a 0.09a 0.11a 0.14a 0.09a

Colon weight/length (mg/cm) 6.41 18.62 10.59 11.48 10.34

± ± ± ± ±

0.18a 0.51a 0.23a,b 0.25a,b 0.26a,b

Macroscopic damage score (mean ± SD) 0.016 4.85 3.23 3.63 2.8

± ± ± ± ±

0.01a 0.08a 0.12a 0.10a 0.10a

Mortality (%) 0 0 0 0 0

Data were expressed as mean ± SEM. a P < 0.001 vs control group. b P < 0.05 vs 3.0% acetic acid group (one-way ANOVA followed by Dunnett’s test).

Fig. 5. Photomicrograph of argyrophilic nucleolar organizing region (AgNOR) staining in the colon of acetic acid-treated mice (A). Normal stratified colonic epithelium with poor AgNOR dots (B). Colitis-induced group showing dispersed and numerous irregularly shaped differentiated AgNORs dots in the nuclei (C). Ranitidine-treated animals showing moderately differentiated AgNORS dots in colonic epithelium. E. triplinerve extracts-treated animals (100 mg and 200 mg/kg b.wt.) showing poorly diiferentiated nuclear organization region of varying sizes (D and E) (magnification: 10×).

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Fig. 6. Representative histological slides of ulcerative colitis-induced mice. A. Normal colonic tissue receiving saline. B. Acetic acid (AA)-induced mucosal damage with chronic ulceration, loss of epithelial cells (colored arrow) and goblet cell depletion in neutrophil infiltration (bold arrow). C. Ranitidine (50 mg/kg) significantly improved AA-induced colonic damage. D. E. triplinerve extract (100 mg/kg) treated shows the normal recovery of colonic epithelial cells. E. E. triplinerve extract (200 mg/kg) treated shows significant recovery of colon damage when compared to the control (H and E stain, magnification: ×40).

of ranitidine and E. triplinerve (100 mg/kg, p.o.) extract treatment. Antioxidant enzyme plays a virile role in scavenging the direct elimination of free radicals. The level of antioxidant enzymes (GST, GPx and CAT) assessed in all experimental animals was evinced in Fig. 4A–C. It was observed that the activity of enzymes GST, GPx and CAT were found to be significantly (P < 0.05) declined in acetic acidinduced group as compared to control animals, while treatment with E. triplinerve predominates the levels of these antioxidants to normal levels as compared to acetic acid-induced animals. Based on the leads, we suggest that E. triplinerve extract at 200 mg/kg showed acceptable free radical scavenging effect as compared to 100 mg/kg extract treated group.

3.3. Effect of E. triplinerve on Microscopic pathological studies The silver-staining pattern represents the rate of cell proliferation as small black dots or clustered nucleus under diseased condition. The data on average number of AGNORs/nucleus in all experimental groups are represented in Fig. 4D. The mean of AgNORs/nucleus in acetic acid-induced group was significantly (P < 0.05) increased as compared to control group. By contrast, the colonic tissues of mice showed significant (P < 0.05) decline of AgNORs/nucleus in E. triplinerve and ranitidine-treated groups. It was observed that treatment with E. triplinerve extract has the ability to reduce the rate of cell proliferation by decreasing the

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nuclear localization staining when compared to rantidine treatment (Fig. 5). The histopathological features of colitis in the colon of control and experimental groups are shown in Fig. 6. The colon of control mice showed normal morphology of epithelial cells. Sections of mice instilled with acetic acid showed loss of epithelial cells, tissue necrosis followed by mucosal edema, diffusion of inflammatory cell infiltration in the mucosa. Pre-treatment of mice with E. triplinerve extract (100 and 200 mg/kg) and ranitidine after induction with acetic acid showed recovery of ulceration mediated mucosal damage (Fig. 6A–E).

4. Discussion Acetic acid-induced ulcerative colitis triggers severe mucosal immune response through reactive oxygen species (ROS) mediated activation of macrophages and neutrophils [30,31]. Several reports on experimental rodents posted that ROS is the main effectors molecule which destroys the membrane barrier and simultaneously terminates process of cell permeable regeneration [32,33]. The present study outlines the antioxidant and antiulcer effect of E. triplinerve methanolic fraction against acetic acid-induced experimental colitis and it was further affirmed by macroscopic, microscopic and biochemical estimations in colon tissues. Considerable increase in colon weight, weight/length ratio, body weight, macroscopic score, rectal bleeding, histological alterations and shearing of epithelial cells was well debated as an indicator for the severity of inflammation in colitis [34,35]. In this study, instillation of acetic acid (3.0%) alone causes significant elevation in colon weight, weight/length ratio, macroscopic gross lesion score, diarrhea and severe histological alterations. Treatment with E. triplinerve extract leads to dose-dependent decrease in the abovementioned parameters which indicate its antiulcer activity. In the present study, nuclear organization region was used for assessing cellular behaviour and proliferation levels [36]. Certain reports predict the thickening of epithelial cells could trigger proliferation rate by liberating excess mucin in colonic region [37,38]. It was observed that acetic acid-induced group showed increased numbers of AgNORs in colon tissues of mice because of enhanced transcriptional and cellular activity. Treatment with ET extract significantly recovered the effect of acetic acid thereby decreasing the number of AgNORs count. Although the underlying inception of UC remains unclear, increase in MPO and MDA levels are the prime biomarker for colon damage have been reviewed elsewhere [39]. MPO, an enzyme released from storage vesicles of neutrophils, catalyzes the formation of cytotoxic oxidants such as hypocholorus acid from chloride ions, hydrogen peroxide (H2 O2 ), and N-chloramine during inflammatory stimuli [40]. The increased MPO levels also enhance the levels of MDA (an indicator of lipid peroxidation) in colonic tissues and can initiate a cascade cycle of free radicals, which favors the development of inflammation and ultimately exhausts cellular antioxidant levels [41]. In this report, intrarectal instillation of acetic acid in mice elicits significantly increased level of MPO and MDA enzymes due to migration of acetic acid into colonic microflora, it could rupture the epithelial lining by producing superoxide anion and ROS. Treatment with E. triplinerve at both doses significantly restores the elevated MPO and MDA levels due to its potent anti-inflammatory effect. The imbalance between the production of ROS and their removal by antioxidants are the major causes for oxidative stress. Immune system often responds to mild oxidative stress by producing more antioxidants. However, extreme oxidative state depletes the cellular antioxidant of tissues due to overutilization to salvage the products of lipid peroxidation [42].

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It is well cognized that CAT, GST and GPx play a crucial role as protective enzymes against oxidants. CAT is an enzyme containing heme group localized in subcellular organelles of peroxisome which commutes hydrogen peroxide (H2 O2 ) to water and oxygen [43]. GPx and GST remove fatty acid hydroperoxides by conjugation its reduction with oxidation of GSH [44]. In the present work, there was depletion in the antioxidant (GST, GPx and CAT) enzyme levels was observed in the colon tissue of acetic acid-induced group. The decrease in activities of GST, GPx and CAT levels was due to depletion in antioxidant reserves of tissues in extent of the oxidative damage. Hence, supplementation of E. triplinerve extract decreased the lipid peroxidation thereby significantly increased levels of antioxidant enzymes and reduced the severity of inflammation. The results from the present study are also presumed with earlier study of its ability to enhance the activities of enzymatic antioxidants by scavenging physiologically relevant oxidative stress [45]. Our results concluded that E. triplinerve extracts possess virile activity against various inflammatory actions caused by 3.0% acetic acid in mice. Furthermore, E. triplinerve (200 mg/kg) was found to be more effective in acetic acid molds by forming a cuticle over the mucosal membrane and also recovered the colonic damage against lipid peroxidaiton. Hence, it was observed that E. triplinerve offers a promising means for the treatment of UC due to its radical scavenging action. However, further study is required to elucidate the compounds responsible for this effect and to elucidate its liable therapeutic mechanism. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements We thank University Grants Commission (UGC), New Delhi, India for financial grant in the form of Non-SAP project grant and stipend for the academic year (2011–2012). References [1] Silva da MS, Fidalgo SS, Talero E, Cardeno A, da Silva MA, Villegas W, et al. Antiinflammatory intestinal activity of Abarema cochliacarpos (Gomes) Barneby and Grimes in TNBS colitis model. J Ethnopharmacol 2010;128(2):467–75. [2] Owczarek D, Cibor D, Szczepanek M, Mach T. Biological therapy of inflammatory bowel disease. Pol Arch Med Wewn 2009;119:84–8. [3] Loftus Jr EV. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology 2004;126:1504–17. [4] Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998;115(1):182–205. [5] Nugent FW, Haggitt RC, Gilpin PA. Cancer surveillance in ulcerative colitis. Gastroenterology 1991;100:1241–8. [6] Rogler G, Andus T. Cytokines in inflammatory bowel disease. World J Surg 1998;224:382–9. [7] Nitta M, Hirata I, Toshima K, Murano M, Maemura K, Hamamoto N, et al. Expression of the EP4 prostaglandin E2 receptor subtype with rat dextran sodium sulphate colitis; suppression by a selective agonist, ONO-AEI-329. Scand J Immunol 2002;56(1):66–75. [8] Kannan N, Guruvayoorappan C. Protective effect of Bauhinia tomentosa on acetic acid induced ulcerative colitis by regulating antioxidant and inflammatory mediators. Int Immunopharmacol 2013;16:57–66. [9] Dincer Y, Erzin Y, Himmetoglu S, Gunes KN, Bal K, Akcay T. Oxidative DNA damage and antioxidant activity in patients with inflammatory bowel disease. Dig Dis Sci 2007;52(7):1636–41. [10] Yao J, Wang JY, Liu L, Li YX, Xun AY, Zeng WS, et al. Anti-oxidant effects of resveratrol on mice with DSS-induced ulcerative colitis. Arch Med Res 2010;41(4):288–94. [11] Keshavarzian A, Morgan G, Sedghi S, Gordon JH, Doria M. Role of reactive oxygen metabolites in experimental colitis. Gut 1990;31(7):786–90. [12] Xu CT, Meng SY, Pan BR. Drug therapy for ulcerative colitis. World J Gastroenterol 2004;10:2311–7. [13] Medhi B, Prakash A, Avti PK, Saikia UN, Pandhi P, Khanduja KL. Effect of manuka honey and sulfasalazine combination to promote antioxidant defense

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Please cite this article in press as: Krishnan M, et al. Antioxidant mediated antiulcer effect of Eupatorium triplinerve Vahl against acetic acid induced ulcerative colitis in mice. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2013.12.002