Protective effect of Bauhinia tomentosa on acetic acid induced ulcerative colitis by regulating antioxidant and inflammatory mediators

International Immunopharmacology 16 (2013) 57–66

Contents lists available at SciVerse ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Protective effect of Bauhinia tomentosa on acetic acid induced ulcerative colitis by regulating antioxidant and inflammatory mediators Narayanan Kannan, Chandrasekharan Guruvayoorappan ⁎ Department of Biotechnology, Karunya University, Karunya Nagar, Coimbatore, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 13 January 2013 Received in revised form 28 February 2013 Accepted 6 March 2013 Available online 26 March 2013 Keywords: Bauhinia tomentosa Ulcerative colitis Myeloperoxidase iNOS TNF-alpha

a b s t r a c t Inflammatory bowel diseases (IBD), including Crohn's disease and Ulcerative colitis (UC), are life-long and recurrent disorders of the gastrointestinal tract with unknown etiology. The present study is designed to evaluate the ameliorative effect of Bauhinia tomentosa during ulcerative colitis (UC). Three groups of animals (n = 6) were treated with B. tomentosa (5, 10, 20 mg/kg B.wt respectively) for 5 consecutive days before induction of UC. UC was induced by intracolonic injection of 3% acetic acid. The colonic mucosal injury was assessed by macroscopic scoring and histological examination. Furthermore, the mucosal content of lipid peroxidation (LPO), reduced glutathione (GSH), nitric oxide (NO), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity confirms that B. tomentosa could significantly inhibit colitis in a dose dependent manner. The myeloperoxidase (MPO), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS) expression studies and lactate dehydrogenase (LDH) assay also supported that B. tomentosa could significantly inhibit experimental colitis. The effect was comparable to the standard drug sulfasalazine. Colonic mucosal injury parallels with the result of histological and biochemical evaluations. The extracts obtained from B. tomentosa possess active substances, which exert marked protective effects in acute experimental colitis, possibly by regulating the antioxidant and inflammatory mediators. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Inflammatory bowel disease (IBD) is a chronic inflammatory disorder involving primarily the gastrointestinal tract and also includes Crohn's disease and ulcerative colitis. The pathology of IBD is characterized by poly-morphonuclear leukocyte (PMNL) infiltration, oedema, erythema, ulceration and lipid mediator release [1]. Oxidative stress has been implicated in the pathogenesis of ulcerative colitis [2,3]. Excess production of reactive oxygen metabolities such as superoxide, hydroxyl radical, hydrogen peroxide, hypochlorous acid and oxidant derivatives N-chloramines, are detected in the inflamated mucosa and they are pathogenic in inflammatory bowel disease [4]. Sustained production of reactive oxygen metabolites during colonic inflammation may overwhelm the endogenous antioxidant defense system that regulates their production leading to oxidative injury [5]. Although ulcerative colitis etiology is largely unknown the current literature suggests that multiple immune, genetic, and environmental factors influence both the initiation and progression of colitis [6]. There is evidence for an intense local immune response associated ⁎ Corresponding author at: Department of Biotechnology, Karunya University, Karunya Nagar, Coimbatore-641114, Tamil Nadu, India. Tel.: +91 9894337418. E-mail addresses: [email protected], [email protected] (C. Guruvayoorappan). 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.03.008

with recruitment of lymphocytes and macrophages followed by release of soluble cytokines and other inflammatory mediators. Subsequent activation of these cells causes a self-augmenting cycle of cytokine production, cell recruitment and inflammation [7,8]. This uncontrolled immune system activation results in a sustained massive production of cytokines such as tumor necrosis factor (TNF)-α and interleukins (IL-1β and IL-8) [9]. In addition to cytokines, leukotrienes, thromboxane, platelet-activating factor, nitric oxide and reactive oxygen species are also released from activated mucosal cells [10,11]. Most of the current therapies for inflammatory bowel diseases involve treatment with glucocorticosteroids and 5-aminosalicylic acid [10,12]. Immunosuppressive drugs have also been used to control severe illness, regardless of the more serious complications and toxic side effects associated with them [8]. Although many types of treatments have been proposed and clinically proven, additional therapeutic approaches are needed because many patients do not respond to the currently available options which demonstrate significant side effects. Plants and plant based drugs have been used to treat various diseases from time immemorial which is proved to be less toxic and free from side effects. The genus Bauhunia belongs to Leguminosae family, subfamily Caesalpiniaceae which comprises about 300 species distributed in tropical and subtropical regions. Several therapeutic properties are attributed to Bauhinia species, which includes anti-ameboic,

58

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

anti-diabetic, analgesic, anti-rheumatic and hypocholesteromic activities [13]. Bauhinia tomentosa is a shrub prevalent among south India and is widely used in ayurvedic preparations. This plant has been reported to possess antioxidant [14], gastroprotective [15], hepatoprotective [16] and antimicrobial effect [17]. Aderogba and his colleagues [18] reported that the ethanolic extract of B. tomentosa leafs contain kaempferol-7-O-rhamnoside, kaempferol-3-O-glucoside, quercetin-3-O-glucoside and quercetin-3-O-rutinoside. We had previously reported that B. tomentosa could stimulate the immune system and could act as a potential anti-inflammatory agent [19]. In the present study we had evaluated the protective effect of B. tomentosa during experimental ulcerative colitis and the possible mechanism of action. 2. Materials and methods 2.1. Preparation and administration of plant extract B. tomentosa leaves were collected from Coimbatore. The plant specimen was authenticated from Botanical survey of India, TNAU campus, Coimbatore (BSI/SRC/5/23/2011-12/Tech-756). B. tomentosa leaves were shade-dried, powdered and extracted (10 g) with 100 ml of 70% methanol by stirring overnight. The supernatant was collected after centrifugation at 5000 rpm for 10 min. The solvent was removed by evaporation and yield of the extract was found to be 8%. For in vivo studies, the extract was re-suspended in 1% gum acacia and administered at a concentration of 5 mg, 10 mg and 20 mg/kg Body weight (i.p.) respectively. 2.2. Animals Adult male Wistar rats (200–250 g) (n = 36) were purchased from Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur. The animals were kept under controlled conditions and fed with normal mouse chow (Sai Durga Feeds, Bangalore, India) and water ad libitum. All animal experiments were performed after getting prior approval from the Institutional Animal Ethics Committee (Approval No. IAEC/KU/BT/12/017), Karunya University, Coimbatore. 2.3. Chemicals Sulfasalazine was procured from Wallace pharmaceuticals, Goa, India. Lactate dehydrogenase (LDH) assay kit was procured from Bio vision, California, USA. Myeloperoxide (MPO), Tumor Necrosis Factor (TNF-α) and Inducible Nitric Oxide Synthase (iNOS) kits were purchased from USCN Life science Inc, Houston, USA. All other chemicals used were analytical reagent grade. 2.4. Experimental design Rats were divided into 6 groups (n = 6 per group). Group I were kept as normal and received no treatment. Group II, III, IV, V and VI were subjected to the induction of ulcerative colitis by intra-colonic injection of 2 ml acetic acid (3% v/v). Group II served as ulcerative colitis control group. Group III was treated with standard drug sulfasalazine (100 mg/kg. B.wt) intra-peritonealy and Group IV, V, VI were treated with B. tomentosa extract (5, 10 and 20 mg/kg. B.wt (i.p.) respectively) for 5 consecutive days before induction of ulcerative colitis. 2.5. Induction of colonic inflammation in rats All the animals (except group I) were kept fasting overnight, with access to water ad libitum and anesthetized by ether inhalation before induction of colitis. 2 ml of acetic acid (3%, v/v) were infused for 30s using a polyethylene tube (2 mm in diameter), inserted through

rectum into the colon up to a distance of 8 cm. 24 h later, blood and colon were collected after sacrificing the animals. For separation of serum, blood samples were centrifuged at 3500 rpm (10 min) and the serum collected were stored at − 20 °C. Portions of colon specimens were dissected out, washed with ice cold PBS (pH 7.2) and kept in 10% formalin for microscopic and histopathological studies. The remaining portion of colon specimens were used for biochemical studies. 2.6. Assessments of colitis 2.6.1. Macroscopic scoring For each animal, the distal 10 cm portions of the colon were removed and cut longitudinally, cleaned with physiological saline to remove fecal residues. Macroscopic inflammation scores are assigned based on the clinical features of the colon using an arbitrary scale ranging from 0-4 as follows: 0 (no macroscopic changes), 1 (mucosal erythema only), 2 (mild mucosal oedema, slight bleeding or small erosions), 3 (moderate oedema, slight bleeding ulcers or erosions) and 4 (severe ulceration, edema and tissue necrosis) [20]. 2.7. Biochemical assays The colon tissue were homogenized in 10 mmol Tris–HCl buffer (pH 7.4) and the homogenate were used for the measurement of Nitric oxide (NO), Myeloperoxidase (MPO), Lipid peroxidation (LPO), reduced glutathione (GSH) and TNF-α. 2.7.1. Determination of the effect of B. tomentosa on serum and tissue Nitric oxide (NO) level Serum and colonic NO level were estimated by using Griess reaction according to the standard method described by Green et al. [21]. 2.7.2. Determination of the effect of B. tomentosa on colon lipid peroxidation Lipid peroxidation, an indicator of mucosal injury induced by reactive oxygen species were measured as described by Ohkawa et al. [22]. Briefly, 0.5 ml of colon tissue homogenate is mixed with 2 ml of TBA reagent containing 0.375% TBA (Thiobarbituric acid), 15% trichloroacetic acid and 0.25 N HCl. The mixture was then boiled for 15 min, cooled and centrifuged (2000 rpm; 15 min). Absorbance of the supernatant was measured at 532 nm. TBARS concentration was calculated using 1,3,3,3 tatra-ethoxypropane as a standard. The results were expressed as μmol/g wet tissue weight. 2.7.3. Determination of the effect of B. tomentosa on colonic Glutathione (GSH) level The Colon GSH level was determined using the standard method explained by Moron et al. [23]. GSH in colonic tissue homogenate were allowed to react with Ellman's reagent (5, 5-Dithio-2nitrobenzoic acid) in phosphate buffer saline (PBS, pH 8.0) and the absorbance was measured at 412 nm. GSH concentration were calculated using the standard GSH. The results are expressed as nmol/g wet tissue weight. 2.7.4. Determination of the effect of B. tomentosa on superoxide dismutase (SOD) activity The determination of SOD were measured by using standard method of Marklund and Marklund [24]. Enzyme activity was calculated from the inhibition of the reduction of NBT (Nitroblue Tetrazolium). The tissue homogenate were incubated with 0.2 ml EDTA/NaCN, 0.1 ml NBT, 0.05 ml riboflavin and the absorbance was measured at 560 nm before and after illumination. The values are expressed as nmol per milligram protein.

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

2.7.5. Determination of the effect of B. tomentosa on glutathione peroxidase activity The GPx activity was measured using the standard method of Paglia and Valentine [25]. The mixture of 0.2 ml Tris–HCl buffer (pH 7), 0.2 ml EDTA, and 0.1 ml sodium azide and tissue homogenate were prepared. GSH (0.2 ml) was added to this followed by the addition of H2O2 (0.1 ml). The contents were mixed well and incubated at 37 °C for 10 min. The reaction was arrested by the addition of 0.5 ml of 10% TCA. The tubes were centrifuged and the absorbance of the supernatant was measured at 340 nm. The tissue GPx activity was expressed as μmoles of GSH utilized/min/mg protein. 2.7.6. Determination of the effect of B. tomentosa on the tissue TNF-α, iNOS and MPO level The colon tissue homogenate were used for the measurement of TNF-α, iNOS, and MPO levels according to manufacturers' instructions (USCN Life science Inc, Houston, USA). The values were expressed as pg/mg tissue (for TNF-α) and ng/g tissue (for iNOS and MPO respectively). 2.8. Histopathology analysis The Colon specimens (2 cm) collected from the animals were fixed in 10% formalin, embedded in paraffin and cut into 4 μm sections. The paraffin sections were deparafinized with xylene, hydrated and stained with hematoxylin and eosin for studying mucosal damage assessment. 2.9. Statistical analysis The results are expressed as mean ± S.D. Statistical analysis was carried out using one-way analysis of variance followed by Dunnet's

59

test using GraphPad Instat (version 3.0 for Windows 95; Graph Pad Software, San Diego, CA). p values b 0.05 were considered as statistically significant. All the experiments were performed at least three times. 3. Results 3.1. Effect of B. tomentosa on the colon morphology, macroscopic scoring and wet weight during ulcerative colitis The effect of B. tomentosa on colon morphology is presented in Fig. 1. Acetic acid caused severe colon mucosal injury and macroscopic edematous colon inflammation. The wet colon weight from the animals treated with B. tomentosa (20 mg/kg B.wt.) were found to be significantly (p b 0.01) decreased (110 mg/cm) compared with the colitis control group (172 mg/cm) where as the standard drug Sulfasalazine (100 mg/kg B.wt.) treatment showed a wet colon weight of 145 mg/cm. The wet colon weight of the normal animals were found to be 70.5 mg/cm (Table 1). Similarly the macroscopic inflammation score also showed that treatment with B. tomentosa (20 mg/kg B.wt.) could inhibit ulcerative colitis (Table 1). 3.2. Effect of B. tomentosa on colon lipid peroxidation (LPO) level during ulcerative colitis The effect of B. tomentosa on the colon lipid peroxidation level is shown in Fig. 2. Treatment with B. tomentosa (20 mg/kg B.wt.) significantly decreased the colon lipid peroxidation (3.39 nmol/mg protein) compared to colitis control group (6.43 nmol/mg protein). The lipid peroxidation in normal animals was found to be 2.36 nmol/mg protein (Fig. 2).

Fig. 1. Effect of B. tomentosa on colon morphology during ulcerative colitis. A) Normal B) ulcerative colitis control C) UC + sulfasalazine (100 mg/kg B.wt.) and D) UC + B. tomentosa (5 mg/kg B.wt) E) UC + B. tomentosa (10 mg/kg B.wt.) F) UC + B. tomentosa (20 mg/kg B.wt.).

60

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

Table 1 Effect of B. tomentosa on the macroscopic scoring and wet weight during ulcerative colitis. Colitis was induced using 3% of acetic acid and treated with B. tomentosa (5, 10, 20 mg/kg B.wt.) or standard drug sulfasalazine (100 mg/kg b.wt.). After the experimental regimen the colon was removed and the wet colon weight and the macroscopic score were noted. Values are expressed as mean ± S.D. **p b 0.01 compared with colitis alone group. Treatment

Macroscopic score

% Reduction

Wet colon weight (mg/cm)

Normal Colitis alone Colitis + sulfasalazine Colitis + Bauhinia tomentosa 5 mg/kg B.wt Colitis + Bauhinia tomentosa 10 mg/kg B.wt Colitis + Bauhinia tomentosa 20 mg/kg B.wt

– 3.90 ± 0.10 2.25 ± 0.25 2.65 ± 0.40

– 0 42.30 32.05

70.5 172 145 152

2.15 ± 0.20

44.87

125 ± 8.5**

1.95 ± 0.30**

50.00

110 ± 8.0**

± ± ± ±

4.1 11.4 9.5** 11.6**

3.3. Effect of B. tomentosa on colon GSH, SOD and GPx level during ulcerative colitis Effect of B. tomentosa on Colon GSH, SOD and GPx level is shown in Table 2. The decreased colonic GSH in the colitis control group (33.91 nmol/mg protein) was found to be significantly (p b 0.05) increased (47.53 nmol/mg protein) after B. tomentosa treatment (20 mg/kg B.wt.). Similarly the decreased SOD (943.01 nmol/mg protein) and GPx (27.08 μg of GSH utilized/min/mg protein) level in the colitis control group was found to be increased to 1193.59 nmol/mg protein and 45.10 μg of GSH utilized/min/mg protein respectively. The normal level of colon GSH, SOD and GPx was found to be 60.53 nmol/mg protein, 1239.20 nmol/mg protein and 59.24 μg of GSH utilized/min/mg protein respectively (Table 2).

Table 2 Effect of B. tomentosa on colon GSH, SOD and GPx level during ulcerative colitis. Colitis was induced using 3% of acetic acid and treated with B. tomentosa (5, 10, 20 mg/kg B.wt) or standard drug sulfasalazine (100 mg/kg b.wt.). After the experimental regimen the colon was removed and used for the estimation of GSH, SOD and GPx. Values are expressed as mean ± S.D. *p b 0.05, **p b 0.01 compared with colitis alone group. Treatment

GSH (nmol/mg protein)

Normal Colitis alone Colitis + sulfasalazine Colitis + Bauhinia tomentosa 5 mg/kg B.wt Colitis + Bauhinia tomentosa 10 mg/kg B.wt Colitis + Bauhinia tomentosa 20 mg/kg B.wt

60.53 33.91 56.10 37.90

± ± ± ±

SOD (nmol/mg protein)

GPx (μg of GSH utilized/min/mg protein)

7.60 1239.20 ± 155.90 59.24 6.10 943.01 ± 120.20 27.08 8.20** 1184.00 ± 76.10** 41.40 6.60 985.00 ± 67.50 34.33

± ± ± ±

3.80 1.80 0.90** 1.20**

42.37 ± 13.30

1023.48 ± 97.10

41.99 ± 0.70**

47.53 ± 9.76*

1193.59 ± 54.30** 45.10 ± 1.80**

NO production and iNOS expression in the colon during ulcerative colitis. Treatment with B. tomentosa significantly inhibited acetic acid induced NO production (both in tissue and serum) and iNOS expression in colon. The increased level of NO in the colon tissue was found to be 211.4 μM where as treatment with B. tomentosa significantly (p b 0.01) inhibited the NO production (175.8 μM) (Fig. 4). Similarly the serum nitric oxide level was also found to be decreased to 50.74 μM after B. tomentosa treatment compared with colitis control group (78.08 μM) (Fig. 3). The iNOS expression studies showed significant inhibition after B. tomentosa treatment in a dose dependent manner (Fig. 5).

3.4. Effect of B. tomentosa on the NO level and iNOS expression during ulcerative colitis

3.5. Effect of B. tomentosa on colon tumor necrosis factor-α (TNF-α), Myeloperoxidase (MPO) and lactate dehydrogenase (LDH) level during ulcerative colitis

Nitric acid (NO) production and inducible nitric oxide synthase (iNOS) expression in colonic tissue appears during active ulcerative colitis, and these are present in excess to play the proinflammatory role [26]. Consequently, we estimated the effect of B. tomentosa on

Effect of B. tomentosa on colon TNF-α, MPO and LDH level is shown in Figs. 6, 7 and 8 respectively. The increased TNF-α production during ulcerative colitis (135.41 pg/mg tissue) was found to be decreased by the treatment with B. tomentosa in a dose dependent manner. B. tomentosa

Fig. 2. Effect of B. tomentosa on colon lipid peroxidation (LPO) level during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. *p b 0.05, **p b 0.01 compared with colitis control group.

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

61

Fig. 3. Effect of B. tomentosa on the tissue NO level during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. *p b 0.05, **p b 0.01 compared with colitis control group.

at a concentration of 20 mg/kg B.wt. could reduce the TNF-α level to 40.91 pg/mg tissue. Similarly the MPO and LDH level was also found to be decreased to 26.30 ng/g tissue and 787.67 U/L respectively after B. tomentosa treatment (20 mg/kg B.wt). The MPO and LDH level in colitis control group was found to be 92.18 ng/g tissue and 2046.67 U/L respectively. The standard drug sulfasalazine could significantly reduce the TNF-α, MPO and LDH level to 42.47 pg/mg tissue, 27.17 ng/g tissue and 742.67 U/L respectively. 3.6. Effect of B. tomentosa on histological changes during ulcerative colitis Effect of B. tomentosa on histological changes during colitis is shown in Fig. 9. The normal colon mucosa (Fig. 9A) showed no inflammation or necrosis. The colitis control animals showed ulceration

of colonic mucosa, diffuse inflammatory cell infiltration in the mucosa. The crypts were found to be distorted and there was loss of epithelium (Fig. 9B). Treatment with B. tomentosa (Fig. 9C) and sulfasalazine (Fig. 9D) showed marked reduction in the severity of cell damage. 4. Discussion Inflammatory bowel diseases (IBD), including Crohn's disease and Ulcerative colitis, are life-long and recurrent disorders of the gastrointestinal tract with unknown etiology. Although ulcerative colitis etiology is largely unknown literature suggests that multiple immune, genetic and environmental factors influence both the initiation and progression of colitis [12]. Ulcerative colitis has been shown to increase the risk of colorectal cancer [27]. There is evidence for an intense local immune response associated with infiltration of lymphocytes and

Fig. 4. Effect of B. tomentosa on the serum NO level during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. **p b 0.01 compared with colitis control group.

62

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

Fig. 5. Effect of B. tomentosa on the iNOS expression during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. Colon tissues were dissected out, washed with ice cold phosphate buffered saline (pH 7.4) and used for the estimation of iNOS expression studies. **p b 0.01 compared with colitis control group.

macrophages into the mucosa followed by release of reactive oxygen species, cytokines and other inflammatory mediators resulting in tissue damage, which recruits additional inflammatory cells to the site of repair [27]. The increase in NO during colitis leads to an increased production of peroxynitrite which ultimately results in oxidation of lipids and proteins, DNA strand breaks, depletion of adenosine triphosphate and tissue damage [28]. Antioxidants such as SOD, GPx and GSH impart protection against oxidative damage produced by reactive oxygen species and nitrogen species [29]. In the present study, the effect B. tomentosa against acetic acid-induced colitis was examined. The decrease in colon wet weight can be directly correlated with the degree of inflammation [30]. The present study showed that treatment with B. tomentosa could reduce the colon wet weight and macroscopic lesion score compared with colitis control group. Recently, many

evidences have indicated that a common link between inflammatory bowel disease may be related with oxidative stress [31]. The free radicals produced during oxidative damage may lead to biological membrane lipid peroxidation, resulting in severe cell damage and play a significant role in the pathogenesis of disease. Therefore receiving free radical scavengers and reducing oxidative stress may benefit for reducing ulcerative colitis [31]. In this study, colitis control animals exhibited increased levels of LPO in colon tissue. B. tomentosa treatment significantly reduced the increase of LPO levels. The results could explain the inhibited activity of ulcerative colitis by B. tomentosa treatment may be related, partially at least, to the antioxidant and free radical scavenging ability. GSH is an important intracellular defense antioxidant agent in mammalian gut. GSH is involved in the repair mechanism, inhibits free radical damage, and enhances the antioxidant

Fig. 6. Effect of B. tomentosa on colon Tumor necrosis factor-α (TNF-α) during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. Colon tissues were dissected out, washed with ice cold phosphate buffered saline (pH 7.4) and used for the estimation of TNF-α. **p b 0.01 compared with colitis control group.

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

63

Fig. 7. Effect of B. tomentosa on colon myeloperoxidase (MPO) during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. Colon tissues were dissected out, washed with ice cold phosphate buffered saline (pH 7.4) and used for the estimation of MPO. **p b 0.01 compared with colitis control group.

activity of Vitamin C. Intracellular GSH is the main reducing agent for colonic epithelial cells which neutralize hydrogen peroxide. During inflammation, GSH level depletes resulting in severe degradation of jejunum and colon mucosa. Depletion of GSH may result in increased MDA, an end product of lipid peroxidation which ultimately results in cellular damage. Therefore, GSH plays a vital role in protecting the intestinal cells and as a defense mechanism against inflammation [32]. Treatment with B. tomentosa significantly increased the colonic GSH levels, preventing the manifestation of mucosal injury during inflammatory bowel disease. Superoxide dismutase (SOD) is a key enzyme which inactivates superoxide ion by transforming it into more stable metabolite, hydrogen peroxide. This H2O2 is further converted to water by

catalase or GPx. The activities of these enzymes usually are balanced which maintains a steady state of ROS. The SOD level was found to be decreased during ulcerative colitis. Treatment with B. tomentosa significantly increased the colon SOD level in a dose dependent manner. This increased level could prevent the adhesion of circulating leucocytes to intestinal endothelium by blocking the expression of adhesion molecules such as vascular cell adhesion molecule and intracellular adhesion molecule and this needs further investigation. GPx is an antioxidant enzyme that helps in the scavenging and inactivation of free radicals thereby protecting the body against oxidative stress. GPx catalyze the reduction of H2O2 and a wide variety of organic peroxides (R-OOH) to the corresponding stable alcohols (R-OH) and water using

Fig. 8. Effect of B. tomentosa on colon lactate dehydrogenase (LDH) during ulcerative colitis. Values are expressed as mean ± SD. B. tomentosa was administered once daily for 5 days before induction of colitis and 24 h after fasting colitis was induced with 3% of acetic acid. Colon tissues were dissected out, washed with ice cold phosphate buffered saline (pH 7.4) and used for the estimation of LDH. **p b 0.01 compared with colitis control group.

64

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

Fig. 9. Effect of B.tomentosa on colon histology. A) Specimen from a normal rat showing colon with normal mucosa. B) Control specimen from acetic acid induced host not related with extract showing colitis with large necrotic destruction of epithelial cells, areas of hemorrhage, submucosal edema and inflammatory cellular infiltration. C) Colitis + B. tomentosa (20 mg/kg B.wt). D) Colitis + sulfasalazine (100 mg/kg B.wt.) (40× magnification).

cellular GSH as the reducing agent. There are reports which show that the cytosolic GPx activity in rat colon tissue is altered in response to oxidative stress with associated disturbances in prostaglandin synthesis that are modulated by anti-inflammatory salicylates [33]. The present study shows that B. tomentosa could significantly decrease the colon GPx, thereby preventing oxidant mediated colon tissue damage. Myeloperoxidase (MPO) is an enzyme most abundantly secreted in neutrophils and stored in azurophilic granules of the neutrophils. It is also secreted at lower concentration in monocytes and macrophages [34]. The level of MPO activity is directly proportional to tissue neutrophil content, thereby it produces more hypochlorous acid which is cytotoxic [35,36]. The present study showed that oxidative damage associated with induction of acetic acid resulted in an increased in MPO activity, as reported previously [37–39]. A significant reduction in MPO by treatment with B. tomentosa extract exhibits the intestinal anti-inflammatory effect in the experimental colitis model. Nitric oxide (NO) is an important proinflammatory mediator that is largely produced by three isoforms of NOS: neuronal NOS (NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (NOS3) [40]. The nitric oxide and iNOS has been reported as potential mediators for colitis [41,26]. However the exact role of NO in intestinal inflammation is unknown. The uncontrolled inflammatory response and oxidative stress could induce dramatic changes in colon tissue morphology and intestinal microvascular function during colitis. The most noticeable changes associated with colitis are enhanced interstitial edema, increased arteriolar blood flow, fluid exudation across intestinal capillaries, thickening of the intestinal wall, and colon inflammation, all of

which are associated with inflammatory mediators such as NO [42]. The over production of NO through iNOS upregulation by the intestinal epithelium has a direct role in the regulation of disease-specific, NO-related genes that are involved in NO-related inflammation during ulcerative colitis [43]. The present study showed that administration of B. tomentosa significantly inhibited the NO production and iNOS expression, which ultimately results in the prevention of peroxynitrite formation from inflammatory cells such as macrophages, neutrophils and endothelial cells, which counter inflammation during ulcerative colitis. Ulcerative colitis has been associated with an intense local immune response which is associated with the recruitment of lymphocytes and macrophages followed by release of soluble cytokines and inflammatory mediators [9]. TNF-α, an important proinflammatory cytokine released from the macrophages and lymphocytes in the early inflammatory response plays an important role to activation and nuclear translocation of NF-kB, a heterodimeric transcription factor that translocates to the nucleus and mediates the transcription of a vast array of proteins involved in cell survival and proliferation as well as inflammatory response. TNF-α has been reported to play an integral role in the pathogenesis of inflammatory bowel disease [44]. Blocking TNF- α has been shown to inhibit colitis and colorectal cancer in animal models [45]. Treatment with B. tomentosa significantly inhibited the TNF- α production in a dose dependent manner. B. tomentosa has been reported to possess phytochemical constituents such as Kaempferlol-3-O-rhamnoside, Kaempferlol-3O-rutinoside, quercerin 3-O-glucoside and quercerin 3-O-rutinoside (rutin). Kaempferlol-3-O-rhamnoside, one of the active components

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

present in B. tomentosa has been shown to inhibit cancer cell proliferation and promote apoptosis by the activation of the caspase signaling cascade [46]. Another compound Quercetin 3-O-rutinoside commonly known as rutin has been shown to exhibit antitumour activity by targeting mitochondrial apoptotic pathway [47]. Rutin has been reported to inhibit B16F-10 induced experimental lung metastasis in C57BL/6 animals [48] and could reduce NF-kappa B, TNF-α and caspase-3 expression during renal inflammation [49]. Kwon et al. [50] had shown that rutin could be useful for the prevention and treatment of inflammatory bowel disease and colorectal carcinogenesis through the attenuation of proinflammatory cytokine production. Further rutin (Quercetin 3-O-rutinoside) has been reported to inhibit experimental colitis by inhibiting TNF-α dependent NF kappa B activation [51]. In conclusion, the present study shows that B. tomentosa could inhibit colitis by regulating antioxidant mediators and inhibiting the myeloperoxidase, nitric oxide (NO) production, inducible nitric oxide (iNOS) expression and TNF-α production. Previously we were able to show that plant based drugs such as β-carotene and amentoflavone could alter proinflammatory cytokine production and could inhibit the activation and nuclear translocation of p65, p50, c-Rel subunits of nuclear factor-kappa B, and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein (CREB) [52,53]. Similarly the presence of phytochemicals such as Kaempferlol-3-O-rhamnoside, Kaempferlol-3-O-rutinoside, Quercetin 3-O-glucoside and quercerin 3-O-rutinoside might have multifunctional targets on the activation and nuclear translocation of transcription factors thereby inhibiting ulcerative colitis which needs further investigation. Acknowledgment We are grateful and thank Indian Council of Medical Research (ICMR), Government of India, New Delhi for their financial support in the Senior Research Fellowship (Ref no; 45/33/2009/BMS/TRM). The valuable support of Dr. V.M. Berlin Grace and Dr. M. Patrck Gomez from the School of Biotechnology and Health Sciences, Karunya University is gratefully acknowledged. References [1] Frettland DJ, Djuric SW, Gaginella TS. Eicosanoids and inflammatory bowel disease: regulation and plgspects for therapy. Prostaglandins Leukot Essent Fat Acids 1990;4l:215–33. [2] Keshavarzian A, Morgan G, Sedghi S, Doria M. Role of reactive oxygen metabolites in experimental colitis. Gut 1990;31:786–90. [3] Kitahora T, Suzuki K, Asakura H, Yoshida T, Suematsu M, Watanabe M, et al. Activeoxygen species generated by monocytes and polymorphonuclear cells in Crohn's disease. Dig Dis Sci 1988;33:951–5. [4] Keshavarzian A, Sedghi S, Kanofsky J, List T, Robinson C, Ibrahim C. Excessive production of reactive oxygen metabolites by inflammed colon: analysis by chemiluminescence probe. Gastroenterology 1992;103:117–85. [5] Blau N, Thony B, Renneberg A, Penzien JM, Hyland K, Hoffmann G. Variant of dihydropteridine reductase deficiency without hyperphenylalaninemia: effect of oral phenylalanine loading. J Inherit Metab Dis 1999;22:216–20. [6] Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998;115(1):182–205. [7] Sartor RB. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases. Am J Gastroenterol 1997;92:5–11. [8] Shanahan P, Hutchinson M, Bohan A, O' Donoghue D, Sheahan K, Owens A. Hemichorea, moya-moya, and ulcerative colitis. Mov Disord 2001;16(3):570–2. [9] Ogata H, Hibi T. Does an elemental diet affect operation and/or recurrence rate in Crohn's disease in Japan? J Gastroenterol 2003;38:1019–21. [10] Podolsky DK. Inflammatory bowel disease.N Engl J Med 1991;325:928–37 [26]. [11] Woywodt A, Ludwig D, Neustock P, Kruse A, Schwarting K, Jantschek G, et al. Mucosal cytokine expression, cellular markers and adhesion molecules in inflammatory bowel disease. Eur J Gastroenterol Hepatol 1999;11:267–76. [12] Strober W. Interactions between epithelial cells and immune cells in the intestine.Ann N Y Acad Sci 1998;859:37–45 [17]. [13] Viana EP, Santa-Rosa RS, Almeida SSMS, Santos LS. Constituents of the stem bark of Bauhinia guianensis. Fitoterapia 1999;70(1):111–2. [14] Khalil NM, Pepato MT, Brunetti IL. Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides. Biol Res 2008;41:165–71.

65

[15] Gonzalez-Mujica F, Motta N, Márquez AH, Capote-Zulueta J. Effects of Bauhinia megalandra aqueous leaf extract on intestinal glucose absorption and uptake by enterocyte brush border membrane vesicles. Fitoterapia 2003;74:84–90. [16] Bodakhe SH, Ram A. Hepatoprotective properties of Bauhinia variegata bark extract. Yakugaku Zasshi 2007;127:1503–7. [17] Gopalakrishnan S, Vadivel E. Antibacterial and antifungal activity of the bark of Bauhinia tomentosa linn. Int J Pharm Sci 2011;2:104–9. [18] Aderogba MA, Mc Gaw LJ, Ogundaini AO, Eloff JN. Cytotoxicity study of antioxidant flavonoids from Bauhinia tomentosa leaf extract. Niger J Nat Prod Med 2008;12:50–4. [19] Kannan N, Renitta RE, Guruvayoorappan C. Bauhinia tomentosa stimulates the immune system and scavenges free radical generation in vitro. J Basic Clin Physiol Pharmacol 2010;21(2):157–68. [20] Millar AD, Rampton DS, Chander CL, Claxson AWD, Blake DR. Evaluating the antioxidant potential of new treatments for inflammatory bowel disease in a rat model of colitis. Gut 1996;39:407–15. [21] Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131–8. [22] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [23] Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979;582(1):67–78. [24] Marklund S, Marklund G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and convenient assay for superoxide dismutase. Eur J Biochem 1974;47:69–74. [25] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70(1): 158–69. [26] Guslandi M. Nitric oxide and inflammatory bowel diseases. Eur J Clin Invest 1998;28:904–7. [27] Sunkara S, Swanson G, Forsyth CB, Keshavarzian A. Chronic inflammation and malignancy in ulcerative colitis. Ulcers 2011:1–8. [28] Kimura W, Makuuchi M, Kuroda A. Characteristics and treatment of mucinproducing tumor of the pancreas. Hepatogastroenterology 1998;45:2001–8. [29] Danese S, Mantovani A. Inflammatory bowel disease and intestinal cancer: a paradigm of the Yin-Yang interplay between inflammation and cancer. Oncogene 2010;29:3313–23. [30] Rachmilewitz D, Simon PL, Schwartz LW, Griswold DE, Fondacaro JD, Wasserman MA. Inflammatory mediators of experimental colitis in rats. Gastroenterology 1989;97:326–37. [31] Jena G, Trivedi PP, Sandala B. Oxidative stress in ulcerative colitis: an old concept but a new concern. Free Radic Res 2012;46(11):1339–45. [32] Chavan S, Sava L, Saxena V, Pillai S, Sontakke A, Ingole D. Reduced glutathione: importance of specimen collection. Indian J Clin Biochem 2005;20:150–2. [33] Drew JE, Arthur JR, Farquharson AJ, Russel WR, Morrice PC, Duthie GG. Salicylic acid modulates oxidative stress and glutathione peroxidase activity in the rat colon. Biochem Pharmacol 2005;70:888–93. [34] Klebanoff SJ. Myeloperoxidase: friend and foe. J Leukoc Biol 2005;77:598–625. [35] Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J Pharmacol Methods 1985;14:157–67. [36] Higa A, Eto T, Nawa Y. Evaluation of the role of neutrophils in the pathogenesis of acetic acid induced colitis in mice. Scand J Gastroenterol 1997;32:564–8. [37] Galvez J, Garrido M, Merlos M, Torres MI, Zarzuelo A. Intestinal anti inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat. Br J Pharmacol 2000;130:1949–59. [38] Paiva LA, Gurgel LA, De Sousa ET, Silveira ER, Silva RM, Santos FA, et al. Protective effect of Copaifera langsdorffii oleo-resin against acetic acid-induced colitis in rats. J Ethnopharmacol 2004;93(1):51–6. [39] Islam MS, Murata T, Fujisawa M, Nagasaka R, Ushio H, Bari AM, et al. Antiinflammatory effects of phytosteryl ferulates in colitis induced by dextran sulphate sodium in mice. Br J Pharmacol 2008;154:812–24. [40] Prabhu VV, Guruvayoorappan C. Nitric oxide: pros and cons in tumor progression. Immunopharmacol Immunotoxicol 2010;32:387–92. [41] Rachmilewitz D, Karmeli F, Okon E, Bursztyn M. Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. Gut 1995;37:247–55. [42] Gillberg L, Varsanyi M, Sjostrom M, Lordal M, Lindholm J, Hellstrom PM. Nitric oxide pathway-related gene alterations in inflammatory bowel disease. Scand J Gastroenterol 2012;47:1283–97. [43] Kolios G, Valatas V, Ward SG. Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle. Immunology 2004;113:427–37. [44] Sands BE, Kaplan GG. The role of TNF alpha in ulcerative colitis. J Clin Pharmacol 2007;47:930–41. [45] Popivanova BK, Kitamura K, Wu Y, Kondo T, Kagaya T, Kaneko S, et al. Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis. J Clin Invest 2008;118(2):560–70. [46] Diantini A, Subarnas A, Lestari K, Halimah E, Susilawati Y, Supriyatna Julaeha E, et al. Kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway. Oncol Lett 2012;3(5):1069–72. [47] Samanta SK, Bhattacharya K, Mandal C, Pal BC. Identification and quantification of the active component quercetin 3-O-rutinoside from Barringtonia racemosa, targets mitochondrial apoptotic pathway in acute lymphoblastic leukemia. J Asian Nat Prod Res 2010;12(8):639–48.

66

N. Kannan, C. Guruvayoorappan / International Immunopharmacology 16 (2013) 57–66

[48] Menon LG, Kuttan R, Kuttan G. Inhibition of lung metastasis in mice induced by B16F10 melanoma cells by polyphenolic compounds. Cancer Lett 1995;95(1–2): 221–5. [49] Arjumand W, Seth A, Sultana S. Rutin attenuates cisplatin induced renal inflammation and apoptosis by reducing NFκB, TNF-α and caspase-3 expression in wistar rats. Food Chem Toxicol 2011;49(9):2013–21. [50] Kwon KH, Murakami A, Tanaka T, Ohigashi H. Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression. Biochem Pharmacol 2005;69(3):395–406.

[51] Kim H, Kong H, Choi B, Yang Y, Kim Y, Lim MJ, et al. Metabolic and pharmacological properties of rutin, a dietary quercetin glycoside, for treatment of inflammatory bowel disease. Pharm Res 2005;22(9):1499–509. [52] Guruvayoorappan C, Kuttan G. Beta-carotene inhibits tumor-specific angiogenesis by altering the cytokine profile and inhibits the nuclear translocation of transcription factors in B16F-10 melanoma cells. Integr Cancer Ther 2007;6(3):258–70. [53] Guruvayoorappan C, Kuttan G. Inhibition of tumor specific angiogenesis by amentoflavone. Biochemistry (Mosc) 2008;73(2):209–18.