Inflammopharmacol DOI 10.1007/s10787-015-0234-3
Inflammopharmacology
RESEARCH ARTICLE
Evaluation of the protective effect of Pterocarpus marsupium on acetic acid-induced ulcerative colitis in rats Merin Maria Mathew1 • Nguyen Vinh Han1 • A. Murugesan1 • E. Arun Raj1 K. G. Prasanth1
•
Received: 12 March 2015 / Accepted: 17 April 2015 Springer Basel 2015
Abstract The aim of the study was to evaluate the effect of Pterocarpus marsupium (PM) on acetic acid (AA)-induced ulcerative colitis (UC) in rats. The rats were divided into five groups, each having six rats. PM extract 100 mg and 200 mg/ kg was given orally to groups four and five, respectively, and standard drug sulfasalazine (100 mg/kg, p.o) to group three. Group two served as UC control animals, and group one control animals received vehicle for 7 days. UC was induced by administering AA (3 % v/v of 2 ml) to all the animals except group one. After 72 h, the animals were killed and the colon was dissected out for microscopic, clinical evaluation, histopathological study and biochemical estimation. PM (100 and 200 mg/kg)-treated group had significantly reduced colon inflammation and mucosal damage. The treatment also normalized the altered antioxidant enzyme levels (LPO, SOD and GSH). Histopathological studies support the effect. The protective effect of PM may be due to antiinflammatory and antioxidant properties. Keywords Acetic acid Ulcerative colitis Pterocarpus marsupium Mucosal damage
Introduction Inflammatory bowel disease (IBD) comprises a group of inflammatory conditions of the colon and small intestine. The major types of IBD are Crohn’s disease and ulcerative
& K. G. Prasanth [email protected] 1
Department of Pharmacology, PSG College of Pharmacy, PO BOX NO 1674, Peelamedu, Coimbatore 641004, Tamilnadu, India
colitis (UC). Both are common in urban areas rather than rural areas with an unknown etiology. The chance of getting colon and bowel cancers are high in patients with UC (Ponder and Long 2013). The main difference between Crohn’s disease and UC is the location and nature of the inflammatory changes. Crohn’s disease can affect any part of the gastrointestinal tract, from mouth to anus, although a majority of the cases start in the terminal ileum. Ulcerative colitis, in contrast, is restricted to the colon and the rectum (Sood et al. 2003). Ulcerative colitis is treated with antiinflammatory drugs, immunosuppressants and biological therapy targeting specific components of the immune response. Glucocorticoids and aminosalicylate, immunomodulators, antibiotics and anti-TNF therapy have been widely used for the treatment of inflammatory bowel diseases (Pravda 2005). But these drugs cause major side effects. Aminosalicylates produce nausea, vomiting, headaches, rash, fever, agranulocytosis, pancreatitis, nephritis and male infertility. In addition, the sulfa portion of the drug interferes with folic acid absorption. The side effects of steroids used for short term include weight gain, mood swings and fluid retention and for long term include osteoporosis, risk of cataract, myopathy, adrenal insufficiency and immune suppression (Habal and Greenberg 1988). Antibiotics have been prescribed for ulcerative colitis, but it is mostly largely ineffective. Immunomodulatory drugs produce pancreatitis, fever, nausea, rashes, arthralgias and diarrhea. Infliximab, a TNF therapy, is associated with risk for infections, which may be fatal (Rajinikanth et al. 2014; Mahgoub 2003). The side effects of chemical drugs remain a major clinical problem. So, plant remedies have become increasingly popular worldwide, and medicinal plants are believed to be an important source of new chemical substances. Thus, emphasis is now given on the
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standardization of herbal medication by screening of biological activities of medicinal plants and isolating active principles from them (Rahman et al. 2006). The use of herbal medicine plays an important role in the therapy of many inflammatory disease conditions, including inflammatory bowel disease (Medhi et al. 2008). Pterocarpus marsupium Roxb. (Leguminosae) is a plant drug used in the preparation of rasayana in the Ayurvedic system of medicine. Rasayana preparations are used as immunomodulators and relieve stress in the body by the Ayurvedic system of treatment (Gairola et al. 2010). In Ayurveda, an aqueous extract of heartwood of P. marsupium is used in the treatment of diabetes. P.marsupium was not tested for its protective action against inflammatory bowel disease, though it has been reported to possess potent antioxidant (Mohammadi et al. 2009) and antiinflammatory (Hougee et al. 2005) activities. Various animal models of experimental colitis to screen drugs effective against inflammatory bowel disease have been established, and acetic acid-induced colitis is an animal model mimics some of the acute inflammatory responses seen in ulcerative colitis (Thippeswamy et al. 2011). Hence, the present study was designed to evaluate the protective effect of Pterocarpus marsupium marketed capsules in the rat model of colitis induced by acetic acid.
Materials and method Pterocarpus marsupium capsules which contain freeze-dried aqueous extract of wood of Pterocarpus marsupium were purchased from a medical shop. Sulfasalazine (SRINIVASA Medicals, Coimbatore), Hemoccult kit (HEMOSPOT, Carewell Scientific Distributors, Coimbatore), 5,5-dithiobis-2nitrobenzoic acid, hexadecyltrimethylammonium bromide, dianisidine hydrochloride, thiobarbituric acid and lactate dehydrogenase were purchased. All other chemicals used were of analytical grade. Female Wistar albino rats (200–250 g) were obtained from the animal house of PSGIMS&R, Coimbatore, Tamilnadu. All the protocols were approved by the institutional animal ethical committee of PSGIMS&R. The animals were maintained under standard conditions (12 h light/dark cycle; 25 ± 3 C, 45–65 % humidity) and had free access to standard rat feed and water ad libitum. All the animals were adjusted to laboratory conditions for a week before commencement of the experiment. The experiments were performed during the light portion between 8:00 a.m and 12:00 a.m. to avoid circadian influences. Animal studies were performed according to the prescribed guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (235/2014/IAEC).
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Different doses of P.marsupium and the standard sulfasalazine were prepared as suspensions in distilled water using sodium carboxymethyl cellulose (sodium CMC, 0.3 % w/v). Two doses of P.marsupium (100 and 200 mg/ kg) were selected for administration in oral route based on an earlier study (Halagappa et al. 2010). Experimental animals were divided into five groups of six animals each. • • • • •
Group I served as normal control and received vehicle (sodium CMC, 0.3 % w/v). Group II served as colitis control and received only vehicle (sodium CMC, 0.3 % w/v). Group III was treated with standard sulfasalazine (100 mg/kg, p.o). Group IV was treated with P.marsupium (100 mg/kg, p.o). Group V was treated with P.marsupium (200 mg/kg, p.o).
All these treatments were given orally for 7 days by oral gavage. On the 4th day of the treatment, the animals were fasted overnight with access to water ad libitum. On the 5th day after 1 h of the aforementioned treatments, the animals (groups II, III, IV and V) were anesthetized by ether inhalation and a polypropylene tube with 2 mm diameter was inserted through the rectum into the colon to a distance of 8 cm. A solution of 2 ml of AA (3 %, v/v) in 0.9 % saline was instilled into the lumen of the colon and maintained in a supine Trendelenburg position for 30 s to prevent the leakage of the intracolonic instillation. After 72 h of single dose administration of AA (8th day), (Mascolo et al. 1995), clinical activity scores were measured and the animals were anesthetized with ether and blood was collected by retroorbital puncture for biochemical estimation. The animals were killed by cervical dislocation and the colon was dissected out. The colon was cleaned gently with saline and weighed. It was used for macroscopic scoring and histopathological and biochemical estimations.
Evaluation of ulcerative colitis Clinical activity score Colitis was evaluated with a clinical score assessing weight loss, stool consistency and bleeding of the colon (measured by guaiac reaction; Hemoccult test) as described previously (Thippeswamy et al. 2011). No weight loss was counted as zero point, weight loss of 1–5 % as one point, 5–10 % as two points, 10–20 % as three points and 20 % as four points. For stool consistency, the stool were collected in a thick paper and observed, zero points were given for wellformed pellets, two points for pasty and semi -formed stools that did not stick to the anus and four points for
Evaluation of the protective effect of…
liquid stools that did stick to the anus. Bleeding was scored zero points for no blood in Hemoccult, two points for positive Hemoccult and four points for gross bleeding. These scores were added and divided by three, forming a total clinical score that ranged from 0.0 (healthy) to 4.0 (maximal activity of colitis).
addition of 0.25 ml of n-butanol. The mixture was allowed to stand for 10 min and centrifuged. 1.5 ml of n-butanol alone was used as blank. The color intensity of the chromogen was read at 560 nm using spectrophotometer (Kakkar et al. 1984). Measurement of glutathione
Macroscopic characters For each animal, a 10 cm portion of the colon was removed and cut longitudinally and slightly cleaned in physiological saline to remove fecal residues. The colon was then weighed. Macroscopic inflammations were scored using the following scoring pattern. No visible change was counted as zero point, hyperemia at sites as one point, lesions having diameter l mm or less counted as two points, lesions having diameter 2 mm or less as three points and lesions more than 2 mm as four, five and six points, respectively (Jagtap et al. 2004; Thippeswamy et al. 2011). Scoring for rat cecum and colon: Rat cecum and colon (5 cm long) were scored using the following scoring pattern. If totally unaffected, it was counted as zero point, 1–5 % as one point, 5–10 as two points, 10–25 as three points, 25–50 as four points, 50–75 as five points and 75–100 as six points, respectively (Jagtap et al. 2004; Minaiyan et al. 2008). Biochemical estimation Colon tissues were stored immediately at -80 C for the enzyme analysis. The tissue homogenate was prepared (10 %w/v) with 0.1 M Tris–HCl buffer (pH 7.4) and deionized water, respectively. The homogenate was used to measure the LPO, SOD and GSH. Measurement of lipid peroxides Measurement of LPO was done from the quantifiable amount of thiobarbituric acid reactive substance using a standard protocol. The homogenate was centrifuged and the supernatant portion was mixed with an equal volume of TBARS (0.67 %) and heated to 90 C for 15 min. The absorbance was measured at 532 nm against the blank solution. The results were expressed as lmol/mg protein.(Ohkawa et al. 1979).
Glutathione content was estimated by following (Moran et al.’s 1979) method. 0.25 ml of homogenate was added to an equal volume of ice cold 5 % TCA. The precipitate was removed by centrifugation at 4000 rpm for 10 min. To 1.0 ml aliquot of the supernatant, 0.25 ml of 0.2 M phosphate buffer (pH 8.0) and 0.5 ml of DTNB (0.6 mM in 0.2 M phosphate buffer, pH 8.0) were added and mixed well. The absorbance was read at 412 nm using a UV spectrophotometer. The values were expressed in lM/g tissues. Histopathological study A portion (2 cm) of the colonic specimen from each rat was fixed in 10 % formalin, cut into 5 lm thickness, stained using hematoxylin–eosin and histopathological observations were made. The stained sections of colon were examined for any inflammatory changes such as infiltration of the cells, necrotic foci and damage to tissue structures like Peyer’s patches, damage to nucleus, etc. Statistical analysis The values were expressed as mean ± SEM. The statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by multiple comparison test of Tukey. Multiple comparison values P \ 0.05 were considered as significant.
Result Intra-rectal administration of acetic acid caused severe inflammatory reactions in the colon. The inflammation occurred in the distal colon and the rectum. The clinical score and the macroscopic characters were significantly increased (P \ 0.001).
Measurement of superoxide dismutase
Macroscopic characters
Superoxide dismutase was assayed by taking 100 ll of tissue homogenate followed by addition of 0.3 ml of sodium pyrophosphate buffer (Ph 8.3), 0.025 ml of PMS and 0.075 ml of nitroblue tetrazolium (NBT). The reaction was started by addition of 0.075 ml of NADH. After incubation, at 30 C for 90 s, the reaction was stopped by the
The colon of the rats was examined macroscopically for signs of hemorrhage and lesions. After 7 days, colon from acetic acid-administered rats showed considerable amount of damage and the results were significant when compared with the normal animals (P \ 001). In the group pretreated with 100 mg and 200 mg of Pterocarpus marsupium and
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Table 1 Clinical and macroscopic characters of P.marsupium-treated rats with acetic acid-induced colitis Treatment Normal control
Wt. loss (%) 0.0 ± 0.0
Stool consistency Bleeding of colon Lesion score (%) (%) (%) 0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
Area affected (%) 0.0 ± 0.0
Wet colon wt. (mg/ cm) 83.67 ± 7.94
Acetic acid control
2.50 ± 1.04** 3.00 ± 1.09***
3.00 ± 1.09***
3.00 ± 1.09*** 3.16 ± 0.75*** 178.00 ± 15.49***
Sulfasalazine 100 mg/kg b.w
1.50 ± 0.54#
2.33 ± 0.81
2.00 ± 1.54
2.33 ± 0.81
Pterocarpus marsupium 100 mg/kg b.w
2.67 ± 0.51
3.67 ± 0.81
Pterocarpus marsupium 200 mg/kg b.w
1.00 ± 0.00## 1.33 ± 1.03###
1.33 ± 0.51### #
3.33 ± 1.03
2.67 ± 1.50
2.00 ± 0.89
1.33 ± 1.03#
0.33 ± 0.51##
0.33 ± 0.51###
118.67 ± 16.28### 170.00 ± 41.18 95.33 ± 11.84###
Values are given as mean ± SEM for groups of six animals each. Tukey–Kramer values are statistically significant at *** P \ 0.001, ** P \ 0.01, * P \ 0.05 between the normal and colitis control, and ### P \ 0.001, ## P \ 0.01, # P \ 0.05 between the colitis control and the treated group
standard drug sulfasalazine, the area of lesion was significantly decreased (P \ 0.001). The rectal administration of acetic acid produced ulcer in the colon when compared with normal animals. The mean ulcer area of the acetic acid group was 3.16 ± 0.75 mm2, showing the high damage produced by acetic acid. The animals pretreated with Pterocarpus marsupium (100 mg and 200 mg/kg, p.o.) for 7 days seemed to have the colon protected from inflammatory damage and ulcer formation produced by acetic acid (P \ 0.05 and P \ 0.001, respectively). The results are summarized in Table 1. The colonic weight of the colitis group was significantly higher than the control group of animals, 178 mg/cm ± 15.49 (P \ 0.01). Animals pretreated with pterocarpus marsupium (200 mg/kg) showed significant decrease in (P \ 0.001) the wet weight of colon, 96.3 mg/cm ± 11.84, but for the 100 mg/kg dose-treated group there was a reduction in the wet weight of the colon, but no statistical difference from the control animals. Similarly, we observed a significant (P \ 0.001) colon weight reduction in the sulfasalazine treated group. Clinical score After the administration of acetic acid, severe increase in the clinical score (2.83 ± 0.02) such as weight loss, stool consistency and bleeding of the colon was observed when compared with the control group of animals (0.33 ± 0.00) (2.5 ± 1.04, 3 ± 1.09, 3 ± 1.09, respectively). The pretreated group of animals and the standard drug-treated group showed significant amount of protection when compared with the acetic acid group. The animals treated with 200 mg/kg of Pterocarpus marsupium had significantly reduced clinical score of 1.22 ± 0.59 when compared with the untreated group (P \ 0.001), but there was no statistical difference in the 100 mg/kg dose-treated group. However, the standard drug showed significant amount of protection when compared with the control animals. The results are summarized in Table 1.
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Biochemical studies After the administration of acetic acid through the intrarectal route, there was a significant increase in the LPO level (13.83 ± 0.99) lmol/g of wet tissue (P [ 0.001) when compared with normal animals. The tissue antioxidant enzymes such as SOD and GSH (13.5 ± 0.87 and 1.86 ± 0.43) were lowered significantly (P [ 0.001) in the acetic acid alone-administered group than in normal animals (23.5 ± 2.4 and 9.3 ± 0.53). Oral administration of PM for 7 days significantly restored (P [ 0.001) the elevated LOP levels in both 100 and 200 mg/kg dose groups (7.4 ± 0.56 and 5.6 ± 0.68). The extract-treated group with 200 mg/kg showed significant (P [ 0.001) increases in the GSH and SOD enzyme levels (20.4 ± 0.93 and 5.3 ± 0.50, respectively). All these parameters were compared with the standard drug and the results are summarized in Fig. 1. Histopathology Acetic acid-induced colitis showed massive necrotic destruction of the epithelium, submucosal edema, areas of hemorrhages and inflammatory cellular infiltration. Animals pretreated with Pterocarpus marsupium for 7 days at low doses showed less protection when compared with normal animals, but the 200 mg/kg group showed much better protection against necrotic destruction of epithelium, submucosal edema, areas of hemorrhage and inflammatory cellular infiltration. The standard drug-treated animals also showed less cellular changes when compared with the control group. The results are shown in Fig. 2.
Discussion Acetic acid-induced colitis is a model used to screen various drugs for colitis (Mohsen et al. 2011) which is characterized by severe inflammation, oxidative stress,
Evaluation of the protective effect of… SOD
GSH
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control Acetic acid STD 100mg extract 200 mg extract
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Fig. 1 The effect of P. marsupium on biochemical profile in acetic acid-induced colitis in rats. Values are represented as mean ± SEM for a group of six rats each. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Tukeys–multiple
comparison test. P \ 0.05 was considered as significant. *** P \ 0.001 between normal and colitis control and ### P \ 0.001 between colitis and treatment group
neutrophil infiltration and release of different inflammatory mediators that are the hallmarks of this model (Carty et al. 2000). The present study was conducted to understand the role of PM in controlling and protecting against the damage produced by the instillation of 3 % AA into the colon. In acetic acid-induced colitis, the wet weight of the inflamed colon tissue is considered as one of the reliable and sensitive indicators of the rigorousness and level of inflammatory response. The elevation of the colon weight to length ratio in the acetic acid group indicates the damage produced by AA (Rachmilewitz et al. 1989). In the present study, pretreatment with PM in acetic acid-induced colitis significantly reduced the wet weight of the colon, clinical activity, gross lesion score and percentage of the affected area compared with the colitis control, indicating the protective effect of PM against ulcerative colitis. Imbalance between the antioxidant and oxidant substance is a typical character of experimentally induced ulcerative colitis model (Dro¨ge 2002). The vasodilation and white blood infiltration will increase the blood flow and oxygen supply, respectively thereby promoting the overproduction of oxidative free radicals is an ideal nature acetic acid-induced of ulcerates colitis model (Hartmann
et al. 2012); (Closa and Folch-Puy 2004). Different studies prove that free radicals play a major role in the pathogenesis of mucosal injury. It is well known that depletion of GSH leads to major oxidative damage to tissues. The first line of defense to oxidative reaction is by the GSH (sulfhydryls groups), which is widely distributed in all tissues and acts as a non-enzymatic antioxidant.(chavan et al. 2005) GSH also facilitates the recycling of vitamin C and E and improves antioxidant activity (isozaki et al. 2006). SOD, an enzymatic antioxidant, is an important antioxidant enzyme which coordinates with GSH in the antioxidant effect (Balaji et al. 2014). Pretreatment with PM improves the depleted GSH and SOD enzyme level in the colon of AA-induced animals to the normal level. Reactive oxygen species are known to cause damage in the lipid content of the cell membrane which activates LPO (Balaji et al. 2015). Elevated LPO levels in the colon can trigger a cascade of reactions that initiate more and more free radical generation, which wears out antioxidant defense and results in the development of ulceration and inflammation (Yoshikawa et al. 1989). PM treated with both doses reduced the LPO levels which were elevated after the treatment of the AA group of animals.
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Fig. 2 Histopathological changes in the colon of experimental animals. a Normal intact mucosa from control animals. b Acetic acid-induced colitis showing massive necrotic destruction of the epithelium, submucosal edema, areas of hemorrhages and inflammatory cellular infiltration. c Acetic acid with PM extract, 200 mg/ kg, showing significant protection of mucosa from acetic acid-induced colitis damage. d Acetic acid with PM extract, 10 mg/kg, showing minimal damage of the mucosa with slight submucosal edema and mild infiltration. e Acetic acid with sulfasalazine showing normal mucosa (images A–E 9400 magnification) (color figure online)
Histopathological studies showed the protective effect of PM on AA-induced damage. 200 mg/kg dose-treated group of animals showed less inflammation and leukocyte infiltration than the 100 mg/kg dose-treated animals.
Conclusion In our study, we found that the pretreatment of PM prevented acetic acid-induced colitis in rats, and this protective effect may at least in part be due to its antioxidant and antiinflammatory actions. The plant PM has proven antioxidant and antiinflammatory properties (Acharya and Ghaskadbi 2013). This effect of the plant may be the reason for the protective effect against the acetic acid-induced ulcerates colitis. However, further investigations are necessary to evaluate the mechanism of
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action and the role of PM on inflammatory markers and a similar efficacy can be achieved in other models of experimental colitis that simulate human inflammatory bowel disease. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical statement All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.
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Evaluation of the protective effect of… Balaji B, Rajendar B, Ramanathan M (2014) Quercetin protected isolated human erythrocytes against mancozeb-induced oxidative stress. Toxicol Ind Health 30:561–569 Balaji B, Kumar EP, Kumar A (2015) Evaluation of standardized Bacopa monnieri extract in sodium fluoride-induced behavioural, biochemical, and histopathological alterations in mice. Toxicol Ind Health 31:18–30 Carty E, De-Brabander M, Feakins RM, Rampton DS (2000) Measurement of in vivo rectal mucosal cytokine and eicosanoid production in ulcerative colitis using filter paper. Gut 46:487–492 Chavan S, Sava L, Saxena V (2005) Reduced glutathione: importance of specimen collection. Indian J Clin Biochem 20:150–152 Closa D, Folch-Puy E (2004) Oxygen free radicals and the systemic inflammatory response. IUBMB Life 56:185–191 Dro¨ge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95 Gairola S, Gupta V, Singh B (2010) Phytochemistry and pharmacological activities of Pterocarpus marsupium: a review. Int Res J Pharm 1:100–104 Habal FM, Greenberg GR (1988) Treatment of ulcerative colitis with oral 5-aminosalicylic acid including patients with adverse reactions to sulfasalazine. Am J Gastroenterol 83:15–19 Halagappa K, Girish HN, Srinivasa BP (2010) The study of aqueous extract of Pterocarpus marsupium Roxb. on cytokine TNF-a in type 2 diabetic rats. Indian J Pharmaco 42:392–396 Hartmann RM, Morgan Martins MI, Tieppo J (2012) Effect of Boswellia serrata on antioxidant status in an experimental model of colitis rats induced by acetic acid. Dig Dis Sci 57:2038–2044 Hougee S, Faber J, Sanders A (2005) Selective COX-2 inhibition by a Pterocarpus marsupium extract characterised by pterostilbene and its activities in healthy human volunteers. Planta Med 71:387–392 Isozaki Y, Yoshida N, Kuroda M (2006) Effect of a novel watersoluble vitamin E derivative as a cure for TNBS-induced colitis in rats. Int J Mol Med 17:497–502 Jagtap AG, Shirke SS, Phadke AS (2004) Effect of polyherbal formulation on experimental models of inflammatory bowel diseases. J Ethnopharmacol 90:195–204 Kakkar P, Das B, Viswanathan PN (1984) A Modified spectrophotometric assay of superoxide dismutase. Indian J Bio Chem Biophys 21:130–132 Mahgoub AA (2003) Thymoquinone protects against experimental colitis in rats. Toxicol Lett 143:133–143 Mascolo N, Izzo AA, Autore G et al (1995) Acetic acid-induced colitis in normal and essential fatty acid deficient rats. J Pharmacol Exp Ther 272:469–475
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Inflammopharmacology
RESEARCH ARTICLE
Evaluation of the protective effect of Pterocarpus marsupium on acetic acid-induced ulcerative colitis in rats Merin Maria Mathew1 • Nguyen Vinh Han1 • A. Murugesan1 • E. Arun Raj1 K. G. Prasanth1
•
Received: 12 March 2015 / Accepted: 17 April 2015 Springer Basel 2015
Abstract The aim of the study was to evaluate the effect of Pterocarpus marsupium (PM) on acetic acid (AA)-induced ulcerative colitis (UC) in rats. The rats were divided into five groups, each having six rats. PM extract 100 mg and 200 mg/ kg was given orally to groups four and five, respectively, and standard drug sulfasalazine (100 mg/kg, p.o) to group three. Group two served as UC control animals, and group one control animals received vehicle for 7 days. UC was induced by administering AA (3 % v/v of 2 ml) to all the animals except group one. After 72 h, the animals were killed and the colon was dissected out for microscopic, clinical evaluation, histopathological study and biochemical estimation. PM (100 and 200 mg/kg)-treated group had significantly reduced colon inflammation and mucosal damage. The treatment also normalized the altered antioxidant enzyme levels (LPO, SOD and GSH). Histopathological studies support the effect. The protective effect of PM may be due to antiinflammatory and antioxidant properties. Keywords Acetic acid Ulcerative colitis Pterocarpus marsupium Mucosal damage
Introduction Inflammatory bowel disease (IBD) comprises a group of inflammatory conditions of the colon and small intestine. The major types of IBD are Crohn’s disease and ulcerative
& K. G. Prasanth [email protected] 1
Department of Pharmacology, PSG College of Pharmacy, PO BOX NO 1674, Peelamedu, Coimbatore 641004, Tamilnadu, India
colitis (UC). Both are common in urban areas rather than rural areas with an unknown etiology. The chance of getting colon and bowel cancers are high in patients with UC (Ponder and Long 2013). The main difference between Crohn’s disease and UC is the location and nature of the inflammatory changes. Crohn’s disease can affect any part of the gastrointestinal tract, from mouth to anus, although a majority of the cases start in the terminal ileum. Ulcerative colitis, in contrast, is restricted to the colon and the rectum (Sood et al. 2003). Ulcerative colitis is treated with antiinflammatory drugs, immunosuppressants and biological therapy targeting specific components of the immune response. Glucocorticoids and aminosalicylate, immunomodulators, antibiotics and anti-TNF therapy have been widely used for the treatment of inflammatory bowel diseases (Pravda 2005). But these drugs cause major side effects. Aminosalicylates produce nausea, vomiting, headaches, rash, fever, agranulocytosis, pancreatitis, nephritis and male infertility. In addition, the sulfa portion of the drug interferes with folic acid absorption. The side effects of steroids used for short term include weight gain, mood swings and fluid retention and for long term include osteoporosis, risk of cataract, myopathy, adrenal insufficiency and immune suppression (Habal and Greenberg 1988). Antibiotics have been prescribed for ulcerative colitis, but it is mostly largely ineffective. Immunomodulatory drugs produce pancreatitis, fever, nausea, rashes, arthralgias and diarrhea. Infliximab, a TNF therapy, is associated with risk for infections, which may be fatal (Rajinikanth et al. 2014; Mahgoub 2003). The side effects of chemical drugs remain a major clinical problem. So, plant remedies have become increasingly popular worldwide, and medicinal plants are believed to be an important source of new chemical substances. Thus, emphasis is now given on the
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standardization of herbal medication by screening of biological activities of medicinal plants and isolating active principles from them (Rahman et al. 2006). The use of herbal medicine plays an important role in the therapy of many inflammatory disease conditions, including inflammatory bowel disease (Medhi et al. 2008). Pterocarpus marsupium Roxb. (Leguminosae) is a plant drug used in the preparation of rasayana in the Ayurvedic system of medicine. Rasayana preparations are used as immunomodulators and relieve stress in the body by the Ayurvedic system of treatment (Gairola et al. 2010). In Ayurveda, an aqueous extract of heartwood of P. marsupium is used in the treatment of diabetes. P.marsupium was not tested for its protective action against inflammatory bowel disease, though it has been reported to possess potent antioxidant (Mohammadi et al. 2009) and antiinflammatory (Hougee et al. 2005) activities. Various animal models of experimental colitis to screen drugs effective against inflammatory bowel disease have been established, and acetic acid-induced colitis is an animal model mimics some of the acute inflammatory responses seen in ulcerative colitis (Thippeswamy et al. 2011). Hence, the present study was designed to evaluate the protective effect of Pterocarpus marsupium marketed capsules in the rat model of colitis induced by acetic acid.
Materials and method Pterocarpus marsupium capsules which contain freeze-dried aqueous extract of wood of Pterocarpus marsupium were purchased from a medical shop. Sulfasalazine (SRINIVASA Medicals, Coimbatore), Hemoccult kit (HEMOSPOT, Carewell Scientific Distributors, Coimbatore), 5,5-dithiobis-2nitrobenzoic acid, hexadecyltrimethylammonium bromide, dianisidine hydrochloride, thiobarbituric acid and lactate dehydrogenase were purchased. All other chemicals used were of analytical grade. Female Wistar albino rats (200–250 g) were obtained from the animal house of PSGIMS&R, Coimbatore, Tamilnadu. All the protocols were approved by the institutional animal ethical committee of PSGIMS&R. The animals were maintained under standard conditions (12 h light/dark cycle; 25 ± 3 C, 45–65 % humidity) and had free access to standard rat feed and water ad libitum. All the animals were adjusted to laboratory conditions for a week before commencement of the experiment. The experiments were performed during the light portion between 8:00 a.m and 12:00 a.m. to avoid circadian influences. Animal studies were performed according to the prescribed guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (235/2014/IAEC).
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Different doses of P.marsupium and the standard sulfasalazine were prepared as suspensions in distilled water using sodium carboxymethyl cellulose (sodium CMC, 0.3 % w/v). Two doses of P.marsupium (100 and 200 mg/ kg) were selected for administration in oral route based on an earlier study (Halagappa et al. 2010). Experimental animals were divided into five groups of six animals each. • • • • •
Group I served as normal control and received vehicle (sodium CMC, 0.3 % w/v). Group II served as colitis control and received only vehicle (sodium CMC, 0.3 % w/v). Group III was treated with standard sulfasalazine (100 mg/kg, p.o). Group IV was treated with P.marsupium (100 mg/kg, p.o). Group V was treated with P.marsupium (200 mg/kg, p.o).
All these treatments were given orally for 7 days by oral gavage. On the 4th day of the treatment, the animals were fasted overnight with access to water ad libitum. On the 5th day after 1 h of the aforementioned treatments, the animals (groups II, III, IV and V) were anesthetized by ether inhalation and a polypropylene tube with 2 mm diameter was inserted through the rectum into the colon to a distance of 8 cm. A solution of 2 ml of AA (3 %, v/v) in 0.9 % saline was instilled into the lumen of the colon and maintained in a supine Trendelenburg position for 30 s to prevent the leakage of the intracolonic instillation. After 72 h of single dose administration of AA (8th day), (Mascolo et al. 1995), clinical activity scores were measured and the animals were anesthetized with ether and blood was collected by retroorbital puncture for biochemical estimation. The animals were killed by cervical dislocation and the colon was dissected out. The colon was cleaned gently with saline and weighed. It was used for macroscopic scoring and histopathological and biochemical estimations.
Evaluation of ulcerative colitis Clinical activity score Colitis was evaluated with a clinical score assessing weight loss, stool consistency and bleeding of the colon (measured by guaiac reaction; Hemoccult test) as described previously (Thippeswamy et al. 2011). No weight loss was counted as zero point, weight loss of 1–5 % as one point, 5–10 % as two points, 10–20 % as three points and 20 % as four points. For stool consistency, the stool were collected in a thick paper and observed, zero points were given for wellformed pellets, two points for pasty and semi -formed stools that did not stick to the anus and four points for
Evaluation of the protective effect of…
liquid stools that did stick to the anus. Bleeding was scored zero points for no blood in Hemoccult, two points for positive Hemoccult and four points for gross bleeding. These scores were added and divided by three, forming a total clinical score that ranged from 0.0 (healthy) to 4.0 (maximal activity of colitis).
addition of 0.25 ml of n-butanol. The mixture was allowed to stand for 10 min and centrifuged. 1.5 ml of n-butanol alone was used as blank. The color intensity of the chromogen was read at 560 nm using spectrophotometer (Kakkar et al. 1984). Measurement of glutathione
Macroscopic characters For each animal, a 10 cm portion of the colon was removed and cut longitudinally and slightly cleaned in physiological saline to remove fecal residues. The colon was then weighed. Macroscopic inflammations were scored using the following scoring pattern. No visible change was counted as zero point, hyperemia at sites as one point, lesions having diameter l mm or less counted as two points, lesions having diameter 2 mm or less as three points and lesions more than 2 mm as four, five and six points, respectively (Jagtap et al. 2004; Thippeswamy et al. 2011). Scoring for rat cecum and colon: Rat cecum and colon (5 cm long) were scored using the following scoring pattern. If totally unaffected, it was counted as zero point, 1–5 % as one point, 5–10 as two points, 10–25 as three points, 25–50 as four points, 50–75 as five points and 75–100 as six points, respectively (Jagtap et al. 2004; Minaiyan et al. 2008). Biochemical estimation Colon tissues were stored immediately at -80 C for the enzyme analysis. The tissue homogenate was prepared (10 %w/v) with 0.1 M Tris–HCl buffer (pH 7.4) and deionized water, respectively. The homogenate was used to measure the LPO, SOD and GSH. Measurement of lipid peroxides Measurement of LPO was done from the quantifiable amount of thiobarbituric acid reactive substance using a standard protocol. The homogenate was centrifuged and the supernatant portion was mixed with an equal volume of TBARS (0.67 %) and heated to 90 C for 15 min. The absorbance was measured at 532 nm against the blank solution. The results were expressed as lmol/mg protein.(Ohkawa et al. 1979).
Glutathione content was estimated by following (Moran et al.’s 1979) method. 0.25 ml of homogenate was added to an equal volume of ice cold 5 % TCA. The precipitate was removed by centrifugation at 4000 rpm for 10 min. To 1.0 ml aliquot of the supernatant, 0.25 ml of 0.2 M phosphate buffer (pH 8.0) and 0.5 ml of DTNB (0.6 mM in 0.2 M phosphate buffer, pH 8.0) were added and mixed well. The absorbance was read at 412 nm using a UV spectrophotometer. The values were expressed in lM/g tissues. Histopathological study A portion (2 cm) of the colonic specimen from each rat was fixed in 10 % formalin, cut into 5 lm thickness, stained using hematoxylin–eosin and histopathological observations were made. The stained sections of colon were examined for any inflammatory changes such as infiltration of the cells, necrotic foci and damage to tissue structures like Peyer’s patches, damage to nucleus, etc. Statistical analysis The values were expressed as mean ± SEM. The statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by multiple comparison test of Tukey. Multiple comparison values P \ 0.05 were considered as significant.
Result Intra-rectal administration of acetic acid caused severe inflammatory reactions in the colon. The inflammation occurred in the distal colon and the rectum. The clinical score and the macroscopic characters were significantly increased (P \ 0.001).
Measurement of superoxide dismutase
Macroscopic characters
Superoxide dismutase was assayed by taking 100 ll of tissue homogenate followed by addition of 0.3 ml of sodium pyrophosphate buffer (Ph 8.3), 0.025 ml of PMS and 0.075 ml of nitroblue tetrazolium (NBT). The reaction was started by addition of 0.075 ml of NADH. After incubation, at 30 C for 90 s, the reaction was stopped by the
The colon of the rats was examined macroscopically for signs of hemorrhage and lesions. After 7 days, colon from acetic acid-administered rats showed considerable amount of damage and the results were significant when compared with the normal animals (P \ 001). In the group pretreated with 100 mg and 200 mg of Pterocarpus marsupium and
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Table 1 Clinical and macroscopic characters of P.marsupium-treated rats with acetic acid-induced colitis Treatment Normal control
Wt. loss (%) 0.0 ± 0.0
Stool consistency Bleeding of colon Lesion score (%) (%) (%) 0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
Area affected (%) 0.0 ± 0.0
Wet colon wt. (mg/ cm) 83.67 ± 7.94
Acetic acid control
2.50 ± 1.04** 3.00 ± 1.09***
3.00 ± 1.09***
3.00 ± 1.09*** 3.16 ± 0.75*** 178.00 ± 15.49***
Sulfasalazine 100 mg/kg b.w
1.50 ± 0.54#
2.33 ± 0.81
2.00 ± 1.54
2.33 ± 0.81
Pterocarpus marsupium 100 mg/kg b.w
2.67 ± 0.51
3.67 ± 0.81
Pterocarpus marsupium 200 mg/kg b.w
1.00 ± 0.00## 1.33 ± 1.03###
1.33 ± 0.51### #
3.33 ± 1.03
2.67 ± 1.50
2.00 ± 0.89
1.33 ± 1.03#
0.33 ± 0.51##
0.33 ± 0.51###
118.67 ± 16.28### 170.00 ± 41.18 95.33 ± 11.84###
Values are given as mean ± SEM for groups of six animals each. Tukey–Kramer values are statistically significant at *** P \ 0.001, ** P \ 0.01, * P \ 0.05 between the normal and colitis control, and ### P \ 0.001, ## P \ 0.01, # P \ 0.05 between the colitis control and the treated group
standard drug sulfasalazine, the area of lesion was significantly decreased (P \ 0.001). The rectal administration of acetic acid produced ulcer in the colon when compared with normal animals. The mean ulcer area of the acetic acid group was 3.16 ± 0.75 mm2, showing the high damage produced by acetic acid. The animals pretreated with Pterocarpus marsupium (100 mg and 200 mg/kg, p.o.) for 7 days seemed to have the colon protected from inflammatory damage and ulcer formation produced by acetic acid (P \ 0.05 and P \ 0.001, respectively). The results are summarized in Table 1. The colonic weight of the colitis group was significantly higher than the control group of animals, 178 mg/cm ± 15.49 (P \ 0.01). Animals pretreated with pterocarpus marsupium (200 mg/kg) showed significant decrease in (P \ 0.001) the wet weight of colon, 96.3 mg/cm ± 11.84, but for the 100 mg/kg dose-treated group there was a reduction in the wet weight of the colon, but no statistical difference from the control animals. Similarly, we observed a significant (P \ 0.001) colon weight reduction in the sulfasalazine treated group. Clinical score After the administration of acetic acid, severe increase in the clinical score (2.83 ± 0.02) such as weight loss, stool consistency and bleeding of the colon was observed when compared with the control group of animals (0.33 ± 0.00) (2.5 ± 1.04, 3 ± 1.09, 3 ± 1.09, respectively). The pretreated group of animals and the standard drug-treated group showed significant amount of protection when compared with the acetic acid group. The animals treated with 200 mg/kg of Pterocarpus marsupium had significantly reduced clinical score of 1.22 ± 0.59 when compared with the untreated group (P \ 0.001), but there was no statistical difference in the 100 mg/kg dose-treated group. However, the standard drug showed significant amount of protection when compared with the control animals. The results are summarized in Table 1.
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Biochemical studies After the administration of acetic acid through the intrarectal route, there was a significant increase in the LPO level (13.83 ± 0.99) lmol/g of wet tissue (P [ 0.001) when compared with normal animals. The tissue antioxidant enzymes such as SOD and GSH (13.5 ± 0.87 and 1.86 ± 0.43) were lowered significantly (P [ 0.001) in the acetic acid alone-administered group than in normal animals (23.5 ± 2.4 and 9.3 ± 0.53). Oral administration of PM for 7 days significantly restored (P [ 0.001) the elevated LOP levels in both 100 and 200 mg/kg dose groups (7.4 ± 0.56 and 5.6 ± 0.68). The extract-treated group with 200 mg/kg showed significant (P [ 0.001) increases in the GSH and SOD enzyme levels (20.4 ± 0.93 and 5.3 ± 0.50, respectively). All these parameters were compared with the standard drug and the results are summarized in Fig. 1. Histopathology Acetic acid-induced colitis showed massive necrotic destruction of the epithelium, submucosal edema, areas of hemorrhages and inflammatory cellular infiltration. Animals pretreated with Pterocarpus marsupium for 7 days at low doses showed less protection when compared with normal animals, but the 200 mg/kg group showed much better protection against necrotic destruction of epithelium, submucosal edema, areas of hemorrhage and inflammatory cellular infiltration. The standard drug-treated animals also showed less cellular changes when compared with the control group. The results are shown in Fig. 2.
Discussion Acetic acid-induced colitis is a model used to screen various drugs for colitis (Mohsen et al. 2011) which is characterized by severe inflammation, oxidative stress,
Evaluation of the protective effect of… SOD
GSH
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Control Acetic acid STD 100 mg extract 200 mg extract
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control Acetic acid STD 100mg extract 200 mg extract
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Drug treatment
SOD
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Fig. 1 The effect of P. marsupium on biochemical profile in acetic acid-induced colitis in rats. Values are represented as mean ± SEM for a group of six rats each. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Tukeys–multiple
comparison test. P \ 0.05 was considered as significant. *** P \ 0.001 between normal and colitis control and ### P \ 0.001 between colitis and treatment group
neutrophil infiltration and release of different inflammatory mediators that are the hallmarks of this model (Carty et al. 2000). The present study was conducted to understand the role of PM in controlling and protecting against the damage produced by the instillation of 3 % AA into the colon. In acetic acid-induced colitis, the wet weight of the inflamed colon tissue is considered as one of the reliable and sensitive indicators of the rigorousness and level of inflammatory response. The elevation of the colon weight to length ratio in the acetic acid group indicates the damage produced by AA (Rachmilewitz et al. 1989). In the present study, pretreatment with PM in acetic acid-induced colitis significantly reduced the wet weight of the colon, clinical activity, gross lesion score and percentage of the affected area compared with the colitis control, indicating the protective effect of PM against ulcerative colitis. Imbalance between the antioxidant and oxidant substance is a typical character of experimentally induced ulcerative colitis model (Dro¨ge 2002). The vasodilation and white blood infiltration will increase the blood flow and oxygen supply, respectively thereby promoting the overproduction of oxidative free radicals is an ideal nature acetic acid-induced of ulcerates colitis model (Hartmann
et al. 2012); (Closa and Folch-Puy 2004). Different studies prove that free radicals play a major role in the pathogenesis of mucosal injury. It is well known that depletion of GSH leads to major oxidative damage to tissues. The first line of defense to oxidative reaction is by the GSH (sulfhydryls groups), which is widely distributed in all tissues and acts as a non-enzymatic antioxidant.(chavan et al. 2005) GSH also facilitates the recycling of vitamin C and E and improves antioxidant activity (isozaki et al. 2006). SOD, an enzymatic antioxidant, is an important antioxidant enzyme which coordinates with GSH in the antioxidant effect (Balaji et al. 2014). Pretreatment with PM improves the depleted GSH and SOD enzyme level in the colon of AA-induced animals to the normal level. Reactive oxygen species are known to cause damage in the lipid content of the cell membrane which activates LPO (Balaji et al. 2015). Elevated LPO levels in the colon can trigger a cascade of reactions that initiate more and more free radical generation, which wears out antioxidant defense and results in the development of ulceration and inflammation (Yoshikawa et al. 1989). PM treated with both doses reduced the LPO levels which were elevated after the treatment of the AA group of animals.
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Fig. 2 Histopathological changes in the colon of experimental animals. a Normal intact mucosa from control animals. b Acetic acid-induced colitis showing massive necrotic destruction of the epithelium, submucosal edema, areas of hemorrhages and inflammatory cellular infiltration. c Acetic acid with PM extract, 200 mg/ kg, showing significant protection of mucosa from acetic acid-induced colitis damage. d Acetic acid with PM extract, 10 mg/kg, showing minimal damage of the mucosa with slight submucosal edema and mild infiltration. e Acetic acid with sulfasalazine showing normal mucosa (images A–E 9400 magnification) (color figure online)
Histopathological studies showed the protective effect of PM on AA-induced damage. 200 mg/kg dose-treated group of animals showed less inflammation and leukocyte infiltration than the 100 mg/kg dose-treated animals.
Conclusion In our study, we found that the pretreatment of PM prevented acetic acid-induced colitis in rats, and this protective effect may at least in part be due to its antioxidant and antiinflammatory actions. The plant PM has proven antioxidant and antiinflammatory properties (Acharya and Ghaskadbi 2013). This effect of the plant may be the reason for the protective effect against the acetic acid-induced ulcerates colitis. However, further investigations are necessary to evaluate the mechanism of
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action and the role of PM on inflammatory markers and a similar efficacy can be achieved in other models of experimental colitis that simulate human inflammatory bowel disease. Conflict of interest of interest.
The authors declare that they have no conflict
Ethical statement All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.
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