|Year : 2011 | Volume
| Issue : 6 | Page : 656-661
Protective effect of aqueous extract of Oroxylum indicum Linn. (root bark) against DNBS-induced colitis in rats
Shrikant V Joshi1, Bhavin A Vyas1, Payal D Shah1, Dinesh R Shah1, Shailesh A Shah1, Tejal R Gandhi2
1 Department of Pharmacology, Maliba Pharmacy College, Gopal Vidyanagar, Bardoli-Mahuva Road, Tarsadi - 394 350, Surat, India
2 Department of Pharmacology, Anand College of Pharmacy, Opp. Town Hall, Anand, Gujarat, India
|Date of Submission||22-Dec-2010|
|Date of Decision||09-Aug-2011|
|Date of Acceptance||31-Aug-2011|
|Date of Web Publication||14-Nov-2011|
Shrikant V Joshi
Department of Pharmacology, Maliba Pharmacy College, Gopal Vidyanagar, Bardoli-Mahuva Road, Tarsadi - 394 350, Surat
Source of Support: None, Conflict of Interest: None
Objective : Aqueous root extract of Oroxylum indicum was evaluated in rats against dinitrobenzene sulfonic acid (DNBS) induced colitis.
Materials and Methods : Rats were pre-treated orally for seven days and continued for four days after the induction of colitis with OI aq (100, 200, and 400 mg/kg) or vehicle. Colitis was induced by intracolonic instillation of 25 mg of DNBS per rat dissolved in 50% alcohol and 4 days later, the colonic mucosal damage was analyzed along with food intake, body weight, colon weight, spleen weight, histological damage, myeloperoxidase (MPO) activity, malondialdehyde (MDA) levels, reduced glutathione (GSH), and nitric oxide levels in colonic tissue homogenate.
Results : Significant reduction in gross damage area, weight loss and increase in colonic and spleen weight were evident in test substance-pretreated animals' dose dependently as compared to vehicle treated control. These effects were confirmed biochemically by a reduction in colonic myeloperoxidase activity, malondialdehyde levels, nitric oxide levels, and increase in reduced glutathione (GSH) levels. Furthermore, microscopic examination revealed diminution of inflammatory cell infiltration and submucosal edema in colon segments of rats treated with OI aq .
Conclusion : The results demonstrate the protective effect of OI aq in the animal model of acute colitis possibly through an antioxidant, anti-lipoperoxidative or due to reduction in nitric oxide generation.
Keywords: Oroxylum indicum, DNBS, colitis, rats
|How to cite this article:|
Joshi SV, Vyas BA, Shah PD, Shah DR, Shah SA, Gandhi TR. Protective effect of aqueous extract of Oroxylum indicum Linn. (root bark) against DNBS-induced colitis in rats. Indian J Pharmacol 2011;43:656-61
|How to cite this URL:|
Joshi SV, Vyas BA, Shah PD, Shah DR, Shah SA, Gandhi TR. Protective effect of aqueous extract of Oroxylum indicum Linn. (root bark) against DNBS-induced colitis in rats. Indian J Pharmacol [serial online] 2011 [cited 2021 Oct 25];43:656-61. Available from: https://www.ijp-online.com/text.asp?2011/43/6/656/89821
| » Introduction|| |
Inflammatory bowel disease (IBD) comprises Crohn's disease (CD) and ulcerative colitis (UC) which are defined as chronic and relapsing inflammations of the gastrointestinal tract caused by variable pathophysiological mechanisms  characterized by clinical manifestations including diarrhea, blood in the stool, abdominal pain, and weight loss. Recent advances in the field of IBD research suggest several areas of possible importance, such as genetic, infectious, immunological, and inflammatory factors in the pathogenesis of disease,  accentuating mainly on the production of cytokines, chemokines, growth factors, arachidonic acid metabolites (e.g. prostaglandins and leukotrienes) and reactive oxygen metabolites (ROM). 
Oroxylum indicum (Syonakh) is a traditional herbal medicine in India, China, and Japan belonging to the family Bignoniaceae, used as an astringent, carminative, diuretic, stomachic, aphrodisiac, anti-diarrheal/anti-dysenteric and has high potential for stimulating digestion, curing fevers, coughs and preventing other respiratory disorders. It has been used as an analgesic, antitussive, and anti-inflammatory agent for treatment of cough, bronchitis, and other diseases.  In India, it is one of the important ingredients in most commonly used Ayurvedic preparation, named as "Dasamula". Root bark is also been used in other Ayurvedic formulation, such as Amartarista, Dantyadyarista, Narayana Taila, Dhanawantara Ghrita, Brahma Rasayana, Chyavanaprasa Awalwha, etc. The plant is reported in Indian ancient text "Ayurveda" to possess diuretic, anti-helminthic, anti-leucodermic, anti-anorexic, anti-arthritic, antifungal, antibacterial activity and used for the treatment of leprosy and tuberculosis. 
The stem bark and leaves of this plant were reported to contain flavonoids namely chrysin, oroxylin-A, scutellarin, and baicalein. Seeds of this plant are reported to contain ellagic acid.  Earlier studies suggested presence of chrysin, baicalein, biochanin-A, and ellagic acid phytoconstituents in the root bark of Oroxylum indicum  Baicalein is reported to possess an anti-inflammatory,  anti-ulcer,  antioxidant,  hepatoprotective  and immunomodulatory  activity, while chrysin and baicalein both are reported to have antibacterial, antifungal, and antiviral activity. , Furthermore, biochanin-A possesses anti-fungal action and inhibition of tumor necrosis factor-α.  Ellagic acid is an important polyphenolic compound. 
The anti-inflammatory, immunomodulatory, anti-oxidant, gastroprotective, analgesic and anti-diarrheal or anti-dysenteric properties, therefore, form a good basis for its use in IBD. Therefore, the aim of the present study was to evaluate the potential of Oroxylum indicum as a curative drug against experimentally induced (DNBS induced) inflammatory bowel disease in rats.
| » Material and Methods|| |
Wistar albino rats of either sex (200-250 g) were obtained from animal house, Department of Pharmacology, Maliba Pharmacy College, Tarsadi. Animals were divided into group of six animals, housed in PVC cages under standard condition (12:12 hour light/ dark cycle at 25±2 0 C, humidity 70-75%). The experimental protocol (MPC0905) was approved by Institutional animal ethics committee (Reg. No. 717/02/a/CPCSEA) as per the guidance of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.
The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced by administration of appropriate volumes of the dosing vehicle.
Root bark of Oroxylum indicum was collected from Bardoli region and authenticated by Dr. Minoo Parabia, Department of Bioscience, VNSGU, Gujarat, India. The plant was submitted in the herbarium of the Pharmacognosy department of Maliba Pharmacy College (Voucher specimen no. is MPC/2009/04).
Root bark of Oroxylum indicum was dried, powdered, and extracted by maceration with water for 7 days. Dried extract was dissolved in distilled water (OI aq ) and administered in dose 100, 200, and 400 mg/kg/day p.o. for seven days prior to induction of colitis and continued for next four days after DNBS treatment.
The extract was expected to have flavonoids and saponins. Phytochemical investigation was carried out to determine the presence of alkaloids, saponins, phenolic compounds, and flavonoids 5 .
UV - Visible Spectrophotometric Analysis
The crude extract was dissolved in distilled water and scanned in UV-Visible range (200-800 nm) (Shimadzu UV 1800).
Thin Layer Chromatography (TLC)
TLC co-chromatography of OI aq and standard baicalein, chrysin, biochanin-A, and ellagic acid was performed using TLC aluminum sheets pre-coated with silica gel 60 F 254, thickness 0.2 mm, (20×20 cm) (E Merck, Germany) as stationary phase, while mobile phase consisted of chloroform: ethyl acetate: acetic acid (5:4:1). Derivatization was done with natural product poly ethylene glycol (NP-PEG).
Acute Toxicity Test
Acute oral toxicity study was performed as per Organization for Economic Co-operation and Development (OECD) - 425 guidelines. , Female Wistar rats were selected by random sampling technique and were acclimatized for five days to the laboratory conditions prior to dosing. The animals were fasted overnight (food but not water withheld) prior to oral administration of OI aq . Test substance was administered to single animals in a sequential manner following the progression slope two throughout the study with the starting dose of 175 mg of OI aq per kg body weight and observed for 14 days.
Colitis was induced using the technique of acid induced colon inflammation, as described by Cuzzocrea et al.  In fasted rats lightly anesthetized with ether, a catheter was inserted into the colon via the anus until approximately the splenic flexure (8 cm from the anus). DNBS (25 mg/rat) was dissolved in 50% ethanol. Thereafter, the animals were kept for 15 minutes in a Trendelenburg position to avoid reflux. Six animals were kept as normal. Body weight and food intake of the rats were measured daily throughout the entire experimental period. In addition, the appearance of stools (normal, pasty and partially formed pellets, liquid pellet, blood in hemoccult, and gross bleeding from the rectum) was also observed as part of index to determine disease activity. After colitis induction, the animals were observed for three days. On day four, the animals were sacrificed and abdomen was opened by a midline incision. The colon was removed, freed from surrounding tissues, opened along the antimesenteric border, rinsed, weighed, and processed for histology.
Evaluation of the Damaged Lesion Area
Lesion area was measured as described by Khan.  In brief, the opened colonic samples were flattened and carefully sandwiched between the two layers of a transparent plastic folder of A4 size. The specimens within the plastic folder were scanned and using Scion Image software the lesion area was measured.
Colon tissues were fixed in 10% neutral buffered formalin solution and embedded in paraffin. The tissues were then cut in to 3 μm sections with uniform shape and size, mounted on silane-coated glass slides and stained with hematoxylin eosin, periodic acid Schiff reagent. Selected tissue sections were fixed on the glass slide with the help of egg albumin.
Myeloperoxidase (MPO) Activity
Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation was determined as previously described.  Four days after intracolonic injection of DNBS the colon was removed and weighed. The colon was homogenized in a solution containing 0.5% hexadecy -trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged (REMI C24) for 30 minutes at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H 2 O 2 . The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol of peroxide/minute at 37°C and was expressed in milliunits per 100 gram weight of wet tissue.
Malondialdehyde (MDA) Measurement 
One ml of supernatant was mixed with 0.2 ml 4%w/v sodium dodecyl sulfate, 1.5 ml 20% acetic acid in 0.27M hydrochloric acid (pH 3.5), and 1.5 ml of 0.8% thiobarbituric acid (TBA) in test tube. The mixture was heated in a water bath at 85°C for 1 hr. The intensity of pink color developed was measured against a reagent blank at 532 nm. Malondialdehyde was calculated using molar extinction coefficient 1.56x105 M -1 cm -1 or from standard curve and reported as millimoles per mg of wet tissue.
Nitrite Analysis 
Accumulated nitrite (NO 2 - ) in the homogenate will be spectrophotometrically determined based on the Griess reaction. The samples (100 μl) were incubated with 100 μl Griess reagent (6 mg/ml) at room temperature for 10 min and then NO 2 - concentration will be determined by the absorbance at 540 nm. The standard curve was obtained using the known concentrations of sodium nitrite.
Measurement of GSH Level in the Colonic Tissue 
Colonic tissues were homogenized in ice-cold 125 mmol/l sodium phosphate buffer with 6.3 mmol/l EDTA (pH 7.5, 3 μl/mg tissue) for 30 s. The crude homogenate was centrifuged at 30,000 g at 4 0 C for 30 min. Then, 200 μl of 40 g/l sulfosalicylic acid was added to 100 μl of supernatant and allowed to stand on ice for 5 min to precipitate protein. The mixture was centrifuged again at 5,000 r/min at 4 0 C for 10 min. Subsequently, 100 μl of the de-proteinized supernatant was mixed well with 300 μl of 125 mmol/l sodium phosphate buffer (pH 8), and 2 μl of 10 mmol/l 5,5'-dithiobis-(2-nitrobenzoic acid). The solution was allowed to stand at room temperature for 15 min to develop yellow color. The absorbance was read against the reagent blank at 412 nm in a spectrophotometer. A standard curve of reduced GSH was used for the calculation of the concentration of GSH in the colonic tissues. The final values were expressed as nanomole per milligram protein.
All values in the figures and text are expressed as mean ± S.E.M. of n (number of animals) observations. The results were analyzed by one-way analysis of variance followed by a Dunnet post hoc test. P value less than 0.05 were considered significant.
| » Results|| |
The qualitative analysis of Oroxylum indicum revealed the presence of saponins, phenolics compounds, and flavonoids whereas alkaloids were found to be absent.
UV - Visible Spectrophotometric Analysis
UV - visible spectrophotometric analysis revealed peaks in the region 280-300 and 220-240 nm [Figure 1].
Thin Layer Chromatography
The TLC study revealed presence of baicalein, chrysin, biochanin-A, and ellagic acid in the root bark of Oroxylum indicum [Figure 2] which correlates with the previous findings.
|Figure 2: Thin Layer Chromatography of OIaq and standards. A – Chrysin, B – Baicalein, C – Biochanin A, D – Ellagic acid and E – OIaq|
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Acute Toxicity Test
Acute toxicity study showed that OI aq did not cause any mortality up to 5000 mg/kg and was considered safe. 
Gross macroscopic inspection of cecum, colon, and rectum of DNBS treated rats' revealed presence of mucosal congestion, erosion, hemorrhagic ulcerations, and sever inflammation. The colon appeared flaccid and filled with liquid stool. The rats pre-treated with OI aq significantly attenuated the extent and severity of the symptoms in a dose dependent manner. [Figure 3] and [Figure 4].
|Figure 3: Photographs of rat colon after four days of induction of DNBS colitis. 1 – Normal Photograph captured, 2 – Lesion area shown after subjecting to Scion Image Software. A – colon of rat treated with DNBS alone, B – colon of Rat treated with OIaq 100 mg/kg, C – colon of Rat treated with OIaq 200 mg/kg, D - colon of Rat treated with OIaq 400 mg/kg|
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|Figure 4: Histological sections of rat colonic mucosa after four days of induction of DNBS colitis. Normal (a), Control, treated only with DNBS (b), Animal pre-treated with 100 mg/kg OIaq (c), Animal pre-treated with 200 mg/kg OIaq (d), Animal pre-treated with 400 mg/kg OIaq (e)|
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Four days after colitis induction by DNBS treatment, all rats had diarrhea and a significant reduction in body weight (compared with the normal group of rats) [Table 1]. A significant increase in the weight of spleen (an indicator of inflammation), distal colon (an indicator of tissue edema) along with decrease in food intake was also been noted in vehicle treated rats who had received DNBS. There was a significant reduction in these symptoms by pretreatment of OI aq in a dose-dependent manner [Table 1].
|Table 1: Effect of aqueous root extract of Oroxylum indicum (OIaq) on physical parameters of rats|
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The histopathological features included a trans-mural necrosis along with extensive morphological disorientation, edema and a diffuse leukocyte cellular infiltrate as well as lymphocyte in the submucosa of colon section from DNBS-treated rats [Figure 4]. Colonic tissues in all OI aq groups demonstrated dose dependent significant reduction in histological damage, as represented by well-organized mucosal architecture and less infiltrated leukocytes in the mucosal and submucosal layers [Figure 4]c-e.
Lesion area measured with Scion Image software were in accordance with the other evaluation parameters and shows significant reduction (dose dependently) in OI aq treated rats [Figure 3].
The notable inflammatory response on day four after the induction of colitis by administration of DNBS in control group was confirmed by the significant rise in MDA, MPO, and NO levels, while decrease in GSH levels [Table 2]. Pretreatment with OIaq dose dependently shows significant reduction in MDA, MPO, and NO levels as compared with control in conjunction with increased GSH levels.
|Table 2: Effect of aqueous root extract of Oroxylum indicum (OIaq) on biochemical parameters|
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| » Discussion|| |
IBD is characterized by dysfunction of mucosal immune response, abnormal cytokine production with increase in tumor necrosis factor (TNF)-α, interleukin (IL)-1, augmentation in adhesion molecules expression and massive infiltration of polymorphonuclear and mononuclear leukocytes which may produce large amounts of free radicals that ultimately lead to epithelial cell apoptosis and mucosal damage. 
The hapten model of colonic inflammation induced by dinitrobenzene sulfonic acid (DNBS) delivered intrarectally causes a substantial degree of inflammation and tissue injury in the rat colon, resembling human IBD in terms of its various histological features including polymorphonuclear colonic infiltrate and predominant NF-kB-dependent Th1 activation. 
The present study showed that DNBS induced significant degree of inflammation and tissue injury (increased lesion area and damage in cytoskeleton) in the rat colon. Induction of IBD was also been supported by decreased food intake, body weight, increased colon weight, and changed stool consistency in control group animals when compared with normal. Furthermore, DNBS treated rats showed significant increase in the weight of spleen which is a good indicator of inflammation.  A decrease in progression of the disease pathogenesis following the treatment of OI aq can be characterized by decrease in lesion area [Figure 3] and tissue necrosis [Figure 4] which was supported by increased food intake, body weight, colon weight, and improved stool consistency.
Myeloperoxidase (MPO) is an enzyme mainly found in azurophilic granules of neutrophils. It can serve as a good marker of inflammation, tissue injury and neutrophil infiltration in gastrointestinal tissues.  Pretreatment with OI aq exhibits decrease in polymorphonuclear infiltration demonstrated by significant reduction in MPO activity and decreased appearance of neutrophils in the histology. However, concentrations of endogenous antioxidants, such as reduced glutathione (GSH) which was found to be significantly decreased in IBD patients along with alpha-tocopherol and cysteine resulting in the loss of balance between antioxidants and ROM.  Treatment with OI aq inhibits this decrease of GSH level in rat treated with DNBS which may be because of protection against the progression of the disease.
Oxidative damage may represent crucial pathogenic factor in IBD because intestinal inflammation is accompanied by increased production of reactive oxygen and nitrogen species, such as superoxide (O 2Ϳ), nitric oxide (NO·), peroxynitrite (ONOO·Ϳ), and hydroxyl radicals (·OH) in conjunction with an imbalanced antioxidant response. Besides nitric oxide (NO) which reacts with superoxide to form peroxynitrite, a highly cytotoxic oxidant, is mainly induced in the infiltrated inflammatory cells, such as neutrophils, macrophages, and mononuclear cells by various stimulants like cytokines.  In present study, DNBS treated rats showed significant increase in levels of nitric oxide compared with normal. Treatment with OI aq significantly reduces the rise in NO levels dose dependently compared with control. Reduction in NO level may contribute to decrease mucosal damage as well as other symptoms of IBD.
Malondialdehyde (MDA) which is an endogenous product of enzymatic and oxygen radical-induced lipid peroxidation was found to be increased in rats treated with DNBS.  Rats pretreated with OIaq showed protection against enzymatic and oxygen radical induced lipid peroxidation characterized by significant decrease in MDA levels.
UV spectrum provides useful means of detecting conjugated unsaturated chromophores within molecules, such as polyenes, α-, β-unsaturated ketones, and aromatic compounds. Within particular families of compounds, the position of maximum absorption can reflect the degree of substitution of the chromophore. UV absorption is associated with the chromophore and not the whole molecule, thus it would not distinguish between the compounds which often co-occur. The spectrum may be due to summation of chromophores from different parts of a polyfunctional molecule and hence it will only provide a rough estimation about the contents. 
In present study, UV - visible spectrophotometric analysis revealed a peak in the region 260-300 nm which was probably due to presence of flavonoids, phenolic compounds, and organic acids.  The peak in the region 220 to 240 nm may be due to the presence of some of the phenolic class of compounds and tannins.  This data supports the qualitative phytochemical analysis of OI aq which confirmed the presence of saponins, phenolics compounds, and flavonoids. TLC confirms the presence of chrysin, baicalein, biochanin-A, and ellagic acid in aqueous extract of root bark of Oroxylum indicum.
In conclusion, aqueous extract of root bark of Oroxylum indicum significantly attenuates the symptoms of experimentally induced colitis in rats which may be attributed to presence of baicalein, chrysnin, biochanin-A, and saponins present in it. Baicalein and saponins may inhibit the damage to the mucosa due to their antioxidant and anti-inflammatory properties. While Baicalein and biochanin-A may crucially be involved in rarefying the progression of the symptoms due to its immunomodulatory (specifically TNF-α inhibition) and gastroprotective activity.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]