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 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 »  References
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 Table of Contents    
RESEARCH ARTICLE
Year : 2012  |  Volume : 44  |  Issue : 5  |  Page : 614-618
 

Effect of aqueous extract of Aegle marmelos unripe fruit on inflammatory bowel disease


1 Department of Pharmacology, MKCG Medical College, Berhampur, Odisha, India
2 Department of Pharmacology, Officer Grade II, Sun Pharma, Sikkim, India

Date of Submission30-Nov-2011
Date of Decision12-Jun-2012
Date of Acceptance01-Jul-2012
Date of Web Publication31-Aug-2012

Correspondence Address:
Jayanti P Behera
Department of Pharmacology, MKCG Medical College, Berhampur, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0253-7613.100389

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 » Abstract 

Objective: The present study was designed to evaluate the effect of Aegle marmelos unripe fruit extract (AMFE) on inflammatory bowel disease (IBD) in Wistar albino rats.
Materials and Methods: Effect of AMFE was studied on acetic acid induced ulcerative colitis (1 ml of 4% acetic acid solution, transrectal) and indomethacin-induced enterocolitis (10 mg/kg, single dose, p.o) in Wistar albino rats. The extract was administered orally at different dose of 150, 200 and 250 mg/kg body weight. Disease pathogenesis was assessed by measuring disease activity index (DAI), macroscopic score, microscopic score, mesenteric mast cell protection, superoxide dismutase (SOD), and malonaldehyde (MDA) levels in the above two models.
Results: The results showed a dose dependent decrease in intestinal inflammation following treatment with AMFE. Significant protection in mast cell degranulation was observed in acetic acid and indomethacin-induced IBD models. Treatment with AMFE significantly decreased the MDA levels and increased SOD activity.
Conclusion: In our study, AMFE produced anti-inflammatory, antioxidant, and mast cell stabilizing effects demonstrating protective effect in inflammatory bowel disease.


Keywords: Aegle marmelos (bael), aqueous extract, inflammatory bowel disease


How to cite this article:
Behera JP, Mohanty B, Ramani Y R, Rath B, Pradhan S. Effect of aqueous extract of Aegle marmelos unripe fruit on inflammatory bowel disease. Indian J Pharmacol 2012;44:614-8

How to cite this URL:
Behera JP, Mohanty B, Ramani Y R, Rath B, Pradhan S. Effect of aqueous extract of Aegle marmelos unripe fruit on inflammatory bowel disease. Indian J Pharmacol [serial online] 2012 [cited 2020 Oct 27];44:614-8. Available from: https://www.ijp-online.com/text.asp?2012/44/5/614/100389



 » Introduction Top


Inflammatory bowel disease (IBD), a spectrum of chronic idiopathic inflammatory intestinal conditions, [1] is the second most common chronic inflammatory disease [2],[3] after rheumatoid arthritis. It causes significant morbidity in the population of European origin with two major forms like Crohn's disease (CD) and ulcerative colitis (UC) having a combined prevalence of 150-250/100,000 population. [4],[5] The prevalence of hospitalization is estimated to be 50.1 and 50.6 per 100,000 population for CD and UC, respectively. [6] IBD is a multi-factorial inflammatory disease of the intestine having immunological, genetic, and environmental origin. [6] Mediators like TNF-α, IL-6, and LB 4 play an important role in the inflammation of the intestinal mucosa in animal models of IBD. [7] Other findings with IBD are abnormal histopathological features and increased intestinal permeability. [8] Oxidative stress also plays an important role in the pathophysiology of IBD. [9],[11]

Herbal remedies are increasingly being used for the treatment of IBD. Aegle marmelos (Bael), one of the oldest and most popular species in the world belonging to the family Rutaceae, is found abundantly throughout India. Its fruits are useful in gastrointestinal (GI) disorders like diarrhea, dysentery, and constipation. It is also used as a medicine for its antiinflammatory, immunomodulatory, antibacterial, and antioxidant properties. [10] Hence, the present study was undertaken to evaluate the effect of Aegle marmelos unripe fruit extract (AMFE) on IBD in Wistar albino rats.


 » Materials and Methods Top


Fresh powder form of A. marmelos pulp extract (unripe fruit) was collected from the Indian Herbs Research and Supply Co. Ltd. Sharda Nagar, Saharanpur, Uttar Pradesh [Ref. No. R &D/2008-09/5456]. Adult albino Wistar rats of either gender (200-250 g) were housed under standard condition of temperature (22°C ± 2°C), relative humidity of 55 ± 5% and 12 h light/dark cycles. The animals were given standard pellet diet and water ad libitum. The study protocol was approved by Institutional Animal Ethics Committee according to the CPCSEA guidelines [472/CPCSEA]. The aqueous extract of unripe fruit of A. marmelos was studied for their phytoconstituents using different phytochemical tests as mentioned in [Table 1].
Table 1: Qualitative phytochemical screening of aqueous extract of Aegle marmelos unripe fruit

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An acute oral toxicity study was performed as per OECD-423 guidelines. Adult albino Wistar rats were administered 5, 50, 300, and 2000 mg/kg b.w. of aqueous extract of unripe fruit of A. marmelos orally. The control group received distilled water. The animals were observed for 14 days. There was no mortality or behavioral changes observed during the study period.

Induction of UC [12]

An 8Fr (2.7 mm) soft pediatric catheter was advanced 6 cm from the anus under low dose ether anesthesia and 1 ml of 4% acetic acid solution (pH 2.4) was slowly administered transrectally. The rat was maintained in head down position for 30 s to prevent leakage. The rest of the solution was aspirated out after which 2 ml of phosphate buffer solution with pH7 was administered transrectally.

Induction of Enterocolitis [13]

Around 10 mg/kg indomethacin was administered orally as a single dose. Enterocolitis developed after 24 h.

The study comprised of 12 groups of 6 animals each as follows:

Group 1: Normal control (no disease)

Group 2: UC control (treated with normal saline)

Group 3 to 5: A. marmelos (AMFE) 150, 200, and 250 mg/ kg dose was administered orally for 10 days, respectively. UC was induced on the 10 th day.

Group 6: Prednisolone (1 mg/kg) was administered orally for 3 days. On the 3 rd day, UC was induced.

Group 7: Normal control (no disease)

Group 8: Enterocolitis control (treated with normal saline)

Group 9 to 11: A. marmelos (AMFE) 150, 200, and 250 mg/ kg dose was administered orally for 10 days. Enterocolitis was induced on the 10 th day.

Group 12: Prednisolone (1 mg/kg) was administered orally daily for 3 days. On the 3 rd day enterocolitis was induced.

In all groups, animals were observed for decrease in body weight, stool consistency, and rectal bleeding for 48 h after inducing IBD and disease activity index [14] was scored. After 48 h, the rats in all the groups were sacrificed by cervical dislocation. A 10 cm length of colonic segment (UC groups) and ileum segment (enterocolitis groups) were resected out along with mesenteric attachments. The mesentries were preserved separately in Ringer Locke solution to study mast cell degranulation. The contents of the intestinal segments were cleaned gently with normal saline by hand and then dried. The distal 5 cm of the segments were scored macroscopically and preserved with 4% formalin for microscopic evaluation. The remaining 5 cm was used for biochemical analysis (malonaldehyde [MDA] and superoxide dismutase [SOD] levels).

Parameters Evaluated

Disease activity index (DAI)


The DAI during the study period (48 h) was scored as follows : 0-no weight loss, normal stool consistency, and no rectal bleeding; 1-weight loss (1-5%), normal stool consistency with no rectal bleeding; 2-weight loss (5-10% ), loose stool with no rectal bleeding; 3-weight loss (10-20%), normal stool consistency with no rectal bleeding; and 4-weight loss (>20% ), diarrhea with gross rectal bleeding.

Macroscopic score [15]

After resection of the portion of intestine (colon/jejunum), the mucosal surface is fixed over a wax tray and graded with the help of a magnifying lens as follows: Grade 0-Normal mucosal pattern, Grade 1-Scattered erosions, Grade 2-Linear ulcerations, Grade 3-Diffuse inflammatory tissue featuring small lesions of less than 5 mm, and Grade 4-Diffuse ulceration with wide lesions.

Microscopic score [16]

A piece of colon/ileum was fixed in phosphate-buffered formaldehyde, embedded in paraffin, sectioned (4 μm thick), and stained with hematoxylin and eosin. Each sample was observed and evaluated under light microscopy by two independent observers. The histopathological score was assessed as follows: 0-no lymphoid hyperplasia, neutrophil infiltration, crypt damage, or submucosal inflammation, 1-mild lymphoid hyperplasia, neutrophil infiltration, crypt damage with no submucosal inflammation, 2-moderate lymphoid hyperplasia, neutrophil infiltration, crypt damage with or without submucosal inflammation, and 3-severe lymphoid hyperplasia, neutrophil infiltration, crypt damage, and submucosal inflammation.

Biochemical estimations

Tissues (colon/ileum) were thoroughly washed and homogenized in 2 ml of normal saline using a glass-teflon homogenizer for 5 min at 5000 rpm. Both MDA (nmol/mg of protein) and SOD levels were measured in the homogenate as described in literature. [17],[18] The protein concentration of the homogenate was determined according to Lowry's method.

Percent of mesenteric mast cell protection

The percent mast cell protection is suggested by the degree of its degranulation. Mesenteric pieces were placed in Ringer Locke solution (NaCl 0.9%, KCl 0.042%, CaCl 2 0.024%, NaHCO 3 0.015%, and dextrose 0.1%) and then stained and fixed with 4% formaldehyde containing 0.1% toluidine blue. The percentage of mast cell protection was evaluated under light microscope at 40× magnification. [19]

Statistical Analysis

The data were analyzed by one way ANOVA followed by Tukey's multiple comparison test for MDA level and SOD activity and Kruskal-Wallis test followed by Dunn's multiple comparison test for disease activity index score, macroscopic and microscopic score using Graph Pad Prism software (version 5.0).


 » Results Top


Preliminary phytochemical screening of aqueous extract of unripe fruit of A. marmelos revealed the presence of alkaloids, carbohydrates, glycosides, tannins, phenolics, gums, mucilage, saponins, flavins, flavinoids, steroids, sterols, and terpinoids [Table 1].

Disease activity index was assessed by changes in body weight, stool consistency, and rectal bleeding during the 48 h period following colitis induction. The groups treated with AMFE in all the doses produced significant improvement in the disease activity index in comparison to disease control in both the models. There was no significant difference between the three doses of AMFE in both the models [Table 2] and [Table 3]. The parameters used for evaluating the degree of inflammation in both the models were changed in mucosal pattern and the severity of lesions. The groups treated with AMFE at all the doses produced significant improvement in macroscopic score in comparison to disease control, which is comparable to that of normal control group in both the IBD models. There was no significant difference between the three doses of AMFE [Table 2] and [Table 3].
Table 2: Effect of aqueous extract of Aegle marmelos unripe fruit on acetic acid induced ulcerative colitis

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Table 3: Effect of aqueous extract of Aegle marmelos unripe fruit on indomethacin induced enterocolitis

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Histopathological analysis of the colon/ileum in acetic acid induced UC as well as in indomethacin induced enterocolitis clearly showed that there was a significant degree of lymphoid hyperplasia, neutrophillic infiltration, crypt damage, and sub mucosal inflammation compared with normal control. In the groups treated with AMFE, the progression of IBD was less prominent characterized by significant dose dependent decrease in lymphoid hyperplasia, neutrophillic infiltration, and crypt damage and sub mucosal inflammation [Figure 1] and [Figure 2].
Figure 1: Histological changes seen with aqueous extract of Aegle marmelos unripe fruit on acetic acid induced ulcerative colitis (a) normal control (b) disease control (c) prednisolone (1 mg/kg) (d) AMFE (250
mg/kg)


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Figure 2: Histological changes seen with aqueous extract of Aegle marmelos unripe fruit on indomethacin induced enterocolitis (a) normal control (b) disease control (c) prednisolone (1 mg/kg) (d) AMFE (250 mg/kg)

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Transrectal administration of acetic acid and high oral dose of indomethacin used in the disease models produced significant oxidative stress as evident by significant increase in the MDA levels and decrease in SOD activity. AMFE at all the doses produced significant antioxidant effect [Table 2] and [Table 3]. The animals treated with acetic acid and indomethacin showed 9.44% and 8.94% protection of mast cell degranulation, respectively, whereas a protection of 80% was observed in normal control. AMFE (150, 200, and 250 mg/kg) produced dose dependent decrease in the mast cell degranulation [Table 2] and [Table 3]. The percentage protection offered by AMFE 250 mg/ kg was comparable to the protection given by the standard, prednisolone (1 mg/kg) in both the models of IBD.


 » Discussion Top


UC and CD are the common IBDs seen in day to day clinical practice. Acetic acid induced UC is an easily inducible model which produces mucosal injury similar to UC due to inflammation and generation of reactive oxygen species. Indomethacin induced enterocolitis is the standard procedure for induction of CD. In the present study, the effect of AMFE was observed on the above experimental animal models of IBD, in albino rats. The pathogenesis of the disease was assessed by evaluating the different parameters like disease activity index, macroscopic score, microscopic score, mesenteric mast cell degranulation, and oxidative stress markers like MDA level and SOD activity. Prednisolone was used as the standard drug which produced no significant difference in the above parameters in comparison to AMFE at all doses.

The results showed a decrease in severity of intestinal inflammation following treatment with AMFE. This effect may be due to inhibition of inflammatory mediators like IL 1 , IL 6 , IL 8 , and TNF-α. [20] The phytochemical constituents of AMFE like flavinoids, phenolic compounds, and steroids [21] may be responsible for this antiinflammatory effect. AMFE showed increase in SOD activity and decrease in MDA levels in the tissue homogenate. This effect may be due to presence of antioxidants like carotene, thiamine, riboflavin, niacin, and vitamin C in the A. marmelos fruit. [10] Many studies report that antioxidants show beneficial effect in experimental colitis. [22],[23] Mast cell degranulation causes mucosal edema, mucus secretion, increased gut permeability, and release of inflammatory mediators, which contribute to the clinical signs and symptoms of IBD. [24],[25] A marked increase in mast cell degranulation was observed in acetic acid and indomethacin induced IBD models which were attenuated by AMFE (250 mg/kg) similar to the standard drug prednisolone. This finding is suggestive of the mast cell stabilization activity of AMFE.

It is evident from this study that AMFE produces anti-inflammatory, antioxidant, and mast cell stabilizing effects. A. marmelos contains a number of polar and nonpolar phytoconstituents, which are the key factors in the medicinal value of this plant. Therefore it can be concluded that aqueous extract of unripe fruit of A. marmelos has protective effect against IBD.

 
 » References Top

1.Sellin JH, Pasricha PJ. Pharmacotherapy of inflammatory bowel disease. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 11 th ed. McGraw-Hill Publications,NewYork; 2006. p. 1009.   Back to cited text no. 1
    
2.Bickson SJ. Treatment of chronic disease at the turn of the century. N Engl J Med 1998;339:401-2.  Back to cited text no. 2
    
3.Hana Strul MD, Nadir Arber MD. Inflammatory bowel disease, ulcerative colitis, Crohn's disease, pathophysiology, new therapies. IMAJ 2000;2:607-9.  Back to cited text no. 3
    
4.Parkes M, Jewell D. Ulcerative Colitis and Crohn's disease: Molecular genetics and clinical implications. Expert Rev Mol Med 2001;2001:1-1800391-Xa.pdf   Back to cited text no. 4
    
5.Calkins BM, Mendeloff AI. The epidemiology of idiopathic inflammatory bowel disease. In: Kirsner JB, Shorter RG, editors Inflammatory Bowel Disease. 4 th ed. 1995; p. 31-68.,Williams and wilkins, Baltimore.  Back to cited text no. 5
    
6.Button LA, Roberts SE, Goldacre MJ, Akbari A, Rodgers SE, Williams JG. Hospitalized prevalence and 5-year mortality for IBD: Record linkage study World. J Gastroenterol 2010;16:431-8.  Back to cited text no. 6
    
7.Giriþ M, Depboylu B, Doðru-Abbasoðlu S, Erbil Y, Olgaç V, Aliþ H, et al. Effect of taurine on oxidative stress and apoptosis-related protein expression in trinitrobenzene sulphonic acid-induced colitis. Clin Exp Immunol 2008;152:102-10.  Back to cited text no. 7
    
8.Razavi A, Khodadadi A, Eslami MB Eshraghi S. Therapeutic effect of sodium alginate in experimental chronic ulcerative colitis. Iran J Allergy Asthma Immunol 2008;7:13-8.  Back to cited text no. 8
    
9.Patel MA, Patel PK, Patel MB. Effect of ethanol extract of Ficus begalensis on inflammatory bowel disease. Indian J Pharmacol 2010;42:214-8.   Back to cited text no. 9
    
10.Dhankhar S, Ruhil M, Balhar M. Aegle marmelos (Linn.) Correa: A potential source of Phytomedicine. J Med Plants Res 2011;5:1497-507.  Back to cited text no. 10
    
11.Narushima S, Spitz DR. Evidence for oxidative stress in NSAID- induced colitis in IL-10 mice. Free Radic Biol Med 2003;34:1153-66.  Back to cited text no. 11
    
12.Jagtap AG, Shirke SS, Phadke AS. Effect of polyherbal formulation on experimental model of IBD. J Ethnopharmacol 2004;90:195-204.  Back to cited text no. 12
    
13.Fedorak RN, Empey LR, MacArthur C, Jewell LD. Misoprostal provides a colonic mucosal protective effect during Acetic acid induced colitis in rats. Gastroenterology 1990;98:615-25.  Back to cited text no. 13
    
14.Kakimoto K, Takai S, Murano M, Ishida K, Yoda Y, Inoue T, et al. Significance of Chymase-Dependent Matrix Metalloproteinase-9 Activation on Indomethacin-Induced Small Intestinal Damages in Rats. J Pharmacol Exp Ther 2010;332:684-9.  Back to cited text no. 14
    
15.Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulphate sodium experimental murine colitis. Lab invest 1993;69:238-49.  Back to cited text no. 15
    
16.Zeytunlu M, Korkut M, Akgün E, Firat O, Aynaci M, Içöz G, et al. The comparative effects of calcium channel blockers in an experimental colitis model in rats. Turk J Gastroenterol 2004;15:243-9.  Back to cited text no. 16
    
17.Ayan f, Aydin S, Uzun H. Effects of clinoptilolite (Froximun Cama) on Trinitrobenzene sulfonic acid (TNBS)-induced colitis colitis model in the study as a new agent for Prevention and Treatment. Available from: http://www.froximun.at/pdfs/13.pdf. [24.02.2012]  Back to cited text no. 17
    
18.Dahel LK, Hill EG, Holman RT. The thiobarbituric acid reaction and autooxidation of polyunsaturated fatty acid methyl esters. Arch Biochem Biophys 1962;98:253-61.  Back to cited text no. 18
    
19.Kakkar P, Das B, Viswanathan PN. Mechanisms of Gastric Mucosal Hemorrhagic Ulceration in Salmonella typhimurium-infected Rats: Protection by Several Drugs. Indian J BiochemBiophys 1984;21:130-2.  Back to cited text no. 19
    
20.Norton S. Quantitative determination of mast cell fragmentation by compound 48/80. Br J Pharmacol Chemother 1954;9:494-7.  Back to cited text no. 20
    
21.Rajan S, Gokila M, Jency P, Brindha P, Sujatha RK. Antioxidant and phytochemical properties of Aegle marmelos fruit pulp. Int J Curr Pharm Res 2011;3:65-70.  Back to cited text no. 21
    
22.Harputluoglu MM, Demirel U, Yücel N, Karadað N, Temel I, Firat S. The effects of Gingko biloba extract on acetic acid induced colitis in rats. Turk J Gastroenterol 2006;17:177-82.  Back to cited text no. 22
    
23.Kurutas EB, Cetinkaya A, Bulbuloglu E, Kantarceken B. Effects of Antioxidant Therapy on Leukocyte Myeloperoxidase and Cu/Zn-Superoxide Dismutase and Plasma Malondialdehyde Levels in Experimental Colitis. Mediators Inflamm 2005;2005:390-4.  Back to cited text no. 23
    
24.MacDonald TT, Muresh SH. Aetiology and pathogenesis of chronic inflammatory bowel disease. Baillieres Clin Gastroenterol 1994;8:1-34.  Back to cited text no. 24
    
25.Goldsmith P, McGarity B, Walls AF, Church MK, Millward-Sadler GH, Robertson DA. Corticosteroid treatment reduces mast cell numbers in inflammatory bowel disease. Dig Dis Sci 1990;35:1409-13.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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