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

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.

Introduction

Inflammatory bowel disease (IBD), a spectrum of chronic idiopathic inflammatory intestinal conditions,[1] is the second most common chronic inflammatory disease[23] 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.[45] 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 LB4 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.[911]

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

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

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 10th day.

Group 6: Prednisolone (1 mg/kg) was administered orally for 3 days. On the 3rd 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 10th day.

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

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.[1718] 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%, CaCl2 0.024%, NaHCO3 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

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 [Tables 2 and 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 [Tables 2 and 3].

Table 2

Effect of aqueous extract of Aegle marmelos unripe fruit on acetic acid induced ulcerative colitis

Table 3

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

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 [Figures 1 and 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)

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)

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 [Tables 2 and 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 [Tables 2 and 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

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 IL1, IL6, IL8, 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.[2223] 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.[2425] 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.