Effect of Aqueous Leaves Extract of Melia Azedarach Linn. on Experimental Colitis in Rats

Introduction

Inflammatory bowel disease (IBD) is a spectrum of chronic idiopathic inflammatory intestinal conditions causes significant gastrointestinal symptoms that include diarrhoea, abdominal pain, bleeding, anaemia and weight loss. IBD conventionally is divided into two major subtypes: Ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is characterized by confluent mucosal inflammation of the colon starting at the anal verge and extending proximally for a variable extent e.g., proctitis, left-sided colitis, or pancolitis. Crohn's disease, by contrast, is characterized by transmural inflammation of any part of the gastrointestinal tract but most commonly the area adjacent to the ileocecal valve.¹ Both diseases increase the risk of adenocarcinoma of the colon in the affected area.² Ulcerative colitis is most prevalent in North America, North Europe and Australia. The prevalence is roughly 10 times lower in southern and Eastern Europe, Africa, Asia and South America. Some reports have indicated that disease is now seen with increased frequency in parts of Asia such as India, Bangladesh and Japan.³ Specific goals of pharmacotherapy in IBD include controlling acute exacerbations of the disease, maintaining remission, and treating specific complications such as fistulas.¹ The main drugs used in the treatment of Ulcerative colitis and Crohn’s disease are the amino salicylates and corticosteroids.⁴ Although many types of treatment have been proposed and clinically proven, additional therapeutic approaches are needed because many patients do not satisfactorily respond to the currently available options or show significant side effects due to their prolonged use. Therefore, there is need to develop safe and effective alternative therapeutic agents for treatment of IBD.⁵

The epidemiological studies say that the incidence of IBD is increasing rapidly all over the world, which affects more than 396/100,000 persons per year⁶ both sexes are affected equally. Which affects young people’s between the ages around 20 to 30 years. In the United States, it is currently estimated that about 1 –1.3 million people suffer from IBD.^{7, 8}. There is a need to develop safe and effective therapeutic methods to IBD particularly of herbal therapies. Considering oxidative stress and inflammatory mediators are the major factors in IBD. Several natural products have been reported to be effective in IBD due to their antioxidant⁹ and inflammatory property¹⁰.These properties could be expected to provide relief from IBD. The pathogenesis of IBD is multifactorial process. One of the earliest factors involved are destruction of the gastrointestinal epithelial barrier by unknown mechanisms¹¹. It is histologically characterised by infiltration of polymorphonuclear leukocytes, monocytes, macrophages, pronounced epithelial hyperplasia, depletion of mucin secreting goblet cells and finally ulceration. Oxidative stress is one of the most important etiological and/or triggering factors for IBD. They are activated by various inflammatory mediators including prostaglandins, leukotrienes, platelet-activating factors and cytokines to synthesize and liberate reactive oxygen species (ROS). It is now accepted that ROS are produced in excess by the inflamed mucosa and may play an important role in IBD etiopathogenesis. Under physiological conditions, colonic mucosa contains relatively low tissue levels of endogenous antioxidants and oxidative stress may easily overcome the endogenous defence systems regulating ROS production. In cells from IBD patients, ROS are produced in abnormally high levels and leads to oxidative stress which may cause chronic and pre-treatment damage. Several cytokines like interleukins IL-1, IL-6 and tumour necrosis factor alpha (TNF-α) chemokines are also known to contribute to the pathogenesis of IBD. Inflammation overwhelms intestinal motility, which induces abnormal growth of intestinal flora. This disruption in intestinal flora causes mucosal inflammation and in turn increases the intestinal dysmotility^12,13

Melia azedarach Linn (Melia azedarach) family: meliaceae, commonly known as mahanimba, having traditional and medicinal repute. Phyto-pharmacological data unveiled that Melia azedarach contains polyphenolic flavonoids, other phenolic compounds, tannins, and it has been reported to possess anti-bacterial¹⁴, anti-pyertic¹⁵, anti-oxidant¹⁶, and anti-malarial¹⁷ activity. No scientific data regarding the activity of Melia azedarach aqueous leaf extract on IBD is available. Hence, the present study is proposed to evaluate the effect of Melia azedarach. aqueous leaf extract on experimental models of IBD in rats.

Materials and Methods

Chemicals

Indomethacin, carbochol, Sulphasalazine (SLZ)were purchased from Hi-media Laboratories Pvt. Ltd, Mumbai, India. All the other chemicals used were of analytical grade.

Animals

Adult male albino wistar rats (180-220 g) were used for the present study. The animals were housed in animal house of Sree Siddaganga College of Pharmacy, Tumkur, Karnataka, India. The animals were maintained under controlled condition of temperature (23±2˚C), humidity (50±5%) and 12 h light-dark cycle. The animals were acclimatized for seven days before the study. The animals were randomized into groups and placed housed individually in a sanitized polypropylene cage. Animals were habitude to laboratory conditions for 48 hours to minimize if any non-specific stress. The animals had free access to standard pellets as a basal diet and water ad libitum. The experimental protocol was approved by the institutional animal ethics committee and conducted according to CPCSEA guidelines, govt, of India. 

Collection of plant material

The leaf of M. azedarach was collected locally around Tumkur, The plant was identified and authenticated by Prof. K. Siddappa, Department of Botany, Sree Siddaganga College of Arts, Science and Commerce, Tumkur, Karnataka, India and a voucher specimen of the same was kept in the college herbarium.

Preparation of plant extract

The aqueous leaf extract of M. azedarach (Aq MA) was prepared by cold maceration process. After extraction the extract was concentrated in vacuum and then lyophilized.

Preparation of Suspension

The plant extract, SLZ and Indomethacin were suspended in water using suspending agent sodium carboxy methyl cellulose (sodium CMC, 0.3%). The plant extract and SLZ suspension were administered orally to animal using oral feeding tube while Indomethacin (7.5 mg/kg) suspension was administered via. s.c route. 

Indomethacin induced Enterocolitis in rats

Animals were pre-treated with Aq leaf extract of M.azedarach to Group IV - V respectively. Group VI receives sulphasalazine. Group I serves as normal control which receives vehicle only, Group II receives aq M. azedarach (200,100 mg/kg) for 7 days. The Indomethacin (7.5 mg/kg) will be given s.c on 8^th and 9^th day of treatment to Group III - VI. Drug treatment will be continued till 11^th day. On the 12^th day after treatment, the animals will be sacrificed by cervical dislocation and dissected. Ileum and colon will be taken out to assess inflammation, based on macroscopic and microscopic features. Quantification of inflammation will be done by assessing the parameters like Myeloperoxidase (MPO), Lipid peroxidase (LPO) and Glutathione (GSH). 

Macroscopic scoring¹⁸

The animals were sacrificed, after blood withdrawal and the small intestines were excised and opened. The proximal 10 cm portion of the ileum removed and opened longitudinally, pinned out on card, slightly cleaned in physiological saline to remove faecal residues and scored visually. Macroscopic inflammation scores were assigned based on clinical features of the ileum using an arbitrary scale as follows: 

Ulcerative Colitis¹⁸

The distal 10 cm portion of the colon was removed and cut longitudinally, slightly cleaned in physiological saline to remove faecal residues. Mucosal injury was assessed macroscopically using the grading scale ranging from 0-8 as follows: 

Biochemical estimations (for both UC and CD)

A portion of ileum/colonic tissue (remaining from histopathological study) were homogenized in 10% (w/v) of ice-cold potassium phosphate buffer (pH 7.4) using tissue homogenizer and the homogenate was used for the measurement of myeloperoxidase activity (MPO) and lipid peroxidation (LPO).

Myeloperoxidase (MPO) estimation¹⁹

The measurement of Myeloperoxidase activity is the first quantitative assessment to quantify the severity of colitis. It is reliable index of inflammation caused by infiltration of activated neutrophils to the inflamed tissue. The tissue homogenate was centrifuged at 3500 rpm for 30 min at 4^o C. The supernatant was used for LPO and the pellet for the MPO activity. To the pellet 10 ml of ice-cold 50 mm potassium phosphate buffer (pH 6.0), containing 0.5% hexadecyl Trimethyl ammonium bromide (HETAB) and 10 mm EDTA was added. It was then subjected to one cycle of freezing, thawing and brief period (15 s) of sonication. After sonication, the solution was centrifuged at 15,000 rpm for 20 min. The MPO activity was measured spectrophotometrically. 0.1 ml of supernatant was combined with 2.9 ml of 50 mm phosphate buffer (pH 6) containing 0.167 mg/ml of O-dianisidine hydrochloride and 0.0005% hydrogen peroxide. The change in absorbance was measured at 460 nm over period of 5 minutes. One unit of MPO activity is defined as the change in absorbance per min by 1.0 at room temperature, in the final reaction.

Calculation of MPO activity

MPO activity (U/g) =X / weight of the piece of tissue taken

Where X=10×change in absorbance per min / volume of supernatant taken in the final concentration.

Antioxidant parameters

Lipid peroxidation (LPO) ²⁰

Lipid peroxidation was evaluated as an index of oxidative damage and was assessed by measuring Malondialdehyde (MDA) in intestinal tissues. MDA is an end product of the lipid peroxidation process. MDA level is commonly known as a marker of oxidative stress. Briefly, 1 ml supernatant of tissue homogenate; 500 µL of TBA reagent (3.7 g/L in mol/L HCI); and ml of % TCA (in mol/L Weight loss HCI) were combined in a ml screw-cap Pyrex centrifuge tube, mixed, and heated for min in boiling water. After cooling in an ice bath, ml of n-butanol was added, mixed and centrifuged, and the chromogen extracted. The absorbance of the pink coloured organic phase was determined spectro- photometrically at 532 nm against a blank. The amount of lipid peroxidation was determined by using the formula

ε= 1.56 × 10^-1 M cm^-1 and expressed as nmol of MDA per g of wet tissue.

Clinical activity score²¹

Colitis was quantified with a clinical score accessing weight loss, stool consistency and bleeding of colon.

These scores were added and divided by 3, forming a total clinical score that ranged from 0.0 (healthy) to 4.0 (maximal activity of colitis).

Weight loss

Stool consistency

Bleeding

Colon Weight/Length index²²

The rats were sacrificed. It was followed by opening their abdomen and examining the appearance of colon. Thereafter, distal colon was removed, opened longitudinally and gently cleaned of faecal content using normal saline. Wet weight (mg) of the distal colon (10 cm from the anus) and weight/length ratio (mg/cm) were recorded for each specimen.

Colonic Contractility Studies²³

Colitis is associated with dysmotility and decrease colonic contractility. The lowest part of distal colon was cut and placed in Kerb‘s solution at pH 7.4 containing NaCl, KCl, CaCl₂ , MgSO₄ , KH₂PO₄ , NaHCO₃ , and glucose. The solution was saturated with carbogen gas. The muscle strip was set up in 40 ml organ bath and contraction is recorded using fixed force transducer-Biopac data Acquisition system. A resting tension of 1 g was applied and the tissues were allowed to stabilize for 30 min, during which the bath fluid was changed at least once. After stabilization tissue viability was checked by adding 80 mm KCl. A cumulative dose response curve was studied with different concentration of Carbachol concentrations (10^-8-10^-4 M); the contractile response was allowed to reach a peak before washing out and left to relax to the base line. This procedure was continued until maximum response was achieved. The maximum response was expressed as mg/mg tissue weight.

Histopathological Studies

To process for histopathological studies, ileum/colonic specimens were fixed in 10% formalin in phosphate buffered saline, embedded in paraffin and cut into 5 μm sections. Paraffin sections were deparaffinised with xylene, hydrated and stained with haematoxylin and eosin. The stained sections were assessed for any inflammatory changes including infiltration of cells, necrosis or damage to nucleus or tissue structures.

Statistical analysis:

The analysis of data was done by using one way analysis of variance (ANOVA), followed by Tukey‘s multiple comparison test by using GraphPad prism 5.0 software (GraphPad, San Diego, CA).

Results.

Macroscopic score.

Macroscopic scores which are indicative of tissue damage were significantly increased (P<0.001) increased with two successive doses of indomethacin (7.5 mg/kg) treated group as compared to normal control group. Pretreatment of Aq.MA (200 and 400 mg/kg) for 12 days to indomethacin treated rats showed significant (P<005; P<0.001) decrease compared to indomethacin alone treated rats. In addition SLZ treatment (100 mg/kg) exhibited better results than tested extract as evident by higher protection by 25%. Pretreatment with higher dose of Aq. MA alone did not exhibited significant changes in tissue damage (Table 1 & Figure 1)  

Serum LDH level

The effect of Aq.MA on serum LDH levels in normal and treated groups were presented in Table 1 and Figure 2. LDH levels were significantly (P<0.001) higher in indomethacin (7.5 mg/kg) alone group compared to normal control, while the results exhibited significant reduction in LDH levels (P <0.05 & P <0.01) and (P <0.001) on Aq.MA (200 & 400 mg/kg) and SLZ (100 mg/kg). Pre-treatment respectively in comparison with Indo alone group. No significant alterations were observed in serum LDH levels in Aq.MA (400 mg/kg) alone treated rats compared to normal control group.

MPO Activity

MPO activity (quantitative index of mucosal inflammation) was significantly increased (P<0.001) in Indo group when compared to normal group while Aq.MA (200 & 400 mg/kg) and SLZ (100 mg/kg) treated group significantly (P<0.01; P<0.001) reduced MPO activity as shown in Table 1 and Figure 2. 

Each bar represent represents mean±S.E.M (n=5), ###P<0.001 compared to Normal control; *P<0.05, **P<0.01 and ***P<0.001 compared to Indo alone group (One-way ANOVA followed by Tukey‘s posthoc test).

(NORMAL) Normal control, (EXT ALONE) Extract alone (400mg/kg), INDO ALONE (7.5 mg/kg), (LOW DOSE) Indo + Aq.MA (200 mg/kg) (HIGH DOSE) Indo + Aq.MA (400 mg/kg) (STANDARD) Indo + SLZ (100 mg/kg)

Each value represents the mean± S.E.M (n=5). Values in parentheses indicate percent protection against ulcer formation and ###p<0.001 compared to normal control; *p<0.05, **p<0.01, ***p<0.001 compared to indomethacin alone group. Statistical evaluation by One way ANOVA followed by Tukey‘s posthoc test

Discussion

IBD is a non-infectious, chronic intestinal inflammation which is comprised of Ulcerative Colitis and Crohn‘s Disease²⁴ and has become the serious threat in all the age groups, sex and races mostly in developing countries.

Melia azedarach linn. is a genus Melia of belonging to the family Meliaceae . Pharmacological evaluation of MA has shown its efficacy as antipyretic, antimicrobial, anti-inflammatory, wound healing and antioxidant agent. In recent years, there has been a global trend toward the use of natural phytochemicals present in natural resources, such as fruits, vegetables and herbs, as antioxidants and functional foods. Herbal plant products play important role in disease management without side effects. The present study has outlined the effectiveness of Aq MA as a therapeutic agent for IBD in experimental animal models of IBD.

Indomethacin induced Enterocolitis in rats

Rats injected with Indo through s.c route affects the middle portion of small intestine (jejunum and proximal ileum). The inflammation was not continuous and develop acute transmural inflammation of the small intestine characterised by wall thickening, mesenteric haemorrhage and multiple mucosal ulcers. Histopathologically the ulcers showed necrosis of mucosa with several submucosal inflammations, with neutrophils and macrophages infiltrate. These findings suggest that this experimental model resembles Chron’s diseases.²⁵

The mechanism of Indo induced Enterocolitis have not been fully illustrated, but previous reports suggest that, multi factors are involved such as depletion in prostaglandins (PGs), bacterial flora, bile acids and oxygen free radicals. Indo inhibits both isoforms of cyclooxygenase (COX-1 & COX-2) enzymes leading to depletion of endogenous, protective prostaglandins PGE1, PGE2 and prostacyline (PGI2) may be a major factor in the pathogenesis since exogenous PGs prevent gastrointestinal lesions associated with Indo may be one of the mechanism by which Indo induces injury. COX-1 is a constitutive enzyme present in various tissues including small intestine maintains integrity of gastrointestinal mucosa while COX-2 is an inducible enzyme which are not expressed in tissues. Both external and internal stimuli leads to higher levels of inflammatory mediators like growth factors and cytokines resulted in up-regulation of COX-2 lesions. And also by inhibition of cyclooxygenase enzyme leading to depletion of formation of protective prostaglandins PGE1, PGE2 and prostacyline (PGI2). As a result, the protective action of PGs is lost which is a predisposing factor for gastric ulceration and erosions Further, due to the non–selectivity of Indo may causes marked reduction in synthesis of cytoprotective PGs (PGE1, PGE2) and prostacyclin (PGI2) required for effective mucosal defence resulting intestinal damage, whereas selective COX inhibitors do notshow such phenomenon.^26,27 In the present study, treatment with Indomethacin produced hyperaemia, haemorrhagic spots, bowel thickening, and gross ulceration with skip lesions mostly in proximal ileum. Treatment with Aq MA and SLZ showed reduced intensity of lesions as evident by the lesser macroscopic score. The reduction in mucosal injury and inflammation with AqMA treatment may be due to increase in the muco-protective PGs synthesis or its antibacterial and membrane stabilizing activity.

Conclusion

The present investigation demonstrates that pretreatment of aqueous extract of Melia Azedarch Linn. can protect Indomethacin induced Enterocolitis (CD) Ulcerative Colitis (UC) in experimental rats. The results may be comparable to that of Sulfasalazine. This beneficial effect might be partly due to suppression of inflammatory reaction and oxidative stress, antibacterial, radical scavenging and membrane stabilizing activities. Overall, the titled plant could be promising therapeutic option for the management of IBD. Further investigation is required to explore the different possible mechanisms of action and proper clinical investigation should be carried out to confirm the same activity in human disease.

Histopathological study of ileum

Histological section of rat ileum treated with Indo showed massive inflammation, necrosis and goblet cells depletion. Furthermore, inflammatory infiltrate and mucosal ulcers as evident by villous loss were also observed. Pretreatment with Aq.MA and SLZ decreased the extent and severity of damage as shown in Table 2 and Figure 3 

Normal control, Indo alone (7.5 mg/kg,), Low dose (Indo + Aq.MA, 200 mg/kg), High dose (Indo + Aq.MA , 400 mg/kg), Standard (Indo + SLZ 100 mg/kg), Extract alone (Aq.MA, 400 mg/kg).

The microscopic images revealed regular normal intact colonic mucosa with epithelium, crypts, submucosa and muscularis propria in normal control and extract alone group. A.A induced colitis group demonstrated massive necrotic erosion of surface epithelium, crypt distortion, inflammation, goblet cells destruction and diffused inflammatory cells infiltration. The SLZ and high and low dose Aq.MA pretreatment had subsided extent and severity of histological signs of cell damage

Colonic Contractility Studies

The addition of Carbachol (cholinergic receptor agonist) in cumulative fashion at the concentration range of 10^-8-10^-2 M into the organ bath containing isolated distal colon produced dose dependant contraction of colonic specimens from all the groups. The Emax (maximal contractile response) and pD2 (negative logarithm of the concentration for the half-maximal response, ED₅₀) values were significantly reduced (P<0.001; P<0.01) in A.A alone group compared to normal control, indicating reduced contractility and sensitivity of the tissue. The induced reductions in the contractile responsiveness to carbachol were significantly improved with all the treatments as demonstrated by significant increment of Emax and pD2 values as shown in Table 3 and Figure 3

Each value represents mean±S.E.M (n=5), ## P<0.01, ###P<0.001 compared to Normal control; * P<0.05, ** P<0.01 and *** P<0.001 compared to Acetic acid alone group One-way ANOVA followed by Tukey‘s Posthoctest)

(A)Normal Control, (B) Indo Alone, (C) Indo+ Aq.MA (200 mg/kg), (D) Indo+ Aq.MA (400mg/kg), (E) )Indo+SLZ (100 mg/kg), (F) Aq.MA (400 mg/kg),