Introduction

Inflammatory bowel disease is an idiopathic and chronic intestinal inflammatory condition. ^{Ulcerative colitis and Crohn's disease are the two major types of inflammatory bowel disease.Developed countries have the highest rates of inflammatory bowel disease [1].}

The major symptoms of inflammatory bowel disease are diarrhoea, rectal bleeding, tenesmus and crampy abdominal pain [1]. Extra intestinal manifestations like arthritis, mucocutaneous, ophthalmic and hepatobiliary lesions are frequent. In severe cases perforations, peritonitis, strictures and tendencies to form fistulae are common [1].

The use of sulfasalazine and steroids in the treatment of inflammatory bowel disease has been established but these drugs only help in remission of the disease with no permanent cure [2]. For such a disease one can always explore the possibility of evaluating drugs used in alternative medicine.

An Indian Ayurvedic drug which required testing was holarrhena antidysenterica. It is a deciduous shrub found in India [2]. Around 30 alkaloids have been isolated from the bark of the plant [3].The drug is found useful in amoebic dysentery, chronic diarrhoea, flatulence and abdominal colic and is widely known for its antinflammatory property [4, 5].The drug is also useful in diseases of spleen and skin [6]. There was no scientific efficacy data available for its use in inflammatory bowel disease. Therefore, an animal study to prove its efficacy was essential. The dextran sulphate induced colitis in albino rats resembles inflammatory bowel disease of moderate degree[7] ^{.The present study was, therefore, planned to evaluate the efficacy of holarrhena antidysenterica in dextran sulphate induced colitis in albino rats.}

Materials and Methods

48 male wistar rats weighing 150 – 250 gms were randomly divided in to four groups (n=12) and were caged indivually at room temperature. Control Group A received no drug, Group B received oral sulfasalazine 50 mg /kg and prednisolone 0.75 mg/kg [8, 9], Group C received oral holarrhena antidysenterica 13.5 mg / 200 grams body weight per day and Group D received orally holarrhena antidysenterica 13.5 mg / 200 grams body weight per day, sulfasalazine 50 mg /kg body weight and prednisolone 0.75 mg/kg body weight. The Institutional Animal Ethics Committee permission was taken prior to the study.

Dextran sulphate sodium (molecular weight. ^{40000) was dissolved in drinking water to a final concentration of 4% for nine days to all the rats [10]. All rats were allowed free access to food and water mixed with dextran sulphate except during the 12 hour fast preceding the colitis induction [10].}

The medications were started from the day 10 of the experiment.Holarrhena antidysenterica was given as oral suspension dissolved in 2% gum acacia and water. Kuthaj ghanavati (Hindi name for holarrhena antidysenterica tablet) 250 mg tablet (preparation of aqueous extract) was taken from Rudra ayurvedic pharmaceutical company, Pune, India for preparation of the final product. Sulfasalazine and prednisolone were also given as oral suspension dissolved in 2% gum acacia and water in appropriate dose.

All rats were sacrificed on the day 19^th from the start of the experiment by giving ether anesthesia. Gross and histological assessment of intestinal tissue of the rats was done at the end of the experiment by an expert who was unaware of the treatment groups. Inflammatory changes on gross morphology were assessed, using a visual analogue scale (minimum injury 0, maximum injury 100) [11]. Microscopic inflammatory changes were scored from 0 to 4 depending on the severity of inflammation and associated crypt damage(0 being absence of inflammation and 4 being extensive inflammation with crypt damage) [11].Myeloperoxidase levels, which is an indicator of polymorphonuclear leukocytic activity within the intestinal tissue was measured and expressed as milliunits per gram weight of the tissue [12]. Liver function tests and renal function tests were estimated from the blood samples of the rats by enzymatic method at the end of the experiment.

Statistical analysis

Results

The results showed that macroscopic, microscopic examination scores and myeloperoxidase levels were comparatively higher in Group A as compared to Group B, C & D.This confirmed the induction of colitis in albino rats. No statistically significant difference was noted when macroscopic, microscopic examination scores and myeloperoxidase levels in the Group B, C & D where compared in between the groups. But when the same scores in Group B, C & D were compared with Group A indivually statistically significant values were obtained.

Figure 1

Table 1-Mean myeloperoxidase values in the intestinal tissue (Mean ± S.D)

Myeloperoxidase levels were higher in group A as compared to group B, C & D.

Figure 2

Table 2-Mean values of Pathological assessment of intestines in all groups (Mean ± S.D)

Pathological assessment (microscopic and macroscopic examination) scores were higher in Group A as compared to Group B, C & D.

Figure 3

Table 3- t and p values for comparison in between groups for different parameters

P values were highly significant when Group A values were compared with Group B, C and D individually for pathological and myeloperoxidase assessment of the intestinal tissues. P values were insignificant when comparison was done in-between Groups B, C and D respectively for the same parameters as mentioned above.

Figure 4

Figure 1-Histology findings of rat intestinal tissue in all groups

The above figure shows that leukocytic infiltration which is a good marker of inflammation is more in group A as compared to intestinal tissue slides of group B, C & D. The microscopic scores for the above slides are in table number 2.

Discussion

Inflammatory bowel disease is an important health problem in the today’s world [13]. The absence of exact experimental colitis models imitating inflammatory bowel disease makes it difficult to search for newer modalities of treatment [13].Various animal models of colitis have been shown to imitate the human inflammatory bowel disease [10]. According to the study conducted by Holma R the dextran sulphate induced colitis in albino rats closely resembles inflammatory bowel disease of moderate severity and therefore is a suitable animal model for the evaluation of a new drug. The mortality associated with the dextran sulphate model is less as compared to other methods for induction of colitis [10]. There are various other methods to induce colitis but these models induce severe colitis and require special procedures for induction [12]. In the present study, experimental colitis was induced by using dextran sulphate and the severity and type of inflammation was in accordance with studies done earlier.

Sulphasalazine (5-ASA) and prednisolone are used as drugs for medical treatment in inflammatory bowel disease. Combination therapy of these drugs is clinically found to be beneficial in inflammatory bowel disease. In two different studies carried out by Kimura I et al and Gyorgy R et al it was found that prednisolone and sulfasalazine were highly effective in remission of the experimental colitis [15, 16].A review article by Axelsson & Ahlstedt stated that sulphasalazine has therapeutic effect on the experimental colitis animal models [17]. The findings of the present study are in accordance to the reports earlier.

In a study carried out by Biesel and Bugra it was found that myeloperoxidase levels were directly proportional to the number of leukocytes present within the tissue. In the present study, the higher levels of myeloperoxidase were comparable to the severity of inflammation and number of leukocytes in the intestinal tissues of the rats and the results of the study were similar to those in the studies done earlier [18].

Holarrhena antidysenterica is indicated in inflammatory bowel disease like condition (Pittavrutta apaana, a disease having clinical resemblance to inflammatory bowel disease) described in Ayurvedic system of medicine [14]. There is no literature available in modern medicine about its possible use in inflammatory bowel disease. Holarrhena antidysenterica showed beneficial effect in experimental colitis as monotherapy. No additional benefit was achieved when holarrhena antidysenterica was tried in combination with steroids and sulfasalazine in the treatment of experimental colitis as compared to monotherapy. This indicated the efficacy of holarrhena antidysenterica in experimental colitis in albino rats.

Conclusion

Dextran sulphate induced colitis in albino rats is a good animal model to study drug action on experimental colitis.Sulphasalzine and prednisolone has a beneficial effect on experimental colitis in albino rats.Holarrhena antidysenterica has therapeutic benefit when used as monotherapy in the treatment of experimental colitis in albino rats. No additional benefit was achieved when holarrhena antidysenterica was tried in combination with steroids and sulfasalazine in the treatment of experimental colitis as compared to monotherapy.Therefore; it is recommended that a further clinical study be carried out.

Acknowledgement