INTRODUCTION:

The inflammatory bowel diseases (IBD), ulcerative colitis and Crohn’s disease, are characterized by chronic idiopathic intestinal inflammation¹. There has been much debate regarding whether CD and UC are distinct entities or if they exist along a continuum of the same disease process. Crohn's disease is characterized by transmural inflammation involving any part of the gastrointestinal tract, with skip lesions often present². The inflammation in UC involves only the mucosa and submucosa of the colon and extends proximally from the rectum in a continuous fashion².

It is estimated that 1-2 million Americans suffer from IBD; approximately half of these have ulcerative colitis. In India, ulcerative colitis was first reported in 1964 and Crohn's disease was considered almost nonexistent till 1986. During last 10 years, Crohn's disease is being reported more frequently from different parts of India, especially southern India³.

Dysfunctions of the intestinal immune system and cross-reactivity against host epithelial cells have been implicated as major mechanisms by which inflammation occurs⁴. In addition, many cytokines that are consideredcentral to the pathogenesis of IBD, such as TNF-a,interleukin-1, and interleukin-6, are expressed in the intestinalepithelium. Aberrant secretion of these proinflammatory chemokinesand cytokines by epithelial cells is an integral part of thedysregulated immune response that initiates or perpetuates intestinalinflammation^5,6.

It was reported that there was an activation of genetic factor NFkB (Nuclear transcription factor) in inflammatory bowel disease. It is a key regulator of the inducible expression of many genes involved in immune and inflammatory responses in the gut. Stimuli like oxidative stress, cytokines (IL-1, IL-6, TNF α), bacteria and viruses can also release NFkB from their inactive cytoplasmatic form to the nucleus⁷. The production of chemokines is normally dependenton the coordinated activation of a number of signaling pathwaysthat converge on the transcription factor NFkB. Recently newer agent against TNF a like Infliximab is under investigation as a treatment option for IBD. Most patients can be adequately treated using a combination of aminosalicylates, antibiotics, and corticosteroids, though many patients with Crohn's disease will require immunomodulators, such as azathioprine (AZA) or 6-mercaptopurine (6MP)⁸.

Long-term use of glucocorticoids is associated with high rates of relapse and unacceptable toxicity. On the other hand, 6-mercaptopurine and its pro drug azathioprine are effective in maintaining remission, however, a significant number of patients are resistant or intolerant to thiopurines.

Quercus infectoria is reputed plant in ayurvedic system of medicine and commonly known as “majuphul” in ayurvedic literature. Quercus infectoria has antioxidant and anti-inflammatory actions⁹. Quercus infectoria contains mainly Gallotannin penta-O-galloyl-B-D-glucose (PGG). PGG, a major constituent, which can inhibit IL-8 gene expression by a mechanism involving inhibition of NFkB activation.

In pathogenesis of IBD involvement of NF-kappaB and oxidative stress is already reported. As well as anti-inflammatory drugs are also beneficial in the treatment of IBD. With this background Querus infectoria is selected as test drug for present study. In the light of above reports present investigation is undertaken to study the potential of Quercus infectoria in the treatment of inflammatory bowel disease using N-ethylmaleimide (NEM) induced colitis model.

MATERIALS AND METHOD:

Plant material:

Quercus infectoria Olivier (Syn: Majuful, Family: Fagaceae) galls were obtained from a commercial supplier of Ahmedabad. It was identified and authenticated by Dr. Hitesh A. Solanki, Reader Department of Botany, Gujarat University, Gujarat, India. Voucher specimen was submitted to Anand Pharmacy College.

Preparation of extract:

The galls of Quercus infectoria was extracted by a water miscible solvent, ethyl alcohol and water mixture with an alcohol content of 90%. The ground plant matter was extracted at a temperature of 50 °C for 30 mins with shaking. The solvent was subsequently evaporated and by this manner a crude extract(8%) was obtained.

Animals:

Healthy adult Sprague-Dawley rats of both sexes, weighing 250-300 gms, housed in a temperature conditioned room (22-24°C) with a 12-h light dark cycle, allowed free access to standard rat chow and water ad libitum. The study protocol was approved by Institutional Animal Ethical Committee as per the guidance of committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Drug treatment protocol for NEM induced Model:

Male Sprague dawley rats (250-300gm) were randomly allocated to 6 groups containing 6 animals. All groups were fasted for 24hrs prior to study, given access to water ad libitum.

Group I served as the normal control group throughout 18 days study period. Group II served as vehicle control group, which received 0.1ml of 1-% Methylcellulose intracolonically on 11 days of the study. Animals of Group-IV, Group-V and Group-VI were given the Std drug (5-ASA 100 mg/kg), Quercus infectoria (300 mg/kg) and Quercus infectoria (450 mg/kg) respectively for 18 days once a day orally. On 11^th day of the study, Colitis was induced with N-ethylmaleimide 3% in animals of Group-III-VI. During the study total food intake, water intake and body weight of each group was measured daily and average daily food intake, water intake and body weight per group was calculated from the above data. Stool consistency was measured for each group daily and scored from 0 to 2(Normal stool-0, Soft stool-1, and Liquid stool-2), the average score was calculated from above data. On 18^th day, the animals were weighedand anaesthetized with ether,and the abdomen was opened by a midline incision. The colon was removed, freed from surrounding tissues, rinsed and length and weight of it was measured. Colon was opened along the antimesentericborder and fixed on a wax block and scored for histological parameters like Macroscopic scoring, Microscopic scoring, Colon mucosal damage index (CMDI), Disease activity index (DAI). After scoring colon homogenate was prepared. From each group one colon of randomly selected animal was stored in the formalin (10%) and was used for the histopathology study in which Colon tissues was fixed in 10% neutral buffered formalin solution and embedded in paraffin. The tissues was then cut into 3 µm sections with uniform shape and size, mounted on glass slides, and were stained with haematoxylin. Selected tissue sections were fixed on the glass slide with the help of egg albumin.

RESULT:

Physical and histological parameters:

Pretreatment with Quercus infectoria reduced the severity of colitis induced in rats by NEM compared with Model control rats. Significant improvement in stool consistency score, CMDI score, DAI score, Macroscopic score, microscopic score, colon weight, colon length and colon weight-to-length ratio was observed with Quercus infectoria treatment as compared to model control group. A trend towards reduction in body weight loss, improved water intake and food intake was observed in all groups although these differences did not reach statistical significance as compared to model control group. (See Table1)

Biochemical parameters:

Tissue Malondialdehyde (MDA), Nitric oxide (NO), Myeloperoxidase (MPO), Superoxide dismutase (SOD) levels showed a statistically significant difference among the groups tested (P<0.001). By performing pair wise comparisons among the groups we can infer that the mean values of model control were significantly increased as compare to normal control group. Treatment with standard and various doses of Quercus infectoria (300 and 450 mg/kg) significantly reduced MDA, MPO and NO levels and increase the levels of SOD. (See Table 2).

Table 1: Effect of Quercus infectoria on various physical and histological parameters.

Parameter	Normal control	Vehicle control	Model control	5-ASA 100mg/kg	Ethanolic extract 300mg/kg	Ethanolic extract 450mg/kg
Physical Parameters
Food intake	139.11± 2.92	139.67±3.94	103.33± 10.67^**	122.78± 6.81	122.78± 6.81	130.56± 5.33^##
Water intake	174.67 ±7.06	171.67± 7.81	136.37±12.31^**	160.33 ±9.87	151.53 ±10.21	150.37 ±11.85
%Decrease in Body wt	-8.298 ±1.88	-5.242 ±2.067	8.739 ±1.615^*	4.29 ±0.579^##	5.433 ±1.495	4.931 ±0.7950
Colon wt	1.078 ±0.023	1.085 ±0.014	1.778 ±0.029^*	1.25 ±0.028^#	1.31 ±0.037^#	1.298 ±0.031^#
Colon length	16.167± 0.447	15.083± 0.602	9.85 ±0.433^*	14.967 ±0.749^#	13.867 ±0.416^#	14.083 ±0.39^#
Colon wt/l	0.067 ±0.002	0.073 ±0.003	0.182 ±0.008^*	0.084 ±0.003^#	0.095 ±0.004^#	0.092 ±0.002^#
Stool consistency	0.167 ±0.141	0.333± 0.178	2±0.0^*	0.5 ± 0.189^#	0.667 ±0.178^#	0.333 ±0.178^#
Histological Parameters
CMDI	0.167± 0.167	0.333± 0.211	3.833± 0.167^*	1.167± 0.167^#	1.333± 0.211^#	1.333± 0.211^#
DAI	0.167± 0.167	0.333± 0.211	3.667± 0.211^*	0.833± 0.167^#	1.5± 0.224^#	1.333± 0.211^#
Macroscopic Scoring	0.167± 0.167	0.167± 0.167	3.83± 0.167^*	0.667± 0.211^#	1.167± 0.167^#	1±0.0^#
Microscopic scoring	0±0	0.167± 0.167	3±0^*	0.5± 0.224^#	0.667± 0.211^#	0.5± 0.224^#

* Significantly different from Normal control group at p < 0.001; ** Significantly different from Normal control group at p < 0.05.

# Significantly different from NEM control group at p < 0.001; # #Significantly different from NEM control group at p < 0.05

Table 2: Effect of Quercus infectoria on various biochemical parameters in colonic tissue homogenate.

Parameter	Normal control	Vehicle control	Model control	5-ASA 100mg/kg	Ethanolic extract 300mg/kg	Ethanolic extract 450mg/kg
MDA	2E-07± 1.03E-08	2.03E-07± 5.65E-09	4.83E-07± 2.85E-08^*	2.25E-07± 1.27E-08^#	3.57E-07± 3.94E-08^#	2.8E-07± 5.19E-08^#
NO	0.713± 0.037	0.744± 0.019	3.53± 0.054^*	0.81± 0.017^#	0.966± 0.033^#	0.912± 0.03^#
MPO	3.63E-06± 1.14E-07	4E-06± 1.4E-07	1.03E-05± 2.52E-07^*	5.15E-06± 1.65E-07^#	6.53E-06± 2.02E-07^#	6.21E-06± 2.5E-07^#
SOD	15.76± 1.78	15.4± 2.43	5.10± 0.82^*	12.61± 2.44^##	8.08± 0.92^##	9.07± 1.22^##

* Significantly different from Normal control group at p < 0.001; #Significantly different from NEM control group at p < 0.001.

# #Significantly different from NEM control group at p < 0.05

Histopathology:

In normal control rats epithelial crypts of the mucosal layer were preserved and there is no infiltration of inflammatory cells. In contrast in model contol rats, epithelial crypts were completely lost and there was a marked inflammatory cell infiltration affecting the laminapropria and submucosa along with submucosal edema. In Quercus infectoria treated colitic rat, epithelial crypts were shortened but still present, and the inflammatory infiltration and edema had decreased (original magnification 10). Similar result was obtained in standard control animal and there was no significant histopathological change observed in vehicle control group as compared to normal control animal. (See figure 1)

DISCUSSION:

In the present study IBD was induced by intrarectal administration of SH blocker, N-ethylmaleimide (NEM 3%, 0.1 ml, single dose) to the colon. This compound produces an erosion and ulcer and the necrosis is accompanied by acute and chronic inflammation. This condition correlates well with Inflammatory Bowel Disease, mainly ulcerative colitis in mammals, particularly humans. Satoh et al. (1997) also reported that NEM induced ulcerative colitis is useful for studying the pathogenesis of chronic ulcerative disease, prevention and treatment thereof and for evaluating drugs suspected of being useful in the treatment of same¹⁰.

The common symptoms observed in IBD patients are reduction in food intake and water intake due to inflammation which results in decrease body weight¹¹. This inflammation and necrosis of colon tissue leads to increase in colon weight and decrease in colon length in animal models¹². Craven et al (2004) showed reduced stool consistency and diarrhea due to IL-1B expression in IBD patients. Similarly in rats, NEM administration caused significant reduction in water intake, food intake, and body weight and reduced stool consistency¹³.Treatment with 5-aminosalicylic acid and test drugs results in regaining normal body weight, food intake and water intake. These treatments also prevent the alteration in colon weight, colon length and stool consistency. This is indirect evidence of reduction in inflammation of the colon with test and standard drug treatment.

Colitis induced in rats showed marked increase in various histological indices like CMDI¹⁴, DAI¹⁴ as well as Macroscopic scoring¹⁵, Microscopic scoring¹⁶, which all reflects a high levels of inflammation. In our study we also observed increased histological profile in model control animals. Test dugs and Std drug treatment showed significant improvement in inflammation reflected from histological indices. These results were also supported by histopathological study results.

Figure: 1 Histologic damage evaluated in colonic samples stained with hematoxylin and eosin.

Normal

Vehicle control

Model control

5-Amino salicylic acid

300 mg/kg GALLS

450 mg/kg GALLS

Perner et al (2002) suggest that there is greatly increased production ratesof nitric oxide (NO) into the colonic lumen of patients with inflammatory bowel disease and provided evidence for the hypothesisthat the enzyme, inducible NO synthase (iNOS), is the sourceof excess NO production¹⁷. One of the major transcriptional inducers of iNOS gene expressionis the transcription factor nuclear factor kB (NFkB)¹⁸. Increased production of reactive metabolites of nitrogen hasa significant pathophysiological role in the development ofinflammation. It may react with superoxide anion, resulting in oxidative tissuedamage through production of peroxynitrite which is believedto mediate many of the destructive effects of NO in colon inflammation¹⁹.Increased luminal activities of NO have also been detected in IBD patients.

In our study we also found significant increase in the colon NO level with NEM administration. Kaur et at 2003 showed decreased in NO levels by ethanolic extract of Quercus infectoria we also observed consistent results with the treatment of both doses of Quercus infectoria (300 mg/kg, 450 mg/kg)⁹. Galls of Quercus infectoria is rich source of tannins(Phytochemical and Ethnobotanical Databases). One type of tannin PGG, a major constituent of Quercus infectoria inhibit IL-8 gene expression by a mechanism involving its inhibition of NF-kappaB activation, because of this activity it also regulates the expression of iNOS and controls the over production of NO which is increased in IBD. Thus inhibition of NFkB may be one of the mechanisms by which test drug showed beneficial effect in inflammatory bowel disease.

Malondialdehyde is final product of lipid peroxidation and is good indicator for extent of oxidative stress²⁰. Myeloperoxidase catalyses the conversion of proportionally more stable hydrogen peroxide to unstable hydrochlorus acid. Thus, it promotes oxidative stress and additionally it induces neutrophil infiltration on mucosal area causing further damage to the tissue²¹. In NEM induced IBD we found significant increased in malondialdhyde as well myeloperoxidase levels in the colon. We found the protective results in context to level of malondialdhyde as well as myeloperoxidase with the treatment of both the doses of Quercus infectoria (300 mg/kg, 450mg/kg).

Preventive anti-oxidant, such as superoxide dismutase (SOD) enzyme is the first line of defense against reactive oxygen species²². Superoxide dismutase (SOD) is widely distributed in cells with high oxidative metabolism and has been proposed to protect such cells against the deleterious effect of superoxide anion²³. SOD catalytically scavenges the superoxide radicals and thus renders cytoprotection against free radical damage. In the present study significant decrease in preventive antioxidant level was observed and this change was prevented with the treatment of both the doses of Quercus infectoria (300 mg/kg, 450mg/kg). Above results revealed that anti-inflammatory and anti-oxidant activity of Quercus infectoria is responsible for improvement of IBD in rats. Besides that inhibition of NFkB may be also contributing factor for protective effect of Quercus infectoria. Still direct evidence of this mechanism should be elucidated and which will open up a new way for the treatment of IBD.

In conclusion, Quercus infectoria showed beneficial effect in NEM induced IBD in rats.

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Received on 27.12.2010 Modified on 27.01.2011

Research J. Pharm. and Tech. 4(5): May 2011; Page 782-786