Protective Effect of Colocasia esculenta on TNBS Induced Ulcerative Colitis in Mice

 

Srikanta Chandra1, Tathagata Roy2*, Preeta Bose3, Somsubhra Ghosh4, Susanta Paul5,

Victor roychowdhury6, Anannya Bose7, Ankita Acharya8

1Assistant Professor, Jakir Hossain Institute of Pharmacy, Miapur, Murshidabad,742225.

2Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

3Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

4Assistant Professor, School or pharmacy, Techno India University, Salt Lake, Kolkata-700091, W.B.

5Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

6Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

7Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

8Assistant Professor, Department of Pharmaceutical Technology, JIS University, Kolkata-700109.

*Corresponding Author E-mail: troy@jisuniversity.ac.in

 

ABSTRACT:

Objective:  Ulcerative colitis (UC) is a recurrent ulceroinflammatory disease that is mainly restricted to colonic mucosa with variable colonic architecture distortion. Different types of drugs are used for management of UC namely Salfasalazine, Balsalazide, Mesalamine etc. However, they are associated with different types of adverse effects such as nausea, vomiting, skin rash, headache. Hence, we narrowed in on Colocasia esculenta and its available in India, Bangladesh, Pakistan. In our present study the anticolitic effects Colocasia esculenta in treatment the TNBS induced model for UC in mice were examined.

Methods: Male mice, 7 weeks of age, are selected. They are divided into various categories:Negative control groups treated with normal saline solution ,positive control group treated with TNBS5%, standard group treated with Balsalazide 4mg/kg & TNBS 5% , Test(1) group treated with Colocasia esculenta 100mg/kg, Test(2) group treated with Colocasia esculenta 150 mg/kg, Test(3) group treated with Colocasia esculenta 250 mg/kg then we were performed various parameters such as DAI, Oxidative damage assessment, MPO Assessment.

Results: The results have been showed that Colocasia esculenta has significant activity against TNBS (5%) induced colitis when compared to the experimental control, with near normalization of colon architecture. Tissue oxidative stress was reduced with significant improvement in tissue levels of MDA &GSH. Furthermore, significant improvement in levels of myeloperoxidase (MPO) was observed.

Conclusion: It is concluded that Colocasia esculenta(250mg/kg) has got potent activity against TNBS (5%) induced ulcerative colitis (UC) due to its anti-inflammatory& antioxidant properties.

 

KEYWORDS: Ulcerative colitis, Anti-inflammatory, Antioxidant, Colocasia esculenta, Myeloperoxidase.

 

 


INTRODUCTION:

Inflammatory bowel disease is an incurable chronic inflammatory intestinal disorder of the gastrointestinal tract that dramatically impacts quality of life. American Gastrological Society states that IBD is defined as acute or chronic ulceroinflammatory idiopathic disease of the bowel which may or may not have any transmural stretch.1 IBD encompasses two important  clinicopathological condition Ulcerative colitis and Crohn’s disease.2 Ulcerative colitis is a recurrent ulceroinflammatory disease confined mainly to the colonic mucosa with variable colonic architectural distortion whereas Crohn’s disease is a chronic ulcerative inflammatory condition similar to ulcerative colitis but having a transmural infestation.3Multiferrous expansion from rectal bleeding, weight loss, stomach pain and malaise are the characteristics observed in the clinical course of the disease.In the year of 1875 two scientists are discovered such as Wilks and Moxon who distinguished it from diarrheal diseases caused by infections agents. Few year later three doctors zero in on Crohn’s disease namely Burnil1 Crohn, Leon Ginzberg and Gordon. D. Oppenheimer.5 Briefly, we can manage ulcerative colitis by two ways one is non pharmacological aspects another one is pharmacological aspects. Under non pharmacological aspects life style modifications like monitored dieting, exercise may play a crucial role in this therapy and psychological intervention also deployed to control the functional aspects of the disease .6 On the other hand Salfasalazine and Balsalazide plays a desire role in management of ulcerative colitis as per as pharmacological aspects.7 However these pharmacological moieties are too much associated with different types of adverse effects such as nausea, vomiting, headache, hypersensitivity reactions, fever, dizziness etc.8 To avoid these adverse effects we narrowed in on Colocasia esculenta which is available in India, Bangladesh, Pakistan. Colocasia leaves contains phytochemicals such as anthraquinones, apigenin, catechins, cinnamic acid derivatives, vitexin &isovitexin already reported by Patil R Bhagyashree et al 2011. The present study was performed to investigates the anticolitic potential of Colocasia esculenta on TNBS induced colitis in rats.

 

MATERIAL AND METHODS:

Plant material:

The fruits of Colocasia esculenta were collected from Rampurhat (W. B), India in August 2020 and were authenticated by the Head of Botany Department of Government College, Rampurhat, Birbhum, West Bengal.

Authentication:

An herbarium sheet was prepared and sent for the authentication of the study plant to the head of the botany department of government college, Rampurhat, Birbhum, West Bengal. Ref No: Rph/BOT/2020/32.

 

Extraction:

Colocasia esculenta fruits were shade dried and powdered. Extraction of the dried powdered material (500g) with ethanol was carried out in a Soxhlet apparatus while the residual marc was extracted with water in a reflux condenser. Both extracts were vacuum dried to yield 5.8% of ethanol extract and 7.5% of aqueous extract.9

 

Animal Husbandry & Statutory Approval:

All animal protocols used in this study were approved by the Institutional Animal Ethics Committee (IAEC). Protocol no: GCTS/IAEC/2018/04. Male C57BL/6J mice at 7 weeks age (average body weight :20-22 gm) were taken & acclimatized to the animal facility for 1 week with ad libitium access to drinking water& a rodent chow diet prior to experiments. Mice were initially housed 4 animals per cage and maintained under standardized conditions (22±2°C, 12 hours light/dark cycle, and 50-60 % humidity)10

 

Acute Oral Toxicity Test in Rats:

After overnight fasting of rats, Colocasia esculenta was given orally in doses of 50, 100, 200, 500 and maximum dose of 1000 mg/kg body weight and observed carefully for the first 2-3 hours for signs of toxicity. The behavioral changes and percentage mortality were documented beginning with 24 hours up to a period of 14 days.11

 

Experimental Design:

Male mice aged 7 weeks are selected for the experiment. They are allocated into various groups: Negative control groups treated with normal saline solution, positive control group treated with TNBS%, standard group treated with Balsalazide 4mg/kg & TNBS 5%, Test (1) group treated with Colocasia esculenta 150 mg/kg, Test (2) group treated with Colocasia esculenta 200 mg/kg, Test (3) group treated with Colocasia esculenta 250 mg/kg.

 

Estimation of Disease Activity Index (DAI):

Onset of disease was assessed by estimating disease activity index by the technique mentioned previously by Cooper et al (2015) with necessary alterations. Severity of colitis was assessed by estimation of colon length and weight ratio. DAI was scored based on parameters like body weight, frequency of defecation, volume of faecal matter and stool consistency throughout the dosing period.12

 

MPO Assessment:

To measure Myeloperoxidase (MPO)activity colonic samples were taken in ice and was homogenized in 10 ml of ice –cold 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyl trimethyl ammonium bromide (HETAB).  Homogenates were then sonicated and centrifuged for 20 min at 12,000 g. MPO activity was measured spectrophotometrically at 460nm.With respect to blank, the concentration was measured& expressed as u/g.13

 


RESULTS:

Table 1: Effect of Colocasia esculenta on experimentally induced ulcerative colitis

Group

DAI

Tissue MPO(u/g)

Tissue MDA(u/g)

Tissue GSH (nmol/mg)

Negative control

0.34±0.21

0.31±0.01

0.512±0.122*

0.005±0.012*

Positive control

4.099±3.015**

2.801±0.004**

2.043±0.133***

1.002±0.113**

Standard

3.112±2.002

1.405±0.003

1.002±0.105

1.226±0.114

Test(1)

3.166±2.014

1.022±0.0121

1.031±0.176

1.276±0.167

Test(2)

2.244±1.165*

1.051±0.0112

1.021±0.124

1.879±0.189

Test(3)

2.065±1.014

0.387±0.002*

0.899±0.111*

2.677±0.197***

Values are expressed as mean ±SEM, n=6. aP<0.05 and p>0.05 when compared to experimental control; ap<0.05 and  bp>0.05when compared to Balsalazide; ANOVA followed by Dunnett’s –test. DAI: Disease activity index, ANOVA: Analysis of variance, SEM: Standard error of mean.

 


Oxidative Damage Assessment:

Tissue was taken and itis homogenized. About 3mL of 1 % phosphoric acids and 1ml of 0.6% TBA (Thiobarbituric acids) solution were added to 0.5mL of colon homogenate. The mixture was heated for 45 min on a boiling water bath. After cooling, 4 mL of n –butanol was added and mixed vigorously. Then butanol phase was separated by centrifugation at 5000 rpm for 10 min and absorbance was measured at 535 and 520 nm. 14 GSH content was measured using the Bioxytech GSH -420 assay Kit (OXIS Health Products, Inc, Portland, or USA). Briefly, the colon was suspended in 10 mM PBS (pH 7.4) mixed with ice –cold 7.5% trichloroacetic acid solution, then homogenized on ice. The homogenates were centrifuged at 30000 rpm for 10 min. Then GSH content was measured at 420 nm using a spectrophotometry with respect to blank, the concentration was measured & expressed as nmol/mg of proteins15.

 

Statistical Analysis:

Results were reported as mean ± SEM. One-way ANOVA used for comparing the most of the parameters except DAI which has been analyzed by two-way ANOVA because of its composite nature. For the identification of groups with major variations in one-way ANOVA, post hoc tests were used (Efstathios Antoniou et al. 2016). The multiple range test from turkey was used for comparisons whereas for post hoc analysis two-way anova bonferronies test was used. The important groups were identified by designated alphabets on the figure.

 

DISCUSSIONS:

Inflammatory bowel disease (IBD) is an emergent problem in present day. IBD encompasses two important pathological condition one is Ulcerative colitis (UC) & Crohn’s disease. Ulcerative colitis is a chronic ulceroinflammatory condition primarily confined within colonic mucosa with variable distortion of the colonic architecture. Whereas Crohn’s disease (CD) is a chronic ulceroinflammatory condition analogue to ulcerative colitis (UC) but having a transmural infestation. Briefly, Sulfasalazine &Balsalazide are the pivotal pharmacological agents and they play a desire role behind the management of ulcerative colitis (UC). However, they are associated with different types of adverse effects such as hypersensitivity reaction, nausea, vomiting, headache etc. Hence, we found a good rational behind probing for the anticolitic potential of the drug in pipeline that is Colocasia esculenta which is available in India, Bangladesh and Pakistan. Briefly, we take the opportunity to test the comparative features of two most commonly used models of Ulcerative colitis (UC) TNBS induced model and DSS induced model.

 

TNBS model shares many histopathological and clinical features of IBD and is useful for the study of the aetiopathogenesis of chronic colonic inflammation as well as providing an inexpensive model suitable for assessing therapeutic agents. In IBD body weight, volume of fecal matter, stool consistency are the important indicators of the severity of the disease. In our study we found that the treatment with Chloroquine decreased weight loss. Since, animal treated with TNBS 5% showed high colon weight compare to negative control group and decrease their body weight. Treatment with Colocasia esculenta 250mg/kg reduced colon weight of animals treated with TNBS 5% so its projects Chloroquine as a potential controller of inflammation and edema associated with ulcerative colitis. In our study, we induced IBD by per oral administration of DSS in animals.

 

The severity of colonic inflammation in the disease was evaluated by measuring standard parameters like DAI, MPO, GSH, MDA activity. In present study, we found that decreased in progression of the disease pathogenesis following treatment with Colocasia esculenta significantly declined the score of DAI compared to the positive control group.

 

MPO is an enzyme found in the neutrophils, and since there happens to be a high infiltration of neutrophils in the inflamed region hence MPO may serve as a quantitative index of inflammation in colonic tissue. MPO activity may be regarded as an index of inflammation damage. This is in the line with the conclusion published by David R .H et al. (2016).  The main pathological feature of IBD is transmural infiltration of polymorphonuclear neutrophils and MPO is released from these neutrophils. Treatment with Colocasia esculenta 250 mg/kg significantly decreased the level of MPO compared to the positive control group which shows that Colocasia esculenta 250 mg/kg decrease the infiltration of the inflammatory cells which are responsible for the increasing the progression of the disease condition.

 

Many studies (Tian Tian et al. 2017, Ritu Chauhan et al. 2014) have revealed that the increase of oxidative stress & MDA & GSH activity has been a notable feature of IBD, which resulted in a pathological cascade of free radical reactions & further yielding more oxidative free radicals. Failures of the endogenous antioxidant defense mechanisms promote formation of excessive free radicals and consequent tissue damage. Parameters such as MDA, GSH activity can be indicative of oxidative stress status of the disease. MDA level can be determining by the thiobarbituric acid reacting substance (TBARS) As observed in our study the increase in MDA levels in the colon affected by the TNBS 5% administration suggests enhanced lipid peroxidation that could be responsible for the tissue damage. This is in the line with the conclusion published by Salim S AL –R. et al (2016).

 

In the present study, animal group treated with TNBS 5% suffered an increase in the oxidative stress indicated by higher MDA and GSH activity which are responsible for the tissue damage and amelioration of inflammation respectively. In our study we found that animals treated with Colocasia esculenta 250mg/kg had reduced MDA expression and increased GSH activity which was significant in comparison to the positive control group thus suggesting its antioxidant property.

 

CONCLUSION:

It can be concluded that Colocasia esculenta(250mg/kg) has got potential against TNBS (5%) induced ulcerative colitis. Our research is the pioneer in providing unequivocal proof of the anticolitic efficacy of Colocasia esculenta (250 mg/kg) an activity that may be largely attributed to its antioxidant and anti-inflammatory potential.

 

ACKNOWLEDGEMENT:

Thanks to department of Pharmaceutical technology, JIS University, Kolkata for provided a good infrastructure to carry out this work.

 

FUNDING: 

This research did not receive any specific grant from any funding agencies in the public commercial or not for profit sectors.

 

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

 

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Received on 11.12.2020            Modified on 17.02.2021

Accepted on 22.03.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):385-388.

DOI: 10.52711/0974-360X.2022.00063