Protective effect of Cod Liver Oil in Experimentally Induced Gastric Ulceration in Rats

 

Kiran P. Narkhede¹, Trilochan Satapathy²*, Bibhas Pandit²

¹Department of Pharmacology, PRIST University, Thanjavur-614904, Tamilnadu, India.

²Columbia Institute of Pharmacy, Near Vidhan Sabha, Tekari, Raipur, Chhattisgarh, Pin-493111, India.

 

ABSTRACT:

Background: Gastric ulcer is a life style disease, observed when gastric mucosa is exposed to the acid-pepsin mixture (APM) for prolonged period of time. The reason to undertake the present study is based on the scientific evidence that the cod liver oil found to possess a significant antioxidant and protective action against inflammation. However, there was no scientific proof established regarding the antiulcer activity of cod liver oil. As inflammation is one the causes of gastric ulcer, the present work was designed to evaluate the anti-ulcer efficiency of cod liver oil in three different ulcerated rat models. Methods: Ulcer was induced through pyloric ligation, ethanol, and aspirin. The effect of Cod liver oil was evaluated by estimating the pH of the gastric juice, volume of gastric juice, ulcer scores, free acidity, total acidity as well as mucin content in the ulcerated rat models. The degree of ulcer protective consequence was explored by comparing the data with standard drugs i.e. omeprazole and sucralfate. Results: Cod liver oil exhibited a significant mucosal protection and anti-secretory action in all three models. Cod liver oil showed percentage ulcer protection of  >50% in pyloric ligation, ethanol-induced and aspirin-induced ulcer model when compared to standard drug omeprazole and sucralfate. Conclusion: The findings from the present study indicated that the use of cod liver oil in gastric ulcer may be beneficial as it showed significant mucoprotective and anti-secretory activity in ulcerated rat models.

 

Graphical Abstract

 

 

 

 

1. INTRODUCTION:

Gastric ulcer is considered as conglomerate of heterogeneous disorder generally acknowledged due to the imbalance between the offensive factors such as gastric acid secretion, pepsin activity, non-judicious use of alcohols, prolonged NSAIDs use, smoking, stress, Helicobacter pylori infection and defensive factors such as gastric mucosal prostaglandin (PG) content, nitric oxide (NO), gastric mucosal blood flow, efflux of bicarbonate, increasing of antioxidant levels, sulfhydryl and others [1-3]. Gastric ulcer is observed when the gastric mucosa is exposed to the acid-pepsin mixture (APM) for prolonged periods of time that results increasing severity associated with ulceration, erosions and stricture formations. Numerous therapeutic agents have been used for the treatment of gastric ulcers including antacids, H₂-receptor antagonists, Proton Pump Inhibitors (PPIs) and others. Amongst them proton pump inhibitors and H₂-receptor antagonists used as first-line therapy for gastric as well as duodenal ulcers by exerting potent and sustained acid inhibitory effect but, it has been hypothesized that, both categories of drugs are responsible for gastrin release by the elevation of intragastric pH [4-5]. Furthermore, the rate of recurrence of ulcers within the one year after the suspending of the treatment is between 40 to 80% [6]. Developing drugs or supplement with balanced cytoprotective and anti-secretory potencies would be desirable. The reason to undertake the present study is based on the scientific evidence that cod liver oil found to possess a significant antioxidant and protective action against inflammation. However, there was no scientific evidence established regarding the antiulcer activity of cod liver oil. As inflammation is one the causes of gastric ulcer, the present study was undertaken to evaluate the ulcer protective efficiency of cod liver oil in three different ulcerated rat models.

 

2. MATERIALS AND METHODS:

2.2 Drugs and chemicals:

All chemicals used in this study were of analytical grade. CLO was procured from Coastal Aquatic Proteins, Mangalore, India; Omeprazole was obtained from Dr. Reddy’s Laboratories, Hyderabad, India; and Sucralfate was collected from Strassenburg pharmaceuticals limited, India.

 

2.2 Experimental animals:

Healthy sixty male Wister rats weighing in between 150 to 200 g were selected for this study. All animals were housed in standard environmental condition at 23±2 ⁰C within a controlled light-dark cycle for 12 h in institutional animal house facility. They were fed with standard pellet diet (Lipton India Ltd., Mumbai) and tap water ad libitum. They were provided humane care in compliance with the CPCSEA guidelines. All experimental protocols were approved by the IAEC.

 

2.3 Grouping of animals and treatments:

Sixty male Wistar rats were used in this study. Out of which six were given sterile placebo (distilled water) and designated as normal control. There were eighteen animals in each model, i.e. pyloric ligation, ethanol-induced, and aspirin-induced gastric ulceration models. The eighteen animals of each model were selected randomly and divided into three groups as control, standard, and CLO-treated, containing six animals in each group (n=6). 1 ml of 1% w/v Carboxymethylcellulose (CMC) solution, 1 ml of omeprazole at a dose of 20 mg/kg body weight [7], and 1 ml of CLO at a dose of 10 ml/kg body weight [8] were administered to the rats selected for pyloric ligation and ethanol-induced model as control, standard, and CLO-treated group respectively. In case of aspirin-induced ulceration model, 1 ml of 1% w/v CMC solution, 1 ml of sucralfate at a dose of 20 mg/kg bw. [9], and 1 ml of CLO at a dose of 10 ml/kg bw were administered to the rats of control, standard, and CLO-treated group.  1% w/v CMC solution was used as a vehicle to dissolve omeprazole, sucralfate, and CLO for its easy administration. Drugs were administered via per oral (P.O.) route.

 

2.4 Antiulceronegic study:

Antiulcerogenic activity of cod liver oil was evaluated in three different models i.e. pyloric ligation, ethanol-induced and aspirin-induced ulcer model.

 

2.4.1 Ulcer induced by Pyloric ligation:

Gastric ulcer was induced by pylorus ligation in Wister rats as described by Shay et al. (1945). Animals used in this model were kept in fast for 24 h. They were kept in Individual cages to avoid cannibalism. Proper attention was given to avoid coprophagy. The rats in the control group received 1% w/v CMC solution, whereas rats in the standard and CLO-treated group received omeprazole (20 mg/kg) and CLO 10 ml/kg) 30 min before and 2 h after the pylorus ligation respectively. Varying degrees of ulceration was manifested 4 h after the pyloric ligation [10].

 

2.4.2 Ethanol induced ulcer in rats:

Ethanol-induced ulcer model was developed as described by Faith MB et al. and Robert A el al. The rats were deprived of food for 18 h before the study. During this time the animals were kept in individual cages to avoid cannibalism and coprophagy. 1% w/v CMC solution, omeprazole (20 mg/kg) and CLO (10 ml/kg) were administered orally 30 min prior to the oral administration of 1 mL 80% ethanol for control, standard and CLO-treated group respectively. Varying degrees of ulceration was examined 1 h after the administration of ethanol [11-12].

 

2.4.3 Aspirin induced ulcer in rats:

The ulcer protective effect of CLO was evaluated using aspirin-induced ulcer model described by Lekha Saha et al. (2016). 1% w/v CMC solution, CLO (10 ml/kg) and sucralfate (20 mg/kg) were administered orally 1 h prior to oral administration of aspirin to the rats of control, CLO-treated and standard groups. Varying degree of ulceration was evaluated 6 h after the administration of aspirin [13-15].

 

2.4.4 Excision of stomach and collection of gastric juice:

After the specific periods of time the animals were sacrificed by cervical dislocation. The midline incision was made and abdomen was opened. The stomach was excised, opened along the greater curvature and the gastric content was collected into a centrifuge tube. 5 mL of distilled water was added into it. The resultant solution was subjected to centrifuge at 1000 rpm for the period of 10 min. The supernatant was collected and used for further biochemical analysis.

 

2.4.5 Quantification of ulceration and macroscopic evaluation of the excised stomach:

The glandular portion of the stomach was opened along with the greater curvature and the severity of hemorrhagic erosions was assessed on a grade scale of 0 to 3 as 0: normal coloured mucosa resembling that of normal rat; 0.5: red colour mucosa; 1: Red spots; 1.5: hemorrhagic streaks (1.5 to 2 mm) patches; 2: 3 > 5 small patches of ulcers; 3: more than 5 small patches of ulcers. The areas of mucosal erosion or damage were measured as a percentage of the total surface area of the glandular stomach in mm². Mean ulcer score for each animal is expressed as Ulcer Index (UI). 

 

UI =(Ulcerated area - Total stomach area)x100

The percentage ulcer inhibition was calculated using the following formula:

 

% Ulcer Inhibition = [Uc – Ut] x 100/ Uc

 

Where

Uc = ulcer index of the control group and

Ut = ulcer index of treated group.

 

The appearance of the stomachs for each groups of all three models were macroscopically observed and degree of ulceration was noted.

 

2.4.6 Estimation of free acidity and total acidity:

1 ml of gastric juice was pipetted out into a 100 ml conical flask. 2 to 3 drops of Topfer’s regent was added into it and titrated with 0.01N sodium hydroxide until the red colour of the solution changed to yellow colour. The added volume of the alkali to neutralize the gastric juice was noted. This volume corresponds to the free acidity of gastric juice. Thereafter, 2 to 3 drops of phenolphthalein indicator were added to the same solution and titration was continued until the end point appeared. Again the total volume of alkali added to reach endpoint was noted. This volume corresponds to the total acidity [11,15-16].

Acidity was calculated by using the following formula:

Acidity = (Volume of NaOH x Normality of NaOH)/0.1 x 100 x mEq/L/100 g

 

2.4.7 Estimation of mucin level:

After the collection of gastric juice, the glandular portion of the stomach was excised and the lesser curvature was opened down. Then the reverted stomach was subjected to soak for 2 h in 0.1% alcian blue 8GX dye along with 0.16 M sucrose buffer and 0.05 M sodium acetate solution.

 

The unbound dye was then removed by 0.25 M sucrose solution successively after 15 and 45 min time intervals. Dye complex with mucus was diluted by immersing it in 10 ml of 0.5 M magnesium chloride for 2 h. The resulting blue solutions were shaken briefly with an equal volume of diethyl ether and concentration of the aqueous phase was measured at 605 nm in Uv-Visible spectrophotometer (Shimadzu Corporation, Japan). The mucin content of the sample was determined from the standard curve of mucin and has been expressed in mcg/g of wet gland tissue [17-18].

 

2.5 Data analysis:

All data were expressed as mean ± standard error of means (S.E.M). Comparison between groups were made by one-way analysis of variance (ANOVA) followed by Student‘s t-test.  The value of p<0.05 was considered to be statistically significant.

 

3. RESULTS:

Data obtained from the experimental with respect to the effect of CLO on UI and percentages of ulcer inhibition in all three models were shown in Table 1.  In pyloric ligation model, CLO exhibited the mean ulcer protection of 64.61%; whereas the standard drug (omeprazole) showed 87.69% of ulcer protection. In ethanol-induced ulcer model, the extent of gastric mucosal protection of CLO was found to be 37.76%; whereas omeprazole showed a mucosal protection of 57.95%. In aspirin-induced ulcer model, CLO produced a significantly protects the gastric mucosa up to 60.42% and sucralfate showed 89.58% of ulcer protection. A significant (p<0.001) change was observed in the volume of gastric juice, pH, free acidity, total acid and mucin content shown in Table 2.

 

 

 

Table 1: Effect of CLO on ulcer indices of ulcerated rats

Pyloric ligation ulcer model
Treatments	Ulcer Index	% Ulcer Inhibition
Normal control	1.09 ± 0.24	NA
Control (Ulcerated )	5.42 ± 0.51	NA
Standard (OMP)	0.67 ± 0.11a	87.69
CLO-treated	1.92 ± 0.30b	64.61
Ethanol-induced ulcer model
Normal control	2.00 ± 0.18	NA
Control (Ulcerated )	8.92 ± 0.27	NA
Standard (OMP)	3.75 ± 0.21a	57.95
CLO-treated	5.55 ± 0.30b	37.76
Aspirin-induced ulcer model
Normal control	2.00 ± 0.18	NA
Control (Ulcerated )	4.00 ± 0.29	NA
Standard (SCF)	0.42 ± 0.18a	89.55
CLO-treated	1.56 ± 0.24b	60.43

n=6, mean ± SEM. ^a,bSignificantly different from the ulcerated control group (p<0.05) and from each other (p<0.05). OMP: omeprazole (20 mg/kg b.w.), SCF: sucralfate (20 mg/kg b.w.), and CLO: cod liver oil (10 ml/kg b.w.), NA= not applicable.

 

 

Table 2: Effects of CLO on gastric volume, pH, free acidity, total acidity and mucin content of ulcerated rats

Treatments	Pyloric ligation ulcer model
	Gastric volume (mL)	pH	Total acidity (mEq/mL)	Free acidity (mEq/mL)	Mucin content (mL)
Normal control	1.40±0.09	1.90±0.11	53.50±1.03	31.17±0.66	403.09±5.27
Control (Ulcerated )	2.22±0.13	2.59±0.17	152.17±2.92	77.50±3.34	261.54±3.69
Standard (OMP)	1.45±0.09	4.70±0.15	67.50±2.37	33.50±2.65	374.54±5.09
CLO-treated	1.80±0.04	3.42±0.14	85.67±2.63	45.50±2.15	286.44±2.14
Treatments	Ethanol-induced ulcer model
Normal control	1.40±0.09	1.90±0.11	53.50±1.03	31.17±0.66	403.09±5.27
Control (Ulcerated )	2.40±0.12	1.90±0.11	160.00±3.78	75.67±1.63	186.72±3.54
Standard (OMP)	1.75±0.08	3.42±0.09	117.67±3.43	59.34±1.12	371.12±4.34
CLO-treated	1.65±0.09	2.74±0.12	132.50±4.71	64.84±1.45	265.70±2.67
Treatments	Aspirin-induced ulcer model
Normal control	1.40±0.09	1.90±0.11	53.50±1.03	31.17±0.66	403.09±5.27
Control (Ulcerated )	1.69±0.07	2.82±0.13	144.84±2.53	70.50±1.57	266.29±2.79
Standard (SCF)	0.97±0.11	3.77±0.13	121.50±2.31	54.00±1.53	426.54±3.37
CLO-treated	1.30±0.06	3.57±0.17	133.84±2.41	63.17±1.54	394.75±3.21

n=6, mean ± SEM. Significantly different from the ulcerated control group (p<0.05) and from each other (p<0.05). OMP: omeprazole (20 mg/kg b.w.), SCF: sucralfate (20 mg/kg b.w.), and CLO: cod liver oil (10 ml/kg b.w.).

 

 

Fig. 1: Effects of CLO on ulcer indices of different ulcerated rat models.

 

 

Fig. 2:  Effects of CLO on volume gastric juice in different ulcerated rat models

 

Fig. 3:  Effects of CLO on pH of gastric juice in different ulcerated rat models

 

 

Fig. 4:  Effects of CLO on total acidity of stomach in different ulcerated rat models

 

 

Fig. 5:  Effects of CLO on free acidity of stomach in different ulcerated rat models

 

Fig. 6:  Effects of CLO on mucin content in different ulcerated rat models.

 

Fig. 7: Macroscopic appearance of stomach of the (a) Ulcerated control, (b) Standard and, (c) CLO treated rats in three different ulcer models. The dark areas represent bleeding portions signifying ulcerated regions.

 

4. DISCUSSION:

Gastric lesions in rats were induced in three different experimental models i.e. through pyloric ligation, using ethanol and aspirin. CLO showed a significant reduction in the extent of gastric mucosal damage in all experimental models. To elucidate the effect of CLO in gastric acid secretion, the volume of gastric juice, pH, free acidity and total acidity were estimated. In pyloric ligation model, mucosal damage was observed as a consequence of decrease in mucosal defence due to increase in vagal stimulation and starvation followed by the degranulation of parietal cells and release of histamine in the gastric tissues [19]. Ethanol is well known necrotizing agent, having capability to alter the gastric secretory activity, cell permeability and volume of gastric mucous [20], by producing free radicals [21]. The protective effect of CLO might be resulted because of its antiperoxidative potential, reported earlier [7,22]. Aspirin is known to cause mucosal injury and hemorrhage based on the direct irritant action by permitting back diffusion of hydrogen ions through the mucosa [23-24]. The gastric mucosal damage by aspirin mediates through the inhibition of Cyclooxygenase enzyme (Wallace 2008). CLO was shown to (p<0.01) increase the mucin content in all the models which indicated a good mucoprotective property of CLO.

 

 

 

6. CONCLUSION:

In this study, CLO was screened to establish its capacity to protect gastric ulcer in rats. Pyloric ligation model was selected to elucidate the antisecretory effect of CLO; whereas other two models were selected to realize the mucoprotective activity of CLO. Our study suggested that, CLO can be used to protect the mucosal damage caused by multifactorial causes like worry, hurry and curry. Findings from this study can provide some additional support to the use of CLO in the treatment of gastric ulcers. The Pharmacological relevance of these findings deserves further extensive investigation.

 

7. CONFLICT OF INTEREST:

Author(s) declare no conflict of interest.

 

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Accepted on 12.07.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 05-10.