INTRODUCTION:

For more than a century, peptic ulcer disease has been a major cause of morbidity and mortality. The Pathophysiology of peptic ulcer disease has centered on an imbalance between aggressive and protective factors in the stomach. Research advancement during the last few years have offered new insights in the therapy and prevention of gastroduodenal ulceration, by measures directed at strengthening the mucosal defense system rather than by attenuating the aggressive acid-pepsin factors held responsible for the induction of ulcers¹. Inflamed break in the lining of the stomach or the duodenum caused due to either increased acid production or damage to the mucus lining of the stomach leads to formation of peptic ulcer, a term that includes both gastric as well as duodenal ulcer.

Peptic ulcer arises when the normal mucosal defensive factors (mucus, mucosal blood flow, formation of HCO₃^- and PGE₂) are impaired or over powered by the aggressive factors (acid, pepsin, epithelial cell restoration)²^-4. Apart from the common myths about the causes of peptic ulcers, it has also been found that caffeine containing beverages, alcohol, nicotine, and acetaminophen may also cause mucosal damage and peptic ulcers^5-8. All NSAIDs and drugs like aspirin are important contributing factor in the genesis of duodenal and especially gastric ulcers⁹ in addition to the microbial factor Helicobacter pylori.

Free radicals are defined as chemical species possessing unpaired electrons in their outer orbital which are generally reactive¹⁰. If a radical reacts with a non radical, another free radical must be produced. This implication is reflected continuously in cells either during phagocytosis or pathological condition. The oxygen derived free radicals are capable of damaging reversibly or irreversibly the compounds of all biochemical classes, including nucleic acids, proteins, free amino acids, lipids, lipoproteins, carbohydrates and connective tissue macromolecules¹¹.

Chemically melatonin is also known as N-acetyl-5-methoxytryptamine. Melatonin directly scavenges and neutralizes O₂^-, which reduces the formation of peroxynitrite anion (ONOO^-). This in turn prevents the activation of poly (ADP ribose) synthase and associated tissue injury. Melatonin was found to reduce the side effects and increase the efficacy of drugs like ranitidine and omeprazole in treatment of peptic ulcer¹².

Omeprazole, which prevents acid production by blocking the parietal cell H⁺/K⁺-ATPase, is administered as a racemic mixture of its two optical isomers, S-omeprazole and R-omeprazole¹³. The S-isomer of omeprazole is esomeprazole. Esomeprazole is claimed to have higher oral bioavailability and to produce better control of intragastric pH than omeprazole in GERD patients because of longer t½. Higher healing rates of erosive esophagitis and better GERD symptom relief have been reported in comparative trials with omeprazole¹⁴. Side effects and drug interaction profile is similar to the racemic drug. In addition, less inter patient variability in acid inhibition is seen with esomeprazole¹³.

Hence an attempt has been made to study the efficacy of combination of the same antioxidant (melatonin) with esomeprazole to treat peptic ulcer.

MATERIALS AND METHODS:

Esomeprazole and melatonin were procured from Lee Pharmaceuticals, Hyderabad and Aristo Pharmaceuticals Pvt. Ltd. Andheri, Mumbai as gift samples. Wistar albino rats of either sex weighing between 180-200g were purchased from the local licensed animal seller. The animals were allowed to acclimatize to the laboratory conditions for a period of one week prior to the beginning of the experiment. The animals were fed with standard food pellets (Hindustan Pvt. Ltd., Mumbai) and water ad. libitum. Prior approval of the Institutional Animal Ethics Committee (IAEC) was obtained for conduction of experiment. The dose calculations were extension of human dose based on body surface area as suggested by Laurence and Bacharach, 1964.

EXPERIMENTAL DESIGN:

Each screening model consists of Wistar albino rats of either sex randomly divided into 4 groups of 6 animals in each. The animals were administered with the drugs in the following sequence:

Group I - Control

Group II - Standard (Esomeprazole 0.54mg/200g; p.o.)

Group III - Melatonin (0.27 mg/200g; p.o.)

Group IV - Esomeprazole + Melatonin (0.54mg + 0.27mg)/200g; p.o

PYLORUS LIGATION MODEL¹⁵:

The drugs were administered for 7 consecutive days. On 7^th day, after the drug administration the rats were fasted for 48h and care was being taken to avoid coprophagy. At the end of 48h, the rats were subjected for pylorus ligation under ether anesthesia. Abdomen was opened by a midline incision. The stomach was lifted and a ligature was placed at the pyloric sphincter without causing any damage to its blood supply. The stomach was replaced carefully and abdominal wall was sutured in two layers. After 6 h, the rats were euthanized with excess of anesthetic ether and the stomachs were dissected out. Gastric juice was collected and subjected to biochemical investigations. The gastric juice was collected and subjected to the following biochemical investigations.

Measurement of Volume (ml) of Gastric Juice and determination of pH¹⁶:

The gastric contents drained into the test tubes were centrifuged at 1000rpm for 10m. The supernatant liquid obtained was measured for its volume (ml). The pH of the gastric juice was measured using digital pH meter, by keeping the tip of the electrode in contact with the gastric fluid.

Determination of Ulcer Index¹⁶:

The stomachs were opened along the greater curvature and washed under running water to observe the ulcers in the glandular portion of the stomach. The number of ulcers per stomach was noted and scoring was done microscopically with the help of hand lens (10X). The ulcers were scored using the following scale -

0.0 = Normal stomach

0.5 = Red coloration

1.0 = Spot ulcers

1.5 = Hemorrhagic streaks

2.0 = Ulcer ≥ 3 ≤ 5

3.0 = Ulcer > 5

Mean ulcer score for each animal is expressed as ulcer index and the percentage protection was calculated by using the formula¹⁷:

% Protection	UI _Control – UI _Test	X 100
	UI_Control

Determination of free acidity and total acidity¹⁶:

1 ml of gastric juice was pipette out into 100 ml conical flask. It was diluted to 10 ml with distilled water and 2 –3 drops of Topfer’s reagent was added and titrated against 0.01N sodium hydroxide (NaOH) until all traces of red color disappears and the color of the solution turns to yellowish orange. The volume of the alkali utilized was noted. This volume corresponds to free acidity.

Then 2 – 3 drops of phenolphthalein indicator was added and titration was continued until a definite red tinge reappears. Again the total volume of alkali added was noted. The volume corresponds to total acidity. Acidity was calculated by using the formula:

Acidity =	Volume of NaOH consumed x Normality of NaOH	X 100 mEq/L/100gm
	0.1

Ethanol induced ulcer model¹:

The drugs were administered for 07 consecutive days. On 7^th day after drug administration, the rats were fasted for 48h. Care was being taken to avoid coprophagy. At the end of 48h, ethanol (1ml/200gm; p.o.) was administered to the rats of all the Groups (I to VI) to induce ulcer. After 2h of ethanol administration the rats were sacrificed and stomachs were excised out and subjected to determination of ulcer index and percentage protection as described above.

Aspirin induced ulcers^{18, 19}:

The selected drugs were administered orally for 07 consecutive days and on 7^th day after drug administration; the animals were fasted for 48h. At the end of 48h, oral aspirin in a dose of 20mg/kg suspended in 1% CMC was administered. 4h later, the rats were sacrificed and their stomachs were excised out. Formol-saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs were opened along the greater curvature, then washed in warm water and examined under a 3-fold magnifier. Ulcer index and percentage protection were evaluated as described above.

Statistical analysis:

Results were expressed as mean ± SEM, (n=6). Statistical analysis was performed using one way ANOVA by Dunnett’s test. P < 0.05 was considered to be statistically significant. *P<0.05, **<0.01 and ***<0.001, when compared with standard esomeprazole and treatment group as applicable.

RESULTS:

Interpretation of the results clearly indicates that the combination of esomeprazole and melatonin was found to be synergistic in nature, as both enhanced their actions and the resulting effect was maximum antiulcer effect (Table 1, 2, 3). The combination was found to have significantly effective in pylorus ligation and ethanol models by imparting 91.9% (Table. 1, 2) protection upon comparison with groups treated with esomeprazole, melatonin individually. In aspirin induced ulcer model the percentage protection was significantly enhanced to 93.3% (Table. 3) upon comparison with other treatment groups.

Table.1: Influence of Melatonin-Esomeprazole combination in Pylorus Ligation induced ulcers

Treatment	Volume of gastric juice (ml)	pH	Free Acidity (mEq/L)	Total Acidity (mEq/L)	Ulcer Index	% Protection
Control	6.017±0.11	1.717±0.03	121.0±1.02	140.4±0.51	3.583±0.15	----
Esomeprazole	1.600±0.11	4.833±0.12	16.67 ± 0.66	33.67 ± 0.88	0.593 ± 0.16	83.4 %
Melatonin	1.105±0.03	5.045±0.06	71.75±0.35	86.40±0.60	0.916±0.08	74.4%
Esomeprazole + Melatonin	0.893±0.09	6.012±0.02	12.65±0.10	30.45±1.03	0.290±0.01	91.9%

Values are the Mean ± S.E.M, n=6, Significant *P <0.05 combination compared with standard esomeprazole

Table.2: Influence of Melatonin-Esomeprazole combination in Ethanol induced ulcers

Treatment	Ulcer Index	% Protection
Control	5.583±0.23	----
Esomeprazole	1.000±0.10	82%
Melatonin	1.417±0.20	74.6%
Esomeprazole + Melatonin	0.451±0.12	91.9%

Values are the Mean ± S.E.M, n=6, Significant *P <0.05 combination compared with standard esomeprazole

Table.3: Influence of Melatonin-Esomeprazole combination in Aspirin induced ulcers

Treatment	Ulcer Index	% Protection
Control	5.002±0.09	----
Esomeprazole	1.000±0.16	80%
Melatonin	1.800±0.28	64%
Esomeprazole + Melatonin	0.333±0.53	93.3%

Values are the Mean ± S.E.M, n=6, Significant *P <0.05 combination compared with standard esomeprazole

DISCUSSION:

The extremely high concentration of H⁺ in the gastric lumen requires robust defense mechanisms to protect the esophagus and the stomach. The primary esophageal defense is the lower esophageal sphincter, which prevents reflux of acidic gastric contents into the esophagus. The stomach protects itself from acid damage by a number of mechanisms that require adequate mucosal blood flow, perhaps because of the high metabolic activity and oxygen requirements of the gastric mucosa. One key defense is the secretion of a mucus layer that protects gastric epithelial cells. Gastric mucus is soluble when secreted but quickly forms an insoluble gel that coats the mucosal surface of the stomach, slows ion diffusion, and prevents mucosal damage by macromolecules such as pepsin. Mucus production is stimulated by prostaglandins E₂ and I₂, which also directly inhibit gastric acid secretion by parietal cells. A second important part of the normal mucosal defense is the secretion of bicarbonate ions by superficial gastric epithelial cells. Bicarbonate neutralizes the acid in the region of the mucosal cells, thereby raising pH and pre-venting acid-mediated damage³.

If these defenses are disrupted, a gastric or duodenal ulcer may form. The treatment and prevention of these acid-related disorders are accomplished either by decreasing the level of gastric acidity or by enhancing mucosal protection. A peptic ulcer is a sore on the lining of the stomach or duodenum, which is the beginning of the small intestine²⁰. Peptic ulcer is one of the major ailments affecting about 60% human adults and nearly 80% child population in topical countries²¹. Acid peptic diseases includes hyperacidity, gastroesophageal reflux diseases (GERD’s), stress induced mucosal erosions and peptic ulcers (gastric as well as duodenal). Uncontrolled acid secretion and ulceration of stomach mucosa due to several reasons have posed serious problems to the human health all over the globe²².

Many natural products and modern synthetic drugs have been used to treat the peptic ulcer disease but so far a complete cure has not been discovered and exploration of new antiulcer drugs has remained a field of active research. Different types of antiulcer agents are being prescribed to overcome the disease problem. For example, a natural product sucralfate that binds to proteinaceous materials in the ulcer crater and prevents further digestion of the mucosa by gastric acid and pepsin has been tried. Similarly, colloidal bismuth salt that forms a precipitate and binds to the surface of ulcer to provide a barrier against corrosive effects of gastric acid and pepsin has been used²³. Many antacids that neutralize gastric acid and anticholinergic agents that decrease acid secretion by blocking parasympathetic activity have long been used²⁴. The antacids are not recommended these days due to their low potency and interference with the electrolyte balance of body while anticholinergics have become obsolete due to their ill effects on eyes, heart and brain, etc.²⁵ Only pirenzipine (a selective antimuscarinic) that exerts antisecretory and ulcer healing properties is still clinically used²⁶. The histamine H₂-receptor antagonists like ranitidine and famotidine, etc., have markedly decreased morbidity and mortality of peptic ulcer patients but they also exert many adverse effects including cardiac arrhythmias, impotence, gynaecomastia and hematopoietic changes as well as high recurrence rates^{27, 28}. The modern drugs like proton pump inhibitors like omeprazole, lansoprazole and rabeprazole decrease the gastric acid secretions from parietal cells^{29, 30}. Metronidazole and antibiotics such as amoxicillin and clarithromycin have also been used to treat the peptic ulcers in different combinations as double, triple and quadruple therapy regimens^{31, 32, 33, 34} In spite of all these developments, recurrence of peptic ulcer disease occurs even after long-term therapy.

The aim of the present study was to investigate the combination effect of the drugs on the gastric secretion activities such as volume, pH, free acidity, total acidity and ulcer index using the pylorus ligation, ethanol and aspirin induced ulcer models.

Melatonin directly scavenges and neutralizes O₂^-, which reduces the formation of peroxynitrite anion (ONOO^-). This in turn prevents the activation of poly (ADP ribose) synthase and associated tissue injury. Melatonin was found to reduce the side effects and increase the efficacy of drugs like ranitidine and omeprazole in treatment of peptic ulcer.

The investigated report shows that pretreatment with combination of drugs i.e.

In pylorus ligation induced ulcer model, the combination group - Esomeprazole and melatonin, showed decrease in ulcer score that indicates it can be due to possible mechanisms like increase in the gastric mucosal blood flow, production of protective mucus and inhibition of acid secretion etc. While control group showed severe ulcer and hemorrhagic streaks, the standard group showed some red spots and hemorrhage. Table.1 indicate combination treated group has significantly reduced the volume of gastric secretion (P < 0.001), free acidity (P < 0.0001), total acidity (P < 0.0001), ulcer index (P < 0.001) and increased the gastric pH (P < 0.0001) and the percentage protection is 91.9%. When compared with control and standard.

In ethanol induced ulcer model, the significant inhibitory effect of combination group (esomeprazole and Melatonin) was produced mainly due to biosynthesis of cytoprotective prostaglandins, inhibition of lipid per-oxidation, gastric mucosal permeability (to H⁺ and Na⁺ ions) and excess acid secretion etc. Table.2 shows, esomeprazole and melatonin combination has significantly reduced the ulcer index (P < 0.001) and the percentage protection was 91.9%, when compared with control and standard. In aspirin induced ulcer model, oral administration of combination of melatonin and esomeprazole showed significant reduction in ulcer index as compared to the control group and standard esomeprazole. The percentage protection of combination group was found to be 93.3% when compared to control and standard esomeprazole alone. The results are shown in Table.3. This indicates that antioxidants play a vital role in the body and can act synergistically with beneficial drugs like esomeprazole. This can help in designing regimens with a lower dose of drug or can help in reducing the duration of regimen and alleviate side effects.

CONCLUSION:

From the results obtained (Table.1, 2 , 3), it can be clearly concluded that the combination of esomeprazole and melatonin is synergistic in nature as the group treated with the combination exhibited significant percentage protection by imparting maximum ulcer protection in all the antiulcer models used in the study and upon comparison with control and standard treatment groups.

REFERENCES:

1. Ronald Fernandes. Ph.D. Dissertation work submitted to Mangalore University. Gastroprotective and antisecretory effect of selenomethionine in albino rats. 2008:1.

2. Sharma HL and Sharma KK. Principles of Pharmacology. 1^st Edition, Paras Publications. 2007:368.

3. Goodman and Gilman. The Pharmacological basis of therapeutics. 10^th Edition, New York, McGraw Hill. 2001:1005-6.

4. Barar FSK. Essentials of Pharmacotherapeutics. 3^rd Edition. 2003:538-9.

5. Parmar NS, Jagruti K Desai. A Review of the current methodology for the evaluation of gastric and duodenal antiulcer agents. Indian J Pharmacol. 1993; 25:120-35.

6. Morris GP and Wallace JL. The role of ethanol in acid production of gastric mucosal erosions in rats. Virchow’s Arch. 1981; 46:239-51.

7. Qui BS, Cho CH and Ogle CW. Chronic nicotine treatment intensifies gastric ulceration by cold resistant stress in rats. Agents Actions. 1991; 3:367-70.

8. Smura H and Marumo F. Effect of acetaminophen on stress induced gastric mucosal lesion in rats. Res. Comm. Mol. Path. Pharmacol. 1994; 86(3):297-310.

9. Rubin R, Schwartong R, Straer DS. Pathology, Clinicopathologic foundation of medicine. 4^th Edition. Philadelphia: Lippincott. Williams and Wilkins. 2005; 40-52.

10. Langseth. L. Oxidants, antioxidants and disease prevention. International Life Sciences Institute.1-26.

11. http://www.merckmanuals.com

12. Reiter RJ, Tan DX, Sainz RM, Mayo JC, Lopez Burillo S. Melatonin reducing the toxicity and increasing the efficacy of drugs. Journal of Pharmacy and Pharmacol. 2002; 54(10):1299-1321.

13. Pommerien W, Schultze V, Braden B, Lembcke B, Wrangstadh M, Londong W. Dose response of omeprazole combined with amoxicillin on duodenal ulcer healing and eradication of Helicobacter pylori. Ailment Pharmacol. Ther. 1996; 10:303-8.

14. Tripathi KD. Essentials of Medical Pharmacology. 6^th Edition. Jaypee Brothers Medical Publications (P) Ltd., New Delhi. 2009; 633.

15. Shay M, Komarov SA, Fels D, Meranze D, Gruenstein H and Siplet: A simple method for uniform production of gastric ulceration in rat. Gastroenterology. 1945; 5:43-61.

16. Kulkarni SK. Hand book of experimental pharmacology 3^rd Edition. Vallabh Prakashan, New Delhi. 2005:148-50.

17. Fathihah B, Mahmood AA, Sidik K and Salmah I. The antiulcer and cytoprotective effect of ageratum conyzoides–honey combination in rats, Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, JUMMEC 2003-2005; 8:28-32.

18. R. K. Goel, S. Gupta, R. Shankar, and A. K. Sanyal. J Ethnopharmacol. 1986; 18:33.

19. Patil S.S., Bhide A.A. and Gorle A.M. Antiulcer activity and Ant inflammatory Studies on acacia catechu. Indian Drugs. 2010; 47(2):52-53.

20. http://www.wikipedia.org/wiki/pepticulcers.html

21. Dandagi PM, Patil MB, Mastiholimatu VS, Gadad AP, Kulkarni AR. Antiulcer activity of extracts of calotropis gigantean root extracts in pylorus ligated rat model. Int J Pharmacol. Biol. Sci. 2008; 2(1):127-32.

22. Bandyopadhyay D, Biswas K, Bandyopadhyay U, Reiter RJ and Banerjee RK. Melatonin protects against stress-induced gastric lesions by scavenging the hydroxyl radical. J Pineal Res. 2000; 29(3):143-151.

23. Bandyopadhyay D, Biswas K, Bhattacharyya M, Reiter R J and Banerjee RK (2001). Gastric toxicity and mucosal ulceration induced by oxygen-derived reactive species: protection by melatonin. Curr Mol Med; 1(4):501-513.

24. Spiro HM. Pharmacology clinical efficacy and adverse effects of sucralfate, an non synthetic agent for peptic ulcer. Pharmacotherapy. 1982; 2(1):67-71.

25. Barbezat GO. Drugs in peptic ulcer disease. Med Prog. 1986; 61-72.

26. Kawamura T, Torrii A, Nozawa H, Onizawa and Kameda H. Comparison of effects of Pirenzipine and other antiulcer agents on gastric mucosal blood flow and mucus syrup, Pirenzipine: new aspects in research and therapy. Excerpta Medica. 1984; 1000BC:48.

27. Kikuko A, Shinichi K, Hiroshi Y and Susumu O. effects of the novel histamine H₂receptor antagonist (±)-(E)-1-[2-hyderoxy-2-(-4-hydroxyphenyl)ethyl]-3[2-[[[5-(methyl amino)methl-2-furyl]methyl]thio]ethyl]-2-(methylsulfonyl)guanidine on gastric secretion and gastroduodenal ulcers in rats. Arzneim-forech / Drug Res. 1996; 46(1) Nr.2:177-85.

28. Maaike Berbee M D and Qiang Fu M D. Reduction of radiation induced vascular nitrosative stress by the vitamin E analog γ-Tocotrienol: Evidence of a role for tetrahydrobiopterin. Int. J. of Radiation Oncology. 2011; 79(3):884-91.

29. Hopkins A.M, McDonnell C, Breslin NP, O'Morain CA and Baird AW. Omeprazole increases permeability across isolated rat gastric mucosa pre-treated with an acid secretagogue. J Pharm Pharmacol. 2002; 54(3):341-47.

30. Ariyoshi I, Toshihara A, Sugimura F, Abe M, Matsuo Y and Honda T. Recceurrence during maintenance therapy with H2-receptor antagonists in cases of gastric ulcers. Folia Pharmacol JPN. 1986; 87(5):551.

31. Wong WM, Wong BC, Hung WK, Yee YK, Yip AW, Szeto ML, Fung FM, Tong TS, Lai KC, Hu WH, Yuen MF, Lam SK. Double blind, rando-mised, placebo controlled study of four weeks of lansoprazole for the treatment of functional dyspepsia in Chinese patients. Gut. 2002; 51(4):502-06.

32. Sener-Muratoglu G, Paskaloglu K, Arbak S, Hurdag C, Ayanoglu-Dulger G. Protective effect of famotidine, omeprazole, and melatonin against acetylsalicylic acid-induced gastric damage in rats. Dig Dis Sci. 2001; 46(2):318-30.

33. Fakheri H, Malekzadeh R, Merat S, Khatibian M, Fazel A, Alizadeh BZ, Mas-sarrat S. Clarithromycin vs. furazolidone in quadruple therapy regi-mens for the treatment of Helicobacter pylori in a population with a high me-tronidazole resistance rate. Aliment Pharmacol Ther. 2001; 15(3):411-16.

34. Silva FM, Zaterka S, Eisig JN, Chehter EZ, Chinzon D, Laudanna AA. Factors affecting Helicobacter pylori eradication using a seven-day triple therapy with a proton pump inhibitor, tinidazole and clarithromycin, in Brazilian patients with peptic ulcer. Rev Hosp Clin Fac Med Sao Paulo. 2001; 56(1):11-16.

Received on 25.12.2014 Accepted on 15.02.2015

Asian J. Pharm. Res. 5(1): Jan.-Mar. 2015; Page 10-14

DOI: 10.5958/2231-5691.2015.00002.7

Synergistic Antiulcer Effect of Melatonin and Esomeprazole Combination in Pylorus Ligation, Ethanol, Aspirin induced Peptic Ulcers

*Corresponding Author E-mail: sayeedhasan1986@gmail.com