Evaluation of Gastroprotective Influence of Vitamin C, Melatonin, Rabeprazole and their Combination in Ethanol Induced Ulcer Model

Nitin Mahurkar*, Sayeed Ul hasan S.M., Arati Malpani A., Chetan Manjunath

HKES College of Pharmacy, Sedam Road, Gulbarga – 585 105 Karnataka, India

ABSTRACT:

Aim: To evaluate the gastroprotective effect of the antioxidants (vitamin C and melatonin), rabeprazole and their combination in ethanol induced peptic ulcers.

Methodology: The study was divided into 2 dosage groups – single and multiple dose study groups. Each of the dosage group consists of wistar albino rats of either sex (180 – 220g) divided into 4 groups of 6 animals each.

In single dose study, The animals were fasted for 48h. At the end of 48h, the animals were administered with the drugs. 1h after drug administration, absolute ethanol (1ml/200g p.o.) was administered to all groups. 2h after ethanol administration, the animals were euthantised and stomachs were excised out. In multiple dose study, the animals were administered with drugs for 7 consecutive days. On 7^th day following drug administration, the animals were fasted for 48h. At the end of 48h, the aimals were administered with absolute ethanol (1ml/200g p.o.) 2h after ethanol administration, the animals were euthantised, stomachs were excised out. The stomachs excised out from both the study groups were evaluated for ulcer index and percentage protection. However, for multiple dose study group histopathological study was also performed to evaluate cytoprotection.

Result: The results were interpreted statistically using one way ANOVA. The combination group (rabeprazole + vitamin C and rabeprazole + Melatonin) showed significant antiulcer activity in both single and multiple dose study groups, on comparison with rabeprazole, vitamin C and melatonin individually.

Conclusion: The effect of combination groups was found to be synergistic in nature and more effective in treating peptic ulcer disease.

KEYWORDS: Antiulcer activity; Rabeprazole; Vitamin C; Melatonin; Antioxidants; Ethanol induced ulcers

INTRODUCTION:

Gastric acid secretion is a complex, continuous process in which multiple central and peripheral factors contribute to a common endpoint: the secretion of H⁺ by parietal cells. Neuronal (acetylcholine), paracrine (histamine), and endocrine (gastrin) factors all regulate acid secretion. 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 cells¹.

A peptic ulcer is a sore on the lining of the stomach or duodenum, which is the beginning of the small intestine². The pathophysiology of peptic ulcer disease has centered on an imbalance between aggressive and protective factors in the stomach. Peptic ulcer represent a major health problem, 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 ulcers³. The usual signs and symptoms of peptic ulcers include abdominal pain with a burning or gnawing sensation, Pain 2 - 3 hours after eating, Pain is often made worse by an empty stomach, Heartburn, Indigestion (dyspepsia), Belching, Nausea, Vomiting, Poor appetite, Weight loss⁴.

The usual cause of peptic ulceration is too much secretion of gastric juice in relation to the degree of protection afforded to the mucosa by the mucus. While other causes include the use of conventional non-steroidal anti inflammatory drugs (NSAIDs), Helicobacter. Pylori, Caffeine containing beverages⁵, alcohol⁶, nicotin⁷, acetaminophen⁸ may also cause mucosal damage and peptic ulcers. However, the sex has no impact on gastric ulceration. Genetic predisposition also seems to play some role in peptic ulceration, person with blood “group O” are more prone to develop an ulcer due to inherent collagen effect compared to persons with blood “group A or B”⁹.

Among the various causes of gastric ulceration, lesions caused by alcohol consumption have been shown to be mediated largely through the generation of reactive oxygen species, especially the hydroxyl radical¹⁰. There is growing indirect evidence indicating that oxygen derived free radicals are responsible for cellular injury¹¹. Free radicals are chemical species possessing unpaired electrons that can be considered as part of the molecule which are generally very reactive¹².

Free radicals are defined as chemical species possessing unpaired electrons in their outer orbital which are generally reactive¹³. 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 most important free radicals include: superoxide anion (O₂^-), alkoxyl, hydroxyl radical (HO^-), and alkoxyl singlet oxygen (¹O₂^-) ¹⁴. These 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 macromolecules¹⁵.

Melatonin chemically 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 ulcer¹⁶.

Vitamin C neutralizes hydroxyl, alkoxyl and peroxyl radicals by hydrogen donation and also neutralizes the radical form of other antioxidant like glutathione and vitamin E¹⁵. Vitamin C is proven to exert gastroprotective by heme oxygenase-1-dependent mechanism. Gastric epithelial cells require vitamin C to translate HO-1 mRNA into active protein, which then may exert gastroprotection by its antioxidant and vasodilative properties¹⁷.

Rabeprazole is a new proton pump inhibitor claimed to be fastest acid suppressive (due to higher pKa), is more rapidly converted to the active species and to aid gastric mucin synthesis and use to heal peptic ulcer. Rabeprazole is potent, has longer duration of action, better bioavailability and lesser drug interaction¹⁸.

Rabeprazole suppresses gastric acid secretion by specific inhibition of H⁺/K⁺ ATPase in the gastric parietal cell. By acting specifically on the proton pump, rabeprazole blocks the final step in acid production, thus reducing gastric acidity. This effect is dose-related up to a daily dose of 20 to 40 mg and leads to inhibition of gastric acid secretion. The antisecretory effect begins within one hour after oral administration of rabeprazole and reaches its maximum within 2-4h¹⁹.

MATERIALS AND METHODS:

Albino wistar rats of either sex weighing between 180 - 220g were procured from central animal house, MR. Medical College, Gulbarga. The animals were acclimatized for seven days and housed under standard conditions of temperature (252⁰C) and relative humidity (30-70%) with a 12:12 light-dark cycle and were fed with standard pellet diet (Hindustan liver co., Mumbai) and water ad libitum. Approval of Institutional Animal Ethical Committee (IAEC) of H.K.E.S College of pharmacy, Gulbarga was taken for conducting antiulcer activity. The registration number is HKECOP/IAEC/34/2010-11 and the animal studies were performed in accordance to guidelines of CPCSEA. The dose calculations were extension of human dose based on body surface area (Laurence and Bacharach, 1964).

Methodology: Single dose and multiple dose study by Ethanol induced ulcer model²⁰:

Albino wistar rats of either sex weighing between 180 – 220g were divided into 6 groups of 6 animals each.

Group I – Control

Group II- Standard (Rabeprazole 0.36mg/200gm b.w. in distilled water orally.)

Group III-Melatonin (0.27mg/200gm b.w. in distilled water orally.)

Group IV-Vitamin C (4.5mg/200gm b.w. in distilled water orally.)

Group V - Rabeprazole + Melatonin (0.36mg + 0.27mg)/ 200gm b.w. orally. Group VI - Rabeprazole + Vitamin C (0.36mg + 4.5mg)/ 200 gm b.w. orally.

Single dose study:

The rats were fasted for 48h. Care was being taken to avoid coprophagy. At the end of 48h, the animals were administered with the selected drugs and their combinations. After 2h of drug administration, ethanol (1ml/200gm b.w orally) was administered to the rats of all the Groups (I to VI) to induce ulcer. After 2 h of ethanol administration, the rats were euthantised and stomachs were excised out.

Multiple dose study:

The drugs were administered for 7 consecutive days. On 7^th day, the rats were fasted for 48h. Care was being taken to avoid coprophagy. At the end of 48h, ethanol (1ml/200gm b.w orally) was administered to the rats of all the Groups (I to VI) to induce ulcer. After 2 h of ethanol administration the rats were sacrificed and stomachs were excised out.

Determination of ulcer index²¹ :

The stomachs were opened along the greater curvature then washed with running water to see for 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)

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 formula²²:

% protection = [UI _control – UI _treated / UI _control X 100]

Histopathological evaluation²³:

The stomachs were immersed in 10% formalin solution for histopathological examination. These tissues were processed and embedded in paraffin wax. The central part of damaged or ulcerated tissue (if present) was cut on half along the long diameter. If the stomach was protected from the damage then the section was taken from basal part using a rotary microtome, sections of thickness of about 5μm were cut and stained with haemotoxylin and eosin. These were examined under the microscope for histopathological changes such as congestion, hemorrhage, necrosis, inflammation, infiltration, erosion and ulcer and photographs were taken.

Statistical analysis:

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

RESULTS:

Single dose studies:

The ulcer index in rabeprazole treated group was decreased significantly to 1.16±0.10, in vitamin C treated group it was 2.16±0.10. With melatonin ulcer index was found to be 2.08±0.15 compared to control group and the percentage protection was 79.2% after treatment with rabeprazole, 62.7% after treatment with vitamin C and 61.2% after treatment with melatonin. In groups treated with combination of rabeprazole and vitamin C, rabeprazole and melatonin the ulcer index was reduced to 0.58±0.08 and 0.91±0.08 and the percentage protection was found to be 89.6 % and 83.6% respectively.

Multiple dose studies:

The ulcer index in rabeprazole treated group was decreased significantly to 0.833±0.23, with vitamin C it was 1.250±0.11, with melatonin ulcer index was found to be 1.417±0.20 compared to control group in which the value was 5.583±0.23 and the percentage protection was 85%, 77.6%, 74.6% respectively. In groups treated with rabeprazole and vitamin C, rabeprazole and melatonin the ulcer index was reduced to 0.333±0.10 and 0.250±0.11 and the percentage protection was 95.2% and 94% respectively.

1A) Stomach epithelium of normal albino rat

1B) Stomach epithelium of control group

1C) Stomach epithelium of rabeprazole treated group.

1D) Stomach epithelium of vitamin C treated group

1E) Stomach epithelium of melatonin treated group

1F) Stomach epithelium of combination (Rabeprazole + Vitamin C) treated group

1G) Stomach epithelium of combination (Rabeprazole + Melatonin) treated group

Figure.1: Stomach epithelium of albino rats treated with single dose regimen

Histopathological study: (Fig.2)

In the control group, gastric mucosa showed ulceration, extensive congestion and cyst formation (Fig. 2B) While in standard rabeprazole the gastric mucosa showed focal ulceration and mild congestion (Fig. 2C). In vitamin C treated group gastric mucosa showed normal scanty inflammatory cells (Fig. 2E). In melatonin treated group the gastric mucosa was found to exhibit focal ulceration, very mild congestion and very few inflammatory cells (Fig. 2D). In the combination group (Rabeprazole + Vitamin C) gastric mucosa appeared normal very few inflammatory cells (Fig. 2G). While in Rabeprazole + Melatonin, the gastric mucosa showed no ulceration mild congestion and very mild inflammation (Fig. 2F)

2A) Histopathological slide of normal rat

2B) Histopathological slide of control group

2C) Histopathological slide of rabeprazole treated group

2D) Histopathological slide of Melatonin treated group

2E) Histopathological slide of Vitamin C treated group

2F) Histopathological slide of Combination (Rabeprazole + Melatonin) treated group

2G) Histopathological slide of Combination (rabeprazole + vitamin C) treated group

Fig. 2: Histopathological slide of multiple dose treated stomachs.

DISCUSSION:

H. pylori peptic ulcers are treated with drugs that kill the bacteria and reduce stomach acid and protect the stomach lining. Antibiotics are used to kill the bacteria. Two types of acid-suppressing drugs might be used: H₂ blockers and proton pump inhibitors. H₂ blockers work by blocking histamine, which stimulates acid secretion. They help reduce ulcer pain after a few weeks. Proton pump inhibitors suppress acid production by halting the mechanism that pumps the acid into the stomach. H₂ blockers and proton pump inhibitors have been prescribed alone for years as treatments for ulcers. But used alone, these drugs do not eradicate H. pylori and therefore do not cure H. pylori related ulcers. Bismuth subsalicylate, a component of Pepto-Bismol, is used to protect the stomach lining from acid. It also kills H. pylori.

Treatment usually involves a combination of antibiotics, acid suppressors and stomach protectors. Antibiotic regimens recommended for patients may differ across regions of the world because different areas have begun to show resistance to particular antibiotics. The use of only one medication to treat H. pylori is not recommended. At this time, the most proven effective treatment is a 2 week course of treatment called triple therapy. It involves taking two antibiotics to kill the bacteria and either an acid suppressor or stomach lining shield. 2 week triple therapy reduces ulcer symptoms, kills the bacteria and prevents ulcer recurrence in more than 90% of patients. Unfortunately, patients may find triple therapy complicated because it involves taking as many as 20 pills a day. Also, the antibiotics used in triple therapy may cause mild side effects such as nausea, vomiting, diarrhoea, dark stools, metallic taste in the mouth, and dizziness, headache, and yeast infections in women. (Most side effects can be treated with medication withdrawal) Nevertheless, recent studies show that 2 weeks of triple therapy is ideal. Early results of studies in other countries suggest that 1 week of triple therapy may be as effective as the 2 week therapy, with fewer side effects. Another option is 2 weeks of dual therapy. Dual therapy involves two drugs: an antibiotic and an acid suppressor. It is not as effective as triple therapy. Two weeks of quadruple therapy, which uses two antibiotics, an acid suppressor, and a stomach-lining shield, looks promising in research studies. It is also called bismuth triple therapy.

The aim of the present study was to investigate the combination effect of the drugs on the ulcer index using ethanol induced ulcer models in both single dose and multiple dose study including histopathological studies.

Rabeprazole, being a potent proton inhibitor, decreases the excess acid secretion, by irreversibly blocking the H⁺/K⁺ ATPase of the parietal cell. Vitamin C, being an antioxidant neutralizes all the free radicals, nitrates, nitrites that cause cellular damage. An oral supplement of vitamin C is sufficient to maintain the gastric blood flow, intragastric vitamin C levels, antioxidant enzyme activities, which is impaired due to peptic ulcer disease. Further it translates HO-1 mRNA into active protein, which then may exert gastroprotection by its antioxidant and vasodilative properties.

On the other hand melatonin chemically 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 ulcer.

In ethanol induced ulcer model, Single dose studies exhibits that Rabeprazole + vitamin C has significantly reduced the ulcer index (P < 0.0001) and the percentage protection is 89.6 % whereas in rabeprazole + melatonin the percentage protection was 83.6 % when compared with control and standard 79.2 % indicating antioxidants play a vital role in the body and can act synergistically with beneficial drugs like rabeprazole. The results are shown in Table. 1.

Table No. 1: Influence of single dose of Rabeprazole, Vitamin C, Melatonin and their combinations on ulcer Index.

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

While in the multiple dose studies, the significant inhibitory effect of combination groups (Rabeprazole+Vitamin C & Rabeprazole+Melatonin) was produced mainly due to biosynthesis of cytoprotective prostaglandins, inhibition of lipid peroxidation, gastric mucosal permeability (to H⁺ and Na⁺ ions) and excess acid secretion etc.

Table.2. shows, rabeprazole + vitamin C has significantly reduced the ulcer index (P < 0.001) and the percentage protection was 95.2% whereas in rabeprazole + melatonin the percentage protection was 94% when compared with control and standard 85% indicating antioxidants play a vital role in the body and can act synergistically with beneficial drugs like rabeprazole. This can help in designing regimens with a lower dose of drug or can help in reducing the duration of regimen.

Histopathological examination using haematoxylene and eosin staining (H & E) also revealed the protective activity of combination groups (Rabeprazole + Melatonin & Rabeprazole + Vitamin C) when compared to control and standard rabeprazole. However groups treated with combinations showed much protective activity when compared to standard and control groups indicating antioxidants play a vital role in the body and can act synergistically with beneficial drugs like rabeprazole. (Fig.2)

Table No.2: Influence of multiple dose of Rabeprazole, Vitamin C, Melatonin and their combinations on ulcer index.

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

CONCLUSION:

From the results it can be concluded that the effect of combination groups was found to be synergistic in nature and more effective in treating peptic ulcer disease when compared to standard rabeprazole alone. Single and multiple dose treatment with vitamin C alone have produced beneficial effect in ulcer protection in ethanol induced ulcers. Single and multiple dose treatment with melatonin also have produced beneficial effect in ulcer protection in ethanol induced ulcers. The results of histopathological studies besides above biochemical parameter have also supported the results.

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Received on 23.02.2013

Modified on 06.03.2013

Accepted on 14.04.2013

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Research J. Pharmacology and Pharmacodynamics. 5(3): May–June 2013, 155-161