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. 20 years have elapsed since Marshall and Warren's discovery of the link - a bacterium called at that time Campylobacter pylori and peptic ulcer disease¹. Research advancement during the last few years have offered new insights in the therapy and prevention of gastro duodenal 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 ulcers².

Peptic ulcer is one of the major ailments affecting about 60% human adults and nearly 80% child population in topical countries³. 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)⁴^-6.

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; nighttime pain is common, Pain may be relieved by antacids or milk, Heartburn, Indigestion (dyspepsia), Belching, Nausea, Vomiting, Poor appetite, Weight loss⁷.

Among the various causes of peptic ulcers are the free radicals which are chemical species containing unpaired electrons in their outer orbital which are generally reactive⁸. 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 (OH^-), and alkoxyl singlet oxygen (¹O₂)⁹. 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 macromolecules¹⁰.

Vitamin C is well known antioxidant that defends against cell damage. Vitamin C neutralizes hydroxyl, alkoxyl and peroxyl radicals by hydrogen donation and also neutralizes the radical form of other antioxidant like glutathione and vitamin E⁹. 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 properties¹¹.

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 interaction¹². Hence an attempt has been made to study the efficacy of combination of the same antioxidant (vitamin C) with rabeprazole to treat peptic ulcer.

MATERIALS AND METHODS:

Rabeprazole was received as gift sample from Lee Pharmaceuticals, Hyderabad and Vitamin C was procured from Wockhardt Ltd; Aurangabad, Maharashtra. Albino Wistar rats of either sex weighing between 180 to 220 g were procured from central animal house, MR. Medical College, Gulbarga. The animals were acclimatized for 07 days and housed under standard conditions of temperature (25±2ºC) and relative humidity (30-70%) with a 12:12 light-dark cycle. The animals were fed with standard pellet diet (Hindustan liver co., Mumbai) and water ad libitum. Approval at the Institutional Animal Ethics Committee (IAEC) of H.K.E.S College of pharmacy, Gulbarga was taken for conducting antiulcer activity. The registration number is HKE-COP/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:

Pylorus Ligation¹³:

Each study group consists of albino wistar rats of either sex weighing between 180–220 g were divided into 4 groups of 6 animals each. The animals were administered with the drugs in the following order:

Group I – Control

Group II - Standard (Rabeprazole 0.36 mg/200 g b.w. in distilled water p.o.)

Group III – Vitamin C (4.5mg/200gm b.w. in distilled water p.o.)

Group IV - Rabeprazole + Vitamin C (0.36 mg + 4.5 mg)/200 g b.w. p.o.

Single Dose Study:

The rats were fasted for 48h prior to the experiment with water ad libitum. During experimentation water also was withdrawn. At the end of 48 h, the rats were administered with drugs. After 1 h of drug administration, pylorus ligation was performed 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.

Multiple Dose Study:

The drugs were administered for 7 consecutive days. On 7^th day, the rats were fasted for 48 h following drug administration and care was being taken to avoid coprophagy. At the end of 48 h, the rats were subjected for pylorus ligation as described above in single dose studies. The gastric juice was collected and subjected to biochemical investigations. The following biochemical parameters were evaluated for both the study groups:

Measurement of Volume (ml) of Gastric Juice and Determination of pH¹⁴:

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

Determination of Ulcer Index¹⁴:

The stomachs were opened along the greater curvature then 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)³⁴. The stomachs were scored using the following scale -

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 was expressed as ulcer index and the percentage protection was calculated by using the formula¹⁵:

% Protection = [(Ulcer Index Control – Ulcer Index Treated) /Ulcer Index Control] X 100

Determination of free acidity and total acidity¹⁴:

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 x Normality of NaOH x 100 / 0.1 mEq/L/100 gm

Histopathological evaluation¹⁶:

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 haematoxylene 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<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 combination treatment with rabeprazole and vitamin C was found to be significant and synergistic when compared to the standard rabeprazole. The ulcer index in the combination treated group effectively reduced to a much significant extent and the percentage protection was found to be 90.7% on comparison with rabeprazole treated group where it was 83.7%. (As seen in Fig.1) The combination treatment also reduced the volume of the gastric juice (ml) secreted, free acidity (mEq/L) and total acidity (mEq/L) and enhanced the pH of the gastric juice significantly, indicating effective gastroprotection. (Table.1)

Multiple dose studies:

Treatment with rabeprazole and vitamin C combination exhibited much significant reduction in ulcer index on comparison with rabeprazole treated group. The percentage protection was 93% with combination group and 88.3% with rabeprazole treated group. The combination also reduced the total acidity (mEq/L), free acidity (mEq/L) and the volume of the gastric juice (ml) and enhanced the pH of the gastric juice significantly, indicating significant ulcer protection (Table.2)

Histopathological studies of stomachs treated with multiple dose regimens: (Fig. 2):

The histopathological reports revealed that the stomachs treated with multiple dose regimen of rabeprazole showed gastric mucosa appearing normal with very few inflammatory cells (Fig.2C), while in vitamin C treated group the stomachs showed few ulcers (Fig.2D), however, the combination group revealed that the gastric mucosa exhibited no congestion, no ulceration and very mild inflammation(Fig.2E) upon comparison to the control group which exhibited gastric mucosa with extensive ulcerations, congestion and extensive inflammation (Fig.2B)

1A) Stomach epithelium of normal albino rat

1B) Stomach epithelium of control group rat

1C) Stomach epithelium of rabeprazole treated rat

1D) Stomach epithelium of vitamin C treated rat

1E) Stomach epithelium of combination treated rat

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

2A) Histopathological slide of normal rat stomach

2B) Histopathological slide of control group rat

2C) Histopathological slide of rabeprazole treated rat

D) Histopathological slide of vitamin C treated rat

2E) Histopathological slide of combination treated rat

Figure.2: Histopathological slides of stomachs of albino rats treated with multiple dose regimens

DISCUSSION:

Among the various causes of gastric ulceration lesions caused by stress, alcohol, H.pylori and due to use of NSAIDs have been shown to be mediated largely through generation of reactive oxygen species (ROS), especially the hydroxyl radical. A number of excellent drugs, developed over the decades have proven useful in controlling hyperacidity and ulceration but their long term use is reported to have various side effects. Hence the investigations continue with an objective to find a therapy with more efficacy and lesser side effects. Antioxidants neutralize the ROS involved in the pathogenesis of peptic ulcer. Hence an attempt was made to study the combination effect of antioxidants with antisecretory drugs (PPIs).

In the present study a detailed investigation of antisecretory and gastroprotective activity of combination therapy of drugs i.e. rabeprazole + vitamin C and rabeprazole + melatonin was carried out in albino wistar rats using following ulcer induction models namely:

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 and 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.

In both single and multiple dose studies the combination group (rabeprazole + vitamin C) 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.

Single dose studies:

Table no 1.

Shows, rabeprazole + vitamin C 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 90.7%. When compared with control and standard 83.7%.

Multiple dose studies:

Table no 2.

Shows, rabeprazole + vitamin C group has significantly reduced the volume of gastric secretion (P < 0.0001), 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 93% when compared with control and standard 88.3%. Histopathological examination using haematoxylene and eosin staining (H and E) also revealed the protective activity of combination group (Rabeprazole + Vitamin C) when compared to control and standard rabeprazole. However group treated with rabeprazole + vitamin C showed much protective activity when compared to group rabeprazole and vitamin C indicating antioxidants play a vital role in the body and can act synergistically with beneficial drugs like rabeprazole.

CONCLUSION:

The combination of rabeprazole + vitamin C has shown better effect compared to rabeprazole alone. The parameters like free acidity, total acidity, volume of gastric juice, pH, and ulcer index have shown significant reduction in their values when given in combination compared to rabeprazole alone. Thus, from the results it can be concluded that the effect of combination group of these drugs was found to be synergistic in nature and more effective in treating peptic ulcer disease when compared to standard rabeprazole alone. The results of histopathological studies besides above biochemical parameters of gastrointestinal secretions have also supported the results.

Table.1: Influence of single dose regimen of vitamin C, rabeprazole and combination

Groups	Volume of gastric juice (ml)	pH	Free acidity (mEq/L)	Total acidity (mEq/L)	Ulcer Index	% Protection
Control	6.017±0.11	1.717±0.3	121.0±1.02	140.4±0.51	3.583±0.15	--
Rabeprazole	1.517±0.03	5.363±0.07	63.15±0.73	75.90±1.03	0.583±0.15	83.7%
Vitamin C	1.233±0.04 **	4.332±0.07 ***	82.35±0.84 ***	99.98±0.41***	1.333±0.10	62.7%
Combination	1.200±0.02**	6.383±0.09***	20.08±0.57***	50.93±0.69***	0.333±0.16**	90.7%

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

Table.2: Influence of multiple dose regimen of vitamin C, rabeprazole and combination

Groups	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	-
Rabeprazole	1.293±0.06	6.033±0.04	50.90±0.44	62.40±0.65	0.416±0.08	88.3%
Vitamin C	1.105±0.03*	5.045±0.06***	71.75±0.35 ***	86.40±0.60 ***	0.916±0.08	74.4%
Combination	0.983±0.03***	6.722±0.0 ***	15.83±0.25 ***	34.37±0.25***	0.250±0.10 **	93.0%

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