Comparative Influence of Selected Antioxidants with Lansoprazole in Aspirin Induced Ulcer Model in Rats
Nitin M.*, Girish M., Chetan M., Ather Javed, Krunal S. and Krishna Kumar
Department of Pharmacology, HKES College of Pharmacy, Sedam Road, Gulbarga-585105, Karnataka, India.
*Corresponding Author E-mail: girish.madri24@gmail.com
ABSTRACT:
Gastric hyperacidity and ulcers are common gastrointestinal complications that affect a considerable number of people in the world. A number of excellent drugs, developed over the decades have proven useful in controlling hyperacidity and ulceration but still the problem of relapse has eluded success. Hence an attempt was made to find a therapy with more efficacy and better results. The present study was designed to evaluate the combination effect of selected antioxidants with lansoprazole against aspirin induced gastric ulcer model in rats. The experiment was conducted using 6 groups of albino rats with 6 animals each and the dose calculations were based on body surface area as described by Laurence and Bacharach. The antiulcer effect of the combination of antioxidants with lansoprazole was compared with the reference standard lansoprazole alone. The ulcer index and percentage protection was calculated. All the combination groups showed reduction in ulcer index and the percentage protection was found to be 92.1% for vitamin C + lansoprazole, 89.5% for coenzyme Q10 + lansoprazole, 90.8% for vitamin E + lansoprazole, 88.1% for melatonin + lansoprazole when compared to standard lansoprazole 82.8%. Thus all the combination groups of selected antioxidants with lansoprazole showed significant enhancement of antiulcer activity and were found to be synergistic in nature when compared to standard lansoprazole alone.
KEYWORDS: Antiulcer activity, vitamin C, coenzyme Q10, vitamin E, melatonin, lansoprazole, ulcer index.
INTRODUCTION:
The peptic ulcer disease refers to painful sores or lesions in the mucosal lining of stomach (gastric ulcer) or first part of small intestine (duodenal ulcer).The pathophysiology of peptic ulcer has been centralized on an imbalance between aggressive and protective factors in the stomach such as acid-pepsin secretion, mucosal barrier, mucus secretion, blood flow, cellular regeneration, prostaglandins and epidermal growth factors1. Various causes of gastric ulceration include stress, smoking, alcohol, nutritional deficiencies, Helicobacter pylori and due to use of NSAIDs have been shown to be mediated largely through generation of reactive oxygen species (ROS) 2.
The term “reactive oxygen species” (ROS) collectively denotes oxygen centered free radicals such as super oxide (O- 2) and hydroxyl (OH) as well as nonradical species derived from oxygen such as hydrogen peroxide (H2O2), singlet oxygen (1O2) and hypochlorus (HOCl) acid. They play an important role in the pathogenesis of a wide variety of clinical disorders and gastric damage. They attack essential cell constituents, such as proteins, lipids and nucleic acids, leading to the formation of toxic compounds3.
The other fundamental factor in the pathogenesis of gastric and duodenal ulcers, besides ROS, is the hypersecretion of acid4. Therefore, the control of acid secretion and neutralization of ROS may be essential for the treatment of peptic ulcer. This can be done by combination therapy of antioxidants with antisecretory drugs like lansoprazole.
Lansoprazole is a well known proton pump inhibitor that suppresses the gastric acid secretion by inhibiting H+K+ ATPase pump. The FDA has approved it for treatment and prevention of recurrence of NSAIDs associated gastric ulcers in patients who continue NSAID use5. It is more potent, has longer duration of action, better bioavailability and lesser drug interaction than other drugs6.
Antioxidants are compounds which help to defend the body against cell damage caused by various free radicals. They neutralize the free radicals by accepting or donating an electron7. There are number of antioxidants like vitamin C, vitamin E, coenzyme Q10, melatonin etc. which prevent cellular damage and ulceration by one or more mechanism.
Vitamin-C is a water soluble, chain breaking antioxidant which neutralizes various reactive oxygen species like superoxide radical, singlet oxygen, hydrogen peroxide8, alkoxyl, hydroxyl radical directly by hydrogen donation and also neutralizes the radical form of other antioxidants like glutathione & vitamin-E7. Apart from scavenging free radicals vitamin C is proven to exert gastroprotection by hemeoxygenase-1-dependent (HO-1) 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 properties 9.
Coenzyme Q10 is a lipid soluble antioxidant and an excellent membrane stabilizing agent. It acts as a cofactor in electron transport chain and involved in the synthesis of ATPs. Since free radical damage is believed to be one of primary mechanisms to induce gastric injury and peptic ulceration, coenzyme Q10 being antioxidant prevents this ulceration and helps in the production of protective mucus by supplying ATPs during cellular repair10. The literature survey also shows that hypoxia and other adverse affect in the healing of gastric ulcer can be prevented by administration of coenzyme Q1011.
Vitamin E is a family of α-, β-, γ-, and δ-tocopherols. Of these, α-tocopherol is the most important lipid-soluble antioxidant, and that it protects cell membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction12. It scavenges oxygen free radicals, lipid peroxy radicals, and singlet oxygen13 and protects the gastric mucosa by increasing mucus secretion14. An increase in the synthesis of prostaglandins and high level of glutathione in tissues of vitamin E treated animals has been suggested as a possible mechanism of anti-ulcer activity15.
Melatonin, a close derivative of serotonin, is a hormone produced by the pineal gland and intestinal enterochromaffin cells which control sleep and gastrointestinal motility16. It is also a powerful scavenger of free radicals and scavenges OH radical and offers gastroprotection by maintaining endogenous prostaglandin levels17. It also inhibits hydrochloric acid18 and pepsin secretion and acts as an immunostimulant19.
Therefore, the present study was designed to evaluate the combination effect of above antioxidants with lansoprazole against aspirin induced gastric ulcer model.
MATERIALS AND METHODS:
Animal:
Albino wistar
rats of either sex weighing between 180 to 220 g were selected for the present
study. 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. Standard animal
pellet food procured from Amrut laboratories, Pranav Agro Industries Ltd.,
Sangli was provided in adequate quantity, with drinking water ad libitum.
The experimental protocol (HKE COP/IAEC/16/2009-10) was prior approved by
Institutional Animal Ethics committee (IAEC) of H.K.E.S’s College of Pharmacy,
Gulbarga for conduction of experiments.
Drugs:
Pure drug samples of lansoprazole, vitamin C, coenzyme Q10, vitamin E and melatonin were procured from Lee Pharmaceuticals (Hyderabad.), Wockhardt Ltd. (Maharashtra.), Elder Health Care Ltd. (Orissa), Zydus Cadila (Ahmedabad) and (Aristo Pharmaceuticals (Mumbai) respectively. Aspirin was procured from Nice Chemicals Pvt Ltd. (Cochin.)
Experimental procedure:
In aspirin induced gastric ulcer model20, albino wistar rats of either sex weighing between 180 to 220 g were divided into 6 groups of 6 animals each. The dose calculations were extension of human dose based on body surface area21.
Group I - Control (distilled water 0.5ml orally)
Group II - Standard (Lansoprazole 0.54mg/200 g b.w in 2% gum acacia orally.)
Group III - Vitamin-C + Lansoprazole (4.5mg +0.54mg)/200 g b.w orally.
Group IV - Coenzyme Q10 + Lansoprazole (5.4mg +0.54mg)/200 g b.w orally.
Group V - Vitamin E + Lansoprazole (0.9mg +0.54mg)/200 g b.w orally.
Group VI - Melatonin + Lansoprazole (0.108mg +0.54mg)/200 g b.w orally.
The drugs were administered daily for 5 days orally with the help of oral feeding needle. On 5th day, the rats were fasted for 24 h. Care was being taken to avoid coprophagy. At the end of 24 h, Aspirin (200mg/kg b.w in 2% gum acacia orally) was administered to the rats of all the Groups (I to VI) to induce ulcer. After 6 h rats were sacrificed and stomach was dissected out. The stomachs were opened along the greater curvature then washed under running water to see 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)22. The recording was ‘0’ for normal colored stomach, ‘0.5’ for red coloration, ‘1’ for spot ulcer, ‘1.5’ for hemorrhagic streaks, ‘2’ for ulcer ≥ 3 ≤ 5 and ‘3’ for ulcer > 5. The mean ulcer score for each animal is expressed as ulcer index and the percentage protection was calculated by using the following formula23:
% Protection = [(UI control – UI treated) /UI control] x 100
Statistical analysis:
The results were expressed as mean ± SEM, (n=6). Statistical analysis was performed using student‘t’ test. P value less than 0.05 was considered to be statistically significant.
RESULTS:
It is evident from Table 1, Graph 1 and Fig.1 to Fig.7 that the effect of combination groups of selected antioxidants with lansoprazole showed reduction in ulcer index and the percentage protection was found to be 92.1% for vitamin C + lansoprazole, 89.5% for coenzyme Q10 + lansoprazole, 90.8% for vitamin E + lansoprazole, 88.1% for melatonin + lansoprazole when compared to standard lansoprazole 82.8%.
Table no. 1- Effect of Antioxidants (Vitamin C, Coenzyme Q10, Vitamin E and Melatonin) with Lansoprazole in aspirin induced gastric ulcer model in rats:
Group No. |
Treatment |
Dose/200g rat p.o |
Ulcer Index |
% Protection |
1. |
Control |
Distilled water 0.5 ml |
6.333 ± 0.21 |
- |
2. |
Lansoprazole |
0.54mg |
1.33 ± 0.27 |
78.9% |
3. |
Vitamin C + Lansoprazole |
(0.54+4.5)mg |
0.500 ± 0.18* |
92.1% |
4. |
Coenzyme Q10 + Lansoprazole |
(0.54+5.4)mg |
0.833 ± 0.21 |
89.5% |
5. |
Vitamin E + Lansoprazole |
(0.54+0.9)mg |
0.6667 ± 0.10 |
90.8% |
6. |
Melatonin + Lansoprazole |
(0.54+0.108)mg |
0.7500 ± 0.17 |
88.1% |
p.o: per oral.; g: gram.; ml: milliliter.; mg: milligram.
Values are the mean ± S.E.M. of 6 rats per treatment; Significant *P < 0.05 compared with standard lansoprazole.
Graph No. 1- Effect of Antioxidants (Vitamin C, Coenzyme Q10, Vitamin E and Melatonin) with Lansoprazole in aspirin induced gastric ulcer model in rats:
Fig. 1 - shows stomach epithelium of normal rat in aspirin induced ulcer model.
Fig. 2 - shows stomach epithelium of control rat in aspirin induced ulcer model
Fig. 3 - shows stomach epithelium of standard lansoprazole treated rat in aspirin induced ulcer model.
Fig. 4 - shows stomach epithelium of vitamin C and lansoprazole treated rat in aspirin induced ulcer model
Fig. 5 - shows stomach epithelium of coenzyme Q10 and lansoprazole treated rat in aspirin induced ulcer model
Fig. 6 - shows stomach epithelium of vitamin E and lansoprazole treated rat in aspirin induced ulcer model
Fig. 7 - shows stomach epithelium of melatonin and lansoprazole treated rat in aspirin induced ulcer model
DISCUSSION:
Gastric bleeding and the formation of gastrointestinal ulcers and erosions are the most adverse reactions in patients subjected to therapy with nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin. Gastric lesions induced by aspirin in the stomach are multiple in nature. The administration of aspirin results in the production of gastric mucosal damage mainly in the glandular portion of the stomach. Aspirin causes mucosal damage by interfering with prostaglandin synthesis (PGE2 and PGI2), enhanced acid secretion, increased back diffusion of H+ ions and breaking up of the mucosal barrier 24. This is accompanied by the activation of neutrophils that lead to excessive release of oxygen radicals. In addition, aspirin induces lipid peroxidation, myeloperoxidase activity that result in suppression of gastric blood flow, intragastric vitamin C levels, superoxide dismutase and glutathione peroxidase activities (antioxidizing enzymes) which further cause gastric damage25.
In the present study, from Table 1, Graph 1 and Fig. 1 to Fig. 7 the reduction in ulcer index by combination groups of selected antioxidants with lansoprazole suggests that its cytoprotective mechanism may be due to inhibition of gastric secretion and neutralization of reactive oxygen species by one or more mechanisms. Lansoprazole, being a potent proton pump inhibitor, decreases the excess acid secretion, by irreversibly blocking the H+, K+-ATPase of the parietal cell. Vitamin C, being an antioxidant neutralizes the free radicals like nitrates, nitrites that cause cellular damage. It maintains the gastric blood flow, intragastric vitamin C levels, antioxidant enzyme activities, which is impaired due to peptic ulcer disease. The study led by researchers also showed that the lower level of vitamin-C in the blood, the more likely a person will become infected by Helicobacter pylori, the bacteria that can cause peptic ulcers and stomach cancer26. Coenzyme Q10 prevents ulceration by reducing the amount of free radical damage and helps in the production of protective mucus and rapid cell turnover of gastric mucosa by supplying ATPs during cellular repair. Vitamin E is a membrane stabilizer and multifaceted antioxidant that scavenges oxygen free radicals, lipid peroxy radicals, and singlet oxygen. It increases the synthesis of protective prostaglandins (PGs) and glutathione level in the tissues and helps in the treatment of peptic ulcer. Melatonin inhibits acid and pepsin secretion and protects gastric epithelium. It also inhibits nitric oxide biosynthesis which may explain the regression of GERD symptoms 27.
CONCLUSION:
From the present study and available results, it can be concluded that the combination group of different antioxidants with lansoprazole was found to be synergistic in nature and showed significant enhancement of antiulcer activity. Hence the combination has more cytoprotective and antisecretory effect when compared to the standard lansoprazole alone.
ACKNOWLEDGMENTS:
The authors wish to thank the authorities of H.K.E. Society College of Pharmacy for providing the necessary facilities to carry out the research work. We are grateful to Lee Pharmaceuticals, Wockhardt Ltd, Elder Health Care Ltd, Zydus Cadila, Aristo Pharmaceuticals and Nice chemicals Pvt Ltd, for providing the gift samples of pure drug.
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Received on 30.04.2011 Modified on 19.05.2011
Accepted on 31.05.2011 © RJPT All right reserved
Research J. Pharm. and Tech. 4(8): August 2011; Page 1273-1277