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 (25
20C) 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%.
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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