Antiulcer Effect of Vitamin E with Lansoprazole in
Treating Peptic Ulcer in Rats
Prasad K.1, Nitin
M.2*, Chetan M.2,
Girish M.2 and Krishna
Kumar2
1Shri Vishnu
College of Pharmacy, Bhimavaram-534202, A.P.
2HKES College of Pharmacy, Department of Pharmacology,
Gulbarga-585105, Karnataka, India.
ABSTRACT:
Peptic ulcer is a
chronic and appalling disease. Today, it is dominant among the diseases that
affect the world’s population. The principal factors causing this disease are
inadequate dietetic habits, prolonged use of non-steroidal anti-inflammatory
drugs, stress and infection by Helicobacter pylori, in addition to other
factors of genetic origin. The present study was designed to evaluate the
combination effect of vitamin E with lansoprazole against pylorus ligation
induced ulcer model in rats. The antiulcer effect of the combination of vitamin
E 0.9mg/200g and lansoprazole 0.54mg/200g b.w orally was compared with the
reference standard lansoprazole 0.54mg/200 g b.w orally. The ulcer index and
other biochemical parameters like volume, pH, free acidity and total acidity of
gastric juice were estimated. The ulcer index and other biochemical parameters
like volume (***P < 0.001), free acidity (***P < 0.001), total acidity
(***P < 0.001) and pH (**P < 0.01), of gastric juice showed reduction when
compared to standard lansoprazole. The percentage protection of combination was
92.9% as compared to standard lansoprazole 82.8%. Thus the combination group
was found to be synergistic in nature when compared to lansoprazole alone.
KEYWORDS: Antiulcer activity,
vitamin E, lansoprazole, pylorus ligation model, ulcer index.
INTRODUCTION:
Peptic
ulcer is an excoriated area of the gastric or duodenal mucosa caused by action
of the gastric juice. It is a chronic and recurrent disease, and is the most
predominant of the gastrointestinal diseases1. It is generally
recognized that peptic ulcer is caused by a lack of equilibrium between the
gastric aggressive factors and the mucosal defensive factors2. A number of drugs including proton pump
inhibitors and H2 receptors antagonists are available for the
treatment of peptic ulcer, but clinical evaluation of these drugs has shown
incidence of relapse, side effects, and drug interactions. This has been the
rational for the development of new antiulcer drugs and the search for novel
molecule or drug therapy that offer better protection and decreased relapse.
Vitamin E is a fat-soluble antioxidant that scavenges
oxygen free radicals, lipid peroxy radicals, and singlet oxygen3.
Since the reactive oxygen species play an important role in formation of ulcer,
vitamin E helps to neutralize these reactive oxygen species and helps in ulcer
prevention. Lansoprazole is a 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
use4. It is more potent, has longer duration of action, better
bioavailability and lesser drug interactions than other drugs5.
Therefore, the present study was designed to evaluate the combination of
vitamin E and lansoprazole against pylorus ligation induced ulcer model.
MATERIALS AND
METHODS:
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 seven days and housed under
standard conditions of temperature (25
20C)
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 protocol number, HKECOP/IAEC/17/2009-10 and the animal studies
were performed in accordance to guidelines of CPCSEA. Pure drug samples of
lansoprazole and vitamin E were procured from Lee Pharmaceuticals (Hyderabad.)
and Zydus Cadila, (Ahmedabad) respectively. The dose calculations were
extension of human dose based on body surface area6.
In pylorus ligation induced ulcer model7,
the rats were divided into 3 groups of 6 animals each. The animals of Group I
were treated with vehicle and the animals of Group II were treated with
standard, i.e., lansoprazole 0.54mg/200 g b.w orally. Similarly Group III was
treated with vitamin E and lansoprazole i.e., 0.9mg and 0.54mg per 200 g b.w
orally respectively. The drugs were administered daily for 5 days. On 5th
day, the rats were fasted for 24 h before pyloric ligation. Care was taken to
avoid coprophagy. At the end of 24 h, the rats were anaesthetized with
anesthetic ether. Abdomen was opened by a midline incision. The stomach was
lifted out and a ligature was placed at the pyloric sphincter without causing
any damage to its blood supply. The stomach was replaced carefully and abdomen
wall was sutured in two layers. After 6 hours, the rats were sacrificed with
excess of anesthetic ether, and the stomachs were dissected out. Gastric juice
was collected and drained into test tubes and then centrifuged at 1000 rpm for
10 min and the volume of supernatant was noted. The pH of the gastric juice was
recorded by pH meter. Then the contents were subjected for the analysis of free
and total acidity. The stomachs were opened along the greater curvature then
washed under running water to see 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)8. The recording was
0 for normal coloured stomach, 0.5 for red colouration, 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 formula9:
% Protection =
[(UI control – UI treated) /UI control] x 100
Determination
of free acidity and total acidity8:
1 ml of
gastric juice was pipetted into 100 ml conical flask. It was diluted to 10ml
with distilled water and added 2 –3 drops of Topfer’s reagent and titrated with
0.01 N sodium hydroxide until all traces of red color disappear and the color
of the solution turns to yellowish orange. The volume of the alkali added was
noted. This volume corresponds to free acidity. Then 2 – 3 drops of phenolphthalein
solution 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:
Volume of NaOH x Normality of NaOH x
100
Acidity = meq/L/100gm
0.1
Histopathological
evaluation10:
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 5 µm were cut and stained with haemotoxylin and eosin.
These were examined under the microscope for histopathological changes such as
congestion, haemorrhage, necrosis, inflammation, infiltration, erosion and
ulcers. The micro photographs were taken for publication.
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 and Graph 1 that the effect
of combination group i.e., vitamin E and
lansoprazole showed reduction in ulcer index and all biochemical
parameters like volume, free acidity, total acidity and increase in pH of
gastric juice when compared to control and standard lansoprazole. The
percentage protection of combination group was found to be 92.9% when compared
to standard lansoprazole alone (82.8%). The histopathological examination using
haematoxylene and eosin staining also revealed the protective activity of
combination group when compared to control and standard lansoprazole (Figure
5).
DISCUSSION:
A peptic ulcer
results from an imbalance between some endogenous aggressive factors that is hydrochloric
acid, pepsin, refluxed bile, leukotrienes, reactive oxygen species and
defensive factors, which include the function of the mucus-bicarbonate barrier,
surface active phospholipids, prostaglandins (PGs), mucosal blood flow, cell
renewal and migration, non enzymatic and enzymatic antioxidants and some growth
factors 11.Although impairment of the gastric mucosal barrier plays
a crucial role in the development of gastric ulcers and has only a relatively
small role in the development of duodenal ulcers, intragastric pH and peptic
activity are important in the development of both gastric ulcer and duodenal
ulcer disease12.
Prostaglandins
(PG) offer protection to stomach by increasing mucosal resistance and
decreasing aggressive factors like acid and pepsin 13.
According to Goel and Bhattacharya (1991)14,
pylorus ligation ulcers may be due to autodigestion of gastric juice, decreased
mucosal blood flow and breakdown of mucosal barrier. The Shay model7
is simple, reproducible and highly predictable model for the screening and
evaluation of antiulcer drugs. It utilizes neither the exogenous ulcerogens nor
the induced exogenous interfering factors. In case of pyloric ligation, ulcer
formation is mainly due to the stasis at the
gastric juice and stress15 or there is an excess of acid for a given
degree of mucosal defense. Therefore the reduced gastric ulcer in this model
may be due to the reduction in acid secretion and increased gastric pH.
Figure 1: shows normal stomach epithelium of rat in
pylorus ligation model
Figure
2: shows stomach of control rat in pylorus ligation model
Figure
3: shows stomach of standard lansoprazole treated rat in pylorus ligation model
Figure
4: shows stomach of vitamin E and lansoprazole treated rat in pylorus ligation
model:
Figure 5: Histopathological studies of
Pylorous ligation induced ulcer model in rat
a. Control negative: Gastric mucosa shows normal histology.
b. Control positive: Gastric mucosa shows redness, congestion,
hemorrhagic streaks, edema, ulceration, necrosis and dilation of blood vessels
c. Standard Lansoprazole: Gastric mucosa shows redness, congestion,
hemorrhage, mild edema and dilation of blood vessels.
d. Vitamin
E and Lansoprazole: Gastric mucosa shows mild redness,
congestion, mild edema and dilation of blood vessels.
In the present
study, from Table 1, Graph 1 and Fig. 1 to Fig. 5 the reduction in ulcer index
and other biochemical parameters of gastric juice like volume, free acidity,
total acidity and increase in pH by combination group suggests that its
cytoprotective mechanism may be due to inhibition of gastric secretion and
neutralization of reactive oxygen species by one or more mechanism.
Lansoprazole, being a potent proton inhibitor, decreases the excess acid
secretion, by irreversibly blocking the H+,K+-ATPase of
the parietal cell. Vitamin E being an antioxidant scavenges OH radical that
cause cellular damage.
CONCLUSION:
From the present
study and available results, it can be concluded that the combination group of
vitamin E and lansoprazole was found to be synergistic in nature and have 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 and Zydus Cadila, for providing the gift
samples of pure drug.
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