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Nigerian Journal of Physiological Sciences
Physiological Society of Nigeria
ISSN: 0794-859X
Vol. 23, Num. 1-2, 2008, pp. 79-83
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Nigerian
Journal Of Physiological Sciences, Vol. 23, No. 1-2, 2008, pp. 79-83
Methanolic Extract Of Tetracera potatoria, An Antiulcer Agent Increases Gastric Mucus Secretion And
Endogenous Antioxidants
F. S. Oluwole, J. A. Ayo, B.
O. Omolaso, B. O. Emikpe1 And J. K. Adesanwo2
Department
of Physiology, College of Medicine,
1Department of Veterinary
Pathology, University of Ibadan, and
2Department of Chemistry,
Obafemi Awolowo University, Ile-ife, Osun-state, Nigeria. E-mail: franwole@yahoo.com
Code Number: np08016
Summary
In this study, the possible mechanism underlying the
antiulcer activity of the methanolic extract of the root of Tetracera
potatoria (MeTp) was studied in albino rats. Misoprostol and omeprazole were
used as reference drugs. The animals had MeTp administered to them at varying
doses of 100, 400 and 800 mg/kg for 15 days. MeTp significantly (P<0.05) increased
gastric mucus secretion and gastric mucus cell counts when compared to control.
MeTp treated animals also showed significant (P<0.05) increase in the
activity of superoxide dismutase (SOD) with concurrent decrease in the level of
malonialdehyde (MDA) with respect to control. These findings suggest that part
of the gastroprotective property of MeTp is associated with the ability of the
extract to cause stimulation of gastric mucus secretion through increased
number of gastric mucus cells. Increased SOD-activity and decreased MDA-levels
further lend support to its gastroprotective effect.
Key
words: Tetracera potatoria, gastroprotective,
gastric mucus secretion, superoxide dismutase
Introduction
Tetracera potatoria Afzel, family Dillenniaceae is known as liane a eau in
France and water tree in Sieerra-leone (Burkil,1985).It is found in wooded
areas of Senegal, Southern part of Nigeria, Central and Eastern Africa
(Dalziel,1937).
The leaves of the plant boiled in its own sap are used
for the treatment of gastrointestinal sores (Burkil, 1985). Adesanwo et al (2003)
reported the antiulcer activity of the methanolic extract of the root of Tetracera
potatoria. Two doses (400mg/kgBW and 800mg/kgBW) administered to albino
rats completely inhibited gastric ulceration and significantly reduced gastric acidity.
Previous antiulcer drugs were designed to inhibit gastric acid secretion.
However, it is not in all cases of hyperacidity that ulceration develops.
Gastric ulcer has been discovered to develop in patients with normal gastric
acid output (Lawrence, 2000).
Some other factors implicated in gastric ulcers are
oxygen derived radicals, pepsinogen and blood flow (Desai et al, 1997).
These radicals are eliminated by the scavenging action of natural antioxidants
(Halliwell and Gutteridge, 1990). Some well established endogenous antioxidants
are superoxide dismutase, catalase, glutathione reductase and glutathione
peroxidase (Kelly, 1998). Flavonoids, another major phytochemical of Tetracera
potatoria were reported as effective gastroprotective agents due to their
antioxidant activity (Mirossay et al, 1996a, b; 1999).
Though the exact anti-ulcerogenic mechanism of Tetracera
potatoria is not fully understood, mucus secretion is regarded as a crucial
defensive factor in the protection of the gastric mucosa from gastric lesions
(Mallika et al, 2005). In this study, we examined the influence of the
methanolic extract of the root of Tetracera potatoria on gastric mucus
secretion, gastric mucus cell counts, and the level of production of gastric
anti-oxidant enzymes.
Materials
and methods
Extract
preparation:
Fresh roots of Tetracera potatoria were
purchased from Ago-Iwoye, Ogun State, Nigeria and were authenticated by Mr. T.K.
Odewo of Forestry Research Institute of Nigeria (FRIN), Ibadan. The roots were
air-dried for six weeks, sawn into tiny pieces and later ground, weighing about
5.2kg. A large quantity (3.4kg) of the grinded material was soxhlet-extracted
with methanol for about 72h. The MeTp was then dried in the Gallenhamp oven at
30°C for three days. The starting sample gave a mean yield of 7.1%. The extract
was reconstituted in distilled water to make up the required concentrations and
was stored at 4°C until use.
Animals:
Adult Wistar strain rats (180-220g) obtained from the
Pre-Clinical Animal house, College of Medicine, University of Ibadan, were used for this study. There were four experimental study groups namely; gastric
mucus secretion, gastric mucus cell count, superoxide dismutase activity and
malondialdehyde level. Control animals were not given any treatment but were
fed normally and given water ad libitum. MeTp-treated rats were given
MeTp at different doses (100mg/kg BW, 400mg/kg BW and 800mg/kg BW) for 15 days.
Some rats pretreated with Omeprazole (0.67mg/kg) and Misoprostol (0.875µg/kg)
served as positive controls.
Drugs
used
Chemicals used were Adrenaline, Magnesium chloride
(sigma), Carbonate buffer (sigma), Potassium chloride (sigma), Thiobarbituric acid
(sigma), Trichloroacetic acid (sigma), Sodium acetate and Alcian blue. Stock
solutions were prepared in distilled water.
Determination
of gastric mucus secretion
Gastric mucus secretion was estimated using the earlier
method described by Oluwole et al, (2007) after two weeks of
pretreatment with the drugs.
Gastric
mucus cell count
The gastric mucus cells were counted using an
improvised calibrated microscope. This was an improvement over the foremost
blind manner approach for counting (Li et al, 2002). Twenty-five
squares, each measuring 2mm by 2mm, were drawn faintly on a transparent nylon.
The nylon was then affixed onto the eyepiece of the microscope. The gastric
mucus cells were counted as cells that stained for Haematoxylin and Eosin
indicated as red patches. The mucus cells were counted in five squares during
each view. The number of gastric mucus cells in each microscopic view was
recorded and the mean number of gastric mucus cells in each square millimeter
of gastric tissue was calculated.
Determination
of superoxide dismutase activity
The assay method involves the inhibition of
autooxidation of adrenaline to adrenochrome by SOD. The rate of autoxidation of
adrenaline and the sensitivity of this inhibition of autooxidation by SOD were
both augmented as the pH was raised from 7.8 10.2. The animals were bled
through the eye and the blood samples were centrifuged in a cold centrifuge.
Plasma samples were stored at 4°C until use. 0.2ml of the test sample was added
to 2.5ml of 0.05M carbonate buffer. It was allowed to equilibrate in the
spectrophotometer. 0.3ml of freshly prepared 0.3mM adrenaline was added to the
buffer-supernatant mixture, which was quickly mixed by inversion. The reference
cuvette contained 2.5ml of the buffer, 0.1ml of adrenaline and 0.2ml of water.
The increase in absorbance at 480nm was monitored every 30 seconds for 150
seconds.
Calculation:
Change
in absorbance/min (∆A/min) = A2-A1
Where
A2 = Final absorbance after 150 seconds
A1 = Initial absorbance after 30 seconds
t = 2.5 min
%
inhibition = 1- (∆A Sample/min) X 100
∆A blank/min
∆A
blank/min = constant = 0.025/min
Using
the above calculations, a standard curve for SOD activity was plotted and the
percentage SOD activity of each experimental group was deduced from the curve.
Estimation
of lipid peroxidation
Lipid peroxidation was assessed by the method
described by Gutteridge and Wilkins(1982), This is based on the reaction
between 2-thiobarbituric acid (TBA) and malonialdehyde(MDA) which is the
end-product of lipid peroxidation.
Statistical
analysis
Results were expressed as Mean ± SEM. Statistical
analysis was performed using students t-test and significant differences were
accepted at P<0.05.
Results
Gastric mucus secretory
activity
From Table 1, the mean gastric mucus secretion in
control animals was 4.16±0.08 as against 4.55±0.09, 6.44±0.13, and 5.67±0.08 in
low dose, medium dose and high dose pretreated animals respectively. The
observed increase with each of the doses was significant (P<0.05).
Similarly, Misoprostol significantly increased gastric mucus secretion compared
with the control (P<0.05). However, omeprazole significantly reduced gastric
mucus secretion comparable to control (P<0.05).
Effect of Tetracera
potatoria on gastric mucus cell counts
Methanolic extract of Tetracera potatoria
significantly increased the number of mucus cell count in animals pretreated
with medium dose (MD) and High dose (HD) when compared with the mean value
obtained from the control rats (Table 2) (P<0.05). There was however, a
significant reduction in low dose (LD) animals. Omeprazole and Misoprostol at
various doses used caused significant increase in gastric mucus cell counts
compared to control (P<0.05).
Table 1: Mean gastric mucus secretion in control and
animals treated with Methanolic extract of the root of Tetracera potatoria (MeTp)
Animal Treatment |
No of Animals |
Gastric mucus secretion (mg/g)
Mean ±S.E.M |
Control (non-treated) |
5 |
4.16 ± 0.08 |
Low-dose (100mg/kg) |
5 |
4.55±0.09* |
Medium-dose (400mg/kg) |
5 |
6.44±0.13* |
High Dose (800mg/kg) |
5 |
5.67±0.08* |
Omeprazole (0.67mg/kg) |
4 |
2.61± 0.02* |
Misoprostol (0.875µg/kg) |
4 |
5.54± 0.02 * |
P-value
at P< 0.05 *Significantly different from control.
Table
2: The mean gastric mucus cell count (mm2) in control and animals
treated with the Methanolic Extract of the Root of Tetracera potatoria (MeTp)
Group Treatment |
No of Animals |
Gastric mucus cell count(mm2) Mean±SEM |
Control (non-treated) |
5 |
1.98 ± 0.00 |
Low-dose (100mg/kg) |
5 |
1.83±0.01* |
Medium-dose(400mgkg) |
5 |
2.34±0.02* |
High dose (800mg/kg ) |
5 |
2.93±0.00* |
Omeprazole (0.67mg/kg |
5 |
2.09±0.05* |
Misoprostol(0.875µg/kg) |
5 |
2.08±0.01* |
P-value
at P< 0.05. *Significantly different from control.
Effect of Tetracera
potatoria on Superoxide dismutase (SOD) activity
Methanolic extract of Tetracera potatoria
increased SOD activity in a dose dependent fashion in all the groups (Table 3).
The increase demonstrated in each group was significant conpared to the control
(P<0.05).
Effect of Tetracera
potatoria on malonialdehye concentration
The extract reduced the concentration of assayed
maloniaaldehye from 1.888±0.011 in the control to 1.714±0.009 (LD), 1.561±0.005
(MD) and 1.304±0.005 (HD). The reduction in the MD and HD pretreated animals
were significant (P<.0.05). This is illustrated in Table 4.
Table
3: The Mean superoxide dismutase activity in (µg/ml) control and animals
treated with Methanolic Extract of the root of Tetracera potatoria (MeTp).
Group
Treatment |
No
of Animals |
Superoxide
dismutase activity(µg/ml) Mean±SEM |
Control
(non-treated) |
5 |
19.90
± 1.25 |
Low-dose
(100mg/kg) |
5 |
21.86±0.64* |
Medium-dose(400mg/kg) |
5 |
32.08±1.50* |
High
Dose (800mg/kg) |
5 |
49.40±4.40* |
P-value
at P< 0.05. *Significantly different from control.
Table
4: Mean malonialdehyde
concentration in control and animals treated with the Extract of the root of Tetracera
potatoria (MeTp)
Group Treatment |
No of Animals |
Malonialdehye concentration (mmol/l) x10-6
Mean±SEM |
Control
(non-treated)
|
5 |
1.888 ± 0.011 |
Low-dose
(100mg/kg)
|
5 |
1.714±0.009 |
Medium-dose (400mg/kg) |
5 |
1.561±0.005* |
High dose (800mg/kg)
|
5 |
1.304±0.005* |
P-value
at P< 0.05. *Significantly different from control.
Discussion
The role of MeTp as an antiulcer agent had earlier
been reported by Adesanwo et al (2003), having discovered a reduction in
gastric acidity in animals treated with MeTp. Acute pretreatment of rats with
MeTp and Misoprostol (15days) caused significant increase in gastric mucus
secretion in all doses administered (100,400, and 800mg/kg) in comparison to
4.16±0.08mg/g in the control group (P<0.05). However, omeprazole, a proton
pump inhibitor significantly reduced gastric mucus secretion (P< 0.05).
Gastric mucus cells counts also increased significantly at doses of 400 and 800
mg/kg compared with the control (Table 2) (P<0.05). This finding is
indicative of the fact that MeTp enhances the growth of mucus secreting cells
and thus agree with the report of Mojzis et al. (1995) that gastric
mucus is an important factor in gastric mucosal defense. Other reports of the
gastro-protective property of mucus opined that a decrease in gastric mucus
secretion renders the mucosa more susceptible to injury induced by various
factors with the converse being very correct (Nosalova et al, 1991;
Farre et al, 1995).
These stimulatory effects of MeTp
on gastric mucus cells and gastric mucus secretion may be similar to that of
known drugs such as sucralfate and misoprostol (Slomiany et al, 1991,
Takahashi and Okabe, 1996). Percentage increase in gastric mucus has been
reported to be associated with graded doses of misoprostol in man (Wilson et
al, 1986). Misoprostol, by virtue of its ability to stimulate mucus
secretion, is an anti-ulcer agent in man (Poonam et al, 2003). Cellular
antioxidant enzymes such as superoxide dismutase, glutathione peroxide and
catalase normally challenge oxidative stress. In this study, MeTp significantly
increased the concentration of superoxide dismutase (an antioxidant enzyme) from
19.90±1.25 in the control to 49.40± 4.40 in high dose treated animals
(P<0.05). Increasing doses of MeTp (LD, MD and HD) significantly decreased
the level of malondialdehyde (MDA), a marker of lipid peroxidation (P<0.05).
The findings support other studies that demonstrated a reduction in lipid
peroxidation of the gastric mucosa shown to be associated with increased
activities of antioxidant enzymes (Melchiorri et al, 1997; Dela et al,
1999).
The mechanism of action of MeTp in ameliorating ulcer
might be due to increased gastric mucus secretion as a result of increased
number of gastric mucus cells through cell-proliferation, a mucogenic effect.
The extract raises the concentration of one of the primary endogenous enzymes;
superoxide dismutase which improves free radical scavenging property in the
stomach.
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© Physiological Society Of Nigeria, 2008.
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