Healing Effect of Alginate Liquid against HCl-Induced Gastric Mucosal Lesions in Rats 287 296V9N3PT
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International Journal of PharmTech Research
CODEN (USA): IJPRIF, ISSN: 0974-4304
Vol.9, No.3, pp 287-296,
2016
Healing Effect of Alginate Liquid against HCl-Induced Gastric
Mucosal Lesions in Rats
Anayanti Arianto and Hakim Bangun*
Faculty of Pharmacy, University of Sumatera Utara, Jl. Tri Dharma No. 5, Kampus
USU, Medan, Indonesia
Abstract: Background: Hyperacidity of stomach is a common global problem. The
pathophysiology of this disorder focuses on the imbalance between destructive and protective
factors of gastric mucosa.
Purpose: The purpose of this study was to determine the healing effect of gastric mucosal
lesions of alginate liquid compared to sucralfate suspension against HCl-induced in rats.
Methods: Sixty Wistar rats weight150-200 g were adapted under standard environmental
conditions for 2 weeks before used. Rats were fasted for 36 hours before the test, then the rats
were orally induced by 1 ml of 1 M HCl. Six animals were killed after 1 hour to confirm the
gastric mucosal lesions by macroscopic (number of lesions and lesion index) and microscopic
(histopathology) observation. The remaining, 54 rats were divided equally in three groups and
each group was divided in three subgroups according to time schedule of treatment (3, 7, and
10 days). First group without treatment (negative control), received 1 ml distilled water only
once a day. Second group (standard treatment group), received 1 ml sucralfate suspension once
a day. Third group (test treatment group), received 1 ml alginate liquid once a day.
Results: On macroscopic examination, the third group of rats (given alginate liquid) had no
gastric lesion and the lesion index was zero compared to second group (received sucralfate
suspension) which the number of gastric lesions was 1.67 and ulcer index was 0.01 after 7 days
treatment. Microscopic examination, the third group showed the intact mucosa compared to
second group after 7 days treatment.
Conclusion: The experimental results indicates that alginate liquid is faster in healing of
gastric mucosal lesions than with sucralfate suspension.
Keywords:Gastric lesions, healing, alginate liquid, sucralfate suspension.
Introduction
Peptic ulcer is a lesion that occurs in the mucosa and muscularis mucosa of the gastrointestinal tract.
Peptic ulcers are ulcers that occur frequently as gastritis and duodenal ulcer. Ulcers caused by an imbalance
between aggressive factors (hydrochloric acid, pepsin, Helycobacter pylory, NSAIDs, gastric acid) with
protective factors (antioxidant enzymatic, non-enzymatic antioxidants, blood flow, the regeneration of cells,
mucin, bicarbonate, prostaglandins), which eventually leads to mucosal damage. 1-2 Peptic ulcer is a common
disease that clinically occurs at any age. There are two types of peptic ulcer which are gastric ulcer, damage the
lining of the stomach, and duodenal ulcer, damage the duodenum. Gastric ulcer is one of the widespread
diseases.3 Major risk factors that cause gastric ulcer include bacterial infection (Helycobacter pylory), certain
drugs (NSAIDs), chemicals (HCl/ethanol), gastric cancer and low risk factors include the state of stress,
smoking, spicy foods and nutritional deficiencies[1]. Gastric acid production will increase in a state of stress,
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
288
such as the heavy workload, panic and haste. Increase stomach acid levels in the state of stress can irritate
gastric mucosa and if this is allowed, after a long time can cause gastritis.
The parietal cells of stomach secrete HCl. The basal acid output is about 1 mEq/hour in normal
subjects and 2 to 4 mEq/hour in duodenal ulcer patients.4 A strong emotional stimulate can improve the basal
acid secretion through the parasympathetic nerves (vagus) and thought to be one of the factors cause peptic
ulcers.2 Hydrochloric acid causes the change of pepsinogen into pepsin which will lower the barrier function
of the stomach. Gastric barrier function loss will result in the destruction of the capillary and venous blood so
that there will be bleeding. The high acid also triggers the release of histamine, causing vasodilatation which
increases bleeding.2
On average, patients with duodenal ulcers produce more acid than control subjects, particularly at night
(basal secretion). Although patients with gastric ulcers have normal or even diminished acid production, ulcers
rarely if ever occur in the complete absence of acid. Presumably, a weakened mucosal defense and reduced
bicarbonate production contribute to the injury from the relatively lower levels of acid in these patients. H.
pylori and exogenous agents such as nonsteroidal anti-inflammatory drugs (NSAIDs) interact in complex ways
to cause an ulcer.5
Currently, increasing the frequency of occurrence of gastric ulcer, development of effective treatment
becomes a challenge. Treatment with H2 receptor antagonist, proton pump inhibitors agents that enhance the
mucosal defence and sucralfate has become an alternative in the treatment of ulcers. Sucralfate is commonly
used as agent to enhance mucosal defence as gastroprotective agent. But, sucralfate has some side effects. The
most common side effect of sucralfate is constipation (about 2%). As some aluminum can be absorbed,
sucralfate should be avoided in patients with renal failure who are at risk for aluminum overload. Likewise,
aluminum-containing antacids should not be combined with sucralfate in these patients. Sucralfate forms a
viscous layer in the stomach that may inhibit absorption of other drugs, including phenytoin, digoxin,
cimetidine, ketoconazole, and fluoroquinolone antibiotics.5
Alginate is a polymer derived from brown algae (Phaeophyceae). Alginate is an anionic copolymer
consisting of residue β-D-mannuronic acid and α-L- guluronic in bond 1,4.6 Alginate has been known to to be
non-toxic, cause no allergies, biodegradable and biocompatible.7 Alginate can be used for the preparation of
gastroretentive and periodental drug delivery systems. 8-11 Beside, alginate can be utilized in various
biomedical applications, including wound healing, cartilage repair, bone and tissue regeneration. 12
Sodium alginate also has a protective effect to the stomach.13
Based on the protective effect of alginate to the stomach, we studied the usage of sodium alginate
for healing rats gastric lesions that was induced by excessive hydrochloric acid. The alginate was used in liquid
preparation.
Materials and Methods
Materials
Sodium alginate 300-400 cP (Wako Pure Chemical Industries, Ltd Japan), nipagin HCl, liquid paraffin,
xylol, xylene, hematosillin, and eosin were the product of Merck. Canada balsam (Entellan), sucralfate
suspension (Combiphar), and formaldehyde obtained from P.T. Rudang. Sucralfate suspension product of
Combiphar, Indonesia).
Animals
Sixty healthy male of Wistar rats weigh 150-200 g were maintained in standard animal house, given
standard pellet diets and tap water ad libitum. All rats were fasted from all medications at least 2 weeks before
treatment was given.
Preparation of liquid
A certain amount of alginate was dissolved in distilled water with the addition of preservative and
simplex syrup.
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289
Induction of gastric lesion by 1 M HCl solution and healing of gastric lesion using alginate liquid
All rats were fasted for 36 hours, then all of rats were induced by 1 ml of 1 M HCl solution to produce
gastric lesion. After 1 hour of induction, six rats were sacrificed by ether, then the stomach was opened and
washed with 0.9% NaCl solution, and the lesions were observed macroscopically and microscopically
(histopatology).
The remaining rats (54 rats) were divided into 3 groups, each group consisting of 6 rats. Group I:
Without treatment (given 1 ml distilled water instead of alginate liquid or sucralfate suspension); Group II:
Standard treatment group (given orally 1 ml sucralfate suspension every day); Group III: Test treatment group
(given orally 1 ml alginate liquid every day).
The group without treatment were given orally 1 ml distilled water instead of sucralfate suspension or
alginate liquid. Alginate liquid and sucralfate suspension were given once a day. The rats were given standard
pellet and tap water adlibitum.Then, the stomachs of rats were opened and washed with 0.9% NaCl solution.
Then, rats were sacrificed using ether inhalation on the third, seventh, and tenth days. The gastric mucosa were
observed macroscopically about the number of lesions, length and width of lesions. For microscopic
observation, the gastric mucosa was immersed in 10% formalin for histopathology processed with hematoxylin
Eosin staining and observed using a microscope.
Measurement of lesion index
Lesion index was determined based on method reported by Ganguly and Bhatnagar; total area of lesions
(mm2) divided by the area of gastric mucosa (mm2).14 The length and width of each lesion in mm were
measured by a calipers. The sum of the area of all lesions for each stomach was used in the calculation of the
total lesions area.
Results and Discussions
Macroscopic and microscopic observation of normal stomach of rats
A
Normal stomach
B
Normal gastric mucosa
C
Epithelial cell
Lamina
propria
Mucosa
Muscularis
mucosae
Sub mucosa
Histological of normal
gastric mucosa
Figure 1. Normal stomach of rats A : Normal stomach; B, Normal gastric mucosa; and C, histological of
normal gastric mucosa.
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290
The alimentary canal comprises of four concentric layers: mucosa, submucosa, muscularis externa, and
serosa. The mucosa of the fundic stomach is composed of the usual three components: (1) an epithelium lining
the lumen; (2) an underlying lining connective tissue, the lamina propria; and the smooth muscle layers forming
the muscalaris mucosae.15. Figure 1 shows a rat's normal stomach (A), gastric mucosa (B), and histological
of partially gastric mucosa (C).
Induction of gastric mucosa lesions
The initial condition of rats used on the study of healing effect of alginate liquid compared to sucralfate
suspension was considered the condition of rats after induction with 1 ml 1 M HCl solution. Gastric mucosa
has a special barrier against noxious agents, such aspirin, ethanol, bile, indomethacin, or hydrochloric acid. As
a barrier, sodium bicarbonate is secreted from the surface of epithelial cells and incorporated into mucus layer
might play role in the mucosal barrier against gastric acid and other noxious substances that are ingested. When
the barrier is broken by noxious agent the gastric mucosa then allows a back diffusion of gastric acid into the
mucosal cells, leading to mucosal damage.
The induction of fasted rats with 1M HCl solution caused swelling of the rats' stomach due to the
accumulation of gas (Figure 2A). This gas is thought come from the result of the reaction between hydrochloric
acid and sodium carbonate that is secreted from the surface of epithelial cells. Hydrochloric acid caused
damage of mucosal barrier. The damages of mucosal barrier cause the back diffusion of hydrochloric acid and
lead the destruction of the capillary and venous blood so that resulting stomach bleeding. High acid also
trigger the release of histamine causing vasodilation that increases bleeding. 2 Furthermore, hydrochloric acid
will change pepsinogen to pepsin which will lower the barrier function of the stomach. The effect of the
administration of 1 ml 1 M HCl solution is shown in Figure 2 (B and C).
Gas accumulation
in the stomach
Gastric mucosal
lesions
Gastric mucosa with
lesions
Swelled stomach
Submucosa
Muscularis mucosae
Erosion of
mucosa
Histological of mucosal erosion
Figure 2: Effect of 1 ml 1 M HCl solution on the rats' stomach
A: Swelled of stomach due to gas accumulation; B: Gastric mucosa with lesions, and C: Histological of
mucosal erosion
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291
Beside the swelling of stomach, the gastric mucosa lesions were formed after the induction with 1 ml 1
M of HCl solution. The average number of lesions and lesion index were 4.50 ± 2.02 and 0.61 ± 0.89,
respectively (Table 1).
Figure 2 (C) shows the histological section of rats stomach after induction with 1 ml 0.6 M HCl
solution. The microscopically observation showed that the administration of 1 ml 1M HCl solution caused the
erosion of rats' mucosa.
Macroscopic observation of rats stomach after three days treatment
Figure 3 shows the gastric mucosa of rats that received preparation for three days. There were still
lesions, either treated with alginate liquid or sucralfate suspension. But, the number of lesions were higher on
without treatment.
.
Lesions
Lesions
Lesions
Figure 3. Gastric mucosa of rat after 3 days treatment. A: Without treatment, B: Given sucralfate
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after three days treatment
In addition to macroscopic observation, it was also conducted the microscopic or histological
observations. Figure 4 shows hystological section of without treatment, given sucralfate suspension, and given
alginate liquid. All of rats showed erosion of mucosa, but erosion of mucosa was deeper in rats without
treatment.
Erosion of mucosa
Erosion of mucosa
Erosion of mucosa
Figure 4. Histological section of rats gastric mucosa (10x10) after 3 days treatment. A: Without
treatment; B: Given sucralfate suspension; C: Given alginate liquid.
Macroscopic observation of rats' stomach after 7 days treatment
Figure 5 shows the gastric mucosa of rats after 7 days treatment. Rats that were treated with alginate
liquid showed no lesion of stomach, but those were treated with sucralfate suspension still showed lesions of
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
292
stomach. In without treatment rats were found more number of lesions. Therefore, it can be concluded that the
administration of alginate liquid was faster in healing gastric lesions than sucralfate suspension.
Lesions
Lesions
Normal (no lesion)
Figure 5. Gastric mucosa of rats after 7 days treatment. A: Without treatment, B: Given sucralfate
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after 7 days treatment
Figure 6 shows the histological section of rats' stomach after 7 days treatment. The cohesion between
epithelial cells was damaged and the epithelial cells were eroded in the rats without treatment (Fig. 6A). Like
wise, rats that were treated with sucralfate suspension still showed the erosion of epithellial cells (Fig. 6B).
However, that rats treated with alginate liquid showed good cohesion between the epithellial cells, no erosion
of epithellial cells, and showed the intact mucosa after 7 days of treatment (Fig. 6C). From the result of
microscopic observation we can conclude that alginate liquid can heal gastric lesion after 7 days treatment.
A
Erosion of mucosa
B
C
Erosion of mucosa
Intact mucosa
Figure 6. Histological section of rats gastric mucosa (10x10) after 7 days treatment.
treatment ; B: Given sucralfate suspension, and C: Given alginate liquid
A: Without
Macroscopic observation of rats' stomach after 10 days treatment
The macroscopic observation of rats' stomach after 10 days treatment shows in Figure 7. After 10 days
treatment, it showed that there were lesions in stomach of untreated rats (Fig. 7A). However, there was no
lesion found in rats that were treated with sucralfate suspension (Fig. 7B), like rats treated with alginate liquid
(Fig. 7C). From the result of macroscopic observation, we can conclude that alginate liquid can heal gastric
ulcer after 7 days, while sucralfate suspension after 10 days treatment.
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293
Normal
Normal
L
Figure 7. Gastric mucosa of rats after 10 days treatment. A: Without treatment, B: Given sucralfate
Lesions
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after 10 days treatment
The histological section of rats gastric mucosa shows in Figure 8. There was still erosion on mucosa of
rats without treament (Fig. 8A). However, there was no erosion on mucosa in rats' gastric mucosa that were
treated with sucralfate suspension (Fig. 8B) like rats which were treated with alginate liquid (Fig. 8C). After 10
days treatment with sucralfate suspension.
A
B
Erosion of mucosa
C
Intact mucosa
Intact mucosa
Figure 8. Histological section of rats gastric mucosa (10x10) after 10 days treatment. A: Untreated, B:
Given sucralfate suspension, and C: Given alginate liquid
Comparison of healing effect of alginate liquid on gastric mucosal lesions
and lesion index
based on number of lesions
Number of lesions
Table 1 lists the average number of lesions before and after treatment with sucralfate suspension and
alginate liquid and the graph is shown in Figure 9. The initial average number of lesions was 4.50 ± 2.02. The
number of lesions decreased with the longer time of treatment. After 7 days treatment, there was no lesion
observed in the rats that were treated with alginate liquid. However, in the rats that were treated with sucralfate
suspension, there were still lesions in the stomaches. The rats that were treated with sucralfate suspension was
found no lesion after 10 days treatment. The rats without treatment haven't healed after 10 days, it was
observed as many as 2.50 ± 2.90 lesions in the stomach.
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294
Table 1. Number of gastric mucosa lesions of rats in each groups after certain days of treatment.
Day
0
Mean number of lesions (n = 6)
(X ± SD)
Standard treatment group Test treatment group
Without treatment
(Given sucralfate
(Given alginate
suspension)
liquid)
4.50 ± 2.02
4.50 ± 2.02
4.50 ± 2.02
3
3.50 ± 3.56
1.00 ± 2.10
1.17 ± 1.92
7
3.17 ± 2.84
1.17 ± 1.24
0 ± 0.00
10
2.50 ± 2.90
0 ± 0.00
0 ± 0.00
Figure 9. The comparison of the healing effect between alginate liquid, sucralfate suspension, and
without treatment based on number of lesions (n=6).
Lesion index
The value of lesion index without and after the treatment using sucralfate suspension and alginate liquid
is listed in Table 2 and the graph is shown in Figure 10. The lesion index is the ratio of the total area of lesions
and total area of mucosa. After the treatment, the value of lesion index decreased. The decrease of lesion index
was faster with the treatment using alginate liquid, where the lesion index became zero after 7 days of
treatment. But, the treatment with sucralfate suspension, the lesion index became zero after 10 days, while
without treatment the ulcer index rats was 0.02 ± 0.02 after 10 days.
In healing experiment, the doses of alginate liquid and sucralfate suspension once a day each 1 ml.
But, the concentration of alginate was lower than the concentration of sucralfate. The concentration of sodium
alginate in alginate liquid was 10 mg/ml, while the concentration of sucralfate in sucralfate suspension was 100
mg/ml. Sucralfate has mucoprotective effect, in an acid environment (pH < 4) sucralfate undergoes extensive
cross-linking to produce a viscous, sticky polymer that adheres to epithelial cells and ulcer craters for up to 6
hours after a single dose. This provides physical protection against irritant substances such as excessive
stomach acid.5 In the stomach, in the presence gastric acid, sodium alginate was changed to alginic acid gel.
This alginic acid gel has a mucoadhesive properties. 8-9 Therefore, alginic acid adhered to the surface of gastric
mucosa and acted as a mucoprotective agent by providing a physical barrier of hydrochloric acid and pepsin
contact to the gastric mucosa, thereby improve the healing effect of lesion or ulcer. The healing of lesions was
faster by alginate liquid than sucralfate was thought to be due to alginate increased the hexosamine levels,
which are glycoprotein constituting of gastric mucus, 16 in addition of protective effect of alginate.
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295
Table 2. Mean lesion index of each group
Day
Without treatment
0
3
7
10
0.61 ± 0.89
0.07 ± 0.07
0.05 ± 0.10
0.02 ± 0.02
Mean lesion index (n = 6)
(X ± SD)
Standard group
(Given sucralfate
suspension)
0.61 ± 0.89
0.01 ± 0.02
0.01 ± 0.01
0 ± 0.00
Test treatment group
(Given alginate
liquid)
0.61 ± 0.89
0.01 ± 0.01
0 ± 0.00
0 ± 0.00
Figure 10. The comparison of healing effect between alginate liquid, sucralfate suspension, and without
treatment based on value of lesion index (n=6).
Conclusion
Based on the results, it can be concluded that alginate liquid is potential to be used for healing gastric
lesions or ulcers.
Acknowledgment
This research was supported by Directorate General of Higher Education of Indonesia through the
schme of competitive research (Hibah bersaing) 2014.
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mucoadhesive, and release of ranitidine from spherical matrices of alginate-chitosan, Journal of
PharmTech Research, 2015, 8(4), 653-665.
Mariadi, Bangun H., and Karsono, Formulation and in vitro evaluation of gastroretentive drug delivery
system of antacids using alginate-chitosan films, International Journal of PharmTech Research, 2015,
8(9), 1-12.
Aryana, Sinurat D., Ervina I., and Bangun H., Formulation of alginate based metronidazole periodental
gel, Asian Journal of Pharmaceutical and Clinical research, 2014, 7 (1), 224-227.
Sun J and Huaping T., Alginate-based biomaterial for regenerative medicine applications, Journal
Materials, 2013, 6, 1285-1309.
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studies of sodium aginate. I. Protective effects of sodium alginate on mucous membranes of uppergastrointestinaal tract, Yakugaku Zasshi, 1981, 101(5), 452-457.
Ganguly A.K. and Bhatnagar O.P., Effect of bilateral adrenalotomy on production of restraint ulcers in
stomach of albino rats. Canadian Journal of Physiology and Pharmacology, 1973, 51, 748-750.
Gartner L. P. and Hiatt J. L., Color Texbook Histology, Third Edition, Phyladelpia: Saunders Elsevier,
2007, p. 381-403.
Daigo K., Yamaji M., Yamada C., Matsutani K., Yagi M., Nakajima Y., Curative effect of sodium
alginate on experimental hydrochloric acid and tetra-gastrin induced ulcer, Japanese Pharmacol Ther.,
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International Journal of PharmTech Research
CODEN (USA): IJPRIF, ISSN: 0974-4304
Vol.9, No.3, pp 287-296,
2016
Healing Effect of Alginate Liquid against HCl-Induced Gastric
Mucosal Lesions in Rats
Anayanti Arianto and Hakim Bangun*
Faculty of Pharmacy, University of Sumatera Utara, Jl. Tri Dharma No. 5, Kampus
USU, Medan, Indonesia
Abstract: Background: Hyperacidity of stomach is a common global problem. The
pathophysiology of this disorder focuses on the imbalance between destructive and protective
factors of gastric mucosa.
Purpose: The purpose of this study was to determine the healing effect of gastric mucosal
lesions of alginate liquid compared to sucralfate suspension against HCl-induced in rats.
Methods: Sixty Wistar rats weight150-200 g were adapted under standard environmental
conditions for 2 weeks before used. Rats were fasted for 36 hours before the test, then the rats
were orally induced by 1 ml of 1 M HCl. Six animals were killed after 1 hour to confirm the
gastric mucosal lesions by macroscopic (number of lesions and lesion index) and microscopic
(histopathology) observation. The remaining, 54 rats were divided equally in three groups and
each group was divided in three subgroups according to time schedule of treatment (3, 7, and
10 days). First group without treatment (negative control), received 1 ml distilled water only
once a day. Second group (standard treatment group), received 1 ml sucralfate suspension once
a day. Third group (test treatment group), received 1 ml alginate liquid once a day.
Results: On macroscopic examination, the third group of rats (given alginate liquid) had no
gastric lesion and the lesion index was zero compared to second group (received sucralfate
suspension) which the number of gastric lesions was 1.67 and ulcer index was 0.01 after 7 days
treatment. Microscopic examination, the third group showed the intact mucosa compared to
second group after 7 days treatment.
Conclusion: The experimental results indicates that alginate liquid is faster in healing of
gastric mucosal lesions than with sucralfate suspension.
Keywords:Gastric lesions, healing, alginate liquid, sucralfate suspension.
Introduction
Peptic ulcer is a lesion that occurs in the mucosa and muscularis mucosa of the gastrointestinal tract.
Peptic ulcers are ulcers that occur frequently as gastritis and duodenal ulcer. Ulcers caused by an imbalance
between aggressive factors (hydrochloric acid, pepsin, Helycobacter pylory, NSAIDs, gastric acid) with
protective factors (antioxidant enzymatic, non-enzymatic antioxidants, blood flow, the regeneration of cells,
mucin, bicarbonate, prostaglandins), which eventually leads to mucosal damage. 1-2 Peptic ulcer is a common
disease that clinically occurs at any age. There are two types of peptic ulcer which are gastric ulcer, damage the
lining of the stomach, and duodenal ulcer, damage the duodenum. Gastric ulcer is one of the widespread
diseases.3 Major risk factors that cause gastric ulcer include bacterial infection (Helycobacter pylory), certain
drugs (NSAIDs), chemicals (HCl/ethanol), gastric cancer and low risk factors include the state of stress,
smoking, spicy foods and nutritional deficiencies[1]. Gastric acid production will increase in a state of stress,
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
288
such as the heavy workload, panic and haste. Increase stomach acid levels in the state of stress can irritate
gastric mucosa and if this is allowed, after a long time can cause gastritis.
The parietal cells of stomach secrete HCl. The basal acid output is about 1 mEq/hour in normal
subjects and 2 to 4 mEq/hour in duodenal ulcer patients.4 A strong emotional stimulate can improve the basal
acid secretion through the parasympathetic nerves (vagus) and thought to be one of the factors cause peptic
ulcers.2 Hydrochloric acid causes the change of pepsinogen into pepsin which will lower the barrier function
of the stomach. Gastric barrier function loss will result in the destruction of the capillary and venous blood so
that there will be bleeding. The high acid also triggers the release of histamine, causing vasodilatation which
increases bleeding.2
On average, patients with duodenal ulcers produce more acid than control subjects, particularly at night
(basal secretion). Although patients with gastric ulcers have normal or even diminished acid production, ulcers
rarely if ever occur in the complete absence of acid. Presumably, a weakened mucosal defense and reduced
bicarbonate production contribute to the injury from the relatively lower levels of acid in these patients. H.
pylori and exogenous agents such as nonsteroidal anti-inflammatory drugs (NSAIDs) interact in complex ways
to cause an ulcer.5
Currently, increasing the frequency of occurrence of gastric ulcer, development of effective treatment
becomes a challenge. Treatment with H2 receptor antagonist, proton pump inhibitors agents that enhance the
mucosal defence and sucralfate has become an alternative in the treatment of ulcers. Sucralfate is commonly
used as agent to enhance mucosal defence as gastroprotective agent. But, sucralfate has some side effects. The
most common side effect of sucralfate is constipation (about 2%). As some aluminum can be absorbed,
sucralfate should be avoided in patients with renal failure who are at risk for aluminum overload. Likewise,
aluminum-containing antacids should not be combined with sucralfate in these patients. Sucralfate forms a
viscous layer in the stomach that may inhibit absorption of other drugs, including phenytoin, digoxin,
cimetidine, ketoconazole, and fluoroquinolone antibiotics.5
Alginate is a polymer derived from brown algae (Phaeophyceae). Alginate is an anionic copolymer
consisting of residue β-D-mannuronic acid and α-L- guluronic in bond 1,4.6 Alginate has been known to to be
non-toxic, cause no allergies, biodegradable and biocompatible.7 Alginate can be used for the preparation of
gastroretentive and periodental drug delivery systems. 8-11 Beside, alginate can be utilized in various
biomedical applications, including wound healing, cartilage repair, bone and tissue regeneration. 12
Sodium alginate also has a protective effect to the stomach.13
Based on the protective effect of alginate to the stomach, we studied the usage of sodium alginate
for healing rats gastric lesions that was induced by excessive hydrochloric acid. The alginate was used in liquid
preparation.
Materials and Methods
Materials
Sodium alginate 300-400 cP (Wako Pure Chemical Industries, Ltd Japan), nipagin HCl, liquid paraffin,
xylol, xylene, hematosillin, and eosin were the product of Merck. Canada balsam (Entellan), sucralfate
suspension (Combiphar), and formaldehyde obtained from P.T. Rudang. Sucralfate suspension product of
Combiphar, Indonesia).
Animals
Sixty healthy male of Wistar rats weigh 150-200 g were maintained in standard animal house, given
standard pellet diets and tap water ad libitum. All rats were fasted from all medications at least 2 weeks before
treatment was given.
Preparation of liquid
A certain amount of alginate was dissolved in distilled water with the addition of preservative and
simplex syrup.
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
289
Induction of gastric lesion by 1 M HCl solution and healing of gastric lesion using alginate liquid
All rats were fasted for 36 hours, then all of rats were induced by 1 ml of 1 M HCl solution to produce
gastric lesion. After 1 hour of induction, six rats were sacrificed by ether, then the stomach was opened and
washed with 0.9% NaCl solution, and the lesions were observed macroscopically and microscopically
(histopatology).
The remaining rats (54 rats) were divided into 3 groups, each group consisting of 6 rats. Group I:
Without treatment (given 1 ml distilled water instead of alginate liquid or sucralfate suspension); Group II:
Standard treatment group (given orally 1 ml sucralfate suspension every day); Group III: Test treatment group
(given orally 1 ml alginate liquid every day).
The group without treatment were given orally 1 ml distilled water instead of sucralfate suspension or
alginate liquid. Alginate liquid and sucralfate suspension were given once a day. The rats were given standard
pellet and tap water adlibitum.Then, the stomachs of rats were opened and washed with 0.9% NaCl solution.
Then, rats were sacrificed using ether inhalation on the third, seventh, and tenth days. The gastric mucosa were
observed macroscopically about the number of lesions, length and width of lesions. For microscopic
observation, the gastric mucosa was immersed in 10% formalin for histopathology processed with hematoxylin
Eosin staining and observed using a microscope.
Measurement of lesion index
Lesion index was determined based on method reported by Ganguly and Bhatnagar; total area of lesions
(mm2) divided by the area of gastric mucosa (mm2).14 The length and width of each lesion in mm were
measured by a calipers. The sum of the area of all lesions for each stomach was used in the calculation of the
total lesions area.
Results and Discussions
Macroscopic and microscopic observation of normal stomach of rats
A
Normal stomach
B
Normal gastric mucosa
C
Epithelial cell
Lamina
propria
Mucosa
Muscularis
mucosae
Sub mucosa
Histological of normal
gastric mucosa
Figure 1. Normal stomach of rats A : Normal stomach; B, Normal gastric mucosa; and C, histological of
normal gastric mucosa.
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
290
The alimentary canal comprises of four concentric layers: mucosa, submucosa, muscularis externa, and
serosa. The mucosa of the fundic stomach is composed of the usual three components: (1) an epithelium lining
the lumen; (2) an underlying lining connective tissue, the lamina propria; and the smooth muscle layers forming
the muscalaris mucosae.15. Figure 1 shows a rat's normal stomach (A), gastric mucosa (B), and histological
of partially gastric mucosa (C).
Induction of gastric mucosa lesions
The initial condition of rats used on the study of healing effect of alginate liquid compared to sucralfate
suspension was considered the condition of rats after induction with 1 ml 1 M HCl solution. Gastric mucosa
has a special barrier against noxious agents, such aspirin, ethanol, bile, indomethacin, or hydrochloric acid. As
a barrier, sodium bicarbonate is secreted from the surface of epithelial cells and incorporated into mucus layer
might play role in the mucosal barrier against gastric acid and other noxious substances that are ingested. When
the barrier is broken by noxious agent the gastric mucosa then allows a back diffusion of gastric acid into the
mucosal cells, leading to mucosal damage.
The induction of fasted rats with 1M HCl solution caused swelling of the rats' stomach due to the
accumulation of gas (Figure 2A). This gas is thought come from the result of the reaction between hydrochloric
acid and sodium carbonate that is secreted from the surface of epithelial cells. Hydrochloric acid caused
damage of mucosal barrier. The damages of mucosal barrier cause the back diffusion of hydrochloric acid and
lead the destruction of the capillary and venous blood so that resulting stomach bleeding. High acid also
trigger the release of histamine causing vasodilation that increases bleeding. 2 Furthermore, hydrochloric acid
will change pepsinogen to pepsin which will lower the barrier function of the stomach. The effect of the
administration of 1 ml 1 M HCl solution is shown in Figure 2 (B and C).
Gas accumulation
in the stomach
Gastric mucosal
lesions
Gastric mucosa with
lesions
Swelled stomach
Submucosa
Muscularis mucosae
Erosion of
mucosa
Histological of mucosal erosion
Figure 2: Effect of 1 ml 1 M HCl solution on the rats' stomach
A: Swelled of stomach due to gas accumulation; B: Gastric mucosa with lesions, and C: Histological of
mucosal erosion
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
291
Beside the swelling of stomach, the gastric mucosa lesions were formed after the induction with 1 ml 1
M of HCl solution. The average number of lesions and lesion index were 4.50 ± 2.02 and 0.61 ± 0.89,
respectively (Table 1).
Figure 2 (C) shows the histological section of rats stomach after induction with 1 ml 0.6 M HCl
solution. The microscopically observation showed that the administration of 1 ml 1M HCl solution caused the
erosion of rats' mucosa.
Macroscopic observation of rats stomach after three days treatment
Figure 3 shows the gastric mucosa of rats that received preparation for three days. There were still
lesions, either treated with alginate liquid or sucralfate suspension. But, the number of lesions were higher on
without treatment.
.
Lesions
Lesions
Lesions
Figure 3. Gastric mucosa of rat after 3 days treatment. A: Without treatment, B: Given sucralfate
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after three days treatment
In addition to macroscopic observation, it was also conducted the microscopic or histological
observations. Figure 4 shows hystological section of without treatment, given sucralfate suspension, and given
alginate liquid. All of rats showed erosion of mucosa, but erosion of mucosa was deeper in rats without
treatment.
Erosion of mucosa
Erosion of mucosa
Erosion of mucosa
Figure 4. Histological section of rats gastric mucosa (10x10) after 3 days treatment. A: Without
treatment; B: Given sucralfate suspension; C: Given alginate liquid.
Macroscopic observation of rats' stomach after 7 days treatment
Figure 5 shows the gastric mucosa of rats after 7 days treatment. Rats that were treated with alginate
liquid showed no lesion of stomach, but those were treated with sucralfate suspension still showed lesions of
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
292
stomach. In without treatment rats were found more number of lesions. Therefore, it can be concluded that the
administration of alginate liquid was faster in healing gastric lesions than sucralfate suspension.
Lesions
Lesions
Normal (no lesion)
Figure 5. Gastric mucosa of rats after 7 days treatment. A: Without treatment, B: Given sucralfate
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after 7 days treatment
Figure 6 shows the histological section of rats' stomach after 7 days treatment. The cohesion between
epithelial cells was damaged and the epithelial cells were eroded in the rats without treatment (Fig. 6A). Like
wise, rats that were treated with sucralfate suspension still showed the erosion of epithellial cells (Fig. 6B).
However, that rats treated with alginate liquid showed good cohesion between the epithellial cells, no erosion
of epithellial cells, and showed the intact mucosa after 7 days of treatment (Fig. 6C). From the result of
microscopic observation we can conclude that alginate liquid can heal gastric lesion after 7 days treatment.
A
Erosion of mucosa
B
C
Erosion of mucosa
Intact mucosa
Figure 6. Histological section of rats gastric mucosa (10x10) after 7 days treatment.
treatment ; B: Given sucralfate suspension, and C: Given alginate liquid
A: Without
Macroscopic observation of rats' stomach after 10 days treatment
The macroscopic observation of rats' stomach after 10 days treatment shows in Figure 7. After 10 days
treatment, it showed that there were lesions in stomach of untreated rats (Fig. 7A). However, there was no
lesion found in rats that were treated with sucralfate suspension (Fig. 7B), like rats treated with alginate liquid
(Fig. 7C). From the result of macroscopic observation, we can conclude that alginate liquid can heal gastric
ulcer after 7 days, while sucralfate suspension after 10 days treatment.
Hakim Bangun et al / International Journal of PharmTech Research, 2016,9(3),pp 287-296.
293
Normal
Normal
L
Figure 7. Gastric mucosa of rats after 10 days treatment. A: Without treatment, B: Given sucralfate
Lesions
suspension, and C: Given alginate liquid
Microscopic observation of rats' stomach after 10 days treatment
The histological section of rats gastric mucosa shows in Figure 8. There was still erosion on mucosa of
rats without treament (Fig. 8A). However, there was no erosion on mucosa in rats' gastric mucosa that were
treated with sucralfate suspension (Fig. 8B) like rats which were treated with alginate liquid (Fig. 8C). After 10
days treatment with sucralfate suspension.
A
B
Erosion of mucosa
C
Intact mucosa
Intact mucosa
Figure 8. Histological section of rats gastric mucosa (10x10) after 10 days treatment. A: Untreated, B:
Given sucralfate suspension, and C: Given alginate liquid
Comparison of healing effect of alginate liquid on gastric mucosal lesions
and lesion index
based on number of lesions
Number of lesions
Table 1 lists the average number of lesions before and after treatment with sucralfate suspension and
alginate liquid and the graph is shown in Figure 9. The initial average number of lesions was 4.50 ± 2.02. The
number of lesions decreased with the longer time of treatment. After 7 days treatment, there was no lesion
observed in the rats that were treated with alginate liquid. However, in the rats that were treated with sucralfate
suspension, there were still lesions in the stomaches. The rats that were treated with sucralfate suspension was
found no lesion after 10 days treatment. The rats without treatment haven't healed after 10 days, it was
observed as many as 2.50 ± 2.90 lesions in the stomach.
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294
Table 1. Number of gastric mucosa lesions of rats in each groups after certain days of treatment.
Day
0
Mean number of lesions (n = 6)
(X ± SD)
Standard treatment group Test treatment group
Without treatment
(Given sucralfate
(Given alginate
suspension)
liquid)
4.50 ± 2.02
4.50 ± 2.02
4.50 ± 2.02
3
3.50 ± 3.56
1.00 ± 2.10
1.17 ± 1.92
7
3.17 ± 2.84
1.17 ± 1.24
0 ± 0.00
10
2.50 ± 2.90
0 ± 0.00
0 ± 0.00
Figure 9. The comparison of the healing effect between alginate liquid, sucralfate suspension, and
without treatment based on number of lesions (n=6).
Lesion index
The value of lesion index without and after the treatment using sucralfate suspension and alginate liquid
is listed in Table 2 and the graph is shown in Figure 10. The lesion index is the ratio of the total area of lesions
and total area of mucosa. After the treatment, the value of lesion index decreased. The decrease of lesion index
was faster with the treatment using alginate liquid, where the lesion index became zero after 7 days of
treatment. But, the treatment with sucralfate suspension, the lesion index became zero after 10 days, while
without treatment the ulcer index rats was 0.02 ± 0.02 after 10 days.
In healing experiment, the doses of alginate liquid and sucralfate suspension once a day each 1 ml.
But, the concentration of alginate was lower than the concentration of sucralfate. The concentration of sodium
alginate in alginate liquid was 10 mg/ml, while the concentration of sucralfate in sucralfate suspension was 100
mg/ml. Sucralfate has mucoprotective effect, in an acid environment (pH < 4) sucralfate undergoes extensive
cross-linking to produce a viscous, sticky polymer that adheres to epithelial cells and ulcer craters for up to 6
hours after a single dose. This provides physical protection against irritant substances such as excessive
stomach acid.5 In the stomach, in the presence gastric acid, sodium alginate was changed to alginic acid gel.
This alginic acid gel has a mucoadhesive properties. 8-9 Therefore, alginic acid adhered to the surface of gastric
mucosa and acted as a mucoprotective agent by providing a physical barrier of hydrochloric acid and pepsin
contact to the gastric mucosa, thereby improve the healing effect of lesion or ulcer. The healing of lesions was
faster by alginate liquid than sucralfate was thought to be due to alginate increased the hexosamine levels,
which are glycoprotein constituting of gastric mucus, 16 in addition of protective effect of alginate.
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295
Table 2. Mean lesion index of each group
Day
Without treatment
0
3
7
10
0.61 ± 0.89
0.07 ± 0.07
0.05 ± 0.10
0.02 ± 0.02
Mean lesion index (n = 6)
(X ± SD)
Standard group
(Given sucralfate
suspension)
0.61 ± 0.89
0.01 ± 0.02
0.01 ± 0.01
0 ± 0.00
Test treatment group
(Given alginate
liquid)
0.61 ± 0.89
0.01 ± 0.01
0 ± 0.00
0 ± 0.00
Figure 10. The comparison of healing effect between alginate liquid, sucralfate suspension, and without
treatment based on value of lesion index (n=6).
Conclusion
Based on the results, it can be concluded that alginate liquid is potential to be used for healing gastric
lesions or ulcers.
Acknowledgment
This research was supported by Directorate General of Higher Education of Indonesia through the
schme of competitive research (Hibah bersaing) 2014.
References
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