Gastro Protective and Antioxidant Activity of Solanum nigrum Linn. against Aspirin and Cold Restraint Stress induced Ulcerated Rats
Present study investigated gastroprotective and antioxidant activity of Solanum nigrum Linn. using aspirin and cold resistant stress induced ulcer rats. The collected plant leaves extracted with methanol by Soxhlet method for further analysis. Test animals were acclimatized in laboratory conditions and divided into six groups (Positive control (aspirin treated), negative control (normal), S. nigrum Extract (SNE) administered and aspirin, Cold Restraint Stress (CRS), S. nigrum with cold restraint stress and famotidine (standard drug) administered). The test animals were treated upto 20 days, after treatment gastric juice obtained from stomach used for biochemical and enzymatic analysis. The standard methodologies were adopted to found the parameters such as level of protein, carbohydrate, ulcer index, % of inhibition, ulcer severity, DPPH scavenging activity and reducing sugar etc. The gastric walls of all groups were used for histopathological studies. From the findings the groups that were treated to aspirin and CRS produced less gastric mucus secretion in gastric juice (101.39 and 91.67 mg g-1, respectively). The groups pretreated with S. nigrum extract exhibited a percentage inhibition of about 77.85% in aspirin and 66.67% in CRS-treated, groups, respectively. However, in groups that were pretreated with famotidine the percentage inhibition was 88.91%. Higher percentage of inhibition (88.91%) observed in groups pretreated with Famotidine. The reduction capability of DPPH radicals was determined from the decreasing absorbance values at 517 nm with increasing concentration, which is induced by anti oxidants present in the extracts. The plant extracts remarkably change all the parameters screening in test animals. Biochemical, antioxidants, histological and enzymological results also show the gastro protective and antioxidant efficiency of test plant. In nutshell the plant methanolic extracts have potential drug against gastro protective and Ulcer.
to cite this article:
S. Saravanan, P. Dhasarathan, V. Indira and R. Venkatraman, 2011. Gastro Protective and Antioxidant Activity of Solanum nigrum Linn. against Aspirin and Cold Restraint Stress induced Ulcerated Rats. Research Journal of Immunology, 4: 1-11.
Gastric ulcer disease is a chronic inflammation, raw eroded area in the lining
of the stomach, where parietal cells are found that secrete hydrochloric acid
and pepsin. The anatomic sites where ulcers, commonly found are stomach and
duodenum, causing gastric and duodenal ulcer, respectively (Kanner
and Lapidol, 2001). Pathophysiology of ulcer is due to an imbalance between
aggressive factors (acid, pepsin, Helicobacter pylori and NSAIDs) and
local mucosal defensive factors (mucin bicarbonate, blood flow and prostagladins).
Integrity of gastric mucosa is maintained through a homeostatic balance between
these factors (Hoogerwerf and Pasricha, 2001). One major
cause of gastric ulceration in human and experimental animals is the chronic
use of NSAIDs in the treatment of mild to moderate pain.
Aspirin is one of such NSAIDs and its main undesirable side effect is that it will induce gastrointestinal ulcers, stomach bleeding and tinnitus, especially in higher dosage. Several drugs are widely used to prevent or treat gastric ulcer, which include H2 receptor antagonists such as Cimetidine, Famotidine, Ranitidine and proton-pump inhibitors such as Sucralfate, Tetracycline and Pepto-Bismol. Due to problems associated with recurrence after treatment, there is the need to seek alternative drug sources against gastric ulcers.
Traditional systems of medicine continue to be widely practiced on many accounts.
Population rise, inadequate supply of drugs, prohibitive cost of treatments,
side effects of several allopathic drugs and development of resistance to currently
used drugs for infectious diseases have led to increased emphasis on the use
of plant materials as a source of medicines for a wide variety of human ailments
(Arrieta et al., 2001; Manjula
et al., 2009). Global estimates indicate that 80% of about 4 billion
population cannot afford the products of the Western Pharmaceutical Industry
and have to rely upon the use of traditional medicines, which are mainly derived
from plant material (Brindha et al., 2008). There
are scanty reports on its antibacterial activities of chosen test plant. In
order to demonstrate the antibacterial efficacy, test were conducted against
human pathogenic bacteria including those responsible for causing inflammation.
Solanum nigrum is an herbal plant which is distributed as a weed throughout
dry parts of India. It has the properties such as emollient, diuretic and laxative
(Al-Daihan, 2008). The leaves are used for the treatment
of diabetes, heart problems and jaundice. This study was therefore undertaken
to analyze gastro-protective effect and in vitro antioxidant activity
of S. nigrum and its effect on acid-pepsin secretion mucus content, acidity
in aspirin-induced ulcerogenesis and Cold Restraint Stress (CRS) induced ulcerated
Plants also need to protect themselves from free radical damages with the help of their own metabolites, so they develop a number of different classes of antioxidants. In this study, those antioxidant activities of S. nigrum Linn. extracts were analyzed by various methods such as reducing power determination.
MATERIALS AND METHODS
Plant sample: In the present study, Solanum nigrum Linn. were selected as the plant sample and collected at Sri Paramakalyani Medicinal garden, Alwarkurichi (Tirunelveli district, Tamilnadu, India) in early morning during second week of January, 2010. The collected samples were immediately transported to the Chemistry Laboratory, Alwarkurichi for plant extraction and phytochemical screening. Animal study was carried out in Biotechnology Laboratory, Prathyusha Institute of Technology and Management, Tiruvallur using plant extracts. The leaves of S. nigrum were dried and coarsely powdered. It was then extracted using methanol as solvent in Soxhlet apparatus. The extract obtained was then filtered through Whatman filter paper No. 3 and was then evaporated at 40-50°C with reduced pressure. Dosage of about 500 mg kg-1 b.wt. S. nigrum extract was given to the rats of group III and group V animals. Dose was administered orally everyday for 20 consecutive days.
Animals: Albino wistar rats weighed about 140-150 g were used in the present investigation. The rats were fed with standard laboratory rat pellet feed (Lipton Ltd., Mumbai) and water provided to ad libitum. After acclimatization, the rats are divided into six groups (Each groups contains six animals) used for analysis.
||Group I: Serves as negative control, which was provided
with normal feed with ad libitum water
||Group II: Serves as positive control, which was fed with normal
feed and on the last day, aspirin (200 mg kg-1 b.wt.) dissolved
in 1% CMC was administered orally
||Group III: Pretreated orally with S. nigrum Linn. Extract
(500 mg kg-1 b.wt.) for consecutively 20 days and on the last
day aspirin (200 mg kg-1 b.wt.) dissolved in 1% CMC was administered
||Group IV: Provided with normal feed and on the last day, the animals
were fasted for 6 h and then subjected to Cold Restraint Stress for 2 h
||Group V: Pretreated orally with S. nigrum extract (500
mg kg-1 b.wt.) for consecutively 20 days and on the final day,
the animals were fasted for 6 h and then subjected to Cold Restraint Stress
for 2 h
||Group VI: Pretreated orally with famotidine for 20 consecutive
days and on the final day, aspirin (200 mg kg-1 b.wt.) dissolved
in 1% CMC was administered orally
By the next day, animals of all the groups were fasted and were sacrificed.
The stomach was cut along the greater curvature and the contents were collected
and centrifuged at 3000 rpm for 10 minutes and the supernatant (gastric juice)
was used for biochemical and enzymatic analysis. The gastric walls of all groups
were used for histopathological studies, like Ulcer index determination, percentage
inhibition and Non-Protein Sulphydryl Group (NPSH) estimations.
The biochemical estimations of total proteins, total carbohydrates, gastric
mucus determination (Corney et al., 1974), non-protein
sulphydryl groups (Sedlak and Lindsay, 1968), total
acidity (Cannon, 1969), volume and pH (Parmer
and Desai, 1993), DPPH radical scavenging activity (0.0025 g of 2, 2-Diphenyl-1-picryl
hydrazyl (DPPH) was dissolved in 25 mL of methanol. The content should be made
and kept in dark condition, because DPPH is light sensitive and reducing sugar
were analyzed by standard methods, ascorbic acid used as a standard for both
The phytochemicals present in the extract was analyzed by Sofowara
(1993), Ulcer index, percentage inhibition (Kaukmann
and Grosmann, 1978) and ulcer severity (Varley et al.,
1991) were carried out to screen the efficiency.
The enzymatic analysis was performed by using hemoglobin method and the enzymes
analyzed include pepsin, trypsin and Cathepsin (Anson, 1938).
Histological studies: The gastric wall cut along the greater curvature from each group of rats was fixed in 10% formalin for 24 h. The formalin fixed specimen was embedded in paraffin, sectioned and stained with hematoxylin and eosin. The histochemical sections were evaluated by light microscopy.
The present study suggests that pretreatment with S. nigrum extract
ameliorated the ulcer index, histological and biochemical changes induced by
aspirin and cold restraint stress gastric ulceration in rats. The level of proteins
and carbohydrates were found in the treated animals were 361.05 and 195.71 mg
dL-1 in group I, 342.63 and 90.05 mg dL-1 in group II,
345.79 and 123.57 mg dL-1 in group III, 340.78 and 104.29 mg dL-1
in group IV, 347.63 and 138.57 mg dL-1 in group V, 354.21 and 167.86
mg dL-1 in group VI (Table 1). It is proved that
there is a little effect in the amount of proteins and carbohydrates during
|| Effect of Solanum nigrum Linn extract on gastric mucosal
factors in experimental gastric ulcer model
|-: Nil value, +: Less severity of ulcer, +++: Moderate severity
of ulcer, ++++: High severity of ulcer
Protein malnutrition results when the bodys needs for protein cannot
be satisfied by the diet. Since all normal metabolic processes require proteins;
all tissues will be affected by a state of protein deprivation. This protein
deprivation due to stress and adequate usage of NSAIDs was overcome by an increase
in protein level in the groups that were pretreated with S. nigrum Linn.
The groups that were treated to induce ulcer produced less gastric mucus secretion in gastric juice, whereas the groups that were pretreated with plant extract and antiulcer drug showed an increase in gastric mucus secretion. The test animal secretion of gastric mucus was reduced in positive control aspirin treated group have 101.39 mg g-1 tissue and cold resistant stress exposed group show 91.67 mg g-1 compared with negative control group (136.11 mg g-1 tissue) and Famotidine treated group (133.33 mg g-1 tissue) (Table 1). Ulcer induced however, plant extract treated groups show moderate changes in between positive and negative control animal groups (116.67 and 113.89 mg g-1 tissue of gastric mucus obtained in group III and group V, respectively). It reported that the gastric mucosa consists of mucin-type glycoprotein, which can be detected by the amounts of Alcian blue. Mucin is a viscous glycoprotein, with physicochemical properties producing relatively resistant acid barrier. In pretreated groups, there is an increase in gastric mucosal secretion indicating the anti secretary effect of S. nigrum Linn. extract and famotidine.
There is a decrease in the concentration of NPSH group in pretreated groups such as group III (315 nM g-1), group V (307 nM g-1) and group VI (438 nM g-1) than that in ulcerated groups II (158 nM g-1) and group IV (132 nM g-1), when compared with normal animal (427 nM g-1) (Table 1). Gastric mucosal NPSH group was measured to analyze the oxidant/antioxidant balance. The basal gastric secretion and titratable acidity were suppressed in pretreated groups than the ulcerated group (Table 1). Therefore, it appears this extract is more effective and possesses cyto secretory capacity. Suppression in the total acidity and the pH of the gastric juice of pretreated groups was higher than the ulcerated group that was reported. The normal animal (Untreated) gastric juice total acidity was 31 mEq L-1 and pH was 2.8, aspirin administered animal gastric juice total acidity was 45 mEq L-1 and pH was 1.7, plant extract with aspirin administered animal gastric juice total acidity was 35 mEq L-1 and pH was 4.3, cold resistant stress induced animal gastric juice total acidity was 47 mEq L-1 and pH was 1.3, plant extract with cold resistant stress induced animal gastric juice total acidity was 47 mEq L-1 and pH was 1.3 and foamatidine administered animal gastric juice total acidity was 32.3 mEq L-1 and pH was 3.8 (Table 1). This is due to the fact that the increased amount of gastric mucus secretion gets combined with low acid content; hence the pH is high in pretreated groups.
Ulcer index is in one of the better confirmation test for ulceration, next to the endoscopy/histopathlogy studies. The pretreated rats showed significant reduction in the ulcer index values, which suggests that the S. nigrum extract posses cytoprotective activity. The groups pretreated with S. nigrum extract exhibited a percentage inhibition of about 77.85% in aspirin and 66.67% in CRS-treated, groups, respectively. However, in groups that were pretreated with famotidine the percentage inhibition was 88.91%. Higher percentage of inhibition (88.91%) observed in groups pretreated with Famotidine it cannot be used to arrive at a conclusion that famotidine has higher ulcer protection ability as many commercial drugs produced many side effects than the extract. Increase in the concentration of the extract may improve the percentage of inhibition without any side effects.
A histological study revealed that, the pretreatment with S. nigrum extract
helped to preserve the cyto-architecture of the entire gastric mucosa of test
animals. S. nigrum extract treatment not only maintenance of gastric
mucosa helped but also the regeneration of gastric mucosa in the damaged regions.
These findings confirmed the gastro protective activity of S. nigrum
extract (Fig. 1-6). The normal animal gastric
mucosa histological section was showed clear and proper arrangement of cells
(Fig. 1). The cells were damaged and created opaque region
in gastric mucosa of both (Aspirin and cold resistant stress) ulcer induced
animal groups (Fig. 2, 3). The standard
gastroprotective drug of formatidine effectively replaces damaged cells in ulcer
induced animals gastric mucosa layer (Fig. 4). The plant extract
administered animal shows replacement of ulcerative cells and moderately arrange
proper layer in gastric mucosa (Fig. 5, 6).
The methanolic fraction of S. nigrum was found to contain 64.7 μg
pyrocatechol equivalents of phenols. The phenolic compounds may contribute directly
to the antioxidant actions. Oxidative stress plays a major role in the pathogenesis
of various diseases including gastric ulcers. Oxidative stress can damage many
biological molecules. Anti-oxidants have been found to play a significant role
in preventing gastric ulcers.
|| Histological section of gastric mucosa from negative control animals
|| Histological section of gastric mucosa from aspirin treated
|| Histological section of gastric mucosa from CRS treated animals
It has appeared that antioxidants may be an important contributory factor in
the protection of gastric mucosa. DPPH is a stable free radical at room temperature
that accepts an electron or H2 radical to become a stable diamagnetic
molecule. The reduction capability of DPPH radicals was determined from the
decreasing absorbance values at 517 nm with increasing concentration, which
is induced by anti oxidants present in the extracts. The maximum (79.84%) percentage
DPPH scavenging activity was obtained 1000 μg mL-1 plant extract
administered animal, followed by 800 μg mL-1 (58.68%), 600 μg
mL-1 (33.06%), 400 μg mL-1(28.93%) and 200 μg
mL-1 (25.62%). The Fe3+-Fe2+ transformation
has been investigated (Table 2). The reducing power of methanol
extract of plant was increased with the increased amount of the sample. The
predominant amount of reducing sugar obtained in 1000 μg mL-1
followed by 800, 600, 400 and 200 μg mL-1 concentrations of
extracts (Table 3).
|| Histological section of gastric mucosa from Famotodine +ASP
|| Histological section of gastric mucosa from Solanum nigrum
linn, +ASP treated animals
Phenolic compounds are known as powerful chain breaking anti oxidants, due to the presence of their hydroxyl groups. As a result of anti oxidant analysis, it was suggested that the increased amount of antioxidants present in the extract might be responsible for its gastro protective and antiulcer activity.
|| Histological section of gastric mucosa from Solanum nigrum
linn, + CRS treated animals
Peptic Ulcer Disease (PUD) is one of the common diseases. The causes of peptic ulcer disease are increased gastric acid secretion and reduced gastric cytoplasm. PUD occurs mainly due to consumption of Non Steroidal Anti Inflammatory Drugs (NSAIDs), infection by Helicobacter pylori, stress or due to pathological conditions such as Zollinger-Ellison syndrome. Non steroidal Anti-inflammatory drugs associated gastric ulceration occurs in 30% of users that led to hospitalization and also associate with high mortality. Hence, there is a need for more effective and less toxic antiulcer agents, plants extract are some of the most attractive sources.
In this study, the methanolic extract of S. nigrum was used for the
treatment of ulcer. The reducing power of methanol extract of plant was increased
with the increased amount of sample. Phenolic compounds are known as powerful
chain breaking anti oxidants, due to the presence of their hydroxyl groups.
It was suggested that the increased amount of antioxidant present in the extract
might be responsible for its gastro protective and anti ulcer activity. The
similar results of the present study found in ethanolic extract of Amla have
the capacity to significantly inhibit the basal gastric secretion and ulcerogenicity
induced by pylorus ligation, indomethacin and noxious chemicals and by hypothermic
restraint stress in rats (Jose and Kuttan, 2000; Yesilada
et al., 2000). Pepsin is one of three principal protein-degrading,
or proteolytic, enzymes in the digestive system, the other two being chymotrypsin
and trypsin and HCl are important for the formation of pylorus ligated ulcers
(John and Onabanjo, 1990; Tan
et al., 2000). The gastric protective effect of the extract be related
to an antacid effect or cytoprotective properties of the plants (Rifat-uz-Zaman
et al., 2004; Naseri and Mard, 2007). It
is possible that the inhibitory effect of the plants is due, at least partly,
to the presence of tannins, terpenes and fatty acids since these compounds were
associated with anti-ulcerogenic activity in other plants (Campos
et al., 2003; Hiruma-Lima et al., 2001;
Sehirli et al., 2008; Khanahmadi
et al., 2010).
Our findings are supported with Bandyopadhyay et al.
(2000), as our results showed a significant reduction in non-protein sulfhydryls
(NP-SH) content of gastric mucosa after 80% ethanol administration. This result
suggests that S. nigrum extract has active substance(s) that increase
the mucosal sulfhydryl groups content. In another hypothesis, this activity
could be attributed to antioxidant compounds found in the alcoholic extract
(Dias et al., 2000; Mahmoud
et al., 2006; Akah et al., 2007; Gill
et al., 2009). The results on histopathological investigation on
the gastric mucosa of rats revealed that the pretreatment with S. nigrum
extract absolutely inhibited the ethanol-induced congestion, hemorrhage, edema,
necrosis, inflammatory and dysplastic changes, erosions and ulceration. Present
results are in corroboration with the antigastric ulcer activity of the extract
observed under the studies on pharmacological and biochemical evaluation.
The extracts of the leaves of S. nigrum is potentially an anti ulcerogenic agent that protects and strengthens the gastric mucosa. The exact mechanism was not known, but it could be due to secretion of large quantity of mucous by the gastric mucosa that acts as a mechanical barrier. From the results, percentage of inhibitory effect and ulcer index, it is confirmed and concluded that S. nigrum has the anti-ulcer activity. Moreover, there is a need to conduct toxicity studies, using the plant extract on both laboratory and target animal species, to justify clinical investigation in other animals and in humans.
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