Evaluation of anti-ulcer activity of Samanea saman (Jacq) merr bark on ethanol and stress induced gastric lesions in albino rats.

OBJECTIVE: To evaluate the antiulcer activity of Samanea saman (Jacq) Merr bark on ethanol and stress induced gastric lesions in albino rats.
MATERIALS AND METHODS: Gastric lesions were induced in rats by oral administration of absolute ethanol (5 ml/kg) and stress induced by water immersion. The antiulcer activity of methanolic extract of Samanea saman (Jacq) Merr bark (100 mg/kg, 200 mg/kg, 400 mg/kg) was compared with standard drugs. The parameters studied were ulcer index, gastric juice volume, pH, free acidity and total acidity. RESULT: Samanea saman (Jacq) Merr showed a dose dependent curative ratio compared to ulcer control groups. The extract at 400 mg/kg showed significant anti ulcer activity which is almost equal to that of the standard drug in both models. The volume of acid secretion, total and free acidity was decreased and pH of the gastric juice was increased compared to ulcer control group.
CONCLUSIONS: The present study indicates that Samanea saman (Jacq) Merr bark extracts have potential anti ulcer activity.

Introduction

Use of medicines of plant origin is based upon the premise that plants contain natural substances that can promote health and alleviate illness.[1] Gastric hyperacidity and gastro duodenal ulcers are very common global health problems today. It is now generally agreed that gastric ulcers develop when the delicate balance between some gastro protective and aggressive factors is lost. Major aggressive factors are acid, pepsin, Helicobacter pylori and bile salts. Defensive factors mainly involve mucus-bicarbonate secretion and prostaglandins.[2] The modern approach to control gastric ulcer is to inhibit gastric acid secretion, to promote gastro protection, to block apoptosis and to stimulate epithelial cell proliferation for effective healing.[3]

Many experimental investigations have been undertaken to elucidate the etiology of development of ulcers induced by various means, however ulcer healing consists of reconstruction of mucosal architecture and is a dynamic, active process of filling the mucosal defects with epithelial and connective tissue cells. It encompasses cell proliferation, division and migration. Prostaglandins (PGs) and growth factors play an important role in healing of ulcers. Synthesis of PGs is governed by the expression of inducible cyclooxygenase-2 (COX-2) isoenzyme in gastric mucosa during healing process. Further, COX-2 expression is enhanced in gastric epithelial cells after treatment with growth factors in vitro and in vivo during acetic acid induced gastric damage. Among clinically established drugs, H 2 receptor blockers (ranitidine etc) and proton pump inhibitors (omeprazole etc) are most widely used as anti-ulcer drugs in addition to the cytoprotective agents like sucralfate and misoprostol. Efficacy of sucralfate is due to its protective effect by forming a coat over ulcer base, which prevents the direct effect of acid on the ulcer. It neither affects acid secretion nor generates prostaglandins, which could be the possible reason for sucralfate being effective in ulcer healing.[2]

Samanea saman (Jacq) Merr is a large umbraculiform tree growing over 20 meters height with a stout trunk about 1.5 meters in diameter, and with a large spreading canopy providing shade. The bark is rough and furrowed. It is valuable as a shade tree in pastures and stimulates grass growth. The leaves fold together on the approach of rain and hence it is named as RAIN TREE. Saponin-like alkaloid pithecolobin has been isolated from the bark and the seed. Alkaloids are said to be abundant in the barks, stems, leaves, and seeds. Leaves and stems have Saponin and tannin; gum is present in the trunk. Additionally steroids, cardiac glycosides, terpenoids are also present in the plant. The plants are used to cure acute bacillary dysentery, enteritis, diarrhea, colds, sore throat and headache. A decoction of the inner bark or fresh cambium and leaves are used to treat anaphylactic dermatitis, eczema, skin pruritus. In Venezuela, rain tree is a traditional remedy for colds, diarrhea, headache, intestinal ailments and stomach ache. Root decoction is used in hot baths for stomach cancer. The alcoholic extract of the leaves are used to treat tuberculosis. Gastric lesion is among the major diseases of gastrointestinal tract (GIT), for which a large number of traditional and modern medicines are being utilized. Among these, the medicines of plant origin are more popular because of lesser adverse effect.[4]

Previously, no scientific work has been reported on the anti-ulcerogenic activity of this plant. The present study was therefore undertaken to evaluate the anti-ulcerogenic activity of Samanea saman (Jacq) Merr bark extract on albino rats. In this study, various parameters like ulcer index, gastric juice volume, pH, free acidity and total acidity were determined.

Materials and Methods

Collection of plant

The fresh barks of Samanea saman (Jacq) Merr were collected in the month of July from Ambattur, Chennai, Tamil Nadu state, India, and authenticated by Prof. P Jayaraman, Ph.D., Plant Anatomy Research Centre, Chennai, Tamil Nadu. (Registration number: PARC/2010/567). The voucher specimen was deposited at the department for future reference.

Extraction of plant material

About 400 g of air dried powdered bark was taken in 1000 ml soxhlet apparatus and extracted with petroleum ether for 2 days. At the end of second day, the powder was taken out and it was dried. After drying, it was again packed and extracted by using methanol as solvent, till the color disappeared. The temperature was maintained at 55 °C – 65 °C. After that, the extract was concentrated by distillation and solvent was recovered. The final solution was evaporated to dryness and dry residue was obtained.

Animals

Male albino rats, (150-200 g) and albino mice (20-25 g) were used in the present study. All the rats were kept at room temperature (24 °C ± 2) in the animal house. All the animals were housed and treated as per the internationally accepted ethical guidelines for the care of laboratory animals. Prior to the experiments, rats were fed with standard food and were acclimatized to laboratory conditions. All the experimental procedures were performed on animals after approval from the ethics committee and in accordance with the recommendations for the proper care and use of laboratory animals.

Acute Oral Toxicity Study

Three animals (Albino mice, 20-25 g) were selected for studies. The dose level of methanolic extracts of Samanea saman (Jacq) Merr in different concentrations were observed for any symptoms of toxicity for 48 hours as per Organisation for Economic Co-operation and Development (OECD) guidelines 423 (Acute toxic class method) and median lethal dose (LD 50 )was estimated to be >2000 mg/kg. Based on the results obtained from this study, the doses of further pharmacological studies were fixed to be 100 mg/kg, 200 mg/kg and 400 mg/kg, per orally.

Ethanol induced ulcer

Male albino rats weighing 150-200 g were divided into six groups, of six animals each, as given below. They were fasted for 24 hours prior to the experiment in perforated steel cages to avoid coprophagy.

Group I - received 1% acacia (1.0 ml/kg per oral) as normal control.

Group II - received 1% acacia (1.0 ml/kg per oral) as vehicle control.

Group III - received 100 mg/kg, per oral methanol extract of Samanea saman (Jacq) Merr bark.

Group IV - received 200 mg/kg, per oral methanol extract of. Samanea saman (Jacq) Merr bark.

Group V - received 400 mg/kg, per oral methanol extract of. Samanea saman (Jacq) Merr bark.

Group VI - received 100 mg/kg, per oral sucralfate as standard

One hour after the drug treatment, the animals belonging to groups II, III, IV, V and VI were treated with absolute ethanol [5 ml/kg] to induce lesions. The animals were sacrificed after 1 hour and the stomach was opened. The ulcer index and percentage inhibition of ulcer was determined.[56] The stomach was carefully excised keeping esophagus closed and opened along greater curvature, and luminal contents were removed. The gastric contents were collected in a test tube and centrifuged at 3000 revolutions per minute (rpm) for 10 minutes. The volume of supernatant was measured and expressed as ml/100 g body weight. The pH of the supernatant was measured using digital pH meter.[78] An aliquot of 1.0 ml of gastric juice was pipetted out to a 50 ml conical flask and 2/3 drops of Topfer's reagent were added to it and titrated with 0.01 N Sodium Hydroxide (NaOH) until all traces of the red color disappeared, and the color of the solution turned yellowish orange. The volume of 0.01 N NaOH was noted, which corresponded to the free acidity. Then 2-3 drops of phenolphthalein were added and titration was continued until a permanent pink color was developed. The volume of total alkali consumed was noted which corresponded to the total acidity. The mucosa was flushed with saline and stomach was pinned on board.

The lesion in the glandular portion was examined under a 10 × magnifying glass and length was measured using a divider and scale and gastric lesion was scored as follows:

0 – Normal colored stomach,

0.5 – Red coloration,

1– Spot ulceration,

1.5 – Hemorrhagic streak,

2 – ulcers

3 – Perforations

Ulcer index of each animal was calculated by adding the values and their mean values were determined and percentage inhibition was calculated.[9]

Swimming stress-induced ulcer

Stress ulcers were induced by forced swimming in a glass cylinder[10] (height 45 cm, diameter 25 cm) containing water to the height of 35 cm and maintained at 25 °C for 3 hours. The animals were fasted for 24 hours prior to the experiment and divided into 6 groups of 6 animals in each group. Group I served as normal control, Group II served as vehicle control and received 1% acacia (1 ml/kg per oral), Group III, IV and V received 100 mg/kg, 200 mg/kg and 400 mg/kg per oral of methanol extracts of Samanea saman (Jacq) Merr bark, respectively. Group VI received 20 mg/kg per oral omeprazole as standard. After the drug treatment, animals were allowed to swim in water for 3 hours. Then the animals were sacrificed and the stomach was opened. The ulcer index and percentage inhibition of ulcer was determined

Statistical Analysis

All the values are expressed as mean ± standard error of the mean (S.E.M) for groups of six animals each The data was analyzed by one way Analysis of Variance (ANOVA) and compared by using Tukey- Kramer multiple comparison test.

Results

Effects of methanol extract of Samanea saman (Jacq) Merr on ulcer index induced by ethanol in rats are shown in Table 1 and Figure 1. Samanea saman (Jacq) Merr showed a dose dependent curative ratio compared to ulcer control groups. The extracts exhibited an inhibition percentage of 65.67, 72.43 and 86.49 at doses of 100, 200 and 400 mg/kg doses respectively; and, the standard drug, sucralfate, exhibited an inhibition percentage of 92.16. The effects of methanolic extract of Samanea saman (Jacq) Merr on acid parameters showed significant (P<0.001) effect at 200 mg/kg and 400 mg/kg doses compared to ulcer control animals. The volume of acid secretion, total and free acidity was decreased and pH of the gastric juice was increased compared to ulcer control group [Table 2].

Table 1

Effect of Samanea saman (Jacq) Merr on Ulcer Index in ethanol induced gastric lesions in the rats during the study

Figure 1

Effect of Samanea saman (Jacq) Merr on ethanol induced ulcers in the rats in the study (a) Normal Control (b) Ulcer Control (c) S. saman (100 mg/kg) treated (d) S. saman (200 mg/kg) treated (e) S. saman (400 mg/kg) treated (f) Sucralfate (100 mg/kg) treated

Table 2

Effect of Samanea saman (Jacq) Merr on volume of acid secretion, total and free acidity, and pH of the gastric juice in ethanol induced ulcers in the rats during the study

In water immersion stress induced ulcers, the mean score value of ulcer inhibition was found to be significant (P<0.001) for 200 mg/kg and 400 mg/kg of the extract. The percentage ulcer inhibition was 53.30, 71.11 and 87.69 for 100 mg/kg, 200 mg/kg and 400 mg/kg respectively, and that of the standard was found to be 92.68. [Table 3 and Figure 2].

Table 3

Effect of Samanea saman (Jacq) Merr on Ulcer Index in water immersion stress induced gastric lesion in the rats during the study

Figure 2

Effect of Samanea saman (Jacq) Merr on stress induced ulcers in the rats in the study (a) Normal Control (b) Ulcer Control (c) S. saman (100 mg/kg) treated (d) S. saman (200 mg/kg) treated (e) S. saman (400 mg/kg) treated (f) Omeprazole (20 mg/kg treated)

Discussion

Ethanol induced gastric damage showed gross mucosal lesion, including long hemorrhage bands and petechial lesion. Animals pretreated with methanol extract of Samanea saman (Jacq) Merr and standard drug sucralfate showed very mild lesions and sometimes no lesion at all, when compared to ulcer control group. Ethanol produces severe gastric hemorrhagic lesions. Ethanol increases super oxide anion and hydroxyl radical production and lipid peroxidation in the gastric mucosa. These and other reactive metabolites react with most of the cell components, changing their structures and functions, or contributing to other mechanisms that finally promote enhanced oxidative damage. Ethanol-induced gastric mucosal injury is associated with extensive damage to mucosal capillaries and increased vascular permeability.[11] Samanea saman (Jacq) Merr has a very good antioxidant activity.[1213] Because of this antioxidant activity, the extract may inhibit tissue derived mediators which cause cellular necrosis, thereby exhibiting anti ulcer activity. Hence the cytoprotective mechanism of the Samanea saman (Jacq) Merr extract may therefore include mechanisms other than simple acid neutralization, and the anti secretory activity might not be the main mechanism of action of these extracts.

Water immersion stress is one of the best models of stress in rats to induce ulcer. This model provides both emotional stress as well as physiological stress to the animal. In case of water immersion induced stress in rats, the extract showed significant ulcer inhibition in a dose dependant manner. One of the close species of Samanea saman (Jacq) Merr has free radical-scavenging and hydrogen cation (H+), Potassium cation (K+) and ATPase inhibition activity. Thus it may be possible that Samanea also exhibit its anti ulcer activity through H+, K+ and ATPase inhibition.[14]

The phytoconstituents like flavonoids, tannins, terpenoids, and Saponin have been reported in several anti-ulcer literatures as possible gastro protective agents. Flavonoids, tannins and triterpenes are among the cytoprotective active materials for which anti ulcerogenic efficacy has been extensively confirmed.[15] It is suggested that these compounds will be able to stimulate mucus, bicarbonate and prostaglandin secretion, and counteract with the deteriorating effects of reactive oxidants in gastrointestinal lumen.[16] Tannins may prevent ulcer development due to their protein precipitating and vasoconstriction effects. Their astringent action can help precipitating micro proteins on the ulcer site, thereby forming an impervious layer over the lining that hinders gut secretions and protects the underlying mucosa from toxins and other irritants.[17-20] Alkaloids and terpenoids prevent ulcers induced by stress.

Similarly, the methanol extract of Samanea saman (Jacq) Merr bark, showed the presence of flavonoids, glycosides, tannins, triterpenoids, alkaloids and Saponin. These phytoconstituents present in the extract could be the possible agents involved in the prevention of gastric lesion in rats. Further, studies are under process to isolate the possible phytoconstituents responsible for the anti ulcer activity. Also, many active phyto constituents have been isolated from similar plant species Pithecellobium dulce Benth.[21]

Conclusion

The present study showed that pretreatment with methanol extract of Samanea saman (Jacq) Merr bark caused a beneficial effect on ethanol induced and water immersion stress induced gastric lesion in rats, as evidenced by the reduction in the ulcer scores.