INTRODUCTION:

Peptic ulcer is one of the most common gastrointestinal diseases¹. T he exact causes of peptic ulcer disease is not known but it may be result from an imbalance between acid-pepsin secretions and mucosal defense factors². Peptic ulcer disease occurs mainly due to consumption of NSAIDS, infection by H. pylori, stress or due to pathological condition such as Zollinger-Ellison Syndrome³.

Couroupita guianensis is a large deciduous evergreen tree growing to a height of 20 meters. Leaves are alternate, oblong up to 20 cm long, entire to slightly serrate and hairy on the veins beneath. Inflorescence is racemose, arising from the trunk and other large branches. Flowers are reddish with a yellow tinge on the outside, fragrant, with stamens borne on an overarching androphore. Fruit is a large, reddish-brown globose, 15 to 24 cm, with a woody capsule, and each containing 200 to 300 seeds. This plant is used for treating mange and other skin conditions. The pulp of the fruit of the cannon ball tree is rubbed on the infected skin of mange dog.

It is claimed that when the dog licks its skin, this medicine will also work internally. The flowers are used to cure cold, intestinal gas formation and stomach ache. The leaf has been found to show anti oxidant activity, anthelmintic activity, immuno modulator and anti-nociceptive activity^4-7. So for no systematic study has been reported for antiulcer properties of Couroupita guianensis leaf extracts. In the present study effort has been made to establish the scientific validity to the anti ulcer property of Couroupita guianensis leaves extracts using pyloric ligation and ethanol induced ulceration models in albino rats.

MATERIALS AND METHODS:

Plant materials:

The leaves of Couroupita guianensis were collected in Venkittampalayam village near Thiruvannamalai district, Tamil Nadu in the month of Dec 2011. The specimen was identified and authenticated by Prof. Dr. P. Jayaraman, Director, Plant Anatomy Research Center (PARC), Tambaram, Chennai. The specimen was deposited to herbarium of Anurag Pharmacy College. After authentication, fresh leaves collected in bulk from plants, washed, shade dried and then milled to a coarse powder by a mechanical grinder,

Preparation of extract:

The powders of dried leaves were packed in to soxhlet column and extract with ethanol. The extract was filter through a Whatman filter paper no.1 and concentrated under reduced pressure (yield of extract was 9.40% with respect to dry material). Just prior to use, the substance was dissolved in physiological saline solution.

Animals:

The study was conducted on male Wister rats (175-200gm) housed in polypropylene cages under standard conditions of temperature (22 ± 2°C), relative humidity (60 ± 5%) and light (12h light/dark cycle) were used. They were fed with standard diet and water. The food was withdrawn 18 hours before the experiment but allowed free access of water. All animal experiments were carried out in accordance with the guidelines of CPCSEA.

Acute oral toxicity studies:

Acute toxicity was carried out according to Organization of Economic Co-Operation and Development (OECD) guidelines⁸, No mortality was observed and all the test doses were found to be safe.

Pyloric ligation in rats:

The animals were divided into 5 groups, each consisting of six rats. Control group received distilled water only. Second group of rats are pyloric ligated. Third and fourth groups received EECG in a dose of 150 and 300 mg/kg. The fifth group of animals received Ranitidine in the dose of 20mg/kg as a reference drug for ulcer protective studies. After 45 min of the treatment, pyloric ligation was done by ligating the pyloric end of stomach of rats of respective groups under ether anesthesia at a dose of 35mg/kg of body weight. Ligation was done without causing any damage to the blood supply of the stomach. Animals were allowed to recover and stabilize in individual cages and were deprived of water during post-operative period. Rats were sacrificed after 4hr of surgery and ulcer scoring was done. Gastric juice was collected and gastric secretion studies were performed according to the standard procedure⁹.

Ethanol induced ulcer model:

The ulcer was induced by administering absolute ethanol (1ml/200g). All the animals were fasted for 36 hours and then ethanol was administered to induce ulcer. The animals were divided into five groups, each consisting of six rats. The control group received distilled water, second group received ethanol. Third and fourth groups received EECG in a dose of 150 and 300 mg/kg. The fifth group of animals received Ranitidine in the dose of 20 mg/kg as a reference drug. They were kept in specially constructed cages to prevent coprophagia during and after the experiment. The animals were anaesthetized 1 hr later with anaesthetic ether and stomach was incised along the greater curvature and ulceration was scored. A score for the ulcer was studied to pyloric ligation induced ulcer model¹⁰.

Scoring of ulcers:

Normal stomach -0

Red coloration -0.5

Spot ulcer -1

Hemorrhagic streak -1.5

Ulcers (< 2mm) -2

Ulcers (>2 < 4 mm) perforation -3

Ulcers (< 4mm) -4

Mean ulcer score for each animal was expressed as ulcer index. The percentage of ulcer protection was determined by

Control mean ulcer index – Test mean ulcer index

% of ulcer protection = ----------------------------------------- X 100

Control mean ulcer index

Determination of free acidity:

Volume of sodium hydroxide x Normality x 100mEq/L/100g

Acidity = -----------------------------------------------------------------

0.1

Statistical analysis:

The values are represented as mean ± S.E.M, and Statistical significance between treated and control groups was analyzed using of one way ANOVA, followed by Dennett’s test where P<0.05 was considered statistically significant.

RESULTS:

Pyloric ligation induced gastric ulcer:

In pyloric ligation induced ulcer model, oral administration of EECG in two different doses showed significant reduction in ulcer index, gastric volume, free acidity, total acidity compared to the control group. EECG exhibited a protection index of 68.7% and 81.2% at the dose of 150 and 300 mg/kg respectively, where as Ranitidine as reference standard exhibited a protection index of 85.2% (Table 1).

Ethanol-induced gastric ulcer:

In control animal, oral administration of absolute ethanol produced characteristic lesions in the glandular portion of rat stomach which appeared as elongated bands of thick, blackish red lesions. EECG has shown significant protection index of 67.7% and 71.2% with the dose of 150 and 300 mg/kg respectively whereas Ranitidine as reference standard showed protection index of 79.6% (Table 2).

Table1: Effect of EECG on various parameters in pyloric ligation induced gastric ulcers.

Group	Treatment	Ulcer index	Free acidity meq/ltr	P^H of gastric juice	Gastric juice	Total acidity meq/ltr	Protection (%)
I	Normal (distilled water)	---	41.3 ± 0.3	5.41 ± 0.3	3.8 ± 0.4	62.3 ± 0.2	---
II	Control (pyloric ligation)	14.2 ± 1.2	95.6 ± 1.4	2.51 ± 0.2	8.2 ± 0.2	112.5 ± 0.2	---
III	EECG (150mg/kg)	4.5 ± 0.5	43.7 ± 0.3	4.87 ± 0.2*	5.3 ±1.2	75.3 ± 0.4	68.7 %
IV	EECG (300mg/kg)	2.8 ± 0.4*	39.8 ± 0.2*	5.51 ± 0.4*	4.2±0.4*	61.7 ± 0.6*	81.2%
V	Ranitidine (20mg/kg)	2.2 ± 0.5*	37.4 ± 0.2*	5.71 ± 0.4*	3.9 ± 0.2*	60.1 ± 1.4*	85.2%

Table 2: Effect of EECG on various parameters in ethanol induced gastric ulcers.

Group	Treatment	Ulcer index	P^H of gastric juice	Protection (%)
I	Normal (distilled water)	---	5.42 ± 0.3	---
II	Control (pyloric ligation)	12.3 ± 0.2	2,83 ± 0.6	---
III	EECG (150mg/kg)	4,3 ± 0.5	3.68 ± 0.6	67.7%
IV	EECG (300mg/kg)	3.6 ± 0.4*	4.86 ± 0.7*	71.2%
V	Ranitidine (20mg/kg)	2.7 ± 0.4*	5.62 ± 0.7*	79.6%

Values are expressed as mean ± SEM of observations, Statistical comparisons as follows: Significant *P <0.005 compared to control group.

DISCUSSION:

The etiology of peptic ulcer is unknown in most of the cases, it is generally accepted that gastric ulcer results from an imbalance between aggressive factors and the maintenance of the mucosal integrity through the endogenous defense mechanism¹¹. Different therapeutic agents are used to inhibit the gastric acid secretion or to boost the mucosal defense mechanisms by increasing mucosal production, stabilizing the surface epithelial cells or interfering with the prostaglandin synthesis¹². The prostaglandins can provide gastric cytoprotection in rats against strong necrotizing irritants without reducing gastric acid secretion¹³.

The causes of gastric ulcer by pyloric ligation are believed to be due to stress induced increase in gastric hydrochloric acid secretion and/or stasis of acid and the volume of secretion is also an important factor in the formation of ulcer due to exposure of the unprotected lumen of the stomach to the accumulating of acid. The ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach. This increase in the gastric acid secretion causes ulcers in the stomach. The lesions produced by this method are located in the lumen region of the stomach^14.

Ethanol induced gastric lesion formation may be due to stasis in gastric blood flow which contributes to the development of the hemorrhage and narcotic aspects of tissue injury¹⁵. Alcohol rapidly penetrates the gastric mucosa apparently causing cell and plasma membrane damage leading to increased intra cellular membrane permeability to sodium and water. The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium¹⁶.

In the present study EECG showed protection against gastric lesions in the experimental rats, reduced gastric volume, free acidity, total acidity and ulcer index thus showing the anti-secretary mechanism involved in the extracts for their anti-ulcerogenic activity. Ulcer index parameter was used for the evaluation of anti ulcer activity since ulcer formation is directly related to factors such as gastric volume, free and total acidity¹⁷.

The protection of EECG against characteristic lesions may be due to both reductions in gastric acid secretion and gastric cycloprotein or enhancement of the mucosal barrier through the increase production of prostaglandin and this may be due to the presence of glycosides. Further studies are needed for their exact mechanism of action on gastric acid secretion and gastric cytoprotection.

ACKNOWLEDGEMENT:

The authors express their gratitude to Department of Pharmacognosy, Anurag Pharmacy College for providing necessary facilities.

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Received on 03.04.2012 Accepted on 10.05.2012

Asian J. Pharm. Tech. 2(2): April-June 2012; Page 64-66