Fresh Eruca sativa leaves were purchased from a local vegetable market in Riyadh, and the identity of these leaves was confirmed by an expert taxonomist of the Department of Pharmacognosy, where a voucher specimen (No. 8208) of the plant has been kept in the Herbarium of the College of Pharmacy, KSU, Riyadh. Shade-dried, coarsely pulverized rocket leaves were placed in a glass percolator with ethanol and were allowed to stand at room temperature for about 72 h. The percolate was collected and dried under reduced pressure in vacuo. The extract obtained was later used and dissolved in distilled water for evaluation of anti-ulcer activity.

Albino Wistar rats of either sex, approximately the same age, weighing 150 to 200 g and fed on a diet of standard chow were used in this study. They were randomly divided into experimental groups of 6 rats each. Aqueous solutions of ulcerogens and EER were freshly prepared before administration. EER at doses of 250 and 500 mg/kg were given orally in the anti-ulcer studies and intraperitoneally for gastric secretion evaluation. The rats were sacrificed, and the stomachs removed and opened along the greater curvature. After washing with saline, the gastric lesions were quantified by a person unaware of the treatments. The animal study protocol was approved by the Research and Ethics Committee of the Experimental Animal Care Society, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Rats were fasted for 36 h with access to water ad libitum before pylorus ligation under ether anesthesia was carried out. Care was taken not to cause bleeding or to occlude blood vessels[20]. EER was administered intraperitoneally immediately after pylorus ligation (Shay). The rats were sacrificed at 6 h after pylorus ligation. The stomachs were removed, the contents were collected, volumes measured, centrifuged and analyzed for titratable acidity against 0.01 mol/L NaOH at pH 7.

Each rat was administered 1 mL of a necrotizing agent (80% ethanol, 0.2 mol/L NaOH or 25% NaCl). Rocket extract was given 30 min before the administration of necrotizing agents. One hour after the administration of ethanol and the alkalis, the rats were sacrificed and examined for stomach lesions. The scoring of stomach lesions was as follows: Patchy lesions of the stomach induced by ethanol were scored according to the method described by Robert et al[21] using the following scale: 0 = normal mucosa; 1 = hyperemic mucosa or up to 3 small patches; 2 = from 4 to 10 small patches; 3 = more than 10 small or up to 3 medium-sized patches; 4 = from 4 to 6 medium-sized patches; 5 = more than 6 medium-sized or up to 3 large patches; 6 = from 4 to 6 large patches; 7 = from 7 to 10 large patches; 8 = more than 10 large patches or extensive necrotic zones. “small” was defined as up to 2 mm across (max. diameter), “medium-sized” between 2 and 4 mm across and “large” more than 4 mm across.

Indomethacin was suspended in 1.0% carboxy-methylcellulose (CMC) in water (6 mg/mL) and administered orally to the 36 h fasted rats at a dose of 30 mg/kg body weight. Control rats were treated similarly with an equivalent amount of vehicle[22]. The rocket extract was given 30 min prior to indomethacin administration at a dose of 250 and 500 mg/kg. The animals were sacrificed 6 h after treatment. The stomachs were excised, rinsed with normal saline and examined for ulceration.

The method described by Senay et al[23] was adopted with slight modifications. Animals were fasted for 36 h but had access to water ad libitum. Thirty minutes after the oral administration of EER (250 and 500 mg/kg), the rats were immobilized in restraint cages and placed inside a ventilated refrigerator maintained at 3 ± 1°C for 3 h. The animals were then sacrificed and the stomachs were excised. They were examined for ulceration and the severity of intraluminal bleeding according to the following arbitrary scale described by Chiu et al[24]. 0 = no blood detectable; 1 = thin blood follows the rugae; 2 = thick blood follows the rugae; 3 = thick blood follows the rugae with blood clots in certain areas and 4 = extensive covering of the whole gastric mucosal surface with thick blood.

Gastric wall mucus was determined according to the modified procedure of Crone et al[25]. The glandular segment of the stomach was separated from the rumen of the stomach, weighed, and transferred immediately to 10 mL of 0.1% w/v Alcian blue solution (in 0.16 mmol/L sucrose solution buffered with 0.05 mL sodium acetate at pH 5). Tissue was stained for 2 h in Alcian blue, and excess dye was removed by two successive rinses with 10 mL of 0.25 mmol/L sucrose, firstly after 15 min and then after 45 min. Dye complexed with the gastric wall mucus was extracted with 10 mL of 0.5 mmol/L magnesium chloride which was intermittently shaken for 1 min at 30 min intervals for 2 h. Four milliliters of blue extract were then vigorously shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged at 4000 r/min for 10 min and the absorbance of the aqueous layer was recorded at 580 nm. The quantity of Alcian blue extracted per gram of wet glandular tissue was then calculated.

Gastric mucosal non-protein sulfhydryls were measured according to the method of Sedlak and Lindsay[26]. The glandular part of the stomach was homogenized in ice-cold 0.02 mmol/L ethylenediaminetetraacetic acid (EDTA). Aliquots of 5 mL of the homogenates were mixed in 15 mL test tubes with 4 mL of distilled water and 1 mL of 50% trichloroacetic acid (TCA). The tubes were shaken intermittently for 10 min and centrifuged at 3000 r/min. Two milliliters of supernatant were mixed with 4 mL of 0.4 mol/L Tris buffer at pH 8.9. 0.1 mL of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was added and the sample was shaken. The absorbance was measured within 5 min of DTNB addition at 412 nm against a reagent blank.

The method reported by Utley et al[27] was followed. The animals were killed 1 h after ethanol administration. The stomachs were removed and each was homogenized in 0.15 mol/L KCl (at 4°C) in a Potter-Elvehjem type C homogenizer to give a 10% w/v homogenate. Aliquots of homogenate 1 mL in volume were incubated at 37°C for 3 h in a metabolic shaker. Then 1 mL of 10% aqueous TCA was added and mixed. The mixture was then centrifuged at 800 g for 10 min. One milliliter of the supernatant was removed and mixed with 1 mL of 0.67% w-thiobarbituric acid in water and placed in a boiling water bath for 10 min. The mixture was cooled and diluted with 1 mL distilled water. The absorbance of the solution was then read at 535 nm. The content of malondialdehyde (nmol/g wet tissue) (index of the magnitude of lipid peroxidation) was then calculated, by reference to a standard curve of malondialdehyde solution.

Gastric tissue samples were fixed in neutral buffered formalin for 24 h. Sections of gastric tissue were histopathologically examined to study the ulcerogenic and/or anti-ulcerogenic activity of EER. The tissues were fixed in 10% buffered formalin and processed using a VIP tissue processor. The processed tissues were embedded in paraffin blocks and sections about 5 &mgr;m thick were cut using an American optical rotary microtome. These sections were stained with haematoxylin and eosin using routine procedures[28]. The slides were examined microscopically for pathomorphological changes such as congestion, hemorrhage, edema, and erosions using an arbitrary scale for severity assessment of these changes.

Values in tables and figures are given as mean ± SE. Data were analyzed by using one-way analysis of variance (ANOVA) followed by Student’s t-test.

When the rats were subjected to pylorus ligation for 6 h, a considerable amount of basal gastric acid secretion was noted (10.83 ± 1.16 mL) in the control group. In the same control group, the titratable acidity was found to be 196.57 ± 15.50 mEq/L and the ulcer index was recorded as 2.33 ± 1.5. EER at both doses (250 and 500 mg/kg) significantly reduced gastric acid secretion, titratable acidity and ulcer formation (6.33 ± 1.63 mL, 4.16 ± 1.16 mL P < 0.05, P < 0.01; 132.77 ± 17.43, 55.55 ± 10.46 mEq/L P < 0.05, P < 0.001, 0.66 ± 0.51, 0.50 ± 0.54, P < 0.05, P < 0.05), respectively (Table 1).

The treatment of rats with 80% ethanol, 0.2 mol/L NaOH and 25% NaCl produced extensive gastric lesions in the glandular mucosa of the stomach in all the control rats. The ulcer index was 6.00 ± 0.89, 6.66 ± 1.36 and 6.66 ± 0.51, respectively in control rats 1 h after administration of the necrotizing agents. Pretreatment of rats with EER at doses of 250 mg/kg (ulcer index in 80% ethanol, 0.2 mol/L NaOH and 25% NaCl = 2.00 ± 0.89, P < 0.01, 2.66 ± 1.21, P < 0.05 and 2.83 ± 0.98, P < 0.001); 500 mg/kg (ulcer index = 1.66 ± 1.03, P < 0.01, 1.50 ± 0.54, P < 0.01 and 2.16 ± 0.75, P < 0.001), respectively, significantly inhibited the formation of gastric lesions as shown in Table 2.

The oral administration of indomethacin induced marked damage in the rat glandular stomach. EER at the 500 mg/kg dose significantly prevented the development of gastric lesions in the rat stomach (P < 0.01), however, no significant preventive effect of EER at the 250 mg/kg dose, in indomethacin-treated rats was observed (Table 3).

Table 4 shows that EER at a dose of 500 mg/kg body weight significantly inhibited intraluminal bleeding and ulcer formation induced by hypothermic restraint stress (P < 0.05). Although the intraluminal bleeding and ulcer index was reduced at a dose of 250 mg/kg body weight, this reduction was not found to be statistically significant.

Rats treated with ethanol showed a significant decrease in the Alcian blue binding capacity of gastric wall mucus (148.41 ± 18.81 &mgr;g/g of tissue, P < 0.001) as compared to control (normal) rats (426.73 ± 39.15 µg/g). Pretreatment of rats with EER at doses of 250 mg/kg (263.87 ± 32.65 &mgr;g/g, P < 0.05) and 500 mg/kg (313.90 ± 24.30 &mgr;g/g, P < 0.001) significantly enhanced the Alcian blue binding capacity of gastric mucosa (Figure 1).

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Figure 1 Effect of EER on the changes in gastric wall mucus induced by 80% ethanol.

The level of NP-SH in the gastric mucosa of control rats was 5.26 ± 0.63 mmol/g of tissue, which was significantly decreased to 2.38 ± 0.33 mmol/g (P < 0.001) following the administration of 80% ethanol. Pretreatment of rats with EER at both doses (250 and 500 mg/kg) significantly replenished the ethanol-induced depletion of NP-SH (3.63 ± 0.22, P < 0.001; 4.53 ± 0.56, P < 0.01), respectively (Figure 2).

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Figure 2 Effect of EER on NP-SH concentration in gastric ulcer induced by 80% ethanol.

As depicted in Figure 3, MDA levels in the gastric mucosa used as an index of lipid peroxidation were significantly higher in the ethanol only treated group than in the untreated control group (7.09 ± 0.60 &mgr;mol/g of tissue; 2.77 ± 0.19 &mgr;mol/g of tissue), respectively. EER at both doses (250 and 500 mg/kg) significantly decreased the MDA content (4.55 ± 0.66 &mgr;mol/g and 3.58 ± 0.49 &mgr;mol/g), respectively.

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Figure 3 Effect of EER on MDA concentration in gastric ulcer induced by 80% ethanol.

Histopathological studies (Figure 4) further confirmed that pretreatment with rocket extract prevented ethanol-induced necrosis in the superficial layers of the gastric mucosa with congestion.

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Figure 4 Light micrographs showing the effect of EER on ethanol-induced gastric lesions of rats. A: Normal mucosa; B: Ethanol-induced gastric mucosal congestion and necrosis; C: Pretreatment of rats with EER 250 mg/kg; D: Pretreatment of rats with EER 500 mg/kg.

Gastric ulcer is an illness that affects a considerable number of people worldwide. The etiological factors of this disorder include: stress, smoking, nutritional deficiencies, infections, frequent and indiscriminate use of nonsteroidal anti-inflammatory drugs (NSAIDs). Herbs, medicinal plants, spices, vegetables and crude drug substances are considered to be a potential source to combat various diseases including gastric ulcer. In the scientific literature, a large number of medicinal plants with gastric anti-ulcer potential have been reported.

Although the introduction of proton-pump inhibitors to the classic anti-ulcer therapy has revolutionized treatment of peptic ulcers and other gastrointestinal disorders, there is still no complete cure for this disease. It has been shown that long term use of these drugs leads to various adverse and side effects. Relapses of the malady, ineffectiveness of different drug regimens and even resistance to drugs are emerging. Thus, there is an urgent requirement to identify more effective and safe anti-ulcer agents. During the past few decades, a widespread search has been launched to identify new anti-ulcer therapies from natural sources.

The anti-secretory, cytoprotective and antioxidant properties of the rocket extract caused an inhibition of chemically and stress-induced gastric ulceration in rats.

The authors examined an ethanolic extract of rocket “Eruca sativa L.” for its claimed beneficial effects on gastrointestinal disorders. The results are interesting and may provide a better understanding and give a clue for further investigations and innovations for an effective and safe phytotherapy for peptic ulcer disease.