Animals
Male albino BALB/c mice 25–35 g were obtained from the animal house of School of Pharmacy, Mashhad University of Medical Sciences. Animals were housed in colony room 12/12 hr light/dark cycle at 24 ± 1°C. After 24 h fasting, the mice were used for the experiments but were allowed drinking water during the 24 h fasting period. All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee acts.
Plant material
The seed was collected at Bojnord (a town in Khorassan province, the northeastern of Iran). All samples collected were dried in shade and then powdered. Ferdowsi University properly identified the plant and voucher samples were preserved for reference in the herbarium of Department of Pharmacognosy, School of Pharmacy, Mashhad (293-0107-18).
The preparation of extracts
The seed powder was extracted using maceration with ethanol (80 v/v) or water for 3 days and, subsequently, the mixture was filtered and concentrated under reduced pressure (by a rotaevaporator) at 40°C. The yield (w/w) of the aqueous and ethanolic extracts was 6.46% and 8.5%, respectively.
HCl or ethanol-induced mucosal membrane lesions
Gastric mucosal lesions were induced by the modified method of Mizui and Doteuchi [17]. The mice were divided into groups of 6 animals. After 24 h fasting, the extracts and drugs were administered orally to the mice. 30 min thereafter, each mouse received 0.2 ml of 1 N HCl or absolute ethanol by oral administration. 60 min after administration of the necrotizing agent, each animal was killed by ether, and the stomach was excised, inflated by injecting 2 ml of normal saline and then fixed for 30 min in 5% formalin solution. After opening along the greater curvature, HCl induced gastric damage was observed in the gastric mucosa as elongated black-red lines parallel to the long axis of the stomach of the mice. The lesion index was determined as the sum of erosion length per mouse [18]. Ethanol induced lesion was assessed and scored for severity according to, (0) absence of lesion, (1) superficial 1–5 hemorrhagic points, (2) superficial 6–10 hemorrhagic points, (3) submucosal hemorrhagic lesions with small erosions (4) severe hemorrhagic lesion and some invasive lesions.
Antisecretory study
One hour after extract or test drug treatment, mice were anesthetized (xylazine 10 mg/kg plus ketamine 100 mg/kg, i.p.) and the pylorus was ligated. The animals were killed 3 h later and their stomach content was drained into a tube which was centrifuged 2000 rpm for 10 min. The pH was recorded with a digital pH meter. Total acid content of gastric secretion was determined by titration against 0.05 N NaOH [19].
ED50 values
ED50 values and the corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4).
Acute toxicity
Different doses of extracts were injected intraperitoneally into groups of six mice. The number of death was counted at 24 h after treatment. LD50 values and the corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4).
Statistical analysis
The data were expressed as mean values ± S.E.M. and tested with analysis of variance followed by the multiple comparison test of Tukey-Kramer. Ethanol induced lesion was assessed by Dunn's test.
