This experiment was approved by the animal ethic committee of animal experimentation, Faculty of Medicine, UM, ethic NO.PM/29/06/2012/SMD (R).
Adult healthy Sprague Dawley (SD) rats were used for gastro-protective and (ICR) mice for acute toxicity evaluation. The animals were supplied from the animal house, Faculty of Medicine, UM, Kuala Lumpur. All experiments were designed to use the minimum number associated with valid statistical analysis. Study animals were housed at least 3 days before experiment under standard condition (individual plastic cages with wide mesh bottom for preventing coprophagia and temperature of 24 ± 2°C and lighting). Animals were fed with commercial diet and tap water . The experimental animals were sacrificed after being anesthetized by 0.1 ml/100 gram body weight of a mixture of ketamine (8.75 ml, 100 mg/ml) and xylazine ( 1.25 ml, 100 mg/ml) ,.
(1-(4-hydroxy-phenyl)-3-m-tolyl-propenone (HPTP) chalcone was obtained from Pharmacy Department, Faculty of Medicine, UM. HPTP was suspended in 5 ml/kg body weight of 0.5% w/v of Carboxylmethylcellulose (CMC) 30 minutes prior to the experiment. Indomethacin was purchased from sigma chemicals and used as ulcerogenic agent to induce gastric ulcers. Omeprazole was used as standard ulcer preventing agent, it was provided from University Malaya Medical Centre. Experimental chemicals were suspended in 0.5% carboxyl methyl cellulose (CMC) 30 minutes before feeding the rats and were administered orally by orogastric tube. All chemicals for laboratory experimentation were purchased from sigma chemical. Laboratory kits for prostaglandin E2 (PGE2), Glutathione peroxidase (GPX), superoxide dismutase (SOD) and Malondialdehyde (MDA) were purchased from Cayman chemicals. Blood samples were analysed in the Clinical Diagnostic Laboratory (CDL), University Malaya Medical Centre (UMMC).
This test was performed to identify the safe dose for HPTP based on the OECD 423 guideline. HPTP was administered at 250, 500 and 1000 mg/kg body weight. All mice were deprived from food for 24 hours and water was withdrawn from the mice 2 hour before commencement of the experiment. Forty mice were equally divided in to four groups 5 male and female mice each:
Group 1 (normal control) received vehicle (5 ml/kg CMC)
Group 2 received (a dosage of 250 mg/kg) HPTP chalcone
Group 3 received 500 mg/kg HPTP chalcone
Group 4 received 1000 mg/kg HPTP chalcone
A single dose of the compound was administered to mice. Following administration , the animals were observed for abnormal behaviour (sedation, convulsions, respiratory distress, salivation, changes in skin, fur and etc.) for first 3 hours continuously and at 24 hours for mortality and at least once daily thereafter for 14 days. Feeding was replaced approximately 3–4 hours after dosing . The animals were sacrificed on the 15th day; blood sample was collected for biochemical assay including liver and kidney function. Liver and kidneys were preserved in buffered formalin 10% for 24 hours for histological study.
In this study, indomethacin as a non-steroidal anti-inflammatory drug (NSAID) was used as ulcerogenic agent . A total of 30 adult female SD rats weighting (200–250 gram) were assigned to 5 groups. All groups were fasted for 24 hours prior to the experiment, but allowed free access to drinking water until 2 hours before beginning the experiment. Group 1 and 2 were received CMC. HPTP at either dose of (50, 100 mg/kg body weight) was administered orally to groups 3 and 4, respectively. Omeprazole was administered to group 5 at a dose of 20 mg/kg. After 30 minutes all groups, except group 1, received indomethacin (100 mg/kg Six hours later, the animals were anesthetized by ketamine and xylazine then sacrificed by cervical dislocation . The stomachs were excised quickly after tying the pyloric and cardiac ends and were kept in normal saline. The protective effects of HPTP were, as well, compared with gastric ulcerogenic model observed in rats that were treated with PPI (omeprazole).
Measurement of gastric acidity:
The gastric juice was collected and centrifuged at 4000 rpm for 10 minutes in room temperature. The supernatant was used to measure the acidity of gastric juice by titration with 0.1 N NaOH solution using digital PH meter and the acid content was measured in mEq/L .
Evaluation of macroscopic gastric mucosal lesions
The stomach was opened out along the greater curvature and washed slightly with normal saline. The erosive gastric damage formed on the inner surface of the stomach were counted under a dissecting microscope (1.8X) with square –grid eye piece to assess the ulcers. Ulcer area was then calculated based on the formula proposed by Salga et al. . The lesion area was measured as (lesions area = lesion number x 1.8 x4).
Mucus barrier estimation
Gastric wall mucus was determined based on the modified procedure of Corne et al. . The glandular portion of the stomach was immersed in 10 ml of 0.1% alcian blue solution which contains 1 g/L alcian blue, 0.16 M/L sucrose and sodium acetate 0.05 M/L, PH adjusted to 5.6 with HCL for 2 hours. The sample was then washed by 0.25 M/L sucrose for 15 minutes and then washed for 45 minutes to remove unbounded dye. Magnesium chloride (MgCl2) (10 ml of 0.5 M/L) was used to elute bound dye. For this purpose the sample was immersed in MgCl2 for 2 hours. The resulting blue solution was mixed and shaken with equal volume of diethyl ether for 2 min. The resulting emulsion was then centrifuged at 3000 rpm for 10 min. at room temperature. The absorbance of the aqueous layer was recorded at 605 nm using spectrophotometer. The standard curve was used to calculate the quantity of the extracted alcian blue per gram of glandular tissue (as μg alcian blue/gram tissue weight).
Biochemical investigation of stomach tissues
Tissue homogenate was prepared by homogenizing 0.5 gram of gastric tissue on ice with 5 ml of phosphate buffered saline (PBS) using an ultra- turrax homogenizer for 15 minutes. The mixture was then centrifuged using refrigerated centrifuge at 4°C for 20 minute. The supernatants were collected in eppendorf tubes and used for enzymatic and non enzymatic assessments of the stomach tissue. SOD, GPX and MDA were assessed using the Cayman’s kits (Cayman chemical, USA) according to the manufacture’s protocols. While for measurement of Prostaglandin E2 (PGE2) stomach mucosal tissue (100 mg) was homogenized in1 ml homogenization buffer, which includes 0.1 phosphate buffer, PH7.4, 1 mM EDTA and 10 μM indomethacin. The sample was then centrifuged at 8000 rpm for 10 minutes to eliminate the particular matter. The supernatant was transferred to a clean eppendorf tube to evaluate PGE2 using prostaglandin E2 enzyme immune assay kit (Cayman Chemical Co.).
Stomach tissue samples were fixed in 10% buffered formalin for 48 hours and were then dehydrated by washing by ascending grades of ethanol. Samples were then cleared with xyline and embedded in paraffin wax. Then 5 μm thick slides were sectioned and stained with haematoxylin and eosin using conventional method and examined by light microscope. Following the method of McManus & Mowry  the glandular portion of the rat stomach sections were stained with Periodic Acid-Schiff (PAS) to observe the changes of glycoprotein.
The immunohistochemical staining was conducted according to manufacturer’s protocol (Dako Cytomation, USA) to detect immunohistochemical antibodies such as HSP70, Bax and TGF-β. The tissue sections were place on Poly-L-lysine coated slides, then kept in oven at 60 (C°) for 24 hours in order to increase section adherence to the slide. The slides were deparaffinized by xylene and rehydrated by graded concentrations of alcohol, then placed in retrieval solution and incubated in microwave at high power for 15 min., the slides were cooled then washed with wash buffer. After that the tissue was covered with the (HSP70 1:1000), (TGF 1: 1000), and (Bax 1:50) biotinylated primary antibody and incubated for 30 minutes in dark and humid place, followed by rinsing the slides gently with wash buffer. Subsequently the tissue sections were covered with streptovidin peroxidase and incubated for 30 minutes, then rinsed gently by wash buffer and placed in humid and dark box. After wards the slides were covered with Diaminobenzidine (DAB) chromogen solution and incubated for10 minutes followed by washing with wash buffer, then by tap water. The next step is counter staining with Hematoxylin for 5 minutes followed by washing with tap water until the blue colour disappeared. Finally the tissue sections were dehydrated by graded alcohol, cleared by xylene and mounted as mentioned in the last steps in H&E staining method. The positive result was appeared brown in color.
Descriptive analysis was performed for continuous variables and these variables were shown as mean ± standard error of measurement (SEM). One-way analysis of variance (ANOVA) was used to analyse the study variables between experimental groups in normally distributed data. P value less than 0.05 was considered as statistically significant at the confidence level of 95%.