In-vivo antiulcer activity 
This study aimed to evaluate the protective potential of ethyl acetate 
extract and isolated pure compounds against stomach ulcers caused by 
pylorus-ligation and ethanol-induced ulcers. The researchers induced 
gastric ulceration in animals by administering ethanol (1 mL/200 g b. 
w.) orally and by pylorus ligation using pentobarbitone (35 mg/kg i.p.). 
2.7.1. Ethanol-induced ulcer 
Disease control (ethanol), positive control (ranitidine) and, test 
samples {ZA/Ea, ethylacetate extract (250 mg/kg); ZA/Eb, ethylacetate 
extract (500 mg/kg); ZA/1a, tambulin (25 mg/kg); ZA/1b, tambulin (50 
mg/kg); ZA/2a, ombuin (25 mg/kg); ZA/2b, ombuin (50 mg/kg)} were 
given to the rats twice a day orally for 5 days prior to ulcer induction. 
According to the method described by Hollander et al., 1985, rats were 
administered with ethanol orally on 6th day (1 mL/200 g, 1 h) to induce 
uniform gastric ulcers and ranitidine was given as positive control at the 
dose of 50 mg/kg, orally. After the rats were euthanized, their stomachs 
were examined along the greater curve to assess the presence of ulcers. 
To determine the ulcer index, the length and width of ulcers in the 
glandular part of the stomach were multiplied (mm
 2
 /rat). This method 
provides a reliable and efficient way of analysing the effects of ethanol 
on the development of gastric ulcers in rats. Ulcer index protection was 
determined by the following formula: 
%Ulcerinhibition = (Ulcerindex
 Control
 ×100/Ulcerindex
 Ulcerindex
 Control  
2.7.2. Pylorus ligated- ulcers 
Test
 )
 Disease control (induced due to accumulation of hydrochloric acid), 
positive control (ranitidine) and test samples, {ZA/Ea, ethylacetate 
extract (250 mg/kg); ZA/Eb, ethylacetate extract (500 mg/kg); ZA/1a, 
3
JournalofKingSaudUniversity-Science36(2024)103326
 4
 tambulin, (25 mg/kg); ZA/1b, tambulin (50 mg/kg); ZA/2a, ombuin 
(25 mg/kg); ZA/2b, ombuin (50 mg/kg)} were given to the rats for 5 
days, and then they were kept in a cage for 18 h without food. Pento
barbitone (35 mg/kg, i.p.) was used for sedation of animals. The belly 
was then opened and the pylorus was tied off without hurting the ani
mals’ blood supply. It was carefully brought back in place, and the wall 
of the belly was closed in two layers with stitches that were broken so 
often. After the surgery, the animals were not given any water (Shay 
et al., 1945). After 4 h, the animals were sacrificed, and the stomach was 
carefully taken out and cut along the larger curve. It was then carefully 
washed with 5.0 mL of 0.9 % NaCl, and ulcers were scored in the 
glandular part of the stomach by someone who wasn’t part of the 
experiment. The ulcer index was estimated by adding the number of 
ulcers on each stomach and how bad each ulcer was. The method given 
by Sanyal et al. (1982) was used to figure out the ulcer score for the 
whole group. 
2.8. Measurement of inflammatory cytokines 
2.8.1. IL-1β, TNF-α, and IL-6 
Were measured using an ELISA kit within 2 hrs, and the concentra
tion was calculated by following the standard procedure. Leukocyte 
infiltration was observed in the deep mucosal layer. A homogenate was 
prepared from the detached scarred fraction. The supernatant collected 
from the homogenate was used for further studies of interleukins 
(Almasaudi et al., 2016). 
2.9. Histopathology 
Histopathology was examined by rats’ stomach which were fixed in 
10 % neutral formalin, then embedded in paraffin blocks. The eosin and 
hematoxylin stains were used for the section visibility. The degree of 
stomach damage was determined using the standard procedure 
mentioned in MICRON desquamative changes, mucosal defects of 
varying depths, and the degree of infiltrative changes. 
2.10. Gastric wall mucus measurement 
Corne et al. (1974) developed a method for measuring gastric wall 
mucus in ethanol-induced ulcerated mice. Glandular segments from 
stomachs were isolated, weighed, and then incubated for 2 h in a 1 % 
Alcian blue solution (0.16 M sucrose in 0.05 M sodium acetate, pH 5.8) 
in test tubes. After centrifuging (100 g) the Alcian blue binding extract 
for 10 min, the absorbency of the supernatant was determined at 498 
nm. The amount of Alcian blue taken from the glandular tissue was then 
determined (µg/g of glandular tissue). 
2.11. Statistical analysis 
The obtained data were processed to determine the significant dif
ference between the groups. Standard mean and error were optimized. 
To summarise the results, a one-way analysis of variance (ANOVA) was 
employed. Version 5.01 of GraphPad PRISM (GraphPad Software, Inc., 
USA) was used. The value p