The present paper relates to the pharmacological validation of the antiulcer efficacy of ethanol leaf extract of
In many parts of the world the indigenous systems of medicines are still used effectively and claimed to have cured innumerable diseases which sans complete medical recordings. The origin of this indigenous medicine system dates back before 2000 years. Current modern medicine systems and the standardized principles can be used to test the hypothesis of traditional claims. The use of indigenous medicines is being limited to small tribal and geographical areas as in many parts of Africa and other countries. Peptic ulcer disease (PUD) is one of the oldest diseases known to human kind. The term PUD generally refers to a spectrum of disorders that includes gastric ulcer (GU), pyloric channel ulcer, duodenal ulcer (DU), and postoperative ulcers at or near the site of surgical anastomosis [
Long-term use of NSAIDs (nonsteroidal anti-inflammatory drugs) is the second most common cause of ulcers, and the rate of NSAID-caused ulcers is increasing. About 20 million people take prescription NSAIDs regularly, and more than 25 billion tablets of over-the-counter brands are sold each year in the US alone. The most common NSAIDs are aspirin, ibuprofen, and naproxen [
Thus, the present study pertains to the evaluation of antiulcer efficacy of ethanol leaf extract of
The leaves of
Freshly collected leaves of
Phytochemical screening of the EEMT extract was performed using the reagents and chemicals as follows. Alkaloids with Mayer’s, Hager’s, and Dragendorff’s reagent. Flavonoids with the use of sodium acetate, ferric chloride, and amyl alcohol. Phenolic compounds and tannins with lead acetate and gelatin. Carbohydrate with Molish’s, Fehling’s, and Benedict’s reagent [ Proteins and amino acids with Millon’s, and Biuret Xanthoprotein test. Saponins test using the hemolysis method. Sterols with 5% potassium hydroxide. Steroids with Libermann Burchard’s test. Saponins with foam and lead acetate test. Terpenes with thionyl chloride. Glycosides with ferric chloride, acetic acid, and concentrated sulphuric acid. Gum tested using Molish’s reagent and ruthenium red. Coumarin by 10% sodium hydroxide and quinones by concentrated sulphuric acid.
These were identified by characteristic color changes using standard procedures [
The screening results were as follows: alkaloids +; carbohydrates +; proteins and amino acids +; steroids −; sterols +; phenols +, flavonoids +; gums and mucilage +; glycosides +; saponins −; terpenes +, and tannins −ve.
Where + and − indicates the presence and absence of compounds.
This was performed for the extracts to ascertain safe dose by the acute oral toxic class method by the Organization of Economic Cooperation and Development (OECD). A single administration of starting dose of 2000 mg/kg body weight/p.o. of the EEMT was administered to three female rats, and the rats were observed for three days to evaluate considerable changes in body weight and other signs of toxicity. There was no considerable change in body weight before and after treatment and no sign of toxicity was observed. When the experiment was repeated again with same dose level, 2000 mg/kg body weight/p.o. of plant extract for 7 more days and observed for fourteen days no change was observed from the experiments.
Colony inbred strains of Wistar rats weighing 250–300 g, obtained from C. L. Baid Metha College of Pharmacy were used for the pharmacological studies. The animals were kept under standard conditions maintained at 23–25°C, 12 hr light/dark cycle, and given water and standard pellet diet (Hindustan lever, Bangalore) provided
Wistar rats of either sex weighing 180 to 250 g were divided into five groups of six animals each. Animals were placed in cages with grating floor to avoid coprophagy in fasting period.
Vehicle control. The animals received 1% CMC (carboxy methyl cellulose) served as control. + PL (pylorus ligated).
The animals received aspirin (200 mg/kg body wt./p.o. only from day 8–10) + PL.
The animals received aspirin (200 mg/kg body wt./p.o.) and EEMT suspended in water at a dose of (200 mg/kg body wt./p.o.) + PL.
The animals received aspirin (200 mg/kg body wt./p.o.) EEMT suspended in water at a dose of (400 mg/kg body wt./p.o.) + PL.
The animals received aspirin (200 mg/kg body wt./p.o.) and ranitidine (50 mg/kg body wt./p.o.) + PL.
Groups III, IV, and V received the assigned drug treatment for the respective 1–10 days daily. From days 8 to 10 (3 days), animals of groups II, III, IV, and V received aspirin orally as an aqueous suspension at the dose of 200 mg/kg., 2 h after the administration of the drugs. Animals in all groups were fasted for 18 h after the assigned treatment, anaesthetized, and the pylorus was ligated. The rats were sacrificed after 4 h by excess anesthesia (ether). The stomach was cut open along the greater curvature and the contents drained into small beaker, centrifuged, and then subjected to analysis for following acid secretory and biochemical parameter. The mucosa was flushed with saline and the stomach was pinned on frog board and the ulcer score was calculated [
After sacrificing the rat, the stomach portion was removed. The gastric contents were transferred in to centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant liquid was then transferred to a measuring cylinder, and the volume was measured.
1 mL of the gastric juice was collected, and pH was directly measured by using pH meter.
1 mL of gastric juice was pipette out into a 100 mL conical flask. 2 to 3 drops of Topfer’s reagent were added and titrated with 0.01 N NaOH (which was previously standardized with 0.01 N of oxalic acid) until all the trace of the red color disappeared and the color of the solution was yellowish orange. The volume of the alkali added was noted. This volume corresponds to free acidity. Then 2 to 3 drops of phenolphthalein solution were added and titration was continued until a definite red tinge reappears. Again the total volume of alkali was noted. This volume corresponds to total acidity. Acidity was calculated by using the formula:
(i) Alkaline copper reagent. Solution A: 2% sodium carbonate in 0.1 N sodium hydroxide. Solution B: 0.5% copper sulphate in 1% sodium potassium tartrate. 50 mL of solution A was mixed with 1 mL of solution B just before use. (ii) Folin’s phenol reagent. One volume of Folin’s reagent was diluted with two volumes of distilled water just before use. (iii) Standard bovine serum albumin. 20 mg of bovine serum albumin was dissolved in 100 mL of distilled water. Few drops of NaOH was added to aid complete dissolution of bovine serum albumin and to avoid frothing, it was allowed to stand overnight in a refrigerator.
The dissolved proteins in gastric juice were estimated in the alcoholic precipitate obtained by adding 90% of alcohol with gastric juice in 9 : 1 ratio respectively. Then 0.1 mL of alcoholic precipitate of gastric juice was dissolved in 1 mL of 0.1 N NaOH and from this 0.05 mL was taken in another test tube. To this 4 mL of alkaline copper reagent was added and kept for 10 minutes. Then 0.5 mL of Folin’s phenol reagent was added and again 10 minutes was allowed for color development. Reading was taken against blank prepared with distilled water at 640 nm. The protein content was calculated from standard curve prepared with bovine albumin and has been expressed in terms of
Sodium stock solution was prepared by dissolving 0.584 g NaCl (equivalent to 0.23 gm of sodium) in 100 mL of distilled water. 1, 2, 3, 4, and 5 mL were pipette out in 5 different 100 mL volumetric flask, and volume is made up to 100 mL. This solution will contain 2.3, 6.9, 8.2, and 11.5 mg of sodium in 100 mL or 1, 2, 3, 4, and 5 millimoles (ppm) of sodium, respectively. Appropriate filter is chosen and the flame intensity is adjusted to 100 units by spraying the highest concentration of the stock solution. The concentration of sodium present in the gastric juice was determined by using a Systronics Mediflame 127. The flame intensity of the gastric juice was noted. The concentration of sodium was calculated from the graph. A standard curve was plotted taking conc. in
Potassium stock solution was prepared by dissolving 0.74 g KCl (equivalent to 0.39 gm of potassium chloride) dissolved in 100 mL of distilled water. 0.5, 1, 2, 3, 4, and 5 mL were pipette out in 6 different 100 mL volumetric flask, and volume is made up to 100 mL. This solution will contain 1.95 mg, 3.9 mg, 5.85 mg, 7.8 mg, and 9.75 mg of potassium in 100 mL or 0.5, 1, 1.5, 2, and 2.5 millimoles of potassium respectively. The concentration of potassium present in the gastric juice was determined by using a Systronics Mediflame 127. The flame intensity corresponding to the concentration of stock solution was noted by using appropriate filters. The results were plotted in a graph. The flame intensity of the gastric juice was noted. The concentration of potassium ions was calculated from the graph. The results are expressed in terms of mg/L.
Wistar rats of either sex weighing 180 to 250 g were divided into five groups of six animals each. Animal were placed in cages with grating floor to avoid coprophagy in fasting period.
The animals received water and served as control.
The animals received cysteamine HCl dissolved in normal saline (30 mg/kg body weight/s.c).The animals received cysteamine HCl dissolved in normal saline (30 mg/kg body weight/s.c).
The animals received EEMT suspended in water (200 mg/kg body weight./p.o.) + cysteamine HCl (30 mg/kg body weight/s.c).
The animals received EEMT suspended in water (400 mg/kg body weight./p.o.) + cysteamine HCl (30 mg/kg body weight/s.c).
The animals received pantoprazole in normal saline (10 mg/kg body weight./p.o.) + cysteamine HCl (30 mg/kg body weight/s.c).
Groups III, IV, and V received the assigned drug treatment for the respective 5 days daily. On the 5th day of drug treatment the animals were kept on fast from 10:00 h after the administration of morning dose and last dose of the test drug was then administered at 15:00 h, 1 h prior to administration of Cysteamine HCl (30 mg/kg body weight/s.c.). The fasting was then continued overnight and the animals were sacrificed at 10:00 h on the following day, and the duodenum was exposed and scored for the presence or absence of ulcers on the anterior and posterior wall of the duodenum near the pyloric end.
Stomach and duodenum obtained from pharmacological studies were immersed in 10% formalin for 24 h for histopathological examination. After standard processing, the cut tissue was embedded in paraffin (Automatic Tissue processor, Lipshaw) and cut into 5
The data represents mean ± SEM. Results were analyzed statistically using one-way ANOVA followed by Dunnett’s test. The minimum level of significance was set at
The gastric volume was significantly increased (
Effect EEMT on gastric volume in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Gastric volume (mL/100 gm) |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Aspirin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The pH level was significantly decreased (
Effect EEMT on pH in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | pH |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The ulcer score was significantly increased (
Effect EEMT on ulcer score in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Ulcer score |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The ulcer severity was significantly increased (
Effect EEMT on ulcer severity in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Ulcer severity |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard–ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The free acidity (mEq/l/100 g) was significantly increased (
Effect EEMT on free acidity in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Free acidity (mEq/l/100 g) |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The total acidity (mEq/l/100 g) was significantly increased (
Effect EEMT on total acidity in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Total acidity (mEq/L/100 g) |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The total protein (
Effect EEMT on total protein in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Total protein ( |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The sodium ion concentration in the gastric juice (mg/L) was significantly decreased (
Effect EEMT on sodium ion in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Sodium ion mg/L |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The potassium ion concentration in the gastric juice (mg/L) was significantly decreased (
Effect EEMT on potassium ion in aspirin + pylorus ligated gastric ulcer.
Group | Treatment | Potassium ion mg/L |
---|---|---|
I | Control (0.5% SCMC) + PL | |
II | Asprin (200 mg/kg) + PL | |
III | Suspension of EEMT (200 mg/kg) + PL | |
IV | Suspension of EEMT (400 mg/kg) + PL | |
V | Standard-ranitidine (50 mg/kg) + PL |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
The ulcer score was significantly increased (
Effect EEMT on ulcer scores in cysteamine HCl induced duodenal ulcer.
Group | Treatment | Ulcer score DU |
---|---|---|
I | Control (water) | |
II | Cysteamine HCl (30 mg/kg) in n.s | |
III | Suspension of EEMT (200 mg/kg) + Cysteamine HCl (30 mg/kg) | |
IV | Suspension of EEMT (400 mg/kg) + Cysteamine HCl (30 mg/kg) | |
V | Pantoprazole (10 mg) + cysteamine HCl (30 mg/kg) |
The values are expressed as mean ± SEM of 6 animals.
Comparisons were made between agroup I with group II and bgroup II with III, IV, and V.
Statistical significant test for comparison was done by ANOVA, followed by Dunnett’s
Pylorus ligation and aspirin-treated (200 mg/kg) rats showed sharply defined mucosal ulcer in stomach. Damaged mucosal epithelium and cellular debris were found in the ulcerated wall of stomach (Figure
Histopathology of stomach after exposure to aspirin plus pylorus ligation EEMT.
Group I
Group II
Group III
Group IV
Group V
EEMT at both dose levels was found to preserve the functional cytoarchitecture of the duodenum in the damaged regions (Figures
Histopathology of duodenum in Cysteamine-induced duodenal ulcer EEMT.
Group I
Group II
Group III
Group IV
Group V
Preliminary phytochemical analysis of the EEMI shows rich possession of phytochemical such as alkaloids, carbohydrates, proteins and amino acids, sterols, phenols, flavonoids, gums and mucilage, glycosides, and terpenes which are potent antioxidants and most of them have been reported for antiulcer activity and antibacterial activity [
Treatment with EEMT at the dose of 200 and 400 mg/kg b.w showed significant reduction in volume of gastric juice (
EEMT at both the dose levels of 200 and 400 mg/kg b.w showed significant increase in pH of the gastric juice to near normal (
Administration of EEMT at the dose of 200 and 400 mg/kg b.w showed significant increase in sodium and potassium ions in gastric juice (
EEMT at the dose of 400 mg/kg b.w showed significant reduction in duodenal ulcer (
The authors are grateful to Dr. S. Venkataraman (director of C.L. Baid Metha Foundation for Pharmaceutical Education and Research, Chennai) for his technical and secretarial assistance. The authors have no conflict of interest to report.