INTRODUCTION:
Ulcer usually represents an open sore in the epithelial lining of stomach due to the presence of oxidative stress on gastric mucosa or inhibition of prostaglandin synthesis(1). The causative agents of ulcer are alcohol consumption, smoking, stress, obesity, diet, zollinger-ellison syndrome, presence of H.pylori bacteria and excessive consumption of NSAIDs(2). It is usually diagnosed by endoscopy and some specific tests like invasive and non-invasive tests(3). Generally, some gastroprotective medicines are used to treat peptic ulcer disease includes H2 receptor antagonists, proton pump inhibitors and drugs used in triple and quatrlet therapy(4).
Due to side effects from chemical drugs, several herbal drugs came into existence for the treatment of ulcer disease without providing any severe ADR or side effects(2).
Barleria buxifolia L is a perennial herb belonging to the family Acanthaceae. The synonyms for this plant are Barleria acanthodes and Dicranacanthus buxifolia. In Sanskrit and Telugu it is called as Iksura and Erramullugoranta(5). It is usually used as folk medicine in many places of India. The plant had been reported for activities like antibacterial, antifungal, antiurolithiatic, anxiety and antidepressant activity(6). Screening of MEBB showed the presence of alkaloids, flavonoids, saponins, terpenoid and acid compounds. The recent study aimed to evaluate the antiulcer activity of methanolic extract of Barleria buxifolia, yet there is no work performed on this plant regarding to ulcer activity.
MATERIAL AND METHODS:
Plant Material:
The whole plant of Barleria buxifolia Linn was procured from Tirupathi, Chittoor district, Andhra Pradesh. The plant material was identified and authentificated by resident Botanist, Prof. K. Madhava Chetty, Sri Venkateswara University, Tirupathi.
Preparation of Extract:
Freshly collected plants were washed, shade dried under room temperature for three weeks, powdered into a coarse powder and passed through 22 sieve number. Then, 300g of coarse powdered plant was extracted with methanol solvent using soxhlet apparatus. Before and when each extraction, the marc was utterly dried and weighed. Solvent recovery was done with the help of distillation. The filtrate was dried by keeping it in a dessicator.
Experimental Animals:
Albino wistar rats of either sex, weighing 150-200g were selected to study the present research work. The animals were housed in a polypropylene cage. Each cage had 6 animals. The animals were fed with standard rat diet and water ad libitum for one week before and during the experiment. The protocol of experiment was approved by Institutional Animal Ethics Committee (IAEC) of PRRM College of Pharmacy (1423/PO/Re/S/11/CPCSEA).
Drugs and Chemicals:
Drugs like Omeprazole (Sun Pharmaceuticals Pvt. Ltd.), Aspirin (Pallav Chemicals and Solvents Pvt. Ltd. Mumbai) and reagents like Topfer’s reagent and Phenolphthalein reagents (Finar Chemicals Ltd. Ahemdabad), 0.01N NaOH, 1% CMC and Distilled water were used as analytical grade.
Acute Toxicity Studies:
Acute toxicity studies were performed as per the procedure described in OECD guidelines 429. Animals were divided into groups and each group had 3 animals. The animals were fasted for 4hrs with free access of water before the experiment. Then the methanolic extract of plant were given to animals at a dose of 5, 50, 100, 500, 1000, 1500, 2000mg/kg and 4000mg/kg and observed for 24hrs for mortality, physical/behavioural changes(7).
Pylorus Ligation Method:
Albino wistar rats of either sex were selected, having weight of 150-200g. Each group had received 6 animals. Control group of animals received 1% CMC and treatment group of animals were received 200mg/kg and 400mg/kg methanolic extract of B.buxifolia. Omeprazole (20mg/kg) used as standard drug. On 28th day of experiment, the animal stomach was opened, ligated and sutured. After 4hrs, the animals were sacrificed, stomach content was collected, centrifuged and parameters were estimated. Before the day of experiment, animals were fasted for 24hrs by only supply of water.
Aspirin Induced Ulcers:
Albino wistar rats of either sex were selected, weighing 150-200g. Each group had received 6 animals. As per treatment scheduled, all animals were received methanolic extract of plant suspended in 1% CMC for 28 days except control and normal group. On 28th day of experiment, control, standard and treatment groups receives aspirin (250 mg/kg) suspended in 1%CMC with the help of gavage. After 4hrs, the animals were sacrificed, stomach content was collected, centrifuged and parameters were estimated. Before the day of experiment, animals were fasted for 24hrs with free access of water(8).
Evaluation of Parameters:
Gastric Juice Volume:
The stomach was excised carefully, opened along the greater curvature and the gastric contents were removed. The gastric contents were collected in a graduated tube and centrifuged at 2000 rpm for 10mins. The supernatant liquid were collected and expressed as ml/100gm body weight.
Ulcer Score, Ulcer Index and % Inhibition:
After rats were sacrificed, the stomach of a rat was opened along the greater curvature, washed it slowly under running tap water and put it on the slide and observed under 10X magnification for ulcer. Score of ulcer was determined as:
0 = Normal coloured stomach, 0.5 = Red colouration, 1 = Spot ulcers, 1.5 = Haemorrhagic streaks, 2 = Ulcers ≥ 3 but ≤ 5, 3 = Ulcers > 5
The mean ulcer score for each animal was described as Ulcer index. The formula for ulcer index is as follows:
UI = UN + US + UP × 10-1
Where
UI = Ulcer index,
UN = Average number of ulcers as per animal,
US = Average number of severity score in animals,
UP = Percentage of animals with ulcers.
Percentage of ulcer inhibition was calculated as:
Ulcer index control – Ulcer Index test
% Inhibition of ulcer = ---------------------------------× 100
Ulcer index control
Free Acidity and Total Acidity:
The content of gastric juice was centrifuged at 1000rpm for 10 mins. Pipette out 1ml of supernatant liquid and dilute it to 10ml of distilled water. Note the pH of the solution with the help of pH meter. Titrate the solution against 0.01N NaOH solution using topfers reagent as an indicator. Titrate to end point when the solution turns to orange colour. Note the quantity of NaOH that corresponds to free acidity. Titrate further till the solution regains its pink colour by using phenolphthalein as an indicator. Note the total volume of NaOH, that represents total acidity. It can be expressed as mEq/l/100g. Following formula was used to calculate acidity:
Volume of NaOH ×Normality
Acidity = ----------------------------- ×100 mEq/l/100g (7)
0.1
Gastric Juice pH:
The pH of gastric juice was recorded with the help of digital pH meter.
Total Acid Output:
Total acid output = 
It is expressed as µEqL-1/100g body weight(9)
Statistical Analysis:
The results of the experiment were expressed as mean ± SEM. Statistical analysis between different groups were performed by using one way ANOVA followed by Dunnett’s comparison test T with the help of computer software GraphPad Prism 7.01.
RESULTS:
Acute Toxicity Studies:
By performing preliminary phytochemical screening on methanolic extract of plant B.buxifolia L, it showed the presence of certain bioactive compounds like alkaloids, flavonoids, saponins, terpenoid and acid compounds. Acute toxicity study was carried out as per OECD guidelines. The present study states that there was no mortality rate in animals upto the dose of 4000mg/kg. Based on this, 200mg/kg and 400mg/kg doses were selected to carry out the research work.
Pylorus Ligation Induced Ulcer:
Pre-treatment of MEBB at a dose of 200mg/kg and 400 mg/kg to treatment group of animals effectively reduces gastric juice volume, free acidity, total acidity, total acid output, ulcer score and ulcer index while increased in gastric juice pH when compared to control group of animals. But, the MEBB at a dose of 400mg/kg produced significant results.
Aspirin Induced Ulcer:
On 28th day of experiment, treatment group of animals received 200mg/kg and 400mg/kg of MEBB along with 250mg/kg aspirin drug which produced significant results by reducing gastric juice volume, free acidity, total acidity, total acid output, ulcer score and ulcer index by simultaneously increased in gastric juice pH when compared to control group of animals. Pylorus ligation model produced effective results when compared to aspirin induced ulcer model.