BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Gastric ulcer is an illness that affects a considerable number of people worldwide1. The development and progression of gastric ulcer depends to some extent on the type of the food consumed by the patient. It has been shown that spicy food, fatty food or foods containing caffeine stimulates acid secretion in stomach and increase the risk of ulcer formation2. It is among the most serious diseases in the world. The etiology of gastro-duodenal ulcers is influenced by various aggressive and defensive factors such as acid-pepsin secretion, parietal cell, mucosal barrier, mucus secretion, blood flow, cellular regeneration and endogenous protective agents (prostaglandins and epidemic growth factors)3. The gastric mucosa is continuously exposed to potentially injurious agents such as acid, pepsin, bile acids, food ingredients, bacterial products (Helicobacter pylori) and drugs4. On the other hand, food containing flavonoids like apples, celery, cranberries and onion may inhibit the growth of H. pylori and may reduce the development of H. pylori induced gastric ulcers. High fiber diets such as potatoes, banana, peas, and beans etc, also reduce the development of duodenal ulcers and the same is true with fruits rich in vitamin C5. These agents have been implicated in the pathogenesis of gastric ulcer including enhanced gastric acid, pepsin secretion, inhibition of prostaglandin synthesis, cell proliferation growth, diminished gastric blood flow and gastric motility6. Drug treatment of peptic ulcers is targeted at either counteracting aggressive factors or stimulating the mucosal defences7.
Nelumbo nucifera Gaertn belongs to the family of Nelubonaceae, which has several common names: e.g. Indian lotus, Chinese water lily and sacred lotus Synonyms – Nelumbium nelumbo, Nelumbo speciosum and Nymphaea nelumbo. Lotus is a perennial, large and rhizomatous aquatic herb with slender, elongated, branched, creeping stem consisting of nodal roots. Leaves are membranous, peltate (60-90 cm and above), orbicular and concave shaped, petioles are long, rough with small distinct prickles and flowers are white to rosy, sweet scented8.
It is reported to possess a wide variety of pharmacological actions, among which, the best known are its effect on the anti inflammatory activity and as anti emetic9. It is believed in the traditional medicine that consumption of aqueous extract of Lotus can cure stomach problems10.
Ulcer therapy has progressed from vagectomy to anti-cholinergic drugs, histamine-H2 receptor antagonists, antacids and more recently to proton pumps inhibitors which revolutionized the treatment of peptic ulcers and other gastro intestinal disorders, but there is still no complete cure for this disease11. It has been shown that long term use of these drugs leads to various adverse and side effects. Relapses of the malady, ineffectiveness of different regimens and even resistance to drug are emerging. In recent years, there has also been growing interest in alternative therapies and the use of natural products, especially those derived from plants12, 13.
Plant extracts are shown to have more attractive sources of new drugs and results for the treatment of gastric ulcer. Thus, there is an urgent requirement to identify more effective and safe anti- ulcer agent14.
Since, Nelumbo nucifera Gaertn is widely used as medicine for the treatment of various diseases, the present study was undertaken to evaluate its effect on the development and healing of experimentally induced gastric and duodenal ulcers in rats.
7.2 Method of Collection of Data:
The data collected will be based on animal experimentation as per the parameters studied under each animal model, which are mentioned under the objectives of the study.
Source of animals :
Wistar albino rats of either sex weighing between 150-200 gm will are obtained from animal house of Nimhans, IISC and Veterinary Hospital.
Preparation of extract:
The Methanolic extract of Nelumbo nucifera Gaertn will be tested for different phyto constituents like alkaloids, tannins, steroids, saponins, flavonoids, terpenoids, glycosides, amino acids and carbohydrates39.
Acute toxicity study40:
Overnight fasted rats will be used. Each dose group should consist of 10 rats. Drug will be injected by intraperitoneal route and will be observed for 2 hours for death due to acute toxicity. The percent mortality values will be converted to probit values by reading the corresponding probit units. Plot the probit values against log doses and read LD50 from the graph.
Lotus Rhizome Extract:
The rhizomes of Nelumbo nucifera Gaertn will be collected from the medicinal garden and forest and dried. The shade dried rhizomes were powdered. About 500gm of dried powder will be extracted with methanol by continuous hot percolation, using soxhlet apparatus the resultant extract was concentrated up to 100 ml on Rota vapour under reduced pressure. The concentrated crude extracts will be lyophilized in to powder and used for the study.
Acetic acid- induced chronic gastric ulcer41, 42.
The animals (n=4) will be fasted for 24 h prior to the experiment. Under light ether anesthesia, the abdomen will be opened by midline incision below the xiphoid process and the stomach will be exposed. Glacial acetic acid (0.05 ml) will be added to the cylindrical mould of 6.0 mm diameter placed tightly over the anterior serosal surface of the stomach and this will be allowed to remain over there for 60 sec. The acid solution was removed by rinsing the mould with normal saline twice or thrice to avoid damage to the surrounding tissues. The stomach was placed back carefully and the abdominal wall will be closed. The animals will be treated with vehicle, methanolic extract of Nelumbo nucifera Gaertn or ranitidine once daily for 10 days after induction of ulcer while the control group received only vehicle. Rats were sacrificed on the 10th day, stomach will be removed and cut opened along the greater curvature. The volume of gastric juice will be measured and this will be used for estimation of free acidity35, total acidity35, pepsin content43 and total proteins38. The ulcer index will be determined using the formula44.
Ulcer index = 10/X
Where X = Total mucosal area / Total ulcerated area.
The ulcers will be given scores based on their intensity as follows
0 = no ulcer, 1 = superficial mucosal erosion, 2 = deep ulcer or transmural necrosis,
3 = perforated or penetrated ulcer.
2. Pylorus ligation induced ulcers (Shay rat)45,46
The animals (n=4) will be fasted with water for 24 h before pylorus ligation. Normal saline (1 ml/rat, p.o.) will administered twice daily to all the animals. Under light ether anesthesia, the abdomen was opened by midline incision below the xiphoid process. The pyloric portion of the stomach will be slightly lifted out and ligated, avoiding damage to its blood supply. The methanolic extract of Nelumbo nucifera Gaertn, ranitidine or normal saline will be administered intraduodenally immediately after pylorus ligation. The stomach will be placed back carefully and the abdominal wall will be closed with sutures. Animals will be sacrificed 6 h after pylorus ligation. The stomach will be isolated and the content of the stomach was collected and centrifuged. The volume of the gastric juice was measured and this will be used for estimation of free acidity47, total acidity47, pepsin content43 and total proteins48. The ulcer index will be determined as mentioned above and the gastric mucus content will be estimated49.
3. Ethanol induced ulcers50
All the animals (n=4) will be fasted for 36 h before administration of ethanol. The standard drug (misoprostol 100 µg/kg, p.o.) or methanolic extract of Nelumbo nucifera Gaertn will be administered 1 h before ethanol administration. Ethanol (90%) will be administered to all the animals at a dose of 1ml/200 g and after 1h, the animals will be sacrificed, stomach will be isolated and ulcer index will be determined as mentioned above.
Cysteamine induced duodenal ulcers51
Duodenal ulcers will be induced by administering cysteamine hydrochloride (400 mg/kg, p.o.) twice at an interval of 4h. Methanolic extract of Nelumbo nucifera Gaertn or ranitidine (50 mg/kg, p.o.) will be administered 30 min prior to each dose of cysteamine hydrochloride. After 24 h, all the animals (n=4) will be sacrificed by over dose of ether anesthesia and the duodenum will be excised carefully and cut opened along the antimesentric side. The duodenal ulcer area, ulcer score and ulcer index will be determined.
The ulcers will be given scores based on their intensity as follows
0 = no ulcer, 1 = superficial mucosal erosion, 2 = deep ulcer or transmural necrosis, 3 = perforated or penetrated ulcer.
The ulcer index will be calculated using the following equation52
U.I. = Arithmetic mean of + number of ulcer positive animals x 2
intensity in a group Total number of animals
Statistical Analysis: All data will be expressed as mean ± SD. Student’s t-test will be performed for each experimental group. Data will be compared by analysis of variance (ANOVA) and only values with P<0.05 will be considered as significant.
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