Gastric and duodenal ulcer in rats. Synopsis for m. Pharm dissertation




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EFFECT OF NELUMBO NUCIFERA GAERTN METHANOLIC EXTRACT ON

GASTRIC AND DUODENAL ULCER IN RATS.

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE


BY
Mrs. SUJITHA P. J.

DEPARTMENT OF PHARMACOLOGY
UNDER THE GUIDANCE OF
Mrs. Ruby. K. Koshy M. Pharm (PhD)

Asst. Professor

DEPARTMENT OF PHARMACOLOGY



THE OXFORD COLLEGE OF PHARMACY

HONGASANDRA, BANGALORE-68

KARNATAKA
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE II



PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION



1.

Name of the Candidate

and Address

  1. Permanent Address

Sujitha P. J.

D/o S.Prabhakaran,

Jeya Scientific,

TC 37/1905,

West Fort,

Trivandrum-23.



b.Postal Address

Oxford College of Pharmacy ,

No.6/9, 1st Cross, Begur Road,

Hongasandra,

Bangalore-560068,

Karnataka.


2.

Name of the Institute


The Oxford College of Pharmacy,

No.6/9, 1st Cross, Begur Road,

Hongasandra,

Bangalore-560068,

Karnataka.


3.

Course of Study and Subject


Master in Pharmacy

[Pharmacology]

4.

Date of Admission to Course





06 September 2010

5.

Title of the Topic:

EFFECT OF NELUMBO NUCIFERA GAERTN METHANOLIC EXTRACT ON GASTRIC AND DUODENAL ULCER IN RATS”



6.

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.






6.2 Review of Literature:

Tender rhizomes, stems and leaves of lotus are edible and can be cooked along with other vegetables or soaked in syrup15. Rhizomes consist of 1.7% protein, 0.1% fat, 9.7% carbohydrate and 1.1%ash16. Lotus stem consist of 6mg/100gm of calcium, 2.4mg/100gm iron and 0.2mg/100gm of zinc17. Lotus (Nelumbo nucifera Gaertn) is a perennial aquatic crop with stout creeping yellowish white coloured rhizomes. It is both an ornamental plant and a dietary staple in eastern asia, particularly in China18. All parts of Nelumbo nucifiera Gaertn are used for various medicinal purposes in oriental medicine19.



  • The seed of Nelumbo nucifera Gaertn is used in folk remedies as diuretic, cooling agent, antiemetic and antidote in the treatment of tissue inflammation, cancer, skin disease, leprosy and poisoning10, 20.

  • Experimental studies demonstrated that seed of Nelumbo nucifera Gaertn has hepatoprotective and antifertility activities as well as free radical scavenging activity21, 22.

  • It is reported that seed of Nelumbo nucifera Gaertn could suppress cell cycle progression, cytokine genes expression, and cell Proliferation in human peripheral blood mononuclear cells23.

  • The leaf of Nelumbo nucifera Gaertn is considered best for ‘over-coming body heat’, and stopping bleeding24. It is used as a drug for hematemesis, epistaxis, haemoptysis, hematuria and metrorrhagia in traditional chinese medicine25.

  • The leaf of Nelumbo nucifera Gaertn contains several flavonoids and alkaloids, and is effective in the treatment of hyperlipidaemia in rodents 26, 27.

  • The 95% ethanol extract of the leaf of Nelumbo nucifera Gaertn was found to display significant anti-HIV activity19.

  • The stalk extract of Nelumbo nucifera Gaertn showed anti-pyretic effect, while leaf and stamen extracts showed anti-oxidant effect and strong radical scavenging activity28, 29, 30.

  • In China, lotus rhizome is a common vegetable; it can be cooked into different dishes or eaten raw. Especially, it has been applied in Chinese herbal prescriptions to alleviate tissue inflammation, cancer, and liver cirrhosis for a long time31.

  • Lotus alkaloids dilate the blood vessels and reduce the blood pressure. Leaves are bitter, sweet and consist of several flavonoids and alkaloids32.

  • The embryos possess small amount of alkaloids, which are antispasmodic for the intestines and alleviates diarrhoea. The embryos within lotus seeds possess an alkaloid isoquinoline, which is sedative, antispasmodic and beneficial to heart33.

  • Work was done on the rat lens aldose reductase (RLAR) (a principal enzyme of polyol pathway associated with diabetes) inhibitory constituents of stamens of Nelumbo nucifera Gaertn in rat lens. Methanol extract of the stamens exert an inhibitory effect on RLAR. Thirteen flavonoids and seven of its glycosides were isolated from lotus plants along with four non-flavonoid compounds. Among the isolated flavonoids, those possessing 3-O-alpha-lrhamnopyranosyl - (1 → 6) – beta – d - glucopyranoside groups in their C rings, including kaempferol3-O-alpha-l-rhamnopyranosyl-(1→6)-beta-d glucopyranoside and isorhamnetin 3-O-alpha-l-rhamnopyranosyl - (1→6) - beta d- glucopyranoside exhibited the highest degree of RLAR inhibitory activity in vitro evidencing IC50 of 5.6 and 9.0 μM respectively34. nnnnn




    1. Objective of study:

Objective of study:

The objective of the proposed study is to investigate antiulcer activity of Nelumbo nucifera Gaertn extracts in rats using various animal models.



Specific objectives:

  1. Preparation of Nelumbo nucifera Gaertn Methanol extract to study the effect on gastric ulcer healing in acetic acid induced chronic gastric ulcers.

2. Study the antiulcer effect in other models of gastric ulcers and duodenal ulcer to

evaluate effect on gastric secretion and gastric cytoprotection



  • Pylorus ligation induced gastric ulcers.

  • Ethanol induced gastric ulcers.

  • Cysteamine induced duodenal ulcers.

3. Biochemical studies

  • Determination of total acidity35

  • Acid volume 36

  • pH 37

  • Estimation of total protein 38.


7.


MATERIALS AND METHODS:

7.1 Source of Data:

Data will be obtained from CD-rom, Internet facilities, Literatures and related articles from libraries of The Oxford College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and Journals.






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.



EXPERIMENTAL MODELS

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:

Procedure

Qualitative analysis:

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:

Procedure

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.



Experimental Models

  1. 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.



  1. 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.





7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

YES


Study requires investigation on animals. The effects of the drug / extract will be studied on various parameters using rats as experimental animal model.




7.4 Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed.



8.

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  2. Crawford JM. The Gastrointestinal Tract in Robin’s Pathologic Basis of Disease. Cotran, Kumar and Collin (Eds), Saunders, New Delhi 2003; pp.787-802.

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7. Borelli F, Izzo AA. The plant kingdom as a source of anti-ulcer remedies. Phytother Res 2000; 14: 581-91.

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9. Xu Li, Wang Wei. Chinese Materia Medica. Jiangsu New Medical College, Peoples Publishing House, Shanghai, China 1977; pp 147.

10. Chopra RN, Nayar SL, Chopra IC. Glossary Of Indian Medicinal Plants. Council of Scientific Industrial Research, New Delhi, India 1956; pp.174.

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Referring to therapy that relies on drugs.

Mentioned in: Pain Management

pharmacological, pharmacologic

pertaining to pharmacology.
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15. Phillips R, Rix M. Vegetables. Macmillan Reference Books, London 1999; pp.146.

16. Reid BE. Famine Foods of the Chiu-Huang Pen-ts'ao. Southern Materials Centre, Taipei 1977; pp.147

17. Ogle BM, Dao HTA, Mulokozi G, Hambraeus L. Micronutrient composition and nutritional importance of gathered vegetables in Vietnam. International Journal of Food Science and Nutrition 2001; 52: 485-499.

18. Hu M, Skibsted LH. Antioxidative capacity of rhizome extract and rhizome knot extract of edible lotus. Food Chem 2002; 76: 327–333.

19. Kashiwada Y, Aoshima A, Ikeshiro Y, Chen YP, Furukawa H, Itoigawa M, Fujioka T, Mihashi K, Cosentino LM, Morris-Natschkeg SL, Leeg KH. Anti- HIV benzylisoquinoline alkaloids and flavonoids from leaves of Nelumbo nucifera Gaertn, and structure-activity correlations with related alkaloids. Bioorg Med Chem 2005; 13: 443–448.

20. Liu CP, Tsai WJ, Lin YL, Liao JF, Chen CF, Kuo YC. The extracts from Nelumbo nucifera Gaertn suppress cell cycle progression, cytokine genes expression, and cell proliferation in human peripheral blood mononuclear cells Life Sciences2004; 75:699-716.

21. Sohn DH, Kim YC, Oh SH, Park EJ, Li X, Lee BH. Hepatoprotective and free radical scavenging effects of Nelumbo nucifera Gaertn. Phytomed 2003; 10: 165–169.

22. Mazumder UK, Gupta M, Pramanik G, Mukhopadhyay RK, Sarkar S. Antifertility activity of seed of Nelumbo nucifera Gaertn in mice. Indian J Exp Biol 1992; 30: 533–534.

23. Jung HA, Kim JE, Chung HYA, Choi JS. Antioxidant principles of Nelumbo nucifera Gaertn stamens. Archives of Pharmacological Research 2003; 26: 279- 285.

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25. Ono Y, Hattori E, Fukaya Y, Imai S, Ohizum Y. Antiobesity effect of Nelumbo nucifera leaves extract in mice and rats. J Ethnopharmacol 2006; 106: 238–244.

26. Onishi E, Yamada K, Yamada T, Kaji K, Inoue H, Seyama Y, Yamashita S. Comparative effects of crude drugs on serum lipids. Chem Pharm Bul 1984; 32: 646–650.

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28. Sinha S, Pulok K, Mukherjee PK, Mukherjee K, Pal M, Mandal SC, Saha BP. Evaluation of antipyretic potential of Nelumbo nucifera Gaertn stalk extract. Phytother Res. 2000; 14: 272–274.

29. Wu MJ, Wang L, Weng CY, Yen JH. Antioxidant activity of methanol extract of the lotus leaf (Nelumbo nucifera Gaertn). Am J Chinese Med 2003; 31: 687–698.

30. Cho EJ, Yokozawa T, Rhyu DY, Kim SC, Shibahara N, Park JC. Study on the inhibitory effects of Korean medicinal plants and their main compounds on the 1, 1- diphenyl- 2-picrylhydrazyl radical. Phytomed 2003; 10: 544–551.

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32. Shoji N, Umeyama A, Saito N, Iuchi A, Takemoto T, Kajiwara A, Ohizumi Y. Asimilobine and lirinidine, serotonergic receptor antagonists, from Nelumbo nucifera Gaertn. Journal of Natural Products 1987; 50: 773-774.

33. Tomita M, Furukawa H, Yang TH.Alkaloids of Nelumbo Nucifera Gaertn I. Yakugaku Zasshi 1961; 81:469-473

34. Lim SS, Jung YJ, Hyun SK, Lee YS, Choi JS. Rat lens aldose reductase inhibitory constituents of Nelumbo nucifera Gaertn stamens. Pyhtotherapy Research 2006; 20: 825-830.

35. Garrick T, Buack S, Bass P. Gastric motility is a major factor in cold restraint induced lesion formation in rats. Am J Physiol 1986; 250: 6191-99.

36. Takeuchi K, Nobuhara Y. Inhibition of gastric motor activity by 16, 16-dimethyl prostaglandin E2: a probable explanation of cytoprotective. Dig Dis Sci 1985; 32: 1181-1188.

37. Shetty R, Kumar KV, Naidu MUR, Ratnakar KS. Effect of Ginkgo biloba extract on ethanol induced gastric mucosal lesions in rats. Indian J Pharmacol 2000; 32: 313-17.

38. Vanisree AJ, Mitra K, Shyamala Devi CS. Antiulcerogenic effect of UL-409 against experimentally induced gastric ulcer in rats. Indian J Pharmacol 1996; 28: 265-68.

39. Dongmei Yang ME1, Qiushuang Wang ME1, Leqin Ke BE1,2, Jianmei Jiang BE1 and Tiejin Ying PhD, Antioxidant activities of various extracts of lotus (Nelumbo nuficera Gaertn) rhizome Asia Pac J Clin Nutr. 2007;16 (Suppl 1):158- 163.

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41. Asad M, Shewade DG, Koumaravelou K, Abraham BK, Vasu S, Ramaswamy S. Gastric anti-secretary anti-ulcer activity of oxytocin in rats and guinea pigs. Life Sci 2001; 70:17-24.

42. Okabe S, Pfeiffer CJ, Roth JL. Experimental production of duodenal and antral ulcers in rats. Fed Proc 1970; 29:25.

43. Debnath PK, Gore KO, Govinda DD, Sanyal AK. Effect of propranolol on gastric secretion in rats. Br J Pharmacol 1974; 51:213-16.

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45. Shay H, Komarov SA, Fele SS, Meranze D, Gruenstein H, Siplet H. A simple method for uniform production of gastric ulceration in rat. Gastroenterology 1945; 5:43-61.

46. Kulkarni S.K., Hand Book Of Experimental Pharmacology (Third Revised and Enlarged Edition) Vallabh Prakashan 1999; pp. 148-150.

47. Hawk PB, Oser BL, Summerson HW. Practical Physiological Chemistry 12th ed. London: Churchill 1947; pp.347.

48. Lowry CH, Rose borough NI, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951; 193:265-75.

49. Prabha T, Babu MD, Priyambada S, Agrawal VK, Goel RK. Evaluation of Pongamia pinnata root extract on gastric and mucosal offensive and defensive factors in rats. Indian J Exp Biol 2003; 41(4):304-310.

50. Brzozowski T, Konturek SJ, Kwiecien S, Pajdo R, Brzozowski I, Hahn EG et al. Involvement of endogenous cholecystokinin and somatostatin in gastro protection induced by intra duodenal fat. J Clin Gastroenterol 1998; 27:125-137.

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52. Desai JK, Goyal RK, Parmar NS. Characterization of dopamine receptor subtypes involved in experimentally induced gastric and duodenal ulcers in rats. J Pharm Pharmacol 1999; 51:187-92.


9.


SIGNATURE OF THE CANDIDATE



SUJITHA P.J.

10.

REMARKS OF THE GUIDE:

Recommended and Forwarded


11.





    1. Name and Designation of Guide

Mrs. Ruby. K. Koshy

Asst. Professor

Department of Pharmacology.





11.2 Signature








11.3 Head of the Department




Dr. S. Jai Kumar

Professor and HOD

Department of Pharmacology




11.4 Signature





12.


12.1 Remarks of the Principal




Recommended and Forwarded







12.2 Signature


Dr. Padmaa M. Paarakh

Principal

The Oxford College of Pharmacy

Hongasandra, Bangalore-68.









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