SYED MAHBOOB AHMAD.pdf

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“A STUDY ON ANTI-ULCER AND ANTI-INFLAMMATORY

ACTIVITIES OF TUBER EXTRACTS OF SOLANUM TUBEROSUM

(SOLANACEAE) IN RATS”

By

SYED MAHBOOB AHMAD

B. Pharm.

Dissertation Submitted to the Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru

In partial fulfillment of the requirements for the degree of

MASTER OF PHARMACY

IN

PHARMACOLOGY

Under the Guidance of

Dr. T. SHIVARAJ GOUDA Associate Professor

Department of Pharmacology

V. L. College of Pharmacy Raichur- 584103

2011

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Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru.

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation/thesis entitled “A STUDY ON ANTI-ULCER

AND ANTI-INFLAMMATORY ACTIVITIES OF TUBER EXTRACTS OF SOLANUM

TUBEROSUM (SOLANACEAE) IN RATS” is a bonafide and genuine research work

carried out by me under the guidance of Dr. T. Shivaraj Gouda, Associate Professor,

V.L. College of Pharmacy, Raichur.

Date: Place: Raichur (Syed Mahboob Ahmad)

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Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru. Dr. T.Shivaraj Gouda Dept of Pharmacology M.Pharm., Ph. D. V.L. College of Pharmacy Associate Professor Raichur – 584 103.

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “A STUDY ON ANTI-ULCER AND

ANTI-INFLAMMATORY ACTIVITIES OF TUBER EXTRACTS OF SOLANUM

TUBEROSUM (SOLANACEAE) IN RATS” is a bonafide research work done by

Syed Mahboob Ahmad in partial fulfillment of the requirement for the degree of Master

of Pharmacy in Pharmacology.

Date: Signature of the Guide Place: Raichur

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Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru.

Dr. N. Venkat Rao Dr. S.M. Shantha Kumar M.Pharm., Ph D. M.Pharm., Ph. D, F.I.C Professor & HOD Principal Dept. of Pharmacology V.L. College of Pharmacy V.L. College of Pharmacy Raichur – 584 103. Raichur – 584 103.

ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE

INSTITUTION

This is to certify that the dissertation entitled “A STUDY ON ANTI-ULCER AND

ANTI-INFLAMMATORY ACTIVITIES OF TUBER EXTRACTS OF SOLANUM

TUBEROSUM (SOLANACEAE) IN RATS” is a bonafide research work done by

Syed Mahboob Ahmad under the guidance of Dr. T. Shivaraj Gouda, Associate

Professor, V.L. College of Pharmacy, Raichur.

Dr. N. Venkat Rao Dr. S.M. Shantha Kumar M. Pharm., Ph. D. M.Pharm., Ph. D, F.I.C

Date: Date:

Place: Raichur Place: Raichur

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COPYRIGHT

DECLARATION BY THE CANDIDATE

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka

shall have the rights to preserve, use and disseminate this dissertation in print or

electronic format for academic / research purpose.

Date:

Place: Raichur (Syed Mahboob Ahmad)

© Rajiv Gandhi University of Health Sciences, Karnataka.

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Dedicated To My Beloved Family

Members

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Acknowledgement

First of all, I am grateful to the omnipresent, omniscient

and omnipotent, the Almighty ALLAH without whose grace I

could never have done the work and to whom I am

accountable for my every act done in this mundane world.

This thesis is the end of my journey in obtaining my

postgraduate degree in Pharmacology. I have not travelled in

a vacuum in this journey and some people who made this

journey easier with words of encouragement to expand my

thoughts and ideas.

It is an exciting, unforgettable and memorable moment

of my life, to express my profound and heartful thanks to my

beloved research guide Dr. T. SHIVARAJ GOUDA, Associate

Professor, Department of Pharmacology, V.L. College of

Pharmacy , Raichur, Karnataka, who has provided excellent

guidance. I am highly indebted to him for his valuable

presence, which helped me to complete this work successfully.

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I express my sincere gratitude, thanks and respect to

Dr. N.VENKAT RAO, Professor and Head Department of

Pharmacology, V.L. College of Pharmacy, Raichur, excellent

guidance during thesis work.

With pride and pleasure, I wish to express my thanks to

Dr. S.M SHANTHA KUMAR, Principal, V.L. College of Pharmacy,

Raichur, for his valuable help to carry out this work with great

ease and precision.

I express my heartful thanks to Dr. D. JEEVAN MANI BABU,

Asst. Professor, Dept. of Pharmacology for his help during the

work.

I wish to offer my respectable thanks to Dr. V. Hemanth

Kumar, Vice principal, V.L. College of Pharmacy, Raichur for

his support and kind cooperation.

I wish to offer my respectable thanks to Dr. K. M. SWAMY,

Dr. MD SALAHUDDIN, Dr. SREENIVAS, Dr. AYESHA, Mrs. HAFSA

MOHAMMADI, Dr. SHALAM, Sri MD SHAMSUDDIN V.L.College of

Pharmacy, Raichur for their encouragement during the course

of study.

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I express my sincere and heartful thanks to my seniors

Mr. BASHA, Mr. YASAR, Mr. NAZEER, Mr. HASNUDDIN,

Mr. RAJESH, Mr. NISHANT, Mr. VENU and Ms. MINHAZ. I thank

them all for their timely support and words of encouragement.

I would like to place on record the constant

encouragement and moral support to my batch mates

Mr. RAJESH, Mr. FASIH, Mr. BASHEER, Mr. MANOJ, Mr. RIZWAN,

Mr. SHIV, PETRICEA, RADHIKA and SWOMYA for completing this

task.

I express my thanks to all my juniors Mr. Vinod,

Mr. Karthik, Mr. Snehal, Mr. Riyaz, Mr. Promod, Mr. Lakshmi

Kanth, Mr. Maruthi, Ms. Manisha, Ms. Aruna, Ms. Sunanda,

Ms. Regina and Ms. Soumya for their constant, affectionate,

encouragement throughout my project work.

I express my sincere and heartful thanks to my Dy. Sales,

RSM and Dy. RSM of Macleod Pharmaceutical Ltd.

Mr. Sandeep Nayyar, Mr. Vivek Shael Mishra, and Mr. Omesh

Bhat, Delhi. I thank them all for their timely support and

words of encouragement.

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I thank Smt Vasundhra, Sri Satyanarayan, Sri RK Patil,

Smt Vijaylakshmi, Sri Kantacharya, Sri Shekhar, Sri Gopal Rao

and Mareppa, for their cooperation and help throughout the

project work.

I extend my profound respect and heartful gratitude to

my beloved father JB SYED MAHMOOD AHMAD who always

covered me under the shade of love, affection and blessings

and my warmest of warm and the most important

acknowledgement, by far, is to my mother UMME HABIBA who is

the constant source of love, care, support, encouragement and

blessing. I am indebted for them for whole of my life.

I also thank my brothers JB HABIB AHMAD, SYED ZAFAR

ALI and SYED IMRAN SHAHID, my loving sisters GULRANA

PARVEEN, GUFRANA PARVEEN & ZEENAT FATIMA, Brother-In-

Law MD IDREES SIDDIQUE, SUHAIL SIDDIQUE and MASROOR

HUSSAIN my nephew AMAN & UMAR neice ZOYA and MAHIRA.

Whose moral and mental support, encouragement and prayers

have been the driving force behind my efforts and are

responsible for whatever I am today.

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Finally, I thank all those people who directly or indirectly

helped me and encouraged me during each and every step of

my life.

Date:

Place: Raichur (SYED MAHBOOB AHMAD)

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LIST OF ABBREVIATIONS

1. OC - Degree centigrade

2. ANOVA - Analysis of variance

3. Kg - Kilogram

4. LD50 - Lethal dose

5. mg - milligram

6. ml - millilitre

7. µg - microgram

8. Min - minutes

9. OECD - Organization for Economic Co-operation and Development

10. p.o. - Per Oral

11. S - Seconds

12. SEM - Standard Error Mean

13. i.p - Intra peritoneal

14. ED50 - Effective dose50

15. AOT - Acute Oral Toxicity

16. Fig. - Figure

17. IAEC - Institutional Animal Ethical Committee

18. NSAID’s - Nonsteroidal anti-inflammatory drugs

19. NaOH - Sodium hydroxide

20. %ROV - Percentage reduction in oedema volume

21. SD - Standard Deviation

22. vs - Versus

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23. WHO - World Health Organization

24. Vol - Volume

25. S. tuberosum - Solanum tuberosum

26. NaCl - Sodium Chloride

27. H. pylori - Helicobacter pylori

28. AETST - Alcoholic Extract of Tubers of Solanum tuberosum

29. AQETST - Aqueous Extract of Tubers of Solanum tuberosum

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ABSTRACT

Objectives:

To evaluate the anti-ulcer and anti-inflammatory activities of tubers extracts of

S. tuberosum in rats.

Background:

In Ayurvedic texts it was reported that tubers of S. tuberosum are used for anti-

ulcer, anti-gout, anti-inflammatory, anti-arthritic, diuretic, anti-scurvy and to increase

milk in lactating mothers. For external use, the grated raw S. tuberosum is applied

locally in cases of arthritis, itching, neuralgia and in mild burns. Since no scientific data

is available on anti-ulcer and anti-inflammatory activities of tubers extracts (alcoholic

and aqueous) of this plant, hence the present work is planned to evaluate the above

mentioned activities in experimental animal, rats.

Materials and methods:

For assessing anti-ulcer and anti-inflammatory activities, ulcer models like

pylorus ligation induced ulcer and stress induced ulcers by cold water immersion

models and carrageenan, histamine and formalin induced inflammatory models are

used. Standard reference compounds like Ranitidine and Ibuprofen respectively are

used in the above mentioned two models.

Resuts:

When AETST and AQETST are subjected for LD50 study none of them produced

behavior abnormalities or mortality even at the dose level of 2000 mg/kg body weight.

Preliminary phytochemical investigation reveals the presence of tannins, carbohydrate,

sterols, flavonoids, glycosides, alkaloid and triterpines in both the extracts.

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In pylorus ligation induced ulcer model, selected doses like low (100 mg/kg),

medium (200 mg/kg) and high (400 mg/kg) doses (AETST and AQETST) of both the

extracts significantly reduced the ulcers (P < 0.05*, 0.01** and 0.001***). In stress

induced ulcers by cold water immersion model also both the extracts with the doses

mentioned above significantly reduced the ulcers (P < 0.05*, 0.01** and 0.001***).

In carrageenan, histamine and formalin induced paw oedema inflammatory

models both the extracts with the 3 selected doses mentioned above significantly

reduced inflammation.

Conclusion:

The present study reveals that both the AETST and AQETST possessed both anti-

ulcer and anti-inflammatory activities. Phytochemical constituent like tannins,

flavonoids and triterpines are already reported for their anti-ulcer and anti-inflammatory

activities. These phytochemical constituents are present in both the extracts, hence

responsible for the observed activities.

Key words:

S. tuberosum, tubers, alcoholic and aqueous extracts, anti-ulcer activity, pylorus

ligation, stress, anti-inflammatory activity, carrageenan, histamine and formalin.

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TABLE OF CONTENTS

S. No.

Contents

Page No.

1

Introduction

1-3

2

Objectives of the study

4

3

Review of Literature

5-47

4

Methodology

48-62

5

Results

63-88

6

Discussion

89-91

7

Conclusion 92

8

Summary

93

9

Bibliography 94-103

10

Annexures

104-107

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LIST OF TABLES

S. No. Tables Page No.

1 Table No: 3.1 Name of the plants reported for anti-ulcer activity. 28

2 Table No: 3.2 Summary of the principal substances released in inflammation. 35

3 Table No: 3.3 Name of the plants reported for anti-inflammatory activity. 45

4 Table No: 3.4 Name of the plants reported for anti-ulcer and anti-inflammatory activities. 47

5 Table No: 4.1 List of materials and equipments used during experiment. 48

6 Table No: 4.2 Nature and Percentage yield of the extracts. 50

7 Table No: 4.3 Details of qualitative phytochemical tests. 51

8 Table No: 4.4 Ulcer scores. 56 9 Table No: 5.1 Antiulcer effects of AETST and AQETST in

pylorus ligation model in rats.

66

10 Table No: 5.2 Antiulcer effects of AETST and AQETST in pylorus ligation model in rats.

67

11 Table No: 5.3 Antiulcer effects of AETST and AQETST in stress induced ulcer model in rats.

68

12 Table No: 5.4 Antiulcer effects of AETST and AQETST in stress induced ulcer model in rats. 69

13 Table No: 5.5 Antiulcer effects of AETST and AQETST in different ulcer models in rats. 70

14 Table No: 5.6 Anti-inflammatory effects of AETST and AQETST in Carrageenan induced paw oedema in rats. 77

15 Table No: 5.7 Anti-inflammatory effects of AETST and AQETST in Carrageenan induced paw oedema in rats at different time intervals.

78

16 Table No: 5.8 Anti-inflammatory effects of AETST and AQETST in Histamine induced paw oedema in rats.

81

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17 Table No: 5.9 Anti-inflammatory effects of AETST and AQETST in Histamine induced paw oedema in rats at different time intervals.

82

18 Table No: 5.10 Anti-inflammatory effects of AETST and AQETST in Formalin induced paw oedema model in rats.

85

19 Table No: 5.11 Anti-inflammatory effects of AETST and

AQETST in Formalin induced paw oedema model in rats at different time intervals.

86

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LIST OF FIGURES

S. No. Figures Page No.

1 Fig No.3.1 Gastric acid secretion. 17

2 Fig No.3.2 Events in Acute and Chronic Inflammation. 34

3 Fig No.3.3 Mechanism of action of NSAIDs. 42

4 Fig No.5.1 Anti-ulcer activity of AETST and AQETST in pylorus ligation induced ulcer model in rats.

71

5 Fig No.5.2 Anti-ulcer activity of AETST and AQETST on free acidity in pylorus ligation induced ulcers in rats.

71

6 Fig No.5.3 Anti-ulcer activity of AETST and AQETST on total acidity in pylorus ligation induced ulcers in rats.

72

7 Fig No.5.4 Anti-ulcer activity of AETST and AQETST in stress induced ulcer model in rats.

73

8 Fig No.5.5 Anti-ulcer activity of AETST and AQETST on free acidity in stress induced ulcers in rats.

73

9 Fig No.5.6 Anti-ulcer activity of AETST and AQETST on total acidity in stress induced ulcers in rats.

74

10 Fig No.5.7 Anti-inflammatory activity of AETST and AQETST in Carrageenan induced paw oedema model in rats.

87

11 Fig No.5.8 Anti-inflammatory activity of AETST and AQETST in Histamine induced paw oedema model in rats.

87

12

Fig No.5.9 Anti-inflammatory activity of AETST and AQETST in Formalin induced paw oedema model in rats.

88

Dept. of Pharmacology, V.L.C.P Raichur Page 1

1. INTRODUCTION

Plants have been one of the important sources of medicine since dawn of human

civilization. These are the gifts of nature to mankind for treating different types of diseases.

Almost from prehistoric period herbal medicine are used for alleviation of suffering caused

by different diseases in human. Further they are well documented in India and other

countries: even today they are in great use in these countries. There are several beliefs or

claims regarding the therapeutic utility of herbs and herbal formulation i.e;

• Herbal medicines exhibit fewer side effects and are safe.

• Herbs and herbal formulations are cheaper and easily available.

• For certain disease like hepatitis, herbs and herbal drugs are the only remedies.

• Certain chemical constituents from the herbs are serving as prototype molecules for

the discovery of more effective drugs than existing ones.

Medicinal plants are treated as a subject of serious study and undergoing intense

research all over the World to identify the lead molecules. The immense surge of scientific

interest in natural products as a potential source of drugs has contributed to the

development of phytopharmacology research. In many developing countries, phyto-

pharmaceuticals form the main base of National Health Care Programmed e.g. the use of

herbal extracts in the treatment of various diseases in different countries and they are also

known to possess a rich traditional medicine. In South Africa it is estimated that 80% of

the black population consults traditional practitioners, perhaps this is not one and only

reason for the developing countries to invest in plant based pharmaceuticals but also has

been a global resurgence of interest in plant based drugs.


Herbal therapy provides rational means for the treatment of many diseases which are

considered to be obstinate and incurable in other system of medicine. It lays a great deal of

emphasis upon the maintenance of positive health of an individual and thus aims at both

the prevention and cure of diseases.

Hyperacidity / Ulcers which require the treatment for chronic period of time and the

usage of such drugs may cause various side effects, sometimes with permanent damage of

vital organs. The ulcer is one of chronic disease which is normally treated with H2-receptor

blockers (Cimetidine, Ranitidine) and proton pump inhibitors (Omeprazole, Pentaprazole).

These drugs are known to effect microsomal enzyme system of the liver resulting in the

pharmacokinetic type of drug- drug interaction with other drugs. But in herbal / traditional

medicines, there are several preparations prescribed to treat hyperacidity and ulcers.

Inflammation is a normal protective response to tissue injury caused by physical

trauma, noxious chemical or microbiological agents. It is body’s response for tissue repair

and is triggered by the release of chemical mediators from injured tissue and migrating

cells. The mechanism of anti-inflammatory drugs is considered to be inhibition of

prostaglandins synthesis at the site of injury. The anti-inflammatory potency of different

compounds roughly corresponds with their ability to inhibit cyclooxygenase enzyme

(COX).

The traditional medicine is largely gaining popularity over allopathic medicine

because of the following reasons.

• Rising cost of medical care.

• Natural products are free from side effects.


• No reoccurrence after the treatment.

• Easy availability of the drugs from natural sources especially in Developing

countries.

• Cure of disease by the changes in life style and social pathology.

• Renewable sources.

In the present study, we have chosen a plant S. tuberosum (Solanaceae) for which

different pharmacological activities are reported with different parts of the plant. From the

literature Ayurvedic texts we found that tubers of S. tuberosum have been traditionally

indicated for the treatments of anti-ulcer and anti-inflammatory condition. However anti-

ulcer and anti-inflammatory activities of this plant are not reported. But for its medicinal

properties reported in the Ayurvedic texts prompted us to select tubers extracts of this plant

for the study of anti-ulcer and anti-inflammatory activities in different experimental animal

models1-6.


2. OBJECTIVES OF THE STUDY

Since the plant S. tuberosum has not explored to significant extent and on the

background of available information of the plant, the present work was planned with the

following objectives.

1. To prepare various extracts (alcoholic and aqueous) with tubers of S. tuberosum, family

Solanaceae, by successive extraction technique and to analyze the extracts for the

phytochemical constituents.

2. To establish the pharmacological profile of the extracts of tubers of S. tuberosum.

• To assess the acute toxicity (LD50) of the two extracts.

• To screen for the anti-ulcer and anti-inflammatory activities in rats.

o To evaluate anti-ulcer activity of the tubers extracts in various

experimental animal models like-

a) Pylorus ligation induced ulcers in rats (SHAY) with the

estimation of parameters like volume of gastric juice, pH, free and

total acidity and ulcer index.

b) Stress induced ulcers (cold-water immersion induced ulcers).

o To evaluate anti-inflammatory activity of the tubers extracts in various

experimental animal models like-

a) Carrageenan induced paw oedema model in rats.

b) Histamine induced paw oedema model in rats.

c) Formalin induced paw oedema model in rats.


3. REVIEW OF LITERATURE

Plant name: S. tuberosum Family: Solanaceae

S. tuberosum plant


S. tuberosum tuber

3.1 Description of the Plant:

Family : Solanaceae

Genus : Solanum

Species : S. tuberosum

Synonym : Hindi – Alu

Sanskrit– Golakandah

Kannada – Batate

Marathi – Batata

Distribution: It grows throughout India and World.

The Plant: The S. tuberosum is a starchy, tuberous crop from the perennial S. tuberosum

of the Solanaceae family (also known as the nightshades).

Leaves: Leaves are very variable 2-6 in a whorl and green in colour.

Flowers: They bear white, pink, red, blue or purple flowers with yellow stamens.

Tubers: The tubers of varieties with white flowers have white skins, while those of

varieties with colored flowers tend to have pinkish skins.

Roots: Roots are reddish brown.


Major chemical constituents:

S. tuberosum contains starch, sugar (glucose, sucrose and fructose), cellulose (10-

20%), crude fiber, pectin substances (0.7-1.5% of dry wt), hemicelluloses (1%), fat (1.1%)

and vitamin C. The proteins of S. tuberosum tuber are comprised of about 60-70% globulin

and 20-40% glutelin with no albumin. 21 amino acids have been identified as normal

constituents of the alcohol - soluble nitrogen of S. tuberosum tuber tissue which is cystine,

aspartic, glutamic acids, serine, glycine, asparagine, theonine, alanine, glutamine, alpha

amino n-butyric acid, histidine, arginine, lysine, proline, methionine sulfoxide, valine,

isoleucine, phenylalanine, trylophane and tyrosine. Citric, isocitric, ascorbic, lactic, malic,

tartaric, succinic, oxalic, hydroxymalonic, aconitic, phytic, alpha ketoglutaric, guinic,

caffeic and chlorogenic organic acids are present in S. tuberosum.

The inorganic constituents or mineral content of S. tuberosum vary with variety.

Medicinal uses:

S. tuberosum tubers are used as anti-ulcer, anti-gout, anti-inflammatory, anti-arthritic,

diuretic, anti-scurvy and to increase milk in lactating mothers. For external use, the grated

raw S. tuberosum is applied locally in cases of arthritis, itching, neuralgia and in mild

burns7-8.


3.2 ULCER

Ulcer is defined as the erosion in the lining of the stomach or duodenum and is

caused by the disruptions of the gastric mucosal defense and repair systems. Ulcer in the

stomach is called gastric ulcer and in the duodenum is called duodenal ulcer and together

peptic ulcer. In clinical practice, peptic ulcer is one of the most prevalent gastrointestinal

disorders, commonly occurs in developed countries (Gregory M et al, 2009) 9.

Peptic ulcer, also known as PUD or peptic ulcer disease is a break in the lining

(mucosa) of the digestive tract produced by digestion of the mucosa by pepsin and acid,

occurs when pepsin and acid are present in high concentration or when some other

mechanism reduces the normal protective mechanism of the mucosa; bile salts may play a

part, especially in stomach ulcer10.

It was once commonly thought that stress, smoking and diet were the principal causes

of stomach ulcers. However, the Helicobacter pylori (H. pylori) bacterium is now known

to be responsible for most duodenal ulcers and 60 per cent of stomach ulcers. The H. pylori

a gram -ve bacterium also produce many symptoms of dyspepsia or indigestion. Treatment

for stomach ulcers includes acid-suppressing drugs and the use of antibiotics to kill the

infection.

3.2.1 Symptoms:

Symptoms of a peptic ulcer are

• Abdominal pain, classically epigastric with severity relating to mealtimes, after

around 3 hours of taking a meal (duodenal ulcers are classically relieved by food,

while gastric ulcers are exacerbated by it).


• Bloating.

• Water brash (rush of saliva after an episode of regurgitation to dilute the acid in

esophagus)

• Nausea and copious vomiting.

• Loss of appetite and weight.

• Hematemesis (vomiting of blood); this can occur due to bleeding directly from a

gastric ulcers or from damage to the esophagus from severe/continuing vomiting.

• Melena (tarry, foul-smelling feces due to oxidized iron from hemoglobin).

• Rarely, an ulcer can lead to a gastric or duodenal perforation, which leads to acute

peritonitis. This is extremely painful and requires immediate surgery.

3.2.2 Complications:

• Gastrointestinal bleeding is the most common complication and sudden large

bleeding can be life-threatening, occurs when the ulcer erodes one of the blood

vessels.

• Perforation (a hole in the wall) often leads to catastrophic consequences. Erosion of

the gastro-intestinal wall by the ulcers leads to spillage of stomach or intestinal

content into the abdominal cavity. Perforation at the anterior surface of the stomach

leads to acute peritonitis, initially chemical and later bacterial peritonitis. The first

sign is often sudden intense abdominal pain. Posterior wall perforation leads to

pancreatitis; pain in this situation often radiates to the back.

• Penetration is when the ulcer continues into adjacent organs such as the liver and

pancreas.


• Scarring and swelling due to ulcers causes narrowing in the duodenum and gastric

outlet obstruction. Patient often presents with severe vomiting.

• Cancer is included in the differential diagnosis (elucidated by biopsy), H pylori as

the etiological factor making it 3 to 6 times more likely to develop stomach cancer

from the ulcers11.

3.2.3 Classification of peptic ulcer:

Peptic ulcer may arise at various locations:

a) Stomach (called gastric ulcer)

b) Duodenum (called duodenal ulcer)

c) Esophagus (called esophageal ulcer) and

d) Meckel's diverticulum12.

a) Stomach Ulcer (Gastric Ulcer):

A stomach ulcer is small erosion (hole) in the stomach are called gastric ulcers. An

ulcer is contagious or cancerous. Duodenal ulcers are almost always benign, while stomach

ulcers may become malignant.

Stomach ulcer disease is common, affecting millions of peoples yearly. The size of a

stomach ulcer can range between 1/8 to 3/4 of an inch. Children too develop stomach

ulcers.

Stomach ulcers may be a symptom of another disease or condition and are often

common in mastocytosis. Bleeding from stomach ulcers may cause iron deficiency anemia.


The direct cause of peptic ulcers is the destruction of either gastric or intestinal

mucosal lining of the stomach/ intestine by hydrochloric acid, normally present in the

digestive juices of the stomach. Infection with the bacterium H. pylori is thought to play an

important role in causing both gastric and duodenal ulcers, may be transmitted from person

to person through contaminated food and water. Antibiotics are the most effective

treatment for H. pylori induced peptic ulcers13.

Causes of Stomach Ulcers:

• Family history of ulcers, smoking, excess alcohol consumption, improper diet,

skipped meals and Zollinger-Ellison syndrome.

• Use of nonsteroidal anti-inflammatory medications or corticosteroids.

• Type O blood (for duodenal ulcers).

• Stress does not cause an ulcer, but may be a contributing factor.

• Chronic disorders such as liver disease, emphysema, rheumatoid arthritis may

increase vulnerability to ulcers13.

Symptoms of ulcers in stomach:

The major symptom of an ulcer is a burning or gnawing feeling in the stomach area

that lasts between 30 min to 3 h. This pain is often interpreted as heartburn, indigestion or

hunger usually occurs in the upper abdomen, but sometimes it may occur below the

breastbone. In some individuals the pain occurs immediately after eating. In other

individuals, the pain may not occur until hours after eating and frequently awakens the

person at night13.

b) Duodenal ulcer:


Stomach normally produces acid to help the digestion of food and to kill bacteria and

is corrosive and some cells on the inside lining of the stomach and duodenum produce a

natural mucus barrier which protects the lining of both the stomach and duodenum. There

is normally a balance between the amount of acid and the mucus defense barrier. An ulcer

may develop with an alteration in this balance allowing the acid to damage the lining of the

stomach or duodenum14.

Causes Duodenal Ulcer:

• Infection with H. pylori.

• Anti-inflammatory drugs - including aspirin, ibuprofen, diclofenac etc.

• Zollinger-Ellison syndrome.

• Other factors such as smoking, stress and drinking heavily may possibly increase

the risk of having a duodenal ulcer14.

Symptoms of Duodenal Ulcer:

• Pain in the upper abdomen just below the sternum (breastbone) is the common

symptom. On and off occurs most before meals or in hunger conditions. It may be

eased with food or antacid tablets.

• Other symptoms which may occur include: bloating, retching and a feeling of

sick. Sometimes food makes the pain worse.

• Complications occur in some cases and can be serious. These include:

Bleeding ulcer. This can range from a 'trickle' to a life-threatening

bleed.


Perforation. This is where the ulcer goes right through (perforates)

the wall of the duodenum. Food and acid in the duodenum then leak

into the abdominal cavity. This usually causes severe pain and is a

medical emergency14.

c) Esophageal ulcer:

Esophageal Ulcers are defined as open sores or lesions in the lining of the esophagus,

usually cause pain felt behind or just below breastbone, similar to heartburn symptoms.

Healing is slow, chronic and severe recurrences can result in a narrowing of esophagus

after healing15.

Causes:

The direct cause of esophageal ulcers is the destruction of the lining of the esophagus

H. pylori bacteria usually found in the stomach.

Other common causes of esophageal ulcers are: Chronic use of anti-inflammatory

medications, smoking cigarettes, chewing tobacco, Gastro Esophagal Reflux Disorder

(GERD) and bulimia15.

Symptoms of Esophageal Ulcers:

Heartburn, inflammation of the esophagus.

A slight bleeding, blood vomiting bright red in colour and coffee ground black or

dark tarry stools.

Include nausea, abdominal indigestion and cramping15.

d) Meckel's Diverticulum:


It was first described by Fabricius Hildanus in the 16th century and later named after

Johann Friedrich Meckel, who described the embryological origin of this type of

diverticulum in 1809.

Meckel's diverticulum a true congenital diverticulum is a small bulge in the small

intestine present at birth. It is a vestigial remnant of the omphalomesenteric duct (also

called the vitelline duct) and is the most frequent malformation of the gastrointestinal tract.

It is present in approximately 2% of the population, with males more frequently

experiencing the symptoms.

Presentation:

Meckel's diverticulum is located in the distal ileum, usually about 60-100 cm of the

ileocecal valve. It is typically 3-5 cm long, runs antimesenterically and has its own blood

supply. It is a remnant of the connection from the umbilical cord to the small intestine

present during embryonic development. It can also present as an indirect hernia, where it is

known as a "Hernia of Littre". Furthermore, it can be attached to the umbilical region by

the vitelline ligament with the possibility of vitelline cysts, or even a patent vitelline canal

forming a vitelline fistula when the umbilical cord is cut. Torsions of intestine around the

intestinal stalk may also occur, leading to obstruction, ischemia and necrosis.

Symptoms:

Approximately 98% of people affected with Meckel's diverticulum are

asymptomatic. If symptoms do occur, they typically appear before the age of two.

The most common presenting symptom is painless rectal bleeding, followed by

intestinal obstruction, volvulus and intususception. Occasionally, Meckel's diverticulitis

may present with all the features of acute appendicitis. Also, severe pain in the upper


abdomen is experienced by the patient along with bloating of the stomach region. At times,

the symptoms are so painful such that they may cause sleepless nights with extreme pain in

the abdominal area16.

3.2.4 Gastrointestinal Tract:

In order to digest food, absorb nutrients and excrete unabsorbed waste products, the

gastrointestinal tract has to perform a number of coordinated activities and to provide the

whole body with a continual supply of water, electrolytes and nutrients. In order to achieve

these objectives, several organs have to integrate with each other and regulated by nervous

and hormonal system, as well as the central nervous system17.

3.2.5 Control and Co-ordination of GI Tract:

The blood vessels and the glands (exocrine, endocrine and paracrine) that comprise

the gastrointestinal tract are under the control of both neuronal and hormonal

mechanisms.

a) Neuronal Control:

There are two principal intramural plexuses in the tract: the myenteric plexus

(Auerbach's plexus) between the outer, longitudinal and the middle, circular muscle layers

and the submucous plexus (Meissner's plexus) on the luminal side of the circular muscle

layer. These plexuses are interconnected and their ganglion cells receive preganglionic

parasympathetic fibres from the vagus, which are mostly cholinergic and excitatory,

although a few are inhibitory. Incoming sympathetic fibres are largely postganglionic and

these in addition to innervating blood vessels, smooth muscle and some glandular cells

directly may terminate in these plexuses, where they inhibit acetylcholine secretion. The

neurons within the plexuses constitute the enteric nervous system and secrete not only


acetylcholine and noradrenaline (norepinephrine), but also 5-hydroxytryptamine, purines,

nitric oxide and a variety of pharmacologically active peptides. The enteric plexus also

contains sensory neurons, which respond to mechanical and chemical stimuli.

B) Hormonal Control:

The hormones of the gastrointestinal tract include both endocrine and paracrine

secretions. The endocrine secretions (i.e. substances released into the bloodstream) are

mainly peptide in nature and are synthesised by endocrine cells in the mucosa. Important

examples include gastrin and cholecystokinin. The paracrine secretions include many

regulatory peptides released from special cells found throughout the wall of the tract.

These hormones act on nearby cells, and in the stomach the most important of these is

histamine. Some of these paracrine factors also function as neurotransmitters18.

3.2.6 Physiology of Gastric Secretion:

The parietal cell contains receptors for gastrin, histamine (H2) and acetylcholine

(muscarinic, M3). When acetylcholine or gastrin bind to the parietal cell receptors, they

cause an increase in cytosolic calcium, which in turn stimulates protein kinases that

stimulate acid secretion from a H+/K+ ATPase (the proton pump) on the canalicular

surface.

In close proximity to the parietal cells are gut endocrine cells called enterochromaffin-like

(ECL) cells. ECL cells have receptors for gastrin and acetylcholine and are the major

source for histamine release. Histamine binds to the H2 receptor on the parietal cell,

resulting in activation of adenylylcyclase, which increases intracellular cyclic adenosine

monophosphate (cAMP). cAMP activates protein kinases that stimulate acid secretion by

the H+/K+ ATPase. In humans, it is believed that the major effect of gastrin upon acid


secretion is mediated indirectly through the release of histamine from ECL cells rather than

through direct parietal cell stimulation19.

Fig No.3.1 Gastric acid secretion

3.2.7 Pathophysiology:

Peptic ulcers are defects in the gastric or duodenal mucosa that extend through the

muscularis mucosa. H. pylori infection and NSAID use are the most common etiologic

factors. Other, less common causes are hypersecretory states such as Zollinger-Ellison

syndrome.


Under normal conditions, a physiologic balance exists between peptic acid secretion

and gastroduodenal mucosal defense. Peptic ulcer occurs when the balance between the

aggressive factors and the defensive mechanisms is disrupted. Aggressive factors, such as

NSAIDs, H. pylori, alcohol, bile salts, acid and pepsin can alter the mucosal defense

mechanisms19.

3.2.8 H. pylori in the pathogenesis of peptic ulcer disease:

H. pylori are a spiral shaped Gram -ve bacterium with flagella that has a urease

enzyme which hydrolyzes urea into ammonia and bicarbonate. The alkaline

microenvironment produced by this action protects the organism from gastric acid. Active

motility of the organism by virtue of its flagella allows it to penetrate the mucus layer of

the gastric mucosa. Pathogenesis of gastroduodenal injury due to H. pylori infection is

explained by ‘leaking roof hypothesis’. H. pylori infection is believed to injure submucosal

tissue by causing a 'leak' in its protective coating of mucin gel and epithelial cells (the roof)

thereby making it susceptible to gastric acid (the rain). Secretion of urease, specific

adhesion receptor interaction, cytotoxins (including hemolysin), superoxide dismutase,

heat shock proteins, mucinase, lipase and phospholipase are some of bacterial virulence

determinants. H. pylori strains collected from patients ulcers with are genetically different

from those isolated from asymptomatic individuals20, 21.

Clinical manifestations of H. pylori:

H. pylori, are the primary cause for gastric inflammation, resides exclusively in

gastric mucosa, but can be found in remote areas that have undergone metaplastic changes

in which gastric epithelial cells are present such as esophagus, duodenum or Meckel's


diverticulum. A higher concentration of bacteria is found in the antral area and the major

risk factors for bleeding are H. pylori infection NSAID’s use and stress22, 23.

3.2.9 Pathologic changes in peptic ulcers:

Peptic ulcers are solitary (80%), small (1-2.5 cm in diameter) and round to oval. The

mucosal folds converge towards the ulcer and vary in depth from superficial (confined to

mucosa) to deep ulcers (penetrating into the muscular layer). Chronic duodenal ulcer never

turns malignant, while chronic gastric ulcer turns to carcinoma. Malignant gastric ulcers

are larger, bowl-shaped with elevated and indurate mucosa of the margin24.

Macroscopical appearance:

Gastric ulcers are most often localized on the lesser curvature of the stomach, round

to oval parietal defect (hole) 2 to 4 cm diameter, with a smooth base and perpendicular

borders. These borders are not elevated or irregular in the acute form of peptic ulcer,

regular but with elevated borders and inflammatory surrounding in the chronic form. In the

ulcerative form of gastric cancer the borders are irregular. Surrounding mucosa may

present radial folds, as a consequence of the parietal scarring25.

Microscopical appearance:

Gastric / peptic ulcer is a mucosal defect which penetrates the muscularis mucosa and

muscularis propria, produced by acid-pepsin aggression. Ulcer margins are perpendicular

and present with chronic gastritis. Microscopically, chronic peptic ulcers have 4

histological zones, from outside to inside are-

i) Necrotic zone- It lies in the floor of the ulcer and is composed of fibrous exudates

containing necrotic debris and a few leucocytes.


ii) Superficial exudative zone- It lies underneath the necrotic zone. The tissue elements

show coagulative necrosis giving eosinophilic, smudgy appearance with nuclear debris.

iii) Granulation tissue zone- It is seen merging into the necrotic zone, composed of non-

specific inflammatory infiltrate and proliferating capillaries.

iv) Zone of cicatrisation- It is seen merging into thick layer of granulation tissue,

composed of dense fibro-collagenic scar tissue over which granulation tissue rests.

Thrombosis or sclerotic arteries cross the ulcer, which on erosion results in hemorrhage26.

3.2.10 Clinical features:

1. Pain- In gastric ulcer, epigastric pain occurs immediately or within 2 h after food

and never occurs at night. In duodenal ulcer, pain is severe, occurs late night

(hunger pain) and is usually relieved by food.

2. Age- The peak incidence of duodenal ulcer is in 5th decade while that for gastric

ulcer is a decade later.

3. Periodicity- The attacks in gastric ulcer last from 2-6 weeks, with interval of

freedom from 1-6 months. The attacks of duodenal ulcer are classically worsened

by ‘work, worry and weather’.

4. Vomiting- Vomiting which relieves the pain is a conspicuous feature in patients of

gastric ulcer. Duodenal ulcer patients rarely have vomiting but instead get heart-

burn (retrosternal pain) and water brash (burning fluid into the mouth).

5. Hematemesis and melaena - Hematemesis and melaena occur in gastric ulcers in

gastric ulcers in the ratio of 60:40, while in duodenal ulcers in the ratio of 40:60.


Both may occur together more commonly in duodenal ulcer than in gastric ulcer

patients.

6. Appetite- The gastric ulcers patients, though have good appetite but are afraid to

eat, while duodenal ulcer patients have very good appetite.

7. Diet- Patients of gastric ulcer commonly get used to a bland diet consisting of milk,

eggs etc and avoid taking fried foods, curries and heavily spiced foods. In contrast,

duodenal ulcer patients usually take all kinds of diet.

8. Weight- Loss of weight is a common finding in gastric ulcer patients while patients

of duodenal ulcer tend to gain weight due to frequent ingestion of milk to avoid

pain.

9. Deep tenderness- Deep tenderness is present in both types of peptic ulcers. In the

case of gastric ulcer it is in the midline of the epigastrium, while in the duodenal

ulcer it is in the right hypochondrium26, 27.

3.2.11 Complications:

If ulcers remain untreated they may lead to:

Gastrointestinal bleeding: It is the most common complication and sudden large bleeding

can be life threatening, occurs when the ulcer erodes one of the blood vessels.

Perforation (hole in the wall): It often leads to catastrophic consequences. Erosion of the

gastro-intestinal wall by the ulcer leads to spillage of stomach or intestinal content into

abdominal cavity. Perforation at the anterior surface of stomach leads to acute peritonitis,

initially chemical and later bacterial peritonitis. Often first sign is sudden intense


abdominal pain. Posterior wall perforation leads to pancreatitis; pain in this situation often

radiates to back.

Penetration: It occurs when the ulcer continues into adjacent organs such as liver and

pancreas.

Obstruction: Scarring and swelling due to ulcers causes narrowing in the duodenum and

gastric outlet obstruction. Patient often presents with severe vomiting26,27.

3.2.12 Treatment of Peptic Ulcers:

Classification of Antiulcer Drugs:

A. Reduction of gastric acid secretion -

1. H2 receptor antagonist -

Ex. - Cimetidine, Ranitidine, Famotidine.

2. Proton pump inhibitors -

Ex. – Omeprazole, Pantoprazole, Lansoprazole, Rabeprazole.

3. Anticholinergics -

Ex. – Pirenzepine.

4. Prostaglandins analogue -

Ex. – Mesoprostol.

B. Neutralizing of gastric acid -

1. Systemic antacid -


Ex. – Sodium bicarbonate, Sodium citrate.

2. Non systemic antacid-

Ex. – Alluminium hydroxide, Magnesium hydroxide, Magaldrate, Calcium carbonate.

C. Ulcer protective -

Ex. – Sucralfate.

D. Anti- H. Pylori drugs -

Ex. - Amoxicillin, Clarithromycin, Metronidazole, Tinidazole.

A. Reduction of gastric acid secretion:

1. H2 receptor antagonists:

Mechanism of action - Drugs like cimetidine, ranitidine, famotidine are act on the H2

receptors and they inhibit the gastric acid secretion.

H2 receptor antagonists competitively blocking the binding of histamine to H2

receptors and reduce intracellular concentration of cyclic AMP and there by secretion of

gastric acid.

Pharmacokinetic-

Cimetidine - It is given orally, distribute throughout the body, excreted mainly in the

urine, bioavailability is 60-80% due to first pass metabolism. About 2/3rd of dose is

excreted unchanged in the urine and bile. The elimination t ½ is 2-3 h.


Ranitidine - Compared to cimetidine, ranitidine is 5 to 10 times more potent and minimal

side effects and the elimination t ½ is 2-3 h.

Famotidine- Famotidine is 20-160 time more potent than ranitidine and the oral

bioavailability is 40-50%, excreted through kidney; elimination t ½ is 2.5 to 3.5 h.

Adverse effects - The most common side effect is headache, dizziness, diarrhoea and

muscular pain. CNS side effects are confusion, hallucination, reduce of sperm count and

also inhibit cytochrome P-450 which can slow the metabolism of several drugs.

Dose – Cimetidine 400 mg BD, Ranitidine 150 mg BD, Famotidine 20 mg BD.

2. Proton pump inhibitors:

Mechanism of action - (Omeprazole, Pantoprazole, Lansoprazole, Rabeprazole) These

drugs bind to the H+ / K+ ATPase enzyme system (Proton pump) of parietal cells

suppressing the secretion of hydrogen ions into the gastric lumen. The membrane bound

proton pump is the final step in the secretion of gastric acid.

Omeprazole, Pantoprazole, Lansoprazole and Rabeprazole inhibit the basal and

stimulated gastric acid secretion (more than 90% acid suppression) begins within 1-2 h

after first dose.

Pharmacokinetic - Plasma t ½ is 1-2 h and metabolites of these agents are excreted in

urine and faeces.


Adverse effects - These drugs are generally well tolerated. Minimal side effects are

nausea, vomiting, loose stool, headache, abdominal pain, muscle and joint pains and

dizziness are complained by 3-5% of patients.

Dose- Omeprazole 20 mg daily, Pantoprazole 40 mg daily, Lansoprazole 15-30 mg daily,

Rabeprazole 20 mg daily.

3. Anticholinergics - (Pirenzepine) Muscaranic receptor stimulation increases

gastrointestinal motility and secretory activity. Cholinergic antagonist such as pirenzepine

is used in the management of peptic ulcer, which reduces the volume of gastric juice

without raising the gastric pH but is reduced by 40-50% without producing intolerable side

effects.

4. Prostaglandin analogues - (Mesoprostol) Prostaglandins produced by the gastric

mucosa, inhibit secretion of Hcl and stimulate secretion of mucus and bicarbonate.

Deficiency of PG involved in the pathogenesis of peptic ulcer. Mesoprostol, an

analogue of PG E is the only agent approved for prevention of gastric ulcer induced by

NSAIDs.

It is less effective than H2 antagonist for acute treatment of peptic ulcers and

clinically effective only at higher doses that diminish gastric acid secretion.

Adverse effects - Diarrhoea, abdominal cramps, uterine bleeding are the adverse effect and

it is contraindicated in pregnancy.

Dose - 400 μg 4 times a day.

B. Neutralizing of gastric acid (Antacids):


Antacids are weak bases that react with gastric acid to form salts and water there by

diminishing gastric acidity. Since pepsin is inactivated at pH > 4.0 antacids also reduce

gastric acidity. Antacids are of two types –

a) Systemic Antacids.

b) Non systemic antacids.

a) Systemic Antacids –

Sodium bicarbonate is an example of systemic antacid, act rapidly and raises the pH

to 7.4. On neutralizing of gastric acid it form NaCl and CO2. NaCl remain unchanged and

is unable to neutralize the bicarbonate in the intestine therefore later absorbed causing

systemic alkalosis. Formation of CO2 can dangerous if the ulcers is near perforation.

b) Non systemic antacids –

They are of two types-

i) Buffer type.

ii) Non buffer type.

i) Buffer type –

Alluminium hydroxide - They have a slow onset of action but longer duration of action

and raise the pH 3.5- 4.0. It has astringent and demulcent properties by which it forms a

protective coating over the ulcer crater.

Adverse effects - Constipation is only major side effect because of formation of an

Alluminium phosphate and mucosal astringent action of Alluminium salt.


Magnesium trisilicate - They have also a slow onset of action but longer duration of

action. Hydrated silicon dioxide has adsorbent properties similar to those of Alluminium

hydroxide.

Adverse effects - It may cause mild diarrhoea because of formation of Magnesium salt.

ii) Non buffer type –

Calcium carbonate - This is a very powerful antacid with fast onset of action and raise the

pH >7.

Adverse effects - Excessive doses of calcium carbonate if given along with milk can lead

to hypercalcaemia. It causes constipation due to formation of calcium stearate in intestine.

Magnesium hydroxide - It is a quick acting antacid and the action is prolonged.

Adverse effects - Magnesium hydroxide has a mild cathartic action which can be

countered by combining with alluminium based antacid. Small amount of magnesium is

absorbed into the blood; it produces toxicity only in the presence of impaired renal

function.

C. Ulcer protective drugs–

Sucralfate – It is a complex of an Octa-alluminium sulphate of sucrose. At low pH it

forms a sticky paste in the stomach and forms a barrier for acid and also binds to pepsin

and bile salts to reduce peptic activity.

Dose- 1 g tablet before a meal and at bed time.

D. Anti-H. Pylori drugs -


(Amoxicillin, Clarithromycin, Metronidazole, Tinidazole) H. pylori are a gram –ve

bacteria found in the stomach, which attach to the surface of epithelium beneath the mucus

has high urease activity can form a microenvironment around the bacteria.

It has accepted that it is the main contribution to the causation of chronic gastritis,

dyspepsia and peptic ulcer. Anti-H. pylori therapy is recommended for those patients

whose test are positive for H. pylori26,27.

Table No: 3.1 Name of the plants reported for anti-ulcer activity

S. No. Name of Plants Family Chemical constituents References

01 Tectona grandis (Trunk Bark and wood chips)

Verbenaceae Phenolic acids, β-sitosterol

& triterpenoids.

02. Wedelia calendulacea (Leaves)

Compositae Saponin, phytosterol &

isoflavonoids.

03. Glycyrrhiza glabra (Root)

Papilionaceae Glycyrrhizin, triterpene,

saponin & isoflavonoids.

Goel and Sairam28,

2002.


04. Ficus religiosa (Bark)

Moraceae β-sitosteryl-D-glucoside,

Vitamin K & Lanosterol,

stigmasterol.

05. Bacopa monniera (Whole plant)

Scrophulariaceae Saponins, monnierin,

hersaponin, bacosides A

and B,brahmine &

herpestine

06. Convolvulus pluricaulis (Whole plant)

Papilionaceae N-methylconiene, flavone

glycoside, diosmin &

chlorogenic acid.

07. Emblica

officinalis (Leaves)

Euphorbiaceae Vitamin C, minerals &

amino acids.

Sairam et al29, 2002.

08. Asparagus racemosus (Root)

Liliaceae Neoprazerigenin A-3-O- β-

lycotetraoside, steroidal

saponins & sibiricoside A

and B.


09. Centella asiatica (Whole plant)

Umbelliferae Saponins, monnierin,

hersaponin & bacosides A

and B.


10. Ocimum sanctum (Leaves)

Labiatae Camphor, pinene,

limonene, terpinolene,

myrcene, linalool &

borneol.

Dharmani et al32,

2006.

11. Allophylus serratus (Leaves)

Sapindaceae saponin & isoflavonoids Dharmani et al33,

2005.

12. Desmodium gangeticum (Leaves)

Leguminosae Pterocarpanoids—

gangetin, gangetinin,

desmodin & several

alkaloids.

Dharmani et al 34,

2005.


13. Terminalia pallida (Leaves)

Combretaceae Glycosides, flavones,

tannins, oligomeric &

proanthocyanidins.

Gupta et al 35, 2005.

14. Musa sapientum (Leaves)

Scitaminaceae Acylsterylglycoside &

sitoindoside IV.

Sanyal et al 36, 1998.

15 Bidens pilosa (Hole plant)

Compostiae Phenylheptatriyne. Alvarez et al 37,

1999. 16. Hemidesmus

indicus (Hole plant)

Asclepiadaceae Coumarino-lignoids,

hemidesmine, hemidesmin-

1, 2 & pregnane glycosides.

Anoop and Jegadees

38, 2003.

17. Maytenus ilicifolia (Leaves)

Celastraceae Alkaloids, tannins &

flavones.

Martins et al 39,

2003.

18. Phyllanthus niruri (Aerial part)

Euphorbiaceae Niuride. Okoli et al 40, 2009.

3.3 INFLAMMATION

Inflammation (Latin, inflammare, to set on fire) is part of the complex biological

response of vascular tissues to harmful stimuli, such as pathogens, damaged cells or

irritants. Inflammation is a protective attempt by the organism to remove the injurious

stimuli and to initiate the healing process. Inflammation is not a synonym for infection,

even in cases where inflammation is caused by infection. Infection is caused by an

exogenous pathogen, while inflammation is one of the responses of the organism to the

pathogen41.

Causes of inflammation:


The numerous causes of inflammation may be classified as follows:

• Microbes, e.g. bacteria, viruses, protozoa and fungi.

• Physical agents, e.g. heat, cold, mechanical injury, ultraviolet and ionising

radiation.

• Chemical agents: organic, e.g. microbial toxins and organic poisons such as weed

killers. Inorganic, e.g. acids, alkalis.

• Antigens that stimulate immunological responses.

Inflammation can be classified as:

1. Acute Inflammation.

2. Chronic Inflammation.

3.3.1 Acute Inflammation:

Episodes of acute inflammation are usually of short duration e.g. days to a few weeks

and may range from mild to very severe. The cardinal signs of inflammation are: redness,

heat, pain, swelling and loss of function.

The acute inflammatory response is described in a series of overlapping stages:

A. Increased blood flow

B. Increased formation of tissue fluid

C. Increased permeability of small blood vessel walls


D. Increased hydrostatic pressure

E. Migration of leukocytes

F. Chemotaxis

A. Increased blood flow:

Following injury, both the arterioles supplying the damaged area and the local

capillaries dilate, increasing blood flow to the site. This is caused mainly by the local

release of a number of chemical mediators from damaged cells e.g. histamine and

serotonin. Increased blood flow to the area of tissue damage provides more oxygen and

nutrients for the increased cellular activity that accompanies inflammation. Increased blood

flow causes the increased temperature and reddening of an inflamed area.

B. Increased formation of tissue fluid:

One of the cardinal signs of inflammation is swelling (oedema) of the tissues

involved, which is caused by fluid leaving local blood vessels and entering the interstitial

spaces.

C. Increased permeability of small blood vessel walls:

This is caused by inflammatory mediators, e.g. prostaglandins, histamine and

serotonin, which are released by injured cells and cause the cells that form the single

layered venule wall to pull apart from one another. This opens channels that allow the

movement of:

• Excess fluid, which leaves the blood and enters the tissues and

• Plasma proteins, which are normally retained within the bloodstream and contribute

to the osmotic pressure of the blood. When plasma proteins leave the blood, as in


inflammation, the osmotic pressure of the blood falls and water moves from the

bloodstream into the tissues.

D. Increased hydrostatic pressure:

The increased blood flow into the capillary bed forces fluid out of the vessels and

into the tissues. Some interstitial fluid returns to the capillaries but most of the

inflammatory exudate, phagocytes and cell debris are removed in lymph vessels because

the pores of lymph vessels are larger and the pressure inside is lower than in blood

capillaries.

E. Migration of leukocytes:

Loss of fluid from the blood thickens it, slowing flow and allowing the normally fast-

flowing white blood cells to make contact with and adhere to the vessel wall. In the acute

stages, the most important leukocyte is the neutrophil, which adheres to the blood vessel

lining, squeezes between the endothelial cells and enters the tissues, where its main

function is in phagocytosis of antigens.

Later in the inflammatory response, after about 24 h, macrophages become the

predominant cell type at the inflammed site and they persist in the tissues if the situation is

not resolved, leading to chronic inflammation. Macrophages are larger and longer lived

than the neutrophils. They phagocytose dead/dying tissue, microbes and other antigenic

material and dead/dying neutrophils.

F. Chemotaxis:

This is the chemical attraction of leukocytes to an area of inflammation. The role of

chemoattractants and the way in which they work is not fully understood.

It may be that chemoattractants act to retain passing leukocytes in the inflammed

area, rather than actively attracting them from distant areas of the body. Known


chemoattractants include microbial toxins, chemicals released from leukocytes,

prostaglandins from damaged cells and complement proteins42.


Fig No.3.2 Events in Acute and Chronic Inflammation

Table No: 3.2 Summary of the principal substances released in inflammation


Substances Made by Trigger for release Main pro-inflammatory

actions

Histamine Mast cells (in most

tissues), basophils

(blood); stored in

cytoplasmic granules.

Binding of antibody

to mast cells and

basophils.

Vasodilatation, itching,

vascular permeability,

degranulation, smooth

muscle contraction (e.g.

bronchoconstriction).

Serotonin (5-HT) Platelets, Mast cells

and basophils (stored

in granules). Also in

CNS (acts as

neurotransmitter).

When platelets are

activated, and when

mast cells/basophils

degranulate.

Vasoconstriction,

vascular permeability.

Prostaglandins (PGs) Nearly all cells; not

stored, but made from

cell membranes as

required.

Many different

stimuli,e.g. drugs,

toxins, other

inflammatory

mediators,

hormones, trauma.

Diverse, sometimes

opposing, e.g. fever,

pain, vasodilatation or

vasoconstriction,

vascular permeability.

Heparin Liver, mast cells,

basophils (stored in

cytoplasmic granules).

Released when cells

degranulate.

Anticoagulant (prevents

blood clotting), which

maintains blood supply

(nutrients, O2) to injured

tissue and washes away

microbes and wastes.

Bradykinin Tissues and blood. When blood clots, in

trauma and

inflammation.

Pain, Vasodilatation.


Outcomes of acute inflammation:

Resolution:

When the injury is limited or short-lived, when there has been no or minimal tissue

damage and when the tissue is capable of replacing any irreversibly injured cells, the usual

outcome is restoration to histologic and functional normalization. This involves

neutralization or removal of the various chemical mediators, normalization of vascular

permeability and cessation of leukocyte emigration with subsequent death (by apoptosis)

of extravasated neutrophils. Eventually, the combined efforts of lymphatic drainage and

macrophage ingestion of necrotic debris lead to the clearance of the edema fluid,

inflammatory cells and detritus from the battlefield.

Scarring or fibrosis:

Scarring or fibrosis results after substantial tissue destruction or when inflammation

occurs in tissues that do not regenerate. In addition, extensive fibrinous exudates (due to

increased vascular permeability) may not be completely absorbed and are organized by in

growth of connective tissue with resultant fibrosis. Abscess formation may occur in the

setting of extensive neutrophilic infiltrates or in certain bacterial or fungal infections (these

organisms are then said to be pyogenic or "pus forming"). Due to the extensive underlying

tissue destruction (including the extracellular matrix), the only outcome of abscess

formation is scarring.

Development of chronic inflammation:

Progression to chronic inflammation may follow acute inflammation, although signs

of chronic inflammation may be present at the onset of injury (e.g., in viral infections or


immune responses to self-antigens). Depending on the extent of the initial and ongoing

tissue injury, as well as the capacity of the affected tissues to re-grow, chronic

inflammation may be followed by regeneration of normal structure and function

(regeneration) or may lead to scarring.

3.3.2 Chronic Inflammation:

Chronic inflammation can be considered to be inflammation of prolonged duration

(weeks to months to years) in which active inflammation, tissue injury and healing proceed

simultaneously. In contrast to acute inflammation, which is distinguished by vascular

changes, edema and a largely neutrophilic infiltrate, chronic inflammation is characterized

by the following:

• Infiltration with mononuclear (chronic inflammatory) cells, including macrophages,

lymphocytes and plasma cells.

• Tissue destruction is largely directed by the inflammatory cells.

• Repair, involving new vessel proliferation (angiogenesis) and fibrosis.

Chronic inflammation arises in the following settings:

• Viral infections, intracellular infections of any kind typically require lymphocytes

(and macrophages) to identify and eradicate infected cells.

• Persistent microbial infections, most characteristically by a selected set of

microorganisms including mycobacteria (Tubercle bacilli), Treponema pallidum

(causative organism of syphilis) and certain fungi. These organisms are of low

direct pathogenicity, but typically they evoke an immune response called delayed

hypersensitivity, which may culminate in a granulomatous reaction.


• Prolonged exposure to potentially toxic agents. Examples include nondegradable

exogenous material such as inhaled particulate silica, which can induce a chronic

inflammatory response in the lungs (silicosis and endogenous agents such as

chronically elevated plasma lipid components, which may contribute to

atherosclerosis).

• Autoimmune diseases, in which an individual develops an immune response to

self-antigens and tissues. Because the responsible antigens are in most instances

constantly renewed, a self-perpetuating immune reaction results (e.g., Rheumatoid

arthritis or Multiple sclerosis).

Mediators of chronic inflammation:

Macrophages:

They constitute the critical mainstay and heart of chronic inflammation. Macrophages

secrete a wide variety of biologically active products that if unchecked, can result in the

tissue injury and characteristic of chronic inflammation.

Complement components and coagulation factors:

Activated macrophages may release significant amounts of these proteins locally into

the extracellular matrix. These include complement proteins C1 to C5, properdin,

coagulation factors V and VIII.

Arachidonic acid metabolites.

Cytokines, such as IL-1 and TNF, as well as a variety of growth factors that influence

the proliferation of smooth muscle cells and fibroblasts and the production of extracellular

matrix. At the sites of acute inflammation when the process is resolved, macrophages


eventually die or wander off into lymphatic. In chronic inflammatory sites, however

macrophage accumulation persists and macrophages can proliferate. Other types of cell

present in chronic inflammation are lymphocytes, plasma, cells, eosinophils and mast cells.

Granulomatus Inflammation:

Granulomatus inflammation is a distinctive pattern of chronic inflammation

characterized by aggregates of activated macrophages that assume a squamous cell like

(epitheloids) appearance43.

3.3.3 Non-Steroidal Anti-Inflammatory Drugs:

Nonsteroidal anti-inflammatory drugs, usually abbreviated to NSAIDs, but also

referred to as nonsteroidal anti-inflammatory agents/analgesics (NSAIAs), are drugs with

analgesic and antipyretic (fever-reducing) effects and which have in higher doses, anti-

inflammatory effects.

The term "nonsteroidal" is used to distinguish these drugs from steroids, which,

among a broad range of other effects, have a similar eicosanoid-depressing, anti-

inflammatory action. As analgesics, NSAIDs are unusual in that they are non-narcotic.

The most prominent members of this group of drugs are aspirin, ibuprofen and

naproxen all of which are available over the counter in many areas44.


Classification of NSAIDs:

NSAIDs can be classified based on their chemical structure or mechanism of action.

1. Salicylates:

Aspirin (acetylsalicylic acid), Diflunisal, Salsalate.

2. Propionic acid derivatives:

Ibuprofen, Naproxen, Fenoprofen, Ketoprofen, Flurbiprofen.

3. Acetic acid derivatives:

Indomethacin, Sulindac, Etodolac, Ketorolac, Diclofenac, Nabumetone.

4. Enolic acid (Oxicam) derivatives:

Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam.

5. Fenamic acid derivatives:

Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid.

6. Selective COX-2 inhibitors:

Celecoxib, Rofecoxib, Valdecoxib, Parecoxib, Lumiracoxib, Etoricoxib, Firocoxib.

7. Sulphonanilides:

Nimesulide (systemic preparations are banned by several countries for the potential

risk of hepatotoxicity)

8. Others:

Licofelone, acts by inhibiting LOX (lipooxygenase) & COX and hence known as 5-

LOX/COX inhibitors.


Mechanism of action:

Most NSAIDs act as nonselective inhibitors of the enzyme cyclooxygenase (COX),

inhibiting both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)

isoenzymes. COX catalyzes the formation of prostaglandins and thromboxane from

arachidonic acid (itself derived from the cellular phospholipid bilayer by phospholipase

A2)45.

NSAIDs have antipyretic activity and can be used to treat fever, caused by elevated

levels of prostaglandin E2, which alters the firing rate of neurons within the hypothalamus

that control thermoregulation. Antipyretics work by inhibiting the enzyme COX, which

causes the general inhibition of prostanoid biosynthesis (PGE2) within the hypothalamus.

PGE2 signals to the hypothalamus to increase the body's thermal set point. Ibuprofen has

been shown to be more effective as an antipyretic than acetaminophen. Arachidonic acid is

the precursor substrate for cyclooxygenase leading to the production of prostaglandins F, D

& E46,47.

Pharmacokinetics:

Most nonsteroidal anti-inflammatory drugs are weak acids, with a pKa of 3-5,

absorbed well from the stomach and intestinal mucosa. They are highly protein-bound in

plasma (typically >95%), usually to albumin, so that their volume of distribution typically

approximates to plasma volume. Most NSAIDs are metabolised in the liver by oxidation

and conjugation to inactive metabolites which are typically excreted in the urine, although

some drugs are partially excreted in bile. Metabolism may be abnormal in certain disease

states and accumulation may occur even with normal dosage.


Ibuprofen and diclofenac have short half-lives (2–3 h). Some NSAIDs (typically

oxicams) have very long half-lives (e.g. 20–60 h).

Fig No.3.3 Mechanism of action of NSAIDs48

Adverse effects:

1. Cardiovascular –

A recent meta-analysis of all trials comparing NSAIDs found an 80% increase in the

risk of myocardial infarction with both newer COX-2 antagonists and high dose traditional

anti-inflammatories compared with placebo49.


2. Gastrointestinal –

The main adverse drug reactions (ADRs) associated with use of NSAIDs relate to direct

and indirect irritation of the gastrointestinal (GI) tract. NSAIDs cause a dual insult on the

GI tract: the acidic molecules directly irritate the gastric mucosa and inhibition of COX-1

and COX-2 reduces the levels of protective prostaglandins. Inhibition of prostaglandin

synthesis in the GI tract causes increased gastric acid secretion, diminished bicarbonate

secretion, diminished mucus secretion and diminished trophic effects on epithelial mucosa.

Common gastrointestinal ADRs include:

Nausea/vomiting, dyspepsia, gastric ulceration/bleeding50 and diarrhea.

3. Inflammatory bowel disease (IBD) –

NSAIDs are never to be used in individuals with inflammatory bowel disease (e.g.

Crohn's disease or ulcerative colitis) due to their tendency to cause gastric bleeding and

form ulceration in the gastric lining. Pain reliever drug such as codeine (which slows down

bowel activity) are safer medications for pain relief in IBD.

4. Renal –

NSAIDs are also associated with a relatively high incidence of renal adverse drug

reactions (ADRs). The mechanism of these renal ADRs is due to changes in renal

haemodynamics (blood flow), ordinarily mediated by prostaglandins, which are affected by

NSAIDs. Prostaglandins normally cause vasodilation of the afferent arterioles of the

glomeruli. This helps maintain normal glomerular perfusion and glomerular filtration rate

(GFR), an indicator of renal function. This is particularly important in renal failure where

the kidney is trying to maintain renal perfusion pressure by elevated angiotensin II levels.

At these elevated levels, angiotensin II also constricts the afferent arteriole into the

glomerulus in addition to the efferent arteriole one it normally constricts. Prostaglandins


serve to dilate the afferent arteriole; by blocking this prostaglandin-mediated effect,

particularly in renal failure, NSAIDs cause unopposed constriction of the afferent arteriole

and decreased renal perfusion pressure.

5. Other –

Common adverse drug reactions (ADR) include: raised liver enzymes, headache,

dizziness and photosensitivity. Uncommon ADRs include: hyperkalaemia, confusion,

bronchospasm, rash, rapid and severe swelling of the face and body. Ibuprofen may also

rarely cause symptoms of irritable bowel syndrome.

Uses:

NSAIDs are generally indicated for the symptomatic relief of the following conditions:

• Rheumatoid arthritis, osteoarthritis, acute gout, metastatic bone pain, headache,

migraine, postoperative pain and dysmenorrhoea (menstrual pain).

• Inflammatory arthropathies (e.g. ankylosing spondylitis, psoriatic arthritis, Reiter's

syndrome).

• Mild-to-moderate pain due to inflammation and tissue injury.

• They are also given to neonate infants whose ductus arteriosus is not closed within

24 h of birth51.

Selective COX-2 inhibitors:

The discovery of COX-2 in 1991 by Daniel L. Simmons at Brigham Young

University raised the hope of developing an effective NSAID without the gastric problems

characteristic of these agents. It was thought that selective inhibition of COX-2 would

result in anti-inflammatory action without disrupting gastroprotective prostaglandins.


Table No: 3.3 Name of the plants reported for anti-inflammatory activity

S. No. Plant Family Chemical constituent Reference

1. Cassia fistula Caesalpinaceae Flavone glycoside,

oxyanthraquinones,

chrysophanol &

chrysophanein.

Ilavarasan et al52,

2005.

2. Margaritaria

discoidea

Euphorbiaceae Phylochrysine &

Securinine.

Adeolu et al53,

2009.

3. Eclipta prostrata Astearaceae Triterpenoid saponins.

Arunachalam et

al54

2009.

4. Curcuma amada Zingiberaceae Hydroxyl, ester, carbonyl

& olefinic groups.

Mujumdar et al55,

2000.

5. Borassus

flabellifer

Arecaceae Alkaloids, terpenoids,

saponins & phenolic

compounds.

Paschapur et al56,

2006.

6. Vitex negundo Verbenaceae Alkaloids, saponins &

tannins.

Dharmasiri et

al57, 2003.

7. Securidaca

longipedunculata

Polygalaceae Elymoclavine,

dehydroelymoclavine,

ergoline, cinnamonic

acid, flavonoids &

xanthones.

Okoli et al58,

2005.

8. Gongronema

latifolium

Asclepiadaceae Alkaloids, saponins,

tannins & flavanoids.

Akuodor et al59,

2010.


9. Teucrium

buxifolium

Lamiaceae Sugars, phenolic

compound, flavones,

saponins, steroids &

resins.

Puntero et al60,

1997.

10. Arecastrum

romanzoffianum

Arecaceae Galactomannoglucan. Silva et al61,

2010.

11. Stachytarpheta

cayennensis

Verbenaceae

Iridoid ipolamide &

phenylethanoid glycoside

aceteoside.

Schapoval et al62,

1998.

12. Zizyphus lotus Verbenaceae Cyclopeptide alkaloids,

dammarane saponins &

flavonods.

Borgi et al63,

2007.

13. Solanum nigrum Solanaceae

Flavones, saponins,

steroids & alkaloids.

Ravi et al64,

2009.

14. Anthurium

cerrocampanense

Arecaceae Terpenoids, saponins and

alkaloids.

Segura et al65,

1998.

15. Byrsocarpus

coccineus

Connaraceae Alkaloids, saponins,

tannins, phlobatannins,

anthraquinones,

glycosides & simple

sugars.

Akindele et al66,

2007.


Table No: 3.4 Name of the plants reported for anti-ulcer and anti-inflammatory

activities

S. No. Plant Family Chemical constituent Reference

1. Bryophyllum

pinnata

Crassulaceae glycosides - quercetin

and kaempferol &

fumaric acid.

Pal et al67, 1991.

2. Calligonum

comosum

Verbenaceae Flavonoids, alkaloids

and saponins.

Liu et al68, 2001.

3. Calotropis

procera

Asclepiadaceae Alkaloids & glycosides Basu et al69 1997.

4. Psidium guajava Myrtaceae linoleic acid & Vitamin

C.

Kulkarni et al70,

1999.

5. Mikania cordata Compositae Stigmasterol, β-

sitosterol, sesquiterpene

& scandenolide

Bhattacharya et

al71, 1988.

6. Rhamnus

procumbers

Rhamnaceae kaempferol, kaempferol

-7-O-methyl ether

kaempferol-4-O-methyl

ether & flavones.

Goel et al72,

1988.

7. Rhamnus

triquerta

Rhamnaceae Emodin, kaempferol &

8 β-D-glucoside.

Goel et al73,

1991.


4. METHODOLOGY

Table No: 4.1 List of materials and equipments used during experiment

S. No. Name of the materials and equipment

1 Anaesthetic ether (Sigma Solvents and Pharmaceuticals, Mumbai)

2 Ibuprofen (Cipla, Mumbai)

3 Carrageenan (Hi-Media Laboratory, Mumbai.)

4 Ethanol (Nice-Cochin)

5 Formalin (Nice-Cochin)

6 Ranitidine (Glaxo Smithkline Ltd, Mumbai)

7 Absolute alcohol (Nice-cochin)

8 Distilled water (Mysore Petro Chemicals, Raichur, India)

9 Microscope (R-motion Opik – India)

10 Evans blue (S.D. Fine chemicals, Mumbai)

11 Lead Mercury (S.D. Fine chemicals, Mumbai)

12 Histamine (S.D. Fine chemicals, Mumbai)

13 Restraint cages (Shri. Venkateswara Enterprises, Bangalore)

14 Chloroform (Nice-Cochin)

15 Refrigerator (Whirlpool-Kelvinator India Ltd, India)

16 Sodium Hydroxide (Nice-Cochin)


EXPERIMENTAL

4.1 Preparation of Different Extracts74:

A. Preparation of alcoholic extract:

Tubers of S. tuberosum were collected in the month of August from the Hind

Pharmacy, Sultanpur (U.P) and dried in shade at room temperature then subjected to size

reduction to a fine powder with the help of mixer grinder.

The tubers powder was packed in a soxhlet apparatus and extracted with 95% alcohol

for 18 h. Appearance of colourless solvent in the siphon tube was taken as the termination

of extraction. The extract was then transferred into the previously weighed empty beaker

and evaporated to a thick paste on the water bath, maintained at 50oC to get alcoholic

(AETST) extract. The extract was finally air dried thoroughly to remove all traces of the

solvent and the percentage yield was calculated.

B. Preparation of aqueous extract:

About 100 g of tubers powder was taken in a round bottom flask (2000 ml) and

macerated with 500 ml of distilled water with 10 ml of chloroform (preservative) for 24 h

with shaking for every hour in a closed vessel. Then the marc was removed by filtering the

extract (AQETST) and then it was concentrated on a water bath maintained at 50oC.

These two extracts were stored in airtight containers in a refrigerator below 10oC.

The two extracts were examined for their colour and consistency. Their percentage yield

was calculated with reference to air-dried powder sample used for the extraction.


Table No: 4.2 Nature and Percentage yield of the extracts

Both the AETST and AQETST were subjected to the following investigations:

1. Preliminary phytochemical screening.

2. Pharmacological activities

a. Toxicity studies (LD50).

b. Anti-ulcer activity.

c. Anti-inflammatory activity.

4.2 Preliminary phytochemical investigations:

The preliminary phytochemical investigations were carried out with AETST and

AQETST for qualitative identification of phytochemical constituents present with each

extract and test were carried out by following standard methods74,75,76. All the chemicals

and reagents used were of analytical grade.

S. No. Name of the Extract Nature Colour %Yield

( w/w) g

1. Alcohol

(AETST) Sticky Reddish Brown 12.50

2. Aqueous

(AQETST) Solid Dark brown 12.00


Table No: 4.3 Details of qualitative phytochemical tests

Test AETST AQETST

I. Tests for sterols

1. Test solution + Conc H2SO4 + +

2. Salkowski’s test + +

3. Test solution + sulphur + +

4. Libermann Burchard’s test + +

II. Tests for Glycosides

1. Baljet’s test + +

2. Keller – killiani test + +

3. Bromine water test. + +

4. Legal’s test + +

III. Test for Saponins

1. Foam test _ _

IV. Tests for Carbohydrates

1. Molisch’s test + +

2. Barfoed’s test + +

3. Benedict’s test + +

4. Fehling test + +

V. Tests for Alkaloids

1. Mayer’s test. + +

2. Wagner’s test. + +

3. Hager’s test + +

4. Dragendorff’s test + +

VI. Test for Ascorbic acid

1. Dichlorophenolindophenol test _ _


VII. Tests for Flavonoids

1. Ferric chloride test + +

2. Shinoda test _ _

3. Zn-Hcl reduction test + +

4. Sodium hydroxide test + +

5. Lead acetate test. + +

VIII. Tests for Tannins

1. Ferric chloride test. + +

2. Gelatin test _ _

IX. Tests for Proteins 1. Million’s test _ _

2. Biuret’s test _ _

3. Ninhydrin test _ _

X. Test for fixed oils and fats

1. Spot test _ _

XI. Tests for triterpines

1. Salkowski’s test + +

2. Libermann- Burchard test + +

(+) Indicates positive result and (–) Indicates negative result.


4.3 Pharmacological activities:

Experimental animals:

Albino rats (Wistar strain) of either sex weighing between 120-200 g and Albino

mice 18-22 g were procured from National Centre for Laboratory Animal sciences, C/0

Shri. Venkateswara Enterprises, Bengaluru for experimental purpose and the animals were

acclimatized for 7 days under standard husbandry condition as:

Room temperature - 26 ± 20 C

Relative humidity - 45-55%

Light/ dark cycle - 12:12 h

The animals were fed with synthetic standard pellet diet purchased from Amrut

Laboratories & Pranav Agro industries Ltd. Sangli (MS) and water was allowed ad libitum

under strict hygienic conditions. All animal studies were performed in accordance to

CPCSEA Guidelines No. 425 and Institutional Animal Ethical Committee (IAEC) of V.L.

College of Pharmacy, Raichur (Karnataka) with CPCSEA Registration Number

557/02/e/CPCSEA. The procedures were followed as per rules and regulations.

4.3.1 Determination of acute toxicity (LD50)77

Method:

The acute toxicity of AETST and AQETST was determined in albino mice of either

sex weighing between 18-22 g those maintained under standard husbandry conditions. The

animals were fasted 3 h prior to the experiment and “up and down” (OECD Guideline No.

425) method of CPCSEA was adopted for toxicity studies. Animals were administered


with single dose of extracts and observed for its mortality during 48 h study period (short

term) toxicity. Based on the short-term toxicity profile of the extracts the doses of the next

animals were determined as per as OECD Guidelines No: 425. All the animals were

observed for long term toxicity (7 days) and then 1/5th, 1/10th and 1/20th of the maximum

dose tested for LD50 of the individual extract was taken as effective dose ED50 and were

used throughout the experimental studies.

4.3.2 Determination of Anti-ulcer Activity:

1. Pylorus Ligation Model78-81:

Albino rats weighing between 150-200 g and each group containing 6 animals were

divided into 8 groups.

Group A: Normal animals treated with vehicle only

Group B: Standard Ranitidine (30 mg/kg i.p)

Group C: AETST (100 mg/kg p.o)

Group D: AETST (200 mg/kg p.o)

Group E: AETST (400 mg/kg p.o)

Group F: AQETST (100 mg/kg p.o)

Group G: AQETST (200 mg/kg p.o)

Group H: AQETST (400 mg/kg p.o)


Experimental Procedure:

Albino rats weighing between 150-200 g were divided into 8 groups of 6 rats in

each. They were fasted in individual cages for 24 h prior to the experiment with free

access to water with measures to avoid coprophagy. Group A served as normal control,

which was given with vehicle only. Group B with standard drug, groups C, D, E and F,

G, H treated with low, medium and high doses of AETST and AQETST respectively.

The various groups were treated with vehicle/extracts 30 min prior to pylorus ligation.

Under light ether anesthesia, the abdomen was opened and the pylorus was ligated

and sutured. 4 h after ligation all the animals were sacrificed with excess of anaesthetic

ether and the stomach were dissected out. Gastric juice was collected into tubes and

centrifuged at 1000 rpm for 10 min and volume was noted. The pH of the gastric juice will

be recorded by pH meter. The gastric content was subjected for analysis of free and total

acidity. The glandular portion of the stomach was opened along the greater curvature and

the severity of hemorrhagic erosions in the acid secreting mucosa was assessed on a scale

of 0 to 3, i.e. ulcer index was determined by the following formula,

Ui = UN+US+UP×10-1

Where Ui is Ulcer index

UN = average of number of ulcers per animal

US = average of severity score

UP = percentage of animals with ulcers


Mean ulcer score for each animal is expressed as Ulcer Index. The percentage ulcer

protection was calculated using the formula

Percentage ulcer protection = Uc - Ut / Uc X 100

Where Ut = Ulcer index of treated group

Uc = Ulcer index of the control group

Table No: 4.4.Ulcer scores

Reagents for biochemical estimations of free and total acidity of gastric juice

1) Reagents for estimation of free and total acidity

a. Freshly prepared 0.01N oxalic acid solution was used to standardize Sodium

hydroxide.

b. Freshly prepared 0.01N Sodium hydroxide

c. Topfer’s reagent. It is Dimethylamino azobenzene 0.5% in absolute Ethanol available

in 100 ml package.

d. Freshly prepared 1% Phenolphthalein solution prepared in 50% absolute Ethanol.

S. No. Stomach colour Ulcer score

1 Normal colour 0

2 Red colour 0.5

3 Red spots 1

4 Hemorrhagic streaks 1.5

5 3 > 5 ulcers 2

6 < 5 ulcers 3


Methods for biochemical estimation of free and total acidity:

Collection of gastric juice82,83

Gastric content collected from pylorus ligated rats was centrifuged and the volumes

of gastric juice as well as pH of gastric juice were noted. Further the gastric juice was

subjected to biochemical estimations as follows:

Determination of free and total acidity

1 ml of gastric juice was pipetted into a 100 ml conical flask, 2 or 3 drops of Topfer’s

reagent was added and titrated with 0.01N Sodium hydroxide until all traces of red colour

disappears and the colour of the solution was yellowish orange. The volume of alkali

added was noted. This volume corresponds to free acidity. Then 2 or 3 drops of

Phenolphthalein solution was added and titration was continued until a definite red tinge

appears. Again the total volume of alkali added was noted now this volume corresponds to

total acidity.

Acidity was calculated by using the formula

Volume of NaOH × Normality of NaOH × 100

Acidity = --------------------------------------------------------------------- m eq/lt/100 g

0.1


2. Stress induced ulcers by cold water immersion84:

Albino rats weighing between 150-200 g and each group containing 6 animals were

divided into 8 groups.

Group A: Normal control treated with vehicle only

Group B: Standard Ranitidine (30 mg/kg i.p)








Albino rats of either sex weighing between 150-200 g were divided into 8 groups

of 6 rats in each. Group A served as normal control, which was given with vehicle only.

Group B with standard drug. Groups C, D, E and F, G, H treated with low, medium and

high doses of AETST and AQETST respectively. After 30 min of oral administration of

the vehicle/Std/extracts, rats were placed vertically in individual restraint cages in cold

water maintained at 220C for 1 h. Then, they were taken out, dried and injected with 30

mg/kg Evans blue i.v. via the tail vein. 10 min later, sacrificed with ether and their

stomachs were removed. Formol-saline (2%v/v) is then injected into the totally ligated

stomachs for overnight storage. The next day, the stomachs were opened along the

greater curvature, washed in warm water and examined for ulcers microscopically with

the help of hand lens (10x). Mean ulcer score for each animal is expressed as ulcer index.


4.3.3 Determination of anti-inflammatory activity:

1. Carrageenan induced paw edema85-87:

Group A: Toxicant control (0.1 ml of 1% w/v Carrageenan, hind paw)

Group B: Standard (Ibuprofen 40 mg/ kg, p.o)








Male albino rats (125-150 g) of 8 groups were housed as groups of six, fasted

overnight prior to and during the experiment but have free access to water. Group A was

served as normal toxicant control treated with toxicant carrageenan, group B with Ibuprofen

(40 mg/kg p.o.) served as standard, groups C, D and E administered with AETST and

groups F, G and H with AQETST (low, medium and high doses p.o) respectively. The rats

in Groups B, C, D, E, F, G and H were administered with 0.1 ml of 1% w/v of carrageenan

into sub plantar region of right hind paw of rats 1 h after the administration of

Ibuprofen/extracts. Immediately thereafter the oedema volumes of the injected paws were

measured plethysmographically at prefixed time intervals.


2. Histamine induced paw edema85-87:

Group A: Toxicant control (0.1 ml of 1% w/v histamine, hind paw)

Group B: Standard (Ibuprofen 40 mg/ kg)








Male albino rats (125-150 g) of 8 groups were housed as groups of 6, fasted overnight

prior to and during the experiment but have free access to water. Group A was served as

toxicant control treated with inflammogen histamine, group B with Ibuprofen (40 mg/kg

p.o.) that served as standard. Groups C, D, E and F, G, H will administered with AETST and

AQETST (low, medium and high dose p.o) respectively. The rats of groups B, C, D, E, F, G

and H were administered with 1% w/v of histamine into sub plantar region of right hind paw

of rats 1 h after administration of Ibuprofen/extracts. Immediately thereafter the oedema

volumes of the injected paws were measured plethysmographically at prefixed time

intervals.


3. Formalin Induced Paw Oedema84-87:

Group A: Toxicant control (Formalin 1%, hind paw)

Group B: Standard (Ibuprofen 40 mg/ kg)








Male albino rats (125-150 g) of 8 groups were housed as groups of 6, fasted overnight

prior to and during the experiment but have free access to water. Group A was served as

toxicant control treated with toxicant Formalin, group B with Ibuprofen (40 mg/kg p.o.) that

served as standard. Groups C, D, E and F, G, H were administered with AETST and

AQETST (low, medium and high dose p.o) respectively. The rats of groups B, C, D, E, F, G

and H were administered with 1% of Formalin into sub plantar region of right hind paw of

rats 1 h after administration of Ibuprofen/extracts. Immediately thereafter the oedema

volumes of the injected paws were measured plethysmographically at prefixed time

intervals.


For comparison purpose, the volume of oedema was measured at prefixed time

intervals. The difference between paw volumes of the treated animals was measured and the

mean oedema volume was calculated. Percentage reduction in oedema volume was

calculated by using the formula,

Vo - Vt

Percentage reduction = x 100

Vo

Where, Vo = Volume of the paw of control at time‘t’.

Vt = Volume of the paw of drug treated at time‘t’.

Statistical analysis:

All results will be expressed as mean ± SEM from 6 animals. Statistical difference in

mean will be analyzed using one-way ANOVA (analysis of variance) followed by Post hoc

test (Dunnett‘s‘t’ test). P< 0.05*, 0.01** and 0.001*** will be considered as statistically

significant.


5. RESULTS

5.1 Preliminary Phytochemical studies

Preliminary phytochemical screening:

AETST and AQETST were subjected for phytochemical screening and found to

contain tannins, carbohydrate, sterols, flavonoids, glycosides, alkaloid and triterpines both

in alcoholic and aqueous extracts.

5.2 Pharmacological activities

5.2.1 Acute oral toxicity study:

The mice treated with AETST and AQETST at a dose of 2000 mg/kg, p.o.

exhibited normal behaviour, without any signs of passivity, stereotypy and vocalization.

Their motor activity and secretory signs were also normal and no sign of depression.

AETST and AQETST even up to the dose level of 2000 mg/kg body weight did not

produce any behavioural symptoms or mortality. So 1/5th, 1/10th and 1/20th doses of LD50

(maximum dose tested for each extract) was selected as low, medium and high doses and

were used in the present study to explore anti-ulcer and anti-inflammatory activities.

5.2.2 Anti-ulcer activity:

1. Pylorus ligation induced ulcer model in rats:

In pylorus ligation induced ulcer model in rats (positive control) a significant

increase in ulcer number (5.00 ± 0.51), ulcer score (2.41 ± 0.27) and ulcer index (10.74)

are noted. In the same model a significant increase in volume of gastric juice (6.70 ± 0.10

ml), free acidity (31.33 ± 1.15 m eq/L) and total acidity (82.33 ± 1.89 m eq/L) are noted.


In the group of rats treated with standard drug ranitidine (30 mg/kg) has

significantly reduced ulcer number (0.33 ± 0.21), ulcer score (0.50 ± 0.12), ulcer index

(3.41), volume of gastric juice (4.28 ± 0.13 ml), free acidity (17.33 ± 0.66 m eq/L), total

acidity (43.50 ± 1.17 m eq/L) and the ulcers are inhibited by 68.24 %.

AETST with low, medium and high dose treatment showed a significant decrease

in ulcer number (3.66 ± 0.42, 1.33 ± 0.33 and 0.83 ± 0.16), ulcer score (2.33 ± 0.21, 1.58 ±

0.08 and 0.83 ± 0.10), ulcer index (10.59, 10.29 and 8.49), volume of gastric juice (6.03 ±

0.07 ml, 5.01 ± 0.07 ml and 4.75 ± 0.07 ml), free acidity (25.00 ± 0.85, 22.33 ± 0.66 and

20.83 ± 1.01 m eq/L), total acidity (59.67 ± 1.05, 47.33 ± 2.01and 46.67 ± 0.80 m eq/L)

and the ulcers are inhibited by 1.39, 4.18 and 20.94 % respectively.

AQETST with low, medium and high dose treatment showed a significant decrease




20.00 ± 0.73 m eq/L), total acidity (58.00 ± 1.39, 46.67 ± 1.99 and 46.00 ± 0.96 m eq/L)


The order of potency in antiulcer activity is Ranitidine >AQETST > AETST.

The results are shown in Table Nos.5.1 and 5.2 and represented graphically in Fig Nos.

5.1, 5.2 and 5.3.

2. Stress induced ulcer model in rats:

In stress induced ulcer model in rats (positive control) a significant increase in

ulcer number (4.67 ± 0.33), ulcer score (2.67±0.21), ulcer index (10.73), volume of gastric


juice (6.77 ± 0.09 ml), free acidity (33.33 ± 0.66 m eq/L) and total acidity (84.83 ± 1.19 m

eq/L) are noted.

In the group of rats treated with standard drug ranitidine (30 mg/kg) has significantly

reduced ulcer number (0.50 ± 0.22), ulcer score (0.75 ± 0.11), ulcer index (5.12), volume

of gastric juice (4.38 ± 0.10 ml), free acidity (19.00 ± 0.68 m eq/L), total acidity (45.67 ±

0.91 m eq/L) and the ulcers are inhibited by 52.28 % .

AETST with low, medium and high dose treatment showed a significant decrease in

ulcer number (3.50 ± 0.22, 1.67 ± 0.21 and 0.83 ± 0.16), ulcer score (2.50 ± 0.22, 1.83 ±





AQETST with low, medium and high dose treatment showed a significant decrease






The order of potency in antiulcer activity is Ranitidine >AQETST > AETST.

The results are shown in Table Nos.5.3 and 5.4 and represented graphically in Fig Nos.

5.4, 5.5 and 5.6.


Table No: 5.1 Antiulcer effects of AETST and AQETST in pylorus ligation induced ulcer model in rats

n = 6, Significant at P < 0.05*, 0.01** and 0.001***, ns = not significant.

AETST- Alcoholic Extract of Tuber of S. tuberosum, AQETST- Aqueous Extract of Tuber of S. tuberosum.

Ulcer number

Animals Control Ranitidine 30 mg/kg

AETST 100 mg/kg

AETST 200 mg/kg

AETST 400 mg/kg

AQETST 100 mg/kg

AQETST 200 mg/kg

AQETST 400 mg/kg

H 5 0 3 1 0 4 1 0 B 4 0 3 1 1 4 1 1 T 6 0 3 3 1 3 1 1

HB 7 1 5 1 1 3 2 0 HT 4 0 3 1 1 3 1 1 BT 4 1 5 1 1 3 1 1

mean±SEM 5.00±0.51 0.33±0.21*** 3.66±0.42* 1.33±0.33*** 0.83±0.16*** 3.33±0.21** 1.16±0.16*** 0.66±0.21***

Ulcer Score H 3.0 0.0 2 1.5 0.5 3.0 1.5 0.5 B 2.0 0.5 2 1.5 0.5 2.0 1.5 0.5 T 3.0 0.5 2 2.0 1.0 2.0 1.5 1.0

HB 3.0 1.0 3 1.5 1.0 2.0 1.5 0.5 HT 2.0 0.5 2 1.5 1.0 1.5 1.0 1.0 BT 1.5 0.5 3 1.5 1.0 2.0 1.0 1.0

mean±SEM 2.41±0.27 0.50±0.12*** 2.33±0.21ns 1.58±0.08** 0.83±0.10*** 2.08±0.20ns 1.33±0.10*** 0.75±0.11***


Table No: 5.2 Antiulcer effects of AETST and AQETST in pylorus ligation induced ulcer model in rats



VOLUME OF GASTRIC JUICE (ml)


AETST 100 mg/kg

AETST 200 mg/kg

AETST 400 mg/kg

AQETST 100 mg/kg

AQETST 200 mg/kg

AQETST 400 mg/kg

H 6.5 4.5 5.9 5.0 5.0 6.2 5.1 4.3 B 6.5 4.8 6.0 4.8 4.5 5.5 4.8 4.2 T 7.0 4.0 5.8 5.2 4.7 5.4 4.8 5.0

HB 6.4 3.9 6.2 4.9 4.9 5.9 4.9 4.5 HT 6.8 4.3 6.0 5.3 4.8 5.7 4.7 4.8 BT 7.0 4.2 6.3 4.9 4.6 5.6 4.8 4.6

mean±SEM 6.70±0.10 4.28±0.13*** 6.03±0.07*** 5.01±0.07*** 4.75±0.07*** 5.71±0.11*** 4.85±0.05*** 4.56±0.12*** FREE ACIDITY (m eq/L)

H 35 16 27 20 22 27 20 19 B 30 15 25 22 21 25 22 21 T 28 19 23 24 18 20 24 18

HB 32 18 28 23 20 28 23 20 HT 34 17 23 24 19 21 22 19 BT 29 19 24 21 25 22 20 23

mean±SEM 31.33±1.15 17.33±0.66*** 25.00±0.85*** 22.33±0.66*** 20.83±1.01*** 23.83±1.35*** 21.83±0.65*** 20.00±0.73*** TOTAL ACIDITY (m eq/L)

H 76 41 56 53 46 55 52 43 B 82 44 58 50 48 54 50 47 T 88 48 59 42 44 56 42 44

HB 86 40 60 41 49 60 41 49 HT 78 45 62 47 45 62 44 45 BT 84 43 63 51 48 61 51 48

mean±SEM 82.33±1.89 43.50±1.17*** 59.67±1.05*** 47.33±2.01*** 46.67±0.80*** 58.00±1.39*** 46.67±1.99*** 46.00±0.96***


Table No: 5.3 Antiulcer effects of AETST and AQETST in stress induced ulcer model in rats



Ulcer number


AETST 100 mg/kg

AETST 200 mg/kg

AETST 400 mg/kg

AQETST 100 mg/kg

AQETST 200 mg/kg

AQETST 400 mg/kg

H 4 1 4 2 1 4 2 1 B 4 0 3 2 1 3 1 1 T 6 0 3 1 0 3 2 1

HB 5 1 3 1 1 3 1 1 HT 5 0 4 2 1 3 2 0 BT 4 1 4 2 1 3 1 1

mean±SEM 4.67±0.33 0.50±0.22*** 3.50±0.22** 1.67±0.21*** 0.83±0.16*** 3.17±0.16*** 1.50±0.22*** 0.83±0.16***

Ulcer score H 2.0 1.0 3.0 2.0 1.0 3.0 2.0 1.0 B 3.0 0.5 2.0 2.0 1.5 2.0 1.5 1.0 T 3.0 0.5 2.0 1.5 1.0 3.0 1.5 1.0

HB 3.0 1.0 2.0 1.5 1.0 2.0 1.5 0.5 HT 3.0 0.5 3.0 2.0 1.0 2.0 2.0 1.0 BT 2.0 1.0 3.0 2.0 1.0 3.0 1.5 1.0

mean±SEM 2.67±0.21 0.75±0.11*** 2.50±0.22 ns 1.83±0.10** 1.08±0.08*** 2.50±0.22 ns 1.67±0.10*** 0.91±0.08***


Table No: 5.4 Antiulcer effects of AETST and AQETST in stress induced ulcer model in rats



VOLUME OF GASTRIC JUICE (ml)


AETST 100 mg/kg

AETST 200 mg/kg

AETST 400 mg/kg

AQETST 100 mg/kg

AQETST 200 mg/kg

AQETST 400 mg/kg

H 6.8 4.6 6.2 5.2 5.1 6.2 5.1 4.6 B 6.6 4.8 6.0 5.0 4.7 5.9 4.9 4.8 T 7.0 4.1 6.1 5.1 4.8 5.8 4.8 5.0

HB 6.4 4.2 6.2 5.2 5.0 6.2 5.1 4.5 HT 6.8 4.3 6.4 5.3 4.9 5.9 4.7 4.8 BT 7.0 4.3 6.3 5.1 4.8 6.1 5.1 4.9

mean±SEM 6.77±0.09 4.38±0.10*** 6.20±0.05*** 5.15±0.04*** 4.88±0.06*** 6.02±0.07*** 4.95±0.07*** 4.77±0.07*** FREE ACIDITY (m eq/L)

H 31 20 25 24 23 26 20 21 B 33 17 26 22 24 28 24 24 T 32 21 27 28 20 24 23 22

HB 34 20 29 23 22 28 25 19 HT 35 19 26 26 22 23 23 20 BT 35 17 28 24 25 25 25 23

mean±SEM 33.33±0.66 19.00±0.68*** 26.83±0.60*** 24.50±0.88*** 22.67±0.71*** 25.67±0.84*** 23.33±0.76*** 21.50±0.76*** TOTAL ACIDITY (m eq/L)

H 80 44 58 50 48 60 52 45 B 83 43 62 52 50 60 51 50 T 86 48 60 48 48 58 44 44

HB 88 48 64 45 51 61 45 49 HT 87 47 62 47 47 62 44 48 BT 85 44 64 52 48 61 51 47

mean±SEM 84.83±1.19 45.67±0.91*** 61.67±0.95*** 49.00±1.15*** 48.67±0.61*** 60.33±0.55*** 47.83±1.57*** 47.17±0.94***


Table No: 5.5 Antiulcer effects of AETST and AQETST in different ulcers models in rats


AETST- Alcoholic Extract of Tuber of S. tuberosum, AQETST- Aqueous Extract of Tuber of S. tuberosum

Pylorus ligation model Stress induced ulcer model

Groups Treatment Ulcer

Number Ulcer Score

Incidence of Ulcers

(%)

Ulcer Index

Inhibition of Ulcers

(%)

Ulcer Number

Ulcer score

Incidence of Ulcers

(%)

Ulcer Index

Inhibition of Ulcers

(%)

Control vehicle

10 ml/kg p.o 5.00

±0.51 2.41

±0.27 100 10.74 - 4.67

±0.33 2.67

±0.21 100 10.73 --

Standard Ranitidine 30 mg/kg

0.33 ±0.21***

0.50 ±0.12***

33.33 3.41 68.24 0.50 ±0.22***

0.75 ±0.11***

50 5.12 52.28

AETST 100 mg/kg

p.o 3.66

±0.42* 2.33

±0.21ns 100 10.59 1.39 3.50 ±0.22**

2.50 ±0.22 ns

100 10.60 1.21

AETST 200 mg/kg

p.o 1.33

±0.33*** 1.58

±0.08** 100 10.29 4.18 1.67

±0.21*** 1.83

±0.10** 100 10.35 3.54

AETST 400 mg/kg

p.o 0.83

±0.16*** 0.83

±0.10*** 83.33 8.49 20.94 0.83

±0.16*** 1.08

±0.08*** 83.33 8.52 20.59

AQETST 100 mg/kg

p.o 3.33

±0.21** 2.08

±0.20ns 100 10.54 1.86 3.17 ±0.16***

2.50 ±0.22 ns 100 10.56 1.58

AQETST 200 mg/kg

p.o 1.16

±0.16*** 1.33

±0.10*** 100 10.24 4.65 1.50

±0.22*** 1.67

±0.10*** 100 10.31 3.91

AQETST 400 mg/kg

p.o 0.66

±0.21*** 0.75

±0.11*** 66.66 6.80 36.68 0.83

±0.16*** 0.91

±0.08*** 83.33 8.50 20.78

Dept. of Pharmacology, V.L.C.P Raichur

Fig No.5.1

Fig No.5.2


Fig No.5.3


Fig No.5.4

Fig No.5.5


Fig No.5.6


5.2.3 Anti-inflammatory activity:

1. Anti-inflammatory activity of AETST and AQETST in Carrageenan induced paw

oedema model in rats:

The AETST and AQETST with three selected doses i.e. 100, 200 and 400 mg/kg

have exhibited a significant reduction in paw oedema volume in carrageenan induced paw

oedema in rats at different time intervals. Results are tabulated in Table No. 5.7. Ibuprofen

(40 mg/kg) was used as standard reference and it has significantly reduced paw oedema

volume by 42.85% at 1st h, 61.43% at 2nd h, 79.29% at 3rd h and 85.33% at 4th h, thus

standard drug has exhibited time dependent reduction in oedema volume.

During 1st h of study AETST and AQETST with low, medium and high doses have

significantly reduced oedema volume by 4.85%, 14.28%, 24.00% and 9.71%, 19.14%,

28.57% respectively, which was found to be a time dependent effect.

During 2nd h of study AETST and AQETST with low, medium and high doses have


38.33% respectively a time dependent effect.

During 3rd h of study AETST and AQETST with low, medium and high doses have


44.72% respectively a time dependent effect was noted.

During 4th h of study AETST and AQETST with low, medium and high doses have


66.66% respectively a time dependent effect was noted and result are graphically

represented in Fig No.5.7.


ANOVA indicates a significant difference among the AETST and AQETST treated

groups. Dunnett’s‘t’ test confirms a significant anti-inflammatory activity with both the

extracts, but more with AQETST than AETST.


Table No: 5.6 Anti-inflammatory effects of AETST and AQETST in Carrageenan induced paw oedema model in rats

Time after Paw oedema volume (ml) Treatment Animals Toxicant Standard AETST

100 mg/kg AETST

200 mg/kg AETST

400 mg/kg AQETST 100 mg/kg

AQETST 200 mg/kg

AQETST 400 mg/kg

H 0.35 0.20 0.35 0.25 0.25 0.30 0.25 0.30 B 0.40 0.25 0.40 0.30 0.30 0.30 0.30 0.25 T 0.30 0.10 0.25 0.30 0.25 0.35 0.20 0.20

HB 0.35 0.30 0.35 0.35 0.30 0.30 0.30 0.30 BT 0.35 0.20 0.30 0.30 0.30 0.35 0.35 0.25

1 h

HT 0.35 0.15 0.35 0.30 0.20 0.30 0.30 0.20 mean±SEM 0.350±0.012 0.200±0.028*** 0.333±0.021ns 0.300±0.012ns 0.266±0.016* 0.316±0.010ns 0.283±0.021ns 0.250±0.018**

H 0.50 0.15 0.35 0.25 0.25 0.35 0.30 0.20 B 0.40 0.25 0.40 0.40 0.30 0.40 0.30 0.25 T 0.45 0.10 0.35 0.35 0.35 0.40 0.25 0.30

HB 0.40 0.25 0.45 0.40 0.30 0.35 0.40 0.30 BT 0.45 0.15 0.45 0.30 0.25 0.45 0.30 0.25

2 h

HT 0.40 0.10 0.40 0.30 0.35 0.35 0.35 0.30 mean±SEM 0.433±0.016 0.167±0.027*** 0.400±0.018ns 0.333±0.024** 0.300±0.018*** 0.383±0.016ns 0.317±0.021** 0.267±0.016***

H 0.50 0.10 0.40 0.30 0.30 0.40 0.40 0.20 B 0.45 0.15 0.45 0.40 0.30 0.40 0.35 0.25 T 0.50 0.00 0.40 0.35 0.35 0.40 0.30 0.30

HB 0.50 0.15 0.45 0.40 0.25 0.35 0.30 0.30 BT 0.45 0.10 0.40 0.35 0.30 0.45 0.35 0.35

3 h

HT 0.50 0.10 0.40 0.30 0.30 0.40 0.30 0.20 mean±SEM 0.483±0.010 0.100±0.022*** 0.417±0.010* 0.350±0.018*** 0.300±0.012*** 0.400±0.012** 0.333±0.016*** 0.267±0.024***

H 0.50 0.10 0.30 0.30 0.20 0.30 0.30 0.15 B 0.45 0.00 0.40 0.35 0.15 0.35 0.30 0.10 T 0.40 0.15 0.35 0.30 0.30 0.30 0.25 0.20

HB 0.50 0.10 0.40 0.25 0.10 0.25 0.30 0.15 BT 0.40 0.05 0.35 0.35 0.15 0.40 0.25 0.20

4 h

HT 0.45 0.00 0.30 0.25 0.20 0.30 0.20 0.10 mean±SEM 0.450±0.018 0.066±0.024*** 0.350±0.018** 0.300±0.018*** 0.183±0.027*** 0.317±0.021*** 0.267±0.016*** 0.150±0.018***


AETST- Alcoholic Extract of Tuber of S. tuberosum, AQETST- Aqueous Extract of Tuber of S. tuberosum .


Table No: 5.7 Anti-inflammatory effects of AETST and AQETST in Carrageenan induced paw oedema model in rats at different time intervals

n = 6, Significant at P < 0.05*, 0.01** and 0.001***, ns = not significant. ROV- Reduction of Oedema Volume.


S.No. Groups Treatment 1 h %

ROV 2 h % ROV 3 h % ROV 4 h % ROV

A Toxicant Carrageenan

(1% w/v) 0.350 ±0.012

-- 0.433 ±0.016

-- 0.483 ±0.010

-- 0.450 ±0.018

--

B Standard Ibuprofen 40 mg/kg

0.200 ±0.028***

42.85

0.167 ±0.027***

61.43 0.100 ±0.022***

79.29 0.066 ±0.024***

85.33

C AETST 100 mg/kg 0.333 ±0.021ns

4.85 0.400 ±0.018ns

7.62 0.417 ±0.010*

13.66 0.350 ±0.018**

22.22

D AETST 200 mg/kg 0.300 ±0.012ns

14.28

0.333 ±0.024**

23.09 0.350 ±0.018***

27.53 0.300 ±0.018***

33.33

E AETST 400 mg/kg 0.266 ±0.016*

24.00

0.300 ±0.018***

30.71 0.300 ±0.012***

37.88 0.183 ±0.027***

59.25

F AQETST 100 mg/kg 0.316 ±0.010ns

9.71

0.383 ±0.016ns

11.44 0.400 ±0.012**

17.14 0.317 ±0.021***

29.55

G AQETST 200 mg/kg 0.283 ±0.021ns

19.14

0.317 ±0.021**

26.78 0.333 ±0.016***

31.05 0.267 ±0.016***

40.66

H AQETST 400 mg/kg 0.250 ±0.018**

28.57

0.267 ±0.016***

38.33 0.267

±0.024*** 44.72 0.150 ±0.018***

66.66


2. Anti-inflammatory activity of AETST and AQETST in Histamine induced paw



have exhibited a significant reduction in paw oedema volume in histamine induced paw

oedema in rats at different time intervals. Results are tabulated in Table No. 5.9. Ibuprofen

(40 mg/kg) was used as standard reference and it has significantly reduced paw oedema

volume by 56.39% at 1st h, 70.44% at 2nd h, 86.80% at 3rd h and 92.93% at 4th h, thus

exhibited a time dependent reduction in oedema volume.



47.78% respectively, which was found to be a time dependent effect.



51.77% respectively noted a time dependent effect.



63.40% respectively a time dependent effect was noted.



74.94% respectively a time dependent effect was noted and result are graphically

represented in Fig No.5.8.


Table No: 5.8 Anti-inflammatory effects of AETST and AQETST in Histamine induced paw oedema model in rats


100 mg/kg AETST

200 mg/kg AETST


AQETST 200 mg/kg

AQETST 400 mg/kg

H 0.35 0.15 0.40 0.25 0.20 0.30 0.25 0.20 B 0.40 0.10 0.35 0.25 0.25 0.40 0.30 0.15 T 0.40 0.20 0.30 0.35 0.30 0.40 0.35 0.20

HB 0.40 0.20 0.40 0.40 0.20 0.35 0.30 0.20 BT 0.40 0.20 0.35 0.30 0.25 0.30 0.25 0.25

1 h

HT 0.35 0.15 0.40 0.35 0.20 0.35 0.25 0.20 mean±SEM 0.383±0.010 0.167±0.016*** 0.367±0.016ns 0.317±0.024* 0.233±0.016*** 0.350±0.018ns 0.283±0.016** 0.200±0.012***

H 0.45 0.20 0.45 0.30 0.20 0.35 0.35 0.25 B 0.50 0.15 0.30 0.30 0.25 0.40 0.30 0.20 T 0.40 0.10 0.30 0.35 0.30 0.40 0.35 0.25

HB 0.50 0.15 0.50 0.35 0.20 0.30 0.30 0.20 BT 0.45 0.10 0.45 0.35 0.25 0.35 0.30 0.15

2 h

HT 0.40 0.10 0.40 0.35 0.30 0.40 0.30 0.25 mean±SEM 0.450±0.018 0.133±0.016*** 0.400±0.034ns 0.333±0.010*** 0.250±0.018*** 0.367±0.016* 0.317±0.010*** 0.217±0.016***

H 0.45 0.10 0.45 0.30 0.20 0.30 0.30 0.15 B 0.50 0.15 0.40 0.30 0.20 0.35 0.30 0.15 T 0.50 0.00 0.40 0.35 0.25 0.40 0.25 0.20

HB 0.55 0.00 0.40 0.30 0.20 0.35 0.30 0.20 BT 0.50 0.05 0.45 0.35 0.25 0.40 0.25 0.15

3 h

HT 0.50 0.10 0.40 0.30 0.20 0.40 0.30 0.25 mean±SEM 0.500±0.012 0.066±0.024*** 0.417±0.010** 0.317±0.010*** 0.217±0.010*** 0.367±0.016*** 0.283±0.010*** 0.183±0.016***

H 0.45 0.00 0.40 0.20 0.15 0.25 0.20 0.10 B 0.40 0.10 0.35 0.25 0.15 0.30 0.20 0.10 T 0.50 0.00 0.30 0.30 0.20 0.30 0.15 0.15

HB 0.50 0.00 0.30 0.25 0.10 0.30 0.25 0.10 BT 0.50 0.00 0.40 0.30 0.15 0.30 0.20 0.10

4 h

HT 0.45 0.10 0.35 0.20 0.15 0.35 0.30 0.15 mean±SEM 0.467±0.016 0.033±0.021*** 0.350±0.018*** 0.250±0.018*** 0.150±0.012*** 0.300±0.012*** 0.217±0.021*** 0.117±0.010***




Table No: 5.9 Anti-inflammatory effects of AETST and AQETST in Histamine induced paw oedema model in rats at different

time intervals



S.No. Groups Treatment 1 h % ROV 2 h % ROV 3 h % ROV 4 h % ROV

A Toxicant Histamine (1% w/v)

0.383 ±0.010

-- 0.450 ±0.018

-- 0.500 ±0.012

-- 0.467 ±0.016

--

B Standard Ibuprofen 40 mg/kg

0.167 ±0.016***

56.39 0.133 ±0.016*** 70.44 0.066

±0.024*** 86.80 0.033

±0.021*** 92.93

C AETST 100 mg/kg 0.367 ±0.016ns

4.17 0.400 ±0.034ns

11.11 0.417 ±0.010**

16.60 0.350 ±0.018*** 25.05

D AETST 200 mg/kg 0.317 ±0.024*

17.23 0.333 ±0.010*** 26.00 0.317

±0.010*** 36.66 0.250

±0.018*** 46.46

E AETST 400 mg/kg 0.233 ±0.016***

39.16 0.250

±0.018**

*

44.44 0.217 ±0.010***

56.66 0.150 ±0.012*** 67.88

F AQETST 100 mg/kg 0.350 ±0.018ns

8.61 0.367 ±0.016*

18.44 0.367 ±0.016***

26.60 0.300 ±0.012*** 35.76

G AQETST 200 mg/kg 0.283 ±0.016**

26.10 0.317 ±0.010*** 29.55 0.283

±0.010*** 43.40 0.217

±0.021*** 53.53

H AQETST 400 mg/kg 0.200 ±0.012***

47.78 0.217 ±0.016*** 51.77 0.183

±0.016*** 63.40 0.117

±0.010*** 74.94


3. Anti-inflammatory activity of AETST and AQETST in Formalin induced paw



have exhibited a significant reduction in paw oedema volume in Formalin induced paw

oedema in rats at different time intervals. Results are tabulated in Table No. 5.11.

Ibuprofen (40 mg/kg) was used as standard reference and it has significantly reduced paw

oedema volume by 54.25% at 1st h, 64.23% at 2nd h, 83.90% at 3rd h and 89.64% at 4th h,

which was found to be a time dependent effect.



41.75% respectively noted as time dependent effect.



50.10% respectively noted as time dependent effect.



61.31% respectively recorded as time dependent effect.



72.46% respectively which was recorded as time dependent effect and result are

graphically represented in Fig No.5.9.


Table No: 5.10 Anti-inflammatory effects of AETST and AQETST in Formalin induced paw edema model in rats


100 mg/kg AETST

200 mg/kg AETST


AQETST 200 mg/kg

AQETST 400 mg/kg

H 0.40 0.20 0.40 0.30 0.25 0.40 0.35 0.25 B 0.40 0.20 0.40 0.35 0.30 0.35 0.35 0.20 T 0.45 0.15 0.25 0.30 0.20 0.40 0.25 0.20

HB 0.40 0.25 0.40 0.40 0.25 0.35 0.30 0.25 BT 0.35 0.20 0.45 0.30 0.30 0.30 0.30 0.30

1 h

HT 0.40 0.10 0.40 0.35 0.20 0.40 0.35 0.20 mean±SEM 0.400±0.012 0.183±0.021*** 0.383±0.027ns 0.333±0.016ns 0.250±0.018*** 0.367±0.016ns 0.317±0.016* 0.233±0.016***

H 0.50 0.20 0.50 0.30 0.30 0.50 0.30 0.20 B 0.45 0.15 0.40 0.40 0.25 0.35 0.35 0.25 T 0.50 0.20 0.45 0.35 0.30 0.40 0.30 0.20

HB 0.45 0.20 0.40 0.40 0.20 0.35 0.35 0.30 BT 0.40 0.15 0.45 0.40 0.25 0.40 0.30 0.20

2 h

HT 0.50 0.10 0.40 0.35 0.30 0.50 0.40 0.25 mean±SEM 0.467±0.016 0.167±0.016*** 0.433±0.016ns 0.367±0.016** 0.267±0.016*** 0.417±0.027ns 0.333±0.016*** ±0.233±0.016***

H 0.55 0.10 0.45 0.40 0.30 0.5 0.25 0.15 B 0.50 0.05 0.50 0.35 0.25 0.45 0.30 0.20 T 0.50 0.10 0.40 0.30 0.20 0.40 0.35 0.20

HB 0.50 0.10 0.45 0.30 0.25 0.30 0.35 0.25 BT 0.50 0.10 0.40 0.35 0.20 0.45 0.35 0.20

3 h

HT 0.55 0.05 0.50 0.40 0.20 0.40 0.30 0.20 mean±SEM 0.517±0.010 0.083±0.010*** 0.450±0.018ns 0.350±0.018*** 0.233±0.016*** 0.417±0.027** 0.317±0.016*** 0.200±0.012***

H 0.50 0.10 0.35 0.30 0.20 0.40 0.20 0.10 B 0.45 0.00 0.40 0.25 0.20 0.30 0.20 0.15 T 0.50 0.00 0.30 0.20 0.15 0.30 0.30 0.10

HB 0.45 0.10 0.40 0.25 0.20 0.25 0.20 0.20 BT 0.50 0.10 0.35 0.30 0.10 0.35 0.25 0.10

4 h

HT 0.50 0.00 0.40 0.30 0.15 0.30 0.25 0.15 mean±SEM 0.483±0.010 0.050±0.022*** 0.367±0.016** 0.267±0.016*** 0.167±0.016*** 0.317±0.021*** 0.233±0.016*** 0.133±0.016***




Table No: 5.11 Anti-inflammatory effects of AETST and AQETST in Formalin induced paw oedema model in rats at different

time intervals



S.No. Groups Treatment 1 h % ROV 2 h % ROV 3 h % ROV 4 h % ROV

A Toxicant Formalin (1% w/v)

0.400 ±0.012

-- 0.467 ±0.016

-- 0.517 ±0.010

-- 0.483 ±0.010

--

B Standard Ibuprofen 40mg/kg

0.183 ±0.021***

54.25 0.167 ±0.016***

64.23 0.083 ±0.010***

83.90 0.050 ±0.022***

89.64

C AETST 100 mg/kg 0.383 ±0.027ns

4.25 0.433 ±0.016ns

7.28 0.450 ±0.018ns

12.95 0.367 ±0.016**

24.01

D AETST 200 mg/kg 0.333 ±0.016ns

16.75 0.367 ±0.016**

21.41 0.350 ±0.018***

32.30 0.267 ±0.016***

44.72

E AETST 400 mg/kg 0.250 ±0.018***

37.50 0.267 ±0.016***

42.82 0.233 ±0.016***

54.92 0.167 ±0.016***

65.42

F AQETST 100 mg/kg 0.367 ±0.016ns

8.25 0.417 ±0.027ns

10.70 0.417 ±0.027**

19.34 0.317 ±0.021***

34.36

G AQETST 200 mg/kg 0.317 ±0.016*

20.75 0.333 ±0.016***

28.69 0.317 ±0.016***

38.68 0.233 ±0.016***

51.75

H AQETST 400 mg/kg 0.233 ±0.016***

41.75 ±0.233 ±0.016***

50.10 0.200 ±0.012***

61.31 0.133 ±0.016***

72.46


Fig No.5.7

Fig No.5.8


Fig No.5.9


6. DISCUSSION

Peptic ulcer is a conglomerate of most common heterogenous disorders, present as a

crater in the lining of the GIT mucosa due to acid, pepsin, bile acid, pancreatic enzyme and

bacteria. It is due to an imbalance between aggressive (acid and pepsin) and defensive

(bicarbonates, mucin, PG etc) factors. Peptic ulcer disease also occurs due to

administration of NSAIDs, stress, H. pylori or pathological condition like Zollinger –

Ellison syndrome. NSAIDs causes erosions, petechiae, type C gastritis,

ulceration/combination with interference of ulcer healing. Further they also induce damage

of the mucosa with imbalance between aggressive and defensive factors. Though a very

good numbers of anti-ulcers drugs like antisecretory drugs, H2 receptor antagonists, proton

pump inhibitors, antimuscaranic, cytoprotectants and Prostaglandins analogues are

available, the side effects associated with these drugs limit their use. Many herbal drugs

from Ayurveda of Indian traditional system of medicine are advocated for the management

of peptic ulcer. Herbal medicines used as whole plant powders/extracts from different parts

are now a days consider as safe medication for the treatment of a numbers of diseases as it

is a general notion that plant based drugs are safer without any side effects28,79,88.

Though the presently available anti-ulcer drugs have remarkable effects in ulcer

therapy the efficacy is still incomplete as there are many a number of incidences of relapse

with adverse effects and drug-drug interaction are reported with the therapy. Hence there is

a need for ideal anti-ulcer drugs with extended action from herbal source with a calibre of

better protection and low incidence of relapse of ulcers89,90.

In Indian system of medicine a very good numbers of herbs are reported to produce

antiulcer and anti-inflammatory activities. Hence in the present study a plant by name


S. tuberosum has considered to evaluate its antiulcer and anti-inflammatory activities

scientifically. For this alcohol and aqueous extracts prepared from the tubers of the plant

were tested against different ulcer models in rats and inflammatory models also in rats8.

In pylorus ligation induced ulcer model, ulcers are produced due to accumulation of

acid at the pyloric end that causes ulcers. Both the extracts AETST and AQETST

significantly reduced ulcers by decreasing gastric volume and increasing the pH, thereby

reducing the severity of ulcers i.e. ulcers numbers and ulcers index91.

Stress induced ulcer are due to release of histamine with an increase in acid secretion

and reduction in mucus production. Stress stimulate adenohypophysial axis and causes

release of endogenous opiates and also produces severe gastric erosion by the activation of

central vagal discharge which release endogenous opiates that cause mucosal congestion

by peripheral mechanism to develop gastric ulcers. Both the extracts have significantly

reduced gastric secretion thus prevented gastric mucosa from the development of ulcers.

Several studies reported that gastroduodenal protection by prostaglandins is due to increase

in mucosal resistant and decrease in aggressive factors like acid and pepsin92.

Tannins89, carbohydrate93, flavonoids94, glycosides95 and triterpines96 are reported for

their anti-ulcers activity. Both AETST and AQETST contained all the above mentioned

phytoconstituents and hence these might have contributed for the anti-ulcer activity. The

anti-ulcerogenic effect of AETST and AQETST may be related to their antisecretory action

since acid is a major factor in the development of peptic ulcers.

Carrageenan induced paw oedema model is used for screening of NSAIDs and

inflammation produced by its biphasic in nature with the release of serotonin, bradykinin

and histamine at I Phase followed by release of prostaglandins in II Phase.


Histamine being an important mediator of inflammation and also a potent vasodilator

that causes increase in vascular permeability. In both phases due to release of these

mediators cause pain and fever and both the extracts significantly reduced paw oedema in

II Phase of the inflammation indicating there effect on prostaglandins97.

Formalin induced paw oedema model consist of two phases too with nociception in

neurogenic and inflammatory phases. Drugs that primarily act on central nervous system

are capable to inhibit both phases equally and by inhibiting the late phase, peripherally

acting drugs produce there anti-inflammatory activity. The neurogenic and inflammatory

phases are due to release of substance P, serotonin, histamine, prostaglandins and

leukotrienes respectively. Both the extracts significantly reduced both phases of

inflammation in formalin induced paw oedema model in rats87.

Tannins, sterols, flavonoids, alkaloids and triterpines are reported for their anti-

inflammatory activity. Both AETST and AQETST contained all the above mentioned

phytoconstituents and hence these might have contributed for the anti-inflammatory

activity54,98.


7. CONCLUSION

Preliminary phytochemical evaluation of both AETST and AQETST revealed the

presence of tannins, carbohydrate, sterols, flavonoids, glycosides, alkaloids and

triterpines in both the extracts. Acute oral toxicity studies no mortality recorded with

either of the extracts even at the dose level of 2000 mg/kg body weight.

Anti-ulcer and anti-inflammatory activities confirmed with both the extracts in

experimental animals, rats with different ulcer and inflammatory models. In both ulcer

and inflammatory models both the extracts at low, medium and high doses produced a

significant anti-ulcer and anti-inflammatory activities (P < 0.05*, 0.01** and 0.001***).

Phytochemical constituents like tannins, flavonoids and triterpines are already

reported for their anti-ulcer and anti-inflammatory activities and both the extracts

contained the above mentioned constituents. Hence these can be accounted for the

observed anti-ulcer and anti-inflammatory activities in rats.


8. SUMMARY

S. tuberosum is mentioned for its anti-ulcer and anti-inflammatory activities in

Ayurvedic texts without an available scientific data. Hence the present study is planned to

explore anti-ulcer and anti-inflammatory activities of AETST and AQETST.

Preliminary phytochemical investigations identified with tannins, carbohydrate,

sterols, flavonoids, glycosides, alkaloid and triterpines in both extracts. Acute oral

toxicity studies revealed that there is no mortality with both the extracts even upto the

dose level of 2000 mg/kg.

In pylorus ligation induced ulcer model, selected doses like low (100 mg/kg),

medium (200 mg/kg) and high (400 mg/kg) doses (AETST and AQETST) of both the

extracts significantly reduced the ulcers (P < 0.05*, 0.01** and 0.001***). In stress

induced ulcers by cold water immersion model also both the extracts with the doses

mentioned above significantly reduced the ulcers (P < 0.05*, 0.01** and 0.001***).

In carrageenan, histamine and formalin induced inflammatory models both the

extracts significantly reduced inflammation (P < 0.05*, 0.01** and 0.001***).


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methanolic extract of Anogeissus acuminata leaf. Int J Pharm Biomed Res 2010;

1(3): 98-100.

INSTITUTIONAL ETHICAL COMMITTEE

V.L.COLLEGE OF PHARMACY, RAICHUR-584103

KARNATAKA, INDIA.

Institutional ethical committee of V.L. College of Pharmacy, Raichur met on 16-11-

2009 to discuss about the details of animal experiments required to be carried out by PG

students of different Departments during their project work of 2010-2011. The following

members were present.

1. Dr. S.M. SHANTA KUMAR CHAIRMAN

2. Dr. N.VENKAT RAO CONVENER

3. Dr. LAKSHMAYYA MEMBER

4. Dr. SOMASHEKAR SHYALE MEMBER


5. Dr. Y.C.MAYUR MEMBER

6. Dr. T.SHIVARAJ GOUDA MEMBER

7. Dr. SRINIVAS MEMBER

The requirement for different experiments of each PG student were discussed in

detail. After thorough discussion the experiments to be carried out by the students of

individual Department were approved. The details are as follows.

1) Department of Pharmaceutics

S. No. Name Title of the work Type of work Animals required

Number

1. Shashikala

Development and characterization of

transdermal films of flurbiprofen

In-vitro Permeation studies Rats 24

2. Prashant Development and evaluation

of TTS containing few antihypertensive agents

Skin permeation Rats 20

3. Azhar

Development and evaluation of mucoadhesive buccal drug delivery systems of few Anti-

emetic drugs

Mucosal permeation Bovine mucosa

20

2) Department of Pharmacology

S. No. Name Title of the work Type of work Animals required Number

1. Radhika

A study on drug-drug interaction between

Candesartan and Glimepiride in rats

Drug-Drug interaction Rats 100

2. Syed Mahboob Ahmad

A study on antiulcer and antinflammatory activities of

tuber extracts of Solanum tuberosum (Solanaceae) in

rats

Antiulcer and antinflammatory activities

Rats 180

3. C.Yasar Ahmaed A study on anti-arthritic and antiulcer activities of Saraca

indica in rats/mice

Anti-arthritic and antiulcer activities

Rats/mice 150/40

4. Sowmya U.

A study on drug-drug interaction between

Carvedilol and Gliclazide in rats


5. J.Manoj Antiobesity activity of

Citrullus vulgaris (Cucurbitaceae) seed

Antiobesity activity Rats 160


extracts in rats.

6. Petricia Regina Irene

A study on Nootropic activity of fruit extracts of

Litchi chinensis Sonn(Sapindaceae) in mice

and rats

Nootropic activity Rats/mice 50/200

7. Patel Shivabhai

A Study on Nootropic activity of root extracts of

Vanda tessallata(Orchidaceae) in

mice and rats

Nootropic activity Rats/mice 150/150

8. Md. Basheer

Food-drug interactions between Capsicum

frutescens L.(Solanaceae)fruit extract and glimepiride in diabetic

rats

Food-drug interaction Rats 200

9. Venu Kola

Evaluation of anti arthritic and antiulcer activity of

root etracts of Momordia charantia in rats and mice

Anti-arthritic and antiulcer activities

Rats /mice 220/40

10. Rajesh Kumar

Calcium channel blockers as hepatoprotective agents

in different hepatotoxic models in rats

Hepatoprotective activity Rats 260

11. Rizwan A. Sheikh A study on hepatoprotective activity of bark extracts of Bassia latifolia roxb.in rats

Hepatoprotective activity Rats 260

12. Md. Fasih Ahmad

A study of herb-drug interaction between

Ocimum sanctum and Glimepiride in diabetic rats


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