i

ANTI-ULCERATIVE EFFECT OF AQUEOUS EXTRACT OF CUCUMIS

MELO ON NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

(NSAIDS) INDUCED GASTRIC ULCERATION IN MALE WISTAR RATS

A PROJECT

BY

ZAINAB SHEHU UTHMAN

BU/17C/BMS/2902

BAZE UNIVERSIRTY, ABUJA, DEPARTMENT OF HUMAN

PHYSIOLOGY, FACULTY OF BASIC MEDICAL SCIENCES,

DECEMBER, 2020

ii

ANTI-ULCERATIVE EFFECT OF AQUEOUS EXTRACT OF CUCUMIS

MELO ON NON-STEROIDAL ANTI-INFLAMMATORY DRUGS

(NSAIDS) INDUCED GASTRIC ULCERATION IN MALE WISTAR RATS

A PROJECT

BY

ZAINAB SHEHU UTHMAN

BU/17C/BMS/2902

BAZE UNIVERSIRTY, ABUJA, DEPARTMENT OF HUMAN

PHYSIOLOGY, FACULTY OF BASIC MEDICAL SCIENCES,

A PROJECT SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE AWARD OF BACHELOR OF MEDICAL

SCIENCES.

DECEMBER, 2020

iii

DECLARATION

I hereby declare that this project work titled: “Anti-ulcerative effect of aqueous extract of

Cucumis melo on non-steroidal anti-inflammatory drugs (NSAIDS) induced gastric ulceration

in male Wistar rats” is the product of my own research efforts undertaken by me under the

supervision of DR.GRACE ADEBAYO-GEGE, and has not been presented here or anywhere for

the award of a degree or certificate. Where the views of others have been expressed, they have

been duly and appropriately acknowledged.

_________________________ ______________________

Zainab Shehu Uthman Date

BU/17C/BMS/2902

iv

APPROVAL PAGE

This Project has been read by the faculty of Basic Medical Sciences, Baze University, Abuja in

partial fulfilment of requirement of the award of a bachelor of science (BSc. Hons.)

________________________ _______________________

DR. G. I. ADEBAYO-GEGE DATE

(Supervisor)

_______________________ _______________________

DR. G. I. ADEBAYO-GEGE DATE

(Acting, Head of department)

_______________________ _______________________

Professor F.A. Om’Iniabohs DATE

(Dean Faculty of Basic Medical Sciences)

v

CERTIFICATION

This Project has been read by the faculty of Basic Medical Sciences, Baze University, Abuja in

partial fulfilment of requirement of the award of a bachelor of science (BSc. Hons.)

________________________ _______________________

DR.G.I. ADEBAYO-GEGE DATE

(Supervisor)

_______________________ _______________________

DR.G.I. ADEBAYO-GEGE DATE

(Acting, Head of department)

_______________________ _______________________

Professor F. O. Om’Iniabohs DATE

(Dean Faculty of Basic Medical Sciences)

__________________________ _________________________

External Examiner DATE

vi

TURNITIN CITATION PAGE

vii

DEDICATION

I dedicate this work to my family and The Faculty of Basic Medical Sciences, Baze University,

Abuja, Nigeria.

viii

ACKNOWLEDGEMENT

My profound appreciation to my parents without whom this project would not have been possible.

I thank them for their moral and financial support throughout the course of this project. I will also

like to appreciate my whole family for their encouragement and prayers.

My deepest appreciation goes to my supervisor Dr. Adebayo- Gege Grace, who I highly

acknowledged for everything she has done to make this project work a success. I appreciate all

her suggestions and guidance. My gratitude also goes to Mr. Michael Ochayi who has been a

mentor and a friend throughout my academic years at the university. I would also like to appreciate

all my lecturers in the Department of Human Physiology, and the Faculty of Basic Medical

Sciences. I am also grateful to Mr. Jibrin Yakub and all the technologists for their supports

throughout my stay in Baze University.

ix

ABSTRACT

Ibuprofen, a strong analgesic and an anti-inflammatory drug used over a long period caused side

effects such as peptic ulcer and constipation. Honeydew melon has been shown to possess anti-

secretory, antiulcer and angiogenic. Hence, this study investigated the anti-ulcer effect of aqueous

extract of honeydew melon on ibuprofen induced gastric ulcer in male Wistar rats.

Wistar rats (n=35,150-200g) were randomly divided into seven groups. They were treated as

follows; I (control- distilled water),II- ibuprofen induced ulcer untreated (400mg/kg ibuprofen,

p.o),III(ibuprofen induced ulcer + misoprostol (200μg/kg),IV-VII induced with ibuprofen and

treated with 25%, 50%, 75%, and 100%aqueous extract of honeydew melon (HDM) for 3 weeks

prior to gastric ulcer induction, respectively. Gastric secretion was measured by titrimetric

method, and the ulcer score was measured. Assessment of activities of Superoxide Dismutase

(SOD), Catalase, and protein level, Malondialdehyde, H+/K+ ATPase were determined by

spectrophotometry. Prostaglandins synthesis was measured using Enzyme linked immunosorbent

assay kit. Data were expressed as Mean + SEM, analyzed using one-way ANOVA, with P<0.05

The total gastric acidity was significantly increased in group II (2.80±0.12) compared to group I

(0.50± 0.00mEq/L),P<0.001, pretreatment with aqueous extract of HDM significantly decrease

the total gastric acidity in IV, V, VII compared to group II, P<0.05. The ulcer score and index was

significantly decrease in all the treatment compared to group II, P<0.05. The percentage inhibition

increased significantly in all treatment groups compared to the group II. The Lipid peroxidation

(MDA) increase significantly in the group II (untreated compared to the treated groups, p<0.05.

The protein level and activities of the catalase, superoxide dismutase significantly increased in

treatment groups compared with group II, P<0.05. Prostaglandin E2 expression increased

significantly in the all treatment groups with much increase in VII, compared with other groups,

p<0.05. Activities of H+/K+ ATPase was significantly decreased in the treatment groups

x

compared to the group II(P<0.05).In conclusion, the findings from this study suggests that extract

of HDM possess antiulcer properties due its ability to increase antioxidant activities, synthesis of

prostaglandins and reduce the activities of H+/K+ATPase .

Keywords: honeydew melon, total gastric acidity, prostaglandin E2, antioxidants, H+/K+ATPase

Word count = 300.

TABLE OF CONTENTS

Title Page

Declaration Page i

Approval Page ii

Certification iii

Turnitin Page iv

Dedication v

Acknowledgement vi

Abstract vii

Table of Content ix

CHAPTER ONE: INTRODUCTION

1.0 Introduction 1

1.1 Statement of problem 3

1.2 significance of study 4

1.3 Aims and objectives 4

1.3.1 Aim 4

1.3.2 Objectives 4

CHAPTER TWO: LITERATURE REVIEW

2.0 Literature review 5

2.1.1 The stomach 8

2.1.2 The small intestine 11

2.2 Peptic ulcer 13

2.2.1 Epidemiology 13

2.2.2Etiology 14

2.2.3 Signs and Symptoms 16

2.2.4 Conventional Treatment 17

2.2.5Cyclooxygenase and prostaglandins 19

2.3 Honeydew melon 23

2.3.1. Composition of Honeydew melon 24

CHAPTER THREE: MATERIALS AND METHODS

3.0 Materials 25

3.1 Chemicals 25

3.2 Animals 25

3.2.1 Animal Groupings 26

3.3 Methods 26

3.3.1 Preparation of aqueous extract of honeydew melon 26

3.3.2 Ibuprofen Induced Ulcer 26

3.3.3 Ulcer scoring 27

3.3.4 Measurement of Gastric Acidity 27

3.3.5 Homogenization 28

3.3.6. Determination of Total Protein 28

3.3.7. Determination of Superoxide Dismutase (SOD) 28

3.3.8. Determination of Oxidative Stress 28

3.3.9. Determination of catalase activity 29

3.3.10. Determination of prostaglandins e level 31

3.3.11. ASSESSMENT OF H+/K+-ATPase 31

3.4 Statistical Analysis 32

CHAPTER FOUR: DATA PRESENTATION AND ANALYSIS

4.1. Effects of aqueous extract of honeydew melon on total gastric acidity 33

4.2. Effects of aqueous honeydew melon extract on ulcer score, ulcer index 36

4.3. Effects of aqueous extract of honeydew melon on percentage inhibition 36

4.4. Effects of aqueous extract of honeydew melon on malondialdehyde 36

4.5. Effect of aqueous extract of honeydew melon on catalase 40

4.6. Effect of aqueous extract of honeydew melon on protein level 42

4.7. Effect of aqueous extract of honeydew melon on superoxide dismutase level 44

4.8. Effect of aqueous extract of honeydew melon on H+/K +-ATPASE 46

4.9. Effect of aqueous extract of honeydew melon on prostaglandin levels 48

4.10. Effect of aqueous extract of honeydew on the stomach mucosa 50

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMENDATION

5.0 Discussion 52

5.1 Conclusion 56

5.2 Recommendation 56

REFERENCES 57

LIST OF TABLES

Table 2.1: Composition of Honeydew Melon 23

Table 3.1: Criteria for Ulcer Scoring 27

Table 4.1. Effect of aqueous extract of honey dew melon on ulcer score, ulcer index and percentage

inhibition 37

LIST OF FIGURES

Fig 2.1. A Diagram of the gastrointestinal tract showing functions of the gastrointestinal organs.

7

Figure 2.2. A diagram of the cross section of the layers of the gut. 7

Figure 2. 3. A diagram of the stomach 10

Figure 2.4: A diagram of the small intestines. 12

Figure 2.4. Causes of peptic ulcer 21

Figure 2.5. Honeydew melon 22

Figure 4.1: effect of aqueous extract of honeydew melon o the total gastric acidity Figure 2.5:

Honeydew melon 35

Figure 4.2. Effect of aqueous extract of honeydew melon on lipid peroxidation (MDA) 39

Figure 4.3. Effect of aqueous extract of honeydew melon on catalase level. 41

Figure 4.4. Effect of aqueous extract of honeydew melon on protein level. 43

Figure 4.5. Effect of aqueous extract of honeydew melon on superoxide dismutase level 45

Figure 4.6. Effect of aqueous extract of honeydew melon on activity of H+/K+ATPase. 47

Figure 4.7. Effect of aqueous extract of honeydew melon on activity of Prostaglandins. 49

LIST OF ABBREVIATIONS

PUD Peptic Ulcer Disease

GIT Gastrointestinal Tract

HCL Hydrochloric Acid

SOD Superoxide Dismutase

MDA Malondialdehyde.

H/K- ATPase Hydrogen Potassium ATPase

PPI Proton Pump Inhibitor

COX-1 Cyclooxygenase-1

COX-2 Cyclooxygenase-2

HDM Honeydew Melon

ROS Reactive Oxygen Species

SEM Standard Error of Mean

RBC Red Blood Cells

CHAPTER ONE

1.0 INTRODUCTION

The gastrointestinal tract (GIT) primary function is to serve as a portal whereby nutrients and water

can be absorbed into the body (Barret and Barman, 2016). The gastrointestinal tract starts from the

mouth to the anus, its length is about 30 feet in adult (Zimmerman, 2016), and its organs includes

the mouth, esophagus, stomach, small and large intestines, anus. It is a specialized system that

provides the body with nutrients and water by performing the following functions, digestions,

transportation, absorption , elimination of waste products(Cheng et al., 2014).

There are several diseased conditions affecting the GIT which affect the overall health of the body.

The common diseases of the GIT includes celiac diseases, Crohn’s disease or inflammatory bowel

diseases , Zollinger Ellison disease, Constipation , Diarrhea, tropical sprue ,achalasia,

gastroesophageal reflux diseases ( Falko, 2012).

Other digestive disorders include; Irritable bowel syndrome, lactose intolerance, malabsorption

syndromes, peptic ulcer, ulcerative colitis, vomiting.

Peptic ulcer is a discontinuation or abrasion of the stomach/intestinal mucosa or upper part of the

small intestine by digestive juice or intestinal secretion. It is a combination of diverse disorder

which is evident as erosion or sore on the gastrointestinal mucosa lining. It usually occur in the

fundic part of the stomach or lower esophageal sphincter or the upper part of duodenum (Hall and

Guyton, 2013). It is common and the percentage distribution is global. Peptic ulcer arises as a

result of imbalance between the offensive and protective factors (Malik et al., 2019). The offensive

factors includes acid, pepsin, bile acids, depressed secretion of prostaglandins, reduced blood flow

to gastric mucosa, impeded mucosal growth and cell proliferation, and alteration of gastric

motility(Qureshi, 2015).

The gastric protective factors include prostaglandin secretion, bicarbonate secretion, expression of

growth factors, etc (Silva and Sousa, 2011).Several factors are involved in pathogenesis of peptic

ulcer such as helicobacter pylori, use of non-steroidal anti-inflammatory drugs, life style.

Approximately 70–90% of ulcers are said to be related to Helicobacter pylori, a spiral-shaped

bacterium that lives in the acidic environment of the stomach.

From studies, the importance of gastric H+/K+ -ATPase in the parietal cell of the gastric mucosa

cannot be exempted, as it controls the movement transport of HCl through membrane by H for K

exchange catalyzed by ATP driven phosphorylation/dephosphorylation (Shin et al., 2009). Its

expression during gastric injury cannot be over emphasized. The inhibition of this enzyme has

been a therapeutic index during healing of gastric ulceration or injury

From studies, it has also been found that disequilibrium between oxidant and antioxidants

contributes to inflammation and this can lead to ulceration of the gastric mucosa (Kisaoglu et al.,

2013). Increase in expression of Reactive Oxygen Species has been one of the major culprit that

directly results in oxidative damage, including lipid peroxidation, protein oxidation, and DNA

damage, which can lead to cell death. In the illness state, oxidative stress of the stomach may occur

and result in an elevation of mucosal lipid peroxide that are generated from the reaction of oxy-

radicals and cellular poly unsaturated fatty acid, while GSH may act to prevent this aggressive

action that can damage gastric mucosal cells. Malondialdehyde (MDA) is an end product resulting

from peroxidation of polyunsaturated fatty acids and related esters within cell membranes, and the

measurement of this substance represents a suitable index of oxidative tissue damage. Other

antioxidants such as Superoxide dismutase, catalase function to prevent the buildup of free radicals

or reactive species in cells. They act very quickly to deactivate chain reactions that might lead to

production of free radicals (Ighadaro et al., 2018). The cytoprotective action of prostaglandins is

as a result of its complex ability to stimulate mucosal mucus and bicarbonate secretion, to increase

mucosal blood flow and sulfhydryl compounds and limit back diffusion of acid into the epithelium

in the stomach (Tarnawski et al., 1985,Farhadi et al.,2003;Kato et al.,2005).

Numerous studies have demonstrated that herbal medicines can effectively treat gastric ulcer in

humans and animals via divergent mechanisms (Bi et al., 2014). Studies have demonstrated that

the efficacy of herbal medicines is comparable or even superior to that of drugs and that herbal

medicines display less adverse effects (Bi et al., 2014). Researches have proved that flavonoids or

bioflavonoids which are naturally occurring compounds in fruits and vegetables may be an

effective additional treatment for stomach ulcers (Krans and Carey 2019). Other naturally

occurring ulcer remedies include; probiotics, honey, garlic, cranberry etc.

Cucmis melo otherwise known as honeydew melon is a delicious oval shaped fruit with vast

nutritional values. (Zhang et al., 2020). Honeydew melon like most melons is rich in fiber,

vitamins, mineral sand plant phenols. Consumption of polyphenol rich plants may decrease

inflammation and oxidative damages (Shoemaker, 2019).

1.1. STATEMENT OF PROBLEM

Ibuprofen is a strong analgesic and an anti-inflammatory drug. Due to its availability in the market,

ibuprofen has become very common painkiller. It is often consumed by different individuals to

alleviate pains, fever, inflammations, cramps etc. (Ngo and Bajaj 2019).The main side effects of

ibuprofen especially when used over a long period of time include gastric ulcer, duodenal ulcer

and indigestion etc. (Liu et al., 2016). Over the years, the rate at which ibuprofen is being

administered increased steadily due to its important as strong analgesics.

Cucumis melo or honeydew melon has been found to be effective in treatment of experimentally

induced ulceration by indomethacin (Adebayo-Gege et al., 2019). It was reported Cucumis melo

possess antisecretory properties and possesses angiogenic properties by increasing expression of

CD31 (Platelette endothelial cell adhesion molecule) which should be responsible for its antiulcer

properties. NSAIDs used in large doses over a long period of time cause ulcer by interfering with

the ability of the stomach cells to protect themselves from aggressive factors(Rogoveanu et al.,

2015).NSAIDs cause ulcers by inhibiting cyclooxygenase-1, a key enzyme in the synthesis of

prostaglandins (Drini, 2017).

HYPOTHESIS 0: Honeydew melon will prevent pathogenesis of ibuprofen induced gastric injury.

HYPOTHESIS 1: Honeydew melon will not prevent pathogenesis of ibuprofen induced gastric

injury.

1.1.1. AIM

The aim of this study was to investigate the antiulcer properties of aqueous extract of honeydew

melon on ibuprofen induced gastric ulceration in male Wistar rats

1.1.2. OBJECTIVES

Study its anti-ulcerative effects on ibuprofen induced gastric ulceration by

Evaluating its effect on total gastric acidity, Ulcer score , ulcer index

Assessing the Percentage inhibition, oxidative stress and antioxidant status

Assessing its regulatory role on the H+/K+-ATPase and Prostaglandins

CHAPTER TWO

LITERATURE REVIEW

2.0.OVERVIEW OF GASTROINTESTINAL TRACT

The gastrointestinal tract is medium that connect the body to the external environment, it is

very important in delivery of Delivery of nutrients, electrolyte, water, destruction of toxic agents,

and excretion of waste materials. Its major function is digestion, absorption, movement,

protection and excretion (Ebneshahidi , 2006). It composes digestive organs (such as mouth,

esophagus, stomach, small and large intestine, and anus) and the accessory organs (Liver,

pancreas, spleen, salivary gland) (Constantine et al., 2014).

2.0.1THE LAYERS OF THE GASTROINTESTINAL TRACT

The gastrointestinal tract consists of five layers starting from the outermost to the innermost

are; the serosa, the longitudinal smooth muscle layer, the circular muscle layer, the submucosa

and the mucosa. These layers perform the motor functions of the gastrointestinal tract (Hall

and Guyton, 2011, pg 797).

a. The serosa is the outermost layer consisting of simple squamous epithelia that produces

serous fluid which lubricates the outer wall of the stomach.

b. The longitudinal smooth muscle layer produces churning movements required for

mechanical digestion.

c. The circular muscle layer has two thin layers of smooth muscle. The function of the

longitudinal smooth muscle layer is to aid in expelling gastric gland secretions into the

lumen of the stomach.

d. The gastric submucosa is a thick layer of thick, flexible yet mobile connective tissues that

houses the Meissner’s plexus.

e. The Meissner’s plexus carries parasympathetic innervation to the blood vessels and smooth

muscles of the stomach wall (Pirie, 2020).

f. Auerbach’s plexus otherwise known as the myenteric plexus is a group of ganglia that

innervates the smooth muscle of the gastrointestinal tract. The myenteric plexus and the

meissner’s plexus make up the enteric nervous system (Shashrestani and Das, 2020).

g. The gastric mucosa, which is the innermost layer of the gut is formed by a layer of surface

epithelium and other underlying structures. The surface epithelium is made of simple

columnar epithelium. It lines the interior of the stomach and forms numerous tiny

invaginations known as gastric pits.

These gastric pits are very important because they are connected to the various glands of

the stomach (Pirie, 2020).

The intestines displays a variety of motility patterns that serve to mix the meal with digestive

secretions and move it along the length of the gastrointestinal tract. Ultimately, residues of the

meal that cannot be absorbed along with cellular debris are expelled from the body. The

gastrointestinal tract has evolved a large number of regulatory mechanisms that act both locally

and over long distances to coordinate the function of the gut. The gastrointestinal tract

comprises of multiple organs but this study will mainly focus on the Stomach and the

duodenum of the small intestines.

.

2.1.1THE STOMACH

Figure 2.2. A diagram of the cross section of the layers of the gut.

(https://drbeeneducation.files.wordpress.com/2016/05/shayan.jpg. Retrieved on 23/07/2020

Fig 2.1. A Diagram of the gastrointestinal tract showing functions of the gastrointestinal organs.

https://lh6.ggpht.com/AmG53XX_uJs/UqmjtKmkS6I/AAAAAAAAAq8/KnNQDak3Uks/human-

digestive-system-diagram_thumb%25255B5%25255D.jpg?imgmax=800 . Retrieved on 23/07/2020.

The stomach is an abdominal muscular organ with the primary function of storing food ,mixing it

with gastric secretions and digesting the food (Hoofman , 2009). The stomach is located between

the esophagus and the duodenum of the small intestine (Pirie , 2020)

The stomach is divided anatomically(body and antrum) and functionally(orad and caudad ) in to

two; the orad portion which comprises of the first two thirds and the caudad portion which

comprises of the remaining body of the stomach and the antrum (Hall and Guyton, 2011, pg 810).

The stomach receives food from the esophagus and stores it before it is converted in to chime , it

is then mixed with gastric secretion . It‘s slowly empties in to the small intestine for digestion and

absorption of the other parts of chime.

Gastric glands of the stomach also known as the oxyntic gland secretes digestive juices. The

oxyntic gland area covers up to 80% of the stomach and is composed of mostly of gastric acid

producing parietal cells (Vakil, 2020).

I. THE OXYNTIC GLANDS AND ITS CELLS

The oxyntic glands are found in the fundic part of the stomach. It covers about 80% of the stomach

and houses the parietal cells that produces the gastric acid. They secrete the oxyntic mucosa. The

oxyntic mucosa is composed of specialized, complex and differentiated lineages that initiate

digestion and offer protection to the epithelial lining of the GIT (Goldenring and Nam, 2015). The

acid secreting parietal cells, the chief cells (that secrete zymogen), surface mucous cells and neck

cells form oxyntic mucosa. It also houses neuroendocrine cells that secrete paracrine hormones

capable of modifying parietal cell activities (Vakil, 2020).

II. GASTRIC SECRETION

Gastric secretion is stimulated by three different phases; the first phase is cephalic phase (When

the bolus of enters the mouth)., the second is the gastric phase (when the food enters the stomach)

and the final phase which is the intestinal phase when the food leaves the stomach and enters the

intestines (Camilleri and Vazquez, 2014). The gastric mucosa is composed of specialized cells that

secrete essential substances like prostaglandins, hormones, bicarbonate e.t,c. Secretion of gastric

juices, peristalsis and other functions of the stomach are controlled by the parasympathetic nervous

system and the enteric nervous system (Hsu and Lui, 2018). Stomach emptying is promoted by intense

peristaltic contraction in the stomach antrum. At the same time, emptying is opposed by varying degrees of

resistance to passage of chyme at the pylorus.

Figure 2. 3. A diagram of the stomach

.https://www.pharmatips.in%2FArticles%2FHuman-Anatomy%2FHuman-Anatomy-Physiology-

Of-The-Stomach. Retrieved on 23/07/2020

2.1.2 THE SMALL INTESTINE

The small intestine is primarily for digestion and absorption of nutrients (Hall and Guyton,

2011, pg 812). It’s divided into duodenum, jejunum and ileum. The small intestine is a 7-meter

long tubular organ that begins at the end of the stomach and terminates at the large intestine. It

receives pancreatic secretion and bile.

The small intestines has four main layers; mucosa, submucosa, muscularis, externa and

adventitia. The small intestine possesses small projections known as microvilli to further increase

the surface area as well as absorption (Turiki, 2018).

Carbohydrates, protein and lipids are digested and absorbed by the small intestine.

Carbohydrates must be broken down into monosaccharides by enzyme amylase. The

monosaccharides are then absorbed through carrier-mediated transport. Protein digestion `occurs

by breaking peptide bonds through hydrolysis by a proteolytic enzyme known as pepsin. Proteins

are broken into amino acids. Amino acids absorption involves many carrier-mediated active and

facilitated transport proteins (Fish and Burns, 2019).

Dietary fats come in these form of triglycerides. They are broken down into 2-

monoglycerides and fatty acids. Lipid digestion requires enzymes like phospholipase, lipase,

cholesterol, ester hydrolase, etc. Emulsification is an important process in digestion of fats and

Absorption of fat is a passive process (Fish and Burns, 2019).

Figure 2.4. A diagram of the small intestines.

https://www.google.com/url?sa=i&url=https%3A%2F%2Fsocratic.org%2Fquestions%2Fwhat-

is-the-name-of-the-middle-part-of-the-small-intestine&p. Retrieved on 23/07/2020

2.2. PEPTIC UL CER

Peptic ulcer disease (PUD) is defined as mucosal erosions equal to or greater than 0.5 cm

(1/5"). Many peptic ulcers arise in the duodenum and the stomach (Malik et al., 2019 ). Peptic

ulcer can be defined as the erosion of the mucosal lining of gastrointestinal tract. These erosions

can occur at the esophagus, stomach and intestines.

2.2.1. EPIDEMIOLOGY

Peptic ulcer incidence is more intense in tropical countries like India and Nigeria. From

previous reports, the pooled incidence of peptic ulcer disease (PUD) was approximately one case

per 1000 person-years in the general population, and the incidence of ulcer complications was

approximately 0.7 cases per 1000 person-years.

Peptic ulcer affects 4 million of the human population annually (Zibima et al., 2020).The

incidence and prevalence of PUD varies based upon the presence of Helicobacter pylori. Higher

rates are found in countries where H. Pylori infection is higher. The incidence of PUD in H.Pylori

infected individuals is approximately 1% per year, a rate that is 6 to 10 fold higher than for

uninfected individuals (Vakil, 2020).

The incidence of peptic ulcer has decreased steadily in western countries as a result of

improvement in hygienic conditions. (Fujinami et al., 2012). Although PUD has significantly

decreased, it has not disappeared (Malfertheiner and Schulz, 2020). The predominance of PUD

has shifted from H. pylori to NSAIDs and other gastroelusive substances ( Malfertheiner and

Schulz, 2020). The risk of developing peptic ulcers by usage of NSAIDs varies between drugs.

The risk for drugs like aceclofenac, ibuprofen and celecoxib is relatively low while the relative

risk for naproxen, indomethacin and diflunisal is high (Drini , 2017).

2.2.2. ETIOLOGY

The digestive tract is coated with a mucous layer that normally protects against acid. If the

amount of acid is increased, or the amount of acid decreases, it can cause development of ulcer.

Peptic ulcer develops when there is an imbalance between the ‘‘aggressive’’ and ‘‘protective’’

factors at the luminal surface of the epithelial cells (Silva and Sousa, 2011, Anand, 2020).

Aggressive factors includes HCl, pepsins, bile acids, ischemia, hypoxia while defensive factors

include bicarbonate, mucus layer, mucosal blood flow, Prostaglandins and growth factors (Harold

et al., 2007).

Peptic ulcer can be caused by other factors such as Helicobacter Pylori, Use of Non-steroidal anti-

inflammatory drugs (NSAIDs), life style of individual such as smoking, taking alcohol, taking

spicy foods (Prabhu and Shivani, 2014).

a. HELICOBACTER PYLORI

Almost half of the world’s population suffered from colonization of H. Pylori which

remains one of the most common causes of peptic ulcer disease. (Kuna et al., 2019). H. Pylori

causes epithelial cell degeneration and injury, which is usually more severe in the antrum by the

inflammatory process with neutrophils, lymphocytes, plasma cells and macrophages. After H.

pylori enters the body, it attacks the lining of the stomach. Once the bacteria has done enough

damage, acid can get through the lining which leads to ulcer. H. Pylori can be gotten from food,

water or contact with saliva or body fluids of infected people. H. pylori is more common in

countries or communities that lacks clean water or good sewage system. (Khatri, 2018). Before

Warren and Marshall’s discovery of H pylori in the gastric mucosa, the gastric environment was

believed to be sterile due its high acidity.

H. pylori uses a wide range of mechanisms (e.g. crucial flagellar motility, chemotaxic

action, surface receptor etc.) To be able to adapt and survive the acidity of the gastric environment

and be able to perpetuate infection (Bittencourt et al., 2019). Development of peptic ulcer caused

by presence of H pylori are influenced by some bacterial hosts and factors.

b. NON-STEROIDAL ANTI INFLAMMATORY DRUGS (NSAIDS)

Non-steroidal anti-inflammatory drugs (NSAIDs) is a class of analgesics such as

ibuprofen Aspirin, indomethacin, naproxen, and piroxicam, nabumetone medication that reduces

pain, fever and inflammation. NSAIDs are a less common but steadily increasing in importance,

cause of peptic ulcer (Tresca, 2019).

Peptic ulcer can occur with higher doses of NSAIDs used for a long period of time.

NSAIDs can cause ulcers by interfering with the stomach’s ability to protect itself from gastric

acids. Peptic ulcer disease is a well-recognized complication of complication of NSAIDs use.

Inhibition of COX-1 in the gastrointestinal tract leads to a reduction of prostaglandin secretion

and its cytoprotective effect in gastric mucosa. This therefore increases the susceptibility to

mucosal injury. Inhibition of COX-2 may also play a role in mucosal injury.

Pathogenesis of NSAIDs induced gastric injuries occur due to the interaction between

NSAIDs and the surface in relation to the detergent properties. This is how bleedings and lesions

often occurs. NDAIDs, due to their acidic structure are unable to biochemically dissociate in the

acidic environment of the gastrointestinal tract they remain in lipophilic form .This aids their

penetration in the gastric epithelium (Rogoveanu et al., 2015).

c. LIFESTYLE :

Smoking is presumed to cause ulcer due to increased nervous stimulation of the gastric

secretory glands while consumption of alcohol causes ulcer because it tends to breakdown the

mucosal barrier.(Hall and Guyton, 2011, pg 845). Smoking is believed to increase gastric

emptying while simultaneously decreasing pancreatic bicarbonate level. Other studies have

shown that there is no conclusive evidence to connect alcohol consumption to pathogenesis of

peptic ulcer. (Anand, 2020). However maintaining a balanced diet, limiting alcohol intake,

cutting bad habits like smoking and getting enough sleep are important in prevention and healing

of peptic ulcer (Yegen, 2020).

d. EMOTIONAL STRESS

Emotional stress causes ulcer because emotional stimuli is believed to increase gastric

secretions. This increases in response to stimuli is believed to contribute to development of peptic

ulcer..(Hall and Guyton, 2011, pg 845). The prevalence of PUD and mental health diseases are

directly proportional i.e, prevalence of PUD increases with increased mental health problems (Lee

et al., 2017).

2.2.3. SIGNS AND SYMPTOMS

Common symptoms of ulcer include; Burning stomach pain Bloating or belching Fatty food

intolerance, Heartburn and Nausea. Other symptoms may include; vomiting or vomiting blood

which may appear red or black. (Kusterset al.,2006).

Dark blood in stools

Troubled breathing

Feeling faint

Unexplained weight loss

Appetite changes

Complications

An ulcer can cause serious problems like stomach and bleeding if it is not treated .it may

lead to formation of holes in the stomach that might require surgery (Kusterset al.,2006).

2.2.4 CONVENTIONAL TREATMENT

Peptic ulcer treatment is normally based on the cause of the ulcer (Ambardekar, 2019). The

treatment of peptic ulcer varies depending on the etiology and clinical presentation. (Anand, 2020).

If peptic ulcer is as a result of H. pylori, it can be treated by using a combination of antibiotics to

kill the H.Pylori bacteria and reduce the acidity in the stomach. Some of these drugs include proton

pump inhibitors. Examples of these antibiotics are; amoxicillin, clarithyromycin and

metronidazole. If the ulcer is caused by taking NSAIDs, the ulcer is treated by PPI or H2 receptors

antagonist. Antacids may be used to relieve symptoms of peptic ulcer.

Furthermore, lifestyle changes are also essential in treatment of ulcer example; quitting

smoking, drinking less alcohol and caffeine. Previous studies carried out on both animal and

human models propose that herbal medicines exert their advantageous effect on the gastric ulcer

by antioxidant activities, elevated mucous production, reversing of inflammation and mucosal

proliferation (Bi et al.2014). furthermore, due to manifestation of various adverse effects caused

by usage of conventional drugs for numerous diseases, medicinal plants are regarded as reservoirs

of potentially new drugs ( Kuna et al.2019).The efficacy of herbal medications in treatment of

gastric ulcer is similar to that of famotidine ( Histamine H2 receptor inhibitor) (Ping et al.2014).

I. SIDE EFFECTS OF CONVENTIONAL TREATMENTS

Although conventional drugs have proved to very effective therapeutic agents in

tackling peptic ulcers, they are not without limitations and side effects. It has been reported

that the use of proton pump inhibitors has resulted in unanticipated adverse effects such as

myocardial infarction, kidney related disorders, stroke etc. (Kuna et al.2019). Use of antibiotics

to treat ulcers often result in gastrointestinal adverse effects and skin irritation, use of antacid

and cytoprotective agents can result in diarrhea and constipation while the use of Histamine 2

receptor antagonists can result in dizziness, tiredness and headaches.

II. DEFENSE MECHANISM, OF THE STOMACH

Despite continuous exposure to injurious factors, under normal conditions large number of

defense mechanisms prevent local damage and aids in maintaining the structural and functional

integrity of the gastric mucosa (Tulassay&Herszényi, 2010). Disruption of these defensive factors

leads to mucosal damage (Qureshi, 2015). Defensive factors of the stomach include; prostaglandins,

bicarbonate, mucus production, mucosal blood flow and growth factors.

a. Prostaglandins

Prostaglandins are lipid autacoids derived from arachnoidic acid produced at sites of tissue

damage or infection. Prostaglandins are any group of physiologically active substances having

diverse hormone-like effects in animals (Utiger, 2019). Prostaglandins sustain homeostatic

functions and mediate pathogenic mechanisms including the inflammatory response. (Riccioti and

Fitzgerald 2011). They are generated from arachidonate by the actions of cyclooxygenase

isoenzymes and their biosynthesis is blocked by NSAIDs, including those selective for inhibition

of COX-2.

Prostaglandin can affect blood pressure by acting as vasodilators. They play pivotal roles

in inflammation. Prostaglandins also plays a role in ovulation and uterine muscle contraction.

It controls acid secretion, mucous production, blood flow and maintenance of mucosal

integrity. Thus, prostaglandins offer protection to the mucosa of the GIT against offensive

agents such as NSAIDs and stress (Takeuchi and Amagase, 2017).

b. Mucous secretion

The mucous contains glycoprotein that swells when they come in contact with

water thus forming the protective mucous layer. This mucous layer prevents ulceration by

reducing the rate of diffusion of HCl in the bicarbonate of the mucosa (Qureshi, 2015).

Decreased duodenal bicarbonate is seen in ulcer due to to abnormalities in Secretin

synthesis or release (Qureshi, 2015).

c. Mucosal blood flow:

Gastric mucosal blood flow is another very important factor in mucosal defense

against ulceration. Reduced mucosal blood flow is a common denominator in animal model

experimentation. Ischemia reduces protective capacity of the gastric mucosa to neutralize

the acid entering the tissue. (Paxton et al., 2016). Ischemia may also increase vulnerability

of the gastric mucosa by reducing energy. (Paxton et al., 2016). Gastroduodenal mucosal

cell proliferation and renewal are essential defensive factors of the stomach. Acute mucosal

lesions or damages to the surface epithelium are often accompanied by mucosal cell

restitution leading to immediate repair (Otsuka et al., 2018).

d. Expression of growth factor, tight intercellular junctions, epithelial renewal and cellular

restitution (Anand 2020).

2.2.5. THE ROLE OF CYCLOOXYGENASE IN PROSTAGLANDIN SYNTHESIS

The cyclooxygenase isoenzymes COX-1 and COX-2 catalyze the formation of

prostaglandins, thromboxane and levuloglandins (Fitzpatrick 2004). COX-1 and COX-2 have been

defined as monotropic integral membrane proteins located primarily in the endoplasmic reticulum

(Griffing, 2019). Evidence suggests that COX-1 and COX-2 are similar in structure and function

but they exist as two distinct enzymatic entities. Cox enzymes are clinically important because

they are inhibited by aspirin and numerous NSAIDs. This inhibition of COX confers relief from

inflammatory, pyretic, thrombotic, neurodegenerative and oncological maladies. COX-2 is

unexpressed under normal conditions in most cells. The ability to selectivity inhibit COX-2 allows

bypassing of COX-1 blockade which prevents peptic ulceration (Mestre et al., 2014).

Figure2.4. Causes Of Peptic Ulcer.

https://d45jl3w9libvn.cloudfront.net/jaypee/static/books/9789351526735/Chapters/images/658-

1.jpg

A: Showing the defense system of the stomach. Protective factors (e.g. bicarbonate, prostaglandin,

mucosal blood flow etc.)And hostile factors (e.g. gastric acid, ischemia, bile acids etc.)

B: Showing imbalance between hostile factors and protective factors.

.

Figure 2.5. Honeydew melon

https://www.google.com/url?sa=i&url=https%3A%2F%2Ffreshpointlocal.co.uk%2Fproduct%2Fh

oneydew-melon-eacRetrieved on 25/08/ 2020.

2.3 HONEYDEW MELON

Honeydew melon, also known as sweet melon, is a member of the indorus group of cucumis melo

(Mercola, 2017). Its size usually ranges from small to medium and it comes in a round or oval

shape with a weight of around 4 to 8 pounds. Although this fruit commonly known for having pale

green flesh and yellow rind, it is available in other colors and textures as well. Several varieties of

honeydew have a bright yellow skin due to a mutation while others have an extra sweet orange

flesh (Mercola, 2017).

Unlike the variety of melons that thrive in tropical climate, honeydew is best grown in warm and

dry regions. Although, honeydew melon is consumed in many parts of Nigeria and it has numerous

nutritional and commercial values, its production is low and on a small scale (Mohammed, 2011).

If planted in humid regions, it may produce smaller fruits with poor resistance against pests and

diseases. In Nigeria, melon is an annual crop which is normally planted at the beginning of the

raining season and then harvested and sold in the market till the end of the year (Mohammed,

2011).

2.3.1 COMPOSITION OF HONEYDEW MELON

Honeydew melon is rich in important vitamins such as riboflavin, thiamin and folic acid It is also

a good source of pro vitamin A, vitamin C and beta carotene. (Zeb, 2016). i. . Its content of vitamin C

and other biologically active compounds such as phenolic compounds and vitamin C have positive

effects on human health (Lester and Hodges, 2008). Studies have shown that honeydew melon

prevents dehydration, promotes heart health ( due to low sodium and high potassium content),

reduces risks of diabetes and helps in management of diabetes, protects eyesight ( due to carotenoid

leutin and zeaxanthine contents) and hydrates the skin and the body.

Despite the name, honeydew melons are not highly concentrated with sugar. The high percentage

of water in the melon dilutes the high sugar content in the melon (Cervoni, 2020).

Studies have been carried out on honeydew melon to study the quality parameters that contribute

to the quality of the melon (Brouwer et al., 2019), concentration of antioxidant, sugar and

phytonutrients across the edible tissues of honeydew melon (Lester, 2008).Superoxide dismutase

activity in mesocarp tissue from divergent Cucumis melo genotypes (Lester et al., 2009).

Tale 2.1: Composition of Honeydew Melon

Calories 64

Fat 0.3 g

Sodium 32 mg

Carbohydrates 16 g

Fiber 1.4 g

Sugars 14 g

Protein 1g

The nutritional information above is provided by the USDA for 177g of balled honeydew melon

(Cervoni, 2020).

CHAPTER THREE

MATERIALS AND METHODS

3.0. Materials

Microscopes, syringes and needles, conical flask, dissecting sets, cotton wool, dissecting board,

weighing balance, hand gloves ,blade ,slides ,animal cages ,EDTA bottles, rubber catheter,

tefflon homogenizer, cold centrifuge, spectrumlab32A spectrophotometer, UV/VIS Micro Elisa

Plate, Reference Standard, ,Stop solution Plate Sealer

3.1. CHEMICALS

10% formalin, Normal saline, phenolphthalein, 0,1NaOH and ketamine (900-B-

2370,Arendonk,Belgium) , 75μg/kg misoprostol (Naari, PTE, Singapore), 400mg/kg Nurofen/

Ibuprofen, (Reckitt Healthcare International ). Sodium tartarate, potassium iodide (KI),

thiobarbituricacid (TBA), trisbase, potassium chloride (KCl) (BDH, England), stock bovine serum

albumin(standard)(Sigma Chemical Co., USA), trichloroaceticacid (TCA) (Oxford laboratory

reagent ,India).Reference Standard and Sample diluents, Concentrated Biotinylated Detection Ab,

Biotinylated Detection Ab Diluent, Concentrated HRP Conjugate, Concentrated Wash Buffer,

Substrate Reagent.

3.2.0. ANIMALS

Thirty-five male Wistar rats (weighing 150g-200 g) were obtained from the Department of Human

Physiology, Faculty Of Basic Medical Sciences, College Of Medical Sciences, Ahmadu Bello

University, Zaria LGA of Kaduna state Nigeria. They were housed in the physiology laboratory of

the Faculty of Basic Medical Sciences, Baze University to acclimatise for two weeks, fed with

standard feed and water ad labtium. They were maintained under standard laboratory conditions

and were fed with commercially formulated rat pellets and water.

3.2.1. ANIMAL GROUPING

After the acclimatisation period, thirty male Wister rats were randomly divided into seven

experimental groups (n=5) viz

Group I–control,

Group II- ibuprofen induced ulcer untreated (400mg/kg ibuprofen-induced gastric ulcer),

Group III- ibuprofen induced ulcer +misoprostol(200μg/kg)

IV-VII were treated with 25%, 50%, 75%, and 100%aqueous extract of honeydew melon(HDM)

for3weeks prior to gastric ulcer induction, respectively.

3.3.0. METHODS

3.3.1. PREPARATION OF AQUEOUS EXTRACT OF HONEYDEW MELON

Honeydew melon fruits were purchased from Gwarimpa model city gate,Galadima. The honeydew

melon was extracted, washed to remove dirt, then the thin yellow outermost pericarp and the almost

white fleshy mesocarp was cut into smaller pieces, and the seeds removed. The pericarp and mesocarp

were blended and filtered with a clean sifter to separate the juice from the solid particle. The

concentrated juice was diluted with distilled H2O to give 75%, 50%, and 25% v/v solutions. Fresh

preparations were made daily.

3.3.2. IBUPROFEN-INDUCED ULCER

The animals were pre-treated with different aqueous extract concentrations of C. Melo for three weeks.

Prior to ulceration by oral gavage with 400 mg/kg body weight of ibuprofen, the animals fasted for 24

hours.

After 4 h, the animals were sacrificed, and gastric lesions in the fundic stomach were scored and

expressed as ulcer index using the method described by Biswas et al., 2003. The sum of the total

scores divided by the number of animals is expressed as the mean ulcer index.

3.3.3. ULCER SCORING

Ulcer was scored according to the method described by Biswas et al., 2003.

Table 3.1: Criteria for Ulcer Scoring

CODE MEANING

0 No ulcer

1 5 petechial hemorrhage

2 5 petechial hemorrhage with erosion of 1mm

depth

3 10 petechial hemorrhage with erosion of 1mm

depth

4 10 petechial hemorrhage with erosion of above

1mm depth

3.3.4. MEASUREMENT OF GASTRIC ACIDITY

Gastric acidity was measured according to the method of Blandizzi et al., 2005 with

modification. On the day of gastric ulcer induction, the animals were sacrificed with overdose of

anesthesia; the abdomen were opened to remove the stomach. The stomach was opened along

the greater curvature and the gastric content was drained into a centrifuge tube. Samples with

more than 0.5ml was discarded and the result and the solution was centrifuged at 3,000rpm for

10 minutes. Gastric acid output was determined in the supernatant by titration with 0.01N

NaOH.0.5ml of gastric juice pipetted into a 25mL conical flask, 2 drops of phenolphthalein

solution was added and titrated with 0.01NaOH until a purple color appears. The volume of

NaOH added was also noted. Acidity was calculated by using the formula below;

Acidity=Volume of NaOH xNormality of NaOH x mEq/L/100g

3.3.5 .HOMOGENIZATION

The stomach tissue was homogenized in 10 vol. of 50 mmol/l phosphate buffer, pH 7.4. (e.g. 0.5g

tissue in 5ml phosphate buffer).

3.3.6. DETERMINATION OF TOTAL PROTEIN

Protein concentration of the homogenate was determined by means of the Biuret reaction as

described by Gornal et al., 1949 with some modification.

3.3.7. DETERMINATION OF SUPEROXIDE DISMUTASE (SOD)

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O2.-)

into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative

enzymes.

PRINCIPLES

The enzyme Superoxide dismutase has the ability to inhibit the autoxidation of pyrogallol. The

autoxidation of pyrogallol in the presence of EDTA in the pH 8.2 is 50%. The principle of this

method is based on the competition between the pyrogallol autoxidation by O2•¯ and the

dismutation of this radical by SOD .The homogenates was centrifuge for 20 min at 3000 rpm, and

the supernatant was collected and used in the assay.

SOD CHROMOGEN SOLUTION PREPARATION

The SOD Chromogen Powder was reconstituted by adding all of the SOD chromogen diluent to

the SOD chromogen powder. 50ul of the homogenate / sample was added into a clean cuvette

and 1ml of SOD assay was added as a buffer. 1ml of SOD chromogen solution was added to the

solution and mixed.the absorbance was read immediately at 420nm, and after 1 minute.

SOD ACTIVITY IN (U/ml) = % inhibition of Pyrogallol autoxidation

50%

3.3.8. DETERMINATION OF OXIDATIVE STRESS

Lipid peroxidation was determined by measuring the thiobarbituric acid reactive substances

(TBARS) produced during lipid peroxidation. This was carried out by the method of Varshney

and Kale (1990).

Principle:

This method is based on the reaction between 2-thiobarbituric acid(TBA) and Malondialdehyde

:an end product of lipid peroxide during peroxidation. On heating in acidic pH, the product is a

pink complex which absorbs maximally at532nm and which is extractable into organic solvents

such as butanol.

Procedure:

0.4ml of reaction mixture (that is, sample already quenched with 0.5ml of 30% TCA) was added

to 1.6M of Tris HCL. Addition of 0.5ml TBA (0.75%) and incubated for 45 minutes at 80°C.

This was then cooled in ice and centrifuged at 3000g for15minutes.The absorbance of the clear

pink supernatant was then read at 532nm and the absorbance was measured against the blank of

distilled water at 532nm.The MDA level was calculated according to the method of Adam-

Viziand Seregi (1982). Lipid peroxidation in units/mg protein orgram tissue was computed with

a molar extinction coefficient of 1.56x105M-1Cm-1

.

Calculation:

MDA(Units/mg protein)=Absorbance x Volume of mixture

E532 x Volume of sample x mg protein

WhereE532 ismolar absorptivity at 532nm

3.3.9. DETERMINATION OF CATALASE ACTIVITY

Catalase activity was determined according to the method of Claiborne (1985).

Principle

The methods is based on the loss of absorbance observed at 240nm as catalase splits hydrogen

peroxide .Despite the fact that hydrogen peroxide has no absorbance maximum at this

wavelength, its absorbance correlates well enough with concentration to allow its use for a

quantitative assay. An extinction coefficient of 0.0436mM-1cm-1(Noble and Gibson, 1970) was

used. Hydrogen peroxide (2.95ml of 19mM solution) was pipetted into a1cm quartz cuvette

and50µl of sample added .The mixture was rapidly inverted to mix and placed in a

spectrophotometer. Change in absorbance was read at 240 nm every minute for 5 min.

Calculation

Catalase activity= ΔA240/min × reaction volume × dilution factor

0.0436 ×sample volume ×mg protein/ml

=µmole H2O2/min/mg protein

3.3.10. DETERMINATION OF PROSTAGLANDINS E LEVEL

The Elisa KIT (Enzyme linked immunosorbent assay kit) was used for the assaying of

prostaglandin E. This was ordered from Elab Science official website (elabscience.com).

Principle: The Elisa Kit uses a Competitive –Elisa as the method. The microtiter plate provide

in the kit has been pre-coated with PGE. During the reaction, PGE in the sample or standard

competes with a fixed amount of PGE on the solid phase supporter for sites on the Biotinylated

Detection Ab specific to PGE.

Excess conjugate and unbound sample or standard are washed from the plate, and Avidin

conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated.

Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated

by addition of a sulphuric acid solution and the color change is measured spectrophotometrically

at a wavelength of 450nm. The concentration of PGE in the sample is then determined by

comparing the OD of the samples to the standard curve. Assay Procedures: 50ul of standard of

sample was added.

3.3.11. ASSESSMENT OF H+/K+-ATPase.

The sample was thawed and then diluted to yield 500g protein/ml solution. ATPase activity was

assay by measuring the amount of inorganic phosphate (Pi) liberated from ATP during incubation

of the microsomal fraction in the presence of appropriate activators. H+/K+ -ATPase was

measured as K+ -stimulated ATP hydrolysis. The assay medium (1 ml) contained 5 mMKCl, 10

mM MgCl2, 1 mM EGTA, 5 mMTris/ATP and 25 mMTris/HCl (pH 7.4), as described by Buffin-

Meyer et al. (1997).

3.4. STATISTICAL ANALYSIS

The statistical analysis for this study was done using Graph pad software. The results were

expressed as Mean ±SEM (Standard Error of Mean). One-way ANOVA (Analysis of Variance)

was used to analyze the differences among them. The statistical difference was taken to be

significant at P<0.05.

CHAPTER FOUR

4.0. RESULTS

4.1 EFFECTS OF AQUEOUS EXTRACT OF HONEYDEW MELON ON TOTAL

GASTRIC ACIDITY

The result of the study shows significant difference (p<0.05) in total gastric acidity after treatment

with honeydew melon extract. A significant decrease in total gastric acidity was seen in the group

III-VII compared to group II, P<0.05. Total gastric acidity in groups I, III, V and VII were not

statistically different as shown in figure 4.1.

Figure 4.1: Effect of aqueous extract of honeydew melon on the total gastric acidity. All values

are expressed as mean ± SEM, P<0.05.

I I I I II

IV V VI

VII

0

1

2

3

4

a

a ,b ,c

b ,c

b , c

b

g ro u p s

tota

l g

as

tric

ac

idit

y(m

Eq

/L)

b

a- Significant compared to group I

b- Significant compared to group II c- Significant compared to group III

Figure 4.1: Effect of aqueous extract of honeydew melon on the total gastric acidity. All values

are expressed as mean ± SEM, P<0.05.

4.2. EFFECTS OF AQUEOUS HONEYDEW MELON EXTRACT ON ULCER SCORE,

ULCER INDEX

From table 4.1, there was significant increase in the ulcer score post ibuprofen induced ulceration

in group II. There was significant decrease in ulcer score in group III-VII compared to group II,

P<0.05.

The ulcer index was significantly increased in group II compared to group I, post ibuprofen

induced ulceration. Administration of aqueous extract of honeydew melon significantly decrease

the ulcer index in group III-VII, P<0.05 compared to group II as shown in table 4.1.

4.3. EFFECTS OF AQUEOUS EXTRACT OF HONEYDEW MELON ON PERCENTAGE

INHIBITION

The aqueous extract of honeydew melon significantly increase the percentage inhibition in group

IV-VII compared to group I and group II, P<0.05 as show in table 4.1. In group III, the percentage

Inhibition was not statistically different from all the treatment groups.

4.4. EFFECTS OF AQUEOUS EXTRACT OF HONEYDEW MELON ON

MALONIDIALDEHYDE (LIPID PEROXIDATION).

The MDA level in group II was significantly increased compared to group I (p<0.05).

Administration of aqueous extract of honeydew melon significantly decreased the rate of lipid

peroxidation in groups IV- VII compare to group II, P<0.05 as shown in figure 4.2 .

Table 4.1. Effect of aqueous extract of honey dew melon on ulcer score, ulcer index and

percentage inhibition

GROUP DOSE OF

EXTRACT

ULCER

SCORE

ULCER

INDEX

PERCENTAGE

INHIBITION (%)

group I Positive control 0.75 ± 0.07219

2.27 61.27

group II Negative control 3.0a,c ± 0.1443

5.86 a,c 0.00

group III Standard 1.0b ± 0.0 1.96 a,b 55.29

group IV 25% 2.3a,b,c ± 0.0 3.47 a,b,c 52.71

group V 50% 1.17a,b,c±0.07219 2.43 a,b,c 54.50

group VI 75% 2.0 a,b,c ± 0.2887 3.60 a,b,c 52.48

group VII 100% 0 a,b,c c ± 0.0 0b,c

100

All values are expressed as mean ± SEM, P<0.05.

a- Significant compared to group I

b- Significant compared to group II c- Significant compared to group III

I II III IV V VI

VII

0

5

10

15

b,c

a,c

b b b

groups

malo

nd

iald

eh

yd

e U

/mg

pro

tein

Figure 4.2. Effect of aqueous extract of honeydew melon on lipid peroxidation (MDA). All values

are expressed as mean ± SEM, P<0.05.

a- Significant compared to group I b- Significant compared to group II

c- Significant compared to group III

b

4.5. EFFECT OF AQUEOUS EXTRACT OF HONEYDEW MELON ON CATALASE.

From figure 4.3, the catalase level was significantly decreased in the group II compared to group

I, P<0.05. There is significant increase in the catalase level after treatment with aqueous extract of

honeydew melon in groups III-VII compared to group II ,p<0.05.

I II III IV V VI

VII

0

1

2

3

4cata

lase (u

/mg

pro

tein

)

a,b

b,c

b

b,cb,c

groups

Figure 4.3. Effect of aqueous extract of honeydew melon on catalase level. All values are

expressed as mean ± SEM, P<0.05.

a- Significant compared to group I b- Significant compared to group II

c- Significant compared to group III

4.6 EFFECT OF AQUEOUS EXTRACT OF HONEYDEW MELON ON PROTEIN LEVEL

The protein level was significantly increased I the group IV, VI ad VII compared to group II,

P<0.05. In the group III and V, the protein level was not statistically different from group I and

group II, p>0.05as shown in figure 4.4.

I II III IV V VI

VII

0

1

2

3

4

5P

rote

in(m

g/m

l) b b b

groups

Figure 4.4. Effect of aqueous extract of honeydew melon on protein level. All values are

expressed as mean ± SEM, P<0.05.

b- Significant compared to group II

4.7. EFFECT OF AQUEOUS EXTRACT OF HONEYDEW MELON ON SUPEROXIDE

DISMUTASE LEVEL

From figure 4.5, the superoxide dismutase level was significantly decreased in group II compared

to group I, P<0.05. However, there was significant increase in group III- VII compared with the

group II, p<0.05. The SOD level in the treatment groups were not significant compared to the

group I, P<0.05.

I II III IV V VI

VII

0.0

0.5

1.0

1.5

2.0

2.5

Sup

ero

xid

e d

imu

tase

level

(U/m

g p

rote

in)

b

b

bb

b b

a

groups

Figure 4.5. Effect of aqueous extract of honeydew melon on superoxide dismutase level. All

values are expressed as mean ± SEM, P<0.05.

a- Significant compared to group I

b- Significant compared to group II

4.8. EFFECT OF AQUEOUS EXTRACT OF HONEYDEW MELON ON H+/K+-ATPASE

From figure 4.6, a significant increase was observed in the group II compared to group I,

p<0.05.There was a significant decrease in the activity of H+/ K+ - ATPase in groups III-VII

compared to group II,P<0.05.

I II III IV V VI

VII

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

H-K

AT

pa

se

(mm

ol p

ho

sp

ho

rus/ g

pro

tein

/m

ins)

b

a,b,c

b

a,b,ca,b

a

groups

Figure 4.6. Effect of aqueous extract of honeydew melon on activity of H+/K+ATPase. All values

are expressed as mean ± SEM, P<0.05.

a- Significant compared to group I b- Significant compared to group II c- Significant compared to group III

4.9. EFFECT OF AQUEOUS EXTRACT OF HONEYDEW MELON ON

PROSTAGLANDIN LEVELS

From figure 4.7, the prostaglandin level was significantly increased in groups V and VII

compared to group II, P<0.05. There was an increase in group III and IV but not statistically

different from group II (p>0.05).

I II III IV V VIVII

0.0

0.5

1.0

1.5

a,b,ca

a,b,c

groups

pro

stag

lan

din

(n

g/m

l)

Figure 4.7. Effect of aqueous extract of honeydew melon on activity of Prostaglandins. All

values are expressed as mean ± SEM, P<0.05.

a- Significant compared to group I

b- Significant compared to group II c- Significant compared to group III

4.10. EFFECT OF AQUEOUS EXTRACT OF HONEYDEW ON THE STOMACH

MUCOSA: HISTOLOGICAL ANALYSIS

From plate 4.1, the mucosal layer of the stomach of animals in group II was severely eroded, with

increased infiltration of inflammatory cells, hemorrhage which extends in to the basal lamina post

ibuprofen induced gastric ulceration compared to the group I. There was moderate erosion in the

group III of the mucosal layer extending in to submucosal with infiltration of inflammatory cells.

Administration of aqueous extract of HDM, decrease the severity of erosion of the mucosal layer

and the hemorrhage occurring at the submucosal layer.

I II III

IV V

VI

Plate 4.1: the photomicrograph of stomach mucosa showing effect of aqueous extract of honeydew melon

on ibuprofen induced gastric ulceration .I: control (mild erosion with few inflammatory cells). II. Negative

group (severe erosion with strong infiltration of inflammatory cells, Hemorrhage at the submucosa) III.

Standard group treated misoprostol) + ibuprofen induced gastric ulceration: moderate mucosal erosion with

infiltration of inflammatory cells at the submucosa level. IV pretreated with 25% of aqueous extract of

HDM + ibuprofen induced gastric ulceration. There was moderate erosion of the epithelial extending to the

submucosa with hemorrhage at the submucosal layer; there was moderate infiltration of inflammatory of

cells V. pretreated with 50% of aqueous extract of HDM + ibuprofen induced gastric ulceration. VI:

pretreated with 75% of aqueous extract of HDM + ibuprofen induced- gastric ulceration. There was a

mild erosion at the mucosal layer with infiltration of inflammatory cells VII: pretreated with 100% of

aqueous extract of HDM + ibuprofen induced gastric ulceration. There was mild erosion with infiltration

of inflammatory cells.

VII

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.0. DISCUSSION

Reports have shown that the prolonged use of nonselective NSAIDs such as aspirin, indomethacin,

ibuprofen results into gastric mucosal damage .Several therapeutic methods have been employed

to curb the side effect of the anti-inflammatory drug which includes the use of H2 antagonists,

calcium channel blockers , etc.(Masato et al., 2001) . The use of plant extracts cannot be an

exception; hence, this study investigated the antiulcer property of honeydew melon (cucumis melo)

and its ability to regulate prostaglandin synthesis. This extract was selected based on the previous

study carried out by Adebayo-gege et al., 2019, that evaluates the potentials of honeydew melon

as antiulcer agents and its ability to regulate CD-31 (platelet endothelial cell adhesion molecule)

expression.

NSAID induced gastric damage has been via direct or topical irritation of the gastric epithelium

and systemic inhibition of endogenous mucosal prostaglandin synthesis (Wallace et al., 2000,

Kansara et al., 2013), a direct toxic effect on gastric epithelial cells such as apoptosis or necrosis

(Alderman et al., 2000) and expression of superoxide or cytokines (Ding et al., 1998). The actual

basis of gastric mucosal damage remains unclear, they are said to increase free radical during the

course of gastric damage (Desai et al., 1997).

Ibuprofen treatment increased the total gastric acidity significantly, upon treatment with the

extracts it significantly decrease the total gastric acidity. Upregulation of offensive factors such as

gastric acidity has been associated with the high secretion of H+ ion in the gastric juice which is

the basic cause of gastric ulceration (Befrits et al 1984 cited by Adebayo- gege et al, 2019). Gastric

ulceration arises as results of decrease in the defensive factors such as decrease in mucous

secretion, blood flow, reduced prostaglandin secretion, etc with increase in the gastric acidity,

pepsin influence by external aggressive factors (Adhikary et al., 2011, Adebayo- gege et al.,

2019).

The ability of the extract of honeydew melon to decrease the total gastric acidity (figure 4.1)

explains its antisecretory properties, its role in restoring the altered hydrophobicity and reduced

protective ability of mucosal membrane. This is in line with the reports of Adebayo-Gege et al.,

2019.

The ulcer score, ulcer index increases with ibuprofen administration, this explains the compromise

in the integrity of the mucosal protection as result of damage caused by ibuprofen. This agrees

with the previous studies (Narayan et al., 2005, Liu et al., 2015). Administration of honeydew

melon extract significantly decrease the ulcer score and ulcer index. This agrees with the report of

Adebayo-gege et al., 2019. The percentages inhibition increased significantly in the group treated

with 100% of the extract, which explains its ability to inhibit the detrimental effect of the ibuprofen

on the gastric mucosa.

The results of this study showed aqueous extract of HDM had the potential to reduce the gastric

injury caused by ibuprofen, possibly through its active antioxidant constituents such as caffeic

acids, vanillic acid derivatives, ellagitanins, Quercetin-3-rutinoside, derivatives of syringic acid

and ellagic acid (Zeb, 2016). The activity of antioxidants increased significantly post

administration of aqueous extract of HDM. The increased activity of lipid peroxidation in the

gastric mucosa could be traceable to the release of MDA, the end product of lipid peroxidation as

found in the ibuprofen induced model. Oxygen derived free radicals cause tissue injury through

lipid peroxidation. Oxygen handling cells have different systems such as superoxide dismutase,

peroxidases and catalases which are able to protect them against the toxic effects of oxygen derived

free radicals. If the generation of free radical exceeds the ability of free radical, scavenging

enzymes to dismute the radicals, this will damage the gastric mucosa. The ability to preserve the

cell membrane integrity could be proven by the increased activity of antioxidants (SOD, catalase,

Protein level) and in this study (Figure 4.3-4.5), HDM increased the antioxidants activity and

decreased the lipid peroxidation , restoring the integrity of the gastric mucosa.

It has been documented that the ATPases plays important roles in maintenance of cellular

electrolyte concentrations and trans-membrane electrochemical gradient. The Maintenance of

the integrity of the mitochondrial membrane depends on the proper functioning of pumps such

as H+/K+ ATPases, Na+/K+ ATPase and Ca2+- ATPase in the gastric mucosal tissue (Kreydiyyeh,

2000).

The gastric H+-K+ ATPase/pump gastric which composed of α,β- heterodimeric enzyme and

its action cause gastric acid secretion ,due to the action of the ATP-dependent hydrogen–

potassium exchanger .It is the final step of acid secretion ,any inhibitor of the pump would be

more effective in suppressing the gastric acid secretion other than a receptor antagonist (Shin et

al. 2009,Fellenius et al.,1981,Sachs et al.,1976).

The results obtained in this study showed an increase in H+/K+ATPases activity upon ibuprofen

induction (Figure 4.6). This ascertained the increase in gastric acidity as shown in figure 4.1

However, HDM treatment of ibuprofen induced rats showed a decrease in the ATPase activity. It

has also been suggested that increase in ATPase activity on ibuprofen induction could be as a result

of free radicals generated in the process of lipid peroxidation and alterations to membrane fluidity.

This change in ATPase activity further results in an increase in the influx of Ca2+ ions, resulting

in reduced membrane integrity of the surface epithelial cells, thereby generating gastric ulcers, as

reported earlier (Bandyopadhyay et al., 1999, Narayan et al., 2005). The stimulation for acid

secretion increases sodium concentration and potassium decreases in the gastric mucosa (Feldman

and Goldschmiedt, 1991; deBeus et al., 1993). A decrease in the activity of upon H+/K+ -ATPase

treatment with Honeydew melon, contributes to protect the gastric mucosa.

Prostaglandins are hormone-like active lipids that play different functional roles in the body.

They are especially found at sites of tissue damage or illness (Clanton et al., 1999). Prostaglandins

can stimulate bicarbonate production, increase gastric blood volume and inhibit gastric secretion

(Jackson et al., 2000).

The mechanism of NSAID induced gastric damage or injury involves prostaglandin (PG)

deficiency as major significance response of gastric mucosa to the NSAIDs, although the process

has been shown to be more complex due to involvement of several relating factors such as

hypermotility, neutrophils, free radicals, and so on (Asako et al., 1995, Takeuchi, 2012 ).

The deficiency of prostaglandins by NSAIDs is a result of inhibition of cyclooxygenase (COX).

COX are of two isozymes, COX-1 and COX-2; the former is constitutively expressed in various

tissues, including the stomach, while the latter appears to be expressed in most tissues in response

to growth factors and cytokines (O’Neill and Ford-Hutchinson, 1993, Takeuchi, 2012).

From figure 4.7, Prostaglandin levels increased significantly in the group V and VII which is an

indication that honeydew melon increases prostaglandin which in turn prevents ulcer. This further

explains the antiulcer property of extract of HDM on ibuprofen induced gastric ulceration. it is

therefore suggested that extract of HDM probably upregulated the expression of COXs which in

turns increases the prostaglandin synthesis.

The basis of gastrointestinal damage induced by NSAIDs (such as ibuprofen) remains insufficient,

although the Gastric injury as characterized by hemorrhage, edema, inflammatory infiltration, and

loss of epithelial cells, were observed through microscopic examination in ibuprofen treated gastric

tissue) (plate 4.1). This was evidenced in the ibuprofen induced models. In the present study, this

was consistent with previous reports (Liu et al., 2016, Adebayo-gege etal, 2019). Studies suggested

that ibuprofen could induce apoptosis in gastric mucosal cells, due to increase leukocyte

infiltration into the gastric mucosa, which is followed by ROS production (Golbabapour et al.,

2013). Damage to the cell is said to be caused by expression of ROS which was principal factor in

NSAID induced gastric ulceration.

The present study confirmed the characteristics of damages caused by ibuprofen on the gastric

mucosa. Extract of HDM significantly reduced the impact of the ibuprofen on the gastric mucosa.

In summary, the extract of HDM significantly protected the integrity of gastric mucosa by

decreasing the total gastric acidity, ulcer score, ulcer index ,lipid peroxidation and activities of

H+/K+ATPases the gastric mucosa, . Also, Increases the percentage inhibition, antioxidants

activities and prostaglandin synthesis.

5.1. CONCLUSION

Findings in this study revealed that Extract of HDM effectively inhibited ibuprofen induced

gastric ulceration in rats via increased antioxidants, prostaglandin synthesis, increases

percentage inhibition, reduced H+/K+ATPases and reduced inflammations

5.2. RECOMMENDATION

The results obtained from the study suggested that aqueous extract of honeydew melon at 100%

could be more effective for management of gastric ulceration compared to conventional drugs. it

is therefore recommended that the consumption of honeydew melon should be encouraged among

people and patients with peptic ulcer.

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