EFFECT OF CITRUS LEMON JUICE EXTRACT ON ACETIC ACID INDUCED

ULCERATIVE COLITIS ON PLATELET INDICES IN LEAD EXPOSED MALE

WISTAR RATS

BY

ABATAN OLUWADARA SIMEON

182349

SUBMITTED TO

THE DEPARTMENT OF PHYSIOLOGY,

FACULTY OF BASIC MEDICAL SCIENCES, LADOKE AKINTOLA

UNIVERSITY OF TECHNOLOGY, OGBOMOSO, OYO STATE, NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

BACHELOR OF TECHNOLOGY ([Link]) DEGREE IN PHYSIOLOGY

MARCH, 2024
 CERTIFICATION
This is to certify that this project titled ‘’Effect of citrus lemon juice extract on acetic acid
induced ulcerative colitison platelet indices in lead exposed male wistar rats’’ was carried out by,
ABATAN OLUWADARA SIMEON of the department of Physiology, Matric no 182349,
Faculty of Basic Medical Science, Ladoke Akintola University of Technology, Ogbomoso.

__________________ _________________
Prof. (Mrs). S.F Ige Date
PROJECT SUPERVISOR

___________________ __________________
Dr. W. A. Saka Date
HEAD OF DEPARTMENT

___________________ __________________
External Examiner Date

ii
 DEDICATION
This review is dedicated to the Almighty GOD, the Alpha and Omega who in HIS infinite love
and mercy helped me this far and to my lovely parents for their untiring support.

iii
 ACKNOWLEDGEMENT
I give all praises to God for the grace I have received thus far throughout my program.
My profound gratitude goes to Almighty God for the opportunity HE gave and to my project
supervisor Prof. (MRS) S.F. Ige for her guidance and her support during the course of writing
this Project Report.
My profound gratitude goes to my parents for everything theve set in place for me right from the
beginning. I am Grateful.
My appreciation goes to my course mates and my friend Benjamin ogunniran who was very
helpful throughout the completion of this project. God bless you.

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 ABSTRACT
Platelets are colorless blood cells that assist in blood clotting by forming plugs in blood vessels
walls and they are produced in the bone marrow. There are about 15000- 40000 platelets per
microliter of blood. Ulcerative Colitis is a chronic mediated inflammatory disorder of the colon
caused by complex contribution of Genetics, environmental and etiological factors. Lead is a
toxic metal and is also an environmental pollutant than can cause neurological, renal and
reproductive damage on exposure.
In this study, forty adult male wister rats were assigned into Control, colitis, Lead, citrus lemon,
lead + colitis, lead+ citrus lemon, lead + colitis+ citrus lemon groups. Lead Acetate was
administered by 100mg/kg of body weight and colitis was induced by intrarectal administration
of 2% acetic acid (2ml/100g of body weight). Citrus lemon was also administered orally using
the juice extract and the total period for this procedures was for 25 days. After this, animals
were sacrificed using cervical dislocation and blood was collected via cardiac puncture into the
EDTA bottles platelet indices assay. These samples were then analysed using the
autoheamotology analyser and the data were then interpreted using the one way ANOVA
followed by tukey's post hoc test for multiple comparisons.
In this Study, citrus limon increases platelet count. In this study, colitis induction resulted in a
significant decrease in Platelet crit (PCT) compared to the control group . Additionally, the
colitis group exhibited a significant increase in rectal temperature on day 3 compared to the
control group. Furthermore, induction of colitis led to significant reductions in platelet count,
platelet distribution width, and platelet crit.
In this study, Lead significantly reduced platelet count,platelet distribution width and
plateletcrit .
This findings suggest that citrus lemon juice extract may effectively alleviate ulcerative colitis
symptoms and counteract the impact of toxin exposure.

Keywords : lead, Ulcerative colitis, Citrus lemon, Platelet indices

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 TABLE OF CONTENTS
Title Page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of contents vi
List of Table viii
Chapter One 1
1.0 Introduction 1
1.1 Background of study 1
1.3 Aim of Study 2
1.4 Objective of Research 2
Chapter Two 4
2.1 Lead 4
2.1.2 Possible pathway of lead poisoning 4
2.1.3 Signs and symptoms 5
2.1.4 Reproductive system 6
2.1.5 Nervous system 7
2.1.6 Diagnosis 7
2.2 Colon 8
2.2.1 Histology of the colon 8
2.3 Ulcerative colitis 9
2.4 Etiology of Ulcerative Colitis 10
2.4.1 Genetics
10
2.4.2 Environmental factors 11
2.4.3 Diet 12
2.4.4 Diagnosis 14
2.4.5 Ischemic colitis 15

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2.4.6 Infectious colitis 15
2.5 Citrus limon 16
2.6 Platelet indices 16
2.7 Factors that affect Mean platelet volume 22
2.8 Platelet Indices as Diagnostic and prognostic markers 23
Chapter Three 26
3.0 Materials Used 26
3.1 Animal Procurement 26
3.2 Experimental Design 27
3.3 Procurement of Citrus limon 28
3.4 Preparation of Citrus limon 28
3.5 Preparation of Lead acetate 28
3.6 Preparation of 6% Acetic acid 28
3.7 Induction of Colitis 28
3.8 Method of Sacrifice 28
3.9 Evaluation of Hematological Parameters 29
Chapter Four 31
RESULTS 31
Chapter Five 40
DISCUSSION, CONCLUSION AND RECOMMENDATION 40
5.1 Discussion 40
5.2 Conclusion 40
5.3 Recommendation 41
REFERENCES 42

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 List of Figures
Fig 4.1 The rectal temperature of effect of Citrus limon (lemon) juice extract on platelet indices
in acetic acid – induced ulcerative colitis in lead exposed male wistar rats 33
Fig 4.2 The platelet count of effect of Citrus limon (lemon) juice extract on platelet indices in
acetic acid – induced ulcerative colitis in lead exposed male wistar rats 36
Fig 4.3 The mean platelet volume of effect of Citrus limon (lemon) juice extract on platelet
indices in acetic acid – induced ulcerative colitis in lead exposed male wistar rats
37
Fig 4.4 The mean platelet distribution width of effect of Citrus limon (lemon) juice extract on
platelet indices in acetic acid – induced ulcerative colitis in lead exposed male wistar rats 38
Fig 4.5 The plateletcrit of effect of Citrus limon (lemon) juice extract on platelet indices in acetic
acid – induced ulcerative colitis in lead exposed male wistar rats 39

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 LIST OF TABLES
Table 2.1: Ulcerative Colitis Severity Index 15
Table 2.2: Platelet indices with their normal range and their diagnostic and prognostic value
in selected conditions. 25
Table 3.1: Illustration of the grouping of the rats. 27
Table 4.1. The body weight changes of effect of Citrus limon (lemon) juice extract on acetic acid
- induced ulcerative colitis on platelet indices in lead exposed male wistar rats 32

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 CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF STUDY
Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is a chronic, remitting and
relapsing inflammatory disorder encompassing ulcerative colitis and Crohn’s disease. Now IBD
has become almost a global disease affecting masses of almost all ages including the pediatric
population (Molodecky et al.,2012) Although, the etiology of IBD is not yet fully clarified, there
is now general consensus that etiologic factors of IBD possibly include genetic predisposition,
immunologic abnormalities (disturbances in the innate and adaptive immune responses), and
environmental influences that eventually result in colonic Miniflammation (Koch et al.,2000)
Both disorders have certain distinct and overlapping immunological, histopathological and
clinical features.
Ulcerative colitis is featured by inflammatory lesions usually affecting the large intestine,
involving the rectum (proctitis), extending proximally to cover the sigmoid colon
(proctosigmoiditis), descending colon (leftsided colitis), and the entire colon (pancolitis) (carter
et al.,2004). Inflammatory bowel disease (IBD) is a group of intestinal disorders, whose main
clinical characterized by chronic inflammation of the gastrointestinal tract. Antibiotics,
corticosteroids, immunosuppressant and tumor necrosis factor (TNF) inhibitors therapy are the
main therapeutic approach for the treatment of IBD (Bernstein 2015). Nevertheless, these drugs
are accompanied by several side effects (Siegel 2011) that shifted the interest of the scientific
community towards natural products. During the past 25 years, the employment of natural
remedies exponentially increased in order to prevent and treat several illnesses (Micali et al.
2014; Miroddi et al. 2014; Saab et al. 2018). In this frame, several studies evaluated the
beneficial properties of phytochemicals in the management of IBD (Hur et al. 2012; Yang et al.
2018). Manifestations are represented by Crohn’s disease and ulcerative colitis (UC) that are
Citrus Lemon.
Representing one of the main sources of flavonoids, Citrus fruits and their juices have been
widely studied for their beneficial properties, including their anticancer, neuroprotective and
anti-inflammatory activities (Cirmi, Ferlazzo, Lombardo, Maugeri, et al. 2016; Citraro et al.,
2016; Cirmi et al., 2017; Cirmi et al. 2018). Several studies claimed that flavonoids could be
helpful for the management of IBD (Vezza et al., 2016; Salaritabar et al., 2017), although others

1
questioned their real applicability (Farzaei et al., 2015) and this underlines the necessity of
further investigations. In this line, considering the richness of Citrus fruits in flavonoids, the aim
of the present review is to provide new insights on the potential of Citrus fruits and their
flavonoids for the prevention and treatment of IBD. (Cardile et al, 2015)
Lead is a common environmental pollutant. Exposure to lead occurs mainly at occupational sites,
production of lead-acid batteries or pipes, metal recycling and foundries (Woolf et al., 2007).
Children living near such places are also at risk of elevated blood lead levels. In August of 2009,
2000 children living near zinc and manganese smelters were found to be poisoned with lead, an
incident which resulted in riots (Watts, 2009). Other common things which cause lead exposure
are lead in the air, household dust, soil, water, and commercial products (Rossi, 2008).
In cases of chronic exposure, lead often sequesters in the highest concentrations first in the bones
then in the kidneys. A blood lead level of 10 μg/dL or above is a cause for concern. However
there is no threshold value below which lead exposure can be considered safe. It has been found
to impair development and have harmful effects even at lower levels (Rossi, 2008; Barbosa et al.,
2005). A variety of compounds formed by lead exists in the environment in different forms
(Grant, 2009). Poisoning and its features also differ between organic and inorganic lead (Kosnett,
2007). Organic lead poisoning is now very rare around the world because of withdrawal of
organic lead compounds as gasoline additives. Nevertheless, such compounds are still used in
industrial settings. Organic lead compounds cross the skin and respiratory tract easily and
quickly, affecting predominantly the central nervous system.
Platelet indices (PI) — plateletcrit, mean platelet volume (MPV) and platelet distribution width
(PDW) and platelet count (PLT)— are a group of derived platelet parameters obtained as a part
of the automatic complete blood count.
1.2 AIM OF THE STUDY
The research was designed to investigate the potential therapeutic effect of Citrus limon (lemon)
juice extract on platelet indices in acetic acid – induced ulcerative colitis in lead exposed male
wistar rats.
1.3 OJECTIVES OF THIS RESEARCH
The objectives of this research include the following;
I. To examine the effect of Citrus limon (lemon) juice extract on body weight changes of
animals.

2
 II. To evaluate the effect of Citrus limon (lemon) juice extract on rectal temperature of
animals.
III. To examine the effect of Citrus limon (lemon) juice extract on platelet indices.

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 CHAPTER TWO
LITERATURE REVIEW
2.1 Lead
Lead is the most important toxic heavy element in the environment. Due to its important
physico-chemical properties, its use can be retraced to historical times. Globally it is an
abundantly distributed, important yet dangerous environmental chemical (Mahaffay, 1990). Its
important properties like softness, malleability, ductility, poor conductibility and resistance to
corrosion seem to make difficult to give up its use. Due to its non-biodegradable nature and
continuous use, its concentration accumulates in the environment with increasing hazards.
Human exposure to lead and its compounds occurs mostly in lead related occupations with
various sources like leaded gasoline, industrial processes such as smelting of lead and its
combustion, pottery, boat building, lead based painting, lead containing pipes, battery recycling,
grids, arm industry, pigments, printing of books, [Link] its widespread use has discontinued
in many countries of the world, it is still used in many industries like car repair, battery
manufacturing and recycling, refining, smelting, etc. Lead is a highly poisonous metal affecting
almost every organ in the body. Of all the organs, the nervous system is the mostly affected
target in lead toxicity, both in children and adults. The toxicity in children is however of a
greater impact than in adults. This is because their tissues, internal as well as external, are softer
than in adults. Long-term exposure of adults can result in decreased performance in some tests of
cognitive performance that measure functions of the nervous system. Infants and young children
are especially sensitive to even low levels of lead, which may contribute to behavioural
problems, learning deficits and lowered IQ (Rubin & Strayer, 2008). Long-time exposure to lead
has been reported to cause anaemia, along with an increase in blood pressure, and that mainly in
old and middle aged people. Severe damage to the brain and kidneys, both in adults and children,
were found to be linked to exposure to heavy lead levels resulting in death. In pregnant women,
high exposure to lead may cause miscarriage. Chronic lead exposure was found to reduce
fertility in males (Sokol & Berman, 1991). Blood disorders and damage to the nervous system
have a high occurrence in lead toxicity.
2.1.2 Possible pathway of lead poisoning
Poisoning due to lead occurs mainly by ingestion of food or water contaminated with lead.
However accidental ingestion of contaminated soil, dust or lead based paint may also result in

4
poisoning. Lead is thought to be quickly absorbed in the blood stream and is believed to have
adverse effects on certain organ systems like the central nervous system, the cardiovascular
system, kidneys, and the immune system (Bergeson, 2008). Most pharmaceutical companies
have set a limit for maximum daily intake of lead as 1.0 μg/g, however prolonged intake of even
this low level of lead is hazardous to human beings. Occupational exposure also results in
elevated blood lead levels. Increased blood levels are associated with delayed puberty in girls
(Schoeters et al., 2008). There is no threshold value for the level of lead present in blood below
which its concentration can be considered safe. Extremely low yet permanent levels of lead
exposure were found to reduce the cognitive capacity of children (Needlemann, 2004). Dangers
from lead poisoning due to paint pigments mainly in children have sharply reduced the use of
lead in paints. However old houses may still contain substantial amounts of lead paint (Levin et
al., 2008). This sometimes causes an accidental contamination of lead in children. (Marino et al.,
1990).Lead toxicity may be caused through fruits and vegetables contaminated with high lead
levels from the soils where they were grown. Lead from water pipes coming into homes is one of
the major sources (Merill et al., 2007.) For ingested lead, the rate of absorption by the body is
very high with almost 20–70% and in children it is even higher. However the rate of skin
absorption for inorganic lead is low.
2.1.3 Signs and symptoms
Lead poisoning causes a variety of symptoms, including abnormal behaviour which varies from
person to person, while time of exposure plays an important role (Kosnett, 2005). There are also
studies which show no symptoms of lead poisoning even with elevated levels of lead inthe body
(Mycyk et al., 2005). The question what makes such differences in the human body is an issue of
major concern. Why are there such differences in attaining toxicity of lead or other toxic
substances. Why for one group of people any increase in the concentration gives toxicity andfor
another the same level has no effect.
Blood lead levels from 25 and 60 μg/dL give rise to neuropsychiatric effects such as delayed
reaction times, irritability, and difficulty in concentrating, as well as slowed down motor nerve
conduction and headache (Merill et al., 2007). Anaemia may appear at blood lead levels higher
than 50 μg/dL (Merill et al., 2007). In adults, abdominal colic, involving paroxysms of pain, may
appear at blood lead levels higher than 80 μg/dL (Kosnett, 2005). High blood lead levels which
exceed 100 μg/dL cause very severe manifestations, like signs of encephalopathy (condition

5
characterized by brain swelling) accompanied by increased pressure within the skull, delirium,
coma, seizures, and headache (Henritig, 2006). However such manifestations appear in children
at lead levels of 70 μg/dL and more.
As lead disrupts the maintenance of the cell membrane, red blood cells with a damaged
membrane become more fragile, which results in anaemia (White et al., 2007). Lead is also
speculated to alter the permeability of blood vessels and collagen synthesis (Needlemann, 2004).
Damaged activity of cells of the immune system, such as polymorphonuclear leukocytes, results
in decreasing immune activity (Kosnett, 2006).
One of the main reasons for lead poisoning causing anaemia is that lead interferes with the
activity of an essential enzyme called delta-aminolevulinic acid dehydratase, or ALAD, which is
important in the biosynthesis of heme, the cofactor found in haemoglobin (Patrick et al., 2006).
Heme precursors, such as aminolevulinic acid, have been found to build up due to their
interference with lead, which may be directly or indirectly harmful to neurons (Fujita et al.,
2002). In a recent finding accumulation of organic lead in the liver was found to induce oxidative
imbalance and protein impairment that may result in ER stress followed by liver injuries (Fang
et al., 2014). In another recent study the effect of lead exposure on C57BL/6J mice was
observed. Early chronic low-level lead exposure yielding BLLs ranging from 1.98 to 14.84
μg/dL was found to disrupt exploratory activity in pre-adolescent mice (Flores and Sobin, 2014).
Lead poisoning affect various normal function of systems in the body;
2.1.4 Reproductive system
The reproductive system of both males and females is affected by lead. In males sperm count is
reduced and other changes occur in the volume of sperm when blood lead levels exceed 40
μg/dL. Activities like motility and the general morphology of sperm are also affected at this level
(Navas-Acien et al., 2007). The problems with the reproductivity of females due to lead
exposure are more severe. Toxic levels of lead can lead to miscarriages, prematurity, low birth
weight, and problems with development during childhood (Park et al., 2008). Low doses of lead
were found to significantly reduce the number of sperms within the epididymis of mice, while
high doses reduced both the sperm count and percentage of motile sperms and led to an increased
percentage of epididymal abnormal sperms. Lead directly targets testicular spermatogenesis and
also the sperms in the epididymis inducing reproductive toxicity (Wadi & Ahmad, 1999). In lead
treated groups suppressed rates of serum testosterone, intratesticular sperm counts, and sperm

6
production rates were reported (Apostoli et al., 1998; Sokol, 1989). In rats exposed to lead, an
80% reduction in plasma and testicular testosterone and a 32% reduction in the plasma
luteinizing hormone (LH) were reported.. The detailed mechanism of how lead induces male
infertility was reviewed (Mohsen et al., 2011). Long-term low-dose lead exposure was shown to
alter the signalling system between the hypothalamus and pituitary gland of male rats. This
signalling is disrupted by long-term exposure, altering thereby the gonadotropin-releasing
hormone system in the male rat (Sokol et al., 2002).
2.1.5 Nervous system
The brain is the most sensitive organ to lead exposure (Cleveland et al., 2008). In a child's
developing brain, synapse formation is greatly affected in the cerebral cortex by lead. Lead also
interferes with the development of neurochemicals, including neurotransmitters, and organisation
of ion channels (Casarett et al., 2007). Lead poisoning also causes loss of neuron myelin sheath,
reduction in the number of neurons, it interferes with neurotransmission and decreases neuronal
growth (Pearson & Schonfeld, 2003). The brain of adults exposed to increased lead levels during
their childhood also shows a decreased volume, especially in the prefrontal cortex on MRI
(Cleveland et al., 2008). Lead is able to pass through the endothelial cells at the blood brain
barrier because it can substitute for calcium ions and be taken up by calcium-ATPase pumps,
thereby interfering with synapse formation. Children with blood lead concentration greater than
10μg/dL are at higher risk for developmental disabilities (Brunton et al., 2007). The effect of
lead on children´s cognitive abilities takes place at very low levels (Xu et al., 2009; Park et al.,
2008; Sanders et al., 2009). Between the blood lead levels of 5 and 35 μg/dL, an IQ decrease of
2–4 points for each μg/dL increase was reported in children (Brunton, 2007). In. An increase in
the blood lead levels from 50 to about 100 μg/dL in adults was found to be associated with more
severe conditions, like permanent impairment of central nervous system function (Bellings et al.,
2004). The hippocampus is a part of the brain involved in learning and memory. The main
reasons for lead interfering with learning particularly in children is that it damages the cells
within the hippocampus. In rats exposed to lead, structural damage such as irregular nuclei and
denaturation of myelin were reported (Mycyk et al., 2005).
2.1.6 Diagnosis
In order to prevent lead poisoning and toxicity, proper diagnosis is a primary and rather
important issue. In order to make a proper diagnosis, an inquiry about the possible routes of

7
exposure is a must (Nevin, 2007). The inquiry should include medical history and determination
of clinical signs. The involvement of proper staff, i.e. clinical toxicologists and medical
specialists, can help in establishing proper diagnosis and treatment. Basophilic stripping is an
important sign of lead poisoning. This stripping makes dots in red blood cells visible through the
microscope (Patrick, 2006). Thus an examination of blood film for such signs could be effective
in detecting lead poisoning. Lead poisoning is associated with iron deficiency anaemia. Lead
poisoning can also be evaluated by measuring erythrocyte protoporphyrin (EP) in blood samples
(Patrick, 2006). EP is known to increase when the amount of lead in the blood is high, with a
delay of a few weeks (Kosnett, 2007). However, the EP level alone is not sensitive enough to
identify elevated blood lead levels below approximately 35μg/dL (Patrick, 2006). Whole body
lead can be measured in bones noninvasively by X-ray fluorescence; this may be the best
measure of cumulative exposure and total body burden (Kosnett, 2006). X-rays may also reveal
lead-containing foreign materials such as paint chips in the gastrointestinal tract (Kosnett, 2007;
Grant, 2009).
2.2 Colon
The large intestine is part of the digestive tract. The digestive tract includes the mouth,
esophagus, stomach, small intestine, large intestine, and rectum. The large intestine is
approximately 5 feet long, making up one-fifth of the length of the gastrointestinal (GI) tract.
The large intestine is responsible for processing indigestible food material (chyme) after most
nutrients are absorbed in the small intestine. The large intestine is composed of 4 parts. It
includes the cecum and ascending colon, transverse colon, descending colon, and sigmoid colon.
The large intestine performs an essential role by absorbing water, vitamins, and electrolytes from
waste material (Sulaiman and Marciani, 2019). The large intestine has 3 primary functions:
absorbing water and electrolytes, producing and absorbing vitamins, and forming and propelling
feces toward the rectum for elimination. By the time indigestible materials have reached the
colon, most nutrients and up to 90% of the water has been absorbed by the small intestine. The
role of the ascending colon is to absorb the remaining water and other key nutrients from the
indigestible material, solidifying it to form stool. The descending colon stores feces that will
eventually be emptied into the rectum. The sigmoid colon contracts to increase the pressure
inside the colon, causing the stool to move into the rectum. The rectum holds the feces awaiting
elimination by defecation.

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2.2.1 Histology of the colon
Adequate contractile function of smooth muscle layers of the gut wall is pivotal for complete
digestion process and finally to expel the undigested materials through the anal canal. During the
post-natal period, changes in the histological picture of National Journal of Physiology,
Pharmacy and Pharmacology gut may be responsible for alteration in gut motility during
developmental process. The principle changes have been observed in gut neuronal elements and
smooth muscle (Wester et al., 1999 ; Burns et al., 2010) The functions of gastrointestinal tract
(GIT) is under control of an intricate network of neurons embedded in the wall of the GIT known
as enteric nervous system (ENS) (Gershon et al., 1999) It has been reported that there are more
neurons in the gut than present in the spinal cord (The ENS is composed of an inner plexus
known as Meissner’s or submucosal plexus located in the submucosal layer, mainly implicated in
regulation of secretion, and an outer plexus known as Auerbach’s or myenteric plexus located
between the intestinal muscular layers and involved in regulation of smooth muscle contractility.
(Furness, 2006) Interactions between these plexuses in different parts of the gut help in motility.
The ENS autonomously regulates various functions of the gut including motility, secretion,
vascular tone, and release of hormones. However, the autonomic nervous system can modulate
these functions. Thus, in humans and other mammalian species such as albino rats, specific
contraction, and relaxation requires coordinated actions of smooth muscle, neurons of the ENS,
and autonomic nervous system (Wallace et al., 2005; Fu et al., 2004).
2.3 Ulcerative Colitis
Ulcerative colitis is a chronic disease characterized by diffuse mucosal inflammation of the
colon. Ulcerative colitis always involves the rectum (i.e., proctitis), and it may extend proximally
in a contiguous pattern to involve the sigzmoid colon (i.e., proctosigmoiditis), the descending
colon (i.e., left-sided coltitis), or the entire colon (i.e., pancolitis). Ulcerative colitis is a chronic
inflammatory disease affecting the colon, and its incidence is rising worldwide. The pathogenesis
is multifactorial, involving genetic predisposition, epithelial barrier defects, dysregulated
immune responses, and environmental factors. Patients with ulcerative colitis have mucosal
inflammation starting in the rectum that can extend continuously to proximal segments of the
colon. Ulcerative colitis usually presents with bloody diarrhoea and is diagnosed by colonoscopy
and histological findings. The aim of management is to induce and then maintain remission,
defined as resolution of symptoms and endoscopic healing. Treatments for ulcerative colitis

9
include 5-aminosalicylic acid drugs, steroids, and immunosuppressants. Some patients can
require colectomy for medically refractory disease or to treat colonic neoplasia. The therapeutic
armamentarium for ulcerative colitis is expanding, and the number of drugs with new targets will
rapidly increase in coming years. Understanding the daiagnosis and treatment of ulcerative
colitis from a primary care perspective is important. (Carter et al., 2004)
The onset of ulcerative colitis is most common between 15 and 40 years of age, with a second
peak in incidence between 50 and 80 years. The disease affects men and women at similar rates.
The precise etiology of ulcerative colitis is not well understood. A current hypothesis suggests
that primary dysregulation of the mucosal immune system leads to an excessive immunologic
response to normal microflora (Strober et al., 2007). Cigarette smokers have a 40 percent lower
risk of developing ulcerative colitis than do nonsmokers; however, compared with those who
have never smoked, former smokers are approximately 1.7 times more likely to develop the
disease and there is no consistent link between diet and the development of ulcerative colitis has
been found. Although an association between the use of nonsteroidal anti-inflammatory drugs
and the development of ulcerative colitis has been suggested (kornbluth et al., 2004).
2.4 Etiology of Ulcerative Colitis
2.4.1 Genetics
A number of genetic variables have been associated with ulcerative colitis. There are 163
susceptibility disease-associated loci associated with IBD. Thirty of them are associated with
Crohn’s disease (CD), Ventham et al., (2013) with ulcerative colitis and most of the remaining
ones are common to both CD and UC, as well as other conditions: psoriasis, celiac disease
[Jostin et al.,2012;Dobre et al., 2017). The unique genes associated with UC can be divided into
genes that affect epithelial barrier (ECM1, HNF4A, CDH1, LAMB1, and GNA12), genes that
are immune-mediated (IL8RA / IL8RB, IL2 / IL21, IFNG / IL26, IL7R, TNFRSF9, TNFRSF14,
IRF5, LSP1, FCGR2A) and others (OTUD3 / PLA2G2E, PIM3, DAP, CAPN10, JAK2). The
genes that overlap with Crohn’s can also be divided into those that are immune-mediated: IL10,
CARD9, MST1, ICOSLG, IL1R2, YDJC, PRDM1, TNFSF15, SMAD3, PTPN2, TNFRSF6B,
HLA: DRB1*03 and others: ORMDL3, RTEL1/SLC2A4RG, PTGER4, KIF21B, NKX2-3,
CREM, CDKAL1, STAT3, ZNF365, PSMG1, IL23R, IL12B, AK2, FUT2, and TYK2 (Jostins
et al., 2012;De lange and Barett,2015;Andersen et al., 2011). Recent studies have shown that the
genes of the major histocompatibility complex are major genetic determinants of susceptibility to

10
UC. The human leukocyte antigen (HLA) region on chromosome 6 is connected with the
greatest genetic signals within UC-specific loci. (Justin et al.,2012; Kaur and Goggolidou,
20202.4.2

11
2.4.2 Environmental factors
in UC The rapid growth in the incidence of ulcerative colitis in newly industrialized nations
implies that environmental factors have a role in disease initiation (Kaplan, 2017;Wang et al.,
2013; Benchimol et al.,2017; Amarapurkar et al.,2018). Ulcerative colitis comes initially in
urban locations, with a quick rise in incidence followed by a slowing period. After that period,
Crohn’s disease grows in frequency, finally approaching that of UC. Industrialization is
associated with a new urban lifestyle, pollution exposure, dietary changes, antibiotic access,
improved cleanliness, and fewer infections, all of which are considered general contributory
factors (Kaplan,2017). Urbanization is no longer regarded as a risk factor, according to studies
from both Western and developing jurisdictions (Wang et al.,2013; Amarapurkar et al.,2018).
Adolescent influences that involve smoking has been linked to an increased risk of ulcerative
colitis (Bernstein et al., 2016). The pathophysiology of how smoking causes ulcerative colitis or
protects a person from developing the condition is unknown. Active or passive smoking
produces milder forms of the disease in the case of UC, requiring fewer surgeries throughout its
development and less need for immunosuppressive drugs. It is unclear whether the rise in
ulcerative colitis is related to smoking cessation patterns. Indian research shows that there is no
link between quitting smoking and the development of ulcerative colitis. As for the association
between active smoking and the incidence of Crohn’s disease, there is also no evidence in their
studies (Amarapurkar et al.,2018 ;Wang et al.,2018). Studies proposed the divergent effect of
appendectomy on UC suggesting inflammation of appendix might have protective interplay with
the disease (Andersen et al.,2001). The dietary habits of adolescents are characterized by
excessive consumption of meat and fat and an insufficient intake of fiber, fruits, and vegetables.
There is also a tendency to frequently consume processed food and high sugary or soft drinks
that increase the risk of developing IBD (Kikut et al.,2021). Psychological comorbidity is three
times higher in those with IBD than in the general population. More than a quarter of people with
IBD will have depression at some point in their lives, and more than a third will experience
anxiety. Not only does having this chronic disease cause an increase in anxiety and depression
but it is also possible that having these psychiatric disorders makes you more likely to develop
IBD. It is unclear how much depression and IBD have in common in terms of gene alterations,
epigenetic changes, or immunological responses. When they coexist, it has a detrimental

12
influence on health-related quality of life regardless of which arrives first, impaired mental
health or IBD (Bernstein, 2016; Guthrie et al., 2002).
Other factors NSAIDs are among the most commonly used drugs, and their link to ulcers in the
stomach or duodenum is well known. They have, however, been associated with the
development of IBD. Several theories have been proposed as possible mechanisms for the link
between NSAIDs and IBD. A prospective cohort study assessed the link between aspirin and
nonsteroidal anti-inflammatory drug (NSAID) use and the occurrence of Crohn’s disease and
ulcerative colitis. A higher risk of both conditions was observed with the highest frequency of
NSAID use (Ananthakrishnan et al.,2012). In urban areas, air pollution has been related to a
variety of health problems. In mice, acute exposure to high levels of airborne particulate matter
increases gut permeability and heightens the innate immune response in the small intestine, while
chronic exposure results in increased expression of pro-inflammatory cytokines and changes in
colon microbiota composition and function (Kish et al.,2013). Particulate matter exposure has
given inconsistent results in epidemiological studies evaluating the link between air pollution and
ulcerative colitis, showing that when there is a link, other components of air pollution may play a
role in disease development. People who lived in locations with greater SO2 concentrations were
more likely to develop ulcerative colitis than people who lived in areas with lower SO2
concentrations (Bernstein et al.,2016).
2.4.3 Diet
Multiple epidemiological researchers have concluded a link between nutrition and ulcerative
colitis. In recent decades, significant changes in food intake have been related to an increase in
the incidence of UC. Consumption of soft drinks and sucrose was linked to an increased chance
of acquiring the condition. On the other hand, the consumption of fruits and vegetables was
related to a decrease in UC development (Wang et al., 2017;Preda et al.,2019).There is a
significant association between red meat intake and ulcerative colitis risk (Ge et al.,2015).
Furthermore, whereas dietary n-3 polyunsaturated fatty acids (PUFAs) were linked to a lower
risk of UC (odds ratio: 0.56) (John et al.,2010), dietary arachidonic acid (an n-6 PUFA) assessed
in adipose tissue was linked to a higher risk of UC (relative risk: 4.16) (Da silva et al.,2010).
Although there is no evidence of the mechanisms involved in the diet role in IBD development,
there are several plausible explanations such as the effects on composition of gut microbiota, the
microbial Differences in food patterns between African and European children were related to

13
increased Bacteroidetes and decreased Firmicutes and Enterobacteriaceae (Brown et al.,2012). A
high fat/high sugar diet can result in intestinal mucosal dysbiosis characterized by an overgrowth
of pro-inflammatory proteobacteria and a decrease in protective bacteria. Dietary factors have
significant effects on microbial composition and can also affect the metabolic functions of gut
microbiota. In both small and large intestines, commensal bacterial fermentation of indigestible
food fibers produces short chain fatty acids (SCFA). SCFA changes gene expression, cellular
differentiation, chemotaxis, proliferation, and apoptosis in epithelial and/or immunological cells
(Sun et al.,2012) . Some UC patients have a lower amount of SCFA-producing bacteria such as
Faecalibacterium prausnitzii, which is inversely connected to disease activity. Furthermore,
experimental investigations have linked a western diet high in sugar and fat and low in dietary
fiber to lower SCFAs and greater colitis susceptibility (Agus et al.,2016). There have been
demonstrated links between dietary PUFA content and inflammatory processes in IBD. Dietary
n-3 polyunsaturated fatty acid (PUFA) intake has been linked to a lower risk of ulcerative colitis,
while dietary n-6 PUFA intake has been linked to a higher risk of ulcerative colitis (Da silva et
al., 2010; Chan et al., 2014).
HISTHOPATHOLOGY OF ULCERATIVE COLITIS
UC starts from the rectum, spreads proximally and in continuity, involving a variable length of
the colon. Pancolitis normally stops abruptly at the ileocecal valve, but in some cases a limited
distal ileitis, called backwash ileitis, is observed.( These ileal lesions are in continuity with
colonic lesions and characterized microscopically by diffuse inflammatory lesions with regular
shortening of the villi. In patients with limited UC, the transition from diseased to normal
mucosa is usually gradual and only occasionally abrupt. Atypical presentations can be observed.
Some patients have left-sided involvement of the colon and cecal (cecal patch) or appendiceal
involvement.(Geboes et al.,1997) . The affected sites are separated by normal mucosa. Diffuse
duodenitis and extensive involvement of the upper small bowel can occur in severely ill UC
patient (Valdes et al., 2000). The gross appearance varies with the activity of the disease.
Lesions are usually limited to the mucosa. Stenoses, fistulas and significant thickening of the
wall are rare. In the acute form, the mucosal surface is wet and glaring from blood and mucus
with numerous petechial hemorrhages. Ulcers of various sizes can appear. They may be small,
rounded and superficial or more irregular and somewhat geographic in configuration. Fissuring
ulcers are not seen, except in some cases of toxic megacolon. Ulcers can become more extensive

14
and undermine the mucosa so that mucosal bridges with an underlying inflammatory infiltrate
develop. Multiple confluent ulcerations provoke denudation of the mucosa. The serosal side may
appear congested, often to a larger extent than the mucosal lesions. Following healing of mucosal
ulcers, elevated sessile reddish nodules – pseudopolyps – appear in an otherwise flat surface.
There is some variability in the prevalence of the lesions depending upon the colonic segment
involved (Tanaka et al., 2000) Pseudopolyps are common in the sigmoid and descending colon
and rare in the rectum where mucosal friability predominates. In the more advanced stages, the
entire bowel becomes fibrotic, narrowed and shortened. During remission, the mucosa may
become normal again. Healing often occurs in an irregular way leading to a discontinuous,
heterogeneous aspect of the mucosa, which can be confused with CD.
Treatment may increase the heterogeneous nature of the lesions. In children, lesions may initially
be heterogeneous. Topical treatment of the rectum can induce complete rectal healing. UC with
rectal sparing, although rare, must therefore not be confused with CD.( Kleer et al., 1998). In
severe, active forms, the entire colon, or a segment, may become dilated (toxic megacolon) and
Inflammation may extend towards the submucosa.
2.4.4 Diagnosis
The differential diagnosis of ulcerative colitis includes any condition that produces chronic,
intermittent diarrhea, such as Crohn’s disease, ischemic colitis, infectious colitis, irritable bowel
syndrome (IBS), and pseudomembranous colitis (Kefalides et al.,2002).
The clinical history can be used to differentiate the various etiologies of chronic diarrhea in
patients who have not previously been diagnosed with ulcerative colitis. For example, recent
antibiotic use might suggest pseudomembranous colitis; recent travel may indicate infectious
colitis; and abdominal pain that is relieved with bowel movements could represent IBS. For the
patient with established ulcerative colitis, the presence of constitutional symptoms and
extraintestinal manifestations, particularly arthritis and skin lesions, may provide clues to the
severity of the disease (Das,1999) Physical examination should target the gastrointestinal,
dermatologic and ocular systems. The presence of finger clubbing increases the likelihood of
ulcerative colitis in patients with bowel symptom.

15
Table 2.1: Ulcerative colitis severity index
Sign or symptom Mild disease Moderate disease Severe disease
Albumin (g per dL [g Normal 3.0 to 3.5 [30 to 35] < 3.0
per L])
Body temperature Normal 99 to 100°F (37.2 to >100°F
37.8°C
Bowel movements < 4 per day 4 to 6 per day >6 per day
ESR (mm per hour) < 20 4 to 6 per day >30
Hematocrit (%) Normal 30 to 40 <30
Pulse (beats per < 90 90 to 100 >100
minute)
(Cohen et al., 2004)

2.4.5 Ischemic colitis

This is more common in the elderly and occurs when there is decreased blood flow to the colon.
Decreased blood flow can cause inflammation or injury to the colon. Some causes of ischemic
colitis are atherosclerosis of arteries, low blood pressure, blood clots, and bowel obstruction.
(Tanaka et al., 2000)
2.4.6 Infectious Colitis
Infectious colitis can occur from many different viruses, bacteria, or parasites. Infectious colitis
most commonly occurs due to ingestion of contaminated food or water, introducing the
infectious organism into the colon. The most common causes are Escherichia coli,
Campylobacter, Shigella, and Salmonella. These infectious organisms invade the colon, cause
inflammation, and affect the normal function, causing abdominal pain and diarrhea. Clostridium
difficile is another organism that can cause colitis in association with antibiotic use. C. difficile is
part of healthy, normal flora in the colon but can cause problems if it overgrows. Antibiotic use
can destroy other susceptible normal flora in the colon, allowing overgrowth and invasion of C.
difficile. (Click and Regueiro, 2019)

16
2.5 Citrus lemon
Citrus fruit belongs to a group of fruits that have notable socio-economic and cultural
significance in our society and thus in high demand (Iglesias et al., 2007). There are different
kind of fruit in the citrus group, and lemon (Citrus Limon Burm.f.) is one of the most important
species after orange and mandarin (Perez-Perez et al., 2005). Vitamin C from flavonoids which
is obtained from citrus spp family is beneficial because vitamin C is water soluble, it can be
easily absorbed by the body and will immediately help in restoring the body’s platelet count.
Sufficient intake of vitamin C and flavanoids from natural foods like lemon, broccoli, spinach,
bell pepper and kiwi are also beneficial for low platelet counts (Swati Burungale ,2016) .Lemon
is a preferred fruit because of its unique flavor and acidity, which make it a value-added food
product, and its utility in industrial applications. Lemon fruit contains various important natural
compounds, including phenolics (mainly flavonoids), Vitamins, minerals, dietary fibres, essential
oils and carotenoids (Gonzalez et al., 2010). The health promoting effect of lemons are mainly
associated with Vitamin C and Flavonoids, which give provide natural antioxidant characteristics
(Vinson et al., 2001). The whole citrus fruit plant, including crude extracts of leaves, peels,
seeds, flowers, possesses anticancer activities and antimicrobial potential (Kawali et al., 2000).
The fruit are used primarily in juice processing industries while seeds and peels are generally
discarded as waste. A lemon without its peel is capable of giving 17 calories whereas with peel it
contains 22 calories. Lemon juice contains 3 calories per 1 table spoon and according to
“World’s Healthiest Foods a quarter cup of it contains 31 percent of recommended vitamin C
intake and 3 percent of Folate and 2 percent of potassium which make up total 13 calories .
When mixed with water, lemon juice can provide 1 calorie that is very helpful in reducing
weight by reducing calorie intake in human body (Charturverdi,2016). it means water reduces
the sourness and calories of lemon juice and can reduce the excessive fat layer of body
(Patel,2015).
2.6 Platelet indices
Platelets are cytoplasmatic fragments of bone marrow megakaryocytes, with a diameter of 3-5
μm and a volume of 4.5–11 fL (Hoffbrand et al.,2006). A single megakaryocyte releases 1500–
2000 of them to the bloodstream, where they circulate for 7–10 days. Inactivated platelets in the
blood are discoid shaped and do not contain a nucleus. Their cytoplasm contains three different
types of granules (i.e. alpha granules, dense granules, and lysosomal granules), secretory vesicles

17
that contain preformed molecules, and a complex membranous system. Platelets are dynamic
blood particles whose primary function, along with the coagulation factors, is haemostasis, or the
prevention of bleeding. Platelets interact with each other, as well as with leukocyte and
endothelial cells, searching the vascular bed for sites of injury, where they become activated.
When stimulated, platelets undergo a shape change, increasing their surface area and bioactive
molecules stored within their alpha and dense granules’ molecules are rapidly secreted (Lopez et
al.,2015). In addition to their important role in haemostasis and thrombosis, accumulating
evidence demonstrates that platelets contribute to the inflammatory process, microbial host
defence, wound healing, angiogenesis, and remodeling (Golobiewska et al., 2015).
Platelets release > 300 proteins and small molecules from their granules (chemokines, cytokines
like interleukin-1β, CD40 ligands, β-thromboglobulin, growth factors etc.), which can influence
the function of the vascular wall and circulating immune cells (Margetic, 2012). Platelets also
secrete microbicidal proteins and antibacterial peptide (Mariani et al., 2014 ;Tang et al., 2002)
Platelets also mediate leukocyte movement from the bloodstream through the vessel wall to
tissues. Platelets are capable of forming reactive oxygen species; the oxidative stress that
accompanies inflammation can also activate platelets (Monteiro et al.,2012, Magwenzi et
al.,2015). Platelets’ ability to influence other cells means that they can also play many principal
roles in the pathophysiology of disease
Complete blood count (CBC) tests with automated haematology analysers are one of the most
commonly ordered tests in clinical laboratories. Modern haematology analysers in routine
diagnostic use, which measure platelet indices (PIs), use impedance counting or optical light
scatter counting techniques. The measurement principle influences the results, and the results
from different analysers are not comparable (Lippi et al., 2015). Platelet count in the blood can
be rapidly measured using an automated haematologic analyser and they are biomarkers of
platelet activation. They allow extensive clinical investigations focusing on the diagnostic and
prognostic values in a variety of settings without bringing extra costs. Among these platelet
indices, plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW)
are a group of platelet parameters determined together in automatic CBC profiles. The volume of
platelets in the bloodstream is heterogeneous, and their structures and metabolic functions differ.
Typically, the average mean cell volume is 7.2–11.7 fL in healthy subjects (Demirin et al.,2011,
Wiwanitkit, 2004)

18
Mean platelet volume (MPV), the most commonly investigated platelet parameter, signifies the
average size of platelets in the blood. In healthy subjects it typically ranges between 7.2 and 11.7
fL (Demirin et al.,2011) . MPV of over 13 fL tends to occur in hyperdestruction, in which case
become bigger and increase in activity, whereas MPV lower than 8 fL is a sign of platelet
hypoproduction (Demirin et al.,2011) levels are not only affected by the abnormality in platelet
count, but are additionally related to the used method of laboratory analysis (Vasudeva et
al.,2019). Many factors including race, age, smoking, alcohol consumption, and physical activity
can modify MPV (Budak et al., 2016). MPV has been analyzed as a potential biomarker of
patient's prognosis, with most studies associating its higher value to worse clinical outcome. A
study conducted by Lembeck et al.,(2019) showed a significant association between high MPV
and worse prognosis in patients with pancreatic ductal adenocarcinoma regardless of the
normalized level of other well-known prognostic markers. This was also observed in patients
suffering from myocardial infarction, where those with higher MPV level seemed to be more
often associated with poor clinical outcome (Vasudeva et al.,2019). Lower MPV level can be
related to low-grade inflammation, such as rheumatoid arthritis (Margetic, 2012).
2.6.1 Platelet Distrubution width (PDW)
Platelet distribution width (PDW) is a marker of platelet anisocytosis, which describes the size
distribution of platelets produced by megakaryocytes and increases upon platelet activation
(Osselear et al., 1997). Although review by (Budak et al., 2014) suggests that PDW reference
levels range between 8.3 and 56.6%, there is little to no evidence for such wide variation
reported in the literature. Analyzed studies found this parameter to vary between 10 and 18% in
healthy individuals (Negash et al., 2016) and observed PDW change in patients suffering from
numerous diseases (Amin et al., 2004). It allows for this parameter to be considered as a
potential biomarker. PDW seems to be proportionally related to MPV in healthy individuals
(Vagdatli et al.,2010) however, under non-physiological conditions, such as, threatened preterm
labor, they show significant dissonance – a rise in PDW and decrease in MPV (Ulkumen et
al.,2014).
Increased PDW values have been reported in patients with diabetes mellitus (Jindal et al., 2011)
cancer (Yu et al., 2017,Fu et al., 2017), and cardiocerebrovascular and respiratory disorders.
Moreover, higher PDW levels have recently been reported to be associated with increased
morbidity and mortality among patients with critical illness (Sezgi et al., 2015], coronary artery

19
disease (Rechsinski et al.2014,) cancer (Zhang et al.,2017), pulmonary embolism (Sevuk et
al.,2015), and chronic obstructive pulmonary disease. Most previous studies on the clinical
significance of PDW involved patients with specific discord.
PDW is Also an indicator of volume variability in platelets size and is increased in the presence
of platelet anisocytosis. PDW is a distribution curve of platelets measured at the level of 20%
relative height in a platelet-size distribution curve, with a total curve height of 100% (Sachdev et
al., 2014). The PDW reported varies markedly, with reference intervals ranging from 8.3 to
56.6% (maluf et al.,2015;Yang et al.,2006) directly measures variability in platelet size, changes
with platelet activation, and reflects the heterogeneity in platelet morphology (Wiwanitkit,2004;
Vagdatli et al.,2010). Under physiological conditions, there is a direct relationship between MPV
and PDW; both usually change in the same direction (Vagdatli et al., 2010). Meanwhile, there
are conflicting reports in the literature about the relationship between platelet volume and
numbers, which suggests that they are affected by different mechanisms (Mariani et
al.,2014;Yang et al., 2005; Chandrashekar,2013)
2.6.2 Mean Platelet volume (MPV)
In MPV, the analyser-calculated measure of thrombocyte volume is determined directly by
analysing the platelet distribution curve, which is calculated from a log transformation of the
platelet volume distribution curve, to yield a geometric mean for this parameter in impedance
technology systems. In some optical systems, MPV is the mode of the measured platelet volume
(Senaran et al.,2001) MPV is determined in the progenitor cell, the bone marrow megakaryocyte.
The platelet volume is found to be associated with cytokines (thrombopoietin, interleukin-6 and
interleukin-3) that regulate megakaryocyte ploidy and platelet number and result in the
production of larger platelets (Larsen et al., 2014). When platelet production is decreased, young
platelets become bigger and more active, and MPV levels increase. Increased MPV indicates
increased platelet diameter, which can be used as a marker of production rate and platelet
activation. During activation, platelets’ shapes change from biconcave discs to spherical, and a
pronounced pseudopod formation occurs that leads to MPV increase during platelet activation.
MPV acts as a negative or positive acute phase reactant in different inflammatory conditions.
High MPV levels are associated with high-grade inflammation owing to the presence of the large
platelets in circulation. MPV might decrease in high grade inflammation due to the consumption
and sequestration of these large platelets in the vascular segments of the inflammatory region.

20
Low MPV is associated with low-grade inflammation, like rheumatoid arthritis and attacks of
familial Mediterranean fever. MPV decreases and increases in acute and chronic disorders,
respectively (Mariani et al.,2014). MPV shows the activity of disease in systemic inflammation,
acute pancreatitis, unstable angina, and myocardial infarction (Beyazit et al., 2011, Gunes et al.,
2015; Safak et al.,2014; Chu et al.,2010) MPV can be a modifiable marker in identifying patients
with active ankylosing spondylitis and rheumatoid arthritis, which is thought to be due to
increased consumption of platelets in the inflammation area and MPV increases with therapy in
these patients (Kim, 2011;Yazici et al., 2010).
2.6.3 MPV in Cardiovascular Diseases.
It is currently suggested that changes in MPV can be considered and used as a prognostic factor
in a number of inflammatory diseases. It has been shown that hyperreactivity of blood platelets
markedly increases individual susceptibility of patients to acute cardiac incidents. A study
conducted by Endler et al., (2002) revealed that irrespective of how advanced coronary disease
is, the increased platelet volume is associated with a higher risk of acute cardiac incidents. The
presence of large and reactive platelets also increases the risk of thrombus formation after
atherosclerotic plaque rupture (Elsenberg et al.,2017). Potential effects of diabetes, smoking, and
hypertension on MPV have also been considered in the study. The authors suggest that MPV ≥
11 6 fl can also be an independent risk factor of heart infarct in patients with coronary disease
and designate patients threatened with acute cardiac incidents. Also, Slavka et al. (2011)
observed that elevated MPV can be an independent high risk factor of death in patients after
acute ischemic cardiac incident. They showed that standard treatment, i.e., aspirin, does not
affect MPV, whereas clopidogrel significantly decreases the ADP-dependent MPV increase
(Skavka et al.,2011; Huczek et al.(2005) indicate that in patients with STEMI infarct treated with
transdermal cardiac intervention, MPV is a potent and independent predictor/factor of increased
number of restenosis after cardiac angioplasty and higher mortality rate. After the procedure,
over 25% of patients have elevated platelet counts and MPV, which enhance the risk of
cardiovascular incidents as compared to subjects with normal platelet count and size.
2.6.4 MPV in Cerebrovascular Ischemia.
High MPV values are also encountered in patients with acute cerebrovascular ischemia. Patients
with high MPV were more at risk of acute stroke than those with normal MPV (Greisnegger et
al.,2004). Butterworth and Bath (2005) showed increased platelet volume 3 months after severe

21
stroke. O’Malley et al. (1999) demonstrated a statistically significant increase in MPV and a drop
in platelet count in patients with acute cerebral ischemia as compared to the control group.
Moreover, MPV did not correlate with a sixmonth survival of patients.
2.6.5. MPV in Respiratory Diseases.
Differences in platelet size have also been observed in respiratory diseases, accompanied by an
inflammatory condition. In active tuberculosis, cellular body immunity is stimulated. Bacillus-
activated macrophages and lymphocytes produce such cytokines as IL-6 or TNF-α, which affect
maturation of megakaryocytes and platelet release. Therefore, reactive thrombocytosis is
observed in tuberculosis, in which increased platelet count and size are associated with
inflammation intensity (Yang et al., 2011; Unsal et al.,2005). Contrary to the stable form, during
disease exacerbation, the MPV is significantly reduced. The decrease in MPV can be related to
the formation of microthrombi in tuberculous cavities, which are to inhibit the disease spread and
are considered a defense reaction (Gunlouglou et al., 2014). It has been suggested that MPV can
be a negative marker of the acute phase of inflammation, and a decrease in MPV can be caused
by increased platelet count and their accelerated wear. In chronic sinusitis, as reported by Koç et
al. (2011), the platelet count is at the upper normal limit as compared to healthy subjects. Also,
MPV is significantly higher in this group of patients. This may suggest the involvement of blood
platelets in the development and course of the disease( Koç et al.,2011).
2.6.6 MPV in Carcinomas of the Gastrointestinal Tract.
Hepatocellular carcinoma (HCC) is one of the most common primary cancers of the liver. The
first symptoms of tumor growth are masked by chronic liver diseases (CLD), such as cirrhosis or
viral infections (Kurt et al., 2012). Kurt et al. (2012) suggest that in patients with chronic liver
diseases, MPV can be a potential marker of liver cancer. The authors showed that MPV levels in
patients with HCC were significantly higher as compared to patients with chronic hepatitis and in
healthy subjects. Moreover, the level of MPV was significantly higher in patients with liver
cirrhosis and hepatocellular carcinoma as compared to patients suffering only from liver cirrhosis
(Kurt et al., 2012).. Thrombocytopenia and a significantly increased MPV in these patients may
result from decreased activity of thrombopoietin and bone marrow suppression associated with
chronic HCV infection and antiviral therapy application (Afdhal et al.,2008). Also, Cho et al.,
(2012) aimed to assess the changes in the platelet count and MPV/PLT ratio in patients with
hepatocellular carcinoma. The authors showed that both the MPV value and the MPV/PLT ratio

22
were significantly higher in patients with carcinoma than in healthy patients. In the study group,
the marker was the highest among women.
2.7 FACTORS THAT AFFECT MEAN PLATELET VOLUME COUNT
Some researchers indicate that MPV should always be assessed together with platelet count as
there is a nonlinear inverse relationship between PLT and MPV (Butkiewicz et al.,2006).
However, Mediators of Inflammation 7 also other factors like age, gender, race and ethnicity,
lifestyle (including diet), and genetic factors may strongly influence MPV (Yacizi et al., 2009).
A high heritability of 84% and of 75% for PLT and MPV, respectively, has been showed due to
genetic variations . Vasudeva and Munshi (2019) in their review strongly highlight the role of
genetic variants in platelet reactivity response at the site of injury of the vessel wall.
Interindividual genetic variations of platelet reactivity (in terms of PLT and MPV) significantly
modulate the course of thrombotic events. Therefore, an identification of genetic effects on PLT
and MPV may become a potential therapeutic target . Genetic variants are classified into rare
mutations (variants) with large effect, common polymorphisms with small effects, and
polymorphisms with major effects.(Slavka et al., 2011.) According to Kunicki et al. (2012)
polymorphisms with small effects are mainly responsible for hyperactive platelet phenotype. By
means of high-throughput techniques, e.g., GWAS (genome-wide association studies), WES
(whole exome sequencing), WGS (whole genome sequencing), PheWAS (phenome-wide
association studies), large-scale Exomechip genotyping array, and multiomic analysis, different
genetic variants influencing PLT and MPV so far have been recognized (Eicher et al.,2016;
Vaudeva et al.,2019) For example, GWAS studies have indicated 68 distinct loci associated with
PLT and MPV (Eicher et al., 2016). Candidate genes related with platelet reactivity response
include genes associated with megakaryopoiesis, megakaryocyte/platelet adhesion, platelet
formation, regulation of the cell cycle, or platelet surface receptors (Kunicki et al.,2010).
However, the heritability of these genetic variations still remains not fully understood (Eicher et
al.,2016)
2.7.1 Platelet larger cell ratio (P-LCR)
Another marker of platelet activity, is the percentage of all platelets with a volume measuring
over 12 fLcirculating in the bloodstream. It normally ranges between 15 and 35% (Hong et
al.,2014). P-LCR seems to be more susceptible to alterations in platelet size in comparison to
MPV, despite their correlation, it is also used to evaluate the health of a patient blood, it is

23
measured using a relative number of platelets and large information in the sample and this ratio
can provide imoportant information about an individual risk of developing certain disease and
they can identify the number of blood clots. Higher platelets count can be a sign of cancer
infections risk and other health issues (Gao et al., 2014). Platelet larger cell ratio (P-LCR) also
indicates circulating larger platelets (> 12 fL), which is presented as percentage. The normal
percentage range is 15–35%. It has also been used to monitor platelet activity (Hong et al.,2014).
The platelet large–cell ratio (P-LCR) is also an index representing the percentage of platelets
larger than 12 fl in the circulating pool (Turk et al.,2013), generated by the automatic blood cell
counter, thus probably identifying those platelets that are metabolically and enzymatically more
active than small platelets, and it is easily available at an affordable cost. It is determined based
on flow cytometry and automated blood cell analysis techniques. The P-LCR was the best tool to
assess megakaryocyte activity and a good monitoring tool for platelet activity. Evidence has
indicated that the P-LCR contributes to the diagnosis of certain diseases (Yan et
al.,2015;Wyokinski and Szcszepocka,2016.) For instance, the P-LCR was proven higher in
patients with chronic myeloid leukemia than patients with reactive thrombocytosis, essential
thrombocythemia, or polycythemia vera (Kabumatori, 2001).
2.7.2 Plateletcrit (PCT)
This measures total platelet mass as a percentage of volume occupied in the blood. The normal
range for PCT is 0.22–0.24% (Budak et al., 2016; Gao et al., 2014). Plateletcrit is an indicator
of platelet activity in blood, low plateletcrit reflects low platelet activity, it seems to play an
effective screening role in detecting platelet quantitative abnormalities (Baig, 2015). The PCT is
nonlinearly correlated to the platelet count and indicates comparable clinical implication (Gao et
al., 2014; Tang et al.,(2015) investigated PCTs potential as a novel biomarker of active Crohn's
disease in patients with low high sensitivity C-reactive protein (hs-CRP).
2.8 Platelet indices as diagnostic and prognostic markers
Platelet indices are currently under thorough investigation to be applied as potential novel
biomarkers in terms of diagnostics and prognosis in various, both acute and chronic diseases. PI
can be measured inexpensively and are accessible at hand during routine blood counts (Tang et
al., 2015) Despite many attempts to establish both significant and applicable clinical
correlations, these parameters are still not used extensively mainly because of the
methodological problems in terms of standardization and determination of reference values

24
(Giovanetti et al.,2011). Simultaneous measurement of all of the platelet indices will provide us
a valid instrument for measuring disease severity and an insight into the potential etiology that
resulted in platelets’ indices changes. Platelet volume heterogeneity occurs during its production
and increases MPV and PDW comparatively, suggesting that bone marrow produces platelets
and rapidly releases them into circulations (Sachdev et al., 2014) A simultaneous reduction of
platelet count and PCT indicates that platelets have been excessively consumed ( Zhang et
al.,2015). Platelets play an important role in inflammation, and recently, several additional
functions for platelets in the process of inflammation were defined. A substantial number of
studies have demonstrated crucial roles for platelets in the pathogenesis of various inflammatory
clinical conditions where inflammation is important (Thachil,2015). Numerous research groups
have found a relationship between the changes in platelet indices and the activation of the
coagulation system, severe infection, trauma, systemic inflammatory reaction syndrome, and
thrombotic diseases (Thachil,2015). Platelet indices have been shown to have diagnostic value in
certain inflammatory diseases, such as inflammatory bowel diseases, rheumatoid arthritis,
ankylosing spondylitis, ulcerative colitis, and atherosclerosis (Margetic, 2012; Purnak et
al.,2011;Ozturk et al., 2013;Kim,2011; Kisacik at al.,2008; Takeyama et al., 2015). Since these
systems are closely linked, septic patients are observed to have low platelet count due to
production of many cytokines, endothelial damage and bone marrow suppression. In patients
with septic shock, the rise in MPV, and to a lesser extent an increase in P-LCR and PDW,
indicates a worse prognosis (Margetic,2012 ;Kim et al.,2015; Gao et al., 2014). In the emergency
department, surgeons frequently use CBC to determine inflammatory pathologies and as part of
routine preoperative assessment. Platelet indices especially MPV, may be a simple way to
provide valuable information during routine blood counts without increasing the cost of
diagnosis or differentiating non-traumatic abdominal surgery patients.

25
Table 2.2 Platelet indices with their normal range and their diagnostic and prognostic
value in selected conditions.
Platelet Indices Platelet Indices Conditions with platelet indices Conditions with platelet
(PI) (PI) above normal range indices below normal
range

MPV 7.2-11.7 Fl Immune thrombocytopenia -Non-complicated acute

purpura (ITP) appendicitis
Diabetes mellitus – DM Related
retinopathy and nephropathy –
Skeptic shock
Heart disease malignant tumors
complicated acute appendicitis
PDW 8.3-56.6% D-M related retinopathy and Non- malignant tumors
nephropathy
ST-elevation myocardial
infarction
Acute cholecystitis
P-LCR 15-35% -Immune thrombocytopenia Myeloid insufficiency
purpura (ITP)
DM related retinopathy and
nephropathy
PCT 0.22-0.24% Active Crohn’s Disease with low Immune
hs-CRP thrombocytopenia
purpura (ITP)

(Pogorzelska et al., 2020)

26
 CHAPTER THREE
METHODOLOGY
3.0 Materials Used
The materials used for the project work are;
 Plastic cages
 Feed
 Feeders
 Hand gloves
 Distilled water
 Cannula
 Weighing scale
 Sensitive weighing scale
 Feeding plates
 Citrus lemon
 Wood shavings
 Syringe (2ml and 5ml)
 Feeding plates
 Drinkers
Chemical used
 Lead acetate
 Acetic acid
3.1 Animal Procurement and Maintenance
Forty (40) adult male Wistar rats, weighing 200 ± 20g, were utilized in this study. They were
procured from the animal facility at the Department of Physiology, Ladoke Akintola University
of Technology, Ogbomoso. The rats underwent a two-week acclimatization period within the
animal facility before the commencement of the research.
The rats were housed in spacious plastic cages furnished with dry wood shavings as bedding
material. They were kept under normal environmental conditions, following a 12-hour light-dark
cycle. Throughout the course of the research, the rats had unrestricted access to standard
pelletized rat feed and water of good quality source ad libitum, ensuring their comfort and well-

27
being. The bedding in each cage was changed daily to maintain hygienic conditions and prevent
disease or contamination.
3.2 Experimental Design
Fourty male rat (40), were distributed into eight (8) groups with each group containing five (5)
animals. Each experimental groups were given different doses of administration through oral
gavage and some through rectal route. The selection of experimental doses was based on
previous researches as well as literature data (Ige and Adekola, 2021). The different treatment
was applied as follows:
TABLE 3.1: illustrates the grouping of the rats
Group 1 (CONTROL) They were given feeds and water daily for twenty one (21) days.

Group 2 (Citrus limon juice) The rats received 7.5ml/Kg of Citrus limon extract orally for
twenty one (21) days.

Group 3 (Lead Acetate) The rats received 60mg/Kg of lead acetate orally for twenty one
(21) days.

Group 4 (Colitis) On the 15th day, the rats underwent a fasting period, and from
the 16th to the 18th day, they were induced with 2% acetic acid
via intra-rectal administration at a dosage of 2 mL per 100 g of
body weight.
Group 5 (Citrus limon juice + The rats received an oral dosage of 7.5 mL per kilogram of
Colitis) Citrus limon extract for a duration of twenty-one (21) days. On
the 15th day, the rats underwent a fasting period, and from the
16th to the 18th day, they were induced with 2% acetic acid via
intra-rectal administration at a dosage of 2 mL per 100 g of body
weight.
Group 6 (Lead Acetate + Colitis) The rats received an oral dosage of 60 mg per kilogram of lead
acetate for a duration of twenty-one (21) days. On the 15th day,
the rats underwent a fasting period, and from the 16th to the 18th
day, they were induced with 2% acetic acid via intra-rectal
administration at a dosage of 2 mL per 100 g of body weight.
Group 7 (Citrus limon juice + The rats were receiving 7.5ml/Kg of Citrus limon extract orally
Lead Acetate ) for twenty one (21) days and They were receiving 60mg/Kg of
lead acetate orally for twenty one (21) days.

Group 8 (Citrus limon juice + The rats were administered 7.5 mL per kilogram of Citrus limon
Lead Acetate + Colitis) extract orally for a duration of twenty-one (21) days.
Additionally, they received 60 mg per kilogram of lead acetate
orally for the same twenty-one (21) day period. On the 15th day,
the rats underwent a fasting period, and from the 16th to the 18th
day, they were induced with 2% acetic acid via intra-rectal

28
 administration at a dosage of 2 mL per 100 g of body weight.
3.3 Procurement Of Citrus Lemon
CITRUS LEMON (Citrus limon) were purchased from Waso market in Ogbomoso, Oyo State,
Nigeria.
3.4 Preparation of Citrus Lemon
The Citrus Lemon Juice was extracted daily by squeezing of the citrus lemon fruit. The rats were
given the liquid extract orally according to their Body Weight using 7.5ml/Kg
3.5 Preparation of Lead Acetate
In preparing the Lead Acetate, 15g of Lead Acetate was diluted in 250ml of Distilled
water. The animals were given lead acetate orally according to their body sizes with the dosage
of 60mgPb/Kg.
3.6 Preparation Of 6% Acetic Acid
A 2% acetic acid solution was prepared by the dilution method. Initially, 98 mL of distilled water
was measured using a measuring cylinder. Then, 2 mL of 100% acetic acid was added to the
water. Additional distilled water was incrementally added to the mixture until it reached a total
volume of 100 mL. The resulting mixture was thoroughly stirred to ensure uniformity.
3.7 Induction of Colitis
Acetic acid induced colitis is commonly employed and easily inducible model (Sasaki et
al.,2000). Acetic acid-induced colitis is a model of IBD that bears close resemblance to human
IBD in terms of pathogenesis, histopathological features and inflammatory mediator profile
(Gonzalez et al.,1999, Hartmann et al.,2012). The animal is fasted for 24 hours before induction,
but has free access to water. Before induction animals will undergo rectal flushing of which 2ml
of distilled water is passed into the animal intra rectally, holding the animal in a trendbulg
position for about 50sec to avoid reflux. The rrectal flushing is done to make sure there is no
faeces in the passage way to the colon so that the chemical induced intra-rectally could reach the
targeted organ which is the colon. Then Acetic Acid is induced intra rectally using 2ml/100g,
also holding in a trendbulg position for about 50sec carefully to avoid reflux.
3.8 Method of Sacrifice
After a period of twenty-one (21) days following the administration of lead acetate, Citrus limon
extract, and 2% acetic acid for three days spanning from the 16th day to the 18th day, the
animals were euthanized using the cervical dislocation method. Following euthanasia, each rat

29
underwent dissection, and their blood was collected through cardiac puncture. The blood was
collected into ethylene diamine tetraacetic acid (EDTA) tubes and promptly mixed with an
anticoagulant.
PARAMETERS
3.8.1 Measurement Of Body Weight Changes
Weekly body weight of the rats for all the groups was weighed with the aid of a digital weighing
balance to assess weekly gain or weight loss during the experiment.
Body weight changes (g) = Final weight (g) – Initial weight (g)
3.8.2 Colitis Assessment
After the animals were sacrificed, the colons were excised, and the colonic tissues were
longitudinally cut open. They were then washed with chilled normal saline solution and
subsequently weighed. A significant portion of each rat's colonic tissue was homogenized in
phosphate buffer solution (PBS) and then centrifuged at a speed of 16,000 RPM at 4°C for 20
minutes. The resulting supernatant was stored at a temperature between 20°C and 4°C for
biochemical assays (Ige and Adio, 2020).
3.8. Colitis Stool Scoring
Stool scoring method of Ige et al, 2020 was used to access the stool consistency. 0 for Normal
stool, 1 for loose stool without Blood, 2 for Stool with visible Blood, 3 for Diarrhea with Blood.
3.9 Evaluation of Hematological Parameters
The following hematological parameters were examined; plateletet count,Mean platelet volume,
platelet distribution width and plateletcrit.
3.9.1 Flow cytometry
This is based on ejecting cells from nozzle at high speed in a fluid; each cell passes through
several laser beams so that optical different optical properties can be measured.
3.9.2 Florescent Dye
Biochemical properties were recently classified using Florescent dyes and labelling occurs using
florescent Dyes and a laser excites this florescent molecules and emit light at various
wavelength.
3.9.3 Electrical Impedance
A stream of cells passes through a small aperture across which an electrical current is applied.
each cell that passes through the electrical impedance can thus be counted and sized. Particles

30
such as blood cells are non conducted but are suspended in an electrically conductiv diluent. As
the dilute suspension of a cell is dream the aperture, the passage of each cell momentarily
increases the impedance of the path between the two electrodes that are located on the side of the
aperture which determine information about blood cell size, surface change, concentration of cell
and shape of cell.
3.9.4 Optical Scalar
In this stage, a single cell passes across a laser light beam into three stages
* Diffraction : Bending around corners
* Refraction : Bending due to changes in speed
* Reflection : light Rays turned back by cell obstruction.
Light scalar correlates to cell volume, sizes and provide information about cell structure, shape
reflectivity.

31
 CHAPTER FOUR
4.1.1 Effect of Citrus limon (lemon) juice extract on body weight changes in acetic acid -
induced ulcerative colitis in lead exposed male wistar rats
There was significant decrease in the body weight gain of colitis, lead, lead +colitis, Colitis +
limon, Lead + colitis + limon groups when compared to control. There was significant increase
in body weight gain of Lead + limon group when compared to control. There was significant
gain in body weight change of Lead + limon group when compared to lead. There was
significant increase in body weight gain of Lead + limon group when compared to limon. There
was significant decrease in the body weight gain of Colitis + limon, Lead + colitis + limon
groups when compared to limon.
4.1.2 Effect of Citrus limon (lemon) juice extract on rectal temperature in acetic acid -
induced ulcerative colitis in lead exposed male wistar rats
Day 1 There was a Significant increase in rectal temperature of Rectal temperature of Colitis+
citrus Limon group when compared to control. There was a significant Decrease in Rectal
temperature of lead , lead+ colitis, lead + citrus limon ,lead + colitis + citrus limon when
compared to control.
Day 3 There was Significant increase in Rectal temperature of Colitis ,lead+ colitis + citrus
limon groups compared to control.
Day 5 There was significant increase in Rectal temperature of lead, citrus limon, lead +
colitis,Colitis + Citrus limon, lead + colitis+ citrus limon groups when compared to control
Day 7 There was Significant increase in rectal temperature of lead + colitis compared to control.
There was Significant Decrease in Rectal temperature of lead, colitis ,citrus limon, colitis +
citrus limon ,lead + citrus limon, lead+ colitis+ citrus limon groups when compared to control.

32
Table 4.1. The body weight changes of effect of Citrus limon (lemon) juice extract on acetic
acid - induced ulcerative colitis on platelet indices in lead exposed male wistar rats
Groups Initial body weight Final body weight Body weight changes
Control 207.20 ± 8.74 213.00 ± 10.82 9.25 ± 3.30
Colitis 214.20± 12.81 210.06± 15.39 -8.75 ± 5.15 a
Lead 203.40± 20.97 201.00 ±16.88 - 2.40 ± 7.14 a
Limon 235.60± 5.94 247.20 ± 7.85 11.60 ±4.65
Lead + colitis 232.40± 15.63 211.40 ± 16.64 -25.00± 16.03 a
Lead + limon 207.60± 12.60 211.60 ± 21.15 -12.75 ± 2.45 a,c,d
Colitis + limon 196 ± 16.06 187.60 ±10.12 -16.00 ± 2.80 a,d
Lead + colitis + 210.20 ± 11.31 193.80 ±7.32 -16.40± 13.28 a,d
limon

a -significant (p>0.05) when compared to control

c -significant (p>0.05) when compared to lead
d -significant (p>0.05) when compared to limon

33
 38.5

38

37.5

37

36.5 Day 1
Day 3
Day 5
36 Day 7

35.5

35
l s s
tro liti ad on iti on on on
n Co Le lim col lim lim lim
Co us + iu
s
iu
s us
tri ad itr itr itr
Ci Le + c + c + c
s s
ad liti ol
iti
Le Co c
+
ad
Le

Fig 4.1 The rectal temperature of effect of Citrus limon (lemon) juice extract on platelet
indices in acetic acid – induced ulcerative colitis in lead exposed male wistar rats

34
4.2 Effect of Citrus limon (lemon) juice extract on platelet indices in acetic acid – induced
ulcerative colitis in lead exposed male wistar rats
4.2.1 Effect of Citrus limon (lemon) juice extract on Platelet count (PLT) in acetic acid -
induced ulcerative colitis in lead exposed male wistar rats
There was significant decrease in PLT of lead, citrus limon ,lead+ colitis,lead+citrus limon,lead+
colitis +citrus limon juice group when compared to control. There was significant decrease in
PLT of lead+colitis, lead+colitis+ Citrus limon compared to colitis. There was Significant
increase in PLT of lead+citrus limon, colitis+ citrus limon group when compared to lead. There
was a significant increase in PLT of lead+colitis+citrus limon when compared to lead. There was
a significant decrease in PLT of Lead+colitis+citrus Limon group when compared to Citrus
limon. There was a Significant decrease in PLT of Lead + colitis+ citrus group when compared
to Lead +colitis. There was a Significant decrease in PLT of Lead+colitis+ citrus compared to
Lead + Citrus Limon group. There was a Significant decrease in PLT of Lead+ colitis + Citrus
limon when compared to colitis + citrus Limon group
4.2.2 Effect of Citrus limon (lemon) juice extract on Mean platelet volume in acetic acid -
induced ulcerative colitis in lead exposed male wistar rats
There was no significance
4.2.3 Effect of Citrus limon (lemon) juice extract on Platelet distribution (PDW) width in
acetic acid - induced ulcerative colitis in lead exposed male wistar rats.
There was a Significant increase in PDW of Lead+ citrus Limon group compared to control.
There is significant decrease in PDW of Lead+ colitis + citrus limon group when compared to
colitis. There was a Significant increase in PDW of Colitis + citrus limon group compared to
citrus limon. There was a Significant decrease in PDW of lead + Colitis+ citrus limon group
when compared to colitis + citrus limon.
4.2.4 Effect of Citrus limon (lemon) juice extract on Platelet crit (PCT) in acetic acid -
induced ulcerative colitis in lead exposed male wistar rats
There was a Significant decrease in PCT of Lead + citrus Limon group when compared to
Control. There was a Significant decrease in PCT of Lead + colitis+ Citrus Limon compared to
Control. There was a Significant decrease in PCT of Lead + colitis + citrus compared to Colitis.
There was a significant decrease in PCT of Lead + colitis+ Citrus Limon compared to Lead .
There was a Significant decrease in PCT of lead + coltis+ citrus compared to citrus Limon juice.

35
There was a Significant decrease in PCT of lead + coltis + citrus Limon compared to Lead +
colitis. There was a Significant decrease in PCT of lead + colitis+ citrus Limon group when
compared to lead + citrus Limon. There was a Significant decrease in PCT of lead+ Colitis+
citrus juice group when compared to colitis + citrus limon.

36
Fig 4.2 The platelet count of effect of Citrus limon (lemon) juice extract on platelet indices
in acetic acid – induced ulcerative colitis in lead exposed male wistar rats
a – significant at (p < 0.05) compared to control
b – Significant at (p < 0.05) compared to colitis
c – significant at (p < 0.05) compared to lead
d – significant at (p < 0.05) compared to citrus limon juice
e – significant at (p < 0.05) compared to lead plus colitis
f – significant at (p < 0.05) compared to lead plus citrus limon juice
g – significant at (p < 0.05) compared to colitis plus citrus limon juice

37
Fig 4.3 The mean platelet volume of effect of Citrus limon (lemon) juice extract on platelet
indices in acetic acid – induced ulcerative colitis in lead exposed male wistar rats
No significance

38
Fig 4.4 The mean platelet distribution width of effect of Citrus limon (lemon) juice extract
on platelet indices in acetic acid – induced ulcerative colitis in lead exposed male wistar rats
a – significant at (p < 0.05) compared to control
b – significant at (p < 0.05) compared to colitis
c – significant at (p < 0.05) compared to citrus limon juice
d – significant at (p < 0.05) compared to colitis plus citrus limon juice

39
4.6 PCT

PCT

Fig 4.5 The plateletcrit of effect of Citrus limon (lemon) juice extract on platelet indices in
acetic acid – induced ulcerative colitis in lead exposed male wistar rats

a – significant at (p < 0.05) compared to control

b – Significant at (p < 0.05) compared to colitis
c – significant at (p < 0.05) compared to lead
d – significant at (p < 0.05) compared to citrus limon juice
e – significant at (p < 0.05) compared to lead plus colitis
f – significant at (p < 0.05) compared to lead plus citrus limon juice
g – significant at (p < 0.05) compared to colitis plus citrus limon juice

40
 CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION
5.1 Discussion
In this Study, Citrus limon significantly increased plateletet count which is in correlation with
previous study, Azra et al., (2013) which stated there was Significant increase in platelet count
by citrus limon, punica granatum combination may be beneficial in the management of dangi
fever suggesting the need to work on more different combination doses to obtain desired increase
in platelet count.
In this study, Lead significantly reduced platelet count,platelet distribution width and plateletcrit
and this is in correlation with a study conducted by Barman et al.,(2014) who stated that lead
exposure may impair coagulation functional through endothelial cell injury. Also,Damaged
activity of cells of the immune system, such as polymorphonuclear leukocytes, results in
decreasing immune activity (Kosnett, 2006).
In this Study, there's was also a significant decrease in plateletcrit count across groups induced
with colitis compared with control. Tang et al., (2015) investigated PCTs potential as a novel
biomarker of active Crohn's disease in patients with low high sensitivity C-reactive protein (hs-
CRP).
In this study, Colitis significantly reduced MPV level which is in correlation with the previous
study, Öztürk et al., (2013) which stated that MPV level in active Ulcerative colitis were lower
than normal population and inactive Ulcerative colitis and MPV can also be influenced by
inflammation.
In this study, rectal temperature significantly increases in temperature between day 3 of the
induction, which is in correlation with previous study, Tagne et al.,(2021) which stated that The
increase in rectal temperature observed in the negative control would result from the increase in
the temperature of the hypothalamic thermostat under the efect of pyrogens (Milton 1989). In
fact, intrarectal administration of acetic acid induces activation of the lymphoid system and
therefore of the paying plaques and GALTs, which are responsible for the signifcant production
of endogenous pyrogens and prostaglandins (Milton 1989).
5.2 Conclusion
These findings underscore the potential of citrus-derived compounds as novel therapeutic agents
for managing inflammatory bowel diseases and mitigating the adverse effects of environmental

41
toxins on gastrointestinal health. Further research in this area holds promise for the development
of effective interventions to improve clinical outcomes in patients with ulcerative colitis and
environmental toxin exposure.
5.3 Recommendation.
Despite the promising findings, this study has certain limitations that need consideration. Firstly,
the experimental design focused solely on male Wistar rats, limiting the generalizing of the
results to other populations. Future studies should explore the effects of citrus lemon juice
extract in diverse animal models and human subjects to establish more understanding.
Additionally, further studies are needed to find the underlying molecular pathways through
which citrus lemon juice extract exerts its therapeutic effects on Platelet indices in ulcerative
colitis and lead exposure.

42
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