THE OUTCOMES OF Ocimum gratissimum ON GLYCATED

HAEMOGLOBIN IN STREPTOZOTOCIN INDUCED DIABETIC MALE

WISTAR RATS

BY

IBRAHIM ISMAIL ABIOLA

MATRIC NUMBER: 171101022

A PROJECT SUBMITTED TO THE DEPARTMENT OF PHYSIOLOGY,

FACULTY OF BASIC MEDICAL SCIENCES, LAGOS STATE UNIVERSITY
COLLEGE OF MEDICINE

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD

OF BACHELOR OF SCIENCE (BS.c) DEGREE IN PHYSIOLOGY

JULY, 2022

I
 CERTIFICATION

This is to certify that IBRAHIM ISMAIL A. has satisfactorily completed the

requirement for research dissertation for the award of Bachelor of Science (B.Sc.)
degree in the department of physiology of the Lagos state university

….……………………………. ….…………………………….
Supervisor Head of Department
Prof. I.I Olatunji-Bello Dr. S.A Salami

II
DEDICATION

I dedicate this project to Almighty Allah, the Most Beneficent and the Most Merciful.

I also dedicate this to my parents and siblings for the financial and moral support

throughout my stay in the university and throughout this work.

III
ACKNOWLEDGEMENT

I would like to first of all give my special acknowledgement to my supervisor Prof.

Ibiyemi Olatunji-Bello for her guidance and mentorship. And to Dr. O.M Olumide, I

thank her for corrections, mentorship, accessibility, mental and academic support

towards this project. I am extremely grateful ma.

To the Head of Department, of the physiology department, Dr Salami for his guidance

throughout our course of study.

To Mr Murtala, thank you sir for your technical advice, guidance, support and

encouragement. Thank you for sacrificing your time for us.

I would also like to acknowledge my parents (Mr and Mrs Ibrahim), my siblings for

their relentless support as regards this project. I want to specially appreciate my

project partner Edun Fiyinoluwa and roommates Adetayo Ayodeji, Ojolola Damilola

and Oreagba AbdulFatah for the support and teamwork.

I appreciate all my classmates, we made it to the final lap, God bless you all.

IV
TABLE OF CONTENTS

Title Page.........................................................................................................................i

Approval Page................................................................................................................ii

Certification.................................................................................................................. iii

Acknowledgement.........................................................................................................iv

Table of Content.............................................................................................................v

List of Table.................................................................................................................. ix

List of Figures................................................................................................................ x

Abstract....................................................................................................................... xiii

CHAPTER ONE

1.0 Introduction.............................................................................................................. 1

1.1 Aim of study ............................................................................................................2

1.2 Justification of study ............................................................................................... 2

1.3 Objectives of study ..................................................................................................2

CHAPTER TWO

2.1 Diabetes Mellitus .....................................................................................................3

2.1.1 Prevalence of Diabetes ......................................................................................... 3

2.1.2 Types of Diabetes .................................................................................................3

2.1.3 Complications of Diabetes Mellitus ..................................................................... 3

V
2.1.4 Risk factors of Diabetes Mellitus ....................................................................... 11

2.1.5 Diagnosis of Diabetes Mellitus .......................................................................... 12

2.3 Anaemia .................................................................................................................14

2.3.1 Classification of Anaemia .................................................................................. 15

2.3.2 Anaemia and Diabetes ........................................................................................16

2.3.3 Prevalence of Anaemia in Diabetes ....................................................................19

2.4 Ocimum Gratissimum ............................................................................................20

2.4.1 Ethnopharmacology ............................................................................................21

2.4.2 Morphology ........................................................................................................ 22

2.4.3 Phytochemistry ...................................................................................................23

2.4.4 Pharmacological Properties ................................................................................25

2.5 Streptozotocin ........................................................................................................30

2.5.1 Diabetogenic Mechanism of STZ .......................................................................31

2.5.2 Effect of streptozotocin on various body organs ................................................34

2.5.3 Glibenclamide .................................................................................................... 36

CHAPTER THREE

3.0 Materials and methodology ................................................................................... 37

3.1 Plant Materials .......................................................................................................37

3.2 Ethical Approval ....................................................................................................37

3.3 Preparation of Ocimum Gratissimum aqueous leaf extract ................................... 37

3.4 Experimental Animal ............................................................................................ 38

VI
3.5 Experimental design .............................................................................................. 38

3.6 Body weight changes .............................................................................................39

3.7 Induction of diabetes in male wistar rats ...............................................................39

3.8 Collection of blood samples .................................................................................. 39

3.9 Organ Collection ................................................................................................... 40

3.10 Determination of blood glucose and glycated haemoglobin (HbA1c) level ....... 40

3.11 Determination of Complete Blood Count ........................................................... 40

CHAPTER FOUR

4.0 Result .....................................................................................................................41

4.1 Effect of Aqueous Extract of Ocimum gratissimum on body weight changes in

male Wistar rats ...........................................................................................................41

4.2 Effect of Aqueous Extract of Ocimum gratissimum on Glycated Haemoglobin in

male Wistar rats ...........................................................................................................43

4.3 Effect of Aqueous Extract of Ocimum gratissimum on Blood Glucose Level in

male Wistar rats ...........................................................................................................45

4.4 Effect of Aqueous Extract of Ocimum gratissimum on Red Blood Cell,

Haemoglobin Concentration, Packed Cell Volume in male Wistar rats ..................... 47

4.5 Effect of Aqueous Extract of Ocimum gratissimum on Total White Blood Cell

Count and Platelets in male Wistar rats .......................................................................49

4.6 Effect of Aqueous Extract of Ocimum gratissimum on Differential White Blood

Cells in male Wistar rats ............................................................................................. 51

VII
4.7 Effect of Aqueous Extract of Ocimum gratissimum on RBC Indices in male

Wistar rats ....................................................................................................................53

4.8 Effect of Aqueous Extract of Ocimum gratissimum on Platelets Indices in Male

Wistar Rats .................................................................................................................. 54

4.9 Effect of Aqueous Extract of Ocimum gratissimum on the relative weight of the

Pancreas, Kidney and Liver in male Wistar rat ...........................................................56

CHAPTER 5

5.0 Discussion ............................................................................................................. 58

5.1 Conclusion .............................................................................................................61

VIII
LIST OF TABLES

Table 1: The Effect of Aqueous Extract of Ocimum gratissimum on RBC Count, PCV

and Haemoglobin Concentration

Table 2: The Effect of Aqueous Extract of Ocimum gratissimum on Differential White

Blood Cells

Table 3: The Effect of Aqueous Extract of Ocimum gratissimum On RBC and

Platelets Indices

Table 4: The Effect of Aqueous Extract of Ocimum gratissimum on the Relative organ

weight on experimental animals.

IX
LIST OF FIGURES

Figure 1: Diagram showing the leaves of Ocimum gratissimum.................................... 24

Figure 2: Graph showing body weight changes between the initial and final weights of

the experimental rats ................................................................................................... 42

Figure 3: Graph showing effect of STZ induced diabetes and Aqueous Extract of

Ocimum gratissimum on Glycated Haemoglobin level in male Wistar rat .................44

Figure 4: Graph showing effect of Aqueous Extract of Ocimum gratissimum on Blood

glucose level ................................................................................................................ 46

Figure 5: Graph showing comparison of platelets count in different experimental group

.................................................................................................................................... 50

Figure 6: Graph showing ccomparison of total white blood cell (WBC) count in

different experimental groups...................................................................................... 50

X
APPENDIX

ADA - American Diabetes Association

AEOG - Aqueous Extract of Ocimum gratissimum

ANG II - Angiotensin II

ATP - Adenosine Triphosphate

CNS - Central Nervous System

CVD - Cardiovascular Disease

CVS - Cardiovascular System

DNA - Deoxyribonucleic Acid

DOCA - Deoxycorticosterone acetate

FBG - Fasting blood glucose

GLUT2 - Glucose Transporter 2

Hb - Haemoglobin

I.C.V - Intracerebroventricular

LADA - Latent autoimmune diabetes in adults

MAPK - Mitogens activated protein kinase

MCHC - Mean corpuscular hemoglobin concentration

MCV - Mean Corpuscular Volume

MPV - Mean Platelet Volume

NADH - Nicotinamide adenine dinucleotide

NF-kB - Nuclear factor kappa beta

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

NO - Nitric Oxide

OGTT - Oral glucose tolerance test

PDW - Platelet Distribution Width

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P–LCR - Platelet-Large Cell Ratio

RBC - Red blood cell

RDW - RBC distribution width

RDW-CV - RBC Distribution Width–Coefficient Of Variation

RDW-SD - RBC Distribution Width–Standard Deviation

RNS - Reactive nitrogen species

ROS - Reactive Oxygen Stress

STZ - Streptozotocin

WBC - White blood cell

XOD - Xanthine oxidase

XII
ABSTRACT

This study was aimed at investigating Outcomes of Ocimum gratissimum on glycated

haemoglobin in streptozotocin induced diabetic male Wistar rats.

Twenty-five adult male rats were selected into five groups. Control rats were group 1.

Group 2 rats were treated with 300 mg/kg Ocimum gratissimum leaf extract only.

Group 3 were given 60 mg/kg Streptozotocin alongside 300 mg/kg B.W Ocimum

gratissimum leaf extract. Diabetic control rats and diabetic rats treated with 5 mg/kg

B.W glibenclamide were group 4 and 5 respectively.

Streptozotocin (60 mg/kg) was given intraperitoneal, while leaf extracts were given

through oral gavage. Blood glucose level were monitored before streptozotocin

injection, 72 hours after injection and after 4 weeks of treatment. Body weights were

monitored weekly. After 4 weeks of treatment, the liver, pancreas and kidneys weight

and glycated haemoglobin level were measured.

The results show that body weight was significantly decreased in diabetic + Ocimum

and diabetic + glibenclamide group after two weeks of treatment compared to control.

After 4 weeks of treatment, body weight of diabetic + Ocimum and diabetic +

glibenclamide group was slightly increased compared to diabetic control group.

Although there was no significant difference in the weight when compared to diabetic

group.

The levels of blood glucose, glycated haemoglobin and platelet count were

significantly decreased in diabetic + Ocimum and diabetic + glibenclamide group

when compared with control. While the counts of red blood cell, packed cell volume,

neutrophils and haemoglobin concentration were significantly increased in the

diabetes and Ocimum compared to control.

XIII
In conclusion, results show that aqueous extract of Ocimum gratissimum has a

potential therapeutic effect of decreasing glycated haemoglobin level which is used as

an objective measure of glycaemic control that diminishes hyperglycaemia actions of

streptozotocin. Therefore, protecting the pancreatic islets against destruction from

diabetes mellitus.

XIV
 CHAPTER 1
1.0 Introduction

The Lamiaceae plant Ocimum gratissimum (OG), often known as Africa basil or

sweet basil, is also known in Nigeria as efinrin, Nehonwu, and ai daya ta guda by the

Yoruba, Igbo, and Hausa, respectively (Ayinla et al., 2011). Its extract has become

more well-known due to its effectiveness in treating a variety of illnesses. While leaf

oil has been demonstrated to have antiseptic, antibacterial, and antifungal properties,

crushed leaf juice is used to treat convulsions, stomach pain, and catarrh (Ezekwesili

et al., 2004). Its leaves have antimicrobial and grain preservative potentials, (Mann

2012) and used traditionally to treat diabetes mellitus (Bailey 1989).

Diabetes Mellitus is a group of metabolic diseases characterized by hyperglycemia

which is as result of defects in insulin secretion, insulin action, or both. (American

Diabetes Association, 2010). The eyes, kidneys, nerve, blood arteries, and heart are

particularly vulnerable to long-term damage, inflammation, malfunction, and failure

caused by the chronic hyperglycemia in diabetes that results from impaired insulin

secretion and action (American Diabetes Association, 2007).

Numerous groups have found that OG has the ability to lower blood sugar. (Aguiyi

2000). Furthermore, its antioxidant and hematological properties have been reported

in normal rats;(Aprioku 2008) In an in vitro investigation, this antioxidant activity

was discovered to be comparable to that of gallic acid and ascorbic acid

(Akinmoladun 2007).

The quantity of sugar in the blood is measured by glycated hemoglobin. Blood is a

tissue comprised of fluid plasma in which various created constituents are suspended.

The blood cells' relatively steady levels imply the existence of a regulatory feedback

1
system. The activity and impact of Ocimum gratissimum on glycated hemoglobin are

not supported by scientific research due to its widespread use. This study aims to

advance our understanding of the relationship between Ocimum gratissimum's effects

on glycated hemoglobin and hematological variables, particularly red blood cells,

hemoglobin concentration, platelet count, and packed cell volume, which are key

factors in anemia.

1.1 Aim of Study

This study is aimed at investigating the effect of Ocimum gratissimum leaf extract on

glycated haemoglobin level in streptozotocin-induced diabetic male Wistar rats.

1.2 Justification of Study

The use of Ocimum gratissimum has grown over the years, the action of Ocimum

gratissimum on the glycated haemoglobin and diabetes induced anaemia using

streptozotocin is not fully understood.

1.3 The Objective of the Study

This Study

1. Investigate the effect of STZ induced diabetes mellitus and Ocimum gratissimum

leaf extract treatment on the glycated haemoglobin in male Wistar rats

2. Determine the effect of Ocimum gratissimum on hematological properties

3. Determine the effect of Ocimum gratissimum on glucose level in male Wistar

rats.

4. Determine the effect of Ocimum gratissimum on body weight changes

2
 CHAPTER 2

2.0 LITERATURE REVIEW

2.1 Diabetes Mellitus

Diabetes Mellitus is a group of metabolic diseases characterized by hyperglycemia

which is as result of defects in insulin secretion, insulin action, or both (American

Diabetes Association, 2010). The chronic hyperglycemia in diabetes resulting from

insulin secretion and action dysfunction is associated with long term damage,

inflammation, dysfunction and failure of various organs, especially the eyes, kidneys,

nerve, blood vessels and heart (American Diabetes Association, 2007).

2.1.1 Prevalence of Diabetes Mellitus

Nigeria is known as Africa’s most populous country with approximately 201.6 million

inhabitants, therefore accounts for one-sixth of the continent’s total population

(Ogbera and Ekebegh 2014). In Nigeria, the prevalence of diabetes mellitus is

reported to be about 1.7% among adults aged 20 - 69 years (Uloko et al.,2018).

Worldwide, the prevalence of diabetes for age-groups was estimated to be 2.8% in the

year 2000 and is expected to increase to about 4.4% in 2030, which indicates the total

number of people with diabetes will increase from 171 million in 2000 to 366 million

in 2030 (Wild et al., 2004).

2.1.2 Types of Diabetes Mellitus

Type 1 Diabetes Mellitus

Type 1 diabetes occurs when the body does not produce insulin. It is often referred to

as insulin-dependent diabetes, juvenile diabetes or early onset diabetes. (Suresh

3
2016). This form of diabetes, accounts for only 5-10% of those with diabetes and

results from a cellular-mediated autoimmune destruction of the β-cells of the pancreas.

The rate of β-cells destruction varies in individuals, it is rapid some individuals

mainly infants and children and slow in some individuals mainly adults (American

Diabetes Association, 2010).

Type 1 diabetes is usually diagnosed in people before the age of 40 years, often in

childhood or early adulthood. Patients with type 1 diabetes usually require treatments

with insulin injections for the rest of their life. They must also ensure a balance in

blood-glucose level by carrying out blood test regularly accompanied with special

diets (Suresh 2016).

Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (formerly known as non–insulin-dependent diabetes or adult-

onset diabetes) accounts for about 90-95% of all cases of diabetes. In this form

diabetes, the response to insulin is declined and therefore, defined as insulin resistance

(Goyal & Jialal 2021). A condition characterized by inability of the body to produce

enough insulin for proper function or inability of body cells to utilize insulin produced

(Suresh 2016).

Majority of the patients with this type of diabetes are obese, and obesity is known to

cause some degree of insulin resistance. People who are not obese by traditional

weight criteria may have an increased in the percentage of body fat distribution

predominately in the abdominal region. In this kind of diabetes, ketoacidosis rarely

happens on its own; when it does, it typically happens in conjunction with the stress

of another sickness, like an infection. Because the hyperglycemia develops gradually

and is frequently not severe enough in the early stages for the patient to detect any of

4
the classic signs of diabetes, this type of diabetes commonly goes untreated for many

years. Nevertheless, such patients are at increased risk of developing macrovascular

and microvascular complications (American Diabetes Association, 2010). Patients

with many features of type 2 diabetes have some type 1 characteristics such as the

presence of islet cell autoantibodies are classified as distinct type of diabetes called

latent autoimmune diabetes in adults(LADA). People diagnosed with LADA do not

require insulin treatment (Kharroubi and Darwish 2015).

Gestational Diabetes Mellitus

Gestational diabetes affects female during pregnancy. During pregnancy some

females have very high blood glucose, therefore their body is unable to produce

enough insulin to transport all the glucose available into their cells (Suresh 2016).

According to the American Diabetes Association (ADA), Gestational Diabetes

complicates 7% of pregnancies. Women with gestational diabetes and their offspring

have an increased risk of developing type 2 diabetes mellitus in the future.

Gestational diabetes can be complicated by hypertension, preeclampsia and

hydraminos and may also lead to increased operative interventions. the fetus can have

increased weight and size (macrosomia) or congenital anomalies. Such newborns may

already have respiratory distress syndrome, which can lead to childhood and

adolescent obesity. Older age, obesity, excessive gestational weight gain, history of

congenital anomalies in previous children, or stillbirth, or a family history of diabetes

are risk factors for gestational diabetes (Goyal & Jialal 2021).

Other Types of Diabetes Mellitus

5
a. Genetic defects of the β cell: Different forms of diabetes are associated with

monogenetic defects in β cells function. This type of diabetes are commonly

characterized by onset of hyperglycemia at an early age (generally before the age

of 25 years). They are commonly referred to as maturity-onset diabetes of the

young (MODY) and are characterized by impaired insulin secretion with minimal

or no defects in insulin action (American Diabetes Association, 2010).

b. Genetic defects of insulin action: Some unusual causes of diabetes results from

genetically determined abnormalities that alters insulin action in the body. The

metabolic abnormalities in the mutation of insulin receptors range from

hyperinsulinemia and modest hyperglycemia to severe diabetes (American

Diabetes Association, 2010).

c. Diseases of the exocrine pancreas: Any process that injures the pancreas can

lead to diabetes. Acquired process that can injure the pancreas include;

pancreatitis, infection, trauma, pancreatectomy, and pancreatic carcinom. With

exception of that caused by cancer, damage to the pancreas must be extensive for

diabetes to occur; adenocarcinomas that involve only a small portion of the

pancreas have been associated with diabetes. This implies a mechanism other

than simple reduction in β cells mass (American Diabetes Association, 2010).

d. Drug- or chemical-induced diabetes: Many drugs can disrupt insulin secretion.

These drugs don’t cause diabetes themselves, but they precipitate diabetes in

individual with insulin resistance. In some situations, the order or relative

importance of -cell malfunction and insulin resistance are uncertain, making

classification difficult. Numerous medications and hormones can potentially

make insulin less effective. Examples include nicotinic acid and glucocorticoids

(American Diabetes Association, 2010).

6
2.1.3 Complications of Diabetes Mellitus

Diabetes is associated with a number of complications, ranging from acute metabolic

complications to chronic vascular complications. Acute metabolic complications are

associated with mortality included ketoacidosis from exceptionally hyperglycemia

and coma as a result of hypoglycemia (Forbes and Cooper 2013).

The chronic complications of diabetes can be broadly divided into mircovascular and

macrovascular complications. Microvascular complications include neuropathy,

nephropathy, and retinopathy, while macrovascular complications include

cardiovascular disease, stroke and peripheral artery disease (Papatheodorou et al.,

2018).

Nephropathy

Diabetic nephropathy is categorized as the major cause of end-stage renal failure in

western societies. Clinically nephropathy is characterized by the development of

proteinuria with a subsequent decrease in glomerular filtration rate, which progresses

over a long period of time, often over 10-20 years. Eventually if left untreated, the

resulting uremia is fatal. Importantly, kidney disease is also major risk factor for the

development of macrovascular complications such as cardiac arrest and strokes.

Once nephropathy has developed, blood pressure is frequently seen to increase, but

paradoxically, due to lower renal insulin clearance by the kidney, glycemic

management may improve in the short term. Given the diversity of cell types found

within the kidney and the different physiological functions of this organ, the

development and progression of nephropathy is exceedingly complex. Indeed, aside

from the filtration of toxins from the blood for excretion, it is difficult to pinpoint

7
which other functional aspects of the kidney are most affected by diabetes. These

include the release of hormones such as erythropoietin, activation of vitamin D, and

acute control of hypoglycemia, in addition to maintenance of fluid balance and blood

pressure via salt reabsorption. Certain biological changes brought on by high glucose

concentrations affect a variety of resident kidney cells, including endothelial cells,

smooth muscle cells, mesangial cells, podocytes, cells of the tubular and collecting

duct system, and inflammatory cells and myofibroblasts (Forbes and Cooper 2013).

Retinopathy

Diabetic retinopathy is characterized by a spectrum of lesions within the retina and is

the leading cause of blindness among adults aged 20–74 years. Vascular permeability

alterations, capillary microaneurysms, capillary degeneration, and an abnormal rate of

blood vessel growth are a few of these (neovascularization). The neural retina is also

dysfunctional with death of some cells, which alters retinal electrophysiology and

results in an inability to discriminate between colors. Clinically, the stages of

proliferative and nonproliferative diabetic retinopathy are distinguished. IEarly on,

hyperglycemia can result in intramural pericyte mortality and thickening of the

basement membrane, which affect the integrity of blood vessels within the retina and

affect vascular permeability and the blood-retinal barrier. Most persons do not

experience any visual impairment during this early stage of nonproliferative diabetic

retinopathy (NPDR).

Degeneration or occlusion of retinal capillaries are strongly associated with worsening

prognosis, which is most likely the result of ischemia followed by subsequent release

of angiogenic factors including those related to hypoxia. This progresses the disease

into the proliferative phase where neovascularization and accumulation of fluid within

the retina, termed macula edema, contribute to visual impairment. In more severe

8
cases, there can be bleeding with associated distorting of the retinal architecture

including development of a fibrovascular membrane which can subsequently lead to

retinal detachment (Forbes and Cooper 2013).

Neuropathy

More than half of all individuals with diabetes eventually develop neuropathy, with a

lifetime risk of one or more lower extremity amputations estimated in some

populations to be up to 15%. Diabetic neuropathy is a syndrome which encompasses

both the somatic and autonomic divisions of the peripheral nervous system. There is,

however, a growing appreciation that damage to the spinal cord and the higher central

nervous system can also occur and that neuropathy is a major factor in the impaired

wound healing, erectile dysfunction, and cardiovascular dysfunction seen in diabetes.

The classic clinical definition of neuropathy disease progression was the emergence

of vascular anomalies, such as capillary basement membrane thickness and

endothelial hyperplasia, followed by a decrease in oxygen tension and hypoxia. In the

therapeutic setting, nerve conduction velocities are improved by renin-angiotensin

system inhibitors and 1-antagonists, which is thought to be due to an increase in

neuronal blood flow. Diabetes-related nerve fiber degeneration causes advanced

neuropathy, which is characterized by increased vibration and heat threshold

sensitivities that eventually lead to sensory perception loss. Hyperalgesia, paresthesias,

and allodynia also occur in a proportion of patients, with pain evident in 40–50% of

those with diabetic neuropathy. Pain is also seen in some diabetic individuals without

clinical evidence of neuropathy (∼ 10–20%), which can seriously impede quality of

life (Forbes and Cooper 2013).

Cardiovascular Disease

9
There is increased risk of cardiovascular disease (CVD) in diabetes, such that an

individual with diabetes has a risk of myocardial infarction equivalent to that of

nondiabetic individuals who have previously had a myocardial infarction. CVD

accounts for more than half of the mortality seen in the diabetic population, and

diabetes equates to an approximately threefold increased risk of myocardial infarction

compared with the general population. In type 1 diabetes, it is not common to see

progression to CVD without an impairment in kidney function. In type 2 diabetes,

kidney disease remains a major risk factor for premature CVD, in addition to

dyslipidemia, poor glycemic control, and persistent elevations in blood pressure.

Premature atherosclerosis, which manifests as myocardial infarction and stroke as

well as reduced heart function, primarily diastolic dysfunction, are cardiovascular

illnesses associated with diabetes. Intensive treatment approaches for diabetics at risk

for CVD include stringent glucose control and the prescription of blood pressure-

lowering medications., such as aspirin.

Atherosclerosis is a complex process involving numerous cell types and important

cell-to-cell interactions that ultimately lead to progression from the “fatty streak” to

formation of more complex atherosclerotic plaques. These complex atherosclerotic

plaques may then destabilize and rupture, resulting in myocardial infarction, unstable

angina, or strokes. The precise initiating event is unknown; however, dysfunction

within the endothelium is thought to be an important early contributor. The

endothelium is crucial for maintenance of vascular homeostasis, ensuring that a

balance remains between vasoactive factors controlling its permeability, adhesiveness,

and integrity such as ANG II and nitric oxide, but this balance appears compromised

by diabetes. Atherogenesis is triggered by local abnormalities, and immune cells like

10
macrophages and T cells might adhere to the vessel wall as a result. Low-density

lipoprotein begins to travel into the subendothelial area as a result, causing the

production of foam cells and fatty streaks, which are frequently observed at turbulent

flow sites such bifurcations, branches, and curves. In the end, smooth muscle cell

proliferation and matrix deposition, frequently accompanied by associated necrosis,

lead to the formation of a complex atherosclerotic plaque. This plaque may obstruct

the blood vessel at the site of formation, such as in the coronary or femoral circulation,

or it may develop into an embolus that obstructs blood vessels at distant sites,

frequently starting from within carotid vessels and reaching the cerebral circulation.

(Forbes and Cooper 2013).

2.1.4 Risk factors of diabetes mellitus

Biomakers, lifestyle and environmental factors, dietary factors, medical history and

psychosocial factors are all risk factors of type 2 diabetes mellitus. Previous studies

have identified several risk factors for Type 2 diabetes such as age, body mass index

(BMI), waist circumference, sex, ethnicity, low physical activity, smoking, diet

including low amount of fiber and high amount of saturated fat, ethnicity, family

history of diabetes, history gestational diabetes mellitus, elevated blood pressure,

dyslipidemia and different drug treatments (diuretics, unselected b-blockers, statins)

(Chatterjee et al., 2018).

2.1.5 Diagnosis of diabetes mellitus

For decades, diagnosis of diabetes has based on fasting blood glucose(FBG), or the

75-g oral glucose tolerance test (OGTT). In 1997, the first Expert Committee on the

Diagnosis and Classification of Diabetes Mellitus reviewed the diagnostic criteria, by

11
utilizing the observed association between fasting blood glucose levels and the

presence of diabetic retinopathy as major factor to detect threshold glucose level. The

Committee looked at data from three cross-sectional epidemiologic studies that

measured glycemia as FPG, 2-h PG, and A1C and evaluated retinopathy using fundus

photography or direct ophthalmoscopy. These studies showed glycemic ranges below

which retinopathy was not commonly occurring and above which it grew in an

apparently linear manner. These investigations verified the long-standing diagnostic

2-h PG value of 200 mg/dl (11.1 mmol/l) and contributed to the development of a new

diagnostic cut point of 126 mg/dl (7.0 mmol/l) for FPG. Glycated haemoglobin is a

widely used marker of chronic glycemia, reflecting average blood glucose levels over

a 2- to 3-month period of time. The test plays an improtant role to help in the

management of the patient with diabetes, since it correlates well with both

microvascular and, to a lesser extent, macrovascular complications and is widely used

as the standard biomarker for the adequacy of glycemic management (American

Diabetes Association, 2010).

Fasting Plasma Glucose Test

Fasting plasma glucose test is used to measure blood glucose at a single point in time.

To achieve the most reliable results, it is best to perform the test in the morning, after

about 8 hours of fasting over the night (NIDDK | National Institute of Diabetes and

Digestive and Kidney Diseases 2022).

Glucose Tolerance Test

A glucose tolerance test is used to measure an individual’s ability to handle glucose

load. This test helps to show how a person can metabolize a standardized measured

12
amount of glucose. The results obtained from this test can therefore be classified as

normal, abnormal or impaired. Glucose tolerance test is important in the diagnosis of

type 1 diabetes, type 2 diabetes and gestational diabetes.

To perform glucose tolerance test, patients should be instructed to consume a normal

diet of at least 150grams of carbohydrates for at least 3 days before the test. On the

day of the test, the patient not have eaten anything prior to the time the test would be

carried out. A fasting sample is taken either by phlebotomy or intravenous access to

establish a baseline glucose level. Then, the patient will drink the glucose (comes in 2

formulas, either 75 grams or 100 grams). The amount is dosed by weight in pediatric

patients at 1.75 g/kg of body weight, while the maximum dose for all nonpregnant

patients is 75 grams.

Patients are asked to fast throughout the test except for drinking the glucose. Samples

are then taken at various timepoints ending at either 60 or 120 minutes post-

consumption of glucose. Throughout the test, patients should remain inactive, and

excess hydration with water should be discouraged as these can impact the results of

the test (Eyth et al., 2022).

1. Normal Results for Type 1 Diabetes or Type 2 Diabetes

a. Fasting glucose level 60 to 100 mg/dL

b. One-hour glucose level less than 200 mg/dL

c. Two-hour glucose level less than 140 mg/dL

2. Impaired Results for Type 1 Diabetes or Type 2 Diabetes

a. Fasting glucose level: 100 to 125 mg/dL

13
b. Two-hour glucose level 140 to 200 mg/dL

3. Abnormal (Diagnostic) Results for Type 1 Diabetes or Type 2 Diabetes

a. Fasting glucose level greater than 126 mg/dL

b. Two-hour glucose level greater than 200 mg/dL

4. Normal Results for Gestational Diabetes

a. Fasting glucose level less than 90 mg/dL

b. One-hour glucose level less than 130 to 140 mg/dL

c. Two-hour glucose level less than 120 mg/dL

5. Abnormal Results for Gestational Diabetes

a. Fasting glucose level greater than 95 mg/dL

b. One-hour glucose level greater than 140 mg/dL

c. Two-hour glucose level greater than 120 mg/dL

2.2 Glycated Haemoglobin(HbA1c)

Glycated haemoglobin can be used in the diagnosis of diabetes, it provides the exact

quality assurance tests are in place and assays are standardized to criteria aligned to

the international reference values, and there are no conditions present which preclude

its accurate measurement. About 40 years ago, glycated haemoglobin was identified

as “unsual” haemoglobin in diabetic patients. After this discovery, quite a number of

small studies were carried out in relations to glucose measurements, this eventually

brought about the idea that HbA1c is important and can be used as an objective

measure of glycemic control (World Health Organization, 2011).

14
In diagnosis of diabetes and montoring one of the primary tests conducted is glycated

haemoglobin test. This test is a measure of β-N-(1-deoxy)-fructosyl hemoglobin that

is contained in the red blood cell which is glycated in varying amounts depending on

blood glucose levels over time (Canadian Agency for Drugs and Technologies in

Health, 2014).

The protein found within the red blood cell has an expected life span of 120 days. In

monitoring and diagnosis of HbA1c analysis is much easier for patients than other

test carried out for blood glucose testing as it does not require prolonged period of

dietary restriction. In addition, this test is rapildy completed. It only requires blood

sample as compared to oral glucose tolerance test that requires 3 days strict diet

before the test is carried out. Additionally, HbA1c testing has no overt requirement

from the patient and is not dependent on any sort of prandial status which means that

it may be taken at any time day or night. Finally, glucose testing must be sent to the

laboratory for measurement within thirty to sixty minutes from the time it was

sampled. This is due to the red blood cells continuing to metabolize glucose post-

withdrawal at a rate of 7% per hour. The protein analyzed in HbA1c testing is capable

of remaining stable for over a week if kept refrigerated (Canadian Agency for Drugs

and Technologies in Health, 2014).

2.3 Anaemia

Anaemia can be described as the decrease in haemtocrit, or haemoglobin or red blood

cell count. Anaemia is not diagnosed, but it is a presentation of an underlying

condition (Turner et al., 2022). Therefore, anaemia is a condition in which the red

blood cell count and/or haemoglobin concentration is below normal and inadequate to

meet an individual’s physiological needs (Chaparro, and Suchdev, 2019).

15
Anaemia is known to roughly affects one-third of the world’s population. Anaemia

can be associated with increased mortality and morbidity in women and children and

poor birth outcomes, it is also associated with a decrease in productivity in adults and

impaired cognitive and behavioral development in children. Anaemia is known to be

prevalent in Preschool children and women of reproductive age (Chaparro, and

Suchdev, 2019).

2.3.1 Classification of Anaemia

Anaemia can be classified in 3 ways; Red cell morphology, Pathogenesis and

Etiological classification.

All are important diagnosis. Pathogenic mechanism involved in the production of

anaemia are very simple: loss of erythrocyte and inadequate production which results

from loss of blood by bleeding or hemolysis. Pathogenic mechanism can be classified

into two classes: Hypo-regenerative and Regenerative (Moreno Chulilla et al., 2009).

Based on Red cell morphology, anaemia can be classified into; microcytic,

normocytic or macrocytic, depending on mean corpuscular volume (MCV) (Moreno

Chulilla et al., 2009).

On the basis of etiology, anaemia can be divided into 5 types: Hemorrhagic anaemia,

hemolytic anaemia, nutrition deficiency anaemia, aplastic anaemia and anaemia of

chronic diseases (Sembulingam and Sembulingam, 2016).

Pathogenic Classification

Regenerative Anaemia: This is characterized by the increase in erythropoietin

production in response to a decrease in haemoglobin concentration, and it eventually

results in loss of erythrocytes, due to bleeding or hemolysis. In both cases, there is an

16
increase in reticulocytes. The blood loss can be intense which can lead to severe

decrease in haematocrit and obvious clinical signs or it can be of small intensity but

chronic which results in a progressive decrease in haematocrit and mean corpuscular

volume, that can be unnoticed (Moreno Chulilla et al., 2009).

Hypo-regenerative Anaemia: This is as a result of alteration of bone marrow

progenitor cells, which can be located at different stages of differentiation and

maturation. Impairment of pluripotent stem cells usually produces pancytopenia

(anaemia, leukopenia and thrombocytopenia). Pancytopenia may be caused by

intrinsic [bone marrow aplasia, leukemia, myelodysplastic syndrome (MDS) or

myelofibrosis] or extrinsic (metastasis, Gaucher disease and other thesaurismosis,

tuberculosis, histoplasmosis, viral and parasitic infections). All of them are capable of

displacing normal hematopoiesis or changing the microenvironment necessary for

regeneration, differentiation and proliferation of stem cells (Moreno Chulilla et al.,

2009).

Morphological Classification

Morphological classification depends upon the size and color of RBC. Size of RBC is

determined by mean corpuscular volume (MCV). Color is determined by mean

corpuscular hemoglobin concentration (MCHC)

Mircocytic Anaemia: This is characterised by the production of red blood cells that

are smaller than normal. This small size is as a result of decrease in haemoglobin

production and haemoglobin is the major constituent of the red blood cell. The causes

of microcytic is as a result of lack of globin product (thalassemia), restricted iron

delivery to the heme group (iron deficiency anaemia), and defects in the synthesis of

the heme group(sideroblastic anaemia) (DeLoughery, 2014).

17
Normocytic Anaemia: In normocytic normochromic anaemia, the size of circulating

red blood cells are normal (normocytic) and the colour of the red blood cell are

normal(normochromic). This type of anaemia differs from other forms of anaemia

because the average size and haemoglobin content of the RBCs are typically within

normal limits. It usually appear normal under microscopic examination, though in

some cases, there may be variations in size and shape that equalize one another,

resulting in average values within the normal range. Most of the normochromic,

normocytic anemias are resulting from other diseases; a minority reflects a primary

disorder of the blood. This could be brought on by acute blood loss, polymyalgia

rheumatica, renal failure, endocrine failure (hypothyroidism, hypopituitarism),

marrow failure (pure red-cell aplasia, aplastic anemia, infiltration), anemia of chronic

disease (inflammation, neoplasia), or anemia of chronic disease (inflammation,

neoplasia). (Yilmaz and Shaikh, 2021).

Macrocytic Anaemia: When there is anemia (hemoglobin less than 12 g/dL or

hematocrit less than 36% in non-pregnant females, hemoglobin less than 11 g/dL in

pregnant females, or hemoglobin less than 13 g/dL or Hct less than 41% in males),

there is a condition known as macrocytosis, which is defined as mean corpuscular

volume (MCV) greater than 100 fL. Megaloblastic anemia and non-megaloblastic

anemia are the two subtypes of this anemia.

Megaloblastic anaemia: Folic acid and vitamin B12 are to blame for this. Folic acid

deficiencies are caused by decreased intake (due to malnutrition and alcohol use),

increased intake (due to hemolysis or pregnancy), and malabsorption (familial, gastric

bypass, or medications like cholestyramine or metformin). Atrophic gastritis, whether

autoimmune or non-autoimmune from Helicobacter pylori or Zollinger-Ellison

18
syndrome, Diphyllobothrium tapeworm infection, gastric bypass, ileal resection, or

the presence of antagonists are all signs of vitamin B12 deficiency (nitrous oxide).

Non-megaloblastic anaemia, occurs in various ways. Benign conditions are alcohol

consumption (RBC toxicity) hereditary spherocytosis (impaired volume regulation

increases red cell size), hypothyroidism and liver disease (due to lipid deposition in

the cell membrane), and marked reticulocytosis from states of excess RBC

consumption such as hemolysis or turnover in pregnancy or primary bone marrow

disease (reticulocytes are larger than the average RBCs) (Moore and Adil, 2021).

2.3.2 Anaemia and Diabetes Mellitus

Patients with type 2 diabetes are twice the chances vulnerable to anaemia. Bosman et

al (2001) identified anemia as a risk factor for cardiovascular and end-stage renal

diseases in diabetic patients. Keane and Lyle (2003) further proved that reduced

hemoglobin (Hb) level identifies diabetic patients at increased risk for hospitalization

and premature death. Regardless of these facts, Anaemia is not noticeable in 25% of

diabetic patients. It was observed in recent studies that, the incidence of anaemia is

mostly associated with the condition of renal insufficiency. Thus, diabetic patients

have a greater degree of anemia for their level of renal impairment than non-diabetic

patients presenting with other causes of renal failure (AlDallal and Jena, 2018).

Diabetic patients with renal insufficiency in some studies have shown that these

patients are at a higher risk of developing anemia than normal diabetics as the ability

of their kidneys to produce erythropoietin reduces. Also, the hormone responsible for

the production of red blood cells is affected by diabetic nephropathy resulting in

anemia. Subjects with diabetes also have some nutritional deficiencies for

19
cyanocobalamin, folate and iron which may result in different types of anemia, such

as megaloblastic anaemia (AlDallal and Jena, 2018).

2.3.3 Prevalence of Anaemia in Diabetes

People with diabetes are more likely to experience the effects of renal impairment

than patients without diabetes. Anaemia commonly occurs in the early stages of

diabetes nephropathy and is typically more severe than in cases of non-diabetic renal

disorders. Endocrinologists are frequently the primary line of care for patients with

diabetic nephropathy because there is typically no overt renal impairment in these

patients, and they are frequently unaware of the crucial need of screening for anemia

in this population. As a result, anemia is frequently ignored or left untreated (Deray et

al., 2004). Socio—demographic characteristics and glycemic index are statistically

correlated with anemia among type II diabetic patients. An increase in evidence

suggests that anemia in the diabetic population, whether type I or type II, is a potent

and independent predictor of the increased risk for macro-vascular and micro-vascular

complications of diabetes (Solomon et al., 2022).

Anemia is another common condition among those with type 2 diabetes, with

prevalence rates ranging from 11% to 55%, with Saudi Arabia reporting the greatest

frequency. the prevalence of anemia among those with type 2 diabetes, which was

recently reported to be 41.4% in Cameroon. Patients with type 2 diabetes were found

to have a similar prevalence (46.4%) in Trinidad and Tobago. The prevalence of type

2 diabetes in Ethiopians was substantially lower, at 19%. In Benin, a southern

Nigerian city, people with type 2 diabetes had a substantially lower prevalence of

20
15.3%. A significant risk factor for anemia is only including patients with renal illness

who have "normal" blood creatinine levels (133 mol/L) (Awofisoye et al., 2019).

2.4 Ocimum gratissimum

Ocimum gratissimum is an herbaceous plant, which belongs to the family of Labiatae,

this plant is indigenous to tropical regions such as West Africa and India. In Nigeria,

it is found in the Savannah and coastal areas. It is given various names in different

parts of the world. In the southern part of Nigeria, it is called “effinrin” by the

Yorubas, it is called “Ahuji” by the Igbos. In the nothern part of Nigeria it is called

“Daidoya” by the Hausas (Prabhu et al., 2009).

2.4.1 Ethnopharmacology

Traditional Uses

Ocimum gratissimum is a plant with wide range of traditional uses and it is used to

cure quite some number ailments (Pandey, 2017b). In North east of Brazil, it is used

for medicinal, culinary and condiment purpose. It has also been proven that the leaves

and flower of this plant are rich in essential oils so it is used in the preparation of teas

and infusion (Prabhu et al., 2009). In traditional medicine practice, it is used in

treatment of diarrhoea as a febrifuge and it is also part of the components used in anti-

malaria remedies, mosquitoes/insects repellant, stomachic and general tonic,

antiseptic, in wound dressing, skin infections, conjunctivitis, and bronchitis (Okoli, C.

O. et al., 2010). In the south eastern part of Nigeria, the Igbos uses O. gratissimum in

the management of baby’s cord to keep wound surface sterile and free from infections

and it is used in the treatment of fever, cold, catarrh and fungal infections (Prabhu et

al., 2009). The Ighala community in Kogi state, Nigeria uses the leaves and root of

21
this plant in the treatment of diabetes, gastrointestinal problems and gonorrhea. In

South West of Nigeria, the plant leaves are used in the treatment of sexually

transmitted diseases, while in some regions the water and ethanol extracts of the plant

are used for several microbial and non-microbial associated diseases (Pandey, 2017b).

The infusion of O. gratissimum leaves is used as pulmonary antisepticum,

antitussivum and antispasmodicum (Prabhu et al., 2009).

Alternative and Complementary Medicinal Uses

Out of various species of Ocimum, O. gratissimum is clinically relevant and used

extensively throughout the world. Preparation of leaf essential oil of O. gratissimum

(Ocimum oil) have been applied in a variety of bases as topical anti-septics and for

use in the treatment of minor wounds, boils and pimples (Prabhu et al., 2009).

Ijeh I.I. (2005) reports that O. gratissimum and Xylopia aethiopica in combination are

used in the preparation of potions and teas for women during peuperium (Prabhu et al.,

2009).

2.4.2 Morphology

Ocimum gratissimum is a shrub with a height of approximately 1.9m, it consists of

stems that are branched. The leaves measure up to 10 by 5cm in size, these leaves are

ovate to ovate-lanceolate, sub-acuminate to acuminate at apex, cuneate and decurrent

at base with a coarsely crenate, serrate margin, pubscent and dotted on both sides

(Prabhu et al., 2009).

The cross section of hexagonal stem is divided into parts; the bark and the central

cylinder. The bark of plant is thin and has three primary tissues(epidermis, cortical

parenchyma and collenchyma) (Rawat et al., 2016).

22
The leaves shows the presence of glandular trichomes. The presence of stromata are

rare and absent on upper surface, but they often present in the lower surface. The long

trichomes up to 6-celled are present on the margins mostly, while the ordinary

trichomes are few. The 2-celled trichomes which are the short ones are present in the

lamina. The racemes are about 18cm long while the petioles are 6cm long. The calyx

measures up to 5mm long, the campanulate 5-7mm long and are greenish-white to

greenish yellow in colour (Prabhu et al., 2009).

2.4.3 Phytochemistry

The phytochemistry study of Ocimum gratissimum have shown the presence of some

bioactive compounds such as steroids, tannins, flavonoids, saponins, terpenoids

alkaloids, inulins, phenolic compounds, B-carotene, glycosides, carotenoids, reducing

sugars, phlobatannins, anthraquinones and cardiac glycosides with steroidal ring and

deoxy–sugar, which are found in the aqueous leaf extract of this plant. The

methanolic leaf extract of this plant shows the presence of flavonoids, alkaloids,

tannins, terpenoids, phlobatannins and cardiac glycosides with steroidal ring, while

the ethanolic extract shows the presence alkaloids, steroids, tannins, flavonoids,

phlobatannins and terpenoids (Pandey, 2017b).

Thin layer chromatography of this plant leaf extract indicated the presence of some

polar compounds. One of the components was analyzed using mass spectra showed

the presence of an unknown compound with a molecular mass of at least 353 daltons

containing carbon, hydrogen, oxygen and possibly nitrogen. It has tentative molecular

formula, of C21 H 37 O 4 or C19 H 35 N 3 O3 as deduced from computer analysis.

23
https://www.researchgate.net/figure/Fig-1-a-Flowering-tops-of-O-gratissimum-b-Whole-plant-O-gratissimum_fig1_228691892

Figure 1: Whole plant Ocimum gratissimum.

Eugenol, citral, ethyl cinnamate, linalool, methyl eugenol, pinene, camphor, cis-

ocimene, transocimene, trans-carypohyllene, germacrene-Dfarnesene and l-bisabolene,

thymol, bisaboline, oleanolic acid, along with the volatile oil, limonene, terpinolene

Ȗ-terpinene, p-cymene, and 1,8-cineole are all present in the essential oil collected

from the fresh leaves (Pandey, 2017b).

2.4.4 Pharmcological Properties

The effect of Ocimum gratissimum as an antifungal and antibacterial agents have

been proven by numerous studies. The pharmacology effect of the aqueous extract of

this plant on rabbits shows the inhibition of the jejunum spontaneous pendular

movement in rabbit, in rats, it shows noncompetitive stomach strip blocking in rat and

non-toxic analgesic effect in mice (Pandey, 2017b).

24
Sofowara gave reports that the leave extracts of Ocimum gratissimum have been used

by several tribes for different purposes in West Africa and Nigeria. The

pharmacological effects of this plant have been studied and reported by various

researchers including antibacterial activity, antioxidant activity, antifungal activity,

antidiarrhoeal activity, antihelmintic activity, anticancer and antitumour potentials,

antibacterial activity, antimutagenic activity, antihypertensive property,

anticonvulsant activity and nematicidal activity (Imosemi, 2020).

Antibacterial Activity

The essential oil extract of this plant, show a high activity of antibacterial effects on

human pathogenic gram positive and gram negative bacteria. (Pandey, 2017a). The

basis of antibacterial activity is due the presence of phyto compounds like saponin ,

tannin, anthraquinone which had been reported to have antimicrobial and medicinal

activity (Omodamiro and Jimoh, 2015).

The antibacterial activity of different extracts from the leaves of this plant was tested

against Staphylococcus aureus, Escherichia coli, Salmonella typhi and Salmonella

typhimurium. Extract of these leaves were evaluated using cold water extract, hot

water extract and steam distillation extract. It was observed for that the steam

distillation extract to have inhibitory effects on selected bacteria and the average

inhibitory concentration ranged from 0.1% to for Staphylococcus aureus, to 0.01% for

Escherichia coli, and Salmonella typhimurium and finally 0.001% for Salmonella

typhi (Prabhu et al., 2009).

Antifugal Activity

Recent researches have shown Ocimum gratissimum to have inhibitory effect on the

growth of some fungi such as Penicillum and Rhizoctonia (Mohr, et al. 2017).

25
A study on the antifugal effect on the essential oil was obtained in a yield 1.10% (w/w)

from dried aerial parts of this plant using steam distillation extract and of an ethanol

extract, the steam-distillation residue was then carried out using agar diffusion method.

The results from this study shows that the essential oil inhibited the growth of all

fungi that was tested, which include the following; the phytopathogens,

Botryosphaeria rhodina, Rhizoctonia sp. and two strains of Alternaria sp. Species of

Alternaria were isolated from tomato and Penicillium chrysogenum, and eugenol

antifungal activity was evalutaed on these species. Eugenol gave the minimal

inhibitory concentration of 0.16 and 3.1 mg/disc for Alternaria sp and Penicillium

chrysogenum respectively (Prabhu et al., 2009; Faria et al., 2006).

For human pathogens, several studies have shown the effect of the essential oil

inhibiting Candida albicans which causes skin infection in humans.

Proven by another study, the hexane fraction and eugenol extract of Ocimum

gratissimum leaf at a concentration of 125-micron ml (-1) suppresses 100% and 80%

growth of dermatophytes Microsporum gypseum, M. canis, Trichophyton

mentagrophytes and T. rubrum (Pandey, 2017a).

Antidiarrhoeal Effect

The aqueous extract of the leaf of this plant was examined for antidiarrhoeal effect,

this study shows that Ocimum gratissimum aqueous leaf extract inhibits castor oil-

induced diarrhea in rats, which was judged by a decrease in wet faeces in the extract

treated animals. The extract was also observed to inhibit propulsive intestinal

movement of the contents present in the intestine. The leaf extract of this plant have

been proven extensively to be effective against a number of aetiologic agents of

diarrhea such as Shigellae (Prabhu et al., 2009).

26
Anti-Inflammatory Effect

A study report shows the inhibitory effect produced by chemical constituents of the

essential oil of three different plants known to have anti-inflammatory and analgesic

effects. This study was carried in vitro on soybean lipoxygenase L-1 and

cyclooxygenase function of prostaglandin H synthase, the two enzymes are involved

in the production of mediators of inflammation. Among the essential oil of these three

plants, the essential oil of Ocimum gratissimum inhibited the two enzymes with IC50 =

125μg/ml for cyclooxygenase function of prostaglandin H synthase, and 144μg/ml for

lipoxygenase L-1 (Prabhu et al., 2009).

Antinociceptive Activity

A study was carried out to evaluate the antinociceptive activity of Ocimum

gratissimum extract on both peripheral and central nervous system analgesic models.

The study was used to determine the different nociceptive stimuli namely thermal (hot

plate test), radiant (tail flick test) and chemical visceral nociceptive stimuli (acetic

acid). Using more than one test is essential to test for antinociceptive action, as it has

been shown that some ‘false positive’ activity can be observed with agents that are not

normally considered as analgesic.

During this study, it was observed that three doses of Ocimum gratissimum extract

administered for seven days was effective in producing analgesic activity in hot plate

test; this effect was dose dependent and statistically significant. Ocimum gratissimum

extract was also observed to effective in the tail flick test with a significant analgesic

activity. In acetic acid induced writhing, using three doses of Ocimum gratissimum

extract, all three doses produced a dose dependent inhibition of the number of

abdominal writhing (Sarral et al., 2014).

27
Anti-hypertensive Effect

Intravenously administering the essential oil of Ocimum gratissimum extract to

conscious deoxycorticosterone acetate DOCA-salt hypertensive rats was found to

have a hypotensive effect that is comparable to an active vascular relaxation. This

investigation focused on the vascular effects of EOOG and eugenol, its primary

component (EUG). The EOOG-induced hypotension in DOCA-salt hypertensive

conscious rats was reversible and did not change after intravenous pretreatment with

propranolol (2 mg/kg). EOOG (1-1000 g/mL) and EUG (0.006-6 mM) both reduced

the phenylephrine-induced contraction in isolated aorta preparations from DOCA-salt

hypertensive rats, with IC50 values of 226.9 g/mL and 1.2 (0.6-2.1) mm, respectively.

Removal of the vascular endothelium considerably changed the vasorelaxant effects

of EOOG (IC50 = 417.2 g/mL). At 300 and 1000 g/mL, respectively, EOOG greatly

decreased and even completely prevented CaCl2-induced contractions in a calcium-

free media, but it had no discernible impact on caffeine-induced contractions. With

EUG (1.8 and 6 mM), similar outcomes were seen for CaCl2 and caffeine-induced

contractions, respectively. The results indicate that active vascular relaxation—which

is partly dependent on the health of the vascular endothelium and appears to be

mediated primarily through an inhibition of plasmalemmal Ca2+ influx rather than

Ca2+-induced Ca2+ release from the sarcoplasmic reticulum—is responsible for the

hypotensive responses to EOOG in DOCA-salt hypertensive rats (Prabhu et al., 2009).

Immunostimulatory Effect

The immune system is a sophisticated bodily mechanism that guards the host against

outside viruses and gets rid of illnesses. (Nahak and Sahu 2014)

28
Using albino rats, the ethanolic leaf extract of Ocimum gratissimum was examined for

its ability to stimulate the immune system. Based on immunologic and hematologic

parameters, the study. The rats were given oral doses of standard E. coli inoculum

during this study, and the level of infection was determined by examining the

hematologic indices before to, during, and after treatment. At the conclusion of this

investigation, it was found that Ocimum gratissimum ethanolic leaf extract was

capable of lowering excessive red blood cell lysis and neutralizing toxins produced by

the organism in addition to effectively suppressing the disease condition following

infection (Prabhu et al., 2009).

Anti-diabetic Effect

On streptozotocin-induced diabetic rats, the considerable hypoglycemic effect of an

aqueous extract of Ocimum gratissimum was examined. The extract was given to the

rats in doses of 250, 500, and 1000 mg/kg body weight. After 24 hours of extract

treatment, the diabetic rats' blood glucose levels significantly decreased (P 0.05) by

81.3% due to the 500 mg/kg dose of aqueous extract. Reducing sugars, cardiac

glycosides, resin, tannins, saponins, glycosides, flavonoids, glycerin, and steroids

were all found during the preliminary phytochemical screening. The computed

median lethal dose (LD50) for rats was 1264.9 mg/kg body weight. In streptozocin-

induced diabetic rats, the leaves extract of O. gratissimum was found to have

antidiabetic action (Prabhu et al., 2009).

29
2.5 STREPTOZOTOCIN

Streptozotocin (2-deoxy-2-(((methylnitrosoamino)carbonyl)amino)-D-glucopyranose),

this is an antimicrobial agent originally derived from the soil microoraganism

Streptomycin Achromogenes (White, 1963; Herr et al., 1967; Weiss 1982).

Streptozotocin is one of the most important diabetogenic chemical agent in

experimental diabetes research among several chemicals that can be used to induce

diabetes, it is most preferred model human diabetes in animals, it’s structural,

functional and biochemical effects resembles that which is usually seen with diabetes

in human (Wu and Human., 2008). It was discovered in 1994 (Schnedl et al., 1994)

that certain insulin producing cell that lack the low affinity GLUT2 expression are

resistant to the actions of STZ (Elsner et al., 2000; Schnedl et al., 1994), whereas in

cells that express GLUT2 such as the liver and kidneys, STZ had ability to destroy

their cells (Lenzen., 2008). The diabetogenic properties of STZ are mainly

characterized by selective destruction of the beta cells, insulin deficiency, polydisia,

polyuria and hyperglycemia, all of which are similar to that seen in human diabetes.

Several studies have proven that STZ has the ability to induce both Type 1 diabetes

and Type 2 diabetes (Goud et al., 2015; Akbarzadeh et al., 2007; Kwon et al., 1994).

the dose and duration of STZ are important in the type of diabetes that is induced.

The chemical properties of STZ includes the following:

a. It is a glucosamine derivative

b. It is a toxic beta cell glucose analogue

c. It is a cytotoxic methyl nitrosourea moiety (N-methyl-N-nitrosourea) attached to

the glucose (2 deoxyglucose) molecule

d. It is an alkylating agent

e. It is relatively stable at pH 7.4 and 30oC at least up to 1 hour

30
f. It has a biological half-life of 5-15 minutes

2.5.1 Diabetogenic Mechanism of STZ

Increase in blood glucose levels is the major characterization of diabetes, other

characterization includes dysfunctions and complications of organs. Type 2 diabetes

is characterized mainly by hyperglycemia and insulin resistance while Type 1

diabetes is caused by destruction of the pancreatic beta cells through apoptosis (Schuit

et al., 2001).

Reactive oxygen species, which are free radicals are generated by excessive

metabolism of glucose results in glycolysis and glucose auto oxidation. This increases

beta cells oxidative stress which favors necrosis and apoptosis. Also insulin

resistance can be caused by inflammatory pathways such as mitogens activated

protein kinase (MAPKs) and nuclear factor kappa beta (NF-kB) (Schuit et al., 2001;

Damasceno et al., 2014). All of these factors therefore contributes to the development

of diabetes (Goyal et al., 2016).

Aconitase inhibition

Aconitase, this is a mitochondrial enzyme that protect mitochondrial DNA from

degradation. Aconitase inhibited by Reactive oxygen species such as superoxide (02)

and hydrogen peroxide (H202) and reactive nitrogen species including peroxynitrite

(Raza et al.,2011). Inactivation of aconitase and protein migration, is potentiated by

increased mitochondrial superoxide. Likewise, the ion that is released from aconitase

enhances mitochondrial oxidative stress (Liochev and Fridovich., 1997; Radi et al.,

2002). Study shows most reactive peroxynitrite and hydroxyl radicals are responsible

for causing extensive oxidative damage (Green et al., 2004).

31
Nitric Oxide (NO) and Nitrosative stress

Nitric oxide is one of the crucial second messenger involved in physiological and

pathological processes of the body (Goud et al., 2015). Investigations have shown

streptozotocin to increase the activity of guanyl cyclase and the formation cGMP,

which are characteristic actions of NO. Pancreatic beta cells are sensitive to damage

caused by nitric oxide and free radicals because of their low levels of free radical

scavenging enzymes (Spinas., 1999). Streotozotocin regulates mitochondrial

respiratory complexes. It stimulates increase in Ca2+ uptake and transformed

mitochondrial transmembrane potential in diabetic animals. Quite a number of studies

have confirmed the role of NO in streptozotocin-induced diabetes but there are

conflicting results from various studies are available concerning its source. Studies

have suggested that nitric oxide synthase is responsible for streptozotocin-induced NO

overproduction (especially iNOS). While some other studies have suggested that STZ

acts as an nitric oxide donor and important amounts of NO are liberated during

intracellular metabolism of streptozotocin to diazomethane (Szkudelski., 2001;

Kroenke et al., 1995).

Reactive Oxygen Species (ROS) and Oxidative Stress

Oxidative stress is the imbalance between antioxidant defense system of the body and

the pro-oxidants as a result of steady state reactive oxygen species. Atalay and

Laaksonen have proven that oxidative stress is responsible, at least in part, for

pancreatic beta cell dysfuction caused by glucose toxicity seen in hyperglycemia.

Different reaction mechanism has been shown to be involved in the genesis of

oxidative stress in both diabetic patients and diabetic animals and they include:

glucose auto-oxidation, protein glycation, formationj of advanced glycation products

and the polyol pathway (Atalay and Laaksonen., 2002). Initial stages of streptozotocin

32
induced diabetes in rats is characterized by free radical generation which includes

reactive oxygen and reactive nitrogen species (ROS and RNS). Reactive species such

as superoxide radical (O2e) hydrogen peroxide (H2O2), hydroxyl radical (OH) and

peroxynitrite (ONOO) induce oxidative stress in diabetic animals (Raza et al., 2011;

Aboonabi et al., 2014).

Reports have shown that uric acid is formed as consequence of adenosine triphosphate

(ATP) degradation by xanthine oxidase (XOD) during streptozotocin metabolism.

Both NO pathway and oxidative stress are linked in that they regulate each other,

causing pancreatic beta cell destruction after streptozotocin administration (Gonzalez

et al., 2000).

2.5.2 EFFECT OF STREPTOZOTOCIN ON VARIOUS BODY ORGANS

Cardiovascular System

Cardiovascular complications is one of the most occurring complications of diabetes.

STZ have proven to contribute to CVS complications independently of diabetogenic

action. Streptozotocin at a dose of 50-60mg/kg in rats leads to dysfunction in the

autonomic system by increasing vagal tone and decreasing sympathetic activity. This

leads to bradycardia, decreased force of myocardial muscles contraction, hypovolemia

and hypertension (Schaan et al., 2004).

At 100mg/kg dose of STZ, it causes decreased cardiac output, thinning of the left

ventricular wall, decreased NADH oxidase activity, increased oxidative stress and

apoptosis were observed (Yu et al., 2007). Diabetic cardiomyopathy is important in

morbidity and mortality among cardiovascular disorder related complications (Akhtar

et al., 2015). The administration of STZ to animals result in development of

cardiomyopathy, which is a clinical complication associated with human diabetes.

33
Respiratory System

There is a decrease in sensitivity of the lungs to streptozotocin as compared to other

organs of the body, respiratory toxicity streptozotocin is rare. Studies have showed

that a normal dose of STZ (60mg/kg) increases the level of nitric oxide, oxidative

stress and inflammatory mediators in the bronchoalveolar lavage fluid of the lungs

which then lead to asthma and lung damage (Samarghandian et al., 2014).

Nervous System

Streptozotocin is known to affect both central and peripheral nervous system.

Impaired motor and sciatic nerve conduction velocity are parts of the effect of the

administration of streptozotocin for 2 months. In the CNS after 3 months of

administration, STZ have been shown to cause impairment of spinal, auditory and

visual pathways (Biessels et al., 1999). Research studies shows that

intracerebroventricular (I.C.V) administration of STZ to rodents leads to an insulin

resistant brain state. This is often characterized by cognitive and cholinergic deficits,

glucose hypo-metabolism, oxidative stress and neurodegeneration in time dependent

manner similar to to sporadic Alzheimer’s disease in humans (Haluzik and

Nevidkova., 2000).

At a dose 75mg/kg administered intraperitoneally to animals result in a decrease in

reaction thresholds to hot and cold noxious stimuli causing allodynia and hyperalgesia.

The use of sreptozotocin is recommended to create a model of chronic neuropathic

pain (Salkovic-Petrisic et al., 2013). Elevation of angiotensin and oxidative stress

which causes neuronal apoptosis and visual function impairment have been observed

during administration of STZ (Ozawa et al., 2011).

34
Kidneys

Renal toxicity is seen in animals administered with STZ. At a dose of 65mg/kg,

streptozotocin causes marked renal toxicity and tubular necrosis. Lesions of the

glomerulus resembling those in human glomerular disease are seen animals treated

with streptozotocin (Martin et al., 2004). Renal toxicity caused by STZ is

characterized by progressive histological changes like tubular and glomerular

hypertrophy, mesangial expression and glomerular lesions (Kazumi et al., 1979).

Renal toxicity caused by administration of STZ may be attributed to increased

aminotransferase levels due to cellular DNA damage, infiltration by inflammatory

macrophages, T-lymphocytes and eicosanoids in kidneys tissues (Kazumi et al.,1979).

In rats, a dose of 55mg/kg induces diabetes with no toxicity to the kidneys (Blease

and Young., 1982).

Reproductive System

Streptozotocin causes indirect effects on accessory sex organs and direct effects on

sex glands and hormone levels. At dose between 45-60mg/kg, STZ causes temporary

decrease in serum testosterone level, impaired testicular function, testicular

degeneration, reduction in sperm count, loss of libido and erectile dysfunction (Ansari

and Ganaie., 2014; Navaro-casado et al., 2010).

In females, ovarian disruption is connected to a decrease in viable oocyte and delayed

oocyte maturation. Streptozotocin is rapidly absorbed into fetal circulation following

intravenous administration which leads to a depleted insulin reserve and mild

hyperglycemia in male offspring of laboratory rodents (Vikram et al., 2008).

35
2.5.3 GLIBENCLAMIDE

This is a second generation sulphonylurea which has been widely used in

management of non-insulin dependent diabetes mellitus.

Sulphonylureas are known to be one of the first-line oral anti-diabetic agents for the

treatment of type 2 diabetes mellitus. This drug is widely most prescribed of this class

and its hypoglycaemic efficacy and positive effects on clinically relevant endpoints

have been confirmed (UK prospective Diabetes Study Group 1998).

Several researches have shown that glibenclamide is a potent insulin-like agent that

promotes glucose uptake, glucose oxidation and activation of glycogen synthase in rat

liver and adipocytes (Altan et al., 1985; Altan et al., 1994; Atalay et al., 1994).

36
3.0 Methodology

3.1 Plant Material

Fresh leaves of Ocimum grassitiimum were collected from open grassland of Lagos

State University College of Medicine, Ikeja, Lagos and other locations around Lagos

State, Nigeria. Identification of the plant was carried out by a Taxonomist of the

Forestry Research Institute, Dr S.K Oluwa. Following identification, a specimen

voucher number LSH 001207 of the plant was deposited in the herbarium of the

Forestry Research Institute Lagos, Nigeria.

3.2 Ethical Approval

Ethical approval (AREC/2022/048) was obtained from Animal Research Ethics

Committee of Lagos State University College of Medicine, Ikeja.

3.3 PREPARATION OF OCIMUM GRASSITIIMUM AQUEOUS LEAF

EXTRACT

The fresh leaves of Ocimum grassitiimum were plucked to get the leaves, the leaves

were then air-dried at room temperature for four weeks. The dried leaves were

blended into a fine powder with a blender.

A total 270g of the powdered leaves was soaked in 1.5L of water for 72hours and

stirred at intervals to create an even dissolution before filtration using a dry Whatman

filter paper into a measuring cylinder. The filtrate was then evaporated to dryness in

rotary evaporator at 45oc to produce a semisolid mass and stored in an air tight

container at 4oc until used.

37
3.4 Experimental Animals

A total of twenty-five male rats aged eight-week-old male rats were obtained at

FellyGem Concepts, Ikorodu, Lagos. The rats were kept at 25 ± 2°C and 50-60%

humidity, with 12 h light/dark cycle and acclimatized for two weeks. They were

housed in separate cages and were randomly divided into five groups of five animals

each (Kim et al., 2020). The rats were fed with normal rats chow diet and water ad

libitum.

5 rats in each group;

1. Group 1: These are control group

2. Group 2: The diabetic rats control group.

3. Group3: The diabetic rats to be treated with O. grassitimum extract group

(300mg/kg)

4. Group 4: These were given O. grassitimum extract (300mg/kg)

5. Group 5: The diabetic rats to be treated with glibenclamide group.(5mg/kg)

3.5 Experimental Design

Rats of first group for normal male healthy control were given only distilled 10ml

water, second group for normal rats, administered orally with O. grassitiimum extract

at a dose of 300 mg/kg body weight/day, the third group for male diabetic rats,

administered orally with O. grassitiimum extract at a dose of 300 mg/kg body

weight/day. The fourth group for diabetic rats were untreated and the fifth group for

diabetic rats were treated with 5 mg/kg body weight of glibenclamide. Glibenclamide

was manufactured by May and Baker Plc, Nigeria. The administration of the doses

lasted for 4 weeks.

38
At the end of four weeks, the experimental animals were fasted for 12 h, water not to

be restricted, and then blood samples drawn, and test for blood glucose level, glycated

haemoglobin level and complete blood count were determined (Al-Attar and Alsalmi

2019).

3.6 Body weight changes

Rats body weights were evaluated at the start of the experimental duration and every

week using a HCB 1002 scale. (Adams Equipment, UK). Body weights was measured

at the same time during the morning throughout the experiments (Al-Attar and Zari,

2010). The experimental animals were observed for signs of abnormalities and

monitored weekly throughout the period of study.

3.7 Induction of Diabetes to Experimental Rats

Diabetes was induced by a single intraperitoneal injection of a freshly buffered (0.1 M

citrate, pH 4.5) solution of STZ at a dosage of 60 mg/kg body weight (b.w.). Their

blood glucose level was checked after 72 hours, by collecting blood from the tail vein

to detect the blood glucose level using fact-check glucometer (from Germany) and

strips (Carvalho et al., 2003). Diabetes in the animals was detected based on loss of

body weight, polyphagia and blood glucose levels (Ganong 2001). Rats with serum

glucose level of ≥200 mg/dl was considered diabetic and used in this study (Lee et al.,

2013).

3.8 Collection of blood samples

Blood samples was collected from the tail of the rats after 72 hours of administration

of STZ. The animals were held by their heads and their tails were rubbed with xylene.

39
Xylene cause dilation of the vessels, ensuring the collection of large volume of blood

by skinning a length of 1 cm from the tail with very sharp scissors. The wounded tails

were rubbed with absolute ethanol to prevent infection. The blood collected was used

for blood glucose analysis (Nwaoguikpe 2010).

Blood samples were rapidly collected from the heart after sedation with diethyl ether into

EDTA bottles. The blood samples are immediately refrigerated at 4oc for full blood count and

glycated haemoglobin level analysis.

3.9 Organ Collection

After sedation with diethyl ether, the following organs; Liver, Pancreas, Kidneys were

removed and weighed, the pancreas was stored in a bottle containing formaline.

3.10 Determination of blood glucose and glycated haemoglobin (HbA1c) level

Blood samples from all were analyzed for glucose level using glucometer. Glycated

haemoglobin was determined according to manufactuer’s instruction using HbA1c

determination kit by Sigma-Aldrich.

3.11 Determination of Complete Blood Count

Blood samples were analyzed using an automated cell counter (Coulter Electronics,

Luton, Bedfordshire, UK) with standard calibration, according to the manufacturer's

instructions for analysis of human blood and accurately programmed for the analysis

of red blood cell (RBC) count, total white blood cell (WBC) count, haemoglobin (Hb),

packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular

haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), RBC

distribution width (RDW), MPV, PDW, and P–LCR (Ofem et al., 2012).

40
 CHAPTER 4
4.0 Results

4.1 Effect of Aqueous Extract of Ocimum gratissimum on body weight changes in

male Wistar rats

The initial weight of experimental rats showed no significant difference (P>0.05)

between the diabetic + Ocimum, diabetic + glibenclamide, control, diabetic control

and Ocimum only group when compared with each other before induction of diabetes.

After 2 weeks of induction of diabetes there was a decrease in weight in all induced

experimental rats, a significant decrease in weight (p<0.05) in diabetic + Ocimum

(118.2 ± 6.15 grams), diabetic + glibenclamide (115.0 ± 6.27 grams) when compared

with control was noticed. The untreated and treated diabetic rats showed a weight

reduction when compared to control.

After 4 weeks, there was a slight increase in weight in the diabetic treated groups

(diabetic + Ocimum and diabetic + glibenclamide) when compared with the diabetic

control group. Although there was no significant difference in the weight when

compared to the diabetic group(P>0.05).

41
Figure 4.1: Body weight changes between the initial and final weights of the
experimental rats.
Key: IW = Initial weight
FW = Final weight after 4 weeks
Initial weight: N=5, ns= P>0.05
Final weight: N=5, *= P<0.05 vs Control
ns = P>0.05 vs Diabetic Control

42
4.2 Effect of Aqueous Extract of Ocimum gratissimum on Glycated Haemoglobin

in male Wistar rats

The mean glycated haemoglobin of the Diabetic + Ocimum, Diabetic Control, and

Diabetic + Glibenaclamide groups were 6.10 ± 0.30%, 6.74 ± 0.52% and 4.50 ±

0.24 % respectively. The Diabetic control group had a significantly (p<.0024) higher

glycated haemoglobin compared with the Diabetic + Ocimum and Diabetic +

Glibenaclamide groups.

The mean glycated haemoglobin of the Normal + Ocimum and Control groups were

3.51 ± 0.32% and 3.36 ± 0.34% respectively. The glycated haemoglobin Normal +

Ocimum group was not significantly different when compared with the Control group.

43
Figure 4.2: Effect of STZ induced diabetes and AEOG on Glycated Haemoglobin
level in male Wistar rat. Values expressed in Mean ± SEM
N=5 *=(p<0.0024) vs Diabetic control
N=5 ns=(p>0.05) vs Control

44
4.3 Effect of Aqueous Extract of Ocimum gratissimum on Blood Glucose Level in

male Wistar rats

The Blood glucose level was significantly higher in the diabetic rats throughout the

period of study (from week 1 to week 4) when compared to control.

After four weeks of treatment, the blood glucose level in the diabetic treated rats were

significantly higher when compared to control (p<0.0001). But the diabetic treated

groups had a significant reduced blood glucose level when compared with the diabetic

rats. (p<0.0002).

45
Figure 4.3: Effect of AEOG on Blood glucose level. Values expressed in Mean ±
SEM
Key: IBG: Initial Blood Glucose Level
ABG: Blood Glucose Level After Induction
FBG: Final Blood Glucose Level after 14 days
N=5 * (p<0.0002) vs Diabetic Control
N= 5~ (p<0.0001) vs Control

46
4.4 Effect of Aqueous Extract of Ocimum gratissimum on Red Blood Cell,

Haemoglobin Concentration, Packed Cell Volume in male Wistar rats.

The mean RBC count of the Diabetic + Ocimum, Control, and Diabetic +

Glibenaclamide groups were 8.72 ± 0.28, 7.18 ± 0.28 and 7.62 ± 0.28 × 1012/L

respectively. The Diabetic + Ocimum group had a significantly (p<0.006) higher RBC

count compared with the control and Diabetic + Glibenaclamide groups.

The mean RBC count of the Normal + Ocimum and Control groups were 7.76 ± 0.12

and 7.18 ± 0.28 × 1012/L respectively. The RBC count of Normal + Ocimum group

was not significantly different when compared with the Control group.

The Diabetic + Ocimum, Control, and Diabetic + Glibenaclamide groups had mean

PCV of 51.38 ± 1.25%, 47.62 ± 0.94% and 48.20 ± 1.54 % respectively. The mean

PCV of the Diabetic + Ocimum group when compared to the control group had a

significantly (p<0.043) higher PCV but it was not significantly different when

compared to the Diabetic + Glibenaclamide group.

The mean Hb concentration of the Diabetic + Ocimum group (14.96 ± 0.49 g/dL) was

significantly (p<0.009) higher when compared to the control group (13.24 ± 0.10

g/dL). But had no significantly difference when compared to the Diabetic +

Glibenaclamide group.

47
TABLE 1: SHOWING THE EFFECT OF AEOG ON RBC COUNT, PCV AND
HAEMOGLOBIN CONCENTRATION

Parameters Control Diabetic Diabetic + Ocimum Diabetic +

Control Ocimum Only Glibenaclamide
RBC 7.18 ± 0.28 8.34 ± 0.58 8.72 ± 0.28* 7.76 ± 0.12ns 7.62 ± 0.28
( 1012/L )
Hb (g/dL) 13.24 ± 0.10 14.78 ± 1.01 14.96 ± 0.49* 14.02 ± 0.42 13.70 ± 0.35
PCV (%) 47.62 ± 0.94 50.40 ± 2.72 51.38 ± 1.25* 50.14 ± 1.11 48.20 ± 1.54

Data represented as MEAN±SEM

Key: RBC - Red Blood Cell count
Hb - Haemoglobin concentration
PCV - Packed Cell Volume

RBC: N=5 *=P<0.006 vs Control

PCV: N=5 *=P<0.043 vs Control
Hb: N=5 *=P<0.009 vs Control

48
4.5 Effect of Aqueous Extract of Ocimum gratissimum on Total White Blood Cell

Count and Platelets in male Wistar rats

For the mean platelets counts, the Diabetic + Ocimum group (450.3 ± 48.92 × 109/L))

when compared with the control and Diabetic + Glibenaclamide groups were not

significantly different.

The diabetic untreated rats had significantly (p<0.0363) higher mean platelet count

when compared to the Diabetic + Ocimum group.

Following the administration of aqueous extract of Ocimum gratissimum, the total

WBC counts remained significantly unaltered.

49
Figure 4.5a: Comparison of platelets count in different experimental groups
N=5 *P<0.0363 vs Diabetic + Ocimum.

Figure 4.5b: Comparison of total white blood cell (WBC) count in different
experimental groups
N=5, ns=P>0.05 vs Control

50
4.6 Effect of Aqueous Extract of Ocimum gratissimum on Differential White

Blood Cells in male Wistar rats

Following the oral administration of Ocimum gratissimum, Neutrophils were

significantly (p<0.0411) increased in Diabetic + Ocimum group (1.44 ± 0.35) when

compared with the control (0.78 ± 0.08) and Diabetic + Glibenaclamide (0.62 ± 0.048)

groups. When compared with diabetic group no significant difference was observed.

However, no significant difference was observed in the proportion of Lymphocytes,

Monocytes, Esinophils.

51
TABLE 2: SHOWING THE EFFECT OF AEOG ON DIFFERENTIAL WHITE
BLOOD CELLS

Parameters Control Diabetic Diabetic + Ocimum Diabetic +

Control Ocimum Only Glibenclamide
Neutrophils 0.78 ± 0.08 1.38 ± 0.78 1.44 ± 0.35* 1.12 ± 0.11 0.62 ± 0.048*
Lymphocytes 3.04 ± 0.54 2.69 ± 0.80 3.28 ± 0.71ns 3.37 ± 0.95 2.32 ± 0.73ns
Monocytes 0.53 ± 0.03 0.72 ± 0.32 0.83 ± 0.23ns 0.36 ± 0.12 0.40 ± 0.12ns
Esinophils 0.02 ± 0.01 0.01 ± 0.01 0.02 ± 0.01ns 0.02 ± 0.01 0.01 ± 0.003ns

Data represented as MEAN±SEM

Key: ns - Not Significant

Neutrophils: N=5, *= P<0.0411 vs Control and Diabetic + Glibenclamide

Lymphocyte: N= 5, ns = P>0.05 vs Control
Monocytes: N= 5, ns = P>0.05 vs Control
Esinophils: N= 5, ns = P>0.05 vs Control

52
4.7 Effect of Aqueous Extract of Ocimum gratissimum on RBC Indices in male

Wistar rats

The mean MCV of Diabetic + Ocimum, Control, and Diabetic + Glibenclamide

groups were 59.08 ± 1.34 fl , 66.52 ±1.62 fl and 63.34 ± 0.79 fl respectively

The mean MCV of Control group was significantly (p<0.0056) elevated when

compared with Diabetic + Ocimum and Diabetic + Glibenclamide groups

The treated diabetic rats were compared with the untreated diabetic rats; no significant

difference were observed.

When compared, MCH and MCHC values showed no significant difference from one

another. The RBC distribution width–standard deviation (RDW–SD) and RBC

distribution width–coefficient of variation (RDW–CV) of the groups when compared

with one another showed no significant differences.

53
4.8 Effect of Aqueous Extract of Ocimum gratissimum on Platelets Indices in

Male Wistar Rats

Following the oral administration of the extract, the mean MPV of the Diabetic +

Ocimum, Control, and Diabetic + Glibenclamide groups were 6.50 ± 0.15, 6.05 ± 0.37

and 6.60 ± 0.07 respectively. When these groups were compared to one another, no

significant differences were observed.

The P-LCR values when compared to one another showed no significant differences.

The mean PDW values of the Diabetic + Ocimum, Control, and Diabetic +

Glibenclamide groups were 16.15 ± 0.25, 16.48 ± 0.16 and 15.85 ± 0.13 respectively.

These groups showed no significant differences when compared with one another.

54
TABLE 3: SHOWING THE EFFECT OF AEOG ON RBC AND PLATELETS
INDICES

Parameters Control Diabetic Diabetic + Ocimum Diabetic +

Control Ocimum Only Glibenclamide
MCV (fl) 66.52 ±1.62* 60.63 ± 1.21 59.08 ± 1.34 64.66 ± 1.09 63.34 ± 0.79
MCH (pg) 18.58 ± 0.82 17.73 ± 0.18 17.20 ± 18.12 ± 0.36 18.00 ± 0.47
0.23ns
MCHC 278.6 ± 6.88 292.5 ± 6.61 291.0 ± 280.0 ± 3.96 284.2 ± 4.76
(g/dl) 4.64ns
RDW-CV 0.18 ± 0.01 0.22 ± 0.01 0.22 ± 0.01ns 0.22 ± 0.01 0.21 ± 0.01
(%)
RDW-SD 52.73 ± 2.00 53.58 ± 0.98 55.23 ± 57.78 ± 2.35 52.88 ± 0.28
(fl) 2.24ns
MPV(fl) 6.05 ± 0.37 6.58 ± 0.24 6.50 ± 0.15ns 6.68 ± 0.17 6.60 ± 0.07
P-LCR (%) 0.15 ± 0.01 0.09 ± 0.01 0.11 ± 0.02ns 0.10 ± 0.01 0.10 ± 0.004
PDW (fl) 16.48 ± 0.16 16.38 ± 0.25 16.15 ± 16.10 ± 0.19 15.85 ± 0.13
0.25ns

Data represented as MEAN±SEM

Key:
ns - Not Significant
MCV - Mean Corpuscular Volume
MCH - Mean Corpuscular Hemoglobin
MCHC - Mean Corpuscular Hemoglobin Concentration
RDW-SD - RBC Distribution Width–Standard Deviation
RDW-CV - RBC Distribution Width–Coefficient of Variation
MPV - Mean Platelet Volume
P-LCR - Platelet-Large Cell Ratio
PDW - Platelet Distribution Width

MCV - N= 5, *= P<0.0056 vs Diabetic + Ocimum

MCH- N= 5, ns = P>0.05 vs Control
MCHC - N= 5, ns = P>0.05 vs Control
RDW-CV - N= 5, ns = P>0.05 vs Control
RDW-SD - N= 5, ns = P>0.05 vs Control
MPV - N= 5, ns = P>0.05 vs Control
P-LCR - N= 5, ns = P>0.05 vs Control
PDW - N= 5, ns = P>0.05 vs Control

55
4.9 Effect of Aqueous Extract of Ocimum gratissimum on the relative weight of

the Pancreas, Kidney and Liver in male Wistar rat.

The mean values of the weight of the pancreas were 0.37 ± 0.07 grams for Diabetic +

Ocimum group, 0.39 ± 0.04 grams for Diabetic + Glibenclamide group and 0.46 ±

0.05 grams for control group as shown in table 4. When compared the mean value of

the weight of the pancreas was significantly (p<0.0019) higher in control than

Diabetic + Glibenclmide and Diabetic + Ocimum group. The pancreas weight was

slightly decreased in the diabetic groups compared to the non-diabetic groups.

Treatment with AEOG and Glibenclamide restored the pancreas weight close to

normal when compared to the controlled.

The mean of the values of the weight of the liver were 4.29 ± 0.12 grams for Diabetic

+ Ocimum group, 3.27 ± 0.06 grams for Diabetic + Glibenclamide group and 3.44 ±

0.18 grams for control group as shown in table 4. The liver weight was slightly

decreased in treated diabetic groups than in non-treated diabetic group.

Comparing the mean value of the liver weight, the mean value of the diabetic group

4.77 ± 0.26 was significantly (p<0.0252) elevated compared to the treated diabetic

groups (Diabetic + Glibenclamide and Diabetic + Ocimum). But when these groups

were compared to the control group no significant difference was observed.

The mean weight of the kidneys in control group 0.75 ± 0.03 grams and 1.06 ± 0.06

grams in the diabetic group. It shows AEOG slightly increases the size of the kidney

compared to glibenclamide.

56
TABLE 4: SHOWING THE EFFECT OF AEOG ON THE RELATIVE
ORGAN WEIGHT ON EXPERIMENTAL ANIMALS
Groups Control Diabetic Diabetic + Ocimum Diabetic +
/ Control Ocimum Only Glibenaclamide
Organs
Pancreas(g) 0.46 ± 0.05 0.29 ± 0.02 0.37 ± 0.07* 0.45 ± 0.04 0.39 ± 0.04
Kidney(g) 0.75 ± 0.03 1.06 ± 0.06 0.99 ± 0.05 ns
0.90 ± 0.05 0.80 ± 0.02
Liver(g) 3.44 ± 0.18 4.77 ± 0.26 4.29 ± 0.12* 3.75 ± 0.12 3.27 ± 0.06

Data represented as MEAN±SEM

Key:
ns - Not Significant

Pancreas: N = 4, * = P<0.0019 vs Control

Kidney: N = 4, ns = P>0.05 vs Control
Liver: N= 4, * = P<0.0252 vs Control

57
 CHAPTER 5

5.0 DISCUSSION

In this study, the Outcomes of Ocimum gratissimum on glycated haemoglobin in

STZ induced diabetic male Wistar rats were investigated, Gilbenclamide was also

used as a reference drug.

Diabetes mellitus have been known to be associated with different complications such

as nephropathy, retinopathy atherosclerosis, and myocardial infarction etc. (Pushparaj

et al., 2007). These complications are usually associated with increase in blood

glucose level. Strepotozotocin has been used as a diabetogenic agent in experimental

animals.

This study demonstrated STZ induced significant decrease in body weight gain of rats

after 4 weeks. A significant increase was observed in the 4th week in the Ocimum and

glibenclamide diabetic treated group compared to the first week, while that of control

was stable. Body weight change is one of the important parameters to ascertain the

effect of treatment on diabetic state. An increase in body weight signifies that

anabolic effects have overridden the catabolic effects. No variation means protection

against weight loss. Decrease in body weight would mean that catabolism has

persisted (Al-Attar and Zari, 2010).

The most potent effect of diabetes; Hyperglycaemia was seen to be subdued after the

administration of AEOG in the diabetic treated rat, while an increase in the blood

glucose level was observed in the diabetic rat. Preliminary phytochemical screening

revealed the presence of reducing sugars, cardiac glycosides, resin, tannins, saponins,

glycosides, flavonoids, glycerin and steroids, which are important factors present in

Ocimum gratissimum for lowering blood glucose level (Prabhu et al., 2009).

58
Hypoglycemic effect of O. gratissimum have been observed by several experiments

(Okoduwa et al., 2017; Oguanobi et al., 2012; Mohammed et al., 2007).

As a result of effect of diabetes on muscle wasting and reduction in tissue protein, it is

believed to affect the other organs of the body, which causes weight loss in specific

organs. Induction of diabetes caused a significant decrease in the weight of pancreas

as result of the diabetogenic effect of STZ, which leads to destruction of the beta cells

of the pancreas resulting in the loss of its ability to produce insulin, leading to a

decrease in the size of pancreas.

The pancreas weight was reduced in the diabetic group, compared to the control group,

although in the Ocimum group and the diabetic group, the pancreatic weight is slight

increased than the diabetic group. This could suggest that Ocimum could help

preserve the weight of specific organs and seen in its ability to preserve the weight of

the body in diabetes.

In this study, after administration of AEOG, glycated haemoglobin was significantly

decreased in diabetic treated group, while an increase in glycated haemoglobin was

observed in diabetic rat. HbA1c is important and can be used as an objective measure

of glycemic control (World Health Organization, 2011). In diagnosis of diabetes and

montoring one of the primary tests conducted is glycated haemoglobin test. This test

is a measure of β-N-(1-deoxy)-fructosyl hemoglobin that is contained in the red blood

cell which is glycated in varying amounts depending on blood glucose levels over

time (Canadian Agency for Drugs and Technologies in Health, 2014).

59
The effect of Ocimum grastissimum on hematological parameters in male wistar rats

was investigated in this study. The Diabetic + Ocimum group showed an increase in

the erythrocyte count. This was confirmed by the increased hematocrit (PCV) and

percentage Hb in the Diabetic + Ocimum group. In normal circumstances, local tissue

anoxia primarily results in formation of erythropoietin, which stimulates increased

production of erythrocytes. (Bowman and Rand., 1980). Therefore, it is likely that the

leaves of Ocimum grastissimum extract contains erythropoeitin-like agent which is

responsible for the increased production of erythrocytes.

On the thrombocytes(platelets). There was a decrease in thrombocytes in the Diabetic

+ Ocimum group. This implies that it is likely that the extract contains some

compounds that inhibits the production of thrombopoietin. This which contradicts the

experiment carried out by Ofem et al., 2012. Platelets play important role in the

maintenance of normal homeostasis and also MPV is an indicator of platelet function,

including platelet aggregation; release of thromboxane A2. (Ofem et al., 2012). In this

study the MPV was significantly unaltered. Studies have shown that increase in MPV

is one of the risk factor for myocardial infarction, cerebral ischemia and chronic

vascular disease (Khandekar et al., 2006). The total WBC(leucocyte) counts were not

significantly altered following the administration of the extract. However, in

differential counts, there was an increase in neutrophil count in Diabetic + Ocimum

group of the male wistar rats. It is likely that the extract contains agents that

stimulates the bone marrow to produce neutrophils and release them into the blood.

Neutrophils are known as the major granulocytes to be activated when the body is

invaded by bacteria and they are known to provide the first line of defence against

invading microorganisms (Ofem et al., 2012).

60
This study is similar to earlier study by Jimoh et al. who reported decrease in

hematological parameters such as platelets following administration of Ocimum

grastissimum extract in wistar rats (Jimoh et al., 2010). This decrease was attributed

to the presence of saponins in the extract.

5.1 CONCLUSION

The outcome of this study implies that Ocimum grastissimum is an important

multipurpose therapeutic plant possessing some pharmacotherapeutic roles to

diminish hyperglycemia, stabilize body weight, decrease platelet counts, and increase

some hematological parameters such as red blood cell count, packed cell volume, and

hemoglobin concentration. It also protects the pancreatic islets against destruction by

diabetes.

61
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