ABSTRACT
Cardiotoxicity has become a major challenge for cancer patients undergoing chemotherapy. Examples of potentially cardiotoxic anticancer agents are 5-fluorouracil and anthracyclines. The leaves of Pterocarpus santalinoides have been shown to possess among other pharmacological properties, the ability to lower serum levels of low density lipoprotein cholesterol (LDL-C) which suggests a cardio protective potential. This study was aimed at experimenting the cardioprotective potential of the ethanol leaf extract against cardiotoxicity side effect of 5-fluorouracil in wistar rats. The study was done in two phases. 36 albino rats were employed for the first phase of the study, they were separated into 6 groups (groups 1, 2, 3, 4, 5 and 6) of 6 animals which received (distilled water, distilled water, 25 mg/kg Aspirin, 34.54 mg/kg crude extract, 69.28 mg/kg crude extract and 103.92 mg/kg crude extract for groups 1, 2, 3, 4, 5 and 6 respectively) orally for a period of 14 days. On the 15th day, groups 2, 3, 4, 5, and 6 were given 5-fluorouracil (150 mg/kg) via intra-peritoneal route to induce cardiotoxicity. After a period of 24 hours, the animals were sacrificed, the sera were analyzed for troponin T, CK-MB, Lipid profile, the whole blood samples were subjected to haematology while the hearts were subjected to histopathological scrutiny, the result obtained revealed significant (p<0.05) increase in HDL-C and decrease in LDL-C, Troponin T and CK-MB across groups compared to positive control which indicates cardioprotective potential, hence the second phase was conducted. In the second phase, the crude extract was fractionated into n-butanol, ethyl acetate and chloroform fractions, 36 albino wistar rats were also used for the study. They were also separated into 6 groups of 6 animals each. The animals were equally given treatments (distilled water, distilled water, 25 mg/kg Aspirin, 69.28 mg/kg n-butanol fraction, 69.28 mg/kg ethyl acetate fraction and 69.28 mg/kg chloroform fraction for groups 1, 2, 3, 4, 5 and 6 respectively) orally for 14 days. On the 15th day, groups 2, 3, 4, 5 and 6 were given 5-FU (150 mg/kg) via intraperitoneal route to induce cardiotoxicity. The animals were sacrificed after 24 hours, and the sera analyzed for troponin T level, CK-MB activity; lipid profile; urea, creatinine and electrolytes concentrations; ALP, AST and ALT activity; malondialdehyde concentration, catalase, glutathione peroxidase, superoxide dismutase activity; the whole blood samples were haematologically analyzed and the heart, kidney and liver tissues were histologically studied. Result revealed significant decrease (p<0.05) in TnT, CK-MB, LDL-C, TG, WBC, platelets, ALP, AST, ALT, urea, creatinine, Na+, K+, Cl-, HCO-3 and significant (p<0.05) increase in HDL-C, RBC, Ca2+, catalase, SOD and GSPx activity across groups as compared to the positive control. Histopathological results revealed that the low and medium dose of crude extract and chloroform fraction yielded more effective protection from cardiotoxicity more than the other doses and fractions. The crude ethanol extract of P. santalinoides and its fractions conferred on the albino rats appreciable protection against 5-FU induced cardiotoxicity with the medium dose and chloroform fraction producing the overall highest cardioprotective effects.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xii
List of Figures xiii
List of Plates xii
List of Appendixes xv
List of Abbreviations xvii
Abstract xix
CHAPTER 1: INTRODUCTION
1.1 Background of the
Study 1
1.2 Pterocarpus santalinoides (PS) 7
1.3 Scope of the Study 10
1.4 Statement of Problem 11
1.5 Justification of the
Study 12
1.6 Relevance of the
Study 13
1.7 Aim of the Study 14
1.8 Objectives of the
Study 14
CHAPTER 2: REVIEW OF RELATED LITERATURE
2.1 Phytochemical and
Proximate Composition of Pterocarpus santalinoides 15
2.1.1 Phytochemistry of Pterocarpus
santalinoides 15
2.1.2 Proximate composition
of Pterocarpus
santalinoides 16
2.2 Pharmacological
Activities of Pterocarpus santalinoides 17
2.2.1 Analgesic activity 17
2.2.2 Insecticidal activity 17
2.2.3 Antitrypanosomal
activity 19
2.2.4 Haematological effects 19
2.2.5 Hypolipidemic activity 19
2.2,6 Anti-cancer activity 20
2.2.7 Antimicrobial activity 21
2.2.8 Anti-diarrhoeal and
antispasmodic activity 22
2.3 Cardioprotective
Effect of Aspirin 24
2.4 Anti-Cancer agents
and their Cardiotoxicity Potential 25
2.4.1 5-Fluorouracil and
other antimetabolites 25
2.4.2 Trastuzumab
(Herceptin) 27
2.4.3 Anthracyclines 28
2.4.4 Taxanes 30
2.4.5 Interleukin-2 (IL-2)
therapy 31
2.4.6 Tyrosine kinase
inhibitors (TKIs) 31
2.4.7 Checkpoint inhibitors immune therapy 32
2.4.8 Angiogenesis
inhibitors 33
2.4.9 Radiation therapy 34
2.5 Biomarkers of
Cardiotoxicity 35
2.5.1 Cardiac function
status 35
2.5.1.1 Troponin 35
2.5.1.2 Creatine kinase (EC
2.7.3.2) 36
2.5.1.3 Lactate dehydrogenase
(EC 1.1.1.27) 37
2.5.1.4 Natriuretic peptide 38
2.5.2 Peripheral blood
mononuclear cell gene expression profile 40
2.5.3 Lipid profile
(cardiovascular risk factor assessment) 41
2.5.3.1 Cholesterol 41
2.5.3.2 Triglycerides 43
2.5.4 Liver function status 43
2.5.4.1 Alkaline Phosphatase
(ALP) (EC
3.1.3.1) 44
2.5.4.2 Aminotransferases: ALT
(EC 2.6.1.2) and AST (EC 2.6.1.1) 45
2.5.5 Oxidative stress
status 47
2.5.5.1 Malondialdehyde (MDA) 47
2.5.5.2 Catalase
(EC 1.11.1.6) 48
2.5.5.3 Superoxide
dismutase (EC 1.15.1.1) 50
2.5.5.4 Glutathione
peroxidase (EC.1.11.1.9) 51
2.5.6 Haematological
indices 52
2.5.6.1 Red blood cell
(erythrocytes) count and haemoglobin (Hb) 54
2.5.6.2 White blood cell (WBC)
count 56
2.5.5.3 Platelet count 56
2.5.7 Kidney function status 57
2.5.7.1 Serum potassium 57
2.5.7.2 Serum sodium 59
2.5.7.3 Serum bicarbonate 60
2.5.7.3 Serum creatinine 61
2.5.7.4 Serum urea
62
CHAPTER 3: MATERIALS AND
METHODS
3.1 Materials 64
3.1.1 Chemicals and reagents 64
3.1.2 Equipment 64
3.1.3 Research hypothesis 65
3.2 Methods 65
3.2.1 Plant leaf collection, identification and
extraction of
the crude ethanol extract 65
3.2.2 Extract
fractionation by partitioning 66
3.2.3 Determination of median lethal dose (LD50)
for the crude ethanol
extract 66
3.2.4 Phytochemical screening 68
3.2.4.1 Test for alkaloids 68
3.2.4.2 Test for saponins 68
3.2.4.3 Test for tannins 68
3.2.4.4 Test for
flavonoids 69
3.2.4.5 Test
for cardiac glycosides 69
3.2.4.6 Test for anthraquinones 69
3.2.5 Experimental design 70
3.2.6 Experimental animals treatment 71
3.2.7 Animal sacrifice and
preparation of sera for phase one studies 72
3.2.8 Animal sacrifice and
preparation of sera for phase two studies 73
3.3 Biochemical Analysis 75
3.3.1 Estimation of serum troponin-T concentration 75
3.3.2 Estimation of CK-MB
concentration 76
3.3.3 Estimation of alkaline
phosphatase activity 78
3.3.4 Estimation of serum
superoxide dismutase activity 79
3.3.5 Estimation of serum
glutathione peroxidase activity 80
3.3.6 Estimation of serum
catalase activity 82
3.3.7 Estimation of serum
malondialdehyde (MDA) level 83
3.3.8 Estimation of total
serum cholesterol 84
3.3.9 Estimation of serum
high density lipoprotein cholesterol 85
3.3.10 Estimation of serum
level of total triglyceride 86
3.3.11 Estimation of
haematological indices 88
3.3.12 Estimation of serum
electrolytes level 89
3.3.13 Determination of
aspartate amino transferase (AST) activity in serum 90
3.3.14 Estimation of alanine aminotransferase
(ALT/SGPT) activity 91
3.3.15 Estimation of urea (BUN) 92
3.3.16 Estimation of serum
creatinine concentration 92
3.3.17 Histological
procedures 94
3.3.18 Statistical analysis 94
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Results 96
4.1.1 Phytochemical
screening 96
4.1.2 Phase one
results 98
4.1.2.1 Effect
of CELEPS on serum troponin-T concentration and
CK-MB activity
in albino rats 98
4.1.2.2 Effect
of CELEPS on serum lipid profile in albino rats 99
4.1.2.3 Effect
of CELEPS on haematological parameters in albino
rats 102
4.1.2.4 Effect
of CELEPS on the histopathology of the heart tissues 104
4.1.3 Phase two
results 112
4.1.3.1 The effect of FELEPS on cardiovascular indices in albino
rats 112
4.1.3.1.1 The
effect of FELEPS on serum troponin T concentration in
albino rats 112
4.1.3.1.2 The effect
of FELEPS on serum CK-MB activity in albino
rats 114
4.1.3.2 The
effect of FELEPS on serum lipid profile in albino rats 116
4.1.3.3 The effect
of FELEPS on liver function status in albino rats 120
4.1.3.3.1 The effect
of FELEPS on serum ALP, AST and ALT activity 120
4.1.3.4 The effect of FELEPS on the
oxidative stress status in albino
rats 122
4.1.3.4.1 The
effect of FELEPS on the serum MDA
concentration in
albino rats 122
4.1.3.4.2 The
effect of FELEPS on serum levels of oxidative enzymes;
Catalase, Superoxide Dismutase and Glutathione Peroxidase in
Albino Rats 124
4.1.3.5 The
effect of FELEPS on the
haematological Indices in albino
Rats 125
4.1.3.6 The effect of FELEPS on kidney function status in albino
rats 132
4.1.3.6.1 The
effect of FELEPS on the serum electrolytes concentration in
albino rats 132
4.1.3.6.2 The
effect of FELEPS on the serum urea and creatinine
concentration in albino rats 135
4.1.3.7 Histopathological
results 137
4.2 Discussion 155
CHAPTER 5: SUMMARY,
CONCLUSION AND RECOMMENDATIONS
5.1 Summary 180
5.2 Conclusion 182
5.3 Recommendation 183
References 184
Appendix 220
LIST OF TABLES
3.1 Dose
Regimen for Phase One Studies (Treatment with Crude Ethanol
Extract) 70
3.2 Dose Regimen for Phase Two (Treatment
with Fractionated Portions of the
Crude ethanol extract) 72
3.3 Sample and Blank Regimen for Determination
of SOD activity 79
3.4 Reagent Blank, Standard, Controls and
Samples Regimen for Estimation
of Serum Creatinine Concentration 92
4.1 Result of Phytochemical Screening 95
4.2 Effect
of CELEPS on Serum of Troponin
Concentration and
CK-MB Activity in Albino
rats 97
4.3 Effect of CELEPS on Serum
Lipid Profile in Albino Rats 99
4.4 Effect of CELEPS on Haematological Parameters in Albino Rats 101
4.5 The Effect of FELEPS on Serum Lipid Profile in Albino Rats 116
4.6 The Effect of FELEPS on Lipid Profile
Assessment (cardiovascular risk index) 117
4.7 The Effect of FELEPS on Serum ALP, ALT
and AST Activities in
Albino Rats 119
4.8 The Effect of FELEPS on
the Serum MDA Concentration in Albino Rats 121
4.9 The
Effect of FELEPS on Serum Activities of Oxidative Enzymes; Catalase, Superoxide Dismutase
and Glutathione Peroxidase in Albino Rats 123
4.10A The Effect
of FELEPS on the Haematological
Indices in Albino Rats 127
4.10B The Effect of FELEPS on the haematological Indices in albino
rats continued 128
4.11 The Effect of
Fractions of Ethanol Leaf Extract of P.
santalinoides on the
Serum Electrolytes in Albino Rats. 131
4.12: The
Effect of FELEPS on the Serum Urea and
Creatinine Concentration in Albino Rats in Wistar rats 133
LIST OF FIGURES
4.1 The
effect of fractions of ethanol leaves extract of p. santalinoides
(FELEPS) on serum troponin levels (ng/ml) in albino
rats. 111
4.2 The effect of fractions of ethanol
leaf extract of P. santalinoides
(FELEPS) on serum CK-MB activity (u/l) in albino rats 113
LIST OF PLATES
1.1 Pterocarpus santalinoides 7
4.1 Photomicrograph of Heart Tissue of
Normal Control Rats (Treated with Distilled
Water Only) 104
4.2 Photomicrograph of Heart Tissue
Treated with Distilled Water and 150
mg/kg bw 5-Fluorouracil (Positive Control) 105
4.3 Photomicrograph of Heart Tissue
Treated with 25 mg/kg bw Aspirin (AS)
and 150 mg/kg bw 5-Fluorouracil (Standard Control) 106
4.4 Photomicrograph
of Heart Tissue Treated with Low Dose (LD) (34.64 mg/kg bw) of CELEPS and 150 mg/kg
bw 5-Fluorouracil (Low Dose) 107
4.5: Photomicrograph of Heart Tissue Treated
with Medium Dose (69.28
mg/kg bw) of CELEPS and 150 mg/kg bw 5-Fluorouracil 108
4.6 Photomicrograph of Heart Tissue
Treated with High Dose (HD) (103.92
mg/kg bw) CELEPS and 150 mg/kg bw 5-Fluorouracil. 109
4.7 Photomicrograph of the Longitudinal Section of the Distilled
Water
(Control) Heart
Tissue 134
4.8 Photomicrograph of the Longitudinal
Section of the Heart Tissue of the Distilled
Water + 150 mg/kg bw 5-Fluorouracil Treated Rats (Positive Control) 135
4.9 Photomicrograph of the Longitudinal
Section of the Heart tissue of the 25mg/kg
bw aspirin + 150mg/kg bw 5-Fluorouracil Treated Rats (Standard Control) 136
4.10 Photomicrograph of the Longitudinal
Section of the Heart Tissue of the
N-Butanol Fraction + 150mg/kg bw 5-Fluorouracil Treated Rats (Group 4) 137
4.11 Photomicrograph of the Longitudinal
Section of the Heart Tissue of the Ethyl-acetate
Fraction + 150mg/kg bw 5-Fluorouracil Treated Rats (Group 5) 138
4.12 Photomicrograph
of the Longitudinal Section of the Heart Tissue of the Chloroform
Fraction + 150mg/kg bw 5-Fluorouracil Treated Rats (Group 6) 139
4.13 Photomicrograph of the Longitudinal
Section of the Kidney Tissue of the Distilled
Water Treated Rats (Normal Control) 140
4.14 Photomicrograph of the Longitudinal
Section of the Kidney Tissue of the Distilled
Water + 150 mg/kg bw Treated Rats (Positive Control). 141
4.15 Photomicrograph
of the Longitudinal Section of the Kidney Tissue of the 25mg/kg bw Aspirin + 150mg/kg 5-Fluorouracil
Treated Rats (Standard Control) 142
4.16 Photomicrograph of the Longitudinal
Section of the Kidney Tissue of the
n-Butanol Fraction + 150 mg/kg bw Treated Rats (Group 4) 143
4.17 Photomicrograph of the Longitudinal
Section of the Kidney Tissue of the Ethyl-acetate
Fraction + 150 mg/kg bw Treated Rats (Group 5) 144
4.18 Photomicrograph of the Longitudinal
Section of the Kidney Tissue of the Chloroform
Fraction + 150 mg/kg bw Treated Rats (group 6) 145
4.19 Photomicrograph of the Transverse
Section of the Liver Tissue of the Distilled
Water Treated Rats (Normal Control). 146
4.20 Photomicrograph of the Transverse
Section of the Liver Tissue of the Distilled
Water + 150 mg/kg bw 5-Fluorouracil Treated Rats (Positive Control) 147
4.21 Photomicrograph of the Transverse
Section of the Liver Tissue of the 25mg/kg
bw Aspirin + 150 mg/kg bw 5-Fluorouracil Treated Rats (Standard Control) 148
4.22 Photomicrograph of the Transverse
Section of the Liver Tissue of the N-Butanol
Fraction + 150 mg/kg bw 5-Fluorouracil Treated Rats (Group 4) 149
4.23 Photomicrograph of the Transverse
Section of the Liver Tissue of the Ethyl-acetate
Fraction + 150 mg/kg bw 5-Fluorouracil Treated Rats (Group 5) 150
4.24 Photomicrograph of the Transverse
Section of the Liver Tissue of the
Chloroform Fraction + 150 mg/kg bw 5-Fluorouracil Treated Rats
(Group 6) 151
LIST OF APPENDIXES
I Estimation of Serum Troponin 217
II Estimation of Serum CK-MB Concentration 218
III Estimation
of Serum Level of Triglyceride (TG) 220
IV Estimation
of Total Serum Cholesterol 221
V Estimation of Serum Level of HDL-Cholesterol 221
VI Estimation of Haematological Indices 222
VII Estimation of Serum Malondialdehyde (MDA)
Level 223
VIII Estimation
of Serum SOD Activity 224
IX Estimation of Serum
Glutathione Peroxidase (GSPx) Activity 224
X Estimation
of Serum Catalase (CAT) Activity 225
XI Estimation of Alkaline
Phosphatase (ALP) Activity 225
XII Estimation of Aspartate Amino Transferase
(AST) Activity 226
XIII Estimation
of Alanine Amino Transferase (ALT) Activity 226
XIV Estimation of Serum Urea Nitrogen (BUN)
Concentration 227
XV Estimation of Serum Creatinine
Concentration 227
XVI Preparation of Extracts
Doses (mg/kg bw) 228
XVII Preparation of Aspirin 25 Mg
(mg/kg bw) 228
XVIII Calculation of 5-Fluorouracil
Doses (mg/kg bw) 228
LIST OF ABBREVIATIONS
5-FU 5-Fluorouracil
ACE Angiotensin
Converting Enzyme
ALP Alkaline Phosphatase
ALT Alanine Transaminase
AST Aspartate
Transaminase
BUN Blood Urea Nitrogen
CAD Coronary Artery
Disease
CAT Catalase
CBC Complete Blood Count
CDC Center for Disease
Control and Prevention
CELEPS Crude ethanol extract of Pterocarpus santalinoides
CHF Congestive Heart
Failure
CKD Chronic Kidney
Disease
CK-MB Creatine Kinase-MB
Fraction
CVD Cardiovascular
Disease
DNPH Dinitrophenyl Hydrazone
FBC Full Blood Count
FDA Food and Drug
Administration
FELEPS Fractions of Ethanol
Extract of Pterocarpus santalinoides
GGT Gamma-Glutamyl
Transferase
GSPx/GPx Glutathione Peroxidase
H &E Hematoxylin and Eosin
Hb/HGBS Haemoglobin
HDL-C High Density Lipoprotein
Cholesterol
HER2 Human Epidermal Growth
Factor Receptor 2
HF Heart Failure
HIV Human
Immunodeficiency Virus
LD50 Lethal
Dosage
LDL-C Low Density
Lipoprotein Cholesterol
LV Left Ventricle
LVD Left Ventricular
Dysfunction
LVEF Left Ventricular
Ejection Fraction
Mabs Monoclonal Antibodies
MDA Malondialdehyde
MEPS Methanol Leaf Extract
of Pterocarpus santalinoides
MI Myocardial
Infarction
NO Nitric Oxide
NSAID Nonsteroidal
Anti-inflammatory Drug
PMBC Peripheral blood
Mononuclear Cells
PS Pterocarpus santalinoides
RBC Red Blood Cell
SGOT Serum Glutamic
Oxaloacetic Transaminase
SGPT Serum Glutamic Pyruvic
Transaminase
SOD Superoxide Dismutase
TC Total Cholesterol
TKIs Tyrosine Kinase
Inhibitors
TnI Troponin I
TnT Troponin T
UA Uric Acid
VEGF Vascular Endothelial
Growth Factor
VLDL Very Low Density
Lipoprotein
WBC White Blood Cell
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
In many nations of the world, cardiovascular
diseases (CVD) are leading causes of deaths despite several advancements in the
medical interventions. Some of the CVDs include: coronary heart disease,
peripheral arterial disease, rheumatic heart disease, congenital heart disease,
strokes, myocardial ischemia and myocardial infarction (heart attack) (Arnett et al., 2014). Myocardial ischemia (MI)
remains a major pathological cause of death globally despite rapid advancements
continously made in the treatment of coronary diseases (Murray and Lopez, 1997;
Boudina et al., 2002). Most cancer therapies have been linked with
risk of causing cardiotoxicity (Curgliano et
al., 2010; Hahn et al., 2014). Over the years and even in recent times, the number of
deaths caused by cardiovascular diseases (CVD) have been on the increase even
in developed nations such as USA, UK, Germany and others. It’s a leading cause
of death in the United States. According to the Centers for Disease Control and
Prevention (CDC), one American dies from cardiovascular disease every 37 seconds. The most common factors that
can increase the risk for cardiovascular disease include high blood pressure,
high blood cholesterol, diabetes, smoking, sedentary lifestyle (physical
inactivity), and obesity.
Cardiotoxicity
refers to toxicity that affects the heart. It can be defined as new onset or worsening of
myocardial damage or ventricular function from baseline during follow-up (Sanz
and Zamorano, 2020). It can also be
referred to as the damage to the myocardium induced by medication, which can precipitate
pathological conditions such as heart failure (HF), hypertension and structural
damage. Cardiotoxicity can also be
defined as either the presence of symptoms of heart failure with greater or
equal to five percent (≥5%) reduction in ejection fraction to less than fifty -
five percent (<55%) or the absence of symptoms with an ejection-fraction
reduction ≥10% to <55% (Csapo and Lazar, 2014). Cardiotoxicity is a known adverse effect
of many conventional chemotherapeutic agents. many of the new cancer drugs also
interact with cardiovascular signalling and have important side effects,
particularly during times of increased cardiac stress (Suter and Ewe, 2013). Cardiac dysfunction and heart failure are
among the most serious cardiovascular side effects of systemic cancer treatment
(Suter and Ewe, 2013). When cancer patients undergo chemotherapy or
radiotherapy they eventually suffer from cardiotoxicity as a result of the
treatment, their quality of life and overall survival is severely affected, but
suspending the treatment of cancer for fear of the cardiotoxicity risk is
actually not the best option, therefore early detection of such condition by
pharmacists and physicians during chemotherapy is very crucial. Most already established and newer anticancer
drugs have this cardiotoxicity side effect.
In other not to deny a patient the the ability to benefit from cancer
treatment, a new field of medical specialty known as Cardio-oncology has been
established in other to manage the cardiotoxicity side effect while the patient
continues with the cancer treatment (Suter and Ewe, 2013). Cardio-oncology has
emerged as a sub-specialty to meet the challenges posed by a complex
interaction between cancer and the cardiovascular system and the cardiotoxicity
of conventional and newly developed cancer therapies (Fiuza et al., 2016; Diwakar et al., 2017).
Cardiotoxic drugs are divided into four categories:
1) Drugs that cause direct cytotoxicity on the heart resulting to
cardiac dysfunction: examples of such drugs include the alkylating agents,
anthracyclines, tyrosine kinase inhibitors (TKIs), interferon alfa and
monoclonal antibodies (Mabs)
2) Drugs that can cause cardiac ischemia:
examples include antitumor antibiotics, fluorouracil (5-FU), topoisomerase
inhibitors.
3) Drugs that can precipitate
cardiac arrhythmias: e.g. anthracyclines, etc.
4) Drugs that can cause pericarditis: e.g.
bleomycin, cyclophosphamide, cytarabine (Csapo and Lazar, 2014)
According to the system
proposed by Ewer et al. (2013),
cardiotoxicity of cancer drugs can be categorized based on their potential to
cause irreversible damage (type 1) or reversible damage (type 2). Type 1 damage
is usually caused by a cumulative dose; type 2 damage is not related to a
cumulative dose. Examples of anti-cancer
agents that can cause irreversible toxicity include anthracyclines
(daunorubicin, idarubicin, doxorubicin, epirubicin); taxanes (docetaxel,
paclitaxel, cabazitaxel); topoisomerase inhibitors (etoposide, tretinoin, vinca
alkaloids); alkylating agents (busulfan, carboplatin, mitomycin, carmustine,
chlormethine, cisplatin, cyclophosphamide); and antimetabolites (cladribine,
cytarabine, 5-FU) (Csapo and Lazar, 2014; Romond et al., 2005).
The most common chemotherapy
agents associated with irreversible damage to the heart are the anthracyclines.
Anthracyclines, especially doxorubicin, are used to treat several types of
cancer, including breast, gynecologic, sarcoma, and lymphoma. Csapo and Lazar
(2014) reported that the mechanisms by which anthracyclines cause
cardiotoxicity is by inducing necrosis and apoptosis of cardiac myocytes and
subsequent myocardial fibrosis. Doxorubicin-induced cardiotoxicity
involves several processes, such as the formation of iron-dependent oxygen free
radicals and peroxidation of lipids in the membrane of myocardial mitochondria,
which results in suppression of DNA, RNA, and proteins; thereby causing altered
adenylyl cyclase activity and disrupted calcium homeostasis (Romond et al., 2005). Cumulative doses are responsible for increasing
the risk of anthracyclines-induced cytotoxicity.
Monoclonal antibodies (Mabs)
are the main cause of type 2 damage.
They are commonly used in the management of many types of cancer.
Chemotherapy agents that can cause reversible cardiotoxicity are trastuzumab,
lapatinib, sunitinib and bevacizumab. These anticancer agents can also cause
hypertension. The mechanism could be by causing a decrease in vascular
endothelial growth factor (VEGF) which in turn results in a reduction of nitric
oxide (NO) in the arteriolar wall hence causing increased vascular resistance. In
breast cancer, aggressive disease and a worse prognosis have been attributed to
human epidermal growth factor receptor 2 (HER2)–positive receptors (Suter and
Ewer, 2013). Csapo and Lazar (2014) reported in their review
that trastuzumab, a humanized Mab, has shown a 50% reduction in recurrence
rates and a 33% improvement in survival (Csapo and Lazar, 2014).
Available data suggest that the cadiotoxicity induced by trastuzumab is
as a result of blockage of HER2 receptors. HER2 receptors are
important for embryonic cardiac development and for protection of the heart
from cardiotoxins, hence they are present on cardiac myocytes (Groarke and
Nohria, 2015). As a result, when the gene of these receptors are suppressed,
dilated cardiomyopathy develops. This helps identify the difference between
type 1 and type 2 cardiotoxicity; type 1 has a greater association with cardiac
dysfunction and clinical heart failure (HF), and type 2 leads to an increased
loss of contractility and less death of cardiomyocytes and can reverse (Suter
and Ewer, 2013).
Suter and Ewer (2013)
proposed a system to identify drugs that cause the different types of damage, and
they defined cardiotoxicity as a serial decline in left ventricular ejection
fraction (LVEF). The American Society of Echocardiography
defined cardiotoxicity as an LVEF drop from >10% to <53%. The Cardiac
Review and Evaluation Committee proposed a definition of left ventricular
dysfunction (LVD) to be “a decline in cardiac LVEF; presence of symptoms of
congestive heart failure (CHF); associated signs of CHF, including but not
limited to S3 gallop, tachycardia, or both; and decline in LVEF of at least 5%
to less than 55% with accompanying signs or symptoms of CHF, or a decline in
LVEF of at least 10% to below 55% without accompanying signs or symptoms
(Groarke and Nohria, 2015).
Many cancer survivors are living
with long-term adverse effects of cancer therapy with pathological organ
systems. Cardiovascular toxicity of
anticancer drugs is one of the most life-threatening adverse effects. Several
anticancer agents, such as anthracyclines (daunorubicin, doxorubicin,
epirubicin, idarubicin), trastuzumab, cyclophosphamide, antimetabolites
(cladribine, cytarabine, 5-fluorouracil), alkylating agents (busulfan,
carboplatin, carmustine, chlormethine, cisplatin, cyclophosphamide, mitomycin),
angiogenesis inhibitors, taxanes (docetaxel, cabazitaxel, paclitaxel),
topoisomerase inhibitors (vinca alkaloids, etoposide, tretinoin,) and tyrosine
kinase inhibitors (TKIs) have been linked with an increase in the risk of
cardiovascular morbidity and mortality (Panjrath and Jain, 2007; Saif et al., 2009; Frickhofen et al., 2002; Cardinale et al., 2015; Rowinsky, et al., 1991; Dutcher, et al., 2001;Chu, et al., 2007).
Childhood cancer survivors face a
high lifetime risk of late cardiovascular disease (Reulen et al., 2010; Lipshultz, et
al., 2012). Patients with pre-existing cardiovascular diseases are at
higher risk of cardiotoxicity compared to those that have healthy
cardiovascular systems. The spectrum of cardiovascular complications of cancer
therapy includes left ventricular (LV) dysfunction, congestive heart failure
(CHF), coronary vasospasm, angina, myocardial infarction (MI), arrhythmias,
systemic hypertension, pericardial effusion, pulmonary fibrosis and pulmonary
hypertension (Schwartz et al., 2013).
A newly emerging subspecialty known as cardio-oncology is designed to address
the complex interaction between cancer and cardiovascular system through
monitoring, early detection, prevention and treatment of cardiotoxicity of
cancer therapies; development of newer therapies with lower or no
cardiotoxicity; and careful planning of cancer therapy in patients with
pre-existing cardiovascular disease to avoid overt cardiotoxicity and heart
failure (Albini et al., 2010; Russell
et al., 2016).
About 80% of worldwide populations depend on
traditional medicines to meet their primary health-care needs (Ullah et al., 2010). Plants are primary
sources of medicines, food, shelters and other items that are daily made use of
by humans. Their stems, leaves, flowers, roots, fruits and seeds provide food
for humans (Amaechi, 2009). Nigeria is
not left out in the medicinal application of plants as plants are employed in
Nigeria for the treatment of many kinds of disease conditions. Infact public
opinion has it that nature has given the cure of every disease in one way or
another (Tiwari et al., 2011). Pterocarpus santalinoides is one of such
plant species used for the management and treatment of ailments (Iwu, 1993;
Dieye et al., 2008). The
people of South Eastern part of Nigeria use the leaves of this plant to prepare
soup especially for women who just gave birth, the leaves are also used to
treat gastro-intestinal diseases, diabetic syndrome and skin diseases (Anowi et al., 2012). In North Central Nigeria, the leaves of this
plant are used in treatment of pain and inflammation of lower abdomen, stomach
ache and other infectious diseases (Igoli et
al., 2003). In India, fruit extracts
of this plant are used traditionally in treating skin diseases, boils, fevers,
headache, etc (Jain et al., 2013). The stem bark extracts of Pterocapus santalinoides were reported to have anti-diabetic,
antibacterial and hepatoprotective activities (Jain et al., 2013).
Various parts of Pterocarpus
santalinoides are employed in
traditional medicine in many African countries, to treat an array of human
ailments. The ethno-medical use of leaves of Pterocarpus santalinoides in the treatment of diarrhoea and other
gastrointestinal disorders as well as its triglyceride and glucose lowering
properties has been experimentally verified (Prado, 2000; Okpo et al., 2011). The leaves of Pterocarpus santalinoides have antimicrobial activity and contain
phytochemicals such as alkaloids, tannins, saponins, terpenoids, flavanoids and
anthraquinones which might be responsible for this activity (Ukwueze et al., 2018). Ethanol extract of the leaves of Pterocarpus santalinoides has been shown to significantly increase the levels
of haematological parameters such as haemoglobin, packed cell volume and
platelets (Offor et al., 2015). The
leaves of Pterocarpus santalinoides
are used in the treatment of skin diseases such as eczema, candidiasis and acne,
the bark extracts are used in the treatment of diabetes, cough and sore (Osuagwu
and Akomas, 2013; Igoli et al., 2005;
Ama, 2010; Okwuosa et al., 2011). Methanol leaf extract of Pterocarpus santalinoides (MEPS) does not cause significant
toxicity in albino rats, when administered for a short duration, rather long
term therapy with the extract could precipitate liver and kidney damage
(Madubuike et al., 2020).
1.2 Pterocarpus santalinoides
Plate 1.1: Pterocarpus
santalinoides
Source: https://tropical.theferns.info
Pterocarpus santalinoides, of the Leguminosae: papilionoideae family is an evergreen
tree with a dense crown of drooping branches as shown in Plate 1.1. It is
essentially bi-continental in distribution being native to tropical Western
Africa and South America (Prado, 2000) and usually called red sandal wood in
English (Offor, et al., 2015). The plant grows in the bush in Nigeria
and is known in various Nigerian vernaculars as nturukpa (Igbo); okumeze (Edo);
nja (Efik); gbengbe (Yoruba); gunduru or gyadar kurmi (Hausa); maganchi (Nupe);
ikyarakya or kereke (Tiv) (Odeh and Tor-Anyiin, 2014). A fully grown tree of Pterocarpus santalinoides is usually very
tall about 9 -15 m tall, the trunk of the tree is up to 1 m in diameter and has
a flaky bark, pinnate leaves (10–20 cm long) with 5–9 leaflets, the flowers are
orange-yellow and produced in panicles; bears fruit in pods 3.5 - 6 cm long,
with a wing extending three-quarters around the margin (Prado, 2000; Keay,
1989).
Taxonomy:
Domain: Eukaryot
Kingdom: Plantae
Subkingdom: viridaeplanntae
Phylum: Magnoliophyta
Subphylum: Euphyllophytina
Infraphylum: Radiatopses
Class: Magnoliopsida
Subclass: Rosidae
Superorder: Fabanae
Order: Fabale
Family: Fabaceae
Subfamily: Faboideae
Tribe: Dalbergieae
Genus: Pterocarpus
Specie: Pterocarpus
santalinoides (ILDIS, 2005; WAC,
2008)
Synonym(s):
Pterocarpus amazonicus Huber; Pterocarpus
esculentus Schum. & Thonn.; Pterocarpus grandis Cowan; Pterocarpus
michelii Britton.
Common
names: (English): red wood sandal; (Hausa):
gunduru, gyadar kurmi; (Igbo): nturukpa; (Yoruba): gbengbe; (Ibibio): mkpafere
idim
Botanic description: the name Pterocarpus is derived from the Greek words ‘pteran’ meaning a wing
and, ‘karpos’ meaning’ fruit. The specific epithet ‘santalinoides’ refers to
its likeness to P. santalinus found in Asia. P. santalinoides is a shade tolerant
tree commonly found along riverine forests in Africa and tropical South
America. Pterocarpus
santalinoides is a tree 9-12 m tall with thin and flaking bark, exuding
drops of red gum. They possess compound
leaves containing 5-9 leaflets. Their leaf stalks are slender, about 10-20 cm
long, leaflet stalk stout 2-5 mm long. Their fruits are light brown pod of
about 3.5-6 cm. Their flowers are orange-yellow with narrowly cup-shaped calyx
and petals that are densely hairy on the outside (WAC, 2008).
Uses: The young shoots and leaves can serve
as fodder for livestock, the leaves are eaten by humans as vegetable. The shoot
of mature Pterocarpus santalinoides is a source of termite resistant
wood and red gum can be obtained from the stem when given a cut. Tannins and
dyes obtained from the bark can be used for dyeing. The leaves and tree bark are
ethnomedically used for the treatment of several ailments such as skin disease,
stomach ache, diarrheoa, diabetic syndrome, inflammation of the lower abdomen
and infectious diseases (Prado, 2000; Okpo et
al., 2011; Osuagwu and Akomas, 2013; Igoli et al., 2005; Ama, 2010; Okwuosa et al., 2011). This plant
is an important species for soil conservation in water catchment areas and good
source of shade around settled areas and farms where it provides protection
from wind. The nitrogen fixing activity in its root nodules coupled with the
decomposition of its leaf litter improves soil fertility. The plant is used as
an ornamental tree and its poles have been used for fencing (Orwa et al., 2009). Its flowers can also
provide ecstatic view beautifying the environment (WAC, 2008). It has been
listed among the species threatened by extinction (IUCN, 2023).
1.3 SCOPE OF THE STUDY
This study is
limited to:
i.
Collection and preparation of leaves of Pterocarpus santalinoides
ii.
Preparation of ethanol crude extract of the leaves
iii.
LD50 Determination of the ethanolic crude extract
iv.
Qualitative phytochemistry
v.
Collection and weighing of the experimental animals.
vi.
Daily administration of standard drugs and extract to the
animals.
vii.
Induction of cardiac injury using 5-fluorouracil
viii.
Sacrificing of experimental animals for collection of sera,
blood and heart samples
ix.
Assay of lipid profile
x.
Assay of troponin
xi.
Assay of haematological indices
xii.
Assay of creatine kinase (CK-MB)
xiii.
Histological studies of the hearts
xiv.
Fractionation of the ethanol crude extract into n-butanol,
ethyl acetate and chloroform fractions
xv.
Treatment of animals using the different fractions and
standard drug
xvi.
Induction of cardiac injury using 5-fluorouracil
xvii.
Sacrificing of the animals
xviii.
Evaluation of the cardiac function biomarkers: lipid
profile, troponin, CK-MB
xix.
Assay of oxidative status biomarkers: Malondialdehyde
(MDA), glutathione peroxidase (GSPx), superoxide dismutase (SOD), catalase (CAT)
xx.
Assay of renal function biomarkers: Electrolytes, urea and creatinine
xxi.
Assay of liver function biomarkers: Alkaline phosphatase
(ALP), aspartate amino transferase (AST) and alanine amino transferase (ALT)
activities
xxii.
Haematological indices determination
xxiii.
Histopathological studies of their hearts, livers and
kidneys
1.4 STATEMENT OF PROBLEM
Several important therapeutic agents have been linked with risks of
cardiotoxicity, for example most anti-cancer agents have been proven to
precipitate this ugly side effect.
Treatment of disease conditions is a major way of reducing casualties
and mortalities from these diseases.
Side effect is a phenomenon common to most drugs and considering the
expediency of these therapeutic agents, it becomes necessary to come up with an
additional substance that can confer a protective effect on the heart from the
cardiotoxicity potential of these chemotherapeutic agents and that is why we
chose to study the effectiveness of the leaves of Pterocarpus santalinoides in this regard.
Pterocarpus
santalinoides is a tropical flowering tree that is widely distributed in
Nigeria and in other tropical countries such as Cameroon, Ghana, Senegal and
Brazil. It has many applications as
earlier mentioned but our interest bothers around the medicinal uses. In the South Eastern part of Nigeria, the
leaves are used to cure gastro-intestinal diseases, diabetic syndrome and skin
diseases (Anowi et al., 2012; Ama,
2010; Osuagwu and Akomas, 2013). In
North Central part of Nigeria, the leaves are used to treat lower abdominal
pain and inflammation, stomach ache and other infectious diseases (Igoli et al., 2005). In India, fruit extracts
are used traditionally for the treatment of skin diseases, boils, fevers,
headache, etc. The stem bark extracts
were reported to have hepatoprotective, antibacterial and anti-diabetic activities
(Jain et al., 2013). The ethnomedicinal
use of leaves of Pterocarpus santalinoides in the treatment of
diarrhea and in lowering blood triglyceride and glucose levels has been experimentally
verified (Iwu, 1993; Ojiako and Nwanjo, 2006).
It is believed that nature has provided the cure of every disease in one
way or another (Okpo et al., 2011). Hence, this research was carried out in other
to experimentally verify if the ethanol leaf extract of this plant could have a
cardioprotective impact on 5-fluouracil induced cardiotoxicity in wistar rats
which could provide insight on the possibility of using it as porphylatic
substance that could be used by cancer patients undergoing chemotherapy to
prevent cardiotoxicity side effects of the anti-cancer drugs.
1.5 JUSTIFICATION
OF THE STUDY
Among other health
benefits attributed to the various parts of Pterocarpus
santalinoides, is its ability to lower blood LDL-cholesterol and enhance
its HDL-cholesterol level, factors which are important indices for measuring
cardiovascular health, this effect has been experimentally verified (Ihedioha et al.,
2018; Okwuosa et al., 2011). In a similar vein, the authenticity of the
cardiotoxicity risk of many chemotherapeutic agents most especially as it
applies to anti-cancer drugs is non – negotiable and an existing fact. Therefore, considering the expediency of
these therapies and the lethality of the cardiotoxicity potential, it will be
more beneficial to identify another harmless but cardioprotective substance
that would be administered to patients before subjecting them to chemotherapy. This could be done in order that the
additional substance can help prevent or significantly reduce the
cardiotoxicity potential of these very important therapeutic agents. This will in turn prevent the prohibition of
the sale or use of such drugs on account of the lethal risks associated with
them, but will encourage their continued usage in as much as the heart and the
components of the cardiovascular system are appreciably protected from their
harmful effects. Because of the
aforementioned medicinal uses of the leaves of Pterocarpus santalinoides, it became expedient to subject its possible
cardioprotective potential against 5-fluorouracil induced cardiotoxicity to
experimental scrutiny and verification. With the aim that if it comes out very
effective compared to aspirin (a known cardioprotective drug), and considering
the low toxicity profile of the tender leaves of this plant, it may be a
preferred adjuvant to chemotherapeutic agents which can protect the heart of
the cancer patients from the cardiotoxicity adverse effects of such drugs.
1.6 RELEVANCE OF
THE STUDY
The assessment of the cardio-protective potential of ethanol leaf extract
and fractionated products of Pterocarpus
santalinoides is necessary because of the following reasons:
1.
It will reveal if the crude
ethanol leaf extract of the Pterocarpus
santalinoides has cardioprotective protective potential on patients on
5-fluoracil
2.
It will reveal whether or not
the fractionated version of the extract will have stronger cardioprotective
potential on the wistar rats than the crude extract
3.
It will show whether or not the
crude ethanol leaf extract and the fractionated portions of the extract will
have stronger cardioprotective potential on the wistar rats than aspirin
4.
It will reveal the harmful
effects 5-fluorouracil treatment on the heart, liver and kidneys as well the
protective effect of the ethanol leaf extract against these toxicities.
5.
This study will reveal the
possible mechanism of cardiotoxicity of 5-fluorouracil as well as the probable
mechanism of cardioprotectiveness of ethanol leaf extract and their n-butanol,
ethyl-acetate and chlroform fractions.
1.7 AIM OF THE
STUDY
The aim of this study is to assess the
cardioprotective potentials of the ethanol leaf extracts and fractions of Pterocarpus santalinoides in Wistar rats.
1.8 OBJECTIVES OF
THE STUDY:
Specifically, the
study sought to:
i.
Carry out qualitative phytochemical
screening of the crude ethanol leaf extract
ii.
Determine the LD50 of
the crude ethanol leaf extract in albino mice
iii.
Determine cardiovascular
indices (serum troponin concentration and CK-MB Activity)
iv.
Determine serum lipid profile
(TC, LDL-C, HDL-C, VLD-C and TG)
v.
Determine haematological
indices
vi.
Determine cardiovascular risk
index
vii.
Determine liver function status
(serum ALP, ALT and AST activity)
viii.
Determine kidney function
status (serum electrolytes, urea and creatinine levels)
ix.
Determine levels of oxidative
damage (serum malonyldialdehyde levels)
x.
Determine serum activities of
antioxidant enzymes (SOD, CAT, GSPx)
xi.
Carry out histopathological
evaluations of the organs (heart, liver and kidney)
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