Assessment Of The Cardioprotective Potential Of The Ethanol Leaf Extract Of Pterocarpus santalinoides In Wistar Rats
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 (
) 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 () 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.
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
0.0
BLESSING, U (2023). Assessment Of The Cardioprotective Potential Of The Ethanol Leaf Extract Of Pterocarpus santalinoides In Wistar Rats. Repository.mouau.edu.ng: Retrieved Jul 27, 2024, from https://repository.mouau.edu.ng/work/view/assessment-of-the-cardioprotective-potential-of-the-ethanol-leaf-extract-of-pterocarpus-santalinoides-in-wistar-rats-7-2
UCHECHI, BLESSING. "Assessment Of The Cardioprotective Potential Of The Ethanol Leaf Extract Of Pterocarpus santalinoides In Wistar Rats" Repository.mouau.edu.ng. Repository.mouau.edu.ng, 14 Aug. 2023, https://repository.mouau.edu.ng/work/view/assessment-of-the-cardioprotective-potential-of-the-ethanol-leaf-extract-of-pterocarpus-santalinoides-in-wistar-rats-7-2. Accessed 27 Jul. 2024.
UCHECHI, BLESSING. "Assessment Of The Cardioprotective Potential Of The Ethanol Leaf Extract Of Pterocarpus santalinoides In Wistar Rats". Repository.mouau.edu.ng, Repository.mouau.edu.ng, 14 Aug. 2023. Web. 27 Jul. 2024. < https://repository.mouau.edu.ng/work/view/assessment-of-the-cardioprotective-potential-of-the-ethanol-leaf-extract-of-pterocarpus-santalinoides-in-wistar-rats-7-2 >.
UCHECHI, BLESSING. "Assessment Of The Cardioprotective Potential Of The Ethanol Leaf Extract Of Pterocarpus santalinoides In Wistar Rats" Repository.mouau.edu.ng (2023). Accessed 27 Jul. 2024. https://repository.mouau.edu.ng/work/view/assessment-of-the-cardioprotective-potential-of-the-ethanol-leaf-extract-of-pterocarpus-santalinoides-in-wistar-rats-7-2