I TITLE PAGE ANTI-MALARIAL ACTIVITY AND

TITLE PAGE

ANTI-MALARIAL ACTIVITY AND PHARMACOKINETIC PROFILES OF METHANOL EXTRACT OF Strophanthus hispidus LEAVES

A PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D) IN BIOCHEMISTRY (PHARMACOLOGY)

UKEGBU, CHIMERE YOUNG PG/Ph.D/14/76645

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA

SUPERVISORS: DR. PARKER E. JOSHUA PROF O.F.C NWODO

NOVEMBER, 2018

CERTIFICATION

Ukegbu, Chimere Young, a postgraduate student of the Department of Biochemistry, Faculty of Biological sciences, University of Nigeria, Nsukka with Registration Number PG/Ph.D/14/76645 has satisfactorily completed the requirements for the research work for the degree of Doctor of Philosophy (Ph.D) in Pharmacological Biochemistry. The work embodied in this report is original and has not been submitted, in part or full, for any other diploma or degree of this or any other University.

------Dr. P. E. Joshua Prof. O. F. C. Nwodo (Project supervisor) (Project supervisor)

------

Prof. F. C. Chilaka External examiner (Head of Department)

DEDICATION

This research work is dedicated to God-Almighty, the father of my lord Jesus Christ, the Source of my strength, inspiration and wisdom.

iii

ACKNOWLEDGEMENTS

I wish to express my profound gratitude to the Almighty-God who has blessed me and brought me thus far. I sincerely appreciate my Supervisors, Prof. OFC Nwodo and Dr. Parker E. Joshua, whose love for excellence and speed spurred me into action. I am convinced to say that you are more than academic Supervisors. You are role models! I also wish to thank the lecturers in the Department of Biochemistry for their dedication and selflessness in discharging their duties. First I want to thank the Head, Prof F. C. Chilaka and to mention just a few, Prof. L. U. S. Ezeanyika, Prof. I. N. E. Onwurah, Prof. F. C. Chilaka, Prof. O. U. Njoku, Prof. S. O. O. Eze, Prof. H. A. Onwubiko, Prof. B. C. Nwanguma, Dr. V. N. Ogugua, Dr. C. S. Ubani, Dr. (Mrs.) C. A. Anosike and Dr (Mrs) U. O. Njoku, for their constructive criticism during my seminar presentations which helped to validate the scientific content of my research work. I deeply appreciate my family whose support has been matchless. Worthy of mention is my father, Mr. Ukegbu, Chinyere Young, who believed in me and kept on telling me “you will go places, you have the qualities to stand anywhere in the world and lead any group in the world”; my sweet and hardworking mother, Mrs. Ukegbu, Hellen who has sacrificed a lot to ensure that my dreams are fulfilled; my only brother, Ukegbu Uzoukwu is greatly appreciated for always being there for me and my sisters, Nkechi, Ugoma, and Precious Oluebube is also thanked for their numerous calls to know the progress of my studies. I will not forget to express my profound gratitude to the chief laboratory scientist at Department of Pharmacognosy, Faculty of Science, Amadu Bello University (ABU) Zaria, Mallam Ibrahim Kaibiru for his contribution to this research work and other post-graduate students who worked with me in the laboratory, just to mention a few, Atinga, Garuba and Emmanuel. Special thanks to natural product laboratories, Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS), University of Strathclyde, Glascow, United Kingdom for the 1H NMR and 13C NMR spectra analysis. I wish to appreciate Mr Nathaniel Friday who accommodated me in all my stay at Zaria without any cost and every member of Post-graduate Students’ Fellowship, Zaria and also members of Agape Choir Chapel of Redemption ABU Zaria. I also wish to appreciate the kind gesture of Mr Lanre Durojaiye and Mr Tobechukwu Paul Okoli who laboured in the in silico pharmacokinetics study and reference formatting respectively. Also worthy of note is the morale support and scientific judgement at different phases of this research work by my colleagues, Mr Abonyi Obiora, Casmir Ezeagu and Solomon Odiba. The happy moments I shared with the following people also provided the needed energy to endure till the end. They include my friends

– Okechukwu Iroha, Onosakponome, Nwambam Ernest, Precious Chris ; my roommates past and present; Tosin, Irrenonsen, Samplus, Don, Wilson, Paul, Papa Bello, Bishop, Mamud, Bro Mike, Cyrill, Tony and Maxwel. Finally My choir members both in fellowship and church (Christ Church Chapel, Franco Outreach) just to mention a few; Victor, Joseph, Miracle, Nene, Chidinma, Ogechi, Prisca, Joy, Nkwachukwu, Ese, Jibrin, Faith, and my brethren at Graduate Students’ Fellowship. My spiritual fathers at home and in school here are not forgotten Rev. Uche, Rev. Prince, Rev. Prof. Christopher okeke, Pst. Don and members of Assemblies of God Church No. 1 Riverlayout Aba. To you all I say a big “thank you” for your love, companionship and prayers.

ABSTRACT

Strophantus hispidus is a plant known to be used locally in the treatment of several diseases including malaria. Currently, there is sperse scientific reports found on the anti-malarial activity and bioactive compounds of the plant leaves. Thus the aim of the study was to investigate the antimalarial properties of the methanol extract of Strophanthus hispidus (MESH) leaves and the possible elucidation of the bioactive compound(s). The percentage yield of the methanol extract of Strophanthus hispidus leaves obtained was 12.53%. The phytochemical compositions of the plant extract showed the presence of bioactive organic compounds such as alkaloids, flavonoids, terpenoids, steroids, phenols, saponins, tannins, soluble carbohydrates, reducing sugars and glycosides. The LD50 of the extract in mice was found to be above 5000 mg/kg b.w. Antimalarial activity of the crude extract of Strophanthus hispidus leaves showed that the extract was effective at various doses of 200, 400 and 800 mg/kg b.w. and showed a dose-dependent significant (p < 0.05) reduction in the parasitemia count of all the treatment groups when compared to the positive (untreated) control group. The P.berghei infected mice treated with various doses of the extract showed a significantly (p < 0.05) higher packed cell volume, red blood cell count and haemoglobin concentration compared to the positive control group. Conversely the packed cell volume, red blood cell count and haemoglobin concentration of the treatment groups showed significant (p < 0.05) decrease when compared to the negative control group. The mice treated with various doses of the extract showed significant (p < 0.05) reduction in the activities of liver marker enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) compared to the positive control group. Partitioning of the extract with different solvents gave the following percentage yields: n-Hexane (22%), dichloromethane (20%) and ethyl acetate (35.38%). Treatment with various doses (400 and 800 mg/kg b.w) of the fractions showed a dose dependent significant (p < 0.05) reduction in the parasitemia count of all the groups when compared to the untreated group. The P. berghei-infected mice treated with various doses of the fractions showed significantly (p < 0.05) higher packed cell volume, red blood cell count and haemoglobin concentration compared to the positive control group. The mice treated with various doses of the fractions showed significant (p < 0.05) reduction in the activities of liver marker enzymes (ALT, AST and ALP) compared to the positive control group. The animals treated with the n-Hexane fraction showed a non-significant difference (p > 0.05) in the AST and ALP activities when compared to the Negative control. Activities of some antioxidant enzymes such as superoxide dismutase (SOD) and catalase activity were found to be significantly (p < 0.05) higher in the groups treated with the fractions when compared to the positive control. There was a significant (p < 0.05) reduction in the formation of hemozoin in the groups treated with the non- polar fractions when compared to the positive control group. However comparatively the non- polar fractions were more effective than the polar fraction with n-Hexane fraction being the most active fraction. The calculated LC50 of the S. hispidus n-hexane fraction was found to be 245.5 µg/ml using Brine shrimp lethality Assay. Ninety-five fractions were obtained from column chromatography and combined based on their TLC profiles to give 9 fractions. The 9 fractions showed various levels of potency by inhibiting β-hematin synthesis. However, fractions 7-9 were the most potent fractions with percentage inhibition of 93%. Beta-setosterol-d-glucoside a steroid was identified as the active compound using 1H NMR, 13C NMR and IR spectra. ADME based on the Lipinski’s rule of 5 analysis revealed molecular weight (MW) of 576.86, hydrogen bond acceptor (HBA) of 6.0, hydrogen bond donor (HBD) of 4.0 and logP o/w 5.51. Beta-setosterol-d- glucose as the most active compound could take a lead in the discovery of new anti-malarial agent or used in combination therapy with artemisinin (ACT). vi

TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgements iv

Abstract vi

Table of Contents vii

List of Figures xiv

List of Tables xvii

CHAPTER ONE: INTRODUCTION 1

1.1 Strophanthus hispidus 2

1.1.1 General Description of Strophanthus hispidus 2

1.1.2 Taxonomy of Strophanthus hispidus 4

1.1.3 Pharmacological/ Medicinal Importance of Strophantus hispidus 4

1.2 Phytochemicals 5

1.2.1 Alkaloids 5

1.2.2 Phenols 6

1.2.3 Flavonoids 7

1.2.4 Tannins 8

1.2.5 Terpenoids 8

1.2.6 Steroids 9

1.2.7 Saponins 9

1.2.8 Glycosides 10

vii

1.3 Malaria 11

1.3.1 Geographical Distribution and Populations at Risk 12

1.3.2 Causative Agents 13

1.3.3 Transmission and Biology of P. falciparum 14

1.3.4 Liver Stage 14

1.3.5 Erythrocytic Stage 15

1.3.6 Gametocyte Differentiation 16

1.3.7 Mosquito Stage 16

1.3.8 Oxidative stress in Malaria and Symptoms 18

1.3.9 Treatment of Malaria 19

1.3.9.1 Artemesinin-based Combinations 19

1.3.9.2 Traditional Antimalarial Herbs 21

1.3.10 Diagnosis 22

1.3.10.1Microscopy 23

1.4 The Blood 23

1.4.1 Haemoglobin Estimation 24

1.4.2 Packed Cell Volume (PCV) 25

1.4.3 Red Blood Cell (RBC) Count 25

1.4.4 White Blood Cell (WBC) Count 25

1.5 Liver Function Tests 26

1.5.1 Alkaline Phosphatase (ALP) 26

1.5.2 Aspartate Aminotransferase 27

1.5.3 Alanine Aminotransferase 27

viii

1.5.4 Bilirubin 27

1.6 Antioxidants 28

1.6.1 Superoxide Dismutase 28

1.6.2 Catalase 29

1.7 Lipid Peroxidation and Tissue Damage 30

1.8 Haemozoin 30

1.9 Calcium ion 31

1.10 Brine shrimp lethality bioassay 32

1.11 Chromatography 32

1.11.1 Thin Layer Chromatography 33

1.11.2 Column Chromatography 34

1.12 Infrared Spectroscopy 34

1.13 Nuclear Magnetic Resonance 35

1.13.1 Proton and Carbon Nuclear Magnetic Resonance 36

1.14 Pharmacokinetics 37

1.14.1 In silico Pharmacokinetics 39

1.14 Rationale of the study 39

1.14.1 Aim of the Study 40

1.14.2 Specific Objectives of the Study 40

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials 41

2.1.1 Animals 41

2.1.2 Plant Materials 41

2.1.3 Rodent Malaria Parasite 41

2.1.4 Drugs 41

2.1.5 Instruments/Equipment 41

2.1.6 Chemicals and Reagents 42

2.2 Methods 42

2.2.1 Collection of Plant Materials 42

2.2.2 Preparation of Plant Extracts 42

2.2.3 Determination of Extract Yield 42

2.2.4 Solvent-Solvent Partitioning of Crude Extract 42

2.2.5 Determination of Fraction Yield 43

2.2.6 Qualitative Phytochemical Analysis of the Different Fractions of Strophantus hispidus Methanol Seed Extract 43

2.2.7 Quantitative Phytochemical Analysis 44

2.2.8 Animal Studies 46

2.2.9 Solvent-Solvent Partitioning of Crude Extract 49

2.2.10 Determination of Fraction Yield 49

2.2.11 Preparation of Sample Solutions 49

2.2.12 Microscopic Examination 50

2.2.13 Determination of Haemoglobin Concentration 50

2.2.14 Determination of Red Blood Cell (RBC) Count 51

2.2.15 Determination of Packed Cell Volume 51

2.2.16 Determination of Total White Blood Cell Count 51

2.2.17 Assay of Alkaline Phosphate Activity 51

2.2.18 Assay of Aspartate Aminotransferase Activity 52

2.2.19 Assay of Alanine Aminotransferase Activity 54

2.2.20 Determination of Total Bilirubin Concentration 55

2.2.21 Assay of Superoxide Dismutase (SOD) Activity 56

2.2.22 Assay of Catalase Activity 56

2.2.23 Determination of Malondialdehyde 57

2.2.24 Determination of Plasma Calcium Concentration 58

2.2.25 Determination of Haemozoin Concentration 59

2.2.26 Histopathological Examination 59

2.2.27 Brine shrimp lethality bioassay 61 2.2.29 Bioassay guided fractionation, isolation and characterization of pure compound 62

2.2.28 In Vitro Anti-Plasmodial Investigation using β-hematin Inhibitory Assay (BHIA) 62

2.2.30 Identification of pure compounds 63

2.2.31 In silico “Drug-likeness” Analysis and Pharmacokinetic Prediction 63

2.2.32 Statistical Analysis 63

CHAPTER THREE: RESULTS

3.1 Percentage Yield of Extract 64

3.2 Outcome of Acute Toxicity Studies (LD50) 64

3.3 Qualitative Phytochemical Composition of Strophantus hispidus Methanol Leaf Extract 64 3.4 Quantitative Phytochemical Composition of Strophantus hispidus Methanol Leaf Extract 64 3.5 Biochemical Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on parasitemia count (PC) of mice passaged with Plasmodium berghei 67

3.6 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on the Packed cell Volume of mice passaged with Plasmodium berghei 69 3.7 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Haemoglobin Concentration (Hb Conc) of mice passaged with Plasmodium berghei 71

3.8 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on the Red blood cell (RBC) count of mice passaged with Plasmodium berghei. 73

3.9 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on White blood cell (WBC) count of mice passaged with Plasmodium berghei 75

3.10 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase (ALT) activity of mice passaged with Plasmodium berghei 77

3.11 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of mice passaged with Plasmodium berghei 79

3.12 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Alkaline phosphatase activity of mice passaged with Plasmodium berghei 81

3.13 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Total bilirubin concentration (T.BIL conc) of mice passaged with Plasmodium berghei 83

3.14 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of mice passaged with Plasmodium berghei 85

3.15 Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase activity of mice passaged with Plasmodium berghei 87

3.16 Percentage yield of the Sequential Partitioning of the methanol extract 89

3.17 Phytochemical analysis of the different partitions of Methanol Extract of Strophantus hispidus Leaves 89

3.18 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the mean parasitemia count (PC) of mice passaged with Plasmodium berghei 91

xii

3.19 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Packed cell Volume of mice passaged with Plasmodium berghei 93

3.20 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Haemoglobin conc. of mice passaged with Plasmodium berghei. 95

3.21 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Red blood cell count of mice passaged with Plasmodium berghei. 97

3.22 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on White blood cell count of mice passaged with Plasmodium berghei 99

3.23 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase activity of mice passaged with Plasmodium berghei 101

3.24 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of mice passaged with Plasmodium berghei 103

3.25 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Alkaline phosphatase activity of mice passaged with Plasmodium berghei 105

3.26 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Total bilirubin conc of mice passaged with Plasmodium berghei 108

3.27 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of mice passaged with Plasmodium berghei 110

3.28 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase activity of mice passaged with Plasmodium berghei 112

3.29 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Catalase activity of mice passaged with Plasmodium berghei 114

xiii

3.30 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Haemozoin concentration of mice passaged with Plasmodium berghei 116

3.31 Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on plasma calcium (PC) ion of mice passaged with Plasmodium berghei 118

3.32 In vitro Cytotoxicity test of Strophantus hispidus N-hexane fraction using brine shrimp lethality assay (BSLA) 124

3.33 Column chromatography and fractions obtained based on their thin layer chromatography profiles 124

3.34 Thin layer chromatography (TLC) profiles of different fractions 124

3.35 In vitro antimalarial activity of different fractions of n-hexane partition of methanol extract of Strophantus hispidus leaves using β-hematin inhibition assay (BHIA) 124

3.36 Preparative Thin Layer Chromatography of Fraction 9 124

3.37 In vitro Antimalarial activity of isolated compounds from fraction 9 using β-hematin inhibition assay (BHIA) 129

3.38 Thin Layer Chromatography of the active compound 129

3.39 IR Spectrum of Active Compound 129

3.40 Proton Nuclear Magnetic Resonance (1H NMR) Analysis 132

3.41 13C NMR Spectral Analysis of Active Compound 134

3.42 Matching of the NMR data using APT (Attached Proton Test) 136

3.43 Structure of Identified Compound 136

3.44 In silico pharmacokinetics of the isolated compounds and the structural analogues 139

3.45 Structural Modifications of the Active Compound 139

CHAPTER FOUR: DISCUSSION

4.1 DISCUSSION 143

xiv

4.2 CONCLUSION 165

4.3 Recommendations 165

References 166

Appendices

LIST OF FIGURES

Figure 1: A pictorial view of the leaves of Strophanthus hispidus 3 Figure 2: Life cycle of Plasmodium Species 17 Figure 3: Oxidative stress in Malaria 18 Figure 4: Biochemical effect of treatment with Methanol Extract of Strophantus hispidus Leaves on parasitemia count (PC) of mice passaged with Plasmodium berghei 68

Figure 5: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on the Packed cell Volume of mice passaged with Plasmodium berghei 70

Figure 6: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on the Haemoglobin Concentration (Hb Conc) of mice passaged with Plasmodium berghei 72

Figure 7: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on the Red blood cell (RBC) count of mice passaged with Plasmodium berghei 74

Figure 8: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on White blood cell (WBC) count of mice passaged with Plasmodium berghei 76

Figure 9: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase (ALT) activity of mice passaged with Plasmodium berghei 78

Figure 10: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of mice passaged with Plasmodium berghei 80

Figure 11: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Alkaline phosphatase activity of mice passaged with Plasmodium berghei 82

Figure 12: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Total bilirubin concentration (T.BIL conc) of mice passaged with Plasmodium berghei 84

Figure 13: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of mice passaged with Plasmodium berghei 86

Figure 14: Effect of treatment with Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase activity of mice passaged with Plasmodium berghei 88

xvi

Figure 15: Shows the effect of treatment with different fractions of Methanol Extract of Strophantus hispidus Leaves on the mean parasitemia count of mice passaged with Plasmodium berghei 92

Figure 16: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Packed cell Volume of mice passaged with Plasmodium berghei 94

Figure 17: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Haemoglobin conc. of mice passaged with Plasmodium berghei 96

Figure 18: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on the Red blood cell count of mice passaged with Plasmodium berghei 98

Figure 19: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on White blood cell count of mice passaged with Plasmodium berghei 100

Figure 20: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase activity of mice passaged with Plasmodium berghei 102

Figure 21: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of mice passaged with Plasmodium berghei104

Figure 22: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Alkaline phosphatase activity of mice passaged with Plasmodium berghei 107

Figure 23: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Total bilirubin conc of mice passaged with Plasmodium berghei 109

Figure 24: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of mice passaged with Plasmodium berghei 111

Figure 25: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase activity of mice passaged with Plasmodium berghei 113

Figure 26: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Catalase activity of mice passaged with Plasmodium berghei 115

Figure 27: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on Haemozoin concentration of mice passaged with Plasmodium berghei 117

xvii

Figure 28: Effect of treatment with different partitions of Methanol Extract of Strophantus hispidus Leaves on plasma calcium (PC) ion of mice passaged with Plasmodium berghei 119

Figure 29: Thin layer chromatography (TLC) profiles of different fractions 126

Figure 30: Preparative thin layer chromatography of Fraction 9 128

Figure 31: Tin Layer chromatogram of the active compound 130

Figure 32: IR SPECTRUM of Active Compound 131

Figure 33: Proton Nuclear Magnetic Resonance (1H NMR) Analysis of active compound 133

Figure 34: 13C NMR Spectral Analysis of Active Compound 135

Figure35: Attached Proton Test of Active Compound (APT) (in CDCl3, 400MHz) 137

Figure 36: Extended APT of Active Compound downfield (in CDCl3, 400MHz) 137

Figure 37: Structure of Identified compound 138

Figure 38: Isolated bioactive compounds and some structural modifications based on in silico pharmacokinetics study 141

xviii

LIST OF TABLES

Table 1: Taxonomic Hierarchy of Strophanthus hispidus 4

Table 2: Procedure for Lipid Peroxidation Assay 58

Table 3: Percentage Yield of Extract 65

Table 4: Result of Acute Toxicity Studies (LD50) 65

Table 5: Qualitative Phytochemical Composition of Strophantus hispidus Methanol leaf Extract 65 Table 6: Quantitative Phytochemical Composition of Strophantus hispidus Methanol Leaf Extract 66 Table 7: Percentage yield of the Sequential Partitioning of the Methanol Extract 90 Table 8: Phytochemical analysis of the different partitions of Methanol Extract of Strophantus hispidus Leaves 90

Table 9: In vitro Cytotoxicity test of S. hispidus N-hexane fraction using Brine Shrimp Lethality Assay (BSLA) 125 Table 10: Column Chromatography and fractions obtained based on their Thin Layer Chromatography Profiles 125 Table 11: In vitro antimalarial activity of different fractions of n-hexane partition of methanol extract of Strophantus hispidus leaves using β-hematin inhibition assay (BHIA) 127 Table 12: In vitro anti-malarial activity of isolated compounds from fraction 9 using β-hematin Inhibition Assay (BHIA) 129 Table 13: In silico pharmacokinetics of the isolated compounds and the structural Analogues 140

xix

LIST OF ABBREVIATIONS

AAS: Anabolic Androgenic Steroid

ACT: Artemisinin Combination Therapy

ADME: Absorption, Distribution, Metabolism and Excretion

AIDS: Acquired Immunodeficiency Syndrome

ALP: Alkaline Phosphatase

ALT: Alanine Aminotransferase

AST: Aspartate Aminotransferase

BDCP: Bio-resource Development and Conservation Programme

CB: Conjugated Bilirubin

CHF: Congestive Heart Failures

CO: carbon Monoxide

COPD: Chronic Obstructive Pulmonary Disease

DNA: Deoxyribonucleic Acid

DOX: 1-deoxy-D-xylulose

EDTA: Ethylenediaminetetraacetic acid

FTIR: Fourier-transform Infrared Spectroscopy

Hb: Haemoglobin

HBA: Hydrogen Bond Acceptor

HBD: Hydrogen Bond Donor

HIV: Human Immunodeficiency Virus

IPP: Isopentenyl Diphosphate

KDa: Killo Dalton

LFT: Liver Function Test

Log p: An Octanol-water partition Coefficient

MDA: Malondialdehyde

MEP: Methylerythritolphosphate

MP: Malaria Parasite

MVA: Mevalonic Acid

MW: Molecular Weight

NAFDAC: National Agency for Food and Drug Administration and Control

NMR: Nuclear Magnetic Resonance

PCV: Packed Cell Volume

Pf: Plasmodium falciparum

PK: Pharmacokinetic

PUFA: Poly Unsaturated Fatty Acid

QBCTM:Quantitative Buffy Coat Method

RBC: Red Blood Cell

SH: Strophanthus hispidus

SOD: Superoxide Dismutase

TLC: Thin Layer Chromatography tR: Retention time

US: United States

UV: Ultravoilet

WBC: White Blood Cell

WHO: World Health Organization

xxi

CHAPTER ONE

INTRODUCTION

Plants, the first medicine of humans, have played a remarkable role in health care since the ancient times. Traditionally plant-based medicines still exert a great deal of importance to the people living in developing countries and also lead to the discovery of new drugs for a variety of diseases that threatens human health. Plants are the rich sources of organic compounds, many of which have been used for medicinal purposes. Medicinal plants are the plants whose parts (leaves, seeds, stems, roots, fruits, foliage etc), extracts, infusions, decoctions or powders are used in the treatment of different diseases of humans, plants and animals (Ntie-Kang et al., 2014). There is a wide spectrum of trees, plants and shrubs whose seeds, roots, barks and leaves are used by humans throughout the globe due to their nutritional or medicinal value (Onguéné et al., 2013). The importance of herbs in the management of human ailments cannot be over emphasized.

The first recorded treatment of malaria dates back to 1600 when the bark of the Cinchona tree was first used by the native Peruvian Indians to treat the intermittent fevers associated with this illness (Crawford, 2014). It was not until 1889 that Alphonse Laveran discovered the protozoal (single celled parasite) cause of malaria and not until 1897 that Ronald Ross demonstrated that the Anopheles mosquito was the vector for the disease (Cox, 2010). His pioneering work on establishing the main features of the parasitic life cycle earned Ross the Nobel Prize in Medicine in 1902. Over the next century significant advances were made towards attempts to eradicate malaria particularly with respect to controlling mosquitoes, understanding the parasite and developing drugs to treat the disease (Karunamoorthi, 2014). But despite this, malaria has proven to be one of the biggest killers in the world. Between 300 and 500 million people have been infected annually and nine out of ten of these cases have occurred in sub-Saharan Africa (World Health Organisation, 2010).

However, plants have been prime in discovering new anti-malaria drugs, for example, Chloroquine was discovered from Cinchona tree while Artemisinin the prime drug of all ACTs was discovered from Chinese salad plant, Artemisia annua (Willcox and Bodeker, 2004). Strophanthus hispidus is a well-known plant among traditional medicine providers in West Africa and eastern Nigeria It belongs to the family of plants known as Apocynaceae, they are popularly known as poison arrow vine, brown strophanthus and hairy strophanthus in western part of Africa

3 including Nigeria (Beentje, 1982). It is commonly known among traditional medicine providers in eastern Nigeria as “Osisi Ikaguru”. The latex and seeds of Strophanthus hispidus are used as arrow poison, while decoctions of root, stem bark or leaf are used to treat skin diseases, leprosy, ulcers, malaria, dysentery, gonorrhoea (Burkill, 2000). Research has also shown its usefulness in the treatment of arthritis, stroke, heart failure, rheumatism (Ayoola et al., 2008), inflammatory (Agbaje and Fageyinbo, 2011) and anti-nociceptive (Agbaje and Fageyinbo, 2014). In Nigeria and Ghana, the root decoction is ingested to treat rheumatic diseases, while in Togo; the root bark macerate is employed for treating oedema (Agbaje and Fageyinbo, 2014).

1 Strophantus hispidus

2 General Description of Strophanthus hispidus Strophanthus is a genus of flowering plants in the family Apocynaceae, first described as a genus in 1802. It is native primarily to tropical Africa, extending to South Africa, with a few species in Asia, from southern India to New Guinea and southern China. Strophanthus hispidus DC belongs to the family of plants known as Apocynaceae, they are popularly known as poison arrow vine, brown strophanthus and hairy strophanthus in western part of Africa including Nigeria. The name (strophos anthos, "twisted cord flower") derives from the long twisted threadlike segments of the corolla, which in one species (S. preussii) attain a length of 30–35 cm. The genus includes vines, shrubs and small trees. The leaves are opposite or whorled, simple broad lanceolate, 2–20 cm long, with an entire margin. A deciduous shrub of 5 m tall and up to 100 cm wide, having its stem bark dark grey in colour, with few lenticels, has been reported to have diverse medicinal uses; for example, in the Savannah Zone of West Africa,

Figure 1: A pictorial view of the leaves of Strophanthus hispidus

3 Taxonomy of Strophanthus hispidus Table 1: Taxonomic classification of Strophanthus hispidus

Class Sub-class Kingdom Plantae – plantes, planta, vegetal, plants

Subkingdom Viridaeplantae – green plants

Infrakingdom Streptophyta – land plants

Division Tracheophyta – vascular plants, tracheophytes

Subdivision Spermatophytina - spermatophytes, seed plants, phenerogames

Infradivision Angiospermae – flowering plants, angiosperms

Class Eudicots

Superorder Asterids

Order Gentianales

Family Apocynaceae

Sub family None

Genus strophantus

Specie Strophantus hispidus DC. poison arrow vine, brown strophanthus hairy strophanthus

Source: Beentje, (1982)

4 Pharmacological/ Medicinal Importance of Strophantus hispidus In the Ayurvedic medicine, the latex and seeds of Strophanthus hispidus are used as arrow poison, while decoctions of root, stem bark or leaf are used to treat skin diseases, leprosy, ulcers, malaria, dysentery, gonorrhea (Burkill, 2000). Research has also shown the usefulness of the leaves and roots in the treatment of arthritis, stroke, heart failure, rheumatism (Ayoola et al., 2008), inflammatory (Agbaje and Fageyinbo, 2011) and anti-nociceptive (Agbaje and Fageyinbo, 2014). In Nigeria and Ghana, the root decoction is ingested to treat rheumatic diseases, while in Togo;

6 the root bark macerate is employed for treating edema (Agbaje and Fageyinbo, 2014). Resent work by Agbaje and Fageyinbo, 2011 showed the presence of some important phytochemicals such as alkaloids, tannins, anthraquinones, flavonoids, and cardiac glycosides in the roots of Strophantus hispidus which could be the reason for the high efficacy of the plant.

5 Phytochemicals Phytochemicals (from the Greek word phyto, meaning plant) are biologically active naturally occurring chemical compounds found in plants, which provide health benefits for humans (Mamta et al., 2013). They protect plants from disease and damage and contribute to the plant’s color, aroma and flavor. In general, the plant chemicals that protect plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack are called phytochemicals (Narasinga, 2003). Phytochemistry is the study of natural bioactive products found in plants that work with nutrients and dietary fibre to protect against diseases (Doughari et al., 2009). Recently, it is clearly known that they have roles in the protection of human health, when their dietary intake is significant. Wide-ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices. Broccoli, cabbage, carrots, onions, garlic, whole wheat bread, tomatoes, grapes, cherries, strawberries, raspberries, beans, legumes, and soy foods are common sources (Mathai, 2000).

These phytochemicals are present in a variety of plants utilized as important components of both human and animal diets, and they are found in different parts of the plant which include; fruits, flower, bark seeds, root and stem (Tiwari et al., 2011). They are chemical compounds formed during the plant normal metabolic processes. These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols, tannins, terpenes and terpenoids .

6 Alkaloids Alkaloids are natural products that contain heterocyclic nitrogen atoms and are basic in character. The name, alkaloids derives from the “alkaline” and it was used to describe any nitrogen- containing base (Mueller-Harvey and McAllan, 1992). These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen.

Previous studies have shown that plant-derived alkaloids have a great potential for anti-malarial drug development (Waako et al., 2007). An example is quinine. Quinine is an alkaloid that acts as a blood schizonticid and weak gametocide against Plasmodium species. As an alkaloid, it is accumulated in the food vacuoles of Plasmodium, especially P. falciparum. It acts by inhibiting the hemozoin biocrystallization, thus facilitating an aggregation of cytotoxic heme (Foley and Tilley, 1998).

Alkaloids are significant for the protecting and survival of plant because they ensure their survival against micro-organisms (antibacterial and antifungal activities), insects and herbivores (feeding deterrens) and also against other plants by means of allopathically active chemicals (Molyneux et al., 1996). The use of alkaloids containing plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization. Alkaloids have many pharmacological activities including antihypertensive effects (many indole alkaloids), antiarrhythmic effect (quinidine, sardine), antimalarial activity (quinine), and anticancer actions (dimeric indoles, vincristine, and vinblastine). These are just a few examples illustrating the great economic importance of this group of plant constituents (Wink et al., 1998). Some alkaloids have stimulant property as caffeine and nicotine, morphine are used as the analgesic and quinine as the antimalarial drug (Rao et al., 1978).

7 Phenols Phenolic phytochemicals (phenolics) occupy a unique position in the area of natural products due to their ubiquitous distribution throughout the plant kingdom and in products (fruits, vegetables, beverages, herbs, cosmetics and nutraceuticals) consumed by the general population on a regular basis (Quideau, 2009). Phenolics are biosynthesized by plants during normal development and in response to stress conditions such as exposure to UV radiation, pest attack, and wounding (Luthria, 2006; Naczk and Shahidi, 2004). Phenolic compounds are known to provide protection against a wide range of diseases such as coronary heart disease, stroke, and certain types of cancers (George et al., 2009; Aggarwal, 2009). Chemically, phenolics are defined as a class of aromatic organic compounds with at least one hydroxyl group attached directly to a benzene ring (Luthria, 2006). Over 8000 phenolics with wide structural diversity and polarities have been isolated from plants (Robbins, 2003). Phenolics can be chemically grouped into three broad categories: polyphenols (tannins and flavonoids), simple phenols (phenolic acids) and a miscellaneous group (Luthria, 2006). Phenolic acids are chemically defined as carboxylic acid derivatives of phenols, whereas

8 no such clear definition for polyphenols is provided in the literature. Rather, polyphenols are described as a group of chemical substances found in plants, characterized by the presence of more than one phenol unit or building block per molecule. Polyphenols serve as antioxidants as they tend to prevent or neutralize the damaging effects of free radicals. They also give flowers, fruits, and vegetables their color. Polyphenols can be arranged into two broad classes: tannins and flavonoids. 8 Flavonoids Flavonoids are important group of polyphenols widely distributed among the plant flora. Structurally, they are made of more than one benzene ring in their structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. They are organic compounds that have no direct involvement with the growth or development of plants, they are plant nutrients that when consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the human body. Flavonoids are poly-phenolic compounds that are ubiquitous in nature (Harborne and Baxter, 1999). More than 4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960).

Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids, flavanones, flavonols and anthocyanidines (Nijveldt et al., 2001). Flavones are characterized by a planar structure because of a double bond in the central aromatic ring. Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli and berries. Flavonones are mainly found in citrus fruit, an example is narigin. Flavonoid is involved in scavenging of oxygen derived free radicals (Harborne and Baxter, 1999). It has been identified as a potent hypolipidemic agent in a number of studies (Tapas et al., 2008). It has also been established that flavonoids from medicinal plants possess a high antioxidant potential due to their hydroxyl groups and protect more efficiently against free radical related diseases like arteriosclerosis (Kris-Etherton et al., 2002).

9 Tannins Tannins are astringent polymerized phenols with high molecular weight (500-3000 KDa) and defensive properties. Their name comes from their use in tanning rawhides to produce leather. In

9 tanning, collagen proteins are bound together with phenolic groups to increase the hide’s resistance to water, microbes and heat (Hans-Walter and Fiona, 2005). Two categories of tannins that are of importance are the condensed and hydrolysable tannins. Though widely distributed, their highest concentration is in the bark and galls of oaks (Hans-Walter and Fiona, 2005). They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007).

Many human physiological activities, such as stimulation of phagocytic cells, host-mediated tumour activity, and a wide range of anti-infective actions, have been assigned to tannins (Haslam, 1996). One of their biological actions is to compete with proteins through non-specific forces such as hydrogen bonding and hydrophobic interactions, as well as by covalent bond formation (Haslam, 1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope, transport proteins etc.

10 Terpenoids

Terpenoids which are sometimes called isoprenoids, are a large and diverse class of naturally occurring organic chemicals similar to terpenes, derived from five-carbon isoprene units assembled and modified in thousands of ways (Firn, 2010). Terpenes are hydrocarbons resulting from the combination of several isoprene units. Terpenoids can be thought of as modified terpenes, wherein methyl groups have been moved or removed, or oxygen atoms added (Ourisson, and Nakatani, 1994). Most are multicyclic structures that differ from one another not only in functional groups but also in their basic carbon skeletons. These lipids can be found in all classes of living things, and are the largest group of natural products. About 60% of known natural products are terpenoids.

Plant terpenoids are used extensively for their aromatic qualities and play a role in traditional herbal remedies especially malaria (McGarrey and Croteau, 1995). Artemisinin a known antimalarial is an isoprene terpenoid whose prime source is Artemisia annua. Terpenoids are vital for life of most organisms in exerting metabolic control and in mediating intra- and inter-species interactions, for example, pollination and defense in plants. In plant terpenoid biosynthesis two different pathways synthesize the main building block, isopentenyl diphosphate (IPP); the

10 methylerythritolphosphate (MEP) pathway (also named 1-deoxy-D-xylulose (DOX) pathway) in the chloroplast forms IPP for mono- and diterpenoids, and the mevalonic acid (MVA) pathway in the cytosol, produces IPP for sesquiterpenoids (Connolly and Hill, 1992).

Terpenoids contribute to the scent of eucalyptus, the flavors of cinnamon, cloves, and ginger, the yellow color in sunflowers, and the red color in tomatoes (Eisenrich et al., 1998). Well-known terpenoids include citral, menthol, camphor, salvinorin A in the plant Salvia divinorum, the cannabinoids found in cannabis, ginkgolide and bilobalide found in Ginkgo biloba, and the curcuminoids found in turmeric and mustard seed. The steroids and sterols in animals are biologically produced from terpenoid precursors. Sometimes terpenoids are added to proteins, e.g., to enhance their attachment to the cell membrane; this is known as isoprenylation.

11 Steroids Sterols are triterpenes which are based on the cyclopentane hydrophenanthrene ring system (Harborne, 1998). Sterols were at one time considered to be animal substances (similar to sex hormones, bile acids, etc) but in recent years, an increasing number of such compounds have been detected in plant tissues. Sterols have essential functions in all eukaryotes. For example, free sterols are integral components of the membrane lipid bilayer where they play an important role in the regulation of membrane fluidity and permeability (Galm and Shen, 2007). While cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants, with sitosterol usually predominating. Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol, stigmasterol and campesterol (Harborne, 1998).

12 Saponins Saponins are bioactive compounds produced mainly by plants. Saponins are a group of secondary metabolites, non-volatile surfactants that are widely distributed in the plant kingdom and marine animal (Vincken et al., 2007). Chemically, they generally occur as glycosides of steroids or polycyclic triterpenes (kensil, 1996). Because of their lyobipolar properties, they are able to interact with cell membranes and are also able to decrease the surface tension of an aqueous solution. This activity is the reason for the name “saponin”, derived from the Latin word “sapo”, which refers to the formation of a stable soap-like foam in aqueous solution (Melzig et al., 2001). The formed foam is a result of the formation of a colloidal solution, having a stable and lasting action of dilute mineral acids, differing from ordinary soaps. Another important feature of this

11 class of substances is related to the ability of precipitating cholesterol by forming insoluble complexes. Both features are correlated to the above amphipathic or amphiphilic nature of these molecules, since they are formed by one hydrophilic and one lipophilic moiety (Augustin et al., 2011). Saponins have a range of properties due to their extensive structural diversity, which includes certain features, bitter sweeteners, detergents and emulsifying properties, in addition to the biological, medical and pharmacological properties, such as haemolytic activity, antimicrobial, insecticides and molluscicides. Also noteworthy are their applications in pharmaceutical industries as raw material for the synthesis of steroidal drugs such as birth control piles, besides their intense use in the cosmetic industry (Sparg et al., 2004). The usage of plants containing saponins was accomplished by early civilizations because of their detergents to fish and toxic properties. The early study of biological activities of this class of substances was conducted through extracts rich in saponins. Currently, the occurence in nature, the ethnopharmacological use and the various biological activities of saponins, scientifically proven, have aroused the interest of mankind and, especially, of the scientists worldwide. 13 Glycosides Glycosides are a group of organic compounds generally obtained from plant origin that bind with sugar protein linked to a non-sugar moiety in a particular manner. All the glycosides have an aglycone (gennin) part attached to one or more than one part of the sugar moieties which is responsible for most of its pharmacological activity. The attached sugar has a modified solubility and cell permeability, we can also say that agycone have short-live and less potent. The cardiac glycosides are an important class of naturally occurring drugs which actions include both beneficial and toxic effects on the heart, and have played an outstanding role in the therapy of congestive heart failures (CHF) (Ersoz et al., 2002). The aglycone may be a terpene, a flavonoid, a coumarine or any other natural product. Among the sugars found in natural glycosides, D-glucose is the most abundant one, L rhamnose and L-fructose also occur quite ferequently. Of the pentoses: L-arabinose is more common than D-xylose. They consist of one or more hydroxyl and other substitution on the aglycone which is responsible for determination of its polarity. For example- Digoxigenin has an additional hydroxyl (OH) group than Digitoxigenin and is more polar (Dembitsky, 2005)

In the nomenclature of glycosides the trivial names have an “in” ending, and the names indicate the source of glycoside, for example, digitoxin from Digitalis, salicin from Salix, and prunasin from Prunus. The systematic names are usually formed by replacing the “ose” suffix of the parent sugar with “oside”. The anomeric prefix (α- or β-) and the configurational prefix (D or L) immediately precede the sugar stem name, and the chemical name of the aglyconee precedes the name of the sugar. For example the name of salicin is o-hydroxy-methylphenyl β-D- glycopyranoside. (Ersoz et al., 2002)

14 Malaria Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium and is presently endemic in a broad band around the equator, in areas of the Americas, many parts of Asia and much of Africa, however, it is in sub-Saharan Africa that 85– 90% of malaria fatalities occur (Hay et al., 2004). It is estimated that up to 124 million people in Africa live in areas at risk of seasonal epidemic malaria, and many more in areas outside Africa where transmission is less intense (Hay and Snow, 2006). Each year, it is estimated to cause disease in approximately 650 million people and kills between one and three million, mostly young children in Sub-Saharan Africa (Hay et al., 2004). It is also a cause of poverty and a major hindrance to economic development (Sachs and Malaney, 2002). The economic impact includes costs of health care, working days lost due to sickness, days lost in education, decreased productivity due to brain damage from cerebral malaria, and loss of investment and tourism (WHO, 2001). Moreover, it remains one of the leading causes of death in Sub-Sahara regions where Human Immunodeficiency Virus (HIV) infection is endemic (Korenromp et al., 2005). Malaria causes about 500 million clinical cases each year (10% of the world population), and more than 1 million, mostly children, die as a result of this disease (Breman, 2001). This translates into a death from malaria every 30 seconds, rendering it an eminent disease in tropical countries and ranking it the third killer among communicable diseases behind HIV/AIDS and tuberculosis (Greenwood and Mutabingwa, 2002). Malaria has been a common disease and it continues to be one of the most widely spread health hazards in tropical and subtropical regions. More than half of the world's population lives in the areas where they remain at risk of malarial infection.

Antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today and has also been implicated in the spread of malaria to new areas and re-emergence of

13 malaria in areas where the disease had been eradicated (Bloland, 2001). Drug resistance has also played a significant role in the occurrence and severity of epidemics in some parts of the world. Population movement has introduced resistant parasites to areas previously free of drug resistance. The emergence and spread of P. falciparum resistance to antimalarial drugs is now one of the greatest challenges facing the global effort to control malaria in Africa (WHO, 2003). Moreover, in recent years the situation has worsened due to malaria parasite becoming resistant to several antimalarial drugs.

Acalypha fruticosa, Azadirachta indica, Cissus rotundifolia, Echium rauwalfii, Dendrosicyos socotrana, Boswellia elongate (Merlin, 2004; Clarkson et al., 2004; Alshwash et al., 2009), Cymbopogon giganteus and Morinda lucida (Olajide et al., 1997; Azas et al., 2002) have all been used in the treatment of malaria based on scientific reports.The use of these local herbs for the treatment of malaria has helped to reduce mortality and morbidity rates especially in the rural areas of the developing world where antimalarial drugs are not readily available. One way to prevent drug resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents (Azas et al., 2002). Traditional healers claim that some medicinal plants are more efficient to treat infectious diseases than synthetic antibiotics. Medicinal plants might represent an alternative treatment in non-severe cases of infectious diseases. They can also be a possible source for new potent antibiotics to which pathogenenic strains are not resistant (Elujoba et al., 2005; Ogbonna et al., 2008). Malaria remains uncontrolled and requires newer drugs and vaccines.

15 Geographical Distribution and Populations at Risk of Malaria Malaria occurs in over 90 countries worldwide. WHO estimated that 36% of the global population live in areas where there is risk of malaria transmission, 7% reside in areas where malaria has never been under meaningful control and 29% live in areas where malaria was once transmitted at low levels or not at all, but where significant transmission has been re-established (WHO, 1996). The development and spread of drug-resistant strains of malaria parasites has been identified as a key factor in this resurgence (Bloland, 2001) and is one of the greatest challenges to malaria control today. Malaria transmission occurs primarily in tropical and subtropical regions in sub-Saharan Africa, Central and South America, the Caribbean island of Hispaniola, the Middle East, the Indian subcontinent, South-East Asia, and Oceania (Hay and Snow, 2006). It has been estimated that

14 more than 90% of the 1.5 to 3.0 million deaths attributed to malaria each year occur in African children (Hay et al., 2004). Other estimates based on a more rigorous attempt to calculate the burden of disease in Africa support this level of mortality (Snow et al., 1999). In addition to its burden in terms of morbidity and mortality, the economic effects of malaria infection can be tremendous. These include direct costs for treatment and prevention, as well as indirect costs such as lost productivity from morbidity and mortality; time spent seeking treatment, and diversion of household resources (Sachs and Malaney, 2002). The annual economic burden of malaria infection in 1995 was estimated at US $ 0.8 billion, for Africa alone (Bloland, 2001). This heavy toll can hinder economic and community development activities throughout the region. More than ever before, malaria is both a disease of poverty and a cause of poverty (Bourdy et al., 2008).

Nigeria is known for high prevalence of malaria and it is a leading cause of morbidity and mortality in the country (Ademowo et al., 2006). Available records show that at least 50 per cent of the population of Nigeria suffers from at least one episode of malaria each year and that malaria accounts for over 45% of all patient visits. It was reported that malaria prevalence (notified cases) in year 2000 was about 2.4 million (Sowunmi et al., 2004). The disease accounts for 25 per cent of infant mortality and 30 per cent of childhood mortality in Nigeria. Therefore, it imposes great burden on the country in terms of pains and trauma suffered by its victims as well as loss in outputs and cost of treatments (Ogungbamigbe et al., 2005).

16 Causative Agents of Malaria Malaria is caused by intracellular protozoan parasites of the genus Plasmodium. The parasite belongs to Kingdom Protista, Phylum Apicomplexa, Class Aconoidasida, Order Haemosporida, Family Plasmodiidae, Genus Plasmodium and Species falciparum. The most serious forms of the disease are caused by P. falciparum and P. malariae, but other related species (P. ovale, P. vivax) can also infect humans. This group of human-pathogenic Plasmodium species is usually referred to as malaria parasites (Trampuz et al., 2003). P. falciparum, P. vivax, P. ovale, and P. malariae differ in geographical distribution, microscopic appearance, clinical features, periodicity of infection, potential for severe disease, ability to cause relapses, and potential for development of resistance to antimalarial drugs. To date, drug resistance has only been documented in two of the four species, P. falciparum and P. vivax (Bloland, 2001).

17 Transmission and Biology of P. falciparum Malaria can be transmitted by several species of female anopheline mosquitoes that differ in behavior (Greenwood et al., 2005). There are about 460 species of the Anopheles mosquito, but only 68 transmit malaria. Anopheles gambiae, found in Africa is one of the best malaria vectors since it is long-lived, prefers feeding on humans, and lives in areas near human habitation (Cowman, 2006). Prior to transmission, P. falciparum resides within the salivary gland of the mosquito. The parasite is in its sporozoite stage at this point. As the mosquito takes its blood meal, it injects a small amount of saliva into the skin wound. The saliva contains anti-hemostatic and anti-inflammatory enzymes that disrupt the clotting process and inhibit the pain reaction (Bruce- Chwatt, 1985). Typically, each infected bite contains 5-200 sporozoites which proceed to infect the human (Gilles et al., 1993). Once in the human bloodstream, the sporozoites circulate for a few minutes before infecting liver cells.

18 Liver Stage After circulating in the bloodstream, the P. falciparum sporozoites enter hepatocytes to initiate the exoerythrocytic stage. At this point, the parasite loses its apical complex and surface coat, and transforms into a trophozoite. Within the parasitophorous vacuole of the hepatocyte, P. falciparum undergoes schizogonic development. In this stage, the nucleus divides multiple times with a concomitant increase in cell size, but without cell segmentation. This exoerythrocytic schizogony stage of P. falciparum has a minimum duration of roughly 5.5 days. After segmentation, the parasite cells are differentiated into merozoites (Miller et al., 1994). After maturation, the merozoites are released from the hepatocytes and enter the erythrocytic portion of their life-cycle. The released merozoites do not re-infect hepatocytes and infection continues.

19 Erythrocytic Stage After release from the hepatocytes, the merozoites enter the bloodstream prior to infecting red blood cells. At this point, the merozoites are roughly 1.5µm in length and 1 µm in diameter, and use the apicomplexan invasion organelles (apical complex, pellicle and surface coat) to recognize and enter the host erythrocyte (Bruce-chwatt, 1985).The parasite first binds to the erythrocyte in a

16 random orientation. It then reorients such that the apical complex is in proximity to the erythrocyte membrane. A tight junction is formed between the parasite and erythrocyte. As it enters the red blood cell, the parasite forms a parasitophorous vesicle, to allow for its development inside the erythrocyte (Gilles et al., 1993). After invading the erythrocyte, the parasite loses its specific invasion organelles (apical complex and surface coat) and differentiates into a round trophozoite located within a parasitophorous vacuole in the red blood cell cytoplasm. The young trophozoite (or "ring" stage, because of its morphology on stained blood films) grows substantially before undergoing schizogonic division (Arora and Arora, 2005). The growing parasite replicates its DNA multiple times without cellular segmentation to form a schizont. These schizonts then undergo cellular segmentation and differentiation to form roughly 16-18 merozoite cells in the erythrocyte (Gills et al., 1993). The merozoites burst from the red blood cell, and proceed to infect other erythrocytes. The parasite then stays in the bloodstream for roughly 60 seconds before invading another erythrocyte. This infection cycle occurs in a highly synchronous fashion, with roughly all of the parasites throughout the blood in the same stage of development. This precise clocking mechanism has been shown to be dependent on the human host's own circadian rhythm. Specifically, human body temperature changes, as a result of the circadian rhythm, seem to play a role in the development of P. falciparum within the erythrocytic stage (Bruce-chwatt, 1985). Within the red blood cell, the parasite metabolism depends greatly on the digestion of haemoglobin. Infected erythrocytes are often sequestered in various human tissues or organs, such as the heart, liver and brain. This is caused by parasite-derived cell surface proteins being present on the red blood cell membrane and it is these proteins that bind to receptors on human cells. Sequestration in the brain causes cerebral malaria, a very severe form of the disease, which increases the victim's likelihood of death. The parasite can also alter the morphology of the red blood cell, causing knobs on the erythrocyte membrane (Miller et al., 1994).

20 Gametocyte Differentiation During the erythrocytic stage, some merozoites develop into male and female gametocytes in a process called gametocytogenesis (Billker et al., 1998). These gametocytes take roughly 810 days to reach full maturity and remain within the erythrocytes until taken up by the mosquito host.

21 Mosquito Stage The gametocytes of P. falciparum are taken up by the female Anopheles mosquito as it takes its blood meal from an infected human. Upon being taken up by the mosquito, they leave the erythrocyte shell and differentiate into gametes. The female gamete maturation process entails slight morphological changes, as it becomes enlarged and spherical. On the other hand, the male gamete maturation involves significant morphological development. The male gamete's DNA divides three times to form eight nuclei and concurrently, eight flagella are formed. Each flagella pairs with a nucleus to form a microgamete, which then separates from the parasite cell in a process referred to as exflagellation (Gilles et al., 1993).Gametogenesis (formation of gametes) has been shown to be caused by: 1) a sudden drop in temperature upon leaving the human host, 2) a rise in pH within the mosquito, and 3) xanthurenic acid within the mosquito (Billker et al., 1998). Fertilization of the female gamete by the male gamete occurs rapidly after gametogenesis. The fertilization event produces a zygote. The zygote then develops into an ookinete. The zygote and ookinete are the only diploid stages of P. falciparum. The diploid ookinete is an invasive form of P. falciparum within the mosquito. It traverses the peritrophic membrane of the mosquito midgut and crosses the midgut epithelium. Once through the epithelium, the ookinete enters the basil lamina, and forms an oocyst. During the ookinete stage, genetic recombination can occur. This takes place if the ookinete was formed from male and female gametes derived from different populations. This can occur if the human host contained multiple populations of the parasite, or if the mosquito fed on multiple infected individuals within a short time-frame (Bruce-chwatt, 1985). Over the period of 1-3 weeks, the oocyst grows to a size of tens to hundreds of micrometres. During this time, multiple nuclear divisions occur. After maturation, it divides to form multiple haploid sporozoites in a process referred to as sporogony. Immature sporozoites break through the oocyst wall into the haemolymph, then migrate to the salivary glands and complete their differentiation. Once mature, the sporozoites can proceed to infect a human host during a subsequent mosquito bite (Gilles et al., 1993).

Figure 2: Life cycle of Plasmodium Species Source: Bruce-chwatt, (1985)

22 Oxidative stress in Malaria and Symptoms

Figure 3: Oxidative stress in Malaria Source: Rifkind et al., 2014

Malaria is a complex disease that varies widely in epidemiology and clinical manifestation in different parts of the world. This variability is due to factors such as the species of malaria parasites that occur in a given area, their susceptibility to commonly used or available antimalarial drugs, the distribution and efficiency of mosquito vectors, climate and other environmental conditions and the behavior and level of acquired immunity of the exposed human populations (Greenwood et al., 2005; Mockenhaupt et al., 2000). The parasites multiply within the red blood cells, causing symptoms that include symptoms of anemia (light headedness, shortness of breath, tachycardia

20 etc.), as well as other general symptoms such as fever, chills, nausea, flu-like illness, arthralgia (joint pain), vomiting, anemia caused by hemolysis, haemoglobinuria, and convulsions and in severe cases, coma and death (WHO, 1991). The classical symptom of malaria is cyclical occurrence of sudden coldness followed by rigor and then fever and sweating lasting four to six hours, occurring every two days in P. vivax and P. ovale infections, while it occurs every three days in P. malariae (Boivin, 2002). P. falciparum can have recurrent fever every 36-48 hours or a less pronounced and almost continuous fever (Trampuz et al., 2003). For reasons that are poorly understood, but which may be related to high intracranial pressure, children with severe malaria frequently exhibit abnormal posturing, a sign indicating severe brain damage (Idro et al., 2007). Malaria has been found to cause cognitive impairments, especially in children. It causes widespread anemia during a period of rapid brain development and also direct brain damage. This neurologic damage results from cerebral malaria to which children are more vulnerable (Boivin, 2002). Consequences of severe malaria include coma and death if untreated, young children and pregnant women are especially vulnerable. Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly (enlarged liver), hypoglycemia, and haemoglobinuria with renal failure may occur (Trampuz et al., 2003). Renal failure may cause blackwater fever, where haemoglobin from lysed red blood cells leaks into the urine (Idro et al., 2007). Severe malaria can progress extremely rapidly and cause death within hours or days. In the most severe cases of the disease fatality rates can exceed 20%, even with intensive care and treatment (Kain et al., 1998). In endemic areas, treatment is often less satisfactory and the overall fatality rate for all cases of malaria can be as high as one in ten (Mockenhaupt et al., 2004). Over the longer term, developmental impairments have been documented in children who have suffered episodes of severe malaria (Trampuz et al., 2003). Chronic malaria is seen in both P. vivax and P. ovale, but not in P. falciparum. Here, the disease can relapse months or years after exposure, due to the presence of latent parasites (hypnozoites) in the liver (Kain et al., 1998). Severe malaria is almost exclusively caused by P. falciparum infection and usually arises 6-14 days after infection. 23 Treatment of Malaria 24 Artemesinin-based combinations

Artemesinin has a very different mode of action from conventional anti-malarials. This makes it particularly useful in the treatment of resistant infections (Mueller et al., 2000). However, in order

21 to prevent the development of resistance to this drug it is only recommended in combination with another non-artemesinin based therapy. It produces a very rapid reduction in the parasite biomass with an associated reduction in clinical symptoms and is known to cause a reduction in the transmission of gametocytes thus decreasing the potential for the spread of resistant alleles. Artemisinin combination therapy (ACT) has been widely adopted as first-line treatment for uncomplicated falciparum malaria (Ashley et al., 2007; Nosten and White, 2007). Although these drug combinations appear to be safe and well-tolerated, experience with their use in Africa is still limited (Talisuna et al., 2006; Staedke et al., 2008). Artesunate and chloroquine combination has been thoroughly tested in randomized controlled trials and has demonstrated that it is well tolerated with few side effects (Nosten and White, 2007). However, in one study there was less than 85% cure in areas where Chloroquine resistance was known. It is not approved for use in combination therapy and is unadvised in areas of high P. falciparum resistance. Artesunate and Amodiaquine combination has also been tested and proved to be more efficacious and similarly well tolerated than the Chloroquine combination. The cure rate was greater than 90%, potentially providing a viable alternative where levels of Chloroquine resistance are high (Sirima et al., 2009). The main disadvantage is a suggested link with neutropenia (Mutabingwa et al., 2005).

Artesunate and mefloquine have been used as an efficacious first-line treatment regimen in areas of Thailand for many years (Adjuik et al., 2004). Mefloquine is known to cause vomiting in children and it induces some neuropsychiatric and cardiotoxic effects, interestingly these adverse reactions seem to be reduced when the drug is combined with Artesunate, it is suggested that this is due to a delayed onset of action of Mefloquine. This is not considered a viable option to be introduced in Africa due to the long half-life of Mefloquine, which potentially could exert a high selection pressure on parasites (Bloland, 2001). Artemether and Lumefantrine (Coartem®, Riamet®, and Lonart®) is a combination that has been extensively tested in 16 clinical trials, proving effective in children less than 5years and has been shown to be better tolerated than Artesunate plus Mefloquine combinations (Mutabingwa et al., 2005). There are no serious side effects documented but the drug is not recommended in pregnant or lactating women due to limited safety testing in these groups. This is the most viable option for widespread use and is available in fixed-dose formulae thus increasing compliance and adherence (Lefevre et al., 2001).

Artesunate and Sulfadoxine-Pyrimethamine is a well-tolerated combination but the overall level of efficacy still depends on the level of resistance to Sulfadoxine and Pyrimethamine thus limiting its usage (WHO, 2001). Piperaquine-dihydroartemisinin-trimethoprim (Artecom®) alone and in combination with Primaquine has been studied in resistant areas of China and Vietnam (Yeka et al., 2008). The drug has been shown to be highly efficacious (greater than 90%) even to strains resistant to Primaquine. More information is required on safety and tolerability in pregnant women and children and toxicology data. Pyronaridine and Artesunate has been tested and was shown to have a clinical response rate of 100% in one trial in Hainan (an area with high levels of P.falciparum resistance to Pyronaridine) (Nosten and White, 2007). Chlorproguanil-Dapsone and Artesunate (Lapdap plus) is the most tested drug currently under development and could be introduced in African countries imminently.

25 Traditional Antimalarial Herbs The use of plants for therapeutic purposes dates back to the human history (Ogbonna et al., 2008). Medicinal plants, since time immemorial, have been used in virtually all cultures as a source of medicine (Hoareau and Dasilva, 1999) and for a long time, natural products were the only sources of medication (Bourdy et al., 2008). Several medicinal plants have been used locally to treat malaria infection. Some of such plants are Enantia chloranta, Nauclea natifolia, Salacia Nitida (Ogbonna et al., 2008), Acalypha fruticosa, Azadirachta indica, Cissus rotundifolia, Echium rauwalfii, Dendrosicyos socotrana, Boswellia elongate (Merlin, 2004; Clarkson et al., 2004; Alshwash et al., 2007), Cymbopogon giganteus and Morinda lucida etc.

The urgency generated by drug-resistant strains of malaria parasites has accelerated antimalarial drug research over the last two decades. While synthetic pharmaceutical agents continue to dominate research, attention has increasingly been directed to natural products (Etkin, 2003). The success of artemisinin, isolated from Artemisia annua and its derivatives for the treatment of resistant malaria has focused attention on the plants as a source of antimalarial drugs (Tan et al., 1998). Moreover, plants have been the basic source of sophisticated traditional medicine systems for thousands of years and were instrumental to early pharmaceutical drug discovery and industry (Elujoba et al., 2005). The world's poorest are the worst affected, and many treat themselves with traditional herbal medicines. These are often more available and affordable, and sometimes are perceived as more effective than conventional antimalarial drugs (Merlin, 2004).

Ethnobotanical information about antimalarial plants used in traditional herbal medicine, is essential for further evaluation of the efficacy of plant antimalarial remedies and efforts are now being directed towards discovery and development of new chemically diverse antimalarial agents (Clarkson et al., 2004). Several rural dwellers depend on traditional herbal medicine for treatment of many infectious diseases including malaria (Ali et al., 2004). The reputed efficacies of these plants have been recognized and passed on from one generation to the other.

About 75% of the population in Africa does not have direct access to chemical treatment, such as chloroquine, but they have access to traditional medicine for treating fevers. Treatment with these remedies has suffered a number of deficiencies; diagnosis is often a problem, identification of plant extracts may be insecure and the chemical content of extracts may vary considerably (Azas et al., 2002). Natural products isolated from plants used in traditional medicine, which have potent anti-plasmodial action in vitro, represent potential sources of new antimalarial drugs. It had been advocated that direct crude drug formulation of the herbs following toxicological absolution may not only produce dosage forms faster but will also lead to cheaper and more affordable drugs for the communities that need them (Elujoba, 1998). Also, there is a belief that these medicines are safe because they are natural and have been used traditionally over a period of time (Sofowora, 1993; Willcox et al., 2003). Plant materials remain an important resource to combat serious diseases in the world (Tshibangu et al., 2002) and pharmacognostic investigations of plants are carried out to find novel drugs or templates for development of new therapeutic agents (König, 1992). Moreover herbs can be highly effective for treating malaria if government can educate those involved in the practice regarding the normal dose to be taken before getting well. Therefore, government should provide subvention for the Ministry of Health incorporating National Agency for Food and Drug Administration and Control (NAFDAC) to go into more Malaria research in local herb just to develop new and more effective drug for prevention and control, particularly in view of the rapid spread of drug resistance. Nevertheless, much work need to be done to educate the community and the producers of indigenous herbal products to strictly adhere to environmental hygiene.

26 Diagnosis of Malaria Direct microscopic examination of intracellular parasites on stained blood films is the gold standard for definitive diagnosis in nearly all settings. However, several other approaches exist or are in development, some of which are discussed here.

27 Microscopy Simple light microscopic examination of Giemsa stained blood films is the most widely practiced and useful method for definitive malaria diagnosis. Advantages include differentiation between species, quantification of the parasite density and ability to distinguish clinically important asexual parasite stages from gametocytes which may persist without causing symptoms (WHO, 1991). These advantages can be critical for proper case-management and evaluating parasitological response to treatment. Specific disadvantages are that slide collection, staining, and reading can be time-consuming and microscopists need to be trained and supervised to ensure consistent reliability. While availability of microscopic diagnosis has been shown to reduce drug use in some trial settings (Chanda et al., 2009). Any programme aimed at improving the availability of reliable microscopy should also retrain clinicians in the use and interpretation of microscopic diagnosis. Another method is a modification of light microscopy called the Quantitative Buffy Coat Method (QBCTM, Becton-Dickinson). Originally developed to screen large numbers of specimens for complete blood cell counts, this method has been adapted for malaria diagnosis (Levine et al., 1989). The technique uses micro haematocrit tubes pre-coated with fluorescent acridine orange stain to highlight malaria parasites. With centrifugation, parasites are concentrated at a predictable location. Advantages to QBC are that less training is required to operate the system than for reading Giemsa-stained blood films and the test is typically quicker to perform than normal light microscopy. Disadvantages are that electricity is always required, special equipment and supplies are needed, the per-test cost is higher than simple light microscopy, and species-specific diagnosis is not reliable. Field trials have shown that the QBC system may be marginally more sensitive than conventional microscopy under ideal conditions (Levine et al., 1989; Tharavanij, 1990).

28 The Blood Blood is the only fluid tissue in the body. It appears to be a thick, homogeneous liquid, but the microscope reveals that blood has both cellular and liquid components (Hart, 2001). Blood accounts for approximately 8% of body weight. Its average volume in healthy adult males is 5–6

L (about 1.5 gallons), somewhat greater than in healthy adult females (4–5 L) (Waugh and Grant, 2014). Blood performs a number of functions such as; delivering oxygen from the lungs and nutrients from the digestive tract to all body cells, transporting metabolic waste products from cells to elimination sites (to the lungs for elimination of carbon dioxide, and to the kidneys for disposal of nitrogenous wastes in urine) and transporting hormones from the endocrine organs to their target organs (Widmaier et al ., 2015). If we spin a sample of blood in a centrifuge, the heavier formed elements are packed down by centrifugal force and the less dense plasma remains at the top. Most of the reddish mass at the bottom of the tube are erythrocytes (e˘-rith_ro-sı¯ts; erythro = red), the red blood cells that transport oxygen. A thin, whitish layer called the buffy coat is present at the erythrocyte-plasma junction. This layer contains leukocytes (leuko = white), the white blood cells that act in various ways to protect the body, and platelets, cell fragments that help stop bleeding. Erythrocytes normally constitute about 45% of the total volume of a blood sample, a percentage known as the haematocrit (he-mat_o-krit; “blood fraction”). Normal hematocrit values vary. In healthy males the norm is 47% ± 5%; in females it is 42% ± 5%. Leukocytes and platelets contribute less than 1% of blood volume (Waugh and Grant, 2014). Plasma makes up most of the remaining 55% of whole blood. 29 Haemoglobin Estimation Haemoglobin contains the red pigment that gives the red cells their colour and also carries oxygen from the lungs to the tissues and carries carbon dioxide (the waste products) from the tissues to the lungs. This test is primarily used to determine the presence of anemia or, its reverse, polycythaemia, or to monitor a patient’s response to treatment. Capillary blood or EDTA anti- coagulated venous blood can be used. The haemoglobin content in a solution may be estimated by several methods: by measurement of its colour, its power of combining with oxygen or carbon monoxide and by its iron content (Yared et al., 2006). A low haemoglobin level means that less oxygen is being delivered round your body, leading to symptoms of anemia such as fatigue, breathlessness, pallor, and palpitations. The patient may need to have a blood transfusion to help relieve these symptoms. In this case the patient may need to have additional blood tests, in order to match the transfusion to your own blood as closely as possible (Cheesbrough, 2000).

30 Packed Cell Volume (PCV) Packed cell volume is a measure of the proportion of blood volume that is occupied by red blood cells. It is normally about 45% in men and 40% in women (Yared et al., 2006). It is considered an integral part of a person complete blood count results along with haemoglobin concentration, white blood cell count and platelet count. Low levels of PCV can be seen in the case of anemia, inflammation, kidney damage, malnutrition and pregnancy. However increase in PCV may be seen in myeloproliferative disorder, chronic obstructive pulmonary disease (COPD), capillary leak syndrome and anabolic androgenic steroid (AAS) (Lewis et al., 2002).

31 Red Blood Cell (RBC) Count Red blood cell count (RBC) measures the number of red cells in the blood. Red blood cells carry oxygen to the tissues and remove waste products from the body’s tissues. These cells also contain haemoglobin. Red blood cells are measured in millions per cubic millimeter (mil/uL) of blood (Lewis et al., 2002). A low count often accompanies anemia, excess body fluid and blood loss. A high count is commonly seen in dehydration but could also mean some other complications such as polycythaemia, lung disease, alcoholism, smoking, kidney disease, dehydration, burns, sweating, diarrhea, carbon monoxide (co) exposure, etc while low RBC might indicate anemia, sickle cell disease, cancer, peptic ulcer, lead poisoning, heavy menstrual bleeding etc depending on the aim of the test (Cheesbrough, 2000).

32 White Blood Cell (WBC) Count White blood cell count (WBC) is a blood test carried out in the laboratory that measures the number of white blood cells per liter of blood. White cells protect against infection and allergies. High counts are seen during infection, after exercise and with stress. Low counts may be seen if there is suppression of the immune system (Yared et al., 2006). An increase above the normal range could imply some other complications such as, leukemia, inflammation, tissue damage, stress, malnutrition, burns, lupus, kidney failure, rheumatoid arthritis, tuberculosis, thyroid gland problems while low WBC count might indicate alcoholism, AIDS, enlarged spleen, viral infection, malaria, that the patient is undergoing chemotherapy, depending on the aim of the test.

33 Liver Function Tests Liver function tests a broad range of normal functions performed by the liver. The diagnosis of liver disease depends upon a complete history, complete physical examination and evaluation of

27 liver function test and further invasive and non-invasive tests (Rajiv et al., 2012). The liver performs different kinds of biochemical, synthetic and excretory functions of the liver. An initial step in detecting liver damage is a simple blood test to determine the presence of certain liver enzymes in the blood. Under normal circumstance, these enzymes are resided in the cells of the liver. But when the liver is injured these enzymes are spilled into the blood stream (Sultana et al., 2004). Among the most sensitive and widely used of these liver enzymes are the amino-transferase (ALT). These enzymes are normally contained within liver cells. When the liver is injured, the cells spill the enzymes into the blood stream, raising the enzymes level in the blood and signaling their damage (Rajiv et al., 2012).

34 Alkaline Phosphatase (ALP) Alkaline phosphateses are a family of zinc metaloenzymes, with a serine residue at the active centre; they release inorganic phosphate from various organic orthophosphates and are present in nearly all tissues (Thapa and Anuj, 2007). ALP is produced in the lower bile duct, bone and gut and is widely distributed in the body. In liver, alkaline phosphatase is found histo-chemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes. In liver, two distinct forms of alkaline phosphatase are also found but their precise roles are unknown. ALP is a hydrolase enzyme responsible for removing phosphate group from many types of molecules, including nucleotides, protein and alkaloids. Alkaline phosphatase lines the cells in the biliary ducts of the liver. ALP levels in plasma will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. It is present in the bone and placenta, so it is higher in growing children (as their bones are being remodeled) and elderly patients with Paget’s disease (Manson, 2004). Elevations occur as a result of both intrahepatic and extra-hepatic obstruction to bile flow. ALP is also raised in cirrhosis and liver cancers, but levels can be within the reference range or with a slight increase in acute hepatitis.

35 Aspartate Aminotransferase Aspartate aminotransferase (AST) is more widely distributed than Alanine aminotransferase (ALT). It is present in the liver, heart, kidneys, skeletal muscle and red blood cells. AST levels are raise in shock. It is less specific for liver disease and is not included in liver function profile by all laboratories because the enzyme is not localized in the liver. AST levels are also raised in

28 pregnancy and after exercise. Ratios between ALT and AST are useful to physicians in assessing the etiology of liver enzyme abnormalities and also useful in differentiating between causes of liver damage (Manson, 2004). ALT exceeds AST in toxic hepatitis, chronic active hepatitis and cholestatic hepatitis. The ratio is characteristically elevated in alcoholic liver disease (Thapa and Anuj, 2007). The AST and ALT levels are increased to some extent in almost all liver diseases. The highest elevations occur in severe viral hepatitis, drug or toxin induced hepatic necrosis and circulatory shock.

36 Alanine Aminotransferase The enzyme ALT is present in high concentration in the liver. It is also found cardiac and skeletal muscle (Manson, 2004). However, ALT is considered as specific marker of hepatocellular damage because levels are generally only significantly raised in liver damage. ALT is the heart and muscles in much lower concentrations – only marginal elevations occur in acute myocardial infarction. People with acute liver damage have particularly high ALT levels and those with chronic liver disease and obstructive jaundice have more modestly raised levels. Low ALT (and AST) levels suggest vitamin B6 deficiency. The levels of ALT abnormality are increased in conditions where cells of the liver have been inflamed or undergone cell death. As the hepatocytes are damaged, the ALT leaks into the blood stream leading to a rise in the serum level (Manson, 2004). Any form of hepatic cell damage can result in an elevation in the ALT. ALT is the most sensitive marker for liver cell damage (Manson, 2004). Elevations are often measured in multiples of the upper limit of normal (ULN). Reference range 5 to 40 IU/L (Reitman and Frankel, 1957).

37 Bilirubin Bilirubin is an endogenous anion derived from haemoglobin degradation from the red blood cell (RBC). The classification of bilirubin into direct and indirect bilirubin is based on the original van der Beigh method of measuring bilirubin. Bilirubin is a yellow fluid produced in the liver when worn-out red blood cells are broken down at the end of their 120 day lifespan (Manson, 2004). Bilirubin is a major product of haemoglobin. During splenic degradation of red blood cells, haemoglobin is separated out from iron and cell membrane components. Haemoglobin is transferred to the liver where it undergoes further metabolism in a process called conjugation. Conjugation allows haemoglobin to become more water-soluble. The water insoluble bilirubin were excreted into bile (Rajiv et al., 2012). In the blood, unconjugated (or indirect) bilirubin is

29 carried by albumin to the liver. It is conjugated to make it more water soluble, before it is excreted in bile. Conjugated bilirubin is also called direct bilirubin. The concentration of bilirubin in the serum therefore reflects the balance between the amount produced by erythrocyte destruction and that removed by the liver. As the liver becomes irritated, the total bilirubin may rise.

38 Antioxidants An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that involves the transfer of electrons from a substance to an oxidizing agent. Antioxidants are often reducing agents such as thiols, polyphenols or ascorbic acid (Seaver and Imlay, 2004). Antioxidants are intimately involved in the prevention of cellular damage which is the common pathway for a variety of diseases. Although, oxidation reactions are crucial for life, they can also be damaging, however insufficient levels of antioxidants or inhibition of the antioxidant enzymes (e.g. superoxide dismutase), causes oxidative stress which will subsequently lead to inflammation and cellular damage. Antioxidants are widely used in dietary supplements and has been investigated for the prevention of diseases such as cancer, coronary heart disease and other sickness (Bjelakovic et al ., 2007).

39 Superoxide Dismutase Superoxide dismutases (SOD, EC 1.15.1.1) are enzymes that catalyze the dismutation of superoxide (O2−) into oxygen and hydrogen peroxide. Thus, they are an important antioxidant defense in nearly all cells exposed to oxygen. Superoxide is one of the main reactive oxygen species in the cell. Consequently, SOD serves a key antioxidant role. The physiological importance of SODs is illustrated by the severe pathologies evident in mice genetically engineered to lack these enzymes (Hijora et al., 2005).

Superoxide dismutase is an enzyme whose function is to protect against the potentially damaging activities of the superoxide radical generated by aerobic metabolic reactions. Two types of SOD have been found in all mammalian cells except erythrocytes. Cu, Zn-SOD was present in both the cytosol and the intermediate membrane space of the mitochondria, and Mn-SOD was present in the mitochondrial matrix (Bjelakovic et al., 2007). Non-sulfur Fe enzyme known as superoxide dismutase (SOD) catalyze disproportion of FeSOD is found in bacteria, especially the more primitive ones, the chloroplast of plants, a few protists and possibly eukaryotes. The homologous MnSODs and found in bacteria and mitochondria, and are believed to protect DNA from

endogenous oxidative stress, whereas FeSOD may serve as a housekeeping enzyme and provide

resistance to environmental oxidatives, SOD caused by the chemical progeny of O2-: H2O2 and OH. Atomic absorption spectroscopy reveals that SOD monomer contains one metal ion and no other cofactors (Bjelakovic et al., 2007).

40 Catalase Catalase is a ubiquitous enzyme associated with the micro bodies in all aerobic cells. It is one of the three enzymes that interact with reactive oxygen species (ROS). Catalase specifically degrades hydrogen peroxide (H2O2) to H2O and O2. Production of H2O2, in low concentrations, occurs during the metabolism of most cells. If concentrations of H2O2 accumulate, oxidative modification of enzymes, lipid peroxidation, and other damaging reactions can occur (Foote and Hare, 2001). Reactive oxygen species (ROS) are highly reactive and damaging towards DNA, lipids, and proteins. Hydrogen peroxide (H2O2) is one of the most important cellular ROS and has crucial regulatory and signaling functions. Within cells, H2O2 can be produced by the mitochondrial respiratory chain, NADPH oxidases, through the enzymatic detoxification of superoxide radicals by superoxide dismutase, or in P. falciparum during hemoglobin degradation reported that erythrocytes infected with P. falciparum produced significantly higher amounts of hydroxyl (OH) radicals and H2O2 when compared to uninfected erythrocytes (Rahbari et al., 2017). Catalase activities decrease under conditions that suppress photorespiration, such as elevated CO2 (Luna, 2004). Studies indicated that erythrocytes which are infected with Plasmodium tend to undergo increased endogenous oxidative stress as a result of the malaria infection. Evidence from a number of studies has indicated that oxidative stress induced physicochemical changes in the membrane of the erythrocytes is the cause of the lipid peroxidation and hemolysis observed in malaria. Reactive oxygen intermediates and hydrogen peroxides produced by neutrophils and macrophages do play important role in the infected person’s defense against malaria infection. Further studies showed that a reduced catalase level of 43.30 IU mL-1 was observed among severe malaria patients compared to 46.86 IU mL-1 of controls (Sakyi et al., 2012).

41 Lipid Peroxidation and Tissue Damage Lipid peroxidation is a known mechanism of cellular injury in human and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides derived from PUFA are unstable and

31 decompose to form a complex series of compounds. These include reactive carbonyl compounds which is the most abundant is malondialdehyde (MDA) (Sarka and Rautary, 2009). Lipid peroxidation is one of the molecular mechanism for cell injury and is associated with a decrease of cellular antioxidants such as glutathione, superoxide dismutase (SOD) and catalase (CAT) (Hijora et al., 2005). Free radicals are released by activated leucocytes which cause peroxidation of membrane lipids. There is a rupture of the liposomal membranes, the release of lysosomal enzymes, necrosis of the cell and destruction of parenchymal tissue. All these processes culminate in an increase in serum MDA levels. Hence, increased serum MDA could be used as a marker for the free radical mediated destruction of liver parenchymal cells. Liver disease is accompanied by an increased production of free radicals. MDA has the ability to interact with lipoproteins and so has received particular attention in pharmacological studies (Sarkar and Rautava, 2009).

42 Haemozoin

Malaria parasites (Plasmodium spp.), digest haemoglobin and release high quantities of free heme, which is the non-protein component of haemoglobin. A heme is a prosthetic group that consists of an iron atom contained in the center of a heterocyclic porphyrin ring. The free heme is oxidatively active and toxic to both the host cell and the malarial parasite, and it causes parasite death. Due to the absence of heme oxygenase, the parasite is unable to cleave heme into an open-chain tetrapyrrole, which is necessary for cellular excretion (Eckman et al., 1977). To protect itself, the malarial parasite detoxifies free heme via neutralization with histidine-rich protein 2 (Huy et al., 2003), degradation with reduced glutathione (Atamna and Ginsburg, 1995), or converts it in the food vacuole into an insoluble crystallization called hemozoin also known as β-hematin or the malarial pigment (Bohle et al., 1997).

Since the formation of hemozoin is essential to the survival of these parasites, it is an attractive target for developing drugs and is much-studied in Plasmodium as a way to find drugs to treat malaria (malaria's Achilles' heel). Several currently used antimalarial drugs, such as chloroquine and mefloquine, are thought to kill malaria parasites by inhibiting hemozoin bio-crystallization (Egan et al., 2000). Thus measuring the level of hemozoin in the blood is also a great tool to checkmate the efficacy of a drug or plant material in fighting the malaria parasite. The normal

32 range for hemozoin is below 0.1 mg/ml according to the method described by (Oliveira et al., 2005) and modified by (Dibua et al., 2013).

43 Calcium ion Calcium is essential for living organisms, particularly in cell physiology, and is the most common metal in many animals. Calcium is also known to be involve in so many vital metabolic processes in the body. Physiologically, it exists as an ion in the body. Calcium ions (Ca2+) are relevant for several vital functions in malaria parasites including host cell invasion, parasite motility, and differentiation. Phospholipase A2, reactive oxygen species, arachidonic acid metabolites, calcium and microvascular constrictors are released by both the host and parasite during malaria attack.

During malaria parasite attack on the human body, red blood cells are destroyed as the parasites complete their growth and as much as 70% of red cell haemoglobin may be digested by parasite secretions (Greenbaum et al., 2002) to produce large amounts of amino acids for osmotic and nutrient purposes. The malaria parasite (trophozoites) during its growth as forms a parasitophorous vacuole which shields it from the attack of the host immune defense system (leucocytes).

Phospholipase A2 liberates free fatty acids from membrane phospholipids. The parasite forms the parasitophorous vacuole by utilizing the enzyme phospholipase A2 by causing inflammation of the cell membrane. Calcium ion is needed for the activation of phospholipase A2 because it binds to the zymogen of phospholipase A2 (Drakenberg et al., 1984). A research work done by (Nwodo et al., 2010) demonstrated that anti-malarial treatment lowered the human plasma concentrations of both calcium (ions) and free fatty acids and inhibited hypotonicity induced haemolysis and phospholipase A2 activity. Due to the need for calcium ion is high in malaria condition thus the levels of calcium ion is needful to be measured in a malaria condition. The normal level for calcium ion level in the blood is 2.0 – 4.0 mg/dl

44 Brine shrimp lethality bioassay Brine shrimp, (genus Artemia), any of several small crustaceans of the order Anostraca (class Branchiopoda) inhabiting brine pools and other highly saline inland waters throughout the world. Artemia salina, the species that occurs in vast numbers in Great Salt Lake, which was used in this study is of commercial and scientific importance. Brine shrimp lethality assay is a useful tool for preliminary assessment of toxicity in plant extract and food items. These days brine shrimp

(Artemia salina, fairy shrimp or sea monkeys) lethality assay is commonly used to check the cytotoxic effect of bioactive chemicals. It is a preliminary toxicity screening of plant extracts (Kibiti and Afolayan, 2016; Oberlies et al., 1998), heavy metals (Saliba and Krzyz, 1976), cyanobacteria toxins (Hisem et al., 2011), pesticides (Michael et al., 1956), cytotoxicity testing of dental material (Pelka et al., 2000) and nanostructures (Maurer-Jones et al., 2013). Other bench top assays are crown gall tumors inhibition on discs of potato tubers (Galsky et al., 1980), frond proliferation inhibition in duckweed (Mclaughlin et al., 1998) and yellow fever mosquito larvae lethality assay (Spielman and Williams, 1966). Among them, brine shrimp lethality test is the simplest one, low cost and effective. Freshly hashed larvae (nauplii; singular nauplius), which is about 22 mm long are used in this study and they are large enough to observe without high magnification and small enough for hatching in enormous amount without extensive workspace in a laboratory.

This assay was first proposed by Michael et al. (1956). Subsequently, it was further developed by other researchers. This lethality assay has been successively employed as a bioassay guide for active cytotoxic and antitumor agents in 1982 (Meyer et al., 1982).

For the bioactive compound of either natural or synthetic origin, this is a rapid and comprehensive test. It is also an inexpensive and simple test as no aseptic techniques are required. It easily utilizes a large number of organisms for statistical validation and requires no special equipment and relatively small amount of sample (2-20 mg or less) is necessary.

45 Chromatography Chromatography is a physicochemical method or group of methods for separation and identification of different components of a complex mixtures. It was discovered at the very beginning of the twentieth century by Russian–Italian botanist M. S. Tswett. The basic principle is that components in a mixture have different tendencies to adsorb on a surface or dissolve in a solvent and this differences is based on their chemical properties. Thus chromatography is a system consisting of a stationary (adsorption) and a mobile phase (solvent) is necessary for chromatographic separation. The stationary phase is where the substance binds and shortly releases the molecules moving through the system by the introduction of a solvent to the system. The particles can move through the system due to the mobile phase, which can be for example a liquid (eluent) or a gas (carrier gas) that carries the molecules through the stationary phase. The

34 chromatographic separation process is based on the different mobility of different components in the chromatographic system (column, plate, etc.). The compounds that are more like the stationary phase (have higher affinity towards it), move slower that the compounds that are more like the mobile phase. All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The time spent on going through the chromatographic system is called the retention time (tR). Due to the different mobilities different compounds also have different retention times.

46 Thin layer chromatography TLC is a type of chromatography, where the stationary phase is a thin layer of sorbent such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic. Eluent moves through the stationary phase due to capillary forces. The process of thin layer chromatography includes;

1.) The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry (stationary phase). 2.) The plate is placed in a closed vessel containing solvent (the mobile phase) so that the liquid level is below the spot. The solvent ascends the plate by capillary action, the liquid filling the spaces between the solid particles. This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapour and that evaporation from the plate is minimised before the run is complete. 3.) The plate is removed when the solvent front approaches the top of the plate and the position of the solvent front recorded before it is dried (this allows the Rf value to be calculated).

Thus after drying depending on the visibility of the components of the mixture separated a detection equipment such as ultra-violet lamp is used to view the various components or the plate is stained with reagents such as P-anisaldehyde or dragendorf reagent in other to view the various components of the mixture. TLC has applications in industry and scientific research in determining the progress of a reaction by studying the components present; and in separating reaction intermediates.

47 Column Chromatography The chromatographic column consists of glass or metal column filled with porous sorbent such as silica gel or sephadex gel. The surface of the sorbent acts as a stationary phase. Gravity or a special eluent pump/solvent forces the eluent flow through the column in the mobile phase. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube or at the end of the tube inform of a tap. As the mobile phase flows, it carries the substances with it. Every substance has its characteristic velocity, which depends on the time that particles spend in mobile phase before it out of the tap. A detecting device usually follow the column in order to detect the separated substances, however most times substances are collected in batches based on their similarity in retention time after which it is identified using a photometer or thin layer chromatography.

48 Infrared spectroscopy Infrared (IR) spectroscopy is one of the most common and widely used spectroscopic techniques employed mainly by inorganic and organic chemists due to its usefulness in determining structures of compounds and identifying them. Infrared Spectroscopy is the analysis of infrared light interacting with a molecule. This can be analyzed in three ways by measuring absorption, emission and reflection. Infrared (IR) spectroscopy deals with the interaction between a molecule and radiation from the IR region of the EM spectrum (IR region = 4000 - 400 cm-1). The fundamental measurement obtained in infrared spectroscopy is an infrared spectrum, which is a plot of measured infrared intensity versus wavelength (or frequency) of light (Mukamel, 2000). The method or technique of infrared spectroscopy is conducted with an instrument called an infrared spectrometer (or spectrophotometer) to produce an infrared spectrum (Smith, 2011). Stronger bonds and light atoms will vibrate at a high stretching frequency (wavenumber). IR radiation causes the excitation of the vibrations of covalent bonds within that molecule. These vibrations include the stretching and bending modes. Chemical compounds have different chemical properties due to the presence of different functional groups. An IR spectrum can be visualized in a graph of infrared light absorbance (or transmittance) on the vertical axis vs. frequency or wavelength on the horizontal axis. Typical units of frequency used in IR spectra are reciprocal centimeters (sometimes called wave numbers), with the symbol cm−1. Units of IR wavelength are

36 commonly given in micrometers (formerly called "microns"), symbol μm, which are related to wave numbers in a reciprocal way (Demirdöven et al., 2004).

49 Nuclear magnetic Resonance Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique used in quality control and research for determining the content and purity of a sample as well as its molecular structure. For example, NMR can quantitatively analyze mixtures containing known compounds. For unknown compounds, NMR can either be used to match against spectral libraries or to infer the basic structure directly. Once the basic structure is known, NMR can be used to determine molecular conformation in solution as well as studying physical properties at the molecular level such as conformational exchange, phase changes, solubility, and diffusion.

The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is applied, an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned.

A key feature of NMR is that the resonance frequency of a particular simple substance is usually directly proportional to the strength of the applied magnetic field. It is this feature that is exploited in imaging techniques; if a sample is placed in a non-uniform magnetic field then the resonance frequencies of the sample's nuclei depend on where in the field they are located. 50 Proton and Carbon nuclear magnetic resonance. Nuclei with an odd mass or odd atomic number have "nuclear spin" (in a similar fashion to the spin of electrons). This includes 1H and 13C (but not 12C). The spins of nuclei are sufficiently different that NMR experiments can be sensitive for only one particular isotope of one particular element. The NMR behavior of 1H and 13C nuclei has been exploited by organic chemist since they provide valuable information that can be used to deduce the structure of organic compounds.

Since a nucleus is a charged particle in motion, it will develop a magnetic field. 1H and 13C have nuclear spins of 1/2 and so they behave in a similar fashion to a simple, tiny bar magnet. In the

37 absence of a magnetic field, these are randomly oriented but when a field is applied they line up parallel to the applied field, either spin aligned or spin opposed. The more highly populated state is the lower energy spin state spin aligned situation.

Proton NMR, hydrogen-1 NMR, or 1H NMR is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules (Silverstein et al., 1991). In samples where natural hydrogen (H) is used, practically all the hydrogen consists of the isotope 1H (hydrogen-1; i.e. having a proton for a nucleus). A full 1H atom is called protium.

Simple NMR spectra are recorded in solution, and solvent protons must not be allowed to interfere. Deuterated (deuterium = 2H, often symbolized as D) solvents especially for use in NMR are preferred, e.g. deuterated water, D2O, deuterated acetone, (CD3)2CO, deuterated methanol,

CD3OD, deuterated dimethyl sulfoxide, (CD3)2SO, and deuterated chloroform, CDCl3. However, a solvent without hydrogen, such as carbon tetrachloride, CCl4 or carbon disulfide, CS2, may also be used (Gottlieb et al., 1997).

The precise resonant frequency of the energy transition is dependent on the effective magnetic field at the nucleus. This field is affected by electron shielding which is in turn dependent on the chemical environment. As a result, information about the nucleus' chemical environment can be derived from its resonant frequency. In general, the more electronegative the nucleus is, the higher the resonant frequency. Other factors such as ring currents (anisotropy) and bond strain affect the frequency shift. It is customary to adopt tetramethylsilane (TMS) as the proton reference frequency. This is because the precise resonant frequency shift of each nucleus depends on the magnetic field used. The frequency is not easy to remember (for example, the frequency of benzene might be 400.132869 MHz) so it was decided to define chemical shift as follows to yield a more convenient number such as 7.17 ppm. Also worthy of note is Spin-spin coupling which has to do with the effective magnetic field is also affected by the orientation of neighboring nuclei.

The 13C NMR is generated in the same fundamental was as proton NMR spectrum. Only 1.1 % of naturally occurring carbon is 13C and actually an advantage because of less coupling. The 13C NMR is directly about the carbon skeleton not just the proton attached to it. The number of signals tell

38 us how many different carbons or set of equivalent carbons while the splitting of a signal tells us how many hydrogens are attached to each carbon. (N+1 rule). The chemical shift tells us the hybridization (sp3, sp2, sp) of each carbon. Each carbon nucleus has its own electronic environment, different from the environment of other, non-equivalent nuclei; it feels a different magnetic field, and absorbs at different applied fields strength. 13C chemical shift range 0-250 ppm. In 13C NMR spectrum, the more electronegative group bonded to carbon atom deshielding increases (Derome, 2013).

51 Pharmacokinetics

Pharmacokinetics is the branch of pharmacoogy that deals with the movement of bioactive molecules in the body - the time course of its absorption, distribution, metabolism, and excretion. Pharmacology is the branch of biology concerned with the study of drug action, where a drug can be broadly defined as any man-made, natural, or endogenous (from within body) molecule which exerts a biochemical or physiological effect on the cell, tissue, organ, or organism (Ruiz-Garcia et al., 2008). Pharmacokinetics of a drug depends on patient-related factors as well as on the drug’s chemical properties. Some patient-related factors (eg, renal function, genetic makeup, sex, age) can be used to predict the pharmacokinetic parameters in populations. For example, the half-life of some drugs, especially those that require both metabolism and excretion, may be remarkably long in the elderly (Wesolowski et al., 2016). Other factors are related to individual physiology. Because of individual differences, drug administration must be based on each patient’s needs— traditionally, by empirically adjusting dosage until the therapeutic objective is met. This approach is frequently inadequate because it can delay optimal response or result in adverse effects. Knowledge of pharmacokinetic principles helps prescribers adjust dosage more accurately and rapidly.

The development of new drug candidates is often limited by initial compounds lacking reasonable chemical and biological properties for further lead optimization. Huge libraries of compounds are frequently selected for biological screening using a variety of techniques and standard models to assess potency, affinity and selectivity (Hsieh and Korfmacher, 2006). This complex process has evolved from classical methods into an integration of modern technologies and innovative strategies addressed to the design of new chemical entities to treat a variety of diseases. Recent

39 advances have been made in the collection of data and the development of models to assess and predict pharmacokinetic properties (ADME - absorption, distribution, metabolism and excretion) of bioactive compounds in the early stages of drug discovery projects (Lipinski et al., 1997): . The evolution of in silico ADME tools, addressing challenges, limitations, and opportunities in medicinal chemistry.

52 In silico Pharmacokinetics

The computational approach is one of the newest and fastest developing techniques in pharmacokinetics, ADME (absorption, distribution, metabolism, excretion) evaluation, drug discovery and toxicity. However, to date, the software packages devoted to ADME prediction, especially of metabolism, have not yet been adequately validated and still require improvements to be effective (Lipinski et al., 1997). Most are ‘open’ systems, under constant evolution and able to incorporate rapidly, and often easily, new information from user or developer databases. Quantitative in silico predictions are now possible for several pharmacokinetic (PK) parameters, particularly absorption and distribution (Hsieh and Korfmacher, 2006). In addition, and of critical importance, it is possible to screen virtual compounds. Some packages are able to handle thousands of molecules in a few hours.

Lipinski's rule of five is a rule of thumb to evaluate druglikeness or determine if a chemical compound with a certain pharmacological or biological activity has chemical properties and physical properties that would make it a likely orally active drug in humans(Lipinski et al., 2001). The rule was formulated by Christopher A. Lipinski in 1997, based on the observation that most orally administered drugs are relatively small and moderately lipophilic molecules. The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion (ADME). However, the rule does not predict if a compound is pharmacologically active (Lipinski et al., 2004). The rule is important to keep in mind during drug discovery when a pharmacologically active lead structure is optimized step-wise to increase the activity and selectivity of the Active Compounds well as to ensure drug-like physicochemical properties are maintained as described by Lipinski's rule (Oprea et al., 2001). Candidate drugs that conform to the RO5 tend to have lower attrition rates during clinical trials and hence have an increased chance of reaching the market. Lipinski's rule states

40 that, in general, an orally active drug has no more than one violation of the following criteria (Lipinski et al., 1997):

• Not more than 5 hydrogen bond donors (the total number of nitrogen–hydrogen and oxygen–hydrogen bonds) • Not more than 10 hydrogen bond acceptors (all nitrogen or oxygen atoms) • A molecular mass less than 500 daltons • An octanol-water partition coefficient log P not greater than 5

53 Rationale of the Study The spread of Plasmodium falciparum in sub-Saharan Africa and its resistance to some antimalarial drugs such as sulfadoxine/pyrimethamine, mefloquine, chloroquine and some ACTs especially those recommended by WHO (Cui et al., 2015) have necessitated the need for the development of new anti-malarial drugs especially from plant sources that are very effective, since plants have been prime in the discovery and development of such anti-malarial drugs as chloroquine and artemisinin. Strophantus hispidus leaves is a plant known to be used locally in the treatment of several diseases including malarial. Currently there is sperse scientific reports found on the anti-malarial activity and bioactive compounds of the plant leaves. 54 Aim of the Study This study was designed to investigate the antimalarial properties of the methanol extract of Strophanthus hispidus leaves and possible identification of the bioactive compound(s).

55 Specific Objectives of the Study. The Study had the following objectives;

➢ To identify and quantify the phytochemicals present in the methanol extract of Strophanthus hispidus leaves.

➢ To determine the median lethal dose (LD50) of the methanol extract of Strophanthus hispidus leaves. ➢ To determine the in vivo anti-malarial activity of the methanol extract of Strophanthus hispidus leaves on Plasmodium berghei in mice. ➢ To determine the in vivo anti-malarial activity of the various partitions of the methanol extract of Strophanthus hispidus leaves on Plasmodium berghei passaged mice.

➢ To determine the cytotoxicity of the partition with the highest activity using brine shrimp lethality test. ➢ To fractionate the partition of the methanol extract with the highest in vivo anti-malarial activity and determine the TLC profiles of the fractions. ➢ To determine the in vitro anti-plasmodial activity of the various fractions and sub-fractions. ➢ To identify the bioactive compound(s) of the fraction with the highest activity. ➢ In silico “drug-likeness” analysis and pharmacokinetic prediction of the active compound.

CHAPTER TWO

MATERIALS AND METHODS

57 Materials

58 Animals The animals (Wistar albino mice) used for this study were between 3 and 7weeks old weighing 28-38g. They were obtained from the Animal House of the Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria. These animals were fed standard animal feed (Vital feed ®) and water ad libitum and were acclimatized to laboratory conditions for 2 weeks.

59 Plant Materials The leaves of Strophantus hispidus Linn Fabaceae were collected from Nsukka environs and authenticated by Mr. Alfred Ozioko of Bioresources Development and Conservation Programme (BDCP), Nsukka, Nigeria.

60 Rodent Malaria Parasite Malaria parasites (Plasmodium berghei) ANKA strains were obtained from malaria-infected mice which were sourced from the Department of Veterinary Medicine, University of Nigeria, Nsukka.

61 Drugs The drugs used for this study were purchased from Elofex Pharmaceutical Shop in Nsukka, Enugu State of Nigeria.

62 Instruments and Equipment Rotary evaporator (Model Modulyo 4k, Edward England), Water bath (Gallenkamp England), Refrigerator (Kelvinator Germany), Colorimeter (E1 312 Model, Japan), Microlux micro pipette, Centrifuge (4000 rpm Abman), Analytical Chemical Balance (Ohaus Corp AR 3130, China), Glass wares (Pyrex, England), Microscope (B. brand specificity) (Sigma Aldrich, Germany), Spectrophotometer (unicotm UV-2101 PC) (Perfect, USA), Chromatographic tank (Shandon, England), Water bath (Chikkpas, England), Improved Neubuer counting chamber (Gallen Kanp, England), Shimadzu FTIR-84000S spectrophotometer (Rheinstetten, Germany)

Bruker DRX 500 NMR Bruker Biospin (Rheinstetten, Germany), Capillary tubes, Hot Plate, test tubes syringe and aluminum foil.

63 Chemicals and Reagents All chemicals used in this study were of analytical grade and were products of Sigma Aldrich, (USA), British Drug House (BDH) (England), Burgoyne, (India), Harkin and Williams, (England), Qualikems (India), Fluka (Germany), May and Baker, (England). Reagents used for all the experiments were commercial kits and products of Randox, (USA) and Teco (TC), (USA). Sterile distilled water was used in the preparation of the chemicals and/or reagents.

64 Methods

65 Collection of Plant Materials The plant materials, Strophanthus hispidus leaves were collected from Opi market in Nsukka, Enugu State, Nigeria and authenticated by a taxonomist, Mr. Alphred Ozioko of Bioresource Development and Conservation Programme (BDCP), Nsukka, Nigeria.

66 Preparation of Plant Extracts The plant material was air-dried, powdered (800 g) and macerated in 4.8 L of 50 % methanol in water for 48 hours at room temperature. The soaked plant material was filtered with Whatman no. 4 filter paper, and concentrated using a magnetic stirrer (70oc) to obtain crude extract (197.62 g) which was stored in the refrigerator until further use.

67 Determination of Extract Yield of Strophantus hispidus The percentage yield of extract of Strophantus hispidus was calculated by weighing the leaves before extraction and after concentration of the extract. It was calculated using the formula below: 푤푒𝑖푔ℎ푡 표푓 푒푥푡푟푎푐푡 Percentage yield % = × 100 푤푒𝑖푔ℎ푡 표푓 푔푟𝑖푛푑푒푑 푙푒푎푣푒푠

68 Solvent Solvent Partitioning of Crude Extract Twenty grams of the extract was suspended in 500 ml of 10% methanol in water and the resulting mixture was successively partitioned against n-hexane, dichloromethane and ethyl acetate to separate constituents of the crude extract of varying polarity. This was done repeatedly in other to get the desired quantity of the fractions needed for the research work.

69 Determination of Fraction Yield The percentage yield of the various fractions of aqueous-methanol extract of Strophantus hispidus was calculated by weighing the extract before fractionation and after concentration of the fractions. It was calculated using the formula below:

푤푒𝑖푔ℎ푡 표푓 푓푟푎푐푡𝑖표푛 Percentage yield % = × 100 푤푒𝑖푔ℎ푡 표푓 푒푥푡푟푎푐푡

70 Qualitative Phytochemical Analysis of the Different Fractions of Strophantus hispidus Methanol Seed Extract Qualitative phytochemical analysis of the extracts was done to determine the presence of secondary metabolites present according to standard procedure (Harborne, 1984).

71 Test for Alkaloids The samples were each extracted with 2% HCl (5 ml) and the mixture filtered. A known quantity 1 mL of the filtrates was treated with 2 drops of Wagner's reagent. The presence of alkaloids was confirmed by the reddish-brown colouration of the solution.

72 Test for Flavonoids The samples were extracted separately with distilled (5 ml) water. A known quantity 5 mL of 10% aqueous ammonia solution was added to 1 ml of each filtrate followed by three drops of concentrated H2SO4. Presence of flavonoids was confirmed by yellow colouration which disappeared on addition of H2SO4. 73 Test for phenols A known quantity, 1g of the extract was boiled with 10 ml of ether for 15 minutes. A known quantity, 5 ml of the extract was pipette into a 50 ml flask and 10 ml of distilled water added into it. A known quantity, 2 ml, of ammonium hydroxide solution and 5 ml of concentrated amyl alcohol were also added. The mixture was allowed to react for 30 minutes for colour development. Formation of bluish black colour indicates the presence of phenol.

74 Test for Tannins The samples were extracted separately with 5 ml of distilled water and filtered. Two drops of 0.1%

FeCl3 was added to each of the filtrates and observed for colour change. A blue-black colouration was taken as evidence for the presence of tannins.

75 Test for Saponins The extracts, 0.50 g was extracted with 5 ml of distilled water and filtered. 10 ml of distilled water was added to 2 mL of the filtrates and shaked vigorously for 5 minutes. Formation of froath that persist indicates the presence of saponins.

76 Test for Terpenoids The samples were extracted with 5 mL of chloroform and filtered. A known volume, 2 ml of concentrated H2SO4 was added to 1 ml of the filtrates and heated. Formation of brown ring at the interface indicates the presences of terpenoids.

77 Test for Steroids The samples were extracted with 5 ml of chloroform and filtered. A known volume, 2 mL of a o cold (4 C) mixture of concentrated H2SO4 and acetic anhydride (1:19) was added to 1 ml of the filtrates and heated. Formation of bluish-green colouration indicates the presences of steroids.

78 Test for Glycosides The samples were dissolved in 5 mL of MeOH and mixed with 2 ml of glacial acetic acid, having one drop of 0.1% FeCl3. A known volume, 1 ml of concentrated H2SO4 was added to each mixture. Presence of brown ring at the interface indicates the presence of deoxy sugar characteristic of a glycoside. 79 Quantitative Phytochemical Analysis 80 Determiation of Alkaloids One gram (1 g) of the extract was macerated with 10 ml of ethanol and 10 ml of 20 % sulphuric acid for 10 minutes and centrifuged for 5 minutes and then 0.5 ml of the supernatant each were transferred into three test tubes. To the tubes were added 2.5 ml of 60 % sulphuric acid and 2.5 ml

46 of 0.5 % formaldehyde in 60 % sulphuric acid, mixed thoroughly and allowed to stand for 3 hours. Absorbance at wavelength of 565 nm against the blank was read. The concentration of the alkaloid was extrapolated from the standard curve.

81 Determiation of Flavonoids One gram (1 g) of the extract was taken and macerated with 20 ml of ethyl acetate for 10 minutes and centrifuged for five minutes. Five milliliter (5 ml) of the supernatant were each transferred into three test tubes and 5 ml of 1 M ammonium hydroxide added and tubes were shaken vigorously for 2 to 5 minutes. The upper layers were discarded, and the absorbance of the lower layer taken at 470 nm. The blank was 1 N ammonia solution. Calculation of flavonoid was done using standard curve. The standard curve was prepared in the same way the test sample was done. The absorbance of the standard was plotted against concentration of the standard flavonoid. The slope was taken and used to calculate in the quantifying of flavonoid in the test sample using the following:

Abs of Sample x dilution factor Slope (Standard)

82 Determination of Total Phenolics The Folin-Ciocalteau’s reagent method was used to determine the total phenolic contents of the crude extract, and the partitions. Folin-Ciocalteau reagent is a mixture of phosphomolybdate and tungenstate used for the colorimetric assay of phenolic compounds. It works by measuring the amount of substance being tested needed to inhibit the oxidation of the reagent. The samples were oxidized with Folin-Ciocalteau reagent and the reaction was neutralized with sodium carbonate. About 0.2 g of samples were measured and macerated with aqueous methanol, then 100 µl were put triplicate tubes and mixed with 0.5 ml of Folin-Ciocalteau reagent (1/10 dilution) and 1.5 ml of dilute sodium carbonate. The blend was incubated in the dark at room temperature for 15 minutes. The absorbance of the blue-colored solution of all samples were measured at 765 nm. Gallic acid was used as the standard.

83 Determination of Total Tannins Tannin content of the samples were estimated by following standard procedure. About 0.2 g of the samples were measured and macerated with aqueous methanol and 1ml was mixed with 0.5ml Folin-Ciocalteau’s reagent. This was followed by the addition of 1ml of saturated sodium carbonate solution and 8ml of water. The reaction mixtures were allowed to stand for 30 minutes at room temperature. The supernatants were obtained by centrifugation and the absorbance were recorded at 725 nm using visible spectrophotometer. Tannic acid was used as standard.

84 Determination of Terpenoids The sample (0.2 g) was macerated with about 5ml absolute ethanol. Then 0.5 ml of the filtrate was mixed with 0.5 ml of aqueous phosphomolybdic acid solution and 0.5ml of concentrated H2SO4 was added and mixed. The mixture was left to stand for 30 minutes and then made up to 2.5 ml with ethanol. The absorbance was taken at 700 nm and compared to the standard curve. Ursolic acid was used as the standard.

85 Determination of Steroids A known quantity, 0.2 g of sample was measured and marcerated with 5 ml of ethanol. A known volume 1 ml was transferred to triplicate tubes followed by the addition of a chromogen. Thirty minutes incubation followed and absorbance taken at 550 nm and compared against the standard curve plotted with testosterone.

86 Determination of Saponin The sample (0.2 g) was measured and marcerated with 5 ml of petroleum ether and decanted. Another 5 ml of petroleum ether was added into the beaker and the filtrate evaporated into dryness. The residue was then dissolved with about 6ml of ethanol and 2 ml was transferred into triplicate tubes mixed with 2 ml of chromogen and absorbance taken at 550 nm after thirty minutes of incubation at room temperature. Diosgenin was used as the standard.

87 Determination of Glycosides One gram (1 g) of the extract was macerated with 20 ml of water and 2.5 ml of 15% lead acetate for 10 minutes, filtered, and 10 ml of chloroform poured to the filtrate. After shaking for 5minutes, centrifugation for 5 minutes was done. The lower fraction (2.5 ml) wastransferred into three test tubes and was evaporated to dryness in water bath. After cooling, 3 ml of acetic acid was added to

48 dissolve the residue after which 0.1 ml of 5% ferricchloride was added, mixed and 0.25 ml of concentrated sulphuric acid added, mixed and keptin the dark for 2 hours. The absorbance of the solution was read at 530 nm.The concentration of glycoside was extrapolated from the standard curve.

88 Animal studies 89 Toxicological Studies (Acute Toxicity Test)

Acute toxicity (LD50) of the methanol extract of Strophantus hispidus was carried out by the method of Lorke (1983) to define the safe dose for the extract. A total of 18 Wistar albino mice of either sex weighing 18-22g were used for this investigation. Wistar albino mice were starved of food for 6 hours (overnight fast) but allowed to water prior to the study and were grouped into five groups of three mice each. The animals were administered orally at the dose levels of 10, 100 and 1000 for phase 1 acute toxicity studies while in phase 2 the animals were administered orally at the dose levels 1600, 2600 and 5000 mg/kg b.w.. The animals were then observed closely for 24 hrs for nervousness, dullness, incoordination and death.

Calculation of LD50

LD50 = √(D0 × D100)

D0 = the highest dose that gave no mortality

D100 = the lowest dose that produced mortality

90 Induction of Malaria Parasitemia was maintained in the laboratory by serial blood passage from mouse to mouse. Albino mice previously passaged with P. berghei having variable parasitemia were used as donors. Blood was collected from parasitized mice from the retro-bulbar plexus of the medial canthus of the eye of mice using a micro-hematocrit tube and diluted with normal saline in the ratio of 1:10 (1 ml of blood in 10 ml of normal saline). Parasitized erythrocyte in the volume of 0.2 ml was used to passage the mice intraperitoneally.

91 Experimental Design of the phase 1 In vivo anti-malarial studies

A total of 30 mice were used for this study. The mice were divided into 6 groups of 5 mice each.

Group 1: Normal control (were not inoculated with malaria parasite but treated with distilled water)

Group 2: Negative control (were inoculated with malaria parasite treated with distilled water)

Group 3: Standard control (were inoculated with malaria parasite treated with artesunate 10mg/kg)

Group 4: were inoculated with malaria parasite and treated with 200 mg/kg b.w. of the extract

Group 5: were inoculated with malaria parasite and treated with 400 mg/kg b.w. of the extract

Group 6: were inoculated with malaria parasite and treated with 800 mg/kg b.w. of the extract

After 3 days (Day 4 ) of inoculation blood were collected from the retro-bulbar plexus of the medial canthus of the eye of mice using a microhematocrit tube and used for haematological analysis and also blood from the tail nip were used to make blood films that were viewed under the microscope. Once malaria is confirmed, the animals were treated for 4 days and blood was collected from the retro-bulbar plexus of the medial canthus of the eye of mice using a microhematocrit tube for biochemical analysis. The animals were observed for 4 days after treatment (post-treatment) after which blood was collected for biochemical analysis.

92 Experimental Design of the Phase 2 In vivo Anti-malarial Studies Using Different Fractions A total of 54 mice were used for this study. The mice were divided into 9 groups of 6 mice each.

Group 1: Negative control (were not inoculated with malaria parasite (m.p); but treated with distilled water)

Group 2: Positive control (were inoculated with MP treated with distilled water)

Group 3: Standard control (were inoculated with MP and treated with the standard drug)

Group 4: Were inoculated with MP and treated with 400 mg/kg b.w. of the n-hexane fraction

Group 5: Were inoculated with MP and treated with 800 mg/kg b.w. of n-hexane fraction

Group 6: Were inoculated with MP and treated with 400 mg/kg b.w. of Dichloromethane fraction

Group 7: Were Inoculated with MP and treated with 800 mg/kg b.w. of Dichloromethane fraction

Group 8: Were Inoculated with MP and treated with 400 mg/kg b.w. of the ethyl acetate fraction

Group 9: Were Inoculated with MP and treated with 800 mg/kg b.w. of the ethyl acetate fraction

93 Solvent Solvent Partitioning of Crude Extract Twenty grams of the extract was suspended in 500 ml of 10 % methanol in water and the resulting mixture was successively partitioned against n-hexane, dichloromethane and ethyl acetate to separate constituents of the crude extract of varying polarity. This was done repeatedly in other to get the desired quantity of the fractions needed for the research work.

94 Determination of Fraction Yield The percentage yield of the various fractions of aqueous-methanol extract of Strophantus hispidus was calculated by weighing the extract before fractionation and after concentration of the fractions. It was calculated using the formula below:

푤푒𝑖푔ℎ푡 표푓 푓푟푎푐푡𝑖표푛 Percentage yield % = × 100 푤푒𝑖푔ℎ푡 표푓 푒푥푡푟푎푐푡

95 Preparation of Sample Solutions 96 Preparation of Normal Saline This was prepared by dissolving 0.9 g of sodium chloride in 100 ml of water and the volume is made up to 100 ml of water.

97 Preparation of Stock Solution A known weight of the methanol extract of Strophantus hispidus was dissolved in a known volume of distilled water.

98 Preparation of Drug Solution Artemisinin (50 mg) was dissolved in a known volume (15 ml) of distilled water

99 Preparation of Serum Samples Whole blood collected from the animals were introduced into two clean non-anti-coagulant and ant-coagulant blood sample containers. The blood samples were centrifuged at a speed of 300 rpm in order to get the supernatant (serum and plasma for non-anti-coagulant and ant-coagulant respectively). The different serum and plasma gotten was used immediately for biochemical analysis. Whole anti-coagulant blood samples were used for the haematological analysis.

100 Microscopic Examination Thin films stained with Giemsa were prepared for the microscopic examination of the malaria parasite. The thin films were fixed with methanol and stained with 3% Giemsa stain of pH 7.0 for 30 min as recommended by WHO (WHO, 2000). Blood films were examined microscopically using 100X (oil immersion) objectives as described by Cheesbrough (2000). The thick films were used to determine the parasite densities while thin films were used to identify the parasite species and infective stages. The result is presented as average prasitaemia count/field in 10 fields counted for every film prepared. 101 Determination of Haemoglobin Concentration Principle

Haemoglobin concentration was determined by the method described by Dacie and Lewis (1991) in which blood from EDTA is diluted in a Drabkin’s solution containing potassium cyanide and potassium ferricyanide. As a result, RBCs are hemolyzed and the haemoglobin is released. The released haemoglobin is oxidized in the following reaction.

Haemoglobin (Hgb) + ferricyanide → methaemoglobin

Methaemoglobin + cyanide → cyanmethaemoglobin (or also called HiCN)

Absorbance of the HiCN solution is read in a spectrophotometer at 540 nm

Absorbance of the HiCN solution is compared to the reference HiCN standard solution

Procedure

The blood (EDTA) was diluted in Dradkin’s solution by 1: 20 (20 µL of blood in 4000 µL). The tube was covered, inverted several times and left to stand for 5-10 minutes to ensure complete conversion. The HiCN solution was poured into a cuvette and read spectophotometrically at the wavelength of 540 nm using the Drabkin’s solution as blank. HiCN is used as standard. Haemoglobin concentration is calculated using the following equation:

Abs. of Test Sample Dilutionfactor x Concentration of STD in mg/l x Abs. of Standard 1000

Result is expressed in g/dl

102 Determination of Red Blood Cell (RBC) Count This was done using standard method as described by Cheesbrough (2005). The blood sample was diluted in the ration of 1:20 with 10% NaCO3. The diluted sample was loaded into the Neubaer chamber with the aid of a Pasteur pipette. The RBC was counted from appropriate squares on the chamber under an electronic microscope.

103 Determination of Packed Cell Volume (PCV) This was done using standard technique as described by Ochei and Kolhartar (2008) Blood sample were collected into PCV tubes heparinized using capillary action. One end of the tube was sealed with plasticine and then centrifuged using the haematocrit centrifuge for 5 mins at 2500 gram. The test result was read using a PCV haematocrit reader.

104 Determination of Total White Blood Cell (WBC) Count The white blood cell count was determined following the standards technique as described by Cheesbrough (2008). The blood sample was diluted 1:20 with Turks solution, which is 2% glacial acetic acid. The diluted sample was loaded into a Neubaer counting chamber with the aid of pasture pipette. The total WBC was calculated by counting the required number of squares on the counting chamber under a microscope.

105 Assay of Alkaline Phosphatase Activity This was done using the QCA Commercial enzyme kit which is based on the phenolphthalein monophosphate method of Klein et al. (1960), Babson (1965) and Babson et al. (1966).

Principle

Serum alkaline phosphatase hydrolyses a colourless substrate of phenolphthalein monophosphate giving rise to phosphoric acid and phenolphthalein which at alkaline pH values, turns to pink colour that can be spectrophotometrically determined.

The concentrations in the reagent solution are;

2-Amion-2-methly-1- propanol - - - 7.9 N

Phenolphthalein monophosphate - - - 63 mM

Na2PO4 - - - 80 mM

Stabilizers and preservatives

Procedure

Distilled water (1 m1) was pipetted into 2 sets of test tubes labelled SA sample and ST standard respectively. Then one drop of each the chromomeric substrates was added to the distilled water in the two sets of test tubes. Their contents were mixed and incubated at 37oC for 5 min. A standard solution of 0.1M (alkaline phosphate) was added to the standard test tube (ST) only, followed by the addition of 0.1m1 of the serum sample to the sample test tube (SA). The contents of the test tubes were mixed and incubated at 370C for 20 min in a water bath. A colour developer (phenolphthalein monosulphate) (5 ml each) was added to both sets of test tubes. Absorbance of the sample against the blank (water) was read at a wavelength of 550 nm. The activity of alkaline phosphates in the serum was obtained from the formula below.

SAO.D X 30 = U/L of Alkaline phosphates STO.D

SAO.D = sample Optical Density

STO.D= Standard Optical Density

Normal values

Adults= 9-35 U/L

Children =35-100 U/L

106 Assay of Aspartate Aminotransferase Activity A Randox Commercial Enzyme Kit according to the method of Reitman and Frankel (1957) was used. This method is based on the principle that oxaloacetate is formed from the reaction below:

α- Oxoglutarate + L-Aspartate L-glutamate+ Oxalocacetate

Glutamic- oxaloacetic acid aminotransferase (aspartate aminotransferase) activity was measured by monitoring the concentration of oxalocetate hydrazone formed with 2, 4-dinitrophentl hydrazine.

Reagents

Contents Initial concentration of reagents

Phosphate buffer - - 100 mmol/, pH 7.4

L-Aspartate - - 100 mmol/1

α-Oxoglutarate - - 2 mmol/l

2, 4-dinitrophnyl hydrazine - 2 mmol/l

Sodium hydroxide solution - 0.4 mol/l

Measurement against Reagent Blank

The AST substrate phosphate buffer (0.5 ml each) was pipette into both the reagent blank (B) and sample (T) test tubes respectively. The serum sample (0.6 ml) was added to the sample (T) test tubes only and mixed thoroughly. Then 0.1 ml of distilled water added to the reagent blank (B). The entire reaction medium was well mixed and incubated for 30 min in a water bath at 370C.

Immediately after incubation, 2, 4-dinitrophenyl- hydrazine (0.5 ml) was added to the reagent blank (B) and the sample (T) test tubes, mixed thoroughly and allowed to for exactly 20 min at 250C. Finally, 5.0 ml of sodium hydroxide (0.4 mol/l) solution was added to both the blank and the reagent test tubes respectively and thoroughly.

The absorbance of sample was read at a wavelength of 550 nm against the reagent blank after 5 min.

Measurement against Sample Blank

The AST substrate phosphate buffer (0.5 ml each) was pipette into the sample blank (B) and sample (T) test tubes respectively. The serum sample (0.1 ml) was added to the sample test (T) only and mixed immediately then incubated in a water bath for exactly 30min at 37oC. 2, 4- Di- nitrophenlhyrazine was added to both the sample blank (B) and sample (T) test tubes immediately after incubation. Also, 0.l ml of the sample was added to the sample blank (B) only. Each medium was mixed and allowed to stand for exactly 20 min at 250C. Finally, 5.0 ml of sodium hydroxide (NaOH) solution 0.4 mol/l was added to both the sample blank (B) and sample (T) test and mixed thoroughly. Absorbance of the sample was read at a wavelength of 550 nm against the sample blank after 5min.

Normal values in human, serum up to 30 U/L

107 Assay of Alanine Aminotransferase Activity A Rondox Commercial Enzyme Kit based on the methods of Reitman and Frankel (1957) was used. Alanine aminotransfrase assay is based on the principle that pyruvate is formed from: α- Oxoglutarate+ L-Alanine L-Glutamate+ Pyruvate. Alanine aminotransferase is measured by monitoring the concentration of pyruvate hydrazine formed with 2, 4-dinitrophenyl hydrazine. Reagents Contents Initial concentrations of solutions Buffer Phosphate buffer - - 100 mmol/l pH 7.4 L-alanine - - 200 mmol/l &-oxoglutarate - - 2.0 mmol/l 2, 4-dinitrophenyl hydrazine - 2.0 mmol/l Sodium hydroxide solution - 0.1 mol/l

Procedure

Measurement against Sample Blank

The ALT substrate phosphate buffer (0.5 ml each) was pitted into two sets of test tubes labeled B (Sample blank) and T (Sample test respectively. The serum (0.l ml) sample was added to the sample test (T) only and mixed properly, then incubated for exactly 30min in a water bath at a temperature of 370C. 2,4 – dinitrophentyl hydrazine (0.5 ml) was added to both test tubes labeled T (sample test) and B sample was (Sample blank) immediately after the incubation. Also, 0.1 ml of serum sample was added to the sample blank (B) only. The entire medium was mixed thoroughly and allowed to stand for exactly 20 min at 250C. After which, 5.0ml of sodium hydroxide (NaOH) solution (0.4 mol/l) was added to both test tubes and also mixed thoroughly. Absorbance of the sample was read at a wavelength of 550 nm after 5 min.

Measurement against Reagent Blank

The ALT substrate phosphate buffer (0.5 ml each) was pipetted into both the reagent blank (B) and sample (T) test tubes respectively. The serum sample 0.lml was added to the sample (T) test tube only and mixed thoroughly. Then 0.l ml of distilled water was added to the reagent blank (B). The entire medium were mixed and incubated for exactly 30 min in a water bath at 370C. Immediately after incubation, 2, 4- dinitrophenyl- hydrazine (0.5 ml) was added to both reagent blank and sample (T) test tubes. The contents of the tubes were mixed thoroughly and allowed to stand for exactly 20 min at 250 C. Finally, 5.0ml of sodium hydroxide solution (0.4 mol) was added to both blank and reagent test tubes respectively. Each was mixed thoroughly and absorbance of sample was read at a wavelength of 550 nm against the reagent blank after 5 min.

Normal Values in humans Serum up to 20 U/L

108 Determination of Total Bilirubin Concentration A colorimetric method with a kit supplied by Randox was used based on the method described by Jendrassik and Grof (1938).

Principle

Direct conjugated bilirubin reacts with diazotized sulphanilic acid in alkaline medium to form a blue coloured complex. Total bilirubin is determined in the presence of caffeine, which releases albumin bound bilirubin, by the reaction with diazotized sulphanilic acid.

Reagent composition

Contents Initial concentration of solution

Sulphanilic acid - - 29 mmol/L

Hydrochloric acid - - 0.17 N

Sodium Nitrate - - 25 mmol/L

Caffeine - - 0.26 mol/L

Sodium benzoate - - 0.52 mol/L

Tartrate - - 0.93 molL

Sodium hydroxide - - 1.9 N

Procedure

Reagent 1 (sulphanilic acid, hydrochloric acid,) 0.20 ml, was pipetted into two different cuvettes labelled sample blank (B) and sample (A) respectively, then a drop (0.05 ml) of reagent was introduced. Then a drop of 0.05 ml of reagent was pipette into the cuvette containing sample (A) only. Afterwards, 1.0 ml of reagent 3 (caffeine, sodium benzoate) was pipetted into the cuvettes containing samples B and A respectively. Serum sample (0.2ml) was then pipetted into both cuvettes, sample blank (B) and sample (A). Their contents were separately mixed and allowed to stand for 10min at 250C.

This was followed by addition of 1ml of reagent 4 (Tartrate, sodium hydroxide) into both cuvettes containing sample blank and sample. They were mixed and allowed to stand for 30min at 250C. Finally, absorbance of bilirubin values were obtained using the calculation below:

Total bilirubin (µmo/L) =184 x ATB (560 nm)

Total bilirubin (mg/dl) =10.8 x ATB (560 nm)

Normal value in serum

Total bilirubin up to 1.7 µmol/L OR up to 1 mg/Dl

109 Assay of Superoxide Dismutase Activity The activity of SOD was evaluated by the method of Xin et al. (1991). It is based on the inhibition of epinephrine auto-oxidation to adenochrome in alkaline environment. Auto-oxidation of epinephrine was initiated by adding 1ml of Fenton reagent to a 4ml mixture of 0.3 mM epinephrine,

1 mM solution of Na2CO3, 0.3 mM EDTA and 1m1 of distilled water at a final volume of 6 ml. The auto-oxidation was monitored spectrophotometrically at 480 nm every 30 secs for 5 min. The experiment was repeated with 1.0ml of the serum. A graph of absorbance against time was plotted for each sample and initial rate of anti-oxidation was calculated. One unit of SOD activity was defined as the concentration of the enzyme in the sample that caused 50% reduction in the auto- oxidation of epinephrine. SOD activity was then calculated for each sample and expressed in lu/L.

110 Assay of Catalase Activity The catalase activity of the serum was determined according to the method of Sinha. (1972). The standard curve of H2O2 was prepared. To prepare the curve, different amounts of 0.2 M H2O2 ranging from 10 to 100 micromoles were taken in test tubes. Dichromate/acetic acid (2 ml) was added to each. On adding the dichromate/acetic acid, an unstable blue precipitate of perchromic acid was instantaneously produced. Heating for 10 min in a boiling water bath changed the colour of the solution to stable green due to the formation of chromic acetate. After cooling at room temperature, the volumes of the samples were made to 3 ml with distilled water and the absorbance measured at 570 nm. The curve was obtained by plotting absorbance on the vertical axis against concentration on the horizontal axis.

The serum was properly diluted 50 times. 4 ml of 0.2 M H2O2 was added to 5 ml of 0.1 M phosphate buffer pH 7.0. Thereafter 1.0 ml of the properly diluted enzyme preparation (the diluted serum) was added to the H2O2 /buffer mixture and gently mixed at room temperature. Some portion (1.0 ml) of the fraction mixture was withdrawn and blown into 2 ml dichromate/acetic acid reagent at one minute interval and the steady absorbance reading taken at 570 nm.

The monomolecular velocity constant K; for the decomposition of H2O2 by catalase was determined by using the equation for a first order reaction

Where S0 is the initial H2O2 concentration and S is the concentration of H2O2 at a particular time interval given as t (minutes). The values of K are plotted against t, and the velocity constant of catalase K (0) at 0 minute determined by extrapolation (that is the intercept on the vertical axis). The catalase contents of the sample are expressed in terms of mg/dl.

111 Determination of Malondialdehyde Concentration Lipid peroxidation was determined spectrophotometrically by measuring the level of the lipid peroxidation product, malondialdehyde (MDA) as described by Wallin et al. (1993)

Principle

Malondialdehyde (MDA) reacts with thiobarbituric acid to form a red or pink coloured complex which, in acid solution, absorbs maximally at 532 nm.

MDA + 2TBA MDA: TBA adduct + H2O

Reagent Preparation

1. 1.0% Thiobarbituric acid (TBA): A known quantity, 1.0 g, thiobarbituric acid was dissolved in 83 ml of distilled water on warming. After complete dissolution the volume was made up to 100 ml with distilled water.

2. 25% Trichloracetic acid (TCA): A known quantity, 12.5 g, of trichloroacetc acid was dissolved in distilled water and made up to 50 ml in a volumetric flask with distilled water.

3. Normal saline solution (NaCI): A known quantity, 0.9 g, of NaCI was dissolved in 10 ml of distilled water and made up to 100 ml with distilled water.

Procedure

To 0.1 ml of plasma in test tube was added 0.45 ml of normal saline and mixed thoroughly before adding 0.5 ml of 25% trichloroacetic acid (TCA) and 0.5 ml of 1% thiobarbituric acid. The same volume of tricholoracetic acid, and saline was added to the blank. 0.1 ml of distilled water was also added to the blank instead of plasma. Then, the mixture was heated in a water bath at 950C for 40 min. Turbidity was removed by centrifugation. The mixture was allowed to cool before reading the absorbance of the clear supernatant against reagent blank at 532 nm. Thiobarbituric acid reacting substances were quantified as lipid peroxidation product by referring to a standard

60 curve of (MDA) concentration (i. e. equivalent generated by acid hydrolysis of 1,1,3,3- tetraethoxypropane (TEP) prepared by serial dilution of a stock solution).

Table 2: Procedure for lipid peroxidation assay

Blank Test

Plasma --- 0.10 ml

Distilled water 0.10 ml ---

Normal saline 0.45 ml 0.45 ml

25%TCA 0.50 ml 0.50 ml

1% TBA 0.50 ml 0.50ml

Then, the absorbance was taken at wavelengths of 532 nm and 600 nm against the blank.

112 Determination of Plasma Calcium Concentration Blood samples were collected from the veins of adult Negroes of various categories i.e. normal (uninfected individuals), untreated infected and treated-infected individuals respectively. Plasma calcium (ion) amounts were determined by the method of Clark and Collip. (1925) in human blood plasma obtained as the supernatant after centrifuging human blood samples at 3000X G for 15 min.

113 Determination of Haemozoin Concentration The formation of haemozoin was determined using the method of Dibua et al. (2013).

Haemozoin Extraction: A 2 ml EDTA blood sample was centrifuged for 5 minutes using bucket centrifuge (Model 80-2). The supernatant was discarded and the pellets suspended in normal saline (NaCl) and further centrifuged for 5 minutes and the supernatant discarded. About 0.5 ml of phosphate solution, pH 7.6 was added to each tube and vigorously shaken mechanically for 2 seconds to haemolyse the erythrocytes. The tubes were then kept on ice for 10 minutes to avoid excess haemolysis and then centrifuged for 5 minutes before discarding the supernatant. Approximately 1 ml of Tris buffered solution of pH 7.2 was dispensed into the pellets in the tubes, centrifuged for 10 minutes, and the supernatant discarded. The insoluble pellets were re-suspended in 0.5 ml of 2.5% Sodium dodecyl sulphate solution (SDS), buffer with Tris buffer solution, pH

7.8 and kept at room temperature for 1 hour before centrifuging for 10 minutes. The supernatant was again discarded and the pellets once more resuspended in 0.5 ml of 2.5% Sodium dodecyl sulphate (SDS) solution buffered with Tris buffer solution pH 7.8 and kept at room temperature for 1 hour. The suspension was then centrifuged for 10 minutes, and the supernatant discarded before harvesting the SDS insoluble pellets (haemozoin) as previously described (Orji, 2001). Concentration by Spectrophotometry: The weight of extracted haemozoin was determined using the Mettler weighing balance and the various masses obtained recorded. The concentration of haemozoin was calculated by completely dissolving known masses of haemozoin (in mg/ml) in 0.5ml of diluted sodium hydroxide, and the solution of haemozoin analyzed spectrophotometrically using Spectrophotometer S23A, at 400nm wavelength (Bohle et al., 2005). 114 Histopathological Examination Histopathological studies were done on the liver and spleen tissues of the mice according to the method of Drury et al. (1967) and Bancroft and Gamble. (2002).

A. Fixation and Washing

Formalin (10%) was used as the fixative and for the purpose of preservation. A thin section of the tissue (about 1 to 2 cm in diameter) was trimmed with a sharp razor blade. The small pieces of the tissue was placed in the 10% formalin, the container was shaken gently several times to make sure that the fluid had reached all surfaces and that pieces were not sticking to the bottom. This was then incubated at 250C for 24 hours, to allow proper fixing. The fixed tissue pieces were washed with running water for 24 hours to free them from excess fixatives.

B. Dehydration

Water was removed from the tissue before embedding the tissue in paraffin. The dehydration was achieved by immersing the thin sections of the tissue in automatic tissue processor containing 12 jars. The first three (3) jars contained 70%, 90% and 95% absolute alcohol respectively. This was done to remove the water content in the tissues. The absolute alcohol reduced the shrinking that occurred in the tissue. The time for each step was 30 minutes. A second change of absolute alcohol was included to ensure complete removal of water. This was achieved in the second three (3) jars of the automatic tissue processor.

C. Clearing

Solutions of xylene was used for clearing the tissue sections. This step was achieved in the third three (3) jars of the automatic tissue processor. Because the alcohol (ethanol) used for dehydration will not dissolve or mix with molten paraffin, the tissue was immersed in xylene solution which is miscible with both alcohol and paraffin before infiltration could take place. Clearing was done to remove opacity from dehydrated tissue. A period of 15 minutes was allowed to elapse before the tissue was removed from the solution for infiltration with paraffin.

D. Infiltration with Paraffin

Paraffin wax at 50 to 52oC was used to infiltrate the tissue. The tissue was transferred directly from the clearer to a bath containing melted paraffin. After 30 - 60 minutes incubation in the first bath, the tissue was removed to a fresh dish of paraffin contained in the fourth three jars of the automatic tissue processor for a similar length of time.

E. Embedding (Blocking) with Paraffin

As soon as the tissue was thoroughly infiltrated with paraffin, it (paraffin) was allowed to solidify around and within the tissue.

F. Paraffin Sectioning

The embedded blocks was trimmed into squares and fixed in the microtome knives for sectioning after which the sections will be floated on a water bath.

G. Mounting

Glass slides were thoroughly cleaned and a thin smear of albumen fixative was made on the slides. The albumenized slide was used to collect the required section from the rest of the ribbon in the water. The section on the glass slide was kept moist before staining.

H. Staining with Haematoxylin

The slides were passed through a series of jars containing alcohols of decreasing strength and various staining solutions.

I. Microscopic Observation of Slide

The slides prepared were mounted on photomicroscope, one after the other and viewed at different magnification power of the microscope. Photograph of each of the slides was taken.

115 Brine shrimp lethality bioassay The N-hexane fraction of Strophantus hispidus was evaluated for lethality using the brine shrimp lethality bioassay as described by McLaughlin. (1991) and modified by Sirintorn et al. (2004). Artificial seawater was prepared by dissolving 40 g of sea salt (Sigma-Aldrich, UK) in 1 litre of distilled water. This was then autoclaved at 121 °C for 20 min, allowed to cool to room temperature before used for hatching the shrimp eggs. Brine shrimp eggs (Sanders Great Salt Lake, Brine Shrimp Company L.C., U.S.A.) were then hatched in the sea water by incubating under light for 48 hours at room temperature (25-29 °C). N-hexane fraction of S. hispidus was solubilized by dissolving in dimethyl sulfoxide (DMSO; Sigma-Aldrich, UK). Serial dilutions of the fraction in sea water were then made in triplicate in the 24-well culture plate (Linbro; Flow Lab. Inc., VA, U.S.A). Toxicity of the fraction was tested at 0.1, 1.0, 10, 100 and 1000 ppm in 1 mL sea water solutions with 1% DMSO (v/v). Each well contained ten live nauplii (larvae). Control wells (0 ppm of test fraction) containing only 1% DMSO (v/v) in sea water was included in the assay. After 48 hours incubation at room temperature (25-29 °C), the culture plate was examined using a magnifying lens to determine the number of dead (non-motile) nauplii in each well. The LC50 was calculated using probit analysis.

116 Bioassay guided fractionation, isolation and characterization of pure compound The n-hexane fraction was subjected to a silica gel (60-200 mesh) column chromatography eluted with gradients of n-hexane-acetone-methanol as described by zofou et al. (2011). The active compound was isolated using preparative TLC and further purified with Sephadex LH- 20. An amount of 2 g of the n-hexane fraction was subjected to a silica gel (60-200 mesh) column chromatography eluted with gradients of n-hexane-Acetone (10:0; 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9; 0:10) and Acetone-MeOH (10:0; 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9; 0:10). Ninety-five fractions of 20 mL each were collected and combined on the basis of their TLC profiles into nine major fractions F1-F9 (F1: 1-15, 0.23 g; F2: 16-24, 0.07 g ; F3: 25-31, 0.03; F4: 32-36, 0.07; F5: 37-50, 0.17 g; F6: 51-60, 0.21 ; F7:61-69, 0.17; F8:70-85, 0.32 g; F9: 86- 95, 0.21 g). All the Fractions were screened for invitro-antiplasmodial activity and fraction 7-9 were considered for further investigation given the outcome of the biological screening. Fraction

F7-9 were spotted on a TLC plate and further developed using preparative TLC glass plate of 0.2mm thickness using solvent system (Hex 6:4 Chl) to isolate the various compounds present in the fractions. The various isolates were screened for invitro-antiplasmodial activity and the most active was further purified on Sephadex LH-20 using Hex-chloroform (1:1) to give Active Compound (30 mg, Rf = 0.88; n-hexane-chloroform 60:40). Column chromatography was run with Merck silica gel 60 and Sephadex LH-20. Analytical TLC was carried out on silica gel (Merck GF254) precoated plates with spots detected with an UV lamp at 254 and 366 nm and further visualized by spraying with P-anisaldehyde-sulphuric acid spray, followed by heating at 100°C.

117 In Vitro Anti-Plasmodial Investigation using β-hematin Inhibitory Assay (BHIA) In Vitro Anti-Plasmodial Investigation using β-hematin Inhibitory Assay (BHIA) was done by the method described by (Basilico et al., 1998). Heme polymerization inhibitory activity test was conducted using hematin solution and the test sample. A total of 100 μL solution of 1 mM hematin in 0.2 M NaOH was put into the microtube, then added 50 μL of test material with various dose levels, 50 and 100 µg/ml. Replication was conducted for 3 times for each dose. To initiate the polymerization reaction heme, 50 μL glacial acetic acid solution (pH 2.6) was added in the microtube which already contains hematin solution and sample, then were incubated at 37 °C for 24 h. Positive control used was artemisinin (10 µg/ml) , whereas the negative control was distilled water. After incubation, the microtube was centrifuged at 8000 rpm for 10 minutes. Supernatant was removed and the precipitate was washed 3 times with 200 μL DMSO. Each microtube was washed by centrifugation speed 8000 rpm for 10 minutes. The precipitate obtained was added with 200 μL 0.1 M NaOH. Each 100 μL of the solution obtained were put in microplate 96. Absorbance was read at wavelength of 405 nm. The results were recorded as % inhibition (I%) of heme crystallization compared to negative control

(DMSO) using the following equation:

AN − AS I% = × 100 AN where, AN = absorbance of negative control,

AS = absorbance of test samples.

118 Identification of Pure Compounds IR spectra were recorded on a Shimadzu FTIR-84000S spectrophotometer while 1H NMR and 13C NMR were recorded at natural product laboratories, Strathclyde institute of pharmacy and biomedical sciences (SIPBS), University of Strathclyde, Glascow, United Kingdom and was recorded on a bruker DRX 500 NMR (Bruker Biospin, Rheinstetten, Germany).

119 In silico “Drug-likeness” Analysis and Pharmacokinetic Prediction. Pharmacokinetic prediction was done based on lipinski’s rule of 5 using the Swiss ADME software. Modifications on the compound was done using Marvin Sketch software. 120 Statistical Analysis The results were expressed as means ± SD and tests of statistical significance were carried out using one way analysis of variance (ANOVA) with repeated measures. The Statistical Package for Social Sciences (SPSS), version 20 was used. P values < 0.05 were considered significant.

CHAPTER THREE

RESULTS

122 Percentage Yield of Extract As shown in table 3, the percentage yield of the methanol leave extract of Strophantus hispidus was found to be 16.92 %.

123 Outcome of Acute Toxicity Studies (LD50) Oral administration of the aqueous-methanol extract of Strophanthus hispidus leaves to mice at doses ranging from 10 – 5000 mg/kg b.w caused death of only one animal at the dose of 5000 mg/kg b.w. Therefore the oral LD50 of the extract in mice was above 5000 mg/kg b.w. as shown in table 4.

124 Qualitative Phytochemical Composition of Strophantus hispidus Methanol Leaf Extract As shown in table 5, bioactive compounds such as alkaloids, phenols, tannins and saponins were highly present in the extract while flavonoids, glycosides, Steroids, and terpenoids were moderately present in the extract.

125 Quantitative Phytochemical Composition of Strophantus hispidus Methanol Leaf Extract Table 6 shows the quantitative phytochemical composition of the methanol leaf extract of Strophantus hispidus. Bioactive compounds such as alkaloids were found to be highest (903.06 ± 67 mg/100g) compared to phenols, (276.48 ± 14mg/100g) flavonoids, (149.27 ± 22mg/100g) and terpenoids (69.27 ± 4 mg/100g) that were moderately present in the extract. However saponins, (18.50 ± 11 mg/100g), glycosides, (3.01 ± 0.5 mg/100g) steroids, (2.35 ± 0.5 mg/100g) and tannins (1.84 ± 2 mg/100g) were found least in the extract.

Table 3: Percentage Yield of Extract

Weight of leaves (g) Extraction yield (g) Percentage yield 800.00 135.36 16.92

Table 4: Acute Toxicity Studies (LD50)

Phase 1 Dosage mg/kg b.w Mortality Group 1 10 0/3 Group 2 100 0/3 Group 3 1000 0/3 Phase 2 Group 1 1600 0/3 Group 2 2900 0/3 Group 3 5000 1/3

Table 5: Qualitative Phytochemical Composition of Methanol Extract of Strophantus hispidus Leaves

Phytochemicals Bioavailability Alkaloids +++ Flavonoids ++ Terpenoids ++ Steroids ++ Phenols +++ Tannins +++ Saponins +++ Cardiac Glycosides ++ Key: ++ Moderately present +++ Highly present

Table 6: Quantitative phytochemical composition of Methanol Extract of Strophantus hispidus Leaves

Phytochemicals Quantity (mg/100g) Alkaloids 903.06 ± 67 Flavonoids 149.27 ± 22 Terpenoids 69.27 ± 4 Steroids 2.35 ± 0.5 Phenols 276.48 ± 14 Tannins 1.84 ± 2 Saponins 18.50 ± 11 Glycosides 3.01 ± 0.5 Values were expressed as Mean ± S.D (n=3)

126 Biochemical Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Parasitemia Count (PC) of Mice Passaged with Plasmodium berghei As shown in Figure 4, significant (p < 0.05) increase was observed in the mean PC of all the animals in groups (2 to 6) passaged with Plasmodium berghei on Day 4 when compared to the negative control group (group 1) that was not passaged. After Day 4 of treatment, there was a significant (p < 0.05) decrease in the mean PC of all the animals in the test group (4-6) when compared to the positive control group 2 and a non-significant (p ˃ 0.05) difference was observed between the test groups and the standard control. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the mean PC between all the test groups. Nonetheless there was a significant (p < 0.05) increase in the mean PC of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the mean PC of all the test groups was observed when compared to the positive control group. Among the test groups there was a significant (p < 0.05) increase in the mean PC of group 4 when compared to group 5 and 6. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the mean PC of group 5 and 6 when compared to the standard control group 3. Conversely there was a significant increase in the mean PC of group 4 when compared to the standard control group. It was observed that the positive control group showed a statistically significant (p < 0.05) increase in the mean PC when compared to the standard control group.

Among various groups, it was observed that after Day 4 of treatment there was a significant (p < 0.05) decrease in the mean PC of all the animals in the test groups when compared to the mean PC after Day 4 of passaging but a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. The positive control group showed a significant (p < 0.05) increase in the mean PC after Day 4 of treatment when compared to the mean PC after Day 4 of passaging. Conversely the standard control group showed a significant (p < 0.05) decrease in the mean PC after Day 4 of treatment when compared to the mean PC after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment

140 Day 4 After passaging On day 4 of treatment

116 Day 4 of Post-treatment 120

100 80.3

39.33

37.7

38.7 34.7

Mean Parasitaemia Count Parasitaemia Mean 40

32 16

20 15.5

9.33

8.66

8 8

7.5

5.5

0 0 0 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups

Figure 4: Effect of treatment with methanol extract of Strophantus hispidus Leaves on the mean parasitaemia count of mice passaged with Plasmodium berghei

Group 1 = Negative control (not passaged but was administered with the vehicle 5 ml/kg of normal saline) Group 2 = Positive control (passaged but not treated.) Group 3 = Standard control (passaged and treated with artesunate 10 mg/kg b.w.) Group 4 = Test group low-dose (passaged and treated with 200 mg/kg b.w. of Strophantus hispidus leaf extract) Group 5 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Strophantus hispidus leaf extract) Group 6 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Strophantus hispidus leaf extract)

127 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on the

Packed Cell Volume of Mice Passaged with Plasmodium berghei From Figure 5 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the PCV of animals in groups (2 to 6) when compared to the negative control group (group 1). Nevertheless after Day 4 of treatment, there was a significant (p < 0.05) increase in the PCV of all the animals in the test groups (4-6) when compared to the positive control group 2. The test groups (4 and 5) showed a significant (p < 0.05) decrease in the PCV when compared to the standard control except group 6. Nonetheless there was a significant (p < 0.05) decrease in the PCV of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the PCV of all the test groups was observed when compared to the positive control group. Among the test groups there was a significant (p < 0.05) decrease in the PCV of group 4 when compared to group 5 and 6. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the PCV of group 4 when compared to the standard control group 3, however groups 5 and 6 showed a statistically significant (p < 0.05) increase. It was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the PCV when compared to the standard control group.

Among the test groups, group 4 showed no statistically significant (p ˃ 0.05) difference in all. However group 5 showed a significant (p < 0.05) increase in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging and a significant (p < 0.05) increase after Day 4 of post-treatment. Group 6 showed a significant (p < 0.05) increase in the PCV after Day 4 of post-treatment when compared to the PCV after Day 4 of passaging and after Day 4 of post- treatment. The positive control group showed a significant (p < 0.05) decrease in the PCV after Day 4 of treatment when compared to the PCV concentration after Day 4 of passaging and also a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) increase in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Thus in the negative control group a significant (p < 0.05) increase was observed in the PCV only at day four of post-treatment.

Day 4 After passaging 50 On day 4 of treatment Day 4 of Post-treatment

45 43

40.5

39.5

38.67 39

40 37

33.5 32.67

35 31.67

29 27.67

30 26.33

26 24.67

25 20.33

17 Mean ( PCV %) Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups

Figure 5: Effect of treatment with methanol extract of Strophantus hispidus Leaves on the packed cell volumn of mice passaged with Plasmodium berghei

128 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on the Haemoglobin Concentration (Hb Conc) of Mice Passaged with Plasmodium berghei. From Figure 6 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the Hb Conc of animals in all groups (2 to 6) when compared to the negative control group (group 1). However after Day 4 of treatment, there was a significant (p ˂ 0.05) increase in the Hb Conc of all the animals in the test groups (4-6) when compared to the positive control group 2 and no significant (p > 0.05) difference when compared to the standard control group3. Nevertheless there was a significant (p < 0.05) decrease in the Hb Conc of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the Hb Conc of all the test groups was observed when compared to the positive control group. Comparatively all the test groups showed no significant (p > 0.05) difference in the Hb Conc and a non-significant (p > 0.05) difference in comparison to the standard control group. Nevertheless a (p < 0.05) significant decrease was observed in the Hb Conc of the test group when compared to the negative control group 1. Also it was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the Hb Conc when compared to the standard control group.

Across the groups, the negative control group showed a non-significant (p > 0.05) increase nor decrease in the Hb Conc after treatment when compared to the Hb Conc after passaging and treatment. Among the test groups 4, 5 and 6, there was a significant (p < 0.05) increase in the Hb Conc after treatment when compared to the Hb Conc after passaging but a non-significant (p > 0.05) difference in comparison to the Hb Conc after post-treatment. The positive control group showed a significant (p < 0.05) decrease in the Hb Conc after Day 4 of treatment when compared to the Hb Conc concentration after Day 4 of passaging and also a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) increase in the Hb Conc after Day 4 of treatment when compared to the Hb Conc after Day 4 of passaging and non-significant (p ˃ 0.05) difference in comparison to the Hb Conc on Day 4 of post-treatment.

16 Day 4 After passaging On day 4 of treatment Day 4 of Post-treatment

14 13.5 12.9

12 11.36

10.7

10.06

10.4

10.45

10.43

10.1 9.73

10 9.5

8.1

7.73

8 7.66

6.6 6

6 Mean Hb Conc. (g/dl) Hb Conc.Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 6: Effect of treatment with methanol extract of Strophantus hispidus Leaves on the Hemoglobin concentration of mice passaged with Plasmodium berghei.

Group 1 = Negative control (not passaged but was administered with the vehicle 5 ml/kg of normal saline) Group 2 = Positive control (passaged but not treated) Group 3 = Standard control (passaged and treated with artesunate 10 mg/kg b.w.) Group 4 = Test group low-dose (passaged and treated with 200 mg/kg b.w. of Strophantus hispidus leaf extract) Group 5 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Strophantus hispidus leaf extract) Group 6 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Strophantus hispidus leaf extract)

129 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on the Red Blood Cell (RBC) Count of Mice Passaged with Plasmodium berghei. From Figure 7 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the RBC count of animals in all groups (2 to 6) when compared to the negative control group 1. However after Day 4 of treatment, there was a significant (p ˂ 0.05) increase in the RBC count of all the animals in the test groups (4-6) when compared to the positive control group 2 and a non-significant (p > 0.05) difference when compared to the standard control group 3. Nevertheless there was a significance (p ˂ 0.05) decrease in the RBC count of the positive control group 2 when compared to the standard control group.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the RBC count of all the test groups was observed when compared to the positive control group. Comparatively all the test groups showed no significant (p > 0.05) difference in the RBC count but the test groups showed a significant (p < 0.05) increase in comparison to the standard control group. Nevertheless a non- significant (p < 0.05) difference was observed in the RBC count of the test groups when compared to the negative control group 1. Also it was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the RBC count when compared to the standard control group.

Across the groups, the negative control group showed a non-significant (p > 0.05) increase nor decrease in the RBC count after treatment when compared to the RBC count after passaging and treatment. Among the test groups 4, 5 and 6 showed a significant (p < 0.05) increase in the RBC count after treatment when compared to the RBC count after passaging but a non-significant (p > 0.05) difference in comparison to the RBC count after post-treatment. The positive control group showed a significant (p < 0.05) decrease in the RBC count after Day 4 of treatment when compared to the RBC count concentration after Day 4 of passaging and also a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) increase in the RBC count after Day 4 of treatment when compared to the RBC count after Day 4 of passaging and a significant (p ˃ 0.05) decrease in comparison to the RBC count on Day 4 of post-treatment.

Day 4 After passaging On day 4 of treatment Day 4 of Post-treatment

10.7

10.52

10.73

10.58

10.29

10.54

10.48

10.22

10.36 9.65

10 9.54

8.73

8.88

8.56

8.47 7.95

8 6.65

6 4.55

4 Mean RBC Count (x109/l) Count RBC Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 7: Effect of treatment with methanol extract of Strophantus hispidus Leaves on the Red blood cell count of mice passaged with Plasmodium berghei.

130 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on White Blood Cell (WBC) Count of Mice Passaged with Plasmodium berghei After passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the WBC count of animals in groups (2 to 6) when compared to the negative control group (group 1) as can be seen in Figure 8. However after Day 4 of treatment, there was a significant (p ˂ 0.05) reduction in the WBC count of all the animals in the test groups (4-6) when compared to the positive control group 2 but a non-significant (p > 0.05) difference was observed when compared to the standard control and negative control group 1. Nonetheless there was a significant (p < 0.05) increase in the WBC count of the positive control group 2 when compared to the standard control group 3.

As observed within the test groups, there was a significant (p < 0.05) decrease in the WBC count of groups 4, 5 and 6 after Day 4 of treatment when compared to the WBC count after Day 4 of passaging, however a non-significant (p ˃ 0.05) difference was observed after Day 4 of post- treatment in the WBC count. The positive control group showed a significant (p < 0.05) increase in the WBC count after Day 4 of treatment when compared to the WBC count after Day 4 of passaging but a non-significant (p ˃ 0.05) difference was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in the WBC count after Day 4 of treatment when compared to the WBC count after Day 4 of passaging however a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment in comparison with the WBC count after Day 4 of treatment. Thus in the negative control group a non-significant (p > 0.05) difference was observed all through the experiment.

Day 4 After passaging On day 4 of treatment

14000 Day 4 of Post-treatment

12,500.00

12,333

11,566.70

11,166.70 11,400.00

12000 11,233.30

11,033.30

10,566.70

10,600.00

10,433.30

10,750.00

10,366.70

10,400.00

10,300.00

10,233.00

9800

9766.7 9,950.00

) 10000

3 -

8000

6000

Mean WBC Count (mm WBC Count Mean 4000

2000

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 8: Effect of treatment with methanol extract of Strophantus hispidus Leaves on White blood cell count of mice passaged with Plasmodium berghei

131 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase (ALT) Activity of Mice Passaged with Plasmodium berghei From Figure 9 a significant (p < 0.05) increase was observed in the ALT activity of all the animals in groups (2 to 6) after passaging with Plasmodium berghei when compared to the negative control group 1. After Day 4 of treatment, there was a significant (p ˂ 0.05) diminution in the ALT activity of the animals in test group 5 and 6 when compared to the positive control group 2 while test group 4 showed no significant difference (p ˃ 0.05) in comparison with the positive control group. However a non-significant (p ˃ 0.05) difference was observed between test group 5 and 6 and the standard control while test group 4 showed a significant (p < 0.05) increase in comparison to the standard control group. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the ALT activity between test group 5 and 6 and the negative control group however test group 4 showed a significant increase in comparison with the negative control group. Nonetheless there was a significant (p < 0.05) increase in the ALT activity of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the ALT activity of all the test groups was observed when compared to the positive control. The test groups showed no significant (p > 0.05) difference in the ALT activity. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the ALT activity of the test groups when compared to the standard control. It was observed that the positive control group showed a statistically significant (p < 0.05) increase in the ALT activity when compared to the standard control.

Within the various groups, it was observed that the negative control group showed no significant difference in the ALT activity in all through the experiment. However test groups 5 and 6 showed a significant (p < 0.05) decrease in the ALT activity after Day 4 of treatment when compared to the ALT activity after Day 4 of passaging but a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. However group 4 showed a significant (p < 0.05) decrease only on Day 4 of post treatment. The positive control group showed a significant (p < 0.05) increase in the ALT activity after Day 4 of treatment when compared to the ALT activity after Day 4 of passaging but a non- significant (p ˃ 0.05) difference on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in the ALT activity after Day 4 of treatment when

81 compared to the ALT activity after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment.

80 Day 4 After passaging

On day 4 of treatment 70

70 Day 4 of Post-treatment

63.66

56.33

55.33

55.33 54.66

60 55.33

54.66

47.67

45.33

45 44.33

50 44.5

42 40.33 40

30 Mean ALT Activities (IU/L) Activities ALT Mean 20

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 9: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Alanine Aminotransferase activity of mice passaged with Plasmodium berghei

132 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of Mice Passaged with Plasmodium berghei As observed from Figure 10 a significant (p < 0.05) increase was observed in the AST activity of all the animals in groups (2 to 6) after passaging with Plasmodium berghei when compared to the negative control group 1. After Day 4 of treatment, there was a significant (p ˂ 0.05) decrease in the AST activity of the animals in test group 4, 5 and 6 when compared to the positive control group 2. However a significant (p < 0.05) increase was observed in the AST activity of test groups 4, 5 and 6 when compared to the standard control. Moreso there was a significance (p ˂ 0.05) increase in the AST activity of the test groups 4, 5 and 6 when compared to the negative control group Nonetheless there was a significant (p < 0.05) increase in the AST activity of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the AST activity of all the test groups was observed when compared to the positive control. Among the test groups there was no significant (p > 0.05) difference in the AST activity. Conversely a significant (p < 0.05) increase was observed in the AST activity of the test groups when compared to the standard control. It was observed that the positive control showed a statistically significant (p < 0.05) increase in the AST activity when compared to the standard control. However the standard control group showed no significant (p > 0.05) difference in comparison to the negative control.

Within the various groups, it was observed that the negative control group showed no significant difference in the AST activity in all through the experiment. Also the test groups 4, 5 and 6 showed neither a significant (p > 0.05) decrease nor increase in the AST activity all through the experiment. Nevertheless the standard control group showed a significant (p < 0.05) decrease in the AST activity on Day 4 of treatment when compared to the AST activity after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Notwithstanding the positive control showed a significant (p < 0.05) increase in the AST activity after Day 4 of treatment when compared to the AST activity after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment.

90 Day 4 After passaging

82 On day 4 of treatment 78.67

80 Day 4 of Post-treatment

65.67

66 66 64.33

70 64.67

62.67 62.33

53.33

51.67

50 50 50

30 Mean AST Activities (IU/L) Activities AST Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 10: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity of mice passaged with Plasmodium berghei

133 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Alkaline Phosphatase Activity of Mice Passaged with Plasmodium berghei As observed from Figure 11, there was statistically a significant (p < 0.05) increase in the ALP activity of all the animals in groups (2 to 6) after passaging with Plasmodium berghei when compared to the negative control. However after Day 4 of treatment, there was a significant (p ˂ 0.05) decrease in the ALP activity of the animals in test groups 4, 5 and 6 when compared to the positive control. However there was neither a significant (p ˃ 0.05) increase nor decrease in the ALP activity of the test groups 4, 5 and 6 when compared to the standard control. Nevertheless there was a non-significant (p > 0.05) difference in the ALP activity of test groups 4, 5 and 6 when compared to the negative control. All the same the positive control group showed a significant (p < 0.05) increase in the ALP activity when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the ALP activity of all the test groups was observed when compared to the positive control. Among the test groups there was no significant (p > 0.05) difference in the ALP activity. Nonetheless a non-significant (p > 0.05) difference was observed in the ALP activity of the test groups when compared to the standard control. However the positive control showed a statistically significant (p < 0.05) increase in the ALP activity when compared to the standard control. However the standard control group showed no significant (p > 0.05) difference in comparison to the negative control.

Within the various groups, the positive control showed no significant (p > 0.05) difference in the ALP activity all through the experiment. However test groups 5 and 6 showed a significant (p < 0.05) decrease in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging and a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Nevertheless group 4 showed no significant (p > 0.05) difference in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging but a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. However the standard control showed a significant (p < 0.05) decrease in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging and a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Nonetheless the negative control showed no significant (p > 0.05) difference in the ALP activity all through the experiment.

120 Day 4 After passaging On day 4 of treatment

Day 4 of Post-treatment

93 95

100 96

92.33

90.66

83.33

80.66

79 79

78.3

79.66

78.66 77 80 76

40 Mean (IU/L) Activities ALP Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 11: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Alkaline phosphatase activity of mice passaged with Plasmodium berghei

134 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Total Bilirubin Concentration (T.BIL conc) of Mice Passaged with Plasmodium berghei As observed from figure 12, there was statistically significant (p < 0.05) increase in the T.BIL conc of all the animals in groups (2 to 6) when compared to the negative control after passaging with Plasmodium berghei. However after Day 4 of treatment, there was a significant (p ˂ 0.05) decrease was observed in the T.BIL conc of the animals in test groups 4, 5 and 6 when compared to the positive control. However there was neither a significant (p ˃ 0.05) increase nor decrease in the T.BIL conc of the test groups 4, 5 and 6 when compared to the standard control. Nevertheless there was a non-significant (p > 0.05) difference in the T.BIL conc of test groups 4, 5 and 6 when compared to the negative control. All the same the positive control group showed a significant (p < 0.05) increase in the T.BIL conc when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the T.BIL conc of all the test groups was observed when compared to the positive control. Among the test groups there was no significant (p > 0.05) difference in the T.BIL conc. Nonetheless a non-significant (p > 0.05) difference was observed in the T.BIL conc of the test groups 5 and 6 when compared to the standard control and the negative control group however group 4 showed a significant (p < 0.05) increase in comparison to the standard control and negative control group. However the positive control showed a statistically significant (p < 0.05) increase in the T.BIL conc when compared to the standard control.

Within the various groups, test groups 5 and 6 showed a significant (p < 0.05) decrease in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Nevertheless group 4 showed no statistically significant difference (p > 0.05) all through the experiment. However the standard control showed a significant (p < 0.05) decrease in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Nonetheless the negative control showed no significant (p > 0.05) difference in the T.BIL conc all through the experiment. The positive control showed a significant (p < 0.05) increase in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and also a significant (p < 0.05) increase was observed on Day 4 of post-treatment.

9 Day 4 After passaging

On day 4 of treatment 7.7 8 Day 4 of Post-treatment

7 6.5

5 4.5

4.51

4.49

4.45

4.51

3.8 3.75

3.03

3.23

3.13

2.85

2.85 2.7

3 2.65

2.55

2.45 Mean Total Bilirubin Conc. (mg/dl)Conc.Bilirubin Total Mean 2

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Group

Figure 12: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Total bilirubin conc on mice passaged with Plasmodium berghei

135 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of Mice Passaged with Plasmodium berghei From Figure 13 after passaging the animals with Plasmodium berghei, it was observed that after Day 4 of treatment there was a significant (p ˂ 0.05) decrease in the MDA conc of animals in test groups 4, 5 and 6 when compared to the positive control group 2. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the MDA conc between all the test groups. However a non-significant (p ˃ 0.05) difference was observed between all the test groups in comparison to the standard control. Nonetheless there was a significant (p < 0.05) increase in the MDA conc of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the MDA conc of all the test groups was observed when compared to the positive control group. Among the test groups there was no significant (p > 0.05) difference in the MDA conc of all test groups. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the MDA conc of groups 4, 5 and 6 when compared to the standard control group 3 and the negative control group. However there was a significant increase in the MDA conc of the positive control compared to the standard control group.

Within various groups, it was observed that after Day 4 of treatment there was no significant (p > 0.05) difference in the MDA conc of all the animals in the test groups when compared to the MDA conc after Day 4 of post-treatment. The positive control group showed a significant (p < 0.05) increase in the MDA conc after Day 4 of post-treatment when compared to the MDA conc after Day 4 of treatment. Conversely the standard control group showed no significant (p > 0.05) difference in the MDA conc after Day 4 of treatment when compared to the MDA conc after Day 4 of post-treatment

7 On day 4 of treatment

Day 4 of Post-treatment

6 5.46

5 4.21

2.49

2.41

2.04

2.25

1.7

Mean MDA (mg/ml)Conc MDA Mean

1.79 1.82

2 1.79

1.46 1.21

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups Figure 13: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Concetration of mice passaged with Plasmodium berghei

136 Effect of Treatment with Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase Activity of Mice Passaged with Plasmodium berghei From figure 14 after Day 4 of treatment there was a significant (p < 0.05) increase in the SOD activity of the test groups 5 and 6 when compared to the positive control group however test group 4 showed no significant (p > 0.05) difference in comparison to the positive control group. However after Day 4 of treatment, there was a non-significant difference (p ˂ 0.05) in the SOD activity of all the animals in the test groups (4-6) when compared to the standard control group. Nevertheless there was a significance decrease (p < 0.05) in the SOD activity of the positive control group 2 when compared to the standard control group.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the SOD activity of all the test groups was observed when compared to the positive control group. Comparatively all the test groups showed no significant (p > 0.05) difference in the SOD activity. Nevertheless all the test groups showed a non-significant (p > 0.05) difference in comparison to the standard control group. Nevertheless a non-significant (p > 0.05) difference was observed in the SOD activity of the test group 6 when compared to the negative control group 1. Also it was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the SOD activity when compared to the standard control group.

Within the groups, the negative control group showed a non-significant (p > 0.05) increase nor decrease in the SOD activity after treatment when compared to the SOD activity after passaging and treatment. Among the test groups 4, 5 and 6, there was a non-significant (p > 0.05) difference in the SOD activity after treatment when compared to the SOD activity after Day 4 of post- treatment. The positive control group showed a non-significant (p > 0.05) difference in the SOD activity after Day 4 of treatment when compared to the SOD activity after Day 4 of post-treatment. Nevertheless the standard control group showed neither a significant (p > 0.05) increase nor decrease in the SOD activity after Day 4 of treatment when compared to the SOD activity after Day 4 of post-treatment.

25 On day 4 of treatment 21.79

21.48 Day 4 of Post-treatment

20 18.91

18.65

17.71

18.09

17.65

18.09

17.04 16

13.2 11.63

10 Mean SOD Activities (IU/L) SOD ActivitiesMean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Treatment Groups

Figure 14: Effect of treatment with methanol extract of Strophantus hispidus Leaves on Superoxide Dismutase activity on mice passaged with Plasmodium berghei

137 Percentage Yield of the Sequencial Partitioning of the Methanol Extract of Strophantus hispidus Leaves As shown in table 7, the percentage yield of different solvents from the methanol leave extract of Strophantus hispidus was found to be 20 %, 22% and 35 % for n-hexane, dichloromethane and Ethyl acetate respectively.

138 Phytochemical Analysis of the Different Partitions of Methanol Extract of Strophantus hispidus Leaves As shown in Table 8, Alkaloids, tannins and saponin were abundantly present in the ethyl acetate while flavonoids terpenoids steroids phenols and glycosides were moderately present in the dichloromethane and n-hexane fraction.

Table 7: Shows the Percentage Yield of Different Solvent Partitions

Weight of Extract (g) SOLVENT Yield (g) Percentage (%) 105 n-Hexane 23.1 22 105 Dichloromethane 21 20 105 Ethyl acetate 36.75 35

Table 8: Phytochemical Analysis of the Different Partitions of Methanol Extract of Strophantus hispidus Leaves Phytochemicals Different Partitions

n-hexane Dichloromethane Ethyl acetate

Alkaloids + + +++

Flavonoids ++ + -

Phenols ++ ++ -

Tannins + + +++

Saponins - + +++

Terpenoids ++ ++ -

Steroids + ++ -

Glycosides + + -

Key: + Slightly present ++ Moderately present +++ Highly present - Not detected

139 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on the Mean Parasitemia Count (PC) of Mice Passaged with Plasmodium berghei From Figure 15 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the mean PC of all the animals in groups (2 to 9) when compared to the negative control group. After Day 4 of treatment, there was a significant (p ˂ 0.05) decrease in the mean PC of all the animals in the test group (4-9) when compared to the positive control group. However among the test groups, groups 5, 7, 8 and 9 showed a significant (p ˂ 0.05) decrease in the mean PC when compared to test groups 6 and 4. Nevertheless test groups 5, 7, 8 and 9 showed a non-significant (p ˃ 0.05) difference in the mean PC when compared to the standard control group. Nonetheless there was a significant (p < 0.05) increase in the mean PC of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the mean PC of all the test groups was observed when compared to the positive control group. Among the test groups there was a significant (p < 0.05) decrease in the mean PC of group 7, 8 and 9 when compared to group 4, 5 and 6. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the mean PC of group 9 when compared to the standard control group 3. Conversely there was a significant increase in the mean PC of group 4, 5, 6, 7, and 8 when compared to the standard control group. It was observed that the positive control group showed a statistically significant (p < 0.05) increase in the mean PC when compared to the standard control group.

Within various groups, the positive control group showed a significant (p < 0.05) increase in the mean PC after Day 4 of treatment when compared to the mean PC after Day 4 of passaging and also a significant (p < 0.05) increase after Day 4 of post-treatment. Nontheless test groups 4, 5, 6, 7, 8 and 9 showed a significant (p < 0.05) decrease in the mean PC after Day 4 of treatment when compared to after Day 4 of passaging but a non-significant difference was observed on Day 4 of post-treatment. In the same vein the standard control group showed a significant (p < 0.05) decrease in the mean PC after Day 4 of treatment when compared to the mean PC after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment.

Day 4 After passaging 140 On day 4 of treatment

Day 4 of Post-treatment

120 115.7

100 93.7

50.7

52.3

50 53.3

60 50.7

49 Mean Parasitaemia count Mean Parasitaemia

28.7

24.3

23.7

19 16.7

20 15.3

14.7

12.3

11.7

11.3

5.7

0 0 0 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9

Treatment Groups Figure 15: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on the Packed cell Volume on mice passaged with plasmodium berghei

Group 1 = Negative control (not passaged but was administered the vehicle 5 ml/kg of normal saline) Group 2 = Positive control (passaged but not treated.) Group 3 = Standard control (passaged and treated with artesunate 10 mg/kg b.w.) Group 4 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Ethyl Acetate fraction) Group 5 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Ethyl Acetate fraction) Group 6 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Dichloromethane fraction) Group 7 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Dichloromethane fraction) Group 8 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of n-Hexane fraction)

Group 9 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of n-Hexane fraction) 140 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on the Packed cell Volume of Mice Passaged with Plasmodium berghei From Figure 16 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant diminution (p < 0.05) in the PCV of animals in groups (2 to 9) when compared to the negative control. Nevertheless after Day 4 of treatment, there was a significant increase (p ˂ 0.05) in the PCV of all the animals in the test groups (4-9) when compared to the positive control. Among the test groups, groups 4 and 6 showed a statistically significant (p < 0.05) decrease in the PCV when compared to group 9 however test groups 4, 5, 6, 7, and 8 showed no significant diference (p > 0.05) among them. Nevertheless test group 9 showed no significant (p > 0.05) difference in the PCV when compared to the standard control. Comparatively there was a significance decrease (p < 0.05) in the PCV of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the PCV of all the test groups was observed when compared to the positive control. Among the test groups there was a significant (p < 0.05) decrease in the PCV of group 4, 5, 6 when compared to group 8 and 9 however test group 5, 6 and 7 showed no significant difference (p ˃ 0.05) same as group 7 and 8. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the PCV of group 9 when compared to the standard control group 3, however groups 4, 5, 6, 7 and 8 showed a statistically significant (p < 0.05) decrease when compared to the standard control. It was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the PCV when compared to the standard control group.

Within the groups, the negative control showed no statistically significant difference (p ˃ 0.05) in all. However group 7, 8 and 9 showed a significant (p < 0.05) increase in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging and also a significant (p < 0.05) increase after Day 4 of post-treatment. Groups 4 and 6 showed a non-significant (p > 0.05) difference in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging but after Day 4 of post-treatment there was a significant (p < 0.05) increase. However groups 5 showed a significant (p < 0.05) increase in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging but a non-significant (p ˃ 0.05) difference after Day 4 of post- treatment. The positive control group showed a significant (p < 0.05) decrease in the PCV after

Day 4 of treatment when compared to the PCV concentration after Day 4 of passaging and also a significant decrease (p ˃ 0.05) was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) increase in the PCV after Day 4 of treatment when compared to the PCV after Day 4 of passaging and also a significant increase (p ˃ 0.05) on Day 4 of post-treatment.

Day 4 After passaging On day 4 of treatment

50 Day 4 of Post-treatment

43 42

45 42

41.7

40.7

39.7 38.7

40 37

37.7

36.3

36.7

35.3

35.7

31.6

33.3

32.6 32.6

35 32

31.6 31.6

31 30.6 30

20 18 Mean (%) PCV Mean

15 13.7

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 16: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on the Packed cell Volume on mice passaged with Plasmodium berghei

Group 7 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Dichloromethane fraction) Group 8 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of n-Hexane fraction) Group 9 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of n-Hexane fraction) 141 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on the Haemoglobin conc. of Mice Passaged with Plasmodium berghei. It was observed from Figure 17 that after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the Hb Conc of animals in all groups (2 to 9) when compared to the negative control group. However after Day 4 of treatment, there was a significant increase (p ˂ 0.05) in the Hb Conc of all the animals in the test groups (4-9) when compared to the positive control. Among the test groups, group 4, 5, 6, 7 and 8 showed no significant difference however group 9 showed a significant increase (p < 0.05) when compared to group 7 but a non- significant (p > 0.05) difference when compared to group 8. Nonetheless group 9 showed a non- significant (p > 0.05) difference in comparison to the standard control. Nevertheless there was a significance decrease (p < 0.05) in the Hb Conc of the positive control group 2 when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the Hb Conc of all the test groups was observed when compared to the positive control group. Comparatively test groups 8 and 9 showed a significant (p < 0.05) increase in the Hb Conc when compared to test group 4, 5, 6 and 7. Nevertheless a significant (p < 0.05) decrease was observed in the Hb Conc of the test group when compared to the negative control and the standard control. Also it was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the Hb Conc when compared to the standard control.

Within the groups, the negative control group showed a non-significant (p > 0.05) difference in the Hb Conc all through the experiment. However test groups 4, 5, 6, 7, 8 and 9, showed a significant (p < 0.05) increase in the Hb Conc after treatment when compared to the Hb Conc after passaging, more so a significant (p < 0.05) increase was observed after post-treatment nevertheless group 5 showed a statistically non-significant (p > 0.05) difference after post treatment. The positive control showed a significant (p < 0.05) decrease in the Hb Conc after Day 4 of treatment when compared to the Hb Conc concentration after Day 4 of passaging and also a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. Conversely the standard control showed a significant (p < 0.05) increase in the Hb Conc after Day 4 of treatment when compared to the Hb

Conc after Day 4 of passaging and also a significant (p < 0.05) increase in the Hb Conc on Day 4 of post-treatment.

Day 4 After passaging On day 4 of treatment 14

12.9 Day 4 of Post-treatment

12.33

12.16 12.07

11.19

10.85

10.23

9.9

10.15

9.65

9.59 9.44

10 9.13

9.36

9.1

8.9

8.86

8.06

8.03

8.13 8.13

8.06

8.03 8

8 6.83

6 5.67

4 Mean Hemoglobin Conc. (g/dl)Hemoglobin Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups

Figure 17: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on the Hemoglobin conc. on mice passaged with Plasmodium berghei.

100

142 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on the Red blood Cell Count of Mice Passaged with Plasmodium berghei. As was observed from Figure 18 after passaging the animals with Plasmodium berghei, there was a significant (p < 0.05) decrease in the RBC count of animals in all groups (2 to 9) when compared to the negative control group. However after Day 4 of treatment, there was a significant increase (p ˂ 0.05) in the RBC count of all the animals in the test groups (4-9) when compared to the positive control. Among the test groups, group 4, 5, 6, 7 and 8 showed no significant difference however group 9 showed a significant increase (p < 0.05) when compared to group 4, 5, 6 and 7 but a non-significant (p > 0.05) difference when compared to group 8. Nonetheless group 9 showed a non-significant (p > 0.05) difference in comparison to the standard control. Nevertheless there was a significance decrease (p < 0.05) in the RBC count of the positive control group 2 when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the RBC count of all the test groups was observed when compared to the positive control group. Comparatively test groups 8 and 9 showed a significant (p < 0.05) increase in the RBC count when compared to test group 4, 5, 6 and 7. Nevertheless a significant (p < 0.05) decrease was observed in the RBC count of all the the test groups when compared to the negative control and the standard control. Also it was observed that the positive control group showed a statistically significant (p < 0.05) decrease in the RBC count when compared to the standard control.

Within the groups, all through the experiment the negative control group showed a significant increase (p > 0.05) in the RBC count after Day 4 of post-treatment. However test groups 4, 5, 6, 7, 8 and 9, showed a significant (p < 0.05) increase in the RBC count after treatment when compared to the RBC count after passaging, more so a significant (p < 0.05) increase was observed after post-treatment nevertheless group 4 and 6 showed a statistically non-significant (p > 0.05) difference after Day 4 of post treatment. The positive control showed a significant (p < 0.05) decrease in the RBC count after Day 4 of treatment when compared to the RBC count concentration after Day 4 of passaging and also a significant (p < 0.05) decrease was observed on Day 4 of post-treatment. Conversely the standard control showed a significant (p < 0.05) increase in the RBC count after Day 4 of treatment when compared to the RBC count after Day 4 of passaging and also a significant (p < 0.05) increase in the RBC count on Day 4 of post-treatment.

101

Day 4 After passaging 14 On day 4 of treatment Day 4 of Post-treatment

12 11.64

11.23

11.1

11.06

10.24

10.09

9.65

9.56

9.49 9.23

10 9.39

8.85

9.06

8.81

8.76

8.66

7.7

7.6

7.72

7.58

7.67 7.67 7.66 8 7.42

6 5.39 4.05

4 Mean Red Blood Cell Count (x109/l) Count Cell Blood Red Mean 2

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 18: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on the Red blood cell count on mice passaged with Plasmodium berghei.

Group 1 = Negative control (not passaged but was administered the vehicle 5 ml/kg of normal saline) Group 2 = Positive control (passaged but not treated) Group 3 = Standard control (passaged and treated with artesunate 10 mg/kg b.w.) Group 4 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Ethyl Acetate fraction) Group 5 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Ethyl Acetate fraction) Group 6 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of Dichloromethane fraction) Group 7 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of Dichloromethane fraction) Group 8 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of n-Hexane fraction) Group 9 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of n-Hexane fraction)

102

143 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on White Blood Cell Count of Mice Passaged with Plasmodium berghei After passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the WBC count of animals in groups (2 to 9) when compared to the negative control as can be seen in Figure 19. However after Day 4 of treatment, there was a significant (p ˂ 0.05) reduction in the WBC count of all the animals in the test groups (4-9) when compared to the positive control. However among the groups there was a non-sigificannt difference (p > 0.05) in the WBC count but a non-significant (p > 0.05) difference was observed in the WBC count of group 9 when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the WBC count of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) diminution in the WBC count of all the test groups was observed when compared to the positive control group. Nevertheless among the test groups, group 4, 5 and 6 showed a significant (p < 0.05) increase in the WBC count when compared to test group 8 and 9 however test group 7 showed a non-significant (p > 0.05) difference when compared to group 4, 5, 6, 8 and 9. Nonetheless test group 7, 8, and 9 showed a non- statistically significant difference difference (p > 0.05) in the WBC count when compared to the standard control and the negative control. However it was observed that the positive control group showed a statistically significant (p < 0.05) increase in the WBC count when compared to the standard control group.

As observed within the test groups, there was a significant (p < 0.05) decrease in the WBC count of groups 4, 5, 6, 7, 8 and 9 after Day 4 of treatment when compared to the WBC count after Day 4 of passaging, however a non-significant (p ˃ 0.05) difference was observed after Day 4 of post- treatment in the WBC count except for group 7 that showed a statistically significant decrease on Day 4 of post-treatment. The positive control group showed a significant (p < 0.05) increase in the WBC count after Day 4 of treatment when compared to the WBC count after Day 4 of passaging and also significant increase (p < 0.05) was observed on Day 4 of post-treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in the WBC count after Day 4 of treatment when compared to the WBC count after Day 4 of passaging however a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment in comparison with the WBC count after Day 4

103 of treatment. Thus in the negative control group a non-significant (p > 0.05) difference was observed all through the experiment.

Day 4 After passaging On day 4 of treatment Day 4 of Post-treatment 18000

16000

14900

14733

13233.33

13166.66

13300 13300

13100 13066.66

14000 13133.33

13066.66

)

11500

11366

11200 11133.33

12000 11133

10966.66 10966.66

10900

10833.33

10833

10733.33

10600

10433.33

10400

10300 10300 10033.33 10000

8000

6000

Mean WBC Count (mm WBC Count Mean 4000

2000

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 19: Effect of treatment with different fractions of Methanol Extract of Strophantus hispidus Leaves on White blood cell count on mice passaged with Plasmodium berghei

104

144 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase Activity of Mice Passaged with Plasmodium berghei From Figure 20, a significant (p < 0.05) increase was observed in the ALT activity of all the animals in groups (2 to 9) after passaging with Plasmodium berghei when compared to the negative control. After Day 4 of treatment, there was a significant (p ˂ 0.05) diminution in the ALT activity of the animals in test group 4, 5, 6, 7, 8 and 9 when compared to the positive control. Among the groups there was a significant increase in the ALT activity of test group 4, 5 and 6 when compared to group 8 and 9 however test group 7 showed a non-statistically significant difference when compared to group 4, 5, 6, 8 and 9. However a non-significant (p ˃ 0.05) difference was observed between test group 9 and the standard control while test groups 4, 5, 6, 7 and 8 showed a significant (p < 0.05) increase in comparison to the standard control. Nonetheless there was a significant (p < 0.05) increase in the ALT activity of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the ALT activity of all the test groups was observed when compared to the positive control. Among the test groups there was a significant (p < 0.05) increase in the ALT activity of test group 4 when compared to group 7, 8 and 9 however test group 5 and 6 showed a non significant difference (p ˃ 0.05) when compared to group 7 and 8. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the ALT activity of the test group 9 when compared to the standard control. It was also observed that the positive control group showed a statistically significant (p < 0.05) increase in the ALT activity when compared to the standard control.

Within the various groups, it was observed that the negative control group showed no significant difference in the ALT activity in all through the experiment. However test groups 4, 5, 6, 7, 8 and 9 showed a significant (p < 0.05) decrease in the ALT activity after Day 4 of treatment when compared to the ALT activity after Day 4 of passaging and also a significant (p < 0.05) decrease on Day 4 of post-treatment however group 4 showed a non-significant difference on Day 4 of post- treatmet. The positive control group showed a significant (p < 0.05) increase in the ALT activity after Day 4 of treatment when compared to the ALT activity after Day 4 of passaging and also a significant (p < 0.05) increase on Day 4 of post-treatment. Nevertheless the standard control group showed a significant (p < 0.05) decrease in the ALT activity after Day 4 of treatment when

105 compared to the ALT activity after Day 4 of passaging and also a significant (p < 0.05) decrease on Day 4 of post-treatment.

90 Day 4 After passaging On day 4 of treatment

80 77 Day 4 of Post-treatment 70.7

59.7

60.3

60.7

60.3

58.7

60.3

59.7 57.7

52.3

51.3

47.3 46.3

50 46

44.3

44.7

43.3

43.3 42.3 40

30 Mean ALT Activities (IU/L) Activities ALT Mean 20

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 20: Effect of treatment with different fractions of methanol Extract of Strophantus hispidus Leaves on Alanine Aminotransferase activity on mice passaged with Plasmodium berghei

106

145 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Aspartate Aminotransferase Activity of Mice Passaged with Plasmodium berghei As observed from Figure 21, after passaging with Plasmodium berghei a significant (p < 0.05) increase was observed in the AST activity of all the animals in groups (2 to 9) when compared to the negative control. After Day 4 of treatment, there was a significant (p ˂ 0.05) decrease in the AST activity of the animals in all test groups when compared to the positive control. Among the test groups, test group 4 showed a significant increase in the AST activity when compared to test group 9 however group 5, 6, 7 and 8 showed a non-signnificant difference (p > 0.05) when compared to group 4 and 9. Nevertheless test group 5, 7, 8 and 9 showed a non-significant (p > 0.05) difference in the AST activity when compared to the standard control however test groups 4 and 6 showed a significant increase (p ˂ 0.05) in the AST activity when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the AST activity of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the AST activity of all the test groups was observed when compared to the positive control. Among the test groups there was no significant (p > 0.05) difference in the AST activity. Conversely a significant (p < 0.05) increase was observed in the AST activity of the test group 4 when compared to the standard control while group 5, 6, 7, 8, and 9 showed a non-significant (p > 0.05) difference when compared to the standard conntrol. It was observed that the positive control showed a statistically significant (p < 0.05) increase in the AST activity when compared to the standard control. However the standard control group showed no significant (p > 0.05) difference in comparison to the negative control.

Within the various groups, it was observed that the negative control group showed no significant difference in the AST activity in all through the experiment. Nevertheless the test groups 4, 5, 6, 7, 8 and 9 showed a significant (p < 0.05) decrease in the AST activity after Day 4 of treatment when compared to the AST activity after Day 4 of passaging more so there was a significant (p < 0.05) decrease in the AST activity on Day 4 of post-treatment. Nevertheless the standard control group showed a significant (p < 0.05) decrease in the AST activity after Day 4 of treatment when compared to the AST activity after Day 4 of passaging and also a significant (p < 0.05) decrease

107 on Day 4 of post-treatment. Notwithstanding the positive control showed a significant (p < 0.05) increase in the AST activity after Day 4 of treatment when compared to the AST activity after Day 4 of passaging and non-significant (p ˃ 0.05) difference on Day 4 of post-treatment.

120 Day 4 After passaging On day 4 of treatment Day 4 of Post-treatment

100 89.7 84.3

69.3

70.3

64.3

63.3

62.7

61.7

60.3

56.7

55.3 53.3

60 54.7

53.7

53 51.7

40 Mean AST Activities (IU/L) Activities AST Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 21: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on Aspartate Aminotransferase activity on mice passaged with Plasmodium berghei

108

146 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Alkaline Phosphatase Activity of Mice Passaged with Plasmodium berghei From Figure 22, after passaging with Plasmodium berghei a significant (p < 0.05) increase was observed in the ALP activity of all the animals in groups (2 to 9) when compared to the negative control. After Day 4 of treatment, there was a significant (p ˂ 0.05) decrease in the ALP activity of the animals in all test groups when compared to the positive control. Among the test groups, there was a non-significant (p > 0.05) difference in ALP activity. Nevertheless test group 5, 7, 8 and 9 showed a non-significant (p > 0.05) difference in the ALP activity when compared to the standard control however test groups 4 and 6 showed a significant increase (p ˂ 0.05) in the ALP activity when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the ALP activity of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the ALP activity of all the test groups was observed when compared to the positive control. Among the test groups there was no significant (p > 0.05) difference in the ALP activity. Nevertheless a significant (p < 0.05) increase was observed in the ALP activity of the test group 4 when compared to the standard control while group 5, 6, 7, 8, and 9 showed a non-significant (p > 0.05) difference when compared to the standard control and the negative control. It was observed that the positive control showed a statistically significant (p < 0.05) increase in the ALP activity when compared to the standard control.

Within the various groups, it was observed that the negative control and group 6 showed a non- statistically significant difference in the ALP activity in all through the experiment. Nevertheless the test groups 4, 5, and 9 showed a significant (p < 0.05) decrease in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging more so there was a significant (p < 0.05) decrease in the ALP activity on Day 4 of post-treatment however group 4 and 5 showed no significant (p > 0.05) difference on Day 4 of post-treatmennt. However test groups 7 and 8 showed a non-significant (p > 0.05) difference in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging but there was a significant (p < 0.05) decrease in the ALP activity on Day 4 of post-treatment when compared to the ALP

109 activity after Day 4 of passaging. Nevertheless the standard control group showed a significant (p < 0.05) decrease in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging and also a significant (p < 0.05) decrease on Day 4 of post-treatment. However the positive control showed a significant (p < 0.05) increase in the ALP activity after Day 4 of treatment when compared to the ALP activity after Day 4 of passaging and non- significant (p ˃ 0.05) difference on Day 4 of post-treatment.

110

140 Day 4 After passaging

On day 4 of treatment 121.3 Day 4 of Post-treatment 120 113

100

88.3

86.7

89.7

87.6

88.3

84.7

83.7 83.7

83.3

82.3 82.3

80.3

79.7

79 78.6 80 78

Mean (IU/L) Activities ALP Mean 40

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 22: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on Alkaline phosphatase activity on mice passaged with Plasmodium berghei

111

147 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Total bilirubin Conc. of Mice Passaged with Plasmodium berghei As observed from Figure 23, there was statistically significant (p < 0.05) increase in the T.BIL conc of all the animals in groups (2 to 9) when compared to the negative control after passaging with Plasmodium berghei. However after Day 4 of treatment, a significant (p ˂ 0.05) decrease was observed in the T.BIL conc of the animals in all test groups 4 to 9 comparatively to the positive control. Nonetheless among the test groups, test group 4 and 5 showed a significant increase (p ˂ 0.05) in the T.BIL conc when compared to test group 8 and 9 however test group 6 and 7 showed a non-significant (p > 0.05) difference when compared to test group 5, 8 and 9. However there was neither a significant (p ˃ 0.05) increase nor decrease in the T.BIL conc of the test groups 6, 7, 8 and 9 when compared to the standard control. Nevertheless there was a significant (p < 0.05) increase in the T.BIL conc of test groups 4 and 5 when compared to the standard control. All the same the positive control group showed a significant (p < 0.05) increase in the T.BIL conc when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the T.BIL conc of all the test groups was observed when compared to the positive control. Among the test groups, group 9 showed a significant (p < 0.05) decrease in the T.BIL conc when compared to group 4 and 6 however group 5, 7 and 8 showed a non-significant difference when compared to group 4, 6 and 9. Nonetheless a significant (p < 0.05) increase was observed in the T.BIL conc of all test groups when compared to the standard control and the negative control However the positive control showed a statistically significant (p < 0.05) increase in the T.BIL conc when compared to the standard control.

Within the various groups, the negative control group showed a non-significant difference all through the experiment. However test groups 6, 7, 8 and 9 showed a significant (p < 0.05) decrease in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and a non-significant (p ˃ 0.05) difference on Day 4 of post-treatment. Comparatively group 5 showed a non-significant difference in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging however a significant decrease was observed on Day 4 of post-treatment when compared to the T.BIL after passaging. Nevertheless group 4 showed a statistically significant (p < 0.05) decrease only on Day 4 of post-treatment. However

112 the standard control showed a significant (p < 0.05) decrease in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and also a significant (p < 0.05) decrease on Day 4 of post-treatment. Nonetheless the positive control showed a significant (p < 0.05) increase in the T.BIL conc after Day 4 of treatment when compared to the T.BIL conc after Day 4 of passaging and also a significant (p < 0.05) increase was observed on Day 4 of post- treatment.

12 Day 4 After passaging On day 4 of treatment Day 4 of Post-treatment

10 9.6 8.6

5.88

5.5

5.4 5.4

5.5

5.6

5.4 5.4

6 5.4

4.88

4.47

4.56

4.37

4.29

4.41

4.25

4.34

4.16

4.27

4.11 3.95

4 3.44

2.5

2.4 2.4

Mean Total Bilirubin Conc. (mg/dl)Conc.Bilirubin Total Mean 2

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 23: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on Total bilirubin conc on mice passaged with Plasmodium berghei

113

Group 8 = Test group mid-dose (passaged and treated with 400 mg/kg b.w. of n-Hexane fraction) Group 9 = Test group high-dose (passaged and treated with 800 mg/kg b.w. of n-Hexane fraction) 148 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. of Mice Passaged with Plasmodium berghei From Figure 24, it was observed that after Day 4 of treatment there was a significant (p ˂ 0.05) decrease in the MDA conc of animals in all test groups (4 to 9) when compared to the positive control. Nevertheless test group 4, 5, 6 and 7 showed a significant increase in the MDA conc when compared to group 9 however group 8 showed a non-significant different when compared to group 5, 6, 7 and 9. However a non-significant (p ˃ 0.05) difference was observed in the test groups 8 and 9 in comparison to the standard control. Nonetheless there was a significant (p < 0.05) increase in the MDA conc of the positive control group 2 when compared to the standard control group 3.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the MDA conc of all the test groups was observed when compared to the positive control group. However among the test groups, test group 4, 5, 6 and 7 showed a significant (p < 0.05) increase in the MDA conc when compared to group 9 however group 8 showed a non-significant different when compared to group 5, 6, 7 and 9. Comparatively, all the test groups showed a significant (p < 0.05) decrease in MDA conc when compared to the standard control and the negative control. However there was a significant increase in the MDA conc of the positive control compared to the standard control group.

Within various groups, it was observed that on Day 4 of post-treatment there was a significant (p < 0.05) decrease in the MDA conc of all the animals in the test groups when compared to the MDA conc after Day 4 of treatment however group 4 showed a non-sigificant all through the experiment. The positive control group showed a significant (p < 0.05) increase in the MDA conc on Day 4 of post-treatment when compared to the MDA conc after Day 4 of treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in MDA conc on Day 4 of post-treatment when compared to the MDA conc after Day 4 of treatment

114

7 On day 4 of treatment 6.41

Day 4 of Post-treatment

6 5.25

5 3.93

4 3.65

3.54

3.46 3.46

3.28

2.97

3.11 3.11

2.98 2.98

3 2.8

2.56

2.43

2.31 2.09

Mean MDA (mg/ml)Conc MDA Mean 2

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups

Figure 24: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on Malondialdehyde (MDA) Conc. on mice passaged with Plasmodium berghei

115

149 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Superoxide Dismutase Activity of Mice Passaged with Plasmodium berghei From Figure 25, it was observed that after fourth day of treatment there was a significant (p < 0.05) increase in the SOD activity of all the test groups when compared to the positive control. Test groups 4 and 6 showed a significant (p < 0.05) decrease in the SOD activity when compared to group 9, however group 5, 7 and 8 showed no significant (p > 0.05) difference when compared to group 9 while group 5 and 7 showed no significant (p > 0.05) difference when compared to group 4 and 6. However, there was a non-significant (p > 0.05) difference in the SOD activity of animals in test groups 5, 7, 8 and 9 when compared to the standard control. Nevertheless there was a significance decrease (p < 0.05) in the SOD activity of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the SOD activity of all the test groups was observed when compared to the positive control. Comparatively, test groups 7, 8 and 9 showed a significant increase (p > 0.05) in the SOD activity when compared to group 4, 5 and 6, however groups 5 and 6 showed a significant increase (p > 0.05) in the SOD activity when compared to group 4 . Nevertheless all the test groups showed a significant (p < 0.05) decrease in comparison to the standard control. All the same the positive control showed a statistically significant (p < 0.05) decrease in the SOD activity when compared to the standard control.

Within the groups, the negative control group showed a non-significant (p > 0.05) increase nor decrease in the SOD activity on Day 4 of post-treatment when compared to the SOD activity after treatment. Among the test groups, group 5, 6, 7 and 8, showed a significant (p < 0.05) increase in the SOD activity on Day 4 of post-treatment when compared to the SOD activity after Day 4 of treatment. However test group 4 showed a non-significant difference in the experiment. The positive control group showed a significant (p < 0.05) decrease in the SOD activity at the Day 4 of post-treatment when compared to the SOD activity after Day 4 of treatment. Nevertheless the standard control showed a significant (p < 0.05) increase in the SOD activity at fourth day of post- treatment when compared to the SOD activity after Day 4 of treatment.

116

20 On day 4 of treatment 17.66 18.09 Day 4 of Post-treatment

16.75

16.05 16.05

15.95

15.35

15.28 15.24

16 14.92

14.89

14.62

14.48

14.31

14.14 13.92 14

12 11.24

10 9.95

6 Mean Mean SOD(IU/L) Activities

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 25: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on Superoxide Dismutase activity on mice passaged with Plasmodium berghei

117

150 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Catalase Activity of Mice Passaged with Plasmodium berghei From Figure 26, it was observed that after fourth day of treatment there was a significant (p < 0.05) increase in the catalase activity of all the test groups when compared to the positive control. However test groups 4 and 6 showed a significant (p < 0.05) decrease in the catalase activity when compared to group 8 and 9, however group 5 and 7 showed no significant (p > 0.05) difference when compared to group 4 and 6. However, there was a significant (p < 0.05) decrease in the catalase activity of animals in all test groups when compared to the standard control. Nevertheless there was a significance decrease (p < 0.05) in the catalase activity of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) increase in the catalase activity of all the test groups was observed when compared to the positive control. Comparatively, test group 9 showed a significant increase (p > 0.05) in the catalase activity when compared to group 4, 5, 6, 7 and 8, however groups 5, 7 and 8 showed a significant increase (p > 0.05) in the catalase activity when compared to group 4 and 6. Nevertheless all the test groups showed a significant (p < 0.05) decrease in comparison to the standard control except group 9 which showed a non-significant difference. However all the test groups showed a significant (p < 0.05) decrease in the catalase activity when compared to the negative control. All the same the positive control showed a statistically significant (p < 0.05) decrease in the catalase activity when compared to the standard control.

Within the groups, the negative control group showed a non-significant (p > 0.05) increase nor decrease in the catalase activity on Day 4 of post-treatment when compared to the catalase activity after treatment. Among the test groups, group 4, 5, 6, 7, 8 and 9, showed a significant (p < 0.05) increase in the catalase activity on Day 4 of post-treatment when compared to the catalase activity after Day 4 of treatment. The positive control group showed a significant (p < 0.05) decrease in the catalase activity at the Day 4 of post-treatment when compared to the catalase activity after Day 4 of treatment. Nevertheless the standard control showed a significant (p < 0.05) increase in the catalase activity at fourth day of post-treatment when compared to the catalase activity after Day 4 of treatment.

118

1 On day 4 of treatment

0.9 0.84 Day 4 of Post-treatment

0.82 0.78

0.8 0.77

0.72

0.7 0.7

0.7 0.69

0.61

0.59

0.58 0.57

0.6 0.55

0.54

0.52 0.51

0.5 0.44

0.4 0.32

0.3 Mean Catalase mg/dlactivity Catalase Mean 0.2

0.1

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups Figure 26: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on catalase activity of mice passaged with Plasmodium berghei

119

151 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Hemozoin Concentration of Mice Passaged with Plasmodium berghei From Figure 27 after passaging the animals with Plasmodium berghei, after fourth days of treatment there was a significant (p < 0.05) decrease in the hemozoin conc of all the animals in groups (4 to 9) when compared to the positive control. Among the test groups, test groups 8 and 9 showed a significant decrease in the hemozoin conc when compared to group 4, 5, 6 and 7. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the hemozoin conc of test groups 8 and 9 when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the hemozoin conc of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the hemozoin conc of all the test groups was observed when compared to the positive control group. Among the test groups there was a significant (p < 0.05) increase in the hemozoin conc of group 4, 5, 6 and 7 when compared to group 8 and 9. Nevertheless test goup 4 and 6 were significantly higher (p < 0.05) than group 5 and 7 in the hemozoin conc. notwithstanding test groups 8 and 9 showed a non- significant (p ˃ 0.05) difference in the hemozoin concentration when compared to the standard control. However it was observed that the positive control group showed a statistically significant (p < 0.05) increase in the hemozoin conc when compared to the standard control group.

Within various groups, it was observed that on Day 4 of post-treatment there was a significant (p < 0.05) decrease in the hemozoin conc of all the animals in the test groups 4 to 9 when compared to the hemozoin conc after Day 4 of treatment. The positive control group showed a significant (p < 0.05) increase in the hemozoin conc on Day 4 of post-treatment when compared to the hemozoin conc after Day 4 of treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in the hemozoin conc on Day 4 of post-treatment when compared to the hemozoin conc after Day 4 of treatment.

120

1.2 On day 4 of treatment

Day 4 of Post-treatment

1 0.906

0.8 0.747

0.6 0.395

0.4 0.353

0.321

Mean Hemozoin Conc. Hemozoin Mean

0.292

0.288

0.267

0.197

0.194

0.181 0.175

0.2 0.166

0.129

0.107

0.106

0 0 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9

Treatment Groups Figure 27: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on hemozoin conc. of mice passaged with Plasmodium berghei

121

152 Effect of Treatment with Different Partitions of Methanol Extract of Strophantus hispidus Leaves on Plasma Calcium (PC) Ion of Mice Passaged with Plasmodium berghei From figure 28 after passaging the animals with Plasmodium berghei, after fourth days of treatment there was a significant (p < 0.05) decrease in the PC ion of all the animals in groups (4 to 9) when compared to the positive control. Among the test groups, test groups 8 and 9 showed a significant decrease in the PC ion when compared to group 4, 5, 6 and 7. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the PC ion of test groups 8 and 9 when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the PC ion of the positive control when compared to the standard control.

On Day 4 of post treatment, a statistically significant (p < 0.05) decrease in the PC ion of all the test groups was observed when compared to the positive control group. Among the test groups there was a significant (p < 0.05) increase in the PC ion of group 4, 5, 6 and 7 when compared to group 9. Nevertheless test goup 5, 6 and 7 showed a non-significant (p > 0.05) difference in the PC ion when compared to group 8. Notwithstanding all the test groups showed a significant (p < 0.05) increase in the PC ion when compared to the standard control. However it was observed that the positive control group showed a statistically significant (p < 0.05) increase in the PC ion when compared to the standard control group.

Within various groups, it was observed that on Day 4 of post-treatment there was a significant (p < 0.05) decrease in the PC ion of the animals in the test groups 4, 7 and 9 when compared to the PC ion after Day 4 of treatment. Conversely it was observed that on Day 4 of post-treatment there was a non-significant (p > 0.05) difference in the PC ion of the animals in the test groups 5, 6 and 8 when compared to the PC ion after Day 4 of treatment. The positive control group showed a significant (p < 0.05) increase in the PC ion on Day 4 of post-treatment when compared to the PC ion after Day 4 of treatment. Conversely the standard control group showed a significant (p < 0.05) decrease in the PC ion on Day 4 of post-treatment when compared to the PC ion after Day 4 of treatment.

122

10 On day 4 of treatment 8.66

9 Day 4 of Post-treatment 7.7 8

7 6

5.3 4.9

4.56

4.43

4.2

4.07 3.97

4 3.77

3.4

3.2 3.2

3.03 2.8

3 2.83 2.23

2 Mean Plasma calcium ion conc. mg/dl ionconc. Plasma calcium Mean

0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Treatment Groups

Figure 28: Effect of treatment with different fractions of methanol extract of Strophantus hispidus Leaves on plasma calcium ion conc. of mice passaged with Plasmodium berghei

123

PT 1 2

PT 3

4 5

Plates 1 to 5. A photomicrograph of liver sections from experimental groups 1 (5 ml/kg 3% tween 80 solution), 2 (untreated), 3 (10 mg/kg Artesunate), 4 (400 mg/kg Ethyl acetate fraction) and 5 (800 mg/kg Ethyl acetate fraction) showing the portal tract (PT) with varying degrees of inflammatory cells infiltration in 2, 4 and 5 (arrows) stained with Haematoxylin and Eosin (H and E) at X 400 magnification.

124

PT PT

6 7

PT PT

8 9

Plates 6 to 9. A photomicrograph of liver sections from experimental groups 6 (400 mg/kg dichloromethane), 7 (800 mg/kg dichloromethane), 8 (400 mg/kg n-hexane fraction) and 9 (800 mg/kg n-hexane fraction) showing the portal tract (PT) with varying degrees of inflammatory cells infiltration shown by the arrows in group 6, 7 and 8 stained with Haematoxylin and Eosin (H and E) at X 400 magnification.

125

10 11 00 00 00 wp 0

13 14

Plates 10 to 14. A photomicrograph of spleen sections from experimental groups 1 (5 ml/kg b.w 3% tween 80 solution), 2 (untreated), 3 (10 mg/kg b.w. Artesunate), 4 (400 mg/kg Ethyl acetate fraction) and 5 (800 mg/kg b.w. Ethyl acetate fraction) showing areas of hyperplastic white pulp (wp) group 2, 3, 4 and 5. Note hematopoietic blast cells (signs of blood formation) in group 1 and 3 (white arrows) and also signs of severe hemosiderosis in group 2. H and E x 400.

126

15 16

wp 17 18

Plates 15 to 18. A photomicrograph of spleen sections from experimental groups 6 (400 mg/kg b.w. dichloromethane fraction), 7 (800 mg/kg b.w. dichloromethane fraction), 8 (400 mg/kg b.w. n-hexane fraction) and 9 (800 mg/kg b.w. n-hexane fraction) showing hematopoietic blast cells such as the megakaryoblasts in (white arrows) which is present in 9. H and E X 400.

127

153 In Vitro Cytotoxicity Test of Strophantus hispidus N-hexane Fraction Using Brine Shrimp Lethality Assay (BSLA) Table 9 shows the results of the invitro cytotoxicity test using brine shrimp lethality assay (BSLA) where the calculated LC50 is 245.5 µg/ml.

154 Column Chromatography and Fractions Obtained Based on their Thin Layer Chromatography Profiles As shown in Table 10, Ninety-five fractions of 20 mL each were collected and combined on the basis of their TLC profiles into nine major fractions F1-F9 (F1: 1-15, 0.23 g; F2: 16-24, 0.07 g ; F3: 25-31, 0.03; F4: 32-36, 0.07; F5: 37-50, 0.17 g; F6: 51-60, 0.21 ; F7:61-69, 0.17; F8:70-85, 0.32 g; F9: 86-95, 0.21 g).

155 Thin Layer Chromatography (TLC) Profiles of Different Fractions Figures 29 shows the TLC profiles of the different fractions fractions 2, 3, 4 and 5 showed similar chromatograms however fraction 1 and 6 had a unique chromatogram. In the same vein fractions 7, 8, and 9 showed similar chromatogram.

156 Invitro Antimalarial Activity of Different Fractions of n-Hexane Partition of Methanol Extract of Strophantus hispidus Leaves Using β-hematin Inhibition Assay (BHIA) Results shown in Table 11 reveals the in vitro antiplasmodial activity of different fractions of n- hexane partition of methanol extract of Strophantus hispidus leaves using β-hematin inhibition assay (BHIA). Fraction 1 showed the least activity while fractions 2 to 6 hade moderate activity however the highest activity was found in fractions 7 to 9. Nevertheless fraction 9 had the highest activity.

157 Preparative Thin Layer Chromatography of Fraction 9 As shown in figure 30, the most active compound was isolated using preparative chromatography. The fraction was developed on a TLC plate to isolate the active compound.

128

Table 9: In vitro cytotoxicity Test Using Brine Shrimp Lethality Assay (BSLA)

Mortality of Artemia salina (%) Concentration Log10 Conc Mean (ppm) Replicate Replicate Replicate Mortality Probit 1 2 3

0.0 (control) -10 0 10 0 3.3 3.3

0.1 -1 0 0 10 3.3 3.12

1.0 0 30 10 20 20 4.16

10.0 1 40 30 10 26.7 4.36

100.0 2 50 30 40 40 4.75

1000.0 3 50 70 60 60 5.25

Thus the calculated LC50 of the S. hispidus N-hexane fraction is 245.5 µg/ml using probit analysis

Table 10: Shows the Result of the Column Chromatography and Fractions Obtained Based on their Thin Layer Chromatography Profiles FRACTIONS PROPORTION WEIGHT (g) Fraction 1 1-15 0.23 Fraction 2 16-24 0.07 Fraction 3 25-31 0.03 Fraction 4 32-36 0.04 Fraction 5 37-50 0.17 Fraction 6 51-60 0.21 Fraction 7 61-69 0.17 Fraction 8 70-85 0.32 Fraction 9 86-95 0.21

129

Fraction 1 Fraction 2 Fraction 3 Fraction 4

Fraction 5 Fraction 6 Fraction Fraction 8 Fraction 9

130

Figures 29: Thin layer chromatography of fractions 1-9

Table 11: Invitro Antimalarial Activity of Different Fractions of n-Hexane partition of Methanol Extract of Strophantus hispidus leaves using β-hematin Inhibition Assay (BHIA)

Fractions Concentration (µg/ml) Abs (405) % Inhibition

1 100 2.762 ± 0.005 9.33

2 100 2.079 ± 0.06 31.72

3 100 1.641 ± 0.03 46.09

4 100 1.405 ± 0.07 53.84

5 100 1.293 ± 0.01 57.52

6 100 0.957 ± 0.04 68.56

7 100 0.259 ± 0.01 91.49

8 100 0.211 ± 0.02 93.06

9 100 0.195 ± 0.01 93.59

Standard Control 10 0.018 ± 0.00 99.41

Positive Control 0 3.044 ± 0.07 0

Absorbance values are expressed as mean ± SD (n=3)

131

Figure 30: Isolation of Bio-active Compound from fraction 9 using preparative TLC

132

158 In vitro Antimmalarial Activity of Isolated Compounds from Fraction 9 Using β- hematin Inhibition Assay (BHIA) Table 12 shows the results of β-hematin inhibition assay of the two isolated compounds in which the most active Compound showed the highest percentage inhibition.

159 Thin Layer Chromatogram of the Active Compound As shown in Figure 31 the Tin Layer chromatogram of the active compound Fractions with RF value of 0.88 with DEE- HEX 4:7

160 IR SPECTRUM of Active Compound The IR spectrum showed absorption bands at 2929.7, 2959.5, 2858.9, 1461.1 and 1379.1cm-1

Table 12: In vitro antimmalarial Activity of Isolated Compounds from Fraction 9 Using β- hematin Inhibition Assay (BHIA) Constituents Concentration (µg/ml) Absorbance 405 nm % Inhibition Active Compound 50 0.187 93.86 Compound B 50 0..725 76.18 Standard Control 10 0.018 99.41 Positive Control 0 3.044 0

133

Figure 31: Tin Layer chromatogram of the Active Compound

134

C-H of CH3 symmetry germinal dimethyl C-H

C-H def, sym C-O of cyclic C-H of CH3

C-H of CH2

Figure 32: IR Spectrum of Active Compound

135

161 Proton Nuclear Magnetic Resonance (1H NMR) Analysis of the Bio-active Compound

1 The H NMR spectrum as shown in Figure 33 showed peaks at δH 7.26, 4.39, 4.35, 4.31, 4.23,

4.18, 4.17, 4.10, 4.07, 4.03, 3.77, 3.74, 3.67, 3.67, 3.64, 3.60, 3.58, 3.57, 3.55, 3.53, 2.35, 2.27,

2.27, 2.26, 2.20, 2.19, 2.17, 2.09, 2.08, 2.00, 1.92, 1.77, 1.74, 1.72, 1.65, 1.64, 1.61, 1.59, 1.57,

1.56, 1.55, 1.54, 1.52, 1.50, 1.33, 1.28, 1.26, 1.09, 1.08, 1.07, 1.05, 1.03, 1.03, 1.00, 0.98, 0.96,

0.92, 0.90, 0.88, 0.86, 0.85, 0.84, 0.84, 0.83.

136

Figure 33: Proton NMR of Active Compound (in CDCl3, 400MHz)

137

162 13C NMR Spectral Analysis of Active Compound

13 The C-NMR ((δ ppm, 500MHz, CDCl3) analysis as shown in Figure 34 revealed signals at C6,

C6, C1, C3, C3, C5, C4, C5, C17, C14, C9, C13, C4, C10, C20, C28, C10, C23, C10, C20, C28,

C10, C23, C18, C11, C28, C19, C21, C28, C18. 146.67, 122.40, 101.52, 79.43, 77.38, 76.31,

73.96, 70.69,62.21, 57.19, 56.37, 51.14, 46.27, 42.68, 40.46, 39.02 37.64, 37.07, 36.49, 34.31,

32.28, 32.26, 29.73, 29.24, 28.36,26.44, 24.63, 23.41, 21.32, 19.95, 19.53, 19.20, 18.99, 12.32 and

12.07

138

13 Figure 34: C NMR of Active Compound (in CDCl3, 400MHz)

139

163 Matching of the NMR Data using APT (Attached Proton Test) As shown in figure 35 and 36, the APT and 13C NMR spectra revealed the presence of three (3) quaternary carbon viz: C-5, C-10 and C-13, four oximethine (4), fourteen methine (14), tewel (12) methylene and six methyl (6) carbons at 12.07, 12.32, 18.99, 19.20, 19.53 and 19.95.

164 Structure of Identified Compound Figure 37 shows the structure of the bio-active compound which was obtained based the spectroscopic data obtained in the analysis.

140

APT

C 6' C25 C11

C C2 17,14 C20 C18/29 Region of sugar moiety C19,21,26,27

Fig 35: Attached Proton Test of Active Compound (APT) (in CDCl3, 400MHz)

141

Figure 36: Extended APT of Active Compound downfield (in CDCl3, 400MHz)

29 21 28 20 22 19 12 24 25 27 11 17 23 13 18 16 1 9 26 6' 2 14 15 HOH C 10 8 2 O 1' 7 4' 5' 2' 3 5 O 4 6 HO OH HO 3'

Figure 37: Beta-sitosterol-d-glucoside (3-O-[β- glucopyranoyl] –β-Sitosterol )

142

165 In silico Pharmacokinetics of the Isolated Compound and the Structural Analogues As shown in Table 13, ADME of the various structural analogues based on the Lipinski’s rule of 5 analysis revealed molecular weight (MW) of 576.86, hydrogen bond acceptor (HBA) of 6.0, hydrogen bond donor (HBD) of 4.0 and logP o/w 5.51. However, with slight modification in the structure MW = 410.50, 422.55, 448.59, 444.54 and 452.58; HBA = 7, 6 and 1; HBD = 5, 4 and 1 and log P o/w 0.80, 1.42, 2.90, 2.39 and 7.25; High Gastrointestinal absorption was recorded in the modifications.

166 Structural Modifications of the Active Compound Figures 37 and 38 shows the structures of the various structural analogues as modified using the SWISS ADME software.

143

Table 13: Summary of the In silico Pharmacokinetics of the Isolated Compounds and the Structural Analogues I.C. MOD 1 MOD 2 MOD 3 MOD 4 MOD 5 Molecular weight (Da) 576.86 410.50 422.55 448.59 444.54 452.58 Hydrogen bond 6.0 1 6 6 6 7 acceptors Hydrogen bond donors 4.0 1 4 4 4 5 Log P o/w 5.16 7.25 2.39 2.90 1.42 0.80 Log S (SILICOS-IT) -4.02 -6.19 -1.49 -1.69 -1..32 -1.06 GI absorbtion low low high high high high lipinski Yes; 1 v Yes; 1 v Yes; 0 v Yes; 0 v Yes; 0 v Yes; 0 v

144

Isolated compound (I.C.) Modification 1 (MOD 1) Modification 2 (MOD 2)

Figure 38: Isolated Bioactive Compounds and Some Structural Modifications Based on In Silico Pharmacokinetics Study

145

Modification 3 (MOD 3) Modification 4 (MOD 4) Modification 5 (MOD 5)

Figure 38: Some Structural Modifications based on In silico Pharmacokinetics Study

146

CHAPTER FOUR

DISCUSSION

The findings of this study are based on anti-malarial activity and pharmacokinetic profiles of methanol extract of Strophanthus hispidus leaves. These will be examined using, qualitative and quantitative phytochemical analysis of leaves of Strophanthus hispidus, invivo and invitro antimalarial properties of the leaves of Strophanthus hispidus, partitioning, column chromatography, tin layer chromatography, NMR spectroscopy, FTIR and insilico pharmacokinetics studies.

Extraction involves solvent penetration into herb cells/tissues, solubilization of secondary metabolites and finally release of the dissolved secondary metabolites in solvent of extraction. Solvents of varying polarity are used alone or in combinations for extraction depending on component. Methanol dissolves most of the secondary metabolites and enhancing their release from cellular matrix/cell surface (Stahl, 2005) however increasing the polarity by adding H20 would increase the dissolution of the secondary metabolites contained in the plant hence an increased yield. From Table 3 the percentage yield of the aqueous-methanol (1:1) extract of Strophantus hispidus is 12.53%. Oral administration of the aqueous-methanol extract of Strophanthus hispidus leaves to mice at doses ranging from 10 – 5000 mg/kg b.w caused death only at the dose of 5000 mg/kg b.w. Therefore the oral LD50 of the extract in mice was 3807.89 mg/kg b.w.

Phytochemicals are pharmacologically bio-active compounds contained in plants that have shown some levels of medicinal properties against any disease. However these phytochemicals are of different classes based on their bio-activity and functional groups. In this study, qualitative phytochemical analysis revealed the presence of bioactive organic compounds such as alkaloids, flavonoids, terpenoids, steroids, phenols, saponins, tannins and glycosides as can be seen in Table 5 and 6 while the quantitative phytochemicals analysis of the crude extract was observed in the order: alkaloids > phenol ˃ flavonoids > terpenoids > saponnin > glycosides > steroid > tannins. Alkaloids were abundantly present in the extract however phenols, flavonoids and terpenoids were moderately present while others such as saponnin, glycosides, steroids and tannins were present in minimal form. Previous researchers have shown that phytochemicals are the major reason why African medicinal plants exhibit numerous pharmacological effect (Ferreira et al., 2010; Elfawal

147 et al., 2015). Alkaloids and alkaloid containing plants have been used as CNS stimulant, topical anaesthetic in ophthalmology, powerful painkillers, antimalarial and antipyretic action among other use. The pharmacological activities of alkaloids are quite diverse however they are important natural products with a wide range of medicinal properties including relief of pain (e.g., morphine), analgesic (e.g.,codeine), antiarrhythmic (e.g., quinidine), antibacterial (e.g., chelerythrine), antiasthma (e.g., ephedrine), cholinomimetic (e.g., galantamine), bebeerine (e.g., antiparasitic), quinine, mefloquine (antimalarial) and vasodilatory (e.g., vincamine) (Sayhan et al., 2017). Thus the presence of alkaloid in Strophantus hispidus shows a possibility of the plant having the above mentioned wide range of pharmacological activities and as such should be investigated. Phenolic compounds are known to provide protection against a wide range of diseases such as coronary heart disease, stroke, malaria and certain types of cancers (George et al., 2009), nonetheless the works of Lin et al. (2016) reveals the OH radical scavenging ability and Fe2+ chelating ability of phenols. These effects are attributed in general to the potential ability of phenolic compounds to reduce, counteract or also repair damages resulting from oxidative stress and inflammation associated with diseases conditions. P-hydroxy-cinnamic acid a phenolics was isolated from Kigelia africana (Bignoniaceae) as an anti-malarial compound (Zofou et al., 2012). The presence of phenols in a plant such as Strophantus hispidus indicates that the plant could be useful in management and treatment of ailment.

Lipid peroxidation is a common consequence of oxidative stress which is a general effect in disease condition. Flavonoid protects lipids against oxidative damage by various mechanisms (Kumar et al., 2013). Free metal ions enhance ROS formation by the reduction of hydrogen peroxide with generation of the highly reactive hydroxyl radical. Due to their lower redox potentials flavonoids (Fl-OH) are thermodynamically able to reduce highly oxidizing free radicals such as superoxide, peroxyl, alkoxyl, and hydroxyl radicals by hydrogen atom donation. Because of their capacity to chelate metal ions (iron, copper, etc.), flavonoids also inhibit free radical generation (Mishra et al., 2013). For example, quercetin in particular is known for its iron-chelating and iron-stabilizing properties (Kumar and Pandey, 2013) however, the use of flavonoids in combination with artemisinin might provide a more effective treatment for malaria. In that regard, flavonoids could serve as artemisinin synergists by reacting with iron and converting Fe+3 to Fe+2, the latter being important in the bioactivity of artemisinin, leading to the release of short-lived toxic free radicals that are part of the antimalarial and anticancer mode of action of artemisinin (Ferreira et al., 2010).

148

Nonetheless its presence in the plant studied could be significant in the numerous pharmacological activities exhibited by the plant. Tannins are astringent polyphenols which react with proteins through hydrogen bonds and/or hydrophobic interactions when not oxidized; however, they are converted into quinone when oxidized, generating covalent bonds with some functional groups of proteins such as the sulfidric groups of cysteine and the amino groups of lysine (Sgarbieri, 1996). The tannins bind to the proteins and adhesions present in mucosal cells with this action being attributed to the poly- phenolic hydroxyl groups situated on the surface of this molecule (Lim et al., 2014). They form a protective cover through interaction, inhibiting the action of pathogenic enzymes, which promotes the disruption of the plasma membrane and deprivation of substrates required for the microbial growth by forming a tannin-protein and/or polysaccharide complex, thus preventing the growth of pathogens (Rodrigues et al., 2014). Nonetheless the presence of tannins in a plant such as Strophantus hispidus could result to the plant exhibiting protective ability against pathogens and related diseases.

Terpenoid-derived drugs have contributed significantly to human disease therapy and prevention however the presence of terpenoids in a plant such as Strophantus hispidus could prognosticate the likelihood of the plant having numerous pharmacological effect. Some terpenoid drugs have provided tremendous benefits for patients and for the pharmaceutical industry. The works of Zhao et al. (2016) reports that terpenoids group of phytochemicals have shown significant pharmacological activities, such as anti-viral, anti-bacterial, anti-malarial, anti-tumor activity, ameliorates acute myocardial infarction anti-aggregatory effect, anti-coagulative effect, analgestic effect, anti-diabetic anti-inflammatory, inhibition of cholesterol synthesis and anti-cancer activities. Molecular basis for these pharmacological activities have revealed copious mode of action which includes inhibition of nitric oxide production, cytotoxicity against cancer cell lines, inhibited against cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes, significant scavenging capacity against the DPPH free radical, ROS, the superoxide anion radical, and the hydroxyl radical, inhibitory effect on collagen, endotoxin and adenosine diphosphate (ADP)- induced blood platelet coagulation and Inhibitory effect on neuromuscular junction in phrenic nerve diaphragm preparations of mice (Zhao et al., 2016 ).

149

Steroids are a class of pharmacologically active phytochemicals that are hormone like in nature. Steroids are commonly used in patients with rheumatoid arthritis, chronic obstructive pulmonary disease, systemic lupus erythematosus, inflammatory, bowel disease, regulate metabolisim, and regulate sodium water levels (Curtis et al., 2006). Nonetheless more specifically steroids are known for their anti-inflammatory effect thus the anti-inflammatory properties of steroids have been attributed to their inhibitory effects on the action of phospholipase A2, an enzyme critical to the production of inflammatory compounds (Wallner et al., 1986). Research has shown that steroids are active in affecting gene expression, translation, and enzyme activity (Rhen and Cidlowski, 2005). In short, they bring about their physiologic effects through a multitude of biochemical pathways (Rhen and Cidlowski, 2005). One such pathway is through their induction of the production of proteins called lipocortins. Particularly, glucocorticoids stem the production of inflammatory mediators such as leukotrienes and prostaglandins and effectively halt the inflammatory cascade (Blackwell et al., 1980)

Saponins have been ascribed a number of pharmacological actions, the important ones being permeability of the cell membrane, lowering of serum cholesterol levels, stimulation of luteinizing hormone release leading to abortifacient properties, immunomodulatory potential via cytokine interplay, cytostatic and cytotoxic effects on malignant tumour cells, adjuvant properties for vaccines as immunostimulatory complexes, and synergistic enhancement of the toxicity of immunotoxins (Fuchs et al., 2009; Francis et al., 2002). This pharmacological activities might be due to the amphiphilic behavior of saponins and the ability to form complexes with steroids, proteins and membrane phospholipids determined a number of different biological properties for these substances, especially the action on cell membranes, changing their permeability, or causing their destruction (Barbosa, 2014). The presence of saponins in Strophantus hispidus leaves suggest the ability of the plant several pharmacological activities as mentioned above.

The presence of glycosides in Strophantus hispidus leaves suggest the possibility of the plant to be effective against cardiac heart failure, tumor and malarial. The studies done by Shitlani et al. (2016) and Khan et al. (2017) have shown that apart from cardiac heart failure, some cardiac glycosides were shown to possess antitumor activity, inhibitory activity against rhinovirus and antimalarial activity. Nonetheless the most important use of the cardiac glycosides is its effects in treatment of cardiac failure. In cardiac failure, or congestive heart failure, heart cannot pump

150 sufficient blood to maintain body needs. During each heart contraction, there is an influx of Na+ and an outflow of K+. Before the next contraction, Na+, K+‐ATPase must re-establish the concentration gradient pumping Na+ into the cell against a concentration gradient. This process requires energy, which is obtained from hydrolysis of ATP to ADP by Na+, K+‐ATPase. Cardiac glycosides inhibit Na+, K+‐ATPase, and consequently increase the force of myocardial contraction (Prassas and Diaandis, 2008). From Figure 4 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the mean Parasitemia Count (PC) of all the animals in groups (2 to 6) when compared to the negative control group (group 1) that was not passaged. This showed the animals were infected with Plasmodium berghei that notwithstanding, after Day 4 of treatment with methanol extract of Strophathus hispidus (MESH) leaves, there was a significant (p ˂ 0.05) decrease in the mean PC of all the test groups (4-6) when compared to the positive control (untreated group). This reduction could be as a result of the astringent polyphenols (tannins) contained in the plant which react with merozoites through hydrogen bonds and/or hydrophobic interactions thereby inhibiting further increase in the parasites and consequently reducing the parasitic load of the mice under treatment. The results of this work is in agreement with the works of Builders et al. (2014) where a phenol extract from Parkia biglobosa elicited potent activity against the rodent malaria parasite. More so a non-significant (p ˃ 0.05) difference was observed between the treatment groups and the standard control, this goes ahead to prove the effectiveness of the extract. On Day 4 of post-treatment there was a further decrease but not significant (p ˃ 0.05) in the mean PC when compared to the mean PC after treatment. This shows that there is no possibility of fall back as the mean PC showed a further decrease and is still below the viable parasitemia.

Packed cell volume (PCV) is the measurement of the proportion of blood that is made up of cells especially the red cells. Red blood cell count (RBC) checks to find out how many red cells that are available in the blood. From Figures 5 and 7 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the PCV and RBC of animals in groups (2 to 6) when compared to the negative control group (group 1). This decrease in PCV and RBC could be practically said to be as a result of anemia which is majorly the first symptom experienced in malaria patient because the malaria parasites attacks majorly the red blood cells in the blood. Nevertheless after Day 4 of treatment with MESH leaves there was a significant increase (p ˂ 0.05)

151 in the PCV and RBC of all the animals in the test groups (4-6) when compared to the positive control, this increase could be as a result of inhibition of the breakdown of the red cells and consequently increased red cell production which could be caused by the actions of some bioactive compounds inherent in MESH leaves. The increase in PCV and RBC could be due to the hormone like actions of the steroids acting like erythropoietin or assisting the kidneys to produce and release erythropoietin, a hormone that stimulates the bone marrow to produce more red blood cells. However the obtained result is in consonance with the works done by Joshua et al. (2016) where the haematological responses and percentage parastaemia in malaria-infected mice treated with ethanol extract of Zapoteca portoricensis roots were estimated. The test groups (4 and 5) showed a significant (p ˂ 0.05) decrease in the PCV when compared to the standard control except group 6 more so this shows that group 6 took a more effective dose when compared to the other treatment groups. Nonetheless on Day 4 of post-treatment a further significant decrease (p ˂ 0.05) was observed in the PCV and RBC of the animals in the treatment groups this proves an improvement in the animal health state after treatment and no possibility of fall back.

Haemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells. From figure 6 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) decrease in the Haemoglobin Concentration (Hb Conc) of animals in all groups (2 to 6) when compared to the negative control group (group 1). These decrease is as a result of plasmodium attack in which the parasites feeds on the protein (globulin) component of the haemoglobin thus degrading the haemoglobin. However after Day 4 of treatment with MESH leaves, there was a significant (p ˂ 0.05) increase in the Hb Conc of all the animals in the test groups (4-6) when compared to the positive control group 2 and no significant (p > 0.05) difference when compared to the standard control group3. The significant increase experienced after treatment could be as a result of the phytochemical components of the plant inhibiting the further breakdown of the haemoglobin by binding to free heme just like artemisinin and other antimalarials which is toxic to the parasite thereby stimulating parasiticidal action which will inhibit further breakdown of the haemoglobin and in turn boost haemoglobin level. This result is in agreement with the research done by Omodeo-Salè et al. (2009). More so on Day 4 of post- treatment there was a further significant (p < 0.05) increase in the Hb conc of the treatment group this shows that the animals treated are recovering from the ailment and no possibility of fall back.

152

White blood cells (WBCs), also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. After passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the WBC count of animals in groups (2 to 6) when compared to the negative control group (group 1) as can be seen in Figure 8. This increase is as a result of infestation by the parasite and the body system through its defense mechanism releasing more WBCs to fight the foreign bodies. This is consistent with work of Okochi et al. (1999) who reported increase in WBC and decrease in RBC, Hb and PCV in rodents after parasite infestation. However after Day 4 of treatment, there was a significant (p ˂ 0.05) reduction in the WBC count of all the animals in the test groups (4-6) when compared to the positive control group 2 but a non-significant (p > 0.05) difference was observed when compared to the standard control and negative control group 1. This reduction could be as a result of the body system returning to normalcy after treatment with the MESH leaves however this reduction was within the normal range as was observed a non- significant (p > 0.05) difference when compared to the negative control group. All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Nonetheless there was a significant (p < 0.05) increase in the WBC count of the positive control group 2 when compared to the standard control group 3. However on Day 4 of post- treatment the WBC count of the animals in the treatment group were still within the normal range which is a positive sign of recovery. The results of this work is in line with the work done by Dawet et al. (2012) where the In vivo antimalarial activity of the ethanolic leaf extract of Hyptis suaveolens poit on Plasmodium berghei in mice.

Aspartate aminotransferase (AST) and alanine aminotransferaes (ALT) are the major liver marker enzymes; however, AST is more widely distributed than ALT it is present in the liver, heart, kidneys, skeletal muscle and red blood cells (Manson, 2004). However, ALT is considered as specific marker of hepatocellular damage because its levels are only raised significantly in liver damage. From Figure 9 and 10 a significant (p < 0.05) increase was observed in the ALT and AST activities of all the animals in groups (2 to 6) after passaging with Plasmodium berghei when compared to the negative control group 1. This increase is as a result of hepatocellular damage caused by the parasitic actions of the sporozoites and the trophozoites in the liver tissue causing damage of the cells and consequently the release of these enzymes into the blood stream. However after Day 4 of treatment with MESH leaves, there was a significant (p ˂ 0.05) diminution in the

153

ALT activity of the animals in test group 5 and 6 when compared to the positive control group 2 while test group 4 showed a decrease but was not significantly (p ˃ 0.05) different in comparison with the positive control group. This significant decrease suggest that the phyto-active constituents of the MESH leaves suppressed the development of the trophozoites in the parasitophorous vacuole of the hepatocyte, in addition to enhanced immune response in the mice and probably abrogated the hepatic phase of development of the protozoa. However a non-significant (p ˃ 0.05) difference was observed between test group 5 and 6 and the standard control while test group 4 showed a significant (p < 0.05) increase in comparison to the standard control group. This also suggest that group 5 and 6 dose had a better activity when compared to the group 4 dose. Moreso there was neither a significance (p ˃ 0.05) increase nor decrease in the ALT activity between test group 5 and 6 and the negative control group even at the fouth day of post-treatment however test group 4 showed a significant increase in comparison with the negative control group. This also shows that the extract was able to inhibit further damage of the hepatocytes and thus return it to normal functionality and sustain it. . This results obtained is in agreement with the scientific reports of Umar et al. (2018) where the antimalarial and liver function potentials of methanol extract of Chrysophyllum albidum stem bark in Plasmodium berghei -infected mice was evaluated.

As observed from Figure 11, there was a significant (p < 0.05) increase in the ALP activity of all the animals in groups (2 to 6) after passaging with Plasmodium berghei when compared to the negative control. Higher than normal levels of ALP in the blood may indicate a problem with the liver cells or gallbladder however in the present research this increase could be as a result of inflammation of the liver cells by the parasites. However after Day 4 of treatment with MESH leaves, there was a significant (p ˂ 0.05) decrease in the ALP activity of the animals in test groups 4, 5 and 6 when compared to the positive control. This reduction could be as a result of healing of the sinusoidal surface of hepatocytes by the phytochemicals inherent in the extract or a resultant effect of the parasiticidal action of the extract as was seen in Figure 4. The results of this research is in consonance with the works of Peter et al. (2011). However there was neither a significant (p ˃ 0.05) increase nor decrease in the ALP activity of the test groups 4, 5 and 6 when compared to the standard control. Nevertheless there was a non-significant (p > 0.05) difference in the ALP activity of test groups 4, 5 and 6 on Day 4 of post-treatment when compared to the negative control. This is evident that the extract had some components that is able to alleviate the ailment and return the body system to normalcy.

154

Bilirubin is the terminal product of heme metabolism. The majority (80%) of the bilirubin formed in the body comes from the heme released from senescent red blood cells. The remainder originates from various heme-containing proteins found in other tissues, notably the liver and muscles. In the current research it was observed from figure 12 a statistically significant (p < 0.05) increase in the T.BIL conc of all the animals in groups (2 to 6) when compared to the negative control after passaging with Plasmodium berghei. This increase in bilirubin concentration is as a result of the breakdown of haemoglobin by the merozoites which in turn increases heme breakdown to bilirubin. However after Day 4 of treatment with MESH leaves, there was a significant (p ˂ 0.05) decrease was observed in the T.BIL conc of the animals in test groups 4, 5 and 6 when compared to the positive control. This significant reduction could be as a result of the elimination of the malaria parasites from the system which inturn causes a reduction in the breakdown of haemoglobin probably by the terpenoids contained in the plant extract or binding of some bioactive components of the plant to the heme which is toxic to the parasite cell consequently causing mortality of the parasites. The results of this work is in agreement with the works done by Nwankwo et al. (2015). All the same the positive control group showed a significant (p < 0.05) increase in the T.BIL conc when compared to the standard control. However there was neither a significant (p ˃ 0.05) increase nor decrease in the T.BIL conc of the test groups 4, 5 and 6 when compared to the standard control. This suggest that the extract could be as effective as the standard drug used. Nevertheless there was a non-significant (p > 0.05) difference in the T.BIL conc of test groups 4, 5 and 6 when compared to the negative control on Day 4 of post-treatment. This goes further to prove the ability of the bioactive compounds of the plant to reduce parasite load, inhibit further breakdown of haemoglobin and probably stimulate the liver to maintain a healthy state.

Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. None withstanding in malaria conditions the parasite utilizes the activity phospholipase A2 to enable it form a parasitophorous vacuole where it can reside and develops and ruptures the cell membrane. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of carbonyl compounds, which is the most abundant malondialdehyde (MDA). From Figure 13, it was observed that after Day 4 of treatment with MESH leaves there was a significant (p ˂ 0.05) decrease in the MDA conc of animals in test groups 4, 5 and 6 when compared to the positive control group 2. The increase in MDA observed in the positive control is as a result of increase in

155 lipid peroxidation by the merozoites and consequently increase in MDA concentration however the significant decrease observed in the treatment groups could be as a result of inhibition of formation of parasitophorous vacuole by the parasite and consequently reduction in the MDA concentration. Thus this could be one of the mechanisms through which the extract exerts its antimalarial activity. However a non-significant (p ˃ 0.05) difference was observed between all the test groups in comparison to the standard control. Nevertheless a non-significant (p ˃ 0.05) difference was observed in the MDA conc of groups 4, 5 and 6 when compared to the standard control group 3 and the negative control group on Day 4 of post-treatment. The results of this work is in agreement with the scientific report of Sulistyaningsiha et al. (2017).

Superoxide dismutase (SOD) is a major detoxifying enzyme and most powerful antioxidant in the cell that acts against reactive oxygen species (ROS). It catalyzes the dismutation of two molecules of superoxide anion (O2) to hydrogen peroxide (H2O2) and molecular oxygen (O2), consequently rendering the potentially harmful superoxide anion less hazardous. From figure 14 after four day of treatment with MESH leaves there was a significant (p < 0.05) increase in the SOD activity of the test groups 5 and 6 when compared to the positive control (untreated group). The decrease in the SOD activity of the positive control could be as a result of increase in breakdown of haemoglobin and cell injury thereby generation high levels of free radicals which in turn depletes the antioxidant level of the rats in the untreated group (positive control). Phytochemical analysis of the plant under study revealed the presence of alkaloids and flavonoids which are rich antioxidants that could mop up free radicals and consequently inhibit cell damage thus treatment with the plant extract led to increase in the antioxidant level of the animals. The results shown in accord with the works of Iyawe and Onigbinde, (2009). The test group 4 showed no significant (p > 0.05) difference in comparison to the positive control group. This suggests that the dose of 200 mg/kg b.w is not effective enough to cause a statistically significant change in the antioxidant level of the mice nevertheless the dose of 400 and 800 mg/kg b.w were more effective. That notwithstanding a non-significant (p > 0.05) difference was observed in the SOD activity of the test group 6 when compared to the negative control group 1 on Day 4 of post-treatment. This indicates that though the doses of 400 and 800 mg/kg b.w were effective in improving the antioxidant level only the dose of 800 mg/kg b.w was able to bring the antioxidant level to normalcy and enable the body sustain it even at post-treatment.

156

Several scientific reports have recorded a wide range of phytochemicals in plants and plant fractions. Some are non-polar, slightly polar, polar and highly polar. This polarity differences is based on majorly their individual functional groups which directly affects their solubility however these differences make it easy to isolate individual bioactive compound(s) or a class of phytochemicals. Hence no single solvent can extract all the phytochemicals in a plant nevertheless a solvent might extract majority of the phytochemicals not all. Extraction of these class of phyto active compounds starts from Non Polar Solvent to Polar e.g hexane or petroleum ether then chloroform then ethyl acetate then ethanol/methanol then water. As shown in Table 7, the percentage yield of different solvents from the methanol leave extract of Strophantus hispidus was found to be 20 %, 22% and 35 % for n-hexane, dichloromethane and ethyl acetate respectively. Phytochemical analysis as shown in table 8 reveals that alkaloids, tannins and saponin were abundantly present in the ethyl acetate while flavonoids terpenoids steroids phenols and glycosides were moderately present in the dichloromethane and n-hexane fraction.

From Figure 15 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the mean Parasitemia Count (PC) of the animals in groups 2 to 9 when compared to the negative control (group 1). This revealed the Plasmodium berghei parasite rapid invasion of the mice body system thus resulting to the increase in the parasitemia level in the mice. Nevertheless after 4 days of treatment with different fractions; 400 and 800 mg/kg b.w of ethyl acetate fraction groups 4 and 5, 400 and 800 mg/kg b.w of dichloromethane fraction groups 6 and 7, and 400 and 800 mg/kg b.w of n-hexane fraction group 8 and 9 of the MESH leaves, the mean PC load showed a dose dependent significant (p < 0.05) reduction in the parasitemia count of all the treatment groups when compared to the positive control. However, among the test groups 5, 7, 8 and 9 showed a significant (p ˂ 0.05) decrease in the mean PC when compared to test groups 6 and 4. This is evident that non-polar fractions were more effective than the polar fraction in reducing the parasitemia load of the mice. A possible mechanism of action might be that due to the non-polar nature of the n-hexane and dichloromethane fraction which is synonymous to the cell membrane of the parasite thus penetrating the cell membrane and probably inhibiting the Plasmodium falciparum Choline Kinase activity of the Kennedy pathway which is a necessary pathway in the asexual reproduction of the parasite in the red blood cell. A similar mechanism of action was seen in the scientific report of Choubey et al. (2007) where they demonstrated inhibition of Plasmodium falciparium choline kinase by Hexadecyltrimethylammonium Bromide as a

157 possible antimalarial mechanism of action because phosphatidylcholine is a necessary requirement for the asexual reproduction of the malaria parasite in the blood. Nonetheless there was a significant (p < 0.05) increase in the mean PC of the positive control when compared to the standard control. This proves that the standard drug was effective in treatment of malaria conditions. At 4th day of post-treatment, among the test groups there was a significant (p < 0.05) decrease in the mean PC of groups 7, 8 and 9 below the viable parasitemia when compared to groups 4, 5 and 6. This established that there was no possibility of fall back and that the anti- plasmodia activity was found in the non-polar region of the extract.

From figure 16 and 18 after passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) diminution in the PCV and RBC count of animals in groups (2 to 9) when compared to the negative control respectively. A significant (p < 0.05) decrease in PCV and RBC count is a sign of anemia which is caused by the malaria parasite (Langhorne et al., 2002). Nevertheless after Day 4 of treatment with different fractions of MESH leaves, there was a significant increase (p ˂ 0.05) in the PCV and RBC count of all the animals in the test groups (4- 9) when compared to the positive control. This is evident that all the fractions where potent in boosting the RBC count and PCV. A possible mechanism of action could be that the fraction had a membrane stabilization activity thus inhibiting further breakdown of the red cell and stimulating the bone marrow to produce red cells. Among the test groups, groups 4 and 6 showed a statistically significant (p < 0.05) decrease in the PCV when compared to group 9 while test groups 4, 5, 6, 7, and 8 showed no significant (p > 0.05) difference among them. This further states that though activity was found in all fraction but group 9 that received n-hexane fraction 800 mg/kg b.w. was the most potent. Phytochemical analysis of the different fractions revealed the presence of flavonoids to be more abundant in the n-hexane fraction when compared to other fractions. The works of Ferreira et al. (2010) suggested that antioxidant compounds such as flavonoids due to their redox properties can delay or inhibit lipid peroxidation and other molecules by inhibiting the initiation or propagation of oxidizing chain reactions. This could be a possible mechanism why fraction was most potent. Nevertheless test group 9 showed no significant (p > 0.05) difference in the PCV when compared to the standard control. Comparatively there was a significance decrease (p < 0.05) in the PCV of the positive control when compared to the standard control. On Day 4 of post treatment it was observed a statistically significant (p < 0.05) increase in the RBC count of all the test groups when compared to the positive control group. Comparatively test groups 8 and

158

9 showed a significant (p < 0.05) increase in the RBC count when compared to test groups 4, 5, 6 and 7. This is evident that the n-hexane fraction contains bio-active compounds that could boost PCV and RBC count and sustain it without a fall back.

It was observed from Figure 17 that after Day 4 of passaging the animals with Plasmodium berghei, there was a significant (p < 0.05) decrease in the Hb Conc of animals in all groups (2 to 9) when compared to the negative control group. This decrease is as a result of increase in the breakdown of haemoglobin by the malaria parasite. Nevertheless after Day 4 of treatment with different fractions of MESH leaves, there was a significant increase (p ˂ 0.05) in the Hb Conc of all the animals in the test groups (4-9) when compared to the positive control. Aforementioned suggest that the different fractions were potent at inhibiting haemoglobin breakdown through various mechanisms. Among the test groups, the n-hexane fraction group was more potent because it showed a significant (p < 0.05) increase when compared to other groups. A possible mechanism as discussed by Basore et al. (2015) could be that the bioactive components of the n-hexane fraction was able to permeate the lipid bilayer and and also was transported through the ion channel of the parasitophorous membrane and act on the parasite digestive vacuole just as chloroquine and mefloquine consequently inhibiting breakdown of the haemoglobin. Nevertheless there was a significant (p < 0.05) decrease (p < 0.05) in the Hb Conc of the positive control group 2 when compared to the standard control. On Day 4 of post treatment it was observed a statistically significant (p < 0.05) increase in the Hb Conc of all the test groups when compared to the positive control group. Comparatively among the test groups, groups 8 and 9 showed a significant (p < 0.05) increase in the Hb Conc when compared to test groups 4, 5, 6 and 7. This further proves that the n-hexane was the most potent of all the fraction and could contain the antimalarial agent(s) of Strophantus hispidus.

After passaging the animals with Plasmodium berghei, on Day 4 there was a significant (p < 0.05) increase in the WBC count of animals in groups (2 to 9) when compared to the negative control as can be seen in Figure 19. The bone marrow also produces and releases more white blood cells in response to infections this could be the reason for this increase in WBC. However after Day 4 of treatment with different fractions of MESH leaves, there was a significant (p ˂ 0.05) reduction in the WBC count of all the animals in the test groups (4-9) when compared to the positive control. This could be a biochemical prove that the animals are responding to treatment and recovering

159 from the ailment. Nonetheless there was a significant (p < 0.05) increase in the WBC count of the positive control when compared to the standard control. On Day 4 of post treatment the test groups, groups 4, 5 and 6 showed a significant (p < 0.05) increase in the WBC count when compared to test group 8 and 9 while test group 7, 8, and 9 showed a non-statistically significant (p > 0.05) difference in the WBC count when compared to the standard control and the negative control. This result is scientifically evident that the non-polar fractions had a better activity than the polar fraction by its ability return the WBC to normalcy on Day 4 of post-treatment. The findings of this work is in accordance with the scientific report of Bello et al. (2009) who evaluated the antimalarial activity of various fractions of Morinda lucida leaf extract and Alstonia boonei stem bark and concluded that the anti-plasmodia activity of both plants were found to reside majorly in the N-Hexane and chloroform fractions. From Figure 20 and 21, a significant (p < 0.05) increase was observed in the ALT and AST activity of all the animals in groups (2 to 9) after passaging with Plasmodium berghei when compared to the negative control. The proliferation of the liver maker enzymes in the blood is as a result of the malaria parasite attack on the hepatocytes causing a lickage of ALT and AST into the blood stream. After Day 4 of treatment with different fractions of MESH leaves, there was a significant (p ˂ 0.05) diminution in the ALT and AST activity of the animals in test groups 4, 5, 6, 7, 8 and 9 when compared to the positive control. This suggest that the various fractions had some bioactive compounds that could ameliorate the malaria condition through various mechanisms. Among the test groups there was a significant increase in the ALT and AST activity of test groups 4, 5 and 6 when compared to group 8 and 9 however test group 7 showed a non-statistically significant difference when compared to group 9. This further explains that beyond the activity that was found in the various fractions, n-hexane and dichloromethane fractions had a better activity when compared to the ethyl acetate fraction. However a non-significant (p ˃ 0.05) difference was observed between test group 9 and the standard control while test groups 4, 5, 6, 7 and 8 showed a significant (p < 0.05) increase in comparison to the standard control. The scientific reports of Pessi et al. (2004) proves that the activity of P. falciparum phosphoethanolamine methyltransferase (Pfpmt) a monopartite enzyme with a single catalytic domain that is responsible for the three-step methylation reaction of the malaria was inhibited by its product phosphocholine and by the phosphocholine analog, miltefosine an alkyl phospholipid compound. The research further illustrated how phosophocholine analogs can also inhibit parasite proliferation which

160 makes Pfpmt a potential target for malaria chemotherapy. The above described mechanism of action could be a possible mechanism of action of this fractions based on the fact that the non- polar fractions were more active than the polar fraction and they are lipophilic in nature. Nonetheless there was a significant (p < 0.05) increase in the ALT activity of the positive control when compared to the standard control. On Day 4 of post treatment it was observed a statistically significant (p < 0.05) decrease in the ALT activity of all the test groups when compared to the positive control. Among the test groups there was a significant (p < 0.05) increase in the ALT activity of test group 4 when compared to groups 7, 8 and 9 however test groups 5 and 6 showed a non-significant (p ˃ 0.05) difference when compared to groups 7 and 8. The above described proves that the activity of the fractions were sustained even on Day 4 of post-treatment and there was no possibility of a fall back, however the non-polar fractions had a better activity. From Figure 22, after passaging with Plasmodium berghei a significant (p < 0.05) increase was observed in the ALP activity of all the animals in groups (2 to 9) when compared to the negative control. High levels of ALP in the blood beyond the normal range may indicate a compromise in the hepatocytes or the blockage of the bile ducts, nevertheless in the present research this increase is as a result of breakdown of the hepatocytes by the sporozoites in the liver. After Day 4 of treatment with different fractions of MESH leaves, there was a significant (p ˂ 0.05) decrease in the ALP activity of the animals in all test groups (4-9) when compared to the positive control group 2. Among the test groups, there was a non-significant (p > 0.05) difference in ALP activity. Nevertheless test groups 5, 7, 8 and 9 showed a non-significant (p > 0.05) difference in the ALP activity when compared to the standard control however test groups 4 and 6 showed a significant (p ˂ 0.05) increase in the ALP activity when compared to the standard control. A possible mechanism could be that the different fractions contain some phytochemicals that could target the schizogonic development of the trophozoites in the liver cells. This mechanism of action was proven by Green et al. (2016) where they demonstrated that different classes of imidazopyridazines inhibits calcium-dependent protein kinase 1 CDPK1 at different stages of the asexual cycle of P. falciparum. Furthermore, they established that Class 2 imidazopyridazines with a non-pyrimidine moiety caused parasite death at the trophozoite stage, probably by inhibiting the activity of Plasmodium falciparum Heat Shock Protein 90 (pfHSP90). On Day 4 of post treatment it was observed a statistically significant (p < 0.05) decrease in the ALP activity of all the test groups when compared to the positive control. Among the test groups there was no significant (p

161

> 0.05) difference in the ALP activity. This further explains that the parasiticidal activity of the different fraction was irreversible.

As observed from Figure 23, there was a statistically significant (p < 0.05) increase in the T.BIL conc of all the animals in groups (2 to 9) when compared to the negative control after passaging with Plasmodium berghei. Bilirubin is the bye product of the normal catabolic pathway of heme however this increase is as a result of increase in the breakdown of haemoglobin by the parasite protein (merozoites). However after Day 4 of treatment with different fractions of MESH leaves, a significant (p ˂ 0.05) decrease was observed in the T.BIL conc of the animals in all test groups 4 to 9 comparatively to the positive control group 2. Nonetheless among the test groups, test groups 4 and 5 showed a significant (p ˂ 0.05) increase in the T.BIL conc when compared to test groups 8 and 9. A possible mechanism of action for this activity could be that the fractions had some bioactive compounds that binds to free heme which is toxic to the parasite thus inhibiting the formation of hemozoin consequently resulting to mortality of the malaria parasites which is one of the main mechanism of action of the first set of antimalarials chloroquine (CQ) and quinine. CQ and quinoline antimalarial drugs are thought to kill parasites by inhibiting the process of crystallization necessary to detoxify ferriprotoporphyrin IX (FP) into hemozoin (HZ) (Pandey et al., 2001 and Bray et al., 2006). However the n-hexane fraction showed a better activity and this could suggest that the n-hexane had more active compounds that could act in synergy at other sites such as the erythrocytic asexual reproduction of the malaria parasites thus reducing the parasitic load of the animals in the treatment group which will in turn reduce the breakdown of heme an formation of bilirubin. The above mechanism was described in the works of Omodeo-Salè et al. (2009) where two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM- 1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Nevertheless there was a significant (p < 0.05) increase in the T.BIL conc of test groups 4 and 5 when compared to the standard control. All the same the positive control group showed a significant (p < 0.05) increase in the T.BIL conc

162 when compared to the standard control. On Day 4 of post treatment it was observed still a statistically significant (p < 0.05) decrease in the T.BIL conc of all the test groups when compared to the positive control. Among the test groups, group 9 showed a significant (p < 0.05) decrease in the T.BIL conc when compared to groups 4 and 6. This further proves the efficacy of the n-hexane fraction above other fractions and suggest the presence of the major bioactive compounds in it.

From Figure 24, it was observed that after Day 4 of treatment with different fractions of MESH leaves there was a significant (p ˂ 0.05) decrease in the MDA conc of animals in all test groups (4 to 9) when compared to the positive control. Low levels of MDA signifies low or minimal lipid peroxidation in the treatment group and vice versa. Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) are the major phospholipids of cellular membranes. Scientific reports from Mi-Ichi et al. (2006) demonstrated inhibition of lipid peroxidation and membrane stabilization effect to impede Plasmodium falciparum activity because it utilizes a broad range of serum-derived fatty acids with limited modification for their growth. More so these two phospholipids represent 40–50% and 35–40% of the total phospholipids in P. falciparum (Vial and Ancelin, 1998) and has been proven to be the vital phospholipids in the Kennedy pathway (a major asexual reproduction pathway) of the parasites as demonstrated by Lykidis and Jackowski, (2001) thus inhibition of lipid peroxidation will resultantly cause death of the malaria parasites. However a non-significant (p ˃ 0.05) difference was observed in the test groups 8 and 9 (that received n-hexane fraction) in comparison to the standard control. This suggests that apart from inhibition of lipid peroxidation that the bioactive compounds in the n-hexane fraction were able to move into affected red cells through facilitated diffusion or active transport by organic solute carriers such as parasite aquaglyceroporin, PfAQP, at the parasite plasma membrane (Hansen et al., 2002; Hediger et al., 2004). On Day 4 of post treatment it was observed a statistically significant (p < 0.05) decrease in the MDA conc of all the test groups when compared to the positive control group. However among the test groups, test groups 4, 5, 6 and 7 showed a significant (p < 0.05) increase in the MDA conc when compared to group 9 however group 8 showed a non-significant different when compared to group 9. This further establish the fact that the bioactive compounds is inherent in the n-hexane fraction that was given to group 8 and 9 treated animals.

163

From figures 25 and 26, it was observed that after fourth day of treatment with different fractions of MESH leaves there was a significant (p < 0.05) increase in the SOD and catalase activity of all the test groups when compared to the positive control. Plants are known to be a major source of antioxidants especially those that are rich in phenols and flavonoids which makes them efficacious against several ailments (Maldini et al ., 2011). Phytochemical analysis of the different fractions revealed the presence of flavonoids and phenols to be moderately present in the dichloromethane and n-hexane fraction and slightly present in the ethyl acetate fraction. In malarial conditions several activities of the malaria parasites such as breakdown of haemoglobin, free heme (fe2+) and conversion of heme to hemozoin are known to generate a lot of reactive oxygen and nitrogen species (ROS and RNS) in the body (Percário et al ., 2012). Thus moping of free radicals is an important mechanism in the treatment of malarial. In this study a one of the possible mechanism of action could be mopping up of free radicals generated in the body by the malaria parasites. However test groups 4 and 6 showed a significant (p < 0.05) decrease in the catalase activity when compared to group 8 and 9. This further suggest that the n-hexane fraction contained more antioxidants than other fractions thus the significant increase. Nevertheless there was a significant (p < 0.05) decrease in the SOD activity of the positive control when compared to the standard control. On Day 4 of post treatment it was observed still a statistically significant (p < 0.05) increase in the SOD activity of all the test groups when compared to the positive control. Comparatively, test groups 7, 8 and 9 showed a significant (p > 0.05) increase in the SOD activity when compared to group 4, 5 and 6 which illustrates that the n-hexane and the dichloromethane were more active than the ethyl acetate fraction.

From Figure 27 after fourth days of treatment with different fractions of MESH leaves there was a significant (p < 0.05) decrease in the hemozoin conc of all the animals in groups (4 to 9) when compared to the positive control. Haemozoin the malaria pigment is known to be in high concentration during plasmodium falciparum infestation in the human body. Scientific reports of Slater and Cerami, (1992) showed that antimalarial drugs such as Chloroquine and quinoline kill parasites by inhibiting the process of crystallization of heme by heme polymerase into haemozoin (HZ). Heme in the food vacuole of the parasite is considered the target of quinoline anti-malarials (Fitch and Russell, 2006). The inhibition of HZ formation leads to the accumulation of free heme in the food vacuole of the parasite which is potentially toxic to the parasite for its pro-oxidant and lytic activities (Omodeo-Salè et al., 2005). Among the test groups, test groups 8 and 9 showed a

164 significant decrease in the hemozoin conc when compared to groups 4, 5, 6 and 7. The above results suggest that reduction in hemozoin concentration in all groups could be a resultant effect of the parasiticidal and therapeutic effect of the bioactive components of the different fractions. However for fractions 8 and 9 that received n-hexane fraction showing a very low concentration of haemozoin could suggest inhibition of hemozoin formation as a major mechanism of its action against the parasite. Moreso there was neither a significant (p ˃ 0.05) increase nor decrease in the hemozoin conc of test groups 8 and 9 when compared to the standard control. Nonetheless there was a significant (p < 0.05) increase in the hemozoin conc of the positive control when compared to the standard control. On Day 4 of post treatment a similar trend was observed such that a statistically significant (p < 0.05) decrease in the hemozoin conc of all the test groups when compared to the positive control group. Also among the test groups there was a significant (p < 0.05) increase in the hemozoin conc of groups 4, 5, 6 and 7 when compared to groups 8 and 9. Nevertheless test groups 4 and 6 were significantly (p < 0.05) higher than groups 5 and 7 in the haemozoin conc. Notwithstanding test group 8 and 9 showed a non-significant (p ˃ 0.05) difference in the haemozoin concentration when compared to the standard control. These results further establishes the fact that inhibition of hemozoin formation is a major mechanism of action of the n-hexane fraction. Calcium is required at two intra erythrocytic stages of the asexual cycle of the malaria parasites during which the erythrocytes are invaded and during normal parasite development (Waserman et al., 1982; Tanabe, 1990). More so Gazarini et al. (2003) showed that reduction in extracellular calcium amounts resulted in slow decrease in parasitophorous vacuoles and that a transient fall in calcium concentration even for two hours inhibited maturation of the parasites within the erythrocytes. From figure 28 after fourth days of treatment with different fractions of MESH leaves there was a significant (p < 0.05) decrease in the plasma calcium (PC) ion of all the animals in groups (4 to 9) when compared to the positive control. This result is in consonance with the major finding of the study carried out by Nwodo et al. (2010) where they stated that antimalarial treatment causes the plasma calcium concentration to fall in humans hence reduction in calcium concentration is a major mechanism by which membrane stabilization is achieved. In the present study groups 8 and 9 that received n-hexane fraction showed a significant decrease in the PC ion when compared to other treatment groups this could suggest that the n-hexane fraction was acting via the two mechanisms which are during the asexual cycle and normal parasite development as

165 reported by Withers-Martinez et al. (2014) which stated that egress of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Also minutes before egress, an essential parasite serine protease called SUB1 a calcium dependent enzyme is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion

The histo-morphological changes in the liver sections of the different groups of mice (positive/untreated control, negative/normal control, standard control and treated groups) are shown in Plates 1-9. There is infiltration of mononuclear cells (inflammatory cells) around the portal tract (PT) (portal hepatitis) (white arrow) in groups 2, 4, 5, 6, 7 and 8 while group 1, 3 and 9 shows normal PT and hepatocytes (black arrow). The severity of inflammatory cells infiltration is more in 2, 4, 6 than 5, 7 and 8. Histopathological studies on the hepatic tissues of the treated mice showed lesser inflammatory cells around the portal areas than the untreated. Results obtained from the histopathological studies further support the efficacy of the plant extract on Plasmodium berghei infection since tissue biopsy may be a valuable tool to establish diagnosis when other diagnostic methods are inconclusive considering the fact that histopathological alterations due to malarial infection in the liver are specific (kamal, 1996 and Shittu et al., 2011). Micrograph of treatment groups shows progressive clearance of K¨upffer’s cells-laden malaria pigment.

The liver was congested with black pigmentation as a result of haemoglobin metabolism by the parasite which leads to the production of hemozoin which consists of iron and protein moiety. The iron porphyrin complex has been reported to be phagocytized and processed by the macrophages in the tissues resulting in the dark pigmentations on the liver (Baheti et al., 2003). The above result is in consonance with the works of Shittu et al. (2011) where the anti-malaria effect of the ethanolic stem bark extracts of Ficus platyphylla Del was evaluated.

The spleen is the largest secondary immune organ in the body and is responsible for initiating immune reactions to blood-borne antigens and for filtering the blood of foreign material and old or damaged red blood cells. These functions are carried out by the 2 main compartments of the spleen, the white pulp (including the marginal zone) and the red pulp, which are vastly different in their architecture, vascular organization, and cellular composition (Cesta, 2006). The changes in the morphology of spleen in the different groups 1-9 of mice (positive/untreated control, negative/normal control, standard control and treated groups) are presented in Plates 10 – 18. The

166 photomicrograph of spleen sections from experimental groups 1 -9 showed areas of hyperplastic white pulp (wp) groups 2, 3, 4, 5, 6, 7 and 8. Note hematopoietic blast cells (signs of blood formation) in group 1, 3 and 9 (white arrows). Plates 11-18 showed different degrees of hemosiderosis which is a sign of increase in the breakdown of haemoglobin, however group 2 showed signs of severe hemosiderosis while the treatment groups showed mild hemosiderosis. The above results is in consonance with works of Mubaraki et al. (2016).

Table 13 shows the results of the invitro cytotoxicity test using brine shrimp lethality assay (BSLA) where the calculated LC50 using probit analysis was 245.5 µg/ml. Significant lethality (LC50) of the crude plant extracts with values less than 1000 ppm or μg/mL using brine shrimp lethality assay (BSLA) is indicative of the presence of potent cytotoxic, antitumor agents and probably insecticidal compounds which warrants further investigation Patil and Magdum, (20120. However for the present study the LC50 was done to determine a safe dose for the in vitro antimalarial studies using the n-Hexane partition that was the most active.

The n-Hexane partition was subjected to silica gel column chromatography and was eluted with n- Hexane, acetone and methanol in increasing polarity to obtain 95 fractions which were combined based on their TLC profiles as shown in table 14 to get 9 fractions.

From Figure 29 TLC profiles of the different fractions showed that fractions 2, 3, 4 and 5 had similar chromatograms however fraction 1 and 6 had a unique chromatograms. In the same vein fractions 7, 8, and 9 showed similar chromatogram. This could be explained that the fractions with similar chromatograms had similar or same compounds while the fractions with unique or different chromatograms had different compounds in them.

This process of hemozoin formation is vital for Plasmodium falciparum survival in hosts and can be reproduced in vitro with hematin under acidic conditions, leading to the formation of beta- hematin (BH), a crystal product showing physicochemical properties identical to those of hemozoin (Pagola et al ., 2000). Table 14 showed the different invitro antimalarial activity of the different fractions using beta hematin inhibitory assay (BHIA). Fraction 1 showed the least activity while fractions 2 to 6 hade moderate activity however the highest activity was found in fractions 7 to 9. Nevertheless fraction 9 had the highest activity. There are two prominent mechanisms known with antimalarial compounds inhibiting heme polymerization; alteration of polymerization conditions and sequestration of hemin to form toxic drug-hemin complexes as in the case of

167 artemisinin (Pandey et al., 1999). The former is commonly ascribed to interactions between the compounds present in the fractions and the haem electronic systems; oxo-bridge between hematin ferric iron and the functional groups of the compounds present in the fractions (Adams et al., 1996). The different percentage inhibition suggests that the compounds present had varying binding constant and bond energy with hematin, which vary with factors such as functional group, affinity, pH, temperature and medium which govern the reaction. This could suggest that the compounds present in fractions 7-9 had a higher binding constant to hematin than the other compounds present in the other fractions. Based on the similarity in the chromatogram and also in anti-malarial activity of fraction 7-9 only fraction 9 was used in preparative chromatography to obtain two Active Compound and B. Both compounds where subjected to invitro antimalarial activity using beta hematin inhibitory assay (BHIA). However Active Compound was the most effective and was subjected infrared spectroscopy, 1H and 13C NMR to determine the structure of the active compound.

-1 The IR spectrum showed absorption bands at 2929.7 cm due to C-H stretch of CH2 (asymmetrical) -1 of an alkane, 2959.5 cm which is due to C-H stretch of CH3 (asymmetrical), 2858.9, 1461.1 and -1 -1 1379.1cm , 2858.9 cm is due to C-H stretch of CH3 (symmetrical). Absorptions bands at 1461.1 cm-1 is due to C-O of cyclic Active Compoundnd 1379.1 cm-1 which is due to germinal dimethyl C-H.

1 The HNMR spectrum showed the presence of six methyl’s at δH one doublet at δH 0.83, 0.84,

0.86, 0.92, 0.96, and 0.98. The carbonylic proton resonance at δH 3.67 was centered at C3 as a doublet (1H, d, J=2.0, 6.0 Hz) of a triterpenoid nucleus (Mouffok et al., 2012) due to the coupling constants which revealed β- and equatorial configuration to an hydroxyl group at C3. The olefinic proton resonance was observed at δH 7.26 (1H, d), while proton resonance at δH 4.39 (1H, 5.0, 10.0 Hz) could be attributed to β-O, by comparison with 3-O-[ β- glucopyranoyl] –β-Sitosterol (Sultana and Alolayan, 2007).

The 13CNMR spectral analysis revealed the presence of 35 carbon signals including the 29 carbon signals due to the triterpenoid skeleton. The oxygenated (quaternary) carbon signal at 73.96, 77.38, 70.69 and 76.31 are due to hydroxyl of glucose sugar moiety (Mouffok et al., 2012). Carbon signal at δC 146.67 was due to olefinic carbon and carbon signals at δC 46.27 is due to influence of germinal methyl carbons.

168

The APT experiment further confirmed the multiplicities of the carbon atoms which revealed 3 quaternaries, 14 methine, 12 methylene and 6 methyl’s. With the APT, it further supported the total number of carbon in the 13C NMR of the isolated compound.

Utilization of IR spectra, 1H and 13C NMR and in comparison with literature data allowed us to propose the structure as Beta-sitosterol-d-glucoside (3-O-[ β- glucopyranoyl] –β-Sitosterol) in Figure 36.

Beta-sitosterol-D-glucoside or Sitosterol beta-d-glucoside belongs to stigmastanes and derivatives class of compounds. Those are sterol lipids with a structure based on the stigmastane skeleton, which consists of a cholestane moiety bearing an ethyl group at the carbon atom C24. Sitosterol beta-d-glucoside is practically insoluble (in water) and a very weakly acidic compound (based on its pKa). Beta-sitosterol-D-glucoside has other family names such as sitosterol 3-O-β-D- glucopyranoside, Sitogluside or daucosterol. It has been previously isolated but not tested for antiplasmodial activity by Sultana, and Afolayan, (2007) from Arctotis arctotoides and Ramiarantsoa et al. (2008) from the leaves of Ravanda madagascariensis. Nevertheless this is the second time based on scientific reports that the compound is being ascribed to have anti-plasmodial activity. The first was by Zofou et al. (2013) where the compound exhibited high anti-plasmodial activity against Chloroquine-sensitive and resistant strains of P. falciparum and the second most active compound behind methyl 3,4,5-trihydroxybenzoate.

In silico pharmacokinetics study using lipinski’s rule of five revealed that the isolated compound had two violations which are high molecular weight and low lipophilicty which contributed to its low solubility. Structural modifications was done by first displacing the glucose molecule attached to the steroid to see if we can generate a structure which has no violation of the lipinski’s rule of five, however we still had one violation of the lipinski rule in the structural modification 1 (MOD 1) which is low lipophilicity thus poor solubility. However in the MOD 2 with the displacement of the akyl group only we had zero violation high lipophilicity and high solubility thus we could say that the akyl group was responsible for the violation of lipophilicity (log p o/w) of the Active Compound which is poor solubility. In the other modifications we substituted the akyl group with an alkene, a hydroxyl group and sodium respectively and thus we still had zero violation and an improve hidrogen bond donor aceptor ratio. Thus conclusively from the insilico pharmacokinetics studies we deduce that the compound despite being active violated lipinski rule of 5 based on the

169 molecular weight and the long akyl was responsible for these two violations cause the removal of the akyl group gave us a zero violation making the molecule fit to go through clinical trials.

168 CONCLUSION The results obtained from this research work suggest that the methanol extract of Strophantus hispidus has antimalarial properties, however, further investigation into the active principle via sequential partitioning and in vivo antimalarial studies showed the ability of the n-hexane fraction in boosting packed cell volume, red blood cell count, haemoglobin concentration while inhibiting haemozoin formation and plasma calcium ion concentration. Column chromatography, in vitro antimalarial studies, tin layer chromatography and spectroscopic studies revealed active compound as beta – Setosterol-d-glucoside. Beta-setosterol-d-glucose as the most active compound showed activity against β-hematin synthesis and could take a lead in the discovery of new anti-malarial agent or even work in combination therapy with artemisinin just as the current Artemisinin Combination Therapies (ACTs). 169 RECOMMENDATIONS 1. Antimalarial studies should be carried out on the isolated compound in combination with Artemisinin since ACTs have been proven to be highly effective anti malarials. 2. Quantitative structural activity relationship should be carried out on the isolated Active Compound and the various modifications made via in silico pharmacokinetics study.

170

REFERENCES

Adams, P. A., Berman, P. A., Egan, T. J., Marsh, P. J. and Silver, J. (1996). The iron environment in heme and heme–antimalarial omplexes of pharmacological interest. Journal of Inorganic Biochemistry, 63: 69–77.

Ademowo, O. G., Nnej, C. M. and Adedapo, A. D. A. (2006). In vitro antimalarial activity of methylene blue against field isolates of Plasmodium falciparum from children in Southwest Nigeria. Indian Journal of Medical Research, 126: 45-49.

Adjuik M, Babiker A, Garner P, Olliaro P, Taylor W, White N (2004). International Artemisinin Study Group: Artesunate combinations for treatment of malaria: metal analysis. Lancet, 363: 9-17.

Agbaje, E. O. and Fageyinbo, M. S. (2011). Evaluating anti-inflammatory activity of aqueous root extract of Strophanthus hispidus (D.C) (Apocynaceae). International Journal of Applied Research in Natural Products, 4 (4): 7–14.

Agbaje, E. O. and Fageyinbo, M. S. (2014). Anti-Nociceptive activity of fractionated root extract of Strophanthus hispidus DC (Apocynaceae). Journal of Natural Remedies, 14 (2): 164- 173.

Aggarwal, B. B. (2009). Targeting inflammatory pathways for chronic diseases by phytochemicals derived from spices, fruits, vegetables, and traditional remedies. Acta Horticulturae, 841, 33–46.

Ali, A. N., Al-rahwi, A. K. and Lindequist, U. (2004). Some medicinal plants used in Yemeni herbal medicine to treat malaria. African Journal of Traditional Medicine, 1: 72– 76.

Alshawsh, M. A., Mothana, R. A., Al-shamahy, H. A., Alsllami, S. F., and Lindequist, U. (2009). Assessment of antimalarial activity against Plasmodium falciparum and phytochemical screening of some Yemeni medicinal plants. Evidence-based Complementary and Alternative Medicine, 6(4): 453-456.

Arora, D. R. and Arora. B (2005). Protozoa. Medical Parasitology 2nd Edn, CBS publishers, India. pp. 67-98

Ashley, E. A., Stepniewska, K., Lindegårdh, N., McGready, R., Annerberg, A., Hutagalung, R., Singtoroj, T. Hla, G., Brockman, A., Proux, S., Wilahphaingern, J., Singhasivanon, P., White, N. J. and Nosten, F. (2007). Pharmacokinetic study of artemether–lumefantrine given once daily for the treatment of uncomplicated multidrug‐resistant falciparum malaria. Tropical medicine and international health, 12(2): 201-208.

Atamna, H. and Ginsburg, H. (1995). Heme degradation in the presence of glutathione A proposed mechanism to account for the high levels of non-heme iron found in the membranes of haemoglobinopathic red blood cells. Journal of Biological Chemistry, 270(42): 24876- 24883.

171

Augustin, J. M., Kuzina, V., Andersen, S. B. and Bak, S. (2011). Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry, 72(6): 435-457.

Ayoola, G. A., Folawewo, A. D., Adesegun, S. A., Abioro, O. O., Adepoju-Bello, A. A. and Coker, H. A. B. (2008). Phytochemical and antioxidant screening of some plants of apocynaceae from South West Nigeria. African Journal of Plant Science, 2 (9): 124-128.

Azas, N., Laurencin, N., Delmas, F., Di-Giorgio, C., Gasquet, M., Laget, M. and Timon-David, P. (2002). Synergistic in vitro antimalarial activity of plant extracts used as traditional herbal remedies in Mali. Parasitology Research, 88: 165-171.

Babson, L. A. (1965). Alkaline phosphatase. Clinical Chemistry, 2: 789-795.

Babson, L. A., Greeley, S. J., Coleman, C. M. and Philips, G. D. (1966). Alkaline phosphatase determination. Clinical Chemistry, 12: 482- 490.

Baheti, R., Laddha, P. and Gehlot, R. S. (2003). Liver involvement in falciparum malaria–A Histo- pathological analysis. Journal, Indian Academy of Clinical Medicine, 4(1): 34-8.

Bancroft, J. D. and Gamble, M. (2002). Theory and Practice of Histological Techniques. Churchill Livingstone, Edinburgh, pp. 16-64.

Barbosa, E. A. (2014). An overview on the biological and pharmacological activities of saponins. International Journal of Pharmacy and Pharmaceutical Sciences, 6(8): 47-50. Basilico, N., Pagani, E., Monti, D., Olliaro, P. and Taramelli, D. (1998) A microtitre-based method for measuring the haem polymerization inhibitory activity (HPIA) of antimalarial drugs. Journal of Antimicrobial Chemotherapy, 42, 55–60.

Basore, K., Cheng, Y., Kushwaha, A. K., Nguyen, S. T. and Desai, S. A. (2015). How do antimalarial drugs reach their intracellular targets? Frontiers in pharmacology, 6(91): 1-7.

Beentje, H. J. (1982). A monograph on Strophanthus DC. (Apocynaceae). Wageningen, Netherlands, pp. 55-67.

Bello, I. S., Oduola, T., Adeosun, O. G., Omisore, N. O. A., Raheem, G. O. and Ademosun, A. A. (2009). Evaluation of antimalarial activity of various fractions of Morinda lucida leaf extract and Alstonia boonei stem bark. Global Journal of Pharmacology, 3(3): 163-165.

Billker, O., Lindo, V., Panico, M., Etienne, A. E., Paxton, T., Dell, A., Rogers, M. and Morris, H. R. (1998). Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature, 392(6673): 289-307.

Bjelakovic, G., Nikolova, D., Gluud, L., Simonetti, R. and Gluud, C. (2007). Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: Systematic review and meta-analysis. The Journal of the American Medical Association, 297(8): 842-857.

172

Blackwell, G. J., Carnuccio, R., Di Rosa, M., Flower, R. J., Parente, L. and Persico, P. (1980). Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature, 287(5778): 147-149.

Bloland B. P. (2001). Drug Resistance in Malaria. World Health Organization Geneva, Switzerland. pp 51-62.

Bohle, D. S., Dinnebier, R. E., Madsen, S. K. and Stephens, P. W. (1997). Characterization of the products of the heme detoxification pathway in malarial late trophozoites by X-ray diffraction. Journal of Biological Chemistry, 272(2): 713-716.

Bohle, D. S., Dimebier, R. E. and Madsen, S. K. (2005). Characterization of the products of the heme detoxification pathway in malarial late trophozoites by x-ray diffraction. Journal of Biological Chemistry, 272: 713 – 716.

Boivin, M. J. (2002). Effects of early cerebral malaria on cognitive ability in Senegalese children. Journal of Developmental and Behavioral Pediatrics, 23(5): 353–364.

Bourdy, M., Willcox, L., Ginsburg, H., Rasoanaivo, P., Graz, B. and Deharo, E. (2008). Ethnopharmacology and malaria: New hypothetical leads or old efficient antimalarials? International Journal for Parasitology, 38: 33–41.

Bray, P. G., Mungthin, M., Hastings, I. M., Biagini, G. A., Saidu, D. K., Lakshmanan, V., Johnson, D. J., Hughes, R. H., Stocks, P. A., O’Neil, P. M., Fidock, D. A. and Ward S. A. (2006). PfCRT and the trans‐vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX. Molecular microbiology, 62(1): 238-251.

Breman, J. (2001). The ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. American Journal of Tropical Medicine and Hygiene, 64: 1-11.

Bruce Chwatt, L. J. (1985). Essential Malariology. 2nd Edn, Heinemanna. London, pp. 61-270.

Builders, M., Alemika, T. and Aguiyi, J. (2014). Antimalarial activity and isolation of phenolic compound from parkia biglobosa. IOSR Journal of Pharmacy and Biological Sciences, 9(3): 78-85.

nd Burkill, H. M. (2000). The Useful Plants of West Tropical Africa. 2 Edn. Kew, Richmond, United Kingdom, pp. 129-136.

Cesta, M. F. (2006). Normal structure, function, and histology of the spleen. Toxicologic pathology, 34(5), 455-465.

Chanda, P., Hamainza, B., Mulenga, S., Chalwe, V., Msiska, C. and Chizema-Kawesha, E. (2009). Early results of integrated malaria control and implications for the management of fever in under-five children at a peripheral health facility: A case study of Chongwe rural health centre in Zambia. Malaria Journal, 8: 49-56.

173

Cheesbrough, M. (2000). District laboratory practice manual in Tropical Countries pt 2. Cambridge University press. pp: 45-62.

Cheesbrough, M. (2008). Counting white cells and platelets in district laboratory practice in tropical countries part 2. The Edinburgh: Cambridge University Press, United Kingdom. pp 314-329.

Chessbrough, M. (2005). District Laboratory Practice in Tropical Countries (Part 1). 2nd Edn. Cambridge University Press. pp. 340-349.

Choubey Vinay, Pallab Maity, Mithu Guha, Sanjay Kumar, Kumkum Srivastava, Sunil Kumar Choubey, V., Maity, P., Guha, M., Kumar, S., Srivastava, K., Puri, S. K. and Bandyopadhyay, U. (2007). Inhibition of Plasmodium falciparum choline kinase by hexadecyltrimethylammonium bromide: a possible antimalarial mechanism. Antimicrobial agents and chemotherapy, 51(2): 696-706.

Clark, E. P. and Collip, J. B. (1925). A study of the Tisdall method for the determination of blood serum calcium with a suggested modification. Journal of Biological Chemistry, 63(2), 461- 464.

Clarkson, C., Maharaj, V. J., Crouch, N. R., Grace, O. M., Pillay, P., Matsabisa, M. G., Bhagwandin, N., Smith, P. J. and Folb, P. I. (2004). In vitro antiplasmodial activity of medicinal plants native to or naturalised in South Africa. Journal of ethnopharmacology, 92(2-3): 177-191.

Connolly, J. D. and Hill, R, A. (1992) Dictionary of Terpenoids. Chapman and Hall. New York, pp. 143-156.

Cowman, A. F. (2006). Invasion of Red Blood Cells by Malaria Parasites. Cell, 124: 755–766.

Cox, F. E. (2010). History of the discovery of the malaria parasites and their vectors. Parasites and vectors, 3(1): 5-12.

Crawford, M. J. (2014). An Empire’s xtract: Chemical manipulations of Cinchona bark in the eighteenth-century spanish atlantic world. Osiris, 29(1), 215-229. Cui, L., Mharakurwa, S., Ndiaye, D., Rathod, P. K. and Rosenthal, P. J. (2015). Anti-malarial Drug Resistance: Literature Review and Activities and Findings of the ICEMR Network. The American Society of Tropical Medicine and Hygiene, 93(3): 57-58

Curtis, J. R., Westfall, A. O., Allison, J., Bijlsma, J. W., Freeman, A., George, V. and Saag, K. G. (2006). Population‐based assessment of adverse events associated with long‐term glucocorticoid use. Arthritis Care and Research, 55(3): 420-426.

Dacie, J. V. and Lewis, S. M. (1991). Practical Haematology. 7th Edn. Churchill Livingstone, Edingburgh, pp. 535-544.

174

Dawet, A. I. Anyanwu, G., Dede, M. P., Uzoigwe N.R. and Onyekwelu, N.A. (2012). In vivo antimalarial activity of the ethanolic leaf extract of Hyptis suaveolens poit on Plasmodium berghei in Mice. International Journal. Biological. Chemical. Sciences. 6(1): 117-127.

Dembitsky, V. (2005). Astonishing diversity of natural surfactants: 5 biologically active glycosides of aromatic metabolites. Lipids, 40: 869-900.

Demirdöven, N., Cheatum, C. M., Chung, H. S., Khalil, M., Knoester, J. and Tokmakoff, A. (2004).Two-dimensional infrared spectroscopy of antiparallel beta-sheet secondary structure. Journal of the American Chemical Society. 126 (25): 7981–90.

Derome, A. E. (2013). Modern NMR techniques for chemistry research (Vol. 6). Elsevier. pp. 103- 7

Dibua, U. M. E., Kalu, A., Attama, A. A., Esimone, C.O. and Eyo, J. E. (2013). In vivo and in vitro evaluation of the inhibitory effect of some medicinal plant extracts on hemozoin concentration. Animal Research International, 10(2): 1699 – 1712.

Doughari, J. H., Human, I. S., Bennade, S. and Ndakidemi, P. A. (2009). Phytochemicals as chemotherapeutic agents and antioxidants: Possible solution to the control of antibiotic resistant verocytotoxin producing bacteria. Journal of Medicinal Plants Research, 3(11): 839-848.

Drakenberg, T., Andersson, T., Forsen, S. and Wieloch, T. (1984). Calcium ion binding to pancreatic phospholipase A2 and its zymogen: a calcium-43 NMR study. Biochemistry, 23(11): 2387-2392.

Drury, R. A., Wallington, A. and Cameroun, S. R. (1967). In: Carlleton’s Histological Techniques. 1st Edn. Oxford University Press, New York. pp 1- 420.

Eckman, J. R., Modler, S., Eaton, J. W., Berger, E. and Engel, R. R. (1977). Host heme catabolism in drug-sensitive and drug-resistant malaria. The Journal of laboratory and clinical medicine, 90(4): 767-770.

Egan, T. J., Hunter, R., Kaschula, C. H., Marques, H. M., Misplon, A. and Walden, J. (2000). Structure− function relationships in aminoquinolines: effect of amino and chloro groups on quinoline− hematin complex formation, inhibition of β-hematin formation, and antiplasmodial activity. Journal of medicinal chemistry, 43(2): 283-291.

Eisenrich, W., Schwarz, M. and Cartayrade, A. (1998) The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chemistry and Biology, 5 (9): 221– 233.

Elfawal, M. A., Towler, M. J., Reich, N. G., Weathers, P. J. and Rich, S. M. (2015). Dried whole- plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin. Proceedings of the National Academy of Sciences, 112(3): 821-826.

175

Elujoba, A. A. (1998). The role of pharmacognosy in phytotherapy, the challenges of our time. Nigerian Journal of Natural Products and Medicine, 2(1): 5-8. .

Elujoba, A. A., Odeleye, O. M. and Ogunyemi, C. M. (2005). Traditional medicine development for medical and dental primary health delivery systems in Africa. African Journal of Traditional Medicine, 21:46-61.

Ersoz, T., M. Ozalp, M. Ekizoglu, M. and Calis. I. (2002). Antimicrobial Activitiеs of the Phenylethanoid Glycosides from Scutellaria galericulata. Hacettepe University Journal of the Faculty of Pharmacy publishes original Turkey, 22: 1-8. Etkin, N. L. (2003). The co-evolution of people, plants, and parasites: biological and cultural adaptations to malaria. Proceedings of the Nutrition Society, 62(2): 311-317.

Ferreira, J. F., Luthria, D. L., Sasaki, T. and Heyerick, A. (2010). Flavonoids from Artemisia annua L. as antioxidants and their potential synergism with artemisinin against malaria and cancer. Molecules, 15(5): 3135-3170.

Firn, R. (2010). Nature's Chemicals: The natural products that shaped our world. Oxford University Press on Demand. pp. 25-30.

Fitch, C. D. and Russell, N. V. (2006). Accelerated denaturation of haemoglobin and the antimalarial action of chloroquine. Antimicrobial agents and chemotherapy, 50(7): 2415- 2419.

Foley, M. and Tilley, L. (1998). Quinoline antimalarials: Mechanisms of action and resistance and prospects for new agents. Pharmacology and therapeutics, 79(1): 55-87. Foote, R. H. and Hare, E. (2001). Blood catalase and haematocrit values in a breeding colony of Dutch-belted rabbits. Laboratory Animals, 35(2): 140–146.

Francis, G., Kerem, Z., Makkar, H. P. and Becker, K. (2002). The biological action of saponins in animal systems: a review. British journal of Nutrition, 88(6): 587-605.

Fuchs, H., Bachran, D., Panjideh, H., Schellmann, N., Weng, A., Melzig, M. F. and Bachran, C. (2009). Saponins as tool for improved targeted tumor therapies. Current drug targets, 10(2): 140-151.

Galm, U. and Shen, B. (2007). Natural product drug discovery: The times have never been better. Chemical Biology, 14: 1098–1104.

Galsky, A. G., Wilsey, J. P. and Powell, R. G. (1980). Crown gall tumor disc bioassay: a possible aid in the detection of compounds with antitumor activity. Plant Physiology, 65(2): 184- 185.

176

Gazarini, M. L., Thomas, A. P., Pozzan, T. and Garcia, C. R. (2003). Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. The Journal of cell biology, 161(1): 103-110.

George, T. W., Niwat, C., Waroonphan, S., Gordon, M. H., Lovegrove, J. A. and Paterson, E. (2009) Effect of chronic and acute fruit and vegetable juice consumption on cardiovascular disease risk factor. Acta Horticulturae, 841: 201–206.

Gilles, H. M., Warrell, D. A. and Edward, A. (1993). Diagnostic Methods in Malaria. Bruce Chwatt’s Essential Malariology 3rd ed London pp 78-95.

Gottlieb, H. E., Kotlyar, V. and Nudelman, A. (1997). NMR chemical shifts of common laboratory solvents as trace impurities. The Journal of organic chemistry, 62(21), 7512-7515.

Green, J. L., Moon, R. W., Whalley, D., Bowyer, P. W., Wallace, C., Rochani, A., Nageshan, R. K., Howell, S. A., Grainger, M., Jones, H. M., Ansell, K. H., Chapman, T. M., Taylor, D. L., Osborne, S. A., Baker, D. A., Tatu, U. and Holdera A. A. (2016). Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development. Antimicrobial Agents and Chemotherapy, 60(3): 1-12.

Greenbaum, D. C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky, K. F., Engel, J. DeRisi, J., Holder, A. A. and Bogyo, M. (2002). A role for the protease falcipain 1 in host cell invasion by the human malaria parasite. Science, 298(5600): 1-13.

Greenwood, B. and Mutabingwa, T. (2002). Malaria in 2002. Nature, 415(6872): 670-688.

Greenwood, B. M., Bojang, K., Whitty, C. J. and Targett, G. A. (2005). Malaria. Lancet, 365: 1487-1498.

Hansen, M., Kun, J. F., Schultz, J. E. and Beitz, E. (2002). A Single, Bi-functional Aquaglyceroporin in Blood-stage Plasmodium falciparum Malaria Parasites. Journal of Biological Chemistry, 277(7): 4874-4882.

Hans-Walter, H. and Fiona, H. (2005). Plant Biochemistry. 3rd Edn. Academic Press, San Diego. pp. 403-413.

Harborne, B. I. (1984). Phytochemical Methods: A Grade to Modern Technology of Plant analysis. 2nd Edn. Chapman and Hall, New York, pp. 88-185.

Harborne, J. B. (1998). Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 3rd Edn. Chapman and Hall, London, pp. 40-138.

Harborne, J. B. and Baxter. H. (1999). The Handbook of Natural Flavonoids, Volume 1 and 2. John Wiley and Sons. Chichester, pp. 50-158.

177

Hart, G. D. (2001). Descriptions of blood and blood disorders before the advent of laboratory studies. British Journal of Haematology, 115(4): 719-728.

Haslam, E. (1996). Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. Journal of Natural Products, 59(2): 205-215.

Hay, S. I. and Snow, R. W. (2006). The malaria atlas project: Developing global maps of malaria risk. PLoS Medicine, 3(12): 473-484.

Hay, S., Guerra, C., Tatem, A., Noor, A. and Snow, R. (2004). The global distribution and population at risk of malaria: past, present, and future. Lancet 4(6): 327-336.

Hediger, M. A., Romero, M. F., Peng, J. B., Rolfs, A., Takanaga, H. and Bruford, E. A. (2004). The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. European Journal of physiology, 447(5): 465-468.

Hijora, E., Nistar, F. and Sipulova, A. (2005). Changes in ascorbic acid and malonaldehyde in rats after exposure to mercury. Bratisl Lek Listy, 106(8-9): 248-251.

Hisem, D., Hrouzek, P., Tomek, P., Tomšíčková, J., Zapomělová, E., Skácelová, K. Lukešová A. and Kopecký, J. (2011). Cyanobacterial cytotoxicity versus toxicity to brine shrimp Artemia salina. Toxicon, 57(1): 76-83.

Hoareau, L. and Dasilva, E. J. (1999). Medicinal plants: a re-emerging health aid. Electronic Journal of Biotechnology, 2(2): 56-69.

Hsieh, Y. and Korfmacher W. A. (2006). Increasing speed and throughput when using HPLC- MS/MS systems for drug metabolism and pharmacokinetic screening. Current Drug Metabolism, 7(5): 479–89.

Huy, N.T., Serada, S., Trang, D.T., Takano, R., Kondo, Y., Kanaori, K., Tajima, K., Hara, S. and Kamei, K. (2003). Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2. Journal of Biochemistry, 133(5): 693–698.

Idro, R., Otieno, G., White, S., Kahindi, A., Fegan, G., Ogutu, B., Mithwani, S., Maitland, K., Neville, B. G. and Newton, C. R. (2007). Decorticate, decerebrate and opisthotonic posturing and seizures in Kenyan children with cerebral malaria. Malaria Journal 4:57-69.

Iyawe, H. O. T. and Onigbinde, A. O. (2009). The role of ascorbic acid in the treatment of Plasmodium berghei infected mice. African Journal of Biochemistry Research, 3(11): 375- 378.

Jendrassik, L. and Gróf, P. (1938) Simplified photometric methods for the determination of bilirubin. Biochem Zschr, 297 (8): 1 - 9.

Joshua. P. E., Ugwuoke M. C, Nwodo O. F. C. and Uroko R. I. (2016). Haematological responses and percentage parastaemia in malaria-infected mice treated with ethanol extract of Zapoteca portoricensis roots. Biochemistry and Analytical Biochemistry, 5(1): 1-5.

178

Kain, K., Harrington, M., Tennyson, S. and Keystone, J. (1998). Imported malaria: prospective analysis of problems in diagnosis and management. Clinical Infectious Diseases, 27(1): 142-149.

Kamal, G. I. (1996). Rodney, Anderson’s Pathology, Vol. 2, Mosby, St. Louis, Mo, USA, 10th edition. pp: 125-139.

Kar, A. (2007). Pharmaocgnosy and Pharmacobiotechnology. 2nd Edn. New Age International Limted Publishres. New Delhi, pp. 332-600.

Karunamoorthi, K. (2014). Malaria vaccine: A future hope to curtail the global malaria burden. International journal of preventive medicine, 5(5): 529-538.

Kensil, C. R. (1996). Saponins as vaccine adjuvants. Critical reviews in therapeutic drug carrier systems, 13(1-2): 1-55.

Khan, H., Mushtaq, G., Amjad Kamal, M., and Ahmad, I. (2017). Glycosides as Possible Lead Antimalarial in new drug discovery: Future perspectives. Current Drug Metabolism, 18(2): 163-171.

Kibiti C, and Afolayan A. (20 16). Antifungal activity and brine shrimp toxicity assessment of Bulbine abyssinica used in the folk medicine in the Eastern Cape Province, South Africa. Bangladesh Journal of Pharmacology, 11: 469-77.

Klein, B., Read, P. A. and Babson, L. A. (1960). Rapid colorimetric method for the quantitative determination of serum alkaline phosphatase. Clinical Chemistry, 6: 269 - 275. König, G. M. (1992). Marine organisms as a source of pharmaceutically important natural substances. German pharmacist newspaper, 132(14): 673–683.

Korenromp, E., Williams, B., De Vlas, S., Gouws, E., Gilks, C., Ghys, P. and Nahlen, B. (2005). Malaria attributable to the HIV-1 epidemic, sub-Saharan Africa. Emerging Infectious Diseases, 11(9): 1410.

Kris-Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E., Hilpert, K. F., Griel, A. E. and Etherton, T. D. (2002). Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. American Journal of Medicine, 113: 715–885.

Kumar, S. and Pandey, A. K. (2013) Chemistry and Biological Activities of Flavonoids: An Overview. Hindawi Publishing Corporation. The Scientific World Journal, 16: 1-16.

Kumar, S., Mishra, A. and Pandey, A. K. (2013). Antioxidant mediated protective effect of Parthenium hysterophorus against oxidative damage using in vitro models. BMC complementary and alternative medicine, 13(1): 120-132.

179

Langhorne, J., Quin, S. J. and Sanni, L. A. (2002). Mouse models of blood-stage malaria infections: Immune responses and cytokines involved in protection and pathology. Malaria immunology, 80: 204-228. Lefevre, G., Looareesuwan, S., Treeprasertsuk, S., Krudsood, S., Silachamroon, U., Gathmann, I., Mull, R. Bakshi, R. (2001). A clinical and pharmacokinetic trial of six doses of artemether- lumefantrine for multidrug-resistant Plasmodium falciparum malaria in Thailand. The American journal of tropical medicine and hygiene, 64(5): 247-256.

Levine, R. A., Wardlaw, S. C. and Patton, C. L. (1989). Detection of haematoparasites using Quantitative Buffy Coat analysis tubes. Parasitology Today 5:132–133.

Lewis, S.M., Bain, B.J., Bates, I. and Dacie, L. (2002). Practical Haematology. 9th Edn. Churchill living stone. Edinburgh, pp. 1-668.

Lim, S. H., Darah, I. and Jain, K. (2014). Chemical properties of tannic extracts from bark of Pinus oocarpa and their use as adhesive. Cerne, 20: 47-54.

Lin, D., Xiao, M., Zhao, J., Li, Z., Xing, B., Li, X., Kog, M., Li, L., Zhang, Q., Liu, Y., Cheng, H., Qin W., Wu, H. and Chen, S. (2016). An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules, 21(1374): 1-19.

Lipinski, C. A. (2004). Lead- and drug-like compounds: the rule-of-five revolution. Drug Discovery Today: Technologies, 1(4): 337–341.

Lipinski, C. A., Lombardo, F., Dominy, B. W. and Feeney, P. J. (1997). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews, 23(1-3): 3-25.

Lipinski, C. A., Lombardo, F., Dominy, B. W. and Feeney, P. J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug. Delivery. Reviews. 46: 3–26.

Lorke, D. (1983). A new approach to practical acute toxicity testing. Archives of Toxicology, 55: 275-287. Luna, C. M. (2004). Drought controls on H2O2 accumulation, catalase (CAT) activity and CAT gene expression in wheat. Journal of Experimental Botany, 56(411): 417–423.

Luthria, D. L. (2006). Significance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods. Journal of the Science of Food and Agriculture, 86(14): 2266-2272.

Lykidis, A. and Jackowski, S. (2001). Regulation of mammalian cell membrane biosynthesis. Progress in Nucleic Acid Research and Molecular Biology, 65: 361–393.

Maldini, M., Montoro, P., Hamed, A. I., Mahalel, U. A., Oleszek, W., Stochmal, A. and Piacente, S. (2011). Strong antioxidant phenolics from Acacia nilotica: profiling by ESI-MS and

180

qualitative–quantitative determination by LC–ESI-MS. Journal of pharmaceutical and biomedical analysis, 56(2): 228-239.

Mamta, S., Jyoti, S., Rajeev, N., Dharmendra, S. and Abhishek, G. (2013). Phytochemistry of medicinal plants. Journal of Pharmacognosy and Phytochemistry, 1(6): 168-182.

Manson, P. (2004). Blood test used to investigate liver, thyroid or kidney function and disease. Pharmaceutical Journal, 272: 446-448.

Mathai, K. (2000). Nutrition in the Adult Years in Krause’s Food, Nutrition, and Diet Therapy. 10th Edn. Escott-Stump Press, Washington, D.C., pp. 274-275.

Maurer-Jones, M. A., Love, S. A., Meierhofer, S., Marquis, B. J., Liu, Z. and Haynes, C. L. (2013). Toxicity of nanoparticles to brine shrimp: An introduction to nanotoxicity and interdisciplinary science. Journal of Chemical Education, 90(4): 475-478.

McGarrey, D. J. and Croteau R. (1995) Terpenoid metabolism. Plant Cell, 7: 1015–1024.

McLaughlin, J. L. (1991). Assays for bioactivity. In: Hostettmann K (Ed). Methods in Plant Biochemistry. Academic Press, London. pp. 1-33.

Mclaughlin, J. L., Rogers, L. L. and Anderson, J. E. (1998). The use of biological assays to evaluate botanicals. Drug information journal, 32(2): 513-524.

Melzig, M. F., Bader, G. and Loose, R. (2001). Investigations of the mechanism of membrane activity of selected triterpenoid saponins. Planta Medica, 67(1): 43-48.

Merlin, L. W. (2004). Traditional Medicinal Plants and Malaria. Buckingham, Gerard Bodeker, University of Oxford England, UK: University of Oxford, pp. 125-138.

Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E., and McLaughlin, J. L. (1982). Brine shrimp: a convenient general bioassay for active plant constituents. Planta medica, 45(5): 31-34.

Michael, A. S., Thompson, C. G. and Abramovitz, M. (1956). Artemia salina as a Test Organism for Bioassay. Science (New York, NY), 123(3194): 464-472.

Mi-Ichi, F., Kita, K. and Mitamura, T. (2006). Intraerythrocytic Plasmodium falciparum utilize a broad range of serum-derived fatty acids with limited modification for their growth. Parasitology, 133(4): 399-410.

Miller, C. H., Good, M. F. and Milon, G. (1994). Malaria pathogenesis. Science, 264:1878-1883.

Mishra A., A. K. Sharma, S. Kumar, A. K. Saxena, and A. K. Pandey. (2013). Bauhinia variegata leaf extracts exhibit considerable antibacterial, antioxidant and anticancer activities, BioMed Research International, 9:,1- 10.

181

Mockenhaupt, F. P., Ehrhardt, S., Burkhardt, J., Bosomtwe, S. Y., Laryea, S., Anemana, S. D., Otchwemah, R., Cramer, J., Dietz, E., Gellert, S. and Bienzle, U. (2004). Manifestation and outcome of severe malaria in children in northern Ghana. The American journal of tropical medicine and hygiene, 71(2): 167-172.

Mockenhaupt, F. P., Rong, B., Günther, M., Beck, S., Till, H., Kohne, E. Thompson, W. N. and Bienzle, U. (2000). Anaemia in pregnant Ghanaian women: importance of malaria, iron deficiency, and haemoglobinopathies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 94(5): 477-483.

Molyneux, R. J., Nash, R. J. and Asano, N. (1996) Alkaloids: Chemical and Biological Perspectives, Vol. 11, Edn. Pergamon, Oxford, pp. 303.

Mouffok, S., Haba, H., Lavaud, C., Long, C. and Benkhaled, M. (2012). Chemical constituents of centaurea omphalotricha Coss. and Durieu ex Batt. and Trab. Record of Natural Products, 6(3): 292-295.

Mubaraki, M. A., Hafiz, T. A., Dkhi, M. A. and Al-Quraishy S. (2016). Beneficial effect of Punica granatum peel extract on murine malaria-induced spleen injury. BMC Complementary and Alternative Medicine, 16: 221-230.

Mueller, M. S., Karhagomba, I. B., Hirt, H. M., Wemarkor, E., Li, S. M. and Heide, L. (2000). The potential of Artemisia annua L. as a locally produced remedy for malaria in the tropics: agricultural, chemical and clinical aspects. Journal of Ethnopharmacology 74:487-493.

Mueller-Harvey, I. and McAllan, A. B. (1992) Tannins: Their Biochemistry and Nutritional Properties and Advances in Plant Cell Biochemistry and Biotechnology. Vol. 1 Morrison IM, Edn. JAI Press Limited, London. pp. 151-217.

Mukamel, S. (2000). Multidimensional fento second correlation spectroscopies of electronic and vibrational excitations. Annual Review of Physical Chemistry. 51(1): 691–729.

Mutabingwa, T. K., Anthony, D. and Heller, A. (2005). Amodiaquine alone, amodiaquine+sulfadoxine-pyrimethamine, amodiaquine+artesunate, and artemether- lumefantrine for outpatient treatment of malaria in Tanzanian children: a four-arm randomised effectiveness trial. Lancet, 365(9469): 1474–1480.

Naczk, M. and Shahidi, F. (2004). Extraction and analysis of phenolics in food. Journal of Chromatography A, 1054(1-2): 95-111.

Narasinga, R. (2003) Bio-active phytochemicals in Indian foods and their potential in health promotion and disease prevention. Asia Pacific Journal of Clinical Nutrition, 12(1): 9-22.

Nijveldt, R. J., Van-Nood, E., Van-Hoorn, D. E., Boelens, P. G., Van-Norren. K. and Leeuween, P. A. (2001). Flavonoids: A review of probable mechanisms of action and potential applications. American Journal of Clinical Nutrition, 74(4): 418-425.

182

Nosten, F. and White, N. J. (2007). Artemisinin-based combination treatment of falciparum malaria. American Journal of Tropical Medicine and Hygiene, 77:181-192.

Ntie-Kang, F., Onguéné, P. A., Lifongo, L. L., Ndom, J. C., Sippl, W. and Mbaze, L. M. A. (2014). The potential of anti-malarial compounds derived from African medicinal plants, part II: a pharmacological evaluation of non-alkaloids and non-terpenoids. Malaria journal, 13(1): 81.

Nwankwo, N. E., Nwodo, O. F. C., Amalunweze, A. E., Agbo K. U. and Abugu, S. C. (2015). Liver and kidney function tests and histological study on malaria parasite infected mice administered with seed extract of Picralima nitida. International Journal of Biochemistry Research and Review, 8(2): 1-14.

Nwodo, O. F. C., Omeje, E. O., Obiezue, R. N. and Ozoemena, N. F. (2010) Inhibition of plasma calcium availability may contribute to anti-malarial activity. Nigerian Journal of Biochemistry and Molecular Biology, 251(1): 39-43.

Oberlies, N. H., Rogers, L. L., Martin, J. M. and McLaughlin, J. L. (1998). Cytotoxic and insecticidal constituents of the unripe fruit of Persea americana. Journal of Natural Products, 61(6): 781-785.

Ochei, J. and Kolhatkar, A. (2008). Medical Laboratory Sciences: Theory and Practice. Tata McGraw Hill, New York. pp. 663-665. Ogbonna, D. N., Sokari, T. G. and Agomuoh, A. A. (2008). Antimalarial activities of some selected traditional herbs from Southeastern Nigeria against Plasmodium species. Research Journal of Parasitology, 3(1): 25-31.

Ogungbamigbe, T., Ogunro, P., Elemile, P., Egbewale, B., Olowu, O. and Abiodun, O. (2005). Presciption patterns of antimalarial drugs among medical practitioners in Osogbo Metropolis, South-West Nigeria. Tropical Medicine and Health 33: 201-208.

Okochi, V. I., Gbenie, G. O., Kazeem, A. A., Fagbenro-Beyioku, A. F., Igbodudu, H. E. and Arukwe, U. (1999). Effect of water extract of Tetrapleura tetraptera (Aidon) on haematological and biochemical parameters in Rats infected with Trypanosoma brucei. Nigerian Quarterly Journal of Hospital Medicine, 9(1): 66-70. Olajide, O. A., Awe, S. O. and Makinde, J. M. (1997). Pharmacological studies on Newbouldia leaves stem bark. Fitoterapia, 68: 439-443.

Oliveira, M. F., Kycia, S. W., Gomez, A., Kosar, A. J., Bohle, D. S., Hempelmann, E., Menezes, D., Vannier-Santos, M. A., Oliveira, P. L. and Ferreira, S. T. (2005). Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus. Febs letters, 579(27): 6010-6016.

Omodeo-Salè, F., Cortelezzi, L., Basilico, N., Casagrande, M., Sparatore, A. and Taramelli, D. (2009). Novel antimalarial aminoquinolines: heme binding and effects on normal or

183

Plasmodium falciparum-parasitized human erythrocytes. Antimicrobial agents and chemotherapy, 53(10): 4339-4344.

Omodeo‐Salè, F., Motti, A., Dondorp, A., White, N. J. and Taramelli, D. (2005). Destabilisation and subsequent lysis of human erythrocytes induced by Plasmodium falciparum haem products. European journal of haematology, 74(4): 324-332.

Onguéné, P. A., Ntie-Kang, F., Lifongo, L. L., Ndom, J. C., Sippl, W. and Mbaze, L. M. A. (2013). The potential of anti-malarial compounds derived from African medicinal plants. Part I: A pharmacological evaluation of alkaloids and terpenoids. Malaria journal, 12(1): 449.

Oprea, T. I., Davis, A. M., Teague, S. J. and Leeson, P. D. (2001). Is there a difference between leads and drugs? A historical perspective. Journal of chemical information and computer sciences, 41(5): 1308-1315.

Orji, A. U. (2001). Toxic heme in sickle cells: An explanation for death of malaria parasites. American Journal of Tropical Health Sciences, 34: 223 – 227.

Ourisson, G. and Nakatani, Y. (1994). The terpenoid theory of the origin of cellular life: the evolution of terpenoids to cholesterol. Chemistry and biology, 1(1): 11-23.

Pagola, S., Stephens, P. W., Bohle, D. S., Kosar, A. D. and Madsen, S. K. (2000). The structure of malaria pigment β-haematin. Nature, 404(6775): 307-314.

Pandey, A. V., Bisht, H., Babbarwal, V. K., Srivastava, J., Pandey, K. C., and Chauhan, V. S. (2001). Mechanism of malarial haem detoxification inhibition by chloroquine. Biochemical Journal, 355(2): 333-338.

Pandey, A. V., Tekwani, B. L., Singh, R. L. and Chauhan, V. S. (1999). Artemisinin, an endoperoxide antimalarial, disrupts the haemoglobin catabolism and heme detoxification systems in malarial parasite. Journal of biological chemistry, 274(27): 19383-19388.

Patil S B. and Magdum. C. S. (2012). Brine Shrimp Lethality Activity of Euphorbia Hirta Linn. International Journal of Pharmacy and Pharmaceutical Sciences, 4(3): 347-348.

Pelka, M., Danzl, C., Distler, W. and Petschelt, A. (2000). A new screening test for toxicity testing of dental materials. Journal of dentistry, 28(5): 341-345.

Percário, S., Moreira, D. R., Gomes, B. A., Ferreira, M. E., Gonçalves, A. C. M., Laurindo, P. S. and Green, M. D. (2012). Oxidative stress in malaria. International Journal of Molecular Sciences, 13(12): 16346-16372.

Pessi, G., Kociubinski, G. and Mamoun, C. B. (2004). A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation. Proceedings of the National Academy of Sciences, 101(16): 6206-6211.

184

Peter, G., Manuel, A. L. and Shetty, A. (2011). Study comparing the clinical profile of complicated cases of Plasmodium falciparum malaria among adults and children. Asian Pacific Journal of Tropical Disease, 1(1): 35-37.

Prassas, I. and Diamandis, E. P. (2008). Novel therapeutic applications of cardiac glycosides. Nature reviews Drug discovery, 7(11): 926.

Pridham, J. B. (1960) In: Phenolics in Plants in Health and Disease. Pergamon Press, New York, pp. 34-35.

Quideau, S. (2009) Why bother with polyphenols? In Groupe Polyphenols: The International Society Dedicated to the Promotion of Research on Polyphenols; 1st Edn. Bordeaux, France. pp: 55-72. Rahbari, M., Rahlfs, S., Jortzik, E., Bogeski, I. and Becker, K. (2017). H2O2 dynamics in the malaria parasite Plasmodium falciparum. Plos One, 12(4): 37-49.

Rajiv, J., Pere, G., Jody, C. O., Rajeshwar, P. M., Richard, M., Guadalupe, G., Vicente. A. and Patrick, S. K. (2012). Acute-on chronic liver failure. Journal of Hepatology, 57: 1336–1348.

Ramiarantsoa, H., Attioua, B. K., Kouamé, M. A. and Djakouré, L. A. (2008). Le O-β-D-glucoside du β-sitostérol Isolé. Journal de la Société Ouest-Africaine de Chimie, 26: 99-103.

Rao, R. V. K., Ali, N. and Reddy, M. N. (1978) Occurrence of both sapogenins and alkaloid lycorine in Curculigo orchioides. Indian Journal Pharmaceutical Science, 40: 104- 105.

Reitman, S. and Frankel, S. (1957). A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic aminotransferases. American Journal of Clinical Pathology, 28: 56-63.

Rhen, T. and Cidlowski, J. A. (2005). Antiinflammatory action of glucocorticoids—new mechanisms for old drugs. New England Journal of Medicine, 353(16): 1711-1723. Rifkind, J. M., Mohanty, J. G. and Nagababu, E. (2015). The pathophysiology of extracellular hemoglobin associated with enhanced oxidative reactions. Frontiers in physiology, 5: 500 - 507

Robbins, R. J. (2003). Phenolic acids in foods: an overview of analytical methodology. Journal of agricultural and food chemistry, 51(10): 2866-2887.

Rodrigues, C. G., Ferreira, P. R. B., Oliveira, M., Junior, R. R., Valerio, H. M., Brandi, V. I. and Oliveira, D. A. (2014). Antibacterial activity of tannins from Psidium guineense Sw. (Myrtaceae). Journal of Medicinal Plant Research, 8: 1095-100.

Ruiz-Garcia, A., Bermejo, M., Moss, A. and Casabo V. G. (2008). Pharmacokinetics in drug discovery. Journal of Pharmaceutical Sciences, 97 (2): 654–90.

185

Sachs, J. and Malaney, P. (2002). The economic and social burden of malaria. Nature 415(7): 680- 685.

Saliba, L. J. and Krzyz, R. M. (1976). Effect of heavy metals on hatching of brine-shrimp eggs. Marine Pollution Bulletin, 7(10): 181-182. Sakyi, S. A., Ephraim, R. K., Antoh, E. O., Obirikorang, C. and Berchie, G. O. (2012). Lipid peroxidation and catalase levels among children presenting with severe falciparum malaria in the Sefwi Wiaso Municipality, Ghana. Journal of Medical Sciences, 12: 141–147.

Sarkar, P. D. and Rautava, S. S. (2009). A study of serum malonaldehyde levels and peroxidase activity in ischemic stroke patients. Biomedical Research, 20(1): 64-66.

Sayhan, H., Beyaz, S. G. and Çeliktaş, A. (2017). The Local Anesthetic and Pain Relief Activity of Alkaloids. In Alkaloids-Alternatives in Synthesis, Modification and Application, 5: 13- 17

Seaver, L. C. and Imlay, J. A. (2004). Are respiratory enzymes the primary sources of intracellular hydrogen peroxide? The Journal of Biological Chemistry, 297(47): 48742- 50.

Sgarbieri, V. C. (1996). Proteins in protein foods, degradation properties and modifications. Sao Paulo Varela, 14: 517-524.

Shitlani, D., Choudhary, R., Pandey, D. P. and Bodakhe, S. H. (2016). Ameliorative antimalarial effects of the combination of rutin and swertiamarin on malarial parasites. Asian Pacific Journal of Tropical Disease, 6(6): 453-459.

Shittu, I. I., Emmanuel, A. and Nok, A. J. (2011). Antimalaria effect of the ethanolic stem bark extracts of Ficus platyphylla del. Journal of parasitology research, 11;1-5.

Silverstein, R. M. Bassler, G. C. and Morrill, T. C. (1991). Spectrometric Identification of Organic Compounds, 5th Edn. Wiley. London, pp: 89-94.

Sinha, A. K. (1972). Colorimetric assay of catalase. Analytical biochemistry, 47(2), 389-394.

Sirintorn, P., Pinyupalianbangchangb, N. P., Siriluk, R. and Onrudee M. (2004) Brine shrimp lethality activity of Thai medicinal plants in the family Meliaceae, Naresuan University Journal, 12(2): 13-18. Sirima, S. B., Tiono, A. B., Gansané, A., Diarra, A., Ouédraogo, A., Konaté, A. T., Kiechel, J.R., Morgan, C.C., Olliaro, P.L. and Taylor, W. R. (2009). The efficacy and safety of a new fixed-dose combination of amodiaquine and artesunate in young African children with acute uncomplicated Plasmodium falciparum. Malaria Journal, 8(1), 48.

Slater, A. F. G., and Cerami, A. (1992). Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature, 355(6356): 167-169.

Smith, J. G. (2011). Chapter 13 Mass Spectrometry and Infrared Spectroscopy. Organic chemistry (Book) (3rd ed.). NY: McGraw-Hill. New York, pp. 463–488.

186

Snow, R. W., Craig, M., Deichmann, U. and Marsh, K. (1999). Estimating mortality, morbidity and disability due to malaria among Africa’s non-pregnant population. Bulletin of the World Health Organization 77:624–640.

Sofowora, A. (1993). Medicinal Plants and Traditional Medicine in Africa. 2nd Edn: Spectrum Books Limited, Nigeria. pp. 32-45.

Sowunmi, A., Fehintola, F. A., Adedeji, A. A., Gbotosho, G. O., Falade, C. O., Tambo, E. Fateye, B.A., Happi, T.C. and Oduola, A. M. J. (2004). Open randomized study of pyrimethamine– sulphadoxine vs. pyrimethamine–sulphadoxine plus probenecid for the treatment of uncomplicated Plasmodium falciparum malaria in children. Tropical Medicine and International Health, 9(5): 606-614.

Sparg, S., Light, M. E. and Van Staden, J. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94(2-3): 219-243.

Spielman, A. and Williams, C. M. (1966). Lethal effects of synthetic juvenile hormone on larvae of the yellow fever mosquito, Aedes aegypti. Science, 154(3752): 1043-1044.

Staedke, S. G., Jagannathan, P., Yeka, A., Bukirwa, H., Banek, K., Maiteki-Sebuguzi, C., Clark, T.D., Nzarubara, B., Njama-Meya, D., Mpimbaza, A. Rosenthal, P. J. (2008). Monitoring antimalarial safety and tolerability in clinical trials: a case study from Uganda. Malaria Journal, 7(1), 107-18.

Stahl, E. (2005). Thin-Layer Chromatography: A Laboratory Handbook, 2nd Edn. Spinger; New York, pp: 77-82.

Sulistyaningsiha, E., Amaliaa, T. Y. and Kartikasaria, R. (2017). Antioxidant and antimalarial activity of Leea indica leaf extract against malaria-mice model. Journal of Applied Pharmaceutical Science, 7(12): 163-168.

Sultana, N. and Afolayan, A. J. (2007). A novel daucosterol derivative and antibacterial activity of compounds from Arctotis arctotoides. Natural product research, 21(10): 889-896. Sultana, R., Newman, S., Mohmmad-Abdul, H., Keller, J. N. and Allan Butterfield, D. (2004). Protective effect of the xanthate, D609, on Alzheimer's amyloid β-peptide (1–42)-induced oxidative stress in primary neuronal cells. Free radical research, 38(5): 449-458.

Talisuna, A. O., Staedke, S. G. and D'Alessandro, U. (2006). Pharmacovigilance of antimalarial treatment in Africa: is it possible?. Malaria Journal, 5(1), 50-62

Tan, R. X., Zheng, W. F. and Tang, H, Q. (1998). Biologically active substances from the genus. Artemisia. Planta Medica, 64: 295–302.

Tanabe, K. (1990). Ion metabolism in malaria-infected erythrocytes. Blood cells, 16(2-3): 437- 449.

187

Tapas, A. R., Sakarkar, D. M. and Kakde, R. B. (2008) Flavonoids as nutraceuticals: A review. Tropical Journal of Pharmaceutical Research, 7: 1089-1099.

Thapa, E. M, and Anuj, W. (2007). Liver function test and their interpretation. Indian Journal of Pediatrics, 74(7): 663-671.

Tharavanij, S. (1990). New developments in malaria diagnostic techniques. Southeast Asian Journal of Tropical Medicine and Public Health, 21(1): 3-16.

Tiwari, P., Kumar, B., Kaur, M., Kaur, G. and Kaur, H. (2011). Phytochemical screening and extraction: A review. Internationale Journal of Pharmaceutica Sciencia, 1: 98-106.

Trampuz A, Jereb M, Muzlovic, I. and Prabhu R (2003). Clinical review: Severe malaria. Critical Care 7(4): 315–23.

Tshibangu, J. N., Chifundera, K., Kaminsky, R., Wright, A. D. and König, G. M. (2002). Screening of African medicinal plants for antimicrobial and enzyme inhibitory activity. Journal of Ethnopharmacology, 80(1): 25-35.

Umar, M. B., Ogbadoyi, E. O., Folarin, A. A., Adamu, Z. and Abubakar, A. N. (2018).Evaluation of the Antimalarial and Liver Function Potentials of Methanol Extract of Chrysophyllum albidum stem bark in Plasmodium berghei -Infected Mice. Journal of Biomedical and Pharmaceutical Sciences, 1(1): 1-5.

Vial, H. J. and Ancelin, M. L. (1998) in Malaria: Parasite Biology, Pathogenesis, and Protection, ed. Shermann, I. W. (Am. Soc. Microbiol. Press, Washington, DC), pp: 159–175.

Vincken, J. P., Heng, L., De Groot, A. and Gruppen, H. (2007). Saponins, classification and occurrence in the plant kingdom. Phytochemistry, 68(3): 275-297.

Waako, P. J., Katuura, E., Smith, P. and Folb, P. (2007) East African medicinal plants as a source of lead compounds for the development of new antimalarial drugs. African Journal of Ecology, 45(1): 102–106.

Wallin, B., Rosengren, B., Shertzer, H. G. and Camejo, G. (1993). Lipoprotein oxidation and measurement of TBARS formation in a single microliter plate: Its use for evaluation of antioxidants. Analytical Biochemistry, 208: 10-15.

Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K. and Pepinsky, R. B. (1986). Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity. Nature, 320(6057): 77-81.

Wasserman, M., Alarcón, C. and Mendoza, P. M. (1982). Effects of Ca++ depletion on the asexual cell cycle of Plasmodium falciparum. The American Journal of Tropical Medicine and Hygiene, 31(4): 711-717.

188

Waugh, A. and Grant, A. (2014). Ross and Wilson Anatomy and Physiology in Health and Illness. Elsevier Health Sciences. 12th Edn. Churchill Livingstone. London, United Kingdom, pp. 312-314.

Wesolowski, C. A., Wesolowski, M. J., Babyn, P. S. and Wanasundara, S. N. (2016). Time varying apparent volume of distribution and drug half-lives following intravenous bolus injections. PloS one, 11(7): 1-9.

WHO (1996). A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO informal consultation on recent advances in diagnostic techniques and vaccines for malaria. Bulletin of the World Health Organization 74:47–54.

WHO (2000). Expert Committee on Malaria, 20th Report, World Health Organisation Technical Report Series, Geneva. Switzerland; pp. 892-905.

WHO (2003). Global defence against the infectious disease threat. WHO/CDS/2003/15.18:178- 181.

Widmaier, E., Raff, H. and Strang, K. (2015). Vander's Human Physiology. 14th Edn. McGraw- Hill Education, New York City, New York, United States, pp. 102-105. Willcox, M. L., and Bodeker, G. (2004). Traditional herbal medicines for malaria. BMJ: British Medical Journal, 329(7475): 1156-67.

Willcox, M. L., Gamaniel, S. K., Matsabisa, M. G., Randriasamimanana, J. R., Wambebe, C. O. N. and Rasoanaivo, P. (2003). Guidelines for the pre-clinical evaluation of the safety of traditional herbal antimalarials. Traditional Medicinal Plants and Malaria, 16: 2004-15.

Wink, M., Schmeller, T. and Latz-Briining, B. (1998). Modes of action of allele-chemical alkaloids: Intraction with neuroreceptors, DNA and other molecular targets. Journal of Chemical Ecology, 24: 1888-1937.

Withers-Martinez, C., Malcolm, S., Fiona, H., Lesley, F. H., Steven, A. H., Philip, A. W., Evangelos, C., Guy, G. D. and Michael, J. B. (2014). The malaria parasite egress protease SUB1 is a calcium-dependent redox switch subtilisin. Nature Communications, 5: 3726- 36.

World Health Organization (1991). Basic Laboratory Methods in Medical Parasitology. World Health Organization Geneva. Switzerland; pp. 72-85.

World Health Organization (2001). In vitro micro test (Mark III) for the assessment of the response of Plasmodium falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine artemisinin. Geneva, Switzerland; pp 4- 17.

World Health Organization. (2010). Guidelines for the treatment of malaria. 3rd Edn. Geneva, Switzerland. pp 31-71.

189

Xin, Z., Waterman, D. E., Henken, R. M. and Harmon, R. J. (1991). Effects of copper status on neutrophil function, superoxide dismutase and copper distribution in steers. Journal of Diary Science, 74: 3078-3080.

Yared, A., Alemayehu, A. and Zewdneh, S. (2006). Haematology. Ethiopia Public Health Training Initiative. Addis Ababa, pp. 569-578.

Yeka, A., Dorsey, G., Kamya, M. R., Talisuna, A., Lugemwa, M., Rwakimari, J. B., Staedke, S. G., Rosenthal, P. J., Wabwire-Mangen, F. and Bukirwa, H. (2008). Artemether- lumefantrine versus dihydroartemisinin-piperaquine for treating uncomplicated malaria: a randomized trial to guide policy in Uganda. PLoS One, 3(6): 1-12.

Zhao, D. D., Jiang, L. L., Li, H. Y., Yan, P. F., and Zhang, Y. L. (2016). Chemical components and pharmacological activities of terpene natural products from the Genus Paeonia. Molecules, 21(10): 1362-74.

Zofou, D., Kowa, T. K., Wabo, H. K., Ngemenya, M. N., Tane, P. and Titanji, V. P. K. (2011). Hypericum lanceolatum (Hypericaceae) as a potential source of new anti-malarial agents: a bioassay-guided fractionation of the stem bark. Malaria Journal, 10 (1): 167- 173.

Zofou, D., Tematio, E. L., Ntie-Kang, F., Tene, M., Ngemenya, M. N., Tane, P., and Titanji, V. P. (2013). New antimalarial hits from Dacryodes edulis (Burseraceae)-Part I: Isolation, in vitro activity, in silico drug-likeness and pharmacokinetic profiles. PloS one, 8(11): 1-9.

Zofou, D., Tene, M., Tane, P. and Titanji, V. P. (2012). Antimalarial drug interactions of compounds isolated from Kigelia africana (Bignoniaceae) and their synergism with artemether, against the multidrug-resistant W2mef Plasmodium falciparum strain. Parasitology research, 110(2): 539-544.

190