ABSTRACT
In recent times, interest in the use of medicinal plants (ethnomedicine or
ethnobotanic medicine) as alternative to modern medicines is increasing globally,
especially in developing countries where traditional beliefs and high cost of, or
limited access to conventional medical treatment may all constitute important
factors. The use of Sarcocephalus latifolius for the control of various illnesses
especially in Africa is widely documented. This study therefore is designed to
establish the toxicity potentials and /or the safety margin of the plant’s root extract
by monitoring the effects of its constituents on some haematological and
biochemical parameters in rats. The haematological parameters were analysed
using the Diatron automated haematological analyser (ABACUS). Liver and
kidney function indices, some electrolytes, and lipid parameters, in serum, were
determined using analytical kits and standard methods, as appropriate. Na+ was
analysed by flame photometry. Antimalarial screening was done by microscopy.
The phytochemical analysis of the root revealed the presence of alkaloids,
saponins, tannins and anthraquinones in extractable quantities; flavonoids are also
present. The results of the study indicates that the observed changes in
haematological parameters – white blood cell (WBC), red blood cell (RBC),
platelets (PLT) and their indices (except in very few cases) were non-significant (p
> 0.05). The effects of the extract on concentrations of different electrolytes in
serum were varied. Elevations were recorded for potassium with a few cases being
significant (p < 0.05). Serum calcium concentration was significantly reduced (p <
0.05) in a dose-dependent manner. Reductions were also observed for serum
chloride concentration, with higher doses causing significant reductions (p < 0.05).
Serum bicarbonate concentration appears almost completely unaffected by the
extract; apparently, no changes were observed when compared with that of the
control. Serum sodium concentrations for the 500 and 800 mg doses and the
control were 139.22 ± 1.02 mmol/L, 134.5 ± 1.50 mmol/L, and (140.66 ± 1.12
mmol/L), respectively, showing a significant reduction for the higher dose. Liver
function studies showed insignificant reductions in mean activities of alkaline
phosphatase (ALP) and aspartate aminotransferase (AST), with significant
reduction in the activity of alanine aminotransferase (ALT), (p < 0.05). Bilirubin,
creatinine, and urea were apparently unaffected by the extract (p > 0.05). For the
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acute toxicity studies, the lethal dose (LD50) of the extract was found to be 2236.07
mg/kg body weight. Effects on liver function parameters – ALP, AST, and ALT
were nonsignificant. Increased acitivities were recorded for gamma
glutamyltransferase (13.90 ± 6.40; 14.48 ± 5.38; 4.34 ± 2.8 iu/l), for 2000 mg,
1500 mg/kg body weight, and control, respectively. Total bilirubin decreased
nonsignificantly (p > 0.05), while conjugated bilirubin decreased significantly (p <
0.05), comparative to the control.Creatinine and urea were decreased significantly
(p < 0.05) by the extract at a single high dose of 2000 mg/kg body weight. Single
dose schedules decreased both cholesterol and triglyceride insignificantly (p >
0.05), but uric acid was significantly reduced (p < 0.05). Results of antimalarial
studies indicate that the ethanol extract of the root is capable of eliminating
malarial parasites as well as suppressing their growth. These studies have shown
that Sarcocephalus latifolius root extract is relatively of very low toxicity and
could conserve the integrity of the blood and essential organs, thus justifying its
wide application in traditional medicine practices.
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TABLE OF CONTENTS
CHAPTER ONE
1.1 Introduction 1
1.2 Aims and objectives 4
CHAPTER TWO
Literature Review 6
2.1 Overview of medicinal plants 6
2.2 Global strategy on conservation 9
2.3: Medicinal plants – Evidence of traditional uses 10
2.4: Use of medicinal plants for specific disease conditions 16
2.5: Sarcocephalus latifolius 19
2.6: Taxonomy of Sarcocephalus latifolius 25
2.7: Overview of malaria and drug resistance 27
2.8: Enzymes of diagnostic importance 29
2.9: Phytochemicals 31
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CHAPTER THREE
Materials and methods
3.0: Materials 40
3.1: Identification of plant specimen – Sarcocephalus latifolius 40
3.1.1: Sample collection 40
3.1.2: Experimental animals
3.1.3: Reagents and equipments 41
3.2: Methods 44
3.2.1: Phytochemical analysis 44
3.2.2: Preparation of root extract 49
3.2.3: Determination of lethal dose 50
3.2.4: Haematological analyses 51
3.2.5: Determination of serum electrolytes concentrations 53
3.2.6: Determination of ALP, ALT (SGPT), AST (SGOT) 59
3.2.7: Assay of bilirubin and sodium concentrations 64
3.2.8: Assay of creatinine and urea levels 68
3.2.9: Acute studies with selected parameters 72
3.2.10: Antiplasmodial screening 79
CHAPTER FOUR
Results 83
4.1: Phytochemical analysis 83
4.2: Lethal dose (LD50) 84
4.3: Haematological studies 85
4.4: Electrolytes 101
4.5: Liver and kidney function markers 105
4.6: Acute dose (toxicity) studies 111
4.7: Antimalarial screening 121
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CHAPTER FIVE
Discussions / Conclusion 124
5.1: Discussions 124
5.2: Conclusion 137
5.3: Suggestions for further studies 138
References 140
Appendices 169
Appendix 1: Summary of data for different analyses 169
Appendix 2: Raw data for different analyses 172
Appendix 3: Acute studies data 191
Appendix 4: Raw data on haematocrit (PCV) 198
Appendix5: Reference tables for the determination of AST
and ALT activities 199
Appendix 6: Dose determination 201
Appendix 7: Reagents for Assays 203
Appendix 8: Original data from full blood count 213
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LIST OF FIGURES
Figure 4.1: Mean WBC and Differential Counts 85
Figure 4.2: Mean Percentage WBC Differential Counts 87
Figure 4.3: Mean Red Blood Cell Counts 89
Figure 4.4: Mean Haemoglobin and Mean Cell Haemoglobin Concentration 90
Figure 4.5 HCT and RDWcv 92
Figure 4.6: MCV and RDWs 94
Figure 4.7: MCH 95
Figure 4.8: PLT 96
Figure 4.9: PCT 97
Figure 4.10: MPV and PDWs 98
Figure 4.11: PDWcv 100
Figure 4.12: Mean concentrations of potassium and calcium 101
Figure 4.13: Mean concentrations of chloride and bicarbonate 103
Figure 4.14: Mean Sodium levels 104
Figure 4.15: Mean enzyme activities (ALP, ALT, and AST) 105
Figure 4.16: Mean bilirubin concentrations 107
Figure 4.17: Mean creatinine and urea concentrations 109
Figure 4.18: Mean ALP, ALT, AST and GGT activities (Acute doses) 111
Figure 4.19: Mean bilirubin concentrations 114
Figure 4.20: Mean concentrations of creatinine 116
Figure 4.21: Mean concentration of urea 117
Figure 4.22: Mean concentration of cholesterol 118
Figure 4.23: Mean concentrations of triglyceride 119
Figure 4.24: Mean concentrations of uric acid 120
Figure 4.25: Percentage PCV levels for chemotherapeutic group 122
Figure 4.26: Percentage PCV levels for prophylactic group 123
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LIST OF TABLES
Table Page
Table 4.1: Summary of Results of phytochemical analysis 83
Table 4.2: Parasite density determined from chemotherapeutic studies 121
Table 4.3: Parasite density determined from prophylactic studies 121
Table 4.4: Parasite density determined for the control group 121
Appendices
Table 1.1: Mean WBC and Differentials 169
Table 1.2: Mean red cells and red blood cell indices 169
Table 1.3: Mean platelets and platelet indices 169
Table 1.4: Mean concentration of various electrolytes 169
Table 1.5: Mean concentrations of Sodium 170
Table 1.6: Mean activities of ALP, ALT, and AST 170
Table 1.7: Mean concentrations of (TB , CB, and UB) 170
Table 1.8: Mean concentrations of Creatinine and Urea 170
Table 1.2.1: Mean activities of ALP, ALT, AST and GGT 171
Table 1.2.2: Mean concentrations of Creatinine and Urea 171
Table 1.2.3: Mean concentrations of Bilirubin (TB , CB) 171
Table 1.2.4: Mean activities of Cholesterol, Triglyceride, and Uric acid 171
Table 1.3.1: Mean percentage PCV from antimalarial screening 171
Table 2.1: Full blood count, Group A (300mg/kg body weight) 172
Table 2.2: Full blood count, Group B (350mg/kg body weight) 173
Table 2.3: Full blood count, Group C (400mg/kg body weight) 174
Table 2.4: Full blood count, Group D (450mg/kg body weight) 175
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Table 2.5: Full blood count, Group E (500mg/kg body weight) 176
Table 2.6: Full blood count, Group F (Control) 177
Table 2.7: Potassium concentration 178
Table 2.8: Calcium concentration 179
Table 2.9: Chloride concentration 180
Table 2.10: Bicarbonate concentration 181
Table 2.11: Sodium concentration 182
Table 2.12: Alkaline phosphatase activities 183
Table 2.13: Alanine aminotransferase 184
Table 2.14: Aspartate aminotransferase 185
Table 2.15: Total bilirubin 186
Table 2.16: Direct (Conjugated) bilirubin 187
Table 2.17: Summary of bilirubin concentrations 188
Table 2.18: Creatinine 189
Table 2.19: Urea 190
Table 3.1: Alkaline phosphatase (ALP) 191
Table 3.2: Alkaline phosphatase (ALP) 191
Table 3.3: Aspartate aminotransferase (AST) 192
Table 3.4: L- γ-Glutamyltransferase (γ-GT, GGT) 192
Table 3.5: Total bilirubin 193
Table 3.6: Direct (Conjugated) bilirubin 193
Table 3.7: Summary of bilirubin concentrations – total , conjugated , and
Unconjugated 194
Table 3.8: Mean concentrations of (TB , CB, and UB) 194
Table 3.9: Creatinine 195
Table 3.10: Urea 195
Table 3.11: Cholesterol 196
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Table 3.12: Triglyceride 196
Table 3.13: Uric acid 197
Table 4.1: Percentage PCV levels for chemotherapeutic group on
days 5 and 10 198
Table 4.2: Percentage PCV levels for prophylactic group on days 1 and 6 198
Table 4.3: Percentage PCV levels for control group on days 1, 6, and 10 198
Table 5.1: AST reference table 199
Table 5.2: ALT reference table 200
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1
CHAPTER ONE
1.1: INTRODUCTION
Through ages, plants have been used as medicine because they are important
source of many biologically active products. Several drugs in use today have
been developed from plants and some examples include digoxin from
Digitalis spp, quinine and quinidine from Chinchona spp, vincristine and
vinblastine from Catharanthus roseus, atropine from Atropa belladonna,
morphine and codeine from Papaver somniferum (Rates, 2001).
Ethnopharmacological studies have resulted in the discovery of many
interesting properties of plants which mainly are based on the studies of how
traditional healers (herbalists) use plants therapeutically (Barrett, 1994; Coe
and Anderson, 1999). Different communities and cultures often use the same
plant in different ways. Therefore, it is important to investigate the use and
the risks that these products may pose to health (Andrade et al, 2008).
The use of herbal-based therapies for the treatment of diseases has been
rapidly gaining acceptance globally. Many of such herbal-based remedies
are already being massively imported into Nigeria from different parts of the
world. Herbal medicine is alternatively called botanicals or phytotherapy,
and locally here in Nigeria, the term traditional medicine is a common
description. According to the US National Institute of Health, an herb is a
plant or plant part used for its scent, flavor or therapeutic properties. Herbal
medicine products are dietary supplements that people take to improve their
health. Many herbs have been used for a long time for various claimed
health benefits. They are being sold as tablets, capsules, powders, teas,
extracts and fresh or dried plants. However, some can cause health
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problems, some are not effective and some may interact with other drugs one
may be taking (Medline Plus). Many species of plants have been in use
medicinally for centuries. Various indigenous people have learned which
species of plants may help alleviate certain ailments such as toothaches,
induce labour, or cure malaria (Abruzzo, 2005).
It is now believed that over 50% of all modern clinical drugs are of natural
products origin (Suffness et al, 1982) and natural products play an important
role in drug development programmes of the pharmaceutical industry (Baker
et al, 1995). Investigations into the chemical and biological activities of
plants during the the past two centuries have yielded compounds for the
development of modern synthetic organic chemistry and the emergence of
medicinal chemistry as a major route for the discovery of novel and more
effective therapeutic agents (Roja et al, 2000). In spite of the vast array of
information on the composition and biological activity of many plant
substances, there has been little effort devoted to the development of
chemotherapeutic and prophylactic agents from these plants (Momoh e
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