ABSTRACT
So many plants have been discovered to possess anti-sickling agents as a result of their usage in the management of sickle cell disease (SCD) in rural African homes. There is the need for further investigation into other plants that can be used in the treatment of sickle cell disease. This study investigated the methanol extracts of the seeds of B. coriacea (sample A) and M. pruriens (sample B) for their possible in vitro anti-sickling effects. Preliminary investigations of the constituents of the plant seeds were done. Amino acid analyses of both samples showed the presence of anti-sickling amino acids: phenylalanine, lysine, leucine, aspartate, serine, arginine and tyrosine. Minerals and vitamins were quantitatively analysed. Samples A and B contained potassium (18.30 ± 2.00 mg/100g and 15.37 ± 0.55 mg/100g), calcium (37.90 ± 1.51 mg/100g and 35.50 ± 1.80 mg/100g), zinc (123.43 ± 1.07 mg/100g and 110.60 ± 3.57 mg/100g), iron (32.90 ± 2.14 mg/100g and 114.37 ± 37.6.47 mg/100g), magnesium (20.37 ± 0.38 mg/100g and 20.27 ± 0.41 mg/100g) and sodium (38.60 ± 0.92 mg/100g and 35.80 ± 1.68 mg/100g) respectively. They also contained vitamin A (35.73 ± 1.06 µg/ml and 73.49 ± 0.51 µg/ml), vitamin B1 (52.31 ± 22.96 µg/ml and 147.35 ± 50.73 µg/ml), vitamin B2 (10.01 ± 2.28 µg/ml and 44.47 ± 3.94 µg/ml), vitamin B3 (14.70 ± 0.40 µg/ml for sample B) and vitamin C (0.878 ± 0.02 µg/ml and 0.490 ± 0.01 µg/ml) respectively. Qualitative and quantitative phytochemical analyses were carried out. Both samples had tannins, saponins, alkaloids, flavonoids, terpenoids, acidic compounds, phenolics, steroids, reducing sugars, carbohydrates and glycosides with M. pruriens sample having higher concentrations of each of them except glycosides. Proximate analysis of samples A and B also showed high percentage of carbohydrates (56.86% and 61.43%) respectively. Sickle cell blood from SCD patients were obtained from a hospital and treated with sodium metabisulphite (2%) to induce further sickling. The red blood cells were treated with varying concentrations (50%, 25%, 12.5% and 6.25%) of the extracts. Both extracts significantly (P<0.05) inhibited sickling, reversed sickling of erythrocytes and reduced the rate of polymer formation at all the concentrations used with M. pruriens extract showing higher effects. The solubility of haemoglobin S was significantly (P<0.05) increased when treated with M. pruriens extract at all the concentrations used while B. coriacea extract did not increase the solubility at 6.25% concentration. Osmotic fragility graphs show that these plant extracts may have the ability to reduce haemolysis as well as protect red cell integrity (at lower concentrations). The Fe2+/Fe3+ ratio was increased at all the concentrations used by both extracts with M. pruriens extract giving rise to higher ratios. These results indicate that B. coriacea and M. pruriens have the potentials of being used in the management of sickle cell disease.
TABLE OF CONTENTS
Title Page
i
Declaration
ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures x
Abstract xi
CHAPTER
1: INTRODUCTION
1.1 Background of the Study 1
1.2
Statement of the Problem 1
1.3 Aim
of the Study 2
1.4 Objectives
of the Study 2
1.5 Justification
for the Study 3
CHAPTER 2: REVIEW OF RELATED LITERATURE
2.1 Sickle Cell 5
2.1.1 Biochemical basis of sickling 6
2.1.2 Pathophysiology of sickle cell disease 7
2.1.3
Symptoms of sickle cell disease 7
2.1.4 Therapeutic
approach to sickling 9
2.2 Haematology of Sickle Cell Blood 11
2.2.1 Red blood cells in sickle
cell disease 11
2.2.2 White blood cells in sickle
cell disease 12
2.2.3 Platelets in sickle cell disease 12
2.3
Haemoglobin 13
2.3.1
Structure of haemoglobin 13
2.3.2
Function of haemoglobin 15
2.4 Anti-sickling
Agents from Plants 15
2.5 M.
pruriens 17
2.6 B.
coriacea 18
CHAPTER 3:
MATERIALS AND METHODS
3.1 Materials 21
3.1.1 Instruments 21
3.2 Methods 21
3.2.1 Preparation of the test samples 21
3.2.2 Buffer preparation 22
3.2.3 Dilution of the extracts 22
3.2.4 Preparation of 2% sodium metabisulphite solution 22
3.2.5 Collection of the
blood samples 23
3.2.6 Amino acid analyses 23
3.2.7 Determination
of the mineral contents of the samples 23
3.2.8 Determination
of the vitamin contents of the samples 26
3.2.9 Phytochemical analyses 28
3.2.9.1 Qualitative phytochemical analyses 28
3.2.9.2
Quantitative phytochemical
analyses 30
3.2.10 Proximate analysis 33
3.2.11 Sickling inhibition test 35
3.2.12 Sickling reversal test 36
3.2.13 Solubility test 37
3.2.14 Osmotic fragility test 37
3.2.15 Polymerisation studies 38
3.2.16 Determination
of the Fe2+/Fe3+ ratio in sickle cell blood 38
3.3 Statistical Analysis 39
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Results 40
4.1.1 Amino acid analyses of the samples 40
4.1.2 Mineral analyses of the samples 42
4.1.3 Vitamin contents of the samples 43
4.1.4 Qualitative phytochemical analyses of the
samples 44
4.1.5 Quantitative phytochemical analyses of the
samples 45
4.1.6 Proximate analysis of the samples 46
4.1.7 Effect of the extracts on sickling
inhibition 48
4.1.8 Effect of the extracts on sickling reversal 49
4.1.9 Effect of the
extracts on the solubility of haemoglobin S 50
4.1.10 Effect of the extracts on osmotic
fragility 52
4.1.11 Effect of the
extracts on polymerisation of haemoglobin S 54
4.1.12 Effect of the extracts on Fe2+/Fe3+
ratio in sickle cell blood 56
4.2 Discussion 58
CHAPTER 5:
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 75
5.2 Recommendations 75
References 76
Appendix 91
LIST OF TABLES
4.1 Amino acid (AA) constituents of the samples 41
4.2 Concentration
of some mineral elements in the samples 42
4.3 Concentration
of some vitamins in the samples 43
4.4 Phytochemical
constituents of the samples 44
4.5 Concentration
of some phytochemicals in the samples 45
4.6 Percentage
haemoglobin, percentage methaemoglobin and
Fe2+/Fe3+
ratio in sickle blood treated with B.
coriacea
seed
extract 56
4.7 Percentage
haemoglobin, percentage methaemoglobin and
Fe2+/Fe3+
ratio in sickle blood treated with M.
pruriens
seed
extract 57
LIST OF FIGURES
1:1 Sickle cell disorder inheritance pattern 5
1.2 The normal and sickled red blood cells 6
1.3 Structure of the haem group and haemoglobin
molecule 14
1.4 Picture
of Mucuna pruriens plant, seed pod and seed 18
1.5 Pictures
of Buchholzia coriacea tree, leaves and seeds 20
4.1 Proximate compositions of B. coriacea seed 46
4.2 Proximate compositions of M. pruriens seed 47
4.3
Percentage sickling inhibition of extracts of B. coriacea seed
(sample A), M. pruriens seed (sample B), a
combination of
samples A and B, and M. pruriens seed coating (sample C) 48
4.4 Percentage
sickling reversal of extracts of B.
coriacea seed
(sample A), M. pruriens seed (sample B), a combination of
samples A and B
and M. pruriens seed coating (sample
C) 49
4.5
Effect of extract of B. coriacea
seeds (sample A) on the
solubility of
haemoglobin S 50
4.6
Effect of extract of M. pruriens
seeds (sample B) on the
solubility of
haemoglobin S 51
4.7 Osmotic fragility
graph of sickle cell blood after supplementation
with different concentrations
of B. coriacea seed extract
(sample A) 52
4.8 Osmotic fragility
graph of sickle cell blood after supplementation
with different concentrations of M. pruriens seed extract
(sample B) 53
4.9 Polymerisation
of sickle cell blood treated with different
concentrations of extracts of B. coriacea seed (sample A) 54
4.10 Polymerisation
of sickle cell blood treated with different
concentrations of extracts of M. pruriens seed (sample B) 55
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Sickle
cell anaemia is a genetic blood disorder which arises from a point mutation in
the β-globin gene that leads to the replacement of glutamic acid residue with
valine at the sixth position of the β-chain of the haemoglobin (Ingram, 1958; Njoku,
2007; Imaga et al., 2009). Glutamic
acid has a hydrophilic side chain and bears a negative charge while valine has a
hydrophobic side chain and is nonpolar. This single amino acid
substitution causes a significant reduction in the solubility of the deoxy form
of sickle haemoglobin (deoxy-HbS), causing polymer formation inside the red
blood cells (RBCs). The gelation and sickling phenomenon can be regarded as one
of relative solubilities under physiological conditions (Njoku, 2007). A characteristic
property of the gelation of deoxy-HbS is the existence of a delay time prior to
polymerisation (Imaga et al., 2009). It
is believed that the delay time represents the time required for the formation
of nuclei (Imaga et al., 2009). Thus,
any pharmacologically active compound that could increase the delay time before
polymerisation would help ameliorate the severity of the sickle cell disease
(Njoku, 2007).
1.2 STATEMENT
OF THE PROBLEM
The major problem in sickle cell anaemia is that the substitution
of glutamic acid with valine results in haemoglobin tetramers that aggregate
upon deoxygenation in the tissues (Mehanna, 2001). In low oxygen pressure
condition, HbS aggregate into intracellular polymers that give the erythrocytes
a sickle shape. This shape modification makes the red blood cells fragile and
less flexible. This gives rise to many complications of sicklers (Buchanan et al., 2004). Rigid sickled
cells obstruct capillaries, causing tissue and organ damage (Buchanan et al.,
2004).
Although there are some compounds that prove to be anti-sickling
in nature, most of them are not affordable to the patients and also there are
compatibility issues. The required medication
to treat sickle cell is often not readily available to most rural Africans
especially in the poorest countries. Most of the rural people with this
challenge cannot afford the available treatment as they always need continuous
medication. Sickle cell disease (SCD) cannot be cured; it can only be
controlled so far. Because of the expensive tests, medication and treatment,
rural Africans often rely on traditional medicine to treat this disease which
comes in the form of plant extracts. Several plants have been indicated to have
anti-sickling effects (Iwu et al.,
1988; Imaga et al., 2009; Egba et al., 2012; Imaga, 2013). There is the
need to screen more plants. Moreover, screening for plant extracts that could increase
the solubility of sickle cell haemoglobin is highly desireable since the
problem of sickle cell is associated with altered solubility. This forms the
basis of this investigation.
1.3 AIM OF THE STUDY
The
aim of this research work was to determine the anti-sickling effects of the methanol extracts of B. coriacea and M. pruriens seeds.
1.4 OBJECTIVES OF THE STUDY
The specific objectives of this work
were:
1. To
determine sickling inhibition ability of the methanol extracts of the seeds.
2. To
determine sickling reversal ability of the methanol extracts of the seeds.
3. To
determine the effect of the methanol extracts of the seeds on the polymerisation
of sickled erythrocytes.
4. To
determine the effects of the extracts on the solubility of haemoglobin S.
5. To
determine the effects of the extracts on the osmotic fragility of the sickled
blood.
6. To
determine the effects of the extracts on the Fe2+/Fe3+ ratio
in sickled erythrocytes.
7. To
analyse for the phytochemical, amino acid and vitamin constituents of the
seeds.
1.5 JUSTIFICATION FOR THE STUDY
There
have been a lot of problems encountered in attempt to manage sickle cell
disease. So many chemotherapeutic agents and nutrients have been used on SCD
patients to know if they have any effect on normalisation rate of sickle
erythrocytes and improvement of oxidant status of the RBCs (Ekeke et al., 2000; Ekeke et al., 2001;
Nwaoguikpe and Uwakwe, 2005; Uwakwe and Nwaoguikpe,
2008). There is no end to the search on agents that can be used to treat sickle
cell anaemia. As a result, there is the need for a research that reflects back
to nature. Based on this, several researchers have attempted management of SCD
using plant extracts (Iwu et al.,
1988; Imaga et al., 2009; Egba et al., 2012; Imaga, 2013). The
overwhelming therapeutic effects of the plant, B .coriacea have been reported in the cure of ulcer, waist pain,
asthma, fibroid, impotency (Erhirhie
et al., 2015). Oral tradition
has lent support to these therapeutic effects and thus the use of the name
“wonderful kola” for the plant. Also M.
pruriens has been used for blood boosting in so many African homes (Nebedum
et al., 2015). Research done by Nwaoguikpe et al. (2014) showed that M. pruriens seeds contained antioxidant
vitamins and phytochemicals. However, the anti-sickling effects of these plants
have not been reported. This forms the basis of this research work. Emphasis
was also on whether the extracts could increase the solubility of the HbS as a
possible mechanism of exerting their anti-sickling effects. These findings
prompted the investigation of the anti-sickling effects of these plants.
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