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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.


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



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



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



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 Qualitative phytochemical analyses                                                                28 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



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



5.1       Conclusion                                                                                                      75

5.2       Recommendations                                                                                          75

References                                                                                                      76

Appendix                                                                                                        91








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







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










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).



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.


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.


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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