SICKLING ACTIVITIES of BIOACTIVE COMPOUNDS from SEEDS of Picralima Nitida STAPF (APOCYNACEAE)

PHARMACOGNOSTIC STANDARDIZATION AND ANTI- SICKLING ACTIVITIES OF BIOACTIVE COMPOUNDS FROM SEEDS OF Picralima nitida STAPF (APOCYNACEAE)

OSUALA FELIX NGOZICHUKWUKA PG/Ph.D./08/49833

DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA

JANUARY 2016

TITLE PAGE

PHARMACOGNOSTIC STANDARDIZATION AND ANTI-SICKLING ACTIVITIES OF BIOACTIVE COMPOUNDS FROM SEEDS OF Picralima nitida Stapf (APOCYNACEAE)

OSUALA FELIX NGOZICHUKWUKA PG/Ph.D./08/49833

A THESIS PRESENTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D)

SUPERVISOR: PROF. S.I. OFOEFULE PROF. S.I. INYA-AGHA

DEPARTMENT OF PHARMACOGNOSY AND ENVIRONMENTAL MEDICINES FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA

JANUARY 2016

CERTIFICATION

This project report titled “pharmacognostic standardization and anti-sickling activities of bioactive compounds from seeds of Picralima nitida Stap (Apocynaceae)” is hereby certified as the requirement for the award of Doctor of Philosopher (Ph.D) in the Department of Pharmacognosy and Environmental Medicines, Faculty of Pharmaceutical Sciences, University of Nigeria Nsukka.

------Prof. S.I. Ofoefule Date Department of Pharmaceutics University of Nigeria

------Prof. S.I. Inya-Agha Date Department of Pharmacognosy University of Nigeria

------Prof. C. O. Ezugwu Date Head of Department Pharmacognosy and Environmental Medicine University of Nigeria.

DEDICATION This work is dedicated to God Almighty the provider of good health.

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ACKNOWLEDGEMENTS

I heartily acknowledge with deep appreciation all those who in one way or the other have sacrificed some efforts towards the success of this work. I am very grateful to my supervisors Prof S I Ofoefule whose dynamism led to the start of the Ph. D program as early as possible, and his constant pressure to see the work neatly done. I thank Prof. S. I. Inya-Agha in whose guidance the sketch of the work was made and her sharp advices in the progress of the work. My thanks go to Dr. Mrs. U. E. Odoh who offered time immensely to correct this work, and in her guidance the work was organized and brought to its logical end.I appreciated the roles played by Prof. C. O. Ezugwu at start and on the progress of this programe. I thanked the staff and management of STRATECH LAB Enugu, for allowing me access to their laboratory facilities where fractionation of the methanol extract and anti-sickling activities of the fractions were carried out. I acknowledge the efforts of staff of Madonna University Teaching Hospital LAB Elele, MLS Lab Nnamdi Azikiwe University, Springboard Lab Awka, Viva laboratory Nnewi, Biotechnological Laboratory Institute Nnamdi Azikiwe University Awka and Lionel Hill, John Innes Centre (JIC) United Kingdom where Spectral data were done and analyzed are herein acknowledged I must express my gratitude to my wife Lolo, Dr (Mrs) Eunice O.Osuala, Head of Department of Nursing, Nnamdi Azikiwe University, Awka, and my children Kingsley, Henrietta, Prisca and Rosemary for their efforts; they were accommodative even when inconvenienced in the course of this work. I pray that God will grant you all abundant blessings.

ABSTRACT

This present study was to establish the pharmacognostic standards of the Picralima nitida, to investigate the anti-sickling effects of crude methanol seed extract and fractions of Picralima nitida, to isolate and elucidate the structure of the bioactive constituents. Phytochemical analysis of the extract, fractions and isolates were carried out using standard procedures. Pharmacognostical profile and proximate/numerical standards were also evaluated. The anti-sickling effect was studied using sodium methabisulphite-induced sickling of the HbSS red blood cells obtained from confirmed non-crisis state (steady state) sickle cell patients. The effects of P. nitida on biochemical parameters of HbSS blood were studied. The effect of the extract on the rheology and viscosity of sickle cell blood were studied using various concentrations (2.5, 5 and 10 mg/ml) at 30 minutes intervals through 180 minutes of incubation. Comparison of the anti-sickling effect of methanol seed extract of P. nitida, fractions and isolates on sickled red blood cells was done using a known standard drug, p-hydroxybenzoic acid. The microscopy of the powdered seeds revealed the presence of sclereids, parenchyma and epidermal cells, calcium oxalate crystal and fat globules. Qualitative and quantitative phytochemical analyses of the extract showed the presence of alkaloids (5.84 g), tannins (0.065), flavonoids (7.03 g), glycosides (4.58 g), terpenoids (4.08 g), protein (11.63 g), steroids, resins, reducing sugars, carbohydrates, fats and oils. The analytical standards gave 3.0, 11.5, 3.5, 4.0, 12.5, 20.4 and 8.0 % for moisture content, total ash, acid insoluble ash, water soluble ash, sulphated ash, alcohol soluble and water soluble extractives respectively. The acute toxicity test showed that the extract was safe at dose 3500 mg/kg. The study showed significant (p < 0.05) antisickling activities of the fractions on HbSS blood and were given to be in order CHCl3 ≤ CH2CL2 < EtOAc < Aqueous at the tested concentrations. The Aqueous Fraction showed highest degree of inhibition (85%) and was comparable to P- hydroxybenzoic acid (100%). The negative control (normal saline) showed no change, Compound 1 at 2.5 mg/ml showed the best anti-sickling activity (100% reversal of sickling at 120 min) compared to that of the standard drug p-hydroxybenzoic acid (100 % reversal of sickling at 180 min). The anti-sickling activity of the compounds are in the order CP1 (100 %) > CP3 (95 %) > CP2 (92 %) unsickling at 120 min. However compound 3 and compound 2 at 5 mg/ml showed 100 % reversal of sickling at 180 min.The isolated compounds are Ajmalicine → (19 α)-16, 17-didehydro – 19 – methyl – 2 –oxayohimbin – 16- carboxylic acid methyl ester, was obtained as white powder. Its molecular formula was derived as C21H24O3 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 353.1867. (1) And Ajmalicine Oxindole B → (19 α) -19-methyl -2-oxoformosanan -16- carboxylic acid methyl ester, was obtained as light yellow powder. Its molecular formula was derived as C21H24O4 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 369.1815. (2). The results of the study suggest that the methanol extract and fractions of P. nitida possess antisickling effect and justify’s the ethnomedicinal claims

TABLE OF CONTENTS

Title page i Certification Page ii Dedication iii Acknowledgements iv Abstract v Table of Contents vi List of Plates vii List of Tables viii List of Figures ix

CHAPTER ONE: INTRODUCTION 1.1 Background of the study 1 1.2 Phytotherapeutic uses of African Medicinal plants in Disease Management. 6 1.3 Statement of Problem 7

CHAPTER TWO: LITERATURE REVIEW 2.0 Review of Related Literature 8 2.1 Herbal Medicine 8 2.2 The African Medicinal Plants 9 2.2.1 Traditional management of Sickle Cell Disease 10 2.2.2 Research update on plants used in treatment of sickle cell disease 11 2.2.3. The family – Apocynaceae 15 2.2.4 Ethnomedicinal uses of P. nitida, (Stapf) Durand & H. Durand 17 2.3 Sickle Cell Disease 33 2.3.1 Signs and symptoms 38 2.3.2 Clinical Manifestation 42 2.3.3 Management 49 2.3.5 Transfusion therapy 51 2.3.6 Management of chronic complications 53 2.3.7 Epidemiology 54 2.4 Electrophoresis 55 2.4.1 Packed Cell Volume (PCV) 56 2.4.2 Erythrocyte Sedimentation Rate (ESR) 56

2.4.3 Hemoglobin 58 2.4.4 Red Blood Cells Count 59 2.4.5 Plasma Calcium 61 2.4.6 Conductivity 64 2.4.7 Total dissolved solids (TDS) 65 2.5. Objectives of the Study 67 2.6. Research Hypothesis 67 CHAPTER THREE: MATERIALS AND METHOD

3.1 Collection and Preparation of Plant Materials 68 3.2 Chemicals/Reagents 68 3.3 Macroscopic and microscopic analysis 68 3.4 Extraction and Fractionation of P. nitida P. nitida seed extract 68 3.5 Purification and Isolation of P. nitida seed extract 69 3.6 3.6 Characterization of isolated compounds 69

3.7 Phytochemical Screening 71

3.7.1 Quantative Phytochemical Analysis 75 3.7.2 Determination of the Mineral Content of the extract of P. nitida. 76 3.7.2.0 Determination of the Analytical Standards 3.7.2. 1. Determination of Total Ash Values 76 3.7.2.2 Determination of Acid Insoluble Ash Value 76 3.7.2.3 Determination of Water Soluble Ash Value 77 3.7.2.4 Determination of Water Soluble Extractive Value 77 3.7.2.5 Determination of Alcohol Soluble Extractive Value 77 3.7.2.6. Determination of Moisture Content 78 3.7.3 Chromatographic Analysis of the Extract and Fractions of Picralima nitida (STAPF) 78

3.7.4 Acute Toxicity Study (LD50) 79 3.7.5 Blood Collection and Preparation 80 3.7.6 Determination of Packed Cell Volume (PCV) 80 3.7.7 Determination of Erythrocyte Sedimentation Rate (ESR) 80 3.7.8 Hemoglobin Determination. 80 3.7.9. Red Blood Count 81 3.7.10. Determination of Plasma Calcium 81 vii

3.7.11 Conductivity 81 3.7.12. Measuring total dissolved solids (TDS) 82 3.7.13 Measurement of Viscosity 82 3.7.14 Observation of normal cell morphology of collected blood sample 3.8 Anti Sickling Evaluation 83 3.8.1 Induction of complete sickling in normal Blood (Genotype As) using Sodium methabisulphite 83

3.8.2 Calculation of % reversal of sickling in HbAs red blood cells 83

3.8.3. Antisickling activity of crude methanol extract, fractions and isolates of P.Nitida 84 3. 9. Statistical analysis 84

CHAPTER FOUR: RESULTS

4.1: Macroscopic and Microscopic analysis 85 4.2: Yield of Extraction 88 4.3: Phytochemical analysis 89 4.4: Mineral content analysis 90 4.5: Acute toxicity test 90 4.6: Antisickling Evaluation 91 4.7: Chromatographic analysis 104 4.8: Analytical standards 105 4.9: Characterization of isolated compounds 106

CHAPTER FIVE: DISCUSSION AND CONCLUSION Discussion 123 Conclusion 131 References 132 Appendices 151

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LIST OF PLATES

Plate 1: Features of blood Cells in minor blood vessel 3 Plate 2: The plant Picralima nitida (Stapf) Fam. Apocynaceae 23 Plate 3: The matured fruits of Picralima nitida. (Stapf) Fam. Apocynaceae 24 Plate 4: The dried seeds of Picralima nitida (Stapf) Fam. Apocynaceae 25 Plate 5: Microscopy of the Powdered Seed of P. nitida 86 Plate 6: Microscopy of the Powdered Seed of P. nitida 87 Plate 7: Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida 114 Plate 8: Incubation of HbSS blood in MeOH extract 5 mg/ml of P.nitida 115 Plate 9: Incubation of HbSS blood in MeOH extract 10 mg/ml of P.nitida 116 Plate 10. Example of normal blood cells 117

Plate 11: TLC of P.nitida seeds extract, solvent system: MeOH : CHCl3 117 Plate 12: TLC of P.nitida seeds extract, solvent system: Chloroform Methanol (45:15) Adsorbent Silica gel, UV at 254 nm reagent Dragendorff’s 118

Plate 13: TLC of P. nitida seed extract, Solvent system Chloroform CHCl3l:

H2O (65:35:10) 118 Plate 14: TLC of MeoH extract and fractions of P.nitida seed, β-carboxylic acid, PHBA Solvents MeOH and CHCl3 (65 : 35), adsorbent: silica gel, UV at 357 nm 119

Plate 15: TLC of Sub-fraction (AqSF1 – 3) of P.nitida seed and PHBA 119

Plate 16: TLC of CP 1 - 3 of P.nitida seed and PHBA . Solvents MeOH and CHCl3 ( 65 : 35), adsorbent: silica gel, UV at 357 nm 120

Plate 17: TLC of Isolates compound 1 compound 2; and pure sample (Ajmaciline) Solvents MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm 120

Plate 18: Two dimension TLC of the isolate compound 1: Solvents MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm 121

Plate 19 Two dimension TLC of the isolate compound 2: Solvents MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm 122

LIST OF TABLES Table 1: Common names of P. nitida (Burki1l, 1985) 19 Table 2: Ethnomedicinal uses of P, nitida in African countries 20 Table 3: ESR reference range putting age into consideration is shown below 58 Table 4: Organoleptic indices of P. nitida. 85 Table 5: Yield % of extract and fraction from P. nitida seed 88 Table 6: Phytochemical analysis of P. nitida seed 89 Table 7: Quantitative phytochemical analysis of P. nitida 90 Table 8: Mineral analysis of P. nitida 90

Table 9: Acute toxicity study (LD50) of P. nitida 90 Table 10: Antisickling effect of methanol extract of P. nitida seeds on HbSS red blood cells 91

Table 11: Antisickling effect of chloroform fraction of P. nitida on HbSS red blood cells 91

Table 12: Antisickling effect of ethyl acetate fraction of P. nitida on HbSS red blood cells 92

Table 13: Antisickling effect of aqueous fraction of P. nitida on HbSS red blood cells 92

Table 14: Antisickling effect of dichloromethane fraction of P. nitida on HbSS red blood 93

Table 15: Antisickling effect of methanol extract of P. nitida on HbAS red blood cells 93

Table 16: Antisickling effect of aqueous of Fraction of P. nitida on HbAS red blood cells. 94

Table 17: Antisickling effect of Chloroform fraction of P .nitida on HbAS red blood cells 94

Table 18: Antisickling effect of ethyl acetate fraction of P .nitida on HbAS red blood cells 95

Table 19: Antisickling effect of dichloromethane fraction of P. nitida on HbAS red blood 95

Table 20: Effect of methanol extract of P.nitida on Viscosity of HbSS red blood 96 Table 21: Effects of chloroform fractions of P.nitida on HbSS red blood 96 Table 22: Effect of aqueous fraction of P. nitida on viscosity of HbSS red blood 97 Table 23: Effect of Ethyl acetate fraction of P. nitida on Viscosity of HbSS red blood 97

Table 24: Effect of Dichloromethane fraction of P. nitida on viscosity of HbSS red blood 98

Table 25: Effect of Methanol extract(2.5mg/ml) of P. nitida on some biochemical Parameters 98

Table 26: Effect of Methanol extract (5 mg/ml) of P. nitida on some biochemical Parameters 99

Table 27: Effect of Methanol extract (10mg/ml) of P. nitida on some biochemical Parameters 99

Table 28: The effects of aqueous fraction (2.5 mg/ml) of P. nitida on some Biochemical 100

Table 29: Effect of aqueous fraction (5 mg/ml) of P. nitida on the biochemical Parameters 100

Table 30:Effect of aqueous fraction (10 mg/ml) of P. nitida on some biochemical Parameters 101

Table 31: Effect of Dichloromethane fraction (2.5 mg/ml) of P. nitida on some biochemical parameters 101

Table 32: Effect of Dichloromethane fraction(5 mg/ml) of P. nitida on the biochemical parameters 102

Table 33: Effect of Dichloromethane fraction (10 mg/ml) of P. nitida on some biochemical parameters 102

Table 34: Antisickling effect of isolated pure compounds from P. nitida on HbSS red blood cells. 103

Table 35: TLC of Methanol extracts and Fractions 104 Table 36: TLC of sub-fractions (AqSF1 -3) of P. Nitida 105 Table 37: TLC of aqueous sub-fraction 34, 23, 21 of P. nitida,and PHBA Solvent MeOH 65 : CHCl3 35, adsorbent: silica gel 40 mesh 105

Table 38. TLC of Isolates compound 1 compound 2; and pure sample (Ajmaciline) Solvents MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm 105 Table 39: Analytical standards of Picralima nitida 105 1 Table 40: H -NMR (δH in ppm, 500MHz) Data of Compounds 1(Ajmaciline) and 2 (Ajmaciline oxindole B) 112

13 Table 41: C -NMR (δC in ppm, 125MHz) Data of Compounds 1(Ajmaciline) and 2 (Ajmaciline oxindole B) 113

LIST OF FIGURES

Fig 1: The chemical structures of the alkaloids isolated from P. nitida 27

Fig 2: Coumestans and coumestan glycosides isolated from the roots of P. nitida 29

Fig 3: Flow chart of the purification and isolation of compounds of P.nitida seed extract 70

Fig 4: Structure of Compound 1-Ajmaciline The Electron Ionization (EI) 106

Fig 5: Structure of Compound 2-Ajmaciline oxindole B 106

Fig. 6: The Mass spectrumbase peak chromatogram of compound 1 (Negative mode) 107

Fig. 7: The Mass spectrum base peak chromatogram of compound 1 (Positive mode) 107

Fig. 8: The Mass Spectrum base peak chromatogram of compound 2 (Negative mode) 108

Fig .9: The Mass spectrum base peak chromatogram of compound 2 (Positive mode) 108

Fig. 10: The UV chromatograms of Compound 1 109

Fig .11: The UV chromatogram of compound 1 (Ajmaciline) at 257nm in 50% MeOH 109

Fig.12: The 13C-NMR Spectrum of Compound 1-Ajmaciline 110

Fig.13: The 13C-NMR Spectrum of Compound 2-Ajmaciline oxindole B 110

Fig.14: The 1H-NMR Spectrum of Compound 2-Ajmaciline oxindole B 111

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CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

The prevalence of sickle cell disease globally, and more especially, among the developing countries calls for a great concern. Wonkam et al., (2012) reported that in Cape Town, South

Africa, over the past 10 years, the frequency of sickle cell disease has increased considerably, imposing a significant burden and new challenges to the health services in Cape Town. Sickle cell disease resulting from abnormal functional sickled red blood cell is a chronic hereditary blood disease that affects virtually every organ, with liver, spleen, heart and kidneys as the major susceptibles (Gillian and Christopher, 1999).

The disease state which is characterized by severe anemic manifestation, vaso- occlusiveness, painful crises and acute organ damage (Serjeant et al., 1974), is one of the non- communicable diseases with serious socio-economic and health consequences. Owing to the debilitating effects, a sickle cell patient has a lifelong battle against a cocktail of health problems, cutting across pains, infections, anemia, and organ damage to heart failure and stroke.

According to Platt et al (1994), sickle cell shortens life expectance to an average of 42 and

48 years for males and females respectively. This age bracket is the active productive age, making sickle cell disease an economic antagonistic disease. Again, within the short life span of a sickle cell patient, most of the time and much of money are spent in management of one crisis or the other with the effect that a significant economic burden is imposed on the patient

(Graham et al., 1982).

This situation becomes aggravated in developing countries where per capita income is low and the level of education low, with its associated poor hygiene and nutrition is also poor

(Wonkam et al., 2012). The high depressive and often dependent life of a poor sickle cell

1 patient, most especially during the crisis period affects the entire personality of the victim, resulting in some cases in mental ill health (Thomas and Taylor, 2002).Ironically, depression and other forms of mental illness affect hormonal balance and entire haemostatic status of the individual, and thereby lowering the immune system. This will in turn result to the development of secondary diseases in the sickle cell patient, a cyclic affair (Wilson et al.,

1999).

Sickle cell disease can be described as a condition or disease state characterized by severe disease manifestation, vaso-occlusiveness, painful crisis and acute organ damage resulting from abnormalities in the haemoglobin of red blood cell (Serjeant et al., 1974). Sickle cell disease changes normal, round red blood cells into cells that can be shaped like crescent moon. The name 'sickle -cells' comes from the crescent shape of the cells. A sickle is a farm tool with a curved blade that can cut crops like wheat. When in normal condition red blood cells move easily through the blood vessels taking oxygen to every part of the body, but when the red blood cells are sickled they can get stuck and block blood vessels, which stops the oxygen from getting through; that can cause a lot of pains. It causes harm to organs, muscles and bones. Features of blood cells are shown in plate 1.

Plate 1: Features of blood Cells in minor blood vessel

A sickle cell patient usually has a lifelong battle against these health problems: pains, infection, anaemia, organ damage and stroke. Sickle cell disease (drepanochytosis) is a genetic life-long disorder characterized by red blood cells that assume an abnormal rigid, sickle shape. Sickling decreases the flexibility of the red blood and results in a risk of various complications.

Sickling occurs as a result of mutation in the haemoglobin gene. Sickle cell in childhood, occur more commonly in people from parts of tropical and sub-tropical regions where malaria is common.

About one third (1/3) of all indigenous inhabitants of sub-Sahara Africa carry the gene because in areas where malaria is common there is a survival value in carrying only one of the two alleles of the sickle cell disease which are more resistant to malaria since the infestation of malaria plasmodium is halted by the sickling of the cell which it infests.

Sickle cell is an inheritable disease which occurs where a pair of homozygous sickle shaped cells; SS are inherited from both parents, each donating one sickle gene. Various types of sickle disease result from abnormality in haemoglobin and they include: Sickle cell anaemia, a homozygous pair of abnormal hemoglobin is the most common form of abnormal haemoglobin found in the general population of sufferers.

Haemoglobin C disease: this is another type of haemoglobin described after haemoglobin S

(Hbs). It is rare.

Haemoglobin D disease: This is abnormal haemoglobin. It exists in a heterozygote pair with normal haemoglobin (Sergeant et al., 1994). The other rare forms of sickle cell disease are compound heterozygous states in which the person has only one copy of the mutation that causes Hbs and one copy of the other abnormal haemoglobin allele.

The flow of blood through the vascular system is proportional to the fourth power of the radius of the vessel and is inversely proportional to the viscosity as stated in Poiseuille’s law

(Gillian and Christopher, 1999).

The flow of blood is not uniform across the diameter of a blood vessel. The layer of fluid next to the vessel wall tends to adhere to it and the neighboring layer tends to adhere to this static layer and so on. Thus the velocity of flow is fastest in the middle of the vessel and slowest next to the wall. When the different layers slip smoothly past each other this is known as Laminar, but when it is broken up by irregularity in the vessel wall such as an athermanous plague, eddies form and the flow is said to be turbulent.

Other factor that may promote irregularity of blood flow is the morphology of the red blood cell. A sickled red blood cell exhibits high viscosity along the arterial wall, and since the blood flow is inversely proportional to its viscosity, it then follows that the more viscous a blood cell the lesser its flow. This is the fundamental basis for hypoxia experienced by sickle cell patient, during crisis. Again, the more viscous a blood, the more it will stick to the arterial walls, thereby encouraging narrowing of the blood capillaries, and promoting vascular pressure (Berne and Levy, 1992; Guyton and Hall, 1996).

When measured in a conventional viscometer, the apparent viscosity of blood is about 2.5 times that of water which is 1.05, which is to say blood is about 2.63 poseuliues. In living tissues, however, this apparent viscosity of the blood is about half of this (1.32). This anomalous behaviour is due to the tendency of red blood cell to flow along the central axis of the smaller blood vessels- a phenomenon known as axial streaming (Levick, 1995). The mechanisms responsible are not fully understood, and it appears that the flexibility of the red blood cells is an important factor. At the low velocities found in the microcirculation, rigid particles tend to remain uniformly distributed across the vessel while the flexible particles migrate towards the central axis.

In sickle cell crises, the sickled red blood cells loose their flexibility because of the assumed sickled morphology, making the conventional axial flow impossible. This reduces flow rate and flow volume, with the effects that organ systems are denied their normal requirement of

5 blood, leading to organ damage and heart failure (Junquieira et al., 1995). While many drug requirements involved in sickle cell treatment are targeted towards the management of associated or secondary diseases, the most important treatment mechanism seem to be that which will reverse the sickled red blood cell.

1.2 Phytotherapeutic Uses of African Medicinal Plants in Disease Managements

The use of medicinal plants in disease management is fast gaining grounds in developing countries. The holistic use of plants in health care affords the benefits of maximizing all the essential ingredients that nature has endowed to humanity. Some of these have antioxidants, anti-inflammatory, anti-microbial and anti-adhesion properties while others may be involved in boosting the immune system, working as analgesic, aphrodisiac or aiding general metabolism.

The use of extracts of the following plants, Piper guineensis, Pterocarpa osun, Eugenia caryophyllala and Sorghum bicolour in treatment of sickle cell disease have been reported,

(Wambebe et al., 2001). Ugbor (2006) reported the increase of the gelling time of sickle cell disorder by the extracts of Pterocarpus santolinoides and Aloe vera, while Sofowara, (1981) reported the reversal of sickling by the root extracts of Fagara zenthoxyloides. The use of

Scoparia dulcis in the management of sickle cell disease was speculated (Orhue et al, 2005;

Orhue, 2006).

Owing to the many uses of P. nitida in various disease management as reported in local and international literatures, there is need to explore its further phytotherapeutic applications.

This work is predicated on the above need.

1.3 Statement of the Problem

The economic burden and psychosocial pressure associated with sickle cell disease management is of global health concern, calling for concerted efforts of health planners and health care takers at all levels. In developing countries like Nigeria, the burden is manifested in the much money that is spent in procurement of synthetic drugs and in the psychological crises resulting from infant mortality. The drugs are not only expensive to the average rural community dweller but are also not readily available, thereby compounding the problems of sickle cell disease.

A recent WHO report estimated that around 2 % of newborns in Nigeria were affected by sickle cell anaemia, giving a total of 150,000 affected children born every year in Nigeria alone. The carrier frequency ranges between 10 % and 40 % across equatorial Africa, decreasing 1-2 % on the North African coast and less than1 % in South Africa, (WHO, 2010).

Thus, sickle cell disease is a significant health problem in Nigeria and its managements should go beyond conventional synthetic chemotherapy.

The use of herbal medicine needs to be encouraged. P. nitida, a multi potent plant, used in various disease managements in West Africa, holds a strong promise as a native medicinal plant with potentials for sickle cell disease management.

CHAPTER TWO

REVIEW OF RELATED LITERATURE

2.1 Herbal Medicine

Herbal medicines are referred to as ‘Drugs’ because they have therapeutic benefits and are used to treat either the cause or the symptoms of illness. Herbal medicines are also known as natural medicines. Phytomedicines are according to the world health organization (WHO)

1984, finished labeled medicinal products that contain active ingredients, aerial or underground parts of plants or other plant material, or combinations thereof, whether in the crude state or as plant preparations. Plant material includes juices, gums, fatty oils, essential oils, and any other substance of this nature. Herbal medicines may contain excipients in addition to the active ingredient. Medicines containing plant material combined with chemically defined active substance, including chemically defined isolated constituents of plants are not considered to be herbal medicines. Exceptionally, in some countries, herbal medicines may also contain by tradition, natural, organic or inorganic active ingredients which are not of plant origin (Shariff, 2001).

Herbs have been used for several generations and their traditional knowledge is irreplaceable.

The medicinal herbs are nature’s remedies and have been placed there by the creator of the universe. They are God’s remedial agents for afflicted humanity. Thus herbal treatment is designed to return body to its healthy state by stimulating natural metabolism and maintaining system functions. Being natural, they are less aggressive than synthetic ones and they have no significant side effects (Sharrif, 2001).

A World Health Organization (WHO) survey indicates that about 70-80 % of the world population particularly in the developing countries rely on non-conventional medicines mainly of herbal sources in their primary health care (Aderele 1993; WHO 1998). The WHO has described traditional medicine as one of the surest means to achieve total health care

8 coverage of the world’s population. In pursuance of its goal of providing accessible and culturally acceptable health care for the global population, the WHO has encouraged the rational use of traditional plant based medicines by member states and has developed technical guidelines for assessment of herbal medicine WHO (2000).

Since the advent of antibiotics in 1950s, the use of plant derivatives as antimicrobials has been virtually of no existence. In recent decades, scientists have come to realize that the life span of antibiotic is limited. Worldwide spending on finding new anti-infective agents

(including vaccines) is expected to increase 60% from the spending levels in 1993 (Alper,

1998). Thus new sources, especially plant sources are also being investigated.

2.2 The African Medicinal Plants

The plant kingdom holds many species containing substances of high medicinal value. In the

African continent alone over 5000 species of plants are known to occur in the forest region and most of them have been used for several centuries in traditional medicine for prevention and treatments of diseases (Iwu, 1993)

Healing has existed in Africa as far back as at the 3200 BC and associated with the reign of

Menes the first pharaoh in Egypt whose son had many preparations from herbs used in traditional healing. The honor of the first African physician in a scientific sense actually belongs to the great Imhotep, who live about 2980 BC had been called the gold of medicine

(Ghalio-Ungui 1973). It is unfortunate that only few African medicinal plants are recognized in the modern day Pharmacopeias even when there are numerous African varieties of such official drugs that are of high medicinal value.

The general knowledge of African medicinal plants is very limited and their documentation is becoming increasingly threatened by deforestation and anthropogenic activities. It is obvious that many species of plants of potent medicinal values in Africa are yet to be

9 discovered and many of them are being screened for possible Pharmacological activities

(lwu, 1993).

2.2.1 Traditional management of Sickle Cell Disease

The practice of herbalism or botanical medicine using medicinal plants is as old as man

(Evans, 2002).The growing sophistication in life style among world population makes it imperative to refer to herbal practice as alternatives or complementary medicine to appeal to a cross section of people irrespective of their cultural affiliation.

In the African world these type of herbal practices which are not of western or orthodox medicine existed and has remained largely unrecognized. This was attributed to the socio cultural, socio economic heritage, lack of basic health care and personnel to take charge of every nook and cranny of the rural populations (Adebanjo et al., 1983).

There is a belief by traditional healers, that the bioactive ingredients that have therapeutic activities in plants have holistic nature of treatment. Substances found in medicinal plant containing the healing property of the plant are known as the active principle (Adebanjo et al., 1983). It defers from plant to plant and examples of active principles can be anthraquinones, flavonoids, glycosides, saponins, tannins etc and other compounds such as morphine, atropine, codeine, steroids, lactones and volatile oils which possess medicinal value for the treatment of different diseases (Chevalier, 2000).

Recently these active principles have been extracted and used in different forms such as infusion, syrups, concoctions, decoctions, infuse oils, ointments and cremes (Sofowora,

1993). Since most plants have medicinal effect, it is of utmost importance that their efficiency and toxicity risk are evaluated (Evans, 2002).

Due to the challenges faced by the scientists in developing countries, so many sources of constituents capable of ameliorating the sickle cell crises have been investigated with a view to contribute to search for substances that would be effective in solving the sickle cell disease

10 problem. Based on this; the anti -sickling effects of different substances have been investigated.

It was established that extracts of dried fish (Tilapia) and dried prawns (Astacus red) have the ability to inhabit polymerization of sickle cell hemoglobin (HBS), improves the Fe2t/Fe3t status and lower the activity of lactate dehydrogenase(LDH) in the blood plasma. The level of

LDH in sickle cell blood, determines the severity of crises (Nwaoguike et al., 2005) as it is sensitive indicator of haemolysis.

The following preparations hydroxyurea, erythropoitien and tucaresol have been found to reduce LDH activities and bilirubin level in serum, as well as increase the level of fetal haemoglobin (HBF) (Goldberg et al., 1992; Roopen, et al., 1996).

There have been reports on effective management of sickle cell disease patients during pregnancy (Mou Sun et al., 2001; Hassel, 2005) In developing countries in the world including Nigeria, some known medicinal plants have been used in the treatment of painful crises associated with sickle cell diseases. The sources of information on the use of medicinal plants are local herb sellers, unorthodox doctors and those whose knowledge were passed down to them by their ancestors. Scientific researches on the claims of the traditional healers are being undertaken to evaluate and authenticate the traditional use of the plants.

2.2.2 Research update on plants used in treatment of sickle cell disease.

The lack of sophistication in medicinal plant research in most places that have high incidence of sickle cell disease, has not allowed an in-depth study on the bioactive constituents of the medicinal plants for the management of sickle cell disease. Medicinal plant researchers in their quest to identify the active components of the medicinal plant which have shown great potential in sickle cell disease managements are hindered by this.

However, much has been done in phytochemical examinations. Phytochemical examinations of the extract of herbal formula ‘Jajawaron’ whose main constituents are roots of Cissus

11 populneal, was found to contain anthraquinone derivatives, steroidal and cardiac glycosides.

Alkaloids and tannins were however absent in Cissus Populneal K extracts (Moody et al.,

2003). Vanhaelen-Fastre et al., 1999 carried out an in vitro antisickling activity of a rearranged limonoid isolated from Khaya senegalensis.

The compound 2-dehydroxybenzoic acid was isolated from Zanthoxyllum macrophylea, while in the Nigerian Fagara zanthoxyloides, the bioactive compounds responsible for antisickling effects were identified as vanillic acid, P-hydroxy benzoic acid and P-fluorobenzoic acid Sofowora et al (1983).

Zanthoxylum macropylum (root), Elekwa et al., (2005), identified Z-hydrobenzoic acid content, mechanism of action-membrane stabilization lower than phenylalanine, Sofowora

(1981) reported the reversal of sickling by the roots of Fagra zanthoxyloides. In the antisickling properties of Zanthoxyloides root extract it was observed that the aqueous extract preserved the colour of red blood cells during a screen for its antimicrobial activity (El-Said et al., 1971). The extract was later shown to revert sickled HbAs and HbSS and crenated

HbAA blood cells to normal in vitro (Sofowora et al., 1979). The activity was also demonstrated in the root of other Zanthoxylum species. Zanthoxylum gillet was found to be as active as Zanthoxylum Xanthoxyloides (Adesanya and Sofowora, 1983). Active principles were found to be hydroxybenzoic acid derivatives, vanillic acid and P-hydroxybenzoic acid.

In 1961, the determination of the X-ray crystal structure of quaterinary salt echitamine

(Hamilton et al., 1961) allowed the final establishment of the structure and absolute configuration of Pseudo-akuammingine, Akuammine and Akuammiline based on their chemical correlation with echitammine

Phytochemical and antisickling activities screening of Entandrophragma utile, Chenopodium ambrosioides and Petiveria alliacea were done by Adejumo et al., 2011.

Adejumo et al., 2010 O.E., carried out in vitro antisickling activities and phytochemical evaluation of Plumbago zeylanica and Uvaria chamae.

Adejumo et al., 2012.carried out an in vitro study of antisickling activity of Moringa oleifera

Lam. (Moringaceae) grown in Nigeria on deoxygenated erythrocyte cells.

Afolabi et al 2012. screened Solenostemon monostachyus, Ipomoea involucrata and Carica papaya seed oil versus Glutathione, or Vernonia amygdalina: methanolic extracts of novel plants for the management of sickle cell anemia disease.

Akinsulie et al 2005 did Clinical evaluation of extract of Cajanus cajan (Ciklavit) in sickle cell anaemia.

Wambebe et al, (2001). Double-blind, placebo-controlled, randomized cross-over clinical trial of NIPRISAN in patients with sickle cell disorder.

Elekwa et al., 2003 carried out Studies on the effect of aqueous extracts of Garcinia kola seed on the human erythrocytes adenosine triphosphatase of HbAA, HbAS, and HbSS genotypes.

Elekwa et al., 2005. in vitro effects of aqueous extracts of Zanthxylum macrophyla roots on adenosine triphosphatases from human erythrocytes of different genotypes and in vitro effects of aqueous extracts of Zanthxylum macrophyla roots on adenosine triphosphatases from human erythrocytes of different genotypes.

Phytochemical and antioxidant nutrient constituents of Carica papaya and Parquetina nigrescens extracts were screened by Imaga et al., 2010.

Mgbemene C.N, Ohiri F.C, 1999.carried out a study on Anti-sickling potential of

Terminalia catappa leaf extract.

Moody et al, 2003. Anti-sickling potential of a Nigerian herbal formula (ajawaron HF) and the major plant component (Cissus populnea L. CPK).

Mpiana et al, 2013 worked on in Vitro Sickling Inhibitory Effects and Anti-Sickle

Erythrocytes Hemolysis of Dicliptera colorata.

Mpiana et al, 2007 carried out an in vitro study on Antisickling Activity of Anthocyanins from Ocimum basilicum L. (Lamiaceae). Mpiana et al in 2008 carried out a study on

Antisickling activity of anthocyanins from Bombax pentadrum, Ficus capensis and Ziziphus mucronata; photodegradation effect.

Mpiana et al, 2009. Antisickling activity of anthocyanins of Jatropha curcas;. And in 2010. in vitro effects of anthocyanin extracts from Justicia secunda Vahl on the solubility of haemoglobin S and membrane stability of sickle erythrocytes.

Nwaoguikpe, R.N., Uwakwe, A.A., 2008. In vitro antisickling effects of Xylopia Aethiopica and Monodora Myristica..

Oduola et al, 2006 worked on Antisickling agent in an extract of unripe pawpaw (Carica papaya).

In vitro effects of AGED garlic extract and other nutritional supplements on sickle cell erythrocytes.

Okpuzor, J, Adebesin, O., 2006. Membrane stabilizing effect and antisickling activity of

Senna podocarpa and Senna alata.

Onwubiko, H.A., 2010 worked on the Anti-Sickling Properties of Ethanol Extracts of

Euphorbia heterophylla and Moringa oleifera Leaves.

Ouattara et al, 2004. LC/MS/NMR analysis of isomeric divanilloylquinic acids from the root bark of Fagara zanthoxyloides Lam. and Ouattara et al., 2009 investigated on Antisickling properties of divanilloylquinic acids isolated from Fagara zanthoxyloides Lam. (Rutaceae).

Thomas, K.D., Ajani, B., 1987. Antisickling agent in an extract of unripe pawpaw fruit

(Carica papaya).

Ugbor C, 2006. The effect of vegetable extracts on the antisickling potential of Aloe vera.

2.2.3. The family - Apocynaceae

The family Apocynaceae consists of about 250 genera and 2000 species which is closely related to Asclepiadaceae (Nyunai et al., 2006). It is distributed primarily in the tropics and subtropics, poorly represented in the temperate regions. In China about 44 genera and 145 species are present, one genus and 38 species are endemic and nearly 95% of the taxa grow in the southern and south western parts of the country (Tsiang et al., 1977) the important genera are arranged under two sub-families namely: Plumierideae and Apocynoideae.

The genus of Plumierideae is futher classified into the following subfamilies ie: Plumeria,

Alstonia, Aspidosperma, Cataranthus, Vinca, Rauwolfia, Carissa, Lochnera, Picralima, etc; while in Apocynoideae exist the following: Strophantus, Funtumia and Dipladenia (Evans,

2000).The known features of the Apocynoideae are that they grow as trees; shrubs of vine rarely sub shrubs. The leaves are simple, opposite rarely whorled or alternate with pinnate veins (Tsiang et al., 1977). The leaves are entire margined. The flowers are gamoseparlous and are made up to four to five lobes. Most of the plants are hermaphrodites. The calyx is 5- or 4- partice with basal glands usually present. The plant contains latex in non -articulated branches or un-branched laticifers and the vascular bundles are bicollateral (Evans, 1996).

The ovaries are superior, rarely half inferior, connate or distinct; l-or 2- lobular, one stile, pistil head capitates, conical or lampshade-shaped based stigmatic. The family produces different types of fruits which include berry, drupe capsule or follicle. The seeds are with or without coma; endosperm is thick and often hairy, scanty, sometimes absent. The embryo is straight or nearly so, the colydons are often large radical teret (Tsiang et aI., 1977).

The major constituents of the family include a vast range of Cyanogenetic glycosides,

Saponins, Coumarins, Tanins, Indole alkaloids, leucoanthocynnins, Triterpenoids, Cyclitols and Phenolic acids; Cardio active glycosides and steroidal alkaloids (Kouitcheu et al, 2005).

The plants of the family Apocynaceae are often poisonous but some species are valuable

15 source of medicine, insecticides fibres and rubber. Picralima exists in about six known species: P. elliotti, P. klaineana, P. laurifolia, P. macro-carpa, P. umbellate, P. nitida.

Plant description of P. nitida

Plant Description

When fully grown, the tree has a height up to 15-30 m. The stem is glabrous. The girth is about 60 cm or more with a dense crown and dark-brown or blackish brown. The leaves are opposite, simple and entirely with stipules. The bark is hard, brittle and pale to dark grayish black or brown and smooth to slightly rough or finely striped (Nyunai et al., 2006). The leaves are oblong-Ianceolate to broadly oblong elliptic, shortly acuminate, blade, rounded to slightly cuneate. It is shiny above with numerous parallel lateral veins, 10-26 cm by 2-13 cm wide.

The apex is abrumptly acuminate. The plant flowers white between April and August in the rainy season. The corolla is about 2-5 cm long, glabrous outside with ribbed tube. The calyx is leathery and lobed, keeled shaped and about 2-5 cm long. The ovary is superior. The flowers are bisexual (Burkill, 1985).

The fruits occur usually in pairs hanging at the end of a long stalk. It is smooth and has round apex. It is about 11-20 cm long and 8-10 cm in diameter.

The fruit is glabrous and leafy green when unripe but yellow to orange in colour when ripe. It has latex and no rubber in the pericap

The seeds are embedded in the white soft pulp. The seeds are obliquely ovate, obovate to oblong, flattened 2.5-4.5 cm long. The seeds are brown in colour and are dicotyledonous

(Burkill 1985).

2.2.4 Ethnomedicinal uses of P. nitida, (Stapf) Durand & H. Durand.

P. nitida (Apocynaceae) is a potent medicinal plant commonly used in treatment of infections in West Africa (lwu, 1982.). It is found as an under-storey plant in the rain forests and sparsely distributed within the tropics where it grows in a home-stead or nearby bushes

(Inya-Agha, 1992). The tropical rain forest plant is distributed to Ivory Coast, Congo,

Uganda, and is sparsely found in Nigeria, Ghana and Upper Volta (Irvine, 1961). In Nigeria, it is mostly found in Okija and Ihiala Local Government Areas of Anambra State. It is also found in Idoma and Obubra in Benue and Cross River states respectively. The plant is sometimes cultivated because of its medicinal and economic values in the East Central States of Nigeria (NNMDA, 2008). The plant is known with different names in different parts of

Africa, where it is also used in different forms for ethno medicine.

Herbalists in Nigeria use leaves, seeds and stem backs of P. nitida in the treatment of fever, jaundice, hypertension, malaria, and gastro intestinal disorders (Dalziel, 1961; Iwu, 1993). In traditional medicine, the seeds of P. nitida are used as good substitutes for quinine in treatment of malaria (Francois, et aI., 1996). Decoction of the crushed seed in traditional medicine in Ghana acts as Enema (Dalziel, 1961), while the crushed seeds are eaten as a cure for chest complaints and pneumonia. The seeds are widely used in West Africa especially in

Nigeria, Cote-d’ivoire and Ghana as antipyretic, aphrodisiac, for treatment of malaria, pneumonia and other chest conditions (Nkere et al, 2005; Kouitcheu et al, 2008) .

In clinical studies, a cream formulation of the methanol extract of the stem bark of P. nitida has been investigated and found to have healing effects on skin conditions like; Pityriasis,

Versicolor, Tinea pedis interdigitalisis (atheletic foot), Tina capitis (ringworm of the head), and Tinea corporis (ringworm of the skin). In some parts of Africa, the roots, barks and seeds of P. nitida are used for treatment of all forms of fever while stem barks of the same plant are used in the treatment of sexually transmitted disease.

A methanol extract of the stem bark was also found to be active against a visceral leishmania, isolate at a concentration of 50 mg/ml or less. A hot water extract of the stem bark, has effect against Tryponosoma brucei. The bark and the seed extracts have been tested in both normal and alloxan-induced diabetic rabbits for their hypoglycaemic activivities by mechanism, independent of the availability of insulin from pancreatic β-cells. The hypoglycaemic activity of the extract was reported (Aguwa et al, 2001). The seeds, stems and roots of P. nitida are effective as cough suppressant and aphrodisal (Ayensu, 1978). The cough suppressant activity and anodyne for topical injuries qualities of extracts from the barks of P. nitida was also evaluated (Iwu, 1982). In Ihiala, Anambra state, Nigeria, P. nitida is used in traditional healing of hepatitis, worms, malaria and sleeping sickness (Ifebuzor, 1989). The analgesic properties of aqueous extract of P. nitida have been evaluated (Asomuah and Ayim, 1983).

Acute toxicity test in rats showed a dose-dependent acute intraperitoneal toxicity (Nyunai et al., 2006).

Common names of P. nitida

Table 1: Common names of P. nitida (Burki1l, 1985).

Nigeria Names

Igbo Osu Igwe, Nkpokiri, Osu abua

Yoruba Abere, Agege-arin, Erin.

Idoma Oozy

Bini Osu

Ethnomedicinal uses of P. Nitida plant parts

Plant parts Uses Fruit Its decoction is taken to cure cough or typhoid fever Is dripped into the ear to treat otitis Leaf Its decoction is taken by mouth or used as a lotion against measles. Its decoction is used as an enema and analgesic. It is chewed as a tonic and stimulant. Seeds They are widely used as an alternative to quinine in the treatment of fever, pneumonia, and chest condition. They are crushed and eaten with lemon juice to treat hernia, vomiting or diarrhea. Also applied to abscesses. A paste of the pulverized seeds and shea butter is rubbed on the abdomen to treat leucorrhoea in women. Bark Boiled with sugar and the decoction is drunk against food poisoning or veneral diseases. Wood Used to make incence holders, combs, small objects, for carvings, paddles, bows and arrows, walking-sticks, weaver,s shuttles, dolls, plane-blocks and handles for carpenter,s tool

Table 2. Ethnomedicinal uses of P, nitida in African countries

Country Plant parts Uses Benin/ Dahomey Leaves Its decoction is taken as an anti-helminthic, purgative Bark and taken orally to cure sterility in men. Seeds It is also used to treat hernia and with other drug plants, relieve gonorrhea and blennorrhea. Wood Used to make incence holders, combs and small objects (Burkil, 1985). Cameroon Seeds It is crushed and taken orally for chest-complaint, pneumonia, etc and acute stomach troubles are not considered purgative. It is also used extensively in place of quinine to treat fevers. (Burkill 1955) Leaves (dry) Its decoction is used as an enema and analgesic. It is chewed as a tonic and stimulant.

Congo Fruits Its decoction is taken to cure cough or typhoid fever Bark Its decoction is taken to cure sterility in men. Leaf sap Is dripped into the ear to treat otitis (Burkil 1985, Nyunai et al, 2006). Cote D’ivoire Barkof root Its decoction is taken against jaundice and yellow fever (Burkill 1985)

Its decoction is taken by mouth or used as a lotion Leaves against measles.

They are crushed and eaten with lemon juice to treat Seed hernia, vomiting or diarrhea. Also applied to abscesses.

A paste of the pulverized seeds and shea butter is rubbed

on the abdomen to treat leucorrhoea in women.

Used to make incence holders, combs and small objects Wood (Burkil, 1985). Democratic Seeds,roots,or As ingredients for arrows poisons republic of Congo fruitpulp(all crushed) It is also used extensively in place of quinine to treat fevers. Wood Used for arrow and spoons

Chewed by pahouin tribe to allay hunger while on the long marches in the bush, Gabon Fruits (immature) Boiled in water and taken to treat guinea worm infestation. Pounded and thrown in the water as a fish poison (Nyunai et al, 2006) Wood Used for walking-sticks, weaver,s shuttles, dolls, plane- blocks and handles for carpenter,s tool Ghana Seeds They are widely used as an alternative to quinine in the treatment of fever, pneumonia, and chest condition. Wood Used for spade handles (Burkill, 1985) Nigeria Fruit /seed Its decoction is taken against jaundice and yellow fever (Burkill 1985) Uganda Its decoction is taken Boiled with sugar and the decoction is drunk against against jaundice and food poisoning or veneral diseases (Burkil, 1985) yellow fever (Burkill 1985)

Wood Used as vermifuge They are recognized as being toxic and the use appears to be restrictrd to external treatment for abscesses, Used for carvings, paddles, incense holders etc and bows and arrows.

Taxonomy of P. nitida (Trease and Evans 1983)

Kingdom Plantae

Sub kingdom Viridae Plantae

Phylum Angiosperm

Sub Phylum Dicotyledon

Class Angiospermae

Sub-class Sympetatae

Order Gentianles

Family Apocynaceae

Sub-family Plumeroideae

Genus Picralima

Species: nitida.

Geographical source: Eastern Nigeria.

Common names Nkpokiri , Osu abua (Ibo), Akuamma (Ghana) .

Plate 2: The plant Picralima nitida (Stapf) Fam. Apocynaceae

Plate 3: The matured fruits of Picralima nitida. (Stapf) Fam. Apocynaceae.

Plate 4: The dried seeds of Picralima nitida (Stapf) Fam. Apocynaceae

Chemical Constituents of Picralima nitida

The plant contains alkaloids, glycosides, saponins and alkaloids are contained in the root and the stem barks, fruits, leaves and mature and immature seeds. The barks and leaves yielded only amorphous alkaloids. The first finding was the presence of alkaloids in seeds, leaves and stem. The plant contains 3.5% of total alkaloid. The seeds have been found to contain ten alkaloids, seven of which have been crystallized and characterized. The first alkaloidal isolation of seed was akuammine from the bark. Other alkaloids that were isolated include: akuammigine, pseudo-akuammigine, akuamimmenine, pseudo-akuammingine, Akuammine hydrate. A number of alkaloids have been isolated and re-isolated from the plant. Among the alkaloids isolated are picraline, picraphyline, picracine, picralicine, picratidine, picratine, burnamine and pericine was done (GuyLeodouble et al, 1964; Arens et al, 1982; Tane et al,2002). Pericine and pericalline were isolatede from the cell suspension culture.

Triterpenoid, Saponin, B-amyrin was isolated from the stem bark (Ezekwesili, 1982). Most of these alkaloids were found not only in one morphological part the plant but they occurred in different parts of the plant at the same time.The chemical structures of the alkaloids isolated from P.nitida are as shown below:

Figure 1: The chemical structures of the alkaloids isolated from P.nitida

Three coumestan glycosides were isolated from the roots of P. nitida by Kouam et al, and these glycosides afforded three coumestan derivatives. The cumastan glycosides are: 3- hydroxy-9-methoxy-2-[2’ (E)-3’-methyl-4’-0 -β-D-3’-methyl-0-β-D –glucopyranosylbutenyl]

-8 –[ 2” (E)- 3”- methyl- 4”- oxobutenyl] coumestan (i) and 3-hydroxy-9-methoxy-4-[2’ (E)-

3’-methyl-4’-0 - β- D – 3’ –methyl -0 - β -D-glucopyranosylbutenyl] -8 –[ 2” (E)- 3”- methyl- 4”- oxobutenyl] coumestan (ii). The coumestan glycosides derivatives are:

3-hydroxy-9-methoxy-2-[2’(E)-4’-hydroxy-3’-methylbutenyl]-8-isoprenylcoumestan (I);

3-hydroxy-9-methoxy-2-[2’(E)-4’-hydroxy-3’-methylbutenyl]-8-[2”(E)-3”-methyl-4” oxobutenyl]coumestan (ii) and

3-hydroxy-9-methoxy-4-[2”(E)-3”-methyl-4”-oxobutenyl]coumestan (III). The structures of coumestan glycosides and their derivativesare shown below:

Figure 2: Coumestans and coumestan glycosides isolated from the roots of P. nitida

Phytochemistry and investigation of P. nitida.

P. nitida contains many alkaloids. The main alkaloid is akuammine which has local anaesthetic action. Its action can be compared to the anaesthetic action of cocaine (Ansa-

Asamoah 1990). In a higher dose, it has strong inhibitory effect on intestinal peristaltic movement. It has hypertensive activity, which is weak and last longer in effect than

Yohimbine Menzies et al 1998

Akuammidine was established to have a sympatholitic and hypotensive actions and its local analgesic action is established to be three times that of cocaine hydrochloride (Menzies et al

1998). Akuammidine also has hypotensive, local anesthetic and muscle relaxant effect. It opposes Akuammine. Akuamigine has sympatholitic effect and antagonizes the effect of adrenaline on the heart, vessels and the regulatory centres of circulatory system. Psuedo-

Akuammigine is a reversible and competitive parasympathomimetic. In high doses, it has the capacity of inhibiting the central nervous system, respiration and also, contraction of skeletal and smooth muscles. It has local analgesic, anti inflammatory effects (Dawiejua et al., 2002).

It has hypotensive and cholinesterase-inhibiting activities. It increases the hexobarbitol- induced sleeping time. Picracine and Picralline have shown in-vitro opium antagonist activity

(Menzies et al 1998). In experiments with mice, Alstonine has shown antipsychotic effects in the treatment of schizophrenia, without some commonly side effects as in Clozapine.

The alkaloids akuammine, akuammidine, akuammicine and pseudo-akuammigine have varying degrees of agonist and antagonist activities at opioid receptors in-vitro. The extracts of the roots, the stem barks, and fruit-rind showed highly significant inhibitory effect in-vitro against Plasmodium falciparum, including chloroquine resistant strain, even in low concentrations W2 strain with IC50 value of (10.9± 1.1) µ g/mL (Bickii et al, 2007).

The root, stem bark nand fruit-rind extracts displayed significant inhibitory activities against asexual erythrocytic form of Plasmodium falciparum with IC50 values of 0.188, 0.545 and

1.581 µ g/mL respectively (Francois et al,1996).

An in vitro a,plasmodial activity of the ethanol seed extract of P. nitida was evaluated in chloroquine-sensitive Plasmodium berghei berghei infected mice. The result of this study showed that the ethanol seed extract of P. nitida exhibited significant in vitro antiplasmodial activity in both early (4-Day chemosuppressive test) and established infections (Curative test). Ethanol seed extract of P. nitida produced a dose dependent chemosupressive effect of

65.5 %, &0.4 %, and 115 mg/kg/day doses (Okokon et al, 2007).

Chloroform extract of the seed of P.nitida was evaluated for possible antileishmanial activity using a radiorespirometric microtest technique and the result of the study confirmed activity against Leishmania donovani at 50 µ g/mL (Iwu et al, 1992).

The basic fraction of the methanol extract of the sterm back exhibited significant antimicrobial activity against a wide range of gram-positive bacteria and fungi, but limited activity against gram negative bacteria (Fakeye et al, 2000).

The antimicrobial activity of the methanol extract of P.nitida fruit-rinds against 15 pathogenic strains of enteric bactaria and two yeast strains implicated in infective diarrhoea was investigated in vitro and the antidiarrhoea effect of the extract against Shiglla dysenteriae type 1 (sd 1)-induced diarrhoea was determined in vivo (Kouitcheu et al, 2013).

The result of the study showed that the extract was effective against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) Shigella dysenteriae, Proteus vulgaris (P. vulgaris),

Enterobacter cloaceae, Streptococcus feacalis, Pseudomonas aeruginosa (P. aeruginosa),

Proteus mirabilis, Salmonella typhi, Bacillus cereus and Candida albicans (C. albicans) with

P vulgaris being the most sensitive.

The methanol fruit extract of P. nitida showed potent and dose-dependent anti-inflammatory activity. The extract when administered intraperitoneally inhibited carrageenan induced rat paw oedema with IC50 value of 102 mg/kg, and with the highest dose tested (300 mg/kg) producing 72.2 % inhibition (Ezeamuzie et al,1994)

The antipyretic activity of P. nitida fruit has been demonstrated. The result of the study showed that the methanol fruit extract at a dose of 50 mg/kg produced a mean percentage antipyrexia of 38.7 % on lopopolysaccharide-induced pyrexia in rabbits, which was comparable to aspirin (29.0 % at 200 mg/kg), Ezeamuzie et al,1994.

According to Dibua et al, the aqueous and methanol extracts of the leaf and seed of P. nitida also have a concentration and time dependent larvicidal activity in the third and early fourth instar larve of Anopheles gambiae with 72 h IC50 values of 0.164, 0.333 and 0.150 mg/mL for aqueous leaf extract, methanol leaf extract and methanol seed extract respectively (Dibua et al, 2013

In a study pseudo-akuammigine was found to exhibit analgesic effect in vivo The ED50 value for this test was 10 mM which was 3.5 and 1.6 times less potent than morphine and indomethacin respectively. In the study , naloxone inhibited the effect of pseudo- akuammigini suggesting that the analgesic actions are mediated via interaction with opioid receptors (Duwiejua et al, 2002).

Pulverized and encapsulated seeds of P. nitida are in sale in Ghana markets for medicinal purposes. Picralima nitida has mainly indole alkaloids and others in less quantity. The main alkaloid is akuammine which has local anaesthetic action. Its action can be compared to the anaesthetic action of cocaine and has strong inhibitory effect on intestinal peristaltic movement (Hamet et al., 1940, Ansa-Asamoah 1990). It has hypertensive activity which is weak and last longer in effect than yohimbine. Akuammindine was established to have a

32 sympatholytic and hypotensive action. Its local analgesic action is established to be three times that of cocaine hydrochloride (Menzies et al, 1998).

The effect of alkaloids and glycosides extracts of the seed of P. nitida on mean fasting blood sugar in alloxanized diabetic rats were evaluated by Okonta and Aguwa. Their findings showed that the glycosides extract have more potent hypoglycaemic effect than the alkaloids extract (Okonta et al, 2007). Ouattara et al 2004 carried out the study on LC/MS/NMR analysis of isomeric divanilloylquinic acids from the root bark of Fagara zanthoxyloides

Lam. Ouattara et al 2009 screened the antisickling properties of divanilloylquinic acids isolated from Fagara zanthoxyloides Lam. The hypoglycaemic effect of the methanol extracts of the seed, pulp and rind of P. nitida were investigated by Inya-Agha et al the result showed a significant (p < 0.01) hypoglycaemic effect of all extracts at 300 and 900 mg/kg in alloxan-induced diabetic in rats (Inya-Agha et al 2006).

Mehanna, A. S., 2001. Sickle Cell Anemia and Antisickling Agents Then and Now.

Osayemwenre et al investigated the antipoliferative and apoptotic effects of fractionated extracts of P. nitida on humam breast cancer ceii line (Osayemwenre et al, 2011). Salihu et al the comparative study of the hypoglycaemic effects of coconut water extract of P. nitida seeds (Apocynaceae) and Daonil in alloxan induced diabetic albino rats (Salihu et al 2009)

Safety evaluation of ethanol leaf extract of P. nitida (Stapf) was screened (Ilodigwe et al,

2012)

2.3 Sickle Cell Disease

Sickle cell disease is an inherited blood disorder that attacks the haemoglobin molecule in red blood cells. Sickle cell disease is the most common of the hereditary blood disorders. It is estimated that sickle cell disease occurs more in black Americans to the tune of 3 in every

1000.

Estimates indicate that severe form of sickle cell anemia in the medical records was in 1910 in Chicago when a male student from the West Indies had the health problems of 'shortness of breath, palpitations, and episodes of icterus (yellow eyes) (Web MD Inc, 2010) with profound evidence of anemia. Dr. Herrick described the student's blood smear as showing thin, sickle-shaped and crescent-shaped red cells, hence the name sickle cell.

Red blood cells deliver oxygen to working or active tissues, in the lungs where hemoglobin takes oxygen, and at the same time, releases carbon dioxide. This process is called oxygenation. In the tissues the activity reversed where the same hemoglobin molecule releases oxygen and takes on carbon dioxide. This process is called de oxygenation.

So in sickle cell disease, some red blood cells become crescent-shaped and fragile, thereby loosing their flexibility with the effect that a sickle-cell patient is prone to infections because the damaged cells eventually clog the spleen. Other forms of ill health associated with sickle cell crisis include non localized pains and organ damages. Sickle cell disease can be described as a condition or disease state characterized by severe disease manifestation, vaso- occlusiveness, painful crisis and acute organ damage resulting from abnormalities in the haemoglobin of red blood cell. (Serjeant et al., 1974).

Sickle cell disease changes normal, round red blood cells into cells that can be shaped like crescent moons. The name 'sickle -cells' comes from the crescent shape of the cells. A sickle is a farm tool with a curved blade that can cut crops like wheat. When in normal condition red blood cells move easily through the blood vessels taking oxygen to every part of the body, but when the red blood cells are sickled they can get stuck and block blood vessels, which stops the oxygen from getting through; that can cause a lot of pains. It causes harm to organs, muscles and bones.

A sickle cell patient usually has a lifelong battle against these health problems: pains, infection, anaemia, organ damage and stroke. Sickle cell disease (drepanochytosis) is a

34 genetic life-long disorder characterized by red blood cells that assume an abnormal rigid, sickle shape. Sickling decreases the flexibility of the red blood and results in a risk of various complications.

Sickling occurs as a result of mutation in the haemoglobin gene. Sickle cells shortens life expectancy; studies have reported average life expectancy of 42 and 48 years for males and females respectively (Platt et al., 1994). Sickle cell in childhood, occurs more commonly in people from parts of tropical and sub-tropical regions where malaria is common.

Sickle cell anaemia, a homozygous pair of abnormal hemoglobin is the most common form of abnormal haemoglobin found in the general population of sufferers.

Sickle cell trait: this is heterozygous pair and has only one sickle gene and one normal adult haemoglobin gene. It is referred to as 'HbAS' or 'sickle cell trait. It is estimated that over 30 percent of the general population are carriers of this disease. Haemoglobin C disease: this is another type of haemoglobin described after haemoglobin S (Hbs). It is rare. Haemoglobin D disease: This is abnormal haemoglobin. It exists in a heterozygote pair with normal haemoglobin (Sergeant et al., 1994).

Pathophysiology

Sickle-cell disease is normally caused by a point mutation in the β-globin chain of haemoglobin, causing the hydrophilic glutamic acid to be replaced with the hydrophobic valine at the sixth position. The β-globin gene is found on chromosome 11 (Lazarus et al.,

2011). The association of two wild-type a-globin subunits with two mutant β-globin subunits forms haemoglobin S (HbS).

Under low-oxygen conditions, the absence of a polar amino acid at position six of the β- globin chain promotes the non-covalent polymerisation (aggregation) of haemoglobin, which distorts red blood cells into a sickle shape and decreases their elasticity.

The loss of red blood cell elasticity is central to the pathophysiology of sickle-cell disease.

Normal red blood cells are quite elastic, which allows the cells to deform to pass through capillaries. In sickle-cell disease, low-oxygen tension promotes red blood cell sickling, and repeated episodes of sickling damages the cell membrane and decrease the cell's elasticity.

These cells fail to return to normal shape when normal oxygen tension is restored. As a consequence, these rigid blood cells are unable to deform as they pass through narrow capillaries, leading to vessel occlusion and ischaemia.

The anaemia associated with sickle cell disease is caused by haemolysis, the destruction of the red cells, because of their misshaped. Although the bone marrow attempts to compensate by creating new red cells, it does not match the rate of destruction (Powers et al., 1991).

Healthy red blood cells typically live 90-120 days, but sickle cells only survive 10-20 days

(Platt et al., 1994).

Prognosis

About 90 % of sickle cell patients survive to age 20, and close to 50 % survive beyond the fifth decade (Kuma et aI., 2009). In 2001, according to one study, the estimated mean survival for sickle cell patients was 53 years old for men and 58 years old for women with homozygous sickle cell disease. (Wierenga et al., 2001).

Genetics

In people heterozygous for HgbS (carriers of sickling haemoglobin), the polymerisation problems are minor, because the normal allele is able to produce over 50 % of the haemoglobin. In people homozygous for HgbS, the presence of long chain polymers of HbS distort the shape of the red blood cell from a smooth doughnut-like shape to ragged and full of spikes, making it fragile and susceptible to breaking within capillaries. Carriers have symptoms only if they are deprived of oxygen (for example, while climbing a mountain) or while severely dehydrated.

The allele responsible for sickle-cell anaemia is autosomal recessive and can be found on the short arm of chromosome 11. A person that receives the defective gene from both father and mother develops the disease; a person that receives one defective and one healthy allele remains healthy, but can pass on the disease and is known as a carrier.

If two parents who are carriers have a child, there is a 1-in-4 chance of their child developing the disease and a 1-in-2 chance of their child being just a carrier.

Due to the adaptive advantage of the heterozygote, the disease is still prevalent, especially among people with recent ancestry in malaria-stricken areas, such as Africa, the

Mediterranean, India and the Middle East (Kwiatkowski, 2005). Malaria was historically endemic to southern Europe, but it was declared eradicated in the mid-20th century, with the exception of rare sporadic cases (Poncon et al, 2007).

Inheritance

Sickle-cell conditions are inherited from parents in much the same way as blood type, hair color and texture, eye colour, and other physical traits. The types of haemoglobin a person makes in the red blood cells depend on what haemoglobin genes are inherited from his parents. If one parent has sickle-cell anaemia (SS) and the other has sickle-cell trait then there is a 50 % chance of a child's having sickle-cell disease and a 50 % chance of a child's having sickle-cell trait. When both parents have sickle-cell trait a child has a 25 % chance of sickle- cell disease.

Diagnosis

In HBSS, the full blood count reveals haemoglobin levels in the range of 6-8 g/dl with a high reticulocyte count (as the bone marrow compensates for the destruction of sickle cells by producing more red blood cells). In other forms of sickle-cell disease, Hb levels tend to be higher. A blood film may show features of hyposplenism (target cells and Howell-Jolly bodies).

Sickling of the red blood cells on a blood film, can be induced by the addition of sodium metabisulfite. The presence of sickle haemoglobin can also be demonstrated with the "sickle solubility test". A mixture of haemoglobin S (HbS) in a reducing solution (such as sodium dithionite) gives a turbid appearance, whereas normal Hb gives a clear solution.

Abnormal haemoglobin forms can be detected on haemoglobin electrophoresis, a form of gel electrophoresis on which the various types of haemoglobin move at varying speeds. Sickle- cell haemoglobin (HgbS) and haemoglobin C with sickling (HgbSC)-the two most common forms-can be identified from there. The diagnosis can be confirmed with high-performance liquid chromatography (HPLC). Genetic testing is rarely performed, as other investigations are highly specific for HbS and HbC (Clark and Higgins, 2000).

An acute sickle-cell crisis is often precipitated by infection. Therefore, a urinalysis to detect an occult urinary tract infection, and chest X-ray to look for occult pneumonia should be routinely performed (Best Bet; 2010).

People who are known carriers of the disease often undergo genetic counseling before they have a child. A test to see if an unborn child has the disease takes either a blood sample from the fetus or a sample of amniotic fluid. Since taking a blood sample from a fetus has greater risks, the latter test is usually used.

2.3.1 Signs and symptoms

Sickle cells in human blood, both normal red blood cells and sickle-shaped cells are present

Sickle-cell disease may lead to various acute and chronic complications, several of which have a high mortality rate (Malowany; Butany 2012).

Sickle cell crisis

The term "sickle cell crisis" is used to describe several independent acute conditions occurring in patients with sickle cell disease. Sickle cell disease results in anemia and crises

38 that could be of many types including the vaso-occlusive crisis, aplastic crisis, sequestration crisis, haemolytic crisis and others. Most episodes of sickle cell crises last between five and seven days, (Best Bets, 2010) Although infection, dehydration, and acidosis (all of which favor sickling) can act as triggers, in most instances no predisposing cause is identified.

(Kumar et al., 2009).

Vaso-occlusive crisis

The vaso-occlusive crisis is caused by sickle-shaped red blood cells that obstruct capillaries and restrict blood flow to an organ, resulting in ischaemia, pain, necrosis and often organ damage. The frequency, severity, and duration of these crises vary considerably.

Splenic sequestration crisis

Because of its narrow vessels and function in clearing defective red blood cells, the spleen is frequently affected (Anie, and Green 2012). It is usually infracted before the end of childhood in individuals suffering from sickle cell anemia. This autosplenectomy increases the risk of infection from encapsulated organisms (Paerson, 1971; Wong et al., 1992,) preventive antibiotics and vaccinations are recommended for those with such asplenia.

Splenic sequestration crises: are acute, painful enlargements of the spleen, caused by intrasplenic trapping of red cells and resulting in a precipitous fall in hemoglobin levels with the potential for hypovolemic shock. Sequestration crises are considered an emergency. If not treated, patients may die within 1-2 hours due to circulatory failure.

Aplastic crisis

Aplastic crises are acute worsening of the patient's baseline anaemia, producing pallor, tachycardia, and fatigue. This crisis is normally triggered by parvovirus B19, which directly affects erythropoiesis (production of red blood cells) by invading the red cell precursors and multiplying in them and destroying them.

(Kumar et al., 2009). Parvovirus infection nearly completely prevents red blood cell production for two to three days. In normal individuals, this is of little consequence, but the shortened red cell life of sickle-cell patient’s results in an abrupt, life-threatening situation.

Reticulocyte counts drop dramatically during the disease (causing reticulocytopenia), and the rapid turnover of red cells leads to the drop in haemoglobin. This crisis takes 4 days to one week to disappear. Most patients can be managed supportively; some need blood transfusion

(Slavov et al., 2011).

Haemolytic crisis

Haemolytic crises are acute accelerated drops in haemoglobin level. The red blood cells break down at a faster rate. This is particularly common in patients with co-existent G6PD deficiency (Balgir, 2012). Management is supportive, sometimes with blood transfusions.

(Glassberg, 2011). One of the earliest clinical manifestations is dactylitis, presenting as early as six months of age, and may occur in children with sickle trait (Jadavji and Prober 1985)

The crisis can last up to a month. Another recognized type of sickle crisis is the acute chest syndrome, a condition characterized by fever, chest pain, difficulty breathing, and pulmonary infiltrate on a chest X-ray.

Complications

Sickle-cell anaemia can lead to various complications, including:

Overwhelming post- (auto) splenectomy infection (OPSI), this is due to functional asplenia caused by encapsulated organisms such as Streptococcus pneumonia and Haemophilus influenzae. Daily penicillin prophylaxis is the most commonly used treatment during childhood, with some hematologists continuing treatment indefinitely. Patients benefit today from routine vaccination for H. influenzae, S. pneumoniae, and Neisseria menengitidis.

Stroke can result from a progressive narrowing of blood vessels, preventing oxygen from reaching the brain. Cerebral infarction occurs in children and cerebral hemorrhage in adults.

Silent stroke is a stroke that causes no immediate symptoms but is associated with damage to the brain. Silent stroke is probably five times as common as symptomatic stroke.

Approximately 10-15 % of children with sickle cell disease suffer strokes, with silent strokes predominating in the younger patients (Adams et al., 2001; Adams, 2007).

Cholelithiasis (gallstones) and cholecystitis, may result from excessive bilirubin production and precipitation due to prolonged haemolysis are common.

A vascular necrosis (aseptic bone necrosis) of the hip and other major joints, may occur as a result of ischaemia. (Marti-Caracals et al., 2004). There is also decreased immune reactions due to hyposplenism (malfunctioning of the spleen). (Kenny et al., 1980). Priapism and infarction of the penis have been reported (Chrouseret al., 20ll).

Osteomyelitis (bacterial bone infection); the most common cause of osteomyelitis in sickle cell disease is Salmonella (especially the non-typical serotypes Salmonella typhimurium,

Salmonella enteritidis, Salmonella choleraesuis and Salmonella paratyphi B), followed by

Staphylococcus aureus and Gram-negative enteric Bacilli perhaps because intravascular sickling of the bowel leads to patchy ischaemic infarction (Almeida, 2005).

Opioid tolerance can occur as a normal physiologic response to the therapeutic use of opiates.

Addiction to opiates occurs no more commonly among individuals with sickle-cell disease than among other individuals treated with opiates for other reasons.

In the eyes, background retinopathy, proliferative retinopathy, vitreous haemorrhages and retinal detachments, resulting in blindness are also common (Elagouz et al., 2010). Regular annual eye checks are recommended. During pregnancy, intrauterine growth retardation, spontaneous abortion, and pre-eclampsia are also associated with sickle cell disease.Even in the absence of acute vaso occlusive pain, many patients have chronic pain that is not reported

(Smith et al., 2008).

Pulmonary hypertension (increased pressure on the pulmonary artery), leading to strain on the right ventricle and a risk of heart failure is often manifested as shortness of breath, decreased exercise tolerance and episodes of syncope (Gladwin et al., 2004). Chronic Sickle cell nephropathy-manifests itself with hypertension (high blood pressure), proteinuria

(protein loss in the urine), haematuria (loss of red blood cells in urine) and worsened anaemia. It progresses to end-stage renal failure; it carries a poor prognosis (Powers et aI.,

1991).

2.3.2 Clinical Manifestation

Patients with sickle cell anemia have a variety of clinical problems. Signs and symptoms of this problem do not manifest until after six months of life when the greater percentage of the foetal hemoglobin (HbF) might have been replaced by Bhs. Clinical manifestation of sickle cell disease are diverse and fall into three major categories: Anemia manifestation, Vaso- occlusive manifestation and Constitutional manifestation.

Anemic Manifestation: Sickle cells have severe hemolytic anemia with hematocrit values of between 18-30 percent.

Vaso-Occlusive Manifestation: The Vaso-occlusive manifestations are of two main groups.

1. Micro infracts: which leads to acute painful crisis. Periodic episodes of pains, called crisis are major symptoms of sickle cell disease. Pains develop when sickle shaped blood cells block blood flows through tiny blood vessels to the chest, abdomen and joints. Pain can also occur in bones. The pain may vary in intensity and can last from a few hours to a few weeks.

Some sickle cell patients experience a dozen or more crisis in a year.

Micro infracts are present and may lead to chronic organ damage and increased susceptibility to infection. Occasionally, people who have sickle cell anemia have some degree of jaundice because the liver, which filters harmful substances from the blood, is over stressed by occlusion of its blood vessels by pack of sickled cells and this leads to ischemia also. Sickle

42 cell can damage the spleen, an organ that fights infection. This makes the patient more vulnerable to infections. Some people with sickle cell anemia experience vision problems.

This is because of the tiny blood vessels that supply the eyes with blood may become plugged with sickled cells; and in consequence the retina is damaged (Mayo Foundation for Medical

Education and Researches, MFMER 1998-2010).

Constitutional Manifestation: The constitutional manifestation includes delayed growth and development. Red blood cells provide the body with the oxygen and nutrients needed for growth. A shortage of healthy red blood cells can slow growth in infants and children, and delayed puberty in teenagers.

General Features

The initial sign in the study of clinical symptoms of sickle cell disease is the intracellular polymerization of sickle hemoglobin (HbS) that occurs when sickle erythrocytes are partially deoxygenated under the hypoxic conditions of the microcirculation (Poillon et al., 1998).

This, in turn, makes sickle RBCs less deformable, invariably resulting in the debilitating micro vascular occlusions, haemolytic anemia and chronic inflammation characteristic of the disease (Edwards et al.,2005).

Studies have revealed that the morbidity of sickle cell disease varies with genotype. Patients with the homozygous SS disease and SpO-thalassaemia suffer greater morbidity and earlier mortality than patients with other genotypes (Manci et al., 2003; Platt et al., 1994).

In the US the life expectancy of patients with the homozygous SS disease is 42 years for males and 48 years for females (Bonds, 2005; Platt et al., 1994) while in Jamaica it is 53 and

58.5 years respectively (Wierenga et al., 2001); whereas for those with sickle cell disease only, life expectancy is 60 years in males and 68 years in females (Platt et al., 1994). Some of the established predictors of this phenotypic heterogeneity are the levels of HbF present,

43 presence of a-thalassaemia, and the p-haplotype associated with the HbS gene (Steinberg,

2005).

The clinical symptoms of the disease include repeated painful vaso-occlusive, haemolytic, aplastic episodes, and sequestration crises. The complications affect various organs and systems mainly skeletal, genito-urinary, gastrointestinal, spleen, hepato-biliary, cardiopulmonary and central nervous system (Meshikhes and Al-Faraj, 1998).

Clinical manifestations of dactylitis, acute splenic sequestration and increased susceptibility to infection are common before age 5 years. Painful crises, delayed growth and sexual maturation, and leg ulcerations and priapism become issues of concern starting in the adolescent period; and chronic end organ complications such as sickle nephropathy and chronic sickle lung disease with pulmonary hypertension become more manifest after age 30 years (Alexander et al., 2004).

Stroke: Next to acute chest syndrome and one of the most common killer of patients with sickle cell disease who are over three years of age is stroke, Between 8 % and 10 % of patients suffer strokes, typically at about age seven (Sergeant, 2001). Stroke prevention has been intensified by the introduction of non-invasive testing to assess cerebral blood flow by

Tran cranial Doppler velocity measurements that detect areas of vascular narrowing (Stuart and Nagel, 2004).

Immediate blood transfusion to the sickle cell disease patient at onset of stroke followed by sequential transfusion programme has been proved successful in reducing recurrence of stroke.

Painful Crisis

The most primary and know clinical manifestation of sickle cell disease is the acute vaso- occlusive event, or painful episode. This unique type of pain can start as early as 6 months of age, recur unpredictably over a lifetime, and require treatment with opioid analgesics (Smith et al., 2005). In children, painful crises often present as dactylitis of the hands and feet

(Chand-foot syndrome') and this in turn may result in premature closure of the affected epiphysis, leading to bone malformation of shortened deformed bones (Sergeant et al., 1994).

Manifested effects on Major Organ Systems

The Respiratory System

One of the commonest set of complications in sickle cell disease is the pulmonary complications. They are also a major cause of acute morbidity and mortality in sickle cell disease (Thomas et al., 1982, Gray et al., 1991; Platt et al., 1994). Acute chest syndrome

(ACS) is made up of a combination of signs and symptoms which include dyspnoea, chest pain, fever, cough, multimodal pulmonary infiltrates on the chest radiograph, and a raised white cell count.

Acute chest syndrome is a form of lung injury that can develop to adult respiratory distress syndrome (ARDS). It is estimated that half of all patients with sickle cell anaemia will develop Acute Chest Syndrome at least once in their lives. ACS is the second most common cause of hospital admission after painful vaso-occlusive crises (Mak and Davies, 2003).

Amongst the chronic cardiopulmonary complications of sickle cell disease, pulmonary hypertension is one amongst of the major threats to the well-being and longevity of patients with sickle cell disease (Machado and Gladwin, 2005).

The Cardiovascular system

Sickle cell disease patient has blood pressure lower than that of non-sickle cell disease person, what is a 'normal' blood pressure in non-sickle cell disease persons, may be clinically quite high for those with the disease (Grell et al., 1981; Homi et al., 1993; Johnson and

Giorgio, 1981; Pegelow et al., 1997). Various theories for this have included defects in renal tubular sodium and water conservation, lowered peripheral vascular resistance, and stimulation of renal prostaglandin synthesis (Homi et al., 1993). In addition the expected age- related increase in blood pressure is there. Notwithstanding the concept of 'relative hypertension' in sickle cell disease has emerged where blood pressure levels considered otherwise normal in individuals without sickle cell disease have been reported to be associated with increased risk of stroke, as well as mortality. (Rodgers et al., 1993; Pegelow et al., 1997; Gordeuk et al., 2008). In addition, increasing blood pressure is also an important predictor of chronic renal disease in sickle cell disease (Powers et al., 1991).

The chronic anaemia of sickle cell disease translated into higher cardiac output, cardiomegaly starting as early as age 5 years, and a systolic murmur which may be related to the increase in stroke volume (Covitz et al., 1995). Invariably there is hypertrophy affecting both left and right ventricles (Sergeant and Sergeant, 2001).

The liver and spleen

Acute splenic sequestration crisis is a life-threatening complication of homozygous sickle cell disease. It is rare in adults due to development of splenic fibrosis caused by repeated infarctions and occurs mainly in infants and young children aged less than 8 years (Solanki et al., 1986) thirty percent (30 %) are under the age of 5 years (Emond et al., 1985). It may occur in patients who have not developed splenic fibrosis and adult patients with sickle cell thalassaemia. It can also occur in persons with sickle cell-haemoglobin C (Hb-SC) and also in patients with high levels of foetal haemoglobin, Hb-F (Sergeant, 1970). These abnormalities

46 in the function of the spleen affect the immune status of patients with sickle cell disease, causing greater susceptibility to infections.

The Musculoskeletal System

The painful crisis which can affect any part of the body, especially the extremities, back or chest is a common future during sickle cell crises. Its severity, location and duration may vary within groups of patients and within each individual. Moreover, the frequency and severity of each pain crisis may change as a person grows from childhood to adulthood, with the break point being the late teens (Niscola et al., 2009). The frequency of the painful crises shows the gravity of the sickling disorder; three or more crises annually indicate severe disease (Ballas, 2005).

The increased susceptibility of sickle cell disease patients to infections, including osteomyelitis, is known with several symptoms including hyposplenism, impaired complement activity and the presence of infracted or necrotic bone (Almeida and Roberts,

2005).

Leg ulcers are common in sickle cell disease (Cumming et al., 2008; Koshy et al., 1989;

Sergeant, 1974; Sergeant et al., 2005). It is rare in patients before the age of 5 years. The incidence of leg ulceration in tropical countries can further be complicated by the high occurrence of tropical and chronic non-specific ulcers (Akinyanju and Akinsete, 1979).

The Nervous System

Cerebrovascular accidents occur in 8 to 17 % of patients with SS disease. Cerebral infarction is common in children and intracranial hemorrhage is commoner in adults. The overall incidence of first overt infarction in HbSS patients by age 20 years is 11% and by age 45 years is 24 % (Wong and Powers, 2005).

Neurocognitive dysfunction has been shown to exist in children with sickle cell disease

.Studies show behavioural problems, low self-esteem and disturbances in body image perception being more evident in them (Brown et al., 1993b). Developmental processes may be slow in children with sickle cell disease as well, and the presence of clinically silent brain damage has been documented (Sergeant and Sergeant, 2001). Silent infarcts have been reported to signal future cerebrovascular events.

The Genito-Urinary system

Priapism is a painful failure of detumescence which could be as a result of excess release of contractile neurotransmitters, obstruction of draining venules, malfunction of the intrinsic detumescence mechanism, or long-lasting relaxation of intracavernosal smooth muscle

(Sergeant and Sergeant, 2001). Low-flow priapism is more common, and is characterized by a reduction in venous outflow, hypoxia, acidosis, stasis, and tissue ischaemia. A sustained attack of priapism can lead to impotence (Stuart and Nagel, 2004).

Sickle nephropathy is common in sickle cell disease. It is quite similar to diabetic nephropathy (Ataga and Orringer, 2000), and at older ages is a major cause of illness and death (Thomas et al., 1982). Microalbuminuria is also found in sickle cell disease, it has been reported in 6.2% of pediatric patients with HbSS genotype; and 10 % in teenagers with HbSS

(Wigfall et al., 2000).

In adults, there is rises to 17-49 % of those with HbSS genotype (Falk et al., 1992; Sesso et al., 1998). The clinical importance and the pathogenic mechanism(s) underlying MA in sickle cell anaemia are unclear but the fact remains that increased glomerular filtration rate and renal plasma flow exhibit in early years in individuals with sickle cell disease (Allon, 1990;

Schmitt et al.,1998). It has been asserted that this hyper-filtration leads to gradual sclerosis of the glomerular capillaries and predisposes to renal insufficiency in these patients (Ataga and

Orringer, 1998; Schmitt et al.,1998).

2.3.3 Management

Management of sickle cell anemia can be achieved through prophylactic treatment, and management of the crisis and complications. The management of sickle cell anemia and its complications can exist in four main methods, namely: Psychotherapy, Transfusion, Bone marrow transplant, and drug therapy.

Management of psychosocial issues

Health care professionals should understand the importance of various psychosocial issues, such as coping, stress, depression, and reduced quality of life, which are of vital importance for anyone living with a chronic illness. Since no cure is possible yet for sickle cell disease, it is recommended that psychological interventions should be incorporated into protocols for the management of patients and offered as standard care (Anie, 2005).

Transfusion:

Blood transfusion is given when the need arises. It can be given when the patients have severe anemia. It can be given on prophylactic basis to patients having frequent crisis or who have major organ damage.

Bone Marrow Transplant:

This is rarely used because of the great risk which supersedes its advantage. In recent years, researchers have proved that there is hope of a cure to sickle cell anemia through giving mini

Bone marrow transplants.

2.3.4 Drug management

Folic acid and penicillin

Children born with sickle-cell disease will undergo close observation by the pediatrician and will require management by a hematologist to assure they remain healthy. These patients will take a 1 mg dose of folic acid daily for life. From birth to five years of age, they will also

49 have to take penicillin daily due to the immature immune system that makes them -more prone to early childhood illnesses.

Malaria chemoprophylaxis

The protective effect of sickle cell trait does not apply to people with sickle cell disease; in fact, they are uniquely vulnerable to malaria, since the most common cause of painful crises in malarial countries is infection with malaria. It has therefore been recommended that people with sickle cell disease living in malarial countries should receive anti-malarial chemoprophylaxis for life (Oniyangi and Omari, 2006).

Painful Crises

Clinical management includes rapid administration of analgesics, mainly opioids, treatment of underlying infections if needed, oxygen supplementation and rest. Use of incentive spirometry and advice about deep breathing exercises is crucial, especially with presence of chest pains, to avoid complications with acute chest syndrome or pneumonia.

Vaso-occlusive crisis

Most people with sickle-cell disease have intensely painful episodes called vaso-occlusive crises. The frequency, severity, and duration of these crises, however, vary tremendously.

Painful crises are treated symptomatically with analgesics; pain management requires opioid administration at regular intervals until the crisis has settled. For milder crises, a subgroup of patients manage on NSAIDs (such as diclofenac or naproxen). For more severe crises, most patients require inpatient management for intravenous opioids; patient-controlled analgesia

(PCA) devices are commonly used in this setting. Diphenhydramine is also an effective agent that is frequently prescribed by doctors in order to help control any itching associated with the use of opioids.

Acute chest syndrome

Acute chest syndrome is also a frequent cause of morbidity and mortality in sickle cell disease and multiple episodes may lead to chronic lung disease and pulmonary hypertension.

Antibiotics including macrolides, liberal use of analgesics and incentive spirometry are mainstay of treatment.

Supplemental oxygen therapy and simple or exchange transfusions will need to be employed for more severe cases (Stuart and Nagel, 2004).

Hydroxyurea

Hydroxyurea, a rib nucleotide reductase inhibitor, has been used to reduce the incidence of painful crises and acute chest syndrome, as well as the need for transfusion in those with more severe disease. It has been shown to be effective in both paediatric and adult patients, but it still has multiple side-effects and requires close monitoring of the patient.

The role and effects of the drug as stroke prevention and organ preservation is still being established (Wiles and Howard, 2009). It is the first approved drug for the causative treatment of sickle-cell anaemia, Hydroxyurea was shown to decrease the number and severity of attacks (Charache et al.,1995) and also to possibly increase survival time in a study by Steinberg et a.l ,(2003). This is achieved, in part, by reactivating fetal haemoglobin production in place of the haemoglobin S that causes sickle-cell anaemia. Hydroxyurea had previously been used as a chemotherapy agent, and there is some fears that long-term use may be harmful, but this risk has been shown to be either absent or very small. It's benefits may outweigh the risks (Platt, 2008).

2.3.5 Transfusion therapy

Blood transfusions are often used in the management of sickle cell disease in acute cases and to prevent complications by decreasing the number of red blood cells that can sickle by adding normal red blood cells (Drasar et aI., 2011).

In children prophylactic chronic red blood cell transfusion therapy is very effective to a certain extent in reducing the risk of first stroke or silent stroke when trans- cranial doppler

(TCD) ultrasonography shows abnormal increased cerebral blood flow velocities. In those who have sustained a prior stroke event it also reduces the risk of recurrent stroke and additional silent strokes (Mirre et al., 2001; Gyang et aI., 2011).

Stem Cell Transplantation

Haemopoietic cell transplantation is the only available potentially curative therapy for sickle- cell disease. Studies show that the estimated risk of death from HLA-identical-stem-cell transplantation in the disease is 5 %. The goal is to successfully replace the host's marrow with normal genotype cells before development of organ dysfunction (Stuart and Nagel,

2004). Gene therapy has been explored successfully in animal models, but is not likely to benefit patients in the near future.

Bone marrow transplants

Bone marrow transplants have proven to be effective in children. Bone marrow transplants are the only known cure for sickle cell disease. (Walterset aI., 1996).

Acute splenic sequestration

The immediate treatment of acute splenic sequestration (ASS) is the correction of hypovolemia with red blood cell transfusion. In case of severe ASS being fatal within a few hours, emergency transfussion is required. If there is a recurrence of the sequestration" splenectomy is usually recommended.

Priapism

Priapism is also one of the clinical syndroms of sickle cell disease. It is treated to prevent partial or complete impotence, which can be caused by poor erections that last several hours to days.

Drugs used to prevent priapism include terbutaline and phenylephrine, (these help restrict blood flow to the penis) and hormonal treatments, such leuprolide and diethylstilbestrol, to avoid repetitive and prolonged episodes. Surgical procedures including direct aspiration of the corpora and shunting procedures can be employed to alleviate a major attack of priapism

(Sergeant, 2001).

Anemic Epesodes

2.3.6 Management of chronic complications

Leg ulcers

Leg ulcerations, can cause severe disability and psychosocial effects on those afflicted due to their chronicity (Serjeant, 2005). Simple treatment like moist dressings, rest with elevation of the affected leg and bandaging with elastic compression bandages are usually effective. In severe cases, skin grafting will be required.

Sickle Retinopathy

Sickle cell disease patients are also more prone to retinopathy and need at least annual eye examinations. If there is proliferative retinopathy development" treatment is needed as retina are then at risk of bleeding and consequent retinal detachment. Techniques such as diathermy, cryotherapy and laser photocoagulation have been used to cause involution of neovascular lesions.

Cardiopulmonary dysfunction

Congestive heart failure as a result of worsening anemia tends to respond well to diuretics and intermittent red cell transfusions. Digoxin and diuretics are employed to manage it if there is no fall in haemoglobin.

2.3.7 Epidemiology

The highest frequency of sickle cell disease is found in tropical regions, particularly sub-

Saharan Africa, India and the Middle-East. (Wheatheral and Clegg, 2001). Migration of substantial populations from these high prevalence areas to low prevalence countries in

Europe has dramatically increased in recent decades and in some European countries sickle cell disease has now overtaken more familiar genetic conditions such as haemophilia and cystic fibrosis. (Roberts et al., 2007).

Three quarters of sickle-cell cases occur in Africa. A recent WHO report estimated that around 2 % of newborns in Nigeria were affected by sickle cell anaemia, giving a total of

150,000 affected children born every year in Nigeria alone. The carrier frequency ranges between 10 % and 40 % across equatorial Africa, decreasing to 1-2 % on the north African coast and <1 % in South Africa.(WHO, retrieved, 2010).

The prevalence of the disease in the United States is approximately 1 in 5,000, mostly affans of Sub-Saharan African descent, according to the National Institutes of Health (National

Heart, Lung and Blood institute, retrieved, 2010). ln the United States, about 1 out of 500

African-American children and 1 :36,000 Hispanic-American born will have sickle-cell anaemia (CDC retrieved, 2011) It is estimated that Sickle Cell Disease (sickle cell disease) affects 90,000 Americans. (Sickle cell disease: Data and Statistics, retrieved, 2011)

Most infants with sickle cell disease born in the United States are now identified by routine neonatal screening. Forty-four states along with the District of Columbia, Puerto Rico and the

Virgin Islands currently provide universal neonatal screening for sickle cell disease

(American Academy, Committee on Genetics, 2002; Pass et al., 2000).

Sickle' Cell trait occurs among about 1:12 African-Americans and 1:100 Hispanic-

Americans." Sickle cell disease" March of Dimes, It is estimated that 2.5 million Americans are heterozygous carriers for the sickle cell trait. (Cinnchinsky et al., 2011).

Sickle cell disease is prevalent in many parts of India, where the prevalence has ranged from

9.4 to 22.2 % in endemic areas (Awasthy et al., 2008).

2.4 Electrophoresis

The test is performed in the laboratory, the blood sample is placed on the special paper and an electric current is applied on it (the paper). The haemoglobins move on the paper and form bands that show the amount of each type of haemoglobin. The aim of haemoglobin electrophoresis is to examine if there is any form of abnormal haemoglobin

(hemoglobinopathy).

Electrophoresis uses electric current to separate normal and abnormal types of haemoglobin in the blood. Haemoglobin types have different electrical charges and move at different speeds. The amount of each haemoglobin type is measured. In abnormal amount of normal haemoglobin or abnormal types of haemoglobin in the blood may mean that disease is present. Abnormal haemoglobin types may be present without any other symptoms, may cause mild disease that do not have symptoms, or cause diseases that can be life-threatening.

For example haemoglobin S is found in sickle cell disease which is a serious abnormality of the blood and causes serious problems.

Many different types of hemoglobin (Hb) exist. The most common ones are HbA, HbA2,

HbF, HbS, HbC, HbH and HbM. Healthy adults only have significant levels of HbA and

HbA2

Some people may have small amount of HbF, the main type of hemoglobin in an unborn baby’s body. Certain diseases are associated with high HbF levels (when HbF is more than

2% of the total hemoglobin). HbS is an abnormal form of hemoglobin associated with sickle cell anemia. In people with HbS, the red blood cells sometimes have a crescent or sickle shaped. The cells easily break down and can block small blood vessels.

HbC is an abnormal form of hemoglobin associated with hemolytic anemia. It has symptoms less than that of sickle cell anemia. Other abnormal hemoglobin molecules cause anemia.

HbS and HbC are the most common types of abnormal hemoglobins that may easily be found by an electrophoresis test.

The physical, biological and physiological parameters (ESR, PVC, RBC, HBS, Plasma

Calcium Conductivity and TDS) were measured before and after incubation with the 2.5 mg,

5 mg and 10 mg concentrations of the methanol extract, ethanol fraction and dichloromethane fraction of P. nitida for 180 minutes.

2.4.1 Packed Cell Volume (PCV)

Packed cell volume (PCV) or erythrocyte volume fraction (EVF), is the volume expressed in percentage (%) of red blood cells in the blood. It is normally about 45 % for men and 40 % for women (Purves et al., 2004). It is also called Haematocrit. It is an integral part of a person’s complete blood count results, along with haemoglobin concentration, white blood count, and platelet count.Red blood cells can be obtained from whole blood by centrifugation, which separates the cells from the blood plasma in a process known as blood fractionation.Packed red blood cells, which are made in this way from whole blood with the plasm aremoved, are used in transfusion medicine (First red blood cells grown in the lab,2008).Red blood cells can be conserved for five weeks at – 79 oC or – 110 oF .

2.4.2 Erythrocyte Sedimentation Rate (ESR)

The erythrocyte sedimentation rate (ESR), also called a sedimentation rate, is the rate at which red blood cells sediment within a period of one hour. It is one of the hematology tests.

It is a non-specific measure of inflammation. The test is used to be performed with an anti- coagulated blood traditionally placed in an upright tube, known as a Westergen tube and the rate at which the red blood cells fall was measured and reported in mm/h

Now the new ESR tests are performed automatically with automated analyzers. The balance between pro-sedimentation factors mainly fibrinogen and those factors resisting sedimentation, namely the negative charge of the erythrocyte (Zeta potential) governs the principles of ESR. When an inflammatory process is present, the high proportion of fibrinogen in the blood causes red blood to stick to each other.

The red cells form skacks called ‘rouleaux’ which settle faster. Lymphoproliferation disorders in which one or more immunoglobulins are secreted in high amount can also exhibit rouleaux formation. It was observed that ESR is increased by any cause of inflammation. The values of ESR is more in pregnancy, inflammation, anemia or rheumatoid arthritis, and less in polycythemia, sickle cell anemia, hereditary spherocytosis and congestive heart failure.

It is increased in kidney cancer. The basal ESR is slightly higher in females (‘ESR’ Medline plus: Retrieved 8 July 2013)

ESR is useful in the diagnosis of some diseases like multiple myeloma, temporal arthritis, polymyalgia rheumatic, various auto-immune disease, systemic lupus erythromatosus, rheumatoid arthritis, inflammatory bowel disease (Liu et al., 2013) and chronic kidney disease. In many of the mentioned diseases the ESR may exceed 100 mm/h (‘’Sedimentation

Rate’’ Web MD, 2006)

There are three stages in erythrocyte sedimentation.

Rouleaux formation (within the first 10 minutes)

Sedimentation or settling stage (within 40 minutes)

Stage of pack in (10 minutes), sedimentation shows and cells start to pack at the bottom of the tube.

Men have 3 mm/h while women have 7 mm/h. ESR is higher in women (Westergen, 1975;

Bottiger and Svedberg, 1967). ESR value increases in state of anemia and also in black population (Gillum, 1993)

The widely used rule calculating normal maximum ESR values in adults (98 % confidence limit) is given by a formula devised in 1983 (Miller et al., 1983).

Age(in years) +10(in female) ESR (mm/h) ≤ 2

This formula is no longer credited.

Table 3: ESR reference range putting age into consideration .

Age 20 51 90

Men 5 % 12 14 19

Women 5 % 18 21 23

Children normal values of ESR have been quoted as 1 to 2 mm/h at birth, rising to 4 mm/h 8 days after delivery (Ibson et al., 1980) and then to 17 mm/h by the 14th day, newborn 0 to 2 mm/h (Medline plus Encyclopedia ESR), neonatal to puberty 3 to 20 (Mack et al., 2007;

Bauchner, 2007).

2.4.3 Hemoglobin.

Hemoglobin is a molecule comprised of four subunits. Each subunit contains an iron containing pigment (heme) and a protein (globulin). There are two types of subunits, alpha and beta. A gram of hemoglobin can carry 1.34 ml of oxygen. The oxygen carrying capacity of blood is directly proportional to its hemoglobin concentration (Mosby’s Medical Dictionary.

(2009)

Erythrocytes consist mainly of hemoglobin, a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules in the lungs or gills and release them throughout the body. Oxygen can easily diffuse through the red blood cell’s cell membrane.Hemoglobin in the erythrocytes also carrries some of waste products carbon dioxide back from the tissues; most waste carbon dioxide , however ,is transported back to

58 the pulmonary capillaries of the lungs as bicarbonate (HCO3) dissolved in the blood plasma.

Myoglobin, a compound related to hemoglobin, acts to store oxygen in muscle cells (Anthis,

2008).The numbers of the red cells does not indicate blood’s oxygen content because some cells may contain more hemoglobin than others.

The colour of erythrocytes is due to the heme group of hemoglobin.The blood plasma alone is straw-colored, but the red blood cells change colour depending on the state of the hemoglobin: when combined with oxygen the resulting oxyhemoglobin is scarlet, and when oxygen has been released the resulting deoxyhemoglobin is of a dark red burgundy colour.

However, blood can appear bluish when seen through the vessel wall and skin (Synder and

Sheafor.1999) Hemoglobin also has a very high sffinity for carbon monoxide, forming carboxyhemoglobin which is a very bright red in colour, Hemoglobin is used in diagnosis, to screen for anemia to identify the severity of anemia, and to assist in evaluating the patient’s response to anemia therapy. Hemoglobin also serves as an important pH buffer in the extracellular fluid.

Normal hemoglobin values are:

Adult (males): 13.5- 17 g/dl

(Females): 12- 15 g/dl

Pregnancy: 11- 12 g/dl

Newborn: 14- 24 g/dl 77 % of this value is fetal hemoglobin which drops to approximately

23% of the total at 4 months of age.Children: 11- 16 g/dl

2.4.4 Red Blood Cells Count

In humans, mature red blood cells are flexible and oval biconcave disks. They lack cell nucleus and most organelles, in order to accommodate maximum space for hemoglobin.

Approximately 2.4 million new erythrocytes are produced per second (Sackmann, ed vol 1,

Elsevier, 1995). The cells develop in the bone marrow and circulate for about 100–120 days

59 in the body before their components are recycled by macrophages. Each circulation takes about 20 seconds. Approximately a quarter of the cells in the human body are red blood cells

( Pierige et al., 2008)

Adult humans have roughly 2-3 x 1013 (20–30 trillion) red blood cells at any given time, comprising approximately one quarter of the total human body cell number (women have about 4 to 5 million erythrocytes per microliter (cubic millimeter) of blood and men about 5 to 6 million;

Erythropoiesis is the development process by which new erythrocytes are produced; it lasts about 7 days. The erythrocytes are continuously produced in the red bone marrow of large bones, at a rate of about 2 million per second in a healthy adult. (In the embryo, the liver is the main site of red blood cell production.) The production is stimulated by the hormone erythropoietin (EPO), synthesised by the kidney. Just before and after leaving the bone marrow, the developing cells are known as reticulocytes; these comprise about 1% of circulating red blood cells.

Complete Blood Count is performed in hospital using an Abbott Cell-Dyn 1700 automatic analyzer. The blood sample was collected into a test tube containing an anticoagulant (EDTA sometimes citrate). Sometimes the sample is drawn off a finger prick using a Pasteur pipette for immediate processing by an automated counter.

In the past, counting the cells in a patient's blood was performed manually, by viewing a slide prepared with a sample of the patient's blood under a microscope (peripheral smear).

Presently, this process is generally automated by use of an automated analyser, with only approximately 10–20 % of samples now being examined manually.

In addition to counting, measuring and analyzing red blood cells, white blood cells and platelets, automated hematology analyzers also measure the amount of haemoglobin in the blood and within each red blood cell. This is achieved by the addition of a diluent that lyses

60 the cells which is then pumped into a spectro-photometric measuring cuvette.The change in color of the lysate equates to the hemoglobin content of the blood. This information can be very helpful in diagnosis, for example, to identify the cause of a patient's anemia. If the red cells are smaller or larger than normal, or if there is a lot of variation in the size of the red cells, this data can help guide the direction of further testing and expedite the diagnostic process so patients can get the treatment they need quickly.

Hemoglobin S result from the substitution of an unusual form of hemoglobin in the red cell and interfering oxygenation and subsequent sickling of red cell and hemolysis. The repeated sickling and unsickling damages the red cell membrane leading to irreversible sickled red cell even when the oxygen pressure is increased. The resulting hemolysis consequent to the damaged red cell membrane may occur intravascularly or extravascularly (Test et al, 1991,

Allan et al, 1982; Platt, 1982)

Extravascular hemolysis occurs by phagocytosis of red cells that have undergone sickling and physical entrapment of rheological compromised red cells (Galili et al,1986, Green and

Kalra, 1988, Kaul et al, 1986). Increased susceptibility to mechanically induce cell fragmentation has been documented in vitro and in sickle cell patients undergoing vigorous exercise (Platt, 1982)

2.4.5 Plasma Calcium

Ca2+ is a universal and ubiquitous signaling molecule (Begrudge et al 2000; Bootman et al,

1997), regulating cell cycle and fate, metabolism and structural integrity motility and volume.

Most of the Ca2+ in the cytosol is bound and buffered by numerous Ca2+binding proteins, phospholipids and inorganic phosphate. When bound and buffered Ca2+ are included, total intercellular Ca2+ in red blood cell (RBCs) reaches 5.7 uM (Bookchin and Lew, 1980). Basal free Ca2+ concentration in RBCs of health human beings under physiological conditions is estimated to be in the range of 30 to 60 nM (Tiffert et al 2003)

Calcium metabolism or calcium homeostasis is the mechanism by which the body maintains adequate calcium levels (Brini et al., 2013). Derangements of this mechanism lead to hypercalcemia or hypocalcemia, both of which can have important consequences for health.

In humans, when blood calcium level rises above a set point, the thyroid gland releases calcitonin, causing the blood calcium level to fall. When blood calcium level falls below a set point, the parathyroid gland releases parathyroid hormones (PTH), causing the blood calcium level to rise. Most of the calcium in adult human body is extracellular and 99 % of it exists as crystalline hydroxyapatite in bones and teeth where it confers rigidity. Calcium exist in the serum in three forms: protein-bond (45 %); ionized (45 %) and 10 % is in complex with small diffusible ligands such as citrate, lactate, phosphate and bicarbonate.

It plays a major role in the mechanisms of nerve impulse transmission, muscle contraction and blood coagulation. Calcium is also involved in regulating the activity of adenylate cyclase and phosphodiesterase through reversible combination with calmodlin. The ionized extracellular calcium on the cell surfaces of the parathyroid glands, thyroid C cells and pancreatic B cells controls their secretions. Independent of its origin, hereditary hemolytic anemia is often associated with an increase in the intercellular Ca2+levels (Tiffert et al 2003)

Most of the information on Ca2+ transport in diseased RBCs was so far obtained for SCD patients. In these cells relatively high rates of Ca2+uptake in RBCs are partially compensated for by sequestration of Ca2+ into intercellular inside-out vesicles, in which Ca2+ is pumped actively by PMCA (Bookchin et al, 1985)

Furthermore, part of Ca2+ take into the cells is immobilized be the intercellular proteins.

Sickle cell transformation associated with polymerization of deoxygenated mutated S- hemoglobin is amplified by 20-40 folds by dehydration (Eaton and Hofrichter,1990).

Deoxygenating promotes Ca2+uptake and release of ionized Ca2+ from the intracellular proteins (reduction in buffering capacity) (Tiffert et al 1993, 1997,Vandorpe et al 2010). In

62 deoxygenated SCD RBC, an acute increase in the intercellular free Ca2+ RBCs causes opening of the Ca2+ sensitive Gardos channel and anion channels (Oritz et al 1990, Clark and

Rossi,1990)

Calcium acts structurally as supporting material in bones as calcium phosphate. Calcium also plays important role in cellular signaling pathways. Intracellular calcium functions as a second messenger in the secretion of many hormones and neurotransmitters. An example being, the influx of calcium into the neuron causes the release of Acetylcholine from pre- synaptic terminals into the neural synapse. Calcium also acts as an intracellular permeation regulator and mediator of muscle contraction. Calcium acts in the contraction of muscles by removing the Triosephosphate isomerase (TPI) subunit from Myosin heads, which has

ATPase activity. Calcium also acts as an enzyme cofactor for some clotting factors (enzymes) in the coagulation cascade.

About 25 mmol of calcium enters the body in a normal daily diet. Of this, about 40%

(10 mmol) is absorbed in small intestine and 5 mmol leaves the body in feces, 5 mmol of calcium remains per day (Barrett et al, 2009)

The kidney excretes 250 mmol a day in pro-urine, and resorbs 245 mmol, leading to a net loss in the urine of 5 mmol/d. In addition to this, the kidney processes Vitamin D into calcitriol, the active form that is most effective in assisting intestinal absorption. Both processes are stimulated by parathyroid hormone.

The plasma level of calcium is closely regulated with a normal total calcium of 2.2-

2.6 mmol/L (9-10.5 mg/dL) and a normal ionized calcium of 1.1-1.4 mmol/L (4.5-

5.6 mg/dL). The amount of total calcium varies with the level of serum albumin, a protein to which calcium is bound. The biologic effect of calcium is determined by the amount of ionized calcium, rather than the total calcium. Ionized calcium does not vary with the albumin level, and therefore it is useful to measure the ionized calcium level when the serum

63 albumin is not within normal ranges, or when a calcium disorder is suspected despite a normal total calcium level.

2.4.6 Conductivity

Other than red blood cells, plasma is the majority of whole blood. As a liquid with ions suspended in it, plasma is more conductive than red blood cells. Therefore, the more plasma by volume, the more conductivity, which is an inverse relation to the hematocrit.

The concentration of electrolytes in blood will affect its conductivity. The more electrolytes, the higher the conductivity.

The numerical value of conductivity is assumed to be varying with several factors. These are concentration of erythrocytes, hematocrit volume and moreover the liquid conducts electricity due to the presence of ions (salt and proteins) (Kubasova and Tamara, 1984)

An understanding of connectivity of blood is essential parameter for determination of interfacial potentials during the flow of blood. A thorough study of electrical conductivity of human blood has been done with respect to the effect of laser radiation. Literature show that the erythrocytes almost behave like a perfect nonconductor of direct current (Vladimirov et al

2004). Later studies showed that only the surface layer of the erythrocytes is nonconductive which offers the resistance to flow of blood while the inner structure of the erythrocyte had a conducting medium which is almost half of the conductivity of the plasma (Rubinov, 2003;

Niemz and Markolf, 2007). Literature also revels that the fact that erythrocyte dose not conduct direct current and act as a dielectric medium at sufficiently high frequency.

Influence the electrical conductivity of the whole blood is: erythrocyte sedimentation rate

(ESR), red blood cell count, hematocrit volume, plasma constituents (salt, proteins etc).

The concentration is determined by one or more ion measurements, with sodium being of the greatest interest since it is the primary electrolyte in plasma.

Erythrocytes or red blood cells have low conductivity. Erythrocytes are concave on both sides to help with the diffusion of oxygen and carbon dioxide, their surfaces are resistant to electric current, and their orientation affects blood conductivity. At low frequencies of electric current, the erythrocytes align with the flow, reducing their cross sections and increasing the conductivity of blood in the direction of flow.

2.4.7 Total dissolved solids (TDS)

A common use for conductivity sensors is to measure the concentration of total dissolved solids (TDS) in water samples. Water can be classified by the level of TDS in water.

Fresh water: less than 5000 mg/L TDS

Brackish water: 500 to 30,000 mg/L TDS

Saline water: 30,000 to 40,000 mg/L

Hypersaline: greater than 40,000 mg/L

Some industries such as water treatment have adopted a measurement expressed as TDS ( total dissolved solids). TDS is approximated with conductivity using a multiple factor and is expressed in parts per milliom (ppm)

An aquarium at Bristol Zoo, England was cited where maintenance of the filters became costly with high TDS. High levels of total dissolved solids do not correlate to hard water, as water softeners do not reduce TDS. Water softeners remove magnesium and calcium ions, which cause hard water, but these ions are replaced with an equal number of sodium or potassium ions.This leaves overall TDS unchanged, Sigler and Bauder (2015)

Usually there's a roughly linear relationship between conductivity and the concentration of ions in a solution, at least until very great ion concentrations are attained. In particular, for salts, an average of 2 μS/cm is produced for each ppm (by weight, or mg/L) of dissolved solids. Most acids and bases are much more conductive than their salts because of the vastly greater mobilities of the H+ and OH- ions (Mosaic Documentation Web 1985)

Viscosity

Blood viscosity is a measure of the resistance of blood to flow,. It can also be described as the thickness and stickiness of blood

Flow resistance is also directly proportional to the viscosity of blood flowing in that segment.

The rheology (i.e. flow behavior) of a fluid can physically be described by its viscosity. In lamiar fluid flow as described by Newton (Lowe and Barbenel, 1988). Viscosity is the ratio of the force that moves the fluid layers or laminae (shear stress) to the velocity gradient in the fluid (shear rate), representing internal resistance between the laminae (Lowe and Barbenel,

1988, Merrill, 1969)

The most important determinants of blood viscosity are hematocrit, red blood cell deformity, red blood cell aggregation, and plasma viscosity. Plasma’s viscosity is determined by water- content and macromolecular components, factors that affect blood viscosity are the plasma protein concentration and types of proteins in the plasma.[ Kemarky et al (2008). Hematocrit has cause up to a 4% increase in blood viscosity (Baskurt and Meiselman 2003). This relationship becomes more sensitive as hematocrit increases. When the hematocrit rises to 60 or 70%, which it often does in polycythemia, Tefferi (2003), the blood viscosity can increase as 10 times that of water, and its flow through blood vessels is greatly retarded because of increased resistance to flow, Lenz et al (2008). This will lead to decreased oxygen delivery,

Kwon et al (2008) Other factor influencing blood viscosity is temperature, (where an increase in temperature results in a decrease in viscosity). This is particularly important in hypothermia, where an increase in lood viscosity will cause problems with blood circulation.

2.5. Objectives of the Study

Establishment of the antisickling properties of P. nitida will be of tremendous health significance in sickle cell disease management as it will bring the disease management to the grass root. Since P. nitida is native to many communities in the country it will be readily

66 available and easily accessible. The cost of sickle cell disease management using conventional synthetic chemotherapy will be reduced and the psychosocial pressure and infant mortality inherent in sickle cell disease will also be reduced.The specific objectives of the study therefore were to:

(i) evaluate the anti-sickling properties of Picralima nitida seeds extract and fractions.

(ii) evaluate the biochemical parameters associated with sickle cell disease.

(iii) determine the phyto-constituents present.

(iv) isolate and characterize the active principle(s) responsible for the anti-sickling activity.

2.6. Research Hypothesis

The following hypotheses are developed in the study:-

Ho1: P. nitida plant seed extract and fractions have anti sickling properties.

Ho: P. nitida plant seed extract and fractions have no anti sickling properties.

Ho2: P. nitida plant seed extract and fractions improve rheological properties of sickled red

blood cells.

Ho: P.nitida plant seed extract and fractions do not improve rheological properties of

sickled red blood cells.

CHAPTER THREE

MATERIALS AND METHOD

3.1 Collection and Preparation of Plant Materials.

Matured and riped fruits of P. nitida were collected in its fruit bearing season from Dim

Anozie village in Isu Imo state in the month of May 2010. The plant was authenticated by a taxonomist at the Botany Herbarium of the University of Nigeria Nsukka where Voucher specimen was maintained. The fresh matured fruits (8kg) were cut into transverse section to expose the seeds, the pulp and the rinds. The seeds were air-dried for three weeks and then pulverized with laboratory hammer mill into a homogenous powder, and stored inside an airtight container until used.

3.2 Reagents

The following materials and reagents were used as procured from their supplies: Chloroform, methanol, ethanol (May & Baker, England), hexane dichloromethane and ethyl acetate

(BDH, England), silica gel 60 microns (Hopkins & Williams, England), parahydr oxybenzoic acid, Sodium methabisulphite A/R (BDH, England)

3.3 Organoleptic tests

The organoleptic properties (taste, colour, odor, texture) of the powdered P. nitida were determined using the sensory organs.

3.4 Extraction and fractionation of P. nitida seeds.

A 2.5 kg pulverized seeds were extracted with methanol in a soxhlet apparatus The extract was concentrated in a rotary evaporator to yield a dried residue. The methanol extract of

Picralima nitida was then defatted using n-hexane. The defatted methanol extract (13.8 g) was mixed with dry silica gel at a ratio of 1:2, put into a tall column cylindrical tube packed with silica gel up to half the volume. A cotton fiber was packed at the bottom of the column to prevent the silica gel from coming out of the tap. The extract was successively fractionated

68 with different solvents, chloroform, dichloromethane, ethyl acetate and 50% ethanol in increasing order of polarity to obtain the chloroform fraction (CHCl3), dichloromethane fraction (CH2Cl2), ethyl acetate fraction (EtOAc) and aqueous fraction. Antisickling activity tests were carried out on all the fractions at the same time, comparing their antisickling activities with that of p-hydroxybenzoic acid (positive control).

3.5. Purification and isolation

The most active fraction (aqueous fraction) was subjected to bioassay guided fractionation and isolation using several chromatographic techniques. A 6 g portion of the aqueous fraction

(Aq F) was subjected to column chromatography over silica gel (230 – 400 mesh) eluting with n-hexane (100 %), n-hexane: ethyl acetate (2:1), n-hexane:ethylacetate (1:1) ethyl acetate 100 %, ethyl acetate:methanol (19-1), ethyl acetate: methanol (9:3) and ethyl acetate : methanol (4:2) in increasing order of polarity to obtain 32 sub-fractions of 100 ml each. The sub fractions were pooled based on their thin layer chromatographic (TLC) characteristics. A series of silica gel column chromatography was carried out of fractions 19-23 with mixture of

EtOAc:MeOH in increasing order of polarity, followed by a preparative TLC using

EtOAc:MeOH (2:3) as solvent system to get compounds 1 and 2. Fractions 30-34 were also subjected to the same purification process to obtain compound 3 (Fig. 3)..

3.6 Characterization of isolated compounds

The isolated compounds were characterized by the following spectroscopic technicques:

ESI-MS, UV, NMR. The mass Spectra was recorded on Bruker TOF Mass Spectrometer

(Shimadzu, Japan) using electrospray ionization (ESI). The UV Spectra (ƛmax: nm) were recorded on Shimadzu UV- 2700 Spectrometer (Shimadzu, Japan) in MeOH. The 1H-NMR and 13C NMR spectra were recorded on a Bruker DPX- 400 NMR Spectrometer (Billerica,

1 13 USA) (500 MHz for H and125 MHz for C-NMR) using CDCl3 as solvents.

FRACTIONATION AND ISOLATION

Aqueous fraction Aq F 6.62 g

Fractionation with solvents/ solvent mixtures : n- hexane 100 %, n-hexane : EtOAc (2:1), n-hexane : EtOAc(1:1), EtOAc 100 %, EtOAc:MeOH (19:1), EtOAc : MeOH (9:10), EtOAc: MeOH (4:12)

N-hexane N-hexane N-hexane EA 100 % EA : Me EA : Me EA : Me 100 % EA(2:1) :EA (1:1) (19 : 1) (9 : 10) (4 : 12)

N-hexane 100 % 1-6 sub- 7-12 sub- 13-18 sub- 19-23 sub- 24-29 sub- 130-34 sub- 35-40 sub- fractions fractions fractions fractions fractions fractions fractions

(i) Antisickling bioassay. (ii) CC (Si gel 300-400 mesh) (i) Antisickling bioassay. Solvent: (EtOAc : MeOH. (ii)CC(Si gel 300-400 mesh) iii)TLC monitoring. : solvent (EtOAc : MeOH). Adsorbent Si gel. iii)TLC monitoring.: Spray with Dragendorff’s reagent Adsorbent Si gel. Spray with Dragendorff’s reagent

AqSF 1 AqSF 2 AqSF 3 2.3 g 1.5 g 1.7 g

PreparativeTLC, Preparative TLC, Preparative TLC, (RP18),Solvent: (RP 18), Solvent: (RP 18), Solvent EtOAc:MeOH EtOAc:MeOH AtOAc:MeOH.

CP 1 CP 2 CP 3 1.1 g 1.4 g 1.9 g

Fig.3 : Flow chart of the purification and Isolation of compounds of P.nitida seed extract.

Key: EA ( Ethyl acetate), Me (Methanol extract), AqF (Aqueous fraction), AqSF (Aqueous sub-fraction, CP (Compound).

3.7 Phytochemical Screening

Chemical tests were carried out on the methanol extract, fractions and isolates using standard procedures involving the identification of the constituents by characteristic colour changes as described by Sofowara (1993), Trease and Evans (1989).

Test for Tannins

Ferric chloride test

A 0.5 g quantity of dried powdered sample, plus 20 mL of distilled water was boiled in a test tube and filtered. Few drops of ferric chloride was added to the filtrate and colour change or precipitate was observed.

Lead Acetate test

A little of the filtrate was added Lead Acetate Solution and observed.

Test for Steroids

A 2 mL volume of acetic anhydride was added to 0.5 g of the methanol extract, followed by addition of 2mL conc 0.1 N sulphuric acid, and the colour change was observed

Test for Terpenoids

A five mililitre volume of the extract was added to 20 mL chloroform. Then concentrated

Sulphuric acid (3 mL) was carefully added to form a layer of redish brown colouration interface.

Test for Flavonoids

A 5 mL volume of dilute ammonia solution was added to a portion of the aqueous extract followed by addition of concentrated sulphuric acid. Colour change was observed.

Test for Phlobatannins

The aqueous extract of the sample was boiled with 1% aqueous hydrochloric acid. The presence or absence of coloured precipitate was noted..

Test for Saponins

Frothing test

A 2 g quantity of the powdered sample was boiled with 20 mL of distilled water in a water- bath and filtered. A 10, mL volume of the filtrate was mixed with 5 ml of distilled water and shacked vigorously for stable persistent froth.

Emulsion Test

In a 5 ml of filtrate in a test tube 2 drops of olive oil were added and shaken vigorously. The formation of emulsion indicated the presence of saponins.

Haemolytic test

A small quantity of the powdered drug was shaken with 3ml of normal saline, filtered and

2mL of the filtrate was added to a 2mL of 10 % v/v blood in normal saline. The mixture was centrifuged, the bright red colour of the blood was compared with the blood in normal saline which serves as control.

Test for Glycosides

A 5 mL volume dilute sulphuric acid was added to 0.1 g of the powdered drug and boiled for

15 minutes on a water bath. It was allowed to cool, then neutralized with 20 % potassium hydroxide solution. A 10 mL volume of the mixture of Fehling’s solution 1 and 11 in equal portions was added and boiled for 5 minutes. Brick red colour was observed

Test for Cardiac Glycosides (Keller-Killani Test)

A five mililitre volume of the extract was treated with 2 mL glacial acetic acid containing one drop of ferric chloride solution.The mixture was observed for presence or absence of brown ring on addiotion of 1 mL concentrated sulphuric acid

Test for Alkaloids

A 20 mL volume of dilute sulphuric acid and 50 % ethanol were added to 2g of the extract.

This was heated in the boiling water bath for 10 minutes. It was then cooled and filtered. A 2

72 mL volume of the filtrate was taken in four different test tubes and tested with a few drops of

Mayer’s reagent (potassium mercuric iodide solution), Dragendorff’s reagent (bismuth potassium iodide solution), Wagner’s reagent (iodine in potassium iodide solution, and Picric acid solution (1 %). The remaining filtrate was made alkaline with dilute ammonia solution in a 100 mL separating funnel.

The aqueous alkaline solution was separated and extracted with two 5 ml portions of dilute sulphuric acid. The extract was again tested for presence of alkaloids with the Mayer’s reagent, Dragendorff’s reagent, Wagner’s reagent and Picric acid solution.

Test for Resins

Precipitation Test

A 0.2 g quantity of extract was extracted with 15 mL of 96 % ethanol. The alcoholic extract was then poured into 20 mL of distilled water in a beaker and presence or absence of precipitate was observed.

Colour Test

A 0.2 g quantity of the extract was extracted with chloroform to obtain chloroform extract and this was concentrated to dryness in a water bath. The residue was then re-dissolved in 3 mL of acetone and 3 mL of concentrated hydrochloric acid was added. The mixture was heated for 30 minutes in water bath, formation and change of the colour was observed.

Test for Reducing Sugars

A 5 mL volume of a mixture of Fehling’s solution I and II in equal portions was added to 5 mL of the aqueous extract in a beaker. The beaker was heated on a water bath for 5 minutes, brick red precipitates were observed.

Test for Proteins

A 0.5 g quantity of the extract was extracted with 20 mL of distilled water and the resultant extract was used for the following tests.

(i) Millon’s Test

A 2 mL volume of the filtrate was taken in a test tube and 2 drops of Millon’s reagent were added, white precipitates were observed.

(ii)Xanthoproteic Reaction Test

A 5 mL volume of the extract in a test tube was heated on a water bath and few drops of concentrated nitric acid were added. There was formation of yellow colour which changed to orange colour on addition of sodium hydroxide.

Picric Acid Test

Few drops of picric acid were added to 2 mL of the extract, formation of yellow precipitates was observed.

Biuret Test

To 2 ml volume of the extract, a crystal of copper sulphate was added after which 2 drops of potassium hydroxide solution was added, appearance of purple or pink colour was observed

Test for Carbohydrates

Molisch Test

A 1.0 g quantity of the extract was boiled with 2 mL of distilled water and filtered. Few drops of Molisch’s reagent (naphtol solution in ethanol) were added. Concentrated sulphuric acid was then poured down the side of the test tube in such a way that a lower layer was formed. Appearance of a purple interfacial ring shows the presence of carbohydrates.

Test for Fats and Oils:

A 0.1g quantity of the extract was pressed between filter paper and the paper was observed.

Two drops of olive oil were dropped on a filter paper and the colour was observed. This served as a control.

3.7.1 Quantitative Phytochemical Analysis of P. nitida seed extract

Alkaliods determination

A method described by Harborne (1973) was used. A portion (5 g) of sample was weighed into a 250 mL beaker and 200 ml of 10 % acetic acid in ethanol was added, covered and allowed to stand for 2 h. This was filtered and the extract was concentrated on a water bath to one – quarter of the original volume. Concentrated ammonium hydroxide was added drop- wise to the extract and the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed.

Flavonoids determination

This was done following the method of Boham & Koupai-Abyazan (1994). A 10 g quantity of the sample was extracted repeatedly with 100 mL of 80 % aqueous methanol at room temperature. The whole solution was filtered through Whatman filter paper No 42 (125 mm).

The filtrate was later transferred into a crucible and evaporated into dryness over a water bath and weighed to a constant weight.

Saponins determination

The method of Obadoni and Ochuko (2001) was used. A portion (20 g) of the sample was put into a conical flask and 100 mL of 20 % aqueous ethanol was added. The sample was heated over a hot water bath for 4 h with continuous stirring. The mixture was filtred and the residue re-extracted with 200 ml of 20 % ethanol. The combined extracts were reduced to 40 mL over water bath at 90 oC . The concentrate was transferred into a 250 mL separating funnel and 20 ml of diethyl ether was added and shaken vigorously. The aqeous layer was recovered while the ether was discarded. The purification process was repeated.sixty mililitres of n- butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5 % aqeous sodium chloride. The remaining solution was heated on a water bath. After

75 evapouration the samples were dried in the oven to a constant weight. The saponins content was calculated in percentage of the 20 g extracted sample

Tannins determination

Tannin determination was done by Van-Burden and Robinson (1981) method. A portion (500 mg) of the sample was weighed into a 50 ml plastic bottle. A 50 mL volume of distilled water was added and shaken for 1 h on a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to mark. A 5 mL volume of the filtrate was pipette out into a test tube and mixed with 2 ml of 0.1 M FeCl3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured at 120 nm within 10 min.

3.7.2 Tests for mineral constituents of the extract of P. nitida.

The extract was tested for mineral ions. Heavy metals analysis was conduct using Varian

AA240 Atomic Absorption Spectrophotometer according to the method conytained in

American Public Health Association (APHA) (1995)

3.7.2 1. Determination of Total Ash Values

A tarred nickel crucible was heated dry, cooled and kept in a desiccator. A 2 g quantity of the powdered drug was weighed into the crucible and heated until there was no change in weight.

The heating continued until all the carbon content was evaporated to produce residue free of carbon. The residue was cooled in a desiccator and weighed. The process of heating and cooling was repeated until constant weight was obtained. The total ash value was calculated from Equation 1.

% Ash value = W3- W2 x 100 / W1- W2………………….Eq. 1.

3.7.2.2 Determination of Acid Insoluble Ash Value

The total ash obtained from the experiment above was transferred to a beaker containing 250 ml dilute hydrochloric acid and heated on a boiling water bath for five minutes. It was then filtered through ashless filter paper. The beaker and the crucible were washed repeatedly

76 until they are free from acid. The ashless filter paper was dried in the oven. It was later folded into a tarred crucible and heated completely to ash. The residual ash was then heated intensively and after cooling in desiccators, it was weighed.

3.7.2.3 Determination of Water Soluble Ash Value

In a heated and tarred nicked crucible, 2 g quantity of powdered material was spread over the bottom. The crucible was ignited at low heat initially to burn off the carbon content. The heat was gradually increased until all the carbon content was burnt off. The content of the crucible was transferred into a small beaker and 250 ml of water was added and this was allowed to boil for five minutes. The mixture was filtered through an ashless filter paper. The filter paper together with the residue was dried in the oven after which it was folded into the crucible and the heating continued until the ashless filter paper was eliminated and the weight of the residue was taken.

3.7.2.4 Determination of Water Soluble Extractive Value

A 5 g quantity of the powdered material was weighed into 250 ml stoppered conical flask.

Hundred mililitre of chloroform-water mixture was added and the flask firmly stoppered and was agitated mechanically for 6 hours. After that, the maceration continued for the next 18 hours. A 20 mL volume of the filtrate was evaporated to dryness in a 100 mL beaker over a hot water bath. The residue was dried to a constant weight at 1050C. The water extractive was calculated using equation 2.

% Extractive value = {(W3-W2) x 100 / (W1-W2) x W4} x 100 …………Eq. 2.

3.7.2.5. Determination of Alcohol Soluble Extractive Value

A 5 g quantity of powdered material was weighed into a 250 ml stoppered flask and100 mL of 95 % ethanol was added. The stopper was firmly placed and the contents of the flask were agitated mechanically for six hours. The maceration continued in the flask for the next 18 hours. A 20 ml volume of the filtrate was evaporated to dryness in a boiling hot water bath.

The residue was dried to a constant weight at 105 oC and the alcohol extractive was calculated from

% Extractive value ={(W3-W2) x 100 / (W1-W2) x W4} x 100 ……….Eq. 3.

3.7.2.6. Determination of Moisture Content

A tarred evaporating dish was heated to a constant weight and stored in a deciccator. A 3 g quantity of the pulverized material was added and the dish was heated in oven at constant temperature of 105 OC. It was allowed to dry until a constant weight was obtained. The moisture content was calculated from equation 4.

% Moisture Content = (W1-W2) x 100 / W3 ------Eq 4.

3.7.3 Chromatographic Analysis of the Extract and Fractions of Picralima nitida

(STAPF)

Procedure:

The TLC plate was a factory made sheet of aluminium foil which was coated with a thin layer of a solid adsorbent thickness made of silica gel G60. A very little capilliary drop of the extract and fractions of P. nitida were spotted on the start line near the bottom of the plate.

On the same start line, the same volume of the positive controls Beta-Carboxylic acid and

Para-hydroxybenzoic acid were spotted.

The TLC plate was then placed in a shallow pool of a solvent or mixture of solvents

(methanol:chloroform (65:35) (50:10) in a developing chamber so that only the very bottom of the plate was in the liquid. The solvent mixture slowly rose up the TLC plate by capillary action. The plate was removed at the end of the elution and dried.

The air-dried developed plate was observed under UV 254 nm rays and RF values of the spots of P. nitida seed fractions and the positive controls were determined after taking measurements from the spots to the start line in each case.

Some plates were treated with reagents: iodine vapour, ammonia vapour, Dragendorff’s reagent and spots produced were noted. The distance moved by each spot from the start line was noted. The Rf values were calculated from equation 5.

Rf = Distance traveled by the spot/ Distance traveled by the mobile phase….….Eq 5.

TLC and 2D TLC analysis was carried out on the isolated compounds were (compound 1 and compound 2) using MeOH : CHCl3 (50:10) as the solvent system, silicagel 40 mesh as the adsorbent and watched under UV at 254 nm.

3.7.4 Acute Toxicity Study (LD50).

The acute toxicity study was carried out using the method employed by Dietrich Lorke

(1983). A total of 13 rats were used and the study was carried out in two stages. Different concentrations of the extract were prepared according to their groups. The weights of the rats were used to determine the dose for each rat. Stage one was done using 9 rats and they were grouped into 3 groups of animals per group. Group 1, ll and lll received single oral dose of

10, 100 and 1000 mg/kg of the extract respectively. The animals were monitored intermittently over a period of 24 hours to record death.

The second stage employed 4 rats grouped into 4 groups of 1 animal each. The doses were given according to the weight of the animals. The first group received single oral dose of

1500 mg/kg, second group received single oral dose of 2500 mg/kg and third group received single oral dose of 3500 mg/kg. The forth group received single oral dose of 5000 mg/kg. The

LD50 was calculated using equation below:

LD50 = x b ………………………..Eq 6.

Where a is the lowest dose that brought death and b is the highest dose that did not cause death.

3.7.5 Blood Collection and Preparation

Five milliliters of HbSS red blood was obtained by venipuncture from a volunteer sickle cell disease patient in steady state from the out-patient Department of Madonna University

Teaching Hospital, Elele, Rivers State. Blood was collected into sodium EDTA bottles (to prevent coagulation), the content was thoroughly mixed by gently rolling the bottle. The sample was used within 24 hours. The blood sample was centrifuged to remove serum and the packed erythrocyte obtained was washed with normal saline.The blood samples were subjected to electrophoresis to determine the genotype

3.7.6 Determination of Packed Cell Volume (PCV)

(i) The packed cell volume (PCV) was determined by centrifuging heparinized blood in a capillary tube (also known as a micro hematocrit tube) at 10,000 rpm for five minutes

(Estridge H.B, Reynolds.A, 2008; “Hematocrit”. Encyclopedia of Surgery). This separates the blood into layers. The ratio of packed red blood cells to the total volume of the blood sample and expressed as a percentage represents the PCV.

3.7.7 Determination of Erythrocyte Sedimentation Rate (ESR).

The sample was an anti-coagulated blood placed in an upright tube, known as a Westergen tube and the rate at which the red blood cells fall was measured and reported in mm/h.

3.7.8 Hemoglobin Determination.

Method: The blood sample was mixed with Drabkins reagent which contains 200 milligrams potassium fericyanide, 50 milligrams of KCN and one gram sodium bicarbonate per litre. The solution was scaned in typical spectrophotometer and the hemoglobin content value was observed at the height of the 530 millimicron peak. Numerous point-of-care testing (POCT) devices are currently available (McMahon and Carpenter , 1990; Lardi et al

1998) that provide blood hemoglobin and hematocrit (H/H) determinations to assist in rapid

80 patient assessment, including the need for blood transfusion (Rippmann et al, 1997, Wu et al, 2003).

3.7.9 Red Blood Count

The automated hematology analyzer was used to measure the amount of haemoglobin in the blood. There was the addition of a diluents to the blood sample that lyses the cells, the mixture was later pumped into the spectro-photometric measuring cuvette.The change in color of the lysate equates to the value of hemoglobin content of the blood.

3.7.10 Determination of Plasma Calcium

The blood was tested for calcium ions using Varian AA240 Atomic Absorption

Spectrophotometer according to the method of American Public Health Association (APHA)

1995. The sample was aspirated into the flame and atomized when the AAS’s light beam is directed through the flame into the monochromator, and onto the detector that measures the amount of light absorbed by the atomized element in the flame, a source lamp composed of calcium element is used, The amount of energy of the characteristic wavelength absorbed in the flame is proportional to the concentration of the calcium in the sample.

3.7.11 Conductivity

As a liquid with ions suspended in it, plasma is more conductive than red blood cells.

Therefore, the more plasma by volume, the more conductivity, which is an inverse relation to the hematocrit.The concentration of electrolytes in blood affect its conductivity. The more electrolytes, the higher the conductivity.

For measurement of conductivity a digital conductivity meter 909, (Digisum Electronoics) has been used, (Shikha and Basharath, 2014). The equipment consists of a chamber and two electrodes which are separated with a small gap of the order of micrometer. Before measuring the conductivity of the given sample the digital meter has to be calibrated by using 0.01N potassium chloride solutions. After calibration the electrodes should br removed from the

KCl and washed with distilled water. Then the conductivity meter can measure the given sample.

3.7.12. Measuring total dissolved solids (TDS)

The TDS was determined by the Direct Reading Engineering Method (DREM) using Hach C

050 meter, the probe which had been pre-formulated to match blood sample was used and the

TDS mode button pressed. The TDS value was read off as displayed on the instrument panel.

3.7.13. Measurement of Viscosity

The viscosity of the blood was determined by the Osawald capillary viscosity method, using water sample as the standard. The distilled water sample was aspirated into the upper bulb of the u-tube viscometer.

The time taken for the water to flow from one mark on the bulb to the other mark was taken as t-water

Normal and sickled cell bloods were also aspirated as described above respectively, to the mark. The time taken by each of the blood samples to flow to the last mark was also taken as t-sample. Since the viscosity of distilled water is 1.025 poise at 25 OC, the viscosity of the blood samples were calculated from equation 7.

η water η sample = ………………………. Eq. 7 t water t sample

Preparation of the concentration of the extract/ fractions of P.nitida

Three concentrations of the extract and P. nitida 2.5, 5 and 10 mg were prepared and used to treat the SS blood. The system was incubated at 37oC and occasionally shacked.

Measurement of viscosity as was described above, were taken at the end of every 30 minutes through 180 minutes for each concentration. The same viscosity test was carried out on ss genotype with the three concentrations 2.5, 5 and 10 mg of P. nitida fractions. The three concentrations of the methanol extract (2.5, 5, and 10 mg), Para-hydroxybenzoic acid (5, 50 82 and 500 mg) and normal saline were prepared and used to treat the SS blood. The system was incubated and measurement of viscosity as was described above were taken at 30 minutes intervals in to 180 minutes

3.7.14 Observation of the normal cell morphology of collected blood sample

A 5 ml volume of normal red blood (HbAA) sample was pipeted into a sample tube and made up to mark with normal saline, mixed gently and centrifuged at 2000-3000 rpm for 10 minutes to remove the serum and the packed erythrocytes were obtained and washed with normal saline as described by Egunyomi et al. (2009) to remove the supernatant.

A thin film of the erythrocytes was made on a clean microscopic slide, covered with cover slid and then examined under oil immersion.

3.8 Anti Sickling Evaluation Evaluation of antisickling activities of the extract/fractions was carried out using a modified method of Sofowora et al. (1979).

3.8.1 Induction of complete sickling in normal Blood (Genotype As) using

Sodium methabisulphite

Complete sickling was induced on a venous blood collected from a voluntary donor, a staff of

Stratech Research Laboratory. A 2 % solution of sodium metabisulphite prepared by dissolving 2 g of sodium metabisulphite in 100 ml of normal saline was labelled solution 1. A

0.5 ml volume of solution 1, was added to 0.5 ml of centrifuged blood sample, and a drop of the mixture was mounted on a slide and examined under x 100 magnification.

3.8.2 Calculation of % reversal of sickling in HbAS red blood cells.

A smear of the HbAS red blood sample (Peripheral cells) was made on a clean slide and viewed under x 100 magnification. The number of the sickled cells and unsickled cells were counted and noted as described by Egunyomi et al. (2009). The percentage of the unsickled cells was then calculated, from equation 8. 83

N ×100 1 ……………………. Eq 8 N 2

Where N1 is the number of unsickled cells, N2 is the total number of cells.

3.8.3 Antisickling activity of crude methanol extract, fractions and isolates of P. nitida

The extract and fractions of P. nitida at different concentrations were screened for antisickling activity. The plant extract/fractions for sickling reversal activity was carried out according to the procedure of Oduola et al. (2006). A 0.5 ml volume of the washed erythrocytes was mixed with 0.5 ml of different concentrations of extract in the uncovered test tubes. A 0.5 ml volume of 2 % sodium metabisulphite was added to deoxygenate the blood sample. The system was mixed thoroughly and sealed with liquid paraffin. Samples were taken from different mixtures immediately at zero time and the remaining mixtures were incubated at 37 °C, in a shaking incubator. Samples were taken in triplicate subsequently at 30 minutes interval (0, 30, 60, 90, 120, 150 and 180 minutes) from the different mixtures. Thin film of each sample was made and examined under oil immersion light microscope 100 x magnification. Counting of 100 sickled and unsickled red blood cells of the sample was carried out and the percentage of unsickled cells determined. The same experimental procedure was repeated for both positive and negative controls i.e parahydroxybenzoic acid and normal saline.

3.9 Statistical analysis

Data obtained were analyzed SPSS version 14 software results obtained were expressed as mean ± SEM. While difference in means was analysed using One Way Analysis of Variance

ANOVA Differences between means were regarded significant at P < 0.05.

CHAPTER FOUR

RESULTS

4.1: Macroscopic and Microscopic analysis

Table 4: Organoleptic indices of P. nitida.

Parameter Seed Powdered seed

Colour Light Brown Light brown

Odour Characteristic Characteristic

Taste Bitter Strong bitter taste

Shape Obovoid Powder

Surface texture Smooth Fine grain

Solubility Not soluble in water/ alcohol. not soluble in water/ alcohol.

Epidermal cells

Fat globules

Photomicrograph of the powdered seed Photomicrograph of the powdered seed sample sample of Picralima nitida showing the of Picralima nitida showing oil globules on the epidermal cells epidermal cells

Calcium oxalate prism crystals

Photomicrograph of the powdered seed Photomicrograph of Picralima nitida sample of Picralima nitida showing Prism shape calcium oxalate crystals and oil powdered seed sample showing globules on the epidermal cells parenchymatous cells

Sclereid cells (Stone cells)

Photomicrograph of Picralima nitida Photomicrograph of Picralima nitida powdered seed sample showing powdered seed sample showing sclereids Collenchymatous cells (stone cells)

Plate 5: Microscopy of the Powdered Seed of P. nitida

Cork cells

Spheroid Calcium

oxalent crystal

Parenchyma cells

Spheroid Calcium oxalate crystal Cluster of cork cells from the testa of the

powdered seeds of P. nitida

Parenchyma cell

Fat globules

Parenchyma cells of the powdered seed of P. nitida Fat globules dissolving in chloral hydrate. From the powdered seed of P. nitida

Plate 6: Microscopy of the Powdered Seed of P. nitida

4.2:Yield of Extraction

Table 5: Yield % of extract and fraction from P. nitida seed

Solvent Yield (g) Yield (% w/w)

Methanol Extract (ME) 345.0a 13.80

Chloroform Fraction (CF) 103.5b 4.14

Dichloromethane Fraction (DCMF) 41.2b 1.65

Ethyl Acetate Fraction (EAF) 33.5b 1.38

Aqueous Fraction ( AF) 165.5b 6.620

a = 2.5 kg of powdered seeds b = 34.5 g methanol extract

4.3: Phytochemical analysis

Table 6: Phytochemical analysis of P. nitida seed

Chemical compounds Observation Inference ME CF DCMF EAF AF Tannins Brownish, green or blue black colouration + _ _ + + Steroids Colour change from violet to blue or green + + + _ _ Terpenoids Redish brown colouration of interface + + + _ _ Flavonoids Yellow colouration which disappears on + + + + + standing Starch + _ _ _ + i.Molisch reagent Appearance of purple interface. ii.Chloral hydrate solution blue black colouration Saponins + _ + + + i.Frothing test Persistent froth ii.Emulsion test Formation of emulsion iii.Haemolysis test Bright red colour of blood Alkaloids + _ _ + + i.Mayer’s reagent Milky precipitate ii.Dragendorff‘s Brick red precipitate reagent iii.Wagners’s reagent Redish brown precipitate iv.Picric acid solution (1%) Yellow precipitate Glycosides + _ + + + Cardiac glycosides Brown ring interface + _ + + + Resins + + _ _ _ i.Precipitation test Precipitation formed ii.Colour test Pink colour which changes to magenta red Reducing sugars + _ _ _ + Fehlings solution Brick red precipitate Proteins + _ _ _ + i.Million test White precipitate ii.Santhoproteic Appearance of Yellow colour which Reaction changes to Orange colour in addition of sodium hydroxide iii.Picric acid test Yellow precipitate. Biuret test Purple or pink colour Fats and oil No translucency + + - - - Phlobatanins Red precipitate _ _ + + + HCL Test Key: + (present), - (Absent), ME (Methanol extract), CF (Chloroform fraction), DCMF

(Dichloromethane fraction), EAF (Ethyl acetate fraction), AF (Aqueous fraction),.

Table 7: Quantitative phytochemical analysis of P. nitida Constituents Value (mg/100) Tannins 0.065± 1.23 Flavonoids 25.48± 0.33 Saponins 7.03± 0.12 Glycosides ±2.15 Terpenoids 4.08± 1.46 Proteins 11.63 ± 0.35 Alkaloids 5.84± 1.18 Values are Mean ± SEM, n=3

4.4: Mineral content analysis Table 8: Mineral analysis of P. nitida Parameter Concentration Calcium 12.686 ppm Magnesium 11.665 ppm Potassium 42.537 ppm Chlorine 159 mg/l

4.5: Acute toxicity test

Table 9: Acute toxicity study (LD50) of P. nitida Stage Dose (mg/kg) No of Death 1 10 0/3 100 0/3 1000 0/3 II 1500 0/1 2500 0/1 3500 0/1 5000 1/0

4.6: Antisickling Evaluation Table 10: Antisickling effect of methanol extract of P. nitida seeds on HbSS red blood cells Incubati N/ Parahydroxybenzoic acid (mg/ml) Methanol Extract (mg/ml) on time S % reversal of siclkling % reversal of sickling (min) 5 50 500 2.5 5 10 0 43 38 ± 0.50* 60 ± 1.22* 83 ± 1.06* 42 ± 1.12* 50 ± 1.04* 54 ± 0.69* 30 43 40 ± 0.40* 65 ± 0.69* 87 ± 0.42* 45 ± 0.22* 50 ± 1.80* 57 ± 1.40* 60 43 42 ± 0.68* 66 ± 0.46* 95 ± 1.82* 47 ± 0.41* 56 ± 2.10* 59 ± 0.80* 90 43 50 ± 1.06* 68 ± 0.43* 98 ± 0.28* 51 ± 0.70* 64 ± 1.60* 63 ± 0.39* 120 43 56 ± 0.64* 76 ± 1.09* 100 ± 0.18* 53 ± 0.44* 65 ± 0.62* 65 ± 0.54* 150 43 56 ± 1.34* 83 ± 0.60* 100 ± 0.60* 57 ± 1.52* 66 ± 0.36* 67 ± 0.66* 180 43 58 ± 0.42* 95 ± 132* 100 ± 0.90* 60 ± 0.58* 66 ± 1.32* 67 ± 1.20* Values are Mean ± SEM, *p < 0.05, n =3

Table 11: Antisickling effect of chloroform fraction of P. nitida on HbSS red blood cells Incub Neg.c Para hydroxy benzoic acid Chloroform fraction concentration ation ontrol (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling. time N/sal 5 50 500 2.5l 5 10 (min) 0 43 38 ± 0.24* 40 ±0.44* 45 ±086* 31 ±1.38* 41 ±1.26* 43 ±0.48* 30 43 40 ± 0.47* 43 ± 0.63* 65 ± 0.59* 42± 0.93* 46 ± 0.44* 52 ± 0.82* 60 43 44 ±0.27* 60 ±0.98* 100 ±1.04* 48±0.96* 48 ±0.78* 57 ±0.34* 90 43 50 ±1.23* 79 ±0.42* 100 ±0.60* 62 ±0.58* 54 ±0.44* 57 ±0.26* 120 43 56 ±0.37* 88 ±0.20* 100 ±0.45* 74 ±0.72* 57 ±1.44* 58 ±0.48* 150 43 56 ±0.56* 88 ±2.36* 100 ±1.83* 78 ±0.52* 58 ±0.48* 63 ±1.09* 180 43 58 ±0.97* 98 ±0.32* 100 ±0.78* 78 ±030* 64 ±0.56* 64 ±0.70* Values are Mean ± SEM, *p < 0.05, n =3

Table 12: Antisickling effect of ethyl acetate fraction of P. nitida on HbSS red blood cells time Neg Para hydroxy benzoic acid Ethyl acetate fraction concentration (min) cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling N/S 5 50 500 2.5 5 10 0 43 38 ± 0.21* 40 ± 0.82* 45 ± 0.78* 30 ± 0.38 33 ± 1.32* 36 ± 0.74* 30 43 41 ± 1.43* 58 ± 0.98* 100 ± 0.30* 36 ± 0.42 39 ± 0.54* 42 ± 0.86* 60 43 44 ± 0.88* 60 ± 1.41* 100 ± 0.22* 38 ± 0.66 46 ± 0.44* 48 ± 0.82* 90 43 50 ± 1.03* 79 ± 0.47* 100± 0.25* 44 ± 0.20 48 ± 1.15* 55 ± 0.46* 120 43 56 ± 0.42* 88 ± 1.12* 100 ± 0.46* 57 ± 0.87 59 ± 0.28* 61 ± 0.32* 150 43 56 ± 0.50* 88 ± 0.64* 100 ± 1.40* 60 ± 1.24 63 ± 0.90* 65 ± 0.52* 180 43 58 ± 0.56* 98 ± 1.44* 100 ± 0.34* 65 ± 0.26 65 ± 0.58* 66 ± 0.40* Values are Mean ± SEM, *p < 0.05, n =3

Table 13: Antisickling effect of aqueous fraction of P. nitida on HbSS red blood cells time Neg. Para hydroxy benzoic acid Aqueous fraction concentration (min cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling N/S 5 50 500 2.5 5 10 0 43 38 ± 0.80* 40 ± 0.42* 45 ± 0.60* 30 ± 0.64* 32 ± 0.63* 45 ± 1.25* 30 43 41± 1.22* 58 ± 0.82* 100 ±1.07* 43 ± 0.34* 44 ± 0.45* 50 ± 0.92* 60 43 44± 0.67* 60 ± 1.84* 100 ± 0.72* 45 ± 0.89* 58 ± 1.52* 55 ± 0.62* 90 43 50 ± 0.24* 79 ± 0.32* 100 ± 1.08* 45 ± 0.63* 64 ± 0.38* 67 ± 2.06* 120 43 56 ± 1.25* 88 ± 0.45* 100 ± 0.54* 57 ± 0.22* 67 ± 0.60* 76 ± 1.76* 150 43 56 ± 0.54* 88 ± 0.86* 100 ± 0.16* 70 ± 1.44* 72 ± 0.93* 85 ± 0.45* 180 43 58 ± 0.88* 98 ± 0.63* 100 ± 1.18* 81 ± 0.78* 83 ± 0.64* 85± 0.82* Values are Mean ± SEM, *p < 0.05, n =3

Table 14: Antisickling effect of dichloromethane fraction of P. nitida on HbSS red blood cells time Neg. Para hydroxybenzoic acid Dichloromethane fraction concentration (min cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling N/S 5 50 500 2.5 5 10 0 43 38 ± 0.51* 40 ± 0.48* 45 ± 0.63* 25 ± 1.32* 33 ± 0.69* 38 ± 0.56* 30 43 41 ± 2.00* 58 ± 1.25* 100 ± 0.30* 44 ± 0.44* 42 ± 0.52* 46 ± 0.34* 60 43 44 ± 0.62* 60 ± 0.55* 100 ± 1.02* 48 ± 1.81* 60 ± 0.32* 64 ± 0.34* 90 43 50 ± 0.27* 79 ± 0.69* 100 ± 0.60* 50 ± 0.75* 65 ± 0.90* 72 ± 0.16* 120 43 56 ± 0.86* 88 ± 0.33* 100 ± 0.39* 55 ± 0.31* 74 ± 0.55* 77 ± 0.63* 150 43 56 ± 0.47* 88 ± 0.81* 100 ± 0.47* 60 ± 0.62* 74 ± 0.22* 82 ± 1.18* 180 43 58 ± 0.38* 98 ± 0.62* 100 ± 2.01* 77 ± 0.92* 82± 0.68* 82 ± 0.32* Values are Mean ± SEM, *p < 0.05, n =3

Table 15: Antisickling effect of methanol extract of P. nitida on HbAS red blood cells time Neg, Para hydroxy benzoic acid Methanol extract concentration (min cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling N/S 5 50 500 2.5 5 10

0 45 38 ± 0.45* 43 ± 0.54* 45 ± 0.42* 42 ± 0.68* 41 ± 0.44* 33 ± 1.24* 30 45 45 ± 0.46* 48 ± 0.86* 90 ± 0.92* 48 ± 0.43* 43 ± 0.65* 65 ± 0.42* 60 45 46 ± 0.37* 52 ± 0.61* 100 ± 1.52* 45 ± 0.45* 47 ± 0.39* 88 ± 0.55* 90 45 46 ± 0.36* 59 ± 0.52* 100 ± 0.70* 44 ± 0.81* 53± 0.57* 89 ± 0.43* 120 45 51 ± 0.73* 62 ± 0.68* 100 ±0.83* 40 ± 0.47* 53 ± 0.69* 89 ± 0.20* 150 45 52 ± 0.63* 75 ± 0.42* 100 ± 0.30* 47 ± 0.74* 57 ± 0.93* 100 ±0.63* 180 45 56 ± 2.00* 87 ± 0.85* 100± 0.13* 53 ± 0.75* 60 ± 0.49* 82 ± 0.78* Values are Mean ± SEM, *p < 0.05, n =3

Table 16: Antisickling effect of aqueous of Fraction of P. nitida on HbAS red blood cells. time Neg. Para hydroxy benzoic acid Aqueous Fraction concentration (min cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling ) rol 5 50 500 2.5 5 10 N/S 0 43 38± 1.63* 43 ± 0.57* 45 ± 0.44* 43 ± 0.34* 40 ± 0.85* 17 ± 0.13*

30 45 45± 0.36* 48 ± 0.66* 90 ± 0.21* 43 ± 0.73* 46 ± 0.48* 67 ± 0.43* 60 45 46 ± 1.42* 52 ± 0.76* 100 ± 0.38* 43 ± 0.57* 51 ± 0.35* 100 ± 1.35* 90 45 46 ± 1.45* 59 ± 0.72* 100 ± 0.33* 45 ± 0.71* 52 ± 0.75* 93 ± 0.45* 120 45 51 ± 0.83* 62 ± 0.31* 100 ± 0.37* 50 ± 0.38* 57 ± 0.67* 100 ± 0.21* 150 45 52 ± 1.05* 75 ± 1.73* 100 ± 0.31* 50 ± 0.52* 59 ± 0.47* 100 ± 0.33* 180 45 56 ± 0.35* 87 ± 0.64* 100 ± 0.34* 52 ± 0.44* 64 ± 0.52* 100 ± 0.36* Values are Mean ± SEM, *p < 0.05, n =3

Table 17: Antisickling effect of Chloroform fraction of P .nitida on HbAS red blood cells Time Neg. Para hydroxy benzoic acid Chloroform fraction concentration (min) control (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling (N/S 5 50 500 2.5 5 10 0 43 38 ± 0.83* 43 ± 2.03* 45 ± 0.64* 44 ± 0.92* 42 ± 0.54* 17 ± 2.43*

30 45 45± 0.42* 48 ± 0.46* 90 ± 0.64* 45 ± 0.48* 44 ± 0.33* 65 ± 069* 60 45 46 ± 0.47* 52 ± 0.96* 100 ±1.52* 47 ± 0.43* 51 ± 0.97* 65 ± 0.58* 90 45 46 ± 0.21* 59 ± 0.75* 100 ±0.61* 48 ± 0.58* 53 ± 0.49* 95 ± 0.40* 120 45 51 ± 0.37* 62± 0.79* 100 ±0.33* 50 ± 0.38* 57 ± 0.45* 100 ±0.55* 150 45 52 ± 2.00* 75 ± 1.52* 100 ±0.32* 51 ± 0.13* 59 ± 0.68* 100 ±0.72* 180 45 56 ± 0.51* 87 ± 0.35* 100 ±0.37* 54 ± 0.46* 65 ± 1.08* 100 ±0.33* Values are Mean ± SEM, *p < 0.05, n =3

Table 18: Antisickling effect of ethyl acetate fraction of P .nitida on HbAS red blood cells Neg. Para hydroxy benzoic acid Ethyl Acetate Fraction conc. (mg/ml) time con (mg/ml) % reversal of sickling % reversal of sickling (min) (N/S) 5 5 500 2.5 5 10 0 43 38 ± 0.82* 43 ± 0.62* 45 ± 0.73* 36 ± 0.42* 77 ± 1.22* 36 ± 0.94*

30 45 45± 0.75* 48 ± 0.47* 90 ± 0.34* 35 ± 0.86* 45 ± 0.38* 52 ± 1.42* 60 45 46 ± 0.45* 52 ± 0.33* 100 ± 0.91* 42 ± 1.41* 41 ± 0.55* 49 ± 0.83* 90 45 46 ± 0.35* 59 ± 0.61* 100 ± 0.59* 40 ± 0.49* 50 ± 0.37* 100 ± 28* 120 45 51 ± 1.09* 62 ± 0.72* 100 ± 1.63* 48 ± 0.97* 53 ± 0.64* 87 ± 0.44* 150 45 52 ± 0.25* 75 ± 0.58* 100 ± 0.71* 50 ± 0.31* 66 ± 0.76* 96 ± 0.19* 180 45 56 ± 0.21* 87 ± 0.49* 100 ± 0.51* 56 ±0.63* 58 ± 0.82* 100± 0.44* Values are Mean ± SEM, *p < 0.05, n =3

Table 19: Antisickling effect of dichloromethane fraction of P. nitida on HbAS red blood cells. time Neg. Para hydroxy benzoic acid Dichloromethane Fraction conc (min) cont (mg/ml) % reversal of sickling (mg/ml) % reversal of sickling (N/S 5 50 500 2.5 5 10

0 45 38 ± 0.46* 43 ± 0.92* 45 ± 0.73* 43 ± 0.22* 42 ± 0.38* 41 ± 0.41

30 45 45 ± 0.52* 48 ± 0.13* 90 ± 0.29* 46 ± 0.77* 45 ± 0.14* 53 ± 0.86* 60 45 46 ± 0.96* 52 ± 0.33* 100 ± 1.47* 27 ± 0.77* 43 ± 0.29* 70 ± 0.43* 90 45 46 ± 0.11* 59 ± 0.19* 100 ± 0.31* 35 ± 0.24* 48 ± 0.17* 90 ± 0.88* 120 45 51 ± 0.59* 62 ± 0.14* 100 ± 0.21* 46 ± 0.73* 60 ± 0.47* 91 ± 1.09* 150 45 52 ± 1.61* 75 ± 0.52* 100 ± 0.66* 49 ± 0.19* 61 ± 0.78* 100 ±1.73* 180 45 56 ± 0.56* 87 ± 0.80* 100 ± 1.83* 53 ± 0.20* 59 ± 0.49* 100 ±0.50* Values are Mean ± SEM, *p < 0.05, n =3

Table 20: Effect of methanol extract of P.nitida on Viscosity of HbSS red blood Incu Negative P-hydroxybenzoic acid (Positive control) Methanol extract concentration (mg/ml) Min control (mg/ml) (N/saline) 5 50 500 2.5 5 10 0 3.00 ± 0.29* 3.00 ± 0.22* 3.00 ± 0.66* 3.00 ± 0.17* 3.02± 0.15* 3.01 ±0.31* 3.01 ±0.41* 30 3.00 ± 0.41* 3.00 ± 0,37* 3.00 ±0.347* 2.90 ± 0.59* 3.01 ±0.53* 3.00 ±0.47* 3.00± 0.53* 60 3.00 ± 0.38* 2.96 ± 0.64* 2.90 ± 0.50* 2.88 ± 0.32* 3.00 ±0.14* 3.00 ±0.52* 2.99 ±0.26* 90 3.00 ± 0.46* 2.90 ± 0.71* 2.64 ± 0.26* 2.76 ± 0.11* 3.00 ±0.76* 2.99 ±0.73* 2.84 ±0.59* 120 3.00 ± 0.12* 2.86 ± 0.19* 2.78 ± 0.69* 2.62 ± 0.35* 2.99± 0.80* 2.96 ± 027* 2.70 ±0.11* 150 3.00 ± 0.27* 2.80 ± 0.38* 2.75 ± 0.55* 2.62 ± 0.83* 2.96 ±0.19* 2.80 ±0.41* 2.66 ±0.72* 180 3.00 ± 0.93* 2.78 ± 0.17* 2.72 ± 0.28* 2.62 ± 0.77* 2.90 ±0.18* 2.80 ±0.87* 2.61 ±0.62* Values are Mean ± SEM, *p < 0.05, n =3

Table 21: Effects of chloroform fractions of P.nitida on HbSS red blood Incu Neg. control P-hydroxybenzoic acid (mg/ml) Chloroform fraction concentration time (N/Saline) (mg/ml) (min 5 50 500 2.5 5 10 0 3.00 ± 0.31* 3.00 ± 0.18* 3.00 ± 0.98* 3.00 ±0.30* 3.00 ±0.47* 3.00 ±0.48* 3.00 ±0.23* 30 3.00 ± 0.52* 3.00 ± 0.71* 3.00 ± 0.79* 2.90 ±0.43* 3.00 ±0.19* 3.00 ± 099* 3.00 ±0.37* 60 3.00 ± 0.39* 2.96 ± 0.13* 2.90 ± 0.81* 2.88 ±0.73* 3.00 ±0.26* 2.96 ±0.41* 2.90 ±0.30* 90 3.00 ± 0.44* 2.90 ± 0.33* 2.64 ± 0.84* 2.76 ±0.94* 2.82 ±0.41* 2.80 ±0.37* 2.76 ±0.98* 120 3.00 ± 0.62* 2.86 ± 0.17* 2.78 ± 0.22* 2.62 ±0.89* 2.80 ±0.73* 2.78 ±0.17* 2.70 ±0.79* 150 3.00 ± 0.59* 2.80 ± 0.72* 2.75 ± 0.63* 2.62 ±0.37* 2.80 ±0.16* 2.70 ±0.68* 2.60 ±0.53* 180 3.00 ± 0.19* 2.78± 0.91* 2.72 ± 0.31* 2.62 ±0.44v 2.70 ±0.53* 2.65 ±0.29* 2.60 ±0.57* Values are Mean ± SEM, *p < 0.05, n =3

Table 22: Effect of aqueous fraction of P. nitida on viscosity of HbSS red blood Incu Neg. control P-hydroxybenzoic acid (mg) Aqueous fraction concentration (mg) time (N/saline) (min 5 50 500 2.5 5 10 0 3.00 ± 0.11* 3.00 ± 0.29* 3.00 ±0.64* 3.00 ±0.88* 3.00 ±0.71* 3.00 ±0.55* 2.99 ± 0.44* 30 3.00 ± 0.72* 3.00 ± 0.52* 3.00 ±0.41* 2.90 ± 081* 3.00 ±0.50* 3.00 ±0.61* 2.96 ± 0.66* 60 3.00 ± 0.,8* 2.96 ± 0.37* 2.90 ±0.71* 2.88 ±0.59* 3.00 ±0.49* 2.96 ±0.84* 2.84 ± 0.14* 90 3.00 ± 0.93* 2.90 ± 0.25* 2.64 ±0.39* 2.76 ±0.16* 2.80 ±0.36* 2.72 ± 060* 2.69 ± 0.94* 120 3.00 ± 0.13* 2.86 ± 0.22* 2.78 ±0.28* 2.62 ±0.38* 2.80 ±0.49* 2.66 ±0.39* 2.60 ± 022* 150 3.00 ± 0.67* 2.80 ± 0.75* 2.75 ±0.19* 2.62 0.28* 2.80 ±0.93* 2.60 ±0.15* 2.60 ± 0.76* 180 3.00 ± 0.53* 2.78 ± 0.57* 2.72 ±0.43* 2.62± 0.84* 2.75 ± 035* 2.60 ±0.14* 2.60 ± 0.79* Values are Mean ± SEM, *p < 0.05, n =3

Table 23: Effect of Ethyl acetate fraction of P. nitida on Viscosity of HbSS red blood incu N/Sal P-hydroxybenzoic acid (mg/ml) Ethyl acetate fraction concentration (mg/ml) time Negative (min control 5 50 500 2.5 5 10

0 3.00 ± 0.16* 3.00± 0.13* 3.00± 0.71* 3.00± 0.55* 3.02 ± 0.44* 3.01 ± 0.55* 3.00 ± 0.34* 30 3.00 ± 0.16* 3.00 ±0.67* 3.00± 0.55* 2.90± 0.63* 3.01 ± 0.27* 3.00 ± 0.18* 3.00 ± 0.81*

60 3.00 ± 0.77* 2.96 ±0.32* 2.90± 0.27* 2.88± 0.13* 3.00 ± 0.88* 3.00 ± 0.66* 2.99 ± 0.31* 90 3.00 ± 0.68* 2.90 ±0.58* 2.64± 0.20* 2.76± 0.18* 3.00 ± 0.79* 2.98 ± 0.19* 2.95 ± 0.65* 120 3.00 ± 0.49* 2.86 ±0.31* 2.78± 0.69* 2.62± 0.84* 2.98 ± 0.35* 2.94 ± 0.38* 2.90 ± 0.97* 150 3.00 ± 0.52* 2.80 ±0.47* 2.75±0.4* 2.62± 0.30* 2.96 ± 0. 77* 2.90 ± 0.49* 2.80 ± 0.29* 180 3.00 ± 0.91* 2.78± 0.82* 2.72±0.3* 2.62± 0.51* 2.95 ± 0.83* 2.91 ± 0.39* 2.80 ±0. 21* Values are Mean ± SEM, *p < 0.05, n =3

Table 24: Effect of Dichloromethane fraction of P. nitida on viscosity of HbSS red blood Incu Normal P-hydroxybenzoic acid (mg/ml) Dichloromethane fraction concentration tim saline (mg/ml) 5 50 500 2.5 5 10 0 3.00 ± 0.43* 3.00 ± 0.50* 3.00 ±0.44* 3.00 ±0.39* 3.00 ±0.77* 3.00 ±0.53* 2.98± 0.36* 30 3.00 ± 0.58* 3.00 ± 0.47* 3.00 ±0.31* 2.90 ±0.49* 3.00 ±0.18* 3.00 ±0.38* 2.6 ± 0.51*

60 3.00 ± 0.38* 2.96 ± 0.23* 2.90 ±0.17* 2.88 ±0.12* 3.00 ±0.88* 2.98 ±0.33* 2.86 ± 0.24* 90 3.00 ± 0.94* 2.90 ± 0.74* 2.64± 0.29* 2.76 ±0.78* 2.82 ±0.51* 2.78 ±0.42* 2.70 ± 0.73* 120 3.00 ± 0.49* 2.86 ± 0.11* 2.78 ±0.53* 2.62 ±0.41* 2.80 ±0.39* 2.76 ±0.21* 2.69 ± 0.80* 150 3.00 ± 0.31* 2.80 ± 0.40* 2.75 ±0.96* 2.62 ±0.89* 2.80 ±0.64* 2.66 ±0.72* 2.62 ± 0.61* 180 3.00 ± 0.75* 2.78 ± 61* 2.72 ±0.87* 2.62 ±0.27* 2.78 ±0.22* 2.62 ±0.73* 2.61 ± 0.48* Values are Mean ± SEM, *p < 0.05, n =3

Table 25: Effect of Methanol extract(2.5mg/ml) of P. nitida on some biochemical parameters Paramete AA AS SS

Pre Post Pre Post Pre Post PCV (%) 35 ± 0.67* 37± 0.46* 35 ± 0.81* 35.5 ± 0.45* 21 ± 0.43* 23 ± 1.80 ESR mm/h 5 ± 0.54* 6 ± 0.78* 6 ± 0.27* 6.7 ± 0.87* 5 ± 0.76* 5.2 ± 0.51 Hbs mg/dl 12.0 ± 0.66* 12. ± 0.10* 11. ±0 .46* 11.5 ± 0.55* 6.2 ± 0.63* 6.7 ± 0.17 RBC mm3 4.1 ± 0.26* 3.3 ± 0.74* 4.4.6±0.68* 4.9 ± 0.71* 4.7 ± 0.59* 4.6 ± 0.42 Plasma Ca 6.95 ± 0.32* 6.5 ± 0.59* 6.95± 0.16* 4.2 ± 1.64* 8.2 ± 0.98* 7.2 ± 0.17 mg/dl Conductivi 5440 ±0.33* 5187 ± 0.26* 6138±0.48* 6528 ± 0.52* 7424 ± 0.97* 7619 ± 0.20 tyµs/m3 TDS mg/L 27.20±0.82* 29.11± 0.27* 30.69±.47* 32.43 ± 0.18* 39.12 ± 0.26* 39.29± 0.43 Viscosity/p 2.55 ± 2.02* 2.51 ± 0.74* 2.67 ±0.32* 2.60 ± 0.56* 3.05 ± 0.66* 2.71 ± 0.15 oise Values are Mean ± SEM, *p < 0.05, n =3

Table 26: Effect of Methanol extract (5 mg/ml) of P. nitida on some biochemical parameters Paramete AA AS SS

Pre Post PRE Post Pre Post PCV (%) 36 ± 0.32* 36 ± 0.88* 30 ± 0.44* 31 ± 0.63* 20 ± 0.20* 21 ± 0.66* ESR 5 ± 0.55* 6 ± 0.87* 6 ± 0.84* 7 ± 0.78* 5 ± 0.41* 5.8 ± 0.89* mm/h Hbs mg/dl 12.0 ± 0.68 12. 2=0.68* 11.0 ± 0.41* 11.8 ± 0.98* 6.2 ± 0.55* 6.7 ± 0.63* RBC mm3 5.5 ± 0.49* 5.4 ± 2.06* 5.1 ± 0.66* 5.9 ± 0.59* 5.4 ± 0.83* 5.4 ± 1.62* Plasma ca 6.95 ± 1.08* 6.3 ± 0.43* 6.95 ± 0.92* 8.1 ± 0.46* 8.2 ± 0.52* 6.3 ± 0.92* mg/ dl Conductiv 5440 ± 0.44* 5903 ± 0.93* 6138 ± 0.75* 6523 ± 0.18* 7424 ±0.61* 7703 ±0.34* ityµs/m3 TDS mg/L 27.20 ± 0.15* 28.51 ± 0.74* 30.69 ± 0.53* 33.54 ± 0.14* 37.12 ± 55* 3951 ±0.67* Viscosity/ 2.55 ± 0.65* 2.54 ± 0.69* 2.67 ± 0.57* 2.63 ± 1.06* 3.05 ± 0.63* 2.80 ± 0.90* poise Values are Mean ± SEM, *p < 0.05, n =3

Table 27: Effect of Methanol extract (10mg/ml) of P. nitida on some biochemical parameters AA AS SS

PRE Post Pre Post Pre Post PVC (%) 35 ± 0.87* 35 ± 0.64* 35 ± 0.88* 36 ± 0.42* 21 ± 0.55* 21.5 ± 0.38* ESR mm/h 5 ± 0.22* 6.4 ± 0.51* 6 ± 0.51* 7.2 ± 0.39* 5 ± 0.73* 6.1 ± 0.31* Hbs mg/ 12.0 ± 0.75* 12.6 ± 0.54* 11.0 ± 0.19* 11.8 ± 0.52* 6.2 ± 0.44* 6.7 ± 0.49* dl RBC mm3 3.0 ± 0.57* 3.1 ± 0.89* 3.9 ± 0.26* 4.0 ± 0.90* 4.5 ± 017* 4.4 ± 0.80* Plasma Ca 6.95 ± 0.69* 3.8 ± 0.33* 6.95 ± 0.23* 5.0 ± 0.14* 8.2 ± 0.81* 7.4 ± 0.42* mg/ dl Conductiv 5440 ± 0.32* 5565 ± 0.59* 6138 ±0.44 * 6604 ± 0.27* 7424 ± 0.32* 7504 ± 0.13* ity µs/ M3 TDS mg/L 27.20± 0.55* 27.82 ± 0.44* 3069 ± 0.36* 32.51 ± 0-.35* 3712 ± 0.58* 3927 ± 0.39* Viscosity/ 10 ± 0.84* 2.55 ± 0.63* 2.52 ± 0.72* 2.67 ± 0.40* 3.05 ± 0.70* 2.61 ± 0.51* poise Values are Mean ± SEM, *p < 0.05, n =3

Table 28: The effects of aqueous fraction (2.5 mg/ml) of P. nitida on some biochemical parameters A A A S S S

Pre Post Pre Post Pre Post PCV (%) 40 ± 0.55* 39 ± 0.64* 32 ± 0.38* 33 ± 0.43* 19 ± 0.34* 19 ± 0.49* ESR mm/h 5 ± 0.42* 5.1 ± 0.38* 6 ± 0.74* 6.4 ± 0.49* 5 ± 0.11* 6.2 ± 0.39* Hbs mg/ dl 12.0 ± 0.97* 12.6± 0.73* 11.0 ± 9.3* 11.8 ± 0.41* 6.2 ± 0.37* 6.8 ± 0.14* RBC mm3 5 ± 0.88* 5.0 ± 0.66* 4.9 ± 0.41* 4.7 ± 0.97* 4.7 ± 0.27* 4.7 ± 0.28* Plasma Ca 6.95 ± 0.52* 3.2 ± 0.54* 6.95± 0.52* 5.2 ± 0.75* 8.2 ± 0.49* 8.2 ± 0.42* mg/dl Conductivi 5440± 0.77* 6128 ± 0.44* 6138± 0.87* 6149 ± 0.17* 7424± .40* 5820 ± 49* ty µs/M3 TDS mg/ 27.20± 0.51* 30.64 ±0.49* 30.69± 0.29* 32.00 ±0.39* 37.12±0.22* 37.50± 0.32* L Viscosity/ 2.55 ± 0.19* 2.53 ± 0.87* 2.67 0.43* 2.65 *± 0.63 3.05 ± 0.42* 2.75 ± 0.73* Poise Values are Mean ± SEM, *p < 0.05, n =3

Table 29: Effect of aqueous fraction (5 mg/ml) of P. nitida on the biochemical parameters Paramete AA AS SS

Pre Post Pre Post Pre Post PCV 35 ± 0.87* 36 ± 0.20* 30 ± 09* 30.9 ± 0.21* 20 ± 1.82* 21 ± 0.86* ESR mm/h 5 ± 0.42* 5.5 ± 0.50* 6 ± 0.61* 7 ± 0.65* 5 ± 0.58* 7 ± 0.37* Hbs mg/dl 12.0 ± 0.53* 12.8 ± 0.55* 11.0 ± 0.57* 11.9 ± 0.33* 6.2± 0.19* 6.8 ± 0.72* RBC mm3 5.5 ± 0.17* 5.0 ± 0.26* 4.8 ± 0.13* 6.0 ± 0.39* 4.6 ± 0.25* 4.5 ± 0.63* Plasma Ca 6.95 ± 0.46* 5.0 ± 0.19* 6.95 ± 0.43* 6.8 ± 0.34* 8.2 ± 0.14* 7.4 ± 0.64* mg /dl Conductivi 5440 ± 0.41* 6882 ± 0.18* 6138 ± 0.18* 6327 ± 0.87* 7424± 0.40* 7444± 0.51* ty µs/ M3 TDS mg/m 27.20 ± 0.54* 34.41 ± 0.42* 30.69± 0.29* 32.31± 0.69* 37.12 ± 0.39* 39.72±2.14* L Viscosity/ 2.55 ± 0.66* 2.53 ± 0.31* 2.67± 0.27* 2.63 ± 0.44* 3.05± 0.24* 2.60 ± 0.52* poise Values are Mean ± SEM, *p < 0.05, n =3

100

Table 30:Effect of aqueous fraction (10 mg/ml) of P. nitida on some biochemical parameters Parameter A A A S S S

Pre Post Pre Post Pre SS2 PCV (%) 34 ± 0.64* 34 ± 0.58* 36 ± 0.55* 36.9 ± 0.33* 21 ± 0.38* 21.6 ± 0.31* ESR mm/h 5 ± 0.66* 6 ± 0.51* 6 ± 0.23* 7.4 ± 0.35* 5 ± 0.33* 7 ±0.33* Hbs mg/dl 12.0 ± 0.69* 12.9± 0.32* 11.0 ±0.34* 11.9 ± 0.69* 6.2 ± 0.42* 6.9 ± 0.28* RBC mm3 3.4 ± 0.30* 3.6 ± 0.29* 4.8 ± 0.57* 5.0 ± 0.62* 5.0 ± 0.51* 5.0 ± 0.39* Plasma Ca 6.95 ± 0.52* 5.0 ± 0.36* 6.95 ±0.49* 6.4 ± 0.60* 8.2 ± 0.49* 5.7 ± 0.24* mg/dl

Conductivity 5440 ± 0.18* 7464±0.31* 6138±0.39* 5178 ± 0.61* 7424± 0.56* 6040 ± 0.47* µS/ M3 TDS mg/L 27.20 ±0.45* 37.32±0.11* 30.69±0.3* 34.89± 0.41* 37.12± 0.19* 39.80 ± 0.84* Viscosity / 2.55 ± 0.33* 2.50 ± 0.45* 2.67 ±0.90* 2,61 ± 0.52* 3.05 ± 0.17* 2.60 ± 0.76* poise Values are Mean ± SEM, *p < 0.05, n =3

Table 31: Effect of Dichloromethane fraction (2.5 mg/ml) of P. nitida on some biochemical parameters Paramete AA AS SS rs Pre Post Pre Post Pre Post PCV (%) 40 ± 0.80* 41 ± 0.45* 28 ± 0.61* 32 ± 0-.14* 19 ± 0.44* 19.8 ± 0.56* ESRmm/h 5 ± 0.69* 5.2 ± 0.73* 6 ± 0.97* 7 ± 0.51* 5 ± 0.74* 5.8 ± 0.14* Hbs mg/dl 12.0± 0.55* 12.3 ± 0.62* 11.0 ± 0.38* 11.6 ± 0.53* 6.2 ± 0.62* 6.7 ± 0.71* RBC mm3 4.6 ± 0.61* 5.3 ± 0.41* 5.0 ± 0.45* 4.9 ± 0.77* 5.0 ± 0.19* 5.0 ± 0-.18* Plasma Ca 6.95± 0.58* 4.0 ± 0.69* 6.95 ± 0.55* 4.4± 0.18* 8.2 ± 0.41* 7.9 ± 0.61* mg/dl Conductiv 544 ± 0.47* 6333 ± 0.15* 6138 ± 0.69* 6528 ± 0.43* 7424 ± 0.18* 7026 ± 0.66* ity µs/m3 TDS mg/L 27.2±0.27* 37.32 ± 0.85* 30.69 ± 0.34* 34.89 ±0.64* 37.12 ± 0.44* 39.80 ± 0.30* Viscosity/ 2.55± 0.22* 2.53 ± 0.49* 2.67 ± 0.19* 2.63 ± 1.58* 3.05 ± 0.17* 2.82 ± 0.76* poise Values are Mean ± SEM, *p < 0.05, n =3

101

Table 32: Effect of Dichloromethane fraction (5 mg/ml) of P. nitida on the biochemical parameters parameters AA AS SS

Pre Post Pre Post Pre Post PVC (%) 35 ± 0.55* 34 ± 0.40* 29 ± 0.48* 31 ± 0.19* 19 ± 0.72* 20 ± 0.15* ESR mm/h 5 ± 0.41* 6.4 ± 1.04* 6 ± 0.41* 6.9 ± 0.92* 5 ± 0.33* 6 ± 0.29* Hbs mg/dl 12.0 ± 0.1*7 12.7 ± 0.53* 11.0 ± 0.42* 11.8 ± 0.17* 6.2 ± 0.26* 6.7 ± 0.18* RBC mm3 5.5 ± 0.72* 5.7 ± 0.47* 4.9 ± 0.69* 5.0 ± 0.28* 5.2 ± 0.38* 5.2 ± 0.63* Plasma Ca. 6.95 ± 0.44* 6.4 ± 0.93* 6.95 ± 0.55* 6.7 ± 0.15* 8.2 ± 0.24* 7.8 ± 0.85* mg/dl Conductivit 5440 ± 0.19* 5895± 0.37* 6138 ± 0.52* 6501± 0.88* 7424 ± 0.69* 7703 ± 0.66* y µs/m3 TDS mg/L 2720 ± 0.39* 2947. ± 0.59* 3069 ± 0.18* 32.40 ± 0.39* 39.12 ± 0.22* 39.33 ± 0.39* Viscosity/p 2.55 ± 0.65* 2.53 ± 0.46* 2.67 ± 2.09* 2.63 ± 0.71* 3.05 ± 0.34* 2.82 ± 0.45* oise Values are Mean ± SEM, *p < 0.05, n =3

Table 33: Effect of Dichloromethane fraction (10 mg/ml) of P. nitida on some biochemical parameters Parameter AA AS SS

Pre Post Pre Post Pre Post PCV (%) 35 ± 0.52* 37 ± 0.53* 35 ± 0.27* 35 ± 0.44* 21 ± 0.77* 21 ± 0.41* ESR mm/h 5 ± 0.71* 6 ± 0.61* 6± 0.67* 7.2 ± 0.51* 5 ± 0.61* 7 ± 0.94*

Hbs mg/dl 12.0 ± 0.49* 12.7± 0.77* 11.0 ± 0.76* 11.9± 0.63* 6.2 ± 0.67* 6.8 ± 0.17* RBC mm3 4.1 ± 0.43* 3.3 ± 0.59* 4.6 ± 0.64* 4.9 ± 0.72* 4.7 ± 0.44* 4.6 ± 0.29* Plasma Ca 6.95 ± 0.46* 6.5 ± 0.56* 6.95 ± 0.47* 4.2 ± 0.93* 8.2 ± 0.89* 7.2 ± 0.37* mg/dl Conductivit 5440 ± 0.68* 5187 ± 0.64* 6138 ± 0.41* 6528±0.61* 7424 ±0.50* 7619 ± 0.14* y µs/m3 TDS mg/L 27.20 ± 0.53* 29.11 ± 0.24* 30.69 ± 0.72* 32.43±0.8* 39.1±0.60* 39.29 ± 0.19* Viscosity 2.55 ± 0.56* 2.51 ± 0.50* 2.67 ± 0.51* 2.60 ±0.69* 3.05 ± 0.38* 2.71 ± 1.47* (poise) Values are Mean ± SEM, *p < 0.05, n =3

102

Table 34: Antisickling effect of isolated pure compounds from P. nitida on HbSS red blood cells. Incu N/ Parahydroxybenzoic acid Pure compounds % reversal of sickling, concentrations (mg/ml) bati sal Concentrations (mg/ml) time ine % reversal of sickling Compound 1 Compound 2 Compound 3 5 50 500 2.5 5 10 2.5 5 10 2.5 5 10 0 43 38 ± 40± 0.44* 45 ± 0.73* 73± 0.29* 84±0.2* - 78 ± 79± 0.83* -- 73 ± 86 ± 0.23* -- 0.62* 0.17* 0.26* 30 43 41 ± 58± 0.57* 100 ± 0.61* 95± 0*.55 - - 78 ± 0.85 79± 0.51* -- 86± 0.14* 90 ± 0.38* -- 0.41* 60 43 44 ± 60± 0.63* 100 ± 0.70* 97± 0.39* - - 91± 0.77* 92± 0.68* -- 92± 0.37* 95 ± 0.74* -- 0.88* 90 43 50± 0.47* 79± 0.19* 100 ± 0.03* 97 ± 0.13 - -- 90 ± 0.45 92± 0.59* -- 92± 0.50* 97 ± 0.18* -- 120 43 56 ± 88± 0.68* 100 ± 0.28* 100±0.36 - - 92± 0.31* 94± 0.48* -- 95± 0.98* 97 ± 0.32* -- 0.16* * 150 43 56 ± 88± 0.48* 100 ± 0.60* 100±0.84 - - 93 ± 0.49 97± 0.11* -- 97 ± 97 ± 0.67* -- 0.53* * 0.31* 180 43 58 ± 98± 0.29* 100 ± 0.15* -- - - 100±0.27 100±0.66 -- 100 ± 100 ± 0.49 -- 0.71* * * 9.44*

Values are Mean ± SEM, *p < 0.05, n =3

103

4.7: Chromatographic analysis Table 35: TLC of Methanol extracts and Fractions. Solvent system: methanol and chloroform is (65:35) Adsobent: silica gel 40 mesh.

Fraction/standard Spot distance Rf values p-hydroxybenzoic acid 10.2 0.80 Β-carboxylic acid 9.6 0.71 Chloroform fraction 4.3 0.32 5.4 0.40 5.6 0.41 6.3 0.47 8.5 0.63 9.4 0.71 Dichloromethane 4.3 0.32 5.41 0.40 5.7 0.42 8.45 0.63 9.6 0.72 Aqueous fraction 4.12 O.31 5.4 0.41 7.5 0.60 8.5 0.63 9.61 0.71 10.2 0.80 Ethyl Acetate fraction 4.1 0.31 5.7 0.41 7.5 0.60 8.6 0.63 9,56 0.71 10.2 0.80 Methanol extract 2.4 0.20 4.9 0.36 5.4 0.40 7.5 0.63 8.62 0.64 9.5 0.70

104

Table 36: TLC of aqueous sub-fractions 34, 23 and 21 of P. nitida, PHBA Solvent MeOH 65: CHCl3 35, adsorbent: silica gel 40 mesh, Sub-fractions/ PHBA Rf values Sub-fraction 34 0.81 Sub-fraction 23 0.79 Sub-fraction 21 0.74 p-hydroxybenzoic acid 0.70

Table 37: TLC of aqueous sub-fraction 34, 23, 21 of P. nitida,and PHBA Solvent MeOH 65 : CHCl3 35, adsorbent: silica gel 40 mesh, Solvent MeOH 65 : CHCl3 35 Rf values SF 34 (Cp 3) 0.96 SF 23 (Cp2) 0.88 SF 21 ( Cp1) 0.86 p-hydroxybenzoic acid 0.83

Table 38. TLC of Isolates compound 1 compound 2; and pure sample (Ajmaciline) Solvents MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm Pure sample, Isolates Rf values Pure sample (Ajmalicine) 0.86 Isolate (compound 1) 0.86 Isolate (compound 2) 0.91

4.8: Analytical standards Table 39: Analytical standards of Picralima nitida Parameter Concentration (%)

Moisture content 3.0

Total ash value 11.5

Acid insoluble ash 3.5

Water soluble ash 4.0

Sulphated ash 12.5

Alcohol soluble extractive 20.4 Water Soluble extractive 8.0

105

4.9: Characterization of isolated compounds

NH OH N H O O

OMe

Fig 4: Structure of Compound 1-Ajmaciline

Fig 5: Structure of Compound 2-Ajmaciline oxindole B

106

Inten. (x1,000,000) 8.0

144.0797 7.0

6.0

5.0

4.0 222.1103

3.0 210.1109 2.0 354.1894 178.0852 1.0 321.1580 162.0912 190.0832 251.1537 127.0525 144.1815 234.1238 277.1504 293.1686 309.1684 336.1595 0.0 354.1345 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 m/z

Fig 6: Electron Ionization (EI) showing fragmentation MS-MS data for the most abundant ions, 50% collision energy, and 50% collision gas, and 10msec ion accumulation time. The spectrum contains the major ions masses (144, 178, 210 and 222) characteristic of ajmaciline amongst other peaks.

Inten. (x10,000,000) 8.0 353.1867

7.0

6.0

5.0

4.0

3.0

2.0

1.0

280.0948 0.0 550.6981 676.3729 792.7972 887.4573 1163.4948 1347.0396 1465.5286 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z

Fig. 7: The The Mass Spectrum base peak chromatogram of compound 1 (Ajmaciline,empirical formula C21H24N2O3,Mwt 353.1867) in positive mode, peak at 4.07min. The expected mass was 353.1860; found 353.1867; error 0.7mDa, 1.98ppm.

107

Inten. (x100,000)

7.0 352.1759

6.0 223.1197 5.0

4.0

3.0

2.0 351.1694 184.0979 337.1566 1.0 132.0807 151.0776 210.1039 251.1617 265.0942 292.1396 323.1920 0.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 m/z

Fig. 8: The Electron Ionization (EI) showing fragmentation MS-MS data for the most abundant ions, 50% collision energy, and 50% collision gas, and 10msec ion accumulation time. The spectrum contains the major ions masses (184 223, 337 and 352) characteristic of ajmaciline oxindole B amongst other peaks.

Inten. (x10,000,000) 1.50 369.1815

1.25

1.00

0.75

0.50

0.25

351.1731 369.3461 468.2926 716.4541 0.00 234.9920 831.4080 966.3354 1125.7402 1499.2061 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z

Fig. 9: The The Mass Spectrum base peak chromatogram of compound 2 (Ajmaciline oxindole B, Empirical formula C21H24N2O4,Mwt. 353.1867) in positive mode, peak at 4.35min. The expected mass 369.1809; found 369.1815, error 0.6mDa, 1.63ppm.

108

mAU 4.00/ 1.00/bgnd 125 219

100

0 412 416 463 467

200 300 400 500 nm

Fig. 10: The UV chromatogram of compound 1 (Ajmaciline) at 257nm in 50% MeOH

(x100,000,000) 1:BPC (1.00) 1:369.1812 (10.00) 1:275.1538 (10.00) 1.25

1.00

0.75

0.50

0.25

0.00

3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9

Fig. 11: The UV chromatograms showing Compound 1- Ajmaciline in blue and Compound 2 (Ajmaciline oxindole B (brown) at 257nm in 50% MeOH

109

Fig. 12: The 13C-NMR Spectrum of Compound 1-Ajmaciline.

Fig. 13: The 13C-NMR Spectrum of Compound 2-Ajmaciline oxindole B ((19 )-19-methyl- 2-oxoformosanan-16-carboxylic acid methyl ester)

110

Fig. 14: The 1H-NMR Spectrum of Compound 2-Ajmaciline oxindole B((19 )-19-methyl-2- oxoformosanan-16-carboxylic acid methyl ester)

111

Physical and Spectroscopic Data of Compounds 1 and 2

1 Table 40: H -NMR (δH in ppm, 500MHz) Data of Compounds 1(Ajmaciline) and 2 (Ajmaciline oxindole B) Compound 1 Compound 2

Position δH δH 1 1.60 (1H, m) 0.82 (1H, s, J.1.8 ) 2 - - 3 3.20 (1H,dd, J.2.1) 3.49(1H, td, J.5.5, 11.1) 4 - - 5 0.95(2H, s) 2.27(2H, m) 6 1.38 (2H, d, J. 7.3) 5.33(2H,d, J, 5.1) 7 - - 8 - - 9 1.95 (1H, s) 0.80 (1H, d) 10 1.91(1H, d, J.6.5) 1.64(1H,d, J.11.5) 11 1.87(1H, dd, J, 1.57 1.25(1H, dd, J.7.3, 14.7) 12 5.16 (1H,t, J.3.6) 1.16(1H,m) 13 - - 14 0.97 (2H, s) 1.97( 2H, s) 15 1.50(1H, s, J.1.88 2.21(1H, s) 16 - - 17 1.11(1H, s J.2.9) 0.90 (1H, s, J.6.5) 18 0.81 (4H, d, J.3.4) 0.67 (4H, d, J.9.1 19 0.77 (3H,d, J.12.5) 0.99 (3H,d, J.6.5) 20 - - 21 0.85 (3H,s, J. 7.2) 1.83(3H, d, J.3.8, 9.8)

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13 Table 41: C -NMR (δC in ppm, 125MHz) Data of Compounds 1(Ajmaciline) and 2 (Ajmaciline oxindole B) Compound 1 Compound 2

Position δC δC

1 38.90 32.15

2 28.31 (C) 29.43(C)

3 81.18(CH) 72.05(CH)

4 39.95 40.04

5 55.48(CH2) 140.97(CH2)

6 18.43(CH2) 122.15(CH2)

7 32.72(C) 32.15(C)

8 40.27(CH) 31.90(CH)

9 47.89(CH) 50.37(CH)

10 37.34(CH) 34.19(CH)

11 23.87(CH) 21.32(CH)

12 124.56(CH) 37.49(CH)

13 139.90(CH) 42.53 (CH)

14 42.31(CH2) 57.01(CH2)

15 28.31(CH) 24.53(CH0

16 23.62(C) 28.46(C)

17 33.08(CH) 56.40(CH)

18 59.30(CH3) 12.10(CH3)

19 39.97(CH) 20.03(CH)

20 31.42(CH2) 36.78(CH2)

21 37.03(CH3) 19.62(CH3)

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Sickled cells

Normal cells

Incubation of HbSS blood in MeOH extract 2.5 Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida at 0 min (X 400) mg/ml of P.nitida at 30 min (X 400)

Incubation of HbSS blood in MeOH extract 2.5 Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida at 90 min (X 400) mg/ml of P.nitida at 60 min (X 400)

Incubation of HbSS blood in MeOH extract 2.5 Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida at 120 min (X 400) mg/ml of P.nitida at 150 min (X 400)

Reverted cells

Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida at 180 min (X 400) Plate 7: Incubation of HbSS blood in MeOH extract 2.5 mg/ml of P.nitida

114

Sickled cells

Normal cells

Incubation of HbSS blood in MeOH extract 5 mg/ml at 0 min (X 400) Incubation of HbSS blood in MeOH extract 5 mg/ml of P.nitida at 30 min (X 400)

Normal cells

Incubation of HbSS blood in MeOH extract 5 mg/ml Incubation of HbSS blood in MeOH extract 5 mg/ml of P.nitida at 60 min (X 400) of P.nitida at 90 min (X 400)

Normal cells

Incubation of HbSS blood in MeOH extract 5 mg/ml of Incubation of HbSS blood in MeOH extract 5 mg/ml P.nitida at 150 min (X 400) of P.nitida at 120 min (X 400)

Reverted cells

Incubation of HbSS blood in MeOH extract 5 mg/ml of P.nitida at 180 min (X 400) Plate 8: Incubation of HbSS blood in MeOH extract 5 mg/ml of P.nitida

115

Sickled cells

Incubation of HbSS blood in MeOH extract Incubation of HbSS blood in MeOH extract 10 mg/ml of P.nitida at 30 min (X 400) 10 mg/ml of P.nitida at 0 min (X 400)

Normal cells

Incubation of HbSS blood in MeOH Incubation of HbSS blood in MeOH extract extract 10 mg/ml of P.nitida at 60 min 10 mg/ml of P.nitida at 90 min (X 400)

Normal cells

Incubation of HbSS blood in MeOH Incubation of HbSS blood in MeOH extract 10 mg/ml of P.nitida at 150 min extract 10 mg/ml of P.nitida at 120 (X 400) min (X 400)

Reverted cells

Incubation of HbSS blood in MeOH extract 10 mg/ml of P.nitida at 180 min (X

Plate 9: Incubation of HbSS blood in MeOH extract 10 mg/ml of P.nitida

116

Normal cell

Plate 10. Picture of normal HbAA blood cells

Plate 11: TLC of MeOH extract and fractions of P.nitida seed, p-hydroxybenzoic acid and beta-carboxylic acid Solvents MeOH : CHCl3 (65: 35), adsorbent: silica gel, UV at 254 nm, reagent: ammonia vapour

117

Solvent Front PHBA

β carboxylic acid

CHCl3 fraction

CH2Cl2 Fraction

Aqueous fraction EtOAc fraction MeOH Extract Start line

Plate 12: TLC of MeOH extract and fractions of P.nitida seed, p-hydroxybenzoic acid and beta-carboxylic acid. Solvents MeOH and CHCl3 (65 : 35), adsorbent: silica gel, UV at 254 nm, reagent Dragendorff’s

Plate 13: TLC of MeOH extract and fractions of P.nitida seed, p-hydroxybenzoic acid. β- carboxylic acid,Solvents MeOH and CHCl3( 65 : 35), adsorbent: silica gel, UV at 254 nm,Reagent: iodine vapour

118

Plat 14: TLC of MeOH extract and fractions of P.nitida seed. p-hydroxybenzoic acid β- carboxylic acid, Solvents MeOH and CHCl3 (65 : 35), adsorbent: silica gel, UV at 254 nm

front line

SF 34

SF 23 SF 21

PHBA

Start line

Plate 15.: TLC of aqueous sub-fractions (34, 23, and 21) of P.nitida seed and PHBA . Solvents MeOH and CHCl3 ( 65 : 35), adsorbent: silica gel 40 mesh, UV at 254 n

119

Solvent front SF 34 SF 23 SF 21

PHBA

Start line

Plate 16: TLC of aqueous sub-fractions (34, 23, and 21) of P.nitida seed and PHBA . Solvents MeOH and CHCl3 ( 65 : 35), adsorbent: silica gel 40 mesh, UV at 254 nm

Front line

Compound 2

Compound 1

Pure sample (Ajmalicine

Start line

Plate 17: TLC of Isolates compound 1 compound 2; and pure sample (Ajmaciline) Solvents

MeOH and CHCl3 (50 : 10), adsorbent: silica gel, UV at 254 nm

120

2nd solvent front

1st solvent front

Isolate compound 1

Start line

2nd solvent front

Plate 18: Two dimension TLC of the isolate compound 1: Solvents MeOH and CHCl3 (50 :

10), adsorbent: silica gel, UV at 254 nm

121

2nd solvent front

1st solvent front

Isolate compound 2

Start line

2nd solvent front

Plate 19 Two dimension TLC of the isolate compound 2: Solvents MeOH and CHCl3 (50 :

10), adsorbent: silica gel, UV at 254 nm

122

CHAPTER FIVE

DISCUSSION

The bio-active ingredients that have therapeutic activities in plants have holistic nature of treatment. Substances found in the medicinal plant containing the healing property of the plant are known as the active principles. It defers from plant to plant (Adebanjo et al.,

1983).Phytochemical screening revealed that the extract was rich in secondary metabolites

(Table 4), These active principles: alkaloids, flavonoids, glycosides, saponins, tannins etc and other compounds such as morphine, atropine, codeine, steroids, lactones and volatile oils possess medicinal value for the treatment of different diseases (Chevalier, 2000). The main alkaloid in P. nitida is akuammine has local anaesthei action. Its action can be compared to the anaesthetic action of cocaine (Ansa-Asamoah 1990). The anti-sickling activities of the extracts of the roots of a plant Cissus populnea L. (CPK) (a major constituent of a herbal formula Ajawaron HF used in the management of sickle cell disease in south-west Nigeria) has been examined. Phytochemical examination of the extract showed the presence of anthraquinone derivatives, steroidal glycosides and cardiac glycosides Moody et al, (2003), the glycosides in combination with other active principles were responsible for antisickling activity of the plant.

Phytochemical and antisickling activities of Entandrophragma utile, Chenopodium ambrosioides and Petiveria alliacea as investigated by Adejumo et al, (2011) revealed the presence of alkaloids, tannins and saponins while free and combined anthraquinones were absent. This result is similar to that reported by Egunyomi et al. (2009) for Plumbago zeylanica and Uvaria chamae recipes used to manage sickle cell disease (SCD) in south-west

Nigeria. In a related development, Ibrahim et al. (2007) reported that saponins, in addition to carboxylic acids and flavonoids may be responsible for the antisickling activity of H. acida leaves. Additionally, alkaloids are nerve stimulants, convulsants and muscle relaxants

123

(Kenner and Yves, 1996) hence; the presence of alkaloids in the investigated plant parts is an indication that they may be useful in alleviating some of the symptoms associated with pains.

Phytochemical screening of P. nitida, showed the presence of those secondary metabolites.

The results of antisickling assay of the extract/fractions of P.nitida in the present study showed that they exhibited substantial antisickling activity. This may give a rational explanation for the use of the plant in managing sickle cell disease (SCD) by traditional healers.

The effect of incubation of HbAA, HbAS and HbSS blood with different concentrations (2.5,

5 and 10 mg/ml) of crude methanol extract, fractions and isolates of P. nitida on the blood genotypes showed positive indication of antisickling activities . The percentages of reversal of sickling blood cells obtained from 0.5 ml HbSS red blood when mixed with 0.5 ml of the

2.5, 5.0, and 10.0 mg of methanol extract, incubated at 30 - 180 min, were compared with percentages of reversal of sickling blood cells obtained from 0.5 ml HbSS red blood, mixed with 0.5 ml of different concentrations 5; 50 and 500 mg/ml of p-hydroxybenzoic acid

(positive control), incubated at 30 minutes intervals up to 180 min (Table 10).

The results in Table 10 showed that p-hydroxybenzoic acid exhibited 100 % reversal of sickling red blood cells at 500 mg/ml in 120 min while the MeOH extract of P. nitida showed

67 % reversal of sickling red blood cells at 10 mg/ml, in 180 min.

The results obtained were compared with that of the positive control p-hydroxybenzoic acid at 120 minutes at concentration 5 mg/ml (p-hydroxybenzoic acid) and 2.5 mg/ml (MeOH extract) 56 and 53% of reversal of sickling respectively. At 180 minutes p-hydroxybenzoic acid at (50 mg/ml) and 5 mg/ml (MeOH extract) exhibited 95 and 67 % reversal of sickling respectively. The reversal of sickling effect of the aqueous fraction on HbSS red blood cells was greater than those of the other fractions. At 180 minutes incubation, and at 10 mg/ml

124 concentration the percentage reversal were 85, 82, 66 and 64 % respectively for aqueous,

Dichloromethane, ethyl acetate and chloroform fractions

The aqueous fraction showed higher antisickling effect compared to PHBA at 5 mg/ml concentrations throughout the period of 180 min incubation (Table 13). The antisickling effects of the three concentration levels of aqueous, ethyl acetate and dichloromethane fractions of P. nitida on HbSS red blood cells indicate that the antisickling effects of aqueous fraction was higher than that obtained with the ethyl acetate or dichloromethane fractions at equal concentrations (Table12-14). CHCl3 fraction showed dose-dependent reversal of sickling (antisickling activity) at all tested concentrations (2.5, 5, 10 mg/ml) at 0 – 60 min incubation period. However, all other fractions (CH2Cl2, EtOAc and Aqueous) gave dose- dependent results also at tested concentrations through out entire incubation period (0 -180 min). The antisickling activities of the fractions on HbSS blood were given to be in order

CHCl3 ≤ CH2CL2 < EtOAc < Aqueous at the tested concentrations.

The antisickling effect of MeOH extract, aqueous, CHCl3, EtOAc and CH2CH2 fractions of

P. nitida on HbAS red blood cells are presented in Tables 15 – 19. The antisickling effect of the aqueous fraction was better than the effects produced by MeOH extract, CHCl3, EtOAc and CH2Cl2 fractions at equivalent concentrations. The aqueous fraction caused a 100 % reversal of sickling of the HbAS blood cells within 60 minutes at concentration of 10 mg, reversal by CHCl3, EtOAc, and CH2Cl2 fractions occurred at 120, 180 and 150 minutes respectively. At 5 mg/ml concentration, the aqueous fraction of P.nitida and PHBA were comparable, the antisickling activity during incubation of the HbAS blood (30 – 180 min) was slightly higher than that of the positive control (PHBA) It is evident from Tables 10 -

19, that at concentrations of 2.5, 5.0 and 10.0 mg/ml methanol extract, aqueous, chloroform ethyl acetate and dichloromethane fractions of P. nitida exhibited progressive and in most cases dose dependent antisickling effects on HbAs blood cells. The antisickling activities of

125 the fractions on HbAS red blood cells were also given in the order CHCl3 ≤ CH2CL2 <

EtOAc < Aqueous at the tested concentrations

In comparison, higher concentrations of CHCl3, Aqueous fractions gave greater inhibition or reversal of sickling of HbAS blood while the HbSS blood cells were more susceptible to lower concentrations of tested agents.

The Isolated compounds also have very significant (p < 0.05) degrees of anti-sickling activity

(Table 34).

Compound 1 was obtained as white powder. Its molecular formula was derived as

C21H24N2O3 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z

= 353.1867. The expected mass was 353.1860, error 0.7mDa, 1.98ppm. The M.p. 253-254 oC., UV/ (MeOH) ƛmax : 257 nm (4.07), 1H-NMR (Measured in CDCL3) (Table 40 ) and

13 C-NMR (Measured in CDCL3) (Table 41). Based on the spectral analysis (UV, chemical shifts, coupling constant of the 1H-NMR and 13C-NMR spectrum experiments) the complete structural elucidation of compound 1 was derived as (19 )-16,17-didehydro-19-methyl- oxayohimbin-16-Carboxylic acid methyl ester (Ajamalicine). It showed the best anti-sickling activity (100 % reversal of sickling at 120 min) and comparable to that of the standard drug parahydroxybenzoic acid at 500 mg/ml (100 % reversal of sickling at 30 min).

Compound 2 was obtained as light yellow powder. Its molecular formula was derived as

C21H24N2O4 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z

= 369.1815.The expected mass 369.1809, error 0.6mDa, 1.63ppm, mass about 16 heavier than ajmalicine. The M.p. 265-266 oC., UV/ (MeOH) ƛmax: 257 nm (4.35), 1H-NMR

13 (Measured in CDCL3) (Table 39) and C-NMR (Measured in CDCL3) (Table 40). The fragmentation does show a loss of 32.0249Da (to 337.1566); this is appropriate for MeOH

(expected neutral mass 32.0262Da), which could account for the esterified methyl group on

126

-COOMe). The structure of Compound 2 was finally concluded as (19 )-19-methyl-2- oxoformosanan-16-carboxylic acid methyl ester (Ajmalicine Oxindole B).

Compound 3 is a cream coloured amorphous powder yet unidentified but has anti-sickling activity more than compound 2. The anti –sickling activity of the compounds is in the order

CP1 > CP3 > CP2 at concentration 2.5 mg/ml at 120 min (Table 34).

The extract and fractions also exhibited significant changes on some of the biochemical parameter (PCV, ESR, HB, RBC Count, Plasma Calcium, Conductivity, TDS , Viscosity).

The packed cell volume (PCV) in sickle-cell blood (HbSS) is lower than that of the normal

HbAA blood as observed from this work. This may be because normal blood cell is disc-like and has larger volume than the sickled cell whose internal content has been depleted leaving banana shape sickled cell. The PCV of the HbAS blood in pre-drug treatment was very slightly lower than that of the normal HbAA blood cells. In post-drug treatment with the aqueous fraction of P .nitida, it was observed that PCV of HbAS and HbSS blood cells were very slightly higher than the pre-drug value (Tables 25 - 33). The slight changes in the post- drug value of PCV may be due to the new volumes of the oxygenated reverting cells.

The erythrocyte sedimentation rate (ESR) showed that the post-drug treatment value was higher than the pre-drug treatment value in all the groups (HbAA, HbAS and HbSS). This may be due to the effect of the drug on the viscosity of the blood. When the viscosity of blood is less, the sedimentation rate of the blood increases, the time taken by the sedimentation becomes less. In Table 26 the values of ESR are; 5 and 6 (HbAA) , 6 – 7

(HbAS) and 5 – 5.8 (HbSS). It was observed that ESR was increased in each case in the post-drug treatment (Tables 25 – 33).

The Hemoglobin (Hb) is less in HbAS blood and least in HbSS blood (Tables 25 – 33). Data obtained from post-drug treatment showed some increase in Hb for all concentrations

127 methanol extract, CH2Cl2 and aqueous fractions, of P. nitida on the blood genotypes. This may be due to the ability of the drug to penetrate the cell membrane of the blood cells and making it possible for oxygen absorption into the blood cells.

There was no increase in the red blood count (RBC) as a result of the application of crude methanol extract, aqueous, and dichloromethane fractions of P. nitida. After treatment with the methanol extract, aqueous fraction of P. nitida, the plasma calcium were less in HbAS and HbSS blood genotypes (Tables 25 – 33). The reduction of plasma calcium is a factor that indicates reduction of sickling effect on HbSS blood.

The Conductivity of blood types showed remarkable increases after treatment with the crude methanol extract, aqueous, and dichloromethane fractions of P. nitida on the HbAA,HbAS and HbSS blood as shown in Tables 25 - 33. Erythrocytes have low conductivity, their surfaces are resistant to electric current. The application of the crude methanol extract and fractions at different concentrations showed increase in the conductivity as the blood plasma were enriched with ion content of the drug. The numerical value of conductivity is assumed to be varying with several factors. These are concentration of erythrocytes, hematocrit volume and moreover the liquid conducts electricity due to the presence of ions (salt and proteins) (Kubasova and Tamara, 1984) An understanding of connectivity of blood is essential parameter for determination of interfacial potentials during the flow of blood. A thorough study of electrical conductivity of human blood has been done with respect to the effect of laser radiation. Literature show that the erythrocytes almost behave like a perfect nonconductor of direct current (Vladimirov et al 2004). Later studies showed that only the surface layer of the erythrocytes is nonconductive which offers the resistance to flow of blood while the inner structure of the erythrocyte had a conducting medium which is almost half of the conductivity of the plasma (Rubinov, 2003; Niemz and Markolf, 2007).

128

The application of the crude methanol extract and fractions at different concentrations on the blood genotypes showed an increase in the total dissolved solids (TDS) (Tables 25 – 33). The conversion of some salts in the extracts to soluble bases and acids aided conductivity.

A common use for conductivity sensors is to measure the concentration of total dissolved solids (TDS) in water samples. TDS is approximated with conductivity using a multiple factor and is expressed in parts per million (ppm)

The viscosity of sickle cell blood is higher than the viscosity of normal blood. This high viscosity of sickle cell blood quickly helps in the vaso-occlusive action of the sickle cell blood in minor circulatory blood vessels. Another factor that promotes irregularity of blood flow is morphology of the red blood cell. A sickled red blood cell exhibits high viscosity along the arterial wall, and since the blood flow is inversely proportional to its viscosity, then it follows that the more viscous a blood is, the lesser it’s flow. This is the fundamental basis for hypoxia experienced by sickle cell patient during crisis. The more viscous a blood is, the more it will stick to the arterial walls, thereby encouraging narrowing of the blood capillaries and promoting vascular pressure (Berne and Levy, 1992; Guyton and Hall, 1996). The extract and fractions of Picralima nitida showed reduction of viscosity of HbSS blood. The aqueous fraction showed to be the most effective. The reductions of the viscosity of sickle cell blood by P. nitida extract and fractions increased with increase in their concentrations and also with increase in period of incubation (Table 20 - 24). Blood viscosity is a measure of the resistance of blood to flow,. It can also be described as the thickness and stickiness of blood.

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Flow resistance is also directly proportional to the viscosity of blood flowing in that segment.

1988, Merrill, 1969) The most important determinants of blood viscosity are hematocrit, red blood cell deformity, red blood cell aggregation, and plasma viscosity. Plasma’s viscosity is determined by water-content and macromolecular components. Factors that affect blood viscosity are the plasma protein concentration and types of proteins in the plasma.(Kemarky et al, 2008). Hematocrit has cause up to a 4% increase in blood viscosity (Baskurt and

Meiselman, 2003). This relationship becomes more sensitive as hematocrit increases. When the hematocrit rises to 60 or 70%, which it often does in polycythemia, Tefferi (2003), the blood viscosity can increase as 10 times that of water, and its flow through blood vessels is greatly retarded because of increased resistance to flow, (Lenz et al,2008). This will lead to decreased oxygen delivery, (Kwon et al, 2008). Other factor influencing blood viscosity is temperature, (where an increase in temperature results in a decrease in viscosity). This is particularly important in hypothermia, where an increase in blood viscosity will cause problems with blood circulation.

In Methanol: Chioroform (65:35) as the solvent system, the TLC of the aqueous and ethyl acetate fractions showed the likely presence of p-hydroxybenzoic acid as evidenced in their

Rf values (Table 35, Plate 14). The TLC of MeOH extract and fractions in Methanol:

Chloroform (65:35) solvent system, gave yellow colour spot with ammonia vapour and this indicates the presence of flavonoids in the extract and fractions (Plate (11). Spraying of the chromatogram (Plate 12) with Dragendoff’s reagent, orange-brown colour spots were observed indicating presence of alkaloids. Brown colour spots in iodine vapour was observed

130 which indicates presence of alkaloids (Plate 13). TLC of aqueous sub-fractions (34, 23 and

21) and PHBA showed single spot each (Plates 15 and 16) using Solvents MeOH and CHCl3

( 65 : 35), adsorbent: silica gel 40 mesh, UV at 254 nm

The TLC of Isolates, compound 1,and 2 of P .nitida seed and pure sample (Ajmalicine) are as shown in (Plate 17, Table 37) using MeOH : CHCl3 (50:10) as the solvent system, silica gel 40 mesh as the adsorbent and UV at 254 nm. The two dimensional TLC of the pure

compounds produced single spot (Plate 18 and 19) using MeOH : CHCl3 (50: 10) solvent system and observed using UV 254 nm. This confirms the purity of the isolated compounds of seeds of P nitida.

CONCLUSION

This study has established the anti-sickling properties of P. nitida extract, fractions and isolates. The isolated compounds ajmalicine (19 )-16,17-didehydro-19-methyl- oxayohimbin-16-carboxylic acid methyl ester.(1) and ajmalicine Oxindole B (19 )-19- methyl-2-oxoformosanan-16-carboxylic acid methyl ester (2) were responsible for the antisickling effect.

This is of tremendous health significance in sickle cell disease management to the grass root.

Since P-nitida is native to many communities in the country, it will be readily available and easily accessible. The cost of sickle cell disease management using conventional synthetic chemotherapy will be reduced and the psyco-social pressure and infant mortality inherent in sickle cell disease will also be reduced. The data obtained from this work can form the basis for further investigations and discoveries, and could serve as a lead for production of antisickling drugs from P. nitida.

131

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APPENDIX 1 RESULT OF THE NUMERICAL VALUES

EXTRACTION

The powdered seed 2.5 kg was macerated respectively methanol for 24 hours with occasional shaking. The filtrates was concentrated in vacuum rotary evaporator. The alcohol extract was freeze-dried.

Extractive value

Weight of the powder = 2.5 kg Weight of the extract = 345 g Percentage of the extract = 13.8

Moisture Content

Weight of the dish + Powder + moisture = W1

Weight of the dish + Powder = W2

Weight of Moisture = W1- W2

Weight of Powder = W3

% Moisture Content = (W1-W2) x 100 / W3 Weight of Petri-dish + drug (g) 25.45 Final Weight of Petri dish + drug (g) 24.36 Amount of moisture content (g) 0.09 Weight of powdered drug used (g) 3.00 Percentage of moisture (%) 0.09 x 100/3 = 3 %

Total Ash Value Weight of the crucible + powder = W1, Weight of the crucible alone = W2

Weight of the powder alone = W1 – W2

Weight of the crucible + Ash = W3

Weight of Ash alone = W3 – W2

% Ash value = W3- W2 x 100 / W1- W2.

151

Weightof the crucible + ash (g) 16.10 Weight of the crucible (g) 15.87 Weight of the ash (g) 0.23 Weight of the powder drug used (g) 2.00

Percentage Ash (%) 0.23 x 100/2 = 11.5 %

Acid-Insoluble Ash Value Weight of Crucible + Acid –insoluble ash (g) 15.70 Weight of the crucible (g) 15.63 Weight of Acid-insoluble ash (g) 0.07 Weight of powdered drug used (g) 2.00 Percentage of Acid-insoluble ash (%) 0.07 x 100/2 = 3.5 %

Sulphated Ash Value Weight of the crucible + Ash (g) 15.71 Weight of crucible (g) 15.46 Weight of Sulphated Ash (g) 0.25 Weight of the powdered drug (g) 2.00 Percentage of Sulphated Ash (%) 0.25 x 100/2 = 12.5

Water Soluble Ash Value Weight of crucible + Ash (g) 15.72 Weight of crucible + Insoluble ash (g) 15.64 Weight of water soluble ash (g) 0.08 Weight of powdered drug used (g) 2.00 Percentage of water soluble Ash (%) 0.08 x100/2 = 4 %

Alcohol Soluble Extractive Value

Weight of the dish + 20 ml volume filtrate = W1

Weight of the dish = W2

Weight of the20 ml volume filtrate = W1- W2

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Weight of the dish + Residue = W3

Weight of Residue from 20 ml volume filtrate = W3-W2

Weight of residue from100 ml volume filtrate = (W3-W2) x100 / (W1-W2)

Weight of the powder Macerated = W4

% Extractive value = {(W3-W2) x 100 / (W1-W2)

% Extractive value ={(W3-W2) x 100 / (W1-W2) x W4} x 100

Weight of beaker + Extract (g) 73.45 Weight of beaker (g) 72.43 Weight of Extract (g) 1.02 Volume of filtrate evaporated (ml) 20 ml Weight in 100 ml (g) 1.02 x 100/20 = 5.1% Weight of powdered drug used (g) 5.00 Percentage Yield (%) 1.02 x 100/5 = 20.4 %

Water Soluble Extractive Value

Weight of the dish + 20 ml volume filtrate = W1

Weight of the dish = W2

Weight of the20 ml volume filtrate = W1- W2

Weight of the dish + Residue = W3

Weight of Residue from 20 ml volume filtrate = W3-W2

Weight of residue from100 ml volume filtrate = (W3-W2) x100 / (W1-W2)

Weight of the powder Macerated = W4

% Extractive value = {(W3-W2) x 100 / (W1-W2) x W4} x 100

Weight of Beaker + Extract (g) 76.35 Weight of Beaker (g) 75.95 Weight of Extract (g) 0.40 Volume of filtrate Evaporated (ml) 20 ml Weight in 100 ml (g) 0.4 x 100/20 = 2.0 g Weight of powdered drug used (g) 5. 0 g Percentage yield (%) 0.4 x 100/5 = 8 %

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Analytical standards

The analytical standards of P. nitida are shown in Table 4.

Table 28: Results of analytical standards of Pricralima nitida Parameter Concentration (%)

Moisture content 3.0

Total ash value 11.5

Acid insoluble ash 3.5

Water soluble ash 4.0

Sulphated ash 12.5

Alcohol soluble extractive 20.4

Water soluble extractive 8.0

METHODOLOGY. STEP 1: Preparation of different concentrations of methanol extract of Pricralima nitida using normal saline to dilute the fraction to the concentrations required. 2.5 mg/ml = 2.5 mg of the extract in 1ml of n.saline 5 mg/ml = 5 mg of the extract in 1ml of saline 10 mg/ml = 10 mg of the extract in 1ml of saline

1g in 100 ml saline 5 mg/ml of P- hydroxybenzoic acid in normal saline =5 mg in 1 ml of saline

50 mg of the P-hydroxybenzoic acid in 100ml of normal saline

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METHANOL EXTRACT OF P.nitida, HbAS BLOOD SAMPLE. Methanol extract of P. nitida was prepared in concentrations 2.5 mg/ml, 5 mg/ml and 10 mg/ml. With each concentration seven result readings of percentag reversal of sickling of HbAS blood cells were obtained. The percentage reversal of sickling of HbAS blood cells was examined in peripheral blood cells and noted.

PERIPHERAL CELLS Sickled cells = 3 Reversal of sickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 %

The washed erythrocytes were treated for 180 minutes with 2 % solution of Sodium Metabisulphite to convert more cells to sickle cell and a sample smear was taken and examined. The estimate of % reversal of sickling noted.

CELL + SOLUTION 1 CNA Sickled cells = 23 Unsickled cells = 11 Total number of cells = 34 % reversal of sickling = 32%

The erythrocytes and the Solution 1 in equal volume of 0.5 ml were mixed with 0.5 ml of 2.5 mg/ml concentration of Methanol extract P. nitida. The mixture was covered with liquid paraffin and incubated at 37 oC with occasional shaking. Sample smears were taken at intervals of 30 minutes through 180 minutes.

2.5 mg/ml methanol extract on HbAS Blood cells at 0 minutes incubation, Sickled cells = 11 Unsickled cells = 8 Total number of cells = 19 % reversal of sickling = 42 %

2.5 mg/ml methanol extract on HbAS Blood cells at 30 minutes incubation 155

Sickled cells = 21 Unsickled cells = 18 Total number of cells = 39 % reversal of sickling = 46 %

2.5 mg/ml methanol extract on HbAS Blood cells at 60 minutes incubation Sickled cells = 12 % reversal of sickling = 10 Total number of cells = 22 % unsickled cells = 45 %

2.5 mg/ml methanol extract on HbAS Blood cells at 90 minutes incubation Sickled cells = 14 Unsickled cells = 11 Total number of cells = 25 % reversal of sickling = 44 %

2.5 mg/ml methanol extract on HbAS Blood cells at 120 minutes incubation. Sickled cells = 18 Unsickled cells = 12 Total number of cells = 30 % reversal of sickling = 40 %

2.5 mg/ml methanol extract on HbAS Blood cells at 150 minutes incubation. Sickled cells = 24 Unsickled cells = 23 Total number of cells = 47 % reversal of sickling = 49 %

2.5 mg/ml methanol extract on HbAS Blood cells at 180 minutes incubation. Sickled cells = 16 Unsickled cells = 18 Total number of cells = 34 % reversal of sickling = 53 %

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A 5 mg/ml concentration was prepared from Methanol extract of P. nitida. A 0.5ml of the concentration was mixed with a 0.5ml of the washed erythrocytes and a 0.5ml of 2 % solution of Sodium Metabisulphite (solution 1). The whole mixture was in test tube which later was covered with liquid paraffin. The system was incubated at 37 oC and occasionally shaked. Smears were made in clean slides at 30 minutes intervals through 180 minutes. Estimate of % reversal of sickling were determined. 5 mg/ml methanol extract on HbAS Blood cells at 0 minutes Sickled cells = 28 Unsickled cells = 20 Total number of cells = 48 % reversal of sickling = 41 %

5 mg/ml methanol extract on HbAS Blood cells at 30 minutes Sickled cells = 25 Unsickled cells = 19 Total number of cells = 44 % reversal of sickling = 43 %

5 mg/ml methanol extract on HbAS Blood cells at 60 minutes incubation. Sickled cells = 19 Unsickled cells = 20 Total number of cells = 39 % reversal of sickling = 51 %

5 mg/ml methanol extract on HbAS Blood cells at 90 minutes incubation Sickled cells = 18 Unsickled cells = 20 Total number of cells = 38 % reversal of sickling = 53 %

5 mg/ml methanol extract on HbAS Blood cells at 120 minutes incubation Sickled cells = 30 Unsickled cells = 39 Total number of cells = 69 % reversal of sickling = 56 %

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5 mg/ml methanol extract on HbAS Blood cells at 150 minutes incubation Sickled cells = 31 Unsickled cells = 42 Total number of cells = 73 % reversal of sickling = 57 %

5 mg/ml methanol extract on HbAS Blood cells at 180 minutes incubation. Sickled cells = 12 Unsickled cells = 18 Total number of cells = 30 % reversal of sickling = 60 %

A 10mg/ml concentration was prepared from methanol extract of P. nitida. A 0.5ml of the washed erythrocytes and the same volumes of solution 1 and the methanol extract of P. nitida were mixed and incubated at 37 oC Estimates of the % reversal of sickling were taken at 30 minutes intervals through 180 minutes.

10 mg/ml methanol extract on HbAS Blood cells at 0 minutes Sickled cells = 12 Unsickled cells = 6 Total number of cells = 18 % reversal of sickling = 33 %

10 mg/ml methanol extrat on HbAS Blood cells at 30 minutes incubation Sickled cells = 18 Unsickled cells = 34 Total number of cells = 52 % reversal of sickling = 65 %

10 mg/ml methanol extract on HbAS Blood cells at 60 minutes incubation Sickled cells = 2 Unsickled cells = 15 Total number of cells = 17 % reversal of sickling = 88 %

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10 mg/ml methanol extract on HbAS Blood cells at 90 minutes incubation. Sickled cells = 1 Unsickled cells = 8 Total number of cells = 9 % reversal of sickling = 89 %

10 mg/ml methanol extract on HbAS Blood cells at 120 minutes incubation Sickled cells = 3 Unsickled cells = 25 Total number of cells = 28 % reversal of sickling = 89 %

10 mg/ml methanol extract on HbAS Blood cells at 150 minutes incubation Sickled cells = 0 Unsickled cells = 15 Total numbers of cells = 15 % reversal of sickling= 100 %

10 mg/ml methanol extract on HbAS Blood cells at 180 minutes incubation. Sickled cells = 2 Unsickled cells = 9 Total number of cells = 11 % reversal of sickling = 82 % Summary The percentage of the unsickled cells was shown as well as the % reversal of sickling obtained from the examinations done at the time intervals from different concentrations of the methanol extract of Picralima nitida,through the incubation of 180 minutes. Peripheral Concentrations At 0 At 30 At 60 At 90 At 120 At 150 At 180 cells mg/ml min min min min min min min without the drug 83 2.5 mg/ml 42 46 45 44 40 49 53 83 5 mg/ml 41 43 47 53 56 57 60 83 10 mg/ml 33 65 88 89 89 100 82

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AQUEOUS FRACTION OF P. nitida Peripheral cells Sickled cells = 3 Unsickled cells = 15 Total numbers of cells = 18 % reversal of sickling = 83 %

The conversion of more blood cells to sickled cells was done by the addition of 2 % solution of sodium metabisulphate represented as Solution 1. A smear sample was made also and % reversal of sickling calculated.

RED CELLS + SOLUTION 1 Sickled cells = 23 Unsickled cells = 11 Total numbers of cells = 34 % reversal of sickling = 32 %

AQUEOUS FRACTION OF P. nitida 2.5 mg/ml Ethanol fraction on HbAS blood cells at 0 minute incubation Sickled cells = 9 Unsickled cells = 7 Total number of cells = 16 % reversal of sickling = 43 % 2.5 mg/ml aqoeous fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 19 Unsickled cells = 17 Total numbers of cells = 36 % reversal of sickling = 47 %

2.5 mg/ml aqoeous fraction on HbAS blood cells at 60minutes incubation. Sickled cells = 9 Unsickled cells = 7 Total numbers of cells = 16 % reversal of sickling = 43 %

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2.5 mg /ml aqoeous fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 12 Unsickled cells = 10 Total numbers of cells = 22 % reversal of sickling = 45 % 2.5 mg/ml aqoeous fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 21 Unsickled cells = 21 Total numbers of cells = 42 % reversal of sickling = 50 %

2.5 mg/ml aqoeous fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 19 Unsickled cells = 19 Total numbers of cells = 38 % reversal of sickling = 50 %

2.5 mg/ml aqoeous fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 12 Unsickled cells = 13 Total numbers of cells = 25 % reversal of sickling = 52 %

Thewashed erythrocytes and the 2 % solution of Sodium Metabisulphite in equal volume of 0.5 ml were mixed with 0.5 ml of 5 mg/ml concentration of Aqoeous Fraction of P. nitida and incubated at 37 oC. Sample smears and observations under the microscope were made at 30 minutes intervals and % reversal of sickling calculated. 5 mg/ml aqoeous fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 30 Unsickled cells = 20 Total numbers of cells = 50 % reversal of sickling = 40 %

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5 mg/ml aqoeous fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 20 Unsickled cells = 12 Total numbers of cells = 37 % reversal of sickling = 46 % 5 mg/ml aqoeous fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 21 Unsickled cells = 22 Total numbers of cells = 43 % reversal of sickling = 51 %

5 mg/ml aqoeous fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 9 Unsickled cells = 10 Total number of cells = 19 % reversal of sickling = 52 %

5 mg/ml aqoeous fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 33 Unsickled cells = 43 Total numbers of cells = 76 % reversal of sickling = 57 %

5 mg/ml aqoeous fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 29 Unsickled cells = 42 Total numbers of cells = 71 % reversal of sickling = 59 %

5 mg/ml aqoeous fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 9 Unsickled cells = 16 Total numbers of cells = 25 % reversal of sickling = 64 % 162

A 0.5 ml of 10 mg/ml concentration Aqoeous Fraction of P. nitida was mixed with 0.5 ml of the washed erythrocytes and 0.5 ml of solution 1 and incubated 37 oC with occasional shaking. Sample smears were taken, examined and the % reversal of sickling were calculated from the smears made at 30 minutes interval

10 mg/ml aqoeous fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 5 Unsickled cells = 1 Total numbers of cells = 6 % reversal of sickling = 17 %

10 mg/ml aqoeous fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 10 Unsickled cells = 20 Total numbers of cells = 30 % reversal of sickling = 67 %

10 mg/ml aqoeous fraction on HbAS blood cells at 60 minutes incubation Sickled cells = 0 Unsickled cells = 22 Total numbers of cells = 22 % reversal of sickling = 100 %

10mg/ml aqoeous fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 1 Unsickled cells = 13 Total numbers of cells = 14 % reversal of sickling = 93 %

10 mg/ml aqoeous fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 0 Unsickled cells = 24 Total numbers of cells = 24 % reversal of sickling = 100 %

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10 mg/ml aqoeous fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 0 Unsickled cells = 10 Total numbers of cells = 10 % reversal of sickling = 100 %

10 mg/ml aqoeous fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 0 Unsickled cells = 12 Total numbers of cells = 12 % reversal of sickling = 100 %

Summary A table was made below showing the % reversal of sickling obtained from different concentrations of aqoeous fractions of P.nitida mixed in equal volume of 0.5 ml of washed erythrocites and 0.5 ml of solution 1 incubated at 37oC with occasional shaking and samples examined at 30 minutes interval. Drug At 0 At 30 At 60 At 90 At At At Peripheral concentrations min min min min 120 150 180 readings mg/ml min min min without drug 2.5 mg/ml 43 43 43 45 50 50 52 83 5 mg/ml 40 46 51 52 57 59 64 81 10 mg/ml 17 67 100 93 100 100 100 81

HBAS BLOOD SAMPLE CHLOROFORM FRACTION OF Picralima nitida ON (HbAS BLOOD GENOTYPE) The sample smear of the washed blood cells (peripheral cells) of the HbAS blood was made on a clean slide and the % reversal of sickling was calculated after the examination under the microscope.

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PERIPHERAL CELLS Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 %

Conversion of more blood cells to sickled cells was done by the addition of 2 % solution of Sodium Metabisulphite. A sample smear was done and the examination of the sickled and unsickled cells was done. % reversal of sickling was estimated.

CELL + SOLUTION 1 (NA2S205) Sickled cells = 23 Unsickled cells = 11 Total numbers of cells = 34 % reversal of sickling = 32 % Equal volumes of 0.5 ml of the washed erythrocytes HbAS, Solution 1, and the 2.5 mg/ml concentration of Chloroform Fraction of P. nitida were incubated at 37 OC with occasional shaking. Smears were made on clean slides at 30 minutes intervals starting from zero, The % reversal of sickling was calculated.

2.5 mg/ml Chloroform fraction on HbAS Blood cells at 0 minute incubation. Sickled cells = 10 Unsickled cells = 8 Total numbers of cells = 18 % reversal of sickling = 44 %

2.5 mg/ml Chloroform fraction on HbAS Blood cells at 30 minutes incubation Sickled cells = 20 Unsickled cells = 17 Total number of cells = 37 % reversal of sickling = 45 %

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2.5 mg/ml chloroform fraction on HbAS Blood cells at 60 minutes incubation Sickled cells = 10 Unsickled cells = 9 Total number of cells = 19 % reversal of sickling = 47 %

2.5 mg/ml chloroform fraction on HbAS Blood cells at 90 minutes incubation Sickled cells = 16 Unsickled cells = 12 Total number of cells = 31 % reversal of sickling = 48 %

2.5 mg/ml chloroform fraction on HBAS Blood cells at 120 minutes incubation. Sickled cells = 21 Unsickled cells = 21 Total number of cells = 42 % reversal of sickling = 50 %

2.5 mg/ml chloroform fraction on HbAS Blood cells at 150 minutes incubation. Sickled cells = 13 Unsickled cells = 14 Total number of cells = 27 % reversal of sickling = 51 %

2.5 mg/ml chloroform fraction on HbAS blood cells at 180 minutes incubation Sickled cells = 16 Unsickled cells = 19 Total number of cells = 35 % reversal of sickling = 54 %

A 5 mg/ml concentration was prepared from methanol extract of P. nitida. A 0.5ml of the concentration was mixed with a 0.5ml of the washed erythrocytes and a 0.5ml of 2 % solution of Sodium Metabisulphite (solution 1). The whole mixture was in test tube which later was covered with liquid paraffin. The system was incubated at 37 OC and occasionally shaked. 166

Smears were made in clean slides at 30 minutes intervals through 180 minutes. Estimate of % reversal of sickling were determined.

5 mg/ml methanol extract on HbAS Blood cells at 0 minutes Sickled cells = 28 Unsickled cells = 20 Total number of cells = 48 % reversal of sickling = 41 %

5 mg/ml methanol fraction on HbAS Blood cells at 30 minutes Sickled cells = 25 Unsickled cells = 19 Total number of cells = 44 % reversal of sickling = 43 %

5 mg/ml methanol fraction on HbAS Blood cells at 60 minutes incubation. Sickled cells = 19 Unsickled cells = 20 Total number of cells = 39 % reversal of sickling = 51 %

5 mg/ml methanol fraction on HbAS Blood cells at 90 minutes incubation Sickled cells = 18 Unsickled cells = 20 Total number of cells = 38 % reversal of sickling = 53 %

5 mg/ml methanol fraction no HbAS at 120 minutes incubatio Blood cells n Sickled cells = 30 Unsickled cells = 39 Total number of cells = 69 % reversal of sickling = 56 %

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5 mg/ml methanol fraction on HbAS Blood cells at 150 minutes incubation Sickled cells = 31 Unsickled cells = 42 Total number of cells = 73 % reversal of sickling = 57 % 5 mg/ml Methanol fraction on HbAS Blood cells at 180 minutes incubation. Sickled cells = 12 Unsickled cells = 18 Total number of cells = 30 % reversal of sickling = 60 % A 5 mg/ml concentration was prepared from methanol extract of P. nitida. A 0.5 ml of the concentration was mixed with a 0.5 ml of the washed erythrocytes and a 0.5 ml of 2 % solution of Sodium Metabisulphite (solution 1). The whole mixture was in test tube which later was covered with liquid paraffin. The system was incubated at 37 OC and occasionally shaked. Smears were made in clean slides at 30 minutes intervals through 180 minutes. Estimate of % reversal of sickling was determined.

5 mg/ml methanol fraction on HbAS blood cells at 0 minutes Sickled cells = 28 Unsickled cells = 20 Total number of cells = 48 % reversal of sickling = 41 %

5 mg/ml methanol fraction on HbAS blood cells at 30 minutes Sickled cells = 25 Unsickled cells = 19 Total number of cells = 44 % reversal of sickling = 43 % 5 mg/ml Methanol fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 19 Unsickled cells = 20 Total number of cells = 39 % reversal of sickling = 51 %

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5 mg/ml Methanol fraction on HbAS blood cells at 90 minutes incubation Sickled cells = 18 Unsickled cells = 20 Total number of cells = 38 % reversal of sickling = 53 %

5 mg/ml Methanol fraction on HbAS blood cells at 120 minutes incubation Sickled cells = 30 Unsickled cells = 39 Total number of cells = 69 % reversal of sickling = 56 %

5 mg/ml Methanol fraction on HbAS blood cells at 150 minutes incubation Sickled cells = 31 Unsickled cells = 42 Total number of cells = 73 % reversal of sickling = 57 % 5 mg/ml Methanol fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 12 Unsickled cells = 18 Total number of cells = 30 % reversal of sickling = 60 %

5mg/ml OF Picralima nitida CHLOROFORME FRACTION MIXED WITH HbAS BLOOD GEOTYPE A smear of the erythrocytes (peripheral cells) only on a clean slide was made from which the % reversal of sickling was estimated after examination of the cells under microscope.

PERIPHERAL CELLS Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 %

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More cells were converted to sickle cells with solution 1.

CELL + SOLUTION 1 (NAS205) Sickled cells = 23 Unsickled cells = 11 Total number of cells = 34 % reversal of sickling = 32 %

Equal volumes of 0.5ml each from washed blood cells, solution 1 and the 5 mg/ml concentration of Chloroform Fraction of P. nitida were incubated at 37 OC. Samples of smears were made at intervals of 30 minutes starting from zero, % reversal of sickling was estimated by calculations after careful examination of the cells under the microscope.

5mg/ml concentration of Chloroform fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 29 Unsickled cells = 21 Total number of cells = 50 % reversal of sickling = 42 %

5mg/ml concentration of Chloroform fraction on HbAS blood cells at 30 minutes incubation Sickled cells = 25 Unsickled cells = 20 Total number of cells = 45 % reversal of sickling = 44 %

5mg/ml concentration of Chloroform fraction on HbAS blood cellsat 60 minutes incubation. Sickled cells = 20 Unsickled cells = 21 Total number of cells = 41 % reversal of sickling = 51 % 5mg/mlconcentration of Chloroform fraction on HbAS blood cells at 90 minutes incubation Sickled cells = 20 Unsickled cells = 23 Total number of cells = 43 % reversal of sickling = 53 % 170

5mg/ml concentration of Chloroform fraction on HbAS blood cells at 120 minutes incubation Sickled cells = 34 Unsickled cells = 45 Total number of cells = 79 % reversal of sickling = 57 %

5mg/ml concentration of Chloroform fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 28 Unsickled cells = 40 Total number of cells = 48 % reversal of sickling = 59 %

5mg/ml concentration of Chloroform fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 7 Unsickled cells = 15 Total number of cells = 33 % reversal of sickling = 21%

10 mg/mlConcentration of Chloroform Fractionof Picralima Nitida In HbAS Blood Genotype. The % reversal of sickling in the peripheral blood cell was calculated from a sample smear examined under the microscope.

PERIPHERAL CELLS Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 %

A 0.5 ml of solution 1 (2 % solution of sodium metabisulphite) and equal volume of washed erythrocytes of HbAS blood were mixed to produce more sickled cells.

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CELL + SOLUTION 1 (NA2S205) Sickled cells = 23 Unsickled cells = 11 Total number of cells = 34 % reversal of sickling = 32 %

A 10 mg/ml concentration was prepared from chloroform Fraction of Picralima nitida and a 0.5 ml of it was mixed with a 0.5 ml of solution 1 (2 % solution of sodium Metabisulphite) and equal volume of washed erythrocytes of HbAS blood. Sample smears were taken at 30 minutes interval of incubation at 37 OC from zero to 180 minutes and % reversal of sickling calculated

10 mg/mlconcentration of the Chloroform fraction on HbAS blood cells at 0 minutes incubation Sickled cells = 8 Unsickled cells = 5 Total number of cells = 13 % reversal of sickling = 38 %

10 mg/ml Chloroform fraction on HbAS blooa cells at 30 minutes incubation Sickled cells = 12 Unsickled cells = 22 Total number of cells = 34 % reversal of sickling = 65 %

10 mg/ml concentration of Chloroform fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 1 Unsickled cells = 20 Total number of cells = 21 % reversal of sickling = 95 %

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10 mg/ml concentration of chloroform fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 0 Unsickled cells = 22 Total number of cells = 22 % reversal of sickling = 100 %

10 mg/ml concentration of Chloroform fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 0 Unsickled cells = 22 Total number of cells = 22 % reversal of sickling = 100 %

10 mg/ml concentration of Chloroform fraction on HbAS blood cells at 150 minutes incubation Sickled cells - 0 Unsickled cells - - 12 Total number of cells - 12 % reversal of sickling - - 100 %

10 mg/ml concentration of Chloroform fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 0 % reversal of sickling = 10 Total number of cells = 10 % reversal of sickling = 100 %

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Summary The recorded % reversal of sickling in the smears taken at different times of incubation at 370C of different concentrations of the Chloroform fraction of P. nitida and HbAS blood Peripheral Concentrations At 0 At 30 At 60 At 90 At 120 At 150 At 180 cells mg/ml min min min min min min min without the drug 83 2.5 mg/ml 44 45 47 48 50 51 54 83 5 mg/ml 42 44 51 53 57 59 65 83 10 mg/ml 65 65 95 100 100 100 100

HbAS BLOOD SAMPLE ON ETHYL ACETATE FRACTION EXTRACTS P.nitida A smear of the peripheral cells was made from where the % reversal of sickling was estimated

PERIPHERAL CELLS Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 8 3%

Solution 1 and the washed erythrocytes were mixed and incubated. A smear was done from which % reversal of sickling was estimated

CELL + SOLUTION 1 (Na2S205) Sickled cells = 23 Unsickled cells = 11 Total number of cells = 34 % reversal of sickling = 32 %

A0.2.5 mg/ml concentration from Ethyl Acetate fraction of P. nitida was prepared. A 0.5 ml of the washed red blood cells mixed with 0.5 ml solution 1 and a 0.5 ml of the prepared concentration. The mixture was incubated at 37 OC. Smears were done at 30 minutes interval from where the % reversal of sickling was estimated.

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2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 0 minutes incubation Sickled cells = 9 Unsickled cells = 5 Total numberof cells = 14 % reversal of sickling = 36 % 2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 30 minutes Sickled cells = 11 Unsickled cells = 6 Total number of cells = 17 % reversal of sickling = 35 %

2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 60 minutesincubation Sickled cells = 25 Unsickled cells = 18 Total number of cells = 43 % reversal of sickling = 42 %

2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 90 minutes incubation Sickled cells = 21 Unsickled cells = 14 Total number of cells = 35 % reversal of sickling = 40 %

2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 120 minutes incubation Sickled cells = 12 Unsickled cells = 11 Total number of cells = 23 % reversal of sickling = 48 %

2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 150 minutes incubation Sickled cells = 15 Unsickled cells = 15 Total number of cells = 30 % reversal of sickling = 50 % 175

2.5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 14 Unsickled cell = 18 Total number of cells = 32 % reversal of sickling = 56 %

5mg/ml Ethyl Acetate fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 37 Unsickled cells = 14 Total number of cells = 51 % reversal of sickling = 77 %

5mg/mlEtrhyl Acetate fraction on HbAS blood cells at 30 minutes incubation Sickled cells = 28 Unsickled cells = 20 Total number of cells = 48 % unsickled cells = 42 %

5 mg/ml Ethyl Acetate fraction fraction on HbAS blood cells at 60 minutes incubation Sickled cells = 38 Unsickled cells = 27 Total number of cells = 65 % reversal of sickling = 41 %

5mg/ml Ethyl Acetate fraction fraction on HbAS blood cells at 90 minutes incubation Sickled cells = 8 Unsickled cells = 8 Total number of cells = 16 % reversal of sickling = 50 % 5mg/ml Ethyl Acetate fraction of the fraction on HbAS blood cells at 120 minutes incumbent Sickled cells = 13 Unsickled cells = 15 Total number of cells = 28 % reversal of sickling = 53 % 176

5 mg/ml Ethyl Acetate fraction fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 5 Unsickled cells = 10 Total number of cells = 15 % reversal of sickling = 66 %

5 mg/ml Ethyl Acetate fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 11 Unsickled cells = 15 Total number of cells = 26 % reversal of sickling = 58 %

A 10 gm/ml concentration of Athyl Acetate fraction of Picralima nitida was prepared. A 0.5ml of washed erythrocytes of HbAS blood was mixed with 0.5 ml of 2% solution of sodium metabisulphite and also 0.5 gm/ ml of the prepared concentration of the extract. The mixture was well shaken and covered with liquid paraffin. The system was incubated at 37oC sample smears were taken at intervals of 30 minutes through 180 minutes.

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 0 minutes incubation Sickled cells = 7 Unsickled cells = 4 Total number of cells = 11 % reversal of sickling = 36 %

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 12 Unsickled cells = 13 Total number of cells = 25 % reversal of sickling = 52 %

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10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 4 Unsickled cells = 9 Total number of cells = 13 % reversal of sickling = 69 %

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 0 Unsickled cells = 10 Total number of cells = 10 % reversal of sickling = 100 %

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 120 minutes incubation Sickled cells = 2 Unsickled cells = 14 Total number of cells = 16 % reversal of sickling = 87 %

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 1 Unsickled cells = 22 Total numberof cells = 23 % reversal of sickling = 96 %

10 mg/ml concentration of Ethyl Acetate fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 0 Unsickled cells = 5 Total number of cells = 5 % reversal of sickling = 100 %

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Summary Ethyl Acetate Fraction of Picralima nitida incubated in HbAS Blood A Table showing % reversal of sickling in peripheral blood, and also the % reversal of sickling in the three concentration samples of the Ethyl Acetate fraction of P. nitida incubated at 30 minutes interval from zero minute –to- 180 minutes. Peripheral Concentration At 0 At 30 At 60 At 90 At 120 At 150 At 180 cells with0ut mg/ml min min min min min min min the drug 83 2.5 mg/ml 36 35 42 40 48 50 56 5 mg/ml 77 45 41 50 53 66 58 10 mg/ml 36 52 49 100 87 96 100

HbAS BLOOD SAMPLE ON DICHLOROMETHANE FRACTION EXTRACT OF Picralima nitida The peripheral blood of the HbAS was smeared on a clean slide and observed under the microscope and the % reversal of sickling was noted to showed the condition of the blood before the incubation in the Dichloromethane fraction of P. nitida

PERIPHERAL CELLS Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 %

A 0.5 g/ml of washed erythrocytes was mixed with equal volume of 2 % solution of sodium Bisulphite (solution 1).

CELL + SOLUTION 1 Sickled cells = 23 Unsickled cells = 11 Total number of cells = 34 % reversal of sickling = 32 %

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A 0.5g/ml of washed erythrocytes was mixed with equal volume of 2 % solution of sodium Bisulphite (solution 1) and a 2.5 mg/ml of Dichloromethane fraction of P. nitida was added. The mixture was covered with liquid paraffin, incubated at 37 OC with occasional shaking. From the mixture a smear was taken at intervals of 30 minutes. % reversal of sickling at the intervals were calculated

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 0 minutes incubation Sickled cells = 12 Unsickled cells = 9 Total number of cells = 21 % reversal of sickling = 43 %

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 14 Unsickled cells = 12 Total number of cells = 26 % reversal of sickling = 46 %

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 60 minutes Sickled cells = 8 Unsickled cells = 3 Total number of cells = 11 % reversal of sickling = 27 %

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 90 minutes incubation Sickled cells = 20 Unsickled cells = 11 Total number of cells = 31 % reversal of sickling = 35 %

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2.5 mg/ml dichloromethane fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 13 Unsickled cells = 11 Total number of cells = 24 % reversal of sickling = 46 %

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 20 Unsickled cells = 19 Total number of cells = 39 % reversal of sickling = 49 %

2.5 mg/ml concentration of dichloromethane fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 8 Unsickled cells = 9 Total number of cells = 17 % reversal of sickling = 53 %

5 mg/ml dichloromethane fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 33 Unsickled cells = 24 Total number of cells = 57 % reversal of sickling = 42 %

5mg/ml dichloromethane fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 33 Unsickled cells = 27 Total number of cells = 60 % reversal of sickling = 45 %

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5mg/ml dichloromethane fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 12 Unsickled cells = 9 Total numbers of cells = 21 % reversal of sickling = 43 %

5mg/ml dichloromethane fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 18 Unsickled cells = 17 Total number of cells = 35 % reversal of sickling = 48 %

5mg/ml dichloromethane fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 8 Unsickled cells = 12 Total number of cells = 20 % reversal of sickling = 60 % 5mg/ml dichloromethane fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 9 Unsickled cells = 14 Total number of cells = 23 % reversal of sickling = 61 %

5mg/ml dichloromethane fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 14 Unsickled cells = 20 Total number of cells = 34 % reversal of sickling = 59 %

10 mg/ml dichloromethane fraction on HbAS blood cells at 0 minutes incubation. Sickled cells = 13 Unsickled cells = 9 Total number of cells = 22 % reversal of sickling = 41 % 182

10 mg/ml dichloromethane fraction on HbAS blood cells at 30 minutes incubation. Sickled cells = 14 Unsickled cells = 16 Total numbers of cells = 30 % reversal of sickling = 53 %

dichloromethane fraction on HbAS blood cells at 60 minutes incubation. Sickled cells = 5 Unsickled cells = 12 Total number of cells = 17 % reversal of sickling = 70 % 10 mg/ml dichloromethane fraction on HbAS blood cells at 90 minutes incubation. Sickled cells = 2 % reversal of sickling = 18 Total number of cells = 20 % unsickled cells = 90 %

10 mg/ml dichloromethane fraction on HbAS blood cells at 120 minutes incubation. Sickled cells = 1 Unsickled cells = 10 Total number of cells = 11 % reversal of sickling = 91 %

10 mg/ml dichloromethane fraction on HbAS blood cells at 150 minutes incubation. Sickled cells = 0 Unsickled cells = 17 Total number of cells = 17 % reversal of sickling = 100 %

10 mg/ml dichloromethane fraction on HbAS blood cells at 180 minutes incubation. Sickled cells = 0 Unsickled cells = 10 Total number of cells = 10 % reversal of sickling = 100 % 183

Summary The table below showed the % reversal of sickling in the peripheral HbAS blood, and the % reversal of sickling readings taken when the HbAS blood was treated with the different concentrations of the dichloromethane fraction of Picralima nitida at 37 oC incubation at 30 minutes interval through 180 minutes. Peripheral Concentrations At 0 At 30 At 60 At 90 At 120 At 150 At180 cells mg/ml min min min min min min min without the drug 83 2.5 mg/ml 43 46 27 35 46 49 53 83 5 mg/ml 42 45 43 48 60 61 59 83 10 mg/ml 41 53 70 90 91 100 100

METHANOL EXTRACT OF Picralima nitida IN SS BLOOD GEONTYPE PERIPHERAL CELLS The sample of the HbSS blood before the treatment with the 2.5 mg/ml concentration of the methanol extract P. nitida. Sickled cells = 15 Unsickled cells = 5 Total number of cells = 20 % reversal of sickling = 25 %

2.5 mg/ml methanol extract on HbSS blood cells at 0 minutes incubation Sickled cells = 29 Unsickled cells = 21 Total number of cells = 50 % reversal of sickling = 42 % .5 mg/ml methanol extract on HbSS blood cells at 30 minutes incubation. Sickled cells = 20 Unsickled cells = 17 Total number of cells = 37 % reversal of sickling = 45 %

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2.5 mg/ml methanol extract on HbSS blood cells at 60 minutesincubation. Sickled cells = 10 Unsickled cells = 9 Total number of cells = 19 % reversal of sickling = 47 %

2.5 mg/ml methanol extract on HbSS blood cells at 90 minutesincubation. Sickled cells = 13 Unsickled cells = 14 Total number of cells = 27 % reversal of sickling = 51 %

2.5 mg/ml methanol extract on HbSS blood cells at 120 minutes incubation. Sickled cells = 13 Unsickled cells = 15 Total number of cells = 28 % reversal of sickling = 53 %

2.5 mg/ml methanol extract on HbSS blood cells at 150 minutes incubation. Sickled cells = 34 Unsickled cells = 45 Total number of cells = 79 % reversal of sickling = 57 % 2.5 mg/ml methanol extract on HbSS blood cells at 180 minutes incubation. Sickled cells = 12 Unsickled cells = 18 Total number of cells = 30 % reversal of sickling = 60 %

5mg/ml of Pricralima nitida methanol extracts on HbSS blood sample The concentration 5 mg/ml of Methanol extract was prepared, incubated at the same 37 oC when mixed and shacked with the washed SS blood red cells. Sample smears were taken at 30 minutes interval through 180 minutes.

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PERIPHERAL CELLS: The sample smear was taken before the treatment of the SS blood cells with the 5mg/ml of the Methanol extract. Sickled cells = 10 Unsickled cells = 3 Total number of cells = 13 % reversal of sickling = 23 %

5mg/ml methanol extract on HbSS blood cells at 0 minutes incubation. Sickled cells = 12 Unsickled cells = 12 Total number of cells = 24 % reversal of sickling = 50 %

5mg/ml methanol extract on HbSS blood cells at 30 minutes incubation. Sickled cells = 15 Unsickled cells = 15 Total number of cells = 30 % reversal of sickling = 50 %

5mg/ml methanol extract on HbSS blood cells at 60 minutes incubation. Sickled cells = 14 Unsickled cells = 18 Total number of cells = 32 % reversal of sickling = 56 %

5mg/ml methanol extract on HbSS blood cells at 90 minutes incubation. Sickled cells = 9 Unsickled cells = 16 Total number of cells = 25 % reversal of sickling = 64 %

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5mg/ml methanol extract on HbSS blood cells at 120 minutes incubation. Sickled cells = 12 Unsickled cells = 22 Total number of cells = 34 % reversal of sickling = 65 %

5mg/ml methanol extract on HbSS blood cells at 150 minutes incubation. Sickled cells = 5 Unsickled cells = 13 Total number of cells = 15 % reversal of sickling = 66 %

5mg/ml methanol extract on HbSS blood cells at 180 minutes incubation. Sickled cells = 9 Unsickled cells = 18 Total number of cells = 27 % reversal of sickling = 66 % 10 mg/ml of Picralima nitida extract on HbSS blood cells Peripheral cells: % reversal of sickling reading from sample smear of the washed HbSS blood cells Sickled cells = 12 Unsickled cells = 3 Total number of cells = 15 % reversal of sickling = 20 %

A10 mg/ml methanol extract was prepared from which 0.5 ml volume was taken and mixed with 0.5 ml washed erythrocytes of the HbSS blood. The mixture was shacked and incubated at 37 oC. Smears were made at 30 minutes intervals through 180 minutes.

10 mg/ml methanol extract on HbSS blood cells at 0 minutes incubation. Sickled cells = 16 Unsickled cells = 19 Total number of cells = 35 % reversal of sickling = 54 % 187

10 mg/ml Methanol extract on HbSS blood cells at 30 minutes incubation. Sickled cells = 34 Unsickled cells = 45 Total number of cells = 79 % reversal of sickling = 57 %

10 mg/ml methanol extract on HbSS blood cells at 60 minutes incubation Sickled cells = 14 Unsickled cells = 20 Total number of cells = 34 % reversal of sickling = 59 % 10 mg/ml methanol extract on HbSS blood cells at 90 minutes incubation. Sickled cells = 10 Unsickled cells = 17 Total number of cells = 27 % reversal of sickling = 63 %

10 mg/ml methanol extract on HbSS blood cells at 120 minutes incubation. Sickled cells = 8 Unsickled cells = 15 Total number of cells = 23 % reversal of sickling = 65 %

10 mg/ml methanol extract on HbSS blood cells at 150 minutesincubation Sickled cells = 8 Unsickled cells = 16 Total number of cells = 24 % reversal of sickling = 67 %

10 mg/ml methanol extract on HbSS blood cells at 180 minutes incubation. Sickled cells = 5 Unsickled cells = 10 Total number of cells = 15 % reversal of sickling = 67 %

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Summary The % reversal of sickling of peripheral cells taken before each concentration of the methanol extract, % reversal of sickling calculated from the sample smears made at different times of incubation from different concentrations of the extract were as shown. Peripheral cells concentration At 0 At 30 At 60 At 90 At 120 At 150 At 180 without the drug min min min min min min min 25 2.5 mg/ml 42 45 47 51 53 57 60 23 5 mg/ml 50 50 56 64 65 66 66 20 10 mg/ml 54 57 59 63 65 67 67

AQUEOUS FRACTION Picralima nitida IN HbSS BLOOD GEONTYPE PERIPHERAL CELLS The sample of the HbSS blood before the treatment with the 2.5 mg/ml concentration of the aqueous fraction of the extract P. nitida. Sickled cells = 18 Unsickled cells = 6 Total number of cells = 24 % reversal of sickling = 25 %

2.5 mg/ml aqoeous fraction of the extract at 0 minutes incubation Sickled cells = 16 Unsickled cells = 7 Total number of cells = 23 % reversal of sickling = 30 % 2.5 mg/ml aqoeous fraction of the extract at 30 minutes incubation. Sickled cells = 12 Unsickled cells = 9 Total number of cells = 21 % reversal of sickling = 43 %

2.5 mg/ml aqoeous fraction of the extract at 60 minutesincubation Sickled cells = 11 Unsickled cells = 9 Total number of cells = 20 % reversal of sickling = 45 %

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2.5 mg/ml aqoeous fraction of the extract at 90 minutesincubation Sickled cells = 11 Unsickled cells = 9 Total number of cells = 20 % reversal of sickling = 45 %

2.5 mg/ml aqoeous fraction of the extract at 120 minutes incubation. Sickled cells = 9 Unsickled cells = 12 Total number of cells = 21 % reversal of sickling = 57 %

2.5 mg/ml aqoeous c fraction of the extract at 150 minutes incubation. Sickled cells = 6 Unsickled cells = 14 Total number of cells = 20 % reversal of sickling = 70 % 2.5 mg/ml aqoeous fraction of the extract at 180 minutes incubation. Sickled cells = 4 Unsickled cells = 18 Total number of cells = 22 % reversal of sickling = 81 %

5mg/ml OF Picralima nitida AQUEOUS FRACTION ON HbSS BLOOD SAMPLE The concentration 5mg/ml of aqoeous fraction of the extract was prepared, incubated at the same 37 oC when mixed and shacked with the washed HbSS blood red cells. Sample smears were taken at 30 minutes interval through 180 minutes.

Peripheral Cells: The sample smear was taken before the treatment of the HbSS blood cells with the 5mg/ml of the aqoeous fraction of the extract. Sickled cells = 20 Unsickled cells = 6 Total number of cells = 26 % reversal of sickling = 23 %

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5mg/ml aqoeous fraction of the extract at 0 minutes incubation. Sickled cells = 17 Unsickled cells = 8 Total number of cells = 25 % reversal of sickling = 32 %

5mg/ml aqoeous fraction of the extract at 30 minutes incubation. Sickled cells = 14 Unsickled cells = 11 Total number of cells = 25 % reversal of sickling = 44 %

5mg/ml aqoeous fraction of the extract at 60 minutes incubation. Sickled cells = 10 Unsickled cells = 14 Total number of cells = 24 % reversal of sickling = 58 % 5mg/ml aqoeous fraction of the extract at 90 minutes incubation. Sickled cells = 9 Unsickled cells = 16 Total number of cells = 25 % reversal of sickling = 64 %

5mg/ml aqoeous fraction of the extract at 120 minutes incubation. Sickled cells = 6 Unsickled cells = 12 Total number of cells = 18 % reversal of sickling = 67 %

5mg/ml aqoeous fraction of the extract at 150 minutes incubation. Sickled cells = 5 Unsickled cells = 13 Total number of cells = 18 % reversal of sickling = 72 %

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5mg/ml aqoeous fraction of the extract at 180 minutes incubation. Sickled cells = 3 Unsickled cells = 15 Total number of cells = 18 % reversal of sickling = 83 % Aqoeous Fraction on HbSS blood 10 mg/ml of Picralima nitida Peripheral cells: The % reversal of sickling reading of sample smear of the washed HbSS blood cells Sickled cells = 16 Unsickled cells = 4 Total number of cells = 20 % reversal of sickling = 20 % A 10 mg/ml aqoeous fraction of the extract was prepared from which 0.5 ml volume was taken and mixed with 0.5 ml washed erythrocytes of the HbSS blood. The mixture was shacked and incubated at 37oC. Smears were made at 30 minutes intervals through 180 minutes. 10 mg/ml aqoeous fraction of the extract at 0 minutes incubation. Sickled cells = 11 Unsickled cells = 9 Total number of cells = 20 % reversal of sickling = 45 %

10 mg/ml aqoeous fraction of the extract at 30 minutes Sickled cells = 10 Unsickled cells = 10 Total number of cells = 20 % reversal of sickling = 50 %

10 mg/ml aqoeous fraction of the extract at 60 minutes incubation Sickled cells = 9 Unsickled cells = 11 Total number of cells = 20 % reversal of sickling = 55 % 10 mg/ml aqoeous fraction extract at 90 minutes incubation.

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Sickled cells = 5 Unsickled cells = 10 Total number of cells = 15 % reversal of sickling = 67 % 10 mg/ml aqoeous fraction of the extract at 120 minutes incubation. Sickled cells = 3 Unsickled cells = 14 Total number of cells = 17 % reversal of sickling = 76 %

10 mg/ml aqoeous fraction of the extract at 150 minutesincubation Sickled cells = 2 Unsickled cells = 12 Total number of cells = 14 % reversal of sickling = 85 %

10 mg/ml aqoeous fraction of the extract at 180 minutes incubation. Sickled cells = 2 Unsickled cells = 12 Total number of cells = 14 % reversal of sickling = 85 %

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Summary In the summary table, the % reversal of sickling from the peripheral cells taken before each concentration of the aqoeous fraction of the extract, % reversal of sickling calculated from the sample smears made at different times of incubation from different concentrations of the extract were as shown below.

Peripheral Concentration At 0 At 30 At 60 At 90 At At At 180 cells without mg/ml min min min min 120 150 min the drug min min 25 2.5 mg/ml 30 43 45 45 57 70 81 23 5 mg/ml 32 44 58 64 67 72 83 20 10 mg/ml 45 50 55 67 76 85 85 Chloroform Extract on SS Genotype A sample smears of the washed HbSS erythrocytes before treatment with 2.5 mg/ml chloroform fraction of of Picralima nitida.

Peripheral cells Sickled cells = 20 Unsickled cells = 6 Total number of cells = 26 % reversal of sickling = 23 %

A2.5 mg/ml of chloroform fraction of the extract was prepared from which a 0.5 ml was taken and mixed with 0.5 ml of washed HbSS erythrocytes. The system was the covered with liquid paraffin and shacked occasionally and incubated at 37oC. Smears were made at intervals of 30 minutes through 180 minutes and % reversal of sickling was calculated.

2.5 mg/ml chloroform fraction of the extract on HbSS blood cells at 0 minute Sickled cells = 18 Unsickled cells = 8 Total number of cells = 26 % reversal of sickling = 31 %

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2.5 mg/ml of chloroform fraction on HbSS blood cells at 30 minutes. Sickled cells = 14 Unsickled cells = 9 Total number of unsickled cells = 23 % reversal of sickling = 37 %

2.5 mg/ml chloroform fraction on HbSS blood cells at 60 minutes Sickled cells = 12 Unsickled cells = 11 Total number of cells = 23 % reversal of sickling = 48%

2.5 mg/ml chloroform fraction on HbSS blood cells at 90 minutes Sickled cells = 9 Unsickled cells = 15 Total number of cells = 24 % reversal of sickling = 62 %

2.5 mg/ml chloroform fraction on HbSS blood cells at 120 minutes Sickled cells = 6 Unsickled cells = 17 Total number of cells = 23 % reversal of sickling = 74 %

2.5 mg/ml of chloroform fractionon HbSS blood cells at 150 minutes Sickled cells = 5 Unsickled cells = 23 % reversal of sickling = 78 %

2.5 mg/ml of chloroform fraction on HbSS blood cells at 180 minutes Sickled cells = 5 Unsickled cells = 18 Total number of cells = 23 % reversal of sickling = 78 % 195

A 5mg/ml of chloroform fraction was prepared. From the prepared concentration a 0.5 ml was mixed with 0.5 ml of the washed HbSS red blood cells. The system was covered with paraffin and subjected to occasional shacking and incubated at 37oC. Smears were made at 30 minutes intervals through 180 minutes from which % reversal of sickling was calculated.

5mg/ml chloroform fraction on HbSS blood cells at 0 minute Sickled cells = 28 Unsickled cells = 20 Total number of cells = 48 % reversal of sickling = 41 %

5mg/ml chloroform fraction on HbSS blood cells at 30 minutes Sickled cells = 26 Unsickled cells = 19 Total number of cells = 45 % reversal of sickling = 42 %

5mg/ml chloroform fraction on HbSS blood cells at 60 minutes Sickled cells = 22 Unsickled cells = 20 Total number of cells = 42 % reversal of sickling = 48 % 5mg/ml chloroform fraction on HbSS blood cells at 90 minutes Sickled cells = 20 Unsickled cells = 24 Total number of cells = 44 % reversal of sickling = 54 %

5 mg/ml chloroform fraction on HbSS blood cells at 120 minutes Sickled cells = 33 Unsickled cells = 44 Total number of cells = 77 % reversal of sickling = 57 %

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5mg/ml chloroform fraction on HbSS blood cells at 150 minutes Sickled cells = 29 Unsickled cells = 38 Total number of cells = 67 % reversal of sickling = 58 %

5mg/ml chloroform fraction on HbSS blood cells at 180 minutes Sickled cells = 8 Unsickled cells = 14 Total number of cells = 22 % reversal of sickling = 64 %

Peripheral blood cells before treatment with chloroform fraction of Picralima nitida 10 mg/ml concentration. Peripheral cells Sickled cells = 18 Unsickled cells = 7 Total number of cells = 25 % reversal of sickling = 28 % The 10 mg/ml concentration of the extract was prepared and a 0.5 ml of it was mixed with a 0.5 ml of washed HbSS red blood cells, the system was covered with liquid paraffin and shacked occasionally. Sample smears were taken at intervals of 30 minutes through 180 minutes from which % reversal of sickling was observed and calculated.

10 mg/ml chloroform fraction on HbSS blood cells at 0 minutes Sickled cells = 13 Unsickled cells = 10 Total number of cells = 23 % reversal of sickling = 43 % 10 mg/ml chloroform fraction on HbSS blood cells at 30 minutes Sickled cells = 11 Unsickled cells = 12 Total number of cells = 23 % reversal of sickling = 52 % 197

10 mg/ml chloroform fraction on HbSS blood cells at 60 minutes Sickled cells = 9 Unsickled cells = 12 Total number of cells = 21 % reversal of sickling = 57 %

10 mg/ml chloroform fraction on HbSS blood cells at 90 minutes. Sickled cells = 10 Unsickled cells = 13 Total number of cells = 23 % reversal of sickling = 57 %

10 mg/ml chloroform fraction on HbSS blood cells at 120 minutes Sickled cells = 10 Unsickled cells = 14 Total number of cells = 24 % reversal of sickling = 58 % 10 mg/ml chloroform fraction on HbSS blood cells at 150 minutes Sickled cells = 9 Unsickled cells = 15 Total number of cells = 24 % reversal of sickling = 63 %

10 mg/ml chloroform fraction on HbSS blood cells at 180 minutes Sickled cells = 9 Unsickled cells = 16 Total number of cells = 25 % reversal of sickling = 64 %.

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Summary The percentages of unsickled cells before the treatment with the chloroform fraction of the extract on HbSS blood at different concentrations, % reversal of sickling from different concentrations at different times of 30 minutes interval were as shown below. Peripheral concentration At 0 At 30 At 60 At 90 At 120 At 150 At 180 cells min min min min min min min without the drug 23 2.5 mg/ml 31 37 48 62 74 78 78 23 5 mg/ml 41 42 48 54 57 58 64 28 10 mg/ml 43 52 57 57 58 63 64

2.5 mg/ml Ethyl acetate fraction on HbSS blood celle. The peripheral cells count was taken to know the number of the sickled cells and unsickled cells and the % reversal of sickling when the ss blood was not incubated in Ethyl Acetate fraction at zero minute.

Peripheral cells Sickled cells = 16 Unsickled cells = 5 Total number of cells = 21 % reversal of sickling = 23.81 taken as 24 %

A 2.5 mg/ml Ethyl Acetate fraction concentration was prepared. Equal volume of 0.5 ml of the washed HbSS red blood cells and the Ethyl Acetate fraction of P. nitida were mixed in a tube. The mixture was covered with paraffin oil and subjected to occasional shacking and incubated at 37 oC. The first reading of the slide was taken when the Ethyl Acetate fraction with concentration 2.5 mg/ml and the ss blood cells are incubated at zero minute.

2.5 mg/ml ethyl acetate fraction on HbSS blood cells at zero minutes Sickled cell = 16 Unsickled cells = 7

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Total number of cells = 23 % reversal of sickling = 30 %

2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 30 minutes incubation. Sickled cells = 14 Unsickled cells = 8 Total number of cells = 22 % reversal of sickling = 36 % 2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 60 minutes incubation. Sickled cells = 13 Unsickled cells = 8 Total number of cells = 21 % reversal of sickling = 38 %

2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 90 minutes incubation. Sickled cells = 14 Unsickled cells = 11 Total number of cells = 25 % reversal of sickling = 44 %

2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 120 minutes incubation. Sickled cells = 10 Unsickled cells = 13 Total number of cells = 23 % reversal of sickling = 57 %

2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 150 minutes lncubation. Sickled cells = 10 Unsickled cells = 15 Total number of cells = 25 % reversal of sickling = 60 %

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2.5 mg/ml ethyl acetate fraction on HbSS blood cells at 180 minutes Sickled cell = 8 Unsickled cells = 18 Total number of cells = 26 % reversal of sickling = 65 %.

Reading of Peripheral blood cells before treatment with 5mg/ml conc Peripheral cells Sickled cells = 13 Unsickled cells = 4 Total number of cell = 17 % reversal of sickling = 24 %

A 5mg/ml concentration of the Ethyl Acetate fraction of the extract was prepared. A 0.5 ml of the washed erythrocytes of the HbSS blood was mixed with the 0.5 ml of the prepared fraction of the extract and incubated, keeping all the conditions. Sample smears and their readings were taken at 30 minutes intervals.

5mg/ml ethyl acetate fraction on HbSS blood cells at Zero minutes Sickled cells = 18 Unsickled crlls = 8 Total number of cells. = 21 % reversal of sickling = 33 %

5mg/ml ethyl acetate fraction on HbSS blood cells at 30 minutes Sickled cells = 14 Unsickled cells = 9 Total number of cells = 23 % reversal of sickling = 39 %

5mg/ml ethyl acetate fraction on HbSS blood cells at 60 minutes Sickled cell = 12 Unsickled cells = 10 201

Total number of cells = 22 % reversal of sickling = 46 %

5mg/ml Ethyl ethyl acetate fraction on HbSS blood cells at 90 minutes Sickled cells = 12 Unsickled cells = 11 Total number of cells = 23 % reversal of sickling = 48 %

5mg/ml ethyl acetate fraction on HbSS blood cells at 120 minutes Sickled cells = 9 Unsickled cell = 13 Total number of cell = 22 % reversal of sickling = 59 %

5mg/ml ethyl acetate fraction on HbSS blood cells at 150 minutes Sickled cells = 9 Usickled cells = 15 Total number of cells = 24 % reversal of sickling = 62.5 5mg/ml ethyl acetate fraction on HbSS blood cells at 180 minutes Sickled cells = 8 Unsickled cells = 15 Total number of cell = 23 % reversal of sickling = 65 %

The readings of peripheral cells of the HbSS red blood before the treatment with 10 mg/ml Ethyl Acetate fraction of the extract. Peripheral cells Sickled cell = 14 Unsickled cells = 4 Total number of cells = 18 % reversal of sickling = 22 %

202

10 mg/ml ethyl acetate fraction on HbSS blood cells at zero minute Sickled cells = 12 Unsickled cell = 7 Total number of cells = 19 % reversal of sickling = 37 %

10 mg/ml ethyl acetate fraction on HbSS blood cells at 30 minutes Sickled cells = 12 Unsickled cells = 9 Total number of cells = 21 % reversal of sickling = 43 %

10 mg/ml ethyl acetate fraction on HbSS blood cells at 60 minutes Sickled cells = 11 Unsickled cell = 10 Total number of cells = 21 % reversal of sickling = 48 %

10 mg/ml ethyl acetate fraction on HbSS blood cells at 90 minutes Sickled cells = 9 Unsickled cells = 11 Total number of cells = 20 % reversal of sickling = 55 %

10 mg/ml ethyl acetate fraction on HbSS blood cells at 120 minutes Sickled cell = 9 Unsickled cell = 14 Total number of cells = 23 % reversal of sickling = 61 %

10 mg/ml ethyl acetate fraction on HbSS blood cells at 150 minutes Sickled cell = 9 Unsickled cell = 17 Total number of cells = 26 % reversal of sickling = 65 %

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10 mg/ml ethyl acetate fraction on HbSS blood cells at 180 minutes incubation. Sickled cells = 8 Unsickled cells = 16 Total number of cells = 24 % reversal of sickling = 67 % Summary The table showed the % reversal of sickling of the peripheral blood smears before the use of each drug concentration, % reversal of sickling obtained from the smears made at different concentrations of the ethyl Acetate fraction and at different times of 30 minutes interval through 180 minutes.

Peripheral concentration At 0 At 30 At 60 At 90 At 120 At 150 At 180 cells min min min min min min min without the drug 24 2.5 mg/ml 30 36 38 44 57 60 65 24 5 mg/ml 33 39 46 48 59 63 65 22 10 mg/ml 36 42 48 55 61 65 67

DICHLOROMETHANE FRACTION IN HbSS GENOTYPE BLOOD The percentage of the unsickled cells of the peripheral blood smear was taken, Peripheral cells Sickled cells = 17 Unsickled cells = 6 Total number of cells = 23 % reversal of sickling = 26 %

The concentration of 2.5 mg/ml of the dichloromethane fraction was made from which a 0.5 ml was taken and mixed with 0.5 ml of washed erythrocytes of HbSS genotype, and was covered with liquid paraffin. The system was shacked occasionally during the incubation at 37 oC. Smears were made from drop of the mixture taken at 30 minutes intervals and the % reversal of sickling were obtained.

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2.5 mg/ml dichloromehane fraction in HbSS blood cells at zero minutes Sickled cells = 15 Unsickled cells = 8 Total number of cells = 23 % reversal of sickling = 25 %

2.5 mg/ml dichloromethane fraction in HbSS blood cell at 30 minutes Sickled cells = 13 Unsickled cells = 10 Total number of cells = 23 % reversal of sickling = 44 %

2.5 mg/ml dichloromethane fraction in HbSS blood cells at 60 minutes Sickled cells = 11 Unsickled cells = 10 Total number of cells = 21 % reversal of sickling = 48 %

2.5 mg/ml dichloromethane fraction in HbSS blood cells at 90 minutes Sickled cells = 11 Unsickled cells = 11 Total number of cells = 22 % reversal of sickling = 50 %

2.5 mg/ml dichloromethane fraction in HbSS blood cells at 120 minutes Sickled cells = 10 Unsickled cells = 12 Total number of celols = 22 % reversal of sickling 55 % 2.5 mg/ml dichloromethane fraction in HbSS blood cells at 150 minutes Sickled cells = 8 Unsickled cells = 13 Total number of cells = 21 % reversal of sickling = 62 % 205

2.5 mg/ml dichloromethane fraction in HbSS blood cells at 180 minutes Sickled cells = 5 Unsickled cells = 17 Total number of cells = 22 % reversal of sickling = 77 %

Before treatment with 5mg/ml dichloromethane fraction in HbSS blood cells Peripherial cells Sickled cells = 19 Unsickled cells = 5 Total number of cells = 24 % reversal of sickling = 21 %

5mg/ml dichloromethane fraction in HbSS blood cells at zero minute Sickled cells = 16 Unsickled cells = 8 Total number of cells = 24 % reversal of sickling = 33 %

5mg/ml dichloromethane fraction in HbSS blood cells at 30 minutes Sickled cells = 14 Unsickled cells = 10 Total number of cells = 24 % reversal of sickling = 42 % 5mg/ml dichloromethane fraction in HbSS blood cells at 60 minutes Sickled cells = 10 Unsickled cells = 15 Total number of cells = 25 % reversal of sickling = 60 %

5mg/ml dichloromethane fraction in HbSS blood cells at 90 minutes Sickled cells = 8 Unsickled cells = 15 Total number of cells = 23 % reversal of sickling = 65 %

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5mg/ml dichloromethane fraction in HbSS blood cells at 120 minutes Sickled cells = 6 Unsickled cells = 17 Total number of cells = 23 % reversal of sickling = 74 %

5mg/ml dichloromethane fraction in HbSS blood cells at 150 minutes Sickled cells = 6 Unsickled cells = 17 Total number of cells = 23 % reversal of sickling = 74 %

5mg/ml dichloromethane fraction in HbSS blood cells at 180 minutes Sickled cells = 4 Unsickled cells = 18 Total number of cells = 22 % reversal of sickling = 82 % Reading obtained before the treatment of the peripheral cells with 10 mg/ml dichloromethane fraction of Picralima nitida. Peripherial cells Sickled cells = 17 Unsickled cells = 5 Total number of cells = 22 % reversal of sickling = 23 %

A 10 mg/ml concentration of dichloromethane fraction was prepare. A 0.5 ml of it was mixed with the washed erythrocytes of HbSS genotype. The system was subjected to occasional shacking and covered with liquid paraffin. Smears were made from drops taken at 30 minutes interval starting from zero time through 180 minutes incubation.

10 mg/ml dichloromethane fraction in HbSS blood cells at zero minute Sickled cells = 15 Unsickled cells = 9 Total number of cells = 24 % reversal of sickling = 38 %

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10 mg/ml dichloromethane fraction in HbSS blood cells at 30 minutes Sickled cells = 13 Unsickled cells = 11 Total number of cells = 24 % reversal of sickling = 4 6%

10 mg/ml dichloromethane fraction in HbSS blood cells at 60 minutes Sickled cells = 9 Unsickled cells = 16 Total number of cells = 25 % reversal of sickling = 64 % 10 mg/ml dichloromethane fraction in HbSS blood cells at 90 minutes Sickled cells = 7 Unsickled cells = 18 Total number of cells = 24 % reversal of sickling = 72 %

10 mg/ml dichloromethane fraction in HbSS blood cells at 120 minutes Sickled cells = 5 Unsickled cells = 17 Total number of cells = 22 % reversal of sickling = 77 %

10 mg/ml dichloromethane fraction in HbSS blood cells at 150 minutes Sickled cells = 4 Unsickled cells = 18 Total number of cells = 22 % reversal of sickling = 82 %

10 mg/ml dichloromethane fraction in HbSS blood cells at 180 minutes Sickled cells = 4 Unsickled cells = 18 Total number of cells = 22 % reversal of sickling = 82%

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Summary % reversal of sickling from the peripheral cells as well as those obtained from the smears made at different concentrations of the fraction and at different time interval of times. Peripheral Concentration At zero At 30 At 60 At 90 At 120 At 150 At 180 min min min min min min min 26 2.5 mg/ml 25 44 48 50 55 62 77 21 5 mg/ml 33 42 60 65 74 74 82 23 10 mg/ml 38 46 64 72 77 82 82

The actions of the positive control and the negative control on HbSS and HbAS blood samples

The table below shows the % reversal of sickling in SS blood cells obtained when a 0.5 ml of SS blood was treated consecutively with 0.5 ml of 5, 50 and 500 mg/ml concentrations of the (positive control) P-Hydroxybenzoic acid at 37 0C incubation and sample smear readings were taken at 30 minutes intervals. Summary: P-Hydroxybenzoic acid (positive control) on SS blood cells concentration 0 min 30 min 60 min 90 min 120 min 150 min 180 min 5 mg/ml 38 41 44 50 56 56 58 50 mg/mg 40 58 60 79 88 88 98

500 mg/ml 45 100 100 100 100 100 100

The table below shows the percentage of unsickled cells obtained when a 0.5 ml of HbAS blood in each case was incubated in (5, 50 and 500 mg/ml) concentrations of P- Hydroxybenzoic acid at 370C and sample smears readings taken at 30 minutes interva

Summary: P-Hydroxybenzoic acid (positive control) on HbAS blood cells concentration 0 min 30 min 60 min 90 min 120 min 150 min 180 min 0.005 g 45 46 46 48 51 52 56 0.05 g 43 48 52 59 62 75 87 0.5 g 42 90 100 100 100 100 100

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Table obtained when 0.5ml of washed HbSS blood cells were treated with a 0.5 ml of Normal saline and incubated at 37 oC taken at 30 minutes intervals

Summary: Normal saline, (the negative control) on HbSS blood cells. s0 min 30 min 60 min 90 min 120 min 150 min 180 min

43 43 43 43 43 43 43

Viscosity (Rheology of the HbAA, HbAS And HbSS Blood Cells) Since the viscosity of distilled water is 1.025 at 25 OC, the viscosity of the blood samples were calculated from the relationship: ηwater twater =ηsample tsample ηwater × twater/ tsample = ηsample It can be represented by ηwt w /ts =ηs Where η = viscosity, t = time, s = sample, w = water

210

The table gave the illustration of the average viscosity of the given three blood samples- the normal blood, the HbAS genotype and the HbSS genotype.

HbAA HbAS Blood HbSS Blood 2.54 poise 2.68 poise 3.04 poise 2.56 poise 2.66 poise 3.06 poise 2.55 poise average Average 2.67 poise Average 3.05 poise

The concentrations of the Methanol extract 2.5 mg/ml 5 mg/ml and 10 mg/ml were prepared and used to treat the HbSS blood. The system was incubated at 37 oC and occasionally shaked. Measurements of viscosity were taken at intervals of 30 minutes as described above.

Effect of Methanol extract of P. nitida on the viscosity of HbSS Blood. G T 0 min T 30 min T 60 min T 90 min T 120 T 150 T 180 min min min 2.5 3.00 2.99 2.86 2.72 2.64 2.60 2.60 mg/ml 5mg/ml 3.00 2.97 2.86 2.70 2.62 2.62 2.60 10 mg/ml 2.99 2.96 2.81 2.66 2.60 2.69 2.60

Effects of extracts of Picralima nitida on the rheology of HbSS genotype Aqoeous Fractions The three concentrations of the aqoeous fraction P. nitida 2.5 mg/ml, 5mg/ml and 10 mg/ml were prepared and used to treat the HbSS blood. The system was incubated at 37oc and occasionally shaked. Measurement of viscosity as was described above, were taken at the end of every 30 minutes through 180 minutes for each concentration. Viscosity at time t zero to t n Mg t o min t 30 min t 60 t 90 min t 120 min t 150 min t 180 mjn min 2.5 3.00 3.00 3.00 2.80 2.80 2.80 2.75 5 3.00 3.00 2.96 2.72 2.66 2.60 2.60 10 2.99 2.96 2.84 2.69 2.60 2.60 2.60

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Methanol Extract The methanol extract was prepared in the three concentrations. The HbSS genotype in three separate tubes was treated with the methanol fraction according to the concentrations. The measurements were taken at intervals of 30 minutes through 180 minutes. Concentrations viscosity at t n Mg/ml t 0 t 30 min t 60 min t 90 min t 120 t 150 min t 180 min 2.5 mg 3.01 3.00 3.00 3.00 2.99 2.97 2.96 5 mg 3.01 3.00 3.00 2.99 2.98 2.90 2.94 10 mg 3.1 3.00 2.98 2.96 2.95 2.94 2.94

The Chloroform Fraction was handled in the similar manner as described above. Concentrations viscosity at t0-tn Mg/ml t 0 t 30 min t 60 min t 90 min t 120 t 150 t 180 min min min 2.5 3.00 3.00 3.00 2.82 2.80 2.80 2.70 5 3.00 3.00 2.96 2.80 2.78 2.70 2.65 10 3.00 3.00 2.90 2.76 2.70 2.60 2.60

Ethyl Acetate Fractions: The same procedure was followed as described above. Viscosity at tn. Mg/ml t 0 min t 30 min t 60 min t 90 min t 120 min t 150 min t 180 min 2.5 3.02 3.01 3.00 3.00 2.98 2.96 2.95 5 3.o1 3.00 3.00 2.98 2.94 2.90 2.91 10 3.00 3.00 2.99 2.95 2.90 2.80 2.80

Dichloromethane Fractions: The same method above was used. Concentrations viscosity at tn. Mg/ml t 0 min t 30 min t 60 min t 90 min t 120 min t 150 min t 180 min 2.5 3.00 3.00 3.00 2.82 2.80 2.80 2.78 5 3.00 3.00 2.98 2.78 2.76 2.66 2.62 10 2.98 2.96 2.86 2.70 2.69 2.62 2.61

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213

214

215

216

217

218

43 43 43 45 50 50 52 100 100

40 46 51 52 57 59 64 80 0.1g 80 0.1g 17 67 100 93 100 100 100 60 0.3g 60 0.3g 0.5g 0.5g 40 40

20 concentration concentration 20 concentration 0 0 time (mins) timetime (mins)(mins) 0 30 60 90 120 150 180 0 30 60 90 120 150 180

Fig.1a: The graph of percentages of unsickled cell when AS Fig.1b: thethe graphgraph obtainedobtained fromfrom thethe percentagespercentages ofof unsickledunsickled cells whenwhen blood was treated with Ethanol Fraction of P. nitida: treatedtreated withwith EthanolEthanol fractionfraction ofof P.P. nitidanitida

120 0 30 60 90 120 150 180 120 100 44 45 47 48 50 51 54 100 80 42 44 51 53 57 59 65 80 60 65 65 95 100 100 100 100 60 0.1g 40 0.1g 0.3g 40

concentration 20 0.3g

0.5g concentration 20 0.5g 0 0 0 30 60 90 120 150 180 time (mins) 0 30 60 90 120 150 180 time (mins) Fig.2a:The graph of percentages of unsickled cells when AS blood was treated with Chloroform Fraction Fig.2b: The graph obtained from the percentages of unsickled cells of P. nitida when AS blood was treated with Chloroform fraction of P. nitida

0 30 60 90 120 150 180 42 46 45 44 40 49 53 120 120 41 43 47 53 56 57 60 33 65 88 89 89 100 82 100 100 80 0.1g 80 0.1g 60 0.3g 60 0.3g 40 0.5g 40 0.5g concentration 20 concentration 20 0 0 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Fig.3a: The graph of percentages of unsickled cellstime (mins)when time (mins) AS blood was treated with Methanol Fraction of P. nitida Fig.3b: The graph obtained from the percentages of unsickled cells when AS blood was treated with Methanol Fraction of P. nitida

120 120 100 100 0 30 60 90 120 150 180 80 80 36 35 42 40 48 50 56 0.1g 60 0.1g 60 77 45 41 50 53 66 58 0.3g 0.3g 40 40 0.5g 36 52 49 100 87 96 100 0.5g concentration concentration 20 20 0 0 0 30 60 90 120 150 180 0 30 60 90 120 150 180 time (mins) time (mins) Fig.4a: The graph of percentages of unsickled cells when AS blood was treated with Eyhyl Acetate Fraction Fig.4b: The graph of percentages of unsickled cells when ASblood of P. nitida was treated with Ethyl Acetate Fraction of P. nitida

120 120 30 60 90 120 150 180 100 100 46 27 35 46 49 53 80 45 43 48 60 61 59 80 60 0.1g 53 70 90 91 100 100 60 40 0.3g 0.1g

concentration 0.5g 40 0.3g

20 concentration 0.5g 20 0 0 30 60 90 120 150 180 time (mins) 0 0 30 60 90 120 150 180 Fig.5a:The graph of percentages of unsickled cells time (mins) when AS blood was treated with Dichloromethane Fraction of P. nitida Fig.5b: The graph of percentages of unsickled cells when AS blood was treated with Dichloromethane Fraction of P. nitida

100 100 90 90 0 30 60 90 120 150 180 80 80 30 43 45 45 57 70 81 70 70 60 60 32 44 58 64 67 72 83 0.1g 0.1g 45 50 55 67 76 85 85 50 50 0.3g 0.3g 40 40 30 0.5g 30 0.5g concentration concentration 20 20 10 10 0 0 0 Fig.6a:30 The60 graph90 of percentages120 150 of unsickled180 time (mins) cells 0 30 60 90 120 150 180 time (mins) when SS blood was treated with Ethanol Fraction of Fig.6b: The graph of percentages of unsickled cells when SS blood P. nitida. was treated with Ethanol Fraction of P. nitida.

90 9090 0 30 60 90 120 150 180 80 8080 39 43 47 53 62 63 64 70 7070 42 46 50 56 65 67 67 60 6060 43 52 57 59 62 65 67 50 0.1g 5050 0.1g0.1g 40 0.3g 4040 0.3g0.3g 30 0.5g 3030 0.5g0.5g concentration concentration concentration 20 2020 10 1010 0 00 0 Fig.7a:30 The60 graph90 of percentages120 150 of unsickled180 time cells(mins) 0 3030 6060 9090 120120 150 180180 timetime (mins)(mins) when SS blood was treated with Methanol Fraction of Fig.7b:Fig.7b: TheThe graphgraph ofof percentagespercentages ofof unsickledunsickled cells whenwhen SSSS bloodblood P. nitida waswas treatedtreated withwith MethanolMethanol Fraction ofof P. nitidanitida

90 0 30 60 90 120 150 180 90 80 31 37 48 62 74 78 78 80 70 41 42 48 54 57 58 64 70 43 52 57 57 58 63 64 60 60 50 50 0.1g 0.1g 40 40 0.3g 0.3g 30 30 0.5g

0.5g concentration concentration 20 20 10 10 0 0 time (mins) 0 30 60 90 120 150 180 time (mins) 0 Fig.8a:30 The60 graph of90 percentages120 of150 unsickled180 cells when SS blood was treated with Chloroform Fraction of P. nitida Fig.8b: The graph of percentages of unsickled cells when SS blood was treated with Chloroform Fraction of P. nitida.

80 80 70 70 0 30 60 90 120 150 180 60 60 30 36 38 44 57 60 65 50 50 33 39 46 48 59 63 65 0.1g 36 42 48 55 61 65 67 0.1g 40 40 0.3g 0.3g 30 30 0.5g 0.5g concentration 20 20concentration 10 10 0 0 0 30 60 90 120 150 180 time (mins) 0 Fig.9a:The30 60 graph90 of percentages120 150 of unsickled180 time cells (mins) Fig.9b: The graph of percentages of unsickled cells when SS blood when SS bloodFig.9a: was treated with Ethyl Acetate Fraction Fig.9b: of P. nitida was treated with Ethyl Acetate Fraction of P. nitida

100 100 90 90 80 80 0 30 60 90 120 150 180 70 70 25 44 48 50 55 62 77 60 60 33 42 60 65 74 74 82 50 0.1g 50 0.1g 38 46 64 72 77 82 82 40 0.3g 40 0.3g 30 0.5g

concentration 30 0.5g concentration 20 20 10 10 0 time (mins) 0 0 Fig.10a:30 The60 graph90 of percentages120 150 of unsickled180 cells 0 30 60 90 120 150 180 time (mins) when SS blood was treated with Dichloromethane Fig.10b:The graph of the percentages of unsickled cells when SS Fig.10a: Fig.10b: Fraction of P. nitida blood was treated with Dichloromethane Fraction of P. nitida

3.2 3.2 3.1 3.1 3 3 2.9 2.9 0 30 60 90 120 150 180 2.8 2.8 0.1g 0.1g 3 3 3 2.8 2.8 2.8 2.75 2.7 2.7 3 3 2.96 2.72 2.66 2.6 2.6 0.3g 0.3g 2.6 2.6 2.99 2.96 2.84 2.69 2.6 2.6 2.6 0.5g 0.5g concentration concentration 2.5 2.5 2.4 2.4 2.3 2.3 time (mins) time (mins) 0 Fig.11a:30 60Histogram90 of viscosity120 150of SS blood180 when 0 30 60 90 120 150 180 incubated with concentrations of Ethanol Fraction of Fig.11b The graph of viscosity when SS blood was incubated with Fig.11a: Fig.11b: P.nitida through 180 minutes concentrations of Ethanol fraction of P. nitida through 180 min

3.1 3.1 0 30 60 90 120 150 180 3.05 3.05 3.01 3 3 3 2.99 2.97 2.96 3 3 3.01 3 3 2.99 2.98 2.9 2.94 2.95 2.95 3.01 3 3 2.99 2.96 2.88 2.8 2.9 219 2.9 0.1g 0.1g 2.85 0.3g 2.85 0.3g 2.8 0.5g 2.8 0.5g 2.75concentration concentration 2.75 2.7 2.7 2.65 2.65 time (mins) time (mins) 0 Fig.12a:30 60 Histogram90 of viscosity120 150 of SS blood180 when 0 30 60 90 120 150 180 incubated with concentrations of Methanol Fraction of P. Fig.12b:Graph of viscosity when SS blood was incubated with Fig.12a: Fig.12b: nitida through 180 minutes concentrations of Methanol fraction of P .nitida through 180 min

3.2 3.2