ABSTRACT
Malaria is the world’s most important parasitic disease especially when Plasmodium falciparum is the cause of most of the mortality and morbidity. Malaria has been challenging human health and losing the lives of many people since long period of time. Malaria has been one of the most extensively studied parasitic infectious diseases for millennia and the recent failure of chemotherapy through parasite resistance to drugs and alternative therapy is paramount. The present study was aimed at evaluating the effect of ethanol extracts of vernonia amygdalina leaf in cerebral and cerebellar cortices of young mice inoculated with plasmodium berghei. Sixty (60) young mice aged 5-7 weeks, weighing between 10-15g were divided into three categories in which category A, B, C contain twenty mice each which represent the Suppressive, Curative, and the Prophylactic design. Each category contains five groups with four mice in each group. In all the categories each Group 1 was administered distilled water, Group 2 was inoculated with P. berghei parasite, Group 3 was treated with P. berghei + ELVA 2500mg/kg, Group 4 was treated with P. berghei + ELVA 1250mg/kg and Group 5 was treated with P. berghei + 10mg/kg of Chloroquine respectively where ELVA is Ethanol Leaf extract of Vernonia amygdalina. In the Suppressive design the mice were inoculated with P. berghei parasite three hours after then parasite load is quantified follow by treatment with ELVA and on the 4th day parasite quantification and sacrifice is done. In the Curative design the mice were inoculated with P. berghei parasite 48 hours after then parasitemia level was taken follow by treatment with ELVA, on the 7th day parasite quantification and sacrifice was carried out. In the prophylactic design the mice were treated with ELVA for 72 hours thereafter inoculation with P. berghei parasite was carried out with parasite quantification noted up till the 8th day before sacrifice. The mice were sacrificed and brain tissues were removed and fixed in Bouin’s fluid, processed for histopathological studies using Haematoxyline
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and Eosin (H and E), and Cresyl Echt Violet stains. The result for the suppressive design indicates that the mean percentage parasitemia in group 3 mice treated P. berghei + ELVA 2500mg/kg (2.84±0.10) was significantly lower (P˂0.001) compare to Group 2 (4.44±0.16), group 4 (3.28±0.12) and group 5 (3.34±0.13). The result from the curative design shows that the parasitemia level of the mice in group 3 (2.48±0.22) was significantly lower compare to Group 2 (4.34±0.57) and group 4 (3.76±0.35) group 5 (2.64±0.10) with a (P value) of 0.015. The ethanol leaf extract of V. amgydalina exhibited different degrees of suppression at the doses (1250 and 2500) mg/kg and Pyrimethamine 1.2mg/kg which were (72.7, p<0.057; 61, p<0.057 and 76.2%, p<0.057) respectively as compared to normal control group. For the mean calculation of the level of parasitemia the result indicates that there is no significant difference observed in the group. In the suppressive and curative design, the histopathological changes in cerebral and cerebellar cortices of the young mice inoculated with P. berghei parasites and the effects were ameliorated when treated with ELVA when compared with the control. However, in the prophylactic design ELVA doesn’t not ameliorate the effects of the inoculated parasite when compare to the suppressive and curative. Thus, the present study has established that ethanol extract of Vernonia amygdalina leaf at doses of 2500 mg/kg and 1250 mg/kg was able to ameliorate the effects of Malaria parasite inoculated and may justify the local usage by the traditionalist and the mass population in endemic areas that are exposed to the malaria parasite.
TABLE OF CONTENTS
Cover page ……………………………………………………………………………………………………………. i
Fly leaf ………………………………………………………………………………………………………………… ii
Title page …………………………………………………………………………………………………………… iii
DECLARATION …………………………………………………………………………………………………. iv
CERTIFICATION ………………………………………………………………………………………………… v
DEDICATION …………………………………………………………………………………………………….. vi
ACKNOWLEDGEMENTS ………………………………………………………………………………….. vii
TABLE OF CONTENTS ……………………………………………………………………………………. viii
LIST OF TABLES ………………………………………………………………………………………………. xii
LIST OF FIGURES …………………………………………………………………………………………… xiii
LIST OF PLATES ……………………………………………………………………………………………… xiv
ABSTRACT ……………………………………………………………………………………………………… xxii
CHAPTER ONE …………………………………………………………………………………………………… 1
1.0 INTRODUCTION …………………………………………………………………………………………… 1
1.1 Background of the Study ………………………………………………………………………………….. 1
1.2 Medicinal Plants………………………………………………………………………………………………. 3
1.3 Statement of Research Problem …………………………………………………………………………. 4
1.4. Significance of the Study …………………………………………………………………………………. 4
1.5 Justification of the Study ………………………………………………………………………………….. 5
1.6 Scope of the Study …………………………………………………………………………………………… 5
1.7 Aim and Objectives of the Study ……………………………………………………………………….. 6
1.7.1 Aim of the study ……………………………………………………………………………………………………. 6
1.7.2 Objectives of the study ………………………………………………………………………………………….. 6
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1.8 Research Hypothesis ………………………………………………………………………………………… 6
CHAPTER TWO ………………………………………………………………………………………………….. 7
2.0 LITERATURE REVIEW …………………………………………………………………………………. 7
2.1 Related Literature…………………………………………………………………………………………….. 7
2.2 Traditional Medicine and Anti-Malarial Drugs from Medicinal Plants …………………… 9
2.3 Description of the Study Plant …………………………………………………………………………. 11
2.4 Historical Background ……………………………………………………………………………………. 13
2.5 The Global Burden of Malaria …………………………………………………………………………. 15
2.6 Life Cycle of Malaria ……………………………………………………………………………………… 18
2.7 Cerebellum ……………………………………………………………………………………………………. 23
2.7.1 Functions of the cerebellum ………………………………………………………………………………… 23
2.7.2 Morphology of the cerebellum …………………………………………………………………………….. 24
2.7.3 Cellular layers of the cerebellum …………………………………………………………………………. 24
2.8 Cerebrum………………………………………………………………………………………………………. 28
2.8.1 The morphology of the cerebrum ………………………………………………………………………… 28
2.8.2 Anatomy frontal lobe …………………………………………………………………………………………… 29
2.8.3 Cellular layers of the cerebral cortex……………………………………………………………………. 31
2.8.4 Variations of cortical structure …………………………………………………………………………….. 35
CHAPTER THREE …………………………………………………………………………………………….. 37
3.0 MATERIALS AND METHODS ……………………………………………………………………… 37
3.1 Materials ………………………………………………………………………………………………………. 37
3.2 Plant Collection and Extraction ……………………………………………………………………….. 37
3.3 Ethanol Extract Preparation …………………………………………………………………………….. 37
3.4 In Vivo Acute Toxicity Tests …………………………………………………………………………… 38
3.5 Experimental Animals Preparation …………………………………………………………………… 38
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3.6 In Vivo Evaluation of the Anti-Malarial Activity of Plant Extracts ………………………. 39
3.6.1 Four-day suppressive test …………………………………………………………………………………….. 39
3.7 Animal Sacrifice ……………………………………………………………………………………………. 46
3.8 Tissue Processing …………………………………………………………………………………………… 48
3.8.1 Haematoxylin and eosin (H and E) staining method …………………………………………….. 48
3.8.2 Cresyl Echt Violet method …………………………………………………………………………………… 48
3.9 Statistical Analyses ………………………………………………………………………………………… 49
3.10 Ethical Consideration ……………………………………………………………………………………. 49
CHAPTER FOUR ……………………………………………………………………………………………….. 50
4.0 RESULTS …………………………………………………………………………………………………….. 50
4.1 Acute Toxicity Test for the Plant Material (Vernonia amygdalina) ………………………. 50
4.2 Physical Observations …………………………………………………………………………………….. 52
4.3 Four Day Suppressive Test ……………………………………………………………………………… 52
4.3.1 Percentage parasitaemia and suppression on a four day suppressive test ……………… 52
4.3.2 Body weight expression on the fourth day of suppressive test ……………………………… 54
4.3.3 Packed cell volume determination on 4 day suppressive test ……………………………….. 56
4.3.4 Temperature of the mice before, during and after the treatment with Vernonia amygdalina ………………………………………………………………………………………………………. 58
4.4 Seven-Day Curative Test (Rane’s test) ……………………………………………………………… 60
4.4.1 Body weight determination of the mice in the curative test ………………………………….. 62
4.4.2 Packed cell volume determination on a seven day curative test ……………………………. 64
4.4.3 Temperature of the pups before, during and after the treatment with Vernonia amygdalina ………………………………………………………………………………………………………. 66
4.5 Prophylactic Test (Repository Test) …………………………………………………………………. 68
4.5.1 Body Weight Determination on Prophylactic Test ……………………………………………….. 70
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4.5.2 PCV Determination on Day 5 and Day 8 of the Prophylactic Test ……………………….. 72
4.5.3 Temperature of the mice before, during and after the treatment with Vernonia amygdalina ………………………………………………………………………………………………………. 74
4.6 Histological Studies ……………………………………………………………………………………….. 76
4.6.1 Histopathological studies of cerebral cortex on a suppressive test, curative test and prophylactic test ……………………………………………………………………………………………….. 76
4.6.2 Histopathological studies of cerebellar cortex on a suppressive test, curative test and prophylactic test ……………………………………………………………………………………………….. 92
CHAPTER FIVE ………………………………………………………………………………………………. 109
5.0 DISCUSSION ……………………………………………………………………………………………… 109
CHAPTER SIX …………………………………………………………………………………………………. 113
6.0 CONCLUSION, RECOMMENDATIONS AND CONTRIBUTION TO KNOWLEDGE ………………………………………………………………………………………. 113
6.1 Conclusion ………………………………………………………………………………………………….. 113
6.2 Recommendations ………………………………………………………………………………………… 114
6.3 Contribution to Knowledge……………………………………………………………………………. 114
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
Malaria is the world’s most important parasitic disease especially when Plasmodium falciparum is the cause of most of the mortality and morbidity (Cropper et al., 2004). Malaria has been challenging human health and losing the lives of many people since long period of time. It is one of the most prevalent; devastating parasitic infectious diseases in the world (Moss et al., 2008). Malaria has been one of the most extensively studied parasitic infectious diseases for millennia. In 2012, there were around 627,000 malaria deaths worldwide, 90% of which were in the African region, followed by Southeast Asia (7%) and the Eastern Mediterranean (3%) (WHO, 2013). Most of these deaths were due to Plasmodium falciparum. However, Plasmodium vivax is now increasingly recognized as a cause of severe malaria and death (WHO, 2013b).
Malaria infections are characterized by defective immune responses with poor efficacy against infection and in some cases with immunopathology (Mackintosh and Beesonk 2004). Severe malaria is a complex multisystem disorder with complications such as cerebral malaria, anaemia, acidosis, jaundice, respiratory distress, renal insufficiency, coagulation anomalies and hyperparasitemia (Penet et al., 2005).
Most cases of endemic P. falciparum malaria remain uncomplicated; however, some develop a number of severe complications including cerebral malaria, severe anaemia and placental malaria. Severe malaria occurs when the parasite reaches the erythrocytic phase of the infection and starts to proliferate inside the erythrocytes. A characteristic feature of P. falciparum malaria is the ability of the parasite infected red blood cells (pRBCs) to
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adhere to host endothelium (cytoadherence) and to non-infected erythrocytes (rosetting). These binding events eventually lead to the occlusion of the microvasculature in various tissues and organs, such as the brain in cerebral malaria (Jerrard et al., 2002) hence contributing directly to the pathogenesis of severe malaria disease. A number of host molecules have been implicated as receptors for parasite adhesion and P. falciparum erythrocyte membrane protein 1 (PfEMP1) has been suggested as the key adhesive ligand of pRBCs.
Approximately 300,000 foetal and infant’s deaths and 2,500 deaths of pregnant women are attributable to malaria in the endemic areas (Brabin et al., 1990). Both mother and foetus are at risk of poor outcome with pregnancy associated malaria and complications like premature delivery, anaemia, abortion, intrauterine growth retardation, low birth weight and perinatal and maternal mortality are well (Brabin et al., 1991 and Menendez 1995) known. The frequency and severity of malaria is increased (Brabin et al., 1990) during pregnancy. Pregnancy complicates the clinical course, diagnosis, and treatment of malaria. Pregnancy is associated with downregulation of maternal immune responses, to protect the foetus from rejection. This altered immunity explains, in part, the association of pregnancy with more severe malaria (Abrams et al., 2009). Anaemia is more pronounced in pregnant patients due to haemolysis from rupture of infected erythrocytes exacerbated by erythrocyte sequestration in the spleen and liver, as well as iron deficiency (Steketee et al., 2001; Jerrard et al., 2002; Desai and Steketee, 2003; Danquah et al., 2008). Thrombocytopenia, another key hematologic feature of malaria, is more common in the pregnant patient (Jerrrd et al., 2002; Trampuz et al., 2003; Greenbaum et al., 2009). Hypoglycemia is more commonly seen in the pregnant patient with malaria, due to increased consumption by host and parasite, as well as glycogen depletion and impaired gluconeogenesis (Nayak et al., 2009). This may be exacerbated by drug treatments for malaria (Whitty et al., 2005). Plasmodium
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parasites uniquely sequester in the placenta in the case of falciparum malaria, contributing to miscarriage, premature labour, and low-birth-weight infants (Jerrard et al., 2002; Whitty et al., 2005; Mali et al., 2007; Abrams et al., 2009).
1.2 Medicinal Plants
Medicinal plants have formed the basis of healthcare worldwide. Hence, the use of plant derived natural products as part of herbal preparations for alternative sources of medicine continues to play a major role in chemotherapy especially in third world countries (Joy et al., 1998). Plants by means of secondary metabolism contain a variety of herbal and non-herbal ingredients that can ameliorate a disease condition by acting on a variety of targets in host organism. Vernonia amygdalina have been known and identified by many researchers to possess herbal ingredients for the treatment of malaria parasite infection and have also been studied for their effect on hematology (Akinnuga et al., 2011; Oyedeji et al., 2013).
Vernonia amygdalina is commonly known as bitter leaf which is a shrub or small tree of 2-5m belonging to the family; “Asteraceae”. The leaves are green with characteristic odour and a bitter taste (Singha et al., 1996). The bitter taste is due to anti-nutritional factors such as alkaloids, saponins, tannins and glycosides (Ologunde et al., 1999). Vernonia amygdalina produces no seeds. Hence, they are distributed through cutting (Tende et al., 2013). It is known as “Ewuro” in Yoruba, “Etidot” in Ibibio, “Onugbu” in Igbo and “Chusa”in Hausa tribes of Nigeria (Egedigwe et al., 2010). The leaves are mostly used as vegetables for preparation of soup to stimulate the digestive system and also used for treatment of fever in South Southern and South Eastern Nigeria. It contains a wide variety of phytochemicals such as oxalates, phytates and tannins (Eleyimi et al., 2008), as well as high amount of flavonoid (Igile et al., 1994). It also contains major minerals like calcium,
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potassium, magnesium (Mensah et al., 2008), and vitamins A, C and E, as well as niacin, riboflavin, thiamine (Atangwho et al., 2009). It grows under a range of ecological zones in Africa and produces large mass of foliage and is tolerant to drought (Bonsi et al., 1995).
1.3 Statement of Research Problem
Malaria is the world most important parasitic infection (Hay et al., 2004) and it remains a major impediment to health in Africa, south of the Sahara (Snow, 1999). It is reported to be a major cause of morbidity and mortality amongst Nigeria’s children population (Ayoola et al., 2005). Plasmodium falciparum is responsible for 13–28% of deaths in children under 5 years of age (Tulu et al., 1993). It is estimated that 1-2 million people die yearly as a result of malaria (Sudhanshu et al., 2003). Efforts to reduce the high malaria morbidity and mortality rates have been hampered by the development to resistance, particularly by falciparum to long-standing conventional drugs such as chloroquine (Okoyeh et al., 1993; Ejov et al., 1999; Wongsrichanalai et al., 2002).
Nigeria has the highest prevalence of malarial cases in Africa (WHO, 2008a) as transmission of the disease occur all year round in the southern part of the country while in the northern part, the disease is more seasonal mostly occurring during the rainy season. As reported by (Davidson et al., 2000), pregnant women, their unborn foetus and children below the age of 5 years are more vulnerable to malaria which serves as the major cause of maternal and infant anaemia.
1.4. Significance of the Study
Of all populations at risk of acquiring malarial disease, children 5 years or younger in Sub-Saharan Africa are most susceptible to developing malaria and the severe form (Cerebral Malaria CM) (Dorovini-Zis et al., 2011) as a manifestation of clinical malaria. Due to
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immunological factors children under five years and pregnant women have a higher risk for both uncomplicated and severe malaria. In high transmission areas the risk of severe Falciparum malaria developing is greatest among young children. (WHO, 2011). Hence this study will provide more information on the established potency or therapeutic value of ethanol extract of Vernonia amygdalina on young children malaria.
1.5 Justification of the Study
Despite the intense efforts made by the research community and the Global Eradication program (Khadjavi et al., 2010), no effective vaccines or adjuvant therapies are available for malaria. There are evidences that Vernonia amgydalina has benefitting effects on induced malaria in mice but little is known of its effects on the nervous system of under aged mice i.e. young mice. The study seeks to provide information on administered effects of Vernonia amgydalina on the cerebral and cerebellar cortices in young ones. The study outcome could serve as the basis on which further studies could be carried out to broaden the understanding of paediatric malaria management.
1.6 Scope of the Study
The study was focused on the parasitic load indices and histology of the cerebrum and cerebellum as a result of young mice exposure to Plasmodium berghei malaria parasite. Haematoxylin and Eosin (H and E) and Cresyl Echt Violet was employed in the study of the tissues.
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1.7 Aim and Objectives of the Study
1.7.1 Aim of the study
To study the effect of Ethanol leaf extract of Vernonia amgydalina on cerebral and cerebellar cortices in young mice inoculated with Plasmodium berghei parasite.
1.7.2 Objectives of the study
The objectives of this study were to:
i. Determine the suppressive effects of the ethanol leaf extract of Vernonia amygdalina against Plasmodium berghei inoculated in young mice.
ii. Determine the curative effects of the ethanol leaf extract of Vernonia amygdalina against Plasmodium berghei inoculated in young mice.
iii. Determine the prophylactic effects of the ethanol leaf extract of Vernonia amygdalina against Plasmodium berghei inoculated in young mice.
iv. Investigate the histological changes in cerebral and cerebellar cortices of the Plasmodium berghei inoculated young mice.
1.8 Research Hypothesis
Ethanol leaf extract of Vernonia amygdalina have an in vivo modulatory effect on induced experimental malaria in young mice.
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