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AND Ocimum Gratissimum Linn (Lamiaceae) in CYCLOPHOSPHAMIDE INDUCED UROTOXICITY and MYELOSUPPRESSION

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AND Ocimum Gratissimum Linn (Lamiaceae) in CYCLOPHOSPHAMIDE INDUCED UROTOXICITY and MYELOSUPPRESSION PROTECTIVE ROLE OF AQUEOUS LEAF EXTRACT OF Vernonia amygdalina Del. (Asteraceae) AND Ocimum gratissimum Linn (Lamiaceae) IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY AND MYELOSUPPRESSION BY IKEH, CHIBUEZE PG/MSC/09/51359 A RESEARCH SUBMITTED IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (M. Sc) DEGREE IN PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. FEBRUARY, 2013. 1 CERTIFICATION PAGE PROTECTIVE ROLE OF AQUEOUS LEAF EXTRACT OF Vernonia amygdalina Del. (Asteraceae) AND Ocimum gratissimum Linn (Lamiaceae) IN CYCLOPHOSPHAMIDE INDUCED UROTOXICITY AND MYELOSUPPRESSION BY IKEH, CHIBUEZE PG/MSC/09/51359 A RESEARCH SUBMITTED IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (M. Sc) DEGREE IN PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. CERTIFIED BY ------------------------------ --------------------------------- DR. A. C. EZIKE PROF P. A. AKAH (PROJECT SUPERVISOR) (PROJECT SUPERVISOR) ------------------------------------- DR. T. C. OKOYE (Ag. HEAD OF DEPARTMENT) 2 DEDICATION This work is dedicated to all patients currently suffering from various human malignancies and metastasizing cancers throughout the globe. I believe this work has provided yet another convenient solution to alleviate their suffering. 3 ACKNOWLEDGEMENT Without any reservation, I wish to express my warmest appreciation to the Almighty God for His eternal mercies and guidance throughout the course of this work without whom it wouldn’t have seen the light of the day. With utmost sincerity, many thanks to my supervisors Dr. (Mrs.) A. C. Ezike and Prof P.A Akah for their invaluable contributions; May God continue to bless you abundantly. I will forever be indebted to my God given parents Rev. Emmanuel and Mrs. Abigail Ike whose kindness and foundational education they provided me with has seen me through in the course of life’s journey. I will not fail to appreciate my lovely wife Mrs. Patricia E. Ikeh for unalloyed contribution in running the biochemical assay with high proficiency as a chemical pathologist. To my humble self, I say “well done” for also taking the pain to type and edit this work effectively. 4 ABSTRACT Cyclophosphamide (CP) is one of the most potent and widely used alkylating anticancer agents. Urotoxicity and myelosuppression is known as the most prevailing dose-limiting toxicity associated with CP. In the present study, the protective potential of Vernonia amygdalina and Ocimum gratissimum aqueous leaf extracts in CP-induced urotoxicity and myelosupression were evaluated using biochemical and histopathological approaches. Sodium -2-macarptoethane sulfonate (MESNA) was used as a positive control. Forty (40) male Sprague-Dawley outbred albino rats weighing between 130 g – 200 g were randomly separated into eight different groups (n=5). Rats in group 1 received only normal saline orally for gavage for ten consecutive days. Animals in group two were injected with CP only on the tenth day intraperitoneally (i.p) at 200 mg/kg body weight. Animals in group 3 were given MESNA (67 mg/kg) and CP (200 mg/kg) i.p on the tenth day at 5 minutes interval. Rats in groups 4 and 5 received two different doses of O. gratissimum orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group 6 and 7 received different doses of V. amygdalina orally by gavage at 250 mg/kg and 500 mg/kg respectively for ten consecutive days before administering CP (200 mg/kg) on the tenth day. Rats in group (8) received combination of V. amygdalina and O. gratissimum at a dose of 250 mg/kg each for ten consecutive before administering CP (200 mg/kg) on the tenth day. Results showed that the extract of V. amygdalina protected significantly (P < 0.05) the urothelium and the myeloid system as observed in the biochemical and hematological parameters evaluated. This protection is comparable to MESNA, but MESNA protection was not adequate to prevent myelosupression as observed with V. amygdalina. O. gratissimum did not show significant protection of the urothelium and myeloid system. The protective effects of V. amygdalina was further evident through decreased histopathological alteration of the urinary bladder, kidney and liver tissues unlike the CP and O. gratissimum treated groups. The result of the present study revealed that aqueous leaf extract of V. amygdalina has the potential to prevent urotoxicity and myelosuppression induced by CP and thus can be used as therapeutic adjuvant in the management of CP and other oxazaphosphorine toxicities. 5 TABLE OF CONTENT Title page i Certification page ii Dedication iii Acknowledgement iv Abstract v Table of content vi Tables and figures vii CHAPTER ONE 1 1.0 Introduction 1 1.1 Pharmacology of cyclophosphamide 2 1.1.1 Pharmacodynamics/mechanism of action of cyclophosphamide 3 1.1.2 Pharmacokinetic profile of cyclophosphamide 4 1.1.3 Pharmacokinetic variability 9 1.1.4 Therapeutic uses 12 1.1.5 Drug Interaction 14 1.2.0 Mechanism of cyclophosphamide toxicity 15 1.2.1 Outcomes of cyclophosphamide toxicity 16 1.2.2 Pathophysiology and consequences of urotoxic effects of cyclophosphamide 17 1.2.3 Pathophysiology and consequences of myelosuppressive effects of cyclophosphamide 20 1.3 Chemoprevention and amelioration of cyclophosphamide -induced toxicities 24 1.3.1 The Role of MESNA (sodium 2-mercaptoethane sulfonate) in 6 amelioration of cyclophosphamide -induced toxicities 27 1.3.2 Mechanism of action of MESNA 27 1.3.3 Other potential uroprotective agents 29 1.3.4. Medicinal plants used in preventing or ameliorating cyclophosphamide-induced toxicity 29 1.4. Botanical profile of Vernonia amygdalina Del 39 1.4.1 Taxonomy 39 1.4.2 Description 40 1.4.3 Geographical Distribution 40 1.4.4 Ethnomedicinal Uses 40 1.4.5 Documented research findings on V. amygdalina 40 1.5. Botanical profile of Ocimum gratissimum Linn. 45 1.5.1 Taxonomy 45 1.5.2 Description 46 1.5.3 Geographical Distribution 46 1.5.4. Ethnomedicinal uses. 46 1.5.5 Documented research findings on O. gratissimum. 47 1.6 Aim of the Study 48 CHAPTER TWO 49 2.0 Materials and Methods 49 2.1 Animals 49 2.2 Drugs and Chemicals 49 2.3 Preparation of extracts 50 2.4 Induction of cyclophosphamide -induced toxicity 50 2.5 Blood sample collection 51 7 2.6 Haematological test 52 2.7 Biochemical analysis 52 2.7.1 Analysis of glutathione (GSH) 52 2.7.2 Superoxide dismutase analysis 52 2.7.3 Catalase assay 53 2.7.4 Malondialdehyde (MDA) Assay 53 2.8 Histopathological Examination 53 2.9 Statistical Analysis 53 CHAPTER THREE 54 3.0 Results 54 3.1. Effects of extracts on cyclophosphamide-induced urotoxicity 54 3.1.1 Effects of extract on glutathione 54 3.1.2 Effects of extracts on Superoxide dismutase analysis activity. 54 3.1.3 Effects on catalase activity 54 3.1.4 Effects of extracts lipid peroxidation (LPO) 55 3.2. Effects of extract on cyclophosphamide -induced myelosupression. 55 3.2.1. Effect on total red blood cell count, total Leucocyte count, absolute neutrophil and absolute lymphocyte counts. 55 3.2.2 Effect on platelet count, absolute basophils and eosinophils 56 3.3 Effect on histopathology of tissues 60 CHAPTER 4 66 4.0 Discussion 66 4.1 Conclusion 71 References 72 8 List of Tables Table 1: Normal reference values of blood cells in adult humans and male rats 23 Table 2: Some plants documented to have ameliorating properties in cylophosphamide-induced toxicity. 38 Table 3: Phytochemical constituents of V. amygdalina. 42 Table 4: Documented pharmacological properties of V. amygdalina. 43 Table 5: Effects of O. gratissimum and V. amygdalina compared to MESNA on glutathione, superoxide dismutase and catalase activities in cyclophosphamide-induced toxicity. 57 Table 6: Effects of O. gratissimum and V. amygdalina compared to MESNA on lipid peroxidation (LPO) induced by cyclophosphamide 58 Table 7: Effects of O. gratissimum and V. amygdalina on hematological parameters of peripheral blood in cyclophosphamide-induced toxicities. 59 List of figures Figure 1: Chemical structure of Cyclophosphamide 2 Figure 2: Chemical structure of Sodium 2-mercaptoethane sulfonate 27 Figure 3: Vernonia amygdalina in its natural habitat 39 Figure 4: Ocimum gratissimum in its natural habitat 45 9 Figure 5: Photomicograph of organ sections from control rats given normal saline 62 Figure 6: Photomicrograph of sections of organs from rats given 200mg/kg of cyclophosphamide 62 Figure 7: Histologic sections of organs from rats treated with 67mg/kg of MESNA and 200mg/kg of cyclophosphamide 63 Figure 8: Histologic sections of organs from rats treated with 250mg/kg of O. gratissimum and 200mg/kg of cyclophosphamide. 63 Figure 9: Histologic sections of organs from rats treated with 500mg/kg of O. gratissimum and 200mg/kg of cyclophosphamide. 64 Fig.10. Photomicrograph of sections of organs from rats treated with 250mg/kg of V. amygdalina and 200mg/kg of O. gratissimum 64 Figure 11: Photomicrograph of sections of organs from rats given 500mg/kg of V. amygdalina and 200mg/kg of cyclophosphamide 65 Figure 12: Histologic sections of organs from rats treated with 250mg/kg of V. amygdalina 250mg/kg of O. gratissimum and cyclophosphamide 200mg/kg. 65 10 CHAPTER ONE 1.0 INTRODUCTION It is a well-known fact that neoplasms are deleterious and reduce quality of life. Many alkylating cytotoxic agents, which cyclophoshpamide (CP) is a member, have been well documented to be effective in management of many human malignancies in order to improve quality of life and extend patients life span (Philip et al., 1961; Colvin, 1978; Friedman et al., 1979; Carter and Livingston, 1982). Despite its adverse effects, many clinicians have continued to use CP either alone or in combination with other agents in cancer chemotherapy due to its efficacy.
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