ASSESSMENT OF THE ANTI PARASITIC AND HISTOPATHOLOGICAL ACTIVITIES OF SELECTED MEDICINAL PLANTS ON Rattus albus EXPERIMENTALLY INFECTED WITH HUMAN INTESTINAL PARASITES IN IMO STATE, NIGERIA. BY ULOGU, IMMACULATA OGOCHUKWU (12/Ph. D/1082) DEPARTMENT OF MEDICAL LABORATORY SCIENCE, FACULTY OF HEALTH SCIENCES, IMO STATE UNIVERSITY, OWERRI, NIGERIA. SEPTEMBER, 2017.
ASSESSMENT OF THE ANTI PARASITIC AND HISTOPATHOLOGICAL ACTIVITIES OF SELECTED MEDICINAL PLANTS ON Rattus albus EXPERIMENTALLY INFECTED WITH HUMAN INTESTINAL PARASITES IN IMO STATE, NIGERIA. BY ULOGU, IMMACULATA OGOCHUKWU (12/Ph. D/1082) (HND, A. IMLS, PGD, M.Sc) A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, IMO STATE UNIVERSITY, OWERRI, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY (Ph. D) IN MEDICAL MICROBIOLOGY IN THE DEPARTMENT OF MEDICAL LABORATORY SCIENCE, FACULTY OF HEALTH SCIENCES. SEPTEMBER, 2017. ii
DEDICATION To: My little Angels: CHINEMEZU and KOSI MARISFAVOUR and My beloved Husband: Godwin Uduchi Whose love, presence and care motivated me to do more. iii
ACKNOWLEDGEMENT I humbly wish to appreciate the Most High God for His abundant grace, mercies and love that have taken me this far. My profound gratitude goes to my supervisors, Prof. Nnaemeka J.C. Okolie and Prof. A.U. Opara for their contributions and corrections toward making this research work a reality. I acknowledge the commitment of Dean, Faculty of Health Sciences, Prof. H. U. Nwanjo towards the actualization of the programme. My appreciation also goes to my Head of Department, Prof. D. C. Nwosu, for his immense support and encouragement and to the co-ordinator of the programme, Dr. (Mrs.) J.N. Dike- Ndudim. I appreciate the tireless efforts, contributions and steadfastness of Elder Obiajuru, I.O.C. and Dr. & Dr. (Mrs.) Elo Ilo. May the good Lord reward you abundantly. Also, am grateful to Mr. & Mrs. Titus Mmirikwe, Mr. Amara Offorego, Mr. Darlinton Onyemekara, Mr. Babatunde Olaleye, Mr. Sunday Azubuike, Mrs. Kate Nnadozie, Mrs. Salome Onyenama and Miss Jovita Uwaezuoke, who really assisted in the field work. To the typist, Maryann and Chinwendu, I say, may God bless you. My sincere regard goes to my beloved husband, Mr. G.C. Uduchi, little Chinemezu and Kosi Uduchi and Nzube Okorie for their love, concern and disturbances. To my siblings, and course mates, I say, thank you and remain blessed.. Amen. iv
CERTIFICATION This is to certify that this work titled : Assessment of the Anti Parasitic And Histopathological Activities of Selected Medicinal Plants on Rattus albus Experimentally Infected with Human Intestinal Parasites in Imo State, Nigeria. was carried out by ULOGU, IMMACULATA OGOCHUKWU (12/Ph.D/1082) under the supervision of Prof. Nnaemeka J. C. Okolie and Prof. A.U. Opara. Prof. Nnaemeka J. C. Okolie...... (Supervisor) Signature Date Prof. A.U. Opara....... (Supervisor) Signature Date Prof. D. C. Nwosu...... ( Head of Department) Signature Date......... External Examiner Signature Date v
ABBREVIATIONS AND ACRONYMS ANOVA CD CI DNS ED EPG FCR GOPD IMSU IMSUTH KG LC LD MCC MCD MG NAFDAC NIH SPP SPSS WHO Analysis of Variance Curative Dose Chemotherapeutic Index DinotroSallcyclic Acid Effective Dose Egg Per Gram Folin-ciocalteau Reagent General Out-Patient Department Imo State University Imo State University Teaching Hospital Kilogram Lethal Concentration Lethal Dose Minimal/Maximum Convenient Concentration Minimal/Maximum Convenient Dose Milligram National Agency for Food and Drug Administration and Control National Institute of Health Specie Statistical Package for Social Sciences World Health Organization vi
ABSTRACT Anti Parasitic and Histopathological effects of selected plant extracts: (Napleonaea imperalis, Sida acuta and Vernonia amygdalina) on Rattus albus experimentally infected with human intestinal parasites was studied from October 2014 to March 2016. The aim was to determine the anti-parasitic and histopathological effects of the plant extracts on Rattus albus experimentally infected with human intestinal parasites. Two hundred and thirty (230) male and female patients presenting with symptoms of gastroenteritis and abdominal discomfort at GOPD, IMSUTH, Orlu were selected for the study. Their willingness to participate in the study was sought for and ethical permit obtained from IMSUTH Orlu. Stool samples were collected from the patients and examined for intestinal parasites, using stool concentration techniques and direct wet mount. The Harada- Mori cultural method was used to isolate and differentiate between hookworm species, Necator americanus and Ancylostoma duodenale. The isolates were inoculated on healthy, uninfected Rattus albus. Eight weeks after, the infected Rattus albus were treated with 3 selected plant extracts: Vernonia amygdalina, Sida acuta and Napoleonae imperialis and observed for 3 weeks. The active principles of the selected medicinal plants were extracted with ethanol at 78 o C using soxhlet extraction method. Fifty four laboratory animals of about 150g body weight each were used for toxicity test. The different treatment groups and controls were selected for histopathological studies using paraffin wax embedding method. The results showed that 157 (68.26%) Rattus albus were successfully infected with different human intestinal parasites. The most prevalent intestinal parasite in Orlu was Necator americanus (23.90%) while the least prevalent was Hymenolepsis nana (1.74%). Statistical analysis of the data using Chi square showed significant difference (p < 0.05) in the prevalence of infection between different age groups of patients. Out of four hundred laboratory animals (Rattus albus), pre - examined for intestinal parasites, 19.50% were infected, 80.50% were not infected. The most prevalent intestinal parasites (9.0%) in laboratory animals was Hymenolepsis diminuta, and the least prevalent (1.5%) was Entamoeba species. Three hundred and twenty two uninfected laboratory animals used for the study were inoculated with 7 human intestinal parasites: Ascaris lumbricoides, Necator americanus, Hymenolepsis nana, Trichuris trichiura, Entamoeba histolytica, Taenia species and Trichomonas hominis. It was observed that the mean body weights of the laboratory animals increased over the period. Post treatment stool analysis of laboratory animals inoculated with the different human intestinal parasites showed that, Necator americanus, was isolated from the stool of 50% male and female laboratory animals aged 2months with mean body weight 100 and treated with Napoleonaea imperialis, Ascaris lumbricoides and Hymenolepsis nana were isolated from the stool of 50% male and female laboratory animals aged 2months with mean body weight 101 150g and treated with Sida acuta. Entamoeba histolytica was isolated from stool of 50% male and female laboratory animals aged 3 months with body weights 151g and treated with Vernonia amygdalina. Trichomonas hominis was isolated from stool of 100% male and female laboratory animals aged 3 months, with mean body weight 151g, treated with Vernonia amygdalina. The test plant extracts yielded 100% parasite clearance in all 12 laboratory animals infected with Trichuris trichuira, 10 (83.3%) laboratory animals inoculated with Necator americanus, Entamoeba histolytica and Ascaris lumbricoides respectively. All 3 selected plant extracts contain Tanins, Saponins, Alkaloids, Flavonoids, Cardiac Glycosides, Phytate, Oxalate, Phenol, Steroids, Terpenoids and Cyanide. Toxicity test showed that all the plant extracts vii
exhibited anti parasitic effects against human intestinal helminthes and protozoa at 10µg/150g body weight. Napoleonaea imperialis and Vernonia amygdalina extracts exhibited toxicity resulting to death of laboratory animals (LD50) at 50µg/150g while Sida acuta extract exhibited toxicity resulting to death of laboratory animals (LD50) at 40µg/150g. Three types of impairment: inflammatory changes, degenerative changes and distortion were observed on the intestine of the laboratory animals. On the kidneys, 5 types of impairment: lymphocytic infiltration, degenerative changes, necrosis, vacuolation, and distortion of stroma and glomerulus were observed. On the liver, 4 types of impairment: inflammatory changes, hepatocytic degernerative changes, necrosis and distortion of hepatocytes (interwoven and fragmented) were observed. The nature and types of impairment caused on the different organs differ remarkably. Whereas the frequency of impairment was higher in liver organs 9 (20.0%) than kidneys 4 (8.9%), the types of impairment observed were higher in kidneys than liver organs. This study has shown that Napoleonae imperialis, Sida acuta and Vernonia amygdalina extracts exhibit anti parasitic effects on human intestinal parasites at low concentrations. At higher concentration, they exhibit toxic effects on host organs: intestine, kidney and liver. The plant extracts are likely to be sources of novel drugs or analogues of synthetic drugs capable of treating human intestinal parasitic infections. viii
TABLE OF CONTENTS Cover page Title page Dedication Acknowledgement Certification Abbreviations and Acronyms Abstract Table of Contents List of Tables List of Figures List of Plates List of Appendices i ii iii iv v vi vii ix xii xiv xv xviii CHAPTER ONE: INTRODUCTION 1.0 Background Information 1 1.1 Justification 6 1.2 Aim and Objectives 9 1.3 Hypotheses 10 CHAPTER TWO: LITERATURE REVIEW 2.1 History of Napoleonaea imperialis P. Beauv 12 2.1.1 History of Sida acuta 12 2.1.2 History of Vernonia amygdalina Del 13 2.2 Scientific Classification of Napoleonaea imperialis 14 ix
2.2.1 Scientific Classification of Sida acuta 15 2.2.2 Scientific Classification of Vernonia amygdalina 16 2.3 Botany of Napoleonaea imperialis 17 2.3.1 Botany of Sida acuta 20 2.3.2 Botany of Vernonia amygdalina Del 23 2.4 Chemical Constituents and Phytochemistry of 26 Napoleonaea imperialis 2.4.1 Chemical Constituents and Phytochemistry of Sida acuta 26 2.4.2 Chemical Constituents and Phytochemistry of Vernonia amygdalina Del 28 2.5 Biological Activities of V. amygdalina based on their Phytochemical Constituents 30 2.6 Animal Data on Phytochemical Analysis 33 2.7 Common Intestinal Parasites of Laboratory Animals 36 2.7.1 Laboratory Animal Data 37 2.8 Pharmacology of the Medicinal Plants 39 2.8.1 Pharmacology of Napoleonaea imperialis 40 2.8.2 Pharmacology of Sida acuta 41 2.8.3 Pharmacology of Vernonia amygdalina Del 46 2.9 Clinical Trial of Vernonia amygdalina Del as Antiparasitic 54 Agent Using Animal Models 2.9.1 Treatment of Intestinal Parasites Using Medicinal 56 x
Plants (Concentrations and Dosage Used in Treatment) 2.9.2 Egg-Per-Gram (EPG) Reduction and Worm Load Reduction 57 of Intestinal Parasites 2.9.3 Dose-Dependent Toxicity of the Medicinal Plants 60 2.9.4 Toxicological Evaluation of the Plant Extracts 63 CHAPTER THREE: MATERIALS AND METHODS 3.1 Materials 70 3.2 Methods 74 CHAPTER FOUR: RESULTS 4.0 Results 86 CHAPTER FIVE: DISCUSSION AND CONCLUSION 5.0 Discussion 167 5.1 Conclusion 180 5.2 Recommendations 181 References 183 Appendices 203 xi
LIST OF TABLES Page Table 1: Prevalence of Test Organisms (Intestinal Parasites) 89 Isolated from Selected Patients 2: Age related Prevalence of Intestinal Parasites 90 Infection amongst Patients 3: Gender related Prevalence of Infection 91 4: Prevalence of Intestinal Parasites in Laboratory Animals 95 5: Parasite Egg Count Per Gramme of Lab. Animal Faeces 96 6: Quantitative and Qualitative Composition of Selected 100 Medicinal Plants 7: Quantitative Pytochemical Analysis of Different Plant Extracts 101 8: Pre Innoculation Weights of Uninfected Laboratory Animals 106 9: Post - Parasite Innoculation Stool Analyses 107 of Laboratory Animals 10: Post - Parasite Innoculation Body Weights of Laboratory Animals 108 11: Mean Body Weights of Lab Animal 3 weeks Post 109 Medicinal Plants Treatment 12: Mean Body Weights of Lab Animal 3 weeks Post Medicinal Plants Treatment 110 13: Mean Body Weights of Lab Animal 3 weeks Post Medicinal Plants Treatment 111 xii
14: Physical Impact of Innoculation of Ascaris lumbricoides and Treatment with Med. Plant Extracts 113 15: Physical Impact of Innoculation of Necator americanus 114 and Treatment with Med. Plant Extracts 16: Physical Impact of Innoculation of Trichuris trichiura and 115 Treatment with Med. Plant Extracts 17: Physical Impact of Innoculation of Taenia 116 species in Laboratory Animals 18: Physical Impact of Innoculation of Hymenolepsis nana 117 and Treatment with Med. Plant Extracts 19: Physical Impact of Innoculation of Entamoeba histolytica 118 and Treatment with Med. Plant Extracts 20: Physical Impact of Innoculation of Trichomonas hominis 119 and Treatment with Med. Plant Extracts 21: Post - Treatment Stool Analyses of Laboratory 121 Animals Innoculated with Human Intestinal Parasites xiii
LIST OF FIGURES Page Fig 1: Comparative Analysis Direct Wet Mount and 92 Concentration Methods of Stool Analysis 2: Comparative Analyses of Intestinal Parasitic 97 Infection in Laboratory Animals 3: Compostion of the Phytochemical components 102 of the Medicinal plants used xiv
LIST OF APPENDICES Appendix: Page 1 Research Questionnaire 203 2 Examination of stool sample using Normal saline and 205 iodine wet preparation methods 3 Examination of stool sample using formol ether concentration method 206 4 Quantification of helminth eggs in faecal samples using modified McMaster egg counting technique 208 xv