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SPERMACOCE VERTICILLATA LINN (Family: Rubiaceae)

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SPERMACOCE VERTICILLATA LINN (Family: Rubiaceae) i ANTIMICROBIAL AND WOUND HEALING PROPERTIES OF LEAF EXTRACTS, FRACTIONS AND OINTMENT FORMULATIONS OF SPERMACOCE VERTICILLATA LINN (Family: Rubiaceae) BY ONWULIRI, EDITH ADANNA PG/Ph.D/08/48221 DEPARTMENT OF PHARMACEUTICS FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA FEBRUARY, 2014 ii ANTIMICROBIAL AND WOUND HEALING PROPERTIES OF LEAF EXTRACTS, FRACTIONS AND OINTMENT FORMULATIONS OF SPERMACOCE VERTICILLATA LINN (Family: Rubiaceae) BY ONWULIRI, EDITH ADANNA B. Pharm. (UNIJOS), M. Sc. (UNIJOS) PG/Ph.D/08/48221 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE (Ph.D) OF THE DEPARTMENT OF PHARMACEUTICS OF THE UNIVERSITY OF NIGERIA, NSUKKA FEBRUARY, 201 iii CERTIFICATION Onwuliri Edith Adanna, a post-graduate student in the Department of Pharmaceutics with registration number PG/Ph.D/08/48221, has satisfactorily completed the requirements for the award of Doctor of Philosophy degree in Pharmaceutical Microbiology of the Department of Pharmaceutics, University of Nigeria, Nsukka. The research work embodied in this thesis is original and has not been submitted in part or full for any other diploma or degree in this or any other university. Prof. E. C. Ibezim Prof. A. A. Attama Supervisor Supervisor Date________________ Date________________ Prof. K. C. Ofokansi Head of Department Date________________ iv DEDICATION This work is dedicated to my darling husband, Prof. Festus Chukwuemeka Onwuliri and our lovely children for their constant love, encouragement, and daily prayers towards the success of the work. To my parents, Chief and Lolo Amanze, my siblings and their families, my parents-in-law, Chief and Lolo Anyanwu Onwuliri and to the evergreen memory of my beloved brother-in-law Prof. Celestine Onwuliri. I miss you so much. v ACKNOWLEDGEMENT I am grateful to you, God Almighty for your guidance and protection throughout the period of this study. I, specially, appreciate my supervisors, Prof. E. C. Ibezim and Prof. A. A. Attama for their immense contribution and assistance towards the completion of this study. To the Head of Department of Pharmaceutics at the University of Nigeria, Prof. K. C. Ofokansi, I wish to express my profound gratitude for all the advice and official assistance I received from him throughout the period of this study. I am also grateful to Prof. C. N. Aguwa, Prof. G.C. Onunkwo and Prof. P. Chigbo for all the assistance they rendered to me. To the following persons in the Department of Pharmaceutics, University of Nigeria; Pharm. J. D. N. Ogbonna, Mr. D. C. Okechukwu, Pharm. F. Kenechukwu for all the advice, assistance and encouragement which I received from them, I am indebted. I am very grateful to Prof. I. G. Okafor of the Department of Pharmaceutics, University of Jos, for his spiritual and academic assistance and to my Dean, Prof. J. C. Aguiyi for his academic encouragement. I appreciate the contributions made by the staff members of Faculty of Pharmaceutical Sciences, University of Jos, especially Mr. S. Ojorinde, Prof. T. E. Alemika, Prof. N. A. Ochepa, Mrs. V. Idyu, Mr. A. Adolong, Mr. S. Francis, Mr. N. Lamsil and Mr. P. Adah. I am grateful to Mr. S. Gotom of Anatomy Department. I appreciate my friends: Mrs. V. Wannang, Mr. J. C. Nwaokoro, Mr. M. C. Chukwuma, Mr. F. Ikechukwu, Mrs. T. Sambo, Mrs. T. Dabit, Mrs. U Okafor, Mrs. A. Ogbonna, Mrs. N. Ibezim, Mrs. J. Nnah, Mrs. J. Okeahilam, Mr. S. Pakbamlar and Mrs. C. Oguikpe for their encouragement. vi I deeply appreciate members of my beloved family, especially my lovely husband whose undying love, relentless support and encouragement spurred me on to completing this study, you were a pillar of strength to me; God bless you and bless our home.; and my lovely children: Chinwendu, Chiazor, Ikechukwu, Uzoamaka, Nkechi and Onyinyechi for all the warmth and distractions I received from them during the period of this study. I must not fail at this juncture to appreciate my mate, Prof. (Mrs.) V. A. Onwuliri for all the encouragement and support I received from her during the course of this study. Finally I am grateful to my dear parents, Chief and Lolo M. O. Amanze and my siblings Ferdinard, Philip, Gerald, Noel and their families for the support I received from them. vii TABLE OF CONTENTS Title page i Certification iii Dedication iv Acknowledgement v Table of Contents vii List of Tables xxiv List of Figures xxvi List of Plates xxvii Appendices xxix Abstract xxx CHAPTER ONE: INTRODUCTION 1 1.1 The human skin 1 1.1.1 Functions of the human skin 2 1.1.2 Disorders of the skin 4 1.2 Microorganisms 4 1.2.1 Microorganisms, the human skin and skin infection 5 1.2.2 Features and classification of test microorganisms 6 1.2.2.1 Staphylococcus aureus 8 viii 1.2.2.2 Bacillus subtilis 9 1.2.2.3 Escherichia coli 9 1.2.2.4 Pseudomonas aeruginosa 10 1.2.2.5 Microsporum audouinii 11 1.2.2.6 Trichophyton rubrum 11 1.2.2.7 Candida albicans 11 1.3 Microbial infections of the skin 11 1.3.1 Bacterial infections 12 1.3.1.1 Impetigo 12 1.3.1.2 Folliculitis 12 1.3.1.3 Erysipelas 13 1.3.1.4 Ecthymas 13 1.3.1.5 Furuncles and Carbuncles 13 1.3.1.6 Paronychia 13 1.3.2 Fungi Infections 13 1.3.2.1 Tinea pedis (Athletes’s foot) 14 1.3.2.2 Tinea capitis (Head infection) 14 1.3.2.3 Tinea cruris (Tinea of the groin) 15 1.3.3.4 Candidiasis 15 1.3.4 Viral infections 15 1.3.4.1 Herpes simplex 15 ix 1.3.4.2 Herpes zoster 16 1.3.4.3 Molluscum contagiosum 16 1.3.5 Parasitic skin infections 16 1.3.5.1 Scabies 16 1.3.5.2 Jiggers 17 1.3.6 Non Infectious Skin Diseases 17 1.3.6.1 Dermatitis 18 1.3.6.2 Infective Dermatitis 18 1.3.6.3 Endogenous Dermatitis 19 1.3.6.4 Atopic dermatitis 19 1.3.6.5 Seborrheic eczema 19 1.3.6.6 Neurodermatitis (Lichenification) 20 1.3.6.7 Cross sensitization 20 1.3.6.8 Contact Dermatitis 21 1.3.6.9 Irritant Dermatitis 22 1.3.6.10 Allergic dermatitis 22 1.4 Factors influencing skin irritation 23 1.5 Patients attitude to skin infections 23 x 1.6 Wounds 24 1.6.1 Open wounds 24 1.6.1.1 Incisions 25 1.6.1.2 Lacerations 25 1.6.1.3 Punctures 25 1.6.1.4 Abrasions 25 1.6.1.5 Avulsion 26 1.6.1.6 Amputation 26 1.6.2 Closed Wounds 26 1.6.3 Microbial Contamination of Wounds 26 1.6.4 Wound Healing 27 1.6.4.1 Clotting/Inflammation stage/phase 27 1.6.4.2 Proliferative phase 28 1.6.4.3 Angiogenesis 28 1.6.4.4 Fibroplasia 28 1.6.4.5 Epithelialisation 29 1.6.4.6 Re-modeling Phase 29 1.6.5 Factors Affecting Wound Healing 29 1.6.5.1 Oxygenation 30 1.6.5.2 Infection 30 1.6.5.3 Age 32 1.6.5.4 Wound size 32 xi 1.6.5.5 Depth of wound type 32 1.6.5.6 Medication 33 1.6.5.7 Nutrition 34 1.6.5.8 Obesity 35 1.6.5.9 Host Immunity 35 1.6.5.10 Health Status of an individual 35 1.6.6 Models for the evaluation of wound healing activity 36 1.6.6.1 In vivo models 36 1.6.6.1.1 Excision wound models 36 1.6.6.1.2 Incision wound model 36 1.6.6.1.3 Dead space analysis 36 1.6.6.1.4 Burn wound model 37 1.6.6.2 In vitro models 37 1.6.7 Wound healing study parameters 37 1.6.7.1 Wound closure 37 1.6.7.2 Epithelialisation period 38 1.6.7.3 Tensile strength 38 1.6.7.4 Increase in granulation tissue 38 1.6.8 Existing therapy of wound healing 38 1.6.9 Types of wound Healing 39 1.6.9.1 Healing by first intention 39 1.6.9.2 Healing by secondary intention 39 1.6.9.3 Healing by third intention 39 xii 1.7 Natural products as sources of medicine 39 1.8 Plants with potential wound healing properties 41 1.8.1 Plant phytochemicals of wound healing importance 45 1.8.1.1 Flavonoids 45 1.8.1.2 Tannins 46 1.8.1.3 Terpenes and Terpenoids 46 1.8.1.4 Saponins 46 1.8.1.5 Alkaloids 47 1.8.1.6 Plant vitamins 47 1.8.1.7 Cardiac glycosides 48 1.9 Antimicrobial agents 48 1.9.1 Determination of an antimicrobial agent’s spectrum of activity 49 1.9.1.1 Carpet plate method 50 1.9.1.2 Cup agar plate method 50 1.9.2 Biostatic action of antimicrobial agents 51 1.9.3 Minimum inhibitory concentration (MIC) 51 1.9.3.1 Broth dilution method 52 1.9.3.2 Agar dilution method 52 1.9.3.3 Concentration gradient technique 53 xiii 1.9.4 Biocidal Activity 54 1.9.4.1 Cell- killing rate 54 1.9.4.2 D-Value 55 1.9.4.3 Extinction time 55 1.9.5 Minimum biocidal (bactericidal) concentration (MBC) 56 1.10 The use of antibiotics in managing microbial infections 57 1.10.1 Plant as source of antimicrobials 58 1.11 Antimicrobial resistance 58 1.12 Drug delivery systems 60 1.12.1.
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