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. Topical drug delivery systems 60

1.12.1.1 Advantages of topical drug delivery systems 60

1.12.1.2 Disadvantages of topical delivery system 61

1.12.1.3 Classification of topical drug delivery systems 61

1.12.2 Permeation of topical drugs through the skin 61

1.13 Routes of drugs administration through the skin 62

1.13.1 Transepidermal 62

1.13.2 Transfollicular (shunt pathway) absorption 62

1.14 Factors affecting topical permeation of drugs 66 xiv

1.15 Fractionation of leaf extracts 67

1.16 Ointments 67

1.16.1 Uses of ointments 68

1.16.2 Classification of Ointment Bases 68

1.16.3 Ideal properties of an ointment base 69

1.16.4 Preparation of ointments 70

1.16.5 Effective drug release of antimicrobial agents from ointment bases 70

1.16.6 Factors affecting the release and absorption of medicaments from

ointment bases 71

1.16.6.1 Factors connected to the antimicrobial agents’ concentration 71

1.16.6.1.1 Solubility in water 71

1.16.6.1.2 Ionization constant 71

1.16.6.1.3 Lipid/water distribution characteristics 72

1.16.6.1.4 Inherent Antimicrobial Action 72

1.16.6.2 Factors connected to the organisms 72

1.16.6.2.1 Microbial density 72

1.16.6.2.2 Presence of protective structures 73 xv

1.16.6.2.3 Physiological state of the organisms 73

1.16.6.3 Factors connected to the environment 73

1.16.6.3.1 Temperature 73

1.16.6.3.2 pH 74

1.16.6.3.3 Organic matter 74

1.16.6.3.4 Surface activity 75

1.17 Response of microorganisms to antimicrobial agents 75

1.17.1 Agar diffusion method 76

1.18 Review of studied plant: Spermacoce verticillata 77

1.18.2 Classification of the plant 78

1.18.3 Common names 79

1.18.4 Geographical distribution 79

1.18.5 Bioactive constituents 79

1.18.6 Medicinal uses of Spermacoce verticillata 80

1.18.7 Ethnomedical properties 81

1.18.8 Chemical constituents and some of their biological activities 83

1.18.9 Biological activities of crude extracts 85 xvi

1.19 Aim of the present study 86

1.20 Objectives of the study 86

CHAPTER TWO: MATERIALS AND METHODS 88

2.1 Materials 88

2.1.1 Reagents and solvents 88

2.1.2 Equipment and apparatus 88

2.1.3 Animals 89

2.1.4 Microorganisms (Clinical isolates) 89

2.2 Methods 89

2.2.1 Experimental design 89

2.2.1.1 Design for studies on inhibition zone diameter 89

2.1.1.2 Design for studies on wound diameter 90

2.2.2 Collection and identification of plant material 91

2.2.3 Preparation of plant material 91

2.2.4 Extraction 91

2.2.5 Preliminary phytochemical tests 91

2.2.5.1 Test for alkaloids 92 xvii

2.2.5.2 Test for phenols 92

2.2.5.3 Test for resins 92

2.2.5.4 Test for saponins (Frothing test) 92

2. 2.5.5 Test for tannins 92

2. 2.5.6 Test for flavonoids 93

2. 2.5.7 Test for steroids and terpenes 93

2. 2.5.8 Test for glycosides 93

2. 2.5.9 Test for balsam 93

2. 2.5.10 Test for volatile oils 94

2.2.6 Characterization of microorganisms using selective media and gram –

reactions 94

2.2.6.1 Bacteria 94

2.2.6.1.1 Escherichia coli 94

2.2.6.1.2 Pseudomonas aeruginosa 94

2.2.6.1.3 Staphylococcus aureus 95

2.2.6.1.4 Bacillus subtilis 95

2.2.6.1.5 Biochemical reactions for identification of bacterial culture 95 xviii

2.2.6.1.6 Maintenance and standardization of bacteria stock cultures 96

2.2.6.2 Fungi 96

2.2.6.2.1 Microsporum audouinii 97

2.2.6.2.2 Trichophyton rubrum 97

2.2.6.2.3 Candida albicans 97

2.2.6.2.4 Aspergillus niger 97

2.2.7 Antimicrobial studies 98

2.2.7.1 Antimicrobial screening test (Kirby-Bauer Method) 98

2.2.7.2 Biostatic action of extracts 98

2.2.7.3 Minimum inhibitory concentration (MIC) determination 99

2.2.7.4 Biocidal activity 99

2.2.7.5 Minimum bactericidal concentration (MBC) 99

2.2.8 Fractionation of acetone extract 100

2.2.8.1 Phytochemical screening of AGC fractions A and B 101

2.2.8.2 Preparation of stock solution and serial dilution of fractions A and B 101

2.2.9 Standardization of bacterial cultures 102

2.2.10 Antimicrobial studies of fractions A and B 102

2.2.10.1 Antimicrobial sensitivity test – using the cup in seeded plate method 102 xix

2.2.10.2 Minimum inhibitory concentration (MIC) of fraction 102

2.2.10.3 Minimum bactericidal concentration (MBC) 103

2.2.10.4 Antimicrobial sensitivity test of the extracting solvent (Acetone) 103

2.2.11 Formulation of ointments of various concentrations 103

2.2.12 Wound healing studies 104

2.2.12.1 Grouping of rats for various treatments 104

2.2.12.2 Creation and infection of experimental wounds on Albino rats 104

2.2.12.3 Application of the ointment on the infected wound 104

2.2.13 Toxicological studies 105

2.2.14 Histological studies 105

2.2.14.1 Fixation 106

2.2.14.2 Mounting of sections 107

2.2.15 Data presentation and statistical analysis 107

CHAPTER THREE: RESULTS AND DISCUSSION 109

3.1 Phytochemical constituents of the leaf extracts 109

3.2 Antibacterial activities of various leaf extracts on test microorganisms 111

3.2.1 Antibacterial activity of acetone leaf extract on test microorganisms 111

3.2.2 Antibacterial activity of ethanol leaf extract on test microorganisms 113 xx

3.2.3 Antibacterial activity of aqueous leaf extract on test microorganisms 115

3.2.4 Relationship between various concentrations of acetone leaf extract and IZD on test

microorganisms 118

3.2.5 Relationship between various concentrations of ethanol leaf extract and

inhibition zone diameter on test microorganisms 121

3.2.6 Relationship between various concentrations of aqueous leaf extract and IZD on test

microorganisms 124

3.2.2 Performance of the three leaf extracts 127

3.3. Minimum inhibitory concentrations (MIC) of ethanol, acetone and aqueous

extracts on test bacteria 132

3.3.1 Minimum MIC of acetone extracts on test bacteria 132

3.3.2 MIC of ethanol extracts on test bacteria 134

3.3.3 MIC of aqueous extracts on test bacteria 136

3.3.4 Minimum bactericidal concentration (MBC) of the Leaf Extracts 139 xxi

3.3.4.1 MBC of the acetone leaf extracts 139

3.3.4.2 MBC of the ethanol leaf extracts 141

3.3.4.3 MBC of the aqueous leaf extracts 143

3.3.5 Antifungal studies of the leaf extracts on test organisms 145

3.4 Antibacterial activity of acetone alone on Test Organisms 145

3.4.1 Antibacterial activities of extracts fractions A and B 147

3.4.1.1 The phytochemical constituents of fraction A 147

3.4.1.2 The phytochemical constituents of fraction B 147

3.4.2 Antibacterial activities of fractions A and B on test microorganisms 151

3.4.2.1 Antibacterial activities of fraction A on test microorganisms 151

3.4.2.2 Antibacterial activities of fraction B on test microorganisms 153

3.4.2.3 Relationship between various concentrations of fraction A leaf extract

and inhibition zone diameter on test microorganisms 156

3.4.2.4 Relationship between various concentrations of fraction B leaf extract

and inhibition zone diameter on test microorganisms 159

3.4.3 Minimum inhibitory concentrations (MIC) of fractions A and B 163 xxii

3.4.3.1 MIC of fraction A 163

3.4.3.2 MIC of fraction B 165

3.5 Antimicrobial wound healing studies 167

3.5.1 Effect of various concentrations of fraction B ointment of Spermacoce

verticillata on Escherichia coli infected wound 167

3.5.2 Effect of various concentrations of fraction B ointment of Spermacoce

verticillata on Pseudomonas aeruginosa infected wounds 169

3.5.3 Effect of various concentrations of fraction B ointment of Spermacoce

verticillata on Bacillus subtilis infected wound 180

3.5.4 Effect of various concentrations of fraction B ointment of Spermacoce

verticillata on Staphylococcus aureus infected wound 182

3.5.5 Interaction among the treatment means in a factorial experiment for wound

diameter 185

3.5.6 Reliability test of treatment of mean for wound diameter 185

3.5.7 Performance of fraction B ointment in relation to number of days, strength of the

ointment and its effect on wound healing infected by test microorganisms 187

3.6 Toxicity study of fraction B for the determination of its lethal dose - 50 (LD50) 189

3.7 Histological Studies 192

xxiii

CHAPTER FOUR: SUMMARY, CONCLUSION AND RECOMMENDATION 203

4.1 Summary and conclusion 203

4.2 Recommendation 204

REFERENCES 206

APPENDICES 218

xxiv

LIST OF TABLES

1: Phytochemical constituents of acetone, aqueous and ethanol leaf

extracts of Spermacoce verticillata 110

2: Performance of the different leaf extracts with respect to the various test

microorganisms according to their coefficient of determinant (r2) 128

3: F-values of the analysis of variance of treatments (extract solvent, extract

concentration and microorganisms) effects on zone of inhibition 129

4: Mean values of zones of inhibition 130

5: MIC of acetone leaf extract of Spermacoce verticillata on test microorganisms 133

6: MIC of ethanol leaf extract of Spermacoce verticillata on test microorganisms 135

7: MIC of aqueous leaf extract of Spermacoce verticillata on test microorganisms 138

8: MBC of acetone leaf extract of Spermacoce verticillata on test microorganisms 140

9: MBC of ethanol leaf extract of Spermacoce verticillata on test microorganisms 142

10: MBC of aqueous leaf extract of Spermacoce verticillata on test microorganisms 144

11: Antimicrobial spectra of the extracting solvent (acetone) 146

12: Phytochemical constituents of fraction A of acetone extract 149

13: Phytochemical constituents of fraction B of acetone extract 150

14: MIC of fraction A of Spermacoce verticillata on test microorganisms 164

15: MIC of fraction B of the leaf extract of Spermacoce verticillata leaf

extract on test microorganisms 166

16: Effect of various concentrations of fraction B ointments of

Spermacoce verticillata on Escherichia coli infected wounds 168

xxv

17: Effects of various concentrations of fraction B ointments of Spermacoce

verticillata on P. aeruginosa infected wounds 170

18: Effects of various concentrations of fraction B ointments of Spermacoce

verticillata on Bacillus subtilis infected wounds 181

19: Effects of various concentrations of fraction B ointments

on Staphylococcus aureus infected wound sizes 183

20: F-values of the analysis of variance of treatments (days, strength of

ointment and microorganisms) effects on wound diameter (µm) 186

21: Mean values of wound diameter 188

22: Result of toxicological study 190

23: Mortality of animals administered graded doses of acetone extract of

Spermacoce verticillata 191

xxvi

LIST OF FIGURES

1.1: A cross section of human skin 3

1.2: The plant Spermacoce verticillata Linn Rubiaceae 77

3.1: IZD of various concentrations of the acetone leaf extract of Spermacoce

vertillata on test microorganisms 112

3.2: IZD of various concentrations of ethanol leaf extract of Spermacoce

vertillata on test microorganisms 114

3.3: IZD of various concentrations of aqueous leaf extract of

Spermacoce vertillata on test microorganisms 116

3.4: Inhibition zone diameters of various concentrations of acetone leaf

extract of Spermacoce verticillata against the test microorganisms 120

3.5: IZD of various concentrations of ethanol leaf extract of Spermacoce

verticillata against test organisms 123

3.6: IZD of various microorganisms against different concentrations of

aqueous leaf extract of Spermacoce verticillata 126

3.7: IZD of various concentrations of fraction A leaf extract of Spermacoce

vertillata on test microorganisms 152

3.8: IZD of various concentrations of fraction B leaf extract of

Spermacoce vertillata on test microorganisms 155

3.9: IZD of various concentrations of fraction A leaf extract of Spermacoce

verticillata against the test microorganisms 159

3.10: IZD of various concentrations of fraction B leaf extract of Spermacoce

verticillata against the test microorganisms 160 xxvii

LIST OF PLATES

1: Wound infected with Pseudomonas aeruginosa on day 1 prior to treatment

with 2 % fraction B ointment 171

2: Pseudomonas aeruginosa infected wound treated with 2 % fraction B

ointment on Day 4 172

3: Pseudomonas aeruginosa infected wound treated with 2 % fraction B

ointment on Day 7 173

4: Pseudomonas aeruginosa infected wound treated with 2 % fraction B

ointment on Day 10 174

5: Pseudomonas aeruginosa infected wound prior to treatment with 5 %

fraction B ointment on Day 1 175

6: Pseudomonas aeruginosa infected wound treated with 5 %

fraction B ointment on Day 4 176

7: Pseudomonas aeruginosa infected wound treated with 5 %

fraction B ointment on Day 7 177

8: Pseudomonas aeruginosa infected wound treated with 5 %

fraction B ointment on Day 10 178 xxviii

9: Pseudomonas aeruginosa infected wound treated with blank ointment on Day 10 179

10: Photomicrograph of liver of untreated (control) rat at x 6.3 Magnification 194

11: Photomicrograph of liver of untreated (control) rat at x 25 magnification 195

12: Photomicrograph of liver of rat treated with bulk B fraction at x 6.3

magnification 196

13: Photomicrograph of kidney of untreated (control) rat at x 25 Magnification 197

14: Photo micrograph of kidney of rat treated with bulk B fraction at x 25

magnification 198

15: Photomicrograph of skin of untreated (control) rat at x25 magnification 199

16: Photomicrograph of skin of rat treated with bulk B fraction (x 25 magnification) 200

17: Photomicrograph of heart of untreated (control) rat at x 25 magnification 201

18: Photomicrograph of heart of rat treated with bulk B fraction at x 25 magnification 202

xxix

LIST OF APPENDICES

1: Preparation of media 218

2: Zones of inhibition of various concentrations of ethanol leaf extract

of Spermacoce verticillata on test micro organisms 219

3: Zones of inhibition of various concentrations of acetone leaf extract

of Spermacoce verticillata on test microorganisms 220

4: Zones of inhibition of various concentration of aqueous leaf extract

of Spermacoce verticillata on test microorganisms 220

5: Antibacterial activity of fraction A 220

6: Antibacterial activity of Fraction B 221

7: Formula for preparation of Bulk—B 0intment 222

8: ANOVA result for inhibition zone diameter 223

9: ANOVA result for wound diameter 225

10: Ethical clearance 229

xxx

ABSTRACT

This study investigated the antimicrobial and wound healing properties of leaf extracts and ointment formulations of Spermacoce verticillata Linn (Family: Rubiaceae). Three different solvents: acetone, ethanol and water were used in the extraction of phytochemicals from the leaves of Spermacoce verticillata. The bioactive constituents of acetone, aqueous and ethanol extracts were determined using standard phytochemical analytical methods. The cup plate agar diffusion method was used to evaluate the antimicrobial activities of the three crude extracts at

100 µg/ml, 200 µg/ml and 400 µg/ml concentrations, in comparison with the control- gentamycin at 40 µg/ml concentration. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts were obtained using agar dilution method for determination of the most efficacious extract. Acetone extract was observed to be the most potent. Accelerated gradient chromatography (AGC) was used to fractionate the acetone extract into two; fractions A and B. Phytochemical analysis of fractions A and B were carried out using standard laboratory methods. The cup plate agar diffusion method was used to assess the antimicrobial activities of the fractions at 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400

µg/ml concentrations in comparison with the control -gentamycin at 40 µg/ml concentration.

w Fraction B which had better antimicrobial properties was formulated into ointments of 0.1% /w,

w w w w 0.2% /w, 1% /w, 2% /w and 5% /w concentrations and assessed for wound healing studies on wounds inflicted on albino rats. The relationship between types of microorganisms, concentrations of ointment, and wound healing time were studied. Acute oral toxicity studies for the determination of the medium lethal dose (LD50) of the acetone extract was calculated using the Kerber's method. Histological studies that would indicate toxic effect(s) of the acetone xxxi

extracts on probable target organs (skin, kidney, liver, and heart) were also undertaken. Data generated were subjected to analysis using analysis of variance (ANOVA) which was arranged in a randomized complete block design (RCBD) and means were compared or separated using least significant difference (LSD) at 5% confidence interval. Correlation and regression analysis was also used in which case coefficient of simple determinant (r2) was obtained which was used to measure the relationships that existed between inhibition zones/wound diameters and various levels of concentrations of extracts. Descriptive statistics was employed to measure the level skewness or variability of data that were obtained in the study. The following phytochemicals: balsam, terpenes and steroids were present in all the three leaf extracts. Acetone and ethanol extracts both had alkaloids, phenols and volatile oils. The acetone extract had glycosides and saponins in addition; all extracts however lacked flavonoids and resins. All the extracts possessed antibacterial activity but lacked antifungal activity. Minimum inhibitory concentration

(MIC) was 50 µg/ml for acetone leaf extract on S. aureus and E. coli, which was the least MIC; ethanol extract had an MIC of 100 µg/ml concentration on S. aureus and E. coli; while the aqueous extract had an MIC of 200 µg/ml on B. subtilis and E. coli. The aqueous extract had a minimum bacterial concentration (MBC) of 400 µg/ml on S. aureus, while the ethanol extract had an MBC of 400 µg/ml on E. coli. The best result was however obtained by acetone extract with 200 µg/ml of MBC on E. coli and P. aeruginosa. The acetone extract was seen to have more pronounced antibacterial activity than the other extracts. Antibacterial studies of the extracts (A and B) fractions on the test organisms showed that the B fraction had better antibacterial activity. All the strengths of the ointments promoted wound healing on rats infected with test microorganisms with best result achieved on S. aureus, followed by E. coli, B. subtilis and lastly, P. aeruginosa. Comparing the progress of healing of the infected wounds treated with xxxii

the blank ointment and those treated with ointments of fraction B, suggests that the wound healing effect of fraction B was attributed to its antibacterial property especially when the extract ointment at 2 % concentration promoted wound healing better than 0.1% gentamycin ointment.

Complete healing was achieved on the 10th day (on S. aureus, E. coli and P. aeruginosa infected wounds); however, all the concentrations achieved complete healing on the 14th day, except the blank ointment. The toxicity study of fraction B indicated that 100 µg/g dose did not kill or adversely affect any animal, while 250 µg/g dose killed just one rat. Since the MICs of fraction B were between 50-100 µg/g, a safe dose for administration would be between 100 µg/g – 250

µg/g. From the histopathological study, the photomicrographs of organs from rats administered with LD50 acetone extract, food and water, showed no histological abnormalities.

CHAPTER ONE

INTRODUCTION

1.1 The human skin

The human skin is the largest organ that covers and protects the internal part of the body from external substances. It is made of three layers -epidermis, dermis and subcutaneous layer and there is a wide variation in the structure of the skin (1).

Epidermis

This is the outermost layer of the skin, and consists of four layers, namely; the horny, granular, prickle cells and the basal layer. The basal cells give rise to the prickle cells by mitotic division; then the prickle cells move upwards but as new cells are formed beneath them; they change their polyhedral shape to a flattened shape. As they continue to move upwards they produce a protein, keratin. The granular layer is filled with granules of keratin. The skin releases lytic-enzymes that xxxiii

destroy the cell nucleus and the granules of keratin are distorted, the unbound keratinocytes that are now on top of the skin die and harden into the horny layer.

The horny layer is thus formed of dead epidermal cells. It takes about 28 days from the formation of a prickle cell to its loss from the skin surface. In this way, the skin renews itself once every four weeks. Melanin is produced in this layer by certain pigment cells (melanocytes) that protect the skin from UV rays. There are langerhans cells which play some part in the immune function of the skin (1).

Dermis

This region is thicker than the epidermis. It is made up of the relatively thin papillary layer and a thicker reticular layer. The surface of the papillary layer has many bumps (papillae) that interlock with the base of the epidermal layer. Each papilla is supplied by a capillary vessel. The dermis has a great capacity for retaining water and is a reservoir of body fluid, that contains collagen formed by fibroblast, two fibers- reticulum and elastin, and these three give the dermis its elastic nature. It contains hair follicles, sweat ducts, blood vessels, and nerve endings; the sweat gland is situated deep in the dermis and opens on the skin surface as the sweat duct (1).

Figure 1.1 shows the cross section of the human skin.

Subcutaneous layer

The layer is below the dermis and consists of connective and fatty tissues. It serves as a fat storage layer and as a padding, shock absorber and insulator for the body (2).

1.1.1 Functions of the human skin

The skin allows man to adapt to a wide variation in the environment. The skin is the largest human organ and it accomplishes a wide variety of tasks. With many nerve endings in the skin, it xxxiv

is able to perceive pain and vibration; the skin can also absorb substances from the environment into the body (medicated creams). The skin prevents germs and pathogens from entering the body and prevents evaporation of tissue fluids. By excreting sweat, it protects the body from overheating (1).

The skin is a barrier against cold. On exposure to cold, there is a reduction of its blood flow and this insulates and maintains the body temperature. Increased blood flow and evaporation of sweat enables man to remain cool in hot climates. The presence of pigment in the skin helps to filter out most of the harmful ultraviolet radiations. This functions/integrity of the skin can be compromised by skin diseases and trauma (wounds) (1, 2).

xxxv

Fig. 1.1: A Cross section of human skin

xxxvi

1.1.2 Disorders of the skin

The skin is susceptible to diseases which could be as a result of genetic disorders like neurofibromatosis, icthyosis, tuberose, sclerosis and xanthomatasis. Skin diseases/conditions can be caused by hypersensitivity reaction of the skin e.g. dermatitis or eczema. Dermatitis is the inflammation of the epidermis; also skin diseases can be caused by microbial infection of the skin (1).

1.2 Microorganisms

Microorganisms are ubiquitous in nature that is to say, they can be found almost everywhere, on land, in water, in the air, clothing, manufacturing equipment, in and on man and anywhere that can support its growth. Examples of microorganisms are bacteria, fungi, viruses, protozoa, etc.

They are capable of producing diseases in many of its hosts (man inclusive) but they can also at the same time, synthesize useful materials to man. So, some by- products of microbial metabolisms can be useful to man, though there is no consideration as to the usefulness of these by-products to man from the microbe’s point of view. For them, it is just a way for survival in an environment (6).

xxxvii

Microorganisms are grown in culture media in the laboratory. The media are designed to supply all the nutrients required to support the growth of the organisms in question. Very small quantities of pure culture of the study organism are aseptically transferred into a sterile liquid or solid medium and incubated at a suitable temperature, which is optimally 25 oC for fungi and 37 oC for bacteria. On solid media, the organisms grow as visible colonies, while in a clear broth it becomes increasingly turbid as the organisms grow in it.

It is very important that the organisms used in any research must be pure cultures, so that each organism can be studied as individual species, because the effects of microorganisms growing as a mixed culture cannot be ascribed with certainty to any particular member of the mixture. They are examined carefully for details of their colony sizes, texture and colour (7).

1.2.1 Microorganisms and the human skin and skin infection

The human skin is a natural host for many microorganisms, some of which are normal flora.

Some microorganisms that are often encountered on the skin, include Staphylococcus aureus,

Streptococcus pyogenes, Corynebacterium spp., Propionibacterium spp., Mycobacterium, spp., yeast -like Candida albicans and viruse like herpes simplex. Bacteria like Brevibacterium spp.,

Acinetobacter spp., Neiseria spp., Erysipelothrix insidiosa, and Haemophilus spp. Others include Helicobacter Pylori, Klebsiella rhinoscleromatis, Pseudomonas aeruginosa,

Calymmatobacterium, granulomatis, Bacillus anthracis, Clostridium perferingens, Treponema spp., Mycobacterium spp., Yersinia pestis and even Serratia marcescens. Some of these microorganisms found on the skin are harmless while others are pathogenic depending on the predisposing factors of the host (3). xxxviii

Staphylococci bacteria are a common type of bacteria that live on the skin and mucous membranes (e.g. in nose) of humans. Staphylococcus aureus (S. aureus) is the most important of these bacteria in human disease. Other Staphylococci including S. epidermidis are considered commensals, or normal inhabitants of the skin surface. Staphyloccocal skin infection includes impetigo, ecthyma, cellulits, folliculitis, boils (furuncles and carbuncles) sycosis and Scaled Skin

Syndrome (SSS). Staphylococci are becoming increasingly resistant to much commonly used antibiotics including penicillin, macrolides such as erythromycin, tetracycline and amino glycosides (3).

Some skin infections have fungal origin, the most popular being the dermatophytes. The three major genera that are recognized to cause fungal infections include the Epidermophyton spp.,

Microsporum spp., and Trichophyton spp. (3).

Dermatophytes are types of fungi that cause skin, hair and nail infections. Infections caused by these fungi are known as “tinea”. They cause diseases such as athlete’s foot and jock itch.

Trichophyton rubrum and Trichophyton tonsurans are two common dermatophytes that can be transmitted from person to person, i.e. anthrophillic; others include Microsporum audounii,

Trichophyton interdigitale, Trichophyton violaceum, Microsporum ferrugineum, Trichophyton schoenieinii, Trichophyton megninii, Trichophyton sandanense and Trichophyton yaoundei.

Other common dermatophytes are transmitted from animals such as cats and dogs to people i.e. zoophillic. They include: Microsporum canis (From cats and dogs) Trichophyton equinum (from horses), Trichophyton erinacei (from hedgehogs), Trichophyton verrucosum (from cattle),

Microsporum nanum (from pigs) and Microsporum distortum (a variant of Microsporum canis). xxxix

The geophillic dermatophytes are transmitted from soil to people; they include: Microsporum gypseum and Microsporum fulvum (4, 5).

1.2.2 Features and classification of test microorganisms

Most microorganisms are free-living and can perform activities that are useful to animals and plants but some are capable of causing diseases and are called pathogens such as bacteria, fungi, viruses and protozoa (7).

Bacteria

Bacteria are essentially unicellular although some are chains of cells. They are prokaryote that is, they do not have true nucleus and exhibit a variety of forms, habitat, metabolic path-ways and pathogenicity. They are divided into two groups, namely; Gram-positive and Gram-negative.

These are microscopic organisms that are devoid of a well defined nucleus and mitochondria; they have a simple rigid cell wall which allows them to have a more or less independent existence (7).

Bacterial cells are divided into two groups, namely; Gram-positive and Gram-negative bacteria.

They differ in the strength and structure of their cell walls. The Gram-positive bacteria are nutritionally exacting organisms that take up complex molecules from their environment and because complex molecules are on their own capable of generating considerable osmotic pressure, a higher internal osmotic pressure is required in the Gram-positive cells to create an osmotic gradient along which nutrients could be taken up into their cells. Gram-negative bacteria on the other hand, do not need complex molecules for their nutritional requirement, so there is no xl

need for them to generate high internal osmotic pressure to absorb the simple molecules they survive on. In view of these, Gram-positive bacteria have a very robust cell wall to contend with its high internal osmotic pressure. The cell wall is made up of single layer of repeating units of mucopeptides. Mucopeptides are composed of alternating units of N-acetylmuramic acid and N- acetalyglucoseamine, each strip of mucopeptide is connected to the next by polypeptide cross link. The mucopeptides layers and their polypeptide cross link can be very extensive which is why Gram-positive bacteria possess robust cell walls. The bacterial cell wall functions are for mechanical support and to protect the cells from osmotic damage. The cell wall has no physiological function but a bacteria cell normally cannot survive the loss or malfunction of its cell wall, which is why most antibacterial agents are targeted towards it (6, 7).

The cell wall of Gram-negative bacteria in comparison to that of the Gram-positive bacteria is very thin but more sophisticated. The Gram-negative cell walls are made up of lipoprotein, liposaccharide, protein and peptidoglycan. The Gram-negative bacterium, with its less robust cell wall, is capable of giving efficient protection against lethal chemical (7).

Gram-stain is the most important staining for identification of bacterial cells. It was described by

Christian Gram in 1884, and involves treatment of fixed bacterial smears with gentian violet and methyl violet as primary stains, then Lugol’s iodine. It acts as a mordant by fixing the primary stains to the bacterial cell well. This is followed by discolourising the stain with alcohol or acetone and washing with water, before counter-staining with safranin. Gram-positive bacteria cells will retain the violet colour of the primary stain while the Gram-negative ones will turn purple or red colour of the counter stain (6). xli

In Gram-negative bacteria, alcohol can penetrate the thin cell wall to cause leakage of primary stain-iodine complex, so their cell wall would be free to accept the counter stain hence they take up the red colour of the counter stain (7).

Apart from the cell wall, a bacteria cell is made up of cytoplasmic membrane, ribosomes, nucleus (nuclear bodies), mesosome, capsule and flagella. Some Gram-positive bacteria, examples bacilli and clostridia have developed a very effective means of surviving adverse conditions, through the formation of spores. The bacteria of interest here are Bacillus subtilis,

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

1.2.2.1 Staphylococcus aureus

These are non-motile Gram-positive cocci that occur in groups of grape-like clusters (staphylo), hence the name staphylococcus. They are non-capsulated, coagulase positive, DNase positive and catalase positive (8, 9).

1.2.2.2 Bacillus subtilis

Bacillus is a genus of Gram-positive rod-shaped bacteria and a member of the division

Firmicutes. Bacillus can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species.

Under stressful environmental conditions, the cells produce oval endospores that can stay dormant for extended periods. These characteristics originally defined the genus, but not all such xlii

species are closely related, and many have been moved to other genera. Bacillus subtilis is one of the best understood prokaryotes, in terms of molecular biology and cell biology (8). Its superb genetic amenability and relatively large size have provided the powerful tools required to investigate a bacterium from all possible aspects. Two Bacillus species are considered medically significant; Bacillus anthracis, which causes anthrax, and B cereus, which causes a food borne illness similar to that of Staphylococcus aureus (7, 8, 9). A third species, Bacillus thuringiensis, is an important insect pathogen, and is sometimes used to control insect pests. The typed specie is Bacillus subtilis, an important model organism. It is also a notable food spoiler, causing ropiness in bread and related food.

1.2.2.3 Escherichia coli

This is Gram-negative rod shaped bacterium, commonly found in the lower intestine of warm- blooded animals, in soil and in water. They are sometimes referred to as coliforms, and are usually non-motile but can be capsulate. They can be identified biochemically by their positive reaction indole test (8, 9).

1.2.2.4 Pseudomonas aeruginosa

It is Gram-negative, non-sporing motile rod, which can sometimes encapsulate. Pseudomonas aeruginosa is found in the intestinal tract, water, soil, sewage, in hospitals, moist environments such as sinks and buckets. It can equally grow in some eye drops. Many infections are xliii

opportunistic hospital–acquired, affecting those in already poor health and immune-suppressed conditions. Pseudomonas aeruginosa is oxidase positive and produces acid from glucose only, with no gas production (8, 9).

Fungi

They are non-photosynthetic organisms that grow either as singles (yeast) or as colonies of multicellular filaments. They are saprophytic, parasitic or commensal organisms. Fungi are eukaryotes, i.e. their nucleus is enclosed by a nuclear membrane. Their cell wall consists of polysaccharides, polypeptides and chitin, while the cell membrane contains sterols which prevent most antibacterial agents from being effective against them (7).

Fungal infection does not cause widespread and dangerous diseases like bacteria but are major causes of individual distress. Fungal infections are called mycoses and based on the site of the body affected, mycoses can be classified as:

Systemic mycoses: This is acquired by inhalation, and may affect the lung to involve other parts of the body (10).

Subcutaneous mycoses: This is acquired when the fungal pathogen gets access into the body through cuts on the skin (8).

Superficial mycoses: Here, the pathogen is confined to the body surfaces like the hair, skin and nails, and does not directly involve living tissues. This class of fungi are called the dermatophytes. When there is a break in the integrity of the skin via wound or trauma, these pathogens access such sites to probably cause secondary infections (8). xliv

The fungal pathogens used in this research work were the dermatophytes Microsporum audouini and Trichophyton rubrum and a unicellular fungal Candida albicans.

1.2.2.5 Microsporum audouinii

It grows slowly on Sabouraud agar as gray colony with a radially folded surface. The centre of the colony is reddish on the reverse. It is associated with the disease known as tinea, an infection of keratinzed tissues like epidermis, hair and nails (11).

1.2.2.6 Trichophyton rubrum

This causes athletes’ foot and ringworm. It grows slowly in the laboratory. Its texture is waxy, smooth and cottony texture. The colour is bright yellow or red violet. It is the most common dermatophytes that causes finger nail fungus infections, and scalp infections (10).

1.2.2.7 Candida albicans

This is the most common causative organism of candidiasis. It occurs as a commensal of the gastrointestinal tract. Skin infections occur too, especially in people whose natural defences are impaired by diseases, wounds and drug therapy. Candida albicans grows well on Sabouraud dextrose agar at 35 – 37 ºC for 2 – 3 days. Its wet preparation (Microscopy) shows budding yeasts and hyphae with buds, they are Gram-positive (8, 12).

1.3 Microbial infections of the skin

The normal skin is inhabited by some microorganisms called normal flora. These microorganisms grow on intact skin without causing any harm to the host. These same microorganisms can however become opportunistic and cause diseases when the skin integrity is xlv

compromised through trauma like wounds, burns, pre-existing skin diseases and poor hygiene

(6). Skin infection can be caused by bacteria, fungi, viruses or parasites.

1.3.1 Bacterial infections

Examples of bacterial skin infections are:

1.3.1.1 Impetigo

It is caused by Streptococcus and/or Staphylococcus species; it is a superficial skin infection mainly involving the surface areas of the skin. Direct contact with the lesions or with exudates from the infected sites is required for transmission. The lesions appear initially as small red spot, which then become vesicles (a small collection of fluid in the epidermis or between the epidermis and dermis) that are filled with an amber fluid.

Eruption of the vesicles releases the amber fluid that dries into a brown or yellow crust on the skin surface. Impetigo is very contagious and re-infection of any exposed part of the body is possible if the infection is not controlled. The incidence is most common in children and could increase the risk of glomerular nephritis if left untreated. There is primary impetigo (Impetigo vulgaris) which is caused by the bacteria directly while secondary impetigo (Bockhart’s impetigo) occurs as a secondary infection to other infections or injuries (13).

1.3.1.2 Folliculitis xlvi

This is a bacterial infection of the hair follicles. They may be superficial or deep, and involve the hair shafts. They are caused by S. aureus, although P. aeruginosa is also implicated

(13).

1.3.1.3 Erysipelas

This is an infection of the superficial skin caused by Streptococcal species. The infected area is often red and raised with local warmth and edema. It occurs mostly on the face and scalp and is usually accompanied by fever and chill (13).

1.3.1.4 Ecthymas

It is caused by the same organisms that cause impetigo i.e. Staphylococcus and / or

Streptococcal species, but the lesion of ecthymas is deeper. The legs are most affected. The lesions begin with vesicles that rapidly erode and become crusted, healing with scarring. This condition occurs mostly as a secondary infection to mild trauma or injury/ wound to the skin

(13).

1.3.1.5 Furuncles and Carbuncles

Superficial infection of the hair follicle is termed folliculitis; a deeper involvement is called a furuncle (small boil). Furuncles are the initial redness and inflammation of the area followed by thinning of the skin around the primary follicle; central ulceration and scarring often occurs. A carbuncle forms when adjacent hair follicles are involved. Both infections are caused by Staphylococcal and Streptococcal organisms (1, 13).

1.3.1.6 Paronychia xlvii

This is an infection of the nails caused by Streptococcus and Staphylococcus species. The nails become irregularly shaped and application of mild pressure may exude pus (1, 13).

1.3.2 Fungal Infections

Fungi exist as unicellular organisms called yeast or as multi-cellular filamentous forms called mould; very complex forms which grow into large structures like mushrooms also exist. The basic unit of a mould is the hypha. Hypha is a branching tubular structure and it is of two forms.

Some hypha project upwards from the surface of the growth media, and are called the aerial hypha, bearing the reproductive cells, while the other form of hypha penetrates the growth media, and are called the vegetative hypha, concerned with absorption of nutrients. Both the aerial and vegetative hypha can assume certain characteristic features that are used to identify them (7).

Yeasts: They are oval unicellular organisms, though sometimes they seem attached to each other to form chains or pseudo-hypha. The fungal cell wall is made up of N-acetylglucoseamine residues, linked together by B-1-4-glycosidic bonds. Some yeasts of medical importance are

Candida albicans, Trichosporon beigeli, and Cryptococcus neoformans (7).

Moulds: The hyphae of many pathogenic moulds are septa that are divided into cells by cross-walls called septa. Hypha without septa are referred to as aseptate. Moulds of medical importance are dermatophytes (7).

1.3.2.1 Tinea pedis (Athletes’ foot) xlviii

This is commonly caused by Trichophyton species and Epidermophyton species. The first signs of Tinea pedis are ulceration, scaling and fissuring on the webs of the little toes. This condition may get mild in the cold weather to recur fully in the warm seasons. As the fungal infections spread, secondary bacterial infection may set in at this stage and the infection sites become purulent and exude an odoriferous serum (1).

1.3.2.2 Tinea capitis (Head infection)

This is transmitted by direct contact with infected persons or animals. The infection is caused by

Microsporum and Trichophyton species. Infection is presented as non-inflamed areas of hair loss to deep, crusted lesions, which may be scarred and with permanent hair loss (1, 2).

1.3.2.3 Tinea cruris (Tinea of the groin)

This is caused by Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes. It affects the upper part of the thighs and the pubic area. Tinea cruris is more common in males than females. The margins of the lesions are slightly elevated and more inflamed than its central part. Small vesicles appear at the margins. The lesions are bright red in acute condition and turn brown in chronic cases (13).

1.3.3.4 Candidiasis

This is transmitted by Candida albicans. When it affects the mucous membranes, it is called thrush; at the anus, it is called pruritus ani, while it is vaginal cadidiasis in the vagina.

There is Candidia paronychia (nails) that is common in people who routinely immerse their hands in water.

xlix

Other fungal skin infections include Tinea barbe (of the beard), Tinea manum (hands), Tinea versicolor - where there is partial discolorations of pigmented skin and Tinea unguium in which the nails become hypertrophic, discolored and scaly (2).

1.3.4 Viral infections

These may occur in or on the skin and may present as warts. Warts are human tumors caused by virus and like other tumors, are due to a group of altered cells that can proliferate uncontrollably.

An example is plane warts of the face and back of the hands, plantar warts occur mainly on the soles (1, 13).

1.3.4.1 Herpes simplex

This is a viral infection of the skin and mucous membranes. It is caused by herpe virus hominis (HVH) which is made up of two strains. HVH-1causes cold sores on the lips and is transmitted by contact from sufferers while HVH-2 causes genital lesions and is sexually transmitted (1).

1.3.4.2 Herpes zoster

This is caused by the same virus that causes chicken pox, Zoster- varicella. Localized and painful shingles are called zosters, and are caused by the activation of chicken pox virus which had lain latent in hosts, years before (1).

1.3.4.3 Molluscum contagiosum

This is caused by a DNA- containing pox virus and it is contracted by direct contact with an infected person or formites. The lesions are seen as pinkish nodules with a slight depression on its top. It has a soft core that can be easily squeezed to express a white curd-like substance

(1).

1.3.5 Parasitic skin infections l

Besides bacteria, fungi (even yeast) and viruses, parasites such as insects or worms can burrow into the skin, and cause skin infection. Some parasites live in the skin for part of their life cycle, while others for their entire life cycle. Parasitic skin infections frequently cause severe itching and inflammation (14) and include:

1.3.5.1 Scabies

This is a mite infestation of the skin that produces tiny reddish bumps and severe itching.

Scabies usually spread from infested persons through physical contact. People with scabies have severe itching, even if there are just few mites on the body. Scabies is caused by Sarcoptes scabiei.

1.3.5.2 Jiggers

This is caused by Sandflea (Tunga penetrans), larva migrans (dog hook-worm,

Ancylkostoma brasiliensis) Cutaneous larva migrans (creeping eruption) and is a hookworm infection transmitted from warm, moist soil to exposed skin. The hookworm normally inhabits dogs and cats. The eggs of the parasite are deposited on the ground in dog and cat feeler. When bare skin touches the ground, which appears when a person walks barefoot or sun bathes, the hookworm gets into the skin. Starting from the site of infection, usually the feet, legs, buttocks, or back, the hookworm burrows along a haphazard tract, leaving a winding, threadlike, raised, red rash. The eruption itches intensely (14, 15). li

1.3.6 Non Infectious Skin Diseases: Eczema/ Dermatitis

Dermatitis and eczema are terms which are often used interchangeably to describe an inflammatory condition of the skin produced by a variety of external and endogenous factors of which the characteristic feature is oedema of the epidermis (15, 16). It is regarded as a reaction pattern rather than a specific disease and can have many external or internal causes – Genetic, immunological, infective, vascular, traumatic and emotional factors. Eczema caused by external factors are termed contact dermatitis and those with internal causes are called endogenous eczema (16)

Eczematous patches have a poorly defined edge and at various stages may show erythema, oedema, scaling, papules, vesicles, weeping and pustules. Eczema most commonly causes dry, reddened skin that itches or burns, although the appearance of eczema varies from person to person and according to the specific type of eczema. While any region of the body may be affected by eczema, both in children and adult, it typically occurs on the face, neck and inside the elbows, knees and ankles. In infant eczema typically occurs on the foreheads, cheek, forearms, legs, scalp and neck.

1.3.6.1 Dermatitis

This is as a result of acquired sensitization to substances on the skin from outside the body. The sensitizer penetrates the epidermis of the skin through the horny layer, sweat ducts or hair follicles and keratins. In the epidermis, the sensitizer combines with protein to form a stable antigen. The antigen sensitizes lymphocytes to cause a specific cell-mediated reaction to occur, lii

after which any further contact with the sensitizer will be followed by inflammatory reaction of the epidermis. This type of cell-mediated reaction is termed delayed hypersensitivity (13).

There is always a latent interval between the first exposure to a sensitizer and the development of sensitization. It may be as short as 5 days to months and even years.

Hypersensitivity to a sensitizer is confirmed by patch-testing, which consists of application of a small amount of a suspected sensitizer to an area of normal skin. The test is positive if that area develops dermatitis beneath the patch after 24-48 hours. There may be a gradual lessening of the person’s sensitivity to it but most times, sensitivity is life long.

The ability of various substances to cause dermatitis varies as the ability of different individuals to same substances varies too.

1.3.6.2 Infective Dermatitis

This condition is caused by the action of microbial toxins, and not the organism itself.

When the skin of susceptible individuals is inoculated with bacterial culture or its filtrate, such conditions develops. The condition responds favorably to systemic and topical antibiotics (1, 13).

1.3.6.3 Endogenous Dermatitis

This is dermatitis caused by unknown internal causes, its symptoms generally lasting longer than those of exogenous dermatitis and examples are atopic and neurodermatitis (13).

1.3.6.4 Atopic dermatitis liii

This skin condition occurs primarily during childhood, around folds of the arms or knees, the symptoms are erythema, scaling and weeping with severe pruritus. Secondary- associated infections are common. The etiology of the condition is unknown but patients usually have asthma or hay fever in addition. Atopic dermatitis is a chronic skin disease characterized by itchy, inflamed skin and is the most common cause of eczema. The conditions tend to come and go, depending upon exposures to triggers or causative factors. The causative factors include environmental agents like molds, pollen, or pollutants; contact irritants like soaps, detergents, nickel (jewelry), or perfumes; food allergies or other allergies. When it occurs in infancy, it is termed infantile eczema.

1.3.6.5 Seborrheic eczema

This is a form of skin inflammation of unknown cause. The signs and symptoms are patches of inflammation on the skin, on the scalp, face and occasionally other parts of the body. Dandruff and “cradle cap” in infants are examples of seborrheic eczema. It inflames the face on the cheeks and/or the nasal folds, though it is not always associated with itching and runs in families.

Emotional stress, oily skin, infrequent shampooing and weather conditions may all increase a person’s risk of developing seborrheic eczema (1).

Nummular eczema is characterized by coin-shaped patches of irritated skin, most commonly located on the arms, back, buttocks and lower legs, and may be crusted, scaling and extremely itchy.

1.3.6.6 Neurodermatitis (Lichenification) liv

Also known as lichen simplex, it is a chronic skin inflammation caused by a scratch – itch cycle that begins with a localized itch (such as an insect bite) that becomes intensively irritated when scratched. This form of eczema results in scale, patches of the skin on the head, lower legs, wrists or forearms (13).

The normal treatment of allergy such as avoidance of contact with allergens and administration of antihistamines does not bring relief to the patient. When inflamed eczematous skin markings become exaggerated and the skin becomes thickened and hardened. This is known as lichenified skin and since it is itchier than normal skin, a vicious cycle develops. Emotional stress plays a role in this disorder which is why an alternative name for this disorder is neurodermatitis. Most patients who suffer from it are tense, excitable and the urge to scratch is more of a bad habit like nail biting, than a disease (13).

1.3.6.7 Cross sensitization

A person who has reacted to one substance is most likely going to develop reactions to other materials even when the substances are chemically un-related. Sensitivities are usually specific but sometimes the body cannot distinguish chemicals of different structures.

Dermatitis can be caused by irritants or by true sensitization. Irritants like detergents remove lipids from keratin and allow the skin to dry excessively and split. This makes the epidermis more permeable to more irritants and sensitivity may occur. When skin eruption occurs, it is important to determine if it was due to exogenous contact or a specific hypersensitivity.

History of cause of dermatitis from a person is of very vital importance. It is important to determine the site of onset of the eruption since this will give a lead to the probable cause of lv

dermatitis. A band of erythema round the forehead will of necessity suggest a scarf as the sensitizer. Sensitivity of a person to a brand of face powder that has been in usage for a very long time is possible, because the user may just have taken years to become sensitive to the powder or the makers of the powder may have changed the constituents of the powder. Some example of areas of body prone to dermatitis and their likely sensitizers are:

• Scalp-hair dyes and scalp lotions

• Neck-ties, scarves, necklaces, perfume.

• Ears -hair nets, ear clips, hearing aids, ear drops and Glass frames.

• Trunk-clothing

• Genitals -clothing, contraceptives and deodorant.

• Arm-pits -deodorants, shaving powder and shaving sticks

• Thigh -suspender, clothing

• Ankles and feet-socks, stockings, shoe

Medicaments applied to the skin are a common cause of sensitization dermatitis and every application is capable of sensitizing someone (13).

1.3.6.8 Contact Dermatitis

These include irritant dermatitis and allergic dermatitis. Skin diseases are the most common of all reported occupational diseases and the majority of the causes are contact dermatitis, in which the sensitizer irritates the skin on first or multiple exposures; in either case, the result is skin inflammation (13). The clinical feature of contact dermatitis is violent inflammation of the epidermis and oedematous swelling which may stimulate urticaria (13). lvi

1.3.6.9 Irritant Dermatitis

Irritant dermatitis can be primary or secondary. A primary irritant such as a strong acid usually causes a response on first exposure, secondary irritants like soap, cosmetics cause an inflammatory response only when the irritant is used repeatedly (1, 2). Primary irritants cause pruritic erythema and ulceration while secondary irritants cause slow grade inflammation that stays for long periods.

1.3.6.10 Allergic Dermatitis

They could be classified as immediate (anaphylactic), intermediate (arthus) or delayed

(tuberculin) the most prominent being delayed hypersensitivity reactions. Allergy cannot occur on first exposure to an allergen, but some people can react abnormally with skin irritations to substances like shellfish on first exposure to them. These are not allergic reactions but idiosyncrasies of such persons.

In immediate allergic dermatitis or anaphylactic reaction, the allergen on first contact causes the production of antibodies, which sensitize tissue cells passively, such that subsequent administration of the allergens reaches the sensitized tissue cell, causing their injury and release of endogenous agents like histamines, kinins and prostaglandins and these agents cause further lvii

local changes that include contraction of smooth muscles, increased vascular permeability and oedema. The cells injured usually recover, though some may die (1).

In intermediate allergic dermatitis or arthus, the antigen combines with the antibodies in tissue spaces or in the circulation, to produce a complex. This causes a primary change which is massive infiltration of the extra vascular tissue. Then a secondary change occurs that changes the tissue and this depends on the composition and strength of the allergen.

Delayed (tuberculin) reaction is the major mechanism involved in allergic contact dermatitis. It occurs days after the first contact with an allergen is made; sometimes it may take months or even years to develop. Once the reaction is initiated it builds up in severity. Susceptibility to this type of sensitization may last a life time though it can be overcome in some cases (1, 13).

1.4 Factors influencing skin irritation

These include the sensitizer itself, climate and the host. The degree of skin irritation is a function of the intrinsic irritation potential of the test material, its concentration, its ability to remain bound to the skin and the texture of the exposed skin. Environmental conditions play a role in skin texture and its resistance to irritant substances. High humidity allows improved skin hydration and thus faster penetration of irritants; occlusion has same effects as it keeps the skin hydrated. Age and colour of the skin also influence irritant dermatitis. Aged skin is less prone to irritation than youthful skin, possibly because it is more difficult to penetrate an older skin than a younger one. Dark skinned persons seem less susceptible to irritants than lighter skinned individuals (2). lviii

Administration of more than one substance promotes skin irritation. A secondary irritant that is not irritating to the skin when applied alone may cause irritation when used as a surfactant or a keratolytic substance. Damaged or traumatized skin encourages skin irritation (1).

1.5 Patients attitude to skin infections

Most diseases of man would need subjective and objective information to be diagnosed but the skin is one organ which when diseased or traumatized can be noticed by all without asking.

People with skin diseases or conditions are very disturbed by their complaint in comparison with other medical conditions because skin diseases tend to make their victim have a leper like complex, a feeling of disgust and shame as most skin infections are on an organ which can be seen by all, as well as the fear that the contagious diseases may spread to family and friends who might on their own part try to avoid the sufferer. Skin infections are still a serious threat in the developing countries, Nigeria inclusive. This is more so as the issue of drug resistance of many of the causative organisms are on the increase (17).

It is a great challenge to treat skin infections as many patients with skin diseases believe that because the lesion is on the surface, it should be easy to cure and it is very difficult, almost impossible to convince a patient into thinking that his compliant has improved when he and others can obviously see it has not (1). A healthy, good looking skin usually implies a healthy person while an un-healthy, sick looking skin is the reverse. Looking good is said to be good business so most people would spend a fortune to keep a healthy radiant skin.

1.6 Wounds lix

A wound is a break in the skin, wounds are injuries usually caused by cut or scrapes that disrupts the continuity and integrity of the external surface of the body. This compromises the normal functioning of the skin. Wound healing is a response to the injury that sets into motion a sequence of events. With the exception of bone, all tissues heal with some scarring. The objective of proper wound care is to minimize the possibility of wound infection and it’s scarring

(18).

Types of Wounds

Wounds are divided into two types: - open and closed wounds.

1.6.1 Open Wounds

Open wounds vary with the type of object that caused it and with the manner in which the skin tissue is broken, there are six kinds of open wounds, incisions, lacerations, punctures, avulsion, abrasions and amputations, sometimes there could be a combination of these six types

(18).

1.6.1.1 Incisions

Incisions are commonly called cuts, and are wounds caused by shape-edged objects like razor, broken glass, knives, or surgical blades. Incision wound are cut neatly with smooth edges.

There is little damage to the surrounding tissue, they are the least most infected wound of all open wound types because the free flow of blood washes away many of the microorganisms that cause infections away from it (18, 19).

1.6.1.2 Lacerations

This type of wound are torn rather then cut. The edges are irregular with torn tissues below; such wounds are usually created by blunt objects like blunt knives. Apart from tearing lx

the tissues, they are also crushed. Lacerations are usually contaminated with dirt and other types of foreign materials ground into them so that they are likely to become infected.

1.6.1.3 Punctures

Punctures are caused by sharp objects that penetrate the skin and tissue to create a small surface opening. They can be created by nails, needles or bullets; the risk of infection is real in puncture wounds, especially if the penetrating object has tetanus bacteria on it.

1.6.1.4 Abrasions

Abrasions are sometimes called grazes, and are superficial wounds caused mostly by a sliding fall on a rough surface in which the top skin is scrapped off. Parts of the body with thin skin like the knees and elbows are most prone to abrasion. This kind of wound can be infected easily because dirt and germs are usually embedded in the tissues from the rough surfaces (18, 19).

1.6.1.5 Avulsion

Avulsion is tearing away of tissue partially or completely from the body part. Sometimes, the torn tissue may be surgically re-attached to the body part.

1.6.1.6 Amputation

This is the non-surgical removal of a limb from the body. Bleeding is usually heavy and shock may occur. Like in a vulsed tissue, the tissue can be surgically re-attached.

1.6.2 Closed Wounds

Closed wounds are also called contusions or bruises; they are caused by a blunt forceful blow/trauma to the skin and soft tissue, leaving the tissue under the skin damaged but the outer layer of skin intact. These injuries may require minimal care as there is no opened wound but lxi

hematoma may develop and this demands evacuation. Hematomas occur when blood vessels are damaged such that it causes blood to gather under the skin (18, 19).

1.6.3 Microbial Contamination of Wounds

Open wounds are prone to infections especially infection by bacteria, these infections may provide an entry point for systemic infections. Microbial infected wounds heal slowly and often result in the production of offensive smelling exudates and toxins that kill regenerating wound cells. Antibacterial and antifungal compounds of natural origin may help prevent this from occurring (20).

Infection is the presence of microbial pathogens proliferating in a wound, causing tissue damage and eliciting inflammatory responses (21). A number of microorganisms are found to infect wounds among which are P. aeruginosa, S. aureus, S. faecalis, E.coli, Clostridum perfringes, C. tetran , Coliform bacilli, Herbal enterococcus (18). Use of herbal extracts may prevent infection that may lead to sepsis (22).

1.6.4 Wound Healing

There are stages of the wound healing process.

1.6.4.1 Clotting/Inflammation stage/phase

This begins with the injury itself. In this phase, is bleeding, immediate narrowing of the blood vessels, clot formation and release of various chemical substances into the wound that will begin lxii

the healing process, occur, and specialized cells clear the wound of debris over the course of several days (20, 23).

Clotting is the first step in the healing of a wound, prevents any further blood loss. Clotting or coagulation is a rapid response to bleeding that initiates homeostasis to stop excessive loss of blood. When injury occurs, the vascular integrity of the injury area is broken; there will be extravasation of the blood into the wound site (24). Platelets are the highest number of blood cells at an injury site. When blood from the wound comes into contact with collagen of the torn muscle fibres, the blood platelets adhere to the collagen leading the platelets to secret fibrinogen, which is converted to fibrin by thrombosis. Also released are monocytes which in turn release growth factors and cytokines that are important in the maintenance of the inflammatory reaction and stimulate cell proliferation to enhance wound healing. Thromboxane, histamine, prostacyclines, prostaglandins, serotonin and neutrophils are also released (24).

Prostaglandins and thromboxanes cause vasoconstriction of the blood vessel to prevent blood loss but histamine, also in the extravasted blood, can counteract this constriction, and causes vasodilation thus making the blood vessels porous. Blood proteins leak out of the porous blood vessels into extravascular spaces, increase its osmolarity and in a bid to balance this raised osmolar load, draws water into the wound site, hence making it oedematous (20, 23).

The neutrophils clean the wound area by secreting enzymes that break down the damaged or injured tissue into wound debris. They also phagocytose the wound debris and contaminating bacteria.

Platelets attract monocytes to the wound sites where they mature into macrophages. The macrophages phagocytose bacteria and wound debris, and also release growth factors and lxiii

cytokines that instill inflammatory reactions and stimulate healing by production of new tissue cells to re-epithelialise the wound.

1.6.4.2 Proliferative phase

In the proliferative phase, a matrix of cell forms. On this matrix, new skin cells and new blood vessels form and it is these new blood vessels known as capillaries that give a healing wound its pink or purple-red appearance. The capillaries supply the rebuilding cells with oxygen and nutrient to sustain the growth of the new wound cells, and also promote the production of the protein- collagen. Collagen acts as the framework upon which the new tissue is built.

1.6.4.3 Angiogenesis

Endothelial cells that originate from the blood of uninjured wound area migrate through the extracellular matrix to the wound area. They become capillaries that supply the rebuilding wound cells with oxygen and nutrients (25). The endothelial cells are attracted to the wound area by the presence of growth factors and fibrin present and by shortage of O2 (26), the endothelial cells continue to grow and proliferate in the wound area, a process that decreases as O2 supply to the site is increased.

1.6.4.4 Fibroplasia

After the development of new blood capillaries, fibroblasts in the normal tissue adjacent to the wound tissue, proliferate and migrate to the wound site. They mingle with the wound and produce reticular fibres which progress into collagen fibres. Fibroplasia takes about 3-4 days after the injury. After fibroplasia is the granulation process, in this process, the new blood vessels, inflammatory cells, growth factors, endothelia cells and fibroblasts attach and grow on the collagen matrix that had been laid down by fibroblasts (26). lxiv

1.6.4.5 Epithelialisation

This is the process of laying down new skin or epithelial cells. The skin forms protective barriers between the wound and the environment. Epithelialisation begins within a few hours of the injury to 48 hours in a clean sutured wound; open wounds take longer time because the inflammatory phase is prolonged (27).

The epithelial cells originate from keratinocytes of the wound edges, hair follicles and sebaceous glands. The epithelial cells proliferate over and across the wound and when they meet, proliferation stops.

1.6.4.6 Re-modeling phase

This begins after 2-3 weeks or months depending on the type of wound. The collagen frame is more organized as there is continual accumulation of collagen.

The blood vessel density becomes less and the wound losses its pinkish colour over time depending on the size of the wound; the wound area increases in strength, and eventually reaches about 50%- 80% of the strength of uninjured wound (28).

1.6.5 Factors affecting wound healing

For a wound to heal successfully, its stages of healing- hemostasis, inflammation, proliferation and remodeling must occur in the right sequence at appropriate time frame. Any factor that disrupts this sequence of healing causes improper wound healing or an impaired wound healing.

These factors can be local or systemic. Local factors directly influence the characteristics of the wound while systemic factors are the health /diseases status of the individual that affects his/her wound healing ability (28).

lxv

Local factors

1.6.5.1 Oxygenation

Adequate oxygenation is essential to wound healing, because O2 is necessary for cell metabolism and production of adenosine triphosphate (ATP) which is critical for all wound- healing processes. Oxygen prevents wound infection, induces angiogenesis, increases keratinocytes differentiation and re-epithelialisation. It enhances fibroblast proliferation, collagen synthesis and wound contraction (28). Wound disrupts the vascular distribution in the wound area that subsequently depletes its oxygen content. Depletion of O2 (hypoxia) after injury triggers wound healing, hypoxia induces cytokines and growth factor production from macrophages, keratinocytes and fibroblasts but prolonged hypoxia delays wound healing (28). Wounds on the neck and face which are greatly supplied with blood heal rapidly while those on the extremities heal slowly. Diseases that compromise blood supply/circulation like diabetes slow down healing because proper oxygen level is crucial for optimal wound healing though initial hypoxia at wound areas stimulates wound healing by the release of growth factors and angiogenesis.

Oxygen is important for sustenance of the healing process (28).

1.6.5.2 Infection

Intact skin usually has microorganisms sequestered on its surface and once the skin is broken by injury or diseases, these microorganisms get access to the underlying tissues to cause contamination. Contamination is the presence of non-replicating organisms on a wound.

Replication of organisms in the wound is termed colonization; there is usually no tissue damage at this stage. If the host reaction to the presence of an organism on it is negligible, then the organism is said to be colonizing the wound. Colonized wounds heal without the need for antibiotics as the host immune system can counteract the activities of the organisms (28). When lxvi

the tissues around/ local to the wound begins to respond to the continuous replication of microorganism by eliciting local damaging tissue responses, there is local infection. Invasive infection is the presence of these replicating organisms within the wound that is accompanied by a subsequent overall host injury (28).

Wound infection (Local/Invasive) occurs when the virulence factors expressed by the organisms in the wound out -competes the host immune system. This is evidenced by purulent drainage or exudates, erythema and fever.

Wound infection is a problem because the infection stops a wound from healing by prolonging inflammatory phase. The pathogenic microorganisms in the wound will compete with macrophages and fibroblast for the limited nutritional resources available at the wound site.

Wound inflammation is a normal stage of the wound-healing process and is vital for the removal of contaminating microorganisms because when these microorganisms are not effectively removed (decontaminated), inflammation stage is prolonged. Bacteria and their toxins can lead to the prolonged elevation of pro-inflammatory cytokines like iterleukin-1 and this elongates the inflammatory phase. If this continues, the wound may enter a chronic stage and fail to heal.

Prolonged inflammation increases the level of matrix metallo proteases, a group of proteases that degrade the intracellular matrix. With the increased protease content, a decreased level of the naturally occurring protease inhibitor occurs. This shift in protease balance causes growth factors that appear in chronic wounds to be rapidly degraded (28). The bacteria in infected wound occur as biofilms. Biofilms formation usually begins with the pioneer cells attaching to the wound surfaces, through adhesion. Once established on the wound, these cells grow and divide to produce micro-colonies which eventually coalesce to produce a bioflim. The resident cells within the biofilm are not exposed to attack by the immune system. Bacteria biofilms are less lxvii

susceptible to antimicrobial agents, (6). Mature biofilms develop protective microenvironment and are more resistant to conventional antibiotic treatment. Staphylococcus aureus,

Pseudomonas aeruginosa, B. hemolytia Streptococci and Escherichia coli are common bacteria in infected wounds (6, 28).

Many chronic ulcers probably do not heal because they have biofilms containing P. aeruginosa that shield them from phagocytic activity of invading polymorphonuclear, neutrophile and antibiotics (28).

1.6.5.3 Age

Aging causes delay in wound healing but not an actual impairment in terms of the quality of wound healing (6, 28). Age delayed wound healing is associated with an altered inflammatory response like delayed T-cell infiltration into the wound area. Delayed re-epithelialisation, collagen synthesis and angiogenesis were observed in animal studies of aged mice as compared to young mice (28).

Every stage of wound healing undergoes characteristic age-related changes like increased secretion of inflammatory mediators, delayed infiltration of macrophages and lymphocytes, impaired macrophage function, decreased re-epithelization of growth factors, delayed angiogenesis and collagen deposition, reduced collagen turnover and remodeling (6, 28).

1.6.5.4 Wound size

The healing time of a wound is related to its size. A small sized wound heals at a faster rate than a larger one.

1.6.5.5 Depth of wound type

The depth of a wound is proportionate to its healing rate; surface or superficial wounds heal faster than deep wounds. In deep wounds the injury affects much more tissue than a surface lxviii

wound and this disrupts a larger vascularisation, reducing blood supply and oxygen supply to the wound site, leading to a greater and more prolonged hypoxia. This insufficient perfusion and prolonged hypoxia amplifies the inflammatory stage causing impaired collagen synthesis and inadequate angiogenesis. Accumulation of metabolites in the hypoxic conditions of the wound increases their susceptibility to infection and this also impairs its healing (20).

1.6.5.6 Medication

Medications that interfere with clot formation, inflammatory response and cell proliferation have the ability to affect wound healing, e.g. drugs like glucocorticoid- steroids, non-steroidal anti-inflammatory drugs and chemotherapeutic drugs.

Glucocorticoid Steroids: Glucocorticoid steroids used as anti-inflammatory drugs inhibit wound repair by their anti-inflammatory effects, which suppresses cellular wound responses, fibroblast proliferation and collagen synthesis. Systemic steroids cause wounds to heal with incomplete granulation tissue and reduced wound contraction thereby increasing the risk of wound supra- infection. However, topical application of corticosteroids on wound, accelerates its healing, reduces pain and exudates (20, 28).

Non-Steroidal anti-inflammatory drugs: Non-steroidal anti-inflammatory drugs like Ibuprofen are used for treatment of inflammation and pain management. Animals wound healing studies suggest that systemic Ibuprofen has an anti-proliferative effect on wound healing; it decreased epithelialisation, reduced wound contraction, and impaired angiogenesis. Thus the majority of surgical patients are recommended to discontinue NSAIDS so that they do not have significant

NSAID activity at the time of their surgical wound repair; exception are patients on low –dosage of aspirin for cardiovascular diseases (20, 28). lxix

Chemotherapeutic Drug: Most chemotherapeutic drugs inhibit cellular metabolism, rapid cell division and angiogenesis. They also inhibit DNA, RNA, protein synthesis, resulting in decreased fibroblast and vascularization of wounds. They delay cell migration into the wound area, lower collagen formation, reduce proliferations of fibroblast. They do also inhibit contraction of wounds (20).

1.6.5.7 Nutrition

Nutrition affects rates of wound healing. Individuals with non-healing wounds often require special nutrients to improve the wound healing. Nutrients like carbohydrates, proteins, fats, minerals and vitamins affect healing process (28).

Glucose from carbohydrates is the main source of ATP; it provides energy for angiogenesis and deposition of new tissue.

Proteins are needed for capillary formation, fibroblast proliferation, collagen synthesis and wound -remodeling. Deficiency of protein affects all these processes and also affects the immune system, resulting in a decline in leukocytes, phagocytes and increased susceptibility to infection.

The major protein important for wound healing is collagen; it is composed of glycine, proline and hydroxyproline. Collagen is synthesized by the hydroxylation of glycine and proline in the presence of co-factors like ferrous ion and vitamins (29).

Arginine is a precursor to proline, which means that adequate amount of it, will be needed for collagen synthesis (29). Arginine stimulates wound healing by supporting collagen deposition, angiogenesis, wound contraction; it also improves immunity of the host. Another amino acid of importance in wound-healing is glutamine; which stimulates the inflammatory response that occurs in early wound healing (29). lxx

Vitamins: Vitamin C (L-ascorbic acid) is a very powerful antioxidant, that has anti- inflammatory effect too. Vitamin C is needed for collagen synthesis, fibroblast proliferation, angiogenesis and improved capillary fragility. A deficiency of vitamin C affects all these processes that are vital to proper wound healing, and reduces host immunity, thus increasing its susceptibility to wound infection. Vitamin A is an effective antioxidant that hastens collagen synthesis and proliferation (29).

Vitamin E (tocopherol) is an effective anti-oxidant that helps to maintain the integrity of cellular membranes by providing protection to it against oxidation. It also has anti-inflammatory properties. Topical application of vitamin E prevents scar formation in chronic wounds (29).

Minerals: Minerals are vital for adequate wound healing and their deficiency impairs wound healing. For example, magnesium is a co-factor for many enzymes needed for protein and collagen synthesis; copper is a co-factor for the optimal cross-linking of collagen; zinc is a co- factor for DNA and RNA polymerase, while iron is a co-factor too, involved in the hydroxylation of proline and lysine (29).

Systemic factors

1.6.5.8 Obesity

Obesity increases the risk of many diseases like coronary heart disease, type 2 diabetes, hypertension, dyslipidemia, stroke, respiratory problems. Obese people heal slowly because fat does not have a good supply of oxygen thus wound healing is impaired. Their numerous skin folds harbor microorganisms that contribute to infection and even administered antibiotics when given, do not help much as there is decreased delivery of the drug as well (29).

1.6.5.9 Host Immunity lxxi

Diseases like HIV infection and tuberculosis that compromise host immunity, impair the rate of wound healing. Inflammatory stage of wound healing is un-duly prolonged in such individuals because their body defense mechanisms are unable to accelerate the inflammatory phase of wound healing (30).

1.6.5.10 Health Status of an individual

Diseases that compromise blood supply such as diabetes, slow down wound healing.

Diabetic individuals exhibit impaired healing of acute wounds, are prone to develop chronic non- healing diabetic foot ulcers that are often caused by hypoxia. Hypoxia lengthens the inflammatory stage due to increased level of oxygen radicals in the wound that prolong healing.

Hypoxia also causes inadequate angiogenesis (30).

1.6.6 Models for the evaluation of wound healing activity

There are two models for studying wound healing namely, in vitro and in vivo models.

1.6.6.1 In vivo models

In vivo models are carried out with small rodents like rats and guinea pigs, and include:

1.6.6.1.1 Excision wound models

They are used to study the rate of wound contraction and epitheliazation (31). The wound is created by excising the full thickness of circular skin from an anaesthetized animal (32).

Wound contraction is assessed by measuring the wound diameter using translucent ruler. The edges of excised wounds are not in contact so contraction and epitheliazation are necessary for its healing process (33). This model studies two parameters - contraction and epithelialisation

(34).

1.6.6.1.2 Incision wound model lxxii

Longitudinal incisions are made on the shaved skin of selected animals under mild anesthesia. The parted skin is brought together by suturing. The skin breaking strength of the wound can be determined (35).

1.6.6.1.3 Dead space analysis

Dead space wound are created by making a pouch through a small opening in the skin of a rat (36). A polypropylene tube is implanted into the pouch beneath the skin and the wound is sutured. After about 10 days, the polypropylene tube is removed and the granulation tissue surrounding it is harvested. These regenerated tissues are cut in the form of squares along with the normal tissues on sides of the wound and both are studied histologically. The physical and mechanical breaking strengths of the tissues are studied (36). Hydroxyproline content of the tissues is studied and histological studies are carried out to examine the pattern of lay down for collagen (37).

1.6.6.1.4 Burn wound model

Burn inflicts extreme damage to the skin, causing tissue necrosis and body fluid exudation, which create a perfect medium for bacterial growth. (39). Partial thickness wound is inflicted upon anesthesied animals, by pouring hot molten wax of about 80oC into a metal cylinder with circular opening and placing on the back of the animal. Wound contraction and epithelialisation are then studied (37).

1.6.6.2 In vitro models

In vitro models are now widely used in wound healing research studies because of ethical reason, since they do not involve inflicting pain on live animals and for their usefulness in bioactive guided fractionation and determination of active compounds (20). An example of in vitro parameter studied is antimicrobial activity. lxxiii

1.6.7 Wound healing study parameters

1.6.7.1 Wound closure

Collagen makes up more than 50% of sutured wounds; hence, any substance that promotes collagen maturation enhances the process of wound healing (39). Wound closure or contraction is part of the proliferate phase of wound healing which is mediated by mainly fibroblasts (40).

Contraction of wounds can be studied by observing the wound healing and wound contraction percentage (%) is calculated using the following formula (41).

WDo – WDt x 100 …Equ. 1 Wound Contraction (%) =

WDo

WDo= wound diameter on day Zero

WDt= wound diameter on day t.

1.6.7.2 Epithelialisation period

This is the period of epithelial renewal after injury. It involves the proliferation and migration of epithelial cells towards the center of the wound (41).

1.6.7.3 Tensile strength

This indicates the quality of the repaired tissue and studies how the repaired tissue resists breaking under tension (40, 41). Tissues from the treated and control animals can be loaded between the upper and lower holders of a tensile testing machine and a load is applied that pulls the tissues apart. The load/weight that breaks the tissues is obtained and compared (42).

1.6.7.4 Increase in granulation tissue

Increased granulation tissue is associated with enhanced collagen maturation in dead space wounds (41). These wounds heal by laying down connective tissue, where more than 50% lxxiv

of the connective tissue is made up of collagen. Collagen is a fibrous protein component of connective tissue, and is made up of hydroxyproline mainly hydrolysine and glycine (39).

Increased levels of hydroxyproline suggest increased collagen turnover and subsequent increase in granulation tissues (43).

1.6.8 Existing therapy of wound healing

Topical antimicrobial therapy is one of the most important method of wound care (44).

Neomycin- bacitracin powder, (CicatrinR), gentamycin ointment, tetracycline ointment and nitrofurazone ointment are among the standard antibiotics used in wound healing (31, 42, 44).

Povidone-iodine cream is also used for wound healing purpose (42). Wound healing is not improved/affected by drug usage alone but by factors like nutritional status of the victim and his/ her clinical conditions like diabetes, obesity and anemia. Therefore wound management must involve a holistic approach (44).

1.6.9 Types of wound Healing

Once an injury has occurred and platelets from the damaged blood vessels come in contact with exposed collagen, its healing starts (45). Healing of wounds can be of three types- healing by first intention (primary healing), healing by second intention (secondary) and healing by third intention (Tertiary). This classification is based on the nature of the wound edges as it heals (44).

1.6.9.1 Healing by first intention

This occurs when the wound edges close with little or no inflammation resulting into a scar-less healed wound (44), surgical incision is targeted towards this type of healing where little or no post surgical tissue necrosis occurs. Primary healing occurs within hours of repairing a full- lxxv

thickness surgical incision by firmly suturing the wound edges together, which prevents granulation tissue from being visible, thus leaving little or no scar (28, 43).

1.6.9.2 Healing by secondary intention

There is formation of granulation tissues, which fill up the gap between the wound edges.

In this type of healing, significant loss of tissue occurs leaving the wound edge open to heal with scarring (44).

1.6.9.3 Healing by third intention

This occurs when the wound is left open until granulation form and falls before the wound edges are united together, this is more of late closure of a primary wound and it heals with scarring (43).

1.7 Natural products as sources of medicine

Traditional medicine is a major African socio-cultural heritage. It had been in existence for several hundreds of years, and was once believed to be primitive and wrongly challenged with animosity, especially by foreign religion dating back to the colonial days in Africa and subsequently by the conventional or orthodox medical practitioners. Today, traditional medicine has been brought into focus for meeting the goals of a wider coverage of primary health care delivery system, not only in Africa but also to various extents in all countries of the world (46).

Traditional medicine is defined by World Health Organization (47) as the sum total of knowledge or practice whether explicable or inexplicable, used in diagnosing, preventing or eliminating a physical, mental or social disease which may rely exclusively on past experience or observation handed down from generations, verbally or in writing. It also comprises therapeutic lxxvi

practices that have been in existence for hundreds of years before the development of modern medicine and are still in use today without any documented evidence of adverse effects.

The explicable form of traditional medicine can be described as the simplified scientific and direct application of animal or plant materials for healing purposes and which can be investigated, rationalized and explained scientifically. The use of Salia alba the willow plant

(containing the Salicylates) for fever and pains which led to the discovery of aspirin belongs to this form of traditional medicine. Herbal medicine is regarded by WHO as finished and labeled medicinal products that contain, as active ingredients, aerial or underground parts of some plants identified and proven in crude form or as plant preparations. They include plant juice, gums, fatty oils, essential oils e.t.c. (48).

There are several other official modern drugs today which were originally developed like aspirin through traditional medicine e.g. morphine, digoxin, quinine, ergometrine, reserpine, atropine etc, all of which are currently being used by orthodox medicine in modern hospitals all over the world.

The inexplicable form of traditional medicine, on the other hand, is the spiritual, supernatural, magical, occult, mystical or metaphysical form that cannot be easily investigated or explained scientifically e.g. the use of incantations for healing purposes, oracular consultation in diagnosis and treatment of diseases. The explanation is beyond the ordinary scientific, human intelligence or intellectual comprehension (48).

The WHO has since urged developing countries of the world to utilize the resources of traditional medicine for achieving the goals of primary healthcare. This injunction stems from lxxvii

the various advantages of traditional medicine namely: low cost, affordability, ready availability, accessibility, acceptability and low toxicity (49).

Antimicrobial and wound healing activities of traditional medicines have been employed in folk medicine for wound care. Most of these plants exhibit wound healing activities or possess antimicrobial and other related activities that improve the wound care (44). The different plants used for wound care do contain active constituents that are nutritive in action (50).

The use of plants for wound care/healing is gaining a lot of attention and about 1-3 % of traditional medicine in use are for the treatment of skin problems and wounds (33). Spermacoce verticillata is an example of a local plant that is claimed by herbalists to be useful in the treatment of skin infections, the leaf extracts being used to treat leprous conditions, furuncles, ulcers and gonorrheal sores (51). It has thus become necessary that plants should be formulated into biologically active ointments with wound healing properties for local application at wound sites (52).

1.8 Plants with potential wound healing properties

Medicinal plants have been in use in folk medicine for wound care, as they possess the ability to directly improve wound healing or have its antimicrobial activities that are beneficial to wound care (53). There are certain plants that can improve wound care by a combination of these properties (44).

Ageratum conyzoides L (Asteracege): The leaves when applied to wounds act as antiseptics that aid its quick healing (54). It contains alkaloids and tannins. The root extract ointment showed significant wound healing activity and this activity is attributed to the antimicrobial and haemostatic action of the plant’s individual phytochemical or their combined actions (54). lxxviii

Allium cepa L. (Liliaceace): Studies of the alcohol extract of tubes of Allium cepa (onions) showed that it contains tannins and flavonoids that exhibited wound healing action in excision and dead space wound models. Their action was attributed to the presence of free radicals, scavenging and antibacterial action of their phytochemicals (55).

Aloe vera (Asphodelaceae): Topical application and oral administration of Aloe vera gel to rats with dermal wounds increased the collagen content of the granulation tissue (56). It seems that

Aloe-vera improves first and second burn wound healing but impairs wound healing of severe burns (57).

Alternanthera brasiliana Kuntz (Amaranthaceae): Photochemical screening of Alternanthere brasilian a revealed the presence of alkaloids, steroids and terpenes. Research has shown that topical application of leaves of this plant improved fibroblastic deposition, angiogenesis and wound contraction (58).

Anthocleista nobilis G. don (Loganiaceae): Studies on the plant show that it had wound healing activities; inhibited bacterial growth and protected the fibroblast cells from oxidants injury (42).

Areca catechu L (Arecaceae): Studies on Areca catechu revealed the presence of alkaloids, and the alkaloidal fraction was shown to improve the healing of incision wounds by increasing the breaking strength of the wound (39).

Azadirachtha indica (Meleaceae): Studies have shown that alcohol leaf extract of Neem

(Azadirachtha indica) was useful in treating ring worm, eczemas and scabies. Its leaf extract and oil from its seeds showed antimicrobial activity that kept wound treated with it, free from secondary microbial infections. It also inhibits wound inflammation as effectively as cortisone acetate and this aids wound healing (57). lxxix

Calotropis gigantea L. (Asclepiadaceae): A study made on topical application of latex of

Calotropis gigantean showed that it promoted collagenation of wound (31). It also increased breaking strength and hydroxyproline of wounds (59).

Carica papaya L. (Caricaeae): The latex of Carica papaya contains cysteine endopeptidases- papain, caricain, chymopapain and endopeptidase. These antioxidants aid wound healing (60,

61).

Catharanthus roseus L (Apocynaceae): Research has shown that ethanol extract of this plant is vital in aiding the wound healing of diabetics (45).

Centella asiatica (Makinlayoideae): Phytochemical analysis of the plant revealed the presence of triterpenes and asiaticoside. Its aqueous extract promoted wound healing when applied topically on open wounds in rats (57).

Cocos nucifera L. (Arecaceae): Cocos nucifera significantly promotes wound contraction and decreases epithelialisation period in burn wound model (45).

Cordial dichotoma (Boraginacea): Studies on this plant showed that it had wound healing potential as claimed traditional medical practitioners (62).

Dissotis theifolia (Melastomataceae): Studies on Dissotis theifolia showed that it possesses antibacterial and wound healing effect when formulated as ointment, on infected excision wound model. Its methanol stem extracts upon phytochemical screening revealed the presence of saponins, tannins, glycosides, flavonoids, terpenoids, carbohydrates, alkaloids and steroids (53).

Other plants of importance in wound healing that have been scientifically proven are Elaeis guineensis Jacg (Mackinlayoidae) that improves the different phases of wound repair (40),

Euphorbia heterophylla whose aqueous and ethanol extracts showed significant wound healing upon topical application in rats (63). lxxx

Ficus religiosa leaf extracts ointment improved healing of wounds (52). Ginkgo biloba contains flavonoids and terpenes and these constituents provides its wound healing activity (57).

Helianthus annus formulated as ointments, upon application on wounds, hastened its healing

(57). Hoslundia opposita has antibacterial and antioxidant properties which inhibit bacterial growth and protect fibroblast cells against oxidant injury (42).

Studies by Raina et al (57) on Hydrocarpus wightiana paste applied on wounds showed that it hastened epithelialisation period. Raina et al. (57) again confirmed the antibacterial wound healing activity of Hypericum prolificum. The juice of Jasminum auriculatum when applied topically on excised wounds in rats promoted wound healing (57). Research carried out on

Jatropha curcas by Shetty et al. (64) showed that it hastened wound healing.

The phytochemical analysis of Lantana camara revealed flavonoids and triterpenoids whose antimicrobial effect is thought to increase the rate of burn wound contraction (45). Flavonoids, tannins, steroids and saponins were found in Lawsonia inermis and this plant showed significant wound healing activities on incision and excision wound models (65). Mimosa pudica is found to be rich in tannins and its aqueous extract increases the rate of wound contraction (66).

Napoleona imperialis formulated into ointment showed wound healing activity comparable to that of Cicatrin®, a wound healing antibiotic (44).

Phytochemical screening of Ocimum kilimandscharicum revealed the presence of flavonoids, tannins and proteins and its aqueous leaf extract possesses wound healing property that is attributed to its ability to increase the rate of wound contraction and epithelialisation (36). lxxxi

Studies carried out on alcohol and aqueous extracts of Ocimum sanctum showed that they significantly increased wound breaking strength (67, 68).

Some other plants that possess wound healing activities are Phyllanthus niruri (41), Quercus infectoria (69), Bubia cordiofolia (32), Trichosanthes dioica (70), Tridax procumbens (57), and

Verononia arborea (43).

1.8.1 Plant phytochemicals of wound healing importance

Plants with medicinal properties perform these activities through their phytochemical constituents called active principles. Some phytochemicals shown to possess wound healing activities include:

1.8.1.1 Flavonoids

They are widely distributed in nature (69), occurring in fruits, vegetables, herbs, beverages, tea, beer and chocolates (70). They have a C6-C3-C6 backbone, with many structural varieties due to their conjugation to sugars at different sites of the molecule (68). They are known to possess free radical scavenging effect and a potent antioxidant effect too. These properties are believed to be important components of wound healing (55). The flexibility of the electron in the benzene nucleus of flavonoids accounts for their antioxidant and free radical scavenging properties and the structural resemblance between the flavonoids aglycone and many substances inherent in the biochemistry of human biological cells of nucleic acids coenzymes, steroids, neurotransmitters. This is why they can inhibit receptors, enzymes and neurotransmitters (71).

The antimicrobial activities of many plants have been attributed to their flavonoids content. (71), hence they have the ability to prevent wound infection. Quercetrin isolated from Hypericum lxxxii

perforatum inhibits the growth of microorganisms (72). Santin, a flavonoid from Tanacetum parthenium exhibits anti-inflammatory activity by inhibiting the cyclo-oxygenase and 5- lipoxygenase pathways.

1.8.1.2 Tannins

Plants used for their wound healing and anti-inflammatory properties are known to contain a high amount of tannins (70). Tannins are phenol, i.e. compounds found in most herbal products used for wound healing. They possess antimicrobial and astringent properties which are responsible for wound contraction and epithelialisation (43).

1.8.1.3 Terpenes and Terpenoids

Terpenes are known for promotion of rapid wound healing (57). Terpenoids promote wound healing via their astringent and antimicrobial properties that improve wound contraction and increased rate of epithelization (40).

Asiaticoside is a terpene found in the plant, Centell asiatica, and is known to improve wound healing and duodenal ulcers (71). Terpenes are natural products, derived from plants that have medicinal properties and biological activities. They are widespread in nature, mainly in plants as constituents of essential oil, particularly conifers. They are large and varied class of hydrocarbons, but oxygen – containing compounds such as alcohol, aldehydes or ketones

(terpenoids) are also found (73). The structure of terpene is repeated isoprene unit (C5H8)n and they are grouped according to the number of such repeated units.

1.8.1.4 Saponins

Saponins possess antioxidant and antimicrobial activities that are known to promote wound healing (54). Triterpene saponin is known to possess immunomodulatory properties (71). lxxxiii

The plant Centella asiatica contains asiaticoside a triterpene saponins. When this is applied topically, twice daily, for seven days on wound, 56 % increase in hydroproline resulted. There was increased tensile strength and collagen content with better epithelization (56). Saponins are glycosides with a distinctive foaming characteristic, bitter and acrid taste (56, 71). They are phytochemicals which are found in most vegetables, beans and herbs (74). Saponins are structurally related to steroid hormones and vitamin D. They consist of polycyclic aglycone that is either a choline steroid or triterpenoids attached via C3 and an ether bond to a sugar side chain.

The aglycone is referred to as sapogenin. They are derivable from plants like soap worth

(Saponeria spp) and soap berry (Sapindus spp). They are also found in Lobelia inflata, Urginea maritima and Bellis perennis (74).

Saponins are used in sneezing powder, emetics and cough syrups. Some are diuretics while others have the ability to reduce serum cholesterol by preventing its re-absorption after it has been excreted in the bile. They are anti inflammatory and anti cancerous, and have high antimicrobial activities. Saponins can cure eczema (74, 75).

The saponins in official saponin drugs are mainly triterpene derivatives, with a smaller number of steroids. All triterpene saponins possess hemolytic activity, which varies from strong to weak, depending on the type of substitution. Steroid saponins are non-hemolytic. Saponins are detected by exposure to UV-254 nm or Uv-365 nm, but with vanillin - Sulphuric acid reagent, saponins form mainly blue or blue – violet and sometimes yellowish zones (76).

1.8.1.5 Alkaloids

Alkaloids have antioxidant and antimicrobial properties which are known to promote and improve wound healing process (53). An alkaloid allantion, found in Symphytum asperum and

Symphytum caucasicum, is thought to be responsible for their wound healing property (77). lxxxiv

1.8.1.6 Plant vitamins

Vitamins A is necessary for epithelial and bone tissue development, immune defense and cellular differentiation (56). Vitamin C stimulates the synthesis of collagen (40), is an antioxidant, enhances neutrophil function and increases angiogenesis. Vitamin E minimizes/prevents scarring (40, 56).

1.8.1.7 Cardiac glycosides

These are drugs that contain steroids, used in the treatment of congestive heart failure and cardiac arrhythmia (76). They are found as secondary metabolites in several plants and a few animals. The plant sources include Digital purpurea (Foxglove), Digitalis lanata, Strophanthus gratus and Strophanthus kombe (78).

Structurally, glycosides consist of a glucose moiety attached to a steroid component called aglycone. They are structurally derived from the tetra cyclic 10, 13, - dimethylcylopentanoperhydrophnanthrene ring system (76, 78). Before exhibiting their cardio tonic effect, the aglycone a molecule that is bioactive in its free form but inert when conjugated must be detached from the carbohydrate, by the breakage of the glycoside bond by water and enzymes. The cardiotonic agents increase the force of heart muscles contraction without a concomitant increase in oxygen consumption. The myocardium thus becomes a more efficient pump and is better able to meet the demands of the circulating system. Example of cardiac glycosides from natural products includes: quabain, cymarin, oleandrin, theretoxin, digitoxin, tecomin, digitalin and cheiranthin (76).

1.9 Antimicrobial agents lxxxv

These are compounds that can kill or inhibit the growth of microorganisms, and may have activity against a wide variety of microorganisms like bacteria, fungi, viruses etc. Such an agent is called a broad spectrum antibiotic. A narrow spectrum antibiotic on the other hand, exerts its activity on just few microorganisms and may not be very effective as an antimicrobial agent (7,

79).

The least acceptable effect of any antimicrobial agents is the inhibition of growth of microorganisms. An antimicrobial agent that inhibits growth of microbes is termed a microbiostatic agent while it is called microbiocidal if it kills the microorganisms. Every drug possesses some level of unwanted effects but an ideal antimicrobial agent should have tolerable levels of side effects on man and animal so it is very important that it is selectively toxic to the microorganisms only and not irritant nor sensitize the animal or man it is used on. Many microorganisms have the ability to develop resistance to the cidal or static effects of antimicrobial agents and no antimicrobial agent will be of much use if microorganisms can in- activate it rapidly. Hence, it is desired that an antimicrobial agent must have the capacity to resist the harmful effects of microorganisms. Medicinal products of natural and chemical origin can sometimes undergo physical and chemical changes such as oxidation, reduction, photolysis, hydrolysis, and these physicochemical changes can bring about degradation of the drug product.

It is desired that any ideal antimicrobial agent should retain its physicochemical and antimicrobial properties while in use or on storage (7, 79).

1.9.1 Determination of an antimicrobial agent’s spectrum of activity lxxxvi

An antimicrobial agent can inhibit or kill microorganisms. The extent to which it does this can be estimated by the determination of their spectrum of activity. This involves bringing different microorganisms in contact with the test antimicrobial agent.

The spectrum of activity of the antimicrobial agent is assessed by the number and type of organisms killed or inhibited by it (6). The carpet agar plate and the cup agar plate methods can be used to determine the agents’ spectrum of activity.

1.9.1.1 Carpet plate method

A known volume of the test organism is used to streak the surface of an already solidified nutrient media in a Petri dish. Sterile paper discs are then impregnated with the antimicrobial agent, placed in the plates and incubated at appropriate temperature and time. Operational conditions used for microbiological techniques are normally specific so that the experiments can be reproduced in any repeat test (8, 80).

The results are taken after a specified time as zones of growth inhibition.

Organisms not susceptible to the antimicrobial agent will grow close to paper while those sensitive to it grow away from it. The distance of the organisms from the paper discs are called inhibition zone diameters (IZDs) and the higher the IZD, the more sensitive is the organism to the antimicrobial agent and vice versa (81). lxxxvii

Organisms can be categorized as sensitive, intermediate or resistant to an antimicrobial agent based on interpretation of IZDs from National Committee for Clinical Laboratory Standards

(NCCLS) IZDs guide-lines chart (8).

A regression analysis can be done on the obtained IZDs data and a line of best fit drawn.

Regression equations are formed and from the straight line charts, approximate 1ZDs corresponding to various sample concentration can be estimated.

1.9.1.2 Cup agar plate method

The microorganisms are streaked on already solidified agar or mixed with the molten agar just before it gets cold and poured into plates. Then instead of using paper discs as the reservoir for the antimicrobial agent, a cork borer is used to bore holes in the solidified agar to create cups into which the antimicrobial solutions are filled (79).

In both plate and cup agar methods the antimicrobial agent diffuses from their reservoir into the medium, where it inhibits the growth of sensitive microorganisms. Sensitive microorganisms will have appreciable zones of growth inhibition that are seen as clear zones or areas of no visible microbial growth around the cups/discs, while resistant microorganisms will show no appreciable zone of growth inhibition (8, 79).

1.9.2 Biostatic action of antimicrobial agents

Antimicrobial agents are termed biostatic when they inhibit the growth of microbial cells without necessarily killing the microbes. To assess such antimicrobial agent, a quantitative comparison of the biostatic actions of the agent and a reference agent that involves the determination of the minimum inhibitory concentration of both antimicrobial agents (test and lxxxviii

reference) that inhibits the visible growth of a specified test microorganism under identical experimental conditions will be carried out. The assessment parameter for biostatic activity of an antimicrobial agent is the minimum inhibitory concentration (MIC) (7).

1.9.3 Minimum inhibitory concentration (MIC)

This is the least concentration of a particular antimicrobial agent that can inhibit the visible growth of a specific microorganism at specified experimental conditions (79). MIC can be determined by various methods, like

- The broth dilution method

- Agar dilution method.

- Agar diffusion method

- Concentration gradient method

1.9.3.1 Broth dilution method

A known concentration of an antimicrobial agent is diluted serially in an arithmetic order called serial doubling dilution. This produces dilutions of antimicrobial agent in decreasing order from the first tube to the last, such that the concentration of antimicrobial agent decreases by half from one tube to the next (79).

A known volume of the microbial test culture is added into the broth/antimicrobial agent tubes and incubated at an ideal temperature for the type of test organism involved. lxxxix

Microbial growth in the test tube is seen as the presence of turbidity in the incubated tubes, in- contrast to a clear sterile nutrient broth. The concentration of the antimicrobial agent that inhibits growth of the test organism is taken as MIC of that agent on a specific organism under stated experiment condition (79).

1.9.3.2 Agar dilution method

This method is preferred to the broth dilution for assessing herbal extracts and coloured substances which would not be feasible with broth dilution method.

Serial dilutions of the antimicrobial agent are made with sterile water instead of nutrient broth.

Each dilution is then mixed with equal volume of double strength sterile molten nutrient agar. By this the final mixture is thus brought back to a normal strength nutrient agar. They are then poured into Petri dishes and allowed to solidify. Known volume of the test microorganism is then streaked on the surface of the agar and kept for one hour for pre – diffusion before being incubated at appropriate temperature and time conducive for the test organism. Microbial growth is observed as colonies of microbial cells on the surface of the agar. The least concentration of the agar plates that inhibits growth of visible microbial cells is taken as the MIC (7, 8, 79).

1.9.3.3 Concentration gradient technique

Two layers of agar are formed in a plate in the form of wedges. The lower wedge is first formed; containing a known concentration of the test antimicrobial agent. The upper wedge is then formed on the lower wedge free of the antimicrobial agent. After formation of the wedges, a pre – diffusion period is observed so that the agent in the lower wedge can diffuse into the upper wedge to reach its upper surface, because of the slant nature of the lower agar wedge. Varied xc

concentrations of the antimicrobial agent are delivered to the surface of the upper wedge such that a concentration gradient is formed. A known volume of the microbial test suspension is then spread on the surface of the upper wedge and the plate is incubated at appropriate conditions of incubation for that particular test organism. The area of the surface of the plate with sub- inhibitory concentrations of the antimicrobial agent will show growth while part of the inhibitory concentration will show no growth (7).

If a sensitive microorganism is exposed to a concentration gradient of an antimicrobial agent on an agar medium, the zone of microbial growth formed along the concentration gradient will terminate at a point corresponding to the MIC.

XYZ ….Equ. 2 MIC = Y

Where Z= Concentration of the antimicrobial agent in the lower wedge

X= Length of zone of growth

Y= Length of possible zone of growth

Another version of the concentration gradient technique using a performed concentration gradient of an antimicrobial agent on a strip of paper is called the E-test.

The strip is placed on the medium and the antimicrobial agent in it diffuses into the medium to inhibit growth of the microorganisms. The point along the strip where growth terminates is known as the MIC point and can be read directly from the already calibrated concentration strip

(7). xci

1.9.4 Biocidal Activity

This is the measure of the ability of an agent (antimicrobial) to kill all the microbial cells in an enclosed environment (79). The effectiveness of biocidal action can be measured by:

• Cell - killing rate

• D – Value

• Extinction time

• Minimum bactericidal concentration (MBC)

1.9.4.1 Cell- killing rate

This is a measure of the rate at which a known concentration and known volume of an antimicrobial agent kills the microbes in an enclosed environment/area.

Microorganisms do not die in singles but as batches, similar to their growth that occurs in batches and not singles. The number of microorganisms that die or reproduce will always be dependent on the entire number of microorganisms in that system, and this occurs in an exponential pattern (7, 79).

In Nt = In No – kt. ….Equ. 3

No = Population of microbial cells at zero time.

Nt = Population of microbial cells surviving at time t.

K = Death rate constant. xcii

When a graph of In Nt/No is plotted against time (t), a linear graph is obtained. The slope of the graph is the death rate constant (k).

The ks of different antimicrobial agents can be obtained under same experimental conditions and compared. The higher the killing rate constant, the better is biocidal activity (79).

1.9.4.2 D-value

This is the time it takes to reduce the population of microorganisms in a system by one logarithmic cycle or the time it takes to reduce the population of the microorganisms by 90% of their original population. D–values of different antimicrobial agents can be determined under same experimental conditions and compared. The smaller the D-value, the better the biocidal action.

1.9.4.3 Extinction time

This is the time it would take a known volume and concentration of an antimicrobial agent to kill all the microorganisms in an enclosed environment. The lower the extinction time, the better the microbiocidal effect of an antimicrobial agent. Under standardized experimental conditions, the concentration of the antimicrobial agent C will have an exponential relationship with the extinction time (79).

Cn t = k ….Equ. 4

Taking logarithm of both sides

n log C + log t = log k ….Equ. 5

Log t = log k- n log C ….Equ. 6 xciii

A plot of log t against log C gives a straight line graph whose slope is n, known as dilution coefficient. Changing the concentration of n by dilution, changes extinction time. Dilution produces increase in extinction time for antimicrobial agents with high n-value while for antimicrobial agents with small n-values, dilution produces little or no significant changes in their extinction time.

1.9.5 Minimum biocidal (bactericidal) concentration (MBC)

Minimum bactericidal concentration (MBC) is an extension of the MIC; it is the minimum concentration of antimicrobial agent that can kill all the microbial cells in an enclosed environment, while MIC inhibits growth of microbial cells (8).

The MBC of an agent can thus be determined from the broth dilution or agar dilution method of determining MIC. The MIC agar plates or broth tubes that showed no growth are used in determination of MBC. A loopful of the reaction mixture is taken from the broth tubes or a disc of the agar is taken from the agar plates and transferred into fresh nutrient broth without antimicrobial agent and incubated at appropriate experimental conditions for 48 hours. After this period, the tubes are observed for microbial growth or no growth. Turbidity of the broth is seen as growth while a clear broth signifies no growth of microbial cells. The minimum concentration

(from the tubes or plates) that produces complete cell death is the minimum biocidal concentration (MBC).

Cultures from MIC tubes or plates that are used for MBC carry along with them some of the antimicrobial agent, and effect of the agent needs to be stopped so that the microbial cells are freed from further action of the antimicrobial agent (79). xciv

Antimicrobial culture in the recovery medium inactivates the antimicrobial agent while for others, specific inactivating agents are required. For example if the antimicrobial agent used is an antibiotic of penicillin origin, then the enzyme penicillinase is used as its in-activator (7, 8, 79).

1.10 The use of antibiotics in managing microbial infections

The term “antimicrobial” is a general term used to refer to all substances that can systematically inhibit (microstatic) or kill (microcidal) microbial cells regardless of their origin (7), while

“antimicrobial agent” refers to any chemical substance produced by plants and all other microorganisms either in-vivo (in the body of the host) or in-vitro (outside the host). These antimicrobial substances have least toxicity to the host cells (selective toxicity). They could be in various forms such as antibacterial, antiviral, antifungal, antiprotozoa and antihelmintic. The antibacterial and antifungal activities vary with the species of the plants. Although, hundreds of plant species have been tested for antimicrobial properties, the vast majority has not yet been adequately evaluated (82-84). In recent years, antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world (49, 84).

An ideal antimicrobial drug exhibits selective toxicity (82). This implies that the drug is harmful to the parasite without being harmful to the hosts. The mechanisms of antimicrobial activity include the following;

i. Inhibition of synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)

which are both nucleic acids. Example is trimethoprim/sulphanamides.

ii. Inhibition of cell wall synthesis. Example is penicillin/cephalosporins

iii. Inhibition of protein synthesis. Example is aminoglycosides/chloramphenicol. xcv

iv. Alteration in the permeability of cell membrane component leading to leakage of

intracellular component of the cell. Example is tetracycline.

v. Inhibition of ergosterol synthesis in the fungal cell membrane. Example is imidazoles.

vi. Interaction with ergosterol, a fungal cell membrane that leads to pores formation

through which essential fungal cell constituents are lost. Example is amphotericin.

vii. Interference with microtubule function. Example is griseofulvin.

However, it is possible that antimicrobial agents can act through more than one of the mechanisms mentioned above.

1.10.1 Plant as source of antimicrobials

Plants use as medication is as old as man’s origin. Man through careful observation and use has identified various medicinal plants that can treat and prevent various ailments. Chaulmoogra oil from species of Hydrocarpus gaertrn was one of the earliest records of use of plants for medicinal purpose. It was used for treatment of leprosy and its record was found in the

Pharmacopoeia of the Emperor Shen Nung of China between 2700 and 3000 B.C. By this period, man was aware of the medicinal uses and properties of most plants in his environment along with their toxic effects. Hippocrates, a Greek medical doctor was referred to as the father of medicine.

He was the first to regard medicine as a science and his Materia medica consisted essentially of herbal recipes (84).

1.11 Antimicrobial resistance

Sometimes microorganisms do not respond to hitherto sensitive antimicrobial agents. This is called resistance. The spread of drug resistance pathogens is one of the most serious threats to the successful treatment of microbial disease (85). Reports have it that bacteria have the ability to xcvi

evolve defense mechanisms against antibiotics and can become resistant to their effects (86). The more an antibiotic is used, the more likely that bacterium will learn how to evade the affects of antibacterial agents on it (87). Most of the antimicrobial resistance, which is now making it difficult to treat infections, is due to extensive use and misuse of antimicrobial drugs, which have favored the emergence and survival of resistant strains of micro-organisms. Drug resistant strains are common, among which are Gonococci, Pneumococci, Pseudomonas, Meningococci,

Staphylococci, Enterococcci, Shigella, Mycobacterium tuberculosis, and Salmonella (8).

Remington reported that bacteria resistance to antibiotics has been recognized since the first drugs were introduced in 1935 and approximately 10 years later, 20% of clinical isolates of

Neisseria gonorrhea had become resistant to the action of penicillin (86). Penicillin was first introduced in 1941, where less than 1% Staphylococcus aureus isolates was resistant to it.

Bacteria become resistant to antimicrobials in several different ways. The type of resistance mechanisms is not confined to a single class of drugs. Different types of bacteria may use different mechanisms to withstand the same chemotherapeutic agent (85). These include;

i. Change in permeability to the drug; Modification of the structured target so that it

no longer interacts with the antimicrobial.

ii. Development of altered enzymes that still perform their metabolic functions but are

much less affected by drugs.

iii. Development of altered metabolic pathways that by passes the reaction inhibited by

the drug.

iv. Production of enzymes that destroy the active antimicrobial.

xcvii

Cheesbrough (8) reported that for bacteria to acquire this new property, they must undergo genetic change. Such a change may occur by mutation or by the acquisition of new genetic properties. The transfer of resistance genes (located on plasmids and transposons) from one bacterium to another, requires new genetic material. Some plasmids encode for resistance to several antibiotics and can be transferred between bacteria species, for instance, Escherichia coli to Shigella dysenterae.

1.12 Drug delivery systems

Substances with medicinal properties are usually formulated into forms called drug delivery systems before they are administered. The dosage forms can be liquid, solid or semi-solids and their intended routes of administration vary from simple to very complex preparation.

The purpose of drug delivery is to formulate the active drug principle such that it will target exactly areas of need in the body so that the drugs are more efficient and their excipients can solubilise, emulsify, thicken, preserve, impact colour and flavour and also preserve and stabilize the drug products. It is desired that with drug delivery systems, accurate drug dosages can always be reproduced with same therapeutic effects too (88-91,168).

1.12.1. Topical drug delivery systems

Topical drug delivery is the application of a drug-containing formulation on the skin so as to directly place the active principles in the formulation onto the surface of the skin or within the skin. Topical preparations are used for their localized effect at the site of their application. This is by virtue of drug penetration into the underlying layers of skin or mucous membranes. The main xcviii

advantage of topical delivery system is to bypass first pass metabolism; there is also avoidance of inconveniences and risk of systemic route of drug administration and also the avoidance of pH changes, presence of enzymes and gastric emptying time associated with oral preparations. Most topical formulations are dominated by semi solids but foams, sprays, medicated powers, solutions and medicated adhesives are also used (88, 89)

1.12.1.1 Advantages of topical drug delivery systems

- Avoidance of first pass metabolism

- Convenient and easy to apply

- Avoidance of risks and inconveniences of systemic therapy

- Achievement of efficacy with lower total daily dosage of drug by continuous drug input.

- Avoids fluctuation in drug levels

- Ability to easily terminate the medication, when needed.

- A relatively large area of application in comparison with buccal or nasal cavity.

- Ability to deliver drug more selectively to a specific site

- Avoidance of gastro-intestinal incompatibility

- Improved patient compliance

- Suitable for self-medication.

1.12.1.2 Disadvantages of topical delivery system

- Skin irritation may occur due to the drug and/ or excipients

- Poor permeability of some drugs through the skin

- Possibility of allergic reactions.

- Can be used for only drugs which require very small plasma concentration for action xcix

- Enzymes in epidermis may denature the drugs

- Drugs of large particle sizes are not easy to absorb through the skin.

1.12.1.3 Classification of topical drug delivery systems

Classification of topical drug delivery systems based on physical state: Solids, powders, aerosols, plasters, liquids, lotions, liniments, solutions, emulsions, suspensions, semi-solids, ointments, creams, pastes, jelly, suppositories.

1.12.2 Permeation of topical drugs through the skin

Most topical formulations are meant to be applied on the skin, so a basic knowledge of the skin and its physiology, function and biochemistry is necessary for designing topical preparations.

The skin is the largest organ of the body and continues with the mucosal lining of the respiratory, digestive and urogenital tracts to form a capsule, which separates the internal body structure from the external environment. The pH of the skin varies forms between 4.0 to 5.6. Sweat and fatty acids secreted from sebum, influence the pH of the skin surface. It is thought that acidity of the skin helps in limiting or preventing the growth of pathogens and other organisms (88, 89)

1.13 Routes of drugs adsorption through the skin

There are two routes of absorption through the skin - Transepidermal and Transfollicular absorption (88, 89).

1.13.1 Transepidermal

This is the principal pathway responsible for diffusion across the skin. Permeation by this route involves partitioning the drug into the Stratum corneum. Diffusion takes places across the

Stratum corneum through the intercellular lipoidal route. This route is a tortuous pathway of c

limited volume. There is another microscopic path though which polar compounds and ions pass.

Because their oil-in-water distributing tendencies will not allow them to permeate at rates that are measurable since the epidermis has no direct blood supply, the drug in it is forced to diffuse across it to reach the vasculature immediately beneath (dermis). Permeation through the dermis is through the interlocking channels of the ground substance. Diffusion through the dermis is without molecular selectivity, since gaps between the collagen fibers are far too wide to filter large molecules (88, 89).

1.13.2 Transfollicular (shunt pathway) absorption

The follicular route is an important route for absorption of drugs via the follicular pore. Sebum aids in diffusion of penetrants into sebum, followed by diffusion through the sebum to the depths of the epidermis. Blood vessel serving the hair follicle located in the dermis is the likely point of systemic entry (88, 89).

The driving force of drugs across a membrane is a concentration gradient. The membrane it diffuses through is a diffusional resistor and this resistance (R) is proportional to the thickness of the membrane (h). R is inversely proportional to the diffusive ability/mobility of the drug molecules within the membrane and it is referred to as its diffusion coefficient (D). It is inversely proportional to the fractional area of a route where there is more than one route (F) and inversely proportional to the carrying capacity of a phase (k).

R = h/fDk ….Equ. 7

ci

Dissolution of drug in vehicle

Diffusion of drug through vehicle to skin surface

______

Transepidermal route Transfollicular route

. cii

Partitioning into stratum corneum Partitioning into sebum

Diffusion through protein-lipid Diffusion through lipids Matrix of stratum corneum in sebaceous pore

Partitioning through epidermis

Diffusion through dermis

Capillary uptake and systemic dilution

Scheme 1.1: Kinetics of permeation

Knowledge of skin permeation is vital to the successful development of topical formulation.

Permeation of a drug involves the following steps

- Absorption by stratum corneum

- Penetration of drug through viable epidermis ciii

- Uptake of the drug by the capillary network in the dermal papillary layer.

This permeation can be possible only if the drug possess certain physicochemical properties. The rate of permeation across the skin (dQ/dt) is given by:

dQ/dt = Ps (Cd-Cr) ….Equ. 8

Where Cd and Cr are the concentrations of skin penetrant on the surface of the stratum corneum.

(donor compartment) and in the body (receptor compartment).

Ps is the overall permeability coefficient of the skin tissues to the penetrant.

Permeability coefficient (Ps) is given by

Ks Dss ….Equ. 9 Ps = Hs

Where Ks is the partition coefficient for the interfacial partition of the penetrant molecule from a solution medium on to the Stratum corneum, Dss is the apparent diffusivity for the steady state diffusion of the drug (penetrant) molecule through a thickness of skin tissues and Hs is the overall thickness of skin tissues (88, 89).

As Ks, Dss and Hs are constants under given conditions, the permeability coefficient (Ps) for a skin penetrant can be considered to be constant.

The rate of drug permeation can be constant when Cd > Cr, that is, the drug concentration at the surface of the Stratum corneum (Cd) is consistently and substantially greater than the drug concentration in the body (Cr), and the rate of skin permeation dG/Dt is also constant provided the magnitude of Cd remains fairly constant throughout the course of skin permeation. For civ

keeping Cd constant, the drug should be released from the device (drug formulation) at a rate

(Rr) that is either constant or greater than the rate of skin uptake (Ra) that is Rr > Ra (88, 89).

1.14 Factors affecting topical permeation

Percutaneous absorption can be improved by chemical or physical enhancer methods. Chemical penetration enhancers are chemicals that increase the skin permeability by altering the nature of the Stratum corneum to reduce its diffusional resistance. They increase the hydration of the

Stratum corneum and change the lipids and lipoproteins structures of intercellular channels by denaturation (88, 89). Examples of such chemicals are:

Solvents: Solvents are thought to increase penetration by swelling the polar pathway and, or by fluidizing lipids. Examples include water and alcohols.

Surfactants: These compounds are proposed to enhance transport of drugs across the skin by improved penetration, thought to be as a function of their polar head and hydrocarbon chain length. Examples of surfactants are: anionic surfactant, cationic surfactants and nonionic surfactants (88, 89).

Physicochemical properties of the drug substances: partition coefficient, pH, drug solubility, drug concentration, particle size, polymorphism, molecular weight

1.15 Fractionation of leaf extracts

Most of the active principles found in plants are secondary metabolites, which are products of plant metabolism that are secreted or stored in parts of the plant (leaves, backs, lactex, etc). cv

These products are not necessarily required by the plants but are useful as protective mechanisms to them. Some secondary metabolites are toxins. Example, phytoalexins protect against bacterial and fungal attacks (88, 89). Fractionation of plant extracts is believed to optimize the potencies of their secondary metabolites by extending the spectrum of antimicrobial activities of some (88,

89), though the spectrum of activity may be reduced in other plants depending on the active principle isolated by the fractionation process. This has rekindled the interest in drugs of plant origin, especially since drugs of natural origin are easily metabolized, have fewer side effects and less toxicity levels (88, 89).

Accelerated gradient chromatography (AGC) is a medium pressure liquid chromatographic method developed by Peter Baeckstron of the Organic Chemistry Department, Royal Institute of

Technology, Stockholm, Sweden. The AGC minimizes the time spent on running preparative columns by the use of continuous accelerating gradients. The gradients were obtained by continuous use of the solvents of different polarities (hexane, ethyl acetate, methanol) to effect separation of the extracts constituents (88, 89).

1.16 Ointments

Ointments are semi-solid preparations that are applied on the skin or mucosa. They are used for medication or emollient effect on the skin and for the protection of skin lesions (92).

1.16.1 Uses of ointments cvi

Medicated ointments can be grouped according to their uses, examples are

Antibiotics ointment (neomycin), antifungal (benzoic acid), acne ointment (sulphur), anti- inflammatory ointment (hydrocortisone), anti-pruritus (benzocaine), antiseptics (zinc oxide), astringents (calamine), eczema ointment (salicylic acid).

1.16.2 Classification of Ointment Bases

There are four main classes of ointment.

Hydrocarbon bases/Oleaginous bases: These are anhydrous, hydrophobic, are insoluble in water and not removable by water. They are earliest ointment bases, which consist of vegetable and animal fat, petroleum hydrocarbon like soft, hard and liquid paraffin. They are almost inert as they consist of saturated hydrocarbon with very few incompatibility and little tendency to rancidity. Instances of skin sensitization are rare and they do not promote the growth of microorganisms in them (92). They are readily available and cheap.

Absorption bases: The term absorption refers to the water absorbing properties of these bases.

They are anhydrous but are hydrophilic, so they can absorb several times their own weight of water, to form water-in-oil (w/o) type of ointment. This class of bases can be formulated into ointment with an equal solution of medicated substance added (92).

They fall into two classes; non-emulsified bases and water in oil emulsions

The non – emulsified bases can absorb water and aqueous solution. An example is wool fat, which can absorb upto 50% of its weight in water. cvii

The water in oil emulsion can absorb more water than non-emulsified bases. An example is the hydrous wool fat.

Water miscible bases: Ointments made from water-miscible bases are easily removed from the skin after use unlike the absorption base which though are hydrophilic in nature are rather difficult to wash from the skin (92).

There are three official anhydrous water miscible bases; emulsifying ointment B.P., centrimide emulsifying ointment B.P. and cetomacrogol emulsifying ointment B.P. which are anionic, cationic and non-ionic, respectively. Water-miscible bases also have good miscibility with exudates from lesion, reduce interference with skin functions, have high cosmetic stability, hence there is good patient compliance, are easily removed from hair, unlike hydrocarbon or absorption ointment that are not ideal for scalp condition due to difficulty on their removal.

Water soluble bases: These bases are prepared from mixture of low and high molecular weight polyethylene glycols (Macrogols) which range in their consistency from viscous liquids to waxy solids. The liquids are clear and colorless, with a characteristic odour. The solids are white or creamy hard lump or flakes that are soluble in water and alcohol in the ratio of 1:3 and 1:2, respectively. The macrogols are non-volatile. They are greasy, water soluble and as such can easily be removed from the skin; they are very well absorbed by the skin which can have deleterious effect by exaggerated toxic and side effects. They would not hydrolyze, deteriorate nor support microbial growth (92).

1.16.3 Ideal properties of an ointment base cviii

Ointment bases are vehicles into which a drug is incorporated. An ideal ointment base is expected to have the following attributes (92-93):

• Non-gritty to touch

• Non-greasy to prevent staining of clothes

• Non-irritant to user (on the mucous membranes)

• Has the ability to retain the physicochemical properties of a drug when formulated

• Stable on storage

• Should have the ability to absorb exudates from sites of application if present

• Should have the ability to release the drugs contained in it to desired sites in quantities

sufficient enough to elicit therapeutic effects

1.16.4 Preparation of ointment

Ointment can be prepared either by litigation or incorporation or by fusion methods (92, 93).

Preparation of ointment by mechanical incorporation can be achieved by the use of

Mortar and pestle

Ointment slab and spatula

Ointment mill

Preparation of ointment by slab and spatula

The insoluble medicaments are mechanically mixed or triturated with a spatula on a slab. The powder is first mixed with a small quantity of the base to form a concentrated ointment base containing finely divided powder uniformly distributed in it. The concentrated medicament- cix

ointment is gradually diluted with the remaining quantity of the base by titrating with a spatula

(81, 92, 93).

Fusion: The ingredients are melted together and stirred to ensure homogeneity (92).

1.16.5 Effective drug release of antimicrobial agents from ointment bases

Antimicrobial ointments must, as a necessity, release their active constituents in order to exert the desired antimicrobial action. The release of these active principles can be measured by agar diffusion technique. Two variations of the test can be used. The agar cup diffusion and the surface plate test (94, 103).

1.16.6 Factors affecting the release and absorption of medicament from ointment bases

The following factors affect the rate and degree of antimicrobial action of medicaments from bases.

1.16.6.1 Factors connected to the antimicrobial agents’ concentration

The rate of antimicrobial action varies directly with concentration of the active constituents.

There is a level/concentration of the active principle below which no significant antimicrobial activity is noticed. There is a maximum concentration above in which no significant increase in antimicrobial activity is noticed. Because the receptor site has been saturated with the active constituents so long as the concentration of the ointment falls between these two extremes, a situation exist where the rate of microbial death/inhibition increase with the concentration of the ointment (79).

1.16.6.1.1 Solubility in water cx

Some active principles depend on electrolytic dissociation for action and this can occur only in the presence of water.

1.16.6.1.2 Ionization constant

Antimicrobial agent can be active in the ionized states i.e. as cations or anions and also in the unionized form. The activity of the agent thus depends on the degree of ionization that is influenced by the pH of the ointment.

1.16.6.1.3 Lipid/water distribution characteristics

Antimicrobial agents that are formulated as multiphase products distribute themselves between the aqueous and oil phases depending on their partition coefficient (79).

1.16.6.1.4 Inherent Antimicrobial Action

Antimicrobial agents vary considerably in their ability to tackle microorganisms. Broad spectrum antibiotics exert powerful antimicrobial effects on all or most classes of bacteria (Gram positive and Gram negative), while the narrow spectrum antibiotics are effective on a class of bacteria

(79).

1.16.6.2 Factors connected to the organisms

Different microorganisms respond differently to the same antimicrobial agent. Some microorganisms may be very sensitive to the action of a particular antimicrobial while others are cxi

less positive or even resistant to it. Gram positive bacteria are more sensitive to antimicrobials than Gram-negative ones; also effects of antimicrobials are more drastic on vegetative organisms than on spores (79).

1.16.6.2.1 Microbial density

The more the number of organisms a product is exposed to, the less is the proportion of the active constituent that is made available to the individual microbial cell, and if it falls below the

MIC value, it would have no significant bacteriostatic effect on the cell. Also large population of organisms would be heterogeneous. Some of the individual cells may be sensitive to the antimicrobial agent while others will be resistant to it. It then means that the greater the microbial population the higher will be the number of resistant cells (79).

1.16.6.2.2 Presence of protective structures

Vegetative cells are more susceptible to antimicrobial agents than bacterial spores. This is because the bacterial spores posses protective structures around them that make them very resistant to antimicrobial agents (79).

1.16.6.2.3 Physiological state of the organisms

The growth stage of organisms affects its responsiveness to an antimicrobial agent. If the antimicrobial agent like the penicillin acts on cells undergoing cell division, then organisms in the stage would be very sensitive to them. If the antimicrobial agent acts by interference with cxii

metabolism, then cells that are actively metabolizing would be more rapidly destroyed than dormant cells (spores) (7, 79).

1.16.6.3 Factors connected to the environment

1.16.6.3.1 Temperature

The bactericidal activity of most disinfectants increase with increased temperature. As the temperature is increased in arithmetic progression, the rate of inhibition/killing of the microbial cells increases in geometric progression. The effect of temperature on the rate of antimicrobial activity of an agent, under specified condition, is expressed in a term called temperature coefficient (Q). Q is the change in the rate of an antimicrobial action, say, an ointment per degree rise in temperature of that ointment , a 10o rise in temperature coefficient is usually employed

(Q10) (79).

Thus, Q10 values can be calculated by determining the extinction time at two temperatures differing exactly by Q10

Q10= (Time required to kill at To )/(Time required to kill at (T + 10)o) ....Equ. 10

1.16.6.3.2 pH

This affects the antimicrobial activity of an antimicrobial agent and it influences the type and rate of microbial growth in it. Most bacteria grow best at pH of 6.0 to 8.0 while lower pH values favour growth of yeast and fungal cells. Sometimes the proliferative growth of one type of organisms at its optimal pH can change the pH of its immediate environment to a pH that flavours the growth of secondary organisms. Yeast would thrive well in a medium containing cxiii

organic reagents which metabolizes these acids in raising the pH of the medium and the now higher pH favours the growth of bacterial cells (79). pH affects the potency of antimicrobial agents. If the agent is an acid or base its degree of ionization will be dependent on pH of the medium/ointment. Antimicrobials like phenol are effective in their non-ionized states. When present in an alkaline environment that favors the formation of ions, it will have decreased antimicrobial activity. Change in pH of the medium may alter the microbial cell surface electric charge and such a change affects the amount of antimicrobial agent absorbed by the organism. Increasing the external pH renders the microbial cell surface more negatively charged and this enhances the attraction of cationic compounds like chlorhexidine to bind to them, thus eliciting more antimicrobial activity (79).

1.16.6.3.3 Organic matter

Antimicrobial agents are used in practice at sites where organic matter in form of blood, pus, faeces, urine and organic wastes will be present. These contaminants reduce the antimicrobial activity of the agents by adsorbing some of their active principle thus reducing the concentration of the agent made available to the microorganisms themselves or the contaminant may be adsorbed on microorganisms to prevent or reduce the diffusion of the antimicrobial agent into the microbial cells or the contaminant may react with active principle to neutralize its antimicrobial activity (79).

1.16.6.3.4 Surface activity

The antimicrobial activity of antimicrobials depends also on their surface activity. The higher the surface activity, the more powerful is the microbial cell membranes to the antimicrobial cxiv

action of the agent. As the concentration of the surfactant increases, the antimicrobial activity is enhanced due to increased uptake of the agent by the microbial cell until a concentration at which increasing surfactant concentration brings about no increased activity but rather a decrease. This concentration is called the critical micelle concentration. (79).

1.17 Response of microorganisms to antimicrobial agents

A microorganism be a single cell (unicellular) or a multicellular organism. Microorganisms are very diverse, and include the prokaryotes comprising of the protozoa, fungi and algae. Most microorganisms are microscopic that cannot be seen with un-aided eyes while some, like the

Thiomagarita namibiensis are macroscopic and are visible to the naked eyes (79).

Microorganisms are ubiquitous i.e. they are found everywhere, soil, air, water, on plants and animals. Useful microbes are exploited for production of food and drugs. However there are non- useful microorganisms called pathogens that are harmful to plant and animals as they cause diseases and even death.

An agent can only be used clinically as an antimicrobial agent if it at least possesses the ability to inhibit the growth of microbial cells; though it is preferable that its antimicrobial effect is micro- biocidal i.e. it is capable of killing the entire microbial cell in a system.

The response of microorganisms to antimicrobial is determined when it is brought in contact with the test antimicrobial agent. This must be done under specified experimental conditions. If the microorganisms are inhibited/killed by the agent, they said to be sensitive to that agent and if the organisms are not killed or their growth inhibited, they are termed resistance to the agent. cxv

The methods below are employed to determine the response of microorganisms to pharmaceutical products (79).

1.17.1 Agar diffusion method

In this method, agar plate is seeded with organisms that are challenged with known concentration(s) of the antimicrobial agents which have been impregnated into paper disc or filled into holes punched out of the agar. The response of the microorganisms to the test agent is related to the sizes of the zones of inhibition surrounding the paper disc or holes. The more sensitive an organism is to the agent, the further away it grows to the hole or disc while resistant organism grow almost into the hole or disc, with no appreciable zones of inhibition. Different organisms respond differently to different antimicrobial agents and this response varies when the concentration of the agent varies (79).

cxvi

1.18 Review of study plant: Spermacoce verticillata

1.18.1 Spermacoce verticillata

Fig. 1.2: The plant Spermacoce verticillata Linn Rubiaceae

This plant is commonly called white head broom; it is also called African borreria, false button weed. It is synonymous to Borreria verticillata Linn (51). It is called Obi-na-ezi by the Igbos,

Wawa kage magori/Alkamar tururuwa by the Hausas and the Yorubas call it Irawo ile (95). cxvii

Spermacoce verticillata is a woody, bushy, fine stemmed scrambling shrub. It is 1-1.2 m in height, and it has herbaceous or semi woody square stems in the first year which becomes rounded in the following year (96). The brown stem reaches a maximum diameter of about 8 mm; they have solid pith and lack visible annual rings. Spermacoce verticillata produces weak tap roots and a moderate amount of fine roots. The leaves are opposite but appearing with two or more clusters of smaller leave whorls at the nodes. The leaves are sessile or nearly so, linear or linear-lanceolate, 2-6 cm long and pointed at both ends. The tiny white flower grows through the centre of the inflorescence so that the fruits develop at nodes in mid-stem. The capsules are oblong with two carpels, each with one seed. The seeds are ellipsoidal, brown and about 1 mm long. The embryo is either straight or curved. (83, 90, 96 and 97)

1.18.2 Classification of the plant

Plant Spermacoce verticillata

Synonyms Borreria podocephala D.C.

Borreria verticillata Linn

Borreria stricta D.C.

Spermacoce globosa

Kingdom: Tracheosbinota (Vascular plants)

Super division: Spermatophytes (Seed Plants)

Family: Rubiaceae cxviii

Genus: Spermacoce

Species: verticillata

Taxonomy: Spermacoce verticillata

Herbarium Specimen – It is deposited in the Forest Research Institute of Nigeria (FRIN) Ibadan with the number 107445.

1.18.3 Common names

Igbo name : Obi na ezi

Hausa name : Wawa kaje magori

Alkamar tururuwa

Karya garma

Yoruba name: Irawo ile

1.18.4 Geographical distribution

Spermacoce verticillata is a very common tropical plant most common in humid areas and blooms during the wet season. It occurs in agricultural areas, grass lands and urban areas, and is found in Africa but its main origin is uncertain. It grows as a native or naturalized species from

Florida through the West Indies and Texas through Central and South America to Argentina and through the moist portions of tropical Africa and Madagascar including Nigeria. It is found in cxix

both Northern and Southern Zones of Nigeria e.g. Jos, Bauchi, Calabar, Nsukka, Lagos, Ile Ife

(97, 98)

1.18.5 Bioactive constituents

Spermacoce verticillata contains certain bioactive Constituents that confer on it its medicinal/therapeutic properties, such as emetine, flavones, irioids, caryophylene, tannins and sesquiterpenes (99).

It also contains an alkaloid called borreverrine which has in vitro antimicrobial actions (100).

The African Borreria contains indole alkaloids like emetine, borrerine, borreverrine and volatile oil (sesquiterpenes and phenolic compounds) (51). The volatile oil, borreverrine has antibacterial activity (101, 102). Iridoids compounds are also isolated from Spermacoce verticillata, and include daphlylloside, asperulocide, feretoside (103, 104). Borreriagenim is another iridoids compound found in Spermacoce verticillata (105).

1.18.6 Medicinal uses of Spermacoce verticillata

Spermacoce verticillata has some medicinal uses, mostly on skin conditions (51, 83). In Senegal, the roots are used as an anti leprosy agent, anti-paralytic, diuretic, anti bilharzias, and as an abortive agent (106).

The leaf extracts are used to treat leprous conditions, furuncles, ulcers and gonorrheal sores (51).

A lotion was prepared with the plant for relief of skin pruritus (97). It is used to treat diarrhoea, also used as a diuretic in the treatment of schistosomiasis (51). It is used to lower blood pressure cxx

and as an abortificient. Spermacoce verticillata is used in the treatment of asthma, bronchitis, haemorrhage, diabetes mellitus, cough, dysentery and erysipelas (99)

The volatile oil of Spermacoce verticillata inhibits the growth of Gram positive and Gram negative bacteria (101). This oil inhibits the growth of Staphylococcus aureus and Escherichia coli (51).

Some species of these genera play an important role in traditional medicine in Africa, Asia,

Europe, and South America, where they are used in the treatment of malaria, diarrheal and other digestive problems, skin diseases, fever, haemorrhage, urinary and respiratory tract infections, headache, and inflammation of the eye and gums. To date, more than 60 components have been reported from Borreria and Spermacoce species. Studies have confirmed that extracts from

Borreria and Spermacoce as well as their isolated compounds possess diverse biological activities, including anti-inflammatory, antitumor, antimicrobial, larvicidal, antioxidant, gastrointestinal, antiulcer, and hepato-protective, with alkaloids and iridoids as the major active principles.

The Rubiaceae family comprises one of the largest angiosperm families, with 650 genera and approximately 13,000 species, distributed not only in tropical and subtropical regions, but also reaching the temperate and cold regions of Europe and Canada (107, 108). In Brazil, this family comprises about 130 genera and 1500 species distributed across different vegetation formations, with great occurrence in the Atlantic forest (108, 109). This family is currently classified into three subfamilies and over 43 tribes (107). The genera Borreria G. F. W. Meyand Spermacoce

L., are characterized by a herbaceous habitat, with over 1000 species having mainly pantropic distribution, but a few genera extend into temperate regions, excluding New Zealand (110,111). cxxi

Based on their true morphology, they are considered by many authors to be distinct genera, and most others however prefer to combine the two taxa under the generic name Spermacoce (108,

112).

1.18.7 Ethnomedical properties

Borreria and Spermacoce are used medicinally in various manners and are reputed in traditional medicine of Latin America, Asia, Africa and West Indies. The species most used as medicinal are described below:

B. alata (Aubl.) DC. [Syn.: S. alata Aubl.,S. latifolia Aubl., B. latifolia Aubl., K. Schum] is a herbaceous species native to South America (113, 114).

S. articularis commonly known in Brazil as “poaia” is originally native to the temperate and tropical Asia regions and naturalized in Africa and Australia (115). The leaves are used in inflammation of eyes and gums, blindness, cataract, fever, spleen complaints, sore, conjunctivitis, haemorrhage, gallstones, dysentery, and diarrhea (115, 116), and the decoction of the leaves, roots, and seed is used in India for dropsy (117).

S. centranthoides known in Brazil as “sabugueirinho do campo” is a perennial herb originating from fields in southern Brazil, and possibly Uruguay and Argentina. In Brazil, these plants have been used for the treatment of liver ailments (118, 119), kidney disorders (120), and in Argentina as an abortifacient (121).

S. hispida L. is being used as an alternative therapy for diabetes (122). In India, decoction of the plant is used for headache (123) and the seeds as stimulant (124) and for the treatment of internal injuries of nerves and kidneys (125). cxxii

S. princeae [(K. Schum.) Verdec], is a scrambling or decumbent perennial herb, native to Africa where it is used for the treatment of skin diseases (101).

S. pusilla Wall, is an annual erect herb native to tropical Africa and Asia. In India, the fresh buds associated with the flowers are used in cuts and wounds (126) and the crushed leaves are applied to the affected areas for bone fractures and scabies and for snake and scorpion bites (127).

S. verticillata L.), known in Brazil as “poaia”, ”poaia preta”, “poaia miuda”, “coroa-de-frade” and “vassourinha”, is a small perennial and erect herb, originating from South and Central

Americas and distributed by the Old World, Southern United States to South America (128,

129). In Brazil, the infusion of the leaves is used as antipyretic and analgesic (130, 131), the roots as emetic, and the leaves as antidiarrheal and for treatment of erysipelas and haemorrhoids

(132). In West Indies, the decoction of the plant is used for diabetes and dysmenorrhoea, and when prepared with Cuscuta and Zebrina schinizlein is used for amenorrhea (133); while in

Senegal, it is used to treat bacterial skin infections and leprosy (100). In Nigeria, fresh aerial part juice is applied for eczema (101) and in Jamaica, the decoction of the endocarp, prepared jointly with Iresine P. Browne, and Desmodium, is used as diuretic and as a remedy for amenorrhea mixed with Cuscuta and Zebrina (134).

1.18.8 Chemical constituents and some of their biological activities

The widespread use of Spermacoce species in traditional medicine has resulted in considerable chemical investigation of the plant and their active principles. The first phytochemical report was published in 1961 and revealed the detection of (-)-emetina (7) from roots of S. verticillata (135).

Today, over 60 compounds distributed in different classes have been isolated. Alkaloids, cxxiii

iridoids, flavonoids, and terpenoids are the main groups of constituents. Among them, alkaloids and iridoids displayed in vivo or in vitro biological activities. A total of 11 alkaloids containing indole [borrecapine (1), borrecoxine (2), borreline (3), borrerine (4), dehydroborrecapine (6), verticillatine A (10) and verticillatine B (11)], bis indole [borreverine (5), isoborreverine (8) and spermacoceine (9)] and tetrahydroisoquinoline [(-)-emetina(7)] skeletons have been isolated from S.verticillata (135-137). Phytochemical screening indicated the presence of emetine in

S.verticillata (136). Among isolated alkaloids, borreverrine tartrate showed in vitro antibacterial activity against Sarcina lutea (MIC 3.0 µg/mL), Vibrio cholera (MIC 12.5 µg/mL), and

Staphyloccocus aureus (MIC 100 µg/mL) (100). The first use of emetine in medicine was as emetic and expectorant (138). Later, other properties were being discovered and today several important biological activities are reported for this compound. Among which are anticancer (139,

140), antiparasitic (142-144), antiviral (145, 146), contraceptive (147), inhibition of protein,

DNA and RNA synthesis reduction of T-2 toxin toxicity association with cells, and inhibition of the non-mediated MRNA decay (NMD) pathway (146). However its medicinal use has been discouraged due to its toxicity (146).

Iridoids

Thirteen iridoids have been isolated from B. verticillata (136, 148). Among these compounds, asperuloside (12) was claimed as muscle anabolic steroid (149), inhibited TNF-α, decreased IL-

1β production, reduced formation of PGE2, and treated rheumatoid arthritis in mice (22). This compound along with deacetylasperulosidic acid (18) and scandoside (24) exhibited in vitro activity against the Epstein–Barr virus (150). Deacetylasperulosidic acid (18, 63.8 ± 1.5%) and scandoside (24, 62.2 ± 1.6%), inhibited LDL-oxidation, at 20 µg/mL (151). Compounds 12, 18 cxxiv

and methyl deacetylasperulosidate showed purgative effects in mice and lowered the blood glucose level in normal mice (152). Asperulosidic acid (13) showed weak inhibition against

TPA-induced inflammation in mice (ID50> 1.0 mh/ear) and exhibited moderate effects against the EBV-EA activation induced TPA (IC50 578 mol) (153).

Terpenoids

The Borreria species also contain pentacyclic triterpenoids. From the essential oil and aerial parts of B. verticillata two sesquiterpenes, caryophyllene (39) and guiaene (40) were isolated respectively (101, 102).

A recent study on verticillata roots has led to the isolation of mixtures of aliphatic acids, tri-O- acylglycerols and sucrose, and glucose and sucrose (131).

Volatile components

Some fatty acids and terpenoids such as linalol, eugenol, β-bisabolene, E-β-farnesene, phytol and terpineol (154), guaiene (101), and phytol, 1, 8-cineole, α-pinene, and p-cymene (155) were identified by GC-MS from the aerial parts of B. verticillata.

1.18.9 Biological activities of crude extracts

Spermacoce species possess a wide variety of medicinal properties. So far, a few species have been screened for confirmation of their biological activities. Experimental results have shown some species as antimicrobial, antitumor, antioxidant, anti-inflammatory, hepatoprotective, larvicidal, etc. cxxv

No definite conclusion can be drawn about chemical relationships among Spermacoce species.

However, the classes of compounds found are suggestive of chemical patterns in the tribe

Spermacoceae. The most representative classes of compounds found were alkaloids and iridoids which have been found in species from America, (e.g. B. capitata and B. verticillata) Europe and

Africa (e.g. B. verticillata). Flavonoids were found only in species from Asia (S. hispida, S. stricta, S. laevis). Therefore, a molecular phylogeny of Spermacoce including plants with a well- known chemistry would be extremely helpful to clarify trends in the chemical evolution of the genera.

The World Health Organization (WHO) has estimated that about 80% of people in the developing countries (Nigeria inclusive) rely extremely on plants as drugs for their health needs

(49). With such a vast dependence on plants for medical purposes, it thus has become very necessary that scientific evaluations of plants known to be used locally for treatment of certain disease be carried out. This would ascertain the therapeutic claims of such plants, and would provide a lead in exploiting antimicrobial compounds which may be present in the plants.

Plants–based antimicrobial compounds are known to be very effective and are not usually associated with the many side effects that are observed when taking synthetic antimicrobials

(49).

The emergence of drug resistant bacteria has made the search for and development of new antimicrobial compounds with improved activity, very critically important, because man is at the verge of losing control over the diseases of microbial origin. Recent reports have shown that microbial resistance to antimicrobial agent is a global health problem. Almost half of all deaths in the tropics were caused by infective diseases. Even in the developed nations despite the cxxvi

progress they have made to understand microorganisms and control them, incidence of epidemics due to drug resistant microorganisms keep emerging (49).

Shortly after celebrating the successful eradication of bacteria infectious disease by using antibacterial agents, resistance to them emanated dreadfully. Mycobacterium tuberculosis and

Staphylococcus aureus had become resistant to most antibiotics then available except for

Vancomycin. (49). Within the period 1997 – 1998, strains of Staphylococcus aureus and

Staphylococcus pneumonia have been found to be resistant to vancomycin (49). This threat has been on since the boom of antibiotics era. Microorganisms develop resistance to an antimicrobial agent when the said organism is no longer susceptible to the agent at concentrations safe for the host but was hitherto lethal to the organism. Since bacteria have always devised means of developing resistance to known antibiotics, it then becomes imperative to continuously search for new antibacterial compounds with better antibacterial activity.

1.19 Aim of the present study

The aim of the present study is to evaluate the antimicrobial activity of Spermacoce verticillata and to formulate the leaf extracts into ointments that promote wound healing.

1.20 Objectives of the study

The objectives of the study are to:

• Determine the phytochemical constituents of the leaf extract.

• Determine the leaf extracts’ activity against test microorganisms as compared to that of a

standard antimicrobial agent. cxxvii

• Evaluate the fractions of the acetone extracts for their spectrum of activity on the test

microorganisms.

• Determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC) on test organism.

• Formulate the most potent fraction into ointments of different concentrations.

• Determine the spectrum of activities of the ointment formulation of the most potent

fraction on wounds infected with the test microorganisms.

• Study the toxicity of this fraction for the determination of its lethal dose - 50 (LD50) on

the animals.

• Determine the histopathological effects of the potent fraction on some organs of the

experimental animals.

cxxviii

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Reagents and solvents

Dragendorff’s reagent [Combination of tartaric acid (Analar, England), bismuth nitrate (Analar,

England) and distilled water), Wagner Raff Reagent [Combination of iodine (Analar, England), potassium iodide (Analar, England), and distilled water], Lead Acetate (Signa, England), 10% Iron

III chloride (FeCl3) [British Drug House, England], Chloroform (Analar, England), Concentrated

Tetraoxosulphate IV (H2SO4) [Analar, England], Alcoholic ferric chloride (British Drug House,

England), Acetic anhydride (Riedel-De Haen Ag Seelze-Hannover, Germany), 70% Ethanol

(British Drug House, England), Gentamycin injection B.P (80 mg/2 ml) [Yanzhou Xier Kangtai

Pharm, China], Ketoconazole tablets (200 mg) [Drugfield, Nigeria], Nutrient broth (Fluka

Biochemika, Germany), Nutrient agar (Fluka Biochemika, Germany), Sabouraud Dextrose Agar

(SDA) (Fluka Biochemika, Germany).

2.1.2 Equipment and apparatus

Autoclave (B. Brand Scientific and Instrument, England), Petri dishes (Pyrex, England), Cork borer (Rexalogy), Nigeria, Thermostat hot plate (Gallenkamp, USA), Incubator (Harrow Scientific

Ltd, USA), Freeze dryer – Model: Lyovac GT40 (Leybold Heraeus, Germany), Spirit lamp (Pyrex, cxxix

England), Harvard trip balance (Ohaus, Fisher Scientific, USA), Refrigerator (Thermocool), Hot

Air Oven (Gallenkamp, USA), Soxhlet apparatus (Pyrex, England), Glass funnel (Pyrex, England).

2.1.3 Animals

White albino rats (adult mice) of 4 weeks were obtained from the University of Jos Animal

House. Guidelines were followed before the use of the animals. These guidelines were according to the Economic Co-operation and Development (ECoD) guidelines for acute oral toxicity studies (9). Accordingly, the University’s ethics form with guidelines was obtained, duly followed and completed. The form was subsequently submitted to the Ethical Committee of

University of Jos Animal House for approval. The approved form is attached at the appendix.

2.1.4 Microorganisms (Clinical Isolates)

Gram-positive bacteria; Staphylococcus aureus, Bacillus subtilis: Gram-negative bacteria;

Pseudomonas aeruginosa, Escherichia coli: Fungal species; Trichophyton rubrum,

Microsporum audouinii, Aspergillus niger, Candida albicans. These clinical isolates were obtained from National Veterinary Research Institute, Vom, Plateau State.

2.2 Methods

2.2.1 Experimental design cxxx

The experiment was arranged in a randomized complete block design (RCBD) which gave room for an analysis of variance in a three factor factorial structure; and was thereafter replicated thrice (156).

2.2.1.1 Design for studies on inhibition zone diameter

The first factor was the solvents used in extracting from the plant (which were ethanol, acetone and water); the second factor was various concentrations of extract that were applied on the microorganisms (Control –gentamycin at 40 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml) and the third factor was various microorganisms that were used in the study which include: Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

Factor 1: Extract solvents 3 places

Factor 2: Concentrations of extract 4 places

Factor 3: Microorganisms 4 places

Therefore, 48 samples (3 x 4 x 4) were collected for each parameter that was measured. Each of the 48 samples was replicated thrice; that is, 48 x 3 = 144 samples in all for each inhibition zone diameter that was measured.

2.1.1.2 Design for studies on wound diameter

The first factor was the number of days used in infected wound (which were one, four, seven, ten and fourteen); the second factor was various concentrations (strength) of ointment used in treating the wounds of rats infected by test microorganisms (0.1 %, 0.2 %, 1 %, 2%, 5 % and 0.1

% gentamycin) and the third factor was various microorganisms that were used in the study cxxxi

which include: Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and

Escherichia coli.

Factor 1: Number of days 5 places

Factor 2: Strength of ointment 6 places

Factor 3: Microorganisms 4 places

Therefore, 120 exposures (5 x 6 x 4) were collected for each parameter that was measured on the wound healing studies. Each of the 120 samples was replicated thrice; that is, 120 x 3 = 360 results in all for each wound diameter that was measured.

2.2.2 Collection and identification of plant material

The fresh leaves of Spermacoce verticillata were collected from Lamingo, Jos South Local

Government Area of Plateau State, and was Identified by Dr. Ebenezer Mbakwe of the

Department of Plant Science and Technology, University of Jos as Spermacoce verticillata of the family Rubiaceae.

2.2.3 Preparation of plant material

The leaves of Spermacoce verticillata were air-dried under a shade for five days after which they were milled using mortar and pestle into a coarse powder.

2.2.4 Extraction

Fifty grams (50 g) of the powdered sample was weighed and soaked in 300 ml of the extracting solvent (acetone/water/ ethanol) for five days while stirring at intervals. Five milliliters of cxxxii

ethanol was added to the extraction flask containing powdered leaf sample in water to prevent deterioration of the product. After five days, this was filtered through a filter paper and the filtrate evaporated to dryness by freeze-drying. The dried extract was then stored in a sample bottle (9, 82).

2.2.5 Preliminary phytochemical tests

The acetone, aqueous and ethanol extracts were subjected to the following phytochemical analysis using established methods (9, 82).

2.2.5.1 Test for alkaloids

A 20 ml volume of sulphuric acid in 50 % ethanol was added to 2 g of the extract. This was heated on a boiling water bath for 10 minutes, cooled and filtered. To 2 ml of the filtrate a few drops of Mayers’ reagent was added. In order to confirm test for alkaloids, the standard laboratory procedures indicate that alkaloids give milky precipitate with Mayers’ reagent (9, 82).

2.2.5.2 Test for phenols

Three drops of FeCl3 was added to a 2 ml of the extract and the mixture was properly shaken. In order to confirm test for phenols, the standard laboratory procedures state that the appearance of a deep bluish green colour indicates the presence of phenols (9, 82).

2.2.5.3 Test for resins cxxxiii

To a 2.0 ml volume of the extract, 2 ml of acetic anhydride and a few drops of concentrated

H2SO4 were added and the mixture shaken. In order to confirm test for resins, the standard laboratory procedures state that a violet colour indicates the presence of resins.

2.2.5.4 Test for saponins (Frothing test)

A 0.5 g quantity of the leaf extract was diluted with 10 ml of distilled water and shaken vigorously; In order to confirm test for saponins, the standard laboratory procedures state that stable foam upon standing indicates the presence of saponins (9, 82).

2. 2.5.5 Test for tannins

A 1.0 g quantity of the leaf extract was boiled with 20 ml of water and this was filtered; few drops of ferric chloride were added to 3 ml of the filtrate. In order to confirm test for tannins, the standard laboratory procedures state that a greenish black precipitate indicates the presence of tannins.

2. 2.5.6 Test for flavonoids

Two or three drops of 10 % lead acetate solution were added to 2 ml of the extract and observed.

In order to confirm test for flavonoids, the standard laboratory procedures indicate that a cream colour confirms the presence of flavonoids in the extract (9, 82).

2. 2.5.7 Test for steroids and terpenes

A 2.0 ml volume of the extract was added to 2 ml of H2SO4 and carefully observed, in order to confirm test for steroids, the standard laboratory procedures state that a reddish brown interphase indicates the presence of steroids. cxxxiv

2. 2.5.8 Test for glycosides

A 5.0 g quantity of the extract was dissolved in 2 ml of acetic anhydride and cooled in ice.

Sulphuric acid was carefully added along the side of the test tube. In order to confirm test for glycosides, the standard laboratory procedures indicate that a colour change from violet to blue to green confirms the presence of glycosides.

2. 2.5.9 Test for balsam

Three drops of alcoholic ferric chloride was added into 2 ml volume of the extract and warmed.

In order to confirm test for balsams, the standard laboratory procedures state that a dark green colouration indicates the presence of balsam.

2. 2.5.10 Test for volatile oils

To 2 ml volume of the extract, 2 ml of 90 % ethanol and 2 drops of FeCl3 were added and the mixture shaken. In order to confirm test for volatile oils, the standard laboratory procedures state that the presence of a dark green colour indicates the presence of volatile oils.

2.2.6 Identification of microorganisms using selective media and Gram –reactions

2.2.6.1 Bacteria

2.2.6.1.1 Escherichia coli cxxxv

A loopful of E. coli colony was inoculated into 5 ml peptone water and incubated for 24 hours at a temperature of 35 0C -37 0C. After the incubation period, 3 ml of peptone culture was pipetted into a test tube and equal volume of Kovac’s reagent was added .Appearance of a pink ring is the indication of the presence of E. coli.

Gram reaction (Gram negative rods)

A 1.0 ml volume of the E. coli stock was diluted in 9 ml of MacConkey broth and was incubated at 37 0C for 18 h. Then 1.0 ml of this was inoculated on MacConkey agar media and further incubated for 24 h at 35-37 oC. Growth of red, non-mucoid colonies that are Gram negative rods on Gram –staining indicated the presence of Escherichia coli (8, 9).

2.2.6.1.2 Pseudomonas aeruginosa

A loopful of Pseudomonas aeruginosa sample was sub-cultured into nutrient broth and incubated at 35 0C overnight. A 1.0 ml volume of this sample was plated on a cetrimide agar and incubated at 35 0C for 48 h. Greenish colonies that are Gram–negative rods on Gram-stained microscopy indicate the presence of Pseudomonas aeruginosa (8, 9).

2.2.6.1.3 Staphylococcus aureus

A colony of the test organism was emulsified on a drop of distilled water on a microscopic slide.

The organisms had been previously checked to be Gram positive cocci in clusters. A loopful of plasma was added unto the emulsified colony and observed. Coagulation/clumping of the organisms within 10 seconds indicated the presence of Staphylococcus aureus (8, 9). cxxxvi

2.2.6.1.4 Bacillus subtilis

2.2.6.1.5 Biochemical reactions for identification of bacterial culture

Catalase test (Bacillus species)

Catalase is an enzyme that catalyses the decomposition of toxic hydrogen peroxide into harmless oxygen and water. Catalase is produced by aerobic organisms like Bacillus subtilis; catalase test differentiates it from closely related but anaerobic Clostridium species and aerobic

Staphylococcus species. A drop of H2O2 was added to emulsified test culture on a microscope slide and the evolution of gas bubbles (oxygen) confirms the presence of catalase (6, 8, 9, and

12).

Indole test (Escherichia coli)

The test organism was inoculated into a Bijou bottle containing 3 ml of sterile tryptone water.

This was incubated at 35-37 0C for up to 48 h, then 0.5 ml of Kovac’s reagent was added to this with gentle shaking. A red colour confirms the presence of indole, Escherichia coli can convert tryptophan to indole, unlike Klebsiella and Enterobacter that are indole negative (6, 8, 9, and 12).

Oxidase test (Pseudomonas aeruginosa)

A piece was filter paper was placed in a clean petri dish and 2 or 3 drops of freshly prepared oxidase reagent was added to it. A colony of the test organism was the smeared on the filter cxxxvii

paper. The development of a blue-purple colour within 10 s indicate a positive oxidase test, while no blue-purple colour within 10 s indicate negative oxidase test (8, 9).

Coagulase test (Staphylococcus aureus)

A drop of distilled water was placed on each end of a microscopic slide, and then a colony of the test organism was emulsified in each of the drops to make two thick suspensions. A loopful of plasma was added to one of the suspensions and mixed gently. Clumping of the organism was checked within 10 s, no plasma was added to the second suspension, and it served as a control (8,

9).

2.2.6.1.6 Maintenance and standardization of bacteria stock cultures

The cultures were activated by successive daily sub- culturing into fresh agar slants for 3 days.

Gram positive organisms were standardized by diluting 0.02 ml of the test organism to 20 ml of distilled water to obtain a dilution of 1:1000; while Gram negative organisms were standardized by diluting 0.02 ml of the test organism to 30 ml of distilled water to obtain a dilution of 1:5000, to obtain population densities of approximately 106 cfu/ml (19).

2.2.6.2 Fungi

A drop of cotton blue lactophenol was placed on a clean microscopic slide with a scalpel blade, a tiny piece of the Sabouraud dextrose agar (SDA) medium on which the fungus was growing was cut out. The top segment was removed and transferred with the aid of a needle to the top of the cotton blue lactophenol stain, and a cover slip carefully placed over it. The slide was then examined under a microscope using the x10 and x40 objectives. cxxxviii

2.2.6.2.1 Microsporum audouinii

The identification and culturing of Microsporum audouinii followed standard laboratory procedure which goes as follows: On Sabouraud dextrose agar, the colony was allowed to grow as grayish white disc of closely matted mycelium. A central knob and radiating furrows developed after 7 days. On the reverse, the center of the colony was reddish brown. On microscopy macro-conidia were rarely seen but terminal chlamydospores were seen (10, 12).

This procedure was carefully followed for the identification of Microsporum audouinii. The

Microsporum audouinii that were identified were isolated for this study.

2.2.6.2.2 Trichophyton rubrum

In a bid to identify and culture Trichophyton rubrum for this study, standard laboratory procedures were followed. The of culture Trichophyton rubrum grew as a white, cottony growth which after about two weeks of incubation turned wine red with time and this pigment diffused into the medium. Microscopy showed that macro-conida were rare in the cottony colonies while micro-conidia were borne singly along the hyphae and were numerous (10, 12).

2.2.6.2.3 Candida albicans

The identification and culturing of Candida albicans for this study followed standard laboratory procedures. Cultures of Candida albicans appeared as creamy colonies and microscopy showed budded yeast. They appeared as Gram- positive cocci on Gram staining (8, 12).

2.2.6.2.4 Aspergillus niger cxxxix

Aspergillus niger culture appeared white, and then turned black as the colonies matured. The steps taken in identification and culturing of Aspergillus niger for this study were according to standard laboratory procedures. Microscopy showed large conidiophores and globose bearing two series of sterigmata, its conidia were brown to black (8, 12).

2.2.7 Antimicrobial studies

2.2.7.1 Antimicrobial screening test (Kirby-Bauer Method)

The sensitivity of the test organisms to the acetone, aqueous and ethanol extracts of Spermacoce verticillata leaf was evaluated by the cup-plate agar diffusion method. The extracts were dissolved in water to obtain a solution of 400 µg/ml concentration. Double serial dilution was employed to obtain concentrations of 200 µg/ml, 100 µg/ml and 50 µg/ml (79).

Twenty milliliter of molten nutrient agar was seeded with 0.1 ml of the standardized broth cultures. Five wells of 6 mm in diameter were made in the agar using sterile cork borer. Two drops (0.02 ml per drop) of each of the extracts were placed into 4 of the wells, two drops (0.02 ml per drop) of gentamycin (4 µg/ml) were put in the center well as positive control.

The plates were left un-disturbed on the work bench for 2 hours at room temperature for pre- diffusion. After 2 hours, the plates were then incubated upright at 37 oC for 24 hours. Zones of inhibitions were observed around the wells, their diameters (1ZDs) were measured. The mean of the replicate determinations was taken (12, 79).

2.2.7.2 Biostatic action of extracts cxl

Biostatic activity of an antimicrobial agent /extract is a quantitative determination of the concentration of the extract that inhibits the growth of test microorganisms. The method used to determine biostatic action was the MIC (79).

2.2.7.3 Minimum inhibitory concentration (MIC) determination

The MIC of the acetone, aqueous and ethanol leaf extract of Spermacoce verticillata against the test bacteria were determined using the agar dilution method, serial doubling dilution of the extract was carried with molten nutrient agar to obtain concentrations of 400 µg/ml, 200 µg/ml,

100 µg/ml, 50 µg/ml and 25 µg/ml. The mixtures (zone) were dispersed into Petri-dishes and allowed to solidify uniformly. Two drops (0.02 ml) of the overnight microbial culture were streaked over the surface of the set agar. The inoculated plates were allowed to stand at room temperature for 2 h until the inocula have been completely absorbed by the medium. The media plates were inversely placed in the incubator at 37 oC for 24 h before the plates were read. The least concentration of the extract that inhibited the growth of an organism (no growth observed) was taken as its MIC.

2.2.7.4 Biocidal activity

Biocidal activity is a measure of the ability of the extract /antimicrobial agent to kill microorganisms in an enclosed environment. The minimum bactericidal concentration method was used to evaluate the biocidal activity of the leaf extracts (6, 7, 8, 12 and 79).

2.2.7.5 Minimum bactericidal concentration (MBC) cxli

The plates that showed no growth in the MIC test were used for the determination of the MBC.

Discs were cut from such plates, transferred into tubes of sterile nutrient broth, and incubated at

37 oC for 48 h. The absence of growth (no turbidity) in the medium was evidence of total cell death. The least concentration of the extract that produces total cell death was taken as the MBC

(12, 79).

Sensitivity test for fungal cultures

The sensitivity of the test fungal cultures to the acetone, aqueous and ethanolic extracts of

Spermacoce verticillata leaf was evaluated by the cup plate agar diffusion method (12, 79).

The extracts were dissolved in distilled water to obtain concentrations of 400 µg/ml and these were serially diluted to give concentrations of 200 µg/ml, 100 µg/ml and 50 µg/ml. A 10 ml volume of normal saline was added to 0.5 ml of Tween-80; 3 glass beads were added to this and

1 ml of fungal spores that had stayed for 7 days was added to the mixture and mixed thoroughly.

Then, 1.0 ml of this mixture was measured into molten Saboraud dextrose agar in Bijou bottles, shaken and then poured into Petri dishes and allowed to set. Five wells of 6 mm diameter were made in the agar using sterile cork borer. Two drops of each of the extract (0.02 ml per drop) were placed into 4 of the wells (12, 79).

Two drops of ketoconazole (50 mg/ml) were placed in the center as positive control. The plates were left undisturbed for 2 hours of pre-diffusion at room temperature. After pre-diffusion they cxlii

were incubated upright at 250C for 7 days. The zones of inhibition around the wells produced by the extract were measured.

2.2.8 Fractionation of acetone extract

An accelerated gradient chromatography (AGC) column is made up of a column of silica gel, a pump of medium pressure with a flow rate of about 10 mml/min was used. A gradient former, the proportion of the non-polar solvent, intermediate solvent and polar solvents were adjusted gradually to produce the stock solutions used to effect separation with the gradient mixer (158).

The elution was started with the non-polar solvent (hexane), then the intermediate solvent, and the concentration of the intermediate solvent was increased gradually as that of the non-polar solvent decreased. After this, the polar solvent was added gradually and the volume of the intermediate solvent decreased until the final mobile phase had a composition approaching that of a polar solvent (methanol). The eluates were collected into various test tubes. Any tube with a sharp color change was noted. After elution, the four eluates in the marked tubes were subjected to thin layer chromatography (TLC) (9, 82).

The TLC plate was made up of a stationary phase of silica gel on an aluminum foil paper and mobile phase of ethyl-acetate solvent that could migrate across the surface of the stationary phase by capillary action. The four eluates were applied on the plates as spots using capillary tubes. The spots were allowed to evaporate between successive applications. The chromatoplates were placed in the development tank containing the mobile phase. When the mobile phase

(ethylacetale) reached two-thirds of the plates, they were removed from the tank, and the solvent front marked. The plates were left to air dry. The chromatogram showed that the four spots cxliii

traveled two equal distances. Fractions that showed similar values were bulked together to obtain two major fractions namely fraction A and fraction B.

2.2.8.1 Phytochemical screening of the AGC fractions A and B

The standard phytochemical procedures carried out on the acetone, aqueous and ethanol extracts were repeated on the fractions (A and B) to determine their bioactive constituents.

2.2.8.2 Preparation of stock solution and serial dilution of fractions A and B

Four milligrams of a fraction was weighed and dissolved in 10 ml of distilled water to obtain concentration of 400 µg/ml serial dilution was done with these stock solutions of 400 µg/ml to get concentrations of 200 µg/ml, 100 µg/ml and 50 µg/ml, these were poured into sterile bottles, and labeled appropriately, for antimicrobial testing.

2.2.9 Standardization of bacterial cultures

Stock culture of each clinical isolate was stored in nutrient agar slant at 4 oC. Prior to use, the cultures were activated by successive daily sub-culturing into fresh agar slants for a period of 3 days. The overnight cultures were standardised by diluting Gram positive microorganisms in nutrient broth to 1:1000 and Gram negative organisms to 1:1500 to obtain population densities of approximately 106 cfu/ml (8).

2.2.10 Antimicrobial studies of fractions A and B

2.2.10.1 Antimicrobial sensitivity test – using the cup in seeded plate method

Specimen bottles containing 20 ml of single strength nutrient agar were retrieved from the refrigerator where they had been stored after sterilization. The contents were melted and allowed cxliv

to cool until they were warm to the cheek. A loopful (0.1 ml) of overnight culture of the test organisms was added to the bottle of the melted agar, mixed by rolling over the palms, then poured into a sterile Petri- dish and the agar was allowed to set firmly. Five wells of 6 mm in diameter were made in the agar using sterile cork borer. Two drops of the fraction were dropped into 4 of the wells each and 2 drops of gentamycin were placed in the center well, as positive control. The plates were left undistributed for two hours of pre-diffusion after which they were incubated upright at 37 0C for 24 h. The zones of inhibition observed around the wells, and were measured. Replicates were made and their mean taken as the IZDs (79).

2.2.10.2 Minimum inhibitory concentration (MIC) of fraction

The MIC of fractions A and B of the leaf extract of Spermacoce verticillata was determined using the agar dilution method. Concentrations of 400 µg/ml, 200 µg/ml, 100 µg/ml, 50 µg/ml and 25 µg/ml were obtained by serial dilution of the fractions with molten nutrient agar. These were poured into Petri-dishes and allowed to solidify uniformly. Two drops (0.02 ml) of overnight culture were streaked over the surface of the set agar. Then the inoculated plates were allowed to stand at room temperature for 2 hours, before they were incubated, inverted, at 37 0C for 24 hours. The least concentration that inhibited the growth of the test bacteria was taken as its

MIC (79).

2.2.10.3 Minimum bactericidal concentration (MBC) cxlv

To determine the MBC, the plates that showed no growth in the preceding MIC testing were used. A cork borer was used to cut out discs from such plates which are transferred into nutrients broth tubes, shaken and incubated at 37 0C for 48 hours.

The absence of growth (clear broth) in the tubes is evidence of total cell death. The least concentration of the extract (from the MIC plates) that produces total cell death was taken as its

MBC of that particular organism within the experiment conditions.

2.2.10.4 Antimicrobial sensitivity test of the extracting solvent (Acetone)

Twenty milliliters of molten nutrient agar was seeded with 0.1 ml of standardized test bacterial broth cultures. Five wells of 6 mm in diameter were made in the agar using sterile cork borer.

Two drops of acetone were placed in four of the wells, and then 2 drops of gentamycin (4 µg/ml) were placed in the center well as positive control. The plates were Left for 2 hours at room temperature for pre diffusion, after which they were incubated upright at 37 oC for 24 hours.

2.2.11 Formulation of ointments of various concentrations

Ointments of varying concentrations/strengths of (0.1 %, 0.2 %, 1 %, 2 % and 5 % w/w), were prepared with the acetone extract using the titration technique. White soft paraffin was used as the ointment base. The ointments prepared were dispensed into jars and stored in the refrigerator prior to use (92).

2.2.12 Wound healing studies

2.2.12.1 Grouping of rats for various treatments

Thirty five albino rats were anaesthetized using chloroform (with the aid of a glass dessicator).

An area of the skin intended for the wound creation was disinfected using methylated spirit. cxlvi

Experimental wounds were then created on the rats by excising their skin using sterile surgical blades. The rats were appropriately labeled according to the test microorganisms and the corresponding strength of the ointment formulations to be used in treating the wounds. The diameters of the wounds were measured and recorded.

2.2.12.2 Creation and infection of experimental wounds on Albino rats

The rats were separated into five cages. Each cage contained seven Albino rats. One of the rats in each cage was treated with 0.1 % gentamycin ointment (positive control), the second rat was treated with a blank ointment (negative control), while each of the remaining five rats was treated with a particular ointment strength (9).

The overnight culture of the selected test organisms (E.coli, B. subtilis, P. aeruginosa and S. aureus) was used for the infection of the wounds. Sterile swab sticks were dipped into the overnight cultures and rubbed all over the surface of the wounds of the appropriate animals. The animals were kept for three days for the infection of wounds to be established (19).

2.2.12.3 Application of the ointment on the infected wound

Various strengths (concentrations) of the ointments were used to treat the infected wounds of the corresponding labeled albino rats. A 0.1% concentration of gentamycin ointment was used as the positive control for treating infected rats while a blank ointment was used as the negative control.

The ointments were applied daily at the infected sites until healing was completed or when the animal dies. The diameters of the wounds were measured using a transparent ruler at regular time intervals.

2.2.13 Toxicological studies

A pilot toxicological study was carried out with three concentration of fraction B (10 µg/ml, 100

µg/ml and 1000 µg/ml). The mouse injected with 1000 µg/ml concentration of the Bulk B cxlvii

fraction died, while the other mice remained alive. From this result, five doses were deduced for the toxicity studies. These doses were 250 µg/g, 500 µg/g, 750 µg/g, 1000 µg/g and 1250 µg/g of their body weights respectively.

Thirty mice divided into six groups of 5 mice each, were kept in separate cages and provided with adequate food and water. The first five groups were randomly selected, weighed and assigned to each dose level of the extract as follows: 250, 500,750, 1000 and 1250 µg/g of their body weights respectively. The extracts were administered orally; the 6th group served as the control group and was provided with food and water only. The number of deaths in each group within 24 hours to 14 days, were noted. The lethal dose – 50 (LD50) was calculated with the arithmetical method of Karber 1931(159).

2.2.14 Histological studies

Four adult Albino mice, five weeks old, were randomly selected, weighed and kept in two groups of two animals to each cage. One group was administered 670 µg/g of body weight of the extract fraction, while the other group was provided with food and water alone and served as the control group. The test mice were administered 670 µg/g on the first day and no unusual reaction was observed. On the second day a repeat dosing was administered and one of the mice died shortly after administering the extract. The other test mouse lived on till the end of the study. The jugular vein of the dead mouse was immediately cut and its blood collected into an EDTA bottle.

The heart, kidney, skin and liver were extracted and stored in formalin. One of the control mice was swirled until it got dizzy. While still dazed, its jugular vein was cut and blood was collected.

The same organs extracted from the test mouse were collected and preserved in formalin (9). cxlviii

2.2.14.1 Fixation

The tissues extracted from the animals were immersed in 10 % formal-saline (formaldehyde and sodium chloride) solution for seven days. The volume of the fixative used was about 50 times that of the tissues.

The formalin component of the fixative is a very powerful reducing agent that acts on the proteinous elements of the tissues; this prevents post-mortem changes to achieve their structural stabilisation, while saline helps to maintain the physiological structure of the tissues; but because the fixation solution formalin was not miscible with paraffin, the tissues were dehydrated with ethyl alcohol (160, 161).

Ethyl-alcohol was removed from the tissues by immersing them in chloroform in a process called de-alcoholisation. Chloroform has the advantage of being miscible with both alcohol and paraffin wax. The tissues were transferred from chloroform to molten paraffin wax in an oven.

During this stage, the chloroform is eliminated from the tissues by diffusion into the surrounding melted wax and the wax in turn diffuses into the tissues to replace it. Plastic ice trays were used for blocking out or embedding the tissues in the paraffin wax. When set, the wax blocks were easily removed by flexing the plastic tray. The microscope was used to study the slices of tissues, obtained by cutting the tissues into thin sections using a microtome (160, 161). Then, the sections are stained but before staining, the paraffin wax was removed. The section was freed from paraffin by immersing the slide in xylene until the paraffin started to melt out of the slide.

Then the slide was placed in absolute alcohol (for 60 s) to remove the xylene. The step above was repeated and the slide immersed in 90% and 70% alcohols for 30 seconds each. After this, cxlix

the slide was thoroughly washed in distilled water. The section was stained using haematoxylin

(160, 161).

After staining, the section was passed back through alcohol (70% and 90%) and two changes of absolute alcohol, washing it very thoroughly in the absolute alcohol. Then the slide was washed in two changes of xylene. After the section had been stained, it was then mounted in Canada balsam under a cover-slip (160, 161).

2.2.14.2 Mounting of sections

A provided cover-slip was cleaned and placed on a sheet of Whatman filter paper. The slide was whipped to remove excess xylene from it with the filter paper. The Canada balsam (the mounting medium) was placed on the section, which was quickly inverted and then placed unto the cover- slip with gentle pressure until the mounting medium flowed out evenly from the edges of the cover-slip. The slide was then turned over and kept in an incubator at 37 ºC for 24 h to harden the mounting medium. After this period, the slide was removed from the incubator and left to air- dry until the edges of the cover-slip were no longer sticky to touch.

The cell structures of the stained sections from the control and test organs were studied microscopically and comparison made between them (160, 161).

2.2.15 Data presentation and statistical analysis

Data were presented in tables and charts. Generated data were subjected to analysis using analysis of variance (ANOVA) which was arranged in a randomized complete block design

(RCBD) and means were compared or separated using least significant difference (LSD) at 5% confidence interval (156). This was carried out using GenStat statistical software (162). cl

Correlation and regression analysis were also used in which case coefficient of simple determinant (r2) was obtained which was used to measure the relationships that existed between inhibition zones/wound diameter and various levels of concentrations of extract using SPSS

Statistics version 17.0 (163). In addition, descriptive statistics was employed to measure the level of skewness or variability of data that were obtained in the study.

cli

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 Phytochemical constituents of the leaf extracts

Table 1 shows the phytochemical constituents of the acetone, aqueous and ethanol leaf extracts of Spermacoce verticillata. Phytochemical analysis revealed the presence of balsam, terpenes and steroids in all the extracts. Acetone and ethanol extracts had alkaloids, phenols and volatile oils. Glycosides, saponins and tannins were absent in ethanol and aqueous leaf extracts, but present in the acetone extract. Flavonoids and resins were totally absent in the three leaf extracts.

Considering the eleven (11) phytochemicals identified, nine (9) phytochemical constituents

(alkaloids, balsam, phenol, glycosides, saponins, tannins, terpenes, steroids, and volatile oils) were present in the acetone leaf extract making it the extract with the highest number of phytochemicals. This was followed by the ethanol leaf extract with 6 phytochemicals (alkaloids, balsam, phenol, terpenes, steroids, and volatile oils) The aqueous leaf extract had the least number of phytochemicals present, namely; balsam, terpenes and steroids.

Previous studies have shown that many plants extracts which contain these phytochemicals, are very helpful as antimicrobial agents. For example, methanol extract of Dissotis theifolia plant, upon phytochemical screening revealed the presence of saponins, tannins, glycosides, flavonoids, terpenoids, carbohydrates, alkaloids and steroids (53, 164, and 165). This plant (Dissotis theifolia), possessed antibacterial and wound healing effect when formulated as ointment, on clii

infected excision wound model (53). Gingko biloba plant contains flavonoids and terpenes and these constituents account for its wound healing activity (57). The phytochemical constituents of the three leaf extracts of Spermacoce verticillata, the extracts have potential antimicrobial and wound healing agents.

Table 1: Phytochemical constituents of acetone, aqueous and ethanol leaf extracts of Spermacoce verticillata

Extracts Phytochemicals Ethanol Aqueous Acetone

Alkaloids + - + Balsam + + + Phenol + - + Glycosides - - + Flavonoids - - - Saponins - - + Tannins - - + Terpenes + + + Steroids + + + Volatile oils + - + Resins - - - + = present; - = absent

cliii

3.2 Antibacterial activities of various leaf extracts on test microorganisms

The results of the antibacterial activities of ethanol, acetone and aqueous leaf extracts of

Spermacoce verticillata on test microorganisms are shown in Figs. 1- 3.

3.2.1 Antibacterial activity of acetone leaf extract on test microorganisms

Figure 1 shows the inhibition zone diameters of various concentrations of acetone leaf extract of

Spermacoce verticillata on test microorganisms. The inhibition zone diameters ranged between

0.0 mm (for Bacillus subtilis at 100 µg/ml and 200 µg/ml) and 28.5 mm (for Escherichia coli at

400 µg/ml). Acetone leaf extract seemed to have the greatest effect on Escherichia coli with a mean of 23.3 ± 4.5 mm inhibition zone diameter and least on Bacillus subtilis with a mean of 3.8

± 6.6 mm inhibition zone diameter. Its effect on Escherichia coli was highest at 400 µg/ml concentration. The inhibition zone diameters of the acetone extracts were however within susceptible limits (greater or equal to 15 mm) published by interpretative chart of zone sizes for

Gentamycin (50 µg/ml) (8).

The order of antibacterial activity of the acetone leaf extracts against the test organisms was as follows: Escherichia coli ˃ Staphylococcus aureus > Pseudomonas aeruginosa > Bacillus cliv

subtilis. Comparing results with the control –gentamycin ointment, it was found that acetone leaf extract (at 400 µg/ml concentration) performed better than gentamycin ointment (at 40 µg/ml concentration). Thus, acetone leaf extract, at 400 µg/ml concentration will achieve the best antibacterial activity against Escherichia coli when compared to all the other three organisms. A regression analysis showed that with increasing extract concentration, the different levels of inhibition zone diameter for the various microorganisms equally increased (Fig. 4).

clv

80

70 70

62.5

60 56.5

50

40

30 28.5

24.5 23.5 23.3 21.5 21 21.5 21.5 20.8 20 18.8 18 18 20 17 16

11.5 11.5 11.5

Inhibition zone diameter (mm) 10 6.6 6.4 3.8 3.8 4.5

0 0 0 40 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml Sum Mean St. Dev. Genta Concentration of the extracts (µg/ml) Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa

Fig. 3.1: IZD of various concentrations of the acetone leaf extract of Spermacoce vertillata on test microorganisms

clvi

3.2.2 Antibacterial activity of ethanol leaf extract on test microorganisms

The antibacterial activity of the ethanol leaf extract of Spermacoce verticillata on the test microorganisms is presented in Fig. 2. The IZD indicates that Staphylococcus aureus showed the greatest susceptibility to ethanol leaf extract (20 mm) at 400 µg/ml concentration; followed by

Escherichia coli, with an IZD of 14 mm at 400 µg/ml concentration. Next was Bacillus subtilis with an IZD of 13.5 mm at 400 µg/ml concentration; and Pseudomonas aeruginosa, with an IZD of 12.50 mm also at 400 µg/ml concentration. Generally, the IZDs ranged between 0.0 mm (at

100 µg/ml concentration for Pseudomonas aeruginosa) and 20 mm (at 400 µg/ml concentration for Staphylococcus aureus). Comparing this result with the control (40 µg/ml concentration of gentamycin ointment), it is important to note that the control, equally performed best on

Staphylococcus aureus with IZD of 23.1 mm and least on Pseudomonas aeruginosa with IZD of

15 mm. The IZD of the control (gentamycin ointment), was generally higher than those of the ethanol leaf extracts (Fig. 2), unlike the result with acetone leaf extract (Fig. 1).

clvii

zone diameter (mm) 60

55

Inhibition 50

40 37 34.5

30

24.5 23.1

20 20 18 18.5 18 18.3 17 15 13.5 14 12 12.5 12.3 11.5 11 11.5 11.5 10 8.2 10 7.1

1.8 1.5 1.4 0 0 40 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml Sum Mean St. Dev. Genta Concentration of the extracts (µg/ml) Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa

Fig.3.2: IZD of various concentrations of ethanol leaf extract of Spermacoce vertillata on test microorganisms

clviii

3.2.3 Antibacterial activity of aqueous leaf extract on test microorganisms

The antibacterial activity of the aqueous leaf extract on the test microorganisms is represented in

Fig. 3. The results showed that Staphylococcus aureus and Escherichia coli had the greatest susceptibility with 17 mm diameter each at 400 µg/ml concentration. At 100 µg/ml concentration, the extract had no activity on any of the organisms. Pseudomonas aeruginosa (at

400 µg/ml) did not equally exhibit any antibacterial activity. Comparing the various mean IZDs of the aqueous leaf extract on the test organisms, Staphylococcus aureus had the highest activity with a mean IZD of 10.3 ± 9.1 mm, followed by Escherichia coli with 10.2 ± 8.9 mm, Bacillus subtilis with 8.5 ± 7.4 mm and lastly Pseudomonas aeruginosa with 4.2 ± 7.2 mm.

Susceptibility of the test organisms to the aqueous leaf extracts decreased with increasing concentrations of the extracts for Pseudomonas aeruginosa. In contrast, the susceptibility of

Staphylococcus aureus, Escherichia coli and Bacillus subtilis, increased with increase in the concentrations of the aqueous leaf extract (Fig. 6). The results showed that the extracts had activities against Gram-positive and Gram-negative bacteria, but were not very effective against

Pseudomonas aeruginosa. clix

The control (gentamycin ointment at 40 µg/ml) when compared with the aqueous leaf extracts performed better, with the highest IZD of 22.1 mm obtained for Staphylococcus aureus as against that of aqueous leaf extract which had a highest IZD value of 17 mm obtained for

Escherichia coli and S aureus, respectively, at 400 µg/ml concentration. Against Escherichia coli, the control equally had higher IZD of 19.5 mm, as against the 17 mm obtained for aqueous leaf extract at 400 µg/ml (Fig. 3). This goes further to show that acetone leaf extract as antibacterial agent performed best on the test organisms than ethanol and aqueous leaf extracts

(Table 2).

clx

35

31 30.5 30

25.5 25

22.1

20 20 19.5 18 17 17

14 15 13.5 13.5 12.5 12.5 12

10.3 10.2 9.1 8.9 10 8.5 7.4 7.2

5 4.2 Inhibition zone diameter (mm)

0 0 0 0 0 0 40 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml Sum Mean St. Dev. Genta Concentration of the extracts (µg/ml) Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa

Fig. 3.3: IZD of various concentrations of aqueous leaf extract of Spermacoce vertillata on test microorganisms

clxi

This is consistent with an earlier study which observed that Spermacoce verticillata possesses antibacterial action at different concentrations depending on the bacteria species (100).

clxii

3.2.4 Relationship between various concentrations of acetone leaf extract and IZD on test microorganisms

E. coli

IZD (mm) = 16.7 + 0.028 Conc. of extract r² = 0.980**

Ninety-eight per cent (98%) of variation caused on the zone of growth inhibition was due to the various concentration of acetone leaf extract of Spermacoce verticillata on E.coli while 2% of was due to chance. The slope of the graph was a positive one, indicating that as the concentration of leaf extract increased, the zone of inhibition equally increased. This relationship was highly significant at p<0.01 (Fig. 4).

S. aureus

IZD (mm) = 18.47 + 0.013 Conc. of extract r² = 0.49*

Forty-nine per cent (49%) of variation on the 1ZD was due to the various concentrations of the acetone extract, and 1ZD increased as the concentration of extract increased, this is evident by its positive slope. There was a positive significant relationship (p<0.05) between zone of inhibition and different concentrations of leaf extract on Staphylococcus aureus. clxiii

P. aeruginosa

IZD (mm) = 14.19 + 0.023 Conc. of extract r² = 0.514*.

The different concentrations of acetone leaf extract caused 51% of variation on the 1ZD in relation to Pseudomonas aeruginosa while the remaining 49% was due to other sources. There was a significant positive relationship at p<0.05 between acetone leaf extract and zones of inhibition.

Bacillus subtilis

IZD (mm) = 6.497 + 0.002 Conc. of extract r² = 0.001NS.

Zero point one per cent (0.1%) variation on 1ZD was due to the concentration of acetone on it, the other 99.9% was due to other sources. The r2 shows that there was no significant effect

(p=0.05) of acetone leaf extract on the inhibition zone diameter of Bacillus subtilis. The relationship between various levels of concentration against the IZD of Bacillus subtilis was the poorest. Therefore, effectiveness of acetone leaf extract on the various microorganisms was in the following decreasing order of performance: Escherichia coli > Pseudomonas aeruginosa >

Staphylococcus aureus > Bacillus subtilis.

clxiv

clxv

30 IZD (mm) = 16.7 + 0.028 Conc. of extract r² = 0.980**, for E. coli

25 IZD (mm) = 18.47 + 0.013 Conc. of extract r² = 0.49*, for S. aureus

20

IZD (mm) = 14.19 + 0.023 Conc. of extract r² = 0.514*, P. aeruginosa BacillusB. subtilis

15 Staphlococcocus aureus (mm)

E. coli Zone ofinhibition

P. aeruginosa 10

Linear (Bacillus)B. subtilis

IZD (mm) = 6.497 + 0.002 Conc. of extract Linear (Staphlococcocus aureus) r² = 0.001NS, for B. subtilis 5 Linear (E. coli)

Linear (P. aeruginosa)

0 0 100 200 300 400 500 Conc. of extract (μg/ml)

clxvi

Fig. 3.4: Inhibition zone diameters of various concentrations of acetone leaf extract of Spermacoce verticillata against the test microorganisms

3.2.5 Relationship between various concentrations of ethanol leaf extract and inhibition zone diameter on test microorganisms

S. aureus

IZD = 20.08 - 0.003 Conc. of extract r² = 0.031NS

With reference to Staphylococcus aureus, 3.1% of the variation on the IZD was due to the effect of various concentration of the ethanol extract. The regression line graph is negative; so increase in concentration of ethanol leaf extract resulted in the IZD. The relationship was not significant at p < 0.05 (Fig. 5).

Escherichia coli

IZD = 15.05 - 0.006 Conc. of extract r² = 0.093NS

On Escherichia coli, 9.3% of the observed IZD variation was due to ethanol leaf extract. The relationship was equally negative and was not significant at p < 0.05.

Bacillus subtilis

IZD = 14.09 - 0.005 Conc. of extract r² = 0.054NS clxvii

Given the above relationship, 5.4% of the variation in IZD was due to effect of the various concentrations of the ethanol leaf extract of Spermacoce verticillata on Bacillus subtilis. This variation is not significant at p<0.05. The regression line is negative. This implies that at increasing concentration of leaf extract, the IZD was decreasing.

Pseudomonas aeruginosa

IZD = 8.16 + 0.009 Conc. of extract r² = 0.047NS

Ethanol leaf extract accounted for 4.7% of the variation on the IZD with reference to

Pseudomonas aeruginosa, while 99.5% was due to chance. The equation show a positive slope, as concentration of the extract increased, the IZD on Pseudomonas aeruginosa equally increased.

Considering the effect of ethanol leaf extract on the IZD with regard to the four test microorganisms; Staphylococcus aureus, Escherichia coli and Bacillus subtilis line graphs had negative slopes, so increased concentrations of the extracts caused decreased IZDs on the organisms, while that of Pseudomonas aeruginosa had a positive relationship.

clxviii

clxix

25

IZD = 20.08 - 0.003 Conc. of extract 20 r² = 0.031NS, for S. aureus

B.subtilis 15 IZD = 15.05 - 0.006 Conc. of extract r² = 0.093NS, forE. coli S.aureus

E.coli IZD = 14.09 - 0.005 Conc. of extract

(mm) NS r² = 0.054 , for B. subtilis P.aeruginosa

Linear Zone ofinhibition 10 (B.subtilis) IZD = 8.16NS + 0.009 Conc.of extract Linear r² = 0.047NS , for P.aeruginosa (S.aureus) Linear (E.coli)

Linear (P.aeruginosa) 5

0 0 100 200 300 400 500 Concentrations of extract (μg/ml)

Fig. 3.5: IZD of various concentrations of ethanol leaf extract of Spermacoce verticillata against test organisms

clxx

3.2.6 Relationship between various concentrations of aqueous leaf extract and IZD on test microorganisms

Staphylococcus aureus

IZD = 11.63 + 0.008 Conc. of extract r² = 0.021 NS

The coefficient of simple determinant, r2, shows that 2.1% of the variation caused on the zone of inhibition was due to the various concentrations of aqueous extract on Staphylococcus aureus.

This variation was not significant at p< 0.05. The graph had a positive slope, so increased concentration of the extract caused increased IZDs (Fig. 6).

Escherichia coli

IZD = 9.949 + 0.013 Conc. of extract r² = 0.062 NS

The aqueous leaf extract caused 6.2% of the variation on the IZD, the relationship was positive, though it was not significant at p<0.05.

Bacillus subtilis

IZD = 10.92 + 0.002 Conc. of extract r² = 0.002NS clxxi

With reference to Bacillus subtilis, 02% of the variation caused on the IZD was due to various concentrations of the aqueous leaf extract which was not significant at p<0.05. The regression line graph was positive.

Pseudomonas aeruginosa

IZD = 13.62 - 0.032 Conc. of extract r² = 0.317 NS

The regression line graph had a negative slope, increasing concentration did not increase IZD on

Pseudomonas aeruginosa, and its r2 shows that 31.7% of the variation on IZD was due to the concentration of the aqueous extract.

All the relationships that were positive actually indicated that they had good effect on the microorganisms with regard to the IZDs. Though their effects were not generally significant at p<0.05, they, however, performed in the following decreasing order: Escherichia coli <

Staphylococcus aureus < Bacillus subtilis. That of Pseudomonas aeruginosa had a negative relationship implying that the aqueous leaf extract did not have much effect of the IZD. It can be surmised that the other three organisms can be treated with aqueous leaf extract.

clxxii

clxxiii

25

20

IZD = 11.63 + 0.008 Conc. of exract 15 r² = 0.021NS, for S. aureus B.subtilis S.aureus IZD = 9.949 + 0.013 Conc. of extract r² = 0.062NS, for E. coli E.coli (mm) P.aeruginosa IZD = 10.92 + 0.002 Conc. of extract r² = 0.002NS, for B. subtilis Linear (B.subtilis) Zone ofinhibition 10 Linear (S.aureus) Linear (E.coli) Linear (P.aeruginosa)

5

IZD = 13.62 - 0.032 Conc. of extract r² = 0.317NS, for P. aeruginosa

0 0 100 200 300 400 500 Concentrations of extract ( μg/ml)

Fig. 3.6: IZD of various microorganisms against different concentrations of aqueous leaf extract of Spermacoce verticillata clxxiv

3.2.2 Performance of the three leaf extracts

Comparing the three leaf extracts, acetone leaf extract performed best as compared to ethanol and aqueous leaf extracts (Figs. 4-6 and Tables 2 and 4). Considering the performances of ethanol and aqueous leaf extracts in relation to their r2 (coefficient of simple determinant), one cannot exactly draw a line between the two; but looking at the mean values of the extract liquids in Table 4, one can conclusively say that ethanol leaf extract performed better than aqueous leaf extract. This can be accepted since ethanol leaf extract had more phytochemicals (Table 1) than aqueous leaf extract. Similarly, acetone leaf extract had the best effect on Escherichia coli, followed by Pseudomonas aeruginosa, Staphylococcus aureus and lastly Bacillus subtilis (Fig. 4 and Table 2). The rest leaf extract liquids, even when they had positive relationship, were all not significant at p<0.05 (Table 4). Therefore, it was concluded that the antimicrobial effect of

Spermacoce verticillata leaf extracted with acetone is preferred to ethanol and aqueous leaf extracts. In addition, Spermacoce verticillata leaf extracted with acetone performed best as an antibacterial agent on Escherichia coli. In view of this finding, further work was carried out on the acetone extract alone.

clxxv

Table 2: Performance of the different leaf extracts with respect to the various microorganisms tested according to their coefficient of determinants (r2)

Regression line Microorganism r2

Acetone leaf extract

IZD (mm) = 16.7 + 0.028 Conc. of extract E. coli 0.980** IZD (mm) = 14.19 + 0.023 Conc. of extract P. aeruginosa 0.514* IZD (mm) = 18.47 + 0.013 Conc. of extract S. aureus 0.49* IZD (mm) = 6.497 + 0.002 Conc. of extract B. subtilis 0.001NS

Aqueous leaf extract

IZD = 9.949 + 0.013 Conc. of extract E. coli 0.062NS IZD = 11.63 + 0.008 Conc. of extract S. aureus 0.021NS IZD = 10.92 + 0.002 Conc. of extract B. subtilis 0.002NS IZD = 13.62 - 0.032 Conc. of extract P. aeruginosa 0.317NS

Ethanol leaf extract

IZD = 8.16 + 0.009 Conc. of extract P. aeruginosa 0.047NS IZD = 20.08 - 0.003 Conc. of extract S. aureus 0.031NS IZD = 14.09 - 0.005 Conc. of extract B. subtilis 0.054NS IZD = 15.05 - 0.006 Conc. of extract E. coli 0.093NS **=highly significant at p<0.01, *=significant at p<0.05, NS=not significant at p<0.05, r2=coefficient of simple determinant clxxvi

Table 3: F-values of the analysis of variance of treatments (extract solvent, extract concentration and microorganisms) effects on zone of inhibition

Source of variation Degree of Freedom F-values LSD (p<0.001)

Extract liquid 2 55.77*** 0.963 Extract concentration 3 127.72*** 1.112 Microorganisms 3 85.44*** 1.112 Extract liquid x Extract conc. 6 15.33*** 1.926 Extract liquid x Microorganisms 6 35.18*** 1.926 Extract conc. x Microorganisms 9 7.62*** 2.224 Extract liq. x Extract conc. x Microorg. 18 4.40*** 3.85 Error 96 Total 143

Grand mean 14.46 Std. Error 2.376 Relative Std. Error (%) 16.4 ***=very highly significant at p<0.001

clxxvii

Table 4: Mean values of zones of inhibition

Mean Extract liquid Acetone 17.19 Aqueous solution 12.10 Ethanol 14.09 LSD (p<0.001) 0.963***

Extract concentration 40 18.75 100 8.38 200 14.12 400 16.60 LSD (p<0.001) 1.112***

Microorganisms B. subtilis 10.60 E. coli 16.28 P. aeruginosa 12.33 S. aureus 18.64 LSD (p<0.001) 1.112*** ***=very highly significant at p<0.001

clxxviii

Interaction among the treatment means in a factorial experiment for inhibition zone

Table 3 shows the effect of treatment means, that is; extract solvent, extract concentration and microorganisms, and their interactions on zone of inhibition. Where there is significant difference among the interactions vis-à-vis the treatments (extract solvent, extract concentration and microorganisms) then, there is an acceptable effect/impact on inhibition zone, which allows discussion to take place (156). Results indicated that whether individual treatment or combined, there was a very high significant difference (p<0.001) among treatment means. This therefore, means that whether singly or combined, the treatments had a very high significant difference

(p<0.001) on the zone of inhibition (Table 4). clxxix

The implication of the above result is that any of the treatment in a single or combined state will give a very good result on the study with respect to zone of inhibition. However, best result will be achieved when an interaction or a combination of extract solvent, extract concentration and microorganisms is used.

Reliability test of treatment mean for zone of inhibition

With reference to Table 3, zone of inhibition had a grand mean of 14.46, with a standard error of

2.376 and relative standard error (r.s.e.) of 16.4%. This r.s.e. is below 30% limit that was set as acceptability of reliability of mean. The US National Center for Health Statistics had giving 30% value of r.s.e. as the highest acceptable limit for reliability of data (167).

3.3. MIC of ethanol, acetone and aqueous extracts of Spermacoce verticillata on bacterial isolates tested

The MICs of the ethanol, acetone and aqueous extracts of Spermacoce verticillata on the test bacteria are presented in Tables 5-7. The MIC values for the different bacteria were obtained using agar dilution experiment.

3.3.1 MIC of acetone extracts on test bacteria

clxxx

Table 5 shows MIC of acetone leaf extracts of Spermacoce verticillata on the test microorganisms. All the test microorganisms had growth at 25 µg/ml concentrations, but none had, at 400 µg/ml concentration. At 50 µg/ml concentrations, Staphylococcus aureus and

Escherichia coli had no growth, while Bacillus subtilis and Pseudomonas aeruginosa had. The following microorganisms: Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa had no growth at 100 µg/ml and 200 µg/ml concentrations, respectively. However, at both 100 µg/ml and 200 µg/ml concentrations, Bacillus subtilis had growth. Staphylococcus aureus and Escherichia coli, therefore, both had MIC of 50 µg/ml. Conversely, Bacillus subtilis and Pseudomonas aeruginosa had MIC of 400 and 100 µg/ml, respectively; showing that acetone leaf extract of Spermacoce verticillata was more effective against Staphylococcus aureus and Escherichia coli, followed by Pseudomonas aeruginosa and lastly Bacillus subtilis.

Table 5: MIC of acetone leaf extract of Spermacoce verticillata on test microorganisms

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MIC Bacillus subtilis - + + + + 400 Staphylococcusaureus - - - - + 50 Escherichia coli - - - - + 50 P.aeruginosa - - - + + 100 - = No growth, + = Growth

clxxxi

3.3.2 MIC of ethanol extracts on test bacteria

Table 6 shows the MIC of ethanol leaf extracts of Spermacoce verticillata on microorganisms tested. All the test microorganisms had growth at 50 µg/ml and 25 µg/ml concentrations, respectively. On the contrary, none of the test microorganisms had growth at 400 µg/ml and 200 clxxxii

µg/ml concentrations. However, at 100 µg/ml concentration of ethanol leaf extract, Bacillus subtilis and Pseudomonas aeruginosa had growth. While Staphylococcus aureus and

Escherichia coli had no growth. Therefore, Bacillus subtilis and Pseudomonas aeruginosa had a minimum inhibitory concentration of 200 µg/ml as against 100 µg/ml for Staphylococcus aureus and Escherichia coli.

Table 6: Minimum inhibitory concentration (MIC) of ethanol leaf extract of Spermacoce verticillata on microorganisms tested

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MIC clxxxiii

Bacillus subtilis - - + + + 200 Staphylococcus aureus - - - + + 100 Escherichia coli - - - + + 100 P. aeruginosa - - + + + 200 - =No growth, + = growth

3.3.3 MIC of aqueous extracts on test bacteria clxxxiv

Table 7 shows the MIC of aqueous leaf extracts of Spermacoce verticillata on the test microorganisms. All the test microorganisms had growth at 25 µg/ml, 50 µg/ml and 100 µg/ml concentrations. At 400 µg/ml concentration however, Bacillus subtilis, Staphylococcus aureus and Escherichia coli, had no growth. At 200 µg/ml concentration, Bacillus subtilis and

Escherichia coli, had no growth. At 200 µg/ml concentration, Staphylococcus aureus had growth. It is noteworthy that Pseudomonas aeruginosa had growth at all the concentrations of the extract. Thus, aqueous leaf extract of Spermacoce verticillata had little or no effect on

Pseudomonas aeruginosa. On the other hand, the extract was more effective against Bacillus subtilis and Escherichia coli, followed by Staphylococcus aureus.

Comparing the three extracts of Spermacoce verticillata, acetone leaf extract performed best given the MICs, followed by ethanol leaf extract and lastly aqueous leaf extract. According to

Cheesbrough (8), the lower the value of the MIC of an antimicrobial agent, the better its activity.

In view of the observation made by Cheesbrough (8), acetone extract showed the least MIC on the test bacteria when compared to the MICs of the other extracts suggesting that it had the best antimicrobial activity among the three extracts.

Corroborating the above findings, Benjamin (102) had also shown that the volatile oils obtained from Spermacoce verticillata inhibited the growth of Gram -positive and Gram –negative bacteria. It would be recalled that, acetone leaf extract and ethanol leaf extract contained volatile oils (Table 1). This may have contributed to the better performance of acetone leaf extract and ethanol leaf extract compared to the aqueous leaf extract which did not contain volatile oils

(Table 1). There is strong evidence in support of the use of this plant in skin diseases (often clxxxv

involving Staphylococcus aureus) and some bacterial infections (102). In addition, De Sa

Peixoto Neto et al. (29) noted that extract of Spermacoce verticillata exhibited a broad antibacterial activity against multi-resistant strains of bacteria.

clxxxvi

Table 7: MIC of aqueous leaf extract of Spermacoce verticillata on test microorganisms

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MIC Bacillus subtilis - - + + + 200 Staphylococcus aureus - + + + + 400 Escherichia coli - - + + + 200 Pseudomonas aeruginosa + + + + + None - = no growth, += growth

clxxxvii

3.3.4 Minimum bactericidal concentration (MBC) of the Leaf Extracts

The MBC of the ethanol, acetone and aqueous extracts of Spermacoce verticillata against the bacterial isolates tested are presented in Tables 8-10.

3.3.4.1 MBC of the acetone leaf extracts

Table 8 shows the MBC of acetone extracts of Spermacoce verticillata on the test organisms.

Bacillus subtilis and Staphylococcus aureus had growth at 25 µg/ml, 50 µg/ml, 100 µg/ml, 200

µg/ml and 400 µg/ml concentrations, respectively. At 25µg/ml, 50 µg/ml, 100 µg/ml concentrations, respectively, Escherichia coli and Pseudomonas aeruginosa had growth, but did not grow at 200 µg/ml and 400 µg/ml concentrations, respectively.

The MBC values of the extracts on the test organisms were mostly unattainable as increasing the concentration through 25 µg/ml to 400 µg/ml had no cidal effect on B. subtilis, E. coli and P. aeruginosa. This same extract had MIC values of 200 µg/ml on B. subtilis and no value for P. aeruginosa. It was observed that the MBC values obtained were many times more than the MIC values. When MBC and MIC values are close or almost equal, the antibacterial agent is bactericidal because the cidal effect can be attained without causing undue harm on the host. clxxxviii

When the MBC is many times the value of MIC, the antibacterial agent is bacteriostatic because it is clinically unachievable to attain a cidal effect without causing damage to the host at concentration far above the MIC value.

Insistence to obtain MBC values would mean increased concentrations of the extracts. These increased concentrations kills the microorganisms as intended but it is accompanied with toxic damaging effects on the host. The toxic effect outweighs the benefits of a cidal action, so the best antibacterial concentration for the acetone use is the MIC concentration. The acetone extract is thus termed to be bacteriostatic in action (6, 79, 70).

Table 8: MBC of acetone leaf extract of Spermacoce verticillata on test microorganisms.

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MBC Bacillus subtilis + + + + + none Staphylococcus aureus + + + + + none Escherichia coli - - + + + 200 Pseudomonas aeruginosa - - + + + 200 - = no growth, + = growth

clxxxix

3.3.4.2 MBC of the ethanol leaf extracts

Table 9 shows the MBC of ethanol leaf extracts of Spermacoce verticillata on the test organisms.

Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa had growths at all the extract concentrations (25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml). However,

Escherichia coli had growth at 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml concentrations, respectively. However, at 400 µg/ml concentration, Escherichia coli had no growth.

These results showed that the extracts had no bactericidal activity on most of the test bacteria even at their highest concentrations. Generally, acetone leaf extract proved more effective than ethanol and aqueous leaf extracts of Spermacoce verticillata. It was observed that ethanol and aqueous leaf extracts of Spermacoce verticillata had similar performance considering the minimum bacteria concentrations. In summary, looking at the occurrence of very high MBC cxc

values that were close to their respective MIC values, it is suggestive that the bioactive principles of the plant extracts had little or no bactericidal effects on the test organisms. Though, further increasing the concentrations of the extracts could elicit bactericidal effect on the microorganisms, this increase could lead toxicity too, which would hamper the usefulness of such extract at higher concentrations. The extracts could thus be said to have bacteriostatic effects on the organisms.

Table 9: MBC of Ethanol leaf extract of Spermacoce verticillata on test microorganisms

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MBC Bacillus subtilis + + + + + none Staphylococcus aureus + + + + + none Escherichia coli - + + + + 400 Pseudomonas aeruginosa + + + + + none - = no growth, + = growth

cxci

3.3.4.3 MBC of the aqueous leaf extracts

Table 10 shows the minimum bactericidal concentration of aqueous extracts of Spermacoce verticillata on the test organisms. The following test microorganisms: Bacillus subtilis,

Escherichia coli and Pseudomonas aeruginosa all had growth at all the extract concentration

(25µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml), while Staphylococcus aureus had growth at 25 µg/ml, 50 µg/ml, 100 µg/ml and 200 µg/ml concentrations, but had none at 400

µg/ml. cxcii

Table 10: MBC of aqueous leaf extract of Spermacoce verticillata on test microorganisms

Test microorganisms Extract concentration (µg/ml) 400 200 100 50 25 MBC Bacillus subtilis + + + + + none Staphylococcus aureus - + + + + 400 cxciii

Escherichia coli + + + + + none Pseudomonas aeruginosa + + + + + none - = no growth, + = growth

3.3.5 Antifungal studies of the leaf extracts on test organisms cxciv

The result of this study indicated that the aqueous, acetone and ethanol leaf extracts of

Spermacoce verticillata had no antifungal activity against all the tested fungal isolates, namely:

Microsporum audouinii, Trichophyton rubrum, Candida albicans, Aspergillus niger.

3.4 Antibacterial activity of acetone alone on test organisms

Table 11 shows the antibacterial activity of acetone and the control (gentamycin at 40 µg/ml).

Acetone had no inhibitory effects on the organisms. This suggests that the extracting solvent - acetone, had no antimicrobial action of its own on the organisms. Hence, all growth inhibition of the organisms using the acetone extract was due to the effects of the plant extract alone on the bacteria.

cxcv

Table 11: Result of antimicrobial test of the extracting solvent (acetone)

Inhibition Zone (mm) Test Organisms Acetone Gentamycin (40 µg/ml) Bacillus subtilis 0.00 19.00 Staphylococcus aureus 0.00 21.00 Escherichia coli 0.00 18.00 P. aeruginosa 0.00 18.00

cxcvi

3.4.1 Antibacterial activities of the fractions A and B

The phytochemical constituents of fractions A and B of the acetone extract of Spermacoce verticillata are presented in Tables 12 and 13, respectively.

3.4.1.1 The phytochemical constituents of fraction A

Tables 12, the following phytochemicals: alkaloids, tannins, terpenes and steroids, balsam, phenol and volatile oils were present in fraction A, while flavonoids, saponins and resins were absent. Out of the 9 phytochemicals, 6 were present while 3 were not present in fraction A.

3.4.1.2 The phytochemical constituents of fraction B

In Table 13, the results of phytochemical analysis of fraction B of the extract are shown.

Alkaloids, tannins, saponins, terpenes and steroids, balsam, phenol and volatile oils were present in fraction B, while flavonoids and resins were absent. Comparing both fractions, alkaloids, tannins, terpenes and steroids, balsam, phenol and volatile oils were present in both, while flavonoids and resins were absent. However, fraction A did not contain saponins, which was present in fraction B. These phytochemicals as was found in fractions A and B of the extract have also been found in other plant extracts; and have been shown to be effective in wound healing. Roy et al. (52), and James and Friday (63) had discovered that extract ointments of

Ficus religiosa and Euphorbia heterophylla, which were rich in phytochemicals such as alkaloids, tannins, saponins, terpenes and steroids, balsam, phenol and volatile oils (as was found in Spermacoce verticillata leaf extract ointment of fractions A and B showed significant wound healing activity upon topical application and improved healing of wounds on rats. cxcvii

Furthermore, phytochemical screening of Ocimum kilimandscharicum revealed the presence of tannins whose leaf extract possesses wound healing property that is attributed to its ability to increase the rate of wound contraction and epithelization (36). This therefore motivated the formulation of ointment with fraction B extract in this study. This ointment effectively improved wound healing of infected rat at 2 % concentration on the 10th day for the various microorganisms (Tables 16 - 19).

cxcviii

Table 12: Phytochemical constituents of fraction A of acetone extract

Metabolites Results Alkaloids + Flavonoids - Tannins + Saponins - Terpenes and steroids + Balsam + Phenol + Resins - Volatile oils + - = not present, + = present

cxcix

Table 13: Phytochemical constituents of fraction B of acetone extract

Metabolites Results Alkaloids + Flavonoids - Tannins + Saponins + Terpenes and steroids + Balsam + Phenol + Resins - Volatile oils + - = not present, + = present

cc

3.4.2 Antibacterial activities of fractions A and B on test microorganisms

The antibacterial activities of fractions A and B on test microorganisms are represented in Figs. 7 and 8, respectively.

3.4.2.1 Antibacterial activities of fraction A on test microorganisms

In Fig. 7, the IZDs of various concentrations of fraction A leaf extract of Spermacoce verticillata on test microorganisms are presented. The IZD of fraction A leaf extract on test organisms varied between 0.0 mm (on all the microorganisms at 50 µg/ml concentration) and 18 mm (on Bacillus subtilis and Pseudomonas aeruginosa at 400 µg/ml concentration). Fraction A extract had the greatest effect on Pseudomonas aeruginosa with a mean IZD of 11 ± 8.1mm, followed by

Bacillus subtilis, Escherichia coli, and lastly, Staphylococcus aureus with the following sum of inhibition zone diameter of 9.5 ± 7.5 mm, 8.5 ± 6.6 mm and 7.3 ± 8.6 mm, respectively. The control (gentamycin at 40 µg/ml concentration), however, had greater inhibition zone diameter cci

of 25 mm on Escherichia coli and Pseudomonas aeruginosa, than that of fraction A’s

greatest -18 mm (at 400 µg/ml concentration), for Bacillus subtilis and Pseudomonas

aeruginosa. De Sa Peixoto Neto et al. (29) had noted that extract of Spermacoce

verticillata exhibit a broad antibacterial activity against multi-resistant strains of

Pseudomonas aeruginosa.

Inhibition zone diameter (mm)

50

45 44

40 38

35 34

30 29

25 25 25 22 21

20 18 18 17 16 16 15 12 12 11 10 10 9.5 8.6 10 8 8 8.5 8.1 7.3 7.5 6.6 5

0 0 0 0 0 0 40 µg/ml 50 µg/ml 100 200 400 Sum Mean St. Dev. Genta µg/ml µg/ml µg/ml Concentration of the extracts (µg/ml) Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa

ccii

Fig. 3.7: IZD of various concentrations of fraction A leaf extract of Spermacoce vertillata on test microorganisms

3.4.2.2 Antibacterial activities of fractions B on test microorganisms

The IZDs of various concentrations of fraction B leaf extract of Spermacoce verticillata on test microorganisms are shown in Fig. 8. The various IZD of the fraction B on test microorganisms ranged between 0.0 mm (at 50 µg/ml concentration) on Bacillus subtilis, Escherichia coli and

Pseudomonas aeruginosa and 30 mm (at 400 µg/ml concentration) on Escherichia coli. This value of 30 mm (at 400 µg/ml concentration) on Escherichia coli exceeded that of the control

(gentamycin at 40 µg/ml concentration) which was highest of 25 mm on Pseudomonas aeruginosa. This performance is better than that of fraction A (Fig.7). Generally, fraction B was most effective against Staphylococcus aureus with a mean IZD of 18.5 ± 6.4 mm. The IZDs against other organisms were: 17.5 ± 12.6 mm, 14.2 ± 10.7 mm and 13.3 ± 9.1 mm for

Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, respectively. This finding corroborates the observation of Benjamin (102) who showed that extract of Spermacoce cciii

verticillata had strong evidence in support of its use in skin diseases (often involving

Staphylococcus aureus) and for some other bacterial infections.

Improved zones of inhibition against the test organisms were observed with both fractions A and

B as the extract concentrations increased. Both fractions A and B exhibited antibacterial activity against Gram positive and Gram negative bacteria. At same concentrations, the zones of inhibition seen with fraction B were higher than those of fraction A against the same organisms.

It could be that the saponin content of fraction B enhanced its antimicrobial activity. Considering the phytochemical contents of both fractions A and B, saponins happened to be the only phytochemical that was present in fraction B, but was absent in fraction A (Tables 13 and 14).

Saponins are surface active agents identifiable by their ability to froth (79).

Surface active agents lower the surface tension between the microorganisms and the antimicrobial agents/bioactive components of the fraction, thereby enhancing the permeability of the cell membrane of the organisms to the extract fraction. Increased uptake of the fraction into the microbial cells increases the concentration of the active principles of the fraction moving to the target sites on the bacteria thus eliciting increased antimicrobial effects of the fraction B on the cells (54). This could have accounted for the higher zones of inhibition noticed with fraction

B. Similarly, saponins possess antioxidant and antimicrobial activities that are known to promote wound healing process (54).

cciv

ccv

80 74

70 70

60 59

53 50

40 ion zone diameter (mm)

30 30

Inhibit 25 25 24 22 21 21 21 21 20 20 20 18.5 20 18 18 17.5 15 14 14.2 13.3 12.6 10 10.7 9.1 10 6.4

0 0 0 0 40 µg/ml 50 µg/ml 100 200 400 Sum Mean St. Dev. Genta µg/ml µg/ml µg/ml

Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Concentration of the extracts (µg/ml) Fig. 3.8: IZD of various concentrations of fraction B leaf extract of Spermacoce vertillata on test microorganisms

ccvi

3.4.2.3 Relationship between various concentrations of fraction A leaf extract and inhibition zone diameter on test microorganisms

The regression line graphs for the relationships between various inhibition zone diameters against the different concentrations of fraction A leaf extract of Spermacoce verticillata on all the test microorganisms are as shown in Fig. 3.9.

Staphylococcus aureus

The coefficient of simple determinant, r², shows that only 13.7% of the variation caused on the zone of growth inhibition of Staphylococcus aureus was due to the various concentrations of fraction A leaf extract which was not significant at p=0.05. Its slope of 0.024 is positive which implied that at increased concentrations of fraction A, there was a corresponding increase of inhibition zone diameter on Staphylococcus aureus, though the relationship was not significant at p=0.05.

Escherichia coli

The line graph for the zone of inhibition of Escherichia coli by different concentrations of fraction A extract show that 2.4% of the variation caused on the inhibition zone diameter of

Escherichia coli was due to the different concentrations of the fraction A which was not significant at p=0.05. The line graph has a positive slope of 0.009, which implied that increasing concentration of fraction A leaf extract, led to increased inhibition zone diameters for

Escherichia coli. ccvii

Bacillus subtilis

The r2 value showed that 12.2 % difference that was observed on the inhibition zone diameter was due to the extract concentrations of fraction A. The relationship was not significant at p=0.05, though it was positive, indicating a corresponding increase on the inhibition zone ccviii

30

25 IZD= 8.814 + 0.020 Conc. of extract r² = 0.122NS, for B. subtilis IZD = 11.14 + 0.016 Conc. of extract r² = 0.071NS, for P. aeruginosa IZD = 10.24 + 0.009 Conc. of extract r² = 0.024NS, for E. coli 20 IZD = 6.212 + 0.024 Conc. of extract r² = 0.137NS, for S .aureus

B.subtilis 15 S.aureus (mm) E.coli P.aeruginosa Zone ofinhibition Linear (B.subtilis)

10 Linear (S.aureus) Linear (E.coli) Linear (P.aeruginosa)

5

0 0 100 200 300 400 500 Concentrations of extract (μg/ml)

ccix

Fig. 3.9: IZD of various concentrations of fraction A leaf extract of Spermacoce verticillata against the test microorganisms

diameter with increasing concentrations of the fraction A leaf extract of Spermacoce verticillata.

Pseudomonas aeruginosa

The regression line graph shows that the relationship between inhibition zone diameter and the concentration of fraction A leaf extract of Spermacoce verticillata was positive with an r2 value of 0.071. This value shows that 7.1% variation that was caused on the inhibition zone diameter of Pseudomonas aeruginosa was due to the concentration of fraction A leaf extract. The relationship was not significant at p=0.05 confidence interval.

Comparing the coefficient of simple determinant, r2, of all the line graphs, fraction A had the greatest effect on Staphylococcus aureus with 13.7% variation on the inhibition zone diameter; this was followed by Bacillus subtilis, Pseudomonas aeruginosa, and lastly Escherichia coli with the following values: 12.2%, 7.1% and 2.4%, respectively. Although all the relationships were not significant at p=0.05 confidence interval, they were all positive indicating a positive correlation between concentration of fraction A and the zones of inhibition.

ccx

3.4.2.4 Relationship between various concentrations of fraction B leaf extract and inhibition zone diameter on test microorganisms

The regression line graphs for the relationships between various inhibition zone diameters and the different concentrations of fraction B leaf extract of Spermacoce verticillata on all the test microorganisms are shown in Fig. 10.

Staphylococcus aureus

The line equation for zones of inhibition of the different concentrations of fraction B leaf extract of Spermacoce verticillata on Staphylococcus aureus is:

IZD = 14.87 + 0.026 Conc. of extract r² = 0.482*.

The relationship between inhibition zone diameter of Staphylococcus aureus and concentrations of fraction B extract is positive with 48.2 % variation on the inhibition zone diameter of

Staphylococcus aureus caused by the extract concentrations. The relationship was significant at p=0.05. This shows that at increasing concentration of fraction B extract, there was a corresponding increase in the inhibition zone diameter; which shows that extract of fraction B actually inhibited the growth of test microorganisms which was significant at p=0.05 confidence level, and the increase was concentration dependent. ccxi

ccxii

35

30

IZD = 10.71 + 0.048 Conc. of extract r² = 0.43*, for E. coli IZD = 14.87 + 0.026 Conc. of extract 25 r² = 0.482*, for S. aureus

20 IZD = 11.61 + 0.032 Conc. of extract r² = 0.225NS, for P. aeruginosa B.subtilis (mm) S.aureus Zone ofinhibition 15 E.coli IZD = 12.41 + 0.015 Conc. of extract r² = 0.069NS, for B. subtilis P.aeruginosa

Linear 10 (B.subtilis) Linear (S.aureus) Linear (E.coli) 5 Linear (P.aeruginosa)

0 0 100 200 300 400 500 Conc. of extract (μg/ml)

ccxiii

Fig. 3.10: IZD of various concentrations of fraction B leaf extract of Spermacoce verticillata against the test microorganisms

Escherichia coli

The line graph of zones of inhibition of fraction B leaf extract on Escherichia coli did also have a positive slope.

The linear equation derived is:

IZD = 10.71 + 0.048 Conc. of extract r² = 0.43*

The extract concentration of fraction B had 43% variation on the inhibition zone diameter of

Escherichia coli with a positive relationship which was significant at p=0.05 confidence interval.

This indicates that at increasing concentrations of fraction B leaf extract, there was a corresponding increase in the inhibition zone diameter of Escherichia coli. Consequently, it can be noted that the fraction B extract inhibited the growth of Escherichia coli.

Bacillus subtilis

The line graph of zones of inhibition of fraction B leaf extract on Bacillus subtilis did also have a positive slope.

The linear equation derived is:

IZD = 12.41 + 0.015 Conc. of extract r² = 0.069NS ccxiv

The relationship between inhibition zone diameter and extract concentrations of fraction B is positive with 6.9% change in inhibition zone diameter of Bacillus subtilis caused by the extract concentration of fraction B, which was however not significant at p=0.05. Though the relationship was not significant at p=0.05, it was evident that fraction B actually inhibited the growth of Bacillus subtilis because of the positive relationship that existed between the two parameters, and the inhibition was concentration dependent.

Pseudomonas aeruginosa

The line equation for zones of inhibition of the different concentrations of fraction B leaf extract of Spermacoce verticillata on Pseudomonas aeruginosa is:

IZD = 11.61 + 0.032 Conc. of extract r² = 0.225NS

The regression line graph above shows that the relationship between inhibition zone diameter and the concentration of fraction B leaf extract of Spermacoce verticillata was positive with an r2 value of 0.225. This value shows that 22.5% variation that was caused on the inhibition zone diameter of Pseudomonas aeruginosa was due to the concentration of fraction B leaf extract. The relationship was not significant at p=0.05 confidence interval, however, the result showed that fraction B really inhibited the growth of Pseudomonas aeruginosa.

Comparing the regression line graphs for both fractions, it is evident that they all had positive slopes, meaning that as the concentration of the fractions A and B extract increased, the accompanying 1ZDs increased too. The effect of Fraction B effect on the organisms were in the following order: Staphylococcus aureus (48.2%) > Escherichia coli (43%) > Pseudomonas ccxv

aeruginosa (22.5%) > Bacillus subtilis (6.9%), the same pattern, though more intense. The effect of fraction A.

Considering their significant levels, the effect of fraction B on the first two microorganisms above were significant at p=0.05, but was not significant in the last two microorganisms at p=0.05 confidence interval. In contrast to the significance of fraction B, fraction A had no significant effect on the inhibition zone diameter of all the test organisms at p=0.05 confidence level. Therefore, it can be inferred that fraction B had a better antimicrobial activity on the test organisms than fraction A.

3.4.3 Minimum inhibitory concentration (MIC) of fraction A and B

The MIC of fractions A and B of acetone leaf extract of Spermacoce verticillata are presented in

Tables 14 and 15, respectively.

3.4.3.1 MIC of fraction A

The minimum inhibitory concentration (MIC) of fraction A on test organisms as presented in

Table 15 showed that at 100 µg/ml, 200 µg/ml and 400 µg/ml concentrations, respectively, fraction A inhibited the growth of the organisms except for Staphylococcus aureus which had a growth at 100 µg/ml concentration. Aside this, all the microorganisms had growth at 25 µg/ml and 50 µg/ml concentrations, respectively, implying there was no inhibition at those concentrations. The better minimum inhibitory concentration (MIC) was achieved on Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli at 100 µg/ml, respectively; this was followed by that of Staphylococcus aureus which had 200 µg/ml. ccxvi

Table 14: MIC of fraction A of Spermacoce verticillata on test microorganisms

Test organism Concentration of fraction A (µg/ml) 25 50 100 200 400 MIC Staphylococcus aureus + + + - - 200 Bacillus subtilis + + - - - 100 Pseudomonas aeruginosa + + - - - 100 Escherichia coli + + - - - 100 + control (S. aureus broth) + + + + + - control (Sterile broth) ------= no Growth, + = growth

ccxvii

3.4.3.2 MIC of fraction B

The MIC of fraction B on test organisms as presented in Table 16 showed that at 50 µg/ml, 100

µg/ml, 200 µg/ml and 400 µg/ml concentrations, respectively, fraction B inhibited the growths of the organisms except for Bacillus subtilis and Escherichia coli which had growth at 50 µg/ml concentrations. In contrast, all the microorganisms had growth at 25 µg/ml concentrations, implying that there was no inhibition at those concentrations. The greatest activity was achieved on Staphylococcus aureus and Pseudomonas aeruginosa at 50 µg/ml concentration. This was followed by Bacillus subtilis and Escherichia coli which had MIC of 100 µg/ml concentration.

Comparing the two fractions, fraction A did not have any inhibition at 100 and 50 µg/ml concentration on Staphylococcus aureus unlike all fraction B, which inhibited Staphylococcus aureus at both concentrations. ccxviii

Fraction B did exhibit an improved MIC on Staphylococcus aureus and Pseudomonas aeruginosa. In addition, it had better antibacterial action on Gram positive and Gram negative organisms. The lower antimicrobial activity of Fraction A was thought to be due to the fact that this fraction was devoid of saponins (Tables 13 and 14). It could be that saponins was very vital for the inhibitory action of Spermacoce verticillata on Staphylococcus aureus and Pseudomonas aeruginosa. Corroborating this finding, Sachin et al. (54) observed that saponins possess antioxidant and antimicrobial activities that are known to promote wound healing process.

Furthermore, it has been noted that the lower the MIC of an antimicrobial agent, the better is its activity (8). As fraction B had the least MIC on all test bacteria (Table 16), it was therefore, chosen for the formulation of ointments used in the wound healing studies

Table 15: MIC of fraction B of the leaf extract of Spermacoce verticillata on test microorganisms.

Test organisms Concentration of fraction B (µg/ml) 25 50 100 200 400 MIC Staphylococcus aureus + - - - - 50 Bacillus subtilis + + - - - 100 Pseudomonas aeruginosa + - - - - 50 Escherichia coli + + - - - 100 + control (S. aureus broth) + + + + + - control (Sterile broth) ------= no growth, + = growth

ccxix

3.5 Antimicrobial wound healing studies

Tables 16 - 19 show results of infected wound healing studies with fraction B ointments at different concentrations on the various organisms, with time.

3.5.1 Effect of various concentrations of fraction B ointment of Spermacoce verticillata on Escherichia coli infected wound

The result on Table 16 shows that after 14 days of dressing wounds infected with Escherichia coli with different concentrations of ointments, 2 % of fraction B had achieved complete healing of the wound on rat number 4 by day 10, while 0.1 %, 0.2 % , 1 %, 5 % concentrations and ccxx

gentamycin recorded complete healing on day 14. Rat number 7 that was treated with blank ointment died on the 7th day. This implied that fraction B had better healing effect as compared to gentamycin.

The acetone extract ointment exhibited wound healing ability at all formulated concentrations.

All wounds treated with it eventually healed unlike the wound treated with the blank ointment in which the rat died on the 7th day. The wound treated with the 2 % ointment healed fastest on day

10.

Table 16: Effect of various concentrations of fraction B ointments of Spermacoce verticillata on Escherichia coli infected wounds

Strength of ointment Rat number Wound diameters (mm)

Day 1 Day 4 Day 7 Day 10 Day 14 0.1% 1 25 15 9 3 0 0.2% 2 25 17 8 2 0 1% 3 25 16 7 2 0 2% 4 25 12 6 <1 0 ccxxi

5% 5 25 17 12 2 0 0.1% gentamycin 6 25 18 8 2 0 Blank ointment 7 25 24 Died - -

3.5.2 Effect of various concentrations of fraction B ointment of Spermacoce verticillata on Pseudomonas aeruginosa infected wounds

Table 17 shows that 2% of fraction B recorded complete healing on wound infected with

Pseudomonas aeruginosa just after day 7. The 0.1 %, 0.2 %, 1 %, 5 % concentrations, as well as ccxxii

gentamycin which recorded complete healing by day 10. Rat number 3 treated with 1 % of the ointment got completely healed by day 14 while no improvement was noticed on the control rat, treated with blank ointment. This shows that the fraction B ointment of Spermacoce verticillata had better healing effect on Pseudomonas aeruginosa than gentamycin ointment.

All the wounds healed in response to the application of fraction B extract at all formulated concentrations. The wound treated with the blank ointment never showed much improvement until the 14th day. The 2 % ointment gave a better wound healing than other ointments at 0.1, 0.2,

1, 5 % and 0.1 % gentamycin ointment.

Table 17: Effects of various concentrations of fraction B ointment of Spermacoce verticillata on P. aeruginosa infected wounds

Strength of ointment Rat number Wound diameters (mm)

ccxxiii

Day 1 Day 4 Day 7 Day 10 Day 14 0.1% 1 25 14 2 1 0 0.2% 2 25 11 1 1 0 1% 3 25 12 1 4 0 2% 4 25 10 1 0 0 5% 5 25 13 2 1 0 0.1% gentamycin 6 25 11 2 1 0 Blank ointment 7 25 25 24 23 23

ccxxiv

Plate 1: Wound infected with Pseudomonas aeruginosa on day 1 prior to treatment with 2 % fraction B ointment

ccxxv

Plate 2: Pseudomonas aeruginosa infected wound treated with 2 % fraction B ointment on Day 4

ccxxvi

Plate 3: Pseudomonas aeruginosa infected wound treated with 2 % fraction B ointment on Day 7

ccxxvii

Plate 4: Pseudomonas aeruginosa infected wound treated with 2 % fraction B ointment on Day 10

ccxxviii

Plate 5: Pseudomonas aeruginosa infected wound prior to treatment with 5 % Fraction B ointment on Day 1

ccxxix

Plate 6: Pseudomonas aeruginosa infected wound treated with 5 % fraction B ointment on Day 4

ccxxx

Plate 7: Pseudomonas aeruginosa infected wound treated with 5 % fraction B ointment on Day 7

ccxxxi

Plate 8: Pseudomonas aeruginosa infected wound treated with 5 % fraction B ointment on Day 10

ccxxxii

Plate 9: Pseudomonas aeruginosa infected wound treated with blank ointment on Day 10

ccxxxiii

3.5.3 Effect of various concentrations of fraction B ointment of Spermacoce verticillata on Bacillus subtilis infected wound

Table 18 shows the diameter of wounds infected with Bacillus subtilis. By day 4, the 2% ointment had the best wound healing activity when compared to other ointments. Gentamycin was the least wound healing promoter on same day, this implied that fraction B had an accelerated healing effect more than gentamycin. By the 10th day however, they all followed the same trend, and by day 14, all wounds had healed. In contrast, the rat treated with the blank ointment died on day 10.

All acetone extract ointment exhibited wound healing abilities on B. subtilis infected wounds; the wound got completely healed on the 14th day. It seemed that the B. subtilis infected wound did not respond as good as E. coli and P. aeruginosa infected wounds to the extract ointment. The 2

% ointment had the best wound healing effect on the 10th day for E. coli and P. aeruginosa but this was not seen with B. subtilis.

ccxxxiv

Table 18: Effects of various concentrations of fraction B ointments of Spermacoce verticillata on Bacillus subtilis infected wounds

Strength of ointment Rat number Wound diameter (mm)

Day 1 Day 4 Day 7 Day 10 Day 14 0.1% 1 25 17 9 3 0 0.2% 2 25 17 8 2 0 1% 3 25 16 7 2 0 2% 4 25 12 6 4 0 5% 5 25 17 12 2 0 Gentamycin ointment 0.1% 6 25 21 11 2 0 Blank ointment 7 25 24 22 Died

ccxxxv

3.5.4 Effect of various concentrations of fraction B ointment of Spermacoce verticillata on Staphylococcus aureus infected wound

Table 19 shows the wound healing effects of fraction B ointments on the wounds infected with

Staphylococcus aureus. The 2% ointment had completely healed the wound on rat number 4 by day number 10, while wounds on rat numbers 2, 3 and 5, were almost healed. These wounds were completely healed by day 14. Rat number 7 treated with the blank ointment however, died on Day 10.

Tables 16 - 19 show that the ointment formulated with fraction B of the acetone leaf extract of

Spermacoce verticillata possessed antimicrobial wound healing effect with the best result achieved at 2 % strength of ointment where infected wounds were healed by the 10th day. The activity of this concentration was superior to that of gentamycin ointment. The wound healing activity of the fraction B becomes more evident when compared to effects of the blank ointment that did not promote any wound healing. The poor performance of fraction B ointment on

Bacillus subtilis -infected wounds is in agreement with the earlier observed IZDs among the test organisms in which Bacillus subtilis also was least susceptible. Bacillus subtilis has the ability ccxxxvi

to form a tough protective endospore that allows the bacterium to tolerate certain antimicrobial substances (7, 81, 165).

It is important to note that increasing the concentration of the ointments increased their ability to promote wound healing. However, increasing the concentrations of this ointment beyond 2 % did not seem to increase the rate of wound healing. This could be related to the concentration of saponins in the extracts. Hugo and Russel (6) and Okore (79) reported that small amounts of surfactants below their critical micelle concentrations (CMC) enhance the antimicrobial actions of ointments by increasing the liberation and uptake/permeability of the

Table 19: Effects of various concentrations of fraction B ointments on Staphylococcus aureus infected wound sizes

Strength of Ointment Rat number Wound diameters (mm)

Day 1 Day 4 Day 7 Day 10 Day 14 0.1% 1 25 15 10 7 0 0.2% 2 25 16 4 1 0 1% 3 25 16 4 1 0 2% 4 25 17 5 0 0 5% 5 25 15 6 1 0 Gentamycin ointment 0.1% 6 25 16 3 4 0 Blank ointment 7 25 24 23 Died -

ccxxxvii

antimicrobial agent from the ointment into the organism but concentrations of the surfactant above the CMC often diminish the activity of the formulation.

The reduction in the activity of the ointment could be due to the entrapment of the antimicrobial compounds/bioactive principles of the fraction within the micelles of the surfactant. This reduces the amount of the antimicrobial agent made available for uptake by the organisms, which is seen as a reduction in antimicrobial activity of the 5 % extract ointment. Besides, the receptor sites on the microorganism may have been significantly saturated with the antimicrobial agent.

Increasing the concentration of the ointment above 2 % therefore, would not be of advantage as it would only increase the probable toxic effects of the ointment on the animals with no accompanying increased antimicrobial activity. Therefore, the best strength of ointment concentration was achieved at 2 % concentration with grand mean of 9.00 µm performing better than that of control (0.1% gentamycin) which had a grand mean of 9.60 µm (Table 21). ccxxxviii

3.5.5 Interaction among the treatment means in a factorial experiment for wound diameter

In Table 20, the interaction among the treatment means in a factorial experiment for wound diameter is represented. It shows the effect of treatment means, that is; days, strength of ointment and microorganisms, and their interactions on wound diameter. Where there are significant differences among the interactions vis-à-vis the treatments (days, strength of ointment and microorganisms) then, there is an acceptable effect on the wound diameter which allows discussion to take place (156). Therefore, the analysis of variance results from this study showed that whether individual treatment or combined, there was a very high significant difference at p<0.001 confidence level among treatment means (Table 22).

The implication of the above result is that any of the treatments (e.g. days, strength of ointment or microorganisms) in a single or combined state will give a very good result on the study with ccxxxix

respect to wound diameter. However, best result will be achieved when an interaction or a combination of days, strength of ointment and microorganisms is used.

3.5.6 Reliability test of treatment of mean for wound diameter

As seen in Table 20, the wound diameter had a grand mean of 10.46, with a standard error of

0.925 and a relative standard error of 8.8 % (r.s.e). It is obvious that this r.s.e. is below 30 % limit that was set as acceptability of reliability of mean. The United States National Centre for

Health Statistics had given 30 % value of r.s.e. as the highest acceptable limit for reliability of data (167).

Table 20: F-values of the analysis of variance of treatments (days, strength of ointment and microorganisms) effects on wound diameter (µm)

Source of variation Degree of Freedom F-values LSD (p<0.001)

Days 4 10,000.31*** 0.2804 Strength of ointment 9 175.67*** 0.5256 Microorganisms 3 39.47*** 0.2714 Days x Strength of ointment 36 33.75*** 1.1752 Days x Microorganisms 12 64.00*** 0.596 Strength of oint. x Microorganisms 15 171.24*** Days x Strength of oint. x Microorg. 60 36.20*** 0.6069 Error 279 Total 418

Grand mean 10.462 Std. Error 0.9249 ccxl

Relative Std. Error (%) 8.80 ***=very highly significant at p<0.001

3.5.7 Performance of fraction B ointment in relation to number of days, strength of the ointment and its effect on wound healing infected by test microorganisms

A critical examination of Table 21 revealed that, given the number of days the wound healing was monitored, best healing was observed on the 14th day. Thus, wound healing followed the following increasing order of performance according to the number of days the wounds took to heal: 1 < 4 <7 < 10 < 14. Although by the 10th day (Tables 17 - 20), some wounds had completely healed, all the wounds at all levels of concentrations healed on the 14th day except for ccxli

the blank which had no extract in it. In relation to the strength of ointment, best result was achieved at 2 % ointment concentration. The performance of the strength of the ointment was in the following increasing order: 5 % < 0.1 % < 0.2/1 % < 0.1% Gentamycin < 2 % ointment concentration.

On the test microorganisms, Staphylococcus aureus had an overall best susceptibility to the fraction B ointment on wound healing. This was followed by Escherichia coli. Therefore, the overall performance of the ointments on the wounds infected by test microorganisms was in the following increasing order: Pseudomonas aeruginosa < Bacillus subtilis < Escherichia coli <

Staphylococcus aureus. The better performance of Escherichia coli and Staphylococcus aureus conforms to the findings of Burkill (51), who noted that volatile oils (which were also present in fraction B) inhibits the growth of Staphylococcus aureus and Escherichia coli. Corroborating the performance of fraction B ointment of the leaf of Spermacoce verticillata in this study, earlier workers had observed that Spermacoce verticillata has some medicinal uses mostly on skin conditions (51, 83). While this study advocates the use of Spermacoce verticillata leaf ointment in the healing of wounds, other authors have equally noted that plant–based antimicrobial compounds are known to be very effective and are not usually associated with the many side effects that are observed when taking synthetic antimicrobials (49).

Table 21: Mean values of wound Diameter

Mean Days One 25.00 Four 16.36 Seven 7.31 Ten 2.82 Fourteen 0.82 LSD (p<0.001) 0.2809*** ccxlii

Strength of ointment 0.1 % 10.30 0.2 % 9.40 1 % 9.40 2 % 9.00 5 % 10.60 0.1% Genta 9.60 LSD (p<0.001) 0.5256***

Microorganisms B. subtilis 10.95 E.coli 10.21 P.aeruginosa 10.98 S.aureus 9.71 LSD (p<0.001) 0.2714*** ***=very highly significant at p<0.001

3.6 Toxicity study of fraction B for the determination of its lethal dose - 50 (LD50) ccxliii

A summary of the result of the study carried out with three doses (10 µg/g, 100 µg/g and 1000

µg/g) of the acetone fraction B of Spermacoce verticillata in order to determine its lethal dose is presented in Table 22. It shows that the rat injected with 1000 µg/g of body weight of the fraction B died, while those injected with 10 µg/g and 100 µg/g, lived on.

The toxicological study suggests that the lethal dose of fraction B would exceed 100 µg (of acetone leaf extract)/g of the animal’s body weight. The 100 µg/g was used as a guide in the acute toxicity study for calculation of the LD50 using the Karbers method. The LD50 was calculated to be 688 µg/g. The LD50 is the dose of a drug that is required to kill or adversely affect 50% of the experimental animals under investigations.

ccxliv

Table 22: Result of toxicological study

Group Dose (µg/g) No of rats adversely affected

1 10 -

2 100 -

3 1000 1(died)

- = No casualty

ccxlv

Table 23: Mortality of animals administered graded doses of acetone extract of Spermacoce verticillata

Group Dose (µg/g) No. of deaths % Mortality

1 250 1 20

2 500 3 60

3 750 4 80

4 1000 4 80

5 1250 5 100

6 (negative control) - - -

- = No casualty

ccxlvi

3.7 Histological Studies

Plates 12-20 show the photomicrographs of the skin, kidney, heart and liver of untreated

(control) experimental animals in comparison with the test experimental animals, treated with the

LD50 of Fraction B. Comparing the photomicrographs of test animals to those of the control animals showed no abnormal findings.

Toxicological studies became necessary after the tragedy of thalidomide that occurred in Great

Britain where mothers that had taken this drug to combat morning sickness gave birth to babies with malformed upper limbs. Although, herbal drugs are less toxic, compared to synthetic drugs, as was also noted by Iwu (49) that plants–based antimicrobial compounds are known to be very effective and are not usually associated with the many side effects that are observed when taking synthetic antimicrobials; it is pertinent to state that countries/studies embarking on new drugs development should go for toxicity testing so as to prevent such tragedies.

Toxicity studies are carried out on experimental animals with the assumption that man will respond to drugs in the same manner as the test animals (9). The acute toxicity studies were carried out to determine the median lethal dose of the extract. It provides guidance on the doses ccxlvii

of the extract that can be used for further investigations. Part of the toxicity test involves studies that may indicate the probable target organ(s) and the specific toxic effect on such organs

(histological studies). Sections from the heart, kidney, skin and liver of the animals administered with the LD50 (668 µg/mg) of the extract were studied histologically and compared to sections of the negative control animal. No abnormality was observed.

This suggests that at LD50 (668 µg/mg), the fraction B extract had no toxic effects on the skin, heart, kidney and liver of the test animals but since this dose could kill 50% of the experimental animals, it is not a safe dose for further investigation. The 100 µg/g dose did not kill or adversely affect any animal, while 250 µg/g killed just one rat and since MIC values of fraction B extract were between 50-100 µg/g, a safer dose for administration would be between 100 µg/g – 250

µg/g (Table 23).

ccxlviii

ccxlix

Plate 10: Photomicrograph of liver of untreated (control) rat at x 6.3 magnification Section shows no abnormal hepatocytes

ccl

Plate 11: Photomicrograph of liver of untreated (control) rat at x 25 magnification. Section demonstrating bile duct lined by normal hepatocytes

ccli

Plate 12: Photomicrograph of liver of rat treated with bulk B fraction at x 6.3 magnification Sections from the test liver show similar changes as control Liver (plates 9 and 10)

cclii

Plate 13: Photomicrograph of kidney of untreated (control) rat at x 25 magnification Section shows tubules, glomeruli and intervening vascular channels with no abnormality ccliii

Plate 14: Photo micrograph of kidney of rat treated with bulk B fraction at (x 25 magnification) Section from the kidney shows no significant inflammatory changes.

ccliv

Plate 15: Photomicrograph of skin of untreated (control) rat at x25 magnification Section shows scattered adipocytes. No inflammatory cells seen. cclv

Plate 16: Photomicrograph of skin of rat treated with bulk B fraction (x25 magnification) Section shows similar changes as control (plate 15)

cclvi

Plate 17: Photomicrograph of heart of untreated (control) rat at x 25 magnification Section shows essentially normal heart muscle comprising of myocytes.

cclvii

Plate 18: Photomicrograph of heart of rat treated with bulk B fraction at (x 25 magnification) Section shows similar changes as control (plate 19)

cclviii

CHAPTER FOUR

SUMMARY, CONCLUSION AND RECOMMENDATIONS

4.1 Summary and conclusion

Acetone, aqueous and ethanol extracts of the leaf of Spermacoce verticillata have been studied and found to possess antimicrobial activity.

Phytochemical analysis carried out on these extracts revealed the presence of balsam, saponins, terpenes and steroids in all the three leaf extracts. The acetone and ethanol extracts both had alkaloids, phenols and volatile oils. The acetone extract had glycosides in addition; all extracts however lacked flavonoids and resins.

All the extracts possessed antibacterial activity but lacked antifungal activity. The acetone extract was seen to have better pronounced antibacterial activity than the other extracts. The acetone extract was fractioned into two, namely, fractions A and B. Antibacterial studies of these fractions on the test organisms showed that the B fraction had better antibacterial activity.

Fraction B also had the least minimum inhibitory concentrations on the test organisms and was therefore chosen for formulation of antimicrobial ointments at various concentrations (0.1 %, 0.2

%, 1 %, 2 % and 5 % w/w). All the strengths of the ointments promoted wound healing on rats infected with test microorganisms with the best result achieved on Staphylococcus aureus, followed by Escherichia coli, Bacillus subtilis and lastly, Pseudomonas aeruginosa. cclix

Comparing the extent of healing of the infected wounds treated with the blank ointment and those treated with ointments of fraction B, suggests that the wound healing effect of fraction B was attributed to its antibacterial property especially when the extract ointment at 2 % concentration promoted wound healing better than 0.1% gentamycin ointment. Complete healing was achieved on the 10th day (on Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa infected wounds). However, all the concentrations achieved complete healing by the

14th day, except the blank ointment.

In the toxicity studies, the 100 µg/g dose did not kill or adversely affect any animal, while the

250 µg/g dose killed just one rat and since results of the MIC values of fraction B extract were between 50-100 µg/g, a safer dose for administration would be between 100 µg/g – 250 µg/g.

4.2 Recommendations

Following the findings of this study, the following recommendations are made:

f In making a choice of extracting solvent for Spermacoce verticillata among the three

solvents: acetone, ethanol and water, for antimicrobial and wound healing ointment

preparations, it is strongly recommended that acetone be used.

f Similarly, acetone leaf extract of Spermacoce verticillata as an antimicrobial agent is

recommended for use against the microorganisms: Staphylococcus aureus, Escherichia

coli, Bacillus subtilis and Pseudomonas aeruginosa. cclx

f Acetone leaf extract of fraction B ointment at a concentration of 2 % is recommended for

use in healing wounds infected by Staphylococcus aureus, Escherichia coli, Bacillus

subtilis and Pseudomonas aeruginosa. f It is recommended that a safer dose of administration of acetone leaf extract of

Spermacoce verticillata would be between 100 µg/g and 250 µg/g. f Further study is recommended for the use of acetone leaf extract of Spermacoce

verticillata on other microorganisms that are of pathogenic importance, especially those

implicated in wound and skin infections. f Further study could be carried out in order to elucidate the chemical structure of the

active constituents of the plant (Spermacoce verticillata) to aid commercial production of

the plants for availability.

cclxi

REFERENCES

1. Sneddon, I.B. and Church, R. E. (1983). Practical Dermatology. Edward Arnold (Publishers) Ltd. London. pp. 1 – 66, 79 – 100.

2. Canizares, O. (1982). A Manual of Dermatology for Developing countries. Oxford University Press. Oxford. pp. 1 - 21, 164 – 181.

3. Oakley, A. (2010). Bacterial infection. New Zealand Dermatological Society Incorporated. www.derment.org.nz/bacterial_skin_ infection/html. Accessed on 11th of February, 2011.

4. CDC (2008). Dermatophytes (Ringworm). National Center for Zoonotic, Vector – Borne and Enteric Diseases. Center for Disease Control and prevention. 16000 Clifton Rd, Atlanta. pp 6232. 5. New Zealand Dermatological Society Incorporated (NZDSI) (2010). Molluscum contagiosum. http://www.dermet.org.nz/viral/molluscum_contagiosum.html. Accessed on the 3rd of January, 2011.

6. Hugo, W.B. and Russell, A.D. (2004). Pharmaceutical Microbiology. Blackwell Scientific Publication. London. pp 187 – 201. 7. Laminkanra, A. (1999). Essential microbiology. Amkra Books, Lagos, Nigeria. pp 1 – 142.

8. Cheesbrough, M. (2000). District Laboratory Practice in Tropical Countries. Part 2. Cambridge University Press. pp. 45 – 62, 63 – 70, 132 – 143.

9. Agrawal, S. S. and Paridhari, M. (2009): Herbal Drug Technology. University Press, India. pp. 607–616. cclxii

10. Mbakwem-Aniebo, C. (2010). The Dermatophytes. Pearl Publishers, Port Harcourt. pp 1 – 27.

11. Gupte, S. (2010). The short text book of medical microbiology. Jaypee Brothers Medical Publishers (P Ltd), New Delhi, India. p 337.

12. Baker, F. J., Silverton, R. E., and Pallister, C. J. (2001). Baker & Silverton’s Introduction to Medical Laboratory Technology. 7th Ed. Nigerian Ed. Sam-Adex Printers, Ibadan. p 323.

13. Zanowiak, P and Jacob, M. R. (1982). Topical Anti-infective Products. In: Handbook of Nonprescription Drugs. 8th Edition. American Pharmaceutical Association. Washington DC. pp 557 – 582.

14. Dinulos, J.G.H. (2008). Parasitic Skin Infections: Introduction Skin Disorder. www.march.com. Accessed on the 10th of July 2012. 15. Parry, E.H.O. (1976). Principles of Medicine in Africa. Oxford University Press. Ibadan. p. 1294.

16. Pegum, J.S. and Baker, H. (1979). Dermatology. Baillere Tindall. London 3rd Ed. pp 925.

17. Lewis, W. H. and Elvin – Lewis, M. P.F. (1977). Medical Botany. John Wiley and Sons, Inc. New York. pp 1354.

18. Odimegwu, D.C. (2007). Antimicrobial Wound Healing Effect and Thermal Stability Studies of Crude Methanolic Extract of Dissotis theifolia (FAM: MELASTOMATACEAE) formulated as Cream and Ointment. Masters Degree Thesis Submitted to the Department of Pharmaceutics, University of Jos. 19. Okafor, O. J. (2008). Effect of mucinated-honey on healing and protease activity in full thickness experimental wounds in rats. A Ph.D Thesis submitted to the Department of Pharmaceutics, University of Nigeria, Nsukka.

20. Houghton, J. P., Hylands, J. P., Mensa, Y. A., Hensel, A. and Deters, M. A. (2005), In vitro tests and ethnopharmacological investigations: Wounds healing as an example. Curr Drug Deliv: pp 100 – 107.

21. Bowler, P., Duerden, B, Armstrong, D. (2001). Wound microbiology and associated approaches to wound management. Clin. Microbiol. Rev. 14(2): 244 – 69.

22. Li, B. (2007). Application of asperuloside for preparing drugs for rheumatoid arthritis. Faming Zhuanli Shenqing. CN 1915236.

cclxiii

23. Nwome, B. C., Yager, D. R., and Cohen, I. K., (1998). Physiology of the Chronic Wound. Plast. Surg. pp 25:341 – 354.

24. Dasu, M. R. K., Barrow, R. E., Spies, M. and Herndon, D. N. (2003). Matrix Metalloproteinase expression in cytokine stimulated human dermal.

25. Mulder M., Small, N., Botma, Y., Ziady, C. and Mackenzie, J. (2002). Basic Principles of Wound Care. 1 Ed Maskew Miller Longman, Pearson Education. South Africa.

26. Li, W. and Li, V. (2002). Therapeutic Angiogenesis for wound healing. J. Wounds, 15 (9 Suppl) 2S–12S.

27. Santoro, M. M. and Gaudino, G. (2005), Cellular and Molecular Facets of Keratinocyte re-epithelization during wound healing. Exper. Cell Res. pp 304: 274–286.

28. Thomas, J. C. (1997). Veterinary Pathology. 6th Ed. Williams and Wilkins, Maryland, U.S.A. pp 150–156.

29. De Sa Peixoto Neto, P. A., Vladimilsa Silva, M., Corrente Campos, N. V. and Porfirio, Z. (2002). Antibacterial activity of Borreria verticillata roots. Fitoterapia. 73 (6):319-22.

30. Zhu, J., Nathan, C., Jin, W., Sim, D., Ashcroft, G. S., Wahl, S. M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Wright, C. D. and Ding, A. (2002), Conversion of Proepitthelin to epithelins: roles of SLPI and elastase in host defence and wound repair, J. Cell 111: 867-878.

31. Nalway, N., Pokharnai, G., Deb L., Kumar, N. (2009). Wound healing activity of latex of Calotropis gigantean. Int. J. Pharm. and Pharm. Sc. 1:176-181.

32. Karodi, R., Jadhar, M., Rub, R., Bafna, A. (2009). Evaluation of the wound healing activity of a crude extract of Rubia cordifolia L. in mice. Int. J. Applied Resi in Natural Products. 2:12-18.

33. Ghasemi, A., Koohpayeh, A., Karimi, I. (2010). The wound healing activity of flower extracts of Punica granatum and Achillea kellalensis in wistar rats. Acta Poloniiae Pharmaceutica-Drug Res. 67:107-110.

34. Malviya, N., Jains, S. (2009). Wound healing activity of aqueous extracts of Radix paeoniae root. Acta Poloniae Pharmaceutica-Drug Res. 67:543-547.

35. Barua, C., Talukdar, A., Begum, S., Sarma, D., Pathak, D., Barua, A. (2009). Wound healing activity of methanolic extracts of leaves of Alternanthera brasiliana Kuntz using in vivo and in vitro model. Indian J. of Experimental Biology. 47:1001-1005. cclxiv

36. Paschapur, M., Patil, M., Kumar, R., Patil, S. (2009). Evaluation of aqueous extract of leaves of Ocimum kilimandscharicum on wound healing activity in albino wistar rats. Int. J. Pharm. Tech. Res. 1:544-550.

37. Veerapur, V. P., Palkar, M. B., Srinivasa, H., Kumar, M. S., Patra, S., Rao, P. G. M. and Srinivasan K. K. (2004). Effect of Ethanol extracts of Wrightia tinctoria bark on wound healing in rats. Journal of Natural Remedies. 4(2): 155-159.

38. Fengy, cheng G, Chen S, Yang H, Huang W. (2010). Evaluation of the burn healing properties of oil extracted from housefly larva in mice. Journal of Ethnopharmacology 130: 586-592.

39. Azeez, S., Amudhan, S., Adiga, S., Rao, N., Udupa, L. (2007). Wound healing profile of Areca catechu extracts on different wound models in wistar rats. Kuwait Med. J., 30:48- 52.

40. Sasidharan, S., Nilawatyi, R., Xavier, R., Yoga, L., Amala, R. (2010). Wound healing potential of Elaesi guneesis Jacq leaves in an infected albino rat model. Molecules. 15:3186-3199.

41. Okoli, C., Ezika, A., Udegbunam, S., Mbanu, T., Ugwu, E. (2009). Studies on wound healing and antiulcer activities of extract of aerial parts of Phyllanthus niruri L. (Euphorbiaceae). Am. J. Pgarm. and Toxicol. 4:118-126.

42. Anna, K. and Dickson, R. (2008). Evaluation of wound healing actions of Hoslundia opposita vahi, Anthocleista nobilis g. Don. and Balanites aegyptiaca. J. Sc. Tech. 28:26- 38. 43. Panda, P., Tripathy, G. (2009). Wound healing activity of aqueous and methanolic bark extract of Vernonia arborea in wistar rats. Nat. Product Radiance. 8:6-11.

44. Esimone, C.O., Grunwold, T., Wildner, O., Nchinda, G., Tippler, B., Proksch, P. and Uberla, K. (2005). In vitro pharmacodynamic evaluation of anti-viral medicinal plants using a vector based assay technique. Journal of Applied Microbiology 99(6): 134-1355.

45. Nayak, S. (2006). Influence of ethanol extract on Uncial rosea on wound healing in diabetic rats. Online J. Bio. Sc. 6:51-55.

46. Sofowora, E. A. (1993), Medicinal Plants and Traditional Medicine in Africa. Spectrum. Ibadan. Nigeria. pp. 150-153.

47. WHO (2005). National policies on traditional medicine and regulation of herbal medicines. Report of a WHO global survey. World Health Organisation, Geneva.

cclxv

48. WHO (1978). The promotion and development of traditional medicine. Report of a WHO meeting, WHO Technical Report Series No. 622. World Health Organisation, Geneva.

49. Iwu, M. M. (1999). Handbook of African Medicinal Plants. CRC Press, Boca Raton.

50. Menakaya, U. C. (1979). A Phytochemical Study of the leaves of Vermonia amygdalina Var. amygdalina. B. Pharm. Thesis, University of Nigeria, Nsukka.

51. Burkill, H.M. (2000). The Useful Plants of West Tropical Africa. Royal Botanic Gardens. Kew, UK. pp 686.

52. Roy, K., Shivakumar, H., Sarkar, S., (2009). Wound healing potential of leaf extracts of Ficus religiosa on wistar albino rats. Int. J. of Pharm. Tech. Res. 1:506-508.

53. Odimegwu, O., Ibezim, E., Esimone, C., Nworu C., Okoye. (2008). Wound healing and antibacterial activities of Dissotis theifolia stem formulated in a simple ointment base. J. Med. Plants Res. 2:11-16.

54. Sachin, J., Neetesh, J., Balekar, T., Jain, O. (2009). Simple evaluation of wound healing activity of Polyherbal formulation or roots of Ageratum conyzoides L. Asian J. Res. Chem. 2:135-138.

55. Shenoy, M. (2007). Animal biotechnology. (http://books.google.com.et/books?id=3d7unzyaDREC&pg=PA24&dg=trypsinization&h l=en&ei=jJZOTNFHYRjAecjsZUBw&yed=X&oi=bookresult&ct=result&resnum=3&ye d=OCDAQ6AEwAg£y=onepage&q=trysinization&f=false). Accessed: 27/07/2010.

56. Mackay, D. and Miller, J. (2003). Nutritional support for wound healing. Alternative Med. Rev. 8:359-377. 57. Raina, R., Prawez, S., Verma, P., Pankaj, N. (2008). Medicinal plants and their role in wound healing. Vetscan 3:1-24.

58. Barua, C., Talukdar, A.: Begum, S., Surma, D., Pathak, D., Barua, A., Bora, R. (2009). Wound healing activity of methanolic extract of leaves of Alternanthera brasiliana Kuntz using in vivo and in vitro model. Indian J. Exp. Bio. 47:1001-1005.

59. Deshmukh, P., Fernandes, J., Atul, A., Toppo, E. (2009). Wound healing activity of Calotropis gigantean root bark in rats. J. Ethopharmacology. 125:178-181.

60. Anuar, N., Zahari, S., Taib, I., Rahman, M. (2008). Effect of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food and Chemical Toxicology. 46:2384-2389.

cclxvi

61. Gurung, S. and Skalko-Basnet, N. (2009). Wound healing properties of Carica papaya latex in vivo evaluation in mice burn model. J. Ethnopharm. 121:338-341.

62. Kuppast, I. and Nayak, P. (2005). Wound healing activity of Cordia dichotoma Forst F. fruits. Nat. Product Radiance. 5:99-102.

63. James, O. and Friday, E. (2010). Phytochemical composition, bioactivity and wound healing potential of Euphorbia heterophylla (Euphoribiaceare) leaf extrac. Int. J. Pharm. & Bio. Res., 1:54-63.

64. Shetty, S., Udupa, S., Udapa, L. (2006). Wound healing activity of bark extract of Jatrophs curcas L. in albino rats. Saudi Med. 27:1473-1576.

65. Sakarkar, D., Sakarkar, U., Shrukhanse, V., Viyas, J., Mandsvhade, S., Jaiswal, S., Purohit, R. (2004). Wound healing properties of Henna leaves. Nat. Products Radiance. 3:406-412.

66. Kokane, D., More, R., Kale, M., Nehete, M., Mehendale, P., Gadgoli, C. (2009). Evaluation of wound healing activities of root Mimosa pudica. J. Ethnopharmacology, 124:311-315.

67. Udupa, S., Shetty, S., Udapa, A., Somayaji, J. (2005). Effect of Ocimum sanctum L. on normal and dexamethanone suppressed wound healing. Indian J. Exp. Biol. 44:49-54.

68. Shetty, S., Udupa, S., Udapa, L. (2008). Evaluation of antioxidant and wound healing effects of alcohol and aqueous extracts of Ocimum sanctum L. I rats. Am. Exp. Biol. 5:95-101.

69. Umachigia, S., Jayaveerab, N., Kumars, A., Kumard, S., Vrushabendra, S., Kumarf, K. (2008). Studies on wound healing properties of Quercus infectoris. Tropical J. Pharm. Res., 7:913-919.

70. Shivhare, Y., Singour, P., Patil, U., Pawa, R. (2010). Wound healing potential of methanolic extract of Trichosathes dioic Roxb (Fruits) in rats. J. Ethnopharmacology, 127:614-619.

71. Havsteen, B. (2002). The biochemistry and medical significance of the flavonoids. Pharmacology and Therapentics. 96:67-202.

72. Kuster, R., Arnold, N., Wessjohann, L. (2009). Antifungal flavonoids from Tibouchina grandifolia. Biochemical Systematics and Ecology, 37:63-65. 73. Institute of Chemistry Fu Berlin (2003). Terpenes. Department of Biology, Chemistry and Pharmacy, FU Berlin. www.chemie.fu-berlin.edu/chemistry/oc/terpenes_en. Accessed on the 14th of March, 2011. cclxvii

74. Herbs 2000.com (2010). Saponins. www.herbs2000com/h-menu/saponins.htm. Accessed on the 16th of February, 2011.

75. Davidson, M. and Florida State University (2009), Molecular Expressions: Phytochemical Galary. 76. Wagner, H., Bladt, S. and Zgainski, E.M. (1984). Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer – Verlag. Berlin.pp356. 77. Barbakadze, V., Mulkijanyan, K., Gogilashvili, L., Amiranash, L., Merlani, M., Novikova, Z., Sulakvelidze, M. (2009). Allantoin and purrolizidine Alkaloids –free wound healing compositions from Sumphytum asperum. Bulletin of the Georgian Academy of Sciences. 3:159-164. 78. Foye, Williams O. (1990). Medicinals of Plant Origin: Historical Aspect In: Principles of Medicinal Chemistry. 3rd ed., Lea and Fibiger. Pennsylvania. Pp1124. 79. Okore, V. C. (2005). Pharmaceutical microbiology, principles of the pharmaceutical applications of antimicrobial agents. El ‘Demark Ltd, Enugu. P 51, 55 – 120. 80. Lamikanra, A. (1999). Essential microbiology. Amkra Books, Lagos. Pp 1 – 92. 81. Carter, S.J. (ed) (2005). Tutorial pharmacy by Cooper and Gunn. CBS Publishers and Distributors, 6th Ed. New Delhi, India. pp 299 82. Trease, G. and Evans, W .C. (1989). Pharmacognosy. Univ. of Nottingham. Britain. 83. Sofowora, A. (1982). Medicinal plants and Traditional medicine in Africa. John Wiley and Sons. New York. Pp1067. 84. Sofowora, A. (2008). Medicinal Plants and Traditional Medicine in Africa. 3rd ed. Spectrum Books, Ibadan. pp. 153-161, 306-310.

85. Prescott, J. L. M., Harley, J. P. and Klenin, D.A. (1999). Microbiology. 4th ed. McGraw Hill Companies, USA. pp960.

86. Remington, S. (1990). Pharmaceutical Science. 16th ed. Mack Publishing Company, Eastern Pennsylvania. pp 842.

87. Gibile, Z. O. (1998). Pharmaceutical potentials of Nigeria flora. 2nd Annual Conference of Botanical Society of Nigeria. Ahmadu Bello University Press. pp 7 88. Aulton, M. E. (1999). Pharmaceutics: The Science of Dosage Form Design. 1st Ed. Churchill Livingstone, Edinburgh. 89. Coben, L. J., Liberman, H. A. and Kanig, J. L. (eds) (1987), The Theory and Practice of Industrial Pharmacy, 3rd Ed. Varghese Publishing House, Bombay. pp 564-588. cclxviii

90. Howard, C. A. (1981), Introduction to Pharmaceutical Dosage Forms, 3rd Ed. Lea and Febiger Philadelphia. 91. Rawlins, E. A. (1977), Bently’s Textbook of Pharmaceutics. 8th Ed. Bailliere Tindal, London. pp. 32-84. 92. Carter, S.J. (ed.) (2008). Dispensing for Pharmaceutical Students by Cooper and Gunn. CBS Publishers and Distributors, 12th Edn New Delhi, India. pp 192 - 199 93. Senior, N. (1971). Ointment composition for potent medicaments. Pharm. J., 206: 99 – 100, 120. 94. Blank, I. H. (1952). Factors which influence the water content of the stratum corneum. J. Invest. Derm. 18:433 – 440. 95. Nwude, N. and Ibrahim, M. A. (1980). Plants used in traditional veterinary medicine practice in Nigeria. Journal of Veterinary Pharmacology and therapeutics 3:261 – 273. 96. Cornell, D. S. and Johnston, M.C. (1970). Manual of the Vascular Plants Of Texas. Texas Research Foundation Renner. TX. Pp1, 881. CDC (2008): Dermatophytes (Ringworm). National Center for Zoonotic, Vector – Borne and Enteric Diseases. Center for Disease Control and prevention. 16000 Clifton Rd, Atlanta. Pp6232. 97. Liogier, H. A. (1997). Descriptive Flora of Puerto Rico and adjacent Islands: Vol. 6. Editorial de’la Universidad de Puerto Rico, Rio Piedras, Porto Rica. pp 436. 98. Natural Resources Conservation Service (2002). Plant Profile: Spermacoce Verticillata L. http://plants.usda.gov/cgi_bin/plant_profile.cgi?simbel=spve2.2p. 99. Got-Fredsen, E. (2010). The incomplete reference guide to Herbal medicine: Borreria verticillata (L) G.F.W Meyer. Liber Herbarium II. Page wise. 100. Maynart, G., Pouset, J. L., Mboups, D. F., and Dennis, F. (1980). Antibacterial Effect of Borreverine, and Alkaloid isolated from Borreria verticillata. C. R Séances Soc. Biol. 33: 713-718. 101. Benjamin, T. V. (1979). Investigation of Borreria Verticillata, an antieczematic plant of Nigeria. Quart J Crude Drug Res. 17:135-6. 102. Benjamin, T. V. (1980), Analysis of the volatile constituents local plants used for skin disease. J Afr Med Pl. 3:135-9. 103. Hellier, F. F. (1969). Creams, ointments and lotions. Practitioner, 202:23 – 26. 104. Sainty D., Bailleul, F., Delaveau, P, Jacquemin, H. (1981). Iridoid of Borreria verticillata. Planta medical. 42(7): 260-264. 105. Swarbrick, J. T. (1997). Weed of the Pacific Island. Technical paper No. 209. South Pacific Commission, Noumea, New Coledonia. pp124. cclxix

106. Kerhazo, J. and Adam, J.G. (1974). La pharmacopee Senegalese Traditional. Vigot freres, Paris. 107. Bremer, B. and Manen, J. F. (2009). Phylogeny and Classification of the subfamily Rubioideae (Rubiaceae) Plant Syst Evol, 225: 43 – 72. 108. Margalho, L. F., da Rocha A. E. Sousa, Secco, R. S. (2009). Rubiaceae Juss. Da resting da APA de algodoal/Maiandeua, Maracana, Para, Brazil. Bol Mus Para Emilio Goeldi. Cienc Nat. 4:303–39 109. Souza, V. C. and Lorenzi, H. (2005). Botanica Systematica: Guia Ilustrado para identifacacao das familias de angiospermas da flora brasiliera, baseado em APG II. Soa Paolo: Nova Odessa. 110. Chaw, S. M. and Srivarajan, V. V. (1989). Seed coat Micromorphology of some Asiatic spermacoce (Rubiaceae) Bot Bull Acad Sin. 30:15–24. 111. Karehe, J., Groeninckx, I., Dessein, S., Motly, T. J. and Bremer, B. (2008). The Phylogenetic Utility of Chloroplast and Nuclear DNA markers and the Phylogeny of the Rubiaceae tribe Spermaceae. Mol Phylogenet Evol. 49:843–66 112. Dessein, S. Huysmans, S., Robrecht, E. and Smet, E. (2002). Pollen of African Spermacoce species (Rubiaceae). Morphology and Evolutionary aspects, Grana. 41:69-89 http://www.micro- magnet.fsu.edu/phytochemicals/pages/saponins.htm 113. Machado, I. C. and Loiola, M. I. (2000). Fly Pollination and Pollinator sharing in two synchronopatric species: Cordia Multispicata (Boraginaceae) and Borreria alata (Rubiaceae) Rev Bras Bot. 23:305-11 114. Funk, V., Hollowell, T., Berry, P., Kellof, C. and Alexander, S. N. (2007). Checklist of the plants of Guiana Shield (Venezuela: Amazonas, Bolivar, Delta Amacuro; Guyana, Surinam, French Guiana) 55:1-585 Available from http://botany.si.edu/bdg/pdf/vol55web.pdf 115. Vadivelan, S., Sinha, B. N., Betanabhatla, K. S., Christina, A. J. and Pillai, R. N. (2007). Anti-inflammatory activity of Spermacoce articularis Linn on carrageenan induced paw edema in Wistar male rats. Pharmacology Online. 3:478–84 116. Sultana, R., Rahman, M. S., Bhuiyan, M. N., Begum, J., and Anwar, M. N. (2008). In vitro antibacterial and antifungal activity of Borreria articularis. Bangladesh J Microbiol. 25:95-8 117. Jain, S. P. and Verma, D. M. (1981). Medicinal Plants in the folklore of Northeast Haryana. Nat’l Acad. Sci. Lett (India) 4:269-71 118. Lucas, V. and Machado, O. (1944). Medical Rubiaceae of Brazil. Rev Flora Med. 11:3-36. cclxx

119. Brandao, M. G., Cosenza, G. P., Grael, C. F. Netto, N. L., Junior, and Monte- Mor R. L. (2009). Traditional Uses of American Plant species from the 1st edition of Brazilian Official Pharmacopoeia. Rev Bras Farmacogn. 19:478-87 120. Steefeld, C. (1968). What the Herbalist of Pelotas (Brazil) Sell Pharm Biol. 8:1300-3 121. Bandoni, A. L., Mendiondo, M. E., Rondina, R. V. and Coussio, J. D. (1976). Survey of Argentina medicinal plants. Folklore and Phytochemical Screening. II. Econ Bot. 30:161-85 122. Vasanthi, H. R., Mukherjee, S., Lekli, I., Ray, D. and Veeraraghavan, G. and Das, D. K., (2009). Potential Role of Borreria Hispida in ameliorating cardiovascular risk factors. J Cardiovasc Pharmacol. 53:499-06 123. Khan, S. S., Changhtai, S. A. and Oommachan M. (1984). Medicinal Plants of Rubiaceae of Bhopal – An Ethnobotanical Study. J Sci. Res (Bhopal). 6:37– 9. 124. Prurshothaman, K. K. and Kalyani, K. (1979). Isholation of isorhamnetin from Borreria Hispida Linn. J. Res. Indian Meg Yoga Homeop. 14:131-2 125. Kaviarasan, K., Kalaiarasi, P. and Pugalendi, V. (2008). Anti-oxidant efficacy of flavonoid-rich fraction from Spermacoce hispida in hyperlipidermic rats. J Appl. Biomed. 165-76 126. Shah, G. L. and Gopal, V. (1985). Ethnomedical notes from the tribal inhabitants of the North Gujarat (India) J Econ Taxon Bot. 6:193-201. 127. Rahman, M. A., Uddin, S. B. and Wilcock, C. C. (2007). Medicinal Plants used by Chakma tribe in Hill distance of Bangladesh. Indian J Tradit Knowl. 6:508-17. 128. Chiquieri, A., Di Maio, F. R. and Peixoto, A. L. (2004). A Distribuicao geografica da familia Rubiaceae Juss. na Flora Brasiliensis de Martius, Rodriguésia. 55:47-57.

129. Burger, W. and Taylor, C. M. (1993). Flora Costaricensis. Fieldiana. 33:1-333. 130. Vieira, I. J., Mathias, L., Braz-Filho, R. and Schripsema, J. (1999), Iridoids from Borreria vericillata. Org Lett. 1:1169-71. 131. Moreira, V. F., Oliveira, R. R., Mathias L., Braz-Filho, R. and Vieira, I. J. (2010). New Chemical Constituents from Borreria Verticillata (Rubiaceae) Helv Chim Acta. 93:1751-7 132. Lorenzi, H. and Matos, F. J. (2002). Plantas medicinais do Brasil. Sao Paulo: Nova Odessa. cclxxi

133. Ayensu, E. S. (1978). Medicinal Plants of the West Indies. Unpublished Manuscript. 110 134. Asprey, G. F. and Thornton, P. (1955). Medicinal Plants of Jamaica. IV. West Indian Med J. 4:145-65. 135. Willaman, J. J. and Schubert, B. G. (1961). Alkaloid-bearing plants and their contained alkaloids. US Department of Agriculture, Technical Bulletin No. 1:234 – 287. 136. Moreira, E. A. (1964). Identification of Emetine in some Borreria Species (paper chromatography) Trib Farmac (Brazil). 32:9-30 137. Ferreira, M. A., Branco, C. S. and Sliwoski, J. K. (1978). Chemical Study of Alkaloids in Borreria verticillata (L.) G. F. W. Meyer Lloydia. 41:655 138. Grollman, A. P. (1966). Structural Basis for inhibition of protein synthesis by emetine an cycloheximide based on an analogy between ipecac alkaloids and glutarminide antibiotics. Proc Natl Acad Sci USA. 56:1867-74. 139. Mastrangelo, M. J., Grage, T. B., Bellet, R. E. and Weiss, A. J. (1973). A phase I study of emetine hydrochloride (NSC 33669) in solid tumors. Cancer. 31:1170-5 140. Siddiqui, S., Firat, D. and Olshin, S. (1973). Phase II study of emetine (NSC- 33669) in treatment of solid tumors. Cancer Chemother Rep. 57: 423—8. 141. Moertel, C. G., Schutt, A. J., Hahn, R. G. and Reitemeier, R. J. (1974). Treatment of advanced gastrointestinal cancer with emetine (NCS 33669) Cancer Chemother Rep. 58:229-32. 142. Muhammad, I., Dunbar, D. C., Khan, S. I., Tekwani, B. L., Bedir, E., Takamatsu, S., et al., (2003). Antiparasitic alkaloids from Psychotria Klugii. J Nat Prod. 66:962-7 143. Cavin, J. C., Krassner, S. M. and Rodriguez, E. (1987). Plant-derived alkaloids active against Trypanosoma cruzi. J Ethnopharmacol. 19:89-4 144. Mackey, Z. B., Baca, A. M., Mallari, J. P. Apsel, B.,Shelat, A., Hansell, E. J., et al. (2006). Discovery of Trypanocidal compounds by whole cell HTS of Trypanosoma brucei. Chem Biol Drug Des. 67:355-63. 145. Low, Y. J., Chen, K. C., Wu, K. X., Mah-Lee, N. M. and Chu, H. J. (2009). Antiviral activity of emetine dihydrochloride against dengue virus infection. J. Antivir Antiretrovir. 1:62-71. 146. Akinboye, E. S. and Bakare, O. (2011). Biological Activities of Emetine. Open Nat. Prod. J. 4:8-15. cclxxii

147. Moyer, D. L., Thompson, R. S. and Berger, I. (1977). Anti-implantation action of a medicated intrauterine delivery system (MIDS) Contraception. 16:39-49. 148. Kamiya, K., Fujita, Y., Saiki, Y., Hanani, E., Mansur, U. and Satake, T. (2002). Studies on the constituents of Indonesian Borreria latifolia. Heterocycles. 56:537- 44 149. Fujikawa, T. and Kawamura, N. (2009). Asperuloside and its analogs from Eucommia ulmoides extracts as muscle anabolic steroids. Jpn. Kokai Tokyo Koho. JP 2009209088. 150. Kapadia, G. J., Sharma, S. C., Tokuda, H., Nishino, H. and Ueda, S. (1996). Inhibitory effect of Iridoids on Epstein-Barr virus activation by a short-term in vitro assay for anti-tumor promoters. Cancer Lett. 102: 223-6. 151. Kim, D. H., Lee, H. J., Oh, Y. J., Kim, M. J., Kim, S. H., Jeong, T. S., et al. (2005). Iridoid glycoside Isolated from Oldenlandia diffusa inhibits LDL- oxidation. Arch. Pharm. Res. 28:1156-60. 152. Miura, T., Nishiyama, Y., Ichimaru, M., Moriyasu, M. and Kato A. (1996). Hypoglycemic activity and structure activity relationship of iridoidal glycosides. Biol. Pharm. Bull. 19:160-1 153. Akihisa, T., Matsumoto, K., Tokuda, H., Yasukawa, K., Seino, K., Nakamoto, K. et al. (2007). Anti-inflamatory and potential cancer chemopreventive constituents of the fruits of Morinda citrifolia (Noni). J Nat Prod. 70:754-7. 154. Ekpendu, T. O., Ekundayo, O. and Laakso, I. (2001). Constituents and antimicrobial activity of the volatile oil of Spermacoce verticillata Linn – a Nigerian medicinal rubiaceous weed. J. Chem. Soc Nigeria. 26; 194 – 8. 155. Ogunwande, I. A. Walker T. M., Bansal, A., Setzer, W. N. and Essien, E. E. (2010). Essential Oil Constituents and Biological Activities of Peristrophe Bicalyculata and Borreria verticillata. Nat. Prod. Comm. 5: 1815–8. 156. Wahua, T.A.T. (1999): Applied statistics for scientific studies. Africa Links Books, Nigeria. 157. http://en.wikipedia.org/wiki/Bacillus. Accessed 13/03/2013. 158. Ogungbamila, F. O. (Coord.) (1997). The antimicrobial plant research group manual. Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. 159. Aguiyi, J. C., Johnson, P.B., Obi, C. I., Onwukeme, K. and Dafur, S. J. (1996). Studies on the rodenticidal activities of indomethacin. West Afr. J. Pharmacol. Drug Res. 12:37 - 40 160. Eroschenko, P.V. (2005). Atlas of histology with functional correlations. Indian Edition. Lippincott Williams and Wilkins. Pp 153 -165, 273 – 279, 309 – 327. cclxxiii

161. Ham, A. W. (1974). Histology. 7th Ed. J. B. Lippincott Company. Philadelphia and Toronto. Pp 3 – 22. 162. Payne, R, W., Murray, D. A., Harding, S.A., Baird, D.B. and Soutar, D.M. (2007): GenStat for windows (12th Edition). VSN International, Hemel Hempstead.

163. SPSS (2009): SPSS Statistics for windows. Version 17.0. SPSS Inc., Chicago

164. Onwuliri, F. C. and Mabitine, N. (2005). Antimicrobial activities of the leaf extract of Acacia ataxacantia dc. African J. Nat. Sc. p10. 165. Taylor, C. M., Steyermark, J. A., Delprete, P. G., Vicentini, A., Cortés, R., Zappi D., et al., (2004), Rubiaceae. In Steyermark, J. A., Steyermark, J. S., Berry, P. E., Holst B. K. editors. Flora of the Venezuelan Guayana. St. Louis: USA: Missouri Botanical Garden Press; pp. 497 – 848.

166. William, R (2000). Antimicrobial Resistance; a global threat. WHO Essential Drugs monitor (28 and 29). Pp7. 167. Klein, R.J.; Proctor, S.E.; Boudreault, M.A.; Turczyn, K.M. (2002): Criteria for data suppression. Statistical Notes. In: Health People 2010. National Center for Health Statistics, No. 24, Hyattsville, Maryland.

168. Attama, A. A. et al. (2012). Nanomedicine. J. Nanotech. Progress Int. 3(2):1- 41

cclxxiv

APPENDICES

Appendix 1: Preparation of media

Sabouraud Dextrose Agar (SDA)

Formulation g/1

Balanced petone No. 1 10.0

Dextrose 40.0

Agar No. 2 12.2

Method of preparation

A 6.2 g quantity of Sabouraud’s dextrose agar was accurately weighed using an analytical balance then transferred into a 250 ml conical flask and made up to 100 ml with distilled water. The solution was heated to boil on a hot plate and dispensed into dispensing bottles, 20 ml each and sterilized in an autoclave at 121oC for 1 hour 30 minutes.

Nutrient agar

Formulation g/1

Peptone 5.0 cclxxv

Beef extract 3.0

NaCl 8.0

Agar No.2 12.0

Method of Preparation

A 2.8 g quantity of the nutrient agar was accurately weighed and transferred into a conical flask and made up to 100 ml with distilled water. This was heated until a clear solution was obtained. It was then distributed into 20 ml dispensing bottles and sterilized in an autoclave at 121 oC for 1 hour 30 minutes.

Nutrient Broth

Formulation g/1

Beef extract 10.0

Balance peptone 10.0

NaCl 5.0

Method of Preparation

A 0.5 g quantity of nutrient broth was successfully weighed using analytical balance and then transferred to a conical flask. This was made up to 200 ml mark with distilled water, stirred with a glass rod to dissolve and brought to boiling using a hot plate. It was then dispensed into 5 ml dispensing bottles and sterilized in an autoclave for 1 hour 30 minutes at 121 oC.

cclxxvi

Appendix 2: Zones of inhibition of various concentrations of the ethanol leaf extract of Spermacoce verticillata on test microorganisms

Test Microorganism Concentration of Extract (µg/ml)/Zones of Inhibition (mm) µg/ml 400 200 100 Gentamycin (40) Bacillus subtilis 13.50 11.00 10.00 18.00

Staphylococcus aureus 20.00 18.00 17.00 23.10

Escherichia coli 14.00 11.50 11.50 18.50

Pseudomonas aeruginosa 12.50 12.00 0.00 15.00

.

Appendix 3: Zones of inhibition of various concentrations of the acetone leaf extract of Spermacoce verticillata on test microorganisms. Test Microorganism Concentration of Extract (µg/ml)/Zones of Inhibition (mm) µg/ml 400 200 100 Gentamycin (40) Bacillus subtilis 11.50 0.00 0. 00 16.00

Staphylococcus aureus 24.50 21.00 17.00 21.50

Escherichia coli 28.50 21.50 20.00 18.00

Pseudomonas aeruginosa 23.50 21.50 11.50 18.00

Appendix 4: Zones of inhibition of various concentration of the aqueous leaf extract of Spermacoce verticillata on test micro organisms

cclxxvii

Test Microorganism Concentration of Extract (µg/ml)/Zones of Inhibition (mm) µg/ml 400 200 100 Gentamycin (40) Bacillus subtilis 13.50 12.00 0. 00 20.00

Staphylococcus aureus 17.00 14.00 0. 00 22.10

Escherichia coli 17.00 13.50 0. 00 19.50

P. aeruginosa 0.00 12.50 0. 00 18.00

Appendix 5: Antibacterial activity of fraction A

Test Micro-organism Concentration of Extract (µg/ml)/Zones of Inhibition (mm) µg/ml 50 100 200 400 Gentamycin (40) Staphylococcus aureus - - 12 17 21

Bacillus subtilis - 8 12 18 22

Pseudomonas aeruginosa - 10 16 18 25

Escherichia coli - 8 10 16 25 - = No Growth

Appendix 6: Antibacterial activity of Fraction B

Test Microorganism Concentration of Extract (µg/ml)/Zones of Inhibition (mm) µg/ml 50 100 200 400 Gentamycin (40) Staphylococcus aureus 10 15 18 20 21

Bacillus subtilis - 10 15 18 21

Pseudomonas aeruginosa - 14 17 22 25

Escherichia coli - 13 1 19 22 cclxxviii

- = No Growth

Appendix 7: Formula for preparation of Bulk—B 0intment Ingredients Amount prescribed Amount Required (1/5 of prescribed)

w Formula for preparation of 0.1 % /w of Bulk- B ointment Fraction B 100 mg (0.1 g) 20 mg (0.2 g) cclxxix

Ointment Base 99.900 mg (99.9 g) 19.98 g 20 g

w Formula for preparation of 0.2 % /w of Bulk- B ointment

Fraction B 200 mg (0.2 g) 40 mg (0.04 g) Ointment Base 99.8 g 19.96 g 20 g

Formula for preparation of 1 %w/w of Bulk- B ointment

Fraction B 1000 mg (1 g) 200 mg (0.2 g) Ointment Base 99 g 19.8 g 20 g

w Formula for preparation of 2 % /w of Bulk B ointment

Fraction B 2000 mg (2 g) 0.4 g Ointment Base 98 g 19.6 g 20 g

w Formula for preparation of 5 % /w of Bulk B ointment

Fraction B 5 g 1.25 g Ointment Base 95 g 18.75 g 20 g

cclxxx

Appendix 8: ANOVA result for inhibition zone diameter Analysis of variance Variate: Zone_of_Inhibition

Source of variation d.f. s.s. m.s. v.r. F pr. Extract_liquid 2 629.920 314.960 55.77 <.001 Extract_conc 3 2164.005 721.335 127.72 <.001 Microorganisms 3 1447.602 482.534 85.44 <.001 Extract_liquid.Extract_conc 6 519.594 86.599 15.33 <.001 Extract_liquid.Microorganisms 6 1192.247 198.708 35.18 <.001 Extract_conc.Microorganisms 9 387.488 43.054 7.62 <.001 Extract_liquid.Extract_conc.Microorganisms 18 447.517 24.862 4.40 <.001 Residual 96 542.167 5.648 Total 143 7330.540

Tables of means

Variate: Zone_of_Inhibition

Grand mean 14.46

Extract_liquid Acetone AqueousSolution Ethanol 17.19 12.10 14.09

Extract_conc 40 100 200 400 18.75 8.38 14.12 16.60

Microorganisms B E P S 10.60 16.28 12.33 18.64

Extract_liquid Extract_conc 40 100 200 400 Acetone 18.33 12.29 16.17 21.96 AqueousSolution 19.29 3.21 13.08 12.83 Ethanol 18.62 9.62 13.12 15.00

Extract_liquid Microorganisms B E P S Acetone 6.87 22.00 18.71 21.17 AqueousSolution 11.79 12.96 8.42 15.25 Ethanol 13.12 13.88 9.88 19.50 cclxxxi

Extract_conc Microorganisms B E P S 40 17.67 18.56 16.56 22.22 100 4.44 11.67 3.94 13.44 200 7.44 15.39 15.44 18.22 400 12.83 19.50 13.39 20.67

Extract_liquid Extract_conc Microorganisms B E P S Acetone 40 16.00 18.00 18.00 21.33 100 0.00 19.67 11.83 17.67 200 0.00 21.83 21.50 21.33 400 11.50 28.50 23.50 24.33 AqueousSolution 40 19.00 19.17 16.67 22.33 100 3.33 3.83 0.00 5.67 200 11.33 12.83 12.83 15.33 400 13.50 16.00 4.17 17.67 Ethanol 40 18.00 18.50 15.00 23.00 100 10.00 11.50 0.00 17.00 200 11.00 11.50 12.00 18.00 400 13.50 14.00 12.50 20.00

Standard errors of differences of means

Table Extract_liquid Extract_concMicroorganisms Extract_liquid Extract_conc rep. 48 36 36 12 d.f. 96 96 96 96 s.e.d. 0.485 0.560 0.560 0.970

Table Extract_liquid Extract_conc Extract_liquid Microorganisms Microorganisms Extract_conc Microorganisms rep. 12 9 3 d.f. 96 96 96 s.e.d. 0.970 1.120 1.940

Least significant differences of means (5% level) cclxxxii

Table Extract_liquid Extract_concMicroorganisms Extract_liquid Extract_conc rep. 48 36 36 12 d.f. 96 96 96 96 l.s.d. 0.963 1.112 1.112 1.926

Table Extract_liquid Extract_conc Extract_liquid Microorganisms Microorganisms Extract_conc Microorganisms rep. 12 9 3 d.f. 96 96 96 l.s.d. 1.926 2.224 3.852

Estimated stratum variances

Variate: Zone_of_Inhibition

variance effective d.f. variance component 5.648 96.000 5.648

Stratum standard errors and coefficients of variation

Variate: Zone_of_Inhibition d.f. s.e. r.s.e. 96 2.376 16.4 Appendix 9: ANOVA result for wound diameter

Analysis of variance

Variate: Wound_Diameter

Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Days 4 34218.5095 8554.6274 10000.31 <.001 Strength_of_Ointment 9 1352.4571 150.2730 175.67 <.001 Microoganism 3 101.3000 33.7667 39.47 <.001 Days.Strength_of_Ointment 36 1039.2571 28.8683 33.75 <.001 cclxxxiii

Days.Microoganism 12 656.9500 54.7458 64.00 <.001 Strength_of_Ointment.Microoganism 15 2197.3000 146.4867 171.24 <.001 Days.Strength_of_Ointment.Microoganism 60 1857.9500 30.9658 36.20 <.001 Residual 279 (1) 238.6667 0.8554 Total 418 (1) 41552.6778

Tables of means

Variate: Wound_Diameter

Grand mean 10.462

Days Four Fourteen one Seven Ten 16.357 0.821 25.000 7.310 2.821

Strength_of_Ointment 0.001 0.002 0.01 10.300 9.400 9.400 rep. 60 60 60

Strength_of_Ointment 0.02 0.05 0.1%Genta6 9.000 10.600 9.600 rep. 60 60 15

Strength_of_Ointment 0.1%Genta7 0.1%Genta8 0.1%Genta9 11.800 7.800 11.333 rep. 15 15 15

Strength_of_Ointment Blank Ointment 14.400 rep. 60

Microoganism B.subtilis E.coli P.aeruginosa S.aureus 10.945 10.212 10.979 9.712

Days Strength_of_Ointment 0.001 0.002 0.01 Four 15.500 15.250 15.000 rep. 12 12 12 Fourteen 0.000 0.000 0.000 rep. 12 12 12 one 25.000 25.000 25.000 rep. 12 12 12 Seven 7.500 5.250 4.750 cclxxxiv

rep. 12 12 12 Ten 3.500 1.500 2.250 rep. 12 12 12

Days Strength_of_Ointment 0.02 0.05 0.1%Genta6 Four 12.750 15.500 16.000 rep. 12 12 3 Fourteen 0.000 0.000 0.000 rep. 12 12 3 one 25.000 25.000 25.000 rep. 12 12 3 Seven 4.500 10.750 3.000 rep. 12 12 3 Ten 2.750 1.750 4.000 rep. 12 12 3

Days Strength_of_Ointment 0.1%Genta7 0.1%Genta8 0.1%Genta9 Four 21.000 11.000 18.000 rep. 3 3 3 Fourteen 0.000 0.000 0.000 rep. 3 3 3 one 25.000 25.000 25.000 rep. 3 3 3 Seven 11.000 2.000 11.667 rep. 3 3 3 Ten 2.000 1.000 2.000 rep. 3 3 3

Days Strength_of_Ointment Blank Ointment Four 24.000 rep. 12 Fourteen 5.750 rep. 12 one 25.000 rep. 12 Seven 11.500 rep. 12 Ten 5.750 rep. 12

Days Microoganism B.subtilis E.coli P.aeruginosa S.aureus Four 17.024 16.857 14.190 17.357 Fourteen -0.137 -0.137 3.696 -0.137 one 25.000 25.000 25.000 25.000 Seven 10.601 6.935 6.935 4.768 cclxxxv

Ten 2.238 2.405 5.071 1.571

Standard errors of differences of means

Table DaysStrength_of_Ointment Microoganism Days Strength_of_Ointment rep. 84 unequal 105 unequal d.f. 279 279 279 279 s.e.d. 0.3377 0.7552 min.rep 0.1427 0.2670 0.1379 0.5970 max-min 0.1689 0.3776 max.rep

Table Days Microoganism rep. 21 s.e.d. 0.3027 d.f. 279 Except when comparing means with the same level(s) of Days 0.3083 d.f. 279

(Not adjusted for missing values)

Least significant differences of means (5% level)

Table DaysStrength_of_Ointment Microoganism Days Strength_of_Ointment rep. 84 unequal 105 unequal d.f. 279 279 279 279 l.s.d. 0.6648 1.4866 min.rep 0.2809 0.5256 0.2714 1.1752 max-min 0.3324 0.7433 max.rep

Table Days Microorganism rep. 21 l.s.d. 0.5960 d.f. 279 Except when comparing means with the same level(s) of Days 0.6069 cclxxxvi

d.f. 279

(Not adjusted for missing values)

Stratum standard errors and coefficients of variation

Variate: Wound Diameter d.f. s.e. r.s.e. 279 0.9249 8.8