i

AGBOKE, AYODEJI AKEEM PG/Ph.D/10/57874

ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON

CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

FACULTY OF PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACEUTICS

Ebere Omeje Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

ii

ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT

BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

BY

AGBOKE, AYODEJI AKEEM PG/Ph.D/10/57874

DEPARTMENT OF PHARMACEUTICS

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

FEBRUARY 2015

CERTIFICATION

This is to certify that Agboke Ayodeji Akeem, a postgraduate student in the Department

of Pharmaceutics with the Reg. No. PG/Ph.D/10/57874 has satisfactorily completed the

degree of Doctor of Philosophy (Ph.D) in Pharmaceutical Microbiolgy and that the work

iii

embodied in this thesis is original and has not been submitted in part or full for any other

diploma or degree of this or other university.

..……………………… ………………………….. Prof. A. A. Attama Prof. K. C. Ofokansi Supervisor Head of Department

iv

DEDICATION

This thesis is dedicated to God Almighty, my other half, Pastor (Mrs.) Abimbola

Labake Agboke and my children (Precious, Gift and Emmanuel) for their moral support and

encouragement.

v

ACKNOWLEDGEMENT

I owe my immeasurable appreciation to Almighty God for His wisdom, love, mercy,

presence, guidance and provision throughout the period of this work. I also wish to express my

profound gratitude to my supervisor Prof. A. A. Attama for his care, concern and direction that was

needed for the successful completion of the work. My profound appreciation goes to the Head of

Department of Pharmaceutics, Prof. K. C. Ofokansi and Prof. V. C. Okore also of Pharmaceutics

for their encouragement and contributions to this study. Let me use this opportunity to appreciate

the Dean of Pharmaceutical Sciences, University of Nigeria Nsukka, Prof. Emmanuel Ibezim, Prof.

S. I. Ofoefule and Prof. C. O. Ezugwu (Faculty Rep., SPGS) for their encouragement and support.

My thanks also go to my professional colleagues in the Faculty of Pharmaceutical Sciencess

U.N.N; Dr. M. A. Momoh, Dr. Paul Akpa, Dr. Petra Nnamani, Dr. E. O. Omeje, Pharm. John

Ogbonna, Pharm. Frank Kenechukwu, Prof. J. Onyechi, Dr. N. Obitte, Dr. Agubata, Mr. Chijioke

Muogbo. I thank Prof. Paul A. Nwafor of Faculty of Pharmacy, University of Uyo for his

contribution to the toxicology aspect of the study. I thank my beloved wife Pastor (Mrs) Abimbola

Agboke, all members, workers and ministers of The Redeemed Christian Church of God, Chapel of

Light Parish and the leadership of Akwa Ibom Province 2, for their prayers. Surely God will reward

all of you. I remain grateful to my friends and well wishers of Faculty of Pharmacy, University of

Uyo, Dr. M. O. Adedokun, Dr. Stephen Majekodunmi, Pharm. Clement Jackson, Pharm.

Olorunshola Samuel, Dr. U .S. Ekong, Dr. (Mrs) Peace Ubulom, Dr. C. Udobi and my Ag Head of

Dept., Pharm. (Mrs.) E. I. Akpabio, Ag. Dean of Pharmacy, Dr. A.C. Igboasoiyi for their

encouragement and support. I also thank Mr. Fadare Olatomide of Central Science Lab., O. A. U.

Ile-Ife for his assistance in the GC-MS analysis.

February 2015

vi

TABLE OF CONTENTS

TITLE PAGE -- -- -- -- -- -- -- -- -- -- i

CERTIFICATION -- -- -- -- -- -- -- -- -- ii

DEDICATION -- -- -- -- -- -- -- -- -- -- iii

ACKNOWLEDGEMENT-- -- -- -- -- -- -- -- -- iv

TABLE OF CONTENTS -- -- -- -- -- -- -- -- v

LIST OF TABLES-- -- -- -- -- -- -- -- -- vi

LIST OF FIGURES -- -- -- -- -- -- -- -- -- vii

ABSTRACT -- -- -- -- -- -- -- -- -- -- viii

CHAPTER ONE

1.0. INTRODUCTION -- -- -- -- -- -- -- -- 1

1.1. History -- -- -- -- -- -- -- -- -- -- 2

1.2. Morphology -- -- -- -- -- -- -- -- -- 3

1.3. Taxonomic classification -- -- -- -- -- -- -- 5

1.4. Synonyms -- -- -- -- -- -- -- -- -- 5

1.5. Range and habitat -- -- -- -- -- -- -- -- 6

1.6. Geographical sources -- -- -- -- -- -- -- -- 7

1.7. Ethnomedical uses -- -- -- -- -- -- -- -- 8

vii

1.7.1 Moringa leaves -- -- -- -- -- -- -- -- -- 8

1.7.2 Moringa flowers -- -- -- -- -- -- -- -- -- 9

1.7.3. Moringa pods -- -- -- -- -- -- -- -- -- 9

1.7.4. Moringa seeds -- -- -- -- -- -- -- -- -- 9

1.7.5. Moringa roots, bark and gum -- -- -- -- --- -- -- 9

1.7.6. Moringa oil -- -- -- -- -- -- -- -- -- 10

1.8 Uses in Ayurvedic medicine -- -- -- -- -- -- -- 10

1.9 Uses in Siddha-- -- -- -- -- -- -- -- -- 10

1.10 Pharmacological properties -- -- -- -- -- -- -- 10

1.10.1 Antihypertensive, diuretic and cholesterol lowering activites - - -- 10

1.10.2 Antispasmodic, antiulcer and hepatoprotective activities-- -- -- -- 11

1.10.3 Antibacterial and antifungal activities -- -- -- -- -- -- 12

1.10.4 Antitumor and anticancer activities -- -- -- -- -- -- 13

1.10.5 Coagulant activities -- -- -- -- -- -- -- -- 13

1.10.6 Moringa seeds as biosorbent -- -- -- -- -- -- -- 14

1.10.7 Other diverse activities -- -- -- -- -- -- -- -- 15

1.11 Non- pharmacological uses -- -- -- -- -- -- -- -- 16

1.12 Phytochemisty -- -- -- -- -- -- -- -- -- 17

1.13 Bacteriology of staphylococci -- -- -- -- -- -- -- 20

1.13.1 Taxonomy-- -- -- -- -- -- -- -- -- 20

1.13.2 Identification of staphylococci in the clinical laboratory-- -- -- -- 21

1.13.3 Epidemiology of S. aureus Infections-- -- -- -- -- -- 23

1.13.4 Clinical manifestations of S. aureus-- -- --- -- -- -- 24

1.13.5 Pathogenesis of S. aureus Infections -- -- -- -- -- -- 24

viii

1.13.6 Infections Associated with Medical Devices-- -- -- -- -- 25

1.13.7 Virulence factors of S. aureus -- -- -- -- -- -- -- 25

1.13.8 Membrane damaging toxins -- -- -- -- -- -- -- 28

1.13.9 Other extracelluar proteins -- -- -- -- -- -- -- 32

1.14 Coagulase Negative Staphylococci (CNS) -- -- -- -- -- 33

1.15 Antimicrobial Resistance. -- -- -- -- -- -- -- -- 35

1.15.1 Mechanism of Bacterial Resistance -- - -- -- -- -- -- 36

1.15.2 Intrinsic resistance-- -- -- -- -- -- -- -- -- 37

1.15.3 Acquired resistance -- - -- -- -- -- -- -- -- 37

1.15.4 Prevention of antimicrobial access to their targets -- -- -- -- 38

1.16 Selected antimicrobial agents according to mechanisms of Action-- -- -- 41

1.16.1 Bacterial cell wall biosynthesis-- -- -- -- -- -- -- 43

1.16.2 Nucleic acid biosynthesis-- -- -- -- -- -- -- -- 47

1.16.3 Rifamycins, RNA transcription -- -- -- -- -- -- -- 49

1.16.4 Protein biosynthesis -- -- -- -- -- -- -- -- 49

1.16.4.1 Inhibitors of 30S subunit -- -- -- -- -- -- -- 50

1.16.4.2 Inhibitors of 50S subunit-- -- -- -- -- -- -- 53

1.16.5 Miscellaneous targets-- -- -- -- -- --- -- -- 58

1.16.5.1 Follic acid metabolism: sulphonamedes and trimethoprim -- -- -- 58

1.16.5.2 Cell membrane disruptors -- -- -- -- -- -- -- 60

1.17 Methicillin Resistant Staphylococcus aureus (MRSA) -- -- -- -- 61

1.17.1 Brief timeline-- -- -- -- -- -- -- -- -- 62

1.17.2 Methicillin-- -- -- -- -- -- -- -- -- -- 63

1.17.3 Mechanism of resistance-- -- -- -- -- -- -- -- 63

ix

1.17.4 Detection of resistance-- -- -- -- -- -- -- -- 65

1.17.5 Phenotypic detection systems-- -- -- -- -- -- -- 66

1.17.6 Genotypic detection system-- -- -- -- -- -- -- 70

1.17.7 Interpretation of genotypic detection methods-- -- -- -- -- 73

1.18 Evolution of methicillin-resistant S. aureus clones-- -- -- -- 73

1.19 Historical origins and mechanisms of evolution of MRSA-- -- -- 75

1.20 Treatment options-- -- -- -- -- -- -- -- -- 77

1.20.1 Cytotoxic effect of commonly available antimicrobial agents-- -- -- 78

1.20.2. Plants and plant product as sources of antimicrobial agents-- -- -- 78

1.21 Future prospects-- -- -- -- -- -- -- -- -- 79

1.21.1 Antimicrobial drugs--- -- -- -- -- -- -- -- 79

1.21.2 Vaccines and new approaches to combatting nosocomial infections -- -- 80

1.22 Antimicrobial evaluation of a new agent-- -- -- -- -- -- 81

1.22.1 Strip- agar – diffusion -- -- -- -- -- -- -- -- 81

1.22.2 Ditch agar diffusion -- - -- -- -- -- -- -- -- 82

1.22.3 Determination of minimal inhibitory concentration (MIC) of extracts -- -- 82

1.22.4 Determination of minimal biocidal concentration (MBC) of extracts -- -- 83

1.23 Aim of the Study-- -- -- -- -- --- -- -- -- 84

1.24 Objectives of the Study -- -- -- -- -- -- -- -- 84

CHAPTER TWO

2.0. MATERIALS AND METHODS -- -- -- -- -- -- -- 85

2.1. Materials -- -- -- -- -- -- -- -- -- -- 85

2.1.1 Sample Collection -- -- -- -- -- -- -- -- -- 85

x

2.1.2 Media -- -- -- -- -- -- -- -- -- -- 85

2.1.3 Reagents -- -- -- -- -- -- -- -- -- -- 85

2.1.4 Solvents -- -- -- -- -- -- -- -- -- -- 86

2.1.5 Equipments -- -- -- -- -- -- -- -- -- 86

2.1.6 Animals -- -- -- -- -- -- -- -- -- -- 86

2.2 METHODS -- -- -- -- -- -- -- -- -- 86

2.2.1 Collection, authentication and processing of plant materials -- -- -- 86

2.2.2 Extraction of root extract-- -- -- -- -- -- -- -- 87

2.2.3 Fractionation of methanol crude extract using column chromatography

- -- -- -- -- -- -- -- -- -- -- 87

2.2.4 Qualitative phytochemical analysis -- -- -- -- -- -- -- 87

2.2.5 Media preparation -- -- -- -- -- -- -- -- 93

2.2.6 Standardization of inoculums-- -- -- -- -- -- -- 93

2.2.7 Characterization of the clinical isolates-- -- -- -- -- -- 93

2.2.8 Antimicrobial susceptibility testing-- - -- -- -- -- 94

2.2.9 Penicilin-binding protein (PBP2ˈ) latex agglutination test for MRSA

confirmation-- -- -- ---- -- -- -- -- -- 95

2.3 Determination of MIC and MBC of the extracts and fractions on

MRSA clinical Isolates -- -- -- -- -- ---- -- -- 96

2.3.1 Preparation of stock solution-- -- -- -- -- -- -- 96

2.3.2 Preparation of extract and fractions solutions for agar dilution MIC tes - -- 96

2.3.3 Determination of MIC of methanol extract and fractions -- -- -- -- 97

2.3.4 Determination of MBC of methanol extract and fractions -- -- -- -- 97

2.3.5 GC-MS Determination of bioactive components of methanol extract

fractions -- -- - -- -- -- -- -- -- -- -- 99

xi

2.3.6 Identification of compounds in methanol extract fractions -- -- -- 99

2.3.7 Structures of some compouds identified in methanol extract fractions -- -- 100

2.4 Preliminary toxicology evaluation of the methanol crude extract

and n-hexane extract fraction-- -- -- -- -- -- -- 100

2.4.1 Acute toxicity study -- -- -- -- -- -- -- -- 100

2.4.2 Sub-acute toxicity study-- ---- -- -- -- -- -- 100

2.5 Stastical analysis -- -- -- -- -- -- -- -- -- 102

CHAPTER THREE

3.0 RESULTS AND DISCUSSIONS-- -- -- -- -- -- -- 103

3.1 Results-- -- -- -- -- -- -- -- -- -- -- 103

3.1.1 The Percentage yield of the methanol extracts and fractions -- -- -- 103

3.1.2 Qualitative phytochemical analysis of the extracts and fractions-- -- -- 105

3.2 Characterization of clinical isolates-- -- -- -- -- --- 106

3.3 Antimicrobial susceptibility test-- -- -- -- --- -- 107

3.4 Penicilin-binding protein (PBP2') latex agglutination test results -- -- 109 3.5 Prevalence rate of clinical isolates of S.aureus, MSSA, MRSA -- - 109

3.6 Results of the MIC and MBC of the extracts and fractions on MRSA clinical isolates- -- -- -- -- -- -- -- - -- 112 3.7 GC-MS identification of bioactive compounds of methanol

extract fractions- -- -- -- -- -- --- -- 120

3.8 Structure of some phytochemical compounds identified in

Moringa oleifera root bark -- -- -- -- -- -- -- -- 127

xii

3.9. Preliminary evaluation of toxicity of crude methanol extract and

n-hexane extract fraction-- --- -- -- -- -- -- -- -- 130

3.9.1 Acute toxicity test of crude methanol extract extract and n-hexane

extract fraction -- -- -- -- -- -- -- -- -- 130

3.9.2 Sub-acute toxicity study of crude methanol extract and n-hexane

extract fraction of M. oleifera root--- -- -- -- -- -- -- 130

3.9.3 Effects of graded doses of crude methanol extract and n-hexane

extract fraction on body weights of rats-- ---- -- -- -- -- 131

3.10 Discussion -- -- -- -- -- -- -- -- -- 138

3.10.1 Percentage yield of extracts and fractions-- - -- -- -- -- 138

3.10.2 Qualitative phytochemical analysis of methanol extract and fractions -- 138

3.10.3 Prevalence rate of clinical isolates of S. aureus -- -- -- -- -- 140

3.10.4 Antimicrobial susceptibility pattern of clinical isolates -- ---- 141

3.10.5 Penicilin – binding protein (PBP2') latex agglutination test -- -- -- 143

3.11 MIC and MBC of methanol extract and fractions -- -- -- -- 144

3.11.1 MIC and MBC of methanol crude extract -- -- -- -- -- 145

3.11.2 MIC and MBC of ethyl acetate fraction-- -- -- -- -- 145

3.11.3 MIC and MBC of n-hexan extract fraction -- -- -- -- -- 146

3.11.4 MIC and MBC of Dichloromethane fraction -- -- -- -- -- 146

3.11.5 MIC and MBC of Methanol fraction -- -- -- -- -- -- 147

3.12 GC-MS identification of bioactive compounds of methanol extract fractions -- -- -- -- -- -- -- -- -- 147

xiii

3.12.1 Gas chromatography-mass spectrometry (GC-MS)

of ethyl acetate fraction -- -- -- -- -- -- -- -- 149

3.12.2 Gas chromatography-mass spectrometry (GC-MS)

of dichloromethane fraction -- -- -- -- -- -- -- 149

3.12.3 Gas chromatography-mass spectrometry (GC-MS) of n-hexane fraction -- -- -- -- -- -- -- -- 150

3.12.4 Gas chromatography-mass spectrometry (GC-MS)

of methanol fraction -- -- -- -- -- -- -- -- 150

3.12.5 Structure of some phytochemical compounds identified in

Moringa oleifera root bark -- - -- -- -- -- -- -- 150

3.13 Acute toxicity test of crude methanol extract and n-hexane

extract fraction-- -- -- -- -- -- -- -- 151

3.13.1 Sub-acute toxicity study of the crude methanol extract and n-hexane

extract fraction -- -- -- -- --- -- --- -- 151

3.13.2 Effects of graded doses of crude methanol extract and n-hexane

extract fraction on body weights of rats -- -- ---- -- -- -- 151

CHAPTER FOUR

4.0 CONCLUSIONS AND RECOMMENDATIONS -- -- --- -- 152

4.1 Conclusions -- -- -- -- -- -- -- -- -- 152

4.2 Recommendations -- - -- -- -- -- -- -- -- 154

4.3 Contributions to knowledge -- -- -- -- -- -- -- 155

References -- -- -- -- --- -- -- -- -- -- -- 156

Apendices -- -- -- -- -- -- -- -- -- -- -- 198

xiv

LIST OF TABLES

Table 1 Preparation of extract and fractions concentration for agar dilution MIC tests-- -98

Table 2 The percentage yield of crude methanol extracts and fractions of

M. oleifera root bark-- -- -- -- -- -- -- -- 104

Table 3 Results of Phytochemical Analysis of crude methanol extracts and fractions- - -- -- -- ---- -- -- -- 105 Table 4 Antimicrobial susceptibility test -- -- -- -- -- - 108

Table 5 Penicilin-binding protein (PBP2') latex agglutination test -- -- -- 110

Table 6 Prevalence rate of clinical isolates of Staphylococcus aureus --- - 111

Table 7 MIC and MBC of crude methanol extract in mg/ml -- -- -- -- 115

Table 8 MIC and MBC of ethyl acetate fraction in mg/ml -- -- -- -- 116

Table 9 MIC and MBC of n-hexane fraction in mg/ml -- -- -- -- 117

Table 10 MIC and MBC of dichloromethane fraction in mg/ml -- -- -- 118

Table 11 MIC and MBC of methanol fraction in mg/ml -- -- -- 119

Table 12 Acute toxicity test of crude methanol extract and

n-hexane extract fraction-- -- -- -- -- -- --- -- 132

Table 13 Effects of the graded doses of crude methanol extract and

n-hexane fraction on haematological parameters of rats -- -- -- 135

Table 14 Effects of the graded doses of crude methanol extract and

n-hexane extract fraction on biochemical parameters of rat--- -- 136

Table 15 Effects of graded doses of crude methanol extract and

n-hexane extract fraction on body weights of rats -- -- -- -- 137

xv

LIST OF FIGURES

Fig. 1 Compuond leaf; paler lower surfaces of leaflets of Moringa oleifera -- -- -- 4

Fig.2 Flower panicle of Moringa oleifera targets for antimicrobial agents. -- -- ---4

Fig. 3 Summary of the resistance mechanisms to the main antibiotimicrobial classes - - 40

Fig. 4 Structures of some representatives of the discussed antimicrobial classes - - - 43

Fig. 5 Inhibitor of β-lactams and the glycopeptides -- -- -- -- --- --44

Fig. 6 Inhibitors of nucleic acid biosynthesis -- -- -- -- -- --48

Fig. 7 The process of protein biosynthesis inhibition -- -- ---- -- - --51

Fig. 8 Sulphonamides and trimethoprim inhibit distinct steps in folate metabolism --61

Fig. 9 Evolution of MRSA clones in Latin American countries -- -- -- --76

Fig. 10 MS Fragment of ethyl acetate fraction composition -- -- -- -123

Fig. 11 MS Fragment of dichloromethane fraction composition -- -- -- -124

Fig. 12 MS Fragment of n-hexane fraction composition -- -- -- -- -125

Fig. 13 MS Fragment of methanol fraction composition -- -- -- -- -126

Fig. 14 Structure of some components identified in methanol extract fractions -- -143

Fig. 15 Determination of LD50 value of methanol extract of M. oleifera root bark in rats -147

Fig. 16 Determination of LD50 value of n-hexane extract of M. oleifera root bark in rats -151

xvi

APPENDICES

Appendix 1: Identification of methicillin resistant S. aureus (MRSA)- -- -- - - - - 198

Appendix 2: Effect of methanol crude extract extracts on methicillin-resistant S. aureus—199

Appendix 3: Effect of ethyl acetate fraction methicillin-resistant S. aureus (MRSA) -- 200

Appendix 4: Effect of dichloromethane fraction on methicillin-resistant S. aureus --- ------201

Appendix 5: Effect of n-hexane extract fraction on methicillin-resistant S. aureus--- 202

Apendix 6: Effect of methanol fraction on methicillin-resistant S. aureus-- -- -- 203

Appendix 7: GC-MS report and activity of components of ethyl acetate fraction ----- 204

Appendix 8: A GC-MS report of dichloromethane fraction -- -- -- -- 207

Appendix 9: Activity of components identified in the sample of dichloromethane fraction-209

Appendix 10: A GC-MS report of n-hexane fraction ------ -- -- -- -- -214

Appendix 11: Activity of components identified in the sample n-hexane fraction -- 215

Appendix 12: A GC-MS report of methanol fraction-- -- -- -- 218

Appendix 13: Activity of components identified in the methanol fraction --- -- 218

Appendix 14: All statistical analysis with spss version 17 software -- -- -- 220

xvii

ABSTRACT

Development of antimicrobial resistance by bacteria is now a world wide health issue, as

infection is one of the leading causes of death in the world today. This fact is also as a result

of the emergence of multiple antibiotic resistant bacteria known as methicillin resistant

Staphylococcus aureus (MRSA) with potential of cross resistance to other antibiotics of

choice like vancomycin. MRSA is often referred to as a potential killer and one of the tree top

superbugs in hospitals multidrug resistant organisms (MDRO). The aim of this study was to

evaluate the phytochemical components and antimicrobial activity of methanol extract and

fractions of Moringa oleifera root bark as possible remedy for MRSA infections.

Staphylococcus aureus isolates from 3 different hospitals in South-east geopolitical region of

Nigeria were confirmed by coagulase/staphylase test using Oxoid® reagents kits (DR0595A).

The characterised S. aureus isolates were further identified as Methicillin resistant

staphylococcus aureus by disc diffusion method as recommended by the Clinical Laboratory

Standards Institute (CLSI), using standard antibiotic discs containing oxacillin (5 µg/ml),

vancomycin (30 µg/ml), cephalexin (30 µg/ml), levofloxacin (5 µg/ml), ciprofloxacin (5

µg/ml), tetracycline (30 µg/ml), cotrimoxazole (25 µg/ml), gentamicin (30 µg/ml),

clindamycin (2 µg/ml) and rifampicin (5 µg/ml). Methicillin resistant staphylococcus aureus

confirmation was done using Oxoid® DR0900 penicillin binding protein (pbp2ˈ) latex

agglutination test kits. Pulverised Moringa oleifera root bark was defatted with n-hexane to

yield hexane fraction (HEF). The dried marc was extracted with methanol using Soxhlet

extractor to obtain crude methanol extract (ME). Methanol extract was adsorbed on Silical gel

(60-200 mesh) and eluted in succession to obtain dichloromethane fraction (DMF), ethyl

acetate fraction (EAF) and methanol fraction (MEF). Qualitative phytochemical analyses of

the extracts were carried out using standard procedures. The antimicrobial activities of ME,

HEF, DMF, EAF and MEF were evaluated on the MRSA, the minimum inhibitory

xviii

concentrations (MICs) and minimum bactericidal concentrations (MBCs) were recorded and

compared with the standard disc antimicrobial test results. The extract fractions were analysed

using gas chromatographic-mass spectrometry (GC-MS) for their bioactive compounds. The

preliminary acute toxicity and sub-acute toxicity of ME and HEF were evaluated. Statistical

analysis was done with ANOVA followed by Duncan post Hoc test using SPSS v 17 software.

Characterised clinical isolates yielded 58 S. aureus strains. Antibiotic susceptibility tests

indicated varied percentages of MRSA that were resistant to various antibiotics thus: oxacillin

(62.1 ± 3.2%), vancomycin (60.4 ± 3.8%), cephalexin (55.2 ± 1.2%), levofloxacin (56.9 ±

2.2%), ciprofloxacin (56.9 ± 0.9%), tetracycline (65.5 ± 2.3%), cotrimoxazole (68.9 ± 0.8%),

gentamicin (67.2 ± 1.3%), clindamycin (62.1 ± 3.3%) and rifampicin (62.1 ± 4.1%). Latex

agglutination test confirmed 39 strains of the clinical isolates to be MRSA. The S. aureus

isolates resistant to all the antibiotics including vancomycin at 30 µg/ml were sensitive to the

extract and all its fractions: ME: MIC (3.0 ± 0.1 to 5.0 ± 0.5 mg/ml) and MBC (3.0 ± 0.1 to

6.0 ± 0.5 mg/ml); EAF: MIC (5.0 ± 1.1 to 8.0 ± 0.5 mg/ml) and MBC (5.0 ± 0.5 to 8.0 ± 0.5

mg/ml); DMF MIC (8.0 ± 1.1 to 10 ± 0.5 mg/ml) and MBC (8.0 ± 0.5 to 10 ± 0.5 mg/ml);

HEF: MIC (7.0 ± 0.5 to 8 ± 1.1 mg/ml) and MBC (7.0 ± 0.5 to 9 ± 0.5 mg/ml), MEF: MIC

(9.0 ± 1.1 to 10.0 ± 0.5 mg/ml) and MBC (9.0 ± 0.5 to 10.0 ± 0.5 mg/ml). Phytochemical

analysis of the extracts showed the presence of alkaloids, glycosides, steroids, terpenoids,

flavonoids, saponins, tannins, resins, reducing sugars, proteins, fats and oil and carbohydrates.

GC-MS analysis revealed over 100 distinct compounds, some of which are stigmasterol

(C29H48O), eugenol (C10H12O2), oxime (C3H7NO ) and ergosta-4, 22-dien-3-one (C28H44O).

The oral acute toxicity test showed the LD50 of ME as 3663.96 mg/kg and HEF as

1934.15mg/kg, with no significant change (P > 0.05) in the hematological, serum biochemical

parameters and weight of the rats.

1

CHAPTER ONE

1.0 Introduction

In the last few decades there has been an exponential growth in the field of herbal

medicine. It is getting popularized in developing and developed countries owing to its natural

origin and lesser side effects [1]. Herbal drugs constitute a major share of all the officially

recognized systems of health in India viz. Ayurveda, Yoga, Unani, Siddha, Homeopathy and

Naturopathy, except Allopathy. More than 70% of India's 1.1 billion population still use these

non-allopathic systems of medicine [2].

In many developing countries, a large proportion of the population relies on traditional

practitioners and their armamentarium of medicinal plants in order to meet health care needs.

Although modern medicines may exist side-by-side with such traditional practice, herbal

medicines have often maintained their popularity for historical and cultural reasons. Such

products have become more widely available commercially, especially in developed

countries. Use of herbal medicines in developed countries has expanded sharply in the latter

half of the twentieth century. In India, herbal drugs are an integral part of the Indian system

of medicine (Ayurveda), which is an ancient and mainstream system [3].

The evaluation of various plant products according to their traditional uses and

medicinal value based on their therapeutic efficacy leads to the discovery of newer and recent

drugs for treatingvarious ailments. This fact forms the basis for the development of new

drugs from various plant sources. One of such plants of medicinal value is Moringa olifera,

belonging to the family Moringaceae, commonly known as ‘sahajan’ in Hindi, Horse radish

in English. It is a small, fast, growing, evergreen, or deciduous tree that usually grows up to

10 or 12 m in height. It is distributed among Sub Himalayan Tracts, Assam, Bengal and

Peninsular India [4]. Various properties are attributed to it like antispasmodic, diuretic,

expectorant and abortifacient [5].

2

1.1 History of Moringa oleifera

It is a small or medium-sized tree, attractive enough to be a focal point in the tropics

and sub-tropics owing to its creamy – white, sweetly scented flowers and light –green,

tripinnately compound foliage [1-3]. It is a native to India, occurring wild in the sub-

Himalayan regions of Northern India and cultivated throughout the country. It is commonly

known as Sajina, sajna (Bengali); horseradish tree, drumstick tree (English); Sahinjan,

mungna (Hindi); murinna, muringa, tishnagandha (Malyalam); sevaga, segata (Marathi);

Sohanjana (Punjabi); Sobhanjana, sigru, murungi, dvishiguru (Sanskrit) and Sehjan(Urdu) in

varied Indian languages and regions [4,5]. It also thrives well in Pakistan, Bangladesh, Sri

Lanka, tropical Africa, Arabia, Philippines, Cambodia and Central, North and South America

[6-10]. Described as “one of the most amazing trees God has created”, almost every part of

drumstick viz. bark, root, fruit, flowers, leaves, seed and gum is a rich repository of proteins,

vitamins and minerals including potassium, calcium, phosphorus, iron, folic acid as well as β

carotene. Leaves can be eaten fresh, cooked or stored as dry powder for many months

without refrigeration, without loss of nutritional value. Almost all the parts of this plant have

been used for various ailments in the indigenous medicine of South Asia [11, 12]. The named

varieties of moringa include Jaffna or Yazhpanam, grown in various parts of South India,

(producing 60-90 cm long pods), Chavakacheri murungai, (producing pods 90-120 cm long),

Chemmurungai (with red tipped fruits), Kadumurungai, Palmurungai, Puna murungai (with

thick pulp and bitter taste), Kodikkal Murungai etc. [13,14].

The Horticultural College & Research Institute of Tamil Nadu Agricultural University

has released two improved annual moringa varieties (PKM1, PKM2) within a span of 10

years, for commercial cultivation [15, 16]. The folklore claims and ancient literature report

moringa to be an abortifacient antidote, antirheumatic, bactericide, diuretic, ecbolic, emetic,

expectorant, purgative, rubefacient, stimulant, tonic, vermifuge and vesicant [17-20].

3

(Pharma Products Pvt Ltd, Thayavur, India) and Livospin (Herbals APS Pvt. Ltd., Patna,

India), which are available for a variety of ailments [21]. Ayurvedic preparations include

Ratnagiri Rasa, Sarasvata Ghrta, Sudarsana churna, Sarsapadi Pralepa, Visatimduka Taila etc

[4, 5].

Leaves of moringa are applied as poultice to sores and in treatment of anemia and

menstrual irregularities. Young leaf paste with curd, is used internally for stomachache while

externally for sprains. Leaf juice or bark paste is used as a drink for constipation and piles

[22, 23]. The root juice is applied externally as rubefacient or counter irritant, in hiccups,

lumbago, enlarged spleen or liver. Bark, leaves and roots are acrid and pungent, taken to

promote digestion. A reddish gum exuded from the bark possess anti diarrhoeal,

emmenagogue, antiscorbic and abortifacient properties. According to Materia Medica, a

compound spirit made from equal parts of roots of Moringa and orange peel acts as

carminative and stimulant in nervous debility, paralytic afflictions, epilepsy and hysteria [24-

26]. Not only this, moringa is glorified as a ‘traditional mother care plant’, for the leaves are

highly nutritious for pregnant women [27].

Until now, only a very few attempts have been made to compile the myriad of potential

uses of this “miracle tree”. In view of a number of recent findings of ethnopharmacological

importance, an updated appraisal was much needed. So, the present research is an attempt to

explore the claims so far and prepare the ground for development of effective novel herbal

formulations of M. oleifera. in the treatment of infections caused by much dreaded Methicilin

resistant Staphylococcus aureus [25-27].

1.2 Morphology

Moringa oleifera is a small, fast-growing evergreen or deciduous tree that usually

grows as high as 9 m, with a soft and white wood and corky and gummy bark. Roots have the

taste of horseradish. Leaves are longitudinally cracked leaves, 30-75 cm long main axis and

4

Fig. 1 Compound leaf; paler lower surfaces of leaflets of Moringa oleifera

Fig. 2 Flower panicle Moringa oleifera

5

its branch jointed, glandular at joints, leaflets are glabrous and entire. The leaflets are finely

hairy, green and almost hairless on the upper surface, paler and hairless beneath, with red-

tinged mid-veins, with entire (not toothed) margins, and are rounded or blunt-pointed at the

apex and short-pointed at the base. The twigs are finely hairy and green. Flowers are white,

scented in large axillary down panicles, pods are pendulous, ribbed, seeds are 3-angled [4, 6].

1.3 Taxonomic classification

Kingdom - Plantae

Sub kingdom - Tracheobionta

Super Division - Spermatophyta

Division - Magnoliophyta

Class - Magnoliopsida

Subclass - Dilleniidae

Order - Capparales

Family - Moringaceae

Genus - Moringa

Species - oleifera

1.4 Synonyms

Latin - Moringa oleifera

Sanskrit - Subhanjana

Hindi - Saguna, Sainjna

Gujarati - Suragavo

Tamil - Morigkai

Telugu - Mulaga, Munaga

Malayalam - Murinna, Sigru

Punjabi - Sainjna, Soanjna

6

Unani - Sahajan

Ayurvedic - Akshiva, Haritashaaka, Raktaka, Tikshnagandhaa

Arabian - Rawag

French - Moringe à graine ailée, Morungue

Spanish - Ángela, Ben, Moringa

Portuguese - Moringa, Moringueiro

Chinese - Laken

English - Drumstick tree, Horseradish tree, Ben tree

Yoruba - Ewe igbale, ewe ile, adagba maloye, igi agunmaniye

Igbo - Okwe oyibo, odudu oyibo, okochi egbu

Hausa - Zogale, bagagruwa maka [4-7].

1.5 Range and habitat

The moringa tree is grown mainly in semiarid, tropical, and subtropical areas,

corresponding in the United States to USDA hardiness zones 9 and 10. It grows best in dry

sandy soil and tolerates poor soil, including coastal areas. As with all plants, optimum

cultivation depends on producing the right environment for the plant to thrive. Moringa is a

sun and heat-loving plant, and thus does not tolerate freeze or frost. Moringa is particularly

suitable for dry regions, as it can be grown using rainwater without expensive irrigation

techniques. The following conditions are reported to be ideal for cultivation of M. oleifera [8-

10].

Parameter Requirement/Range

Climate

Grows best in tropical or sub-tropical

Altitude/ Height

0 – 2000 meters

7

Rainfall

250 – 2000 mm.

Irrigation needed for leaf production if rainfall < 800 mm

Soil Type

Loamy, sandy or sandy-loam

Soil pH

pH 5 – 9

1.6 Geographical sources

The tree is wild in the Sub-Himalayan tracts from Chenab to Oudh. It grows at

elevations from sea level to 1400 m. It is very commonly cultivated near houses in Assam,

Bengal and peninsular India. It is a prolific coppice [4]. It is also cultivated in north-eastern

Pakistan, north-eastern Bangladesh, Sri Lanka, West Asia, the Arabian Peninsula, East and

West Africa, throughout the West Indies and southern Florida, in Central and South America

from Mexico to Peru, as well as in Brazil and Paraguay [6].

As of 2010, cultivation in Hawaii, for commercial distribution in the United States, is in

its early stages.[9] "India is the largest producer of moringa, with an annual production of 1.1

to 1.3 million tones of tender fruits from an area of 380 km². Among the states, Andhra

Pradesh leads in both area and production (156.65 km²) followed by Karnataka (102.8 km²)

and Tamil Nadu (74.08 km²). In other states, it occupies an area of 46.13 km². Tamil Nadu is

the pioneering state in·so·much as it has varied genotypes from diversified geographical areas

and introductions from Sri Lanka.[9 -10]

Moringa is grown in home gardens in Odisha and as living fences in Southern India and

Thailand, where it is commonly sold in local markets.[11] In the Philippines, it is commonly

grown for its leaves which are used in soup. Moringa is also actively cultivated by the World

Vegetable Center in Taiwan, a center for vegetable research with a mission to reduce poverty

and malnutrition in developing countries through improved production and consumption of

8

vegetables. Tamil nadu, Southern India has Moringa in its folk stories and use in home

gardens. In Haiti it is grown as wind breaks and to help reduce soil erosion [11-12].

1.7 Ethnomedical uses

Moringa oleifera has an impressive range of medicinal uses with high nutritional value and

medicinal benefits. Different parts of Moringa contain a profile of important minerals and are

good source of protein, vitamins, beta-carotene, amino acids and various phenolics. Moringa

provides a rich and rare combination of zeatin, quercetin, beta-sitosterol, caffeoylquinic acid

and kaempferol [13]

Moringa can act as cardiac and circulatory stimulants, possess antitumor, antipyretic,

antiepileptic, antiinflammatory, antiulcer, antispasmodic, diuretic, antihypertensive,

cholesterol lowering, antioxidant, antidiabetic, hepatoprotective, antibacterial and antifungal

activities, and are being employed for the treatment of different ailments in the indigenous

system of medicine.

1.7.1 Moringa leaves [14, 15]

Leaves rubbed against the temple can relieve headaches and stop bleeding from a shallow

cut. There is an anti-bacterial and anti-inflammatory effect when applied to wounds or insect

bites, extracts can be used against bacterial or fungal skin complaints. The Leaf tea treats

gastric ulcers and diarrhea, eating Moringa food products is good for those suffering from

malnutrition due to the high protein and fibre content. Leaves of this plant treat fevers,

bronchitis, eye and ear infections and inflammation of the mucus membrane.

The iron content of the leaves is high, and they are reportedly prescribed for anemia in the

Philippines. Dried Moringa leaves treat diarrhoea in Malawi, Africa. The powder ground

from the seeds is also used in the treatment of scurvy skin diseases (common bacterial

infections of the skin).

9

1.7.2 Moringa flowers

Flower juice improves the quality and flow of mothers’ milk when breast feeding, it is also

useful for urinary problems as it encourages urination. In Haiti, villagers boil Moringa

flowers in water and drink the tea as a powerful cold remedy.

1.7.3 Moringa pods

If eaten raw, pods act as a de-wormer and treat liver and spleen problems and pains of the

joints. Due to high protein and fibre content pods can play a useful part in treating

malnutrition and diarrhoea.

1.7.4 Moringa seeds

Used for their antibiotic and anti-inflammatory properties to treat arthritis, rheumatism, gout,

cramp, sexually transmitted diseases and boils. The seeds are roasted, pounded, mixed with

coconut oil and applied to the problem area. Seed oil can be used for the same ailments.

Roasted seeds and oil can encourage urination and can also be used as a relaxant for epilepsy.

Moringa seeds are effective against skin-infecting bacteria Staphylococcus aureus and

Pseudomonas aeruginosa. They contain the potent antibiotic and fungicide terygospermin

[16].

1.7.5 Moringa roots bark and gum

The roots and the bark have all of the properties described above but are more concentrated,

therefore much more care should be taken if using them as medicines. Roots bark are used for

cardiac and circulatory problems, as a tonic and for inflammation. The bark is an appetizer

and digestive.

In Senegal and India, roots are pounded and mixed with salt to make a poultice for treating

rheumatism and articulars pains. This poultice is also used to relieve lower back or kidney

pain, an alkaloid spirachin (a nerve paralysant) has been found in the roots.

The gum is diuretic, astringent and abortifacient and is used against asthma.

10

1.7.6 Moringa oil

Oil of Ben is used for hysteria, scurvy, prostate problems and bladder troubles. Villagers in

Oman use Moringa oil to treat stomach disorders. They also use it in perfume and hair oil.

1.8 Uses in ayurvedic medicine

Uses every part of the Moringa tree and considers it one of the most valuable and useful

plants. The ayurvedic medicine of India has many uses for moringa tree products, such as a

natural antibiotic, an aid in childbirth, for treating liver disorders, and many other uses.

1.9 Uses in Siddha medicine

In Siddha medicine says that the leaves are full of medicinal properties. The drumstick seeds

are used as a sexual virility drug for treating erectile dysfunction in men and also in women

for prolonging sexual activity [17-21].

1.10 Pharmacological properties

Moringa oleifera also has numerous medicinal uses, which have long been recognized in the

Ayurvedic and Unani systems of medicine [22]. The medicinal attributes and

pharmacological activities ascribed to various parts of moringa are detailed below.

1.10.1 Antihypertensive, diuretic and cholesterol lowering activities

The widespread combination of diuretic along with lipid and blood pressure lowering

constituents make this plant highly useful in cardiovascular disorders. Moringa leaf juice is

known to have a stabilizing effect on blood pressure [The Wealth of India, 1962; [23]. Nitrile,

mustard oil glycosides and thiocarbamate glycosides have been isolated from Moringa

leaves, which were found to be responsible for the blood pressure lowering effect [23]. Most

of these compounds, bearing thiocarbamate, carbamate or nitrile groups, are fully acetylated

glycosides, which are very rare in nature [24]. Bioassay guided fractionation of the active

ethanol extract of Moringa leaves led to the isolation of four pure compounds, niazinin A,

niazinin B, niazimicin and niazinin A B which showed a blood pressure lowering effect in

11

rats mediated possibly through a calcium antagonist effect [25]. Activity-guided fractionation

of the ethanol extract of pods of M. oleifera has led to the isolation of thiocarbamate and

isothiocyanate glycosides which are known to be the hypotensive principles [25]. Methyl

phydroxybenzoate and β-sitosterol investigated in the pods of M. oleifera have also shown

promising hypotensive activity [26], Moringa roots, leaves, flowers, gum and the aqueous

infusion of seeds have been found to possess diuretic activity [27,28] and such diuretic

components are likely to play a complementary role in the overall blood pressure lowering

effect of this plant. The crude extract of Moringa leaves has a significant cholesterol lowering

action in the serum of high fat diet fed rats which might be attributed to the presence of a

bioactive phytoconstituent, i.e. β-sitosterol [29]. Moringa fruit has been found to lower the

serum cholesterol, phospholipids, triglycerides, low density lipoprotein [LDL], very low

density lipoprotein [VLDL] cholesterol to phospholipid ratio, atherogenic index lipid and

reduced the lipid profile of liver, heart and aorta in hypercholesteremic rabbits and increased

the excretion of fecal cholesterol [30].

1.10.2 Antispasmodic, antiulcer and hepatoprotective activities

M. oleifera roots have been reported to possess antispasmodic activity [31]. Moringa

leaves have been extensively studied pharmacologically and it has been found that the

ethanol extract and its constituents exhibit antispasmodic effects possibly through calcium

channel blockade [32-35]. The antispasmodic activity of the ethanol extract of M. oleifera

leaves has been attributed to the presence of 4-[α-[L-rhamnosyloxy] benzyl]-o-methyl

thiocarbamate [trans], which forms the basis for its traditional use in diarrhea [35]. Different

constituents provide pharmacological basis for the traditional uses of this plant in

gastrointestinal motility disorder [36].The methanol fraction of M. oleifera leaf extract

showed antiulcerogenic and hepatoprotective effects in rats. Aqueous leaf extracts also

showed antiulcer effect [37] indicating that the antiulcer component is widely distributed in

12

this plant. Moringa roots have also been reported to possess hepatoprotective activity. The

aqueous and alcohol extracts from Moringa flowers were also found to have a significant

hepatoprotective effect which may be due to the presence of quercetin, a well known

flavonoid with hepatoprotective activity [38].

1.10.3 Antibacterial and antifungal activities

Moringa roots have antibacterial activity [39] and are reported to be rich in

antimicrobial principles. These are reported to contain an active antibiotic principle,

pterygospermin, which has powerful antibacterial and fungicidal effects. A similar compound

is found to be responsible for the antibacterial and fungicidal effects of its flowers [40]. The

root extract also possesses antimicrobial activity attributed to the presence of 4-α-L-

rhamnosyloxybenzyl isothiocyanate [41]. The aglycone of deoxy-niazimicine [N-benzyl, S-

ethyl thioformate] isolated from the chloroform fraction of an ethanol extract of the root bark

was found to be responsible for the antibacterial and antifungal activities [42]. The bark

extract has been shown to possess antifungal activity [43], while the juice from the stem bark

showed antibacterial effect against Staphylococcus aureus [44]. The fresh leaf juice was

found to inhibit the growth of microorganisms [Pseudomonas aeruginosa and Staphylococcus

aureus], pathogenic to man [45].

The seeds also possess antimicrobial properties [46, 47] reported that a recombinant protein

in the seed is able to flocculate Gram-positive and Gram-negative bacterial cells. In this case,

microorganisms can be removed by settling in the same manner as the removal of colloids in

properly coagulated and flocculated water [47]. On the other hand, the seeds may also act

directly upon microorganisms and result in growth inhibition. Antimicrobial peptides are

thought to act by disrupting the cell membrane or by inhibiting essential enzymes. It was

reported that the seeds could inhibit the replication of bacteriophages [47-49].

13

The antimicrobial effects of the seeds are attributed to the compound 4[α-L-rhamnosyloxy]

benzyl isothiocynate [49].

1.10.4 Antitumor and anticancer activities

Makonnen et al [31] found Moringa leaves to be a potential source for antitumor

activity. O-Ethyl- 4-[α-L-rhamnosyloxy]benzylcarbamate together with 4[α-L-

rhamnosyloxy]-benzylisothiocyanate, niazimicin and 3-O-[6′-O-oleoyl-α-D-glucopyranosyl]-

β-sitosterol have been tested for their potential antitumor promoting activity using an in vitro

assay which showed significant inhibitory effects on Epstein–Barr virus-early antigen.

Niazimicin has been proposed to be a potent chemo preventive agent in chemical

carcinogenesis [50]. The seed extracts have also been found to be effective on hepatic

carcinogen metabolizing enzymes, antioxidant parameters and skin papillomagenesis in mice

[51]. A seed ointment had a similar effect to neomycin against Staphylococcus aureus

pyodermia in mice [31, 52]. It has been found that niaziminin, a thiocarbamate from the

leaves of M. oleifera, exhibits inhibition of tumor-promoter-induced Epstein–Barr virus

activation. On the other hand, among the isothiocyanates, naturally occurring 4-[4′-O-acetyl-

α-i-rhamnosyloxybenzyl], significantly inhibited tumor-promoter induced Epstein–Barr virus

activation, suggesting that the isothiocyano group is a critical structural factor for activity

[53].

1.10.5 Coagulant activities

Moringa seeds are one of the best natural coagulants discovered so far [54]. Crushed

seeds are a viable replacement of synthetic coagulants [55]. In Sudan, seed crude extract is

used instead of alum by rural women to treat the highly turbid Nile water because of a

traditional fear of alum causing gastrointestinal disturbances and Alzheimer’s disease [56-

59]. The seeds are very effective for high turbidity water and show similar coagulation effects

to alum [60]. The coagulation effectiveness of M. oleifera varies depending on the initial

14

turbidity and it has been reported that it could reduce turbidity by between 92% and 99%

[61]. The seeds also have softening properties in addition to being a pH correct ant [alkalinity

reduction], as well as exhibiting a natural buffering capacity, which could handle moderately

high to high alkaline surface and ground waters. The seeds can also be used as an antiseptic

in the treatment of drinking water [62]. It is believed that the seed is an organic natural

polymer [63]. The active ingredients are dimeric proteins with a molecular weight of about

1300 Da and iso-electric point between 10 and 11 [63].

The protein powder is stable and totally soluble in water. Moringa coagulant protein can

be extracted by water or salt solution [commonly NaCl]. The amount and effectiveness of the

coagulant protein from salt and water extraction methods vary significantly. In crude form,

the salt extract shows a better coagulation performance than the corresponding water extract

[64]. This may be explained by the presence of higher amount of soluble protein due to the

salting-in phenomenon. However, purification of the M. oleifera coagulant protein from the

crude salt extract may not be technically and economically feasible. The coagulation

mechanism of the M. oleifera coagulant protein has been explained in different ways. It has

been described as adsorption and charge neutralization [65, 66] and interparticle bridging

[67]. Flocculation by inter-particle bridging is mainly characteristic of high molecular weight

polyelectrolytes. Due to the small size of the M. oleifera coagulant protein [6.5–13 kDa], a

bridging effect may not be considered as the likely coagulation mechanism. The high positive

charge [IP above 10] and small size may suggest that the main destabilization mechanism

could be adsorption and charge neutralization [68].

1.10.6 Moringa seeds as biosorbent

Moringa seeds could be used as a less expensive biosorbent for the removal of

cadmium (Cd) from aqueous media [69]. The aqueous solution of the seed is a heterogeneous

complex mixture having various functional groups, mainly low molecular weight organic

15

acids (amino) Kumar et al. [70]. These amino acids have been found to constitute a

physiologically active group of binding agents, working even at a low concentration, which

because of the ability to interact with metal ions is likely to increase the sorption of metal

ions [70]. The proteineous amino acids have a variety of structurally related pH dependent

properties, generating a negatively charged atmosphere and play an important role in the

binding of metals [70, 71].

1.10.7 Other diverse activities

Moringa oleifera has also been reported to exhibit other diverse activities. Aqueous

leaf extracts regulate thyroid hormone and can be used to treat hyperthyroidism and exhibit

an antioxidant effect [72-74]. A methanol extract of M. oleifera leaves conferred significant

radiation protection to the bone marrow chromosomes in mice [72]. Moringa leaves are

effective for the regulation of thyroid hormone status [74]. A recent report showed that M.

oleifera leaf may be applicable as a prophylactic or therapeutic anti-HSV [Herpes simplex

virus type 1] medicine and may be effective against the acyclovir-resistant variant [75]. The

flowers and leaves also are considered to be of high medicinal value with antihelmintic

activity [76]. An infusion of leaf juice was shown to reduce glucose levels in rabbits [77]. M.

oleifera is coming to the forefront as a result of scientific evidence that Moringa is an

important source of naturally occurring phytochemicals and this provides a basis for future

viable developments. Different parts of M. oleifera are also incorporated in Kumar et al. [70]

various marketed health formulations. The seeds have specific protein fractions for skin and

hair care. Two new active components for the cosmetic industry have been extracted from oil

cake Purisoft® consists of peptides of the Moringa seed. It protects the human skin from

environmental influences and combats premature skin aging. With dual activity, antipollution

and conditioning/strengthening of hair, the M. oleifera seed extract is a globally acceptable

innovative solution for hair care [77, 78].

16

1.11 Non- pharmacological uses

Moringa oleifera possesses a multitude of non – pharmacological uses as well. The

defatted seed meal is an excellent additive in sheep diet as it is reported to improve rumen

fermentation [79]. Milk production in cows was found to increase on administration of

Moringa as a protein supplement with low quality diets [80].

Biodiesel derived from M. oleifera oil by alkali-catalyzed transesterification with

methanol is reported to be an acceptable substitute for petrodiesel. Its cetane number was

found to be [81], the highest reported for a biodiesel fuel with much better oxidative stability

[82, 83].

The seeds serve as one of the best natural coagulants for water treatment and a cheap and

feasible alternate to the synthetic ones. The seed extract is an effective natural clarification

agent for highly turbid and untreated pathogenic surface water [84-86].

Ben oil, a non drying oil obtained from the seeds is employed in the manufacture of

perfumes, hairdressings etc and as a lubricant for fine machinery. As it is resistant to

rancidity, it is extensively used in the ‘enfleurage’ process whereby delicate fragrances are

extracted from flower petals [87]. The chemical properties of protein fraction of M. oleifera

permit their use in a wide variety of skin care, hair care and cosmetic formulations such as

purisoft, puricare etc. [88].

Shelled moringa seeds possess potential to eliminate toxic metals such as cadmium from

water resources. The sorption was found to occur due to amino acid-Cd interactions, as

revealed by Fourier transform infrared spectrometry [89]. The bark too has an excellent bio-

sorbent property for removal of heavy metal ions from waste water or effluents [90].

Similar investigations have revealed the removal of zinc ions and sodium lauryl sulphate (up

to 80 %) from aqueous solutions [91, 92].

17

1.12 Phytochemistry

Moringa oleifera is rich in compounds containing the simple sugar, rhamnose and a

fairly unique group of compounds called glucosinolates and isothiocyanates [92-95]. The

stem bark has been reported to contain two alkaloids, namely moringine and moringinine

[96]. Vanillin, β-sitosterol [97], sitostenone, 4-hydroxymellin and octacosanoic acid have

been isolated from the stem of M. oleifera [98]. Purified, whole-gum exudate from M.

oleifera has been found to contain L-arabinose, galactose, glucuronic acid, and L-rhamnose,

mannose and xylose, while a homogeneous, degraded-gum polysaccharide consisting of L-

arabinose, D-galactose, D-glucuronic acid, L-rhamnose, D-mannose has been obtained on

mild hydrolysis of the whole gum with acid. Flowers contain nine amino acids, sucrose, D-

glucose, traces of alkaloids, wax, quercetin and kaempferat; the ash is rich in potassium and

calcium. They have also been reported to contain some flavonoid pigments such as alkaloids,

kaempherol, rhamnetin, isoquercitrin and kaempferitrin [98-102]. Antihypertensive

compounds thiocarbamate and isothiocyanate glycosides have been isolated from the acetate

phase of the ethanol extract of moringa pods [103]. The cytokinins have been shown to be

present in the fruit. A new O-ethyl-4-(ά-L-rhamnosyloxy)benzyl carbamate together with

seven known bioactive compounds, 4(ά-L-rhamnosyloxy)-benzyl isothiocyanate3,

niazimicin4, 3-O-(6′-O-oleoyl-D-glucopyranosyl)-β sitosterol [103,104], β-sitosterol-3-O-D-

glucopyranoside, niazirin, β-sitosterol and glycerol-1-(9-octadecanoate)have been isolated

from the ethanol extract of the Moringa seed [105]. Lately, interest has been generated in

isolating hormones/growth promoters from the leaves of M. oleifera. Nodulation of black-

gram (Vigna munga L.) has been shown to increase vigorously with the application of an

aqueous-ethanol extract of M. oleifera leaves, although the nature of the active ingredient is

still unknown. Moringa leaves act as a good source of natural antioxidant due to the presence

of various types of antioxidant compounds such as ascorbic acid, flavonoids, phenolics and

18

carotenoids [105-110]. The high concentrations of ascorbic acid, oestrogenic substances and

β-sitosterol [111], iron, calcium, phosphorus, copper, vitamins A, B and C, α-tocopherol,

riboflavin, nicotinic acid, folic acid, pyridoxine, β-carotene, protein, and in particular

essential amino acids such as methionine, cystine, tryptophan and lysine present in Moringa

leaves and pods make it a virtually ideal dietary supplement [112-114].

The composition of the sterols of Moringa seed oil mainly consists of campesterol,

stigmasterol, β-sitosterol, avenasterol and clerosterol accompanied by minute amounts of 24

methylenecholesterol, campestanol, stigmastanol and isoavenasterol. The sterol composition

of the major fractions of Moringa seed oil differs greatly from those of most of the

conventional edible oils. The fatty acid composition of M. oleifera seed oil reveals that it falls

in the category of high oleic oils (67.90 % –76.00 %) among the other component fatty acids.

Moringa oleifera is also a good source of different tocopherols the concentration of those is

reported to be 98.82–134.42, 27.90–93.70 and 48.00– 71.16 mg/kg, respectively [115-118].

Shanker et al. [119] isolated nitrile glycosides (niaziridin & niazirin) from the leaves, pods

and bark of Moringa oleifera by reverse phase HPLC. Forty four compounds from the

essential oil isolated from the leaves of Moringa oleifera by GC-MS analysis [120].

Yammuenart et al. [121] isolated seven compounds, β-sitosterol-3-O-β-D-glucopyranoside,

β-sitosterol, linoleic sitosteroate, linoleic acid, 1,2,3-triolein, a mixture of 1,3-dilinoleoyl-2-

olein, 1,3-dioleoyl-2-linolein and 1,2,3-trilinolein and isothiocyanatomethylbenzene from the

ethylene chloride extract of Moringa oleifera [122].

Sashidhara et al [123] from the roots of Moringa oleifera isolated and characterized

aurantiamide acetate 4 and 1, 3-dibenzyl urea. Both these compounds were isolated for the

first time from this genus [123]. Ogunbinu et al. [124] isolated monoterpenoid compounds

(81.8 %) from the essential oil of Moringa oleifera extracted by hydrodistillation and

analysed by GC and GC-MS. The oil consists of alpha-phellandrene with highest percentage

19

(25.2 %) along with p-cymene (24.9%) [125]. Singh et al. [126] reported presence of gallic

acid, chlorogenic acid, ellagic acid, ferulic acid, kaempferol, quercetin and vanillin from the

aqueous extracts of leaves, fruits and seeds of Moringa oliefera. All compounds were

analyzed by HPLC and MS/MS techniques. Verma et al. [127-128] reported presence of

phenolic acids like gallic acid, chlorogenic acid, ellagic acid, ferulic acid and flavonoids like

kaempferol, quercetin and rutin from the leaves of Moringa oleifera by HPLC techniques.

Makkar and Becker [129] investigated various phytochemicals present in the leaves of

ethanolic extract of Moringa oleifera by GC-MS. The leaves contain fifteen components. The

major compounds were hexadecanoic acid, ethyl palmitate, palmitic acid ethyl ester, 2, 6-

dimethyl-1, 7-octadiene-3-ol, 4-hexadecen-6-yne, 2-hexanone, 3-cyclohexyliden-4-ethyl -

E2-dodecenylacetate, hi-oleic safflower oil. The major compounds from the seeds were

roridin E, veridiflorol, 9-octadecenoic acid. Presnt in the flowers were, 9-octadecen –1- ol,

cis - 9 – octadecen – 1 –ol, oleol, satol, ocenol, sipo, decanoic acid, dodecanal were identified

as major compounds [130-135].

The seeds also contain Moringyne, 4-(α-L-rhamnosyloxy) benzyl isothiocyanate &

several amino acids [135-138]. The roots also contain benzyl isothiocyanate. The plant also

contains antibacterial principles such as spirochin and pterygospermin, which are effective

against both gram negative and gram positive bacteria [139]. The gum contains

aldotriouronic acid which is obtained from the acid hydrolysis of gum and is characterized as

O-(β-D-glucopyranosyluronic acid) (1→6)-β-D-galactopyranosyl (1→6)-D-galactose. The

leaves contain aspartic acid, glutamic acid, glycine, threonine, alanine, valine, leucine,

isoleucine, histidine, lysine, phenylalanine, tryptophan, cysteine and methionine [139-143].

The stem contains 4-hydroxy mellein, vanillin, octacosonoic acid, β-sitosterol and β-

sitosterone [144] & Kaempferol-3-rutinoside was identified in flowers [145].

20

1.13 Bacteriology of staphylococci

Bacteria in the genus Staphylococcus are pathogens of man and other mammals [146].

Traditionally they were divided into two groups on the basis of their ability to clot blood

plasma (the coagulase reaction). The coagulase-positive staphylococci constitute the most

pathogenic species S aureus. The coagulase-negative staphylococci (CNS) are now known to

comprise over 30 other species [147]. The CNS are common commensals of skin, although

some species can cause infections. It is now obvious that the division of staphylococci into

coagulase positive and negative is artificial and indeed, misleading in some cases. Coagulase

is a marker for S aureus but there is no direct evidence that it is a virulence factor. Some

natural isolates of S. aureus are defective in coagulase. Nevertheless, the term is still in

widespread use among clinical microbiologists [147].

Staphylococcus aureus expresses a variety of extracellular proteins and

polysaccharides, some of which are correlated with virulence. Virulence results from the

combined effect of many factors expressed during infection [146]. Antibodies will neutralize

staphylococcal toxins and enzymes, but vaccines are not available. Both antibiotic treatment

and surgical drainage are often necessary to cure abscesses, large boils and wound infections

[147,148]. Staphylococci are common causes of infections associated with indwelling

medical devices. These are difficult to treat with antibiotics alone and often require removal

of the device [149]. Some strains that infect hospitalized patients are resistant to most of the

antibiotics used to treat infections; vancomycin being the only remaining drug of choice has

started to have resistance to some new resistant strains [150-153].

1.13.1 Taxonomy

DNA-ribosomal RNA (rRNA) hybridization and comparative oligonucleotide

analysis of 16S rRNA has demonstrated that staphylococci form a coherent group at the

21

genus level. This group occurs within the broad bacillus-lactobacillus-streptococcus cluster

defining Gram-positive bacteria with a low G + C content of DNA [152].

At least 30 species of staphylococci have been recognized by biochemical analysis

and in particular by DNA-DNA hybridization. Eleven of these can be isolated from humans

as commensals [153]. Staphylococcus aureus (snares) and S. epidermidis (snares, skin) are

common commensals and also have the greatest pathogenic potential. S. saprophyticus (skin)

is also a common cause of urinary tract infection. Staphylococcus haemolyticus, S. simulans,

S. cohnii, S. warneri and S. lugdunensis can also cause infections in man [154].

1.15.1 Identification of staphylococci in the clinical laboratory

(a) Isolation

The presence of staphylococci in a lesion might first be suspected after examination

of a direct Gram stain. However, small numbers of bacteria in blood preclude microscopic

examination and require culturing first [155-158].

The organism is isolated by streaking material from the clinical specimen (or from a

blood culture) onto solid media such as blood agar, triptich soy agar or heart infusion agar.

Specimens likely to be contaminated with other microorganisms can be plated on mannitol

salt agar containing 7.5% sodium chloride, which allows the halo-tolerant staphylococci to

grow [158].

Ideally a gram stain of the colony should be performed and tests made for catalase and

coagulase production, allowing the coagulase-positive S. aureus to be identified quickly

[159]. Another very useful test for S. aureus is the production of thermostable

deoxyribonuclease. Staphylococcus aureus can be confirmed by testing colonies for

22

agglutination with latex particles coated with immunoglobulin G and fibrinogen which bind

protein A and the clumping factor, respectively, on the bacterial cell surface [160]. These are

available from commercial suppliers (e.g., Staphaurex). The most recent latex test (Pastaurex)

incorporates monoclonal antibodies to serotype 5 and 8 capsular polysaccharide in order to

reduce the number of false negatives. (Some recent clinical isolates of S. aureus lack

production of coagulase and/or clumping factor, which can make identification difficult.)

[161].

The association of S. epidermidis (and to a lesser extent of other coagulase-negative

staphylococci) with nosocomial infections associated with indwelling devices means that

isolation of these bacteria from blood is likely to be important and not due to chance

contamination, particularly if successive blood cultures are positive. Nowadays, identification

of S epidermidis and other species of Staphylococcus is performed using commercial biotype

identification kits, such as API Staph Ident, API Staph-Trac, Vitek GPI Card and Microscan

Pos Combo. These comprise preformed strips containing test substrates [161-163].

(b) Structure

Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow in

clusters, pairs and occasionally in short chains [155]. The clusters arise because staphylococci

divide in two planes. The configuration of the cocci helps to distinguish micrococci and

staphylococci from streptococci, which usually grow in chains. Observations must be made

on cultures grown in broth, because streptococci grown on solid medium may appear as

clumps. Several fields should be examined before deciding whether clumps or chains are

present [156].

23

(c) Catalase Test

The catalase test is important in distinguishing streptococci (catalase-negative)

staphylococci which are catalase positive. The test is performed by flooding an agar slant or

broth culture with several drops of 3% hydrogen peroxide. Catalase-positive cultures bubble

at once. The test should not be done on blood agar because blood itself will produce bubbles

[157].

1.13.3 Epidemiology of Staphylococcus aureus infections

Staphylococcus aureus is a major cause of nosocomial and community-acquired

infections, it is necessary to determine the relatedness of isolates collected during the

investigation of an outbreak [162]. Typing systems must be reproducible, discriminatory,

easy to interpret and to use. The traditional method for typing S. aureus is phage-typing. This

method is based on a phenotypic marker with poor reproducibility. It does not type many

isolates (20% in a recent survey at the Center for Disease Control and Prevention), and it

requires maintenance of a large number of phage stocks and propagating strains and

consequently can be performed only by specialist reference laboratories [163].

Many molecular typing methods have been applied to the epidemiological analysis of S

aureus, in particular, of methicillin-resistant strains (MRSA). Plasmid analysis has been used

extensively with success, but suffers the disadvantage that plasmids can easily be lost and

acquired and is thus inherently unreliable [164]. Methods designed to recognize restriction

fragment length polymorphisms (RFLP) using a variety of gene probes, including rRNA

genes (ribotyping), have had limited success in the epidemiology of MRSA [165]. In this

technique the choice of restriction enzyme used to cleave the genomic DNA, as well as the

probes, is crucial. Random primer PCR offers potential for discriminating between strains but

24

a suitable primer has yet to be identified for S. aureus. The method currently regarded as the

most reliable is pulsed field gel electrophoresis, where genomic DNA is cut with a restriction

enzyme that generates large fragments of 50-700 kb [166].

1.13.4 Clinical manifestations of S. aureus

Staphylococcus aureus is notorious for causing boils, furuncles, styes, impetigo and

other superficial skin infections in humans. It may also cause more serious infections,

particularly in persons debilitated by chronic illness, traumatic injury, burns or

immunosuppression [167]. These infections include pneumonia, deep abscesses,

osteomyelitis, endocarditis, phlebitis, mastitis and meningitis, and are often associated with

hospitalized patients rather than healthy individuals in the community. Staphylococcus

aureus and S. epidermidis are common causes of infections associated with indwelling

devices such as joint prostheses, cardiovascular devices and artificial heart valves [168].

1.13.5 Pathogenesis of S. aureus infections

Staphylococcus aureus expresses many cell surface-associated and extracellular

proteins that are potential virulence factors. For the majority of diseases caused by this

organism, pathogenesis is multifactorial. Thus, it is difficult to determine precisely the role of

any given factor. This also reflects the inadequacies of many animal models for

staphylococcal diseases [169].

However, there are correlations between strains isolated from particular diseases and

expression of particular factors, which suggests their importance in pathogenesis. With some

toxins, symptoms of a human disease can be reproduced in animals with pure proteins. The

application of molecular biology has led to recent advances in the understanding of

pathogenesis of staphylococcal diseases [170]. Genes encoding potential virulence factors

25

have been cloned and sequenced and proteins purified. This has facilitated studies at the

molecular level on their modes of action, both in in vitro and in model systems. In addition,

genes encoding putative virulence factors have been inactivated, and the virulence of the

mutants compared to the wild-type strain in animal models. Any diminution in virulence

implicates the missing factor. If virulence is restored when the gene is returned to the mutant

then “Molecular Koch's Postulates” have been fulfilled. Several virulence factors of S aureus

have been confirmed by this approach [171].

1.13.6 Infections associated with medical devices

Infections associated with indwelling medical devices ranging from simple

intravenous catheters to prosthetic joints and replacement heart valves can be caused by S

aureus and S epidermidis. Very shortly after biomaterial is implanted in the human body it

becomes coated with a complex mixture of host proteins and platelets [174]. In one model

system involving short-term contact between biomaterial and blood, fibrinogen was shown to

be the dominant component and was primarily responsible for adherence of S aureus in

subsequent in vitro assays. In contrast, with material that has been in the body for longer

periods (e.g human intravenous catheters) the fibrinogen is degraded and no longer promotes

bacterial attachment. Instead, fibronectin, which remains intact, becomes the predominant

ligand promoting attachment [176].

1.13.7 Virulence factors of Staphylococcus aureus.

The receptor which promotes attachment to collagen is particularly associated with

strains that cause osteomyelitis and septic arthritis. Interaction with collagen may also be

important in promoting bacterial attachment to damaged tissue where the underlying layers

have been exposed [172].

26

Evidence that these staphylococcal matrix-binding proteins are virulence factors has

come from studying defective mutants in vitro adherence assays and in experimental

infections. Mutants defective in binding to fibronectin and to fibrinogen have reduced

virulence in a rat model for endocarditis, suggesting that bacterial attachment to the sterile

vegetations caused by damaging the endothelial surface of the heart valve is promoted by

fibronectin and fibrinogen. Similarly, mutants lacking the collagen-binding protein have

reduced virulence in a mouse model for septic arthritis. Furthermore, the soluble ligand-

binding domain of the fibrinogen, fibronectin and collagen-binding proteins expressed by

recombinant methods strongly blocks interactions of bacterial cells with the corresponding

host protein [173-175].

(a) Adherence to endothelial cells

Staphylococcus aureus can adhere to the surface of cultured human endothelial cells

and become internalized by a phagocytosis-like process [177]. It is not clear if attachment

involves a novel receptor or a known surface protein of S aureus. Some researchers think that

S. aureus can initiate endocarditis by attaching to the undamaged endothelium. Others feel

that trauma of even a very minor nature is required to promote attachment of bacteria [178].

(b) Avoidance of host defenses

Staphylococcus aureus expresses a number of factors that have the potential to interfere with

host defense mechanisms. However, strong evidence for a role in virulence of these factors is

lacking [176].

27

(c) Capsular polysaccharide

The majority of clinical isolates of S. aureus express a surface polysaccharide of

either serotype 5 or 8. This has been called a microcapsule because it can be visualized only

by electron microscopy after antibody labeling [177], unlike the copious capsules of other

bacteria which are visualized by light microscopy. S. aureus isolated from infections

expresses high levels of polysaccharide but rapidly loses it upon laboratory subculture [179].

The function of the capsule is not clear, it may impede phagocytosis, but in in vitro tests this

was only demonstrated in the absence of complement. Conversely, comparing wild-type and

a capsule defective mutant strain in an endocarditis model suggested that polysaccharide

expression actually impeded colonization of damaged heart valves, perhaps by masking

adhesins [180].

(d) Protein A

Protein A is a surface protein of S. aureus which binds immunoglobulin G molecules

by the Fc region. In serum, bacteria will bind IgG molecules the wrong way round by this

non-immune mechanism. In principle this will disrupt opsonization and phagocytosis. Indeed

mutants of S aureus lacking protein A are more efficiently phagocytozed in vitro, and studies

with mutants in infection models suggest that protein A enhances virulence [181].

(e) Leukocidin

Staphyloccocus aureus can express a toxin that specifically acts on

polymorphonuclear leukocytes. Phagocytosis is an important defense against staphylococcal

infection so leukocidin should be a virulence factor. This toxin is discussed in more detail in

the next section [182].

28

(f) Damage to the host

Staphylococcus aureus can express several different types of protein toxins which are

probably responsible for symptoms during infections. Some damage the membranes of

erythrocytes, causing hemolysis; but it is unlikely that hemolysis is relevant in vivo [183].

The leukocidin causes membrane damage to leukocytes and is not hemolytic. Systemic

release of α-toxin causes septic shock, while enterotoxins and TSST-1 cause toxic shock

[184].

1.13.8 membrane damaging toxins

(a) α-toxin

The best characterized and most potent membrane-damaging toxin of S. aureus is α-

toxin. It is expressed as a monomer that binds to the membrane of susceptible cells. Subunits

then oligomerize to form hexameric rings with a central pore through which cellular contents

leak [185]. Susceptible cells have a specific receptor for α-toxin which allows low

concentrations of toxin to bind, causing small pores through which monovalent cations can

pass. At higher concentrations, the toxin reacts non-specifically with membrane lipids,

causing larger pores through which divalent cations and small molecules can pass. However,

it is doubtful if this is relevant under normal physiological conditions [184-186].

In humans, platelets and monocytes are particularly sensitive to α-toxin. They carry high

affinity sites which allow toxin to bind at concentrations that are physiologically relevant. A

complex series of secondary reactions ensue, causing release of eicosanoids and cytokines

which trigger production of inflammatory mediators. These events cause the symptoms of

septic shock that occur during severe infections caused by S. aureus [187]. The notion that α-

toxin is a major virulence factor of S. aureus is supported by studies with the purified toxin in

29

animals and in organ culture. Also, mutants lacking α-toxin are less virulent in a variety of

animal infection models [187].

(b) β-toxin

β -toxin is a sphingomyelinase which damages membranes rich in this lipid. The classical

test for β-toxin is lysis of sheep erythrocytes. The majority of human isolates of S. aureus do

not express β-toxin. A lysogenic bacteriophage is inserted into the gene that encodes the

toxin. This phenomenon is called negative phage conversion. Some of the phages that

inactivate the β-toxin gene carry the determinant for an enterotoxin and staphylokinase. In

contrast the majority of isolates from bovine mastitis express β-toxin, suggesting that the

toxin is important in the pathogenesis of mastitis. This is supported by the fact that β-toxin-

deficient mutants have reduced virulence in a mouse model for mastitis [187-190].

(c) δ-toxin

The δ-toxin is a very small peptide toxin produced by most strains of S. aureus. It is also

produced by S. epidermidis and S. lugdunensis. The role of δ-toxin in disease is unknown

[188].

(d) γ-toxin and leukocidin

The γ-toxin and the leukocidins are two-component protein toxins that damage

membranes of susceptible cells. The proteins are expressed separately but act together to

damage membranes. There is no evidence that they form multimers prior to insertion into

membranes. The γ-toxin locus expresses three proteins. The B and C components form a

leukotoxin with poor hemolytic activity, whereas the A and B components are hemolytic and

weakly leukotoxic [189]. The classical Panton and Valentine (PV) leukocidin is distinct from

30

the leukotoxin expressed by the γ-toxin locus. It has potent leukotoxicity and, in contrast to γ-

toxin, is non-hemolytic. Only a small fraction of S. aureus isolates (2% in one survey)

express the PV leukocidin [190], whereas 90% of those isolated from severe dermonecrotic

lesions express this toxin. This suggests that PV leukocidin is an important factor in

necrotizing skin infections [190].

PV-leukocidin causes dermonecrosis when injected subcutaneously in rabbits.

Furthermore, at a concentration below that causing membrane damage, the toxin releases

inflammatory mediators from human neutrophils, leading to degranulation. This could

account for the histology of dermonecrotic infections (vasodilation, infiltration and central

necrosis) [189-190].

(e) Super antigens: enterotoxins and toxic shock syndrome toxin (TSST)

Staphylococcus aureus can express two different types of toxin with superantigen

activity, enterotoxins, of which there are six serotypes (A, B, C, D, E and G) and toxic shock

syndrome toxin (TSST-1) [191]. Enterotoxins cause diarrhea and vomiting when ingested and

are responsible for staphylococcal food poisoning. When expressed systemically,

enterotoxins can cause toxic shock syndrome (TSS) - indeed enterotoxins B and C cause 50%

of non-menstrual TSS [192]. TSST-1 is very weakly related to enterotoxins and does not

have emetic activity. TSST-1 is responsible for 75% of TSS, including all menstrual cases.

TSS can occur as a sequel to any staphylococcal infection if an enterotoxin or TSST-1 is

released systemically and the host lacks appropriate neutralizing antibodies [193]. Tampon-

associated TSS is not a true infection, being caused by growth of S aureus in a tampon and

absorption of the toxin into the blood stream. TSS came to prominence with the introduction

of super-absorbent tampons; and although the number of such cases has decreased

31

dramatically, they still occur despite withdrawal of certain types of tampons from the market

[194].

Super antigens stimulate T cells non-specifically without normal antigenic

recognition. Up to one in five T cells may be activated, whereas only 1 in 10,000 are

stimulated during antigen presentation. Cytokines are released in large amounts, causing the

symptoms of TSS. Superantigens bind directly to class II major histocompatibility complexes

of antigen-presenting cells outside the conventional antigen-binding grove. This complex

recognizes only the Vβ element of the T cell receptor. Thus any T cell with the appropriate

Vβ element can be stimulated, whereas normally antigen specificity is also required in

binding super antigens and the non-specific stimulation of T cells [194-196].

(f) Epidermolytic (exfoliative) toxin (ET)

This toxin causes the scalded skin syndrome in neonates, with widespread blistering and

loss of the epidermis. There are two antigenically distinct forms of the toxin, ETA and ETB

[197]. There is evidence that these toxins have protease activity. Both toxins have a sequence

similarity with the S. aureus serine protease, and the three most important amino acids in the

active site of the protease are conserved. Furthermore, changing the active site of serine to a

glycine completely eliminated toxin activity [198]. However, ETs do not have discernible

proteolytic activity but they do have esterase activity. It is not clear how the latter causes

epidermal splitting. It is possible that the toxins target a very specific protein which is

involved in maintaining the integrity of the epidermis [199].

32

1.13.9 Other extracellular proteins

(a) Coagulase

Coagulase is not an enzyme. It is an extracellular protein which binds to prothrombin

in the host to form a complex called staphylothrombin [200]. The protease activity

characteristic of thrombin is activated in the complex, resulting in the conversion of

fibrinogen to fibrin. This is the basis of the tube coagulase test, in which a clot is formed in

plasma after incubation with the S. aureus broth-culture supernatant [201]. Coagulase is a

traditional marker for identifying S. aureus in the clinical microbiology laboratory. However,

there is no evidence that it is a virulence factor, although it is reasonable to speculate that the

bacteria could protect themselves from host defenses by causing localized clotting. Notably,

coagulase deficient mutants have been tested in several infection models but no differences

from the parent strain were observed [202].

There is some confusion in the literature concerning coagulase and clumping factor,

the fibrinogen-binding determinant on the S aureus cell surface [200]. This is partly due to

loose terminology, with the clumping factor sometimes being referred to as bound coagulase.

Also, although coagulase is regarded as an extracellular protein, a small fraction is tightly

bound on the bacterial cell surface where it can react with prothrombin [202]. Finally, it has

recently been shown that the coagulase can bind fibrinogen as well as thrombin, at least when

it is extracellular [203]. Genetic studies have shown unequivocally that coagulase and

clumping factor are distinct entities. Specific mutants lacking coagulase retain clumping

factor activity, while clumping factor mutants express coagulase normally [203].

33

(b) Staphylokinase

Many strains of S. aureus express a plasminogen activator called staphylokinase. The

genetic determinant is associated with lysogenic bacteriophages [204]. A complex formed

between staphylokinase and plasminogen activates plasmin-like proteolytic activity which

causes dissolution of fibrin clots. The mechanism is identical to streptokinase, which is used

in medicine to treat patients suffering from coronary thrombosis [205]. As with coagulase

there is no evidence that staphylokinase is a virulence factor, although it seems reasonable to

imagine that localized fibrinolysis might aid in bacterial spreading [206].

(c) Enzymes

Staphylococcus aureus can express proteases, a lipase, a deoxyribonuclease (DNase)

and a fatty acid modifying enzyme (FAME). The first three probably provide nutrients for the

bacteria, and it is unlikely that they have anything but a minor role in pathogenesis. However,

the FAME enzyme may be important in abscesses, where it could modify anti-bacterial lipids

and prolong bacterial survival. The thermostable DNase is an important diagnostic test for

identification of S aureus. [204]

1.14 Coagulase negative staphylococci (CNS)

Staphylococci other than S. aureus can cause infections in man. S epidermidis is the

most important coagulase-negative staphylococcus (CNS) species and is the major cause of

infections associated with prosthetic devices and catheters [207]. CNS also causes peritonitis

in patients receiving continuous ambulatory peritoneal dialysis and endocarditis in those with

prosthetic valves. These infections are not usually nosocomially acquired. Other species such

as S. haemolyticus, S. warneri, S. hominis, S. capitis, S. intermedius, S. schleiferi and S.

simulans are infrequent pathogens. S. lugdunesis is a newly recognized species. It is probably

34

more pathogenic than are other CNS species, with cases of endocarditis and other infections

being reported. It is likely that the incidence of infections caused by these organisms is

underestimated because of difficulties in identification [205-208]. Diagnosis of CNS

infections is difficult. Infections are often indolent and chronic with few obvious symptoms.

This is due to the smaller array of virulence factors and toxins compared to those in the case

of S aureus. S epidermidis is a skin commensal and is one of the most common contaminants

of samples sent to the diagnostic laboratory, while S lugdunensis is often confused with S

aureus. Precise identification of CNS species requires the use of expensive test kits, such as

the API-Staph [209].

In contrast to S. aureus, little is known about mechanisms of pathogenesis of S.

epidermidis infections. Adherence is obviously a crucial step in the initiation of foreign body

infections [210]. Much research has been done on the interaction between S. epidermidis and

plastic material used in implants, and a polysaccharide adhesion (PS/A) has been identified

[211]. Mutants lacking PS/A are less virulent in an animal model for foreign body infection,

and immunization with purified PS/A is protective. Bacteria-plastic interactions are probably

important in colonization of catheters through the point of entry [212]. However, host

proteins are quickly deposited on implants. S. epidermidis does not bind to fibrinogen but

most isolates bind fibronectin, albeit less avidly than S. aureus. However, it is not known if a

protein analogous to the fibronectin binding protein of S. aureus is involved [213].

A characteristic of clinical isolates of S. epidermidis is the production of “slime.” This is

a controversial topic. Some feel that slime is an in vitro manifestation of the ability to form a

biofilm in vivo, for example on the surface of a prosthetic device, and is thus a virulence

marker [214]. In vitro, slime is formed during growth in broth as a biofilm on the surface of

the growth vessel. The composition of this slime is probably influenced by the growth

35

medium. One study with defined medium showed that the slime was predominantly secreted

teichoic acid, a polymer normally found in the cell wall of staphylococci. Some

polysaccharides in slime from bacteria grown on solid medium are derived from the agar

[213-215].

1.15 Antimicrobial resistance

Biological resistance refers to changes that result in the organism being less

susceptible to a particular antimicrobial agent than has been previously observed. When

antimicrobial susceptibility has been lost to such an extent that the drug is no longer effective

for clinical use, the organism is then said to have achieved clinical resistance. It is important

to note that often, biologic resistance and clinical resistance do not necessarily coincide.

From a clinical laboratory and public health perspective it is important to realize that biologic

development of antimicrobial resistance is an ongoing process, while clinical resistance is

dependent on current laboratory methods and established cut-offs. Our inability to reliably

detect all these processes with current laboratory procedures and criteria should not be

perceived as evidence that they are not occurring [216].

Many plasmid-encoded determinants have recently become inserted into the

chromosome at a site associated with the methicillin resistance determinant. There may be an

advantage to the organism having resistance determinants in the chromosome because they

will be more stable.There are essentially four mechanisms of resistance to antibiotics in

bacteria: (1) enzymatic inactivation of the drug, (2) alterations to the drug target to prevent

binding, (3) accelerated drug efflux to prevent toxic concentrations accumulating in the cell,

and (4) a by-pass mechanism whereby an alternative drug-resistant version of the target is

expressed [215].

36

Hospital strains of S. aureus are often resistant to many different antibiotics. Indeed

strains resistant to all clinically useful drugs, apart from the glycopeptides vancomycin and

teicoplanin, have been described [214-216]. The term MRSA refers to methicillin resistant

staphylococcus aureus. Plasmid-associated vancomycin resistance has been detected in some

enterococci and the resistance determinant has been transferred from enterococci to S. aureus

in the laboratory and may occur naturally [217]. Staphylococcus epidermidis nosocomial

isolates are also often resistant to several antibiotics including methicillin. In addition, S.

aureus expresses resistance to antiseptics and disinfectants, such as quaternary ammonium

compounds, which may aid its survival in the hospital environment [218]. Since the

beginning of the antibiotic era S. aureus has responded to the introduction of new drugs by

rapidly acquiring resistance by a variety of genetic mechanisms including (1) acquisition of

extrachromosomal plasmids or additional genetic information in the chromosome via

transposons or other types of DNA insertion and (2) by mutations in chromosomal genes

[219].

1.15.1 Mechanism of bacterial resistance

The history of human kind can be regarded from a medical point of view as a struggle

against infectious diseases. Infections were the leading cause of death worldwide at the

beginning of the 20th century. Since the discovery of penicillin by Alexander Fleming in

1929 and the first introduction of the sulpha drugs by Domagk in 1932, the number of new

antimicrobials available has increased tremendously between 1940 and 1960. ‘The era of

antibiotics’ led to optimism till the early 1970s that infectious diseases can be controlled and

prevented and mankind felt confident that modern medicine would prevail. However,

infections are still the second-leading cause of death worldwide, causing over 13 million

deaths each year. This fact is the result of the emergence of new diseases, the re-emergence

37

of diseases once controlled and more specifically of the development of antimicrobial

resistance [220-222].

Bacteria have a remarkable ability to adapt to adverse environmental conditions,

which is an example of the ancient law of nature of ‘survival of the fittest’. It appears that the

emergence of antimicrobial resistant bacteria is inevitable to most every new drug and it is

recognized as a major problem in the treatment of microbial infections in hospitals and in the

community [223].

1.15.2 Intrinsic resistance

Bacteria may be inherently resistant to an antimicrobial. This passive resistance is a

consequence of general adaptive processes that are not necessary linked to a given class of

antimicrobials. An example of natural resistance is Pseudomonas aeruginosa, whose low

membrane permeability is likely to be a main reason for its innate resistance to many

antimicrobials [224]. Other examples are the presence of genes affording resistance to self-

produced antibiotics, the outer membrane of Gram-negative bacteria, absence of an uptake

transport system for the antimicrobial or general absence of the target or reaction hit by the

antimicrobial [225].

1.15.3 Acquired resistance

Active resistance, the major mechanism of antimicrobial resistance, is the result of a

specific evolutionary pressure to develop a counterattack mechanism against an antimicrobial

or class of antimicrobials so that bacterial populations previously sensitive to antimicrobials

become resistant [226]. This type of resistance results from changes in the bacterial genome.

Resistance in bacteria may be acquired by a mutation and passed vertically by selection to

daughter cells. More commonly, resistance is acquired by horizontal transfer of resistance

genes between strains and species. Exchange of genes is possible by transformation,

transduction or conjugation [227].

38

The major mechanisms of active antimicrobial resistance (Figure 1) are (1) prevention

of accumulation of antimicrobials either by decreasing uptake or increasing efflux of the

antimicrobial from the cell via a collection of membrane-associated pumping proteins, (2)

qualitative drug target site alteration, which reduces the affinity for antimicrobials either by

mutation or by target modification, or quantitative drug target alteration by overproduction of

the target and (3) inactivation of antibiotics either by hydrolysis or by modification [226-

228].

1.15.2 Prevention of antimicrobial access to their targets

(a) Permeability barriers

The cytoplasmic membrane is a barrier to hydrophilic compounds. Entry of cytoplasmatically

targeted compounds is usually through carrier-mediated transport mechanisms or via

channels in the outer membrane of Gram-negative bacteria formed by porins (e.g. OprD

porin). Antibacterial compounds transported in this way may be subject to resistance by loss

of non-essential transporters, by lack of porins or by mutations that are able to modify the

structure of these channels and thus decreasing the influx [229]. Some microbes possess

impermeable cell membranes that prevent drug influx as exemplified by P. aeruginosa.

Furthermore, many large molecule antimicrobials are naturally inactive against certain groups

of bacteria because they simply cannot pass into the bacterial cell [230].

(b) Efflux pumps

Increasing the efflux also plays a role, especially with hydrophobic compounds that

presumably enter the cell via diffusion [229-231]. At the same speed where these

antimicrobials are entering the cell, efflux mechanisms are pumping them out again, before

they reach their target [232]. A mutation resulting in over expression of a multidrug efflux

pump leads to resistance to a wide variety of structurally unrelated antimicrobials [233].

39

Multidrug resistance proteins (MDRs) or multidrug efflux pumps are widespread in bacteria

[234].

(c) Alteration of drug target

Natural variations or acquired changes in the target sites of antimicrobials that prevent

drug binding or action is a common mechanism of resistance. Target site changes often result

from spontaneous mutation of a bacterial gene on the chromosome and selection in the

presence of the antimicrobial [235,236].

They are grouped into five families based on their mechanisms and primary sequence

homologies. The major facilitator super family (MFS), the resistance-nodulation-division

(RND) family, the small multidrug resistance (SMR) family and the multidrug and toxic

compounds extrusion (MATE) family are secondary transporters using either proton motive

force (PMF) or sodium ion motive force (only for the MATE proteins) to expel

antimicrobials from cells. Members of the ATP-binding cassette (ABC) super family are

primary transporters using energy liberated by ATP hydrolysis [237].

(d) Antibiotic inactivation

Some bacteria produce modifying enzymes that reside within or near the cell surface

(Figure 3), which selectively target and inactivate the drug. Enzymatic inactivation either by

hydrolysis or by modification (group transfer and redox mechanisms) is a major mechanism

of resistance to natural antibiotics in pathogenic bacteria [238]. The resistant isolates in most

cases inherit the antibiotic resistance genes on resistance (R) plasmids.

These resistance determinants are most probably acquired by pathogenic bacteria

from a pool of resistance genes in other microbial genera, including antibiotic producing

organisms. No enzymes that hydrolyse or modify manmade antimicrobials have been found

[239].

40

Fig. 3 summarises the resistance mechanisms to the main antimicrobial classes [238].

41

Antibiotic inactivation mechanisms share many similarities with well-characterized

enzymatic reactions and resistance proteins show homologies to known metabolic and

signalling enzymes with no antibiotic resistance activity.Therefore, one can speculate that

these are the original sources of resistance [240]. Either hydrolysis or group transfer

reactions, or alternatively oxidation or reduction reactions, can sign for the inactivation

mechanism.

Many antibiotics possess hydrolytically susceptible chemical bonds (e.g. esters and amides)

whose integrity is central to biological activity. When these vulnerable bonds are cleaved, the

antibiotic activity is destroyed [240]. The most diverse and largest family of resistance

enzymes is the group transferases. Those enzymes covalently modify antibiotics leading to

structural alterations that impair target binding. Chemical strategies include O-acylation and

N-acylation, O-phosphorylation, O-nucleotidylation, O-ribosylation, O-glycosylation and

thiol transfer [240]. The oxidation or reduction of antibiotics has not been frequently

exploited by pathogenic bacteria. Lyases are enzymes that cleave C-C, C-O, C-N and C-S

bonds by non-hydrolytic or non-oxidative routes. These reactions frequently result in double

bond formation or ring closure [240].

1.16 Selected antimicrobial agents according to mechanisms of action.

A key feature of the target sites for antimicrobial agents is their vital role in microbial

growth and survival [241]. Antimicrobials are usually classified on the basis of their mode of

action. The main classes of antimicrobials inhibit four classical targets (Figure 2): (1) cell

wall biosynthesis, (2) protein biosynthesis, (3) nucleid acid biosynthesis and (4) folate

biosynthesis [241,242]. Structures of some representatives for each discussed antimicrobial

class are given in Figure 4 [241,242].

42

Fig. 4 Structures of some representatives for the discussed antimicrobial classes.

43

1.16.1 Bacterial cell wall biosynthesis

Bacterial cells are surrounded by a cell wall or layers of peptidoglycan. This is a

mesh-like carbohydrate polymer, which provides the mechanical support necessary to protect

themselves from osmolysis [243]. Peptidoglycan is composed of linear chains of β-(1,4)-N-

acetyl hexosamine units joined by peptide cross-links. The peptidoglycan undergoes cross-

linking of the glycan strands by the action of transglycosidases and of the peptide strands by

the action of transpeptidases, also called penicillin-binding proteins (PBPs) [244]. Inhibitors

of one of both enzymes, active in the last stage of cell wall biosynthesis, fall into two major

classes withrespect to their mechanism of action, the β-lactams and the glycopeptides (Figure

5) [243, 244].

The cell wall (peptidoglycan matrix) surrounding bacterial cells are a mesh-like

carbohydrate polymer with glycan strands connected bypeptide cross-links.

Transglycosylases catalyse cross-linking of the glycan strands, and the peptide strands

undergo cross-linking by the action of transpeptidases. Transpeptidases are inactivated by β-

lactams and glycopeptides. Glycopeptides also inhibit transglycosylase activity. Interference

with cross-linking results in cell lysis and death. M: N-acetylmuramic acid; G: N-acetyl-D-

glucosamine [264].

i β-lactam antibiotics

The primary targets of the β-lactam agents are the PBPs. Upon nucleophilic attack on the β-

lactam ring by the side chain oxygen atom of a serine residue at the active site of the enzyme,

a relatively stable lethal covalent penicilloyl-enzyme complex is formed in which the serine

is covalently acylated by the hydrolysed β-lactam leading to inactivation of the enzyme and

blocking of the normal transpeptidation reaction [243-246]. This results in weakly cross-

linked peptidoglycan and eventually cell lysis and death [242, 243].

44

Fig. 5 Inhibitor of β-lactams and the glycopeptides

45

Resistance to β-lactam antibiotics

(a) Antibiotic inactivation.

The β-Lactamases are hydrolytic enzymes that disrupt the amide bond of the characteristic β-

lactam ring, before the antibiotic can get to the site of cell wall synthesis, rendering the

antimicrobial ineffective [246]. β-Lactamase expression is a principle mechanism of Gram-

negative resistance [246]. There are four classes of β-lactamases: three serine-dependent

enzyme classes (A, C and D) and one metal-dependent class (B) [247]. Of particular concern

are the enzymes able to target the expanded spectrum β-lactams, such as the AmpC enzymes,

the so-called extended spectrum β-lactamases (ESBL) and the carbapenemases [247].

(b) Target site alteration

There is several penicillin-binding protein (PBP)-mediated mechanisms of β-lactam

resistance, including acquisition of a ‘new’ less-sensitive enzyme, mutation of an endogenous

PBP to lessen the reaction with β-lactams (while maintaining some transpeptidase activity) or

upregulation of PBP expression [248]. PBP alteration is a principle mechanism of Gram-

positive resistance [247]. The most important example is the acquisition and expression of the

mecA gene by S. aureus, encoding a new low-affinity PBP, PBP2a (also called PBP2́). This

gene is found on a mobile element, the staphylococcal cassette chromosome mec (SCCmec),

carrying additional antibiotic (non-β-lactam) resistance genes [249]. Alterations in or

overproduction of other PBPs are also possible [250].

(c) Decreased permeability and increased efflux

Reduced outer membrane permeability to β-lactams as a result of porin loss or changes in

porin structure can promote resistance to these agents [251]. A major contribution to

antibiotic resistance in Gram-negative species is the presence of broad-specificity drug-efflux

pumps. One of the best characterized of these is the drug efflux system MexAB-OprM of P.

aeruginosa [251-253]. To overcome resistance, semisynthetic β-lactamase resistant β-lactams

46

were developed [252,253]. β-Lactamase susceptible β-lactams can be co-administered with β-

lactamase inhibitors, such as clavulanic acid, sulbactam and tazobactam [251,254]. A number

of β-lactam compounds that bind strongly to low-affinity PBPs have been designed as well as

agents that potentiate the activity of existing β-lactams against low-affinity PBP-producing

organisms [255]. Dual action hybrid antimicrobials were designed by fusing β-lactams to

other antimicrobials, harnessing the enzymatic action of β-lactamases, which on their turn

release the second antimicrobial [254 - 256].

ii Glycopeptides

Glycopeptides bind highly specific, non-covalently to the D-Ala -4-D-Ala-5- termini of the

UDP-muramylpentapeptide peptidoglycan precursors. Through this binding, the bound

glycopeptide acts as a steric impediment. The substrates are kept away from transglycosidase

(chain elongation) and transpeptidase (crosslinking). This substrate sequestration leads to the

failure of peptidoglycan cross-links, making the cell wall susceptible to osmolysis [254-257].

Whereas binding to the D-Ala-4-D-Ala-5- peptide motif is crucial for antimicrobial activity,

the mode of action is still more sophisticated and dimerization and membrane anchoring have

been suggested [256]. This stabilizes the binding to the new depsipeptide or facilitates a new

mechanism of action, namely the active site inhibition of transglycosylase activity [257].

Resistance to glycopeptides

(a) Target site alteration and impermeability

The most frequent cause of resistance in enterococci (vancyomycin resistant enterococci,

VRE) is the acquisition of one of two related gene clusters, vanA or vanB, located on

transposable elements [255]. This results in synthesis of peptidoglycan by an alternative

pathway, which produces modified peptidoglycan precursors ending in D-Ala-4-D-Lac-5- or

D-Ala-4-D-Ser-5- instead of D-Ala-4-D-Ala-5- and concomitantly eliminates precursors

ending in D-Ala-4-D-Ala-5 [258]. This causes loss of binding affinity [259]. The VanA

47

phenotype shows resistance to glycopeptide drugs, vancomycin and teicoplanin, while the

VanB phenotype is resistant to vancomycin, but remains susceptible to teicoplanin [260].

Sequestration of the agent in a modified wall structure or in the medium has been noticed.

Vancomycin resistant Staphylococcus aureus (VRSA) strains typically generate multilayered,

thickened cell walls as if more sites for stoichiometric binding of drugs are the cause of

reduced susceptibility [258-260]. The second-generation semi-synthetic lipoglycopeptides

oritavancin, telavancin and dalbavancin as well as chlorobiphenyl vancomycin analogues

retain activity against these resistant strains [260].

1.16.2 Nucleic acid biosynthesis

Two classes of antimicrobials are known to interfere with nucleic acid biosynthesis, (fluoro)

quinolones and rifamycins [241,242].

i. Quinolones

In order to fit inside the bacterium, the DNA is negatively supercoiled and is arranged

around an RNA core. The topological stress during transcription or DNA replication is

relieved and the positive supercoils are removed by a type II topoisomerase, known as DNA

gyrase, which makes double-stranded breaks in the DNA and reduces the linking number by

two [260]. Following DNA synthesis, the daughter chromosomes are unlinked by another

type II topoisomerase, topoisomerase IV, in a process called decatenation [260,261]. (Fluoro)

quinolones inhibit DNA synthesis and at higher concentrations they also inhibit RNA

synthesis [262]. They interact with the complexes formed between DNA and the DNA gyrase

or topoisomerase IV creating conformational changes that result in inhibition of the normal

enzyme activity. DNA gyrase seems to be the primary target for Gram-negative organisms,

while topoisomerase IV is the primary target in Gram-positive organisms [262-263]. There

are two steps to quinolone action: formation of bacteriostatic drug-enzyme-DNA complexes,

followed by the release of lethal doublestranded DNA breaks (Figure 6) [264].

48

Fig. 6 Inhibitors of nucleic acid biosynthesis

49

(Fluoro) quinolones and rifamycins interfere with nucleic acid biosynthesis. (Fluoro)

quinolones inhibit DNA synthesis by interacting with DNA gyrase and topoisomerase IV

resulting in inhibition of normal enzyme activity. Rifamycins act as allosteric inhibitors of the

bacterial DNA-dependent RNA polymerase resulting in inhibition of transcription [264].

1.16.3 Rifamycins, RNA transcription

Transcription is an essential process for decoding genetic information from DNA to

mRNA in all organisms. The RNA polymerase of bacteria, composed of different subunits

with a stoichiometry of α2ββ’ω to form the core enzyme, catalyses transcription [266].

Rifampicin, important in combination therapy in the treatment of Mycobacterium

tuberculosis infections, inhibits bacterial DNA-dependent RNA polymerase by binding to the

β-subunit of the enzyme, encoded by rpoB, at an allosteric site. It apparently blocks the entry

of the first nucleotide, which is necessary to activate the polymerase, thereby blocking

mRNA synthesis [266-268].

Resistance to rifamycins, RNA transcription inhibitors

(i) Antibiotic inactivation

A number of mechanisms (uncharacterised kinase, ADP-ribosyl transferase (ARR),

glycosylation, monooxygenase) can modify the hydroxyl group at position 23 of rifampicin

and presumably interfere with binding to RNA polymerase [267, 268].

(ii) Target site alteration

Resistance due to modification of the β-subunit of the enzyme through chromosomal

mutations in rpoB in M. tuberculosis arises with a high frequency [267].

1.16.4 Protein biosynthesis

Protein biosynthesis is catalysed by ribosomes and cytoplasmic factors. The bacterial

70S ribosome is composed of two ribonucleoprotein subunits, the 30S and 50S subunits

[269].

50

The smaller 30S subunit is made up of 16S rRNA and about 21 ribosomal proteins

(S1 to S21), while the larger 50S subunit consists of two RNA molecules, 5S rRNA and 23S

rRNA and over 36 ribosomal proteins (L1 to L36). The catalytic ribozyme domain of the 23S

rRNA possesses peptidyl transferase activity and catalyses peptide bond formation [270].

Antimicrobials inhibit protein biosynthesis by targeting the 30S or 50S subunit of the

bacterial ribosome (Figure 7).

1.16.4.1 Inhibitors of 30S Subunit

(a) Aminoglycosides

Aminoglycosides interact with the conserved sequences of the 16S rRNA of the 30S

subunit near the A site through hydrogen bonds. They cause misreading and premature

termination of translation of mRNA. The aberrant proteins may be inserted into the cell

membrane leading to altered permeability and further stimulation of aminoglycoside transport

[267-271].

Resistant to Aminoglycosides

(i) Antibiotic inactivation

Inactivation by covalent modification of the key hydroxyl and amine groups on the

aminoglycoside antibiotics is the most significant form of acquired resistance in both Gram-

negative and Gram-positive bacteria [272]. There are three types of

aminoglycosidemodifying enzymes (AMEs), each with many variants: aminoglycoside

acetyltransferases (AAC), aminoglycoside phosphotransferases (kinases) (APH) and

aminoglycoside adenylyltransferases (ANT) [271-273].

(ii) Target site alteration

Many aminoglycoside producing organisms express rRNA methylases

(aminoglycoside resistance family of methyltransferases), which modify the 16S rRNA

molecule at specific positions critical for the tight binding of the drug.

51

Fig. 7 The process of protein biosynthesis inhibition

52

This is highlighted by the finding of the rmtA, rmtB and armA genes [273] causing a

post transcriptional 16S rRNA methylation.

Aminoglycoside resistance can also occur by point mutations in the rrs gene,

encoding the 16S rRNA of the 30S subunit, or by mutations in the rpsL gene, encoding the

30S ribosomal protein S12.These ribosomal mutations are clinically relevant only for

streptomycin in M. tuberculosis [272-274].

(iii) Decreased permeability and increased efflux

Finally, aminoglycoside concentrations can be decreased inside a target cell by reduction of

drug uptake, activation of drug efflux pump or both [273]. The most successful approach to

combat resistance is by development of aminoglycosides that lack sites of inactivation, as

exemplified by amikacin, which is protected from attack by steric hindrance due to the

presence of a side chain [275]. The second approach is the design of inhibitors of the three

classes of aminoglycoside-modifying enzymes. Inhibitor design can be targeted at the

aminoglycoside, cofactor binding sites or both [275].

(b) Tetracyclines

Tetracyclines can be divided into two types based on their mode of action [276]. Typical

tetracyclines, such as tetracycline, chlortetracycline, doxycycline or minocycline, act upon

the conserved sequences of the 16S rRNA of the 30S ribosomal subunit to prevent binding of

tRNA to the A site [276, 277]. Some other tetracycline derivatives, such as chelocardin,

thiatetracycline, anhydrotetracycline, have been shown to act by inserting into the

cytoplasmic membrane [278]. Thirty-eight acquired genetically mobile tetracycline (tet) and

oxitetracycline (otr) resistance (Tcr) genes are known, including genes coding for energy-

dependent.

53

Resistance to tetracycline

Alteration of porin proteins, e.g. OmpF, or other outer membrane proteins limits the

diffusion of tetracycline into the periplasm in Gram-negative bacteria [278]. About 60% of all

tet and otr genes code for energy-dependent membrane-associated transporters belonging to

the MFS, which export tetracycline out of the cell at a rate equal to or greater than its uptake

[278,279]. On the other hand, ribosomal protection proteins promote GTP dependent release

of tetracyclines from the ribosomal A site leading to dissociation of the antibiotic-target

interaction [279,280]. Three classes of ribosome protection resistance genes have been

described, tet (M), tet (O) and tet (Q) genes [278]. Resistance can also arise by point mutation

in ribosomal RNA [30]. Finally, the tet (X) gene encodes an NADPH requiring

oxidoreductase, which oxidizes tetracycline antibiotics. The antibiotic undergoes non-

enzymatic rearrangement into unstable products that polymerise into a black product after

several hours [278,279]. A new generation of tetracyclines, the 9-glycinyltetracyclines or

glycylcyclines (tigecycline, 9-t-butylglycylamido-minocycline) have been developed.

Glycylcyclines have a higher binding affinity for ribosomes than earlier tetracyclines.

Furthermore, the Tet efflux proteins fail to recognise glycylcyclines or are unable to transfer

glycylcyclines [280]. A number of tetracycline efflux pump inhibitors have been discovered

that might be used in combination with earlier tetracyclines.

1.16.4.2 Inhibitors of 50S subunit

(a) Chloramphenicol

Chloramphenicol interacts with the conserved sequences of the peptidyl transferase

cavity of the 23S rRNA of the 50S subunit. It inhibits protein synthesis by preventing binding

of tRNA to the A site of the ribosome. It interacts with various nucleotides of the peptidyl

transferase cavity of the 23S rRNA through hydrogen bonds [280].

54

Resistance to chloramphenicol

Antibiotic Inactivation

The inactivation of chloramphenicol is accomplished by the chloramphenicol

acetyltransferases (CAT) by transferring the acetyl group from acetyl CoA, resulting in a

lower affinity of the antibiotic for the rRNA [280]. Hereto, florfenicol was developed to

overcome CAT-mediated resistance.

(b) Macrolides

Macrolides affect the early stage of protein synthesis, namely translocation, by targeting the

conservedsequences of the peptidyl transferase centre of the 23S rRNA of the 50S ribosomal

subunit [280]. This results in a premature detachment of incomplete peptide chains [280].

Although compounds of considerable structural variety, macrolides, lincosamides and

streptogramins B (MLSB antibiotics) show a similar mechanistic action [280].

Resistance to macrolides

(i) Target site alteration

Any discussion of mechanisms of resistance to macrolide antimicrobials must include

the lincosamide and streptogramin B families as well. This type of cross-resistance has

therefore been referred to as MLSB resistance [281,282] and is generally the result of target

site alteration. The latter results from a post-transcriptional modification of the 23S rRNA

component of the 50S ribosomal subunit involving methylation or dimethylation of A2058

(E. coli numbering) in the peptidyl transferase functional domain. This is catalysed by

adenine-specific N-methyltransferases (methylases, MTases) specified by the erm class of

genes, frequently plasmid encoded [280,281]. This modification reduces the affinity of the

rRNA for the antimicrobials but does not interfere with protein biosynthesis [279]. Mutations

in 23S rRNA close to the sites of methylation can also lead to macrolide resistance [278,281].

55

In addition to multiple mutations in the 23S rRNA, mutations in the L4 and L22 50S

ribosomal proteins have also been seen [282,283].

(ii) Antibiotic inactivation

Macrolides can also be inactivated by specific enzymes inside the cell, such as

proteins that cleave the macrocycle ester, encoded by ereA and ereB genes. Phosphorylation

by MPHs (macrolide kinases) encoded by mphA and mphB from E. coli and mphBM (mphC)

from S. aureus and macrolide glycosylation by the product of the mtg gene [282,283] are also

possible.

(iii) Decreased permeability and increased efflux

Finally, macrolide entrance into bacterial cell can be prevented by changes in the

permeability of the membrane or the cell wall. The active extrusion of antimicrobials from

the bacterial cell by the action of efflux pumps, encoded by mef genes, has also been

observed [283, 284]. The ketolide telithromycin retains activity against isolates resistant by

target modification [284]. Activity of the existing antimicrobial drugs can also be restored by

the design of inhibitors of the Erm MTases [285].

(c) Lincosamides

Lincosamides interact with the conserved sequences of the 23S rRNA of the 50S

subunit [286]. They act by affecting the process of peptide chain initiation and may also

stimulate dissocation of peptidyl-tRNA from ribosomes. In contrast with macrolides,

lincosamides are direct peptidyltransferase inhibitors [287].

Resistance to lincosamides

(i) Target site alteration

The main type of resistance is the MLSB resistance [288]. Recently, methylation of 23S

rRNA at A2503 by the cfr gene product has been seen. Cfr causes resistance by inhibiting

ribose methylation at nucleotide C2498. The phenotype was named PhLOPSA [289].

56

Mutations in the 23S rRNA and in L4 and L22 ribosomal protein genes likewise have been

found [290].

(ii) Antibiotic inactivation and efflux

As inactivation mechanism, three lincosamide O-nucleotidyltransferase genes, linA, linA’

and linB [291] have been characterized. Alternatively, efflux of the antibiotic is the main

resistance mechanism in Gram-negative bacteria [292]. No specific mechanisms to overcome

the ever increasing resistance have been developed, beside the use of combinations of

different antibiotics or of antibiotics with non-antibiotic antimicrobials [293].

(d) Streptogramins

Streptogramins act by binding to the conserved sequences of the 23S rRNA of the 50S

subunit and byinterfering with peptidyltransferase activity [294]. Type A streptogramins

block the substrate site of the peptidyl transferase centre, thus preventing the earliest event of

elongation [293]. Type B streptogramins block peptide bond synthesis and cause a premature

release of incomplete peptide chains [293]. The synergism between types A and B

streptogramins is due to induction by type A streptogramins of conformational changes in

ribosomes that significantly increase the ribosome affinity for type B streptogramins

[293,294].

Resistance to streptogramins

Resistance to streptogramin combinations requires resistance specifically to the SA

component, but it is augmented by the presence of mechanisms conferring SB resistance

[295].

(i) Target site alteration

The main type of resistance is the MLSB resistance. Type A streptogramins are not

affected by this altered residue and the efficacy of the synergistic combination is thus retained

[291-293]. Also, modification of 23 rRNA ribosomal proteins, such as ribosomal protein L4,

57

due to point mutations and small deletions or insertions has been described. Low-level

resistance has been reported resulting from mutations in rplV, which encodes ribosomal

protein L22 [294].

(ii) Antibiotic inactivation

The streptogramin acetyltransferases (VATs) inactivate the type A streptogramins by

O-acetylation. Five acetyltransferases, encoded by vat (A), vat (B) and vat (C) and by vat (D)

and vat (E), have been seen [291, 294]. The producer of the type A streptogramin

virginiamycin M1 protects itself by reducing a critical ketone group, thereby generating an

inactive compound. This reduction is NADPH-dependent and regiospecific [292]. Specific

resistance to type B streptogramins is mediated by lyases, encoded by vgb (A) and vgb (B),

which inactivate the compounds via an elimination mechanism [293, 294].

(iii) Decreased uptake and increased efflux

Alternatively, streptogramin uptake into the periplasm is impaired among most Gram-

negative organisms owing to the impermeable Gram-negative outer membrane (intrinsic

resistance) [294]. Active efflux of type A streptogramins is due to ATP-binding cassette

proteins encoded by plasmid-borne vga (A) and vga (B) genes [21]. Efflux of type B

streptogramins is due to the presence of another ATP-binding transporter encoded by the

msrA, msrSA, msrB and msrC genes [294].

(e) Oxazolidinones

Oxazolidinones inhibit formation of the 70S initiation complex by binding to the P

site at the 50S ribosomal subunit near to the interface with the 30S subunit, thereby blocking

the first peptide-bond forming step [295]. If the 70S initiation complex is already formed,

they inhibit translocation of peptidyl-tRNA from the A site to the P site during formation of

the peptide bond [296]. Recently it was shown that they also inhibit fMet-tRNA binding to

58

the P site [294]. As the action mechanism of oxazolidinones is unique, no cross-resistance

between oxazolidinones and other protein synthesis inhibitors has been observed [294].

Resistance to Oxazolidinones

(i) Target site alteration

Resistance arises by spontaneous mutations in chromosomal genes encoding 23S

rRNA, resulting in decreased affinity for binding, or in protein L4 [294]. Also

posttranscriptional modification of the target site is a possible cause of resistance. Linezolid

resistance is determined by the presence of the cfr gene. Cfr methyltransferase modifies

adenosine at position 2503 in 23S rRNA. The natural function of cfr likely involves

protection against natural antibiotics whose site of action overlaps that of linezolid [295].

1.16.5 Miscellaneous targets

1.16.5.1 Folic acid metabolism: sulphonamides and trimethoprim

Each of these drugs inhibits distinct steps in folic acid metabolism (Figure 8). A

combination of sulpha drugs and trimethoprim acting at distinct steps on the same

biosynthetic pathway shows synergy and a reduced mutation rate for resistance [295].

Sulphonamides inhibit dihydropteroate synthase in a competitive manner with higher affinity

for the enzyme than the natural substrate, p-aminobenzoic acid. Agents such as trimethoprim

act at a later stage of folic acid synthesis and inhibit the enzyme dihydrofolate reductase

[295].

Resistance to sulphonamides and trimethoprim

(i) Target site alteration

Mutations in the d (h)fr gene producing single amino acid substitution in the

dihydrofolate reductase are responsible for trimethoprim resistance. Changes in both the

promoter and coding regions of the dhfr gene have been found [296]. Overexpression or

metabolic bypass of the target has also been observed.

59

Fig. 8 Sulphonamides and trimethoprim inhibit distinct steps in folate

Metabolism

60

1.16.5.2 Cell membrane disruptors

(i) Polymyxin antibiotics

Cationic cyclic peptides with a fatty acid chain attached to the peptide, such as

polymyxins, attack the cytoplasmic membrane of Gram-positive and Gram-negative bacteria

and the outer membrane of Gram-negative bacteria. They bind to phospholipids in the

cytoplasmic membrane, causing loss of membrane integrity, leakage of cytoplasmic contents

and finally cell death [295, 296].

Resistance to polymyxin antibiotics

The key initial interaction between the polymyxins and lipopolysaccharides can be blocked

by modification of the phosphate esters linked to the diglucosamine components of lipid A

[297].

(ii) Lipopeptides: Daptomycin

Daptomycin has a unique mode of action and involves a calcium-dependent insertion

of the lipid side chain into the Gram-positive cell membrane. After this, several molecules

come together to form oligomers that disrupt the cell membrane without entering the

cytoplasm of the cell. This ion-conduction structure results in potassium efflux and associated

membrane depolarisation. This disruption of the bacterial cell membrane function also

appears to trigger inhibition of DNA, RNA and protein synthesis resulting in cell death [295,

296]. The synthesis of lipotheichoic acid, found in Gram-positive organisms, is also inhibited

by daptomycin [36]. Due to its unique mode of action, there is generally no crossresistance

[297]. Spontaneous acquisition of resistance to daptomycin occurs rarely [296].

Resistance to lipopeptides: Daptomycin

i. Impermeability

The failure to cross the outer membrane of Gram-negative bacteria to reach the inner

cell membrane target is likely to explain the lack of daptomycin activity against gram

61

negative bacteria (intrinsic resistance) [297]. Correlation with vancomycin and daptomycin

resistance linked to the thickness of the cell wall suggests that prior use of vancomycin may

predispose to decreased daptomycin susceptibility [296].

This overview has given insight in the many therapeutic possibilities that exist for treatment

of bacterial infections and in the continuous battle between resistance development and

overriding mechanisms.

1.17 Methicillin resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus is responsible for a broad range of clinical infections, most

notable of which are cases of bacteremia and endocarditis [298]. Staphylococcus aureus is an

important cause of serious infections in both hospitals and the community. Methicillin-

resistant Staphylococcus aureus (MRSA) were first reported in 1961 and the first hospital

outbreak of MRSA was reported in 1963 [297]. When MRSA strains first appeared, they

occurred predominantly in the healthcare setting. Cases of community-associated MRSA

(CA-MRSA) infections were first reported in the late 1980s and early 1990s [296]. Health

care-associated MRSA (HA-MRSA) is particularly efficient at developing resistance to

antimicrobial agents. Methicillin resistance among staphylococci has steadily increased

worldwide, especially among cases acquired in hospitals. It is associated with longer hospital

stay and more infections in intensive care units and leads to more antibiotic administration

[298]. Asymptomatically colonized patients and health care workers are the major sources of

methicillin-resistant Staphylococcus aureus (MRSA) in the hospital environment [298].

MRSA-infected patients in burns units are particularly problematic because the big

surface area of denuded skin can produce a large inoculum of organisms that can be easily

transmitted to other patients via the hands of health care workers. Extensive skin lesions also

result in heavy shedders of MRSA [297]. The commonest site of MRSA carriage is the

anterior nares. A significant risk factor for acquisition of MRSA is the duration of hospital

62

stay. Prolonged stay in the hospital is likely with patients in orthopedics and dermatology

wards, which may result in high rates of carriage observed in these patients [296].

HACO (Health care associated MRSA with a community onset) refers to community

onset for a person associated with a hospital environment, e.g., a person living in a residential

home, a health care worker, a dialysis patient, or an individual with a history of

hospitalization within the previous year [298]. Risk factors associated with MRSA

bacteremia include the following: residence in an extended care facility, prior antibiotic

exposure, insulin dependent diabetes, prolonged hospitalization, urinary catheterization,

nasogastric tube placement, prior surgery, and having an underlying disease [299]. The

elderly population (≥ 65 years old) is at a significantly higher risk of death due to MRSA

bacteremia than are younger populations.

MRSA bacteremia has been associated with an increased risk of acute renal failure,

longer hospital and intensive-care-unit stays, development of ventilator dependency, and

increased hospital costs. Fatality rates for patients that develop MRSA bacteremia are

estimated to be between 23 % and 54 %. Nosocomial MRSA is remarkable for its clonal

pattern of spread. Currently, 5 major MRSA clones account for approximately 70 % of

MRSA isolates in hospitals in the United States, South America, and Europe [298,299].

1.17.1 Brief timeline [298-300].

1940 Penicillin introduced

1942 Penicillin-resistant Staphylococcus aureus appears

1959 Methicillin introduced; most S aureus strains in both hospital and community settings

are penicillin resistant

1961 Methicillin-resistant S aureus appears

1963 First hospital outbreak of methicillin-resistant S aureus

1996 Vancomycin-resistant S aureus (VRSA) reported in Japan

63

1.17.2 Methicillin

Meticillin or methicillin is a narrow spectrum beta-lactam antibiotic of the penicillin

class developed in 1959 that was previously used to treat infections caused by beta-lactamase

producing Staphylococcus aureus [300]. Methicillin is no longer manufactured because the

more stable and similar penicillins such as oxacillin, flucloxacillin and dicloxacillin are used

medically [301]. The presence of the ortho-dimethoxyphenyl group directly attached to the

side chain carbonyl group of the penicillin nucleus facilitates the β-lactamase resistance.

Methicillin has recently been renamed meticillin to comply with European law, which

requires the use of the recommended international non-proprietary name (rINN).

International convention has now renamed this agent as meticillin [300].

1.17.3 Mechanism of resistance:

Penicillin-resistant strains of S. aureus appeared as early as the 1940s, but for many

years these remained susceptible to ß-lactamase-stable penicillins [299, 300]. Then, in the

mid-1980s, S. aureus strains emerged that were resistant to the ß-lactamase-stable penicillins.

These strains were termed “methicillin resistant S. aureus” (MRSA), because methicillin was

initially used to detect their resistance to ß-lactamase-stable penicillins (oxacillin, methicillin,

nafcillin) [300]. Even though the drug methicillin is no longer the agent of choice for

treatment, the acronym MRSA continues to be used. Later use of oxacillin as an alternative to

methicillin in susceptibility tests resulted in the term ‘oxacillin-resistant S. aureus’ (ORSA)

[299].

These designations are used interchangeably in the literature and are synonymous. In

S. aureus, resistance to penicillins occurs through two mechanisms: the production of the ß-

lactamase enzyme and the presence of the mecA gene [301]. Majority of S. aureus strains

today produce ß-lactamase and are thus resistant to penicillin. Some of these strains produce

excessive amounts of ß-lactamase, which makes them appear borderline resistant to oxacillin.

64

These strains are termed borderline oxacillinresistant S. aureus (BORSA) and they can be

difficult to differentiate from classic MRSA [300].

Methicillin-resistant isolates with alterations to existing PBPs have been described.

These isolates have been termed ‘moderately resistant S. aureus (MODSA). They are not

frequently reported, the resistance is low-level and their clinical significance is unclear [302].

Methicillin resistance in S. aureus is primarily mediated by the mecA gene, which codes for

the modified penicillin-binding protein 2a (PBP2a or PBP2ʹ) [303]. PBP2́ is located in the

bacterial cell wall and has a low binding affinity for β-lactams. Although all cells in a

population of S.aureus may carry the mecA gene, often only a few of the cells will express

the gene [304].

Thus, both resistant and nonresistant bacteria can exist in the same culture. mecA

expression can be constitutive or inducible [305]. mecA is carried on a mobile genetic

element, the staphylococcal cassette chromosome mec (SCCmec), and at least five types of

SCCmec elements have been reported. Characteristics shared by all SCCmec elements are

carriage of the mec and ccr (cassette chromosome recombinases) gene complexes and

integration in the S. aureus genome at the 3' end of an open reading frame (orfX) with an

unknown function [306].

Four and five types of mec and ccr genes have been identified, respectively, and

SCCmec elements can be differentiated based on various combinations of these alleles [306].

MRSA infections in patients without any established risk factors too is on the rise in the

community where skin and soft-tissue infection, necrotizing fasciitis and serious necrotizing

pneumonia have been reported to cause epidemics[307].

These infections have been attributed to dissemination of genetically distinct clonal

strains, which contain Panton-Valentine leukocidin (lukS-PV and lukF-PV) and possesses

different staphylococcal cassette chromosome mec (SCCmec) genetic elements (type IV a–d

65

and type V). SCCmec types I to IV are annotated primarily by using the multiplex PCR.

Isolates that are nontypeable (NT) by this method are further investigated by PCR to assess

the ccrA, ccrB, and ccrC recombinase genes and the mec class or by multiplex PCR to

distinguish SCCmec subtypes IVa to IVh [306]. These strains tend to be more susceptible to

antimicrobial agents compared with the SCCmec type’s I–III. On the other hand, HA-MRSA

strains mainly harbor SCCmec types I, II, and III and in contrast to CA-MRSA strains tend to

be multidrug resistant with hallmark resistance to fluoroquinolones [307].

1.17.4 Detection of resistance

Several organizations have recommended that patients be screened upon admission to

hospitals and persons identified as colonizers are placed on contact isolation. Nasal

colonization has been shown to be predictive of the likelihood of patients to develop an

infection. Active surveillance cultures from patients for carriage of MRSA facilitate an early

contact isolation (and even treatment), thus preventing spread in the hospital and reducing

costs. The accurate and early determination of methicillin resistance is of key importance in

the prognosis of infections caused by S. aureus. Strains that possess mecA gene are either

heterogeneous or homogeneous in their expression of resistance [307,308]. The

heterogeneous expression occasionally results in minimal inhibitory concentrations that

appear to be borderline and consequently the isolates may be interpreted as susceptible.

MRSA strains expressing heterogeneous resistance (a predominantly low-level resistance

population coexisting with a small proportion of highly resistant cells) are often mistaken for

methicillin-sensitive S. aureus (MSSA) by conventional culture and represent a hidden

reservoir in hospitals [308]. The presence of both resistant and nonresistant bacteria, along

with the fact that the resistant bacteria often grow more slowly, can make it difficult to detect

methicillin resistance. Their detection using methicillin and oxacillin is aided by following

66

changes [309]: Neutral pH, incubation temperature of 33 oC – 5 oC, Mueller-Hinton agar or

broth infused with 2 % - 4 % NaCl, and 24-h incubation time.

Several selective media have been used for screening MRSA, such as mannitol salt agar with

oxacillin, Oxacillin blood agar and CHROM agar. Occasionally, heteroresistant mecA-

positive strain is not detected due to low expression of resistance. Oxacillin agar screen

generally does not detect borderline resistant strains [310].

1.17.5 Phenotypic detection systems

Phenotypic expression of resistance can vary depending on the growth conditions (e.g.

temperature, osmolarity and culture medium supplements such as NaCl or sucrose), making

susceptibility testing by standard microbiological methods potentially difficult.

(a) Agar dilution test

A minimum of four to five colonies isolated from an overnight growth are transferred

to sterile saline. The suspension is adjusted to a 0.5 McFarland standard (108 cfu/ml) and spot

inoculated on Mueller-Hinton agar plates supplemented with 2 % NaCl and containing 2.56 –

0.125 μg oxacillin/ml in serial doubling dilutions. The oxacillin Mueller-Hinton plates are

incubated at 35 oC for 24 h. MIC of ≥ 4 μg/ml is considered resistant and MIC of ≤ 2 is

considered susceptible [310].

(b) Broth microdilution

This involves the use of Mueller-Hinton broth with 2 % NaCl, an inoculum density of 5 x 105

cfu/ml and incubation at 33 oC–35 oC for 24 h.[308 - 310].

Breakpoint methods

Breakpoint methods include both agar and broth methods and are essentially similar to

dilution MIC methods but test only the breakpoint concentration (2 mg/L oxacillin, 4 mg/L

methicillin).[310].

67

(c) Disc diffusion test

A direct colony suspension of each S. aureus isolate is prepared to a 0.5 McFarland

standard and plated on Mueller-Hinton agar containing 2-4 % NaCl. An oxacillin (1 μg) disk

is placed on the surface and incubated at 35 oC for 24 h [310]. Oxacillin disk is more resistant

to degradation in storage and more likely to detect heteroresistant strains. The zone of

inhibition must be read with transmitted light and not reflected light. Zone diameter of ≤ 10

mm is considered as resistant, ≥ 13 mm as susceptible whereas 11-12 mm is considered as

intermediate. If intermediate results are obtained for S. aureus, testing for mecA, PBP2a,

[310]. Cefoxitin disk test, Oxacillin MIC test or Oxacillin-salt agar screen test may be

performed. Any discernable growth within the zone of inhibition when seen using transmitted

light is indicative of oxacillin resistance [310]. It may be possible that some of the oxacillin

disk test positive isolates are hyperbeta-lactamase producers, thereby accounting for non-

mecA-mediated methicillin resistance [311].

In disc diffusion tests, hyper-producers of penicillinase may show small methicillin

or oxacillin zones of inhibition, whereas most true methicillin-/oxacillin-resistant isolates

give no zone. A 5μg methicillin disk can also be used but is not a popular choice. Zone

diameter of ≤ 9 mm is considered resistant, ≥ 14 mm is considered resistant whereas a

diameter of 10-13 mm is considered intermediate [310, 311].

(d) Etest oxacillin MIC test

The inoculum is standardized to 0.5 McFarland turbidity and plated on Mueller-Hinton agar

supplemented with 2 % NaCl [310]. Etest strips are placed and incubation at 35 oC for 24 h.

The Etest has an advantage over other MIC methods in that it is as easy to set up as a disc

diffusion test [311].

68

(e) Oxacillin screen agar

Mueller-Hinton agar (MHA) plates containing 4 % NaCl and 6 μg/ml of oxacillin are

inoculated with 10 μL of 0.5 Mc Farland suspension of the isolate by streaking in one

quadrant and incubated at 35 oC for 24 h. Plates are observed carefully in transmitted light for

any growth. [311].

Any growth after 24 h is considered oxacillin resistant. Induction with oxacillin requires an

extended period for full expression. Hence, oxacillin-containing media achieve sufficiently

high sensitivities only after 48 h of incubation [312].

(f) Cefoxitin disc diffusion test

Cefoxitin, which is a potent inducer of the mecA regulatory system, is being widely

used as a surrogate marker for detection of mecA gene-mediated methicillin resistance.

MRSA strains exhibiting inducible resistance to methicillin grow much more readily in the

presence of cefoxitin than oxacillin [312], due to an enhanced induction of PBP 2a by

cefoxitin. CLSI has recommended cefoxitin disc diffusion method for the detection of

MRSA. A 0.5 Mc Farland standard suspension of the isolate is made and lawn culture done

on MHA plate. A 30 μg cefoxitin disc is placed and plates are incubated at 37 OC for 18 h and

zone diameters are measured [312].

The zone diameter must be measured in reflected light. An inhibition zone diameter of

≤ 21 mm is reported as methicillin resistant and ≥ 22 mm is considered as methicillin

susceptible. In one study a 10 μg cefoxitin disk has been shown to be superior to the 30 μg

disk with IsoSensitest agar and semiconfluent growth. Recent studies indicate that disc

diffusion testing using cefoxitin disc is far superior to most of the phenotypic methods like

oxacillin disc diffusion and oxacillin screen agar testing and is recommended by CLSI.

Cefoxitin will detect only MRSA with a mecA-mediated resistance mechanism [311].

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(g) Chromogenic media for MRSA

Currently available chromogenic media for MRSA detection include ChromID, MRSA

Select, CHROMagar MRSA, Chromogenic MRSA/Denim Blue agar, ORSAB (oxacillin

resistance screening agar base), MRSA Ident agar and Chromogen oxacillin S. aureus

medium. [311].

The chromogen in Chrom-ID targets the α-glucosidase enzyme of S. aureus, and the

inhibition of competing flora is brought about by the incorporation of cefoxitin (4 mg/liter),

resulting in green-colored colonies of MRSA. ORSAB, a modified version of mannitol salt

agar, is made selective by the addition of oxacillin (2 mg/liter) to inhibit MSSA and

polymyxin to suppress gram-negative bacteria [311]. This medium incorporates aniline blue

as a PH indicator, giving MRSA colonies a characteristic blue color. Colonies of MRSA on

MRSA Ident agar are dusky pink or ruby-colored due to a chromogenic phosphatase substrate

and an antibiotic supplement including cefoxitin [311].

Chromogen oxacillin S. aureus medium characterizes MRSA colonies by a pink-

mauve color. MRSA Select incorporates a cephamycin derivative and characterizes MRSA

colonies by a pink color [310]. CHROMagar contains cefoxitin (6 mg/liter) and a chromogen

that also results in rose to mauve MRSA colonies [310]. The chromogen in Chromogenic

MRSA or Denim Blue agar detects phosphatase activity in S. aureus strains and, coupled

with a selection with cefoxitin, produces denim blue colonies of MRSA. The currently

available chromogenic media for MRSA detection show almost uniformly high specificities

after 24 h of incubation, although sensitivities tend to vary widely both between media and

between studies. Prolonging incubation time to 48 h can improve sensitivities; however,

specificities are adversely affected, necessitating confirmatory tests before reporting MRSA

[312].

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(h) Quenching fluorescence method

With the Crystal MRSA method (Becton Dickinson) inhibition of growth of an isolate

by oxacillin is indicated by the quenching of fluorescence of an oxygen-sensitive fluorescent

indicator by oxygen remaining in the broth. The method is reasonably reliable but requires

several hours of incubation [312].

(i) The 3M™ BacLite™ Rapid MRSA test

It is a rapid culture-based test that detects ciprofloxacin-resistant MRSA. The test

measures adenylate kinase (AK) activity, an enzyme in cells that regulates adenosine

triphosphate (ATP) AK catalyzes the conversion of adenosine diphosphate (ADP) to ATP. In

the assay, AK detection is combined with selective broth enrichment [312]. Magnetic

microparticles coupled with a mouse anti-S. aureus monoclonal antibodies are used to

capture MRSA. Lysostaphin is added to lyse the S. aureus and release AK. ADP is provided

as the substrate for the enzyme activity and production of ATP [311]. The ATP is detected by

the addition of firefly luciferin and luciferase, allowing a reaction that emits light when

MRSA is present in the sample. This assay yields results in five hours. Few false positives

due to methicillin resistant coagulase-negative staphylococci (CNS) misidentified as MRSA

have been encountered [312].

1.17.6 Genotypic detection system

(a) PBP2́ latex agglutination kit

The method involves extraction of PBP2´ from suspensions of colonies and detection

by latex agglutination [312]. The kit contains latex particles sensitized with a monoclonal

antibody against PBP2ʹ. Visible agglutination indicates a positive result and the presence of

PBP2́, the mecA gene product [312]. The test is rapid (10 minutes for a single test) and very

sensitive and specific with S. aureus, but may not be reliable for colonies grown on media

containing NaCl. Isolates producing small amounts of PBP2́ may give weak agglutination

71

reactions or agglutinate slowly. Reactions tend to be stronger if PBP2ʹ production is induced

by growth in the presence of penicillin. Rare isolates may give negative reactions [312].

(b) Molecular methods

Detection of mecA gene by PCR is considered as the gold standard. DNA extraction

is performed on the isolate and mecA gene is amplified using specific primers [312]. The

master mix containing PCR buffer, dNTP mix, primer, Taq DNA polymerase, and MgCl2 and

template DNA is subjected to hot start PCR. This is followed by 30 cycles of denaturation at

94 OC for 45 seconds, annealing at 50 oC for 45 s, and extension at 72 oC for 1 minute and

final extension step at 72 oC for 3 min [312]. PCR products are visualized on 2 % agarose gel

with ethidium bromide dye under UV transilluminator. Another method that is designed to

detect MRSA directly from clinical samples uses a frontend immunocapture of S. aureus

followed by MRSA detection using a multiplex PCR that detects S. aureus-specific femA and

mecA. Staphylococcus aureus-specific targets used in various user-defined molecular assays

for detection of MRSA [312]:

• nuc Encodes heat-stable DNA nuclease gene

• femA, femB Encode enzymes important in cross-linking peptidoglycan

• spa Encodes S. aureus-specific protein A [312].

(c) The hyplex StaphyloResist™ and hyplex StaphyloResist™ plus

are qualitative multiplex PCR assays for the direct detection of clinically relevant

staphylococci from swabs of the nose, skin, wounds, and endotracheal specimens[313]. The

assay consists of PCR modules that contain labeled oligonucleotide primers, enabling

simultaneous and specific amplification of different staphylococcal DNA regions in a single

PCR reaction [313]. The PCR is followed by recerse hybridization procedures using single-

stranded specific probes immobilized on microtitre plates. Hybridization of PCR products

with specific probes is detected using the ELISA principle [313].

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(d) The Genotype MRSA direct

In GenoType MRSA Direct, the IDI-MRSA, and the GeneXpert MRSA assay, the

MRSA detection is based on the detection of a single amplicon, which includes the right

junction of the SCCmec downstream of the mecA gene and a part of the adjacent S. aureus-

specific orfX gene [313].

The Genotype MRSA Direct targets SCCmec types I to V in a multiplex PCR using

biotinylated primers followed by a reverse hybridization step. An updated version of the

assay, the Genoquick MRSA dipstick assay, does away with the reverse line hybridization

step to reduce the total assay time from 4 h to 2 h 20 min [313].

(e) The GenoQuick™ MRSA assay.

This is a direct test for MRSA from swabs of the nose, throat, skin, and wounds that

uses a dipstick for qualitative detection of amplicons [313]. Following DNA extraction and

conventional PCR, the single stranded amplicon hybridizes with a fluorescein labeled probe

included in the mastermix. The amplicon-probe complex is selectively labeled with gold and

generated a band on the dipstick that is inserted in a tube that contains the amplicon. The

assay aslo has an amplification control dipstick. It takes approximately 2 h and 20 min to

perform [312].

(f) The BD GeneOhm™ MRSA assay (also called IDI-MRSA).

It is a multiplex assay comprised of six primers that amplify target sequences near the

insertion site of SCCmec. Amplified targets within SCCmec and orfX genes are detected with

four molecular beacons [312]. This assay has an internal control not found in MRSA. The

fluorescence emitted by each beacon is measured and interpreted. Results are displayed as

postive, negative or unresolved. It can differentiate MRSA from MSSA and mecA positive

CNS in clinical sample [313].

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1.17.7 Interpretation of genotypics detection methods

The detection of mecA gene or its product, penicillin binding proteins (PBP2́), is

considered the gold standard for MRSA confirmation [312]. Isolates of S. aureus that carry

mecA gene or that produce PBP2ʹ should be reported as MRSA and isolates lacking mecA

gene or not producing PBP2ʹ should be reported as methicillin susceptible [312] For MRSA

strains, other beta-lactam agents such as penicillins, β-lactam/β-lactamase inhibitor

combinations, cephems and carbapenem must be reported as resistant or not reported at all

even though they may appear susceptible in vitro [313]. This is because most MRSA

infections have responded poorly or convincing data to prove their clinical efficacy are

unavailable [313]. For oxacillin susceptible S. aureus isolates, results for parenteral and oral

cephems, β-lactam/ β-lactamase inhibitor combinations and carbapenems the results should

be reported as routine interpretative criteria. Certain rare isolates of S. aureus that are mecA

& PBP2̍ negative but have oxacillin MIC ≥ 4 μg/ml should be reported as oxacillin resistant

[314]. Penicillin resistant but oxacillin susceptible strains produce β-lactamase and should be

tested using 10 unit penicillin disk and not ampicillin disk. A positive β-lactamase test

predicts resistance to all β-lactamase labile penicillins such as ampicillin, amoxicillin,

carbenicillin, ticarcillin and piperacillin [312]. For oxacillin resistant S. aureus penicillin

susceptibility should not be reported or reported as resistant. The results of oxacillin

resistance can be applied to other penicillinase-stable penicillins such as cloxacillin,

dicloxacillin and flucloxacillin. Cloxacillin disks should not be used to detect MRSA because

they may not detect oxacillin resistance [314].

1.18 Evolution of methicillin-resistant Staphylococcus aureus clones

Methicillin-resistant Staphylococcus aureus (MRSA) clones have emerged to cause a

global public health concern [313-315]. Several major pandemic MRSA clones have been

identified around the world, but despite one such clone having originated in Brazil, the

74

molecular epidemiology of MRSA in Latin America is largely unknown [316]. MRSA clones

(bacterial strains descended from a common ancestor) diversify through point mutations,

recombination, or the acquisition/deletion of mobile genetic elements, giving rise to extensive

genomic and phenotypic diversity [317]. Discriminating and reproducible typing methods are

needed to study the epidemiology of MRSA. Frequently used methods include ribotyping,

pulsed-field gel electrophoresis (PFGE), andpolymerase chainreaction (PCR) with repetitive

element primers, but thesemethods are inherently prone to produce different results in

different laboratories due to the highly variable, rapidly evolving bacterial genome regions

that they target. PFGE has commonly been used in studies of MRSA epidemiology in Latin

America, to show the relatedness of isolates from recent outbreaks [315, 316]. However, the

technique is not well-suited to long-term global epidemiology, which requires a highly

discriminatory procedure to decipher gene variations that accumulate slowly. Newer

molecular typing methods, including multilocus enzyme electrophoresis (MLEE) and

multilocus sequence typing (MLST), are highly discriminatory and easily reproducible [316].

MLST is used to characterize isolates of bacteria and distinguish between clones by targeting

the sequences of internal fragments of housekeeping genes, where genetic variations develop

gradually [315, 316]. MLST allows related strains recovered in different countries to be

readily identified. When MLST results are analyzed using an algorithm known by the

acronym BURST (Based Upon Related Sequence Types), groups of isolates with a defined

level of similarity in allelic profile can be discerned among large MLST datasets, and a

dendrogram can be constructed [316]. BURST has been useful in studying the evolutionary

history of MRSA, to predict the ancestral allelic profile (genotype) of each clonal complex

(CC), and the pattern of evolutionary descent of all isolates in the group [316].

75

1.19 Historical origins and mechanisms of evolution of MRSA

The first MRSA clones had similar genetic and phenotypic properties to methicillin-

susceptible Staphylococcus aureus (MSSA) clones that were epidemic in the early 1960s in

Europe. The initial appearance of MRSA resulted from the acquisition by successful MSSA

clones of mecA, the gene that encodes a penicillin-binding protein conferring resistance to

methicillin, from an unknown heterologous source. The mobile genetic element that carries

mecA, called the staphylococcal cassette chromosome mec (SCCmec) has five forms (I, II,

III, IV and V), which have arisen by the horizontal transfer of mecA in independent events

[317,318].

The five major lineages of MRSA (CC5, CC8, CC22, CC45 and CC30) circulate

internationally and cause most nosocomial MRSA infections worldwide [318, 319] within

each lineage, putative evolutionary pathways have been proposed by an earlier researchers

[318, 319] and were based on sequence types (ST) and characterized by MLST. A relatively

small number of pandemic MRSA clones have caused a majority of MRSA infections. Five

predominant clones (Brazilian, Iberian, Hungarian, pediatric and New York/Japan (NYJ)

clones) were identified among 3000 MRSA strains collected in surveillance studies and

outbreak investigations from 1994 to 2000 (the CENMET initiative); PFGE was used to

characterize these strains. These five clones accounted for 68 % of isolates. The authors

hypothesized that these major clones have a unique ability to cope with changing clinical

environments (Figure. 9) [320].

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Fig. 9 Evolution of MRSA clones in Latin American countries: time line to show where

and when clones were isolated. [318-320].

B = Brazilian MRSA clone and variants; C = Cordobes/Chilean MRSA clone; CA =

CMRSA-6 (Canadian MRSA clone); H = Hungarian MRSA clone; I = Iberian-relatedMRSA

clone;

M = Mexican MRSA clone; MW2 = MW2-related MRSA clone; NY/J = New York/Japan-

related MRSA clone; P = pediatric-related MRSA clone; O = Oceania Southwest Pacific

MRSA clone; U = Uruguayan hospital MRSA clone; UR6 = Uruguayan community outbreak

MRSA clone; U3 = USA-300 MRSA clone; U8 = USA-800 MRSA clone ; W= Western

Australia 1 MRSA clone [319, 320, 321].

*First identification of Brazilian MRSA isolates with vancomycin and teicoplanin

heterogeneous resistance (1996–1998) [321].

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1.20 Treatment options

Daptomycin is an acidic lipopeptide with a mode of action requiring calcium.

Daptomycin has recently demonstrated significantly better bactericidal activity than

vancomycin against S. aureus and enterococci and has activity against a small number of

glycopeptide-intermediate S. aureus strains and vancomycin resistant enterococcus [324].

Mupirocin is a bacteriostatic antibiotic used exclusively as a topical agent. It exerts its

antimicrobial effect by specifically and irreversibly binding to bacterial isoleucyl tRNA

synthetase, thus preventing protein synthesis. It has been used widely for the clearance of

nasal methicillin-resistant S. aureus (MRSA) carriage during outbreaks and has been

recommended for the decolonisation of methicillin-sensitive S. aureus (MSSA) in healthcare

personnel. Intranasal application of mupirocin ointment is effective in reducing surgical site

infections and the likelihood of bronchopulmonary infection [323, 325].

Recently, there have been reports of vancomycin failure due to either relative vancomycin

resistance or MRSA infections in sites that have poor vancomycin penetration [324]. Few

other drugs including linezolid (a synthetic oxazolidinone), tigecycline (a derivative of

minocycline), and daptomycin (a cyclic lipopeptide) appear promising in treatment.

Daptomycin should be avoided in the treatment of MRSA-associated pneumonia because it is

inactivated by pulmonary surfactant [325]. For initial empiric therapy, oral trimethoprim-

sulfamethoxazole is a good choice. Other alternatives include minocycline, clindamycin, or a

macrolide antibiotic, depending on local susceptibility patterns. Staphylococci may develop

resistance during prolonged treatment with quinolones [325]. Therefore isolates initially

susceptible may become resistant within three to four days after initiation of therapy.

Rifampicin should not be used alone for treatment. Macrolide resistance in S. aureus may be

inducible or constitutive and the former can be detected by D-test. [324,325].

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1.20.1 Cytotoxic effect of commonly available antimicrobial agents

A major problem encountered with antibiotics in clinical use is drug resistance, which

mostly leads to treatment failure [325]. Other problems with antibiotics include toxicity high

cost, low cost efficacy, etc. This necessitates a continuous search for new antimicrobial

agents [324]. Medicinal plants have no doubt remained the major sources of traditional

medicine worldwide [325].

1.20.2. Plants and plant product as sources of antimicrobial agents

Plants with their wide variety of chemical constituents offer a promising source of new

antimicrobial agents, with general as well as specific antimicrobial activity [326, 327]. There

are several reports on the presence of anti-microbial compounds in various plant parts like

leaves, bark, fruit, root and flowers [327]. A number of plants have been screened for their

antimicrobial properties especially due to the presence of phenolic compounds like

flavonoids. Antimicrobial properties of medicinal plants are being increasingly reported from

different parts of the world. In recent years, secondary plant metabolites, previously with

unknown pharmacological activities, have been extensively investigated as a source of

medicinal agents [327].

Generally, medicinal herbs are moving from fringe to mainstream use with a

greater number of people seeking for remedies and health approaches free from side effects

caused by synthetic chemicals., Recently, considerable attention has been paid to eco-friendly

and bio-friendly plants, which can prevent and cure different human diseases [328].

According to the World Health Organization reports, the use of traditional medicine in the

first world countries is on the rise due to failure of conventional medicine that can cure

chronic diseases, emergence of multi-drug resistant pathogens and parasites, adverse effects

of chemical drugs, increasing cost and information of herbal medicine. Accordingly, attention

of scientists and researchers have been attracted towards developing new antibiotics that will

79

curtail the increasing drug resistance among microorganisms [329], reported that plants used

for traditional medicine generally contain a number of compounds which may be a potential

natural natural antimicrobial combination and which may serve as an alternative, effective,

cheap and safe antimicrobial agents for treatment of common microbial infections [330].

Antimicrobial potentiality of different medicinal plants is extensively studied all over the

world. However, only a few studies have been carried out in a systematic manner [330].

1.21 Future prospects

Through proper and adequate research into mechanisms of resistance, development of

antimicrobials with specific target of resistant genes and advance study of biotechnology of

natural products. There is hope for combating the revenge mechanism of bacteria called

resistance.

1.21.1 Antimicrobial drugs

Ever since the first use of penicillin, S. aureus has shown a remarkable ability to

adapt. Resistance has developed to new drugs within a short time of their introduction. Some

strains are now resistant to most conventional antibiotics. [331]. It is worrisome that there do

not seem to be any new antibiotics on the horizon. Any recent developments have been

modifications to existing drugs [331]. The original strategy used by the pharmaceutical

industry to find antimicrobial drugs was to screen natural products and synthetic chemicals

for antimicrobial activity, after which the mechanism of action was then investigated [332].

New approaches are being adopted to find the next generation of antimicrobials.

Potential targets such as enzymes involved in an essential function (e.g., in cell division) are

identified based on knowledge of bacterial physiology and metabolism [332]. Screening

methods are then developed to identify inhibitors of a specific target molecule. In addition,

with detailed molecular knowledge of the target molecule, specific inhibitors can be designed

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[333]. To prevent the emergence and dissemination of resistant bacteria, continuing efforts to

develop new antibacterial agents are warranted. Although this is not an easy assignment,

there is still hope and many new avenues are being explored. Indeed, the recent advance in

bacterial genomics has changed the antibacterial therapeutic environment from target-poor to

target-rich; hence many potential targets are awaiting researchers [293-298].

1.21.2 Vaccines and new approaches to combatting nosocoial infections

No vaccine is currently available to combat staphylococcal infections. There may now

be a case for considering methods to prevent disease, particularly in hospitalized patients

[334]. Hyperimmune serum from human volunteer donors or humanized monoclonal

antibodies directed towards surface components (e.g., capsular polysaccharide or surface

protein adhesions) could both prevent bacterial adherence and also promote phagocytosis of

bacterial cells [334, 335]. Indeed a prototype vaccine based on capsular polysaccharide from

S aureus has been administered to volunteers to raise hyperimmune serum, which could be

given to patients in hospital before surgery. A vaccine based on fibronectin binding protein

induces protective immunity against mastitis in cattle and might also be used as a vaccine in

humans. [335].

When the molecular basis of the interactions between the bacterial surface proteins

and the host matrix protein ligands are known it might be possible to design compounds that

block the interactions and thus prevent bacterial colonization. These could be administered

systemically or topically [336]. Continuous development of novel antibacterial is mandatory.

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1.22 Antimicrobial evaluation of a new agent

When a new antimicrobial agent is discovered, there is the need to evaluate it for the

following reasons [337-339].

(a) To estimate the potency. This gives an idea of its basic value. A good result here

makes further test worthwhile. The activity of the new agent is compared to that of a

standard antimicrobial agent.

(b) To determine it’s spectrum of activity. Knowledge of the range of microorganisms

which an antimicrobial agent can kill (or inhibit) helps one to determine its indications. An

agent with a narrow spectrum of activity may be used as antiseptic or as a chemotherapeutic

agent for infections while that with a wide range may be used as disinfectant. It is also to be

determined whether the activity is microbiocidal or microbiostatic “Microbiostatic effect is

unsuitable as disinfectant but may be used as antiseptic, preservative or in chemotherapy.

(c) To determine the efficacy and limitations of the new antimicrobial substance. This

includes test that will determine whether it is more potent than existing standard agent

example phenol coefficient activity when in the type of environment in which it will be used

and whether it is toxic.

(d) This can be done qualitatively and quantitatively by the following methods [340]

1.22.1 Strip- agar – diffusion

A strip of sterile filter paper is dipped into the anti-microbial agent using a sterile

forceps. Excess solution is allowed to drip off and the filter paper placed onto the surface of

a nutrient agar medium (poured into a plate) at the side of the plate each of the cultures of

organisms being used is streak at right angles to the strip of filter paper. The plate is

incubated. The degree of sensitivity of the organisms to the agent is reflected by the length

of streak showing no growth [341].

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1.22.2 Ditch agar diffusion

This is a similar method to the strip agar diffusion method. Here, a ditch is cut into the

agar and the solution of the bacteriostatic agent is poured into the ditch, streaks of various

organisms are made and the plate incubated. The temperature of incubation depends on

whether the organisms are bacteria, yeast or fungi. Hence these are kept separately since the

optimum temperature for them are different, 37 OC for bacteria and 25 OC for fungi and

yeast [342].

1.22.3 Determination of minimal inhibitory concentration (MIC) of extracts

The minimum inhibitory concentration of an antimicrobial substance is the least

concentration that inhibits the growth of the test organism. This can be conducted using

different methods as indicated below [343].

(a) Broth dilution method

Serial dilution of the agent whose biostatic activity is to be determine are made in an

appropriate nutrient medium to get different concentrations. These are then inoculated with

the test organism and incubated. The highest dilution (or lowest concentration) of the agent

which prevents microbial growth is then taken as the minimum inhibitory concentration

(MIC). Optimum temperature and time of incubation (48 hrs for bacteria and several days for

fungi) are used. Organic matter may be added to the medium. Age of the culture and number

of viable cell contained can also affect results. An inoculum containing 105 viable cell / ml is

normally used [344].

(b) Agar dilution method

Serial dilution of the agent whose biostatic activity is to be determined are prepared

and added separately to molten double strength agar medium and mixed well. The agar

83

dilutions obtained are poured into sterile dishes, allowed to solidity and dried. The agar

surface of each dish is inoculated with the organisms and incubated. This method is suitable

for substances which produce turbid solution in broth. The microbial growth is visually

examined for growth [344].

(c) Agar diffusion method

This is generally preferred to serial dilution method because of the ease which

quantitative result can be obtained. It cannot be used when the test substance dose not readily

diffuse through the gel. About 0.1 ml of the culture is added to the tubes containing the

molten nutrient agar (45 OC). This is poured into a sterile petri-dish and mixed thouroughly,

the agar is left for 10 mins to set. The dish is divided into four quadrants and cut is made in

the center of each and the discs are removed. Four different dilution of the bacteriostatic

solution are prepared and two to four drops of each are added in each cup. The plate is

allowed to stand for some time at room temperature for prediffusion to occur and then

incubated at 37 OC for 24 h for bacteria and at 25 OC for 48- 96 h for fungi and yeast [344].

The inhibition zone diameter is calculated by subtracting the diameter of cork borer from the

measured zones of inhibition.

1.24.4 Determination of minimal biocidal concentration (MBC) of extracts

The MBC is defined as the minimal concentration of a biocide, which kills off all the

cells in a microbial population. This can be considered to as an extension of the MIC

procedures. Hence, MBC can be derived from the broth dilution method of agar dilution

method of determining MIC. In either method, the MIC procedure is first completed. The

tubes or agar plates showing no growth in the MIC tests are used for the determination of

MBC [344].

84

(a) Broth dilution method

In this method, a loopful of the reaction mixture is transferred from each tube to

corresponding containers of fresh nutrient broth, acting as the cell recovery medium. The

newly inoculated broth medium is incubated for 48 h at temperature between 32 oC and 35

oC. The absence of growth in the recovery medium is evidence of total cell death. The

minimal concentration of the antimicrobial agent that produces total cell death is taken as the

MBC [344].

(b) Agar dilution method

Similar to broth dilution method, in this test, discs are cut from each agar plate and

transferred into corresponding containers of the fresh nutrient medium. The media are also

incubated at temperature between 32 oC and 35 oC for 48 h, at the end of incubation,

microbial growth can be ascertained in the usual way (turbidity of the medium or acid-base

reaction). The absence of growth in the recovery medium is evidence of total cell death. The

minimal concentration of the antimicrobial agent that produces total cell death is taken as the

MBC. [344].

1.23 Aim of the study

The aim of this study was to evaluate the phytochemical components and

antimicrobial activity of methanol extract and fractions of Moringa oleifera root bark as an

alternative source of antimicrobial agent for the treatment of MRSA infections.

1.24 Objectives of the study

The objectives of the study are to evaluate the antimicrobial activity of the methanol

extract and fractions of Moringa oleifera root bark on clinical isolates of MRSA, to extract

and identify phytochemical components of antimicrobial activity from the plant and to

elucidate the structure of the antimicrobial active compounds of methanol extract and

fractions of Moringa oleifera root bark.

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CHAPTER TWO

2.0. MATERIALS AND METHODS

2.1. MATERIALS

2.1.1. Sample collection

This study was conducted from September 2012 to June 2013 at 3 different hospitals in

the South-East region of Nigeria. The hospitals are Bishop Shanahan Hospital, Nsukka,

University of Nigeria Teaching Hospital, Ituku/Ozalla, Enugu State and Federal Medical

Centre, Abakaliki, Ebonyi State. A sample size of 2,372 hospitalized patients with skin,

throat, open wound, Ear/Nasal infections and Abscess were enrolled for this study. Samples

were taken from sputum, open wound, abscess, ear and nasal swab from male and female

wards in the orthopedic and intensive care departments of the hospitals. 1,230 samples were

taken from University of Nigeria Teaching Hospital, 200 samples from Bishop Shanahan

hospital and 942 samples from Federal Medical Centre, Ebonyi. The samples were distributed

according to their clinical specimens as sputum (376), skin swab (327), absces swab (466),

open wound swab (762) and ear/nasal swab (441).

2.1.2. Media

Nutrient Agar (Fluka) Sigma Aldrich UK, Mueller-Hinton Agar (MHA), Oxoid Ltd, England,

Mannitol Salt Agar (MSA), Oxoid Ltd, England.

2.1.3. Reagents

Penicillin binding protein 2 prime (PBP2ˈ) test kits lot. no. 130422, Oxoid Ltd, Japan,

staphylase test kit (Oxoid Ltd, Wade Road, Basingstoke, Hants, RG24, UK), Oxoid

antimicrobial susceptibility test discs. Hydrogen peroxide (H2O2), Dimethylsulfoxide

(DMSO), distilled water, silical gel (60-200 mesh), (Titan Biotech Ltd, India). 0.5 McFarland

turbidity standard.

86

2.1.4. Solvents

Methanol, (Sigma Aldrich, U.K), n-hexane, (Sigma Aldrich, U.K), ethyl acetate, (Sigma

Aldrich U.K), dichloromethane (Sigma Aldrich, U.K).

2.1.5. Equipment

Soxhlet extractor, glass chromatographic column, autoclave, refrigerator, weighing

balance, incubator, antibiotic disc dispensers, GC-MS equipment with Agilent technologies

7890B for GC systems and Agilent technologies 5975 series for MS system.

2.1.6. Animals

Wistar strain albino rats of both sexes that weighed between 80 -150 g which were bred in

the animal house of the Department of Pharmacology, University of Nigeria, Nsukka. The

animals were housed in standard environmental conditions with 12 h light-dark cycle. The

animals were divided into extract treated groups and the control groups. All the animals were

starved for 12 h, but were allowed free access to water, before commencement of the

experiments [345].

2.2. METHODS

2.2.1. Collection, authentication and processing of plant materials

The root of Moringa oleifera was collected from Nsukka Local Government Area,

Enugu State, Nigeria. The plant materials were identified and authenticated by a Botanist at

the Biological Science Department, University of Nigeria, Nsukka. Taxonomic identity of the

plant was authenticated by Mrs. Immanuela Udoma of the Department of Pharmacognosy,

Faculty of Pharmacy, University of Uyo, Uyo. The plant materials were air-dried in the

laboratory for four weeks. The dried samples were ground to coarse powder with a

mechanical grinder; the powder was stored for future use.

87

2.2.2. Extraction of root extract

The pulverized root of Moringa oleifera (3 kg) was defatted with 10 litres of n-hexane

by cold merceration over night to yield hexane extract fraction (HEF). The marc was dried

and extracted with 20 litres of methanol for 4 hours using Soxhlet extraction technique to

yield crude methanol extract (ME) using established standard procedures [347 - 349].

The crude methanol extract (ME) and hexane extract fraction (HEF) were concentrated

in-vacuo using rotary evaporator and yielded percentage was calculated. The dried extracts

were stored in amber coloured bottles and kept in the refrigerator until use.

2.2.3. Fractionation of crude methanol extract using column chromatography

Crude methanol extract (ME) (850.60 g) was adsorbed on silica gel (60-200 mesh) in

a glass column, was then eluted in succession with dichloromethane, ethyl acetate and

washed finally with methanol to yield dichloromethane fraction (DF), ethyl acetate fraction

(EAF) and methanol fraction (MF). These extract fractions were air dried at room

temperature for 24 h and the percentage yeilds of the fractions were calculated.

2.2.4. Qualitative phytochemical analysis

The hexane extract fraction (HEF), crude methanol extract (ME) and its fractions of Moringa

oleifera root bark was subjected to phytochemical tests using established standard procedures

[347 - 349].

1. Test for carbohydrate

Molisch test

The extract/fraction (0.1 g) was boiled with 2 ml of distilled water and filtered. To the filtrate,

few drops of naphthol solution in ethanol (Molisch’s reagent) were added. Concentrated

sulphuric acid was then gently poured down the side of the test tube to form a lower layer. A

purple interfacial ring indicates the presence of carbohydrate.

88

2. Test for alkaloids

Twenty ml of 3 % sulphuric acid in 50 % ethanol was added to 2 g of the extract and

heated on a boiling water bath for 10 min, cooled and filtered. A 2 ml volume of the filtrate

was tested with a few drops of Mayer’s reagent (potassium mercuric iodide solution),

Dragendorff’s reagent (bismuth potassium iodide solution), Wagner’s reagent (iodine in

potassium iodide solution), and picric acid solution (1 %). The remaining filtrate was placed

in 100 ml separatory funnel and made alkaline with dilute ammonia solution. The aqueous

alkaline solution was separated and extracted with two 5 ml portions of dilute sulphuric acid.

The extract was tested with a few drops of Mayer’s, Wagner’s, Dragendorff’s reagents and

picric acid solution. Alkaloids give milky precipitate with few drops of Mayer’s reagent;

reddish brown precipitate with few drops of Wagner’s reagent; yellowish precipitate with few

drops of picric acid and brick red precipitate with few drops of Dragendorff’s reagent.

3. Test for reducing sugar

Five ml of a mixture of equal parts of Fehling’s solution I and II were added to 5 ml

of aqueous extract and then heated on a water bath for 5 min. A brick red precipitate shows

the presence of reducing sugar.

4. Test for glycosides

Five ml of dilute sulphuric acid was added to 0.1 g of the extract in a test tube and

boiled for 15minutes on a water bath, then cooled and neutralized with 20 % potassium

hydroxide solution. 10 ml of a mixture of equal parts of Fehling’s solution I and II was added

and boiled for 5 min. A more dense brick red precipitate indicates the presence of glycoside.

89

5. Test for saponins

Twenty ml of distilled water was added to 0.25 g of the extract and boiled on a hot water bath

for 2 min. The mixture was filtered while hot and allowed to cool and filtrate was used for the

following tests.

(a) Frothing test

Five ml of the filtrate was diluted with 15 ml of distilled water and shaken vigorously.

A stable froth (foam) upon standing indicates the presence of saponins.

(b) Emulsion test

To the frothing solution was added 2 drops of olive oil and the contents shaken

vigorously. The formation of emulsion indicates the presence of saponins.

(c) Fehling’s test

To 5 ml of the filtrate was added 5 ml of Fehling’s solution (equal parts of Fehling’s

solution 1 and 11) and the contents were heated on a water bath. A reddish precipitate which

turns brick red on further heating with sulphuric acid indicates the presence of saponins.

6. Test for tannins/polyphenols

One g of the powered material was boiled with 20 ml of water, filtered and used for

the following test.

(a) Ferric chloride test

To 3 ml of the filtrate, few drops of ferric chloride were added. A greenish black precipitate

indicates the presence of tannins.

90

(b) Lead acetate test

To a little of the filtrate was added lead acetate solution. A reddish colour indicates

the presence of tannins.

7. Test for flavonoid

Ten ml of ethyl acetate was added to 0.2 g of the extract and heated on a water bath

for 3 min. The mixture was cooled, filtered and the filtrate was used for the following tests.

(a) Ammonium test

Four ml of filtrate was shaken with 1 ml of dilute ammonia solution. The layers were

allowed to separate and the yellow colour in the ammoniacal layer indicates the presence of

flavonoids.

(b) 1 % Aluminium chloride solution test.

Another 4 ml portion of the filtrate was shaken with 1 ml of 1 % Aluminium chloride

solution. The layers were allowed to separate. A yellow colour in the Aluminium chloride

layer indicates the presence of flavonoids.

8. Test for resins

(a) Precipitation test

The extract (0.2 g) was extracted with 15 ml of 96 % ethanol. The alcoholic extract

was poured into 20 ml of distilled water in a beaker to obtain a precipitate indicating the

presence of resins.

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(b) Colour test

The extract (0.2 g) was extracted with chloroform and the extract was concentrated to

dryness. The residue was redissolved in 3 ml of acetone and another 3 ml concentrated

hydrochloric acid was added. This mixture was heated in a water bath for 30 minutes. A

pink colour which changes to magenta red indicates the presence of resins.

9. Test for proteins

Twenty ml of distilled water was added to 0.5 g of the extract and the filtrate was used for the

following tests.

(a) Millon’s test

To a little portion of the filtrate in a test tube, two drops of Million’s reagent were

added. A white precipitate indicates the presence of proteins.

(b) Xanthoproteic reaction test

Five ml of the filtrate was heated with few drops of concentrated nitric acid. A

yellow colour which changes to orange on addition of an alkali (dilute Sodium hydroxide)

indicates the presence of protein.

(c) Picric acid test

To a little portion of the filtrate was added a few drop of picric acid. A yellow

precipitate indicates the presence of proteins.

(d) Biuret test

A crystal of copper sulphate was added to 2 ml of the filtrate, and then 2 drops of potassium

hydroxide solution was added. A purple or pink colour shows the presence of proteins.

92

10. Test for fats and oil

The extract (0.1 g) was pressed between filter paper and the paper was observed. A

control was also prepared by placing 2 drops of olive oil on filter paper. Translucency of the

filter paper indicates the presence of fats and oil.

11. Test for steroids and terpenoids

Nine ml of ethanol was added to 1 g of the extract and refluxed for few minutes and

filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. 5 ml of hot distilled

water was added to the concentrated solution, the mixture was allowed to stand for 1 hour

and the waxy matter was filtered off. The filtrate was extracted with 2.5 ml of chloroform

using separating funnel. To 0.5 ml of the chloroform extract in a test tube was carefully

added 1ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface

shows the presence of steroids.

Another 0.5 ml of the chloroform extract was evaporated to dryness on a water bath and

heated with 3ml of concentrated sulphuric acid for 10 min on a water bath. A grey colour

indicates the presence of terpenoids.

12. Test for acidic compounds

The extract (0.1 g) was placed in a clear dry test tube and sufficient water added. This

was warmed in a hot water bath and then cooled. A piece of water-wetted litmus paper was

dipped into the filtrate and the colour change on the litmus paper was observed. Acidic

compounds turn blue litmus paper red.

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2.2.5. Media preparation

The media used for culturing and sub culturing of the Clinical isolates were prepared

according to manufacturer’s Protocols.

2.2.6. Standardization of inoculum

The inocula were prepared from the stock cultures, which were maintained on nutrient

agar slant at 4 OC and subcultured onto nutrient broth using sterilized wire loop. The density

of suspension inoculated onto the media for susceptibility test was determined by comparison

with 0.5 McFarland standard of barium sulphate solution [344].

2.2.7. Characterization of the clinical isolates

The clinical isolates obtained from the clinical samples collected were characterised

as described.

1. Cultural characterization

The cultural characteristics of the isolates were based on their shapes, opacity, pigments,

surface texture, edge, water emulsification [350].

2. Microscopic characterization

Gram staining reaction and their appearance based on shape, arrangement and spore

formation. [350].

3. Biochemical tests

Catalase Test: The test was performed by covering an agar slant culture of the isolate

with several drops of 3 % hydrogen peroxide [351]. Mannitol Salt Agar (MSA) Test: The

plates were prepared with 7.5 % NaCl [1.28 M, approx 8.5 × isotonic (physiological) saline]

and mannitol agar was prepared according to the manufacturer’s protocol followed by

inoculation of the isolates.

94

4. Slide coagulase/staphylase test

The test was carried out according to manufacturer’s protocol, using the Oxoid

staphylase tests kits.

Test and control reagents were shook vigorously to obtain a homogenous suspension

in order to avoid trapping of reagent cells in the dropping pipette; with the loop 1 to 3 of the

suspected colonies was smeared on a test circle and control circle on the reaction card. 1

drop of the test reagent was added to the test circle and 1 drop of control reagent was also

added to the control circle, the contents of the test circle and the content of the control circle

were mixed separately with the sterile loop and observed for agglutination while mixing. The

reaction cards were disposed safely into disinfectant after recording the test result. A positive

result is obtained if clumping of the test cell suspensions occurs during mixing. This indicates

the presence of Staphylococcus aureus. Results cannot be interpreted if there is any clumping

of the control cell suspension. In these cases, cultural purity and identity should be checked.

Limitations of the test

Occasional false positive results may be found with strains of Staphylococcus sciuri.

Other rarely isolated staphylococci may also give positive Staphylase results. Suspected

isolates should be identified by biochemical tests.

2.2.8. Antimicrobial susceptibility testing

A standard disc diffusion technique for antimicrobial susceptibility testing was

performed as recommended by the Clinical Laboratory Standards Institute (CLSI) [352] on

Mueller Hinton agar. Standard antibiotics disks (Oxoid, Ltd) set were oxacillin (5 µg),

vancomycin (30 µg), cephalexin (30 µg), levofloxacin (5 µg), ciprofloxacin (5 µg),

tetracycline (30 µg), cotrimoxazole (25 µg), gentamycin (30 µg), clindamycin (2 µg),

rifampicin (5 µg).

95

A direct colony method {preferred method for staphylococci}(CLSI) [352] was used,

pure isolated colonies were taken from Mueller Hinton agar into sterile broth tubes and a

suspension equivalent to a 0.5 McFarland standard was prepared and used to inoculate the

test media. The discs were applied to the plate within 15 minutes of inoculation and incubated

for 24 hours at 35 OC (methicillin resistant staphylococci may not be detected at temperatures

above 35 OC) [352].

2.2.9. Penicilin-binding protein (PBP2̍ ) latex agglutination test for MRSA

confirmation

(a) Preparation of culture

The PBP2̍ test should be performed only on Staphylococcus species (Gram+positive

cocci). A coagulase test confirming the isolates used for this test to be S. aureus was done

prior to the PBP2̍ test. A pure clinical isolates of S. aureus were used for this test. MRSA

strain ATCC® 43300 (Oxoid Culti – Loops C9022) was used as positive control.

(b) Test method

(i) The PBP2̍ extraction procedure as recommended by the Manufacturer Oxoid,

Four drops of Extraction Reagent 1 was added to a micro centrifuge tube, an approximately

1.5 x 109 (3-5 μl) cells was then suspended into the micro centrifuge tube to obtain a very

turbid suspension. The tube was placed into a water bath at temperature over 95 oC and

allowed to heat for three minutes, it was removed and allowed to cool to room temperature

before adding a drop of extraction reagent 2 and the mixture was vigorously shook to obtain

homogenous mixture. The mixture was centrifuged at 3000 rpm at 15 cm rotation radius for 5

min to obtain a supernatant solution containing the extracted PBP2ʹ for MRSA.

96

(ii) Latex agglutination procedure

For each supernatant to be tested, one circle of the test card was labeled ‘T’ for testing

with Test Latex and another with ‘C’ for Control Latex. The latex reagent was properly

mixed by inversion several times and a drop of test Latex or Control Latex was added to each

labeled circle accordingly. 50 μl of supernatant was placed on the Test circle and the Control

circle and mixed thoroughly with the latex with the aid of the provided sterile plastic mixing

stick. The mixing was done for three minutes and observed for agglutination under normal

lighting conditions. The results of the Test and Control reactions were recorded before

disposing the reaction card safely into disinfectant. If agglutination is seen with test but not

with Control Latex within three minutes PBP2ʹ is positive (MRSA), if no agglutination in

either latex test and control test within 3 minutes PBP2́ is negative (MSSA). If agglutination

is seen in the latex test and the control test within 3 minutes Intermediate VISA (Vancomycin

intermediate S. aureus.) is positive.

2.3 Determination of MIC and MBC of the extracts and fractions on MRSA

clinical isolates.

2.3.1 Preparation of stock solution

Stock solution (50 mg/ml) of the plant extracts were prepared by dissolving 1250 mg

extracts and fractions in 25 ml of sterile water and DMSO (dimethyl sulfoxide) diluted in 1:5

(DMSO:Water). The DMSO is an organic solvent that aid the dissolution of organic

substance that will not dissolve easily in water alone. The water and DMSO dilution was

carried out to avoid the interference of DMSO with original activity of the extract [352].

Twenty five ml of stock is prepared to allow for 5 ml overage. Dimethyl sulfoxide has been

shown to improve the efficiency of fungicides, to possess anti-inflammatory effects, as well

as additional non-specific biological effects and it is for this reason that control experiments

were conducted in all cases to account for additive effects, if any.

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2.3.2. Preparation of extract and fractions solutions for agar dilution MIC test

The different concentrations of the crude methanol extract and fractions prepared for the MIC

and MBC tests were shown in table 1. Twenty ml volume of Muller Hinton Agar (MHA) was

used in 9 cm Petri dishes for agar dilution MICs. Dilution schemes using formula

C1V1 = C2V2 are given in the Table [352].

C1 = Stock concentration of the extract and fractions = 50 mg/ml

V1 = Volume of the extract and fractions in the agar dilution = to be determined

C2 = Concentration of the extract and fraction in agar dilution (1 mg/ml – 10 mg/ml)

V2 = Volume of reaction mixture in MHA plate = 20 ml

2.3.3. Determination of MIC of methanol extract and fractions

By means of a sterile calibrated micro pipette, 0.002 ml of the MRSA clinical isolates

suspension was streaked with a sterile loop on the surface of the MHA and allowed for 10

minutes for complete absorption of the inoculum by the medium. The plates were incubated

in an inverted position at 37 Oc for 24 h before taking the results. The least concentration that

inhibits the growth of the organism is taken as the MIC (minimal inhibitory concentration).

The control plate without antimicrobial agents was also incubated [344].

2.3.4. Determination of MBC of methanol extract and fractions

The value of MBC is an extension of MIC. The agar plates showing no growth in the

MIC tests were used for the determination of the MBC. Discs were cut from the agar plate of

the MIC concentration and two preceding concentrations and transferred into the

corresponding containers of the fresh Muller Hinton broth (recovery medium). The media

were also incubated at 35 OC for 48h. At the end of incubation the media were observed for

any visible growth or turbidity. The absence of growth in the recovery medium is evidence of

total cell death. The minimal concentration of the antimicrobial agent that produces total cell

death is taken as the MBC [344].

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Table 1: Preparation of extract and fractions concentrations for agar dilution MIC test

S/N C1 (mg/ml ) V1 (ml) C2 (gm/ml) Volume of

MHA (ml)

V2 (ml)

Volume of reaction

mixture

1 50 4.00 10 16.00 20

2 50 3.60 9 16.40 20

3 50 3.20 8 16.80 20

4 50 2.80 7 17.20 20

5 50 2.40 6 17.60 20

6 50 2.00 5 18.00 20

7 50 1.60 4 18.40 20

8 50 1.20 3 18.80 20

9 50 0.80 2 19.20 20

10 50 0.40 1 19.60 20

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2.3.5. Gas Chromatography–Mass Spectrometry (GC-MS) determination of bioactive

components of methanol extract fractions

Gas chromatography-mass spectrometry was performed on the methanol (MEF), ethyl

acetate (EAF), n-hexane (HEF) and dichloromethane (DF) fractions of the Moringa oleifera

root bark. This is to identify the constituents of these fractions. The GC-MS analysis of the

extract fractions were performed on an Agilent technologies model 7890 Series GC System

equipped with an Agilent technologies 5975 MS detector (EI mode, 70 eV). A column type

DB-5 (5 % phenyl methylsiloxane) with a length of 30 m, an inside diameter of 0.25 µm and

a film thickness of 320 μm was used. The temperature of the column was programmed to

increase after 5 min from 50 oC to 280 oC at the rate of 5 oC/min after which the run was left

for 9 min at 280 oC. Helium was used as a carrier gas at a flow rate of 1.4 ml/min. The

injector and detector temperatures were 300 oC and 250 oC, respectively. The components in

the extract samples under investigation were identified by comparing on the basis of gas

chromatographic retention indices, mass spectra from National institute of standards and

technology (NIST) Standard Reference Database 1A (NIST/EPA/NIH Mass Spectral

Database (NIST 11) and NIST Mass Spectral Search Program (Version 2.0 g), Agilent

Technologies, Inc. ChemStation Version) [353 - 357].

2.3.6 Identification of components in methanol extract fractions

The constituents of the methanol extract fractions were identified by comparison of

their mass spectra with those in the MS library (Wiley7Nist) incorporated in the HP

Enhanced ChemStation software and those in literature [356-360].

100

2.3.7 Structures of some compounds identified in methanol extract fractions

The structure of some distinct bioactive compound identified from (MS report) in

Moringa oleifera lam. Fam. Moringaceae root bark with reference to Chem.spider,

Pub.chem, NIST, Look.chem and Chem.book data bases were reported [360-364].

2.4 Preliminary toxicological evaluation of the crude methanol extract (ME) and n-

hexane extract (HE) fraction

2.4.1. Acute Toxicity Study

Acute oral toxicity study was performed in accordance to OECD guidelines (Orga-

nization of Economic Cooperation and Development) 423 guide-line (Acute toxic class

method) [345]. The acute oral toxicity of the extracts was determined using Miller and

Tainter method [365]. Toxicity assay was performed in male albino Wistar rats (80-150g),

the rats were randomly divided into 6 groups with 6 animals in each group. The animals were

kept fasting overnight provided only water, after which the methanol extract of the roots was

administered orally with increasing doses (50 mg/kg, 250 mg/kg, 500 mg/kg, 1000 mg/kg,

5000 mg/kg, 10 g/kg body weight) by intra-gastric tube to determine the safe dose. The

control group (group 7) was treated with orally administered distillated water (2 ml/kg) only.

The animals were observed continuously for 1h, then frequently for 4h and later at the end of

24 h for general behavioral, neurological and autonomic profile.

2.4.2. Sub-acute toxicity study

The animals were divided into four groups with control, given daily oral graded doses

of the extracts 50 mg/kg, 250 mg/kg, 500 mg/kg, 1000 mg/kg bodyweight [366]. The

experiment was carried out for 21 days, with oral administration of methanolic extract and n-

hexane extract fraction. At the end of 21 days, the animals were deprived of food overnight

101

and sacrificed by cervical decapitation for hematological, liver and biochemical parameters.

This experiment was repeated thrice for confirmation of results.

(a) Haematological parameters

A 2.0 ml portion of the blood samples collected in EDTA (Ethylene diamine

tetraacetic acid) was used. The samples were mixed in a Roller mixer at the rate of 30 per

minute for 5 min. The following blood parameters white blood cell (WBC), red blood cells

count (RBC), haemoglobin count (HGB), mean concentration volume (MCV), platelet

counts (PLT), and mean packed volume (MPV), were determined using an automated

haematological analyzer machine (Abacus Junior, Model S/N 111244).

(b) Effect on the liver enzymes

A 3 ml volume of the sample collected in a serum extractor was used for the study.

The sample was allowed to stand undisturbed for 1 h away from sunlight. This was followed

by spinning for 5 min, to separate the serum from the clotted red cells. The resulting

supernatant was used for the assessment of liver integrity. Using a 3.2 microlitre automated

pipette, a sample was dropped on the sample spot of each liver function test kit and tested for

the following enzymes, alanine aminotransferase (ALT), aspatate aminotransferase (AST),

alkaline phosphatase (ALP). Bilirubin concentration was also analysed using a Reflotron-Plus

machine (Model: SN747461 Germany).

(c) Biochemical parameters of the kidney

The effects of the preparations on certain biochemical parameters of the kidney were

compared with those of the control. Serum from the blood of each rat was collected into

lithium heparin bottles, mixed properly and used for the following kidney indices urea and

creatinin within 24 hours of collection using the following procedure.

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(d) Urea concentration

Three different test tubes (labelled as A, B and C) were used for the test. To the test tubes,

10 μl of sample, calculated standard of a known urea and blank (distilled water) were added

respectively. This was followed by addition of 100 μl of reagent (sodium nitroprusside), mixing

thoroughly by shaking and incubating at 37oC for 10 min. About 2.5 ml of diluted phenol and sodium

hypochlorite mixture added into the tubes, mixed and incubated for another 15 min. The

absorbances of the A and B against the C were taken using the spectrophotometer at a wavelength

of 500 nm and the concentration of the urea calculated as follows:

2.5. Statistical analysis

Results were expressed as mean ± SD and differences between sets obtained were

determined using ANOVA followed by Duncan post Hoc Test with the use of SPSS v 17

software. Differences were considered significant at p < 0.05.

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CHAPTER THREE

3.0 RESULTS AND DISCUSSION

3.1. Results

3.1.1 The percentage yield of the methanol extracts and fractions

The percentage yield of the crude extracts and fractions are as presented in Table 2.

The total amount of crude extract obtained shows that methanol was quantitatively a

good solvent for the plant while n-hexane extract fraction had the lowest yield. The

percentage yield of the extracts and fractions is in the order of n-hexane extract fraction

(HEF) 68.80 ± 13.7 g (2.29 %) < methanol crude extract 850.60 ± 20.8 g (28.35 %) and

fractions of Methanol crude are in the order of ethyl acetate fraction 50.90 ± 8.7 g (5.98 %)

< dichloromethane fraction 150.70 ± 9.5 g (17.71 %) < methanol fraction 350.60 ± 14.8g

(31.21 %). There was significant (p < 0.05) difference in the percentage yield of the various

fractions, methanol fraction having the highest yield of 31.21 % and n–hexane, the lowest

yield of 2.29 %.

3.1.2 Qualitative phytochemical analysis of the extracts and fractions

The results of the qualitative phytochemical analyses of methanol crude extract and

fractions as presented in Table 3, showed that for crude methanol extract (ME), alkaloids

were abundantly present, proteins and carbohydrates present in high concentrations, tannins,

resins, glycosides, steroids, flavoinoids, reducing sugars, fats and oil, sapponins are all

present in moderately high concentrations with terpenoids present in low concentrations. The

fractions of the extracts showed varying concentrations of phytochemical constituents; n-

hexane fraction is very rich in steroids, terpenoids, fats and oils are abundantly present in the

fraction.

104

Table 2: The percentage yield of the crude methanol extract and fractions of M. oleifera

root bark

Initial weight of ground

moringa root bark 3000 g

Final weight of extract (g)

Percentage yield of

extract

Crude methanol extract

N-hexane extract fraction

850.60 ± 20.80

68.80 ± 13.70

28.35 %

2.29 %

Percentage yield of methanol fractions

Ethyl acetate fraction

Dichloromethane

Methanol fraction

50.90 ± 8.70

150.70 ± 9.50

350.60 ± 14.80

5.98 %

17.71 %

31.21 %

105

Table 3: Results of phytochemical analysis of the crude methanol extracts and fractions

Chemical constituent

Test Crude methanol extract (ME)

N-hexane fraction (HEF)

Dichloromethane fraction (DF)

Ethyl acetate fraction (EF)

Methanol fraction (MF)

Alkaloids

Dragendorff’s reagent Mayer’s reagent Wagner’s reagent

++++ - + - ++

Glycosides

Fehling’s solution I and II

+ + - - + +

Steroids

General Test + + + + + + + + + -

Terpenoids

General Test + + + + + + -

Flavonoids

Ammonium Test 1 % Aluminium Chloride solution Test.

+ + - + ++++ +

Saponins

Frothing Test Emulsion Test Fehling’s Test

+ + - - _ +

Tannins

Ferric chloride Test Lead Acetate Test

+ + - - _ ++

Resins

Precipitation Test Colour Test

++ + + + + ++ _ +

Reducing Sugar

Fehling’s solution I and II

++ - - _ ++

Proteins Millon’s Test Xanthoproteic Reaction Test Picric Acid Test Biuret Test

+++ - _ _ +

Fats and Oil

General filter paper Test

++ ++++ ++ _ _

Carbohydrate

Molisch’s +++ - - + ++

Key: (-): Not present. (+): Present in small concentration. (++): Present in moderately high concentration. (+++): Present in very high concentration. (++++): Abundantly present

106

Dichloromethane fraction is rich in resins with moderate concentration of steroids and

fats and oil, ethyl acetate fraction is rich in flavonoids which is abundantly present, with low

concentrations of steroids, terpenoids and carbohydrates, methanol fractions is with moderate

concentrations of alkaloids, tannins, resins, glycosides, saponin and low concentration of

flavonoids and no steroids and terpenoids present in the fraction.

3.2 Characterisation of clinical isolates The characterisation of the 2,372 isolates was done in sequence to eliminate the

unwanted isolates and to confirm the S. aureus isolates. The clinical isolates were separated

into two major groups, cocci and non-cocci isolates by cultural and microscopic characters

with gram staining reaction given 1,860 (78.41 %) of the isolates suspected to be

staphylococci.

A total of 1860 clinical isolates suspected to be staphylococci were subjected to

catalase test, catalase positive organisms further revealed 839 out of the 1860 isolates to be

staphylococci 45.1 % of the suspected clinical isolates subjected to the test.

The mannitol salt agar test revealed 218 of the clinical isolates which represent 25.98 % of

the catalase positive to be S. aureus grew in 7.5 % Nacl and fermented the Mannose sugar

giving a yellow acidic color.

The coagulase test confirmed 58 (26.6 %) clinical isolates to be S. aureus, 7 from

sputum, 10 from skin swab, 11 from abscess, 19 from open wound and 11 from ear/nasal

swab.

107

3.3 Antimicrobial susceptibility test

The antimicrobial susceptibility test was done to check the susceptibility pattern of the

isolates to various antibiotics disc used in the study as shown in Table 4. The results showed

susceptibility and resistant pattern of the antibiotics disc used on the 58 clinical isolates of

Staphylococcua aureus. It was observed that out of 58 clinical isolates of S.aureus tested for

resistance, 39 isolates showed resistance in varying degree to the antibiotics, while 19 of the

isolates were sensitive to all the antibitotics. MSSA 32.75 % and MRSA 67.24 %. Cephalexin

having the highest 26.00 ± 1.00 (44.82 %) and tetracycline, the lowest 18.33 ± 0.58 (31.03

%) susceptibility. However, there was no significant (p > 0.05) difference in susceptibility

between cephalexin and levofloxacin and also in the resitance pattern. There was also

significant (p < 0.05) difference among the various antimicrobials resistance, tetracycline

having the highest 39.67 ± 0.58 (68.97 %) and cephalexin, the least 32.33 ± 0.58 (55.18 %)

resistance. Cotrimoxazole had similar resistance to tetracycline (p > 0.05) also with resitance

of 67.25 %. Oxacillin has 37.93 % sensitivity, 62.07 % resistance and vancomycin has 39.65

% sensitivity and 60.35 % resitance by the isolates.

Vancomycin has been the drug of choice for MRSA infections until the first S. aureus

isolate with reduced sensitivity to vancomycin (vancomycin intermediate Staphylococcus

aureus (VISA)) was reported in Japan in 1997 [3]. This result has shown that most of the

MRSA isolates used in this study are vancomycin resistant. Hospital acquired MRSA (HA-

MRSA) and they are also multidrug resistant as seen in the susceptibility pattern in table 4,

with over 60% of the S. aureus resisting vancomycin.

108

Table 4: Antimicrobial susceptibility test

S/N Antimicrobial MSSA % MSSA MRSA % MRSA

1 Oxacilin 5 µg/ml 22.3 ± 0.5 37.93 36.0 ± 0.5 62.07

2 Vancomycin 30 µg/ml 23.0 ± 1.0 39.65 35.6 ± 1.1 60.35

3 Cephalexin 30 µg/ml 26.0 ± 1.0 44.82 32.3 ± 0.5 55.18

4 Levofloxacin 5 µg/ml 25.3 ± 0.5 43.10 32.6 ± 0.5 56.90

5 Ciprofloxacin 5 µg/ml 21.0 ± 0.5 34.48 37.6 ± 0.5 65.52

6 Tetracycline 30 µg/ml 18.3 ± 0.5 31.03 39.6 ± 0.5 68.97

7

Cotrimoxazole

25µg/ml

20.0 ± 0.5 32.75 39.3 ± 0.5 67.25

8 Gentamycin 30µg/ml 21.3 ± 1.0 37.93 36.3 ± 0.5 62.07

9 Clindamycin 2 µg/ml 21.3 ± 0.5 36.20 37.3 ± 0.5 63.79

10 Rifampicin 5µg/ml 21.3 ± 0.5 37.93 36.3 ± 0.5 62.07

Values were expressed as Mean ± SD, N = 3

MSSA – Methicilin sensitive S. aureus

MRSA – Methicilin resitant S. aureus

109

3.4 Penicilin-binding protein (PBP2̍) latex agglutination test results

The Clincal isolates were subjected to latex agglutination test and the results are as

presented in Table 5.

The 39 Clinical isolates of S. aureus with varying percentages of resistance to the 10

antibiotics used for the susceptibility study were subjected to PBP2ˈ latex agglutination test

and result gave different types of resistance strains as indicated by latex agglutination test

into VISA and MRSA. The results confirmed 39 strains of the isolates as MRSA or VISA.

3.5 Prevalence of clinical isolates of MSSA and MRSA

The prevalence of the S. aureus isolates of the different specimen are presented in

Table 6 and it shows the number of staphylase positive and degrees of occurrence of the

MSSA and MRSA from different source of the clinical isolates, staphylase positive S. aureus,

MSSA and MRSA prevalence is as follow: Open wound (coagulase positive 19, MSSA 5,

MRSA 14), greater than Abscess (coagulase positive 11, MSSA 3, MRSA 8), greater than

Ear/Nasal swab (coagulase positive 11, MSSA 4, MRSA 7), greater than Skin swab

(coagulase positive 10, MSSA 4, MRSA 6), greater than Sputum (coagulase positive 7,

MSSA 3, MRSA 4). The results shows open wound as a source with highest prevalence and

sputum with lowest prevalence of the S.aureus and the MRSA.

110

TABLE 5

111

Table 6: Prevalence of clinical isolates of MSSA and MRSA

Samples Staphylase

positive

S. aureus

MSSA % MSSA Latex test MRSA % MRSA

Sputum

Skin swab

Abscess

Open wound

Ear/Nasal

Total

7

10

11

19

11

58

3

4

3

5

4

19

5.17

6.89

5.17

8.62

6.89

32.74

4 PBP2́+ve

6 PBP2́ +ve

8 PBP2́+ve

14 PBP2́+ve

7 PBP2́ +ve

39 PBP2́+ve

4

6

8

14

7

39

6.89

10.34

13.79

24.13

12.06

67.21

112

3.6 Results of the MIC and MBC of the extracts and fractions on MRSA clinical

isolates.

The results of the MIC and MBC of the extracts and fractions are presented in tables

7, 8, 9, 10 and 11 respectively.

The MIC and MBC as displayed in table 7, showed that the methanol crude extract is

highly effective against all the 39 MRSA clinical isolates, with MIC ranging from

(3.0 ± 0.1 - 5.0 ± 0.5) and MBC ranging from (4.0 ± 0.5 - 5.6 ± 1.1). The least MIC is seen in

clinical isolate OW 36 (3.0 ± 0.1) an isolate from the open wound and highest MIC is seen in

EN 208 (5.0 ± 0.5) an isolate from Ear/Nasal swab. The least MBC is seen in AB20 (4.0 ±

0.5) an isolate from abscess and highest MBC (5.6 ± 1.1) is seen with OW 940, OW 1104 AB

1009 and AB 1956 isolates fron open wound and abscess respctively. There is no significance

difference (P < 0.05) in the activities of the extract against the isolates except some

differences in between the isolates that is observed at the lower MIC and MBC and higher

MIC and MBC.

As shown in Table 8, the ethyl acetate fraction (EF) showed better activity next to the

methanol crude extract than remaining fractions, possibly because of the presence of high

concentration of flavonoids which has been shown to enhance antimicrobial property of a

plant [13]. The MIC and minimal MBC as displayed in the table shows that the ethyl acetate

fraction is also effective against all the 39 MRSA clinical isolates, with MIC ranging from

(3.5 ± 0.3 - 6.6 ± 0.5) and MBC ranging from (4.3 ± 0.5 -7.5 ± 0.5). The least MIC is seen in

clinical isolate OW 947 (3.5 ± 0.3) an isolate from open wound and highest MIC (7.5 ± 0.5)

is seen in OW53, AB61, EN127 and AB187 isolates from open wound, abscess and ear/nasal.

The least MBC is seen in OW 819 (4.3 ± 0.5) an isolate from open wound and highest MBC

is seen in EN 127 (6.6 ± 0.5) isolates from ear/nasal swab. There is no significance difference

(p < 0.5) in the response of the isolates to the extract.

113

The MIC and MBC as displayed in Table 9 showed the results of the antibacterial

activity of the hexane extract fraction (HEF) against the MRSA clinical isolates. The MIC

and MBC as displayed in the table showed that the HEF is also effective against all the 39

MRSA clinical isolates, with MIC ranging from (5.3 ± 1.5 - 7.6 ± 0.5 ) and MBC ranging

from (5.6 ± 1.1 - 8.8 ± 0.5). The least MIC is seen in clinical isolate AB20 (5.6 ± 1.1) an

isolate from absces and highest MIC AB570 (7.6 ± 0.5) was also from abscess. The least

MBC is seen in OW 123 (5.6 ± 1.1) an isolate from open wound and highest MBC is seen in

SP 651 (8.8 ± 0.5) isolate from sputum. There is no significance difference (P < 0.5) in the

activities of the extract.

The MIC and MBC as displayed in Table 10, showed the results of the

antibacterial activity of the dichloromethane fraction (DMF) against the MRSA clinical

isolates. DMF shows activity against all the MRSA isolates. The MIC and MBC as displayed

in the table shows that DMF is also effective against all the 39 MRSA clinical isolates, with

MIC ranging from (6.3 ± 1.1 - 9.3 ± 0.5) and MBC ranging from (7.5 ± 1.0 - 9.7 ± 1.1). The

least MIC is in clinical isolate SS8 (6.3 ± 1.1) an isolate from sputum and highest MICs in

AB 841 and OW 1827 (9.3 ± 0.5) were isolates from abscess and open wound. The least

MBC is seen in SS8 (7.5 ± 1.0) an isolate from abscess and highest MBC is seen in AB 187

and OW 940 (9.7 ± 1.1) isolate from abscess and open wound. There is no significance

difference (P < 0.5) in the activities of the extract against the MRSA.

From Table 11, the results of the antibacteriail activity of MF against the MRSA

clinical isolates. MF showed activity against some of the MRSA clinical isolates but not all,

some resisted the MF fraction at the concentration range of 1mg/ml -10mg/ml used for this

study. The methanol fraction MIC ranges from (8.3 ± 1.1- 9.8 ± 1.1) and MBC ranging from

(9.3 ± 0.5 - 10 ± 1.1) in the isolates that are susceptible to the fraction. The least MIC is in

clinical isolate SS8 (8.3 ± 1.1) an isolate from sputum and highest MICs in OW 578 and OW

114

947 (9.8 ± 1.1) were isolates from open wound. The least MBC is seen in SS235 (9.3 ± 0.5)

an isolate from sputum and highest MBC was seen in AB20, OW30, OW36, OW1104 and

EN127 (10 ± 0.5) isolate from abscess, open wound and ear/nasal swab. At the concentration

range of 1 mg/ml to 10 mg/ml used for this study the following isolates were not inhibited by

the MF: MRSA isolates with MIC but no MBC (EN38, EN 208), (OW 53, OW 154, OW 947,

OW 1420,), SP 651. These isolates are, 2 from ear/nasal swab, 4 from open wound and 1

from sputum. MRSA isolates with complete resistance to MF: (EN 390, EN 831), (AB 600,

AB 1009), (SS 310), (SP 1172), (OW 620, OW 940) 2 from ear/nasal swab, 2 from abscess, 1

from skin swab, 1 from sputum, 2 from open wound.

This result showed MF as the least potent of the fraction with weak antibacterial activity

generally against all the isolates considered for the study.

115

Table 7: MIC and MBC of methanol crude extract in mg/ml

Values were expressed as Mean ± SD, N = 3

Key:

SP: Sputum

SS: Skin swab

AB: Abscess

OW: Open wound

EN: Ear/Nasal

S/

N

Clinical

isolates

MIC MBC S/N Clinical

isolates

MIC MBC

1 SP4 4.3 ± 0.5 5.0 ± 0.5 21 EN390 4.3 ± 0.5 5.3 ± 1.1

2 SS8 3.3 ± 0.5 5.3 ± 1.1 22 SS310 3.3 ± 0.3 4.6 ± 0.5

3 AB20 3.3 ± 0.5 4.0 ± 0.5 23 OW417 3.0 ± 0.3 4.3 ± 0.3

4 SP22 4.6 ± 0. 5 4.3 ± 0.5 24 AB570 3.3 ± 0.1 3.6 ± 0.5

5 OW30 4.0 ± 0.5 4.6 ± 0.5 25 OW578 3.3 ± 0.5 4.3 ± 1.0

6 SS33 4.3 ± 0.5 5.3 ± 1.0 26 AB600 3.0 ± 0.5 5.3 ± 1.0

7 EN35 4.0 ± 0.5 5.3 ± 0.5 27 OW620 3.3 ± 0.3 5.0 ± 0.5

8 OW36 3.0 ± 0.1 4.6 ± 0.5 28 SP651 3.3 ± 0.3 4.3 ± 0.5

9 EN38 3.3 ± 0.1 5.3 ± 0.3 29 OW819 3.0 ± 0.1 4.3 ± 0.5

10 SS42 3.3 ± 0.3 4.6 ± 0.5 30 EN831 3.0 ± 0.3 4.6 ± 1.0

11 OW53 3.3 ± 0.5 4.6 ± 0.5 31 AB841 3.6 ± 0.5 4.6 ± 0.5

12 SS57 4.3 ± 0.5 5.0 ± 1.0 32 OW940 4.0 ± 0.5 5.6 ± 0.3

13 AB61 3.3 ± 0.3 4.3 ± 0.5 33 OW947 4.3 ± 0.3 5.3 ± 0.3

14 EN62 3.3 ± 0.5 4.6 ± 0.3 34 AB1009 3.6 ± 0.3 5.6 ± 1.0

15 OW123 3.3 ± 0.5 5.3 ± 1.0 35 OW1104 4.0 ± 0.5 5.6 ± 0.5

16 EN127 4.0 ± 0.3 4.3 ± 0.5 36 SP1172 4.3 ± 0.3 5.3 ± 1.0

17 OW154 4.3 ± 0.5 5.3 ± 0.5 37 OW1420 3.6 ± 0.1 4.3 ± 0.3

18 AB187 4.3 ± 0.3 5.6 ± 1.1 38 OW1827 3.6 ± 0.3 5.0 ± 0.5

19 EN208 5.0 ± 0.5 5.3 ± 0.5 39 AB1956 4.6 ± 0.5 5.6 ± 1.1

20 SS235 4.6 ± 0.3 5.3 ± 0.5

116

Table 8: MIC and MBC of ethyl acetate fraction in mg/ml

Values were expressed as Mean ± SD, N = 3

Key:

SP: Sputum

SS: Skin swab

AB: Abscess

OW: Open wound

EN: Ear/Nasal

S/N Clinical isolates

MIC MBC S/N

Clinical isolates

MIC MBC

1 SP4 5.6 ± 0.5 6.3 ± 0.5 21 EN390 4.6 ± 0.5 5.3 ± 1.1 2 SS8 4.3 ± 1.1 5.6 ± 1.1 22 SS310 5.3 ± 0.3 6.6 ± 0.5 3 AB20 4.6 ± 1.5 6.6 ± 0.5 23 OW417 4.6 ± 0.3 5.3 ± 0.3 4 SP22 5.3 ± 0.5 6.3 ± 0.5 24 AB570 4.8 ± 0.2 5.6 ± 0.5 5 OW30 5.0 ± 0.5 6.6 ± 0.5 25 OW578 5.3 ± 0.5 6.5 ± 1.0 6 SS33 5.3 ± 0.3 6.3 ± 1.0 26 AB600 5.3 ± 1.1 5.3 ± 1.0 7 EN35 5.6 ± 0.5 7.3 ± 0.5 27 OW620 4.5 ± 0.5 5.0 ± 0.5 8 OW36 5.0 ± 0.5 6.3 ± 0.5 28 SP651 5.5 ± 0.5 6.6 ± 0.5 9 EN38 5.3 ± 0.5 6.6 ± 0.3 29 OW819 3.6 ± 0.3 4.3 ± 0.5 10 SS42 5.6 ± 0.3 6.3 ± 0.5 30 EN831 4.3 ± 0.3 4.6 ± 1.0 11 OW53 6.6 ± 0.5 7.3 ± 0.5 31 AB841 5.6 ± 0.5 6.6 ± 0.5 12 SS57 5.3 ± 0.5 6.6 ± 1.0 32 OW940 4.3 ± 0.5 5.6 ± 0.3 13 AB61 6.6 ± 0.3 7.3 ± 0.5 33 OW947 3.5 ± 0.3 5.3 ± 0.3 14 EN62 5.6 ± 0.5 6.6 ± 0.3 34 AB1009 4.5 ± 0.3 5.6 ± 1.0 15 OW123 5.0 ± 0.5 6.3 ± 1.0 35 OW1104 4.3 ± 0.5 5.6 ± 0.5 16 EN127 6.6 ± 0.3 7.5 ± 0.5 36 SP1172 5.6 ± 0.3 7.3 ± 1.0 17 OW154 5.6 ± 0.5 6.5 ± 0.5 37 OW1420 5.6 ± 0.5 6.3 ± 0.3 18 AB187 6.6 ± 0.3 7.2 ± 1.1 38 OW1827 5.3 ± 0.3 6.0 ± 0.5 19 EN208 5.3 ± 0.5 6.5 ± 0.5 39 AB1956 4.6 ± 0.5 5.6 ± 1.1 20 SS235 5.6 ± 0.3 6.3 ± 0.5

117

Table 9: MIC and MBC of n-hexane fraction in mg/ml

Values were expressed as Mean ± SD, N = 3

Key:

SP: Sputum

SS: Skin swab

AB: Abscess

OW: Open wound

EN: Ear/Nasal

S/N

Clinical isolates

MIC MBC S/N Clinical isolates

MIC MBC

1 SP4 6.3 ± 0.5 7.5 ± 0.5 21 EN390 7.5 ± 0.5 8.6 ± 1.1 2 SS8 5.5 ± 1.1 6.5 ± 1.1 22 SS310 6.3 ± 0.5 7.5 ± 0.5 3 AB20 5.3 ± 1.5 6.6 ± 0.5 23 OW417 5.6 ± 0.5 6.6 ± 0.5 4 SP22 6.5 ± 0.5 7.3 ± 0.5 24 AB570 6.5 ± 0.5 7.6 ± 0.5 5 OW30 7.3 ± 1.5 8.5 ± 0.5 25 OW578 6.7 ± 0.5 8.5 ± 1.0 6 SS33 5.6 ± 0.5 6.3 ± 1.0 26 AB600 7.5 ± 1.1 8.7 ± 1.0 7 EN35 7.3 ± 1.1 8.3 ± 0.5 27 OW620 6.8 ± 0.5 8.5 ± 0.5 8 OW36 6.5 ± 1.1 7.6 ± 0.5 28 SP651 7.6 ± 0.5 8.8 ± 0.5 9 EN38 6.7 ± 0.5 8.3 ± 0.5 29 OW819 5.8 ± 0.5 6.6 ± 0.5 10 SS42 7.3 ± 1.0 8.5 ± 0.5 30 EN831 7.5 ± 0.5 7.5 ± 1.1 11 OW53 6.8 ± 0.5 7.5 ± 0.5 31 AB841 6.8 ± 0.5 8.5 ± 0.5 12 SS57 7.3 ± 0.5 8.3 ± 1.0 32 OW940 7.5 ± 0.5 8.7 ± 0.5 13 AB61 5.6 ± 0.5 6.6 ± 0.5 33 OW947 6.9 ± 0.5 8.5 ± 0.3 14 EN62 6.5 ± 0.5 7.3 ± 0.5 34 AB1009 7.5 ± 0.5 7.6 ± 1.1 15 OW123 5.6 ± 0.5 5.6 ± 1.0 35 OW1104 6.3 ± 0.5 7.5 ± 0.5 16 EN127 6.7 ± 0.5 7,5 ± 0.5 36 SP1172 7.5 ± 0.5 8.6 ± 1.0 17 OW154 7.3 ± 0.5 7.3 ± 0.5 37 OW1420 6.8 ± 0.5 7.8 ± 0.5 18 AB187 6.8 ± 0.5 8.5 ± 1.1 38 OW1827 7.5 ± 0.35 7.5 ± 0.5 19 EN208 5.6 ± 0.5 6.5 ± 0.5 39 AB1956 6.5 ± 0.5 7.6 ± 1.1 20 SS235 6.6 ± 0.5 7.3 ± 0.5

118

Table 10: MIC and MBC of Dichloromethane fraction in mg/ml

Values were expressed as Mean ± SD, N = 3

Key:

SP: Sputum

SS: Skin swab

AB: Abscess

OW: Open wound

EN: Ear/Nasal

S/N

Clinical isolates

MIC MBC S/N Clinical isolates

MIC MBC

1 SP4 7.5 ± 0.5 8.7 ± 0.5 21 EN390 7.5 ± 0.5 8.6 ± 1.0 2 SS8 6.3 ± 1.1 7.5 ± 1.1 22 SS310 8.7 ± 1.1 9.5 ± 1.3 3 AB20 6.5 ± 1.5 7.8 ± 0.5 23 OW417 8.3 ± 0.5 9.6 ± 1.0 4 SP22 8.2 ± 0.5 8.2 ± 0.5 24 AB570 8.7 ± 1.1 8.8 ± 0.5 5 OW30 7.5 ± 1.5 9.2 ± 0.5 25 OW578 6.7 ± 0.5 8.5 ± 1.1 6 SS33 7.6 ± 0.5 8.6 ± 1.0 26 AB600 7.5 ± 1.1 8.7 ± 1.0 7 EN35 6.6 ± 1.1 7.8 ± 0.5 27 OW620 8.5 ± 1.5 8.8 ± 0.5 8 OW36 8.7 ± 1.1 8.8 ± 0.5 28 SP651 7.6 ± 0.5 8.8 ± 0.5 9 EN38 6.7 ± 0.5 7.8 ± 1.1 29 OW819 6.2 ± 1.1 7.6 ± 0.5 10 SS42 7.5 ± 1.0 8.7 ± 0.5 30 EN831 8.5 ± 0.5 9.5 ± 1.1 11 OW53 8.2 ± 0.5 9.5 ± 0.5 31 AB841 9.3 ± 0.5 9.5 ± 1.1 12 SS57 7.8 ± 0.5 8.8 ± 1.0 32 OW940 8.5 ± 0.5 9.7 ± 1.1 13 AB61 8.8 ± 0.5 9.5 ± 0.5 33 OW947 7.8 ± 1.1 8.5 ± 1.1 14 EN62 8.7 ± 0.5 9.6 ± 1.1 34 AB1009 7.5 ± 1.1 7.6 ± 1.1 15 OW123 7.8 ± 0.5 8.8 ± 1.1 35 OW1104 8.0 ± 0.5 8.5 ± 0.5 16 EN127 8.5 ± 0.5 9.5 ± 0.5 36 SP1172 8.5 ± 1.1 9.6 ± 1.0 17 OW154 7.8 ± 0.5 8.3 ± 0.5 37 OW1420 8.7 ± 0.5 8.8 ± 0.5 18 AB187 8.5 ± 0.5 9.7 ± 1.1 38 OW1827 9.3 ± 1.1 9.5 ± 0.5 19 EN208 7.8 ± 0.5 8.7 ± 0.5 39 AB1956 8.2 ± 0.5 9.6 ± 1.1 20 SS235 8.8 ± 0.5 9.3 ± 0.5

119

Table 11: MIC and MBC of methanol fraction in mg/ml

Values were expressed as Mean ± SD, N = 3

Key:

SP: Sputum

SS: Skin swab

AB: Abscess

OW: Open wound

EN: Ear/Nasal

S/N

Clinical isolates

MIC MBC S/N Clinical isolates

MIC MBC

1 SP4 9.7 ± 1.1 9.8 ± 0.5 21 EN390 - - 2 SS8 8.03± 1.0 9.5 ± 1.1 22 SS310 - - 3 AB20 9.5 ± 0.5 10 ± 1.1 23 OW417 9.3 ± 0.5 9.6 ± 1.1 4 SP22 8.2 ± 1.1 9.2 ± 0.5 24 AB570 9.7 ± 1.1 9.8 ± 1.1 5 OW30 9.8 ± 0.5 10 ± 0.5 25 OW578 9.8 ± 1.0 - 6 SS33 8.6 ± 1.0 9.8 ± 1.1 26 AB600 - - 7 EN35 9.6 ± 1.1 9.8 ± 0.5 27 OW620 - - 8 OW36 8.7 ± 1.1 10 ± 0.5 28 SP651 9.7 ± 0.5 - 9 EN38 9.7 ± 1.0 - 29 OW819 9.5 ± 1.1 9.6 ± 0.5 10 SS42 8.5 ± 1.1 9.7 ± 0.5 30 EN831 - - 11 OW53 9.2 ± 0.5 - 31 AB841 9.7 ± 0.5 9.9 ± 1.1 12 SS57 8.8 ± 1.1 9.8 ± 1.0 32 OW940 - - 13 AB61 8.8 ± 1.1 - 33 OW947 9.8 ± 1.1 - 14 EN62 8.7 ± 0.5 9.6 ± 1.1 34 AB1009 - - 15 OW123 8.8 ± 0.5 9.8 ± 1.1 35 OW1104 9.0 ± 0.5 10 ± 0.5 16 EN127 8.5 ± 1.1 10 ± 0.5 36 SP1172 - - 17 OW154 9.8 ± 1.0 - 37 OW1420 - - 18 AB187 8.5 ± 1.0 9.7 ± 1.1 38 OW1827 9.7 ± 1.1 9.9 ± 0.5 19 EN208 9.8 ± 0.5 - 39 AB1956 8.9 ± 0.5 9.8 ± 1.1 20 SS235 8.8 ± 0.5 9.3 ± 0.5

120

3.7 GC-MS identification of bioactive components of methanol extract fractions

The four fractions obtained from the fractionation process were analysed for

identification of their bioactive compounds, Ethyl acetate fraction (EAF), dichloromethane

fraction (DF), n-hexane extract fraction (HEF) and methanol fraction (MF). The results of the

GC-MS and the activities of various compounds are displayed in appendices 6-13.

A. Ethyl acetate fraction (EAF)

The result of ethyl acetate fraction GC-MS showed 18 peaks each representing

different functional groups. The structural analogue of each compound is also presented in

each peak. Twenty seven (27) compounds identified in the ethyl acetate fractions, the

molecular weight, molecular formula, nature and activities of the compound are in agreement

with references. Appendix 6 and 7 showed the activities, molecular weight, molecular

formula and nature of the compounds identified in the fraction. All the bioactive principles

have one or more activities in agreement with the previous works [354-358].

The following compounds have the highest percentages, Stigmast-4-en-3-one

(36.95 %), molecular formula C29H48O, M.W 412, Cholest-4-en-26-oic acid (36.95 %),

Molecular formula C27H42O4, M.W 430, 3-oxo- 1,4-Benzenediol (28.23 %), C18H18N2O5,

M.W 342. Other significant constituents were Spinasterone (12.29 %), 5-Bromovaleric acid

(3.19 %), 2-Amino-4-methyl-3-pyridinol (5.8 %), Ergosta-4,22-dien-3-one (10.73 %), Pregn-

4-en-3-one (5.8 %), 2(1H)-Naphthalenone (2.89 %), Pentaene (2.28 %), (5.alpha.)-beta.-

Alanine (3.57 %), Supraene (1.39 %), Oxime (1.49 %) and Citrost-7-en-3-ol (6.7 %).

Figure 10, showed the fragmentation pattern for the identified compounds.

The abundance of each compound, the peak height, percentage area and retention time are

presented in the graph. There is overlapping of fragments, showing that some of the

functional grops can be repeated in another peak.

121

B. Dichloromethane fraction (DF)

The compounds identified in dichloromethane fraction are as shown in the tables in

appendices 8 and 9, with their retention time (RT), Percentage area (% Area), height amd

peak representing each functional group. Forty seven (47) functional groups are identified

from the GC-MS report of DMF according to the peak number, with different compounds

structural analogue in each peak.

A total of 80 compounds were identified in the dichloromethane fractions, the

molecular weight, molecular formula, nature and activities of the compounds in agreement

with references are as displayed in appendix 9. The compounds with high percentages in this

fraction are 5-bromovaleric acid (89.57 %), 2, 6-dimethylnon-1-en-3-yn-5-yl ester (89.57 %),

stigmast-4-en-3-one (12.33 %), Testosterone (12.33 %), Ergosta-4, 22-dien-3-one (11.91 %),

Hexadecanoic acid (7.31 %), beta.-Tocopherol (2.59%).

Fragmentation pattern for the identified compounds are as presented in Figure 11.

The abundance of each compound, the peak height, percentage areas and retention time are

presented in the graph.

C. N-hexane extract fraction (HEF)

The compounds in n-hexane fraction are as shown in appendices 10 and 11.

Forty one (41) functional groups and different structural analogue of the compounds

according to their peak are presented in the tables, with the retention time and percentage

area.

Forty five (45) compounds were identified in the n-hexane fraction, the molecular

weight, molecular formula, nature and activities of the compound are in accordance with the

references. The compounds with high percentages in this fraction are 9, 12-octadecadienoic

acid (41.08 %), 2-Chloroethyl linoleate (41.08 %), n-hexadecanoicacid (17.35 %),

stigmasterol (4.44%), ergost-22-en-3-one (4.44%), Campesterol (3.46%).

122

The mass spectrophotometric fragments in Figure 12 showed the abundance of each

compound, the peak height, percentage areas and retention time. There is an overlapping of

fragments, showing that some of the functional groups are repeated in another peak.

D. Methanol fraction (MF)

The compounds in methanol fraction are as presented in appendices 12 and 13, The

MF is having the least number of peaks, with the least functional groups and structural

analogue of the compounds according to their peak are presented in appendix 13, with the

retention time and percentage area.Only twelve (12) functional groups were identified in the

fraction.

Fourteen (14) compounds were identified in the methanol fraction with their

molecular weight, molecular formula, nature and activities. The compounds with high

percentages in this fraction are 3, 7-dimethyl-1, 6-Octadien-3-ol, (37.09 %), Eugenol (29.12

%), Terpinen-4-ol (7.46 %), Bicyclo[3.1.1]hept-2-ene (7.21 %), (+)-epi-

Bicyclosesquiphellandrene (5.28 %), Naphthalene (5.28 %), 1,3,3 trimethyl- L-Fenchone (1.53

%). The mass spectrophotometric fragments in Figure 13 showed the abundance of each

compound, the peak height, percentage areas and retention time. The methanol fraction gave

the least fragments out of the four fractions with the least compounds.

123

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

T ime-->

Abundance

TIC: EAE2RERUN.D\ data.ms

46.49846.58146.628

48.54548.622

49.97050.49850.522

51.347

52.51052.52854.48754.51655.43056.02456.05359.18159.217

Fig. 10: MS fragment of ethyl acetate fraction composition

124

10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

2000000

4000000

6000000

8000000

1e+07

1.2e+07

1.4e+07

Time-->

Abundance

TIC: OE3.D\data.ms

11.337

16.875 25.979

30.217

32.093

32.35433.57735.41740.21240.711

50.641

52.16053.39553.923

56.07156.267

56.44557.11664.34568.62569.05270.47671.81876.720

79.02380.83481.51681.97385.32786.28287.33388.063

89.57090.757

91.10891.70192.217

93.054

94.09394.11196.05896.08197.577100.669100.711101.156101.192

Fig. 11: MS fragment of dichloromethane fraction composition

125

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

5000000

1e+07

1.5e+07

2e+07

2.5e+07

Time-->

Abundance

TIC: OE4N-PASTORAYO.D\data.ms

19.267 25.22027.32130.41931.87334.01035.06635.14435.701

36.592

37.38737.92138.52138.63939.286

40.30740.33140.384

40.574

43.12645.37646.40346.51646.955

48.11248.534

49.685

49.81651.222

52.91453.01553.329

53.715

53.905

54.326

54.75455.027

55.508

56.344

58.70159.615

Fig. 12: MS fragment of n-hexane fraction composition

126

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

Time-->

Abundance

T IC: SS1-RERUN.D\ data.ms

13.064

13.580

16.115

18.32318.578

21.575

23.70028.965

31.315 41.72643.429

55.430

Fig 13: MS fragment of methanol fraction composition

127

3.8 STRUCTURE OF SOME PHYTOCHEMICALCOMPONENTS IDENTIFIED FROM MASS SPECTOMETRY REPORT IN MORINGA OLEIFERA ROOT BARK WITH REFERENCE TO: CHEMSPIDER, PUBCHEM, NIST, LOOKCHEM AND CHEMBOOK DATABASES [359-364].

Stigmast-4-en-3-one 7 alpha-Hydroxy-3-oxo-4-cholestenoate 3,methyl 5(2,6,6trimethylcyclohex- 1-en-1-yl) pent-1-yn-3-ol

5-bromovaleric acid 3,4-dihydro-1(2H)-Naphthalenone 2,2-Dimethylpropanoic acid, 2,6- dimethylnon-1-en-3-yn-5-yl ester

Phthalimidine Ergosta-4, 22-dien-3-one (E, E, E)-3, 7, 11, 15-Tetramethylhexadeca-1,3,6- 10, 14-pentaene

ETHISTERONE alpha.-amino-Benzene acetic acid 2-Thiazolamine

128

9-Octadecenoicacid,methylester (1,2,6)-phosphadiazine UREA 2-Phenyl-2,3-dihydro-1H- naphtho[1,8de][1,3,2]diazaphosphinine - 2-sulfide

Cis- muurola-3,5-diene Pentadecanoic acid Palmitic Acid

a-alanine 4-methoxy- beta-naphthylamide Dimethyl(ethenyl)silyloxy-3-phenylpropane 1, 3-Dioxolane

Cyclohexanecarboxylic acid Ergost-25-ene-3, 5, 6, 12-tetrol Dodecanoic acid

129

Benzofran-3-one C(14a)-Homo-27-nor-14.beta- Carboxamide 1,3,3trimethyl-L-Fenchon Gammaceran-3.alpha.-ol

7H-pyrazolo [4, 3-e][1,2,4] Triazolo [1, 5-c] pyrimidine, Trimethyl(propoxy)silane, C (14a)-Homo-27- Gammaceran-3.alpha-ol Terpinen-4-ol

Isopropyl 2-benzyl-2- PropenylBenzene Geraniol Benzenemethanamine

Isopropyl 2-benzyl-2-propenyl Ether Benzylisocyanate Eugenol

Fig. 14: The structures of some of the identified components from mass spectometry report were presented with reference to the following data base: Chemspider, Pubchem, National institute of science and technology (NIST), Lookchem and Chembook.

130

3.9 Preliminary evaluation of toxicity of crude methanol extract and n-hexane extract fraction The evaluation of acute toxicity and toxicity indices of crude methanol extract and n-

hexane extract fraction were carried out to ascertain the safety of the extract root bark. The

results are presented in the tables below.

3.9.1 Acute toxicity test of crude methanol extract and n-hexane extract fraction

The acute toxicity test results were as presented in table 12.

The LD50 values of crude methanol extract and n-hexane extract fraction in rats, by Miller

and Tainter method were determined from probit vs Log Conc.graph to be 3663.96 mg/kg for

crude methanol extract and 1934.15 mg/kg for n-hexane extract as presented in Fig. 15 and

16 respectively.

The organisation of Economic Co-operation and Development (OECD, Paris, France)

(Walum, 1998) recommended as follow: very toxic, <5 mg/kg; toxic, >5<50 mg/harmful,

>50<500 mg/kg; no label, >500<2000 mg/kg. This confirmed that methanol extract and n-

hexane extract fraction of the root bark of moringa oleifera are safe for human consumption

at a dose below their LD50. 3663.96 mg/kg and 1934.15 mg/kg.

3.9.2 Sub-acute toxicity study of the crude methanol extract and n-hexane extract

fraction As shown in Tables 13 and 14, the results of the effect of the extracts at graded doses below

the LD50 on the haematological and biochemical parameters of the rats further confirmed the

safety of the extracts for human consumption. There was no significant difference (P > 0.5)

when the values of the parameters were compared with the control rats group. This showed

that the extract had no effect on the liver, kidney and blood of the rat at these doses.

131

3.9.3 Effects of graded doses of crude methanol extract and n-hexane extract fraction

on body weights of rats

As shown in Table 15, there was general increase in the body weight of the animals except at

1000 mg/kg for crude methanol extract when some of the animals in the group showed little

weekness and later recovered. This may explain the little drop in weight at the dose of 1000

mg/kg of the crude methanol extract with little behavioral change.

The n-hexane extract fraction also showed increase in body weight but some of the animals

in the group showed weekness due to loose stools observed at a dose of 1000mg/kg, this was

not obvious with the group of 500 mg/kg but with a marked behavioural change with the

group of 1000 mg/kg.

132

Table 12: Acute toxicity test of crude methanol extract and n-hexane extract Fraction

Group Dose mg/kg N/D % Mortality

Observation Period (h)

Symptoms of Toxicity

Crude methanol extract 1 50 mg 6/0 0 24 No toxic symptoms 2 250 mg 6/0 0 24 No toxic symptoms 3 500 mg 6/1 16.6

24 Slow movement and dullness of animals

4 1000 mg 6/2 33.3 24 Weak and less active 5 5000 mg 6/2 33.3

24 Marked behavioral change, restlessness and gradual death

6 1 g 6/4 66.6 24 restlessness and gradual death 7 (control)

2 ml distlled water 6/0 0 24

No change observed

N-hexane extract fraction 1 50 mg 6/0 0 24 No toxic symptoms 2 250 mg 6/0 0 24 No toxic symptoms 3 500 mg 6/1 16.6 24 Weak, less active 4 1000 mg 6/2 33.3 24 Weak, less active and loose stool 5 5000 mg 6/4 66.6 24 Weak, loose stool and gradual

death 6 1 g 6/6 100 24 Death 7 (control)

2 ml distlled water 6/0 0 24 No change observed

N = no. of rats, D = no. of Deaths

The LD50 for crude methanol extract is 3663.96 mg/kg and n-hexane extract is 1934.15.

Determined from probit graph analysis on SPSS v17.

133

Fig. 15: Determination of LD50 value of crude methanol extract in rats

LD50 = 3663.96 mg/kg

Determined from probit analysis onSPSS v17

134

Fig 16: Determination of LD50 value of n-hexane extract fraction

LD50 = 1934.15 from probit graph. Determined from probit analysis onSPSS v17

135

Table 13: Effects of the graded doses of the extracts on haematological parameters of rats

Parameters Control

(A)

50 mg/kg

(B)

250 mg/kg

(C)

500 mg/kg

(D)

1000 mg/kg

(E)

Crude methanol extract

PCV% 44.6 ± 0.6 42.2 ± 0.9 45.3 ± 0.3 45.1 ± 0.2 46.2 ± 0.4

RBC% 7.1 ± 0.9 7.3 ± 0.2 7.40 ± 0.5 7.4 ± 0.5 7.5 ± 0.5

WBC% 13.2 ± 0.1 13.9 ± 0.1 13.75 ± 0.1 13.8 ± 0.2 14.2 ± 0.2

MCV% 51.2 ± 0.2 51.1 ± 0.1 49.1 ± 0.1 48.8 ± 0.2 50.2 ± 0.1

PLT% 409.1 ± 8.1 409.0 ± 7.5 406.22 ± 8.1 399.1 ± 8.2 388.40 ± 8.2

N-hexane extract fraction

PCV% 44.6 ± 0.2 41.24 ± 0.1 43.20 ± 0.2 44.50 ± 0.4 45.10 ± 0.6

RBC% 7.1 ± 0.1 7.00 ± 0.3 7.21 ± 0.2 7.32 ± 0.2 7.40 ± 0.4 WBC%

13.2 ± 0.2 13.60 ± 0.2 13.67 ± 0.1 13.78 ± 0.2 13.90 ± 0.2 MCV%

51.2 ± 0.1 50.10 ± 0.2 49.50 ± 0.2 49.90 ± 0.2 49.95 ± 0.2 PLT%

409.1 ± 8.2 409.0 ± 7.2 407.22 ± 7.5 405.10 ± 8.1 398.50 ± 8.2

Values were expressed as Mean ± SD, N = 6

136

Table 14: Effects of the graded doses on biochemical parameters of rats

Parameters Control

(A)

50 mg/kg

(B)

250 mg/kg

(C)

500 mg/kg

(D)

1000 mg/kg (E)

Crude methanol extract BILURIBIN 1.60 ± 0.01 1.63 ± 0.02 1.61 ± 0.01 1.60 ± 0.02 1.58 ± 0.01

CREATININE 0.73 ± 0.01 0.69 ± 0.01 0.71 ± 0.01 0.69 ± 0.01 0.69 ± 0.01

ALT 34.11 ± 0.02 33.17 ± 0.02 33.02 ± 0.15 31.40 ± 0.15 32.60 ± 0.02

ALP 129.4 ± 0.15 129.88 ± 0.15 129.31 ± 0.21 130.11 ± 0.22 132.11 ± 0.20

AST 63.1 ± 0.1 66.1 ± 0.2 64.41 ± 0.2 63.22 ± 0.2 66..53 ± 0.2

N-hexane extract fraction BILURIBIN 1.62 ± 0.01 1.62 ± 0.01 1,60 ± 0.02 1.58 ± 0.01 1.59 ± 0.01

CREATININE 0.74± 0.02 0.70 ± 0.01 0.71± 0.02 0.69 ± 0.01 0.68 ± 0.01

ALT 34.11 ± 0.01 33.17 ± 0.02 33.02 ± 0.15 31.40 ± 0.01 32.60 ± 0.01

ALP 129.41± 0.20 129.88± 0.20 129.31 ± 0.15 130.11 ± 0.20 132.11 ± 0.2

AST 63.11 ± 0.2 66.24± 0.2 64.41 ± 0.2 63.22 ± 0.1 66..53 ± 0.2

Values were expressed as Mean ± SD, N = 6

Key:

AST: Aspatate amino transferace

ALTS: Alanine amino transferace

ALP: Alkaline phosphate.

137

Table 15: Effects of graded doses of the extracts on body weights of rats

Parameters Control 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg

Crude methanol extract Weight before extract administration (g)

95.3 ± 4.2 110.2 ± 6.2 115.3 ± 8.2 120.4 ± 9.2 150.7 ± 8.2

Weight after 21days (g)

150 ± 10.3 155 ± 9.2 145 ± 11.4 135 ± 12.5 95 ± 8.2

% weight difference 57.89 % 53.46 % 38.09 % 35 % Weight lost 3 %

N-hexane extract fraction Weight before extract administration (g)

95.2 ± 4.1 101.4 ± 3.2 105.7 ± 4.3 100.6 ± 5.2 98.8 ± 3.1

Weight after 21days (g)

150 ± 7.2 145 ± 7.6 130 ± 9.7 95 ± 8.2 80 ± 12.7

% weight difference 57.89 % 43.56 % 23.80 % Weight lost 5 %

Weight lost 18.36 %

Values were expressed as Mean ± SD, N = 6

138

3.10 Discussion

3.10.1 Percentage yield of extracts and fractions

The extractive values are useful to evaluate the chemical constituents present in the

crude drug and also help in estimation of specific constituents soluble in a particular solvent

(Ozarkar) [103]. In this study, the percentage yield of the extract and fractions indicate a

good yield with methanol crude extract 850.60 ± 20.8 g (28.35 %) compared to n-hexane

extract faction of 68.80 ± 13.7 g (2.29 %). There was significant (p > 0.05) difference in the

percentage yield of the various fractions, methanol fraction having the highest (31.21 %) and

n-hexane, the lowest (2.29 %). The fact that methanol fraction, dichloromethane fraction and

ethylacetate fraction yields are more than n-hexane yield means there are more polar

phytochemical constituents in the roots of Moringa oleifera than non polar [103-105]

3.10.2 Qualitative phytochemical analysis of methanol extract and fractions

Phyto chemical compounds may inhibit bacterial growth by mechanisms different from

presently used treatment regimens, and could be of clinical value in the treatment of resistant

bacteria, including MRSA. Tannins, flavonoids, alkaloids, essential oils and many phenolic

compounds serve as plant defence mechanisms against predation by insects, herbivores and

infection by microorganisms (Cowan) [120]. It is, therefore, not surprising that these

compounds have been found to exhibit profound antimicrobial activities in-vitro against this

multi drug resistant MRSA. Moringa oleifera is already highly esteemed by people in the

tropics and sub-tropics for the many ways it is used nutritionally and medicinally by local

herbalist. Some of these traditional uses reflect the nutritional content of the various tree plant

parts. The following are but some of the ways the tree plant parts is used in Asia, Africa and

America. In recent years, laboratory investigations have confirmed the efficacy of some of

these applications as found in leaves, flowers, pods, roots, root bark and stem bark, gum,

seeds and seed oil [116-120].

139

The roots of Moringa oleifera have been known to be used in the treatment of dental

caries, common cold, fever, diarrhea, flatulence and edema [117]. There is an increasing

awareness that many components of traditional medicine are beneficial while others are

harmful, hence WHO encourages and supports countries to identify and provide safe and

effective remedies for use in the public and private health services [118].

The present study showed that the root bark of Moringa oleifera have pharmacologically

important chemical compounds such as carbohydrates, saponins, cardiac glycosides, terpenes,

steroids, flavonoids and alkaloids. Alkaloids were abundantly present, proteins and

carbohydrates are present in high concentrations, tannins, resins, glycosides, steroids,

flavoinoids, reducing sugars, fats and oil, sapponins are present in moderately high

concentrations with terpenoids present in low concentrations. These findings agrees with the

review of (Fahey) [125]. The fractions of the extracts showed varying concentrations of

phytochemical constituents: n-hexane fraction is very rich in steroids, terpenoids, fats and

oils, dichloromethane fraction is rich in resins with moderate concentration of steroids and

fats and oil and low concentration of steroids, ethyl acetate fraction is rich in flavonoids

which is abundantly present, with low concentrations of steroids, terpenoids and

carbohydrates, methanol fractions is with low concentrations of alkaloids, tannins, resins,

glycosides. Alkaloids have pharmacological effects and are used as local anesthetic and

stimulants [340]. Cocaine, caffeine, nicotine, the analgesic morphine, the anti-bacterial

berberine and antimalarial drug quinine are all alkaloids. The alkaloid spirachin (a nerve

paralysant) has been found in the roots of Moringa oleifera. [341]. Flavonoids, according to

the research by [346] may modify allergens, viruses and carcinogens, thereby, acting like a

biological response modifier. Also, in vitro studies showed that flavonoids could also posses

anti-microbial [346], anti-allergic and anti-inflammatory properties. Steroids are used in the

stimulation of bone marrow and growth. It stimulates lean body mass and also play vital roles

140

in the prevention of bone loss in elderly men [133]. Tannins could be an effective

ameliorative agent of the kidney [130].

Moringa oleifera fats and oil are used traditionally to treat stomach disorders. They are

used in perfume and hair oil [128]. Glycosides from Moringa oleifera were found to be

responsible for the blood pressure lowering effect of the root (Faizi et al) [128].

Carbohydrates, one of the most common groups of natural products, are construction

materials for plant (cellulose, hemicelluloses and pectin) and animal tissues. They are the

sources of energy and storage material in flora (starch, insulin) and fauna (glycogen). Several

specific carbohydrates are utilized in nature, for instance, as water maintaining hydrocolloids,

sex attractants (pheromones), and others formed by photosynthesis and associated processes.

Some of them are subsequently utilized by plants which transform them into other products;

for instance, some plants may turn D-galactose into ascorbic acid [123]. The presence of

these phytochemicals in the extracts and fractions support the antimicrobial activities of the

Moringa oleifera root bark on MRSA (HA-MRSA) isolates used in this study.

3.10.3 Prevalence of clinical isolates of Staphylococcus aureus Staphylococcus aureus was found to be prominent in cases of wound sepsis [350].

Many studies have shown that open wond is a reservoir for transmission of S. aureus. A study

[351] at Ilorin, Nigeria reported wound infections of 38 % as the highest frequency of S.

aureus isolates. This agrees with the result of the present study where the order of the

Coagulase positive S. aureus, MSSA and MRSA prevalence is as follow: Open wound 19

(58) (32.75 %), greater than abscess 11 (58) (18.96 %), greater than Ear/Nasal swab 11 (58)

(18.96 %), greater than Skin swab 10 (58) (17.24 %), greater than Sputum 7 (58) (12.06 %)

except the little difference between the abscess and ear/nasal in the staphylase positive S.

aureus and MRSA prevalence.

141

The association of MRSA with therapeutic challenges, complications, deaths and cost

related to longer hospital stay compared with MSSA has been widely documented [35-351].

More importantly, the multidrug resistant hospital-acquired MRSA (HA-MRSA) strains and

their intrinsic resistance to beta-lactam antibiotics confer limited treatment options to the

most available and less costly antibiotics in developing countries [351]. In agreement with the

previous study by (Olowo) [315]. Open wound has the highest prevalence of S.aureus and

MRSA.

3.10.4 Antimicrobial susceptibility parttern of the clinical isolates

The susceptibility and resistance pattern of the 58 clinical isolates S. aureus were as seen in

the Table 8. Susceptibility test profile revealed a high level of resistance amongst the S.

aureus to all the commonly used antimicrobials, ciprofloxacin, gentamicin, clindamycin,

levfloxacin, cephalexcin, tetracycline, rifampicin cotrimoxazole, oxacillin and vancomycin.

The results were comparable to those in a previous study carried out by Nwankwo and Nasiru

[326], but not as high as in this study. For many years vancomycin was the drug of choice for

MRSA infections, until the first S. aureus isolate with reduced sensitivity to vancomycin

(vancomycin intermediate Staphylococcus aureus (VISA) was reported in Japan in 1997.

Thereafter, several reports of VISA from USA , France , Brazil , Korea , and other parts of

the world [319-323] were published and resulted in increasing concerns about the

effectiveness of vancomycin therapy in serious staphylococcal infections. Reduced sensitivity

to vancomycin in S. aureus occurs due to several genetic and phenotypic alterations in wild-

type bacteria including altered expression of regulatory genetic elements, thickness of cell

wall, changes in the penicillin binding protein (PBP) profiles, and decreased cell wall

autolysis [324]. Since 2002, there have been also several reports of vancomycin resistant

Staphylococcus aureus (VRSA) strains, mainly from the United States, India, and Iran [322-

324]. The majority of the reported VISA isolates are MRSA strains with reduced

142

susceptibility to vancomycin even though a few reports have also shown this phenomenon in

methicillin sensitive Staphylococcus aureus (MSSA) isolates [321-323]. In the present study,

Staphylococcus aureus susceptibility and resistance seems not to conform to usual norm of

having Vancomycin as the most sensitive drug, The susceptibility and resistance parterns of

these clinical isolates in oxacillin and vancomycin was not different, almost no significance

difference (P > 0.05). Hence, most of the MRSA among these isolates were also VISA and

VIRA as it is revealed from table 9. It was discouraging to note that vancomycin resistance

was higly observed among the isolates.

The MRSA showed a high level of resistance to all antimicrobials in general in

comparison to the MSSA. Also most of the MRSA in this study were actually resistant to

many classes of antimicrobials at the same time and thus qualify as multiply drug resistant

Staphylococcus aureus (MDR-MRSA).[325] in agreement with [263]. This mechanism of

resistance to glycopeptides is believed to be due to the presence of the vanA gene

complex[.9] Isolates with this gene complex are noted to have a thicker cell wall with a

decreased production of PBPs.[325]. Heterogeneous vancomycin-intermediate S. aureus

(hVISA) is defined as being susceptible to vancomycin, but contain subpopulations of MRSA

which confer intermediate resistance to vancomycin [319]. It likely precedes the development

of vancomy intermediate S. aureus. Since screening for hVISA is not routinely performed in

the clinical microbiology laboratory, many cases go undetected [320]. In recent studies, the

rate of hVISA was approximately 6-11 percent of all MRSA isolates. [302 - 305] One study

determined that hVISA bacteremia infections were associated with prolonged duration of

bacteremia and increased number of complications [302]. Infection-related mortality due to

hVISA and MRSA was similar. [305]. In this study levfloxacin and cephalexcin are the least

resistant antibiotics, while tetracycline and cotrimoxazole are the highest resisted antibiotics

[324-326].

143

3.10.5 Penicilin – binding protein (PBP2́) latex agglutination test

Staphylococci are a leading cause of nosocomial and community acquired infections

worldwide. In many institutions, approximately 25% - 50% of S.aureus strains and 75% of

coagulase – negative staphylococci (CNS) are resistant to methicillin [266]. MRSA are of

particular concern because of the ease with which certain epidemic strains spread and

colonise debilitated patients. The methicillin-resistant phenotype can be highly

heterogeneous, making it difficult to detect by conventional anti-microbial susceptibility test

methods, such as minimum inhibitory concentration (MIC), disc and agar screen. The

accuracy of these methods is affected by 143noculums size, incubation time and temperature,

medium, pH, salt concentration and other factors [282, 284]. Detection of the mecA gene has

been considered the gold standard in the determination of methicillin-resistance because of its

accuracy, but this method is labour – intensive and expensive to perform [232, 233]. The

Oxoid PBP2́ latex test has the advantage of direct detection of the PBP2́ protein performed

in a rapid timeframe with minimal labour. It has the potential for being even more accurate

than the detection of the mecA gene, as false –positive results will not occur with strains that

possess mecA gene but are unable to produce the protein product of the gene. This is why

PBP21 latex agglutination test is used in this study for the confirmation of the MRSA isolates.

In the present study it was observed that out of 58 clinical isolates of S.aureus tested for

resistance, 39 isolates were confirmed MRSA and 19 MSSA prevalence of 32.75 % and

MRSA 67.24 % using PBP2ʹ latex agglutination test. It is believed that the high percentage of

MRSA is due to long stay of the patient in the hospital as they are vulnerable and easily

infected by MRSA of different clonal structure or hetrogenous MRSA especially when

hospitalized for long period [282]. The specimen used for this study was collected from

hospitalized patient for over six months. Increasing antibiotic resistance in major S. aureus

clones intensifies precautionary policies for public health care systems. In this study, we have

144

shown the upward trend of MRSA probably due to dissemination of resistance by clonal

spread and horizontal transfer of mecA genes and regulatory sequences. Our study suggest

that there are VISA strains from some of the hospitalized patients that serve as reservoir for

the transmission of this MRSA and it has been reported that vancomycin resistance has the

potential to become a widespread problem in both MRSA and MSSA strains [302].

3.11 MIC and MBC of methanol extract and fractions An examination of the phytochemicals of Moringa species affords one the opportunity to

examine a range of fairly unique compounds. In particular, this plant family is rich in

compounds containing the simple sugar, rhamnose, and it is rich in a fairly unique group of

compounds called glucosinolates and isothiocyanates [10, 84]. These components of Moringa

preparations have been reported to have hypo-tensive, anticancer, and antibacterial activity.

The antibacterial activity of Moringa oleifera root bark extract agrees with the findings

of many authors on the antimicrobial activities of this great plant [19-22]. The antimicrobial

activities demonstrated by the extract and fractions in this study is quite remarkable,

particularly as standard antibiotics are in the purified and concentrated form, yet the MRSA

isolates resisted them, whereas the extracts are crude and may 144arbor both

pharmacologically and non-pharmacologically active compounds with the chance of some

compounds having a masking effect over others but the extract and fractions still exhibit a

strong antmicrobial activity over 30 µg/ml vancomycin [23, 194]. There is the possibility of a

number of compounds working synergistically or additively within the extract, thereby

resulting in a significantly greater antimicrobial effect of the crude extract than the fractions,

which is the case in this study. However, this explanation remains highly hypothetical, as the

exact mechanisms of action of many phytomedicines are as yet unknown. Only once a full

explanation of this mechanism is available, will conclusive evidence regarding the identity of

the compounds responsible for biological activity is established. All plant species with MIC

145

values of up to 8 mg/ml are considered to possess some degree of inhibitory effect, and any

concentration exceeding this should not be considered effective, according to Fabry [23]. The

extract and all the fractions except methanol fraction (MF) from the resul showed MIC and

MBC lower than 8 mg/ml with few exceptions that are above 8 mg/ml.The MIC and MBC

result confirms the moringa root bark as a potential herbal remedy for infection by MRSA.

3.11.1 MIC and MBC of crude methanol extract

The results of the MIC and MBC of the crude extract as shown in table 12, confirmed

the antimicrobial activity of the extract on the MRSA isolates, thus, support the traditional

use of Moringa oleifera root bark [20]. The MRSA isolates selected for the study, in addition

to being resistant to oxacillin and vancomycin, also resisted other antibiotics like levfloxacin,

tetracycline, gentamicin, clindamycin, cotrimoxazole, rimfampicin, cephalexcin and

ciprofloxacin, showing that the isolates are multi drug resistant, but this isolates were

effectively inhibited by the methanol crude extract at concentration as low as 3.0 mg/ml as

the least MIC and 4.0 mg/ml as the least MBC and the highest MIC and MBC are 5.0 mg/ml

and 5.6 mg/ml respectively.The result confirm the potency of methanol extract of the root

bark over the standard antibiotics used in the treatment of the infections caused by MRSA in

the hospital and other health care facilities. Probably due to the heterogenous nature of the

isolates and secondary metabolites of present in the fractions [9-12] the isolates shows

different susceptibilities to each fraction.

3.11.2 MIC and MBC of ethyl acetate fraction

The results of the antibacterial testing of the ethyl acetate fraction against all the

MRSA clinical isolate as displayed in Table 14, ethyl acetate fraction (EF) showed better

activity next to the methanol crude extract than the remaining fractions, possibly because of

the presence of high concentration of flavonoids and other secondary metabolites eluted by

the solvent, which has been shown to enhance antimicrobial property of a plant [13-15]. The

146

MRSA isolates for this study restricted all the popular antibiotics used in the hospitals in

varying degrees, but was inhibited by ethyl acetate fraction at concentrations of 3.5 mg/ml as

the least MIC, 4.3 mg/ml as the least MBC and 6.6 mg/ml and 7.5 mg/ml as the highest MIC

and MBC. This confirms the presence of bioactive principles that can be formulated for the

treatment of infections by the MRSA isolates in the hospitals.

3.11.3 MIC and MBC of N-hexan extract fraction (HEF) in mg/ml

The results of the antibacterial testing of the hexane extract fraction against all the MRSA

clinical isolates. HEF shows good activity next to ethyl acetate fraction possibly because of

the presence of high concentration of fatty acids, terpenoids and phenols which was in

agreement with previous studies to enhance antimicrobial property of a plant [22 – 25]. The

fraction as shown in table 14 inhibited the MRSA isolates at least MIC and MBC of 5.3

mg/ml and 5.6 mg/ml while the highest MIC and MBC are 7.6 mg/ml and 8.8 mg/ml. This

confirms the presence of some non-polar contents present in the n-hexane fraction as seen in

the phytochemical analysis and the GC-MS report.

3.11.4 MIC and MBC of dichloromethane fraction (DMF) in mg/ml

The results of the antibacterial testing of the dichloromethane fraction (DMF) against

all the MRSA isolates seems to show low activity compared to the n-hexane and ethyl acetate

fractions, thi can be due to reduction in the concentrations of active principles [23] in the

fraction. The antibacterial results in table 15 indicated that the (DF) still maintain some level

of activities against the MRSA clinical isolates possibly because of the presence of an

antibacterial compound from the GC-MS report known as benzylisocyanate[10,84] in

agreement with previous study of (Fahey) [84] that the compound has potent antibacterial

property. The MIC and MBC of the fraction ranges from 6.3 ± 1.1 – 9.3 ± 0.5 and 7.5 ± 1.0 –

9.7 ± 1.1, respectively.

147

3.11.5 MIC and MBC of methanol fraction (MF) in mg/ml

All the fractions showed varying degrees of inhibition on the tested organisms the

results of the antibacterial testing of the methanol fraction against all the MRSA isolates is

the least potent of the fractions, this is probably as a result of low concentration of active

principles after elutetion of the crude extracts by other solvents [23]. The fraction from the

phytochemical and GC-MS shows the presence of Terpinen-4ol and Eugenol which were

repoted to be strong antibacterial compounds [353]. MF has activity against some of the

isolates but not all, some resisted the MF fraction at the concentration range of 1mg/ml -

10mg/ml used for this study. Ethyl acetate fraction, N-hexane extract fraction,

Dichloromethane fraction clearly demonstrated far more superior effect than methanol

fraction. The change in susceptibility and resistance of these isolates is not well understood

but it might be due to the fact that some of the MRSA may be hVRSA (hetrogenous

vancomycin staphylococcus aureus), that has different resistant ability towards different

antibiotics, since they were from different specimen [302, 320, 321]. The MIC and MBC for

the susceptible isolates ranges from 8.3 ± 1.1 – 9.8 ± 1.1 and 9.3 ± 0.5 – 10 ± 1.1,

respectively. There was no inhibition produced by the dimethyl sulfoxide (DMSO) used as

the solvent, indicating non involvement of the solvent in the activity of the extract and

fractions.

3.12 GC-MS Identification of bioactive compounds of methanol extract fractions

The possible presence of synergistic interactions between the bioactive constituents of the

methanol crude extract is responsible for the strong antibacterial activity exhibited by the

extract against this multi drug resistant MRSA isolated in thi study. These consequences

suggest that M. oleifera root bark used, contain bioactive substances whose antibacterial

potentials are higher than that of antibiotics used in sensitivity test against MRSA isolates.

The activity of plants against microorganisms may be indicative of the presence of broad-

148

spectrum antibiotic compounds in that plant [329, 330]]. Failure of some of the extracts to

exert antibacterial effect on the test organism is not enough to conclude that the extract does

not contain substances that can exert antibacterial activity against the test organism because

the potency of the extract depends on the method used to obtain the extract [320]. Research

has also shown that the difference in antimicrobial properties of the plant extracts might be

attributable to the age of the plant used, freshness of the plant material, physical factor

(temperature, light or water), contamination by field microbes, and incorrect preparation of

the plant etc[337]. Today, most pathogenic organisms are becoming resistant to antibiotic

[333]. Moringa oliefera have been reported to be a good source of natural antioxidants such

as ascorbic acid, phenolic and carotenoids [331]. To overcome this alarming problem, the

discovery of novel active compounds against new targets is a matter of urgency. Thus M.

oleifera Lam. could become promising natural antimicrobial agents with potential

applications in pharmaceutical industry for controlling the pathogenic bacteria.

The GC-MS analysis revealed the content of the fractions that possibly account for

the activity of the methanol extract and its fractions. In this study the nature of the

compounds identified in the methanol extract fractions are listed in the tables of results and

their activities. [354-358] The following compounds are common in all the fractions. This

may explain why all the fractions had one activity or the other. Oxime, stigmast -4-en-3-one,

5- Bromovaleric acid, Ergost-4-22-dien-3-one, Cholest-4-en-26-oic acid, stigmasterol,

Naphthalenone, 4, 22-stigmastadiene-3-one, Pyridine-3-carboxamide.

Methanol fraction (MF) has the least compound, with only oxime and Pyridine-3-

carboxamide in common with others, this may also account for the low activity shown by the

fraction against the MRSA clinical isolates. Over 100 hundred dinstict compounds were

identified from the fractions of the methanol extract of M. oleifera root bark.

149

3.12.1 Gas chromatography-mass spectrometry (GC-MS) of ethyl acetate fraction

In this study a total of twenty seven (27) compounds were identified in the fraction

with these three compounds having the highest percentages, Stigmast-4-en-3-one (36.95 %),

molecular formula C29H48O, M.W 412, a ketone steroid with antimicrobial activities,

antioxidant, anti-inflammatory, antiarthritic, antiasthma and diuretic activities John and

Kumar [355], Jennings and shibamoto [357], Cholest-4-en-26-oic acid (36.95 %) Molecular

formula C27H42O4, M.W 430, Aliphatic acid sesterterpenes that regulates the metabolism of

cholesterol and homeostasis [356], 3-oxo- 1,4-Benzenediol (28.23 %), C18H18N2O5, M.W

342, Oxygenated aldehyde with antimicrobial and antioxidant properties, antimycobacterial

activity [353]. Other significant constituents with bioactivities are Spinasterone (12.29 %), a

seroid compound with antibacterial and antifungal activities [354], 5-Bromovaleric acid

(3.19%), 2-Amino-4-methyl-3-pyridinol (5.8%), Ergosta-4,22-dien-3-one (10.73%), Pregn-4-

en-3-one (5.8%), 2(1H)-Naphthalenone (2.89%), Pentaene (2.28%), (5.alpha.)-beta.-Alanine

(3.57 %), Supraene (1.39 %), Oxime (1.49 %) and Citrost-7-en-3-ol (6.7%), [354-358].

3.12.2 Gas chromatography-mass spectrometry (GC-MS) of dichloromethane fraction

In this study, DF contains the highest number of bioactive principles 80, but next to

ethyl acetate fraction in the antimicrobial susceptibility test, showing that there is a potent

compound in EAF that is probably not in DF or a synergistic effect of the compounds in EAF

but not exhibited in DF. The compounds with high percentages in this fraction are 5-

Bromovaleric acid (89.57 %) , 2, 6-dimethylnon-1-en-3-yn-5-yl ester (89.57 %,) Stigmast-4-

en-3-one (12.33 %), Testosterone (12.33 %), Ergosta-4, 22-dien-3-one (11.91 %),

Hexadecanoic acid (7.31 %) nature and activities of the compound agrees with references

[356, 357].

150

3.12.3 Gas chromatography-mass spectrometry (GC-MS) of n-hexane fraction

The HF fraction activity is also encouraging against the MRSA isolates, 45

compounds identified with the following compounds dominating the fraction 9,12-

Octadecadienoic acid (41.08 %), 2-Chloroethyl linoleate (41.08 %), n-Hexadecanoicacid

(17.35 %), 12-Octadecadienoic acid (8.55 %), Stigmasterol (4.44 %), Ergost-22-en-3-one

(4.44 %), Campesterol (3.46 %). The molecular formula, M.W., nature and activities of all

the compounds are included in Table 21. The fractions contain high content of Fatty acids,

terpenoids and steroids their nature and activities of the compound agrees with references

[354-357].

3.12.4 Gas chromatography-mass spectrometry (GC-MS) of methanol fraction

In this study, 14 compounds were identified in the methanol fraction (MF). It has the

least bioactive compounds and least concentrations of these compounds; this may also

explain the low antibacterial activity displayed against the MRSA isolates. The compounds

with high percentages in this fraction are 3,7-dimethyl-1,6-Octadien-3-ol (37.09 %), Eugenol

(29.12 %), Terpinen-4-ol (7.46 %), Bicyclo[3.1.1]hept-2-ene (7.21 %), (+)-epi-

Bicyclosesquiphellandrene (5.28 %), (6-bromohexyl)- 2-Butanol (3.49 %), Naphthalene (5.28

%), 1,3,3 trimethyl- L-Fenchone (1.53 %). Nature and activities of the compound agrees with

references [356,357].

3.12.5 Structure of some phytochemical components.

The structures of some of the identified components from mass spectometry report in

Moringa oleifera lam. Fam. Moringacea root bark were presented with reference to the

following data base: Chemspider, Pubchem, National institute of Science and Technology

(NIST), Look chem and Chem book.

151

3.13 Acute toxicity test of crude methanol extract

The acute toxicity of the crude methanol extract and n-hexane extract fraction of the root

bark of Moringa oleifera in this study agrees with previous finding of kasolol et al [366]. The

extracts are safe for human consumption at a dose below the LD50 which is 3663.96 mg/kg

for crude methanol extract and 1934.15 mg/kg for n-hexane extract fraction and also with

reference to LD50 values recommended by the organisation of Economic Co-operation and

Development (OECD, Paris, France) (Walum, 1998) are as follow: very toxic, < 5 mg/kg;

toxic, > 5 <5 0 mg/harmful, > 50 <500 mg/kg; no label, > 500 < 2000 mg/kg.

3.13.1 Sub-acute toxicity study of the crude methanol extract and n-hexane extract fraction

The result of this study showed that there was no significant difference (p > 0.05)

when the values of the parameters were compared with the control rat group. This shows that

the extracts had no effect on the liver, kidney and blood of the rat at these doses below the

LD50. This agrees with kasolol et al [365,366].

3.13.2 Effects of graded doses of crude methanol extract and n-hexane extract fraction on body weight of rats

The result showed general increase in body weight of the animals except at

1000mg/kg where some of the animals in the group showed little weekness and later

recovered. This may explain the little drop in weight at the dose of 1000mg/kg with little

behavioural changes [365, 366]. In the n-hexane extract fraction, some of the animals in the

group showed weekness due to loose stools, this was not obvious with the group of 500mg/kg

dose but it was so obvious in the group of 1000mg/kg dose. The weekness due to loose stool

by these animals was possibly due to the high Fats and oil content in n-hexane extract

fraction; these effects are time-dependent and dose-dependent Paul and Didia [366].

152

CHAPTER FOUR

4.0. CONCLUSIONS AND RECOMMENDATIONS

4.1. Conclusions

The discovery of effective antibiotics, vaccines and other products or methods have

decreased the devastating impact of infectious diseases and improved quality of life.

However, the efficacy of many antibiotics is being threatened by the emergence of

microbial resistance to existing chemotherapeutic agents because of their indiscriminate

and inappropriate use [37]. The use of some antibiotics is associated with side effects,

including allergy, immune suppression, and hypersensitivity [38].

Many populations who live in developing countries are deprived of the advantages of

modern medicine because of its high cost; hence, poor people are more vulnerable to

infectious diseases. Besides these, co-infection with multiple diseases is an obstacle to

infection prevention and treatment. For all these reasons, there is a pressing need to

identify new, safe, and cost-effective antimicrobial agents that would help to alleviate the

problems of infectious diseases. Plant-derived natural products represent an attractive

source of antimicrobial agents because they are natural and affordable, especially for rural

societies [39].

Acceptance of medicines from such plant origins as an alternative form of healthcare

is increasing because they are serving as promising sources of novel antibiotic prototypes.

Moreover, these compounds may have different mechanisms of action than conventional

drugs and could be of clinical importance to improve health care [40 - 42]. Some of the

phytochemical compounds e.g., glycoside, saponin, tannin, flavonoids, terpenoid, and

alkaloids, have been reported to have antimicrobial activity [43, 44]. Medicinal plants

have formed the basis of health care since earliest times of humanity and are still being

widely used. The clinical, pharmaceutical and economic value continues to

153

grow, varying between countries. Chemo-diversity in plants has proven to be important in

pharmacological research and drug development, not only for the isolation of bio-active

compounds used directly as therapeutic agents, but also as leads to the synthesis of drugs

or as models for pharmacologically active compounds.

The rapid identification of these bio-active compounds, however, is critical if this

tool of drug discovery is to compete with developments in technology. Plant preparations

are distinguished from chemical drugs by their complexity-mixtures containing large

numbers of bio-active compounds. This brings about the challenge of drug discovery

from natural sources. Approximately 9000 different flavonoids have been reported from

plant sources, and with almost certainty many more are still to be discovered, as they

continue to capture the interests of scientists from numerous disciplines. Based on the 10-

carbon skeleton o flavonoids, they can be substituted by a range of different groups, viz.

hydroxyl, methoxyl, methyl, isoprenyl and bezyl substituents. This study confirmed S.

aureus to be prevalent in open wound than other specimens considered having 24.13 % of

the MRSA isolates out of 67.2 % of the MRSA.

This suggests that in Hospital Acquired MRSA ((HA-MRSA) health personnel are

one of the major source of transmission and this phenomenon should be properly addressed.

The GC-MS revealed the presence of over 100 distinct bioactive compounds among which

are: Stigmast-4-en-3-one, sitosterol, 7alpha-Hydroxy-3-oxo-4-cholestenoate,

3,methyl5(2,6,6trimethylcyclohex-1-en-1-yl)pent-1-yn-3-ol, 5-bromovaleric acid, 3,4-

dihydro-1(2H)-Naphthalenone, Ergosta-4, 22-dien-3-one, Phthalimidine, pentaene,

Ethisterone, alpha.-amino-Benzene acetic acid, 2-Thiazolamine, 9-Octadecenoic acid, Cis-

muurola-3,5-diene, Pentadecanoic acid, Palmitic Acid, a-alanine 4-methoxy-beta-

naphthylamide, Cyclohexanecarboxylic acid, Ergost-25-ene-3, 5, 6, 12-tetrol, Dodecanoic

acid, Benzofran-3-one, Carboxamide, 1,3,3 trimethyl- L-Fenchone, Gammaceran-3.alpha.-ol,

154

Terpinen-4-ol, Propenylbenzene, Geraniol, Eugenol etc. Most of these compounds had been

reported to have pharmacological and antimicrobial activities [354-357]. This can also

explain the antimicrobial activity against the MRSA clinical isolates used for this study. The

LD50 of methanol and n-hexane extracts were 3663.96 mg/kg and 1934.15 mg/kg

respectively showing that the root bark of moringa oleifera is safe for human consumption at

a dose below the LD50, This is in agreement with previous work (Ayi) [86], though his study

was on aqueous extract of Moringa oleifera root bark. .

However, further studies should be undertaken to isolate the extract pure compound,

elucidate the structure and the mechanism of action by which the compound exert their

antimicrobial effect against the MRSA.

4.2 Recommendations

It has been established that MRSA has continued to be a public health threat since it

was first discovered. Currently, MRSA is not only a prominent healthcare-associated

pathogen, but also an important cause of community-associated infections. Populations at-

risk for MRSA have also expanded to include young, healthy individuals, in addition to

vulnerable patients in healthcare facilities. The pathogen itself has evolved and showed its

ability to resist all classes of antibiotics currently used.

It is, therefore, recommended that an effective infection control strategy should

include combination of multiple interventions with a multidisciplinary approach requiring.

participation from all levels, ranging from patients themselves to health workers.

Moreover, many advances in study, methodology, including mathematical modeling

and computer simulations should be made to allow better understanding of transmission

systems and infection control measures.

There is an increasing awareness that many components of traditional medicine are

beneficial while others are harmful, hence WHO encourages and supports countries to

155

identify and provide safe and effective remedies for use in the public and private health

services (Sofowora) [97]. In view of this it is recommended that more research should be

directed towards natural products for remedy to combat this super bug resistant bacteria..

4.3 Contribution to knowledge

This study evaluated the antimicrobial efficiency of the methanol extract and fractions of

Moringa oleifera root bark against MRSA clinical isolates. The extract and fractions were

found to possess significant antimicrobial activity against MRSA clinical isolates used for the

study comparable to the standard antibiotics.

From this study, it was discovered that Moringa oleifera root bark can be used to

develop new drugs for the treatment of infectious diseases with particular reference to those

caused by methicillin resistant Staphylococcus aureus (MRSA), especially hospital acquired

strain.

156

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APPENDICES

Appendix 1. Identification of Methicillin Resistant Staphylococcus aureus (MRSA) Table: Antimicrobial susceptibility test. using Oxoid antimicrobial standard susceptibility test discs. S/n Strain No Oxa.

5 Van. 30µ

Cl. 30µ

Lev. 5µ

Cip. 5µ

Tet. 30µ

Cot. 25µ

Gen. 30µ

Da. 2µ

Rif. 5µ

1 Sample 1 S S S S S S S S S S 2 Sample 2 S S S S S S S S S S 3 Sample 4 R R R R R R R R R R 4 Sample 5 S S S S S S S S S S 5 Sample 6 S S S S S S S S S S 6 Sample 8 R R R R R R R R R R 7 Sample 10 S S S S S S S S S S 8 Sample 12 S S S S S S S S S S 9 Sample 19 S S S S S S S S S S 10 Sample 20 R R R R R R R R R R 11 Sample 22 R R R R R R R R R R 12 Sample 30 S R S S R R R R R S 13 Sample 31 S S S S S S S S S S 14 Sample 33 R R R R R R R R R R 15 Sample 35 R R R R R R R R R R 16 Sample 36 R R R R R R R R R R 17 Sample 38 R R R R R R R R R R 18 Sample 42 R R R R R R R R R R 19 Sample 44` S S S S S S S S S S 20 Sample 46 S S S S S S S S S S 21 Sample 53 R R R R R R R R R R 22 Sample 55 S S S S S S S S S S 23 Sample 57 R R R R R R R R R R 24 Sample 61 R R R R R R R R R R 25 Sample 62 R R R R R R R R R R 26 Sample 66 S S S S S S S S S S 27 Sample 123 R R R S R R R S R R 28 Sample 127 R R R R R R R R R S 29 Sample 154 R S S R R R R S R R 30 Sample 161 S S S S S S S S S S 31 Sample 187 R R R R R R R R R R 32 Sample 208 R R S R R R R S R S 33 Sample 211 S S S S S S S S S S 34 Sample 235 R R R R R R R R R R 35 Sample 270 S S S S S S S S S S 36 Sample 390 S S R S R R R R R R 37 Sample 310 R R R R R R R R R R 38 Sample 417 R R R R R R R R R R 39 Sample 504 S S S S S S S S S S 40 Sample 551 S S S S S S S S S S 41 Sample 570 R S S S R R R R R R

199

42 Sample 578 S R R R R R R R R R 43 Sample 600 R S R R R R R R R R 44 Sample 601 S S S S S S S S S S 45 Sample 620 R R R R R R R R R R 46 Sample 651 R R S S R R S R R R 47 Sample 819 R R R R R R R R R R 48 Sample 831 R R R R R R R R R R 49 Sample 841 R R S R R R R R R R 50 Sample 940 R R S R R R R R R R 51 Sample 947 R R R R R R R R R R 52 Sample 967 S S S S S S S S S S 53 Sample 1009 R R R R R R R R R R 54 Sample 1104 R R R R R R R R R R 55 Sample 1172 R R R R R R R R S R 56 Sample 1420 R R R R R R R R R R 57 Sample 1827 R R R S S R R R R R 58 Sample 1956 R R R R R R R R S R Key: Oxa: oxacillin, Van: vancomycin, Cl: Cephalexin, Lev: levofloxacin, Cip: ciprofloxacin, Tet: tetracycline, Cot: cotrimoxazole, Gen: gentamycin, Da: clindamycin, Rif: rifampicin, R: resistant, S: sensitive Appendix 2. Effect of crude methanol extract extract on methicillin-resistant

Staphylococcus aureus (MRSA)

Extract conc. 1 mg/ml

2 mg/ml

3 mg/ml

4 mg/ml

5 mg/ml

6 mg/ml

7 mg/ml

8 mg/ml

9 mg/ml

10 mg/ml

S/n Strain no 1 Sample 4 + + + - - - - - - - 2 Sample 8 + + + - - - - - - - 3 Sample 20 + + + - - - - - - - 4 Sample 22 + + - - - - - - - - 5 Sample 30 + + - - - - - - - - 6 Sample 33 + + + - - - - - - - 7 Sample 35 + + + - - - - - - - 8 Sample 36 + + - - - - - - - - 9 Sample 38 + + - - - - - - - - 10 Sample 42 + + - - - - - - - - 11 Sample 53 + + - - - - - - - - 12 Sample 57 + + + - - - - - - - 13 Sample 61 + + - - - - - - - - 14 Sample 62 + + - - - - - - - - 15 Sample 123 + + - - - - - - - - 16 Sample 127 + + + - - - - - - - 17 Sample 154 + + + - - - - - - - 18 Sample 187 + + + - - - - - - - 19 Sample 208 + + + + - - - - - - 20 Sample 235 + + + + - - - - - -

200

21 Sample 310 + + + - - - - - - - 22 Sample 390 + + - - - - - - - - 23 Sample 417 + + + - - - - - - - 24 Sample 570 + + - - - - - - - - 25 Sample 578 + + - - - - - - - - 26 Sample 600 + + - - - - - - - - 27 Sample 620 + + - - - - - - - - 28 Sample 651 + + - - - - - - - - 29 Sample 819 + + - - - - - - - - 30 Sample 831 + + - - - - - - - - 31 Sample 841 + + + - - - - - - - 32 Sample 940 + + + - - - - - - - 33 Sample 947 + + - - - - - - - - 34 Sample 1009 + + + + - - - - - - 35 Sample 1104 + + + - - - - - - - 36 Sample 1172 + + + - - - - - - - 37 Sample 1420 + + + - - - - - - - 38 Sample 1827 + + + - - - - - - - 39 Sample 1956 + + + + - - - - - -

Key: + Growth

- No Growth Appendix 3. Effect of Ethyl acetate fraction on methicillin-resistant Staphylococcus

aureus (MRSA)

Extract conc. mg/ml

1 2

3

4

5

6

7

8 9 10

S/n Strain no 1 Sample 4 + + + + + - - - - - 2 Sample 8 + + + + + + - - - - 3 Sample 20 + + + + - - - - - - 4 Sample 22 + + + + - - - - - - 5 Sample 30 + + + + - - - - - - 6 Sample 33 + + + + + - - - - - 7 Sample 35 + + + + - - - - - - 8 Sample 36 + + + - - - - - - - 9 Sample 38 + + + + + - - - - - 10 Sample 42 + + + + + - - - - - 11 Sample 53 + + + + - - - - - - 12 Sample 57 + + + + + + - - - - 13 Sample 61 + + + + + - - - - - 14 Sample 62 + + + + + - - - - - 15 Sample 123 + + + + + + - - - - 16 Sample 127 + + + + + - - - - - 17 Sample 154 + + + + + - - - - - 18 Sample 187 + + + + - - - - - - 19 Sample 208 + + + + + - - - - -

201

20 Sample 235 + + + + + - - - - - 21 Sample 390 + + + + + - - - - - 22 Sample 310 + + + + - - - - - - 23 Sample 417 + + + + - - - - - - 24 Sample 570 + + + + - - - - - - 25 Sample 578 + + + + - - - - - - 26 Sample 600 + + + + - - - - - - 27 Sample 620 + + + + - - - - - - 28 Sample 651 + + + + - - - - - - 29 Sample 819 + + + + - - - - - - 30 Sample 831 + + + + - - - - - - 31 Sample 841 + + + + + - - - - - 32 Sample 940 + + + + + + + - - - 33 Sample 947 + + + + + - - - - - 34 Sample 1009 + + + + - - - - - - 35 Sample 1104 + + + + + - - - - - 36 Sample 1172 + + + - - - - - - - 37 Sample 1420 + + + + - - - - - - 38 Sample 1827 + + + + + - - - - - 39 Sample 1956 + + + + + + - - - -

Key: + Growth

- No Growth

3 Sample 20 + + + + + + + + - - 4 Sample 22 + + + + + + + - - - 5 Sample 30 + + + + + + + + - - 6 Sample 33 + + + + + + + + - - 7 Sample 35 + + + + + + + + - - 8 Sample 36 + + + + + + + - - - 9 Sample 38 + + + + + + + - - - 10 Sample 42 + + + + + + + - - - 11 Sample 53 + + + + + + + - - - 12 Sample 57 + + + + + + + + - - 13 Sample 61 + + + + + + + + - - 14 Sample 62 + + + + + + + + - - 15 Sample 123 + + + + + + + + - - 16 Sample 127 + + + + + + + + - - 17 Sample 154 + + + + + + + + - - 18 Sample 187 + + + + + + + + - - 19 Sample 208 + + + + + + + + + - 20 Sample 235 + + + + + + + + + - 21 Sample 310 + + + + + + + - - - 22 Sample 390 + + + + + + + + - - 23 Sample 417 + + + + + + + + - - 24 Sample 570 + + + + + + + + - - 25 Sample 578 + + + + + + + + - - 26 Sample 600 + + + + + + + + - -

202

27 Sample 620 + + + + + + + - - - 28 Sample 651 + + + + + + + - - - 29 Sample 819 + + + + + + + + - - 30 Sample 831 + + + + + + + + - - 31 Sample 841 + + + + + + + + - - 32 Sample 940 + + + + + + + + - - 33 Sample 947 + + + + + + + + - - 34 Sample

1009 + + + + + + + + - -

35 Sample 1104

+ + + + + + - - - -

36 Sample 1172

+ + + + + + + - - -

37 Sample 1420

+ + + + + + + + - -

38 Sample 1827

+ + + + + + + + - -

39 Sample 1956

+ + + + + + + + - -

Key: + Growth

- No growth Appendix 4. Effect of n- hexane fraction on methicillin-resistant Staphylococcus aureus

(MRSA)

Extract conc. mg/ml

1 2

3

4

5

6

7

8 9 10

S/n Strain no 1 Sample 4 + + + + + + + - - - 2 Sample 8 + + + + + + + - - - 3 Sample 20 + + + + + + + - - - 4 Sample 22 + + + + + + - - - - 5 Sample 30 + + + + + + - - - - 6 Sample 33 + + + + + + + - - - 7 Sample 35 + + + + + + + - - - 8 Sample 36 + + + + + + + - - - 9 Sample 38 + + + + + + - - - - 10 Sample 42 + + + + + + - - - - 11 Sample 53 + + + + + + + - - - 12 Sample 57 + + + + + + + - - - 13 Sample 61 + + + + + + + - - - 14 Sample 62 + + + + + + - - - - 15 Sample 123 + + + + + + + - - - 16 Sample 127 + + + + + + + - - - 17 Sample 154 + + + + + + + - - - 18 Sample 187 + + + + + + + - - -

203

19 Sample 208 + + + + + + + - - - 20 Sample 235 + + + + + + + - - - 21 Sample 310 + + + + + + + - - - 22 Sample 390 + + + + + + + - - - 23 Sample 417 + + + + + + - - - - 24 Sample 570 + + + + + + - - - - 25 Sample 578 + + + + + + - - - - 26 Sample 600 + + + + + + - - - - 27 Sample 620 + + + + + + - - - - 28 Sample 651 + + + + + + - - - - 29 Sample 819 + + + + + + - - - - 30 Sample 831 + + + + + + - - - - 31 Sample 841 + + + + + + + - - - 32 Sample 940 + + + + + + + - - - 33 Sample 947 + + + + + + + - - - 34 Sample 1009 + + + + + + + - - - 35 Sample 1104 + + + + + + - - - - 36 Sample 1172 + + + + + + - - - - 37 Sample 1420 + + + + + + - - - - 38 Sample 1827 + + + + + + + - - - 39 Sample 1956 + + + + + + + - - - Key: + Growth

- No Growth -

Appendix 5. Effect of methanol fraction on methicillin-resistant Staphylococcus aureus (MRSA)

Extract conc. mg/ml

1 2

3

4

5

6

7

8 9 10

S/n Strain no 1 Sample 4 + + + + + + + + + + 2 Sample 8 + + + + + + + + + + 3 Sample 20 + + + + + + + + + + 4 Sample 22 + + + + + + + + + - 5 Sample 30 + + + + + + + + + - 6 Sample 33 + + + + + + + + + + 7 Sample 35 + + + + + + + + + + 8 Sample 36 + + + + + + + + + - 9 Sample 38 + + + + + + + + + - 10 Sample 42 + + + + + + + + + - 11 Sample 53 + + + + + + + + + - 12 Sample 57 + + + + + + + + + + 13 Sample 61 + + + + + + + + + - 14 Sample 62 + + + + + + + + + - 15 Sample 123 + + + + + + + + + - 16 Sample 127 + + + + + + + + + + 17 Sample 154 + + + + + + + + + + 18 Sample 187 + + + + + + + + + +

204

19 Sample 208 + + + + + + + + + + 20 Sample 235 + + + + + + + + + + 21 Sample 310 + + + + + + + + + + 22 Sample 390 + + + + + + + + + - 23 Sample 417 + + + + + + + + - - 24 Sample 570 + + + + + + + + - - 25 Sample 578 + + + + + + + + - - 26 Sample 600 + + + + + + + + - - 27 Sample 620 + + + + + + + + - - 28 Sample 651 + + + + + + + + + + 29 Sample 819 + + + + + + + + + + 30 Sample 831 + + + + + + + + + + 31 Sample 841 + + + + + + + + + + 32 Sample 940 + + + + + + + + + + 33 Sample 947 + + + + + + + + + + 34 Sample 1009 + + + + + + + + - - 35 Sample 1104 + + + + + + + + + - 36 Sample 1172 + + + + + + + + + - 37 Sample 1420 + + + + + + + + + - 38 Sample 1827 + + + + + + + + + - 39 Sample 1956 + + + + + + + + - -

Appendix 6. GC-MS report of ethyl acetate fraction

Structural analogue Rt (min) Area % Peak Stigmast-4-en-3-one , Cholest-4-en-26-oic acid, 3-oxo-Cholest-4-en-3-one

46.49 8.72 1

5-Bromovaleric acid, 2,6-dimethylnon-1-en-3-yn-5-yl ester , Cyclobuta [1,2:3,4]dicyclooctene-1,7(2H,6bH)-dione.

46.58 3.19 2

3-Chloropropionic acid, 2,6-dimethylnon-1-en-3-yn-5-yl ester, 2-Amino-4-methyl-3-pyridinol, Pregn-4-en-3-one.

46.62 5.80 3

4,22-Stigmastadiene-3-one, 24S--Ethyl-5.alpha.-cholesta-2-dien-6-one, Spinasterone

48.54 12.29 4

4,22-Stigmastadiene-3-one, 4,22-Cholestadien-3-one , Ergosta-4,22-dien-3-one

48.62 10.73 5

9,19-Cycloergost-24(28)-en-3-ol, 4 ,14-dimethyl-,(3.beta.,4.alpha.,5 .alpha.)-7-(1,3-Dimethylbuta-1,3-dienyl)-1,6,6-trimethyl-3,8-dioxatricyclo[5. (2,4)]Octane 2(1H)-Naphthalenone.

49.97 2.89 6

11,13-Dimethyl-12-tetradecen-1-ol, Bicyclo[5.2.0]nonane, 4-methylene-2,8,8-trimethyl-2-vinyl(E,E,E)-3,7,11,15-Tetramethylhexadeca1,3,6,10,14-pentaene.

50.49 2.28 7

9,19-Cycloergost-24(28)-en-3-ol ,14-dimethyl-(3.beta.,4 .alpha.,5.alpha.)-9,19-Cyclolanostan-3-ol,

50.52 2.57 8

Stigmast-4-en-3-one, Cholest-4-en-26-oic acid, 3-oxo- 1,4-Benzenediol, 51.34 28.23 9 Supraene, Octasiloxane, 1,1,3,3,5,5,7,7,9,92511,11,13,13,15,15-hexadecamethyl-17-(1,5-Dimethylhexyl)-10,13 dimethyl4vinylhexadecahydrocyclopenta [a]phenanthren-3-ol

52.51 1.39 10

17-(1,5-Dimethylhexyl)-10,13-dimethyl4vinylhexadecahydrocyclopenta [a]phenanthren-3-ol, Stigma sterol , 2,2-Dimethylpropanoic acid, 2,6-

52.52 1.18 11

205

Appendix 7 (a). Activity of components identified in the sample [GC-MS study] of ethyl acetate fraction S/N Name of compounds Molecular.formula M.W Nature Activity

1

Stigmast-4-en-3-one,

C29H48O Chem Spider ID: 504066

412 Ketone, Steroid compound

Antimicrobial, Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

2

4,22-Stigmastadiene-3-one

C29H46O Pub.Chem: ID5364563

410 Ketone, Steroid Compund

Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

3

Cholest-4-en-26-oic acid

C27H42O4

Chem Spider ID:20566404

430 Aliphatic Acid, Sesterterpenes

Metabolism and regulation of cholesterol homeostasis

4

5-Bromovaleric acid C5H9BrO2, CAS No.: 2067-33-6

181 Β-Aroylacrylic acids, Polyether.

Antimicrobial

5

2,6-dimethylnon-1-en-3-yn-5-yl ester,

C16H26O2, NIST MS No 299336

250 Oxime esther derivatives

Antimicrobial, Virucidal activity

6

Cyclobuta [1, 2:3, 4] dicyclooctene-1,7(2H,6bH)-dione.

C16H24O2 Pub. Chem. ID: 579875

248 Hydrocarbons No Activity reported

7

3-Chloropropionic acid C3H5ClO2

Chem. Spider ID: 7611

108 chlorinated aliphatic acids

protocols in organic synthesis including Antibacterial agents

8 2-Amino-4-methyl-3-pyridinol

C5H6N2O ChemSpider ID:

110 Pyridine derivatives,

Antimicrobial, antitumor agents and

dimethylnon-1-en-3-yn-5-yl ester Cholestan-3-one, 4,4-dimethyl-, (5.alpha.)-beta.-Alanine, N-(2-furoyl)-, hep tyl ester, 5.beta.,6.beta.-Epoxy-7.alpha.-bromocholestan-3.beta.-ol

54.48 3.57 12

4-Oxatricyclo[20.(7,16) ]triac onta-1(20)7(16)-diene , 17-(1,5-Dimethylhexyl)-10, 13-dimethyl-4-vinylhexade cahydrocyclopenta [a]phenanthren-3-ol, Cholestan-3-one,

54.51 3.39 13

Pyridine-3-carboxamide, oxime, N-(2-trifluoromethylphenyl) Cyclopropane carboxamide, 2-cyclopropyl-2-methyl-N-(1-cyclopropylethyl)-Benz[e]azulene-3,8-dione.

55.43 1.49 14

1,19-Eicosadiene, 9-Methyl-Z-10-tetradecen-1-ol, 1-Naphthalenepropanol.

56.02 3.51 15

2(1H)-Naphthalenone, octahydro-4a- methyl-7-(1-methylethyl)-, (4a.alpha. 7. beta.,8a.beta.)- 9-Methyl-Z-10-tetradecen-1-ol , 1-Naphthalenepropanol,

56.05 2.03 16

3a-Cholesterol acetate, Cholest-7-en-3-ol, Citrost-7-en-3-ol 59.18 3.96 17 Citrost-7-en-3-ol, Stigmastane-3,6-dione, Cholest-8-en-3.beta.-ol, 59.21 2.78 18

206

26152 Alcohol herbicide

9

-bis (Methylene-dioxy)-Pregn-4-en-3-one

C25H34O6 ChemSpider ID: 4444479

486 Endogenous steroid hormone

Metabolic Intermediate in Production of other endogenous steroid, Treatment of Brain Injury

10

24S--Ethyl-5.alpha.-cholesta-2-dien-6-one, Spinasterone

C29H46O PubChemID: 5364566

410 Steroid compound

Antibacterial and antifungal activities

11

2,2-Dimethylpropanoic acid (Pivalic acid)

C5H10O2

ChemSpider ID: 7611

Pivalic (Carboxylic) acid

Antimicrobial

12

4vinylhexadecahydrocyclopenta[a] phenanthren-3-ol

C29H50O ChemSpider ID: 467796

414 Steroidal compound

Antibiofilm, Insecticidal activity

13

Ergosta-4,22-dien-3-one

C28H44O ChemSpiderID:4941415

396 Triterpenoids and Steroids, Pyridine

Antimicrobial and Antitumor, Anti inflammatory, Anti viral.

14

2(1H)-Naphthalenone. C10H10O Chem Spider ID: 21106584

146 Aliphatic Alcohol, ally amines

Antibacterial and antifungal.

Appendix 7 (b). Activity of components identified in the sample [GC MS study] of ethyl

acetate

S/N Name of compounds Molecular.formula M.W Nature Activity

15

11,13-Dimethyl-12-tetradecen-1-ol

C18H34O2 NIST :MS NO: 130810

282 Fatty Alcohols, Phenolic

Antioxidant

16

Bicyclo[5.2.0] nonane C9H16, Pub. ChemID:524792

124 Sesquiterpene compound

Antimicrobial,Anti-inflammatory, Anti Hyperlipidemic

17

Pentaene C20H32 Chem Spider ID: 391697

272 Polyene Lipids Antimicrobial

18

3.beta.,4 .alpha.,5.alpha.)-9,19-Cyclolanostan-3-ol,

C32H54O2 Pub.Chem:ID 00537304

470 Triterpenes Antimicrobial, Antioxidant

19

3-oxo- 1,4-Benzenediol C18H18N2O5 Chem Spider 677762

342 Oxygenated Aldehyde Derivatives, Indole analogues

Antimicrobial, Antioxidant

20

Supraene C30H50 Pub. Chem.ID: 638072

410 Sesquiterpenoid and triterpenoid

Anticancer, antimicrobial, antioxidant, chemo preventive pesticide, anti- tumor sunscreen

21

Octasiloxane C40H74O13 Sigma Alreich CAS Number

987 Sesquiterpenoid, organosilicon compound

Antibacterial ,Antifungal

207

352538-83-1

22

4, 4-dimethyl-, (5.alpha.)-beta.-Alanine (3-aminopropanoic acid)

C14H16N2O2 ChemSpider ID: 580864

244 Organ peptide macro cyclic compounds

Antimicrobial

23

Pyridine-3-carboxamide

C6H6N218O ChemSpider ID: 9334423

124 Nicotinamide, Nicotine Amines.

Antiviral, Anticancer , antiprotozoa

24

Oxime C3H7NO ChemSpider ID: 60524

73 Natural Phenolic compound

Antioxidant, Antibacteria and anticancer agent

25

N-(2-trifluoromethylphenyl) Cyclopropane carboxamide,

C15H18F3NO Pub. Chem ID 00722477

285 Aliphatic Hydrocarbon

Pesticides in domestic animals

26

Citrost-7-en-3-ol C30H52O Pub. Chem.ID: 541368

428 Aliphatic Alcohol Compound

Antimicrobial, Antioxidants

27

1-Naphthalenepropanol C13 H15 N O CAS No.: 19352-04-6

201 Phenolic compound

Antimicrobial and Antifungal

Appendix 8 (a). GC-MS report of dichloromethane fraction

Structural analogue Rt(min) Area % Peak Benzylamine 11.33 0.46 1 Benzyl isocyanate, Phthalimidin 16.87 1.96 2 3-Methyl-4-isopropylphenol , Thymol , 2-methyl-5-(1-methylethyl) Phenol

25.97 1.57 3

N-Benzylformamide 30.21 0.68 4 Acetamide, N-(phenyl methyl)- 2-Methyl-5-butylpyridine , Dimethyl (1E)-N-hydroxyethanimidoy lphosphonate

32.09 2.78 5

Humulene 32.35 0.56 6 1,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-1H Cyclopenta[1,3]cyclopropa[1,2]benzene, octahydro-7-methyl-3-methylene-4-(1-methylethyl)-[3aS-(3a alpha.,3b.beta.,4.beta.,7.alpha.,7 aS*)]-.beta.-copaene

33.57 0.77 7

1, 2, 3, 5, 6, 8a-hexahydr-4,7-dimethyl-1-(1-methylethyl)-,(1S-cis)-Naphthalene.

35.41 1.06 8

tau.-Muurolol, alpha-Cadinol , cis-muurola-3,5-diene 40.21 0.99 9 .alpha-Cadinol, Cyclohexane, 1-ethenyl-1-methyl-2- (1-methylethenyl)-4-(1-methylethylidene)-Epiglobulol

40.71 1.70 10

Hexadecanoic acid, Pentadecanoic acid, 14-methyl-,methyl ester 50.64 5.89 11 n-Hexadecanoic acid 52.16 1.42 12 Cyclopropaneoctanoic acid, 2- methyl ester 9-Octadecenoic acid, (Z)-methyl Ster,cis-10-Heptadecenoic acid.

53.39 0.67 13

Heptadecanoic acid, Hexadecanoic acid. 53.92 0.39 14 9,12-Octadecadienoic acid, 10,13-Octadecadienoic acid, 56.07 4.29 15 9-Octadecenoic acid , 8-Octadecenoic acid, methyl ester 56.26 3.32 16 9-Octadecenoic acid, 11-Octadecenoic acid, 56.44 0.96 17 Methyl stearate 57.11 1.28 18 Urea, N,N'-bis(phenyl methyl)-Benzeneacetamide, alpha.-amino-Benzene acetic acid,

64.34 1.87 19

Docosanoic acid, methyl ester, Tricosanoic acid, methyl ester 68.62 0.53 20

208

Appendix 8 (b). GC-MS report of dichloromethane fraction

Bis(2-ethylhexyl) phthalate, Di-n-octyl phthalate 21.69 0.37 21 5-Fluoro-1,3-bis[phenylmethyl]-2,4 (1H,3H)-pyrimidinedione, 5-Benzyloxy-6-methoxy-8-nitroquinoline, Ethisterone

70.47 0.39 22

2-Thiazolamine, 4-(4-methoxyphenyl )-N-(4-methylphenyl)-Naphtho[1,8-cd]-(1,2,6)-phosphadiazine, 2-phenyl-2-thioxo-1,3(2H)-di hydro-1-Dimethyl(ethenyl)silyloxy-3-phenylpropane

23.71 1.20 23

Benzene, 1-isocyanato-3-methoxy- 1H-S-Triazolo[1,5-a]pyridin-4-ium, 2-hydroxy-1-methyl-, hydroxide, inner salt, Didodecylphthalate,

76.72 0.73 24

Benzonitrile, 2-fluoro-4-(4'-propyl l[1,1'-bicyclohexyl]-4-yl)-Oxazole, 2-(3-methoxyphenyl)-5-phe nyl-Cyclopropane , carboxamide,

79.02 0.39 25

N-(phenylmethyl)- Acetamide, 80.83 0.69 26 Cedran-diol, (8S,14)- Phthalic acid, hexyl 1-phenylpropy l este, Benzo[1,3]dioxole-5-carboxylic aci d (5-chloro-2-oxo-1,2-dihydro-indo l-3-ylidene)-hydrazide

81.51 0.51 27

Stigmastan-3,5-diene; .beta.-Sitosterol acetate 81.97 0.57 28 Campesterol, (3.beta.)-Ergost-7-en-3-ol, 85.32 0.92 29 Stigmasterol 86.28 1.13 30

Structural analogue Rt(min) Area % Peak Pyridine-3-carboxamide, oxime, N-( 2-trifluoromethylphenyl)-3-n-Heptyl-7-methyl-9-(2,6, thylcyclohex-1-enyl)nona-2,4,6,8- etraenal ,1,3-Dioxolane.

87.33 0.38 31

4,4,6a,6b,8a,11,12,14b-Octamethyl-octadecahydro-2H-picen-3-one, 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-.gamma.-Sitosterol.

88.06 1.63 32

5-Bromovaleric acid, 2,6-dimethylnon-1-en-3-yn-5-yl ester, 6-Bromohexanoic acid,4-methoxyphenyl ester Cyclohexanecarboxylic acid, 4-methoxyphenyl ester

10.14 89.57 33

4,22-Stigmastadiene-3-one, Ethyl-5.alpha.-cholesta -dien-6-one, Ergosta-4,22-dien-3-one

90.75 11.91 34

Cholest-7-en-3-one, 4, 4-dimethyl-(5.alpha.)- Stigmasterol, Ergost-25-ene-3,5,6,12-tetrol

91.10 0.44 35

Benzofran-3-one, 2-[3,4-dihydroxybenzylidene]-6-hydroxy- Stigmasterol, Ergosta-4,6,22-trien-3-one

91.70 1.60 36

C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha.-ol, 17-(1,5-Dimethylhexyl)-10,13-dimet hyl-4 vinylhexadecahydrocyclopenta[a]phenanthren-3-ol, 9,19-Cyclo-25,26-epoxyergostan-3-o l, 4,4,14-trimethyl-, acetate

92.21 2.77 37

Stigmast-4-en-3-one, Testosterone , Androst-4-en-3-one, 93.05 12.33 38 Nickel, cyclopentadienyl-(dicycloh exylphosphino)benzyl-o-yl- Stigmasta-4,6,22-trien-3.alpha.-ol, Stigmasta-4,6,22-trien-3.beta.-ol

94.09 1.53 39

-4,6,22-trien-3.beta.-ol , Stigmasta-4,6,22-trien-3.alpha.-ol, Stigmasta-3,5-dien-7-one

94.11 1.02 40

Cholestan-3-one, 4,4-dimethyl-, (alpha.)-Stigmastan-7-one, 17 (1,5Dimethylhexyl)-10,13-dimethyl-4vinylhexadecahydrocyclopenta[a]phenanthren-3-ol

96.05 2.17 41

Cholestan-3-one, 4,4-dimethyl-Dihydrosarsasapogenin-5,17(20)-die Phenol 96.08 1.98 42 C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha.-ol 22-Stigmasten-3-one ,4-Diethyl thiophosphoryl-3-thiometh 1 yl allophanate

97.57 3.62 43

Stigmastane-3,6-dione, (5.alpha.)- Lanostane, 11,18-epoxy-, (11.beta. 17-(1,5-Dimethylhexyl)-2-(1-hydrox yethylidene)-10,13-dimethylhexadec ahydrocyclopenta[a]phenanthren-3-one

100.66 1.80 44

Stigmastane-3,6-dione , (5.alpha.)- anost-7-en-3-one, (9.beta.,,17.alpha.)- 3,12 Diazatetrabenzo[a,cd,j,lm] per ylene

100.71 1.62 45

Benzenepropanoic acid, 3,5-bis(1,1 -dimethylethyl)-4-hydroxy-, octade Benzenepropanoic acid, -dimethylethyl)-4-hydroxy-,octade cyl ester, beta.-Tocopherol,

101.15 2.59 46

Benzenepropanoic acid, 3,5-bis(1,1 -dimethylethyl)-4-hydroxy-, octade cyl ester

101.19 2.43 47

209

Appendix 9 (a). Activity of components identified in the sample [GC MS study] DCM fraction

S/N Name of Compounds Molecular Formula

M.W Nature Activity

1 Benzylamine C7H9N Chem Spider ID7223 107

Alkaloids Antimicrobial

2 Benzyl isocyanate C10H14O Chem.Bk:2491615 150

Aromatic compound

Antimicrobial, Antitumor, Antiviral

3 Phthalimidin C8H7NO2

Pub. Chem ID171091 149

Organic Compound, phthalic anhydride

Antibacterial, Antifungal

4 3-Methyl-4-isopropylphenol, Thymol, 2-methyl-5-(1-methylethyl) Phenol

C10H14O Lun.Chem:3228-02-2

150

Aromatic Alcohol, Monoterpines, Phenol Derivative

Antiviral, Antibacterial and Antifungal Activities

5 N-Benzylformamide C8H9NO Pub. chem. ID:80654 135

Peptide, Amide Derivatives

Antimicrobial, Antimalaria[

6 Acetamide C2H5NO Pub. Chem: ID 178 59

Peptide, Amide Derivative

Anti cancer, Anti inflammatory, Antioxidant

7 N-(phenyl methyl)- 2-Methyl-5-butylpyridine

C12H11N,Guide.Chem CAS27012-22-2 169

Esther, Pyridine Derivative

Antimicrobial analogues

8 Dimethyl (1E)-N-hydroxyethanimidoy lphosphonate

C10H21ClN2O2

MFCD08457445 236

Piperidine carboxylic acid, Isoprenoid

Antibacterial and Ant parasitic drug

9 Humulene C15H24 Pub Chem:5281520 204

monocyclic sesquiterpene

Antimicrobial and Antitumor activity

10 1,6-Cyclodecadiene C10H16 Chem Spider ID: 4517601 136

Antimicrobial and Antifungal activity

11 1-methyl-5-methylene-8-(1-methylethyl)-1H Cyclopenta[1,3]cyclopropa[1,2]benzene

C15H24 Look Chem

CAS 13744-15-5

204

Aromatic compound

Antioxidant, Antimicrobial and Insecticidal agent,

12 7 aS*)]-.beta-copaene C15H24ChemSpider ID: 10306774 204

A tricyclic sesquiterpene

Antimicrobial

13 1, 2, 3, 5, 6, 8a-hexahydr-4,7-dimethyl-1-(1-methylethyl)-,(1S-cis)-Naphthalene

C15H24 ChemSpider ID: 389830

204

Aliphatic Alcohol Antimicrobial and Antifungal Activity

14 alpha-Cadinol C15H26O ChemSpider ID: 8574094 222

Phenol Antimicrobial, Antifungal, Anticancer

15 cis-muurola-3,5-diene C15H24 (CHEBI:61687) 204

carbobicyclic compound ,sesquiterpene

Antibacterial, Antioxidants

16 Hexadecanoic acid (Palmitic Acid)

C16H32O2 ChemSpider ID: 960

256

Ester, Fatty Acid Antibacterial, Anti-inflammatory, Antitumor

17 Pentadecanoic acid C15H30O2 ChemSpider ID: 13249 242

Saturated Fatty Acid

Antibacterial, Antifungal activity

18 Cyclohexane, 1-ethenyl-1-methyl-2- (1-methylethenyl)-4-(1-methylethylidene)-Epiglobulol

C6H12 ChemSpider ID: 7787

84

Aromatic Phenols Cytotoxic, Antimicrobial, Antifungal, Antioxidant

210

Appendix 9 (b). Activity of components identified in the sample [GC MS study] of DCM fraction

S/N Name of Compounds Molecular Formula

M.W Nature Activity

19 Cyclopropaneoctanoic acid C22H38O2 NIST

CAS 10152-71-3 334 Natural alicyclic fatty acids

Antimicrobial [370]

20 2-methyl ester 9-Octadecenoic acid, Methyl stearate

C19H36O2, NIST

CAS 1937-62-8 296 Ester Antioxidant activity,

Ant carcinogenic,-exist in human blood and urine and serve as endogenous peroxisome proliferatoractivated receptor ligand, dermatitigenic flavor

21 Heptadecenoic acid C17H32O2. Pub. Chem CID 5312435 268

Fatty Acid Antifungal

22 Urea CH4N2O Chem

Spider ID: 1143 60 Organic Compound

Antimicrobial

23

N,N'-bis(phenyl methyl)-Benzeneacetamide

C14H21 N O, Guide Chem CAS. 34251-46-2 219

Endogenous Peptide

Antimicrobials, Herbicidal

24 alpha.-amino-Benzene acetic acid

C8H9NO2. Chemical Book CAS 2835-06-5 151

Carboxylic Acid, Amino Acid, Ester

Antibacterial and Antifungal

25 Docosanoic acid (Behenic Acid)

C22H44O2 Pub. Chem ID 8215 340

Saturated Fatty Acid

Antibacterial

26

Tricosanoic acid C23H46O2. Pub. Chem CID 17085 354

Long Chain Fatty Acid

Antimicrobial and Insecticidal activity

27

5-Fluoro-1,3-bis[phenylmethyl]-2,4 (1H,3H)-pyrimidinedione,

C18H15FN2O2 ChemSpider ID: 309662 310

Peptides Antiglioma, Antimicrobial, Cytotoxic

28

5-Benzyloxy-6-methoxy-8-nitroquinoline

C17H14N2O4.

Pub. Chem 563944 310 Quinolines, Ethyl

Esther Antitumor Antibiotic, Antimalarial, Antileishmanial, Antimicrobial

29 Ethisterone C21H28O2

Pub Chem 5284557 312 Steroid Hormone Antibacterial and Antitumor

30

2-Thiazolamine C3H4N2S CAS 96-50-4

100 Hydrazine derivatives, Heterocyclic Amine

Antimicrobial, Anti-infective and Antioxidant

31

]-(1,2,6)-phosphadiazine

C16H13N2PS Chem Spider 544268

296 Hydrazine derivatives

Antimicrobial(Topical) Agents

32

Dimethyl(ethenyl)silyloxy-3-phenylpropane

C13H20OS Pub. Chem 00576332

220 Pyridine Derivatives

Antimicrobial

33

Didodecylphthalate C24H38O4 ChemSpider ID: 8043

390 Carboxylic Acid Antimicrobial, Food Preservative

34

Cedran-diol C15H26O2

Pub Chem 536384 238 Flavonoids Antimicrobial

anti inflammatory, anticancer

211

Appendix 9 (c). Activity of components identified in the sample [GC MS study] of DCM fraction

S/N Name of Compounds Molecular Formula

M.W Nature Activity

35

Inner salt C11H23COHNSO3

EC N0 226-003-9 292 sulfonium salts Antimicrobial and

Antifunga l

36

2-fluoro-4-(4'-propyl l[1,1'-bicyclohexyl]-4-yl)-Oxazole

C26H40O5, CAS 913258-34-1

432 Carboxylic Acids, Diterpines

Antimicrobial

37

Benzonitrile C7H5N Chem Spider ID: 7224

103 benzo and naphthonitrile derivatives.

Antibacterial and Antifungal

38 Benzene C6H6 78 Aromatic

Compound Antibacterial and Antifungal

39

2-(3-methoxyphenyl)-5-phenyl-Cyclopropan-1- carboxamide

C24H20FNO4

Pub. ChemID 59206456

405 Carboxylic Acid Anticonvulsant, Antituberculosis

40

N-(phenylmethyl)-Acetamide,

C9H11NO ChemSpider ID: 11016

149 Peptide Amide Derivative

Trypanosidal, Antibacterial Antifungal

41

(5-chloro-2-oxo-1,2-dihydro-indo l-3-ylidene)-hydrazide

Isatin, Imidazoline Antibacterial, antifungal, Anticancer, Antihelmintic

42

Stigmastan-3,5-diene C29H48

Pub Chem ID 00525918

396 Steroid compound Antifungal, Antibacterial Antioxidant

43 beta.-Sitosterol acetate C29H50O

CAS 83-46-5 414 Plant Sterols Benign prostatic

hyperplasia, Antimicrobial

44 Campesterol C28H48O 400 Steroidal

compound Antimicrobial, Antileishmanial

45

(3.beta.)-Ergost-7-en-3-ol, C28H48O Pub. Chem ID 86509

400 Fatty Alcohols, Steroid compound

Antibacterial, Anticandidasis, Antioxidant.

46

Stigmasterol C29H48O Chem Spider ID: 504066

412 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

47 Pyridine-3-carboxamide C8H7N3O

Pub.Chem CID 80322 161 Nicotinamide,

Nicotine Amines. Antiviral, Anticancer, antiprotozoa

48

Oxime C3H7NO ChemSpider ID: 60524

73 Natural Phenolic flavonoid

Antioxidant, Antibacteria and anticancer agent

49

N-( 2-trifluoromethylphenyl)-3-n-Heptyl-7-methyl-9-(2,6, thylcyclohex-1-enyl)nona-2,4,6,8- etraenal

Alkyl Esthers useful for inhibiting the delta isoform of PI3K, and for treating disorders mediated by lipid kinases such as inflammation, immunological, and cancer .

50

1,3-Dioxolane C3H6O2 CB 5712494

74 Aprotic solvent, Ether

Intermediate for the preparation of Acyclovir-d4, Antibacterial

51 14b-Octamethyl-octadecahydro-2H-picen-3-

C30H48O Pub.Chem ID 612782

424 Triterpine Antibiotic Prototype, Antioxidant

212

one

Appendix 9 (d): Activity of components identified in the sample [GC MS study] of DCM fraction

S/N Name of Compounds Molecular Formula

M.W Nature Activity

52

14b-Octamethyl-octadecahydro-2H-picen-3-one

C30H48O Pub.Chem ID 612782

424 Triterpine Antibiotic Prototype, Antioxidant

53

2(1H)Naphthalenone C10H10O Chem Spider ID: 21106584

146 Aliphatic Alcohol, ally amines

Antibacterial and antifungal.

54

8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-.gamma.-Sitosterol

Steroid compound Frangrance Ingredient

55 5-Bromovaleric acid C5H9BrO2,

CAS No.: 2067-33-6 181 Β-Aroylacrylic

acids, Polyether. Antimicrobial

56 2,6-dimethylnon-1-en-3-yn-5-yl ester

C16H24O2 Pub. Chem ID 00530940

248 Ester Moderate Antibacterial and Antifungal, Vector control

57 6-Bromohexanoic acid BrCH2(CH2)4COOH 195 Carboxylic Acid As a conjugate in Antitumor

drug

58 Cyclohexanecarboxylic acid

C6H11CO2H CAS Num 98-89-5

128 Carboxylic acid, Ester

Food and Flavor Ingredient, Antifungal

59

4,22-Stigmastadiene-3-one C29H46O Pub.Chem: ID5364563

410 Steroid Compound Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

60

Ethyl-5.alpha.-cholesta -dien-6-one

C29H48O Chem Spider ID: 504066

412 Steroid compound Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

61

Ergosta-4,22-dien-3-one C28H44O Chem Spider ID:4941415

396 Triterpenoids and Steroids, Pyridine

Antimicrobial and Antitumor, Anti inflammatory, Anti viral.

62 Cholest-7-en-3-one C27H44O

Pub. Chem.ID 27296 384 Steroid Compound,

Peptide antibacterial, antifungal, antiviral, and antiprotozoal

63

Ergost-25-ene-3,5,6,12-tetrol

C28H48O4 ChemSpider ID: 473378

448 Steroid Compound, Peptide

Cytotoxic effect

64

Benzofran-3-one C15H10O5 ChemSpider ID: 4444681

270 Triterpenoids and Steroids, Pyridine

Antileishmanial, Antimicrobial

65

2-[3,4-dihydroxybenzylidene]-6-hydroxy- Stigmasterol

C29H48O Chem Spider ID: 504066

412 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

66

Ergosta-4,6,22-trien-3-one C28H42O ChemSpider ID: 4526809

394 Aromatic Steroids Antimicrobial

67

C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha.-ol

C30H52O Pub. ChemID 550124

428 Fatty Alcohols, Steroid compound

Peptide Receptor Targeting in Cancer

213

Appendix 9 (e). Activity of components identified in the sample [GC MS study] of DCM fraction

68

Dimethylhexyl)-10,13-dimet hyl-4 vinylhexadecahydrocyclopenta[a]phenanthren-3-ol

C29H50O ChemSpider ID: 467796

414 Steroid compound Antibiofilm, Insecticidal activity

69

9,19-Cyclo-25,26-epoxyergostan-3-ol4,4,14-trimethyl-, acetate

C33H54O3

Pub Chem 565753 498 Steroid compound Anti inflammatory

70

Stigmast-4-en-3-one C29H48O Chem Spider ID: 504066

412 Steroid compound Antimicrobial Antioxidant Anti-inflammatory, Ant arthritic Antiasthma Diuretic

71 Testosterone, Androst-4-en-3-one

C19H28O2

Pub. Chem:ID 6013 288 Androgenic

Steroid Anti-inflammatory

72

Nickel, cyclopentadienyl-(dicycloh exylphosphino)benzyl-o-yl-

C24H34NiP- Pub. Chem ID 11987286

412 Isoflavones, Flavonoids

Chiral Langand in synthesis of drugs

73

Stigmasta-4,6,22-trien-3.alpha.-ol, Stigmasta-4,6,22-trien-3.beta.-ol, Stigmasta-3,5-dien-7-one , 4,4-dimethyl-, (alpha.)-Stigmastan-7-one

C29H46O Pub. Chem ID 5379793

410 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory

74

Cholestan-3-one C27H46O ChemSpider ID: 77468

386 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory

75

C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha.-ol

C30H52O Pub. Chem ID 550124

428 Chenodeoxycholic acid, steroid acid

cholagogue, a choleretic laxative

76 (5.alpha.)- Lanostane C30H54 Chem Spider

ID:7827588 414 Triterpines

compound Antibacterial, Antiinflammatory

77

10,13-dimethylhexadec ahydrocyclopenta[a]phenanthren-3-one

C23H35NO3 Chem. Book

373 Phenyl Ester Antibiofilm Agent

78 5.alpha.)- anost-7-en-3-one C19H28O

CB1139907 272 Androgenic

Steroid Antimicrobial, Anti-inflammatory

79

Benzenepropanoic acid-dimethylethyl)-4-hydroxy-,octadecyl ester

C37H66O3 Chem. Net CAS 56823-64-4

558 Acid Ester Ester Prodrugs and Antiviral Activity

80 beta.-Tocopherol C28H48O2.

Pub.hem 86052 416 Beta Carotene Cancer Prevention,

Antioxidants

214

Appendix 10 (a). A GC-MS report of n-hexane fraction Structural analogue Rt(min) Area % Peak Dodecane, 2,6,11-trimethyl- Tridecane, 1-iodo- 1,3-Dimethylcyclopentanol

18.26 0.22 1

Heneicosane, Pentadecane, Dodecane 25.22 0.27 2 Dodecanoic acid 27.32 0.29 3 Heneicosane , 2-Bromo dodecane, Nonane, 30.41 0.32 4 Tetradecanoic acid 31.87 0.40 5 Pentadecanoic acid 34.01 0.43 6 Heptacosane Heneicosan, Hexadecane 35.06 0.33 7 Hexadecanoic acid, Pentadecanoic acid, 35.14 0.24 8 Palmitoleic acid, 9-Hexadecenoic acid , 4-Methyl-dodec-3-en-1-ol 35.70 0.31 9 n-Hexadecanoic acid 36.59 17.35 10 n-Hexadecanoic acid , Cyclic octaatomic sulfur7-Amino-7H-S-triazolo[5,1-c]-S-tri azole-3-thiol

37.38 0.25 11

cis-Vaccenic acid, Cyclopentadecanone, 2-hydroxy- 9-Hexadecenoic acid 37.92 0.23 12 9,12-Octadecadienoic acid, methyl ester 38.52 0.56 13 9-Octadecenoic acid 38.63 0.30 14 Hexacosane , Octadecane, 2-methyltetracosane 39.28 0.22 15 9,12-Octadecadienoic acid, 2-Chloroethyl linoleate 40.30 41.08 16 9,12-Octadecadienoic acid (Z,Z)-2-Chloroethyl linoleate 40.33 1.73 17 40.384 8.55 C 9,12-Octadecadienoic acid 40.38 8.55 18 9,12-Octadecadienoic acid 40.57 1.35 19 9,12-Octadecadienoic acid 43.12 0.47 20 Oleic Acid, 2-Methyl-Z,Z-3,13-octadecadienol, 1-Naphthalenol, 1,2,3,4-tetrahydroacetate

45.37 0.38 21

Octadecane, 1-(ethenyloxy)- 9,17-Octadecadienal, (Z)-Z,E-3,13-Octadecadien-1-ol

46.40 0.25 22

Bis(2-ethylhexyl) phthalate 46.51 0.54 23 Dodecanoic acid, Phenyl methyl ester, Undecanoic acid, phenyl methyl ester, Octadecanoic acid, phenyl methyl ester

46.95 0.63 24

Difluoro(methylamino)phosphine sulfide, Cinnamylcinnamate 48.11 0.52 25 Difluoro(methylamino)phosphine sulfide, Cinnamyl cinnamate, Naphthalene, 1,2,3,4-tetrahydro-1

48.53 0.26 26

Morpholine, 4-[2-phenyl-1-(phenylethyl)ethenyl]- (N-(Benzyloxycarbonyl)glycyl)-l-rine hydrazide 1,3-Benzoxazin-2-one,

49.68 1.55 2;7

12-Methyl-E,E-2,13-octadecadien-1-Ol , Cyclohexane ethanol, 4-methyl-.beta-methylene-trans-4,9-Decadienoic acid, 2-nitro-ethyl ester

49.81 0.25 28

Dibut-3-enyl phthalate, Benzothiazole, 2-methyl-Phthalic acid, 3,4-dimethylphenyl isobutyl ester

51.22 0.82 29

1-Bromo-11-iodoundecane, C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha-ol, Oxalic acid, pentadecyl propyl ester

52.91 0.23 30

9-(3-Fluorobenzyl)-9-hydroxy-3,6,10,10-tetramethyl-9,10-dihydrophena, Nthrene, 2,3-Dimethoxy-5-methyl-6-dekaisoprenyl-chinon, 7H-Pyrazolo[4,3-E][1,2,4]triazolo[ 1,5-c] pyrimidine.

53.01 1.13 31

Cyclopropane, carboxamide, 2-cyclopropyl-2-methyl-N-(1-cyclopropylethyl)-2,4,4-Trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-cyclohexene beta. Carotene

53.32 0.41 32

Cholest-5-en-3-ol, (3.beta.)-carbonochloridate, Cholest-5-en-3-ol (3.beta.)-, propanoate1H-Isoindole,

53.71 2.24 33

215

Appendix 10 (b). A GC-MS report of n-hexane fraction

Structural analogue Rt(min) Area % Peak Silane, [[(3.beta.)-lanosta-9(11), 24-dien-3-yl]oxy]trimethyl- 3.beta.-Hydroxy-5-cholen-24-oic acid, 9,19-Cyclolanost-23-ene-3,25-diol,

53.90 0.31 34

Cholesta-6,22,24-triene, 4,4-dimethyl, Stigmasteryl tosylate Stigmastan-6,22-dien

54.32 3.42 35

Stigmastan-3,5-diene, Hexacosanoic acid, A-Norcholestan-3-one

54.75 0.64 36

Stigmasteryltosylate Oxazole, 5-(2-furfurylamino)-2-(4- methyl phenyl)-4-phenylsulfonyl- Stigmasta-5,22-dien-3-ol.

55.02 0.39 37

Stigmastan-3,5-diene, .beta.-Sitosterol acetate, Ergosta-4,6,22-trien-3.beta.-ol

55.50 2.97 38

1-Nonadecene , 1,14-Dibromotetradecane, 2-Dodecen-1-yl(-)succinic anhydrid

56.34 0.26 39

Campesterol 58.70 3.46 40 Stigmasterol, Ergost-22-en-3-one, 59.61 4.44 41 Appendix 11 (a). Activity of components identified in the sample [GC MS study] of HEF

S/N

Name of compounds Molecular Formula M.W Nature Activity

1 2,6,11-trimethyl-Tridecane, 1-iodo-1,3-Dimethylcyclopentanol

C16H34

CAS RN 3891-99-4 226 Aliphatic

Hydrocarbon Antibacterial, cardio toning properties and anti-hyperlipidemic

2 Dodecane C12H26 ChemSpider ID: 7890

170 Aliphatic Hydrocarbon

Antibacterial and Antioxidants

3 Heneicosane, C21H44 CH.BK CAS 629-94-7

296 Aliphatic Hydrocarbon

Antibacterial, Vector control

4 Dodecanoic acid C12H24O2 CHEBI:30813

172 Fatty Acid, Ester

Antibacterial, Anti inflammatory, Plant metabolite

5 Pentadecane, C15H32 ChemSpider ID: 11885

212 Aliphatic Hydrocarbon

Antimicrobial, Antitumor, Antioxidant

6 2-Bromododecane, C12H25Br Pub.ChemID 98299

249 Aliphatic Hydrocarbon, Fatty Acid

Antimicrobial

7 Nonane, C9H20 ChemSpider ID: 7849

128 Aliphatic Hydrocarbon

Antimicrobial and Hemolytic activity

8 Tetradecanoic acid (Myristic Acid)

C14H28O2

Pub. Chem ID 11005 228 Fatty Acid Antimicrobial

9 Pentadecanoic acid C15H30O2

Pub. Chem ID 13849 242 saturated fatty

acid, Methyl Ester

Antibacterial, Antifungal

10 Heptacosane , C27H56 Chem Spider ID: 11146

380 Aliphatic Hydrocarbon, Fatty Acid

Antimicrobial, Anticancer

216

Appendix 11(b). Activity of components identified in the sample [GC MS study] of HEF fraction.

S/N

Name of compounds Molecular Formula M.W Nature Activity

11 Hexadecane C16H34 Pub. ChemID 11006

226 Aliphatic Hydrocarbon

Antimicrobial , Antifungal

12 Hexadecanoic acid, , Palmitoleic acid

C16H32O2 ChemSpider ID: 960

256 Fatty Acid, Methyl Ester

Antimicrobial

13 4-Methyl-dodec-3-en-1-ol

C13H26O ChemSpider ID: 4516810

198 Phenol, Methyl Ester

Antimicrobial, Antileishmanial

14 Cyclic octaatomic sulfur7-Amino-7H-S triazolo[5,1-c]-S-tri azole-3-thiol

S-alkylated-3-mercapto-1,2,4-triazole

Alkylated Pyridine Derivative

Antimicrobial and Insecticidal, Anticancer

15 cis-Vaccenic acid, C18H34O2

Pub.Chem ID 5282761 282 Fatty Acid,

Sterol

Antimicrobial

16 9,12-Octadecadienoic acid, methyl ester

C18H32O2 NIST CAS 2462-85-3

280 Esther Antimicrobial

17 Cyclopentadecanone C15H28O ChemSpider ID: 9980

224 Aliphatic Hydrocarbon

Antimicrobial

18 2-methyltetracosane C25H52 ChemSpider ID: 459700

352 Aliphatic Hydrocarbon

Antimicrobial and Antioxidative

19 2-Chloroethyl linoleate C20H35ClO2Pub. Chem ID 6433897

342 Fatty Acid, Ester

Antimicrobial and Anticancer

20 Oleic Acid (cis-9-Octadecenoic acid)

C18H34O2

Pub.Chem ID 445639 282 Fatty Acid Antimicrobial Agents

21 1-Naphthalenol, 1,2,3,4-tetrahydroacetate

C12H14O2 LookChem CAS 21503-12-8

190 Aliphatic Alcohol, ally amines

Antibacterial and Antioxidant

22 Bis(2-ethylhexyl) phthalate (phthalic acid)

C24H38O4ChemSpider ID: 21106505

390 Fatty Acid Antimicrobial and Cytotoxic agent

23 Difluoro(methylamino)phosphine sulfide (Morpholine)

C4H9NOChemSpider ID: 13837537

87 Pyrimidine, Ether

Antiinflammatory, Analgesic, Antipyretic,Antimicrobial

24 Cinnamylcinnamate C18H16O2 CB N0:5174225

264 Diterpenic Acid, Carboxylic Acid Derivative

Antibacterial, Antifungal, Anticancer

25 Naphthalene C10H8 ChemSpider ID: 906

128 Bicyclic Aromatic Hydrocarbon

Antibacterial and Antiplatyhelmintic, Pesticide

26 (N-(Benzyloxycarbonyl)glycyl)-l-rine hydrazide

C12H15NO5 Look Chem CAS 1676-81-9

Peptide Hydrides, Phenyl Esters

Antitumor Activity

27 1,3-Benzoxazin-2-one C8H7NO2 Pub. Chem ID 121020

149 Aliphatic Hydrocarbon, Ester

Antibacterial, Antifungal

28 Benzothiazole C7H5NS Pub.Chem ID 7222

135 Aromatic heterocyclic compound

Antimicrobial, Anticancer

29 C(14a)-Homo-27-nor-14.beta.-gammaceran-3.alpha-ol

C30H52O Pub. Chem ID 550124

428 Fatty Alcohols, Steroid compound

Peptide Receptor Targeting in Cancer

217

Appendix 11(c). Activity of components identified in the sample [GC MS study] of HEF fraction S/N Name of compounds Molecular

Formula M.W

Nature Activity

30 Oxalic acid C2H2O4 Chem Spider ID: 946

90 Carboxylic Acid

Antibacterial Activity

31 7H-Pyrazolo[4,3-E][1,2,4]triazolo[ 1,5-c] pyrimidine.

C18H13N7 ChemSpider ID: 4090727

327 Primidine Derivatives

Antimicrobial and Antifungal Activity

32 Cyclopropane, C3H6 ChemSpider ID: 6111

42 Aliphatic Hydrocarbon, , Fatty Acid, Cyclic Alkane

Anesthetic, Antimicrobial

33 Carboxamide, 1H-indole-3-carboxamide

C9H8N2O ChemSpider ID: 1636821

160 carboxylic acid Fungicide, Antiviral and Antibacterial Activity

34 cyclohexene beta. Carotene

C40H56 ChemSpider ID:4444129

536 Aromatic Hydrocarbon

Antioxidant, Vitamin, Antimicrobial

35 Cholest-5-en-3-ol, (3.beta.)-carbonochloridate,

C28H45ClO2 Pub. Chem

ID 12451

449 Steroid Alcohol Plasticizer, antihelminthic and antimicrobial activities

36 Cholest-5-en-3-ol (3.beta.)-, propanoate

C30H50O2 Chem.BK CB5243420

442 Steroid Alcohol Antiulcer , anti-inflammatory and antimicrobial activity

37 Stigmastan-3,5-diene, Stigmasta-5,22-dien-3-ol

C29H48 Pub. Chem CID 00525918

396 Steroid Compound

anti-inflammatory and antimicrobial activity

38 A-Norcholestan-3-one C26H44O Pub. Chem ID 242347

372 Steroid Compound

antimicrobial, antioxidant, anticancer activities

39 Stigmasteryltosylate Oxazole, beta.-Sitosterol acetate

C29H50O CAS 83-46-5

414 steroid compounds

Benign prostatic hyperplasia, Antimicrobial

40 Ergosta-4,6,22-trien-3.beta.-ol

C28H42O ChemSpider ID: 4526809

394 Aromatic Steroids

Antimicrobial

41 1,14-Dibromotetradecane

C14H28Br2 ChemSpider ID: 148624

356 Alkyl bis Esters Antimicrobial, Antifungal Activity

42 2-Dodecen-1-yl(-)succinic anhydrid

C16H26O3 Pub. Chem

ID 29772

266 Anhydride Bactericidal and Antifungal Activity

43 Campesterol C28H48O 400 Steroidal compound

Antimicrobial, Antileishmanial

44 Stigmasterol C29H48O Chem Spider ID: 504066

412 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

45 Ergost-22-en-3-one C28H46O ChemSpider ID: 4516831

398 Fatty Alcohols, Steroid compound

Antimicrobial Antioxidant Anti-inflammatory, Antiarthritic Antiasthma Diuretic

218

Appendix 12. GC-MS report of methanol fraction

Structural analogue Rt (min) Area % Peak Bicyclo[2.2.1]heptan-2-one, 1,3,3- trimethyl-Bicyclo[2.2.1]heptan-2-one, 1,3,3 trimethyl- L-Fenchone

13.06 1.53 1

3,7-dimethyl-1,6-Octadien-3-ol, 13.58 37.09 2 Terpinen-4-ol 16.11 7.46 3 Silane, trimethylpropoxy- Silane, 18.32 2.50 4 Geraniol 18.57 1.50 5 Eugenol 21.57 29.12 6 Bicyclo[3.1.1]hept-2-ene, 23.70 7.21 7 (+)-epi-Bicyclosesquiphellandrene , Naphthalene 28.96 5.20 8 Benzenemethanamine 31.31 1.74 9 6-heptynyl- Benzene, (6-bromohexyl)- 2-Butanol. 41.72 3.49 10 isopropyl 2-benzyl-2-propenylBenzene, 43.42 1.57 11 Pyridine-3-carboxamide, oxime, N-(2-trifluoromethylphenyl)- 3-Octadecene.

55.43 1.65 12

Appendix 13 (a). Activity of components identified in the sample [GC MS study] of methanol fraction

S/n Name of compounds Molecular Formula

M.W Nature Activity

1

1,3,3-trimethyl-Bicyclo[2.2.1]heptan-2-one, 1,3,3 trimethyl- L-Fenchone

C10H16O ChemSpider ID: 13869

152 monoterpene

Antimicrobial, Antifungal, Antimalaria

2

3,7-dimethyl-1,6-Octadien-3-ol C10H18O NIST CAS 106-24-1

154 Terpene phenol

Antioxidant and Antimicrobial

3

Terpinen-4-ol C10H18O ChemSpider ID: 10756

154 monoterpine

Antimicrobial

4

trimethylpropoxy- Silane C6H16OSi Pub. Chem CID 74577

132 Saturated Hydrosilicons

An intermediary in Antimicrobial drugs

5

Geraniol C10H18O ChemSpider ID: 13849989

154 monoterpenoid

Antibacterial, Antifungal

6

Eugenol C10H12O2 ChemSpider ID: 13876103

164 Terpene phenol

Antimicrobial

7

Bicyclo[3.1.1]hept-2-ene, C10H14O NIST CAS 564-94-3

150 Phenol Antioxidant, Antibacterial

8

(+)-epi-Bicyclosesquiphellandrene C15H24 Pub. Chem 521496

204 Terpene Antimicrobial, Antifungal, Spasmolytic

9

Naphthalene C10H8 ChemSpider ID: 906

128 Bicyclic Aromatic Hydrocarbon

Antibacterial and Antiplatyhelmintic, Pesticide

219

Appendix 13 (b). Activity of components identified in the sample [GC MS study] of methanol fraction S/n Name of compounds Molecular

Formula M.W Nature Activity

10 Benzenemethanamine C9H13N NIST CAS

103-83-3 135 Antipyretic,

Antimicrobial

11

Isopropyl2-benzyl-2-propenylBenzene

C13H18O ChemSpider ID: 506324

190 Ether, Aromatic Alcohol

Antioxidant, Antibacterial

12

Pyridine-3-carboxamide C6H6N218O ChemSpider ID: 9334423

124 Nicotinamide, Nicotine Amines.

Antiviral, Anticancer , antiprotozoa

13

Oxime C3H7NO ChemSpider ID: 60524

73 Natural Phenolic flavonoid

Antioxidant, Antibacteria and anticancer agent

14 N-( 2-trifluoromethylphenyl)- 3-Octadecene

C25H40O3 CAS 114275-39-7

388 Ester Antioxidant, Antibacterial

220

Appendix 14: Statistical software used is spss version 17 in all the analyses results for

comparison of the percentage yield of the various fractions

ONEWAY Fractions BY VAR00001/STATISTICS DESCRIPTIVES /MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA (0.05).

Oneway

Descriptives

Fractions

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

1.00 3 2.2900 .01000 .00577 2.2652 2.3148 2.28 2.30

2.00 3 5.9800 .02000 .01155 5.9303 6.0297 5.96 6.00

3.00 3 17.7067 .00577 .00333 17.6923 17.7210 17.70 17.71

4.00 3 31.2100 .01000 .00577 31.1852 31.2348 31.20 31.22

Total 12 14.2967 11.80543 3.40793 6.7959 21.7975 2.28 31.22

ANOVA

Fractions

Sum of Squares df Mean Square F Sig.

Between Groups 1533.048 3 511.016 3227469.053 .000

Within Groups .001 8 .000

Total 1533.049 11

221

Post Hoc Tests

Homogeneous Subsets

Fractions

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

N-Hexane 3 2.2900

Ethylacetate 3 5.9800

DCM 3 17.7067

Methanol 3 31.2100

Sig. 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

Interpretation

ANOVA was performed followed by Duncan post Hoc Test with the use of SPSS v 17

software.

There was significant (P< 0.05) difference in the percentage yield of the various

fractions, methanol fraction having the highest (31.21 ± 0.01) and N –hexane, the lowest

(2.29 ± 0.01)

ONEWAY Susceptibility Resistance BY VAR00001 /STATISTICS DESCRIPTIVES /MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA(0.05).

222

Oneway

Descriptives

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum

Maximum

Lower Bound

Upper Bound

Susceptibility

1.00 3 22.3333 .57735 .33333 20.8991 23.7676 22.00 23.00

2.00 3 23.0000 1.00000 .57735 20.5159 25.4841 22.00 24.00

3.00 3 26.0000 1.00000 .57735 23.5159 28.4841 25.00 27.00

4.00 3 25.3333 .57735 .33333 23.8991 26.7676 25.00 26.00

5.00 3 21.0000 1.00000 .57735 18.5159 23.4841 20.00 22.00

6.00 3 18.3333 .57735 .33333 16.8991 19.7676 18.00 19.00

7.00 3 20.0000 1.00000 .57735 17.5159 22.4841 19.00 21.00

8.00 3 21.6667 .57735 .33333 20.2324 23.1009 21.00 22.00

9.00 3 21.3333 .57735 .33333 19.8991 22.7676 21.00 22.00

10.00 3 21.3333 .57735 .33333 19.8991 22.7676 21.00 22.00

Total 30 22.0333 2.31164 .42205 21.1702 22.8965 18.00 27.00

Resistance

1.00 3 36.0000 .00000 .00000 36.0000 36.0000 36.00 36.00

2.00 3 35.6667 1.15470 .66667 32.7982 38.5351 35.00 37.00

3.00 3 32.3333 .57735 .33333 30.8991 33.7676 32.00 33.00

4.00 3 32.6667 .57735 .33333 31.2324 34.1009 32.00 33.00

5.00 3 37.6667 .57735 .33333 36.2324 39.1009 37.00 38.00

6.00 3 39.6667 .57735 .33333 38.2324 41.1009 39.00 40.00

223

7.00 3 39.3333 .57735 .33333 37.8991 40.7676 39.00 40.00

8.00 3 36.3333 .57735 .33333 34.8991 37.7676 36.00 37.00

9.00 3 37.3333 .57735 .33333 35.8991 38.7676 37.00 38.00

10.00 3 36.3333 .57735 .33333 34.8991 37.7676 36.00 37.00

Total 30 36.3333 2.39732 .43769 35.4382 37.2285 32.00 40.00

ANOVA

Sum of Squares df Mean Square F Sig.

Susceptibility Between Groups 142.967 9 15.885 26.475 .000

Within Groups 12.000 20 .600

Total 154.967 29

Resistance Between Groups 158.667 9 17.630 44.074 .000

Within Groups 8.000 20 .400

Total 166.667 29

Post Hoc Tests

Homogeneous Subsets

224

Susceptibility

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

6.00 3 18.3333

7.00 3 20.0000

5.00 3 21.0000 21.0000

9.00 3 21.3333 21.3333

10.00 3 21.3333 21.3333

8.00 3 21.6667 21.6667

1.00 3 22.3333 22.3333

2.00 3 23.0000

4.00 3 25.3333

3.00 3 26.0000

Sig. 1.000 .066 .071 .058 .304

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

225

Resistance

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

3.00 3 32.3333

4.00 3 32.6667

2.00 3 35.6667

1.00 3 36.0000

8.00 3 36.3333 36.3333

10.00 3 36.3333 36.3333

9.00 3 37.3333 37.3333

5.00 3 37.6667

7.00 3 39.3333

6.00 3 39.6667

Sig. .526 .250 .080 .526 .526

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

Key s; 1= Oxacilin, 2= Vancomycin, 3= Cephalexin, 4= Levofloxacin, 5= Ciprofloxacin, 6= Tetracycline, 7 = Cotrimoxazole, 8= Gentamycin,

9 = Clindamycin, 10 = Rifampicin

226

Interpretation

Susceptibility and Resistance

ANOVA was performed followed by Duncan post Hoc Test with the use of SPSS v 17

software.

There was significant (P< 0.05) difference in the susceptibility of the various antimicrobials,

Cephalexin having the highest (26.00 ± 1.00) and Tetracycline, the lowest (18.33 ± 0.58).

However, there was no significant (P>0.05) difference in susceptibility between Cephalexin

and Levofloxacin.

On the other hand, the reverse is the case in the resistance test. There was also significant (P<

0.05) difference among the various antimicrobials, Tetracycline having the highest (39.67 ±

0.58) and Cephalexin, the least (32.33 ± 0.58). Cotrimoxazole had similar resistance to

tetracycline (P>0.05)

227

HEMATOLOGICAL PARAMETERS

ONEWAY PCV RBC WBC MCV PLT BY VAR/STATISTICS DESCRIPTIVES

/MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA (0.05).

Oneway

Descriptives

N Mean Std. Deviation

Std. Error

95% Confidence Interval for Mean Mini

mum Maximum Lower Bound Upper Bound

PCV 1.00 3 44.2633 .61776 .35667 42.7287 45.7979 43.55 44.62

2.00 3 42.7433 .90644 .52333 40.4916 44.9951 42.22 43.79

3.00 3 45.1100 .34641 .20000 44.2495 45.9705 44.71 45.31

4.00 3 45.2400 .22517 .13000 44.6807 45.7993 45.11 45.50

5.00 3 46.4333 .40415 .23333 45.4294 47.4373 46.20 46.90

Total 15 44.7580 1.34892 .34829 44.0110 45.5050 42.22 46.90

RBC 1.00 3 7.1633 .09238 .05333 6.9339 7.3928 7.11 7.27

2.00 3 7.3233 .02309 .01333 7.2660 7.3807 7.31 7.35

3.00 3 7.4333 .05774 .03333 7.2899 7.5768 7.40 7.50

4.00 3 7.4733 .05774 .03333 7.3299 7.6168 7.44 7.54

5.00 3 7.5400 .05196 .03000 7.4109 7.6691 7.51 7.60

Total 15 7.3867 .14593 .03768 7.3059 7.4675 7.11 7.60

WBC 1.00 3 13.3000 .17321 .10000 12.8697 13.7303 13.20 13.50

2.00 3 13.9167 .01155 .00667 13.8880 13.9454 13.91 13.93

3.00 3 13.7333 .02887 .01667 13.6616 13.8050 13.70 13.75

4.00 3 13.8733 .01155 .00667 13.8446 13.9020 13.86 13.88

5.00 3 14.2233 .02309 .01333 14.1660 14.2807 14.21 14.25

Total 15 13.8093 .31847 .08223 13.6330 13.9857 13.20 14.25

MCV 1.00 3 51.2400 .05292 .03055 51.1086 51.3714 51.20 51.30

2.00 3 51.1467 .06429 .03712 50.9870 51.3064 51.10 51.22

3.00 3 49.1600 .06557 .03786 48.9971 49.3229 49.10 49.23

4.00 3 48.7433 .11547 .06667 48.4565 49.0302 48.61 48.81

5.00 3 50.1767 .05774 .03333 50.0332 50.3201 50.11 50.21

Total 15 50.0933 1.04985 .27107 49.5119 50.6747 48.61 51.30

PLT 1.00 3 409.1000

.00000 .00000 409.1000 409.1000 409.10 409.10

228

2.00 3 409.0000

.00000 .00000 409.0000 409.0000 409.00 409.00

3.00 3 406.2200

.00000 .00000 406.2200 406.2200 406.22 406.22

4.00 3 399.1000

.00000 .00000 399.1000 399.1000 399.10 399.10

5.00 3 388.4000

.00000 .00000 388.4000 388.4000 388.40 388.40

Total 15 402.3640

8.14940 2.10417

397.8510 406.8770 388.40 409.10

ANOVA

Sum of Squares df Mean Square F Sig.

PCV Between Groups 22.400 4 5.600 18.213 .000

Within Groups 3.075 10 .307

Total 25.474 14

RBC Between Groups .261 4 .065 17.717 .000

Within Groups .037 10 .004

Total .298 14

WBC Between Groups 1.357 4 .339 53.607 .000

Within Groups .063 10 .006

Total 1.420 14

MCV Between Groups 15.375 4 3.844 688.832 .000

Within Groups .056 10 .006

Total 15.431 14

PLT Between Groups 929.778 4 232.444 . .

Within Groups .000 10 .000

Total 929.778 14

229

Post Hoc Tests

Homogeneous Subsets

PCV

Duncana

VAR N

Subset for alpha = 0.05

1 2 3

2.00 3 42.7433

1.00 3 44.2633

3.00 3 45.1100

4.00 3 45.2400

5.00 3 46.4333

Sig. 1.000 .066 1.000

Means for groups in homogeneous subsets are

displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

RBC

Duncana

VAR N

Subset for alpha = 0.05

1 2 3

1.00 3 7.1633 2.00 3 7.3233

3.00 3 7.4333 7.4333 4.00 3 7.4733

5.00 3 7.5400

Sig. 1.000 .051 .067

Means for groups in homogeneous subsets are

displayed.

230

RBC

Duncana

VAR N

Subset for alpha = 0.05

1 2 3

1.00 3 7.1633

2.00 3 7.3233 3.00 3 7.4333 7.4333

4.00 3 7.4733 5.00 3 7.5400

Sig. 1.000 .051 .067

Means for groups in homogeneous subsets are

displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

WBC

Duncana

VAR N

Subset for alpha = 0.05

1 2 3 4

1.00 3 13.3000

3.00 3 13.7333

4.00 3 13.8733 13.8733

2.00 3 13.9167

5.00 3 14.2233

Sig. 1.000 .057 .520 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

MCV

Duncana

VAR N Subset for alpha = 0.05

231

1 2 3 4

4.00 3 48.7433

3.00 3 49.1600

5.00 3 50.1767

2.00 3 51.1467

1.00 3 51.2400

Sig. 1.000 1.000 1.000 .157

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

BIOCHEMICAL PARAMETERS

ONEWAY BILIRUBIN CREATININE ALT ALP AST BY VAR /STATISTICS

DESCRIPTIVES /MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA(0.05).

Oneway

Descriptives

N Mean Std. Deviation

Std. Error

95% Confidence Interval for Mean

Minimum Maximum

Lower Bound

Upper Bound

BILIRUBIN 1.00 3 1.6000 .02000 .01155 1.5503 1.6497 1.58 1.62

2.00 3 1.6167 .01528 .00882 1.5787 1.6546 1.60 1.63

3.00 3 1.6000 .01000 .00577 1.5752 1.6248 1.59 1.61

4.00 3 1.5900 .01000 .00577 1.5652 1.6148 1.58 1.60

5.00 3 1.5900 .01000 .00577 1.5652 1.6148 1.58 1.60

Total 15 1.5993 .01534 .00396 1.5908 1.6078 1.58 1.63

CREATININE 1.00 3 .7300 .01000 .00577 .7052 .7548 .72 .74

2.00 3 .7000 .01000 .00577 .6752 .7248 .69 .71

3.00 3 .7200 .01000 .00577 .6952 .7448 .71 .73

232

4.00 3 .6900 .01000 .00577 .6652 .7148 .68 .70

5.00 3 .6900 .01000 .00577 .6652 .7148 .68 .70

Total 15 .7060 .01882 .00486 .6956 .7164 .68 .74

ALT 1.00 3 34.1100 .00000 .00000 34.1100 34.1100 34.11 34.11

2.00 3 33.1700 .00000 .00000 33.1700 33.1700 33.17 33.17

3.00 3 33.0200 .00000 .00000 33.0200 33.0200 33.02 33.02

4.00 3 31.4000 .00000 .00000 31.4000 31.4000 31.40 31.40

5.00 3 32.6000 .00000 .00000 32.6000 32.6000 32.60 32.60

Total 15 32.8600 .91223 .23554 32.3548 33.3652 31.40 34.11

ALP 1.00 3 129.4100

.00000 .00000 129.4100 129.4100

129.41 129.41

2.00 3 129.8800

.00000 .00000 129.8800 129.8800

129.88 129.88

3.00 3 129.3100

.00000 .00000 129.3100 129.3100

129.31 129.31

4.00 3 130.1100

.00000 .00000 130.1100 130.1100

130.11 130.11

5.00 3 132.1100

.00000 .00000 132.1100 132.1100

132.11 132.11

Total 15 130.1640

1.05238 .27172 129.5812 130.7468

129.31 132.11

AST 1.00 3 63.1100 .00000 .00000 63.1100 63.1100 63.11 63.11

2.00 3 66.1200 .00000 .00000 66.1200 66.1200 66.12 66.12

3.00 3 64.4100 .00000 .00000 64.4100 64.4100 64.41 64.41

4.00 3 63.2200 .00000 .00000 63.2200 63.2200 63.22 63.22

5.00 3 66.5300 .00000 .00000 66.5300 66.5300 66.53 66.53

Total 15 64.6780 1.47591 .38108 63.8607 65.4953 63.11 66.53

ANOVA

Sum of Squares df Mean Square F Sig.

BILIRUBIN Between Groups .001 4 .000 1.911 .185

Within Groups .002 10 .000

Total .003 14

233

CREATININE

Between Groups .004 4 .001 9.900 .002

Within Groups .001 10 .000

Total .005 14

ALT Between Groups 11.650 4 2.913 . .

Within Groups .000 10 .000

Total 11.650 14

ALP Between Groups 15.505 4 3.876 . .

Within Groups .000 10 .000

Total 15.505 14

AST Between Groups 30.496 4 7.624 . .

Within Groups .000 10 .000

Total 30.496 14

234

Post Hoc Tests Homogeneous Subsets

BILIRUBIN

Duncana

VAR N

Subset for alpha = 0.05

1

4.00 3 1.5900

5.00 3 1.5900

1.00 3 1.6000

3.00 3 1.6000

2.00 3 1.6167

Sig. .053

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

CREATININE

Duncana

VAR N

Subset for alpha = 0.05

1 2

4.00 3 .6900

5.00 3 .6900

2.00 3 .7000

3.00 3 .7200

1.00 3 .7300

Sig. .269 .249

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

235

Descriptives

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum

Lower Bound

Upper Bound

S4 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.7333 2.78944 .72023 5.1886 8.2781 .00 10.00

S8 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.3333 5.50757 3.17980 -7.3482 20.0149 .00 10.00

Total 15 6.9333 2.60403 .67236 5.4913 8.3754 .00 10.00

S20 1.00 3 4.0000 .00000 .00000 4.0000 4.0000 4.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.6667 2.89499 .74748 5.0635 8.2699 .00 10.00

S22 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 6.4000 2.13140 .55032 5.2197 7.5803 3.00 10.00

S30 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 6.0000 1.00000 .57735 3.5159 8.4841 5.00 7.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 7.6667 1.15470 .66667 4.7982 10.5351 7.00 9.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 6.6667 2.25726 .58282 5.4166 7.9167 3.00 10.00

S33 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

236

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.6000 2.58567 .66762 5.1681 8.0319 .00 10.00

S35 1.00 3 4.0000 .00000 .00000 4.0000 4.0000 4.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

5.00 3 6.3333 5.50757 3.17980 -7.3482 20.0149 .00 10.00

Total 15 6.2667 2.54858 .65804 4.8553 7.6780 .00 10.00

S36 1.00 3 3.0000 .00000 .00000 3.0000 3.0000 3.00 3.00

2.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

3.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

4.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 6.3333 2.38048 .61464 5.0151 7.6516 3.00 10.00

S38 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 6.5333 2.19957 .56793 5.3153 7.7514 3.00 10.00

S42 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 6.6667 1.15470 .66667 3.7982 9.5351 6.00 8.00

4.00 3 7.6667 1.15470 .66667 4.7982 10.5351 7.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.0000 2.75162 .71047 4.4762 7.5238 .00 10.00

S53 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.1333 2.82506 .72943 4.5689 7.6978 .00 10.00

S57 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

237

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.7333 2.63131 .67940 5.2762 8.1905 .00 10.00

S61 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.2000 2.80815 .72506 4.6449 7.7551 .00 10.00

S62 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 9.0000 1.00000 .57735 6.5159 11.4841 8.00 10.00

5.00 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00

Total 15 7.0667 2.46306 .63596 5.7027 8.4307 3.00 10.00

S123 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.5333 2.87518 .74237 4.9411 8.1256 .00 10.00

S127 1.00 3 4.0000 .00000 .00000 4.0000 4.0000 4.00 4.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.0000 1.00000 .57735 4.5159 9.4841 6.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.5333 2.72204 .70283 5.0259 8.0408 .00 10.00

S154 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.3333 1.15470 .66667 4.4649 10.2018 6.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 3.3333 5.77350 3.33333 -11.0088 17.6755 .00 10.00

238

Total 15 5.8000 2.95683 .76345 4.1626 7.4374 .00 10.00

S187 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 6.0000 1.00000 .57735 3.5159 8.4841 5.00 7.00

3.00 3 7.0000 1.00000 .57735 4.5159 9.4841 6.00 8.00

4.00 3 8.3333 1.15470 .66667 5.4649 11.2018 7.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.1333 3.06749 .79202 3.4346 6.8321 .00 9.00

S208 1.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 9.0000 1.00000 .57735 6.5159 11.4841 8.00 10.00

5.00 3 3.3333 5.77350 3.33333 -11.0088 17.6755 .00 10.00

Total 15 6.2667 3.03472 .78356 4.5861 7.9472 .00 10.00

S235 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.8000 2.78260 .71846 5.2590 8.3410 .00 10.00

S310 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 3.3333 5.77350 3.33333 -11.0088 17.6755 .00 10.00

Total 15 5.8000 2.93258 .75719 4.1760 7.4240 .00 10.00

S390 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 6.7333 2.31352 .59735 5.4521 8.0145 3.00 10.00

S417 1.00 3 3.0000 .00000 .00000 3.0000 3.0000 3.00 3.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

239

4.00 3 7.6667 1.15470 .66667 4.7982 10.5351 7.00 9.00

5.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

Total 15 6.2667 2.12020 .54743 5.0925 7.4408 3.00 9.00

S570 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

Total 15 6.3333 2.05866 .53154 5.1933 7.4734 3.00 9.00

S578 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.0000 .00000 .00000 7.0000 7.0000 7.00 7.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

Total 15 6.6000 2.16465 .55891 5.4013 7.7987 3.00 9.00

S600 1.00 3 3.0000 .00000 .00000 3.0000 3.0000 3.00 3.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

Total 15 6.5333 2.23180 .57625 5.2974 7.7693 3.00 9.00

S620 1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

Total 15 6.6000 1.95667 .50521 5.5164 7.6836 3.00 9.00

S651 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.3333 5.50757 3.17980 -7.3482 20.0149 .00 10.00

Total 15 6.0667 2.78944 .72023 4.5219 7.6114 .00 10.00

S819 1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

240

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.3333 2.76887 .71492 4.8000 7.8667 .00 10.00

S831 1.00 3 3.3333 .57735 .33333 1.8991 4.7676 3.00 4.00

2.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.2000 2.93258 .75719 4.5760 7.8240 .00 10.00

S841 1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.0000 3.09377 .79881 3.2867 6.7133 .00 9.00

S940 1.00 3 4.0000 1.00000 .57735 1.5159 6.4841 3.00 5.00

2.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.5333 3.35659 .86667 3.6745 7.3921 .00 9.00

S947 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.0000 1.00000 .57735 4.5159 9.4841 6.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.4667 2.69568 .69602 4.9739 7.9595 .00 10.00

S1009

1.00 3 3.6667 1.15470 .66667 .7982 6.5351 3.00 5.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 9.0000 1.00000 .57735 6.5159 11.4841 8.00 10.00

5.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

241

1 TO 5 REP THE GP A TO E

Total 15 6.8000 2.21037 .57071 5.5759 8.0241 3.00 10.00

S1104

1.00 3 4.0000 1.00000 .57735 1.5159 6.4841 3.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.0000 1.00000 .57735 4.5159 9.4841 6.00 8.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.5333 2.89992 .74876 4.9274 8.1393 .00 10.00

S1172

1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

5.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

Total 15 6.3333 2.09307 .54043 5.1742 7.4924 3.00 9.00

S1420

1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

2.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.1333 2.85023 .73593 4.5549 7.7117 .00 10.00

S1827

1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.0000 1.00000 .57735 4.5159 9.4841 6.00 8.00

4.00 3 8.3333 1.15470 .66667 5.4649 11.2018 7.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 4.9333 3.08143 .79562 3.2269 6.6398 .00 9.00

S1956

1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 7.2667 1.70992 .44150 6.3197 8.2136 4.00 10.00

242

ANALYSIS FOR MIC ONEWAY S4 S8 S20 S22 S30 S33 S35 S36 S38 S42 S53 S57 S61 S62 S123 S127 S154 S187 S208 S235 S310 S390 S417 S570 S578 S600 S620 S651 S 819 S831 S841 S940 S947 S1009 S1104 S1172 S1420 S1827 S1956 BY VAR00001 /STATISTICS DESCRIPTIVES /MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA(0.05).

Oneway

ANOVA

Sum of Squares df Mean Square F Sig.

S4 Between Groups 39.600 4 9.900 1.428 .294

Within Groups 69.333 10 6.933

Total 108.933 14

S8 Between Groups 31.600 4 7.900 1.247 .352

Within Groups 63.333 10 6.333

Total 94.933 14

S20 Between Groups 48.667 4 12.167 1.772 .211

Within Groups 68.667 10 6.867

Total 117.333 14

S22 Between Groups 60.267 4 15.067 45.200 .000

Within Groups 3.333 10 .333

Total 63.600 14

S30 Between Groups 64.667 4 16.167 24.250 .000

Within Groups 6.667 10 .667

Total 71.333 14

243

S33 Between Groups 22.933 4 5.733 .811 .546

Within Groups 70.667 10 7.067

Total 93.600 14

S35 Between Groups 28.267 4 7.067 1.128 .397

Within Groups 62.667 10 6.267

Total 90.933 14

S36 Between Groups 77.333 4 19.333 96.667 .000

Within Groups 2.000 10 .200

Total 79.333 14

S38 Between Groups 64.400 4 16.100 48.300 .000

Within Groups 3.333 10 .333

Total 67.733 14

S42 Between Groups 32.667 4 8.167 1.114 .403

Within Groups 73.333 10 7.333

Total 106.000 14

S53 Between Groups 41.067 4 10.267 1.453 .287

Within Groups 70.667 10 7.067

Total 111.733 14

S57 Between Groups 27.600 4 6.900 .995 .454

Within Groups 69.333 10 6.933

Total 96.933 14

S61 Between Groups 39.733 4 9.933 1.406 .301

Within Groups 70.667 10 7.067

244

Total 110.400 14

S62 Between Groups 80.933 4 20.233 50.583 .000

Within Groups 4.000 10 .400

Total 84.933 14

S123 Between Groups 46.400 4 11.600 1.673 .232

Within Groups 69.333 10 6.933

Total 115.733 14

S127 Between Groups 33.733 4 8.433 1.205 .367

Within Groups 70.000 10 7.000

Total 103.733 14

S154 Between Groups 51.067 4 12.767 1.790 .207

Within Groups 71.333 10 7.133

Total 122.400 14

S187 Between Groups 124.400 4 31.100 42.409 .000

Within Groups 7.333 10 .733

Total 131.733 14

S208 Between Groups 58.933 4 14.733 2.105 .155

Within Groups 70.000 10 7.000

Total 128.933 14

S235 Between Groups 39.067 4 9.767 1.409 .300

Within Groups 69.333 10 6.933

Total 108.400 14

S310 Between Groups 51.067 4 12.767 1.841 .198

245

Within Groups 69.333 10 6.933

Total 120.400 14

S390 Between Groups 71.600 4 17.900 53.700 .000

Within Groups 3.333 10 .333

Total 74.933 14

S417 Between Groups 58.267 4 14.567 31.214 .000

Within Groups 4.667 10 .467

Total 62.933 14

S570 Between Groups 56.667 4 14.167 53.125 .000

Within Groups 2.667 10 .267

Total 59.333 14

S578 Between Groups 62.933 4 15.733 59.000 .000

Within Groups 2.667 10 .267

Total 65.600 14

S600 Between Groups 67.067 4 16.767 62.875 .000

Within Groups 2.667 10 .267

Total 69.733 14

S620 Between Groups 50.267 4 12.567 37.700 .000

Within Groups 3.333 10 .333

Total 53.600 14

S651 Between Groups 45.600 4 11.400 1.800 .205

Within Groups 63.333 10 6.333

Total 108.933 14

246

S819 Between Groups 38.000 4 9.500 1.370 .312

Within Groups 69.333 10 6.933

Total 107.333 14

S831 Between Groups 51.733 4 12.933 1.883 .190

Within Groups 68.667 10 6.867

Total 120.400 14

S841 Between Groups 130.000 4 32.500 81.250 .000

Within Groups 4.000 10 .400

Total 134.000 14

S940 Between Groups 154.400 4 38.600 115.800 .000

Within Groups 3.333 10 .333

Total 157.733 14

S947 Between Groups 31.067 4 7.767 1.099 .409

Within Groups 70.667 10 7.067

Total 101.733 14

S1009 Between Groups 61.733 4 15.433 23.150 .000

Within Groups 6.667 10 .667

Total 68.400 14

S1104 Between Groups 45.733 4 11.433 1.588 .252

Within Groups 72.000 10 7.200

Total 117.733 14

S1172 Between Groups 58.000 4 14.500 43.500 .000

Within Groups 3.333 10 .333

247

Total 61.333 14

S1420 Between Groups 44.400 4 11.100 1.601 .249

Within Groups 69.333 10 6.933

Total 113.733 14

S1827 Between Groups 126.933 4 31.733 52.889 .000

Within Groups 6.000 10 .600

Total 132.933 14

S1956 Between Groups 37.600 4 9.400 28.200 .000

Within Groups 3.333 10 .333

Total 40.933 14

Post Hoc Tests

Homogeneous Subsets

S4

Duncana

VAR00001 N Subset for alpha = 0.05

1

1.00 3 4.3333

2.00 3 5.6667

5.00 3 6.6667

3.00 3 8.3333

4.00 3 8.6667

Sig. .094

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

248

S8

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667

5.00 3 6.3333

2.00 3 6.6667

3.00 3 8.3333

4.00 3 8.6667

Sig. .104

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S20

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 4.0000

2.00 3 5.6667 5.6667

5.00 3 6.6667 6.6667

3.00 3 7.6667 7.6667

4.00 3 9.3333

Sig. .141 .141

Means for groups in homogeneous subsets are displayed.

249

S20

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 4.0000

2.00 3 5.6667 5.6667

5.00 3 6.6667 6.6667

3.00 3 7.6667 7.6667

4.00 3 9.3333

Sig. .141 .141

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S22

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 5.3333

3.00 3 6.6667

4.00 3 7.3333

5.00 3 9.3333

Sig. 1.000 1.000 .188 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

250

S30

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 6.0000

3.00 3 6.6667 6.6667

4.00 3 7.6667

5.00 3 9.6667

Sig. 1.000 .341 .165 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S33

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333

2.00 3 6.6667

5.00 3 6.6667

3.00 3 7.3333

4.00 3 8.0000

Sig. .151

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

251

S35

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.0000

2.00 3 5.6667

5.00 3 6.3333

3.00 3 7.6667

4.00 3 7.6667

Sig. .130

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S36

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

1.00 3 3.0000

2.00 3 5.0000

3.00 3 6.3333

4.00 3 7.6667

5.00 3 9.6667

252

Sig. 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S38

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 5.6667

3.00 3 6.6667 6.6667

4.00 3 7.3333

5.00 3 9.6667

Sig. 1.000 .060 .188 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S42

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 3.3333

253

2.00 3 5.6667

3.00 3 6.6667

5.00 3 6.6667

4.00 3 7.6667

Sig. .102

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S53

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 3.3333

2.00 3 5.3333

5.00 3 6.6667

3.00 3 7.3333

4.00 3 8.0000

Sig. .076

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

254

S57

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333

2.00 3 6.6667

5.00 3 6.6667

3.00 3 7.6667

4.00 3 8.3333

Sig. .118

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S61

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 3.3333

2.00 3 5.6667

5.00 3 6.6667

255

3.00 3 7.3333

4.00 3 8.0000

Sig. .076

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S62

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 3.3333

2.00 3 6.3333

3.00 3 6.6667

4.00 3 9.0000

5.00 3 10.0000

Sig. 1.000 .533 .082

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

256

S123

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 3.3333

2.00 3 6.6667 6.6667

5.00 3 6.6667 6.6667

3.00 3 7.3333 7.3333

4.00 3 8.6667

Sig. .113 .405

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S127

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.0000

2.00 3 6.3333

5.00 3 6.6667

3.00 3 7.0000

4.00 3 8.6667

257

Sig. .075

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S154

Duncana

VAR00001 N

Subset for alpha = 0.05

1

5.00 3 3.3333

1.00 3 4.3333

2.00 3 5.6667

3.00 3 7.3333

4.00 3 8.3333

Sig. .061

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

258

S187

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 4.3333

2.00 3 6.0000

3.00 3 7.0000 7.0000

4.00 3 8.3333

Sig. 1.000 1.000 .183 .086

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S208

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

5.00 3 3.3333

1.00 3 5.0000 5.0000

2.00 3 6.3333 6.3333

3.00 3 7.6667 7.6667

4.00 3 9.0000

259

Sig. .091 .115

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S235

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667

2.00 3 5.6667

5.00 3 6.6667

3.00 3 7.6667

4.00 3 9.3333

Sig. .074

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S310

Duncana

VAR00001 N

Subset for alpha = 0.05

1

5.00 3 3.3333

1.00 3 4.3333

2.00 3 5.6667

260

3.00 3 7.3333

4.00 3 8.3333

Sig. .058

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S390

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 5.3333

3.00 3 7.6667

4.00 3 7.6667

5.00 3 9.6667

Sig. 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S417

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.0000

2.00 3 5.3333

261

3.00 3 6.6667

4.00 3 7.6667 7.6667

5.00 3 8.6667

Sig. 1.000 1.000 .103 .103

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S570

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 5.0000

3.00 3 6.6667

4.00 3 8.3333

5.00 3 8.3333

Sig. 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S578

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.3333

2.00 3 5.3333

3.00 3 7.0000

4.00 3 8.6667

5.00 3 8.6667

Sig. 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

262

S600

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.0000

2.00 3 5.3333

3.00 3 7.3333

4.00 3 8.3333

5.00 3 8.6667

Sig. 1.000 1.000 1.000 .448

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S620

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 3.6667

2.00 3 5.6667

3.00 3 6.6667

5.00 3 8.3333

4.00 3 8.6667

Sig. 1.000 .060 .496

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

263

S651

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 3.3333

2.00 3 5.3333 5.3333

5.00 3 6.3333 6.3333

3.00 3 6.6667 6.6667

4.00 3 8.6667

Sig. .161 .161

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S819

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 3.6667

2.00 3 5.6667

5.00 3 6.6667

3.00 3 7.3333

4.00 3 8.3333

Sig. .074

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

264

S831

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 3.3333

2.00 3 5.0000 5.0000

5.00 3 6.6667 6.6667

3.00 3 7.3333 7.3333

4.00 3 8.6667

Sig. .112 .141

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S841

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 3.6667

2.00 3 5.6667

3.00 3 7.6667

4.00 3 8.0000

Sig. 1.000 1.000 1.000 .533

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

265

S940

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 4.0000

2.00 3 7.3333

3.00 3 7.3333

4.00 3 9.0000

Sig. 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S947

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333

2.00 3 5.6667

5.00 3 6.6667

3.00 3 7.0000

4.00 3 8.6667

Sig. .096

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

266

S1009

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.6667

2.00 3 5.3333

3.00 3 7.3333

5.00 3 8.6667 8.6667

4.00 3 9.0000

Sig. 1.000 1.000 .073 .628

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S1104

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 4.0000

2.00 3 5.6667 5.6667

5.00 3 6.6667 6.6667

3.00 3 7.0000 7.0000

4.00 3 9.3333

Sig. .230 .150

Means for groups in homogeneous subsets are displayed.

267

S1104

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 4.0000

2.00 3 5.6667 5.6667

5.00 3 6.6667 6.6667

3.00 3 7.0000 7.0000

4.00 3 9.3333

Sig. .230 .150

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

172

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

2.00 3 3.6667

1.00 3 4.3333

3.00 3 7.3333

4.00 3 7.6667 7.6667

5.00 3 8.6667

Sig. .188 .496 .060

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

268

S1420

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 3.6667

2.00 3 4.6667

5.00 3 6.6667

3.00 3 7.3333

4.00 3 8.3333

Sig. .074

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S1827

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 3.6667

2.00 3 5.6667

3.00 3 7.0000 7.0000

4.00 3 8.3333

Sig. 1.000 1.000 .061 .061

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

269

S1956

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 4.6667

2.00 3 6.6667

3.00 3 7.3333 7.3333

4.00 3 8.3333 8.3333

5.00 3 9.3333

Sig. 1.000 .188 .060 .060

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

270

ANALYSIS FOR MBC

THR

ONEWAY S4 S8 S20 S22 S30 S33 S35 S36 S38 S42 S53 S57 S61 S62 S123 S127 S154 S187 S208 S235 S310 S390 S417 S570 S578 S600 S620 S651 S 819 S831 S841 S940 S947 S1009 S1104 S1172 S1420 S1827 S1956 BY VAR00001 /STATISTICS DESCRIPTIVES /MISSING ANALYSIS /POSTHOC=DUNCAN ALPHA (0.05).

Oneway

Descriptives

N Mean Std. Deviation

Std. Error

95% Confidence Interval for Mean

Minimum

Maximum

Lower Bound

Upper Bound

S4 1.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

2.00 3 6.0000 .00000 .00000 6.0000 6.0000 6.00 6.00

3.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

4.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.0667 2.71153 .70011 5.5651 8.5683 .00 10.00

S8 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

3.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.3333 2.58199 .66667 5.9035 8.7632 .00 10.00

S20 1.00 3 4.0000 .00000 .00000 4.0000 4.0000 4.00 4.00

271

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.6000 3.48056 .89868 3.6725 7.5275 .00 10.00

S22 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 7.1333 1.88478 .48665 6.0896 8.1771 4.00 10.00

S30 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00

Total 15 7.4000 1.88225 .48599 6.3576 8.4424 4.00 10.00

S33 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.0000 1.00000 .57735 3.5159 8.4841 5.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.3333 1.15470 .66667 5.4649 11.2018 7.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.9333 2.60403 .67236 5.4913 8.3754 .00 10.00

272

S35 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.0000 1.00000 .57735 3.5159 8.4841 5.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 6.3333 5.50757 3.17980 -7.3482 20.0149 .00 10.00

Total 15 6.8000 2.48424 .64143 5.4243 8.1757 .00 10.00

S36 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00

Total 15 7.1333 2.06559 .53333 5.9894 8.2772 4.00 10.00

S38 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 7.4000 1.59463 .41173 6.5169 8.2831 5.00 10.00

S42 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 1.15470 .66667 3.7982 9.5351 6.00 8.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

273

Total 15 6.8667 2.64215 .68220 5.4035 8.3298 .00 10.00

S53 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.9333 2.63131 .67940 5.4762 8.3905 .00 10.00

S57 1.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

2.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.2000 2.59670 .67047 5.7620 8.6380 .00 10.00

S61 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.8000 2.70449 .69830 5.3023 8.2977 .00 10.00

S62 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

274

5.00 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00

Total 15 7.4667 1.95911 .50584 6.3818 8.5516 4.00 10.00

S123 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.0000 2.50713 .64734 5.6116 8.3884 .00 10.00

S127 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 7.0000 .00000 .00000 7.0000 7.0000 7.00 7.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.9333 2.71153 .70011 5.4317 8.4349 .00 10.00

S154 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.0000 2.56348 .66189 5.5804 8.4196 .00 10.00

S187 1.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

275

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.7333 3.21751 .83076 3.9515 7.5151 .00 9.00

S208 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 6.0000 3.46410 .89443 4.0816 7.9184 .00 10.00

S235 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.2000 2.67795 .69144 5.7170 8.6830 .00 10.00

S310 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.6000 3.15776 .81533 3.8513 7.3487 .00 9.00

S390 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

276

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00

Total 15 7.4667 1.92230 .49634 6.4021 8.5312 4.00 10.00

S417 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

5.00 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00

Total 15 7.0000 2.07020 .53452 5.8536 8.1464 4.00 10.00

S570 1.00 3 3.6667 .57735 .33333 2.2324 5.1009 3.00 4.00

2.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

Total 15 6.8667 2.16685 .55948 5.6667 8.0666 3.00 9.00

S578 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 7.4667 1.92230 .49634 6.4021 8.5312 4.00 10.00

S600 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

277

2.00 3 6.0000 .00000 .00000 6.0000 6.0000 6.00 6.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 7.5333 1.68466 .43498 6.6004 8.4663 5.00 10.00

S620 1.00 3 5.0000 .00000 .00000 5.0000 5.0000 5.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 7.3333 1.83874 .47476 6.3151 8.3516 5.00 10.00

S651 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.3333 5.50757 3.17980 -7.3482 20.0149 .00 10.00

Total 15 6.5333 2.64215 .68220 5.0702 7.9965 .00 10.00

S819 1.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

2.00 3 5.6667 1.15470 .66667 2.7982 8.5351 5.00 7.00

3.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.6000 2.74643 .70912 5.0791 8.1209 .00 10.00

278

S831 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.8667 2.77403 .71625 5.3305 8.4029 .00 10.00

S841 1.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

2.00 3 6.0000 1.00000 .57735 3.5159 8.4841 5.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.5333 3.29213 .85002 3.7102 7.3565 .00 9.00

S940 1.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

2.00 3 7.6667 .57735 .33333 6.2324 9.1009 7.00 8.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 6.2000 3.46822 .89549 4.2794 8.1206 .00 10.00

S947 1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

279

Total 15 7.2000 2.54109 .65611 5.7928 8.6072 .00 10.00

S1009

1.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

2.00 3 6.6667 .57735 .33333 5.2324 8.1009 6.00 7.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

Total 15 7.8667 1.59762 .41250 6.9819 8.7514 5.00 10.00

S1104

1.00 3 5.6667 1.15470 .66667 2.7982 8.5351 5.00 7.00

2.00 3 6.3333 .57735 .33333 4.8991 7.7676 6.00 7.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 9.3333 .57735 .33333 7.8991 10.7676 9.00 10.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.2000 2.65115 .68452 5.7318 8.6682 .00 10.00

S1172

1.00 3 5.3333 .57735 .33333 3.8991 6.7676 5.00 6.00

2.00 3 4.3333 .57735 .33333 2.8991 5.7676 4.00 5.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00

5.00 3 9.0000 .00000 .00000 9.0000 9.0000 9.00 9.00

Total 15 7.0000 1.92725 .49761 5.9327 8.0673 4.00 9.00

S1420

1.00 3 4.6667 1.15470 .66667 1.7982 7.5351 4.00 6.00

2.00 3 4.6667 .57735 .33333 3.2324 6.1009 4.00 5.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

280

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 6.6000 2.87352 .74194 5.0087 8.1913 .00 10.00

S1827

1.00 3 5.0000 1.00000 .57735 2.5159 7.4841 4.00 6.00

2.00 3 5.6667 .57735 .33333 4.2324 7.1009 5.00 6.00

3.00 3 8.0000 1.00000 .57735 5.5159 10.4841 7.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 .0000 .00000 .00000 .0000 .0000 .00 .00

Total 15 5.4667 3.22638 .83305 3.6800 7.2534 .00 9.00

S1956

1.00 3 5.6667 1.15470 .66667 2.7982 8.5351 5.00 7.00

2.00 3 7.3333 .57735 .33333 5.8991 8.7676 7.00 8.00

3.00 3 8.3333 .57735 .33333 6.8991 9.7676 8.00 9.00

4.00 3 8.6667 .57735 .33333 7.2324 10.1009 8.00 9.00

5.00 3 6.6667 5.77350 3.33333 -7.6755 21.0088 .00 10.00

Total 15 7.3333 2.52605 .65222 5.9345 8.7322 .00 10.00

281

ANOVA

Sum of Squares df Mean Square F Sig.

S4 Between Groups 35.600 4 8.900 1.322 .327

Within Groups 67.333 10 6.733

Total 102.933 14

S8 Between Groups 24.000 4 6.000 .865 .517

Within Groups 69.333 10 6.933

Total 93.333 14

S20 Between Groups 167.600 4 41.900 209.500 .000

Within Groups 2.000 10 .200

Total 169.600 14

S22 Between Groups 47.067 4 11.767 44.125 .000

Within Groups 2.667 10 .267

Total 49.733 14

S30 Between Groups 45.600 4 11.400 28.500 .000

Within Groups 4.000 10 .400

Total 49.600 14

S33 Between Groups 22.267 4 5.567 .766 .571

Within Groups 72.667 10 7.267

Total 94.933 14

S35 Between Groups 20.400 4 5.100 .773 .567

Within Groups 66.000 10 6.600

Total 86.400 14

S36 Between Groups 57.067 4 14.267 53.500 .000

Within Groups 2.667 10 .267

Total 59.733 14

S38 Between Groups 32.933 4 8.233 30.875 .000

Within Groups 2.667 10 .267

Total 35.600 14

S42 Between Groups 25.067 4 6.267 .862 .519

Within Groups 72.667 10 7.267

Total 97.733 14

S53 Between Groups 27.600 4 6.900 .995 .454

282

Within Groups 69.333 10 6.933

Total 96.933 14

S57 Between Groups 25.733 4 6.433 .937 .481

Within Groups 68.667 10 6.867

Total 94.400 14

S61 Between Groups 33.067 4 8.267 1.192 .372

Within Groups 69.333 10 6.933

Total 102.400 14

S62 Between Groups 51.067 4 12.767 47.875 .000

Within Groups 2.667 10 .267

Total 53.733 14

S123 Between Groups 18.667 4 4.667 .673 .626

Within Groups 69.333 10 6.933

Total 88.000 14

S127 Between Groups 32.933 4 8.233 1.176 .378

Within Groups 70.000 10 7.000

Total 102.933 14

S154 Between Groups 21.333 4 5.333 .755 .577

Within Groups 70.667 10 7.067

Total 92.000 14

S187 Between Groups 140.933 4 35.233 88.083 .000

Within Groups 4.000 10 .400

Total 144.933 14

S208 Between Groups 165.333 4 41.333 155.000 .000

Within Groups 2.667 10 .267

Total 168.000 14

S235 Between Groups 31.067 4 7.767 1.120 .400

Within Groups 69.333 10 6.933

Total 100.400 14

S310 Between Groups 136.933 4 34.233 128.375 .000

Within Groups 2.667 10 .267

Total 139.600 14

S390 Between Groups 46.400 4 11.600 21.750 .000

Within Groups 5.333 10 .533

283

Total 51.733 14

S417 Between Groups 55.333 4 13.833 29.643 .000

Within Groups 4.667 10 .467

Total 60.000 14

S570 Between Groups 63.067 4 15.767 59.125 .000

Within Groups 2.667 10 .267

Total 65.733 14

S578 Between Groups 48.400 4 12.100 36.300 .000

Within Groups 3.333 10 .333

Total 51.733 14

S600 Between Groups 37.067 4 9.267 34.750 .000

Within Groups 2.667 10 .267

Total 39.733 14

S620 Between Groups 45.333 4 11.333 56.667 .000

Within Groups 2.000 10 .200

Total 47.333 14

S651 Between Groups 34.400 4 8.600 1.358 .315

Within Groups 63.333 10 6.333

Total 97.733 14

S819 Between Groups 34.267 4 8.567 1.201 .369

Within Groups 71.333 10 7.133

Total 105.600 14

S831 Between Groups 39.067 4 9.767 1.422 .296

Within Groups 68.667 10 6.867

Total 107.733 14

S841 Between Groups 147.733 4 36.933 92.333 .000

Within Groups 4.000 10 .400

Total 151.733 14

S940 Between Groups 165.733 4 41.433 155.375 .000

Within Groups 2.667 10 .267

Total 168.400 14

S947 Between Groups 19.733 4 4.933 .698 .610

Within Groups 70.667 10 7.067

Total 90.400 14

284

S1009 Between Groups 32.400 4 8.100 24.300 .000

Within Groups 3.333 10 .333

Total 35.733 14

S1104 Between Groups 25.733 4 6.433 .885 .507

Within Groups 72.667 10 7.267

Total 98.400 14

S1172 Between Groups 50.000 4 12.500 62.500 .000

Within Groups 2.000 10 .200

Total 52.000 14

S1420 Between Groups 44.267 4 11.067 1.551 .261

Within Groups 71.333 10 7.133

Total 115.600 14

S1827 Between Groups 140.400 4 35.100 65.813 .000

Within Groups 5.333 10 .533

Total 145.733 14

S1956 Between Groups 18.000 4 4.500 .631 .652

Within Groups 71.333 10 7.133

Total 89.333 14

285

Post Hoc Tests Homogeneous Subsets

S4 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.0000

2.00 3 6.0000

5.00 3 6.6667

3.00 3 8.6667

4.00 3 9.0000

Sig. .113

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S8

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333

5.00 3 6.6667

2.00 3 7.3333

3.00 3 8.6667

4.00 3 8.6667

Sig. .184

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

286

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

5.00 3 .0000

1.00 3 4.0000

2.00 3 6.3333 3.00 3 8.3333

4.00 3 9.3333 Sig. 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000.

S22

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 4.3333

2.00 3 6.3333

3.00 3 7.3333

4.00 3 8.0000

5.00 3 9.6667

Sig. 1.000 1.000 .145 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

287

S30

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 4.6667

2.00 3 6.6667

3.00 3 7.6667 7.6667

4.00 3 8.0000

5.00 3 10.0000

Sig. 1.000 .082 .533 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S33 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333

2.00 3 6.0000 5.00 3 6.6667

3.00 3 8.3333

4.00 3 8.3333 Sig. .238

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

288

S35 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333

2.00 3 6.0000

5.00 3 6.3333

4.00 3 8.0000

3.00 3 8.3333

Sig. .217

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S36

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 4.6667

2.00 3 5.3333

3.00 3 7.3333

4.00 3 8.3333

5.00 3 10.0000

Sig. .145 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

289

S38 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 5.3333

2.00 3 6.3333

3.00 3 7.6667 4.00 3 8.0000

5.00 3 9.6667 Sig. 1.000 1.000 .448 1.000

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000.

S42 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667 2.00 3 6.6667

5.00 3 6.6667 3.00 3 8.0000

4.00 3 8.3333

Sig. .156

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S53 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667 2.00 3 6.6667

5.00 3 6.6667 3.00 3 8.3333

4.00 3 8.3333

Sig. .148

Means for groups in homogeneous subsets are displayed.

290

S53 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667

2.00 3 6.6667

5.00 3 6.6667 3.00 3 8.3333

4.00 3 8.3333 Sig. .148

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S57 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.0000 5.00 3 6.6667

2.00 3 7.3333 3.00 3 8.3333 4.00 3 8.6667

Sig. .146

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S61 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333 2.00 3 6.3333

5.00 3 6.6667

3.00 3 8.3333 4.00 3 8.3333

Sig. .118

Means for groups in homogeneous subsets are displayed.

291

S57 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.0000

5.00 3 6.6667 2.00 3 7.3333

3.00 3 8.3333 4.00 3 8.6667

Sig. .146

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S62 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

1.00 3 4.6667

2.00 3 6.3333 3.00 3 7.6667

4.00 3 8.6667 5.00 3 10.0000

Sig. 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S123 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333 2.00 3 6.6667

5.00 3 6.6667

3.00 3 7.6667 4.00 3 8.6667

Sig. .184

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

292

S127 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333

5.00 3 6.6667 2.00 3 7.0000

3.00 3 8.0000 4.00 3 8.6667

Sig. .095

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S154 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333 2.00 3 6.3333

5.00 3 6.6667

3.00 3 8.0000 4.00 3 8.6667

Sig. .188

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S187 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

5.00 3 .0000 1.00 3 5.6667

2.00 3 6.3333

3.00 3 8.0000 4.00 3 8.6667

Sig. 1.000 .226 .226

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

293

S208 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 5.3333

2.00 3 6.6667 3.00 3 8.6667

4.00 3 9.3333 Sig. 1.000 1.000 1.000 .145

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. S235 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333 2.00 3 6.3333

5.00 3 6.6667 3.00 3 8.3333

4.00 3 9.3333

Sig. .118

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S310 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4 5

5.00 3 .0000

1.00 3 5.3333 2.00 3 6.3333

3.00 3 7.6667

4.00 3 8.6667 Sig. 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000.

294

S390 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 4.6667

2.00 3 6.6667

3.00 3 8.0000 4.00 3 8.0000

5.00 3 10.0000 Sig. 1.000 .058 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S417 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 4.3333 2.00 3 5.3333

3.00 3 7.6667

4.00 3 8.0000 5.00 3 9.6667

Sig. .103 .563 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S570 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

1.00 3 3.6667

2.00 3 5.3333 3.00 3 7.6667

4.00 3 8.6667 5.00 3 9.0000

Sig. 1.000 1.000 1.000 .448

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

295

S578 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 4.3333

2.00 3 6.6667

3.00 3 8.3333 4.00 3 8.6667

5.00 3 9.3333 Sig. 1.000 1.000 .070

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. S600 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 5.3333 2.00 3 6.0000

3.00 3 8.3333 4.00 3 8.6667 8.6667

5.00 3 9.3333

Sig. .145 .448 .145

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S620 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

1.00 3 5.0000 2.00 3 5.6667

3.00 3 7.6667 4.00 3 9.0000

5.00 3 9.3333

Sig. .098 1.000 .383

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

296

S651 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333 2.00 3 5.6667

5.00 3 6.3333

3.00 3 7.6667 4.00 3 8.6667

Sig. .081

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S819 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.3333

2.00 3 5.6667 5.00 3 6.6667

3.00 3 7.6667 4.00 3 8.6667

Sig. .098

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S831 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667

2.00 3 5.6667 5.00 3 6.6667

3.00 3 8.3333 4.00 3 9.0000

Sig. .092

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

297

S841 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000

1.00 3 4.6667

2.00 3 6.0000 3.00 3 8.3333

4.00 3 8.6667 Sig. 1.000 1.000 1.000 .533

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. S940 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

5.00 3 .0000 1.00 3 5.6667

2.00 3 7.6667 3.00 3 8.3333

4.00 3 9.3333

Sig. 1.000 1.000 .145 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S947 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.3333 5.00 3 6.6667

2.00 3 7.3333 3.00 3 8.0000

4.00 3 8.6667

Sig. .188

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

298

S1009 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2

1.00 3 5.6667

2.00 3 6.6667

3.00 3 8.3333 4.00 3 9.3333

5.00 3 9.3333 Sig. .060 .070

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000. S1104 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.6667

2.00 3 6.3333

5.00 3 6.6667 3.00 3 8.0000

4.00 3 9.3333 Sig. .156

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S1172 Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3 4

2.00 3 4.3333

1.00 3 5.3333 4.00 3 8.0000

3.00 3 8.3333 8.3333

5.00 3 9.0000 Sig. 1.000 1.000 .383 .098

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000.

299

S1420

Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 4.6667

2.00 3 4.6667

5.00 3 6.6667

3.00 3 8.3333

4.00 3 8.6667

Sig. .122

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000.

S1827

Duncana

VAR00001 N

Subset for alpha = 0.05

1 2 3

5.00 3 .0000

1.00 3 5.0000

2.00 3 5.6667

3.00 3 8.0000

4.00 3 8.6667

Sig. 1.000 .290 .290

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 3.000. S1956 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.6667

5.00 3 6.6667

2.00 3 7.3333

3.00 3 8.3333

4.00 3 8.6667

Sig. .234

Means for groups in homogeneous subsets are displayed.

300

S1956 Duncana

VAR00001 N

Subset for alpha = 0.05

1

1.00 3 5.6667

5.00 3 6.6667

2.00 3 7.3333

3.00 3 8.3333

4.00 3 8.6667

Sig. .234

Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 3.000.