Evaluation of the Antimycobacterial Properties of Plant Extracts From

Evaluation of the antimycobacterial properties of plant extracts from

Vernonia adoensis

Ruvimbo Vicki Tricia Mautsa

R076753C

DISSERTATION IN FULFILLMENT OF THE MASTER OF PHILOSOPHY

DEGREE IN BIOCHEMISTRY

Department of Biochemistry

University of Zimbabwe

2018 DECLARATION

I, Ruvimbo Vicki Tricia Mautsa, a student of the Faculty of Science of University of Zimbabwe, declare that this thesis is the result of my own independent experimental work, carried out from August 2014 to July 2017 at University of Zimbabwe except where otherwise stated. Other sources used are acknowledged in the thesis by explicit references. This work has never been submitted elsewhere to meet the requirements for any other award.

Signed by student……………………...……………………………………………………

Date…………………………………………………………………………………………

DEDICATION

I dedicate this thesis to the four lovely members of my family; my husband Ronald, my two daughters;

Mukudzeishe and Matipaishe; and lastly but not least, my little boy, Kuitakwashe.

There were times when I got tired and frustrated, thank you guys for being my pillars of strength, and thank you for being the supporters that never got tired of inspiring and cheering on me

ACKNOWLEDGEMENTS

I would like to express my sincerest gratitude towards my supervisor, Professor Stanley Mukanganyama, and my co-supervisor, Professor Dexter Tagwireyi, for their persistence, mentorship and support throughout the years as I carried out my research.

My deepest gratitude, through the efforts of my supervisor, similarly go to the sponsors of this study; the

International Foundation for Science (IFS) Stockholm, Sweden and IPICS-ZIM01 project from the

International Program in the Chemical Sciences (IPICS), Uppsala University, Sweden.

Many thanks go to group members of the Biomolecular Interactions Analyses (BIA) for the constructive criticism and sharing of knowledge and to the Biochemistry staff who played their corresponding roles to make this study achievable.

Lastly but not least, I express gratitude towards my family for their staunch support all the way.

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ABSTRACT

Tuberculosis (TB), is a mycobacterial infection resulting from Mycobacterium tuberculosis. Two million individuals die annually due to TB and currently, treating this infection is a massive challenge owing to the emergence of drug-resistant strains and co-infection with HIV. Medicinal plants, having been used since time immemorial for the treatment of TB and TB-related ailments, are a possible lead for the discovery of novel phytocompounds that can be exploited for the invention of more efficacious antimycobacterial agents. Vernonia adoensis is a herbal medicine that is used as a natural therapy for TB. This study examined the antimycobacterial properties of V. adoensis against a model mycobacterial species Mycobacterium smegmatis and the plant’s mode of action. Crude extracts of V. adoensis were prepared from the flowers, leaves, root bark and roots of V. adoensis using solvents of varying polarity. The major biologically active class of phytochemicals were identified. The antimycobacterial activity of the prepared extracts was assessed using the broth microdilution assay with rifampicin as the reference drug. The plant extracts and phytochemicals were tested for their inhibitory and mycobactericidal activities against M. smegmatis. The consequence of the most powerful extract of V. adoensis on the transport of ciprofloxacin across the cell membrane was investigated. The extract's ability to damage membrane integrity resulting in protein and nucleic acid leakage in mycobacterial cells was determined and its ability to scavenge for free radicals was assessed using the DPPH method. In vitro determination of the effect of the plant extract on the survival of mycobacteria inside macrophages was conducted and the cytotoxic effects of the plant extract were investigated using sheep erythrocytes. The most potent extract from V. adoensis was the ethyl acetate leaf extract which had a minimum inhibitory concentration and minimum bactericidal concentration of 63 µg/ml and 125 µg/ml respectively against M. smegmatis. The most active phytochemical class was the terpenoid fraction with an MIC of 250 µg/ml. Nucleic acid and protein leakage in M. smegmatis cells were observed after they were subjected to the leaf extract. The ethyl acetate leaf extract didn’t show neither free radical scavenging activity nor effect on drug transport in mycobacterial cells. The extract showed minimal cytotoxic effects towards the sheep erythrocytes and post-treating M. smegmatis-infected murine macrophages with the leaf extract at a concentration of 126 µg/ml significantly reduced the viability of the mycobacteria. The result of the study supports the traditional use of V. adoensis leaves in the treatment of tuberculosis. It is suggested that cell membrane disruption resultant in protein and nucleic acid leakage could be the plant's mode of action. Since the plant extract was not cytotoxic, it is a potential candidate as a template for new antimycobacterials. Since a model non-pathogenic organism was used, further studies must be done using M. tuberculosis. Further studies may also include isolation of the active constituents of Vernonia adoensis, which may form a basis for discovering new compounds with better antimycobacterial activity than the drugs that are in use.

Keywords: Vernonia adoensis, plant extracts, antimycobacterial, Mycobacterium smegmatis

Table of Contents

Declaration...... i

Dedication……...... ii

Acknowledgements………………………………………………….………………………………….iii

Abstract……...... iv

Table of contents…...... v

List of Abbreviations...... x

List of Tables...... xii

List of Figures...... xiii

List of Appendices...... xv

Chapter One Introduction………………………………………………………………………...... 1

1.1 Tuberculosis………….……………………………………………………………………………1

1.2 Tuberculosis epidemiology in humans ….…………………………………...... 1

1.3. Clinical symptoms of tuberculosis ………………….…………………...... 3

1.4. Treatment options of tuberculosis …………………...... 4

1.5. How the current tuberculosis drugs work…………………………………...... 5

1.5.1 Rifampicin (RIF).……….………………………………………...... 6

1.5.2 Pyrazinamide (PZA) (Pyrazinecarboxamide)……………………………...... 7

1.5.3 Isoniazid (INH) (Isonicotinic acid hydrazide) ……………………...... 8

1.5.4 Ethambutol (EMB) ((+) - ethambutol dihydrochloride) …………………………………….9

1.6 Drug resistance in tuberculosis….….…………………………………...………………………...10

1.6.1 Drug efflux pumps……………………………………………………...... 11

1.6.2 Efflux pump inhibitors………………………………….……………………..……………13

1.7 Mycobacteria.…………………………………………………...... 14

1.7.1 Mycobacterium tuberculosis………...... 17

1.7.2 Non-pathogenic mycobacteria…….…………………………………………..…………….18

1.7.3 Mycobacterium aurum…………………………………………...... 18

1.7.4 Mycobacterium smegmatis………………………………………………………………...... 19

1.7.5 Use of M. smegmatis as a model for M. tuberculosis……………….…………………….....21

1.8 Current challenges in treating tuberculosis...... 22

1.9 The use of traditional medicine as an alternative or complementary

medicine…………………………………………………...... 24

1.10 Plant phytochemicals …………………………………………………………………..…….…...26

1.10.1 Classes of phytochemicals………………………………………………………..……...28

1.10.2 Alkaloids……………………………………………………………………………..…..28

1.10.3 Flavonoids …………………………………………………………………………….....29

1.10.4 Phenolics ………………………………………………………...... 30

1.10.5 Saponins …………………………………………………………...... 31

1.10.6 Tannins ………………………………………………………………………………...... 32

1.10.7 Terpenoids …………………………………………………………………………….....33

1.11 Antioxidants ……………………………………………………………………………………....34

1.11.1 Antioxidants and their role in the treatment of tuberculosis...... 38

1.12 Antimycobacterial susceptibility tests………………………………....……………………….....39

1.12.1 Minimum inhibitory concentration determination using the broth

microdilution method………………………………………….………………...... …..39

1.12.2 Determination of the minimum bactericidal concentration (MBC)...... 41

1.13 Determination of toxicity in mammalian cells ……………….………….………………………..42

1.14 Protein determination……………………………………………………………………………...43

1.15 Murine macrophages……………………………………………...... 44

1.16 Plant used in this study: Vernonia adoensis (Sch. Bip. ex Walp.) …………………………….….46

1.17 Rationale of study…………….…………………………………...... 50

1.18 Research Question………………………………………………...... 51

Chapter Two: Objectives…...... 52

2.1 Main objective…………………………………………...... 52

2.2 Specific objectives…………………………………...... 52

2.3 Design of study……………………………………………………...... 53

Chapter Three: METHODOLOGY…...... 54

3.1 Materials...... 54

3.2 Methods...... 54

3.2.1 Collection of plant sample……...... 54

3.2.2 Preparation of extracts……………………………………………………………………….55

3.2.3 Qualitative analysis of V. adoensis plant extracts …………………………………………..55

3.2.3.1 Test for flavonoids ………………………………………………………………….55

3.2.3.2 Test for alkaloids ……………………………………………….…………………...56

3.2.3.3 Test for saponins ……………………………………………………………………56

vii

3.2.3.4 Test for tannins ……………………………………………………………………...56

3.2.3.5 Test for phenols ………...………………………………………………………...... 56

3.2.3.6 Test for terpenoids …………………………………………………………………..56

3.2.4 Quantitative phytochemical analyses of V. adoensis plant extracts……………………….58

3.2.5 Culturing of Mycobacterium smegmatis...... 59

3.2.6 Determination of MIC and MBC of plant extracts…………...... 59

3.2.7 Determination of effect of V. adoensis on drug transport in M. smegmatis

cells…………………………………………….……...... 60

3.2.8 Determination of effect of V. adoensis extract on cell membrane integrity using th nucleic acid leakage assay………………………………………………………………...62

3.2.9 Determination of V. adoensis extract on cell membrane integrity using the p protein leakage assay ...... 62

3.2.10 Determination of the free radical scavenging potential of V. adoensis extract

using the DPPH antioxidant assay………...……………………………………………….63

3.2.11 Haemolysis assay………………………………...... 64

3.2.12 In vitro determination of viability of mycobacteria inside macrophages…………………65

3.2.13 Phytochemical screening tests for the active ethyl acetate fraction of V. adoensis……….65

3.3 Statistical analysis………………………………………...... 66

Chapter Four: RESULTS...... 67

4.1 Qualitative phytochemical analyses of V. adoensis plant………...... 67

4.2 Quantitative phytochemical analyses V. adoensis plant ……………………………………...67

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4.3 Antimycobacterial susceptibility tests: Determination of MIC and

MBC of V. adoensis plant extract against M. smegmatis cells………………………………..68

4.4 The effect V. adoensis leaf extract on the transport of ciprofloxacin…...... 73

4.5 The effect of V. adoensis extract on cell membrane integrity in the protein leakage assay…..74a

4.6 The effect of V. adoensis extract on leakage of nucleic acids...... 75

4.7 The effect of V. adoensis extract on free radical scavenging activity...... 77

4.8 The effect of V. adoensis extract on haemolysis of sheep erythrocytes…...... 78

4.9 The in vitro determination of viability of mycobacteria inside macrophages…….…………..79

4.10 Phytochemicals present in V. adoensis ethyl acetate plant extract……………………………80

Chapter Five: DISCUSSION...... 81

Chapter Six: CONCLUSION...... 91

FUTURE STUDIES...... 92

REFERENCES...... 93

APPENDICES………………………………………………………………...... 117

LIST OF ABBREVIATIONS

ARV Antiretroviral

ATP Adenosine triphosphate

BCG Bacillus Calmette-Guerin

BSA Bovine serum albumin

CBBG Coomassie Brilliant Blue G

CCCP Carbonyl cyanide 3-chlorophenylhydrazone

CFU Colony forming units

DCM Dichloromethane

DMSO Dimethyl sulfoxide

DOTS Directly Observed Treatment Short course

DPPH 2, 2-diphenyl-1-picrylhydrazyl

EMB Ethambutol

EPI Efflux pump inhibitor

HIV Human Immunodeficiency Virus

INH Isoniazid

MBC Minimum bactericidal concentration

MIC Minimum inhibitory concentration

MTC Mycobacterium tuberculosis Complex

MTT 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS Phosphate Buffered Saline

PI Propidium iodide

PZA Pyrazinamide

RIF Rifampicin x

ROS Reactive oxygen species

SDS Sodium dodecyl sulphate

TB Tuberculosis

WHO World Health Organization

LIST OF TABLES

Table 4.1: Qualitative phytochemical analysis of Vernonia adoensis plant parts……………………..67

Table 4.2: Percentage yields of fractions of V. adoensis plant parts……………………...... 68

Table 4.3: Summary of the MICs of the crude extracts of different plant parts of

V. adoensis prepared from solvents of varying polarity...... 69

Table 4.4: Summary of the MICs of the fractions extracted from V. adoensis………………………..69

Table 4.5: Antimycobacterial activity of the most active extracts and fractions of V. adoensis……...70

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LIST OF FIGURES

Figure 1.1: World map representation of reported cases of TB per 100 000 citizens in 2017…………….2

Figure 1.2: Functioning principle of anti-TB drugs….………………………………………...... 5

Figure 1.3: Structure of rifampicin………………………………………………………………………...6

Figure 1.4: Structure of pyrazinamide…….……………………………………………...... 7

Figure 1.5: Structure of isoniazid………………………...………………………………. ………………9

Figure 1.6: Structure of ethambutol……..…………………………………………………...... 10

Figure 1.7: Schematic representation depicting various mechanisms of how generic bacteria

can acquire resistance to antimycobacterial agents.………………...…………………….…11

Figure 1.8: Illustration of types of efflux pumps found in gram-negative and gram- positive

bacteria……………………………………………………………………………………….12

Figure 1.9: The mycobacterial cell wall.…….…………………………………………………………...16

Figure 1.10: A typical Ziehl-Neelsen stain of mycobacteria. ……………………………………….…...17

Figure 1.11: Colonies of Mycobacterium aurum growing on solid media…………………...... 19

Figure 1.12: Mycobacterium smegmatis streak plate (a) coloured electron micrograph (b) and

electron microscopy (c and d) colonies on plates …………...…...…………………………..20

Figure 1.13: Major classes of phytochemicals……………………………………………………………27

Figure 1.14: Structure of a known alkaloid, nicotine………………………………………………….....29

Figure 1.15: Chemical structure of a flavone (2-phenyl-1,4-benzopyrone)……………………………...30

Figure 1.16: Chemical structure of a common phenol, gallic acid……………………………………….31

Figure 1.17: The general structure of a saponin…………………………………………………………..32

xiii

Figure 1.18: General chemical structure of tannins…………………………………………...…………33

Figure 1.19: General structure of terpenoids………………………………………………...…………..34

Figure 1.20: Structures of some known phytochemical antioxidants……………………………………37

Figure 1.21: Typical mechanism of action of an antioxidant………………………………………...….37

Figure 1.22: Schematic diagram of typical MIC and MBC determination…………………...... 40

Figure 1.23: Picture of Vernonia adoensis taken in Centenary, Zimbabwe.………………...... 47

Figure 4.1 The effect of V. adoensis ethyl acetate leaf extract on the growth of M. smegmatis in liquid

broth.……………………………………………………………………...... 71

Figure 4.2: Image of 96 well plate showing the antimycobacterial activity of V. adoensis ethyl acetate leaf extract leaf extract against M. smegmatis in the MTT assay……………………………….……….72

Figure 4.3: Accumulation of drug in M. smegmatis cells.…………………………………………..…...73

Figure 4.4: Protein leakage in M. smegmatis after exposure to V. adoensis ethyl acetate leaf extract…..75

Figure 4.5: Fluorescence of nucleic acid binding propidium iodide after exposure of M. smegmatis to V. adoensis adoensis leaf extract...... 76

Figure 4.6: DPPH free radical scavenging activity of V. adoensis ethyl acetate leaf extract and ascorbic acid……… acid…………………………………………………………………………………………..77

Figure 4.7: Haemolytic effect of the V. adoensis leaf extract……………………….…………………..78

Figure 4.8: Intracellular survival of M. smegmatis in V. adoensis extract

treated macrophages…….……………………………………………………..…………....80

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LIST OF APPENDICES

Appendix 1. Raw results used to calculate activity of V. adoensis ethyl acetate extract……………….117

Appendix 2. Standard curve for BSA…………………………………………………………………...118

Appendix 3. Publication………………………………………………………………………………...119

Appendix 4. Conference presentations………………………………………………………………….120

Chapter One: INTRODUCTION

1.1 Tuberculosis

Tuberculosis (TB) is a communicable chronic infection caused by bacteria that is both treatable and avertible, and by right, belongs to be a disease of the past, but it now is one of the top causes of human illness and death (Sanusi et al., 2017). Mycobacterial species from the Mycobacterium tuberculosis complex have been discovered to cause TB (Sandhu, 2011), with M. tuberculosis being the root cause of

TB in humans. TB transmission is via exposure to tubercle bacilli in airborne droplet nuclei produced by expiratory efforts such as coughing or sneezing, and the resultant lung disease is known as pulmonary TB

(WHO, 2006). This infection can as well affect other organs of the body like the urinary tract, the abdomen, bones, the kidneys, spine, brain and the skin (Mohajan, 2015). Like other communicable diseases, TB is transmittable to anyone irrespective of age, sex and nationality. An individual suffering from active TB is able to infect an average of 15 people a year (WHO, 2007), with the disease thriving in poverty-laden and overcrowded conditions.

1.2 TB epidemiology in humans

TB became a global health emergency in 1993 (WHO, 1994) and in 2014, the annual total TB deaths surpassed those caused by HIV with 1.5 million deaths as compared to 1.2 million deaths due to HIV

(Global TB report, 2017). TB is now responsible for an estimated 2 million preventable deaths annually and is the main cause of death from a communicable disease in adults (WHO, 2015). The WHO estimates that about a quarter of the world’s population has latent TB and there are new individual TB bacilli infections each second (WHO, 2006a; Dye et al., 1999). The disease attacks young adults particularly those who are in economically disadvantaged areas. Reduction of the reservoir of infection in

1 humans will take many years of study and unrelenting effort of accelerated increase in TB interventions, intensified case finding among at-risk populations, and a much greater focus on upstream prevention.

Tuberculosis and HIV are the world’s major result of mortality (WHO, 2015) and their co- infection results in challenges in diagnosis and therapy and at the same time inciting enhanced disease progression.

Zimbabwe is on the list of the worlds’ 30 high burden TB countries which account for 87% of all roughly calculated incident cases globally (WHO, 2015). The high TB burden country list also includes Kenya,

India, Ethiopia and Indonesia. Figure 1.1 shows a representation of the estimated TB incidence rates that were documented in 2017.

Figure 1.1: World map representation of reported cases of TB per 100 000 citizens in 2017

The TB incidence rate for different countries ranges substantially, with around 300 or more cases for every 100 000 people in countries like Botswana and South Africa and fewer than 24 for every 100 000 population in parts of America, Argentina and Australia (WHO, 2017).

1.3 Clinical symptoms of TB

TB infection is when TB pathogens enter the body but do not make the individual fall sick (Guinn and

Rubin, 2017). This can be explained along these lines: a healthy strong immune system might be unable to do away with the pathogen by itself, but it can keep the bacteria trapped in the alveoli of the lungs and inhibit its spread from one individual to another (Cambier et al., 2014). The function of the body’s main defense, the macrophages, is to actively hunt down, ingest and destroy bacteria (Peters and Smith, 2017), however, after ingestion, some mycobacteria have the ability to evade the macrophages. When ingesting mycobacteria, macrophages form a vesicle that is called a phagosome, which then matures and turns into a lysosome. The environment within the lysosome is acidic and hydrolytic enzymes within, break down the mycobacteria (Cooper, 2000). However, mycobacteria can apprehend phagosome maturation preventing acidification of the inside environment and thus hydrolysis by the enzymes (Kastrinsky et al.,

2010). Information on the precise mechanism of mycobacteria to evade destruction inside macrophages is scanty (Hossain and Norazmi, 2013). The TB bacillus can, therefore, lie idle in the macrophages for ages. Individuals with TB infection, who are immunocompromised or have lung ailments, will most likely suffer from TB (WHO, 2007).

Symptoms of TB differ depending on the specific body part the pathogens multiply in. Common early symptoms of an active TB infection include cough which persists for over 2 weeks, chills, inexplicable loss of weight, consistent fever, bloody sputum, food aversion and excessive night sweating (WHO, 2010,

Curry International Tuberculosis Center, 2011). Some patients feel joint pain just like arthritis when the tuberculosis pathogen stays in the bones.

1.4 Treatment options of tuberculosis

The current preferred treatment for TB is a multi-drug regimen that utilizes ethambutol, rifampicin, isoniazid, and pyrazinamide. The therapy consists of an early rigorous stage in which the aforementioned drugs are taken every day for the initial two months, accompanied by a continuation phase of rifampicin and isoniazid for four months (Huаng et al., 2009). To achieve high cure rates, the shortest duration that the therapy can be taken for is 6 months (van den Boogaard et al., 2009). According to Jasmer and co- writers (2004), TB treatment targets the following goals: i) To cure TB infection and lower the communication of M. tuberculosis to others in the community; ii) To avoid relapse and prevent the advancement of resistance, and iii) To prevent TB or TB-complication related deaths

Treating TB using a cocktail of drugs is grounded on main two principles: to enhance efficacy of the drugs and to avoid the bacilli acquiring drug resistance (Chan and Iseman, 2002).

To effectively treat TB, the Directly observed treatment short course (DOTS) is being used by health workers. The WHO introduced the DOTS strategy to ensure compliance to treatment and, hence, lower the incidence of resistance (McRae et al., 2007). DOTS programs use a nurse or surrogate to deliver and observe patients taking all the dosages of their drugs, instead of relying on patients to take them on their own. DOTS is very efficient in promoting successful treatment, with an evaluation of self-treatment versus various forms of DOTS revealing that completion of treatment is appreciably higher when the treatment is monitored (Chaulk and Kazandjian, 1998).

1.5 How the current TB drugs work

Optimism that TB disease could actually be controlled, if not eradicated, arose when the first TB antibiotics were introduced (WHO, 2003). Chemotherapy for TB basically banks on drugs that are able to inhibit bacterial metabolism, with huge emphasis on inhibitors of mycobacterial cell wall synthesis.

Depending on their mode of action, TB drugs may be categorized as inhibitors of i) protein synthesis ii) nucleic acid synthesis iii) cell wall synthesis of bacteria and iv) electron transport across the bacterial membrane (du Toit et al., 2006; Lаurenzi et al., 2007). Figure 1.2 shows a depiction of how the anti-TB drugs that are currently in use work.

Figure 1.2: Functioning principles of anti-TB drugs. TB drugs target diverse aspects of M. tuberculosis biology, which include preventing nucleic acid synthesis, cell wall, or protein synthesis. (NIAID, 2016)

1.5.1 Rifampicin (RIF) (5,6,9,17,19,21-hexahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8- [N-(4-methyl-1-piperazinyl) formimidoyl]-2,7-(epoxypentadeca[1,11,13]trienimino)-naphtho[2,1- b]furan-1,11(2H)-dione 21-acetate)

Rifampicin is a broad-spectrum, semi-synthetic agent produced by the gram-positive bacterium

Streptomyces mediterranei and since its discovery in 1965, it is still amongst the most powerful anti-TB drugs (Figure 1.3) (El-Tayeb et al., 2004). It can diffuse freely into tissues, viable cells and bacteria and is bactericidal towards extracellular and intracellular gram-negative and gram- positive bacteria such as

M. tuberculosis (Shinnick, 1996; Campbell et al., 2001). Its principle of action is the inhibition of DNA- dependent RNA polymerase activity. Rifampicin interacts with the enzyme polymerase forming a very stable complex, and in the process inhibiting the initiation of RNA synthesis (Wehrli and Staehelin,

1971).

O OH O

OH OH

O NH

N O N

O OH N

Figure 1.3: Structure of RIF

1.5.2 Pyrazinamide (PZA) (Pyrazinecarboxamide)

Pyrazinamide is another first-line drug plays a critical role in TB treatment. The drug can eradicate non- replicating bacilli that other anti-TB drugs fail to get rid of (Shi et al., 2011). Therefore, it is included in drug permutations for treating both drug-susceptible and drug- resistant TB forms like multidrug resistant

TB (MDR-TB), and when combined with rifampicin as well as isoniazid, the drugs decrease the time of treatment from 1 year to six months (Zhang et al., 2013).

Figure 1.4: Structure of PZA

Pyrazinamide binds to ribosomal proteins, and in so doing inhibits translation in bacterial cells (Shi et al.,

2011). Bacterial pyrazinamidase transforms pyrazinamide, the parental form of the drug to pyrazinoic acid (POA), the activated form of the drug (Raynaud et al., 1999; Scorpio and Zhang,

1996). POA goes into the cell using passive diffusion and takes advantage of the poorly defined efflux mechanism in M. tuberculosis to accrue in the cells (Zhang et al., 1999). The accumulation of POA leads to cytoplasmic acidification that can result in inhibition of key enzymes such as fatty acid synthetase I

(FASI) (Zhang et al., 1999). Some studies that were done suggest that pyrazinamide might lack a particular target in mycobacterial cells; instead, the accumulation of POA results in the interruption of

7 membrane potential, hindering the ATP production and inhibiting M. tuberculosis membrane transporters

(Zhang et al., 2003).

1.5.3 Isoniazid (INH) (Isonicotinic acid hydrazide)

Isoniazid was discovered to possess a notably higher anti- TB activity as compared to the other drugs that were in use in the 1950’s making it one of the most potent anti-TB drugs (Bernstein et al., 1952).

Isoniazid enters mycobacterial cells by passive diffusion, and once inside, it can only eradicate actively dividing bacteria (Bardou et al., 1998). The drug has no effect on cells growing under anaerobic conditions, nor on those in stationary phase (Schaefer, 1954). Just like pyrazinamide, isoniazid is also a prodrug, which must be activated by a mycobacterial enzyme catalase-peroxidase hemoprotein, KatG

(Zhang, et al., 1992). For this activation to occur hydrazine must reduce KatG and then the reduced KatG will react with oxygen, forming an oxyferrous enzyme complex. This activated form of isoniazid combines with NAD+ forming a covalent chemical compound, InhA, that is an effective inhibitor of mycobacterial enoyl-acyl protein reductase. InhA plays an important role in the production of mycolic acids and so in this way, isoniazid manages to disrupt the biosynthesis of mycolic acids (Winder, 1982).

Mycolic acids make the cell envelope impervious to xenobiotics thus, form a crucial cell wall component.

This makes mycolic acids important targets for antitubercular agents. (Slayden and Barry, 2000).

Figure 1.5 shows the chemical structure of isoniazid.

Figure 1.5: Structure of INH

1.5.4 Ethambutol (EMB) ((+)-ethambutol dihydrochloride)

Ethambutol is bacteriostatic towards mycobacteria and works against multiplying cells and usually has very little effect on non-multiplying cells. In report by Forbes et al., (1962), it was observed that ethambutol showed indications of impairing the metabolism of glycerol in multiplying cells. Ethambutol prevents the production of metabolites required for differentiation. When these metabolites deplete, multiplication cannot occur, leading to impairment of metabolism, and inevitably, loss of viability of the cells (Belanger et al., 1996). Ethambutol inhibits metabolite synthesis, but the precise nature of these particular metabolites is unknown. Another way that ethambutol works is by inhibiting arabinosyl transferases, enzymes that are included in synthesis of the cell wall (Mikusovа et al., 1995). Production of the cell wall complex in the mycobacteria is then inhibited, leading to increase in permeability of the cell wall (Forbes et al.,1962).

H N N H

Figure 1.6: Structure of the EMB

1.6 Drug resistance in TB

The present TB treatment regime utilizes multidrug therapy, usually, a cocktail of three or four drugs.

The intense battle against TB infection has given rise to MDR strains which have developed numerous defense mechanisms against the antimycobacterial agents. New antibiotics bedaquiline, daptomycin and linezolid penetrated the market around 2000 to treat MDR strains to tuberculosis. Years later, there were reports of resistance against these new therapies (Lewis and Jorgensen, 2005). Resistance to antituberculosis agents can be acquired through a number of ways (Figure 1.7), and the mechanisms that the mycobacteria use include; i) enzymatic inactivation; ii) cell wall impermeability resulting in reduction of intracellular drug concentration; iii) alteration of drug target/s, iv) protection of target site, v) the extrusion of antibiotics by mycobacterial efflux pumps vi) overexpression of antibiotic hydrolyzing enzymes in the bacterial cell (Danilchаnkа et al., 2008; Sаbаtini et al., 2012).

Figure 1.7: Schematic representation depicting various mechanisms of how generic bacteria can become resistant to antibacterial agents.

1.6.1 Drug efflux pumps

Efflux pumps can be defined as transport proteins that are found on cytoplasmic membranes and are associated with the pumping out of foreign toxic substances from inside cells to the outside environment

(Webber and Piddock, 2003). The foreign toxic substances include every class of antibiotics, thus preventing the drug from accessing its target. Some efflux pumps have specificity to one substrate, extruding specific antibiotics only, whilst others transport an array of structurally diverse compounds.

Such pumps are known as multi-drug efflux pumps (MEPs), and are usually linked with multiple drug resistance (Bansal et al., 2003). Depending on factors such as the number of transmembrane spanning regions of the efflux pumps, their composition, energy sources and substrates utilized (ion electrochemical gradient or ATP hydrolysis), generic bacterial efflux pumps are categorized into five

11 super families (Figure 1.8); the major facilitator superfamily (MFS), the ATP (adenosine triphosphate)- binding cassette (ABC) superfamily, the resistance-nodulation-division (RND) family, the small multidrug resistance (SMR) family, and the multidrug and toxic compound extrusion (MATE) family

(Pule et al., 2016). The ABC superfamily are primary active transporters that utilize free energy generated from ATP hydrolysis to energize drug efflux. The other four groups are secondary active transporters, that use proton motive force as their energy source. M. tuberculosis efflux pumps have been characterized, and they belong to the ABC superfamily, SMR and MFS family (Ramon-Garcia et al.,

2006). Efflux pumps are a significant player in conferring resistance to practically all classes of antibiotics (Prаsch and Bucаr, 2015).

Figure 1.8: Illustration of the types of efflux pumps found in gram-negative and gram- positive bacteria. Shown are the five main super families of efflux pumps. “The ABC, the MFS, the MATE, SMR and the RND families of efflux pumps” (Munita and Arias, 2016). The diagrammatic illustration shows the energy source and examples of the drugs that act as substrates.

1.6.2 Efflux pump inhibitors

A means that can be employed to overcome the decrease in sensitivity of mycobacteria to antibiotics is usage of efflux pump inhibitors or EPIs. Co-administering an EPI with an antibiotic decreases the amount of antibiotic required for the same therapeutic effect (Prasch and Bucar, 2015). Efflux pumps represent a potential target for drug development, by blocking the pumps, antibiotic activity can be restored. Various reports claim that EPIs can: i) inhibit the enzymes that maintain the proton motive force, ii) disturb the pump assembly; iii) block the efflux channels; iv) alter the regulation genes; v) form an antibiotic and EPI complex thereby impair access to the binding site for the drug

(Fernandez and Hancock 2012; Amaral et al., 2014). A few EPIs for mycobacteria have been identified so far. Although some experimental compounds, for instance carbonyl cyanide m-chlorophenylhydrazone

(CCCP), chlorpromazine and verapamil have shown to have EPI properties against mycobacteria in vitro and ex vivo, these EPIs have not so far satisfied certain requirements to make them clinically applicable

(Lechner et al., 2008). These requirements include toxicity, immunosuppression, serum concentration, and stability and solubility concerns in human or veterinary medication (Piddock, 2006). Discovery of new EPIs for mycobacteria is thus imperative.

Plants are a promising source for new efflux pump inhibitors owing to the vast compound diversity, tolerability and low toxicity. Reserpine and piperine are plant derived alkaloids that are examples of EPIs obtained from natural resources. They enhance the efficacy of anti-TB agents against M. tuberculosis strains by inhibiting the multi drug transporters of the mycobacteria (Pule et al., 2016). In a report by

Chitemerere and Mukanganyama, (2014) the efflux pumps of Staphylococcus aureus and Pseudomonas

13 aeruginosa were inhibited by a leaf extract from V. adoensis. With an IC50 value that was higher than that of reserpine, a standard EPI, the extract from V. adoensis proved to be a more potent EPI.

Because of this promising finding in previous studies, the potential of leaf extract from V. adoensis to reduce antimycobacterial efflux in mycobacteria was investigated in this study. The efflux pump inhibitory potential of V. adoensis extract was determined whilst comparing its activity to that of reference inhibitor, reserpine. Efflux pump inhibitory activity represents a ground-breaking and promising tool to battle increasing mycobacterial resistance.

1.7 Mycobacteria

The family that mycobacteria belong to is Mycobacteriaceae and the genus is Mycobacterium. Members belonging to the genus Mycobacterium are heritably closer to one another compared to microorganisms belonging to other genera, making identification a complex and challenging job (Tortoli, 2003). With over one hundred and fifty recognized species, members of this genus can be grouped into either fast- growing and slow growing, depending on phenotypic and physiological differences. The mycobacteria fitting in the slow-growing group are more frequently linked with host pathogenicity than those belonging in the fast growing. Pathogens that are members of the M. tuberculosis complex that causes TB, form part of the slow growing group. Those that cause disease conditions other than tuberculosis are called

“non-tuberculous mycobacteria” or NTM (Gopinath and Singh, 2010). The M. tuberculosis complex includes several species, all probably derived from a soil bacterium such as M. tuberculosis, M. caprae,

M. microti, M. mungi, Dassie bacillus, Oryx bacillus (M. orygis), M. surricatae, the attenuated M. bovis

Bacille Calmette–Guerin (BCG) vaccine strain, M. bovis and M. africanum. Except for BCG, these species are obligate parasites, pathogenic and are capable of causing tuberculosis (TB) in mammalian hosts (Silaigwanа et al., 2012; Brown-Elliot et al., 2010). Mycobacteria are slender, curved rods that are 14 acid fast and resistant to acids, alkalis, and dehydration. They are aerobic, non-motile, single celled and non-spore forming bacilli as well (Gupta et al, 2010). The most dominant feature that is distinctive to the

Mycobacteria genus is the complex, lipid-rich cell envelope (Figure 1.9). The mycobacterial cell envelope comprises of an inner layer and an outer layer that surrounds the plasma membrane (Brennan,

2003). The outer compartment comprises of both lipids and proteins. The lipids are usually freely attached to the cell wall, with some long and short-chain fatty acids accompanying the short and long chains found in the inner layer. The arrangement of the mycobacterial cell envelope consists of a plasma membrane, arabinogalactan layer esterified to an uneven mycolate layer, peptidoglycan layer and glycolipid layer. Lipoarabinomannan capped with mannose in addition to a small number of porins cross the width of the mycobacterial envelope (Daffe and Reyrat, 2008). An important feature of the mycobacterial envelope is the lipid layer, which is a thick, hydrophobic waxy cell wall rich in mycolic acids up to fifty percent (Riley, 2006).

Figure 1.9: The mycobacterial cell wall. This diagram shows the main components of the cell wall and their distribution. Mycobacteria are characterized by the presence of a thick mycolate- rich outer layer constituting an extraordinarily efficient permeability barrier. (www.jci.org)

The precise framework of the lipids that connects the interior and outer membrane of the mycobacterial cell envelope is responsible, largely, for its impermeability to drugs, disinfectants and biocides (Jackson,

2014). These chemical structures are also responsible for the exceptional staining properties of mycobacteria that help in the diagnosis of infected samples (Jarlier and Nikaido, 1994). This characteristic of the genus is also responsible for extreme hydrophobicity, acid fastness and resistance to injury such as desiccation. It also owes to the slow growth rate of some species by controlling the uptake of nutrients (Hett and Rubin, 2008).

1.7.1 Mycobacterium tuberculosis

Robert Koch documented Mycobacterium tuberculosis on 24 March in 1882. He was endowed with a

Nobel Prize in physiology and medicine in 1905 (Newton et al., 2000). M. tuberculosis belongs to the M. tuberculosis complex that causes TB, and along with the other species in the same genus, they do not show the biochemical characteristics of gram-negative or gram-positive bacteria. Such that, M. tuberculosis is not categorized as either gram-negative or gram-positive bacteria (Camus et al., 2002).

When gram stain is smeared on M. tuberculosis, its staining is exceptionally poor. Acid- fast bacteria are identified using the Ziehl-Neelsen technique of staining (Figure 1.10). Mycobacterium species are, thus, considered as acid-fast because they are not pervious to some specific dyes and stains. M. tuberculosis has extraordinarily waxy walls, is slow growing and is considered one of the most extremely difficult bacteria to treat. The tubercle bacilli appear as thin and straight or slightly curved rods and according to growth conditions and age of the culture medium; bacilli may differ in morphology from short coccoid bacilli to long filamentous rods (Shepard, 1957).

Figure 1.10: A typical Ziehl-Neelsen stain of mycobacteria. The mycobacteria come out as dark pink straight and curved rods. (Munot et al., 2015).

1.7.2 Non-pathogenic mycobacteria

The Mycobacterium genus contains more than 120 diverse species, with the species dividable into three major groups; the Mycobacterium leprae, non-tuberculous mycobacteria (NTM) and the Mycobacterium tuberculosis-complex (Botha et al., 2013). The NTM group has over 140 diverse species of environmental organisms that reside in soil and water (Velayati et al., 2015). Amongst the NTM are the opportunistic mycobacteria e.g. M. avium and M. simiae and the non-pathogenic (rare) mycobacteria e.g.

M. smegmatis and M. phlei (Rastogi et al., 2001). Non-pathogenic mycobacteria rarely result in disseminating disease, even in immunosuppressed people (Brown-Elliott and Wallace, 2002).

1.7.3 Mycobacterium aurum

Mycobacterium aurum is an environmental Mycobacterium that resides mostly in moist surroundings

(Hartmans et al., 2006). In the laboratory, high-quality growth of the mycobacterial species takes place on solid media forming rounded colonies that have a golden yellow pigment (Figure 1.11). The colonies grow between 1 to 2 mm in diameter, are spherical, dense, and moist (Yokota et al., 1993). M. aurum is gram-positive, acid-fast, a fast grower which has a doubling time of two to three hours (Honarvar et al.,

2012), and is non-pathogenic (Phelan et al., 2015). M. aurum also possesses cell wall and antibiotic susceptibility profiles similar to those of M. tuberculosis (Gupta et al., 2009). The non-pathogenecity and rapid growth of M. aurum have directed to its selection as a surrogate model for the extremely pathogenic

M. tuberculosis in antimycobacterial activity studies of potential anti-TB drugs (Gupta et al., 2009).

Figure 1.11: Colonies of M. aurum growing on solid medium. Viable M. aurum forms orange- pigmented colonies (Tsukamura, 1966).

1.7.4 Mycobacterium smegmatis

Mycobacterium smegmatis is a non-tuberculous causing Mycobacterium that is commonly found in the environment. This mycobacterial species may, however, cause a disease resembling tuberculosis in immuno-compromised individuals (O’Brien et al., 1987). The phylum and genus that M. smegmatis belongs to are Actinobacteria and Mycobacterium respectively. It is an acid- fast bacterial species 3.0 to

5.0 µm long that is known to live in aggregate layers of cells attached to each other in a community called a biofilm (Nguyen et al., 2010). The bacillus shaped bacteria can be stained by the Ziehl-Neelsen technique, and when growing on accessible nutrients, are finely wrinkled and creamy white (Figure

1.12). Usually after 48 hours of growth, the colour of the colonies will change from white to a nonpigmented creamy yellow. The colonies also become waxy due to the unique coating of the cell wall with mycolic acids. The bacteria also range in textures, being smooth, flat and glistening or coarsely folded or finely wrinkled (Gordon and Smith, 1953).

a a b

c d d

Figure 1.12: Mycobacterium smegmatis streak plate (a) coloured electron mic rograph (b) and electron microscopy (c and d) colonies on plates (Science Prof Online, 2014; Stanic, 2013).

1.7.5 Use of M. smegmatis as a model for M. tuberculosis

Mycobacterium tuberculosis is the causative agent of the global tuberculosis epidemic. To fight this successful human pathogen, a better appreciation of the rudimentary biology of mycobacterial pathogenesis and its direct study is vital to understanding its pathogenesis (Russell et al., 2010). On the other hand, the use of this pathogen in the laboratory is labour-intensive for several reasons. To begin with, M. tuberculosis belongs to category 3 of human pathogens. The Advisory Committee on Dangerous

Pathogens (ACDP) classified microbes into four main hazard groups on the following grounds i) infectivity to human beings, ii) hazard to laboratory workers, iii) transmissibility to the community, and

20 iv) availability of effective treatment (www.hse.gov.uk). A category 1 pathogen is one that is most unlikely to result in human disease, whilst a category 2 human pathogen is a pathogen that may result in human disease and may be hazardous to laboratory workers but is not likely to disseminate to the community. A category 3 pathogen is one that may result in acute human disease and present a serious hazard. Such a pathogen may pose a risk of spreading to the community but is treatable. M. tuberculosis being a category 3 pathogen, therefore, requires dedicated biosafety level three laboratory and animal facilities, extensive training before handling, and carries with it a danger of accidental exposure (Alderton and Smit, 2001). Secondly, M. tuberculosis is a slow grower, replicating every 22 hours in liquid medium. As a result, colonies require two to three weeks to form, making each experiment time- consuming (Smith, 2003). However, it is becoming common practice for laboratories to successfully study M. tuberculosis, making use of mycobacterial model systems to fully understand M. tuberculosis virulence mechanisms. The use of these models significantly contributes to knowledge of how M. tuberculosis causes disease (Shiloh and Champion, 2010).

M. smegmatis is particularly useful for the investigative analysis of other species belonging to the genus

Mycobacteria in cell culture research laboratories. It is of interest because it is typically a rapid grower; under favorable conditions it has a doubling time of about 3 hours and colony generation in two to three days (Shiloh and Champion, 2010; Gonzalez-y-Merchand et al., 1998). M. smegmatis is avirulent and so can be safely handled in the laboratory. It as well serves as a good model to understand the physiology of

M. tuberculosis because of the numerous similarities. M. smegmatis shares more than 2000 homologous genes with M. tuberculosis and has the same irregular cell wall structure of M. tuberculosis (King, 2003).

It has the potential to adapt to micro-aerobiosis by altering from active growth to dormant states in the same way that M. tuberculosis does. M. smegmatis can lie dormant in conditions of low oxygen concentrations, surviving for more than 650 days without carbon, nitrogen and phosphorus (Akinola et

21 al., 2013). Both species have similar uses of mycothiol biosynthesis for making an essential thiol that is vital for life. If this thiol is knocked out, the species will be terminated, and a treatment will be found

(Newton and Fahey, 2002). It is, therefore, highly probable that discovery of an antimycobacterial that has activity against M. smegmatis will also have activity against M. tuberculosis.

1.8 Current challenges in treating TB

One of the main challenges in TB control is the length and complexity of treatment. The WHO presently recommends at least six months of treatment for active disease, and twelve months for latent TB (Smieja et al., 1999). The duration of treatment can be trying for patients to adhere to; particularly once they start feeling better. Poor adherence leads to suboptimal treatment response (failure and relapse), the development of drug resistance, and incessant spread of the disease (Karumbi and Garner, 2015).

Drug-associated toxicity is an unfortunate consequence of this tuberculosis treatment owing to the number of antibiotics used and the rather long duration of treatment (Chan and Iseman, 2002).

Occasionally, the harshness of adverse effects experienced by the patient forces the termination of the antibiotic schedule. This then facilitates the development of drug resistant strains of M. tuberculosis (Sarkar et al., 2016; Karumbi and Garner, 2015; Chan and Iseman, 2002). The side-effects of TB medicines include loss of appetite for food, stomach upset, nausea, or vomiting, aching joints, flu- like symptoms, fever, extreme tiredness, light coloured stools or severe diarrhoea, bruises and/or red and purple spots on skin that one cannot explain and skin rash or itchiness amongst others (Mohajan, 2015).

A third challenge associated with TB treatment is the increasing incidence of resistance to drugs. Two main forms of resistance exist; MDR-TB and XDR TB. MDR-TB is resistance to at least rifampicin and isoniazid. Extensively-drug resistant TB (XDR-TB) is resistance to at least rifampicin and isoniazid plus

22 any fluoroquinolone and at least one of three injectable second-line drugs, such as amikacin, kanamycin, or capreomycin (Calligaro et al., 2014). Drug resistant TB is an escalating global problem that is tougher and costlier to treat and cure. These forms of TB can occur when the agents used to treat TB are mishandled or mismanaged (Costa-Gouveia et al., 2017). Mismanagement of drugs includes several factors such as; patients who do not take all the requisite medicines, clinicians prescribing the incorrect treatment, the incorrect dose, or incorrect length of time for taking the drugs, erratic supply of drugs; using poor quality drugs etc. (Mohajаn, 2015). All these factors can cause patient relapse and the spread

TB to others. If a patient stops their treatment prematurely, or takes their medicine at irregular times, the

TB bacteria evolve or mutate and, thus, develop resistance to the TB antibiotics. In a short time, those medicines will not work anymore against the disease. Apart from being expensive, second- line drugs required to treat drug-resistant TB also cause severe adverse effects. For 2 years, it is a must for MDR- patients to take, daily, more than three antibiotics to which the bacteria respond but 40%- 60% of MDR-

TB patients still die in spite of this. HIV infection is the major cause to the rise of TB across the world, with people living with HIV being 20 to 37 times more probable to acquire TB disease during their lifetimes than people who are HIV-negative (WHO, 2010). Of the 9.4 million people who became ill with TB in 2009, an estimated 1.0–1.2 million (11–13%) were HIV positive, with a best estimate of 1.1 million (12%). HIV co-infection with TB creates challenges to effective TB diagnosis. Treatment of patients who have both TB and AIDS raises some crucial issues, which include:

a) Sometimes patients fail to properly absorb the anti-TB drugs, which may intensify the risk of treatment failure, relapses, and acquired drug resistance. b) There are drug-drug interactions that occur that may compromise antiretroviral and /or antituberculosis treatment, in addition to increasing the risk of toxicity.

23 c) Antiretroviral (ARV) therapy reconstitutes CD4 lymphocyte numbers and boosts immune function, patients normally experience a contradictory worsening of symptoms or other manifestations-such as, worsening of infiltrates on chest radiographs, enlarging pleural or pericardial effusions, inflammation on lymph nodes from pre-existent conditions like TB.

d) Risk of relapse in patients is higher.

Evidently there is an imperative need to come up with new and more effective TB drugs, which will not only be active against MDR-TB but will also shorten the length of treatment and treatment with fewer toxic effects (van den Boogaard et al., 2009; Lienhardt et al., 2010).

1.9 The use of traditional medicine as an alternative or complementary medicine

Health is a fundamental human right, and almost every culture has its distinctive herbal traditions, each with its indigenous plants and exclusive practices. In both developed and developing countries, the practices of modern-day medicine exist side-by-side with alternative remedies and traditional approaches

(Briggs, 2014). In some developing economies, herbal remedies and traditional healers are the sole source of available health care for numerous individuals whilst in more developed economies, these methods are used as an optional supplement to modern medicine, motivated by patient inclination

(Birhan et al., 2011). There has been an upsurge in the use of alternative medicines as a form of health care (WHO, 2004), with close to 80% of the inhabitants of low and middle-income countries relying on it for their primary health care needs. Most of this therapy consists of the use of plant extracts, which come in aqueous solution form (Zhang, 2002). Herbal medicines have stood the test of time, with many believing that herbal medicine has better compatibility with the human body, better efficacy and lesser adverse reactions (Pathare and Wagh, 2012).

It is anticipated that roughly one quarter of approved drugs have plant extracts or active ingredients taken from or modeled from plants (Pan et al., 2013; Tripathi and Tripathi, 2003). Significant examples of drugs obtained from medicinal plants include aspirin, digoxin, quinine, morphine, reserpine and vinblastine (Thatoi and Patra, 2011). Despite the remarkable developments made in modern medicine, there are still many ailments for which suitable drugs are yet to be found.

Diseases that were once managed by antibiotics are returning in new leagues, now being resistant to those once effective therapies (Levy and Marshal, 2004). Some of these therapies cause adverse side effects to patients and most of them are costly, being beyond the reach of the average person (Lange et al., 2014).

For this reason, there is a rising interest in the pharmacological evaluation of different plants that are used in traditional systems of medicine. A wide assortment of plants possesses bitter substances that stimulate digestion and have anti- inflammatory chemicals that reduce swelling and pain (Mahomoodally, 2013).

The same plants also have phenolic compounds that work as antioxidants; antifungal and antibacterial tannins that function as natural antibiotics; diuretic substances that assist in getting rid of waste product and poisons; and alkaloids that improve mood and provide sense of well-being (van Wyk & Wink, 2004).

Medicinal plants are available, less costly, safe and efficient to use and do not have side effects, and according to WHO (2012), medicinal plants can be the best source of developing several drugs.

Consequently, plants should be examined thoroughly to better appreciate their properties, safety profiles and efficacy. Phyto-medicines carry unlimited promise in the treatment of infectious diseases such as TB and are potential targets as sources of novel molecular structures that can be employed as lead compounds for new antimycobacterials (Ekor, 2013).

1.10 Plant phytochemicals

Medicinal plants have been the backbone of traditional herbal medicine amongst rural dwellers worldwide since olden days to date (Pan et al., 2014). Over the years they have taken a central stage in modern civilization as natural source of chemotherapy as well as amongst scientists in the search for alternative sources of drugs (Doughari et al., 2009). For a plant to be termed ‘medicinal’, one or more of its organs must contain substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semi synthesis (Demain and Valshnav, 2011). The leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds contain chemical components that are medically active and can be employed in the control or treatment of a disease condition. These non-nutrient plant bioactive components are called phytochemicals (‘phyto-’from Greek - phyto meaning ‘plant’) or phytoconstituents and they protect the plant against microbial infections or infestations by pests (Abo et al., 1991 and

Doughari et al., 2009). Phytochemicals are divided in two major categories; primary and secondary metabolites according to their functions in plant metabolism. The primary metabolites constitutes of substances that are common to living things and essential to cells maintenance that are originated from primary metabolism such as carbohydrates, proteins, chlorophyll and nucleic acids (Vickery and Vickery,

1981). The secondary constitutes of alkaloids, tannins, anthroquinones, steroids, flavonoids, terpenoids and saponins (Maobe, et al., 2013). The secondary metabolites are not directly involved in the normal growth, development, or reproduction of the plant but they are the ones that help the plant to survive in the environment by increasing the fitness of the plant an protecting them against predators (War et al.,

2012).

Research has shown that these secondary metabolites can be used to treat diseases in both animals and humans (Kokwaro, 2009). Different phytochemicals have been reported to have almost all types of medicinal properties which include antioxidant, anti-depressive, anti-inflammatory, antispasmodics, anti-

26 cancer, antifungal, anti-viral, anti-bacterial, anti-malarial, detoxifying, anti-hypertensive, immunomodulatory, clot dissolving, anti-ulcer and many others (Rao, 2003; Mamta, et al. 2013). The following scheme in Fig 1.13 portrays the classes of phytochemicals.

phenols quinones flavonoids

coumarins tannins

volatile oils phytochemicals acids

glycosides steroids

alkaloids lignans terpenoids

Fig 1.13: Major classes of phytochemicals

1.10.1 Classes of phytochemicals

1.10.2 Alkaloids

Alkaloids are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue (Kutchan,

1995). These nitrogenous compounds (Figure 1.14) function in the defense of plants against herbivores

(feeding deterrents) and pathogens (antifungal and antibacterial activities), and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to their potent biological activities. Alkaloids have great antimicrobial activity against bacterial pathogens such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aureginosa and Staphylococcus aureus (Cowan, 1999). They have a wide range of pharmacological activities such as antimalarial, cholinomimetic, antiasthma, vasodilatory, anticancer, analgesic, antibacterial and antihyperglycemic activities (Cushnie and Lamb, 2014). The name alkaloid ends with the suffix - ine and plant-derived alkaloids in clinical use include the analgesics morphine and codeine, the muscle relaxant (+)-tubocurarine, the antibiotics sanguinarine and berberine, the anticancer agent vinblastine, the anti-arrhythmic ajmaline, the pupil dilator atropine, and the sedative scopolamine

(Harvsteen, 1983). Other important alkaloids of plant origin include the addictive stimulants caffeine, nicotine, codeine, atropine, morphine, ergotamine, cocaine, nicotine and ephedrine.

N CH3

Fig 1.14. Structure of a known alkaloid, nicotine

1.10.3 Flavonoids

Flavonoids are polyphenolic compounds widely distributed in nature. Structurally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). They can be divided into a variety of classes such as flavones (e.g., flavone, apigenin and luteolin), flavonols (e.g., quercetin, kaempferol, myricetin and fisetin), flavanones (e.g., flavanone, hesperetin and naringenin), and others

(Saeed et al., 2012). Flavonoids have been reported to exert multiple biological property including antimicrobial, cytotoxicity, anti-inflammatory as well as antitumor activities. The antimicrobial activity works by inhibiting the synthesis of the nucleic acids and tampering with the integrity of the cytoplasmic membrane function and the energy metabolism process (Nazzaro et al., 2013). They induce permeability of the inner bacterial membrane and result in dissipation of the membrane potential of

Gram-negative and Gram-positive bacteria (Cushnie and Lamb, 2005). However, the best described property of almost every group of flavonoids is their capacity to act as powerful antioxidants which can protect the human body from free radicals and reactive oxygen species and reduce their formation by chelating the metals. Flavonoids have been stated to possess many useful activities like enzyme 29 inhibition, oestrogenic, anti-allergic, antioxidant activity, vasodilative, cytotoxic and antitumor

(Havsteen, 1983 and Tapas et al., 2008).

O Fig 1.15. Chemical structure of a flavone (2-phenyl-1,4-benzopyrone)

1.10.4 Phenolics

Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur universally as natural colour pigments and are responsible for the colour of fruits of plants. The most important role phenolics play in plants is defense against pathogens and herbivore predators making them therefore applied in the control of human pathogenic infections (Puupponen-Pimia et al., 2008).

Phenolic compounds from medicinal herbs and dietary plants include phenolic acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, and others (Huang et al., 2009). Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-

30 wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents.

HO O HO OH

Fig 1.16. Chemical structure of a common phenol, gallic acid

1.10.5 Saponins

The term saponin is derived from Saponaria vaccaria (Quillaja saponaria), a plant, which abounds in saponins and was once used as soap. Saponins thus, possess ‘soap like’ behaviour in water, i.e. they produce foam. There are two major groups of saponins, and these include: steroid saponins and triterpene saponins. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give aglycones. They possess a bitter and unpleasant taste, besides causing irritation to mucous membranes. Saponins are extremely poisonous, as they cause haemolysis of blood and are known to cause cattle poisoning (Kar, 2007). They are considered as one of the natural antimicrobial products that make up the defense system of the plants and the antimicrobial activity is attributed mainly to its capability of lysing microorganism’s membranes (Asl, 2008). Saponins are also important

31 therapeutically as they are shown to have hypolipidemic and anticancer activity. The general structure of a saponin in Fig 1.17.

CH3 O-sugar

CH3 CH3 CH3 CH3 H3C HOH2C CH3 sugar-O

Fig 1.17: The general structure of a saponin

1.10.6 Tannins

Tannins are phenolic compounds of high molecular weight and are widely distributed in plant flora.

They are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things into leather. They are used as antiseptics and this activity is due to presence of the phenolic group. Tannin rich medicinal plants are used as healing agents in a number of diseases like leucorrhoea and diarrhea and in the inhibition of growth of many yeasts, fungi, viruses and bacteria (Chung et al., 1998). Some of the bioactive compounds of tannins such as catechin and pyrogallol found in vegetable tannins have been found to be toxic to microorganisms (Cowan, 1999). They are divided in to two groups namely the hydrolysable

32 tannins and condensed tannins (Figure 1.6). Common examples of hydrolysable tannins include theaflavins (from tea), daidezein, genistein and glycitein.

O OH OH O HO OH

HO OH OH

OH OH

Hydrolysable tannins Condensed tannins

Fig 1.18: General chemical structure of tannins

1.10.7 Terpenoids

The fragrance of plants is carried in the essential oil fraction, and these essential oils are the volatile secondary plant metabolites which mainly consist of terpenoids and benzenoids. These oils are highly enriched in compounds based on an isoprene structure and are called terpenes. Their general structure is

C10H16 and they occur as diterpenes, triterpenes and tetraterpenes (C20, C30 and C40), as well as hemiterpenes (C5) and sesquiterpenes (C15) (Ghoshal et al., 1996). Terpenes are among the most widespread and chemically diverse groups of natural products. Examples of commonly important monoterpenes include terpinen-4-ol, thujone, camphor, eugenol and menthol. Diterpenes are classically considered to be resins and taxol, the anticancer agent, is the common example. The triterpenes include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic

33 activity (Taylor et al., 1996). The sesquiterpene acts as irritants when applied externally and when consumed internally their action resembles that of gastrointestinal tract irritant. A number of sesquiterpene lactones have been isolated and broadly they have antimicrobial (particularly antiprotozoal) and neurotoxic action. When the compounds contain additional elements, usually oxygen, they are termed terpenoids. Terpenoids are synthesized from acetate units, and as such they share their origins with fatty acids. They differ from fatty acids in that they contain extensive branching and are cyclised (Cowan, 1999). Terpenenes or terpenoids are active against bacteria, fungi, viruses and protozoa (Taylor et al., 1996 and Ghoshal et al., 1996).

CH3 O O P P O H2C O - O O O Fig 1.19: General structure of terpenoids

1.11 Antioxidants

A free radical is any molecular species that contains an unpaired electron in an atomic orbital and can either accept an electron from or donate an electron to other molecules (Cheeseman and Slater, 1993).

In this way, free radicals can behave as reductants or oxidants and can thus attack important macromolecules in the body leading to homeostatic disruption and damage to proteins, lipids, enzymes and nucleic acids leading to cell or tissue injury (Lobo et al., 2010). A wide range of degenerative diseases including inflammation and cancer are due to these free radicals and oxidative stress. In TB disease excess oxidative stress results in a granulomatous inflammatory disease (Shastri et al., 2018). If

34 the inflammation persists, an alteration of the lung architecture may occur, manifesting as cavitation, fibrosis, or parenchymal lung destruction (Jordan et al., 2010). The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by exposure to oxidants. There occurs depletion of antioxidants during oxidative stress. Free radicals, namely reactive oxygen species (ROS) and reactive nitrogen species (RNS) include various reactive entities namely superoxide O2-, peroxyl (ROO-), hydroxyl (OH-), nitric oxide (NO- ) and peroxynitrite (-ONOO- ) radicals as well as non-free radical species as hydrogen peroxide (H2O2), nitrous acid (HNO2) and hydrochlorous acid (HOCl) (Mavi et al.,

2003).

Antioxidants are bioactive compounds that prevent or delay the oxidation of molecules (Halliwell et al.,

1995). They can be categorized as natural or synthetic antioxidants. Butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, and tertbutylhydroquinine are some of the synthetic antioxidants that are commonly used but of late there have been concerns about their safety

(Pradhananga and Manandhar, 2017). Synthetic antioxidants have been shown to cause liver damage due to their toxicity and carcinogenicity. Because of this, the development of safer antioxidants from natural sources has increased and medicinal plants are a potential source as they are rich in phytochemicals that possess antioxidant activities (Cardozo et al., 2013). In spite of occurrence of antioxidants such as vitamin C and E, the majority of the antioxidant activity of plants may be from compounds such as polyphenols and flavonoids and the activity is considered to be much greater than that of the essential vitamins (Shi et al., 2003). The antioxidant activity of these antioxidant phytochemicals is largely determined by their structures, in particular the electron delocalization over an aromatic nucleus, in those based on a phenolic structure (Tsao and Deng, 2004). Usually, antioxidant phytochemicals possess strong antioxidant and free radical scavenging abilities as well as anti- inflammatory action, which are also the basis of other bioactivities and health benefits. 35

With over 8,000 structural variants, the polyphenols are a group of chemical substances found in plants that are characterized by having aromatic ring(s) bearing one or more hydroxyl moieties. The polyphenols are broadly divided into five classes: flavonoids, phenolic acids, stilbenes, tannins and lignans (Rahman et al., 2006). The structures of flavonoids, stilbenes, lignans and phenolic acids are shown in Fig 1.20. Recent scientific studies have proved that phenolic compounds are capable of protecting cells from free radical damage. Flavonoids and other polyphenols have powerful antioxidant activities in vitro, being able to scavenge a wide range of reactive oxygen, nitrogen, and chlorine species, such as superoxide, hydroxyl radical, peroxyl radicals, hypochlorous acid, and peroxynitrous acid. The antioxidant activity of the phenolic compounds is essentially determined by their structure, in particular the electron delocalization over an aromatic nucleus.

R O O O OH HO OH R B A O

Flavonoid (E)- and (Z)- Stilbene

R O O OH HO OH R B A

Phenolic acid Hydro-benzoic and cinnamic acid Lignan

Fig 1.20: Structures of some known phytochemical antioxidants

When these compounds react with free radicals, the delocalization of the gained electron over the

phenolic antioxidant occurs, and the stabilization by the resonance effect of the aromatic nucleus, which

prevents the continuation of the free radical chain reaction. Furthermore, the significant biological

actions such as subduing oxidative stress, protection from degenerative disease, and reducing risk of

cardiovascular disease could be attributed to their intrinsic antioxidant capabilities (Lee et al., 2017).

OH O O O O -H+

+H+

Antioxidant Free radical Stable compound

Fig 1.21: Typical mechanism of action of an antioxidant

There exists a potential supplemental role for micronutrient and other antioxidants in management of

both drug-sensitive and drug-resistant tuberculosis. The antioxidants may also find a beneficial role in

prevention and treatment of drug toxicity, particularly hepatotoxicity due to anti-tubercular drugs.

1.11.1 Antioxidants and their role in the treatment of TB

Tuberculosis is one such disease that stimulate acute/chronic inflammatory processes that are associated with a high production of chemical mediators leading to creation of free radicals (Gaestel et al., 2009).

Mycobacteria can prompt production of ROS by activating phagocytes, resulting in free radical burst

(McGarvey et al., 2004). Despite the crucial role in host defense played by the phagocytes against mycobacteria, the increased production of ROS gives rise to tissue damage and inflammation (Beers et al., 1952). Patients with advanced tuberculosis display high serum levels of these free radicals and high lipid peroxidation products. The patients are not capable of producing adequate amounts of antioxidants to deal with the elevated oxidative stress in them (Fu et al., 2011; Wiidi et al., 2004). Moses et al.,

(2008) linked the low antioxidant levels to heavy load of free radicals, oxidative stress and lipid peroxidation. For this reason, supplementations with appropriate antioxidant therapy may be necessary to protect TB patients from free radical attack (Reddy et al., 2004). Co-administration of antioxidants and anti- tubercular drugs, therefore, has the possibility of benefiting tuberculosis patients by minimizing oxidative stress and immunosuppression that is brought about by oxidative stress. (Bhattacharyya and

Banerjee, 2011). Oxidative stress that happens in TB patients causes immunosuppression, and immunosuppression in turn increases TB. People suffering from TB are not able to produce sufficient quantities of antioxidants to deal with the elevated levels of oxidative stress in their systems (Wiid et al.,

2004). There have been previous reports of reduced concentrations of vitamin A and of antioxidants vitamins C and E in TB patients. Supplementing antitubercular therapy with antioxidants may be

38 beneficial and may aid in faster recuperation in such cases. Therefore, the discovery of natural antioxidants is presently one of the core topics of rigorous research because the role of antioxidants in tuberculosis is vital. In this study, the plant extracts of V. adoensis were evaluated for their antioxidant potential using the DPPH free radical scavenging assay.

1.12 Antimycobacterial susceptibility tests

Antimycobacterial susceptibility tests are tests used to determine the specific antibiotics certain mycobacteria are sensitive to (Sanchez and Kouznetsov, 2010). The tests are carried out in vitro to evaluate the growth response of the desired organism to a particular extract or drug. The objective of susceptibility assessment is to predict, in vivo or in vitro, the therapeutic outcome of antibiotic or extract therapy (Rex et al., 2001). There are various procedures that are used by microbiologists to investigate the effects of various antimycobacterial agents on the test organisms but the two main methods of investigating the susceptibility of a mycobacterial isolate to an antimicrobial agent. These are disc diffusion and minimal inhibitory concentration (MIC) tests (Tenover, 2009). Any one of several methods, which include agar dilution, broth microdilution or agar gradient dilution, can achieve MIC testing.

1.12.1 MIC determination using the broth microdilution method

The broth microdilution test was the method of choice for antimycobacterial susceptibility testing in this study. This procedure involves subjecting the mycobacterial isolate to a series of concentrations of antimicrobial agents in a broth environment. The concentrations are prepared as two-fold dilutions

(Figure 1.22) of the antimycobacterial agent (e.g. 0.5, 1, 2, 4, 8, 16 µg/ml) in liquid growth medium dispensed in test tubes or microtitre plates (Patel, 2012).

Figure 1.22: Schematic diagram of typical MIC and MBC determination. An organism is cultured in broth and grown to standard density. The organism is exposed to varying concentrations of a serially diluted antimycobacterial agent. The concentration at which there is no visible growth is the MIC and where there is death of cells is the MBC. (Hasan, 2017).

The tubes containing the antimycobacterial agent will be inoculated with a standardised mycobacterial suspension of 1 x 106 CFU/ml. After incubating the tubes or microtitre plates overnight at 37°C, examination for visible mycobacterial growth as evidence by turbidity will then be carried out. The lowermost concentration of the antimycobacterial agent or extract that prevents growth corresponds to the

40 minimal inhibitory concentration (MIC). MIC can also be regarded the lowest concentration of the extract that does not allow any noticeable growth when compared with the control tube (Alade and Irobi,

1993). In this MIC method, cell viability is quantified by the colorimetric MTT assay. A dye named

MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to the wells of the microtitre plate effectively to differentiate between viable and non-viable bacteria as only live bacteria are able to change the dye into an insoluble purple formazan measured at 560 nm (Vipra et al., 2013). This assay measures the reduction of dimethylthiazol diphenyl tetrazolium bromide to formazan by the mitochondrial enzyme succinate dehydrogenase (Ginouves et al., 2014). Dead cells lack the ability to convert MTT into formazan, thus the formation of colour serves as a convenient marker of the viable cells

(Riss and Moravec, 2004). For plant extracts, some authors recommend that MICs of 100 µg/ml and lower should be considered to have significant activity (Rios and Recio, 2005).

1.12.2 Determination of the minimum bactericidal concentration (MBC)

To determine the MBC, test solutions that display no visible growth (that show no turbidity) after

24-hour incubation were sub-cultured onto fresh agar that was not supplemented with drugs. The test solutions are incubated for an additional 24 hours to establish the minimum bactericidal concentration of the extracts needed to kill the mycobacteria. Growth of the test organism on the plate after incubation indicates that the extract or antimycobacterial agent can only inhibit the growth of bacteria (has bacteriostatic effect), whilst the plates that do not exhibit growth after incubation indicate that the extract or agent has the ability to kill bacteria (has bactericidal effect) (Anibijuwon et al., 2010).

1.13 Determination of toxicity in mammalian cells

Compounds derived from natural products have been sources of raw materials for medicine for many

years, with some of the compounds used as drugs, either in their original form or as semi- synthetic forms

(Veeresham, 2012). There has been a great demand for these medicines in primary health care in many

countries because of their purported high therapeutic efficacy, fewer side effects and safety (Sharwan,

2016). Besides being effective, plant derived therapies are also considered to have relatively preferable

safety profiles (Aldaas, 2011). Nevertheless, just because something is natural does not inevitably imply

it is safe or effective. Plant extracts are putative chemicals containing active constituents that are

comparable to those found in purified medications. This implies that they have the same potential to

cause serious adverse effects (Sharwan, 2016). High therapeutic efficacy and minimum side effects are

some of the main concerns in drug development programs, with toxicity testing of the new compounds

being a vital step in the preliminary steps of drug development (Parasuraman, 2011). The antimicrobial

compounds obtained from plants that show no or minimal toxicity to host cells are regarded as prospects

for developing new antimicrobial drugs. Safety is, thus, essential in the preparation of antimicrobials.

For that reason, evaluation of the cytotoxic levels of a candidate antimycobacterial plant will reveal its

safety as a potential therapeutic agent (Morobe et al., 2012). Numerous cytotoxicity assays can be

carried out to confirm whether cell integrity has not been compromised after contact with xenobiotics,

which include; i) Using a tetrazolium salt e.g. MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in

colorimetric assays that measures metabolic activities that depend on the activity of succinic

dehydrogenase and mitochondrial enzymes that become damaged when exposed to toxic compounds

(Morobe et al., 2012; Chiba et al., 1998).

42 ii) Using dyes like propidium iodide and trypan blue. The principle behind the use of these dyes is that

viable cells have intact cellular membranes which exclude such dyes, whereas non-viable cells do not

(Stiefel et al., 2015).

iii) In vitro haemolysis assay using erythrocytes where the release of haemoglobin is an indicator of lysis of

red blood cells subsequent to exposure to the test agent.

Red blood cells of mammals are an excellent model to assess the cytotoxicity of molecules by measuring

cellular damage (Pagano and Faggio, 2015). Erythrocytes are the major cells in circulation that are

responsible for the movement of oxygen; and any modifications to this process could be deadly. If there

is cellular distress resulting in alterations to this structure, there will be signs that indicate damage

(Mohandas and Chasis, 1993). There will be loss of haemoglobin from erythrocytes when the cells are

distressed; therefore, haemolysis is a sign that indicates erythrocyte cell membrane instability (Orsine et

al., 2012; Pagano and Faggio, 2015). Therefore, it can be concluded that the concentration of

haemoglobin found in the supernatant is directly proportional to the number of lysed cells. The

concentration of haemoglobin in the supernatants is measured spectrophotometrically at 540 nm, with

100% haemolysis being defined as the photometric absorption of the supernatant of red blood cells lysed

in water. The in vitro haemolysis assay was the method that was used in this study to investigate the

cytotoxic effects of test plant extract.

1.14 Protein determination

A commonly used endpoint in cellular cytotoxicity testing is based on the interruption of the cellular

permeability barrier (Eisenbrand et al., 2002). This membrane damage can then be estimated by i)

measuring protein release (in the form of nucleosides), ii) dye exclusion tests, iii) the release of

43 intracellular enzymes such as lactate dehydrogenase and iv) measuring pH of the tissue culture medium

(Thelestam and Mollby, 1975; Cho, 2008; Chan et al., 2013). In order to quantify the extent on the damage done to the cell membranes because of the toxicity of the antimycobacterial agent, a protein determination assay can be performed. Numerous protein determination assays can be used which include the Lowry, the Bradford, and the Lowry-TCA to mention a few. In this study, the assay of choice was the Bradford assay (Bradford et al., 1976), a colorimetric method quicker and more sensitive than the

Lowry method. Furthermore, common reagents and non-protein components of biological samples cause less interference when compared with the Lowry method (Kruger, 1994). The interactions between

Coomassie brilliant blue G-250 dye (CBBG) to basic amino acid residues at acidic pH form the basis of the Bradford assay (Redmile-Gordon et al., 2013; Noble and Bailey, 2009). This results in a shift of the absorption maximum of the dye from 465 to 595 nm and thus measures the CBBG complex with the protein.

1.15 Murine macrophages

Macrophages are huge mononuclear cells that form an integral part of the innate and adaptive immune response system, capable of engulfing microbes and other particles. These phagocytes function in controlling and clearing infections and thus, play a crucial role in host defense (Pollard, 2009).

Macrophages are vital in providing immunity to TB with the alveolar macrophages being the chief host cells for M. tuberculosis, since they are the first to encounter the inhaled bacilli. TB infection begins when aerosols containing a small number of M. tuberculosis bacilli are inhaled (Kaufmann, 2001). Upon entering the lung, bacilli are engulfed through phagocytosis by the alveolar macrophages. Activation of the alveolar macrophages by appropriate stimuli results in efficient transferal of the phagocytosed bacilli to the destructive environment of the lysosomes. However, some bacilli evade lysosomal delivery and 44 survive inside the macrophages (Kaufmann, 2001, Russell, 2001). The infected macrophages then either persist in the lungs or are circulated to other organs and tissues in the body. Though, only a marginal percentage of infected people develop tuberculosis (WHO, 2007), because in healthy individuals, the host defense system is robust enough to keep M. tuberculosis in check so that TB disease does not develop.

In susceptible individuals, mycobacteria ingested by alveolar macrophages can subvert the protective mechanisms initiated by macrophages and survive inside macrophages (Ghosh and Saxena, 2004). The reason why a number of pathogens adapted to the environment inside the cell is unknown, but probably it is connected to the accessibility of specific nutrients and because of the protection that the intracellular environment provides against host defense mechanisms. To study the interactions that occur between M. tuberculosis and macrophages, several model systems have been put in place. Ex-vivo assays, using either murine or human macrophages, have been utilized to investigate the intracellular M. tuberculosis killing or sterilizing activity of the test compounds. Murine macrophages can easily be harvested from the mouse peritoneal cavity for use in such assays. M. tuberculosis is a facultative intracellular pathogen capable of surviving and growing within macrophages, therefore, it would be essential to discover a new drug that can penetrate macrophages and inhibit the growth of intracellular bacteria. Anti-TB agents that can also kill bacilli within the granuloma would also probably reduce the duration of treatment and eliminate relapse (Lin and Flynn 2010). In this study, the viability of M. smegmatis cells within murine macrophage in the presence of V. adoensis leaf extract was determined. Extracts from V. adoensis may increase the killing efficiency of macrophages.

1.16 Plants used in this study: Vernonia adoensis (Sch. Bip. ex Walp.)

In this current study, the plant Vernonia adoensis var. kotschyana, Asteraceae (Figure 1.14) plant was selected to evaluate its antimycobacterial activity. Synonyms of the plant name include Baccharoides adoensis (Sch. Bip. ex Walp.) and Vernonia integra. The vernacular name is Musikavakadzi.

The genus Vernonia consists of about a thousand species of herbaceous flowering plants and shrubs and belongs to the Asteraceae family. Vernonia species are found mainly in grassland habitats that have scattered trees, in miombo woodlands and frequently alongside streams. The herbaceous shrubs are prevalent in areas extending from Nigeria and Ethiopia through to the Democratic Republic of Congo and

East African countries like Kenya and downwards to Zimbabwe. V. adoensis is used by various communities to treat numerous illnesses due to lack of resources to access hospitals or even due to inclination of the use of medicinal plants (Swamy et al., 2013). People in various parts of the world grow

Vernonia as a food vegetable and as a culinary herb in soups as well (Nwosu et al., 2013).

People from Nandi and Kamba in Kenya use V. adoensis for oral health, whilst others from the Rift

Valley and Western part of Kenya use the plant roots for treating gonorrhoea (Muhindi et al., 2016). The plant roots are used primarily for the treatment of gonorrhea whilst the decoction of the roots mixed with the bark of other trees is used as a remedy for kidney and heart problems (Kokwaro, 2009 in Swamy et al., 2013). A boiled leaf decoction is also drunk as a medicine to cure tuberculosis (Kisangau et al.,

2007). The Asteraceae is the biggest family of flowering plants (Scott et al., 2004). Members in this family are known for their great diversity in phytochemicals such as flavonoids, polyphenols and diterpenoids, which are responsible for the pharmacological activity (Jisaka et al., 1993; Koc et al., 2015).

Scientifically, the Asteraceae family has been established to possess phytochemical principles, which 46 comprise vernodalin, bitter sesquiterpene lactones, vernomygdin and vernolepin. The sesquiterpene lactones are ascribed to have immunostimulant properties (Hamburger and Hostettmann, 1991). In fact, it has been claimed that most of the traditional remedies used by native communities come from plants belonging to the Asteraceae family (Green, 1991; Rashid et al., 2018). The Asteraceae family is of importance to this study because of their purported use in the treatment of tuberculosis and related ailments. Plants such as Conyza podocephala, Conyza ulmifolia, Eriocephalus africanus have been used traditionally to treat coughs, chest complaints, fevers and respiratory disorders (Lall and Meyer, 1999).

Helichrysum odoratissimum and Helichrysum nudifolium have also been used to treat TB (Scott et al.,

2004).

Figure 1.23 Picture of Vernonia adoensis taken in Centenary, Zimbabwe. (Stanley Mukanganyama)

The Asteraceae (Compositae), is a large family of flowering herbs, shrubs and trees (Angiospermae), that has about 1,620 genera and over 23,600 species. Vernonia is one of the largest genera in the Asteraceae family, with over 1 000 species of forbs and shrubs known for having food, medicinal and industrial uses

(Aobuli et al., 2018). An ethnomedical survey of Vernonia species revealed extensive and assorted medical usage. Usually, this species is used in the treatment of parasitic and infectious diseases. The infectious diseases range from those affecting the skin to gastrointestinal infections. Other main applications include treatment of diabetes, bacterial infections, respiratory diseases, urinary tract infections, gynaecological diseases and complications and venereal diseases (Farombi and Owoeye,

2011). Vernonia amygdalina is the most studied member of the Vernonia genus as well the most prominent specie (Johri and Singh, 1997). Its full binomial name is Vernonia amygdalina Del. V. amygdalina Del, commonly known as bitter leaf, is a small shrub or tree that can grow up to 3 meters which grows predominantly in tropical Africa especially in Nigeria, Zimbabwe and South Africa (Igile et al., 1995; Erasto et al., 2006). It is domesticated in parts of West Africa such as Guinea, Niger, Senegal,

Sierra Leone, Togo (Orwa et al., 2009). The leaves have a distinctive odour and bitter taste but can used as soup condiments after washing and boiling to get rid of the bitter taste (Akpaso et al., 2011). Some tribes regard V. amygdalina as a wonderful gift from God to mankind because of its numerous medicinal values which include cure for stomach-ache, skin infections, diabetes, insomnia, tooth ache, acne, pneumonia, stroke, arthritis, fatigue, cough and bleeding (Habtamu and Melaku, 2018).

It is highly improbable that a single molecule could be responsible for such diverse activities; instead multiple molecules, working alone or in combination with others, are much more likely to be responsible for each of these biological activities. Phytochemicals such as saponins and alkaloids, terpenes, steroids, coumarins, flavonoids, phenolic acids, lignans, glycosides, triterpernoids, xanthones, anthraquinones, 48 edotides and sesquiterpenes have been extracted and isolated from V. amygdalina (Farombi and Owoeye,

2011; Luo et al., 2017; Erasto et al., 2006). Sesquiterpene lactones (vernodalinol, vernolepin, vernomygdin, hydroxyvernolide, vernolide and vernodalol) have been reported to possess antimicrobial properties, bactericidal activity against gram bacteria and inhibit breast cancer cell growth (Jisaka et al.,

1993; Luo et al., 2017). Isolated vernoniosides from V. amygdalina leaves exhibited anti-inflammatory activity. Flavonoids, tannins, saponins, and triterpenoids had been studied to possess antioxidant and

(Erasto et al., 2007; Alara et al., 2017).

Vernonia cinerea also known as little iron weed is a perennial grass that grows mainly in China, India,

Bangladesh, Srilanka, New Zealand, Asia and Africa (Dhanalakshmi et al., 2013). The roots and leaves of this plant is are known to cure fever, hiccups, kidney disease and stomach discomfort (Syed et al.,

2011). The plant has antibacterial, antimicrobial, antihelmintic, antitumour, antioxidant and antihyperglycemic effects and is used to treat nerve disorders and is also a potent analgesic (Lakshmi, 2015;

Thiagarajan et al., 2014). Another Vernonia species which is important in Madagascar is Vernonia polytricholepis which has been widely used for treating fever and respiratory problems (Aliyu et al.,

2011). Asteraceae family plants have been found to possess diverse biological effects referring to in vivo and in vitro researches conducted.

Active primary and secondary metabolites found in one genus in a family will exhibit largely similar active principles. Past studies reported several plants with promising anti-tubercular activity with the most reported activity in the plant families of Asteraceae, Apiaceae, Fabaceae and Lamiaceae and among others (Sanchez and Kouznetsov, 2010; Gautam et al., 2007). It has been reported that Vernonia amygadlina has antioxidant (Odukoya et al., 2007), hepatoprotective (Iwalokun et al., 2006) properties. 49

In this light, V. amygadalina and V. adoensis are in the same genus therefore it can be expected that V. adoensis will have high activity due to the similarity of active principles. Basically, this project investigated the antimycobacterial, antioxidant as well as other pharmacological properties of Vernonia adoensis against M. smegmatis. The results obtained may be beneficial in providing an ethnopharmacognostical approach intended for drug discovery and advancement as well as establish the scientific basis for some of their therapeutic properties in folkloric use.

1.17 Rationale of study

Failure to curb the communicable infection from M. tuberculosis has attracted and continues to attract the attention of governments, microbiologists, researchers, health organizations and the entire public worldwide. The current anti-TB regimen makes use of a cocktail of drugs that were developed mostly in the 1940s and 1980s (Shehzad et al., 2013; Sloan et al., 2013). Treatment is successful for the drug susceptible strains of M. tuberculosis when the advised treatment protocols are observed. However, correctly prescribing and obeying these complex and prolonged protocols is difficult, and the coming of

M. tuberculosis strains resistant to multiple drugs, and the drug-drug interactions that interfere with the treatment of TB and HIV coinfection has created an irresistible need for enhanced TB therapies (Lange et al., 2014).

The present decade has witnessed a resurgence of TB drug research and development, stimulated by an urgent necessity to reduce the upsurge of the disease worldwide and come up with novel, potent treatments that are more effective against drug-susceptible and resistant strains. The main purposes for the current drug researches would be to shorten and reduce the complexity of the treatment of active and latent TB infections, offer safer and more effective treatments for drug- resistant TB and make simpler the

50 treatment of TB/HIV co-infections by eradicating arduous drug-drug interactions (Ginsberg and

Spigelman, 2007).

The urgency for enhanced treatments is motivated by the fact that universally; TB is not being managed effectively with the existing available treatment, particularly in areas where there is limited public health infrastructure, high HIV prevalence, or both (Msuya, 2005; Vitoria et al., 2009). Due to lack of access to proper healthcare, in rural areas particularly, people treat infectious diseases such as TB and HIV using a variety of plants without any systematically documented information (Flint, 2015). In this study, the antimycobacterial activities of the herbal medicine V. adoensis, which is available in Zimbabwe, were examined. The assessment of the biological activity of such plants is essential, as it could be a possible lead for new drugs or herbal preparations. This study would provide valuable information for the effectiveness of the plant extracts, the modes of action against mycobacterial species and its cytotoxicity.

1.18 Research question

Do Vernonia adoensis plant extracts have antimycobacterial activity against Mycobacterium smegmatis.

Chapter Two: OBJECTIVES

2.1 Main objective

The main aim of the study was to investigate the antimycobacterial potential of V. adoensis

against a model Mycobacterium species, M. smegmatis.

2.2 Specific objectives

The specific objectives were:

(a) To establish the minimum inhibitory concentration (MIC) of the plant extracts and minimum

bactericidal concentration (MBC) of the plant extracts.

(b) To examine the effect of the most potent extract on drug transport pumps by carrying out transport assays

on accumulation of ciprofloxacin in mycobacterial cells.

using the protein leakage and nucleic acid leakage assays.

(d) To determine the free radical scavenging capacity of the most potent extract.

(e) To establish the levels of toxicity in the most potent extract by detecting haemolysis in sheep

erythrocytes.

(f) To investigate the effect of the most potent extract on the survival of mycobacteria in murine

macrophages.

2.3 Design of study

Plant collection and authentication

Preparation of plant extracts

Qualitative and quantitative phytochemical analysis

Culturing of mycobacteria

Testing of extracts against mycobacterial species using the broth microdilution assay

Determination of the MIC and MBC of the extracts

Determination of effect of most potent plant extract on drug transport in M. smegmatis

Determination of effect of plant extract on cell membrane permeability using the nucleic acid leakage assay

Determination of effect of plant extract on cell membrane permeability using the protein leakage assay

Antioxidant assay

Haemolysis assay

In vitro determination of viability of mycobacteria inside macrophages

Phytochemical screening tests for the active ethyl acetate fraction of V. adoensis

Statistical analysis

Chapter Three: METHODOLOGY

3.1 Materials

Chemicals namely; Potassium ferricyanide, Bradford reagent, sodium bicarbonate, potassium chloride, disodium hydrogen phosphate, hydrochloric acid, reserpine, dimethyl sulphoxide (DMSO), 2,2-diphenyl-

1-picrylhydrazyl (DPPH), Middlebrook 7H9, barium chloride, 3-(4,5- dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium bromide (MTT), casein acid hydrosylate, ascorbic acid, Middlebrook 7H10, D- glucose, rifampicin, potassium dihydrogen phosphate, glycine, sodium azide, disodium hydrogen phosphate and ciprofloxacin were all procured from Sigma- Aldrich Company (Taufkirchen, Germany).

All supplementary chemicals used were of superior quality and were attained from numerous sources. M. smegmatis mc2 155 was used as a model organism for M. tuberculosis. The mycobacteria were a charitable contribution from Prof. D. Steenkamp of the Department of Clinical Laboratory studies,

University of Cape Town, South Africa. The mycobacterial strain was maintained as stock strains in 50% glycerol in Eppendorf® microtubes and kept at -34 °C until use.

3.2 Methods

3.2.1 Collection of sample

Vernonia adoensis plant was collected in Centenary, (Geographic coordinates, Latitude:

16º43’22'’ S, Longitude: 31º06’52'’ E, Elevation above sea level: 1 156 m, Mashonaland Central of

Zimbabwe Province) and was authenticated by a taxonomist from the National Herbarium and

Botanic Gardens in Harare, Zimbabwe, Mr. Christopher Chapano. Herbarium samples (sample number C1 E7) were kept at the University of Zimbabwe, Department of Biochemistry.

3.2.2 Preparation of extracts

The plant parts; the leaves, flowers, roots and root bark material were dried in an oven at 40℃ and ground to a powder in a Philips electric blender (Philips, Malaysia). Solvents of different polarity were used to perform serial exhaustive extraction on the plant parts in sequence of increasing polarity were hexane, dichloromethane (DCM), acetone, ethyl acetate, ethanol, 70 % ethanol, methanol and distilled water. To 10g of the powdered plant part, 100ml of solvent was added and the mixture was left overnight stirring on a stirrer. Filtration was carried out using a Whatman filter paper No. 1 (Sigma-Aldrich, Taufkirchen, Germany) and the filtrate was concentrated using a rotary evaporator left to evaporate in a fume hood under an air stream. A constant dry weight for each of the extracts was obtained and the residues were put aside at 4 ℃ until they were required.

3.2.3 Qualitative analysis of V. adoensis plant extracts

Preliminary phytochemical analysis for alkaloids, flavonoids, phenols, saponins, tannins and terpenoids were made by following the methods of Prabhavhati et al., (2016) with modifications.

3.2.3.1 Test for Flavonoids

A few drops of 20% sodium hydroxide to 2 ml of each extract. The formation of an intense yellow colour was observed. To this, a few drops of 70% dilute hydrochloric acid were added and yellow colour was disappeared. Formation and disappearance of yellow colour indicated the presence of flavonoids in the sample extract.

3.2.3.2 Test for Alkaloids

To 1 ml of each extract, 1 ml of marquis reagent, 2 ml of concentrated sulphuric acid and a few drops of 40% formaldehyde were added and mixed, appearance of dark orange or purple colour indicated the presence of alkaloids.

3.2.3.3 Test for Saponins

To 2 ml of each extract, 6 ml of distilled water were added and shaken vigorously; formation of bubbles or persistent foam indicated the presence of saponins.

3.2.3.4 Test for Tannins

To 2 ml of each extract, 10% of alcoholic ferric chloride was added; formation of brownish blue or black colour indicated the presence of tannins.

3.2.3.5 Test for Phenols

To 2 ml of each extract, 2 ml of 5% aqueous ferric chloride were added; formation of blue colour indicated the presence of phenols in the sample extract.

3.2.3.6 Test for Terpenoids

Take 1 ml of extract of each solvent and add 0.5 ml of chloroform followed by a few drops of concentrated sulphuric acid, formation of reddish-brown precipitate indicates the presence of terpenoids in the extract.

3.2.4 Quantitative phytochemical analyses of V. adoensis plant extracts

Phytochemical screening of the crude extracts was performed using standard procedures.

Quantitative evaluation of saponins was carried out using the method of Obadoni and Ochuko,

(2001). A mass of 3. 60g of blended plant material powder was weighed and extracted using 30 ml of 75% ethanol. The extract was re-extracted with 100 ml of ethyl acetate and the organic solvent was then evaporated. The content of saponin in the extract was measured as a percent of the original sample weight.

The method of Harborne (1973) was employed to quantitatively determine alkaloids present in the plant samples. The sample was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added. The beaker was covered and allowed to stand for 4 h. The sample was filtered, and the extract was concentrated on a water bath to one quarter of its original volume.

Concentrated ammonium hydroxide was added drop wise to the extract until precipitation was complete. The whole solution was allowed to stand until it settled. The precipitate was easily collected from the solution and was washed with dilute ammonium hydroxide and filtered. The residue was the alkaloid which was weighed after complete dryness and the percentage was calculated.

% Alkaloid = Weight of alkaloid x 100

Weight of sample

Determination of the tannin content was carried out using the method of Van- Burden and

Robinson, (1969), with modifications. The sample and distilled water were together added to a

500 ml conical flask and the resulting solution was put in a shaker. After 1 hour, the next step was filtration of the solution. The obtained filtrate was made to the mark with water in a 50 ml volumetric flask. The solution was put in a clean test tube and 0.1 M FeCl3 in 0.1 N HCl and

0.008 M potassium ferrocyanide was added. Optical density was measured at 605 nm using a spectrofluorometer (Shimadzu RF-1501, Shimadzu Corporation, Kyoto, Japan) within 10 minutes of adding 0.1 M FeCl3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. Tannic acid dilutions

(0 to 0.5mg/ml) were used as standard solutions. The results of tannins are expressed in terms of tannic acid in mg/ml of extract.

To estimate the quantity of flavonoids, the method of Boham and Kocipai-Abyazan (1994) was used. Ten grams of the plant sample was extracted three times with 100 ml of 80% aqueous methanol at room temperature. Whatman No. 1 filter paper was used to filtrate the solution, and the obtained filtrate was placed in a crucible and the solvent was evaporated to dryness to attain a constant mass of the extract.

The quantity of total terpenoids was estimated using the method by Ferguson (1956) in Biradar and Rachetti (2013). Dried plant extract 10 g (W1) was taken and soaked in 90 ml of ethanol for

24 hours. After filtration, the filtrate was extracted with 10ml of petroleum ether using separating funnel. The ether extract was separated in pre-weighed glass vials and waited for its complete drying (W2). Ether was evaporated and the yield (%) of total terpenoids contents was measured by the formula :

(W1-W2/W1 ×100).

3.2.5 Culturing of Mycobacterium smegmatis

The medium for culturing mycobacteria, Middlebrook 7H9, was prepared by supplementing 5.2g of Middlebrook 7H9 base with 1g casein acid hydrosylate and the mixture was made up to 1 litre using distilled water previously boiled to 96 ℃. The media was autoclaved to sterilize it, and 20 ml of this solution was placed in a culturing tube and inoculated with M. smegmatis. In a separate tube, 20 millilitres of the media without inoculum was employed as the sterility control. The tubes were incubated in an incubator (Lab Companion IS 300, Jeio Tech, Korea) overnight at 37 ℃ and the following day after 24 h, the tubes were checked for growth. Mycobacteria were enumerated by determining the optical density at 600 nm using a spectrofluorometer Shimadzu RF-1501,

Shimadzu Corporation, Kyoto, Japan), and comparing it with McFarland turbidity standards (0.1

OD 600, 1.5 × 108 cfu/ml). The concentration of the cells was diluted to 1 x 106 cfu/ml for use in antimycobacterial susceptibility assays.

3.2.6 Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal Concentration (MBC) of plant extracts

To determine the MICs of all the plant extracts, fractions and rifampicin against M. smegmatis, the microbroth dilution method as described by McGaw et al., (2008) with modifications was used.

Serial dilution of the extracts was made from 1 000 µg/ml with media to 2 µg/ml. A volume of

100 µl of the extracts was placed in duplicate wells of a 96- well microtitre plate and to the first double rows of the serially diluted compound, no inoculum was added. Rifampicin, a standard anti-TB drug, was used as the positive control, and was serially diluted down from a concentration of 50 µg/ml to 0.1 µg/ ml. A concentration of 2 x 106 cfu/ml of M. smegmatis in 100 µl aliquots was transferred to the wells, except for the sterility control wells where only media was added.

The microplate was covered and sealed with Parafilm ® (Sigma-Aldrich, Taufkirchen, Germany) before being put in a plastic container lined with absorbent paper saturated with sterile water. The container was wrapped in aluminum foil and incubated overnight in a Lab Companion IS 300 incubator (Jeio Tech, Korea) at 37 °C. MTT was added to each well in aliquots of 30 µl. A Tecan microplate reader (Grodig, Austria) was used to read the optical densities of the wells at 590 nm.

Inhibition of mycobacterial growth was indicated by the concentrations of the plant extract that did not change in colour after the addition of MTT while the wells which showed a change in colour from clear yellow to purple indicated Mycobacterium growth. Middlebrook 7H10 agar was used to establish the MBCs of the compounds and from the wells that did not indicate detectable growth and the controls, aliquots were streaked onto solid agar using a sterile loop. The agar plates were incubated overnight and then checked for growth the following day to establish the MBC of each compound.

3.2.7 Determination of effect of V. adoensis on drug transport in M. smegmatis cells

To investigate the effect of the most effective plant extract on Mycobacterium efflux pumps, accumulation of the fluoroquinolone, ciprofloxacin, inside M. smegmatis cells was carried out.

The methods described by Nyambuya et al., (2017) and Mortimer and Piddock (1991) were performed with some modifications. Three separate flasks containing Middlebrook 7H9 media augmented with casein hydrosylate were inoculated with M. smegmatis and incubated for 48 hours at 37 °C. The cells that grew were centrifuged at 3000 rpm in a Rotofix 32 centrifuge (Hettich

Zentrifugen, Tuttlingen, Germany) for 10 minutes in pre-weighed tubes then the supernatant was thrown away. The harvested mycobacteria were washed two times with 50 mM sodium phosphate buffer (PBS) (pH 7.0). After being washed and weighed, the obtained cells were made up to 40 mg/ml using 10 mM PBS containing sodium azide. This mixture was incubated for 15 minutes at

37 °C after which ciprofloxacin at a concentration of 20 µg/ml was added. The sample was incubated at 37 °C with shaking at 120 rpm for 60 minutes and then divided into two aliquots: tube A containing 1/3 of the suspended mixture and tube B containing 2/3 volume of the sample mixture. Tubes A and B were centrifuged for 5 minutes at 3000 rpm. For tube A, after the supernatant was thrown away, the pellet was weighed and made up to 40 mg/ml by adding PBS.

Tube A represented a sample without glucose. To tube B, a concentration of 40 mg/ml of the pellet was made by adding PBS containing 1 M glucose. Tube B was further sub-divided into 2 equal aliquots, one containing reserpine and the other, the most powerful plant extract from V. adoensis. Reserpine is an alkaloid widely utilized in vitro as an efflux pump inhibitor (EPI)

(Garvey and Piddock, 2008). The activity of the extract as a potential EPI was compared to the activity of reserpine. All the prepared tubes were mixed by gentle vortexing before being incubated for 30 minutes at 37 ℃ with shaking at 120 rpm. The cells inside the tubes were washed using cold PBS and re-centrifuged in a Rotofix 32 centrifuge at 4000 rpm for 10 minutes.

After discarding the supernatant, 3 ml of 0.1 M glycine hydrochloride (pH 3.0) was added to resuspend the cells. The contents were mixed to ensure maximum contact of the cells with the lysis buffer before incubation at 37 ℃ for 24 hours. After that, centrifugation was carried out at

3000 rpm for 10 minutes. The supernatant was transferred to clean separate tubes and centrifuged again for a further 5 minutes. The amount of ciprofloxacin was determined using an RF-1501

Shimadzu spectrofluorometer (Shimadzu Corporation, Kyoto, Japan), at excitation and emission on wavelengths of 270 nm and 452 nm respectively. A standard curve of fluorescence versus the concentration of ciprofloxacin was used to enumerate the amount of the drug in the supernatants.

To determine if there were any interferences from reserpine and the extract, the fluorescence of

61 reserpine and extract was also measured at the same excitation and emission wavelength as ciprofloxacin.

3.2.8 Determination of effect of V. adoensis extracts on cell membrane integrity using the nucleic acid leakage assay

The method described by El-Nakeeb et al., (2011) with modifications, was used to investigate the effect of extract on the bacterial membrane. Mycobacteria were grown and adjusted to an optical density at 600 nm of 1.5 using saline solution. The cell suspension was exposed to the extracts at

MIC, 2 x MIC and 5 x MIC concentrations for a period of 10 minutes. One millilitre of the mycobacterial mixture was centrifuged for 1 minute at 11000 x g (Centrifuge 5415C, Berlin,

Germany). Saline solution was used to wash the pellet and a volume of 3 µl of propidium iodide was added to the mixture which was then kept in darkness for 10 minutes. An F max spectrofluorometer (Molecular Devices, Sunnyvale, USA) at excitation wavelength 490 nm and emission 635 nm was used to measure the fluorescence. The control was the cells not exposed to plant extract.

3.2.9 Determination of effect of V. adoensis extract on integrity of the cell membrane using the protein leakage assay

The method by Kruger (1996) with modifications was used to determine protein leakage. M. smegmatis was sub-cultured in 20 µl of Middlebrook media in a 50 ml centrifuge tube and left to incubate for 18 hours at 37 ℃ with shaking at 120 rpm. The cells were then diluted with 0.9 % saline to (OD 600) = 1.5. Five centrifuge tubes were labelled A, B, C, D and E, and to each respective tube, 6 ml of cells were added. To tubes A, B and C, a final concentration of 63 µg/ml

(MIC), 126 µg/ml (2 x MIC) and 250 µg/ml (MBC) of extract were added. To tubes D and E, sterile water and 0.1 % SDS were added respectively. The tubes were incubated for 2 hours at 37

℃ with shaking in a Lab Companion SI-300 incubator (Jeio Tech, Korea) and then centrifuged for

4 min at 3 500 rpm. After disposal of the pellets, the supernatants were used for the protein determination assays. The positive control was Tube D containing cells only and tube E containing 0.1 % SDS was the negative control. From each tube mentioned above, 0.1 ml was withdrawn and dispensed in sterile test tube. The method as described by Bradford (1976) was employed to determine the protein leaked in cells that were exposed to extract. To each tube, 3 ml of Bradford reagent was added, and the mixture mixed gently. Incubation of the samples was done for 15 minutes at room temperature. The samples were placed in cuvettes and the absorbances measured at 595 nm. The protein-dye complex is stable for up to 60 minutes only so the recording of the absorbances of the samples was done before the 60-minute time limit. A calibration curve of concentration 0-500 µg/ml was created for bovine serum albumin (BSA) versus absorbance to approximate the protein concentration in the unknown.

3.2.10 Determination of the free radical scavenging potential of V. adoensis extract using the

DPPH antioxidant assay

The free radical scavenging ability of V. adoensis leaf extracts against 2, 2-diphenyl-1- picrylhydrazyl (DPPH) radical was investigated using the methods of Mannan et al., (2013) and

Hsu et al., (2003). The antioxidant activity of the extracts was compared with that of ascorbic acid. Two millilitres of various concentrations (25 to 275 µg/ml) of extract were mixed with 3 ml of 0.1 mM DPPH solution. After vigorous shaking, the mixture was incubated in the dark at room temperature for a period of 30 min. The absorbances of the solutions were then measured at 517 nm using a UNICO 2100 Spectrophotometer (United Products and Instruments, Inc. New Jersey,

USA). The following formula was used to calculate the percentage of DPPH radical scavenging activity: ((A0 - A1)/A0) x 100 where, A0 is the absorbance of a DPPH solution without tested sample and A1 is the absorbance of the tested sample. All measurements were done in triplicate and data was presented as mean ± standard deviation (SD). Finally, the percentage DPPH radical scavenging activity was plotted against respective concentrations used and IC50 was calculated from the graph.

3.2.11 Haemolysis assay

The method according to Noudeh et al., (2010) with modifications, was used to carry out the haemolysis assay. Blood was obtained from a full-grown sheep from the Animal House at the

University of Zimbabwe. Sheep blood (50 ml) was aseptically collected and placed in the flask containing an equal volume of Alsevier solution. After centrifuging the blood solution for 10 min at 3000 rpm, the supernatant was discarded, and the erythrocytes washed 3 times with PBS buffer

(pH 7.2) by centrifuging in a Hettich Rotofix 32 centrifuge (Tuttlingen, Germany) for 5 min at

4000 rpm. The volume of PBS buffer used for washing the erythrocytes was five times the volume of the erythrocytes. After washing, the cells were diluted four-fold with PBS and the resultant suspension was used in the determination of haemolysis. The erythrocyte suspension

(200 µl) was incubated with 200 µl of the V. adoensis leaf extract in PBS (pH 7.2) at 37 °C for 60 min. Post incubation, the tubes were centrifuged at 3000 rpm for 60 sec. Three millilitres of

Drabkin's reagent were then added to the resultant supernatant. The amount of haemoglobin liberated from the samples was determined at 590 nm using a Tecan Genios Pro microplate reader

(Grödig, Austria). The positive control was made up of 200 µl each of PBS buffer and uncentrifuged erythrocyte suspension, which was added to 3 ml Drabkin's reagent to obtain a

64 value for total (100%) haemolysis. Additionally, a negative control, included to measure the level of spontaneous hemolysis, was comprised of 200 µl PBS buffer mixed with 200 µl of the supernatant from centrifuged erythrocytes, and added to 3 ml of Drabkin's reagent. Haemolysis percentage for each sample was calculated using the following the equation:

Percentage haemolysis = (sample absorbance / positive control absorbance) X 100

3.2.12 In vitro determination of viability of mycobacteria inside macrophages

To determine if treatment with V. adoensis leaf extract increased the killing efficiency of macrophages, 1 x 105 mouse peritoneal cells were treated with the leaf extract 2 h before and 2 h after M. smegmatis infection at a multiplicity of infection of 10. The cells were termed as 'pretreated' and 'post-treated'. The concentrations of extract used were 32 µg/ml (1/2 MIC), 63 µg/ml

(MIC) and 126 µg/ml (2 x MIC). After treatment, the cells were washed three times with 1 x PBS to remove extracellular bacteria. Macrophages that were infected with mycobacteria were used as a control. After washing, the cells were lysed with 0.05% SDS after 3 hours of incubation.

Serially diluted cultures were plated on 7H10 medium in order to determine the intracellular survival. Enumeration of the cells was done the following day.

3.2.13 Phytochemical screening tests for the active ethyl acetate fraction of V. adoensis

Qualitative chemical screening was carried out to identify the presence of alkaloids, tannins, flavonoids, saponins, phenols and terpenoids according to Prabhavathi et al., (2016) as described earlier.

3.13 Statistical analysis

Statistically significant differences among the mean values were determined by one-way analysis of variance (ANOVA). A Dunnet's post-test was used to determine the mean values that were significantly different from the control values. All graphical and statistical computations were performed using Graph Pad Prism 6® software (Version 6.0, Graph Pad Software Inc., San Diego,

California, USA).

Chapter 4: RESULTS

4.1 Qualitative phytochemical analyses of V. adoensis plant extracts

The qualitative analysis of secondary metabolites revealed the presence of several

phytoconstituents in V. adoensis plant (Table 4.1).

Table 4.1 Qualitative phytochemical tests on Vernonia adoensis plant parts

Name of test Leaf Root Root bark Flower

Flavonoid test + + + +

Saponin test + + + +

Alkaloid test + + _ _

Terpenoids test + + _ _

Tannins test + + _ _

Key: (+) present (-) absent

4.2 Quantitative phytochemical analyses of V. adoensis plant extracts

The quantitative analysis of secondary metabolites revealed the presence of several phytoconstituents in V. adoensis plant. The following phytochemicals were found present in the four assayed plant parts; tannins, flavonoids, alkaloids, saponins and terpenoids. Tannins, alkaloids and terpenoids were, however, not present in the root and root bark of V. adoensis. The formula:

Percentage yield (%) = Weight of extract residue (g) x100 % / Mass of starting plant material (g)

67 was used to calculate the percentage yield for the various fractions.

The percentage yield of the fractions are shown in Table 4.2.

Table 4.2 Percentage yields of fractions of V. adoensis plant parts

Phytoconstituent Leaf Flower Root Root bark class Flavonoids 5.61 7.74 12.54 11.45

Alkaloids 2.81 1.62 - -

Saponins 9.56 11.2 8.44 1.14

Tannins 1.78 0.13 - -

Terpenoids 2.77 0.98 - -

4.3 Antimycobacterial susceptibility tests: Determination of MIC and MBC of V. adoensis plant extracts against M. smegmatis cells

The antimycobacterial potential of V. adoensis solvent extracts, flavonoids, saponins, alkaloids, terpenoids and tannins was determined by the broth microdilution method. Of the 52 plant extracts and fractions (Table 4.3 and Table 4.4), only 8 managed to show noteworthy antimycobacterial activity against M. smegmatis at concentrations of 1 000 μg/ml or less.

Table 4.3 Summary of the MICs of the crude extracts of different plant parts of V. adoensis prepared from solvents of varying polarity

Solvent Leaf extract Flower extract Root extract Root bark extract Hexane 500 µg/ml - - -

Dichloromethane - 500 µg/ml - -

Acetone - 250 µg/ml - -

Ethyl acetate 63 µg/ml - - -

Ethanol - - - -

70 % Ethanol - - - -

Methanol - - - -

Water - - - -

Key: (-) did not show activity

Table 4.4 shows the activities of the fractions extracted from V. adoensis plant.

Table 4.4 Summary of the MICs of the fractions extracted from V. adoensis

Fraction Leaf extract Flower extract Root extract Root bark extract Alkaloid 125 500 n/a n/a

Saponin - - - -

Terpenoid - 250 n/a n/a

Flavonoid - - - -

Tannins - - n/a n/a

N/A standing for absence of phytochemical in the plant

Table 4.5 summarises the results of the antimycobacterial activities of the most active extracts that were prepared from V. adoensis plant parts.

Table 4.5: Antimycobacterial activity of the active extracts and fractions of Vernonia adoensis

Extract/fraction MIC/µg/ml MBC/µg/ml

Ethyl acetate leaf 63 250

Hexane flower 125 500

Alkaloid flower 125 1000

Acetone flower 250 >1000

Hexane flower 500 >1000

Ethyl acetate flower 1000 >1000

Ethyl acetate root >1000 >1000

Terpenoid flower 250 >1000

The most active extract was the ethyl acetate leaf, which had an MIC of 63 μg/ml (Figure 4.1). A photograph of the microplate after incubation of cells with the ethyl acetate extract is shown in

Figure 4.2. The most active phytochemical class against M. smegmatis with an MIC of 125 μg/ml was the alkaloids, followed by the terpenoids with an MIC of 250 μg/ml. However, rifampicin, the reference drug, was still a more potent antimycobacterial agent than all the plant extracts with

MIC of 1. 6 μg/ml. MBC determination showed that the ethyl acetate leaf extract mycobactericidal activity at a concentration of 250 μg/ml.

1.0

0.8

0.6

0.4

0.2

Absorbance at 590 nm 590 at Absorbance

0.0

-0.2

2 4 8 16 32 63 125 250 500 Cells 1000 Media

[Ethyl acetate leaf extract]/g/ml Figure 4.1: The effect of V. adoensis ethyl acetate leaf extract on the growth of M. smegmatis in liquid broth. M. smegmatis was grown in Middlebrook 7H9 broth and exposed to increasing concentrations of the ethyl acetate leaf extract in a 96-well microtiter plate.

MIC of extract Sterility control, media alone

Extract only

Extract + cells

Rifampicin + cells

MIC of Rifampicin Positive control, mycobacterial cells alone

Figure 4.2: Image of a 96 well plate showing the antimycobacterial activity of V. adoensis ethyl acetate leaf extract against M. smegmatis in the MTT assay. Purple coloured wells indicate mycobacterial cell growth whilst yellow coloured wells indicate inhibition of mycobacterial cell growth.

The MICs of all the crude extracts of the different plant parts of V. adoensis which were prepared

using solvents of varying polarity are illustrated in Table 4.3. Minimum inhibitory concentrations

of the crude extracts which were greater than 1000 µg/ml, which was the highest concentration

used, are denoted by the symbol ‘-’

4.4 The effect of V. adoensis leaf extract on the transport of ciprofloxacin

The effects of the ethyl acetate extract on drug transport inside mycobacterial cells were analyzed against M. smegmatis. Reserpine was employed as a positive control for transport inhibition, whilst ciprofloxacin was the probe drug. The results for the drug accumulation assay are illustrated in Figure 4.3. Accumulation of ciprofloxacin in M. smegmatis was highest in the presence of reserpine, with a concentration of 4.03 μg /ml. The cells exposed to glucose only had an accumulation of ciprofloxacin of 2.05 μg/ml. The accumulation caused by exposure to the ethyl acetate extract was 2.1 μg/ml, was not significantly different from that instigated by exposure to glucose alone.

5 **

* g/ml  3

2 [Ciprofloxacin]

Glucose No glucose

Reserpine+glucose

Ethyl acetate extract+glucoseEthyl acetate extract+glucose

Sample of exposure

Figure 4.3: Accumulation of drug in M. smegmatis cells. The graph shows the accumulation of the drug ciprofloxacin in M. smegmatis cells after exposure to V. adoensis leaf ethyl acetate extract. The accumulated ciprofloxacin is measured in µg/ml. N = 2. The test was performed

73 three times. The test for significance was carried out by comparing glucose + reserpine/plant extracts to glucose only. *P < 0.05. **P < 0.01

4.5 The effect of V. adoensis extract on cell membrane integrity in the protein leakage assay

The plant extract caused leakage of proteins from the cells to different extents, depending on the concentration of the extract that was used, and this is shown in Figure 4.4. The cells exposed to

63 μg/ml (MIC) of the plant extract showed a leakage of 17.8 μg/ml of protein. This amount of proteins leaked from the cells was not significant as compared to the leakage in the controls. The concentration 126 μg/ml (2 x MIC) significantly caused a loss of 25.5 μg/ml of protein. The MBC of the plant extract (250 μg/ml) resulted in a very substantial leakage of protein of 47.9 μg/ml

(P<0.0001) when compared to the control. Exposure of cells to the positive control, 0.1 % SDS, caused the greatest protein leakage.

Figure 4.4: Protein leakage in M. smegmatis after exposure to the V. adoensis leaf ethyl acetate extract. The protein concentration is shown in μg/ml. N= 2. The test was conducted two times. The test for significance was carried out by comparing all samples to cells exposed to distilled water only. 0.1 % SDS used as the positive control. * < 0.05. *** < 0.001.

4.6 The effect of V. adoensis leaf extract on leakage of nucleic acids

The nucleic acid leakage assay revealed that there was membrane disruption and damage in the presence of V. adoensis leaf extract at varying concentrations. The fluorescence of propidium iodide in M. smegmatis cells after exposure to the V. adoensis leaf ethyl acetate is shown in

Figure 4.5. Nucleic acid leakage was symbolized by an increase in fluorescence caused by the propidium iodide in the mycobacterial cells as compared to the untreated cells which served as the control. The results indicated that there was membrane disruption and damage to M. smegmatis in the presence of the extract at all the concentrations used. An increase in the concentration of the

75 leaf ethyl acetate extract resulted in increase of fluorescence of propidium iodide. The fluorescence units for the MIC of the leaf extract was significantly different from the untreated cells. The fluorescence units for 16 µg/ml of the extract were 0.05, which though was low but was of statistical significance and those for 32 µg/ml, 63 µg/ml and 126 µg/ml of extract were 0. 148,

0.162 and 0.177 F.U. which were highly significantly different from the control (P<0.0001).

0.20 *** *** *** 0.15

0.10

Fluorescence (au) ** 0.05

0.00

16 32 63 126

Concentration of V. adoensis leaf extract (g/ml)

Control; untreated cells

Figure 4.5: Fluorescence of nucleic acid binding propidium iodide after exposure of M. smegmatis to V. adoensis leaf extract. The V. adoensis extract was tested at 16 µg/ml (¼ the MIC), 32 µg/ml (½ the MIC), 63 µg/ml (MIC) and 126 µg/ml (2-fold the MIC), while untreated cells were used as control. n = 2. The test was conducted twice. The test for significance was conducted by comparing all samples to cells only. ** < 0.01. *** < 0.001

4.7 The effect of V. adoensis extract on free radical scavenging activity

The free radical scavenging potential of V. adoensis ethyl acetate leaf extract was evaluated using

DPPH (2, 2-diphenyl-1-picrylhydrazyl). Results showed that the DPPH radical scavenging ability of the extract was lower than that of ascorbic acid (Figure 4.6). The standard antioxidant ascorbic acid had an IC50 of 7.4 µg/ml, whilst V. adoensis had weak antioxidant activity with an IC50 of

114.8 µg/ml.

150

Ascorbic acid

V. adoensis

100

Scavenging activity % 50

0 0 100 200 300 Concentration (g/ml)

Figure 4.6: DPPH free radical scavenging activity of V. adoensis ethyl acetate leaf extract and ascorbic acid.

4.8 The effect of V. adoensis extract on haemolysis of sheep erythrocytes

The haemolytic assay was performed to determine whether extracts from V. adoensis had cytotoxic effects of sheep erythrocytes. The haemolytic activity of V. adoensis leaf extracts at 4 different concentrations is shown in Figure 4.7. The extracts exhibited low haemolytic effect towards the sheep erythrocytes on all concentrations tested. The haemolysis caused by exposure to concentrations 2 x MIC (125 µg/ml), and MBC (250 µg/ml) is similar to that caused by the higher concentrations of 1 mg/ml and 5 mg/ml.

Figure 4.7: Haemolytic effect of the Vernonia adoensis leaf extract at concentrations 2 x MIC (126 µg/ml) and MBC (250 µg/ml), 1 mg/ml and 5 mg/ml. Each concentration shows the mean of haemolysis percentage for n=3.

4.9 The in vitro determination of viability of mycobacteria inside macrophages

The assumption of this assay was that the extract from V. adoensis may stimulate macrophages, resulting in the intracellular killing of mycobacteria. To investigate this assumption, M. smegmatis was used to infect murine-derived macrophages 2 h before and 2 h after treatment with extract, with cells treated 2 hours before termed 'pretreated' and those treated 2 hours post infection termed 'post treated'. After 4 hours of treatment, it was noted that the mycobacterial burden was less in post treated cells in comparison to pretreated cells (Figure 4.8). The concentration of 32 µg/ml (½ MIC) did not decrease the mycobacterial burden. Post treating the macrophages with V. adoensis extract reduced the survival of M. smegmatis cells within the macrophages, with the greatest effect observed at 126 µg/ml.

Pretreatment of extract 40 Post treatment of extract

30 4

CFU X 10 CFU *

32 63 126 Control Concentration of extract g/ml

Figure 4.8: Intracellular survival of M. smegmatis in V. adoensis leaf extract treated macrophages. Murine macrophages were treated with 32 µg/ml (½ MIC), 63 µg/ml (MIC) and 126 µg/ml (2 x MIC) of extract after infection with M. smegmatis infection. The cells were lysed, and intracellular survival was determined at 30-minute intervals by CFU assay. * P < 0.05

4.10 Phytochemical analysis of V. adoensis ethyl acetate leaf extract

Phytochemical screening of the leaf ethyl acetate extract showed the presence of flavonoids, tannins, alkaloids, terpenoids and saponins.

Chapter Five: DISCUSSION

Drugs for treating tuberculosis have been accessible for over 50 years, and yet, the prevalence of disease globally persists to increase annually (Ballell et al., 2005). TB is notorious for resulting in high numbers of death in developing and undeveloped countries. The emergence of drug resistance has created a solemn need for new anti-TB agents to replace those which have lost efficacy (Buyazan and El-Gabulli, 2012; Zaman, 2010). Because of increasing drug-resistant mycobacterial infections, innovative chemical entities with new modes of action against TB and

MDR-TB are being sought. There is an urgent need of new regimens that can shorten the length and simplify the complexity of the present treatment. The regimen should also be efficacious against MDR strains and are capable of being administrated with antiretroviral drugs to improve therapeutic outcomes (AlMatаr et al., 2017).

Traditional therapies have guided the search for chemotherapeutic alternatives that combat infections and drug-resistant bacteria. The plant V. adoensis has been used traditionally to treat diseases. The plant leaves are boiled, and the decoction is drunk to treat the indications of TB

(Kisanagu et al., 2007). For this study, the antimycobacterial effects of V. adoensis were investigated against model Mycobacterium species M. smegmatis. Solvent extracts from its plant parts – leaves, flowers, roots and root bark were made using solvents of varying polarity ranging from a non-polar solvent, hexane, to a polar one, water. Eight solvents were used in the preparation of extracts, and the use of the broad range of polarity of the solvents was to guarantee extraction of a wide variety of compounds. According to Ncube and co-workers, (2015), during extraction, the solvents enter into plant material and dissolve compounds of similar polarity, and these compounds are usually the active principles or medicinal ingredients within the plant.

Preliminary qualitative phytochemical analysis made for the plant parts of V. adoensis revealed the presence of alkaloids, flavonoids, saponins, tannins and terpenoids. These secondary metabolites are reported to have antimicrobial properties and other biological and therapeutic properties which may support the purported medicinal use of this species (Chung et al., 1998; Cowan, 1999).

Preliminary phytochemical analysis of V. adoensis leaves and roots Swamy et al., (2013) also showed similar results on the type of phytochemicals present in the plant parts. According to

Okoli et al., (2009), phytochemical screening is significant and helpful in revealing the chemical nature of constituents in the plant material and the one that dominates over the others. It may be used to explore for bioactive agents that could be used in the synthesis of pharmacologically active drugs.

Eight extracts from the 4 plant parts used exhibited noteworthy antimycobacterial activity against the M. smegmatis (Table 4.5). The results indicate that the plant medicinal ingredients were mainly restricted to the leaves and the flowers, as can be revealed by the antimycobacterial activities being prominent in these plant parts. Extracts belonging to other plant parts did not display significant antimycobacterial activities. In comparison to other solvent extracts from the different plant parts, the leaf ethyl acetate extract proved to be the most effective in antimycobacterial susceptibility tests with an MIC and MBC of 63 µg/ml and 250 µg/ml respectively. Further phytochemical tests were carried out on the extract that had the highest activity, the leaf ethyl acetate extract and it was found to contain alkaloids, saponins, flavonoids and terpenoids. These phytochemical compounds may be responsible for the antimycobacterial activity of the plant against M. smegmatis.

Bacterial efflux pumps offer potential targets to combat problematic infectious diseases such as those caused by Escherichia coli (Stavri et al., 2007) and M. tuberculosis. To establish if the plant extracts could function as inhibitors of M. smegmatis cell efflux pumps, the accumulation of ciprofloxacin in the mycobacterial cells in the presence of the plant compounds was monitored by a fluorometric method. In this study, the drug transport assay was used to investigate the ability of the leaf extract to cause ciprofloxacin accumulation in M. smegmatis cells whilst comparing its activity to that of reserpine, a common efflux pump inhibitor. Lately, EPIs have been termed as

‘putative’ new drug compounds, as they are capable of restoring susceptibility to existing anti-TB drugs to a certain degree, by hindering the operation of the efflux pumps (Viveiros et al., 2012).

Bacterial efflux pumps present potential targets to combat obstinate infectious diseases (Stavri et al., 2007). Results from this assay showed that drug accumulation cannot be counted to be one of the plant's modes of action. In actual effect, the extract resulted in significant accumulation of ciprofloxacin inside the mycobacterial cells (Figure 4.3). The drug accumulated in the cells was similar to that which accumulated in cells subjected to glucose alone and was not comparable to drug accumulated due to reserpine. Glucose is converted to adenosine triphosphate (ATP) which supplies energy to the mycobacterial efflux pumps resulting in the drug being pumped out

(Sarathy et al., 2012). Though in a different study, an extract of V. adoensis was able to exhibit

EPI activity against Bacillus subtilis and Staphylococcus aureus (Chitemerere and

Mukanganyama, 2011), in this study, V. adoensis did not exhibit EPI activity. The results indicate that the plant extract was not able to inhibit the efflux pumps of the mycobacteria, which resulted in the drug of interest being pumped out of the cell. Hence, ciprofloxacin did not have any antimycobacterial effect on the cells. The V. adoensis leaf extract did not have efflux pump inhibitory activity but was in fact, stimulating the pumping of the drug out of the cell.

Another probable mode of action for the extract that was investigated was the extract’s ability to disrupt mycobacterial membrane integrity such that there is leakage of cellular proteins.

Mycobacteria demonstrate a high level of noteworthy resistance to a great number of antituberculars and chemotherapeutic agents (Jarlier and Nikaido, 1994). Effective TB treatment is, therefore, difficult because of the obstruction of entry of drugs by the thick mycobacterial cell wall, rendering many antibiotics ineffective (Sunduru et al., 2010). Significant leakage of proteins from M. smegmatis cells caused by exposure to the V. adoensis ethyl acetate leaf extract was observed for the concentrations 63 µg/ml and 126 µg/ml. The integrity of the cell membrane/wall was disrupted most probably by the extract’s binding to lipids or proteins on the cell membrane causing increase in membrane permeability, reduction in pH of the cytoplasm, hyperpolarization of the cell membrane, and a reduction in cellular ATP concentration, which all are vital for numerous physiological activities (Sunduru et al., 2010; Sanchez and Kouznetsov, 2010 and Bai et al., 2015). Leaking of cellular products interferes with mycobacterial cell growth, ultimately leading to the cell death. Crude and partially purified plant extracts contain phytochemicals, which differ in chemical organization and structure, and in turn, influence the biological functions of the plant (Saritha et al., 2015). Some known phytochemicals are vital elements in plant defense because they have antimicrobial effects (Cowan, 1999). The major active components like terpenoids and aldehydes can act by compromising the functional and structural integrity of cell cytoplasmic membranes, causing lysis and leaking of cellular contents (Nazzaro et al., 2013). Saponins also make up the defense system of the plants and their antimicrobial activity is attributed mainly to their capability of lysing microorganism’s membranes (Asl, 2008).

So, in this case, activity against the cytoplasmic membrane of M. smegmatis cells may be credited to terpenoids and saponins present in the plant, forming part of the plant defense.

Membrane damaging agents can influence the leaking of proteins, nucleic acids and other intracellular components since the protective barrier will be destroyed. Propidium iodide (PI) is a dye that does not penetrate viable cells yet can enter cellular membranes of dead bacterial cells and stain nucleic acids (Hiraoka and Kimbara, 2002). The fluorescence of PI staining nucleic acids can be quantified to determine membrane damage (Lahiri et al., 2005). It attaches to DNA by infiltrating between base pairs and since PI is not permeant to live cells, it is also normally used to identify dead cells in a population. Leakage of nucleic acids occurred after addition of V. adoensis extract to the mycobacterial cells. Nucleic acid leakage was represented by an intensification in fluorescence in the mycobacterial species when compared to untreated cells, which functioned as the control (Figure 4.5). The greatest disruption of M. smegmatis cellular membranes was experienced at the MIC of V. adoensis extract as further increase in concentration did not result in any noteworthy increase in fluorescence. Leakage of nucleic acids at MIC and 2- fold MIC was almost comparable. This result suggests that the MIC of V. adoensis extract completely damaged the cell membrane causing maximal nucleic acid leakage from the cells, such that any additional increase in concentration did not bring about any additional destruction to the membrane. There are several other extracts from plants that have been investigated that induce bacterial cell nucleic acid leakage. Stem bark extracts of Afzelia africana (Smith) have been investigated to be biocidal to E. coli due to disruption of cell membrane leading to the discharge of bacterial nucleic acids (Akinpelu et al., 2009). Extracts from Piper nigrum and Triumfetta welwitschii also exhibited potential in leakage of cellular nucleic acids (Karsha and Lakshmi,

2010; Moyo and Mukanganyama, 2015). Phenolics such as flavonoids; terpenes and essential oils available in plants react with the cellular membrane, impairing both its functions and integrity

(Nazzaro et al., 2013; Saritha et al., 2015). Saponins also tend to act as detergents through cell

85 membrane disruption (Morrissey and Osbourn, 1999). Using phytochemical analysis, the leaves of V. adoensis possessed saponins, flavonoids and terpenoids, which could be accountable for the nucleic acid leakage.

The antioxidant activity of V. adoensis leaf extract was tested on its capacity to scavenge the stable

DPPH free radical activity. The principle behind this method will be the ability of the alleged antioxidant to quench the DPPH radical through the addition of a radical species that decolourises the DPPH solution. The principle behind this method will be the ability of the alleged antioxidant to quench the DPPH radical through the addition of a radical species that decolourises the DPPH solution. The ability of the compound under investigation to quench a free radical is signified by a decline in the absorbance of the reaction mixture (Krishnaiah et al., 2011). Researchers are studying the potential of medicinal plant extracts to quench free radicals and red lessen inflammatory reactions. This potential of medicinal plants to scavenge for and quench free radicals in so doing, reducing inflammatory reactions in the human body, is currently under scrutiny by scholars and scientists. Free radicals are generated by different biochemical processes in the human system and the body attempts to neutralize their deleterious effects by making use of antioxidants. However, during this course, some imbalances can occur that can activate pathways that promote inflammation

(Rahman, 2007). Most of the potentially harmful effects of oxygen are due to formation of reactive oxygen species/intermediates which are produced within macrophages during the course of TB infection (Sharma et al., 2004). ROS play an important mycobactericidal role, but their extreme production could, contrarily, lead to inflammation. Antioxidant substances that can quench and eliminate ROS may be useful in minimizing oxidation-related complications like TB (Lobo et al.,

2010). This study indicated that the extract from V. adoensis exhibited poor free radical scavenging activity. The standard antioxidant, ascorbic acid that was used had an IC50 of 7.4 µg/ml, whilst V.

86 adoensis had an IC50 of 114.8 µg/ml. Samples with IC50 < 50 μg/ml (extract) are regarded to have significant antioxidant capacity, those with 50 < IC50 < 100 μg/ml (extract) moderate antioxidant capacity and those with IC50 > 100 μg/ml (extract) or IC50 > 20 μg/ml (compounds) low antioxidant capacity (Kuete and Efferth, 2010). Also, according to Karamian and Ghasemlou, (2013) greater antioxidant activity is illustrated by a lower IC50, meaning that ascorbic acid is a more powerful antioxidant in comparison to V. adoensis leaf extract. Though the antioxidant potential of ascorbic acid was higher than that of the extract, the study showed V. adoensis leaf extract has antioxidant activity. The occurrence of flavonoids in the plant could be responsible for the observed antioxidant characteristics of this extract. An explanation for the low antioxidant activity may be because of some hinderances from other chemical components in the extract, such as sugars (Saeed et al.,

2012).

Information obtained from this study, hence, suggests that no relationship exists between the antioxidant and antimycobacterial activities of this plant extract. The leaf ethyl acetate extract had promising antimycobacterial activity but had poor antioxidant activity. A study on the evaluation of antimycobacterial and antioxidant properties of extracts from folk remedies used in Turkey reported similar findings in which the authors established no relation between the two properties

(Orhan et al., 2012). Antimycobacterial activity and antioxidant potential of the leaf extract from this plant are not related. According to Trombetta and co-authors, (2005), terpenoids and phenolic compounds are, instead, two important phytochemicals found in numerous medicinal plants that impart antimicrobial activity and exhibit antioxidant properties. Nevertheless, this is not true for all antioxidants. It is proposed that the way the plant is processed may expose some phytochemicals to heat, thereby, reducing antimicrobial potential but antioxidant activity remaining unchanged.

Compounds possessing potent biological activity may not be profitable in pharmacological preparations if they possess haemolytic effect. The erythrocyte model has been broadly used as it displays a direct indicator for toxicity of injectable formulations and as an overall indication of toxicity of the membrane. Another advantage of the erythrocyte model is that blood is easily accessible and that cells are not difficult to separate from the blood, enabling the reduction of laboratory animals for in vivo studies (Zohra and Fawzia, 2014; Orsine et al., 2012). Destruction of erythrocytes, caused by lysis of the membrane lipid bilayer results in haemolysis, which relates to concentration and effectiveness of extract (Lee et al., 2015; Zohra and Fawziа, 2014).

Consequently, the haemolytic assay was performed to determine if extracts from V. adoensis had haemolytic or cytotoxic effects of sheep erythrocytes. The haemolytic potential of V. adoensis leaf extracts at 4 different concentrations: 125 µg/ml; 250 µg/ml; 1 mg/ml and 5 mg/ml was investigated. The extracts exhibited low haemolytic effect towards the sheep erythrocytes on all concentrations tested. Ralph et al., (2009) reported that haemolytic activity was related to the extent or magnitude of in vitro toxicity according to the observed mortality rates like 0-9% - non- toxic, 10- 49% - slightly toxic, 50-89% - toxic then 90-100% - highly toxic. Consequently, haemolytic activity is a significant measurement and indicator for cell toxicity. The haemolysis instigated by exposure to concentrations 2 x MIC (125 µg/ml), and MBC (250 µg/ml) was comparable to that caused by the higher concentrations of 1 mg/ml and 5 mg/ml, suggesting that the extract exhibited minimal toxicity even at high concentrations. V. adoensis showed slight levels of toxicity on sheep erythrocytes at concentrations as high as 5mg/ml; the highest percentage haemolysis was 18.2 % at 5 mg/ml. These figures suggest the low toxicity effect of the extract towards the erythrocyte membrane, making it a potential compound for developing new agents that can be used in the treatment of TB.

To examine the possibility of V. adoensis extract stimulating macrophages, resulting in the killing of mycobacteria inside them, murine-derived macrophages were infected using M. smegmatis 2 hours before and 2 hours after treating them with the extract. The murine macrophages exposed to the extract 2 hours before mycobacterial infection were termed ‘pre-exposed’ and those exposed 2 hours after infection were termed as ‘post exposed’. Four hours post exposure, it was discovered that the mycobacterial burden was less in post exposed cells in comparison to pretreated cells.

The concentration of 32 µg/ml (½ MIC) intracellular mycobacterial growth was not affected by the concentration of 32 µg/ml (1/2 MIC). Mycobacteria are intracellular pathogens (Meena and

Rajni, 2010), which resides in phagosomal chamber of the macrophages. It is, thus, fundamental to activate the macrophages or deliver therapeutic molecules to the target sites that would kill the intracellular mycobacteria (Naik, 2014). Post treating the cells with 126 µg/ml of V. adoensis extract resulted in significant reduction in survival of intracellular M. smegmatis cells. Treating the infected macrophages with plant extract might have prompted the manufacture of pro- inflammatory cytokines (Spelman et al., 2006) that stimulate the cells such that there was an upsurge in the killing proficiency of macrophages. V. adoensis may act by stimulating the immune response by interacting with various cells of the immune system, but further investigation on this will have to done. According to Spelman et al., (2006), immune stimulatory response may explain how herbs exert their effects on the body's line of defense in addition to other tissues.

They also stated that research done on some medicinal plant extracts revealed them to have effects on several cytokines and the most commonly studied cytokines were IL-1, IFN, IL-6 and TNF.

Future studies on how V. adoensis effects cytokines may be an area of interest.

Many studies on M... tuberculosis have been established based on results from using M. smegmatis mc2155 since they share significant similarities and yet M. smegmatis is non-virulent and has a faster growth rate (Baloni et al., 2014). M. smegmatis may be utilized in preliminary studies to choose compounds that have promising antimycobacterial activity against M. tuberculosis (Baruа et al., 2014). Positive results from studies that use M. smegmatis can be utilized to facilitate the exploitation of M. tuberculosis in future studies. For clinical relevance, working with pathogenic M. tuberculosis in future studies would be considered since the leaf extract has demonstrated to have minimal cytotoxicity, possesses ability to interfere with the integrity of the M. smegmatis cell membranes and reduce intracellular mycobacterial survival in macrophages.

Chapter Six: Conclusion

The leaf extract of V. adoensis had antimycobacterial activity against M. smegmatis and phytochemicals such alkaloids, terpenoids and saponins that were present in the extract, may be liable for this activity. V. adoensis plant displayed low antioxidant activity, thus, may not a play role in minimizing inflammation associated with TB. The plant did not possess activity against transport of compounds by the efflux pumps of mycobacteria as well but was able to disrupt integrity of the cell membranes resulting in leakage of protein and nucleic acids from the cells.

Thus, the possible modes of activity of the plant maybe leakage of protein and nucleic acids. V. adoensis extract also possesses minimal haemolytic activity making it a potential plant for use in development of antimycobacterial agents. Post treating the macrophages with V. adoensis extract increased the killing efficiency of macrophages compared with pretreating them. There was significant decline in intracellular mycobacterial burden at a concentration of 126 µg/ml of extract.

The study findings support the traditional medicinal use of V. adoensis leaves in the treatment of

TB and TB-related ailments. However, more work needs to be done to determine the compounds responsible for the activity as V. adoensis may have the capacity to provide new lead compounds for the development of novel antimycobacterial agents.

FUTURE STUDIES

Further studies of this project would include investigation of the leaf extract in vivo, and to isolate and purify the compounds responsible for the antimycobacterial activity. For clinical relevance of the plant, the use of M. tuberculosis in forthcoming studies should also be considered.

REFERENCES

Abo, K.A.; Ogunleye, V.O. and Ashidi, J.S. (1991). Antimicrobial poteintial of Spondias mombin, Croton zambesicus and Zygotritonia crocea. Journal of Pharmacological Research. 5(13): 494-497.

Akinola, R.O., Mazandu, G.K., Mulder, N.J. (2013) A systems level comparison of Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium smegmatis based onfunctional interaction network analysis. Journal of Bacteriology & Parasitology. 4: 173.

Akinpelu, D.A., Aiyegoro, A.O. and Okoh, A.I. (2009). Studies on the biocidal and cell membrane disruption potentials of stem bark extracts of Afzelia Africana (Smith). Biological Research. 42: 339-349.

Akpaso, M. I., Atangwho, I. J., Akpantah, A., Fischer, V. A., Igiri, A. O. and Ebong, P. E. (2011). Effect of combined leaf extracts of Vernonia amygdalina (Bitter Leaf) and Gongronema latifolium (Utazi) on the pancreatic β-Cells of streptozotocin-induced diabetic rats. British Journal of Medicine & Medical Research. 1(11): 24-34.

Alade, P.I and Irobi, O.N. (1993) Antimicrobial activity of crude leaf extracts of Alcalyhpa wilkesiana. Journal of Ethnopharmacology. 39:171-174.

Alara, O. R., Abdurahman, N. H. and Olalere, O. A. (2017c). Ethanolic extraction of flavonoids, phenolics and antioxidants from Vernonia amygdalina leaf using two-level factorial design. Journal of King Saud University – Science.

Aldaas, S.A. (2011). Cytotoxic and antibacterial activity of an extract from a Saudi traditional medicinal plant Equisetum arvense. MSc thesis, King Abdullah University of Science and Technology, Thuwal.

Alderton, H. and Smith, D. (2001). Safety in the laboratory. In methods in molecular medicine: Mycobacterium tuberculosis protocols. Edited by Parish T, Stoker NG. Humana Press 367-383.

Aliyu, A.B., Musa, A.M., Abdullahi, M.S., Ibrahim, H. and Oyewale, A.O. (2011). Phytochemical screening and antibacterial activities of Vernonia ambigua, Vernonia blumeoides and Vernonia oocephala (Asteraceae). Acta Poloniae Pharmaceutica—Drug Research. 68(1): 67-73.

AlMatar, M., AlMandeal, H., Var, I., Kayar, B. and Koksal, F. (2017). New drugs for the treatment of Mycobacterium tuberculosis infection. Biomedicine and Pharmacotherapy. 91: 546- 558.

Amaral, E.P., Ribeiro, S.C.M., Lanes, V.R., Almeida, F.M., de Andrade, M.R.M., Bomfim, C.C.B., Salles, E.M., Bortoluci, K.R., Coutinho-Silva, R., Hirata, M.H., Alvarez, J.M. et al. (2014). Pulmonary Infection with Hypervirulent Mycobacteria Reveals a Crucial Role for the P2X7 Receptor in Aggressive Forms of Tuberculosis. PLoS Pathogens 10(7): e1004188.

Anibijuwon, I.I., Duyilemi, O.P. and Onifade, A.K. (2010). Antimicrobial activity of leaf of Aspila africana on some pathogenic organisms of clinical origin. Nigerian journal of Microbiology. 24(1): 2048-2055.

Aobuli, A., Maitusong, J., Bakri, M., Lu, X., Maiwulanjiang, M. and Aisa, H.A. (2018). The effect of volatile oil from Vernonia anthelmintica seeds on melanin synthesis in B16 cells and its chemical analysis by GC-QTOF-MS. Evidence-Based Complementary and Alternative Medicine. 1-8.

Asl, M.N., Hosseinzadeh, H. (2008). Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytotherapy Research 22: 709-724.

Bai, J., Wu, Y., Liu, X., Zhong, K., Huang, Y. and Gao, H. (2015). Antibacterial activity of shikimic acid from pine needles of Cedrus deodara against Staphylococcus aureus through damage to cell membrane International Journal of Molecular Sciences. 16: 27145-27155.

Ballell, L., Field, R. A., Duncan, K. and Young, R. J. (2005). New small-molecule synthetic antimycobacterials. Antimicrobial Agents and Chemotherapy. 49(6): 2153–2163.

Baloni, P., Padiadpu, J., Singh, A., Gupta, K.R. and Chandra, N. (2014). Identifying feasible metabolic routes in Mycobacterium smegmatis and possible alterations under diverse nutrient conditions. BMC Microbiology. 14: e276.

Bansal, A., Mallik, D., Kar D. and Ghosh A.S. (2016). Identification of a multidrug efflux pump in Mycobacterium smegmatis. FEMS Microbiology Letters. 363(13): 1-5.

Bardou, F., Raynaud, C.,Ramos, C., Laneelle, M.A. and Laneelle, G. (1998). Mechanism of isoniazid uptake in Mycobacterium tuberculosis. Microbiology. 144: 2539-2544.

Barua, C.C., Singh, A., Sen, S., Barua, A.G., Barua, I.C. (2014). In vitro antioxidant and antimycobacterial activity of seeds of Piper longum Linn: A comparative study. SA Journal of Pharmacy and Pharmacology 1: 101

Beers, R.F. and Sizer, I.W. (1952). A spectrophotometer method of measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry. 195: 133-140.

Belanger, A. E., Besra, G. S., Ford, M. E., Mikusova, K., Belisle, J. T., Brennan, P. J. and Inamine, J. M. (1996). The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterialdrug ethambutol. Proceedings of the National Academy of Sciences of the United States of America, 93(21): 11919–11924.

Bernstein, J., Lott, W.A., Steinberg, B.A. and Yale, H.L. (1952). Chemotherapy of experimental tuberculosis. - V. lsonicotinic acid hydrazide (nydrazid) and related compounds. American Review of Tuberculosis. 65: 357-364.

Bhattacharyya, R. and Banerjee, D. (2011). Glycation of calmodulin binding domain of iNOS may increase the chance of occurrence of tuberculosis in chronic diabetic state. Bioinformation. 7(7): 324–327.

Biradar, S.R. and Rachetti, B.D. (2013). Extraction of some secondary metabolites and thin layer chromatography from different parts of Centella asiatica L. (URB). American Journal of Life Sciences. 1(6): 243-247.

Birhan, W., Giday, M. and Teklehaymanot, T. (2011). The contribution of traditional healers' clinics to public health care system in Addis Ababa, Ethiopia: a cross-sectional study. Journal of Ethnobiology and Ethnomedicine. 7:39

Boham, B.A. and Kocipai-Abyazan. R. (1994). Flavonoids and condensed tannins from leaves of Vaccinium vaticulatum and V. calycinium. Pacific Science, 48: 458 – 463.

Botha, L., van Pittius, N.C.G. and van Helden, P. (2013). Mycobacteria and disease in Southern Africa. Transboundary and Emerging Diseases 60(1): 147-156.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72:248–254.

Brennan, P. J. (2003). Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis (Edinburgh). 83: 91–97.

Briggs, J.P. (2014). The art and science of traditional medicine part 1: TCM Today – A case for integration. Science. 346 (6216 Suppl).

Brown-Elliott, B.A. and Wallace, R.J. (2002). Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clinical Microbiology Reviews. 15(4): 716–746.

Brown-Elliott, B. A., Wallace, R. J., Jr, Petti, C. A., Mann, L. B., McGlasson, M., Chihara, S., … Simmon, K. E. (2010). Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as causes of mycobacteremia. Journal of clinical microbiology, 48(12): 4377–4385.

Buzayan, M.M and Rajab El-Garbulli, F. (2012). Antibacterial activity of medicinal aqueous plant extracts against Mycobacterium tuberculosis. Malaysian Journal of Microbiology. 8(3): 203-206.

Calligaro, G. L., Moodley, L., Symons, G., and Dheda, K. (2014). The medical and surgical treatment of drug-resistant tuberculosis. Journal of Thoracic Disease. 6(3): 186–195.

Cambier, C.J., Falkow, S. and Ramakrishnan, L. (2014). Host evasion and exploitation schemes of Mycobacterium tuberculosis. Cell. 159(7): 1497-1509.

Campbell, E.A., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A. andDarst, S.A. (2001). Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104: 901–912.

Camus, J. C., Pryor, M. J., Medigue, C. and Cole S.T. (2002). Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology 148: 2963-2967.

Cardozo, L.F., Pedruzzi, L.M., Stenvinkel, P., et al. (2013). Nutritional strategies to modulate inflammation and oxidative stress pathways via activation of the master antioxidant switch Nrf2. Biochimie. 95: 1525-1533.

Chandra, P. and Arora, D.S. (2017). Antioxidant compounds derived from plants, description and mechanism of phytochemicals. Journal of Agroecology and Natural Resource Management. 4(1): 55-59.

Chan, E. D. and Iseman, M. D. (2002). Current medical treatment for tuberculosis. The BMJ. 325: 1282-1286.

Chan, F. K.-M., Moriwaki, K. and De Rosa, M. J. (2013). Detection of necrosis by release of lactate dehydrogenase (LDH) activity. Methods in Molecular Biology (Clifton, N.J.). 979: 65–70.

Chaulk, C.P. and Kazandjian, V.A. (1998). Directly observed therapy for treatment completion of pulmonary tuberculosis: consensus statement of the Public Health Tuberculosis Guidelines Panel. Journal of the American Medical Association. 279: 943–948.

Cheeseman, K.H. and Slater, T.F. (1993). An introduction to free radicals chemistry. British Medical Bulletin. 49: 481-493.

Chiba, K., Kawakami, K. and Tohyama, K. (1998). Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells. Toxicology in Vitro. 12: 251–258.

Chitemerere, T.A. and Mukanganyama, S. (2011). In vitro antibacterial activity of selected medicinal plants from Zimbabwe, The African Journal of Plant Science and Biotechnology. 5(1): 1-7.

Chitemerere, T. and Mukanganyama, S. (2014) Evaluation of cell membrane integrity as a potential antimicrobial target for plant products. BMC Complementary Alternative Medicine. 14:278 1-8.

Cho, M. H., Niles, A., Huang, R., Inglese, J., Austin, C. P., Riss, T. and Xia, M. (2008). A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker. Toxicology in vitro: An International Journal Published in Association with BIBRA. 22(4): 1099–1106.

Cooper, G.M. (2000). The cell: A molecular approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Lysosomes. https://www.ncbi.nlm.nih.gov/books/NBK9953/.

Costa-Gouveia, J., Pancani, E., Jouny, S., Machelart, A., Delorme, V., Salzano, G., Iantomasi, R., Piveteau, C., Queval, C.J.J., Song, O.R., Flipo, M., Deprez, B., Saint-André, J.P., Hureaux, J., Majlessi, L., Willand, N., Baulard, A., Brodin, P. and Gref, R. (2017). Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles. Scientific Reports. 7: 5390.

Cowan, M.M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews. 12(4): 564-582.

Curry International Tuberculosis Center (2011). Tuberculosis Infection Control: A Practical Manual for Preventing TB. CITC, USA.

Cushnie, T.P.T., Lamb, A.J. (2005) Antimicrobial activity of flavonoids. International. Journal of Antimicrobial Agents, 26(5): 343-356.

Daffe, M. and Reyrat, J.M. (2008). The Mycobacterial Cell Envelope. Weinheim: Wiley.

Danilchanka, O., Pavlenok, M. and Niederweis, M. (2008). Role of porins for uptake of antibiotics by Mycobacterium smegmatis. Antimicrobial Agents and Chemotherapy. 52: 3127– 3134.

Demain, A. and Valshnav, P. (2011). Natural products for cancer chemotherapy. Microbial Biotechnology 4: 687–699.

Dhanalakshmi P., Priya, A.J.P., Sagadevan, E., Lakshmi, Y.S., Manimaran, A., Sindhu, S. and Arumugam, P. (2013). Evaluation of inhibitory effect of Vernonia cinerea L. leaf extracts on different fungal species. International Journal of Pharmacy and Pharmaceutical 5(2): 414-416.

Doughari, J.H., Human, I.S., Bennade, S. and Ndakidemi, P.A. (2009). Phytochemicals as chemotherapeutic agents and antioxidants: Possible solution to the control of antibiotic resistant verocytotoxin producing bacteria. Journal of Medicinal Plants Research. 3: 839–848. du Toit, L.G., Pillay, V. and Dunkwerts, M.P. (2006). Technique for evaluation of medicinal plant products as antimicrobial agents: current methods and future trends. Journal of Medicinal Plants Research 4(2): 104-111.

Dye, C., Scheele, S., Dolin, P., Pathania, V. and Raviglione, M.C. (1999). Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. Journal of the American Medical Association. 282: 677–686.

Eisenbrand, G., Pool-Zobel, B., Baker, V., Ball, M., Blaauboere, B.J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.C., Pieters, R. and Kleiner, J. (2002). Methods of in vitro toxicology. Food and Chemical Toxicology. 40: 193–236

El-Nakeeb, M.A., Abou-Shleib, H.M., Khalil, A.M., Omar, H.G. and El-Halfawy, O.M. (2011). Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics. Brazilian Journal of Microbiology. 42: 992-1000.

El-Tayeb, O.M., Hussein, M. M. M.2, Salama, A.A. and El-Sedawy, H.F. (2004). Optimization of industrial production of rifamycin B by Amycolatopsis mediterranei. II. The role of gene amplification and physiological factors in productivity in shake flasks. 3(5): 273-280.

Ekor, M. (2013). The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Frontiers in Pharmacology. 4: 177.

Erasto, P., Grierson, D. S. and Afolayan, A. J. (2007). Evaluation of antioxidant activity and the fatty acid profile of the leaves of Vernonia amygdalina growing in South Africa. Food Chemistry. 104: 636-642.

Erasto, P., Grierson, D.S., Afolayan, A.J. (2006). Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina. Journal of Ethnopharmacology. 106: 117-120.

Farombi, E.O. and Owoeye, O. (2011). Antioxidative and chemopreventive properties of Vernonia amygdalina and Garcinia biflavonoid. International Journal of Environmental Research and Public Health. 8: 2533-2555.

Ferguson. N.M. (1956). A textbook of pharmacognosy. The Macmillan Company, New York.

Fernandez, L. and Hancock, R.E.W. (2012). Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance. Clinical Microbiology Reviews. 25: 661–681.

Flint, A. (2015). Traditional healing, biomedicine and the treatment of HIV/AIDS: Contrasting South African and native American experiences. International Journal of Environmental Research and Public Health. 12(4): 4321–4339.

Flynn, J.L. and Chan, J. (2001). Tuberculosis: latency and reactivation. Infection and Immunity. 69(7): 4195–4201.

Forbes, M., Kuck, N A., Peets, E.A. (1962). Mode of action of ethambutol. Journal of Bacteriology. 84: 1099-1103.

Fournier Dit Chabert, J., Marquez, B., Neville, L., Joucla, L., Broussous, S., Bouhours, P., David, E., Pellet-Rostaing, S., Marquet, B., Moreau, N., et al. (2007). Synthesis and evaluation of new arylbenzo[b]thiophene and diarylthiophene derivatives as inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Bioorg. Med. Chem. 15: 4482–4497.

Fu, L., Xu, B.-T., Xu, X.-R., Gan, R.-Y., Zhang, Y., Xia, E.-Q. and Li, H.-B. (2011). Antioxidant capacities and total phenolic contents of 62 fruits. Food Chemistry. 129: 345–350.

Funk, V.A., Bayer, R.J., Keeley, S., Chan, R., Watson, L., et al. (2005) Everywhere but Antarctica: using a super tree to understand the diversity and distribution of the Compositae. Biologiske Skrifter. 55: 343–374.

Gaestel, M., Kotlyarov, A. and Kracht, M. (2009) Targeting innate immunity protein kinase signalling in inflammation. Nature Reviews Drug Discovery. 8: 480–499.

Garvey, M.I. and Piddock, L.J. (2008). The efflux pump inhibitor reserpine selects multidrug- resistant Streptococcus pneumoniae strains that overexpress the ABC transporters Pat A and Pat B. Antimicrobial Agents and Chemotherapy. 52(5): 1677–1685

Gautam, R., Saklani, A. and Jachak, S.M. (2007). Indian medicinal plants as a source of antimycobacterial agents. Journal of Ethnopharmacology. 110: 200–234.

Ghosh, S. and Saxena, R.K. (2004). Early effect of Mycobacterium tuberculosis infection on Mac-1 and ICAM-1 expression on mouse peritoneal macrophages. Experimental and Molecular Medicine. 36: 387–395.

Ghoshal, S., Krishna, P.N., Lakshmi, V. (1996). Antiamoebic activity of Piper longum fruits against Entamoeba histolytica invitro and in-vivo. Journal of Ethnopharmacology. 50:167–170.

Ginouves, M., Carme, B., Couppie, P. and Prevot, G. (2014). Comparison of tetrazolium salt assays for evaluation of drug activity against leishmania spp. Journal of Clinical Microbiology, 52(6): 2131–2138.

Ginsberg, M.A. and Spigelman, M. (2007). Challenges in tuberculosis drug research and development. Nature Medicine. 13(3): 290-294.

Global tuberculosis report (2017). Geneva: World Health Organization; 2017. License: CC BY- NCSA 3.0 IGO.

Gonzalez-y-Merchand, J.A., Colston, M.J. and Cox, R.A. 1998. Roles of multiple promoters in transcription of ribosomal DNA: effects of growth conditions on precursor rRNA synthesis in mycobacteria. Journal of Bacteriology. 180: 5756–5761.

Gopinath, K. and Singh, S. (2010) Non-tuberculous mycobacteria in TB-endemic countries: are we neglecting the danger? PLoS neglected tropical diseases 4: e615.

Gordon, R. and Smith, M. (1953). Rapidly growing acid-fast bacteria I. Species descriptions of Mycobacterium phlei Lehmann and Neumann and Mycobacterium smegmatis (Trevisan) Lehmann and Neumann, Journal of Bacteriology. 66:41-48.

Green, C.M. (1991). A Survey of the Medicinal Plants in the Stellenbosch Region. B. Sc (Hons.) Dissertation, University of Stellenbosch, South Africa.

Guinn, K.M. and Rubin, E.J. (2017). Tuberculosis: just the FAQs. mBio 8: e01910-17. https://doi.org/10.1128/mBio.01910-17.

Gupta, A., Bhakta, S., Kundu, S., Gupta, M., Srivastava, B.S. and Srivastava, R. (2009). Fast- growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high throughput antituberculosis drug screening. Journal of Antimicrobial Chemotherapy. 64: 774–781.

Gupta, S., Bandyopadhyay, D., Paine, S. K., Gupta, S., Banerjee, S., Bhattacharya, S., … Bhattacharya, B. (2010). Rapid identification of Mycobacterium species with the aid of multiplex polymerase chain reaction (PCR) from clinical isolates. The open microbiology journal. 4: 93–97.

Habtamu, A. and Melaku, Y. (2018). Antibacterial and antioxidant compounds from the flower extracts of Vernonia amygdalina. Advances in Pharmacological Sciences. 2018: 1-6.

Hahn, N.I. (1998). Is phytoestrogens nature’s cure for what ails us? A look at the research. Journal of the American Dietetic Association. (98): 974-976.

Hain Life Science. http://www.hain- lifescience.de/en/products/microbiology/mycobacteria/mycobacteria.html. Accessed 20 October 2017.

Halliwell, B. and Gutteridge, J.M.C (1989). Free Radicals in Biology and Medicine (2nd ed.) Clarendon Oxford, UK.

Harborne, J.B. (1973). Phytochemical methods, London. Chapman and Hall, Ltd. pp 49-188.

Hamburger, M. and Hostettmann, K. (1991). Bioactivity in plants: between phytochemistryand medicine, Phytochemistry. 30: 3864– 3874.

Hartmans, S., de Bont, J.A.M. and Stackebrandt, E. (2006). The genus Mycobacterium – nonmedical, in: Dworkin, M., Falkow, S. (Eds.), The prokaryotes: Vol. 3: Archaea. Bacteria: Firmicutes, Actinomycetes, Springer Science & Business Media, New York, 889–918.

Havsteen, B. (1983). Flavonoids, a class of natural products of high pharmacological potency. Biochemical Pharmacology. 32(7): 1141-1148.

Hett, E.C. and Rubin, E.J. (2008). Bacterial growth and cell division: a mycobacterial perspective. Microbiology and Molecular Biology Reviews 72: 126–156.

Hiraoka, Y. and Kimbara, K. (2002). Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry. Applied and Environmental Microbiology. 68: 2031-2035.

Honarvar, B., Movahedan, H., Mahmoodi, M., Sheikholeslami, F.M. and Farnia, P. (2012). Mycobacterium aurum keratitis: an unusual etiology of a sight-threatening infection. Brazilian Journal of Infectious Diseases. 16(2): 204–208.

Hossain, M.M. and Norazmi, M.N. (2013). Pattern recognition receptors and cytokines in Mycobacterium tuberculosis infection-the double-edged sword? Biomed Research International. 2013: 179174.

100

Hsu, C.L., Chen, W., Weng, Y.M. and Tseng C.Y. (2003). Chemical composition, physical properties, and antioxidant activities of yam flours as affected by different drying methods. Food Chemistry. 83: 85-92.

Hasan, S. Standing up against antibiotic resistance with synergistic approach http://slideplayer.com/slide/9080085/ Accessed 21 November 2017.

Huang, C.; Tsai, Y.; Shu, C.; Lei, Y.; Wang, J.; Yu, C.; Lee, L.; Yang, P. and the TAMI Group (2009). Clinical significance of isolation of nontuberculous mycobacteria in pulmonary tuberculosis patients. Respiratory Medicine. 103: 1484-1491.

Igile, G.O., Oleszek, W., Jurzysta, M. (1995) Vernoniosides D and E, two novel saponins from Vernonia amygdalina. Journal of Natural Products. 58: 1438-1443.

Iwalokun, B.A., Efedede, B.U., Alabi-Sofunde, J.A., Oduala, T., Magbagbeola, O.A. and Akinwande, A.I. (2006). Hepatoprotective and antioxidant activities of Vernonia amygdalina on acetaminophen-induced hepatic damage in mice. Journal of Medicinal Food. 9: 524–530.

Jackson M. (2014). The mycobacterial cell envelope-lipids. Cold Spring Harbor perspectives in medicine, 4(10): a021105.

Jarlier, V. and Nikaido, H. (1994). Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiology Letters. 123: 11-18.

Jasmer, R.M., Nahid, P. and Hopewell, P.C. (2004). Clinical practice: latent tuberculosis infection. New England Journal of Medicine. 347: 1860-1866.

Jisaka, M., Ohigashi, H., Takegawa, K., Huffman, M.A. and Koshimizu, K. (1993). Antitumoral and antimicrobial activities of bitter sesquiterpene lactones of Vernonia amygdalina, a possible medicinal plant used by wild chimpanzees. Bioscience, Biotechnology and Biochemistry. 57: 833-834.

Johri, R.K. and Singh, C. (1997). Medicinal uses of Vernonia species. Journal of Medicinal and Aromatic Plant Science. 19: 744-752.

Jordan, T.S., Spencer, E.M. and Davies, P. (2010). Tuberculosis, bronchiectasis and chronic airflow obstruction. Respirology. 15(4): 623–628.

Kahkonen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. The Journal of Agricultural and Food Chemistry. 47(10): 3954-3962.

Kar, A. (2007). Pharmaocgnosy and pharmacobiotechnology (Revised Expanded Second Edition). New Age International Limited Publishers, New Delhi. 332-600

101

Karamian, R. and Ghasemlou, F. (2013). Screening of total phenol and flavonoid content, antioxidant and antibacterial activities of the methanolic extracts of three Silene species from Iran. International Journal of Agriculture and Crop Sciences. 5(3): 305-312.

Karsha, P.V. and Lakshmi, O.B. (2010). Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference to its mode of action on bacteria. Indian Journal of Natural Products and Resources. 213-215.

Karumbi, J. and Garner, P. (2015). Directly observed therapy for treating tuberculosis. Cochrane Database Systematic Reviews 5: CD003343-CD003343.

Karthikeyan, S. C., Velmurugan, S., Donio, M. B. S., Michaelbabu, M. and Citarasu, T. (2014). Studies on the antimicrobial potential and structural characterization of fatty acids extracted from Sydney rock oyster Saccostrea glomerata. Annals of Clinical Microbiology and Antimicrobials. 13(1): 1-11.

Kastrinsky, D.B., McBride, N.S., Backus, K.M., LeBlanc, J.J. and Barry III, C.E. (2010). Mycolic acid/cyclopropane fatty acid/fatty acid biosynthesis and health relations. In Comprehensive Natural Products II, Elsevier, Oxford. 65-145.

Kaufmann, S.H. (2001). How can immunology contribute to the control of tuberculosis? Nature Reviews Immunology 1. 20-30.

King G.M. (2003). Uptake of carbon monoxide and hydrogen at environmentally relevant concentrations by mycobacteria. Applied and Environmental Microbiology. 69(12): 7266–7272.

Kisangau, D.P., Hosea, K.M., Joseph, C.C. and Lyaruu, H.V.M. (2007) In vitro antimicrobial assay of plants used in traditional medicine in Bukoba rural district, Tanzania. African Journal of Traditional, Complementary and Alternative Medicines. 4: 510-523.

Koc, S., Isgor, B.S. Isgor, Y.B., Moghaddam, N.S. and Yildirim, O. (2015) The potential medicinal value of plants from Asteraceae family with antioxidant defense enzymes as biological targets. Pharmaceutical Biology. 53(5): 746-751.

Kokwaro, J.O. (2009). Medicinial Plants of East Africa. Nairobi: University of Nairobi Press

Krishnaiah, D., Sarbatly, R. and Nithyanandam, R.R. (2011). A review of the antioxidant potential of medicinal plant species. Food and Bioproducts Processing. 89: 217-233.

Kruger, N.J. (1994). The Bradford method for protein quantitation. In: Walker J.M. (eds) Basic Protein and Peptide Protocols. Methods in Molecular Biology™. Humana Press 32: 9-15.

Kruger, N.J. (1996). The Bradford method for protein quantitation. In Protein Protocols Handbook (Walker, J.M., ed.). Totowa, NJ: Humana Press. 15-20.

Kuete, V. and Efferth, T. (2010). Cameroonian medicinal plants: pharmacology and derived natural products. Frontiers in Pharmacology. 1: 123.

102

Kutchan, T.M. (1995). Alkaloid biosynthesis-the basis for metabolic engineering of medicinal plants. Plant Cell. 7: 1059-1070.

Lahiri, R., Randhawa, B. and Krahenbuhl, J. (2005). Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad. Journal of Medical Microbiology. 54: 235-242.

Lakshmi P.J. (2015). Therapeutic uses of Vernonia cinereal. International Journal of Pharmaceutical and Clinical Research. 7(4): 323-325.

Lall, N. and Meyer, J.J.M. (1999). In vitro inhibition of drug-resistant and drug-sensitive strains of Mycobacterium tuberculosis by ethnobotanically selected South African plants. Journal of Ethnopharmacology. 66: 347–354.

Lange, C., Abubakar, I., Alffenaar, J.W.C., Bothamley, G., Caminero, J.A., Carvalho, A.C.C., … Cirillo, D.M. (2014). Management of patients with multidrug-resistant/extensively drug-resistant tuberculosis in Europe: a TBNET consensus statement. The European Respiratory Journal. 44(1): 23–63.

Lаurenzi, M., Ginsberg, A. and Spigelman, M. (2007). Challenges associated with current and future TB treatment. Infectious Disorders - Drug Targets. 7: 105-119.

Lee, M.T., Lin, W.C., Yu, B. and Lee, T.T. (2017). Antioxidant capacity of phytochemicals and their potential effects on oxidative status in animals - A review. Asian-Australasian Journal of Animal Sciences. 30: 299-308.

Lee, S.Y., Park, H.J., Best-Popescu, C., Jang, S., Park, Y.K. (2015). The effects of ethanol onthe morphological and biochemical properties of individual human red blood cells. PLoS ONE 10(12): e0145327.

Lechner, D., Gibbons, S. and Bucar, F. (2008). Plant phenolic compounds as ethidium bromide efflux inhibitors in Mycobacterium smegmatis. Journal of Antimicrobial Chemotherapy. 62: 345- 348.

Levy, S.B. and Marshall B. (2004). Antibacterial resistance worldwide: causes, challenges and responses. Nature Medicine Supplement. 10(12): S122-S129.

Lewis, J.S. and Jorgensen, J.H. (2005). Inducible clindamycin resistance in Staphylococci: Should clinicians and microbiologists be concerned? Clinical Infectious Diseases. 40(2): 280–285.

Lienhardt, C., Vernon, A. and Raviglione, M.C. (2010). New drugs and new regimens for the treatment of tuberculosis: review of the drug development pipeline and implications for national programmes. Current Opinion in Pulmonary Medicine. 16(3): 186–193.

Li, S., Chen, G., Zhang, C., Wu, M., Wu, S. and Liu, Q. (2014). Research progress of natural antioxidants in foods for the treatment of disease. Food Science and Human Wellness. 3(3–4): 110-116.

103

Lin, P. L. and Flynn, J. L. (2010). Understanding latent tuberculosis: a moving target. Journal of Immunology. 185: 15–22.

Lobo, V., Patil, A., Phatak, A. and Chandra, N. (2010). Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews. 4(8): 118-126.

Luo, X., Jiang, Y., Fronczek, F. R., Lin, C., Izevbigie, E. B., Lee, S. and Lee, K. S. (2017). Isolation and structure determination of a sesquiterpene lactone (vernodalinol) from Vernonia amygdalina extracts. Pharmaceutical Biology. 49(5): 464-470.

Mahomoodally, M.F. (2013). Traditional medicines in Africa: an appraisal of ten potent African medicinal plants. Evidence-Based Complementary and Alternative Medicine. 1–14

Mamta, S., Jyoti S., Rajeev, N., Dharmendra, S. and Abhishek, G. (2013). Phytochemistry of medicinal plants. Journal of Pharmacognosy and Phytochemistry. 1: 6-8.

Mannan, M.A., Sarker, T.C., Rahman, M.M. and Alam, M.F. (2013). Screening of phytochemical compounds and antioxidant properties in local and HYV of Bangladeshi rice (Oryza sativa L.). International Journal of Biosciences. 3(4): 151-160.

Maobe, M.A.G, Gatebe, E., Gitu, L. and Rotich, H. (2013). Preliminary phytochemical screening of eight selected medicinal herbs used for the treatment of diabetes, malaria and pneumonia in Kisii region southwest Kenya. European journal of applied sciences. 5(10): 1-6.

Marin-Penalver, J.J., Martin-Timon, I., Sevillano-Collantes, C. and Del Canizo-Gomez, F.J. (2016). Update on the treatment of type 2 diabetes mellitus. World Journal of Diabetes. 7(17): 354–395.

Martin, A. (2011). Identification of mycobacteria. Microbiology Focus Edition 2.2. Sigma Aldrich. http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/microbiology- focus/pdf/identification-of0.pdf. Accessed 23 October 2017

Mavi, A., Terzi, Z., Ozoen, U., Yildirim, A., Coskun, M. (2003). Antioxidant properties of some medicinal plants: Prangos ferulacea (Apiaceae), Sedum sempervivoides (Crassulaceae), Malva neglecta (Malvaceae), Cruciata taurica (Rubiaceae), Rosa pimpinellifolia (Rosaceae), Galium verum subsp. verum (Rubiaceae), Urtica dioica (Urticaceae). Biological and Pharmaceutical Bulletin. 27: 702-705.

McGaw, L.J., Lall, N., Meyer, J.J.M. and Eloff, J.N. (2008). The potential of South African plants against Mycobacterium infections. Journal of Ethnopharmacology. 119: 482-500.

McGarvey, J.A., Wagner, D. and Bermudez, L.E. (2004). Differential gene expression in mononuclear phagocytes infected with pathogenic and non-pathogenic mycobacteria. Clinical and Experimental Immunology. 136(3): 490-500.

McRae, J., Yang, Q., Crawford, R. and Palombo, E. (2007). Review of methods used for isolating pharmaceutical lead compounds from traditional medicinal plants. Environmentalist.

104

27: 165-174.

Meenakshi, S., Manicka, G.D., Tamil, Mozhi, S. et al. (2009). Total flavanoid and in vitro antioxidant activity of two seaweeds of Rameshwaram coast. Global Journal of Pharmacology. 3: 59-62.

Meena, L.S. and Rajni. (2010). Survival mechanisms of pathogenic Mycobacterium tuberculosis H37Rv. FEBS Journal. 277: 2416 – 2427.

Mery, B., Guy, J.B., Vallard, A., Espenel, S., Ardail, D., Rodriguez-Lafrasse, C., Rancoule, C. and Magne, N. (2017). In vitro cell death determination for drug discovery: A landscape review of real issues. Journal of Cell Death. 10: 1179670717691251.

Mikusova, K., Slayden, R.A., Besra, G.S. and Brennan, P.J. (1995). Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrobial Agents and Chemotherapy. 39(11): 2484–2489.

Mohajan, H.K. (2015). tuberculosis is a fatal disease among some developing countries of the world. American Journal of Infectious Diseases and Microbiology. 3(1): 18–31.

Mohandas, N., Chasis, J.A. (1993). Red blood cell deformability, membrane material properties and shape: regulation by transmembrane, skeletal and cytosolic proteins and lipids. Seminars in Hematology. 30:171–192.

Munot, P. P., Mhapuskar, A. A., Ganvir, S. M., Hazarey, V. K., Mhapuskar, M. A., and Kulkarni, D. (2015). Detection of acid-fast bacilli in saliva using papanicolaou stain induced fluorescence method versus fluorochrome staining: An evaluative study. Journal of international oral health. 7(7): 115–120.

Morobe, I.C., Mthethwa, N.S., Bisi-Johnson, M.A., Vasaikar, S.D., Obi, C.L., Oyedeji, A.O., Kambizi, L., Eloff, J.N. and Hattori, T. (2012). Cytotoxic effects and safety profiles of extracts of active medicinal plants from South Africa. Journal of Microbiology Research. 2(6): 176-182.

Morrissey, J.P. and Osbourn, A.E. (1999). Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiology and Molecular Biology Reviews. 63: 708-724.

Mortimer, P.G. and Piddock, L.G. (1991). A comparison of methods used for measuring the accumulation of quionolones by Enterobactericeae, Pseudomonas aueruginosas and Staphylococus aureus. Journal of Antimicrobial Chemotherapy. 28: 639-653.

Moyo, B., Mukanganyama, S. (2015). Antibacterial effects of Cissus welwitschii and Triumfetta welwitschii extracts against Escherichia coli and Bacillus cereus. International Journal of Bacteriology http://dx.doi.org/10.1155/2015/162028.

Moses, A.O., Emmanuel, O.O., Ganiyu, A.O., Fidelis, A.A. and Dickson, A.O. (2008). Assessment of antioxidants and nutritional status of pulmonary tuberculosis patients in Nigeria. European Journal of General Medicine. 5(4): 208-211.

105

Msuya, J. (2005). Horizontal and vertical delivery of health services: what are the tradeoffs? Washington, DC, World Bank. 1-28.

Muhindi, S.W., Ngule C.M. and Ramesh F. (2016). Phytochemical and antibacterial potential of Vernonia adoensis stem bark to curb cariogenic microorganisms. American Journal of Phytomedicine and Clinical Therapeutics. 4(01): 019-027.

Munita, J.M. and Arias, C.A. (2016). Mechanisms of antibiotic resistance. Microbiology Spectrum. 4(2): 10.1128/microbiolspec.VMBF–0016–2015.

National Institute of Allergy and Infectious Diseases (NIAID) (2016). https://www.niaid.nih.gov/diseases-conditions/tbdrugs. Accessed 12/10/2017.

Naik, S.K., Mohanty, S., Padhi, A., Pati, R. and Sonawane, A. (2014). Evaluation of antibacterial and cytotoxic activity of Artemisia nilagirica and Murraya koenigii leaf extracts against mycobacteria and macrophages. BMC Complementary and Alternative Medicine. 14(1): 87

Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R. and De Feo, V. (2013). Effect of essential oils on pathogenic bacteria. Pharmaceuticals. 6(12): 1451-1474.

Ncube, N.S., Afolayan, A.J. and Okoh, A.I. (2008). Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. African Journal of Biotechnology. 7(12): 1797-1806.

Newton, G. and Fahey, R. (2002). Mycothiol biochemistry. Archives of Microbiology. 178: 388- 394.

Newton, S.M., Lau, C. and Wright, C.W. (2000). A review of antimycobacterial natural products. Phytotherapy Research. 14: 302-322.

Nguyen, K.T., Piastro, K., Gray, T.A. and Derbyshire, K.M. (2010). Mycobacterial biofilms facilitate horizontal DNA transfer between strains of Mycobacterium smegmatis. Journal of Bacteriology. 192: 5134–5142.

Nikaido, H. (1996). Multidrug efflux pumps of gram-negative bacteria. Journal of Bacteriology. 178(20): 5853–5859.

Noble, J.E. and Bailey, M.J.A. (2009) Methods in enzymology. Elsevier BV 463(C): 73-95.

Noudeh, G.D., Sharififar, F., Khatib, M., Behravan, E., Afzadi, M.A. (2010). Study of aqueous extract of three medicinal plants on cell membrane–permeabilizing and their surface properties. African Journal of Biotechnology. 9: 110-116.

Nyambuya, T., Mautsa, R. and Mukanganyama S. (2017). Alkaloid extracts from Combretum zeyheri inhibit the growth of Mycobacterium smegmatis. BMC Complementary and Alternative Medicine. 17: 124.

106

Nwosu, S.I., Stanley, H.O. and Okerentugba, P.O. (2013). Occurrence, types and location of calcium oxalate crystals in Vernonia amydalina Del (Asteraceae). International Journal of Natural Sciences. 4(3): 533-537.

O'Brien, R.J., Geiter, L.J. and Snider, D.E. Jr. (1987). The epidemiology of nontuberculous mycobacterial diseases in the United States. Results from a national survey. The American Review of Respiratory Disease. 135:1007-1014.

Obadoni, B.O. and Ochuko, P.O. (2001). Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta States of Nigeria. Global Journal of Pure and Applied Sciences. 86: 2003-2008.

Odukoya, O.A., Inya-Agha S.I., Segun, F.I., Sofidiya, M.O. and Ilori, O.O. (2007). Antioxidant activity of selected Nigerian green leafy vegetables. American Journal of Food Technology. 2: 169–175.

Orhan, D.D., Ozçelik, B., Hosbas, S. and Vural, M. (2012). Assessment of antioxidant, antibacterial, antimycobacterial, and antifungal activities of some plants used as folk remedies in Turkey against dermatophytes and yeast-like fungi. Turkish Journal of Biology. 36: 672-686.

Orsine J.V.C, da Costa R.V., da Silva R.C., Santos M.M.A. and Novaes M.R.C.G. (2012). The acute cytotoxicity and lethal concentration (LC50) of Agaricus sylvaticus through hemolytic activity on human erythrocyte. International Journal of Nutrition and Metabolism. 4: 19-23.

Orwa, C., Mutua, A., Kindt, R., Jamnadass, R., Anthony, S. (2009) Agroforestree Database:a tree reference and selection guide version 4.0 (http://www.worldagroforestry.org/sites/treedbs/treedatabases.asp)

Pagano, M. and Faggio, C. (2015). The use of erythrocyte fragility to assess xenobiotic cytotoxicity. Cell Biochemistry and Function. 33: 351–355.

Pan, S.-Y., Zhou, S.-F., Gao, S.-H., Yu, Z.-L., Zhang, S.-F., Tang, M.-K., … Ko, K.-M. (2013). New perspectives on how to discover drugs from herbal medicines: CAM’s outstanding contribution to modern therapeutics. Evidence-Based Complementary and Alternative Medicine: eCAM. 2013: 627375.

Pan, S. Y., Litscher, G., Gao, S. H., Zhou, S. F., Yu, Z. L., Chen, H. Q. et al., (2014). Historical perspective of traditional indigenous medical practices: the current renaissance and conservation of herbal resources. Evidence-based complementary and alternative medicine: eCAM, 2014, 525340.

Parasuraman, S. (2011). Toxicological screening. Journal of Pharmacology & Pharmacotherapeutics, 2(2): 74–79.

Patel, R.M. (2012). The guiding principles on antimicrobial susceptibility testing. Bulletin of Pharmaceutical Research. 2(3): 146-153.

107

Pathare, Y.S. and Wagh, V.D. (2012). Herbal medicines and nutritional supplements used in the treatment of glaucoma: A review. Research Journal of Pharmaceutical, Biological and Chemical Sciences 3(1): 331-339.

Pawlowski, A., Jansson, M., Skold, M., Rottenberg, M.E. and Kallenius, G. (2012). Tuberculosis and HIV co-infection. PLoS Pathogens. 8(2): p. e1002464.

Peters, K. and Smith, J. (2017). The Francis Crick Institute. Clinical medicine (London, England), 17(2): 105–107.

Phelan, J., Maitra, A., McNeney, R., Nair, M., Gupta, A., Coll, F., Pain, A., Bhakta, S. and Clark, T.G. (2015). The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae. International Journal of Mycobacteriology. 4: 207–216.

Piddock, L.J. (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacterial. Clinical Microbiology Reviews. 19: 382-402.

Pollard, K. (2009). Student engagement in interprofessional working in practice placement settings. Journal of Clinical Nursing. 18: 2846e2856.

Prabhavathi, R.M., Prasad, M.P., Jayaramu, M. (2016). Studies on qualitative and quantitative phytochemical analysis of Cissus quadrangularis. Pelagia Research Library. Advances in Applied Science Research. 27(4): 11-7

Pradhananga, M. and Manandhar, P. (2017). Preservative effects of some selected spice oleoresins to stabilize the sunflower oil in comparison to tertiary butylhydroquinone. Food, Science and Nutrition. 00: 1-5.

Prasch, S. and Bucar, F. (2015). Plant derived inhibitors of bacterial efflux pumps: an update. Phytochemical Review. 14: 961-974.

Pule, C.M., Sampson, S.L., Warren, R.M., Black, P.A., van Helden, P.D., Victor, T.M. and Louw, G.E. (2016). Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy. Journal of Antimicrobial Chemotherapy. 71(1): 17–26.

Puupponen-Pimia, R., Nohynek, L., Ammann, S., Oksman-Caldentey, K.M. and Buchert, J. (2008). Enzyme-assisted processing increases antimicrobial and antioxidant activity of bilberry. Journal of Agricultural and Food Chemistry, 56(3): 681-688.

Rahman, I., Biswas, S.K., Kirkham, P.A. (2006). Regulation of inflammation and redox signaling by dietary polyphenols. Biochemical Pharmacology. 72(11): 1439-1452.

Rahman, K. (2007). Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging. 2: 219-236.

108

Ralph A.C.L., Ferreira S.B., Ferreira V.F., Lima E.S. and Vasconcellos M.C. (2009). Cytotoxicity assessment in synthetic naphthoquinones franciscan and erythrocyte artemia model. C. Biological sciences – Pharmacology 2009: 6-4.

Ramon-Garcia, S., Martin, C., Ainsa, J.A., De Rossi, E. (2006). Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. Journal of Antimicrobial Chemotherapy. 57: 252–259.

Rao, B.N. (2003). Bioactive phytochemicals in Indian foods and their potential in health promotion and disease prevention. Asia Pacific Journal of Clinical Nutrition. 12(1): 9-22.

Rashid, N., Gbedomon, R.C., Ahmad, M., Salako, V.K., Zafar, M., Malik, K. (2018). Tradit etional knowledge on herbal drinks among indigenous communities in Azad Jammu and Kashmir, Pakistan Journal of Ethnobiology and Ethnomedicine. 14: 16.

Rastogi, N., Legrand, E. and Sola, C. (2001). The Mycobacteria: an introduction to nomenclature and pathogenesis. Revue scientifique et technique (International Office of Epizootics). 20: 21–54.

Raynaud, C., Laneelle, M.A., Senaratne, R.H., Draper, P., Laneelle, G. and Daffe, M. (1999). Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity. Microbiology. 145(6): 1359.

Reddy, Y.N., Murthy, S.V., Krishna, D.R. and Prabhakar, M.C. (2004). Role of free radicals and antioxidants in tuberculosis patients. Indian Journal of Tuberculosis. 51: 213-218.

Redmile-Gordon, M.A., Armenise, E., White, R.P., Hirsch, P.R. and Goulding, K.W.T. (2013). A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts. Soil Biology and Biochemistry. 67: 166-173.

Rex, J.H., Pfaller, M A., Walsh, T.J., Chaturvedi, V., Espinel-Ingroff, A., Ghannoum, M.A., … Warnock, D.W. (2001). Antifungal susceptibility testing: Practical aspects and current challenges. Clinical Microbiology Reviews. 14(4): 643–658.

Riley, L.W. (2006). Of mice, men, and elephants: Mycobacterium tuberculosis cell envelope lipids and pathogenesis. Journal of Clinical Investigation. 116(6): 1475-1478.

Rios, J.L. and Recio, M.C. (2005). Medicinal plants and antimicrobial activity. Journal of Ethnopharmacology. 100: 80-84.

Riss, T.L. and Moravec, R.A. (2004). Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay and Drug Development Technologies. 2(1): 51-62.

109

Russell, D.G. (2001). Mycobacterium tuberculosis: here today, and here tomorrow. Nature Reviews Molecular Cell Biology. 2: 569–577

Russell, D. G., Barry, C. E. and Flynn, J. L. (2010). Tuberculosis: What we don’t know can, and does, hurt us. Science (New York, N.Y.). 328(5980): 852–856.

Saeed, N., Khan, M.R. and Shabbir, M. (2012). Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and Alternative Medicine. 12: 221.

Sabatini, S., Gosetto, F., Serritella, S., Manfroni, G., Tabarrini, O., Iraci, N., Brincat, J.P., Carosati, E., Villarini, M., Kaatz, G.W. et al. (2012). Pyrazolo[4,3-c][1,2]benzothiazines 5,5- dioxide: A promising new class of Staphylococcus aureus NorA efflux pump inhibitors. Journal of Medicinal Chemistry 55: 3568–3572.

Sanchez, J.G.B. and Kouznetsov, V.V. (2010). Antimycobacterial susceptibility testing methods for natural products research. Brazilian Journal of Microbiology. 41(2): 270–277.

Sandhu, G.K. (2011). Tuberculosis: current situation, challenges and overview of its control programs in India. Journal of Global Infectious Diseases. 3: 143-150.

Sanusi, S. B., Abu Bakar, M. F., Mohamed, M., Sabran, S. F. and Mainasara, M. M. (2017). Southeast Asian medicinal plants as a potential source of antituberculosis agent. Evidence-Based Complementary and Alternative Medicine. 1-39.

Sarathy, J.P., Dartois, V. and Lee, E.J.D. (2012). The role of transport mechanisms in Mycobacterium Tuberculosis drug resistance and tolerance. Pharmaceuticals. 5(11): 1210-1235.

Saritha, K., Rajesh, A., Manjulatha, K., Setty, O.H. and Yenugu, S. (2015). Mechanism of antibacterial action of the alcoholic extracts of Hemidesmus indicus (L.) R. Br. ex Schult, Leucas aspera (Wild.), Plumbago zeylanica L., and Tridax procumbens (L.) R. Br. ex Schult. Frontiers in Microbiology. 6: 577.

Sarkar, S., Ganguly, A. and Sunwoo, H.H. (2016). Current overview of anti-tuberculosis drugs: metabolism and toxicities. Mycobacterial Diseases. 6: 209.

Schaefer, W.B. (1954). The effect of isoniazid on growing and resting tubercle bacilli. American Review of Tuberculosis. 69: 125-127.

Science Prof Online. (2014). Mycobacterium smegmatis bacterial colonies. http://www.scienceprofonline.com/science-image-libr/sci-image-libr-Mycobacterium- bacteria.html. Accessed 28 October 2017

Scorpio, A. and Zhang, Y. (1996). Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nature Medicine. 2: 662-667.

110

Scott, G., Springfield, E.P. and Coldrey, N. (2004) A pharmacognostical study of 26 South African plant species used as traditional medicines. Pharmaceutical Biology. 42(3): 186-213.

Sharma, S., Sharma, M., Roy, S., Kumar, P. and Bose, M. (2004). Mycobacterium tuberculosis induces high production of nitric oxide in coordination with production of tumour necrosis factoralpha in patients with fresh active tuberculosis but not in MDR tuberculosis. Immunology and Cell Biology. 82(4): 377-382.

Shastri, M. D., Shukla, S. D., Chong, W. C., Dua, K., Peterson, G. M., Patel, R. P., … O'Toole, R. F. (2018). Role of oxidative stress in the pathology and management of human tuberculosis. Oxidative medicine and cellular longevity. 2018: 7695364.

Sharwan, G., Jain, P., Pandey, R. and Shukla, S. S. (2016). Toxicity and safety profiles ofmethanolic extract of pistacia integerrima J. L. Stewart Ex Brandis (pi) for wistar rats. Journal of Pharmacopuncture. 19(3): 253–258.

Shehzad, A., Rehman, G., Ul-Islam, M., Khattak, W.A. and Lee, Y.S. (2013). Challenges in the development of drugs for the treatment of tuberculosis. The Brazilian Journal of Infectious Diseases 17(1): 74–81.

Shepard, C.C. (1957). growth characteristics of tubercle bacilli and certain other mycobacteria in Hela cells. The Journal of Experimental Medicine. 105(1): 39–48.

Shi, J., Yu, J., Pohorly, J.E., Kakuda, Y. (2003). Polyphenolics in grape seeds—biochemistry and functionality. Journal of Medicinal Food. 6: 291-299.

Shi, W., Zhang, X., Jiang, X., Yuan, H., Lee, J.S., Barry, C.E. 3rd, Wang, H., Zhang, W. and Zhang, Y. (2011). Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 16, 333(6049): 1630-1632.

Shiloh, M.U. and Champion P.A. (2010). To catch a killer. What can mycobacterial models teach us about Mycobacterium tuberculosis pathogenesis? Current opinion in microbiology. 13: 86–92.

Shinnick, T. (1996). (Ed.), Current Topics in Microbiology and Immunology, Springer- Verlag, New York.

Silaigwana, B., Green, E. and Ndips, R.N. (2012). Molecular detection and drug resistance of Mycobacterium tuberculosis complex from cattle at a dairy farm in the Nkonkobe region of South Africa: A pilot study. International Journal of Environmental Research and Public Health. 9: 2045-2056.

Singh, G. and Kumar, P. (2013). Extraction, gas chromatography–mass spectrometry analysis and screening of fruits of Terminalia chebula Retz. for its antimicrobial potential. Pharmacognosy Research. 5(3): 162-168.

111

Slayden, R.A. and Barry, C.E., III (2000). The genetics and biochemistry of isoniazid resistance in Mycobacterium tuberculosis. Microbes and Infection 2: 659–669.

Sloan, D. J., Davies, G. R. and Khoo, S. H. (2013). Recent advances in tuberculosis: New drugs and treatment regimens. Current Respiratory Medicine Reviews. 9(3): 200–210.

Smieja, M., Marchetti, C., Cook, D. and Smaill, F. M. (1999). Isoniazid for preventing tuberculosis in non-HIV infected persons. In Cochrane Database of Systematic Reviews. John Wiley & Sons, Ltd.

Smith, I. (2003). Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clinical Microbiology Reviews. 16(3): 463–496.

Spelman, K., Burns, J., Nichols, D., Winters, N., Ottersberg, S. and Tenborg, M. (2006). Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Alternative Medicine Review. 11: 128-150.

Stanic, M. (2013). Mycobacteria. http://svet- biologije.com/biologija/mikrobiologija/bakterije/klasifikacija-bakterija/mikobakterije/ Accessed 28 October 2017.

Stavri, M., Piddock, L.V.J. and Gibbons, S. (2007) Bacterial efflux pump inhibitors from natural sources. Journal of Antimicrobial Chemotherapy 59: 1247-1260.

Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K. and Ren, Q. (2015). Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiology. 15: 36.

Sunduru, N., Sharma, M., Chauhan and P.M.S. (2010). Recent advances in the design and synthesis of Heterocycles as anti-tubercular agents. Future Medicinal Chemistry 2(9): 1469-1500.

Surendranath, C.R., Ramana, B. V., Chaudhury, A. and Kumar, A.G. (2014). Functional study of efflux pump inhibitors in clinical isolates of multidrug resistant Klebsiella pneumoniae. Asian Journal of Pharmaceutical and Clinical Research. 7(1): 80-82.

Swamy T.A., Obey J. and Mutuku N.C. (2013). Phytochemical analysis of Vernonia adoensis leaves and roots used as a traditional medicinal plant in Kenya. International Journal of Pharmacy and Biological Sciences. 3(3): 46-52.

Syed, M., Danish, R., Deboshree, B., Jamal, M.A. and Mohd, Z. (2011). In vitro antibacterial and antioxidant potential of leaf extracts and flower extracts of Vernonia conerea and its phytochemical comstituents. International Journal of Pharmaceutical Sciences Review and Research. 9(2): 164.

Tapas, A.R., Sakarkar, D.M. and Kakde, R.B. (2008). Review article flavonoids as nutraceuticals: A review. Tropical Journal of Pharmaceutical Research. 7:1089–1099

112

Taylor, R.S., Edel, F., Manandhar, N.P., Towers, G.H. (1996). Antimicrobial activities of Southern Nepalese medicinal plants. Journal of Ethnopharacology. 50: 97-102

The Approved List of biological agents (2013). http://www.hse.gov.uk/pUbns/misc208.pdf Accessed 13 December 2017

Tenover, F.C. (2009) Antimicrobial susceptibility testing methods for bacterial pathogens. In: Mayers D.L. (eds) Antimicrobial Drug Resistance. Infectious Disease. Humana Press. 1151.

Thatoi, H. and Patra, J. K. (2011). Biotechnology and Pharmacological Evaluation of Medicinal Plants: An Overview. Journal of Herbs, Spices & Medicinal Plants, 17(3): 214–248.

Thelestam, M. and Mollby, R. (1975). Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infection and Immunity. 11(4): 640–648.

Thiagarajan, V.R., Shanmugam, P., Krishnan, U.M., Muthuraman, A. (2014). Ameliorative potential of Vernonia cinerea on chronic constriction injury of sciatic nerve induced neuropathic pain in rats. Anais da Academia Brasileira de Ciências. 86(3): 1435-1450.

Tortoli, E. (2003). Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clinical Microbiology Reviews. 16(2): 319–354.

Tripathi, L. and Tripathi, J.N. (2003). Role of biotechnology in medicinal plants. Tropical Journal of Pharmaceutical Research. 2(2): 243-253.

Trombetta, D., Castelli, F., Sarpietro, M.G., et al. (2005). Mechanisms of antibacterial action of three monoterpenes. Antimicrobial Agents Chemotherapy. 49(6): 2474-2478.

Tsao, R. and Deng, Z. (2004). Separation procedures for naturally occurring antioxidant phytochemicals. The Journal of Chromatography B. 812: 85-99.

Tsukamura, M. (1966). Adansonian classification of mycobacteria. Journal of General Microbiology. 45: 253-273. van-Burden, J.P. and Robinson, W.B. (1969). Formation of complexes between protein and tannin acid. The Journal of Agricultural and Food Chemistry. 17(4): 772-777. van den Boogaard, J., Kibiki, G.S., Kisanga, E.R., Boeree, M.J. and Aarnoutse, R.E. (2009). New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrobial Agents Chemotherapy. 53: 849–862.

Van Wyk, B.E. and Wink, M. (2004). Medicinal Plants of the World. Briza Publications, Pretoria.

Veeresham, C. (2012). Natural products derived from plants as a source of drugs. Journal of Advanced Pharmaceutical Technology & Research. 3(4): 200–201.

113

Velayati, A.A., Rahideh, S., Derakhshani Z., Farnia, P. and Mirsaeidi, M. (2015). Nontuberculous mycobacteria in Middle East: current situation and future challenges. International Journal of Mycobacteriology. 4(1): 7–17.

Vicki, A.F., Alfonso, S., Tod, F.S. and Harold, R. (2009) Classification of Compositae. The International Compositae Alliance.

Vickery, M.L. and Vickery, B. (1981). Compound derived from amimo acids. In Vickery M.L.,Vickery, B. editors. Secondary plant metabolism. London: Springer. 220–254

Vipra, A., Desai, S.N., Junjappa, R.P., Roy, P., Poonacha, N., Ravinder, P., Seriran, P. and Radmanabha, S. (2013). Determining the minimum inhibitory concentration on bacteriophages: Potential advantages. Microbiology Team, Gangagen Biotechnologies Pvt Ltd. 3: 180-190.

Vitoria, M., Granich. R., Gilks. C.F., Gunneberg, C., Hosseini, M., Were. W., Raviglione, M. and De Cock, K.M. (2009). The global fight against HIV/AIDS, Tuberculosis, and Malaria: Current status and future perspectives. American Journal of Clinical Pathology. 131(6): 844– 848.

Viveiros, M., Martins, M., Rodrigues, L. et al. (2012). Inhibitors of mycobacterial efflux pumps as potential boosters for anti-tubercular drugs. Expert Review of Anti-infective Therapy. 10: 983–998.

War, A.R., Paulraj, M.G., Ahmad, T., Buhroo, A.A., Hussain, B., Ignacimuthu, S., and Sharma, H.C. (2012). Mechanisms of plant defense against insect herbivores. Plant signaling & behavior. 7(10): 1306–1320.

Webber, M.A. and Piddock, L.J.V. (2003). The importance of efflux pumps in bacterial antibiotic resistance. Journal of Antimicrobial Chemotherapy. 51(1): 9-11.

Wehrli, W. and Staehelin, M. (1971). Actions of the rifamycins. Bacteriological reviews. 35(3): 290–309.

Wiid, I.S., Edmen, T., Hoal, E.G., Benade, A.J. and Van Helden, P.D. (2004). Total antioxidants levels are low during active TB and rise with anti-tuberculosis therapy. The International Union of Biochemistry and Molecular Biology, Life. 56(2): 101-106.

Winder, F.G. (1982). Mode of action of the antimycobacterial agents and associated aspects of the molecular biology of mycobacteria, In Ratledge, C. and Standford, J. (ed.), The biology of mycobacteria, vol. 1. Academic Press, Inc., New York, 353-438.

World Health Organization (1994). TB A global emergency. http://apps.who.int/iris/bitstream/10665/58749/1/WHO_TB_94.177.pdf (Accessed 10/10/17).

World Health Organization (2003). The current anti-TB drug research and development pipeline. (Accessed 28/05/18).

114

World Health Organization (2004). WHO Guidelines: Developing information on proper use of traditional, complementary and alternative medicine. (Accessed 28/05/18).

World Health Organization (2005). Addressing poverty in TB control – options for national TB control programs, Geneva, World Health Organization, WHO/HTM/TB/2005.352. (Accessed 11/11/17)-.

World Health Organization (2006). The Global Plan to Stop TB 2006-2015: Part I. Strategic directions. Geneva: WHO; 2006. (Accessed on 22/12/17).

World Health Organization (2007). Improving the diagnosis and treatment of smear-negative pulmonary and extrapulmonary tuberculosis among adults and adolescents: Recommendations for HIV-prevalent and resource-constrained settings. (Accesses 31/05/18).

World Health Organization (2010). Treatment of Tuberculosis, Guidelines, 4th Ed., WHO Press, Geneva. ((Accessed 26/10/17).

World Health Organization. (2011). Global tuberculosis control: WHO report 2011. Geneva:World Health Organization. http://www.who.int/iris/handle/10665/44728 (Accessed 22/10/17).

World Health Organization (2015). Global tuberculosis report 2015, 20th ed. (Accesses 4/06/18).

World Health Organization. (2017). Global tuberculosis report 2017. World Health Organization. (Accessed 6/05/18).

Yeap, R.S.K., Ho, W.Y., Beh, B.K., Liang, W.S., Ky, H., Yousr, A.H.N., and Alitheen, N.B. (2010). Vernonia amygdalina, an ethnoveterinary and ethnomedical used green vegetable with multiple bioactivities. Journal of Medicinal Plants Research 4(25): 2787-2812.

Yokota, A., Takeuchi, M., Sakane, T. and Weiss, N. (1993). Proposal of two new species in the genus Microbacterium: Microbacterium dextranolyticum sp. nov. and Microbacterium aurum sp. nov. International Journal of Systematic Bacteriology. 43: 549–554.

Young, I.S. and Woodside, J.V. (2001). Antioxidants in health and disease. Journal of Clinical Pathology. 54:176-186.

Zaman, K. (2010). Tuberculosis: A Global Health Problem. Journal of Health, Population, and Nutrition, 28(2): 111–113.

Zhang, Y., B. Heym, B. Allen, D. Young, and S. Cole. (1992). The catalase peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature (London) 358:591–593.

115

Zhang, Y., Scorpio, A., Nikaido, H. and Sun, Z. (1999). Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. Journal of Bacteriology. 181: 2044–2049.

Zhang, J.-H., Chung, T.D.Y. and Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening. 4(2): 67-73

Zhang, X. (2002). WHO Traditional Medicine Strategy 2002–2005. World Health Organization: Geneva, Switzerland. 1-74.

Zhang, Y., Wade, M.M., Scorpio, A., Zhang, H. and Sun, Z. (2003). Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid. Journal of Antimicrobial Chemotherapy. 52(5):790-795.

Zhang, Y., Shi, W., Zhang, W. and Mitchison, D. (2013). Mechanisms of Pyrazinamide Action and Resistance. Microbiology Spectrum. 2(4): 1–12.

Zimhony, O., Cox, J.S., Welch, J.T., Vilcheze, C. and Jacobs, W.R. (2000). Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Journal of Natural Medicine. 6(9): 1043.

Zohra M. and Fawzia A. (2014). Hemolytic activity of different herbal extracts used in Algeria. International Journal of Pharma Sciences and Research. 5(8): 495-500.

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APPENDICES

Appendix 1: The raw results that were used to calculate activity of V. adoensis ethyl acetate extract

Microplate readings for the most potent extract: Ethyl acetate leaf extract

Microplate reading 1

<> 1 2 3 4 5 6 7 8 9 10 11 12

A 0.1436 0.0958 0.0811 0.0542 0.0428 0.0369 0.0326 0.0350 0.0340 0.0324 0.0314 0.6672

B 0.2609 0.2808 0.0622 0.0493 0.0431 0.0365 0.0350 0.1376 0.0349 0.0325 0.0345 0.6802

C 0.1708 0.1202 0.0803 0.0655 0.3225 0.6247 0.6602 0.7107 0.6829 0.7287 0.0825 0.7543

D 0.2258 0.1191 0.0666 0.0732 0.1981 0.6340 0.7307 0.7333 0.7197 0.7486 0.0391 0.7434

E 0.2331 0.1052 0.0643 0.0685 0.1766 0.5254 0.6666 0.7701 0.7634 0.7392 0.1156 0.7518

F 0.2431 0.1023 0.0649 0.0872 0.1818 0.6715 0.6700 0.6480 0.7260 0.8439 0.0354 0.7728

G 0.0642 0.0481 0.0388 0.0359 0.1662 0.3715 0.4918 0.6940 0.7348 0.7749 0.2115 0.7647

H 0.0661 0.0474 0.5077 0.5379 0.0353 0.3134 0.5369 0.8002 0.8552 0.8048 0.0317 0.7611

Microplate reading 2

<> 1 2 3 4 5 6 7 8 9 10 11 12

A 0.1663 0.1203 0.0813 0.0567 0.0402 0.0371 0.0341 0.0340 0.0336 0.0336 0.0323 0.0350

B 0.1918 0.1209 0.0702 0.0520 0.0417 0.0368 0.0351 0.0341 0.0337 0.0331 0.0331 0.0341

C 0.2358 0.1268 0.0722 0.0500 0.0425 0.0362 0.0349 0.0329 0.0352 0.0318 0.0331 0.0349

D 0.2611 0.1234 0.0607 0.0573 0.0408 0.0371 0.0357 0.0398 0.0317 0.0321 0.0346 0.0350

E 0.2357 0.0930 0.0594 0.0481 0.0392 0.0364 0.0345 0.0340 0.0338 0.0334 0.0330 0.0343

F 0.2416 0.0965 0.0602 0.0475 0.0387 0.0354 0.0347 0.0370 0.0342 0.0323 0.0329 0.0339

G 0.0599 0.0473 0.0397 0.0348 0.0322 0.0327 0.0321 0.0311 0.0314 0.0329 0.0325 0.0353

H 0.0612 0.0470 0.0387 0.0320 0.0315 0.0328 0.0311 0.0309 0.0303 0.0310 0.0332 0.0370

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Appendix 2: Standard curve for BSA

0.6

0.4 R2=0.9856

0.2 Absorbance at 650nm Absorbance

0.0 0 50 100 150 200 250 Protein Concentration(g/ml)

The standard curve that was used in the evaluation of protein

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Appendix 3. Publication

Mautsa, R. and Mukanganyama, S. (2017). Vernonia adoensis leaf extracts cause cellular membrane disruption and nucleic acid leakage in Mycobacterium smegmatis. Journal of *Biologically Active

Products from Nature. 7:2, 140-156, DOI: 10.1080/22311866.2017.1324321

119

Appendix 4. Conference presentations

a) Title of presentation: Evaluation of the antimycobacterial properties of extracts from

Vernonia adoensis

Conference Title and Date: 3 rd. NAPRECA-Zimbabwe Symposium; 9 December 2016

Venue: Cresta Oasis Hotel Harare Zimbabwe

b) Title of presentation: Vernonia adoensis leaf extracts cause cellular membrane disruption and nucleic acid leakage in Mycobacterium smegmatis

Conference title and Date: UZ Research Week; 24-29 July 2017

Venue: UZ Students Union Building.

120