African Potato)

Evaluating the anticancer and antimicrobial properties of extracts from Hypoxis hemerocallidea (African potato)

Xolani Sikhakhane

Evaluating the anticancer and antimicrobial properties of extracts from Hypoxis hemerocallidea (African potato)

Xolani Sikhakhane (200606751)

Dissertation Submitted in fulfilment of the requirements for the degree

Magister Scientiae

Biochemistry

In the

Faculty of Science

at the

University of Johannesburg

Supervisor: Prof Marianne J Cronjé Co-supervisor: Dr Jacinda T James

November 2013

AFFIDAVIT: MASTER AND DOCTORAL STUDENTS

TO WHOM IT MAY CONCERN

This serves to confirm that I______

Full Name(s) and Surname

ID Number/ Passport______

Student number______enrolled for the Qualification ______

______in the Faculty of Science

Herewith declare that my academic work is in line with the Plagiarism Policy of the University of Johannesburg with which I am familiar.

I further declare that the work presented in the ______(minor dissertation/dissertation/thesis) is authentic and original unless clearly indicated otherwise and in such instances full reference to the source is acknowledged and I do not pretend to receive any credit for such acknowledged quotations, and that there is no copyright infringement in my work. I declare that no unethical research practices were used or material gained through dishonesty. I understand that plagiarism is a serious offence and that should I contravene the Plagiarism Policy notwithstanding signing this affidavit, I may be found guilty of a serious criminal offence (perjury) that would amongst other consequences compel the University of Johannesburg to inform all other tertiary institutions of the offence and to issue a corresponding certificate of reprehensible academic conduct to whomever requests such a certificate from the institution.

Signed at Johannesburg ______on this ______of ______2013

Signature ______Print name ______

STAMP: COMMISSIONER OF OATHS

Affidavit certified by a Commissioner of Oaths

This affidavit conforms with the requirements of the JUSTICES OF THE PEACE AND COMMISSIONERS OF OATHS ACT 16 OF 1963 and the applicable Regulations published in the GG GNR 1258 of 21 July 1972; GN 903 of 10 July 1998; GN 109 of 2 February 2001 as amended.

UNIVERSITY OF JOHANNESBURG

FACULTY OF SCIENCE DECLARATION ON SUBMISSION OF FINAL COPIES AND CD

I, (Mr / Mrs / Ms /Dr ):

Student number: ID number / Passport:

declare that the thesis / dissertation / minor dissertation, which I hereby submit for the qualification (e.g. PhD in Zoology, MSc Mathematics, Minor Dissertation in Aquatic Health): at the University of Johannesburg:

 Is the final copy and includes all corrections required by the assessors, supervisor and co-supervisors.  The thesis, dissertation / minor dissertation complies with the requirements of the University of Johannesburg Policy on Plagiarism  The electronic PDF format of the thesis / dissertation / minor dissertation is a true replica of the examined and corrected thesis / dissertation / minor dissertation.  In the case of a MSc Minor dissertation / Dissertation: A manuscript based on a topic of the dissertation in the format of a research article meeting the requirements for publication has been submitted to the supervisor and electronically submitted to the Faculty YES / NO

 In the case of a PhD Thesis: An article based on the thesis accepted for publication by an accredited journal was submitted YES / NO

Evidence of acceptance for publication from the editor or the editorial panel of such an accredited journal was submitted to the Faculty electronically.

I am aware of the fact that I will be subject to criminal prosecution, should this declaration be false or contain a misrepresentation.

SIGNATURE DATE

DECLARATION BY SUPERVISOR AND CO-SUPERVISOR: The submission by the above student of the final corrected thesis/dissertation/minor dissertation, is hereby approved and an electronic manuscript/article is available and has also been submitted to the Faculty.

SUPERVISOR: SIGNATURE DATE

CO-SUPERVISOR: SIGNATURE DATE

Title and copies are in order:

FACULTY OFFICER: POSTGRADUATE STUDIES DATE

“While there’s life, there’s hope” - Marcus Tullius Cicero (106 BC – 43 BC)

“The key to everything is patience. You get the chicken by hatching the egg, not by smashing it” - Arnold Henry Glasow (1905 – 1998)

Throughout my MSc studies I have learned an important life lesson that through hope, patience, persistence, faith and prayer, all things are possible...

I dedicate this to....

My mother

Ntombifikile and aunt

Sibongile for raising me.

ABSTRACT

A rich diversity of medicinal plants is found in Southern Africa and approximately 80% of the population still relies on medicinal plants to fulfil its primary health care needs. Many of these medicinal plants are used to treat ailments such as burns, sores, urinary tract infections, colds, flu, rheumatism, gout, cancer, hypertension, diabetes, human immunodeficiency virus infections and acquired immunodeficiency syndrome. An example of such a plant is Hypoxis hemerocallidea (Fisch & CA Mey), formerly known as Hypoxis rooperi and popularly known as the African potato, from the Hypoxidaceae family. This plant is found across five of the South African provinces and corm extracts are reported to contain bioactive compounds that account for the plant’s medicinal and therapeutic properties.

This study was conducted to investigate the anti-oesophageal cancer and antimicrobial potential of H. hemerocallidea. In cancer patients, the currently used cancer treatments such as radiotherapy and chemotherapy are ineffective in decreasing disease progression, prolonging survival, providing cure and are associated with side-effects such as cytotoxicity to normal body cells and tumour non-specificity. Therefore, current cancer research is aiming at searching for novel plant-based anticancer compounds that can be used for the development and manufacturing of cancer treatment drugs that will have less side-effects and less toxicity towards the normal human body cells, and ultimately provide cure for cancer. In addition to cancer, infectious diseases still contribute to most premature deaths worldwide and are now becoming more difficult to treat due to multidrug resistance developed by pathogens against many of the currently used antibiotics. This multidrug resistance of human pathogens to antibiotics has led to a search for new antimicrobial compounds from plants sources, for use in the production of new affordable antibiotic drugs to effectively treat infections without posing any unwanted toxicity and harm towards the human body.

An oesophageal SNO cancer cell line was treated with H. hemerocallidea extracts and the effect of the extracts on the cancer cells were investigated with cell viability assays (trypan blue dye exclusion and AlamarBlue® viability assays), light microscopy and flow cytometrical analysis (forward and side scatter analysis). The plant extracts were also tested for antimicrobial activities against various microorganisms - Gram-positive and Gram-negative bacteria, yeast and fungi cultures by means of thin layer chromatographic bioautography (TLC-DB), microdilution assays and the BacTiter-GloTM assay. Antimicrobial compounds were then putatively identified and characterised using gas chromatography-mass spectrometry (GC-MS).

No morphological changes were observed in the SNO cells and significant cell death did not occur following treatment with either water or ethanolic H. hemerocallidea extracts from fresh or dried corms or leaves. The ethanolic leaf extracts did not show any significant inhibition against any of the microorganisms tested in contrast to the ethanolic extracts from the corms, which showed microbial growth inhibition against Gram-positive bacteria and fungi and partial inhibition of the Gram-negative bacteria. The bioactive compounds responsible for the antibacterial and antifungal activities were identified as levoglucosan (as the major antimicrobial compound), pyrocatechol and hexahydro-3-(2-methylpropyl)-pyrrolo[1,2- α]pyrazine-1,4-dione. These results show that H. hemerocallidea plant extracts possessed no anticancer effects towards the SNO cell line. In addition, the corm extracts of H. hemerocallidea contain a levoglucosan compound, which may work synergistically with other antimicrobial compounds to exert antimicrobial properties. With more research, the antimicrobial compounds in H. hemerocallidea may hold promise for possible candidates for use in the development of antibiotic or antiseptic products (for example, topical creams and lozenges) to be used in the treatment of skin and soft tissue infections caused by bacterial and fungal infections.

PREFACE

Part of the work in this dissertation was presented at the following symposia:

Biochemistry Symposium hosted by the Department of Biochemistry at the University of Johannesburg - 30th of September 2011.

Cross-Faculty Postgraduate Symposium hosted by the Postgraduate Centre (Research and Innovation Division) at the University of Johannesburg - 26th of October 2011.

AKNOWLWDGEMENTS AND THANKS

I would like to extend a word of thanks to everyone who has contributed positively to my life and personal development. More especially, I would like to thank the following people and institutions for their contribution and support of this project...

 My supervisor (Prof. Marianne Cronjé) and co-supervisor (Dr. Jacinda James) for allowing me the opportunity to pursue my MSc studies under their supervision. Thanks for all their knowledge they shared with me, their support and mentoring (since my undergraduate years), assistance with my experiments, expert advices and for believing in me and in my talents. Thanks for all their effort and inputs in reviewing my work and giving feedback and constructive criticism on how to best improve it. Most importantly, I would like to thank them for dedicating their own personal time to make this project a completed success - all is much appreciated. Thanks to Dr. James for encouraging me never to give up on my studies, for pushing me to work hard, for helping me organise my dissertation till submission. I will not forget her constant text messages, emails, whatsapp messages and phone calls to check up on me and remind me of our own mini deadlines and meetings – words cannot explain how grateful I am for that – I truly appreciate it.

 My friends and colleagues at the Department of Biochemistry for their advices, assistance and encouragement. A special thanks to my dear friend Mr. Londiwe Mgcina for the moral support and encouragement; Mrs. Heather Byth-Illing for assisting me with the flow cytometry; Tanya (from Chemistry) for helping me dry off my samples during the preparation of plant extracts; Mrs. Eloise Ferreira-Van der Merwe, Ms. Tamarisk Horne and Mrs. Nicola Skerman-Van der Walt for their expert advice, assistance with the cancer work and for keeping up with all my thousand questions. To Dr. Edwin Madala, Dr. Mosotho George (who greatly assisted me with GC-MS) and Mr. Fidele Tugizimana for their advice and encouragement. Thanks to everyone else I shared the lab with and made my experience at the lab a fruitful one: Ms. Sherrie-Ann New, Mr. Robert Gerrard, Mrs. Rabia Bhamjee, Mr. John Walters, Mrs. Colette Osmond-Mathiesen and Ms. Zelinda Human.

 My friends (who are more like brothers and sisters to me) for all their support, kind words of encouragement and for providing me with a social life. I am grateful and honoured to have you all in my live. Thank you: Londiwe Siphephise Mgcina (and the gang), Moeketsi Simon Machogo-Sekoto (and the gang), Palesa Luvhengo-Madzivhandila, Mbali Mabuza, Deborah Letseka, Refilwe Modise and Relebohile Ntoi, Itumeleng Rantsu (and the gang), Palesa Diale, Jabulani Mabena and family, Maureen Baloyi, Peggy Maake, Kenny Bobotjane and Thabiso Mampa, Mickey Bryceland Manganye, Lorraine Sefoka, Shaakira Abrahams, Rirhandzu Sibisi and Florence Letswalo, Lesego Motang, Tumelo Maake, Serialong

Moshesh, Cynthia Tshabalala, Paul Tshepo Nthoba, Prudence Nephawe, Grant Mabuza, Tebogo Maleka, Sibongile Nhlapho-Nkosi, Nonhle Mkhize, Gomolemo Moagi, to all my UJ friends, to everyone in my 2009 UJ Biochemistry Honours class, to all my friends from the 2010 UJ Biochemistry Honours class, to all my Discovery Health friends and colleagues, to all my friends and elders at The House Of The Twelve Apostles Church, and to all of my other friends from all spheres of life (you are just too many to mention, but I appreciate all your love and I am greatly honoured to have you as my friends).

 My project funders for the financial assistance: UJ Merit, UJ-CSIR, UJ-CANSA and Capitec Bank. I am very grateful and privileged of the opportunity you have given me.

 My lovely girlfriend Prudence Maseko for the love, support, prayers, motivation, for lending a listening ear and a helping hand and for encouraging me to persevere. You are a blessing to me and I am grateful to have you in my life.

 My beloved family for the love and support. A special thanks to my mother Ntombifikile Sylvia and aunt Sibongile Theresa (whom this dissertation is dedicated to) for all their sacrifices in raising me the way they did, even under difficult circumstances. Thanks for their prayers, support, guidance, encouragements and for believing in my dreams and talents. Thanks for always being there for me, you are my major support structure, my pillar of strength, teachers and best friends. It just amazes me and fills me with joy to see how you would go out of your way just to make us happy. Thanks for always being so understanding and supportive whenever I am going through hard times. You are probably the only people who truly understand what I have been going through over the past years. You have been there for me through it all and you never backed out on me. You kept supporting me, encouraged me, and assured me that all will be okay. I cannot thank you enough for all you have done for me. If you were not around, I would not have achieved most things in my life. Thank you so much for everything. Ngiyabonga! Thanks also to my cousins Siboniso and Nonhlanhla and to Babu Ndlovu for all the support.

 To the LORD GOD Almighty for the unconditional love and source of strength. I would like to praise Him and lift His name up high and give my gratitude of thanks because He simply is an amazing GOD to me. Jeremiah 29:11 (NIV) – For I know the plans I have for you,” declares the LORD, “plans to prosper you and not to harm you, plans to give you hope and a future. With GOD on my side, I know I shall prosper!

 To everyone else whom I have forgotten to mention, but have contributed positively to this project and / or to my life and personal development, thank you so much. All is much appreciated, GOD BLESS!

TABLE OF CONTENTS

List of Acronyms and Abbreviations ...... i List of Figures ...... iv List of Tables ...... vii

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: LITERATURE REVIEW ...... 6 2.1 Cancer 9 2.1.1 Cancer statistics 10 2.1.2 Oesophageal cancer 13 2.1.3 Current cancer treatment regimes 16 2.1.4 Oesophageal cancer therapeutics 16

2.2 Medicinal plants in South Africa 17

2.3 Medicinal plants as source for cancer treatment 18

2.4 Hypoxis plants (Family: Hypoxidaceae) 19 2.4.1 Hypoxis hemerocallidea syntax 20 2.4.2 Taxonomy, macroscopic morphology and habitat of H. hemerocallidea 21 2.4.3 Traditional medicinal uses of Hypoxis hemerocallidea 22 2.4.4 Bioactive compounds in Hypoxis hemerocallidea 25 2.4.4.1 Hypoxoside and rooperol 25 2.4.4.2 Phytosterols 27 2.4.4.3 Lectins 30

2.5 Techniques used in the evaluation of the anticancer properties of the H. hemerocallidea extracts 31 2.5.1 The trypan blue dye exclusion assay 31 2.5.2 The AlamarBlue® dye reduction assay 32 2.5.3 The CytoTox-GloTM cytotoxicity assay 35 2.5.4 Flow cytometry 36

2.6 Medicinal plants as target for use as antimicrobial agents 38

2.7 Antimicrobial properties of medicinal plants and the microorganisms

under investigation from this study 40

2.8 Techniques used in the evaluation of the antimicrobial properties of the H. hemerocallidea extracts 42 2.8.1 Direct TLC-bioautography assay 42 2.8.2 Microdilution assay 44 2.8.3 The BacTiter-GloTM microbial cell viability assay 44

2.9 Overview 45

CHAPTER 3: EXPERIMENTAL PROCEDURES 46 3.1 Preparation of the plant extracts 47

3.2 Cancer studies 47 3.2.1 Culturing of the SNO cancer cell line 47 3.2.2 Dose-dependent studies of plant extracts on the SNO cancer cell line 48 3.2.2.1 Cell viability studies of SNO cells treated with plant extracts 48 3.2.2.1.1 Trypan blue dye exclusion assay analysis 48 3.2.2.1.2 AlamarBlue® dye reduction assay analysis 49 3.2.2.1.3 CytoTox-GloTM cytotoxicity assay analysis 50 3.2.2.1.4 Flow cytometry (forward and side scatter) analysis 51 3.2.2.1.5 Light microscopy analysis of SNO cells treated with plant extracts 52 3.2.2.2 Statistical analysis 52

3.3 Microbial studies 53 3.3.1 Phytochemical analysis of plant extracts by thin layer chromatography (TLC) 53 3.3.2 Culturing of microorganisms 54 3.3.3 Determination of antimicrobial activities using direct TLC-bioautography 54 3.3.3.1 Antibacterial screening 54 3.3.3.2 Anti-mycobacterial screening 55 3.3.3.3 Antifungal screening 56 3.3.3.4 Control samples 56 3.3.4 Quantification of the antimicrobial activity 56 3.3.5 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity 58

3.3.6 The use of GC-MS for partial identification of the antimicrobial compounds 59

CHAPTER 4: RESULTS 62 4.1 Cancer studies 63 4.1.1 Cell viability studies of SNO cells treated with plant extracts 63 4.1.2 CytoTox-GloTM cytotoxicity assay analysis 67 4.1.3 Flow cytometry (forward and side scatter) analysis 67 4.1.4 Light microscopy analysis of SNO cells treated with plant extracts 68

4.2 Microbial studies 71 4.2.1 Phytochemical analysis of plant extracts by TLC 71 4.2.2 Determination of antimicrobial activities using direct TLC-DB (antibacterial, anti-mycobacterial and antifungal screening 73 4.2.3 Quantification of the antimicrobial activity using microdilution assay 78 4.2.4 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity 79 4.2.5 The use of GC-MS for partial identification of the antimicrobial compounds 81

CHAPTER 5: DISCUSSION 87 5.1 Cancer studies 88

5.2 Microbial studies 93 5.2.1 Phytochemical analysis of plant extracts by TLC 93 5.2.2 Determination of antimicrobial activities using direct TLC-DB 93 5.2.3 Control samples used for the identification of the active antimicrobial compounds in the H. hemerocallidea corm extracts 95 5.2.4 Quantification of the antimicrobial activity using microdilution assay and the BacTiter-GloTM microbial cell viability assay 96 5.2.6 The use of GC-MS for partial identification of the antimicrobial compounds 97

CHAPTER 6: CONCLUSION 101

CHAPTER 7: REFERENCES 104

LIST OF ACRONYMS AND ABBREVIATIONS A AC Adenocarcinoma ADP Adenosine 5’-diphosphate AIDS Acquired immune deficiency syndrome AMP Adenosine 5’-monophosphate Apaf-1 Apoptosis protease-activating factor-1 ATP Adenosine 5’-triphosphate

B BPH Benign prostatic hyperplasia BSSG Beta-sitosterol glucoside

C CA-4 Combretastatin A-4 CANSA Cancer Association of South Africa COX Cyclooxygenase CT Computer tomography

D dATP Deoxyadenosine triphosphate DEC Dried ethanolic corm extract DEL Dried ethanolic leaf extract DMEM Dulbecco’s modified eagle medium DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DWC Dried water corm extract DWL Dried water leaf extract

E EUS Endoscopic ultrasonography

F FAD Flavin adenine dinucleotide FCS Fetal calf serum FEC Fresh ethanolic corm extract FEL Fresh ethanolic leaf extract FMN Flavin mononucleotide FWC Fresh water corm extract FWL Fresh water leaf extract

G GC-MS Gas chromatography – mass spectroscopy GORD Gastro-oesophageal reflux disease

H HIV Human immunodeficiency virus

IC50 Inhibitory concentration at 50 percent INT Iodonitrotetrazolium violet salt

M m / z Mass-to-charge ratio MCL-1 Myeloid cell leukemia-1 MIC Minimum inhibitory concentration MRI Magnetic resonance imaging

N NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NCR National cancer registry

O OC Oesophageal cancer

OD600 Optical density at 600 nm wavelength ii

P PCD Programmed cell death PET Positron-emission tomography

R REDOX Oxidation-reduction reaction

Rf Retardation factor RNA Ribonucleic acid Rt Retention time

S SADC Southern Africa development community SAPA South African press association SCC Squamous cell oesophageal cancer

T TLC-DB Direct thin layer chromatography–bioautography

W WHO World health organisation

iii

LIST OF FIGURES

Chapter 2 Figure 2.1 The 2008 estimates of the 20 most commonly diagnosed cancers worldwide 12

Figure 2.2 The worldwide statistics of 2008 estimates of 20 most common causes of death from cancer 12

Figure 2.3 The 2008 global cancer statistics for oesophageal cancer incidence rates in the world 13

Figure 2.4 Estimate incidence and mortality rates per 100 000 populations in males, worldwide 14

Figure 2.5 Worldwide statistical figures of the incidence and mortality rates per 100 000 populations in females 14

Figure 2.6 Phytochemical classes 19

Figure 2.7 Photograph showing the macroscopic view of the H. hemerocallidea plant and corms 22

Figure 2.8 Chemical structure of hypoxoside 25

Figure 2.9 Chemical structure of rooperol 25

Figure 2.10 A chemical reaction showing the conversion of the non-toxic hypoxoside molecule into a cytotoxic rooperol compound 26

Figure 2.11 Chemical structures the well-known sterol molecules 28

Figure 2.12 Chemical structures of β-Sitosterol and β-Sitosterol glucoside 30

Figure 2.13 Chemical structure of trypan blue dye 32

Figure 2.14 AlamarBlue® reaction mechanism 34

Figure 2.15 Chemical reaction representing the mechanism that occurs in the use of The CytoTox-GloTM cytotoxicity assay 36

Figure 2.16 Schematic illustration of a flow cytometer 37

Figure 2.17 A simple diagram showing the principle involved in TLC-DB 43

Figure 2.18 Structural representation of the process involved in the reduction of the MTT tetrazolium salt into a coloured formazan product, indicating bacterial growth 44

Figure 2.19 Firefly luciferase bioluminescence reaction 45

Chapter 3 Figure 3.1 A 96-well plate showing the set-up that was used to conduct the microdilution assay 57

Chapter 4 Figure 4.1 The cell viability of the SNO cells after treatment with H. hemerocallidea plant extracts 64

Figure 4.2 AlamarBlue® SNO cell viability results after treatment with H. hemerocallidea corm extracts. 65

Figure 4.3 Cell viability of the SNO cells obtained following the treatment with H. hemerocallidea leaf extracts 66

Figure 4.4 Cell viability of the SNO cells treated with FWC, FEC, DWC, DEC, FWL and FEL extracts using the CytoTox-GloTM assay 67

Figure 4.5 Dot-plots showing the cytometrical data obtained for the SNO cells treated with plant extracts to indicate viability 69

Figure 4.6 Microscopic view the H. hemerocallidea treated SNO cells in comparison With the untreated control cells 70

Figure 4.7 TLC chromatogram representing the phytochemical separation of the H. hemerocallidea plant extracts: FEC, DEC, FEL, and DEL 71

Figure 4.8 TLC-DB results for all test microorganisms 74

Figure 4.9 TLC-DB results obtained for the commercially known standards, Moducare® and African Potato tuber capsules 77

Figure 4.10 Quantification of antimicrobial activity by means of INT 78

Figure 4.11 Cell viability of microorganisms (B. subtilis, C. albicans, E. faecalis, E. coli, M. smegmatis, and P. aeruginosa) following the treatment with the H. hemerocallidea extracts 80

Figure 4.12 TLC chromatogram for H. hemerocallidea plant extracts FEC, DEC, FEL, and DEL and TLC plate of TLC-DB results for P. aeruginosa 81

Figure 4.13 GC Chromatogram profile of S1 isolated from the FEC extract that was separated using TLC 82

Figure 4.14 Mass spectrum of peak A 82

Figure 4.15 Mass spectrum of peak B 83

Figure 4.16 Chromatogram of the antimicrobial compound S2, which was obtained following the TLC-DB analysis of E. faecalis 84

Figure 4.17 Mass spectrum of peak A 84

Figure 4.18 Mass spectrum of peak C 85

Figure 4.19 TLC-DB results for the levoglucosan compound that was isolated via GC- MS 86 vi

LIST OF TABLES

Chapter 2 Table 2.1 The South African pandemic and epidemic diseases and their causes 24

Chapter 4

Table 4.1 Rf values of the compounds found in the H. hemerocallidea plant after phytochemical analysis by TLC 72

Table 4.2 Summary of the TLC-DB results, obtained after testing the H. hemerocallidea plant extracts DEC, FEC, DEL and FEL against six microorganisms, namely: B. subtilis, E. faecalis, E. coli, P. aeruginosa, M. smegmatis, and C. albicans 75

Table 4.3 MIC values (mg / ml) of the H. hemerocallidea extracts and antibiotics tested 79

vii

CHAPTER 1 - INTRODUCTION

Chapter 1: Introduction

CHAPTER 1 - INTRODUCTION

One of the essential organs that form part of the digestive system is the oesophagus. It is a hollow muscular tube which necessitates the movement of solid foods and liquids from the mouth to the stomach (Martini, 2006). A defective or malfunctioning oesophagus may lead to multiple digestive disorders, such as nutrients failing to reach cells due to oesophageal malfunction, which may lead to the inability of organs to perform their respective physiological functions in order to sustain and keep the human being (organism) alive. One of the main contributing factors to a malfunctioning oesophagus is oesophageal cancer (OC).

Cancer is a disease in which normal cells are transformed to malignancy due to the damage in their genomes (DNA). DNA damage can be caused by endogenous processes (e.g. DNA replication errors, instability in DNA bases or DNA attack by free radicals during metabolism) or by the interaction with exogenous agents (e.g. UV radiation, ionisation radiation and chemical carcinogens) (Bertram, 2001). Statistics show that cancer is still one of the leading causes of deaths worldwide, with new cancer cases contributing to about 2–3% of worldwide cancer deaths each year (Koduru et al., 2007). It is estimated that by 2030, about 13.1 million worldwide deaths will be due to cancer (WHO, 2013a). According to worldwide cancer statistics, of all known cancers, OC was the 8th most common cancer and the 6th most common death-causing cancer in 2008 (Cancer Research UK, 2012). It has a high incidence in southern Africa and is mostly prevalent in black men; in South Africa alone, OC is reported as the 3rd most common cancer in men and the 4th most common in women (Sharma and Kotzen, 2007; CANSA, 2012). OC is caused by oxidative damage to the oesophageal mucosa possibly due to the use of tobacco and high alcohol consumption (De Stefani et al., 1993; Wu et al., 2001); nutritional deficiencies in minerals such as zinc, riboflavin, nicotinic acid, magnesium and selenium (Bird-Lieberman and Fitzgerald, 2009); and the exposure to fungal toxins and spices in food and frequent consumption of very hot beverage drinks (Enzinger and Mayer, 2003; McCabe and Dlamini, 2005).

OC is frequently characterised by symptoms of odynophagia, dyspnea, dysphagia, gastrointestinal reflux and oesophageal ulcers (Elton, 2005; Sharma and Kotzen, 2007), and can be diagnosed by oesophagogram and endoscopic ultrasonography (Enzinger and Mayer, 2003). Treatment usually involves surgery, resection therapy, chemotherapy, radiotherapy or chemoradiotherapy (Elton, 2005); but unfortunately all these therapies are 2

Chapter 1: Introduction linked to many complications such as non-specificity in targeting cancer cells by affecting normal body cells (Hsu et al., 2004; Ashkenazi, 2008), a developed resistance to the treatment (Hannun, 1997; Reed, 2003; Hsu et al., 2004) and adverse cytotoxic side-effects for example mouth sores and tiredness (Love et al., 1989), hair loss (Griffin et al., 1996) and nausea and vomiting (Bergkvist and Wengström, 2006). The currently used cancer therapies are ineffective in prolonging survival, and in providing cure (Ashkenazi, 2008) and thus, there is a high need for better and novel cancer therapeutics, preferably isolated from plants because of their low cytotoxicity effects, high efficacy, easy accessibility and affordability (Gupta and Raina, 1998; Mander, 1998; Haq, 2004).

Since the evasion of apoptosis and the uncontrollable proliferation are a characteristic of cancer (Bird-Lieberman and Fitzgerald, 2009), there is a pursuit for anticancer compounds which will induce apoptosis in cancer cells as a way to cure cancer (Dean et al., 2007; Arlt et al., 2013). Apoptosis is an active form of programmed cell death (PCD) characterised by cell shrinkage, reduction of cellular volume and nuclear volume (pyknosis), deformation and loss of neighbouring-cell contact, pseudopodes retraction (Kerr et al., 1972; Bosman et al., 1996; Gewies, 2003; Kroemer et al., 2009), nuclear condensation and chromatin aggregation (Gewies, 2003), endonucleocytic degradation of DNA into nuclear fragments (karyorrhexis) (Cohen et al., 1994) and cellular fragmentation into membrane bound apoptotic bodies after the controlled systematic destruction of a cell (Kerr et al., 1972; Allen et al., 1997; Elmore, 2007). Other structural changes that occur within the cell during apoptosis include phosphatidylserine translocation from the inner plasma membrane to the outer cell surface (Martin et al., 1995; Allen et al., 1997), protease activation and cleavage of intracellular substrates (Martin and Green, 1995), plasma membrane blebbing and budding, increased mitochondrial outer membrane permeability (Chipuk et al., 2006), cytochrome c release from the mitochondria to the cytosol and cell degradation by phagocytosis but without any occurrence of inflammation (Gewies, 2003; Boyer, 2007; Elmore, 2007; Kroemer et al., 2009).

In addition to cancer contributing to a vast number of worldwide deaths, infectious diseases caused by pathogenic organisms like bacteria, fungi, viruses and parasites (WHO, 2013b) also contribute to these deaths, especially in developing countries (Fauci, 1998; Iwu et al., 1999). Infections such as the human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) are now an epidemic in Africa. In sub-Saharan Africa, infections such as candidemia caused for the most part by Candida albicans, are opportunistic infections especially in HIV patients (Fan-Harvard et al., 1991; UNAIDS, 2012), 3

Chapter 1: Introduction and accounted for morbidity rates of 46% and mortality rates of 69% in South African adults in Soweto between 2005–2007 (Kreusch and Karstaedt, 2013). With such devastating statistics, there is now an urgent search for novel natural plant compounds with antibiotic activities, since many pathogenic microorganisms are rapidly gaining acquired resistance against a variety of commercially available antibiotic drugs (Fauci, 1998; Cowan, 1999; Iwu et al., 1999).

Hypoxis hemerocallidea is an indigenous South African plant, popular for its use in the treatment of HIV / AIDS infections (Southern African Development Community, 2002; Mills et al., 2005; Street et al., 2008), prostatic hypertrophy, testicular cancer and other internal cancers because it is believed to possess anticancer properties (Smith et al., 1995; Nair et al., 2007a; Drewes et al., 2008; Van Wyk, 2008; Abegaz et al., 1999). Corm extracts of the plant contain the compounds hypoxoside and rooperol, which are claimed to be responsible for the anticancer properties of the plant. In vitro studies on the extracts have shown that hypoxoside is non-toxic. It can be converted by the enzyme β-glucosidase to form a cytotoxic anticancer rooperol compound (Albrecht et al., 1995c); and in vivo studies have shown that the non-toxic hypoxoside is catalysed by β-glucuronidase upon oral ingestion, to form non-toxic rooperol metabolites (glucuronides and sulphates) (Smith et al., 1995). In the US, rooperol (an aglycone isolated from hypoxoside) was patented as an anti-cancer agent. Data on the pharmacological properties of rooperol and the potential course of action of hypoxoside, which is used as an oral prodrug for cancer therapy, is now available (Abegaz et al., 1999). In addition to hypoxoside and rooperol, H. hemerocallidea extracts also possess other compounds such phytosterols (Boukes et al., 2008; Nair and Kanfer, 2008b), lectins (Gaidamashvili and Van Staden, 2002; Erlwanger and Cooper, 2008) and cytokinins (Hutchings et al., 1996), which may also contribute to the medicinal properties of the plant. Extracts from H. hemerocallidea have also been shown to have antibacterial activities against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae (Buwa and van Staden, 2006).

In this study, extracts from corms and leaves of the medicinal H. hemerocallidea were evaluated for both its proposed anticancer and antimicrobial properties. The ability of this plant to trigger apoptosis in the SNO oesophageal cancer cell line was investigated and the antimicrobial activity against a variety of microbial cell cultures was examined. Thus, the objectives were:

Chapter 1: Introduction

 To investigate if H. hemerocallidea plant extracts possess any anticancer and antiproliferation properties. Techniques employed to explore this were cell viability assays, light microscopy and flow cytometry.

 To evaluate the antimicrobial potential of H. hemerocallidea against a variety of microorganisms through direct thin layer chromatography biography (TLC-DB). Preliminary photochemical analysis of the plant extracts which showed positive antimicrobial capabilities were further explored by gas chromatography-mass spectrometry (GC-MS).

The finding of this study may contribute to the search for novel plant-based anticancer and antimicrobial compounds for use in the development of cancer-drugs and antibiotics that will be more affordable and effective in providing cure and have less side-effects and toxicity to the human body. Since most medicinal plants are found growing wild as bushes, this makes traditional herbal medicines easily accessible and relatively affordable, especially for those people residing in the rural areas.

Chapter 2: Literature Review

CHAPTER 2 - LITERATURE REVIEW 2.1 Cancer 9 2.1.1 Cancer statistics 10 2.1.2 Oesophageal cancer 13 2.1.3 Current cancer treatment regimes 16 2.1.4 Oesophageal cancer therapeutics 16

2.2 Medicinal plants in South Africa 17

2.3 Medicinal plants as source for cancer treatment 18

2.6 Medicinal plants as target for use as antimicrobial agents 38

2.7 Antimicrobial properties of medicinal plants and the microorganisms under investigation from this study 40

Chapter 2: Literature Review

CHAPTER 2 - LITERATURE REVIEW

By virtue of random trial and error, and through word of mouth, many plants are now clinically used as medicinal remedies (in the form of drugs) to treat a number of acute and chronic human diseases. Examples of such plant-derived drugs include the analgesic drug aspirin isolated from Salix alba; the analgesic drugs morphine, codeine, noscapine or narcotine, and papaverine isolated from P. somniferum; the antimalarial drug quinine isolated from Cinchona officinalis; the antihypertensive agent reserpine from Rauwolfia serpentine, and the anti-asthma agent ephedrine isolated from Ephedra sinica (Chin et al., 2006; Gurib-Fakim, 2006).

Plants have been used by humans as a source of basic needs like food, shelter, clothing and medicines for thousands of years (Cragg and Newman, 2005a). In addition, plants have also been utilized specifically for flavourings, fragrance, poisons, insecticides, nutrients, cosmetics (Abegaz et al., 1999) and as traditional medicines since 2600 BC (Gurib-Fakim, 2006). Medicinal plants such as Vaccinium macrocarpon (cranberry juice) and Melaleuca alternifolia (lemon balm) have long been used to treat infections such as those of the urinary tract, the gastrointestinal tract and of the skin (Rios and Recio, 2005); and plants such as Artemisia annua (Asteraceae), Quillaja saponaria (Rosaceae) and Strychnos myrtoides (Loganiaceae) have been used for the treatment of malaria- causing strains of Plasmodium falciparum (Gurib-Fakim, 2006). Other medicinal plants such as garlic (Allium sativum), onion (Allium cepa), ginger (Zingiber officinalis), tea (Camellia sinensis, Dahanukar et al., 2000), cloves (Syzygium aromaticum, Gurib-Fakim, 2006), aloe (Aloe ferox, Van Wyk, 2008; Van Wyk, 2011), mint (Mentha spicata, Arumugan et al., 2008; Van Wyk, 2011), turmeric (Curcuma longa) and poppy juice (Papaver somniferum, Gurib-Fakim, 2006) are still used even today to treat illnesses such as common colds, fever, infections, inflammations and a variety of other diseases.

With cancer being one of the leading causes of death worldwide, one good example of a South African medical plant studied for cancer treatment is Combretum caffrum (Combretaceae), commonly referred to as the African Bush Willow. An anti-angiogenic and antimitotic agent called combretastatin A-4 (CA-4) [3,4,5-trimethoxy-3’-hydroxy-4’- methoxy-(Z)-stilbene] (Pettit et al., 1989), which is responsible for the anticancer properties of the plant, is isolated from this plant (Pettit et al., 1995). CA-4 is highly cytotoxic, especially against a variety of multidrug resistant cancer cell lines and many

Chapter 2: Literature Review studies are currently conducted to investigate CA-4 as a candidate for a plant-derived anticancer drug (Chin et al., 2006; Gurib-Fakim, 2006). In an in vitro study in the lymphocytic leukemia cell line WSU-CLL, it was found that CA-4 inhibited tubulin polymerization and activated caspase-9 of apoptosis (Nam, 2003; Chin et al., 2006; Gurib-Fakim, 2006). Recently, clinical studies were conducted to assess the dose- limiting toxicity and antitumour effectiveness of a phosphate prodrug of CA-4 (known as combretastatin A-4 phosphate (CA-4P) / ZybrestatTM) against ovarian, lung and thyroid cancers (Garon et al., 2010; ClinicalTrials.gov, 2011; Zweifel et al., 2011). Phase II / III studies were conducted to measure the overall survival rate in anaplastic thyroid cancer (ATC) patients and to evaluate the safety and efficacy of CA-4P combined with paclitaxel and carboplatin in the treatment of ATC and ovarian cancer (Zweifel et al., 2011; Mikstacka et al., 2013). Ovarian cancer studies are still underway (OXiGENE, 2013) and the study on ATC which was completed in 2011, showed that CA-4P effectively increased the overall survival in ATC patients, with a tripling of 1-year survival (Sosa et al., 2011).

An urgent search for better and preferably natural anticancer agents is ongoing because the cancer therapeutics currently in use (primarily chemotherapy and radiotherapy) are associated with adverse side-effects, which include bone marrow suppression, an increased susceptibility to infections, nephrotoxicity, alopecia (hair-loss), loss of taste, loss of appetite, anorexia, sore mouth, dry skin, diarrhoea, difficulty sleeping, tiredness, emesis, pins and needles in limbs, depression, dysphoria, nausea, vomiting and many others (Love et al., 1989, Griffin et al., 1996; Carelle et al., 2002). Both chemotherapy and radiotherapy are palliative and only provide patients with a very short survival rate, and a survival life-span of less than two years after diagnosis (Dean et al., 2007; Arlt et al., 2013). The other major problem associated with chemotherapy and radiotherapy is that they both can be non-specific in targeting tumour cells in the human body, which makes them cytotoxic and harmful even to normal body cells (Hannun, 1997; Bertram, 2001; Steenkamp and Gouws, 2006; Dean et al., 2007). In addition, some cancer patients become insensitive and resistant to both chemotherapy and radiotherapy, leading to the development of a multi-drug resistant type of cancer in these patients (Nguyen and Wells, 2003; Mashima and Tsuruo, 2005). Bearing all this in mind, it is therefore a matter of utmost importance that the search for new agents to be used for the treatment of cancer be explored; and hence now, more than ever, medicinal plants are fast becoming used in traditional medicinal systems to manufacture medicines necessary

Chapter 2: Literature Review for the treatment of a variety of illnesses, as it is believed that the plants possess many therapeutic and healing properties (Steenkamp and Gouw, 2006).

2.1 Cancer

Cancer is a genetic disease whereby normal cells become progressively transformed to malignancy due to the damage in their genomes. The DNA damage can be due to endogenous processes such as DNA replication errors, the intrinsic chemical instability of DNA bases or DNA attack from free radicals during metabolism. Damage to the DNA can also be caused by the cell’s interaction with exogenous agents such as UV or ionising radiation and chemical carcinogens (Bertram, 2001). Cancer cells are simply characterised by an uncontrolled cell growth, thus the cells are continuously replicating and dividing; sometimes, to such an extent that other healthy tissues also become cancerous. There are six main clinical characteristics of cancer, including: a) sustaining proliferative signalling b) evading growth suppressors c) activating invasion and metastasis d) enabling replicative immortality e) inducing angiogenesis and f) resisting cell death (Hanahan and Weinberg, 2000; Bird-Lieberman and Fitzgerald, 2009; Lazebnik, 2010; Hanahan and Weinberg, 2011; Hanahan and Coussens, 2012).

Cancer cells have acquired insensitivities towards the antigrowth and antiproliferation signals known as ‘self-sufficiency’ in growth signals. This is the ability for them to proliferate autonomously without any stimulation from the mitogenic growth signals that normal cells require to proliferate; leading to them evading apoptosis by avoiding programmed cell death (PCD), unlike in normal cells where PCD is essential for the removal of old and damaged cells. Cancer cells have a sustained angiogenesis (the ability for them to connect directly to the body’s vascular system and allow growth of new blood vessels that will supply them with nutrients and oxygen for survival) and the ability to metastasize or move from one body tissue to another in order to create more tumours (Campbell and Farrell, 2006; Hanahan and Weinberg, 2011).

Chapter 2: Literature Review

The main causes of cancer are mutations in the DNA’s tumour suppressor gene and oncogene. The TP53 tumour suppressor gene is responsible for the production of a tumour protein (e.g. the p53 protein) that restricts the cell’s ability to divide. Thus upon its mutation, the cell loses its ability to halt division, and starts dividing uncontrollably. The (proto)-oncogene is responsible for the production of proteins that regulate normal cell growth, cell division and apoptosis in normal cells. When mutated (often by environmental factors or carcinogens) or hyper-expressed, the oncogene stimulates normal cells to turn cancerous (Cooper, 2000; Campbell and Farrell, 2006; Croce, 2008; Hanahan and Weinberg, 2011).

2.1.1 Cancer statistics

The latest world cancer statistics are currently not available. The Cancer Research UK website (2011a, http://www.cancerresearchuk.org/cancer-info/cancerstats) last recorded global statistics for the year 2008 and the Cancer Association of South Africa (CANSA; http://www.cansa.org.za/statistics) stated that they last received statistical reports from the National Cancer Registry (NCR) for 2005; The South African Broadcasting Corporation (SABC) news website (http://www.sabc.co.za/news), published an article by the South African Press Association (SAPA) on the 06 December 2012, confirming that the South African NCR has not been updated for the past few years and the last report was done in 2004. The Statistics South Africa (Stats SA; http://www.statssa.gov.za) currently does not have any cancer statistics shown on the website either. Even though there is lack in current cancer statistics, the NCR together with the Department of Health are working on a strategy that will solve the problem (Adminuser, 2011). The cancer statistics mentioned in this work will therefore be for the year 2008, as they are the latest recorded statistics available (http://www.who.int/mediacentre/factsheets/fs297/en/ index. html; http://www.cancerresearchuk.org/cancer-info/cancerstats/world).

In the United States of America (USA), cancer is the second most common cause of death accounting for nearly 1 of every 4 deaths. In the USA alone, it is estimated that in 2013 there will be about 1 660 290 newly diagnosed cancer cases; and almost 1 600 Americans are expected to die of cancer per day with approximately 580 350 American expected cancer deaths for 2013. Out of the 1 660 290, it is estimated that about 17 990 will be new oesophageal cancer cases, with 15 210 deaths expected due to oesophageal cancer (Siegel et al., 2013). In 2010 in the United Kingdom (UK) alone,

Chapter 2: Literature Review more than one in every four (about 28%) of all deaths was due to cancer. Oesophageal cancer is the 9th most common cancer and the 6th most common (4th most common in males) cause of cancer deaths in the UK, accounting for about 5% of all cancer types (http://publications.cancerresearchuk.org/cancerstats).

New cancer cases contribute about 2–3% of worldwide cancer deaths each year, as reported by the American Cancer Society (Koduru et al., 2007). As reported by the Cancer Research UK (2011b) in 2008, an estimated 12.66 million people were diagnosed with cancer throughout the world and an estimated 7.56 million people died from the disease in the same year (Jemal et al., 2011). The World Health Organisation (WHO) estimates that these numbers will increase drastically by the year 2030, where an estimate of about 13.1 million deaths will be seen worldwide due to cancer and 15.5 million new cancer cases will be reported in the same year (WHO, 20013a).

Looking at southern Africa alone, The Cancer Research UK showed that in 2008, out of a population of about 56 million people, an estimated number of 79 179 people were recorded as new cancer cases; with an estimated number of 54 818 cancer deaths recorded. Furthermore, oesophageal cancer was reported to be the 8th most common cancer worldwide, with over 480 000 people diagnosed (Figure 2.1), more specifically in developing countries where oesophageal cancer is most prevalent.

Chapter 2: Literature Review

Figure 2.1: The 2008 estimates of the 20 most commonly diagnosed cancers worldwide. Oesophageal is the 8th most common type of cancer worldwide (http://www.cancerresearchuk.org/ cancer-info/cancerstats/world/the-global-picture).

Statistics showed that oesophageal cancer is the 6th most common death causing cancer worldwide (Figure 2.2).

Figure 2.2: The worldwide statistics of 2008 estimates of 20 most common causes of death from cancer. Oesophageal cancer is the 6th most deadly type of cancer worldwide (http://www.cancerresearchuk.org/cancer-info/cancerstats/world/the-global-picture).

Chapter 2: Literature Review

2.1.2 Oesophageal cancer

South Africa (SA) was found to have the highest incidence of oesophageal cancer (OC) (Figure 2.3, Figure 2.4 and Figure 2.5), affecting mostly black African males. In 2007, OC was the 3rd most common cancer among all South African males combined, and the 4th most common in SA females (Sharma and Kotzen, 2007); according to CANSA (2012a, 2012b), oesophageal cancer is now the 5th most common cancer in SA men and the 7th most common cancer in SA women, where 1 in 129 men and 1 in 297 women have a lifetime risk of being diagnosed with OC (http://www.cansa.org.za/south-african- cancer-statistics/). Generally, men have the highest incidence of contracting OC, with an incidence rate of 2.4 to 1.0 of that of women (Cancer Research UK, 2012; CANSA, 2012a, 2012b).

Figure 2.3: The 2008 global cancer statistics for oesophageal cancer incidence rates in the world. SA was at the top of the ranking worldwide (Jemal et al., 2011).

Chapter 2: Literature Review

Figure 2.4: Estimate incidence and mortality rates per 100 000 populations in males worldwide. Southern Africa has the highest incidence and mortality rates amongst all other world regions (http://www.cancerresearchuk.org/cancer-info/cancerstats/world/oesophageal-cancer- world).

Figure 2.5: Worldwide statistical figures of the incidence and mortality rates per 100 000 populations in females. The southern African part of the world has both the highest incidence and mortality rates amongst all other parts of the world (http://www.cancerresearchuk.org/cancer- info/cancerstats/world/oesophageal-cancer-world).

Chapter 2: Literature Review

OC is one of the least studied and yet deadliest cancers worldwide and it still remains one of the cancers with the lowest possibilities of cure (Enzinger and Mayer, 2003), while at the same time, it is associated with very high mortality and morbidity rates (Cancer Research UK, 2012). OC is caused by various factors, but mainly by oxidative damage and chronic irritations to the oesophageal mucosa due to tobacco smoking, human papilloma viruses, nitrosamines, fungal mycotoxin contamination in food, the use of Solanum nigrum in diet (De Stefani et al., 1993; McCabe and Dlamini, 2005; Sammon, 2007), excessive alcohol consumption, nutritional deficiencies in vitamins including zinc, nicotinic acid, magnesium, riboflavin and / or selenium, and in general by a lack of fruit and vegetables in the diet (Wu et al., 2001; Sharma and Kotzen, 2007; Bird-Lieberman and Fitzgerald, 2009). OC is more prominent in black men (especially those in the Transkei area) due to genetic, diet and cultural practices such self-induced vomiting as a detoxification remedy (Matsha et al., 2006).

Epidemiologically, OC can occur into two different forms: the squamous cell oesophageal cancer (SCC) and the oesophageal adenocarcinoma (AC). SCC is caused mainly by factors such as the chronic irritation and inflammation of the oesophageal mucosa, too much alcohol intake and smoking. AC is mainly caused by the existence of a gastro-oesophageal reflux disease (GORD) (such as in hiatal hernia or Barrett’s oesophagus), eosophagitis, oesophageal ulcers (Bird-Lieberman and Fitzgerald, 2009) and by the presence of the Helicobacter pylori infection in the gastrointestinal system (Nasrollahzadeh et al., 2012).

The most common symptoms of oesophageal cancer include dysphagia (difficulty in swallowing), weight loss, backache (Sharma and Kotzen, 2007), gastrointestinal reflux, odynophagia (pain on swallowing food and liquids), dyspnea (shortness of breath), iron- deficiency anaemia, gastrointestinal bleeding, chest discomfort, voice hoarseness, unpleasant coughs, ulceration of the oesophagus, and the occurrence of metastatic lesions in the mouth and other surrounding oral areas (Elton, 2005). OC can be diagnosed using oesophago-gastroscopy and endoscopy, and more particularly the endoscopic ultrasonography (EUS), which is used to predict the intensity of tumour invasion and the degree of lymph node association in metastatic cancer. These predictions are known as the tumour and node stages respectively, and are necessary for the determination of the correct prognosis (disease stage). Other techniques used for oesophageal cancer diagnosis include the use of computer tomography (CT) scans,

Chapter 2: Literature Review magnetic resonance imaging (MRI) scans, and the positron-emission tomography (PET) scans (Johnstone et al., 2000; Enzinger and Mayer, 2003; Elton, 2005; Bird-Lieberman and Fitzgerald, 2009).

2.1.3 Current cancer treatment regimes

The treatment of cancer is currently restricted to surgery, radiotherapy, chemotherapy, adjuvant or neoadjuvant treatments, hormone therapy, bisphosphonates, bone marrow (and stem cell) transplants and biological therapies. Most of these cancer treatments are used with great success, but are associated with a number of complications, adverse cytotoxic side-effects and resistance (i.e. chemoresistance and radioresistance) development by patients (Hannun, 1997; Reed, 2003; Hsu et al., 2004). According to Ashkenazi (2008), the currently used cancer therapies are ineffective at improving a patient’s quality of life while reducing disease progression, prolonging survival and providing cure. Faced with such challenges, there is thus a need for better and novel cancer therapeutics (Ashkenazi, 2008). The current research on cancer therapeutics is aimed at discovering and identifying innovative agents that have activities against specific kinds of cancers, and more specificity at targeting only the tumour cells and inhibiting cancer cell growth (both in vitro and in vivo), without exerting any cytotoxic effects to the normal body cells (Hsu et al., 2004; Ashkenazi, 2008). Therefore, the approach is to induce tumour PCD or tumour apoptosis using novel anticancer agents (Cummings et al., 2004; Fischer and Schulze-Osthoff, 2005; Ashkenazi, 2008; Fulda, 2013). Natural sources such as plants are now becoming great resources to use for the discovery of novel anticancer agents for anticancer drug development (Balunas and Kinghorn, 2005; Fouche et al., 2008).

2.1.4 Oesophageal cancer therapeutics

Oesophageal cancer patients are normally treated using surgery or resection therapy, with the hope of fully curing or removing the disease. Unfortunately, resection therapy is only palliative and usually leaves patients with a median survival life-span of only 15 to 18 months. Therefore, in order to counteract these effects, patients who undergo resection are usually also treated using primary therapy in conjunction with surgery, but primary therapy is ordinarily used on patients who do not qualify for surgery. Thus, primary therapy treatments options include chemotherapy, radiotherapy, chemo-

Chapter 2: Literature Review radiotherapy (or combination therapy) and adjuvant or neoadjuvant treatments (Elton, 2005).

It is well known that cancer cells have an ability to evade apoptosis to undergo profound proliferation, which results in uncontrollable tumourigenesis (Dean et al., 2007). Due to this fact, research now involves the identification of key genes involved in the inhibition of tumour apoptosis, as well as the genes responsible for initiating and promoting cancer cell growth (proliferation). This involves manipulating the growth genes by either silencing or inactivating their tumour growth activation functions and activating the apoptotic genes. Apoptotic pathways are thus the focus of a therapeutic strategy for the treatment or curing of cancer, as the currently used primary cancer treatments fail at initiating apoptosis in cancer cells, leading to cancer cell survival (Dean et al., 2007; Arlt et al., 2013). It is believed that the study of apoptosis in oesophageal cancer cells may provide information for designing useful diagnostic and therapeutic strategies for better management of oesophageal cancer to ensure non-reoccurrences and an increased life span for a best quality of life in cancer patients (McCabe and Dlamini, 2005).

As mentioned, cancer is one of the leading diseases contributing to the escalating deaths worldwide, yet the current cancer treatment options available are associated with many complications such as non-specificity in targeting cancer cells in the body and the exertion of toxic side-effects to the body cells. This opens up a new field of research on alternative methods for cancer treatment, which are to be cost effective and efficient for cancer treatment without exerting any side effects to the human body. Medicinal plants such as H. hemerocallidea (Discussed in Section 2.4), Sutherlandia frutescens and Sutherlandia tomentosa are now targeted as alternative treatment options for use as anticancer therapeutics. S. frutescens and S. tomentosa contain promising apoptosis- inducing anticancer agents. It was shown that extracts from these two plants are able to induce caspase-dependent, as well as -independent cell death as a mode of cytotoxicity to cancerous SNO cells in vitro, without posing any cytotoxicity effects to the non- cancerous peripheral blood mononuclear cells (PBMCs) (Skerman et al., 2011).

2.2 Medicinal plants in South Africa

Traditional herbal medicines (as derived from medicinal plants) have a substantial contribution to the economy of developing countries such as SA, as they boost the

Chapter 2: Literature Review world’s health and international trades (Akerele, 1988; Koduru et al., 2007). Many people still use traditional herbal medicines due to cultural, traditional and religious beliefs. Approximately 75-80% of Africans depend on traditional herbal medicines to fulfil their daily healthcare requirements (Ojewole, 2006; Nyinawumuntu et al., 2008; Boukes, 2010).

In contrast to the conventional (modern) or western medicines, which are said to be costly and associated with numerous adverse side effects due to toxicity and inefficiency, as they tend to lose their efficacy over time (Verschaeve and Van Staden, 2008), traditional herbal medicines are regarded as natural and environmental-friendly products which have low toxicities, lower side effects and high efficacy. As most medicinal plants are found growing wild as bushes, this makes traditional herbal medicines easily accessible and relatively affordable, especially for those people residing in the rural areas (Farnsworth et al., 1985; Mander, 1998; Steenkamp, 2003; Shanley and Luz, 2003; Fennell et al., 2004a; Fennell et al., 2004b).

Traditional herbal medicines are mostly supplied by traditional healers and herbalists in informal herbal markets (street vendors and herbal / muthi shops) (Dold and Cocks, 2002; Fyhrquist et al., 2002; Drewes and Khan, 2004; Ndhlala et al., 2011) and are also obtainable in some pharmacies as over-the-counter ready-made remedies or herbal supplements (Drewes and Khan, 2004; Van Wyk, 2011). Not much science was known about indigenous South African medicinal plants except for their use in traditional healing (Lin et al., 1999; Taylor et al., 2001). Therefore, many pharmaceutical companies, government and other private research laboratories and institutes are now encouraged to conduct research that focuses on indigenous medical plants for therapeutic use in medicine (Louw et al., 2002; Owira and Ojewole, 2009).

2.3 Medicinal plants as source for cancer treatment

Medicinal plants produce a variety of therapeutic and bioactive compounds known as phytochemicals (Figure 2.6) (Rowland, 1999), which are now becoming a great source of ingredients for use in manufacturing anticancer drugs. Moreover, the phytochemicals obtained in food as dietary nutrients are necessary for the prevention of cancers (Awad and Fink, 2000; Hsu et al., 2004; Liu, 2004; Bradford and Awad, 2007; Russo, 2007; Link et al., 2010) and other chronic illnesses such as cardiovascular diseases, hypertension,

Chapter 2: Literature Review obesity, diabetes and other aging-related pathologies (Craig, 1997; Russo, 2007; Chen and Blumberg, 2008). Examples of anticancer phytochemicals currently isolated and used for clinical purposes include the vinca alkaloids, epipodophyllotoxins (such as paclitaxel / Taxol®, topotecan, irinotecan, etopophos, teniposide and etoposide), tannins, quinines taxanes and camptothecins (vinblastine and vincristine) compounds (Hartwell, 1976; Cragg and Newman, 2005b). Many of these compounds are obtained and isolated from the medicinal plants such as Catharanthus roseus, Podophyllum peltatum and Taxus brevifolia (Fabricant and Farnsworth, 2001; Balunas and Kinghorn, 2005; Gurib- Fakim, 2006; Cragg and Newman, 2009).

Figure 2.6: Phytochemical classes. Phytochemicals can be classified as alkaloids, carotenoids, organosulfurs, phytosterols and phenols, which can further be classified as stilbenes, phenolics acids and flavonoids. Phytochemicals contribute to the therapeutic properties of medicinal plants (Chen and Blumberg, 2008).

2.4 Hypoxis plants (Family: Hypoxidaceae)

Hypoxis plants are widely used for herbal and medicinal purposes in the Southern Africa Development Community (SADC) regions. Some species that have been identified under the Hypoxis (Hypoxidaceae) genus include H. interjecta, H. multicips, H. nyasica, H. obtuse, H. rooperi (also known as H. hemerocallidea) and H. sobolifera (Abegaz et al., 1999), with H. hemerocallidea being a potential medicinal plant for the development of cancer therapeutics (Albrecht, 1995a; Albrecht et al., 1995c). This indigenous South

Chapter 2: Literature Review

African plant is the most studied and most widely used. Some scientific research has been done on the medicinal benefits of the Hypoxis plants (such as its use for cancer treatment), and it was discovered that the rhizomes of the plant possess some therapeutic properties, which have allowed for the plant to be used to treat diseases such as prostatic hypertrophy and many other internal cancers (Abegaz et al., 1999; Nair and Kanfer, 2006; Drewes et al., 2008; Katerere and Eloff, 2008).

The rhizomes of these plants contain hypoxoside, obtuside A and B, nyasol, nyaside, nyasoside, nyasicoside, mononyasine A and B and interjectin, which are the glycoside molecules believed to be responsible for the pharmacological properties of the Hypoxis plants (Galeffi et al., 1987; Marini-Bettolo et al., 1982a, 1982b; Marini-Bettolo et al., 1985; Messana et al., 1989; Sibanda et al., 1990; Marini-Bettolo et al., 1991; Nicoletti et al., 1992; Boukes, 2010). Hypoxoside, in particular, is converted into an aglycone molecule rooperol, which has been patented in the US as an anticancer agent that can potentially be used as an oral prodrug for cancer therapy (Albrecht et al., 1995c). Besides the glycoside molecules, it has been shown that Hypoxis plants also contain phytosterol compounds (particularly β-sitosterols), that are capable on acting as important energy boosters and immunity modulators, especially to individuals with compromised immune systems such as in acquired immune deficiency syndrome (AIDS) patients. The therapeutic bioactive compounds of H. hemerocallidea are further discussed in Section 2.4.4. Examples of commercial preparations containing the H. hemerocallidea extracts are Moducare®, Harzol®, Hypo-Plus® and Prostone®, which are mostly sold in Europe (Singh, 1999; Van Wyk, 2007; Drewes and Khan, 2004; Erlwanger and Cooper, 2008; Abegaz et al., 1999).

2.4.1 Hypoxis hemerocallidea syntax

The name H. hemerocallidea (previously known as H rooperi) is the scientific name for the commercially known African potato plant (Boukes et al., 2008), but the use of this word is incorrect since the underground portion of the plant is a tuberous rhizome or corm (underground stem developing vertically) and not a tuber (swollen stem, like the potato, developing horizontally). This term may cause confusion as there is another plant, Plectrantus esculentus, which is also called the African potato (Nair and Kanfer, 2008b). The African potato plant has many other vernacular names which are mostly indigenous to most southern parts of Africa, and these include the yellow-star flower or

Chapter 2: Literature Review the star lily in English, sterblom in Afrikaans, lilabatseka or zifozonke in isiSwati, inkomfe, igudu or ilabatheka in isiZulu, inongwe in isiXhosa, tshuka in seTswana, and lotsane or molikharatsa in seSotho (Singh, 1999; Erasto et al., 2005; Mills et al., 2005; Drewes et al., 2008; Katerere and Eloff, 2008).

2.4.2 Taxonomy, macroscopic morphology and habitat of H. hemerocallidea

Taxonomically, the H. hemerocallidea plant falls under the star-lily family or Hypoxidaceae, which consists of 8 genera and 130 species; with 90 of them belonging to SA (Drewes et al., 2008). This family usually consists of monocotyledonous plants, which are normally found as habitants of the savanna regions of SA, Swaziland, Lesotho, Botswana, Mozambique, Zimbabwe and in North-Eastern Africa (Pegel, 1980; Singh, 2007; Drewes et al., 2008; Katerere and Eloff, 2008). In SA, H. hemerocallidea is found growing in the wild areas of the Eastern Cape, KwaZulu-Natal, Gauteng and Limpopo provinces, but it can also be found in the mountainous areas of South America, Australia, and in the coasts of Asia (Drewes et al., 2008). The H. hemerocallidea plant has been described as a stemless, geophytic, perennial herb with large dark brown to black corms (tubers) and bright yellow flowers; the plant is a herbaceous and / or tuberous perennial plant that consists of yellow star-shaped flowers, long strap-like leaves (30 cm long and 3.2 cm wide), brown tuberous rhizomes or corms (up to 10 cm in diameter or length and about a half a kilogram in weight) and lots of adventitious roots that allow them to survive unfavourable conditions (Figure 2.7) (Albrecht et al., 1995b, 1995c; Ndong et al., 2006; Ojewole et al., 2006; Drewes et al., 2008; Boukes, 2010).

Chapter 2: Literature Review

Figure 2.7 Photograph showing the macroscopic view of the H. hemerocallidea plant (left) and the corms (right) (Drewes et al., 2008).

2.4.3 Traditional medicinal uses of Hypoxis hemerocallidea

H. hemerocallidea has always been used in SA for medicinal purposes, which includes its use to treat human immunodeficiency virus (HIV) infections (Albrecht 1995a; Southern African Development Community, 2002; Giraldo, 2003; Mills et al., 2005; Nair et al., 2007b; Street et al., 2008; Verschaeve and Van Staden, 2008). Recently, it is also used in most countries world-wide as a medicinal remedy for malignancies and inflammatory conditions (Albrecht, 1995c; Nair et al., 2007b; Drewes et al., 2008; Van Wyk, 2008). The plant has long been used by traditional healers to treat a variety of general ailments such as cardiac diseases, bad dreams, impotency, apprehension, insanity, depression, barrenness and infertility, intestinal parasites, headaches, dizziness, burns, polyarthritis, hypertension (Drewes et al., 2008; Katerere and Eloff, 2008), diabetes (Erasto et al., 2005), bronchitis, sore throat, rush, lice, allergies, colds and flu, yuppie flu, anxiety, palpitations, skin blemishes, wounds, wasting diseases, Herpes simplex, cancer, tuberculosis, chronic viral diseases, haemorrhoids, asthma, psoriasis, bladder disorders, stomach (gastric and duodenal) and varicose ulcers, vomiting and nausea (Watt and Breyer-Brandwijk, 1962; Pujol, 1990; Albrecht 1995a, Albrecht, 1996; Van Wyk et al., 2002; Hutchings et al., 1996; Steenkamp, 2003; Steenkamp et al., 2006; Van Wyk, 2008).

Chapter 2: Literature Review

To date, the H. hemerocallidea plant is now used to treat diverse types of cancers and tumours such as the urinary diseases, testicular tumours, prostatitis, prostate hypertrophy and benign prostate hyperplasia (BPH) (Abegaz et al., 1999; Nair and Kanfer, 2006; Drewes et al., 2008; Katerere and Eloff, 2008). The plant is also used for many other purposes such as an emetic, enema and as an immune system booster, a CD4 lymphocyte stabiliser in cancer and HIV / AIDS patients, and as a stimulant of muscular and hormonal activities (Albrecht et al., 1995b; Mills et al., 2005; Nair et al., 2007b; Katerere and Eloff, 2008; Verschaeve and Van Staden, 2008). Traditionally, Hypoxis is prepared by being cut into small cubes which are then boiled for 20 minutes and orally consumed (Nair and Kanfer, 2006). A recent review on the medicinal value of Hypoxis has been provided by Ncube et al. (2013).

Chapter 2: Literature Review

Table 2.1: The South African pandemic and epidemic diseases and their causes. The ones marked with a tick can be treated using H. hemerocallidea.

Some pandemic and Epidemiology / Pathogenic Treated by H. epidemic diseases in SA strain hemerocallidea HIV / AIDS HIV1 or HIV2 infection Mycobacterium tuberculosis More research to be TB (Tuberculosis) infection done Plasmodium falciperum Malaria Research to be done infection High Blood Pressure due to Hypertension genetic makeup, diet and lack of exercise Vibrio cholera infection via Cholera faecally contaminated Research to be done drinking or eating matter Hepatitis viral (A, B, C, D & E) Hepatitis Research to be done infections High Blood Sugar Levels due to inadequate insulin Diabetes production or insulin resistance by body cells Environmental pollutants, infection, diet, auto immune Inflammatory disorders Some disorders and psychological stress etc. Diet, lack of physical exercise, tobacco, Cancer environmental carcinogens, Some genetic composition and UV radiation etc. Various pathogenic bacteria, Other infectious diseases Some virus, protozoa or fungi

Chapter 2: Literature Review

2.4.4 Bioactive compounds in Hypoxis hemerocallidea extracts

2.4.4.1 Hypoxoside and rooperol

Many intensive and crucial studies have been done on the H. hemerocallidea plant extracts. Research has shown that the corm extracts of H. hemerocallidea mainly consist of hypoxoside (Figure 2.8), which can be converted into a cytotoxic compound known as rooperol (Figure 2.9).

Figure 2.8: Chemical structure of hypoxoside (Nair and Kanfer, 2006).

Figure 2.9: Chemical structure of rooperol (Nair et al., 2007b).

A study by Albrecht et al. (1995b) on the H. hemerocallidea extracts isolated from the corms showed that the extracts contained a yellow water-soluble crystalline compound named hypoxoside. Hypoxoside is the trivial name for (E)-1,5-bis-(4’-β-D- glucopyranosyloxy-3’-hydroxyphenyl)pent-4-en-1-yne, which is a norlignan diglucoside isolated from the corms of Hypoxis plants (Albrecht et al., 1995b; Albrecht et al., 1995c; Nair and Kanfer 2006). When hypoxoside is exposed to β-glucosidase containing media, it is converted and deconjugated into a lipophilic bioactive dicatechol aglucone (rooperol)

Chapter 2: Literature Review which is cytotoxic and inhibits cell growth of melanoma cells (in both human and murine) in vitro (Figure 2.10).

Figure 2.10: A chemical reaction showing the conversion of the non-toxic hypoxoside molecule into a cytotoxic rooperol compound. The reaction is catalysed by the β-glucosidase enzyme which is normally released in the gastrointestinal tract and in dividing cancer cells (Drewes et al., 2008).

The enzyme β-glucosidase is mainly found in the human gastrointestinal tract (mainly in the large intestines) and is also released by rapidly dividing cancer and bacterial cells (Boukes et al., 2008). The cytotoxic rooperol compound has been proven to exert growth inhibition properties in 60 human cancer cell lines (Nair and Kanfer, 2006; Boukes et al., 2008), and these include the breast, colon, uterus, melanoma and non-small cell lung cancer cell lines (Albrecht et al., 1995b, 1995c; Smith et al., 1995; Boukes et al., 2008). Albrecht et al. (1995b) also showed that rooperol may play a role in the mechanisms involved in the maintenance of chromosome structural integrity and segregation during mitosis. In vivo studies showed that upon oral ingestion of the plant extracts from corms, the non-toxic hypoxoside is converted by β-glucuronidase (present in the gastrointestinal system) to form rooperol phase 2 metabolites (glucuronides / sulphates) which are also non-toxic (Smith et al., 1995).

Chapter 2: Literature Review

Research has further shown rooperol to contain antioxidant (radical-scavenging) capabilities (Laporta et al., 2007a; 2007b), bacterostatic, bactericidal (Drewes and Liebenberg, 1982; Laporta et al., 2007a; 2007b), anti-inflammatory (antiCOX-1 and antiCOX-2 activities) (Jager et al., 1996; Ojewole, 2002, Ojewole, 2006; Steenkamp et al., 2006), analgesic and antinociceptive (Ojewole, 2006), anticonvulsant (Ojawole, 2008), antidiabetic and hypoglycaemic (Zibula and Ojewole, 2000; Ojewole, 2002; Mahomed and Ojewole, 2003; Ojewole, 2006), antiphologistic (Weiss, 1985), antimetastatic (collagen type I synthesis to prevent tumour cell invasion), anticancer (Drewes and Liebenberg, 1987; Nair et al., 2007a; Drewes et al., 2008), cardiodepressant and hypotensive (Ojewole et al., 2006), and bronchorelaxant properties (Ojewole et al., 2009). However, rooperol is also shown to affect the human cytochrome P-450 (Cyp) enzymes, the P-glycoprotein and the pregnane X receptor (PXR, Drewes et al., 2008; Chinsembu and Hedimbi, 2010). Laporta et al. (2007b) showed that rooperol had a phospholipid / water partition coefficient (KP) that is higher than that of hypoxoside, thus indicating that rooperol was mainly responsible for biological activities that are mostly associated with membranes.

2.4.4.2 Phytosterols

In addition to hypoxoside and rooperol, H. hemerocallidea plants also possess plant sterols known as phytosterols, which also contribute to the medicinal and therapeutic properties of the plant (Boukes et al., 2008; Nair and Kanfer, 2008b). Phytosterols (Figure 2.11) are amphiphilic molecules that have hydrophilic heads (hydroxyl groups) and sterane skeletons with side chains that form hydrophobic tails (Heldt, 2005).

Phytosterols have a structure that closely resembles that of the cholesterol molecule, with the exception that the phytosterols are modified with side chains, double bonds and methyl or ethyl groups at the C24 carbon site. Like cholesterol, phytosterols are mainly found in plant cell membranes as membrane stabilisers (Awad and Fink, 2000; Heldt, 2005; Nair et al., 2006; Boukes et al., 2008; Du Plessis-Stoman et al., 2009).

The most commonly known phytosterols are betasitosterol (C29), campesterol (C28) and stigmasterol (C29), which are only produced in plants and not in humans (Figure 2.11). Phytosterols are therefore only obtained by humans through diet, especially from sources like unrefined plant oils, seeds, nuts, legumes, cereals, fruit and vegetables

Chapter 2: Literature Review

(Awad and Fink, 2000; Moghadasian, 2000; Boukes et al., 2008). In nature, phytosterols are found together with phytostanols and phytosterolins (glucosides) which are less abundant in food but are also as important as phytosterols (Nair et al., 2006).

Figure 2.11: Chemical structures the well-known sterol molecules, with cholesterol as the mammalian produced sterol while β-sitosterol, campesterol and stigmasterol as the most commonly produced sterols in plants (Awad and Fink, 2000).

Experimental studies have shown that phytosterols, particularly β-sitosterol, have anticancer activities against several cancer cell lines such as the colon, prostate and breast cancers (Awad and Fink, 2000; Boukes et al., 2008). This anticancer property of

Chapter 2: Literature Review phytosterols is obtained through multiple mechanisms. This protection from cancer by the phytosterols is achieved by the ability of β-sitosterol to affect the membrane integral structure and fluidity mainly by altering the phospholipid concentration, the cholesterol / phospholipid ratio and the fatty acid composition of cancer cells (Awad and Fink, 2000). Moreover, β-sitosterol stimulates the proliferation of the human peripheral blood lymphocyte (human T-cells) (Bouic et al., 1996) and has no effect on estrogen receptors (Awad and Fink, 2000); thus β-sitosterol can be used as an immune system booster and also for the management of testicular tumours and benign prostatic hyperplasia (BPH) (Rhodes et al., 1993; Pegel, 1997; Von Holtz et al., 1998; Berges et al., 2000; Dreikorn, 2000; Lowe and Fagelman, 2000; Steenkamp, 2003; Katerere and Eloff, 2008; Nair and Kanfer, 2008a, 2008b; Wilt et al., 2011; Street and Prinsloo, 2012). Beta (β)-sitosterol also has an effect on membrane-bound enzymes as well as on the signal transduction pathways. Beta-sitosterol stimulates apoptosis in cancer cells by inhibiting cancer cell proliferation and tumour growth (Awad and Fink, 2000).

The Hypoxis plant contains a combination of β-sitosterol and its glucoside β-sitosterol glucoside (BSSG) (Figure 2.12). These compounds are known to treat prostate hypertrophy due to their ability to inhibit the 5α-reductase and aromatase enzymes in order to inactivate the binding of dihydrotestosterone within the prostate gland (Katerere and Eloff, 2008; Van Wyk, 2008; Nair and Kanfer, 2008b).

Chapter 2: Literature Review

Figure 2.12: Chemical structures of β-Sitosterol (MW = 414.65) and β-Sitosterol glucoside (MW = 552.89). These two compounds are used to treat diseases such as the prostate hypertrophy (Nair and Kanfer, 2008b).

In some countries, these phytosterols (because of their capability of lowering both the plasma-cholesterol and LDL-cholesterol levels) are now used to treat mild hypercholesterolaemia and cardiovascular diseases, pulmonary tuberculosis, HIV and immune system suppression (Pegel, 1997; Moghadasian, 2000; Nair et al., 2006; Boukes et al., 2008). Phytosterols have no adverse side-effects beside mild constipation or diarrhoea (Pegel, 1997; Moghadasian, 2000). Other important therapeutic properties of phytosterols include them being used as anti-ulcer, anti-diabetic and anti-inflammatory agents (Pegel, 1997; Boukes et al., 2008; Katerere and Eloff, 2008).

2.4.4.3 Lectins

A few studies have shown that the extracts of H. hemerocallidea contain lectin-like compounds (Gaidamashvili and Van Staden, 2002; Erlwanger and Cooper, 2008) and cytokinins (zeatin, zeatin riboside, zeatin glucoside, Hutchings et al., 1996). Lectins are

Chapter 2: Literature Review non-enzymatic glycoproteins that reversibly and specifically bind carbohydrate moieties on cell membranes (Gurib-Fakim, 2006). Lectins, also called agglutinins, react with surface-exposed carbohydrates of microbes and cause lectin-bacteria agglutination reactions to occur (Gaidamashvili and Van Staden, 2002). Due to their high specificity, lectins are used as diagnostic probes for the identification of bacterial pathogens as well as for the recognition and differentiation of malignant tumours. Studies on lectins have shown that lectins are capable of inhibiting the motility, multiplication and growth of some plant bacterial pathogens by agglutinins; thus lectins are perceived important for plant defence activation (Gaidamashvili and Van Staden, 2002; De Hoff et al., 2009; Ghazarian et al., 2011).

In humans, lectins prevent the adhesion and interaction of bacteria with the epithelial cells of the gastrointestinal tract. Lectin agglutinins can interact with enterocytes and lymphocytes in order to expose pathogens to body tissues, thus allowing for the activation of an immune response. Lectins are also believed to possess some anticancer properties together with apoptosis-inducing capabilities in cancer cells (Fu et al., 2011; Ghazarian et al., 2011). Due to this, most South African medicinal plants are now screened for lectin-like proteins which can be used and exploited as antibacterial (Gaidamashvili and Van Staden, 2002) and anticancer agents (Fu et al., 2011; Ghazarian et al., 2011).

2.5 Techniques used in the evaluation of the anticancer properties of the H. hemerocallidea extracts

2.5.1 The trypan blue dye exclusion assay

The trypan blue dye exclusion staining assay (Kaltenbach et al., 1958) is a simple and inexpensive method of determining growth and cellular viability of treated and untreated cells by measuring the membrane integrity (Allison and Ridolpho, 1980; Perry et al., 1997; Kumi-Diaka et al., 1999; Longo-Sorbello et al., 2006). Trypan blue dye exclusion assay uses the vital diazo dye trypan blue (Figure 2.13), which interacts only with dead cells to allow for the measurement of cell viability (Allison and Ridolpho, 1980; Perry et al., 1997; Tran et al., 2011).

Chapter 2: Literature Review

Figure 2.13: Chemical structure of trypan blue dye (Sarma et al., 2000).

The principle behind the trypan blue dye exclusion assay is based on the ability of viable cells to be impermeable to trypan blue (Longo-Sorbello et al., 2006). In a reaction mechanism, the negatively charged trypan blue only interacts with cells possessing compromised membrane integrity. Viable cells have intact and selectively permeable membranes and are thus able to exclude the dye from entering the cytoplasm. Non- viable cells have damaged membranes that allow for the uptake of the trypan blue dye into the cytoplasm. This makes the non-viable cells to appear dark blue in colour when viewed under a microscope (Sarma et al., 2000; Longo-Sorbello et al., 2006; Trans et al., 2011). The percentage of unstained cells gives the percentage of viable cells (Longo- Sorbello et al., 2006), and the percentage cell viability is calculated by the formula:

Cell viability (%) = Number of viable cells (unstained) x100 Total number of cells (stained and unstained)

(Kumi-Diaka et al., 1999)

2.5.2 The AlamarBlue® dye reduction assay

The AlamarBlue® dye reduction assay is a reductase-based assay that requires live cells with functioning mitochondria to convert the precursor AlamarBlue® dye into a measurable fluorescent coloured product (McMillian et al., 2002). Resazurin (AlamarBlue®) dye is used as an oxidation-reduction indicator of proliferation and cytotoxicity in a cell culture (O’Brien et al., 2000).

Chapter 2: Literature Review

Advantages of using the AlamarBlue® assay include the following:  AlamarBlue® can be washed free from cells, allowing for other assays to be performed or cells to be reused for further experiments (saving money and time)  AlamarBlue® does not interfere with the compounds tested  The assay is less costly to discard as it does not require any special handling or disposal because of no radioactive nor toxic substances used  The assay is sensitive, inexpensive and simple to use, and may be adapted for large scale use in vitro screening  Dye is added directly to the cells in culture towards the end of the incubation period  Dye is nontoxic and nonmutagenic to cells and the user; this is important as it allows for the continuous assessment of cellular proliferation in cultures overtime  No additional reagents or manipulations are required (Nakayama et al., 1997; O’Brien et al., 2000; McMillian et al., 2002).

Resazurin, a phenoxazin-3-one redox dye, acts as an intermediate electron acceptor in the electron transport chain between the last reduction step of oxygen and cytochrome oxidase (oxidoreductase) where it is substituted for a molecular oxygen as an electron acceptor either from NADPH, FADH, FMNH, NADH and cytochromes (Pagé et al., 1993; McMillian et al., 2002; Al-Nasiry et al., 2007). In a chemical reaction (Figure 2.14), the oxidised resazurin (blue and nonfluorescent) is reduced by respiring intracellular mitochondrial reductases of proliferating cells into a deoxygenated resorufin product, which is pink and highly fluorescent at wavelengths greater than 550 nm. Resorufin can be measured calorimetrically (by spectrophotometry) or fluorometrically (by spectrofluorometry) to determine cellular proliferation (Nakayama et al., 1997; O’Brien et al., 2000; McMillian et al., 2002; Perrot et al., 2003; Anoopkumar-Dukie et al., 2005; Longo-Sorbello et al., 2006; Al-Nasiry et al., 2007; Markaki, 2009).

Chapter 2: Literature Review

Figure 2.14: AlamarBlue® reaction mechanism. The non-fluorescent resazurin dye is converted, by the reduction reactions of metabolically active cells, into a highly fluorescent resorufin product, which can be measured via spectrofluorometry at an emission wavelength of 590 nm. The fluorescence produced is proportional to the number of viable cells (Markaki, 2009).

The percentage of AlamarBlue® reduction is calculated by the formula:

® % of AlamarBlue reduction = (ԐOXλ2)(Аλ1) – (ԐOXλ1)(Аλ2) x100

(ԐREDλ1)(А’λ2) – (ԐREDλ2)(А’λ1) where ® ԐOX = molar extinction coefficient constant of AlamarBlue (oxidised form) ® ԐRED = molar extinction coefficient constant of AlamarBlue (reduced form) А = absorbance of test wells А’ = absorbance of negative control wells

λ1 = 570 nm / 540 nm

λ2 = 600 nm / 630 nm (Al-Nasiry et al., 2007; Pettit, 2009).

Chapter 2: Literature Review

2.5.3 The CytoTox-GloTM cytotoxicity assay

Bioluminescence is the emission of electromagnetic radiation in the visible region (i.e. light) as a result of a luciferase-catalysed chemical reaction. The colour of light emitted varies between 560 nm (greenish yellow) and 620 nm (red) wavelengths. The bioluminescence reaction is commonly used as the basis for an enzyme assay due to its high quantum yield, which makes it a highly sensitive technique. The assay is based on the following principle:

(Wilson and Walker, 2005)

Bioluminescent assays are developed to evaluate the cytotoxicity of cytotoxic agents in vitro. An example of a commercial kit available for this purpose is the CytoTox-GloTM Cytotoxicity Assay, which measures the relative number of dead cells in a cell population via a distinct protease activity associated with cytotoxicity (Figure 2.15) (Bedford et al., 2012; Promega, 2012).

Dying cells have compromised cell membranes that allow for the release of the intracellular protease (cell-death protease) to the extracellular space where it binds and cleaves the luminogenic peptide substrate alanyl-alanylphenylalanyl-aminoluciferin (AAF-GloTM) to produce aminoluciferin, which is oxidised by ultra-GloTM recombinant luciferase to give off an intense light signal correlating with the number of dead cells and relatively with the percentage of cells undergoing cytotoxic stress (Figure 2.15) (Niles et al., 2007a, 2007b; Bedford et al., 2012; Promega, 2012). The dead-cell substrate and luciferase are not membrane permeable, thus they cannot cross the intact membrane of viable cells; thus no light signal is generated from live cells (Niles et al., 2007a, 2007b; Promega, 2012). A lysis reagent can be added to the experiment (after the measurement of cytotoxicity) to determine the relative number of viable cells remaining in the assay as well as the total number of cells present. Therefore viability is calculated by subtracting the experimental dead-cell luminescence from the total luminescence value:

Viable cell luminescence = total luminescence – dead-cell luminescence

Chapter 2: Literature Review

Figure 2.15: Chemical reaction representing the mechanism that occurs in the use of the CytoTox-GloTM cytotoxicity assay. Dead cells release a protease, which bind to the AAF- aminoluciferin substrate to produce the aminoluciferin product that is cleaved by the Ultra-GloTM 2+ Recombinant Luciferase (in the presence of Mg ion, ATP and molecular O2) to produce a quantifiable light signal, which is proportional to the percentage of dead cells (Niles et al., 2007; Cho et al., 2008).

2.5.4 Flow cytometry

Flow cytometry (FC) is a simultaneous measurement of multiple physical characteristics of a single cell in a suspension of cells flowing through a flow cytometer (Brown and Wittwer, 2000; Bakke, 2001). The cell characteristics measured include:  Cell size (represented by forward angle light scatter)  Cytoplasmic complexity (represented by right-angle light scatter)  DNA or RNA content  Membrane-bound and intracellular proteins (Brown and Wittwer, 2000; Cram, 2002).

Chapter 2: Literature Review

FC qualitatively and quantitatively measures the optical (or light scattering) and fluorescence properties of single cells in a suspension passed through a focused beam of laser light (Brown and Wittwer, 2000; Bakke, 2001; Otsuki et al., 2003). Light scatter determines the intrinsic size and granularity of the cell and the fluorescence determines the extrinsic features of a cell e.g. specific protein expression and nucleic acid content (Bakke, 2001).

A flow cytometer (Figure 2.16) has three components, namely: (a) optics, (b) fluidics and (c) electronics.

Figure 2.16: Schematic illustration of a flow cytometer. This diagram shows the fluidic and optical parts of a flow cytometer. A single cell suspension is hydrodynamically focused with buffered saline sheath fluid to pass through an argon-ion laser. Light signals are collected by a forward angle light scatter detector, a side angle light scatter detector (1), and multiple fluorescence emission detectors (2–4). The signals are amplified and converted to digital signals for computer analysis and display (Brown and Wittwer, 2000).

A laser beam (e.g. argon-ion laser) is used as the light source to excite a fluorescent tag (e.g. propidium iodide (PI), phycoerythrin (PE), allophycocyanin (APC), fluorescein and Annexin V) bound to cells in a sample (Brown and Wittwer, 2000). The laser beam is passed through focusing and shaping optics to strike cells in specimens of cell suspensions injected in the middle of a flow chamber (Bakke, 2001). In the fluidics system, the sample is injected in a centralised core, surrounded by an outer sheath that contains a faster flowing buffered saline fluid (Bakke, 2001; Rahman et al., 2009). The outer sheath fluid causes hydrodynamic focusing by compressing and localising the core cell sample into a small area to produce a single stream of cells that pass one-by-one

Chapter 2: Literature Review through the focal point, the laser beam for fluorescence and light scatter analysis at multiple wavelengths and angles, respectively (Bakke, 2001; Cram, 2002).

When the laser collides with the cell, light scattering occurs and light is diffracted in all angles around the cell borders. Light scattering can be detected by forward scatter (FSC) (forward angle light scatter, FALS) and by side scatter (SSC) either as right-angle light (RALS) or 90-degree light (90LS) scatters. FALS is used to measure cell size and 90LS the cell structure or granularity, making FC a useful tool to distinguish between viable and dying cells and apoptotic from necrotic cells (Brown and Wittwer, 2000; Bakke, 2001; Otsuki et al., 2003). The emitted light given off in all directions is collected via optics and channelled to a series of filters and dichroic mirrors at particular wavelength bands (Figure 2.16) (Brown and Wittwer, 2000). Light signals are detected by optical detectors (photodiode detectors or photomultiplier tubes) and digitised for computer analysis (Brown and Wittwer, 2000; Bakke, 2001; Cram, 2002).

The digital signal produced is interpreted by the computer as linear or logarithmic amplifications. Linear amplification provides information about scatter signals and fluorescent measurements of DNA; and the logarithmic amplification provides information about most other biologic signals e.g. immunofluorescence (Bakke, 2001). Computer data analysis displays digital data as one-parameter histograms or as two- parameter dot-plot graphs (Jaroszeski and Radcliff, 1999; Bakke, 2001). Histograms are used to obtain the percentages of cells and the mean fluorescence intensity of a cell population. Dot-plots can be divided into four quadrants, each showing a percentage of cells in the total cell population, to distinguish between live and dead cells or fluorescence and non-fluorescence cells (Bakke, 2001).

2.6 Medicinal plants as target for use as antimicrobial agents

Infectious diseases account for most premature deaths worldwide (Ahmad and Beg, 2001) as many pathogenic microorganisms (e.g. vancomycin-resistant Enterococci) have developed resistance against a variety of antibiotics currently used in the healthcare system. This then makes infectious diseases more difficult to treat with the already known antibiotics. It is this multidrug resistance of human pathogens to antibiotics that has led to a search for new antimicrobial agents from sources such as plants (Fauci, 1998; Ahmad and Beg, 2001).

Chapter 2: Literature Review

Traditional medicinal plants are now being investigated for the presence of antimicrobial compounds that can partially or completely inhibit the growth of pathogens without posing any toxic effects to the host cells. The strategy is to isolate the antimicrobial compounds in medicinal plants and further use them to manufacture new antimicrobial drugs (Iwu et al., 1999; Ahmad and Beg, 2001).

The well-known active antimicrobial compounds in medicinal plants include the phenolics (phenols and phenolic acids), which are mostly effective against the growth inhibition of Gram-positive bacteria (Rios and Recio, 2005). Other antimicrobial compounds found mainly in medicinal plants are the alkaloids, lectins, terpenoids, sesquiterpene lactones, diterpens, triterpenes, naphtoquinones, quinones, flavones, flavonoids and flavonols, tannins, sterols, quinines, terpenes, curcuminoids, stilbenes, coumarins, quinines, lignans, flavonoids, saponins, steroid lactones, quassinoids, ansa macrolides, proteins, and alkaloids, which are active against the growth inhibition of a broad-spectrum of microorganisms (Cowan, 1999; Rios and Recio, 2005; Huang et al., 2009).

Currently, many South African researchers are focusing in the search for plants with antimicrobial activities, particularly against the infectious diseases such as HIV, as it is currently a pandemic in the country (UNAIDS, 2011). It has been shown that the well- known indigenous South African medicinal plants, such as H. hemerocallidea and S. frutescens, are now used for the treatment of HIV infections (Mills et al., 2005; Street et al., 2008).

Examples of medicinal plants with active antimicrobial compounds include Felicia arigeroides (Asteraceae) and Brucea antidysenterica (Simaroubaceae). F. arigeroides has tannins, saponins and triterpene steroids that are effective against infectious diseases caused by Pseudomonas aeruginosa and Candida albicans (Salie et al., 1996), and B. antidysenterica has quassinoid-bruceantin compounds, which are active against the infectious disease dysentery (Gurib-Fakim, 2006). A plant which grows in SA, Schotia latifolia, has show to possess antimicrobial activities because of epicatechins and catechins compounds, which were shown to be active against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and P. aeruginosa (Masika et al., 2005; Van Vuuren, 2008).

Chapter 2: Literature Review

2.7 Antimicrobial properties of medicinal plants and the microorganisms under investigation for this study

Infectious diseases are those caused by pathogenic organisms like bacteria, fungi, viruses and parasites and are contagious as they can be spread directly or indirectly from one person to another (WHO, 2013b). When looking at the worldwide statistics for human diseases, besides the noncommunicable diseases (e.g. cardiovascular diseases, cancers, diabetes and chronic lung diseases), infectious diseases still remain the leading cause of deaths killing more people than any other sinlge cause, especially in tropical regions of developing countries and in low-income countries (Fauci, 1998; Iwu et al., 1999; WHO, 2011; MedlinePlus, 2014). Due to the fact that many pathogenic microorganisms are gaining acquired resistance against a variety of antibiotic drugs, there is a current urgent search for novel natural compounds with antibiotic activities (Fauci, 1998; Cowan, 1999; Iwu et al., 1999). Antibiotics are beneficial to humans as they can be used in many sectors such as in the health care sector, biotechnology sector, food sector and in the pharmaceutical sector. Since it has previously been mentioned that medicinal plants are safer and more affordable than the synthetic antimicrobial drugs, medicinal plants now provide a great resource in the field of new antibiotic drug development (Iwu et al., 1999).

The use of medicinal plants as herbs to treat infectious and other diseases is increasing in SA (Louw et al., 2002). Many South Africans are finding themselves seeking alternative methods for treating infectious diseases other than the use of common western or modern medicines, which can cause side-effects, may be expensive, difficult to acquire, misused and over-prescribed, and can become non-effective due to acquired resistance. The South African government is now looking at involving herbalists and traditional healers in the healthcare system in order to help solve the current existing problems associated with the use of modern medications (Drewes and Khan, 2004; Chinsembu and Hedimbi, 2010). It is envisaged that with proper education and more research on the quality, safety and efficacy of plant derived medicines, more people will soon resort to using herbs as preference to treating infectious diseases (Fennell et al., 2004a; Fennell et al., 2004b). Microorganisms used in this study pose environmental as well as human threats as most are human pathogens responsible for the cause of some well known infectious diseases.

Chapter 2: Literature Review

The microorganisms that were tested in this study were  Bacillus subtilis  Enterococcus faecalis  Escherichia coli  Pseudomonas aeruginosa  Mycobacterium smegmatis and  Candida albicans

B. subtilis are a rhizobacteria or Gram-positive endospore-forming bacteria found mostly in soil (Stein, 2005; Dhanapathi, et al., 2008; Earl et al., 2008). In immunocompromised patients, the probiotic strains of B. subtilis can be responsible for the formation septicaemia and severe bacteremia infections (Richard et al., 1988; Oggioni et al., 1998).

Enterococci are Gram-positive, non-spore forming, facultative microorganisms that belong to the genus Enterococcus (Franz et al., 2003; Moreno et al., 2006; Zoletti et al., 2011). Enterococci, more specifically E. faecalis, are opportunistic human pathogens that cause major nosocomial infections such as bacteraemia, endocarditis, urinary tract infections, intra-abdominal infections, meningitis, respiratory tract infections and wound infections, which are hard to treat with antibiotics because of E. faecalis resistance to a variety of antibiotics such as vancomycin (Chenoweth and Schaberg, 1990; Franz et al., 2003; Kau et al., 2005; McBride et al., 2007).

E. coli are a Gram-negative, facultative anaerobe, motile bacillus bacteria that normally resides in the human gut as colonic flora (Nataro and Kaper, 1998). The pathogenic form of E. coli accounts for many urinary tract infections, sepsis or meningitis, gastrointestinal diseases, prostatitis and most enteric or diarrheal diseases, which are a challenge to treat (Nataro and Kaper, 1998; Steenkamp et al., 2006; Vejborg and Klemm, 2009).

P. aeruginosa is a Gram-negative rod shaped flagellated bacterium which is 0.5-0.8 x 1.5-8 µm in size (Bennik, 2004). P. aeruginosa are normally not harmful to healthy individuals but are opportunistic (Ferroni et al., 1998; Iversen et al., 2008). They are responsible for most hospital-acquired infections (that are not easy to treat) such as chronic lung infections (most prevalent in cystic fibrosis patients), bacteraemia,

Chapter 2: Literature Review endocarditis, and infections of the urinary tract, respiratory tract, central nervous system, ear, eye, bone, joints and skin (Ferroni et al., 1998; Hoiby et al., 2005; Iversen et al., 2008).

M. smegmatis are aerobic, nonmobile, acid fast bacteria found in nature as water- and soil-dwelling saprophytes or as pathogens of humans and animals (Rogall et al., 1990; Pierre-Audigier et al., 1997; Röse et al., 2004). Examples of infections caused by these mycobacteria include the skin and soft-tissue infections (granulomatous lesions) in immunocompetent individuals, pulmonary infections, lipoid and aspiration pneumonia, sterna wound infections, breast abscesses, endocarditis, lymphadenitis, osteomyelitis and cellulitis among others (Rogall et al., 1990; Pierre-Audigier et al., 1997).

C. albicans is a germ tube and chlamydospore forming pathogenic yeast that falls under the Protean genus (Pinjon et al., 1998). This microorganism can be found in the normal gastrointestinal microflora of most healthy human beings (Berman and Sudbery, 2002); however, the standard strain of C. albicans, ATCC10231, is the main cause of an infectious disease called candidiasis, which is mostly prevalent in HIV infected patients and / or in immunocompromised patients (Calderone and Fonzi, 2001; Berman and Sudbery, 2002; Naglik et al., 2003; Motsei et al., 2003; Shai et al., 2008).

The pathogenic C. albicans has a capability of causing other nosocomial infectious fungal diseases (fungemia) such as arthritis, pneumonitis, meningitis, peritonitis, pyelonephritis, endophthalmitis and osteomyelitis (Fridkin and Jarvis, 1996). Most of these conditions are chronic and very hard to treat. Posed with the problem of controlling the spread and resistance of infectious microorganisms, it is thus vital that the search of new antibiotics for the treatment of such infections be explored.

2.8 Techniques used in the evaluation of the antimicrobial properties of the H. hemerocallidea extracts

2.8.1 Direct TLC-bioautography assay

Thin layer chromatography (TLC) is used to analyse the chemical composition of extract samples, and together with bioautography, it is used to analyse the antimicrobial

Chapter 2: Literature Review activities in the extract samples (Horváth et al., 2010; Udobi et al., 2010). The direct TLC-bioautography (TLC-DB) is a relatively cheap, rapid, sensitive, quick and simple screening method used for the detection of antimicrobial compounds (antibacterial, antifungal, antitumour and antiprotozoa) on a chromatogram (Horváth et al., 2010; Choma and Grzelak, 2011). The technique has been used in the search for new antibiotics against a variety of human and plant pathogenic microorganisms since 1946 (Hamburger and Cordell, 1987; Horváth et al., 2010; Choma and Grzelak, 2011).

The TLC-Bioautography assay principle is illustrated in Figure 2.17 (Hamburger and Cordell, 1987). After separation of the compounds by means of TLC, a suspension of a particular microorganism is applied onto the developed plate. The plate is then incubated in a humid atmosphere to allow for bacterial growth. Zones of inhibition by the compounds on the plate are then visualised by a dehydrogenase-activity-detecting reagent such as the tetrazolium salts. Examples of tetrazolium salts that can be used include ρ-iodonitrotetrazolium violet (INT), tetranitro blue tetrazolium (TNBT), and methyl thiazolyl tetrazolium (MTT); with INT being the most preferably used in all these, because of its high sensitivity (Marston, 2011).

Figure 2.17: A simple diagram showing the principle involved in TLC-DB. Firstly, phytochemical separation of the test compounds is conducted, then bacteria is directly grown on the TLC plate afterwards. After a certain period of incubation, the plate is then sprayed with a detection reagent, which will then allow for the visualisation and identification of the zones of inhibition. Clear areas will then represent zones of inhibition by some separated compounds and where there is bacterial growth; it will be shown by the coloured background (Marston, 2011).

Metabolically active bacterial organisms convert the tertrazolium salt into the matching strongly coloured formazan product via a reduction process (Figure 2.18). Places of growth inhibition (exerted by the antibacterial compounds) on the bioautogram are

Chapter 2: Literature Review shown by clear or creamy-white spots against the intensely coloured background indicative of growing bacteria (Hamburger and Cordell, 1987; Choma and Grzelak, 2011).

Figure 2.18: Structural representation of the process involved in the reduction of the MTT tetrazolium salt into a coloured formazan product, indicating bacterial growth. Viable, growing, metabolically active bacteria have dehydrogenase enzymes that reduce and convert the yellow MTT molecules into a violet-coloured formazan product which makes it easy for the identification of live bacteria. This process similar in all tetrazolium salts (Marston, 2011).

2.8.2 Microdilution assay

Another method used to test for the presence of antimicrobial activities is the microdilution assay, adapted by Eloff (1998a). This method is usually used to determine the minimum concentration required for an extract to effectively inhibit the growth of a microorganism (Eloff, 1998a; Choma and Grzelak, 2011). The concentrations obtained from this assay are called the minimum inhibitory concentration (MIC) values. Using serial dilutions, different concentrations of the extract sample are tested to determine the optimal MIC value.

2.8.3 The BacTiter-GloTM microbial cell viability assay

BacTiter-GloTM assay is used to determine viable bacterial cells in culture by measuring the quantity of the ATP content in viable cells (Figure 2.19; Promega, 2012).

Chapter 2: Literature Review

Figure 2.19: Firefly luciferase bioluminescence reaction. The luciferin substrate is catalysed 2+ by the luciferase enzyme (in the presence of ATP, O2 and Mg ) to produce a bioluminescent oxyluciferin product, which produces a quantifiable light signal (Promega, 2012).

Since ATP acts as an indicator for metabolically active cells, the assay is based on the firefly luciferase bioluminescent reaction, where ATP produced by live-viable cells binds to substrate-luciferin for catalyses by luciferase to form an excitable bioluminescent oxyluciferin product (Figure 2.19), which emits radiation in the visible region of the electromagnetic spectrum (550–570 nm, Leitão and Esteves da Silva, 2010; Promega, 2012).

2.9 Overview

The anticancer and antimicrobial properties of H. hemerocallidea were explored using the techniques described in this section. The viability of SNO oesophageal cancer were investigated by means of trypan blue dye exclusion assay, AlamarBlue® dye reduction assay, CytoTox-GloTM cytotoxicity assay and forward- / side-scatter FC analysis. Morphological changes due to cytotoxic effects and the inhibition of growth in the cancer cells, following treatment with plant extracts from corms and leaves were studied using light microscopy. The antimicrobial activities of the plant extracts against fungi, mycobacteria, Gram-negative bacteria and Gram-positive bacteria were determined using the direct TLC-DB assay, microdilution assay, and the BacTiter-GloTM microbial cell viability assay on organisms mentioned in Section 2.7.

Chapter 3: Experimental Procedures

CHAPTER 3 - EXPERIMENTAL PROCEDURES

3.1 Preparation of the plant extracts 47

3.3 Microbial studies 53 3.3.1 Phytochemical analysis of plant extracts by thin layer chromatography (TLC) 53 3.3.2 Culturing of microorganisms 54 3.3.3 Determination of antimicrobial activities using direct TLC- bioautography 54 3.3.3.1 Antibacterial screening 54 3.3.3.2 Anti-mycobacterial screening 55 3.3.3.3 Antifungal screening 56 3.3.3.4 Control samples 56 3.3.4 Quantification of the antimicrobial activity 56 3.3.5 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity 58 3.3.6 The use of GC-MS for partial identification of the antimicrobial compounds 59

46 Chapter 3: Experimental Procedures

CHAPTER 3 - EXPERIMENTAL PROCEDURES

3.1 Preparation of the plant extracts

H. hemerocallidea plants were purchased from a local nursery in Johannesburg, South Africa. Plants were uprooted, cleaned and separated into leaves and corms. The plant material was then further divided into two components to provide fresh and dried samples, for both corms and leaves respectively. Dry plant material was obtained by placing the clean material in an oven at 50°C for 72 – 96 h.

Water extracts were prepared by homogenising 1 g of plant material in 10 ml sterile dH2O. The plant material was then boiled for 15 min and centrifuged at 1500 x g for 10 min using the High Performance Avanti® J-30I centrifuge (Beckman Coulter, Germany). The resulting supernatants were filtered through filter paper (Schleicher and Schuell, Germany), then through 0.45 µm cellulose acetate filters, and lastly through 0.2 µm cellulose acetate filters (Sartorius, Germany). The filtrates were concentrated by freeze- drying to obtain four extracts viz. fresh water corm extract (FWC), dried water corm extract (DWC), fresh water leaf extract (FWL), and dried water leaf extract (DWL).

Ethanolic extracts were prepared by homogenising 1 g plant material in 10 ml absolute ethanol (Sigma-Aldrich, Germany), followed by overnight agitation at 80 rpm on an orbital shaker. The homogenate was centrifuged at 1500 x g for 10 min and supernatants were collected and filtered through 0.2 m cellulose acetate filters before being concentrated in a vacuum system. The following four ethanol extracts were made: fresh ethanolic corm extract (FEC), dried ethanolic corm extract (DEC), fresh ethanolic leaf extract (FEL), and dried ethanolic leaf extract (DEL).

3.2 Cancer studies

3.2.1 Culturing of the SNO cell line

The SNO cell line (Highveld Biologicals, South Africa) was maintained in 75 cm3 cell culture flasks (Highveld Biologicals, South Africa) containing 25 ml sterile Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) (heat inactivated at 56°C for 20 min), 5% penicillin / streptomycin / fungizone, and 1% gentamycin (all antibiotics and serum were supplied by Highveld Biologicals, South

47 Chapter 3: Experimental Procedures

Africa). The SNO cell line was cultured at 37°C, 99% atmospheric pressure, 95% humidity and 5% CO2 levels. Experiments were performed with SNO cells at the exponential phase of the cell-growth curve; between passages 2 and 7.

3.2.2 Dose-dependent studies of plant extracts on the SNO cancer cell line

The SNO cell line was treated using different plant extracts with plant extract concentrations ranging from 0 - 500 µg / ml for 24 h. All plant extracts (both water and ethanolic extracts) used for experimentation were prepared from the same stock solution of 20 mg / ml concentration. Ten percent (10%) dimethyl sulphoxide (DMSO, Sigma-

Aldrich, Germany) and sterile dH2O was used (due to their non-toxicity effects on cells) to prepare the plant samples in order to facilitate extract uptake by the cells. SNO cells were used at a concentration of 6x105 cells / ml for all experiments. Upon treatment with plant extracts, the SNO cell line was incubated at 37°C for 24 h. Light microscopy and cell viability analyses of the cell line were performed to study the morphological changes and the rates of cell growth inhibition in the SNO cell line.

3.2.2.1 Cell viability studies of SNO cells treated with plant extracts

Cell viability determines the amount of living-to-dead cell ratio in a sample of cells. For these studies, cell viability was used to calculate the percentages of living and dead SNO cells upon treatment with the plant extracts. Cell viability of the SNO cell line was studied and measured using the following viability assaying techniques: (1) the trypan blue dye exclusion assay (Sigma Aldrich, Germany), (2) the AlamarBlue® dye reduction assay (AbDSerotec, UK), (3) the CytoTox-GloTM cytotoxicity assay (Promega, Germany) and (4) flow cytometry (forward and side scatter analyses).

3.2.2.1.1 Trypan blue dye exclusion assay analysis

The trypan blue dye exclusion assay uses the state of the cell’s plasma membrane integrity to differentiate between live and dead cells. Live cells have their membrane integrity retained in an intact state and any toxic substances are not allowed to pass through the selectively permeable membranes. Dying cells lose membrane integrity and become more porous such that toxic dyes are allowed to easily pass in and out the porous membranes. The assay is principled on the trypan blue dye penetrating the compromised plasma membranes of nonviable or dead cells to stain them blue in colour.

48 Chapter 3: Experimental Procedures

The dye is excluded from the intact membranes of viable cells, leaving them clear and uncoloured. Therefore, the trypan blue cell viability assay allows for the determination of the percentage number of viable cells in a cell sample (Perry et al., 1997; Longo-Sorbello et al., 2006). Low cellular viability percentages indicate potential anticancer properties of the plant extracts on the SNO cell line.

SNO cells were harvested through trypsinisation which involved removing the media from the flasks, washing the SNO cells twice with 10 ml Hank’s balanced salt solution (HBSS) to remove dead cell debri, the addition of 6 ml of 0.25% trypsin / 0.1% versene (w/v) followed by incubation at 37 °C for 4.5 minutes with slight agitation to detach SNO cells from the flask surfaces. Addition of 10 ml supplemented DMEM was used done to inactivate trypsin. The cells were then washed by centrifuging at 1028 x g for 4 minutes at 25 °C, and resuspending the pellet in 1 ml supplemented DMEM medium (supplementation of the DMEM medium was as mentioned in Section 3.2.1). An automated cell counter, Cell Countess™ (Invitrogen, USA), and a haemocytometer test were used to measure the cell viability of the SNO cells following their treatment with plant extracts (as mentioned in Section 3.2.2). Ten microlitres (10 µl) of trypan blue solution (Sigma Aldrich, Germany) was added to 10 µl treated SNO cell suspensions and counted. Cell viability was calculated using the formula:

% cell viability = total number of viable cells per ml x 100 total number of cells per ml

3.2.2.1.2 AlamarBlue® dye reduction assay analysis

The AlamarBlue® dye reduction viability assay is used to test for cellular viability in cell samples by monitoring the activity of the metabolically active mitochondria in cells. Healthy, proliferating, viable cells are metabolically active due to the presence of intact active mitochondria and dead or damaged cells with compromised mitochondria are metabolically inactive. The assay exploits the principles of an oxidation-reduction (REDOX) reaction, where levels of respiratory oxidation are measured. Cell viability is measured by the reduction of AlamarBlue® by proliferating viable cells (Pagé et al., 1993; McMillian et al., 2002; Longo-Sorbello et al., 2006; Pettit, 2009). The AlamarBlue® dye is a fluorogenic REDOX indicator for growing cells in a culture. In an oxidised form, the dye is dark blue in colour and has little to no fluorescence. When taken up by metabolically active cells, the dye is reduced by the components of the electron transport chain (e.g.

FMNH2, FADH2, NADH, NADPH and cytochromes) into a pink and highly fluorescent

49 Chapter 3: Experimental Procedures compound. The reduced AlamarBlue® dye is pink and highly fluorescent (Al-Nasiry et al., 2007). Dye reduction is measured by fluorescence at an excitation wavelength of 544 nm and emission wavelength of 590 nm.

The AlamarBlue® dye reduction assay was used to measure the SNO cell viability, following treatment with plant extracts (as mentioned in Section 3.2.2). After the SNO cells were harvested by trypsinisation (as previously mentioned), 10 µl AlamarBlue® solution was added to 100 µl of treated SNO cell suspensions. Incubation was done in the dark at 37°C for 2 h. Dye reduction was measured using the SynergyTM HT multi- detection microplate reader (Biotek®, USA). Percentage cell viability was determined using the formula:

% cell viability = fluorescent value of the treated SNO cells x 100 fluorescent value of the untreated SNO cells

3.2.2.1.3 CytoTox-GloTM cytotoxicity assay analysis

The CytoTox-GloTM (Promega, USA) assay is a luminescent based assay used to determine the number of dead cells decrease in cell viability and cytotoxicity in a cell population. The assay measures the activity of the dead-cell protease, which is released from cells with lost membrane integrity. Provided in the assay kit is a luminogenic peptide substrate, AAF-GloTM (alanyl-alanyl-phenyalanyl-aminoluciferin), which is cleaved by the released dead-cell protease peptides. This allows for the aminoluciferin component of AAF-GloTM to be converted into a quantifiable luminescent signal (light).

The CytoTox-GloTM cytotoxicity assay analysis was used to measure the decrease in cell viability of the SNO cells after treatment with plant extracts. The manufacturer’s instructions were followed. Briefly, 50 µl of CytoTox-GloTM reagent (mixture of substrate and assay buffer) was added to a 100 µl aliquot of harvested SNO cell suspension in a 96-well microtitre plate and incubated in the dark at room temperature for 15 min. After the incubation period, luminescence was measured using the SynergyTM HT multi- detection microplate reader (Biotek®, USA) at simultaneous multiple ranging wavelengths of between 300 – 700 nm. An additional 50 µl of Lysis Reagent (mixture of Digitonin and assay buffer) was added, and the cells were further incubated in the dark at room temperature for 15 min. After incubation, the total luminescence was measured. The total luminescence determines the total number of dead cells in the sample. Viable cell

50 Chapter 3: Experimental Procedures luminescence was then calculated by subtracting the initial luminescence from the total cell death luminescence. The negative controls for the study were the untreated SNO cells and the positive controls were the SNO cells treated with 3% formaldehyde (Sigma, USA), as a cytotoxic agent (Lovschall et al., 2002).

3.2.2.1.4 Flow cytometry (forward and side scatter) analysis

Flow cytometry is a technique that allows for the qualitative and quantitative analysis of multiple characteristics of single cells. Characteristics that can be measured include cell size, cytoplasmic complexity, the cell’s nucleic acid (DNA or RNA) content and membrane bound or intracellular proteins. Flow cytometry measurements are obtained by studying the optical and fluorescence characteristics of single cells. The physical characteristics of a single cell (such as the cell’s morphological state) can be measured using forward and side scatter analyses. The forward angle light scatter (FSC-A) measures the cell size and the right-angled or side angle light scatter (SSC-A) measures the shape or the internal complexity of a cell; this internal complexity includes measurements of how granular the cell is (Brown and Wittwer, 2000; Nunez, 2001).

For this study, flow cytometry was used to measure the cell viability of the SNO cells, following their treatment with plant extracts. Viable cells are larger and less granular than dead cells. Following SNO cell treatment with plant extracts and incubation at 37°C for 24 h, media was removed from the culture dishes and replaced with phosphate buffered saline (PBS; pH 7.4) in a 1:1 ratio. PBS was made up with 8 g NaCl, 0.2 g KCl, 1.44 g

Na2HPO4, 0.24 g KH2PO4 in 1 L. Using 0.25% typsin / 0.1% versene (w/v) (Highveld Biologicals, South Africa), the SNO cells were removed from the culture plates. The resulting cell solution was centrifuged at 1028 x g for 4 min using the AllegraTM 25R centrifuge (Beckman Coulter, Germany), the supernatants (which contained the dead cells) were kept and the pellets (which contained the live cells) resuspended in new 1X PBS. Both the supernatants and pellets were used for flow cytometry studies to determine the cell viability of the cells. Five hundred microlitres (500 µl) of treated SNO cell samples were read at 1 flow rate / min. Cell analysis was done using the FACS AriaTM Flow Cytometer (BD Biosciences, USA), and data was analysed using the BD FACSDivaTM 6.0 software. Cell viability was determined by calculating the cell percentages of live-to-dead cells, in comparison to the negative control. Untreated SNO cells served as negative controls, SNO cells treated with 3% formaldehyde as a positive control, and both media and PBS as vehicle controls.

51 Chapter 3: Experimental Procedures

Since only the size and granularity of the SNO cells in response to treatment of the plant extracts was measured, there was no need to incoparate a florescence probe. However, although it is not totally accurate to make assumptions based on forward scatter and side scatter alone, a viability stain such as acetomethoxy or diacetate of a fluorescent stain (Calcein-AM, Fluorescein diacetate) should be considered. Another cytotoxicity stain like propidium iodide, which works similarly to trypan blue, can also be used.

3.2.2.1.5 Light microscopy analysis of SNO cells treated with plant extracts

Morphological changes in the SNO cell line were studied using a simple AxioCam MRTM colour inverted light microscope (CarlZeiss, Germany). Cell micrographs were taken using the AxioVisionTM software at 200 x magnification to visualise the morphological differences between the normal-untreated SNO cell line and the plant extract-treated SNO cell line.

3.2.2.2 Statistical analysis

Cell viability studies were done in three biological replicates with two technical repeats for each experiment (n = 6) unless indicated otherwise. The mean viability values, the standard deviation (STDEV) and the standard error of the mean (SEM) were calculated. The error bars on the graphs (Figures 4.1 to 4.5) represent the SEM. An ANOVA single factor test was performed to determine statistically significant differences between the untreated and the treated SNO cells. A probability value (P-value) less than 0.05 signified a statistical significant difference between the untreated and treated SNO cells.

3.3 Microbial studies

Since results indicated that the ethanolic plant extracts (FEC, DEC, FEL, and DEL) were able to induce cell death in SNO cells, for the microbial studies, only these extracts were used. A concentration of 1 mg / ml ethanolic plant extract was used for further experiments. Partial identification and fractionation, using the phytochemical analysis, was conducted to identify the number of compounds present in each crude extract.

Although the plant extracts prepared in dH2O (FWC, DWC, FWL, and DWL) were unable to induce cell death, this does not provide a reason for these extracts not to demonstrate antimicrobial activity. Many antibiotics used today may not likely show any activity

52 Chapter 3: Experimental Procedures against the SNO cell line, but may be excellent antibiotics. Further testing of these extracts need to be done.

3.3.1 Phytochemical analysis of plant extracts by thin layer chromatography (TLC)

TLC was used to study the phytochemistry of the plant extracts. Phytochemical analysis involved the separation of the compounds present in the extracts using TLC plates. Silica coated TLC plates on alumina sheets (20 cm x 20 cm) with a UV254 fluorescent indicator (Merck, Germany) were used as a stationery phase. The mobile phase used consisted of ethyl acetate (Rochelle Chemicals, South Africa), methanol (Associated Chemical

Enterprises, South Africa) and dH2O mixed at a ratio of 80:10:10 (v / v / v).

Experiments were done in duplicate whereby 40 µl of extract sample were applied to the TLC plates, which were then developed. Detection of the compounds present in the extracts was done by means of vanillin (0.1 g vanillin (Saarchem, South Africa) in 97% methanol and 3% sulphuric acid (Associated Chemical Enterprises, South Africa) (w / v / v)). The plates were heated in an oven at 50°C for 10 min to allow for colour development and visualisation.

Retardation factor (Rf) values were calculated using the formula:

Rf = distance migrated by compound distance migrated by the mobile front

3.3.2 Culturing of microorganisms

Strains of Gram-positive bacteria (B. subtilis (ATTC 6051) and E. faecalis (ATTC 29212), Gram-negative bacteria (E. coli (ATTC 1175), P. aeruginosa (ATTC 9027)), yeast (C. albicans (ATTC 10231)) and mycobacteria (M. smegmatis), were used as test for the antimicrobial activity of the compounds in the plant extracts. Microorganisms were originally obtained at the Pharmacology department at the University of the Witwatersrand (Wits) and maintained, preserved and stored as frozen stocks at the Biochemistry Department at the University of Johannesburg.

53 Chapter 3: Experimental Procedures

3.3.3 Determination of antimicrobial activities using direct TLC- bioautography

The plant extracts were used to test for compounds with activities against the microorganisms using a simple and direct assay on TLC plates. The technique, as initially described by Hamburger and Cordell (1987), was used with slight modifications. The principle of the assay involves a suspension of microbial cells in suitable growth medium (as explained in the Sections 3.3.3.1, 3.3.3.2 and 3.3.3.3) being applied to a developed TLC plate. The developed TLC plate is then overlaid with a thin layer of microbial culture solution, and incubated for a specific period of time. The plates are usually incubated at 37°C in humid conditions to allow for optimal microbial growth. The zones of inhibition on the plates are visualised by the conversion of a tetrazolium salt by metabolically active bacteria, into a corresponding intensely coloured formazan product formed on the background of the plates. Hence compounds which are able to retard the growth of the microorganism appear as a clear zone(s) against this coloured background (Hamburger and Cordell, 1987; Horváth et al., 2010; Udobi et al., 2010; Choma and Grzelak, 2011).

3.3.3.1 Antibacterial screening

Cell culturing of the bacteria and C. albicans was done using the tryptone soy broth (TSB) medium, made up from 30 g of tryptone powder (Biolab Diagnostics, Merck South

Africa) and dH2O up to a final volume of 1 L. The lysogeny broth (LB) medium (pH 7.0), made up from 10 g bacto-tryptone (Oxoid Unipath, England), 5 g bacto-yeast extract (Fluka Analytical, USA) and 5 g sodium chloride (Promark Chemicals, South Africa) in dH2O up to a final volume of 1 L, was used for culturing of E. coli. Both the bacteria and C. albicans were maintained as cell stocks by combining 1 ml of established microbial cells (in media) with 1 ml glycerol (v / v) (Merck, South Africa) and stored at -20°C.

Five millilitres (5 ml) of the prepared microbial cell suspensions (OD600  0.2) were applied and spread with a glass rod to the developed TLC plates to ensure an even distribution of the microorganism suspensions on the plates. The plates were incubated in a sealed plastic container lined with wet filter paper at 37C for 8 h. The plates were sprayed with 3 ml of 2 mg / ml solution of -iodonitrotetrazolium violet (INT, iodonitrotetrazolium chloride, 3-[4-iodo-phenyl]-2-[4-nitrophenyl]-5-phenyl-2H-tetrazolium chloride, Sigma-Aldrich, Germany) and further incubated at 37C overnight for colour

54 Chapter 3: Experimental Procedures development. Comparison with a duplicate TLC plate developed under the same conditions but visualized with vanillin reagent (as described in Section 3.3.1) allowed for the detection of the TLC-separated compounds exhibiting antimicrobial properties in the plant extracts.

3.3.3.2 Anti-mycobacterial screening

The Bacto-Middlebrook 7H9 broth medium was prepared from 5.9 g Middlebrook 7H9 broth base (Difco, USA), 1.25 g bacto casitone (Difco, USA), and 3.1 ml glycerol (Merck,

South Africa) made up to 1 L in dH2O. M. smegmatis were maintained in Bacto- Middelbrook 7H9 broth supplemented in 50% glycerol and kept at -20°C.

A similar procedure as described in Section 3.3.3.1 was followed. Five millilitres (5 ml) of the prepared mycobacterial suspensions (OD600  0.2) were applied to the developed TLC plates and spread with a glass rod. The plates were incubated at 37C for 8 h. Visualisation of the antimycobacterial compounds was obtained using 2 mg / ml INT. Comparison of the bioautogram with a duplicate chromatogram developed under the same conditions but visualized with vanillin helped to detect the separated compounds containing antimycobacterial properties.

3.3.3.3 Antifungal screening

The glucose-mineral salt medium used for the preparation of the fungal spore suspension was made up from 1 L 0.7% KH2PO4, 0.3% Na2HPO4·2H2O, 0.4% KNO3,

0.1% MgSO4·7H2O and 0.1% NaCl in dH2O. The solution was autoclaved for 120°C for 20 min and under sterile conditions; 10 ml of 4.3% glucose (D+ glucose monohydrate, Merck, South Africa) was added.

3.3.3.4 Control samples

Commercially known standards of H. hemerocallidea plant extracts, Moducare® capsules (Dischem, South Africa) and the African Potato capsules (Gabby Marketing cc, South Africa), were used for the TLC-DB identification of antimicrobial compounds found in the plant extracts. Moducare® and African Potato capsule samples were prepared by dissolving 1 mg capsule powder in 1 ml absolute ethanol. Forty microlitres (40 µl) of 1

55 Chapter 3: Experimental Procedures mg / ml Moducare® and African Potato capsule samples were loaded and developed as standards on the TLC plates, following the TLC method as described previously.

3.3.4 Quantification of the antimicrobial activity

Quantification of the antimicrobial activity of the plant extracts was determined using the serial microdilution assay described by Eloff (1998a) (Figure 3.1). The minimum inhibitory concentration (MIC) values were calculated for each microorganism and for each plant extract tested. The MIC value was taken as the lowest concentration of the plant extract that displayed inhibition to microbial growth after 24 h incubation.

Using the 96-well microtitre plates (Corning Incorporated, New York, USA), 200 µl of 1 g / 10 ml ethanol plant extracts were added in duplicates as test samples to the wells of the second column and serially diluted 50% to the tenth column using fresh, sterile media (Figure 3.1). The working microbial inoculums were prepared as mentioned in Sections 3.3.3.1, 3.3.3.2 and 3.3.3.3. Hundred microlitres (100 µl) untreated microbial inoculums were added in all wells, except for the wells of columns 11 and 12, which were reserved for the control samples.

The control samples that were included in the assay were:

 200 µl of diluted microbial inoculums (untreated) (OD600  0.2) added into each well of column 1 served as negative controls,  200 µl of absolute ethanol added into each well of column 11 which served as a vehicle control. Ethanol was used as an extractant during the extraction and needs to be factored into the interpretation of the results, and  200 µl of sterile media added into each well of column 12 served as the sterility control

56 Chapter 3: Experimental Procedures

1 2 3 4 5 6 7 8 9 10 11 12

A Rows A and B: Serial dilution of the FEC treated cultures B

Rows C and D: Serial dilution of the DEC treated cultures nol D

E Rows E and F: Serial dilution of the FEL treated cultures Sterile media Absolute etha Absolute F Untreated microbial cultures microbial Untreated G Rows G and H: Serial dilution of the DEL treated cultures H

Highest concentration Lowest concentration

Figure 3.1: A 96-well plate showing the set-up that was used to conduct the microdilution assay as described by Eloff (1998a). The extracts used were: FEC – Fresh Ethanolic Corm extract, DEC – Dried Ethanolic Corm extract, FEL - Fresh Ethanolic Leaf extract, and DEL - Dried Ethanolic Leaf extract.

After adding all the samples onto the plates, as shown in Figure 3.1, the microtitre plates were sealed with an optically adhesive film to prevent evaporation during incubation at 37°C overnight. To indicate microbial growth, 40 µl of 2 mg / ml INT was added to each well and additional incubation step was done at 37°C for 30 min to allow for colour development. In the presence of actively growing microorganisms, the tetrazolium salts are reduced from a yellow to a red colour (Hamburger and Cordell, 1987). An inhibition or a decrease in microbial cell growth was indicated by faintly coloured to a clear solution in the wells. Microdilution assay experiments were repeated three times (three biological repeats) with duplicate technical repeats per experiment. MIC values were obtained by calculating an average of all MIC values.

Antibiotics, tested at a concentration range of 1.04 mg / ml – 26.0 µg / ml, was performed in a separate experiment and served as positive controls. The experiment was done as

57 Chapter 3: Experimental Procedures in the section mentioned above (Section 3.3.4) but with commercial antibiotics instead of using plant extracts.

The stock concentrations used for the preparation of each antibiotic were as follows: a) 10 mg / ml neomycin sulphate (Sigma, Germany), b) 80 mg / ml gentamicin sulfate (Micro Healthcare, South Africa), c) 500 mg / ml kanamycin sulfate (Sigma-Aldrich, St Louis, USA) for Gram-positive and Gram-negative bacteria, and d) 2 mg / ml diflucan (1H-1, 2, 4-trizole-1-ethanol, - (2,4-diflurophenyl)-(1-1,2- riazol-1-ylmethyl)-fluconazole, Pfizer, USA) for C. albicans.

3.3.5 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity

The BacTiter-GloTM assay (Promega, USA) is a luminescent-based assay used to determine the number of viable microbial cells in a culture. Metabolically active cells produce ATP, which can be quantified using luminometry. As explained in the kit’s instructions manual, the BacTiter-GloTM assay exploits the luciferase reaction, where in the presence of ATP and oxygen, the luciferin substrate is converted into oxyluciferin and a quantifiable luminescent light signal. The ATP produced by live-viable cells binds to luciferin and undergoes catalyses by luciferase and to form a bioluminescent oxyluciferin product, which emits radiation at 550 – 570 nm wavelengths (Leitão and Esteves da Silva, 2010; Promega, 2012). Using this principle, cell viability is measured and correlated to the amount of ATP produced by viable microbial cells.

The BacTiter-GloTM assay was used to evaluate the antimicrobial compound activity found in the ethanol-prepared plant extracts of H. hemerocallidea (FEC, DEC, FEL, and DEL). Depending on the microorganism studied, duplicate tests of plant extracts were done in TSB (for B. subtilis, E. faecalis and C. albicans), LB (for E. coli) or Bacto- Middlebrook 7H9 (M. smegmatis) medium. The plant extract MIC values obtained in Section 3.3.4 were used as a starting point for treatments in the BacTiter-GloTM studies. All microbial strains, in appropriate media, were used to prepare the working microbial inoculums by diluting overnight cultures to an OD600 of approximately 0.2 absorbance units. Using three 96-well microtitre plates, duplicate tests of each extract were done. A total volume of 100 µl culture inoculums were added to each well containing plant extract. All three plates were tightly sealed with parafilm® and incubated at 37°C. An

58 Chapter 3: Experimental Procedures incubation time study was conducted using the 3 h, 5 h and 8 h test exposure time intervals. After each time interval, a 100 µl of BacTiter-GloTM reagent was added to all wells of the experiment. Plate contents were briefly mixed on an orbital shaker and further incubated for 15 min at room temperature. Luminescence was determined using the SynergyTM HT multi-detection microplate reader (Biotek®, USA). Two hundred microlitres (200 µl) of the untreated microorganisms of each test strain were included as negative controls in the BacTiter-GloTM assay tests. Absolute ethanol and media were used as vehicle controls, and 200 µl of 500 mg / ml Moducare® capsules against C. albicans and E. faecalis (done in duplicate) were used as positive controls. Moducare® capsules were used because they contain 20 mg plant sterols and 0.2 mg sterolins, which are also known to be present and exhibit medicinal properties in the H. hemerocallidea plants. Microbial cell viability, after each incubation time interval, was determined by comparing the luminescent values of the treated microorganisms to the values of the untreated control organisms. The duplicate BacTiter-GloTM assay experiments were repeated five times (n = 10). Average values were used to calculate the STDEV and the SEM.

3.3.6 The use of GC-MS for partial identification of the antimicrobial compounds

The antimicrobial agents obtained using TLC-DB were isolated and analysed using GC- MS, an analytical method combining the principles of gas chromatography and mass spectrometry to identify chemical compounds in a sample. Gas chromatography allows for the identification of volatile compounds by analysing their retention time (Rt). The Rt is then linked and referenced to a standard compound in order to identify the unknown compounds. In mass spectrometry, the test compounds in a sample are converted to gaseous ions, which are separated in a mass spectrometer according to the mass-to- charge ratio (m / z). This m / z ratio allows for the identification of a compound by referencing its structure and mass to those of the well known standard compounds. The detection of the compounds is by the use of mass analysers, detectors and computer databases (Wilson and Walker, 2005).

Instrument set-up for the GC-MS studies were as follows: A Shimadzu QP2010 GC-MS (Shimadzu, Kyoto Japan) was fitted with a Zebron 5ms column (Phenomenex, Torrance, California, USA) with 30 m × 0.25 mm × 0.25 µm dimensions. Carrier gas (He 99.99% obtained from Afrox SA) was maintained at a constant flow rate of 1 ml / min and the

59 Chapter 3: Experimental Procedures column temperature programmed at 80°C, held for 4 min, then increased to 290°C at the rate of 20°C / min, and then lastly held for 2.5 min up to a total time of 17 min. The injection port was set at 260°C and operated split-less with sampling time of 2 min followed by split ratio of 10. The MS was operated using 70 eV electron impact ionization, with detector voltage run relative to tuning file. The acquisition was carried out on a scanning mode between 60 and 500 m / z values. The ion source temperature was set at 200°C and the interface temperature at 250°C. The MS acquisition was done from 4 min to 15 min.

GC-MS was used to attempt to identify the compounds responsible for the antimicrobial activities in the ethanol extracts of the H. hemerocallidea plant. Samples were obtained by isolating the antimicrobial compounds from the TLC and TLC-DB plates which had not been treated with any microorganism or with the INT. Samples were obtained by scraping off the separated antimicrobial plant extract compounds from the TLC and TLC- DB plates, re-dissolving and reconstituting them in a minimal amount of absolute ethanol. Only the FEC extracts that showed growth inhibition of the Gram-positive E. faecalis were used for GC-MS.

Gas chromatography-flame ionization detector (GC-FID) was used for the separation of the compounds of the TLC-DB-isolated FEC antimicrobial compound. The GC-FID- isolated compounds of FEC antimicrobial compounds (TLC-DB-isolated) were then analysed and identified using mass spectrometry.

One of the prominent compounds was tentatively identified as levoglucosan by GC-MS, which was assumed to be the main compound responsible for the majority of the antimicrobial activities found in FEC and DEC extracts. Thus, a levoglucosan (1,6- anhydro-β-D-glucose, Sigma-Aldrich, Germany) standard was separated on TLC and tested against B. subtilis, C. albicans and E. faecalis (using TLC-DB) for verification. FEC and DEC extracts were also included in the tests. One hundred microlitres (100 µl) of 1 mg / ml levoglucosan and 40 µl of both FEC and DEC were applied as loading concentrations on the TLC and TLC-DB plates. The resulting chromatograms and bioautograms obtained were compared and correlated to each other to determine if the standard levoglucosan had any antimicrobial activities against the test microorganisms used.

To further support the TLC-DB results obtained in the levoglucosan tests, 10 mg of the standard levoglucosan was mixed with 1 ml of either C. albicans or E. faecalis (w / v).

60 Chapter 3: Experimental Procedures

The negative control contained only 1ml of the microorganism (levoglucosan was omitted from the tube). The tubes were incubated at 37°C overnight, with constant agitation. Following incubation, 100 µl of INT was added in all tubes, and all were further incubated for 30 min at 37°C to allow for colour development. Microbial growth was observed by the development of a red colour in the tubes, and the presence of microbial inhibition was expected to be shown by a clear colour in the tubes.

61 Chapter 4: Results

CHAPTER 4 – RESULTS

4.1 Cancer studies 63 4.1.1 Cell viability studies of SNO cells treated with plant extracts 63 4.1.2 CytoTox-GloTM cytotoxicity assay analysis 67 4.1.3 Flow cytometry (forward and side scatter) analysis 67 4.1.4 Light microscopy analysis of SNO cells treated with plant extracts 68

4.2 Microbial studies 71 4.2.1 Phytochemical analysis of plant extracts by TLC 71 4.2.2 Determination of antimicrobial activities using direct TLC-DB (antibacterial, anti-mycobacterial and antifungal screening 73 4.2.3 Quantification of the antimicrobial activity using microdilution assay 78 4.2.4 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity 79 4.2.5 The use of GC-MS for partial identification of the antimicrobial compounds 81

62 Chapter 4: Results

CHAPTER 4 - RESULTS

This Chapter is divided into two main sections. The first part of this project involved the study of the ability of the H. hemerocallidea plant extracts to inhibit cell growth in an oesophageal SNO cancer cell line (CCL-185). The mode of anticancer activities was studied by investigating the viability and morphological changes in the SNO cells treated with the H. hemerocallidea extracts. The second part of this project involved the use of TLC-DB to study the ability of the H. hemerocallidea extracts to inhibit the cell growth of the B. subtilis, E. faecalis, E. coli, P. aeruginosa, M. smegmatis and C. albicans microorganisms in vitro. The active antimicrobial compounds in the extracts were characterised and identified using GC- MS.

4.1 Cancer studies

4.1.1 Cell viability studies of SNO cells treated with plant extracts

The trypan blue and AlamarBlue® assays were used to determine the cell viability of the SNO cells after treatment with H. hemerocallidea extracts. Dose-dependent studies were conducted by using the different plant extract concentrations (10 µg / ml – 500 µg / ml) to treat the SNO cells and determine the optimal concentration for cell growth inhibition. Trypan blue cell viability assay results are shown in Figure 4.1 and the AlamarBlue® cell viability assay results are shown in Figure 4.2 (for the corm extracts) and Figure 4.3 (for the leaf extracts), respectively. In the figure legends, the acronyms used for the extracts are as follows: FWC (fresh water corm), DWC (dried water corm), FWL (fresh water leaf), DWL (dried water leaf), FEC (fresh ethanolic corm), DEC (dried ethanolic), FEL (fresh ethanolic leaf) and DEL (dried ethanolic leaf).

The trypan blue assay showed that the SNO cells are not significantly influenced by the H. hemerocallidea extract treatments, with the exception of the 100 µg / ml DEC treatment, which showed only a 20% decrease in cell viability (Figure 1A). The AlamarBlue® results showed that no statistically significant decrease in cell viability was obtained for the FWC- (Figure 4.2A), DWC- (Figure 4.2C) and FWL- (Figure 4.3A) treated SNO cells, but viability statistically decreased significantly in cells treated with 250 – 500 µg / ml FEC (40% inhibition; Figure 4.2B), 100 – 500 µg / ml DEC (20 – 30% inhibition; Figure 4.2D), 100 µg / ml ml FEL (40% inhibition; Figure 4.3B), 10 µg / ml DWL (60% inhibition; Figure 4.3C) and 200 µg / ml DEL (35% inhibition; Figure 4.3D).

63 Chapter 4: Results

A. B.

C. D.

A Figure 4.1: The cell viability of the SNO cells after treatment with H. hemerocallidea corm extracts: (A) FWC, (B) FEC, (C) DWC and (D) DEC at varying concentrations of between 10 µg / ml and 500 µg / ml, in correlation to the untreated negative control cells. Cell viability of 10µl SNO cells was determined using the trypan blue dye exclusion assay analysis and cell counting was done using the automated cell countess™ counter. Error bars represent the SEM (n = 9) and asterisks show the significantly different values (ANOVA: Single Factor test * P<0.05) between treated and untreated control cells.

64 Chapter 4: Results

A. B.

C. D.

Figure 4.2: AlamarBlue® SNO cell viability results after treatment with H. hemerocallidea corm extracts. Results shown are for the SNO cells treated with (A) FWC, (B) FEC, (C) DWC and (D) DEC extracts at varying concentrations of between 10 µg / ml – 500 µg / ml in correlation to the untreated negative control SNO cells. Error bars represent the SEM (n = 12) and the asterisks indicate significant differences (ANOVA: Single Factor test * P<0.05 and **P<0.01) between the treated and untreated control cells.

65 Chapter 4: Results

A. B.

C. D.

Figure 4.3: Cell viability of the SNO cells obtained following the treatment with H. hemerocallidea leaf extracts: (A) FWL, (B) FEL, (C) DWL and (D) DEL at concentrations between 10 µg / ml and 200 µg / ml). Cell viability was determined using the AlamarBlue® assay. Error bars represent the SEM (n = 18) and the asterisks indicate significant different values (ANOVA: Single Factor test * P<0.05, **P<0.01 and ***P<0.001) between the treated and untreated control cells.

66 Chapter 4: Results

4.1.2 CytoTox-GloTM cytotoxicity assay analysis

CytoTox-GloTM assay is a luminescent based assay used to determine the decrease in ATP levels as a measure of cytotoxicity in a cell population. Due to the inconsistencies between the trypan blue and AlamarBlue® cell viability data, the CytoTox-GloTM assay was elected, to determine the levels of ATP as a measure of cell viability. Again, inconsistent results were obtained (Figure 4.4), and with the exception of the FWC-treated sample, no correlation was observed between these 3 different cell viability / toxicity assays with the differentially-treated SNO cell samples.

Figure 4.4: Cell viability of the SNO cells treated with 250 µg / ml FWC and FEC extracts, 300 µg / ml DWC and DEC extracts, 100 µg / ml FWL and FEL extracts, and 200 µg / ml DWL and DEL extracts. Cell viability was determined using the CytoTox-GloTM assay (n = 1), and is represented as a percentage relative to the untreated control cells (negative control). SNO cells treated with formaldehyde were used as a positive control and the media was used as a vehicle control.

4.1.3 Flow cytometry (forward and side scatter) analysis

Flow cytometry was used to count the number of cells in a sample, as well as to indicate the morphological differences and measure the viability of the SNO cells. The forward light scatter measures cell size and the side scatter measures cellular granularity. The concentrations selected for treatment were based on the AlamarBlue® data, and only the concentrations that would have induced cell death were chosen. In general, no significant decrease in SNO cell viability was obtained for the flow cytometry results (Figure 4.5), as indicated by the absence of a shift in the gated population relative to the untreated control. Slight alterations were observed in the FEC population, correlating to the data in Figure 4.2

67 Chapter 4: Results and Figure 4.4. However, a decrease in cell viability of SNO cells treated with other extracts was not observed in the flow cytometry data, since the population of cells did not shift; indicating a highly viable population based on the absence of increased granularity.

4.1.4 Light microscopy analysis of SNO cells treated with plant extracts

The absence of changes in the differentially-treated cells prompted the observation of morphological changes with light microscopy. It was thus decided to look closer at the potential alterations in cell morphology of the SNO cells following the various treatments. The same different concentrations (indicated in Figure 4.4) were used to treat the cells before light microscopy analysis.

The micrographs of the untreated versus treated samples are shown in Figure 4.6. In general, when viewed under a microscope, the normal healthy SNO cells appear to be attached to the surfaces of the culture plates, with a confluent, transparent, and a round appearance. It is known that SNO cells treated with apoptotic inducers show characteristics of unhealthy, dying cells, which detach themselves from each other and from the culture plate surfaces. Apoptotic and dying cells appear disintegrated with membrane blebbing, spherical, granular and shrunken when viewed under the microscope (Kroemer et al., 2009).

Some inconsistency in the results, as compared to the results of the previously mentioned studies, was also obtained with the microscopic study. In these micrographs, the control cells remained attached to the culture plate and looked normal, confluent and healthy with very limited signs of cell death (Figure 4.6). In comparison to the control cells, the majority of the FWC-, FEL-, DWC- and DWL-treated cells also looked healthy with limited signs of cell death. The FEC-, FWL- and DEL-treated cells also looked healthy with a few morphological changes and a small degree of unhealthy looking cells, where the cells were detached from each other and with a small percentage of loose cell debris and black coloured fragmented cells. Signs of cell death were obtained in the DEC treated cells as some of the cells appeared detached from the culture plate and with a dark granular appearance. Although limited amounts of anomalies were observed (white circles in Figure 4.6), the majority of the treated cells looked healthy, round and identical to the normal untreated cells. This led to the conclusion that the different extracts (water or ethanol leaf and corm) at relatively high dosages, did not cause significant cell death in the SNO cell line.

68 Chapter 4: Results

Figure 4.5: Dot-plots showing the cytometrical data obtained for the SNO cells treated with plant extracts to indicate cell viability. Different populations (viable or dead) were gated on the forward and side scatter plots. The dark coloured areas on the plots represent the viable populations as gated according to the untreated control population. The y-axis on the plots show the side scatter (SSC-A) and the x-axis show the forward scatter (FSC-A). From left to right of the figure is the negative control untreated SNO cells, formaldehyde treated cells as positive control, vehicle controls (phosphate-buffered saline / PBS and media), FWC treated cells, FEC treated cells, DWC treated cells, DEC treated cells, FWL treated cells, FEL treated cells, DWL treated cells and DEL treated cells. Dosages used are the same as in Figure 4.4, whereby the SNO cells were treated with 250 µg / ml FWC and FEC extracts, 300 µg / ml DWC and DEC extracts, 100 µg / ml FWL and FEL extracts, and 200 µg / ml DWL and DEL extracts.

69 Chapter 4: Results

Control 250 µg / ml FWC 250 µg / ml FEC 300 µg / ml DWC 300 µg / ml DEC

Control 100 µg / ml FWL 100 µg / ml FEL 200 µg / ml DWL 200 µg / ml DEL

Figure 4.6: Microscopic view the H. hemerocallidea treated SNO cells in comparison with the untreated control cells. The figure shows the micrographs taken at 200 x magnification. The circles on the graph point out the anomalies. Control cells looked healthy and growing well with no signs of cell death. FWC treated cells looked healthy with a very small insignificant percentage of cell death. FEC treated cells still looked normal but with a few morphological changes as some cells looked detached from one another with a few loose fragmented cell bodies that were seen scattered in the culture plate. DWC treated cells looked slightly more granular. Some apparent apoptotic cells were seen in the DEC treated-SNO cells as some cells looked abnormal or dying and detached from the culture plate, but a high percentage of them still looked healthy and attached to the plate. FWL treated cells looked healthy and growing well with a few unhealthy cells with dark membranes and a few black fragmented patches in the plate. FEL treated cells looked healthy with only a few cells with apoptotic membrane blebs and apoptotic bodies scatted in the plate. DWL treated cells looked alive and healthy without any signs of cell death. DEL treated cells looked healthy with a few abnormally dark coloured cells showing nuclear condensation; no major cell death was observed following treatment with different extracts.

70 Chapter 4: Results

4.2 Microbial studies

4.2.1 Phytochemical analysis of plant extracts by TLC

Phytochemistry is an analytical technique used to study the chemical composition of plants in order to explain the various plant chemical processes. It involves the qualitative detection, isolation and quantitative estimation of plant components (Burrel, 1937). In this study, the phytochemical analysis of the plant extracts was done, using TLC, to separate the compounds found in H. hemerocallidea extracts, namely FEC, DEC, FEL and DEL. Compounds of different colours and migration distances were obtained as shown in Figure 4.7. The retardation factor (Rf) values were calculated as described in Chapter 3.

A. B.

Figure 4.7: TLC chromatogram representing the phytochemical separation of the H. hemerocallidea plant extracts FEC, DEC, FEL, and DEL. The compounds were separated in a mobile phase consisting of ethyl acetate: methanol: dH2O (80:10:10 (v / v / v)) and developed with vanillin. (A) shows the separation of compounds when 40 µl crude plant extract and (B) when 50 µl crude plant extract was used. The band intensity is increased with a higher concentration, however no additional componds were observed.

Twelve (12) corm extract compounds and 10 leaf extract compounds were separated using TLC. All compounds separated in the H. hemerocallidea crude plant extracts are showed in

Table 4.1, together with their Rf values. The corms have a higher concentration of the more non-polar compounds than the leaves, which were separated better with a higher concentration of plant extracts, whereas the leaves (FEL and DEL) had a better resolution of polar compounds at a lower concentration. In addition, similar compounds in the corms and leaves are present in higher concentrations in the corms. There does not seem to be a difference between fresh and dry tissue in respect to the compounds present.

71 Chapter 4: Results

Table 4.1: Rf values of the compounds found in the H. hemerocallidea plant after phytochemical analysis by TLC.

The loading volumes are very high, and a smaller volume may have yielded a better separation and less tailing. However, due to the low concentration of the antimicrobial metabolites in the extracts, a larger volume was needed.

72 Chapter 4: Results

4.2.2 Determination of antimicrobial activities using direct TLC-DB (antibacterial, antimycobacterial and antifungal screening)

The direct TLC-bioautography (TLC-DB) assay is a qualitative analytical technique, which combines principles of TLC and bioautography to detect, identify, screen and localize compounds with antimicrobial activity, in crude plant extracts, for use in the search of new antimicrobial compounds (Esterhuizen et al., 2006; Horváth et al., 2010; Udobi et al., 2010).

TLC-DB assay is quick, easy to perform and relatively cheap. There is no sophisticated equipment required for this assay and it only requires a small amount of test compound; results obtained are easy to interpret and the assay is suitable for testing of all compounds that can be separated by TLC against any organisms that grow directly on the TLC plate surface (Esterhuizen et al., 2006; Horváth et al., 2010; Choma and Grzelak, 2011).

The TLC-DB results are shown in Figure 4.8 for B. subtilis, E. faecalis, E. coli, P. aeruginosa, M. smegmatis and C. albicans microorganisms.

73 Chapter 4: Results

B. subtilis C. albicans E. faecalis E. coli M. smegmatis P. aeruginosa

Figure 4.8: TLC-DB results for all test microorganisms. The bioautograms show results for 40 µl of crude plant extract. Clear zones (shown as rectangles on the figure) indicate areas of microbial inhibition by extract compounds, and the red background shows areas of bacterial growth. Only FEC and DEC presented zones of growth inhibition for all tested microorganism, except for M. smegmatis. These potential antimicrobial compounds had an Rf of between 0.12 and 0.14. FEL only displayed antimicrobial activities against E. faecalis and P. aeruginosa.

74 Chapter 4: Results

Using 40 µl of crude extracts to test for antimicrobial inhibition against the B. subtilis, E. faecalis, E. coli, P. aeruginosa, M. smegmatis and C. albicans microorganisms, all microorganisms except M. smegmatis were susceptible to the inhibitory effects of the corm extracts (FEC and DEC). The compounds responsible for the antibacterial and antifungal inhibition had Rf values ranging from 0.12 to 0.14 (the purple compounds on the chromatograms shown in Figure 4.7). The leaf extracts did not have any antimicrobial effects on all microorganisms except for FEL, which showed bacterial growth inhibition against E. faecalis and P. aeruginosa. The compounds responsible for anti- E. faecalis growth had Rf values between 0.14 and 0.17 and those responsible for the inhibition of P. aeruginosa growth had Rf values between 0.76 and 0.79 (Figure 4.8 and Table 4.1).

When 50 µl crude plant extracts were tested against the microorganisms, both FEC and DEC extacts inhibited the growth of M. smegmatis, which was not obtained when using the 40 µl crude extacts, indicating higher concentration of these extracts were needed to inhibit the growth of this microorganism. Table 4.2 provides a summary of the TLC-DB results obtained after testing the plant extracts DEC, FEC, DEL and FEL against all microorganisms used for this study.

Table 4.2: Summary of the TLC-DB results, obtained after testing the H. hemerocallidea plant extracts DEC, FEC, DEL and FEL against six microorganisms, namely: B. subtilis, E. faecalis, E. coli, P. aeruginosa, M. smegmatis, and C. albicans. Keys on the table denote the following: +microbial inhibition, -no microbial inhibition and ~inconclusive results.

Plant extracts

Microorganisms Corms Leaves

FEC DEC FEL DEL

B. subtilis + + - -

C. albicans + + - -

E. faecalis + + ~ -

E. coli + + - -

M. smegmatis ~ ~ - -

P. aeruginosa + + ~ -

75 Chapter 4: Results

According to the results obtained in the TLC-DB studies, H. hemerocallidea corm extracts inhibited the growth of Gram-positive bacteria (E. faecalis and B. subtilis) and fungi (C. albicans) more than the Gram-negative bacteria and mycobacteria (Table 4.2). There was one major antimicrobial compound (with a distinctive purple colour when visualised using vanillin) from the corm extracts, FEC and DEC, with an Rf value of 0.12 that inhibited the growth of B. subtilis, E. faecalis, and C. albicans; partially inhibited E. coli and P. aeruginosa; and had minimal anti-M. smegmatis activity (Figure 4.8). The leaf extracts (FEL and DEL) displayed partial anti-microbial activity against E. faecalis and P. aeruginosa.

As a positive control, commercially available Moducare® and Hypoxis tuber capsules were subjected to the same procedure. Compounds from Moducare® compounds also had a ® purple colour with vanillin and had a Rf value 0.73. Moducare and Hypoxis tuber capsules did not show any antimicrobial activities against the B. subtilis, C. albicans and E. faecalis; showing that the plant sterols and sterolins were most likely not responsible for the antimicrobial activities obtained for the FEC and DEC extracts (Figure 4.9). This led to further isolation of the FEC and DEC antimicrobial compounds, from the chromatograms and bioautograms, to be characterised using GC-MS.

76 Chapter 4: Results

Βeta-sitosterol glucoside sterolin

Figure 4.9: TLC-DB results obtained for the commercially known standards, Moducare® and African Potato tuber capsules. On the left, the reference plate is the TLC chromatogram showing the components of the Moducare® product (M) at 40 and 50 µl application volumes, Hypoxis tuber capsules (H) and H. hemerocallidea extracts (FEC and DEC) at 40 µl volumes. The circled compounds on the reference plate were separated from the Moducare® product. TLC bioautography results of B. subtilis, C. albicans and E. faecalis showed that the Moducare® product and the Hypoxis tuber capsules did not have any antimicrobial activities against those microorganism. Only the H. hemerocallidea extracts (FEC and DEC) showed microbial inhibition of B. subtilis, C. albicans and E. faecalis.

77 Chapter 4: Results

4.2.3 Quantification of the antimicrobial activity using microdilution assay

The microdilution assay, developed by Eloff, (1998a), is used to test for the presence of antimicrobial activities in plant extracts. The method measures the minimum concentration of plant extract required to effectively cause inhibition of a microorganism (Choma and Grzelak, 2011). The concentrations obtained from this assay are called the minimal inhibitory concentrations (MIC) values; thus, different concentrations (done via serial dilutions) of the antimicrobial sample are tested to determine the MIC value. By means of a tetrazolium salt (INT) for visualisation, wells of microbial growth inhibition are identified by looking at the concentration gradient of the test extracts applied on the plates, ranging from the highest to the lowest concentration, to determine their MIC values (Figure 4.10). Antimicrobial activity is detected by a colourless solution in the wells of the assay (representing no microbial growth), whilst in the wells where microbial growth is present, the tetrazolium salt are turned into an intensely coloured formazan product shown by a red colour (representing the live and metabolically active bacteria).

Figure 4.10: Quantification of antimicrobial activity by means of INT. The colourless tetrazolium salt is reduced to a red product by biologically active organisms; the inhibition of growth was detected when the solution remained clear after incubation with INT.

In all instances, the MIC values of the H. hemerocallidea extracts were lower than that of the positive controls, which were the commercially acquired antibiotics kenamycin, gentamycin and neomycin shown in Table 4.3.

78 Chapter 4: Results

Table 4.3: MIC values (mg/ml) of the H. hemerocallidea extracts and commercially available antibiotics tested.

MIC scores (mg / ml)

Plant extracts Antibiotics FEC DEC FEL DEL Kenamycin Gentomycin Neomycin

Bacillus subtilis 10.4 10.4 10.4 10.4 33.3 Candida albicans 2.6 2.6 5.21 5.21 Enterococcus faecalis 1.3 2.6 2.6 5.21 Escherichia coli 10.4 10.4 10.4 5.21 4.17 Mycobacterium smegmatis 2.6 5.21 2.6 1.3 26 µg / ml 1.04 1.04 Microorganisms Pseudomonas aeruginosa 10.4 10.4 5.21 5.21

The commercially available antibiotics only showed inhibition of microbial growth against B. subtilis, E. coli and M. smegmatis. The microdilution assay results confirmed the TLC-DB results, indicating that H. hemerocallidea plant extracts possess antimicrobial activity against E. faecalis and C. albicans and low MIC values for these microorganisms were obtained. A lower MIC value is indicative of a more antimicrobial active extract (Eloff, 2004). Higher MIC values (10.4 mg / ml) were obtained for all extracts against B. subtilis and even though low MIC values attained for M. smegmatis, this could also be due to the slow growth of this microorganism in comparison to the others. Any antibiotic that produces an MIC over 4 mg / ml shows that it is not susceptible and should not be chosen. Antibiotics, if selected on the basis of susceptibility to the test organism should yield antimicrobial susceptibilities under 1 mg / ml. Thus a comparison of the quantified microdilution results with the control antibiotics needs to be reconsidered.

4.2.4 The use of the BacTiter-GloTM microbial cell viability assay to determine antimicrobial activity

Using the BacTiter-GloTM assay, an incubation time study was done at 3 h, 5 h and 8 h to determine the optimal time period required from the compounds to exert the antimicrobial effects on the microorganisms. Figure 4.11 shows the microbial cell viability results obtained for the four ethanolic plant extracts used: FEC, DEC, FEL and DEL. The results showed that the corm extracts (FEC and DEC) exerted an increasing antimicrobial activity from 3 h to 8h.

79 Chapter 4: Results

A. B.

C. D.

A Figure 4.11: Cell viability of microorganisms (B. subtilis, C. albicans, E. faecalis, E. coli, M. smegmatis, and P. aeruginosa) following the treatment with H. hemerocallidea extracts (A) FEC (B) DEC (C) FEL and (D) DEL. Cell viability is represented as actual luminescent values. A time study of 3h, 5h and 8h was done to observe the time of maximal antimicrobial toxicity of the plant extract on the microbial cells. Data represents five independent replicate experiments of duplicate samples, and error bars indicate the SEM (n = 10).

80 Chapter 4: Results

FEC showed a large extent of inhibition against M. smegmatis, as inhibition of this microorganism was obtained at 3 h, followed by E. faecalis. At 8 h, FEC showed complete inhibition of M. smegmatis, E. faecalis, E. coli and P. aeruginosa (Figure 4.11A). The same results were obtained for DEC extracts. At 8 h, DEC showed great antibacterial activities against E. coli, P. aeruginosa, M. smegmatis, E. faecalis and C. albicans, in that order (Figure 4.11B).

4.2.5 The use of GC-MS for partial identification of the antimicrobial compounds

According to Gurib-Fakim, (2006), spectroscopy coupled with extraction techniques like chromatography can be used in the isolation and characterisation of bioactive molecules. The antimicrobial compounds, obtained using TLC-DB, were isolated by scrapping off from silica plates and resuspended in ethanol. The compounds were dissolved into ethanol and after the silica sedimeted to the bottom of the vial; the supernatants containing the antimicrobial compounds were used to loading samples on the GC-MS (Figure 4.12). The TLC-DB results showed that both FEC and DEC had similar positive antimicrobial results showing the presence of the same antimicrobial compounds of identical Rf value (0.17) and colour (purple).

Figure 4.12: TLC chromatogram for H. hemerocallidea plant extracts FEC, DEC, FEL, and DEL and TLC plate of TLC-DB results for P. aeruginosa. The large purple zone (left) and the zone that showed inhibition of P. aeruginosa growth was also re-extracted and further analysed using GC-MS and labelled S1 and S2 respectively.

81 Chapter 4: Results

Sample 1 (S1) (Figure 4.12) isolated from the one of the compounds separated from the FEC extract showed two peaks (A and B) as shown in by the chromatographic profile in Figure 4.13. Peaks A and B were both identified using mass spectrometry. Compound A was identified as 1,4-Anhydro-d-mannitol (1,4-Anhydrohexitol, Figure 4.14) and compound B was identified as 1,6-Anhydro-β-D-glucopyranose (levoglucosan, Figure 4.15).

Figure 4.13: GC Chromatogram profile of S1 isolated from the FEC extract that was separated using TLC. Two compounds (A and B) were further separated and identified from S1, using GC-MS. Mass spectra of peaks A and B are shown in figures 4.14 and 4.15, respectively. Gas chromatography was carried out with a carrier gas (He 99.99%), which was maintained at a constant flow rate of 1 ml / min. Column temperature was programmed at 80°C for 4 min, and then increased to 290°C at the rate of 20°C / min for 2.5 min.

Figure 4.14: Mass spectrum of peak A (from figure 4.13). For all GC-MS experiments, the MS was operated at 70 eV (electron impact ionization) with a detector voltage run relative to tuning file. The acquisition was carried out on scanning mode between 60 and 500 m / z values. Ion source temperature was set at 200°C and the interface at 250°C. MS acquisition was made from 4 min to 15 min. The antimicrobial compound for peak A was identified to be 1,4-Anhydro-d-mannitol (1,4- Anhydrohexitol) (structure shown in the figure).

82 Chapter 4: Results

Figure 4.15: Mass spectrum of peak B (from figure 4.13). The antimicrobial compound for peak B was identified to be 1,6-Anhydro-β-D-glucopyranose (levoglucosan) (structure shown in the figure). Levoglucosan was found to be the major common compound present in S1.

Figure 4.12 shows a chromatographic profile obtained for the S2 (sample 2) compound isolated from the separated from FEC after the TLC-DB analysis with E. faecalis and other microorganisms. S2 separated into three peaks predominant peaks (A, B and C in Figure 4.16) and identified via mass spectrometry (Figures 4.15, 4.17 and 4.18).

Peak A was identified as 1,2-Benzenediol (Pyrocatechol / o-Benzenediol / o- Dihydroxybenzene) (Figure 4.17), peak B which was identified as levoglucosan, was common in both S1 and S2 (Figure 4.15). Peak C was identified as hexahydro-3-(2- methylpropyl)-pyrrolo[1,2-α]pyrazine-1,4-dione (Figure 4.18). Due to the fact that the compound, levoglucosan, was common in both S1 and S2, it was speculated that it was the main compound that possessed the antimicrobial activities in the corm extracts of the H. hemerocallidea plant; thus the compound was further analysed for antimicrobial activities against B. subtilis, C. albicans, and E. faecalis using TLC-DB (Figure 4.12). Only B. subtilis, C. albicans, and E. faecalis were chosen because they showed major susceptibility against the H. hemerocallidea ethanol corm extracts in the TLC-DB studies (Figures 4.8).

83 Chapter 4: Results

Figure 4.16: Chromatogram of the antimicrobial compound S2 (refer to Figure 4.12), which was obtained following the TLC-DB analysis of E. faecalis. According to the TLC-DB results, S2 was found in the FEC extract and showed major antimicrobial activities against B. subtilis, E. faecalis, and C. albicans.

Figure 4.17: Mass spectrum of peak A (from figure 4.16). The antimicrobial compound for peak A was identified to be 1,2-Benzenediol (Pyrocatechol / o-Benzenediol / o-Dihydroxybenzene) (structure shown in the figure).

84 Chapter 4: Results

Figure 4.18: Mass spectrum of peak C (from figure 4.16). The antimicrobial compound for peak C was identified to be hexahydro-3-(2-methylpropyl)-pyrrolo[1,2-α]pyrazine-1,4-dione (structure shown in the figure).

The commercially acquired levoglucosan compound was analysed alone for antimicrobial activities against B. subtilis, C. albicans, and E. faecalis using TLC-DB to confirm its antimicrobial activities in the FEC and DEC extracts. As shown on the TLC chromatogram (Figure 4.19), the levoglucosan (1,6-anhydro-β-D-glucose) standard did not separate on the TLC plate with these developing conditions, hence, no antimicrobial activity was detected on the bioautograms by levoglucosan. The H. hemerocallidea FEC and DEC (40 µl of sample applied) still showed the presence of antimicrobial activities effective against the test microorganisms, B. subtilis, C. albicans, and E. faecalis, as was previously seen on the bioautograms (Figure 4.8 and Figure 4.19).

85 Chapter 4: Results

Figure 4.19: TLC-DB results for the levoglucosan compound that was isolated via GC-MS. The TLC chromatogram shows the application of 100 µl of levoglucosan and 40 l of the H. hemerocallidea extracts (FEC and DEC). Levoglucosan did not separate on the TLC plate under these conditions, thus the TLC bioautography results of B. subtilis, C. albicans, and E. faecalis showed no antimicrobial activities for the levoglucosan compound.

86 Chapter 5: Discussion

CHAPTER 5 – DISCUSSION

5.1 Cancer studies 88

87 Chapter 5: Discussion

5.1 Cancer studies

H. hemerocallidea has been used in South Africa for many years to treat a variety of illnesses including diabetes, HIV and cancer. Research shows that H. hemerocallidea is a potential medicinal plant for the development of cancer therapeutics (Albrecht et al., 1995b; 1995c). A compound known as rooperol has been isolated from rhizome extracts of the H. hemerocallidea plant and it is believed to have anticancer properties. Rooperol showed promise as an oral prodrug for cancer therapy (Albrecht et al., 1995c) and is now used to treat cancers such as prostate hypertrophy, testicular tumours and urinary cancers (Nicoletti et al., 1992; Abegaz et al., 1999; Nair and Kanfer, 2006; Drewes et al., 2008; Katerere and Eloff, 2008). However, it is still unknown as to how the anticancer compounds in H. hemerocallidea plant extracts exert their growth inhibitory effects towards cancer cell lines, as the mode of action of these compounds is, to our knowledge, not mentioned in literature. However, Albrecht et al. (1995c) observed that rooperol inhibited the in vitro growth of the human melanoma cell line (UCT-Mel 1) via apoptosis.

H. hemerocallidea also contains phytosterols, responsible for its numerous medicinal properties, making it a suitable candidate for the treatment of breast, colon, prostate and testicular cancers (Rhodes et al., 1993; Pegel, 1997; Von Holtz et al., 1998; Awad and Fink, 2000; Berges et al., 2000; Dreikorn, 2000; Lowe and Fagelman, 2000; Steenkamp, 2003; Boukes et al., 2008; Katerere and Eloff, 2008; Nair and Kanfer, 2008a; Nair and Kanfer, 2008b; Wilt et al., 2011; Street and Prinsloo, 2012), hypercholesterolemia, cardiovascular diseases, pulmonary tuberculosis (Pegel, 1997; Moghadasian, 2000; Nair et al., 2006; Boukes et al., 2008) and infectious diseases such as HIV (Albrecht 1995a; Southern African Development Community, 2002; Giraldo, 2003; Mills et al., 2005; Nair et al., 2007b). In the study presented here, plant extracts from H. hemerocallidea corms and leaves were evaluated for possible anticancer properties in the SNO oesophageal cancer cell line using cell viability assays, flow cytometry and microscopy.

According to the American National Cancer Institute (ANCI) guidelines, the set limit of activity for crude extracts at a 50% inhibition (IC50) of proliferation is less than 30 µg / ml after an exposure time of 72 h (Suffness and Pezzuto, 1990). The IC50 value is defined as the concentration of a test compound required to achieve half maximal (50%) inhibition of the cancer cells; it is a parameter that is indicative of the antiproliferative potency of a test compound (Griffiths and Sundaram, 2011).

88 Chapter 5: Discussion

Due to the inconsistencies of data obtained from the trypan blue assay, AlamarBlue® assay, CytoTox-GloTM assay, flow cytometry and light microscopy, it was concluded that the H. hemerocallidea extracts used in this study did not qualify as having potent antiproliferative properties against the SNO cancer cells. Although a statistically significant decrease (P < 0.05) in SNO cells was obtained with the treatment of 100 µg / ml DEC (20% cytotoxicity; Figure 4.1D), 250 – 500 µg / ml FEC (40% cytotoxicity; Figure 4.2B), 100 – 500 µg / ml DEC (20 – 30% cytotoxicity; Figure 4.2D), 100 µg / ml FEL (40% cytotoxicity; Figure 4.3B) and 200 µg / ml DEL (35% cytotoxicity; Figure 4.3D) extracts, the results between the various assays did not correlate. Indeed, with the exception of the 10 µg / ml DWL extract causing more than 40% cell death (Figure 4.3C) shown by AlamarBlue® assay, no other extract (less than 30 µg / ml as per ANCI guidelines) resulted in IC50 values of less than 50%, irrespective of the type of assay used to analyse cell death.

With such low cancer cytotoxicity activities, the extracts prepared in this study do not hold any promise for use as a candidate for anticancer drug development against the SNO oesophageal cancer cell line, as cytotoxicity is only considered highly potent if less than 50% cancer cell survival after exposure time is achieved (Steenkamp and Gouws, 2006).

Even though both the trypan blue assay and the AlamarBlue® assay measure cell viabilty, the differences in the results could have been due to the differences in the reaction mechanisms between the two assays. As mentioned in Chapter 2, the trypan blue assay determines cell viability by measuring membrane integrity and the AlamarBlue® determines cell viability by measuring the activities of a functioning mitochondria in a redox reaction. Viable proliferating cells have active mitochondria and are able to convert resazurin into a quantifiable highly fluorescent compound. This then suggested that the H. hemerocallidea extracts were unable to cause either cell membrane damage or mitochondrial damage as the SNO cells mostly remained viable and proliferating even at high doses of plant extracts.

The CytoTox-GloTM assay, flow cytometry and light microscopy were perfomed to further investigate the anticancer activies of the H. hemerocallidea extracts againts the SNO cancer cells. These studies were conducted using the same extract concentrations obtained from the viability assays that showed ‘promising’ results and potential for SNO cell growth inhibition. However, data obtained from the CytoTox-GloTM assay confirmed that the H. hemerocallidea extracts were, in fact, non-cytotoxic towards the SNO cancer cell line (Figure 4.4).

89 Chapter 5: Discussion

Having tested 3 different types of viability assays, it was decided to evaluate cell morphology in an effort to consolidate or affirm the varying results. The flow cytometry results showed no major significant changes in cell morphology and / or complexity in the treated SNO cells. Compared to the untreated control SNO cells, the majority of treated SNO cells remained viable (Figure 4.5), confirming the results of the preceding studies (trypan blue, AlamarBlue® and CytoTox-GloTM assays) and that indeed, the H. hemerocallidea extracts did not have any anticancer effects towards the SNO cells. The microscopy results (Figure 4.6) supported the flow cytometric data, and showed that the extracts did not significantly alter the cell morphology.

Taken together, it is therefore concluded that the extracts prepared from both the leaves and corms of H. hemerocallidea were unable to exert any significant anticancer activity against the oesophageal SNO cancer cells (CCL-185).

In spite of previous reports promulgating the plant’s anticancer properties, there are other reports indicating the inability of extracts of this plant to effectively induce cell death in cancer cells. For example, Reid et al. (2006) showed that both the dichloromethane and 90% methanol H. hemerocallidea (corm and leaf) extracts had negative antimutagenic properties. Verschaeve et al. (2013) confirmed this using the alkaline comet and cytome assays, and reported that the dried water corm extracts of the H. hemerocallidea plant were neither genotoxic nor cytotoxic to the human hepatocellular liver carcinoma cell line 2 (HepG2), meaning that even though the extracts were not cancer-causing, they also did not have any anticancer activities against the HepG2 cell line. Furthermore, Steenkamp and Gouws (2006) showed that the dried water extracts of H. hemerocallidea stimulated cancer cell proliferation in human DU-145 prostate cancer cells and in MCF-12A non-malignant breast cells following the exposure time of 72 h. The same extracts used by Steenkamp and Gouws (2006) also had no cytotoxicity against the MDA-MB-231 breast cancer cells and no major cytotoxicity in MCF-7 breast cancer cells after 72 h. Indeed, in this study it was also observed that certain H. hemerocallidea extracts stimulated cell growth of the SNO cells as opposed to causing inhibition of cell growth (via trypan blue and AlamarBlue® analyses of FWC (Figure 4.1A and Figure 4.2A), low dosages of FEC (Figure 4.1B and Figure 4.2B), and high dosages of FEL (Figure 4.3B).

The cell line used in this study, the CCL-185 SNO oesophageal cancer cell line, was used by Skerman et al. (2011) to investigate the apoptosis-inducing effects of three different Sutherlandia spp. extracts. These authors showed, through AlamarBlue® studies, that the Sutherlandia spp. extracts, at concentrations 2.5 and 5 mg / ml, induced a decrease in SNO

90 Chapter 5: Discussion cell viability with above 50% cytotoxicity relative to the untreated control cells. This could indicate that the H. hemerocallidea extracts used in this study may not have been used at high enough concentrations in this specific cell line. According to Steenkamp and Gouws (2006), different cell lines have different sensitivities torwards plant extracts. Also, plants have different cytotoxic effects on cancer cells (Kusuge et al., 1985; Alley et al., 1988), while other plant extracts activate certain molecules of the immune system in order to destroy cancer cells (Abuharfeil et al., 2000). It is therefore possible that the SNO cells were particularly resistant to the plant extracts.

In fact, the natural variability in plants may also have played a role (Steenkamp and Gouws, 2006); during seasonal changes, plants experience physiological changes due to changes in temperature, availability of nutrients, light and water (Iason et al., 2012). This physiological change influences the distribution of secondary metabolites within the plant. For example, during summer, metabolites may be located within the aerial parts of the plant such as leaves, where they are needed for photosynthesis and for plant defence against herbivores, insects and pathogens; and in winter, metabolites may be found in the underground parts of the plant where they are needed to reserve the nutrient stores for the survival and sustenance of the plant. In a nutshell, abiotic stress factors have a major influence in the accumulation of plant secondary metabolites, which are important in assisting with the adaptation of plants to the environment and overcoming stress conditions (Ramakrishna and Ravishankar, 2011). The harvesting of the plants used in this study was subject to availability, and the season was not taken into consideration when performing the plant extraction.

Another possible reason for the absence of apparent anticancer activity could be due to the variation in the different extraction methods used. According to Tiwari et al. (2011), the quality of an extract is influenced by the plant part used as starting material, the solvent used for extraction and by the extraction procedure used; and the effect of extracted plant phytochemicals depend on the nature of the plant material, its origin, degree of processing, moisture content and particle size. Extraction methods affect the quantity and secondary metabolite composition of an extract, and this is mainly due to the type of extraction used, time of extraction, temperature, nature of extractant solvent, solvent concentration and polarity. A good solvent to use should have low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, and should have an inability to cause the extract to complex or dissociate. In this study, water and ethanol were used as extractants during plant tissue homogenisation for extract preparation. Since no anticancer activity was obtained using water- and ethanol-prepared extracts, other potential candidate

91 Chapter 5: Discussion extractants to use could include methanol, chloroform, ether and acetone, using other extraction procedures such as sonication and percolation. For example, Boukes and van der Venter (2011) used chloroform to extract the non-polar compounds, such as phytosterols and sterolins, from H. hemerocallidea to test for cytotoxicity against cervical (HeLa)-, colorectal (HT-29)- and breast (MCF-7) cancer cell lines. Boukes and van der Venter (2011) showed that H. hemerocallidea corm extracts had cytotoxic effects against the HeLa and HT-29 cancer cells at 250 and 500 µg / ml concentrations. The cytotoxic mechanism of Hypoxis was exerted through the induction of cell cycle arrest and apoptosis.

The lack of enzymes in the SNO cells in this case, which are responsible for the conversion of the inactive forms of the anticancer compounds to their active cytotoxic form (e.g. rooperol) may have played a role. It is known that ethanol and methanol are more effective in extracting hypoxoside (Louw et al., 2002). Hypoxoside is a norlignan diglucoside isolated from the corms of Hypoxis plants (Marini-Bettolo et al., 1982a; Drewes et al., 1984). Albrecht et al. (1995c) reported that hypoxoside was non-toxic for mouse (BL6) melanoma cells in tissue culture, but the activated form of hypoxoside, rooperol, caused 50% inhibition in BL6 melanoma cells. In human melanoma cell homogenates (UCT-Mel 1) and in vitro (in the presence of fetal calf serum / FCS), the inactive diglucoside (hypoxoside) is deconjugated by the heat-labile beta-glucosidase to form the cytotoxic and lipophilic aglucone, rooperol (Theron et al., 1994; Albrecht et al., 1995c). In this study, SNO cell cultures where prepared using DMEM medium supplimented with 10% FCS (heat inactivated at 56°C for 20 min). This method required FCS to be heat-inactivated in order to remove proteins which might have interfered with the assays. However, according to Albrecht et al. (1995c), heat inactivation destroys the endogenous β-glucosidase activity, thus it is speculated that the hypoxoside, in H. hemerocallidea extracts used in this study, was never converted into the cytotoxic rooperol, which may have led to the non-toxicity of the plant extracts towards the SNO cells. Another possibility is that the SNO cells may naturally lack the endogenous β- glucosidase enzyme. Thus, for future studies, non-heat-inactivated FCS could be used, the presence of endogenous β-glucosidase activity should be confirmed and / or the addition of an exogenous β-glucosidase enzyme could be considered.

The choice of using flow cytometry to determine the cytotoxicity of a plant extract on human cancer cells was gained from literature. For example, a study by Tavakkol-Afshari et al. (2008) showed that the Saffron (dried stigmas of Crocus sativus L.) extracts had apoptotic effects on the cervical cancer (HeLa) cells and epithelial-like human hepatocellular carcinoma (HepG2) cells as determined by flow cytometry. However, together with the microscopy data, it is concluded that the extracts prepared from H. hemerocallidea did not

92 Chapter 5: Discussion inhibit the growth of the CCL-185 SNO oesophageal cancer cells (in vitro) and therefore did not possess any significant anticancer activities against this cell line.

5.2 Microbial studies

Many South African plants are now being investigated for the presence of antimicrobial activities. These include plants such as the Combretum woodii, which showed antibacterial activities against Staphylococcus aureus, P. aeruginosa, E. faecalis and E. coli (Eloff et al., 2005a, 2005b), and Coleonema album, which have been reported to display antimicrobial activities against E. coli, B. subtilis, E. faecalis, P. aeruginosa, S. aureus, M. smegmatis, M. tuberculosis, and C. albicans using TLC-DB (Esterhuizen et al., 2006). Even though H. hemerocallidea plants have been used for therapeutic and medicinal purposes for many years, there are few reports on its antimicrobial activities mentioned in literature.

5.2.1 Phytochemical analysis of plant extracts by TLC

H. hemerocallidea corm extracts have more non-polar compounds than the leaf extracts (Figure 4.7 and in Table 4.1), which were separated better with a larger application volume i.e. higher concentration of plant extracts; whereas the leaves (FEL and DEL) had a better resolution of polar compounds at a lower concentration. The non-polar compounds in the corm extracts migrated with the non-polar mobile phase, while more polar compounds in the leaf extracts were retarded in the stationary phase (consisting of a polar silica coating). Twelve (12) corm extract compounds and 10 leaf extract compounds were separated using

TLC. A compound with an Rf value of 0.57 (in the corm extracts FEC and DEC) correlates to the sterols (stigmasterol and β-sitosterol), which have a reported Rf value of 0.53 (Boukes et al., 2008). No difference was observed in the concentrations of compounds present in extracts from fresh and dry tissue. For compounds that were present in both the corms and leaves, the concentration thereof was higher in the corms.

5.2.2 Determination of antimicrobial activities using direct TLC-DB

By means of TLC-DB, it was determined that two compounds which were able to inhibit microbial growth were present in the corm extracts. These compounds had Rf values between 0.12 and 0.14 (Figures 4.8) and were polar in character since they were close to the origin of application. The Rf value of 0.12 for an antimicrobial compound in FEC and DEC extracts corresponds to that obtained by Muwanga (2006), who obtained an antimycobacterial active compound with an Rf of 0.17 on TLC with a mobile phase composed of

93 Chapter 5: Discussion methanol: ethyl acetate (1:4). The identity of this compound was also unknown. The leaf extracts (FEL and DEL) used showed minimal antimicrobial activity against E. faecalis and P. aeruginosa.

The growth of Gram-positive bacteria was inhibited to a greater extent than Gram-negative bacteria (Table 4.2). This is likely due to the difference in cell wall composition; Gram- negative bacteria have more complex and less permeable cell walls. Gram-negative bacteria are more resistant to antibiotics and cause more serious infections in humans, which are difficult to treat by conventional antibiotics. Therefore, the ability of natural remedies to significantly inhibit Gram-negative bacteria is of great importance (Salie et al., 1996; Atlas 1997). From these results, H. hemerocallidea could be a potential source for new drug development or even topical creams that can be used for the treatment of infectious diseases such as Candida and some B. subtilis and E. faecalis -manifested diseases.

These results, although obtained through in vitro studies, indicate that the leaves of Hypoxis hemerocallidea do not contain a compound, or compounds capable of overcoming the highly resistant cell wall. Another possible reason for the leaf extracts showing no antimicrobial activities is that the leaves may lack the presence of any antimicrobial compounds or these antimicrobial compounds could be present in levels too low to overcome microbial growth, perhaps these antimicrobials are induced by host-pathogen interactions. Reports by Ncube et al. (2011) indicated that seasonal variation may play a role in the production of active compounds. H. hemerocallidea did not show good activity in spring but the activity shifted from the leaf to the corm in summer. There is a shift in the activity during different seasons between plant organs which suggests a possible corresponding alteration in and / or accumulation of some compounds which may be responsible for the activity (Koptur, 1985). Since most of these phytochemicals are produced in response to external stimuli (Derita et al., 2009) such as light intensity, moisture stress and temperature amongst others, it may be possible that depending on the season of the year, the content and presence of these bioactive compounds could vary in parallel with the presence or absence of the stimuli, which results in changing antibacterial properties. In addition to the intrinsic morphological, physiological and biochemical differences between bulbs and leaves, the dynamics in the production of the active compounds in response to stimulation factors could explain the observed differences in the activity between leaves and bulbs of the same plant.

The absence of antimicrobial activities in the leaf extracts may also be linked to factors such as the extractant used during the extraction process, which might have led to the antimicrobial compounds not being isolated from the leaf samples. Studies by Eloff (1998b)

94 Chapter 5: Discussion demonstrated that the activities of antimicrobial compounds in leaf extracts from Anthocleista grandiflora and Combretum erythrophyllum with different extractants, yielded varied degrees of inhibition. Extracts isolated in acetone provided extensive inhibition against S. aureus and P. aeruginosa as shown by TLC-DB. The presence of plant sterols in chloroform extracts and not in water extracts of H. hemerocallidea was reported by Du Plessis-Stoman et al. (2009).

Other parameters that should be taken into consideration before studying antimicrobial activities of plants are the plant material used, techniques employed, growth medium and microorganisms tested. Solvent and extraction systems may influence results of antimicrobial studies, and pH, together with the composition of growth medium can also influence the activity of the tested extracts or compounds, thereby influencing results (Rios et al., 1988; Rios and Recio, 2005). It was noted that M. smegmatis did not grow well on the TLC plates, and it took longer for this microorganism to grow in comparison to the other microorganisms used, which has an effect on the end result. It is likely that the M. smegmatis did not survive the culturing conditions that the TLC plates were subjected to. Hence, TLC-DB results showed no inhibition against M. smegmatis. Furthermore, according to He and De Buck (2010), M. smegmatis is highly resistant because of the cell wall that is responsible for providing these mycobacteria with protection against differences in osmotic pressure of the media and against other physical and chemical disturbances. The cell walls of M. smegmatis consist of thick, waxy and highly impermeable outer surfaces, which enable the mycobacteria to survive extreme environmental conditions and the presence of antibiotics. The cell walls consist of two lipid layers, one making up the inner membrane and the other outer layer consists of mycolic acids, and thus known as the mycomembrane. This mycomembrane is tightly connected to the peptidoglycan and arabinomannan inner layers of the cell wall, and its outer surface consists of sugars lipids and very complex proteins, which are essential for the mycobacterial protection. Although very little literature has been reported regarding the antimicrobial activities of H. hemerocallidea using TLC-DB, the findings of the study does not provide significant results and thus does not warrant further study.

5.2.3 Control samples used the identification of the active antimicrobial compounds in the H. hemerocallidea corm extracts

The active compounds in H. hemerocallidea plant extracts were partially identified by comparing separated compounds in the extracts, with the compounds found in commercially available H. hemerocallidea products (Moducare® capsules and the African Potato

95 Chapter 5: Discussion capsules). Each Moducare® capsule contained 20 mg β-sitosterol and 0.2 mg β-sitosterol glucoside sterols (Du Plessis-Stoman et al., 2009). According to the results obtained (Figure

4.9), a compound with Rf value of 0.73 that stained a light purple colour with vanillin, was detected from the Moducare® capsules. This compound correlate to β-sitosterol glucoside sterolin, which has a reported Rf value of 0.75 (Du Plessis-Stoman et al., 2009) and did not display any antimicrobial activity when compared to the FEC and DEC compound against the tested mircoorganisms B. subtilis, C. albicans and E. faecalis. Thus, the active antimicrobial compounds in FEC and DEC extracts were not sterols or sterolins found in the H. hemerocallidea plant.

5.2.4 Quantification and determination of antimicrobial activity by means of the microdilution and the BacTiter-GloTM microbial cell viability assay

Quantification of the antimicrobacterial activity of H. hemerocallidea extracts was carried out using the serial plate microdilution assay (Eloff, 1998). Figure 4.10 is a representative plate for the assay. The highest concentrations of plant extract are present in the 2 nd column and 200 l of untreated bacteria (column 1) and 200 l of culture medium (column 11) were added as a positive and negative control respectively.

The results showed that all of the H. hemerocallidea plant extracts (from corms and leaves) were able to inhibit the growth of the microorganisms in the low mg / ml MIC range (Table 4.3). It is possible that the separated compounds could be present in concentrations too low to cause growth inhibition on the TLC plates or that there is a need for the compounds to work synergistically together to pose the antimicrobial effects against the microorganisms tested, as opposed to posing the antimicrobial effects on their own. The MIC values obtained in this study did not correlate with MIC values obtained by Katerere and Eloff (2008) and Aremu et al. (2010) reported dried ethanol corm extracts of H. hemerocallidea inhibiting B. subtilis and E. coli at 3.13 mg / ml and 6.25 mg / ml respectively; and the dried leaf extracts inhibiting both B. subtilis and E. coli with an MIC value of 3.13 mg / ml. Even though contradictory results were obtained in this study, according to Fabry et al. (1998), extracts with MIC values below 8.0 mg / ml are considered to possess antimicrobial activity and only those with MIC values below 1 mg / ml are considered important (Gibbons, 2004; Rios and Recio, 2005). According to Rios and Recio (2005), MIC values higher than 1 mg / ml for extracts or 0.1 mg / ml for isolated compounds should not be used and only MIC values for extracts below 100 µg / ml and 10 µg / ml for isolated compounds should be considered to show the presence of antimicrobial activity. It should also be taken into consideration that parameters for these values depend on the growth medium used for the organism and the

96 Chapter 5: Discussion inoculum size. Adequate dilutions of overnight cultures also have to be prepared to allow sufficient growth over 24 h but still provide acceptable MIC values since an excessive amount of bacteria would provide higher MIC values.

The BacTiter-GloTM assay showed that the corm extracts greatly inhibited Gram-negative bacteria and mycobacteria. As mentioned before, it might be necessary for the compounds present in H. hemerocallidea plant extracts to be associated with each other to produce a more potent antimicrobial inhibition than when they are separated into individual compounds. Both FEL and DEL leaf extracts initially activated cell proliferation of all microorganisms at 3 h with the exception of M. smegmatis. The leaf extracts demonstrated increasing antimicrobial activities from 5 - 8 h, with M. smegmatis being the most inhibited microorganism by both FEL and DEL (Figure 4.11A and 4.11B).

The concentrations of crude H. hemerocallidea extracts (from both the corms and leaves) were used in the BacTiter-GloTM experiments. The lowest MIC values obtained were chosen where microorganism growth was inhibited (Table 4.3). The BacTiter-GloTM confirmed the results that H. hemerocallidea crude extracts possess antimicrobial activities against M. smegmatis, P. aeruginosa, E. coli and E. faecalis.

5.2.5 The use of GC-MS for partial identification of the antimicrobial compounds

The active antimicrobial compound in the FEC and DEC H. hemerocallidea corm extracts was characterised and identified as levoglucosan using GC-MS (Figure 4.15). Levoglucosan (1,6-anhydro-b-D-glucopyranose) is a carbohydrate degradation product (Okutucu et al., 2011) and anhydrosaccharides produced by the pyrolysis of cellulose and hemi-cellulose during combustion (Shafizadeh and Yu, 1973; Shafizadeh et al., 1979, Shafizadeh 1984; Simoniet et al., 1999; Mochida and Kawamura, 2004). Levoglucosan has the molecular formula C5H10O5, molecular weight of 162.1 g / mole (Rosenorn et al., 2006) and it is used for the synthesis of stereo-regular polysaccharides that have biological activities, which include anti-HIV and blood anti-coagulant activities (Hattori et al., 1998).

To confirm the findings of the GC-MS results that levoglucosan was the antimicrobial compound in the FEC and DEC extracts, a levoglucosan standard was analysed independently with TLC-DB for antimicrobial activities against B. subtilis, C. albicans, and E. faecalis. The levoglucosan (1,6-anhydro-β-D-glucose) standard did not separate on the TLC plate under these developing conditions (Figure 4.18). This may be due to the solubility

97 Chapter 5: Discussion problem that was experienced when preparing the levoglucosan test samples. Levoglucosan was also not able to completely dissolve in ethanol, and this was unexpected since the compound is a water-soluble atmospheric organic aerosol compound with highly hydrophilic solubility properties (Graham et al., 2002; Nolte et al., 2001; Mochida and Kawamura, 2004), since ethanol is a moderately polar solvent, it would be expected that levoglucosan completely dissolves in the solvent. Given that levoglucosan is an aerosol, it can be presumed that is may also be volatile, thus it may have evaporated before being separated via TLC. It may also be a possibility that the levoglucosan detected was a product of the heating process during GC-MS which requires high temperatures. To ensure that sufficient amounts of levoglucosan were present, 100 µl of the solution was applied on the TLC plates. Due to the fact that levoglucosan did not separate well on the TLC plates, its individual antimicrobial activity was not detected on the bioautograms. The H. hemerocallidea FEC and DEC 40 µl samples (used as a positive control) still showed the presence of antimicrobial activities against the test microorganisms, B. subtilis, C. albicans, and E. faecalis (Figure 4.19).

Water was not used to dissolve levoglucosan since all water H. hemerocallidea extracts (FWC, DWC, FWL and DWL) did not separate well on the TLC plates either, thus they were excluded from the microbial studies. According to Louchouarn et al. (2009) the choice of solvent used for extracting levoglucosan may affect its concentration and may either be underestimated or overestimated, thus polar solvents such as methanol are to be used for extraction (Louchouarn et al., 2009). In addition, the polar silica coating on the TLC plate may have had an influence on the separation of levoglucosan. It has been reported that by adding silica to glucose, levoglucosan is not formed; however, silica added on starch increases the formation of levoglucosan. The effect of silica on the formation of levoglucosan can be due to the hydrogen bonds between silica and carbohydrates (Choi et al., 2011). High temperatures have also been shown to decrease the concentration of levoglucosan (Gunawan et al., 2012), which may have occurred during heating of the TLC plates for vanillin colour development (or during the incubation of the bioautograms for microbial growth purposes), where the levoglucosan concentration was affected and thus it was not detected on the plate.

Besides the solubility issue of levoglucosan during sample preparation, other possible reasons for the isolated antimicrobial levoglucosan compound not showing antimicrobial activities on the B. subtilis, C. albicans, and E. faecalis microorganisms may be due to the fact that it might have a synergistic activity. According to Rios and Recio (2005) some compounds exhibit no antimicrobial activities on their own, and may require the presence of

98 Chapter 5: Discussion other compounds to exert their antimicrobial effects on microorganisms. According to Van Vuuren (2007), even though many publications focus on the isolation and identification of bioactive compounds, it is important to note that plants are complex living organisms. Therefore a single compound may not be responsible for the antimicrobial activity alone, but rather a combination of compounds interacting in an additive or synergistic manner may be responsible for the antimicrobial activity (Van Vuuren, 2008). Thus it can be assumed that the antimicrobial compounds obtained from by TLC-DB and characterised by GC-MS, namely 1,4-Anhydro-d-mannitol (1,4-Anhydrohexitol) (Figure 4.14); 1,2-benzenediol (pyrocatechol / o-benzenediol / o-dihydroxybenzene) (Figure 4.17); 1,6-anhydro-β-D- glucopyranose (levoglucosan) (Figure 4.15) and hexahydro-3-(2-methylpropyl)-pyrrolo[1,2- α]pyrazine-1,4-dione (Figure 4.18), worked synergistically to exert antimicrobial effects against the test microorganisms for this study. Since levoglucosan was the most prominent compound present, it was speculated that it may have contributed greatly to the antimicrobial activities of the H. hemerocallidea corm extracts.

Even though levoglucosan might have synergistic activities with other compounds, it has been identified as one of the compounds that were responsible for the antimicrobial activities in the H. hemerocallidea extracts. Literature has reported levoglucosan being an antimicrobial compound. It could perhaps be used as starting material in the manufacturing of antibiotics (De Wild et al., 2011) or be used to treat inflammatory disorders and infections (Fatima et al., 1996). In a study by Waghmare et al. (2010), it was found that one of the active antimicrobial compounds isolated from the methanol extracts of seed coat of Tamarindus indica was levoglucosan, which was characterised using GC-MS. This however was present with a mixture of other compounds whose presence was necessary for the antimicrobial effect. Levoglucosan and other compounds were found to inhibit growth of S. aureus, Salmonella typhimurium and P. aeruginosa (Waghmare et al., 2010). In addition, levoglucosan has antifungal activities (Prosen et al., 1993); and it was reported that the compound, amongst others, formed part of the water-soluble compounds in bio-oil extracts of the pistachio (Pistacia vera L.) shells, which had fungicidal activities against Aspergillus niger (a saprophytic fungus), Trichoderma viridae (a phytopathogenic fungus), Coriolus versicolor (a white rot fungus), and Trichophyton rubrum (a dermatophytic fungus) (Okutucu et al., 2011). However, to date, the presence of levoglucosan in H. hemerocallidea has not been reported by other authors.

Further studies would need to be conducted to verify the identity of the compound(s) that are responsible for the antimicrobial effects and to investigate each of them individually. From Figure 4.16, peak C is a compound that closely resembles deoxybrevianamide E, an

99 Chapter 5: Discussion antibiotic used in the food industry. The compound identified as 1,4-Anhydro-d-mannitol (1,4- Anhydrohexitol, peak A from Figure 4.13) is also known to interfere with carbohydrate metabolism, and this may have contributed to the inhibition of microbial growth. Not all of the peaks in Figure 4.13 and Figure 4.16 were identified or characterized and these compounds, even though present in low concentrations, may have contributed to the synergetic antimicrobial effect.

100 Chapter 6: Conclusion

CHAPTER 6 - CONCLUSION

101 Chapter 6: Conclusion

CHAPTER 6 - CONCLUSION

H. hemerocallidea is a medicinal plant with a wide spectrum of pharmacological properties such as antimicrobial, antiviral, anti-inflammatory, antidiabetic, antioxidant, anticancer, cardiovascular and anticonvulsant activities. The plant is now actively used in traditional African medicine to treat various ailments like stomach ailments, HIV / AIDS infections, arthritis, cancers, diabetes, urinary tract infections, tuberculosis, prostatitis, benign prostatic hyperplasia, prostate adenoma and psoriasis. Studies have focussed mainly on corm extracts, which have revealed the presence of a bioactive glucoside compound and a cytotoxic aglycone, identified as hypoxoside and rooperol respectively. Furthermore, the corms also contain phytosterols, glycoside sterolins, stanols, secondary metabolites, lectins and cytokinins, and are also claimed to contribute to the therapeutic and medicinal properties of the plant.

No anticancer effects were observed against the CCL-185 SNO oesophageal cancer cells following treatment by either the corm or leaf extracts (fresh or dried) whether prepared in water or ethanol (even at the higher concentrations of 500 g / ml). In addition, no morphological changes were observed in the SNO cells and no features of cell death (either apoptosis or necrosis) were observed. This can be attributed to the fact that the SNO cells may not be susceptible to the extracts and thus the extracts are ineffective at inhibiting the growth of the cancer SNO cells. This SNO cell line may lack an active endogenous β-glucosidase enzyme responsible for the conversion of hypoxoside to the cytotoxic anticancer-active rooperol biocompound which could be investigated by the addition of β-glucosidase or non-heat-inactivated FCS to the assays to allow for the conversion of hypoxoside to rooperol.

The direct TLC-DB assay is a qualitative analytical technique, which combines TLC and bioautography to screen crude plant extracts for compounds with antimicrobial activity. This assay is suitable for the testing of any compound that can be separated by TLC against any organisms that will grow directly on the TLC plate surface. Twelve (12) corm extract compounds and 10 leaf extract compounds were separated using TLC, showing a complex array of compounds present. The compounds shown to have antimicrobial activity had Rf values ranging from 0.12 to 0.14 (white zones were observed on a red background). The leaf extracts display minimal antimicrobial effects against E. faecalis and P. aeruginosa. The compounds responsible for anti- E. faecalis growth had Rf values

102 Chapter 6: Conclusion between 0.14 and 0.17 and those responsible for the inhibition of P. aeruginosa growth had Rf values between 0.76 and 0.79.

The microdilution assay was used to investigate the inhibition of microbial growth quantitatively. H. hemerocallidea plant extracts possess antimicrobial activity against E. faecalis and C. albicans and low MIC values for these microorganisms were obtained. Higher MIC values (10.4 mg / ml) were obtained for all extracts against B. subtilis and even though low MIC values were attained for M. smegmatis, this could be attributed to the slow growth of this microorganism. The BacTiter-GloTM microbial cell viability assay (Promega, Germany) was used to study the cell viability of the microorganism following treatment with plant extracts. This assay showed that corm extracts exerted an increasing antimicrobial activity from 3 h to 8h. FEC showed a large extent of inhibition against M. smegmatis, as inhibition of this microorganism was obtained at 3 h, followed by E. faecalis. At 8 h, FEC showed complete inhibition of M. smegmatis, E. faecalis, E. coli and P. aeruginosa. The same results were obtained for DEC extracts. At 8 h, DEC showed extensive antibacterial activities against E. coli, then P. aeruginosa, M. smegmatis, E. faecalis and finally C. albicans.

GC-MS was used to identify compounds in the plant extracts that displayed antimicrobial activity. The active compounds 1,4-anhydro-d-mannitol, 1,2-benzenediol, hexahydro-3- (2-methylpropyl)-pyrrolo[1,2-α]pyrazine-1,4-dione and levoglucosan were assumed to be the compounds responsible for antimicrobial activities in H. hemerocallidea corm extracts. Levoglucosan is believed to work synergistically with other compounds (pyrocatechol / 1,4-anhydro-d-mannitol and hexahydro-3-(2-methylpropyl)-pyrrolo[1,2- α]pyrazine-1,4-dione) to exert antimicrobial effects on the microorganisms as a commercially available pure sample was unable to be inhibit microbial growth alone.

In conclusion, H. hemerocallidea plant extracts, at these concentrations, are not able to effectively retard the growth of SNO oesophageal cancer cells. This study demonstrated antimicrobial properties of H. hemerocallidea corms using TLC-DB for the first time. Future studies may involve testing for compounds which are able to restrain other microorganisms of other South African epidemic ailments like malaria, cholera, tuberculosis and hepatitis viral infections. Such results may lead to the isolation of antimicrobial compounds, which can be further explored for their use in pharmacology and aid in the development of antibiotics and / or topical creams.

103 Chapter 7: References

CHAPTER 7 - REFERENCES

104 Chapter 7: References

CHAPTER 7 - REFERENCES

AbDSerotec: A Bio-Rad Company. (2012) Redox principle for AlamarBlue®. http://www.abdserotec.com/catalog/alamarblue/redox-principle-for-alamarblue.html. Date accessed: 01 December 2012.

Abegaz BM, Ngadjui BT, Bezabih M, Mdee LK. (1999) Novel natural products from marketed plants of eastern and southern Africa. Pure and Applied Chemistry 71: 919–926.

Abuharfeil NM, Maraqa A, Von Kleist S. (2000) Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan. Journal of Ethnopharmacology 71: 55–63.

Adminuser. (2011) New registry to give major boost to fight against cancer. Medical chronicle: The doctor’s newspaper. http://www.medicalchronicle.co.za/new-registry-to-give-major-boost-to- fight-against-cancer/. Date accessed: 03 October 2013.

Ahmad I, Beg AZ. (2001) Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. Journal of Ethnopharmacology 74: 113–123.

Akerele O. (1988) Medicinal plants and primary health care: An agenda for action. Fitoterapia 59: 355–363.

Albrecht CF. (1995a) A putative prodrug for the treatment of malignancies, HIV-infections, and inflammatory conditions. South African Medical Journal 85: 302–307.

Albrecht CF, Kruger PB, Smith BJ, Freestone M, Gouws L, Miller R, Van Jaarsveld PP. (1995b) The pharmacokinetic behaviour of hypoxoside taken orally by patients with lung cancer in a phase I trial. South African Medical Journal 85: 861–865.

Albrecht CF, Theron EJ, Kruger PB. (1995c) Morphological characterisation of the cell-growth inhibitory activity of rooperol and pharmacokinetic aspects of hypoxoside as an oral prodrug for cancer therapy. South African Medical Journal 85: 853–860.

Albrecht CF (1996) Chemistry, biological and pharmacological properties of African medicinal plants–hypoxoside: a putative, non-toxic prodrug for the possible treatment of certain malignancies, HIV-infections, and inflammatory conditions. Hostettman K, Chinyanganya F, Maillard M, Wolfender JL (eds). Proceedings of the First International IOCD Symposium. University of Zimbabwe Press, Victoria Falls, Harare, Zimbabwe. Page 303–307.

105 Chapter 7: References

Allen RT, Hunter WJ, Agrawal DK. (1997) Morphological and biochemical characterization and analysis of apoptosis. Journal of Palliative Medicine 37: 215–228.

Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Shoemaker RH, Boyd MR. (1988) Feasibility of drug screening with panels of tumor cells using a microculture of tetrazolium assay. Cancer Research 48: 589–601.

Allison DC, Ridolpho P. (1980) Use of a trypan blue assay to measure the deoxyribonucleic acid content and radioactive labelling of viable cells. Journal of Histochemistry and Cytochemistry 28: 700–703.

Al-Nasiry S, Geusens N, Hanssens M, Luyten C, Pijnenborg R. (2007) The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Human Reproduction 22: 1304–1309.

Anoopkumar-Dukie S, Carey J, Conere T, O’Sullivan E, Van Pelt FN, Allshire A. (2005) Resazurin assay of radiation response in cultured cells. The British Journal of Radiology 78: 945–947.

Aremu AO, Ndhlala AR, Fawole OA, Light ME, Finnie JF, Van Staden J. (2010) In vitro pharmacological evaluation and phenolics content of ten South African medicinal plants used as anthelmintics. South African Journal of Botany 76: 558–566.

Arlt A, Müerköster SS, Schäfer H. (2013) Targeting apoptosis pathways in pancreatic cancer. Cancer Letters 332: 346–358.

Arumugan P, Priya NG, Subanthra M, Ramesh A. (2008) Anti-inflammatory activity of four solvent fractions of ethanol extract of Mentha spicata L. investigated on acute and chronic inflammation induced rats. Environmental Toxicology and Pharmacology 26: 92–95.

Ashkenazi A. (2008) Targeting the extrinsic apoptosis pathway in cancer. Cytokine and Growth Factor Reviews 19: 325–331.

Atlas RM. (1997) Principles of Microbiology, 2nd Edition. Wm. C. Brown Publishers, Dubuque, Iowa, USA. Page 109– 110, 112–113.

Awad AB, Fink CS. (2000) Phytosterols as anticancer dietary components: evidence and mechanism of action. The Journal of Nutrition 130: 2127–2130.

Bakke AC. (2001) The principles of flow cytometry. Laboratory Medicine 32: 207–211.

106 Chapter 7: References

Balunas MJ, Kinghorn AD. (2005) Drug discovery from medicinal plants. Life Sciences 78: 431– 441.

Bedford R, LePage D, Hoffmann R, Kennedy S, Gutschenritter T, Bull L, Sujijantarat N, DiCesare JC, Sheaff RJ. (2012) Luciferase inhibition by a novel naphthoquinone. Journal of Photochemistry and Photobiology B: Biology 107: 55–64.

Bennik MHJ. (2004) Pseudomonas aeruginosa. Encyclopedia of Food Microbiology 1867–1871.

Berges RR, Kassen A, Senge T. (2000) Treatment of symptomatic benign prostatic hyperplasia with betasitosterol: an 18-month follow-up. British Journal of Urology International 85: 842–846.

Bergkvist K, Wengström Y. (2006) Symptom experiences during chemotherapy treatment–With focus on nausea and vomiting. European Journal of Oncology Nursing 10: 21–29.

Berman J, Sudbery PE. (2002) Candida albicans: a molecular revolution built on lessons from budding yeast. Nature Reviews Genetics 3: 918–932.

Bertram JS. (2001) The molecular biology of cancer. Molecular Aspects of Medicine 21: 167–223.

Bird-Lieberman EL, Fitzgerald RC. (2009) Early diagnosis of oesophageal cancer. Journal of Cancer 101: 1–6.

Bosman FT, Visser BC, Van Oeveren J. (1996) Apoptosis: pathophysiology of programmed cell death. Pathology, Research and Practice 192: 676–683.

Bouic PJD, Etsebeth S, Liebenberg RW, Albrecht CF, Pege K, Van Jaarsveld PP. (1996) Beta- sitosterol and beta-sitosterol glucoside stimulate human peripheral blood lymphocyte proliferation: implications for their use as an immunmodulatory vitamin combination. International Journal of Immunopharmacology 18: 693–700.

Boukes GJ. (2010) The in vitro biological activities of three Hypoxis species and their active compounds. Doctoral Thesis. Nelson Mandela Metropolitan University.

Boukes GJ, van de Venter M. (2011) Cytotoxicity and mechanism(s) of action of Hypoxis spp. (African potato) against HeLa, HT-29 and MCF-7 cancer cell lines. Journal of Medicinal Plants Research 5: 2766–2774.

107 Chapter 7: References

Boukes GJ, van der Venter M, Oosthuizen V. (2008) Quantitative and qualitative analysis of sterols / sterolins and hypoxoside contents of three Hypoxis (African potato) spp. African Journal of Biotechnology 7: 1624–1629.

Boyer MJ. (2007) Apoptosis: the beginning of the end. Tools for analyzing apoptosis: A kit selection guide. Calbiochem: CB0729–2007 EUR. Merck Chemicals Ltd., Nottingham. Page 2.

Bradford PG, Awad AB. (2007) Phytosterols as anticancer compounds. Molecular Nutrition and Food Research 51: 161–170.

Brown M, Wittwer C. (2000) Flow cytometry: principles and clinical applications in hematology. Clinical Chemistry 46: 1221–1229.

Burrel RC. (1937) Phytochemistry. What it is and how it has developed. Journal of Chemical Education 14: 520 520–527.

Buwa LV, Van Staden J. (2006) Antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases in South Africa. Journal of Ethnopharmacology 103: 139–142.

Calderone RA, Fonzi WA. (2001) Virulence factors of Candida albicans. Trends in Microbiology 9: 327–335.

Campbell MK, Farrell SO. (2006) Biochemistry. 5th edition. Thomson Brooks / Cole, Belmont, USA. Page 403–407.

Cancer Research UK. (2012) Worldwide cancer statistics. http://www.cancerresearchuk.org/cancer-info/cancerstats/world. Date accessed: 03 October 2013.

CANSA. (2012a) Oesopheageal cancer. http://www.cansa.org.za/oesophageal-cancer/. Date accessed: 25 April 2013.

CANSA. (2012b) South African cancer statistics. http://www.cansa.org.za/south-african-cancer- statistics/. Date accessed: 03 October 2013.

Carelle N, Plotto E, Bellanger A, Germanaud J, Thuillier A, Khayat D. (2002) Changing patient perceptions of the side effects of cancer chemotherapy. American Cancer Society 95: 155–163.

Chen CO, Blumberg JB. (2008) Phytochemical composition of nuts. Asia Pacific Journal of Clinical Nutrition 17: 329–332.

108 Chapter 7: References

Chenoweth C, Schaberg D. (1990) The epidemiology of Enterococci. Europian Journal of Clinical Microbiology and Infectious Diseases 9: 80–89.

Chin YW, Balunas MJ, Chai HB, Kinghorn AD. (2006) Drug discovery from natural sources. The American Association of Pharmaceutical Scientists Journal 8: 28.

Chinsembu KC, Hedimbi M. (2010) Ethnomedicinal plants and other natural products with anti- HIV active compounds and their putative modes of actions. International Journal for Biotechnology and Molecular Biology Research 1: 74–91.

Chipuk JE, Bouchier-Hayes L, Green DR. (2006) Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. Cell Death Differentiation 13: 1396–1402.

Cho MH, Niles A, Huang R, Inglese J, Austin CP, Riss T, Xia M. (2008) A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker. Toxicology in vitro 22: 1099–1106.

Choi SS, Kim MC, Kim YK. (2011) Influence of silica on formation of levoglucosan from carbohydrates by pyrolysis. Journal of Analytical and Applied Pyrolysis 90: 56–62.

Choma IM, Grzelak EM. (2011) Bioautography detection in thin-layer chromatography. Journal of Chromatography A 1218: 2684–2691.

ClinicalTrials.gov. (2011) Study of combretastatin and paclitaxel / carboplatin in the treatment of anaplastic thyroid cancer. http://clinicaltrials.gov/ct2/show/NCT00507429. Date accessed: 03 October 2013.

Cohen GM, Sun XM, Fearnhead H, MacFarlane M, Brown DG, Snowden RT, Dinsdale D. (1994) Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes. Journal of Immunological Methods 153: 507–16.

Cooper GM. (2000) The cell: a molecular approach. 2nd Edition. Sinauer Associates Inc. Sunderlands (MA). E-book: Chapter 15.

Cowan MM. (1999) Plant products as antimicrobial agents. Clinical Microbiology Reviews 12: 564–582.

Cragg GM, Newman DJ. (2005a) Biodiversity: a continuing source of novel drug leads. Pure and Applied Chemistry 77: 7–24.

109 Chapter 7: References

Cragg GM, Newman DJ. (2005b) Plants as a source of anti-cancer agents. Journal of Ethnopharmacology 100: 72–79.

Cragg GM, Newman DJ. (2009) Nature: a vital source of leads for anticancer drug development. Phytochemistry Reviews 8: 313–331.

Craig WJ. (1997) Phytochemicals: guardians of our health. Journal of the American Dietic Association 97: S199–S204.

Cram LS. (2002) Flow cytometry, an overview. Methods in Cell Science 24: 1–9.

Croce CM. (2008) Oncogenes and cancer. The New England Journal of Medicine 358: 502–511.

Cummings J, Ward TH, Ranson M, Dive C. (2004) Apoptosis pathway-targeted drugs–from the bench to the clinic. Biochimica et Biophysica Acta 1705: 53–66.

Dahanukar SA, Kulkarni RA, Rege NN. (2000) Pharmacology of medicinal plants and natural products. Indian Journal of Pharmacology 32: S81–S118.

De Hoff PL, Brill LM, Hirsh AM. (2009) Plant lectins: the ties that bind in root symbiosis and plant defense. Molecular Genetics and Genomics 282: 1–15.

De Stefani E, Barrios E, Fierro L. (1993) Black (air-cured) and blond (flue-cured) tobacco and cancer risk III oesophageal cancer. European Journal of Cancer 29A: 763–766.

De Wild PJ, Reith H, Heeres HJ. (2011) Biomass pyrolysis for chemicals. Biofuels 2: 185–208.

Dean EJ, Ranson M, Blackhall F, Holt SV, Dive C. (2007) Novel therapeutic targets in lung cancer: inhibitor of apoptosis proteins from laboratory to clinic. Cancer Treatment Reviews 33: 203–212.

Derita MG, Leiva ML, Zacchino SA. (2009) Influence of plant part, season of collection and content of the main active constituent, on the antifungal properties of Polygonum acuminatum Kunth. Journal of Ethnopharcology 124: 377–383.

Dhanapathi, Prabhakar TG, Prabhakar P. (2008) Antibacterial activity of Bacillus subtilis extract on pathogenic organisms. Tamilnadu Journal of Veterinary and Animal Sciences 4: 150–153.

Dold AP, Cocks ML. (2002) The trade in medicinal plants in the Eastern Cape province, South Africa. South African Journal of Science 98: 589–597.

110 Chapter 7: References

Dreikorn K. (2000) Phytotherapeutic agents in the treatment of benign prostatic hyperplasia. Current Urology Reports 1: 103–109.

Drewes SE, Elliot E, Khan F, Dhlamini JTB, Gcumisa MSS. (2008) Hypoxis hemerocallidea–not merely a cure for benign prostate hyperplasia. Journal of Ethnopharmacology 119: 593–598.

Drewes S, Hall AJ, Learmonth RA, Upfold UJ. (1984) Isolation of hypoxoside from Hypoxis rooperi and synthesis of (E) -1,5-Bis (3’ ,4’ dimethoxyphenyl) pent-4-en-lyne. Phytochemistry 23: 1313–1316.

Drewes SE, Khan F. (2004) The African potato (Hypoxis hemerocallidea): a chemical–historical perspective. South African Journal of Science 100: 425–430.

Drewes SE, Liebenberg RW. (1982) U.K. Patent Application GB 2 120650.

Drewes S, Liebenberg RW. (1987) Rooperol and its derivates. U.S. Patent 4644085.

Du Plessis-Stoman D, Downing TG, van de Venter M, Govender S. (2009) Traditional herbal medicines: potential degradation of sterols and sterolins by microbial contaminants. South African Journal of Science 105: 147–150.

Earl AM, Losick R, Kolter R. (2008) Ecology and genomics of Bacillus subtilis. Trends in Microbiology 16: 269–275.

Elmore S. (2007) Apoptosis: a review of programmed cell death. Toxicology and Pathology 35: 495–516.

Eloff JN. (1998a) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64: 711–713.

Eloff JN. (1998b) Which extractant should be used for the screening and isolation of antimicrobial components from plants. Journal of Ethnopharmacology 60: 1–8.

Eloff JN. (2004) Quantification the bioactivity of plant extracts during screening and bioassay guided fractionation. Phytomedicine 11: 370–371.

Eloff JN, Famakin JO, Katerere DRP. (2005a) Combretum woodii (Combretaceae) leaf extracts have high activity against Gram-negative and Gram-positive bacteria. African Journal of Biotechnology 4: 1161–1166.

111 Chapter 7: References

Eloff JN, Famakin JO, Katerere DRP. (2005b) Isolation of an antibacterial stilbene from Combretum woodii (Combreceae) leaves. African Journal of Biotechnology 4: 1167–1171.

Elton E. (2005) Esophageal cancer. Disease-a-Month 51: 664–684.

Enzinger PC, Mayer RJ. (2003) Esophageal cancer. The New England Journal of Medicine 349: 2241–2252.

Erasto P, Adebola PO, Grierson DS, Afolayan AJ. (2005) An ethnobotanical study of plants used for the treatment of diabetes in the Eastern Cape province. South Africa African Journal of Biotechnology 4: 1458–1460.

Erlwanger KH, Cooper RG. (2008) The effects of orally administered crude alcohol and aqueous extracts of African potato (Hypoxis hemerocallidea) corm on the morphometry of viscera of suckling rats. Food and Chemical Toxicology 46: 136–139.

Esterhuizen LL, Meyer R, Dubery IA. (2006) Antimicrobial compounds Coleonema album (Rutaceae). Verlag der Zeitschrift für Naturforschung C 61: 489–498.

Fabricant DS, Farnsworth NR. (2001) The value of plants used in traditional medicine for drug discovery. Environmental Health Perspectives 109: 69–75.

Fabry W, Okemo PO, Ansorg R. (1998) Antibacterial activity of East African medicinal plants. Journal of Ethnopharmacology 60: 79–84.

Fan-Harvard P, Capano D, Smith SM, Mangia A, Eng RHK. (1991) Development of resistance in Candida isolates from patients receiving prolonged antifungal therapy. Antimicrobial Agents and Chemotherapy 35: 2302–2305.

Farnsworth NR, Akerel O, Bingel AS, Soejarto DD, Guo Z. (1985) Medicinal plants in therapy. Bulletin of the World Health Organization 63: 965–981.

Fatima Favoreto Mendes Rita De, Ina Produtos Farmaceuticos S A, Edilmere Regina Sprada. (1996) The use of levoglucosan for the treatment of infectious or inflammatory conditions. http://www.google.com/patents/WO1996015796A1?cl=en. Date accessed: 10 October 2013.

Fauci AS. (1998) New and reemerging diseases: the importance of biomedical research. Emerging Infectious Diseases 4: 374–378.

112 Chapter 7: References

Fennell CW, Light ME, Sparg SG, Stafford GI, Van Staden J. (2004a) Assessing African medicinal plants for efficacy and safety: agricultural and storage practices. Journal of Ethnopharmacology 95: 113–121.

Fennell CW, Lindsey KL, McGaw LT, Sparg SG, Stafford GI, Elgorashi EE, Grace OM, Van Staden J. (2004b) Assessing African Medicinal plants for efficacy and safety: pharmacological screening and toxicology. Journal of Ethnopharmacology 94: 205–217.

Ferroni A, Nguyen L, Pron B, Quesne G, Brusset MC, Berche P. (1998) Outbreak of nosocomial urinary tract infections due to Pseudomonas aeruginosa in a paediatric surgical unit associated with tap-water contamination. Journal of Hospital Infection 39: 301–307.

Fischer U, Schulze-Osthoff K. (2005) Apoptosis-based therapies and drug targets. Cell Death and Differentiation 12: 942–961.

Fouche G, Cragg GM, Pillay P, Kolesnikova N, Maharaj VJ, Senabe J. (2008) In vitro anticancer screening of South African plants. Journal of Ethnopharmacology 119: 455–461.

Franz CMAP, Stiles ME, Schleifer KH, Holzapfel WH. (2003) Enterococci in foods–a conundrum for food safety. International Journal of Food Microbiology 88: 105–122.

Fridkin SK, Jarvis WR. (1996). Epidemiology of nosocomial fungal infections. Clinical Microbiology Reviews 9: 499–511.

Fu LL, Zhou CC, Yao S, Yu JY, Liu B, Bao JK. (2011) Plant lectins: targeting programmed cell death pathways as antitumor agents. The International Journal of Biochemistry and Cell Biology 43: 1442–1449.

Fulda S. (2013) Targeting apoptosis pathways in childhood malignancies. Cancer Letters 332: 369–373.

Fyhrquist P, Mwasumbi L, Haeggstrom CA, Vuorela H, Hiltumen R, Vuorela P. (2002) Ethnobotanical and antimicrobial investigation of some species of Terminalia and Combretum (Combretaceae) growing in Tanzania. Journal of Ethnopharmacology 79: 169–177.

Gaidamashvili M, Van Staden J. (2002) Interaction of lectin-like proteins of South African medicinal plants with Staphylococcus aureus and Bacillus subtilis. Journal of Ethnopharmacology 80: 131–135.

113 Chapter 7: References

Galeffi C, Multari G, Msonthi JD, Nicoletti M, Marini-Bettolo GB. (1987) Nyasicoside, a new glucoside of Hypoxis nyasica Bak. Tetrahedron 43: 3519–3522.

Garon EB, Kabbinavar FF, Neidhart JA, Neidhart JD, Gabrail NY, Oliveira MR, Lu SP, Balkissoon J, Geffen D. (2010) Randomized phase II trial of a tumor vascular disrupting agent fosbretabulin tromethamine (CA4P) with carboplatin (C), paclitaxel (P), and bevacizumab (B) in stage IIIb/IV nonsquamous non-small cell lung cancer (NSCLC): The FALCON trial. Journal of Clinical Oncology 28: 7587 (abstract).

Gewies A. (2003) Introduction to apoptosis. ApoReview 1–26.

Ghazarian H, Idoni B, Oppenheimer SB. (2011) A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. Acta histochemica 113: 236–247.

Gibbons, S. (2004) Anti-staphylococcal plant natural products. Natural Products Report 21: 263– 277.

Giraldo RA. (2003) Southern African Development Community (SADC) Ministerial Consultative Meeting on Nutrition and AIDS. Brief Report.

Graham B, Mayol-Bracero OL, Guyon P, Roberts GC, Decesari S, Facchini MC, Artaxo P, Maenhaut W, and Köll P, Andreae MO. (2002) Water-soluble organic compounds in biomass burning aerosols over Amazonia: characterization by NMR and GC-MS. Journal of Geophysical Research – Atmospheres 107: 1–40.

Griffin AM, Butow PN, Coates AS, Childs AM, Ellis PM, Dunn SM, Tattersall MHN. (1996) On the receiving end V: patient perceptions of the side effects of cancer chemotherapy in 1993. Annals of Oncology 7: 189–195.

Griffiths M, Sundaram H. (2011) Drug design and testing: profiling of antiproliferative agents for cancer therapy using a cell-based methyl-[3H]-thymidine incorporation assay. Cancer Cell Culture Methods in Molecular Biology 731: 451–465.

Gunawan R, Li X, Larcher A, Hu X, Mourant D, Chaiwat W, Wu H, Li CZ. (2012) Hydrolysis and glycosidation of sugars during the esterification of fast pyrolysis bio-oil. Fuel 95: 146–151.

Gupta LM, Raina R. (1998) Side effects of some medicinal plants. Current Science 75: 897–900.

114 Chapter 7: References

Gurib-Fakim A. (2006) Medicinal plants: traditions of yesterday and drugs of tomorrow. Molecular Aspects of Medicine 27: 1–93.

Hamburger MO, Cordell GA. (1987) A direct bioautographic TLC assay for compounds possessing antibacterial activity. Journal of Natural Products 50: 19–22.

Hanahan D, Coussens LM. (2012) Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21: 309–322.

Hanahan D, Weinberg R. (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674.

Hanahan D, Weinberg RA. (2000) The Hallmarks of Cancer. Cell Press 100: 57–70.

Hannun YA. (1997) Apoptosis and the dilemma of cancer chemotherapy. The Journal of The American Society of Hematology 89: 1845–1853.

Haq I. (2004) Safety of medicinal plants. Pakistan Journal of Medical Research 43: 1–8.

Hartwell JL. (1976) Types of anticancer agents isolated from plants. Cancer Treatment Reports 60: 1031–1067.

Hattori K, Yoshida T, Nakashima H, Premanathan M, Aragaki RR, Mimura T, Kaneko Y, Yamamoto N, Uryu T. (1998) Synthesis of sulfonated amino-polysaccharides having anti-HIV and blood anticoagulant activities. Carbohydrate Research 312: 1–8.

He Z, De Buck J. (2010) Research article cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155. BMC Microbiology 10: 121.

Heldt HW. (2005) Plant Biochemistry. 3rd Edition. Elsevier Academic Press, London. Page 364– 370.

Hoiby N, Frederiksen B, Pressler J. (2005) Eradication of early Pseudomonas aeruginosa infection. Journal of Cystic Fibrosis 4: 49–54.

Horváth G, Jámbor N, Végh A, Böszörményi A, Lemberkovics Ế, Héthelyi Ế, Kovács K, Kocsis B. (2010) Antimicrobial activity of essential oils: the possibilities of TLC–bioautography. Flavour and Fragrance Journal 25: 178–182.

Hsu S, Singh B, Schuster G. (2004) Induction of apoptosis in oral cancer cells: agents and mechanism for potential therapy and prevention. Oral Oncology 40: 461–473.

115 Chapter 7: References

Huang WY, Cai YZ, Zhang Y. (2009) Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutrition and Cancer 62: 1–20.

Hutchings A, Scott AH, Lewis G, Cunningham A. (1996) Zulu medicinal plants—an inventory. University of Natal Press, Pietermaritzburg, South Africa. Page 55–56.

Iason GR, Dicke M, Hartley SE. (2012) The ecology of plant secondary metabolites, ecological reviews: from genes to global processes. Cambridge University Press, USA. Page 35.

Iversen BG, Brantsaester AB, Aavitsland P. (2008) Nationwide study of invasive Pseudomonas aeruginosa infection in Norway: importance of underlying disease. Journal of Infection 57: 139– 146.

Iwu MM, Duncan AR, Okunji CO, Jannick J (ed). (1999) New antimicrobials of plant origin. Perspectives on new crops and new uses. American Society for Horticultural Science Press, Alexandra, Virginia. Page 457–462.

Jager AK, Hutchings A, Van Staden J. (1996) Screening of Zulu medicinal plants for prostaglandin-synthesis inhibitors. Journal of Ethnopharmacology 52: 95–100.

Jaroszeski MJ, Radcliff G. (1999) Fundamentals of flow cytometry. Molecular Biotechnology 11: 37–53.

Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. (2011) Global cancer statistics. CA: A Cancer Journal for Clinicians 61: 69–90.

Johnstone PAS, Takimoto CHM, Goulet R. (2000) Esophageal cancer, a systematic review. Current Problems in Cancer 24: 295–373.

Kaltenbach JP, Kaltenbach MH, Lyons WB. (1958) Nigrosin as a dye for differentiating live and dead ascites cells. Experimental Cell Research. 15: 112–117.

Katerere DR, Eloff JN. (2008) Anti-bacterial and anti-oxidant activity of Hypoxis hemerocallidea (Hypoxidaceae): can leaves be substituted for corms as a conseravation strategy? South African Journal of Botany 74: 613–616.

Kau AL, Martin SM, Lyon W, Hayes E, Caparon MG, Hultgren SJ. (2005) Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J Mice. Infection and Immunity 73: 2461– 2468.

116 Chapter 7: References

Kerr JFR, Wyllie AH, Currie AR. (1972) Apoptosis: a basic biological phenomenon with wide- ranging implications in tissue kinetics. British Journal of Cancer 26: 239–257.

Koduru S, Grierson DS, Afolayan AJ. (2007) Ethnobotanical information of medicinal plants used for treatment of cancer in the Eastern Cape province, South Africa. Current Science 92: 906–908.

Koptur, S (1985) Alternative defenses against herbivores in Inga (Fabaceae: Mimosideae) over an elevational gradient. Ecology 66: 1639-1650.

Kreusch A, Karstaedt AS. (2013) Candidemia among adults in Soweto, South Africa, 1990–2007. International Journal of Infectious Diseases 17: e621–e623.

Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Walorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G. (2009) Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death and Differentiation 6: 3–11.

Kumi-Diaka J, Nguyen V, Butler A. (1999) Cytotoxic potential of the phytochemical genistein isoflavone (4’,5’,7-trihdroxyisoflavone) and certain environmental chemical compounds on testicular cells. Biology of the Cell 91: 515–523.

Kusuge T, Yokota M, Sugiyama K, Yamamoto T, Yan SC. (1985) Studies on antitumor activities and antitumor principles of Chinese herbs. Yakogako Zasshi 105: 791–795.

Laporta O, Funes L, Garzón MT, Villalaín J, Micol V. (2007a) Role of membranes on the antibacterial and anti-inflammatory activities of the bioactive compounds from Hypoxis rooperi corm extracts. Archives of Biochemistry and Biophysics 467: 119–131.

Laporta O, Pérez-Fons L, Mallavia R, Caturla N, Micol V. (2007b) Isolation, characterization and antioxidant capacity assessment of the bioactive compounds derived from Hypoxis rooperi corm extract (African potato). Food Chemistry 101: 1425–1437.

Lazebnik Y. (2010) What are the hallmarks of cancer? Nature Reviews Cancer 10: 232–233.

Leitão JMM, Esteves da Silva JCG. (2010) Firefly luciferase inhibition. Journal of Photochemistry and Photobiology B: Biology 101: 1–8.

117 Chapter 7: References

Lin J, Opoku AR, Geheeb-Keller M, Hutchings AD, Terblance SE, Jäger AK, Van Staden J. (1999) Preliminary screening of some traditional Zulu medicinal plants for ant-inflammatory and anti-microbial activities. Journal of Ethnopharmacology 68: 267–274.

Link A, Balaguer F, Goel A. (2010) Cancer chemoprevention by dietary polyphenols: promising role for epigenetics. Biochemical Pharmacology 80: 1771–1792.

Liu RH. (2004) Potential synergy of phytochemicals in cancer prevention: mechanism of action. Journal of Nutrition 134: 3479S–3485S.

Longo-Sorbello GSA, Saydam G, Banerjee D, Bertino JR. (2006) Cytotoxicity and Cell Growth Assays. Cell Biology 38: 315–324.

Louchouarn P, Kuo LJ, Wade TL, Schantz M. (2009) Determination of levoglucosan and its isomers in size fractions of aerosol standard reference materials. Atmospheric Environment 43: 5630–5636.

Louw CAM, Regnier TJC, Korsten L. (2002) Medicinal bulbous plants of South Africa and their traditional relevance in the control of infectious diseases. Journal of Ethnopharmacology 82: 147– 154.

Love RR, Leventhal H, Easterling DV, Nerenz DR. (1989) Side effects and emotional distress during cancer chemotherapy. Cancer 63: 604–612.

Lovschall P, Eiskjaer M, Arenholt-Bindslev D. (2002) Formaldehyde cytotoxicity in three human cell types assessed in three different assays. Toxicology in Vitro 16: 63–69.

Lowe FC, Fagelman E. (2000) Phytotherapy for chronic prostatitis. Current Urology Reports 1:164–166.

Mahomed IM, Ojewole JAO. (2003) Hypoglycaemic effect of Hypoxis hemerocallidea corm (‘African Potato’) aqueous extract in rats. Methods and Findings in Experimental & Clinical Pharmacology 25: 617–623.

Mander M. (1998) Marketing of indigenous medicinal plants in South Africa. A case study in KwaZulu-Natal. Food and Agriculture Organization of the United Nations, Rome, Italy. Page 162.

Marini-Bettolo GB, Galeffi C, Multari G, Pallazzino G, Messana I. (1991) Interjectin, a derivative of nyasicoside from Hypoxis interjecta and Hypoxis multiceps. Tetrahedron 47: 6717–6724.

118 Chapter 7: References

Marini-Bettolo GB, Nicoletti M, Messana I, Galeffi C, Msonthi JD, Chapya WA. (1985) Glucosides of Hypoxis nyasica Bak. The structure of nyasoside, a new glucoside biologically related to hypoxoside. Tetrahedron 41: 665–670.

Marini-Bettolo GB, Patamia M, Nicoletti M, Galeffi C, Messana I. (1982a) Hypoxoside, a new glycoside of uncommon structure from Hypoxis obtusa Burch. Tetrahedron 38: 1683–1687.

Marini-Bettolo GB, Patamia M, Nicoletti M, Galeffi C, Messana I. (1982b) Research on African medicinal plants – II. Hypoxoside, a new glycoside of unknown structure from Hypoxis obtuse Busch. Tetrahedron 38: 1683–1687.

Markaki AE. (2009) AlamarBlue® assay for assessment of cell proliferation using the FLUOstar OPTIMA. BMG Labtech Application Note: 195 Rev. 06/2009.

Marston A. (2011) Thin-later chromatography with biological detection in phytochemistry. Journal of Chromatography A 1218: 2676–2683.

Martin SJ, Green DR. (1995) Protease activation during apoptosis: death by a thousand cuts? Cell 82: 349–352.

Martin SJ, Reutelingsperger CPM, McGahon AJ, Rade JA, Van Schie RCAA, LaFace DM, Green DR. (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. Journal of Experimental Medicine 182: 1545–1556.

Martini FH. (2006) Fundamentals of Anatomy and Physiology. 7th Edition. Pearson Benjamin Cummings, San Francisco. Page 875–876.

Mashima T, Tsuruo T. (2005) Defects of the apoptotic pathway as therapeutic target against cancer. Drug Resistance Updates 8: 339–343.

Masika PJ, Sultana N, Afolayan AJ. (2005) Isolation of two antibacterial compounds from the bark of Salix capensis. South African Journal of Botany 71: 441–443.

Matsha T, Stepien A, Blanco-Blanco E, Brink LT, Lombard CJ, Van Rensburg S, Erasmus RT. (2006) Self-induced vomiting–risk for oesophageal cancer? South African Medical Journal 96: 209–212.

McBride SM, Fischetti VA, LeBlanc DJ, Moellering RC, Gilmore MS Jr. (2007) Genetic diversity among Enterococcus faecalis. Plos One 7: e582.

119 Chapter 7: References

McCabe ML, Dlamini Z. (2005) The molecular mechanisms of oesophageal cancer. International Immunopharmacology 5: 1113–1130.

McMillian MK, Li L, Parker JB, Patel L, Zhong Z, Gunnett JW, Powers WJ, Johnson MD. (2002) An improved resazurin-based cytotoxicity assay for hepatic cells. Cell Biology and Toxicology 18: 157–173.

MedlinePlus. (2014) Infectious Diseases. http://www.nlm.nih.gov/medlineplus/infectiousdiseases.html. Date accessed: 20 February 2014.

Messana I, Msonthi JD, Di Vicente Y, Multari G, Galeffi C. (1989) Mononyasine A and mononyasine B: two glucosides from Hypoxis nyasica. Phytochemistry 28: 2807–2809.

Mikstacka R, Stefański T, Różański J. (2013) Tubulin-interactive stilbene derivatives as anticancer agents. Cellular and Molecular Biology Letters 18: 368–397.

Mills E, Cooper C, Seely D, Kanfer I. (2005) African herbal medicines in the treatment of HIV: Hypoxis and Sunderlandia. An overview of evidence and pharmacology. Nutrition Journal 4: 19.

Mochida M, Kawamura K. (2004) Hygroscopic properties of levoglucosan and related organic compounds characteristic to biomass burning aerosol particles. Journal of Geophysical Research – Atmospheres 1–27.

Moghadasian HM. (2000) Pharmacological properties of plant sterols in vivo and in vitro observations. Life Sciences 67: 605–615.

Moreno MRF, Sarantinopoulos P, Tsakalidou E, DeVuyst L. (2006) The role and application of Enterococci in food and health. International Journal of Food Microbiology 106: 1–24.

Motsei ML, Lindsey KL, van Staden J, Jäger AK. (2003) Screening of traditionally used South African plants for antifungal activity against Candida albicans. Journal of Ethnopharmacology 86: 235–241.

Muwanga C. (2006) An assessment of Hypoxis hemerocallidea extracts, and actives as natural antibiotic, and immune modulation phytotherapies. Masters Dissertation. University of the Western Cape.

Naglik JR, Challacombe SJ, Hube B. (2003) Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiology and Molecular Biology Reviews 67: 400–428.

120 Chapter 7: References

Nair VDP, Dairam A, Agbonon A, Arnason JT, Foster BC, Kanfer I. (2007a) Investigation of the antioxidant activity of African potato (Hypoxis hemerocallidea). Journal of Agricultural and Food Chemistry 55: 1707–1711.

Nair VDP, Foster BC, Arnason JT, Mills EJ, Kanfer I. (2007b) In vitro evaluation of human cytochrome P450 and P-glycoprotein-mediated metabolism of some phytochemicals in extracts and formulations of African potato. Phytomedicine 14: 498–507.

Nair VDP, Kanfer I, Hoogmartens J. (2006) Determination of stigmasterol, beta-sitosterol and stigmastastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection. Journal of Pharmaceutical and Biomedical Analysis 41: 731–737.

Nair VDP, Kanfer I. (2006) High-performance liquid chromatographic method for the quantitative determination of hypoxoside in African potato (Hypoxis hemerocallidea) and in commercial products containing the plant material and / or its extracts. Journal of Agricultural and Food chemistry 54: 2816–2821.

Nair VDP, Kanfer I. (2008a) Development of dissolution tests for the quality control of complementary / alternate and traditional medicines: application to African potato products. Journal of Pharmacy and Pharmaceutical Sciences 11: 35–44.

Nair VDP, Kanfer I. (2008b) Sterols and sterolins in Hypoxis hemerocallidea (African potato). South African Journal of Science 104: 322–324.

Nakayama GR, Caton MC, Nova MP, Parandoosh Z. (1997) Assessment of the alamar blue assay for cellular growth and viability in vitro. Journal of Immunological Methods 204: 205–208.

Nam NH. (2003) Combretastatin A-4 analogues as antimitotic antitumor agents. Current Medicinal Chemistry 10: 1697–1722.

Nasrollahzadeh D, Malekzadeh R, Aghcheli K, Sotoudeh M, Merat S, Islami F, Kamangar F, Abnet CC, Shakeri R, Pourshams A, Semnani S, Boffetta P, Dawsey SM, Ye W. (2012) Gastric atrophy and oesophageal squamous cell carcinoma: possible interaction with dental health and oral hygiene habit. British Journal of Cancer 107: 888–894.

Nataro JP, Kaper JB. (1998) Diarrheagenic Escheria coli. Clinical Microbiology Reviews 11: 142– 201.

121 Chapter 7: References

Ncube B, Finnie JF, Van Staden J. (2011) Seasonal variation in antimicrobial and phytochemical properties of frequently used medicinal bulbous plants from South Africa. South African Journal of Botany 77: 387–396.

Ncube B, Ndhlala AR, Okem A, Van Staden J. (2013) Hypoxis (Hypoxidaceae) in African traditional medicine. Journal of Ethnopharmacology 150: 818–827.

Ndhlala AR, Finnie JF, Van Staden J. (2011) Plant composition, pharmacological properties and mutagenic evaluation of a commercial Zulu herbal mixture: imbiza ephuzwayo. Journal of Ethnopharmacology 133: 663–674.

Ndong TA, Wadouachi A, Sangwan-Norreel BS, Sangwan RS. (2006) Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace gaza–the African potato. Plant Cell Reports 25: 265–273.

Nguyen JT, Wells JA. (2003) Direct activation of the apoptosis machinery as a mechanism to target cancer cells. Proceedings of the National Academy of Sciences of the United States of America 100: 7533–7538.

Nicoletti M, Galetti C, Messana I, Marini-Bettolo GB. (1992) Hypoxidaceae. Medicinal uses and the norlignan constituents. Journal of Enthopharmacology 36: 95–101.

Niles A, Scurria M, Bernad L, McNamara B, Rashka K, Lange D, Guthmiller P, Worzella T, Riss T, Promega Corporation, Promega Biosciences (2007a) Measure relative numbers of live and dead cells and normalize assay data to cell number. Cell Notes 18:15–20.

Niles AL, Maravec RA, Hesselberth PE, Scurria MA, Daily WJ, Riss TL. (2007b) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Analytical Biochemistry 366: 197–206.

Nolte CG, Schauer JJ, Cass GR, Simoneit BRT. (2001) Highly polar organic compounds present in wood smoke and in the ambient atmosphere. Environmental Science & Technology 35: 1912– 1919.

Nunez R. (2001) Flow cytometry: principles and instrumentation. Current Issues in Molecular Biology 3: 39–45.

Nyinawumuntu A, Awe EO, Ojewole JAO. (2008) Uterolytic effect of Hypoxis hemerocallidea Fisch. & C.A. Mey. (Hypoxidaceae) corm [‘African Potato’] aqueous extract. Journal of Smooth Muscle Research 44: 167–176.

122 Chapter 7: References

O’Brien J, Wilson I, Orton T, Pognan F. (2000) Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry 267: 5421–5426.

Oggioni MR, Pozzi G, Valensin PE, Galieni P, Bigazzi C. (1998) Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. Journal of Clinical Microbiology 36: 325–326.

Ojewole JAO, Kamadyaapa DR, Musabayane CT. (2006) Some in vitro and in vivo cardiovascular effects of Hypoxis hemerocallidea Fisch & CA Mey (Hypoxidaceae) corm (African potato) aqueous extract in experimental animal models. Cardiovascular Journal of South Africa 17: 166– 171.

Ojewole JAO, Olayiwola G, Nyinawumuntu A. (2009) Bronchorelaxant property of ‘African potato’ (Hypoxis hemerocallidea corm) aqueous extract in vitro. Journal of Smooth Muscle Research 45: 241–248.

Ojewole JAO. (2002) Anti-inflammatory properties of Hypoxis hemerocallidea corm (African potato) extracts in rats. Methods and Findings in Experimental & Clinical Pharmacology 24: 685– 687.

Ojewole JAO. (2006) Antinociceptive, anti-inflammatory and antidiabetic properties of Hypoxis hemerocallidea Fisch. & C.A. Mey. (Hypoxidaceae) corm [‘African Potato’] aqueous extract in mice and rats. Journal of Ethnopharmacology 103: 126–134.

Ojewole JAO. (2008) Anticonvulsant activity of Hypoxis hemerocallidea Fisch. & C. A. Mey. (Hypoxidaceae) corm [‘African potato’] aqueous extract in mice. Phytotherapy Research 22: 91– 96.

Okutucu C, Duman G, Ucar S, Yasa I, Yanik J. (2011) Production of fungicidal oil and activated carbon from pistachio shell. Journal of Analytical and Applied Pyrolysis 91: 140–146.

Otsuki Y, Li Z, Shibata MA. (2003) Apoptotic detection methods–from morphology to gene. Progress in Histochemistry and Cytochemistry 38: 275–340.

Owira PMO, Ojewole AO. (2009) ‘African potato’ (Hypoxis hemerocallidea corm): a plant– medicine for modern and 21st century diseases of mankind?–a review. Phytotherapy Research 23: 147–152.

123 Chapter 7: References

OXiGENE. (2013) Clinical trials. http://www.oxigene.com/pipeline/clinical_trials/. Date accessed: 03 October 2013.

Pagé B, Pagé M, Woël C. (1993) A new fluorometric assay for cytotoxicity measurements in vitro. International Journal of Oncology 3: 473–476.

Pegel KH. (1980) Active plant extracts of Hypoxidaceae and their use. United States Patent 4198401.

Pegel KH. (1997) The importance of sitosterol and sitosterolin in human and animal nutrition. South African Journal of Science 93: 263–268.

Perrot S, Duterre-Catella H, Martin C, Warnet JM, Rat P. (2003) A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability. Cytometry Part A 55A: 7–14.

Perry SW, Epstein LG, Gelbard HA. (1997) In situ trypan blue staining of monolayer cell cultures for permanent fixation and mounting. BioTechniques 22: 1020–1024.

Pettit GR, Singh SB, Hamel E, Lin CM, Alberts DS, Garcia-Kendall D. (1989) Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4. Experientia 45: 209– 211.

Pettit GR, Temple C Jr, Narayanan VL, Varma R, Simpson MJ, Boyd MR, Rener GA, Bansal N. (1995) Antineoplastic agents 322. Synthesis of combretastatin A-4 prodrugs. Anti-Cancer Drug Design 10: 299–309.

Pettit RK, Weber CA, Pettit GR (2009) Application of a high throughput alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms. Annals of Clinical Microbiology and Antimicrobials 8: 28.

Pierre-Audigier C, Jouanguy E, Lamhamedi S, Altare F, Rauzier J, Vincent V, Canioni D, Emile JF, Fischer A, Blanche S, Gaillard JL, Casanova JL. (1997) Fatal disseminated Mycobacterium smegmatis infection in a child with inherited interferon gamma receptor deficiency. Clinical Infectious Diseases 24: 982–984.

Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. (1998) Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. Journal of Clinical Microbiology 36: 2093–2095.

124 Chapter 7: References

Promega. (2012a) BacTiter–GloTM microbial cell viability assay. Technical Bulletin Part# TB337. Promega Corporation USA. Available: http://www.promega.com.

Promega. (2012b) CellTiter-Blue® cell viability assay instructions for use of products G8080, G8081 and G8082. Technical Bulletin Part# TB317. Promega Corporation USA. Available: http://www.promega.com.

Prosen EM, Radlein D, Piskorz J, Scott DS, Legge RL. (1993) Communication to the editor microbial utilization of levoglucosan in wood pyrolysate as a carbon and energy source. Biotechnology and Bioengineering 42: 538–541.

Pujol J. (1990) Naturafrica: the herbalist handbook–African flora, medicinal plants. Jean Pujol Natural Healers Foundation, Durban, South Africa. Page 51, 63.

Rahman M, Lane A, Swindell A, Bartram S. (2009) Introduction to flow cytometry. MorphoSys UK Ltd 1: 2–6.

Ramakrishna A, Ravishankar GA. (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant signalling and Behavior 6: 1720–1731.

Reed JC. (2003) Apoptosis-targeted therapies for cancer. Cancer Cell 3: 17–22.

Reid KA, Jäger AK, Light ME, Mulholland DA, van Staden J.V., (2006) Phytochemical and pharmacological screening of Sterculiaceae species and isolation of antibacterial compounds. Journal of Ethnopharmacology 97: 285-291.

Rhodes L, Primka RL, Berman C, Vergult G, Gabriel M, Pierre-Malice M, Gibelin B. (1993) Comparison of finasteride (Proscar), a 5 alpha reductase inhibitor, and various commercial plant extracts in in vitro and in vivo 5 alpha reductase inhibition. Prostate 22: 43–51.

Richard V, Van der Auwera P, Snoeck R, Daneau D, Meunier F. (1988) Nosocomial bacteremia caused by Bacillus species. European Journal of Clinical Microbiology and Infectious Diseases 7: 783–785.

Rios JL, Recio MC. (2005) Medicinal plants and antimicrobial activity. Journal of Ethnopharmacology 100: 80–84.

Rios JL, Recio MC, Villar A. (1988) Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 23: 127–149.

125 Chapter 7: References

Rogall T, Wolters J, Flohr T, Böttger EC. (1990) Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. International Journal of Systematic Bacteriology 40: 323–330.

Röse L, Kaufmann SHE, Daugelat S. (2004) Involvement of Mycobacterium smegmatis undecaprenyl phosphokinase in biofilm and smegma formation. Microbes and Infection 6: 965– 971.

Rosenorn T, Kiss G, Bilde M. (2006) Cloud droplet activation of saccharides and levoglucosan particles. Atmospheric Environment 40: 1794–1802.

Rowland I. (1999) Optimal nutrition: fibre and phytochemicals. Proceedings of the Nutrition Society 58: 415–419.

Russo GL. (2007) Ins and outs of dietary phytochemicals in cancer chemoprevention. Biochemical Pharmacology 74: 533–544.

SABC. (2012) South African Cancer Registry backlogged. http://www.sabc.co.za/news/a/0a4646004db523e2890adf0380ff593a/South-African-Cancer- Registry-backloged-20120612. Date accessed: 03 October 2013.

Salie F, Eagles PFK, Leng HMJ. (1996) Preliminary antimicrobial screening of four South African Asteraceae species. Journal of Ethnopharmacology 52: 27–33.

Sammon AM. (2007) Carcinogens and endemic squamous cancer of the oesophagus in Transkei, South Africa. Environmental initiation is the dominant factor; tobacco or other carcinogens of low potency or concentration are sufficient for carcinogenesis in the predisposed mucosa. Medical Hypotheses 69: 125–131.

Sarma KD, Ray D, Antony A. (2000) Improved sensitivity of trypan blue dye exclusion assay with Ni2+ or Co2+ salts. Cytotechnology 32: 93–95.

Shafizadeh F, Rowell R (ed). (1984) The chemistry of pyrolysis and combustion, in Chemistry of Solid Wood. Advances in Chemistry Series 207, American Chemical Society, Washington DC. Page 489–529.

Shafizadeh F, Fu YL. (1973) Pyrolysis of cellulose. Carbohydrate Research 29: 113–122.

126 Chapter 7: References

Shafizadeh F, Furneaux RH, Cochran TG, Scholl JP, Sakai Y. (1979) Production of levoglucosan and glucose from pyrolysis of cellulosic materials. Journal of Applied Polymer Science 23: 3525– 3539.

Shai LT, McGaw LT, Masoko P, Eloff JN. (2008) Antifungal and antibacterial activity of seven traditionally used South African plant species active against Candida albicans. South African Journal of Botany 78: 677–684.

Shanley P, Luz L. (2003) The impacts of forest degradation on medicinal plant use and implications for health care in Eastern Amazonia. Bioscience 53: 573–584.

Sharma V, Kotzen J. (2007) Cancer of the oesophagus is a distressingly common cancer in men and women. Continuing Medical Education 25: 55–58.

Sibanda S, Ntabeni O, Nicoletti M, Galeffi C. (1990) Nyasol and 1,3(5)-diphenyl-1-pentene related glycosides from Hypoxis angustifolia. Biochemical Systematics and Ecology 18: 481–483.

Siegel R, Naishadham D, Jemal A. (2013) Cancer Statistics, 2013. CA: A Cancer Journal for Clinicians 63: 11–30.

Sigma-Aldrich. (2012) Cell viability and proliferation. http://www.sigmaaldrich.com/technical- documents/articles/biofiles/cell-viability-and-proliferation.html#trypan-blue. Date accessed: 01 December 2012.

Simoniet BRT, Schauer JJ, Nolte CG, Oros DR, Elias VO, Fraser MP, Rogge W, Cass GR. (1999) Levoglucosan, a tracer for cellulose in biomass burning and atmospheric particles. Atmospheric Environment. 33: 173-182.

Singh Y. (1999) Hypoxis: yellow stars of horticulture, folk remedies and conventional medicine. Veld and Flora 85: 123–125.

Singh Y. (2007) Hypoxis (Hypoxidaceae) in southern Africa: taxonomic notes. South African Journal of Botany 73: 360–365.

Skerman NB, Joubert AM, Cronjé MJ. (2011) The apoptosis inducing effects of Sutherlandia spp. Extracts on an oesophageal cancer cell line. Journal of Ethnopharmacology 137: 1250–1260.

Smith BJ, Albrecht CF, Liebenberg RW, Kruger PB, Freestone M, Gouws L, Theron E, Bouic PJD, Atsebeth S, van Jaarsveld PP. (1995) A phase I trial of hypoxoside as an oral prodrug for cancer therapy – absence of toxicity. South African Medical Journal 85: 865–870.

127 Chapter 7: References

Sosa JA, Elisei R, Jarzab B, Bal CS, Koussis H, Gramza AW, Ben-Yosef R, Gitlitz BJ, Haugen B, Karandikar SM, Khuri FR, Licitra LF, Remick SC, Marur S, Lu C, Ondrey FG, Lu S, Balkissoon J. (2011) A randomized phase II/III trial of a tumor vascular disrupting agent fosbretabulin tromethamine (CA4P) with carboplatin (C) and paclitaxel (P) in anaplastic thyroid cancer (ATC): Final survival analysis for the FACT trial. Journal of Clinical Oncology 29: 5502 (abstract).

Southern African Development Community (SADC). (2002) SADC Ministerial Consultative Meeting on Nutrition and HIV/AIDS. SADC: Johannesburg.

Steenkamp V. (2003) Phytomedicines for the prostate. Fitoterapia 74: 545–552.

Steenkamp V, Gouws MC. (2006) Cytotoxicity of six South African medicinal plant extracts used in the treatment of cancer. South African Journal of Botany 72: 630–633.

Steenkamp V, Gouw MC, Gulumian M, Elgorashi EE, van Staden. (2006) Studies on antibacterial, anti-inflammatory and antioxidant activity of herbal remedies used in the treatment of benign prostatic hyperplasia and prostatitis. Journal of Ethnopharmacology 103: 71–75.

Stein T. (2005) Bacillus subtilis antibiotics: structure, synthesis and specific functions. Molecular Microbiology 56: 845–857.

Street RA, Prinsloo G. (2012) Commercially important medicinal plants of South Africa: a review. Journal of Chemistry 2013: 1–16.

Street RA, Stirk WA, Van Staden J. (2008) South African traditional medicinal plant trade– challenges in regulating quality, safety and efficacy. Journal of Ethnopharmacology 119: 705–710.

Suffness M, Pezzuto JM, Hostettmann K (ed). (1990) Assays related to cancer drug discovery. Methods in plant biochemistry: assays for bioactivity. Volume 6. Academic Press, London. Page 71 – 133.

Tavakkol-Afshari J, Brook A, Mousavi SH. (2008) Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines. Food and Chemical Toxicology 46: 3443–3447.

Taylor JLS, Rabe T, McGaw LJ, Jäger AK, Van Staden J. (2001) Towards the scientific validation of traditional medicinal plants. Plant Growth Regulations 34: 23–37.

128 Chapter 7: References

The Cancer Research UK. (2011a) Cancer treatments. http://cancerhelp.cancerresearchuk.org/about-cancer/treatment/. Date accessed: 07 November 2011.

The Cancer Research UK. (2011b) Cancer worldwide–the global picture. http://info.cancerresearchuk.org/cancerstats/world/the-global-picture/#common. Date accessed: 07 November 2011.

Theron E, Albrecht C, Kruger P, Jenkins K, Van der Walt BJ. (1994) Beta-glucosidase activity in fetal calf serum renders the plant glucoside, hypoxoside, cytotoxic towards BL-6 mouse melanoma cells. In vitro Cellular and Developmental Biology 30A: 115–119.

Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. (2011) Phytochemical screening and extraction: a review. Internationale Pharmaceutica Sciencia. 1: 98–106.

Tran SL, Puhar A, Ngo-Camus M, Ramarao N. (2011) Trypan blue dye enters viable cells incubated with the pore-forming toxin Hlyll of Bacillus cereus. PLoS ONE 6: e22876.

Udobi CE, Onaolapo AJ, Abdulsalaam IA. (2010) Bioautographic determination of the antistaphylococcal components of the stem bark of Parkia biglobosa (JACQ) BENTH (MIMOSACEAE). Journal of Pharmacognosy and Phytotherapy 2: 108–112.

UNAIDS. (2012) Global Report: UNAIDS report on the global AIDS epidemic. http://www.unaids.org/en/media/unaids/contentassets/documents/epidemiology/2012/gr2012/201 21120_UNAIDS_Global_Report_2012_with_annexes_en.pdf. Date accessed: 05 May 2013.

Van Vuuren SF. (2008) Antimicrobial activity of South African medicinal plants. Journal of Ethnopharmacology 119: 462–472.

Van Wyk BE. (2007) People’s plants. Briza Publications. Pretoria, South Africa. Page 146–147.

Van Wyk BE. (2008) A broad review of commercially important South African medicinal plants. Journal of Ethnopharmacology 119: 342–355.

Van Wyk BE. (2011) The potential of South African plants in the development of new medicinal products. South African Journal of Botany 77: 812–829.

Van Wyk BE, Van Oudtshoorn B, Gericke N. (2002) Medicinal Plants of South Africa. Briza Publications. Pretoria, South Africa. Page 157–157.

129 Chapter 7: References

Vejborg RM, Klemm P. (2009) Cellular chain formation in Escherichia coli biofilms. Microbiology 155: 1407–1417.

Verschaeve L, Mertens B, Ndhlala AR, Anthonissen R, Gorissen B, Van Staden J. (2013) Investigation into the genotoxicity of water extracts from Hypoxis species and a commercially available Hypoxis preparation. Phytotherapy Research 27: 350–356.

Verschaeve L, Van Staden J. (2008) Mutagenic and antimutagenic properties of extracts from South African traditional medicinal plants. Journal of Ethnopharmacology 119: 575–587.

Von Holtz RL, Fink CS, Awad AB (1998) Beta-sitosterol activates the sphingomyelin cycle and induces apoptosis in LNCaP human prostate cancer cells. Nutrition and Cancer 32: 8–12.

Waghmare SS, Jadhav DY, Ghosh JS, Sahoo AK. (2010) Characterization of some antimicrobial substances from seed coat of Tamarindus indica Linn. British Journal of Pharmacology and Toxicology 1: 29–32.

Watt JM, Breyer-Brandwijk MG. (1962) The Medicinal and Poisonous Plants of Southern and Eastern Africa. 2nd Edition, Livingstone, London. Page 31–41.

Weiss RF. (1985) Lehrbuch der Phytotherapie, Hippokrates. Verlag, Stuttgart. Page 317.

WHO. (2011) The top 10 causes of death. http://www.who.int/mediacentre/factsheets/fs310/en/index2.html. Date accessed: 06 February 2014.

WHO. (2013a) Cancer. http://www.who.int/mediacentre/factsheets/fs297/en/index.html. Date accessed: 03 October 2013.

WHO. (2013b) Infectious diseases. http://www.who.int/topics/infectious_diseases/en/. Date accessed: 03 March 2012.

WHO. (2013c) Drug resistance. http://www.who.int/drugresistance/AMR_Importance/en/. Date accessed: 07 June 2013.

Wilson K, Walker J. (2005) Principles and Techniques of Biochemistry and Molecular Biology. 6th Edition. Cambridge University Press, New York. Page 405–447, 541, 672.

Wilt TJ, Ishani A, MacDonald R, Stark G, Mulrow CD, Lau J (2011) Beta-sitosterols for benign prostatic hyperplasia–review. John Wiley and Sons, Ltd. Minnesota. Abstract.

130 Chapter 7: References

Wu AH, Wan P, Bernstein L. (2001) A multiethnic population-based study of smoking, alcohol and body size and risk of adenocarcinoma of the stomach and esophagus (United States). Cancer Causes Control 12: 721–32.

Zibula SMX, Ojewole JAO. (2000) Hypoglycaemic effects of Hypoxis hemerocallidea (Fisch. And C.A. Mey.) corm ‘African Potato’ methanolic extract in rats. Medical Journal of Islamic Academy of Sciences 13: 75–78.

Zoletti GO, Pereira EM, Schuenck RP, Teixeira LM, Siqueira JF Jr, Dos Santos KRN. (2011) Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals. Research in Microbiology 162: 151–158.

Zweifel M, Jayson GC, Reed NS, Osborne R, Hassan B, Ledermann J, Shreeves G, Poupard L, Lu SP, Balkissoon J, Chaplin DJ, Rustin GJ. (2011) Phase II trial of combretastatin A4 phosphate, carboplatin, and paclitaxel in patients with platinum-resistant ovarian cancer. Annals of Oncology 22: 2036–2041.

131