Investigation of Wild Garlic Plant Extracts As Potential Devulcanizing Agents

INVESTIGATION OF WILD GARLIC PLANT EXTRACTS AS POTENTIAL DEVULCANIZING AGENTS

Mooketsi Mpuputla

A thesis submitted in fulfillment of the requirements of the degree for Magister Scientae in chemistry at the Nelson Mandela University

January 2020

Supervisor: Dr. B.G. Hlangothi

Co-supervisor: Prof. C. Woolard

Co-supervisor: Dr. P.S. Hlangothi

DEDICATION

The work present in this study is dedicated to my son, Hlompo. Though a young growing man and you might have not known it, but I want to thank you for being the wind beneath my wings, reminding me that I have the ability to reaching greater successes, being the role model of the successes that you too are to one day fulfill – this is your first step my son

I LOVE YOU

DECLARATION

ACKNOWLEDGEMENTS

Firstly, I wish to thank my Heavenly Father, God, for all He has done for me the past years throughout my studies. Thanking Him for His endless strength provided and ability to continue in my journey of studies, from undergrad until this very point.

I wish to sincerely extend my gratitude to my supervisor, Dr. B.G. Hlangothi. Thank you for the opportunity and trust in me to work on this project. The road was not easy, but because of your endless support and encouragement, we have made it as a team. THANK YOU SO MUCH!!

My co-supervisor, Prof. C.D. Woolard, thank you so for your support and research expertise in the field of rubber and science technology. Thank you for your time to listen and share new ideas. You are much appreciated!!

Dr. S.P. Hlangothi, I wish to thank you for your continuous support and role that you have played in supporting and motivating me through my education and research. I could never forget the things you have pulled together for me in order to complete this degree too.

My friends, peers, and colleagues in the BGH and CRST research groups, thank you all so much for your moral support and inputs.

Mr Lukhanyo Bolo for his assistance with thermal analysis, guidance and valuable input throughout my research.

I wish to thank the technical staff, Mr H. Schalkenkamp, Mr E. Bashman, and Mrs K. Muller for their assistance. Ms E. Storm from the Horticulture Department for plant harvesting and the Botany Department at the Nelson Mandela University for plant identification.

My parents, I thank you so much for your love and support. I appreciate your patience and understanding that it has not only been about what you wish for me but mostly what I want to achieve. My family, at large, thanks for your endless prayers, believing in me and continuous support

iii

Contents

Dedication ...... i

Declaration ...... ii

Acknowledgements ...... iii

Abstract ...... ix

LIST OF ABBREVIATIONS ...... xi

LIST OF FIGURES Page ...... xiii

LIST OF TABLES ...... xvii

LIST OF SCHEMES ...... xviii

CHEMICAL STRUCTURES OF SOME IMPORTANT SPECIES ...... xix

CHAPTER 1: ...... 1

INTRODUCTION ...... 1

1.1. Aims and objectives ...... 4

CHAPTER 2: ...... 5

LITERATURE REVIEW ...... 5

2.1. THE FAMILY ALLIACEAE ...... 5

2.2. THE GENUS TULBAGHIA ...... 5

2.3. TULBAGHIA VIOLACEA ...... 7

2.3.1 Pharmacological studies of Tulbaghia violacea ...... 9

2.3.2 Bioactive components of T. violacea ...... 11

2.4. VULCANIZATION ...... 14

2.4.1 SULFUR VULCANIZATION REACTION SCHEME ...... 18

2.4.2 MODEL COMPOUND VULCANIZATION ...... 19

2.5. DEVULCANIZATION ...... 22

CHAPTER 3: ...... 27

EXPERIMENTAL METHODS and MATERIALS ...... 27

3.1. REQUIREMENTS ...... 27

3.1.1. Chemicals and solvents ...... 27

3.1.2. Other materials ...... 28

3.1.3. Collection of plant material ...... 28

3.2. Instrumentation ...... 28

3.2.1 High-Performance Liquid Chromatography (HPLC) ...... 28

3.2.2 Reverse-Phase Chromatography ...... 29

3.2.3 Gel Permeation Chromatography (GPC) ...... 29

3.2.4 Nuclear Magnetic Resonance (NMR) ...... 29

3.2.5 Fourier Transform Infrared Spectroscopy (FTIR) ...... 30

3.2.6 Rotavapor ...... 30

3.2.7 Hot Plate Magnetic Stirrer ...... 30

3.2.8 Differential Scanning Calorimetry (DSC) ...... 30

3.2.9 Thermographic Analysis ...... 31

3.2.10 Balances ...... 31

3.3. Experimental procedures ...... 31

3.3.1. Plant extract preparation ...... 31

3.3.2. TLC and phytochemical test of extracts ...... 32

3.3.3. Analysis of crudes by HPLC ...... 32

3.3.4. Purification of MBTS ...... 33

3.3.5. Purification of MBT ...... 34

3.3.6. Thermogravimetric Analysis (TGA) ...... 34

3.3.7. Differential Scanning Calorimetry (DSC) ...... 34

3.3.8. Vulcanization of model compound ...... 34

3.3.9. HPLC analysis of curatives and vulcanized products ...... 36

3.3.10. Gel Permeation Chromatography ...... 37

3.3.11. Devulcanization of Squalene vulcanizates ...... 37

CHAPTER 4 ...... 39

RESULTS AND DISCUSSION ...... 39

4.1. Extraction and analysis of crude extracts of Tulbaghia violacea...... 39

4.1.1. Plant extraction ...... 39

4.1.2. Identification of sulfides ...... 40

4.1.3. Phytochemical analysis of sulfides ...... 42

4.1.4. FTIR analysis of crudes ...... 43

4.1.5. HPLC analysis of crude extracts and quantification of DADS ...... 45

4.2. MBTS accelerated sulfur vulcanization of squalene ...... 50

4.2.1. TLC and NMR analysis of vulcanizates ...... 50

4.2.2. FTIR analysis of vulcanizates ...... 51

4.2.3. RP-HPLC analysis vulcanizates ...... 52

4.2.4. GPC analysis of vulcanizates ...... 54

4.2.5. Thermal analysis and reactions of curatives ...... 58

4.3. devulcanization of squalene products ...... 65

4.3.1. FTIR analysis...... 65

4.3.2. NMR analysis ...... 66

4.3.3. GPC analysis ...... 68

4.3.4. Thermogravimetric analysis ...... 76

4.3.5. DSC analysis ...... 82

Chapter 5 ...... 85

Conclusion ...... 85

Chapter 6 ...... 88

Recommendations ...... 88

Chapter 7 ...... 89

REFERENCES ...... 89

APPENDICES ...... 94

Appendix A ...... 94

Appendix B ...... 95

Appendix C ...... 95

Appendix C1 ...... 95

Appendix C2 ...... 96

Appendix C3 ...... 96

Appendix D ...... 97

Appendix D1 ...... 97

Appendix E ...... 98

Appendix E1(a) ...... 98

Appendix E1(b) ...... 99

Appendix E2(a) ...... 99

Appendix E2(b) ...... 100

Appendix E3(a) ...... 100

Appendix E(b) ...... 101

Appendix F ...... 101

Appendix F1 ...... 101

Appendix F2 ...... 102

Appendix F3 ...... 102

vii

Appendix G ...... 103

Appendix G1 ...... 103

viii

ABSTRACT

Commercially available chemical compounds used as devulcanizing agents have been found to be relatively expensive, and harmful to human health. These include compounds such as diallyl disulfide (DADS) and diphenyl disulfide (DPDS). However, compounds like DADS and other sulfides are found in the readily available natural resource material, T. violacea, which is rich in sulfur-derived compounds that may exhibit potential use as devulcanizing agents. Hence, this study is aimed at examining the efficacy of the extracted sulfur compounds of T. violacea as potential devulcanizing agents.

The sulfides of T. violacea were successfully extracted by means of sequential extraction using chloroform (CHCl3), ethyl acetate (EtOAc), and methanol (MeOH). Identification of present sulfides was done by phytochemical analysis, using the TLC method. The commercially available HPLC grade DADS reference standard was used to quantify the amount of DADS in each extract. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for quantification. The HPLC results showed that only the EtOAc and MeOH extracts contained DADS, while insignificant amount of DADS was seen in the CHCl3 extracts. The EtOAc roots, bulbs and leaf extracts (18.8 × 10-3, 8.84 × 10-3, 7.2 × 10-3 mg/mL) showed greater DADS concentration compared to the MeOH roots, bulbs and leaf extracts (5.3 × 10-3, 8.07 × 10-3, 1.9 × 10-3 mg/mL), respectively.

RP-HPLC and Gel Permeation Chromatography (GPC) were the methods used to monitor and identify crosslink formation and devulcanizing. The vulcanization and devulcanization studies were carried out using the model compound, squalene. All extracts showed a decrease in the molecular weight distribution of the devulcanized products. The leaf extract, CHCl3 and EtOAc, showed the highest devulcanization efficacy overall, while the MeOH extracts showed least devulcanization efficacy as devulcanization agents.

Thermal analysis studies were performed to investigate the interactions of the curatives used in the vulcanization system(s). Isothermal analysis of the vulcanization system

ix was evident in a vulcanization reaction at an optimal time of 20 min, and at onset temperature of 177.6 oC. Upon devulcanization, DSC analysis gave evidence of the glass transition of squalene products. This indicates that no main-chain scission or backbone breakage occurred in reacting the vulcanized products with the devulcanizing agents.

LIST OF ABBREVIATIONS

Tv Tulbaghia violacea TGA Thermogravimetry analysis Ta Tulbaghia alliacea UV-Vis. Ultraviolet-visible DTVL Dry T. violacea leaves DADS Diallyl disulfide DTVB Dry T. violacea bulbs DAS Diallyl sulfide DTVR Dry T. violacea roots

S8 Sulfur TLC Thin layer chromatography ZnO Zinc oxide PTLC Preparative thin layer chromatography MBT 2-Mercaptobenzothiazole

HPLC High-Performance Liquid MWD Molecular weight Chromatography distribution

RP-HPLC Reversed Phase High- MBTS 2-Bis-benzothiazole-2,2’- Performance Liquid disulfide Chromatography MBT 2-mercaptobenzothiazole GPC Gel Permeation ACN Acetonitrile Chromatography CBS N- cyclohexyl-2- FTIR Fourier transform infrared benzothiazyl sulfenamide spectroscopy CPTD N,’N- 1H-NMR Proton Nuclear Magnetic Dipentamethylenethiuram Resonance disulfide 13C-NMR Carbon Nuclear Magnetic CPTM N,’N- Resonance Dipentamethylenethiuram DSC Differential scanning monosulfide calorimetry Sq Squalene

MBTP 2-Bis-benzothiazole-2,2'- ppm Parts per million polysulfide phr Parts per hundred rubber TME 2,3-Dimethyl but-2-ene Rt Retention time Hex Hexane Rf Retention factor PE Petroleum ether min Minute DCM Dichloromethane s Second MeOH Methanol r.p.m Revolutions per minute EtOAc Ethyl acetate b.p. Boiling point EtOH Ethanol C-S Carbon-sulfur THF Tetrahydrofuran S-S Sulfur-sulfur

CHCl3 Chloroform C-C Carbon-carbon C-H Carbon-hydrogen CDCl3 Deuterated chloroform CO2 Carbon dioxide IPA Isopropyl alcohol SBR Styrene-butadiene rubber cm Centimeter EPDM Ethylene propylene diene cm-1 Per centimeter RRM Renewable Resource Material nm Nanometer WSCP World Checklist of Selected Plants µm Micrometer EC Eastern Cape oC Degrees celsius KZN KwaZulu Natal kHz Kilohertz MIC Minimum inhibitory MHz Megahertz concentration mAu Milli adsorption units TB Tuberculosis mL Milliliter @ At mg/mL milligram per milliliter

xii

LIST OF FIGURES PAGE

Figure 2.1: Shows: (a) T. violacea plant, (b) Bulbs, (c) Roots and (d) Leaves 8

Figure 2.2: Examples of harmful compounds upon consumption of T.violacea. 9

Figure 2.3: Major compound alliin found in garlic. 12

Figure 2.4: Enzyme-mediated formation allicin from alliin. 12

Figure 2.5: Organosulfur compounds formed by degradation of thiosulfinates. 13

Figure 2.6: Shows cysteine derivatives. 14

Figure 2.7: Shows a representation of sulfur vulcanization crosslink formation 15

Figure 2.8: A typical Rheometer curve showing the three phases of vulcanization. 16

Figure 2.9: Sulfur vulcanized network. 19

Figure 2.10: Examples of common model compounds. 20

Figure 2.11: Structural similarity of 2,3-methyl-2-butene (TME) and Isoprene monomer unit in polyisoprene. Equivalent allylic hydrogen positions are shown on TME. 21

Figure 2.12: Organic compounds used as devulcanization agents. 23

Figure 3.1: Setup of vulcanization 36

Figure 4.1: TLC plate of T. violacea extracts against reference standards, AS and DADS, under UV short wavelength eluted with 4:1 Hex: EtOAc as mobile phase (where: Cl, El and ML is the extracts of the leaves. Cb, Eb and Mb are extracts of the bulbs. And Cr, Er and Mr is the extracts of the roots). 41

Figure 4.2: TLC plate of extracts after treatment with Wagner and Bladt vanillin reagent.

Figure 4.3: FTIR spectrum overlay of dry EtOAc crude extracts of T. violacea roots, bulbs and leaves. 44

Figure 4.4: RP-HPLC chromatograms of (a) the mobile phase, (b) reference standard, and (c) DTVR-EtOAc extract 46

xiii

Figure 4.5: DADS reference standard calibration curve 48

Figure 4.6: Shows the 1H-NMR spectrums of (a) squalene reaction product and (b) unreacted squalene 51

Figure 4.7: FTIR overlay of the vulcanizate and squalene 52

Figure 4.8: RP-HPLC analysis of the Squalene/MBTS/S8/ZnO (5.65:1.1:1:1:) system heated isothermally @ 150 oC. 53

Figure 4.9: GPC curve for the squalene/sulfur (5.65:1.0) system heated isothermally at 150 oC for 120 min. 55

Figure 4.10: GPC curve for the squalene/MBTS/sulfur (5.65:1.1:1.0) system heated isothermally at 150 oC for 30 min. 56

Figure 4.11: GPC analysis of the squalene/MBTS/S8/ZnO (5.65:1.1:1:1) system heated isothermally @ 150 oC 57

Figure 4.12: Comparison of the molecular weight distribution of reaction products 58

Figure 4.13: DSC thermogram for sulfur heated @ 10 oC/min, sample size = 1.0338 mg

Figure 4.14: DSC thermogram of MBTS heated at 10 oC/min, sample size = 1.0532 mg

Figure 4.15: DSC thermogram of the squalene/MBTS/ZnO system heated at 10 oC/min cycle 61

Figure 4.16: DSC thermogram of the squalene/MBTS/S8/ZnO system heated at 10 oC/min cycle 1, sample size = 2.6560 mg 62

Figure 4.17: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 5 min, sample size = 10.0990 mg 63

Figure 4.18: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 20 min, sample size = 10.6670 mg 63

xiv

Figure 4.19: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 60 min, sample size = 9.5590 mg 64

Figure 4.20: FTIR overlay of sq-vulcanizate and devulcanizates 66

Figure 4.21: 1H-NMR overlay of (a) squalene-98, (b) vulcanizate, (c - e) devulcanizates using EtOAc extracts as devulcanizing agent 67

Figure 4.22: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVR-EtOAc @ 250 nm 68

Figure 4.23: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVB-EtOAc @ 250 nm 70

Figure 4.24: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVL-EtOAc @ 250 nm 71

Figure 4.25: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization using the CHCl3, EtOAc, and MeOH leaves extracts and the 60 min vulcanizate 72

Figure 4.26: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization, using the CHCl3, EtOAc, and MeOH bulb extracts, and the 60 min vulcanizate 73

Figure 4.27: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization, using the CHCl3, EtOAc, and MeOH roots extracts, and the 60 min vulcanizate 74

Figure 4.28: Comparison of the total crosslink MWD decrease and pendent group formation of the MeOH (blue), CHCl3 (orange), and EtOAc (grey) extracts. 75

Figure 4.29: Overlay of the TGA/Hi-Res DTGA curves of the DTVL-EtOAc extract. 77

Figure 4.30: Overlay of the TGA/Hi-Res DTGA curves of the DTVB-EtOAc extract 79

Figure 4.31: Overlay of the TGA/Hi-Res DTGA curves of the DTVR-EtOAc extract 80

Figure 4.32: TGA curves for the EtOAc crude extracts of T. violacea 82

Figure 4.33 (a): DSC thermogram for the squalene devulcanizate from the DTVL-EtOAc extract heated at 10 oC/min showing no temperature curve, sample size = 5.5300 mg xv

Figure 4.33 (b): DSC thermogram for the squalene devulcanizate from the DTVL-EtOAc extract heated at 10 oC/min showing temperature curve, sample size = 5.5300 mg

Figure 8.1: 1H-NMR spectra of (a) MBTS before purification and (b) Purified MBTS

Figure 8.2: FTIR of clean and purified MBTS 94

Figure 8.3: FTIR overlay of CHCl3 extracts 95

Figure 8.4: RP-HPLC chromatograms of (a) DTVL-EtOAc and (b) DTVB-EtOAc extracts

Figure 8.5: RP-HPLC chromatograms of (a) DTVL-MeOH, (b) DTVB-MeOH and (c)

Figure 8.6: RP-HPLC chromatograms of (a) DTVL-CHCl3, (b) DTVB-CHCl3 and (c)

DTVR-CHCl3 96

Figure 8.7: 1H-NMR overlay of squalene, vulcanizate, and devulcanizates using MeOH extracts as devulcanizing agent 97

1 Figure 8.8: H-NMR overlay of squalene, vulcanizate, and devulcanizates using CHCl3 extracts as devulcanizing agent 97

Figure 8.9: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with

DTVL-CHCl3 @ 250 nm 98

Figure 8.10: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVL-MeOH @ 250 nm 99

Figure 8.11: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with

DTVB-CHCl3 @ 250 nm 99

Figure 8.12: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVB-MeOH @ 250 nm 99

xvi

Figure 8.13: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with

DTVR-CHCl3 @ 250 nm 100

Figure 8.14: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVR-MeOH @ 250 nm 100

Figure 8.15: TG/Hi-Res DTGA curves of the DTVL-EtOAc extract, heated at 10 0C/min. sample mass = 4.7860 mg 101

Figure 8.16: TG/Hi-Res DTGA curves of the DTVB-EtOAc extract, heated at 10 0C/min. sample mass = 4.2780 mg 102

Figure 8.17: TG/Hi-Res DTGA curves of the DTVR-EtOAc extract, heated at 10 0C/min. sample mass = 4.3118 mg 102

Figure 8.18: DSC thermogram for the squalene devulcanizate from the DTVB-EtOAc extract heated at 10 oC/min showing temperature curve, sample size = 8.5360 mg

103

LIST OF TABLES

Table 2.1: Shows plant species in the genus Tulbaghia.

Table 2.2: Shows some commonly used accelerators and their efficiency.

Table 3.1: Lists the chemicals and solvents used in the study

Table 3.2: Lists the other materials used in the study

Table 3.3: Shows the styragel high-resolution columns used for GPC experiments

Table 3.4: Mass of dry plant part weighed

Table 3.5: Calculated concentrations of the standards

Table 3.6: Weighed masses of crude extracts for analysis by HPLC

Table 3.7: Squalene/S8/MBTS/ZnO mole ratio for Sulfur vulcanization

Table 4.1: Illustrates the dry masses and calculated percentage yields of the crude extracts

xvii

Table 4.2: Illustrates the retention times of the analytes corresponding to the reference standard.

Table 4.3: Shows the calculated concentration of DADS and percentage in each extract

Table 4.4: Total weight loss of DTVL-EtOAc extract compounds by Hi-Res DTGA

Table 4.5: Total weight loss of DTVL-EtOAc extract compounds by Hi-Res DTGA

Table 4.6: Total weight loss of DTVR-EtOAc extract compounds by Hi-Res DTGA

LIST OF SCHEMES

Scheme 2.1: Generally accepted vulcanization mechanism.

xviii

CHEMICAL STRUCTURES OF SOME IMPORTANT SPECIES

squalene

HS N

2,3-dimethylbut-2-ene 2-mercaptobenzothiazole

N S S S S

Bisbenzothiazole-2,2'-disulfide

xix

CHAPTER 1:

INTRODUCTION

Native to the Central and Southern regions of America, the history of rubber tells it is known to the indigenous rainforest dwellers (Mesoamericans) of America, who collected and used rubber generations before its exploration. Mesoamericans used rubber to make rubber balls to play the Mesoamerican ballgame. They collected the natural latex from the rubber-engendering tree; Hevea brasiliensis (Euphorbiaceae), growing indigenously in the small river towns of the Amazon. It was not until the late 1400s that rubber was included in the records of history when Europeans explorers docked in America and saw Mesoamericans play the ballgame. This led to the weary travelers to be curious about the material used to make these balls, and questions which led to the uprising of rubber. By 1525, the first report about rubber was made public when Padre d'Anghieria (1525) reported seeing Mexican civilians playing with rubber balls.27 In 1735, Charles Marie de La Condamine (1735) performed the first study of rubber, introducing rubber samples to the Académie Royale des Sciences of France.28 He later presented a paper describing the properties of the rubber by the French engineer; Francois Fresneau in 1751, which was then published in 1755.28 In his study, Fresneau concluded that rubber is a similar type of condensed resin oil.27 The use of rubber as an eraser was suggested by the Portuguese traveler Magellan. In 1770, Joseph Priestley popularized the use of rubber as an eraser when he observed the material rub off pencil marks on paper.29

As the years progressed, latex rubber became more popular in many countries. It was used for the manufacturing of a variety of goods including waterproof fabrics and snow boots. Nonetheless, these products did not last for a long time because the non- vulcanized rubber used for their production was not durable and would be easily affected by temperature changes resulting in being brittle in cold weather and sticky in hot weather.27 During the mid-nineteenth century, Charles Goodyear (1839) discovered the process of vulcanization which helped overcome the challenges faced with rubber latex when he accidentally burnt together rubber and sulfur, forming a charred material of greater plasticity and elasticity.29 This was a consequence of the sulfur bridges formed between rubber molecules, resulting in a 3-dimensional 1 crosslinked network structure that has proved insoluble and infusible.30, 31 The improvement of the chemical and physical properties of rubber made it useful in almost all economic areas, namely automobile, civil construction, plastics, footwear, and other daily life areas. Greatly, since the invention of automobiles, natural and synthetic rubber have been broadly utilized in industry to manufacture tyres, strengthened with a mixture of carbon black and silica to prevent incisions and enrich the durability of tyres.32 Regrettably, because of this durability, discarded scrap tyres are major problems the 21st century must confront.30, 31 This is due to the covalent crosslinking bonds present in vulcanized rubber, which makes the rubber materials difficult to remove naturally or decompose.

Owing to the challenges faced with waste management of vulcanized rubber materials, the process of devulcanization is an important topic in the rubber industry, as one of the potential approaches to recycle rubber waste and is of great value and demand to date. Vulcanized rubber contains mono-, di- and polysulfide crosslinking bonds in its three-dimensional structure.33 These bonds are one of the weakest in diene rubbers, responsible for oxidative degradation.34, 35 Thus, during devulcanization, it is best preferred that the C-S and S-S bonds be cleaved, as the process is the reverse of the vulcanization process, which involves the formation of the C-S and S-S bonds. Generally, devulcanization is a process performed either physically, chemically or biotechnologically.31 In this study, chemical devulcanization using organic devulcanizing agents is employed. Common devulcanizing agents include diamyl disulfide, 2-thionaphthol, 2-amino-diphenyl disulfide, diamyl disulfide, dibenzyl disulfide, diallyl disulfide, and diphenyl disulfide.17-19 During devulcanization, these agents have the ability to break the sulfur bridges that form crosslinks, thus deactivating the reactive fragments that may lead to mingling the devulcanized products with additives and the virgin rubber. Hence, devulcanization is a method used to aid the recycling of discarded polymeric materials for re-use, particularly scrap tyres.

In industry, the devulcanization of rubber is performed using commercially available devulcanizing agents such as dially disulfides, which are often expensive. Nevertheless, another suitable and inexpensive method has been found in the extraction of disulfides from plant-based material rich with sulfur compounds, e.g., Allium sativum (garlic). De and colleagues (1999) reported a reclamation study on

2 styrene-butadiene rubber (SBR), using renewable resource material – a vegetable product, having diallyl disulfide as major product for cleaving of the crosslink bonds in cured rubber.17 In a more recent study, Sonti (2017) evaluated the crude extracts of T. violacea (RRM), also rich with sulfur compounds, as devulcanizing agents by reacting them with vulcanized model compound, 2,3-dimethyl-2-butadiene (TME).36 Because RRM's are found naturally compared to commercial agents that are expensive, they are found to lead to the advancement of inexpensive devulcanizing agent sources.

For decades, plant species have been used as a source for a variety of purposes, including use as a food source, for building shelter, as clothing material, and as traditional medicine.37 To date, rural dwellers still depend on indigenous plants as a primary source for medicinal purposes. These plants are easily accessible and affordable than the more expensive and scarce synthetic drugs in those areas. However, because no proper documentation has been done, the knowledge about traditional medicine is at risk of being lost, as it is only verbally passed on from generation to generation.38 Traditional medicines have gained much popularity through the years, playing a significant role in drug development and pharmacological studies due to the beneficial, decorative and medicinal significance.39

Scientific research for new drug development and modification, in concoction with medicinal plants, uses the biologically active secondary metabolites found in these plants40. These include compounds such as the alkaloids. Since the attention of scientific interest was drawn to medicinal plants, alkaloids became the cornerstones of many aspects of drug discovery today.40, 41 In a study by Lewis and O’Neil (1993), they postulated that half of the best pharmaceuticals in 1991 are based on a natural product precursor or phamacophore.42 These phytochemicals are famously known for their defensive role to preserve plants from harmful microorganisms such as bacteria and fungi.43 Also known as non-essential micronutrients, phytochemicals as secondary metabolites found in plants can minimize chronic diseases and maintain the wellbeing of patients. Their classification depends on the nature of the compound – chemical composition, structure and miscibility in different solvents.

Generally, there is not much work published regarding the use of plant-based sulfur compounds as devulcanizing agents. Studies concerning plant-based sulfur compounds have been mostly performed on Allium sativum (garlic), a member of the 3 family Alliaceae, which has been proven to be rich of sulfur-derived compounds and has good medicinal properties.2, 6, 44-46 Tulbaghia is a genus from the family Alliaceae, consisting of species that are closely related to species belonging to the Allium genus. Tulbaghia violacea Harv is the most famous species of the genus Tulbaghia, previously examined in pharmacological studies to test the potency of the sulfur compounds, showing good activity for use in concoction with pharmaceuticals due to the high sulfur content contained in the extracts employed. Consequently, Tulbaghia violacea has been chosen in this study to extract sulfur compounds, which in-turn will be evaluated for their effectiveness as potential devulcanizing agents.

1.1. AIMS AND OBJECTIVES

The aims and objectives of this study are outlined below:

• To extract sulfur-derived compounds of Tulbaghia violacea using sequential extraction and, to perform characteristic studies of the crude extracts using chromatographic and spectroscopic methods such as TLC, HPLC and FTIR. • To vulcanize the model compound squalene and perform characteristic analysis of the reaction products. The techniques TLC, PTLC, FTIR, NMR, HPLC, GPC, TGA and DSC are methods of choice to be employed for characterizing the vulcanized products. • To examine and compare the effectiveness of the of Tulbaghia violacea extracts as potential devulcanizing agents, using TLC, PTLC, FTIR, NMR, HPLC, GPC, TGA and DSC as methods of choice for characterization purposes of the devulcanized products.

CHAPTER 2:

LITERATURE REVIEW

2.1. THE FAMILY ALLIACEAE

The Alliaceae family is a large family consisting of 30 genera and approximately 600 species. As a family that is taxonomically intermediate between the Liliaceae and Amaryllidaceae, Alliaceae, is widely spread in Asia, Mediterranean Europe, Northern and Southern America, and Southern Africa. In Sub-Saharan Africa, three genera are endemic, namely; Allium, Agapanthus and Tulbaghia.44 Of the three endemic genera, the genus Allium is most popular, made famous by its species’ use as food plants, including A. sativum (garlic), A. cepa (onion), A. schoenoprasum (chives), and A. porrum (leeks). Kourounakis and Rekka reported that, though not all members of Allium are of the same flavor, they all have strong onion-like characteristics.47

Species belonging to the Alliaceae family are identified by the sulphur-containing volatile chemical compounds that give rise to their alliaceous odour. Upon tissue disruption, precursors such as the sulphur nonprotein amino acids (S-alk(en)yl cysteine, S-oxides or N-oxides) are formed from the volatile compounds.44 These amino acids are then enzymatically cleaved, resulting in the formation of sensory active compounds (including pungent thiosulfinates [RS(O)SR’] and lachrymatory sulfines (RCH=S=O)) that give rise to the onion- or garlic-like smell.48, 49

In this study, however, attention is mostly drawn to species belonging to the genus Tulbaghia which is endemic and indigenous in South Africa. Scientific research about some Tulbaghia species has not yet been done, though previous investigations that were done on the other species have revealed the genus to be rich in sulphur- containing compounds, which are accelerators of the alliaceous odor of Allium, Agapanthus and Tulbaghia species.50

2.2. THE GENUS TULBAGHIA

The genus Tulbaghia (also known as the wild garlic or society garlic) is a member of the family Alliaceae, named after the one-time Dutch governor of the Cape of Good

Hope, Ryk Tulbagh (1699-1771).49, 51 The genus consists of the highest number of species belonging to the family Alliaceae. On a worldwide spec, the genus compromises of approximately 85 species, where The World Checklist of Selected Plant Families (WCSP) reported only 31 of the species to have been accepted for scientific naming.52, 53

A large population of the species of Tulbaghia is widely distributed in countries in Asia, North and Southern America, Mediterranean Europe and Southern Africa. The distribution of Tulbaghia is extended entirely in Africa, from the Namaqualand through Eastern and Western regions into the Southern parts of Tanzania, in Southern Tropical Africa.

Endemic to the Southern African countries, Tulbaghia species are mostly found in countries including Botswana, Lesotho, South Africa.54 In South Africa, the genus is widely spread across the Eastern Cape, Southern KwaZulu-Natal, Gauteng, Limpopo, and Mpumalanga.54 Vosa et al. (2000) recorded a geographical distribution survey that indicated that the Eastern Cape is the centre of speciation for Tulbaghia species.21

Table 2.1: Shows plant species in the genus Tulbaghia.21-25

Species Common name Tulbaghia pretoriensis Wild garlic Tulbaghia acutiloba Harv. Wild Garlic, Wildeknoffel [Afrikaans], sefothafotha [Southern Sotho], lisela [Swazi], isihaladi lezinyoka [Zulu] Tulbaghia aequinoctialis Welw. Ex Baker Wild garlic Tulbaghia affinis Link Wild garlic Tulbaghia alliacea L.f Wild garlic Tulbaghia bragae Engl. Wild garlic Tulbaghia calcarea Engl. and K.Krause Wild garlic Tulbaghia cameronii Baker Wild garlic Tulbaghia capensis L. Wild garlic, Wildeknoffel Tulbaghia coddii Vosa and Burb. Wild garlic Tulbaghia cominsii Vosa Wild garlic Tulbaghia dregeana Kunth Wildelook, Ajuin [Afrikaans]

Tulbaghia friesii Suess Wild garlic Tulbaghia galpini Schltr. Wild garlic Tulbaghia hypoxidea Sm. Wild garlic Tulbaghia leucantha Baker Wild garlic, sefothafotha [South Sotho] Tulbaghia ludwigana Harv. Scented Wild garlic Tulbaghia luebbertiana Engl. and Wild garlic K.Krause Tulbaghia macrocarpa Vosa Wild garlic Tulbaghia montana Vosa Wild garlic Tulbaghia natalensis Baker Sweet Wild garlic, Iswele lezinyoka [Zulu] Tulbaghia nutans Vosa Wild garlic Tulbaghia pauciflora Baker Wild garlic Tulbaghia rhodesica R.E.Fr. Wild garlic Tulbaghia simmleri P.Beauv. Wild garlic Tulbaghia tenuior K.Krause and Dinter Wild garlic Tulbaghia transvaalensis Vosa Wild garlic Tulbaghia verdoornia Vosa and Burb. Wild garlic Tulbaghia violacea Harv. Wild garlic, Wildeknoffel [Afrikaans], Isihaqa [Zulu] Tulbaghia x alliaceae Vosa Wild garlic

2.3. TULBAGHIA VIOLACEA

Tulbaghia violacea is the most well-known species in the genus Tulbaghia, which consists of approximately 85 species. The plant is classified by several names including wild garlic, wildeknoffel (Afrikaans), Itswele lomlambho (Xhosa), Icinsini (Zulu) and Mothebe (Sesotho).55, 56 It is a small fast-growing bulbous plant that grows to a height of about 50 cm, growing indigenously in rocky grasslands of the Southern African regions such as the KwaZulu Natal (KZN), Gauteng, and the Eastern Cape (EC), where it is used mostly as a medicinal plant in both KZN and EC.57 The plant has evergreen, narrow, hair-less, and strap-shaped leaves that grow to a height of 30 cm and 1.5 cm wide. Upon tissue rupture, the leaves exhibit a characteristic smell like that

7 of garlic, hence in some case, the leaves are used as a substitute for garlic and chives.57 Amongst other species in the genus, T. violacea is identified by the flowers that form umbrels of up to 20 violets, developing during the period from December to April, in the summer. 55

(a) (b)

Figure 2.1: Shows (a) T. violacea plant, (b) Bulbs, (c) Roots and (d) Leaves

T. violacea is a species that has gained much popularity through its existence for several uses, mostly famous for its use as traditional medicine. To date, the bulb of the plant is traditionally used to treat a variety of ailments and diseases including fever and colds, asthma, tuberculosis, and gastrointestinal ailments.6 In the Transkei, Xhosa diviners apply the blubs of T. violacea on their bodies as protection against evil spirits before ritual dancing.50 The leaves are famously used as food culinary herbs, being

8 applied as hot peppery when cooking meat stew, or as a vegetable spinach.48 Furthermore, in some South African cultures such as the Zulus and Xhosas, T. violacea is planted around homes as a snake repellent because of the pungent smell it exhibits.48 However, though T. violacea could have good medicinal properties for use as traditional medicine, too much consumption of it could lead to undesirable side effects including gastroenteritis, inflammation, and abdominal pain.1 These side effects are due to the high sulfur and steroidal saponin content present in the plant.58

S S S S O

S S S S 2,4,5,7-tetrathiaoctane O

2,4,5,7-tetrathiaoctane-2,2-dioxide

Figure 2.2: Examples of harmful compounds upon consumption of T.violacea.1

Bungu et al. (2008) hypothesized that because T. violacea belongs to the same family as A. sativum, the two species possibly have indistinguishable medicinal properties.58 They further postulated that because the two species have the same alliaceous odor, they may have similar phytochemicals, which are in-turn also responsible for the species’ medicinal properties.58 A previous study showed evidence of sulfur compounds corresponding to those of Allium to be present in Tulbaghia, further suggesting that the alliaceous scent exerted is most likely to be due to the similar sulfur compounds found in the two genera.59

2.3.1 Pharmacological studies of Tulbaghia violacea Pharmacological investigations of Tulbaghia violacea has improved over the years. In a study by Burton (1990), she performed a biological study where T. violacea extracts were tested for antibacterial properties on normal muscle contraction. Isolated smooth muscle preparations were treated with T. violacea extracts, indicating the presence of a β-adrenergic agonist having an inhibitory effect on normal muscle contraction. It was suggested from the investigation that the extract(s) obtained from Tulbaghia violacea may be used as an antibacterial agent(s).1 Buwa and Afolayan (2009) tested the 9 aqueous and organic (ethanol and dichloromethane) extracts of Tulbaghia violacea dry bulbs, screening the antibacterial activity of the extracts. The extracts were screened for activity against Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Staphylococcus and Mycobacterium aurum A+ strain, using a two-fold microdilution bioassay.60 The M. aurum strain used is tuberculosis (TB) related strain. From the results obtained, it was concluded that the DCM extract of T. violacea showed the best Minimum Inhibitory Concentration (MIC) values against all the tested bacteria, except for K. pneumoniae.60 The MIC (mg/ml) results for the DCM exctract were; B cereus =

0.780, E. Coli = 0.780, K. pneumoniae = 6.250, S. aureus = 0.780. and M. aurum = 0.780. No activity was reported for the aqueous extract against M. aurum.60

In another study, Ncube et al. (2012) did an in vitro study where they investigated the antimicrobial efficacies of independent and various with-in plant extract combinations of T. violacea. The organic and aqueous extracts of the dried bulbs and leaves were assessed for antimicrobial activity against two gram-positive and two gram-negative bacteria, and Candida albicans using the microdilution method.61 Assays were carried out independently for each extract and synergistically, by combining extracts and testing them for their combined effect. The dried leaves and bulbs were extracted using the organic solvents; petroleum ether (PE), ethanol (EtOH), dichloromethane (DCM), and water (H2O), which were then screened against Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus.61 The independent assays results showed the DCM extracts of T. violacea to have good antibacterial activity against all bacterial strains, excluding Escherichia coli. The MIC’s (mg/ml) for the DCM extracts of the dried bulbs were; B. subtilis = 0.8, E. coli = 3.1, K. pneumonia = 0.8, and S. aureus = 0.8, while that of the dried leaves were; B. subtilis = 0.8, E. coli = 3.1, K. pneumonia = 1.6, and S. aureus = 0.8. The synergistic assays containing the DCM extract showed high activity against almost all bacterial strains for both the bulb and leaf extracts’ combinations, i.e.: PE/DCM, DCM/EtOH, PE/DCM/EtOH, PE/DCM/H2O and DCM/EtOH.61 The recorded MIC’s are tabulated below.

Table 2.2: Antibacterial activity (MIC mg/ml) for synergisitic combinations of the DCM extract of T. violacea.

Plant Plant Bacterial MIC (mg/ml)

part strain P/D P/D/E P/D/W D/E D/W D/E/W P/D/E/W

T. Bulb Bs 0.4 0.1 0.3 0.4 0.8 0.5 0.8 violacea Ec 0.4 0.3 0.3 0.4 0.80 0.5 0.4 Kp 0.8 0.3 0.3 0.4 0.80 0.5 0.4 Sa 0.4 0.3 0.3 0.8 1.6 1.0 0.4 Leaves Bs 0.4 0.3 0.3 0.4 0.8 0.5 0.4 Ec 0.4 0.3 0.5 0.4 0.8 1.0 0.4 Kp 0.2 0.3 0.3 0.2 0.8 1.0 0.2 Sa 0.4 0.3 0.3 0.4 1.6 0.5 0.4

Moodley et al. (2013) assessed the methanol extract of T. violacea for potential against cardiovascular-related diseases. The extract was tested for anti-hypertensive activity against Dahl salt-sensitive laboratory rats in an in vivo study, where captopril was used as the control sample.62 From the assay response, the methanolic extract from the leaves of T. violacea showed positive anti-hypertensive activity.62 The authors further postulated the positive effect to be maybe a secondary effect to a direct negative chronotropic effect on the heart muscle.62

2.3.2 Bioactive components of T. violacea T. violacea is one of the species belonging to the family Alliaceae that has been found rich with sulfur-containing compounds. These constituents are responsible for the characteristic smell and medicinal properties of Tulbaghia and Allium species. Upon tissue rupture of garlic (Allium sativum) and related Alliums, a garlic flavor is released as a result of the enzyme alliinase that hydrolyzes the S-alka(en)yl-L-cysteine sulfoxides (e.g. alliin) to form pyruvate, ammonia, and sulfur-containing volatiles.2, 3

O NH2

CO2H Alliin

Figure 2.3: Major compound alliin found in garlic.2, 3

Alliin is a major sulfur-containing compound within intact garlic bulbs. It is the primary odourless, sulfur-containing amino acid and a precursor of the thiosulfinates; allicin, methiin, (1)-S-(trans-1-propenyl)-L-cysteine sulfoxide, and cycloalliin, which are formed by enzymatic conversion of alliin (figure 4) upon crushing of the garlic bulb.4, 63

O NH2 Allinase S S OH X2, -H O S S 2 CO2H Alliin 2-propenesulfenic acid O Allicin

Figure 2.4: Enzyme-mediated formation allicin from alliin.4

As seen from figure 2.4, the disruption of garlic bulbs causes an enzymatic reaction between the cysteine sulfoxide and the enzyme allinase to take place, thus forming a highly reactive intermediate sulfur-substituted sulfenic acid.4 The sulfenic acid intermediate thus undergoes self-condensation forming thiosulfinates, mainly allicin, which is a very volatile and unstable.4, 64, 65

Thiosulfinates are very unstable compounds that undergo rapid decomposition, resulting in further rearrangements that lead to a variety of sulfur-derived compounds.5, 64 These newly arranged compounds are shown in figure 2.5 including thiosulfonates (1 and 2), di- and tri- sulfur compounds (3 – 8), vinyldithiines (9 and 10), ajoenes (11) and polysulfides (12 and 13).4

O O O O S S S S 1 2

S S S S S S S 3 4 5

S S S S S S S 6 7 8

S S S S S

S O S

9 10 11

S S S S S S

12 13

Figure 2.5: Organosulfur compounds formed by degradation of thiosulfinates.5

Unlike the closely related Allium species, whose chemistry has been widely studied, only a few scientific articles that describe phytochemicals on the species belonging to Tulbaghia have been published. Mostly reported are studies on the species T. violacea. The first publication was by Jacobsen et al. (1968) where they proposed the presence of a carbon-sulfur lyase and three unidentified S-substituted cysteine sulfoxide derivatives in their methanolic extract.59 The authors further suggested that the C-S lyase can act similarly as that of the allinase on L-cysteine sulfoxides described in the allinase reaction in Allium species.59 Burton (1990) performed a chemical investigation 13 on T. violacea where two flavonols; kaempferol and quercetin were isolated.1 In a further study, Burton and Kaye (1992) isolated the two compounds found to be harmful upon too much consumption; 2, 4, 5, 7-tetrathiaoctane-2,2-dioxide and 2, 4, 5, 7- tetrathiaoctane (figure. 2).66 Kubec et al. (2002) performed a study where studied the amino acid precursors and odor formation of T. violacea. They isolated 2, 4, 5, 7- tetrathiaoctane-4-oxide (marasmicin) for the first time and identified the three unidentified cysteine derivatives suggested by Jacobsen et al. (1968); S- (methylthiomethyl)-cysteine-4-oxide, S-methyl cysteine and S-ethyl cysteine derivatives.6 See figure 2.6 below.

NH2 NH2

S OH S OH

O O S-methyl cysteine S-ethyl cysteine

O NH2

S S OH

S-(methylthiomethyl)-cysteine-4-oxide

Figure 2.6: Cysteine derivatives.6

2.4. VULCANIZATION

Rubber polymers are made up of long entangled hydrocarbon chains that are randomly coiled and not chemically bonded. These chains are thus capable of untangling when subjected to an applied force, resulting in the deformation of the polymer's physical properties. Consequently, even upon removal of this force, the rubber polymer loses

its original shape due to chain slippage and alignment of the unbonded chains. However, in 1839 Charles Goodyear developed the process named vulcanization to overcome this deformation.67 Vulcanization is simply the process in which, in the

presence of sulfur (S8), crosslinks are introduced between polymer chains to form a three-dimensional network, preventing chain slippage and alignment (Figure 2.7). Chemical cross-links result in increased elasticity, low plasticity, and eliminates insolubility characteristics of rubber polymers.68

CH3 S CH3 H2 H2 H2 H2 H2 H2 C C C C C C C C C C * H H S S S S S CH3 H3C H3C H CH3 H H2 H2 H2 H2 H2 S S * C C C C C C C C C C C C C C H H H + S S * C H2C C H2C * S H2 H2 n S S S S CH3 H3C H3C H2 H2 H2 H2 C C C C C C C C C C * H H H n S

Figure 2.7: Representation of sulfur vulcanization crosslink formation

Rubber vulcanization that uses sulfur only is a slow and inefficient process. This called for urgent development of new vulcanizing methods, which include accelerated sulfur, peroxide, and polyfunctional coupling agent vulcanization.16 The method popularly used to date is that of accelerated sulfur vulcanization. It gained popularity because of low-cost processes, high ability of sulfur to react with other additives, and improved physical properties of the vulcanizate.16

The curing (vulcanization) process, in the presence of curing agents, is itself a process that takes place by three distinct phases: (i) the induction/scorch phase, (ii) the curing period or crosslink formation phase, and (iii) maturation or modification period.7, 69 The induction period is where the interaction of reciprocal elements that lead to crosslink formation starts to occur. In the case of accelerated sulfur vulcanization, the induction period is affected by the presence of vulcanizing agents: accelerators and vulcanization retarders or pre-cure inhibitors.7 In the second stage, crosslinks between rubber molecules are formed at the rate which the vulcanizing agents affect vulcanization and system. Finally, in the third stage restructuring of formed crosslinks and modification of rubber chains occurs.7 The course of the vulcanization process is measured on different types of rheometers, using the means of vulcanization curves to evaluate the process and its characteristics (Figure 2.8).7

Figure 2.8: A typical Rheometer curve showing the three phases of vulcanization.7

Accelerated sulfur vulcanization and physical properties of the vulcanizate(s) are improved by the presence of organic compound additives termed accelerators.7, 16 Accelerators are the most important additives among others, and their addition results in less sulfur content required for vulcanization, reduced reaction time, and low processing temperatures.7, 69 Also, accelerators increase the curing ratio and 16 effectiveness of sulfur to bond to rubber molecules.7 Most accelerators contain a Nitrogen or Sulfur atom in their chemical structure, are classified and divided according to their chemical composition. Depending on the rubber polymer to be vulcanized, a specific accelerator is chosen for different rubber polymers, depending on the nature of the rubber polymer to be vulcanized and vulcanization conditions. The same accelerator may be used for vulcanizing two or more rubber compounds but produce different results for each.7

Table 2.4: shows some commonly used accelerators and their efficiency.7

Name and abbreviation Efficiency Thiazoles 2-Mercaptobenzothiazole (MBT) Fast, frequently used accelerator; it is used preferably in combination with other accelerators Bis-benzothiazole-2,2’-disulfide (MBTS) Fast accelerator with a slightly delayed start Sulfenamides N-Cyclohexyl-2-benzothiazole Fast accelerator with scorch delay, used sulfenamide (CBS) also in semi EV and EV systems N-Tert. butyl-2-benzothiazole Accelerator with shorter scorch delay sulfenamide (TBBS) and lower cure ratio than CBS Thiurams N,’N-Dipentamethylenethiuram disulfide Very fast accelerator used also in semi (CPTD) EV and systems. Also acts as sulfur donor N,’N-Dipentamethylenethiuram A very fast accelerator also used in semi monosulfides (CPTM) EV and EV systems

2.4.1 SULFUR VULCANIZATION REACTION SCHEME A generally accepted vulcanization system in the presence of sulfur and accelerators is shown in scheme 2.1 below. Vulcanizing ingredients: Sulfur,

Accelerators, Activators

Rubber Hydrocarbon Active sulfurating agent (R-H)

Rubber-bound

intermediate, pendent

group (R-SM-X or R-SM-H)

Initial polysulfidic

crosslink formation.

 Crosslink shortening with additional crosslinking  Crosslink destruction with main

chain modifications  Sulfur-sulfur bond interchange

Final crosslinked network

Aged network vulcanizate

Scheme 2.1: Generally accepted vulcanization mechanism.26

In the first step, a reaction between sulfur, accelerator, and activators occurs to form active sulfurating agents. These agents then react with the rubber hydrocarbon at the α-methylinic or α-methylic hydrogen, forming a rubber bound intermediate pendent group. Pendent groups are crosslink precursors made up of an accelerator and a rubber fragment, which are bound by two or more sulfur atoms.69, 70 The pendent groups then react with each other to form initial polysulfidic crosslinks that are normally long chains that are thermally unstable. This instability results in thermal degradation reactions, thus resulting in shorter crosslinks by the loss of released sulfur atoms, that are in turn used to form new crosslinks. Furthermore, polysulfidic crosslinks are also subject to main chain modifications and S-S bond interchange before the final product vulcanizate.16 In addition, main chain modifications result in cyclic sulfides and conjugated double bonds being formed, where under thermal decomposition of crosslinks cyclic mono- and disulfides are formed, see figure 2.9.16, 69, 70 Van Der Host and Woolard (2001) suggested that conjugated diene and triene groups in a vulcanized network to be a result of the dehydrogenation of the rubber chain16.

S2 S3

Sy S X SX

Figure 2.9: Sulfur vulcanized network8.

2.4.2 MODEL COMPOUND VULCANIZATION The crosslinks formed during rubber compound vulcanization cause the rubber compound to be insoluble. This is a result of the 3-dimensional crosslinked network that strengthens the rubber, converting a weak permanently deformable rubber compound into a highly elastic and deformation-resistant material, called an elastomer.71 These products are hard to analyze by analytical techniques leading to various approaches to study rubber compound vulcanization, where the method of

19 model compound vulcanization became most popular. Examples of model compounds used to mimic the monomer unit of rubbers are shown in figure 2.10.

2-methyl-2-pentene trans-2-hexene Cyclohexene

1-decene 5-decene

cis-3-hexene cyclo-1,5-octadiene

2,3-dimethyl-2-butene

squalene

Figure 2.10: Examples of common model compounds.9-15

A model compound is simply defined as a low molecular organic molecule with a chemical structure that mimics the monomer unit of rubber69, 70. In model compound vulcanization, the low molecular olefin is reacted with sulfur and other curing agents to 20 study their effectiveness as vulcanizing agents. The reaction products that are formed from these reactions are thus easily analyzed by analytical techniques such as TLC, PTLC, HPLC, GC, GPC, MS, and NMR. However, though most model compounds result in simple and analyzable products, they do not allow for reactions such as cyclization, main chain modification and double bond migration to take place.16, 70 Thus, to overcome such shortcomings, more complex model compounds such as squalene are employed. Nevertheless, model compounds such as squalene lead to complex reactions, and because of their complex structure, it makes them less-desirable than model compounds such as 2,3-dimethyl-2-butene (TME), which contains one equivalent allylic hydrogen position (figure 2.11) due to the symmetry of the compound. Furthermore, reactions and products obtained from TME are greatly simplified, reducing the number of reaction products and intermediates to be analyzed.

CH3 CH2 H3C CH3

H2C H H3C CH3

2,3-dimethyl-2-butadiene Isoprene monomer unit

Figure 2.11: Structural similarity of 2,3-methyl-2-butene (TME) and isoprene monomer unit in polyisoprene. Equivalent allylic hydrogen positions are shown on TME16.

However, though squalene may have a complex structure and may lead to complex reactions, it better mimics the complex structure of natural rubber when compared to TME. Sonti (2018) performed a study on TME, where she used wild garlic plant extracts as devulcanizing agents.36 She postulated that wild garlic plant extracts have effectiveness as potential devulcanizing agents.36 Hence in this study, squalene has been used to study the effectiveness of wild garlic plant extracts as potential devulcanizing agents of vulcanized squalene.

2.5. DEVULCANIZATION

The vulcanization of rubber by sulfur results in the formation of sulfur bridges between rubber molecules, to afford an elastic three-dimensional network. These sulfur bridges thus increase the strength of rubber materials, making them strong and resistant against natural decomposition upon disposal.31 This resistance leads to increased amounts of rubber waste, especially rubber tires, which in turn increases the environmental, social, and economic challenge.31, 72

Arthur H. Marks (1899) was the first to report an advancement past the problem(s) faced with vulcanized rubber. He invented a process of reclaiming rubber from rubber waste known as the alkali digester process.73 In his study, ground rubber waste is submerged in alkaline dilute caustic soda in a tightly sealable vessel. The vessel and contents were exposed to high temperatures (173 – 187 oC) for as long as 20 hours.73 At this temperature and time for the experiment, he concluded to have obtained devulcanized rubber by desulfuration and eliminated fabric matter that was contained in the rubber waste. He further postulated that the devulcanized rubber had the same characteristics as fresh rubber and could be used in the same manner for like purposes.73

Waste tyre rubber disposal is one of the major environmental pollutants, since its invention, devulcanization has been of great importance to the rubber industry in overcoming the problems that are faced with rubber tires disposal.74 To date, the devulcanization process is a well-known and most used method for rubber vulcanizate reclamation. The process involves chemical and physical activity to break the intermolecular carbon-sulfur and sulfur-sulfur bonds formed between rubber chains without damaging the carbon backbone.74-76 This breakage results in the formation of polymer chains of shorter chain lengths and rubber products of poorer properties, as compared to the original rubber vulcanizate.74

Generally, devulcanization is a method that is carried out following one of three processes; (1) a physical, (2) chemical, or (3) a biotechnological process must take place.31, 74 The physical process involves devulcanizing of rubber waste in the presence of an external energy, including mechanical,77 cryomechanical,78 microwave79 or ultrasonic.80 However, these processes result in unfavorable random

22 cleaving of crosslinks and polymeric main chain bonds, which in turn results in poor quality rubber vulcanizates upon revulcanization.31 In the chemical process, devulcanization is carried out using organic and inorganic chemical compounds. Commonly used compounds are the organic disulfide compounds (see figure. 2.12) and thiols. Rubber compounds are normally initially swollen in organic solvents such as toluene, cyclohexene and benzene before reclaiming. However, these solvents are difficult to remove after the completion of the reaction. Kojima et al. (2005) studied the efficacy of supercritical CO2 as a swelling solvent for the reclaiming of unfilled and carbon black-filled polyisoprene rubber vulcanizates.81 They postulated that the residual CO2 was easily and rapidly removed from the rubber matrix upon pressure release.81 Nevertheless, because of the use of chemicals, the major challenge faced by the chemical process is the high economic and environmental costs. In the biotechnological process, rubber powder is biodegraded through oxidation-reduction reactions that are catalyzed by species of Thiobacillus, Acidithiobacillus, Narcadia, and more.82-86 In this process, the rate and effect of devulcanization is a function of particle size, thus limiting the process to the surface layers of the rubber polymers.86

S H2N S S NH2 S

diphenyldisulfide 2-aminodiphenyldisulfide

S S S S diallyl disulfide diamyl disulfide

S S S

2-thionaphthol dibenzyl disulfide

Figure 2.12: Organic compounds used as devulcanization agents.17-19

Jana et al. (2005) performed a study where they made use of two different molar concentrations of diallyl disulfide as a devulcanizing agent, in the devulcanization of gum natural rubber. Devulcanization was carried out in a two-roll mixing mill at 110 oC for approximately 10 mins. The devulcanized rubber was revulcanized and both the properties of the devulcanized and revulcanized rubber were analyzed. The results showed a linear increase in devulcanizate properties with an increase in disulfide concentration and an increase of the mechanical properties of the revulcanized rubber with the decrease of sulfur content.30 Pipat et al. (2010) conducted a study where they made use of a mechano-chemical process to devulcanize natural rubber vulcanizates and revulcanized the devulcanized rubber.31 In their study, thiosalicyclic acid was used as a devulcanizing agent, in comparison to diphenyl disulfide as a reference, where revulcanization was achieved by making up composites of virgin natural rubber mixed devulcanized rubber at different ratios.31 Devulcanization was achieved by first grinding natural rubber vulcanizates at room temperature in a two-roll mill, which was then followed by treatment of the resulting rubber sample with the devulcanizing agent at 140 oC for 30 mins. The level of devulcanization was monitored by sol-gel fractions of the devulcanized rubber. The results showed the amount of the sol fraction % to be dependent on the amount of devulcanizing agent added and pretreatment time.31 The properties of the revulcanized rubber show a 5-10% decrease in tensile strength and a 5-10% increase in elongation at break at different amounts (0.5, 1.0, 2.0 phr) of thiosalicyclic acid as devulcanizing agent.31

Microwave devulcanization is one of the most efficient techniques of devulcanization, known for its ability to regain the fluidity of rubber, making it easy to regenerate and be revulcanized. The technique allows one to devulcanize rubber by applying high energies without the use of chemicals.79, 87 Zanchet et al. (2012) reported microwave devulcanization to increase the interfacial adherence between devulcanized styrene- butadiene (SBR) in a virgin SBR matrix. The results showed that the mixture can be used in the manufacturing of rubber products obtained by compression molding.88 In one study, Paulo et al. (2012) conducted a study where they used microwave devulcanization with the addition of inorganic salts and nitric acid. From their results, there was evidence that the presence of the salts and acid promoted both sulfur crosslink breakage and oxidation of SBR.89 Pistor and colleagues (2011) performed studies where they worked devulcanizing different ethylene-propylene-diene (EPDM) 24 polymers using microwave devulcanization. They reported that devulcanization by microwave irradiation promoted crosslink breakage for the different EPDM rubber polymers. They further suggested that the presence of additives and oil in EPDM rubbers have a positive effect during the devulcanization process.90

Boyce (2017) performed a study where the devulcanization of vulcanized natural rubber was studied by model compound vulcanization of TME and squalene. In the study, she made use of several diphenyldisulfides as devulcanizing agents; 2- aminodiphenyldisulfide, 4-aminodiphenyldisulfide, bis(2-benzamidophenyl) disulfides and 2.2'-bithiosalicyclic acid.91 The model compounds were vulcanized with sulfur as curative, in the presence of 2-bisbenzothiazole-2-2’-disulfide (MBTS) and N- Cyclohexyl-2-benzothiazyl sulfenamide (CBS) as organic accelerators. Vulcanization and devulcanization products of the model compounds were monitored by a variety of methods of analysis including HPLC, GPC, Differential scanning calorimetry (DSC), Thermogravimetric analysis (TGA) and Infrared Spectroscopy (IR). The results from the study showed 4-aminodiphenyldisulfide to have a greater devulcanizing efficiency compared to the other devulcanizing agents.91

In a more recent study, Sonti (2018) performed a three-stage study or the devulcanization of model compound TME. In the first part of the study, Tulbaghia violacea (wild garlic) was extracted for diallyl sulfides and other sulfur-containing compounds to use as devulcanizing agents. Crudes were extracted from the plant parts (bulbs, roots, and leaves) by Soxhlet extraction using a mixture of organic solvents, n- Hexane and isopropyl alcohol (98:2).36 The second part involved the vulcanization of TME with sulfur in the presence of MBTS. Vulcanization was carried out on a hot plate with continuous stirring at 140 oC for 60 minutes. In the last stage, TME vulcanized products were devulcanized using the sulfur-containing extracts as devulcanizing agents. The concentration of diallyl disulfide extracted in the bulbs, roots, and leaves was quantified on normal phase HPLC and was found to be 7.74x10-2 mg/mL, 2.93x10- 2 mg/mL and 3.69x10-2 mg/mL respectively36. RP-HPLC was the method of analysis used to monitor the degree of vulcanization and devulcanization. The crosslinking products were detected on the chromatogram, eluting in order of increasing sulfur ranks for the vulcanized products, where the di- and polysulfidic crosslinks were not detected for the devulcanized products.36 The results showed the extracts from the

25 bulbs and leaves to have a greater devulcanizing efficiency than that of the roots. Sonti further suggested that extracts of T. violacea containing sulfur-derived compounds can be used as alternative devulcanizing agents to petroleum-derived agents.36

CHAPTER 3:

EXPERIMENTAL METHODS AND MATERIALS

3.1. REQUIREMENTS

3.1.1. Chemicals and solvents The chemicals and solvents (Table 3.1) used in this study were purchased from local and commercial suppliers.

Table 3.1: lists the chemicals and solvents used in the study

Chemical % Purity Supplier n-Hexane 98.0 Merck Chemicals Ethyl acetate 98.0 Merck Chemicals Ethanol 99 Sigma Aldrich Methanol 99.9 Merck Chemicals Chloroform 99 Merck Chemicals Dichloromethane 99.0 Merck Chemicals Triethylamine 99.0 Merck Chemicals Deuterated chloroform 99.8 Merck Chemicals4

Toluene 99.8 UniLAB Acetone 98 UniLAB Conc. Sulfuric acid 98.0 UniLAB

Diallyl Sulfide 97 Sigma Aldrich Diallyl Disulfide 98 Sigma Aldrich Squalene 99 Sigma Aldrich

HPLC acetonitrile 99.9 Merck Chemicals HPLC water Merck Chemicals

3.1.2. Other materials Table 3.2: Lists the other materials used in the study

Material Supplier Tulbaghia violacea (plant species) NMU Horticulture Department, Port Elizabeth, South Africa. Voucher specimen number: PEU25230. Aluminum TLC plates (silica-coated) Merck Cotton wool Crown Parafilm Bemis Flexible Packaging Heavy-duty aluminum foil Bidvest Foods Glass ampoules (10 ml) Wheaton

3.1.3. Collection of plant material The plant species, T. violacea, was collected from the Nelson Mandela University (NMU) Horticulture Department. The plant species were harvested by Ms. Elana Storm and Miss Jennifer Clark from the university gardens. The fresh plant was immediately washed gently under cold running tap water to remove soil, dust particles, and other contaminants. After washing, a sample of the species was removed and was positively identified by the herbarium curator in the Nelson Mandela University Botany Department. The specimen voucher number recorded as PEU25230. Thereafter, the plant was dissected into its different components - leaves, bulbs, and roots. The plant components were then dried in an oven at 40 oC until dry.

3.2. INSTRUMENTATION

The instruments that were used in this study included HPLC, RP chromatography, NMR-spectroscopy, FTIR-spectroscopy, DSC and TGA.

3.2.1 High-Performance Liquid Chromatography (HPLC) HPLC experiments were conducted for the identification and analysis of crude extracts, also to study the reaction products formed during vulcanization and devulcanization. For this study, HPLC experiments were performed on an Agilent Technologies 1290

28 infinity HPLC unit, operated by an Agilent Chemstation software. The unit is fitted with an Agilent Binary pump, an Agilent infinity thermostat column compartment, and an Agilent infinity detector (Diode Array). The solvents used were HPLC grade, supplied by Merck.

3.2.2 Reverse-Phase Chromatography Two commercially available reverse-phase columns were utilized in this study for analysis by RP chromatography. A Phenomex Luna 5µ C18 (250 × 4.00 × 5µm) column was used for the separation compounds present in the crude extracts, and a Waters Symmetry C18 (250 × 4.00 × 5µm) column for the separation of reaction products. Detection was achieved at a wavelength of 254 nm. In this procedure, the stationary phase is non-polar and the mobile phase is generally more polar, resulting in the elution of polar compounds being weakly retained in the stationary phase and eluted first. The crude extracts were separated gradually, whilst the squalene reaction products were separated isocratically. The experimental procedures/conditions for crudes and reaction products are discussed in section(s) 3.3.3 and 3.3.5.1.

3.2.3 Gel Permeation Chromatography (GPC) Gel permeation chromatography is used to analyze the molecular weight distribution of reaction products and compounds. Molecular weight distribution was achieved by utilizing three High-Resolution Waters Styragel columns of varying pore sizes and effective molecular weight range (table 3.3). The pores act as molecular filters, where smaller molecules fit into the pores and be retained longer for a period while the larger molecules elute first.

Table 3.3: Styragel high-resolution columns used for GPC experiments

Column Pore size Å Effective MW (mAu) Styragel HR 0.5 0.5 0 - 1000 Styragel HR 1 1.0 1000 – 5000 Styragel HR 4 4.0 500 – 600 000

3.2.4 Nuclear Magnetic Resonance (NMR) NMR spectra of isolated compounds and reaction products of the vulcanizates and devulcanizates were recorded in deteriorated chloroform (CDCl3) solvent on a Bruker

Ultrashield Plus 400 MHz spectrometer. The data of the chemical shifts (δ) are expressed in parts per million (ppm). NMR spectroscopy experiments included: 1H and 13C. Data of the NMR spectra were reported relative to the internal reference standard tetramethylsilane (δ = 0.00 ppm). The 1H and 13C reference peaks were recorded at 7.26 ppm and 77.00 ppm respectively.

3.2.5 Fourier Transform Infrared Spectroscopy (FTIR) FTIR was used to characterize the functional groups present in isolated compounds of the crude extracts, vulcanized and devulcanized products. The IR spectra were recorded on a Bruker Platinum ATR Tensor 27 spectrometer fitted with a Bruker Platinum ATR containing a single reflection diamond crystal, and an Opus data collection program. The background scan was done at 16 scans while the samples were done at 32 scans at a scan velocity of 10 kHz/s.

3.2.6 Rotavapor A Buchi Rotavapor (R-210) equipped with a Buchi Heating Bath (B-491) was used for the removal of the extraction solvent and concentrating of the extracts. Solvent evaporation was performed under the aid of a slight vacuum supplied from a benchtop vacuum tap. The temperature of the bath was varied depending on the extraction, to 15 oC less than the boiling point of the extraction solvent.

3.2.7 Hot Plate Magnetic Stirrer Vulcanization and devulcanization samples of the model compound were prepared on a benchtop hot plate magnetic stirrer (Stuart-SD162, 20–350 oC, 200–1300 rpm). The reaction of the ingredients for vulcanization and devulcanization was aided using small magnetic stirrer bars.

3.2.8 Differential Scanning Calorimetry (DSC) DSC was used for the analysis of reaction products between curatives and model compounds. DSC experiments were conducted using a TA Instruments Discovery Series Differential Scanning Calorimeter. High purity nitrogen gas (AFROX, Johannesburg, South Africa), at a flow rate of 50 mL/min, was used as the purge gas. An empty sealed aluminum pan was used as a reference. Mercury (-37.43 oC), Indium (156.6 oC) and zinc (422.93 oC) was used for the temperature and heat flow calibration of the DSC cell. Calibration of the heat capacity was done using a sapphire crystal.

3.2.9 Thermographic Analysis A TA Instruments Discovery thermogravimetric analyzer controlled by Thermal Advantage software was used to determine product mass loss. High purity Nitrogen (AFROX) was used as the purge gas, at a flow rate of 25 mL/min. Calibration of the instrument was done using mass standards for mass loss determinations, and Curie points of Nickel (357.3 oC) and Iron (778 oC) were used for temperature.

3.2.10 Balances Analytical balance Mettler Toledo AB204-S (1×10-4 g)

Microbalance Mettler UM3 (1×10-7 g)

3.3. EXPERIMENTAL PROCEDURES

3.3.1. Plant extract preparation The collected plant material was thoroughly washed under cold running water, cut to separate the components (leaves, bulbs, and roots), and then oven-dried at 40 oC until dry. The dried components were further cut into smaller pieces and ground to a powder. The components were then individually weighed and extracted using solvents of differing polarity levels: chloroform, ethyl acetate, and methanol 1, 57, 66, 92, 93. Extraction was carried out overnight at room temperature by digesting each component in a solvent, maintained under continuous stirring on an overhead mechanical stirrer (Junke and Kunkel IKA-werk: RW-20). The solvent extract was then vacuum filtered to remove plant tissue using a Buchner funnel and cotton wool. The filtered extract was subjected to centrifugation for the removal of any residual matter a 1500 r.p.m for 10 minutes, then it was gravity filtered through a Whatman no. 1 filter paper into a round bottom flask. The solvent was removed from the extract on the rotary vapor under slight vacuum to afford the crude extracts. The masses and percentage yields of the extracts are presented in table 4.1.

Table 3.4: Mass of dry plant part weighed

Plant part Leaves Bulbs Roots Mass weighed (g) 98.3781 144.3284 69.2935

3.3.2. TLC and phytochemical test of extracts TLC is one of the quickest and easiest chromatographic methods, used for the identification of active compounds in an extract and reaction products. The dried crude extracts were completely dissolved in enough extraction solvent to obtain a homogenous liquid extract of workable concentration. 20µl aliquots of liquid extracts were applied 1 cm from the bottom of a UV-light active silica gel 60 F254 TLC plate against standard samples; diallyl sulfide and diallyl disulfide, prepared by dissolving 10.0 mg of each in 2mL dichloromethane. The plate was developed in a conventional glass tank containing 8:2 hexane: ethyl acetate solvent mixture as developing solvent. When the solvent front had traveled to 1 cm below the top of the plate, the plate was removed from the tank and dried in a fume cupboard and viewed under UV-light, both short and wavelength. For the identification of sulfur-derived phytochemicals not active under UV-light, the plate was then sprayed with a vanillin-sulfuric acid reagent, made up by dissolving 800 mg vanillin in 40 mL glacial acetic acid and 2 mL sulfuric acid. The sprayed plate was incubated in an oven (at 100 oC) for 10 minutes to obtain results for UV non-active phytochemicals.

3.3.3. Analysis of crudes by HPLC HPLC analysis of plant crude extracts was performed to separate the active ingredients present in the crude extracts. The samples were prepared by dissolving 0.05 g of extract in 2 mL extraction solvent, and further diluted with 3 mL HPLC grade methanol in a pill vial. The solutions were passed through 0.2 µm fibre filters into amber vials before injecting them into the HPLC unit.

In this study, RP-Chromatography was the method of choice utilized using an RP-Luna Phenomex column (section 3.2.1) packed with silica as stationary phase. The active ingredients were eluted by employing gradient elution using H2O: MeOH (95:5 – 0:100) -1 as the mobile phase at a constant flow rate of 1.0 mL.min . H2O was used as solvent A and MeOH was used as solvent B, and MeOH was also used as the wash solvent at the end of the analysis to rinse the column. The analysis was carried out at a constant temperature of 25 oC, with an injection volume of 1.5 µl and the results were detected at a wavelength of 250 nm on a diode array detector.

3.3.3.1. Preparation of diallyl disulfide (DADS) stock solution and standards The DADS stock solution was prepared by dissolving a pre-weighed amount of 98% pure DADS with methanol in a 10 mL volumetric flask. The resulting mixture was then made to the mark with methanol and was labeled as the primary standard. From the stock solution, further dilutions were prepared by 10× dilution to prepare reference standards of varying concentrations (see table 3.5). The standards were thus filtered through 0.2 µm fibre filters into amber vials before injecting them into the HPLC unit.

Table 3.5: Calculated concentrations of the standards

DADS Standard Concentration mol/L Standard 1 (stock solution) 5.84 x 10-3 Standard 2 5.84 x 10-4 Standard 3 5.84 x 10-5 Standard 4 5.84 x 10-6 Standard 5 5.84 x 10-7

3.3.3.2. Preparation of crude extract samples Extracts from the plant parts were weighed (0.05 g) into small pill vials and dissolved in 2 mL of HPLC methanol. The dissolved samples were then filtered by passing through 0.2 µm fibre filters into amber HPLC vials before injection into the HPLC unit. Table 3.6 below shows the amounts of crude weighed.

Table 3.6: Weighed masses of crude extracts for analysis by HPLC

Extract mass (g)

Plant part CHCl3 EtOAc MeOH Leaves 0.0563 0.0579 0.0573 Bulbs 0.0568 0.0547 0.0528 Roots 0.0595 0.0581 0.0558

3.3.4. Purification of MBTS To purify MBTS, the method used by Morgan et al. was facilitated, where MBTS was dissolved in boiling benzene (b.p. 80.1 oC) at temperatures between 79-81 oC. The

33 mixture was stirred to ensure a homogenous solution and was immediately by gravity filtration filtered through a filter paper for the removal of any undissolved material.94 NMR, HPLC, and IR were methods of analysis used to confirm the recovery of MBTS. See appendix A for the NMR and IR spectra of MBTS.

3.3.5. Purification of MBT MBT was purified similarly to that described for MBTS. The purity was confirmed by RP-HPLC, NMR, and IR analysis

3.3.6. Thermogravimetric Analysis (TGA) TGA was done to determine the mass changes that occur during the vulcanization of squalene. Likewise, the crude extracts of T. violacea were analyzed to determine the degradation stages relevant to the compounds present in each plant extract. Approximately 5 mg of each sample was directly placed in the TGA platinum cradle and heated at 10 oC min-1. The curatives and vulcanization products were heated up to 200 oC and the extracts up to 700 oC of temperature. This temperature for crude extracts was chosen since some of the compounds degrade at such temperatures and beyond.

3.3.7. Differential Scanning Calorimetry (DSC) Samples from the masterbatch were weighed on a microbalance, compacted in DSC pans and encapsulated by crimping the pans. The samples were then compressed to safeguard even heating of the whole sample and provide maximum surface contact between the samples and aluminum pans. Known amounts of each sample were then heated at 20 oC min-1 to 200 oC with a modulation time of 40 s and a modulation amplitude of 0.8 oC.

3.3.8. Vulcanization of model compound The sulfur vulcanization of squalene was conducted using the recipe and procedure by Rajan,18 Boretti,95 Mpahlele69 and Boyce.91 However, because squalene consists of six isoprene units, its molar ratio was adjusted as compared to 2,3-dimethyl-2-butene which consists of one isoprene unit. Squalene (2 mL) and appropriate amounts of curatives were mixed in thick-walled sealable glass ampoules containing a small stirrer bar. The mixture was then degassed by the aid of pumping in nitrogen to remove oxygen, to prevent bursting of the ampoules as the reaction takes place. Degassing was repeated three times, and the ampoules were sealed by applying heat to melt the 34 neck of the ampoule under slight vacuum to exclude excess air. Table 3.7 shows the recipe of the model compound and ingredients used, where sulfur (S8) was used as the curative, 2-bisbenzothiazole-2,2’-disulfide (MBTS) as the accelerator, and zinc oxide (ZnO) used as the activator.

Table 3.7: Squalene/S8/MBTS/ZnO mole ratio for Sulfur vulcanization

Ingredient Amount (mol)

Squalene 5.65

Sulfur 1.0

MBTS 1.1

ZnO 1.0

Vulcanization was performed by fully submerging the sealed ampoules in an oil bath maintained on a benchtop magnetic stirrer set at 150 oC. The reaction was carried out at different reaction times 5, 10, 15, 20, 30, 45 and 60 min. A calibrated thermometer was fitted with the setup to monitor the reaction temperature to be constant. When the reaction time was completed, the reaction was stopped by immersing the ampoule in liquid nitrogen. Figure 3.1 illustrates the vulcanization setup.

Thermometer

Glass ampoule

Oil bath

Hot plate magnetic stirrer

Figure 3.1: Setup of vulcanization

3.3.9. HPLC analysis of curatives and vulcanized products Samples for curative interactions were prepared by weighing the curatives into sealable glass ampoules and adding squalene. The ampoules were then immersed in a silicone oil bath set at 150 oC and continuously stirred until reaction time was reached. When the reaction time was reached, the ampoules were quenched in liquid nitrogen to stop the reaction. 30.0 mg of the samples were then dissolved in 5 mL of dichloromethane (DCM) and made to volume (10 mL) with HPLC grade acetonitrile. The solution mixture was passed through 0.2 µm fibre filters into amber HPLC vials before injecting into the

HPLC unit. The mobile phase consisted of acetonitrile (ACN) and water (H2O), which were degassed before use to eliminate any dissolved gases. The reaction products

were separated via isocratic elution using 97:3 ACN: H2O and were detected at a wavelength of 254 nm. The flow rate was held constant at 1.0 mL/min and the injection volume used was 20 µl to prevent degradation and further reactions to take place.

3.3.10. Gel Permeation Chromatography Reaction product samples for analysis by GPC were prepared similarly as to the RP- HPLC samples. 30.0 mg of each sample was dissolved in 5 mL of HPLC tetrahydrofuran (THF). The solution mixture was then filtered through 0.2 µm fibre filters into amber HPLC vials before injecting into the HPLC unit. The reaction products were thus distributed by isocratic elution using pre-degassed 100% THF as the mobile phase, detected at a wavelength of 254 nm. Molecular weight distribution was achieved by injecting 20 µL of the sample into the unit. GPC analysis was carried out at a constant temperature and flow rate, 25 oC and 1.0 mL/min, for 20 minutes.

Detection of pendent groups in MBTS accelerated sulfur vulcanization of squalene

Coran suggested that MBTS accelerated sulfur vulcanization occurs by pendent group formation.71 In a study by Boretti and Woolard (2002) the presence of pendent group formation in the vulcanization of squalene was detected by comparison of a squalene/sulfur (5.65:1) system and a squalene/sulfur/MBTS (5.65:1:1.1) system. From the study, pendent group formation was evident in the latter MBTS accelerated system, whereas in the squalene/sulfur system, no pendent groups were formed, but only crosslink products – oligomer and dimer crosslinks.95 In the present study, a similar study to Boretti and Woolard’s study has been performed to discover the presence of pendent groups. The reaction products in the presence and absence of MBTS were heated on the oil bath for 20 and 60 minutes at 150 oC. The products were then separated by applying 5 µL on the TLC plates and was developed with 50:50 Hex: DCM. The PTLC plates were prepared in the same way, the bands of interest were lifted by scrapping off the desired bands with a surgical blade and were extracted with DCM.

3.3.11. Devulcanization of Squalene vulcanizates Devulcanization of squalene vulcanized products was done in separate ampoules, using wild garlic extracts as devulcanizing agents. About 0.3 g of vulcanized products were reacted with 0.06 g of the devulcanizing agent. Each ampoule containing the vulcanizate and devulcanizing agent was submerged in an oil bath (Figure 3.1) set at 180 oC for a reaction time of 60 minutes. This devulcanization time is more than the time used by Rajan (2005) and Sonti (2018), who carried out the devulcanization process for 30 minutes at 200 oC .18, 36 The adjustment in time and temperature allowed 37 the efficacy of the different extracts to be explored further as devulcanizing agents, as also squalene contains six isoprene units as compared to TME, which only contains one isoprene unit.

CHAPTER 4

RESULTS AND DISCUSSION

4.1. EXTRACTION AND ANALYSIS OF CRUDE EXTRACTS OF TULBAGHIA VIOLACEA.

4.1.1. Plant extraction The plant extraction process is one of the most important stages in the study, to ensure the extraction of the sulfides of T. violacea, mainly disulfide compounds to enhance the potential of the extracts as potential devulcanizing agents. This requires carefully choosing a suitable extraction solvent and method, targeting the compounds of interest. Furthermore, parameters such as temperature for drying the plant, amount of extract, and chemical composition of the extracts were considered, as they play a significant role in the extract’s potential as devulcanizing agents. The literature suggested the use of the solvents; CHCl3, EtOAc, EtOH and MeOH that readily dissolve DADS and other sulfides of wild garlic and other species of the family Alliaceae.1, 57, 92 Suggested methods included distillation, digestion, solvent extraction, homogenizing and solvent partitioning. Maoela et al. extracted sulfides from fresh rhizomes and dry bulbs of T. alliacea by homogenizing the plant parts in MeOH and 57 collected EtOAc and CHCl3 by solvent partitioning of the MeOH extract. As DADS are the mainly targeted compounds in the present study, Wan and colleagues showed that extraction using MeOH yields greater amounts of DADS compared to other solvents – THF and benzene.93 Due to the challenges faced during vulcanization/devulcanization, such as the bursting of the glass ampoules in the presence of peroxides and water, the extraction of dry plant material was essential to avoid extraction of such compounds. The plant material was first oven-dried to ensure the removal of all excess water, taking care not to use high temperatures that could cause degradation of the targeted sulfur constituents. Sonti (2018) made use of soxhlet extraction, using a mixture of 98:2 n-hexane: IPA as extraction solvent to extract DADS and other sulfides.36 However, this method is considered unfavorable for a few reasons such as; the minute amount of extract, exposure to water should any faults occur, and temperature risks that could cause the extract to char and change in the chemical

39 composition of the extract. Thamburan et al. (2006) performed a study where solvent extraction is employed at room temperature to successfully extract sulfides of wild garlic with high yields.92 Hence, in the present study, T. violacea is extracted by means of gradual solvent extraction (CHCl3 < EtOAc < MeOH) at room temperature to extract sulfur-rich extracts. However, due to polarity differences of the solvents used, from less polar to most polar, focus will be drawn on the results obtained for EtOAc extract as the mid-polar extraction solvent, with a few comparisons of the extracts discussed. Table 4.1 below shows the dry masses and percentage yields of the crude extracts.

Table 4.1: Illustrates the dry masses and percentage yields of the crude extracts

Plant part Solvent used mass of crude (g) Yield (%)

Leaves CHCl3 3.20 3.25 EtOAc 1.41 1.43 MeOH 11.28 11.46

Bulbs CHCl3 0.80 0.56 EtOAc 0.76 0.53 MeOH 7.48 5.18

Roots CHCl3 0.53 0.76 EtOAc 0.39 0.57 MeOH 1.47 2.13

4.1.2. Identification of sulfides Upon tissue rupture, the major sulfur compounds of T. violacea (and other species of the family Alliaceae) undergo enzymatic conversion and chemical degradation compared with when the plant is intact.96 Thus, upon obtaining the dry mass of the crude extracts, TLC was immediately done to evaluate the crude extracts for the presence of sulfides, as a result of the enzymatic change and chemical degradation of the major compound marasmin. As part of the degradation products, commercially available standards of DADS and DAS have been used as reference spotted against the crude extracts for positive identification. About 5 µl of each standard and extract were applied on Merck silica gel 60 F250 TLC plates and eluted gradually with a 4:1 Hex: EtOAc solvent mixture. Figure 4.1 below shows the plate developed with 4:1 Hex:

EtOAc as a suitable solvent mixture for separation of compounds, and elution of the reference standards.

DAS DADS Cl El Ml Cb Eb Mb Cr Er Mr

Figure 4.1: TLC plate of T. violacea extracts against reference standards, DAS and DADS, under UV short wavelength eluted with 4:1 Hex: EtOAc as mobile phase (where: Cl, El and Ml is the extracts of the leaves. Cb, Eb and Mb are extracts of the bulbs. And Cr, Er and Mr is the extracts of the roots).

From the chromatogram, elution of compounds interacting more with the less polar mobile phase is clearly observed for the CHCl3 and EtOAc extracts, whereas for the MeOH extracts, the compounds remained at the baseline. This observation is due to the polarity differences amongst the extraction solvent and elution mixture. MeOH is a polar solvent, thus contains more polar compounds, which in-turn interacted more with the polar silica stationary phase. Consequently, the more non-polar mobile phase partially separates the extracted constituents. Both the CHCl3 and EtOAc extracts contained DAS when compared to reference, whereas the MeOH and root extracts showed very faint spots under short wave UV-light. Furthermore, a reference spot belonging to the DADS standard is observed at the solvent front. Spots of the same

Rf-value are once again clearly seen for the leaves and bulbs CHCl3 and EtOAc extracts. For the leaves, similar spots are seen at the Rf values 0.63, 0.54 and 0.32, while those of the bulbs were seen at a Rf value of 0.78. 41

4.1.3. Phytochemical analysis of sulfides Analysis by TLC allowed only for maximum separation of compounds in the extracts, whereas viewing of the TLC plates under UV-light allowed visualizing of only UV-active compounds, particularly those with extended conjugation such as aromatic rings. Nevertheless, this method of separation is not indicative of the groups/types of compounds separated from either extract. This required employing a method more suitable for positive identification of sulfidic phytochemicals. In this study, the method of Wagner and Bladt has been employed to positively identify sulfides of T. violacea.20 Sonti made use of the same method in her study, where the presence of sulfides in fresh T. violacea extracts was observed as blue spots.36 Figure 4.2 shows the TLC chromatograms of the extracts spotted in comparison to the DADS and DAS standards upon staining with vanillin staining reagent.

DAS DADS Cl El ML Cb Eb Mb Cr Er Mr

Figure 4.2: TLC plate of extracts after treatment with Wagner and Bladt vanillin reagent.20

As per Wagner and Bladt method, the TLC plate was treated with vanillin-glacial acetic acid staining reagent and heated in an oven. Figure 4.2 shows the TLC chromatogram after staining and heating in an oven. As observed, the sulfur-derived compounds are evident by the appearance of blue spots, a similar observation made by Sonti. A similar observation at the solvent front in figure 4.1 is seen for the Rf of DADS in figure 4.2. 42

The spot of the standard DADS is observed at the solvent front for all extracts of T. violacea. A lot of papers in the literature have reported species of the family Alliaceae to contain sulfur-derived phytochemicals. From figure 4.2, an abundance of vanillin- sensitive compounds is evident from the variety of blue spots eluted with the mobile phase.

Though TLC is one of the cheapest, easiest and quickest methods of analysis, it still does not provide full information about the type and nature of the identified, and separated compounds on the TLC plates. This then requires further analysis using more powerful techniques and methods such as IR, HPLC, NMR, etc.

4.1.4. FTIR analysis of crudes Analysis by FTIR spectroscopy is done to provide information about the different functional groups associated/present with/in the chemical structures of the extracted compounds, within the extracts. Shown in figure 4.3, is an overlay of the IR spectra for the EtOAc crude extracts of T. violacea. In the spectral region between 3500 – 3000 cm-1, a broad signal assigned to either an –NH or –OH stretch is seen. This signal may be associated with either an amine/amide and alcohol groups. These are groups that are suggestive of the presence of compounds such as cysteine derivatives found in T. violacea (Section 2.3.1).59 The peak is unlikely to be evidence of the presence of water since the plant material was oven-dried prior use. Further confirmation of such –OH and –NH groups is observed between 1300-1000 cm-1, belonging to the C-O and N-H bonds of hydroxyls and amines.

FTIR of EtOAc extracts

0.95

0.9

0.85 DTVB-EtOAc DTVL-EtOAc 0.8 DTVR-EtOAc 0.75

0.7

0.65 3950 3450 2950 2450 1950 1450 950 450

Figure 4.3: FTIR spectrum overlay of dry EtOAc crude extracts of T. violacea roots, bulbs, and leaves.

The selection of an extraction solvent could lead to the extraction of sulfur compounds of different nature and orientation. However, the sulfur compounds of interest in this study contain similar functional group linkages, such as C=C, C-H (sp, sp2 or sp3),

C=C-H2, CH=CH2, C-S and S-S bonds in the chemical structure. The three peaks that are seen in the region 3000-2850 cm-1 are assigned to C-H bonds, indicating the presence of alkyl groups. Further down at 1500-1400 cm-1, these are signals most likely to be C-H bends correlating to the latter peaks providing evidence of the presence of methylene groups. At approximately 2500 cm-1, no peaks were observed, suggesting the absence of any thiol groups (-SH). In the region 1700-1600 cm-1, sharp stretches (leaves and bulbs) and a broad stretch (roots) can be observed. These signals provide clear evidence for the presence of either alkene C=C bonds or carbonyl C=O bonds. At approximately 1050 cm-1, medium bands suggesting the presence of S=O bonds are likely to be seen, typical of sulfoxide and sulfone constituents.66 Between 700-600 cm-1, vibrations that are related to the stretching of C-S linkages are observed. Furthermore, peaks consistent with S-S linkages are seen around 500-400 cm-1. These vibrations (C-S and S-S) are evidence of the extraction of sulfur-derived compounds. Furthermore, because of the presence of S-S vibrations, one can be confident about

44 the extraction of either di-, tri-, or tetra- sulfides, and/or other possible polysulfidic constituents.

As can be seen in Figure 4.2, the EtOAc and CHCl3 extracts show a similar pattern of eluted spots, meaning they have the same or similar compounds. This pattern has been followed on analysis using FTIR, showing signals that belong to the similar functional group(s) for both extracts. See appendix B for the overlay of CHCl3 extracts FTIR spectra for the different plant parts.

4.1.5. HPLC analysis of crude extracts and quantification of DADS Analysis by FTIR provided evidence of possible functional groups present in the extracted constituents of T. violacea. No information is provided about the quantity, and separation or isolation of single compounds. This required the use of other powerful techniques such as HPLC to separate and quantify constituents individually. However, the isolation of compounds in this study has been sidestepped, as the application of the study makes use of the uncut crude extract. An analysis by HPLC was performed for the identification of some of the sulfur-derived constituents, and quantification of DADS as main target constituent. Organosulfur compounds were successfully separated by employing a reverse-phase column in an Agilent HPLC unit fitted with a diode array detector. Due to the grade differences, only DADS have been used as a reference standard for the quantification of the crude extracts, because it is of HPLC grade. On the other hand, as can be seen in Figure 4.1 (section 4.2), the DAS reference standard shows secondary spots, ensuing it to be considered unfavorable for quantification because it is not of HPLC grade. Figure 4.4 shows the HPLC chromatograms of (a) the solvent, (b) DADS reference standard, and (c) EtOAc roots extract. In Figure 4.4(a), shown is the chromatogram of the mobile phase/solvent used to dilute extract analytes before injection onto the HPLC unit. The peaks observed from this chromatogram and retention times were eliminated in the identification of extracted compounds, as these peaks were noticeable in all prepared samples, depending on the concentration of the analyte diluted.

(a)

(b)

(c)

Figure 4.4: RP-HPLC chromatograms of (a) the mobile phase, (b) reference standard, and (c) DTVR-EtOAc extract

From Figure 4.4 (b), the retention time for DADS is observed at about 3.86 min. This is different from the retention time observed by Sonti – 4.67 minutes.36 The difference could due to several reasons such as (i) the method of preparation of the standards, (ii) the different analysis parameters and mobile phase polarity changes, starting from least polar to more polar, and (iii) the HPLC method employed – reverse phase. In Figure 4.4 (c), shown is the RP-HPLC chromatogram of the DTVR-EtOAc crude extract, where a peak corresponding to the retention time of DADS is observed at 3.89 minutes. This observation is suggestive of the extraction and presence of diallyl disulfide in the EtOAc crude extract of the roots. A similar observation was seen for all remaining EtOAc and MeOH extracts (Appendix C1-2), at a similar retention time. The chromatograms obtained for the CHCl3 extracts (Appendix C3) did not show the presence of diallyl sulfide from the RP-HPLC analysis employed in this study. This observation could be due to negligible amounts of DADS present in the crude extracts, implying that CHCl3 has a low affinity to extract noticeable concentrations of DADS, or simply because CHCl3 is not a proper solvent for the extraction of DADS, but other 46 sulfur-derived constituents of T. violacea. Shown in table 4.2 are the analytes retention times and peak areas that correspond to that of the DADS reference standard.

Table 4.2: Illustrates the retention times of the analytes corresponding to the reference standard.

Reference standards and crude extract Peak area (mAu) Retention time analytes (min) Standard number 1 (0.539 mg/mL) 4819.96 3.86

2 (0.0539 mg/mL) 484.84 3.82 3 (0.00539 mg/mL) 39.22 3.83 4 (0.000539 mg/mL) 6.378 3.82 MeOH Leaves 51.93 3.89 Bulbs 79.93 3.90 Roots 18.19 3.90 EtOAc Leaves 71.68 3.87 Bulbs 87.39 3.88 Roots 185.51 3.89

Using the reference standards’ peak areas (Table 4.2) and concentrations (table 3.4), a calibration curve (Figure 4.5) has been made to obtain a calibration curve equation for calculating the quantity of DADS extracted in each extract.

Absorbance vs Concentration y = 8945.6x - 1.3973 R² = 1 5000 4500 4000 3500 3000 2500 2000 1500 Absorbance (mAU) Absorbance 1000 500 0 0 0.1 0.2 0.3 0.4 0.5 Concentration (mg/mL)

Figure 4.5: DADS reference standard calibration curve

From the equation obtained in the calibration curve plot (Figure 4.5), Table 4.3 was constructed to show the percentage of DADS present in the crude extracts of T. violacea.

Table 4.3: Shows the calculated concentration of DADS and percentage in each extract

Extraction Plant part Concentration Percentage (%) solvent (mg/mL) MeOH Leaves 5.26x10-3 0.98 Bulbs 8.07x10-3 1.50 Roots 1.93x10-3 0.36 EtOAc Leaves 7.21x10-3 1.34 Bulbs 8.84x10-3 1.64 Roots 18.82x10-3 3.49

From table 4.3, it can be observed that the EtOAc extracts contain a higher percentage of DADS when compared to the MeOH extracts. This observation is seen for all plant parts of T. violacea, suggesting EtOAc to have a greater affinity to extract DADS in the 48 present study. A further observation is made with the EtOAc extracts, where there is an increase in the DADS concentration in the order; leaves < bulbs < roots. This pattern for the increase in DADS concentration can be associated with the plant part and environment of growth. For instance, because the leaves grow above ground level, they are more exposed to different atmospheric influences such as drastic weather conditions (dryness and heat) and seasonal changes. Taking into account the effect of weather, it should be noted that too much heat from the sun causes the leaves to lose the water contained in them for survival, causing them to rupture upon dryness, and thus results in the slight cleaving and chemical degradation of the unstable sulfur compounds. This slight cleaving and degradation, in turn, causes a decrease in the availability of the sulfur constituents found in the leaves. On the other hand, the bulbs and roots are buried below ground level, under the soil, being exposed more to the minerals and nutrients the soil is enriched with. These minerals and nutrients act to support and enhance the nature of present chemical constituents in both bulbs and leaves. The roots have been seen to contain more DADS in the EtOAc crude extracts. This observation can be assumed to be because of the solvent (EtOAc) having a greater extraction affinity for the constituents of the roots than that of the bulbs. Furthermore, because the plant parts were cut and dried prior use (section 3.3.1), the longer drying time in the oven and exposure of the bulbs to temperature could have resulted in the low concentrations of DADS present in the bulbs. In comparison to the percent yield of DADS obtained in the previous study by Sonti, the concentration of DADS was found greater for all EtOAc plant part extracts.

Regrettably, marasmicin and other sulfur-derived constituents of T. violacea could not be quantified due to the scarcity of HPLC grade reference standards. These are the compounds that could possibly be correlated with the other eluted peaks seen in the chromatogram shown in Figure 4.4 (c), neglecting the peaks observed for the mobile phase in 4.4 (a). However, as discussed in section 4.4, and seen in Figure 4.2, organosulfur compounds are evident in the DTVR-EtOAc RP-HPLC chromatogram. Owing to the abundant availability of sulfur-derived constituents in the crude extracts, these extracts may be considered for use in the devulcanization process as potential devulcanizing agents, as previous studies show not only DADS to be a devulcanizing agents, but other disulfides such as 2,4,5,7-tetrathiaoctane, isolated by Burton (1990)1

49 may possibly act as a devulcanizing agent since it contains a disulfide linkage as in known devulcanizing agents. Examples of previously used agents include compounds such as bis(2-benzamidophenyl) disulfide and 2-aminodiphenyldisulfide.91

4.2. MBTS ACCELERATED SULFUR VULCANIZATION OF SQUALENE

The vulcanization process is one of the crucial steps of this research study. This section discusses the 2-bisbenzothiazole-2,2’-disulfide (MBTS) accelerated sulfur (S8) vulcanization of the model compound, squalene. Vulcanization was successfully achieved following the method employed by Boretti (2002).95 In their study, they performed a comparative study that followed MBTS accelerated vulcanization of squalene in the presence and absence of zinc oxide (ZnO), where it was postulated that crosslinks were formed earlier in the presence of ZnO.95 This method has been the primary method of choice to complete the study, without looking too much in-depth at other processes, reported in the previous study.95 The results discussed for the vulcanized products were obtained by chromatographic (TLC, PTLC, HPLC and GPC) and spectroscopic (FTIR and NMR) methods, and thermographic analysis (DSC and TGA).

4.2.1. TLC and NMR analysis of vulcanizates Previous research reports that MBTS accelerated sulfur vulcanization of squalene occurs by the formation of pendent groups, thus suggesting the presence of pendent 71 groups in the squalene/MBTS/S8/ZnO. The products of the system were dissolved in dichloromethane (DCM) and injected onto a TLC plate, and then developed with a 50:50 v/v Hex: DCM solvent mixture to obtain a minimum separation. The reaction products were then applied to PTLC to afford two bands between the Rf-values 0.49 and 0.53 which were then extracted with DCM, dissolved in CDCl3, and were subjected to NMR. PTLC allowed maximum separation of the unreacted MBTS and MBT from the squalene products containing pendent groups. From the 1H-NMR spectrum (Figure 4.6 (a)), two peaks were observed downfield between δ 7.35-7.45 and δ 7.77-8.00 ppm suggesting the presence of aromatic hydrogens. These peaks are comparable to those obtained for MBTS, which further suggests that the chemical shifts can be conclusively assigned to the aromatic hydrogens present on the benzothiazole groups of pendent groups bound to squalene. The peaks are seen more upfield represented the signals

50 of squalene and those of the solvent. At approximately δ 7.2 ppm, the peak assigned to the solvent is observed. Around 5 ppm, a broad singlet is observed. This is the peak assigned to the ethylene hydrogens of squalene bonded cis and trans in the chemical formula of squalene. Further upfield, two peaks are observed around δ 2.0 and δ 1.7 ppm. At δ 2 ppm, this is the signal that belongs to the hydrogens of the methylene (-

CH2) groups that are bonded to the -C=C group, in the β form. At δ 1.7 ppm, observed is the methyl (-CH3) groups of squalene that bond in an α form to the -C=C group. Noticeable on the spectrum (a), there is a slight chemical shift and peak distortion on the peaks. This can be assumed to be a result of the chemical change caused by the aromatic rings of the benzothiazole groups that form pendent groups of squalene. In comparison to spectrum (b), it is clear to see that the aromatic groups of the benzothiazole groups have reacted with squalene to form pendent groups.

(a)

Figure 4.6: Shows the 1H-NMR spectrums of (a) squalene reaction product and (b) unreacted squalene

4.2.2. FTIR analysis of vulcanizates FTIR analysis of the vulcanized products was performed to distinguish the difference before and after vulcanization to observe the presence of the carbon-sulfur and sulfur- sulfur signals. These bonds are unlikely to appear on analyzing squalene, as it contains 51 no sulfur in its pure state. Figure 4.6 shows an overlay of the vulcanized product and pure squalene. In the spectrum of the vulcanized product, a sharp band observed at approximately 700 cm-1 is consistent with the existence of C-S functional groups. In the region 670-500 cm-1, signals consistent with the presence of S-S bonds are observed. This is a result owing to the reaction products not only consisting of disulfide bonds but trisulfide, tetrasulfide, and other polysulfide bonds.

FTIR overlay of Sq and Vulc 1 0.9 0.8 0.7 0.6 Sq Vulcanizate

0.5 (%) Intensity Squalene 0.4 0.3 3100 2800 2500 2200 1900 1600 1300 1000 700 400 Wavenumber (cm-1)

Figure 4.7: FTIR overlay of the vulcanizate and squalene

In the region between approximately 1800-1600 cm-1 weak bands be observed. These bands are consistent and characteristic of the absorption patterns of different substitutions of aromatic rings, indictive of the benzothiazole aromatic rings of the pendent groups. From the results obtained in the comparison of the vulcanizate and squalene spectrum, shown in Figure 4.7, FTIR provides evidence of vulcanization, as the squalene spectrum does not contain any of the sulfide and aromatic bands, belonging to crosslink and pendent group formation.

4.2.3. RP-HPLC analysis vulcanizates Reversed-Phase HPLC was performed for the separation and detection of the curative levels in the vulcanized products of squalene. Studied is the consumption of sulfur and MBTS and the formation of MBT. Boretti (2002) performed a study where both the consumption of S8 and MBTS and the formation of 2-bisebenzothiazole-2,2’- polysulfides (MBTP’s) was shown.95 The study showed a gradual decrease in the concentration of both S8 and MBTS, and the increase in the MBTP concentration. It 52 should be noted that Boretti (2002) recorded the results based on different curative reactions, i.e. in the presence and absence of sulfur, and in the presence and absence of ZnO.95 Consequently, the current study only shows the results obtained for the system where sulfur and ZnO are present, the optimal vulcanization system as per 95 Boretti (2002) . Figure 4.7 shows the mol% curves obtained for S8, MBTS, MBT and MBTP’s.

Mol% vs Reaction Time 110

70 Sulfur 50 MBT MBTS 30 MBTP's mol % initial curative

-10 0 20 40 60 Reaction time (min)

Figure 4.8: RP-HPLC analysis of the Squalene/MBTS/S8/ZnO (5.65:1.1:1:1:) system heated isothermally @ 150 oC.

From the RP-HPLC of the squalene/MBTS/S8/ZnO system after heating isothermally at 150 oC, a decrease in MBTS concentration is seen occurring rapidly within the first 5 min, being completely consumed by 15 min. MBT formation (22.9 mol %) was noted during the first 5 minutes, as a result of the transformation of MBTS during crosslink formation because of its high reactivity.97 This is observation is made concurrent with the formation of crosslinking products, a similar result as in the study by Boretti (2002)95 Within the next 10 minutes of the reaction, the bulk of MBT was formed, reaching a maximum concentration by 15 min (43.7 – 45.1 mol %). By 20 min, the MBT concentration dropped to 22.8 mol %. This is assumed to be a result of a percentage 53 of the free MBT forming a zinc metal complex in its reaction with ZnO, which reacts further to complete crosslink formation. However, Zn(MBT)2 is insoluble, thus is not compatible with analysis by RP-HPLC. Nonetheless, the MBT concentration did not increase or decrease above and below 20 mol %. This tells us that approximately 80 mol % has been converted to the insoluble Zn(MBT)2. During the first 10 min, sulfur is gradually decreased (47.2 mol % remaining @ 10 min) and then rapidly decreased during crosslink formation with no sulfur being detected 20 min, the optimal vulcanization time. This result is seen as evident in the thermal analysis of the squalene/MBTS/S8/ZnO system (section 4.2.5.4). It is thus assumed that the cross-link products formed beyond 20 min are a result of the benzothiazole groups (Zn(MBT)2). This is evident in the study by Boretti (2002), where a rapid increase of crosslinks was 95 detected beyond 20 min in the reaction system squalene/Zn(MBT)2/sulfur. 2- bisbenzothiazole-2,2-polysulfides (MBTP’s) eluted at a retention time of 7.7 min, reaching a maximum concentration at 5 min.98

4.2.4. GPC analysis of vulcanizates Due to the complexity of squalene reaction products, separation of the squalene derivatives (i.e. pendent groups and crosslink products) was found difficult by RP- HPLC analysis. Thus, GPC, which is more suitable to study high molecular weight distributions was employed to separate squalene derivatives into their relative categories.18, 95

Initially, the molecular weight (MW) distribution was obtained using three GPC Styragel HR columns of different pore sizes (section 3.3.8.2). However, better peak resolution and distribution, over a limited molecular weight range, were obtained from the Styragel HR 0.5 Å column only. This column covers a MW range between 0 – 1000 mAu, the MW range where the squalene dimer crosslinks and a variety of pendent groups would emerge.91

In a previous study, Coran (1978) postulated that the MBTS accelerated sulfur vulcanization of squalene occurs via pendent group formation.71 Boretti and Woolard (2006) confirmed the postulation by investigating the presence of pendent groups by heating up the squalene/sulfur (5.65:1.0) and squalene/MBTS/sulfur (5.65:1.1:1.0) systems isothermally at 150 oC for 120 and 20 min respectively99. They suggested that, because no pendent groups were detected in the squalene/sulfur system, the shoulder 54 peak observed on the dimer crosslink peak in the squalene/MBTS/sulfur system, may be assigned to the existence of pendent groups.95 Hence, in this study, the systems by Boretti (2002) were studied to detect the presence of pendent groups.

Figures 4.9 and 4.10 show the GPC curves of the systems in the absence and presence of MBTS. Vulcanization of squalene with sulfur (figure 4.9) shows two distinct peaks. These are assigned to oligomer crosslink products (tR = 1.8 min) and the dimer crosslinking products (tR = 3.0 min). No evidence of a third peak suggestive of a pendent group was detected as expected. The molecular weight distribution of the molecules is reflected by the peak width (in minutes), with the bigger molecules eluting first.

Figure 4.9: GPC curve for the squalene/sulfur (5.65:1.0) system heated isothermally at 150 oC for 120 min.

The GPC curve obtained for the squalene/MBTS/sulfur system three peaks, where the first peaks elute at the same retention as that of the squalene/sulfur system, as seen in Figure 4.9. This is easily assigned to the oligomer crosslinks eluting at tR = 1.9 min. Noticeably, a second peak, reflecting the similar retention time of the dimer crosslink products of the squalene/sulfur system, is seen at tR = 3.1 min with a shoulder peak at 2.8 min. Due to the peaks non-existence in the chromatogram of the squalene/sulfur system, this peak is safely assigned to the presence of pendent groups. It must be 55 noted that the pendent groups contain a benzothiazole group that is planar, causing the pendent group compound to be more rigid and have a larger hydrodynamic volume than the squalene dimer. Hence, the pendent group elutes before the squalene dimer products.

Figure 4.10: GPC curve for the squalene/MBTS/sulfur (5.65:1.1:1.0) system heated isothermally at 150 oC for 30 min.

Figure 4.11 shows the GPC curve for the squalene/MBTS/S/ZnO system, heated isothermally @ 150 oC for 60 min. The chromatogram shows two distinct peaks for the vulcanized squalene product. This suggests that the system mixture contains molecules of different MW. The first peak (tR = 1.9 min) is assigned to the oligomer crosslink products of squalene, and the second peak (tR = 3.8 min) is assigned to the dimer cross-linking products of vulcanized squalene. The broad width of the second may suggest a shoulder due to pendents groups between the two peaks

Figure 4.11: GPC analysis of the squalene/MBTS/S8/ZnO (5.65:1.1:1:1) system heated isothermally @ 150 oC.

In GPC, molecules of greater size are eluted first while the smaller size molecules are retained longer in the column. From the chromatogram shown in Figure 4.11, one can conclude that the dimer crosslinks are smaller in size as compared to the oligomer crosslinks. The molecular weight distribution (MWD) of the crosslinked product is thus determined by the width of the peaks suggests of the crosslink products. It is clearly illustrated from the GPC curve that the peak assigned to dimer/trimer crosslinks is broader than that of the oligomer crosslinks. This suggests that dimer crosslinks have a broader molecular weight distribution. In terms of peak maximum, though little difference, the maximum of oligomer crosslink product was reached later than the dimer crosslink product. Figure 4.12 shows the comparison of the peak widths belonging to the categorized reaction products.

PEAK WIDTH VS VULCANIZATE 5 min 10 min 15 min 20 min 30 min 45 min 60 min 2.0135 1.7185 1.3655 1.258 0.6145 0.5819 0.5605 0.4364 0.4216 0.4112 0.401 0.3886 0.3621 0.2958 0.2879 0.2784 0.262 0.2602 0.2397 PEAK WIDTH (MIN) 0 0

OLIGOMER PENDENT DIMER VULCANIZATE

Figure 4.12: Comparison of the molecular weight distribution of reaction products

It is observed that at 5 min, both the cross-link products and pendent groups are formed. However, there is a rapid formation of the pendent groups, having a greater molecular weight distribution (MWD) than either of the cross-link products, reaching a maximum distribution of pendent groups in 20 min. This is the same time where the dimer cross-link products show maximum MWD distribution overall. This further suggests that the pendent group peak is now smaller and hides under the dimer peak, making it look wider. Furthermore, in 30 min, though little difference between the MWD of pendent groups and oligomer cross-links, the pendent groups showed a minimum distribution of formed products. At 15 min, the oligomer crosslink product showed maximum MWD, whereas the dimer/trimer crosslink product shows maximum MWD for the reaction times between 20 – 60 min and at 10 min.

4.2.5. Thermal analysis and reactions of curatives 4.2.5.1. Sulfur The heating of sulfur in a closed DSC pan at 10 oC/min showed three endothermic transitions (Figure 4.13). The first transition at 105 oC is assigned to the α-β solid-solid transition in sulfur, from rhombic sulfur to monoclinic sulfur. The major peak at 118 oC is representative of the melting of the monoclinic sulfur. Previous research showed the

58 nuclei of monoclinic sulfur (Sβ) to be necessary for the solid-solid transition, and that the transformation of single crystals of rhombic to form Sβ did not occur but melted at 112 oC.100 A small peak is seen at 115 oC, attributed to be the melting of a small fraction of the rhombic sulfur without undergoing the solid-solid transition.

o The temperatures of the major peaks compare well with literature; (Sα - Sβ = 95.3 C) o o 100, 101 (Sβ – liquid = 115.2 C) (liquid - Sµ = 169.6 C). The temperature of the first transition is however slightly higher than that obtained in the literature. This is due to the equilibrium temperature in literature because solid-solid transitions are generally observed at slightly elevated temperatures when performing DSC experiments.102

Figure 4.13: DSC thermogram for sulfur heated @ 10 oC/min, sample size = 1.0338 mg

4.2.5.2. 2-Bisbenzothiazole-2, 2’-disulfide (MBTS) Figure 4.14 below shows a DSC thermogram for the heating of 2-bisbenzothiazole- o 2,2’-disulfide. The curve shows a sharp endothermic peak at 173.1 C (Tonset = 168.8 oC and ∆H = 133.5 J/g) which is attributed to the melting of MBTS. This temperature compared well with that reported in the literature (172.5 oC).103 The endotherm is equally composed of components that are reversing and non-reversing. Boyce (2017) suggested that even though MBTS may be purified, its endotherm is significantly broader and less sharp than other purified curatives, suggesting the presence of other species such as MBTM and MBTP’s formed on heating.91

Figure 4.14: DSC thermogram of MBTS heated at 10 oC/min, sample size = 1.0532 mg

4.2.5.3. Squalene/MBTS/ZnO (5.65:1.1:1.0) Figure 4.15 shows the DSC scans for the heating of squalene/MBTS/ZnO at 10 oC/min o o from ambient temperature to 200 C. The first two endotherm peaks at -31.3 C (Tonset o o o = -35.2 C, ∆H = 48.7 J/g) and -7.86 C (Tonset = -11.58 C, ∆H = 81.61 J/g) belong to the solid-solid state transitions of the squalene fingerprint. A third endotherm is seen o o as a very broad peak at 155.74 C (Tonset = 131.7 C, ∆H = 12.79 J/g). This is the peak that shows the melt of the subsumed monocyclic crystals,91 suggesting a melt related to MBTS.103 There is no exotherm suggesting the presence of a crosslink reaction 60 around/near 150 oC. This is consistent with literature that MBTS cannot vulcanize squalene in the absence of sulfur9, 95.

Figure 4.15: DSC thermogram of the squalene/MBTS/ZnO system heated at 10 oC/min cycle 1, sample size = 2.6560 mg

4.2.5.4. Squalene/MBTS/S8/ZnO (5.65:1.1:1.0:1.0)

Figure 4.16 shows cycle 1 of the DSC thermogram of the squalene/MBTS/S8/ZnO system heated at 10 oC/min from -100 oC to 200 oC. The curve was generated by the heat flow against temperature. Three endotherms and exotherms can be seen on the o o curve. The first two endotherm peaks at -31.5 C (Tonset = -34.9 C, ∆H = 41.2 J/g) and o o -8.24 C (Tonset = -11.65 C, ∆H = 82.2 J/g) belong to the melts of the squalene o fingerprint. A third endotherm is seen as a very broad peak at 155.5 C (Tonset = 136.2 oC, 10.6 J/g). This is the peak where the melting of the monocyclic crystals have been subsumed.91 This peak also suggests the melting of MBTS dissolved in the molten sulfur.103 Though the system initially contains sulfur, no melt attributed to sulfur is seen around -100 oC after heating the system for 60 min. At this reaction time, all sulfur is assumed to have dissolved in the squalene to form sulfur crosslinked products of o o squalene. An exotherm is seen at 187.5 C (Tonset = 177.6 C, ∆H = 16.0 J/g). As vulcanization is known to be an exothermic process, the exotherm peak at 187.5 oC is associated with a cure reaction – vulcanization of squalene.

Figure 4.16: DSC thermogram of the squalene/MBTS/S8/ZnO system heated at 10 oC/min cycle 1, sample size = 2.6560 mg

The RP-HPLC analysis of the products obtained from the reaction times, 5 – 60 min, suggested that the formation of crosslinks initiated from 5 min, reaching a maximum concentration of the dimer crosslinks by 20 min. In Figures 4.17 and 4.19 is the DSC o thermograms of the squalene/MBTS/S8/ZnO system heated isothermally at 150 C. The thermograms are plotted against time to monitor the optimal reaction time where curing takes place. Figure 4.17 shows the DSC thermogram recorded for a 5 min reaction time. It is clearly seen from the thermogram that there is an exotherm that rises around 5 min. This peak is representative of a curing reaction starting to take place, with a recorded heat flow of 0.0574 W/g. However, because the reaction is not complete, we observe that the peak is not a full peak, but only the tailing of the actual cure peak. Upon cooling (seen around 6 min to 16 min) and heating (seen around 17 min to 32.5 min) again, the presence of a post-cure squalene fingerprint (-28.2 oC and -5.2 oC) is observed, suggesting that the reaction did not go to completion in 5 min. Furthermore, a peak associated with residual cure is seen near 30 min, further suggesting the incomplete vulcanization reaction at 5 min.

Figure 4.17: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 5 min, sample size = 10.0990 mg

Figure 4.18 shows the thermogram obtained for the squalene/MBTS/S8/ZnO system heated for 20 min at 150 oC. Heating the system for 20 min showed an exotherm indicating a crosslinking reaction by 7.72 min. it is seen that 27.61 J/g were consumed to completely generate crosslinking products at 20 min reaction time. Cooling the reaction generated a crystallization peak. This peak may belong to unreacted MBTS or MBT salts formed in the reaction of MBTS and ZnO (i.e. Zn(MBT)2).

Figure 4.18: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 20 min, sample size = 10.6670 mg

Heating the system again for another 20 min showed no presence of a post-curing exotherm. This showed evidence of a completed reaction in 20 min. Evident was the post-cure melt of the assumed unreacted MBTS or MBTS salts. In addition, as there is no melt for sulfur observed, a correlation with the RP-HPLC analysis of the 20 min reaction product, where sulfur was completely consumed, is seen to be evident.

Figure 4.19 is the thermogram for the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 60 min. The thermogram shows the squalene fingerprint to still be present after 5 min. This observation is in relation to when the system is heated for 5 min (Figure 4.17). A melt representing the solid-solid transition of sulfur is seen around 10 min (113.7 oC). A corresponding result showing the presence of sulfur after 10 minutes is observed in the RP-HPLC analysis, showing the presence of sulfur after 10 min of reaction. An exotherm showing the squalene curing reaction is observed at 21.2 min (150 oC). Curing occurred by consuming 63.45 J/g energy. No peaks are seen beyond 30 min to 60 min, implying that in 20 min squalene completely reacts with pure sulfur to form crosslinks. In a study by Sonti (2018), it was shown that the reaction between MBTS and ZnO to form a zinc metal complex occurs beyond 200 oC,36 whereas in this study the system is heated isothermally at 150 oC, hence no second exotherm indicative of a MBTS and ZnO reaction is observed.

Figure 4.19: DSC thermogram of the squalene/MBTS/S8/ZnO system heated isothermally @ 150 oC for 60 min, sample size = 9.5590 mg

Upon cooling and heating the system for a further 60 min showed a squalene post-cure glass transition (-84.96 oC). The glass transition is assumed to belong to excess squalene. No exotherm was observed at 80 min (20 min after cooling), evident of complete reaction between squalene and sulfur. An endotherm is observed between 90 – 100 min. This peak is associated with the post-cure MBTS melt (153.72 oC). The melting point is observed to be low due to the presence of sulfur, which in its molten- state dissolves MBTS to form MBTP’s.

4.3. DEVULCANIZATION OF SQUALENE PRODUCTS

This section discusses the effectiveness of the crude extracts of T. violacea for their use as potential devulcanizing agents against the vulcanized products of squalene. It should be noted that the results presented are of the whole crude extract consisting of a mixture of compounds, and no single compounds have been isolated and tested against the vulcanized products.

4.3.1. FTIR analysis An overlay of the FTIR spectra of the vulcanizate and the devulcanizates, obtained by treating the vulcanizate with the EtOAc extracts of the leaves, bulbs, and roots is shown in Figure 4.20. It appears that the spectra of the devulcanized products contain peaks resonating in the spectral regions between 700 – 600 cm-1 and 500 – 400 cm-1, suggestive of the presence of the carbon-sulfur and sulfur-sulfur bonds. Regrettably, these bonds exist in the crude extracts and the vulcanized product(s). This rendered analysis of the devulcanized products to be indecisive in verifying whether if and to what degree does devulcanization takes place.

FTIR overlay of Vulcanizate and devulcaizates 1 0.95 0.9 0.85 0.8 Squalene vulc 0.75 DTVR-EtOAc 0.7

0.65 (%) Intensity DTVB-EtOAc 0.6 DTVL-EtOAc 0.55 0.5 3050 2750 2450 2150 1850 1550 1250 950 650 350 Wavenumber (cm-1)

Figure 4.20: FTIR overlay of sq-vulcanizate and devulcanizates

4.3.2. NMR analysis An overlay of the proton NMR (1H-NMR) spectra of squalene, vulcanized product, and the devulcanized product is seen in Figure 4.21 (a) – (e). The spectra are labeled as; (a) squalene, (b) vulcanizate, and (c) DTVR-EtOAc devulcanizate, DTVL-EtOAc devulcanizate, and (e) DTVB-EtOAc devulcanizate, respectively. It is clear from the spectra of the devulcanized products that there are no signals characteristic of the pendent group’s aromatic hydrogens between δ 7.3 ppm – 7.8 ppm. In the devulcanizate spectra ((c) – (e)), signals like that of clean squalene are clearly observed. Form these observations, it may be postulated that the crude extracts obtained from T. violacea have the potential to act as devulcanizing agents against squalene vulcanized products.

(a) (b) (d) (e)

Figure 4.21: 1H-NMR overlay of (a) squalene, (b) vulcanizate, (c - e) devulcanizates using EtOAc extracts as devulcanizing agent

The same results were observed upon devulcanization employing the MeOH and

CHCl3 extracts as potential devulcanizing agents (see appendix D). However, the degree of devulcanization could not be compared from an analysis by NMR. A stronger technique such as HPLC and GPC was employed for such a comparison study.

4.3.3. GPC analysis 4.3.3.1. Devulcanization using EtOAc extracts of T.violacea as Devulcanizing agents A GPC curve for the devulcanization of the 60 min squalene vulcanized product using DTVR-EtOAc as a devulcanizing agent is presented in Figure 4.22. An overall decrease in the concentration of the high molecular weight products is seen upon devulcanization with the DTVR-EtOAc extract. Both peaks of the oligomer and dimer crosslinks are seen to have become narrower and shorter after devulcanizing, showing also a decrease in retention time for both peaks when compared to the vulcanized product. The oligomer crosslinks retention time decreased from tR = 1.9 min to tR =

1.68 min, and dimer crosslinks retention time decreased from tR = 3.78 min to tR = 2.55 min. The difference in the elution time of the oligomer peak from the vulcanizate and devulcanizate is negligible. It is thus assumed that all the oligomer crosslinks associated with this peak are above the exclusion limit of the GPC column (Styragel HR 0.5Å, MW = [0 – 1000 mAu]) employed, and thus elute concurrently.104

Figure 4.22: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVR-EtOAc @ 250 nm

The latter peak, tR= 2.55 min, signifies that there is a decrease in the molecular weight of the dimer/trimer crosslinks of the formerly vulcanized squalene product. Closely looking at the peak at tR = 2.55 min, a shoulder peak is evident around 2.3 min. Because we know pendent groups are eluted before the dimer crosslinks, after oligomer crosslinks, these products are of higher molecular weight than the later eluted oligomer crosslink products. From this knowledge and the decrease in oligomer crosslink MWD, it may be suggested that the shoulder peak indicates a release of the active benzothiazole radicals, pendent groups or 2-bisbenzothiazole-2,2’-polysulfides (MBTP’s) that were initially formed from the thermal decomposition of MBTS during the early stages of vulcanization. Consequently, from the reduction in peak area and peak width of both the oligomer and dimer crosslinks, it may be suggested that DTVR-EtOAc exerts good efficacy as potential devulcanizing agent, as the results obtained compared well with those obtained in a study by Boyce et al. (2017), where 2-amino and 4-aminodiphenyldiphenyldisulfide were used as devulcanizing agents.91

To investigate the effectiveness of each plant part extract, the efficiency of the extract of the roots was compared to the EtOAc extracts of the bulbs and leaves, as other potential devulcanizing agents. Figure 4.23 illustrates the GPC curve for the DTVB- EtOAc devulcanized product of squalene. The curve shows an insignificant change in the molecular weight distribution of the crosslink products. It appears that the peak at tR = 2.5 min is broader, while that of the oligomer products is narrower. Further observation showed that the intensities of the peaks are higher for the DTVB-EtOAc devulcanizate, especially at tR = 2.01 where maximum difference in peak intensity is seen. However, from the results discussed and the similar shift in peak position as in the DTVR-EtOAc devulcanizate, the DTVB-EtOAc extract behaves effectively as a devulcanizing agent, but less effective compared to DTVR-EtOAc.

Figure 4.23: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVB-EtOAc @ 250 nm

Shown in Figure 4.24 is the GPC curve for the squalene devulcanization using DTVL- EtOAc as the devulcanizing agent. It is seen that the DTVL-EtOAc extract reacted in a somewhat different manner with the squalene vulcanizate when compared to the DTVR- and DTVB-EtOAc extracts. The reaction with DTVL-EtOAc resulted in the formation of two extra peaks, at tR = 3.00 min and tR = 4.26 min. The reaction between squalene and sulfur showed the formation of oligomer and dimer crosslinks, where no pendent groups were detected due to the absence of MBTS, with dimer crosslinks being eluted just after tR = 4.00 min. In Figure 4.24, a peak suggesting the presence of dimer crosslinks is seen around this time. This contrasts with the suggestion made by Boyce et al. (2017), that dimer crosslinks are retained longer in the column, only to elute after longer retention times. Furthermore, reversion of the vulcanized may be suggested in the reaction of the vulcanizate with DTVL-EtOAc as a devulcanizing agent, resulting in only the breakage of the S-S bonds only and not the C-S bonds of the vulcanized product.

Figure 4.24: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVL-EtOAc @ 250 nm

4.3.3.2. Comparison of the leaf’s extracts of T.violacea as devulcanizing agents A comparative study of the leaf’s extracts was employed to see the changes, if any, in molecular weight distribution (MWD) when the leaves are extracted using different solvents. Figure 4.25 shows a summary of the comparison of the molecular weight distribution of the devulcanized products using the leaf’s MeOH, CHCl3 and EtOAc extracts as devulcanizing agents. Close observations of the MeOH devulcanizates show only a small change in molecular weight distribution of both the oligomer and dimer crosslinks can be seen, 0.67 min and 0.25 min respectively. Devulcanization using the CHCl3 and EtOAc extracts resulted in the regeneration of pendent groups upon crosslink breakdown. This is assumed to be a result of the broken S-S and C-S bonds allowing a reaction with the free radical MBT to reform pendent groups.

Comparing the two extracts, it is clearly seen that the CHCl3 extract showed greater devulcanizing potency in breaking the crosslinks of oligomer crosslinks (peak width/MWD = 0.093 min), meanwhile, the EtOAc extract showed greater potency against the dimer/trimer crosslinks (peak width/MWD = 0.17 min).

LEAVES EXTRACTS GPC PEAK WIDTHS MeOH CHCL3 EtOAc Vulcanizate @ 60 min 1.2518 1.0022 0.779 0.5355 0.4425 0.401 0.3343 PEAK WIDTH 0.2022 0.1723 0.1394 0.0929 0 0 0 0

OLIGOMER PENDENT GOUP DIMER TRIMER

Figure 4.25: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization using the CHCl3, EtOAc, and MeOH leaves extracts and the 60 min vulcanizate

As previously discussed in section 4.3.3.1, the EtOAc extract separated the dimer and trimer crosslink products. As the trimer crosslink products are larger in molecular weight than the dimer products, the same result is observed after devulcanization. Furthermore, close observation in the MWD of the pendent group in relation to the crosslink products shows that, the more the MWD of the crosslink products decreases, the more the MWD of the pendent groups increase.

4.3.3.3. Comparison of the bulbs extracts of T.violacea as devulcanizing agents Figure 4.26 below shows a bar graph comparing the MWD of the bulbs extracts of T.violacea. There is no separation of the dimer and trimer crosslinks upon devulcanization using the bulbs extracts. Maximum decrease in the MWD of the oligomer crosslink products is seen for the CHCl3 extract (MWD = 0.086 min), while that of the dimer/trimer products is witnessed in the EtOAc devulcanized product (MWD = 0.89 min).

BULBS EXTRACTS GPC PEAK WIDTHS MeOH CHCL3 EtOAc Vulcanizate @ 60 min 1.2518 1.0628 0.9666 0.8902 0.401 0.34 0.2799 0.2517 PEAK WIDTH 0.1547 0.086 0 0 0 0

OLIGOMER PENDENT GOUP DIMER TRIMER

Figure 4.26: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization, using the CHCl3, EtOAc, and MeOH bulb extracts, and the 60 min vulcanizate

As previously seen with the leaves extracts, devulcanization using the bulbs MeOH extract resulted in no formation of pendent groups. This result is due to the bulbs MeOH extract having a low potency in breaking the S-S and C-S crosslink bonds of the oligomer and dimer/trimer products. Furthermore, this may be a result of the concentration of extracted disulfides in the extract, and/or the amount of extract reacted with the vulcanized product, and possibly the synergistic effect of the extracted sulfides. Maximum pendent group formation was obtained from devulcanization using EtOAc as a devulcanization agent.

4.3.3.4. Comparison of the bulbs extracts of T.violacea as devulcanizing agents A comparison of the MWD of the devulcanized products obtained by the devulcanization of the vulcanizate using the roots extracts is represented in figure 4.27. It is clear that there is no separation of the dimer/trimeric products, as observed when bulb extracts are used as devulcanizing agents. The high ability of the CHCl3 extract as a potential devulcanizing agent is evident by the strong decrease in MWD of the oligomer products. Furthermore, pendent group formation is only seen for the CHCl3 extract, showing a high degree of devulcanization using CHCl3 extract in comparison to the MeOH and EtOAc extracts. In terms of the dimer/trimer products, the MeOH 73 extract showed slightly higher efficacy in breaking the dimer/trimer crosslinks compared to the CHCl3 extract.

ROOTS EXTRACTS GPC PEAK WIDTHS MeOH CHCL3 EtOAc Vulcanizate @ 60 min 1.2518 1.027 0.9996 0.8428 0.401 0.3381 0.2029 PEAK WIDTH 0.1758 0.1004 0 0 0 0 0 0

OLIGOMER PENDENT DIMER TRIMER GOUP

Figure 4.27: Comparison of the molecular weight distribution of the devulcanizates, upon devulcanization, using the CHCl3, EtOAc, and MeOH roots extracts, and the 60 min vulcanizate

4.3.3.5. Comparison of the total crosslink MWD decrease and pendent group formation The total crosslink MWD decrease and pendent group formation are shown in Figure 4.28. It is observed that the EtOAc extracts showed greater devulcanization efficacy for all plant parts (i.e. leaves, bulbs, and roots). Maximum crosslink MWD was achieved using the extracts from the leaves, meanwhile increased MWD of pendent groups formed was obtained only upon devulcanization using the leaves CHCl3 and EtOAc extracts. Though the bulb EtOAc extract showed a higher decrease in crosslink MWD, no significant difference was seen when compared to the devulcanization efficacy of the CHCl3 extracts.

TOTAL MWD DECREASE AND P.GROUP FORMATION VULCANIZATE DTVL-MeOH DTVL-CHCl3 DTVL-EtOAC DTVB-MeOH DTVB-CHCl3 DTVB-EtOAC DTVR-MeOH 1.6528 1.4028 1.3391 1.3365 1.1274 1.0526 1.0449 1.0186 0.6284 0.5779 0.5139 0.4425 0.2799 0.2577 0.2026 0 0 0 0 0 TOTAL PEAK WIDTH TOTAL leaves bulbs roots leaves bulbs roots CROSSLINK MWD (MIN) P.GROUP MWD (MIN)

Figure 4.28: Comparison of the total crosslink MWD decrease and pendent group formation of the MeOH (blue), CHCl3 (orange), and EtOAc (grey) extracts.

Pendent group formation is achieved for all CHCl3 extracts of T.violacea, reaching a maximum formation using the leaves extract as the devulcanizing agent. In comparison to the CHCl3 extracts, devulcanization using the leaves and bulbs EtOAc extracts showed less pendent group formation when using the bulbs EtOAc extract as a devulcanizing agent, but more formation of pendent groups when the bulbs extract was employed. However, little difference is observed in the MWD of the pendent groups formed during devulcanization using the bulbs CHCl3 and EtOAc extracts. This suggests that the constituents extracted from the CHCl3 and EtOAc extracts of the leaves and bulbs have an ability to cleave crosslink C-S and S-S bonds, thus forming pendent groups. No pendent group products were formed upon devulcanization using the MeOH extracts when on the other hand, only the CHCl3 extracts were capable to form pendent groups for all its extracts used as devulcanizing agents. From the results obtained it can be seen that pendent group formation increases, upon devulcanization using T.violacea extracts, in the order of; CHCl3 > EtOAc > MeOH. The GPC chromatograms for the devulcanized products using the CHCl3 and MeOH extracts as devulcanizing agents are shown in Appendix E.

4.3.4. Thermogravimetric analysis The crude extracts used as devulcanizing agents were analyzed by means of thermogravimetric analysis to determine the thermal stability of each extract. The analysis was carried out in both the conventional constant heating rate mode (dashed curves) and in the high-resolution mode (solid curves). The high-resolution mode adjusts the heating rate in response to the rate of mass loss, resulting in faster experiments and improved peak resolution of overlapping weight losses. The DTGA curves were obtained by heating the platinum TGA pans at constant and dynamic rates, at a heat flow of 10 oC/min from ambient temperature to 700 oC.

Analysis of the EtOAc crudes extracts by both modes showed a similar degradation pattern on the DTGA curves. The curves showed four degradation stages of the present compounds, classified as compound class A, B, C and D respectively. These can further be described as: thermally unstable (A), moderately thermally stable (B), thermally stable (C) and highly thermally stable (D). It was seen that analysis by the conventional DTGA mode yields a low resolution, non-descriptive profile, while the Hi- Res DTGA yields a profile consisting of distinct peaks of higher resolution, a result of the adjusted heated rate in response to the change in the rate of mass loss, which then results in improved peak resolution. The weight loss changes of the extracts are shown by a comparison of the conventional and Hi-Res DTGA curves in Figures 4.28 – 4.31. Furthermore, analyzing the information provided by the curves, a careful notice of the evaporation of the solvent was highly accounted for. This is seen around 55 – 60 oC, before the thermal degradation of the class A compounds. Thus, the recorded weight losses of compounds have been rationed on a dried extract basis.

4.3.4.1. DTVL-EtOAc extract Figure 4.29 shows the comparison of the conventional(CDTGA) and Hi-Res (HRDTGA) DTGA curves of the DTVL-EtOAc extract used as devulcanizing agent. Two different samples were prepared to account for each experiment, 1.7218 mg, and 4.7860 mg, respectively. On the Hi-Res curve, above 600 oC, a residual signal is seen. At approximately 160 oC, both curves show the degradation of class A compounds. These are assumed to be the most volatile compounds of the extract. They are the compounds that are considered ineffective during vulcanizing because they evaporate at a temperature below the devulcanizing temperature employed (180 oC). Class B compounds decomposed between 200 – 425 oC in a two-step manner, class B1 (200 – 305 oC) and Class B2 (305 – 430 oC), showing two distinct peaks on the Hi-Res curve, while the conventional curve showed the existence of an overlap. Class C compounds were evaporated around 425 oC (Hi-Res) and 450 oC (conventional). Looking closely at the class B and C peaks of the two curves, one can see that the peaks of the conventional curve are broad, evidence of the low/poor resolution on the conventional curve. Furthermore, it can be observed that analysis by the Hi-Res mode increases the intensity of the peaks when at the same time it causes a shift in the position of the peak as a result of the adjusted heating rate.

Figure 4.29: Overlay of the TGA/Hi-Res DTGA curves of the DTVL-EtOAc extract.

A summary of the total weight loss associated with the degradation stages of DTVL- EtOAc is illustrated in Table 4.4 obtained at Hi-Res.

Table 4.4: Total weight loss of DTVL-EtOAc extract compounds by Hi-Res DTGA

Weight loss Weight loss Total Weight loss stage/compound (mg) (%) class A 0.6040 12.62 B 2.783 58.15 C 0.7322 15.30 D 0.5795 12.11

It is clear from Table 4.4 that most of the compounds present in the DTVL-EtOAc extract are moderately thermally stable, evaporating between, 200 – 400 oC. Consequently, the compounds that are active in the devulcanization process belong to the classes B and C, evaporated above the devulcanizing temperature of 180 oC. See appendix F1 for a more detailed Hi-Res thermogram.

4.3.4.2. DTVB-EtOAc extract An overlay of the DTGA curves of the DTVB-EtOAc extract can be seen in Figure 4.30. The same results, in terms of the different employed modes of analysis, are observed. An observation of the class A compounds showed a multistep degradation of the compounds in this class, A1 – A5 (approx. 100 – 250 oC), while class B (approx. 251 – 400 oC), C (approx. 400 – 500 oC), and D (approx. 500 – 650 oC) showed a single- step degradation. This illustrates that most of the compounds extracted from the bulbs belong to the class A compounds. However, looking closely at the class curve(s), we see that a portion of the compounds (A1 – A3) is evaporated by 180 oC (the devulcanizing temperature employed), and not all the compounds of class A are evaporated. Consequently, these compounds are considered as being ineffective as responsible compounds for the extract to act as potential devulcanizing agents. Thus, only the compounds belonging to classes A4, A5, B, C, and D which evaporate beyond 180 oC, were considered as being part of the compounds responsible for the effectiveness of the extract’s potential to act as devulcanizing agent.

Figure 4.30: Overlay of the TGA/Hi-Res DTGA curves of the DTVB-EtOAc extract

A summary of the total weight loss associated with the degradation stages of DTVB- EtOAc is illustrated in Table 4.5 obtained at Hi-Res.

Table 4.5: Total weight loss of DTVL-EtOAc extract compounds by Hi-Res DTGA

Weight loss Weight loss (mg) Total Weight loss (%) stage/compound class A 2.211 51.68 B 0.5605 13.10 C 0.1849 4.32 D 0.9798 22.90 E 0.3312 7.742

From the summary in Table 4.5, it can be seen that most of the compounds present in the DTVB-EtOAc extract belong to the class A compounds. However, only 25.24 % is regarded as ineffective during the devulcanizing process. See appendix F2 for a more detailed Hi-Res thermogram.

4.3.4.3. DTVR-EtOAc extract The thermogravimetric curves for the DTVR-EtOAc extract are shown in Figure 4.31. Similar to the leaves extract, the curves of the roots extract show three weight loss stages, also classified as class A, B, C, and D. It can be observed that all the weight losses are single-step processes. This suggests that the compounds are of the same nature and share similar chemical properties. A high weight loss was observed with class A compounds between 100 – 275 oC. However, though a single profile is observed and similar compound characteristics, the compounds that evaporated below the devulcanizing temperature (180 oC) were considered ineffective as part of the compounds that enhance the effectiveness of the crude extract to act as a potential devulcanizing agent.

Figure 4.31: Overlay of the TGA/Hi-Res DTGA curves of the DTVR-EtOAc extract

A summary of the total weight loss associated with the degradation stages of DTVR- EtOAc is illustrated in Table 4.6 obtained at Hi-Res.

Table 4.6: Total weight loss of DTVR-EtOAc extract compounds by Hi-Res DTGA

Weight loss Weight loss (mg) Total Weight loss (%) stage/compound class A 2.765 64.12 B 0.5717 13.25 C 0.3582 8.31 D 0.4177 9.69

The summary in Table 4.6 shows that there is a rapid weight loss of the class A compounds present in the DTVB-EtOAc extract, while the compounds of classes B and C undergo a gradual weight loss. Like the bulbs extract, only a portion of the class A compounds are evaporated by the devulcanization temperature and are thus regarded ineffective during the devulcanizing process. See appendix F3 for a more detailed Hi- Res thermogram.

Figure 4.32 shows a comparison of the TGA curves for EtOAc extracts of the leaves, bulbs, and roots. The overlay plot has been constructed to monitor the rate of compound evaporation in each extract. Noticeably, class A compounds of the bulbs rapidly decrease, while those of the roots decreases in a slightly gradual manner, and that of the leaves evaporates at a very slow rate. These observations suggest a pattern of increased thermal stability of compounds present in the extracts that goes from the leaves - roots – bulbs.

Between 200 and just above 400 oC, though all extracts undergo a gradual decrease, the extract of the roots shows a large percent weight change and a decrease in thermal stability, falling beneath the curve of the bulbs. This means that the bulbs have more thermally stable compounds in the class B step when compared to the roots. The leaves results do not show much of a change and still dominate containing thermally stable compounds even in the class B stage.

Above 400 oC, the leaves undergo a rapid weight loss, falling way beneath the curve of the bulb and very close to the roots curve. The curves of the leaves and roots show

81 that the plant parts lose up to more than 80 % of their compounds by 600oC, while the bulbs lose only up to about 70 % of its chemical constituents by 600 oC.

Figure 4.32: TGA curves for the EtOAc crude extracts of T. violacea

4.3.5. DSC analysis Figures 4.33 (a) – (b) show the DSC thermogram of the DTVL-EtOAc devulcanized product of squalene. The devulcanized product was obtained by heating a mixture of the vulcanized product and the devulcanizing agent (DTVL-EtOAc) in a DSC pan at 10 oC/min and held isothermally at 180 oC for 30 min. The curve shows a squalene product post-cure glass transition appearing at 2.81 min (-74.19 oC), prior to the devulcanizing temperature, which is immediately followed by a MBTS melt at 9.16 min (∆H = 1.920

J/g). The squalene product glass transition is a result of the attached MBT or Zn(MBT)2 on squalene. No exotherm evident of a curing was observed as expected. During the devulcanization time, there was no clear evidence of an endotherm representing a devulcanization reaction. This is suggestive of the formation of reversion products similarly presented in section 4.3.3, where no cleaving of C-S bonds has occurred but only that of the S-S bonds.

Figure 4.33 (a): DSC thermogram for the squalene devulcanizate from the DTVL- EtOAc extract heated at 10 oC/min showing no temperature curve, sample size = 5.5300 mg

Figure 4.33 (b): DSC thermogram for the squalene devulcanizate from the DTVL- EtOAc extract heated at 10 oC/min showing temperature curve, sample size = 5.5300 mg

Upon cooling and heating up of the reaction mixture for a further 25 mins showed only the post-devulcanization squalene product glass transition and melt of MBTS. This suggests that the presence of the leaves extract as the devulcanizing agent did not affect the nature and backbone of squalene. Again, no curing reaction was observed as expected.

The DSC analysis of the vulcanized products after treatment with DTVB-EtOAc extract as devulcanizing agent is shown in Figure 8.18 (Appendix G1). The same analysis conditions and parameters used for devulcanization using the leaf extract, was employed for devulcanization by the bulbs and roots. The curve of the bulb devulcanization shows a similar pattern to that of the leaves devulcanizate. A squalene product post-cure glass transition is seen at 2.73 min, while a post-devulcanization glass transition is seen at 62.03 min. Evident is also the melting of MBTS at 9.54 min (3.552 J/g), while its crystallisation is seen at 54 min (0.3643 J/g) upon cooling. Expectedly, no curing reaction was observed.

CHAPTER 5

CONCLUSION

The extraction and analysis of the crude extracts obtained from T.violacea were evident of the presence of sulfur-derived constituents and showed effectiveness as potential devulcanizing agents. Analysis of the crude extracts by thin-layer chromatography assisted in positively identifying the present sulfur-derived constituents, and detection of the reference standards, especially Allyl sulfide (AS). Owing to the commercial scarcity of standards such as marasmicin, these constituents could not be identified by TLC. Nonetheless, viewing of the TLC plates under UV light was evident in the separation of such compounds extracted from T.violacea. However, these compounds were not isolated as single compounds for the current study, which is aimed at evaluating the efficacy of the whole extract(s) for potential use as devulcanizing agents. Further evidence for the detection of sulfur-derived compounds was provided by employing the TLC sulfur phytochemical test method.20

In contrast to the plant material being oven-dried, gradual solvent extraction of the pre- dried plant material using CHCl3, EtOAc, and MeOH afforded crude extracts that are free of water. Extraction was carried out in a well-evacuated system to avoid easily oxidizing of the sulfur constituents. Greater extract values for each plant part were obtained using methanol as an extraction solvent. The FTIR analysis of the extracts suggested the presence of sulfur compounds, evident the C-S and S-S bands. Owing to the plant being dried and the absence of water, the broad stretching band around 3500 – 3000 cm-1 was evident of the presence of the N-H and O-H groups belonging to the cysteine derivatives, as suggested by Jacobsen et al.(1968).59

Reversed-phase HPLC was employed to separate and quantify the concentration of DADS extracted in the leaves, bulbs, and roots. All ethyl acetate (EtOAc) extracts showed a greater concentration of diallyl disulfide (DADS) compared to extracts from chloroform (CHCl3) and methanol (MeOH), whereas the EtOAc roots extract had the greater concentration overall and least concentration was found in the MeOH roots extract. Regrettably, all chloroform (CHCl3) extracts showed no/an insignificant concentration of DADS.

Thermal analysis studies of the crude extracts revealed that, at the devulcanization temperature (180 oC), the majority of compounds present in the crude extract were thermally stable and suitable for use as potential devulcanizing agents. Because the whole crude extracts were used, only an insignificant amount of the present compounds were volatile at the devulcanizing temperature.

Experiments using Differential Scanning calorimetry (DSC) were suggestive of reactions between the model compound, sulfur, and the accelerator studied, MBTS. Differential scanning calorimetry was employed for monitoring the curatives reactivity. It was seen that heating the squalene system in the absence of sulfur showed only the melting of MBTS around 150 oC.

Squalene was successfully vulcanized by sulfur (S8) in the presence of 2- bisbenzothizole-2,2’-disulfide (MBTS) and zinc oxide (ZnO). Identification and separation of the reaction products were observed by chromatographic (TLC, PTLC, RP-HPLC, GPC) and characterized by spectroscopic (IR and NMR) methods. RP- HPLC and GPC proved to be ideal in the identification and quantification of the curing agents and squalene vulcanizates. Analysis by RP-HPLC was evident in the decrease in MBTS and S8, being completely consumed by 15 and 20 min, respectively. MBT formation gradually increased to approximately 20%, suggesting that approximately

80% of MBTS was only converted to the insoluble Zn(MBT)2 metal complex. The formation of MBTP’s was noticeable by 5 min, reaching a maximum by 10 min and completely consumed by 20 min.

The large size of squalene and its products requires the use of a less sensitive method of analysis such as Gel Permeation Chromatography.95 Analysis by GPC provided evidence of the squalene crosslinked products and by-products. Separation of the vulcanized products was best obtained using the 0.5 HR column, affording two distinct peaks eluting at tR = 1.9 min and tR = 3.7 min. The narrow peak at 1.9 min is attributed to the oligomeric crosslink products, Sq-(Sx-Sq)n, while the much broader peak at 3.7 min is attributed to the squalene dimer and trimer crosslinked products. Furthermore, GPC analysis of the reaction products of squalene provided evidence of pendent groups in the presence of the accelerator, and no pendent groups were found for the sulfur vulcanization of squalene in the absence of the accelerator - MBTS. As postulated by Boretti et al. (2002), the presence of MBTS and ZnO improved the rate 86 of vulcanization.95 At the same time, the presence of ZnO is seen to be responsible for the rapid formation of the polysulfides of MBTS, which then acct as the active Zn complex vulcanizing agent.

The vulcanized products of squalene were then reacted with the crude extracts of T. violacea in the devulcanization process, to test their potency as potential devulcanizing agents. Results obtained from the FTIR analysis of the devulcanizates were inconclusive in providing evidence of devulcanization occurring. This result is due to the presence of C-S and S-S bonds in the sulfur compounds of T. violacea, in the vulcanized products and devulcanizates. Analysis by 1H-NMR provided evidence of devulcanization occurring. The benzothiazole aromatic ring signals had disappeared, and the chemical shifts representative of squalene were seen on the spectrum of the devulcanized products. Analysis by GPC showed a decrease in molecular weight distribution (MWD) of both the oligomer and dimer/trimer crosslinking products. Greater degree and decrease in crosslink MWD and pendent group formation was seen for the leaf EtOAc and CHCl3 extract, while the least decrease in crosslink MWD was seen for the MeOH extracts. No pendent group formation was observed upon devulcanization using MeOH extracts.

Devulcanization of the vulcanized products by DSC was evident of no curing reaction taking place, as expected, and showed the glass transition of squalene. Owing to no evidence of curing reactions, it may be postulated that DSC does provide evidence of possible devulcanization

CHAPTER 6

RECOMMENDATIONS

• Use of extracts belonging to other species of the family Alliaceae and test their effectiveness as potential agents. These include T. alliacea, T. capensis, A. sativum, A. cepa etc. • The extracts from the different plants and relative plant parts may be used synergistically by means of combinatorial chemistry to test their combined potential devulcanization efficacy. • The different variables (e.g. concentration, time, temperature) involved in the process’s vulcanization and devulcanization may be tested for their individual influence during devulcanization using a factorial design layout. • Single compounds may be isolated and examined for their individual effect on the devulcanized products. These can be compared to the efficacy shown by the whole extract to calculate how much the single compound contributes to the devulcanization. • Study the reactions between the curatives and the devulcanizing agents, extracts. • Consider usage of different techniques such as MALDI-MS and ESI-MS to study better information for molecular weight.

CHAPTER 7

REFERENCES

1. Burton, B. G. A chemical investigation of Tulbaghia violacea. Rhodes Univeristy, Grahamstown, South Africa, 1990. 2. Fritsch, R. M.; Keusgen, M., Occurrence and taxonomic significance of cysteine sulphoxides in the genus Allium L. (Alliaceae). Phytochemistry 2006, 67, 1127-35. 3. Lancaster, J. E.; Shaw, M. L.; Joyce, M. D.; McCallum, J. A.; McManus, M. T., A novel alliinase from onion roots. Biochemical characterization and cDNA cloning. Plant physiology 2000, 122, 1269- 79. 4. Amagase, H., Clarifying the real bioactive constituents of garlic. The Journal of nutrition 2006, 136, 716S-725S. 5. Lanzotti, V., Review: The analysis of onion and garlic. Journal of Chromatography A 2006, 1112, 3-22. 6. Kubec, R.; Velisek, J.; Musah, R. A., The amino acid precursors and odor formation in society garlic (Tulbaghia violacea Harv.). Phytochemistry 2002, 60, 21-5. 7. http://laroverket.com/qp-content/uploads/2015/03/rubber_chemistry.pdf (24 February 2017), 8. Porter, M., The chemistry of the sulfur vulcanization of natural rubber. In The chemistry of sulfides, Tobolsky, A. V., Ed. Interscience Publishers: New York, 1968; pp 165-168. 9. Morgan, B.; McGill, W. J., Benzothiazole-accelerated sulfur vulcanization. I. 1- mercabptobenzothiazole as accelerator for 2,3-Dimetyl-2-Butene. J. Appl. Plym. Sci. 2000, 76, 1377- 1385. 10. Datta, R. N.; Schotman, A. H. M.; Weber, A. J. M.; van Wijk, F. G. H.; van Haeren, P. J. C.; Hofstraat, J. W.; Talma, A. G.; Bovenkamp-Bouwman, A. G. V. D., Biscitraconimides as anti-reversion agents for diene rubbers: Spectroscopic studies on citraconimide-squalene adducts. Rubber Chemistry and Technolgy 1997, 70, 129-145. 11. Gregg, E. C.; Katrenick, S. E., Chemical structures in cis-1, 4-Polybutadiene vulcanizates. Model compound approach. Rubber Chemistry and Technolgy 1970, 43, 549-571. 12. Lattimer, R. P.; Kinsey, R. A.; Layer, R. W.; Rhee, C. K., The Mechanism of phenolic resin vulcanization of unsaturated elastomers. Rubber Chemistry and Technolgy 1989, 62, 107-123. 13. Lautenschlaeger, F. K., Model compound vulcanization-part I. Basic studies. Rubber Chemistry and Technolgy 1979, 52, 213-231. 14. Ross, G. W., The reaction of sulphur and sulphur compounds with olefinic substances. Part X. The kinetics of the reaction of sulphur with cyclohexene and other olefins. J. Chem. Soc. 1958, 580, 2856. 15. Versloot, P.; Haasnoot, J. G.; Nieuwenhuizen, P. J.; Reedijk, J.; van Duin, M.; Put, J., Sulfur vulcanization of simple model olefins, Part V: Double bond isomerization during accelerated sulfur vulcanization as studied by model olefins. Rubber Chemistry and Technolgy 1997, 70, 106-119. 16. Van der Horst, M.; Woolard, C. D.; Hendrickse, K. G. The importance of thiol intermediates in 2-mercaptobenzothiazole accelerated sulfur vulcanization of 2,3-dimethyl-2-butene. Msc thesis, University of Port Elizabeth. , Port Elizabeth. South Afica, 2001. 17. De, D.; Maiti, S.; Adhikari, B., Reclaiming of rubber by a renewable resource material (RRM). II. Comparatve evaluation of reclaiming process of NR vulcanizate by RRm and diallyl disulfide. Journal of Applied Polymer Science 1999, 73, 2951-2958.

18. Rajan, V. V. Devulcanization of NR-based latex products for tyre applications: Comparative investigation of different devulcanization agents in terms of efficacy and reaction mechanism. PhD thesis, Twente University of Technology, Enschede. Netherlands, 2005. 19. Tawfic, M. L.; Younan, A. F., Feasible and economic process for using organi disulfide and organic mercaptan as a reclaiming agent for ground tire powder (GTP). Journal of Elastomers & Plastics 2012, 46, 1-14. 20. Wagner, H.; Bladt, S., Plant drug analysis: a thin layer chromatography atlas.; Springer Science and Business Media 1996. 21. Vosa, C. G., A revised cytotaxonomy of the genus Tulbaghia (Alliaceae). Caryologia 2000, 53, 83-112. 22. Burbidge, R. B., A reision of the genus Tulbaghia (Liliaceae). Notes from the Royal Botanic Garden Edinburgh 1978, 36, 77-103. 23. Vosa, C. G., Notes on Tulbaghia 2. Journal of South African Botany 1980, 46, 109-114. 24. Vosa, C. G.; Condy, G., Tulbaghia acutiloba. Flowering Plants of Africa. 2001, 57, 24-28. 25. Vosa, C. G.; Gillian, C., Tulbaghia pretoriensis, a new species from the Province of Gauteng (South Africa). Caryologia 2006, 59, 164-167. 26. Bateman, L.; Moore, C. G.; Porter, M.; Saville, B., The chemistry and physics of rubber-like substances. Maclaren and Sons: London, 1963. 27. http://www.oringsusa/comRubber_intro.pdf (23/04/2019), 28. Hosler, D.; Burkett, S. L.; Tarkanian, M. J., Prehestoric polymers: Rubber processing in ancient Mesoamerica. Science 1999, 284, 1988 - 1991. 29. https://rainforests.mongabay.com/10rubber.htm (23/04/2019), 30. Jana, G. K.; Das, C. K., Recycling natural rubber vulcanizates through mechanochemical devulcanization. Macromolecular Research 2005, 13, 30-38. 31. Thaicharoen, P.; Thamyongkit, P.; Poompradub, S., Thiosalicylic acid as a devulcanizing agent for mechano-chemical devulcanization. Korean Journal of Chemical Engineering 2010, 27, 1177-1183. 32. Brinke, J. W., Silica reinforced tyre rubbers: Mechanistic aspects of the role of coupling agents. Twente University Press.2002. 33. Saville, B.; Watson, A. A., Structural characterzation of sulfur-vulcanized rubber networks. Rubber Chemistry and Technolgy 1967, 40, 100-148. 34. Tobolsky, A. V., Properties and structres of polymers.; John wiley and sons: New York, 1960. 35. Tobolsky, A. V., Polymer science and materias. International Science 1960. 36. Sonti, T.; Hlangothi, B. G.; Woolard, C. D. Extraction of diallyl sulfides and other similar compounds from Tulbaghi violacea a South African plant for use as devulcanizing agents. MTech thesis, Nelson Mandela University, Port Elizabeth. South Africa, 2018. 37. Maloney, J. R.; Theroit, K. K.; Magee, S. B. D.; Torres, J. E.; JR, W. R. W. Process for producing diallyl disulfide. 016881, 2006. 38. Tortora, G. J.; Cicero, D. R.; Parish, H. I., Plant form and function: an introduction to plant science. Ollier-MacMillan Canada: Toronto 1970. 39. Van Wyk, B. E., The potential of South African plants in the development of new medicinal products. South African Journal of Botany 2011, 77, 812-829. 40. Cordell, G. A., Introduction to alkoloids-A biogenetic approach. Wiley Intercience: New York, 1981. 41. Sneader, W., Drug Discover: the eviction of modern medicines.; Wiley: Chichester, 1985. 42. O'Neil, M. J.; Lewis, J. A., In: Kingdom AD and Balandrin MF. Human Medicinal Agents from Plants, ACS Symposium 534. American Chemical Society: Washington DC, 1993. 43. Group, A. P., 2003. Botanical Journal of the Linean Society 141, 399. 44. Dahlgren, R. M. T.; Clifford, H. T.; Yeo, P. F., The families of the monocotyledons. Springle- Verlang Publishers: Berlin, 1985. 45. Reuter, H. D., Allium sativum and Allium ursinum: Part 2. Pharmacology and medical application. Phytomedicine 1995, 2, 73-91. 90

46. Ross, I. A., Medicinal plants of the world: Chemical constituents, traditional and modern uses., 2nd ed. Humana Press Inc: Totowa New Jersey, 2003; pp 1127-1135. 47. Kourounakis, P. N.; Rekka, E. A., Effect on active oxygen species of alliin and Allium sativum (garlic) powder. Research communications in chemical pathology and pharmacology 1991, 74, 249-52. 48. Margaret Roberts, J.; Jin, F.; Ekman, D.; Kay Adams, M.; Lindsay McDonald, R.; Kathleen Thurloe, J.; Richards, A.; Mary Poynten, I.; Law, C.; Kincaid Fairley, C.; John Hillman, R.; Tabrizi, S. N.; Marie Cornall, A.; James Templeton, D.; Marie Garland, S.; Edwin Grulich, A.; Farnsworth, A., Is there a role for the thinprep imaging system in reporting anal cytology? Diagnostic cytopathology 2016, 44, 384-8. 49. Watt, J. M.; Berger-Brandwijk, M. G., The medicinal plants and poisonous plants of Southern Africa and Eastern Africa. 2nd Ed ed.; Livingstone: London, UK, 1962. 50. Hutchings, A.; Scott, A. H.; Lewis, G.; Cunningham, A., Zulu medicinal plants. An inventory of Natal Press. Durban South Africa, 1996. 51. Pooley, E., A fireld guide to wild flowers: KwaZulu-Natal and the Eastern Regions.; Nata Flora Publications Trust: Durban. South Africa, 1998. 52. Kubec, R.; Krejcova, P.; Mansur, L.; Garcia, N., Flavor precursors and sensory-active sulfur compounds in alliaceae species native to South Africa and South America. Journal of agricultural and food chemistry 2013, 61, 1335-42. 53. WCSP World Checklist of Selected Plant families. http://aps.kew.org/WCSP/ (9 February 2017), 54. Lyantagaye, S. L., Ethnopharmalogical and phytochemical review of Allium species (sweet garlic) and Tulbaghia species (wild garlic) from Southern Africa. Tanz. J. Sci. 2011, 37, 58-72. 55. Dyson, A., Discovering Indigenous Healing Plants of the Herb and Fragrance ardens at Kirstenbosch National Botanical Garden. Natal Botanical Institute: Cape Town. South Africa, 1998. 56. van Wyk, B. E.; Gericke, N., Peoples plants: A guide to useful plants of South Africa. 1 ed.; Brizza Publications: Pretoria. South Africa, 2000. 57. Maoela, M. S. Studies on some biologically active natural products from Tulbaghia alliacea. MSc Dissertation, University of the Westen Cape2005. 58. Bungu, L.; van de Venter, M.; Frost, C., Evidence for an in vitro anticoagulant and antithrombotic activity in Tulbaghia violacea. African Journal of Biotechnology 2008, 7, 681-688. 59. Jacobsen, J. V.; Yamaguchi, Y.; Mann, L. K.; Howard, F. D., An alkyl-cysteine sulfoxide lyase in Tulbaghia violacea and its relation to other alliinase-like enzymes. Phytochemistry 1968, 7, 1099-1108. 60. Buwa, L. V.; Afolayan, A. J., Antimicrobial activity of some medicinal plants used for the treatment of tuberculosis in the Eastern Cape province, South Africa. African Journal of Biotechnology 2009, 8, 6683-6687. 61. Ncube, B.; Finnie, J. F.; Van Staden, J., In vitro antimicrobial synergism within plant extract combinations from three South African medicinal bulbs. Journal of ethnopharmacology 2012, 139, 81- 9. 62. Moodley, K.; Mackraj, I.; Naidoo, Y., Cardiovascular effects of Tulbaghia violacea Harv. (Alliaceae) root methanolic extract in Dahl salt-sensitive (DSS) rats. Journal of ethnopharmacology 2013, 146, 225-31. 63. van Wyk, B. E.; van Oudtshoorn, B.; Gericke, N., Medicinal Plants od South Africa. 2nd ed.; Briza Publications: Pretoria, South Africa, 2013. 64. Bocchini, P.; Andalo, R.; Pozzi, R.; Galletti, G. C.; Antonelli, A., Determination of diallyl thiosulfinate (allicin) in garlic (Allium sativum L.) by high perfomance liquid chromatography with a post-column photochemical reactor. Analytica Chimica Acta 2001, 441, 37-43. 65. Corzo-Martinez, M.; Corzo, N.; Villamiel, M., Biological properties of onions and garlic Trends in Food Science & Technology 2007, 18, 609-625. 66. Burton, S. G.; Kaye, P. T., Isolation and characterisation of sulphur compounds from Tulbaghia violacea. Planta medica 1992, 58, 295-6. 67. Niewenhuizen, P. J. New perspectives on sulfur vulcanization, reactions and homogenous catalysis concerning zinc dithiocarbamates. PhD thesis, Rijks University, Leiden, 1998. 91

68. Duck, E. W., Plastics and Rubbers. Philosophical library Inc: New York, 1971. 69. Mpahlele, M. M.; Woolard, C. D. N,N'-dipentamethylenethiuram disulfide sulfur vulcanization of squalene. MSc thesis, University of Port Elizabeth, Port Elizabeth. South Africa, 2006. 70. Morgan, B.; Mc Gill, W. J. Benzothiazole accelerated sulfur vulcanization of 2,3-dimethyl-2- butene. PhD thesis, University of Port Elizabeth, Port Elizabeth. South Africa, 1998. 71. Coran, A. Y., Vulcanization. In Science and Technology of Rubber, Eldrich, F. R., Ed. Academic Press, Inc.: Orlando, 1978; pp 292-335. 72. EPA, U. Markets for scrap tires; EPA/530-sw-92074A: Washington, D. C, 1991; pp 1-3, 21, 27. 73. Marks, A. H., Process of reclaiming rubber from vulcanized-rubber waste. Patent 635141 1899. 74. Isayev, A. I., Science and technology of rubber. Elsevier. New York 2002. 75. Kim, D., Devulcanization of scrap tire through matrix modification and ultrasonification. Energy Sources 2003, 25, 1099-1112. 76. Shi, J.; Ren, D.; Zou, H.; Wang, Y.; Lv, X.; Zhang, L., Structure and performance of reclaimed rubber obtained by different methods. J. Appl. Plym. Sci. 2013, 129. 77. Phadke, A. A.; bhattacharya, A. K.; Chakraborty, S. K.; De , S. K., Studies of vulcanization of reclaimed rubber. Rubber Chemistry and Technolgy 1983, 56, 726-736. 78. Klingensmith, B., Recycling, production and use of processed rubbers. Rubber world 1991, 203, 16. 79. Novotny, D. S.; Marsh, R. L.; Masters, F. C.; Tally, D. N. Microwave devulcanization of rubber. 104205, 1978. 80. Feng, W.; Isayev, A. I., Recycling of tire-curng bladdeer by ultrasonic devulcanization. Polym. Eng. Sci. 2006, 46, 8. 81. Kojima, M.; Tosaka, M.; Ikeda, Y.; Kohjiya, S., Devulcanization of carbon black filled natural rubber using supercritical carbon dioxide. J. Appl. Polym. Sci. 2005, 95, 137. 82. Holst, O.; Stenberg, B.; Christiansson, M., Biotechnological possibilities for waste tyre-rubber treatment. Biodegradation 1998, 9, 301-10. 83. Sato, S.; Honda, Y.; Kuwahara, M.; Kishimoto, H.; Yagi, N.; Muraoka, K.; Watanabe, T., Microbial scission of sulfide linkages in vulcanized natural rubber by a white rot basidiomycete, Ceriporiopsis subvermispora. Biomacromolecules 2004, 5, 511-5. 84. Kim, J. K.; Park, J. W., Journal of Applied Polymer Science 1999, 72, 1543. 85. Bredberg, K.; Persson, J.; Christiansson, M.; Stenberg, B.; Holst, O., Applied Microbiology and Biotechnology 2001, 55, 43. 86. Tsuchii, A.; Suzuki, T.; Takeda, K., Microbial degradation of natural rubber vulcanizates. Applied and environmental microbiology 1985, 50, 965-70. 87. Isayev, A., Recycling of rubber in science and technology of rubber. Elsevier 2005, 3, 663-701. 88. Zanchet, A.; Carli, L. N.; Giovanela, M.; Brandalise, R. N.; Crespo, J. S., Use of styrene butadiene rubber industrial waste devulcanized by microwave in rubber composites for automotive application. Materials and Design 2012, 39, 437-443. 89. Paulo, G. D.; Hirayama, D.; Saron, C., Mivrowave devulcanization of waste rubber with inorganic salts and nitric acid. Advanced Materials Research 2012, 418-420, 1072-1075. 90. Pistor, V.; Scuracchio, C. H.; Oliveira, P. J.; Fiorio, R.; Zattera, A. J., Devulcanization of ethylene- propylene-diene polymer residues by microwabe - Influence of the presence of paraffinic oil. Polymer Engineering and Science 2011, 51, 697-703. 91. Boyce, A.; Woolard, C. D.; Hlangothi, P. Devulcanization of model compounds by a variety of diphenyldisulfides. MSc thesis, Nelson Mandela Metropolitan University. Port Elizabeth2017. 92. Thamburan, S.; Klaasen, J.; Mabusela, W. T.; Cannon, J. F.; Folk, W.; Johnson, Q., Tulbaghia alliacea phytotherapy: a potential anti-infective remedy for candidiasis. Phytotherapy research : PTR 2006, 20, 844-50. 93. Wan, X.; Polyakova, Y.; Ho Row, K., Determination of diallyl disulfide in garlic by reversed-phase HPLC. Center of Advanced Bioseparation Technology and Dept. of Chem. Eng 2007, 20, 442-447.

94. Morgan, B.; Gradwell, M. H. S.; Mc Gill, W. J. The formation of polysulfides of 2- Bisbenzothiazole-2,2’-disulfide and their reaction with 2,3-dimethyl-2-butene. MSc Dissertation, University of Port Elizabeth. Port Elizabeth, South Africa, 1995. 95. Boretti, L. G.; Woolard, C. D.: N-(Cyclohexylthio) phthalimide inhibition of Bis-benzothiazole- 2,2’ disulphide accelerated vulcanization of squalene. PhD thesis, University of Port Elizabeth, Port Elizabeth, South Africa, 2002. 96. Jager, A. K.; Stafford, G. I., Quality assessment of Tulbaghia rhizomes. South African Journal of Botany 2012, 82, 92-98. 97. Kresja, M. R.; Koening, J. L., The nature of sulfur vulcanization. In Elastomer Technology handbook, Cheremisinoff, P. N., Ed. CRC Press: Boca Raton, Florida, 1993; pp 475-494. 98. Gradwell, M. H.; Mc Gill, W. J., Sulfur vulcanization of polyisoprene accelerated by benzothiazole derivatives. III. The reaction of 2-bisbenzothiazole2.2'-disulfide with sulfur and ZnO in polyisoprene Journal of Applied Polymer Science 1996, 61, 1131-1136. 99. Boretti, L. G.; Woolard, C. D., An appropriate model compound for the accelerated sulfur vulcanization of polyisoprene: I. The mechanism of bisbenzothiazole-2, 2'-Disulfide accelerated vulcanization of squalene in the absence of ZnO. Rubber Chemistry and Technolgy 2006, 79, 135-151. 100. Currell, B. R.; Williams, A. J., Thermal analysis of elemental sulfur. Thermochim.Acta 1974, 9, 255-259. 101. Meyer, B., Elemental Sulfur. Interscience Publishers: New York, 1965. 102. Gallagher, P. K.; Kemp, R. B.; Brown, M. E., Handbook of Thermal Analysis and Calorimetry. Elsvier Science: Amsterdam, The Netherlands, 1999. 103. Kruger, F. W.; Mc Gill, W. J., A study of curative interactins.II. The interaction 2.2'- dibenzothiazole with ZnO, Sulfur, and Stearic acid. Journal of Applied Polymer Science 1991, 42, 2651- 2659. 104. Streigel, A.; Yau, W. W.; Kirkland, J. J.; Bly, D. D., Modern Size-Exclusion Liquid Chromatography: Practice of Gel Permeation and Gel Filtration. 2nd ed.; John Wiley & Sons: New York, USA, 2009.

APPENDICES

APPENDIX A

(a)

(b)

Figure 8.1: 1H-NMR spectra of (a) MBTS before purification and (b) Purified MBTS

FTIR spectra of Clean MBTS and Purified MBTS

1 0.9 0.8 0.7 0.6

0.5 0.4 (%) Intensity 0.3 0.2 2900 2400 1900 1400 900 400

wavenumber (cm-1)

Clean MBTS Purified MBTS Figure 8.2: FTIR of clean and purified MBTS

APPENDIX B

FTIR overlay of CHCl3 extracts

0.9

0.8 DTVL DTVB 0.7 % Intensiy DTVR 0.6

0.5 3400 2900 2400 1900 1400 900 400 Wavenumber (cm-1)

Figure 8.3: FTIR overlay of CHCl3 extracts

APPENDIX C

APPENDIX C1

(a)

(b)

Figure 8.4: RP-HPLC chromatograms of (a) DTVL-EtOAc and (b) DTVB-EtOAc extracts

APPENDIX C2

(a)

(b)

(c)

Figure 8.5: RP-HPLC chromatograms of (a) DTVL-MeOH, (b) DTVB-MeOH and (c) DTVR-MEOH

APPENDIX C3

(a)

(b)

(c)

Figure 8.6: RP-HPLC chromatograms of (a) DTVL-CHCl3, (b) DTVB-CHCl3 and (c)

DTVR-CHCl3

APPENDIX D

APPENDIX D1

Figure 8.7: 1H-NMR overlay of squalene, vulcanizate, and devulcanizates using MeOH extracts as devulcanizing agent

Appendix D2

Figure 8.8: 1H-NMR overlay of squalene, vulcanizate, and devulcanizates using

CHCl3 extracts as devulcanizing agent 97

APPENDIX E

Appendices E1 (a – b) show the devulcanized products using the leaves CHCl3 and MeOH extracts as devulcanizing agents.

APPENDIX E1(A)

Figure 8.9: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVL-CHCl3 @ 250 nm

APPENDIX E1(B)

Figure 8.10: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVL-MeOH @ 250 nm

Appendices E2 (a – b) show the devulcanized products using the bulb CHCl3 and MeOH extracts as devulcanizing agents.

APPENDIX E2(A)

Figure 8.11: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVB-CHCl3 @ 250 nm

APPENDIX E2(B)

Figure 8.12: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVB-MeOH @ 250 nm

Appendices E3 (a – b) show the devulcanized products using the roots CHCl3 and MeOH extracts as devulcanizing agents.

APPENDIX E3(A)

Figure 8.13: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVR-CHCl3 @ 250 nm 100

APPENDIX E(B)

Figure 8.14: Shows the GPC curve for the 60min squalene vulcanizate devulcanized with DTVR-MeOH @ 250 nm

APPENDIX F

APPENDIX F1

Figure 8.15: TG/Hi-Res DTGA curves of the DTVL-EtOAc extract, heated at 10 oC/min. sample mass = 4.7860 mg 101

APPENDIX F2

Figure 8.16: TG/Hi-Res DTGA curves of the DTVB-EtOAc extract, heated at 10 oC/min. sample mass = 4.2780 mg

APPENDIX F3

Figure 8.17: TG/Hi-Res DTGA curves of the DTVR-EtOAc extract, heated at 10 oC/min. sample mass = 4.3118 mg

102

APPENDIX G

APPENDIX G1

Figure 8.18: DSC thermogram for the squalene devulcanizate from the DTVB- EtOAc extract heated at 10 oC/min showing temperature curve, sample size = 8.5360 mg

103