EXTRACTION OF DIALLYL SULFIDES AND OTHER SIMILAR COMPOUNDS FROM TULBAGHIA VIOLACEA, A SOUTH AFRICAN PLANT, FOR POTENTIAL USE AS DEVULCANIZING AGENTS

by

Thembela Sonti

Dissertation

submitted in fulfilment of the requirements for the degree

Magister Technologiae

in

Chemistry

in the

Faculty of Science

at the

Nelson Mandela University

Supervisor: Dr B. G. Hlangothi Co-supervisor: Prof. C. Woolard

April 2018

In memory of my late brother Mzwabantu Vena

DECLARATION

In accordance with Rule G5.6.3,I Thembela Celia Sonti 20236939, hereby declare that the above-mentioned dissertation for Magister Technologiae is my own work and that it has not previously been submitted for assessment to another University or for another qualification.

Thembela Celia Sonti

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ACKNOWLEDGEMENTS

The author would like to thank all the people involved in the successful completion of this dissertation. Particularly the following:

 The Lord for everything that He has done for me these past two years;  My husband, Phindile Sonti, and my sons, Kamvalethu and Khwezi, thank you very much for everything. I appreciate your patience and understanding, love you guys;  My supervisor Dr B.G. Hlangothi for her guidance, encouragement and understanding;  My co-supervisor, Prof C. Woolard for his expertise and guidance;  Mr Lukhanyo Bolo for assistance and DSC analysis;  Dr M Phiri for assistance;  The Recycling and Economic Development Initiative of South Africa (REDISA) and the Nelson Mandela University for funding;  The BGH research group for assistance;  Ms E Storm from the Horticulture department for plant harvesting and the Botany department for plant identification;  Technical staff in the organic chemistry laboratory and physical chemistry laboratories at the Nelson Mandela University; and  My sister Thandi Ndongeni, mom Mildred Vena, dad Thamsanqa Vena, and brother Msimelelo Vena, thank you for being patient with me. I appreciate your love, support and words of encouragement.

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TABLE OF CONTENTS

DECLARATION ...... i ACKNOWLEDGEMENTS ...... ii ABSTRACT ...... vi LIST OF FIGURES ...... viii LIST OF TABLES ...... xi LIST OF ABBREVIATIONS ...... xii CHAPTER ONE ...... 1 INTRODUCTION AND AIMS OF THIS STUDY ...... 1

1.1 Introduction ...... 1

1.2 Aims and a brief description of this study ...... 3

CHAPTER TWO ...... 5 LITERATURE REVIEW ...... 5

2.1 Alliaceae family ...... 5

2.1.1 Tulbaghia violacea ...... 5

2.1.1.1 Medicinal studies of Tulbaghia violacea ...... 7

2.1.2 Tulbaghia violacea chemistry ...... 9

2.2 Extraction methods ...... 12

2.3 Choice of solvent ...... 13

2.4 Vulcanization overview ...... 14

2.4.1 Accelerated sulfur vulcanization ...... 17

2.4.2 Model compound vulcanization ...... 18

2.5 Devulcanization overview ...... 21

2.6 Conclusion ...... 24

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CHAPTER THREE ...... 26 EXPERIMENTAL...... 26

3.1 Materials ...... 26

3.1.1 Plant material ...... 26

3.2 Instruments ...... 28

3.2.1 Balances ...... 28

3.2.2 Fourier-transform infrared spectroscopy (FTIR) ...... 28

3.2.3 High Performance Liquid Chromatography (HPLC) ...... 28

3.2.4 Nuclear Magnetic Resonance Spectrometry (NMR) ...... 28

3.2.5 Differential Scanning Calorimetry (DSC) ...... 29

3.3 Experimental Procedure ...... 29

3.3.1 Plant Extraction ...... 29

3.3.1.1 Method I ...... 29

3.3.1.2 Method II ...... 30

3.3.2 Sulfur-containing compounds phytochemical test ...... 31

3.3.3 HPLC analysis of crude extracts ...... 32

3.3.3.1 Preparation of standard stock solution ...... 32

3.3.3.2 Sample preparation ...... 33

3.3.4 Vulcanization of 2,3 dimethyl-2-butene (TME) ...... 33

3.3.4.1 HPLC analysis of vulcanizates ...... 34

3.3.5 Devulcanization ...... 35

3.4 Data presentation ...... 36

CHAPTER FOUR ...... 37 RESULTS AND DISCUSSION ...... 37

4.1 Identification of disulfides on crude extracts ...... 37

4.1.1 Method I ...... 37

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4.1.2 Method II ...... 37

4.2 Sulfur-containing compounds phytochemical analysis ...... 39

4.3 FTIR of crude extracts ...... 40

4.4 Quantification of DADS by HPLC ...... 42

4.5 Vulcanization process ...... 46

4.5.1 FTIR of vulcanizates ...... 46

4.5.2 Vulcanization results ...... 48

4.5.2.1 Vulcanization reactions ...... 53

4.6 Devulcanization process ...... 59

4.6.1 FTIR of the devulcanizates ...... 59

4.6.2 HPLC and NMR ...... 60

CHAPTER FIVE ...... 68 CONCLUSIONS AND RECOMMENDATIONS ...... 68 REFERENCES ...... 72 APPENDICES ...... 79

Appendix A...... 79

Appendix B...... 81

Appendix C ...... 82

Appendix D ...... 83

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ABSTRACT

Tulbaghia violacea is a plant that is commonly used for traditional medicine in the Eastern Cape and Kwa-Zulu Natal Provinces of South Africa for treatment of ailments, such as fever, colds, asthma, tuberculosis, stomach-ache and cancer of the oesophagus. This plant has been found to be rich in sulfur-containing compounds that may display good potential as devulcanizing agents. Commonly used chemical devulcanizing agents are disulfides, such as diphenyl disulfide. These have been found to be relatively expensive compared to other methods of devulcanization. This study aimed to show that the sulfur- containing compounds extracted from the readily available Tulbaghia violacea plant can be used as devulcanizing agents. The presence of sulfur-containing compounds in the plant was positively identified by qualitative phytochemical analysis.

Extraction of sulfur-containing compounds from the bulk plant material was successfully performed using the Soxhlet extraction method with a 2% 2-propanol in n-hexane solvent mixture. There was a positive identification of sulfur compounds in the crude extracts of the bulbs, roots and leaves. Diallyl disulfides (DADS) extracted from the plant were successfully quantified using normal phase high-performance liquid chromatography (HPLC). The results showed that in a bulb crude extract of 9.89 mg/mL concentration, there was 7.74X10-2 mg/mL concentration of DADS. In the roots crude extract of 10.26 mg/mL concentration, there was 2.93X10-2 mg/mL concentration of DADS; and in leaves crude extract of 10.47 mg/mL concentration, there was 3.69X10-2 mg/mL concentration of DADS. The crude extracts were evaluated for their effectiveness as devulcanizing agents by reacting them with vulcanized 2,3-dimethyl-2-butene (TME), which was used as a model compound.

A reverse phase HPLC method was used to identify crosslink species formed during vulcanization, and to monitor the broken crosslinks during devulcanization. It was observed that the di- and polysulfidic crosslinked vulcanized model compound species were not present in the devulcanized product. Furthermore monosulfidic crosslink

vi

species, a common end species of devulcanization, were observed to increase in concentration from the HPLC chromatograms.

It was observed that the extracts from the bulbs and leaves of Tulbaghia violacea were more effective than the extract from the roots when applied to the vulcanized model compound. This study demonstrates that Tulbaghia violacea can be used as alternatives to petroleum-derived chemicals as devulcanizing agents.

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LIST OF FIGURES

Figure 2.1 Tulbaghia violacea plant with voucher specimen number PEU25230 ...... 6 Figure 2.2 Map of the South African provinces[27] ...... 6 Figure 2.3 Reaction scheme of cysteine sulfoxide with alliinase enzyme to form thiosulfinate[40] ...... 8 Figure 2.4 Conversion of alliin into allicin by the enzyme alliinase and of allicin into various sulfur-containing compounds[43] ...... 9 Figure 2.5 Chemical structures of vinyldithiines[44] ...... 10 Figure 2.6 Chemical structures of ajoene[49] ...... 10 Figure 2.7 Chemical structures of the isolated compounds[50] ...... 11 Figure 2.8 Chemical structure of cysteine derivatives[23] ...... 12 Figure 2.9 Vulcanization process[58] ...... 14 Figure 2.10 Rheometer curve of different vulcanization stages[61] ...... 15 Figure 2.11 The effect of vulcanization on some properties of rubber summary[64] ...... 16 Figure 2.12 Simplified reaction scheme for accelerated sulfur vulcanization[67, 68] ...... 17 Figure 2.13 Structure of the vulcanizate network[61, 72] ...... 18 Figure 2.14 Examples of model compounds[63, 74] ...... 20 Figure 2.15 Crosslinking bond breakage mechanism[79] ...... 22 Figure 2.16 Devulcanization mechanism using disulfides[81] ...... 24 Figure 3.1 Soxhlet extractor apparatus ...... 30 Figure 3.2 Juicer, fresh plant material and juices extracted...... 31 Figure 3.3 Vulcanization setup ...... 34 Figure 4.1 TLC chromatograms for Tulbaghia violacea crude extracts in a 2 % 2- propanol in n-hexane solvent mixture, developed in a hexane:ethyl acetate (80:20) solvent system...... 38 Figure 4.2 TLC chromatograms for dichloromethane extracts of the fresh leaves, bulbs and roots of Tulbaghia violacea ...... 40 Figure 4.3 FTIR spectrum of from the bulb extract ...... 41 Figure 4.4 HPLC chromatogram for 2 % 2-propanol in n-hexane solvent ...... 43

viii

Figure 4.5 Chromatograms of a) DADS reference standard and b) bulbs crude extract ...... 44 Figure 4.6 Graph of peak area versus concentration ...... 45 Figure 4.7 FTIR spectra for a) TME and b) vulcanizate ...... 47 Figure 4.8 HPLC chromatogram of a vulcanizate at 140 oC for 40 minutes ...... 48 Figure 4.9 Sulfur ranks of different crosslinks over the logarithmic retention time ...... 49 Figure 4.10 TME bis(alkenyl) crosslinks[83] ...... 50 Figure 4.11 Mole percentage of a) sulfur and b) MBTS as a function of reaction time . 52 Figure 4.12 HPLC analysis of the TME/MBTS/sulfur/ZnO (33.9:1.1:1.1:1) system heated isothermally at 140 oC ...... 53 Figure 4.13 DSC thermogram of sulfur heated at 2 oC/min, sample mass = 1.0380 mg ...... 54 Figure 4.14 DSC thermogram of MBTS/ZnO (1.1:1) heated at 2 oC/min, sample size = 1.9090 mg ...... 55 Figure 4.15 DSC thermogram for TME/MBTS/ZnO (33.9:1.1:1) heated at 2 oC/min, sample size = 1.3900 mg ...... 56 Figure 4.16 DSC curve of TME/MBTS/ZnO/S8 (33.9:1.1:1:1.1) cycle 1 heated at 2 oC/min, sample size = 10.2320 mg ...... 57 Figure 4.17 DSC curve of TME/MBTS/ZnO/S8 (33.9:1.1:1:1.1) cycle 3 heated at 2 oC/min, sample size = 10.2320 mg ...... 58 Figure 4.18 DSC curve of Natural rubber conventional curing heated at 2 oC/min, sample mass = 11.9460 mg ...... 59 Figure 4.19 HPLC peak percentage areas of the monosulfidic crosslinks after devulcanization with bulb extract...... 60 Figure 4.20 Peak percentage areas of the monosulfidic crosslinks after devulcanization with leaf extract...... 61 Figure 4.21 Peak percentage areas of the TME crosslinks of different sulfur rank after devulcanization with rootextract...... 62 Figure 4.22 Peak percentage areas of the TME crosslinks of different sulfur rank with no extract added ...... 63

ix

Figure 4.23 NMR spectra overlay of a) bulb crude extract, b) devulcanizate and c) vulcanizate ...... 64 Figure 4.24 DSC thermogram for the devulcanizate from the leaf extract heated at 2 oC/min, sample size = 3.9090 mg...... 65 Figure 4.25 DSC thermogram for the devulcanizate from the leaf extract, cooled at 10 oC/min, sample size = 3.9090 mg...... 66 Figure 4.26 DSC thermogram for the devulcanizate from the leaves extract heated at 2 oC/min, sample size = 3.9090 mg ...... 67 Figure A.1 FTIR spectrum of Tulbaghia violacea crude extract from the roots ...... 79 Figure A.2 FTIR spectrum of Tulbaghia violacea crude extract from leaves ...... 80 Figure B.1 1H-NMR spectrum of the crosslinks for bis(alkenyl) product ...... 81 Figure C.1 HPLC chromatogram of the sulfur used for vulcanization ...... 82 Figure D.1 DSC thermogram of pure MBTS heated at 2 oC/min; mass = 1.5460 mg .. 83 Figure D.2 DSC thermogram of TME/MBTS/ZnO heated at 2 oC/min cycle 2; sample mass = 1.39090 mg ...... 84 Figure D.3 DSC thermogram of TME/MBTS/ZnO heated at 2 oC/min cycle 3; sample mass = 1.39090 mg ...... 84 o Figure D.4 DSC thermogram of TME/MBTS/ZnO/S8 heated at 2 C/min cycle 2, sample mass = 10.2320 mg ...... 85

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LIST OF TABLES

Table 3.1 Materials and reagents ...... 27 Table 3.2 Yield of the crude extracts of n-hexane ...... 29 Table 3.3 Yield of the crude extracts of 2 % 2-propanol in n-hexane ...... 31 Table 3.4 HPLC conditions for the analysis of organosulfur compounds...... 32 Table 3.5 Model compound vulcanization recipe ...... 33 Table 3.6 HPLC measurements ...... 35 Table 3.7 The retention time of the initial components table ...... 35 Table 4.1 Analyte, retention time and area of the peaks ...... 44 Table 4.2 DADS concentration and percentages of extracts in different plant parts .. 45 Table C.1 Starting materials and solvent retention times ...... 82

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LIST OF ABBREVIATIONS oC Degrees Celsius

CDCl3 Deuterated chloroform DADS Diallyl disulfide DAS Diallyl sulfide DSC Differential scanning calorimetry FTIR Fourier transform infrared spectroscopy HPLC High performance liquid chromatography kHz Kilohertz mAU Milli adsorption units MBT 2-Mercaptobenzothiazole MBTS 2-Bisbenzothiazole-2,2’-disulfide MBTP 2-Bisbenzothiazole-2,2’-polysulfide MCV Model compound vulcanization mg/mL Milligram per millilitre MHz Megahertz mL Millilitre mm Millimetre min Minute mol Mole nm Nanometre NMR Nuclear magnetic resonance 1H-NMR Proton nuclear magnetic resonance

Rf Retention factor TLC Thin layer chromatography TME 2,3-Dimethyl-2-butene; tetramethylethene UV Ultra violet ZnO Zinc oxide

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CHAPTER ONE INTRODUCTION AND AIMS OF THIS STUDY

1.1 Introduction The recorded history of rubber began in the years 1493-1496 when Christopher Columbus was on his second trip to America. He witnessed a game being played by the inhabitants of Haiti using balls prepared from a gum tree. Since there is no rubber- producing tree indigenous to Haiti, the story was open to question. By about 1521 Spanish explorers had found rubber in tropical Mexico. They saw it being used by the locals in different ways in the activities of their lives[1]. The first Europeans to explore the Amazon Valley were Portugese missionaries in 1731. They found the inhabitants making articles of clothing - shoes, head gear, and other useful articles. The species of the tree found in Amazon Valley is called Hevea brasiliensis which is the primary form of natural rubber used in the commercial rubber industry today[1].

The latex from the rubber tree is the leading source of natural rubber. This type of raw rubber is not suitable for commercial purposes because of limitations that lead to undesirable properties. With temperature changes the rubber would be too soft or brittle. Goodyear and Hancock separately developed the process of vulcanization by heating the rubber with sulfur[2]. During vulcanization, the rubber molecular chains form crosslink bonds with sulfur leading to a rubber material that is more elastic with much better resistance to deformation[3]. This development improved the properties of rubber and brought about a broad scope of application opportunities. For the past two centuries natural and synthetic rubbers have been widely used in tyres, reinforced with carbon black and silica to resist abrasion and to improve durability[3]. The disposal of these durable tyres after their life cycle is a challenge that is faced in the 21st century. Because rubber materials contain covalent crosslink bonds, they are non-biodegradable making them very difficult to decompose.

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Owing to these waste management challenges, devulcanization has been developed to form part of the solution to decrease the amount of rubber material waste. During the devulcanization process it is preferred that only carbon–sulfur (C-S) and sulfur–sulfur (S- S) bond cleavage take places as this is the reverse of the vulcanization process, where only (C-S) and (S-S) bonds are formed[4]. The process makes use of devulcanizing agents like diphenyl disulfide (DPDS), dibenzyl disulfide (DBDS), dipentyl disulfide (PDS) and diallyl disulfide (DADS) to name a few[5]. These agents attack the sulfur bridges and deactivate the reactive fragments leading to the possibility of blending devulcanized rubber with additives and virgin rubber. Thus, the devulcanization process can assist in the recycling of waste rubber material for reuse, e.g., revulcanization of devulcanized tyres.

The rubber industry currently uses commercially available agents to devulcanize rubber which are expensive. Some of these devulcanizing agents like diallyl disulfide are synthesised through long processes[6]. However, an alternative can be found in the extraction of plant-based material. De et al.[5] have used a vegetable product, containing a disulfide major constituent, diallyl disulfide, which cleaves the crosslink bonds in vulcanized rubber. In South Africa, some medicinal plants have been known to contain sulfur compounds. The use of plant material could lead to the development of a green product as plants are considered beneficial for the environment.

People have long depended on plants for food, shelter, clothing and medicine[7]. In many rural areas, plants are still regarded as the source of medicine since they are readily available and affordable. Most communities of South Africa have traditional medicine forming part of their culture and tradition[8, 9]. Traditional medicine knowledge is regularly passed on verbally from era to era and can be lost if the values of the cultures are not properly managed[10, 11]. Medicinal plants have always been of great significance to drug development and pharmacological research, where the plant components are utilised directly as therapeutic agents for the production of drugs[12].

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For the development of new drugs, research can be done using the secondary metabolites derived from plants, to fight diseases like cancer and diabetes[13]. The research work includes the use of traditional knowledge to assist scientists to select plants that may be useful[14]. Secondary metabolites, also known as phytochemicals, take part in a number of general protective roles to defend plants from microorganisms like bacteria, fungi and viruses[15]. They are categorised as non-essential micronutrients and are able to take part in the support of wellbeing and reducing the effect of chronic diseases in the life nature of the patient. Their categorisation is based on their chemical structure, composition and their solubility in different solvents[16]. The identification of plant extracts which are effective for fighting chronic diseases is one of the advantages of the study of natural product chemistry[13].

According to our knowledge, there is little literature published concerning the use of sulfur- containing compounds from a plant-based material applied as a devulcanizing agent. Some studies have been performed on garlic which is part of the Alliaceae family as there is evidence of their richness in sulfur-containing compounds[5, 17]. Tulbaghia violacea which is one of the species from the Tulbaghia genus, previously tested in medicinal studies for sulfur-containing compounds, has been selected for this study due to a high content of sulfur in this plant. This project is aimed at extraction of sulfur-containing compounds from the plant, Tulbaghia violacea, and their use for the devulcanization of a vulcanized model compound. The specific aims and a brief description of the organisation of this dissertation are given in section 1.2.

1.2 Aims and a brief description of this study The aims of this study were:

 to extract sulfur-containing compounds from Tulbaghia violacea and to characterise the crude extract material;  to perform vulcanization of a suitable model compound using existing methods and to characterise the vulcanizate; and

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 to evaluate the effectiveness of the plant crude extract as a devulcanizing agent followed by characterisation of devulcanization products.

It should be noted that the current study is a preliminary study which was not aimed at the isolation of single compounds but rather that of a mixture, hopefully containing sulfides and disulfides, which could be used as a crude extract for use as devulcanizing agents.

This dissertation is divided into five chapters:

 In chapter 2, a literature review of Tulbaghia violacea plant is discussed in detail, together with the reasons for its selection. The Model Compound Vulcanization (MCV) technique is presented with 2,3-dimethyl-2-butene (TME, tetramethyl ethene) being the model compound used in this study. The devulcanization technique is also discussed.  Chapter 3 of this dissertation is the experimental section, where methodology and the instrumental methods employed are discussed in detail.  In chapter 4 results and a discussion thereof is provided.  The conclusion of the study as well as a proposal for future work in this area may be found in chapter 5.

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CHAPTER TWO LITERATURE REVIEW

This chapter provides background on the Tulbaghia violacea plant, its origin, current uses as well as the chemistry behind the plant. There is a discussion on different types of solvents to choose from for extraction, as well as the extraction methods available for plant material. A method that has been employed for quantification of sulfur-containing compounds is discussed. A vulcanization overview is provided, including accelerated sulfur vulcanization and model compound vulcanization. Vulcanization must be performed for devulcanization to be evaluated. In this chapter the devulcanization process, including a proposed mechanism, is presented.

2.1 Alliaceae family The Alliaceae family is widely distributed with about 600 species in 30 genera[17]. Alliaceae is a family that generally has members with long hollow leaves, making it difficult to identify them until the flowers bloom. They also have a fragrance that can be pungent due to their sulfur content[18]. The family is distributed in Mediterranean Europe, Asia, North America, South America and Sub-Saharan Africa[19]. Allium, Tulbaghia, and Agapanthus are the Sub-Saharan genera[20]. Tulbaghia is a small genus with approximately 63 species[21]. Most of the species can be identified by their strong alliaceous smell[22]. This alliaceous smell gave most of the species of Tulbaghia the popular name of “wild garlic”[20, 23]. Indigenous people have used several species of Tulbaghia as food and medicine[24].

2.1.1 Tulbaghia violacea Tulbaghia violacea (Figure 2.1) is commonly known as Wilde knoffel (Afrikaans), Itswele lomlambo (Xhosa), Icinsini (Zulu) and Mothebe (Sotho)[25]. These names are believed to originate from the garlic-like flavour of the plant, which, however, does not lead to bad breath when consumed, as opposed to the effect produced by the consumption of the real garlic, Allium sativum[26]. It is an enduring, fast growing, bulbous plant, up to 0.5 m in

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height. It has violet flowers that form umbels of up to 20 which develop from December to April[25]. Its leaves are long, narrow, grey-green and strap shaped. The plant has fat tuberous roots[27] and is found in rocky grasslands of the Eastern Cape, KwaZulu-Natal and Limpopo (see Figure 2.2).

Figure 2.1 Tulbaghia violacea plant with voucher specimen number PEU25230

Figure 2.2 Map of the South African provinces[28]

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Tulbaghia violacea is commonly used in traditional medicine in the Eastern Cape and KwaZulu-Natal to treat fever, colds, asthma, tuberculosis, stomach-ache and cancer of the oesophagus[29]. The Zulu people utilise its bulb to make an aphrodisiac medicine and they also plant it around their houses to repel snakes[30]. The literature suggests that Tulbaghia violacea and Allium sativum (garlic) may have similar biological activities since they both belong to the Alliaceae family[31]. These plants have the same garlic smell and may therefore have similar secondary metabolites. The activity of Tulbaghia species might be the result of similar compounds to those found in garlic[31]. Garlic is generally employed in the food industry, therefore the compatibility studies performed on the Tulbaghia violacea plant will diminish the threat to the food industry as this plant may be utilised instead of garlic. Garlic has for some time been utilised for flavouring. Garlic can be used as a medicinal plant because of two major classes of flavonoids and organosulfur compounds[14, 32]. These organosulfur compounds are either water or lipid soluble and they give garlic its health beneficial properties[14, 33]. Alliin ((2R)-2-amino-3-[(S)-prop-2- enylsulfinyl]propanoic acid, also known as S-allyl-L-cysteine sulfoxide) is the main active ingredient that contains sulfur in the intact plant[34].

2.1.1.1 Medicinal studies of Tulbaghia violacea Bungu et al. performed a study where Tulbaghia violacea was tested for the inhibition of growth and an induction of apoptosis in cancer cells in vitro[35]. The cells were stained with Hoechst 33342 to indicate fragmented nuclear material and condensed chromatin. It was concluded that the induction of apoptosis by Tulbaghia violacea bulb and leaf extracts was promising for anticancer therapy as it is desirable for anticancer agents to induce apoptosis[35]. Olorunnisola et al. tested the methanolic extracts of the Tulbaghia violacea rhizomes (dried and fresh), evaluating their antioxidant properties and cytotoxicity. The methodology used to determine the antioxidant activity was spectrophotometric methods using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide and lipid peroxidation scavenging activities and ferric reducing power assay. For toxicity evaluation, the brine shrimp cytotoxicity test was carried out. It was concluded that the rhizomes of Tulbalghia violacea may serve as potential source of natural antioxidant, antimicrobial and anticancer agents[36].

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Further studies were done by Davison et al. to investigate its organic extracts for cardiovascular benefits in terms of anticoagulant and anti-platelet properties. In the study the platelets were exposed to various extracts of the Tulbaghia violacea to test their effects on platelet aggregation, adhesion and protein secretion in both in vitro and ex vivo models. Their study demonstrated that an organic extract of the Tulbaghia violacea plant has a beneficial effect on the cardiovascular system similar to that of garlic[37]. Despite its benefits, it has been related with variable undesirable symptoms when extensively consumed, for example, abdominal pain, inflammation, and gastroenteritis[30].

An enzyme whose functional properties are similar to the alliinase found in garlic has been reported as being present in Tulbaghia violacea[38, 39]. In plants, the C-S lyases are involved in the primary and secondary metabolism. They cleave the bonds between sulfur and carbon atoms. They are a part of the larger family of aminotransferases with members like alliinases, cystathionine and cysteine lyases, cysteine desulfhydrase and enzymes that take part in the detoxification of xenobiotics after conjugation with glutathionine, and those that take part in glucosinolate and cyanogenic glucoside biosynthesis[40]. Alliinases are pyridoxal 5’-phosphate dependent α,β-eliminating lyases which catalyse the decomposition of S-alk(en)yl cysteine sulfoxide to ammonia pyruvate and thiosulfinates[24], as shown in Figure 2.3.

Figure 2.3 Reaction scheme of cysteine sulfoxide with alliinase enzyme to form thiosulfinate[41]

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2.1.2 Tulbaghia violacea chemistry The crushing, grinding or cutting of a garlic bulb results in the damage that permits the release of an enzyme, alliinase, which immediately transforms alliin into allicin, the source of the pungent smell of garlic[42]. Allicin is a thiosulfinate compound, said to be responsible for garlic’s biological activity[14]. Allicin decomposes rapidly because of its instability and is transformed into oil-soluble polysulfides, including diallyl sulfides, ajoenes and vinyldithiines[14] as shown in Figure 2.4. Vinyldithiines are thermal degradation products from allicin, as shown by Brodnitz et al.[43].

H N 2 H S S S S allinase O O O HO O S-allyl-L-cysteine sulfoxide 2-propenesulfenic acid allyl 2-propenethiosulfinate (allicin) (alliin)

O S S S S S S Glutathione S S S S

diallyl trisulfide (E)-ajoene 2-vinyl-4H-1,3-dithiin S-allylmercaptoglutathione

SH OH S allyl mercaptan O S S O S S S S NH S S 2 (Z)-ajoene S-allylmercaptocysteine S diallyl disulfide 3-vinyl-4H-1,2-dithiin allyl methyl sulfide

S

diallyl sulfide

S S allyl methyl disulfide

Figure 2.4 Conversion of alliin into allicin by the enzyme alliinase and of allicin into various sulfur-containing compounds[44]

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The identification of the two structures of the vinyldithiines was performed with Gas Chromatography (GC) and Gas Chromatography-Mass spectrometry (GC-MS). They were identified as 2-vinyl-[4H]-1,3-dithiin (1) and 3-vinyl-[4H]-1,2-dithiin (2) (see Figure 2.5)[45]. In 1983 Apitz-Castro et al. isolated ajoene from ether fractions in the extracts of garlic oil[46]. Ajoene assists in the prevention of growth tumour cells in vivo and in vitro[47]. It also has antifungal activities[48, 49]. Block and Ahamed determined the ajoene structure in 1984 as E and Z isomers of 4,5,9-trithiadodeca-1,6,11-triene-9-oxide[50, 44] (3, 4) as shown in Figure 2.6.

S S S CH2

CH2 S

3-vinyl-[4H]-1,2-dithiin 2-vinyl-[4H]-1,3-dithiin 2 1

Figure 2.5 Chemical structures of vinyldithiines[45]

O

S S S

Z-ajoene

3 O

S S S

E-ajoene

4

Figure 2.6 Chemical structures of ajoene[50]

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There are three scientific publications that described the chemical constituents of Tulbaghia violacea. The first one is by Jacobsen et al.[39] where they suggested the presence of three unidentified S-substituted cysteine sulfoxide derivatives. It was also suggested that the C-S lyase could play a role in the biosynthetic pathways that involve sulfur-containing compounds. The second one is from a publication by Burton and Kaye[51] where they isolated 2,4,5,7-tetrathiaoctane-2,2-dioxide (5) and 2,4,5,7- tetrathiaoctane (6) (shown in Figure 2.7) from the leaves of the Tulbaghia violacea plant. These compounds are disulfides which is a known functional group in devulcanizing agents. Kubec et al. [26] being the third publication where S-(methylthiomethyl)-cysteine- 4-oxide (7) (Figure 2.8), S-methyl cysteine (8) and S-ethyl cysteine (9) derivatives were isolated from the rhizomes of Tulbaghia violacea. S-(methylthiomethyl)-cysteine-4-oxide was found to be the major component of the extract.

O

S S S S S S S S O

2,4,5,7-tetrathiaoctane-2,2-dioxide 2,4,5,7-tetrathiaoctane 6 5

Figure 2.7 Chemical structures of the isolated compounds[51]

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O NH2

S S OH

O

S-(methylthiomethyl)-cysteine-4-oxide 7

NH2 NH2

S OH S OH

O O S-methyl cysteine S-ethyl cysteine 8 9

Figure 2.8 Chemical structure of cysteine derivatives[24]

2.2 Extraction methods Plant samples need to be prepared before the actual extraction process can take place. Samples such as leaves, roots, and bulbs, from fresh or dried plant material, can be used for extraction. Grinding and drying of these materials can influence the preservation of phytochemicals in the final extract as was stated by Azwanida[52]. This is brought about by the fact that grinding leads to smaller particles that enhance enzyme action. Fresh plant material deteriorates much more quickly and some phytochemicals will be lost. Both fresh and dried plant material is used in medicinal plants studies[52]. In this study, fresh plant material was utilised for the sulfur compounds identification test and the dried plant material was utilised for the plant extraction test.

There are different types of extraction methods such as maceration, infusion, percolation, digestion, decoction, Soxhlet extraction, sonication, counter current extraction, microwave-assisted extraction, aqueous-alcohol extraction by fermentation, and

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distillation techniques[53]. A juice extractor can be used which squeezes the juice out of a fresh plant material separating the pulp from the juice. There is no universal extraction method as each procedure is appropriate for different plants. Previously optimised methods assist in the selection of an ideal method. These methods also depend on the objectives, the plant material and the target compounds. Amagase et al.[54] mentions steam distillation method as the extraction method used to target diallyl sulfides in garlic.

2.3 Choice of solvent The plant material extract is largely dependent on the type of solvent used in the extraction procedure for successful determination of biologically active compounds. The choice of solvent is determined by what the extract is intended for[53]. Methanol, ethanol and water are solvents used to extract a wide range of compounds. For the purposes of the current study, the required common solvents must be able to extract oil from the plant material as the target compounds are oily liquids. Yoo et al.[55] performed a study for the selection of the extraction solvent. They were targeting the oil-soluble organosulfur compounds from commercial garlic macerated oil. The oil was vortexed and ultrasonicated for 5 minutes at room temperature. The solvents used were as follows: i) 2 – 15 % 2-propanol in n-hexane, ii) 10 – 40 % ethyl acetate in hexane, iii) 100 % n- hexane, and iv) 2 % 2-propanol in n-hexane. The study resulted in them choosing the 2 % 2-propanol in n-hexane solvent system, as the extracts of the mixture showed the highest level of oil-soluble organosulfur compounds and proved to increase the extraction efficiency. This solvent mixture was utilised in the quantification analysis test which was performed with Normal Phase High Performance Liquid Chromatography (NP-HPLC)[55]. In Normal Phase chromatography the stationary phase is polar and the mobile phase is nonpolar. For this type of chromatography, a typical stationary phase is silica. The least polar compounds elute first in NP-HPLC, and the most polar compounds elute last. The mobile phase is made up of non-polar solvents, such as n-heptane or n-hexane, combined with a slightly more polar solvent, such as ethyl acetate, 2-propanol or chloroform. This type of chromatography is used for compounds that are highly hydrophobic or are insoluble in water[56].

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2.4 Vulcanization overview Before Goodyear and Hancock’s discovery of vulcanization, there were limits to using rubber material because of its physical nature. Its properties changed with temperature, where it was hard in winter and soft and sticky in summer due to the large temperature changes[57, 58]. Goodyear developed a process called vulcanization where the rubber is heated with sulfur (Figure 2.9). He also added accelerators to minimise processing time[57]. An accelerator is a chemical added into a rubber compound to allow vulcanization to take place at lower temperatures and to increase the speed of vulcanization. Accelerators also decrease the quantity of sulfur needed for vulcanization; thereby improving the properties of the rubber vulcanizates[59]. In the absence of an accelerator each crosslink needs 40 to 55 sulfur atoms because the chemical reaction between sulfur and the rubber hydrocarbons occurs mainly at the carbon to carbon double bonds[59]. The process is not economically feasible by any production measure as it takes approximately 6 hours at 140 oC to complete[59].

Figure 2.9 Vulcanization process[59]

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Accelerated vulcanization is a process that occurs in 3 stages: i) scorch time or induction where different chemical reactions take place, except crosslinks. At this stage there must be enough delay or scorch resistance to allow mixing, shaping, forming in the mould before vulcanization. ii) Curing or crosslinking stage where the rubber changes from a soft plastic to an elastic material. iii) the post cure region where the modulus can increase, decrease or remain the same depending on the temperature of the actual vulcanization system[60, 61] as illustrated in rheometer curve (Figure 2.10).

Figure 2.10 Rheometer curve of different vulcanization stages[62]

Vulcanized natural rubber is approximately 10 times stronger and more firm than natural rubber. Its elasticity gives it an advantage over unvulcanized rubber by stretching reversibly[63]. The summary of the effects of vulcanization on different mechanical properties of rubber is shown in Figure 2.11. Unvulcanized rubbers possess poor

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properties both physical and chemical. Polymeric networks of unvulcanized rubber are in an unsystematic order and are held jointly by their entangled complex state and by the weak electrostatic London and van der Waals forces[64].

Figure 2.11 The effect of vulcanization on some properties of rubber summary[65]

Currently, there are many different methods used to introduce crosslinks to materials both chemical and physical[64]. These methods consist of the use of sulfur, peroxides, special vulcanizing agents and high-energy irradiation[64, 66]. The most used method is the sulfur accelerated method despite its limits to rubbers with unsaturated carbon bone. It has an advantage of having networks that display better mechanical properties and also suggests the economic feasibility[64].

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2.4.1 Accelerated sulfur vulcanization Accelerated sulfur vulcanization chemistry is complicated because the crosslinking, desulfuration and degradation reactions take place simultaneously[59]. It is generally accepted that during the accelerated sulfur vulcanization process (Figure 2.12) there are reactions between the vulcanization ingredients to produce an active sulfurating agent[67]. The active sulfurating agent reacts with rubber hydrocarbons to form crosslink precursors. Reactions between crosslink precursors and the elastomer chain finally produce crosslinking.

Vulcanization ingredients (sulfur, accelerators and activators)

Active sulfurating agent Rubber hydrocarbon

Rubber bound intermediate (crosslink precursor)

Initial polysulfidic crosslinks Network maturation reaction 1. Crosslinking shortening with additional crosslinking 2. Crosslink destruction with main chain modification (dehydrogenation and cyclic sulfide formation) 3. S-S bond interchange

Vulcanizate network

Figure 2.12 Simplified reaction scheme for accelerated sulfur vulcanization[68, 69]

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Vulcanization of rubber by sulfur is necessary for the production of tyres, suitable for cars and aircrafts which are essential to modern society[70]. The vulcanization process produces the sulfur bridges between rubber molecules resulting in an elastic three- dimensional network[70]. Typical chemical structures, formed in the vulcanizate network during accelerated sulfur vulcanization scheme are shown in Figure 2.13. The cross- linked network structure of vulcanized natural rubber contains cyclic and mono, di and polysulfidic linkages[71]. The cross-linked sulfur-sulfur bonds are weakest bonds in the diene rubber and through an interchange chemical mechanism they are responsible for oxidative degradation[71, 72].

S S S

S S X x

Sy S S

Figure 2.13 Structure of the vulcanizate network[62, 73] (X = accelerator residue, y≥1, x≥3)

2.4.2 Model compound vulcanization Model compound vulcanization is used to simplify the complicated accelerated sulfur vulcanization process, explained in section 2.2.1. It is performed by vulcanizing a model molecule which has a chemical structure similar to that of real rubber. The model molecule has a lower molecular weight than rubber which allows the application of solution-based analytical techniques[74]. The model compound vulcanization samples are still in liquid form after vulcanization making them soluble in most solvents. This allows better sample

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handling and application of a number of analytical techniques[64]. Figure 2.14 shows examples of model compounds for rubber according to van Rooyen, cited from Boretti and Woolard[64, 75].

In model compound vulcanization, not all the ingredients for vulcanization are used, unlike the vulcanization of rubber; thus making it easy to concentrate on the more active role- playing intermediates and products. The whole process of vulcanization is covered from the formation of the active sulfurating agents, to the precursor formation up to the crosslink shortening and the formation of vulcanizate network, mentioned in Figure 2.12[76].

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cyclohexene 2-methyl-2-pentene (2MP) 2-ethylidene norbonane (ENBH)

10 11 12

trans-3-hexene trans-2-hexene 1-decene 13 14 15

OH 2,3-dimethyl-1-butene 2,3-dimethyl-2-butene (TME) geraniol 16 17 18

squalene 19

cycloocta-1,5-diene cyclohexadeca-1,5,9,13-tetraene 20 21

Figure 2.14 Examples of model compounds[64, 75]

Choosing a model compound is important to obtain results that can be analysed with ease as well as a molecule that can be taken as an effective model for the system being studied[64]. 2,3-dimethyl-2-butene (TME) is one of the most used model compounds. It is

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a general model compound even though it is not directly comparable with any specific rubber. Another compound that has been used for vulcanization mechanism studies is squalene[62]. Boretti and Woolard[75] found an effective model for cis-1,4-polyisoprene to be squalene because it has at least two isoprene units allowing for the reaction at the adjacent methylene carbons, and it mimics the polyisoprene well. There is one difference between squalene and polyisoprene rubbers. The double bonds are trans and not cis as in natural rubber and cis-1,4-polyisoprene[61, 62]. Unlike TME, it requires analysis with techniques that are less sensitive than HPLC such as size exclusion chromatography (SEC).

Scheele et al.[77] used geraniol as a model compound in 1956. It has a disadvantage of containing an alcohol group and that can lead to side reactions. Rajan et al.[78] performed a study in two stages where TME was vulcanized using a mixture of sulfur, zinc stearate and N-cyclohexyl-2-benzothiazylsulfenamide(CBS) as an accelerator at 140 oC in the first stage. The results were a mixture of addition products (C6H11-Sx-C6H11). HPLC was used to separate and identify the resulting products with respect to their sulfur rank (number of sulfur atoms in the crosslink bridges). For the purpose of this study, TME was chosen as the best model because it is a highly symmetrical model molecule. TME has a structure that is similar to a polyisoprene monomer[64]. It has a simple product mixture when vulcanized because of the equivalent allylic positions[79]. This model compound was used to check whether the crosslinking took place with the assistance of an HPLC, FTIR and NMR. The devulcanization process was tested using both HPLC and NMR on the vulcanized products of TME.

2.5 Devulcanization overview An important subject for the rubber industry is the devulcanization of rubber waste, particularly car tyres where potential methods are of great value and demand[70]. The first step of the devulcanization process (Figure 2.15) is the conversion of polysulfide and disulfide bonds to monosulfide bonds by heat. Next, there is an addition of shear stress to break the monosulfide bond. Lastly, devulcanized rubber is obtained[80].

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Disulfide Main chain bond Monosulfide H2S SO2 CS2 Polysulfide bond bond

SH S S Heat SH Shear Stress SH S S S Sx S SH S SH SH

Vulcanized rubber Devulcanized rubber

Figure 2.15 Crosslinking bond breakage mechanism[80]

As mentioned in section 2.2.2, Rajan et al. [78] had two stages in their study. The second stage was to devulcanize the vulcanized products using aromatic disulfides and aliphatic amines at 200 oC. It was concluded that the devulcanizing agents decomposed sulfidic vulcanization products with sulfur ranks equal to or higher than 3 quite effectively and with comparable speed. It was also observed that diphenyldisulfide as a devulcanizing agent gave rise to mono- and disulfidic compounds formed during devulcanization, whereas hexadecylamine prevented those lower sulfur ranks from being formed.

Jana and Das conducted a study where they devulcanized the vulcanized natural rubber by mechanical milling at high temperatures utilizing diallyl disulfide as devulcanizing agent. A comparison study of the properties of vulcanized natural rubber and revulcanized natural rubber was performed. They were able to devulcanize vulcanized rubber through a mechano-chemical process. The mechanical properties of the revulcanized natural rubber was found to be dependent on the concentration of the DADS[81]. In a study by De et al.[5] a vegetable product and DADS were used to reclaim sulfur cured natural rubber vulcanizates under various reclaiming conditions. Estimation of the sol and gel portions, determination of sol portions, and Mooney viscosity of the reclaimed rubber was performed to monitor the progress of reclaiming. They observed an influence of reclaiming agent and milling conditions on the molecular weight of the sol, sol content, and viscosity of the reclaim rubber.

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A recent study performed by Joseph et al.[82] on the current status of sulfur vulcanization and devulcanization chemistry with a focus on devulcanization, provided a proposed mechanism employing disulfides as devulcanizing agents. This is illustrated in Figure 2.16. The first step is where diphenyl disulfide undergoes thermal degradation forming reactive radicals that attack the crosslinks. The second step is when the reactive radicals break the sulfur crosslinking bond. Thirdly, there is the formation of a new active crosslinking site to ensure good revulcanization of the devulcanized sample. Lastly, the regenerated rubber can be revulcanized alone or combined with fresh rubber in the revulcanization process. In the current study, the disulfides extracted from the Tulbaghia violacea were tested for potential use as devulcanizing agents.

Boyce[83] performed a devulcanization study of sulfur vulcanized natural rubber utilising a variety of diphenyldisulfides. 2-aminodiphenyldisulfide, 4-aminodiphenyldisulfide, bis(2- benzamido-)diphenyldisulfide and 2,2’-bithiosalicylic acid were utilised as aromatic disulfides devulcanizing agents. TME and squalene were the model compounds used for the study, they were vulcanized by sulfur and 2-bisbenzothiazole-2,2’-disulfide. Thermal analysis, thermogravimetric analysis, and differential scanning calorimetry were used for analysis in this study. It was observed that 4-aminodiphenyldisulfide was the most effective of the devulcanizing agents. An observation on the substitution of basic amino group with an acidic carboxylic acid group had no detrimental effect on the devulcanization efficiency.

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Step I S 120 °C n S S 2n Mechanical shearing Diphenyl disulfide Sulfide radical

Step II CH2 H2C H H C S  2 S  H C H S + 2 S S CH2 S CH2 CH2 + CH2

Step III HC H H S SH CH H2C S + H S + + H2C New active crosslink site

Step IV

HC H S HC H C HC 2 S H + H C H Sulfur + CBS 2 CH H2C 150 °C H H2C

Figure 2.16 Devulcanization mechanism using disulfides[82]

2.6 Conclusion The Tulbaghia violacea plant used for this study is said to be rich in sulfur. Success in finding these compounds depends on the extraction method and solvents employed. The discovery of an effective solvent and an extraction method for sulfur-containing compounds from a Tulbaghia violacea plant, will lead to a crude extract with the targeted compounds required for the study. Scientific evidence of the effectiveness of the sulfur-

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containing compounds in devulcanizing a model compound needs to be provided. The next chapter will outline the research methodology used to carry out the investigation.

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CHAPTER THREE EXPERIMENTAL

This chapter presents an overview of the specific methods that were followed in this research study. Experiments covered in this chapter relate to the aims of the project, which are stated in section 1.2.

3.1 Materials Table 3.1 has a list of the materials and reagents that were used to perform the current study. All solvents and/or reagents were used as purchased.

3.1.1 Plant material Tulbaghia violacea was collected from the Horticulture Department at the Nelson Mandela University in February 2016. The plant was harvested by Ms Elana Storm from the Nelson Mandela University garden. After the plant was washed under running tap water to remove dirt and soil, the specimen for positive identification was prepared. The voucher specimens were then deposited in the Department of Botany Herbarium (Nelson Mandela University) and it was given a voucher specimen number of PEU25230.

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Table 3.1 Materials and reagents

Materials and Reagents Suppliers Tulbaghia violacea (plant material) Nelson Mandela University Horticulture Department, Port Elizabeth, South Africa 2,3-Dimethyl-2-butene (TME) Sigma-Aldrich, St Louis, United States of America Diallyl sulfide (97.0 %) standard Sigma-Aldrich, St Louis, United States of America Diallyl disulfide (98.0 %)standard Sigma-Aldrich, St Louis, United States of America n-Hexane (98.0 %) Merck, Darmstadt, Germany 2-Propanol (99.9 %) Merck, Darmstadt, Germany

TLC Silica gel 60 F254 Aluminium plates Merck, Darmstadt, Germany Glacial acetic acid Merck, Darmstadt, Germany Vanillin BDH Chemicals, Radnor, United States of America Acetonitrile (99.5 %) Merck, Darmstadt, Germany Dichloromethane (99.0 %) Merck, Darmstadt, Germany Methanol (99.9 %) Merck, Darmstadt, Germany Deuterated chloroform (99.8 %) Merck, Darmstadt, Germany Toluene (99.8 %) Sigma-Aldrich, St Louis, United States of America Ethyl acetate (99.5 %) Merck, Darmstadt, Germany Sulfuric acid (98.0 %) Merck, Darmstadt, Germany Sulfur Associated Chemical Enterprises (ACE), Southdale, South Africa 2-Bisbenzathiazole-2,2’-disulfide (MBTS) Orchem, South Africa Zinc oxide (ZnO) Merck, Darmstadt, Germany

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3.2 Instruments

3.2.1 Balances The following three balances were used:

Microbalance - Mettler Toledo MX5 (1x10-6g) Top pan balance - Mettler Toledo BB 240 (1x10-3g) Top pan balance - Mettler Toledo AB 204-S (1x10-4g)

3.2.2 Fourier-transform infrared spectroscopy (FTIR) Infrared spectra of all the compounds were collected using a Bruker Tensor 27 FTIR instrument with a Bruker Platinum ATR which contains a single reflection diamond crystal, and Opus data collection program in an attenuated total reflectance. 16 scans for background and 32 scans for sample with a scanner velocity of 10 kHz.

3.2.3 High Performance Liquid Chromatography (HPLC) HPLC was utilised to analyse, identify, quantify the crude extracts, and to analyse vulcanizates and devulcanizates. An Agilent Technologies 1290 Infinity system, fitted with a 1290 Infinity Binary pump, an Agilent 1290 Infinity thermostatted (10 oC below ambient up to 100 oC) column compartment and an Agilent 1290 Infinity Diode Array Detector controlled by Agilent Chemstation software. Two types of columns were used for analysis in this study, a Waters XSelect HSS C18 5 µm 250X4.6 mm column for reverse phase chromatography and a Luna 5 µm Silica (2) 100 Å 250X4.6 mm column for normal phase chromatography.

3.2.4 Nuclear Magnetic Resonance Spectrometry (NMR) NMR analysis was carried out on a 400 MHz Bruker Avance Ultrashield Plus 400 spectrophotometer for solutions in deuterated chloroform (CDCl3). Tetramethylsilane (TMS) was used as an internal reference standard.

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3.2.5 Differential Scanning Calorimetry (DSC) Differential Scanning Calorimetry experiments were conducted in a TA Instruments Discovery Series DSC. High purity nitrogen gas (Nitrogen baseline 5.0 from AFROX, South Africa, Johannesburg) was used as the purge gas at a flow rate of 50 mL/min. The calibration of this instrument was performed in three steps; firstly the cell constant where there was an empty aluminium pan as a reference. Secondly, temperature and heat flow was calibrated with Indium (melting point 156.60 oC, measured melting point 157.66 oC) and lastly the heat capacity was calibrated with a sapphire crystal of known mass and heat capacity at 20 oC/min.

3.3 Experimental Procedure

3.3.1 Plant Extraction The plant material was cut to separate the leaves, bulbs and roots. They were individually cut into smaller pieces except for the bulbs. These were then dried in an oven at approximately 40 oC for 72 hours. Two extraction methods (I and II) were used.

3.3.1.1 Method I The roots, bulbs and leaves were prepared separately. In this method the plant material was weighed as shown in Table 3.2 below. The plant material was then stirred in n- hexane (500 mL) only for 24 hours at room temperature. The resulting crude extract was filtered with a Buchner funnel and the organic solvent was removed from the extract with the aid of a rotary evaporator to afford crude mass as shown in Table 3.2.

Table 3.2 Yield of the crude extracts of n-hexane

Plant parts Dried plant mass (g) Crude mass (g) Yield (%) Bulbs 91.0289 0.9367 1.03 Leaves 29.0514 0.8654 2.98 Roots 24.0115 0.6345 2.64

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3.3.1.2 Method II The roots, bulbs and leaves were prepared separately. In this method the plant material was placed in a cellulose thimble in an extraction chamber (as illustrated in Figure 3.1), which was placed on top of a collecting flask beneath a reflux condenser. A mixture of 2 % 2-propanol in n-hexane (200 mL) was added to the flask. The solution was then heated under reflux at 65 oC for 24 hours. The solvents were siphoned into the flask beneath when the condensed solvent had accumulated in the thimble. When the extraction process was finished, the solvent being siphoned was checked if it still had a residue by sampling a small amount and placing it on top of a clean watch glass and evaporated against a clean unused solvent. If they both looked the same, then the extraction was finished. The resulting extraction mixture was concentrated with the aid of a rotary evaporator to afford the crude extracts as shown in Table 3.3.

Figure 3.1 Soxhlet extractor apparatus

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Table 3.3 Yield of the crude extracts of 2 % 2-propanol in n-hexane

Plant parts Dried plant mass (g) Crude mass (g) Yield (%) Bulbs 450.6796 5.6450 1.25 Leaves 148.0954 4.5422 3.07 Roots 119.0505 4.7622 4.00

3.3.2 Sulfur-containing compounds phytochemical test Fresh plant leaves, roots and bulbs were cut into smaller pieces. These were extracted using a juicer as shown in Figure 3.2. The resulting plant juice was diluted with dichloromethane in the ratio of (1:10) and then approximately 20 µL of this extract was utilised for Thin Layer Chromatography (TLC). 10 mg of both diallyl disulfide and diallyl sulfide standard samples were dissolved in dichloromethane (2 mL). The solutions were spotted on a silica gel 60 F254 TLC plate. The plate was impregnated in a (10:3) toluene:ethyl acetate solvent system, dried at room temperature and viewed under ultraviolet (UV) radiation prior to the staining. The plate was then sprayed with 10 mL of the stain reagent (vanillin (0.8 g) dissolved in glacial acetic acid (40 mL) and concentrated sulfuric acid (2 mL)) and the TLC plate was heated for 3 to 5 minutes at 100 oC to obtain results.

Figure 3.2 Juicer, fresh plant material and juices extracted.

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3.3.3 HPLC analysis of crude extracts The solvents used for this experiment are HPLC grade. The method from Yoo et al. [55] was adapted for quantitative analysis of the crude extracts. Normal phase chromatography was employed to achieve chromatographic separation of compounds using a silica column at 30 oC. A gradient system using n-hexane (mobile phase A) and 2-propanol (mobile phase B) was utilised. Table 3.4 gives a gradient system with flow rate changing from 0.7 – 1.0 mL/min. The injection volume used was 5 µL and the detection was accomplished at 240 nm using a diode array detector.

Table 3.4 HPLC conditions for the analysis of organosulfur compounds

Time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%) 0 0.7 100 0 20 0.7 100 0 22 0.8 98 2 24 0.9 95 5 26 1.0 92 8 28 1.0 90 10 45 1.0 90 10

3.3.3.1 Preparation of standard stock solution A primary standard of diallyl disulfide (DADS) with 98 % purity was used to prepare standard stock solutions. DADS (104.0 mg) was weighed and directly added into a 10 mL amber volumetric flask (to prevent light from entering the flask as it might degrade the contents in the flask) diluted and made up to volume with 2 % 2-propanol in n-hexane. Four further dilutions (1.04 mg/mL, 0.104 mg/mL, 0.0104 mg/mL and 0.00520 mg/mL) were prepared from the stock solution. These were filtered through a 0.2 µm syringe glass fibre filter and transferred into an amber HPLC vial before injecting into the HPLC instrument.

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3.3.3.2 Sample preparation The bulbs, roots and leaves crude extracts were weighed directly into a 10 mL volumetric flask. Their masses were as follows: i) 98.9 mg bulbs; ii) 102.6 mg roots; iii) 104.7 mg leaves. These were diluted and made up to volume with the mixture of 2 % 2-propanol in n-hexane. The solution was then filtered through a 0.2 µm syringe glass fiber filter and transferred into an amber HPLC vial before injecting into the HPLC instrument.

3.3.4 Vulcanization of 2,3 dimethyl-2-butene (TME) The vulcanization experiment was carried out using a recipe similar to that used by Morgan[84] (see Table 3.5) and the procedure adapted from Rajan et al.[78] and Boyce[83]. TME was washed with aqueous iron (II) sulfate to remove unwanted peroxides prior to mixing. All the components of the recipe were mixed in thick walled glass ampoules with small stirrer bars. Before closing the ampoules, the mixtures were degassed via freeze- thaw pump method. The sample was frozen with liquid nitrogen and vacuum pumped for a minute, the vacuum was then closed for the thawing process. The degassing was repeated three times to make sure there was no oxygen present, as this would cause oxidation of the double bond in TME. The ampoules were then sealed by melting the glass neck under vacuum.

Table 3.5 Model compound vulcanization recipe

Component Amount (mol) TME 33.9 Sulfur 1.1 2-Bisbenzothiazole-2,2'-disulfide (MBTS) 1.1 ZnO 1.0

The vulcanization was done by immersing the ampoules in an oil bath, preheated at 140 oC, as illustrated in Figure 3.3. A calibrated thermometer was fitted to constantly check the temperature of the oil. The ampoules were put inside the oil bath and the vulcanization reactions carried out for 2, 10, 20, 40, and 60 minutes. During the reactions, mixtures

33

were continuously stirred to make sure they stayed homogeneous. After the experiment the reactions were stopped by quenching the mixtures in an ice bath followed by liquid nitrogen. The reaction products were analysed by FTIR, NMR and HPLC.

Figure 3.3 Vulcanization setup

3.3.4.1 HPLC analysis of vulcanizates The ampoules were broken with long nose pliers. Approximately 30.0 mg of the vulcanizates was dissolved in acetonitrile (2.5 mL). The mixture was filtered through a 0.45 µm porous filter before it was transferred into a vial. Table 3.6 shows the HPLC conditions utilized in analysing the reaction products.

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Table 3.6 HPLC measurements

Column Reverse phase column ( section 3.2.3) Mobile phase 97 Acetonitrile : 3 water (vol%) Flow rate 1 mL/min Temperature 23 oC Detector UV Wavelength 254 nm Injected volume 20 µL Length of column 250 mm Internal diameter of the column 4.6 mm

Retention time of the components given in Table 3.7 was measured by injecting a solution of a single ingredient into the HPLC instrument.

Table 3.7 The retention time of the initial components table

Component Retention time (approximate minutes) Mobile phase peak_1 3.0 Mobile phase peak_2 9.4 Mobile phase peak_3 12.4 MBTS 6.2 Sulfur 14.2

3.3.5 Devulcanization For devulcanization experiments, 3 respective portions of the vulcanized product (approximately 0.5 g) were added into different ampoules, and into each a different extract was added (0.7 g). The same setup, as shown in Figure 3.3, was used but the temperature was set at 200 oC. The experiment was carried out for 30 minutes. This choice of experimental conditions was based on Rajan et al.[78].

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3.4 Data presentation The data on the concentration of the extractable products for the HPLC analysis are indicated in the text as a mole percentage of the original curatives added.

.

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CHAPTER FOUR RESULTS AND DISCUSSION

4.1 Identification of disulfides on crude extracts

4.1.1 Method I The ability of a solvent to extract an oil fraction as a requirement is discussed in section 2.3. The solvent choice for the extraction method in this study was based on the findings of Amagase et al.[54] that the oil fraction from garlic cloves can be extracted with n-hexane at room temperature. Literature in section 2.1.1 suggested similarities between garlic and Tulbaghia violacea which are both in the Allium genus; therefore the extraction process for the leaves, roots and bulbs of Tulbaghia violacea was performed using n-hexane. n- Hexane extracts were analysed by TLC using a n-hexane:ethyl acetate (80:20) solvent mixture and then developed using a vanillin locating reagent. Reference standards (diallyl sulfide and diallyl disulfide) were spotted against the crude material on TLC plate. The spots observed from the crude extracts were not clear and did not correspond to the reference standards. This was an indication of the non-detection of diallyl sulfides and diallyl disulfides in the crude extracts. Based on the TLC results, it was then concluded that the method and solvent used were not suitable for the targeted compounds. Since sulfides are the compounds of interest, another solvent and extraction method had to be investigated.

4.1.2 Method II The study performed by Yoo et al.[55] for the selection of the extraction solvent suggested 2 % 2-propanol in n-hexane solvent system as a potential solvent of choice for this study. Nonetheless, their extraction method was deemed not suitable for plant materials as they were extracting from a finished product in a capsule form. In section 2.2 it was noted, based on Amagase et al.[54], that steam distillation can be used to extract DADS. However, in a study performed by Govender[85] in which Soxhlet extraction and steam distillation for the curry leaf plant were compared. It was observed that both techniques

37

showed similar quantities of the extract. Steam distillation extracts will have water content which may cause problems in the devulcanization application step of this study. Consequently, Soxhlet was the extraction method chosen for this study since an appropriate solvent to target DADS had already been chosen. The resulting crude extracts were analysed using TLC. In the TLC chromatogram in (Figure 4.1), the DADS reference standard is denoted by B, DAS reference standard is denoted by A, bulb extract denoted by C, leaves extract denoted by D, and the roots extract is denoted by the letter E. A suitable separation of the compounds was reached when the TLC plate was developed in a n-hexane:ethyl acetate (80:20) solvent system. Reference spots, consistent with A, were clearly seen on the bulbs and leaves extracts, whereas in the roots extract they were faint. This observation might be due to the small quantity of the compounds that correspond to each of the standards in the roots extract. Unfortunately DADS was observed at the solvent front which made its identification in the extracts by TLC uncertain.

Figure 4.1 TLC chromatograms for Tulbaghia violacea crude extracts in a 2 % 2- propanol in n-hexane solvent mixture, developed in a hexane:ethyl acetate (80:20) solvent system

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4.2 Sulfur-containing compounds phytochemical analysis The phytochemical analysis procedure was adapted from Wagner and Bladt[86]. A TLC method using dichloromethane was followed to provide suggestive evidence for relevant sulfur-containing compounds present in the freshly juiced leaves, bulbs and roots. The resulting TLC chromatograms in comparison to the diallyl disulfide (DADS) standard and the diallyl sulfide (DAS) standard are presented in Figure 4.2. The first TLC plate on the left was viewed under ultraviolet light at 254 nm. A spot at a similar Rf (retention factor) to that of the DADS standard was visible in the bulb and leaves extracts but was very faint in the roots of the fresh extract. Another observation made was that of the spot of the DAS standard appeared very intense when compared to that of the DADS. The extracts seem to exhibit at a similar Rf value. A number of other non-polar spots, besides the DAS spot, were observed from the bulbs extract; one of which was particularly intense. The leaf extract gave evidence of a dark green compound-specific zones for chlorophyll with

Rf = 0.667. The intense peak in the bulbs extract was much weaker in the leaves extract. It also provided a faint spot in the roots extract.

The TLC chromatogram was treated with a vanillin-glacial acetic acid stain and heated in an oven (Figure 4.2, plate on the right). Spots corresponding to the DADS were observed at the solvent front as blue spots as per Wagner and Bladt method[86]. Gitin et al.[87] also observed diallyl disulfides to be at the solvent front but brownish in colour. The different observations in colour may be due to the different staining reagent preparation. Gitin et al.[87] prepared the staining reagent without sulfuric acid in it whereas Wagner and Bladt[86] had both the glacial acetic acid and the sulfuric acid. It can be seen that juiced bulbs, roots, and leaves have spots with similar Rf values as the DAS and DADS reference standards.

In order to increase the yield of the crude extract, the freshly juiced extraction method was repeated. The results, however, did not deviate significantly from those of method II. In fact, the Soxhlet extraction proved to be a much better method of extraction with better yields as shown in Table 3.1 (section 3.3.1). The choice of solvent for extraction when the

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plant was juiced resulted in compounds which appeared weak on TLC plate viewed under UV; thus, the method was not investigated further.

Figure 4.2 TLC chromatograms for dichloromethane extracts of the fresh leaves, bulbs and roots of Tulbaghia violacea

In the FTIR spectra, a broad peak at 3348 cm-1 frequency, consistent with the presence of water, was observed. During the juicing process, the plant was not dried in an oven leading to the observation of a water peak in the spectra of freshly squeezed material. Difficulties were also experienced in obtaining significant amounts of juiced material, despite juicing a large amount of bulbs and roots. Therefore, a Soxhlet extraction method was followed to provide a water free extract.

4.3 FTIR of crude extracts An FTIR spectrum of the Tulbaghia violacea bulb extract is shown below in Figure 4.3. There is no –OH stretch in the region of 3100-3700 cm-1 which is indicative of the absence of water as well as other –OH containing compounds such as alcohols and carboxylic

40

acids[88]. The sulfides that were targeted consists of bonds like C=C, =C-H, C-S, S-S, and C-H associated with them. In the region of approximately 1460 cm-1, a C-H bending frequency was observed. This vibration indicates the possible presence of methylene groups. The presence of olefinic vinyl C-H bending is more difficult to discern as this usually occurs at lower frequency (1420-1410 cm-1)[88] which would mean it would be obscured as a shoulder on the C-H bend. Shoulders are present in Figure 4.3 but their indication of the presence of vinyl groups is not definitive. At approximately 710 cm-1, a band, that may be associated with the stretching of C-S bonds[89], can be observed. S-S bonds are typical observed in the region of 450-550 cm-1[89]. A peak can be seen at 490 cm-1, consistent with the presence of disulfidic (S-S) linkages. The three peaks that are in the region of approximately 2800-3000 cm-1 were both an indication of C-H bonds. These bands are likely associated with the presence of alkyl groups. No clear presence of alkene C=C stretches can be seen near 1600 cm-1. Significantly, no thiol (–SH) group stretch near 2550 cm-1.

Figure 4.3 FTIR spectrum of from the bulb extract

41

Strong bands can be seen near 1040 cm-1. Such strong bands are typical, although not definitive, evidence of S=O bonds which may be found in sulfoxide and sulfone compounds. As noted earlier Burton and Kaye[51] isolated 2,4,5,7-tetrathiaoctane-2,2- dioxide, commonly known as marasmicin, from Tulbaghia violacea which contains such groups.

Structures that contain cysteine residues may be eliminated as there is no N-H stretches at 3300-3500 cm-1. These observations were an indication of some of the sulfur- containing and other functional groups that were found in the crude extract of the bulb. The same observations were evident on the leaves and root extracts presented in Appendix A, Figure A.1 and Figure A.2, respectively. There were some similarities observed between the leaves and bulb extract in the 1000-1200 cm-1 region indicating the presence of similar or the same compounds. The FTIR spectrum of the root extract was more different, however. This is consistent with observations of the TLC plates (see Sections 4.1.2 and 4.2). FTIR alone is not sufficient to make a conclusive decision. Therefore, additional tests such as HPLC identification and quantification were performed to support the findings of this experiment.

4.4 Quantification of DADS by HPLC FTIR analysis suggested the presence of sulfur-containing compounds that might be present in the crude extracts of Tulbaghia violacea plant. However, a more powerful tool was needed where the isolation of single compounds would be avoided as the application of the current study utilises the whole crude extract at this stage. A normal phase silica column was employed in HPLC with a diode array detector to separate and quantify some of the sulfur-containing compounds. DADS standard was used as a reference standard. These sulfur-containing compounds are insoluble in water, making normal phase a suitable chromatographic technique (section 2.3). Only the DADS reference standard was used for quantification as it was an HPLC grade standard. The DAS reference standard used for TLC phytochemical analysis on the other hand was not of an HPLC grade. It had secondary spots even as seen in figures above (refer Figure. 4.1 and Figure 4.2). The organosulfur compounds present in the crude extracts of the bulbs, leaves and roots were

42

successfully separated by the use of HPLC. Figure 4.4 shows a chromatogram of the injection of the solvent (retention times of 5.7 minutes and 26.6 minutes) used to dilute the analytes. There are three peaks observed. These peaks were excluded from the identification of compounds as they were detected in all samples depending on the concentration of the analyte. Note though the low response (mAU) compared to the primary peaks in the chromatograms of the extracts that follow.

Figure 4.4 HPLC chromatogram for 2 % 2-propanol in n-hexane solvent

Figure 4.5 (a) and (b) shows the chromatograms of the reference standard and bulbs crude extract, respectively. The peaks, present in the solvent, were detected in both chromatograms, but because of the concentration of the reference standard they are not as pronounced as they are on the crude extract. The retention time for DADS was about 4.67 minutes as observed on the chromatogram on Figure 4.5 a). A corresponding retention time was also observed for the bulbs crude extract at 4.64 minutes. This is an indication of the possibility of the presence of diallyl disulfide in the crude extract. Table 4.1 shows the analytes, retention times and areas corresponding to DADS reference standard. Analytes being the plant bulbs, leaves and roots crude extracts. The crude extracts were injected twice and an average of the two results is reported in Table 4.1. From the standard concentrations in section 3.3.4.1, a calibration curve (Figure 4.6) was constructed with peak area being a dependent variable.

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Figure 4.5 Chromatograms of a) DADS reference standard and b) bulbs crude extract

Table 4.1 Analyte, retention time and area of the peaks

Standards and analytes Retention time (minutes) Area (mAU) Standard 1 (0.00520 mg/mL) 4.65 25 Standard 2 (0.0104 mg/mL) 4.65 51 Standard 3 (0.104 mg/mL) 4.67 342 Standard 4 (1.04 mg/mL) 4.67 3524 Standard 5 (10.4 mg/mL) 4.67 27673 Roots 4.65 78 Bulbs 4.64 207 Leaves 4.62 99

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Figure 4.6 Graph of peak area versus concentration

Peak area = 2668.1[concentration], from Figure 4.6. Table 4.2 gives the concentrations and percentages of the DADS in different parts of the plant material.

Table 4.2 DADS concentration and percentages of extracts in different plant parts

Plant parts Concentration (mg/mL) Percentage (%) Bulbs 7.74X10-2 0.78 Leaves 3.69X10-2 0.35 Roots 2.93X10-2 0.29

Oil-soluble organosulfur compounds were successfully separated. Yoo et al.[55] quantified most of the compounds using reference standards. Due to the unavailability of HPLC grade reference standards other sulfur-containing compounds could not be quantified. The identification and quantification of such compounds, possibly including

45

marasmicin, could form the basis of a follow-up study. In normal phase chromatography polar compounds elute first as mentioned in section 2.3, In Figure 4.5 b) there are two additional peaks that elute in the first 5 minutes, they are associated with the other oil- soluble organosulfur compounds.

Evidence has been provided that suggests the presence of the sulfur-containing compounds in the crude extracts of the Tulbaghia violacea plant. The sulfur-containing compounds discussed above are part of the sulfur compounds that were not quantified due to the unavailability of reference standards. The crude extract containing the collection of sulfur-containing compounds may be able to play a role in the devulcanization process as potential devulcanizing agents. Not only has diallyl disulfide (DADS) been shown to be a devulcanizing agent [5] but it is also likely that other compounds such as 2,4,5,7-tetrathiaoctane-2,2-dioxide, isolated by Burton and Kaye[51] may also act as a devulcanizing agent because it contains a disulfide linkage which is a known functional group in devulcanizing agents.

4.5 Vulcanization process The vulcanization process was included in this study for the purpose of testing the efficiency of the organo-sulfur compounds extracted from the Tulbaghia violacea plant. No detailed explanation is given about the process itself, only crosslink formation is described. This is because the vulcanization of TME, used as a model compound has been widely described elsewhere[90].

4.5.1 FTIR of vulcanizates The vulcanizate was analysed to check for the carbon to sulfur bond which will give an indication of whether the reaction took place. The model compound TME which was vulcanized was also analysed to demonstrate the absence of C-S and S-S bonds. FTIR spectra for both the TME and the vulcanizate are provided in Figure 4.8 (a) and (b), respectively. In the spectrum for the vulcanizate a medium, yet broad, band at approximately 530 cm-1 is consistent with the presence of S-S bonds. The broadness is

46

likely due to the fact that the vulcanizates contains not only disulfides but also trisulfides, tetrasulfides and other polysulfides[64, 75]. At approximately 670 cm-1 a sharp band appears in the spectrum of the vulcanizate, indicative of C-S bonds

Figure 4.7 FTIR spectra for a) TME and b) vulcanizate

47

When comparing the FTIR spectrum of TME with that of the vulcanizate, it can be observed that there are no bands that match any of the sulfides bands found in the vulcanizate spectrum. FTIR provides evidence of changes, consistent with vulcanization.

4.5.2 Vulcanization results The following results provide evidence of the crosslinks formed during the vulcanization reaction. An HPLC chromatogram of the vulcanization of TME, vulcanized with sulfur, in the presence of zinc oxide and 2-bisbenzothiazole-2-2’-disulfide (MBTS) for a reaction time of 40 minutes is shown in Figure 4.8. Table C.1 in Appendix C shows the retention times of the starting materials and solvent peaks.

Figure 4.8 HPLC chromatogram of a vulcanizate at 140 oC for 40 minutes

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The retention times of the MBTS, sulfur, and solvent peaks were observed at 6.3, 14.5 and 12.9 minutes, respectively (Figure C.1 in Appendix C). The unidentified peaks belong to the crosslink products with retention times of 6.7, 7.6, 8.4, 9.4, 11.5 and 14.5 minutes. Hann et al.[91] and van der Horst et al.[92] observed that the sulfur ranks and the logarithm of the retention time have a linear correlation.

The following graph in Figure 4.9, known as a Möckel plot[93], allows the various crosslink species of different sulfur rank (number of sulfur atoms in the crosslinks) to be identified.

In the notation, TME-Sx-TME, x represents the number of sulfur atoms in the crosslink.

Figure 4.9 Sulfur ranks of different crosslinks over the logarithmic retention time

It can be observed from the graph above that the sulfur ranks and the logarithm of the retention time have a linear correlation. The R-squared value of 0.98 indicates a good fit.

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The unidentified peaks now may be extrapolated from the graph as follows:

i) 6.7 min assigned to the monosulfide ii) 7.6 minutes assigned to disulfide iii) 8.4 minutes assigned to the trisulfide iii) 9.4 minutes assigned to tetrasulfide, iv) 11.5 minutes assigned to pentasulfide.

The line displays slight curvature at high sulfur ranks and retention time which is a result of peak asymmetry. The peak at 14.5 minutes may well be the hexasulfide. The peak at 8.8 minutes is due to 2-mercaptobenzathiazole, a decomposition product of MBTS vulcanization.

An additional confirmation study was performed with NMR. 1H NMR spectrum of the single band from section 3.3.5.3 resulted in a bis(alkenyl) product (see Figure B.1 in Appendix B). This spectrum is similar to that reported by van der Horst[94]. Figure 4.10 shows the bis(alkenyl) crosslinked structure.

Sx

Figure 4.10 TME bis(alkenyl) crosslinks[84]

A vulcanizate to be used to perform the devulcanization experiment must be of an appropriate reaction time where all the important crosslinks have been formed. The optimum vulcanization time selected for this study was chosen based on the diode array detector HPLC results. Figure 4.11 a) and b) show the sulfur and MBTS during vulcanization at 140 oC. A decrease in sulfur can be observed from the first 2 minutes of the reaction up to 40 minutes. This is due to 2-bisbenzothiazole-2,2'-polysulfide formation[95]. Initial consumption of MBTS occurs in the first 2 minutes as polysulfides are

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formed. Further consumption of MBTS is evident again from the twentieth minute up to 40 minutes into the reaction when crosslinking occurs[96]. These observations were not investigated any further as they have been well studied elsewhere.

Figure 4.12 shows the crosslink formation. The crosslinking had already started by the tenth minute. Morgan[84] observed rapid vulcanization occurrence after the initiation time of 6 minutes. It should be noted that Morgan performed vulcanization at 150 oC which would have reduced the initiation time. Here it occurs between 20 and 40 minutes as indicated by the drop in MBTS concentration. In his study the maximum concentration of disulfidic crosslinked products was attained after 15 minutes, whereas in the current study the observation was made at 20 minutes. In general, significant crosslinking had occurred 40 minutes. Rajan et al.[78] made a statement that polysulfidic crosslinks act as precursors for the shorter tri- and disulfidic crosslinks. The formation of shorter crosslinks is boosted by the breakdown of this precursor. It is observed from Figure 4.13 that at approximately 40 minutes an optimum concentration of crosslinks is reached. In rubber, curing time is defined as the time required for the maximum amount of crosslinks to form. Therefore, for this study 40 minutes was chosen as the optimum time for the crosslinks formation.

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Figure 4.11 Mole percentage of a) sulfur and b) MBTS as a function of reaction time

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Figure 4.12 HPLC analysis of the TME/MBTS/sulfur/ZnO (33.9:1.1:1.1:1) system heated isothermally at 140 oC

4.5.2.1 Vulcanization reactions This section employs differential scanning calorimetry (DSC) to show the reactions that take place between the curatives. Sulfur was heated in a sealed DSC pan at 2 oC/minute. Three endotherms were observed (as shown in Figure 4.13), a major peak being that of a sulfur melt at 119.7 oC, and the first one at 108.0 oC is associated with the - solid- solid transition in sulfur (rhombic to monoclinic form). Currell and Williams found that the nuclei of monoclinic sulfur (S) were necessary for the solid-solid transition, and that a

[97] single crystal of rhombic sulfur did not transform to S, but melted at 112 °C . The small peak at 115 °C, is thus attributed to a small fraction of the rhombic sulfur melting without undergoing the solid-solid transition. The two peaks in the HPLC chromatogram of the sulfur used (see Figure C.1 in Appendix C) suggests that both molecular and plastic sulfur

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are present. The temperatures are slightly higher than stated in the literature. The temperature of the first transition is slightly higher than the literature value which may be the equilibrium temperature in the literature since solid-solid transitions are typically observed at slightly elevated temperatures during DSC experiments[98].

Figure 4.13 DSC thermogram of sulfur heated at 2 oC/min, sample mass = 1.0380 mg

As seen in Figure 4.14, heating MBTS/ZnO (1.1:1) in a sealed DSC pan at 2 oC/min produced 2 endotherms and 1 exotherm. There is a peak at approximately 112.0 oC whose origin is unclear. There also seems to be a shift on the MBTS melt to 157 oC, it is known to be at about 173.0 oC (see Figure D.1 in Appendix D). Morgan demonstrated that ZnO catalyses the formation of polysulfides from MBTS[84]. These may well have lower melting points. It should also be noted that the endotherm at 157 oC is very broad, indicative of a mixture of compounds.

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There is also an exothermic reaction that can be observed at 228.1 oC with an enthalpy of 7.6 J/g.

Figure 4.14 DSC thermogram of MBTS/ZnO (1.1:1) heated at 2 oC/min, sample size = 1.9090 mg

In Figure 4.15, TME/MBTS/ZnO is heated in a sealed DSC pan at 2 oC/min. One endotherm and exotherm is produced. The endotherm is the resulting MBTS melt that has been established already. There is an exothermic reaction taking place even though sulfur is not present. The reaction taking place is at the approximate region of 239.2 oC which is similar to that seen in MBTS/ZnO. Three cycles of the formulation TME/MBTS/ZnO were scanned but the same reaction remains in the same region (Figure D.2 and Figure D.3 in Appendix D).

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Figure 4.15 DSC thermogram for TME/MBTS/ZnO (33.9:1.1:1) heated at 2 oC/min, sample size = 1.3900 mg

In Figure 4.16 and Figure 4.17, TME/MBTS/ZnO/S8 (33.9:1.1:1:1.1) was heated in a DSC pan at a rate of 2 oC/min. In cycle 1 (Figure 4.16) solid-solid transition for sulfur was observed at about 108 oC. Boyce[83] demonstrated that the presence of MBTS suppressed the melting temperature of sulfur which may be seen as a small shoulder on this peak. The melting of the MBTS is significantly reduced to approximately 132 oC. The presence of sulfur significantly lowers the melting point due to MBTS dissolving in the molten sulfur[99]. It is also known that MBTS reacts readily with sulfur to form MBTPs[100] which may also form part of this endotherm. Note again the broadness, indicative of the melting of a variety of compounds, possibly polysulfides.

A reaction that may be associated with curing is observed at an onset of about 171 oC. Vulcanization is known to be an exothermic process[84]. A reaction between ZnO and MBTS or its polysulfides is postulated in the region of approximately 240 oC with an

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enthalpy of 18.94 J/g. In the third cycle (Figure 4.17) the reaction enthalpy has decreased to 1.958 J/g. This is consistent with less MBTS being able to further react at this stage. Figure D.4 in Appendix D shows DSC curve for cycle 2 which looks similar to cycle 3 with enthalpy of 1.918 J/g.

Figure 4.16 DSC curve of TME/MBTS/ZnO/S8 (33.9:1.1:1:1.1) cycle 1 heated at 2 oC/min, sample size = 10.2320 mg

It can be seen in Figure 4.17 that the melting of MBTS has disappeared consistent with it having been converted into a zinc salt or other compound.

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Figure 4.17 DSC curve of TME/MBTS/ZnO/S8 (33.9:1.1:1:1.1) cycle 3 heated at 2 oC/min, sample size = 10.2320 mg

Figure 4.18 shows the curing of natural rubber when heated at 2 oC/min in a DSC pan. A glass transition at -62.3 oC provides evidence that the sample is a natural rubber. The exothermic peak at 148.7 oC with an onset of 140 oC is associated with crosslink formation. It has an enthalpy of 7.125 J/g. No evidence of curative melting or other peaks can be seen. This, however, is because of the high natural rubber content which swamps any curative signals. Furthermore curatives are known to dissolve in natural rubber during mixing which may also explain why such signals cannot be seen.

From the DSC curves it can be observed that there is a reaction taking place at approximately 240 oC. It is postulated that this reaction is associated with MBTS (or its polysulfides) and ZnO. The reaction was monitored and it may be deduced that there is

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an excess of MBTS and ZnO in the formulation with respect to TME and sulfur. According to this study the recipe may be reworked to accommodate sulfur and TME as they seem to be consumed to completion.

Figure 4.18 DSC curve of Natural rubber conventional curing heated at 2 oC/min, sample mass = 11.9460 mg

4.6 Devulcanization process

4.6.1 FTIR of the devulcanizates In the FTIR spectra of the vulcanizates treated separately with the bulbs, roots, and leaves extract, it can be observed that there are some bands that resemble those of the carbon– sulfur bonds and sulfur-sulfur bonds. Unfortunately such bonds are present in the crude

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extracts as well as the vulcanization products. FTIR experiments are thus inconclusive when providing evidence as to whether and to what extent devulcanization has occurred.

4.6.2 HPLC and NMR Figure 4.19 shows the area percentage of the peaks corresponding to the monosulfides. After devulcanization reaction with bulb extracts employed as devulcanizing agents, the di-, tri-, and polysulfides peaks were not detected by an HPLC system. It was only the monosulfides that were detected; the trend observed was that of a decrease as the reaction time increases. It is known that sulfide bridges (S-S) are more reactive because of their weaker bond strength[77, 82]. The absence of di and higher order polysulfides after exposure to the bulb extract demonstrates that it is a highly effective devulcanizing agent. The fact that no disulfides were observed after as little as 10 min illustrates the extract’s efficacy.

Figure 4.19 HPLC peak percentage areas of the monosulfidic crosslinks after devulcanization with bulb extract.

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Devulcanization reaction with leaf extract as devulcanizing agents produced mixtures in which di-, tri-, and polysulfides peaks could not be detected by an HPLC system. In Figure 4.20 it can be observed from the chromatograms that the area percentage of the peaks correspond to monosulfides only. This extract behaves similar to the bulbs extract, except there was an increase in the monosulfides after 10 minutes, a decrease was then observed after 20 minutes.

Figure 4.20 Peak percentage areas of the monosulfidic crosslinks after devulcanization with leaf extract.

Figure 4.21 below shows the area percentage of the peaks corresponding to the mono-, di-, tri-, and tetrasulfides. The peak at 8.8 min (associated with the vulcanization product 2-mercaptobenzothiazole (MBT)) was absent after reaction with root extracts as devulcanizing agents. MBT is known to undergo exchange reactions with di- and polysulfidic compounds. Given the composition of the all extracts it is no surprise that it has disappeared[92, 101]. The monosulfidic crosslinks still have a high percentage area than the other crosslinks.

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Figure 4.21 Peak percentage areas of the TME crosslinks of different sulfur rank after devulcanization with root extract.

Figure 4.22 shows the area percentages of the sulfidic crosslinks as detected by the HPLC system in a system in which no devulcanizing agent was added. It can be observed that crosslinks of all sulfur ranks are detected, some in very small percentage. The percentage of monosulfidic are also noticed to have a lesser percentage when no extract is employed for devulcanization. This can be explained by the fact that as devulcanization proceeds, polysulfidic crosslinks open and react with devulcanizating agent radicals. These then undergo metathesis reactions which ultimately lead to an increase in monosulfidic species and a decrease in all others [77, 82]. Figure 4.19-4.22 would have been improved if the values at 0 min were included for comparison.

The lower efficacy of the roots extract is consistent with the lower concentration of DADS (see section 4.4) and the less pronounced spots in the developed TLC plates for the 3 extracts (see sections 4.1 and 4.2)

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Figure 4.22 Peak percentage areas of the TME crosslinks of different sulfur rank with no extract added

Figure 4.23 shows an overlay of proton NMR spectra. The green spectrum represents the crude extract, the red spectrum represents the devulcanizate produced by the bulb crude extract, and the blue spectra represents the vulcanizate. It can be observed from the spectra that the devulcanizate has some of the signals from the crude extract as was mentioned earlier on section 4.6.1 with the FTIR. It was also observed that the vulcanizate signals are not as evident on the devulcanizate spectrum. From the information found from these spectra, it may be suggested that there is a possibility of devulcanization that took place.

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a)

b)

c)

Figure 4.23 NMR spectra overlay of a) bulb crude extract, b) devulcanizate and c) vulcanizate

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Figures 4.24-4.26 shows the DSC thermograms of the leaf devulcanizate (product of the devulcanization process). The devulcanizate was heated in a DSC pan at a rate of 2 oC/min. The curves were split into three namely a) heating at 2 oC/min to 280 oC, b) cooling at 10 oC/min to 95 oC, and c) heating again to 280 oC at a 2 oC/min rate. Upon heating in cycle (a) no curing was observed as expected. A possible endotherm at approximately 175 oC is observed and a constantly significant exothermic reaction at approximately 240 oC. This is in a similar region to that observed for vulcanization as well. In cycle (b) during the cooling experiment an endotherm is observed and in the same approximate region. This suggests that the process may well be reversible. Cycle c) shows no reaction between 150 and 200 oC, unlike cycle (a). This suggests that the devulcanizate had reacted with all of a certain type of reactive species, e.g., MBT in cycle (a).

a)

Figure 4.24 DSC thermogram for the devulcanizate from the leaf extract heated at 2 oC/min, sample size = 3.9090 mg.

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b)

Figure 4.25 DSC thermogram for the devulcanizate from the leaf extract, cooled at 10 oC/min, sample size = 3.9090 mg.

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c)

Figure 4.26 DSC thermogram for the devulcanizate from the leaves extract heated at 2 oC/min, sample size = 3.9090 mg

.

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CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS

This project has reported on extraction and characterization of some sulfur-containing compounds from the Tulbaghia violacea plant, which has been demonstrated to be effective as an alternative devulcanizing agent. The TLC technique assisted in the detection of sulfur-containing compounds. The TLC plates of the crude extracts showed similar spots to those of the reference standards, in particular diallyl sulfide (DAS). The other spots that were visible on the plate were not positively identified. These may be compounds such as marasmicin, identified by Burton and Kaye[51]. Their isolation as individual compounds, however, was beyond the scope of this study whose primary aim was to evaluate Tulbaghia violacea extracts as devulcanizing agents.

A sulfur phytochemical test added to the positive detection of sulfur-containing compounds. Soxhlet extraction of the plant material with the 2 % 2-isopropanol in n- hexane resulted in crude extracts with no traces of water which could be detected by FTIR. FTIR spectra indicated this extract as having sulfur-containing compounds as evidenced by the presence of C-S, S-S and S=O stretches. The Soxhlet extraction method was preferred because the application of the resulting crude extracts requires the absence of oxygen, because oxygen influences the reaction. Sulfur-containing compounds can oxidise easily in the presence of oxygen. Greater extract yields were obtained from the leaves and roots than the bulbs. It must, however, be born in mind that the bulbs form significantly more of the plant mass than the roots. The resulting extracts were later employed in devulcanization experiments that were conducted on a vulcanized model compound, 2,3-dimethyl-2-butene (TME).

Some of these sulfur-containing compounds were quantified using a normal-phase HPLC method. This type of chromatography successfully separated and quantified diallyl disulfide (DADS) in the crude extracts from the bulbs, leaves and roots. DADS were found to be present in highest concentration in the bulb extract and least in the root extract.

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Nonetheless the low percentage of DADS (< 1%) in the extract indicates the presence of a host of other compounds.

The progress of model compound vulcanization was followed by use of the HPLC, which effectively allowed crosslink formation and devulcanization to be interpreted. 2,3- dimethyl-2-butene-derived mono-, di- and polysulfidic crosslink products were easily identified as the products of vulcanization. In contrast to Rajan et al.[78] who observed no monosulfidic products in the reaction mixture, monosulfidic products were detected. This is the result of an efficient, rather than a conventional, vulcanization system being used here. An efficient vulcanization system is one which has a low sulfur to accelerator ratio[102]. 40 minutes was found to be the optimum time for vulcanization where a significant quantity of crosslink species was formed.

This vulcanizate was then used in a devulcanization process by heating with root, leaf and bulb extracts of Tulbaghia violacea. FTIR was not suitable for monitoring the progress of devulcanization because of the presence of similar functional groups such as C-S and S-S bonds in Tulbaghia violacea extracts, vulcanization products and devulcanization products.

Differential scanning calorimetry was used to monitor curative interaction behaviour. It is postulated that MBTS and ZnO displayed an on-going reaction throughout the experiments, suggesting that they are in excess in the formulation used. For future studies, it may be useful to heat the same formulation at 140 oC to compare the results with those of an oil bath and to follow the reaction mechanism. For devulcanization experiments carried out with the DSC, there was no conclusive evidence as to whether devulcanization occurred.

The crude extracts were heated with the vulcanized model compound system and evidence, consistent with devulcanization was observed. During the devulcanization reactions the extracts from the bulbs and the leaves removed most of the sulfides except for the monosulfides which could still be detected by HPLC. The roots extract reduced

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the concentration of higher sulfur rank crosslinks but was not as effective as the other extracts. A high, but lower, monosulfide crosslink concentration was observed. These results are consistent with the lower DADS percentage measured as well as the lower concentration on non-polar species as observed by TLC. When no crude extract was heated with the vulcanized model compound system, the concentration of monosulfides was lower than all of the system in which Tulbaghia violacea extracts had been applied. Monosulfidic crosslinks are more stable than di- and polysulfidic crosslinks because of the absence of weaker S-S bonds [77, 82]. This is indicative, too, of the efficacy of the extracts as devulcanizing agents. Interestingly, after devulcanization, the absence of di- and polysulfidic crosslink species, suggests that the crosslink species were more effective than the commercial products used by Rajan et al.[78] and Boyce[83].

A number of recommendations can be made for future studies. These include:

 the individual compounds in the different extracts be isolated and compared;  the efficacy of these species as devulcanizing agents be compared with that of commercial devulcanizing agent such as diphenyl disulfide and diallyl disulfide as well as other natural product derived agents, e.g., that reportedly derived from garlic[5];  a concentration study be performed where higher and lower concentrations of crude extract as devulcanizing agent be used, to investigate more thoroughly the efficacy of the bulbs vs leaves extracts;  the effect of co-devulcanizing agents such as n-hexylamine or natural product amines be investigated to see if devulcanization by natural products can be enhanced still further[78];  the devulcanization of conventionally vulcanized model compound systems, with higher sulfur to accelerator ratios be investigated;  more complex model systems, such as those based on squalene, be investigated to provide further evidence of the efficiency of Tulbaghia violacea extracts[83];

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 devulcanizition with Tulbaghia violacea extracts be performed on vulcanized natural rubber;  other South African species in Amaryllidaceae order such as widely grown Agapanthus species be investigated; and  costing and life-cycle assessments of the use of Tulbaghia violacea vs commercial devulcanizing agents be performed.

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REFERENCES

1. Babcock E., Industrial & Engineering Chemistry, 1939, 31(10), 1196. 2. Loadman M., Analysis of rubber and rubber-like polymers, Springer Science & Business Media, 1999. 3. Brinke J. W., Silica reinforced tyre rubbers: mechanistic aspects of the role of coupling agents, Twente University Press, 2002. 4. Abraham E., Cherian B. M., Elbi P., Pothen L. A. and Thomas S., Recent Developments in Polymer Recycling, 2011, 47, 47. 5. De D., Maiti S. and Adhikari B., Journal of Applied Polymer Science, 1999, 73, 2951. 6. Maloney J. R., Theriot K. J., Magee S. B. D., Torres J. E. and Jr W. R. W., Process for producing diallyl disulfide WO/2006/016881. 2006. 7. Tortora G. J., Cicero D. R. and Parish H. I., Plant form and function: an introduction to plant science, Toronto, Collier-MacMillan Canada, 1970. 8. Fennel C. W., Lindsey K. L., Mcgraw L. J., Sparg S. G., Stafford G. I., Elgorashi E. E., Grace O. M. and Staden J. V., Journal of Ethnopharmacology, 2004, 94(2), 205. 9. Koduru S., Grierson D. S. and Afolayan A. J., Current Science, 2007, 92, 906. 10. Neuwinger H. D., African traditional medicine, Stuttgart, Germany, Medpharm Scientific Publishers, 2000. 11. Zhang X., Journal of manipulative and Physiological Therapeutics, 2000, 23, 139. 12. Levetin E. and Mcmahon K., Plants and Society, Boston, WCB/McGraw-Hill, 1999. 13. Davison C., A biochemical study of the antidiabetic and anticoagulant effects of Tulbaghia violacea, MSc, Nelson Mandela Metropolitan University, Port Elizabeth, 2010. 14. Belewa X. V., The effect of Tulbaghia violacea plant extract on the growth of Aspergillus species, MSc, Nelson Mandela Metropolitan University, Port Elizabeth, 2009. 15. Kennedy D. O. and Wightman E. L., Advances in Nutrition: An International Review Journal, 2011, 2(1), 32.

72

16. Kabera J. N., Semana E., Mussa A. R. and He X., Journal of Pharmacy and Pharmacology, 2014, 2, 377. 17. Lyantagaye S. L., Tanzania Journal of Science, 2011, 37, 58. 18. Sara, Vegetable Plant Families [Online], http://hortusurbanus.blogspot.co.za/2010/03/vegetable-plant-families.html [Accessed 01 Feb 2017]. 19. Apg (Angiosperm Phylogeny Group), Botanical Journal of the Linnean Society, 2003, 141, 399. 20. Dahlgren R. M. T., Clifford H. T. and Yeo P. F., The families of the monocotyledons, Berlin, Springer-Verlag, 1985. 21. Aremu A. O. and Van Staden J., Journal of Ethnopharmacology, 2013, 49, 387. 22. Vosa C. G., Caryologia, 2000, 53(2), 83. 23. Kubec R., Kreji'ova P., Mansur L. and Garcia N., Journal of Agriculture and Food Chemistry, 2013, 61, 1335. 24. Maoela M. S., Studies on some biologically active natural products from Tulbaghia alliacea, MSc, University of the Western Cape, Cape Town, 2005. 25. Dyson A., Discovering Indigenous Healing Plants of the Herb and Fragrance Gardens at Kirstenbosch National Botanic Garden, Cape Town, National Botanic Institute, 1998. 26. Kubec R., Velisek J. and Musah R. A., Phytochemistry, 2002, 60, 21. 27. Department of Agriculture, Forestry and Fisheries, Medicinal Plants of South Africa [Online], www.nda.agric.za/docs/Brochures/Medicin.pdf [Accessed 15 Nov 2015]. 28. Images for map with South African provinces [Online], https://www.google.co.za/search?q=map+with+south+african+provinces&sa=X& biw=1536&bih=718&tbm=isch&source=iu&ictx=1&fir=Ca_WZ4Enthfp- M%253A%252C1tQCkMQBtk2nCM%252C_&usg=__r3- PjCDdyJBlXPWRKZljNE3XBgo%3D&ved=0ahUKEwiy-- u53ZHYAhXGDcAKHYrvAhgQ9QEILDAB#imgrc=Ca_WZ4Enthfp-M: [Accessed 15 Dec 2017]. 29. Van Wyk B. E., People's plants: a guide to useful plants of Southern Africa, Pretoria, Briza Publications, 2000.

73

30. Maoela M. S., Studies on some biologically active natural products from Tulbaghia Alliacea, MSc, University of Western Cape, Cape Town, 2005. 31. Bungu L., Van Der Venter M. and Frost C., African Journal of Biotechnology, 2008, 7, 681. 32. Tsuchiya H. and Nagayama M., Journal of Biomedical Science, 2008, 15, 653. 33. Wargovich M. J., Journal of Nutrition, 2006, 136, 832. 34. Bungu L. C., Anticancer and anticoagulant activities of Tulbaghia violacea, MSc, University of Port Elizabeth, Port Elizabeth, 2005. 35. Bungu L., Frost C. L., Brauns S. C. and Van Der Venter M., African Journal of Biotechnology, 2006, 5(20), 1936. 36. Olorunnisola O. S., Bradley G. and Afolayan A. J., African Journal of Pharmacy and Pharmacology, 2011, 5(22), 2490. 37. Davison C., Levendal R. A. and Frost C. L., Journal of Medicinal Plants Research, 2012, 6(33), 4815. 38. Burton G. B., A chemical investigation of Tulbaghia violacea, MSc, Rhodes University, Grahamstown, 1990. 39. Jacobsen J. V., Yamaguchi Y., Mann L. K. and Howard F. D., Phytochemistry, 1968, 7, 1099. 40. Kiddle G., Bennett R., Hick A. and Wallsgrove R., Plant, Cell & Environment, 1999, 22(5), 433. 41. Manabe T., Hasumi A., Sugiyama M., Yamazaki M. and Saito K., European Journal of Biochemistry, 1998, 257(1), 21. 42. Iciek M., Kwiecien I. and Wlodek L., Environmental and Molecular Mutagenesis, 2009, 50, 247. 43. Brodnitz M. H., Pascale J. V. and Derslice L. V., Journal of Agricultural and Food Chemistry, 1971, 19, 273. 44. Amagase H., Journal of Nutrition, 2006, 136, 716. 45. Kimbaris A. C., Siatis N. G., Daferera D. J., Tarantilis P. A., Pappas C. S. and Polissiou M. G., Ultrasonics Sonochemistry, 2006, 13, 54. 46. Apitz-Castro R., Cabrera S., Cruz M. R., Ledezma E. and Jain M. K., Thrombosis Research, 1983, 32, 155.

74

47. Li M., Ciu J.-R., Ye Y., Min J.-M., Zhang L.-H., Wang K., Gares M., Cros J., Wright M. and Leung-Tack J., Carcinogenesis, 2002, 23(4), 573. 48. Yoshida S., Kasuga S., Hayashi N., Ushiroguchi T., Matsuura H. and Nakagawa S., Applied and environmental microbiology, 1987, 53(3), 615. 49. Davis S. R., Perrie R. and Apitz-Castro R., Journal of Antimicrobial Chemotherapy, 2003, 51(3), 593. 50. Nagawana R., Iwata N., Ishikawa K., Fukuda H., Fujino T. and Suzuki A., Applied and Environmental Microbiology, 1996, 62, 4238. 51. Burton S. G. and Kaye P. T., Planta Medica, 1992, 58, 295. 52. Azwanida N. N., Medicinal and Aromatic Plants, 2015, 4(3), 1. 53. Pandey A. and Tripathi S., Journal of Pharmacognosy and Phytochemistry, 2014, 2(5), 115. 54. Amagase H., Petesch B. L., Matsuura H. and Kasuga S., The Journal of Nutrition, 2001, 131, 955S. 55. Yoo M., Kim S., Lee S. and Shin D., Journal of Chromatographic Science, 2013, 1. 56. Lough W. J. and Wainer I. W., High Performance Liquid Chromatography: Fundamental Principles and Practice, Pin Press, 1995. 57. The Editors of Encyclopaedia Britannica, Vulcanization [Online], https://global.britannica.com/technology/vulcanization, [Accessed 06 Feb 2017]. 58. Swart P. J., Polyisoprene vs Natural Rubber in tyre compounds, MSc, University of Port Elizabeth, 1983. 59. Arvind Mafatlal Group, Vulcanization and Accelerators [Online], http://www.nocil.com/DownloadFile/DTechnicalNote-Vulcanization-Dec10.pdf [Accessed 15 Feb 2017]. 60. Ciesielski A., An introduction to Rubber Technology, Showburry, Rapra Technology Ltd, 1999. 61. Ciullo P. A. and Hewitt N., The Rubber Formularly, New York, William Andrew Inc., 1999. 62. Medrano M. G., Novel vulcanizing ingredients: towards greener rubber formulations, PhD, Universitat Ramon Lull Fundacio Privada, Barcelona, 2012.

75

63. Net Industries, Vulcanization - Rubber as a natural product, vulcanization and properties of vulcanized rubber [Online], http://science.jrank.org/pages/7266/vulcanization.html [Accessed 06 Feb 2017]. 64. Van Rooyen J., A comparison of the vulcanization of polyisoprene by a range of Thiuram disulphides, MSc, Nelson Mandela Metropolitan University, Port Elizabeth, 2007. 65. Coran A. Y., Journal of Applied Polymer Science, 2003, 87(1), 24. 66. Hofmann W., Rubber Technology Handbook, Munich, Carl Hanser, 1989. 67. Reyneke-Barnard C. P., N, 'N-Dipentamethylene thiuram sulphides in accelerated sulphur vulcanization, PhD, University of Port Elizabeth, Port Elizabeth, 1999. 68. Porter M., The chemistry of sulphides, New York, Interscience Publishers, 1968. 69. Kruger F. W. H. and Gill W. J. M., Journal of Applied Polymer Science, 1991, 42, 2669. 70. Thaicharoen P., Thamyongkit P. and Poompradub S., Korean Journal of Chemical Engineering, 2010, 27(4), 1177. 71. Tobolsky A. V., Properties and Structures of Polymers, New York, John Wiley and Sons, 1960. 72. Tobolsky A. V., Polymer Science and Materials, New York, International Science, 1960. 73. Chapman A. V., Reducing zinc in rubber compounds, Maarstricht, IRC 2005, 2005. 74. Vega B., Agullo N., Ventura M., Comellas L. and Borros S., Rubber Chemistry and Technology, 2013, 565, 7. 75. Boretti L. G. and Woolard C. D., Rubber Chemistry and Technology, 2006, 79, 135. 76. Agullo N. and Borros S., Kautschuk Gummi Kunststoffe, 2000, 53(3), 131. 77. Scheele W. and Lorenz O., Rubber Chemistry and Technology, 1956, 28(1), 37. 78. Rajan V., Dierkes W. K., Noordermeer J. W. and Joseph R., Rubber Chemistry and Technology, 2005, 78(4), 572. 79. Geyser M. and McGill W., Journal of Applied Polymer Science, 1995, 55(2), 215.

76

80. Tampere University of Technology, https://www.tut.fi/muo/vert/6_elastomeric_materials/recycling_methods_devulcan ization.htm [Accessed 19 Feb 2017]. 81. Jana G. K. and Das C. K., Macromolecular Research, 2004, 13(1), 30. 82. Joseph A. M., George B., Madhusoodanan K. N. and Alex R., Rubber Science, 2016, 29(1), 62. 83. Boyce A., Devulcanization of model compounds by a variety of diphenyldisulfides, MSc, Nelson Mandela Metropolitan University, Port Elizabeth, 2017. 84. Morgan B., Benzothiazole accelerated sulfur vulcanization of 2,3-dimethyl-2- butene, PhD, University of Port Elizabeth, Port Elizabeth, 1998. 85. Govender H., A comparative study of solvent extraction, steam distillation, headspace analysis and headspace solid phase microextraction for the extraction of volatile terpenoid compounds in the curry leaf plant (Murraya Koenigii), MSc, University of KwaZulu-Natal, Durban, 2010. 86. Wagner H. and Bladt S., Plant drug analysis: a thin layer chromatography atlas, Springer Science and Business Media, 1996. 87. Gitini L., Dinica R., Neagu C. and Dumitrascu L., Journal of Food and Drugs Analysis, 2014, 22, 425. 88. Coates J., Interpretation of infrared spectra, a practical approach. In: Meyers R. A. (ed.) Encyclopedia of Analytical Chemistry. Chichester, United Kingdom: John Wiley and Sons, 2000. 89. Stuart B. H., Infrared spectroscopy: Fundamentals and applications, New York, United States of America, John Wiley and Sons, 2004. 90. Boretti L. G., Bis(Triethoxysilylpropyl) Tetrasulfane as a coupling agent for silica filled vulcanizates, MSc, University of Port Elizabeth, Port Elizabeth, 1999. 91. Hann C. J., Sullivan A. B., Host B. C. and Kuhls Jr G. H., Rubber Chemistry and Technology, 1994, 67(1), 76. 92. Van Der Horst M., Hendrikse K. G. and Woolard C. D., Journal of Applied Polymer Science, 2003, 89(1), 47. 93. Möckel H. J., Journal of Chromatography A, 1984, 317, 585.

77

94. Horst M. V. D., The importance of thiol intermediates in 2-mercaptobenzothiazole accelerated sulfur vulcanization of 2,3-dimethyl-2-butene, MSc, University of Port Elizabeth, 2001. 95. Morgan B. and Mcgill W. J., Journal of Applied Polymer Science, 2000, 76(9), 1395. 96. Morgan B. and Mcgill W. J., Journal of Applied Polymer Science, 2000, 76(9), 1413. 97. Currell B. R. and Williams A. J., Thermochimica Acta, 1974, 9(3), 255. 98. Gallagher P. K., Brown M. E. and Kemp R. B., Handbook of Thermal Analysis and Calorimetry, Amsterdam, Netherlands, Elsevier, 1999. 99. Kruger F. W. and Mcgill W. J., Journal of Applied Polymer Science, 1991, 42, 2651. 100. Gradwell M. H. and Mcgill W. J., Journal of Applied Polymer Science, 1996, 61, 1131. 101. Boretti L. G., N-(cyclohexylthio)phthalimide inhibition of bis-benzothiazole-2,2'- disulfide accelerated vulcanization of squalene., PhD, University of Port Elizabeth, Port Elizabeth, 2002. 102. Mark J. E., Erman B. and Roland M. Eds., The science and technology of rubber., New York, United States of America, Academic Press, 2013.

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APPENDICES

Appendix A

Figure A.1 FTIR spectrum of Tulbaghia violacea crude extract from the roots

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Figure A.2 FTIR spectrum of Tulbaghia violacea crude extract from leaves

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Appendix B

Figure B.1 1H-NMR spectrum of the crosslinks for bis(alkenyl) product

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Appendix C

Table C.1 Starting materials and solvent retention times

Component Retention time (approximate minutes) MBTS 6.2 Sulfur 14.2 Solvent_1 2.6 Solvent_2 12.9

Figure C.1 HPLC chromatogram of the sulfur used for vulcanization

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Appendix D

Figure D.1 DSC thermogram of pure MBTS heated at 2 oC/min; mass = 1.5460 mg

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Figure D.2 DSC thermogram of TME/MBTS/ZnO heated at 2 oC/min cycle 2; sample mass = 1.39090 mg

Figure D.3 DSC thermogram of TME/MBTS/ZnO heated at 2 oC/min cycle 3; sample mass = 1.39090 mg

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o Figure D.4 DSC thermogram of TME/MBTS/ZnO/S8 heated at 2 C/min cycle 2, sample mass = 10.2320 mg

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