STUDIES ON SOME BIOLOGICALLY ACTIVE NATURAL PRODUCTS FROM TULBAGHIA ALLIACEA

MANKI SARAH MAOELA

A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in the Department of Chemistry at the University of the Western Cape.

November 2005

Supervisor: Dr. Wilfred Mabusela Co-supervisor: Prof. Quinton Johnson

i DEDICATION

I would like to dedicate my work to my sisters Palesa and Matshediso for they understood my pressure and helped me through it all. As for my mom, dad and granny thank you for giving me freedom and allowance to take some time off at home to be here to further my studies. My brother Sephiri, your knowledge and intelligence took me very far as you are that big shining star to keep me going. Thank you all.

ii KEYWORDS

Allium

Tulbaghia alliacea

HPLC

ESI/LC-MS/MS

GC-MS

S-alk(en)yl cysteine sulfoxide

Thiosulfinate

Sulfone

Polysulfide

Furanoid derivative

iii DECLARATION

I declare that Studies on some biologically active natural products from Tulbaghia alliacea is my own work, that it has not been submitted for any degree or examination in any other university, and that all the sources I used or quoted have been indicated and acknowledged by complete references.

Manki Sarah Maoela November 2005

Signed: ……………………………

iv ACKNOWLEDGEMENT

First of all I would like to thank God for being with me through everything and for

protecting me all these years. Secondly I am very grateful to my supervisor Dr. W.

Mabusela for the support, encouragement and guidance he gave me throughout my

study. Thank you very much, Doc. I would also like to thank Mr. Timothy Lesch for

teaching me to operate the GC and GC-MS. Thanks to Dr. Marietjie Stander from the

Stellenbosch University for the ESI/LC-MS/MS analyses, may God bless you. Thanks to Prof. Ivan Green for the NMR analyses and doing it for me without hesitation. To

the entire staff and postgraduate students of the Department of Chemistry and South

African Herbal Science and Medicine Institute (SAHSMI) at the University of the

Western Cape, thank you for everything. Financial support from the NRF is greatly

acknowledged.

I would like to thank my family for believing in me especially my mother, my friend

Mrs. Molehali Alinah Maoela. To my close friends Miss Nikkie Hendricks and Miss

Lebogang Manthata, thank you very much for encouraging and loving me.

v ABSTRACT

It is believed that early humans had knowledge of how to use traditional medicinal

plants, but the knowledge has been partially lost as society underwent various changes

leading to new civilizations. Traditional healers and remedies made from traditional

medicinal plants still play an important role in the health of millions of people,

especially rural people and urban poor people in developed countries. Traditional

medicinal plants are important for pharmacological research, and drugs development.

For the past years, scientist began to purify active extracts from the medicinal plants.

The aim of this study was to isolate and identify natural product constituents from

Tulbaghia alliacea. Tulbaghia alliacea is a species from the Alliaceae family and it grows in South Africa. There has not yet been any scientifically conducted investigation on the plant. Tulbaghia alliacea is used for fever and colds, asthma, pulmonary tuberculosis and stomach problems. Because of its garlic smell, Tulbaghia

alliacea was considered to be a sulfur-containing plant. In garlic, alliinase enzymes

catalyse the conversion of odorless S-alk(en)yl cysteine sulfoxide into volatile thiosulfinates, which are presumed to be responsible for the characteristic, garlic smell. Thiosulfinates are very unstable; they form polysulfides if stored over long period.

vi The extraction of the plant material was performed with methanol, ethyl acetate and

chloroform. High performance liquid chromatography (HPLC), electrospray

ionization liquid chromatography-mass spectroscopy (ESI/LC-MS), gas

chromatography (GC) and gas chromatography-mass spectroscopy GC-MS) were

used for separation of components into its individual peaks and characterization was

done using electron impact ionization mass spectroscopy (EI/MS), tandem MS

(MS/MS) and nuclear magnetic resonance (NMR).

Four S-alk(en)yl cysteine sulfoxides were found in the methanol extract; S-

(methylthiomethyl) cysteine-4-oxide (1), S-methyl cysteine sulfoxide (2), S-allyl cysteine sulfoxide (3) and S-propyl cysteine sulfoxide (4) occur as major compounds.

Thiosulfinates were found in the methanol, ethyl acetate and chloroform extracts, namely 2,4,5,7-tetrathiaoctane-2,2-dioxide (5), 2-propenyl methanethiosulfinates (6),

1-propenyl butanethiosulfinate (7), 1-propenyl propanethiosulfinate (8), butyl butanethiosulfinate (9) and 2,4,5,7-tetrathiaoctane-4-oxide (marasmicin) (10).

Polysulfides were found in the ethyl acetate extract, and these were 2,4,5,7-

tetrathiaoctane (11) and 2,4,6-trithiaheptane (12). The furanoid compound, 5-

(hydroxymethyl)-2-furfuraldehyde (13) was identified as an artefact compound

generated by the acid hydrolysis step. This compound occurs as a product from the

acid-catalyzed dehydration of fructose. Fructose and glucose are the carbohydrate

compounds found from the aqueous extract.

O O H2N H2N S S S 3CH CH COOH 3 COOH 1 2

vii

O O H2N H2N S S 2CH 3CH COOH COOH 3 4

O O

S S S CH CH3 CH 3 S CH 2 O S 3 S 5 6

O O

CH 3 S S CH3 CH CH3 S 3 S 7 8

O

3CH S S CH3 9

O

S S S CH S CH 3CH 3 3CH 3 S S S S 10 11

O

O

CH2OH S S S H 3CH CH3 12 13

viii LIST OF ABBREVIATIONS

APCI Atmospheric pressure chemical ionisation ATP Adenosine 5'-triphosphate CoA Coenzyme A d doublet J Coupling constant NAD+ Nicotinamide adenine dinucleotide phosphate

Pί Inorganic phosphate

PPί Inorganic pyrophosphate s singlet tR Retention time

ix TABLE OF CONTENTS

Content Page

DEDICATION ii

KEYWORDS iii

DECLARATION iv

ACKNOWLEDGEMENT v

ABSTRACT vi

LIST OF ABBREVIATIONS ix

LIST OF FIGURES xiii

LIST OF TABLES xiv

LIST OF SCHEMES xv

CHAPTER 1: LITERATURE REVIEW 1

1. Introduction 1

1.1 General 2

1.1.1 Traditional medicine 2

1.1.2 Medicinal plants 5

1.1.3 The Family Alliaceae 6

1.1.3.1 The Genus Allium 7

1.1.3.1.1 Allium sativum 7

1.1.3.1.2 Allium cepa 7

1.1.3.2 The Genus Tulbaghia 8

1.1.3.2.1 Tulbaghia violacea 8

1.1.3.2.2 Tulbaghia alliacea 10

1.2 The chemistry of Allium and Tulbaghia species 11

1.2.1 Structural studies of some sulfur-containing compounds 19

x 1.2.2 Biosynthesis of flavor precursors of Allium species 20

1.3 Pharmacological activity of Allium sativum 31

1.4 Mass spectrometry 33

1.5 Aim of the thesis 34

CHAPTER 2: EXPERIMENTAL WORK 35

2.1 Instrumentation 35

2.2 Plant materials 35

2.3 Extraction 36

2.3.1 Methanol extraction 36

2.3.2 Solvent partitioning fractionation 36

2.3.3 Chloroform extraction 37

2.4 Acid hydrolysis, ethyl acetate extraction and acetylating

of aqueous extract 38

2.5 Chromatography 39

2.5.1 Gas Chromatography (GC) 39

2.5.2 Gas Chromatography-Mass Spectroscopy (GC-MS) 39

2.5.3 High Performance Liquid Chromatography (HPLC) 39

2.5.3.1 Sample preparation 40

2.5.4 Liquid Chromatography-Mass Spectroscopy (LC-MS) 40

2.5.4.1 Sample preparation 40

2.5.4.1.1 Chloroform extract 40

2.5.4.1.2 Ethyl acetate extract 40

2.5.4.1.3 Methanol extract 40

xi CHAPTER 3: RESULTS AND DISCUSSION 41

3.1 Characterization of cysteine sulfoxide structures 41

3.1.1 S-(methylthiomethyl) cysteine-4-oxide (marasmin) (1) 44

3.1.2 S-methyl cysteine sulfoxide (2) 45

3.1.3 S-allyl cysteine sulfoxide (Alliin) (3) 46

3.1.4 S-propyl cysteine sulfoxide (4) 47

3.2 Characterization of sulfone and thiosulfinates structures 48

3.2.1 2,4,5,7-tetrathiaoctane-2,2-dioxide (5) 53

3.2.2 2-Propenyl methanethiosulfinate (6) 54

3.2.3 1-Propenyl butanethiosulfinate (7) 55

3.2.4 1-Propenyl propanethiosulfinate (8) 56

3.2.5 Butyl butanethiosulfinate (9) 56

3.2.6 2,4,5,7-tetrathiaoctane-4-oxide (marasmicin) (10) 57

3.3 Characterization of polysulfide structures 60

3.3.1 2,4,5,7-tetrathiaoctane (11) 60

3.3.2 2,4,6-trithiaheptane (12) 63

3.4 Isolation of a furanoid derivative 64

3.4.1 5-(hydroxymethyl)-2-furfuraldehyde (13) 64

CHAPTER 4: CONCLUSION 69 Reference 72 Appendix

xii

LIST OF FIGURES

Page Figure 1. Alliinase catalysed conversion of cysteine sulfoxide to thiosulfinate 11

Figure 2. Structure of marasmin 15

Figure 3. Structures of disulfide from A. cepa and A. sativum 17

Figure 4. Formation of marasmicin 17

Figure 5. Structures of thiosulfinate 18

Figure 6. Structure of an amino acid glycoside 19

Figure 7. Cysteine sulfoxide reacts with alliinase enzyme to form thiosulfinate 29

Figure 8. Structures of ajoene and vinyldithiin 32

Figure 9. HPLC-UV chromatograms of methanol extract 42

Figure 10. ESI/LC-MS BPI chromatogram of methanol extracts 43

Figure 11. HPLC-UV chromatograms of ethyl acetate extract 49

Figure 12. ESI/LC-MS BPI chromatogram of ethyl acetate extracts 50

Figure 13. HPLC-UV chromatograms of chloroform extract 51

Figure 14. ESI/LC-MS BPI chromatogram of chloroform extracts 52

Figure 15. 1H NMR of marasmicin from Tulbaghia alliacea 59

Figure 16. GC-MS chromatogram of ethyl acetate extract 62

Figure 17. 1H NMR of 5-(hydroxymethyl)-2-furfuraldehyde from Tulbaghia alliacea 67

Figure 18. 13C NMR of 5-(hydroxymethyl)-2-furfuraldehyde from Tulbaghia Alliacea 68

xiii LIST OF TABLES Page

Table 1. Ratio of medical and traditional practitioners to population 3

Table 2. Total content(%) and relative amounts(%) of cysteine sulfoxides of Allium species and T. acutiloba 12

Table 3. Total content(%) and relative amounts(%) of cysteine sulfoxides in different parts of A. siculum 13

Table 4. Total content(%) and relative amounts(%) of cysteine sulfoxide of Allium hybrids 14

Table 5. ESI/LC-MS spectral data of cysteine sulfoxides 44

Table 6. ESI/LC-MS spectral data of sulfone and thiosulfinates from different extracts 53

Table 7. GC-MS spectral data of ethyl acetate extract 60

Table 8. EI/MS spectral data of the ethyl acetate-soluble fraction of the hydrolysed aqueous extract 64

.

xiv LIST OF SCHEMES Page

Scheme1. Biosynthesis of serine 21

Scheme 2. Biosynthesis of methionine 23

Scheme 3. Biosynthesis of cysteine 25

Scheme 4. Biosynthesis of methiin 27

Scheme 5. Biosynthesis of methiin (alternative) 27

Scheme 6. Biosynthesis of the allyl-, propenyl-, and propyl- group of the Allium flavour precursors 28

Scheme 7. Mass spectral fragmentation pathway of compound 11 61

Scheme 8. Mass spectral fragmentation pathway of compound 12 63

xv CHAPTER 1

LITERATURE REVIEW

1. Introduction

In the past, modern science has considered methods of traditional knowledge to be primitive. During the colonial era, traditional medical practices were often declared as illegal by the colonial authorities. Consequently medical doctors and health personnel have in most cases continued to avoid traditional practitioners despite their contribution to meeting the basic health needs of the population, especially the rural people in the developing countries. However, recent progress in the field of environmental science, immunology, medical botany and pharmacognosy have led researchers to appreciate in a new way the precise descriptive capacity and rationality of various traditional medicine as well as the effectiveness of the treatments employed. Traditional medicine or more appropriately, traditional systems of health care, have undergone a major revival in the last twenty years. Even the World Health

Organization (WHO) has described traditional medicine as one of the surest means to achieve total health care coverage of the world’s population.

In spite of the marginalisation of traditional medicine practiced in the past, the attention currently being given by the governments to widespread health care application has given a new drive to research, investment and design of programs in this field in several developing countries. The research into traditional medicines will be beneficial for non-developed countries, since drugs and medicines are becoming more expensive, because current health systems are dependent upon imported technologies and medicine, while the supply of the drugs are also becoming more erratic.

1 1.1 General

1.1.1 Traditional medicine

It is believed that early human kind had an innate knowledge of how to use herbs.

However this knowledge has been partially lost as society underwent various changes leading to new civilizations (Maher, 1992). According to the World Health

Organization (WHO) the definition of traditional medicine may be summarized as the sum total of all the knowledge and practice, whether explicable or not, used in the diagnosis, prevention and elimination of physical, mental or social imbalance and so relying exclusively on practical experience and observation handed down from generation to generation, whether verbally or in writing (Conserve Africa).

Furthermore the term traditional medicine is also used to refer to ways of protecting and restoring health that existed before the arrival of modern medicine.

In Africa, traditional healers and remedies made from plants play an important role in the health of millions of people. Natural medicines are the ‘alternative’ remedies that complement conventional medicine in the west. Many African people especially rural people and urban poor people in developed countries rely on the use of herbal medicine when they are ill. In these communities, traditional medicine is the major source for health care and sometimes the only source.

2 Table 1 shows that in Africa, there are still people who prefer to consult traditional medical practitioners rather than medical doctors. This is due to poverty in these countries, and hence people cannot afford to pay for medication, while on the other hand there is a shortage of medical doctors in the hospitals.

Table 1: Ratio of medical and traditional practitioners to population

Country Medical doctor Traditional References practitioners

Kenya 1: 7,142 (overall) 1: 987 (urban-mathare) World Bank, 1993

1: 833 (urban-mathare) 1: 378 (rural-Kilungu) Good, 1987

Lesotho Licensed TMPs Scott et al. 1996 estimated at 8,579 in 1991

Namibia 1: 1,000 (Katutura) Lumpkin, 1994

1: 500 (Cuvelai)

1: 300 (Caprivi)

South Africa 1: 1,639 (overall) 1: 700-1,200 (Venda) World Bank, 1993

1: 17,400 (homeland areas) Savage, 1985 Arnold and Gulumian, 1987

Swaziland 1: 10,000 1: 100 Green, 1985 Hoff and Maseko

3 The practice of traditional medicine has improved over the past few years. The World

Health Organization (WHO) has adopted a number of resolutions to draw attention to traditional medicine, because most of the populations in various developing countries around the world depend on traditional medicine for primary health care. But the most important concern of the WHO is the limited scientific evidence about the safety and efficacy of traditional medicines. The WHO has published guidelines in order to define basic criteria for evaluating the quality, safety and efficacy of traditional medicines aimed at assisting national regulatory authorities, scientific organizations and manufacturers in this particular area (Calixto,J.B.; 2000;Drew and Myers,

1994;Akerele,O., 1993). Thus, the work force represented by practitioners of traditional medicine is a potentially important resource for delivery of health care, while medicinal plants are of great importance to the health of individuals and communities. Traditional medicine has maintained its popularity in a number of Asian countries (China, India, Japan and Pakistan). China is able to provide adequate and constantly improving health care coverage for its urban and rural population because it harnesses the precious legacy of traditional medicine. In 1993 alone, the total sales of herbal medicines in China amounted to more than 2,5 billion US$ (WHO, 1996).

4 1.1.2 Medicinal plants

Medicinal plants are the oldest known health-care products. The importance of medicinal plants is still growing although it varies depending on the ethnological, medical and historical background of each country. Medicinal plants are also important for pharmacological research, and drug development, not only when the plant constituents are used directly as therapeutic agents, but also when they are used as templates for the synthesis of drugs or as models for pharmacologically active compounds. The earliest known medical document is a 4000-year-old Sumerian clay table, which lists plant remedies for various illnesses, including plants like mandrake for pain relief and garlic for treatment of heart and circulatory disorders (Levetin and

McMahon, 1999).

Scientists began to purify active extracts from medicinal plants as early as the nineteenth century. For example, Friedrich Serturner isolated morphine from the opium poppy in 1806. With the increase in knowledge of active chemical ingredients, the first purely synthetic drugs based on natural products were formulated in the middle of the nineteenth century. William Withering investigated foxglove (1775-

1785) for treatment of dropsy (congestive heart failure). In 1853 salicylic acid was synthesized for the first time, since it had been found to be the active ingredient in a number of plants known for their pain-relieving qualities (Levetin and McMahon,

1999).

5 1.1.3 The Family Alliaceae

The Alliaceae family has 600 species in 30 genera and the family is taxonomically intermediate between the Liliaceae and the Amaryllidaceae. Alliaceae is a widely distributed family and the major places of distribution for the whole family are

Mediterranean Europe, Asia, North and South America and Southern Africa. The

Southern African genera are Agapanthus, Tulbaghia and Allium. Many constituents have been isolated from the Alliaceae family. The strong onion or garlic smells were found in species from the Allium and Tulbaghia genera, while steroidal saponins are found in most of the species (Dahlgren et al., 1985).

For the purpose of this study, our attention will be focused mainly on the Tulbaghia and Allium genera since they are both endemic in South Africa. A number of species in these genera have not yet been scientifically investigated. Those that have been already investigated have been found to be rich in sulfur–containing compounds, which in most cases account for the characteristic odors of these plants.

6 1.1.3.1 The Genus Allium

Allium species contain mostly sulfur compounds, such as allyl sulfides, propionthiol and vinyl disulfide in their essential oils (Dahlgren et al., 1985).

1.1.3.1.1 Allium sativum

Common name: Garlic

Allium sativum is the most widely used herb in the world and is indigenous to Asia

(Hyams, 1971). Allium sativum has linear sheathing leaves, globose umbels of white or reddish flowers. The bulbs are composed of “cloves”, which are wrapped in a shared whitish papery coat. The odor is weak when the plant is intact, when damaged the smell grows strong. Allium sativum is traditionally used to treat minor vascular disorders and to decrease blood cholesterol. It has an antihypertensive effect

(Bruneton, 1995;Martindale, 1993;Reuter, 1995).

1.1.3.1.2 Allium cepa

Common name: Onion

Allium cepa leaves are cylindrical, with subglobose umbels of trimerous flowers initially covered by a membranous spathe. The bulbs are either white or colored and the shape and size differ with each variety of the species. Allium cepa has been used for boils, anthrax or whitlows. It is frequently used in homeopathy (coryza).

7 1.1.3.2 The Genus Tulbaghia

Tulbaghia is a small genus of about twenty species and entirely African in distribution. The distribution extends from Namaqualand and the Western Cape to the southern parts of Tanzania in southern tropical Africa. Two species are commonly grown as ornamentals. Indigenous people used several species as food and medicine

(Vosa, 1975). The bulbs of Tulbaghia cepacea are recommended for phthisis and as an anthelmintic (Watt and Breyer-Brandwijk, 1932).

1.1.3.2.1 Tulbaghia violacea

Common names: Wild garlic or society garlic (English), Wildeknoffel (Afrikaans),

Icinsini (Zulu), Itswele lomlambo (Xhosa) and Mothebe (Sotho) (Dyson, 1998).

The common name “society garlic” apparently originated from the belief that, in spite of its garlic-like flavor, the consumption of Tulbaghia violacea is not accompanied by the development of bad breath, as is the case with consumption of commercial garlic

(Allium sativum). Tulbaghia violacea is found originally in the Eastern Cape,

KwaZulu-Natal, and Northern Gauteng in South Africa, and as far north as

Zimbabwe. It is a bulbous plant, which grows to a height of 50cm. Leaves are narrow, hairless and strap-shaped, and grow to 30cm long and 1.5cm wide, arising from several white bases. The leaves are dark green, leathery in texture and smell strongly of garlic. It has umbels of up to 20 violet flowers (Dyson, 1998).

8 The bulbs of Tulbaghia violacea are used as a remedy for pulmonary tuberculosis and to destroy intestinal worms. The Zulu people use the bulb to make an aphrodisiac medicine. Some of the Rastafarians eat copious amounts of it and chili during winter allegedly “to keep the blood warm” and stop aches and pains. Bulbs and leaves soaked in water for a day can be used for rheumatism, arthritis and to bring down fever. The bulbs are also used for coughs, colds and flu. Zulu people used it to repel snakes away from their houses. It is also used for the treatment of infant and mother in the case of depressed fontanelle. In the Eastern Cape Tulbaghia violacea is used for colic, wind, restlessness, headache and fever, largely for young children. The evergreen leaves exhibit a garlic-like smell when bruised and have been used in some cultures as a substitute for garlic and chive (Dyson, 1998). Like any drug, extensive use can give adverse symptoms such as abdominal pain, gastroenteritis, acute inflammation and sloughing of the intestinal mucosa, cessation of gastro-intestina peristalsis, contraction of the pupils and subdued reactions to stimuli. Tulbaghia simmeleri may be substituted for Tulbaghia violacea, where the latter is not available.

9 1.1.3.2.2 Tulbaghia alliacea

Common name: Wild garlic (English), Wildeknoffel (Afrikaans), Isihaqa (Zulu) and

Moelela (Sotho).

Tulbaghia alliacea is an indigenous species in South Africa, growing particularly in the Eastern Cape and southern KwaZulu-Natal (Robert, 2001). It is a bulbous plant with long, narrow, hairless leaves arising from several white bases. Brownish green flowers occur in-groups of about 10 or more at the tip of a slender stalk (Robert,

2001). The bulbs and leaves of Tulbaghia alliacea are used medicinally. It is used for fever and colds, asthma, pulmonary tuberculosis and stomach problems. In the Cape

Dutch tradition, Tulbaghia alliacea was used as a purgative and for fits, rheumatism and paralysis. A tea can be made from chopped bulbs and roots and used as a purgative. The Khoikhoi and Basotho used to make a brew from the chopped bulbs and roots (Robert, 2001).

10 1.2 The Chemistry of Allium and Tulbaghia species

Volatile sulfur-containing flavor compounds are responsible for the characteristic smell and taste of the Allium and Tulbaghia species. The mainly intact bulbs contain the odorless, nonvolatile precursor S-alk(en)yl-L-cysteine sulfoxides. Hence, only the

L- (+)-isomers of these substances has been found in nature (Koch and Lawson,

1996). Alliinase enzymes catalyze the conversion of the odorless S-alk(en)yl-L- cysteine sulfoxides into volatile thiosulfinates, while pyruvate and ammonia are formed as byproducts. Thiosulfinates are relatively unstable; storage over long periods or steam distillation results in the formation of di-, tri-, and polysulfides, ajoenes and vinyldithiines, as shown in Fig. 1. (Koch and Lawson, 1996;Block, 1992).

O O NH2 Alliinase S RSOH CH COOH + NH 3 H O + 3 R COOH 2 sulfenic acid pyruvic acid ammonia S-alk(en)yl cysteine sulfoxide

1, R = m ethyl 2, R = ethyl O 3, R = propyl S R 4, R = 1-propenyl R S 5, R = allyl thiosulfinate 6, R = butyl

S R R S disulfide

Fig. 1. Alliinase catalyzed conversion of cysteine sulfoxide to thiosulfinate

11 S- methyl-L-cysteine sulfoxide (MCSO, methiin, [1]) has been reported as a constituent of A. sativum, A. cepa, A. porrum, and A. ursinum (Knobloch et al.,

1991;Koch and Lawson, 1996). S-propyl cysteine sulfoxide (PCSO, propiin, [3]) was reported to be present in A. porrum and A. cepa and S- (2-propenyl)-L-cysteine sulfoxide, also known as S-allyl-L-cysteine sulfoxide (ACSO, alliin, [5]) was detected in A. sativum. S- (trans-1-propenyl)-L-cysteine sulfoxide (PeCSO, isoalliin, [4]) is the precursor of the lachrymatory factor of Allium cepa (Block, 1992;Koch and Lawson,

1996;Breu, 1996).

In certain Allium species, compound 1 has been detected as the major component, while compound 3 is less abundant and occurs in considerable amounts in a limited number of species (A. vineale, A. victorialis, A. ursinum, A. triquetrum and T. acutiloba). Compounds 4 and 5 were detected in all of Allium species, but in more significant amount in T. acutiloba. The total amounts of all cysteine sulfoxides for wild species vary between 0.006% and 0.309%, as is shown in Table 2 (Krest et al.,

2000). Compound 5 occurs in the subgenera Allium, Rhizerideum and Amerallium in considerable amounts.

Table 2. Total content (%) and relative amounts (%) of cysteine sulfoxides of Allium species and T. acutiloba Compound no. 1 3 4 5 Total (%) (%) (%) (%) content (%) Species A. sativum (bulb) 15 nd 10 76 0.229 A. vineale (bulb) 24 1 56 16 0.182 A. ursinum (bulb) 52 1 26 19 0.309 A. triquetrum (bulb) 62 6 14 12 0.128 A.victorialis (rhizome) 52 2 nd 44 0.117 T. acutiloba (rhizome) 66 24 8 nd 0.006 nd (not determined due to insufficient amount)

12 The contents of cysteine sulfoxide derivatives differ in all parts of the plant. For example, compounds 1, 4 and 6 were isolated from the bulbs, stem, leaves and flowers of Allium siculum, and the relative distribution in the different plant organs is presented in Table 3. Trace amounts of compound 3 and S- (methylthiomethyl)-L- cysteine sulfoxide were also detected from the plant (Kubec et al., 2002b).

Table 3. Total content (%) and relative amounts (%) of cysteine sulfoxides in different parts of A. siculum Compound no. 1 4 6 Total (%) (%) (%) content (%) Plant organs Stem 18 64 18 0.03 Leaves 23 48 29 0.04 Flowers 73 11 15 nd Bulbs 62 15 23 0.35 nd (not determined due to insufficient amount)

Various Allium hybrids were investigated with respect to their individual profiles of cysteine sulfoxides. For the breeding of hybrids, A. cepa was used as the maternal plant and wild species were used as paternal parents. Compounds 1 and 4 were detected in all investigated Allium hybrids with only low levels of compound 3 being reported. Compound 5 was abundant in A. cepa and A. obliquum. The total amounts of all cysteine sulfoxides for hybrids were shown (Table 4) to be between 0.08% and

0.47%. Hybrids have the potential to increase the yields of the volatile sulfur substances and can produce more potent pharmacological or flavoring effects

(Keusgen et al., 2002).

13 Table 4. Total content (%) and relative amounts (%) of cysteine sulfoxide of Allium hybrids. Compound no 1 3 4 5 Total (%) (%) (%) (%) content (%) Hybrids A.cepa x A. altyncolicum 42 14 48 2 0.18 A.cepa x A.chevsuricum 16 4 82 2 0.20 A.cepa x A.globosum 68 nd 20 14 0.32 A.cepa x A.saxarile 42 4 56 4 0.12 A.cepa x A.obliquum 2 nd 18 48 0.47 A.cepa x A.senescens 46 22 56 nd 0.08 nd (not determined due to insufficient amount)

Compounds 1 and 4 are the most common secondary metabolites occurring in the majority of Allium species (Kubec et al., 1999;Fenwick and Hanley, 1985; Block,

1992;Kubec et al., 2000;Krest et al., 2000;Yoo and Pike, 1998;Thomas and

Parkin, 1994; Ziegler and Sticher, 1989;Lawson, 1996). In Allium tripedale, only compound 1 was detected in the bulbs (Krest et al., 2000). Höerhammer et al. (1968) were the first to report the occurrence of compound 6 in nature. It was detected in commercial garlic (Allium sativum) using a mildly sensitive TLC procedure.

Compound 6 may be present in some other Alliaceae species. Trace quantities of several volatile butyl-containing sulfides presumably derived from compound 6, were found in onion (Allium cepa) (Talyzin et al., 1988) and chinese chive (Allium tuberosum L.) (Pino et al., 2001). Compound 2 has been found in members of several genera of the Brassicaceae in addition to the Alliaceae (Kubec et al., 2000;Kubec and Musah, 2001).

14 Several γ-glutamyl peptide derivatives of cysteine sulfoxide have been detected within the Allium species (Whitaker, 1976). Over 17 types have been isolated (Granroth,

1970), including γ-glutamyl-S-alk(en)yl glutathiones, γ-glutamyl-S-alk(en)yl cysteine and γ-glutamyl-S-alk(en)yl cysteine sulfoxide. All are derived from glutathione (γ- glutamyl cysteinyl glycine). They do not directly contribute to the flavor.

Chemical constituents of Tulbaghia violacea have been described in three scientific articles. Jacobsen et al. (1968) suggested the presence of three unidentified S- substituted cysteine sulfoxide derivatives, as well as a C-S lyase which presumably could play a role in the biosynthetic pathways involving sulfur-containing compounds, while Burton and Kaye (1992) isolated 2,4,5,7-tetrathiaoctane-2,2- dioxide and 2,4,5,7-tetrathiaoctane from the leaves of Tulbaghia violacea. Kubec et al. (2002a) isolated S- (methylthiomethyl)-cysteine-4-oxide, Fig. 2, S-methyl cysteine and S-ethyl cysteine derivatives from the rhizomes of Tulbaghia violacea, where S-

(methylthiomethyl)-cysteine-4-oxide occurred as the major component of the extract.

O NH2 S S 3CH COOH S-(methylthiomethyl)cysteine-4-oxide

Fig. 2. Structure of marasmin

15 S- (methylthiomethyl)-cysteine-4-oxide, trivially named “marasmin” was first isolated, as a γ-glutamyl dipeptide from several basidiomycetous mushrooms of the genus Marasmius (Gmelin et al., 1976). Marasmin was also recently isolated from the fruits of the tropical tree Scorodocarpus borneensis (Olacaceae) (Kubota et al.,

1998). The absolute configuration of marasmin was determined to be RSRC (Broek et al., 1987).

Krest et al. (2000) described compound 1, 3 and 4 as constituents of the extract from

Tulbaghia acutiloba Harv.

There is a correlation that exists between the analyzed cysteine sulfoxide; flavor precursors and each resulting flavor profile. Garlic (Allium sativum) is characterized by the main components: di-2-propenyl disulfide and methyl 2-propenyl disulfide, while onion (Allium cepa) contained predominantly dipropyl disulfide and (E)-1- propenyl propyl disulfide. Flavor substances derived from compound 5 are (Z)-1- methyl propenyl disulfide, di-2-propenyl disulfide and 2-propenyl-propyl disulfide,

Fig. 3 (Keusgen et al., 2002).

16 S S S S 2-propenyl propyl disulfide dipropyl disulfide

S S S S (E)-1-propenyl propyl disulfide di-2-propenyl disulfide

S S methyl 2-propenyl disulfide

Fig. 3. Structures of disulfide from A. cepa and A. sativum

Gmelin et al. (1976) had previously suggested the possible formation of S-

(methylthiomethyl) (methylthio) methanethiosulfinate, as a primary breakdown product from marasmin, Fig. 4. Kubec et al. (2002a) isolated the thiosulfinate from T. violacea and gave it the trivial name “marasmicin”.

.. O H NH2 C-S lyase S SOH S S COOH Marasmin -1/2 H2O

O

S S S S Marasmicin

Fig. 4. Formation of marasmicin

17 The latter author further suggested that marasmicin is a very unstable compound, which decomposes rapidly into various sulfur-containing products, such as 2,4,5,7- tetrathiaoctane; 2,4,5,7-tetrathiaoctane-2,2-dioxide; 2,4,5,7-tetrathiaoctane-4,4- dioxide or 2,4,5,7-tetrathiaoctane-2,2,7,7-tetraoxide, Fig. 5. These compounds have been shown to have strong antimicrobial and antifungal activity as well as antithrombotic properties (Takazawa et al., 1982;Burton and Kaye, 1992;Kubota et al., 1994a,b; Block et al., 1994;Rapior et al., 1997;Lim et al., 1998,1999).

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

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

Fig. 5. Structures of thiosulfinate

18 1.2.1 Structural studies of some sulfur-containing compounds

In a study conducted by Mütsch-Eckner et al. (1993) on Allium sativum, they firstly subjected plant organ (leaves) to 80% aqueous methanol extraction at room temperature. Chromatography of the methanol extract over a strong acidic cation exchanger resin gave amino acids, which were further purified using gel permeation chromatography and then chromatography on high performance liquid chromatography (HPLC) reversed phase C18. On the bases of fast atom bombardment mass spectroscopy (FABMS), infrared (IR), and proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR), the structure was proposed to be (-)-N-(1'- deoxy-1'-β-D-fructopyranosyl)-S-allyl-L-cysteine sulfoxide, Fig. 6 (Mütsch-Eckner et al., 1993).

OH OH O H 4' H N 3' 2' OH 6' 6 S 1' 5' 2 O 5 3 COOH 7 1 OH

Fig. 6. Structure of an amino acid glycoside

19 1.2.2 Biosynthesis of flavor precursors of Allium species

Amino acids are precursors of proteins. Proteins are important in the metabolism of all organisms. Different organisms vary in their ability to synthesize amino acids.

Higher plants and microorganisms can synthesize all 20 amino acids. Amino acids also serve as precursors for other biomolecules having specialized functions.

Flavor precursors of Allium species are the S-alk(en)yl cysteine sulfoxides and their γ- glutamyl peptide relatives. Four non-volatile, odorless S-alk(en)yl cysteine sulfoxides are the precursors of the flavor and odors of the Alliums species. These are S-methyl cysteine sulfoxide, S-allyl cysteine sulfoxide, S-trans-prop-1-enyl cysteine sulfoxide and S-propyl cysteine sulfoxide. Amino acids like cysteine and serine are in turn the precursors for the biosynthesis of the flavor precursors. Serine is a precursor of glycine and cysteine, while cysteine in mammals arises from methionine and serine.

Serine

The major source of serine is 3-phosphoglycerate, which is derived from the glycolytic pathway. The pathway for the biosynthesis of serine is as follows: 3- phospho-D-glycerate is oxidized by phosphoglycerate dehydrogenase at the expense of NAD+ to give 3-phosphohydroxypyruvate. Then, there occurs transamination from glutamate to give 3-phosphoserine, which forms free serine on the hydrolysis by phosphoserine phosphatase, Scheme 1 (Lehninder, A.L., 1975).

20

OH

HPO CH 2 CH COOH 3-Phospho-D-glyceric acid OH O

+ NAD

phosphoglycerate dehydrogenase

NADH

OH

HPO OCH2 C COOH 3-Phosphohydroxy-pyruvic acid O O

glutamate phosphoserine transaminase

α−ketoglutarate

OH

HPOCHO 2 CH COOH 3-Phosphoserine O NH2

H 2O

phosphoserine phosphatase

Pι

3CH CH 2 CH COOH Serine NH2

Scheme 1. Biosynthesis of serine

21 Methionine

The carbon chain of homoserine is derived from aspartic acid in a series of reactions that do not take place in mammals. The succinyl group of succinyl-CoA transfers to homoserine, to give O-succinylhomoserine (enzymatic formation). Cysteine displaces succinate from O-succinylhomoserine, to give cystathionine (thioether compound).

Cystathionine is cleaved hydrolytically to yield homocysteine, pyruvic acid and NH3 by action of cystathionine β-lyase. Methionine was formed when homocysteine is

5 methylated with N -methyl- FH4 (Scheme 2) (Lehninder, A.L., 1975).

22 HOH2CCH2 CH COOH Homoserine NH2

succinyl-CoA

homoserine acyltransferase

CoA

H2COCO

CH2 CH2 O-Succinyl-homoserine 2NH CH CH2

COOH COOH

cysteine

cystathionine γ -synthase succinate

H2CSCH2

CH2 HC NH2 HCHS 2 CH2 CH COOH NH CH COOH 2 Homocysteine NH H2O 2 COOH NH3 cystathionine Cystathionine β-lyase methyl transferase

3CH SCH2 CH2 CH COOH

NH2 Methionine

Scheme 2. Biosynthesis of methionine

23 Cysteine

The pathway for the biosynthesis of cysteine involves transfer of the sulfur atom in methionine to replace the hydroxyl group oxygen atom of serine, thus converting serine to cysteine. The process is called transsulfuration.

The steps in the biosynthetic pathway are as follows:

1. Methionine loses the methyl group atom from its sulfur atom to become

homocysteine. During this process ATP converts methionine into S-

adenosylmethionine (active form). The methyl group attached to the sulfonium

linkage may be donated to any large number of different methyl group acceptors

in the presence of an appropriate enzyme, leaving S-adenosylhomocysteine as a

demethylated product. Hydrolysis then occurs to give free homocysteine.

2. Homocysteine reacts with serine in a reaction catalyzed by cystathionine β-

synthase, to give cystathionine. Cystathionine plays a central role in the

biosynthesis of methionine in plants and bacteria and the biosynthesis of cysteine

in animals.

3. Cystathionine γ-lyase (a pyridoxal phosphate enzyme) catalyzes the cleavage of

cystathionine, to give free cysteine and other products like α-ketobutyrate and

NH3.

Cysteine is an allosteric inhibitor of cystathionine γ-lyase, Scheme 3 (Lehninder,

A.L., 1975).

24 3CH SCH2 CH2 CH COOH Methionine NH2 ATP

methionine adenosyltransferase

Pι + PPι

CH NH2 3 O adenine + HOOC HC CH CH S CH 2 2 2 S-Adenosyl methionine

methyl-group acceptor OH OH

methyltransferase

methylated acceptor

NH2 O adenine NH2 HOOC HC CH2 CH2 SCH2 adenosylhomocysteinase

HOOC HC CH2 CH2 SH

OH OH Homocysteine H O adenosine S-Adenosyl-homocysteine 2 serine cystathionine β-synthase H O cystathionine 2 γ-lyase NH2

HS CH2 CH COOH HOOC HC CH2 CH2 S CH2 CH COOH

NH2 H O NH3 2 NH2 Cysteine L-Cystathionine

Scheme 3. Biosynthesis of cysteine

25 Flavor precursors

Investigation of the biosynthesis of Allium flavor compounds started with the discovery by Stoll and Seebeck (1947) that compound 5 was present within garlic tissue as a stable source of diallyl thiosulfinate (allicin), the major flavor volatile.

Lancaster and colleagues have proposed a pathway requiring γ-glutamyl peptides as intermediates, which has become accepted as the biosynthetic route leading to the production of the flavor compounds (Lancaster and Shaw, 1989;Lancaster et al.,

1989;Randle et al., 1995;Block, 1992).

The pathway is based primarily on radiolabelling studies and analysis of Allium tissues. The biosynthesis of the flavor precursors in Allium proceeds via S- alk(en)ylation of the cysteine moiety in glutathione, followed by transpeptidation to remove the glycyl group. Oxidation to the cysteine sulfoxide is followed by removal of the glutamyl group to yield cysteine sulfoxides, Scheme 4. This pathway has parallels with glutathione degradation (Leustek et al., 2000). Glutathione is synthesized in both cytosol and chloroplasts (Noctor et al., 2002). Biosynthesis of other precursors occurs similarly, but with different alk(en)yl donors.

26 glutathione methyl donor

S-methyl glutathione

glycine

γ-glutamyl S-methyl cysteine

γ-glutamyl S-methyl cysteine sulfoxide

glutamic acid S-methyl cysteine sulfoxide

Scheme 4. Biosynthesis of methiin

An alternative biosynthetic route for flavor precursors involves omission of glutathione in favor of direct alk(en)ylation of cysteine or thioalk(en)ylation of O- acetyl serine followed by oxidation to a sulfoxide, Scheme 5. This has parallels in the production of several secondary metabolites (Ikegami and Murakoshi, 1994).

O -acetyl serine or cysteine S-methyl or methyl donor

S-methyl cysteine

S-methyl cysteine sulphoxide

Scheme 5. Biosynthesis of methiin (alternative)

27 Enzymes like peptidases and γ-glutamyl transpeptidases (except alliinase) are present in onion and garlic tissues providing further opportunities for interchange among the biosynthetic intermediates of the flavor precursors (Lancaster and Shaw, 1991;Ceci et al., 1992;Hanum et al., 1995). Enzymes that catalyse steps in flavor compound biosynthesis in Allium species are members of large families that have diverse functions.

Cysteine is known to react chemically with methacrylic acid to give S-2- carboxypropyl cysteine, to provide a route to the alk(en)yl groups in compounds 3, 4 and 5 (Scheme 6). Granroth (1970) had already shown that 14C-valine gave rise to radiolabelled methacrylic acid, which could react with glutathione to produce S-2- carboxypropyl glutathione. All the alk(en)yl side-chains in the cysteine sulfoxides

(except methyl) were derived from an S-2-carboxypropyl group through decarboxylation and reduction.

methacrylic acid

2-carboxypropyl-

propenyl- allyl-

propyl-

Scheme 6. Biosynthesis of the allyl-, propenyl-, and propyl- group

28 Allinase and C-S lyases

C-S lyases in plants are involved in primary and secondary metabolism, cleaving bonds between sulfur and carbon atoms and they are part of the larger family of aminotransferases. Members include alliinases, cystathionine and cystine lyases, cysteine desulphydrase and enzymes involved in the detoxification of xenobiotics after conjugation with glutathione, as well as others involved in glucosinolate and cyanogenic glucoside biosynthesis (Kiddle et al., 1999).

Alliinases are pyridoxal 5’-phosphate dependent α,β- eliminating lyases, which catalyze the decomposition of S-alk(en)yl cysteine sulfoxide to ammonium pyruvate and thiosulfinates, Fig. 7.

O O NH2 Alliinase S RSOH CH COOH + NH3 H O + 3 R COOH 2 sulfenic acid pyruvic acid ammonia cysteine sulfoxide

R = allyl O

S R R S thiosulfinate

Fig. 7. Cysteine sulfoxide reacts with alliinase enzyme to form thiosulfinate

29 Onion and garlic are the species in the Allium genus which contain high levels of alliinase, and which is estimated to be up to 6% by weight of the soluble protein

(Nock and Mazelis, 1987). Most of the Allium species have highest activity of alliinase toward alliin and isoalliin (Krest et al., 2000). It has been suggested that alliinase is a dimer of molecular mass between 108kDa and 111kDa, its subunits range between 48kDa and 54kDa and it requires a flavincoenzyme in addition to pyridoxal 5’-phosphate for its activity (Jansen et al., 1989). In a study involving protein analysis of Allium species, one enzyme extract exhibited alliinase activity in

o o pH range between 5 and 8, temperature optimum between 36 C and 40 C, and KM values ranging between 0.42mM and 1.79mM (Krest et al., 2000).

It has been reported that onion (Allium cepa) has higher alliinase activity at pH 7.4

(Vitanen and Spare, 1961). In garlic (Allium sativum) there are two modes of alliinase activity that can be distinguished from each other and which can be ascribed to two different enzyme forms of alliinase. One enzyme form acts on alliin and isoalliin, has optimum pH of 4.5 and cleaves 97% of both cysteine sulfoxides, while the other enzyme form acts on methiin, has optimum pH of 6.5 and cleaves 97% of cysteine sulfoxides. Alliinases are irreversibly deactivated at pH 1.5 - 3 (pH of stomach) and pH>9 (Lawson et al., 1992; and Lancaster et al., 1988). Formation of different thiosulfinates and corresponding transformation products depends on the action of alliinase.

30 1.3 Pharmacological activity of Allium sativum.

A number of researchers became interested in garlic’s sulfur compounds, because of the pharmacological activities of sulfur containing drugs such as penicillin, sulfonamide antibiotics, hypotensive captopril and many others. The main pharmacological effects of garlic are bacteriocidal, virucidal, fungicidal and antiparasitic. This is due to the organo-sulfur compounds, especially thiosulfinates among which allicin is the most abundant (Koch and Lawson, 1996). Allicin’s main antimicrobial effect is due to its chemical reaction with thiol groups of various enzymes (Ankri and Mirelman, 1999). Aqueous garlic extract has demonstrated significant in vitro inhibition of a number of drug-resistant bacterial strains, and promising in vivo activity when tested against Shigella flexneri in rabbits (Singh and

Shukla, 1984). Garlic has been reported to have potential anti-viral activity against various herpes viruses (Weber et al., 1992).

Anti-parasitic properties of garlic were successfully employed against Dermatophytes

(Venugopal and Venugopal, 1995), Trypanosoma spp. (Nok et al., 1996;Lun et al

1994) and Hymenolepis and Giardia (Soffar and Mokhtar, 1991). Garlic extract has shown success in treating cryptococcal meningitis (a systemic fungal infection)

(Davis et al., 1990). Garlic also exhibits antioxidant activity due to alliin and allicin, along with other compounds (Rabinkov et al., 1998;Kourounakis and Rekka,

1991).

It has been suggested that allicin is not directly responsible for medicinal effects, but that its transformation products like the sulfides, ajoene and vinyldithiin (Fig. 8) are.

31

S S S Ajoene

S

S 1,3 vinyldithiin

Fig. 8. Structures of ajoene and 1,3 vinyldithiin

Allicin is metabolized to allyl merceptan in blood and hepatic tissue (Egen-Schwind et al., 1992;Lawson and Wang, 1993). Garlic sulfides exhibit antibiotic, anticancer, anti-inflammatory, antioxidant, antiparasitic and antithrombotic effects (Reuter et al.,

1996) and modulatory effects on hepatic detoxification enzymes (Wu et al.,

2002;Loizou and Cocker, 2001;Guyonnet et al., 2001). Ajoene has been shown to have anticancer effect (Ahmed et al., 2001), antibiotic, antioxidant, antithrombotic and hypotensive effects. Vinyldithiins demonstrate antibiotic, anti-aggregatory

(Nishimura et al., 2000), anticancer (Lee et al., 2001) and antihypercholesterolemic effects.

Tulbaghia violacea has been proved to have similar antibacterial and antifungal activities as has been scientifically verified for commercial garlic (Allium sativum)

(Bruneton, 1995;Martindale, 1993).

32 1.4 Mass Spectrometry

Mass spectrometry has progressed extremely rapidly during the last decade: production, separation and detection of ions, data acquisition, data reduction and etc.

This has led to the development of entirely new instruments and applications. A variety of ionization techniques are used for mass spectrometry. Electrospray ionization (ESI) is used as ion source in this report. ESI is a liquid-phase ion source and one example of atmospheric pressure ionization (API) sources. Such sources ionize the sample at atmospheric pressure and then transfer the ions into the mass spectrometer. An electrospray is produced by applying a strong electric field, under atmospheric pressure, to a liquid passing through a capillary tube with a weak flux (1–

10 μl min-1). The electric field is obtained by applying a potential difference of 3-6 kV between this capillary and the counter-electrode, separated by 0.3-2 cm, producing electric fields of the order of 106 Vm-1. This field induces a charge accumulation at the liquid surface located at the end of the capillary, which will break to form highly charged droplets. A gas injected coaxially at a low flow rate allows dispersion of the spray to be limited in space. These droplets then pass either through a curtain of heated inert gas, most often nitrogen, or through a heated capillary to remove the last solvent molecules (De Hoffmann and Stroobant, 2002).

33 1.5 Aim of the thesis

The aim of the thesis is to investigate the structures of bioactive and related natural products from Tulbaghia alliacea. Previous studies on Tulbaghia alliacea

(Thamburan et al., 2005a, b) have shown that the extract of rhizomes from T. alliacea exhibits a wide range of biological activities, which include antimycobacterial and antifungal. Thus, it is anticipated that some or all the compounds that will be identified may be implicated, directly or indirectly, in the various biological activities cited above.

34 CHAPTER 2

EXPERIMENTAL WORK

2.1 Instrumentation

All solvents were of analytical grade and distilled prior to use. The NMR spectra were recorded on a Varian Gemini 2000 instrument (1H at 200MHz and 13C at 50.3MHz) at room temperature. The chemical shifts are reported in δ(ppm) using tetramethylsilane

(TMS) as an internal standard. GC-MS analysis was performed using Finnigan-Matt

GCQ-Gas chromatography equipped with an electron impact ionization source at

70eV over a range of 40-600 mass units. GC analysis was performed using 5890

Series II Gas Chromatography Hewlett Packard equipped with a flame ionization detector. Thin layer chromatography (TLC) was performed on aluminium plates coated with Merck Kieselgel 60 F254. HPLC analysis was performed using a Beckman

System Gold Autosampler 507 equipped with Diode Array detector. LC-MS analyses were performed using Waters API Q-TOF Ultima with positive electrospray ionization source (ESI) and MS/MS using Q-TOF Ultima mass spectrometer from

Waters/Micromass over the range of 150-1500.

2.2 Plant materials

Fresh rhizomes of Tulbaghia alliacea were collected from Durbanville Nature

Reserve at Cape Town on the 27th February 2004 in the morning. Dried bulbs were purchased from the Rastafarian herbal dealers near Bellville Station at Cape Town.

35 2.3 Extraction

2.3.1 Methanol extraction

The freshly harvested rhizomes of Tulbaghia alliacea (315.69g) were homogenized with methanol (700ml) using a blender and the suspension were stirred for 24 hours at room temperature. The suspension was filtered using muslin cloth and then centrifuged (500 R.P.M) for 10 minutes. The clear solution was recovered and the organic solvent was removed from the extract with the aid of a rotavapor. Yield =

21.17g

The same method was repeated for the dried bulbs (213.64g). Yield = 17.89g

2.3.2 Solvent partitioning fractionation

The dry methanol extract (20g) of fresh rhizomes was dissolved in distilled water

(100ml). The resulting aqueous solution was successively extracted with ethyl acetate

(100ml) and butanol (100ml). After the extractions were completed, the aqueous layers was evaporated to a minimal volume and then freeze dried. Yield for ethyl acetate extract = 1.49g, butanol extract = 3.66g and aqueous extract = 7.53g.

36 Flow chart

Fresh rhizomes

Methanol extract

Aqueous solution

Ethyl acetate extract Aqueous layer

Butanol extract Aqueous layer

Aqueous extract

2.3.3 Chloroform extraction

Thamburan et al. reported that the chloroform extract of dried rhizomes of Tulbaghia alliacea showed in vitro antimycobacterial activity against Mycobacterium smegmatis, using disc diffusion assays (Thamburan et al.,2005a).

The dried rhizomes of Tulbaghia alliacea (314.82g) were cut into small pieces in the chloroform solvent (64ml). Plant material and solvent were allowed to stand overnight at room temperature. The suspension was filtered using muslin cloth and the organic solvent was removed from the extract with the aid of a rotavapor. Yield = 260mg.

37 2.4 Acid hydrolysis, ethyl acetate extraction and acetylation of the aqueous extract

Aqueous extract (500mg) collected after sequentially partitioning with ethyl acetate and butanol was heated with 10ml of 2M hydrochloric acid in the water bath (100oC) for 2 hours. The acidic solution was neutralized using anion exchange resin

(Amberlite resin IRA 410). The neutral aqueous solution was extracted with ethyl acetate (100ml). The ethyl acetate layer was evaporated using rotavapor. Yield =

40mg.

The aqueous solution remaining after ethyl acetate extraction was evaporated to dryness and then redissolved in distilled water (5ml). Sodium borodeuteride (120mg) was added to the solution and left to stand for overnight at room temperature. Acetic acid was added dropwise into the mixture until no more effervescence was observed.

Repeated addition and evaporation of methanol was performed to facilitate removal of boric acid. The mixture was evaporated to dryness, and to the residue was added acetic anhydride (5ml). The mixture was heated in a water bath for 2 hours with occasional swirling. Ice water (10ml) was added to the mixture and it was stirred vigorously for 30 minutes. The mixture was extracted with chloroform (20ml). The chloroform layer was washed with distilled water (20ml), dried over anhydrous magnesium sulfate, filtered and concentrated to small volume. The yield of alditol acetate derivatives = 10mg.

38 2.5 Chromatography

2.5.1 Gas chromatography (GC)

For GC analysis, a 30m long ZB225 (0.25mm internal diameter and 25μm film thickness) capillary column with stationary phase based on 50% cyanopropylphenyl- dimethylpolysiloxane was used. The operation conditions employed were as follows: injector, detector and oven temperatures of 250oC, 250oC and 215oC, respectively;

Helium gas as a carrier gas with a flow rate of 3.3 ml/min.

2.5.2 Gas chromatography-Mass spectroscopy (GC-MS)

A 3m long HP-5 (0.25mm internal diameter and 25μm film thickness) capillary column with stationary phase based on 5% phenyl-methylpolysiloxane was used for

GC-MS analysis. The operation conditions employed were as follows: interface temperature 180oC, ion source temperature 200oC and injector temperature 180oC, with Helium as a carrier gas at a flow rate of 0.27 ml/min. The temperature program was set to rise linearly from 180oC to 220oC at 2oC/min, and from 220oC to 260oC at

10oC/min and held at the final temperature for 25 minutes.

2.5.3 High performance liquid chromatography (HPLC)

A reverse phase column: Phenomenex, Luna C18 (2) 5µm Disc (Size: 250 x 4.6mm) was used for HPLC analysis. The mobile phase was methanol and 5% acetic acid, mode: gradient increasing organic phase from 20% to 90% over 18minutes and running time of 25 minutes with flow rate 1ml/min. Ultraviolet (UV) scanning wavelength range from 168nm to 280nm and from 168nm to 325nm. 20µL of a sample was injected and the temperature of a column was 25oC (room temperature).

39 2.5.3.1 Sample preparation

All samples were dissolved in the same solvents they had been extracted with and filtered prior to injection.

2.5.4 Liquid chromatography-Mass spectroscopy (LC-MS)

The column, mobile phase and method used for HPLC were used for LC-MS analysis.

A 10µL aliquot of the sample was introduced into a Waters alliance 2690 instrument with a split ratio 4:1 to a photo diode array detector and (200µL/min) to MS. The flow rate was 0.8 ml/min. The source temperature was 100oC and desolvation temperature was 350oC.

2.5.4.1 Sample preparation

2.5.4.1.1 Chloroform extract

The chloroform extract was dissolved in 2ml of chloroform and then 100µL of the sample were diluted in 900µL of 50% acetonitrile. The milky solution was centrifuged. Two layers were collected separately. The top layer was analyzed.

2.5.4.1.2 Ethyl acetate extract

Ethyl acetate extract was dissolved in 2ml of ethyl acetate and 1ml of the sample was centrifuged. 100µL of sample was diluted in 900µL of 50% acetonitrile.

2.5.4.1.3 Methanol extract

Methanol extract was dissolved in 2ml of methanol and 1ml of the sample was centrifuged. 100µL of sample was diluted in 900µL of 50% acetonitrile.

40 CHAPTER 3

RESULTS AND DISCUSSION

3.1 Characterization of cysteine sulfoxide structures.

Methanol, ethyl acetate and chloroform extracts were analysed by TLC, eluting with a butanol:water:acetic acid (4:1:1) mixture and spraying with freshly prepared ninhydrin reagent. Methanol extract gave one spot with Rf value 0.80 and also at the origin under UV. The methanol extract gave a positive ninhydrin reaction (yellowish- purple colour), suggesting the possible presence of compounds of the amino acid type.

The methanol extract was also examined using reversed-phase high performance liquid chromatography (HPLC) on a C18 column and electrospray ionization (ESI)

LC tandem MS (LC-MS/MS) methods.

The HPLC-UV chromatograms (Fig. 9) show numerous peaks, but no structural information could be retrieved out of the data. However, a total of four S-alk(en)yl cysteine sulfoxides could be detected by the ESI/LC-MS method (Fig. 10). The relevant structural information is discussed below. Molecular ions of the structures discussed below display dimerization in the MS source. There are similarities in fragmentation pathways of cysteine sulfoxide derivatives observed and agreed with that reported in the literature (Van Leuken et al., 1995).

41

Fig. 9. HPLC-UV chromatograms of methanol extract:168-280nm(top);168- 325nm(bottom)

42 Fig.10. ESI/LC-MS BPI chromatogram of methanol extract

43 Table 5. ESI-MS/MS spectral data of cysteine sulfoxides Cpd tR Molecular ion MS/MS spectral data, m/z (100%) no adduct (m/z) 1 2.352 221 137(100);160(4)

2 2.352 303 225(100);165(32)

3 2.599 393 104(100); 264(66);179(64)

4 2.978 382 114(100)

3.1.1. S-(methylthiomethyl) cysteine-4-oxide (marasmin) (1)

O H2N S S 3CH COOH 1

ESI/LC-MS of the methanol extract shows a peak at tR 2.352, corresponding to

+ molecular ion adduct of m/z=221 [M+Na+H] for molecular formula C5H11NO3S2.

MS/MS shows a fragment ion at m/z=160, which is due to loss of NH2 and COOH units from m/z=221 and fragment ion at m/z=137 (base peak) shows loss of sodium

(Na) ion (Appendix A and B).

44 Kubec et al. (2002a) were able to characterise compound 1 from the rhizomes of

Tulbaghia violacea Harv by isolating it among amino acids using ion-exchange chromatography followed by GC-MS analysis, which was preceded by derivatization with ethyl chloroformate (ECF) and reduction with sodium iodide (NaI). This shows that some of the species from the genus Tulbaghia contain compound 1 as one of their cysteine derivatives. Compound 1 was also found to occur in trace amounts in the bulbs of Allium siculum, but in this case derivatization was performed with methyl chloroformate (MCF) (Kubec et al., 2002b). This compound was first isolated as a γ- glutamyl dipeptide from several basidiomycetous mushrooms of the genus Marasmius

Fr. (Gmelin et al., 1976) and from the fruits of the tropical tree of Scorodocarpus borneensis Becc (Olacaceae) (Kubota et al., 1998).

3.1.2 S-methyl cysteine sulfoxide (2)

O H2N S CH 3 COOH

2

On further examination, the ESI/LC-MS of the methanol extract shows a peak at tR

2.352, corresponding to molecular ion adduct of m/z=303 [2M+H]+ for molecular formula C4H9NO3S. MS/MS shows a fragment ion at m/z=225 (base peak) which is due to the loss of hydrogen (H) ion and CH3SOCH2 unit from m/z=303, and fragment ion at m/z=165 shows loss of CHCOOH unit from m/z=225 (Appendix A and B).

45 Compound 2 has been found in Tulbaghia violacea (Kubec et al., 2002a) and

Tulbaghia acutiloba (Krest et al., 2000), suggesting that it is of relatively common occurrence in Tulbaghia species. Compound 2 is also a common secondary metabolite which occurs as a main constituent in the majority of Allium species (Kubec et al.,

1999;Fenwick and Hanley, 1985;Block, 1992;Kubec et al., 2000;Krest et al.,

2000;Yoo and Pike, 1998;Thomas and Parkin, 1994;Ziegler and Sticher,

1989;Lawson, 1996) and it occurs abundantly in species of the Alliaceae family, as well as other families like Brassicaceae (Kubec and Musah, 2001) and Leguminosae

(Giada et al., 1998;Kasai et al., 1986). It has also been detected in bulbs and leaves of Leucocoryne species (Liliaceae) (Lancaster et al., 2000).

3.1.3 S-allyl cysteine sulfoxide (Alliin) (3)

O H2N S 2CH COOH

3

The third component within the methanol extract was detected as a peak at tR 2.599, corresponding to molecular ion adduct of m/z=393 [2M+K]+ for molecular formula

C6H11NO3S. MS/MS suggests a loss of potassium (K) ion and CH2CHCH2SO unit to give a fragment ion at m/z=264 from m/z=393, fragment ion at m/z=179 shows loss of CH2CHCOOHNH2 unit from m/z=264, and fragment ion at m/z=104 (base peak) indicates loss of CHCOOHNH2 unit from m/z=179 (Appendix A and B).

46 Compound 3 was detected as the major compound in garlic (Allium sativum) (Ziegler and Sticher, 1989;Mütsch-Eckner and Sticher, 1992), and it is contained mainly in the bulbs of the plant (Block, 1992). This compound was characterised for the first time independently by Ziegler and Sticher (1989) and Mochizuki et al. (1988). After pre-column derivatization, compound 3 was analyzed as isoindole and a fluorophore derivative, respectively. This compound was the first of cysteine sulfoxides to be isolated and characterised as an antibiotic precursor in garlic (Stoll and Seebeck,

1951).

3.1.4 S-propyl cysteine sulfoxide (4)

O H2N S 3CH COOH

4

ESI/LC-MS of the methanol extract also shows a peak at tR 2.978, corresponding to

+ molecular ion adduct of m/z=382 [2M+Na+H] for molecular formula C6H13NO3S.

MS/MS indicates loss of M and hydrogen (H) ions and CH2CHCOOHNH2 unit from m/z=382 to give fragment ion at m/z=114 (base peak) (Appendix A and B).

Leek (Allium porrum L.) has been reported to contain mainly compound 4 (Block,

1992). It was reported to occur in trace amount in the bulbs of Allium siculum (Kubec et al., 2002b). It has also been found in white onion (Allium cepa L.) in low concentration (Lancaster and Boland, 1990)

47 3.2 Characterization of sulfone and thiosulfinate structures

TLC analysis of the chloroform extract was performed using hexane:ethyl acetate

(2:1) mixture as a solvent system, while analysis of the ethyl acetate and methanol extracts employed ethyl acetate:ethanol:water (7:2:2) mixture. The plates were sprayed with vanillin/sulphuric acid reagent. The methanol extract gave one spot and a short streak from the origin under UV and the spot had an Rf value of 0.84. Ethyl acetate extract gave two separated spots with Rf values 0.78 and 0.70, and one at the origin. The chloroform extract gave one spot with Rf value 0.70 and a faint short streak from the origin. All the extracts showed green colour response against the reagent, and they were then fully characterized using reversed-phase HPLC and

ESI/LC-MS/MS methods.

The HPLC-UV chromatograms of the ethyl acetate (Fig. 11) and chloroform (Fig. 13) extracts show numerous peaks with no structural information. Hence the extracts were also studied by the ESI/LC-MS method (Fig. 12 and 14). The sulfone and thiosulfinates were detected and the relevant structural information is discussed below. The molecular ion of the structures discussed below exhibit dimerization in the

MS source. The dimerization phenomenon has been reported previously in a study by

Ferary and Auger (1996). The fragmentation pathways of sulfone and thiosulfinates structures discussed below showed unique differences among each other as a group.

48

Fig. 11. HPLC-UV chromatograms of ethyl acetate extract:168-280nm(top);168- 325nm(bottom)

49 Fig. 12. ESI/LC-MS BPI chromatogram of ethyl acetate extract

50

Fig. 13. HPLC-UV chromatograms of chloroform extract:168-280nm(top);168- 325nm(bottom)

51 Fig. 14. ESI/LC-MS BPI chromatogram of chloroform extract

52 Table 6. ESI-MS/MS spectral data of sulfone and thiosulfinates from different extracts. a: methanol extract; b: ethyl acetate extract; c: chloroform extract Cpd tR Molecular ion MS-MS spectral data, m/z (100%) no. adduct (m/z) 5 2.978 476 134(100);116(36)a 3.206 460 118(100)a

6 5.119 295 115(100);133(56);151(32)a

7 19.133 397 397(100);284(54);156(22)b 15.183 379 255(100);217(41);298(40)c

8 18.442 368 256(100);368(88);156(58)b

9 20.606 412 411(100);214(30);242(28)c

10 11.410 225 132(100)c

3.2.1. 2,4,5,7-Tetrathiaoctane-2,2-dioxide (5)

O

S S CH CH3 3 S O S

5

ESI/LC-MS of the methanol extract shows a peak at tR 2.978, corresponding to

+ molecular ion adduct of m/z 476 [2M+K+H] for molecular formula C4H10S4O2.

MS/MS shows fragment ion at m/z=134 (base peak) which is due to loss of M ion and

CH3SCH2SS unit from m/z=476, and fragment ion at m/z=116 indicates to loss of

CH2 unit from m/z=134. At tR 3.206 there occurs molecular ion adduct of m/z=460

[2M+Na+H]+ for the same molecular formula. MS/MS indicates fragment ion at m/z=118(base peak) which is due to loss of M ion and CH3SCH2SS unit from m/z=460 (Appendix A and B).

53 Both peaks lost the same first unit from their molecular ion adduct, but the base peaks are different. Compound 5 was isolated and characterized as one of the sulfur compounds from Tulbaghia violacea (Burton and Kaye, 1992). It is a sulfur- containing product produced from decomposition of marasmicin (Kubec et al.,

2002a). This compound has been shown to have strong antimicrobial and antifungal activity, as well as antithrombotic properties (Kubota et al., 1994a,b;Block et al.,

1994;Rapior et al., 1997;Lim et al., 1998,1999). It has been reported (Takazawa et al., 1982) that compound 5 has an interesting relationship with bis[(methylsulfonyl)methyl] disulfide, in that their structures and antimicrobial activities closely resemble each other. Bis[(methylsulfonyl)methyl] disulfide has been isolated as an antifungal compound from Shiitake mushroom. There are also reports about similar oxygen-containing sulfur compounds from other families (Kouokam et al., 2002).

3.2.2 2-Propenyl methanethiosulfinate (6)

O

S CH CH 2 3 S

6

The second component within the methanol extract was detected as a peak at tR 5.119, corresponding to molecular ion adduct of m/z 295 [2M+Na]+ for molecular formula

C4H8S2O. MS/MS shows a fragment ion at m/z=151 to indicates loss of sodium (Na) ion and CH2CHCH2SSO unit from m/z=295, fragment ion at m/z=133 shows loss of

CH3 unit from m/z=151, and fragment ion at m/z=115 (base peak) indicates loss of

CH3 unit from m/z=133 (Appendix A and B).

54 Compound 6 was identified by LC/APCI-MS as one of the major components in supercritical fluid extracts of aqueous homogenates of ramp (Allium tricoccum) and garlic (Allium sativum) (Ferary and Auger, 1996;Calvey et al., 1998).

3.2.3 1-Propenyl butanethiosulfinate (7)

O

CH 3 S CH3 S

7

On examination of ethyl acetate extract, ESI/LC-MS/MS shows a peak at tR 19.133, corresponding to molecular ion adduct of m/z 397 [2M+K+2H]+. MS/MS shows fragment ion at m/z=397 as a base peak, fragment ion at m/z=284 suggests loss of potassium (K) and hydrogen (H) ions and CH3CHCHS unit from m/z=397, whereas fragment ion at m/z=156 shows loss of hydrogen (H) ion, CH3CH2CH2CH2SO and

CH3 units from m/z=284. ESI/LC-MS of chloroform extract shows a peak at tR

15.183, corresponding to molecular ion adduct of m/z 379 [2M+Na]+. MS/MS shows fragment ion at m/z=298 indicates loss of sodium (Na) ion and CH3CH2CH2CH2 unit from m/z=379, fragment ion at m/z=255 (base peak) suggests loss of CH3CHCH unit from m/z=298 (Appendix A and B).

Compound 7 was isolated from Allim siculum (Kubec et al., 2002b). It is the primary breakdown product from S-butyl cysteine sulfoxide. This suggests that Tulbaghia alliacea contains S-butyl cysteine sulfoxide as a precursor.

55 3.2.4 1-Propenyl propanethiosulfinate (8)

O

S CH CH3 3 S

8

On examination of ethyl acetate extract, the ESI/LC-MS shows a peak at tR 18.442, corresponding to molecular ion adduct of m/z 368 [2M+K+H]+ for molecular formula

C6H12S2O. MS/MS shows fragment ion at m/z=368, fragment ion at m/z=256(base peak) suggests loss of potassium (K) ion and CH3CHCHS unit from m/z=368, and fragment ion at m/z=156 shows loss of CH3CH2CH2SO and CH3 units from m/z=256

(Appendix A and B).

Compound 8 was detected by GC-MS method in a variety of Allium species (Block et al., 1992). It was also detected in garlic and leek using HPLC-MS method (Ferary and Auger, 1996).

3.2.5 Butyl butanethiosulfinate (9)

O

3CH S S CH3

9

56 The component within the chloroform extract was detected as a peak at tR 20.626, corresponding to molecular ion adduct of m/z 412 [2M+Na+H]+ for molecular formula C8H18S2O. MS/MS shows fragment ion at m/z=411(base peak) is due to loss of hydrogen (H) ion from m/z=412, fragment ion at m/z=242 indicates loss of

CH3CH2CH2CH2SOSCH2CH2 unit from m/z=411, and fragment ion at m/z=214 suggests loss of CH3CH2 from m/z=242 (Appendix A and B).

Compound 9 contains an n-butyl moiety and has been detected from Allium siculum as one of the six n-butyl-containing thiosulfinates (Kubec et al., 2002b).

3.2.6 2,4,5,7-Tetrathiaoctane-4-oxide (marasmicin) (10)

O

S S CH 3CH 3 S S

10

On further examination, the ESI/LC-MS of chloroform extract shows a peak at tR

11.410, corresponding to molecular ion adduct of m/z 225 [M+Na]+ for molecular formula C4H10S4O. MS/MS shows fragment ion at m/z=132(base peak) which is due to loss of CH3SCH2S unit from m/z=225 (Appendix A and B). Since the TLC profile showed one major spot, which also coincided with antimycobacterial activity from bioautographic studies (Thamburan et al., 2005a), the extract was submitted for

NMR measurements. The 1H NMR spectrum revealed the presence of two isolated methyl groups δ2.28 (3H, s), 2.39 (3H, s) and two pairs of heterosteric methylene protons δ4.15 (2H, d, J=7.6Hz) and 4.25 (2H, d, J=1.2Hz).

57 The 1H NMR spectral data (Fig. 15) agreed well with that from the literature (Kubec et al., 2002a), where this compound was isolated from rhizomes of Tulbaghia violacea. Compound 10 is the primary breakdown product of compound 1.

58 Fig. 15. 1H NMR of marasmicin from Tulbaghia alliacea

59 3.3 Characterization of polysulfide structures

The ethyl acetate extract was examined by the GC-MS method. The chromatogram

(Fig. 16) shows four peaks. The fragmentation pathways of the structures discussed below are similar to that reported from literature (Rapior et al.,1997). The GC-MS chromatogram of ethyl acetate extract shows peak 1 to be chloroform solvent and peak 2 was not analysed, because it was too complex (Fig. 16).

Table 7. GC-MS spectral data of ethyl acetate extract Peak Cpd tR MS spectral data, m/z (%) no. no. (minutes)

3 11 3.25 61(100);45(59);186(8)

4 12 6.16 61(100);45(99);139(96);154(29)

3.3.1. 2,4,5,7-tetrathiaoctane (11)

S S CH 3CH 3 S S

11

The mass spectrum of peak 3 at tR 3.25 shows the relative molecular mass of 186.

Fragment ion at m/z = 61(base peak) indicates loss of CH3SCH2SS unit from the parent ion (Scheme 7) (Appendix C). Compound 11 is a tetrathiaoctane and sulfur atoms are in the 2-, 4-, 5-, and 7- positions. Compound 11 was described by Kubota and Kubayashi (1994). The mass spectrum of compound 11 was in agreement with the literature (Rapior et al., 1997;Burton and Kaye, 1992).

60

S S CH3 3CH S S m/z = 186

- CH3SCH2SS

S + 3CH CH2 m/z -125

Scheme 7. Mass spectral fragmentation pathway of compound 11

The TLC profile of chloroform extract showed one major spot, which coincided with antimycobacterial activity from bioautographic studies (Thamburan et al., 2005a), the extract was run on preparative TLC for purification and then submitted for NMR measurements. The 1H NMR revealed the presence of two methyl groups at δ2.23

(6H,s) and two methylene groups at δ3.94 (4H,s) Fig. 15. It has been identified as a flavor compound in Marasmius species by Gmelin et al. (1976). Sulfides have been oxidized with H2O2 to yield sulfoxides, which may further be converted into sulfones using KMnO4 (Ahern et al., 1997). In this case compound 5 could be the oxidation product of compound 11, which has been previously isolated from Tulbaghia violacea. (Altamura et al., 1963). Thiosulfinates from Allium species are known to decompose on heating or attempted GC analysis (Brodnitz et al., 1971).

61

Fig. 16. GC-MS chromatogram of ethyl acetate extract

62 3.3.2 2,4,6-trithiaheptane (12)

S S S 3CH CH3

12

The GC-MS chromatogram of ethyl acetate extract shows peak 4 at tR 6.16 (Fig. 16.) having a relative molecular ion of 154. Fragment ion at m/z=139 shows loss of CH3 unit from the parent ion, fragment ion at m/z=61(base peak) indicates loss of SCH2S unit from m/z=139 (Scheme 8) (Appendix C).

S S S CH 3 CH3 m/z = 154

- CH3

+ S S S 3CH

m/z -15

SCH2S

S + 3CH CH2

m/z = 61

Scheme 8. Mass spectral fragmentation pathway of compound 12

63 Compound 12 is a dioxygenated product of 2,4,6-trithiaheptane 4,4-dioxide, also known as bis-methylthiomethylsulfone, which has been detected in the bark of the tropical garlic tree Scorodophloeus zenkeri Harms (Kouokam et al., 2002).

3.4 Isolation of a furanoid derivative

The ethyl acetate-soluble fraction obtained by solvent partitioning of the hydrolysed aqueous extract was analysed by TLC, eluting with hexane:ethyl acetate:ethanol

(1:2:1) mixture and spraying with the vanillin-sulfunic acid reagent. The TLC showed a single spot under UV with Rf value 0.51 and the spot gave a green color against the reagent. The aqueous solution remaining after ethyl acetate extraction was analysed by paper chromatography (PC), eluting with ethyl acetate:acetic acid:formic acid:water (18:3:1:4). The paper was dipped in p-anisidine hydrochloride reagent. The alditol acetate derived from the aqueous solution were analysed by gas chromatography (GC).

Table 8. EI/MS spectral data of the ethyl acetate-soluble fraction of the hydrolysed aqueous extract Cpd MS spectral data, m/z (%) no.

13 127(100);189(25);253(18);109(11);97(6)

3.4.1 5-(hydroxymethyl)-2-furfuraldehyde (13)

O

O H CH2OH

13

64 The 1H NMR spectrum revealed one aldehydic proton at δ9.60 (1H,s); two aromatic signals at δ7.23 (1H,d,J=3.6 Hz) and 6.53 (1H,d,J=3.6 Hz), the symmetrical appearance of these doublets as well as their integration relative to the aldehydic proton indicated the possible presence of a furan ring. One oxygenated methylene group was also observed at δ4.73 (2H,s), Fig. 17. The 13C NMR spectra exhibited one aldehyde at δ177.67, two oxygenated olefinic quaternary at δ160.48 and 153.90, two olefinic methine’s at δ122.34 and 110.04, and one oxygenated methylene at δ57.84 for carbon signals, Fig. 18. EI/MS spectrum showed a molecular ion of 127 [M+H]+ as a base peak, fragment ions at m/z=109 indicates loss of water ion (H2O), and fragment ion at m/z=97 shows loss of carbon ion (C). The MS peak at m/z=253 is most due to probably dimerization of molecular ion (Table 9) (Appendix C).

The spectral data of compound 13 agreed with that from the literature, where it was isolated as an anticonvulsant furan derivative from the arils of Euphoria longana L.

(Kim et al., 2005). Several authors have successfully detected compound 13 in several products of plant extracts (Kiridena et al., 1994), pharmaceutical syrups and infusion fluids (Cook et al., 1989), and coffee (Chambel et al., 1997).

Compound 13 is not a true compound found in aqueous extract, but an artefact generated by the acid hydrolysis step. There have been some reports about experimental and theoretical investigation of the acid-catalyzed kinetics of ketose and aldose reactions in liquid water at high temperature (200-250o) and pressure (34.5

MPa) with pH values at NTP (25o,0.1 MPa) ranging from 2 to 7. Compound 13 occurs as a major product with high yield from the acid-catalyzed dehydration of fructose

(Shaw et al., 1967;Antal and Mok, 1990).

65 The GC chromatogram of the derived alditol acetates was compared with that of authentic standards of monosaccharide derivates. The peak at tR 20.04, correspond with that of the glucose standard and it may thus be deduced that glucose was also a product of hydrolysis in this analysis. This may further suggest that compound 13 is probably present as a glucoside in the original extract.

66

Fig. 17. 1H NMR of 5-(hydroxymethyl)-2-furfuraldehyde from Tulbaghia alliacea

67

Fig. 18. 13C NMR of 5-(hydroxymethyl)-2-furfuraldehyde from Tulbaghia alliacea

68 CHAPTER 4

CONCLUSION

Tulbaghia alliacea is considered to be sulfur containing, because of its garlic smell when crushed or bruised. The mainly intact rhizomes of the plant contain the odourless, non-volatile precursor S-alk(en)yl cysteine sulfoxides. Allinase enzymes catalyse the conversion of odourless S-alk(en)yl cysteine sulfoxides into volatile thiosulfinates. Thiosulfinates are relatively unstable, storage over long period results in the formation of polysulfides. Due to the thermal instability of S-alk(en)yl cysteine sulfoxides and thiosulfinates, reversed phase HPLC and ESI/LC-MS/MS methods were used, as the techniques of choice for detecting and identifying these compounds without thermal degradation. ESI/LC-MS/MS gave a facile, reproducible, and accurate determination of S-alk(en)yl cysteine sulfoxides and thiosulfinates.

There have been reports that different members of the genus Tulbaghia contain different cysteine sulfoxides (Krest et al., 2000). In this study, four S-alk(en)yl cysteine sulfoxides were detected and identified from the methanol extracts. S-methyl cysteine sulfoxide (2), S-allyl cysteine sulfoxide (3), S-propyl cysteine sulfoxide (4) and S-(methylthiomethyl) cysteine-4-oxide (marasmin) (1) were isolated from

Tulbaghia alliacea. Species to species variation in relative proportions of S-alk(en)yl cysteine sulfoxides has also been observed in other genera of the Alliaceae family, most notably Allium and Leucocoryne (Fenwick and Hanley, 1985;Block,

1992;Kubec et al., 2000;Lancaster et al., 2000).

69 The volatile flavor components of a number of Allium species were reported to have significant differences in the compositions of thiosulfinates (Freeman and

Whenham, 1975). These organic sulfur compounds are of interest in research, for quantity control of foods, and in the pharmaceutical industry, which is increasingly processing garlic for phytomedicinal preparations. Six volatile thiosulfinates were detected and identified from the methanol, ethyl acetate and chloroform extracts. 2- propenyl methanethiosulfinate (6), 1-propenyl butanethiosulfinate (7), 1-propyl propanethiosulfinate (8), butyl butanethiosulfinate (9), 2,4,5,7-tetrathiaoctane-4-oxide

(marasmicin) (10) and 2,4,5,7-tetrathiaoctane-2,2-dioxide (5) are the thiosulfinates and sulfone found in Tulbaghia alliacea. Sulfur amino acids are of significance with respect to the flavor characteristic of the plant. The thiosulfinates mentioned above were primary breakdown products from compounds 1, 2, 3, and 4. Since compound 9 contains a butyl moiety, this suggests that S-butyl cysteine sulfoxide may be one of the flavor precursors found in this plant. Compound 5 is a sulfone produced from decomposition of compound 10.

Thiosulfinates from Allium species are known to decompose on heating or attempted

GC analysis. 2,4,5,7-tetrathiaoctane (11) and 2,4,6-trithiaheptane (12) were detected and identified by GC-MS from the ethyl acetate extract of Tulbaghia alliacea.

Compound 11 is the deoxygenated analogue of compound 5, while compound 12 has a similar relationship with 2,4,6-trithiaheptane 4,4-dioxide.

70 5-(hydroxymethyl)-2-furfuraldehyde (13) was identified as an artefact generated by the acid hydrolysis step. It is the product from the acid-catalyzed dehydration of fructose. Thus, fructose and glucose were the carbohydrate compounds found from the aqueous extract.

71 References

Ahern,T.P;Hennigar,T.L.;MacDonald,J.A.;Morrison,H.G.;Langler,R.F.;Satyanarayan a,S.;Zaworotko,M.J.1997.Synthesis and chlorination of chloromethyl methylsulfonylmethyl sulfides.Aust. J. Chem.,50,683-687.

Ahmed,N.;Laverick,L.;Sammons,J.;Zhang,H.;Maslin,D.J.;Hassan,H.J..2001.Ajoene,a garlic-derived natural compound,enhances chemotherapy-induced apoptosis in human myeloid leukemia CD34-positive resistant cells.Anticancer Res.,5,3519-3523.

Akerele,O.1993.Summary of WHO guidelines for the assessment of herbal medicines.HerbalGram,28,13-19.

Altamura,M.R.;Hasselstrom,T.;Long,L.1963.Synthesis of di(methylthiomethyl) polysulfides.J. Org. Chem.,28,2438-2440.

Antal,M.J. and Mok,W.S.1990.Mechanism of formation of 5-(hydroxymethyl)-2- furaldehyde from D-fructose and sucrose.Carbohydr. Res.,199,91-109.

Ankri,S.;Mirelman,D.1999.Antimicrobial properties of allicin from garlic.Microbes and Infection,2,125-129.

Block,E.1992.The organosulfur chemistry of the genus Allium-implications for the organic chemistry of sulfur.Angew. Chemie, Int. Ed. Engl.,31,1135-1178.

Block,E.;Putman,D.;Zhao,S.1992.Allium Chemistry:GC/MS analysis of thiosulfinates and related compounds from onion,leek,scallion,chive and chine chive.J. Agric. Food Chem.,40,2431-2438.

Block,E.;DeOrazio,R.;Thiruvashi,M.1994.Simple total syntheses of biogically active pentathiadecane natural products, 2,4,5,7,9-pentathiadecane 2,2,9,9- tetraoxide(dysoxysulfope), from Dysoxylum richii, and 2,3,5,7,9-pentathiadecane 9,9- dioxide, the misidentified lenthionine precursor SE-3 from shiitake mushroom(Lentinus edodes).J. Org. Chem.,59,2273-2275.

Breu,W.1996.Allium cepa(onion).Part1:Chemistry and analysis.Phytomedicine,3,293- 306.

Brodnitz,M.H.;Pascale,J.V;Van Derslice,L.1971.Flavour components of garlic extract.J. Agric Food Chem.,19,273-275.

Broek,L.A.G.M.;Breuer,M.L.;Liskamp,R.M.J.;Ottenheijm,H.C.J.1987.Total synthesis and absolute configuration of the natural dipeptide γ-glutamylmarasmine.J. Org. Chem.,52,1511-1517.

Bruneton,J.1995.Pharmacognosy,Phytochemistry,Medicinal Plants.Intercept,Hampshire.

Burton,S.G. and Kaye,P.T.1992.Isolation and characterization of sulphur compounds from Tulbaghia violacea.Planta Med.,58,295-296.

72 Calixto,J.B.2000.Efficacy,safety,quality control,marketing and regulatory guidelines for herbal medicines phytotherapeutic agents.Braz. J. Med. Biol. Res.,33,179-189.

Calvey,E.M.;White,K.D.;Matusik,J.E.;Sha,D.;Block,E.1998.Allium Chemistry:Identification of organosulfur compounds in Ramp(Allium Triciccum) Homogenates.Phytochemistry,49(2),359-364.

Ceci,L.N.;Curzio,O.A.;Pomilio,A.B.1992.γ-glutamyl transpeptidase/γ-glutamyl peptidase in sprouted Allium sativum.Phytochemistry,31,441-444.

Chambel,P.;Oliveira,M.B.;Andrade,P.B.Seabra,R.M.;and Ferreira,M.A.1997.Development of an high performance liquid chromatography/diode array detector method for simultaneous determination of 5- HMF,fufural,5-O-caffeoylquinic acid and caffeine in coffee.J. liq. Chrom. (in press).

Conserve Africa.Overview on the Medicinal Plants and Traditional Medicine in Africa.[Online].Available http:// www.conserveafrica.org/html.

Cook,A.P.;Macleod,T.M.;Appleton,J.D.;Fell,A.F.1989.Reversed phase high performance liquid chromatographic method for the quantification of 5- hydroxymethylfurfural as the major degradation product of glucose in infusion liquids.J. Chrom.,467,395-401.

Dahlgren,R.M.T;Clifford,H.T. and Yeo,P.F.1985.The Families of the Monocotyledons.Springer-Verlag,Berlin.

Davis,L.E.;Shen,J.K.;and Cai,Y.1990.Antifungal activity in Human cerebrospinal fluid and Plasma after Intravenous Administration of Allium sativum.Antimicrob. Agents and Chemo.,April,651-653.

De Hoffmann,E and Stroobant,V.2002.Mass Spectrometry.Principles and Applications.Second edition.John wiley and sons,Ltd.

Drew,A.K. and Myers,S.P.1994.Safety issues in herbal medicine:implications for the health professions.Drug Saf,11,284-294.

Dyson Alex.1998. Discovering Indigenous Healing plants of the herb and fragrance gardens at Kristenbosch (National Botanical Garden).

Egen-Schwind,C.;Eckard,R.;Jekat,F.W.;and Winterhoff,H.1992.Pharmacokinetic of Vinyldithiins,Transformation products of Allicin.Planta Med.,58,8-13.

Fenwick,G.K.;Hanley,A.B.1985.The genus Allium-Part 2. Critical. Rev. Food Sci. Nutri.,22,273-377.

73 Ferary,S.; and Auger,J.1996.What is the true odour of cut Allium?Complementarity of various hyphenated methods:gas chromatography-mass spectrometry and high- performance liquid chromatography-mass spectrometry with particle beam and atmospheric pressure ionization interfaces in sulphenic acids rearrangement components discrimination.J. Chrom.,750,63-74.

Freeman,G.G.;Whenhan,R.J.1975.A survey of volatile components of some Allium species in terms of S-alk(en)yl-L-cysteine sulfoxides present as flavour precursors.J. Sci. Food Agric.,26,1869-1886.

Giada,M.L.R.;Miranda,M.T.M.;Marquez,U.M.L.1998.Sulfur γ-glutamyl peptides in mature seeds of common beans (Phaseolus vulgaris L.).Food Chem.,61,177-184.

Gmelin,R.;Luxa,H.-H.;Roth,K;Höfle,G.1976.Dipeptide precursor of garlic ador in Marasmius species.Phytochemistry,15,1717-1721.

Granroth,B.1970.Biosynthesis and decomposition of cysteine derivatives in onion and other Allium species.Ann. Acad. Sci. Fen.,154,1-71.

Guyonnet,D.;Belloir,C.;Suschetet,M.;Siess,M;Le Bon,A.2001.Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction.Mu. Res. Tox. Environ. Mut.,495(1-2),135-145.

Hanum,T.;Sinha,N.K.;Cash,J.N.1995.Characteristics of γ-glutamyl transpeptidase and alliinase of onion and their effects on the enhancement of pyruvate formation in onion macerates.J. Food Biochem.,19,51-65.

Höerhammer,L;Wagner,H.;Seitz,M.;Vejdelek,Z.J.1968.Evaluation of garlic preparations I.Chromatographic studies of the actual components of Allium sativum.Pharmazie,23,462-467.

Hyams, F.1971. Plants in the service of man: 10,000 years of domestication. JM Dent & Son, London.

Ikegami,F.;Murakoshi,I.1994.Enzymic synthesis of non-protein β-substituted alanines and some higher homologues in plants.Phytochemistry,25,1089-1104.

Jansen,H.;Muller,B.;Knoblock,K.1989.Alliin lyase from garlic,Allium sativum.Investigation on enzyme/substrate,enzyme/inhibitor interactions and on a new coenzyme.Planta Med.,55,434-439.

Jacobsen,J.V.;Yamaguchi,Y.;Mann,L.K.;Howard,F.D.1968.An alkyl-cysteine sulfoxide lyase in Tulbaghia violacea and its relation to other alliinase-like enzymes.Phytochemistry,7,1099-1108.

Kasai,T.;Cirrosita,Y.;Sakamura,S.1986γ-Glutamyl peptides of Vigna Radiata seeds.Phytochemistry,25,679-682.

74 Keusgen,M.;Sculz,H.;Glodek,J.;Krest,I.;Kruger,H.;Herchert,N.;Keller,J.2002. Characterization of some Allium hybrids by aroma precursors, aroma profiles and Alliinase activity.J. Agric. Food Chem., 50, 2884-2890.

Kiddle,G.A.; Bennett

Kiridena, W. ; Poole, S.K. ; and Poole, C.F.1994.Identification of 5-(hydroxymethyl- 2-furfur in vanilla extracts. J. Planar Chrom.,7,273-277.

Knobloch,K.;Lohmüller,E.;Landshuter,J;Winkler,G.1991.C-S lyasen aus Allium- Arten und ihre biochemischen Aktivitaten Dtsch.Apoth. Ztg.,133(Suppl. 24),5-6 (in German).

Koch, H.P.1996.Biopharmaceutics of Garlic’s Effective Constituents. In Garlic: The Science and Therapeutic Application of Allium sativum L. and Related Species. Koch and Lawson Eds. Williams and Wilkins, Baltimore, 213-220.

Koch,H.P.;Lawson,L.D.1996.Garlic:The Science and Therapeutic Application of Allium sativum and Related Species,Williams &wilkins:Baltimore,MD,1-227.

Kouokam,J.C.;Zapp,J.;Becker,H.2002.Oxygen-containing sulfur rich compounds from the bark of the tropical garlic tree Scorodophloeus zenkeri Harms.Phytochemistry,60,403-407.

Kourounakis,P.N.;Rekka,E.A.1991.Effect on active oxygen species of alliin and Allium sativum(garlic) powder.Res. Commun. Chem. Pathol. Pharmacol,74(2),249- 252.

Krest,I.;Glodek,J.;and Keusgen,M.2000.Cysteine sulfoxides and alliinase activity of some Allium species.J. Agric. Food Chem.,48,3753-3760.

Krest,I. and Keusgen,M.2002.Biosensoric flow-through method for the determination of cysteine sulfoxides.Analy. Chim.,469,155-164.

Kubec,R.;Svobodová,M.;Velišek,J.1999.Gas chromatographic determination of S- alk(en)ylcysteine sulfoxides.J. Chrom.,862,85-94.

Kubec,R.;Svobodová,M.;Velišek,J.2000.Distribution of A-alk(en)ylcysteine sulfoxide in some Allium species.Identification of a new flavor precursor S-ethylcysteine sulfoxide(ethiin).J. Agric. Food Chem.,48,428-433.

Kubec,R.;Musah,R.A.2001.Cysteine sulfoxides derivatives in Petiveria alliacea.Phytochemistry,58,981-985.

75 Kubec,R.;Velisek,J.;Musan,R.A.2002a.The amino acid precursors and odor formation in society garlic(Tulbaghia violacea Harv.).Phytochemistry,60(1),21-25.

Kubec,R.;Kim,V.;McKeon,D.M.;Musah,R.A.2002b.Isolation of S-n-butylcysteine sulfoxide and Six n-butyl-containing Thiosulfinates from Allium siculum.J. Natural Prod.,65,960-964.

Kubec,R.;Kim,S.;Velišek,J.;Musah,R.A.2003.Flavour precursors and aroma formation in society garlic (Tulbaghia violacea Harv.).Flavour research at the Dawn of the Twenty-First Century,Proceedings of the Weurman Flavor Research Symposium,10th,Beaune,France,June 25-28,2002.

Kubota,K.;Matsumoto,M.;Ueda,M.;Kobayashi,A.1994a.New antimicrobial compound from Scorodocarpus borneensis Becc.Biosci. Biotech. Biochem.,58,430-431.

Kubota,K.;Ohhira,S.;Kobayashi,A.1994b.Identification and antimicrobial activity of the volatile flavor constituents from Scorodocarpus borneensis Becc.Biosci. Biotech. Biochem.,58,644-646.

Kubota,K.;Hirayama,H.;Sato,Y.;Kobayashi,A.;Sugawara,F.1998.Amino acid precursors of the garlic-like odour in Scorodocarpus borneenis.Phytochemistry,49,99- 102.

Kubota,K.;Kobayashi,A.1994.Sulfur compounds in wood garlic (Scorodocarpus borneensis Becc.) as versatile food components.In Sulfur Compounds in Foods;ACS Symposium series;American chemical society:Washington,DC,pp 239-249.

Lancaster,J.E.;Dommisse,E.M.;Shaw,M.L.1988.Production of flavour precursors S- alk(en)yl-L-cysteine sulfoxides in photomixotrophic callus of garlic.Phytochemistry,27,2123-2124.

Lancaster,J.E and Shaw,M.L.1989.γ-glutamyl peptides in the biosynthesis of S- alk(en)yl-L-cysteine sulfoxide (flavour precursors) in Allium.Phytochemistry,28,455- 460.

Lancaster,L.D.;Reynolds,P.H.S.;Shaw,M.L.;Dommisse,E.M.;Munro,J.1989.Intercellu lar localization the biosynthetic pathway to flavour precursors in onion.Pytochemistry,28,461-464.

Lancaster,J.E. and Shaw,M.L.1991.Metabolism of γ-glutamyl peptides during development, storage and sprouting in onion bulbs.Phytochemistry,30,2857-2859.

Lancaster,J.E.;and Boland,M.J.1990.Flavour biochemistry,33-72.In:H.D. Rabinowitch and J.L. Brewster(cds).Onions and allied crops.vol.3.CRC Press,Boca Raton,Fla.

Lancaster,J.E.;Shaw,M.L,;Walton,E.F.2000.S-alk(en)yl-L-cysteine sulfoxides,aliinase and aroma in Leucocoryne.Phytochemistry,55,127-130.

76 Lawson,L.D.;Ranson,D.K.;Hughes,B.G.1992.Inhibition of whole blood platelet- aggregation by compounds in garlic clove extracts and commercial garlic products.Thromb. Res.,65(2),141-156.

Lawson,L.D.;and Wang,Z.J.1993.Pre-hepatic Fate of the Organosulfur compounds derived from garlic (Allium sativum).Planta Med.,59,A688.

Lawson,L.D.1996.In the Composition and Chemistry of Garlic cloves and Processed Garlic;Koch,H.P.,Lawson,L.D.,eds.;Williams & Wilkins:Baltimore,37-101. Lee,K.T.;Choi,J.H.;Kim,D.H.;Son,K.H.;Kim,W.B.;Kwon,S.H.;Park,H.J.2001.Constit uents and antitumor principle of Allium victorials var platyphyllum.Archiv. Pharm. Res.,24,44-50.

Lehninger,A.L.1975.Biochemistry:The molecular basis of cell structure and function.The Johns Hopkins University, School of medicine,2nd edition,Biosynthesis of the Nonessential Amino Acids.Worth publishers,inc.,694-718.

Leustek,T.;Martin,M.N.;Bick,J-A.;Davies,J.P.2000.Pathways and regulation of sulfur metabolism revealed through molecular and genetic studies.Annual Review of Plant Physiology and Plant.Molecular Biol.,51,141-165.

Levetin,E. and McMahon,K.1999. Plants and Society,second edition,Boston:WCB/McGraw-Hill.

Lim,H.;Kubota,K.;Kobayashi,A.Sugawara,F.1998.Sulfur-containing compounds from Scorodocarpus borneensis and their antimicrobial activity.Phytochemistry,48,787- 790.

Lim,H.;Kubota,K.;Kobayashi,A.Seki,K.;Ariga,T.1999.Inhibitory effect of sulfur- containing compounds in Scorodocarpus borneensis Becc. On the aggregation of rabbit platelets.Biosci. Biotech. Biochem.,63,298-301.

Loizou,G.D.;Cocker,J.2001. The effects of alcohol and diallyl sulphide on CYP2E1 activity in humans: a phenotyping study using chlorzoxazone.Hum. Exp. Tox.,20(7),321-327.

Lun,Z.R.;Burri,C.;Menzinger,M.;and Kaminsky,R.1994.Antiparasitic Activity of Diallyl Trisulfide (Dasuansu) on Human and Animal Pathogenic Protozoa (Trypanosoma sp.,Entamoeba Histolytica and Giardia Lamblia) In vitro.Ann. Soc. Belge Med. Trop,74,51-59.

Maher, G. 1992.Start a career in Complementary medicine: a manual/directory of course in alternative and complementary medicine, 2nd edition.

Martindale.1993.The Extra Pharmacopoeia.30th edition. Pharmaceutical Press,London.

Mochizuki,E.;Nakayama,A.;Kitada,Y.;Saito,K.;Nakazawa,H.;Suzuki,S.;Fujita,M.198 8.Liquid chromatographic determination of alliin in garlic and garlic products.J. Chrom.,455,271-277.

77 Mondy,N.Duplat,D.;Christides,J.P.;Arnault,I.;Auger,J.2002.Aroma analysis of fresh and preserved onions and leek by dual solid phase microextraction liquid extraction and gas chromatography.J. Chrom.,963,89-93.

Mütsch-Eckner,M;and Sticher,O.1992.Reversed phase high performance liquid chromatography of S-alk(en)yl-L-cysteine derivatives in Allium satinum including the determination of (+)-S-allyl-L-cysteine sulfoxide,γ-L-glutamyl-S-allyl-L-cysteine and γ-L-glutamyl-S-(trans-1-propenyl)-L-cysteine.J. Chrom.,625,183-190. Mütsch-Eckner,M.;Erdelmeier,C.A.J.;Sticher,O.1993.A novel amino acid glycoside and three amino acids from Allium sativum.J. Natural Prod.,56,864-869.

Nishimura,H.;Takahashi,T.;Wijaya,C.H.;Satoh,A.;Ariga,T.2000.Thermochemical transformation of sulfur compounds in Japanese domestic Allium,Allium victorials L.Biofact.,3,257-263.

Nock,L.P.;Mazelis,M.1987.The C-S lyases of higher plants.Direct comparison of the physical properties of homogeneous alliin lyase of garlic(Allium sativum) and onion (Allium cepa).Plant Physiol.,85,1079-1083.

Noctor,G.;Gomez,L.;Vanacker,H.;Foyer,C.H.2002.Interactions between biosynthesis,compartmentation and transport in the control of glutathione homeostasis and signalling.J. Exp. Bo.,3,1283-1304.

Nok,A.J.;Williams,S.;Onyenelwe,P.C.1996.Allium sativum induced Death of African Trypanosomes.Parasitology Res.,82,634-637.

Pino,J.A.;Fuentes,V.;Correa,M.T.2001.Volatile constituents of Chinese Chive (Allium Tuberosum Rottl.ex sprengel) and Rakkyo (Allium Chinense G.Don).J.Agric. Food Chem.,49(3),1328-1330.

Rabinkov,A;Miron,T;Konstantinivski,L;Wilchek,M;Mirelman,D;Weiner,L.1998.Bioc himica et Biophysica Acta(BBA)-General Subjects,1379(2),233-234.

Randle,W.M.;Lancaster,J.E.;Shaw,M.L.;Sutton,K.H.;Hay,R.L.;Bussard,M.L.1995.Qu antifying onion flavor compounds responding to sulfur fertility-sulfur increases levels of alk(en)yl cysteine sulphoxides and biosynthetic intermediates.J.Am. Soc. Hort. Sci.,120,1075-1081.

Rapior,S.;Breheret,S.;Talou,T.;Bessiére,J.-M.1997.Volatile flavour contiuents of fresh Marasmius alliaceus(garlic Marasmius).J. Agric. Food Chem.,45,820-825.

Reuter,H.D.1995.Allium sativum and Allium ursinum:Part 2.Pharmacology and medical application.Phytomedicine,2,73-91.

Reuter,H.D.;Koch,H.P.;Lawson,L.D.1996.In Garlic: The science and therapeutic application of Allium Sativum L. and Related species,Koch,H.P.;Lawson,L.D.Eds.Williams and Wilkins:Baltimore,135-212.

Robert Margaret.2001. Indigenous Healing Plants.

78 Shaw,P.E;Tatum,J.H.;and Berry,R.E.1967.Acid-catalyzed degradation of D- fructose.Carbohydr. Res.,5(3),266-273.

Singh,K.V;and Shukla,N.P.1984.Activity of Multiple Resistant Bacteria of Garlic (Allium sativum) Extract.Fitoterapia,LV(5),313-315.

Soffar,S.A.;and Mokhtar,G.M.1991.Evaluation of the Antiparasitic effect of aqueous garlic(Allium sativum) extract in Hymenolepiasis Nana and Giardiasis.J. Egypt. Soc. Parasitology,21(2),497-502.

Stoll,A. and Seebeck,E.1947.Alliin,the pure mother substances of garlic oil.Experentia,3,114-115.

Stoll,A.;and Seebeck,E.1951.Allium compound.V.The synthesis of natural alliin and its three optically active isomers.Helv. Chim. Acta,34,481-487.

Takazawa,H.;Tajima,F.;Miyashita,C.1982.An antifungal compound from “shutake”(Lentinus edodes).Yakug. Zas.,102,489-491.(Chem. Abstr. 1982,97,69141).

Talyzin,V.V.;Anisimov,V.Ya.;Yakovleva,O.I.;Golovnya,R.V.;Misharima,T.A.;Vitt,S. 1988.Identification of components of Allium cepa L. onion flavour.Zhurm. Anal. Khim.,43(5),911-916.

Thamburan,S.;Adams,J.F.;Klassen,J.;Johnson,Q.2005a.An assessment of Tulbaghia alliacea as a potential antimycobacterial agent.J. Ethnopharmacol.(submitted).

Thamburan,S.;Klassen,L.;Mabusela,W.T.;Cannon,J.F.;Johnson,Q.2005b.African garlic phytotherapy:a potential antifungal remedy.Phytotherapy Res.(in press).

Thomas,D.J.;Parkin,K.L.1994.Quantification of Alk(en)yl-L-cysteine sulfoxides and Related Amino acids in Alliums by High Performance Liquid Chromatography.J. Agric. Food Chem.,42,1632-1638.

Tsuno,S.1958.The nutritional value of Allium plants XVII.Formation of S-{2-[N-(2- methyl-4-amino-5-pyrimidyl methyl)formamidol]-5-hydroxy-2-pentene3-3yl}allyl disulfide with ipheion uniflorum.Bitamin,14,665-670.

Van Leuken,R.G.T.;Duchateau,A.L.L.;Kwakkenbos,G.T.C.1995.Thermospray liquid chromatography-mass spectrometry study of diastereomeric isoindole derivatives of amino acids and amino acid amides.J. Pharm. Biom.Anal.,13,1459-1464.

Venugopal,P.V.;and Venugopal,T.V.1995.Antidermatophytic Activity of garlic(Allium sativum) In vitroa.International J. Dermatology,34(4),278-279.

Vitanen,A.I.;and Spare,C.G.1962.Enzymic splitting of S-(1-propenyl) cysteine sulfoxide and the formation of the lacrymatory factor.Suom.Kemis. B,B35(No 2),,28- 29.

Vosa,C.G.1975.The cytotaxonomy of the genus Tulbaghia.Annali Bot.,34,47-121.

79 Watt,J.M and Breyer-Brandwijk,M.G.1932.The Medicinal and Poisonous plants of Southern Africa:being an account of their medicinal uses,chemical composition,pharmacological effects and toxicology in man and animal.Edinburgh,Livingstone.

Weber,N.D.;Andersen,D.O.;North,J.A.;Murray,B.K.;Lawson L.D.;and Hughes,B.G.1992.In vitro virucidal effects of Allium sativum (garlic) extract and compounds.Planta Med.,58,417-423.

Whitaker,J.R.1976.Development of flavor, odor and pungency in onion and garlic.Advan. Food Res.,22,73-133.

WHO (1996).Fact Sheet N 134, September 1996, Traditional Medicine.[Online]. Available http: //www.who.ch

Wu,C.C;Sheen,L.Y.;Chen,H.W.;Kuo,W.W.;Tsai,S.J.; Lii,C.K..2002. Differential Effects of Garlic Oil and Its Three Major Organosulfur Components on the Hepatic Detoxification System in Rats.J. Agric. Food Chem.,50,378-383.

Yoo,K.S.;Pike,L.M.1998.Determination of flavour precursor compound S-alk(en)yl- L-cysteine sulfoxide by an HPLC method and their distribution in Allium species.Scien. Hort. (Amterdam),75,1-10.

Ziegler,S.J.;Sticher,O.1989.HPLC of S-alk(en)ylcysteine derivatives in garlic including quantitative determination of (+)-S-ally-L-cysteine sulfoxide(alliin).Planta Med.,55,372-378.

80

81 APPENDIX A

ESI/LC-MS

Methanol extract, 10x dil in 50 % acetonitrile WM_UWC_050411_MeOH_Ex 124 (2.352) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (115:130 303.0507 100 1.63e4 319.0311 221.0418

% 301.0207 376.0116

397.9884 447.0608 489.0476 518.0253 560.0001 263.0733 0 m/z 200 250 300 350 400 450 500 550 600

Methanol extract, 10x dil in 50 % acetonitrile WM_UWC_050411_MeOH_Ex 137 (2.599) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (137:146 351.2150 100 3.19e4

369.9268

% 340.9717 392.9205 234.9253 316.9313 294.9423 431.0338 512.8907 578.9249 0 m/z 250 300 350 400 450 500 550

Methanol extract, 10x dil in 50 % acetonitrile WM_UWC_050411_MeOH_Ex 157 (2.978) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (157:159 476.1900 100 6.74e3

490.1918 % 382.1338 527.1747

317.1102 637.2584 818.3314 1160.4523 0 m/z 200 400 600 800 1000 1200 1400

Methanol extract, 10x dil in 50 % acetonitrile WM_UWC_050411_MeOH_Ex 169 (3.206) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (166:171 518.1920 100 6.61e3 365.0842

% 460.2195 519.1998 321.1317 860.3214 0 m/z 300 400 500 600 700 800 900 1000 1100

Methanol extract, 10x dil in 50 % acetonitrile WM_UWC_050411_MeOH_Ex 270 (5.119) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (270:274 405.0418 100 1.17e4

% 295.0546 427.0286 296.0618 510.1369 458.9683 0 m/z 250 300 350 400 450 500 550 600 650 700 750

Ethyl acetate extract, 10x dil in 50 % acetonitrile WM_UWC_050411_EtOAc_Ex 1007 (19.133) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (1007 396.4568 100 3.31e4

% 397.4745

398.4846 0 m/z 200 250 300 350 400 450 500 550 600 650 700 750

Ethyl acetate extract, 10x dil in 50 % acetonitrile WM_UWC_050411_EtOAc_Ex 971 (18.442) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (962:97 326.3839 100 2.37e4

368.4293

% 369.4435 513.3749 323.1656 529.3586 473.3874 595.3698 0 m/z 200 250 300 350 400 450 500 550 600 650 700

Chloroform extract, 10x dil in 50 % acetonitrile, top layer WM_UWC_050411_CHCl3_ExT 800 (15.183) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (792:8 270.9521 100 1.70e3

347.1885378.9240 346.9379 % 503.3106 295.1273 394.9041 330.9207 454.9163 233.9911 428.9538 0 m/z 200 225 250 275 300 325 350 375 400 425 450 475 500

Chloroform extract, 10x dil in 50 % acetonitrile, top layer WM_UWC_050411_CHCl3_ExT 1084 (20.626) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (107 410.4420 100 3.91e4 411.4735

408.9093 % 412.4929 343.3086 365.2920 315.1683 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420

Chloroform extract, 10x dil in 50 % acetonitrile, top layer WM_UWC_050411_CHCl3_ExT 602 (11.410) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (591:6 300.9399 100 9.20e4

224.9476 382.8898 % 226.9504 302.9476 360.9698 384.8882 426.9286 502.9191 255.9113 0 m/z 200 250 300 350 400 450 500 550

APPENDIX B

MS/MS

221 MSMS 26-Apr-2005 11:41:17 WM_UWC_050426_MeOHMSMS1 28 (2.311) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (28:29 137.0352 100 1.35e3

% 101.0079 138.0388 160.0542 0 m/z 100 120 140 160 180 200 220 240 260 280 300

303 MSMS 26-Apr-2005 11:41:17 WM_UWC_050426_MeOHMSMS1 29 (2.383) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (28:29 225.0235 100 41.3

181.0341

% 164.9953 226.0271

261.0409 0 m/z 100 150 200 250 300 350 400 450 500 550 600

319 MSMS 26-Apr-2005 11:41:17 WM_UWC_050426_MeOHMSMS1 31 (2.528) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (31-3 196.9266 100 22.2

% 214.9619 256.9922 100.0770 152.9238 196.8527 235.0784 0 m/z 100 120 140 160 180 200 220 240 260 280 300

393(104) MSMS 26-Apr-2005 11:41:17 WM_UWC_050426_MeOHMSMS1 30 (2.435) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (30:3 100 3.26

178.9665 264.0767 %

0 m/z 100 150 200 250 300 350 400 450 500 550 600

382 MSMS 26-Apr-2005 15:09:03 WM_UWC_050426_MeOHMSMS2 64 (2.963) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (62:66 114.1124 48.5 100 123.1012

% 112.0928 125.1215 141.1322 157.1081 169.1151 101.0812 130.1029 151.0990 0 m/z 100 110 120 130 140 150 160 170

476 MSMS 26-Apr-2005 15:09:03 WM_UWC_050426_MeOHMSMS2 65 (3.014) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (64:67 134.0898 100 183

% 116.0410 135.0965 0 m/z 100 120 140 160 180 200 220 240 260 280 300 320 340

460 MSMS 26-Apr-2005 15:09:03 WM_UWC_050426_MeOHMSMS2 20 (3.221) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (19:22 118.0955 100 118

%

119.0950 127.0479 0 m/z 100 110 120 130 140 150 160 170 295 MSMS 26-Apr-2005 11:41:17 WM_UWC_050426_MeOHMSMS1 31 (5.048) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (30:32 114.9518 100 51.8

132.9668 % 150.9801 116.9252 168.9908

0 m/z 100 120 140 160 180 200 220 240

397 MSMS 26-Apr-2005 13:38:06 WM_UWC_050426_EtOAc 38 (19.193) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (37:39-24:27 396.5020 100 668

284.3665 % 397.5065 156.1915 256.3316 285.3690 398.5100 184.2312 254.3125 0 m/z 150 175 200 225 250 275 300 325 350 375 400 425 450 475

368 MSMS 26-Apr-2005 13:38:06 WM_UWC_050426_EtOAc 27 (18.430) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (26:29-18:20 256.3300 100 182 368.4737

156.1903 % 369.4725 254.3134 257.3329 157.1910 370.4890 0 m/z 150 175 200 225 250 275 300 325 350 375 400 425 450 475

379 MSMS 26-Apr-2005 12:23:06 WM_UWC_050426_CHCl3 9 (15.005) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (9-11) 254.9388 100 5.11

% 216.9823 297.9631

0 m/z 100 150 200 250 300 350 400 450 500 550 600

412 MSMS 26-Apr-2005 12:23:06 WM_UWC_050426_CHCl3 25 (20.541) Cm (25:26-19:21) 31: TOF MSMS 410.40ES+ 410.4988 100 1.20e3

% 411.5159 242.3068 214.2722 243.3110 412.5342 0 m/z 100 150 200 250 300 350 400 450 500 550 600

225 MSMS 26-Apr-2005 12:23:06 WM_UWC_050426_CHCl3 54 (10.402) Cn (Cen,4, 80.00, Ar); Sm (SG, 2x3.00); Cm (52:55-58:59 131.9759 100 114

%

133.9749 0 m/z 100 105 110 115 120 125 130 135 140

APPENDIX C

GC-MS of ethyl acetate

Retention time 3.25

Retention time 6.16

EI/MS of aqueous extract