Bioactive S-Alk(En)Yl Cysteine Sulfoxide Metabolites in the Genus Allium: the Chemistry of Potential Therapeutic Agents

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Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus www.rsc.org/npr Allium: the chemistry of potential therapeutic agents NPR

Peter Rose,*a Matt Whiteman,a Philip K. Mooreb and Yi Zhun Zhu*b a Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore, 117597. E-mail: [email protected]; Fax: (65)-6779-1453; Tel: (65)-6874-4996 b Department of Pharmacology, National University of Singapore, 18 Medical Drive, Singapore, 117597. E-mail: [email protected]; Fax: (65)-6773-7690; Tel: (65)-6874-3676

Received (in Cambridge, UK) 30th March 2005 First published as an Advance Article on the web 10th May 2005

Covering: 1892 to 2004

S-Alk(en)yl cysteine sulfoxides are odourless, non-protein sulfur amino acids typically found in members of the family Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomically important genus Allium. Traditionally, Allium species, particularly the onion (Allium cepa) and garlic (A. sativum), have been used for centuries in European, Asian and American folk medicines for the treatment of numerous human pathologies, however it is only recently that any significant progress has been made in determining their mechanisms of action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from the combination of several academic disciplines including botany, biochemistry and nutrition. During tissue damage, S-alk(en)yl cysteine sulfoxides are converted to their respective thiosulfinates or propanethial-S-oxide by the action of the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinates can decompose to form additional sulfur constituents including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, the vinyldithiins and (E)- and (Z)-ajoene. Recent reports have shown onion and garlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes like glutathione-S-transferases (EC 2.5.1.18) and quinone reductase (QR) NAD(P)H: (quinine acceptor) oxidoreductase (EC 1.6.99.2) in mammalian tissues, as well as also influencing cell cycle arrest and apoptosis in numerous in vitro cancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds in cardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxide metabolites in the context of their biochemical and pharmacological mechanisms.

Peter Rose is a research fellow in the Department of Biochemistry, National University of Singapore. He obtained his BSc in Botany at the University of Nottingham prior to obtaining his PhD at the Institute of Food Research and the John Innes Centre, UK. His research focuses on signaling cascades mediated by natural products. Matt Whiteman is an Assistant Professor in the Department of Biochemistry, National University of Singapore. He obtained his PhD in Medical Biochemistry in 1997 in the Department of Pharmacology, King’s College, University of London. He moved to Singapore in 2000 after completing 3 years’ post-doctoral work at the Centre for Age-Related Diseases, King’s College. His research interests focus on reactive nitrogen and chlorine species in cellular behaviour. Philip Moore is a Professor and Head of the Department of Pharmacology at National University of Singapore. He obtained his PhD from King’s College in London, and has been actively involved in research into the mechanisms underlying inflammatory and cardiovascular disease for over 25 years. Yi Zhun Zhu is a senior research fellow at the Department of Pharmacology, National University of Singapore and Professor of Pharmacology at Fudan University, Shanghai. He obtained his undergraduate medical training in Shanghai and did doctorate and post-doctoral training in Heidelberg and Kiel. His research is focusing on cardioprotective effects of ischemic heart disease using Western drugs and/or extracts from natural products.

Peter Rose Matt Whiteman Philip Moore Yi Zhun Zhu DOI :10.1039/b417639c

This journal is © The Royal Society of Chemistry 2005 Nat. Prod. Rep., 2005, 22, 351–368 351

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1 Introduction Allium species arose from non-volatile precursor substances. It 2 Sulfur storage compounds was in the laboratory of Stroll and Seebrook in 1948 that the 2.1 S-Alk(en)yl cysteine sulfoxides in Allium species first stable precursor compound, (+)-S-allyl-L-cysteine sulfoxide 2.1.1 Alliinase (ACSO), commonly known as alliin, was identified.3 Alliin is the 2.2 Organosulfur compounds in Allium species parental sulfur compound that is responsible for the majority of 2.2.1 Thiosulfinates the odorous volatiles produced from crushed or cut garlic. Three 2.2.2 Decomposition products additional sulfoxides present in the tissues of onions were later 2.2.3 Novel thiosulfinate-derived sulfur compounds identified in the laboratory of Virtanen and Matikkala, these 3 Allium vegetables and human health being (+)-S-methyl-L-cysteine sulfoxide (methiin; MCSO), (+)- 3.1 Anticarcinogenic properties S-propyl-L-cysteine sulfoxide (propiin; PCSO) and (+)-S-trans- 3.1.1 Enzymatic inhibition 1-propenyl-L-cysteine sulfoxide or isoalliin (TPCSO). Isoalliin 3.1.2 Enzymatic induction is the major sulfoxide present within intact onion tissues and is 3.1.3 Apoptosis the source of the A. cepa lachrymatory factor.4,5 With regards 3.2 Anti-inflammatory properties to chemical distribution, (+)-S-methyl-L-cysteine sulfoxide is 3.3 Antioxidant properties by far the most ubiquitous, being found in varying amounts 3.4 Antimicrobial properties in the intact tissues of A. sativum, A. cepa, A. porrum,andA. 3.5 Antifungal and antiparasitic properties ursinum L.(Table 1). 3.6 Cardiovascular disease To date, only the L-(+)-isomers have been described in nature.6 3.7 Metabolism of Allium sulfur compounds A variety of methods have now been established to allow 4 Summary the direct analysis of CSs in plant tissues.7,8 These techniques 5 Abbreviations include HPLC or gas chromatographic (GC) methodologies, 6 References and rely either on the direct measurement of the CS following derivatisation or measurement of their respective degradation 1 Introduction products. One popular method that circumvents the problem of low sensitivity experienced with HPLC techniques is the use of Although formally classified in the family Liliaceae, represented GC. In the studies of Kubec et al.9,10 GC methodologies were by 280 separate genera and 4000 species, recent taxonomic developed to determine the distribution of CSs within the genus revisions have seen members of the genus Allium placed in Allium. Initially, the CSs are derivatised due to their apparent the family Alliaceae. Distributed throughout most regions of thermal instability, using ethyl chloroformate prior to GC the temperate world including Europe, Asia, North America analysis (Fig. 1). Data from these studies provided a quantitative and Africa, Allium species have a long history in common method to measure the nonvolatile CSs in 15 separate Allium folklore and as sources of therapeutic principles. In addition species (Table 2). Moreover, using this technique Kubec and to preventing the nocturnal bloodthirsty pursuits of vampires, colleagues identified S-ethylcysteine sulfoxide (ethiin; ECSO), one of the most famous members of the Alliaceae, garlic Allium not previously reported to occur in Allium species, as a minor sativum L. has been used throughout the centuries to treat animal component of most extracts.11 Similarly, S-n-butylcysteine sul- bites, leprosy, the plague, heart disease and cancer. Indeed, the foxide (BCSO) was identified and isolated from the tissues of A. realisation in 1858 by Louis Pasteur that garlic had potent siculum using this same method, and confirmed the earlier report antibacterial properties later led to its use in the First and by Horhammer¨ et al. on the occurrence of BCSO in garlic (A. Second World Wars to prevent gangrene. Of approximately 700 sativum).12,13 To date, four major and two minor CS have been species, it is the edible members including onion (A. cepa L.), identified and isolated from Allium vegetables. garlic (A. sativum L.), chives (A. schoenoprasum L.), leek (A. After the discovery of stable CSs, attention was next directed porrum L.) and Welsh onion (A. fistulosum L.)thatarehighly towards the elucidation of the biosynthetic pathway. Much of 1 prized. Ordinarily, the vegetative parts are odour-free, and it our knowledge of this area comes from radiolabel feeding studies is only during tissue damage that volatile flavour principles are and direct chemical analysis. What is apparent from these studies generated. Interestingly, these volatile chemicals are produced is that CSs have a common origin in plant sulfur metabolism. through enzymatic hydrolysis of non-volatile sulfur storage 2− In plants, sulfate (SO4 ) is used as the primary source of compounds, termed S-alk(en)yl-L-cysteine sulfoxides (CSs). To sulfur for the biosynthesis of the amino acid cysteine and date, four major and two minor CSs are have been identified 14 2− also the antioxidant glutathione. Initially, SO4 is transported in the genus Allium, and it is from these that approximately 50 across the root plasma membrane, whereupon it accumulates additional sulfur compounds can be generated. It is therefore 2− within plant cells. In order for SO4 to be utilised for cysteine not surprising that the composition and quantity of each CS biosynthesis it must first be converted to the intermediate determines the odour, flavour variation and biological activities compound 5-adenylylsulfate (APS). This reaction is catalysed observed for Allium vegetables. Given such chemical diversity, by the enzyme ATP sulfurylase in the plastids. APS can then be members of the genus Allium have received considerable atten- used by the enzyme APS reductase to form sulfite, prior to its tion from both chemists and biologists alike as new sources of conversion to sulfide by the enzyme sulfide reductase. Following bioactive compounds. Therefore, in the current review we will this, cysteine is formed from the reaction of sulfide with O- describe the biosynthesis, generation and bioactive properties of acetylserine, a process catalysed by the enzyme OAS thiol-lyase. sulfur compounds derived from the genus Allium. O-acetylserine is derived from the acetylation of serine by the action of the enzyme serine acetyltransferase.15 Once formed, 2 Sulfur storage compounds cysteine is rapidly channeled into several metabolic pathways involved in protein synthesis as well as in numerous plant 2.1 S-Alk(en)yl cysteine sulfoxides in Allium species secondary metabolic pathways. The medicinal properties associated with members of Allium With regards to CSs, early investigations using radiolabelled has long been recognised and has provided the impetus for carbon and sulfur sources showed that each were rapidly chemists to determine the active chemical substances.2 Early incorporated into the glutathione cycle and also in the formation investigators identified volatile odour principles in garlic oils, of low molecular weight glutamyl peptides (GPs).16 To date, however, these compounds were only generated during tissue approximately 24 sulfur-containing GPs have been identified damage and preparation. Indeed, the vegetative tissues of Allium in Allium species, where they are considered to function as species are usually odour-free, and it is this observation that led sulfur and nitrogen stores as well as intermediates in CS to the hypothesis that the generation of volatile compounds from biosynthesis. When garlic bulbs were injected with 14C-valine

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Table 1 S-Alk(en)yl cysteine sulfoxides in Allium spp.

Common name Chemical name Chemical structure Representative species

Methiin S-Methyl-L-cysteine sulfoxide A. cepa L. A. sativum L. A. chinense G. Don A. longicuspis Rgl.

Alliin S-Allyl-L-cysteine sulfoxide A. sativum L. A. ursinum L. A. ampeloprasum L. A. longicuspis Rgl.

Propiin S-Propyl-L-cysteine sulfoxide A. cepa L. A. porrum L. A. altaicum Pall. A. fistulosum L.

Isoalliin S-Propenyl-L-cysteine sulfoxide A. cepa L. A. nutans L. A. ascalonicum auct. A. schoenoprasum L.

Ethiin S-Ethyl-L-cysteine sulfoxide A. aflatunense B. Fedt. A. ampeloprasum L. A. ochotense Prokh. A. victorialis L.

Butiin S-n-Butyl-L-cysteine sulfoxide A. siculum

Table 2 S-Alk(en)yl cysteine sulfoxide content in selected Allium spp. (Adapted from Kubec et al.).11

Content of S-alk(en)ylcysteine sulfoxides/mg per 100 g fresh weight Representative species ACSO MCSO PCSO PeCSO Total

Shallot, A. ascalonicum auct. 1.141.117.792.7 155.8 Scallion, A. fistulosum L. trace 5.61.813.1 21.2 Leek, A. porrum L. trace 4.0trace17.6 21.6 Garlic, A. sativum L. 1077 122 trace trace 1199 Chive, A. schoenoprasum L. 21.132.2 606 21.0 72.4 Wild garlic, A. ursinum L. 40.360.01.2 trace 101.9

the radiolabel 14C was found to be incorporated into S- the cysteine residue of glutathione, followed by the removal of 2-carboxypropylcysteine, S-2-carboxypropyl glutathione and the glycyl residue by transpeptidation. Subsequently, the CS methacrylic acid.17 Granroth et al.18 later showed that S- proceeds undergoes oxidation and loss of the glutamyl group, 2-carboxypropyl glutathione could be hydrolysed by onions, leading to the parental CS (Fig. 2). Alternatively, the direct and the S-2-carboxypropylcysteine formed could be utilised S-alk(en)ylation of cysteine or thioalk(en)ylation of O-acetyl in the biosynthesis of TPCSO, suggesting a putative link serine followed by oxidation to the respective CS can occur between GP and CS synthesis. Unequivocal evidence that S- (Fig. 3). The second pathway has been suggested due to the 2-carboxypropylcysteine was indeed the precursor compound observation that cysteine will readily react with methacrylic acid. for TPCSO synthesis was later confirmed by Parry et al.19,20 Granroth et al.18 previously identified methacrylic acid in Allium Further confirmation of the role of GPs in CS biosynthesis and a tissues and showed it to react with thiol compounds. Indeed, S-2- suggested biosynthetic model was later highlighted by Lancaster carboxypropyl cysteine and S-2-carboxypropyl glutathione can et al.21 in model studies using A. cepa, A. sativum and A. siculum. be formed through the direct reaction of methacrylic acid with In onion seedlings a 10 minute pulse of radiolabelled sulfate cysteine or glutathione, providing a route to the formation of 35 2− ( SO4 ) leads to its rapid assimilation into GPs – after 1 hour, ACSO, TPCSO, and PCSO. The relative contribution of both 35% of the administered label was found in the GPs. Using thin pathways in CS biosynthesis has yet to be determined, moreover, layer chromatography the following radiolabelled compounds little is known about the origin of the S-alk(en)yl donor groups were identified: methyl glutathione, c-glutamyl methyl cysteine, or the genes, enzymes and regulatory mechanisms involved in the S-2-carboxypropyl glutathione and c-glutamylpropenyl cysteine biosynthetic pathway. Hopefully, future work will address these sulfoxide. Over a period of several days labelled sulfur appeared areas. For further information with regards to CS biosynthesis in PCSO (5-fold increase), TPCSO (8-fold increase) and MCSO we refer the reader to the excellent and comprehensive works of (1.2-fold increase). A similar pattern of labelling was also Whitaker,16 Block et al.22 and Jones et al.23 confirmed for A. sativum. In additional studies using A. siculum, labelled sulfur was rapidly assimilated into glutathione, c- 2.1.1 Alliinase. Common to all Allium speciesistheen- glutamyl cysteine, methyl glutathione and c-glutamyl methyl zyme alliinase [EC 4.4.1.4], a 50 kDa glycoprotein that catalyses cysteine prior to incorporation into MCSO (12-fold increase). the hydrolysis of CS in the presence of the cofactor pyridoxal 5- These findings led the authors to propose the following: 1) c- phosphate to produce pyruvate, ammonia and sulfenic acids. Glutamyl cysteine and glutathione are the starting compounds In intact tissues alliinase is compartmentalised within plant and, 2) CS biosynthesis can proceed by S-alk(en)ylation of vacuoles and the representative CS located in the cytoplasm.24

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sphaerocephalon L., A. victorialis L., A. hymenorrhizum Ledeb, A. saxatile M. Bieb, A. obliquum L., A. subhirsutum L., A. jesdianum and A. stipitatum Reg) showed that the pH and temperature optima for each species were similar. Moreover, alliinase activities from each species showed the greatest activity towards PCSO. Genes encoding alliinase have been isolated from A. cepa, A. tuberosum (Chinese chives) and Allium ascalonicum auct. (shallot).27,37–40 Analysis of cDNA libraries constructed for A.sativum, A. cepa and A. ascalonicum reveal a high degree of sequence similarity both at the nucleotide and at the amino acid level in the alliinase cDNA for each species.37 In A. tuberosum, functional recombination of the cDNA encoding alliinase in Eschericha coli and Saccharomyces cerevisiae afforded a catalytically active enzyme. Moreover, site-directed mutagenesis revealed that lysine-280 was essential for catalytic activity, and that it was the binding site for the cofactor pyridoxal 5- phosphate. This data is further supported by the presence of the same lysine residue in the pyridoxal 5-phosphate binding site of A. cepa alliinase.41

2.2 Organosulfur compounds in Allium species Upon tissue damage the first chemical compounds to be formed are the sulfenic acids and thiosulfinates. These progenitor compounds are intermediates in the formation of the majority of sulfur volatiles. With regards to the types of chemical components formed in Allium species, Block et al.22 proposed a scheme that provides a means to characterise the origins of many of these compounds. These categories are: 1) head space volatiles; chemicals generated at room temperature following cutting or homogenisation of Allium tissues, 2) decomposition products formed from thiosulfinates at room temperature, and 3) oil components – compounds generated by vigorous preparation such as steam distillation (Fig. 5). For the purpose of the current review we will introduce the thiosulfinates first prior to addressing compounds identified in each of the above three categories.

Fig. 1 (A) 1, Commercially available Allium species consumed in South 2.2.1 Thiosulfinates. In freshly macerated Allium tissues East Asia; 2, Section through the stem tissue of leek (A. porrum); 3, the initial chemical compounds formed are the thiosulfinates, Red onion A. cepa; 4; Garlic A. sativum; 5, Made to make your eyes derived from the condensation of sulfenic acid principles water, A. cepa; 6, Chinese chives A. tuberosum L. (Photos by P.R.) (Fig. 6). All Allium-derived thiosulfinates can be represented (B) Derivatisation and reduction of S-alk(en)ylcysteine sulfoxides with = ethyl chloroformate (adapted from Kubec et al.9). by four types: 1) fully saturated (RS(O)SR (R,R Me or Pr); 2) mono- or di-S-b,c-unsaturated thiosulfinates, AllS(O)SMe, AllSS(O)Me, AllS(O)SAll; 3) mono-a,b-unsaturated thiosulfi- Upon tissue disruption the vacuole and cytoplasmic contents nates; and 4) mixed a, b-andc-unsaturated thiosulfinates.22 mix, promoting the enzymatic hydrolysis of the respective Because of the inherent thermal instability of the thiosulfinates, CS. This catalytic reaction leads to the generation of sulfenic only a few methods for their extraction and detection have acids that condense to form thiosulfinates. Indeed, we are aware been developed.42 The most suitable of these appears to be of the presence of alliinase, as it is this enzyme that catalyses the vacuum distillation of tissue homogenates coupled with HPLC reaction leading to the generation of the lachrymatory principle detection. Using this method the primary compounds formed found in onions and leeks, thiopropanal S-oxide25 (Fig. 4). in Allium homogenates are the thiosulfinates, rather than their Alliinase protein has been isolated and characterised from thermal degradation products such as polysulfides. In a recent several Allium species including A. cepa, A. sativum, A. tubero- study by Block et al.,43 HPLC detection identified 8 individual sum L., A. ursinum,andA. porrum (leek).26–31 Moreover, alliinase thiosulfinates in the tissues of onion (A. cepa), garlic (A. derived from A. sativum has been crystallised and its three- sativum), wild garlic (A. ursinum), leek (A. porrum), scallion (A. dimensional structure has been reported.32,33 Characterisation fistulosum), shallot (A. ascalonicum), elephant (or great-headed) of the catalytic activity of alliinase shows that it is sensitive garlic (A. ampeloprasum L. var. ampeloprasum auct.), chive (A. to pH, pyridoxal-5-phosphate availability and temperature. schoenoprasum), and Chinese chive (A. tuberosum)(Table3). Purified onion alliinase shows optimum activity at pH 7.4 Thiosulfinates containing the 1-propenyl group were the most and preferentially hydrolyses to TPCSO rather than MCSO or abundant in A. cepa, A. fistulosum, A. porrum, A. schoenoprasum, PCSO.26,34 In the case of garlic the situation is more complicated and A. ascalonicum. 72% of the total thiosulfinate content in as it has two distinct alliinase activities. For the hydrolysis A. tuberosum was MeS(O)SMe, while in A. schoenoprasum, of PCSO and TPCSO the pH optimum is 4.5, whereas the 58% of total thiosulfinate content was n-PrS(O)S(n-Pr). In A. second activity is specific to MCSO, with a pH optimum of sativum and A. ursinum the major thiosulfinates present were 6.5. An interesting study by Lancaster et al.35 demonstrated AllS(O)SAll. It is apparent from this study that thiosulfinates that hydrolysis of PCSO and MCSO in onion macerates can be might serve as useful chemo-taxonomic markers, as suggested enhanced by pyridoxal-5-phosphate addition. In a recent study by the authors. Indeed, the types of thiosulfinate compounds by Krest et al.,36 analysis of alliinase activities derived from identified in Allium tissues directly correlate with the composi- crude protein extracts of nine allium species (A. sativum, A. tion of the parental cysteine sulfoxides.

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Nat. Prod. Rep., 2005, 22, 351–368 355 中国科技论文在线 http://www.paper.edu.cn

Fig. 5 Chemical constituents generated in Allium spp. are dependent on the processing methods used.

Fig. 3 Alternative route to S-alk(en)yl cysteine sulfoxides by thioalk(en)ylation of O-acetylserine or direct alk(en)ylation of cysteine (adapted from Block22 and Jones et al.23).

To explain the reported composition of the thiosulfinate in Allium tissues we must highlight the data obtained from model reaction systems in which synthetic CS, alliinase and thiosulfinates are examined. It is apparent that the kinetics of CS hydrolysis and the reactivity of the initial sulfenic acids influence the types of thiosulfinates generated. Thiosulfinates are represented by the following groups: 1) symmetrical thiosulfinates that arise from the condensation of two molecules of sulfenic acid sharing the same alk(en)yl group, or 2) asymmetrical thiosulfinates that are generated by the condensation of two different molecules of sulfenic acid. To further explain these chemical reactions, Shen and Parkin developed novel in vitro models to study the pathway of thiosulfinate production.44,45 The authors found that the rate of hydrolysis of parental CS followed the order TPCSO > ACSO > PCSO > ECSO > MCSO. In additional studies Fig. 6 Structures of common Allium thiosulfinates (adapted from Shen the authors also showed that thiosulfinates can readily react et al.42). with sulfenic acids, leading to the biogeneration of additional thiosulfinate species (Fig. 7). These findings are explained by accumulation of “slow” sulfenic acids allows them to react that fact that individual CSs can be hydrolysed at different rates, with the pre-formed thiosulfinates derived from the “fast” and thus the rapidity by which the sulfenic acid intermediates reaction. These reactions promote the generation of additional are produced differs. Indeed, the “faster” the CS reacts, thiosulfinates. For example, in Allium species two isomeric the quicker is the formation of thiosulfinates. In contrast, thiosulfinates are found: allyl methanethiosulfinate and methyl “slow”-reacting CSs generated sulfenic acids much slower. The 2-propenethiosulfinate. In garlic homogenates propenyl groups

Fig. 4 Alliinase-mediated hydrolysis of S-alk(en)yl cysteine sulfoxides leads to the formation of sulfenic acids that can self-condense to form thiosulfinates, or in the case of S-trans-1-propenylcysteine sulfoxide the lachrymatory compound 1-propanethial-S-oxide.

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Table 3 Thiosulfinate content of selected Allium spp. (adapted from Block et al.43). Quantification was determined by GC-MS analysis (total thiosulfinate content is expressed as lmol g−1 fresh weight)

A. ascalonicum A. schoeno- Thiosulfinate A. cepa L. auct. A. fistulosum L. A. porrum L. A. sativum L. A. tuberosum L. prasum L.

AllSS(O)-(E)-propenyl — — — — 1.6— — AllSS(O)-(Z,E)-propenyl — — — — 5.30.9— AllS(O)SAll — — — — 89 28 — PrSS(O)-(E)-propenyl 9 14 2 8 — — 2.5 PrS(O)S-(Z,E)-propenyl 16 22 17 15 — — 16 PrS(O)SPr 13 26 24 24 — — 58 MeSS(O)-(E)-propenyl 22 9 12 12 1.4— — AllS(O)SMe — — — — 1.4— — MeS(O)S-(Z,E)-propenyl 31 16 22 31 — 0.71 MeSS(O)Pr 1 2.8115—— 5.9 MeS(O)Pr 1 1.28 5——15 AllSS(O)Me — — — — 2.934— MeS(O)SMe — 9 1 3 — 0 1.8

Total MeS (%) 28 23 25 27 2 49 13 Total AllS (%) — — — — 94 50 — Total 1-propenyl (%) 45 32 24 33 3 1 10 Total PrS (%) 27 46 51 40 — — 77 Total thiosulfinate/lmol g−1 0.14 0.25 0.08 0.15 14.3 21 0.19 fresh weight

condense to re-form one molecule of allicin or alternatively, two molecules of thioacrolein. Thioacrolein is highly reactive and can undergo self-condensation by a Diels–Alder reaction to generate the cyclics 2-vinyl-[4H]-1,3-dithin (2VDN) and 3- vinyl-[4H]-1,2-dithin (3VDN) (Fig. 8).2 Both biochemical and physiological parameters can promote thiosulfinate reactivity and decomposition – these include pH, temperature, sample preparation and storage time. The substances generated during the decomposition of storage compounds will be described using the categories highlighted by Block.21

2.2.2 Decomposition products. To date, there have been two main approaches to studying Allium aroma compounds. The first involves analysing volatile constituents generated upon tissue damage in the headspace or alternatively preparing water macerates and collecting the compounds present using solvent extraction. With regards to headspace volatiles, Allium tissues are ho- mogenised prior to GC or GC-MS analysis. Early studies reported the occurrence of disulfides from chopped onion, with dipropyl disulfide (DPDS) accounting for the largest amount.46,47 Similarly, in chopped garlic the major headspace volatile components are diallyl disulfide (DADS) and allyl methyl disulfide (AMDS).25,48 Recent analysis of onion volatiles Fig. 7 Reaction of 2-propenyl thiosulfinate with methylsulfenic acid by Kallio and Salorinne identified 27 separate aroma com- leads to the generation of allyl methanethiosulfinate. pounds, the most prominent of which were DPDS, methyl propyl disulfide (MPDS), 1-propenyl propyl disulfides ((E)- and (Z)- are formed 10 times faster than their methyl counterparts, and PPDS), methyl 1-propenyl disulfides ((E)- and (Z)-MPeDS), 1- thus 2-propenethiosulfinate is formed rapidly. The preformed propanethiol (PT), dipropyl trisulfide (DPTS), methyl propyl 2-propenethiosulfinate is therefore free to react with the slowly trisulfide (MPTS) (Fig. 9A).49 Later, Jarvenp¨ a¨a¨ et al.,usingthe forming methanesulfenic acid, leading to the formation of allyl novel Solid Phase Microextraction (SPME) technique coupled methanethiosulfinate.21 to GC-MS, determined the changes in volatile compound In fresh Allium homogenates thiosulfinates are reported to be composition in chopped onion.50 Initially, thiopropanal S-oxide relatively stable when left at room temperature for 26 hours. was the major sulfur compound identified in the head space Indeed, Block et al.43 reported that the total thiosulfinate with minor levels of DPDS and PPDS. Within 30 minutes concentration in garlic homogenates remained roughly constant thiopropanal S-oxide had disappeared and was replaced by and that the only thiosulfinates that showed any apprecia- diprop(en)yl disulfides. These data indicate that the chemical ble loss were those represented by MeCH=CHS(O)SR and profiles of Allium volatiles change dramatically with time. MeS(O)SMe. Most flavour compounds are derived from the With regards to the chemical components identified in decomposition of thiosulfinates. For example, allicin can react to solvent extracts, GC-MS analysis of methanolic preparations form ajoene, for which the trans and cis isomers are recognised. of chopped garlic identified numerous mixed polysulifdes, In a separate pathway, allicin can react with thiol substrates, including the parental thiosulfinate allicin, diallyl, di-, and including cysteine, to form S-allylmercapto-L-cysteine. Alter- tetrasulfides, and allylmethyl trisulfides. Many of these novel natively, allicin can further decompose to form allyl sulfenic sulfides are generated through the thermal decomposition acid and thioacrolein. Two molecules of allyl sulfenic acid can of thiosulfinate intermediates (Fig. 9B). Indeed, when the

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Fig. 8 Alliinase-mediated hydrolysis of S-2-propenyl cysteine sulfoxide: generation of garlic flavour components.

thiosulfinate allicin is heated, as is likely during food preparation, sulfur dioxide, dimethyl trisulfide (DMTS), diallyl mono-, di-, and trisulfides, and allylmethyl di- and trisulfides are generated. Similar groups of mixed polysulfides have also been identified in dichloromethane extracts of A. fistulosum var. maichuon and A. fistulosum var. caespitosum.51 Thermal decomposition reactions and products are also observed as major components of Allium oils, generated during the steam distillation process. During steam distillation, whereupon the tissues of the selected Allium species are heated to 100 ◦C, oils are gen- erated. Because thiosulfinates and disulfides formed during chopping are thermally unstable, they decompose to form mixed polysulfides. To date, the distilled oils of onion and garlic are highly prized as food and health products. Semmler (1892) was the first to identify DADS and diallyl trisulfide (DATS) as the major flavour compounds of onion oil.52 GC-MS chemical analysis has also revealed the presence of monosulfides, disulfides, and trisulfides in garlic essential oils. Indeed, 28 components were reported in the study of Yu et al.53 The essential oils obtained by steam distillation contained PT, 1,2-epithiopropane (EP), methyl allyl sulfide (MAS), diallyl sulfide (DAS), tetrahydro-2,5-dimethylthiophene (THDMP), MPDS, DADS, 1,2-dimercaptocyclopentane (DMCP), 3VDN, and 2VDN. Analysis of the steam distillates of Chinese chive (A. tubero- sum) and rakkyo (A. chinense G. Don) found that sulfur- containing compounds account for 88 and 94% of the total volatiles. The relative abundance of these volatiles in Chinese chive extracts were AMDS (39.3%), followed by DMDS (15%) and DMTS (12.6%). The most abundant volatiles in the rakkyo extracts were MPTS (9.9%), DMDS (7.3%), DMTS (6%), and MPDS (5.5%). Among the 21 and 34 sulfur compounds identified in Chinese chive and rakkyo respectively, many have not been previously been reported in these plants.54 These included some novel sulfide and polysulfides with ethyl, butyl, and pentyl groups. All of the identified chemical constituents in oils are initially derived from the thermal decomposition of thiosulfinates. The origins of these sulfur components are best highlighted using allicin derived from garlic as an example (Fig. 10A). Previous studies have also shown that at room temperature allicin can rearrange to afford sulfur dioxide, and diallyl mono-, di and trisulfides (Fig. 10B).25 Furthermore, the DADS formed can also thermally decompose to produce at least 25 additional 17 Fig. 9 Sulfides identified in the headspace volatiles of crushed Allium reaction products. It was in the study of Block et al. that the tissues: (A) onion volatiles, and (B) garlic volatiles. mechanism and identities of many of these mixed polysulfides

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Fig. 10 (A) Formation of diallyl disulfide from 2-propenyl thiosulfinate, (B) major thermal decomposition products of diallyl disulfide, and (C) minor decomposition products of diallyl disulfide.

were determined.55 Using GC-MS, Block and colleagues con- methyl 1-(methylsulfinyl)propyl disulfide was isolated from ducted additional experimentation to identify the unknown onion extracts.56 Furthermore, Wagner also reported the components. Heating pure diallyl disulfide at 150 ◦Cfor10 presence of the cepaenes 1-((E)-1-propenylsulfinyl)propyl minutes afforded DAS, DATS, the cyclics 2VDN and 3VDN, propyl disulfide and 1-((E)-1-propenylsulfinyl)propyl (E,Z)- and diallyl tetrasulfide (DATeS) as would be expected, in 1-propenyl disulfide in onions.57 The cepaenes have been addition to 29 other sulfur components (Fig. 10C). shown to have potent anti-inflammatory properties.58,59 Ad- ditional studies have now shown the presence of six new 2.2.3 Novel thiosulfinate-derived sulfur compounds. In ad- cepaenes in the chloroform extracts of onion.60 Furthermore, dition to the presence of thiosulfinates in fresh Allium ex- Calvey and colleagues identified methyl 1-(methylsulfinyl)propyl tracts, additional novel sulfur compounds have also been disulfide, 2-propenyl 1-(2-propenylsulfinyl) propyl disul- identified. In the studies of Kawakishi and Morimitsu, fide, methyl (E)-1-(1-propenylsulfinyl)propyl disulfide, and

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1-(1-methylsulfinyl)propyl (E,Z)-1-propenyldisulfide in super- dietary components that have antimicrobial or antioxidant-like critical fluid extracts of A. tricoccum.61 The cepaenes are formed properties may also play a significant role in the prevention from thiosulfinate intermediates through a mechanism proposed of human pathologies. Not surprisingly, studies conducted by Block.22 This proceeds through a reaction mediated by throughout the 1970s to the present have determined that many carbophilic addition involves thiols and sulfenic acid deriva- dietary botanicals and their phytochemical constituents can tives. In addition, deoxycepaenes have also been identified modulate several of these protective pathways. Therefore, in in the oils of shallot (e.g. ethyl-1-(methylthio)propyl disulfide order to determine the potential beneficial effects of Allium and methyl-1-(methylthio)propyl disulfide) and scallion (e.g. 1- vegetables on human health it will be necessary to collate the propenyl-1-(propylthio)propyl disulfide).51 However, it is not available data from an array of both in vivo and in vitro studies. It known whether these compounds arise from the deoxygenation is with such knowledge that we can then appreciate the potential of cepaenes or are formed during preparative or analytical role of Allium species towards human health. procedures.22 A second group of very unusual sulfur compounds that 3.1 Anticarcinogenic properties are likely to be derived from bis-a-b-unsaturated thiosulfinates The diet plays a fundamental role in the etiology of human has also recently been identified in Allium extracts and shown disease. Lifestyle choices including alcohol consumption, high to be biologically active. Two isomers of 2,3-dimethyl-5,6- intake of dietary fats and smoking can increase the risk of cancer dithiabicyclo[2.1.1]hexane 5-oxide were identified in onions, in humans. Examination of the available epidemiological evi- shallot, scallion and chive extracts.62–64 These compounds are dence by Block et al. indicated that high dietary intake of fruits trivially named cis-andtrans-zwiebelanes (Fig. 11A). Recently, and vegetables was correlated to a reduction in the prevalence research has focused on determining the mechanisms of for- of several forms of human cancers.67 Statistically significant mation of these novel compounds, the chemistry of which is protective effects were associated with fruit and vegetable highly complex.63–66 Hopefully, in the future more information consumption in 128 of 156 dietary studies examined. In human pertaining to these chemical agents will be available. Moreover, studies in which Allium vegetable consumption (particularly a structurally similar (Z,Z)-(±)-2,3-dimethylbutanedithial S,S- garlic and onions) were evaluated, a protective effect towards dioxide was been identified in onion extracts using NMR cancers was observed. In the review of the epidemiological methodologies and subsequently extracted (Fig. 11B). (Z,Z)- literature by Fleischauer and Arab, a high dietary intake of raw (±)-2,3-dimethylbutanedithial S,S-dioxide is the first example or cooked garlic was inversely associated with the development of a bis(thial S-oxide).65,66 of stomach and colo-rectal cancers.68 This data was further supported by the recent study of Steinmetz et al.,inwhichthe consumption of garlic was inversely associated with colon cancer risk.69 Protective effects have also been observed in a Chinese study, in which a reduction in oesophageal and stomach cancer were observed in individuals consuming Allium vegetables like onions, Welsh onions, Chinese chives and garlic.70 Similarly, in a recent population-based, case-control study conducted in Shanghai, China, an association between garlic, onion, scallion, chive and leek intake and reduced risk of prostate cancer was found.71 Onion consumption is also associated with reduced risk of developing brain cancer.72 In contrast, in the Netherlands Cohort Study (comprising 120 852 Dutch men and women aged between 55 and 69), Dorant et al. reported a strong association between onion consumption and a reduction in the incidence of stomach carcinoma.73 However, in the same study group the authors could find no evidence for a protective effect of Allium vegetable consumption and reduced risk of developing colon, rectum carcinoma, lung or female breast cancer. In support of the epidemiological studies are the protective ef- Fig. 11 Zwiebelanes and bis(thial S-oxide), novel Allium sulfur fects observed for Allium vegetables towards chemically induced compounds. cancers in rodent models. Benzo[a]pyrene, a carcinogen derived from tobacco smoke, promotes neoplasia of the forestomach 3 Allium vegetables and human health and lungs in female A/J mice that can be inhibited by allyl methyl trisulfide (AMTS).74 Likewise, DAS was shown to Mammalian cells are continuously exposed to endogenous inhibit 1,2-dimethylhydrazine-induced hepatocarcinogenicity in and exogenous chemicals, either as by-products of metabolism male Fischer 344 rats.75 Accordingly, based on these early such as reactive oxygen species and lipid peroxides or en- reports, numerous publications have now determined Allium vironmental agents like tobacco carcinogens. Many of these phytochemicals to be potent inhibitors of chemically induced compounds are often recognised for their ability to react with tumours in rodent models. To date, DAS,76,77 MPDS,77 AMDS78 key cellular biomolecules, including DNA and proteins, which DATS, and their corresponding saturated propyl analogues, can disrupt normal cellular function. The consequence of dipropyl sulfide (DPS) and DPDS,79 propylene sulfide (PS), these interactions, if not prevented or repaired, is the possible and S-allylcysteine (SAC),80 have all been reported to reduce development of human pathologies including cardiovascular, the levels of chemically induced tumours in rodent models. cancer and inflammatory diseases. Fortunately, through the Considering the chemopreventative effects of Allium vegetables course of mammalian evolution, mechanism(s) have arisen to and constituents, current studies are now revealing the molecular cope with the constant barrage of potential disease-inducing mechanism(s) for the observed prevention of carcinogenesis. agents. These include the synthesis of intracellular antioxidants, e.g. glutathione, detoxification pathways coordinated by phase 3.1.1 Enzymatic inhibition. Cytochrome P450 monoxyge- II detoxification enzymes, glutathione-S-transferases (GST) nases constitute a superfamily of enzymes involved in oxidation [EC 2.5.1.18], quinone reductase (NQO1) [EC 1.6.99.2] and and reduction reactions of both endogenous and exogenous UGT-glucuronosyltransferases (UGDT) [EC 2.4.1.17], cell cycle compounds. It is now accepted that members of the CYP1A, regulatory proteins and programmed cell death. In addition, 1B and 2E subfamilies are responsible for the bioactivation of

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chemical pro-carcinogens to more toxic forms. Consequently, and nitro-compounds. Likewise, GSTs function in the detoxi- the generation of highly electrophilic intermediates that can bind fication process by catalysing the conjugation of electrophilic to important nucleophilic sites is recognised to contribute to species with the intracellular nucleophile glutathione (GSH) (c- human pathologies. Therefore, modulation of enzymes involved glutamyl-cysteinyl-glycine). in the metabolism of carcinogens or toxic chemicals will influ- DAS, DADS, and DATS (compounds derived from garlic), ence their toxicity and carcinogenicity potential.81 For example, and DPS and DPDS (from onions), have been reported to cytochrome P450 2E1 catalyses the oxidation of numerous increase the activity of the phase II detoxification enzymes volatile environmental carcinogens, while CYP2A6 is known NQO1 and GSTs in a variety of rat tissues.95,96 These studies to be involved in nicotine oxidation and the bioactivation of identified DATS, DADS and DPS as potent inducers of phase the genotoxic N-nitrosamines.82,83 Therefore, phytochemically II detoxification enzymes. More recently, the relative abilities targeted inhibition of CYP450 members involved in the bioacti- of additional sulfides, including DPS, DAS, DPDS, DADS, vation of chemical carcinogens may prevent cellular damage in DPTS, DATS, DPTeS and DATeS, to increase the activities this regard. of NQO1 and GST in rat organs have also been reported.

DAS, DASO and DASO2 are inhibitors of rat hepatic CYP450 DATS and DATeS were found to induce NQO1 and GST in 2E1 and inducers of CYP450 2B1 in vivo.84 Indeed, treatment the liver, kidneys, and lungs of rat tissues while also increasing of rats with DAS before exposure to carbon tetrachloride activities in the duodenum, jejunum, ileum and cecum of the and N-nitrosodimethylamine prevented hepatotoxicity, perhaps gastrointestinal tract. These studies suggest an important role due to the inhibition of CYP450 2E1dependent bioactivation for the allyl group in the ability of Allium sulfur compounds to of procarcinogens. DAS can act as a competitive inhibitor induce phase II detoxification enzymes.97 In 2004, the molecular and directly inactivate CYP450 2E1.85 Indeed, the effects of mechanism(s) for the induction of the phase II detoxification

diallyl sulfoxide (DASO) and diallyl sulfone (DASO2)towards enzymes NQO1 and HO1 were determined for the major sulfides acetaminophen-induced hepatotoxicity in mice would suggest present in garlic (including DAS, DADS, and DATS) in an in this to be the case.86,87 In the studies of Kwak et al.,the vitro human hepatoma model. Up-regulation of both NQO1 and effects of allylsulfide (AS), S-allylmercaptan (SAM) and allyl HO-1 occurred through Nrf-2 activation and ARE-mediated methylsulfide (AMS) on the expression of CYP450 2E1 were transcription. As before, this study confirmed previous reports examined in rats. CYP450 2E1 protein content was significantly that the allyl functional group is more potent than that of the decreased and correlated with decreased enzymatic activity.88 propyl moiety for enzymatic induction.98 Interestingly, rats treated with each compound showed no significant changes in the levels of CYP450 2E1 mRNA 3.1.3 Apoptosis. Apoptosis, also known as programmed levels, suggesting that post-transcriptional regulation may be cell death, is a means by which living organisms control associated with the suppression of CYP2E1 apoprotein levels. abnormalities in cells that occur as a result of genetic or envi- Similarly, inhibition of CYP450-mediated bioactivation of the ronmental cues. Characteristic changes including cell shrinkage, tobacco carcinogen benzo[a]pyrene by DAS, DADS and DATS chromatin condensation, plasma membrane blebbing, DNA has also been described. Analysis of the relative protein levels fragmentation and finally cell breakdown with the release of for the benzo[a]pyrene-inducible CYP450 1A2 enzyme were apoptotic bodies are often observed during apoptosis. The observed to be reduced, and corresponded with a reduction in initiation of apoptosis can occur by two major pathways. Firstly, the levels of CYP450 benzo[a]pyrene metabolites.89 interaction of extracellular ligands with membrane-bound re- ceptors (including the TNFa, TRAIL and FADD receptors, 3.1.2 Enzymatic induction. Phase II detoxification en- and also the death receptors such as DR5) leads to the initiation zymes are recognised for their ability to facilitate the excretion of an intracellular signal that promotes apoptosis. Secondly, of carcinogens from the body. Common to the majority of phase apoptosis can be initiated by mitochondria, with the release of II detoxification enzymes including GSTs, NQO1 and UGDTs apoptotic signaling molecules like cytochrome c, smac/Diablo is the presence of a conserved binding region, designated and apoptosis inducing factor.99 Pivotal in the response of the antioxidant responsive element (ARE). Located within cancer cells to apoptotic stimuli are the caspases. Caspases the promoter regions of each gene, this cis-acting regulatory are intracellular cysteine-containing proteases that cleave their element has been identified in the 5-flanking region of GST substrates after an aspartate residue in a tetrapeptide in a Ya, hemoxygenase 1 (HO1), glutamyl cysteine synthetase and sequence-specific manner.100 Caspases are found as pro-enzymes NQO1 genes of several mammalian species. During enzymatic that require activation to their proteolytic forms either through induction, a small basic leucine zipper protein (bZIP) designated cell surface death receptors, mitochondria or a convergence of Nrf-2 interacts with the ARE, promoting detoxification gene both signaling pathways. Apoptotic cues induced cytochrome expression. Nrf-2 is sequestered in the cytoplasm by a redox- c release from mitochondria, leading to the activation and sensitive protein known as Keap-1. Redox signalling promotes formation of the apoptotic protease-activating factor-1 (APAF- Nrf-2 dissociation from Keap-1, allowing for its translocation 1)–caspase-9 holoenzyme complex.101 Subsequently the forma- to the nucleus, interaction with the ARE and the transcriptional tion of the APAF-1–caspase-9 holoenzyme complex leads to activation of phase II detoxification enzymes.90 Indeed, it is caspase-3 activation.102 Caspase-3 proteolytically degrades nu- common for several detoxification enzymes to be up-regulated merous cellular targets including poly(ADP-ribose) polymerase in a coordinated manner by the Nrf-2 signaling pathway. (PARP), Protein Kinase Cd, retinoblastoma protein, lamin, a- The import role Nrf-2 plays is highlighted by the observation fodrin, DNAse and DNA fragmentation factor.103 Intracellular that mice null for Nrf-2(−/−) show reduced expression of degradation of many of these important protein substrates several detoxification enzymes and an increased sensitivity to leads to the appearance of typical apoptotic morphology. It carcinogenesis and liver toxicity.91–94 is of interest that in numerous human pathological conditions The ability of synthetic and plant-derived Allium compounds including cancers, that apoptotic signaling cascades are often to induce cystolic quinone reductase NQO1 [NADP(H):quinone impaired. Indeed, Apaf-1, caspase-9 and caspase-3 null cell lines reductase oxidoreductase] and glutathione-S-transferases have are highly resistant to apoptotic stimuli.104,105 been reported on several occasions. NQO1 is a 32 kDa FAD- Since the 1950s, scientists have been aware of the inhibitory containing flavoprotein that is necessary for the detoxification effects of garlic and its components that are active against of quinones to hydroquinones. The two-electron-mediated re- tumour growth.106 However, it was not until the advent of duction promotes the glucuronidation of hydroquinones and modern techniques in cell tissue culture that major explorations their excretion. NQO1 can also function in the detoxification were conducted to determine their mechanisms of action. Both of several other carcinogens including quinone-imines, azo- Allium extracts and their phytochemical constituents can induce

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apoptosis in several in vitro cell culture models. From the molecule in DATS-induced apoptosis. Using PC-3 and DU145 available data, activation of the proteolytic caspases, changes in human prostate cancer cells as a model, DATS has been intracellular redox homeostasis, generation of reactive oxygen shown to be a significantly more potent apoptosis inducer species and the activation of stress signaling cascades are all than DAS or DADS.115 In hepatoma HepG2 cells, inhibition implicated in the apoptotic response of cancer cells to Allium of activated/phosphorylated mitogen-activated protein kinases sulfur compounds. Studies have shown that the garlic-derived (MAPK) p38 or ERK p42/44 enhances the DADS-induced S-allylmercaptocysteine (SAMC) can inhibit cell proliferation apoptosis, although the mechanism has yet to be determined.116 in human erythroleukaemia HEL and OCIM-1 cell lines by Ajoene, a major compound of garlic, induces apoptosis in hu- inducing apoptosis.107 Likewise, SAMC (but not SAC) can man leukaemic HL-60 cells, but not in peripheral mononuclear prevent human colon SW480 and HT29 cancer cell proliferation blood cells of healthy donors. Ajoene increased the production by inducing a G2/M cell cycle block and promoting apoptosis, of intracellular peroxide in a concentration and time-dependent through a caspase-3 mediated pathway.108 Xiao et al. later fashion, which could be partially blocked by pre-incubation with showed, using immunofluorescent microscopy, that SAMC the antioxidant N-acetylcysteine.117 In the same cell line, (Z)- rapidly induces microtubule (MT) depolymerisation, MT cy- ajoene induced a time- and concentration-dependent increase toskeleton disruption, centrosome fragmentation and Golgi in HL-60 cell apoptosis, a process involving the activation of dispersion in interphase cells. Moreover, SAMC also caused caspase-3 and the cleavage of the anti-apoptotic protein Bcl-2.118 the formation of monopolar and multipolar spindles in mitotic The thiosulfinate alliicin, also from garlic, inhibits the growth cells in SW480 cells and NIH3T3 fibroblasts.109 It was thus of cancer cells of murine and human origin. Allicin induced postulated that the antiproliferative effects of SAMC were due the formation of apoptotic bodies, nuclear condensation and a to its interaction with tubulin, this leading to the activation typical DNA ladder in cancer cells, as well as the activation of the stress-activated c-Jun NH(2)-terminal kinase (JNK) and of caspases-3, -8 and -9 and cleavage of PARP.119 A more activation of a caspase-3 mediated apoptotic cascade. exciting avenue of research is using the clinical therapeutic Similarly, Allium-derived sulfides have also been shown to be drug anti-CD20 mAb rituximab (Rituxan Mabthera), which is potent inducers of apoptosis in cancer cells. Human leukaemia extensively in use in the treatment of non-Hodgkin lymphomas, HL-60 cells exposed to DADS undergo apoptotic cell death, in combination with the enzyme alliinase. By targeting alliinase mediated by the activation of caspase-3 and cleavage of PARP.110 to CD20+ tumour cells the researchers found that when alliin In addition, it was also reported that apoptosis was dependant was added it promoted the in situ generation of the sulfur on the generation of intracellular peroxides, as apoptosis could compound allicin, which was able to kill CD20+ B-CLL cells as be blocked by the antioxidant enzyme catalase.111 Recently, a well as other human B-cell lymphoma cell lines, both in vitro number of sites within mitochondria have been proposed to and in vivo.120 Indeed, the authors conclude that these new be important in production of reactive oxygen species (ROS), techniques may offer a new, powerful and less toxic therapy including complexes I, II, and III. It is feasible therefore, that for B-CLL and other B-cell malignancies. Moreover, combining modulation of mitochondrial integrity by Allium constituents is alliinase with additional antibodies may extend the application likely to contribute to apoptotic signaling. Indeed, pro-apoptotic of this technique to other conditions in which the elimination of bax and bid can interact with mitochondria to promote the a specific cell population is required. release of apoptotic signaling factors including cytochrome c, smac/Diablo and apoptosis inducing factor. In contrast, 3.2 Anti-inflammatory properties anti-apoptotic members including bcl-2 and bclxLcanprevent mitochondrial perturbations and preserve cell viability.Recently, Over-expression of pro-inflammatory enzymes such as inducible several studies have implicated a role of mitochondria in the nitric oxide synthetase (NOS) and cyclooxygenase-II (COX- response of cancer cells to Allium chemicals. II) are observed in numerous human pathologies including Both DATS and DADS induce apoptosis in cultured human cancer, cardiovascular and inflammatory diseases. Increased neoplastic (A549) and non-neoplastic (MRC-5) lung cancer activity of inflammatory enzymes leads to the generation of cells.111 Furthermore, Hong et al. confirmed that lung cancer pro-inflammatory mediators including nitric oxide (NO) and cells exposed to DAS and DADS caused an increase in the pro- prostaglandins (PG). Endogenous production of NO and PG apoptotic proteins p53 and bax, while reducing anti-apoptotic has a beneficial role in the maintenance of blood pressure, bcl-2 protein level in p53-wild type H460 and p53-null type inflammation, wound healing and temperature regulation. H1299 non-small-cell lung cancer cells.112 These data implicate However, overproduction leads to pathophysiological condi- a role of mitochondria in the apoptotic response of cancer cells to tions such as the promotion of colon cancer, atherosclerosis, Allium sulfides. Additional research has also shown that DADS inflammatory bowel disorders, multiple sclerosis, Alzheimer’s can induce growth arrest and apoptosis in neuroblastoma cell disease and septic shock.121–123 Both iNOS and COX-II protein line SH-SY5Y.DADS promoted ROS generation and the release expression are regulated by Nuclear factor kappa B (NF-jB), a of mitochondrial cytochrome c prior to capsase-9 and capase-3 transcription factor activated by carcinogens, toxins and oxida- activation in these cells.113 Moreover, following ROS generation, tive stress. To date, NF-jB has been demonstrated to regulate the stress-signaling kinase JNK was activated, followed by the transcription of over 150 separate genes, many of which the release of pro-apoptotic mitochondrial proteins. Again it are involved in inflammation.124 Recent studies have shown was observed that anti-apoptotic bcl-2 proteins were down- that antioxidants can inhibit NF-jB activation and thus reduce regulated. Interestingly, JNK inhibition reduced the levels of the symptoms of such debilitating disease states.125,126 Indeed, apoptotic cell death in this model system. JNK activation leads when we consider the association between Allium vegetable to the inactivation of the anti-apoptotic bcl-2 protein by a consumption and reduced risk to cardiovascular diseases and mechanism involving phosphorylation. Indeed, the changes in cancers, many of the beneficial effects are perhaps due to the the ratios of anti- and pro-apoptotic proteins have been observed anti-inflammatory properties. in PC-3 and DU145 human prostate cancer cells and human Early studies showed that garlic extracts could inhibit ADP, breast cancer cells.114 For example, DATS-induced apoptosis collagen, epinephrine, arachidonic acid (AA) and calcium in PC-3 cells was associated with phosphorylation of bcl-2, ionophore A23187-induced platelet aggregation in vitro.127 altered bcl-2, bax interaction, and cleavage of procaspase-9 Platelet aggregation has been implicated in coronary heart and procaspase-3. JNK1 and/or JNK2 phosphorylation of disease and stroke.128 The properties of the garlic extracts bcl-2 in DATS-treated PC-3 cells was blocked in the presence were reportedly due to an inhibitory effect in the degradation of JNK-specific inhibitor SP600125 and prevented apoptosis. of platelet phospholipids, reduced formation of thrombox- Again hydrogen peroxide was implicated as a possible signaling ane (TxB2) and lipoxygenase-derived products from labelled

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platelets. In addition, garlic was also observed to inhibit J774 murine macrophages and human umbilical endothelial cyclooxygenase and lipoxygenase enzymes.129 Similar findings cells (HUVECs) can be abolished by treatment with SAC.142 are also reported for extracts of onion.130 Allicin is a potent hydroxyl radical scavenger.143 Oxidation and To date, few detailed studies have been conducted to de- glycation of human LDL can be inhibited by DAS, DADS, termine the molecular mechanisms of Allium-mediated anti- S-ethylcysteine (SEC) and N-acetylcysteine (NAC). All four inflammatory effects. Dirsch et al. reported that low concentra- derivatives found in garlic can inhibit superoxide production

tions of ajoene (IC50 2.5–5 lM) and allicin (IC50 15–20 lM) could generated by xanthine-xanthine oxidase while also being able reduce nitrite accumulation in LPS-stimulated macrophages.131 to chelate copper ions.144 These findings are further supported Garlic and S-allylcysteine can inhibit NO production in murine by the studies that show DAS, DADS, SAC, SEC, SMC, S- RAW264.7 macrophage cells. The suppressive effects were due methylcysteine and S-propylcysteine are able to prevent the to a reduction of iNOS mRNA and protein levels, following oxidation and glycation of LDL in the plasma isolated from lipopolysaccharide (LPS) and IFN-cstimulation. Garlic extracts patients with non-insulin-dependent diabetes.145 In our labo- also inhibited NO production in peritoneal macrophages, rat ratory we recently demonstrated that the novel 3-mercapto-2- hepatocytes, and rat aortic smooth muscle cells stimulated with methylpentan-1-ol (3-MP), of which four possible diastereoiso- LPS and cytokines. The inhibitory effects were determined to be mers can occur in varying amounts in A. cepa (Fig. 12), due to the suppression of NF-jB signaling.132 Garlic extract significantly inhibited peroxynitrite-mediated tyrosine nitration has also been identified as a potent inhibitor of leukocyte andinactivationofa(1)-antiproteinase. Moreover, 3-MP also in- migration through endothelial cell monolayers, suggesting a pos- hibited peroxynitrite-induced cytotoxicity, intracellular tyrosine sible inhibitory role in the inflammatory processes.133 Likewise, nitration, and intracellular reactive oxygen species.146,147 garlic powder extract inhibits NF-jB activity in LPS-stimulated human whole blood, reducing the levels of pro-inflammatory cytokines. These same properties could also be attributed to DADS that could also significantly reduce the secretion of the proinflammatory cytokines IL-1b and TNF-a.134 In contrast, DADS or AM could inhibit TNF-a-induced activation of NF- jB or the NF-jB-regulated endothelial gene product (E)-selectin in human umbilical endothelial cells (HUVECs). This suggests that the inhibitory effects of Allium-derived sulfur compounds on the NF-jB signaling pathway are structurally dependant.135 3.3 Antioxidant properties Antioxidants are of interest to pharmacologists and biochemists because they are reported to inhibit the damaging effects caused by free-radical agents, including reactive oxygen, nitrogen, and chlorine species (ROS, RNS, and RClS). Due to the apparent formation of these toxic entities at sites of tissue injury, free radicals can alter the structural and functional integrity of cells by promoting intracellular redox changes including protein ox- idation, lipid peroxidation, oxidative DNA damage and protein carbonyl formation. Therefore, considerable interest in the iden- tification of natural and synthetic antioxidants that can interfere with free-radical mediated damage has been sought. To date, plant-derived antioxidants have received the most attention. Extracts of A. sativum and also alliin have been reported to have potent reducing abilities when examined by the 1,1- diphenyl-2-picrylhydrazyl stable free radical (DPPH) scavenging Fig. 12 The biosynthetic pathway for the production of 3-mer- assay.136 Aqueous extracts of garlic can also prevent copper- capto-2-methylpentan-1-ol in Allium species remains largely unknown, 137,138 although a hypothetical pathway for the formation of this potent induced lipid peroxidation in vitro. In the study of Rajasree antioxidant has been suggested (adapted from Widder et al.147). et al., garlic and onion extracts were shown to reduce nicotine- induced lipid peroxidation, as assessed by measuring thiobar- 3.4 Antimicrobial properties bituric acid reactive substance, conjugated diene and hydroper- oxide concentrations in the tissues of nicotine-treated rats.139 The antimicrobial properties of Allium species have been the The inhibitory effects appear to be associated with increased focus of several recent review articles and therefore we will activities of the antioxidant enzymes catalase, superoxide dis- only provide brief description herein.148,149 In common folklore mutase and glutathione peroxidase, and increased intracellular medicine, Allium plants have long been associated with the glutathione content. With regard to individual phytochemical treatment of infections.150 It is apparent from recent chemical constituents, several have been shown to act as potent free- characterisation that the therapeutic effects, particularly with radical scavengers and prevent cellular damage. Xiao et al. regards to the antimicrobial properties, are due to constituents recently concluded that the antioxidant potential of crude Allium derived from the parental CSs. Over the last century, garlic has extracts is probably due to additional chemical components been proven to be effective against both gram-positive, gram- rather than thiosulfinates. This assumption was made based negative and acid-fast bacteria. These include Pseudomonas, on the fact that the thiosulfinates were poorer antioxidant Proteus, Escherichia coli, Staphylococcus aureus, Klebsiella, scavengers than conventional antioxidants like ascorbic acid, Salmonella, Micrococcus, Bacillus subtilis, Mycobacterium,and trolox and glutathione.140 Clostridium.151–156 Furthermore, garlic also inhibits the growth of Several organosulfur compounds identified in Allium species strains of S. aureus, E. coli, Proteus mirabilis and Pseudomonas do have antioxidant properties. SAC and SAMC, the major aeruginosa that are multiply resistant to antibiotics including organosulfur compounds found in aged garlic extract, can penicillin, streptomycin, doxycilline and cephalexin.157 Similarly, reduce lipid peroxidation and 1,1-diphenyl-2-picrylhydrazyl the bactericidal effects of extracts of onion towards Streptococ- (DPPH) assays.141 In addition, hydrogen peroxide formation in- cus mutans and S. sobrinus, the main causal bacteria for dental duced by oxidised low-density lipoprotein (ox-LDL) in activated caries, and Porphyromonas gingivalis and Prevotella intermedia,

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considered to be the main causal bacteria of adult periodontitis, strains of Leishmania including L. major, L. major (Pakistan), have been described.158 L. tropica, L. mexicana ssp. mexicana and L. donovani were Recent work has been conducted to determine the antimicro- found to be sensitive to onion juice. The authors suggest that the bial properties of additional Allium sulfur constituents. Cavillato susceptibility of the Leishmania strains is a likely consequence and Bailey discovered that the major antibacterial agent present of the sulfur compounds present within the onion extract.175 in garlic was allicin.151 The inhibitory effect of garlic extract, DAS and DADS against methicillin-resistant S. aureus (MRSA) 3.6 Cardiovascular disease infection in BALB/cA mice was studied. The oral admin- Garlic extracts as well as several additional Allium organosulfur istration of these agents significantly decreased the viability compounds have hypocholesterolemic effects in human and of MRSA in plasma, liver, kidney and spleen. Indeed, the animal models.176–179 Gebhardt demonstrated that water-soluble authors conclude that DAS and DADS are potential therapeutic garlic extracts reduced cholesterol biosynthesis in hepato- agents for the treatment of MRSA infection.159 The thiosul- cytes when exposed to the radiolabelled precursor compound finates 2-propene-1-sulfinothioic acid S-(Z,E)-1-propenyl es- [14C]-acetate, indicating a potential regulation of 3-hydroxy-3- ter [AllS(O)SPn-(Z,E)], 2-propenesulfinothioic acid S-methyl methylglutaryl CoA (HMG-CoA) reductase.180 However, the ester [AllS(O)SMe], and methanesulfinothioic acid S-(Z,E)-1- mechanism(s) by which garlic regulates HMG-CoA reductase propenyl ester [MeS(O)SPn-(Z,E)] isolated from oil-macerated activity has yet to be fully elucidated. Recently, Augusti et al. garlic also have antimicrobial activity.160 1-Methyl methanethio- reported that rats fed with a polar fraction of garlic oil could sufinate and S-methyl-2-propene-1-thiosulfinate isolated from inhibit the hyperlipidemic and elevated tissue levels of HMG- Chinese chive (A. tuberosum), both showed significant antibac- CoA reductase.181 Ajoene is known to be a potent inhibitor terial activity against E. coli O-157:H7.161 Ajoene shows a of arterial smooth muscle proliferation.182 Moreover, chronic broad spectrum of antimicrobial activity towards the growth treatment of rats with garlic extracts is protective against of gram-positive bacteria, including Bacillus cereus, Bacillus isoprenaline-induced cardiac necrosis.183 subtilis, Mycobacterium smegmatis,andStreptomyces griseus. In a recent study by Miron et al., a novel antihypertensive drug The disulfide bond in ajoene appears to be necessary for the was synthesised through the reaction of the pharmaceutical drug antimicrobial activity, since reduction by cysteine, which reacts Captopril with the Allium-derived thiosulfinate allicin (Fig. 13). with disulfide bonds, destroyed its antimicrobial activity.162 Captopril possesses potent antihypertensive properties due to its An exciting finding with regards to Allium sulfur compounds ability to inhibit the angiotensin-converting enzyme. Likewise, is their activity towards Helicobacter pylori. H. pylori infection allicin also inhibits hypertension by reducing serum cholesterol is intimately involved in stomach cancer development. Recent and triglyceride levels. Therefore, the authors proposed that epidemiological studies have indicated that the consumption of a combination of both drugs, each working at two separate Allium vegetables can reduce the risk of gastric neoplasia.163 pharmacological sites of action, may provide better protection Chung and colleagues reported that the garlic oil components against hypertension. The reaction product between captopril DAS or DADS had bacteriocidal affects towards H. pylori and allicin was termed allylmercaptocaptopril (CPSSA), a non- growth.164 Furthermore, garlic extracts can inhibit H. pylori- symmetric disulfide that combines the specific drug activity of induced gastritis in Mongolian gerbils and has been suggested captopril and the beneficial properties of allicin. In fructose- as a useful agent in the prevention of H. pylori pathologies.165 induced hypertensive rats, CPSSA decreased triglyceride levels The crude methanol extract of the leaf tissues of A. ascalonicum and significantly lowered blood pressure.184 In a rat myocardial also have antibacterial activity towards H. pylori strains (ATCC infarct study, garlic oil has also been shown to exert its protective 24376, UCH 97001, UCH 97009, UCH 98026 and UCH effects by modulating lipid peroxidation and enhancing an- 99039).166 Likewise, allicin, allylmethyl- and methylallylthiosul- tioxidant and detoxifying enzyme systems185 (see Sections 3.1.1 finate isolated from acetonic garlic extracts inhibit the in vitro and 3.3). The antioxidant properties of several chemical groups growth of H. pylori.167 of natural products served as one of the important factors to protect against ischemic myocardium in vivo.186 Garlic extracts 3.5 Antifungal and antiparasitic properties contain large amounts of sulfhydryl-containing amino acids, notably cysteine and the S-alk(en)yl derivatives such as SAC, Many fungi have proven susceptible to Allium extracts, partic- SEC and SPC.179 Interestingly, cysteine can be metabolised to ularly those of garlic. These included Candida, Trichophyton, generate hydrogen sulfide (H S) via the catabolic action of the Torulopsis, Rhodotorula, Cryptococcus, Aspergillus,andTri- 2 enzymes cystathionine c-lyase and cystathionine b-synthetase. chosporon.168–170 More recently, the in vitro activities of garlic oil, Recently, H S has been shown to be a novel gasomediator in the Chinese leek oil and four diallyl sulfides towards three Candida 2 cardiovascular system.187 Consequently, it is conceivable that ad- species and three Aspergillus species were also determined. ministration of chemically modified cysteine analogues, particu- The antifungal activities of the garlic and Chinese leek oil larly those found in Allium species, may influence the formation extracts were shown to be dependant on the relative sulfide of endogenous H S in the heart and/or vasculature. However, a concentration. Moreover, of the four diallyl sulfides examined, 2 potential therapeutic role of garlic or indeed other Allium species the antifungal activity followed the order of DATeS > DATS > towards cardiovascular disease requires more research. DADS > DAS.171 The antifungal properties observed for Allium extracts and sulfur compounds appear to be associated with their ability to reduce the growth of and inhibit lipid, protein and nucleic acid synthesis.172 With regards to the use of Allium species and their constituents as antiprotozoals, only a few reports have been published. DATS can inhibit metabolism or growth of parasites, particularly Try- panosoma brucei ssp. brucei, ssp. rhodesiense, ssp. gambiense, ssp. evansi, ssp. congolense and ssp. equiperdum,aswellasEntamoeba histolytica and Giardia lamblia.173 Whole garlic (A. sativum) extracts, allyl alcohol and AM are inhibitory towards the microaerophilic flagellate protozoon Giardia intestinalis,apar- asitic species responsible for causing waterborne diarrhoea.174 Antileishmanial effects of aqueous onion (A. cepa)extracts Fig. 13 Synthesis of the potent antihypertensive drug Allylmercapto- towards leishmanial promastigotes has also been reported. Five captopril.

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Fig. 14 Metabolism of Allium sulfides in mammals. (A) Oxidation of diallyl sulfide and the conjugation of its metabolites to the intracellular antioxidant glutathione (SR), (B) Metabolic oxidation products of diallyl disulfide.

3.7 Metabolism of Allium sulfur compounds are perhaps due to the ability of Allium sulfur compounds to positively modify the antioxidant, apoptotic, inflammatory, There is ample evidence showing the potential beneficial prop- and cardiovascular systems in mammalian systems. However, erties of Allium sulfur compounds on human health. However, much remains to be done in each of these research areas, few studies have dealt with the metabolism or bioavailability thus presenting a challenge for future studies by chemists, drug of these compounds. Undoubtedly, such data would give a development researchers and the pharmaceutical industry. greater understanding of the role of Allium phytochemicals in human health, and more importantly identify biomarkers that could be used to determine an individual’s exposure to 5 Abbreviations such Allium sulfur compounds. Metabolism of mono-sulfides, ACSO, (+)-S-allyl-L-cysteine sulfoxide (alliin); AM, allyl mer- including DAS, in rat models occurs via sequential oxidation that captan; AMDS, allyl methyl disulfide; AMS, allyl methyl sulfide; leads to the formation of the sulfoxide prior to the formation of AMSO, allyl methyl sulfoxide; AMSO , allyl methyl sulfone; the sulfone (Fig. 14A).188,189 Moreover, Jin et al. have also shown 2 AMTS, allyl methyl trisulfide; AS, allyl sulfide; BCSO, S-n- that DAS can readily conjugate to the intracellular antioxidant butyl-L-cysteine sulfoxide; CS, S-alk(en)yl cysteine sulfoxides; glutathione.190 The thiol reactivity of Allium-derived sulfides is DADS, diallyl disulfide; DAS, diallyl sulfide; DASO, diallyl particularly interesting, as this can result in the formation of S- sulfoxide; DASO , diallyl sulfone; DATeS, diallyl tetrasulfide; mercaptoconjugates such as SAMC. Moreover, recent evidence 2 DATS, diallyl trisulfide; DMTS, dimethyl trisulfide; DPDS, has shown that the cysteine conjugates SAC and SAMC are dipropyl disulfide; DPS, dipropyl sulfide; DPTS, dipropyl substrates for the mammalian b-lyase c-cystathionase in rat liver trisulfide; ECSO, S-ethyl-L-cysteine sulfoxide (ethiin); EP, homogenates. However, the biological significance of this has 1,2-epithiopropane; GSH, glutathione (c-glutamyl-cysteinyl- yet to be determined.191 Similar results have also been obtained glycine); LPS, lipopolysaccharide; MAS, methyl allyl sulfide; for the metabolism of dipropyl sulfide and dipropyl sulfoxide MCSO, (+)-S-methyl-L-cysteine sulfoxide (methiin); 3-MP, 3- in the rat. Disulfides, such as DADS, are readily absorbed and mercapto-2-methylpentan-1-ol; MPDS, methyl propyl disulfide; accumulate in the liver within 90 minutes.192 DADS is converted MPeDS, methyl 1-propenyl disulfides (E and Z); MPTS, methyl to AM and allyl methyl sulfide as determined in ex vivo metabolic propyl trisulfide; NAC, N-acetylcysteine; NF-jB, nuclear factor models using perfused rodent liver or rat hepatocytes.193,194 kappa B; PARP, poly(ADP-ribose) polymerase; PCSO, (+)-S- Germain and colleagues have also reported that a single dose propyl-L-cysteine sulfoxide (propiin); PPDS, 1-propenyl propyl of DADS in rats led to the formation of AM and AMS, as well disulfides (E and Z); PS, propylene sulfide; PT, 1-propanethiol; as two oxidative metabolites, allyl methyl sulfoxide (AMSO) and RClS, reactive chlorine species; ROS, reactive oxygen species; allyl methyl sulfone (AMSO ) (Fig. 14B).195 2 RNS, reactive nitrogen species; SAC, S-allylcysteine; SAM, S- allylmercaptan; SEC, S-ethylcysteine; SPC, S-propylcysteine; 4 Summary THDMP, tetrahydro-2,5-dimethylthiophene; TPCSO, (+)-S- trans-1-propenyl-L-cysteine sulfoxide (isoalliin); 2VDN, 2-vinyl- Allium species are an agronomically important genus because [4H]-1,3-dithin; 3VDN, 3-vinyl-[4H]-1,2-dithin. of their sulfur flavour components. The majority of the volatile flavour principles are generated through the enzymatic hydrol- ysis of the non-volatile CS storage compounds. Furthermore, 6 References these compounds may be possible sources of new novel therapeu- 1 G. R. Fenwick and A. B. Hanley, Crit. Rev. Food Sci. Nutr., 1985, tic principles. Allium compounds are reported to be effective in 22(3), 199–271. the prevention of numerous disease states in humans, including 2 E. Block, Sci. Am., 1985, 252(3), 114–119. cancer, cardiovascular and inflammatory disorders. These traits 3 A. Stoll and E. Seebeck, Experentia, 1947, 3, 114–115.

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