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Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genusAllium: the chemistry of potential therapeutic agents

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: bchpcr@nus.edu.sg; Fax: (65)-6779-1453; Tel: (65)-6874-4996

b Department of Pharmacology, National University of Singapore, 18 Medical Drive, Singapore,117597. E-mail: phczhuyz@nus.edu.sg; Fax: (65)-6773-7690; Tel: (65)-6874-3676

Received (in Cambridge, UK) 30th March 2005First 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 thefamily Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomicallyimportant 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 humanpathologies, however it is only recently that any significant progress has been made in determining their mechanismsof action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from thecombination 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 actionof the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinatescan 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 andgarlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes likeglutathione-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 vitrocancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds incardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxidemetabolites 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 atthe University of Nottingham prior to obtaining his PhD at the Institute of Food Research and the John Innes Centre, UK. His researchfocuses 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 PhDin Medical Biochemistry in 1997 in the Department of Pharmacology, King’s College, University of London. He moved to Singapore in2000 after completing 3 years’ post-doctoral work at the Centre for Age-Related Diseases, King’s College. His research interests focuson 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 fromKing’s College in London, and has been actively involved in research into the mechanisms underlying inflammatory and cardiovasculardisease for over 25 years.

Yi Zhun Zhu is a senior research fellow at the Department of Pharmacology, National University of Singapore and Professor ofPharmacology at Fudan University, Shanghai. He obtained his undergraduate medical training in Shanghai and did doctorate andpost-doctoral training in Heidelberg and Kiel. His research is focusing on cardioprotective effects of ischemic heart disease using Westerndrugs and/or extracts from natural products.

Peter Rose Matt Whiteman Philip Moore Yi Zhun Zhu

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View Article Online / Journal Homepage / Table of Contents for this issue

1 Introduction2 Sulfur storage compounds2.1 S-Alk(en)yl cysteine sulfoxides in Allium species2.1.1 Alliinase2.2 Organosulfur compounds in Allium species2.2.1 Thiosulfinates2.2.2 Decomposition products2.2.3 Novel thiosulfinate-derived sulfur compounds3 Allium vegetables and human health3.1 Anticarcinogenic properties3.1.1 Enzymatic inhibition3.1.2 Enzymatic induction3.1.3 Apoptosis3.2 Anti-inflammatory properties3.3 Antioxidant properties3.4 Antimicrobial properties3.5 Antifungal and antiparasitic properties3.6 Cardiovascular disease3.7 Metabolism of Allium sulfur compounds4 Summary5 Abbreviations6 References

1 Introduction

Although formally classified in the family Liliaceae, representedby 280 separate genera and 4000 species, recent taxonomicrevisions have seen members of the genus Allium placed inthe family Alliaceae. Distributed throughout most regions ofthe temperate world including Europe, Asia, North Americaand Africa, Allium species have a long history in commonfolklore and as sources of therapeutic principles. In additionto preventing the nocturnal bloodthirsty pursuits of vampires,one of the most famous members of the Alliaceae, garlic Alliumsativum L. has been used throughout the centuries to treat animalbites, leprosy, the plague, heart disease and cancer. Indeed, therealisation in 1858 by Louis Pasteur that garlic had potentantibacterial properties later led to its use in the First andSecond World Wars to prevent gangrene. Of approximately 700species, it is the edible members including onion (A. cepa L.),garlic (A. sativum L.), chives (A. schoenoprasum L.), leek (A.porrum L.) and Welsh onion (A. fistulosum L.) that are highlyprized.1 Ordinarily, the vegetative parts are odour-free, and itis only during tissue damage that volatile flavour principles aregenerated. Interestingly, these volatile chemicals are producedthrough enzymatic hydrolysis of non-volatile sulfur storagecompounds, termed S-alk(en)yl-L-cysteine sulfoxides (CSs). Todate, four major and two minor CSs are have been identifiedin the genus Allium, and it is from these that approximately 50additional sulfur compounds can be generated. It is thereforenot surprising that the composition and quantity of each CSdetermines the odour, flavour variation and biological activitiesobserved for Allium vegetables. Given such chemical diversity,members of the genus Allium have received considerable atten-tion from both chemists and biologists alike as new sources ofbioactive compounds. Therefore, in the current review we willdescribe the biosynthesis, generation and bioactive properties ofsulfur compounds derived from the genus Allium.

2 Sulfur storage compounds

2.1 S-Alk(en)yl cysteine sulfoxides in Allium species

The medicinal properties associated with members of Alliumhas long been recognised and has provided the impetus forchemists to determine the active chemical substances.2 Earlyinvestigators identified volatile odour principles in garlic oils,however, these compounds were only generated during tissuedamage and preparation. Indeed, the vegetative tissues of Alliumspecies are usually odour-free, and it is this observation that ledto the hypothesis that the generation of volatile compounds from

Allium species arose from non-volatile precursor substances. Itwas in the laboratory of Stroll and Seebrook in 1948 that thefirst stable precursor compound, (+)-S-allyl-L-cysteine sulfoxide(ACSO), commonly known as alliin, was identified.3 Alliin is theparental sulfur compound that is responsible for the majority ofthe odorous volatiles produced from crushed or cut garlic. Threeadditional sulfoxides present in the tissues of onions were lateridentified in the laboratory of Virtanen and Matikkala, thesebeing (+)-S-methyl-L-cysteine sulfoxide (methiin; MCSO), (+)-S-propyl-L-cysteine sulfoxide (propiin; PCSO) and (+)-S-trans-1-propenyl-L-cysteine sulfoxide or isoalliin (TPCSO). Isoalliinis the major sulfoxide present within intact onion tissues and isthe source of the A. cepa lachrymatory factor.4,5 With regardsto chemical distribution, (+)-S-methyl-L-cysteine sulfoxide isby far the most ubiquitous, being found in varying amountsin the intact tissues of A. sativum, A. cepa, A. porrum, and A.ursinum L.(Table 1).

To date, only the L-(+)-isomers have been described in nature.6

A variety of methods have now been established to allowthe direct analysis of CSs in plant tissues.7,8 These techniquesinclude HPLC or gas chromatographic (GC) methodologies,and rely either on the direct measurement of the CS followingderivatisation or measurement of their respective degradationproducts. One popular method that circumvents the problem oflow sensitivity experienced with HPLC techniques is the use ofGC. In the studies of Kubec et al.9,10 GC methodologies weredeveloped to determine the distribution of CSs within the genusAllium. Initially, the CSs are derivatised due to their apparentthermal instability, using ethyl chloroformate prior to GCanalysis (Fig. 1). Data from these studies provided a quantitativemethod to measure the nonvolatile CSs in 15 separate Alliumspecies (Table 2). Moreover, using this technique Kubec andcolleagues identified S-ethylcysteine sulfoxide (ethiin; ECSO),not previously reported to occur in Allium species, as a minorcomponent of most extracts.11 Similarly, S-n-butylcysteine sul-foxide (BCSO) was identified and isolated from the tissues of A.siculum using this same method, and confirmed the earlier reportby Horhammer et al. on the occurrence of BCSO in garlic (A.sativum).12,13 To date, four major and two minor CS have beenidentified and isolated from Allium vegetables.

After the discovery of stable CSs, attention was next directedtowards the elucidation of the biosynthetic pathway. Much ofour knowledge of this area comes from radiolabel feeding studiesand direct chemical analysis. What is apparent from these studiesis that CSs have a common origin in plant sulfur metabolism.In plants, sulfate (SO4

2−) is used as the primary source ofsulfur for the biosynthesis of the amino acid cysteine andalso the antioxidant glutathione.14 Initially, SO4

2− is transportedacross the root plasma membrane, whereupon it accumulateswithin plant cells. In order for SO4

2− to be utilised for cysteinebiosynthesis it must first be converted to the intermediatecompound 5-adenylylsulfate (APS). This reaction is catalysedby the enzyme ATP sulfurylase in the plastids. APS can then beused by the enzyme APS reductase to form sulfite, prior to itsconversion to sulfide by the enzyme sulfide reductase. Followingthis, cysteine is formed from the reaction of sulfide with O-acetylserine, a process catalysed by the enzyme OAS thiol-lyase.O-acetylserine is derived from the acetylation of serine by theaction of the enzyme serine acetyltransferase.15 Once formed,cysteine is rapidly channeled into several metabolic pathwaysinvolved in protein synthesis as well as in numerous plantsecondary metabolic pathways.

With regards to CSs, early investigations using radiolabelledcarbon and sulfur sources showed that each were rapidlyincorporated into the glutathione cycle and also in the formationof low molecular weight glutamyl peptides (GPs).16 To date,approximately 24 sulfur-containing GPs have been identifiedin Allium species, where they are considered to function assulfur and nitrogen stores as well as intermediates in CSbiosynthesis. 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. DonA. 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. 1.1 41.1 17.7 92.7 155.8Scallion, A. fistulosum L. trace 5.6 1.8 13.1 21.2Leek, A. porrum L. trace 4.0 trace 17.6 21.6Garlic, A. sativum L. 1077 122 trace trace 1199Chive, A. schoenoprasum L. 21.1 32.2 606 21.0 72.4Wild garlic, A. ursinum L. 40.3 60.0 1.2 trace 101.9

the radiolabel 14C was found to be incorporated into S-2-carboxypropylcysteine, S-2-carboxypropyl glutathione andmethacrylic acid.17 Granroth et al.18 later showed that S-2-carboxypropyl glutathione could be hydrolysed by onions,and the S-2-carboxypropylcysteine formed could be utilisedin the biosynthesis of TPCSO, suggesting a putative linkbetween GP and CS synthesis. Unequivocal evidence that S-2-carboxypropylcysteine was indeed the precursor compoundfor TPCSO synthesis was later confirmed by Parry et al.19,20

Further confirmation of the role of GPs in CS biosynthesis and asuggested biosynthetic model was later highlighted by Lancasteret al.21 in model studies using A. cepa, A. sativum and A. siculum.In onion seedlings a 10 minute pulse of radiolabelled sulfate(35SO4

2−) leads to its rapid assimilation into GPs – after 1 hour,35% of the administered label was found in the GPs. Using thinlayer chromatography the following radiolabelled compoundswere identified: methyl glutathione, c-glutamyl methyl cysteine,S-2-carboxypropyl glutathione and c-glutamylpropenyl cysteinesulfoxide. Over a period of several days labelled sulfur appearedin PCSO (5-fold increase), TPCSO (8-fold increase) and MCSO(1.2-fold increase). A similar pattern of labelling was alsoconfirmed for A. sativum. In additional studies using A. siculum,labelled sulfur was rapidly assimilated into glutathione, c-glutamyl cysteine, methyl glutathione and c-glutamyl methylcysteine prior to incorporation into MCSO (12-fold increase).These findings led the authors to propose the following: 1) c-Glutamyl cysteine and glutathione are the starting compoundsand, 2) CS biosynthesis can proceed by S-alk(en)ylation of

the cysteine residue of glutathione, followed by the removal ofthe glycyl residue by transpeptidation. Subsequently, the CSproceeds undergoes oxidation and loss of the glutamyl group,leading to the parental CS (Fig. 2). Alternatively, the directS-alk(en)ylation of cysteine or thioalk(en)ylation of O-acetylserine followed by oxidation to the respective CS can occur(Fig. 3). The second pathway has been suggested due to theobservation that cysteine will readily react with methacrylic acid.Granroth et al.18 previously identified methacrylic acid in Alliumtissues and showed it to react with thiol compounds. Indeed, S-2-carboxypropyl cysteine and S-2-carboxypropyl glutathione canbe formed through the direct reaction of methacrylic acid withcysteine or glutathione, providing a route to the formation ofACSO, TPCSO, and PCSO. The relative contribution of bothpathways in CS biosynthesis has yet to be determined, moreover,little is known about the origin of the S-alk(en)yl donor groupsor the genes, enzymes and regulatory mechanisms involved in thebiosynthetic pathway. Hopefully, future work will address theseareas. For further information with regards to CS biosynthesiswe refer the reader to the excellent and comprehensive works ofWhitaker,16 Block et al.22 and Jones et al.23

2.1.1 Alliinase. Common to all Allium species is the en-zyme alliinase [EC 4.4.1.4], a 50 kDa glycoprotein that catalysesthe hydrolysis of CS in the presence of the cofactor pyridoxal 5′-phosphate to produce pyruvate, ammonia and sulfenic acids.In intact tissues alliinase is compartmentalised within plantvacuoles and the representative CS located in the cytoplasm.24

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Fig. 1 (A) 1, Commercially available Allium species consumed in SouthEast Asia; 2, Section through the stem tissue of leek (A. porrum); 3,Red onion A. cepa; 4; Garlic A. sativum; 5, Made to make your eyeswater, A. cepa; 6, Chinese chives A. tuberosum L. (Photos by P.R.)(B) Derivatisation and reduction of S-alk(en)ylcysteine sulfoxides withethyl chloroformate (adapted from Kubec et al.9).

Upon tissue disruption the vacuole and cytoplasmic contentsmix, promoting the enzymatic hydrolysis of the respectiveCS. This catalytic reaction leads to the generation of sulfenicacids that condense to form thiosulfinates. Indeed, we are awareof the presence of alliinase, as it is this enzyme that catalyses thereaction leading to the generation of the lachrymatory principlefound in onions and leeks, thiopropanal S-oxide25 (Fig. 4).

Alliinase protein has been isolated and characterised fromseveral Allium species including A. cepa, A. sativum, A. tubero-sum L., A. ursinum, and A. porrum (leek).26–31 Moreover, alliinasederived from A. sativum has been crystallised and its three-dimensional structure has been reported.32,33 Characterisationof the catalytic activity of alliinase shows that it is sensitiveto pH, pyridoxal-5-phosphate availability and temperature.Purified onion alliinase shows optimum activity at pH 7.4and preferentially hydrolyses to TPCSO rather than MCSO orPCSO.26,34 In the case of garlic the situation is more complicatedas it has two distinct alliinase activities. For the hydrolysisof PCSO and TPCSO the pH optimum is 4.5, whereas thesecond activity is specific to MCSO, with a pH optimum of6.5. An interesting study by Lancaster et al.35 demonstratedthat hydrolysis of PCSO and MCSO in onion macerates can beenhanced by pyridoxal-5-phosphate addition. In a recent studyby Krest et al.,36 analysis of alliinase activities derived fromcrude protein extracts of nine allium species (A. sativum, A.

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 andtemperature optima for each species were similar. Moreover,alliinase activities from each species showed the greatest activitytowards 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 forA.sativum, A. cepa and A. ascalonicum reveal a high degree ofsequence similarity both at the nucleotide and at the amino acidlevel in the alliinase cDNA for each species.37 In A. tuberosum,functional recombination of the cDNA encoding alliinasein Eschericha coli and Saccharomyces cerevisiae afforded acatalytically active enzyme. Moreover, site-directed mutagenesisrevealed that lysine-280 was essential for catalytic activity, andthat it was the binding site for the cofactor pyridoxal 5′-phosphate. This data is further supported by the presence ofthe same lysine residue in the pyridoxal 5′-phosphate bindingsite of A. cepa alliinase.41

2.2 Organosulfur compounds in Allium species

Upon tissue damage the first chemical compounds to be formedare the sulfenic acids and thiosulfinates. These progenitorcompounds are intermediates in the formation of the majorityof sulfur volatiles. With regards to the types of chemicalcomponents formed in Allium species, Block et al.22 proposeda scheme that provides a means to characterise the origins ofmany of these compounds. These categories are: 1) head spacevolatiles; chemicals generated at room temperature followingcutting or homogenisation of Allium tissues, 2) decompositionproducts formed from thiosulfinates at room temperature, and 3)oil components – compounds generated by vigorous preparationsuch as steam distillation (Fig. 5). For the purpose of thecurrent review we will introduce the thiosulfinates first priorto addressing compounds identified in each of the above threecategories.

2.2.1 Thiosulfinates. In freshly macerated Allium tissuesthe initial chemical compounds formed are the thiosulfinates,derived from the condensation of sulfenic acid principles(Fig. 6). All Allium-derived thiosulfinates can be representedby 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-nates; and 4) mixed a, b- and c-unsaturated thiosulfinates.22

Because of the inherent thermal instability of the thiosulfinates,only a few methods for their extraction and detection havebeen developed.42 The most suitable of these appears to bevacuum distillation of tissue homogenates coupled with HPLCdetection. Using this method the primary compounds formedin Allium homogenates are the thiosulfinates, rather than theirthermal degradation products such as polysulfides. In a recentstudy by Block et al.,43 HPLC detection identified 8 individualthiosulfinates in the tissues of onion (A. cepa), garlic (A.sativum), wild garlic (A. ursinum), leek (A. porrum), scallion (A.fistulosum), shallot (A. ascalonicum), elephant (or great-headed)garlic (A. ampeloprasum L. var. ampeloprasum auct.), chive (A.schoenoprasum), and Chinese chive (A. tuberosum) (Table 3).Thiosulfinates containing the 1-propenyl group were the mostabundant in A. cepa, A. fistulosum, A. porrum, A. schoenoprasum,and A. ascalonicum. 72% of the total thiosulfinate content inA. tuberosum was MeS(O)SMe, while in A. schoenoprasum,58% of total thiosulfinate content was n-PrS(O)S(n-Pr). In A.sativum and A. ursinum the major thiosulfinates present wereAllS(O)SAll. It is apparent from this study that thiosulfinatesmight serve as useful chemo-taxonomic markers, as suggestedby the authors. Indeed, the types of thiosulfinate compoundsidentified in Allium tissues directly correlate with the composi-tion of the parental cysteine sulfoxides.

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Fig. 3 Alternative route to S-alk(en)yl cysteine sulfoxides bythioalk(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 thiosulfinatein Allium tissues we must highlight the data obtained frommodel reaction systems in which synthetic CS, alliinaseand thiosulfinates are examined. It is apparent that thekinetics of CS hydrolysis and the reactivity of the initialsulfenic acids influence the types of thiosulfinates generated.Thiosulfinates are represented by the following groups: 1)symmetrical thiosulfinates that arise from the condensationof two molecules of sulfenic acid sharing the same alk(en)ylgroup, or 2) asymmetrical thiosulfinates that are generatedby the condensation of two different molecules of sulfenicacid. To further explain these chemical reactions, Shen andParkin developed novel in vitro models to study the pathwayof thiosulfinate production.44,45 The authors found that therate of hydrolysis of parental CS followed the order TPCSO >

ACSO > PCSO > ECSO > MCSO. In additional studiesthe authors also showed that thiosulfinates can readily reactwith sulfenic acids, leading to the biogeneration of additionalthiosulfinate species (Fig. 7). These findings are explained bythat fact that individual CSs can be hydrolysed at different rates,and thus the rapidity by which the sulfenic acid intermediatesare produced differs. Indeed, the “faster” the CS reacts,the quicker is the formation of thiosulfinates. In contrast,“slow”-reacting CSs generated sulfenic acids much slower. The

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

Fig. 6 Structures of common Allium thiosulfinates (adapted from Shenet al.42).

accumulation of “slow” sulfenic acids allows them to reactwith the pre-formed thiosulfinates derived from the “fast”reaction. These reactions promote the generation of additionalthiosulfinates. For example, in Allium species two isomericthiosulfinates are found: allyl methanethiosulfinate and methyl2-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 formthiosulfinates, 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 (totalthiosulfinate content is expressed as lmol g−1 fresh weight)

Thiosulfinate A. cepa L.A. ascalonicumauct. A. fistulosum L. A. porrum L. A. sativum L. A. tuberosum L.

A. schoeno-prasum L.

AllSS(O)-(E)-propenyl — — — — 1.6 — —AllSS(O)-(Z,E)-propenyl — — — — 5.3 0.9 —AllS(O)SAll — — — — 89 28 —PrSS(O)-(E)-propenyl 9 14 2 8 — — 2.5PrS(O)S-(Z,E)-propenyl 16 22 17 15 — — 16PrS(O)SPr 13 26 24 24 — — 58MeSS(O)-(E)-propenyl 22 9 12 12 1.4 — —AllS(O)SMe — — — — 1.4 — —MeS(O)S-(Z,E)-propenyl 31 16 22 31 — 0.7 1MeSS(O)Pr 1 2.8 11 5 — — 5.9MeS(O)Pr 1 1.2 8 5 — — 15AllSS(O)Me — — — — 2.9 34 —MeS(O)SMe — 9 1 3 — 0 1.8

Total MeS (%) 28 23 25 27 2 49 13Total AllS (%) — — — — 94 50 —Total 1-propenyl (%) 45 32 24 33 3 1 10Total PrS (%) 27 46 51 40 — — 77Total thiosulfinate/lmol g−1

fresh weight0.14 0.25 0.08 0.15 14.3 21 0.19

Fig. 7 Reaction of 2-propenyl thiosulfinate with methylsulfenic acidleads to the generation of allyl methanethiosulfinate.

are formed 10 times faster than their methyl counterparts, andthus 2-propenethiosulfinate is formed rapidly. The preformed2-propenethiosulfinate is therefore free to react with the slowlyforming methanesulfenic acid, leading to the formation of allylmethanethiosulfinate.21

In fresh Allium homogenates thiosulfinates are reported to berelatively stable when left at room temperature for 26 hours.Indeed, Block et al.43 reported that the total thiosulfinateconcentration in garlic homogenates remained roughly constantand that the only thiosulfinates that showed any apprecia-ble loss were those represented by MeCH=CHS(O)SR andMeS(O)SMe. Most flavour compounds are derived from thedecomposition of thiosulfinates. For example, allicin can react toform ajoene, for which the trans and cis isomers are recognised.In a separate pathway, allicin can react with thiol substrates,including cysteine, to form S-allylmercapto-L-cysteine. Alter-natively, allicin can further decompose to form allyl sulfenicacid and thioacrolein. Two molecules of allyl sulfenic acid can

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

2.2.2 Decomposition products. To date, there have beentwo main approaches to studying Allium aroma compounds.The first involves analysing volatile constituents generated upontissue damage in the headspace or alternatively preparing watermacerates and collecting the compounds present using solventextraction.

With regards to headspace volatiles, Allium tissues are ho-mogenised prior to GC or GC-MS analysis. Early studiesreported the occurrence of disulfides from chopped onion,with dipropyl disulfide (DPDS) accounting for the largestamount.46,47 Similarly, in chopped garlic the major headspacevolatile components are diallyl disulfide (DADS) and allylmethyl disulfide (AMDS).25,48 Recent analysis of onion volatilesby Kallio and Salorinne identified 27 separate aroma com-pounds, the most prominent of which were DPDS, methyl propyldisulfide (MPDS), 1-propenyl propyl disulfides ((E)- and (Z)-PPDS), methyl 1-propenyl disulfides ((E)- and (Z)-MPeDS), 1-propanethiol (PT), dipropyl trisulfide (DPTS), methyl propyltrisulfide (MPTS) (Fig. 9A).49 Later, Jarvenpaa et al., using thenovel Solid Phase Microextraction (SPME) technique coupledto GC-MS, determined the changes in volatile compoundcomposition in chopped onion.50 Initially, thiopropanal S-oxidewas the major sulfur compound identified in the head spacewith minor levels of DPDS and PPDS. Within 30 minutesthiopropanal S-oxide had disappeared and was replaced bydiprop(en)yl disulfides. These data indicate that the chemicalprofiles of Allium volatiles change dramatically with time.

With regards to the chemical components identified insolvent extracts, GC-MS analysis of methanolic preparationsof chopped garlic identified numerous mixed polysulifdes,including the parental thiosulfinate allicin, diallyl, di-, andtetrasulfides, and allylmethyl trisulfides. Many of these novelsulfides are generated through the thermal decompositionof 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.

Fig. 9 Sulfides identified in the headspace volatiles of crushed Alliumtissues: (A) onion volatiles, and (B) garlic volatiles.

thiosulfinate allicin is heated, as is likely during foodpreparation, sulfur dioxide, dimethyl trisulfide (DMTS), diallylmono-, di-, and trisulfides, and allylmethyl di- and trisulfidesare generated. Similar groups of mixed polysulfides have alsobeen identified in dichloromethane extracts of A. fistulosumvar. maichuon and A. fistulosum var. caespitosum.51 Thermaldecomposition reactions and products are also observed asmajor components of Allium oils, generated during the steamdistillation process.

During steam distillation, whereupon the tissues of theselected Allium species are heated to 100 ◦C, oils are gen-erated. Because thiosulfinates and disulfides formed duringchopping are thermally unstable, they decompose to formmixed polysulfides. To date, the distilled oils of onion andgarlic 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-MSchemical analysis has also revealed the presence of monosulfides,disulfides, and trisulfides in garlic essential oils. Indeed, 28components were reported in the study of Yu et al.53 Theessential oils obtained by steam distillation contained PT,1,2-epithiopropane (EP), methyl allyl sulfide (MAS), diallylsulfide (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 totalvolatiles. The relative abundance of these volatiles in Chinesechive extracts were AMDS (39.3%), followed by DMDS (15%)and DMTS (12.6%). The most abundant volatiles in the rakkyoextracts were MPTS (9.9%), DMDS (7.3%), DMTS (6%),and MPDS (5.5%). Among the 21 and 34 sulfur compoundsidentified in Chinese chive and rakkyo respectively, many havenot been previously been reported in these plants.54 Theseincluded some novel sulfide and polysulfides with ethyl, butyl,and pentyl groups.

All of the identified chemical constituents in oils are initiallyderived from the thermal decomposition of thiosulfinates. Theorigins of these sulfur components are best highlighted usingallicin derived from garlic as an example (Fig. 10A). Previousstudies have also shown that at room temperature allicin canrearrange to afford sulfur dioxide, and diallyl mono-, di andtrisulfides (Fig. 10B).25 Furthermore, the DADS formed canalso thermally decompose to produce at least 25 additionalreaction products.17 It was in the study of Block et al. that themechanism 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-ducted additional experimentation to identify the unknowncomponents. Heating pure diallyl disulfide at 150 ◦C for 10minutes afforded DAS, DATS, the cyclics 2VDN and 3VDN,and diallyl tetrasulfide (DATeS) as would be expected, inaddition to 29 other sulfur components (Fig. 10C).

2.2.3 Novel thiosulfinate-derived sulfur compounds. In ad-dition to the presence of thiosulfinates in fresh Allium ex-tracts, additional novel sulfur compounds have also beenidentified. In the studies of Kawakishi and Morimitsu,

methyl 1-(methylsulfinyl)propyl disulfide was isolated fromonion extracts.56 Furthermore, Wagner also reported thepresence of the cepaenes 1-((E)-1-propenylsulfinyl)propylpropyl disulfide and 1-((E)-1-propenylsulfinyl)propyl (E,Z)-1-propenyl disulfide in onions.57 The cepaenes have beenshown to have potent anti-inflammatory properties.58,59 Ad-ditional studies have now shown the presence of six newcepaenes in the chloroform extracts of onion.60 Furthermore,Calvey and colleagues identified methyl 1-(methylsulfinyl)propyldisulfide, 2-propenyl 1-(2-propenylsulfinyl) propyl disul-fide, methyl (E)-1-(1-propenylsulfinyl)propyl disulfide, and

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1-(1-methylsulfinyl)propyl (E,Z)-1-propenyldisulfide in super-critical fluid extracts of A. tricoccum.61 The cepaenes are formedfrom thiosulfinate intermediates through a mechanism proposedby Block.22 This proceeds through a reaction mediated bycarbophilic addition involves thiols and sulfenic acid deriva-tives. In addition, deoxycepaenes have also been identifiedin the oils of shallot (e.g. ethyl-1-(methylthio)propyl disulfideand methyl-1-(methylthio)propyl disulfide) and scallion (e.g. 1-propenyl-1-(propylthio)propyl disulfide).51 However, it is notknown whether these compounds arise from the deoxygenationof cepaenes or are formed during preparative or analyticalprocedures.22

A second group of very unusual sulfur compounds thatare likely to be derived from bis-a-b-unsaturated thiosulfinateshas also recently been identified in Allium extracts and shownto be biologically active. Two isomers of 2,3-dimethyl-5,6-dithiabicyclo[2.1.1]hexane 5-oxide were identified in onions,shallot, scallion and chive extracts.62–64 These compounds aretrivially named cis- and trans-zwiebelanes (Fig. 11A). Recently,research has focused on determining the mechanisms of for-mation of these novel compounds, the chemistry of which ishighly complex.63–66 Hopefully, in the future more informationpertaining to these chemical agents will be available. Moreover,a structurally similar (Z,Z)-(±)-2,3-dimethylbutanedithial S,S′-dioxide was been identified in onion extracts using NMRmethodologies and subsequently extracted (Fig. 11B). (Z,Z)-(±)-2,3-dimethylbutanedithial S,S′-dioxide is the first exampleof a bis(thial S-oxide).65,66

Fig. 11 Zwiebelanes and bis(thial S-oxide), novel Allium sulfurcompounds.

3 Allium vegetables and human health

Mammalian cells are continuously exposed to endogenousand exogenous chemicals, either as by-products of metabolismsuch as reactive oxygen species and lipid peroxides or en-vironmental agents like tobacco carcinogens. Many of thesecompounds are often recognised for their ability to react withkey cellular biomolecules, including DNA and proteins, whichcan disrupt normal cellular function. The consequence ofthese interactions, if not prevented or repaired, is the possibledevelopment of human pathologies including cardiovascular,cancer and inflammatory diseases. Fortunately, through thecourse of mammalian evolution, mechanism(s) have arisen tocope with the constant barrage of potential disease-inducingagents. These include the synthesis of intracellular antioxidants,e.g. glutathione, detoxification pathways coordinated by phaseII detoxification enzymes, glutathione-S-transferases (GST)[EC 2.5.1.18], quinone reductase (NQO1) [EC 1.6.99.2] andUGT-glucuronosyltransferases (UGDT) [EC 2.4.1.17], cell cycleregulatory proteins and programmed cell death. In addition,

dietary components that have antimicrobial or antioxidant-likeproperties may also play a significant role in the preventionof human pathologies. Not surprisingly, studies conductedthroughout the 1970s to the present have determined that manydietary botanicals and their phytochemical constituents canmodulate several of these protective pathways. Therefore, inorder to determine the potential beneficial effects of Alliumvegetables on human health it will be necessary to collate theavailable data from an array of both in vivo and in vitro studies. Itis with such knowledge that we can then appreciate the potentialrole of Allium species towards human health.

3.1 Anticarcinogenic properties

The diet plays a fundamental role in the etiology of humandisease. Lifestyle choices including alcohol consumption, highintake of dietary fats and smoking can increase the risk of cancerin humans. Examination of the available epidemiological evi-dence by Block et al. indicated that high dietary intake of fruitsand vegetables was correlated to a reduction in the prevalenceof several forms of human cancers.67 Statistically significantprotective effects were associated with fruit and vegetableconsumption in 128 of 156 dietary studies examined. In humanstudies in which Allium vegetable consumption (particularlygarlic and onions) were evaluated, a protective effect towardscancers was observed. In the review of the epidemiologicalliterature by Fleischauer and Arab, a high dietary intake of rawor cooked garlic was inversely associated with the developmentof stomach and colo-rectal cancers.68 This data was furthersupported by the recent study of Steinmetz et al., in which theconsumption of garlic was inversely associated with colon cancerrisk.69 Protective effects have also been observed in a Chinesestudy, in which a reduction in oesophageal and stomach cancerwere observed in individuals consuming Allium vegetables likeonions, Welsh onions, Chinese chives and garlic.70 Similarly,in a recent population-based, case-control study conducted inShanghai, China, an association between garlic, onion, scallion,chive and leek intake and reduced risk of prostate cancer wasfound.71 Onion consumption is also associated with reduced riskof developing brain cancer.72 In contrast, in the NetherlandsCohort Study (comprising 120 852 Dutch men and women agedbetween 55 and 69), Dorant et al. reported a strong associationbetween onion consumption and a reduction in the incidenceof stomach carcinoma.73 However, in the same study group theauthors could find no evidence for a protective effect of Alliumvegetable consumption and reduced risk of developing colon,rectum carcinoma, lung or female breast cancer.

In support of the epidemiological studies are the protective ef-fects observed for Allium vegetables towards chemically inducedcancers in rodent models. Benzo[a]pyrene, a carcinogen derivedfrom tobacco smoke, promotes neoplasia of the forestomachand lungs in female A/J mice that can be inhibited by allylmethyl trisulfide (AMTS).74 Likewise, DAS was shown toinhibit 1,2-dimethylhydrazine-induced hepatocarcinogenicity inmale Fischer 344 rats.75 Accordingly, based on these earlyreports, numerous publications have now determined Alliumphytochemicals to be potent inhibitors of chemically inducedtumours in rodent models. To date, DAS,76,77 MPDS,77 AMDS78

DATS, and their corresponding saturated propyl analogues,dipropyl sulfide (DPS) and DPDS,79 propylene sulfide (PS),and S-allylcysteine (SAC),80 have all been reported to reducethe levels of chemically induced tumours in rodent models.Considering the chemopreventative effects of Allium vegetablesand constituents, current studies are now revealing the molecularmechanism(s) for the observed prevention of carcinogenesis.

3.1.1 Enzymatic inhibition. Cytochrome P450 monoxyge-nases constitute a superfamily of enzymes involved in oxidationand reduction reactions of both endogenous and exogenouscompounds. It is now accepted that members of the CYP1A,1B and 2E subfamilies are responsible for the bioactivation of

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chemical pro-carcinogens to more toxic forms. Consequently,the generation of highly electrophilic intermediates that can bindto important nucleophilic sites is recognised to contribute tohuman pathologies. Therefore, modulation of enzymes involvedin the metabolism of carcinogens or toxic chemicals will influ-ence their toxicity and carcinogenicity potential.81 For example,cytochrome P450 2E1 catalyses the oxidation of numerousvolatile environmental carcinogens, while CYP2A6 is knownto be involved in nicotine oxidation and the bioactivation ofthe genotoxic N-nitrosamines.82,83 Therefore, phytochemicallytargeted inhibition of CYP450 members involved in the bioacti-vation of chemical carcinogens may prevent cellular damage inthis regard.

DAS, DASO and DASO2 are inhibitors of rat hepatic CYP4502E1 and inducers of CYP450 2B1 in vivo.84 Indeed, treatmentof rats with DAS before exposure to carbon tetrachlorideand N-nitrosodimethylamine prevented hepatotoxicity, perhapsdue to the inhibition of CYP450 2E1dependent bioactivationof procarcinogens. DAS can act as a competitive inhibitorand directly inactivate CYP450 2E1.85 Indeed, the effects ofdiallyl sulfoxide (DASO) and diallyl sulfone (DASO2) towardsacetaminophen-induced hepatotoxicity in mice would suggestthis to be the case.86,87 In the studies of Kwak et al., theeffects of allylsulfide (AS), S-allylmercaptan (SAM) and allylmethylsulfide (AMS) on the expression of CYP450 2E1 wereexamined in rats. CYP450 2E1 protein content was significantlydecreased and correlated with decreased enzymatic activity.88

Interestingly, rats treated with each compound showed nosignificant changes in the levels of CYP450 2E1 mRNAlevels, suggesting that post-transcriptional regulation may beassociated with the suppression of CYP2E1 apoprotein levels.Similarly, inhibition of CYP450-mediated bioactivation of thetobacco carcinogen benzo[a]pyrene by DAS, DADS and DATShas also been described. Analysis of the relative protein levelsfor the benzo[a]pyrene-inducible CYP450 1A2 enzyme wereobserved to be reduced, and corresponded with a reduction inthe levels of CYP450 benzo[a]pyrene metabolites.89

3.1.2 Enzymatic induction. Phase II detoxification en-zymes are recognised for their ability to facilitate the excretionof carcinogens from the body. Common to the majority of phaseII detoxification enzymes including GSTs, NQO1 and UGDTsis the presence of a conserved binding region, designatedthe antioxidant responsive element (ARE). Located withinthe promoter regions of each gene, this cis-acting regulatoryelement has been identified in the 5′-flanking region of GSTYa, hemoxygenase 1 (HO1), glutamyl cysteine synthetase andNQO1 genes of several mammalian species. During enzymaticinduction, a small basic leucine zipper protein (bZIP) designatedNrf-2 interacts with the ARE, promoting detoxification geneexpression. Nrf-2 is sequestered in the cytoplasm by a redox-sensitive protein known as Keap-1. Redox signalling promotesNrf-2 dissociation from Keap-1, allowing for its translocationto the nucleus, interaction with the ARE and the transcriptionalactivation of phase II detoxification enzymes.90 Indeed, it iscommon for several detoxification enzymes to be up-regulatedin a coordinated manner by the Nrf-2 signaling pathway.The import role Nrf-2 plays is highlighted by the observationthat mice null for Nrf-2(−/−) show reduced expression ofseveral detoxification enzymes and an increased sensitivity tocarcinogenesis and liver toxicity.91–94

The ability of synthetic and plant-derived Allium compoundsto induce cystolic quinone reductase NQO1 [NADP(H):quinonereductase oxidoreductase] and glutathione-S-transferases havebeen reported on several occasions. NQO1 is a 32 kDa FAD-containing flavoprotein that is necessary for the detoxificationof quinones to hydroquinones. The two-electron-mediated re-duction promotes the glucuronidation of hydroquinones andtheir excretion. NQO1 can also function in the detoxificationof several other carcinogens including quinone-imines, azo-

and nitro-compounds. Likewise, GSTs function in the detoxi-fication process by catalysing the conjugation of electrophilicspecies with the intracellular nucleophile glutathione (GSH) (c-glutamyl-cysteinyl-glycine).

DAS, DADS, and DATS (compounds derived from garlic),and DPS and DPDS (from onions), have been reported toincrease the activity of the phase II detoxification enzymesNQO1 and GSTs in a variety of rat tissues.95,96 These studiesidentified DATS, DADS and DPS as potent inducers of phaseII detoxification enzymes. More recently, the relative abilitiesof additional sulfides, including DPS, DAS, DPDS, DADS,DPTS, DATS, DPTeS and DATeS, to increase the activitiesof NQO1 and GST in rat organs have also been reported.DATS and DATeS were found to induce NQO1 and GST inthe liver, kidneys, and lungs of rat tissues while also increasingactivities in the duodenum, jejunum, ileum and cecum of thegastrointestinal tract. These studies suggest an important rolefor the allyl group in the ability of Allium sulfur compounds toinduce phase II detoxification enzymes.97 In 2004, the molecularmechanism(s) for the induction of the phase II detoxificationenzymes NQO1 and HO1 were determined for the major sulfidespresent in garlic (including DAS, DADS, and DATS) in an invitro human hepatoma model. Up-regulation of both NQO1 andHO-1 occurred through Nrf-2 activation and ARE-mediatedtranscription. As before, this study confirmed previous reportsthat the allyl functional group is more potent than that of thepropyl moiety for enzymatic induction.98

3.1.3 Apoptosis. Apoptosis, also known as programmedcell death, is a means by which living organisms controlabnormalities in cells that occur as a result of genetic or envi-ronmental cues. Characteristic changes including cell shrinkage,chromatin condensation, plasma membrane blebbing, DNAfragmentation and finally cell breakdown with the release ofapoptotic bodies are often observed during apoptosis. Theinitiation of apoptosis can occur by two major pathways. Firstly,interaction of extracellular ligands with membrane-bound re-ceptors (including the TNFa, TRAIL and FADD receptors,and also the death receptors such as DR5) leads to the initiationof an intracellular signal that promotes apoptosis. Secondly,apoptosis can be initiated by mitochondria, with the release ofapoptotic signaling molecules like cytochrome c, smac/Diabloand apoptosis inducing factor.99 Pivotal in the response ofcancer cells to apoptotic stimuli are the caspases. Caspasesare intracellular cysteine-containing proteases that cleave theirsubstrates after an aspartate residue in a tetrapeptide in asequence-specific manner.100 Caspases are found as pro-enzymesthat require activation to their proteolytic forms either throughcell surface death receptors, mitochondria or a convergence ofboth signaling pathways. Apoptotic cues induced cytochromec release from mitochondria, leading to the activation andformation of the apoptotic protease-activating factor-1 (APAF-1)–caspase-9 holoenzyme complex.101 Subsequently the forma-tion of the APAF-1–caspase-9 holoenzyme complex leads tocaspase-3 activation.102 Caspase-3 proteolytically degrades nu-merous cellular targets including poly(ADP-ribose) polymerase(PARP), Protein Kinase Cd, retinoblastoma protein, lamin, a-fodrin, DNAse and DNA fragmentation factor.103 Intracellulardegradation of many of these important protein substratesleads to the appearance of typical apoptotic morphology. Itis of interest that in numerous human pathological conditionsincluding cancers, that apoptotic signaling cascades are oftenimpaired. Indeed, Apaf-1, caspase-9 and caspase-3 null cell linesare highly resistant to apoptotic stimuli.104,105

Since the 1950s, scientists have been aware of the inhibitoryeffects of garlic and its components that are active againsttumour growth.106 However, it was not until the advent ofmodern techniques in cell tissue culture that major explorationswere conducted to determine their mechanisms of action. BothAllium extracts and their phytochemical constituents can induce

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apoptosis in several in vitro cell culture models. From theavailable data, activation of the proteolytic caspases, changes inintracellular redox homeostasis, generation of reactive oxygenspecies and the activation of stress signaling cascades are allimplicated in the apoptotic response of cancer cells to Alliumsulfur compounds. Studies have shown that the garlic-derivedS-allylmercaptocysteine (SAMC) can inhibit cell proliferationin human erythroleukaemia HEL and OCIM-1 cell lines byinducing apoptosis.107 Likewise, SAMC (but not SAC) canprevent human colon SW480 and HT29 cancer cell proliferationby inducing a G2/M cell cycle block and promoting apoptosis,through a caspase-3 mediated pathway.108 Xiao et al. latershowed, using immunofluorescent microscopy, that SAMCrapidly induces microtubule (MT) depolymerisation, MT cy-toskeleton disruption, centrosome fragmentation and Golgidispersion in interphase cells. Moreover, SAMC also causedthe formation of monopolar and multipolar spindles in mitoticcells in SW480 cells and NIH3T3 fibroblasts.109 It was thuspostulated that the antiproliferative effects of SAMC were dueto its interaction with tubulin, this leading to the activationof the stress-activated c-Jun NH(2)-terminal kinase (JNK) andactivation of a caspase-3 mediated apoptotic cascade.

Similarly, Allium-derived sulfides have also been shown to bepotent inducers of apoptosis in cancer cells. Human leukaemiaHL-60 cells exposed to DADS undergo apoptotic cell death,mediated by the activation of caspase-3 and cleavage of PARP.110

In addition, it was also reported that apoptosis was dependanton the generation of intracellular peroxides, as apoptosis couldbe blocked by the antioxidant enzyme catalase.111 Recently, anumber of sites within mitochondria have been proposed tobe important in production of reactive oxygen species (ROS),including complexes I, II, and III. It is feasible therefore, thatmodulation of mitochondrial integrity by Allium constituents islikely to contribute to apoptotic signaling. Indeed, pro-apoptoticbax and bid can interact with mitochondria to promote therelease of apoptotic signaling factors including cytochromec, smac/Diablo and apoptosis inducing factor. In contrast,anti-apoptotic members including bcl-2 and bclxL can preventmitochondrial perturbations and preserve cell viability. Recently,several studies have implicated a role of mitochondria in theresponse of cancer cells to Allium chemicals.

Both DATS and DADS induce apoptosis in cultured humanneoplastic (A549) and non-neoplastic (MRC-5) lung cancercells.111 Furthermore, Hong et al. confirmed that lung cancercells exposed to DAS and DADS caused an increase in the pro-apoptotic proteins p53 and bax, while reducing anti-apoptoticbcl-2 protein level in p53-wild type H460 and p53-null typeH1299 non-small-cell lung cancer cells.112 These data implicatea role of mitochondria in the apoptotic response of cancer cells toAllium sulfides. Additional research has also shown that DADScan induce growth arrest and apoptosis in neuroblastoma cellline SH-SY5Y. DADS promoted ROS generation and the releaseof mitochondrial cytochrome c prior to capsase-9 and capase-3activation in these cells.113 Moreover, following ROS generation,the stress-signaling kinase JNK was activated, followed bythe release of pro-apoptotic mitochondrial proteins. Again itwas observed that anti-apoptotic bcl-2 proteins were down-regulated. Interestingly, JNK inhibition reduced the levels ofapoptotic cell death in this model system. JNK activation leadsto the inactivation of the anti-apoptotic bcl-2 protein by amechanism involving phosphorylation. Indeed, the changes inthe ratios of anti- and pro-apoptotic proteins have been observedin PC-3 and DU145 human prostate cancer cells and humanbreast cancer cells.114 For example, DATS-induced apoptosisin PC-3 cells was associated with phosphorylation of bcl-2,altered bcl-2, bax interaction, and cleavage of procaspase-9and procaspase-3. JNK1 and/or JNK2 phosphorylation ofbcl-2 in DATS-treated PC-3 cells was blocked in the presenceof JNK-specific inhibitor SP600125 and prevented apoptosis.Again hydrogen peroxide was implicated as a possible signaling

molecule in DATS-induced apoptosis. Using PC-3 and DU145human prostate cancer cells as a model, DATS has beenshown to be a significantly more potent apoptosis inducerthan DAS or DADS.115 In hepatoma HepG2 cells, inhibitionof activated/phosphorylated mitogen-activated protein kinases(MAPK) p38 or ERK p42/44 enhances the DADS-inducedapoptosis, although the mechanism has yet to be determined.116

Ajoene, a major compound of garlic, induces apoptosis in hu-man leukaemic HL-60 cells, but not in peripheral mononuclearblood cells of healthy donors. Ajoene increased the productionof intracellular peroxide in a concentration and time-dependentfashion, which could be partially blocked by pre-incubation withthe antioxidant N-acetylcysteine.117 In the same cell line, (Z)-ajoene induced a time- and concentration-dependent increasein HL-60 cell apoptosis, a process involving the activation ofcaspase-3 and the cleavage of the anti-apoptotic protein Bcl-2.118

The thiosulfinate alliicin, also from garlic, inhibits the growthof cancer cells of murine and human origin. Allicin inducedthe formation of apoptotic bodies, nuclear condensation and atypical DNA ladder in cancer cells, as well as the activationof caspases-3, -8 and -9 and cleavage of PARP.119 A moreexciting avenue of research is using the clinical therapeuticdrug anti-CD20 mAb rituximab (Rituxan Mabthera), which isextensively in use in the treatment of non-Hodgkin lymphomas,in combination with the enzyme alliinase. By targeting alliinaseto CD20+ tumour cells the researchers found that when alliinwas added it promoted the in situ generation of the sulfurcompound allicin, which was able to kill CD20+ B-CLL cells aswell as other human B-cell lymphoma cell lines, both in vitroand in vivo.120 Indeed, the authors conclude that these newtechniques may offer a new, powerful and less toxic therapyfor B-CLL and other B-cell malignancies. Moreover, combiningalliinase with additional antibodies may extend the applicationof this technique to other conditions in which the elimination ofa specific cell population is required.

3.2 Anti-inflammatory properties

Over-expression of pro-inflammatory enzymes such as induciblenitric oxide synthetase (NOS) and cyclooxygenase-II (COX-II) are observed in numerous human pathologies includingcancer, cardiovascular and inflammatory diseases. Increasedactivity of inflammatory enzymes leads to the generation ofpro-inflammatory mediators including nitric oxide (NO) andprostaglandins (PG). Endogenous production of NO and PGhas a beneficial role in the maintenance of blood pressure,inflammation, wound healing and temperature regulation.However, overproduction leads to pathophysiological condi-tions such as the promotion of colon cancer, atherosclerosis,inflammatory bowel disorders, multiple sclerosis, Alzheimer’sdisease and septic shock.121–123 Both iNOS and COX-II proteinexpression are regulated by Nuclear factor kappa B (NF-jB), atranscription factor activated by carcinogens, toxins and oxida-tive stress. To date, NF-jB has been demonstrated to regulatethe transcription of over 150 separate genes, many of whichare involved in inflammation.124 Recent studies have shownthat antioxidants can inhibit NF-jB activation and thus reducethe symptoms of such debilitating disease states.125,126 Indeed,when we consider the association between Allium vegetableconsumption and reduced risk to cardiovascular diseases andcancers, many of the beneficial effects are perhaps due to theanti-inflammatory properties.

Early studies showed that garlic extracts could inhibit ADP,collagen, epinephrine, arachidonic acid (AA) and calciumionophore A23187-induced platelet aggregation in vitro.127

Platelet aggregation has been implicated in coronary heartdisease and stroke.128 The properties of the garlic extractswere reportedly due to an inhibitory effect in the degradationof platelet phospholipids, reduced formation of thrombox-ane (TxB2) and lipoxygenase-derived products from labelled

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platelets. In addition, garlic was also observed to inhibitcyclooxygenase and lipoxygenase enzymes.129 Similar findingsare also reported for extracts of onion.130

To date, few detailed studies have been conducted to de-termine the molecular mechanisms of Allium-mediated anti-inflammatory effects. Dirsch et al. reported that low concentra-tions of ajoene (IC50 2.5–5 lM) and allicin (IC50 15–20 lM) couldreduce nitrite accumulation in LPS-stimulated macrophages.131

Garlic and S-allylcysteine can inhibit NO production in murineRAW264.7 macrophage cells. The suppressive effects were dueto a reduction of iNOS mRNA and protein levels, followinglipopolysaccharide (LPS) and IFN-cstimulation. Garlic extractsalso inhibited NO production in peritoneal macrophages, rathepatocytes, and rat aortic smooth muscle cells stimulated withLPS and cytokines. The inhibitory effects were determined to bedue to the suppression of NF-jB signaling.132 Garlic extracthas also been identified as a potent inhibitor of leukocytemigration through endothelial cell monolayers, suggesting a pos-sible inhibitory role in the inflammatory processes.133 Likewise,garlic powder extract inhibits NF-jB activity in LPS-stimulatedhuman whole blood, reducing the levels of pro-inflammatorycytokines. These same properties could also be attributed toDADS that could also significantly reduce the secretion of theproinflammatory 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)-selectinin human umbilical endothelial cells (HUVECs). This suggeststhat the inhibitory effects of Allium-derived sulfur compoundson the NF-jB signaling pathway are structurally dependant.135

3.3 Antioxidant properties

Antioxidants are of interest to pharmacologists and biochemistsbecause they are reported to inhibit the damaging effects causedby free-radical agents, including reactive oxygen, nitrogen, andchlorine species (ROS, RNS, and RClS). Due to the apparentformation of these toxic entities at sites of tissue injury, freeradicals can alter the structural and functional integrity of cellsby promoting intracellular redox changes including protein ox-idation, lipid peroxidation, oxidative DNA damage and proteincarbonyl formation. Therefore, considerable interest in the iden-tification of natural and synthetic antioxidants that can interferewith 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 reportedto have potent reducing abilities when examined by the 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH) scavengingassay.136 Aqueous extracts of garlic can also prevent copper-induced lipid peroxidation in vitro.137,138 In the study of Rajasreeet al., garlic and onion extracts were shown to reduce nicotine-induced lipid peroxidation, as assessed by measuring thiobar-bituric acid reactive substance, conjugated diene and hydroper-oxide concentrations in the tissues of nicotine-treated rats.139

The inhibitory effects appear to be associated with increasedactivities of the antioxidant enzymes catalase, superoxide dis-mutase and glutathione peroxidase, and increased intracellularglutathione content. With regard to individual phytochemicalconstituents, several have been shown to act as potent free-radical scavengers and prevent cellular damage. Xiao et al.recently concluded that the antioxidant potential of crude Alliumextracts is probably due to additional chemical componentsrather than thiosulfinates. This assumption was made basedon the fact that the thiosulfinates were poorer antioxidantscavengers than conventional antioxidants like ascorbic acid,trolox and glutathione.140

Several organosulfur compounds identified in Allium speciesdo have antioxidant properties. SAC and SAMC, the majororganosulfur compounds found in aged garlic extract, canreduce lipid peroxidation and 1,1-diphenyl-2-picrylhydrazyl(DPPH) assays.141 In addition, hydrogen peroxide formation in-duced by oxidised low-density lipoprotein (ox-LDL) in activated

J774 murine macrophages and human umbilical endothelialcells (HUVECs) can be abolished by treatment with SAC.142

Allicin is a potent hydroxyl radical scavenger.143 Oxidation andglycation of human LDL can be inhibited by DAS, DADS,S-ethylcysteine (SEC) and N-acetylcysteine (NAC). All fourderivatives found in garlic can inhibit superoxide productiongenerated by xanthine-xanthine oxidase while also being ableto chelate copper ions.144 These findings are further supportedby the studies that show DAS, DADS, SAC, SEC, SMC, S-methylcysteine and S-propylcysteine are able to prevent theoxidation and glycation of LDL in the plasma isolated frompatients with non-insulin-dependent diabetes.145 In our labo-ratory we recently demonstrated that the novel 3-mercapto-2-methylpentan-1-ol (3-MP), of which four possible diastereoiso-mers can occur in varying amounts in A. cepa (Fig. 12),significantly inhibited peroxynitrite-mediated tyrosine nitrationand inactivation of a(1)-antiproteinase. Moreover, 3-MP also in-hibited peroxynitrite-induced cytotoxicity, intracellular tyrosinenitration, and intracellular reactive oxygen species.146,147

Fig. 12 The biosynthetic pathway for the production of 3-mer-capto-2-methylpentan-1-ol in Allium species remains largely unknown,although a hypothetical pathway for the formation of this potentantioxidant has been suggested (adapted from Widder et al.147).

3.4 Antimicrobial properties

The antimicrobial properties of Allium species have been thefocus of several recent review articles and therefore we willonly provide brief description herein.148,149 In common folkloremedicine, Allium plants have long been associated with thetreatment of infections.150 It is apparent from recent chemicalcharacterisation that the therapeutic effects, particularly withregards to the antimicrobial properties, are due to constituentsderived from the parental CSs. Over the last century, garlic hasbeen proven to be effective against both gram-positive, gram-negative and acid-fast bacteria. These include Pseudomonas,Proteus, Escherichia coli, Staphylococcus aureus, Klebsiella,Salmonella, Micrococcus, Bacillus subtilis, Mycobacterium, andClostridium.151–156 Furthermore, garlic also inhibits the growth ofstrains of S. aureus, E. coli, Proteus mirabilis and Pseudomonasaeruginosa that are multiply resistant to antibiotics includingpenicillin, streptomycin, doxycilline and cephalexin.157 Similarly,the bactericidal effects of extracts of onion towards Streptococ-cus mutans and S. sobrinus, the main causal bacteria for dentalcaries, and Porphyromonas gingivalis and Prevotella intermedia,

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considered to be the main causal bacteria of adult periodontitis,have been described.158

Recent work has been conducted to determine the antimicro-bial properties of additional Allium sulfur constituents. Cavillatoand Bailey discovered that the major antibacterial agent presentin garlic was allicin.151 The inhibitory effect of garlic extract,DAS and DADS against methicillin-resistant S. aureus (MRSA)infection in BALB/cA mice was studied. The oral admin-istration of these agents significantly decreased the viabilityof MRSA in plasma, liver, kidney and spleen. Indeed, theauthors conclude that DAS and DADS are potential therapeuticagents for the treatment of MRSA infection.159 The thiosul-finates 2-propene-1-sulfinothioic acid S-(Z,E)-1-propenyl es-ter [AllS(O)SPn-(Z,E)], 2-propenesulfinothioic acid S-methylester [AllS(O)SMe], and methanesulfinothioic acid S-(Z,E)-1-propenyl ester [MeS(O)SPn-(Z,E)] isolated from oil-maceratedgarlic also have antimicrobial activity.160 1-Methyl methanethio-sufinate and S-methyl-2-propene-1-thiosulfinate isolated fromChinese chive (A. tuberosum), both showed significant antibac-terial activity against E. coli O-157:H7.161 Ajoene shows abroad spectrum of antimicrobial activity towards the growthof gram-positive bacteria, including Bacillus cereus, Bacillussubtilis, Mycobacterium smegmatis, and Streptomyces griseus.The disulfide bond in ajoene appears to be necessary for theantimicrobial activity, since reduction by cysteine, which reactswith disulfide bonds, destroyed its antimicrobial activity.162

An exciting finding with regards to Allium sulfur compoundsis their activity towards Helicobacter pylori. H. pylori infectionis intimately involved in stomach cancer development. Recentepidemiological studies have indicated that the consumption ofAllium vegetables can reduce the risk of gastric neoplasia.163

Chung and colleagues reported that the garlic oil componentsDAS or DADS had bacteriocidal affects towards H. pylorigrowth.164 Furthermore, garlic extracts can inhibit H. pylori-induced gastritis in Mongolian gerbils and has been suggestedas a useful agent in the prevention of H. pylori pathologies.165

The crude methanol extract of the leaf tissues of A. ascalonicumalso have antibacterial activity towards H. pylori strains (ATCC24376, UCH 97001, UCH 97009, UCH 98026 and UCH99039).166 Likewise, allicin, allylmethyl- and methylallylthiosul-finate isolated from acetonic garlic extracts inhibit the in vitrogrowth of H. pylori.167

3.5 Antifungal and antiparasitic properties

Many fungi have proven susceptible to Allium extracts, partic-ularly those of garlic. These included Candida, Trichophyton,Torulopsis, Rhodotorula, Cryptococcus, Aspergillus, and Tri-chosporon.168–170 More recently, the in vitro activities of garlic oil,Chinese leek oil and four diallyl sulfides towards three Candidaspecies and three Aspergillus species were also determined.The antifungal activities of the garlic and Chinese leek oilextracts were shown to be dependant on the relative sulfideconcentration. Moreover, of the four diallyl sulfides examined,the antifungal activity followed the order of DATeS > DATS >

DADS > DAS.171 The antifungal properties observed for Alliumextracts and sulfur compounds appear to be associated withtheir ability to reduce the growth of and inhibit lipid, proteinand nucleic acid synthesis.172

With regards to the use of Allium species and their constituentsas antiprotozoals, only a few reports have been published. DATScan inhibit metabolism or growth of parasites, particularly Try-panosoma brucei ssp. brucei, ssp. rhodesiense, ssp. gambiense, ssp.evansi, ssp. congolense and ssp. equiperdum, as well as Entamoebahistolytica and Giardia lamblia.173 Whole garlic (A. sativum)extracts, allyl alcohol and AM are inhibitory towards themicroaerophilic flagellate protozoon Giardia intestinalis, a par-asitic species responsible for causing waterborne diarrhoea.174

Antileishmanial effects of aqueous onion (A. cepa) extractstowards leishmanial promastigotes has also been reported. Five

strains of Leishmania including L. major, L. major (Pakistan),L. tropica, L. mexicana ssp. mexicana and L. donovani werefound to be sensitive to onion juice. The authors suggest that thesusceptibility of the Leishmania strains is a likely consequenceof the sulfur compounds present within the onion extract.175

3.6 Cardiovascular disease

Garlic extracts as well as several additional Allium organosulfurcompounds have hypocholesterolemic effects in human andanimal models.176–179 Gebhardt demonstrated that water-solublegarlic extracts reduced cholesterol biosynthesis in hepato-cytes when exposed to the radiolabelled precursor compound[14C]-acetate, indicating a potential regulation of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase.180 However, themechanism(s) by which garlic regulates HMG-CoA reductaseactivity has yet to be fully elucidated. Recently, Augusti et al.reported that rats fed with a polar fraction of garlic oil couldinhibit the hyperlipidemic and elevated tissue levels of HMG-CoA reductase.181 Ajoene is known to be a potent inhibitorof arterial smooth muscle proliferation.182 Moreover, chronictreatment of rats with garlic extracts is protective againstisoprenaline-induced cardiac necrosis.183

In a recent study by Miron et al., a novel antihypertensive drugwas synthesised through the reaction of the pharmaceutical drugCaptopril with the Allium-derived thiosulfinate allicin (Fig. 13).Captopril possesses potent antihypertensive properties due to itsability to inhibit the angiotensin-converting enzyme. Likewise,allicin also inhibits hypertension by reducing serum cholesteroland triglyceride levels. Therefore, the authors proposed thata combination of both drugs, each working at two separatepharmacological sites of action, may provide better protectionagainst hypertension. The reaction product between captopriland allicin was termed allylmercaptocaptopril (CPSSA), a non-symmetric disulfide that combines the specific drug activity ofcaptopril and the beneficial properties of allicin. In fructose-induced hypertensive rats, CPSSA decreased triglyceride levelsand significantly lowered blood pressure.184 In a rat myocardialinfarct study, garlic oil has also been shown to exert its protectiveeffects by modulating lipid peroxidation and enhancing an-tioxidant and detoxifying enzyme systems185 (see Sections 3.1.1and 3.3). The antioxidant properties of several chemical groupsof natural products served as one of the important factors toprotect against ischemic myocardium in vivo.186 Garlic extractscontain large amounts of sulfhydryl-containing amino acids,notably cysteine and the S-alk(en)yl derivatives such as SAC,SEC and SPC.179 Interestingly, cysteine can be metabolised togenerate hydrogen sulfide (H2S) via the catabolic action of theenzymes cystathionine c-lyase and cystathionine b-synthetase.Recently, H2S has been shown to be a novel gasomediator in thecardiovascular system.187 Consequently, it is conceivable that ad-ministration of chemically modified cysteine analogues, particu-larly those found in Allium species, may influence the formationof endogenous H2S in the heart and/or vasculature. However, apotential therapeutic role of garlic or indeed other Allium speciestowards cardiovascular disease requires more research.

Fig. 13 Synthesis of the potent antihypertensive drug Allylmercapto-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 intracellularantioxidant glutathione (SR), (B) Metabolic oxidation products of diallyl disulfide.

3.7 Metabolism of Allium sulfur compounds

There is ample evidence showing the potential beneficial prop-erties of Allium sulfur compounds on human health. However,few studies have dealt with the metabolism or bioavailabilityof these compounds. Undoubtedly, such data would give agreater understanding of the role of Allium phytochemicalsin human health, and more importantly identify biomarkersthat could be used to determine an individual’s exposure tosuch Allium sulfur compounds. Metabolism of mono-sulfides,including DAS, in rat models occurs via sequential oxidation thatleads to the formation of the sulfoxide prior to the formation ofthe sulfone (Fig. 14A).188,189 Moreover, Jin et al. have also shownthat DAS can readily conjugate to the intracellular antioxidantglutathione.190 The thiol reactivity of Allium-derived sulfides isparticularly interesting, as this can result in the formation of S-mercaptoconjugates such as SAMC. Moreover, recent evidencehas shown that the cysteine conjugates SAC and SAMC aresubstrates for the mammalian b-lyase c-cystathionase in rat liverhomogenates. However, the biological significance of this hasyet to be determined.191 Similar results have also been obtainedfor the metabolism of dipropyl sulfide and dipropyl sulfoxidein the rat. Disulfides, such as DADS, are readily absorbed andaccumulate in the liver within 90 minutes.192 DADS is convertedto AM and allyl methyl sulfide as determined in ex vivo metabolicmodels using perfused rodent liver or rat hepatocytes.193,194

Germain and colleagues have also reported that a single doseof DADS in rats led to the formation of AM and AMS, as wellas two oxidative metabolites, allyl methyl sulfoxide (AMSO) andallyl methyl sulfone (AMSO2) (Fig. 14B).195

4 Summary

Allium species are an agronomically important genus becauseof their sulfur flavour components. The majority of the volatileflavour principles are generated through the enzymatic hydrol-ysis of the non-volatile CS storage compounds. Furthermore,these compounds may be possible sources of new novel therapeu-tic principles. Allium compounds are reported to be effective inthe prevention of numerous disease states in humans, includingcancer, cardiovascular and inflammatory disorders. These traits

are perhaps due to the ability of Allium sulfur compoundsto positively modify the antioxidant, apoptotic, inflammatory,and cardiovascular systems in mammalian systems. However,much remains to be done in each of these research areas,thus presenting a challenge for future studies by chemists, drugdevelopment researchers and the pharmaceutical industry.

5 Abbreviations

ACSO, (+)-S-allyl-L-cysteine sulfoxide (alliin); AM, allyl mer-captan; AMDS, allyl methyl disulfide; AMS, allyl methyl sulfide;AMSO, allyl methyl sulfoxide; AMSO2, allyl methyl sulfone;AMTS, allyl methyl trisulfide; AS, allyl sulfide; BCSO, S-n-butyl-L-cysteine sulfoxide; CS, S-alk(en)yl cysteine sulfoxides;DADS, diallyl disulfide; DAS, diallyl sulfide; DASO, diallylsulfoxide; DASO2, diallyl sulfone; DATeS, diallyl tetrasulfide;DATS, diallyl trisulfide; DMTS, dimethyl trisulfide; DPDS,dipropyl disulfide; DPS, dipropyl sulfide; DPTS, dipropyltrisulfide; ECSO, S-ethyl-L-cysteine sulfoxide (ethiin); EP,1,2-epithiopropane; GSH, glutathione (c-glutamyl-cysteinyl-glycine); LPS, lipopolysaccharide; MAS, methyl allyl sulfide;MCSO, (+)-S-methyl-L-cysteine sulfoxide (methiin); 3-MP, 3-mercapto-2-methylpentan-1-ol; MPDS, methyl propyl disulfide;MPeDS, methyl 1-propenyl disulfides (E and Z); MPTS, methylpropyl trisulfide; NAC, N-acetylcysteine; NF-jB, nuclear factorkappa B; PARP, poly(ADP-ribose) polymerase; PCSO, (+)-S-propyl-L-cysteine sulfoxide (propiin); PPDS, 1-propenyl propyldisulfides (E and Z); PS, propylene sulfide; PT, 1-propanethiol;RClS, reactive chlorine species; ROS, reactive oxygen species;RNS, reactive nitrogen species; SAC, S-allylcysteine; SAM, S-allylmercaptan; SEC, S-ethylcysteine; SPC, S-propylcysteine;THDMP, tetrahydro-2,5-dimethylthiophene; TPCSO, (+)-S-trans-1-propenyl-L-cysteine sulfoxide (isoalliin); 2VDN, 2-vinyl-[4H]-1,3-dithin; 3VDN, 3-vinyl-[4H]-1,2-dithin.

6 References

1 G. R. Fenwick and A. B. Hanley, Crit. Rev. Food Sci. Nutr., 1985,22(3), 199–271.

2 E. Block, Sci. Am., 1985, 252(3), 114–119.3 A. Stoll and E. Seebeck, Experentia, 1947, 3, 114–115.

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4 A. L. Virtanen and E. J. Matikkala, Acta Chem. Scand., 1959, 13,1898–1900.

5 A. L. Virtanen and C. G. Spare, Suom. Kemistl., 1961, 34, 72.6 H. P. Koch, in: Garlic: the Science and Therapeutic Application of

Allium sativum L. and Related Species, ed. H. P. Koch and L. D. Law-son, Williams & Wilkins, Baltimore, 1996, 213–220.

7 I. Krest, J. Glodek and M. Keusgen, J. Agric. Food Chem., 2000,48(8), 3753–3760.

8 K. Tsuge, M. Kataoka and Y. Seto, J. Agric. Food Chem., 2002,50(16), 4445–4451.

9 R. Kubec, M. Svobodova and J. Velisek, J. Chromatogr., A., 1999,862(1), 85–94.

10 R. Kubec, M. Svobodova and J. Velisek, J. Agric. Food Chem., 1999,47(3), 1132–1138.

11 R. Kubec, M. Svobodova and J. Velisek, J. Agric. Food Chem., 2000,48(3), 428–433.

12 R. Kubec, S. Kim, D. M. McKeon and R. A. Musah, J. Nat. Prod.,2002, 65(7), 960–964.

13 L. Horhammer, H. Wagner, M. Seitz and Z. Vejdelek, Pharmazie,1968, 23, 462–467.

14 H. Hesse, V. Nikiforova, B. Gakiere and R. Hoefgen, J. Exp. Bot.,2004, 55(401), 1283–1292.

15 T. Leustek, M. N. Martin, J. A. Bick and J. P. Davies, Annu. Rev.Plant Physiol. Plant Mol. Biol., 2000, 51, 141–165.

16 R. Whitaker, Adv. Food Res., 1976, 22, 73–133.17 T. Suzuki, M. Sugii and T. Kakimoto, Chem. Pharm. Bull., 1962,

10, 328–331.18 B. Granroth, Ann. Acad. Sci. Fenn., Ser. A., 1970, 154, 1–71.19 R. J. Parry and G. R. Sood, J. Am. Chem. Soc., 1989, 111, 4514–

4515.20 R. J. Parry and F.-L. Lii, J. Am. Chem. Soc., 1991, 113, 4704–

4706.21 J. E. Lancaster and M. L. Shaw, Phytochemistry, 1989, 28, 455–460.22 E. Block, Angew. Chem., Int. Ed. Engl., 1992, 31, 1135–1178.23 M. G. Jones, J. Hughes, A. Tregova, J. Milne, A. B. Tomsett and

H. A. Collin, J. Exp. Bot., 2004, 55(404), 1903–1918.24 J. E. Lancaster and H. A. Collin, Plant. Sci. Lett., 1981, 22, 169–176.25 M. H. Brodnitz and J. V. Pascale, J. Agric. Food Chem., 1971, 19(2),

269–272.26 L. P. Nock and M. Mazelis, Plant Physiol., 1987, 85, 1079–1083.27 T. Manabe, A. Hasumi, M. Sugiyama, M. Yamazaki and K. Saito,

Eur. J. Biochem., 1998, 257, 21–30.28 J. Landshuter, E. M. Lohmuller and K. Knobloch, Planta Med.,

1994, 60, 343–347.29 M. Mazelis and R. K. Creveling, Biochem. J., 1975, 147, 485–491.30 M. Mazelis and L. Crews, Biochem. J., 1968, 108, 725–730.31 E. M. Lohmuller, J. Landshuter and K. Knobloch, Planta Med.,

1994, 60, 337–342.32 E. B. Kuettner, R. Hilgenfeld and M. S. Weiss, Arch. Biochem.

Biophys., 2002, 402(2), 192–200.33 E. B. Kuettner, R. Hilgenfeld and M. S. Weiss, J. Biol. Chem., 2002,

277(48), 46402–46407.34 T. W. Coolong and W. M. Randle, J. Am. Soc. Hortic. Sci., 128,

776–783.35 J. E. Lancaster, M. L. Shaw and W. M. Randle, J. Sci. Food Agric.,

1998, 78, 367–372.36 I. Krest, J. Glodek and M. Keusgen, J. Agric. Food Chem., 2000,

48(8), 3753–3760.37 E. J. M. Van Damme, K. Smeets, S. Torrekens, F. Van Leuven and

W. J. Peumans, Eur. J. Biochem., 1992, 209, 751–757.38 S. A. Clark, PhD Dissertation, 1993, University of Canterbury,

Christchurch, New Zealand.39 B. J. Gilpin, D. W. Leung and J. E. Lancaster, Plant Physiol., 1995,

110, 336.40 Rabinkov, X. Z. Zhu, G. Grafi and D. Mirelman, Appl. Biochem.

Biotechnol., 1994, 48, 149–171.41 N. Kitamura, N. Shimomura, J. Iseki, M. Honma, S. Chiba, S.

Tahara and J. Mizutani, Biosci., Biotechnol., Biochem., 1997, 61(8),1327–1330.

42 C. Shen, H. Xiao and K. L. Parkin, J. Agric. Food Chem., 2002,50(9), 2644–2651.

43 E. Block, S. Naganathan, D. Putman and S.-H. Zhao, J. Agric. FoodChem., 1992, 1002(40), 2410–2430.

44 C. Shen, Z. Hong and K. L. Parkin, J. Agric. Food Chem., 2002,50(9), 2652–2659.

45 C. Shen and K. L. Parkin, J. Agric. Food Chem., 2000, 48(12), 6254–6260.

46 R. A. Bernhard, A. R. Saghir, J. V. Jacobsen and L. K. Mann, Arch.Biochem. Biophys., 1964, 107, 137–140.

47 A. R. Saghir, L. K. Mann, R. A. Bernhard and J. V. Jacobsen, Proc.Am. Soc. Hortic. Sci., 1964, 84, 386–398.

48 D. M. Oaks, H. Hartmann and K. P. Dimick, Anal. Chem., 1964,36, 1560–1565.

49 H. Kallio and L. Salorinne, J. Agric. Food Chem., 1990, 38, 1560–1564.

50 E. P. Jarvenpaa, Z. Zhang, R. Huopalahti and J. W. King,Z. Lebensm.-Unters.-Forsch. A., 1998, 207, 39–43.

51 M. C. Kuo, M. Chien and C. T. Ho, J. Agric. Food Chem., 1990,38(3), 1378–1381.

52 F. W. Semmler, Arch. Pharm. (Weinheim, Ger.), 1892, 230, 434–443.53 T. H. Yu, C. M. Wu and Y. C. Liou, J. Agric. Food Chem., 1989, 37,

725–730.54 J. A. Pino, V. Fuentes and M. T. Correa, J. Agric. Food Chem., 2001,

49(3), 1328–1330.55 E. Block, R. Iyer, I. Grisoni, C. Saha, S. Belman and F. P. Lossing,

J. Am. Chem. Soc., 1988, 110, 7813–7827.56 S. Kawakishi and Y. Morimitsu, Lancet, 1988, 2(8606), 330.57 T. Bayer, H. Wagner, V. Wray and W. Dorsch, Lancet, 1988, 2(8616),

906.58 W. Dorsch, E. Schneider, T. Bayer, W. Breu and H. Wagner, Int.

Arch. Allergy Appl. Immunol., 1990, 92(1), 39–42.59 H. Wagner, W. Dorsch, T. Bayer, W. Breu and F. Willer,

Prostaglandins, Leukotrienes Essent. Fatty Acids, 1990, 39(1), 59–62.60 T. Bayer, W. Breu, O. Seligmann, V. Wray and H. Wagner,

Phytochemistry, 1989, 28(9), 2313–2371.61 E. M. Calvey, K. D. White, J. E. Matusik, D. Sha and E. Block,

Phytochemistry, 1998, 49(2), 359–364.62 T. Bayer, H. Wagner, E. Block, S. Grisoni and S. H. Zhao, J. Am.

Chem. Soc., 1989, 111, 3085–3086.63 E. Block, T. Bayer, S. Naganathan and S. H. Zhao, J. Am. Chem.

Soc., 1996, 118, 2799–2810.64 E. Block, M. Thiruvazhi, P. J. Toscano, T. Bayer, S. Grisoni and

S. H. Zhao, J. Am. Chem. Soc., 1996, 118, 2790–2798.65 E. Block and T. Bayer T, J. Am. Chem. Soc., 1990, 112, 4584–4585.66 F. Freeman, C. N. Angeletakis, W. Pietro and W. J. Hehre, J. Am.

Chem. Soc., 1982, 104, 1161–1165.67 G. Block, B. Patterson and A. Subar, Nutr. Cancer, 1992, 1, 1–29.68 A. T. Fleischauer and L. Arab, J. Nutr., 2001, 131(3s), 1032S–1040S.69 K. A. Steinmetz, L. H. Kushi, R. M. Bostick, A. R. Folsom and

J. D. Potter, Am. J. Epidemiol., 1994, 139(1), 1–15.70 C. M. Gao, T. Takezaki, J. H. Ding, M. S. Li and K. Tajima,

Jpn. J. Cancer Res., 1999, 90(6), 614–621.71 A. W. Hsing, A. P. Chokkalingam, Y. T. Gao, M. P. Madigan, J.

Deng, G. Gridley and J. F. Fraumeni, Jr., J. Natl. Cancer Inst.,2002, 94(21), 1648–1651.

72 J. Hu, C. La Vecchia, E. Negri, L. Chatenoud, C. Bosetti, X. Jia,R. Liu, G. Huang, D. Bi and C. Wang, Int. J. Cancer, 1999, 81(1),20–23.

73 E. Dorant, P. A. van den Brandt, R. A. Goldbohm and F. Sturmans,Gastroenterology, 1996, 110(1), 12–20.

74 V. L. Sparnins, A. W. Mott, G. Barany and L. W. Wattenberg, Nutr.Cancer, 1986, 8(3), 211–215.

75 M. A. Hayes, T. H. Rushmore and M. T. Goldberg, Carcinogenesis,1987, 8(8), 1155–1157.

76 M. J. Wargovich, Carcinogenesis, 1987, 8(3), 487–489.77 V. L. Sparnins, G. Barany and L. W. Wattenberg, Carcinogenesis,

1988, 9(1), 131–134.78 L. W. Wattenberg, V. L. Sparnins and G. Barany, Cancer Res., 1989,

49(10), 2689–2692.79 S. K. Srivastava, X. Hu, H. Xia, H. A. Zaren, M. L. Chatterjee, R.

Agarwal and S. V. Singh, Cancer Lett., 1997, 118(1), 61–67.80 H. Sumiyoshi and M. J. Wargovich, Cancer Res., 1990, 50(16), 5084–

5087.81 C. Ioannides and D. F. Lewis, Curr. Top. Med. Chem., 2004, 4(16),

1767–1788.82 F. P. Guengerich, D. H. Kim and M. Iwasaki, Chem. Res. Toxicol.,

1991, 4(2), 168–179.83 O. Pelkonen, A. Rautio, H. Raunio and M. Pasanen, Toxicology,

2000, 144(1–3), 139–147.84 J. F. Brady, M.-H. Wang, J.-Y. Hong, F. Xiao, Y. Li, J.-S. Yoo, S. M.

Ning, M.-J. Lee, J. M. Fukuto, J. M. Gapac and C. S. Yang, Toxicol.Appl. Pharmacol., 1991, 108(2), 342–354.

85 J. F. Brady, H. Ishizaki, J. M. Fukuto, M. C. Lin, A. Fadel, J. M.Gapac and C. S. Yang, Chem. Res. Toxicol., 1991, 4(6), 642–647.

86 E.-J. Wang, Y. Li, M. Lin, L. Chen, A. P. Stein, K. R. Reuhl andC. S. Yang, Toxicol. Appl. Pharmacol., 1996, 136(1), 146–154.

87 J. J. Hu, J. S. Yoo, M. Lin, E. J. Wang and C. S. Yang, Food Chem.Toxicol., 1996, 34(10), 963–969.

88 M. K. Kwak, S. G. Kim, J. Y. Kwak, R. F. Novak and N. D. Kim,Biochem. Pharmacol., 1994, 47(3), 531–539.

89 H. S. Chun, H. J. Kim and E. H. Choi, Biosci., Biotechnol., Biochem.,2001, 65(10), 2205–2212.

3 6 6 N a t . P r o d . R e p . , 2 0 0 5 , 2 2 , 3 5 1 – 3 6 8

Publ

ishe

d on

10

May

200

5. D

ownl

oade

d by

CO

NR

iCY

T o

n 16

/10/

2015

11:

51:0

9.

View Article Online

90 M. K. Kwak, N. Wakabayashi and T. W. Kensler, Mutat. Res., 2004,555(1–2), 133–148.

91 Y. Aoki, H. Sato, N. Nishimura, S. Takahashi, K. Itoh and M.Yamamoto, Toxicol. Appl. Pharmacol., 2001, 173(3), 154–160.

92 S. A. Chanas, Q. Jiang, M. McMahon, G. K. McWalter, L. I.McLellan, C. R. Elcombe, C.J. Henderson, C.R. Wolf, G.J. Moffat,K. Itoh, M. Yamamoto and J.D. Hayes, Biochem. J., 2002, 365(2),405–416.

93 M. Ramos-Gomez, M.-K. Kwak, P. M. Dolan, K. Itoh, M.Yamamoto, P. Talalay and T. W. Kensler, Proc. Natl. Acad. Sci.U. S. A., 2001, 98(6), 3410–3415.

94 K. Chan, X. D. Han and Y. W. Kan, Proc. Natl. Acad. Sci. U. S. A.,2001, 98(8), 4611–4616.

95 D. Guyonnet, M. H. Siess, A. M. Le Bon and M. Suschetet, Toxicol.Appl. Pharmacol., 1999, 154(1), 50–58.

96 R. Munday and C. M. Munday, Nutr. Cancer, 1999, 34(1), 42–48.97 R. Munday, J. S. Munday and C. M. Munday, Free Radical Biol.

Med., 2003, 34(9), 1200–1211.98 C. Chen, D. Pung, V. Leong, V. Hebbar, G. Shen, S. Nair, W. Li and

A.-N. T. Kong, Free Radical Biol. Med., 2004, 37(10), 1578–1590.99 S. Gupta, Life Sci., 2001, 69, 2957–2964.

100 T. Fernandes-Alnemri, G. Litwack and E. S. Alnemri, J. Biol. Chem.,1994, 269, 30761–30764.

101 H. Zou, W. J. Henzel, X. Liu, A. Lutschg and X. Wang, Cell, 1997,90, 405–413.

102 G. M. Cohen, Biochem. J., 1997, 326, 1–16.103 M. Woo, A. Hakem, A. J. Elia, R. Hakem, G. S. Duncan, B. J.

Patterson and T. W. Mak, J. Immunol., 1999, 163, 4909–4916.104 M. S. Soengas, R. M. Alarcon and H. Yoshida, Science, 1999, 284,

156–159.105 A. S. Weisberger and J. Pensky, Science, 1957, 126(3283), 1112–

1114.106 J. A. Dipaolo and C. Carruthers, Cancer Res., 1960, 20, 431–434.107 G. Sigounas, J. L. Hooker, W. Li, A. Anagnostou and M. Steiner,

Nutr. Cancer, 1997, 28(2), 153–159.108 H. Shirin, J. T. Pinto, Y. Kawabata, J. W. Soh, T. Delohery, S. F.

Moss, V. Murty, R. S. Rivlin, P. R. Holt and I. B. Weinstein, CancerRes., 2001, 61(2), 725–731.

109 D. Xiao, J. T. Pinto, J.-W. Soh, A. Deguchi, G. G. Gundersen, A. F.Palazzo, J.-T. Yoon, H. Shirin and I. B. Weinstein, Cancer Res.,2003, 63(20), 6825–6837.

110 K.-B. Kwon, S.-J. Yoo, D.-G. Ryu, J.-Y. Yang, H.-W. Rho, J.-S. Kim,J.-W. Park, H.-R. Kim and B.-H. Park, Biochem. Pharmacol., 2002,63(1), 41–47.

111 K. Sakamoto, L. D. Lawson and J. A. Milner, Nutr. Cancer, 1997,29(2), 152–156.

112 Y. S. Hong, Y. A. Ham, J. H. Choi and J. Kim, Exp. Mol. Med.,2000, 32(3), 127–134.

113 G. Filomeni, K. Aquilano, G. Rotilio and M. R. Ciriolo, CancerRes., 2003, 63(18), 5940–5949.

114 H. Nakagawa, K. Tsuta, K. Kiuchi, H. Senzaki, K. Tanaka, K.Hioki and A. Tsubura, Carcinogenesis, 2001, 22(6), 891–897.

115 D. Xiao, S. Choi, D. E. Johnson, V. G. Vogel, C. S. Johnson, D. L.Trump, Y. J. Lee and S. V. Singh, Oncogene, 2004, 23(33), 5594–5606.

116 J. Wen, Y. Zhang, X. Chen, L. Shen, G. C. Li and M. Xu, Biochem.Pharmacol., 2004, 68(2), 323–331.

117 V. M. Dirsch, A. L. Gerbes and A. M. Vollmar, Mol. Pharmacol.,1998, 53(3), 402–407.

118 M. Li, J. M. Min, J. R. Cui, L. H. Zhang, K. Wang, A. Valette,C. Davrinche, M. Wright and J. Leung-Tack, Nutr. Cancer, 2002,42(2), 241–247.

119 S. Oommen, R. J. Anto, G. Srinivas and D. Karunagaran,Eur. J. Pharmacol., 2004, 485(1–3), 97–103.

120 F. D. Arditti, A. Rabinkov, T. Miron, Y. Reisner, A. Berrebi, M.Wilchek and D. Mirelman, Mol. Cancer Ther., 2005, 4(2), 325–331.

121 S. Moncada and E. A. Higgs, Eur. J. Clin. Invest., 1991, 21, 361–374.122 J. Claria, Curr. Pharm. Des., 2003, 9, 2177–2190.123 M. Ali, M. Thomson and M. Afzal, Prostaglandins, Leukotrienes

Essent. Fatty Acids, 2000, 62(2), 55–73.124 A. S. Baldwin, Annu. Rev. Immunol., 1996, 14, 649–681.125 T. C. Theoharides, M. Alexandrakis, D. Kempuraj and M. Lytinas,

Int. J. Immunopathol. Pharmacol., 2001, 14, 119–127.126 M. Irshad and P. S. Chaudhuri, Indian J. Exp. Biol., 2002, 40, 1233–

1239.127 K. C. Srivastava, Prostaglandins, Leukotrienes Med., 1986, 22(3),

313–321.128 R. C. Becker, Am. J. Cardiol., 1999, 83, 3E–6E.129 K. C. Srivastava and U. Justesen, Wien. Klin. Wochenschr., 1989,

101(8), 293–299.130 K. C. Srivastava, Prostaglandins, Leukotrienes Med., 1986, 24(1),

43–50.

131 V. M. Dirsch, A. K. Kiemer, H. Wagner and A. M. Vollmar,Atherosclerosis (Shannon, Irel.), 1998, 139(2), 333–339.

132 K. M. Kim, S.-B. Chun, M.-S. Koo, W.-J. Choi, T.-W. Kim, Y.-G.Kwon, H.-T. Chung, T. R. Billiar and Y.-M. Kim, Free Radical Biol.Med., 2001, 30(7), 747–756.

133 R. Hofbauer, M. Frass, B. Gmeiner, A. D. Kaye and E. A. Frost,Heart Dis., 2001, 3(1), 14–17.

134 H. P. Keiss, V. M. Dirsch, T. Hartung, T. Haffner, L. Trueman,J. Auger, R. Kahane and A. M. Vollmar, J. Nutr., 2003, 133(7),2171–2175.

135 V. M. Dirsch, H. P. Keiss and A. M. Vollmar, Eur. J. Nutr., 2004,43(1), 55–59.

136 P. N. Kourounakis and E. A. Rekka, Res. Commun. Chem. Pathol.Pharmacol., 1991, 74(2), 249–52.

137 G. Lewin and I Popov, Arzneim.-Forsch., 1994, 44(5), 604–607.138 J. S. Munday, K. A. James, L. M. Fray, S. W. Kirkwood and K. G.

Thompson, Atherosclerosis (Shannon, Irel.), 1999, 143(2), 399–404.139 H. A. Rajasree, C. R. Krishnakumar, K. Augusti and P. L.

Vijayammal, Vet. Hum. Toxicol., 1999, 41(5), 316–319.140 H. Xiao and K. L. Parkin, J. Agric. Food Chem., 2002, 50(9), 2488–

2493.141 J. Imai, N. Ide, S. Nagae, T. Moriguchi, H. Matsuura and Y. Itakura,

Planta Med., 1994, 60(5), 417–420.142 S. E. Ho, N. Ide and B. H. Lau, Phytomedicine, 2001, 8(1), 39–46.143 K. Prasad, V. A. Laxdal, M. Yu and B. L. Raney, Mol. Cell.

Biochem., 1995, 148(2), 183–189.144 C. C. Ou, S. M. Tsao and M. C. Yin, Lipids, 2003, 38(3), 219–224.145 C. N. Huang, J. S. Horng and M. C. Yin, J. Agric. Food Chem.,

2004, 52(11), 3674–3678.146 P. Rose, S. Widder, J. Looft, W. Pickenhagen, C. N. Ong and M.

Whiteman, Biochem. Biophys. Res. Commun., 2003, 302(2), 397–402.

147 S. Widder, C. Sabater Luntzel, T. Dittner and W. Pickenhagen,J. Agric. Food Chem., 2000, 48(2), 418–423.

148 J. C. Harris, S. L. Cottrell, S. Plummer and D. Lloyd, Appl.Microbiol. Biotechnol., 2001, 57, 282–286.

149 G. P. Sivam, J. Nutr., 2001, 131(3s), 1106S–1108S.150 K. S. Farbman, E. D. Barnett, G. R. Bolduc and J. O. Klein, Pediatr.

Infect. Dis. J., 1993, 12(7), 613–614.151 C. J. Cavallito and J. H. Bailey, J. Am. Chem. Soc., 1944, 66, 1950–

1951.152 M. G. Johnson and R. H. Vaughn, Appl. Microbiol., 1960, 17, 903–

905.153 L. Jezowa, T. Rafinski and T Wrocinski, Herba Pol., 1966, 12,

3–13.154 V. D. Sharma, M. S. Sethi, A. Kumar and J. R. Rarotra, In-

dian J. Exp. Biol., 1977, 15, 466–468.155 J. C. De Witt, S. Notermans, N. Gorin and E. H. Kampelmacher,

J. Food Prot., 1979, 42, 222–224.156 E. C. Delaha and V. F. Garagusi, Antimicrob. Agents Chemother.,

1985, 27, 485–486.157 K. V. Singh and N. P. Shukula, Fitoterapia, 1984, 15(5), 313–315.158 J. H. Kim, J. Nihon Univ. Sch. Dent., 1997, 39(3), 136–141.159 S. M. Tsao and M. C. Yin, J. Med. Microbiol., 2001, 50(7), 646–649.160 H. Yoshida, H. Katsuzaki, R. Ohta, K. Ishikawa, H. Fukuda, T.

Fujino and A. Suzuki, Biosci., Biotechnol., Biochem., 1999, 63(3),591–594.

161 K. L. Seo, Y. H. Moon, S. U. Choi and K. H. Park, Biosci.,Biotechnol., Biochem., 2001, 65(4), 966–968.

162 R. Naganawa, N. Iwata, K. Ishikawa, H. Fukuda, T. Fujino and A.Suzuki, Appl. Environ. Microbiol., 1996, 62(11), 4238–4242.

163 W. C. You, L. Zhang, M. H. Gail, J. L. Ma, Y. S. Chang, W. J. Blot,J. Y. Li, C. L. Zhao, W. D. Liu, H. Q. Li, Y. R. Hu, J. C. Bravo, P.Correa, G. W. Xu and J. F. Fraumeni, Int. J. Epidemiol., 1998, 27(6),941–944.

164 J. G. Chung, G. W. Chen, L. T. Wu, H. L. Chang, J. G. Lin, C. C.Yeh and T. F. Wang, Am. J. Chin. Med., 1998, 26(3–4), 353–364.

165 M. Iimuro, H. Shibata, T. Kawamori, T. Matsumoto, T. Arakawa,T. Sugimura and K. Wakabayashi, Cancer Lett. (Shannon, Irel.),2002, 187(1–2), 61–68.

166 B. A. Adeniyi and F. M. Anyiam, Phytother. Res., 2004, 18(5), 358–361.

167 P. Canizares, I. Gracia, L. A. Gomez, C. M. de Argila, D. Boixeda,A. Garcıa and L. de Rafael, Biotechnol. Prog., 2004, 20(1), 397–401.

168 G Zubay, Biochemistry, Cummings, California, 1986.169 R. A. Fromtling and G. S. Bulmer, Mycologia, 1978, 70, 397–405.170 H. Hitokoto, S. Morozumi, T. Wauke, S. Sakai and H. Kurata, Appl.

Environ. Microbiol., 1980, 39, 818–822.171 M. R. Tansey and J. A. Appleton, Mycologia, 1975, 67, 409–413.172 M. A. Adetumbi, G. T. Javor and B. H. S. Lau, Antimicrob. Agents

Chemother., 1986, 30, 499–501.

N a t . P r o d . R e p . , 2 0 0 5 , 2 2 , 3 5 1 – 3 6 8 3 6 7

Publ

ishe

d on

10

May

200

5. D

ownl

oade

d by

CO

NR

iCY

T o

n 16

/10/

2015

11:

51:0

9.

View Article Online

173 Z. R. Lun, C. Burri, M. Menzinger and R. Kaminsky, Ann. Soc.Belge Med. Trop., 1994, 74(1), 51–59.

174 J. C. Harris, S. Plummer, M. P. Turner and D. Lloyd, Microbiology,2000, 146, 3119–3127.

175 D. Saleheen, S. A. Ali and M. M. Yasinzai, Fitoterapia, 2004, 75(1),9–13.

176 A. A. Qureshi, Z. Z. Din, N. Abuirmeileh, W. C. Burger, Y. Ahmadand C. E. Elson, J. Nutr., 1983, 113, 1746–1755.

177 M. Steiner, A. H. Khan, D. Holbert and R. I. Lin, Am. J. Clin.Nutr., 1996, 64, 866–870.

178 Y. Y. Yeh, R. I. Lin, S. M. Yeh, and S. Evens, ‘Garlic reducedplasma cholesterol in hypercholesterolemic men maintaining habit-ual diets’, in: Food Factors for Cancer Prevention, ed. H. Ohigashiet al., Springer-Verlag, Tokyo, 1997.

179 L. Liu and Y. Y. Yeh, J. Nutr., 2002, 132(6), 1129–1134.180 R. Gebhardt, Lipids, 1993, 28, 613–619.181 K. T. Augusti, J. Chackery, J. Jacob, S. Kuriakose, S. George and

S. S. Nair, Indian J. Exp. Biol., 2005, 43(1), 76–83.182 N. Ferri, K. Yokoyama, M. Sadilek, R. Paoletti, R. Apitz-Castro,

M. H. Gelb and A. Corsini, Br. J. Pharmacol., 2003, 138(5), 811–818.

183 S. K Banerjee, S. Sood, A. K. Dinda, T. K. Das and S. K. Maulik,Comp. Biochem. Physiol., C: Pharmacol., Toxicol., Endocrinol.,2003, 136(4), 377–386.

184 T. Miron, A. Rabinkov, E. Peleg, T. Rosenthal, D. Mirelman andM. Wilchek, Am. J. Hypertens., 2004, 17(1), 71–73.

185 G. Saravanan and J. Prakash, J. Ethnopharmacol., 2004, 94(1), 155–158.

186 Y. Z. Zhu, S. H. Huang, B. K. Tan, J. Sun, M. Whiteman and Y. C.Zhu, Nat. Prod. Rep., 2004, 21(4), 478–489.

187 P. K. Moore, M. Bhatia and S. Moochhala, Trends Pharmacol. Sci.,2003, 24(12), 609–611.

188 J. F. Brady, H. Ishizaki, J. M. Fukuto, M. C. Lin, A. Fadel, J. M.Gapac and C. S. Yang, Chem. Res. Toxicol., 1991, 4, 642–647.

189 R. M. Nickson and S. C. Mitchell, Xenobiotica, 1994, 24(2), 157–68.

190 L. Jin and T. A. Baillie, Chem. Res. Toxicol., 1997, 10, 318–327.

191 A. J. Cooper and J. T. Pinto, Biochem. Pharmacol., 2005, 69(2),209–220.

192 C. K. Pushpendran, T. P. Devasagayam, G. J. Chintalwar, A. Banerjiand J. Eapen, Experientia, 1980, 36(8), 1000–1001.

193 C. Egen-Schwind, R. Eckard and F. H. Kemper, Planta Med., 1992,58, 301–305.

194 C. Teyssier, L. Guenot, M. Suschetet and M. H. Siess, Drug Metab.Dispos., 1999, 27, 835–841.

195 E. Germain, J. Auger, C. Ginies, M. H. Siess and C. Teyssier,Xenobiotica, 2002, 32(12), 1127–1138.

3 6 8 N a t . P r o d . R e p . , 2 0 0 5 , 2 2 , 3 5 1 – 3 6 8

Publ

ishe

d on

10

May

200

5. D

ownl

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