Molecular Approaches to Genetically Improve the Accumulation of Health-Promoting Secondary

33 Pages·2016·6.3 MB·English

International Journal o f
Molecular Sciences
Review
Molecular Approaches to Genetically Improve the
Accumulation of Health-Promoting Secondary
Metabolites in Staple Crops—A Case Study:
The Lipoxygenase-B1 Genes and Regulation of
the Carotenoid Content in Pasta Products
GraziaM.BorrelliandDanielaTrono*
ConsiglioperlaRicercainAgricolturael’Analisidell’EconomiaAgraria,CentrodiRicercaperlaCerealicoltura,
S.S.673Km25,200,71122Foggia,Italy;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:MarcelloIriti
Received:5June2016;Accepted:11July2016;Published:21July2016
Abstract: Secondary metabolites, also known as phytochemicals, represent a large subset of
plant molecules that include compounds with health-promoting effects. Indeed, a number of
epidemiological studies have shown that, when taken regularly and in adequate amounts, these
moleculescanhavelong-termbeneficialeffectsonhumanhealth,throughreductionoftheincidence
ofdegenerativediseases,suchascardiovasculardiseases,obesity,diabetes,andcancer. Asthedietary
intakeofthesephytochemicalsisofteninadequate, variousstrategiesareinusetoimprovetheir
contentinstaplecrops,andtheend-productsthereof. Oneofthemosteffectivestrategiesiscrop
improvementthroughgeneticapproaches,asthisistheonlywaytogeneratenewcultivarsinwhich
thehighaccumulationofagivenphytochemicalisstablyfixed. Effortstogeneticallyimprovequality
traitsarerapidlyevolving,fromclassicalbreedingtomolecular-assistedapproaches;theserequire
soundunderstandingofthemolecularbasesunderlyingthetraits,toidentifythegenes/allelesthat
controlthem. Thiscanbeachievedthroughglobalanalysisofthemetabolicpathwayresponsiblefor
phytochemicalaccumulation,toidentifythelinkbetweenphytochemicalcontentandtheactivitiesof
keyenzymesthatregulatethemetabolicpathway,andbetweenthekeyenzymesandtheirencoding
genes/alleles. Oncethesehavebeenidentified, theycanbeusedasmarkersforselectionofnew
improvedgenotypesthroughbiotechnologicalapproaches. Thisreviewprovidesanoverviewofthe
majorhealth-promotingpropertiesshowntobeassociatedwiththedietaryintakeofphytochemicals,
anddescribeshowmolecularapproachesprovidemeansforimprovingthehealthqualityofedible
crops. Finally,acasestudyisillustrated,oftheidentificationindurumwheatoftheLipoxygenase-B1
genesthatcontrolthefinalcarotenoidcontentinsemolina-basedfoods,suchaspastaproducts.
Keywords: phytochemicals; human health; metabolic pathway; metabolite analysis; enzymatic
activity;transcriptprofile;candidategene;biotechnologicalbreeding;lipoxygenase;pasta-products
1. Introduction
Thesecondarymetabolitesfromplants,whichareknownasphytochemicals,representalarge
subset of plant chemicals, but their importance in both plant metabolism and human health has
onlybeenrecognizedmorerecently. Thisisbecause,unliketheprimarymetabolites,carbohydrates,
lipidsandproteins,whichareubiquitouslypresentathighconcentrationsinalllivingorganisms,in
whichtheysharethesameessentialfunctions(i.e.,growth,developmentandreproduction),secondary
metabolitesarepresentatmuchlowerconcentrations,arerestrictedtospecificgroupsoforganisms
Int.J.Mol.Sci.2016,17,1177;doi:10.3390/ijms17071177 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2016,17,1177 2of33
andareproducedinresponsetoparticularconditions[1]. Likewise,inhumannutritionattentionhas
beenfocusedforyearsontheenergydemandthatneedstobesatisfiedthroughthecorrectcombination
ofcarbohydrates, lipids, andproteinsinthediet, whereastheimpactofsecondarymetaboliteson
humanhealthhasbeenignoredforalongtime.
Inthelastdecades,evidencehasaccumulatedofthedirectinvolvementofsecondarymetabolites
invariousprocessesrelatedtoplantfitness,suchasdefenseagainstbioticandabioticstress,formation
ofsymbiosisattherootlevel,andreproductiveprocessesthroughattractionofanimalsinvolvedin
pollinationorseeddispersal[2]. Inthesamemanner,therecognitionofthebiologicalpropertiesofa
numberofsecondarymetaboliteshasledtotherevaluationofthesecompoundsalsofromthehealth
pointofview. Althoughphytochemicalsdonothaverolesasnutrients,increasingepidemiological
evidencehasassociatedtheconsumptionoffoodsrichinphytochemicals,suchasfruit,vegetables,
andgrainstoreducedincidenceofcardiovasculardiseases(CVD),obesity,diabetes,cancer,andother
chronicdegenerativediseases[3].
Nowadays,thedietaryintakeofbioactivecompoundsisofteninadequate,andworldwide,people
arebecomingevenmoresusceptibletothedevelopmentofdegenerativediseases[4]. Therefore,the
demand for new effective strategies that are aimed at prevention of these diseases has increased
considerablyinthelastfewdecades. Oneapproachistoincreasethecontentofhealth-promoting
compounds in staple crops and their end products. This can be effectively achieved by genetic
improvementofcropsthroughproperbreedingprograms,whichrepresenttheonlycertainwayto
generatenewcultivarsinwhichadesirablephytochemicalcontentisstablyfixed.
Fromthelate19thcentury,plantbreedingreliedonphenotypicselection. Thustheimprovement
ofaqualitytrait,suchastheaccumulationofadesirablecompound,wasachievedthroughscreeningof
hundreds,oreventhousands,ofplants. Laboratorytestswerethenusedtoevaluatethephytochemical
contentandtoselecttheplantsofsuperiorquality. Theentireprocesswasrelativelylong(>10years)
andlaborious. Fortunately,overtheyears,withtheadventofmoleculargenetics,cropimprovement
hasrapidlyevolvedfromclassicalbreedingapproachestobiotechnologicalbreedingapproaches,such
asmarker-assistedselection(MAS)[5],genetictransformation[6],andveryrecently,genomeediting[7].
Theseadvancedtechnologiesrequiresoundunderstandingofthemolecularbases underlyingthe
accumulationofabioactivecompoundinagivencrop,tobeabletoidentifythecandidategenethat
controlsitslevels. Thechallengeisthentointroducethiscandidategeneintoanelitecultivar,while
retainingitsperformanceattributes.
Twoapproachescanbeusedtoidentifyandvalidatesuchacandidategene: (i)thepositional
approach;and(ii)thefunctionalapproach[8]. Thepositionalapproachisbasedontheidentification
ofapotentialcandidategeneamongthegenesthatmaparoundthequantitativetraitlocus(QTL)
that is associated with the trait. This is followed by its validation through the demonstration that
polymorphic variants of the candidate gene (i.e., alleles) can explain the variability of the trait.
Conversely,thefunctionalapproachbypassestheQTLstep,asitreliesonidentificationofthecandidate
genethroughitsfunctionalrelationtothetrait, andinparticularthroughtheassociationbetween
itsallelicvariationandthevariationinthefluxratethroughthepathwaythatisresponsibleforthe
metaboliteaccumulation. Themetabolicpathwayisoftenamulti-stepeventthatinvolvesdifferent
enzyme-catalyzedreactions,althoughtherateoftheoverallpathwayisgenerallyregulatedbytherate
ofone,orafew,reactionsthatarecatalyzedbytheso-called“rate-limiting”enzymes. Allelicvariation
atthegenethatencodesakeyregulatoryenzymeofthepathwaymighthavethepotentialtomodify
the functioning of the enzyme, and consequently, the accumulation of the desired phytochemical.
Therefore, the combination of target metabolite determination with enzymatic assays as well as
gene-sequenceandexpressionanalysesmightrepresentavaluablemulti-disciplinaryapproachfor
theidentificationoffavorableallelesthat,whenusedasmarkers,canhelpabreedertosuccessfully
achievethegoalofbiofortificationofstaplecrops.
Here,weprovideanoverviewofthemainclassesofplantphytochemicals,andwesummarize
thecurrentknowledgeoftheirhealth-promotingeffects. Then,wefocusonthemolecularstrategies
Int. J. Mol. Sci. 2016, 17, 1177 3 of 31
2. General Overview of Plant Secondary Metabolites
Int.J.Mol.Sci.2016,17,1177 3of33
Secondary metabolites can essentially be divided into three main groups that are based on their
structures and the metabolic pathways responsible for their biosynthesis (Figure 1): phenolic
caonmdptoooulnsdtsh;a ttercpanenboeidasp; panlided nittoroegffeenc-tcivoenltyaimnianngi pcoumlapteouthnedsp,h i.yet.o, cahlkeamloiicdasl alenvde lgsluincoasginroonlaotmesi c[9a]l.l y
importantcrops. Finally,wedescribeafunctionalstudyofthelipoxygenase-B1genefamilyindurum
w2.1h.e Pathetnhoalticc oCnotmroplosutnhdes finalcarotenoidcontentinpastaproducts.
Phenolic compounds represent one of the largest groups of secondary metabolites produced in
2. GeneralOverviewofPlantSecondaryMetabolites
plants (i.e., >8000 different structures), and particularly in edible plants (e.g., vegetables, fruit, cereals).
Secondary metabolites can essentially be divided into three main groups that are based on
These include several structurally different compounds that have at least one aromatic hydroxyl-
theirstructuresandthemetabolicpathwaysresponsiblefortheirbiosynthesis(Figure1): phenolic
substituted ring in common. Phenolic compounds are produced primarily via the shikimate pathway
compounds;terpenoids;andnitrogen-containingcompounds,i.e.,alkaloidsandglucosinolates[9].
that gives rise to phenylpropanoids (Figure 1).

Figure 1. Schematic representation of the biosynthesis of secondary metabolites. E4P, erythrose
Figure 1. Schematic representation of the biosynthesis of secondary metabolites. E4P, erythrose
4-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; DOX5P,
4-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate;
deoxyxylulose 5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate;
DOX5P, deoxyxylulose 5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl
PP, pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP,
pyrophosphate;PP,pyrophosphate;GPP,geranylpyrophosphate;FPP,farnesylpyrophosphate;GGPP,
geranylgeranyl pyrophosphate.
geranylgeranylpyrophosphate.
The combination of the shikimate pathway with the mevalonate pathway leads to the generation
2.1. PhenolicCompounds
of the flavonoids [10]. Condensation and polymerization reactions produce hydrolysable and
condPenhseendo tliacncnoinms,p loigunnadnss,r leipgrneisne, nctutoinne, aonfdt shueblearrigne. sPthgernooulpics coofmspeocounnddas rayrem inevtaoblvoelidte isn par noudmucbeedr
oinf pphlaynstisol(oi.gei.c,a>l 8p0r0o0cedsisfefse rienn ptlsatnrutsc, tsuurcehs) a,sa npdollpinaarttiicounl,a rrelysisitnanecdei btloe ppaltahnotgse(nes.g a.,nvde pgreetdabalteosr,s,f raunidt,
rceesriesatlasn).ceT tho easbeiointiccl ustdreessse [v1e0r].a Al sltthruocutguhra tlhlyeyd cifafne reexnitstc ionm thpeoiur nfrdese tfhoarmt hs,a pveheantolleica sctoomnpeoaurnodms aatriec
choymdrmoxoynll-ysu cbosntjiutugtaetdedri tnog siungcaorms omr opnr.oPtehienns,o alincdc ocmanp aolusnod osccaurer pasro edsutecresd apnrdi mmaertihlyylv eiasttehres,s phriokbimabaltye
pbeacthauwsaey mthoastt goifv tehsermis eartoe tpohxeicn yclopmroppoaunnodids sth(Faitg aurree a1t) .least in part neutralized in their bound form.
Int.J.Mol.Sci.2016,17,1177 4of33
Thecombinationoftheshikimatepathwaywiththemevalonatepathwayleadstothegenerationof
theflavonoids[10]. Condensationandpolymerizationreactionsproducehydrolysableandcondensed
tannins, lignans, lignin, cutin, and suberin. Phenolic compounds are involved in a number of
physiological processes in plants, such as pollination, resistance to pathogens and predators, and
resistancetoabioticstress[10]. Althoughtheycanexistintheirfreeforms,phenoliccompoundsare
commonlyconjugatedtosugarsorproteins,andcanalsooccurasestersandmethylesters,probably
becausemostofthemaretoxiccompoundsthatareatleastinpartneutralizedintheirboundform.
Duetothelargenumberandheterogeneityofstructures,phenoliccompoundscanbeclassifiedin
differentways. Onthebasisoftheircarbonchain,theycanbedistinguishedindifferentclassesthat
rangefromsimplephenolstohighlypolymerizedcompounds(Table1).
Table1.Themainclassesofphenoliccompoundsaccordingtotheircarbonchains.
Class CarbonSkeleton Examples
Catechol,2,6-dimethoxybenzoquinone,
C6 Simplephenolsandbenzoquinones hydroquinone,phenol,pirocatechol
Gallic,p-hydroxybenzoic,salicylic,
C6–C1 Benzoicacids siringicandvanillicacids
3-Acetyl-6-methoxybenzaldehyde,
C6–C2 Phenylaceticacidsandacetophenones tyrosol,p-hydroxyphenylaceticacid
Caffeic,ferulic,p-coumaric,sinapicacids,
C6–C3 Cinnammicacidsandphenylpropanoids aesculetin,bergenon,eugenin,myristicin
C6–C4 Naphtochinones Juglone,plumbagin
C6–C1–C6 Xanthones Mangiferin,mangostin
Astringin,resveratrol,viniferin,
C6–C2–C6 Stilbenesandanthraquinones chrysophanol,emodin
Catechin,cyanidin,
C6–C3–C6 Flavonoidsandisoflavonoids epigallocatechin-3-gallate,naringenin,
quercetin,myricetin,genistein
Lariciresinol,pinoresinol,
(C6–C3)2 Lignansandneolignans eusiderin,magnolianin
(C6)n Catecholmelanins
(C6–C1)n Hydrolysabletannins
(C6–C2)n Lignin,cutinandsuberin
(C6–C3–C6)n Condensedtannins(proanthocyanidins)
Thethreemostimportantclassesofphenoliccompoundsforhumanhealtharephenolicacids,
flavonoids,andtannins.Phenolicacidsarethesimplestphenoliccompoundsfoundinnature,andthey
accountforone-thirdofallofthephenoliccompoundsinthehumandiet. Phenolicacidsincludetwo
majorgroups,benzoicacidsandcinnamicacids,whichhaveseven(C –C )andnine(C –C )carbon
6 1 6 3
atoms,respectively(Figure2). Thesecompoundsarecharacterizedbyanaromaticring,acarboxylic
group,andoneormorehydroxyland/ormethoxylgroups. Cinnamicacidsaremorecommonlyfound
innaturethanbenzoicacids. Amongthecinnamicacids,caffeicacidanditsesterifiedderivativesare
themostabundantinfruit,whereasferulicacidanditsderivativesarethemostabundantincereal
grains[11].
Flavonoidsarethemostwidelydistributedphenoliccompoundsinplants,astheyaccountfor
morethanhalfofthe8000phenoliccompoundsfoundinnature[12]. Theseoccurinvarioustissues
andorgansofediblecrops,suchasfruit,leaves,roots,andtubers,aswellasinlegumes,tea,coffee,
herbs,andspices. Flavonoidshaveacommonstructurethatconsistsoftwoaromaticrings(theAand
Brings),joinedbyaC chainthatisgenerallyintheformofaheterocyclicring(theCring)(Figure2).
3
AromaticringAcomesfromthemalonatepathway,whereasringBcomesfromtheshikimatepathway.
SubstitutionstoringCresultindifferenttypesofflavonoids,suchasflavones,flavonols,flavanones,
Int.J.Mol.Sci.2016,17,1177 5of33
flavanols,anthocyanidins,andisoflavones,whereassubstitutionstoringsAandBgiverisetodifferent
compoundswithineachtypeofflavonoid.
Tanninsarethethirdmostimportantgroupofphenoliccompounds.Theseincludealargenumber
ofoligomersandpolymersthatcanformcomplexeswithstarch, proteins, cellulose, andminerals.
Int. J. Mol. Sci. 2016, 17, 1177 5 of 31
Tannins can be distinguished as two groups: hydrolysable tanninsand condensed tannins, which
qaureenaclshoinkgn oowf nthaessper opahnethnoocliyca ncoidminpso.uHnyddsr. oOlyfs anboletet,a nfnlaivnosnaoreidcso marpeo uanmdosnfgo rmtheed mfroomst gpaollwicearfcuidl
aonrteiopxiigdaallnitcsa tchidat uhnaivtse thbeaetna roebctoanindeedn sfreodmto palacnetnst.r Talhseuirg astrromnogl eacnutlieo,xwidhainlet ccoanpdaceintyse ids tdaunen itnos tahree
pproelsyemnceers ooff hcaytdecrohxinyla ngdroeuppicsa itnec hpions,itoironsism 3il’a arnudn it4s’ (oFfi gruinreg2 B)., Pwrohainchth opcayratinciidpiantes airne tmheo reelwecitdroelny
ddeislotrciabluiztaedtioinn eadnidb lsetapblailniztes tthhaen rahdyidcarol ltyhsaatb ilse ftoarnmneinds,, aansdth oefy aa dreouwbildee bsporneda dbeintwfreuenit ,tlheeg uCm2 easn,dn uCt3s ,
caanrdbomnsin ionr rcinerge aCl,s ,tosguecthhears wsoirtghh au cmarabnodnybla grlreoyu,pa nadt tthhee yC4te cnadrbtoonc,o wncheinchtr mateakine dtheelopceaelilzoaftifornu iotfa annd
etlheectbrroann forofmgr ariinngs. B possible [14].

Figure 2. General structures of the three most important classes of phenolic compounds for human
Figure2. Generalstructuresofthethreemostimportantclassesofphenoliccompoundsforhuman
hheeaalltthh.. BBeennzzooiicc aacciiddss:: pp--hhyyddrrooxxyybbeennzzooiicc aacciidd,, RR3==OOHH;; vvaanniilllliicc aacciidd,, RR2==OOCCHH3 aanndd RR3==OOHH; ;ggaalllilcic aaccidid, ,
3 2 3 3
RR2==RR3=R=4R=O=HO;H s;ysryinrignigc icacaicdid, ,RR2=R=R4=O=COHC3H anadn dRR3=O=HO.H C.Cininnanmamic icacaicdids:s :pp-c-coouummaarirci caacciidd, ,RR3==OOHH; ;
2 3 4 2 4 3 3 3
ccaaffffeeiicc aacciidd,, RR3==RR4==OOHH; ;ffeerruulliicc aacciidd,, RR2==OOCCHH3 aanndd RR3==OOHH;; ssiinnaappiicc aacciidd,, RR2==RR4==OOCCHH3 aanndd RR3==OOHH. .
3 4 2 3 3 2 4 3 3
FFllaavvoonnooiiddss:: RR3––RR6==HH oor rooththeerr ggrroouuppss ssuucchh aass OOHH oorr ooxxoo-- ggrroouuppss.. FFoorr mmoosstt ffoooodd flflaavvoonnooiiddss::R R4’==RR6==HH
3 6 4’ 6
aanndd RR5==OOHH. .InIn bbioiochchaanninin AA, ,RR4’=O=OCHCH3; fo;rfomromnoonnoentient,i nR,4’R= O=COHC3 Handa nRd5=RR6==HR; =gHly;cegtleyicne, tRei5n=,HR a=nHd
5 4’ 3 4’ 3 5 6 5
Ra6n=dORH.= OH.
6
Another class of compounds that contains a phenol ring is represented by the alkylresorcinols.
Phenoliccompoundsareconsideredtobepowerfulantioxidantsduetothereactivityoftheir
However, compared to phenolic compounds, alkylresorcinols represent a very distinct group, as they
phenol moiety. Their main mode of action as antioxidants is radical scavenging via hydrogen
have both polar and nonpolar properties. Indeed, in addition to the phenol ring with two hydroxyl
atoms or electron donation, together with the delocalization of the unpaired electron within the
groups, alkylresorcinols also have a hydrophobic and mostly saturated alkyl chain. The rich sources
aromaticring[13]. Substituentsonthearomaticringsaffecttheirstabilization,andthereforeaffectthe
of alkylresorcinols include cereal grains (i.e., from wheat, rye, triticale, barley), in which these
radical-quenchingofthesephenoliccompounds. Ofnote,flavonoidsareamongthemostpowerful
compounds accumulate in the cuticle layer. There is evidence that alkylresorcinols protect cellular
antioxidants that have been obtained from plants. Their strong antioxidant capacity is due to the
lipids from oxidative damage [15].
presence of hydroxyl groups in positions 3’ and 4’ of ring B, which participate in the electron
delocalization and stabilize the radical that is formed, and of a double bond between the C2 and
2.2. Terpenes
Terpenes and terpenoids (i.e., terpene-like compounds) are lipid-soluble compounds that
represent the most abundant and structurally diverse group of plant secondary metabolites (with
>50,000 different structures) [16]. These are normally produced in vegetative tissues and flowers, and
occasionally in roots [17]. They are also referred to as isoprenoids, as they are all derived from the
repetitive fusion of isoprene (C5H8) units. All isoprenoids are derived from the common precursor
isopentenyl pyrophosphate (IPP), which is ubiquitously synthesized through the mevalonate
Int.J.Mol.Sci.2016,17,1177 6of33
C3carbonsinringC,togetherwithacarbonylgroupattheC4carbon,whichmakedelocalizationof
anelectronfromringBpossible[14].
Anotherclassofcompoundsthatcontainsaphenolringisrepresentedbythealkylresorcinols.
However,comparedtophenoliccompounds,alkylresorcinolsrepresentaverydistinctgroup,asthey
havebothpolarandnonpolarproperties. Indeed,inadditiontothephenolringwithtwohydroxyl
groups,alkylresorcinolsalsohaveahydrophobicandmostlysaturatedalkylchain. Therichsourcesof
alkylresorcinolsincludecerealgrains(i.e.,fromwheat,rye,triticale,barley),inwhichthesecompounds
accumulateinthecuticlelayer. Thereisevidencethatalkylresorcinolsprotectcellularlipidsfrom
oxidativedamage[15].
2.2. Terpenes
Terpenesandterpenoids(i.e.,terpene-likecompounds)arelipid-solublecompoundsthatrepresent
the most abundant and structurally diverse group of plant secondary metabolites (with >50,000
different structures) [16]. These are normally produced in vegetative tissues and flowers, and
occasionally in roots [17]. They are also referred to as isoprenoids, as they are all derived from
the repetitive fusion of isoprene (C H ) units. All isoprenoids are derived from the common
5 8
precursorisopentenylpyrophosphate(IPP),whichisubiquitouslysynthesizedthroughthemevalonate
pathwayorthedeoxyxylulose-5-phosphatepathway[18](Figure1). IPPisomerizestodimethylallyl
pyrophosphate, andthecondensationbetweenIPPanddimethylallylpyrophosphategivesriseto
geranyl pyrophosphate, the precursor of all of the monoterpenes. The IPP additions that follow
generateotherprenyldiphosphates,whicharetheprecursorsofalloftheotherterpenes. Inplants,
the synthesis of monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30),
tetraterpenes(C40),andpolyterpenes(C50–C130)hasbeenreported([19],andreferencestherein).
AsshowninTable2, terpenoidsincludecompoundsthatareimplicatedinbothprimaryand
secondarymetabolism. Gibberellin,cytokinins,abscisicacid,andbrassinosteroidsarehormonesthat
areinvolvedinplantgrowthanddevelopment,whereascarotenoids,andcompoundsthatcontain
anisoprenylsidechain,suchaschlorophyll, phylloquinone, plastoquinone,andtocopherolshave
functionsinphotosynthesis[16]. However,mostoftheterpenoidsaresecondarymetabolitesthathave
crucialrolesaspollinatorattractants,andinthedefenseofplantsagainstherbivoresandmicrobial
pathogens[19].
Table2.Classificationofterpenesandterpenoidsonthebasisoftheirisopreneunits.
Class IsopreneUnits CarbonAtoms Examples
Canphor,eucalyptol,geraniol,lavandulol,
Monoterpene 2 10
limonene,menthol,pinene,thymol
Abscisicacid,bergamotene,cedrol,
Sesquiterpene 3 15
curcumene,patchoulol,vetivone
Abieticacid,cafestol,caffeol,
Diterpene 4 20
carnosol,gibberellin,phytol
Betulinicacid,morolicacid,oleanolicacid,ursolic
Triterpene 6 30
acid,brassinosteroids,saponins
Carotenoids(e.g.,α-carotene,β-carotene,crocin,
Tetraterpene 8 40 licopene)andxanthophylls(e.g.,lutein,zeaxanthin,
α-cryptoxanthin,β-cryptoxanthin)
Polyterpene >8 >40 Cytokinines,gutta-percha,rubber
Int.J.Mol.Sci.2016,17,1177 7of33
Figure3.Structuresofcarotenesandxanthophyllsthatareimportantforhumanhealth.
Int.J.Mol.Sci.2016,17,1177 8of33
Thecarotenoidsareaclassofterpenoidsthathaverecognizedbeneficialfunctionsforhuman
health [20]. These are C40 isoprenoids, and they represent the second most abundant pigments
in nature (with >750 compounds). There are two general classes of carotenoids: carotenes, which
consistexclusivelyofhydrogenandcarbon;andxanthophylls,whichalsocontainoxygen(Figure3).
Asanimalsandhumanscannotsynthesizecarotenoidsdenovo,thedietisreliedonfortheseessential
compounds.Pro-vitaminAcarotenoids,suchasα-carotene,β-carotene,andβ-cryptoxanthin(Figure3),
areimportantprecursorsofretinol(i.e.,vitaminA),retinal,retinoid,andretinoicacidinanimalsand
humans[20,21],whereassomexanthophylls,suchasluteinandzeaxanthin(Figure3),areessential
componentsofmacularpigments. Inaddition,carotenoidsactasantioxidants. Inparticular,theycan
efficientlyquenchsingletoxygen.Thisimpliesenergytransferfromthesingletoxygentothecarotenoid
molecule,toyieldground-stateoxygenandatripletexcitedcarotenoidmolecule;theexcitedcarotenoid
canthenreturntothegroundstatebydissipatingthisenergyasheat[22]. Chemicalquenchingof
singletoxygenhasalsobeenreported,whichleadstooxidationofthecarotenoidmolecule[23].
Amongthevariousradicals,carotenoidsmostefficientlyreactwithperoxylradicalsgenerated
duringlipidperoxidation. Forthisreason,andbecauseoftheirlipophilicity,carotenoidsarebelieved
tohaveimportantrolesintheprotectionofcellmembranesagainstoxidativedamage[22]. Thefood
sourcesofthesecompoundsincludevariousfruitandvegetables.
2.3. Nitrogen-ContainingCompounds
Secondary metabolites that contain nitrogen include alkaloids and glucosinolates. Alkaloids
representalargeclassofnitrogen-containingcompounds(with>12,000)wherethenitrogenisderived
fromanaminoacid,suchastyrosine,tryptophan,lysine,andaspartate(Figure1). Comparedtothe
otherclassesofsecondarymetabolites,alkaloidshavegreatstructuraldiversity,andforthisreason,
thereisnosingleuniformclassificationforthesecompounds.Mostofthemaretoxictootherorganisms,
and therefore they have important roles in plants in terms of their defense against pathogen and
herbivoreattack[24]. Onceproduced,alkaloidsarestoredinthecellvacuoleandthenreleasedwhen
mechanicaldamagebreaksthevacuole. Alkaloidsincludeanumberofmoleculesofpharmacological
interest, such as morphine, codeine, atropine, quinine, vincristine, and vinblastine, which have
importanttherapeuticactivitiesthatincludeantibacterial,antiviral,antimalarial,antiproliferative,and
antimetastaticeffectsinvarioustypesofcancers[25].
Conversely, there is a nutritional interest in the class of glucosinolates, which is a relatively
small, but diverse, group of about 200 compounds. These are substituted esters of thio-amino
acids that are mainly limited to species of the order of Brassicales, such as broccoli, cauliflower,
cabbage,Brusselssprouts,redradish,horseradish,andmustard[26]. Inplants,glucosinolatesactas
pesticidesandasherbivorerepellents[27]. Again,thesecompoundsarestoredinthecellvacuole,
anduponcelldisruptiontheyarerapidlyhydrolyzedbymyrosinaseandconvertedtothiocyanate,
isothiocyanate,nitrile,epithionitrile,andoxazolidine-thione(Figure4),whicharetoxicforpathogens
andherbivores[28].
Basedonthestructureoftheamino-acidprecursor,glucosinolatescanbedefinedasaliphatic,
indole, and aromatic [29,30] (Table 3). Aliphatic glucosinolates are derived from alanine, leucine,
isoleucine,methionine,andvaline,whileindoleglucosinolatesandaromaticglucosinolatesarederived
fromtryptophanandphenylalanineortyrosine,respectively. Thepresenceofglucosinolatesvaries
notonlyamongplantspecies,organs,andtissues,butalsounderspecificenvironmentalconditions.
Ofnote,nearlyallofthepotentialhealthbenefitsofglucosinolatesareduetotheirhydrolyticproducts
and in particular to the isothiocyanates [31]. Mammalian tissues do not contain myrosinases, but
degradation of glucosinolates still occurs in humans through the gut microflora. Indeed, several
studieshaveidentifiedgutbacterialstrainsthatareassociatedwiththeformationofisothiocyanates
fromglucosinolates([32],andreferencestherein).
Int.J.Mol.Sci.2016,17,1177 9of33
Int. J. Mol. Sci. 2016, 17, 1177 8 of 31
Int. J. Mol. Sci. 2016, 17, 1177 8 of 31
IInntt.. JJ.. MMooll.. SSccii.. 22001166,, 1177,, 11117777 88 ooff 3311
Int. J. Mol. Sci. 2016, 17, 1177 8 of 31
InInt.t .J .J .M Mool.l .S Scci.i .2 2001166, ,1 177, ,1 1117777 88 o of f3 311

FFiigguurree 44.. HHyyddrroollyyssiiss ooff gglluuccoossiinnoollaatteess ccaattaallyyzzeedd bbyy mmyyrroossiinnaassee.. MMyyrroossiinnaassee ccaattaallyyzzeess tthhee hh yyddrroollyyssiiss
ooff tthhee tthhiioogglluuccoossiiddee lliinnkkaaggee,, tthhuuss lleeaaddiinngg ttoo tthhee ffoorrmmaattiioonn ooff ddiiffffeerreenntt pprroodduuccttss.. RR rreepprr eess eennttss tthhee
aammiinnooF--FFaaigciicggiiudduur--rreddee e4e 44r.r i..iH vvHHeeydyydddd rssrroiidoodlylleeyys--sscicsiihhss oaa ooifin nffg ..ggl ulluucccooosssiniinnooolallaatetteesss c ccaaatattaalyllyyzzzeeeddd b bbyyy m mmyyyrrrooosssiniinnaaassseee. ..M MMyyyrrrooosssiniinnaaassseee c ccaaatattaalyllyyzzze ees ss t htthheee h hhyyydddrrrooolyllyysssisiiss
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oooaffaam f mmtththTihniieeanneo boo tt-hthla--heaaiico iocci3odgiig.ddg l-lCulud--uddclceacoeeorosrrisssviisiivvididefdeeideecedd a sll ilisstininidiinoddkkekneeaa-a c--ggoccgheefhhea ,,g aa ,i ttniilhthnnu.h uu..cu soss s llieleneaaoaddldiainintnegggs ttootoon tt ththhheeee ffbofooarrrsmmmisaa aottitifoio otnnhn e oooiffr f daddmiififfffiefenerroreee-nnanttc t i ppdprr roopoddrdeuuucccucttstsrs.s. .o RRRr . r rreeeppprrreeessseeennnttstss tththheee
amino-acid-derived side-chain.
aamminiTnoao-ba-lacecidi3d-.d-Cdelerarisvivseiedfid cs asidtidieoe-nc-chohafaigninl.u . cosinolatesonthebasisoftheiramino-acidprecursor.
Class Example Structure
Table 3. Classification of glucosinolates on the basis of their amino-acid precursor.
TTaabbllee 33.. CCllaassssiiffiiccaattiioonn ooff gglluuccoossiinnoollaatteess oonn tthhee bbaassiiss ooff tthheeiirr aammiinnoo--aacciidd pprreeccuurrssoorr..
Table 3. Classification of glucosinolates on the basis of their amino-acid precursor.
CTTlaaabsblsele 3 C3. .lC aClsalasssssifiifcicaatitoionn Eo Eoxfx fag maglumlpuclcpoeoslsieninoolalatetess o onn t hthee b baassisis o of fSt Shtthterreiuurir cca ttamuumrrienienoo-a-accidid p prreeccuurrssoorr. .
AliphatiCCc llaassss SinigrEEinxx aammppllee SSttrruuccttuurree
Class Example Structure
CCllaassss EExxaammppllee SSttrruuccttuurree
AliphatAiAAclillpiipphhhaaatittciicc SSiSSniniiinngirgiiiggrnrriniinn
Aliphatic Sinigrin
AAlilpiphhaatticic SSininigigrrinin
Glucoraphanin

Glucoraphanin
GGGlullcuuoccrooaprraahppahhniaannniinn
Glucoraphanin
GGluluccoorraapphhaanninin
Dehydroerucin

Dehydroerucin
DDDDeehheeyyhhddyyrddrooerreoorureeucrricuuniccnii nn
DDeehhyyddrrooeerruuccinin
Indole Glucobrassicin
Indole Glucobrassicin
IInnddoollee GGlluuccoobbrraassssiicciinn
IndoleIndole GGlluuccoobbrraasssisciicnin
IInnddoolele GGluluccoobbrraassssicicinin

Aromatic Sinalbin

Aromatic Sinalbin
AArroommaattiicc SSiinnaallbbiinn
Aromatic Sinalbin
AromaAAtircroommaatticic SSSiinninaalalbblibninin

Gluconasturtiin

Gluconasturtiin
GGlluuccoonnaassttuurrttiiiinn
Gluconasturtiin
GGGlluluucccooonnnaasaststuturutrirtitiniinin
3. Health Benefits of Secondary Metabolites

Chronic diseases, such as CVD, diabetes, cancer, and neurodegenerative and age-related
3. Health Benefits of Secondary Metabolites
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33. .H HeeaCallhtthrh oB Bneiencn eedffiiitstses ao osffe S sSe, ecscouonncdhda ararysy M MCeVettaDabb, oodlliiitateebsse tes, cancer, and neurodegenerative and age-related
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Chronic diseases, such as CVD, diabetes, cancer, and neurodegenerative and age-related
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diseases are worldwide health problems that cause severe disability and even death. Epidemiological
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studies have shown that due to their significant content of phytochemicals, regular consumption of
ssftffrturruuduuidtiiiei,tt e,,sv svv eh eehgaggaeveevteatteaa bs bbslhellhoeesossw, w,,a naannnn dt ddth hg aggartrrat d aaidniiunnusess e i t siitoss os sstt thrtthrroeooeninnrigr ggs lsylliyygi ga naansisssfissfiociooccaccianaiinaatttett c eedcoddo nw nwwtiteteiinhttnhht t r o rreoeefdf ddp upuuhchcceyeeydtddto or crrichsiihsskekkem om oofiff cit cahttahhlesleess, ss,er eer ec egcchghhururrolaoolnarnnir ciic ccc od oddninsiisssseueeuamaamsssepeepstss.ti ..oi onn o off
fruit, vegetables, and grains is strongly associated with reduced risk of these chronic diseases.
ffrruuitit, ,v veeggeettaabbleless, ,a anndd g grraaininss i sis s sttrroonngglyly a assssoocciaiatteedd w witithh r reedduucceedd r risiskk o off t thheessee c chhrroonnicic d disiseeaasseess. .
Int.J.Mol.Sci.2016,17,1177 10of33
3. HealthBenefitsofSecondaryMetabolites
Chronicdiseases,suchasCVD,diabetes,cancer,andneurodegenerativeandage-relateddiseases
areworldwidehealthproblemsthatcauseseveredisabilityandevendeath. Epidemiologicalstudies
have shown that due to their significant content of phytochemicals, regular consumption of fruit,
vegetables,andgrainsisstronglyassociatedwithreducedriskofthesechronicdiseases.
3.1. CardiovascularDiseases
Cardiovascular diseases are the main cause of death and disability in developed countries.
Epidemiologicalstudieshaveshownthatthereisastronginverseassociationbetweenintakeofsome
phytochemicals and incidence of hypertension, myocardial infarction, stroke, and atherosclerosis.
In particular, consumption of foods rich in phenols has been shown to have a protective effect
uponCVD.Thisisparticularlyconvincingforflavanolsfromcocoa-derivedproductsandtea, the
consumptionofwhichleadstoreducedbloodpressureandimprovedendothelialfunction[33,34].
Although conclusive data have yet to be realized, promising findings have also been obtained for
the effects of resveratrol (from berries, grapes, plums, peanuts) on ischemic heart disease [35], of
anthocyanins (from berries, grapes, citrus fruit) on low-density-lipoprotein cholesterol [36], and
of quercetin (from onions, apples, berries, grapes, tea) on hypertension and atherosclerosis [37].
Asforcarotenoids,crocin(fromsaffron)hasproventobeagoodcandidateforinhibitionofplatelet
aggregationandforprotectionagainsttheapoptosisofplateletsinducedbyoxidativestress[38],while
lycopene(fromtomato)appearstoimproveendothelialfunctioninpatientswithCVD,andtoreduce
incidenceofcoronaryheartdisease[39]. Finally,thereisevidenceoftheinvolvementofthedietary
intakeofisothiocyanates(fromBrassicaspecies)inimprovedpost-ischemicventricularfunction,in
reducedmyocardialinfarctsize,andindecreasedcardiomyocyteapoptosisinmammals[40]. Allicin
(fromgarlic,onion,shallot,leek)isanotherorganosulfurcompound,andithasbeenshowntoprotect
thecardiovascularsystemthroughinductionofvasorelaxation,andtoalleviatevariouspathological
conditionsofCVD[41].
3.2. Obesity
ObesityisassociatedwithincreasedriskofCVD,type2diabetes, andgastrointestinalcancer.
Atthecellularlevel,obesityischaracterizedbyincreasednumberandsizeofadipocytes,whichare
differentiatedfromfibroblasticpre-adipocytesinadiposetissue. Evidencehasbeenaccumulatedthat
the dietary intake of foods rich in phenols can prevent obesity by induction of pre-adipocyte and
adipocyte apoptosis, and by inhibition of adipocyte lipid accumulation. In particular, catechins
and epigallocatechin gallates (from green tea), resveratrol, and curcumin (from the curry spice
turmeric)canreducetheviabilityofadipocytesandproliferationofpre-adipocytes,suppressadipocyte
differentiationandtriglycerideaccumulation,stimulatelipolysisandfatty-acidβ-oxidation,andreduce
inflammation[42]. Moreover,epidemiologicalstudiesinhumanshaveassociatedhigherdietaryintake
andserumlevelsofcarotenoidswithdecreasedadiposity.Thisisprobablyduetotheanti-adiposityand
anti-inflammatoryactionsofthesecompoundsandtheirderivatives. Indeed,cellandanimalstudies
havedemonstratedthatsomecarotenoids,suchasfucoxanthin-,astaxanthin-,andβ-carotene-derived
retinoids, can suppress adipogenesis and activate lipid oxidation and thermogenesis in mature
adipocytes [43]. As for other classes of phytochemicals, animal studies have revealed that an
onion extract has anti-obesity effects [44]. In the same study, sulfur-containing components, such
ascycloalliin,S-methyl-L-cysteine,S-propyl-L-cysteinesulfoxide,dimethyltrisulfide,andespecially
S-methyl-L-cysteine sulfoxide, were reported to be effective for inhibition of the formation of oil
dropletsincells,whichsuggeststhatthesecompoundsareinvolvedintheanti-obesityeffectsofthe
onionextract.