Diterpenes in Coffea canephora.

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Original research article

Diterpenes in Coffea canephora

André Luiz Buzzo Mori

^a,

*, Daneysa Lahis Kalschne

^a

, Maria Amélia Gava Ferrão

^b

,

Aymbiré Francisco Almeida da Fonseca

^b

, Romário Gava Ferrão

^c

, Marta de Toledo Benassi

^a

aDepartamentodeCiênciadeTecnologiadeAlimentos,UniversidadeEstadualdeLondrina,Londrina,Paraná,Brazil

bEmbrapaCafé,Vitória,EspíritoSanto,Brazil

cInstitutoCapixabadePesquisa,AssistênciaTécnicaeExtensãoRural(Incaper),Vitória,EspíritoSanto,Brazil

ARTICLE INFO

Articlehistory:

Received2May2016

Receivedinrevisedform5August2016 Accepted9August2016

Availableonline10August2016

Chemicalcompoundsstudiedinthisarticle:

Kahweol(PubChemCID:114778) Cafestol(PubChemCID:108052) 16-O-methylcafestol(PubChemCID:

68103163) Keywords:

Kahweol Cafestol

16-O-methylcafestol UPLC

Conilon DiamanteES8112 ES8122‘Jequitibá’

CentenáriaES8132 Foodanalysis Foodcomposition

ABSTRACT

Thepresenceofditerpenesincoffeehasreceivedagreatdealofattentioninrecentyears,duetotheir physiologicaleffectsonhumanhealth.SomestudiesrelatedtokahweolandcafestolcontentsinCoffea arabicaareavailableintheliterature;however,informationontheimpactofgeneticvariabilityonthe proﬁleofditerpenesinCoffeacanephoraisscarce.Thisworkevaluatesthecontentsofkahweol,cafestol and16-O-methylcafestolin15genotypesofC.canephora.Coffees correspondedtothreecultivars– DiamanteES8112,ES8122‘Jequitibá’andCentenáriaES8132 –withdifferentfruit-ripeningseasons (early,mediumandlate).CoffeesweregrownattwolocationsinthestateofEspíritoSanto,thelargestC.

canephoragrowingregioninBrazil,resultingin30samples.Kahweolwasabsentin70%ofthesamples andthehighestcontentobservedwas14.1mg100g¹intheJequitibácultivar.Cafestolwaspresentinall samplesanditwasthemainrepresentativeofthediterpeneclass,withcontentsvaryingfrom152mg 100g¹to360mg 100g¹.Contentsof16-O-methylcafestolvariedfrom26.3mg 100g¹to132mg 100g¹.Asigniﬁcantdifferenceamonggenotypeswasobserved,andtherewasaninteractionbetween genotypesandgrowingsiteforthethreediterpenesstudied.

ã2016ElsevierInc.Allrightsreserved.

1.Introduction

The maincompounds ofthe unsaponifiablematterof coffee lipidsarethediterpeneskahweolandcafestol.Theconsumptionof unfilteredcoffeebrewhasbeenassociatedwithapossibleincrease inthelevelsofsericcholesterolandlowdensitylipoproteins;the effectsaretransientafterwithdrawalofthediterpenesandaredue mainly to the hypercholesterolemic activity of cafestol (Cano- Marquinaetal.,2013;HigdonandFrei,2006;Urgertetal.,1995, 1996).However, studies have stressed the beneficial effects of diterpeneingestiononhealth,duetotheiranti-carcinogenic,anti- inflammatoryandantioxidantactivities(Cavinetal.,2002; Kim etal.,2006;Gaaschtetal.,2015;Higginsetal.,2008;Leeetal., 2007;MurielandArauz,2010;Wangetal.,2012),suggestingthat

themoderateconsumptionofcoffeereducestheriskorseverityof severaldiseases(Freedmanetal.,2012),beingassociatedwitha reductioninmortalityrate(Dingetal.,2015).

Amongst the most commercially important coffee species, Coffeacanephorastandsoutforitsvigor,beingadaptedtoregions of low altitude and hightemperatures and resistant to hydric stress.Acoffeebrewwithlowacidity,withmorephenolic,spicy, paperyandwoodyaromasandbittertaste,andhighastringency andbodyisproducedwithC.canephora,whichisuseddirectlyin theproductionofinstantcoffeeandinblendswithCoffeaarabica forroastedcoffee(ClarkeandMacrae,1985;Williamsetal.,1989).

BrazilisthesecondlargestgrowerofC.canephoraintheworld, witha productionof 17millionbags of60kgin the2014/2015 harvest(USDA,2016),concentratedinthestatesofEspíritoSanto (70%)andRondônia(15%)(CONAB,2015;MAPA,2016).

Geneticdiversityhascontributedtothewidevariationinthe proﬁle of diterpenes for different coffee species and varieties (BenassiandDias,2015;Kitzbergeretal.,2013;SpeerandKölling-

*Correspondingauthorat:RodoviaCelsoGarciaCidPR445,km380,CaixaPostal 10011PostalCode:86057970,Londrina,PR,Brazil.

E-mailaddress:buzzo.mori@gmail.com(A.L.B.Mori).

http://dx.doi.org/10.1016/j.jfca.2016.08.004 0889-1575/ã2016ElsevierInc.Allrightsreserved.

ContentslistsavailableatScienceDirect

Journal of Food Composition and Analysis

j o u r n al h o m e p a g e : w w w . el s e v i e r . c o m / l o c a t e / j fc a

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Speer,2006).Diterpeneshavebeenstudiedaspossiblediscrim- inantsofthespeciesC.canephoraandC.arabicainroastedcoffee blends(De Souza and Benassi, 2012; Speerand Kölling-Speer, 2006).DiterpenescontentsinC.arabicaarewellknown,butthe literatureishighlylimitedandprovidesconﬂictinginformationfor C.canephora.Ingeneral,whencomparedtoC.arabica,C.canephora standsoutbecauseofitslowkahweolcontent(Campanhaetal., 2010;DeSouzaandBenassi,2012;SpeerandKölling-Speer,2006) and the presence of a speciﬁc diterpene, 16-O-methylcafestol (Pettitt,1987;Speeretal.,1991).Inarecentreview,Benassiand Dias(2015)studiedC.canephorafromseveralcountriesandunder differentroastingconditions,andfoundcontentsofcafestolfrom 76to363mg100g¹andtheabsenceofkahweolorcontentslower than14mg100g¹.Asfor16-O-methylcafestol,therearelittledata available,withcontentsfrom100.8mg100g¹to198.0mg100g¹ beingreportedinroastedcoffee(Schievanoetal.,2014).

InthebreedingprogramoftheInstitutoCapixabadePesquisa, Assistência Técnica e Extensão Rural (Incaper, Espírito Santo, Brazil),awidevarietyofC.canephoragenotypeshasbeingstudied fordifferentagronomictraits(Ferrãoetal.,2009).Ninecultivars weredevelopedandrecommendedforthestateofEspíritoSanto, outofwhicheightareclonalandoneisseed-propagated.Clonal cultivars are formed by groups of compatible clones of C.

canephora;each cultivar is formedbyat leastnine clonesused in the plantations. Diamante ES8112, ES8122 ‘Jequitibá', and CentenáriaES8132areexamplesof clonalcultivarsthatpresent differentfruit-ripeningseasons(early-maturing,medium-matur- ingandlate-maturing,respectively)(Incaper,2016a,b,c).

Consideringtheinterestinthestudyofditerpenes,thelackof dataonC. canephora matrix,and theimportance ofBrazilas a growerofthis coffee species,theobjectiveof thiswork was to evaluatethecontentsofkahweol,cafestoland16-O-methylcafes- tolin15clones(fromnowonreferredtoasgenotypes)ofthreeC.

canephora cultivars DiamanteES8112, ES8122‘Jequitibá' and CentenáriaES8132–grownintwolocations.

2.Materialsandmethods

2.1.Reagents,standardsandequipment

For extraction of diterpenes and preparation of the mobile phase,potassiumhydroxide(KOH)analyticalgrade(F.Maia,São Paulo, Brazil), ethanol 96% analytical grade (Êxodo Científica, Hortolândia, Brazil), acetonitrile HPLC grade (Fischer Scientific, Bridgewater,NJ)andmethyltert-butylether(MTBE)HPLCgrade (AcrósOrganics,MorrisPlains,NJ)wereused.Kahweolandcafestol (Axxora, San Diego, CA) of 98% purity certified by Alexis Biochemicals (Lausen, Switzerland) and 16-O-methylcafestol (Sigma-Aldrich, Saint Louis, MO) of 98.6% purity wereused as standards.Thewaterusedforthepreparation ofstandardsand solutions was obtained by Elga Purelab Option-Q purification system(VeoliaWaterTechnologies,Saint-Maurice,France).Nylon membranes of 0.22

m

^m ^were ^used ^to ﬁlter the mobile phase (Millipore,Billerica,MA)andsamples(Whatman,Maidstone,UK).

Chromatographicanalyseswereperformedinanultra-perfor- mance liquid chromatograph Waters Acquity (Waters, Milford, MA)equippedwithaﬂow-throughneedleinjector,aquaternary solvent pumping system, column heater/cooler module and photodiodearraydetector,controlledbytheEmpower3program.

For color analysis, a Minolta CR-410 colorimeter (Konica MinoltaSensing Inc.,Osaka,Japan)was usedtoobtainedtheL*

(lightness),andthechromaticcoordinatesa*(red-greencompo- nent)andb*(yellow-bluecomponent)intheCIELabsystem.The analyses were performed under the conditions of standard illuminantCand10observer.

For moisturedetermination, agravimetricmoistureanalyzer MB45(Ohaus,Barueri,Brazil)withhalogenlampwasused.The analysiswasperformedat105C,toaconstantsampleweight.

2.2.Geneticmaterialandpreparation

Fifteen genetic materials (genotypes) from C. canephora originatedfromthebreedingprogramofINCAPER(EspíritoSanto, Brazil)werestudied.Thesegenotypesareagronomicdivergentand correspond to three clonal cultivars that have distinct fruit- ripening seasons: Diamante ES8112–early-maturing (genotype 101E,103E,105E,106E,and108E),ES8122‘Jequitibá’–medium- maturing(genotype201M,202M,203M,207M,and209M)and CentenáriaES8132–late-maturing(genotype301L,302L,303L, 306L,and307L).Fiveoftheninegenotypeswerestudiedforeach cultivar.

Sampleswerecollectedfromdemonstrationcropsgrownunder two distinct experimental conditions and at 36 months: a) Experimental Farm of Marilândia – located in the county of Marilândia,intheNorthwestoftheEspíritoSantostate(1924⁰S, 4031⁰W),altitudeof104m,soilofthered-yellowlatosoltype,dry and hot,withanaverageannualtemperatureof 24.2C, annual rainfall of 1129mm; and b) Experimental Farm of Bananal do Norte, locatedin thecounty ofCachoeirodoItapemirim, inthe SouthernoftheEspíritoSantostate(2075⁰S,4129⁰W),altitude of 146m, soil of the red-yellow latosol type, dry and with an average annual temperature of 23.8C and annual rainfall of 1086mm.

Samples(500g) werecollectedin theyear2014,duringthe harvestfromMaytoJuly.First, theearly maturationgenotypes (May) were harvested manuallyfollowed by those of medium maturation(June)and late maturation(July). Onlycherrystage fruit werecollected. Coffeewerenaturallysun-dried,processed andcleaned.Onlynon-defectiveand16(6.5mm)sieve-sizebeans (Brasil,2003)wereselected.Thegreencoffeebeanswerestoredin plasticbagsatroomtemperatureuntilbeingroastedinOctober 2014.

Coffees(100g)wereroastedinaRodBel(Rod-Bel,SãoPaulo, Brazil)gaspilotroasterfor17–29min,attemperaturesfrom210C to230C(Mendesetal.,2001).Thediversityintheprocesswasdue todifferencesinsizeandcoffeebeancharacteristics.Thedegreeof roasting was standardized in order to achieve weight loss of 16.50.4g100g¹,similartothemethoddescribedbyMendes etal.(2001)foranoptimalroastingdegreeforC.canephora.

SamplesweregroundusingaBurrbenchgrinderGVX2(Krups, Shanghai,China).Roastcoffeewasgroundataﬁnegranulometry (0% retained in sieve size 1.18mm; 70% retained in sieve size 0.60mm,and30%passingasievesize0.60mm),accordingtoABIC (Brazilian Coffee Roasters Association) (ABIC, 2016). Roast and groundcoffeeshadL*of25.31.4,a*of8.20.5,b*of10.61.9, andmoistureof0.80.1g100g¹.Sampleswerestoredinplastic bagsandkeptat8Cuntilanalysis.

2.3.Kahweol,cafestoland16-O-methylcafestoldetermination

TheextractionwasperformedasdescribedbyDiasetal.(2010).

Samples (0.2000g) were saponiﬁed with 2.0mL of potassium hydroxide (2.5M)in ethanol (96% v/v) at80C for 1h. For the extractionoftheunsaponiﬁablematter,2.0mLofdistilledwater and2.0mLofMTBEwereadded.Afteragitationandcentrifugation (3minat3000rpmandroomtemperature),theorganicphasewas collected. This last stage of the procedure was repeated three times. Distilled water (2mL) was added for cleaning and the organicextractwascollectedandevaporatedtodrynessinawater bath (70C). After resuspension in 4.5mL of the mobilephase

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(45:55,v/v,water:acetonitrile),theextractwasﬁltered.Duplicate independentextractionswereperformed.

The analysis was carried out according to the method developedand validatedbyDias et al.(2010) and modiﬁedby Wuergesetal.(2016).Theanalysiswas carriedoutina Kinetex 2.6

m

^m ^C18 ⁽¹⁵⁰^mm4.6mm) (Phenomenex, Torrance, CA) column,at26C,withdetectionatthemaximumwavelengthof each diterpene: 230nm (cafestol and 16-O-methylcafestol) and 290nm(kahweol).Isocraticelutionofwater:acetonitrile(45:55, v/v)ataﬂowrateof1.2mLmin¹andaninjectionvolumeof1.4

m

^L

were applied. The total chromatographic run time was 7min.

Duplicateinjectionsweredone.

Identiﬁcationwas based onretention timesand UV spectra.

Quantiﬁcationwascarriedoutbyexternalstandardizationusing6- pointanalytical curves withtriplicatemeasurements(r0.999, p<0.001) in the concentrationrange of 2–200

m

^g^mL¹^, ^corre-

spondingto4.5mg100g¹,and450mg100g¹,respectively.

Considering the analytical curve parameters (ICH, 2005), a detectionlimit (DL) of 0.5

m

^g^mL¹ ^was ^obtained^for ^the ^three

compoundsandquantiﬁcationlimits (QL)of1.4

m

^g^mL¹^,^1.6

m

^g

mL¹ and 1.5

m

^g^mL¹ (corresponding to3.2mg100g¹,3.6mg 100g¹, and 3.4mg 100g¹) for kahweol, cafestol and 16-O- methylcafestol,respectively.Diterpenecontents wereexpressed onadryweightbasis(db).

2.4.Statisticaltreatment

To evaluatetheeffectofgrowing siteandgenetic variability, resultsweresubmittedtoANOVAandTukeyTest(p0.05)using thefreesoftwareSISVARversion5.6(SISVAR,2016).Growingsite/

experimentalfarm(mainplot)andgenotype(subplot)treatments were considered in a split-plot design. If a signiﬁcant main subplotinteraction(p0.05)wasobserved,theeffectofgenotype wasindependentlystudiedforeachexperimentalfarm.

3.Resultsanddiscussion

Tables1,2and3showthecontentsofkahweol,cafestoland16- O-methylcafestol, respectively, for 15 different genotypes of C.

canephoragrownattwosites.Thetotalcontentofditerpenes(the sumofkahweol,cafestoland16-O-methylcafestol)variedfrom191

to415mg100g¹.Cafestolwas themainrepresentative,witha contributionof66%–90%ofthetotalditerpenes.

Considering Diamante, Jequitibá and Centenária cultivars, levels of kahweol varied from absent (below LQ of 3.2mg 100g¹)to5.3mgkahweol100g¹(Table1).Theaveragecontent ofcafestolforeachcultivarvariedfrom200mg100g¹to264mg 100g¹(Table2).Thesevaluesareinagreementwiththeliterature thatreportstheabsenceofkahweolorcontentsbelow14mgof kahweol

¹⁰⁰^g¹^and^from⁷⁶^to³⁶³^mg^of^cafestol

¹⁰⁰^g¹^for^C.

canephora from several countries and under different roasting conditions(Campanhaetal.,2010;DeSouzaandBenassi,2012;

Lerckeretal.,1996;Sridevietal.,2011).

For kahweol contents, there was a difference (p<0.001) between genotypes but not between growing sites (p=0.117).

Considering the average contents for each cultivar, Jequitibá cultivar (medium-maturing)presented higher contents of kahweol; the highest content was observed for genotype 207M (14.1mg100g¹and 10.3mg100g¹)atthetwogrowing sites.

Kahweolwasabsentin70%ofthe30samplesanalyzed(Table1).

There was an interaction between growing site and genotype (p<0.001),showingthatkahweolcontentineachgenotypewas inﬂuencedbythegrowingsite;however,thiseffectwasgenotype- dependent.

A similar behavior was observed for cafestol: there was a difference (p<0.001) between genotypes but not between growing sites (p=0.149). In general, higher content of cafestol was observed for the Centenária cultivar (late-maturing); the highest valueswereobservedforgenotypes303Land 306L.An interactionbetweengrowingsiteandgenotypealsooccurredfor cafestol(p<0.001).Highercontentsofcafestolforgenotypes303L and306LwereobservedinMarilândia(around355mg100g¹)in comparisonwiththoseobtainedforthesamegenotypesinBananal doNorte(around298mg100g¹)(Table2).Theearly-maturing genotypes101E,103Eand 106E showed thelowest contentsof cafestolinMarilândia(165mg100g¹,173mg100g¹and178mg 100g¹)andgenotype103EshowedthelowestcontentinBananal doNorte,152mg100g¹(Table2).

Itisdifﬁculttocompareourresultswiththeliteraturedueto thelimiteddataavailableforC.canephora;additionallydifferent bases are still used to express diterpene contents. Speer and Kölling-Speer(2001),studyingditerpenesin greenC.canephora

Table1

Kahweolcontent^a(mg100g¹)inCoffeacanephoragenotypesgrownattwosites.

Cultivar Genotypes GrowingSite/ExperimentalFarm

Marilândia BananaldoNorte

Diamante(early-maturing) 101E 0.0^Be0.0 3.7^Ae0.0

103E 0.0^Ae0.0 0.0^Af0.0

105E 0.0^Ae0.0 0.0^Af0.0

106E 0.0^Ae0.0 0.0^Af0.0

108E 0.0^Ae0.0 0.0^Af0.0

Mean^bSD(CV%) 0.00.0(0.0) 0.71.6(228.6)

Jequitibá(medium-maturing) 201M 0.0^Ae0.0 0.0^Af0.0

202M 3.8^Bd0.3 4.7^Ad0.0

203M 8.4^Ab0.1 8.0^Bb0.3

207M 14.1^Aa0.3 10.3^Ba0.2

209M 0.0^Ae0.0 0.0^Af0.0

Mean^bSD(CV%) 5.36.0(113.3) 4.64.6(100.0)

Centenária(late-maturing) 301L 0.0^Ae0.0 0.0^Af0.0

302L 5.0^Bc0.1 6.1^Ac0.3

303L 0.0^Ae0.0 0.0^Af0.0

306L 0.0^Ae0.0 0.0^Af0.0

307L 0.0^Ae0.0 0.0^Af0.0

Mean^bSD(CV%) 1.02.2(220.0) 1.22.7(225.0)

Meansfollowedbythesamecapitalletterinthesamerowshownosigniﬁcantdifferencebetweengrowingsites(Tukey,p0.05).Meansfollowedbythesamelowercase letterinthesamecolumnshownosigniﬁcantdifferencebetweengenotypes(Tukey,p0.05).

aMean(duplicates)SD(standarddeviation)foreachgenotype;ZerovaluecorrespondstocontentsbelowQL(3.2mg100g¹).

b AveragecontentforeachcultivarSD(standarddeviation)andCV(coefﬁcientofvariation)betweengenotypesofthesamecultivar.

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coffeesoriginatingfromVietnam,theIvoryCoast,Indonesia,Zaire, UgandaandNewGuinea,reportedkahweolcontentsbelow10mg 100g¹ of unsaponiﬁable matter and cafestol contents up to 300mg100g¹ofunsaponiﬁablematter.Roosetal.(1997)found kahweolat levels below 8mg 100g¹ and cafestol contents of 239mg 100g¹ and250mg 100g¹in twosamplesof greenC.

canephoracoffeefromtheIvoryCoast.Kahweolwasnotdetected by De Souza and Benassi (2012) in three roasted Brazilian C.

canephora coffees originating from the states of Rondônia and Espírito Santo.Dias etal. (2010), studyingC. canephora Apoatã cultivar,didnotdetectthepresenceofkahweolintheendosperm ofgreen coffee,and reportedcafestolcontentof94mg 100g¹. Campanhaetal.(2010)reportedcontentsfrom163mgto275mg ofcafestol100g¹andtheabsenceofkahweolintwoC.canephora

braziliancoffees,fromRondônia andEspírito Santostates,with differentroastingdegrees.

DespitethehighkahweolvaluesobservedforJequitibácultivar (Table1)andofcafestolfortheCentenáriacultivar(Table2),no effectwasobservedonthecontentofthisditerpenerelatedtothe fruitripeningseason(early-maturing,medium-maturingorlate- maturing),since intra-cultivar variationsweremoresigniﬁcant.

Consideringtheﬁvegenotypesofeachcultivar,greatervariability was observed in thecontents of kahweol,witha coefﬁcient of variation(CV%)ofupto228.6%,comparedtocafestol(CVofupto 32.2%)(Tables1and2).

Theaveragecontentof16-O-methylcafestolfortheDiamante, JequitibáandCentenáriacultivarsvariedfrom52.9mg100g¹to 64.1mg100g¹,withhighvariabilitybetweentheﬁvegenotypes ineachcultivar(CVfrom20.3%to118.9%).Aneffectofthefruit- Table2

Cafestolcontent^a(mg100g¹)inCoffeacanephoragenotypesgrownattwosites.

Cultivar Genotypes Site/ExperimentalFarm

Diamante(early-maturing) 101E 164.8^Bg8.9 234.9^Ae1.4

103E 172.7^Afg2.8 151.7^Bg1.5

105E 230.9^Bc1.4 242.6^Ade4.6

106E 178.1^Befg0.5 235.5^Ae3.2

108E 254.0^Ab0.5 252.4^Acd3.1

Mean^bSD(CV%) 200.139.8(19.9) 223.440.7(18.2)

Jequitibá(medium-maturing) 201M 216.7^Bd0.4 261.4^Ac4.1

202M 231.3^Ac0.8 233.7^Ae5.1

203M 229.7^Bcd3.0 245.4^Ade5.8

207M 226.5^Bcd1.7 239.0^Ade6.7

209M 227.6^Acd0.8 182.0^Bf2.7

Mean^bSD(CV%) 226.45.7(2.5) 232.3729.9(18.2)

Centenária(late-maturing) 301L 237.0^Bc1.8 275.6^Ab4.0

302L 184.7^Aef1.3 174.9^Bf6.0

303L 349.5^Aa0.3 296.3^Ba0.1

306L 359.7^Aa1.7 300.0^Ba2.6

307L 190.3^Ae3.6 178.4^Bf6.1

Mean^bSD(CV%) 264.285.0(32.2) 245.063.1(25.8)

aMean(duplicates)SD(standarddeviation)foreachgenotype.

Table3

Content^aof16-O-methylcafestol(mg100g¹)inCoffeacanephoragenotypesgrownattwosites.

Cultivar Genotypes Site/ExperimentalFarm

Diamante(early-maturing) 101E 26.3^Bg0.7 40.8^Aj0.8

103E 35.9^Bf0.7 42.0^Aij0.6

105E 34.0^Bf0.2 47.1^Ahi1.4

106E 36.3^Bf0.4 53.1^Afg0.1

108E 132.1^Aa2.3 120.8^Ba0.9

Mean^bSD(CV%) 52.944.5(118.9) 60.833.9(55.8)

Jequitibá(medium-maturing) 201M 34.8^Bf0.4 44.2^Ahij2.0

202M 49.3^Ae0.2 46.0^Bhij2.1

203M 49.0^Be1.6 59.7^Ae1.1

207M 68.1^Bc0.5 91.8^Ab3.6

209M 77.7^Ab0.9 78.7^Ac0.2

Mean^bSD(CV%) 55.817.0(30,5) 64.120.7(32.3)

Centenária(late-maturing) 301L 48.9^Be0.6 59.6^Ae1.1

302L 77.2^Bb0.5 83.3^Ac1.3

303L 36.8^Bf0.7 48.7^Agh1.5

306L 54.9^Bd0.3 58.1^Aef2.0

307L 53.9^Bde0.1 65.1^Ad3.6

Mean^bSD(CV%) 54.314.6(26.9) 63.012.8(20.3)

aMean(duplicates)SD(standarddeviation)foreachgenotype.

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ripening season (early-maturing, medium-maturing or late- maturing)onthe16-O-methylcafestolcontentwasnotobserved (Table3).

Thereweresigniﬁcantdifferences(p<0.001) betweengenotypes and growing sites (p<0.029) for the contents of 16-O- methylcafestol.Forkahweolandcafestolnosystematiceffectof growingsitewasobserved.

In general,higher contents of 16-O-methylcafestol were ob- servedfor thecoffees grown in Bananaldo Norte. The highest contentswereobservedingenotype108E(early-maturing)atthe twogrowingsites(132mg100g¹and121mg100g¹)(Table3).

Twoearly-maturing genotypes (101E, 103E) and one medium- maturinggenotype(201M)stoodoutduetotheirlowercontentsof 16-O-methylcafestol(below44.2mg100g¹)atthetwogrowing sites.Literaturedataon16-O-methylcafestolarescarceandthereis no consensus regarding the concentration range. For green C.

canephoracoffees,contentsbetween1.0and5.0mg100g¹were citedbySpeerandKölling-Speer(2006)whileBelitzetal.(2009) reporteda rangefrom60 to180mg 100g¹. Contentsof 16-O- methylcafestolfrom102to154mg100g¹ingreencoffeesfromthe IvoryCoastwerereportedbyRoosetal.(1997).Forroastedcoffee, Schievano et al. (2014) reported contents from 101 to 198mg 100g¹.Pacettietal.(2012)analyzedditerpenesinfourroastedC.

canephoracoffeesfromIndia,VietnamandtheIvoryCoast,and reportedcontentsof16-O-methylcafestolbetween16.2mg100g¹ and2.6210⁴mg100g¹ofunsaponiﬁablematter.

Pettitt (1987) and Speer et al. (1991) described that 16-O- metihylcafestolwaspresentonlyinC.canephoraatlowconcen- trationsandintheCoffeadewevreispecies.Asitisthermallystable, thiscompoundcouldbeusedasanindicatorofthepresenceofC.

canephoraspeciesinroastedcoffeeproducts(Kemsleyetal.,1995).

InGermany,thequantiﬁcation of16-O-methylcafestolisrecom- mended by the norm DIN 10779 (published in 1999) for the evaluation of C. canephora coffee percentage in blends with C.

arabica(Speerand Kölling-Speer,2006).AsBrazilisthesecond largest producer of this coffee species, the contents of 16-O- methylcafestolreportedinthisstudy,inwhichagreatnumberof genotypes were evaluated, can help establish a concentration rangeofthisditerpeneinC.canephoracoffees.

Byevaluatingthewiderangeof16-O-methylcafestolcontents, from26.3to132mg100g¹,itissuggestedthatthesoleuseofthis compound contentcould notbe enoughto safelyestimatethe percentage ofC. canephora in blends withC. arabica.A similar observationwasmadebySchievanoetal.(2014).Theauthorsalso reportedawiderangefor16-O-methylcafestolcontentinroasted coffeeofthreedifferentoriginsandpointedthatsuchvariationisa problemforC.canephoraquantiﬁcationinblendswithC.arabica, regardlessoftheanalyticalmethodused.

DeSouzaandBenassi(2012)proposetheuseoftherelationship kahweol/cafestolasanadditionaltooltoestimatethepresenceof C.canephorainblendswithC.arabica.Theseauthorsstatedthata kahweol/cafestolratioabove1.00isindicativeofC.arabicaandthe additionofC.canephoracoffeeshoulddecreasethisratio.Inour study, a kahweol/cafestol ratio between 0.00 and 0.06 was obtained; in accordance to De Souza and Benassi (2012) that kahweol/cafestol ratio could be indicative of C. canephora.

Considering the results, we propose that the combined use of 16-O-methylcafestolcontentwithkahweol/cafestolratiocouldbe useful in the discrimination of coffee species. Further studies wouldverifyifthesetwoparameterscouldindicatewithefﬁciency theadditionofC.canephoratoC.arabicainroastedcoffeeblends.

4.Conclusions

ThediterpenesproﬁleofC.canephoragenotypesstudiedhere reinforcesthe wide variation of kahweol and cafestol contents

describedintheliteratureandshowsahighvariabilityfor16-O- methylcafestolcontent.Cafestolrepresentsthelargestproportion ofditerpenesinC.canephora,beingthediterpenewiththelowest variabilitybetweengenotypesofthesamecultivar.Kahweolwas absentinmostgenotypesstudied.

The fruit-ripening seasons of the cultivars (early-maturing, medium-maturing or late-maturing) do not affect diterpene content, thevariation betweengenotypes of the same cultivar being more relevant. Only contents of 16-O-methylcafestol presentedasigniﬁcantinﬂuenceofgrowingsite,butaninteraction between growing site and genotype was observed for all diterpenes.

Acknowledgements

The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico, Brazil) and CAPES (Comissão de Aperfeiçoamento de Pessoal do Nível Superior, Brazilforthescholarshipsandﬁnancialsupport(CNPq14/2014– Process:445757/2014-0)andtoIAPAR(InstitutoAgronômicodo Paraná,Brazil)fortheassistanceintheroastingprocess.

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