Diterpenes in Coffea canephora.

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

a

DepartamentodeCiênciadeTecnologiadeAlimentos,UniversidadeEstadualdeLondrina,Londrina,Paraná,Brazil

b

EmbrapaCafé,Vitória,EspíritoSanto,Brazil

c

InstitutoCapixabadePesquisa,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

andthehighestcontentobservedwas14.1mg100g
1intheJequitibácultivar.Cafestolwaspresentinall

samplesanditwasthemainrepresentativeofthediterpeneclass,withcontentsvaryingfrom152mg

100g
1 to360mg 100g
1.Contentsof16-O-methylcafestolvariedfrom26.3mg 100g
1to132mg

100g
1.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;Speerand Kölling-*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

Speer,2006).Diterpeneshavebeenstudiedaspossible discrim-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 76to363mg100g
1andtheabsenceofkahweolorcontentslower than14mg100g
1.Asfor16-O-methylcafestol,therearelittledata available,withcontentsfrom100.8mg100g
1to198.0mg100g
1 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,cafestoland 16-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 puri_fication 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). Chromatographicanalyseswereperformedinan ultra-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-green compo-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 24.2_{C, annual}

rainfall of 1129mm; and b) Experimental Farm of Bananal do Norte, locatedin thecounty ofCachoeirodoItapemirim, inthe SouthernoftheEspíritoSantostate(20₇₅0_S,41₂₉0_W),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 16.50.4g100g
1,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, andmoistureof0.8_0.1g100g
1.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

(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 (150mm4.6mm) (Phenomenex, Torrance, CA) column,at26C,withdetectionatthemaximumwavelengthof each diterpene: 230nm (cafestol and 16-O-methylcafestol) and 290nm(kahweol).Isocraticelutionofwater:acetonitrile(45:55, v/v)ataflowrateof1.2mLmin
1andaninjectionvolumeof1.4

m

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

Identificationwas based onretention timesand UV spectra. Quantificationwascarriedoutbyexternalstandardizationusing 6-pointanalytical curves withtriplicatemeasurements(r0.999, p<0.001) in the concentrationrange of 2–200

m

gmL
1, corre-spondingto4.5mg100g
1,and450mg100g
1,respectively.

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

m

gmL
1was obtainedfor the three compoundsandquantificationlimits (QL)of1.4

m

gmL
1,1.6

m

g mL
1and 1.5

m

gmL
1(corresponding to3.2mg100g
1,3.6mg 100g
1, and 3.4mg 100g
1) 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,cafestoland 16-O-methylcafestol, respectively, for 15 different genotypes of C. canephoragrownattwosites.Thetotalcontentofditerpenes(the sumofkahweol,cafestoland16-O-methylcafestol)variedfrom191

to415mg100g
1.Cafestolwas themainrepresentative,witha contributionof66%–90%ofthetotalditerpenes.

Considering Diamante, Jequitibá and Centenária cultivars, levels of kahweol varied from absent (below LQ of 3.2mg 100g
1)to5.3mgkahweol100g
1(Table1).Theaveragecontent ofcafestolforeachcultivarvariedfrom200mg100g
1to264mg 100g
1(Table2).Thesevaluesareinagreementwiththeliterature thatreportstheabsenceofkahweolorcontentsbelow14mgof kahweol



100g
1andfrom76to363mgofcafestol



100g
1forC. 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 kah-weol; the highest content was observed for genotype 207M (14.1mg100g
1and 10.3mg100g
1)atthetwogrowing sites. Kahweolwasabsentin70%ofthe30samplesanalyzed(Table1). There was an interaction between growing site and genotype (p<0.001),showingthatkahweolcontentineachgenotypewas influencedbythegrowingsite;however,thiseffectwas genotype-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(around355mg100g
1)in comparisonwiththoseobtainedforthesamegenotypesinBananal doNorte(around298mg100g
1)(Table2).Theearly-maturing genotypes101E,103Eand 106E showed thelowest contentsof cafestolinMarilândia(165mg100g
1,173mg100g
1and178mg 100g
1)andgenotype103EshowedthelowestcontentinBananal doNorte,152mg100g
1(Table2).

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

Table1

Kahweolcontenta_(mg100g
1_{)inCoffeacanephoragenotypesgrownattwosites.}

Cultivar Genotypes GrowingSite/ExperimentalFarm

Marilândia BananaldoNorte Diamante(early-maturing) 101E 0.0Be_

0.0 3.7Ae_ 0.0 103E 0.0Ae_ 0.0 0.0Af_ 0.0 105E 0.0Ae_ 0.0 0.0Af_ 0.0 106E 0.0Ae_{0.0 0.0}Af_0.0 108E 0.0Ae_{0.0 0.0}Af_0.0 Meanb SD(CV%) 0.00.0(0.0) 0.71.6(228.6) Jequitibá(medium-maturing) 201M 0.0Ae_

0.0 0.0Af_ 0.0 202M 3.8Bd_ 0.3 4.7Ad_ 0.0 203M 8.4Ab_ 0.1 8.0Bb_ 0.3 207M 14.1Aa_ 0.3 10.3Ba_ 0.2 209M 0.0Ae_{0.0 0.0}Af_0.0 Meanb SD(CV%) 5.36.0(113.3) 4.64.6(100.0) Centenária(late-maturing) 301L 0.0Ae

0.0 0.0Af 0.0 302L 5.0Bc_ 0.1 6.1Ac_ 0.3 303L 0.0Ae_ 0.0 0.0Af_ 0.0 306L 0.0Ae_ 0.0 0.0Af_ 0.0 307L 0.0Ae_ 0.0 0.0Af_ 0.0 Meanb_{SD(CV%) 1.02.2(220.0) 1.22.7(225.0)} Meansfollowedbythesamecapitalletterinthesamerowshownosignificantdifferencebetweengrowingsites(Tukey,p0.05).Meansfollowedbythesamelowercase letterinthesamecolumnshownosignificantdifferencebetweengenotypes(Tukey,p0.05).

a

Mean(duplicates)SD(standarddeviation)foreachgenotype;ZerovaluecorrespondstocontentsbelowQL(3.2mg100g
1).

b

coffeesoriginatingfromVietnam,theIvoryCoast,Indonesia,Zaire, UgandaandNewGuinea,reportedkahweolcontentsbelow10mg 100g
1 of unsaponifiable matter and cafestol contents up to 300mg100g
1ofunsaponifiablematter.Roosetal.(1997)found kahweolat levels below 8mg 100g
1 and cafestol contents of 239mg 100g
1and250mg 100g
1in 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 100g
1.

Campanhaetal.(2010)reportedcontentsfrom163mgto275mg ofcafestol100g
1andtheabsenceofkahweolintwoC.canephora

braziliancoffees,fromRondônia andEspírito Santostates,with differentroastingdegrees.

DespitethehighkahweolvaluesobservedforJequitibácultivar (Table1)andofcafestolfortheCentenáriacultivar(Table2),no effectwasobservedonthecontentofthisditerpenerelatedtothe fruitripeningseason(early-maturing,medium-maturingor late-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, JequitibáandCentenáriacultivarsvariedfrom52.9mg100g
1to 64.1mg100g
1,withhighvariabilitybetweenthefivegenotypes ineachcultivar(CVfrom20.3%to118.9%).Aneffectofthe

fruit-Table2 Cafestolcontenta

(mg100g
1)inCoffeacanephoragenotypesgrownattwosites.

Cultivar Genotypes Site/ExperimentalFarm

Marilândia BananaldoNorte Diamante(early-maturing) 101E 164.8Bg_{8.9 234.9}Ae_1.4

103E 172.7Afg 2.8 151.7Bg 1.5 105E 230.9Bc_ 1.4 242.6Ade_ 4.6 106E 178.1Befg_ 0.5 235.5Ae_ 3.2 108E 254.0Ab_ 0.5 252.4Acd_ 3.1 Meanb_{SD(CV%) 200.139.8(19.9) 223.440.7(18.2)} Jequitibá(medium-maturing) 201M 216.7Bd_{0.4 261.4}Ac_4.1

202M 231.3Ac 0.8 233.7Ae 5.1 203M 229.7Bcd 3.0 245.4Ade 5.8 207M 226.5Bcd_ 1.7 239.0Ade_ 6.7 209M 227.6Acd_ 0.8 182.0Bf_ 2.7 Meanb_ SD(CV%) 226.45.7(2.5) 232.3729.9(18.2) Centenária(late-maturing) 301L 237.0Bc_{1.8 275.6}Ab_4.0

302L 184.7Aef_{1.3 174.9}Bf_6.0 303L 349.5Aa 0.3 296.3Ba 0.1 306L 359.7Aa_ 1.7 300.0Ba_ 2.6 307L 190.3Ae_ 3.6 178.4Bf_ 6.1 Meanb_ SD(CV%) 264.285.0(32.2) 245.063.1(25.8) Meansfollowedbythesamecapitalletterinthesamerowshownosignificantdifferencebetweengrowingsites(Tukey,p0.05).Meansfollowedbythesamelowercase letterinthesamecolumnshownosignificantdifferencebetweengenotypes(Tukey,p0.05).

a

Mean(duplicates)SD(standarddeviation)foreachgenotype.

b

AveragecontentforeachcultivarSD(standarddeviation)andCV(coefficientofvariation)betweengenotypesofthesamecultivar.

Table3 Contenta

of16-O-methylcafestol(mg100g
1)inCoffeacanephoragenotypesgrownattwosites.

Cultivar Genotypes Site/ExperimentalFarm

Marilândia BananaldoNorte Diamante(early-maturing) 101E 26.3Bg_{0.7 40.8}Aj_0.8

103E 35.9Bf 0.7 42.0Aij 0.6 105E 34.0Bf_ 0.2 47.1Ahi_ 1.4 106E 36.3Bf_ 0.4 53.1Afg_ 0.1 108E 132.1Aa_ 2.3 120.8Ba_ 0.9 Meanb_ SD(CV%) 52.944.5(118.9) 60.833.9(55.8) Jequitibá(medium-maturing) 201M 34.8Bf_{0.4 44.2}Ahij_2.0

202M 49.3Ae_{0.2 46.0}Bhij_2.1 203M 49.0Be 1.6 59.7Ae 1.1 207M 68.1Bc_ 0.5 91.8Ab_ 3.6 209M 77.7Ab_ 0.9 78.7Ac_ 0.2 Meanb_ SD(CV%) 55.817.0(30,5) 64.120.7(32.3) Centenária(late-maturing) 301L 48.9Be_

0.6 59.6Ae_ 1.1 302L 77.2Bb_{0.5 83.3}Ac_1.3 303L 36.8Bf_{0.7 48.7}Agh_1.5 306L 54.9Bd 0.3 58.1Aef 2.0 307L 53.9Bde_ 0.1 65.1Ad_ 3.6 Meanb_ SD(CV%) 54.314.6(26.9) 63.012.8(20.3) Meansfollowedbythesamecapitalletterinthesamerowshownosignificantdifferencebetweengrowingsites(Tukey,p0.05).Meansfollowedbythesamelowercase letterinthesamecolumnshownosignificantdifferencebetweengenotypes(Tukey,p0.05).

a_{Mean(duplicates)SD(standarddeviation)foreachgenotype.} b

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

Thereweresignificant differences(p<0.001) between geno-types 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(132mg100g
1and121mg100g
1)(Table3). Twoearly-maturing genotypes (101E, 103E) and one medium-maturinggenotype(201M)stoodoutduetotheirlowercontentsof 16-O-methylcafestol(below44.2mg100g
1)atthetwogrowing sites.Literaturedataon16-O-methylcafestolarescarceandthereis no consensus regarding the concentration range. For green C. canephoracoffees,contentsbetween1.0and5.0mg100g
1were citedbySpeerandKölling-Speer(2006)whileBelitzetal.(2009)

reporteda rangefrom60 to180mg 100g
1. Contentsof 16-O-methylcafestolfrom102to154mg100g
1ingreencoffeesfromthe IvoryCoastwerereportedbyRoosetal.(1997).Forroastedcoffee,

Schievano et al. (2014) reported contents from 101 to 198mg 100g
1.Pacettietal.(2012)analyzedditerpenesinfourroastedC. canephoracoffeesfromIndia,VietnamandtheIvoryCoast,and reportedcontentsof16-O-methylcafestolbetween16.2mg100g
1 and2.62104_mg100g
1_{ofunsaponifiablematter.}

Pettitt (1987) and Speer et al. (1991) described that 16-O-metihylcafestolwaspresentonlyinC.canephoraatlow concen-trationsandintheCoffeadewevreispecies.Asitisthermallystable, thiscompoundcouldbeusedasanindicatorofthepresenceofC. canephoraspeciesinroastedcoffeeproducts(Kemsleyetal.,1995). InGermany,thequantificationof16-O-methylcafestolis recom-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, from26.3to132mg100g
1,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

describedintheliteratureandshowsahighvariabilityfor 16-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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