Polyphenol Diversity and Antioxidant Activity of European Cistus creticus L. (Cistaceae) Compared to Six Further, Partly Sympatric Cistus Species

Abstract

This investigation focused on the qualitative and quantitative composition of polyphe-nolic compounds of Mediterranean northern shore Cistus creticus and six further, partly sympatric Cistus species (C. albidus, C. crispus, C. ladanifer, C. monspeliensis, C. parviflorus, C. salviifolius). Aque-ous extracts of 1153 individual plants from 13 countries were analyzed via high performance liquid chromatography (HPLC). The extracts of C. creticus were primarily composed of two ellagitannins (punicalagin and punicalagin gallate) and nine flavonol glycosides (myricetin and quercetin glyco-sides, with m-3-O-rhamnoside as the dominant main compound). Differences in the proportions of punicalagin derivatives and flavonol glycosides allowed the classification into two chemovariants. Plants containing punicalagin derivatives and flavonol glycosides were especially abundant in the western and central Mediterranean areas and in Cyprus. From Albania eastwards, punicalagin and punicalagin gallate were of much lesser importance and the predominant chemovariant there was a nearly pure flavonol type. With its two chemovariants, C. creticus takes a central position between the flavonol-rich, purple-flowered clade (besides C. creticus, here represented by C. albidus and C. crispus) and the more ellagitannin-rich, white-or whitish-pink-flowered clade (here represented by C. ladanifer, C. monspeliensis, C. parviflorus and C. salviifolius). The median antioxidative capacity of C. creticus plant material was, with 166 mg Trolox equivalents/g dry wt, about half of the antioxida-tive capacity of C. ladanifer (301 mg te/g dry wt), the species with the highest antioxidative potential.

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Plants2021,10,615.https://doi.org/10.3390/plants10040615www.mdpi.com/journal/plants
Article
PolyphenolDiversityandAntioxidantActivityofEuropean
CistuscreticusL.(Cistaceae)ComparedtoSixFurther,Partly
SympatricCistusSpecies
BrigitteLukas*,LauraBragagna,KatharinaStarzyk,KlaudiaLabedz,KlausStolzeandJohannesNovak
InstituteofAnimalNutritionandFunctionalPlantCompounds,UniversityofVeterinaryMedicineVienna,
Veterinärplatz1,1210Vienna,Austria;laura.bragagna@gmail.com(L.B.);a01005378@unet.univie.ac.at(K.S.);
klaudia.lab@gmail.com(K.L.);klaus.stolze@drei.at(K.S.);Johannes.Novak@vetmeduni.ac.at(J.N.)
*Correspondence:Brigitte.Lukas@vetmeduni.ac.at;Tel.:+43‐1‐25077‐3110;Fax:+43‐1‐25077‐3190
Abstract:Thisinvestigationfocusedonthequalitativeandquantitativecompositionofpolyphe‐
noliccompoundsofMediterraneannorthernshoreCistuscreticusandsixfurther,partlysympatric
Cistusspecies(C.albidus,C.crispus,C.ladanifer,C.monspeliensis,C.parviflorus,C.salviifolius).Aque‐
ousextractsof1153individualplantsfrom13countrieswereanalyzedviahighperformanceliquid
chromatography(HPLC).TheextractsofC.creticuswereprimarilycomposedoftwoellagitannins
(punicalaginandpunicalagingallate)andnineflavonolglycosides(myricetinandquercetinglyco‐
sides,withm‐3‐O‐rhamnosideasthedominantmaincompound).Differencesintheproportionsof
punicalaginderivativesandflavonolglycosidesallowedtheclassificationintotwochemovariants.
Plantscontainingpunicalaginderivativesandflavonolglycosideswereespeciallyabundantinthe
westernandcentralMediterraneanareasandinCyprus.FromAlbaniaeastwards,punicalaginand
punicalagingallatewereofmuchlesserimportanceandthepredominantchemovarianttherewas
anearlypureflavonoltype.Withitstwochemovariants,C.creticustakesacentralpositionbetween
theflavonol‐rich,purple‐floweredclade(besidesC.creticus,hererepresentedbyC.albidusandC.
crispus)andthemoreellagitannin‐rich,white‐orwhitish‐pink‐floweredclade(hererepresentedby
C.ladanifer,C.monspeliensis,C.parviflorusandC.salviifolius).Themedianantioxidativecapacityof
C.creticusplantmaterialwas,with166mgTroloxequivalents/gdrywt,abouthalfoftheantioxida‐
tivecapacityofC.ladanifer(301mgte/gdrywt),thespecieswiththehighestantioxidativepotential.
Keywords:Cistuscriticus;Cistaceae;rockrose;HPLC;polyphenols;flavonoids;ellagitannins;anti‐
oxidantactivity;chemotaxonomy

1.Introduction

CistusL.(Cistaceae,Malvales)comprisesabout20frutescentandsuffrutescentshrub
speciesdistributedintheMediterranean,ontheCanaryIslandsandonMadeira.Thege‐
nusistaxonomicallycomplex,andhybridizationandahighdegreeofmorphologicalpol‐
ymorphismcomplicatethedeterminationofspeciesboundaries.Variousmonographs
haverecognizedbetween16and28speciesandhaveproposedconflictingintrageneric
classifications.Thelatesttaxonomictreatmentsbasedonmolecularphylogeneticsand
pollenanalysesrecognizedawell‐supported,purple‐floweredclade(equivalenttosub‐
genusCistusandincludingallpink‐floweredCistusspeciesexceptforC.parviflorus)and
asecond,sometimesweaklysupportedwhite‐andwhitish‐pink‐floweredclade(compris‐
ingthetwosubgeneraLeucocistusandHalimioidesandC.parviflorus)[1,2].Cistuscreticus
L.(syn.C.incanusauct.,C.villosusL.)isaprominentmemberofthesmaller,purple‐flow‐
eredcladeandoneofthefewCistusspecieswidelydistributedintheeasternMediterra‐
nean.Thespeciesseemstobeagoodtaxonomicentitybutappearstobehighlyvariable
withsomegeographicalstructuring.Thehighmorphologicalvariabilityisreflectedinthe
Citation:Lukas,B.;Bragagna,L.;
Starzyk,K.;Labedz,K.;Stolze,K.;
Novak,J.PolyphenolDiversityand
AntioxidantActivityofEuropean
CistuscreticusL.(Cistaceae)
ComparedtoSixFurther,Partly
SympatricCistusSpecies.Plants
2021,10,615.https://doi.org/10.3390/
plants10040615
AcademicEditor:AntonellaSmeriglio
Received:3March2021
Accepted:21March2021
Published:24March2021
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinstitu‐
tionalaffiliations.

Copyright:©2021bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(http://crea‐
tivecommons.org/licenses/by/4.0/).

Plants2021,10,6152of20
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plethoraofscientificnamesgiventothepresentlyrecognizedC.creticustodistinguishthe
variouskindsofvariation.ThespecificnameC.creticusseemstobewidelyaccepted
(Euro+MedPlantBase,[3])buttheputativesynonymsC.incanusandC.villosushavebeen
frequentlyappliedinlocalflorasandtherecentliterature(seelistofreferences).Basedon
morphologicalandphytochemicalcharacteristics,someauthorsrecognizedthreesubspe‐
cies,Cistuscreticussubsp.eriocephalus(Viv.)GreuterandBurdet,C.creticussubsp.corsicus
(Loisel.)GreuterandBurdet(bothpoorinessentialoil)andC.creticussubsp.creticus,rich
inessentialoil(e.g.,[4–6]).
AmongstotherCistusspecies,C.creticushasbeenknownasamedicinalplantsince
ancienttimes.Ladanum,exudatesfromtheglandularhairsofthestemandleaves,or
herbalinfusionsfromleavesandupperstemshavebeenusedtohealeczema,abscesses,
furunclesanddiarrheaortotreathairloss(e.g.,[7,8]).Today,herbalinfusions,extractsor
cremesareconsumedorappliedforthetreatmentof,e.g.,influenza,respiratorydisorders,
borreliosesorskinirritations.Thediversepositivehealtheffectsobservedcanbeat‐
tributedtoawidespectrumofsecondarycompounds.TheessentialoilofC.creticusisrich
inlabdane‐typediterpenes(e.g.,[5,6,9,10]).Additionally,thepresenceofawiderangeof
non‐volatilecompounds,mainlybelongingtophenolicacids,ellagitanninsandflavonols,
wasdescribed[11–14].Thereisthusconsiderableinterestinthisspeciesandavastnum‐
berofpublicationspresentdiversepharmacologicalactivities,e.g.,antioxidant(e.g.,
[12,15]),anti‐inflammatory[16],antiviral(e.g.,[17–19]),antimicrobial(e.g.,[14,20–24]),an‐
ticancer[25–27],cardiovascularprotecting[28]orskinprotecting(e.g.,[29,30])activities.
Pharmacologicalpropertiesand,subsequently,healthbenefitsofplantpreparations,
however,arestronglydependentontheirsecondarycompoundcomposition.Despitethe
increasingknowledgeofpromisingpropertiesofC.creticusplantmaterial,thenatural
variabilityoftheresponsiblecompoundsislargelyunknown.Pharmacologically,me‐
thodicallyormorecompoundcharacterization‐orientatedinvestigationsofC.creticus
plantmaterial(e.g.,[31–33])oftenrelyontradesamplesorplantmaterialfromjustoneor
afewpooledindividualplants.Inmanycases,plantmaterialusedforthestudieswasnot
chemicallycharacterized.Acomprehensiveoverviewisalsohinderedbytheconfusing
taxonomiccircumstancesandtheuseofdifferentmethodicalapproachesthatimpedea
comparisonandsummaryofresults.Thefewpreviousstudiesonnaturalbiodiversityof
C.creticuspresentedeitheruncharacterizedNMRdata[34]orwerefocusedontheessen‐
tialoilcomposition(e.g.,[5,6,9,10]).Notmuchisknownaboutabundanceandintraspe‐
cificvariabilityofnon‐volatilesecondarycompoundspresentinC.creticus.Theauthors
of[11]investigatedpolyphenoliccompoundsofoneorafewindividualplantsoftendif‐
ferentCistusspeciesnativetoSpain(amongthemC.creticus)andprovidedafirstcom‐
parativeoverviewaboutintragenericdiversityandspecies‐specificcharacteristicsofphe‐
nolicacidderivatives,ellagitanninsandflavonoids.Twomorepublicationsdescribedthe
flavonolandpunicalagin/punicalagingallatediversityofCypriotC.creticus[35]orcom‐
parativelydiscussednon‐volatilecompoundsofSardinianC.creticusbelongingtodiffer‐
entsubspecies[14].Themainaimofthisinvestigationwastoexpandthefragmentary
knowledgebycomparativelycharacterizingflavonolcompounddiversityofnaturalpop‐
ulationsofC.creticus,C.albidus,C.crispus(allthreefromthepurple‐floweredclade),C.
ladanifer,C.monspeliensis,C.parviflorusandC.salviifolius(fourspeciesofthewhite‐and
whitish‐pink‐floweredclade).Basedonourspecificexperimentalprocedure,besidefla‐
vonols,twomorecompoundsbelongingtotheellagitannincompoundfamily,puni‐
calaginandpunicalagingallate,wereprominentlypresentinC.creticusandwereincluded
inthecomparativeanalysis.Twoadditionalparameters,antioxidantactivity(2,2‐diphe‐
nyl‐1‐picrylhydrazyl(DPPH))andtotalphenoliccontent,weredeterminedtodefineand
comparativelydescribedrugactivity.Onethousandonehundredandfifty‐threeindivid‐
ualplantsofaltogether127populationsfrom13Mediterraneancountrieswerestudied
fortheirtotalphenoliccontent,compoundcompositionandantioxidantactivity.Addi‐
tionally,15commercialsampleswereanalyzedtoverifythelabelingandqualityofCistus
productsformedicalapplications.Theresultspresentedherecontributetotheknowledge

Plants2021,10,6153of20
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ofchemicalcharacteristicsandinter‐andintraspecificcompounddiversityofpromising
medicinalplants,mayprovideargumentsforchemosystematicconsiderationsaswellas
evidenceforpharmacologistsandmaytherewithfinallyhelptoimprovedrugactivity
andproductsafety.
2.Results
Intotal,onehundredandtwenty‐sevenpopulations(1153individualplants)of8dif‐
ferentCistustaxawereanalyzed.Cistuscreticuswasparamount(72populations,704indi‐
vidualplants),accompaniedbyC.albidus(13populations,131individualplants),C.xca‐
nescens(onepopulation,oneplant),C.crispus(twopopulations,22individualplants),C.
ladanifer(onepopulation,eightplants),C.monspeliensis(eightpopulations,89individual
plants),C.parviflorus(eightpopulations,48individualplants)andC.salviifolius(22pop‐
ulations,150individualplants).
2.1.ExtractCompositionandChemotypes
Chromatogramevaluationwasperformedat354nmfocusingonconspicuousand
recurrentpeakspresentinthespeciesofmaininterest,C.creticus.Altogether,13major
peaks(Table1;FigureS1,SupplementaryMaterial)representingbetween55and100%of
thetotalHPLCpeakarea(75to100%inC.creticus,70to100%inC.albidus,91to97%in
C.crispus,80to96%inC.ladanifer,65to93%inC.monspeliensis,55to90%inC.parviflorus
and60to100%inC.salviifolius;datanotshown)wereanalyzed.Amongthese13peaks
werenineflavonols,fourmyricetinglycosides(m‐3‐O‐galactoside,m‐3‐O‐glucoside,m‐
O‐xylosideorm‐3‐O‐arabinoside,m‐3‐O‐rhamnoside)andfivequercetinglycosides(q‐3‐
O‐rutinoside,q‐3‐O‐galactoside,q‐3‐O‐glucoside,q‐O‐xylosideorq‐3‐O‐arabinoside,q‐3‐
O‐rhamnoside).Basedoncompoundtablesoftherelevantliterature,weinitiallyfocused
onsuchmyricetinandquercetinglycosidesandweresurprisedbytheappearanceoffour
additionalprominentpeaksthatelutedmuchearlierandexhibiteddistinctUVspectra.
Thesefourpeaksweresubsequentlyidentifiedaspunicalaginderivatives,morespecifi‐
callytwopunicalaginisomersandtwopunicalagingallateisomers(Table1).
Amongtheminorcompounds,thepresenceoffurthermyricetinglycosides(onem‐
O‐rhamnoside‐O‐hexoside,oneputativebreakdownproductofm‐3‐O‐rhamnoside),
quercetinglycosides(oneq‐pentoside,q‐3‐O‐rutinoside‐7‐O‐hexosideand/orq‐3‐O‐
(2’caffeoyl)‐rutinosideandonefurtherq‐derivative)andellagitannins(cornusiinBiso‐
mers,bis‐hexahydroxydiphenoyl(HHDP)‐glucoseorpedunculagin,galloyl‐HHDP‐glu‐
coside,probably7‐xylosideellagicacid)wasindicated(datanotshown).Furtherminor
compoundsweretentativelyidentifiedaskaempferolderivatives(k‐3‐O‐galactosideor‐
glucoside,k‐diglucosides(s)ortilirosideisomersandonefurtherputativek‐derivative),
flavanols((epi)gallocatechindimerorprodelphinidinB4,(epi)catechin,(epi)gallocatechin
trimer)andapropiophenonederivative(3,4ʹ‐dihydroxypropiophenone‐3‐β‐D‐glucoside).
Table1.HPLCretentiontimes(Rt)ofthe13evaluatedpeaks,massdata(baseionsatnegativemode([M‐H]‐),mainfrag‐
mentions(MS/MS)),peakidentification(M=myricetin,Q=quercetin)andrelevanceofcompoundswithinaqueousex‐
tractsofC.creticus(rangeofrelativeareapercentagesat354nm).1)Highlightscompoundsthatwereidentifiedviarefer‐
encecompounds;2highlightspeaksthatwereshowntobedoublepeaksinapartoftheaccessions(characteristicsofthe
overlainpeakareindicatedbelowtherespectiveline).Identificationliterature[11–13,26,31,36–38].
PeakRt
(min)
[M‐H]‐
(m/z)
MS/MS
(m/z) ProposedCompoundLiteratureRel.Area
%
110.11083301/541Punicalagin,isomer11[11,13,26,31,36] 0–16%
214.41083301/541Punicalagin,isomer21[11,13,26,31,36] 0–19%
314.81251541/603Punicalagingallate,isomer1[11,26,31,36] 0–10%
422.11251541/603Punicalagingallate,isomer2[11,26,31,36] 0–14%
532.1479316M‐3‐O‐galactoside[11,12,26,31,37,38] 0–47%
632.8479316M‐3‐O‐glucoside[11–13,26,31,37,38]0–6%
737.3449316M‐O‐xylosideand/orm‐3‐O‐arabinoside2[26,31,37,38] 0–50%

Plants2021,10,6154of20
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838.4463316M‐3‐O‐rhamnoside(myricitrin)1[11–13,26,31,37,38]5–83%
939.3609 301 Q‐3‐O‐rutinoside(rutin)1,2[11–13,26,31,37,38]0–17%

521316Putativem‐derivative,ev.anartifact
1040.0463301Q‐3‐O‐galactoside(hyperoside)1[26,31,37,38] 0–12%
1140.8463301Q‐3‐O‐glucoside(isoquercetin)1[11,13,26,31,36–38]0–3%
1246.0433 301 Q‐O‐xylosideand/orq‐3‐O‐arabinoside2[11–13,26,31,36–38]0–9%
1348.9447301Q‐3‐O‐rhamnoside(quercitrin)1[11–13,26,31,37,38]0–35%
Tovisualizetheincidenceandvariabilityofthemajorcompounds,thepeakareasof
thefourmainmyricetinglycosides(m‐3‐O‐galactoside,m‐3‐O‐glucoside,m‐O‐xyloside
orm‐3‐O‐arabinoside,m‐3‐O‐rhamnoside),thefivemainquercetinglycosides(q‐3‐O‐ru‐
tinoside,q‐3‐O‐galactoside,q‐3‐O‐glucoside,q‐O‐xylosideorq‐3‐O‐arabinoside,q‐3‐O‐
rhamnoside)andthetwopunicalaginderivatives(punicalaginandpunicalagingallate)
werequantified(equivalenttotherespectivemajorcompoundm‐3‐O‐rhamnoside,q‐3‐
O‐rhamnosideandpunicalagin),cumulatedandplotted(Figure1).
2.1.1.MyricetinGlycosides
Theoverallhighestcontentsofmyricetinderivativeswerepresentinthethreeflavo‐
nol‐rich,purple‐floweringspecies(C.creticus,C.albidusandC.crispus),whereasthewhite
(C.ladanifer,C.monspeliensis,C.salviifolius)‐orpink(C.parviflorus)‐floweringspeciesex‐
hibitedloweramountsorweredevoidofsomeofthesecompounds(Figure1a).Witha
mediancontentof11mg/gdrywt,C.creticusdifferedsignificantlyfromC.albidus(7mg/g
drywt),C.salviifolius(4mg/gdrywt)andthepairC.ladaniferandC.parviflorus(<LOD
and0.3mg/gdrywt).Withinthepurple‐floweredspecies,C.crispus(9mg/gdrywt)could
notbedifferentiatedfromC.creticusandC.albidus.Cistusmonspeliensis,with5mg/gdry
wt,thewhite‐floweredspeciesrichestinmyricetinglycosides,occupiedacentralposition
betweenC.albidusandC.salviifolius.Withinthethreepurple‐floweringspecies,m‐3‐O‐
rhamnosidewasthemainmyricetinglycosideandmaincompound(upto83%relative
peakareapercentageinC.albidus;datanotshown).Inrarecases,m‐3‐O‐galactosidewas
higherthanm‐3‐O‐rhamnoside(upto47%inanAlbanianaccessionofC.creticus;datanot
shown).Withinthewhite‐orthepink‐floweringspecies,apredominanceofm‐3‐O‐galac‐
tosideorm‐O‐xylosidewasmorefrequentortherule.WithinC.parviflorusandC.salviifo‐
lius,m‐3‐O‐rhamnosidewasgenerallyloworevennotpresent(TableS1,Supplementary
Material).
WithinC.creticus,thehighestamountsofmyricetinglycosidesweredetectedinItaly
andCyprus(mediancontentof13and12mg/gdrywt,respectively;Figure1b).Popula‐
tionsderivingfromthesetwocountriesdifferedsignificantlyfrompopulationsoriginat‐
ingfromGreece,Ukraine,IsraelandJordangrowninthegreenhouse(mediancontents
between5and8mg/gdrywt)thatexhibitedtheoveralllowestamountsofmyricetingly‐
cosides.WildpopulationsofAlbania(12mg/gdrywt)werenotsignificantlydifferent
fromthepopulationsrichestinmyricetinglycosides.WildpopulationsfromSpainand
Croatia(both10mg/gdrywt)andtheLebanesegreenhousepopulation(10mg/gdrywt)
werenotsignificantlydifferentfrompopulationspoorinmyricetinglycosides.
2.1.2.QuercetinGlycosides
Withamediancontentof5mg/gdrywt,C.salviifoliuswasthespecieswiththehigh‐
estcontentofquercetinderivatives(Figure1c).Significantdifferencesweredetectedbe‐
tweenC.salviifolius,C.creticus(3mg/gdrywt),C.albidus(2mg/gdrywt)andthethree
speciescomparativelylowinorcompletelylackingquercetinglycosides(C.ladanifer,C.
monspeliensisandC.parvifloruswithcontentsbetween0and1mg/gdrywt).Cistuscrispus
(1mg/gdrywt)tookacentralpositionbetweenC.albidusandthethreewhite‐flowering
specieslowinquercetinglycosides.TheextraordinaryC.salviifoliuswasthesolespecies
withquercetinglycosidesasthepredominantflavonolcompoundfamily.Intheflavonol‐
richaccessionsofpurple‐floweringC.creticus,C.albidusandC.crispus,thecontentsof

Plants2021,10,6155of20
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
quercetinglycosideswereusuallyconspicuouslylowerthanthoseofthemyricetinglyco‐
sides.WithinC.monspeliensisandC.parviflorus,thepredominanceofthemyricetinglyco‐
sideswasnotthatdistinctive.Cistusladaniferlackedbothmyricetinandquercetinglyco‐
sides.InC.creticusandC.albidus,q‐3‐O‐rhamnosidewasoftenthemainquercetinglyco‐
side(upto34%and80%relativepeakareapercentage;datanotshown).However,the
predominanceofthequercetinrhamnosidewasnotaspronouncedasthatofthemyricetin
rhamnoside(seeabove).Someaccessionsevenshowedq‐3‐O‐rutinosideorq‐3‐O‐galac‐
tosideasthemainquercetinglycosides(about20%oftheC.creticusaccessions,about2%
oftheC.albidusaccessions;datanotshown).WithinC.crispus,q‐3‐O‐galactosidewasthe
mainquercetinglycoside.WithinC.monspeliensis,q‐3‐O‐galactoside,q‐O‐xylosideandq‐
3‐O‐rhamnosidewerepresentinhigheramounts.WithinC.parviflorusandC.salviifolius,
q‐3‐O‐rhamnosidewasonlypresentintraceamountsandq‐3‐O‐galactosideandq‐O‐xy‐
loside(and/ororq‐3‐O‐arabinoside)werethemainquercetinglycosides(TableS1,Sup‐
plementaryMaterial).
WithinC.creticus,theoverallhighestamountsofquercetinglycosideswerepresent
inpopulationsofSpainandItaly(mediancontentsof5and4mg/gdrywt;Figure1d).
BothcanclearlybedifferentiatedfrompopulationsofCyprus(3mg/gdrywt)andthe
greenhousepopulationsfromGreece,UkraineandtheNearEast(allaround1mg/gdry
wt).ThepopulationsfromCroatiaandAlbania(bothwithamediancontentaround2
mg/gdrywt)werenotclearlydifferentiated.
2.1.3.PunicalaginDerivatives
Theoverallhighestamountsofpunicalaginandpunicalagingallatewerepresentin
thealsohereoutstandingC.salviifolius(mediancontentof149mg/gdrywt).Cistussalviifo‐
liusdifferedsignificantlyfromthefurtherthreewhite‐ orpink‐floweringspecies(C.
ladanifer,C.monspeliensisandC.parviflorus,withmediancontentsbetween57and68mg/g
drywt).CistussalviifoliusalsodifferedfromC.creticus(15mg/gdrywt)andtheothertwo
purple‐floweringspecies(C.albidusandC.crispus),whoexhibitednoorsolelytracesof
punicalaginandpunicalagingallate(Figure1e).Cistuscreticuswastheonlypurple‐flow‐
eredspecieswithaccessionsexhibitingnoteworthyamountsofpunicalaginderivatives.
Higherproportionsofpunicalaginandpunicalagingallatewereusuallyalsodetected
withinC.ladanifer.Cistusmonspeliensis,C.parviflorusandC.salviifoliuswererichinpuni‐
calaginbutthetwopunicalagingallateisomerswerenotpresentinhigheramounts(ex‐
ceptasmallernumberofsingleaccessionsfromthewholedistributionarea)(TableS1,
SupplementaryMaterial).Besidethetwopunicalagingallatepeaksevaluated,however,
thechromatogramsofwhite‐ andwhitish‐pink‐floweringspeciesexhibitedtwoaddi‐
tional,veryconspicuouspeakswithaclearpunicalagingallatesignatureindicatingthe
presenceoftwofurtherisomersthatarespecificforthewhite‐andwhitish‐pink‐flowered
clade.
WithinC.creticus,thehighestproportionsofpunicalaginandpunicalagingallate
weredetectedintheItalian,CroatianandCypriotpopulations(mediancontentsbetween
16and19mg/gdrywt,Figure1f).Populationsfromthesethreecountriesdifferedsignif‐
icantlyfromthoseofAlbania,Greece,UkraineandtheNearEast(allwithmediancontents
between0and4mg/gdrywt).ThepopulationsfromSpain(9mg/gdrywt)werebetween
punicalaginderivative‐richand‐poorC.creticuspopulations.Regardingthetwoevalu‐
atedellagitannins,C.creticuswasextremelyvariable.About20%oftheC.creticussamples
weredevoidofpunicalaginandpunicalagingallate(morespecificallyamountsbelowour
calculatedLODof0.2μg/μl).SomeaccessionsofC.creticuscontainedloweramountsof
punicalaginbutnodetectableamountsofpunicalagingallate,andsomeexhibitedlower
amountsofboth.Singleaccessionsreachedamountscomparabletothoseextractedfrom
themoreellagitannin‐richspecies.AhighdiversitywasobservedespeciallyinItalywhere
thepunicalaginderivativecontentsofC.creticusrangedfrom<LODto147mg/gdrywt.

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Figure1.Comparisonof(a)myricetinglycosideamounts(mg/gdrywt),(c)quercetinglycosideamounts(mg/gdrywt)
and(e)amountofpunicalaginderivatives(mg/gdrywt)ofC.creticus,C.albidus,C.crispus,C.ladanifer,C.monspeliensis,
C.parviflorusandC.salviifolius.Specieswiththesameletterontopdonotdiffersignificantlyfromeachother(groupswere
determinedbyTukeyhonestlysignificantdifference(HSD)test,alpha=0.005).Comparisonof(b)myricetinglycoside
amounts(mg/gdrywt),(d)quercetinglycosideamounts(mg/gdrywt)and(f)ellagitanninamounts(mg/gdrywt)ofC.
creticuspopulationsoriginatingfromSpaintoJordan.Colorsdeterminenatural(pink)orgreenhouseorigin(lightpink)of
plantmaterial.Countrieswiththesameletterontopdonotdiffersignificantlyfromeachother(groupsweredetermined
byTukeyHSDtest,alpha=0.005).ES=Spain(threepopulations),IT=Italy(twocultivatedpopulationsandelevennatural
populations),HR=Croatia(16populations),AL=Albania(fourpopulations),GR=Greece(fourpopulations),UA=
Ukraine(onepopulation),CY=Cyprus(28populations),LB=Lebanon(onepopulation),IL=Israel(onepopulation),JO
=Jordan(onepopulation).


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2.2.SampleClassification
Aprincipalcomponentanalysis(PCA)wasperformedbyusingcompositiondataof
the13majorpeaks(relativepeakareapercentages;Figure2a).Thefirsttwodimensions
explainedabout59%ofthevariance.Principalcomponent1(41%)differentiatedpurple‐
fromwhite‐orwhitish‐pink‐floweringspecies.Themostimportantvariablesresponsible
forthisdifferentiationincludethetwoflavonolsmyricetin‐andquercetin‐3‐O‐rhamno‐
sideaswellasthefourpeaksrepresentingthepunicalaginderivatives(Figure2b).More‐
over,PC1separatedC.monspeliensisfromthethreeotherwhite‐orpink‐floweringspecies,
withsomem‐glycosidesasdiscriminatingvariables.Withinthepurple‐floweredclade,
principalcomponent2(18%ofthevariation)distinguishedC.creticusandC.albidusfrom
C.crispus.Themostinfluentialvariableherewasm‐3‐O‐galactoside.Withinthewhite‐
andwhitish‐pink‐floweredclade,PC2providedonlymarginaldistinguishingpower.The
separationofC.ladaniferindividualsalongastraightlinereflectedthecompletelackof
nineofthethirteenincludedpeaks(allninemyricetinandquercetinglycosides).Witha
closerlookattheC.creticuspopulations,noconspicuousintraspecificdifferentiationwas
obvious(Figure2c).Accordingtoprincipalcomponent1(33%),allthecultivatedpopula‐
tionsclusteredcloselytogetherontheleftmarginbasedonthepredominanceofq‐and
m‐3‐O‐rhamnoside(Figure2d).Principalcomponent2(28%)providednonoticeabledis‐
criminationbetweencultivatedandwildpopulationsorpopulationsofdifferentcoun‐
tries.However,thegroupmidpointsofthegeographicallydistantAlbanianandSpanish
populationsclusteredmorecloselytoeachother(andtothegroupmidpointsofthecul‐
tivatedpopulations)thantotheirgeographicallycloserpopulations.

Figure2.Principalcomponentanalysis(PCA)andcorrespondingfactorloadings.(a)Plotofthefirsttwodimensions(dim)
fromPCAperformedoverallsevenspeciesandtheelevenevaluatedcompounds(relativeareapercent).In(b),theinflu‐
enceofeachincludedvariableisindicated.(c)PlotoffirstandsecondprincipalcomponentsfromPCAperformedover

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allsampledC.creticuspopulations(countrywisecombined,groupmidpointswereaccentuated)andtheelevenmaincom‐
poundsusedforquantification(relativeareapercent).(d)Factorloadingsofthethirteenvariablesincluded.Thepercent‐
ageofvarianceexplainedbyeachdimensionisindicatedinparenthesis.Colorsindicateeitherspeciesaffiliation(2a)or
differentiatewild(pink)andcultivated(lightpink)populations(2c).ES=Spain(threepopulations),IT=Italy(twoculti‐
vatedpopulationsandelevennaturalpopulations),HR=Croatia(16populations),AL=Albania(fourpopulations),GR=
Greece(fourpopulations),UA=Ukraine(onepopulation),CY=Cyprus(28populations),LB=Lebanon(onepopulation),
IL=Israel(onepopulation),JO=Jordan(onepopulation).
2.3.ExtractCompositionandClassificationofTradeSamples
Fifteentradesamplesofcoarse‐cutCistusproducts(onetradesamplewaslabeledas
C.creticus,twelveasC.incanusandtwosamplesasCistussp.;Table2)werepurchased
fromdifferentsupplierstocomparetheircompoundcompositionwiththatofthewild
andgreenhousepopulations.Thefourmainmyricetinglycosidessummeduprangedbe‐
tween1and10mg/gdrywt,thatofthefivemainquercetinglycosidesbetween<LOD
and3mg/gdrywtandthatofthetwopunicalaginderivativesbetween<LODand161
mg/gdrywt(Table2).Asthepeakpatternsofthechromatogramsofthetradesamples
appearedratherheterogeneousandexhibitedattributescharacteristicfordifferentspe‐
cies,werefrainedfromincludingthemasonesamplegroupintheprimarystatistical
analysisandplots.Theheterogeneityofthetradesampleswaswelldemonstratedwhen
includedinthePCAanalysis(FigureS2,SupplementaryMaterial).Onlyfivesamples
groupedwithinoratleastclosetosamplesofC.creticus,whereasthecompositionofthe
othertradesamplescorrespondedmoretothatofwhite‐floweringspecies(C.mon‐
speliensisandC.salviifolius).
Table2.Designationandgeographicalorigin(whenindicated)ofthe15commercialtradesamplespurchasedfromdif‐
ferentpharmacies1orhealthretailers2(kbA=controlledorganiccultivation)andtheircontentsofpunicalaginderivatives
andmyricetinandquercetinglycosides(mg/gdryweight).
SampleDeclarationOriginP‐DerivativesM‐GlycosidesQ‐GlycosidesTentativeIdentification
   (mg/gdrywt)(mg/gdrywt)(mg/gdrywt)(viaHPLCprofile)
CHP011)Cistussp.TR161.24.82.4White‐floweredspecies
CHP021C.incanusL. GR<LOD4.00.8C.creticus
CHP031C.incanus‐ 16.810.02.5C.creticus
CHP041C.incanusL. GR<LOD0.70.2C.creticus
CHP052C.incanus‐ 81.14.11.5White‐floweredspecies
CHP062C.incanuskbA‐ 33.10.50.2White‐floweredspecies
CHP072C.incanuskbACY85.38.42.3C.creticus
CHP082C.incanusTR22.18.62.0C.creticus(adulterated?)
CHP092C.incanus‐ 50.75.91.7White‐floweredspecies
CHP102C.incanus‐ 61.54.01.3White‐floweredspecies
CHP112C.incanus‐ 79.63.51.1White‐floweredspecies
CHP122C.creticus‐ 123.33.33.0White‐floweredspecies
CHP132C.incanuskbATR66.03.21.4White‐floweredspecies
CHP142C.incanus‐ 82.95.12.7White‐floweredspecies
CHP152Cistussp.TR29.46.51.5C.creticus(adulterated?)
2.4.AntioxidantActivityandTotalPhenolics
ADPPHradicalscavengingassaywasusedtocharacterizetheantioxidantactivity
oftheCistusplantsamples.ThehighestantioxidantactivitieswerepresentinC.ladanifer
(medianantioxidantcapacityof301milligramTroloxequivalentspergramdryweight)
andC.salviifolius(261mgte/gdrywt;Figure3a).BothdifferedsignificantlyfromC.albidus
(142mgte/gdrywt)withtheoveralllowestantioxidativeactivity.Cistuscrispus(201mg
te/gdrywt)wasnotclearlydifferentiatedfromthetwospecieswiththehighestactivities.
Cistusmonspeliensis(171mgte/gdrywt),C.creticus(166mgte/gdrywt)andC.parviflorus
(147mgte/gdrywt)werenotdifferentfromC.albidus,thespecieswiththelowestanti‐
oxidantactivity.

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WithinC.creticus,thehighestantioxidantcapacitywaspresentintheAlbanianpop‐
ulations(medianantioxidantcapacityof232mgte/gdrywt;Figure3b).Thepopulations
fromAlbaniaweresignificantlydifferentfromgreenhousepopulationsofcloselylocated
Greecethatexhibitedthelowestantioxidantactivity(115mgte/gdrywt).TheLebanese
(230mgte/gdrywt)andIsraeli(198mgte/gdrywt)populationscouldnotbedifferenti‐
atedfromtheAlbanianpopulations.ThewildpopulationsfromItaly(174mgte/gdry
wt),Cyprus(170mgte/gdrywt),Spain(164mgte/gdrywt)andCroatia(160mgte/gdry
wt)aswellasthegreenhousepopulationsoriginatingfromIsrael(198mgte/gdrywt),
Jordan(170mgte/gdrywt)andUkraine(139mgte/gdrywt),werenotsignificantlydif‐
ferentfromtheGreekpopulationswiththelowestantioxidativecapacity.
ThetotalphenoliccontentofCistusplantsampleswasevaluatedspectrophotometri‐
callyusingcaffeicacidasthestandard.Thehighestcontentsofphenoliccompoundswere
againpresentwithinC.ladanifer(121milligramcaffeicacidequivalentspergramdry
weight)andC.salviifolius(105mgcae/gdrywt)thatcanclearlybedifferentiatedfromC.
monspeliensis(68mgcae/gdrywt),C.creticus(65mgcae/gdrywt),C.parviflorus(54mg
cae/gdrywt)andC.albidus(56mgcae/gdrywt;Figure4c).Cistuscrispus(69mgcae/g
drywt)couldnotclearlybedifferentiatedfromboththespecieswithhigherandthespe‐
cieswithcomparativelylowcontentsoftotalphenolics.
WithinC.creticus,theAlbanianpopulations(71mgcae/gdrywt),togetherwith
Spanish(79mgcae/gdrywt),Italian(73mgcae/gdrywt)andCypriotpopulations(62
mgcae/gdrywt;Figure4d),exhibitedthehighestmediancontentsofphenoliccom‐
pounds.TheywerestatisticallydifferentfromthegreenhousepopulationsfromGreece
(45mgcae/gdrywt)withtheoveralllowestcontentsoftotalphenoliccompounds.Pop‐
ulationsoriginatingfromCroatia(64mgcae/gdrywt),theUkraine(63mgcae/gdrywt)
andtheNearEast(mediancontentsoftotalphenolicsbetween47and58mgcae/gdrywt)
werenotsignificantlydifferentfromboth.SimilartotheresultsfromtheDPPHradical
scavengingassay,nosignificantinfluenceoforigin(fromwildorcultivatedpopulations)
wasdetectable(datanotshown).
WhencomparingtheplotsdescribingDPPHandphenoliccontent,ahighsimilarity
betweenthepatternsofFigure3a/bandofFigure3c/dbecameobvious,indicatingasim‐
ilarvariabilityofbothparametersbetweenCistusspeciesandC.creticuspopulationsfrom
differentcountries.Thiswasstatisticallyconfirmedbyastrongpositivecorrelation(r=
0.77)betweenantioxidantcapacityandphenoliccontent(TableS2,SupplementaryMate‐
rial).However,therewasonlyapositivecorrelationbetweenantioxidativeactivity/total
phenolicsandthesummedcontentofpunicalaginandpunicalagingallate(r=0.47/0.43)
andonlyaratherweakpositivecorrelationbetweenantioxidativeactivity/totalphenolics
andthesummedcontentofquercetinglycosides(r=0.27/0.34).Myricetinglycosidesdo
notparticipatemuchinantioxidativeactivity(r=−0.05).

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Figure3.Comparisonof(a)2,2‐diphenyl‐1‐picrylhydrazyl(DPPH)radicalscavengingactivity(mgte/gdrywt)and(c)
totalphenoliccontent(mgcae/gdrywt)ofC.creticus,C.albidus,C.crispus,C.ladanifer,C.monspeliensis,C.parviflorusand
C.salviifolius.Specieswiththesameletterontopdonotdiffersignificantlyfromeachother(groupsweredeterminedby
TukeyHSDtest,alpha=0.005).Comparisonof(b)DPPHradicalscavengingactivity(mgte/gdrywt)and(d)totalphenolic
content(mgcae/gdrywt)ofC.creticuspopulationsoriginatingfromSpaintoJordan.Colorsdetermineeitherspeciesor
natural(pink)orgreenhouseorigin(lightpink)oftheanalyzedplantmaterial.Countrieswiththesameletterontopdo
notdiffersignificantlyfromeachother(groupsweredeterminedbyTukeyhonestlysignificantdifference(HSD)test,
alpha=0.005).ES=Spain(threepopulations),IT=Italy(twocultivatedpopulationsandelevennaturalpopulations),HR
=Croatia(16populations),AL=Albania(fourpopulations),GR=Greece(fourpopulations),UA=Ukraine(onepopula‐
tion),CY=Cyprus(28populations),LB=Lebanon(onepopulation),IL=Israel(onepopulation),JO=Jordan(onepopu‐
lation).
3.Discussion
PlantmaterialofsevenCistusspeciesfrom13Mediterraneancountrieswassampled,
aimingtoprovideaprimaryinventoryofnaturalflavonoidvariabilitywithinC.creticus
andtoallowadirectcomparisonofqualitativeandquantitativeextractcompositionwith
thatofdifferent,partlysympatricCistusspecies.
Tooptimizesamplepreparation,initialexperimentswereperformedtoassessthe
influenceofdifferentsampleweightsandextractiontimesandtocomparethecomposi‐
tionofaqueous(basedontheprotocolof[11])andhydromethanolicextracts(asused,e.g.,
by[32,39]).Thedifferentsolventextractsexhibitedhighlycomparablepeakpatternsbut
differentquantitativecharacteristics(FigureS3,SupplementaryMaterial).Comparedto
hydromethanolicextracts(50%),purewaterextractsexhibitedhigheramountsofthetwo
punicalaginderivativesconsideredinthework(5to30%)andloweramountsofthemain
flavonoidcompounds(20to40%).Basedontheprimaryresults,wedecidedondeionized
waterastheextractionmediumbecauseofthecompositionalsimilarityofwaterextracts

Plants2021,10,61511of20
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toCistusteapreparations,themostcommonpharmaceuticalformofuse.Onefurther,
pragmaticargumentwasthemoresafeandsustainableextractionprocedureforthehigh
numberofsamplesexpected.Theherepresentedvaluesquantifyingtotalphenolicsand
antioxidantcapacitymightbe,bytrend,lowerthanthosewithhydroethanolicorhydro‐
methanolicextracts(seealso[33],whocomparativelydiscussedcharacteristicsofaqueous
andhydroethanolicC.incanusextracts).
Qualitatively,chromatogramsofallthesevenCistusspeciesexhibitedquitestable
and,forthefirmlytrainedeye,verydistinguishingpeakpatterns(FigureS1,Supplemen‐
taryMaterial).Quantitatively,awiderangeofvariationwasobserved.Polyphenolsare
thoughttobeimportantfactorsforplants’abilitytocopewithdifficultenvironmental
conditionsandaresupposedtoaidtheirpersistenceinextremehabitats.Ashighlyre‐
sponsivetospecifichabitatconditions,polyphenoliccompoundlevelsandpatternscan
varysignificantlywithinaspecies(e.g.,[40–43]).Theauthorsof[44]observedamoderate
positiveinfluenceoftemperatureandsolarirradianceonthetotalphenoliccontentofC.
incanus,specificallyonthecontentofquercetinandtanninderivatives.Theauthorsof[45]
describedasuiteofgenesregulatingflavonoidbiosynthesisandtransporttobelargely
overexpressedinsun‐adaptedleavesofC.incanusandreportedalight‐inducedaccumu‐
lationofmyricetinandquercetinglycosides.Backedupbytheseresults,itcanbehypoth‐
esizedthatthelowercompoundlevelsofthepottedpopulationscanbeprimarilyascribed
tolatitude‐relatedeffectssuchaslowermeansolarirradianceandmeantemperatures
duringtheyear.ThewildCistusplantsweresampledalongawidegeographicalgradient
andfromvarioushabitats.Eachindividualplantwasexposedtospecificenvironmental
conditionsandrespondedtotheminanindividualway.Toovercomethisvariabilityand
toobtainstatisticalsignificance,wecollectedplantmaterialfromahighnumberofwide‐
spreadpopulationsandanalyzed,inmostcases,atleasttenindividualplantsperpopu‐
lation.Tominimizeeffectsrelatedtoplantdevelopmentorseason[33,44],plantsexhibit‐
ingacomparablephenologicalstage(fulltoendingbloom)wereharvested.Basedonthis
carefullycollectedandextensivesampleset,persistentoveralltendencieswerefound.
ExtractsofC.creticuswereusuallycharacterizedbypunicalaginasthemaincom‐
poundinthe“ellagitannin‐half”andm‐3‐O‐rhamnoside(inrarecasesm‐3‐O‐galactoside)
asthemaincompoundinthe“flavonol‐half”ofthechromatogram.Inoursampleset,
about80%oftheC.creticusplantsexhibitedbothflavonolglycosidesandpunicalagin
derivates.About20%ofourC.creticussamplesweredevoidofpunicalaginandpuni‐
calagingallate(morespecificallyamounts<LODof0.2μg/μl).Thisnearlypureflavonol
variantwasdetectedfromSpaintotheNearEast,withhigherabundanceinthemost
western(37%inSpain)andmoreeasternmostpopulations(40%inAlbaniaandthe
Ukraine,75%inGreece,upto100%inpopulationsoftheNearEast)andaconspicuously
lesserfrequencyinItaly(9%),Croatia(8%)andCyprus(12%).Therarerflavonolchemov‐
ariantrelatesC.creticustoitscloserelativesC.albidusandC.crispusandseemstobea
specificcharacteristicofthepurple‐floweredclade,aswasalreadypostulatedby[11].
ComparedtoC.creticus,thechromatogramcharacteristicsofC.albidusappearedtobe
morestable,withtheflavonolvariantasthepredominantone(87%ofthesamples)and
fewindividualplantsexhibitingcomparativelysmallamountsofpunicalaginderivatives.
WithinthetwopopulationsofC.crispus,solelytheflavonolvariantwasdetected.The
frequentchemovariantcharacterizedbythepresenceofflavonolglycosidesandpuni‐
calaginderivativesrelatespurple‐floweredC.creticustothewhite‐orwhitish‐pink‐flow‐
eredspeciesC.ladanifer,C.monspeliensis,C.parviflorusandC.salviifolius.ComparedtoC.
creticus,thewhite‐orwhitish‐pink‐floweringspeciesusuallycontainedhigherpercent‐
agesandamountsofpunicalaginand,inmanycases,alsoofpunicalagingallate.Moreo‐
ver,withinthewhite‐andpink‐floweredspecies,twoadditionalpunicalagingallateiso‐
merswereprominentlypresent,indicatingclade‐andspecies‐specificpeculiaritiesinthe
respectivebiosyntheticpathway.Inthewhite‐orwhitish‐pink‐floweringspecies,flavonol
compoundswereusuallyoflessimportancethaninthepurple‐floweringones.Theone
populationofC.ladanifercompletelylackedthemyricetinandquercetinglycosidesand

Plants2021,10,61512of20
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bothcompoundfamilieswerescarcelypresentwithinC.parviflorus.Somehow,higherper‐
centagesofflavonolsweredetectedwithinC.monspeliensisandC.salviifolius,thespecies
thatwasexceptionalregardingitsvariabilityandpartlyoutstandinghighcontentofquer‐
cetinglycosides.Regardingchemotypecomposition,ourresultsresembled,inprinciple,
previousresultsdescribedfromsingleorfewsamplesofSpanishC.creticus,C.albidus,C.
crispus,C.ladanifer,C.salviifoliusandC.monspeliensis[11]andItalianC.creticus[12–14,44].
OnefurthersampleofPortugueseC.ladaniferexhibited,contraryto[11]andourresults,
alsosmalleramountsofquercetinglycosides[36].
Thefirsttwodimensionsoftheprincipalcomponentanalysisvisualizewelltheout‐
comesdiscussedaboveandearlierhypothesesthatwerebasedontheanalysisofsingle
orfewextractsoftendifferentCistusspecies[11].Accordingtotheirpolyphenolicprofiles,
thepurple‐floweredclade(subgenusCistus)couldbewellseparatedfromthewhite‐and
whitish‐pink‐floweredclade(allfourspecieshererepresentingsubgenusLeucocistus).
Thisdifferentiationwasmainlybasedonthepresenceandproportionsofpunicalagin,
punicalagingallateandm‐andq‐3‐O‐rhamnoside,butalsofurthermyricetinandquerce‐
tinderivativeswereinvolved.Withinthepurple‐floweredclade,C.crispuswasclearly
distinct,whereasC.creticusandC.albiduswerenotdifferentiatedfromeachother.These
resultsresembledearlierfindingsbasedonDNAsequenceandpollenanalysisthatpos‐
tulatedacloseevolutionaryrelationshipofC.creticusandC.albiduswithC.crispusasa
moredistantlyrelatedsistertaxon[1,2].Obviously,theseparationofthepurple‐flowered
andthewhite‐andwhitish‐pink‐floweredcladewasnotentirelyperfect,mainlydueto
themanyItalian,CroatianandCypriotaccessionsofC.creticusthatexhibitedcompara‐
tivelyhighpercentagesofpunicalaginderivativesandtendedtowardssubgenusLeucocis‐
tus.Theunequalgeographicaldistributionofplantsrichinpunicalaginderivativeswithin
C.creticuswasalsolightlyindicatedinthePCAplots.Thetwogroupmidpointsrepre‐
sentingC.creticusfromgeographicallydistinctSpainandAlbania,respectively,clustered
morecloselytoeachotherthantothegroupmidpointsrepresentingtheaccessionsfrom
theirgeographicallyclosercountriesItalyandCroatia.Suchanaccumulationofspecial
featuresincertaingeographicalareasofaspeciesdistributionmightconstitutearesponse
tospecifichabitatfactors(seeabove).However,theoveralltendencyofahigherfrequency
ofpunicalaginderivativescontainingplantsinpopulationsoriginatingfromthemid‐
Mediterraneanareaandtheminorimportanceofpunicalaginderivativesinthecontinen‐
taleasternMediterraneanareawere,tosomedegree,reflectedinthegreenhousepopula‐
tionsthatwerecultivatedandharvestedunderuniformconditions.Thesecongruentpat‐
ternswouldarguemoreforthelocalpresenceofgeneticvariantsorforlocalgeneticex‐
change.GeneticanalysisruninparallelindeeddetecteduniqueDNAsequencecharacter‐
isticsintheItalian,CroatianandCypriotpopulations(Lukasetal.,unpublished).Besides
thenon‐gradualclusteringofpopulationsrelatedtogeography,noclusteringclearlyre‐
latedtoourdesignationofsubtaxa(subspeciesorvarieties)wasobserved.Thisisinac‐
cordancewith[35],whoanalyzedflavonoiddiversityofCypriotC.creticusinmoredetail
anddescribedaconspicuouspopulationclustercomparativelypoorinflavonolglyco‐
sidesthatwasinthemostwesternpartoftheisland(geographicallyclosetoorwithinthe
PolisBasin)andincludedpopulationsofbothC.creticusvarietiesvar.tauricusandvar.
creticus.Basedon52compounds,[14]couldnotdetectsignificantqualitativedifferences
inpolyphenoliccompoundpatternsofSardinianC.creticussubsp.corsicus,subsp.erio‐
cephalusandsubsp.creticus.Thesubtaxon‐related,intraspecificdifferentiationbasedon
essentialoilandNMRdata[6,34]andfromgeneticdata[6]seemsnottobereflectedin
thepolyphenolicprofilesofC.creticus.Withinthewhite‐ andwhitish‐pink‐flowered
clade,C.monspeliensisseparatedclearly,whereastheotherthreespecieswerenotsepa‐
rated.Theevolutionaryrelationshipsthatlinkwhite‐andwhitish‐pink‐floweredspecies
weredescribedtoberathercomplexandarestillunresolvedinmanydetails[1,2].Cistus
monspeliensis(sect.LedoniaDunal),C.ladanifer(sect.Ladanium(Spach.)Gren.&Godr.),C.
parviflorus(sect.LedonellaDunal)andC.salviifolius(sect.LedoniaDunal)havebeenas‐
signedtothreedifferentgenericsectionsbutnoneofthesesectionsweresubsequently

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
supportedbycombinedDNAsequenceandpollenanalysis[2].Thepolyphenolprofiles,
asrecordedduringthisinvestigation,didnotprovidedistinctivesignalsinthecaseof
threeofthefourspeciesincluded.However,itmustbeconsideredthat,duetotheprimary
aimofthisinvestigation,ahigherproportionofthetotalHPLCpeakareaofthewhite‐or
whitish‐pink‐floweringspecieswasnotconsidered.Thehereneglectedcompoundsmay
providefurtherdistinctivesignalsforaclearerspeciesdiscrimination.Theauthorsof[11]
defined,besidepunicalagin,hexahydroxydiphenoyl‐glucose,(epi)catechin,(epi)gallocat‐
echinandan(epi)catechin‐(epi)gallocatechindimerascompoundswithhighdiscriminat‐
ingpower.
Theoverallhighestantioxidativeactivitieswereobservedintwospeciesofthewhite‐
andwhitish‐pink‐floweredclade,C.ladanifer(meanvalueof303mgte/gdrywt)andC.
salviifolius(264mgte/gdrywt).Asexpected,theantioxidativecapacitycorrelated
stronglywiththetotalphenoliccontentbuttherewassolelyamoderate(punicalaginde‐
rivatives),weak(quercetinglycosides)orevenno(myricetinglycosides)correlationbe‐
tweenantioxidantactivityandquantifiedcontentsofmaincompoundsandmaincom‐
poundfamilies.Inparticular,theweakorlackingstatisticalcorrelationbetweenantioxi‐
dativeactivityandmyricetinorquercetinglycosideswasrathersurprising.Myricetin,
quercetinandsomeoftheirglycosides,especiallytheirrhamnosides,weredescribedto
bepowerfulantioxidants,withanantioxidantactivitysimilartoorslightlyweakerthan
thatofvitaminE[37,46].However,whencomparingtheplotsoftotal,myricetin,querce‐
tinandpunicalaginderivativecontentsofthedifferentspecieswiththeplotvisualizing
theirantioxidativecapacity,thepatternsareobviouslynotcongruent.Thiswasespecially
obviouswithinC.creticus.Althoughstrikinglypoorerinthesecompounds,theantioxida‐
tivecapacityofplantmaterialfromsomecultivatedC.creticuspopulationswascompara‐
bletothatofmanynaturalpopulations.Thesefindingsimplythat,besidetherecorded
punicalaginderivativesandflavonolglycosides,furthercomponents(possiblynotthat
sensitivetocertaingrowingconditions)mustbesignificantlyinvolvedintheantioxida‐
tivecapacityofCistusplantmaterial.Suchcandidatecompoundswouldbe,e.g.,hexahy‐
droxydiphenoyl‐glucose,gallocatechin,gallicacidandcatechinthatwere,besidesm‐3‐O‐
rhamnoside,identifiedascompoundswithstrongerantioxidantactivityinC.incanus
herbalteainfusions[36].
Withinthepurple‐floweredclade,plantmaterialofC.crispus(206mgte/gdrywt)
andplantmaterialofC.albidus(142mgte/gdrywt)exhibitedaslightlyhigheranda
slightlylowerantioxidativeactivity,respectively,thanthatofC.creticus(170mgte/gdry
wt).Comparedtothewhite‐orwhitish‐pink‐floweredspecies,theantioxidativecapacity
ofC.creticusplantmaterialwasabouthalfofthatofC.ladanifer,abouttwothirdsofthat
ofC.salviifoliusorclosetothatofC.monspeliensis(175mgte/gdrywt)andC.parviflorus
(169mgte/gdrywt).Directcomparisonsofphenoliccontentsandantioxidantactivity
withresultsofpreviousinvestigationsofCistusplantmaterialweredifficultasvarious
experimentalconditionsanddifferentmodesofresultexpressionwereused.Thetotal
phenoliccontentsdeterminedinthecourseofthisinvestigationseemtobesomehow
higherthanthosepublishedby[47](about55mggallicacidequivalents(gae)/gdrywtin
ethanolicextractsofTunisianC.monspeliensisandC.salviifolius),seemtosomehowresem‐
blethoseof[32](about65mggae/gdrywtinanaqueousextractofSyrianC.creticus,
about70mggae/gdrywtinanaqueousextractofC.salviifolius)orseemtobesomehow
lowerthanthosepredictedby[48](about250mggae/gdrywtinanethanolicextractofa
PortugueseC.ladanifersample),[33](upto115mggae/gdrywtinethanolicextractsofa
BulgarianC.creticussample),[49](about500mggae/gdrywtinanethanolicextractofa
TunisianC.salviifoliusleafsample)or[39](about408mggae/gdrywtor335mggae/g
drywtinaqueousextractsofMoroccanC.salviifoliusandC.monspeliensis).Whencompar‐
ingC.creticustoprominentaromaticplantsoftheLamiaceaefamilythatwerepreviously
studiedinourlabbythesamequantificationmethodusedhere,theantioxidativeactivity
ofC.creticuswashigherthanthatofSalviaofficinalis[50]orThymusvulgarisL.[51].How‐
ever,thetotalphenoliccontentofC.creticuswascomparabletothatofSalviaofficinalis[50]

Plants2021,10,61514of20
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andslightlyhigherthanthatofThymusvulgarisL.[51],indicatingspecificcompoundsof
higheractivityinCistus.
Inrecentyears,CistusplantmaterialandCistuspreparationshavebeenincreasingly
usedfordiversemedicinalpurposes.However,uptodate,noCistusmonographisavail‐
ableintheEuropeanPharmacopoeia[52]todefinetargetspeciesandminimumquality
parameters.DespiteitslongtraditionaluseinsomeSouthEuropeancountries,Cistusis
subjecttothe“novelfoodregulation”(EU2015/2283).Acertainvarietylocallynativeto
northernGreece,CistusincanusL.Pandalisherba,hasbeenregisteredinthecategory
herbalinfusions[53].Apartfrompharmacies,differentCistusplantmaterialsareoften
offeredasa“bathadditive”.Inthefaceoflackingguidelinesfordrugqualityrequire‐
mentsandconfusioncausedbytheinconsistentcategorizationofCistusplantmaterial,it
isdoubtfulthatconsumerscanrelyonaconstantactivesubstancecontentandaconsistent
qualityandpurityoftheirhealthremedies.Tovalidatethepolyphenoliccontentandcom‐
positionofcurrentlyavailablecommercialproducts,15tradesamplesofcoarse‐cutCistus
plantmaterialfromdifferenttrademarks,pharmaciesandhealthretailerswereincluded
inouranalysis.TwelveofthesetradesampleswereoriginallylabeledasC.incanusand
oneasC.creticus,andtwofurtherCistusherbal“teas”hadnospeciesdesignationonthe
label.Thecontentofwater‐solublecompoundsofthetradesamplesvariedhighly,from
nearlyzerotowellcomparablewiththatofplantmaterialcollectedduringthisinvestiga‐
tion.Thisstrikingvariabilityinpolyphenoliccompoundlevelsmightreflectdifferences
inindividualsamplecomposition,ageorsamplingandstorageconditionsoftrade
batches.Theauthorsof[31]revealedthatthewoodenfractionoftradesamplescontained
onlysmallamountsofpolyphenolscomparedtotheleafyfraction.Therewasalsohigh
variabilityconcerningthequalitativecompositionoftheaqueousextracts,whatwasim‐
mediatelyvisiblefromthecharacteristicpeakpatternsoftheirchromatograms.Theprin‐
cipalcomponentanalysisthenplacedsixofthetradesampleswithinorclosetotheC.
creticus/C.albiduscluster,whereastheotherninesamplesseemedtoresemblemorethe
typicalqualitativecharacteristicsofwhite‐floweringspecies.Smallwhiteflowerpieces
presentinsomeofthesetradesamplessubsequentlyconfirmedourresults.Theseout‐
comesledtotheconclusionthat(currently)tradebatchesofCistusplantmaterialcandif‐
ferhighlyinqualityandarealmostcertainlynotdesignatedcorrectlyorareatleastad‐
mixturesofC.creticusanddifferentCistusspecies.Thesefindingsshouldbeconsidered
whenpostulating,assigningorcomparingpharmaceuticaleffectsbasedonresultsgained
fromtheanalysisoffewCistustradesamplepreparations.
4.MaterialsandMethods
4.1.PlantMaterial
Onehundredandtwenty‐sevenpopulationsand1153individualplantsofC.creticus
L.(72populations,704individualplants),C.albidusL.(13populations,131individual
plants),C.xcanescensSweet(onepopulation,oneplant),C.crispusL.(twopopulations,22
individualplants),C.ladaniferL.(onepopulationofeightplants),C.monspeliensisL.(eight
populations,89individualplants),C.parviflorusLam.(eightpopulations,48individual
plants)andC.salviifoliusL.(22populations,150individualplants)and15tradesamples
fromdifferentsupplierswereanalyzedforthisinvestigation.Themainpartoftheplant
materialwascollectedinthewildfromnativepopulationsinAlbania,Croatia,Cyprus,
France,Italy,PortugalandSpain,inautumn2016(Albania)orthelatespringof2017and
2018.ExceptforthefiveAlbanianpopulationsandtwoPortuguesepopulationsofC.cris‐
pus,thebiggerpartofthewildplantswasharvestedatacomparablephenologicalstage
(beginningtoendofbloom).Thesecondpartoftheanalyzedplantmaterialwasfrom
pottedC.creticus(tenpopulations)andC.albidus(fivepopulations)plantsgrowninthe
greenhouse(greenhousecultivationinwinter,openlandcultivationfromearlyspringto
lateautumn)attheUniversityofVeterinaryMedicineVienna.Seedsforthegreenhouse‐
grownplantswereobtainedfromtheMilleniumSeedbank(RoyalBotanicGardensKew)

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andtheSeedBankBerlinDahlem.Thecultivatedplantswereinfullbloomwhenthey
wereharvestedintheirsecondvegetationperiod(earlysummer2018).Geographicallo‐
cationsofpopulationscanbeseeninFigure4,geographicalcoordinatesofthenativepop‐
ulationsandseedbankaccessionnumbersaresummarizedinTableS1(Supplementary
Material)andtradesamplesarecharacterizedinTable2.Theincludedpicturegallery
(SupplementaryMaterial)offersaviewofselectednaturalpopulationsandpottedplants.
Allplantmaterialofwildpopulationswassampledinaccordancewiththeguidelinesof
theNagoyaProtocol(https://www.cbd.int/abs/text(accessedon15September2016)).Spe‐
cieswereidentifiedbyfollowingkeysofthelocalfloras(referencesareprovidedinTable
S1,SupplementaryMaterial).Voucherspecimensofwildandcultivatedpopulations,cur‐
rentlykeptattheherbariumoftheInstituteforAnimalNutritionandFunctionalPlant
Compounds,UniversityofVeterinaryMedicineVienna,willbesubmittedtotheHerbar‐
iumoftheInstituteofBotany,UniversityofVienna(WU).Copyrightofpicturesinthe
graphicalabstractbelongstoJohannesNovak,CorinnaSchmiderer,MartinaPettighofer
and,inthecaseofC.crispus,WillemvanKruijsbergen(SaxifragaFoundation;
http://www.freenatureimages.eu(accessedon15October2020).
 
Figure4.GeographicaloriginoftheCistuspopulationsanalyzed(distributionmapwascompiledwithGoogle
Earth(https://www.google.com/earth/download/(accessedon12March2020)).ThethreemarksinCyprusrep‐
resentthe29populationsofC.creticus,eightpopulationsofC.parviflorusandsevenpopulationsofC.salviifoliussampled
(mainly)inSouthernCyprus.GeographicalcoordinatesaresummarizedinTableS1,SupplementaryMaterial.
4.2.SamplingProcedureandHandlingofPlantMaterial
Fromeachindividualsampledplant,onerepresentativebranchwascollectedfrom
thecanopytop.Theplantmaterialwaseitherairdried(wildpopulations)ordriedina
dryingcabinet(30°C,greenhousepopulations).Thedryplantmaterialwasthenkeptin
cartonsatroomtemperature.Foranalysis,allleavesofabranchwereseparatedfromthe
stems.Stemsand,whenpresent,flowersandearlyfruitswereremoved.Immediatelybe‐
foreextraction,arepresentativeportionoftheroughlycrushedleaveswasgroundtoa
finepowderbyusingaballmill(Pulverisette,Fritsch,Germany).
4.3.Extractions
Onehundredandfiftymgoffinelygroundedplantmaterialwasextractedwitheight
mlmilli‐Qwaterat60°C,for120mininashakingwaterbath(basedontheprotocolof
[11]).ThefilteredextractswerealiquotedinHPLCvials(forHPLCanalysis)andin1.5ml
Eppendorftubes(foranalysisofDPPHandtotalphenolics).Thealiquotswerekeptat‐20
°Cuntilanalysis.

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4.4.HPLCandHPLC‐MSAnalysis
HPLCanalyseswereperformedusingaShimadzuNexeraXRchromatograph(Shi‐
madzu,Austria)equippedwithacontroller(CBM‐20A),adegasser(DGU‐20A5R),aqua‐
ternarypump(LC‐20ADXR),anautosampler(SIL‐20AXR),acolumnoven(CTO‐20AC)
andaphotodiodearraydetector(SPD‐M20A).ThesoftwarepackageLabSolutions5.82
(Shimadzu,Austria)wasusedfordatacollectionandprocessing.Separationswereper‐
formedonaXBridge™ShieldRP18column(3.5μm,4,6×150mm;Waters,Austria)
equippedwithaC18guardcolumn(ODSOctadecyl,4mm×0.3mm,Phenomenex,Ger‐
many).Alineargradientelutionwascarriedoutataflowrateof1mL/minandanoven
temperatureof25°Cusingacetonitrile(CarlRoth,Germany;solventA)and2%acetic
acid(CarlRoth,Germany;solventB).Thefollowinggradientwasused:min0–38,6–17%
AinB(lineargradient);min38–53,17–20%AinB(lineargradient);min53–58,100%A
(isocratic);min58–65,0–6%AinB(lineargradient).Injectionvolumewas20μL.Peak
detectionwasperformedat354nm,closetotheUV/Visabsorptionmaximumofmany
flavonoids.
Aftervisualinspectionofthefirst30chromatogramsofCypriotC.creticus,13prom‐
inentand/orrecurrentpeaksweredefinedandsubsequentlyevaluated(Table1andFig‐
ureS1,SupplementaryMaterial).Identificationandverificationoftrans‐speciesoccur‐
renceoftheseprominentcompoundswereconductedvialiteraturedata(referencesare
providedinTable1),bycomparingretentiontimesandUVspectratothoseofavailable
referencechromatographystandards(punicalagin,myricetin‐3‐O‐rhamnoside,quercetin‐
3‐O‐rutinoside,quercetin‐3‐O‐galactoside,quercetin‐3‐O‐glucoside,quercetin‐3‐O‐rham‐
noside;allfromPhytolab,Germany)andbycomparativerunsofselectedaccessionsof
thesevenCistusspecies,commercialstandardsandcharacterizedgreenteasamplesona
HPLC‐MS(Waters,Austria)(Table1).TheHPLC‐MSwasequippedwithaseparation
module(Waters2695),aphotodiodearraydetector(Waters996)andamassspectrometer
(WatersMicromassQuattromicroTM).Variousrunsusingalternativecolumns,sol‐
vents/gradientsandscanmodeswereperformedandcomparativelyanalyzedtotracese‐
lected(mass)componentsandtoverifythepresenceorcompositionofmultiplepeaks.
TheESIsourcewasoperatedinnegativemodeusingthefollowingconditions:capillary
voltage2.5kV,conevoltage35V,extractor3V,RFlens0V,sourcetemperature150°C,
desolvationtemperature350°C.Nitrogenwassetat600L/min.Theunambiguousidenti‐
ficationofsmallerorminorpeaks,however,wasfinallyhinderedbythecombinationof
lowsignalstrengthandslightretentiontimeshiftsandpatternshiftswhencomparing
HPLCandHPLC‐MSchromatograms.
Thequantificationofpunicalaginandpunicalagingallateaswellasthatofmainmy‐
ricetinandquercetinglycosideswasconductedbycomparisonwithexternalstandardsof
punicalagin(y=3345451x,r2=0.995;LOD=0.189μg/μL,LOQ=0.631μg/μL;quantifica‐
tionofpunicalagingallatewasperformedequivalenttopunicalagin),myricetin‐3‐O‐
rhamnoside(y=36317204x,r2=0.998;LOD=0.0199μg/μL,LOQ=0.066μg/μL;quantifi‐
cationofm‐3‐O‐galactoside,m‐3‐O‐glucosideandm‐O‐xylosidewasperformedequiva‐
lenttom‐3‐O‐rhamnoside)andquercetin‐3‐O‐rhamnoside(y=42551527x,r2=0.999;LOD
=0.009μg/μL,LOQ=0.032μg/μL;quantificationofq‐3‐O‐rutinoside,q‐3‐O‐galactoside,
q‐3‐O‐glucosideandq‐O‐xylosideorq‐3‐O‐arabinosidewasperformedequivalenttoq‐3‐
O‐rhamnoside).Quantificationwasexpressedasmilligrampergramdryweight(mg/g
drywt).
4.5.TotalPhenolics
Thetotalphenoliccontentwasevaluatedasdescribedin[50],withsmallmodifica‐
tions.Thewaterextractsweredilutedwithmilli‐Qwater(1:10).Tenμlofthedilutionwas
mixedwith100μlofmilli‐Qwaterand5μlofFolin–Ciocalteu’sphenolreagent(Merck,
Germany)inamicroplatewell.Themixturewaskeptatroomtemperaturefor3minand
then10μlofNa2CO3solution(CarlRoth,Germany;35gin100mlmilli‐Qwater)and125

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μlofmilli‐Qwaterwereadded.After60minofincubationinthedark,theabsorbanceat
750nmwasmeasuredusingamicroplatereader(i‐Mark,Bio‐Rad,Austria).Caffeicacid
(Sigma‐Aldrich,Austria;10mgin100mlmilli‐Qwater)wasusedasstandard.Ablank
wasusedtocorrectthereadings.Calibrationpointsandsampleswerepipettedandmeas‐
uredasquadruplicates.Theresultswereexpressedasmilligramofcaffeicacidequiva‐
lentspergramdryweight(mgcae/gdrywt).
4.6.DPPHRadicalScavengingActivity
TheDPPHradicalscavengingactivitywasevaluatedaccordingto[50].Thewater
extractsweredilutedwithmilli‐Qwater(1:10).OnehundredandfiveμLofthedilution
wasthenmixedwith95μlmethanol(CarlRoth,Germany)and100μLofsolution(2,2‐
diphenyl‐1‐picrylhydrazyl,Sigma‐Aldrich,Germany;0.0038gin25mlmethanol).After
30minincubationinthedarkatroomtemperature,theabsorbanceofthereactionmixture
wasmeasuredat490nmusingamicroplatereader(i‐mark,Bio‐Rad,Austria).Trolox(6‐
hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylicacid,Sigma‐Aldrich,Austria;0,0063g
in10mlpureethanol)wasusedasstandard.Ablankwasusedtocorrectallreadings.
Calibrationpointsandsampleswerepipettedandmeasuredasquadruplicates.There‐
sultswereexpressedinmilligramTroloxequivalentspergramdryweight(mgte/gdry
wt).
4.7.Statistics
Statisticalanalyses(basicstatisticalparameters,correlations,AnalysisofVariance
(ANOVA),Tukeyhonestlysignificantdifference(HSD)test,principalcomponentanaly‐
sis(PCA))wereperformedandvisualizedbyusingR3.5.2[54]andthepackagesagrico‐
lae,corrplot,dplyr,factoextra,FactoMiner,ggplot2,ggpubr,ggsci,Hmisc,
multcompView,PerformanceAnalytics,readxl,tidyverseandRColorBrewer.
5.Conclusions
Cistuscreticusexhibitedanimpressivediversityintotalcontentofwater‐solublecom‐
poundsandincontentsofpunicalaginderivativesandflavonolglycosides.Twochemov‐
ariantsbasedonthepresence/absenceofpunicalaginderivativeswereidentified:amore
frequentonecontainingpunicalaginderivativesandarareronewithoutpunicalaginde‐
rivatives.Punicalaginderivativescontainingplantsaccumulatedregionallyinthewest‐
ernandespeciallythemid‐MediterraneanareasandinCyprus.Inpopulationsoftheeast‐
ernMediterraneanarea,punicalaginandpunicalagingallatewere(almost)absent.Beside
thislarge‐scalepattern,therewasnoobviouscorrelationbetweenpolyphenolicprofiles
andsmall‐scalemorphologicaldiversitysupportingaclassificationofC.creticusvariants
tosubspeciesorvarieties.NaturalandcultivatedC.creticuspopulationsdifferedsignifi‐
cantlyinflavonolglycosideandpunicalaginderivativecontentsbutnotintotalphenolic
contentandantioxidativecapacity.ComparedtotheCistusspecieswiththeoverallhigh‐
estantioxidativecapacity,C.ladanifer,theantioxidativecapacityofC.creticuswasapprox‐
imatelyhalf.ComparedtotwoLamiaceaespeciesoftendeclaredasmedicinalplantswith
highantioxidativecapacity,SalviaofficinalisandThymusvulgaris,theantioxidativecapac‐
ityofC.creticuswashigher.
ThespecificpolyphenoliccompoundcompositionofCistusspeciesseemstobere‐
latedtoevolutionaryevents.Basedonrelativepercentagesofpunicalaginderivativesand
themainflavonolglycosides,purple‐floweredandwhite‐ andwhitish‐pink‐flowered
cladescouldprincipallybewellseparated.Withinthepurple‐floweringsubgenusCistus,
C.crispusdifferentiatedclearlyfromthestronglyoverlappingclustersofC.creticusandC.
albidus.Morepunicalaginderivative‐richplantsofC.creticussegregatedtowardssubge‐
nusLeucocistusandmightindicateanevolutionaryeventdifferentiatingwestern/mid‐
MediterraneanpopulationsandeasternMediterraneanpopulationsofC.creticus.Within

Plants2021,10,61518of20


subgenusLeucocistus,C.monspeliensisseparatedclearly,whereastheotherthreespecies
didnotdifferentiate.
SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/2223‐
7747/10/4/615/s1,FigureS1:(a)ExamplechromatogramofCypriotC.creticusrecordedat354nm.
Redmarksrefertotheretentiontimesofthe13peaksdefinedafterinitialinspectionofrepresenta‐
tivechromatograms.Thepeaknumbersindicatedinthegraphcorrespondtothepeaknumbersand
compoundslistedinTable1andTableS1.(b)Directcomparisonofrepresentativeexamplechro‐
matogramsofC.creticus(SRC580,cre),C.albidus(SRC71,alb),C.crispus(SRC135,cri),C.ladanifer
(SRC59,lad),C.monspeliensis(SRC115,mon),C.parviflorus(SRC595,par)andC.salviifolius(SRC96,
sal).Identificationofthequantifiedmaincompoundsisindicated.,FigureS2:PrincipalComponent
Analysis‐plotofthefirsttwodimensionsfromPCAperformedoverallsevenspeciesandtheeleven
evaluatedmaincompounds(relativeareapercent).Blacksquaresindicatethepositionofthe15
tradesamples.,FigureS3:HPLCchromatograms(354nm)ofSRC170andSRC204(C.creticus,Cy‐
rus),comparisonofhydromethanolicextract(50:50;blackline)andpurewaterextract(pinkline).,
TableS1(sheet1):GeographicaloriginofnaturalandcultivatedCistuspopulations,collectionde‐
tails,numberofplantsanalysed,populationmeanvaluesandstandarddeviationsofquantified
maincomponents,totalphenolicsandantioxidativeactivity.TableS1(sheet2):Speciesminimum
values,maximumvalues,meanvaluesandstandarddeviationsofmaincomponents,totalphenolics
andantioxidantactivity.TableS2:Statisticalcorrelationbetweentotalphenolics(mgcae/gdrywt),
antioxidantactivity(mgte/gdrywt)andsumsofpunicalaginderivatives,myricetin‐andquercetin‐
glycosides(mg/gdrywt).,Picturegallery:ViewofselectednaturalC.creticuspopulationsandpot‐
tedCistusplants.
AuthorContributions:Conceptualization,B.L.andJ.N.;datacuration,B.L.;formalanalysis,B.L.,
K.S.(KlausStolze)andJ.N.;fundingacquisition,B.L.;investigation,B.L.,L.B.,K.S.(Katharina
Starzyk),K.L.andK.S.(KlausStolze);methodology,B.L.andJ.N.;projectadministration,B.L.;
supervision,B.L.,K.S.(KlausStolze)andJ.N.;validation,B.L.,K.S.(KlausStolze)andJ.N.;writ‐
ing—originaldraft,B.L.;writing—reviewandediting,B.L.,L.B.,K.S.(KatharinaStarzyk),K.L.,
K.S.(KlausStolze)andJ.N.Allauthorshavereadandagreedtothepublishedversionoftheman‐
uscript.
Funding:FinancialsupportforthisstudywasprovidedbytheAustrianScienceFoundation
(FWF;grantno.P29305‐B22).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Dataarecontainedwithinthearticle.
Acknowledgments:TheauthorsthanktheProtectedAreasandBiodiversityServiceofCastilla‐La
ManchaforauthorizingthecollectionofsamplesofC.creticus,aprotectedspeciesinSpain.The
authorsthankCorinnaSchmiderer,MartinaPettighoferandLinaMerzafortheirhelpwithplant
collectioninCyprus,ItalyandCroatiaaswellasProf.JoséGómezNavarroandProf.ArturoVal‐
desfortheirhelpwithcollectingandherborizingplantsinSpain.Theauthorswishtoextendtheir
gratitudetoGabrielaDekrout‐SzpusztaandBettinaBein‐Lobmaierfortheirvaluablehelpinthe
greenhouseandwithplantmaintenance,andtoMartinFinsterböckforhistechnicalassistance
withHPLCanalyses.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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