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Alexandre A. Borghi , Cláudia de L. Almeida , Alexandra C.H.F. Sawaya Mikania laevigata leaves Damage and drying modify the composition of Mikania glomerata and

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Short communication

Damage and drying modify the composition of Mikania glomerata and Mikania laevigata leaves

Alexandre A. Borghi

a

, Cláudia de L. Almeida

a

, Alexandra C.H.F. Sawaya

a,b,∗

aInstitutodeBiologia,UniversidadeEstadualdeCampinas,Campinas,SP,Brazil

bFaculdadedeCiênciasFarmacêuticas,UniversidadeEstadualdeCampinas,Campinas,SP,Brazil

a r t i c l e i n f o

Articlehistory:

Received20September2018 Accepted29August2019 Availableonline24October2019

Keywords:

Chlorogenicacid Grandifloricacid Harvestingprocedures Kaurenoicacid o-Coumaricacid Plantstress

a b s t r a c t

Thisstudycomparedtheinfluenceofmechanicaldamageanddryingonthechemicalcomposition ofMikaniaglomerataSpreng.andMikanialaevigataSch.Bip.exBaker,Asteraceae,leaves.Leaveswere collected1–24hafterdamage.Oven-dryingat40Candshade-dryingatambienttemperaturewerecom- paredtolyophilization.Sampleswereextractedin70%ethanolandanalyzedbyultra-high-performance liquidchromatographywithmassspectrometry.Significant(p<0.05)increasesofcaffeoylquinicacids wereobservedindamagedleavesofbothspeciesandcoumarindecreasedinM.laevigata,indicating stress.Althoughthefinalwatercontentwassimilar,thedryingmethodaffectedtheleafcomposition.In shade-driedleavesofM.laevigatacoumarindecreasedandthepresenceofumbelliferonewasobserved;

caffeoylquinicacidcontentsincreasedfor288hininbothspecies.Apparently,enzymeswereinactivated after6hinovendrying,stabilizingtheirchemicalcomposition,whileshadedryingallowedenzymaticand microbialactivitytocontinue;illustratingtheimportanceofpostharvestingproceduresonthequality ofmedicinalplants.

©2019SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TheAsteraceaefamilypresentsamultitudeofvaluablemedic- inalplantsincludingMikaniaglomerataSpreng.andM.laevigata Sch.Bip.exBaker.Thesespecies,knownas“guaco”inBrazil,are frequentlyusedfortheiranti-inflammatory,antimicrobial,bron- chodilatorand even antiophidic effects (Maiorano et al., 2005;

Gasparettoetal., 2011;Collac¸oet al.,2012).Theirdried leaves aremore commonlyemployed andstocked, asfreshleavesare unstableduetotheirwater contentwhich permitstheonsetof enzymaticdegradationandmicrobialdevelopment,leadingtothe lossofactivecompoundsinmedicinalplants(Meloetal.,2004;

Costaetal.,2005;Radünzetal.,2014).

Inspiteoftheimportanceofthedryingprocessofmedicinal plants,fewstudieshaveaddressedthissubject,dealingmainlywith speciescontainingvolatilecompounds(Meloetal.,2004).Themax- imumtemperatureofthedryingprocessisnaturallylimitedby thestabilityoftheplantcompoundsandthestructureswherein theyarestored(Martinazzoetal.,2013).AccordingtoCostaetal.

(2005)temperaturesover45Ccandamageplantstructuresand

Correspondingauthor.

E-mail:achfsawa@unicamp.br(A.C.Sawaya).

composition,however,Radünz(2004)driedM.glomerataandmint leaves(Mentha×villosaHuds.)concludedthat50Cpresentedthe bestresultsforcoumarinandvolatileoilcontents.Theflowofair aroundtheplantmaterialalsoaffectstheefficiencyofthedrying process(Meloetal.,2004).

Lyophilizationconservestheoriginalplantcompositionbetter, duetolowertemperatures,andisconsideredtoeffectivelyreduce microbialcontaminationbyfastmoistureremoval.Thecontentsof naturalpigmentsandvolatileswashigherinlyophilizedleavesof Melissaofficinalis.andUrticadioica(Branisaetal.,2017)andbasil(Di Cesareetal.,2003).However,lyophilizationisnotalwaysfeasible forlargeamountsofplantmaterial,fortechnicaloreconomicrea- sons.Furthermore,theidealdryingproceduresmustbedetermined foreachplantspecies,asmanyvariablesareinvolved.

Thepresentstudyaimedtocomparetheinfluenceofmechan- icaldamageandthedryingprocessonthechemicalcomposition ofM.glomerataandM.laevigataleaves,assomechangesincom- positionmayberelatedtostressresponsemechanismsinducedby harvestinganddryingprocedures.Furthermore,commondrying procedures(ovenandshadedrying)werecomparedtolyophiliza- tion;todeterminewhichdryingmethodbestmaintainstheoriginal plantcompositionforthesespecies.

https://doi.org/10.1016/j.bjp.2019.08.006

0102-695X/©2019SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

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794 A.A.Borghietal./RevistaBrasileiradeFarmacognosia29(2019)793–797 Materialandmethods

Adultplants, growingin thefieldoftheBiologyInstitute, in Unicamp(latitude224911.12S;longitude470415.50W)were used for the damage and drying experiments. Vouchers were depositedin theUEC herbariumunder thenumber 102046 for MikanialaevigataSch.Bip.exBakerandnumber102047forM.glom- erataSpreng.,Asteraceae.Healthystemsofapproximately40cm werecollectedfrombothplantsinthemorningofthesameday forthedryingexperiment,whereasforthedamageexperiment, cuttingsoftheseplantswereallowedtorootinonelpotsinthe greenhouse, withautomatic irrigation.Over six months passed betweenthecollectionofmaterialfortheseexperiments,allowing theplantsinthefieldtostabilize.

Duringthedamageexperiment,leavesofthirtyindividualplants ofeachspeciesweredamagedwithtweezers;tenindividualsof eachspecieswerenotdamagedandtheirleavesimmediatelycol- lectedandfrozeninliquid nitrogen(control).Theleavesoften more individuals were collected 1, 12 and 24h after damage;

immediatelyplacedinliquidnitrogenandstoredat–40Cuntil lyophilization.

For the drying experiment, lyophilization was compared to oven-dryingat40Cwithforcedventilationandshade-dryingat ambienttemperature(approximately25C).Aliquotsofleavesof bothspecieswereimmediatelyweighedandfrozeninliquidnitro- gen(control).Theremainingleavesofbothspecieswererandomly assignedtooneofthemethodsandweighedinitially.Oven-dried leaveswerecollectedafter6,12,18,24,30,36,42and48h.Shade- driedleaveswereplacedonbenchesandallowedtodrynaturally, collectingafter 48, 96, 144, 192, 240, 288and 336h. The col- lectedsampleswereweighedandimmediatelyfrozen,lyophilized, todeterminethe residualwater content, then stored in closed polypropylenetubes(50ml)atroomtemperatureinthedarkuntil extraction.

Alllyophilizedplantmaterialwasgroundmanuallyusingamor- tarandpistil,whilefrozenwithliquidnitrogen.Preliminarytests werecarriedouttodeterminetheproportionofleavestosolvent toassureanexhaustiveextractionusing66.6mgofeachsamplein 1mlof70%ethanolandwatersolution,inanultrasonicbath,for 30min,thencentrifugedat13,000×gfor5min.Analiquotof100␮l ofthesupernatantsolutionwasplacedinavial,addinganother 100␮l70%ethanol and water solutionwasadded,followed by 800␮lofpurifiedwater.Analiquotof4␮lofeachfinalsolution wasanalyzedbyultra-high-performanceliquid chromatography withmass spectrometry(UHPLC-MS), according tothemethod describedin(MeloandSawaya,2015).Commercialstandardswere usedforidentificationandquantification,exceptforgrandifloric acidwhichwasadonatedisolatedcompound.

The areas of the main peaks (ions and retention time), in bothnegativeand positiveion modes,wereextractedfromthe UHPLC–MSchromatogramsforeachsampleandweresubmitted toANOVA,followedbytheFisherLSDtest,usingGraphpadPrism software,version6.01(GraphPadSoftware,LaJollaCaliforniaUSA).

Data wereconsideredsignificantly differentwhen p< 0.05.For thedamageexperiment thereweretenbiologicalreplicatesper treatment.Forthedryingexperimenttherewerethreeforeach collectiontime.

Resultsanddiscussion

Representativecoumarins,terpenesandphenoliccompounds wereevaluated.Theirstructures,retentiontimes,m/zofprecursor andproductionsareinSupplementaryTable1.

Inthedamageexperiment(SupplementaryFig.1),M.glomer- ataextractspresentedsignificantincreaseofbothchlorogenicand

dicaffeoylquinicacids after48h. Therewasan initialreduction inkaurenoic acidafter 1and 12hwhich returned totheorigi- nallevelafter24h.Lowconcentrationsofcoumarinwerefound inthisspecieanddecreasedfurtherafter1and12h.ForM.laevi- gata,coumarinalsodecreasedinthefirsthourbutreturnedtothe originallevelafter12h.Bothchlorogenicanddicaffeoylquinicacids increasedsignificantlyafter12hand24h,whereaskaurenoicacid decreased.

The increase in chlorogenic and dicaffeoylquinic acids is a knownreactionstodamage(Gouveaetal.,2012),althoughboth speciesincreasedtheproductionofphenoliccompoundsatdiffer- entspeeds.Thereductionincoumarinmayberelatedtotheuse ofacommonprecursor(cinnamicacid)tothisend.Thereduction ofkaurenoicacidinbothspecies,althoughrelatedtoadifferent biosyntheticpathwaymaybeanindirecteffectofcarbonallocation todefensemechanismsandtheproductionofphenoliccompounds.

The effect of the drying process on the concentration of coumarin, o-coumaric acid, umbelliferone, chlorogenic acid, dicaffeoylquinic acid, kaurenoic acid and grandifloric acid was determinedinrelationtoacontroloftheoriginallyophilizedleaves ofM.laevigataandM.glomerata:Bothspecieslostwaterasimi- larrate,howevertheshade-driedleavesofM.laevigatalostwater moreslowlyatfirst,possibly duetothethicker waxy-cuticular layerontheleaves(SupplementaryFig.2).Lyophilizationdriedthe leavesmoreefficiently,M.laevigataleaveslost76.7%(m/m)and M.glomeratalost80.4%(m/m).

Thechemicalcompositionvariedsignificantlythroughoutboth dryingprocedures.Theconcentrationofcoumarin,thebest-known activecompoundofbothspecies,wasinitiallymuchhigherinM.

laevigata(266␮g/ml)thanM.glomerata(7.2␮g/ml).Bothoven- dryingandshade-dryingreducedcoumarincontentsofM.laevigata leaves(Fig.1A),shade-dryingreducedM.glomeratacoumarincon- tent(Fig.1D).o-CoumaricacidwasonlydetectedinM.laevigata leaves.Itsconcentrationvariedduringthedryingprocess(being bothprecursoranddegradationproductofcoumarin)andwassig- nificantlyhigherattheendofbothshadeandoven-drying(Fig.1C).

Umbelliferonewasinitiallydetectedinlowconcentrationsonlyin M.laevigataleavesanditsconcentrationdecreasedthroughoven drying. However, in shade-dried M.laevigata and M.glomerata leaves,umbelliferoneconcentrationincreasedsignificantly,possi- blybytheoxidationofcoumarin(Fig.1DandE).Althoughseveral coumarinsarebiosyntheticallyrelated,coumarinitselfdoesnot possessanticoagulantactivity,whileumbelliferonedoes(Jainand Joshi,2012).Therefore,onemustbear inmindthatbadlydried plantmaterialcouldincreasetheundesirableanticoagulantactiv- ity.

Apparently, in M. laevigata, the phenylpropanoid pathway is directed mainly toward the biosynthesis of coumarinvia o- coumaricacid,whereasinM.glomeratathispathwayisdirected mainly toward the biosynthesis of caffeoylquinic acids via p- coumaricacid.Bothchlorogenicanddicaffeoylquinicacidswere initiallyinhigherconcentrationsinM.glomerata(Fig.2BandD).

However,duringthedryingprocedure,leaftissuesrespondedto stress,formingcaffeoylquinicacidsinbothspecies.Thisincreaseis similartothedamageresponseinplantsobservedinthefirstexper- imentindicatingthatenzymesinvolvedinthispathwaycouldstill beactiveafter6hintheovenat40Corupto192hduringshade drying(Fig.2).

Thediterpenesquantifiedhereinarebiosynthesizedthroughan unrelatedpathway.Kaurenoicacidwasdetectedinbothspecies, withaslightlyhigherconcentrationinM.glomerata,andremained relatively stable throughout both drying procedures. A slight increaseinthefirstdaysofshadedryingandat6hovendrying mayberelatedtosenescenceretardingreactionsintheplantmate- rial,throughthebiosynthesisofgibberellin(Skutniketal.,2001;

Trindade,2012).Theincreaseofgrandifloricacidmayberelatedto

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Fig.1.Concentration(␮g/ml)ofcoumarin(A)o-coumaricacid(C)andumbelliferone(D)inleafextractsofMikanialaevigata;Concentration(␮g/ml)ofcoumarin(B)and umbelliferone(E)inleafextractsofM.glomerata;driedintheoven( )orshade().Differentletterswithinthesamegraphindicatesignificantdifferences(p<0.05)n=3.

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796 A.A.Borghietal./RevistaBrasileiradeFarmacognosia29(2019)793–797

Fig.2.Chlorogenicacidanddicaffeoylquinicacidconcentration(␮g/ml)inleafextractsofMikanialaevigata(A)and(C)andM.glomerata(B)and(D);driedintheoven( ) orshade().Differentletterswithinthesamegraphindicatesignificantdifferences(p<0.05)n=3.

thesamereactions,ormayberelatedtotheoxidationofkaurenoic acid(SupplementaryFig.3).

Inspiteofthelowerwatercontentintheshade-driedleaves after2weeks,ovendryingat40Cpreservedthecompositionof theleavesmorecloselytothelyophilizationprocess,althoughthe resultsofenzymaticactivitywerestilldetectedforupto6h.Shade- dryingresultedingreaterdegradationofcoumarinandincreaseof umbelliferone.

Conclusions

Bothspeciesreacttodamagereducingcoumarinsynthesisand increasingcaffeoylquinicacidsinthefirst6h.Inrootedplantsthis tendstoequilibrateafter24–48h.During thedryingprocedure, theharvestedleavessufferhydricanddamage stress,adjusting theirmetabolismtoreacttothesefactors.Theyarealsosubjectto microbialdegradationaslongasthewatercontentsallow.There- fore,dryingproceduresthatquicklyinactivateenzymesandreduce

watercontentarepreferable.Ovendryingat40Cpreservedthe compositionoftheleavesmorecloselytothelyophilizationpro- cess;shadedryingresultedinanincreaseofumbelliferone,which isnotrecommendable.Therefore,forthesespecies,lyophilization orovendryingispreferable.

Authors’contributions

AABcontributedinthecollectionofplantsamples,execution ofthedryingtest andlaboratorywork,chromatographicanaly- sis,dataanalysisandelaborationofthework.CLAhascontributed tothedevelopmentofassemblingandobtainingthedatarelated tothedamagetest.ACHFScontributedtothesupervisionoflab- oratorywork,translatedthearticleintotheEnglishlanguageand contributedtothecriticalreadingofthemanuscript.Allauthors readthefinalmanuscriptandapprovedthesubmission.

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Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorswouldliketothankDr.VeraLuciaGarciaforthe kinddonationoftheisolatedgrandifloricacid.AABwouldliketo thankCAPESfor ascholarship, CLAwouldliketothankFAPESP (grant2013/15962-2)for ascholarshipand ACHFwouldliketo thankCNPQ,andFAPESP.

AppendixA. Supplementarydata

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/

j.bjp.2019.08.006.

References

Branisa,J.,Jomova,K.,Porubska,M.,Kollar,V.,Simunkova,M.,Valko,M.,2017.Effect ofdryingmethodsonthecontentofnaturalpigmentsandantioxidantcapac- ityinextractsfrommedicinalplants:aspectroscopicstudy.Chem.Papers71, 1993–2002.

Collac¸o, Rd.C.O., Cogo, J.C., Rodrigues-Simioni, L., Rocha, T., Oshima-Franco, Y., Randazzo-Moura, P., 2012. Protection by Mikania laevigata (guaco) extractagainstthetoxicityofPhilodryasolfersii snakevenom.Toxicon60, 614–622.

Costa,Ld.B.,Corrêa,R.M.,Cardoso,J.C.W.,Pinto,J.,Bertolucci,S.K.,Ferri,P.H.,2005.

Secagemefragmentac¸ãodamatériasecanorendimentoecomposic¸ãodoóleo essencialdecapim-limão.Hort.Bras.23,956–959.

DiCesare,L.F.,Forni,E.,Viscardi,D.,Nani,R.C.,2003.Changesinthechemicalcom- positionofbasilcausedbydifferentdryingprocedures.J.Agric.FoodChem.51, 3575–3581.

Gasparetto,J.C.,deFrancisco,G.,Thais,M.,Campos,F.R.,Pontarolo,R.,2011.Devel- opmentandvalidationoftwomethodsbasedonhigh-performance liquid chromatography-tandemmassspectrometryfordetermining1,2-benzopyrone, dihydrocoumarin,o-coumaricacid,syringaldehydeandkaurenoicacidinguaco extractsandpharmaceuticalpreparations.J.Sep.Sci.34,740–748.

Gouvea,D.R.,Gobbo-Neto,L.,Lopes,N.P.,2012.TheInfluenceofBioticandAbiotic FactorsontheProductionofSecondaryMetabolitesinMedicinalPlants,Plant BioactivesandDrugDiscovery.JohnWiley&Sons,Inc.,pp.419–452.

Jain,P.,Joshi,H.,2012.Coumarin:chemicalandpharmacologicalprofile.Int.J.Appl.

Pharm.Sci.Res.2,236–240.

Maiorano,V.A.,Marcussi,S.,Daher,M.A.,Oliveira,C.Z.,Couto,L.B.,Gomes,O.A., Franc¸a,S.C.,Soares,A.M.,Pereira,P.S.,2005.Antiophidianpropertiesofthe aqueousextractofMikaniaglomerata.J.Ethnopharmacol.102,364–370.

Martinazzo,A.P.,DeCastro,E.,Demuner,A.,Amorim,P.,2013.Avaliac¸ãodoóleo essencialfolhasdeCymbopogoncitratus(DC.)Stapfapósoprocessodesecagem.

B.Latinoam.CaribePl.12,523–536.

Melo,Ed.C.,Radünz,L.,Melo,Rd.A.,2004.Influênciadoprocessodesecagemna qualidadedeplantasmedicinais–Revisão.Eng.Agric.12,307–315.

Melo,L.Vd.,Sawaya,A.C.,2015.UHPLC–MSquantificationofcoumarinandchloro- genicacidinextractsofthemedicinalplantsknownasguaco(Mikaniaglomerata andMikanialaevigata).Rev.Bras.Farmacogn.25,105–110.

Radünz,L.L.,2004.Secagemdealecrimpimenta,guacoehortelãcomumsobredifer- entestemperaturasesuainfluêncianaquantidadeequalidadedosprincípios ativos.In:ProgramadePós-graduac¸ãoemEngenhariaAgrícola.Universidade FederaldeVic¸osa,Vic¸osa-MG,pp.100f.

Radünz,L.L.,Melo,E.C.,Rocha,R.P.,Barbosa,F.F.,Santos,R.H.S.,Berbet,P.A.,2014.

Avaliac¸ãodascurvasdesecagemdeguaco(MikaniaglomerataSpreng.)em secadordebandejas.Rev.Bras.Pl.Med.16,378–387.

Skutnik,E.,Lukaszewska,A.,Serek,M.,Rabiza,J.,2001.Effectofgrowthregula- torsonpostharvestcharacteristicsofZantedeschiaaethiopica.PostharvestBiol.

Technol.21,241–246.

Trindade,J.L.Fd.,2012.Obtenc¸ãodegiberelinaspelafermentac¸ãodoesteviosídeo edo7␤-hidroxiesteviolporbiotransformac¸ão.In:ProgramadePós-graduac¸ão emProcessosBiotecnológicos.UniversidadeFederaldoParaná,Curitiba-PR,pp.

82f.

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