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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-dryingat40◦Candshade-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)temperaturesover45◦Ccandamageplantstructuresand
∗ Correspondingauthor.
E-mail:achfsawa@unicamp.br(A.C.Sawaya).
composition,however,Radünz(2004)driedM.glomerataandmint leaves(Mentha×villosaHuds.)concludedthat50◦Cpresentedthe 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/).
794 A.A.Borghietal./RevistaBrasileiradeFarmacognosia29(2019)793–797 Materialandmethods
Adultplants, growingin thefieldoftheBiologyInstitute, in Unicamp(latitude22◦4911.12S;longitude47◦0415.50W)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–40◦Cuntil lyophilization.
For the drying experiment, lyophilization was compared to oven-dryingat40◦Cwithforcedventilationandshade-dryingat ambienttemperature(approximately25◦C).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.Analiquotof100l ofthesupernatantsolutionwasplacedinavial,addinganother 100l70%ethanol and water solutionwasadded,followed by 800lofpurifiedwater.Analiquotof4lofeachfinalsolution 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(266g/ml)thanM.glomerata(7.2g/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 beactiveafter6hintheovenat40◦Corupto192hduringshade 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
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.
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,ovendryingat40◦Cpreservedthecompositionof theleavesmorecloselytothelyophilizationprocess,althoughthe resultsofenzymaticactivitywerestilldetectedforupto6h.Shade- dryingresultedingreaterdegradationofcoumarinandincreaseof umbelliferone.
Conclusions
Bothspeciesreacttodamagereducingcoumarinsynthesisand increasingcaffeoylquinicacidsinthefirst6h.Inrootedplantsthis tendstoequilibrateafter24–48h.During thedryingprocedure, theharvestedleavessufferhydricanddamage stress,adjusting theirmetabolismtoreacttothesefactors.Theyarealsosubjectto microbialdegradationaslongasthewatercontentsallow.There- fore,dryingproceduresthatquicklyinactivateenzymesandreduce
watercontentarepreferable.Ovendryingat40◦Cpreservedthe 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.
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.
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