Rev. bras. farmacogn. vol.27 número4

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Qualitative

and

quantitative

analysis

of

the

phenolic

content

of

Connarus

var.

angustifolius

,

Cecropia

obtusa

,

Cecropia

palmata

and

Mansoa

alliacea

based

on

HPLC-DAD

and

UHPLC-ESI-MS/MS

Fernanda

B. Pires

a

_,

_Carolina

_B.

_Dolwitsch

a

_,

_Valéria

_Dal

_Prá

a

_,

_Henrique

_Faccin

b

_,

Débora

Luana

Monego

b

_,

_Leandro

_M.

_de

_Carvalho

a,b

_,

_Carine

_Viana

a

_,

_Osmar

_Lameira

c

_,

Fernanda

O. Lima

d

_,

_Lucas

_Bressan

b

_,

_Marcelo

_B.

_da

_Rosa

a,b,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_de_Santa_Maria,_Santa_Maria,_RS,_Brazil b_Programa_de_{Pós-graduac¸}_ão_em_Química,_Universidade_Federal_de_Santa_Maria,_Santa_Maria,_RS,_Brazil

c_Laboratório_de_{Agrobiotecnologia,}_Embrapa_Amazônia_Oriental,_Belém,_PA,_Brazil d_Curso_de_Química,_Universidade_Federal_da_Fronteira_Sul,_Realeza,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2November2016 Accepted28March2017 Availableonline5May2017

Keywords:

Barbatimão-do-pará Redandwhite-embaúba Cipó-d’alho

Phenoliccontent HPLC-DAD UHPLC-ESI-MS/MS

a

b

s

t

r

a

c

t

ThephenoliccontentofthemedicinalspeciesConnarusperrottettivar.angustifoliusRadlk.,Connaraceae,

CecropiaobtuseTrécul, CecropiapalmataWilld.,Urticaceae;and Mansoaalliacea(Lam.)A.H.Gentry,

Bignoniaceae,collectedinthreedifferentyearswasevaluated.Plantinfusionsandhydroalcoholic,butanol

andethylacetateextractswereanalyzedbyhigh-performanceliquidchromatographywithdiodearray

detection.Inordertoendorsetheseresults,analysisbyelectrospraymassspectrometrywasalso

per-formed.Wereidentified:gallicacid,catechin,caffeicacid,ferulicacid,rutin,quercitrinandresveratrol.

C.perrottettishowedgreaterdiversityofpolyphenols.M.alliaceahadthehigherconcentrationofcaffeic

acideventhoughitwasfoundinallspecies.Catechinwasthemajorantioxidant,butwasnotdetectedin

M.alliacea.However,wediscussthepopularuseofthesespecies,aswellastheirphenolicconstitution

andtheinterannualdistributionofphenoliccompounds.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Theuseofmedicinalplantsforpreventionandrecoveryof infec-tionsaswellasforhealthpromotionisanancientpractice(Veiga etal.,2005;Schmitzetal.,2005;Alvimetal.,2006).Thisactivity grewoutofpopularknowledgeandforalongtimewastheonly alternativetreatmentforhealthproblems(Alvimetal.,2006).

Althoughmodernmedicinehasbeengreatlyadvancinginthe pastdecades,phytotherapyisstillwidelyemployed(Alvimetal., 2006).TheWorldHealthOrganizationestimatesthatabout80%of theworldpopulationusesthistraditionalmedicineinitsprimary healthcareneeds(MS,2006).AccordingtotheInstituteforApplied EconomicResearch(IPEA,2010),about30%ofcommercially avail-abledrugsderivefromnaturalsources,whileLahlou(2013)asserts thatapproximately40%ofalldrugsareeithernaturalproductsor theirsemi-syntheticderivatives.

∗ _{Corresponding}_author.

E-mail:marcelo.b.rosa@ufsm.br(M.B.Rosa).

Brazilholds anexpressiveplantbiodiversity, along withthe largestrainforestontheplanet:theAmazon(Paracampo,2011). Althoughtheemploymentofplantsformedicinalpurposesisvery widespreadinBrazil,knowledgeabouttheirchemical composi-tionisratherlimited,asmostofthemareappliedwithlittleor noscientificproofofaction(Campelo,2006).Inordertochange thissituation,relevantstudiesconcerningthechemical composi-tionofthesespeciesandthearisingpharmacologicalpropertiesare ofconsiderableimportance.

Differentpartsoftheplantmaybeusedinherbalmedicine,such asroots,bark,leaves,fruitsandseeds(RezendeandCocco,2002). Tea,usuallyobtainedbyinfusion,isaverypopularwayofgetting theactivecompoundsofdifferentplantproducts(Schmitzetal., 2005).

Polyphenolshavebeenincreasinglyinvestigatedandconsumed inrecentyearsduetotheirnutritionalpotentialandtherapeutic value(Ajilaetal.,2011).However,numerousspecies of medic-inal interest still need to be studied regarding this and other classesofcompounds.AmongstthemareConnarusperrottettivar.

angustifoliusRadlk.,CecropiaobtusaTrécul,CecropiapalmataWilld., Urticaceae,andMansoaalliacea(Lam.)A.H.Gentry,Bignoniaceae,all

http://dx.doi.org/10.1016/j.bjp.2017.03.004

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nativespeciesoftheAmazonrainforest,wheretheyareextensively usedbythelocalswithlimitedscientificinformation.

The species C. perrottetti var. angustifolius, popularly known as “barbatimão-do-pará”, is a member of the Connaraceae family. This plant present antidiarrheal, anti-bleeding, anti-inflammatory, antibacterial, antifungal, antiviral and healing activities(Paracampo,2011),beingcommonlyusedmacerated,or asteaandsyrup,forthetreatmentofgenitourinaryinfectionsin women,uterinebleeding,vaginaldischarge,headache,gastric dis-easesandcough(Coelho-Ferreira,2009).

ThespeciesC.obtusaandC.palmata,popularlyknowninBrazilas “red-embaúba”and“white-embaúba”respectively,areboth mem-bers of the Urticaceae family (Beringhs et al., 2015). Plants of thisgenusareusuallyemployedastea,andexhibitseveral recog-nizedactivities,suchasantidiabetic(FreitasandFernandes,2006), expectorant,mucolytic,antiseptic,laxative,antimicrobial(Lameira etal.,2004),diuretic,antitussiveandanti-inflamatory(Freitasand Fernandes,2006;Lameiraetal.,2004;Costaetal.,2011).

In Brazil,especiallyin thestateof Pará,M. alliaceais popu-larly knownas “cipó-d’alho” due to the characteristic smell of garlicreleasedbyitsleaveswhenmacerated(Zoghbietal.,2009). ThisplantbelongstotheBignoniaceaefamilyandisusedbyits anti-rheumatic (Ribeiro etal.,2009), antimalarial (Pérez, 2002), antifungalandantiviral(Zoghbietal.,2009),activitiesandtothe treatmentofrespiratorydiseases(Ribeiroetal.,2009).Maceration, infusion,teapreparationanddecoctionarethemainprocesses pop-ularlyusedinthisplanttoobtainitsactivecompounds(Zoghbi etal.,2009).

Severalfactorscancoordinateorchangetherateofproduction ofphenoliccompoundsandothersecondarymetabolitesinplants. Theperiodofcollectionisoneofthem,sincetheconcentrationand evennatureofsuchcompoundsmayvaryconsiderablyoverthe year(Gobbo-NetoandLopes,2007).

Phenoliccompoundsrangefromsimpletohighlypolymerized structures,whichcanwithalbecomplexedtovariousotherplant components.Therefore,differentmethodsofextractioncombined withsolventsofdifferentpolaritiesarerequiredtoobtainthem (NaczkandShahidi,2004).

This paper aims to identify and to quantify phenolic con-tent,namely,gallicacid,catechin,caffeicacid,rutin,ferulicacid, quercitrin, myricetin, fisetin,resveratrol, quercetin, kaempferol, chrysinandflavone,presentinthespeciesC.perrottettivar. angus-tifolius, C. obtusa, C.palmata and M. alliacea.The extractswere obtainedbyultrasoundextractionusingethanol/water,n-butanol andethylacetateassolvents.Waterinfusionswerealsoanalyzed. The separation, identification and quantification of the afore-mentionedcompoundswerecarriedoutusinghigh-performance liquid chromatographywith diodearraydetection (HPLC-DAD). An ultra-high-performance liquid chromatography-electrospray ionization-tandemmassspectrometry(UHPLC-ESI-MS/MS) anal-ysis wasperformed in order to endorse theresults previously obtainedbyHPLC-DAD.

Materialsandmethods

Chemicals

Analytical standards of gallic acid, (+)-catechin, caffeic acid, rutin,quercitrin,myricetin,fisetin,quercetin,kaempferol,chrysin andflavonewerepurchasedfromSigma–Aldrich(St.Louis,MO, USA).FerulicacidwaspurchasedfromFluka(Buchs,Switzerland) andresveratrolfromTedia(RiodeJaneiro,RJ,Brazil).Allstandards usedwereofanalyticalgrade(≥95%purity).

Themobilephasewaspreparedbydilutingan85%(v/v) phos-phoricacid(PA) (F.Maya,Brazil)withultrapurewater(Milli-Q,

MilliporeSynergyUV,Bedford,MA,USA)toaconcentrationof0.1% (w/w)inavolumetricflask.Thesolutionwasthenfilteredunder vacuumsystem(Prismatec,131, 2V)througha0.2␮mcellulose

acetatemembranefilter(Sartorius,Goettingen,Germany). Acetoni-trilewasalsointhecompositionoftheeluentandwasobtained fromPanreacITWcompanis(Germany).

Stocksolutions of1000mg/lwereprepared bydilutingeach phenoliccompoundstudiedinHPLCgrademethanol,suppliedby Tedia(RiodeJaneiro,RJ,Brazil).TheywerestoredinFalcontubes at−30◦_C._These_solutions_were_then_diluted_in_methanol_to_reach

intermediateconcentrations.

Plantsampleswereweighedonadigitalanalyticalbalance (Shi-madzu/AUY220)witha0.0001gprecision.

Plantmaterial

Barks of Connarus perrottetti var. angustifolius Radlk., Con-naraceae(IAN184393)andleavesofCecropiapalmataWilld.(IAN 185556),C. obtusaTrécul,Urticaceae (IAN 185555)and M. alli-acea(Lam.)A.H.Gentry,Bignoniaceae(IAN184394)werecollected in April and May of 2012,2013 and 2014, properly identified, driedandmilled.Allplantsstudiedwereprovidedbythe herbar-iumIANlocatedattheBrazilianAgriculturalResearchCorporation (Embrapa)EasternAmazon,Belém,PA,locatedat1◦₂₇′₂₁′′_S_and

48◦₃₀′₁₄′′_W,_at_an_altitude_of₁₀_m_and_an_average_annual

temper-atureof30◦_C.

AccordingtoAnaniasetal.(2010)theclimateoftheAmazon regionischaracterizedbyadryperiod(July–October)andarainy season(December–May),whileJuneandNovemberareconsidered transitionperiods.In addition,itis consideredahot andhumid climatewithverysmalltemperaturegradients.Therefore,samples studiedwerecollectedduringtherainyseason.

Extractionprocedure

Plant extracts were preparedby ultrasound assisted extrac-tion(Bandelin,SonorexSuperRK510H).Glasstubescontaining approximately0.2gofthesamplesreceived10mloftheextraction solvent(70%hydroethanolic,butanolorethylacetate)and were placedinanultrasonicbathfor4hatroomtemperature.Then,the supernatantwaswithdrewandtheremainingextractwasfiltered ona0.22␮mmembrane(SorblineTecnologie).Butanolandethyl

acetatewereevaporatedat40◦_C_in_a_glass_beaker._These_extracts

werelaterresuspendedinHPLCgrademethanolandfilteredina 0.22␮mmembrane(SorblineTecnologie).Theinitial

concentra-tionsweremaintained.Theextractedsamplesremainedstoredat

−30◦_C_until_the_analysis._Before_injection_into_the_{chromatograph,}

allsamplesweredilutedto0.01g/mlwithHPLCgrademethanol.

Infusions

Avolumeof50mlwaterat90◦_C_were_added_to_1.5_g_of_dried

plant.Thirtyminuteslater,theinfusionswerefiltered,bothbya paperfilterandbya0.22␮mmembrane(SorblineTecnologie)and

thenstoredat−40◦_C_until_used._The_samples_injected_were_first

dilutedat0.01g/mlwithHPLCgrademethanol.

HPLC-DADapparatus

ThechromatographicanalysiswasperformedinaKnauer chro-matograph (Berlin, Germany) equipped with a manual Knauer injector(20␮lloop).ThesystemconsistedofaSmartlinePump

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Table1

DatafromthechromatographicseparationviaHPLC-DAD.

Phenoliccompound/equationofanalyticalcurve tR(min)a (nm)b LOD(mg/kg)c LOQ(mg/kg)d

1.Gallicacid/y=9387.5x+5270.2 4.40 220 0.7 1.0

2.(+)-Catechin/y=13992x+23821 8.65 220 1.7 2.8

3.Caffeicacid/y=14013x+7961.9 10.10 320 0.3 1.0

4.Rutin/y=16178x+1196.8 12.45 254 0.6 2.4

5.Ferulicacid/y=15927x+3286.3 14.75 320 0.5 1.0

6.Quercitrin/y=18225x+5576.9 18.00 254 0.4 0.8

7.Resveratrol/y=24317x+1592.9 28.45 320 0.4 0.8

a_t

R:retentiontime. b _:_wavelength. c _LOD:_detection_limit. d _LOQ:_{quantification}_limit.

Identificationandquantificationofphenoliccompoundswere performedusingthemethodologydevelopedwhich,besidesthe lowlimitsofdetectionandquantificationpresentedbythis chro-matographicmethod(Table1),theanalyticalvalidationconfirmed itsselective,linear,preciseandaccuratecharacter(Table2).

Compoundswereseparatedwithagradientreverse-phase sys-tem.A250mm×4.6mmchromatographic columnpackedwith C18 (5␮m particle diameter, Phenomenex) and a guard

col-umnwithsimilarcompositionwereemployed.Themobilephase consistedoforthophosphoricacidsolution(0.1%,w/w)assolventA andacetonitrileassolventB.Theelutionconditionswere:90–80% Aand10–20%B(0–5min);80–75%Aand20–25%B(5–35min); 75–0%Aand25–100%B(35–55min).Theflowrateofthemobile phasewas0.8ml/min(0–35min)and0.8–1.0ml/min(35–55min). Roomtemperature(21±2◦_C)_was_monitored_during_all

chromato-graphic runs. The wavelengths scanned were 220nm, 254nm, 320nmand360nm.

Identificationofcompoundswasbasedonthecorrespondence ofretentiontimesofchromatographicsignalsofplantextractsand referencematerials,theultraviolet(UV)spectraandbytheaddition ofthreedifferentconcentrationsofthestandardsolutionstothe samples.

Following,compoundswerequantifiedbythestandard addi-tionmethod,whichcorrelatestheconcentrationofthestandard solution(addedin␮mol/l)tothepeakarea.AccordingtoRibani

etal.(2004)theconcentrationof thesamplesanalyzedmaybe determinedthroughstraightlineextrapolation,correspondingto thepointwhereitcutstheaxisoftheabscissae(x).

Table1showstheequationfortheanalyticalcurve,retention timesoftheantioxidantcompoundsfoundinplantspecies,aswell asthedetectionwavelength(nm),limitofdetectionandlimitof quantificationofthechromatographicseparationviaHPLC-DAD.

Thelimitsofdetection(LOD)andofquantification(LOQ)were determinedbythesignal-to-noiseratio,inacordancetoNational Instituteof Metrology (2016). Thearea and standard deviation oftheretention times werecomputedthrough theinjectionof 7replicatesoftheblank(matrixfreeofthecompoundof inter-est), in this case methanol (HPLC grade). LOD=X+t(n−1)·s and

LOQ=X+10·s,where:X=mean ofblanks, s=standarddeviation of blanks,t=number ofinjections (7-1)=6 degrees of freedom. Consideringthedegreesoffreedom,theunilateralt,ata99% con-fidencelevel,is3.143.

Table2showsthelinearrange,thecorrelationcoefficient,the intradayprecision,expressedbytherelative standarddeviation (RSD%),theinter-dayprecision,expressedbythecoefficientof vari-ation(CV%),andtheaccuracy(%),determinedbyrecoveryassays.

UHPLC-ESI-MS/MSmethod

The method developed and applied by Faccin et al. (2016)

to detect the molecular ions through mass spectrometry was employedforconfirmationpurposes.

Resultsanddiscussion

The methodologyemployed to determinethephenolic con-stituentsbyHPLC-DADenablestheseparationandidentification ofthirteenantioxidantsasshowninFig.1(A).Amongthese,seven weredetectedinthestudiedplants,namely,gallicacid(1), cate-chin(2),caffeicacid(3),rutin(4),ferulicacid(5),quercitrin(6)and resveratrol(9).

DeterminationbyHPLC-DADofphenoliccompoundsinplant extractsfromdifferentyearsofcollection

Fig.2 shows the concentrationof antioxidantsfound inthe medicinalspeciesstudiedrelatingtheextractstotherespective col-lectionperiod.Themeancoefficientofvariationoverthetriplicate extractionswas28–30%forallextractsobtained.

Connarusperrottettivar.angustifolius

Amongtheanalyzedspecies,C.perrottettivar.angustifolius pre-sented the highest diversity of phenolic compounds. Although catechin was the predominant antioxidant, corresponding to

Table2

ValidationparametersoftheHPLC–DADmethod.

Phenoliccompound Linearrange(␮mol/l) (r)a Intraday(RSD%)b Interday(CV%)c Accuracy(%)

1.Gallicacid 2.94–588 0.9990 0.6 1.9 104.7

2.(+)-Catechin 8.61–344.4 0.9997 1.2 3.5 83.8

3.Caffeicacid 5.5–550 0.9997 0.8 2.3 103.4

4.Rutin 0.82–164 0.9996 0.9 2.8 88.2

5.Ferulicacid 5.15–515 0.9997 0.5 1.6 97.6

6.Quercitrin 2.23–223 0.9984 1.2 3.7 94.6

7.Resveratrol 4.38–438 0.9996 0.6 1.9 100

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150 135 120 105 90 75 60 45 30 15 0

–15 0 5 10 15 20 25 30 35 40 45 50 55

1 2

3

4 5

6

8 7

9

10

11 12

13

Time / min

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

Intensity / mA

U

A

B

C

D

E

F

H

G

Standard Sample

Standard Sample 80

60

40

20

0

200 250 300 350 400

40 30 20 10 0 30 20 10 10 8 6 4 2 10 8 6 4 2 6 4 2 Wavelength / nm Wavelength / nm Wavelength / nm Wavelength / nm 200 250 300 350 400

Wavelength / nm 200 250 300 350 400

Wavelength / nm 12.5

10.0

7.5

5.0

2.5

Fig.1. (A)ChromatographicseparationofthethirteenantioxidantsstandardsbyDAD(20.5–218.25␮mol/l).1.gallicacid,2.catechin,3.caffeicacid,4.rutin,5.ferulicacid,

6.quercitrin,7.myricetin,8.fisetin,9.resveratrol,10.quercetin,11.kaempferol,12.chrysinand13.flavone;(B)Standardspectrumofgallicacid/sample:Cecropiaobtusa

hydroalcoholicextract2013;(C)Standardspectrumofcatechin/sample:Cecropiapalmatabutanolicextract2013;(D)Standardspectrumofcaffeicacid/sample:M.alliacea

butanolicextract2013;(E)Standardspectrumofrutin/sample:C.perrottettivar.angustifoliusethylacetateextract2014;(F)Standardspectrumofferulicacid/Sample:

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B Cecropia obtusa

A Connarus perrottetti var. angustifolius

C Cecropia palmata

D Mansoa alliacea

10 000

1000

100

10

1

Concentr

ation / (mg/kg)

Concentr

ation / (mg/kg)

Hydroethanolic Butanolic

Butanolic

Butanolic Butanolic

Eth

yl acetate

Eth

yl acetate

Eth

yl acetate

Hydroethanolic Hydroethanolic

2012 2013

Years of collecting - extracts

2014

100

10

1

Concentr

ation / (mg/kg)

100

10

1

Concentr

ation / (mg/kg)

100

10

1 Hydroethanolic

2012

Hydroethanolic 2013

Hydroethanolic 2014 2012

Butanolic Hydroethanolic

2012 2012

Butanolic 2013

Butanolic Butanolic

Hydroethanolic

2012 2012 2013

Butanolic 2014 Ethyl acetate

2013 Butanolic

2013

Butanolic 2014

Gallic acid (+)- Catechin Caffeic acid Rutin Ferulic acid Quercitrin Resveratrol

Gallic acid (+)- Catechin Caffeic acid

Gallic acid (+)- Catechin Caffeic acid Ferulic acid Quercitrin Resveratrol

Gallic acid

Caffeic acid

Fig.2.PhenoliccompoundsfoundinthestudiedspeciesviaHPLC-DAD:(A)C.perrottettivar.angustifolius,(B)Cecropiaobtusa,(C)Cecropiapalmataand(D)M.alliacea; coefficientofvariation:28–30%overextractstriplicate.

approximately80%,allsevencompoundsdeterminedinthestudied plantswereencounteredinthisspecies.

Fig. 2 shows a decrease on the concentration of catechin betweencollectionsof2012and2014.Thisdecreasewasobserved forallextractsanalyzed,buttherewasnoclearcorrelationtothe concentration.

The highly polar phenolic compounds are not completely extracted with pure organic solvent. The addition of water to

ethanol increases thepolarity, facilitating theextraction ofthe polarconstituents (Stalikas,2007), i.e. gallic acid and catechin. Accordingly, the greatest amounts of these compounds were detected in the hydroethanolic extracts. Silveira et al. (2015)

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Theuseofsolventswithlowerpolarities,suchasbutanoland ethylacetate,enabledtheidentificationoflesspolarconstituents. ThisobservationissupportedbyNaczkandShahidi(2006),who reportsthatthesolubilityofphenolic compoundsis affectedby thechemicalnatureoftheplantconstituentsandbythepolarity ofthesolventextractor.Therefore,variousorganicsolventsand aqueousmixtureswithalcoholsarereportedintheextractionof thesemetabolites(Ignatetal.,2013).

Caffeicandferulicacidwerepresentinhigherconcentrations inthesamplecollectedin2013.Rutinandquercitrin,ontheother hand,reachedhigherconcentrationsinplantscollectedin2014, whatissupportedbyFig.1(E)and(G),thatshowsthespectraused toidentifyrutinandquercitrininthespecies.

Therewasnosignificantvariationonresveratrolconcentration whencomparingbutanolicextractsofC.perrottettivar.angustifolius

collectedindifferentyears(2013and2014).Nonetheless,extracts usingethylacetateasasolventexhibitedadecreaseof approxi-mately34%onthisconcentrationfrom2013to2014.Thisisastrong evidenceoftheloweraffinitybetweenthissolventandresveratrol, whosepresenceintheextractissupportedbyFig.1(H).Fig.2(A) showsthatresveratrolwasnotdetectedinplantscollectedin2012.

Cecropiaobtusa

From2012to2014,asignificantincrease(92.2%)wasobserved ontheconcentrationofgallicacidinthehydroethanolicextractof thisspecies,asshowninFig.2(B).Thismaybearesultofsomestress sufferedbytheplant,since,asreportedinliterature,many environ-mentalfactorssuchasseasonality,temperature,wateravailability, ultraviolet radiation, nutrients and altitude may interfere with thebiosynthesisofsecondarymetabolites(Gobbo-NetoandLopes, 2007;Morais,2009).In ordertoendorsetheresultsconcerning theidentificationofgallic acid,thespectrummeasuredfor this specieswascomparedtoitsreferencespectrum.Thiscomparison isreportedinFig.1(B).

Catechin was themajor compound in C. obtusa, but it was detectedonlyinthebutanolicextractofplantscollectedin2013. Theseextractionconditionsalsodetectedcaffeicacidinthesample collectedin2014.

Besidespresentinglittlevarietyof antioxidantsin its consti-tution, C. obtusahasshown very irregularbehavior duringthe analyzedperiod.Thismayberelatedtothedefensestrategy of theplantagainsttheinfluenceofbioticand/orabioticfactors,as mentionedbyMorais(2009)andCoutinhoetal.(2010).

Cecropiapalmata

Regarding the studied compounds, C. palmata exhibited a greaterdiversitywhencomparedtotheotherspeciesofthesame genusanalyzed,asshowninFig.2(C).

Thehydroethanolicsolventextractedthehighestconcentration ofgallicacidonplantscollectedin2012.Thesimilaritybetween polarities(bothhigh)ofsolventandextractedcompoundjustifies thisresult.However,this conditiondidnotextractedanyother compoundofinterest.

ButanolicextractionofC.palmatacollectedin2013,ontheother hand,enabledtheseparationoflesspolarcompoundswhichhave notbeenidentifiedinthisplantsofar,i.e.,inthe hydroethano-licextract.Fig.1(F)showsthatferulicacidisalsopresentinthis species.Inaddition,thefollowingcompoundswereextracted: cate-chin,quercitrinandresveratrol.Thissolventwasalsoabletoextract caffeicacid,despiteitshigheraffinitywithethylacetate.

SimilarlytowhatwasobservedtoC.obtusa,catechinwasthe majorcompound,butwasonlydetectedonbutanolicextractsof thesamplecollectedin2013.Comparingbothspeciesinrelationto thecatechinconcentration,aminordifferenceof1.3%–higherfor

C.obtusa–isobserved.Fig.1(C)showsthespectrumemployedon theidentificationofcatechininC.palmata.

ThereareseveralstudiesregardingspeciesofCecropia genus reportedinliterature.Franck(1998)analyzedphenolicacidsand flavonoidsinextractsoftheleavesofC.hololeuca,C.pachystachaya

andC.glazioviiusingthin-layerchromatography.C.glazioviiwas alsostudiedusingliquidchromatographybyLuengas-Caicedoetal. (2007)who identified catechin,and byArend etal. (2011)and

Beringhs et al. (2015) who detected caffeic acid. These results supporttheonespresentedhereforthetwospeciesofCecropia

investigated.

Mansoaalliacea

Thisspeciesshowedthelowerdiversityofphenoliccompounds, asshowninFig.2(D).Incontrast,itpresentedthehighest concen-trationofcaffeicacid,detectedinthebutanolicextract,Fig.1(D). Consideringtheentireperiodofcollection,i.e.2012–2014,there wasadecreaseof87.9%intheconcentrationofsuchcompound. Similarlytotheotherplantsevaluatedinthisstudy,gallicacidwas detectedinM.alliacea,butonlyinsamplescollectedin2012.

Phytochemical studies involving the leaves of M. alliacea

revealedthepresenceofpolyphenolsandflavonoids(Ribeiroetal., 2009;Pateletal.,2013)andtheabsenceofcatechins(Ribeiroetal., 2009).Also, flavonoidslikeluteolinandapigeninweredetected intheflowers(Zoghbietal.,2009),andphenolsintheroot(Patel etal.,2013).Thechemicalcompositionofthisspeciesmaybe bet-terunderstooddue totheresultspresentedin thisstudy,since theyagreewiththeaforementionedworksandprovideadditional informationonthephenolicacidscomposition,suchascaffeicand gallic.

AccordingtotheNationalInstituteofMeteorology(INM,2015), theaveragerainfallduring theyears ofcollections (2012, 2013 and 2014) was416.7±80.4mmand thecoefficientof variation stayedaround20%.Thetemperaturesvariedbetweenaminimum of23.3±0.3◦_C_and_a_maximum_of_32.8_±_0.3◦_C._Becho_et_al.₍₂₀₀₉₎

showedthatcompoundswithhigherpolaritycanbeeliminated fromplantsbyleaching.Whereasthesamplesanalyzedwere col-lectedduringtherainyseason,thesedatasupportthebehavior observedconcerningtheperiodofcollection.

Althoughthechemicalcompositionofplantsislargely deter-mined by the genetic characteristics of the species, various environmentalfactors(Dinizetal.,2007)andtheemploymentof differentextractionmethods canchangeit (Naczkand Shahidi, 2004). For this reason, studies concerning these variables are paramountinordertoassurethedesiredconcentrationsofactive compounds.

AnalysisofinfusionsbyHPLC-DAD

InvestigatedcompoundsweredetectedonlyoninfusionsofC. perrottettivar.angustifoliusandC.obtusa.Consideringtheuseof thesespeciesintheformoftea,aconcentrationof0.81mg/100ml ofgallicacidwasquantifiedinsamplesofC.perrottettivar. angusti-foliuscollectedin2013and0.58mg/100mlinsamplescollected in 2014. Concentrations of catechin were considerably higher onthisspecies:7.9mg/100mlinsamplescollectedin2013and 3.5mg/100mlinsamplesfrom2014.SamplesofC.obtusacollected in2012and2013showednovariationontheconcentrationofgallic acid,whichwasof0.14mg/100ml.

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Table3

Tandemmassspectrometry(MS/MS)parametersofinvestigatedphenoliccompounds.

Phenoliccompound Fragmentadorvoltage Quantificationtransitiona _Confirmation_transitiona

1.Gallicacid 106 169.0>125.1(10) –

2.(+)-Catechin 134 289.1>245.1(10) 289.1>203.2(15)

3.Caffeicacid 106 179.0>135.1(10) –

4.Rutin 201 609.1>300.1(31) –

5.Ferulicacid 88 193.1>134.1(9) 193.1>178.1(7)

6.Quercitrin 164 447.1>301.1(17) –

a_The_energy_collision₍_V₎_is_given_in_brackets.

pharmacologicaleffectsandespeciallyrecognizedfortheir antiox-idantactivity.

Confirmationoftheidentityofantioxidantcompoundsby UHPLC-ESI-MS/MS

Inordertoendorsetheresultsconcerningthephenolic consti-tutionofthemedicinalplantsstudied,dataobtainedbyHPLC-DAD andbyUHPLC-MS/MSwerecompared.

Negativechemical ionizationwas employed for allphenolic compoundsanalyzedand theretentiontimesrangedfrom0.39 to3.66min.Themassspectraldataobtainedaresummarizedin

Table3.

Morethan 60% of theresultsreportedin Fig.2 are in good agreement with the ones from analysis by liquid chromatog-raphy coupled to tandem mass spectrometry (LC-MS/MS). For samplescontainingcatechin and ferulic acid, thesecompounds weredetectedbyHPLC-DADandLC-MS/MS.

The results not supported by LC-MS/MS were: caffeic acid, rutinandquercitrinonsamplesofC.perrottettivar.angustifolius

collectedin2013,aswellasresveratrolforallperiodsassessed forthisspecies;gallic acidoninfusionandsamplesofC.obtusa

collectedin 2012and 2014; gallic acidonM.alliacea samples; gallic acid, quercitrin and resveratrol on C. palmata samples. Thisdisagreementisbelieved tobeduetothedifferenceinthe extractionmethods used,as allthese wereobtainedfrom cold maceration.

AccordingtoNaczkandShahidi(2006),theextractionof phe-noliccompoundsinplantsisinfluencedbyseveralfactors,suchas chemicalnatureofthespecies,extractionmethod,sampleparticle size,processing,amongstothers.Eachextractionmethodaffects theselectivityofthecompounds,especiallywhenitinvolvesthe studyofantioxidantcomponents,sincetheyaresensitivetothe actionoflight,oxygenandheat(AndreoandJorge,2006;Pereira andMeireles,2010).

Thereis nostandard or fully satisfactory procedure for the extractionofallphenoliccompoundsoraspecificclassofvegetable material(NaczkandShahidi,2004).Giventhevastnumberof stud-iesonthesubject,however,theextractionassistedbyultrasound provestobequiteeffectiveinisolatingpolyphenols(Luqueand Priego-Capote,2007;RoutrayandOrsat,2013).

AccordingtoDalPráetal.(2015),extractionassistedby ultra-soundinvolvestheformationofcavitationbubbles,whichassist therelease ofthevegetablecontent,increasingthemass trans-fer.In studieswithBrassicaoleracea, theextractionassisted by ultrasoundhadapositiveeffect,increasingtheefficiencyin approx-imately36.1±15.5%whencomparedtoconventionalextraction (maceration).

Somephenoliccompoundsareveryphotosensitive.Therefore, time-consumingextraction may result in degradation. Thereby techniquesinvolvingultrasoundhavebeenwidelyusedmainlyin ordertopreventthedegradationofresveratrol(Guamán-Balcázar etal.,2016).Resveratrolbelongstotheclassofstilbenes,which presenthighsensitivitytoexternalagentssuchasair,light and

oxidative enzymes.Accordingly, there are many foodadditives beingdevelopedaimingtheincreaseonsolubilityand photosta-bilityofthesecompounds(Silvaetal.,2014).

Studiesregardingthesolidphaseextractionofpolyphenolsin winedemonstrateda lowrecoveryrateforthegallicacid.Even tough,quantificationwasstillpossible,giventhelargeamountsof thiscompoundpresentinthismatrix(Villiersetal.,2004).Hence, whenoccurringinlowerconcentrations,asinsomeplantspecies, thedetectionmaynotbepossible,dependingontheextraction procedure.

AccordingtoJaquesetal.(2010),whostudiedthechangeon phenoliccompoundcompositionofblackberrypulpsduring stor-age,gallicacidsufferedthemajordegradation.Thisresultisrelated totheredoxreactionsresultingfromitshighlyhydroxylated struc-ture.

Furthermore, according toWaterhouse (2002), gallic acid is widelyusedasastandardintheFolin-Ciocalteumethodforthe determinationoftotalpolyphenols,becauseitisrelatively inex-pensiveand pureanditsdry formis verystable.However,the standardsolutionlosesabout5%ofitseffectivenessafteroneweek atroomtemperatureandthedecompositionisevenfasterwhen thesolutionisexposedtotheair.

Takingintoaccountallparametersinvolvingthedetermination ofthepolyphenols,whichwereheredemonstrated,alongwiththe numerousstudiesonthissubject,wecanaffirmthatboth analy-sisperformedinthisstudy,bydiodearraydetectionandbymass spectrometry,yieldedsatisfactoryresults.

Conclusions

One or more phenolic compounds, e.g. phenolic acids and flavonoids,arepresentinthechemicalconstitutionoftheextracts ofmedicinalplantsanalyzed.Amongtheinvestigatedcompounds, gallicandcaffeicacidweretheonlyantioxidantsfoundinallspecies whenanalyzingtheextractswithdiodearraydetection.InC. per-rottettivar.angustifolius,catechin,rutin,ferulicacid,quercitrinand resveratrolwerealsopresent.Amongthese,onlyrutin wasnot detectedinC.palmata.CatechinwasnotpresentonlyinM.alliacea, beingthemajorcompoundinallotherspecies.Theinfusionwith

C.perrottettivar.angustifoliusmayprovideimportantamountsof gallicacidandcatechin,supportingitsemploymentwithmedicinal purposes.

Whencomparing the results obtainedvia LC-MS/MS tothe onesobtainedbyHPLC-DAD,over 60%agreementwasreached. Ofallcompounds,onlyresveratrolcouldnotbedetectedbymass spectrometry.However,thisresult,aswellasthefactthatother componentswerealsonotdetectedinsomeperiodsofcollectionor eveninsomeplantspecies,maybeaconsequenceoftheextraction methodused.

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Authors’contributions

FBPcarriedouttheresearch.Theseresultsformapartofher Masterdegreework.CBD,DM,andVDPsupportedtheultrasound extraction.HFperformedtheLC-MSanalysis.LMC,CVandMBR cre-atedtheProjectandwereresponsibleforarrangingthescholarship andfinancialsupport.MBRwastheadvisorofFBP.OLcollectedthe plantsandperformedthevoucher.FOLperformedthe chromato-graphicvalidation.Alltheauthorshavereadthefinalmanuscript andapproveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ToCNPqforfinancialsupport.

References

Ajila,C.M.,Brar,S.K.,Verma,M.,Tyagi,R.D.,Godbout,S.,Valéro,J.R.,2011.Analysis ofpolyphenols:recenttrends.Crit.Rev.Biotechnol.31,227–249.

Alvim,N.A.T.,Ferreira,M.A.,Cabral,I.A.,Almeida,J.de,2006.Ousodeplantas medic-inaiscomorecursoterapêutico:dasinfluênciasdaformac¸ãoprofissionalàs implicac¸õeséticaselegaisdesuaaplicabilidadecomoextensãodapráticade cuidarrealizadapelaenfermeira.Rev.Latino-Am.Enfermagem.14,1–9. Ananias,D.dosS.,Souza,E.B.,Souza,P.F.S.,Souza,M.deL.,Vitorino,M.I.,Teixeira,

G.M.,Ferreira,D.daS.B.,2010.Climatologiadaestruturaverticaldaatmosfera emNovembroparaBelém-PA.Rev.Bras.Meteorol.25,218–226.

Andreo,D.,Jorge,N.,2006.Antioxidantesnaturais:técnicasdeextrac¸ão.B.doCEPPA 24,319–336.

Arend,D.P.,Santos,T.C.,Sonaglio,D.,Santos,A.L.G.,Reginatto,F.H.,Campo,A.M., 2011.Experimentaldesignasatooltoevaluatechlorogenicandcaffeicacids extractedfromCecropiaglazioviiSneth.J.Pharm.Biomed.Anal.54,58–66. Becho,J.R.M.,Machado,H.,Guerra,deM.,2009.Rutina–estrutura,metabolismoe

potencialfarmacológico.Rev.Int.Est.Exp.1,21–25.

Beringhs,A.O.,Dalmina,M.,Crecznski-Pasa,T.B.,Sonaglio,D.,2015.Responsesurface methodologyIV–optimaldesignappliedtotheperformanceimprovementofan RP-HPLC-UVmethodforthequantificationofphenolicacidsinCecropiaglaziovii products.Rev.Bras.Farmacogn.25,513–521.

Campelo,P.M.S.,2006.Medicinalplantsanditsextracts:thenecessityofcontinuous studies.Rev.Est.Biol.Amb.Divers.28,1–2.

Coelho-Ferreira,M.,2009.Medicinalknowledgeandplantutilizationinna Amazo-niancoastalcommunityofMaruda,ParáState(Brazil).J.Ethnopharmacol.126, 159–175.

Costa,G.M., Ortmann,C.F.,Schenkel, E.P.,Reginatto, F.H.,2011. AnHPLC-DAD methodtoquantificationofmainphenoliccompoundsfromleavesofCecropia species.J.Braz.Chem.Soc.22,1096–1102.

Coutinho,I.D.,Kataoka,V.M.F.,Honda,N.K.,Coelho,R.G.,Vieira,M.C.,Cardoso,C.A.L., 2010.Influênciadavariac¸ãosazonalnosteoresdeflavonóideseatividade antiox-idantedasfolhasdeCampomanesiaadamantium(Cambess.)O.Berg,Myrtaceae. Rev.Bras.Farmacogn.20,322–327.

DalPrá,V.,Dolwitsch,C.B.,Lima,F.O.,Carvalho,deL.M.,Viana,C.,Nacimento,doP.C., Rosa,daM.B.,2015.Ultrasound-assistedextractionandbiologicalactivitiesof extractsofBrassicaoleraceavar.capitata.FoodTechnol.Biotechnol.53,102–109. Diniz,A.C.B.,Astarita,L.V.,Santaré,E.R.,2007.Alterac¸ãodosmetabólitossecundários emplantasdeHypericumperforatumL.(Hypericaceae)submetidasàsecageme aocongelamento.ActaBot.Bras.21,443–450.

Faccin,H.,Viana,C.,Nascimento,P.C.do,Bohrer,D.,Carvalho,L.M.de,2016.Study ofionsuppressionforphenoliccompoundsinmedicinalplantextractsusing liquidchromatography–electrospraytandemmassspectrometry.J.Chromatogr. A1427,111–124.

Franck,U., 1998. Phytochemischeund pharmakologischeUntersuchungen der kardiovaskulärenWirkprinzipienvonCecropiahololeucaMiq.,Cercropia pachys-tachya Tréc., CecropiaglazioviiSneth.,Musanga cecropioidesR. Brown und CrataegusL.monogyna,oxyacantha.HerdeckeGCA-Verl,Alemanha. Freitas,J.C.,Fernandes,M.E.B.,2006.Usodeplantasmedicinaispelacomunidade

deEnfarrusca,Braganc¸a.Pará.Bol.Mus.Para.EmílioGoeldi,sér.Ciênc.Nat.1, 11–26.

Gobbo-Neto,G.L.,Lopes,N.P.,2007.Plantasmedicinais:fatoresdeinfluênciano conteúdodemetabólitossecundários.Quim.Nova30,374–381.

Guamán-Balcázar,M.C.,Setyaningsih,W.,Palma,M.,Barroso,C.G.,2016. Ultrasound-assistedextractionofresveratrolfromfunctionalfoods:cookiesandjams.Appl. Acoust.103,207–213.

Ignat,I.,Volf,I.,Popa,V.I.,2013.Analyticalmethodsofphenoliccompounds.In: Ramawat,K.G.,Mérillon,J.-M.(Eds.),NaturalProducts–Phytochemistry,Botany andMetabolismofAlkaloids,PhenolicsandTerpenes.Springer, Berlin,pp. 2061–2092.

IPEA,2010.SustentabilidadeambientalnoBrasil:biodiversidade,economiae bem-estarhumano.Institutode PesquisaEconômicaAplicada,http://www.ipea. gov.br/portal/images/stories/PDFs/livros/livros/livro07

sustentabilidadeambienta.pdf/(accessed02.04.16).

INM, 2015. Instituto Nacional de Meteorologia, http://www.inmet.gov.br/sim/ gera graficos.php/(accessed15.11.15).

INM, 2016.NormalizaçãoeQualidade Industrial: Orientaçõessobre Validação de Métodos Analíticos –DOQ-CGCRE-008. Instituto Nacional de Metrolo-gia, http://www.inmetro.gov.br/Sidoq/Arquivos/CGCRE/DOQ/DOQ-CGCRE-8

02.pdf/(acessed13.03.16).

Jaques,A.C.,Pertuzatti,P.B.,Barcia,M.T.,Zambiazi,R.C.,Chim,J.F.,2010.Estabilidade decompostosbioativosempolpacongeladadeamora-preta(Rubusfruticosus) cv.TUPY.Quim.Nova33,1720–1725.

Lahlou,M.,2013.Thesuccessofnaturalproductsindrugdiscovery.Pharmacol. Pharm.4,17–31.

Lameira,O.A.,Paiva,J.S.,OliveiraE.C.P.de,Pinto,J.E.B.P.,2004.Fenologiaeanálise fitoquímicadeplantasmedicinaisdeocorrêncianaAmazônia.Hortic.Bras.22, 1–6.

Luengas-Caicedo,P.E.,deOliveira,A.B.,Brago,F.C.,Brandão,G.C.,2007.Seasonal andintraspecificvariationofflavonoidsandproanthocyanidinsinCecropia glazioviSneth.leaves fromnativeand cultivated specimens.J. Biosci. 62, 701–709.

LuqueM.D.,deC.,Priego-Capote,F.,2007.Ultrasound-assistedpreparationofliquid samples.Talanta72,321–334.

Morais,L.,2009.Influênciadosfatoresabióticosnacomposic¸ãoquímicadosóleos essenciais.Hortic.Bras.27,4050–4062.

MinistériodaSaúde(MS),2006.PolíticaNacionaldePlantasMedicinaise Medica-mentos Fitoterápicos, http://bvsms.saude.gov.br/bvs/publicacoes/politica

nacionalfitoterapicos.pdf/(acessed02.10.15).

Naczk,M.,Shahidi,F.,2004.Extractionandanalysisofphenolicsinfood.J. Chro-matogr.A1054,95–111.

Naczk,M.,Shahidi,F.,2006.Phenolicsincereals,fruitsandvegetables:occurrence, extractionandanalysis.J.Pharm.Biomed.Anal.41,1523–1542.

Paracampo, N.E.N.P., 2011. Connarus perrottetii var. angustifolius Radlk. (Con-naraceae): tradicionalmenteutilizada como barbatimãonoPará. Embrapa AmazôniaOriental,Belém,PA,Brasil,pp.2011.

Patel, I., Sipai,S., Rathod, D., Shrimali, G., Patel, A., Rami, E., 2013. Phyto-chemical studies on Mansoa alliacea (Lam.). Int. J. Adv. Pharm. Res. 4, 1823–1828.

Pereira, C., Meireles, M.A.A., 2010. Supercritical fluid extraction of bioactive compounds:fundamentals,applicationsandeconomicperspectives.Food Bio-processTechnol.3,340–372.

Pérez,D.,2002.EtnobotánicamedicinalybiocidasparaMalariaenlaregiónUcayali. Fol.Amazon13,87–101.

Rezende,H.A.,Cocco,M.I.M.,2002.Utilizac¸ãodefitoterapianocotidianodeuma populac¸ãorural.Rev.Esc.Enferm.USP36,282–288.

Ribani,M.,Bottoli,C.B.G.,Collins,C.H.,Jardim,C.S.F.,2004.Validac¸ãoemmétodos cromatográficoseeletroforéticos.Quim.Nova27,771–780.

Ribeiro,C.M.,Souza,K.G.daS.,Ribeiro,A.B.R.R.,Mendonc¸a,L.C.V.,Barbosa,W.L.R., 2009.Avaliac¸ãodaatividadeantimicrobianadeplantasutilizadasnamedicina populardaAmazônia.Infarma21,45–49.

Routray,W.,Orsat,V.,2013. Preparativeextractionandseparationofphenolic compounds.In:Ramawat,K.G.,Mérillon,J.-M.(Eds.),NaturalProducts– Phy-tochemistry,BotanyandMetabolismofAlkaloids,PhenolicsandTerpenes. Springer,Berlin,pp.2013–2046.

Schmitz,W.,Saito,A.Y.,Saridakis,D.H.O.,2005.Ocháverdeesuasac¸õescomo quimioprotetor.SeminaCienc.Biol.Saude26,119–130.

Silva,F.,Gallardo,A.F.E.,Nerín,C.,Domingues,F.C.,2014.Strategiestoimprovethe solubilityandstabilityofstilbeneantioxidants:Acomparativestudybetween cyclodextrinsandbileacids.FoodChem.145,115–125.

Silveira, da G.D., Motta, M.J., Müller,L.S., Lameira, O., Athayde, M.L., Pianna, M., Rosa, da M.B., Viana, C., Carvalho, de L.M., 2015. Determination of phenolic antioxidants in Amazonian medicinal plants by HPLC with pulsed amperometric detection. J. Liq. Chromatogr. Relat. Technol. 38, 1259–1266.

Stalikas,C.D.,2007.Extraction,separation,anddetectionmethodsforphenolicacids andflavonoids.J.Sep.Sci.30,3268–3295.

Veiga,V.F.J.,Pinto,A.C.,Maciel,M.A.M.,2005.Plantasmedicinais:curasegura?Quim. Nova28,519–528.

Villiers,F.L.,Lynen,A.,Crouch,S.P.,2004.Developmentofasolid-phaseextraction procedureforthesimultaneousdeterminationofpolyphenols,organicacidsand sugarsinwine.Chromatographia59,403–409.

Waterhouse,A.L.,2002.Determinationoftotalphenolics.Curr.Protoc.FoodAnal. Chem.6,1–8.