Rev. bras. farmacogn. vol.25 número2

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

article

UHPLC–MS

quantification

of

coumarin

and

chlorogenic

acid

in

extracts

of

the

medicinal

plants

known

as

guaco

(

Mikania

glomerata

and

Mikania

laevigata

)

Lucilia

V. de

Melo,

Alexandra

C.H.F.

Sawaya

∗

DepartamentodeBiologiaVegetal,InstitutodeBiologia,UniversidadeEstadualdeCampinas,Campinas,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16December2014 Accepted6February2015 Availableonline27March2015

Keywords: Mikaniaglomerata Mikanialaevigata

Coumarin Chlorogenicacid UHPLC–MSanalysis

a

b

s

t

r

a

c

t

InBrazil,MikaniaglomerataSpreng.andM.laevigataSch.Bip.exBaker,Asteraceae,knownpopularly asguaco,arewidelyusedforcoldsandasthma.Althoughcoumarinisadoptedasthechemicalmarker ofbothspecies,itwasnotalwaysdetectedinM.glomerata,forwhichchlorogenicacidwasidentified andquantifiedinstead.Thepurposeofthisstudywastodevelopandvalidateamethodtoquantifyboth coumarinandchlorogenicacidandapplyittoextractsofplantsidentifiedasM.glomerata,M.laevigata,or asguaco,todeterminethepatternofcompositionofthesetwospeciesandtoobservedifferencesbetween oven-driedandlyophilizedleaves.Amethodusingultra-highresolutionliquidchromatography–mass spectrometry(UHPLC–MS)inthefullscanmodewasvalidatedforselectivity,matrixeffect,linearity, lim-itsofdetectionandquantification,precisionandaccuracy.Theconcentrationofcoumarinvariedbetween speciesandsamples,thereforethesetwospeciesshouldnotbeusedinterchangeably.Theconcentration ofchlorogenicacidwasalsodeterminedforallsamples.TheUHPLC–MSmethodpermittedthe quantifi-cationofcoumarinandchlorogenicacidin16samplesofguacoandseveralcommercialsampleswere possiblymisidentified.

Introduction

The therapeutic use of medicinal plants is part of the

his-toryofhumanity.Frequentlythepopulationof underdeveloped

countriesstilldependsstronglyonmedicinalplantsforspiritual,

culturaloreconomic reasons(Quirozetal.,2014).Furthermore,

evenindevelopedcountries,theuseoftraditionalherbal

reme-diesiswidespread.Herbalremediesareevencrossingborders,for

examplethewidespreaduseof traditionalChinese Medicinein

Europe(Hook,2014).

InBrazil,twospeciesoftheMikaniagenus,Asteraceae,popularly

knownasguaco,areusedinsyruporpreparedasteaforcoldsand

otherrespiratoryproblemsduetotheirbronchodilatoreffect(Silva

etal.,2008).MikaniaglomerataSpreng.andM.laevigataSch.Bip.ex

BakermaybefoundintheAtlanticCoastforest,rangingfromthe

stateofBahiaintheNortheastofBrazil,tothesouthernstatesof

Brazil(Gasparettoetal.,2010)andeveninParaguayandArgentina

(Limaetal.,2003).M.glomeratawasincludedinthefirstBrazilian

Pharmacopeia(Brasil,1929)whileM.laevigatawasonlyincludedin

∗ Correspondingauthor.

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

thefourtheditionoftheBrazilianPharmacopeia(Brasil,2005).Both

speciesaremorphologicallysimilarandmaybeeasilymistaken;

theyareoftencommercializedandpreparedindistinctly(Anvisa,

2011).

Studiesoftheircomposition haveledtotheidentificationof

phenoliccompounds,di-andtri-terpenes,tanninsandother

com-ponents (Gasparettoet al.,2010).Although coumarinhas been

adopted asthe chemical markerof both species(Anvisa, 2008,

2011)and theirpharmacologicalproperties areoftenattributed

to this substance,several studiespresent conflicting resultson

theconcentrationofcoumarin.Forexample:Santosetal.(2006)

reportedthattheextractofM.glomeratapresentedtwiceasmuch

coumarinasthatM.laevigata,whereasBolinaetal.(2009)

con-cluded that M. laevigata presented a slightly higher coumarin

content(0.43%)thanM.glomerata(0.30%).Athirdstudyreported

thatcoumarinwasnotpresentinM.glomerata,onlyinM.

laevi-gata(Bertoluccietal.,2009)whichisconsistentwithourresults.

These contradictory resultsmay be due to misidentification of

theplantspecies(duetotheirmorphologicalsimilarity)ortothe

diverseanalyticalmethodsemployed.Theuseoffreshleaves,oven

dried leavesor lyophilizedmaterial mayalsohave affectedthe

results(Santosetal.,2006).Furtherpossibilitiesarevariationsin

secondarymetabolitesduetoseasonalorenvironmentalfactors

http://dx.doi.org/10.1016/j.bjp.2015.02.005

(2)

(Gobbo-NetoandLopes,2007).Preliminarystudiesbyourgroup

detectedthepresenceofchlorogenicacidinM.glomerataleaves,

whichwasasurprisingasonlyonestudywasfoundrelatingthe

presenceofchlorogenicacidin guacoleaves(Silva etal.,2006).

Thereforeitwasnecessarytobeginthisinvestigationbydefining

theplantsamples,thedryingmethodandtheanalyticalmethod

tobeused.Thismethodshouldbeselectiveenoughtoquantify

coumarinand chlorogenicacidcorrectly,but shouldalsodetect

other,stillunidentified,componentsintheextracts.

Severalmethodshave beenfoundinliteratureforthe

quan-tificationof coumarins;fromsimplethin layerchromatography

(Alvarenga et al., 2009) to modern electrochemical methods (Miyanoetal.,2014).Celeghinietal.(2001)quantifiedcoumarin

inextractsofM.glomerataleavesbyhighresolutionliquid

chro-matographywithUVdetection(HPLC–UV),whileMuceneekietal.

(2009)quantifiedo-coumaricacid,coumarinandsyringaldehyde

byHPLC–UV.Parketal.(2009)andChenetal.(2012)usedHPLC

coupledtomassspectrometry(HPLC–MS)intheMRMmodeto

quantifycoumarins.Whilethismethodishighlyselectiveand

sen-sitive,itonlypermitstheanalysisoftheselectedcomponents.In

fact,allthemethods citedabovewouldnothave allowedusto

observethepresenceofchlorogenicacidinM.glomeratasamples,if

theyhadbeendevelopedforcoumarin.Thesamecanbesaidofthe

HPLC–MS(MRM)methodusedtoquantifydicaffeoylquinicacids

(Cliffordetal.,2008)andchlorogenicacidmetabolites(Santosetal.,

2005).However,byusingMSdetectioninfullscanmodeitis

pos-sibletoquantifyselectedionsaswellasdetectothercomponents

oftheextract.

Inordertoevaluatetheconcentrationofcoumarinand

chloro-genicacidinleavesofM.glomerataandM.laevigata,whileallowing

thedetectionoftheothersamplecomponents,a methodusing

ultra-highresolution liquidchromatography–massspectrometry

(UHPLC–MS)wasdevelopedandvalidated.Thismethodwasused

toanalyzetheethanolic extractsof ovendried and lyophilized

leavesofbothspecies,aswellasleavescommercializedsimplyas

guaco.Themethodwasdevelopedforethanolicextractsofleaves

oftwoplantsidentifiedbyspecialistsasM.laevigataandM.

glom-erata;andthenappliedtoothersamplesoffreshanddryleaves

(commercializedasguaco).

Materialsandmethods

Plantsamples

MikaniaglomerataSpreng.andM.laevigataSch.Bip.exBaker,

Asteraceae,plants weredonatedand identified byCPQBA,

Uni-camp(Paulínia,SP)andvoucherspecimensdepositedattheState

UniversityofCampinasHerbarium(UEC)number102046forM.

laevigataandnumber102047forM.glomerata.Theseplantsare

growingintheExperimentalFieldoftheInstituteofBiology.For

thisstudy,leavesofbothspecieswerecollectedthesamemorning

anddriedbytwodifferentmethodsbeforegrindingand

extrac-tion.

Othercommercialsamplesfreshanddryleaveslabeledasguaco,

M.laevigataorM.glomeratawereboughtfromlocalmarketsor

collectedfrominstitutional (CPQBA-Unicamp)or homegardens

(Table2).Allsampleswereextractedwithindaysoftheir

acqui-sition. The dry leaves were extracted in the same way as the

identifiedplant samples;thefresh leaveswerelyophilized and

thenextracted.OneplantwhichwascollectedinthestateofSão

PauloandidentifiedasM.glomeratabyProf.GeorgeY.Tamashiro

oftheBiologyInstituteofUnicamp,presentedconcentrationsof

coumarinandchlorogenicacidsbelowquantificationlevelandwas

thereforedriedandextractedtobeusedasablankplantmatrix

(BPM)forcalibrationcurves.

Dryingandextraction

Approximatelyhalfoftheleavescollectedfromeachplantwere

driedinanovenwithaircirculationat40◦_C_for₅₀_h;_the_other_half

wasdriedbylyophilizationfor50h.Thematerialwasthenground

inamortar,passedthroughasievewith0.84mmspaces,andplaced

in67%ethanol(Ecibra,Brazil)toextract,followingtheproportionof

200gofleavestoatotalof1.0lofsolvent(Brasil,1929).After

filter-ing,a10mlaliquotwastakentoevaluatethetotalsolidsextracted

bydyingthesolventinanovenat105◦_C_until_constant_weight.

Chromatographicmethod

A chromatographic method was developed and validated

using an ultra-high performance liquid chromatographer

cou-pled toa triple quadrupolemass spectrometer.Theequipment

used was an Acquity UPLC-TQD (Micromass, Waters,

Manch-ester,England)and thecolumnwasa C18BEHAcquityWaters

(1.7␮m×2.1mm×50mm),oventemperatureof 30◦_C._The

elu-tionwascarriedoutwithaflowof200␮l/min,SolventA–purified

water(Milli-Q)with0.1%formicacidandSolventB–HPLCgrade

acetonitrile(JTBaker,PA,USA),underagradientstartingwith10%B,

rampingto25%Bin4min,thento100%Bin8min,heldat100%B

until8.5minthereturningtotheinitialconditionsandstabilizing

until10min.

MSdetection wasperformedwithelectrosprayionizationin

bothpositiveandnegativeionmodes,underthefollowing

con-ditions:capillary±3000V,cone±35V,sourcetemperature150◦_C

and desolvation temperature of 300◦_C. _Due _to _their _structure,

chlorogenicacidionizedwellinnegativeionmodeandcoumarin

inthepositiveionmode.

Priortoinjectiontheextractswerefurtherdilutedinpurified

water(Milli-Q)intheproportionof1partextractto2partswater,

2␮lofeachsamplewereinjected.Theconcentrationofcoumarin

andchlorogenicacidintheplantextractswasquantifiedby

com-parisontoexternalcalibrationcurvesofcoumarin(Sigma–Aldrich)

andchlorogenicacid(Sigma–Aldrich)insolutionsof70%ethanol

andinBPM.Themethodwasvalidatedaccordingtotheparameters

describedbelow.

Selectivity.Solutionsofthestandardsofcoumarinand

chloro-genic acid, plant extracts and plant extracts spiked with the

standardswereinjected,evaluatingretentiontimesand

fragmen-tationspectra(MS/MS)oftheionofm/z147inthepositiveionmode

(coumarin)andorm/z353inthenegativeionmode(chlorogenic

acid).

Matrixeffect.ThiseffectwascalculatedaccordingtoEconomou etal.(2009)usingtheformula:C%=100×(1−Sm/Ss);whereC%is

thepercentageofincreaseorsuppressionofthesignal,Smisthe

angularcoefficientofthecalibrationcurveusingBPMandSsisthe

angularcoefficientofthecalibrationcurveusingasolutionof70%

ethanol.

Linearity.Thisparameterwasevaluatedbythecorrelation

coef-ficientofthecurvesofcoumarinandchlorogenicacidinBPM.

Limitofdetection(LD)andquantification(LQ).These

parame-tersweredeterminedbytheinjectionofa seriesofdilutionsof

coumarinandchlorogenicacidinBPM,withLDdeterminedasthe

concentrationthatresultedinapeakareathreetimesgreaterthan

thenoiselevelandLQaconcentrationthatresultedinapeakarea

tentimesgreaterthanthenoiselevel.

Precision.Thisparameterwasevaluatedfor fiveinjectionsof

extractsofM.laevigataandM.glomerataleavesalongonedayfor

theconcentrationofcoumarinandchlorogenicacid.

Accuracy.Asnocertifiedmaterialwasavailable,this

parame-terwasevaluatedbytheaddition(fortification)ofcoumarinand

chlorogenicacidinthreelevelsofconcentration:low(30␮g/ml),

(3)

tothepreparationofthecalibrationcurve.Recovery(R%)was

calcu-latedaccordingtothefollowingequation:R%=(C1−C2)/C3×100;

whereC1istheconcentrationwhichwasdeterminedforthe

forti-fiedsample,C2istheconcentrationofthenon-fortifiedsampleor

matrixandC3istheconcentrationofstandardwhichwasadded

(fortification).

Resultsanddiscussion

Dryingmethod

Leafextractsofbothspecies(driedintheovenorby

lyophiliza-tion)presentedsimilaramountsofsolid residue:1.2%(m/v)for

ovendriedM.laevigataand1.3%forlyophilizedM.laevigata;1.3%

forovendriedM.glomerataand1.4%forlyophilizedM.

glomer-ata.TheUHPLC–MSprofileoftheleafextractsofeachspecieswas

different(Fig.1)butthedryingproceduredidnotaffectthe

gen-eralprofileofthespecies.Howevertheamountofcoumarinand

chlorogenicacidwaslowerfortheovendriedleavesofbothspecies

(Table1),showingthatheataffectedthecontentsofthebioactive

compoundsevaluated.Forthis reason,alltheotherfreshleaves

collectedduringthisstudy(Table2)werelyophilized,ratherthan

ovendried,beforeextraction.

ValidationofUHPLC–MSmethod

Analyticalcurvesofcoumarinandchlorogenicacidstandards

withconcentrationsbetween1ng/mland 800␮g/mlwerebuilt

in 70% ethanol/waterand in BPM tovalidate themethod.This

rangeof concentrationswasnecessarydue tothevariable

con-tentofcoumarinandchlorogenicacidinbothplantspeciesandin

theextractsthatwereanalyzed.Furthermoreallparameterswere

successfullyvalidatedforthisrangeofconcentrations.

Selectivity. This parameter was determined comparing the

retentiontime(RT)andthefragmentationofcoumarinstandard

(positiveionmodem/z147)andchlorogenicacidstandard

(nega-tiveionmodem/z353)withthesameionsinthesamples.Fig.1A

showstheselectedionchromatogram(m/z147positiveionmode)

ofthecoumarinstandardandFig.1Bshowstheselectedion

chro-matogram(m/z147)oftheM.laevigataextract,theretentiontimes

arepracticallyidenticalandbothpresentthesameMS/MS(Fig.1E).

Fig.1Fshowstheselectedionchromatogramofm/z353negative

ion mode.Although threepeaksare present,thefirst retention

time(2.45min)correspondstochlorogenicacidandtheothersare

isomers,presentasimpuritiesinthestandard.Fig.1Gshowsthe

selectedionchromatogram(m/z353)oftheM.glomerataextract,

thepeak atretention timeof 2.51showedthesameMS/MSas

chlorogenicacid(Fig.1J).Smallvariationsin theretention time

wereduetothecomplexmatrixoftheplantextracts.Thesolvent

didnotpresentpeaksofthesecompoundsandinBPM(Fig.2)the

areasofpeaksofthesecompoundswerebelowtheLD.

Matrixeffect.Thisparameterwasevaluatedcomparingcurvesof

bothstandardsinsolventwithcurvesINBPM.Theresultsshowed

thattheplantmatrixresultedinareductionofpeakareaof4.37%

forcoumarinandof19.20%forchlorogenicacidinrelationtothe

sameconcentrationsinsolvent.Thereforeallsubsequentanalytical

curvesusedinthisstudywerebuiltusingBPM.

Linearity.AnalyticalcurvesofbothstandardsinBPMwerebuilt

between1ng/mland800␮g/ml,withtriplicateinjectionsofeach

point. The ideal parameter of linearity (R2_>_0.99) _could_not _be

attainedduetotheinterferenceofthematrixandthewiderangeof

concentrationsused.Theanalyticalcurveforcoumarin(m/z147in

thepositiveionmode)waslinear(R2₌_0.9718)_for_{concentrations}

between1.5␮g/mland730.0␮g/ml,andthecurveforchlorogenic

acid(m/z353inthenegativeionmode)waslinear(R2₌_0.9831)

forconcentrationsbetween10.0␮g/mland550.0␮g/ml.Onlyone

sampleofdryleaves(sample8,Table2)felloutsidetherangeof

thesecurvesandtheconcentrationofcoumarinwasobtainedby

extrapolationofthecurve.

Limitofdetection(LD)andquantification(LQ).Forcoumarinthe

LDwas0.32␮g/mlandtheLQwas3.30␮g/mlinBPM.For

chloro-genicacidtheLDwas4.18␮g/mlandtheLQwas20.38␮g/mlin

theblankplantmatrix.TheLDandLQwerehigherforchlorogenic

acid,possiblybecauseoflessionizationinthenegativeionmode.

Precision.Theareaof thepeaksoffivereplicateinjectionsof

theextractsofM.glomeratapresentedvariationof5%forcoumarin

(m/z147inthepositiveionmode)and4%forchlorogenicacid(m/z

353inthenegativeionmode).FortheextractsofM.laevigatathe

variationwasof1%forcoumarinand4%forchlorogenicacid.

Accuracy.Therecuperation(R)valuesforthethreelevelsof

for-tificationofcoumarinwereintheextractofM.laevigata75.15%

(low),84.60%(medium)and67.04%(high).Forthefortificationwith

chlorogenicacidintheextractofM.glomeratatheRwas:106.42%

(low),88.56%(medium)and95.74%(high).Asonlytheresultforthe

highestconcentrationofcoumarinfellslightlyoutsidetheaccepted

recuperationparameter(70–120%)(Ribanietal.,2004).

AnalyticalresultsforplantsamplesOneadvantageofthemethod

presentedhereinisthatacquisitioninbothpositiveandnegative

modesisobtainedinthesameshortchromatographicrun.This

chromatographic methodwasfirst usedtoanalyzetheextracts

oftheovendriedandlyophilizedleavesofM.glomerataandM.

laevigata.Theextractswerepreparedusing200gofleaves/lof

sol-vent;thereforetheresultsshowninTable1in␮g/mlcorrespondto

200mgofdriedleaves.Inthismannerweobservethattheleavesof

theidentifiedM.glomerataplantcontainedpracticallynocoumarin.

Chlorogenicacidcontentwasbetween0.67%(m/m)forovendried

leavesand0.82%(m/m)forlyophilizedleaves.Incomparison,M.

laevigataovendriedleavescontained0.37%(m/m)coumarinand

lyophilizedleavescontained0.57%(m/m)coumarin.Thisresultis

inagreementwiththecoumarincontentpresentedbyBolinaetal.

(2009)forM.laevigataleaves,andalsowiththeresultspresentedby

Bertoluccietal.(2009)whostatedthatnocoumarinwasdetected inM.glomerata.Furthermore,theUHPLC–MSchromatogramsshow

thatthetwospeciesofguacopresentdistinctchromatographic

pro-filesinbothpositive(Fig.1CandD)andnegative(Fig.1HandI)ion

modes.

The peak of coumarin(RT 4.06) is clearly seenin the

chro-matogramoftheM.laevigataextract (Fig.1C)butabsentinthe

chromatogramoftheM.glomerataextract(Fig.1D).Inversely,the

peakofchlorogenicacid(RT2.51)isabsentinthechromatogram

of theM. laevigata extract (Fig. 1H) but is clearly seen in the

chromatogramoftheM.glomerataextract(Fig.1I).Furthermore,

thechromatographicprofilesofbothspeciesareclearlydifferent.

Althoughclimaticandseasonalvariationscouldaffecttheseresults,

bothspeciesareplantedside-bysideintheExperimentalFieldin

Unicampandcollectedatthesametime,sosubjecttothesame

influences.

Inordertocheckifotherguacosampleswouldbehaveinthe

samemanner,eightsamplesoffreshleavesandeightsamplesofdry

leaveswereacquired,extractedandanalyzedusingthesame

vali-datedchromatographicmethod.TheresultsareshowninTable2.

Freshleaves,samples9–12,identifiedasM.glomerataand

col-lected at CPQBA-Unicamp, presented the same pattern as our

originalM.glomerataleaves,withvaryingamountsofchlorogenic

acidandnodetectablelevelsofcoumarin.Sample13,identifiedas

M.laevigataandcollectedatCPQBA-Unicamp,presented

approx-imately the same amount of coumarin as our original sample

(Table1)andchlorogenicacidbelowtheLQ.Thisconfirmedthe

expectedpattern ofcompoundsexpectedforthesespecies.The

freshleavesofbothspecieswerecollectedseveralmonthsafterthe

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Time

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0 100

T4REPcurva cum 25ug ml 8a 1: Scan ES+

147 6.39e8

4.03

9.61

Time

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0 100

MLF0 2b 1: Scan ES+

147 1.45e9

4.10

2.93

MLF0 2b 1: Scan ES+

BPI 1.34e8

x2 4.06

0.74

0.65 2.20 2.512.93 3.613.85

8.00 6.48

5.91 4.43

7.51 7.40

8.10

9.65 8.64

9.94

MGL 2b TACL 1 1: Scan ES+

BPI 2.62e7

2.51

0.83 0.72

0.11 1.922.08

9.65 3.80

3.44

3.28 3.92 4.48 _5.92

5.12 5.33 _6.536.957.587.938.13_{8.53 8.67} 9.60

m/z 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

%

0

100 91

77

147 103

Time

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

T4curva AC 10ug ml 8b 2: Scan ES-

353 7.36e6

2.45

3.36

3.93 _9.12 _9.62

Time

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

2

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0 100

MGL 2b TACL 1 2: Scan ES-

353 8.70e7

2.51

2.26

3.34 2.73 3.51

MLF0 2b 2: Scan ES-

BPI 1.37e7

2.92 0.80

0.66 0.90 _1.79 2.44

4.28 3.86

3.60 3.24

7.97 5.90

4.42

4.985.17 _{6.16 6.79}6.91 _{7.39 7.52}

9.60 9.40 8.67

8.84

MGL 2b TACL 1 2: Scan ES-

BPI 1.87e7

4.47 3.45

2.51 0.82 2.26

1.81

3.34 2.96

3.83

4.19 4.59 _5.38 5.90 6.596.827.01 7.99 8.68_{8.86 9.60}

m/z

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100 179191 135

161 353

A

J

I

H

G

F

E

D

C

B

Fig.1.PositiveionmodeESI-MSUHPLCof:(A)coumarinstandardm/z147RT4.03min,(B)extractedionm/z147M.laevigataextract,(C)chromatogramofM.laevigata extract,(D)chromatogramofM.glomerataextract,(E)MS/MSofcoumarinm/z147.NegativeionmodeESI-MSUHPLCof:(F)chlorogenicacidstandardm/z353RT2.45min, (G)extractedionm/z353M.glomerataextract,(H)chromatogramofM.laevigataextract,(I)chromatogramofM.glomerataextract,(J)MS/MSofchlorogenicacidm/z353.

coumarinthanchlorogenicacidforM.laevigataandtheopposite,

morechlorogenicacidthancoumarin,forM.glomerata.

Driedleafsamples2,3,5,6,7and8,presentedapattern

simi-lartoM.laevigataleaves,withvaryingconcentrationsofcoumarin

andlessornochlorogenicacid.Thewaythesecommercial

sam-plesweredriedisunknown,butpossiblyaffectedthecontentsof

coumarin/chlorogenicacid.Thesesampleswerelabeledsimplyas

guaco(sample2)orM.glomerataandmayhavebeenmisidentified.

Driedleafsample4,labeledasguacoandacquiredintheAmazon,

presentedanapproximatelyequalconcentrationofcoumarinand

chlorogenicacid,whichisdifferentfromthepatternspreviously

encounteredandcouldevenbelongtoadifferentspeciesof

Mika-nia.Sample1,alsolabeledasguaco,presentedseveralmoldyleaves

andstemsinthepackageandnodetectablelevelsofcoumarinor

chlorogenicacid.Thereforeitisimpossibletoascertainifthis

(5)

Table1

Concentrationofcoumarinandchlorogenicacidinhydro-ethanolicextractsofovendriedandlyophilizedleavesofM.glomerataandM.laevigataplantedintheexperimental field(IB,UNICAMP),triplicateextractions.

Leavesof Coumarincontentinextract (␮g/ml)

CV(%) Chlorogenicacidcontentinextract (␮g/ml)

CV(%)

OvendriedM.glomerata * _– ₁₃₄₈ _9.94

LyophilizedM.glomerata * _– ₁₆₃₄ _5.79

OvendriedM.laevigata 775 3.26 * _–

LyophilizedM.laevigata 1131 0.61 * _–

*_Below_LD.

Table2

Concentrationofcoumarinandchlorogenicacidinhydro-ethanolicextractsofdryandfreshleaveslabeledasM.glomerata,M.laevigataorguaco.

Samplelabeledas Form Boughtorcollectedin Coumarin (␮g/ml)

Chlorogenicacid(␮g/ml)

1.Guaco Dryleaves PortoAlegre–RS a a

2.Guaco/Mikania Dryleaves PortoAlegre–RS 155 b

3.M.glomerata Dryleaves Florianópolis–SC 170 64

4.Guaco Dryleaves Manaus–AM 128 156

5.M.glomerata Dryleaves Brasília–DF 375 b

6.M.glomerata Dryleaves Paulínia–SP 254 a

9.M.glomerata(1)Id Freshleaves CPQBA,UNICAMPPaulínia–SP a 529

13.M.laevigata(1)Id Freshleaves CPQBA,UNICAMPPaulínia–SP 1024 b

14.Guaco Freshleaves Guarapuava–PR 172 b

15.Guaco Freshleaves JoãoPessoa–PB 365 b

16.Guaco Freshleaves SãoPaulo–SP 354 a

a:BelowLD,b:BelowLQ,Id–botanicalidentificationbyCPQBA-UNICAMP.

badconservation.Itisworryingthatthissamplewasbeingsold

atamarketandcouldhavebeenconsumedbychildrenoradults

wishingtoalleviatethesymptomsofacold!

Freshleafsamples14–16,knownsimplyasguacobytheirusers,

presentedvaryingconcentrationsofcoumarinbutchlorogenicacid

contentsbelowLDorLQ,whichissimilartotheresultsofM.

laevi-gatavoucherplantandfreshplantfromCPQBA.

Theonlysampleswhichpresentedconcentrationsofcoumarin

and chlorogenic acid compatible with theidentified sample of

M.glomeratawerethosefreshleavesidentifiedand collectedat

CPQBA-Unicamp.Mostothersamples,regardlessoftheirlabels,had

concentrationsofcoumarinandchlorogenicacidwhichwere

sim-ilartothosefoundinleavesoftheM.laevigatavoucherplant.The

onlytwoguacosampleswhichdidnotfollowthispatternwere

pos-siblydegraded(sample1)orbelongedtoanotherspeciesofMikania

(sample4).

The variation in theconcentration of coumarin and

chloro-genic acid found in identified specimens of M. glomerata and

M.laevigatademonstratesthatthesetwospeciesdonotcontain

similaramounts ofcoumarinand thereforeshould notbeused

interchangeably. Furthermore commercialsamples also contain

variablecontentsofcoumarinandchlorogenicacidandmaybe

misidentifiedordegraded.Furtherstudiesregardingthe

morpho-logicaldescriptionofplantsknownasguacothroughoutBrazilare

underwaytoascertainwhichspeciesisbeingusedregionallyand

ifenvironmentalfactorsaffecttheconcentrationofcoumarinand

chlorogenicacidintheseplants.

By using UHPLC–MS in the full scan mode, not only were

coumarinandchlorogenicacididentifiedandquantified,butalso

othercompounds,whichmake upthecomplexchemicalprofile

oftheseplantextracts,weredetected.AlthoughotherHPLC–MS

methodsintheMRMmodemaybemoresensitive,theydonot

pro-videuswithapanoramicviewofsamplecomposition,whichleads

tonewdiscoveries,suchastheimportanceofchlorogenicacidin

M.glomeratasamples.Thischromatographicmethodallowedus

todistinguishbetweensamplesofleavesfromtwo

morpholog-ically similarspeciesandcan beused forthequalitycontrolof

thedryleavesand extractsofthesespecies.Thisinformationis

paramountforthecorrect useofthesemedicinalplantsby the

population.

(6)

Conflictofinterest

Theauthorshavenonetodeclare.

Authors’contributions

ACHFSplannedtheresearch,LVMperformedtheexperiments

validatedthemethod.Bothauthorshelpedwithwritingthispaper.

Acknowledgements

This study was partially financed by CNPq grant number

473597/2013-6. We would like to thank P. Mazzafera for the

useof theUHPLC–MSequipment(BIOEN-FAPESP grant number

2008/58035-6)andDr.GlynM.Figueira(CPQBA)fortheM.

glom-erataandM.laevigataplantsamples.

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