Rev. bras. farmacogn. vol.25 número3

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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

Analysis

of

flavonoids

in

Rubus

erythrocladus

and

Morus

nigra

leaves

extracts

by

liquid

chromatography

and

capillary

electrophoresis

Luciana

R. Tallini

a

_,

_Graziele

_P.R.

_Pedrazza

a

_,

_Sérgio

_A.

_de

_L.

_Bordignon

b

_,

_Ana

_C.O.

_Costa

c

_,

Martin

Steppe

d

_,

_Alexandre

_Fuentefria

e

_,

_José

_A.S.

_Zuanazzi

a,∗

a_Departamento_de_Produc¸_ão_de_Matéria_Prima,_Faculdade_de_Farmácia,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

b_Centro_{Universitário}_La_Salle,_Canoas,_RS,_Brazil

c_Departamento_de_Ciência_e_Tecnologia_de_Alimentos,_Universidade_Federal_de_Santa_Catarina,_{Florianópolis,}_SC,_Brazil

d_Departamento_de_Produc¸_ão_e_Controle_de_{Medicamentos,}_Faculdade_de_Farmácia,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

e_Departamento_de_Análises,_Faculdade_de_Farmácia,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15January2015 Accepted30April2015 Availableonline17June2015

Keywords:

HPLC-UV UPLC-DAD/MS CE-DAD

Rubuserythrocladus

Morusnigra

Flavonoids

a

b

s

t

r

a

c

t

Thisstudyuseshighperformanceliquidchromatographyandcapillaryelectrophoresisasanalyticaltools toevaluateflavonoidsinhydrolyzedleavesextractsofRubuserythrocladusMart.,Rosaceae,andMorus nigraL.,Moraceae.Forphytochemicalanalysis,theextractswerepreparedbyacidhydrolysisand ultra-sonicbathandanalyzedbyhighperformanceliquidchromatographyusinganultravioletdetectorand bycapillaryelectrophoresisequippedwithadiode-arraydetector.Quercetinandkaempferolwere iden-tifiedintheseextracts.Theanalyticalmethodsdevelopedwerevalidatedandapplied.Quercetinand kaempferolwerequantifiedinR.erythrocladus,with848.43±66.68␮gg−1 and304.35±17.29␮gg−1, respectively,byHPLC-UVand quercetin,836.37±149.43␮gg−1,byCE-DAD.In M.nigrathe quan-tifications of quercetin and kaempferol were 2323.90±145.35␮gg−1 and 1446.36±59.00␮gg−1, respectively,byHPLC-UVand,2552.82±275.30␮gg−1and1188.67±99.21␮gg−1,respectively,by CE-DAD.Theextractswerealsoanalyzedbyultra-performanceliquidchromatographycoupledwitha diode-arraydetectorandmassspectrometer(MS),UPLC-DAD/MS.

Introduction

ThefruitsofRubus(Rosaceae)andMorus(Moraceae)havebeen extensivelystudiedaroundtheworldbecauseoftheirbeneficial effectsonhealth.Rubus,popularlyknownasthegenusof rasp-berriesandblackberries,iswidelydistributedaroundtheworld, bothaswildandcultivatedspecies(Deightonetal.,2000).Morus isknownasthegenusof mulberry,and isfoundfrom temper-ateregionstosubtropicalregionsofthePlanet(Gundogduetal., 2011).InBrazil,plantsbelongingtothegenusMorusarelistedby thegovernmentasinterestingtoresearchbecauseoftheirusein folkmedicine,andtheirpotentialforuseasphytotherapics(Portal daSaúde,2013).However,RubusandMorusareoftenmistakenin thedifferentiation,duetothesimilarityoftheirfruits.Also,there isalotofinformationaboutthefruitsofthesespecies,butlittle informationabouttheirleaves.

∗ _{Corresponding}_author.

E-mail:zuanazzi@ufrgs.br(J.A.S.Zuanazzi).

Studies have shown that Rubus leavesdemonstrate antioxi-dant (Venskutoniset al., 2007;Martini etal., 2009), anticancer (Durgo et al., 2012), gastrointestinal (Rojas-Vera et al., 2002), antiangiogenic(Liuetal.,2006),antithrombotic(Hanetal.,2012), hypoglycemic (Jouad et al., 2002), antimicrobial (Panizzi et al., 2002;ThiemandGoslinska,2004;Martinietal.,2009;Ostrosky et al., 2011) and anxiolytic activities (Nogueira et al., 1998; NogueiraandVassilieff,2000).FortheleavesofMorus,thereare studies reporting antioxidant(Katsube et al., 2010; Choiet al., 2013), anticancer(Dat etal.,2010; Skupienet al.,2008), hypo-glycemic(Volpatoetal.,2011;Chungetal.,2013),anti-obesity(Oh etal.,2009;Tsudukietal.,2013),antimicrobial(Omidiranetal., 2012)andvasodilationactivities(Xiaetal.,2008).

Flavonoids derived from quercetin and/or kaempferol were reportedinextractsofRubusleavesanalyzedbyTLC( Tzouwara-Karayanniand Philianos,1982),HPLC(Venskutonisetal.,2007; Martinietal.,2009;Durgoetal.,2012;GudejandTomczyk,2004) andNMR(Panizzietal.,2002;Hanetal.,2012;Gudej,2003).There arenostudiesaboutanalysisofRubusleavesbyCE,andthespecies erythrocladushasneverbeenstudiedbefore.InMorusleaves, sec-ondarymetabolitesderivedfromquercetinand/orkaempferolhave

http://dx.doi.org/10.1016/j.bjp.2015.04.003

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beenidentifiedbyHPLC(Katsubeetal.,2010;Thabtietal.,2012; Choietal.,2013),andbyNMR(Datetal.,2010;Katsubeetal.,2010), andtherearetwostudiesonthechemicalcompositionofMorus albabyCE(Suntornsuketal.,2003;Chuetal.,2006).

Highperformanceliquidchromatographyisaclassicmethod usedfortheseparationandquantificationofsecondarymetabolites inplantextractsbutsometimesitcanbeanexpensive analyti-caltool(MerkenandBeecher,2000).Capillaryelectrophoresisis characterizedbyhighseparationefficiencyandgoodcompromise betweenanalysistimeandsatisfactorycharacterizationof pheno-liccompoundsinplants.Thelowoperationalcostandthesmall residuegenerationmakethis techniqueanattractiveoption for thedevelopmentofanalyticalmethodsforphytochemicalanalysis (Carrasco-Pancorboetal.,2006).

TheaimofthisstudywastodevelopHPLC-UVandCE-DAD ana-lyticalmethods for theevaluation ofquercetin and kaempferol inRubus erythrocladus Mart.and MorusnigraL.leavesextracts. Theresultswerestatisticallyanalyzedusingvalidationparameters andthemethodswereappliedtodeterminetheamountofthese flavonoidsinthesesamples.

Experimental

Samples

LeavesofRubuserythrocladusMart.,Rosaceae,werecollectedat Embrapa–TemperateClimate(Pelotas,Brazil)andleavesofMorus nigraL.,Moraceae,werecollectedinPortoAlegre(Brazil).Vouchers havebeendepositedintheHerbarium–UFRGS,FederalUniversity ofRioGrandedoSul(179856and176765,respectively).The sam-plesweredriedatroomtemperaturefortwoweeksandkeptinthe dark.

Chemicalsandmaterials

Quercetin (purity≥98%) and kaempferol (purity≥97%)were obtainedfromSigma(USA)andmethylparabenfromFluka(USA). HPLC-grade acetonitrile and methanol were purchased from Merck (Germany). Throughout the study, deionized and ultra-purewaterwereused.Allotherchemicalswereanalyticalgrade. DichloromethaneandhydrochloricacidwereobtainedfromSynth (Brazil),trifluoroaceticacidfromVetec(Brazil),andethylacetate, sodiumhydroxideandsodiumtetraboratedecahydratefromMerck (Germany).

HPLCconditions

HPLCanalysiswereperformedonaWatersAlliance2695(USA) chromatographusingaUVdetector(UV/VISWaters2487,USA)and aC18reversed-phasecolumn(Phenomenex,150×4.6mm,4␮m) withguard-column(C18). TheEmpower softwarewasused for thedataacquisition.Aphotodiodearraydetector(DAD)(UV/VIS Waters996,USA)wasalsousedtoevaluatethespecificity.

Flavonoidswereelutedusingagradientsystem.Mobilephase Aconsistedofwaterwith0.01%(v/v)oftrifluoroaceticacidandB consistedofacetonitrilewith0.08%(v/v)oftrifluoroaceticacid.The gradientprofilewas:0min50%ofB,0–2min60%ofB,2–4min80% Band4–7min95%B.Theflow-ratewas0.6mlmin−1_and_detection

wasat370nm.

UPLCconditions

AnUltraPerformanceLiquidChromatography(WatersAcquity UPLC, USA) equipped with a diode array detector (DAD) cou-pledwitheletrosprayionizationinthepositiveionmode(ESI(+)) quadrupoletime-of-flight(Q-Tof)massspectrometry(MS)(MS Q-TofMicro-Micromass)wasused.Theseparationofthecompounds

wasperformedonaC18reversephasecolumn(AcquityUPLCbeh, 50.0×2.1mm, 1.7␮m). The software program Mass Lynx v.4.1 wasusedfortheanalysisanddataacquisitionandtheprogram AcquityUPLCColumnsCalculatorv.1.1.1wasusedtoadjustthe HPLCmethodtotheUPLCmethod.

MobilephaseAconsistedofwaterwith0.1%(v/v)offormicacid andacetonitrileBwith0.1%(v/v)offormicacid.Thegradientwas 0min50%ofB,0–0.09min60%ofB,0.09–0.38min80%ofBand 0.38–0.80min95%ofB.Columntemperaturewasmaintainedat 25◦_C,_flow_rate_was_0.294_ml_min−1_,_injection_volume_was_1.0_␮_l

anddetectionwasbetween200and400nm.

CEconditions

TheCEassays wereconducted ina capillary electrophoresis system(Agilent Technologies, model 7100, Palo Alto,CA, USA) equippedwithadiodearraydetector,temperature-controldevice (maintainedat25◦_C)_and_data_acquisition_and_analysis_software suppliedbythemanufacturer(HPChemStation®_)._Before_the_first

run,thecapillarycolumnwassequentiallyrinsedwith1moll−1

NaOH(30min)andwater(30min).Betweenruns,thecapillarywas flushedfor1minwithbackgroundelectrolyte(BGE).Atthe begin-ningofeachdaythecapillarywasconditionedbyflushingwith 1moll−1 _NaOH₍₂₀_min),_followed_by_a₂₀_min_flush_with

deion-izedwaterandanelectrolytesolution(20min).Betweenruns,the capillarywasreconditionedwith1moll−1_NaOH₍₂_min),_deionized

water(1min)andelectrolytesolution(2min).Attheendofeach workingday,thecapillarywasrinsedwith1moll−1_NaOH₍₁₀_min)

andwater(10min).

Separations were conducted in an uncoated fused-silica capillary 48.5cm in length (40cm effective length×50␮m I.D.×375␮mO.D.).DirectUVdetectionwassetat210nm,andthe temperaturewasmaintainedat25◦_C._The_standards_and_samples wereintroducedtothecapillarywithahydrodynamicpressureof 50mbar,for3s.Theseparationvoltageappliedwas30kVunder normalpolarity.TheoptimizedBGEusedintheproposedmethod wascomprisedof20mmoll−1_(pH_9.2)_and_10%_(v/v)_{acetonitrile,}

andmethylparabenwasusedasinternalstandard,dilutedtoobtain afinalconcentrationof45.46␮gml−1.

Standardpreparationandcalibrationcurves

Thestandardstocksolutionswerepreparedbydissolving1mg ofeachcompoundin1mlofmethanol.Thesesolutionswerestored in dark glass bottles at 4◦_C. _Working _standard _solutions _were freshly prepared by dissolving a suitable amountof theabove solutions with methanol before injection. Analytical curves for bothquercetinandkaempferolwereprepared,withninepoints between1and100␮gml−1fortheHPLCanalysisandsevenpoints between15and150␮gml−1withincrementsofmethylparaben, 45.46␮gml−1,fortheCEanalysis.

Samplepreparationofextracts

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1.00 0.00

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00 0.02 0.04 0.06 0.08 0.10 0.12

2.00 3.00 4.00 5.00 6.00 7.00 Minutes

A

_Q

K

Q

K

B

AU

8.00 9.00 10.00 11.00 12.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

8.00 9.00 10.00 11.00 12.00

Fig.1. ChromatogramsofRubuserythrocladus(A)andMorusnigra(B)hydrolyzedleafextractsbyHPLC-UV,370nm.Q=quercetinandK=kaempferol.

Validation

TheHPLCandCEmethodswerevalidatedforthequantitative analysisofquercetinandkaempferolpresentinR.erythrocladusand M.nigraleaves,inaccordancewiththeInternationalConferenceon Harmonizationguidelines,usingtheparametersoflinearity, speci-ficity,precision,accuracy(recoverytest),limitofdetection,limit ofquantificationandrobustness(ICH,2005).ForvalidationinCE, wealwaysusedaninternalstandard(IS)–methylparaben–togive greatercertaintytotheresults.

Specificity was determined by checking the peak purity of the quercetin and kaempferol peaks, using a photodiode-array detector by HPLC and CE. Linearity was analyzed using three standardcurves obtainedonthreedifferentdaysat nine differ-entconcentrationsofquercetinandkaempferol(1–100␮gml−1) by HPLC and seven different concentrations of quercetin and kaempferol(15–150␮gml−1)byCE.Forelectrophoresisanalysis, 45.46␮gml−1 ofmethylparabenwasaddedinallthecalibration curvepoints,asinternalstandard.Linearitywasevaluatedby cal-culatingtheregressionlineusingtheleastsquaresmethod.

Precision was performed in two different levels: intra-day precision and inter-day precision. The intra-day precision was determinedafteranalysesofthree solutionsprepared at differ-entconcentrations,intriplicate,injectedinoneday.Theinter-day precisionwasstudiedby analyzing a solutionof quercetinand

kaempferol, preparedin triplicate,over three consecutivedays. Theaccuracy(assayrecovery)ofthemethodwasassessedby ana-lyzingsamplesofR.erythrocladusandM.nigra,byHPLCandCE, respectively–thesampleswerespikedwithknownamountsof quercetinandkaempferolstandardsolutionsatthreedifferent con-centrations:20,40and60␮gml−1ofquercetinandkaempferolfor chromatographyanalysisand30,60and90␮gml−1ofquercetin andkaempferolforelectrophoresisanalysis.Accuracywas deter-minedbythemeanconcentrationrecovered,andtheresultswere expressedasrecoverypercentage.

Detection(LOD)andquantitation(LOQ)limitswerecalculated directlyfromthestandardcurve.LODwascalculatedbythe equa-tion3.3/S,whileforLOQ,theequation10/Swasused,where

istheSDofyinterceptsofregressionlinesandSistheslopeofthe standardcurve.

Robustnesswasavailablefromtheinjectionofaquercetinand kaempferolsolutionbynormalconditionsandbyvaryingthe fol-lowinganalyticalparameters:chromatographiccolumnbatch,pH ofthemobilephase andflowrateforHPLCandtemperatureof cassette,injectiontimeandorganicsolventofelectrolyteforCE.

Resultsanddiscussion

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1 0

5 10 15 20 25 30

25

20

15

10

5

0 mAU

mAU

A

B

2 3 4 5 6 7 Min

1 2 3 4 5 6 7 Min

K Q

K Q IS

EOF + #

EOF + # IS

Fig.2.ElecthropherogramsofRubuserythrocladus(A)andMorusnigra(B)hydrolyzedleavesextractsbyCE-DAD,200nm.EOF+#=electroosmoticflow+unidentifiedpeak, IS=internalstandard,K=kaempferolandQ=quercetin.

Table1

Linearequation,linearity,limitofdetection(LOD)andquantification(LOQ),intra-dayandinter-dayprecision(valuesexpressedinRSD%)andrecoveryofquercetinand kaempferolbyHPLC-UVandCE-DAD.

Flavonoid Linearequation R2 _LOD₍_␮_g_ml−1₎ _LOQ₍_␮_g_ml−1₎ _Precision_intraday _Precision_interday _Mean_recovery_(%)_±_RSD%

HPLC Quercetin y=83082x+42,829 0.9996 0.9519 2.8844 0.30 1.69 95.35±2.88 Kaempferol y=69225x+56,605 0.9995 1.0519 3.1876 0.23 2.02 95.76±2.77 CE Quercetin y=0.0176x+0.0276 0.9979 5.3043 16.0737 2.48 7.76 104.56±1.77 Kaempferol y=0.0203x+0.005 0.9987 4.1887 12.6931 3.68 8.44 104.31±1.66

Table2

DifferentparametersandresultsofrobustnessofquercetinandkaempferolinHPLC-UVandCE-DADmethods.Valuesexpressedinmean(%)±RSD%.

HPLCparameter Quercetin Kaempferol CEParameter Quercetin Kaempferol

Flow0.55mlmin−1 _109.40_±_0.31 _109.40_±_0.45 _25.5◦

C 97.63±0.26 106.27±0.07 Flow0.65mlmin−1 _92.35_±_0.26 _92.38_±_0.02 _24.5◦_C _98.39

±0.63 106.37±0.71 0.005%TFA 95.89±7.70 93.50±11.58 2s 95.76±1.87 107.73±0.14 0.015%TFA 101.57±0.13 100.14±0.39 4s 97.94±0.25 103.19±4.12 0.04%TFA 102.96±0.61 100.89±0.69 5.0% 99.30±3.71 98.73±2.41 0.12%TFA 104.90±0.16 104.28±0.05 15.0% 98.77±1.43 104.76±0.16

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Table3

QuantificationofquercetinandkaempferolinhydrolyzedleavesextractsofRubuserythrocladusandM.nigrabyHPLC-UVandCE-DAD.Valuesexpressedas␮gg−1ofdried

plant(D.P.).

Flavonoid R.erythrocladusMean(␮gg−1)±RSD% M.nigraMean(␮gg−1)±RSD%

HPLC Quercetin 848.43±10.48 2323.90±7.28

Kaempferol 304.35±6.83 1446.36±6.72

CE Quercetin 836.37±9.36 2552.82±7.96

Kaempferol N.Q. 1188.67±9.20

N.Q.=notquantified.

1.9e+1 1.8e+1

1.7e+1

1.6e+1

1.5e+1

1.4e+1 1.3e+1

1.2e+1

1.1e+1

1.0e+1 9.0

AU

8.0

7.0

6.0

5.0 4.0

3.0 2.0

1.0

5.0e+1 4.75e+1 4.5e+1

4.25e+1 4.0e+1

3.75e+1 3.5e+1

3.25e+1 3.0e+1

2.75e+1 2.5e+1

2.25e+1 2.0e+1 1.75e+1 1.5e+1 1.25e+1 1.0e+1

AU

7.5 5.0 2.5 0.0

0.00 0.100.200.30 0.400.50 0.600.700.800.90 1.001.101.201.30 1.401.50 1.601.701.801.902.00 Time

0.0

0.000.100.200.300.400.500.60 0.700.800.901.001.101.201.30 1.401.501.601.701.801.902.00 Time 1.56

1.13 0.89

0.69 0.58

K

0.69

K Q

0.58

Q

A

B

0.85 0.64 0.40

0.47

1.56 0.40

0.47

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100

0

50 100 200 300 400 303.0503

A

209.8139

254.8139

209.8139 287.0564

265.8138

365.8138 370.8138

500 550 600650 700750 800850 9009501000 m/z 450

350 250 150

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 9501000 m/z

%

100

0

B

%

Fig.4. MassandUVspectraofquercetin(A)andkaempferol(B)ofRubuserythrocladushydrolyzedleafextractsbyUPLC-DAD/MS.

chromatography (Fig. 1) and electrophoresis (Fig. 2). The hydrolyzationprocessgivesusclearerchromatogramsthancrude extract and the identification of quercetin and kaempferol in hydrolyzedleavesextractssuggeststhepresenceofquercetinand kaempferolderivativecompoundsintheseleavestoo.

HPLC methods are developed based on the polarity of the molecules.ThisdiffersfromtheCEmethodology,inwhich both flavonoidswereionizedin theelectrolyte usedinthis work,so thattheseparationofquercetinandkaempferolwasdetermined bytheirmolecularweight.Firstweobservedtheelectromigration ofkaempferol(MW=286.24)andthenweobservedthe electromi-grationofquercetin(MW=302.2).

ThepHofthebufferwillinfluencethedegreeofionizationof thesolutes,andhence,theirelectrophoreticmobilities.Giventhat thepKa of quercetinis 7.76and the pKa of kaempferol is7.89

(Tungjaietal.,2008),sodiumtetraborate(pH9.2)waschosenas theelectrolytesystemtoinitiatetheoptimizationprocedure.At

thispH,theelectroosmoticflowisusuallygreaterthanthe elec-trophoreticvelocitiesoftheanions,soquercetinandkaempferol (negatively charged), which are attracted to the positive elec-trode,are carriedtowardthecathode.In thiscase,anionselute ininversesequence totheircharge-to-sizeratios. Initialresults haveshownthatinsufficientresolutionoccurredwithelectrolyte systemcontainingonly tetraborate, sotheuseof anadditional modifierwasrequired.Adramaticimprovementcanbeobtained inselectivity,resolution,andseparationefficiencywhenorganic solvents,suchasmethanol,ethanolandacetonitrileormixtures thereof,areaddedtotheelectrolyte(Baker,1995).The acetoni-trileconcentrationwasstudiedovertherangeof0–15%forafixed amountofsodiumtetraborate,and10%(v/v)wasselectedbecause itrepresentedacompromisebetweenresolutionandanalysistime. Theseparation ofquercetin,kaempferol andI.S. methylparaben under investigation at the optimized conditions is shown in

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50

303.0512

209.8139

255.8139 370.8138

100

0

%

AU

A

287.0557

209.8139

265.8138

366.8138 100

0

%

AU

B

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/Z

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/Z

Fig.5.MassandUVspectraofquercetin(A)andkaempferol(B)ofMorusnigrahydrolyzedleafextractsbyUPLC-DAD/MS.

Methodsvalidation

Alltheparameterstestedinthevalidationmethodsforthe quan-tificationofquercetinandkaempferolinhydrolyzedleafextracts ofR.erythrocladusandM.nigrashowedsatisfactoryresults,with RSD ofless than 15% (Tables 1 and 2).The purityof the chro-matographic and electrophoretic peaks obtained for quercetin andkaempferolwasevaluatedusingaDADdetector.Theresults showedthatothercompoundsdidnotco-elute/migratewiththe standards.

Sampleanalysis

ItwaspossibletoapplythedevelopedmethodsinRubusand Morussamples(Table3).ByCE,kaempferolcouldnotbe quanti-fiedinR.erythrocladus.Thisresultisduetothelowersensitivity ofthistechnique,asshowninTable1.TheCEsensitivityisrelated tothecapillarydetectionwindow,capillarydiameterandlightUV

dispersion(Li,1996).Lessdilutedsamplescouldhaveprovided bet-terresults.Applyingtheanalyticaltoolt-studentitwasobserved thatnosignificantdifferenceswereobservedinthequantitiesof theseflavonoidsbetweenthetwoformsofanalyses–HPLCandCE (p>0.05)–Table3.

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Bothanalyticaltechniquesprovedtobefastandreliable.The HPLC methodcould beapplied as a form of quality control of quercetinand kaempferol inhydrolyzed leafextractsof R. ery-throcladusandM.nigra.EvenwiththelowersensitivityoftheCE method,it ispossibletoobservehigherefficiencyofseparation peaks(Fig.2).Thehighversatility,thelowvolumeofreagentsand sample,andthegoodresultsobtainedforquercetinandkaempferol quantification by CEsuggest that this analytical tool could be appliedasacomplementarytechnicforthequalitycontrolofthese compoundsinnaturalproducts.

Botanicalandchemicalanalysesofplantsareveryimportantfor theidentificationandqualitycontrolofmedicinalplants(Brazilian Pharmacopoeia,2010).ByUPLC-DAD/MS,adifferencewas iden-tifiedbetweenthechemicalqualitativeprofilesoftheRubusand Morusspecies(Fig.3).

Allpeakswerefragmented,butonlyquercetinandkaempferol (at0.58 and 0.69s, respectively)were identified in hydrolyzed leafextracts of R. erythrocladus and M. nigra by UPLC-DAD/MS (Figs.4and5,respectively).

ThepresenceofkaempferolandquercetininRubusandMorus chromatograms,andsmallunidentifiedpeaksinthechromatogram forR.erythrocladus(Fig.3)byUPLC-DAD/MSsuggestthattheRubus leafextractmaybemorecomplexthantheMorusleafextract.

Conclusions

It was possible to determine the flavonoids present in hydrolyzedleavesextractsofR.erythrocladusandM.nigraandit wasshowedtwofastandreliablemethodstoquantifyquercetin andkaempferolintheseextracts,HPLC-UVandCE-DAD.Thesetwo methodswerevalidatedandcouldbeappliedtothequality con-troloftheseextracts.Moreover,we reportchemical differences betweentheMorusandRubusleavesstudiedbyUPLC-DAD/MSand wealsocontributewithnewinformationabouttheMorusgenus fortheBraziliangovernment.

Authors’contributions

LRTcontributedin samplepreparation, chromatography and electrophoresisanalysis,analysisofdataanddraftof thepaper. GPRPcontributedinsamplepreparationandchromatography anal-ysis.ACOCandMScontributedinelectrophoresisanalysisanddraft ofthepaper.SALBcontributedtoplantscollections.AFandJASZ designedthestudy,leadandadvisedthelaboratoryworkand per-formedtheanalysisofthedataanddraftedthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WewouldliketothankCAPES,CNPq,PPGCF-UFRGSandDra. Maria do Carmo Bassols Raseira (Embrapa). JASZ acknowledge CNPqforresearcherfellowship.

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