Rev. bras. farmacogn. vol.25 número4

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

Phytochemical

analysis

of

Vernonanthura

tweedieana

and

a

validated

UPLC-PDA

method

for

the

quantification

of

eriodictyol

Layzon

Antonio

Lemos

da

Silva

_,

_Larissa

_Gabriela

_Faqueti

_,

_Flávio

_Henrique

_Reginatto

_,

Alan

Diego

Conceic¸

ão

dos

Santos

_,

_Andersson

_Barison

_,

_Maique

_Weber

_Biavatti

a_{LaboratóriodeFarmacognosia,DepartamentodeCiênciasFarmacêuticas,CentrodeCiênciasdaSaúde,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil} b_{DepartamentodeQuímica,UniversidadeFederaldoParaná,Curitiba,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29May2015 Accepted12July2015 Availableonline8August2015

Keywords:

Asteraceae Eriodictyol UPLC-PDA

Vernonanthuratweedieana

a

b

s

t

r

a

c

t

Vernonanthuratweedieana(Baker)H.Rob.,Asteraceae,isusedintheBrazilianfolkmedicineforthe

treat-mentofrespiratorydiseases.Inthisworkthephytochemicalinvestigationofitsethanolextractsaswell

asthedevelopmentandvalidationofanUPLC-PDAmethodforthequantificationoftheeriodictyolfrom

theleaveswereperformed.Thephytochemicalstudyforthisspeciesleadtotheidentificationofethyl

caffeate,naringeninandchrysoeriolinmixture,eriodictyolfromleaves,andthemixtureof

3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-oneandevofolinB,apigenin,themixtureofcaffeicand

protocatechuicacidandluteolinfromstemswithroots,beingreportedforthefirsttimeforV.tweedieana,

exceptforeriodictyol.Thestructuralelucidationofallisolatedcompoundswasachievedby1_Hand2D

NMRspectroscopy,andincomparisonwithpublisheddata.AnUPLC-PDAmethodforquantificationof

theeriodictyolinleavesofV.tweedieanawasdevelopedandvalidatedforspecificity,linearity,precision

(repeatabilityandintermediateprecision),limitofdetection(LOD)andlimitofquantification(LOQ),

accuracyandrobustness.Inthisstudy,anexcellentlinearitywasobtained(r2_{=0.9999),goodprecision}

(repeatabilityRSD=2%andintermediateprecisionRSD=8%)andaccuracy(averagerecoveryfrom98.6%

to99.7%).ThecontentoferiodictyolintheextractofleavesofV.tweedieanawas41.40±0.13mg/g.Thus,

thisstudyallowedtheoptimizationofasimple,fastandvalidatedUPLC-PDAmethodwhichcanbeused

tosupportthequalityassessmentofthisherbalmaterial.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Naturalproducts,aswellasmedicinalplants,areconsideredas

agoodsourceforthedevelopmentofmedicinesanddrugsandas

atoolforthediscoveryofinnovativemolecularpatterns(Alvim etal.,2006;BalunasandKinghorn,2005;Craggetal.,2009;Cragg andNewman,2013).

BelongingtotheAsteraceaefamily,Vernonanthuratweedieana

(Baker)H.Rob.isknowninBrazilas“assapeixe”and“mata-pasto”

and theirleavesare popularlyused for thetreatmentof

respi-ratory diseases(Cabrera and Klein, 1980; Trevisanetal., 2012; Zanon, 2006; Zanon et al., 2008).Extracts of this species have

demonstrated antibacterial (Díaz et al., 2008),

immunomodula-tory(Petrietal.,2008),antioxidant(Zanon,2006),antinociceptive andantiedematogenic(Trevisanetal.,2012),antifungal(Portillo et al., 2005) and allelopathic (Olguin et al., 2005) activities.

∗ _{Correspondingauthor.}

E-mail:maique.biavatti@ufsc.br(M.W.Biavatti).

AmongthesecondarymetabolitesalreadydescribedforV.

tweed-ieanaarelupeol,␣-and␤-amyrin,␤-sitosterol,stigmasterol(Díaz et al., 2008; Portillo et al., 2005; Zanon, 2006; Zanon et al., 2008), ␣-spinasterol (Trevisanet al.,2012; Zanon etal., 2008), eriodictyol(Rossatoetal.,2011;Zanon,2006), 6-cinnamoyloxy-1-hydroxyeudesm-4-en-3-one(Portilloetal.,2005)andpalmitic acid(Díazetal.,2008).

For the evaluation of quality of raw materials and

phy-tomedicinesisfundamentaltoknowandtostandardizethecontent oftheirmaincomponents(Baraetal.,2006).Sinceherbalextracts

contain many different chemical components, techniques with

highsensibilityand selectivityaremandatorytotheiradequate

qualitycontrol.Inthecontext,ultraperformanceliquid

chromatog-raphy (UPLC) has shorter run time and higher sensitivity and

resolutionwhencompared toHPLCandisaneleganttechnique

fordetectionandscreeningofcomplexanalytesinalargenumber

ofsamples(SteinmannandGanzera,2011;WalterandAndrews,

2014).

InordertobetterexplorethechemicalcompositionofV. tweed-ieana, ethanol extracts fromstems with roots and from leaves

http://dx.doi.org/10.1016/j.bjp.2015.07.009

(thelatterbeingpreparedaccordingtothepopularuse)were pre-paredandchemicallyfractioned,conductingtotheidentification

ofitsmaincompounds.Additionally,consideringtheimportance

ofqualitycontrolofV.tweedieanaleavesduetoitspopularuse,a

newandsimpleUPLC-PDAmethodforquantificationofitsmajor

compound(eriodictyol)wasdevelopedandvalidatedinthiswork.

Materialsandmethods

Chemicals

The solvents used in chromatographic procedures were

p.a. grade: n-hexane, dichloromethane, ethyl acetate, acetone,

methanol(Vetec®_{QuímicaFina,RiodeJaneiro,RJ,Brazil).For} UPLC-PDAanalyseswereusedacetonitrileHPLCgrade(Tedia®_Brazil,Rio deJaneiro,RJ,Brazil),formicacid85%p.a.grade(Vetec®_Química

Fina,Rio de Janeiro, RJ, Brazil) and thewater was obtainedby

Millipore® _{MilliQ(Bedford,MA,USA)waterpurificationsystem.} AllthesolutionspreparedforUPLCanalyseswerefilteredthrough

a0.22␮mmembraneandsonicatedbeforeuse.ForTLCanalyses

plates of silicagel 60 (SiliCycle®_{, Quebec,QC, Canada) and}

sil-icaRPC18(Macherey-Nagel®,Düren,NRW,Germany)wereused.

ForNMRanalysesmethanol-d4andacetona-d6(CambridgeIsotope

Laboratories,Inc.,Andover,MA,USA)wereused.

Plantmaterial

Vernonanthura tweedieana (Baker) H. Rob., Asteraceae, was collectedduringbloomingseasoninFlorianópolis (27◦₃₃′_54.8′′_S

48◦_25’48.0′′_{W and 27}◦₃₅′_54.7′′_{S 48}◦₃₁′_00.3′′_{W), Santa Catarina,}

BrazilinApril2013,andidentifiedbyProfessorDr.AdemirReis.A

voucherspecimenwasdepositedintheHerbariumRB(RB612274),

attheJardimBotânicodoRiodeJaneiro,RiodeJaneiro/RJ,Brazil.

Extractionandpartition

Thefreshleaves (2.6kg) and stemswith roots(5.9kg) were

extractedbymacerationwithethanol75%atroomtemperature,

for56days.Theextractivesolutionswerefilteredthroughpaper;

thesolventwasremovedunderreducedpressureandusedto

re-extracttheplantmaterial.Theresultingcrudeextractsfromleaves (ELF,172g)andfromstemswithroots(ESR,291.6g)aftersolvent removalwerekeptinadesiccatorinvacuo.

Bothcrude extracts were partitioned withincreasing

polar-itysolvents,yieldingn-hexane(17.9g),dichloromethane(CH2Cl2,

3.6g), ethyl acetate (EtOAc, 10.4g) and the aqueous residual

(104.3g)fractionsfromELF,andn-hexane(10.9g),CH2Cl2(4.2g),

EtOAc(2.5g)andtheaqueousresidual(244.9g)fractionsfromESR.

Phytochemicalstudy

TheCH2Cl2(3.6g)fractionfrompartitionofELFwassubjected

tosilicagel(230–400mesh)liquidchromatographyundervacuum

(flash-LCV)andelutedinagradientsystemconsistingofincreasing concentrationsofCH2Cl2inn-hexane(0–70%),followedbyacetone

inCH2Cl2 (0–80%)andfollowedbymethanol(MeOH)inacetone

(0–100%),yieldingseventeensubfractions(LF1–LF17).Pooled sub-fractionsLF7andLF8(676.6mg)wasalsosubjectedtoflash-LCV, elutedinagradientsystemconsistingofincreasingconcentrations ofCH2Cl2 inn-hexane(40–70%),followedbyacetonein CH2Cl2

(0–70%)andfollowedbyMeOHinacetone(0–100%),yielding

six-teen subfractions, named LF7-8A to LF7-8P. Subfraction LF7-8F

(368.7mg)wassubjectedtosilicagelcolumnchromatography(CC) andelutedinagradientsystemconsistingofincreasing

concentra-tionsofacetoneinhexane(25–100%)followedbyMeOH(100%),

yieldingtensubfractions,namedLF7-8FItoLF7-8FX,inwhichthe fractionLF7-8FVIIIcorrespondstocompound1(37.3mg).

TheEtOAc(10.4g)fractionfrompartitionofELFwassubjectedto flash-LCV,elutedinagradientsystemconsistingofincreasing con-centrationsofCH2Cl2inhexane(30–70%),followedbyacetonein

CH2Cl2(0–80%)andfollowedbyMeOHinacetone(0–100%),

yield-ingeighteensubfractions(LF18–LF35).SubfractionLF25(319mg)

wassubjectedtosilicagelCCelutedinagradientsystem consist-ingof increasingconcentrations of CH2Cl2 inhexane (80–95%),

followed byacetonein CH2Cl2 (0–70%)and followedby MeOH

inacetone(0–100%),resultingelevensubfractions(LF25A–LF25K).

ThesubfractionLF25G(22mg)wasfractionatedthroughSephadex

LH-20withacetoneaseluent,yieldingsevensubfractions,named

LF25GItoLF25GVII,whichfractionLF25GVcorrespondstothe mix-ture(3.4mg)ofcompounds2and3.SubfractionLF26(2079.3mg)

wassubjectedtosilica gelCCeluted ina gradient system

con-sisting of increasing concentrations of CH2Cl2 in hexane (80%),

followedbyacetoneinCH2Cl2(0–30%)andfollowedbyMeOHin

acetone(0–50%),untilwaterinMeOH(0–10%),resulting

seven-teensubfractions(LF26A-LF26Q).PooledsubfractionsLF26Kand

LF26L(262.1mg)wassubjectedtosilicagelCC,elutedina

gradi-entsystemconsistingofincreasingconcentrationsofacetonein

CH2Cl2 (15–70%)followedbyMeOHinacetone(0–100%),

yield-ingninesubfractions,namedLF26K-LItoLF26K-LIX,whichfraction

LF26K-LIVcorrespondstocompound4(104.7mg).

The CH2Cl2 (4.2g) fraction from partition of ESR was

sub-jectedtoflash-LCVandelutedinagradientsystemconsistingof increasingconcentrationsofCH2Cl2 inhexane(0–70%),followed

by EtOAc in CH2Cl2 (0–80%) and followed by MeOH in EtOAc

(0–70%),untilwaterinMeOH(0–50%),yieldingeighteen

subfrac-tions(SR1–SR18).ThesubfractionSR10(112mg)wassubjectedto

silicagelCC,elutedinagradientsystemconsistingofincreasing concentrationsofacetoneinCH2Cl2(3–70%)followedbyMeOHin

acetone(0–100%),yieldingtwelvesubfractions,namedSR10Ato

SR10L.ThesubfractionSR10E(19.7mg)wasfractionatedthrough

SephadexLH-20withacetoneaseluent,yieldingsixsubfractions, namedSR10EItoSR10EVI,whichfractionSR10EVIcorrespondsto themixture(0.8mg)ofcompounds5and6.

TheEtOAc(2.5g)fractionfrompartitionofESRwassubjected toflash-LCV,elutedinagradientsystemconsistingofincreasing concentrationsofEtOAcinCH2Cl2(0–70%)andfollowedbyMeOH

inEtOAc(0–100%),yieldingsevensubfractions(SR19-SR25).The

subfractionSR21(135.9mg)wassubjectedtosilicagelCC,eluted inagradientsystemconsistingofincreasingconcentrationsof

ace-toneinCH2Cl2 (3–70%)followedbyMeOHinacetone(0–100%),

yieldingfifteensubfractions,namedSR21AtoSR21O.The subfrac-tionSR21H(16.7mg)wasalsosubjectedtosilicagelCC,elutedin agradientsystemconsistingofincreasingconcentrationsofMeOH inCH2Cl2 (5–100%),resultingsixsubfractions,namedSR21HIto

SR21HVI.ThesubfractionSR21HV(9.6mg)wassubjectedto

prepar-ativeRPC18 TLC,elutedwithwater-MeOH(30:70,v/v)allowing

theisolationofcompounds4(4.9mg)and7(0.7mg).The

subfrac-tionSR21K(21.3mg)wasfractionatedthroughSephadexLH-20

withacetoneaseluent,yieldingeightsubfractions,namedSR21KI toSR21KVIII,whichfractionSR21KVcorrespondstothemixture

(11mg)ofcompounds8and9,andthefractionSR21KVII

corre-spondstothecompound10(1mg).

The elucidation of thecompounds was achieved by 1_{H and}

2D(HSQCandHMBC)NMR spectroscopy,in Bruker® _Ascend600

(600MHzforH,125MHzforC),andincomparisonwithpublished

data.

Ethylcaffeate(1):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)

J(Hz),7.16(1H,d,J=2.0,H-2),6.87(1H,d,J=8.2,H-5),7.04(1H, dd,J=8.2;2.0,H-6),7.54(1H,d,J=15.9,H-7),6.27(1H,d,J=15.9, H-8),4.18(2H,q,J=7.2,H-1′_{),1.27(3H,t,J=7.2,H-2}′_),8.17(1H,

br,OH-3)and8.41(1H,br,OH-4).13_{CNMR(acetone-d}

TMS)ıC(ppm),127.5(C-1),115.2(C-2),146.4(C-3),148.7(C-4),

116.3(C-5),122.3(C-6),145.2(C-7),115.8(C-8),167.4(C-9),60.5 (C-1′_)and14.7(C-2′_).

Naringenin(2):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)

J(Hz),5.45(1H,dd,J=12.9;3.0,H-2),3.17(1H,dd,J=17.1;12.9, H-3a),2.74(1H,dd,J=17.1;3.0,H-3b),5.95(1H,d,J=2.1,H-6),5.96 (1H,d,J=2.1,H-8),7.39(2H,d,J=8.5,H-2′_,H-6′_{),6.90(2H,d,J=8.5,}

H-3′_,H-5′_{)and12.17(1H,s,OH-5).}13_{CNMR(acetone-d}

6,150MHz,

TMS)ıC(ppm),80.0(C-2),43.5(C-3),197.2(C-4),166.8(C-5),96.4

(C-6),161.8(C-7),95.9(C-8),164.5(C-9),103.2(C-10),130.7(C-1′_),

129.0(C-2′_,C-6′_),116.4(C-3′_,C-5′_{)and158.7(C-4}′_{).Thiscompound}

wasidentifiedinmixturewithcompound3. Chrysoeriol(3):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)

J(Hz),6.69(1H,s,H-3),6.26(1H,d,J=2.0,H-6),6.55(1H,d,J=2.0, H-8),7.63(1H,d,J=2.1,H-2′_{),7.01(1H,d,J=8.3,H-5}′_{),7.60(1H,dd,} J=8.3;2.1,H-6′_{),4.00(3H,s,OCH}

3-1′′)and13.00(1H,s,OH-5).13C

NMR(acetone-d6,150MHz,TMS)ıC(ppm),164.9(C-2),104.5

(C-3),182.9(C-4),163.3(C-5),99.7(C-6),165.0(C-7),94.7(C-8),158.7 (C-9),105.2(C-10),123.6(C-1′_),110.7(C-2′_),148.8(C-3′_),151.3

(C-4′_),116.4(C-5′_),121.4(C-6′_)and56.6(C-1′′_{).Thiscompoundwas}

identifiedinmixturewithcompound2. Eriodictyol(4):1_{HNMR(methanol-d}

4,600MHz,TMS),ıH(ppm)

J(Hz),5.28(1H,dd,J=12.8;3.0,H-2),3.06(1H,dd,J=17.2;12.8, H-3a),2.69(1H,dd,J=17.2;3.0,H-3b),5.88(1H,d,J=2.2,H-6),5.90 (1H,d,J=2.2,H-8),6.91(1H,m,H-2′_{),6.78(1H,m,H-5}′_)and6.79

(1H,m,H-6′_).13_{CNMR (methanol-d}

4,150MHz,TMS)ıC (ppm),

80.7(C-2),44.2(C-3),197.8(C-4),165.6(C-5),97.2(C-6),168.6 (C-7),96.2(C-8),164.9(C-9),103.5(C-10),131.8(C-1′_),114.7(C-2′_),

146.1(C-3′_),147.1(C-4′_),116.6(C-5′_{)and119.2(C-6}′_).

3-Hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-one (5):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)J(Hz),7.34(2H,

s,H-2,H-6),3.17(2H,d,J=6.2,H-8),3.91(2H,m,H-9)and3.91 (6H,s,OCH3-3,OCH3-5).13CNMR(acetone-d6,150MHz,TMS)ıC

(ppm),129.3(C-1),107.0(C-2,C-6),148.5(C-3,C-5),142.0(C-4), 198.2(C-7),41.8(C-8),58.6(C-9)and56.9(OCH3-3,OCH3-5).This

compoundwasidentifiedinmixturewithcompound6.

EvofolinB(6):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)

J(Hz),7.60(1H,d,J=2.0,H-2),6.84(1H,d,J=8.3,H-5),7.66(1H, dd,J=8.3;2.0,H-6),4.80(1H,dd,J=8.5;5.2,H-8),4.23(1H,m, H-9a),3.71(1H,m,H-9b),6.99(1H,d,J=2.0,H-2′_{),6.74(1H,d,J=8.1,}

H-5′_{),6.80(1H,dd,J=8.1;2.0,H-6}′_{),3.88(3H,s,OCH}

3-3)and3.82

(3H,s,OCH3-3′).13CNMR(acetone-d6,150MHz,TMS)ıC(ppm),

130.5(C-1),112.3(C-2),148.2(C-3),152.1(C-4),115.3(C-5),124.5 (C-6),198.2(C-7),55.9(C-8),65.5(C-9),129.1(C-1′_),112.8(C-2′_),

148.5(C-3′_),146.6(C-4′_),115.9(C-5′_),121.9(C-6′_),56.3(OCH

3-3)

and56.4(OCH3-3′).Thiscompoundwasidentifiedinmixturewith

compound5.

Apigenin(7):1_{HNMR(methanol-d}

4,600MHz,TMS),ıH(ppm)J

(Hz),6.60(1H,s,H-3),6.21(1H,d,J=2.1,H-6),6.46(1H,d,J=2.1, H-8),7.85(2H,d,J=8.9,H-2′_,H-6′_{)and6.93(2H,d,J=8.9,H-3}′_,

H-5′_).13_{CNMR(methanol-d}

4,150MHz,TMS)ıC(ppm),166.3(C-2),

102.9(C-3),183.9(C-4),163.2(C-5),100.2(C-6),166.1(C-7),95.0 (C-8),159.5(C-9),105.3(C-10),123.3(C-1′_),129.6(C-2′_,C-6′_),117.1

(C-3′_,C-5′_{)and162.7(C-4}′_).

Caffeicacid(8):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)J

(Hz),7.16(1H,d,J=2.0,H-2),6.87(1H,d,J=8.2,H-5),7.03(1H,dd, J=8.2;2.0,H-6),7.55(1H,d,J=15.9,H-7)and6.27(1H,d,J=15.9, H-8).13_{CNMR(acetone-d}

6,150MHz,TMS)ıC(ppm),127.6(C-1),

115.2(C-2),146.3(C-3),148.7(C-4),116.4(C-5),122.5(C-6),146.1 (C-7),115.9(C-8)and168.3(C-9).Thiscompoundwasidentifiedin

mixturewithcompound9.

Protocatechuicacid(9):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH

(ppm)J(Hz),7.53(1H,d,J=2.0,H-2),6.90(1H,d,J=8.3,H-5)and 7.48(1H,dd,J=8.3;2.0,H-6).13_{CNMR(acetone-d}

6,150MHz,TMS)

ıC(ppm),123.2(C-1),117.6(C-2),145.5(C-3),150.6(C-4),115.8

(C-5),123.7(C-6)and167.7(C-7).Thiscompoundwasidentifiedin

mixturewithcompound8.

Luteolin(10):1_{HNMR(acetone-d}

6,600MHz,TMS),ıH(ppm)J

(Hz),6.57(1H,s,H-3),6.25(1H,d,J=2.1,H-6),6.53(1H,d,J=2.1, H-8),7.50(1H,d,J=2.3,H-2′_{),7.00(1H,d,J=8.4,H-5}′_),7.46(1H,

dd,J=8.4;2.3,H-6′_{)and13.00(1H,s,OH-5).}13_{CNMR(acetone-d}

6,

150MHz,TMS)ıC(ppm),165.3(C-2),104.2(C-3),182.9(C-4),163.3

(C-5),99.6(C-6),164.9(C-7),94.7(C-8),158.9(C-9),105.3(C-10), 123.7(C-1′_),114.1(C-2′_),146.6(C-3′_),150.2(C-4′_),116.6(C-5′_)and

120.1(C-6′_).

Ultraperformanceliquidchromatographyoptimizedconditions

TheUPLCanalyseswerecarriedoutinaWaters®_AcquityUPLC

systemequippedwithphotodiodearray(PDA)detection,anauto

samplerandcolumnoven.Thechromatographicseparationwas

achievedusingaWaters®_{AcquityUPLCBEHC}

18(2.1mm×50mm

i.d.,1.7␮m)at40◦_C(±2◦_{C).Theinjectionvolumewas2␮land}

theflowratewaskeptconstantat0.4mlmin−1_{.Themobilephase}

consisted of a 7min gradient system combining 0.1% aqueous

formicacidadjustedtopH3.0 (solventA)andacetonitrile (sol-ventB)asfollows:0–1mingradientof90–80%A(curve7);1–5min

80–55%A(curve7);5–6min55–35%A(curve9);6–7min35–90%A

(curve9).DatawereprocessedusingtheEmpower3(Waters®₎

soft-ware.Thechromatogramswererecordedatwavelengthof287nm

whiletheUVspectraweremonitoredoverarangeof400–200nm.

PeakswereidentifiedbycomparingretentiontimesandUVspectra withreferencestandards.

Stocksolutionspreparation

Thequantificationoferiodictyolinthesampleswascarriedout

bytheexternalstandardmethod.Eriodictyol(purity≥98%,

veri-fiedbyPeakPuritytoolinEmpower3software)usedinthisworkas analyticalstandardwasobtainedfromphytochemicalinvestigation ofEtOAcfractionfrompartitionofELFofV.tweedieana.Thestock standardsolutionsoferiodictyol(1000and500␮g/gofsolution)

were freshly preparedin 0.1% aqueousformic acid–acetonitrile

(1:1,v/v) mixture.Thestockstandard solutionwasthen

appro-priatelydilutedin0.1%aqueousformicacid–acetonitrile(1:1,v/v) toobtainworkingstandardsolutions.

The sample solutions analyzed by UPLC-PDA were obtained

fromthecrudeextractsELFandESR,resultingofmacerationfrom leavesand fromstemswithrootsofV.tweedieana,respectively.

Bothcrudeextracts,ELFandESR,wereusedtopreparethestock

samplesolutions(1000␮g/gofsolution)in0.1%aqueousformic

acid–acetonitrile(1:1,v/v).Thestocksamplesolutionswerealso appropriatelydilutedin0.1%aqueousformicacid–acetonitrile(1:1, v/v)toobtainworkingsamplesolutions(about200␮g/gof solu-tion).

Thesestocksolutionswerefilteredthrougha0.22␮m

mem-braneandstoredat4◦_C.

ValidationofUPLC-PDAmethod

TheUPLC-PDAmethodwasvalidatedintermsofspecificity, lin-earity,precision(repeatabilityandintermediateprecision),limit ofdetection(LOD)andlimitofquantification(LOQ),accuracyand robustness(Araujo,2009;ICH,2005;Shabir,2003;USP,2007).

Inordertocheckthespecificityofthemethod,thepurityof eriodictyolintheextractofV.tweedieanawasverifiedusingaPDA

detectorandthesoftwareEmpower3(Waters®

).

Linearitywasdeterminedwithcalibrationcurves,whichwere

constructed by plotting themeasurements of areapeak versus

resultswere analyzed by linear regression and the correlation coefficient(r2_{)wascalculated.One-wayANOVAwascalculatedto}

comparethereplicatesofthecalibrationcurve(GraphPadPrism® version6.03).

Theprecisionofthemethodwasassessedinrepeatability

(intra-day) and intermediate precision (inter-day). Repeatability was

establishedbyanalyzingtherelativestandarddeviation(RSD%)of 6independentsamplesolutionsdeterminationsat100%ofthetest

concentration(200␮g/g).Intermediateprecisionwasdetermined

consideringtheRSD%bytheanalysisinsextuplicatebytwo

ana-lystsinthreedifferentdays(analystAperformedtheanalysisin

daysoneandtwo,whileanalystBperformedtheanalysisinday

three).

TheLODandLOQwereassessedatsignal-to-noiseof3and10,

respectively,byinjectingaseriesofdilutesolutionswithknown concentration.

Theaccuracywasevaluatedbytherecoverytest.Threedifferent concentrationsofstockstandardsolution(500␮g/g),

correspond-ingtheadditionof10, 14and 18␮g/gofsolution,werespiked

intoaknownsamplesolution.Thespikedsample,unspikedsample

andcorrespondingaddedstandard solutionsweremeasured, in

triplicate,underthesameconditions.Therecoverywascalculated bythereasonbetweentheconcentrationofspikedsampleanalyzed

andthetheoreticalspikedconcentration(establishedbythesum

ofmeasuredconcentrationsofunspikedsampleandthestandard

added).

Therobustnessofthemethodwasdeterminedrelativetothe

variation of retention time, resolution and tailingfactor of the peak(USP, 2007), and concentrationof eriodictyolfor

determi-nationsin nominalconditionsand againstvariationsofmethod

conditions.Experimentalvariablesweretheflowratevariedby(±) 0.1mlmin−1_{,columnoventemperaturevariedby(±)10}◦_Candthe

percentageoforganicmodifiervariedby(±)0.05%.Theresultswere

evaluatedbyone-wayANOVAfollowedbyBonferroniposthoctest,

withaconfidencelevelof95%(GraphPadPrism®_{version6.03).}

Resultsanddiscussion

Phytochemicalstudy

Thephytochemicalinvestigationallowedcharacterizingten

dif-ferent known phenolic compounds from ethanol extractsof V.

tweedieana.Thesecompoundswereidentifiedby1_Hand2DNMR

experimentsincomparisonwithpublisheddata.Consideringthat

themajorityofthepreviouslypublishedNMRdatapresentedsome

inconsistenciesregardingchemicalshifts(ppm)orwerenotinthe samesolvent,thenewdataarepresentedinMaterialsandMethods

section.Forcompound4differentchemicalshiftsofH-3aandH-3b

werepreviouslydescribedin2.69ppmforboth,andnowassigned

in3.06and2.69ppmrespectively.Forcompound5,thechemical

shiftsforcarbonsC-2,C-4,C-5andC-6werepreviouslypublishedas

115.5,152.9,112.4and124.6ppm,andnowassignedrespectively

in107.0,142.0,148.5and107.0ppm.Forcompound7,thechemical

shiftsH-6andH-8previouslypublishedin6.45and6.16ppmare

nowassignedin6.21and6.46ppm,respectively.Thecompounds

8and9werepublishedbeforeinmethanol-d4andnowacetone-d6

wasused.FromCH2Cl2 fractionofleaveswasisolatedethyl

caf-feate(1)(Uwaietal.,2008),andfromEtOAcfractionwasobtained naringenin(2)(Fatope etal.,2003)andchrysoeriol(3)(Lin and Kong,2006)inmixture,anderiodictyol(4)(Huangetal.,2014).

Fromstemswithrootsextract,theCH2Cl2fractionfurnished

3-hydroxy-1-(4-hydroxy-3.5-dimethoxyphenyl)-propan-1-one (5)

(Jonesetal.,2000)andevofolinB(6)(Wuetal.,1995)inmixture, andfromEtOAcfractionwereobtainedagaintheeriodictyol(4), apigenin(7)(Kimetal.,2014), caffeic(8)(Leeetal.,2012)and protocatechuic(9)(Liaoetal.,2014)acidsinmixtureandluteolin (10)(LinandKong,2006).

Exceptforeriodictyolthatwaspreviouslyreportedfortheleaves ofV.tweedieana(Rossatoetal.,2011;Zanon,2006),allsubstances aredescribedforthefirsttimeinthisspecies.Theseresultsallow increasingtheknowledgeforthespeciesV.tweedieana.

UPLC-PDAanalysesandmethodvalidation

Bothcrudeethanolextracts,fromleavesandfromcombination

ofstemswithrootsofV.tweedieana,wereanalyzedbyUPLC-PDA

andthechromatogramsareshowedinFig.1A,B.

The comparative analysis of the crude extracts with all

isolatedcompoundsallowedcharacterizingfourpeaksinthe chro-matograms: ethyl caffeate (1), eriodictyol(4), apigenin (7) and luteolin(10).

The UPLC-PDA analyses of the two crude ethanol extracts

showeddistinctchromatographicprofiles.Eriodictyol(4)wasthe majorcompoundinELF,whileitwasnotobservedintheESR

chro-matogram,probablybecauseitscontentwasnotdetectable,since

theamountoferiodictyolisolatedfromESRwasverylow(4.9mg, 0.047%ofthecrudeextract).Flavonoidcontentvarieswithin indi-vidualleaves, stems or roots (Julkunen-Tiittoet al., 2015).The

presence of flavonoids within different cells and cellular

com-partmentscanberelatedtotheirfunctionsinplantenvironment

0.00 0.000 0.010 0.020 0.030 0.040 AU0.050 0.060 0.070 0.080 0.090 0.100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.802.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 Minutes

4

A

10 1

7

3.60 3.804.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00

0.00 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 0.060 0.065

AU

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.802.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 Minutes

B

1

7

3.60 3.804.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00

Fig.1. UPLCchromatogramsofcrudeethanolextracts(200␮g/g)fromleaves(A)andfromcombinationofstemswithroots(B)ofV.tweedieana,withdiodearraydetection at287nm.1.ethylcaffeate;4.eriodictyol;7.apigenin;10.luteolin.Forchromatographicconditions,seeMaterialsandmethodssection.

Table1

Linearityandsensitivitydata.

Compound UVmax(nm) Rangeoflinearitya(␮g/g) Linearregressionequation r2 LOD(␮g/g) LOQ(␮g/g)

Eriodictyol 287.6 1.5–30.0 y=16,211x−277.7 0.9999 0.02344 0.06214

r2_{,correlationcoefficient;LOD,limitofdetection;LOQ,limitofquantification.} a_{Sevenlevelsconcentrations(n=3).}

Consideringtheuseofleavesinpopularmedicinalpreparations ofV.tweedieanainBrazil(Trevisanetal.,2012),thecrudeextractof leaveswasanalyzedbyUPLC-PDA,inordertoverifythepotential oferiodictyolaschemicalmarkerforthisplantextract.

Thecalibration curves werefoundto belinear inthe range

1.5–30␮g/gofsolutionforeriodictyol.

CharacteristicparametersforlinearityaregiveninTable1.The

one-wayANOVAcalculatedtocomparethereplicatesofthe

cal-ibrationcurveshowednosignificantdifferencebetweengroups

(F=0.003254;p=0.9968).Thelinearregressionequationand cor-relationcoefficient(r2_{)of0.9999revealedagoodlinearityresponse}

forthemethoddeveloped,sinceaccordingtotheliteraturea

coef-ficient ofdetermination for standard curves greater than0.999

indicatesanevidenceofacceptablefitofthedatatotheregression line(Shabir,2003).

TheLODand LOQvalues(Table1), respectively0.02344and

0.06214␮g/g,weresimilartothosereportedintheliteraturefora HPLCmethodforquantificationoferiodictyolinSorghumbicolour (Taleonetal.,2014).Thepeak purity,obtainedbyPDAdetector analysisoferiodictyolwasinagreementwithitspurityspectral profile.

Theresultsofrepeatability(intra-dayvariation)and

intermedi-ateprecision(inter-dayvariation)showedRSD%valuesof2%and

8%,respectively(Table2).Analyticalproceduresforbiologicaland biotechnologicalproductscanpresentahigherinherentvariation.

Naturalproductshavehighchemicalcomplexity,andconsidering

thatallreplicatesperformedinthisworkinvolvedthepreparation ofnewstockandworksamplessolutions,itcanrepresentasource ofvariation.However,thedataobtainedforprecisionwas accept-ableandisinaccordancewithotheranalyticalstudieswithsame

Table2

Repeatability,intermediateprecisionandaccuracydata.

Compound Repeatability(n=6) Intermediateprecision(n=18) Accuracy

AnalystA AnalystA AnalystB Standardadded(␮g) Recoveryb_{(%) RSD}

Day1a _{RSD Day2}a _Day3a _RSD

Eriodictyol 56.15±1.06 2% 46.66±0.34 53.74±0.57 8%

10 99.7±2.6 3%

14 98.6±1.6 2%

18 99.5±1.4 1%

RSD,relativestandarddeviation.

Table3 Robustnessdata.

Variables Levels Parameters

Concentrationa_{(mg/g) tR}a_{(min) RS}a _T

fa

Nominalconditions 42.56±0.04 3.634±0.002 2.937±0.087 1.075±0.007

Flow 0.3mlmin−1 _{56.62±0.20 4.376±0.015 2.225±0.021 1.143±0.010}

0.5mlmin−1 _{33.93±0.05 3.106±0.003 3.230±0.068 1.076±0.009}

Columntemperature 30◦_{C 42.50±0.02 4.000±0.013 2.262±0.024 1.105±0.007}

50◦_{C 42.22±0.14 3.249±0.017 3.180±0.035 1.092±0.000}

Modifiercontent 0.05% 42.06±0.06 3.583±0.018 2.609±0.046 1.087±0.007

0.15% 42.34±0.01 3.546±0.015 2.653±0.038 1.070±0.008

Nominalconditionsareflowat0.4mlmin−1_{,columntemperatureat40}◦_{Candmodifiercontent(formicacid)at0.1%.tR,retentiontime;RS,resolution(calculatedbetween} peaks4and10);Tf,tailingfactor;ThetR,RSandTfvalueswerecalculatedaccordingUSPharmacopeia(2007).

a_{Average±standarddeviation(n=3).}

naturalsamples(Limetal.,2014;Motilvaetal.,2014;Oketal., 2014;Stincoetal.,2014).

TheaccuracydataaregiveninTable2.Theaveragerecovery

ofthethree amountaddedof eriodictyolrangedfrom98.6%to

99.7%,andtheirRSDvalueswerelessthan3.0%,characterizinggood reliabilityandaccuracyofthemethod.

Thedeliberatevariationsanalyzedforrobustnessofthemethod areshowedinTable3.

The robustness was estimated by one-way ANOVA analysis

between the nominal determination and the different

lev-els of each variable (flow, column temperature and modifier

content),andassessedreferenttodifferentparameters

(concen-tration,retentiontime,resolutionandtailingfactorofthepeak) (Fig.2).

Amongrobustness evaluatedparameters,were notobserved

significantdifferencesintheeriodictyolconcentrationforcolumn temperatureat30◦_{Candmodifiercontentat0.15%variables.The}

parametertailingfactorwastheleastaffectedbythedeliberate variations,toleratingchangesofmodifiercontent(lowerandhigher level),higherlevelofflow(0.5mlmin−1_{)andhigherlevelof}

col-umntemperature(50◦_{C),withnostatisticalsignificantdifferences.}

Theflowvariationwasthemostimpactingvariableinthemethod, exceptforthetailingfactor(Table3,Fig.2).Resultsshowthatall variablesarecriticalforthemethodandshouldbecarefully con-trolledduringtheanalysis.

Thequantitativeanalysisoferiodictyolinleavescrudeextract from of V. tweedieana showed a content of 41.40±0.13mg/g

(RDS=0.31%). Eriodictyolhasbeendescribed and quantifiedfor

60.0

p<0.0001

****

p=0.0001

***

****

p=0.0037

**

40.0

30.0

20.0

Concentr

ation (mg/g)

10.0

0.0

Variables

5.0 p<0.0001

****

****

p<0.0001

****

p<0.0001

****

**

****

4.00

3.0

3.00

2.0

2.00

1.0

1.00

Retention time (min)

0.0

0.00

Nominal determination

Flow at 0.3 ml.min–1 Column temperature at 30ºC Modifier content at 0.05%

Modifier content at 0.15% Column temperature at 50ºC

Flow at 0.5 ml.min–1

Variables

p<0.0001

****

p<0.0001

****

p<0.0001

****

p<0.0001

****

p<0.0001

****

p=0.0003

***

Resolution

Variables

p<0.0001

****

_**

T

ailing f

actor

0.00 0.30 0.60 0.90 1.20 1.50

Variables

someotherplantextracts,butthecontentfoundintheleavesof V.tweedieanaseemstobesuperiortopreviouspublishedstudies (Borosetal.,2010;Linetal.,2007;Renetal.,2008).

Insummary,thephytochemicalinvestigationallowsto

char-acterizeten differentcompounds,ethyl caffeate(1), naringenin (2),chrysoeriol(3),eriodictyol(4), 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-one(5),evofolinB(6),apigenin(7), caffeicacid(8),protocatechuicacid(9)andluteolin(10),reported forthefirsttimeforV.tweedieanaexceptforeriodictyol.Further,a simpleandfastUPLC-PDAmethodforquantificationoferiodictyol

incrudeextractfromleavesofV.tweedieanawasdevelopedand

validated,whichcanbeusedtothequalitycontrolpreparations

thatcontainsV.tweedieana.

Authors’contributions

LALS (MSc student) contributed in collecting plant sample,

phytochemicalinvestigation,elucidationofcompounds,

chromato-graphic and validation analyses, data analysesand draftedthe

paper.LGF(MScstudent)contributed inrunningthelaboratory

work,validationanalysesanddraftedthepaper.FHRcontributed

todesignandinterpretationofthevalidatedanalyticalconditions.

ADCS(PhDstudent)andABcontributedtoNMRanalysesand

elu-cidationofcompounds.MWBdesignedthestudy,supervisedthe

laboratorywork,dataanalysesandcontributedtocriticalreading anddraftofthemanuscript.

Conflictofinterest

Theauthorsdeclarenoconflictofinterest.

Acknowledgments

TheauthorsthankCNPqandCAPESforthefinancialsupportand thescholarships.TheauthorsalsothankProfessorDr.AdemirReis fortheidentificationofplantmaterial.

References

Agati,G.,Azzarello,E.,Pollastri,S.,Tattini,M.,2012.Flavonoidsasantioxidantsin plants:Locationandfunctionalsignificance.PlantSci.196,67–76.

Alvim,N.A.T.,Ferreira,M.A.,Cabral,I.E.,AlmeidaFilho,A.J.,2006.Theuseof medic-inalplantsasatherapeuticalresource:fromtheinfluencesoftheprofessional formationtotheethicalandlegalimplicationsofitsapplicabilityasanextension ofnursingcarepractice.Rev.Lat.Am.Enferm.14,316–323.

Araujo,P.,2009.Keyaspectsofanalyticalmethodvalidationandlinearityevaluation. J.Chromatogr.BAnalyt.Technol.Biomed.LifeSci.877,2224–2234.

Balunas,M.J.,Kinghorn,A.D.,2005.Drugdiscoveryfrommedicinalplants.LifeSci. 78,431–441.

Bara, M.T.F., Ribeiro, P.A.M., Arantes, M.C.B., Amorim, L., Paula, J.R., 2006.

Determinac¸ãodoteordeprincípiosativosemmatérias-primasvegetais.Braz.J. Pharmacogn.16,211–215.

Boros,B.,Jakabova,S.,Dornyei,A.,Horvath,G.,Pluhar,Z.,Kilar,F.,Felinger,A., 2010.Determinationofpolyphenoliccompoundsbyliquid chromatography-massspectrometryinThymusspecies.J.Chromatogr.A1217,7972–7980.

Cabrera,A.L.,Klein,R.M.,1980.FascículoCompostas:3.TriboVernonieae.In:Reitz, R.(Ed.),FloraIlustradaCatarinense.HerbárioBarbosaRodrigues,Itajaí/SC,Brasil, pp.324–327.

Cragg,G.M.,Grothaus,P.G.,Newman,D.J.,2009.Impactofnaturalproductson devel-opingnewanti-canceragents.Chem.Rev.109,3012–3043.

Cragg,G.M.,Newman,D.J.,2013.Naturalproducts:acontinuingsourceofnoveldrug leads.Biochim.Biophys.Acta1830,3670–3695.

Díaz,G.,Nogueira,M.A.,Olguín,C.F.A.,Somensi,A.,Vidotti,G.J.,2008.Estudo fito-químicoebiológicodeVernoniatweedianaBaker(Asteraceae).Lat.Am.J.Pharm. 27,56–61.

Fang,J.,Reichelt,M.,Hidalgo,W.,Agnolet,S.,Schneider,B.,2012.Tissue-specific distributionofsecondarymetabolitesinrapeseed(BrassicanapusL.).PLoSONE 7,e48006.

Fatope,M.O.,Al-Burtomani,S.K.S.,Ochei,J.O.,Abdulnour,A.O.,Al-Kindy,S.M.Z., Takeda,Y.,2003.Muscanone:a3-O-(1′′_,8′′_,14′′_{-trimethylhexadecanyl)} narin-geninfromCommiphorawightii.Phytochemistry62,1251–1255.

Huang,Y.-H.,Zeng,W.-M.,Li,G.-Y.,Liu,G.-Q.,Zhao,D.-D.,Wang,J.,Zhang,Y.-L.,2014.

Characterizationofanewsesquiterpeneandantifungalactivitiesofchemical constituentsfromDryopterisfragrans(L.)Schott.Molecules19,507–513.

ICH,H.T.G.,2005.Validationofanalyticalprocedures:textandmethodology,Q2 (R1).CurrentStep4Version,ParentGuidelinesonMethodologyDated Novem-ber61996.IncorporatedinNovember2005.In:InternationalConferenceon HarmonisationofTechnicalRequirementsforRegistrationofPharmaceuticals forHumanUse,Geneva,Switzerland.

Jones,L.,Bartholomew,B.,Latif,Z.,Sarker,S.D.,Nash,R.J.,2000.ConstituentsofCassia laevigata.Fitoterapia71,580–583.

Julkunen-Tiitto,R.,Nenadis,N.,Neugart,S.,Robson,M.,Agati,G.,Vepsäläinen,J., Zipoli,G.,Nybakken,L.,Winkler,B.,Jansen,M.A.K.,2015.Assessingtheresponse ofplantflavonoidstoUVradiation:anoverviewofappropriatetechniques. Phytochem.Rev.14,273–297.

Kim,A.R.,Jin,Q.,Jin,H.G.,Ko,H.J.,Woo,E.R.,2014.PhenoliccompoundswithIL-6 inhibitoryactivityfromAsteryomena.Arch.Pharm.Res.37,845–851.

Laitinen,M.L.,Julkunen-Tiitto,R.,Rousi,M.,2002.Foliarphenoliccompositionof Europeanwhitebirchduringbudunfoldingandleafdevelopment.Physiol.Plant. 114,450–460.

Lee,S.Y.,Kim,K.H.,Lee,I.K.,Lee,K.H.,Choi,S.U.,Lee,K.R.,2012.Anewflavonol glycosidefromHylomeconvernalis.Arch.Pharm.Res.35,415–421.

Liao,C.-R.,Kuo,Y.-H.,Ho,Y.-L.,Wang,C.-Y.,Yang,C.-S.,Lin,C.-W.,Chang,Y.-S.,2014.

StudiesoncytotoxicconstituentsfromtheleavesofElaeagnusoldhamiiMaxim. innon-smallcelllungcancerA549cells.Molecules19,9515–9534.

Lim,S.J.,Jeong,D.Y.,Choi,G.H.,Park,B.J.,Kim,J.H.,2014.Quantitativeanalysisof matrineandoxymatrineinSophoraflavescensextractanditsbiopesticidesby UPLC.J.Agric.Chem.Environ.3,64–73.

Lin,L-Z.,Mukhopadhyay,S.,Robbins,R.J.,Harnly,J.M.,2007.Identificationand quantificationofflavonoidsofMexicanoregano(Lippiagraveolens)by LC-DAD-ESI/MSanalysis.J.FoodCompos.Anal.20,361–369.

Lin,Y.,Kong,L.,2006.StudiesonthechemicalconstituentsofDesmodium styraci-folium(Osbeck)Merr.AsianJ.Tradit.Med.1,34–37.

Motilva,M.J.,Macia,A.,Romero,M.P.,Labrador,A.,Dominguez,A.,Peiro,L.,2014.

Optimisationandvalidationofanalyticalmethodsforthesimultaneous extrac-tionofantioxidants:applicationtotheanalysisoftomatosauces.FoodChem. 163,234–243.

Ok,H.E.,Choi,S.W.,Kim,M.,Chun,H.S.,2014.HPLCandUPLCmethodsforthe determinationofzearalenoneinnoodles,cerealsnacksandinfantformula.Food Chem.163,252–257.

Olguin,C.d.F.A.,Hamerski,L.,Percio,M.F.,Somensi,A.,2005.Avaliac¸ãodopotencial biológicoalelopáticodosextratosfracionadosdaraizdaVernoniatweediana Baker.Rev.VariaSci.5,137–143.

Petri,R.D., Pletsch,M.U., Zeifert,M.,Schweigert,I.D.,2008. Efeito deextratos hidroetanólicosdeVernoniatweedieanaeVernoniacognatasobreimunidadede camundongos.Rev.Bras.Farmacogn.89,139–141.

Portillo,A.,Vila,R.,Freixa,B.,Ferro,E.,Parella,T.,Casanova, J.,Canigueral, S., 2005.AntifungalsesquiterpenefromtherootofVernonanthuratweedieana.J. Ethnopharmacol.97,49–52.

Ren,D.M.,Qu,Z.,Wang,X.N.,Shi,J.,Lou,H.X.,2008.Simultaneousdetermination ofninemajoractivecompoundsinDracocephalumrupestrebyHPLC.J.Pharm. Biomed.Anal.48,1441–1445.

Rossato,M.F.,Trevisan,G.,Walker,C.I.,Klafke,J.Z.,deOliveira,A.P.,Villarinho,J.G., Zanon,R.B.,Royes,L.F.,Athayde,M.L.,Gomez,M.V.,Ferreira,J.,2011.Eriodictyol: aflavonoidantagonistoftheTRPV1receptorwithantioxidantactivity.Biochem. Pharmacol.81,544–551.

Shabir,G.A.,2003.Validationofhigh-performanceliquidchromatography meth-odsforpharmaceuticalanalysis:Understandingthedifferencesandsimilarities betweenvalidationrequirementsoftheUSFoodandDrugAdministration,the USPharmacopeiaandtheInternationalConferenceonHarmonization.J. Chro-matogr.A987,57–66.

Steinmann,D.,Ganzera,M.,2011.RecentadvancesonHPLC/MSinmedicinalplant analysis.J.Pharm.Biomed.Anal.55,744–757.

Stinco,C.M.,Benítez-González,A.M.,Hernanz,D.,Vicario,I.M.,Meléndez-Martínez, A.J.,2014.Developmentandvalidationofarapidresolutionliquid chromatog-raphymethodforthescreening ofdietaryplantisoprenoids:carotenoids, tocopherolsandchlorophylls.J.Chromatogr.A1370,162–170.

Taleon,V.,Dykes,L.,Rooney,W.L.,Rooney,L.W.,2014.Environmentaleffecton flavonoidconcentrationsandprofilesofredandlemon-yellowsorghumgrains. J.FoodCompos.Anal.34,178–185.

Trevisan,G.,Rossato,M.F.,Walker,C.I.B.,Klafke,J.Z.,Rosa,F.,Oliveira,S.M.,Tonello, R.,Guerra,G.P.,Boligon,A.A.,Zanon,R.B.,2012.Identificationoftheplantsteroid ␣-spinasterolasanoveltransientreceptorpotentialvanilloid1antagonistwith antinociceptiveproperties.J.Pharmacol.Exp.Ther.343,258–269.

USP,2007.621Chromatography.In:UnitedStatesPharmacopeia.USPharmacopeial Convention,Rockville,MD.

Uwai, K., Osanai, Y., Imaizumi, T., Kanno, S., Takeshita, M., Ishikawa, M., 2008.Inhibitoryeffectofthealkylsidechainofcaffeicacidanalogueson lipopolysaccharide-inducednitricoxideproductioninRAW264.7macrophages. Bioorg.Med.Chem.16,7795–7803.

Walter,T.H.,Andrews,R.W.,2014.RecentinnovationsinUHPLCcolumnsand instru-mentation.TrAC–TrendsAnal.Chem.63,14–20.

Wu,T.-S.,Yeh,J.-H.,Wu,P.-L.,1995.TheheartwoodconstituentsofTetradium glabri-folium.Phytochemistry40,121–124.

Zanon,R.B.,2006.MetabólitossecundáriosemVernoniatweedianaBaker.In:Centro deCiênciasdaSaúde,ProgramadePós-Graduac¸ãoemCiênciasFarmacêuticas. UniversidadeFederaldeSantaMaria,SantaMaria/RS,Brasil.