Rev. bras. farmacogn. vol.26 número2

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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

Simultaneous

determination

of

epicatechin

and

procyanidin

A2

markers

in

Litchi

chinensis

leaves

by

high-performance

liquid

chromatography

Liliani

C. Thiesen

a

_,

_Luciana

_C.

_Block

b

_,

_Sabrina

_L.

_Zonta

b

_,

_Christiane

_M.

_da

_S.

_Bittencourt

a,b

_,

Rene

A. Ferreira

b

_,

_Valdir

_Cechinel

_Filho

a,b

_,

_Angélica

_G.

_Couto

a,b

_,

Tania

M.B.

Bresolin

a,b,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_do_Vale_do_Itajaí,_Itajaí,_SC,_Brazil b_Núcleo_de_Investigac¸_ões_{Químico-Farmacêuticas,}_Universidade_do_Vale_do_Itajaí,_Itajaí,_SC,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21July2015 Accepted10November2015 Availableonline31December2015

Keywords: Analyticalvalidation Epicatechin Leafextract LC-UVmethod Litchichinensis ProcyanidinA2

a

b

s

t

r

a

c

t

ThefruitsofLitchichinensisSonn.,Sapindaceae,arerenownedfortheirbiologicalactivities.However, theirleavesarepoorlyexplored,althoughtheyrepresentanimportantsourceofvegetablerawmaterial withbiologicalpropertiesasantioxidant,anti-inflammatoryandantinociceptive.AnHPLCmethodwas developedandvalidatedforthesimultaneousquantificationofepicatechinandprocyanidinA2intheleaf hydroethanolicextractofL.chinensis.Themarkersandotherunidentifiedcomponentswereseparatedon aLunaPhenomenexC18column(250mm×4.6mm,5␮m)withmobilephasecomposedofacetonitrile: waterpH3.0(withsulfuricacid),inagradientrun;at1.0mlmin−1_,₃₀◦_C_and₂₇₈_nm_for_detection.

ThemethodwaslinearoveranepicatechinandprocyanidinA2concentrationrangeof10–100␮gml−1. TheLimitofQuantificationforepicatechinandprocyanidinA2were1.7and2␮gml−1,respectively.The RelativeStandardDeviation(%)valuesformarkers(intra-andinter-dayprecisionstudies)were<4.0%and theaccuracywas100±5%.ThemethodwasappliedtotensamplescollectedinthestateofSantaCatarina (Brazil),whichshowed14.8–44.5and44.8–69.6mgg−1_of_epicatechin_and_procyanidin_A2,_{respectively.}

TheproposedmethodcouldbeavaluabletoolforqualityassessmentofL.chinenisleavesaswellastheir herbalderivatives.

Introduction

LitchichinensisSonn.,Sapindaceae,isanevergreentreethatis

indigenoustothesubtropicsofsouthernChinaandhasbeengrown

thereforover3500years(HolcroftandMitcham,1996).Thisspecie

iswidelydistributedinAsia,AfricaandAmericas,andthemain

economicinteresthasbeenassignedtoitsfruits,whichareused

bothasasourceoffoodasincosmetics(Menzel,2002;Martins,

2005).InBrazil,‘Bengal’and‘Brewster’specieshavebeencultivated

since1810(Martins,2005).

Therearemanyreportsaboutthephytochemicalcomposition

ofL.chinensisfruits,especiallyepicatechinandproanthocyanidins

(Ruenroengklinetal.,2008;Zhouetal.,2011;Lietal.,2012),and

∗ _{Corresponding}_author.

E-mail:tbresolin@univali.br(T.M.B.Bresolin).

pharmacologicalactivities,suchasimmunomodulatoryand

anti-tumoral(Wangetal.,2006;Zhaoetal.,2007),antioxidant(Yang

et al., 2006; Duan et al., 2007)and hepatoprotective (Bhoopat

etal.,2011).Ontheotherhand,leaveshavebeenusedas

poul-ticesforskindiseasetreatment(PandeyandSharma, 1989)and

alsoasa sourceof TraditionalChineseMedicineforheatstroke,

flatulenceanddetoxificationtreatment(Wenetal.,2014).

Anal-gesicandanti-inflammatoryactivitywasfirstlyreportedforthe

petroleum ether leaf extracts in 1996 (Bersa et al., 1996), as

wellas hepatoprotective activityin rats, especially for theleaf

methanolicextract(Basuetal.,2012).Recently,theisolationand

theantioxidant activity (DPPH,ABTS+ _and _lipidic

lipoperoxida-tion)of(−)-epicatechin(1), procyanidinA2 (2),procyanidinB2

(3),frommethanolandethanolcrudeleafextractsofL.chinensis,

werereportedbyourresearchgroup(Castellainetal.,2014),and

inaddition,themethanolextractshowedcentralandperipheral

antinociceptiveeffectinfourpre-clinicalmodels.Thesefindings

http://dx.doi.org/10.1016/j.bjp.2015.11.003

(2)

suggestthepotentialofL.chinensisleavesassourceofmaterial

forpharmaceuticandcosmeticherbalbasedproducts.Despitethe

pharmacologicalinterestinthisherbaldrug,thereisnoreportin

theliteratureofHPLCanalyticalmethodologytosupportits

qual-itycontrol,concerningthequantificationofpotentialmarkers,as

epicatechin(1)andprocyanidinA2 (2),whichhadalreadybeen

reportedinthelitchileaves(Castellainetal.,2014).Polyphenolsas

epicatechinandprocyanidinareknowntohavebeneficialeffects

onthehumanhealthandhavebeenquantifiedinseveral

medic-inal plants by LC-UV methods (Lopeset al., 2010; Negri et al.,

2010;Sousa et al.,2011), however,since herbalmedicines and

extractsareacomplexmatrix,eachspecierepresentsananalytical

challenge.

BasedonthesebiologicalactivitiesrelatedtoL.chinensisleaves,

hydroethanolic and glycolic extracts have been studied by our

groupassourceofactivepharmaceuticalingredientformedicines

andcosmeticherbalbasedproducts.Forthesepurposes,itis

impor-tant to establish markers and analytical methodologies, which

couldbeabletomonitorthechemicalqualityoftheherbalraw

materialanditsderivatives,batchtobatch,andalsoasavaluable

toolfor optimizationofextractionandstability indicating

stud-ies.In this sense, thepresent workaims thedevelopment and

validation ofa LC-UVmethodology toquantifyepicatechin and

procyanidinA2 in hydroethanolicextract of L. chinensis leaves,

applyingthemethodologytodifferentgeographicalsamples,

har-vestedintheSouthofBrazil.

Materialandmethods

Chemicalsandreagents

Epicatechin(1)andprocyanidinA2 (2)wereisolatedfromL.

chinensisleaves.Thepurityoftheisolatedcompoundswas

deter-mined by HPLC using the method described as follow (> 99%

for EPI and >85% for PA2). The chemical structures were

con-firmedbyNMR1_H_and13_C_as_previously_reported₍_Castellain_et_al.,

2014).

Methanoland acetonitrile(HPLC grade)wereobtainedfrom

J.T.Baker (Phillipsburg, New Jersey, USA). Water was purified

using Easy Pure® _equipment _(Waltham, _{Massachusetts,} _USA).

The ethanol was analytical grade, purchased from Dinâmica

(Diadema,São PauloBrazil). Allother solvents were analytical

grade.

O

OH OH

HO

OH OH

1

O

OH OH

HO

OH OH

2

O OH

O HO

HO OH

O

OH OH

HO

OH OH

HO

OH O

OH OH

OH

3

Plantmaterial

BatchesofLitchichinensisSonn.,Sapindaceae,leaveswere

sam-pledfromdifferentcitiesofSantaCatarina,astateintheSouthof

Brazil,inthesummerovertheyearsof2010–2014:Itajaí(yearof

2010,2014),Blumenau(yearof2010),Florianópolis(yearof2014),

Palhoc¸a(yearof2014),Pomerode(yearof2014),andZimbros(year

of2014).Allthesampleswereauthenticated,takingintoaccount

theliterature(Menzel,2002)andcomparedwiththevoucher

speci-men,number52829,whichwasdepositedattheBarbosaRodrigues

Herbarium(BRH,Itajaí-SC,Brazil).Theleavesweremanually

sep-arated, cleanedanddriedinanair ovenat35◦_C _until_moisture

stabilization.Afterdrying,theplantmaterialwasgroundinaknife

mill(outletsieve=2mm)andtheaveragesizeoftheparticleswas

determinedbysieving(FarmacopeiaBrasileira,2010).

Preparationofextractivesolutionandsoftextract

Theextractivesolutionwasobtainedbydynamicmaceration

ofmilledleaveswithethanol:water70:30(v/v)ataplant:solvent

ratioof5:100(w/v),stirredfor4hatroomtemperature,filtered

throughSontara® _paper,_kept_in _amber_glass_bottles _and_stored

atroomtemperature.Thedriedresiduewasdeterminedby

dry-ing5ml(weighed)ofextractivesolutionat100◦_C_until_constant

weightintriplicate.

Thesoftextractwasobtainedbyevaporationofextractive

solu-tionat50◦_C,_with_the_aid_of_vacuum_and_water_bath,_in_order_to

achieve 92%of driedresidue.The lossondryingof softextract

wasdeterminedbydrying0.5gat100◦_C_until_constant_weight_in

triplicate(FarmacopeiaBrasileira,2010).

Samplesolutionspreparation

BothextractivesolutionandsoftextractwereanalyzedbyHPLC

inordertoquantifythemarkers.Theextractivesolutionsamplefor

HPLCanalysiswaspreparedbydissolving1mlwith5mlwith

sol-ventsolution(1:9(v/v)acetonitrile:acidifiedwateratpH3.0using

sulfuricacid).Thedriedresiduewastakenintoaccounttoexpress

themarkerassayinthissample.

ThesoftextractsolutionsampleforHPLCanalysiswasprepared

bydissolving300mgin5mlofmethanol,ina 10mlvolumetric

flask,whichwassonicatedfor15min,andthevolumewas

(3)

storedat−20◦_C._At_the_moment_of_analysis,_the_stock_sample

solu-tionwas thawedat roomtemperature and diluted (1:10)with

solventsolution(1:9 (v/v)acetonitrile:acidifiedwater atpH3.0

usingsulfuricacid)toobtainaconcentrationof3mgml−1_._The_loss

ondryingwastakenintoaccounttoexpressthemarkerassayin

thissample.

Standardsolution

EPIandPA2(10mg)weredissolvedwithmethanoltoproducea

stockstandardsolution(1000␮gml−1).Thisstandardsolutionwas

keptinafreezer(−20◦_C)_and_used_for_the_analyses_and_validation.

Atthemomentofanalysis,thestockstandardsolutionwasthawed

atroomtemperatureanddiluted(1:10)withsolventsolutionto

obtainaconcentrationof50␮gml−1(standardsolution).

HPLCanalysis

AShimadzuLC-10ADLCsystem(Shimadzu,Tokyo,Japan)

con-sistingofabinarypump(LC-10ADvp),columnoven(CTO-10Avp),

anautomaticinjector(SIL-10AF)andaShimadzuSPD-M10Aphoto

diodearraydetectorwasused.The injections(20␮l)were

car-riedoutonaPhenomenex®_(Torrance,_California,_USA)_Luna_C18

5␮m(250mm×4.6mm)at30◦_C,_with_detection_at₂₇₈_nm._For_the

methoddevelopment,differentsolventsystemswereassayedin

isocraticandgradientconditionsusingmethanol,acetonitrileand

acidifiedwater(pH3.0withsulfuricacid),at1mlmin−1_._The_best

gradientwaschosen:gradientacetonitrile(A):acidifiedwaterpH

3.0withsulfuricacid(B)of10:90(A:B)(0–5min);15:85(5–7min);

20:80(7–13min);25:75(13–20min);30:70(20–30min);10:90

(30min)maintainingthiscompositionfor40minthenreturning

totheinitialconditions.

TheEPIandPA2assayinthesampleswasdeterminedby

exter-nalstandardization,bythesimultaneous analysisof astandard

solutionasdescribedabove.Daily,thesuitabilityparameters

(res-olution–R>1.0,tailingfactor–T<1.5,andrepeatabilityofarea–

RSD%<2.0)weredetermined.

Methodvalidation

Themethodwasvalidatedaccordingtothenational(Anvisa,

2003)andinternationalguidelines(ICH,2005),usingtheextractive

solutionpreparedfromthesample(Itajaí–summer,2010).

TheselectivityoftheHPLCmethodwasevaluatedbycomparing

thechromatogramofablank(diluentsolution),themobilephase,

andthesamplesolution,todetectanyco-elutioninterference.The

resolutionbetweenpeakswasanalyzed,andalsothepurityindex

ofmarkerspeaksthroughPDAdetector.

Thelinearitywasevaluatedforanalyticalcurvesusingthree

dif-ferentprocedures.Inthefirstprocedure,theEPIandPA2standard

solutionat1000␮gml−1 wasdilutedintriplicate,intherangeof

10–100␮gml−1,insevenlevels,andinjectedinduplicate(curve

A).CurveAwasspikedwithafixedvolumeofstocksample

solu-tion(1mlinallvolumetricflasksofthestandardanalyticalcurve)

(curveB).Finally,anothercurveofsamplesolutionwascarriedout

bythedilutionofastocksamplesolutionat5mgml−1_in_seven

dif-ferentlevels(1–5mgml−1_),_in_triplicate._All_the_analytical_curves

wereplottedandstatisticallyevaluated.

TheLimitofDetection(LOD)andLimitofQuantification(LOQ)

werecalculatedbasedonstandarddeviationofthey-interceptsof

equationcurvesobtainedbylinearregression(ICH,2005).

Theaccuracyofthemethodwasestimatedthroughtheanalyte

recoverytest(Anvisa,2003;ICH,2005),spikinga20mgml−1_stock

samplesolutionwiththreedifferentlevel(250,500and750␮l)of

stockstandardsolution(at1000␮gml−1)intriplicate.Thethree

levelsofstandardadditionrepresented50,100and150%ofthe

markerstargetconcentration,whichwaspreviouslydetermined

inthesample.Therecoveryofbothmarkerswascalculatedafter

discounting themarkerarea fromthesample solutionwithout

standardaddition.

Repeatability(intra-day)andintermediateprecision(inter-day)

weredeterminedthroughanalysis(sixreplicates)ofthesample

solution,preparedasdescribedabove,withinjectioninduplicate.

TheRSD%of themarkerassaywasdetermined. Thisprocedure

wasrepeatedaftertwodaysinordertoassesstheintermediate

precision.

Therobustnessofthechromatographicmethodwasevaluated

bychangingthemobilephaseflow(0.9,1.0and1.1mlmin−1_),_the

oventemperatureofthecolumn(29,30and31◦_C),_the_pH_of_the

mobilephase,andthestoreconditionsoftheanalyticalsolutions

(0,24and48h,atroomtemperature,fridgeandfreezer).Foreach

experiment,thesampleandthestandardsolutions(preparedas

describedbefore)wereinjectedintriplicate.Thedata(retention

time,resolutionandassayofmarkers)wereanalyzedandtheRSD%

wascalculated.

Statisticalanalysis

Theresultswereexpressedasthemean(RSD%).Statistical

sig-nificance among groups wascalculated by analysis of variance

(ANOVA)inwhichtheresultswereconsideredsignificantwhen

theprobabilitywaslessthan5%(p<0.05),followedbytheTukey

test.Statisticalanalysiswascarriedoutusingthesoftwareprogram

GraphPadPrism5.0.

Resultsanddiscussion

AccordingtoBrazilianand Europeanguidelines,markersare

chemicallydefinedasconstituentsorgroupsofconstituentswhich

are useful to the obtainment of an herbal preparation and an

herbalmedicinalproduct,beingconsideredactivemarkerswhen

contributetothetherapeuticactivity(EMA,2011;Anvisa,2014).

Therefore,PA2andEPImaybeconsideredactivemarkers,based

onpreviousstudiesofL.chinensisleaves(Castellainetal.,2014).

Thedeterminationofthesemarkerslevelsinavarietyofherbal

drugsamplescontributestotheestablishmentofspecificationsfor

thefuturedevelopmentofpharmaceuticalorcosmeticherbalbased

products.

Methoddevelopment

RegardingtheseparationofthephenoliccompoundsoftheL.

chinensisleafextractivesolution(Fig.1),initiallymanygradient

systems were tested, using acetonitrile, methanol and

acidi-fiedwater (pH 2.5–3.0, withsulfuric or trifluoroaceticacid), at

30–40◦_C,_1.0–1.3_ml_min−1_._Also,_different_columns_(XSelect_CSH®

hexylphenyl3.5␮m,4.6mm×100mm,Waters;LunaC183␮m,

4.6mm×150mm and 5␮m, 4.6mm×250mm, Phenomenex)

weretested.TheLunaC185␮m, 4.6mm×250mmPhenomenex

providedasuitablechromatographicprofile,withthebest

sepa-rationofbothsupposedmarkers,aswellastheothercompounds.

TheeluentwasmonitoredbyPDA,andthedetectionwavelength

wassetat278nmwhichrepresentsthewavelengthofmaximum

absorbance(Fig.1).

Inarunof40min,theEPIelutedat17minandPA2at24min

(Fig.2aandb).Anotherminoritycompound,identifiedasbeingPB2

elutedat16min.TheUVprofileofthetwomajormarkersisshown

intheinsertsofFig.1,beingsimilartotheothermajorunknown

peaks(peak1and2),withtypicalUVabsorptionprofileofphenolic

(4)

80

60

40

20

mA

U

0

0 2 4 6 8 10 12 14 16 18 20 22

Minutes

a

EPI

1

200220240260280300320340360

200220240260280300320340360 1

2

2 PA2

PA2

24 26 28 30 32 34 36 38 40

80

60

40

20

mA

U

0

0 2 4 6 8 10 12 14 16 18 20 22

Minutes

b

EPI

200220240260280300320340360 200220240260280300320340360

PA2 PA2

24 26 28 30 32 34 36 38 40

Fig.1. ChromatographicprofileofLitchichinensissamplesolutionat3mgml−1_(a);_EPI_and_PA2_at₁₀₀_␮_g_ml−1_(b).

Methodvalidation

Theselectivityofthemethodwasshown,withoutco-elution

ofthepeaks(Fig.1),purityindex>0.999,andabsenceofsolvent

influence.

TheEPIand PA2 calibrationcurves provedtobelinear over

therange of10–100␮gml−1,asshown bythe linearequations

and regression coefficients (r2_): _y₌₁₄₀₃₁_x₋_8345.3₍_r2_>_0.999),

y=11630x+20,426(r2_>_0.999),_for_the_EPI_and_PA2,_{respectively,}

demonstrating an acceptable data fit to the regression curve.

The standard addition in soft extract (curve B) proved to be

paralleltoisolatedstandards(curveA),withsimilarslopes,

with-out interference of the matrix on the linearity of the method

(y=13715x+60,459,(r2_>_0.996,_y₌₁₁₄₀₅_x₊_58,866,₍_r2_>_0.996)_for

theEPIandPA2,respectively),andrandomdistributionof

residu-als(datanotshown).Inaddition,theanalyticalcurveofsoftextract

(curveC)intherangecorrespondingto1–5mgml−1_of_EPI_and_PA2

alsoshowedsuitablelinearity(r2_>_0.99)._The_method_showed_high

sensitivity,asdemonstratedbythelowestimatedvaluesofLOD

andLOQforbothmarkers,of0.50and1.67forEPI,respectively,

and0.61and2.04␮gml−1,forPA2,respectively.

Themeanrecoveryofbothchemicalmarkerswaswithinthe

rangeof90–107%consideredacceptable(AOAC,2012)inviewof

thelow levelofchemicalmarkersintheanalyzedsamples.The

intra-andinter-dayprecision(Table1)showedRSD%<5%,which

isconsideredappropriate(AOAC,2012).

Therobustnesswasestimatedaftermakingsmallvariationsin

theparametersofthemethodology,andtheresultswereevaluated

basedonthemeanandRSD%(intraandinter)forarea,retention

time, resolutionand markerassayin thesoft extract (Table2).

Althoughthereisinfluenceofdeliberatevariationsinthe

method-ologyimposedinrelationtothechromatographicparameters(area,

retentiontimeand resolution),themethodprovedtoberobust

Table1

AccuracyandprecisionfortheassayofEPIandPA2inleafextractofLitchichinensis. Accuracy

Theoreticalconcentration (␮gml−1)

Recovery(%) RSD(%)

EPI PA2 EPI PA2 EPI PA2

26.13 22.96 102.5 102.2 0.67 2.69

52.43 45.94 102.8 102.2 2.03 0.72

80.05 69.96 104.6 103.8 0.38 2.09

Precision

AmountofEPI (mgg−1₎ (RSD%)

AmountofPA2 (mgg−1₎ (RSD%)

Repeatability(Day1) 19.58(2.96) 23.58(3.71)

Repeatability(Day2) 19.97(1.95) 24.31(2.54)

Intermediateprecision 19.78(2.64) 23.95(3.47)

consideringtheassayofbothmarkersinthesampleswithRSD% inter<5%formostdeterminations.Amongthechromatographic parametersmostaffected,theresolutionshowedthegreatest vari-abilityin thehighestlevelsoftemperature,flow and pHof the mobilephase.Regardingthevariationsimposedonthemethod, theflowofthemobilephaseshowedthegreatestimpactonthe chromatographicparameters,includingthedeterminationofPA2, highlightingthatthisparametershouldbewellcontrolledduring analysis.Thesampleandstandardsolutionsstoredatroom tem-perature,freezersandrefrigeratorshowedtobestablefor 48h, maintainingthepeakareaofbothmarkers(RSD%≤1.0%).

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Table2

RobustnessoftheassayofEPIandPA2inleafextractofLitchichinensis.

Mean(%RSDintra)

Area Rt(min) R Assay(mg/g)

EPI PA2 EPI PA2 EPI PA2 EPI PA2

Temperature(◦ C)

29 709,706(3.60) 711,762(0.92) 17.34(0.82) 23.59(0.11) 2.54(1.67) 1.14(4.40) 19.34(2.94) 16.65(0.92)

30 710,487(0.34) 707,988(1.85) 17.19(0.21) 23.86(0.15) 1.22(0.80) 1.75(2.94) 19.30(0.34) 22.15(1.85)

31 696,520(0.45) 684,428(1.24) 17.00(0.46) 24.02(0.22) 1.29(30.92) 1.60(34.66) 19.03(0.37) 22.12(1.24)

%RSDinter (1.87) (2.16) (0.78) (0.73) (27.06) (23.68) (1.76) (2.20)

Flow(mlmin−1₎

0.9 794,709(1.07) 814,976(2.20) 18.57(1.18) 25.40(0.67) 2.00(29.02) 2.18(4.49) 19.44(0.87) 22.87(2.20)

1.0 710,487(0.34) 707,988(1.85) 17.19(0.21) 23.86(0.15) 1.22(0.80) 1.75(2.94) 19.30(0.34) 22.15(1.85)

1.1 662,000(1.19) 655,750(1.27) 15.88(0.09) 22.30(0.33) 1.05(8.26) 1.67(9.33) 19.79(0.97) 22.08(1.27)

%RSDinter (7.29) (5.74) (6.08) (4.86) (28.71) (34.27) (1.47) (5.74)

pHmobilephase

2.9 727,819(0.08) 722,094(0.27) 16.79(0.23) 23.35(0.17) 1.33(1.98) 2.27(0.25) 19.63(0.08) 21.75(0.61)

3.0 710,487(0.34) 707,988(1.85) 16.74(0.18) 23.86(0.15) 1.21(4.26) 1.75(2.94) 19.30(0.34) 22.15(1.85)

3.1 704,938(2.60) 689,595(1.64) 17.12(0.57) 23.64(1.75) 1.78(3.89) 2.04(17.93) 19.33(2.12) 22.38(1.64)

%RSDinter (1.76) (8.91) (1.10) (5.05) (13.88) (21.33) (1.50) (2.20)

80

60

40

20

a

b

bc bc

bc c

Procy

anidin A2 (mg/g)

0

Flor

ianopolis 2014 Palhoça 2014 Itajai 2010

Itajai 2014

Pomerode 2014Zimbros 2014

80

60

40

20

b

a b

b

c _c

d

Epicatechin (mg/g)

0

Flor

ianopolis 2014Palhoça 2014

Itajai 2014 Itajai 2010

Pomerode 2014 Zimbros 2014

Fig.2.ProcyanidinA2(a)andEpicatechin(b)concentrationandvariationamong samplesofLitchichinensisleaveswhichwereharvestedinthesummerofdifferent citiesofSantaCatarinaState(Brazil).Meansfollowedbythesameletteroverthe barsdonotdifferaccordingTukeytest(˛=0.05).

shown)incomparisonwiththesamplefromItajaí.EPIandPA2 werepresentinallsamples(Fig.2).

TheamountofEPIandPA2mayvarydependingonthecity(five

differentlocation)forthesameseason(Fig.2),withhigher

con-tentofPA2inallsamples.Ontheotherhand,thevariationofEPI

washigherthanthatofPA2,beingthefirstamorediscriminative

markerofenvironmentalconditions.Apartfromtheneedto

estab-lishthemarkersranges,tosupportthequalityspecificationsofraw

plantmaterialitisknownthattanninspossessanabilityas

non-enzymaticantioxidantsandinhighconcentrationstheymayalso

playaroleinUV-protectionofplants(Hagermanetal.,1998).Also,

Lavolaetal.(2003)reportedthatambientrangeofUV-Bradiation

caninfluencethechemicalcomposition(flavonoidsandtannins)of

Scotspineseedlings(Pinussylvestris)andenhancementinnutrients

mayincreasetheirresponsiveness.

Therefore,theseactivecompoundscanbeestablishedasquality

markersforL.chiniensisleavesderivativesemployedinthe

devel-opmentof pharmaceuticals and cosmetics. Itsdetermination in

differentsamplesisacrucialfactorforthefutureestablishment

ofqualityspecificationsforvegetablerawmaterialtobeusedin

thepharmaceuticalandcosmeticsindustries.Afurtheranalysisof

thebiologicalactivityofthedifferentsampleswillestablishthe

minimumlimitsformarkers,contributingtotheproductionofa

monographfortheherbaldrug.

Conclusion

TheLC-UV methodfor theEPIand PA2 assaywasvalidated

andprovedtobesensitive,accurate,linear,precise,reproducible,

repeatableandrobust.Theresultsshowedthatthismethodcould

bereadily appliedas a suitable quality-control methodfor the

quantification ofepicatechin and procyanidinA2 in L.chinensis

leavesderivatives,andmaybeusedinthefuturedevelopmentof

herbalmedicinalproducts,aspotentialmarkers.Furtherstudyis

needbeyondseasonality,locationasparametersforestablishment

ofqualitycontrolspecificationsofthisspecieasherbal

pharmaceu-ticalrawmaterial.

Authorscontributions

LCT,LCBandSLZdevelopedtheresearch,RAFassistedthein

micro-andmacroscopicidentificationofthespecies,CMSBandVCF

(6)

researchandpreparedthemanuscript.Alltheauthorshaveread

thefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbyLaboratóriodeProduc¸ãoeAnálise

deMedicamentos(Univali-Lapam);CNPqandCAPES,Brazil,inthe

formofadoctoral’sdegreescholarshiptoThiesen,L.C.

References

Anvisa,2003.Resoluc¸ãon899de29demaiode2003:GuiaparaValidac¸ãodeMétodo AnalíticoeBioanalíticoAgênciaNacionaldeVigilânciaSanitária.DiárioOficial daUnião,Brasília(accessed02.06.03).

Anvisa,2014.Resoluc¸ãon◦₂₆_de₁₃_de_maio_de_2014._Agência_Nacional_de_Vigilância Sanitária.DiárioOficialdaUnião,Brasília(accessed14.05.14).

AOAC, 2012. Guidelines for Single Laboratory Validation of Chemical Meth-ods for Dietary Supplements and Botanicals., pp. 1–38, Available from: http://www.aoac.org/imis15 prod/AOACDocs/StandardsDevelopment/SLV GuidelinesDietarySupplements.pdf(accessedMay2015).

Basu,S.,Haldar,N.,Bhattacharya,S.,Biswas,S.,Biswas,M.,2012.Hepatoproctive activityofLitchichinensisleavesagainstparacetamol-inducedliverdamagein rats.J.Sci.Res.7,77–81.

Bersa,S.E.,Sharma,R.M.,Gomes,A.,1996.Antiinflammatoryeffectofpetroleum etherextractofleavesofLitchichinensisGaertn.(Sapindaceae).J. Ethnopharma-col.54,1–6.

Bhoopat,L.,Srichairatanakool,S.,Kanjanapothi,D.,Taesotikul,T.,Thsnanchai,H., Bhoopat,T.,2011.Hepatoprotectiveeffectsoflychee(LitchichinensisSonn.):bA combinationofantioxidantandanti-apoptoticactivities.J.Ethnopharmacol. 136,55–66.

Castellain,R.C.L.,Gesser,M.,Tonini,F.,Schulte,R.V.,Demessiano,K.Z.,Wolff,F.R., Delle-Monache,F.,Netz,D.J.A.,Cechinel-Filho,V.,deFreitas,R.A.,deSouza,M.M., Meyre-Silva,C.,2014.Chemicalcomposition,antioxidantandantinociceptive propertiesofLitchichinensisleaves.J.Pharm.Pharmacol.66,1796–1807. Duan,X.,Jiang,Y.,Su,X.,Zhang,Z.,Shi,J.,2007.Antioxidantpropertiesof

antho-cyaninsextractedfromlitchi(LitchichinensisSoon.)fruitpericarptissuesin relationtotheirroleinthepericarpbrowning.FoodChem.101,1365–1371. EMA/CPMP/QWP/2819/00Rev.2:Guidelineonqualityofherbalmedicinal

prod-ucts/traditionalherbalmedicinalproducts,2011.http://www.ema.europa.eu/ docs/enGB/documentlibrary/Scientificguideline/2011/09/WC500113209.pdf (accessedMarch2015).

FarmacopeiaBrasileira,2010.AgênciaNacionaldeVigilânciaSanitária,Brasília,DF, v.1e2,5thed.

Holcroft,D.M.,Mitcham,E.J.,1996.Postharvestphysiologyandhandlingoflitchi (LitchichinensisSonn.).PostharvestBiol.Tech.9,265–281.

Hagerman,A.E.,Riedl,K.M.,Jones,G.A.,Sovik,K.N.,Ritchard,N.T.,Hartzfeld,P.W., Riechel,T.L.,1998.Highmolecularweightplantpolyphenolics(tannins)as bio-logicalantioxidants.J.Agric.FoodChem.46,1887–1892.

ICH,2005.ValidationofAnalyticalProcedures:TextandMethodology.International ConferenceonHarmonization,Geneva.

Lavola,A.,Aphalo,P.J.,Lahti,M.,Julkunen-Tiitto,R.,2003.Nutrientavailabilityand theeffectofincreasingUV-BradiationonsecondaryplantcompoundsinScots pine.Environ.Exp.Bot.49,49–60.

Li,S.,Xao,J.,Chen,L.,Hu,C.,Chen,P.,Xie,B.,Sun,Z.,2012.Identificationof A-seriesoligomericprocyanidinsfrompericarpofLitchichinensisbyFT-ICR-MS andLC–MS.FoodChem.135,31–38.

Lopes,G.C.,Blainski,A.,dosSantos,P.V.P.,Diciaula,M.C.,PalazzodeMello,J.C.,2010. DevelopmentandvalidationofanHPLCmethodforthedeterminationof epicat-echininMaytenusilicifolia(Schrad.)Planch.,Celastraceae.Rev.Bras.Farmacogn. 20,781–788.

Martins,A.B.G.,2005.Lichia.Rev.Bras.Frutic.27,349–520.

Menzel,C.(Ed.),2002.TheLycheeCropinAsiaandthePacific.RegionalOfficefor AsiaandthePacific,Thailand,pp.1–33.

Negri,G.,diSanti,D.,Tabach,R.,2010.Bitteracidsfromhydroethanolicextractsof

HumuluslupulusL.,Cannabaceae,usedasanxiolytic.Rev.Bras.Farmacogn.20, 850–859.

Pandey,R.M.,Sharma,H.C.,1989.ThelitchiICAR.NewDelhi,India,pp.1–79. Ruenroengklin,N.,Zhong,J.,Duan,X.,Yang,B.,Li,J.,Jiang,Y.,2008.Effectsofvarious

temperaturesandpHvaluesontheextractionyieldofphenolicsfromLitchi fruitpericarptissueandantioxidantactivityoftheextractedanthocyanins.Int. J.Mol.Sci.9,1333–1341.

Sousa,S.,Alves,S.F.,dePaula,J.A.M.,Fiuza, T.S.,Paula,J.R.,Bara,M.T.F.,2011. Determinac¸ãodetaninosemetilxantinasnoguaranáempó(Paulliniacupana

Kunth,Sapindaceae)porcromatografialíquidadealtaeficiência.Rev.Bras. Far-macogn.20,866–870.

Wang,X.,Yuan,S.,Wang,J.,Lin,P.,Liu,G.,Lu,Y.,Zhang,J.,Wang,W.,Wei,Y.,2006. Anticanceractivityoflitchifruitpericarpextractagainsthumanbreastcancer invitroandinvivo.Toxicol.Appl.Pharmacol.215,168–178.

Wen,L.,He,J.,Wu,D.,Jiang,Y.,Prasad,K.N.,Zhao,M.,Lin,S.,Jiang,G.,Luo,W.,Yang, B.,2014.Identificationofsesquilignansinlitchi(LitchichinensisSonn.)leafand theiranticanceractivities.J.Funct.Foods8C,26–34.

Yang,B.,Wang,J.,Zhao,M.,Liu,Y.,Wang,W.,Jiang,Y.,2006.Corrigendumto: Iden-tificationofpolysaccharidesfrompericarptissueofLitchichinensisSoon.fruit inrelationtotheirantioxidantactivities.Carbohydr.Res.341,634.

Zhao,M.,Yang,B.,Wang,J.,Liu,Y.,Yu,L.,Jiang,Y.,2007.Immunomodulatoryand anticanceractivitiesofflavonoidsextractedfromlitchi(LitchichinensisSonn.) pericarp.Int.Immunopharmacol.7,62–66.

Zhou,H.,Lin,Y.,Li,Y.,Li,M.,Wei,S.,Chai,W.,Tam,N.,2011.Antioxidantpropertiesof polymericproanthocyanidinsfromfruitStonesandpericarpsofLitchichinensis