Rev. bras. farmacogn. vol.27 número5

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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

Short

communication

Quantification

of

catechin

in

the

spray-dried

extract

of

Pimenta

pseudocaryophyllus

Leonardo

C. Silva

a

_,

_Rúbia

_D.

_Machado

b

_,

_Dayane

_R.

_Silva

a

_,

_Vanessa

_C.S.

_Amaral

a

_,

_José

_R.

_Paula

b

_,

Edemilson

C. Conceic¸

ão

b

_,

_Joelma

_A.M.

_Paula

a,∗

a_Universidade_Estadual_de_Goiás,_Campus_Anápolis_de_Ciências_Exatas_e_{Tecnológicas,}_Anápolis,_GO,_Brazil

b_Laboratório_de_Pesquisa,_{Desenvolvimento}_and_Inovac¸_ão_de_Bioprodutos,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30April2017

Accepted7July2017

Availableonline5August2017

Keywords:

HPLC-PDA

Citralchemotype

Qualitycontrol

Chemicalmarker

a

b

s

t

r

a

c

t

Thispaperdescribesthequantificationofcatechininthespray-driedextractofPimenta pseudocaryophyl-lus(Gomes)Landrum,Myrtaceae,citralchemotypeusingavalidatedHPLC-PDAmethod.Themethod employsaRP-18columnwithacetonitrile:water-orthophosphoricacid0.05%(gradientsystem)andUV detectionat210nm.Themethodwasdemonstratedtobesimple,sensitive,specific,linear,precise, accu-rateandrobust.Theresponsewaslinearoverarangeof5–200␮g/ml(r>0.999).Therangeofrecoveries was92.27–102.54%.Therelativestandarddeviationvaluesforintra-andinter-dayprecisionstudieswere 4.30and3.78%,respectively.Thisassaycanbereadilyutilizedasqualitycontrolmethodforcatechinin thedriedextractofP.pseudocaryophyllus.

Introduction

Pimenta pseudocaryophyllus (Gomes) Landrum, Myrtaceae,

knownas“pau-cravo”,“louro-cravo”,“louro”,“craveiro”,and “chá-de-bugre”istheonlyspeciesofthegenusnativetoBrazil.Infolk medicine,teasandrefreshingdrinkspreparedwithitsleavesare usedastranquilizers,diuretics,digestiveregulators, andforthe reliefofcoldsymptoms,fatigue,fevers,andmenstrualproblems. Theyhavealsobeenusedtotreatarthritis,gonorrheaandsyphilis (Landrum,1986,D’angelisandNegrelle,2014).

Pimentapseudocaryophyllusisanaromaticplantwithessential oilsofvariablechemicalconstitutioninitsleaves.Intraspecific vari-abilitystudieshaveidentifiedthreechemotypesfor thisspecies basedonitsessentialoilchemicalcomposition:citral, caryophyl-leneand(E)-methylisoeugenol(Paulaetal.,2011).

Manystudieshaveshownthebiologicalandpharmacological activitiesofthisplant:anti-nociceptive,anti-inflammatory, anti-fungal,anti-depressive-like,againstUVB-inducedoxidativestress (Paulaetal.,2012;Campaninietal.,2013;Fajemiroyeetal.,2013;

Campaninietal.,2014;Silvaetal.,2014).

The compounds lupeol, ␣-amyrin, ␤-amyrin, quercetin, quercitrin, catechin and afzelin isolated from the fractions of the crude extractsof the leaves of P. pseudocaryophyllus citral

∗ Correspondingauthor.

E-mail:joelma.paula@ueg.br(J.A.Paula).

chemotypemayberelatedtotheanti-inflammatory, antinocicep-tive,antioxidantandantimicrobialactivitiespreviouslymentioned (Paulaetal.,2012;Silvaetal.,2014).Catechincanbeconsidered one of the main compounds because of its considerable con-centrationintheextractanditstherapeuticimportancealready registeredforotherplants(Bansaletal.,2013;SharmaandGoyal, 2015;Ranaetal.,2016).

Traditional HPLC coupled with ultraviolet (UV), photodiode array(PDA),electrochemicaldetection(ECD),andmass spectrom-etry(MS)isamongthemostfrequentlyemployedmethodsinthe identificationofcatechinsinplantmaterials(Novaketal.,2010; Cuevas-Valenzuelaetal.,2014;Baeetal.,2015;Taoetal.,2016). Mostrecently,ultrahighpressureliquidchromatography(UHPLC) hasshownconsiderablepotentialintermsofspeedand separa-tionefficiencyfortheanalysisofcatechins(Rahimetal.,2014). HPLC-MSandHPLC-MS/MSareconsideredmoreusefulthanother methodsbecausetheyprovideinformation aboutthemolecular massandstructuralfeaturesofcomponents.However,sinceUHPLC andHPLC-MSmethodsarequitecostlytopurchaseandmaintain, manylaboratoriesprefertouseHPLC-UVorPDAdetection,which arefoundtobelesscostly,comparablyconvenienttooperate,and remainsuitableforprovidingroutineanalysis(Baeetal.,2015).

Therefore,consideringthepharmacologicalpotentialofP.

pseu-docaryophyllus, the goal of the present work was to quantify

catechininthespray-driedextractofthecitralchemotypefromP. pseudocaryophyllusleaves,usingavalidatedHPLC-PDAmethod,to

http://dx.doi.org/10.1016/j.bjp.2017.07.002

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Table1

Summaryofparametersofcatechinandspray-driedextractcalibrationcurves.

Catechin Spray-driedextract

Linearrange(g/ml) 200–5 4000–100 (219.32–4.36of catechin)

LOD(g/ml) 0.7825 21.3463

LOQ(g/ml) 2.6085 71.1545

Linearregressiondataa

n 7 7

Slope(a) 91367.0161 5064.4

Standarddeviationof

theslope

1658.363 126.1257

Standarddeviationof

theslope(%)

1.8150 2.4904

y-Intercept(b) 32641.3373 −78592

Linearcorrelation

coefficient(r)

0.9998 0.9999

a_y₌_ax₊_b_,_where_x_is_the_{concentration}_of_the_analyte_and_y_is_the_peak_area.

contributetothechoiceofachemicalmarkerandoftheanalytical methodthatismostusefulinitsqualitycontrol.

Materialandmethods

AllreagentsandsolventswereHPLCgrade(J.T.Baker®,USA), exceptorthophosphoricacid(Impex®,P.A.grade).Thewaterwas purifiedusingaMilli-Qsystem(Millipore®,USA).Catechin(Sigma®, USA)of thehighestgrade (purity>98.0%)wasusedas external standard.

LeavesofPimentapseudocaryophyllus(Gomes)Landrum, Myr-taceae,citralchemotypewerecollectedinSãoGonc¸alodoAbaeté (MinasGerais,Brazil)18◦₂₀′_58.4′′_South;₄₅◦₅₅′_23.4′′_West,₈₆₄_m,

inFebruary2014.TheplantmaterialwasidentifiedbyCarolynE. BarnesProenc¸a,fromtheUniversidadedeBrasília.Avoucher spec-imenwasdepositedintheherbariumoftheUniversidadeFederal deGoiásunderthenumberUFG-27.159.

Leavesweredriedinacirculatingairovenat40◦_C,_and

pow-dered.Themilledplantmaterialwassubmittedtopercolationwith 95%(v/v)ethanol.Thefluidextractwasconcentratedinarotary evaporatoruntilplant:solventratioof1:2.

Thespray-dryingoftheconcentratedextractwasperformed usingaminispray-dryermodelLM-MSD1.0(Labmaq®,Brazil)with aconcurrentflowregime.Thedryingconditionswere:setat dry-ingairinlettemperatureof125◦_C,_outlet_temperature_of₆₅◦_C,_the

atomizingairflowratewasmaintainedat40l/minwithapressure of4kgf,dryingairflowrateat3m3_/min_and_flow_rate_of_the

con-centratedextractat4ml/min.Thetemperatureinthedryingroom wasmaintainedat25◦_C_and_the_relative_humidity_at_25%._No_drying

adjuvantswereused.

For quantification of catechin in the spray-dried extract, a Waters® HPLCAlliancesystem(WatersCorp.,USA)consistingof aseparation modulee2695,a 2998photodiodearraydetector, andsoftwareEmpower2.0wereused.Thechromatographicruns withinjectionvolumeof10␮lwerecarriedoutonaZorbaxEclipse Plus(Agilent®)C-185␮m(250mm×4.6mm)columninanoven equilibratedat25◦_C._During_development_of_the_method,_different

solventsystemsweretriedoutandtheanalysesweremonitored from190to400nm.Themobilephasethatdemonstratedthebest resultsconsistedofapreviouslysonicatedmixtureofacetonitrile (A)andwater-orthophosphoricacid0.05%(B);12:88(0–9min); 30:70(9–17min);12:88(17–30min),in gradientconditionand flowrateof1ml/min.Wavelengthsbetween200and280nmare ordinarilyappliedtothedetectionofcatechins(Santagatietal., 2008;Yuetal.,2009;Dhananietal.,2016).Inthisworkthe chro-matogramswasrecordedat210nm.Atthiswavelength,thebest

base-lineseparationswithmaximumabsorbanceingradient con-ditionswereachieved(datanotshowed).

Forpreparationofthesample,1mgofthespray-driedextract wasdissolvedinmethanol(10ml)bysonicationfor10minand filteredthrougha0.45␮mMillipore®membranefilter.

Thecatechinstandard(1mg)wasdissolvedinmethanol(10ml) bysonicationfor10mininordertoprepareaworkingsolution, whichwasusedforthesubsequentdilutionsforvalidationofthe method.

Thevalidationofthemethodwasperformedaccordingtothe InternationalConferenceontheHarmonization(ICH)ofTechnical RequirementsfortheRegistrationofPharmaceuticalsforHuman Use(ICH,2005), andBrazilianResolutionRE899/2003fromthe BrazilianNationalSurveillanceAgency(Anvisa,2003).

Thechromatographicsystemwasevaluatedbeforethemethod validationinordertoassessthesuitabilityparameters(US-FDA, 2001),includingResolution(Rs),tailingfactor(TF)andthenumber oftheoreticalplates(n).Theresultswereexpressedastheaverage (±SD)ofsixdeterminations.

Theselectivityofthemethodwasevaluatedthroughthe com-parisonofthechromatogramsoftheblank(methanol),thesample solutionandthecatechinstandardinordertodetectiftherewere anyco-elutioninterferents.Thespectralsimilarityofthecatechin peakinthestandardandsamplewasalsoevaluatedbycomparing theUVspectraoverarangeof190–400nm.

Thelinearitywasdeterminedbytheanalyticalcurveofseven solutions with concentration of the catechin standard ranging between5.0and200␮g/ml.Thesesolutionswerepreparedin trip-licateandinjectedthreetimes,afterfiltrationthrougha0.45mm Milipore®membrane.Theanalyticalcurveandlinearityequations werecalculatedbylinearregressionanalysisfromthecorrelation betweenthepeakareasandstandardconcentrations,usingExcel software.Thelinearcorrelationcoefficient(r)wascalculated.

BasedonAnvisa’sNormativeInstructionn◦_4,_published_on_June

18th2014(Anvisa,2014),thelinearitywasalsodeterminedforthe spray-driedextractinconcentrationsolutionsrangingfrom4.36to 219.32␮g/mlofcatechin.

Thelimitofdetection(LOD)andlimitofquantification(LOQ) weremathematicallydeterminedbasedonthestandarddeviation (SDb)ofthey-interceptandtheslope(S)ofthecalibrationcurve, accordingtoEqs.(1)and(2).

LOD= SDb×3

S (1)

LOQ= SDb×10

S (2)

Theprecisionwasevaluatedattwolevels:repeatability (intra-day) and intermediate precision (inter-day), using the relative standarddeviation(RSD)asthecriterion.

Therepeatabilitywasdeterminedthroughanalysisofthe sam-plesolutionbyperformingthreeinjectionsatthreelevels(5,50 and200␮g/ml)ofthemarkerintriplicate.

Theintermediateprecisionwasassessedbythesameprocedure overaperiodoftwodaysandbydifferentanalysts.

Theaccuracywasdeterminedthroughtheanalyterecoverytest. Samplesolutionswerepreparedatlow,mediumandhighlevel concentrations(80%,100%,and120%)ofthemarkerintheliner range,intriplicate.Standardconcentrationof25␮g/mlofcatechin wasaddedtothreesamplesolutions.

Theaccuracywascalculatedforeach levelthroughtheratio betweentheaverageexperimentalconcentrationandtheoretical concentrationoftheaddedstandardaccordingtoEq.(3).

Accuracy=samplecon.withstandard−sampleconc.withoutstandard

standardtheoreticalconcentration ×100 (3)

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1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

0.00 2.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Catechin Catechin

8.17 min 8.13 min

14.00

Minutes16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

4.00

AU

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

nm

AU

nm

AU

22.00 24.00 26.00 28.00

A

B

30.00

1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

t_R t_R

Fig.1. ChromatogramofPimentapseudocaryophylluscitralchemotypespray-driedextract(A),catechinstandard(B).Forchromatographicconditions,seeMaterialand

Methodssection.

Table2

ANOVAdatatothelinearityofcatechinandspray-driedextract.

DF SS MS F Ftab

Catechin

Model 1 8.6741×1014 _8.6741

×1014 _19706.89984 _4.38

Residual 19 9.04547×1011 _4.7607741360_×₁₀10 _Linear

Lackoffit 5 2.8833×1011 _5.7665921163_×₁₀10 _1.3101265 _2.95

Error 14 6.16217×1011 _4.4015534288_×₁₀10 _No_lack_of_fit

Spray-driedextract

Model 1 1.03912×1015 _1.03912_×₁₀15 _9056.30325 _4.38

Residual 19 1.75672×1012 _9.2458980207_×₁₀10 _Linear

Lackoffit 5 1.50361×1011 _3.0072203206

×1010 _0.26209 _2.95

Error 14 1.60636×1012 _1.1474

×1011 _No_lack_of_fit

DF,degreesoffreedom;SS,sumofsquares;MS,meansumofsquares;F,calculatedFvalue;Ftab,tabulatedFvalue.

conditions.Thefollowingconditionswerechanged:thebatch

num-berofthecolumn,theoventemperatureofthecolumn(25◦_C,₂₃◦_C,

and27◦_C)_and_the_manufacturer_of_acetonitrile_used_in_the_mobile

phase.Foreachoftheaboveconditions,thesamplesolutionswere

injectedintriplicate.Thedatawereevaluatedusingtherelative

standarddeviation(RSD).

Thequantificationofcatechininthespray-driedextractwas

performedthroughthelinearregressionoftheexternalstandard,

insixreplicates.ThecatechincontentwasdeterminedbyEq.(4).

Content (%)C×100

Ca (4)

C=catechin concentration (␮g/ml) in the extract, calculated throughthelinearregressionequation.

Ca=extractconcentrationinthesamplesolution(␮g/ml).

Resultsanddiscussion

Theanalyticalseparationswereoptimized byevaluating dif-ferent parameters and solventsystems consisting of methanol, acetonitrile, orthophosphoric acid solution (0.05%) and acetic acid (1–2%) as mobile phase, in isocratic and gradient condi-tions.Amongstthetestedmobilephases,thebestpeakresolution for catechin and thebestbase-line separations withmaximum

absorbance were achieved employing a gradient method with mobilephasethathasconsistedofamixtureofacetonitrile(A) andwater-orthophosphoricacid0.05%(B):12(A):88(B)(0–9min); 30(A):70(B) (9–17min); 12(A):88(B) (17–30min), monitoredat 210nmatflowrateof1ml/min.Itisopportunetonotethateven thoughtheretentiontimeofcatechinisabout8.10min,therun timeofthemethodwasproposedtobe30minsothatthe chro-matographicsystemis settoitsfirst conditionslowly. Besides, thereareothercompoundsintheextractwithlongerretention timesthatneedtoberemovedfromthecolumnbeforethenext injection.

Fig.1showsthechromatogramsofthesamplesolution,marker (catechin), and blank (methanol) as well as the UV spectra (190–400nm)ofcatechininthechosenconditionsdescribedabove. Thedevelopedmethodprovedtobeselectiveandspecific.No co-elutioninterferentsweredetectedandtherewasnointerference intheretentiontimeofcatechin.

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The linearity results for catechin and sample in the devel-opedmethodarepresentedinTable1.Thecatechinandsample calibrationcurves provedtobelinearover theproposedrange, 5–200␮g/mland100–4000␮g/mlrespectively,andthedatashow agood linearcorrelationcoefficient(r), whichdemonstrates an acceptabledatafittotheregressioncurve.

InaccordancewiththeAnalyticalMethodsCommittee(AMC),a valueoflinearcorrelationcoefficientclosetotheunitisnotenough datatosupportlinearity,and,consequently,thetestforthelackof fitshouldbeapplied.Thistestassessesthevarianceoftheresidual values(Hadadetal.,2009).AccordingtoTable2,thecalculatedF

valuesforlackoffitwerelowerthanthetabulatedFvaluesat95% confidencelevel(p=0.05);therefore,thelinearregressiondidnot showlackoffitforthemarkerandsample,confirmingthelinearity ofthemethods.Therelativestandarddeviation(%RSD)regarding theslopeofthecalibrationcurveofcatechinwas1.815%.Thisvalue iswithinthelimitssetbyICH(2005)andAnvisa(2003),which shouldnotexceed5%.

TheLODvalueswere0.7825␮g/mland21.3463␮g/mlfor cat-echinstandardandcatechininthesample,respectively.TheLOQ valueswere2.6085␮g/mland71.1545␮g/mlforcatechinstandard andcatechininthesample,respectively.

Theresultsofthemethod’sprecision,atthelevelsof repeatabil-ityandintermediateprecision,bothexhibitedRSDvalueslower than5%.TheRSDforday1,analyst1was4.30%;theRSDforday2, analyst2was2.41%;andtheRSDinter-daywas3.78%.

For the accuracy results, the recovery interval ranged from 92.276%to102.547%,withRSD%of3.16%.Therecoverytest meas-urestheamountoftheanalyte,presentoraddedintheanalytic portionofthetestmaterialthatisrecoveredandcouldbe quan-tified.Theacceptablerecoveryintervalsdependontheanalytical complexityandthesample,andcanrangefrom50to120%with precisionupto±15%(Ribanietal.,2004).

Themodifiedconditionsinthecolumnoventemperature,the acetonitrilemanufacturerandcolumnbatchresultedin%RSD val-uesof3.71,3.90,and4.31forthepeakarea,and3.30,3.44,and3.61 forthecatechincontent,respectively,demonstratingthe robust-nessoftheproposedmethod,sincetherewasnosignificanteffect onchromatographicresolutioninthemethod.

Thecatechincontentinthespray-driedextract,calculatedfrom linearregressionequationwas5.44%(±0.18).

Conclusion

FortheanalysisofcatechininP.pseudocaryophyllus,asensitive andaccurateHPLC-PDAmethodwasdeveloped.Themethodwas validatedaccordingtotheICHguidelinesandBrazilianregulations. Theresultsprovedthatthismethodissuitableforitspurpose,and thuscanbeusedinthequalitycontrolofP.pseudocaryophylluscitral chemotypespray-driedextract.

Authors’contributions

LCS contributed by runningthe laboratorywork, chromato-graphicanalysis, and analysis of the data and also draftedthe paper.RDM contributed tothelaboratorywork and chromato-graphic analysis.DRS contributed to chromatographic analysis. VCSAcontributedtocriticalreadingofthemanuscript.JRPandECC contributedbyprovidingthestandardcatechinandwiththecritical readingofthemanuscript.JAMPcontributedbyrunninglaboratory work,anddesignedthestudy,supervisedthelaboratoryworkand contributedtothedraftingandcriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors are grateful to CNPq, CAPES, and Fundac¸ão de AmparoàPesquisadoEstadodeGoiásfortheirfinancialsupport.

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