Rev. bras. farmacogn. vol.25 número3

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RevistaBrasileiradeFarmacognosia25(2015)208–211

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

Development

of

a

rapid

and

simple

HPLC-UV

method

for

determination

of

gallic

acid

in

Schinopsis

brasiliensis

Felipe

H.A.

Fernandes

a

_,

_Rayanne

_S.

_de

_A.

_Batista

b

_,

_Francinalva

_D.

_de

_Medeiros

a

_,

Fábio

S. Santos

b

_,

_Ana

_C.D.

_Medeiros

a,∗

a_Laboratório_de_{Desenvolvimento}_e_Ensaios_de_{Medicamentos,}_Centro_de_Ciências_Biológicas_e_da_Saúde,_Universidade_Estadual_da_Paraíba,_Campina_Grande,_PB,_Brazil

b_{Laboratórios}_Unificados_de_{Desenvolvimento}_e_Ensaios_de_{Medicamentos,}_Departamento_de_Ciências_{Farmacêuticas,}_Centro_de_Ciências_da_Saúde,_Universidade_Federal_da_Paraíba,

JoãoPessoa,PB,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17December2014 Accepted19May2015 Availableonline11June2015

Keywords:

Gallicacid Chemicalmarker Validation

Schinopsisbrasiliensis

a

b

s

t

r

a

c

t

Theaimofthisworkwastodevelopandvalidateananalyticalmethodfortheidentificationofthe chem-icalmarkerofSchinopsisbrasiliensisEngl.,Anacardiaceae.Itwoulddeterminethetotalpolyphenolsand flavonoidcontentbyspectrophotometricmethodologyinthedriedextractofplant.Thechromatographic profilesofS.brasiliensisweredeterminedusingHPLC-UV.Theliquidchromatographymethodwas con-ductedonaPhenomenexGeminiNXC18column(250×4.6mm,5␮m).Themobilephaseconsisted of0.05%orthophosphoricacid:methanol.Theflowratewas1ml/minandeffluentsweremonitoredat 271nm.Theretentiontimeforgallicacidwas8.5min.Thedescribedmethodhastheadvantageofbeing bothrapidandeasy.Henceitcanbeappliedforroutinequalitycontrolanalysisofherbalpreparation containingS.brasiliensis.

Introduction

Polyphenolsarea groupofsecondarymetabolitespresent in differentplants. In generalpolyphenols are classifiedinto four classes:phenolicacids,flavonoids,lignansandstilbenes.Theseare veryimportanttothepharmaceutical,cosmeticandfood indus-tries,mainlyfortheirantioxidantactionandproperties(Gharras, 2009).

Schinopsis brasiliensis Engl., Anacardiaceae, is a plant which belongs to the Anacardiaceae family, known as “baraúna” or “bráuna,”andcanbefoundintheBraziliansemi-aridregions.Their shellshaveethnopharmacologicalusesforthetreatmentof diar-rheaandcoughs,andcanalsobeusedasanantisepticandanalgesic (Chavesetal.,2013).Severalstudieshavedemonstratedthe antimi-crobialactivityofS.brasiliensisagainstPseudomonasaeruginosa,

Staphylococcusaureus,Escherichiacoli,Klebsiellapneumoniaeand

Candidaalbicans(Chavesetal.,2011;Saraivaetal.,2011;Silvaetal., 2012);Salmonellatyphimurium(Saraivaetal.,2011);Streptococcus oralis,Streptococcusmutans,Streptococcusparasanguinisand Entero-coccusfaecalis(Silvaetal.,2012).Otherstudiesincludedmulti-drug

∗ Correspondingauthor.

E-mail:[email protected](A.C.D.Medeiros).

resistantstrainsfortetracycline,gentamicinandoxacillin(Saraiva etal.,2011,2013)anderythromycin(Silvaetal.,2015).

The current list of compounds found in S. brasiliensis

leavesincludes the presence of tannins and polyphenols, such as methyl gallate, gallic acid, ellagic acid and 5,6,7,8,3′_,4′

hexahydroxy-flavonol (Saraiva et al., 2011). New alkylphenol methyl6-eicosanoyl-2-hydroxy-4-methoxybenzoate and steroid 5␣,8␣-epidioxyergosta-6,22-dien-3␤-olarebothcompounds iso-latedfromthe barkofS. brasiliensis(Cardosoet al.,2005).The essentialoilextractedfromtheleavesofS.brasiliensishasagood amountofmyrceneandlowamountsofothercompoundssuchas ␤-caryophyllene,eucalyptolandguaiol(Donatietal.,2014).

Oneimportantaspectforthequalitycontrolofherbalmedicine productsisthedeterminationofthechemicalmarker,which con-sistsofaconstituent(orgroupsofconstituents)thatischemically definedandpresentintheplantproduct.Theseconstituentsmay ormaynotberelatedtotheirpharmacologicalactivity(Marques etal.,2013).Whileseveraldifferentanalyticaltechniquescanbe usedtoidentifyandquantify thesesubstances,we useda high performanceliquidchromatography(HPLC),whichisthemethod mostcommonlyusedbythevariousofficialstandards(Zollnerand Schwarz,2013).

Theaimofthisworkwastodevelopandvalidateananalytical methodusingHPLCfortheidentificationandquantificationofthe chemicalmarkersofS.brasiliensis.

http://dx.doi.org/10.1016/j.bjp.2015.05.006

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F.H.A.Fernandesetal./RevistaBrasileiradeFarmacognosia25(2015)208–211 209

Materialsandmethods

Rawmaterial

Thestem bark ofSchinopsis brasiliensisEngl., Anacardiaceae, wascollectedin July2013fromtheregionof CampinaGrande, stateofParaiba(7◦₁₃′₅₀′′_S,₃₅◦₅₂′₅₂′′_W)._The_voucher_specimen

waspreparedandidentifiedbyProfessorJaymeCoelhodeMorais Herbarium,UniversidadeFederaldaParaiba,undertheEAN-14049. Theherbaldrugsweredriedat40◦_C_using_an_air_oven._After

drying,thematerialwasreducedinaknifemill(10meshoutlet sieve)toafinepowder.Thehydroalcoholicextractwasobtainedby macerationofthepowderinawater:ethanol(30:70,v/v)solvent systemfor72handthendriedusingSprayDryer(LabPlant®_),_with

onsettemperatureof140◦_C_and_under₃_ml/min_flow_rate._Colloidal

silicondioxide(Aerosil®₂₀₀₎_was_used_as_a_drying_agent_at_20%

(w/w).Thedryingextractwasweighted,protectedfromlightand storedinclosedflasksatroomtemperature.

Determinationoftotalpolyphenols

TheFolin–Ciocalteumethodwasusedtodeterminethetotal polyphenols,usinggallicacidasastandard(Chavesetal.,2013).A samplewasdissolvedinwaterwith0.5mlofFolin–Ciocalteu solu-tion(Sigma–Aldrich)andleftatrestfor2min.Thenitwasaddedto 1mlofsodiumcarbonatesolution20%(w/w)andwaslefttostand for10min.Thecalibrationcurvewaspreparedwithagallicacid standardindifferentconcentrations.Thesamplesweremeasured usingaUV–VisSpectrophotometerShimadzuUV-Mini1240,with awavelengthof757nm.Theresultswereexpressedasmicrograms ofgallicacid/mgofextract(␮gGAE/mgextract).

Determinationoftotalflavonoids

The aluminum chloride method wasused to determinethe flavonoidcontentusingaquercetinstandard(Chavesetal.,2013). Initiallyanaliquotoftheextractwasdissolvedinmethanol.Then theextractwasmixedwithanaluminumchloridesolution(2%in methanol)inaratioof1:1andlefttostandfor10min.The cali-brationcurvewaspreparedwithaquercetinstandardindifferent concentrations.WeusedaUV–VisSpectrophotometerShimadzu UV-Mini1240,withawavelengthof415nmtoanalyzeour sam-ple.Theresultswereexpressedasmicrogramsofquercetin/mgof theextract(␮gQE/mgextract).

Chemicalmarker

Chromatographyanalysisforthequantificationofthechemical markergallicacidinS.brasiliensiswasconductedonaShimadzu®

HPLCmodelLC-10controlledbyClassVPSoftwareandwith qua-ternarypumps(LC-10AD),anon-linedegasser(DGU-14A),andan injectionvalve(Rheodyne®_LLC)_with_a₂₀_␮_l_loop._The_equipment

includeda UV–Visdetector(SPD-10A). The columnused wasa PhenomenexRP-GeminiNXC18(250mm×4.6mm,5␮m)andit wasprotectedbyapre-columnGeminiNXC18(10mm×4mm).

Thechromatographicseparationwascarriedoutusingamobile phasewithphosphoricacid:water0.05%assolventAandmethanol assolventBataflowrateof1ml/min.Thegradientprogramwas asfollows:90–10%B(10min),70–30%B(3min),40–60%B(5min), 60–40%B(3min), 80–20%B(3min) and90–10%B(6min).The peaksweredetectedat271nmandwereidentifiedbycomparing theretentiontimewithstandardgallicacid.

Thevalidationoftheanalyticalmethodhasbeenmadewhile takingintoaccountthefiguresofmeritsuchasprecision,accuracy, linearity,limitofdelectationandlimitofquantification(Marques etal.,2013;Ribanietal.,2004;PedrosoandSalgado,2014).

Table1

PolyphenolandflavonoidcontentsofSchinopsisbrasiliensis.

Totalpolyphenols (␮g/mgextractGAE)

Totalflavonoids (␮g/mgextractQE)

Concentration 24.52 1.43

% 15.08 1.44

The linearity was evaluated from the calibration curve, found=withastandardsolutionofgallicacidwithaconcentration between2.5and15␮g/ml,andobtainedthelinearregression equa-tiony=ax+b.AccordingtoRibanietal.(2004),verificationofthe datafitcanbeobservedbythecorrelationcoefficientand determi-nation.TheOfficialCompendiumrecommendsavaluegreaterthan 0.99.

Resultsanddiscussion

Thetotalpolyphenolcontentisquitehighwhencomparedto thetotal flavonoids(Table1).Boththeseclasses ofcompounds arepresentinvariousmedicinalplantsandarerelatedto antiox-idants,antimicrobial,andanti-inflammatoryactivities(Corradini etal.,2011).Althoughweusedaquantitativemethodtodetermine thesecompounds,thistechniquecannotpredictthecompositionof eachpolyphenol,sinceitmaypossiblyalsomeasurenon-phenolic compounds(Robyetal.,2013).

Thepolyphenolswerepresentingreaterquantityintheplant whenweanalyzedthehydroalcoholicextractofS.brasiliensisusing gallicacidasstandard.Gallicacid(3,4,5-trihydroxybenzoicacid)is animportantpolyphenolpresentinplantsandisacompoundwith importantapplicationsinthesynthesisofthedrugTrimethoprim, asafoodpreservative,andanantioxidantinoilsandproductsrich inlipids(Battestinetal.,2004).

Inthepreliminaryanalysis,wedetectedthepresenceofthese compoundclassesinlargequantities.Basedontheseresults, gal-lic acidwaschosenas a markerandwe proceededtoquantify andvalidatethemethodsbymeansofthestandardaddition.For thistest,weusedhighperformanceliquidchromatographywitha UV–Visdetector,asitisthemethodmostsuitableforthistypeof compounds(Arapitsas,2012).

Fig.1wasobtainedfromthechromatogramoftheextractofS. brasiliensis,whichshowsthepeakofgallicacidanditsstandard. Theretentiontime was8.5min,inadditiontopeakspresentin othercompoundsofthephytocomplex.Goodlinearity(R2=0.9984) wasachievedwithintheinvestigatedrangesintheextractandwas 99.5%oftheexplainablevariance(Fig.2).

Differentstudiesaredescribedintheliterature(Arapitsas,2012) analyzinggallicacidasthemajorcompound;however,the differ-encebetweenthemisthetypeofmobilephaseused.Inastudy ofgallicacidbyDendrophthoefalcataL.f.,DeshmukhandPrabhu (2011)foundaretentiontime ofapproximately12min,usinga mobilephaseofwateracidifiedwithphosphoricacid(0.1%)and acetonitrileintheratioof4:6andaflowrate1ml/min.Inanother workwithSchinusterebinthifoliusRaddi,gallic acidwas quanti-fiedusingtwo phases: onewithwater:methanoland theother with water:acetonitrile. In both cases, the water wasacidified withformicacidtopH2.7.Theretentiontimesunderboth condi-tionswere5min,althoughthephasewithacetonitrilewasslightly shorter(Carvalhoetal.,2009).

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210 F.H.A.Fernandesetal./RevistaBrasileiradeFarmacognosia25(2015)208–211

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Minutes

0.1

0.0 0.2 0.3 0.4 0.5 0.6 0.7

Retention Time Retention Time

OH

HO HO

O

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Minutes

0.00 0.02 0.04 0.06 0.08 0.10

B

V

o

lts

A

V

o

lts

Fig.1.Chromatograms(HPLC/UV)ofextractofSchinopsisbrasiliensis(A)andgallicacid(B)in271nm.

y = 63804x-1280.7

r2_=0.9968

0 7 14

0 500 000 1000 000 1500 000

Area peak

Concentration (μg/ml)

Fig.2. Calibrationplotofgallicacidstandardin271nm.

betterresolutionofthepeaksintheseconditions(Arapitsas,2012; DeSouzaetal.,2002;Lopesetal.,2009).

Through the parameters of the calibration curve it was possible to determine with 95% confidence the limit of detection (LOD=1.22␮g/ml) and the limit of quantification (LOQ=1.82␮g/ml).Theaccuracyofthemethodwasevaluatedby repeatabilityandintermediateprecision(Table2).

Table2

Resultsofprecisionforthetwoanalytes.

Analytes Areapeak RSD(%)

Gallicacid(10␮g/ml) 623,253±14,409.03 2.31

Extract(day1) 616,653.8±17,097.11 2.77

Extract(day2) 627,397±24,066 3.83

Table3

AccuracyofthreerecoverieswithdifferentconcentrationtoextractofSchinopsis brasiliensis.

75% 100% 125%

Recovery(%) 92.83 96.87 111.20

RSD(%) 1.65 0.72 0.65

Theaccuracyofthemethodwasdeterminedbyanalysisofthree fortificationsbyaddingstandardgallicacid(Table3),anditwas determinedthat the methodperforms within acceptablelimits between80%and120%(Limaetal.,2010).AccordingBetzetal., theaccuracytestthroughrecoveryofthematrixiscrucialtothe developmentofmethodologiesforherbalproducts.Therecovery testconsistsofaddingastandardofknownconcentrationafterthe extractionprocessinthematrix(Betzetal.,2011).

Conclusions

Inthisstudy,asimple,accurateandreliablemethodwas devel-opedtodeterminethechemicalmarkerofS.brasiliensisbyusing HPLC-UV.Theresultsdemonstratedthatthemethodshowedgood linearity,recoveryandreproducibilityandlowlimitsofdetection andquantification.Thus,itissuitabletobeusedinthe quantifica-tionofgallicacidinherbalmedicineproducedwithdriedextract ofS.brasiliensis.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

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F.H.A.Fernandesetal./RevistaBrasileiradeFarmacognosia25(2015)208–211 211

researchfellowships.Theauthorsespeciallywishtogivethanks toProfessorIvanCoelhoDantas(inmemoriam)forindicatingthe varietyofplanttobestudiedinthiswork.

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