Bressana, Fábio Vieira da Silva Jr.a, Osmar Lameirac, Marcelo Barcellos da Rosaa,b, ∗

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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

Some

triterpenic

compounds

in

extracts

of

Cecropia

and

Bauhinia

species

for

different

sampling

years

Marcella

Emilia

Pietra

Schmidt

a

_,

_Fernanda

_Brum

_Pires

b

_,

_Lucas

_Paines

_Bressan

a

_,

Fábio

Vieira

da

Silva

Jr.

a

_,

_Osmar

_Lameira

c

_,

_Marcelo

_Barcellos

_da

_Rosa

a,b,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Química,_Universidade_Federal_de_Santa_Maria,_Camobi,_Santa_Maria,_RS,_Brazil

b_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Universidade_Federal_de_Santa_Maria,_Camobi,_Santa_Maria,_RS,_Brazil c_Embrapa_Amazônia_Oriental,_Belém,_PA,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9October2017 Accepted24November2017 Availableonline12December2017

Keywords: HPLC-UV Validation Triterpenes

Whiteandpinkpata-vaca Redandwhite-embaúba Amazonianmedicinalplants

a

b

s

t

r

a

c

t

Theaimofthispaperistoprovideanoverviewonthechemicalcompositionoftriterpenesinwidespread usedfolkmedicinespecies,throughthedevelopmentandvalidationofelevencompoundsusing HPLC-UVdetection.Thecompoundswereseparatedusingisocraticelution,onareversephasecolumn(Kinetex C18,250mm×4.6mm,5␮m)withmobilephaseconsistedofacetonitrile:tetrahydrofuran(90:10,v/v), flow-rateof0.5ml/minanddetectionin210nm.Diversevalidationparametersweresuccessfully eval-uated.ThesamplesofBauhiniavariegataL.,B.variegatavar.candidaVoigt,Fabaceae,Cecropiapalmata Willd.andC.obtusaTrécul,Urticaceae,collectedin2012,2013and2014fromAmazonweretreatedwith twodifferentsolvents(ethylacetateandchloroform)andanalyzedbytheproposedmethod. Stigmas-terol,lupeol,␤-sitosterol,␤-amirinand␣-amirinwerefoundinallthestudiedplants.Highlightingthe presenceofoleanolicacid,maslinicacidinC.obtusaandC.palmataextracts,erythrodiolonlyinC. pal-mata,stigmasteolinB.variegataand␣-amirininB.variegatavar.candida.Overall,ethylacetateshowed betterperformanceastheextractorsolventthanchloroform.Moreover,itcouldbeusedforthequality controlofmedicinalplantsandtoassesspotentialmarkercompounds.

Introduction

Triterpenes,presentsinvegetableoils,cereals,fruitsandbarkof treesarewidespreadinthehumandiet(Rhourri-Frihetal.,2009; Saleem,2009;SiddiqueandSaleem,2011;Szakieletal.,2012)and one of thelargestclasses of secondary metabolites, withmore than30,000differenttriterpenes reported(Muffleret al.,2011; Thimmappaetal.,2014).

Thislargenumberofcompoundsisrelatedtotheversatilityof theirstructure,consistedofacycles,bi-,tri-,tetra-andpentacycles (Dias et al., 2011; Muffler et al., 2011). Among those, penta-cyclictriterpenespresentedpromisingpharmacologicalproperties (Szakieletal.,2012;GhoshandSil,2013;Shanmugametal.,2013) suchas,anti-inflammatory(Saleemetal.,2008;Martelancetal., 2009), hepatoprotector (Kumari and Kakkar, 2012; Pollier and Goossens, 2012), anti-tumor (Saleem, 2009; Shanmugamet al., 2013), anti-viral(Sánchez-Ávilaet al.,2009; Kong etal., 2013),

∗ Correspondingauthor.

E-mail:marcelo.b.rosa@ufsm.br(M.B.Rosa).

anti-HIV(Chenget al.,2011; Wójciak-Kosioret al.,2013), anti-microbial (Pai et al., 2011), anti-fungal (Rocha et al., 2004), anti-diabetic (Manna et al., 2010), gastroprotective (Sánchez et al., 2006; Quílez et al., 2010), anti-hyperlipidemic (Claude et al., 2004), neuroprotector (Silva et al., 2011), antiarthritic (SiddiqueandSaleem,2011),antioxidant(Alloucheetal.,2010), cholesterol-reducingproperties(Chauhanetal.,2013), cardiopro-tective(Somovaet al.,2003)andtrypanocidal activity(Ferreira etal.,2010).

Medicinalplantshavebeenusedforcenturiesinfolkmedicine associatedwithhealthpromotion,preventionandcureofhuman diseases(Laszczyk,2009;Romeroet al.,2010).Withmorethan 50,000plantspecies,Brazilhasthegreatestlevelsofbiodiversity intheplanet(Giuliettietal.,2005)andAmazoniaisaregionwith oneoftherichestfloraintheworldwithalargepotentialdiscovery andresearchofnewdrugs(Giuliettietal.,2005;Coelho-Ferreira, 2009).Despitethediversityandthewidespreaduseof phytother-apicsinBrazilthescientificknowledgeaboutthisfloraproperties islimited(Figueredoetal.,2014).

BauhiniavariegataL.,popularknowninBrazilas“pata-de-vaca” or “unha-de-boi”, member of the Fabaceae family, it has been

https://doi.org/10.1016/j.bjp.2017.11.005

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usedinpopularmedicineasaresultoftheirhypoglycemic(Silva and Filho, 2002;Parekhet al., 2006; Murillo etal., 2007; Silva etal.,2007),anti-cholesterol,anti-elephantiasis(Silvaetal.,2007), antibacterial(SilvaandFilho,2002;Parekhetal.,2006),anti-tumor (Rajkapooretal.,2003),antifungal(SilvaandFilho,2002;Rajkapoor etal.,2003),diuretic,tonic,depurativeactivities(Pizzolattietal., 2003), while alsobeing usefulagainst skindiseases and ulcers (Reddyetal.,2003)inbronchitis,leprosy(Rajkapooretal.,2003), andinthemanagementofinflammatorydiseases(SilvaandFilho, 2002;Raoetal.,2008).

AnothermedicinalplantsincludedinthisstudyisCecropia pal-mata Willd. and C. obtusaTrécul,popularly known in Brazilas “embaúba-vermelha”and“embaúba-branca”fromtheUrticaceae family,traditionalusedasanti-rheumatic(Silvaetal.,2007), anti-inflammatory(Rochaetal.,2007;Costaetal.,2011;Nicasio-Torres etal.,2012;Pelaezetal.,2013),anti-oxidantactivities( Nicasio-Torresetal.,2012),anti-tumor(Rochaetal.,2007),actincentral nervoussystem,includinganxiolyticandantidepressant-like activ-ities(Silvaetal.,2007;Costaetal.,2011),againstasthma,highblood pressure(Costaetal.,2011),aswellusedinthetreatmentoftype 2diabetes(Rochaetal.,2007;Nicasio-Torresetal.,2012;Pelaez etal.,2013).

This wide diversity of pharmacological properties reported inCecropia andBauhiniaisrelated tosecondarymetabolites, as flavonoids,phenolicacids,carotenoids,tocopherols,alkaloids, lig-nans,tannins,salicylates,glucosinolatesandtriterpenes(Szakiel etal.,2012).

Severalpapersdescribedthedetectionandseparationof triter-penes in medicinal plants. Some methods include preparative thin-layerchromatography(TLC)(Martelancetal.,2007; Marte-lancet al.,2009), gaschromatography (GC) withderivatization step(Zhangetal.,2012), capillaryelectrophoresis(CE)(Cheung and Zhang, 2008; Li et al., 2011), evaporative light-scattering detectors (Lesellier et al.,2012), high-performance liquid chro-matography(HPLC)(Martelancetal.,2009;Lietal.,2011;Lesellier etal.,2012;Zhangetal.,2012)withUVdetectorormass spectro-metricdetectorsusingatmosphericpressurechemicalionization (APCI)atmosphericpressurephotoionization(APPI)and electro-sprayionization(ESI)(Sánchez-Ávilaetal.,2009).Thesimultaneous determinationof triterpenes rendera difficulttaskconsidering, theirsimilarstructure,lackofchromophores,verylowUV absorp-tionandsimilarpolarity.Accordingtotheliterature,thereisonly onepublishedreportthatseparatesmorethansixtriterpenesin a singleHPLC run (Bedneret al., 2008;Martelanc et al., 2009; Sánchez-Ávilaetal.,2009;Lietal.,2011;SlavinandYu,2012;Li etal.,2013).

Inthisstudythedevelopmentofa methodforsimultaneous determinationofeleventriterpeneswithisocraticelutionandUV detectionwasproposed. Thedevelopedmethodwasappliedto theanalysisoffourdifferentmedicinalplantsfromtheAmazon region(Cecropiaobtusa,C.palmata,B.variegataandB.variegatavar.

candida)HPLCwithUVdetection.

Materialsandmethods

Chemicals

All the chemical standards used, ␣-amirin (98%), ␤-amirin (98.5%),␤-sitosterol(85%),stigmasterol(95%),lupeol(90%),uvaol (95%),erythrodiol(97%),oleanolicacid(97%),betulinicacid(97%), arjunicacid(88%)and maslinicacid(95%)usedwereof analyti-calgradefromSigma–Aldrich(St.Louis,MO,USA).Thesolvents acetonitrile(ACN),methanol(MeOH)andtetrahydrofuran(THF) wereofHPLCgradefromTedia(Fairfield,OH,EUA).Chloroform (CHCl3)andethylacetate(EtOAc)wereofanalyticalgradefrom

Merck (Darmstadt, Germany). Stock solutions of ␣-amirin, ␤ -amirin,uvaol,erythrodiol,oleanolicacid,arjunicacidandmaslinic acid1000mg/l, stigmasterol481mg/l, lupeol365mg/l, betulinic acid196mg/land␤-sitosterol873mg/lwerepreparedinmethanol. Theworkinganalyticalsolutionsforanalyticalcurvewereobtained bydilutingtheanalyticalsolutionsinacetonitrilewiththe follow-ingconcentrations0.7,6.5,12.2,18.0,23.6,29.3and35.0mg/l.All thesolutionswerestoredat−20◦_C_until_analysis.

HPLC-UVanalysis

ChromatographicmeasurementswereperformedonaDionex®

modelP680(Sunnyvale,CA,USA)liquidchromatographequipped withaUV-visdetectormodelUVD170U,Rheodyne®

injectionvalve model 8125 (Cotati, CA, USA) with loop of 100␮l. The analy-seswerecarriedoutwithaKinetexreversed-phaseC18 column

(250mm×4.6mm,5␮mparticlesize;Phenomenex,Torrance,CA, USA) which was precededby a Security Guard C18.pre-column

(Phenomenex,Torrance,CA,USA).Themobilephaseconsistedof acetonitrile:tetrahydrofuran(90:10,v/v)andtheflow-ratewasset at0.5ml/min.Spectrophotometrydetectionofanalyteswas per-formedat210nmwavelengths.Evaluationandquantificationwere madeonaChromeleon6.7Workstation.Thesamesampleswere previouslystudiedalsobyperforminganultra-high-performance liquid chromatography – atmospheric pressure photoionization sourcemassspectrometry(UHPLC-APPI-MS/MS).Therefore,a com-parisonofthespeciesdetectedusingHPLC-UVwasperformedby UHPLC-APPI-MS/MS,describedindetailsbyGoboetal.(2016).

Plantmaterial

ThemedicinalplantspeciesBauhiniavariegataL.(depositn◦_IAN

185932),B.variegatavar.candidaVoigt(depositn◦_IAN_185831), CecropiaobtusaTrécul(depositn◦_IAN_185555)_and_C._palmata_Willd

(depositn◦ _IAN _185556)_were_obtained_from_the_herbal

collec-tionoftheBrazilianAgriculturalResearchCorporation,Embrapa AmazôniaOriental,Belém,PA,Brazil.Thegeographicallocationof thecollectionsiteis1◦₂₇′₂₁′′_S_latitude_and₄₈◦₃₀′₁₄′′_W_longitude.

TheAmazonregionhasahotandhumidcharacteristicclimate withsmalltemperaturegradients.Thereare twowellestablish seasonsinthatregion,adry-period(July–October)andrainy sea-son (December–May); the months of June and November are considered transition periods (Ananias et al., 2010). According

Gobbo-NetoandLopes(2007)thereisapositiveinfluenceof rain-fall ontheconcentration of secondary metabolites(cyanogenic glycosides,glucosinolates,terpenes,anthocyaninsand alkaloids) therefore,thesamplesstudiedwerecollectedduringtherainy sea-soninthreedifferentyears(2012,2013and2014).

Samplepreparationandextractionprocedure

Thefreshplantspecimenswerecleaned,driedat40◦_C_for₁₂_h,

groundintoafinepowderinalaboratorymillandusedasadry powderedmaterial.Allplantswerereceivedasafinepowdered driedleafmaterial.Driedsampleswerestoredindesiccatorsunder vacuumatroomtemperatureuntilsampletreatment.

Ultrasound-assisted extraction was performed in a reactor thermostaticwater bath(temperatureaccuracy of±1.0◦C).The

experimental setup consists of an ultrasonic bath USC 1800A (UniqueInc.,Brazil,BR)equippedwithatransducerwith longitu-dinalvibrations.Theultrasonicunithasanoperatingfrequencyof 40kHzandamaximum-ratedultrasoundpoweroutputof132W. Theultrasonictransducer(surfaceareaof282.2cm2₎_is_fitted_at_the

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andsonicatedfor30minat37◦_C._Extraction_was_carried_out_three

timeswithfreshportionsofsolventintheaboveconditions(Pai etal.,2011;Wójciak-Kosioretal.,2013).Theextractremainedwas driedwithN2anddissolvedin10mlofmobilephase.Allthe

sam-plesweredilutedtoa2%(m/v)andfilteredthroughChromafilXtra PEFT-20/25filtersfromMacherey-Nagel(Düren,Germany)before injection.Forconstructionofthecalibrationcurves,sevendifferent mixedsolutionswereinjectedinthreereplicates.

Validationprocedure

Theanalyticalmethodwasvalidatedforlinearity,limitof detec-tion(LOD),limitofquantification(LOQ),accuracy,precisionand robustnessfollowingtheREn◦_899/2003₍_Anvisa,₂₀₀₃_)._The

cal-ibration curves were preparedusing sevenconcentrations (0.7, 6.5,12.2,18.0,23.6,29.3and35.0mg/l)ofthe11stockstandard solutionsintherange0.21–40mg/lwhichwereinjectedin trip-licate. The mean peak areas were taken for the construction thecalibrationcurve.Thedata wereanalyzed bylinear regres-sionleastsquaremodel.TheLODandLOQ,underthepresented chromatographicconditions weredeterminedvisually.The pre-cisionwasdetermined byintraand inter-dayvariation andthe recoverywereevaluatedbystandardadditionmethod,the vari-ationswereexpressedbyRSD %asthefollowing formula:RSD (%)=(detected amount−original amount)/amount spiked×100.

Allsolutionswerekeptat−20◦_C_before_analysis.

Resultsanddiscussion

HPLCanalysis

Basedinmethodsreportedintheliterature(Martelancetal., 2007;Martelancetal.,2009;Sánchez-Ávilaetal.,2009;Yangetal., 2009;Lietal.,2013),chromatographicconditionswereoptimized inordertoobtainthebestseparationoftheanalytesintheshortest time.Themainvariablesevaluatedinthechromatographic sepa-rationwereratioofthemobilephase,flow-rateofmobilephase, temperatureoftheoperationalroomanddetectionwavelength.

Table1showsthesevariables,therangesstudiedandtheir opti-mumvaluesaswellasthesolventsusedintheoptimizationofthe mobilephasecomposition.

Table1

Optimization of the chromatographic conditions for separation of triterpenic compounds.

Variable Tastedvalue Optimumvalue

Flowrate(ml/min) 0.5,0.8and1.0 0.5

Roomtemperature(◦_C) _21.0_and_23.0 _21.0

Compositionmobilephase

MeOH:H2O 90:10(pH3.0)

80:20(pH3.0)

ACN:H2O 90:10(pH3.0) ACN:THF(90:10)

THF:ACN:H2O 30:60:10

ACN:THF 90:10

Wavelength(nm) 200,205,210,220

and225

210

ACN,acetonitrile;MeOH,methanol;THF,tetrahydrofuran;H2O,water.

Table2

Molarabsorptivitycoefficient(ε)inl/molcmoftriterpenesindifferentwavelengths.

Compound ε200nm ε210nm ε220nm

Arjunicacid 2.4·104 1.6·104 6.4·103

Betulinicacid 5.0·103 5.7·103 2.2·103

Erythrodiol 7.1·104 2.8·104 1.2·104

Lupeol 1.2·105 6.1·104 3.1·104

Maslinicacid 2.2·105 1.5·105 7.1·104

Oleanolicacid 1.6·105 9.7·104 5.4·104

Stigmasterol 1.1·105 5.0·104 2.8·104

Uvaol 1.1·105 5.1·104 2.9·104

␣-Amirin 1.4·105 8.1·104 4.7·104

␤-Amirin 1.3·105 4.6·104 2.4·104

␤-Sitosterol 1.3·104 7.2·104 4.3·104

ThebestmobilephasewasACN/THF(90:10;v/v).Theoptimum flowrateandroomtemperaturewere0.5ml/minand21◦_C, respec-tively.Thedetectionwavelengthwaschosenat210nmaccordingly toexperimentaldata(Table2)and theliterature(Holen,1985; Schneideretal.,2009;Romeroetal.,2010;SlavinandYu,2012; Xuet al.,2012; Zhanget al.,2012)due tobetterabsorptionat the selected wavelength. The representative chromatogram for standard solutions under the proposed conditions is shown in

Fig.1.Itisnoteworthytomentionthatthecompleteseparation oftheeleventriterpenescouldbeachievedin45min,comparedto otherseparationmethods,itcanbesaidthatthenew methodol-ogydevelopedseparatesmorecompoundsthantheothersreported

120 _mAU

Arjunic acid

Maslinic acid

Betulinic acid

Oleanolic acid

Er

ythrodiol

Lupeol

a-amir

in

b-amir

in

Stigmasteol _{b - stigmasteol}

Uv

aol

100

80

60

40

20

-20 0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

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Table3

AnalyticalparametersforthevalidatedmethodbyHPLC-UV.

Compound LOD(mg/kg) LOQ(mg/kg) Linearrange

(mg/kg)

Correlation

coefficient(r2₎ Intra-day_(RSD%) precision Inter-day_(RSD%) precision Accuracy(%)

␣-Amirin 0.37 0.75 0.74–40.0 0.996 3.60 3.21 95–110

␤-Amirin 1.04 2.12 0.74–40.0 0.996 2.73 2.15 100–105

Oleanolicacid 0.22 0.43 0.74–35.0 0.990 8.92 3.65 98–102

Betulinicacid 0.09 0.20 0.74–35.0 0.996 9.38 3.40 93–97

␤-Sitosterol 0.08 0.16 0.74–40.0 0.992 3.95 3.19 97–110

Stigmasterol 0.06 0.13 0.74–40.0 0.991 2.01 2.12 85–96

Lupeol 0.12 0.25 0.74–40.0 0.996 2.43 2.54 92–102

Uvaol 0.12 0.24 0.74–35.0 0.991 2.38 2.29 95–108

Arjunicacid 0.07 0.15 0.74–35.0 0.990 4.01 3.75 97–103

Maslinicacid 0.13 0.27 0.74–40.0 0.996 4.39 4.40 96–99

Erythrodiol 0.08 0.17 0.74–35.0 0.994 2.16 1.85 90–96

LOD,limitofdetection;LOQ,limitofquantification;RSD,relativestandarddeviation.

A

B

Bauhinia variegata

Bauhinia variegatavar.candida

Maslinic acid

Stigmasterol

Lupeol

Stigmasterol

Lupeol

Chlorof

or

m

Chlorof

or

m

Chlorof

or

m

Eth

yl acetate

Eth

yl acetate

Eth

yl acetate

2012 2013 2014

Chlorof

or

m

Chlorof

or

m

Chlorof

or

m

Eth

yl acetate

Eth

yl acetate

Eth

yl acetate

2012 2013 2014 10000

1000

100

10

1

1000

Concentr

ation (mg/kg)

Concentr

ation (mg/kg)

100

10

1

Fig.2.TriterpeniccompoundsfoundintheBauhiniaspecies viaHPLC-UV(A) Bauhiniavariegataand(B)B.variegatavar.candida.

inliteratureonisocraticmode(Liuetal.,2003;Ávilaetal.,2007; Razborˇseketal.,2007;CheungandZhang,2008).

Methodvalidation

Thecalibrationcurvesshowedgoodlinearityandthe correla-tioncoefficientswerefoundintherangeof0.990–0.996forallofthe testedcompounds.Therecoveriesoftheelevenanalyteswereinthe rangeof85–110%.Therelativestandarddeviation(RSD)between samplemeasurementswasusedasprecision,varyingfrom2.01 to9.37%forintradayprecisionandfrom1.84to4.04%tointerday forprecision.Robustnesswastested intermsofflowdifference (±0.1ml/min),%mobilephase(±5%THF),andwavelength(±1nm).

Thevariationdoesnotexceed20%.Overall,theseresults demon-stratethatthedevelopedmethodhasenoughaccuracy,precision, andsensitivityforthesimultaneouslyquantitativeanalysisofthe elevencompounds.Therecoveryresultsforevaluatingtheaccuracy ofthemethodweresatisfactoryaccordingtotheAnvisa(2003),

Maslinic acid Oleanolic acid

B

Cecropia obtusa

Cecropia palmata

Stigmasterol

Lupeol

Maslinic acid Oleanolic acid

Stigmasterol

Lupeol

Concentr

ation mg/kg

Concentr

ation mg/kg

10000 10000

A

1000

100

10

1 1 1000

100

10

Chlorof

or

m

Chlorof

or

m

Chlorof

or

m

Eth

yl acetate

Eth

yl acetate

Eth

yl acetate

2012 2013 2014

Chlorof

or

m

Chlorof

or

m

Chlorof

or

m

Eth

yl acetate

Eth

yl acetate

Eth

yl acetate

2012 2013 2014

Fig.3.TriterpeniccompoundsfoundintheCecropiaspecies viaHPLC-UV(A) Cecropiaobtusaand(B)Cecropiapalmata.

whichallowsarangeof80–120%foramazonplantsextracts.The obtainedvalidationparameterssuchascorrelationcoefficient, lin-earrange,recovery,LODandLOQaresummarizedinTable3.

ApplicationtoBauhiniasamples

Previous studies in phytochemical analysis reported the presence of glycosides, triterpene, flavonoid,lactones, steroids, alkaloids,coumarinsandsaponinsinBauhiniaspecies(Silvaand Filho, 2002; Pizzolatti et al., 2003; Rajkapoor et al., 2003; Rao etal.,2008).Sampleanalysis(Fig.2)indicatedthepresenceofsix (maslinicacid,stigmasterol,lupeol,␤-sitosterol,␤-amirinand␣ -amirin)outoftheelevencompoundsintheBauhiniaspecies.The presenceofthefourcompoundspreviouslylisted,usingthesame sample, was also confirmed previously by UHPLC-APPI-MS/MS analysis(Goboetal.,2016).

Bauhinia variegata shows greater amounts of triterpenes in

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lupeol (+35%) and ␤-sitosterol (+13%) in comparison with the chloroform.

ApplicationtoCecropiasamples

Regardingthestudiedcompounds,C.obtusa(Fig.3)exhibited a greater diversity when compared tothe otherspecies ofthe samegenus analyzedC. palmata.Among theeleventriterpenes separatedinthismethodoleanolicacid,maslinicacid,␤-amirin, stigsmasterol,lupeoland␤-sitosterolwerefoundinthesesamples.

␤-amirin,lupeoland␤-sitosterolwerealsoidentifiedby UHPLC-APPI-MS/MSanalysis(Goboetal.,2016).

Noteworthy is thepresence of␤-sitosterol (+55%),␣-amirin (+60%),␤-amirin(+60%)andoleanolicacid(+20%),increasing con-centrationsalong theyears, and theethyl acetatewas a better extractsolventthanchloroformforthesecompounds.This obser-vation can be a result of some environmental factors such as ultravioletradiation,rainfall,temperature(Gobbo-NetoandLopes, 2007).

Conclusions

Anewanalyticalmethodhasbeendevelopedforthe simulta-neousidentificationandquantificationoftriterpenescompounds usingHPLC-UVandisocraticelution,toanalyzethesecompounds

inCecropiaand Bauhiniaspecies.TheHPLC-UVmethodis

effec-tivetoseparateandquantifythemedicinalplantsextractswith good validationparameters, suchaslinearity, LOD,LOQ, recov-ery, accuracy, precision,robustness and repeatability.Although triterpenoidscontributesignificantlytothebioactivityand phar-macologyofBauhiniaandCecropia,nostudywasreportedsofar forthequantitativedetermination ofthesecompoundsinthese folkmedicineplants.Triterpenescompoundssuchasmaslinicacid, oleanolicacid,␣-amirin,␤-amirinand␤-sitosterolwerefoundas majorcompoundsinchloroformandethylacetateextracts. Fur-thermore, the presence of these triterpenic compounds in the extractsreinforcesthepharmacologicalaction,andthemedicaluse ofsuchplantsinfolkmedicine.Basedontheresults,thespecies withthehighestvarietyofcompoundsandconcentrationwereB. variegataandC.obtusa.Thepresentmethoddevelopedcanbeused inresearchofchemicalmarkersinmedicinalplantsaswellasin thequalitycontrolofherbalmedicineswidelyusedinBraziland infolkmedicine.TheidentifiedcompoundsinBauhinia(lupeol,␤ -sitosterol,␤-amirinand␣-amirin),aswellinCecropia(␤-amirin,

lupeoland␤-sitosterol)extractswerealsoobservedandusingthe samesampleby HPLC-APCI-MS/MSanalysis,therefore,ratifying theimportanceofthiswork.

Authors’contributions

MEPS,FBPandLPBdevelopedandtestedtheproposedmethod. FVJr. gavea graphical andcalculationsupport.OL collectedthe plantsandperformedthevoucher.MBRwastheadvisorofMEPS, FBP,LPBandFVSJr.Alltheauthorshavereadthefinalmanuscript andapproveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This work was supported by grants from the Brazilian NacionalResearchCouncilCNPq,(processes487028/2012-0and

440132/2014-2).TheauthorsalsothankEmbrapaAmazônia Ori-ental(Belém/PA)forsupplyingthemedicinalplantsstudied.

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