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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

Effects

of

Acmella

oleracea

methanolic

extract

and

fractions

on

the

tyrosinase

enzyme

Alan

F.

Barbosa

a,∗

,

Keila

C.B.

Silva

b

,

Márcia

C.C.

de

Oliveira

b

,

Mário

G.

de

Carvalho

b

,

Armando

U.O.

Sabaa

Srur

a,c

aDepartmentofFoodTechnology,InstitutodeTecnologia,UniversidadeFederalRuraldoRiodeJaneiro,Seropédica,RJ,Brazil

bDepartmentofChemistry,InstitutodeCiênciasExatas,UniversidadeFederalRuraldoRiodeJaneiro,Seropédica,RJ,Brazil

cDepartmentofBasicandExperimentalNutrition,InstitutodeNutric¸ãoJosuédeCastro,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23July2015

Accepted20January2016

Availableonline23February2016

Keywords: Jambu

Acmellaoleracea

Spilanthol Tyrosinase

a

b

s

t

r

a

c

t

TheaimofthecurrentstudyistoevaluatetheeffectofAcmellaoleracea(L.)R.K.Jansen,Asteraceae,

methanolicextract,hexane(84.28%spilanthol)anddichloromethane(approximately100%spilanthol)

fractionsonthetyrosinaseenzyme.Thedehydratedjambuextractwasobtainedthroughmaceration

usingmethanol.TheextractresiduewassolubilizedinMeOH/H2O(8:2)andsubjectedtoliq.–liq.partition inorganicsolvents.Boththeextractionandthepartitionprocedureswereconductedwiththree repli-cates.TheanalyseswereperformedusingGC–MS,1Hand13CNMR.Thehexanefractionprovidedsamples containing84.28,82.91and62.83%spilantholinrepetitions1,2and3,respectively.Thedichloromethane fractionshowed88.55%spilantholinrepetition1,andapproximately100%spilantholinrepetitions2and 3.Thejambuextractaswellasthehexanefraction(84.28%spilanthol)wereabletoactivatethe

oxidiz-ingactivityofthetyrosinaseenzymeforl-DOPA.Thedichloromethanefraction(approximately100%

spilanthol)showedstrongerinhibitioneffectonthetyrosinaseenzymeinthefirst10min.Theresults raisetheinterestinstudyinspilantholformulationsfortopicaluse,sinceitmaypreventand/orslow

skinhyperpigmentationordepigmentationprocesses.Furthermore,spilantholmaybeusedtocontrol

theenzymaticbrowninginfruitsandvegetables.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Acmellaoleracea(L.)R.K.Jansen,Asteraceae,isanative

Amazo-nianplantpopularlyknownasjambu.Itisoftenusedascondiment

intypicaldishesoftheNorthernBraziliancuisine,suchastacacá

andpato-no-tucupi (duck intucupisauce).It is alsousedinfolk

medicinetotreatstomatitis,coldsandgeneralpain(Nascimento

etal.,2013).A.oleraceaisconsideredtobeanimportantnative

plant in the Amazon region, and it is found in large

cultiva-tionareas(Rebelloand Homma,2005).Spilanthol hasbeenthe

mainmetaboliteoftenisolatedfromA.oleracea.Itisanaliphatic

amidedescribed asaburningviscousoil,which produces

anes-thetic effect and tongue tingling (Molina-Torres et al., 1996),

anditisalsoabletopenetratetheskin(Boonenetal.,2010a,b;

Spiegeleer et al., 2013).Besides Spilanthol’s anti-wrinkleeffect (Demarneand Passaro, 2005), it is alsopossiblementioningits

∗ Correspondingauthor.

E-mail:alanfbarbosa@ufrrj.br(A.F.Barbosa).

diuretic(Ratnasooriyaetal.,2004),fungistaticandbacteriostatic

activities(Molina-Torresetal.,2004),sensoryproperties(Leyetal.,

2006),antisepticactivity,immunestimulation(Rojasetal.,2006),

antioxidantandanti-inflammatoryproperties(Diasetal.,2012),

saliva-secretioninduction(Ramsewaketal.,1999;Sharmaetal.,

2011),analgesic(Riosetal.,2007),andacaricideactivity(Castro

etal.,2014),aswellitsuseagainstskindiseasessuchaseczema

(Boonenetal.,2010a,b).

A.oleraceahasimportantchemicalpropertiesthatawakened

theinterestofthepharmaceuticalindustryduetoitsactive

ingre-dient, spilanthol (Borges et al., 2012). Currently the search for

naturalproductswithinhibitoryactiononmelanizationprocess

hasincreased,focusingonthephenoloxidasetyrosinase.

Tyrosinase,alsoknownaspolyphenoloxidase(PPO),iswidely

distributedinmicroorganisms,animalsandplants.Itcatalyzesthe

oxidation of monophenols, o-diphenolsand o-quinones(Karioti

et al.,2007).Tyrosinase is knownas a key enzyme inmelanin

biosynthesis and it is responsible for melanization in animals

and for browningin plants.Tyrosinase is responsiblefor

enzy-maticbrowningreactionsindamagedfruitsduringpost-harvest

http://dx.doi.org/10.1016/j.bjp.2016.01.004

(2)

handlingandprocessing.Thus,controllingtheenzymatic

brown-ingisessentialduringfruitpulpmanufacturingprocesses(Seoetal.,

2003;Khanetal.,2006).

Tyrosinasesynthesisoccursinsidehighlyspecializedorganelles

calledmelanosomes.Studieshaveshownthepresenceof

tyrosi-nase in all the evaluated melanomas; fact that proves this

enzyme’s importance to the development of this cancer type

(Figueiredo,2003).Theincreasedproductionandaccumulationof

melaninhyperpigmentationmayleadtodisorderssuchasmelasma

(Miotetal.,2009).Manychemicalsandfoodhavedemonstrated

inhibitoryeffectonmelanogenesisthroughtheinhibitionofthe

tyrosinaseenzymeactivity.Suchresulthasincreasedthedemand

fornaturalproducts.Thus,theaimofthecurrentstudyisto

eval-uatethe effectof A.oleraceamethanol extract, hexane (84.28%

spilanthol)anddichloromethane(approximately100%spilanthol)

fractionsonthetyrosinaseenzyme.

Materialsandmethods

Jambusamples

Theplantmaterial(leaves,stemsandinflorescences)fromthe

jambu samples was collected in Igarapé-ac¸u County, which is

locatedinBragantinaRegion,intheNortheasternParáState,Brazil,

atthecoordinates:01◦0733′′Sand473727′′W(Oliveiraetal.,

2011).Theplant(MG205534)wasidentifiedasAcmellaoleracea(L.)

R.K.Jansen,Asteraceae,anditwasincorporatedtotheherbarium

ofEmílioGoeldiMuseum,Belém,ParáState.

Theplantwasinitiallywashedinwatertoremovesoilresidues,

therootswereremovedusingstainlessknives,andtheplant’storn

andcrumpledparts,aswellasthosewithdarkenededgeswere

eliminatedfromthedryingprocess.Therawmaterialswere

sani-tizedthroughimmersioninsolutioncontaining200ppm(mgl−1)

offreeresidualchlorine(FRC)derivedfromsodiumhypochlorite

with10%purity,for10min.Thelastrinsewasperformedthrough

immersioninsolutioncontaining5ppm(mgl−1)FRC,for10min,

andsubsequentwaterdrainage.

Thecold-dryingprocesswascarriedoutinacclimatizedroom

usingairconditioning(Midea,modelMS2E-18CR,Brazil)at25◦C,

anddehumidifier(Arsec,model160,Brazil).Theroommeasured

4m2andremainedclosedduringthedryingprocedure.

Extractionprocedure

ThedriedA.oleraceaplantmaterialwascrushedandsubjected

toan exhaustive extraction process through methanol(MeOH)

macerationatroomtemperature(Mbeunkuietal.,2011).The

sol-vent was removedin rotary evaporator at 40◦C under reduced

pressure.Themethanolusedshowed99.8%purityandextraction

wasperformedforabout30dayswithapproximately6lofsolvent.

TheMeOHextractwassolubilizedinMeOH/H2O(8:2)andthe

solutionwassubjectedtosuccessiveextractionsinseparatory

fun-nelwiththesolvents:n-hexane,dichloromethane (CH2Cl2)and

ethylacetate(AcOEt),asshowninFig.1.Threereplicationswere

performedtoobtaintheextractandtheliq.–liq.extraction.

Tyrosinaseenzymeactivity

The reagents employed in the inhibitory investigationwere

obtainedatSigma–Aldrich.

TyrosinaseinhibitionactivitywasmeasuredbyamodifiedPatil

andZucker(1965)UV–vismethod.Themodificationsconsistedon

theuseofa differentconcentrationofEDTA. Also,l-DOPAwas

employedasa substrateandfinally,commercialtyrosinasewas

usedwhereastheauthorsisolatedtheenzyme.

Hexane fraction (0.3217 g)

Residue 1st extration

Residue 2nd extration CH2Cl2fraction

(1.5190 g)

AcOEt fraction (0.2318 g)

Residue 3rd extration (5.2078 g) Methanol extract

MeOH/H2O (8:2)

CH2Cl2

AcOEt Hexane

Fig.1. FractionationoftheAcmellaoleraceamethanolextractinthesecond

repeti-tion.

Methanolicextractfromjambu,hexanefraction(84.28%

spilan-thol)anddichloromethane(containingapproximately100%

spilan-thol)fractionweresolubilizedindimethylsulfoxide(10mgml−1)

anddifferentaliquotsofthesolutionwereaddedtothereaction

mediumcontainingthetyrosinaseenzyme(50–100units),EDTA

(0.022mmoll−1),l-DOPA(0.17mmoll−1)inPBS(50mmoll−1,pH

8.0) in room temperature. The time for this phenolase to

oxi-dizel-DOPA was30min afterthe reaction time readingswere

performedinaspectrophotometer(Shimadzu,modelMini1240,

Japan)UV–visat475nm.

The otherexperiment evaluated the action of themethanol

extractedfromjambubyusingtheenzymereactionmediumwith

directincidenceofultravioletirradiationat312nm(usedUVlamp)

at10minintervalsfor30min.

Theconcentrations0.66and0.16mgml−1(methanolicextract);

0.51–0.05mM(hexanefractioncontainingapproximately84.28%

spilanthol) and 0.53mM (dichloromethane fraction containing

approximately100%spilanthol)usedintheenzymeactivity

eval-uationtest wereusedtodeterminetheenzyme kineticsinthe

presenceofsample.Theevolutionofthereactionwasmonitoredby

readingstakeninUV–visspectrophotometerat475nmfor30min

and60minin10minintervals.

Theactivationandinhibitionvalueswerecalculatedfromthe

belowequation:

%inib=

[(B30B0)(A30A0)]

(B30−B0)

×100 (1)

where B0=absorbance of l-DOPA+tyrosinase at t=0min,

B30=absorbance of l-DOPA+tyrosinase at time=30min, A0=

absorbance of l-DOPA+tyrosinase+inhibitor/activator at time=

0min, and A30=absorbance of l-DOPA+tyrosinase+inhibitor/

activatorattime=30min.

Theaboveequationallowstheevaluationoftheactionofplant

extractsandorganiccompoundsontheenzymetyrosinase,aswhat

int=0min.Thepossibleabsorptionofthetestsamplesat475nm

(whicharerelatedtoproductiondopacromona)issubtracted.

Allexperimentswereperformedintriplicate,andtheresults

wereexpressedasmeans±SD.Thegraphicswasfitofthe

experi-mentaldatainOriginsoftware(ANOVAstatisticalfunction).

Chemicalanalysis

The material was analyzed through a gas chromatograph

coupledtoamassspectrometer–GC/MS(Shimadzu,model

(3)

3.0

a

b

2.0

1.0

100 90 80 70 60 50

30 40 41

53 69 81

85 98

115 126

141

167 178 192 206 221

50 60 70 80 90 100 110 120 130

m/s 140 150 160 170 180 190 200 210 220 40

30 20 10

2.5 5.0 7.5 10.0 12.5 Spilanthol TIC

(x1 000 000)

15.0 17.5 20.0 22.5

Fig.2.Chromatogramofthedichloromethanefraction(F2R32)(a)andthemassspectrumofspilanthol(b).

model advance III, EUA). Dichloromethane (99.9%, HPLC grade,

Sigma–AldrichInc.,EUA)wasusedassolventintheGC/MS

analy-sis.TheCDCl3(99%,Sigma–AldrichInc.,USA)wasusedassolvent

intheNMRanalysis.TheMEOD(99%,Sigma–AldrichInc.,USA)was

usedassolventintheNMRanalysisofthejambumethanolextract.

TheGC/MSwasequippedwithaFactorFour/VF-5msfused-silica

capillarycolumn(30m×0.25mm×0.25␮mfilmthickness),using

heliumascarriergasat1ml/min.Theinitialoventemperaturewas

100◦C;itwaskeptconstantfor40minandincreasedattherate

of10◦Cmin−1to290C;thefinalisotherm(300C)washeldfor

20min.Theinjectionvolumeofthesamplewas1␮l(1:50split

mode).Boththeinjectorandthedetectorweresetat300◦C.The

massspectrawereobtainedintherangeofm/z10–300,usingthe

electronimpacttechniqueat70eV.Thechemicalcompositionof

thesampleswasanalyzedinaHP5890SeriesIIgaschromatograph

withflameionizationdetector(FID).Thesameoperational

condi-tionsandcolumntypewereusedintheGC/MSanalysis,exceptfor

thetemperaturesoftheinjectorandthedetector,whichwere250

and300◦C,respectively.

Thepercentageofeachcomponentwascalculatedthroughthe

integralareaundertherespectivepeaksincomparisontothetotal

areaofallthecomponentsofthesample.

Themajorcomponentwasidentifiedaccordingtoinformation

fromtheaforementionedanalysismethods and fromdata

gen-eratedthrough thecomparison using1HNMR(Bruker 500MHz

spectrometer)andcarbon13CNMR(Bruker125MHz

spectrome-ter).

Resultsanddiscussion

Table1showsthedataoftheA.oleraceamethanolextractand

fractionsfromthreereplications.

Themaincomponentinthehexane(F1R1)anddichloromethane

(F2R32)fractionswasidentifiedthroughthepeaksat12.21and

12.09minintheGCchromatogram(Figs.2aand3).Bothpeaks

pre-sentedmassspectrum,withmainpeaksatm/z(%):221(M+,2),141

(M-C6H8,50),126([C7H12NO]+,40),98([C5H8NO]+,35),81(C6H9+,

100),asitisshowninFig.2b,andthesedatameetthespilanthol

structure.Thespilantholstructurewasalsoconfirmedthrough1H

10 20 30 40 50 60

0.0 1.0 2.0 3.0 4.0

(x1 000 000) TIC

Spilanthol

N-(2-methylbutyl)-(2E,6Z,8E)-decatrienamide

Fig.3. Chromatogramofthejambuhexanefraction(F1R1).

NMRand13CNMRanalysisandthroughitscomparisonwithdata

describedintheliterature(NakataniandNagashima,1992).

Itisimportanttoemphasizethepresenceof

N-(2-methylbutyl)-(2E,6Z,8E)-decatrienamide in the hexane fraction. This amides’

chemicalstructureissimilartothatofspilanthol.Thiscompoundis

alsoknownashomospilanthol,anditisthesecondmostabundant

N-alkylamideintheSpilanthesethanolicextract,sinceitrepresents

9.04%ofthetotalN-alkylamidesamount(Boonenetal.,2010a).

Colorimetryis readilyadaptable toan analysisoftyrosinase

substratesandinhibitors.Substratescanbedeterminedbytheir

enzymatic conversion to darkly pigmented melanin-like

mate-rialswithabsorptionmaximaabout 570␮p. Inthe presenceof

tyrosine-tyrosinase,inhibitorsofthereactionwillpreventcolour

development(Spenceretal.,1956).

Themechanismofactionoftyrosinaseanditsinteractionwith

l-DOPAwasdescribedindetailbyChang(2009,2012).

Sampleanalysesshowthatthemethanolicextractfromjambu

andthehexanefraction(84.28%spilanthol)areabletoactivatethe

oxidizingactionofthetyrosinaseenzymeforl-DOPA(Table2).

Theincreaseddopachrome formationwasassessed afterthe

directultravioletlightradiationoverthemethanolicextractwas

performedat312nm,anditshowedhighpoweractivationinthe

enzyme.Thiswavelengthispartof theUV-Bradiationwhichis

partlyabsorbedbytheozone intheatmosphere.Theportionof

this radiation that actually reachestheEarth is responsiblefor

skininjuries.Thisanalysiswasperformedattwoconcentrations,

namely: 0.66 and 0.16mgml−1, and it resulted in 57% and 9%

(4)

Table1

MethanolextractfractionsfromAcmellaoleracealeaves,stemsandflowerplantmaterial.

Replications(driedjambumass) Methanolextractmass Fractions Code Fractionmass(g) Spilanthol%

1st(66.00) 12.51 Hexane F1R1 0.0821 84.28

Dichloromethane F2R1 0.2264 88.55

Ethylacetate F3R1 0.2662 –

Residue F4R1 4.4151 –

2nd(265.00) 48.45 Hexane F1R2 0.3217 82.90

Dichloromethane F2R2 1.5190 100.00

Ethylacetate F3R2 0.2318 –

Residue F4R2 5.2078 –

3rd(263.90) 40.58 Hexane F1R3 0.3819 62.83

Dichloromethane F2R3 3.1742 79.41

F2R32 0.4569 100.00

Ethylacetate F3R3 0.2791 –

Residue F4R3 6.9670 –

Table2

Tyrosinaseactivationthroughjambumethanolicextractandhexanefraction.

Jambumethanolicextract Hexanefraction

(84.28%spilanthol)

[mgml−1] %ofactivation [mM]a %ofactivation

0.83 357 0.51 40

0.66 297 0.38 23

0.5 107 0.13 10

0.33 63 0.07 5

0.16 39 0.05 1

aRelativespilantholconcentrationinthesamples.

150

120

90

60

30

0 5 10 15 20

0.66 mg/ml 0.16 mg/ml Time in minutes

% activ

ation

25 R2=0.9765

R2=0.9861

30 35

0

Fig.4.TheUV(312nm)radiationeffectontheactivationpowerinjambumethanol

extractonthetyrosinaseenzyme.

impactoftheUVradiationat312nmsignificantlydecreasedthe

poweractivationinthejambumethanolicextract(Fig.4).

Anotherinterestingfactwasobservedduringthekinetic

exper-imentthatusedthehereinadoptedconcentrations.Thepotential

tyrosinaseenzymeactivationthroughhexanefractionat475nm

containing84.28%spilantholreacheditsmaximuminthirtymin;

however,therewasnosignificantpoweractivationdecayupto

60min(Fig.5).

Thedichloromethanefractioncontainingapproximately100%

spilantholshowedtyrosinaseenzymeinhibition(IC50=0.50mM)

duetothehigherenzymeinhibitionpowerwithinthefirst10min.

Afterthistime,theinhibitionpowerdecayed(Figs.6and7).

Intheseexperimentsweobtainedtheincreaseinthepercentage

ofdopachromeproduction,percentageactivation,inadditiontothe

enzymekineticsinthepresenceofthesesamples.

Theabilityofthedichloromethanefractiontoinhibitthe

tyrosi-naseactivitymaybetranslatedintoitspotentialasskinwhitening

agent.Melaninproductionisreducedwhenthetyrosinaseenzyme

activityisinhibited,anditresultsinafairerskin(Karimetal.,2014).

200

150

100

50

0

0 20

0.513 mM 0.385 mM 0.128 mM 0.077 mM 0.051 mM 40

Time in minutes

Absorbance

60 80

Fig.5.Thekineticactionofthetyrosinaseenzymeonl-DOPAinthepresenceof

hexanefraction.

60

50

40

30

20

10

0.13 0.26 0.40

[mM]

% inhibition

0.53 0

Fig.6. Inhibitionactivityofdichloromethane(containingapproximately100%

spi-lanthol)fractiononthetyrosinaseenzyme,withl-DOPAsubstrate.

100

80

60

40

20

0 10 20 30 40

R2=0.999

Time in minutes

% inhibition

50 60 70

0

Fig.7. Kineticsofthetyrosinaseenzymeinhibitionthroughdichloromethane

(5)

Theseresultsindicatethatspilantholcanbeusedtocontrolthe

enzymaticbrowninginfruitsandvegetables,sincethisamide

inac-tivatesthetyrosinaseenzyme(polyphenoloxidase)involvedwith

theseproducts’darkeningprocess.Thus,spilantholmayincrease

theshelf liveandthenutritionalqualityofprocessedfruitsand

vegetables,suchasstandardtyrosinaseinhibitorskojicacid,IC50

valueof0.130mM(Okunjietal.,2007).

Tyrosinaseinhibitorsarechemicalagentsabletoreduce

enzy-maticreactions,suchasfruitandvegetable’sskinbrowningand

humanskinmelanization.Therefore,theseagentshavegood

com-mercialpotentialin both thefoodprocessingand thecosmetic

industries(Limetal.,2009).

Conclusion

The activating or inhibitory actions against the tyrosinase

enzyme dependonthe degreeof purity ofjambu extracts and

fractions.Thejambumethanolicextractresultsonthetyrosinase

enzymecorroboratedthehereincarriedoutanalyses,whichused

hexanefractionof84.28%spilanthol.Suchresultprovedthe

tyrosi-naseenzymeactivationthroughdopachromeincrease.Therefore,

jambu-basedproductsmaybeusedascosmetics(creams,soap,

etc.).Atrandom,theycanacceleratemelaninproduction(which

darkenstheskin),aswellasformmelanomas.However,itsuse

againstlocateddepigmentationprocesses(skinandhair)demands

furtherstudies,sincetheanalysisusingUVBirradiationshowed

viableformulationscontainingthisextractintropicalcountries.

Sincethedichloromethanefraction(containingapproximately

100%spilanthol)showedabilitytoinhibitthetyrosinaseenzyme,

spilantholarousesinterestinthestudybasedonformulationswith

this amide in topicalproducts. Surely it canto preventand/or

reduceskinhyperpigmentationprocesses.Anotherpossible

appli-cationofspilantholisthecontrolofenzymaticbrowninginfruits

andvegetables.Themosttyrosinaseinhibitorsarenotcurrently

commerciallyavailable,especiallythosefromnaturalsources,and

thislimitstheirfurtherevaluationinaninvivostudy,whereusually

alargeamountisneededforaninhibitiontest.

Thusfurtherstudiesneedtobeperformedtofoundinhibitors

withahumanclinicalpointofviewincludinghelpandcooperation

ofcosmeticorbiotechnologygroups.

Authors’contributions

AFB,KCS,MCCO,MGC,andAUOSRallcontributedtothe

writ-ingofthisarticle.AFB,MGC,andAUOSRobtainedandidentified

samples.KCSandMCCOperformedenzymaticanalyzes.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsaregratefultoFAPERJ,CNPq,andtoCAPESfor

grant-ingthescholarshipsandthefinancialsupport.

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Imagem

Fig. 1. Fractionation of the Acmella oleracea methanol extract in the second repeti- repeti-tion.
Fig. 2. Chromatogram of the dichloromethane fraction (F2R32) (a) and the mass spectrum of spilanthol (b).
Fig. 4. The UV (312 nm) radiation effect on the activation power in jambu methanol extract on the tyrosinase enzyme.

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