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

Original

Article

Antiproliferative

effects

of

pinostrobin

and

5,6-dehydrokavain

isolated

from

leaves

of

Alpinia

zerumbet

Walter

A. Roman

Junior

a,b,∗

_,

_Denise

_B.

_Gomes

a

_,

_Barbara

_Zanchet

a

_,

_Amanda

_P.

_Schönell

b

_,

Kriptsan

A.P.

Diel

b

_,

_Thais

_P.

_Banzato

c

_,

_Ana

_L.T.G.

_Ruiz

c

_,

_João

_E.

_Carvalho

c,d

_,

_Angelita

_Neppel

e

_,

Andersson

Barison

e

_,

_Cid

_Aimbiré

_M.

_Santos

f

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_da_Saúde,_Universidade_Comunitária_da_Região_de_Chapecó,_Chapecó,_SC,_Brazil

b_Grupo_de_Pesquisa_em_Fitoquímica_e_Farmacologia_de_Produtos_Naturais,_Universidade_Comunitária_da_Região_de_Chapecó,_Chapecó,_SC,_Brazil

c_Centro_{Pluridisciplinar}_de_Pesquisas_Químicas,_Biológicas_e_Agrícolas,_Divisão_de_Farmacologia_e_Toxicologia,_Universidade_Estadual_de_Campinas,_Campinas,_SP,_Brazil d_Faculdade_de_Ciências_{Farmacêuticas,}_Universidade_Estadual_de_Campinas,_Campinas,_SP,_Brazil

e_Departamento_de_Química,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

f_Laboratório_de_{Farmacognosia,}_Departamento_de_Farmácia,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14April2017 Accepted30May2017 Availableonline29July2017

Keywords:

Medicinalplants Antiproliferativeeffects 5,6-Dehydrokavain Pinostrobin Antitumoragents

a

b

s

t

r

a

c

t

Naturalproductsareamajorsourceofdrugsforthetreatmentofcancer.ThespeciesAlpiniazerumbet

(Pers.)B.L.Burtt&R.M.Sm,Zingiberaceae,iswidelydistributedinBrazilwhereitisknownas“colônia”. Theleavesarecommonlyusedinthetreatmentofhypertensionanddyspepsia,however,theeffectsof

A.zerumbetextractsandisolatedsubstancesonhumancancercellsremaintobeelucidated.Thisstudy wasdesignedtoidentifythechemicalconstituentsofhydroalcoholicanddichloromethaneextractsfrom

A.zerumbetleavesandtoinvestigatetheirinvitroantiproliferativeactivity.Theisolatedphytochemicals includedkaempferol,dihydro-5,6-dehydrokavain,5,6-dehydrokavain,andpinostrobin.The hydroalco-holicextractinhibitedcellularproliferationonlyathighconcentrations,whilethedichloromethane extractshowedamoderateantiproliferativeeffectagainstleukemiaandlungtumorcelllines. 5,6-Dehydrokavainshowedpotentcytostaticactivityagainstglioblastomacellsandamoderateeffectonall othertumorcelllines.Pinostrobinshowedpotentactivityagainstleukemiaandbreasttumorcelllines andmoderatecytostaticeffectagainstovariancell.Furthermore,thisisthefirstreportontheisolationof kaempferolandpinostrobinfromA.zerumbetleaves.Moreover,thepurificationprocessdescribedinthis studywaseffective.TheseresultssuggestthatA.zerumbetleavesareapromisingsourceofanticancer compounds.

Introduction

Cancerisaseriouspublichealthproblemrepresentingthe sec-ondlargestcauseofdeath(Siegeletal.,2016).Overthenexttwo decades,approximately22 millioncases per yearare expected worldwide(McGuire,2016).Canceristhenamegiventoasetof morethan 100diseases thathave incommontheuncontrolled growthof(malignant)cellsthatinvadetissuesandorgansandcan spread(metastasize)tootherpartsofthebody.Dividingrapidly, thesecellstendtobeveryaggressiveanduncontrollable,causing

∗ Correspondingauthor.

E-mail:romanwa@unochapeco.edu.br(W.A.Junior).

theformationoftumorsormalignantneoplasms(Almeidaetal., 2005;Elkadyetal.,2016).

Thetreatmentofcancerisbasedmainlyonsurgicalresection of the tumor mass and/or administration of radiotherapy, immunotherapy,and/or chemotherapy.However,many cancers stillexhibitonlymodestclinicalresponsestoprotocolsdeveloped foreitherprimarytumorsormetastases(Costa-Lotufoetal.,2010). Moreover,manyanticanceragentshavehighratesofadverse reac-tionsandtoxicityaswellasalowselectivityfortumorcells(Prakash etal.,2013;TopculandCetin,2014).Therefore, withthe objec-tiveoffindingmoreeffectiveandsafetreatments,pharmacological studieswithsubstancesisolatedfromplants,aswellassynthetic derivativesbasedonthesenatural compounds,haveintensified (Harveyetal.,2015;NewmanandCragg,2016).

http://dx.doi.org/10.1016/j.bjp.2017.05.007

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Naturalproductshave beenreportedtoact directlyor indi-rectlyviamultiplecellsignalingpathways.Thus,thecombination oftraditionalchemotherapeuticdrugswithextractsand/orisolated substancescouldprovideaneffectivealternativeforcancer treat-mentorovercomechemoresistance(Apayaetal.,2016).Among naturalproducts,phenoliccompounds,includingflavonoids,have beenshowntohaveanarrayofpharmacologicalactivities(Kristo etal.,2016)suchasanti-inflammatory,cancerchemopreventive, andchemotherapeutic(García-Lafuenteetal.,2009;Georgeetal., 2017).

Alpiniazerumbet(Pers.)B.L.Burtt&R.M.Sm,Zingiberaceae,is

nativetoChinaandJapanandcultivatedinBrazilwhereispopularly knownasfalso-cardamomo,pacová,gengibre-concha,andcolônia (Lorenzi and Matos,2002; Lorenziand Souza,2008).Thisplant is herbaceous,tropical perennial, rhizomatoza,with stemshort thatcanreachupto3mtall.Itbearsfunnel-shapedflowersand theleavesarelanceolate,aromatic,andofcoriaceousconsistency (Correaetal.,2010;Sabooetal.,2014).TheA.zerumbetleavesare traditionallyusedindyspepsiatreatmentandasananthelmintic, in addition to theirantimicrobial, anti-inflammatory,and anti-hypertensive properties (Almeida, 1993; Correa et al., 2010). In addition tokava pyrones,dihydro-5,6-dehydrokavain (DDK), and 5,6-dehydrokavain (DK), rutin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucuronide, (+)-catechin, and (−)-epicatechin, have been reported (Mpalatinos et al., 1998; Elzaawely et al., 2007;XuanandTeschke,2015;Kumagaietal.,2016).The phar-macologicalpropertiesattributedtoextractsofA.zerumbetleaves, flowers, seeds, and rhizome include diuretic and hypotensive effects(Laranjaetal.,1991;Mendonc¸aetal.,1991;Lahlouetal., 2003),antioxidant(Elzaawelyetal.,2007),vasodilatory(Pintoetal., 2009;Victórioetal.,2009),hypolipidemic(Linetal.,2008),and antidepressant-likeeffect(Bevilaquaetal.,2016).Further, antineo-plasticeffectsofAlpiniaofficinarumHence(Ghil,2013)andAlpinia

galanga(L.)Willd.(Samarghandianetal.,2014)rhizomeextracts

havebeendescribed.However,therearenoreportsregardingthe antiproliferativeactivityofA.zerumbetextractsandisolated sub-stances.

Inthiscontext,thepresentstudyaimedtoinvestigatethe chem-ical composition and the in vitro antiproliferative effects of A.

zerumbetleafextractsandtheirmajorconstituents.

Materialandmethods

Standardsandchemicals

Allsolventsand reagentswereofanalytical gradeandwater wasdistilledanddeionized.Thesolventsusedwereethylacetate, methylenechloride,ethanol,andhexane(Vetec®,RiodeJaneiro, Brazil).Nuclearmagneticresonance(NMR)experiments(1_H_and 13_C)_were_performed_on_a_Bruker_Avance₄₀₀_spectrometer₍₄₀₀

and100MHz,respectively)usingCDCl3assolvent.Chemicalshifts

of 1_H _and 13_C _NMR _were _expressed _in _ppm _(ı)_using _TMS _at

0.00ppm as aninternal standard, and couplingconstants(J)in Hz.Columnchromatographywasperformedonsilicagel(Merck, Darmstadt,Germany;230–400meshASTM),andanalytical thin layerchromatography(TLC)wasperformedusingsilicagelplates (Kieselgel60F254,Merck).Thespotswerevisualizedusing

ultra-violetlight(366nm)orbysprayingwith10%H2SO4inmethanol

followedbyheatingat110◦_C₍₁₀_min).

Plantmaterial

TheleavesofAlpiniazerumbet(Pers.)B.L.Burtt&R.M.Sm, Zin-giberaceae,werecollectedinChapecó(SC),Brazil(26◦₅₈′_36.06′′_S and52◦₄₄′_27.18′′_W)._The_plant_material_was_identified_by_Osmar

dosSantosRibas,theherbariumcuratoroftheMunicipalBotanical MuseumofCuritiba(PR),whereavoucherspecimenwasdeposited (MBM#306196).

PreparationofextractsofA.zerumbetandchemicalisolation

The leaves of A. zerumbet were dried at room temperature (25±5◦_C), _pounded _in _a _knife _mill _(Ciemlab®_, _CE430),_passed throughsieve(425␮m;35Tyler/Mesh),identified,andstored

pro-tectedfromlight.Theextractswereproducedbymaceration(5 days)atroomtemperatureusingdry-milledleavesofA.zerumbet

(100g)insolvent(1:20,w/v),first,withdichloromethaneand, sub-sequently,70%ethanol.AfterfiltrationthroughBüchnerfunnel,the dichloromethane (DEA)and hydroalcoholic(HEA)extractswere concentratedbyevaporationunderreducedpressure,lyophilized, weighed,andstoredinafreezerat−20◦_C.

AsampleofDEAextract(4g)wasdissolvedinhexaneand sub-mittedtocolumnchromatographyusingastationaryphasesilica gel(Merck,Darmstadt,Germany)and eluted withhexane:ethyl acetate(EtOAc)(90:10,v/v)increasinginpolarityto90%EtOAc(v/v) toyieldfoursubfractions.ThesubfractionswereanalyzedbyTLC usinghexane:EtOAc(80:20,v/v)asthemobilephase,visualizedat 366nm,andrevealedwithH2SO4 (10%inmethanol)followedby

heatingat110◦_C₍₁₀_min)._Subfraction₂_(0.032_g)_was_observed_as_a spotbyTLCanalysisandwasidentifiedascompound1.Subfraction 3(0.316g)wasfurtherseparatedusingflashcolumn chromatogra-phywithdichloromethaneasaneluent,producingthreeadditional subfractions.Subfraction3.1(0.042g)wasidentifiedascompound 2.

TheHEAextract(50g)wasdilutedindistilledanddeionized water(500ml)andpartitionedwithEtOAc(fivetimes;500ml).The fractionEtOAc(5.2g)wasfractionatedusingcolumn chromatog-raphy (SephadexLH-20) withMeOH asan eluent.TLC analysis usingDCM:MeOH(90:10,v/v)asthemobilephasewasusedto identifyfivesubfractions.Subfraction4(0.053g)wasidentifiedas compound3.

FreshleavesofA.zerumbet(2kg)wereextractedbydecoctionin distilledwater(10l)for15min.Afterfiltration,theaqueousextract (AEA)wasreducedto1000mlbyevaporationunderreduced pres-surefollowedbyliquidpartitionwithchloroform(1000ml).The chloroformfraction(CFA)wasconcentratedbyevaporationunder reducedpressureandweighed(0.785g).Forrecrystallization,to CFA(0.785g),100mlofdistilledwaterwasaddedat100◦_C_and_the solutionfilteredthroughaglassfiltrationfunnel.Thefiltratewas immediatelyrefrigeratedat−8◦_C_for₄₈_h,_after_which,_the crys-talswerefilteredusingaBüchnerfunnelandstoredinthehood withanhydrousNa2SO4.Thecrystalsobtained(0.045g)were

ana-lyzedbyTLCusingCHCl3:EtOH(9:1,v/v)asaneluentandvisualized

at366nmorbysprayingwith10%H2SO4 inmethanolfollowed

byheatingat110◦_C₍₁₀_min)₍_Itokawa_et_al.,₁₉₈₁_)._The_crystals isolatedusingthismethodwereidentifiedascompound4.

Antiproliferativeassay

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RPMI-1640supplementedwith5%fetalbovineserum(RPMI/FBS 5%)withpenicillin:streptomycin mixture1000U/ml:1000␮g/ml

(1ml/l;RPMI-1640).

Stocksolutionsofthesamples(5mg)werepreparedinDMSO (50␮l)followedbysuccessivedilutionsinRPMI/FBS5%togivefinal

concentrationsof0.25,2.5,25,and 250␮g/ml.Doxorubicinwas

usedasapositivecontrolatfinalconcentrationsof0.25,2.5,25, and250␮g/ml.

Cells in 96-well plates (100␮l cells/well, cell densities:

3–7×104_cells/ml)_were_incubated_with_each_of_the_four

concentra-tionsofsamplesolutionordoxorubicin(100␮l/well)intriplicate

(n=3),for48hat37◦_C_and_5%_of_CO₂_._Before_(T0_plate)_and_after_(T1 plates)sampleaddition,cellswerefixedwith50%trichloroacetic acid(50␮l well) and stained with sulforhodamine B to

quan-titate cell proliferation using the reading at 540nm. The GI50

(concentrationthatproduces50%cellgrowthorcytostaticeffect) andtheTGI(concentrationthatresultedintotalcellulargrowth inhibition)valuesweredeterminedthroughnon-linearregression appliedtoasigmoidalcurveusingOrigin8.0software(OriginLab Corporation).

Results

ChemicalconstituentsofAlpiniazerumbet

The purification techniques employed allowedthe isolation of four compounds from the A. zerumbet leaf extracts (HEA, DEA, and AEA); thesecompounds were identified by compari-son of theirexperimental spectra (IR, NMR 1_H, _and 13_C) _with

thosepreviouslydescribed:DDK(1),DK(2)(Itokawaetal.,1981; Mpalatinosetal.,1998),3,4′_{,5,7-tetrahydroxyflavone}_(kaempferol; 3)(Markhametal.,1978),and 5-hydroxy-7-methoxy-2-phenyl-2,3-dihydrochromen-4-one(pinostrobin,PTB;4)(Smolarzetal., 2006;Ramirezetal.,2013).

Diidro-5,6-dehydrokavain(DDK;1):Itwasobtainedascolorless needles,meltingpoint96–98◦_C;_C₁₄_H₁₄_O₃_,_IR_n_max_(cm−1_;_KBr_disk)

1725(pyronecarbonyl),1650,1530(C C),1238,1129(C O C), 1600,725, 600(benzenering)cm−1_; _ESI-MS:_231.1_[M+H]+_; 1_H

NMR(400MHz,CDCl3)ppm (ı):2.79(2H,t; J=8Hz, 7-H),2.96

(2H,t;J=8Hz,8-H),3.81(3H,s,MeO-),5.51(1H,d;J=2.2Hz,3-H), 5.92(1H,d;J=2.2Hz,5-H),7.19–7.34(5H,m,aromatic);13_C_NMR

(100MHz,CDCl3)ppm(ı):33.72(C-8),36.13(C-7),56.82(MeO-),

88.21(C-3),101.96(C-5),128.53(C-12),129.44(C-10),129.49 (C-14),129.52(C-11),129.55(C-13),141.23(C-9),166.26(C-6),167.81 (C-2),173.76(C-4).

5,6-Dehydrokavain(DK;2):Itwasobtainedasyellowneedles, melting point 136–138◦_C; _C₁₄_H₁₂_O₃_, _IR _n_max _(cm−1_; _KBr _disk)

1710(pyronecarbonyl),1620,1525(C C),1230,1125(C O C), 1650,700, 620(benzenering)cm−1_; _ESI-MS:_229.1_[M+H]+_; 1_H

NMR(400MHz,CDCl3)ppm(ı):3.87(3H,s,MeO-),5.62(1H,d;

J=2.2Hz,3-H),6.24(1H,d;J=2.2Hz,5-H),6.86(1H,d;J=16Hz, 7-H),7.43(1H,d;J=16Hz,8-H),7.36–7.59(5H,m,aromatic);13_C_NMR

(100MHz,CDCl3)ppm(ı):56.61(MeO-),89.50(C-3),102.73(C-5),

120,12(C-7),128.10(C-14),128,44(C-10),129,41(C-13),129.52 (C-11),130.23(C-12),136.31(C-9),136.74(C-8),160.55(C-2),166.63 (C-6),173.34(C-4).

3,4′_{,5,7-Tetrahydroxyflavone}_(kaempferol;₃_):_It_was_obtained asyellowcrystallinepowder,meltingpoint276–278◦_C;_C₁₅_H₁₀_O₆_, IRnmax(cm−1;KBrdisk)3440(OH),1681(C O),1600(C C),1375

(C C),1335,1303,1267,1165(C O)746,748,705.ESI-MS:286.1 [M+H]+_.1_H_NMR₍₄₀₀_MHz,_CDCl

3)ppm(ı):6.17(1H,d,J=2.1Hz,

6-H),6.37(1H,d,J=2.1Hz, 8-H),6.89(2H, d,J=9.0Hz,3′_,5′_-H), 8.07(2H,d,J=9.0Hz,2′_,6′_-H);13_C_NMR₍₁₀₀_MHz,_CDCl

3)ppm(ı):

99.30(C-6),94.56(C-8),104.67(C-10),116.35(C-3′_),_116.38 (C-5′_),_123.70_(C-1′_),_130.76_(C-6′_),_130.78_(C-2′_),_137.01_(C-3),_148.09

Table1

ValuesofGI50andTGI(␮g/ml)forAlpiniazerumbetleaveshydroalcoholic(HEA)and

dichloromethane(DEA)extractsagainstdifferentcelllines.

Celllines GI50(␮g/ml) TGI(␮g/ml)

HEA DEA HEA DEA

U-251 32.73 22.89 222.00 81.82

MCF-7 27.58 9.44 213.60 69.43

NCI/ADR-RES a _7.79 a a

786-0 202.53 24.51 a _52.80

NCI-H460 12.17 5.85 228.88 62.35

PC-3 226.33 23.05 a _78.34

OVCAR-3 18.39 2.05 a _69.17

HT-29 45.13 23.68 a _60.51

K-562 66.98 6.12 a _55.69

HaCat 0.93 4.44 133.31 74.34

Note:Human tumorcelllines: glioblastoma (U-251),breast (MCF-7), ovarian expressingtheresistancephenotype(NCI/ADR-RES),786-O(kidney),non-small cellslung(NCI-H460),prostate(PC-3),ovarian(OVCAR-3),colon(HT-29),leukemia (K-562);humanimmortalizedkeratinocyte(HaCat).GI50=50%growthinhibition;

TGI,totalinhibitionofgrowth.

a_Effective _{concentration} _higher _than _the _highest _tested _{concentration}

(250␮g/ml).

(C-2),158.35(C-9),160.67(C-4′_),_162.54_(C-5),_165.64_(C-7),_177.41 (C-4).

5-Hydroxy-7-methoxy-2-phenyl-2,3-dihydrochromen-4-one (pinostrobin(PTB);4):Itwasobtainedasyellowneedles,melting point 96–98◦_C;_C

16H14O4,IRnmax (cm−1; KBrdisk) 3432(OH),

1635(C O),1620(C C),1390(C C),1350,1311,1301,1245,1178 (C O)759, 749,700.ESI-MS:271.0[M+H]+_;1_H_NMR₍₄₀₀_MHz,

CDCl3)ppm(ı):2.72(1H,dd,J=16.6,3.2Hz,3-H),2.98(1H,dd,

J=16.6, 12.7Hz, 3-H), 3.82 (3H, s, 7-OMe), 5.24 (1H, dd, 12.7, 3.2Hz, 2-H), 6.05 (1H, d;J=2.2Hz, 6-H), 6.10 (1H, d;J=2.2Hz, 8-H),7.35–7.42(5H,m,aromatic);13_C_NMR₍₁₀₀_MHz,_CDCl

3)ppm

(ı):46.52(C-3),56.31(C-7-OMe),80.20(C-2),94.41(C-8),97.28 (C-6),106.20 (C-10), 127.33(C-5′_), _127.39 _(C-6′_), _129.51_(C-3′_), 129.78(C-4′_), _129,79 _(C-2′_), _141.13_(C-1′_), _164.48 _(C-7),_166.67 (C-5),167.28(C-9),191.88(C-4).

Antiproliferativeeffects

The HEA extract inhibited 50% of the growth of various human tumor cell lines (Fig. 1): immortalized keratinocytes (HaCat; GI50=0.93␮g/ml), lung (NCI-H460; GI50=12.17␮g/ml),

ovarian (OVCAR-3; GI50=18.39␮g/ml), and breast (MCF-7;

GI50=27.58␮g/ml); however,theHEA extract couldnot induce

totalgrowthinhibition(TGI>200.00␮g/ml)(Table1).Incontrast,

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10–3 10–2 10–1 100 101 102 –100

–75 –50 –25 0 25 50 75 100

A

Ce

ll g

ro

w

th

(

%)

Concent

ration

(

µ

g/ml)

U251 MCF7 NCI/ADR-RES 786-0 NCI-H460 HT29 K-562 HaCaT PC-3 OVCAR-3

0.25 2.5 25 250

B

Concent

ration

(

µ

g/ml)

10–3 10–2 10–1 100 101 102

–100 –75 –50 –25 0 25 50 75 100

C

el

l g

ro

w

th

(

%)

0.25 2.5 25 250

Fig.1.InvitroantiproliferativeeffectsofAlpiniazerumbetleavesextracts.(A)Hydroalcoholicextract(HEA);(B)dichloromethaneextract(DEA);concentrationrange: 0.25–250␮g/ml;expositiontime:48h;humantumorcelllines:glioblastoma(U-251),breast(MCF-7),ovarianexpressingtheresistancephenotype(NCI/ADR-RES),786-O

(kidney),non-smallcellslung(NCI-H460),prostate(PC-3),ovarian(OVCAR-3),colon(HT-29),andleukemia(K-562);humanimmortalizedcellline:keratinocytes(HaCat).

ovarian(OVCAR-3;GI50=2.05␮g/ml)andlungtumorcells

(NCI-H460;GI50=5.85␮g/ml)inadditiontototalgrowthinhibitionof

allcelllinesatconcentrationsfrom52.80to81.82␮g/ml(Fig.1

andTable1).

Amongtheisolatedcompounds,DK(2)andPTB(4)were eval-uatedintheantiproliferativeassay(Fig.2).DK(2)showedamore potentantiproliferativeeffectthandidPTB(4)andshowed cyto-staticeffectsagainstalmostallthecelllinestestedwithaGI50from

0.25to5.03␮g/ml(Table2).Interestingly,PTB(4)wasmoreactive

againstthebreastcancercellline(MCF-7)withGI50<0.25␮g/ml

andtheleukemiacellline(K562;GI50=0.91␮g/ml)(Table2).

Discussion

ThefamilyZingiberaceaeisarichsourceofsubstanceshavinga therapeuticvalue,suchasflavonoids,whichhavebeendetectedin severalspecies(Iwashina,2000)andareconsideredphytochemical markersoftheorderZingiberales(Pugiallietal.,1993).Previous studiesonAlpiniarhizomeandseedhavereportedtheisolation of alpinetinfrom Alpiniaspeciosa (Krishna andChaganty,1973; Itokawaetal.,1981);pinocembrinandPTBfromAlpiniarafflesiana

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10–3 10–2 10–1 100 101 102 –100

–75 –50 –25 0 25 50 75 100

Cell

grow

th

(

%

)

Conc

entration

(

µ

g/ml)

Conc

entration

(

µ

g/ml)

U251 MCF7 NCI/ADR-RES 786-0 NCI-H460 PC-3 HT29 K-562 HaCaT

0.25 2.5 25 250

A

10

–3

10

–2

10

–1

10

0

10

1

10

2

–10

0 –75

–50

–25

0

25

50

75

100 Ce

ll gr

ow

th

(%

)

0.25

2.5

25

250 B

Fig.2. Invitroantiproliferativeeffectsof5,6-dehydrokavain(A)andpinostrobin(B)isolatedfromAlpiniazerumbetleaves.A:5,6-dehydrokavain;B:pinostrobin;concentration range:0.25–250␮g/ml;expositiontime:48h;humantumorcelllines:glioblastoma(U-251),breast(MCF-7),ovarianexpressingtheresistancephenotype(NCI/ADR-RES),

786-O(kidney),non-smallcellslung(NCI-H460),prostate(PC-3),ovarian(OVCAR-3),colon(HT-29),andleukemia(K-562);humanimmortalizedcellline:keratinocytes (HaCat).

kaempferol-3-O-glucuronide,andkaempferol-3-O-rutinosidehave beenisolated fromA.zerumbetleaves(Mpalatinos etal., 1998; Victórioetal.,2009).

Inthisstudy,fourcompoundswereisolatedfromA.zerumbet

leaves,namelyDDK(1),DK(2),kaempferol(3),andPTB(4).DDK (1),andDK(2),whicharecommonconstituentsofthegenusAlpinia

(Pugiallietal.,1993;Iwashina,2000),havebeenidentifiedinthe rootsandleavesofA.zerumbet(Mpalatinosetal.,1998;Kusteretal., 1999;Chompooetal.,2011).However,thisisthefirstreportof kaempferol(3)andPTB(4)isolationfromA.zerumbetleavesand,

thus,contributestothechemotaxonomicinformationrelatedto

Alpiniaspecies.

According to the literature, alcoholic extracts from other

Alpinia species have shown antiproliferative effects against

MCF-7 cells. A methanolic extract of A. officinarum rhizomes (85␮g/ml) inhibited MCF-7 cell proliferation by inducing

pro-grammed celldeathand arrestingthecell cycleat theSphase (Ghil, 2013). Moreover, a 48h treatment with an ethanolic extract ofA. galanga (L.) Willd. rhizomesat 250␮g/ml reduced

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Table2

ValuesofGI50andTGI(␮g/ml)for5,6-dehydrokavain(DK)andpinostrobin(PTB)

isolatedfromAlpiniazerumbetleavesagainstdifferentcelllines.

Celllines GI50(␮g/ml) TGI(␮g/ml)

DK PTB DK PTB

U-251 0.25 27.33 4.43 228.40

MCF-7 2.85 <0.25 17.28 17.67

NCI/ADR-RES 4.95 4.35 106.64 a

786-0 5.03 12.11 28.08 221.63

NCI-H460 2.04 27.41 25.96 a

PC-3 4.19 34.81 31.26 a

OVCAR-3 a _3.97 a

HT-29 24.77 89.70 79.03 a

K-562 2.81 0.91 9.79 235.62

HaCat 1.53 30.76 150.60 a

Note:Human tumorcelllines: glioblastoma (U-251),breast (MCF-7), ovarian expressingtheresistancephenotype(NCI/ADR-RES),786-O(kidney),non-small cellslung(NCI-H460),prostate(PC-3),ovarian(OVCAR-3),colon(HT-29),leukemia (K-562);humanimmortalizedkeratinocyte(HaCat).GI50,50%growthinhibition;

TGI,totalinhibitionofgrowth.

a_Effective _{concentration} _higher _than _the _highest _tested _{concentration}

(250␮g/ml).

phosphatidylserineresidueexposureandplasmamembrane per-meation(Samarghandianetal.,2014).

Here,basedontheantiproliferativeactivity,wedemonstrated that successive extraction with an increasingly polar solvent resultedin theconcentrationof bioactive compoundsfromthe DEAextractbutnotfromtheHEAextractofA.zerumbetleaves.A possiblereasonisthatthemorelipophilicchemicalconstituents present in extracts of lower polarity have greater affinity and greatereaseof permeationacrosscellularmembranes (Lee and Houghton,2005).UndertheconditionsdescribedbyFoucheetal. (2008),theHEAextractshowedaweakcytostaticeffectagainst lung (NCI-H460, GI50=12.17␮g/ml) and ovary tumor cell lines

(OVCAR-3,GI50=18.39␮g/ml).However,inourexperiments,the

HEA extractactivelyinhibitedtheproliferationof immortalized keratinocytes(HaCat,GI50=0.93␮g/ml),suggestingpossibletoxic

effectsonnon-tumortissues.Inaddition,theDEAextractshowed moderateantiproliferativeeffectsagainstleukemia(K-562),ovary (OVCAR-3andNCI-ADR/RES),lung(NCI-H460),andbreasttumor celllines(MCF-7)andagainstimmortalizedhumankeratinocytes (HaCat)(Table1).

Amongtheisolatedcompounds,DKandPTBwereevaluated againsttumor and non-tumorcell lines(Table2).Accordingto Fouche et al. (2008), compounds with TGI values<6.25␮g/ml

show potent antiproliferative activity. Thus, DK had a potent antiproliferativeeffectagainstglioblastomacells(U-251)withTGI value=4.43␮g/ml,whichwasabout33-foldhigherthanthatfor

theimmortalizedkeratinocytecellline(HaCat;TGI=150.60),and withtwicethepotencyofdoxorubicinusedasapositivecontrol, indicatingpossibleselectivity.ThemainmetabolicpathwayofDK inratsandhumansisthehydroxylationoftheC-12inthearomatic ringtoproducep-hydroxy-5,6-dehydrokavain.Thismetabolitewas detectedinbloodandurineinthefirstfewhoursafterabsorption, producingbydesmethylationofthe4-methoxygroupofthe lac-toneringthecompoundo-desmethyl-hydroxy-5,6-dehydrokavain. Inrats,approximately50–75%ofadministeredkavalactonesare excretedintheurine, mostly asglucuronideand sulphate con-jugates.Approximately15%is excretedin thebile(Roweetal., 2011).

WithregardtotheGI50results,PTBshowedapotentcytostatic

effect(GI50<0.25␮g/ml)againstbreast carcinomacells(MCF-7)

anda219-foldselectivitywhencomparedtothatforimmortalized keratinocytes(HaCat,GI50=30.76␮g/ml).Accordingtothe

litera-ture,PTBdemonstratedastrongantitumoractivityinmammary carcinomacells,whichwaspartiallyexplainedbytopoisomerase

Iinhibition (Sukardimanet al.,2000).Thisenzymeis responsi-ble for DNAstrandbreak repair, allowingthebroken strandto rotateontheintactoneandreducingthetorsionaltensionofthe molecule(Brandãoetal.,2010)duringtheprocess.The antiprolif-erativeeffectofPTBonmammarytumorscouldalsobeattributed toananti-aromataseeffectratherthanantiestrogenicactivity,as demonstratedbyLeBailetal.(2000).Becausearomataseis respon-siblefortheconversionoftestosteronetoestrogen,inhibitionofthe enzymecouldreduceestrogenlevels,andthereforetheprobability ofhormone-relatedcancer(LeBailetal.,2000).

In addition, PTB presented a potent cytostatic effect on the leukemiacellline(K-562,GI50=0.91␮g/ml)(Table2),whichisin

agreementwiththeresultsdescribedbySmolarzetal.(2006).PTB alsoinhibitedendothelialcellproliferation,probablybypromoting membrane celldepolarization,suggestinga potential antiangio-genic effect (Siekmann et al., 2013). Due to its lipolyphilicity (LogP=3.1),PTBinratsshowedhighabsorptionwithamaximum concentrationtime of about2h. A largevolumeof distribution has been observed, and the metabolism appears to be essen-tiallyhepatic.Basedonclearancevaluesthiscompoundismainly excretedvianon-renal,withserumhalf-lifeofapproximately7h (Sayreetal.,2012,2015).

Cancerchemotherapycanoftenprolonglife,andprovide tem-poraryrelieffromsymptomsandoccasionallycompleteremission. Asuccessfulanticancermoleculeshouldkillorincapacitatecancer cellswhilehavingreducedtoxicitytowardnormaltissues(Sharma etal.,2016).Inthisstudy,DKandPTBshowedhighselectivityand potentantiproliferativeorcytostaticeffectsagainstglioblastoma andbreastcancercelllines,implicatingapotentialrolein inhibi-tingcancerprogression.Theseresultsaddtotheliteratureshowing thatanumberofherbalcompoundsarecytostaticandinducecell cyclearrest,therebyshowinganimportantmechanismusefulfor developmentofnewantineoplasticdrugs(Sharmaetal.,2014).

Conclusions

DKandPTBisolatedfromtheleavesofA.zerumbethad antipro-liferativeeffectsinvitrowithhighpotencyandselectivityagainst glioblastoma(U-251)andbreastcarcinoma(MCF-7)tumorlines and are potential candidates for development of antineoplasic drugs.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authorscontribution

WARJ,DBG,BZ,APS,andKAPDcontributedinallstepsofthis study.TPB,ALTGR,andJECcontributedtoantiproliferativestudies. AN,andABcontributedtochemicalanalyses.CAMScontributedto designofthestudy.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

(7)

Acknowledgments

ThisworkwassupportedbytheUnochapecó[modalityArt.170 and171–FUMDES].

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