Rev. bras. farmacogn. vol.27 número4

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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

Schinus

terebinthifolius

:

phenolic

constituents

and

in

vitro

antioxidant,

antiproliferative

and

in

vivo

anti-inflammatory

activities

Marciane

M. da

Silva

a

_,

_Edna

_K.K.

_Iriguchi

b

_,

_Candida

_Aparecida

_L.

_Kassuya

b

_,

_Maria

_do

_Carmo

_Vieira

c

_,

Mary

Ann

Foglio

d

_,

_João

_Ernesto

_de

_Carvalho

d

_,

_Ana

_Lúcia

_T.G.

_Ruiz

d

_,

_Kely

_de

_P.

_Souza

a

_,

Anelise

S.N.

Formagio

a,b,∗

a_Faculdade_de_Ciências_Biológicas_e_Ambientais,_Universidade_Federal_da_Grande_Dourados,_Dourados,_MS,_Brazil b_Faculdade_de_Ciências_da_Saúde,_Universidade_Federal_da_Grande_Dourados,_Dourados,_MS,_Brazil

c_Faculdade_de_Ciências_Agrárias,_Universidade_Federal_da_Grande_Dourados,_Dourados,_MS,_Brazil

d_Centro_{Pluridisciplinar}_de_Pesquisas_Químicas,_Biológicas_e_Agrícolas,_Universidade_Estadual_de_Campinas,_Campinas,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2August2016

Accepted28December2016

Availableonline28April2017

Keywords:

Anacardiaceae

Biologicalactivity

Flavonoids Galloyl

Pinkpepper

a

b

s

t

r

a

c

t

SchinusterebinthifoliusRaddi,Anacardiaceae,nativetoBrazil,isreferredtoas“pimento-rosa”andisused

totreatinflammatorydiseaseinfolkmedicine.Studieshavereportedimportantpharmacological

prop-erties,buttheseeffectshavestillnotbeenfullyexploited.Thisstudyreportsthatthecrudeextractand

isolatedcompoundsofS.terebinthifolius(leaves)haveinvitroantioxidant,antiproliferative,andinvivo

anti-inflammatoryactivities.Thesampleswereevaluatedforantioxidantactivityusing2,

2-diphenyl-1-picrylhydrazyl,␤-carotene/linoleicacidand2,2′_{-azino-bis-(3-ethylbenzothiazoline)-6-sulphonic}_acid

reagents.Theanti-inflammatoryeffectswereassayedagainstacarrageenan-inducedpawoedemamodel

inmicetotestdosesof10,100and300mg/kgatdifferenttimepointsinadditiontomyeloperoxidase

activityanalysis.Theantiproliferativeactivitywasevaluatedusingtenhumantumourcelllines.Two

derivativesofgallicacidandfourflavonoidswereisolatedandexhibitedconsiderableantioxidantactivity.

Theextractanditscompoundsshowedselectivitytowardsovariancancercells,withgrowthinhibitory

activityvaluesrangingfrom1.9to6.5␮g/ml.Sampleextractsandmethylgallatesignificantlyinhibited

carrageenan-inducedoedemainthemicepawoedemaexperimentalmodel.Thecalculatedtopological

polarsurfaceareaformethylgallate(86.98 ˚A2₎_showed_good_intestinal_absorption._The_effects_reported

hereinareberelatedtothepresenceofflavonoidsandthegalloylphenolicderivativecontent.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/

4.0/).

Introduction

Schinusterebinthifolius Raddi,Anacardiaceae,isan evergreen

shrub that grows in South and Central America. In Brazil,

it is popularly known as “pimenta-rosa”, “aroeira-vermelha”,

“aroeira-pimenteira”, “aroeira-da-praia”, “aroeira-negra” and/or “aroeira-de-minas”andusedinfolkmedicinefortreatmentof sev-eralhealthdisordersaswellanti-inflammatoryprocesses(Morton, 1978;Gazzaneoetal.,2005).Itsbiologicalapplicationshavebeen describedsincethefirsteditionoftheBrazilianPharmacopoeia,

published in 1926. A Brazilian gel-based aqueous bark extract

ofS.terebinthifoliushasbeenmarketedsince1999forthe treat-ment ofvaginitis and cervical vaginitis(Leiteet al.,2011).The

∗ _{Corresponding}_author.

E-mail:aneliseformagio@ufgd.edu.br(A.S.N.Formagio).

Brazilian Pharmacopoeia recommends the decoction of S.

tere-binthifoliusforuseasanaturalanti-inflammatoryagent(Santosand Amorim, 2002).Pharmacological studies withextractsobtained from leaveshave reported antioxidant,anti-allergic, antimicro-bial,anti-inflammatory,antiulcerandantiadherentpropertiesas wellaswound-healingproperties(CasteloBrancoNetodeetal., 2006;Carvalher-Machadoetal.,2008;Gomesetal.,2010;Johann etal.,2010;Carvalhoetal.,2013;Barbierietal.,2014;Ulianaetal.,

2016).Chemicalstudiesshowedthatpolyphenolicandflavonoid

aremajorconstituentsoftheextractsofS.terebinthifoliusleaves (Farag,2008;El-Massryetal.,2009;Santanaetal.,2012).

Investigationsbyourresearchgroupshowthattheessential

oilofS.terebinthifoliusfruitscontainsapredominanceof monoter-penes,with␤-pineneasthemajorconstituent.Thisoilwaseffective

againstpersistentinflammationcausedbyCompleteFreund

Adju-vant(CFA)oracuteinflammationinducedbycarrageenaninthe

paw or in pouches (Formagio et al., 2011). In another study,

http://dx.doi.org/10.1016/j.bjp.2016.12.007

(2)

theextractof theleaveshasanti-inflammatory, immunomodu-latory,chemopreventive,antigenotoxicandantimutageniceffects owingtophenolandflavonoidcompounds(Fedel-Miyasatoetal., 2014a,b).

Theoxidativedamageinducedincellsandtissuesisrelatedto theaetiologyofvariousdiseasesincludinginflammatoryand can-cerdiseases.Thus,thenaturalproductsarecandidatesforthese

testsbecausetheinsertionoffoodcompoundsor

phytopharma-ceuticalsmaybeanimportantalternativetotreatinflammation

andthepreventionofcancer.Althoughcancerisaspecificdisease, ithasbeenonlyslightlydefinedintermsoftraditionalmedicine. Recentcontributionstothearmamentofchemotherapeuticagents, inalliancewithnaturalproductsapprovedasdrugsinthis30-year timeframe,includepaclitaxel(Taxol®),isolatedfromTaxus brev-ifolia;thealkaloidsvincristineandvinblastinefromCatharanthus roseus;camptothecinandderivativesfromCamptothecaacuminata;

combretastatinfromCombretum caffrum(Newman etal., 2005;

Newmanand Gragg,2012); andcurcumin fromtherhizomeof Curcuma longa (Aggarwal and Bharti,2003)in addition to syn-theticderivativesorcombinationsofagentssuchasflavopiridol androscovitine(Newmanetal.,2002;DanceyandSausville,2003), justifyingtheimportanceofthesearchforcancertherapy.Even ifS. terebinthifolius hasbeenproposed asa folk remedyin the treatmentofinflammation,morestudiesmustbereported. There-fore,weevaluatedtheinvitroantioxidant,antiproliferative and

in vivo anti-inflammatory activities of methanolic extracts and

compoundsisolatedfromS.terebinthifolius(leaves).Asa comple-ment,acomputationalstudyforpredictingtheADMEpropertiesof

compoundswasperformedbydeterminingthelipophilicity,

topo-logicalpolarsurfacearea(TPSA),absorption(%ABS)andsimple moleculardescriptorsusingLipinski’srule.

Materialsandmethods

Plantmaterial

The leaves of Schinus terebinthifolius Raddi, Anacardiaceae,

werecollectedattheMedicinalPlantsGardenofFederal Univer-sityofGrandeDourados(22◦₁₁′_43.7′′_S,₅₄◦₅₆′_08.5′′_W_and₄₃₀_m)

in November 2014. A voucher specimen was deposited in the

HerbariumoftheUFGDunderthenumberDDMS4600andwas

identifiedbyDr.MariadoCarmoVieira.Authorizationfor

acces-singand studying samples from the Brazilian genetic heritage

site wasobtainedfromtheBrazilian governmentthrough

Con-selhoNacionaldeDesenvolvimentoCiêntíficoeTecnológico(CNPq

authorizationno.010220/2015-1–CNPq/CGEN/MMA).

Extraction,fractionationandidentificationprocedures

Leaves(860g) weredriedand extractedbymacerationwith

methanol, filtered, concentrated under reduced pressure and

lyophilized to yield the methanolic extract (MEST) (42.7g).

The MEST (30g) was partitioned with hexane, chloroform and

ethyl acetate.The chloroform fraction(1.8g) was submittedto

columnchromatography(CC)silicagel,yieldingsitosterol-3-O-␤ -glucopyranoside(64mg).Fractionationofpartoftheethylacetate

fraction(6.2g) by CCin silica gel wasperformed using a

mix-tureofhexane/EtOAcandEtOAc/MeOH,inincreasingpolarity,to

affordcompounds1(25.4mg),2(19.4mg)and3(12.2mg).The

hydromethanolicfraction(13g)waspurifiedbysuccessiveCCon SephadexLH-20usingH2O,H2O/MeOH7:3–3:7,andMeOHas

elu-ents,yieldingcompounds4(22.8mg),5(17.8mg)and6(26mg). The1_H_and13_CNMR_spectra_were_collected_using_a_Varian_Mercury

PlusBBspectrometeroperatingat300MHzand75.5MHzusing

CD3ODasthesolventandtetramethylsilane(TMS)astheinternal

standard.

HPLCanalysis

TheLCdatawithMESTincludecaffeicacid(Rt=14.78min),p -coumaricacid(Rt=25.82min),luteolin(Rt=62.08min),quercetin (Rt=64.62min)andapigenindata(Rt=66.79min).Thesedatawere

described in a previous report by our research group (

Fedel-Miyasatoetal.,2014a).

Invivoanti-inflammatoryactivity

Animals

Male Swiss mice (25–35g) were used for in vivo

anti-inflammatoryevaluationandwereprovidedbytheUniversidade

Federalda GrandeDourados.The micewerekeptundera 12h

light-darkcyclewithcontrolledhumidity(60–80%)and

temper-ature(22±1◦_C)._Two_hours_before_the_experiments,_the_animals

wereplacedinthelaboratoryandwereusedonlyoncefor experi-ments(n=5/group).Allexperimentalprocedureswereperformed inaccordancewiththeU.S.NationalInstituteofHealthandwere approvedbytheethicscommitteeforresearchonlaboratory ani-malsoftheUFGD(Nbr.005/2010).

Carrageenan-inducedpawoedema

Five groups of Swiss mice were orally treated with MEST

(100 and 300mg/kg) and 2 (100 and 300mg/kg) as well as a

vehicle.Twogroups weretreatedintraplantarly with2 (10and

100mg/kg).Onegroupofmicewastreatedsubcutaneouslywithan anti-inflammatorypositivecontroldrugdexamethasone(1mg/kg). After1h,theanimalsreceivedanintraplantarinjection(50␮l)ofa solutionofcarrageenan(300␮g/paw,dilutedinsterile0.9%saline) intotherighthindpaw.Thecontralateralpawreceivedonlysaline andwasusedasacontrol.

Theoedemawasthedifferenceinthicknessofbothpawsusing

adigitalmicrometre(DIGIMESS110-284)atseveraltimepoints

(0.5,1,2,and4h)aftercarrageenaninjection. Theresultswere expressedin␮m(Kassuyaetal.,2009).

Myeloperoxidaseactivity

Myeloperoxidase (MPO) activity was measured in the paw

after 6h to evaluate indirect neutrophil migration to this tis-sue(De Young et al., 1989).The paw tissue washomogenized

in 5%(w/v) 80mM phosphatebufferat pH5.4 containing0.5%

ofhexadecyltrimethylammoniumbromide.Thehomogenatewas

centrifugedat3200×gand4◦_C_for₂₀_min._Thirty_microliters_of

each supernatant was mixed with100␮l of 80mM phosphate

buffer,85␮lof0.22Mphosphatebufferand15␮lof0.017%H2O2

ina96-wellplate.Thereactionwasinitiatedwith20␮lof

3,3,3-tetramethylbenzidine(dissolvedinN,N-dimethylformamide).The

platewasmaintainedat37◦_C_for₃_min,_after_which_the_reaction

wasinterruptedbyadding30␮lof1.46Msodiumacetate(pH3.0).

Theenzymaticactivitywasdeterminedbymeasuringtheoptical

densityat630nmandwasexpressedasmOD/mgofprotein.

Invitroantiproliferativeactivity

MESTandothercompoundswereassessedinthefollowingten

humantumourcelllinesfromvarioustissues,kindlyprovidedby theNationalCancerInstitute(Frederick,MA,USA):U251(glioma,

CNS),MCF-7(breast),NCI-ADR/RES(ovarianexpressingthe

(3)

K-562(leukaemia)andHaCaT(human keratinocytes,

immortal-ized non-tumouralcell). The test results were measuredusing

thecolorimetricsulphorhodamineBmethod,accordingtotheNCI

standard protocol, and doxorubicin (0.025–25␮g/ml) wasused

aspositivecontrol(Monksetal.,1991).Assayswereperformed

ina 96-wellplateusing fourconcentrationsproduced bya

10-folddilution(0.25–250␮g/ml).Theactivitywasdeducedfromthe

concentrationresponse,and GI50 parameters(growthinhibitory

activity)werecalculated.

Invitroantioxidantactivity

DPPHradicalscavengingassay

SamplestocksolutionsofMEST(1.0mg/ml)andcompounds1–6

(0.1mg/ml)weredilutedtofinalconcentrationsof300,200,125,

50,25,10and5␮g/mlinmethanol.Sampleswereaddedto3ml

of methanolic DPPH (2, 2-diphenyl-1-picrylhydrazyl) (0.1mM)

andwereprepared daily,shaken,and leftatroomtemperature

in the dark for 30min. Absorbance was measured at 517nm

againsta blank containingallreagents exceptthe testsamples

(Brand-Williams et al.,1995).Assayswerecarried outin tripli-cate.ThepercentageofinhibitionofDPPH(I%)wascalculatedusing thefollowingequation:I%=(Ablank−Asample/Ablank)×100;theIC50

concentration,indicating50%inhibitionofDPPH,wasplottedina graphofI%versussampleconcentration.

ˇ-Carotene/linoleicacidassay

A␤-carotene-chloroformsolution(1ml)wasmixedwith20mg linoleicacidand0.2gTween40®,withsubsequentevaporationof thechloroform.Distilledwater(50ml)wasslowlyaddedwith vig-orousagitationtoformanemulsion.Emulsionaliquots(5ml)were

transferredwith0.2ml of extracts(1mg/ml)and isolated

com-pounds (0.1mg/ml) at different concentrations (10–200␮g/ml).

Control samples contained all reagents except the test

sam-ples (Jayaprakasha et al., 2001). An emulsion was added to

each tube, and the absorbance was read at 470nm for zero

time. Tubes were placed in a water bath at 50◦_C, _and

oxida-tionwasmonitoredbyabsorbanceat15-minintervalsuntilthe

colourof␤-caroteneinthecontrolsampledisappeared(105min).

The analyseswere performed in triplicate. Antioxidantactivity

(AA)wascalculated as the %inhibition relative tothe control: %AA=[1−(AsampleT0−AsampleT105)/(AcontrolT0−AcontrolT105)]×100.

ABTSradicalscavengingassay

MESTandcompoundstocksolutions(1mg/ml)weredilutedto

finalconcentrationsof250–5␮g/ml.Briefly,7mMof2,2′

-azino-bis-(3-ethylbenzothiazoline)-6-sulphonicacid(ABTS)and140mM

potassiumpersulphateweremixedandkeptinthedarkfor16h

atambienttemperature.Thereafter,3mlofABTS•+_solution_was

addedto30␮lsampleswithvaryingconcentrations.After5min,

theabsorbancewasmeasuredat734nmusinga

spectrophotome-ter(Djeridaneet al.,2006).TheABTS•+ _scavenging_activity_was

calculatedusingthefollowingequation:ABTSradicalscavenging activity(%)=(A_blank−A_sample/A_blank)×100.

InsilicostudyandLipinski’sruleoffive

Anin silico computational studyof the isolated compounds

(1–6)wasperformedbythedeterminationof Lipinski’s

param-eters, topological polar surface area (TPSA) and percentage of

absorption(%ABS)(Lipinskietal.,1997).Calculationswere

per-formed using the “Molinspiration online property calculation

toolkit” (http://www.molinspiration.com) (Ertl, 2014). The per-centageofabsorptionwasestimatedusingthefollowingequation: %ABS=109−[0.345×TPSA](Lipinskietal.,1997).

Lipinski’s“ruleoffive”(Zhaoetal.,2006)wasusedto evalu-atethedrug-likecharacterofthosecompounds.Generally,orally bioavailabledrugsfollowtheserules:theyhavefewerthanfive

hydrogenbonddonors,nomorethantenhydrogenbondacceptors,

amolecularweightbelow500Da,andanoctanol–waterpartition

coefficientlogPofnomorethan5.

Statisticalanalysis

All data are presented as the mean±S.E.M. The difference

betweengroupswasevaluatedbyanalysesofvariance(one-way

ANOVA)followedbytheTukeyorStudent–Newman–Keulstest.

Thenumberofanimalspergroupisindicatedinthelegends. Sta-tisticaldifferenceswereconsideredsignificantatp<0.05.

Resultsanddiscussion

In the present study, we characterized seven previously

known compounds, including one steroid, sitosterol-3-O-␤

-glucopyranoside; two gallic acid derivatives, 1,2,3,4,6-penta-O -galloyl-␤-glucopyranoside(1)andmethylgallate(2);andthefour followingflavonoids:robustaflavone(3),quercetin(4),quercetrin (5)andluteolin(6)fromS.terebinthifolius.Thestructuresof

com-poundswereelucidatedusing1Dand2DNMRspectraldataand

acomparisonof1_H_and13_C_NMR_reported_data₍_Agrawal,_1989;

Carvalher-Machadoetal.,2008;Ceruksetal.,2007).Thequercetin derivativesofthesugarunitandhydrogen,aswellastheposition oftheirlinkagetotheaglycone,weredeterminedusinga combi-nationof2DNMRexperiments.Tothebestofourknowledge,this studyisthethirdphytochemistrystudyofS.terebinthifoliusleaves andthefirstreportofluteolinfrommethanolicextract,conducted duringthepreviousHPLCdatastudy.

The antioxidant activity was initially evaluated for S. tere-binthifoliusMEST.Theresultsshowedthatthesamplepresented

potent antioxidant activity when tested against DPPH (IC50

12.32±1.50␮g/ml), ␤-carotene/linoleic acid(AA=70.44±0.88%) and ABTS (AA=86.94±2.04%) (Table 1). Fractionation of these extractsbysolventpartitionandpurificationbyachromatographic

columnprovidedsixphenoliccompounds.Theresultsshowedthat

phenolics(2),hydrolysedtannins(1)andflavonoids(4and6)were activeinallassays,comparabletothenaturalantioxidant ascor-bicacid(Table1).Methylgallate(2)and quercetin(4)are also

considered natural antioxidants. Anacardiaceaespecies showed

(4)

Table1

Antioxidant assays of MEST and phenolic compounds (2–7) from Schinus

terebinthifolius.

Sample DPPHIC50a_(95% confidencelimit)

␤-Carotene/linoleic acidAA(%)

ABTSAA(%)

MEST 12.32±1.50c (11.01–14.86)

70.44±0.88b 86.94±2.04ab

1 3.03±0.89a (2.54–3.10)

87.57±2.28a 90.41±1.84a

2 5.54±1.55b (5.30–5.78)

86.26±2.84a 77.65±1.06c

3 19.44±1.89d (18.64–20.11)

68.92±0.50b 65.72±0.83d

4 4.03±1.57ab (3.88–4.12)

84.40±2.13a 83.07±3.34b

5 20.43±0.54d (18.10–21.66)

62.18±0.64c 63.21±1.55d

6 4.57±2.43ab (3.35–5.08)

86.36±2.18a 85.32±1.32b

Ascorbicacid 3.99±0.36ab (14.3–20.1)

88.37±1.91a 87.32±1.33ab

Values are expressed as the mean±SD (n=3); n.d.= not determined; a_IC50₌_{concentration} _resulting _in _50% _inhibition _of _DPPH, _derived _from _the

graphofI%(inhibition percentage)versus concentrationin␮g/ml(MEST and ascorbicacid) and␮M (2–7compounds).%AA=antioxidant activity,evaluated usingthe␤-carotene/linoleicacidandABTSmethods.Differentsuperscriptletters indicatestatisticallysignificantdifferencesineachline(p<0.05)usingtheTukey test.

andHarpephyllumcaffrum(Moyoetal.,2010).Thesecompounds exhibitedasimilarscavengingactivityasourextract,andthis

activ-itywasattributedtovariouscompoundsfoundinthesespecies,

includingphenolicacids,flavonoidsandtannincompoundsintheir crudeextracts.

OurdatashowedthattheIC50andAA%valuesindicated

correla-tionswiththepresenceofthesugarunit,hydroxylorhydrogenof theaglyconelinkedatC-3,whichwereduetothesterichindrance betweentheC-3substituentandtheBringoftheflavonol,whose decreasesignificantlyenhancedfreeradical-scavengingability,as evidencedbycompound5.Thecatechol,␣,␤-unsaturatedcarbonyl moietyand␤-hydroxyketonegroupsconferredhigherstabilityto theradicalformandparticipatedinelectrondelocalization(Pietta, 2000).Compound3,whichbelongstotheflavonesubclass,didnot presentcatecholgroupsinthestructure,whichlikelyexplainsthe inactivityofthesample.

Lipinski’s “rule of five” [molecular weight (MW)≤500Da,

logP≤5, H-bond donors (HBD)≤5 and H-bond acceptors

(HBA)≤10], topologicalpolarsurface area (TPSA)and per cent

absorption (%ABS) were calculated and presented in Table 2.

MoleculesviolatingmorethanoneofLipinski’sparametersmay

haveproblemswithbioavailabilityandahighprobabilityoffailure todisplaydrug-likecharacter.Fromthedataobtained,2and4were

foundtoobeyLipinski’srule,showinggoodpermeabilityinthe

cellularplasmatic membrane(78.79% and 50.33%,respectively).

ThecomputationalTPSAfor3(86.98 ˚A2₎_showed_good_intestinal

absorption,whereas1,3and6violatedthreeparameters. Freeradicalspeciesarealsoresponsibleforactivatingseveral pro-inflammatorytranscriptionfactorsinvolvedinthepromotion

ofinflammatory diseases.Theanti-inflammatory activity(MEST

and 2)was evaluated using carrageenan-induced paw oedema

andassessmentofmyeloperoxidase(MPO).Asshowninthe

lit-eratureresults,carrageenaninjection-inducedpawoedemawas

observedatalltime pointsinthepresentwork(Fig.1A).MEST

treatmentsinhibitedoedemaformationwith33±9(100mg/kg)

0.0 0.5 1.0

***

C

Vehicle 100 300 1 mg/kg MEST (p.o) DEX (s.c.)

6 hours after carrageenan (300µg/paw)

Activity of MPO (D.O)

0 1 2 3 4

0 425 850

Vehicle MEST 300 mg/kg DEX 1mg/kg

*

Time (h) after carrageenan injection (300µg/paw)

A

Pa

w

oede

ma

(

µ

m)

0 500 1000

*

_*

**

Vehicle 100 300 1 mg/kg

MEST (p.o) DEX (s.c.)

B

Fig.1.Anti-inflammatoryeffectofMESTadministrationoncarrageenan-inducedpawoedemainmice.AnimalsreceivedMEST(100or300mg/kg,p.o.),dexamethasone

(DEX,1mg/kg,s.c.)oravehiclecontrol.After1h,anintraplantarinjectionofcarrageenan(300␮g/paw)wasperformed.In(A),thetimecourseofinhibition,inducedby

300mg/kgMESTandDEX,ispresented.In(B),thebarsindicatetheeffectofvariousMESTandDEXdosesinpawoedema(␮m)2haftercarrageenaninjection.In(C),MEST

didnotaltertheincreaseinmyeloperoxidase(MPO)activityinducedbylocalcarrageenaninjections.Thebarsexpressthemean±SEMoffiveanimals.Comparisonswere

(5)

Table2

Lipinski’sparameters,topologicalpolarsurfacearea(TPSA)andpercentageofabsorption(%ABS)ofthecompounds(2–7).

Lipinski’sparameters

Compound %ABS TPSAa_{( ˚A}2₎ _n_HBAa₍_n_ON) _n_HBDa₍_n_OHNH) _log_Pa _MWa _n_violationsa

1 −37.26 423.95 25 14 3.65 938.70 3

2 78.99 86.98 5 3 0.84 184.14 0

3 46.28 181.79 10 6 5.16 538.46 3

4 63.68 131.35 7 5 1.68 302.23 0

5 50.33 170.04 10 6 1.13 432.38 1

6 18.12 263.42 16 10 −1.063 610.52 3

a_{www.molinspiration.com}_;_%ABS₌₁₀₉₋_0.345_×_TPSA;_number_of_hydrogen_bond_acceptors_(NO)₌_n_HBA_≤_10;_number_of_hydrogen_bond_donors_(OHNH)₌_n_HBD_≤_5;

MW≤500;octanol–waterpartitioncoefficient=logP<5.

0 200 400 600 800 1000

**

A

Vehicle 100 300 1 DEX 2

mg/kg Vehicle 100 300 1 mg/kg

Paw oedema (

µ

m)

0.0 0.5 1.0 1.5

B

DEX 2

*

A

c

ti

v

ity

o

f M

P

O

(

D

.O

)

0.0 0.5 1.0 1.5

D

Vehicle 10 100 1 2 DEX

**

mg/kg

Activity of MPO (D.O)

0 200 400 600 800 1000

*

**

Vehicle 10 100 1 2 DEX

C

mg/kg

Paw oedema (

µ

m)

Fig.2.Anti-inflammatoryeffectofmethylgallate(2)oncarrageenan-inducedpawoedemainmice.Animalsreceived2orally(100and300mg/kg,p.o.),2intraplantarly

(10and100mg/kg,p.o.),dexamethasone(DEX,1mg/kg,s.c.)oravehiclecontrol.After1h,themicereceivedanintraplantarinjectionofcarrageenan(300␮g/paw).In(A),

thebarsindicatetheeffectsofvariousdosesof2andDEXinpawoedema(␮m)2haftercarrageenaninjection.In(B),2didnotinhibitacarrageenan-inducedincreasein

myeloperoxidase(MPO)activity.In(C),barsdemonstratetheeffectsofvariousdosesofintraplantarinjectionsof2(10and100␮g/paw)andDEXonpawoedema(mm)2h

aftercarrageenaninjection.In(D),2didnotinhibitcarrageenan-inducedincreaseofMPOactivity.Thebarsexpressthemean±SEMoffiveanimals.Comparisonsweremade

betweenthevehicleandthetreatedgroups.*p<0.05,**p<0.01,***p<0.001,one-wayANOVAfollowedbytheStudent–Newman–Keulsmethod.

and34±6%(300mg/kg)reductions(Fig.1AandB).Moreover,our resultsindicatedthat300mg/kgoftheMESTdisplayedsignificant inhibition2hafteradministration,asindicatedbythetimecourse analysis(Fig.1A).Inaddition,41±5%inhibitionwasobservedin

thedexamethasone-treatedgroup2haftercarrageenaninjection

(Fig.1A).OralMESTtreatment(100and300mg/kg)didnotalter theincreaseinMPOactivityinducedbycarrageenan.Thepositive

control(dexamethasone)inducedinhibitoryactivityintheMPO

analysiswhencomparedwiththecontrolgroup(Fig.1C). Scien-tificworkswithS.terebinthifoliusshowedthatextractsfromleaves exhibitedtopical(dosesof0.1,0.3and1mg/ear)(Fedel-Miyasato etal.,2014a)andsystemicanti-inflammatorypropertiesinanother

inflammatory model including croton oil-induced ear oedema,

arthritis and air pouch models in mice (Fedel-Miyasato et al.,

2014a;Rosasetal.,2015).Previousstudiesusingwound-healing modelsshowedthatextractsfromtheleavesofS.terebinthifolius

wereeffectivefor woundhealing (Nunes etal.,2006; Coutinho

etal.,2006;Martorellietal.,2011).Inrats,Fedel-Miyasatoetal. (2014a)showedthatMEST(80mg/ml)topicalapplication signifi-cantlydecreasedthediameterofthewound.Inthepresentwork, theextractwastestedsystemically(oralroute)inadifferentmodel ofinflammationandcompound3wasassayedviabothanoralroute andlocalinjection(intraplantarinjection).

Compound2alsoexhibitedanti-edematogenicactivity,

inhib-itingapproximately43±9%(100mg/kg)and58±5%(300mg/kg)

(Fig.2A).Dexamethasoneinhibitedoedemaformation(51±4%)2h afterinflammatorystimulus(Fig.2A).Animalstreatedwith intra-plantarcompound2injectionsdisplayed34±2%(10␮g/paw)and 39±8%(100␮g/paw)inhibition(Fig.2C).Oral(100and300mg/kg)

orintraplantar(10and100␮g/paw)administrationofcompound

2didnotalterMPOactivitycomparedtothecontrolgroup(Fig.2B andD).Theanti-inflammatoryeffectsofMESTwereassociatedwith increasedlevelsofmethylgallate(2),whichisalsofoundinother

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A

100

75

50

25

0

–25

–50

–75

–100

100

75

50

25

0

–25

–50

–75

–100

100

75

50

25

0

–25

–50

–75

–100 100

75

50

25

0

–25

–50

–75

–100

10–3 ₁₀–2 ₁₀–1

Cellular growth, %

Cellular growth, % Cellular growth, %

Cellular growth, %

Concentration (µg/ml)

Concentration (µg/ml) Concentration (µg/ml)

Concentration (µg/ml)

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

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

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

0.25 ₁₀02.5 ₁₀125 ₁₀2250

10–3 10–2 10–10.25 1002.5 10125 102250 10–3 10–2 10–10.25 1002.5 10125 102250

10–3 ₁₀–2 ₁₀–10.25 ₁₀02.5 ₁₀125 ₁₀2250

C

D

B

Fig.3.Antiproliferativeactivity.InA:MEST;B:compound1;C:compound5;D:compound6.U251(glioma,CNS),MCF-7(breast),NCI-ADR/RES(ovarianexpressingthe

multipledrugresistancephenotype),786-0(renal),NCI-H460(lung,non-smallcells),PC-3(prostate),OVCAR-3(ovarian),HT-29(colon),K-562(leukaemia)andHaCaT

(humankeratinocytes,immortalizednon-tumouralcells).GI50:concentrationsthatelicit50%inhibitionofcellgrowth(in␮g/ml).

L,andA.saccharumMarsh(Whangetal.,2005;Kimetal.,2006; Abou-Zaidetal.,2009).

The results demonstrated that sample MEST possesses

in vitro anticancer activity, with GI50 values ranging from 6.3

to9.4␮g/mlwithselectivityforprostate(PC-3)(GI50=6.3␮g/ml),

ovarian (OVCAR-3) and resistant ovarian- (NCI-ADR/RES)

(GI50=6.5␮g/ml), breast (NCI/H460) (GI50=7.6␮g/ml), glioma

(U251) (GI50=9.1␮g/ml) and breast (MCF-7) (GI50=9.4␮g/ml)

cancerandforHaCat(GI50=8.1␮g/ml)non-tumouralcells(Fig.3A).

Furthermore, 1,2,3,4,6-penta-O-galloyl-O-␤-glucopyranoside

(1) demonstrated high activity with GI50<5.00␮g/ml against

resistant ovarian (NCI-ADR/RES), breast (NCI/H460)

(GI50=1.9␮g/ml), leukaemia (K-562) (GI50=2.2␮g/ml), ovarian

(OVCAR-3)(GI50=2.5␮g/ml)andcolon(HT-29)(GI50=4.9␮g/ml)

cancercells (Fig.3B). Quercetrin (5)showed inhibitoryactivity

against prostate (PC-3) (GI50=2.5␮g/ml), ovarian (OVCAR-3)

(GI50=4.1␮g/ml), (HaCaT) (GI50=4.3␮g/ml), ovarian

(NCI-ADR/RES) and breast (NCI/H460) (GI50=4.4␮g/ml) cancer

cell lines (Fig. 3CD). Luteolin (6) showed activity against

OVCAR-3 (GI50=1.3␮g/ml), K562 (GI50=4.5␮g/ml) and

HaCaT (GI50=17.4␮g/ml) cell lines (Fig. 3D). Compound 3,

robustaflavone,didnotshowinhibitoryactivityagainstanycell lines(GI50>100␮g/ml)(datanotshown).Ingeneral,S.

terebinthi-foliusand itscompoundsshowedthehighestgrowthinhibitory

activities towards ovarian cancer cells (NCI-ADR/RES;

OVCAR-3) which showed GI50 values in the 1.3–6.5␮g/ml range. The

Anacardiaceae plant species has already been recorded in the

literature, withvaried growth inhibition potential for different cancercelllines(Kimetal.,2013).Studiesreportthat␣-pinene

isolated from S. terebinthifolius leaves induces apoptosis and

confersantimetastaticprotectioninamelanomamodel(Matsuo

etal.,2011).

In another study by our group, the in vitro

antiprolifera-tive activityagainst ten human cancercell lines of a series of galloylderivativesbearingsubstituted-1,3,4-oxadiazoleand

carbo-hydrazidemoietieswasevaluated.Theresultsdemonstratedthat

methylgallateandintermediarygalloylhydrazideshowedgreat

antiproliferative activity,with GI50 values<5.54␮M,against all

humantumourcelllinestested(DaSilvaetal.,2015).Therefore, since2exhibitedseveralactivities,thisantioxidantagentisagood naturalproductleadforstructuralmodificationstudiesyielding newagents(Asnaasharietal.,2014).Thisresultisinaccordance withLipinski’sRuleofFive,TPSAand%ABS,whichareimportant forfurtherdevelopmentofdrugsbaseduponthesemoieties.

Conclusions

ThisstudydemonstratedthatMEST,obtainedfromleaves

col-lectedinDourados-MS,haspotentialanti-inflammatoryactivity,

whichsupportspreviousclaimsregardingthetraditionaluseof

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S.terebinthifoliusleaves.Themethanolicextractandcompounds

showed the highest growth inhibitory activity, with particular

effectivenessagainstovariancancer(NCI-ADR/RES;OVCAR-3)cell lines.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave

fol-lowed theprotocolsof theirworkcenter onthe publicationof

patientdata.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

MCVidentifiedandcollectedplantmaterial.ASNF,MMSand

KPSpreparedthemethanolicextractandphytochemistrystudy,

assessed antioxidant activity and helped to write and edit the

manuscript.CALKandEKKIdesignedtheanti-inflammatoryassays. MAF,JECandALTGRcontributedtotheantiproliferativeassay.All

authorshavereadandapprovedthefinalmanuscriptfor

submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbytheConselhoNacionalde

Desen-volvimento Científico e Tecnológico (CNPq, Brazil). We thank

CAPES,CNPqandFundectforfellowships(A.S.N.F,M.A.F,J.E.C.and M.M.S).

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