Rev. bras. farmacogn. vol.25 número2

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Anti-inflammatory

sesquiterpene

lactones

from

Tithonia

diversifolia

trigger

different

effects

on

human

neutrophils

Aneli

E. Abe

a

_,

_Carine

_E.

_de

_Oliveira

a

_,

_Thalita

_M.

_Dalboni

a

_,

_Daniela

_A.

_Chagas-Paula

b

_,

_Bruno

_A.

_Rocha

b

_,

Rejane

B. de

Oliveira

b

_,

_Thais

_H.

_Gasparoto

a

_,

_Fernando

_B.

_Da

_Costa

b

_,

_Ana

_P.

_Campanelli

a,∗

a_Department_of_Biological_Sciences,_Universidade_de_São_Paulo,_Bauru,_SP,_Brazil

b_AsterBioChem_Research_Team,_Laboratory_of_{Pharmacognosy,}_Department_of_{Pharmaceutical}_Sciences,_School_of_{Pharmaceutical}_Sciences_of_Ribeirão_Preto,_Universidade_de_São

Paulo,RibeirãoPreto,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received13July2014 Accepted19January2015 Availableonline23March2015

Keywords: TagitininA TagitininC TagitininF Humanneutrophils Anti-inflammatoryactivity

a

b

s

t

r

a

c

t

ThetagitininsisolatedofTithoniadiversifolia(Hemsl.)A.Gray,Asteraceae,arethemoststudied

sesquiter-penelactonesduetotheirwidespectrumofpharmacologicactivities,especiallyrelatedwithnuclear

factor-kappaBinhibition.Nevertheless,detailedstudiesaboutthemechanismofactionofitsactive

compoundsarestilllacking.Neutrophilsperformafundamentalroleintheinflammatoryresponseto

severaletiologicfactors.However,theeffectoftagitininsonhumanneutrophilisnotyetclearlyknown.

WeinvestigatedtheroleoftagitininC(1),tagitininF(2)andtagitininA(3)inactivationandsurvivalof

humanneutrophilstoestablishpossibleeffectsintheirmechanismsofinflammation.Humanneutrophils

werepurifiedfromtheperipheralbloodandcultivatedwithtagitininsC(1),F(2)andA(3)inthepresence ornotofEscherichiacolilipopolysaccharide.Theenzymaticactivity,apoptosisandsecretionofcytokines

rateweredeterminedafter18h.Lipopolysaccharide-inducedmyeloperoxidaseactivityofhuman

neu-trophilswassignificantlyinhibitedonlybytagitininF(2).Apoptosisofneutrophilswasincreasedin

thepresenceoftagitininC(1),anditoccurredindependentlyofthepresenceoflipopolysaccharide

ordexamethasone.TagitininsC(1),F(2)andA(3)decreaselipopolysaccharide-inducedinterleukin-6,

interleukin-8andTumornecrosisfactoralphaproductionbyhumanneutrophils.Together,theseresults

indicatethattagitininsexhibitanti-inflammatoryactiononhumanneutrophils.However,tagitininF(2)

wastheonlysesquiterpenelactonethatdecreasedsecretionofinflammatoryproductsbyneutrophils

withoutinducingneutrophilapoptosis.

Introduction

Tithoniadiversifolia(Hemsl.)A.Gray,Asteraceae,isamedicinal plantthatisknownworldwideandseveralbiologicalproperties havebeenreportedforthisspecies,suchasanti-parasitic, antimi-crobialandanti-inflammatory,amongothers(Chagas-Paulaetal., 2012).T.diversifoliaisanimportantsourceofbiologicallyactive natural products such as terpenoids and phenolic compounds (Chagas-Paulaetal.,2012).Amongtheterpenoids,the sesquiter-penelactones(STL)comprisethemoststudiedclassbecausethey are frequentlythemajor compounds present in activeextracts andfractionsfromleavesandflowers(Chagas-Paulaetal.,2012). AlthoughSTLareplant-derivedcompoundswithremarkable anti-inflammatoryandantitumoractivity,theyareusuallyknownfor

∗ Correspondingauthor.

E-mail:[email protected](A.P.Campanelli).

theirtoxicproperties (Chagas-Paula et al.,2011,2012;Merfort, 2011; Ghantous et al., 2010; Schmidt, 1999). Among all com-pounds fromT. diversifoliaalready tested forbiological activity, thetagitinins(Tags),STL,haveshownrelevantactivityindiverse pathologicsituations(Chagas-Paulaetal.,2012;Guetal.,2002;Liao etal.,2011;Sánchez-Mendozaetal.,2011;Zhaoetal.,2012).At themolecularlevel,atleastpartially,theanti-inflammatoryeffect ofSTLcanbeexplainedbytheinhibitionoftheactivationofthe nuclearfactor-kappaB(NF-␬B)(Merfort,2011;Siedleetal.,2004). NF-␬Bfamilycomprisesagroupoftranscriptionfactorsthat reg-ulatesinduciblegeneexpressioninvariousphysiologicalcontexts butitisbestknownforitsfunctionsinimmunity,inflammation, andoncogenesis(GhoshandHayden,2008;Suganumaetal.,2002; Tornatoreetal.,2012).Bacterialproducts,suchas lipopolysaccha-ride(LPS),induceneutrophilactivation,anditislargelyknownto resultinmyeloperoxidase(MPO)releaseand,throughitsbinding withreceptorsonneutrophilmembranetoactivateNF-␬B(ElKebir andFilep,2013;Xiaoetal.,2014).Then,resultingNF-␬Bactivation

http://dx.doi.org/10.1016/j.bjp.2015.01.005

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inthesecellscouldgeneratepro-survivalsignalsanddecreased apoptosis being important to trigger defense strategies of the immunesystem(ChenandChen,2013;KebirandFilep,2013). Neu-trophilsarethefirstleukocytestoberecruitedtoaninflammatory site,andtheyhavediverserolesininfection,inflammationand can-cerimmunology(Borregaard,2010;Gallietal.,2011;Futosietal., 2013).Althoughneutrophilshavelongbeenconsideredexclusively pro-inflammatoryandtheirlifespanisprolongedbyinflammatory cytokines,thereis increasingevidencethat sometypesof neu-trophilsmayexhibitanti-inflammatoryorhealingcharacteristics (KolaczkowskaandKubes,2013).Consideringsuchinformationit ispossibletostatethatthedepletionofneutrophilscouldresultin harmfullevelsofimmunosuppression(KolaczkowskaandKubes, 2013).Forthesereasons,theideaofmanipulatingneutrophilsas aformofimmunotherapyduringinflammatoryprocessesshould beevaluatedcarefully.Therefore,effectsofSTLasattemptingto controlinflammationthroughinhibitionofNF-␬Bactivationmay negativelyinfluenceneutrophilfunctionresultingin immunodefi-ciency.

Inmodernmedicine,theuseofplantsasmedicinaltreatment forseveraldiseaseshasbeenstimulated.Theuseofplantsinorder tomedicatediseaseisalmostuniversalamongnon-industrialized societies since pharmaceuticals are prohibitively expensive for mostoftheworld’spopulation(DaSilvaetal.,2002).

BecausethepharmacologicalpropertiesobservedforT. diver-sifoliaextractsmight beuseful for manipulation of neutrophils (Chagas-Paulaetal.,2012),weinvestigatedtheeffectsoftheTags C(1),F(2)andA(3)onhumanneutrophils.

HO OH

HO

HO O

O

O O

O O O

O

O O O

O

O O O

1 2

3

Materialsandmethods

Material

LPSfromEscherichiacoli,dimethylsulfoxide(DMSO)and dexa-methasone(Dexa)werepurchasedfromSigma–Aldrich(St.Louis, Missouri,MO).AposcreenAnnexinV-FITCwaspurchasedfromR&D Systems(Minneapolis,MN,USA).

Plantmaterial

LeavesfromTithoniadiversifolia(Hemsl.)A.Gray,Asteraceae, werecollectedbyD.A.Chagas-Paulain March2008,inRibeirão Preto,SP, Brazil (S21◦_10.681′_; _W₀₄₇◦_51.541′_; _altitude₅₃₈_m).

A voucher specimen (R.B. Oliveira#497) was deposited in the

herbariumSPFRoftheDepartmentofBiologyFFCLRP,USP,Ribeirão Preto,SP,Brazil.Entireleaveswereairdriedat40◦_C_for_a _week

andkeptinhumidityandlightfreeconditionsuntiltheextraction processwasstarted.

ExtractionandisolationoftheTagsA,CandF

Theextractwasobtainedfrom2.0kgofentireanddriedleaves individually rinsedfor20swithacetone.The freshextract was filteredthroughcommonfilterpaper,andaftersolvent evapora-tionunderreducedpressure,thedriedresiduewasresuspendedin 10mlofMeOH–H2O(7:3,v/v)toprecipitatelipophilicmaterial.The

precipitatewasdiscarded,andthesolventfromthesupernatant wasevaporatedunderreducedpressure.Theextract(20g)was par-titionedwithn-hexanefollowingbyEtOAcpartition. TheEtOAc fraction(5g) was submittedtovacuum liquid chromatography using500gofsilicagel60H(Merck,Brazil,art.no.7736)onaglass column(115mmofdiameter)andelutingwithn-hexanefollowed by increasing concentrations of EtOAc, furnishing 30 fractions [fractions1–2,500mlofn-hexane/fraction;fractions3–6,250ml of n-hexane/EtOAc (75:25, v/v)/fraction; fractions 7–8, 250ml of n-hexane/EtOAc(1:1, v/v)/fraction;fractions 9–12, 125ml of

n-hexane/EtOAc(1:1,v/v)/fraction;fractions13–18,125mlofn -hexane/EtOAc (25:75, v/v)/fraction; fractions 19–24, 125ml of

n-hexane/EtOAc (1:9, v/v)/fraction; fractions 25–30, 125ml of EtOAc/fraction].The fractions wereevaluated and further com-binedbyTLC(pre-coatedaluminumsheetswithsilicagel60F254; Merck,Brazil,art.no.5554)elutingwithn-hexane/EtOAc1:1and 1.5%ofHOAcandrevealedwithvanillin–sulfuricacid.Thefractions 17–20(1.1g)weregroupedandfurtherpurifiedbyRP-HPLC(ODS C18column20mm×250mm,Shimadzu,Japan;Proeminence Shi-madzuchromatographlinkedtoaCBM20controller,UV/visible detectorSPD-20,LC6ADpumpsandautomaticfractioncollector FCR-10,Shimadzu)onisocraticmodewithMeCN–H2O(45:55,v/v),

flowrateof10ml/min,togivepureTagC(1)(20.8mg)andF(2) (6.1mg).OnegramoftheEtOAcfraction(seeabove)was fraction-atedbyflashchromatography(20mm×140mmglasscolumn,17g ofsilicagel0.040–0.063mm,Merck,Brazil,art.no.9385)usingthe followingsolventsasmobilephase(flowrateof5cm/min):30ml ofCHCl3(fractions1–3),120mlofdiethylether(fractions4–15),

and100mlofEtOAc(fractions16–25).Thefractionswereevaluated byTLC(seeabove).Thefractions14–17(42.3mg)werecombined andfurtherpurifiedbyRP-HPLC(seeabove)tofurnish18.2mgof pureTagA(3).Thestructuralelucidationofthethreecompounds wascarriedoutby1_H_and 13_C_NMR _and_the_spectral _data_were

comparedwiththosefromauthenticmaterialanddatafromthe literature(Baruahetal.,1979;Zderoetal.,1987).

Samplepreparation

Priortothebioassays,thecompoundsweredissolvedinDMSO (0.1%inRPMI1640medium).

Neutrophilisolation

Human neutrophilswere isolatedfrom theperipheralblood of healthy donors by Histopaque 1119 and 1083 gradients (Sigma–Aldrich,Germany),asdescribedbyDalbonietal.(2013). Neutrophils purity was assessed by Giemsa staining (phase-contrastmicroscopy)andviablebyTrypanblueexclusion.

Neutrophilsculture

Humanneutrophilsweresuspendedatadensityof1×106_cells

permlandincubatedat37◦_C_in_5%_CO

2.ForSTLstimulationstudies,

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for21hinthepresenceorabsenceofLPS(10ng/ml),DMSO(1␮l) orDexa(100␮M).Dexawasusedaspositivecontrolbecauseithas beenlargelyrelatedtoananti-inflammatorymechanisminvolving neutrophils(Calouetal.,2008;Vigiletal.,2008;Tsuchihashietal., 2002;Chinetal.,2000).

MPOactivity

MPO activity was determined by enzymatic reaction as described byDalboni et al.(2013). Neutrophilswere harvested aftercultureandcentrifugedat350×gfor15min,andthepellet was frozen at −20◦_C. _The _pellet _was _then _liquefied _and

cen-trifugedtwiceat10,000×gfor15minat4◦_C._The_MPO_activity

inthesuspendedpelletwasassayedbymeasuringthechangein absorbanceat450nmusingtetramethylbenzidine(1.6mM)and H2O2(0.5mM).

Detectionofapoptosis

Apoptotic cells were identified by staining with annexin V–fluorescein,aspreviouslydescribed(Tessarollietal.,2010).In addition,theviabilityofneutrophilswasalsoanalyzedby fluores-cencemicroscopy(AxiostarplusHBO50/AC,CarlZeiss,Germany). Thepercentage of apoptotic cells wascalculated fromthe pro-portionofneutrophilspositivityforAnnexin-V-FITCorpropidium iodide(greenand/orredcells)inrelationtothetotalnumberof neutrophils(Tessarollietal.,2010).

Cytokinesandchemokinedetection

Interleukin-6(IL-6),interleukin-8(CXCL8),tumornecrosis fac-toralpha(TNF-␣)productionwerequantifiedbythequantitative ELISAusingcommercialKITS(BDPharmingenCorp.,SanDiego,CA, USA)accordingtothemanufacturer’sinstructions.

Statisticalanalysis

Statistical analysis and graphical representations were per-formedusingGraphPadPrism(version5forWindows,GraphPad Software,SanDiego,CA,USA).One-wayANOVAfollowedby Bon-ferroni’stestwasusedfortheanalysis.

Results

TagF(2)negativelyinfluencedMPOactivityonLPS-stimulated neutrophils

MPOisaheme-containingperoxidasehighlyexpressedby neu-trophilsandreleasedwhentheyarestimulated(Malleetal.,2007). WetheninvestigatedthedirecteffectsofTagsC(1),F(2)andA(3) at100␮Minneutrophils’MPOactivity.Neutrophilsactivationwith LPSinducedsignificantlyincreasedMPOactivity(Fig.1A). Impor-tantly,TagF(2)andDexasignificantlyinhibitedMPOactivityby LPS-stimulatedneutrophils(Fig.1A).Incontrast,treatmentwith TagsC(1)andA(3)increasedtheMPOactivity.Thesedata there-foredemonstratedthatTagF(2)mayaffectenzymaticactivityof neutrophilsmodifyingtheirinflammatorymachinery.Asexpected, DexasignificantlydecreasedMPOactivity.

TodetermineifthereductionofMPOactivityafterTagF(2) stim-ulationcouldbeconsequenceofneutrophildeath,weanalyzedthe apoptosisrate(Fig.1B).Wedidnotdetectsignificantdifferences inapoptosisratein unstimulatedneutrophilsand LPSor Dexa-stimulatedneutrophils.Inaddition,neutrophilsapoptosiswasnot alteredafterstimulationwithDexaplusTags(Fig.1B).More impor-tantly,thestimulationofLPS-stimulatedneutrophilswithTagF(2) didnotinduceincreaseinapoptoticrates(Fig.1B).Ontheother

1500

1000

500

0

No treatment

Tag A Tag C Tag F 20

40 60 80

My

elopero

xidase activity

Medium

LPS Dexa

A

Apoptosis (%)

B

Fig.1.MPOactivityandapoptosispercentageoftagitinins-treatedhuman neu-trophils.Purifiedneutrophils(1×106₎_were_cultivated_with_10%_SFB_RPMI₁₂₄₀

(whitebars),LPS(10ng/ml)(blackbars),dexamethasone(100␮M)(graybars)only (notreatment)orassociatedwithtagitininA(3),C(1)orF(2)(100␮M)and,after 12and21h,respectively,toapoptoticcells(A)andMPO(B)weremeasuredas describedinMaterialsandMethods.Theresultsareexpressedasmean±SEMfor eachvolunteertestedindividually.Theexperimentswereperformedintriplicate. Theresultswereevaluatedbyone-wayANOVAfollowedbyBonferroni’spost-test. *p<0.05whentreatedgroupswerecomparedwithnotreatmentdata.

hand,TagC(1)significantlyinducedneutrophilapoptosis(Fig.1B). SuchanapoptosisrateafterTagC(1)stimulationoccurredeven whenLPSorDexawereaddedintothecultures(Fig.1B).TagA(3) didnotalterapoptoticratesinLPS-stimulatedneutrophils(Fig.1B). NosubstantialdifferencewasfoundinrelationtoMPOactivityand apoptosisratewhenneutrophilswerecultivatedwithTagsat1or 10␮M(datanotshown).

Our resultsindicatethat thedecreased MPOactivityof neu-trophilswasnotaconsequenceoftheirapoptosis.Togetherthese observationsalsoindicatethattheimpactofTagF(2)uponMPO activitydidnotinfluenceneutrophilsurvival.

CXCL8,IL-6andTNF-˛releasefromhumanneutrophilsare reducedinthepresenceofTagsC(1_),_F₍2₎_and_A₍3₎

Activated neutrophils secrete a variety of pro-inflammatory cytokines,forexampleIL-1,IL-6,CXCL8,andTNF-␣(Cassatellaetal., 1997;Dalbonietal.,2013).ToascertainifstimulationwithTagsC (1),F(2)andA(3)alteredhumanneutrophilsactivation,weisolated humanneutrophilsandincubatedthemwithLPSinthepresence orabsenceofTagsfor18h.WedetectedCXCL8,IL-6,andTNF-␣

insupernatantsfromLPS-stimulatedneutrophils.TagsC(1),F(2) andA(3)at100␮MsignificantlydecreasedCXCL8,IL-6andTNF-␣

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0

0 50 100 150 200 250 300 350 20 40 60 75 150 225 300 8 16 24 75 100 250 300 350

CXCL8 (pg/mL)

Medium

LPS

Dexa

A

IL-6 (pg/mL)

B

TNF-α

(pg/mL)

C

No treatment

Tag A Tag C Tag F

Fig.2. Detectionofcytokinesproducedbytagitinin-treatedhumanneutrophils. Purifiedneutrophils(1×106₎_were_cultivated_with_10%_SFB_RPMI₁₂₄₀_(white_bars),

LPS(10ng/ml)(blackbars),dexamethasone(100␮M)(graybars)only(no treat-ment)orassociatedwithtagitininA(3),C(1)orF(2)(100␮M)and,after21h, CXCL8(A),IL-6(B)andTNF-␣(C)werequantifiedbyELISA.Theresultsareexpressed asmean±SEMforeachvolunteertestedindividually.Theexperimentswere per-formedintriplicate.Theresultswereevaluatedbyone-wayANOVAfollowedby Bonferroni’spost-test.*p<0.05,**p<0.01and***p<0.001whentreatedgroups werecomparedwithnotreatmentdata.

inthepresenceorabsenceofTagsC(1),F(2)andA(3).Together theseresultsshowthatTagsC(1)andF(2)wereabletodecrease LPS-inducedproductionofinflammatorycytokinesbyneutrophils. Besides,TagC(1)andF(2)didnotstimulateneutrophilstosecrete CXCL8,IL-6andTNF-␣intheabsenceofinflammatorystimuli.

Discussion

STL, diterpenes, caffeoylquinic acid derivatives as well as flavonoidsarethemostcommonnaturalproductswhichmaybe encounteredintheaerialpartsofT.diversifolia(Chagas-Paulaetal., 2012).Severalbiologicalactivitieshavebeendescribedformostof thesecompounds(Liaoetal.,2011,2013;Chagas-Paulaetal.,2011, 2012;Lin,2012;Zhaoetal.,2012;Sánchez-Mendozaetal.,2011; Guetal.,2002).ThetestedSTLfromT.diversifoliausuallyexhibit expectedbiological activitybecause,ina generalway,theyare knownfortheirkeymedicinalpotential(Chagas-Paulaetal.,2012;

Ghantousetal.,2010).AmongallcompoundsfromT.diversifolia

alreadytestedforanybiologicalactivity,theSTLTagC(1)hasbeen themostfrequentlystudied(Liaoetal.,2011,2013;Chagas-Paula etal.,2012;Lin,2012;Leeetal.,2011;Sánchez-Mendozaetal., 2011;Ghantousetal.,2010;Goffinetal.,2002).Inthepresenceof LPS,allTagswereabletodecreasetherateofneutrophilapoptosis. However,intheabsenceofstimulianincreasedrateofapoptosisof neutrophilscultivatedwithTagC(1)wasobserved.Somestudies haveshownthatTagC(1)inducedcaspase-dependentapoptosis orevenautophagicdeathoftumorcells,andsuchroleofTagC (1)isthoughttobebeneficialforthepreventionand treatment ofcancer(Liaoetal.,2011,2013;Liuetal.,2013).When occur-ringinthesiteofinfectiontheexacerbatedneutrophilapoptosis wouldbedangerousbecauseof thelateeffects inthelocalized immuneresponsesuchasmodulationorevenimmunosuppression (Ortega-Gómezetal.,2013).Althoughmacrophagephagocytosisof apoptoticneutrophilsavoidsneutrophilautolysisacceleratingthe resolutionoftheinflammation,macrophagessecreteVEGF,IL-10 andTGF-␤afterefferocytosis,resultinginapoorprognosis( Ortega-Gómezetal.,2013;Kawakamietal.,2013;Gholaminetal.,2009; OhmandCarbone,2001).Althoughapoptosisisadefaultfateof neutrophils,in theinflammatory microenvironmentneutrophils arelikelyexposedtovariouspro-survivalsignals,suchasLPS(Kebir and Filep, 2013). The defense mechanisms of neutrophils that destroyinvadingpathogensarealsocapableofinflictingdamage tothesurroundingtissue(Nathan,2006);however,itisnot inter-estingthatamedicinemayinduceneutrophildeathsinceitcould becomeanindividualsusceptibletosevereinfections(Tortorella etal.,2001;Salmenetal.,2004;Arefetal.,2011;Breaketal.,2012). Consequently,apoptosisinductionofneutrophilswouldcomprise abadsideeffectofTagC(1).Ontheotherhand,wefoundTagF(2) andTagA(3)didnotinduceneutrophilapoptosis;thisfactmight indicatebetterchancestotheuseofthesecompoundsinaninvivo

system.

Inaddition,TagF(2)significantlydecreasedtheLPS-induced MPOactivityofhumanneutrophils.LPSisabletostrongly acti-vateneutrophils and,asa consequenceoftheiractivation,they releaseMPO.MPOispresentinthecytoplasmicazurophilic gran-ulesofneutrophilsandupondegranulationintothephagosome, MPOcanreactwithhydrogenperoxidetoproducevarious antimi-crobialreactivespecies,alsoculminatinginthehypochlorousacid generation(Amulicetal.,2011).However,studieshavesuggested thatmostofthesespeciesproduced wouldreactwithhost pro-teins before reachingthe pathogen (Amulic et al., 2011). MPO absencedoesnotnecessarilymakeanindividualtobe suscepti-bletotheinfections(Amulicetal.,2011),anditspresencehasbeen correlatedtodifferentdiseases.Besides,excessivegenerationof MPO-derivedoxidanthasbeenlinkedtotissuedamageandinthe initiationandprogressionofdiseases,suchascancer,and cardio-vascularillness,whicharisefromchronicinflammation(Nussbaum etal.,2013;Mika&Guruvayoorappan,2011).Theoxidantactivityof MPOisbelievedtopromotethemetabolismofchemical carcino-gens,causeDNAdamageandcompromise therepairingprocess (Nussbaumetal.,2013;MikaandGuruvayoorappan,2011). There-fore,thecontrolofMPOactivityhasbeenthoughtasatargetfor newdrugsdevelopment(Malleetal.,2007).WespeculatethatTags couldblockMPOactivity,affectingitsactionintheinflammatory environment.Thishypothesiswillhavetobeinvestigatedinthe future.

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(3)significantlyinhibitedLPS-inducedIL-6,CXCL8andTNF-␣by humanneutrophils.However,neutrophilscultivatedwithTagA producedTNF-␣,acytokinewitheffectonneutrophilssurvivalina concentration-dependentway(Crossetal.,2008;Walmsleyetal., 2004).TheseeffectsmightdiscourageinvestigationsusingTagA (3)astherapeuticagentininflammation.

Withregardtothestructuralrequirementsthatmaybeinvolved inthebiologicalactivityofthethreecompounds,itisinteresting toobservethattheTagsbelongtothegermacranolideclassand haveincommona␥-lactoneconjugatedwithanexocyclic methy-lenegroupaswellasanisobutanoyloxysidechaingroupatC8. ThesecommonchemicalfeaturesamongtheTags,especiallythe

␣-methylene-␥-lactonegroup,probableareresponsiblefortheir commoneffectsonneutrophils,suchasinhibitionoftheproduction ofIL-6andCXCL8.However,thecompoundsalsoshowsome dif-ferencesamongthem.Besidesthe␣-methylene-␥-lactonegroup, TagC(1)hasacarbonylgroup(C3)conjugatedwithtwo differ-entdoublebondswhileTagF(2)andA(3)haveanetherlinkage betweenC3-C10andforthattheyarecalledfuranoheliangolides. TagC(1)andF(2)haveacisdoublebondatC4whilethemain skeletonofTagA(3)iscompletelyreduced;finally,besidesthe hydroxylgroupatC3,TagA (3)shows anextrahydroxylatC1 againstonlyoneinTagF(2)(C3)thatinturnhasadoublebond atC1.AllthesesmalldifferencesinthestructuresofthethreeTags certainlyexertgreatinfluenceontheireffectsontheneutrophils (Figs.1and2).Forexample,thefeaturesofTagC(1)(three conju-gatedelements)couldbecorrelatedwithitsundesirableapoptotic inductiononneutrophilsandthoseofTagF(2)shouldprovideits betterprofileonneutrophilsbecauseitwastheonlythatwasable toinhibitTNF-␣productionandMPOactivitywithoutapoptotic inductionbesidestheinhibitionofproductionofIL-6andCXCL8.

Together,theseresultsclearlydemonstrateforthefirsttimethe actionofTagsC(1),F(2)andA(3)fromT.diversifoliaonhuman neutrophils.However,TagF(2)istheonlyonethatexhibits anti-inflammatory potential onneutrophils without significant side effects.

Furtherstudiesareneededtobetterunderstandthemolecular modesofactionofTagF(2)fromT.diversifolia,aswellasto deter-mineitstoxicityandactivityinco-cultureswithimmunologiccells andinvivomodels.

Authors’contributions

APC,FBC,andTHGconceivedanddesignedtheexperiments; AEA, CEO,TMD, and THGforculture-related experiments; BAR, DACP,andRBOforpurificationandcharacterizationofTag;AEA, CEO,FBC,APC,andTHGcontributedtodataanalysis;APCandFBC contributedwithreagents/materials/analysistoolsandAEA,CEO, THG,DACP,BAR,RBO,FBC,andAPCforpaperwriting.Allauthors havereadthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Allauthorshavenonetodeclare.

Acknowledgments

The authors express their gratitude for CAPES, FAPESP and CNPqforfunding,grantsandscholarships.Theauthorswere sup-portedbyFAPESPforgrant#2008/57149-8(scholarshiptoA.E.A. [2011/18174-0], T.M.D. [2010/19317-6], T.H.G.[2009/14127-7]), R.B.O.[2011/01862-0],D.A.C.P.[2008/02185-0and2010/10940-2], CAPESforscholarshiptoC.E.O.andB.A.R.,andCNPqforscholarship toF.B.C.andA.P.C.

TheauthorsthankProf.Dr.MarcusTulliusScotti(Departamento deEngenhariaeMeioAmbiente,UniversidadeFederaldaParaíba, RioTinto,Paraíba,Brazil)forhiscriticalreviewofourstatistical analysis.

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