Rev. bras. farmacogn. vol.25 número6

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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

Antimicrobial

(including

antimollicutes),

antioxidant

and

anticholinesterase

activities

of

Brazilian

and

Spanish

marine

organisms

–

evaluation

of

extracts

and

pure

compounds

Éverson

Miguel

Bianco

a,∗

_,

_Jéssica

_Lenita

_Krug

a

_,

_Priscila

_Laiz

_Zimath

b

_,

_Aline

_Kroger

b

_,

Camila

Jeriane

Paganelli

b

_,

_Ariela

_Maína

_Boeder

b

_,

_Larissa

_dos

_Santos

a,b

_,

_Adrielli

_Tenfen

a,b

_,

Suzi

Meneses

Ribeiro

c

_,

_Kátia

_Naomi

_Kuroshima

d

_,

_Michele

_Debiasi

_Alberton

a,b

_,

Caio

Maurício

Mendes

de

Cordova

a,b

_,

_Ricardo

_Andrade

_Rebelo

a

a_Programa_de_{Pós-graduac¸}_ão_em_Química,_Departamento_de_Química,_Fundac¸_ão_Universidade_Regional_de_Blumenau,_Campus_I,_Blumenau,_SC,_Brazil b_Departamento_de_Ciências_{Farmacêuticas,}_Universidade_Regional_de_Blumenau,_Campus_III,_Blumenau,_SC,_Brazil

c_Museu_Nacional,_Departamento_de_{Invertebrados,}_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de_Janeiro,_RJ,_Brazil d_Laboratório_de_Oceanografia_Química,_CTTMar,_Universidade_do_Vale_do_Itajaí,_Itajaí,_SC,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9April2015 Accepted20July2015 Availableonline29August2015

Keywords: Elatol

Dolastanediterpene Antimollicutesactivity Antioxidantactivity Anticholinesteraseactivity

a

b

s

t

r

a

c

t

Thisworkdescribestheantimicrobial,antioxidantandanticholinesteraseactivitiesinvitrooforganic extractsfromfourteenseaweeds,elevensponges,twoascidians,onebryozoan,andoneseaanemone speciescollectedalongtheBrazilianandSpanishcoast,aswellastheisolationofthediterpene(4R,9S, 14S)-4␣-acetoxy-9␤,14␣-dihydroxydolast-1(15),7-diene(1)andhalogenatedsesquiterpeneelatol(2). Themostpromisingantimicrobialresultsforcellwallbacteriawereobtainedbyextractsfromseaweeds

LaurenciadendroideaandSargassumvulgarevar.nanun(MIC250␮g/ml),andbythebryozoanBugula ner-itina(MIC62.5␮g/ml),bothagainstStaphylococcusaureus.Asforantimollicutes,extractsfromseaweeds showedresultsbetterthantheextractsfrominvertebrates.Almostallseaweedsassayed(92%) exhib-itedsomeantimicrobialactivityagainstmollicutesstrains(Mycoplasmahominis,Mycoplasmagenitalium,

MycoplasmacapricolumandMycoplasmapneumoniaestrainFH).Fromtheseseaweeds,A1(Canistrocarpus cervicornis),A11(Gracilariasp.)andA4(Lobophoravariegata)showedthebestresultsforM.pneumoniae

strainFH(MIC250␮g/ml).Furthermore,compounds1and2werealsoassayedagainstmollicutesstrains

M.hominis,M.genitalium,M.capricolum,M.pneumoniaestrain129andM.pneumoniaestrainFH,which showedMIC>100␮g/ml.Antioxidantactivitiesofextractsfromthesemarineorganismswereinactive, exceptforE7(fromspongeIrciniasp.),whichexhibitedmoderatedantioxidantactivitiesfortwomethods assayed(IC5083.0±0.1␮g/ml,and52.0±0.8mgAA/g,respectively).Finally,fortheanticholinesterase activity,allthe29samplesevaluated(100%)exhibitedsomelevelofactivity,withIC50<1000␮g/ml. Fromthese,seaweedsextractswereconsideredmorepromisingthanmarineinvertebrateextracts[A10 (IC5014.4±0.1␮g/ml),A16(IC5016.4±0.4␮g/ml)andA8(IC5014.9±0.5␮g/ml)].Thefindingsofthis workareusefulforfurtherresearchaimingatisolationandcharacterizationofactivecompounds.

Introduction

Marine organisms produce substances with different well-definedecologicalfunctions.Moreover,someofthesemolecules alsoexhibitpharmacological propertiessuchas antiviral(Sagar etal.,2010;Guimarãesetal.,2013),antifungal(Wattanadiloketal., 2007), antibacterial(Mancinietal.,2007), antiprotozoal(Santos

∗ Correspondingauthor.

E-mail:[email protected](É.M.Bianco).

etal.,2010;Veiga-Santosetal.,2010;Santosetal.,2011),cytotoxic (Moore andScheuer,1971;Friedmanetal.,2008;Wang,2008), anticancer(Rinehartetal.,1981;Simmonsetal.,2005),antioxidant (Utkinaetal.,2010),andothers.Advancesinmarinepharmacology aredemonstratedbyseveralnewcompoundsinpre-orclinical evaluation(Costa-Lotufoetal.,2009;Molinskietal.,2009;Gerwick andMoore,2012).

Despitehavingmorethan8500kmofcoastline,Brazilian stud-iesconcerningmarinenaturalproductsrelatedtodrugsdiscovery arestillveryscarceandhavebeenmainlyconductedinthe south-eastofthecountry.SofaronlyafewclassesofBrazilianmarine

http://dx.doi.org/10.1016/j.bjp.2015.07.018

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organismshavebeeninvestigatedfortheirchemicaland pharma-cologicalproperties(Teixeira,2010).Thereforeitisbelievedthat theidentificationofBrazilianorganismswithsignificant biotech-nologicalpotentialisanimportanttoolforthediscoveryofnew drugs.

ConsideringthegreatbiodiversityofBrazilianmarinespecies, theuseofappropriatemethodologieswhichcouldrapidlyscreen differentmarinesourcesforbioactivecompoundsisofgreat inter-est. In a previous study concerning bioprospecting of marine naturalproductsfromBraziliancoast,wehaveshownthatBrazilian seaweeds,aswellassponges,ascidians,octocoralsandbryozoans, can offer a rich source for the chemical study of novel bioac-tivesecondary metaboliteswithpotentialmedicinalproperties, speciallyas antibacterial,antifungal, antiprotozoaland antiviral (Biancoetal.,2013a).

Inordertocompareandcomplementdifferentspeciesofmarine organisms,fivemarinespongespeciesfromtheSpanishcoastwere collectedatintertidalandsublittoralsitesoftheGalicianlittoral (NWSpain),andalsowerescreenedfortheirbiologicalpotential. Alargenumber of compoundswithunusualchemical diversity andremarkablebiologicalactivityhavebeenisolatedfrommarine sponges(NakaoandFusetani,2010),thediscoveryofnucleoside derivativesfromtheTectitethyacrypta(formerCryptotethyacrypta) inthe1950sbyBergmannandFeeney(1950,1951)beinga success-fulexample.IthasledtoartificialderivativesARA-C(cytarabine) andARA-A(vidarabine),whichareusedasanticancerand antivi-raldrugs,respectively(Molinskietal.,2009;GerwickandMoore, 2012).Eribulinmesylate(anticancer)isanotherrecentexampleof anapproveddrugobtainedasaderivativefromthenatural prod-ucthalichondrinB,isolatedfromthespongeHalichondriaokadai

(Huycketal.,2011).

Withregard tothis work, and complementing our previous screening(Biancoetal.,2013a),thebiotechnologicalpotentialof organicextractsfromBrazilianandSpanishmarinebiodiversity, andthetwoalreadyknowncompounds(1and2)isolatedfromthe BraziliamseaweedsCanistrocarpuscervicornisandLaurencia

den-droidea,respectively,whichwerestillbeinginvestigated,butnow

evaluatedfordifferentproperties,suchasantimicrobial,including antimollicutes(mollicutesarewall-lessbacteria),antioxidantand anticholinesterase.Furthermore,thisworkalsoshowedforthefirst timeascreeningforantimollicutesagentsfromBrazilianmarine organisms.

Materialsandmethods

General

All chemicals used were of analytical grade; 2,2-diphenyl-1-picrylhydrazyl (DPPH), acetylcholinesterase (AChE) type VI-S,

from electric eel 349U/mg solid, 411U/mg protein,

5,50-dithiobis[2-nitrobenzoic acid] (DTNB), acetylthiocholine iodide (AChI),tris[hydroxymethyl]aminomethane(Trisbuffer), dimethyl-sulfoxide(DMSO)andTween40weresuppliedbySigma;solvents werepurchasedfromMerck (Germany),Sinth(Brazil)and Vetec

(Brazil),and usedwithoutfurtherpurification.Solidsupportfor chromatographycolumn(CC):silicagel(SiO2)Vetec(70–230mesh;

230–400mesh);TLC(SiO2GF254–Merck);1HNMR(299.99MHz)

and 13_C _NMR _(70.0_MHz) _spectra _were _recorded _on _a _Varian

UnityPlus300spectrometerusingdeuteratedchloroform(CDCl3)

CambridgeassolventandTMSasinternalstandard.

Collectionofmarineorganisms

Seaweedsspecimens(Rhodophyta,Pheophyceae,and Chloro-phyta)werecollectedinthemidlittoralzoneofthenortheastern

Box1:Marineseaweedscollectedforbiologicalassays.

Species Collectionlocala _Collection_date _Voucher

PhylumOchrophytab Canistrocarpus cervicornis

ParaísoBeach, CabodeSanto Agostinho,PE (8◦21′S; 34◦57′W)

August2009 SP401.485

Dictyota mertensii

ItapuamaBeach, CabodeSanto Agostinho,PE (8◦₁₇′_S;₃₄◦₅₇′_W)

August2009 MOUFPE 000.001

Lobophora variegata

CalhetasBeach, CabodeSanto Agostinho, PE (8◦₂₀′_S;₃₄◦₅₆′_W)

Sargassum vulgarevar. nanun

BoaViagemBeach, Recife,PE(8◦₇′_S; 34◦₅₃′_W)

Sargassum vulgarevar. vulgare

ParaísoBeach, Cabo de Santo Agostinho, PE (8◦₂₁′_S;₃₄◦₅₇′_W)

Padina gymnospora

BoaViagemBeach, Recife,PE(08◦₇′_S; 34◦₅₃′_W)

PhylumRhodophytac

Digeniasimplex Suape Beach, Cabo deSanto Agostinho,PE (8◦₂₂′_S;₃₄◦₅₆′_W)

August 2009 SP 401.477

Gracilariasp. BoaViagemBeach, Recife,PE(8◦₇′_S; 34◦₅₃′_W)

Laurencia dendroidea

Suape Beach, PE (8◦₂₂′_S;₃₄◦₅₆′_W)

August 2009 SP 401.478

Laurencia translucida

ItapuamaBeach, CabodeSanto Agostinho,PE (8◦₁₇′_S;₃₄◦₅₇′_W)

August2009 –

Hypnea musciformis

ParaísoBeach, Cabo de Santo Agostinho,PE (8◦₂₁′_S;₃₄◦₅₇′_W)

Palisada perforata

SuapeBeach,Cabo deSanto Agostinho,PE (8◦22′S; 34◦56′W)

Phylum Chlorophytad Anadyomene saldanhae

Arraial d ´Ajuda Beach,Porto Seguro,BA (16◦₂₉′_S;₃₉◦₀₄′_W)

September 2011

–

Chaetomorpha antennina

SuapeBeach,Cabo deSanto,gostinho PE (8◦22′S; 34◦56′W)

a_All_seaweeds_were_collected_in_the_intertidal_zone. b_Ochrophyta₌_brown_seaweeds.

c _Rhodophyta₌_red_seaweeds. d_Chlorophyta₌_green_seaweeds.

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É.M.Biancoetal./RevistaBrasileiradeFarmacognosia25(2015)668–676

Table1

Marineinvertebratescollectedforbiologicalassays.

Species Collectionlocal Depth Collectiondate Voucher PhylumPorifera

Aplysinafulva FornoBeach,RJ,ArraialdoCabo,RJ(22◦₄₅′_S,₄₂◦₀₀′_W) _1–3_m _July₂₀₀₆ _MNRJ₁₃₅₅₄

Amphimedonviridis Bonfim’sIsland,AngradosReis,RJ(23◦₀₁′_S,₄₄◦₁₉′_W) _1–3_m _July₂₀₀₆ _MNRJ₁₄₅₁₇

Desmapsammaanchorata Bonfim’sIsland,AngradosReis,RJ(23◦₀₁′_S,₄₄◦₁₉′_W) _1–3_m _July₂₀₀₆ _MNRJ₁₄₅₂₀

Desmacidonfruticosum PuntaFornelos,Spain(43◦₂₆′_N,₈◦₁₈′_W) _5–19_m _October₂₀₁₀ _MNRJ₁₄₅₁₄

Dysideacf.fragilis BaixoPereiro,Galicia,Spain(43◦₂₈′₄₈_N,₈◦₁₅′₂₃_W) _5–19_m _October₂₀₀₆ _MNRJ₁₄₅₁₆

Haliclonaoculata PuntaFornelos,Spain(43◦₂₆′_N,₈◦₁₈′_W) _5–19_m _October₂₀₀₆ _MNRJ₁₄₅₁₅

Irciniasp. PuntaFornelos,Spain(43◦₂₆′_N,₈◦₁₈′_W) _5–19_m _October₂₀₀₆ _MNRJ₁₄₅₁₉

Polymastiarobusta PuntaFornelos,Spain(43◦₂₆′_N,₈◦₁₈′_W) _5–19_m _October₂₀₀₆ _MNRJ₁₄₅₃₃

Tethyamaza TaritubaBeach,Paraty,RJ(23◦₀₂′_S,₄₄◦₃₅′_W) ₁_m _April₂₀₀₄ _MNRJ₁₄₅₄₉

Petromicacitrina ArchipelagoofCagarras,RiodeJaneiro,RJ(23◦₀₁′_S,₄₃◦₁₁_W) ₁₀_m _August₂₀₀₈ _MNRJ₁₄₅₃₉

Hymeniacidonheliophila ItaipuBeach,Niterói,RJ(22◦₅₈′_S,₄₃◦₀₃′_W) ₁_m _July₂₀₀₆ _MNRJ₁₄₅₂₈ PhylumUrochordata

Didembunperlucidum Armac¸ãodeItapocoroyBay,Penha,SC(26◦₄₆′_S,₄₈◦₃₆′_W) _2–3_m _July₂₀₁₄ _–

Polyclinumsp. Armac¸ãodeItapocoroyBay,Penha,SC(26◦₄₆′_S,₄₈◦₃₆′_W) _2–3_m _July₂₀₁₄ _– PhylumBryozoa

Bugulaneritina Armac¸ãodoItapocoroyBay,Penha,SC(26◦₄₆′_S,₄₈◦₃₆′_W) _2–3_m _July₂₀₁₄ _– PhylumCnidaria

Bunodosomacaissarum Armac¸ãodeItapocoroyBay,Penha,SC(26◦₄₆′_S,₄₈◦₃₆′_W) ₁_m _July₂₀₁₄ _–

Bay, Penha, SC, at a depth of 1–3m, in July 2014, and the material identification was made by Departamento de OceanografiaatUniversidadedoValedoItajaí(Table1).

Preparationoftheextracts

Air-driedseaweeds(100gperspecies)wereextractedatroom temperaturebystaticmacerationwithdifferentorganicsolvents; ExtractsA1,A3,A4,A5,A6,A7,A8,A9,A10,A11,A13,A15,A16and A17wereobtainedusingamixtureofdichloromethane/methanol (2:1);extractsDEandHEwereacquiredusingdichloromethaneand

n-hexane,respectively.Theprocesswasrepeatedfivetimesduring 15days,using50mlofsolventperextraction.All16crude sea-weedextractsobtainedwereevaporatedunderreducedpressure (<50◦_C).

Thefreeze-driedspongesmaterial (100g),fromeleven spec-imens, were extracted three times with acetone at room temperature,andextractsacquired(E1,E2,E3,E4,E5,E6,E7,E8,E9, E10andE11)wereconcentratedunderreducedpressure(<50◦_C).

Freshfrozensamplesoftwo ascidians(100g),one bryozoan (100g)and oneseaanemone(50g)wereextracted threetimes withmethanolatroomtemperatureandcrudeextracts(AS1,AS2, B1andAN1,respectively)wereevaporatedtodrynessunderlow temperatures(<50◦_C)_on_a_rotary_evaporator.

Allextractsweresubmittedtobiologicalscreeningassays.

Compoundsisolation

Compounds1and2werepreviouslyisolatedfromC.cervicornis

andL.dendroidea,respectively(Bornetal.,2012).NMR

spectro-scopicdata(1_H_and13_C),_and_comparison_with_data_from_literature

confirmedtheiridentity(Garciaetal.,2009;Lhullieretal.,2009).

(4R, 9S, 14S)-4˛-Acetoxy-9ˇ,14˛-dihydroxydolast-1(15),7-diene

(1): yellowishgum(130mg).1_H _NMR _(CDCl

3):ı2.71(ddd, 1H,

J=13.8,13.8and5.4,H-2␣),ı2.20−2.00(m,1H,J=13.8, H-2␤), ı 1.90−1.81(m, 1H, H-3␣), ı 2.05−1.97 (m, 1H,H-3␤), ı 4.85 (t,1H, J=3.0,H-4),ı3.07 (dd,1H,J=15.0 and4.8,H-6␣),ı1.57 (dd,1H,J=15.0and9.6,H-6␤),ı5.54(dd,1H,J=9.6and4.8, H-7),ı1.50−1.47(m,1H,H-10␣), ı 1.77−1.70 (m,1H, H-10␤), ı 1.60−1.53(m,1H,H-11␣),ı1.80−1.78(m,1H,H-11␤),ı1.82(d, 1H,J=14.4,H-13␣),ı1.93(d,1H,J=14.4,H-13␤),ı4.82(s,1H, H-15a),ı4.93(s,1H,H-15b),ı0.89(s,3H,H-16),ı1.95(qq,1H,J=6.9

and6.6,H-17),ı 1.03(d,3H, J=6.6, H-18),ı0.83(d,3H,J=6.9,

H-19),ı1.24s,(3H,H-20),ı2.16(s,3H,C(4) COOCH3),ı3.75(br s,1H,C(9) OH).13_C_NMR_(CDCl₃_):_ı_151.0_(C-1),_ı_26.5_(C-2),_ı_28.0

(C-3),ı81.72(C-4),ı42.4(C-5),ı30.16(C-6),ı117.7(C-7),ı157.1 (C-8),ı86.2(C-9),ı28.0(C-10),ı41.2(C-11),ı45.0(C-12),ı43.0 (C-13),ı79.4(C-14),ı109.6(C-15),ı19.7(C-16),ı34.5(C-17),ı 17.1(C-18),ı18.9(C-19),ı24.1(C-20),ı169.3(C(4) COOCH3),ı

21.2(C(4) COOCH3).

Elatol(2):colorlessoil(19mg).(CDCl3):ı2.08(brs,1H,H-1),

ı1.85(m,1H,H-4␣),ı1.98(d,1H,J=3.0,H-4␤),ı1.63(m,1H, H-5␣),ı1.81(m,1H,H-5␤),ı2.50(dd,1H,J=3.0,14.7,H-8␣),ı2.36 (dm,1H,15.0,H-8_␤),ı4.15(dd,1H,J=3.0,6.6,H-9_␤),ı4.61(d,1H,

J=2.7,H-10␤),ı1.07(s,3H,H-12),ı1.08(s,3H,H-13),ı4.80(brs, 1H,H-14a),ı5.13(brs,1H,H-14b),ı1.71(brs,3H,H-15).13CNMR

(CDCl3):ı38.6(C-1),ı128.0(C-2),ı124.1(C-3),ı29.3(C-4),ı25.6

(C-5),ı49.1(C-6),ı140.7(C-7),ı38.0(C-8),ı72.1(C-9),ı70.8 (C-10),ı43.1(C-11),ı20.7(C-12),ı24.2(C-13),ı115.8(C-14),ı 19.4(C-15).

Evaluationofantibacterialactivity

The samples were evaluated against a different panel of bacteriastrains:onegrampositive strain[Staphylococcusaureus

(ATCC25923)], two gram negative [Pseudomonas aeruginosa

(ATCC27853), and Escherichia coli (ATCC25922)], and five mol-licutes strains [Mycoplasma hominis (ATCC23114), Mycoplasma

genitalium (ATCC33530), Mycoplasma capricolum (ATCC27343),

MycoplasmapneumoniaestrainFHATCC15531),andM.

pneumo-niaestrain129(ATCC29342)].Forthegrowthofcellwallbacterial strains,Müller–HintonbrothwasusedforS.aureus,E.coliandP.

aeruginosa;SP4(glucose)brothwasusedforM.pneumoniaestrain

FH,M.pneumoniaestrain129,M.capricolumandM.genitalium;MLA

(arginine)brothwasforM.hominis.

Themicrodilutionbrothassayswereperformedinsterile 96-wellmicroplates,asrecommendedbytheClinicalandLaboratory StandardsInstitute(CLSI,2012)forcellwallbacteriaandbyBébéar andRobertson(1996)formollicutes.

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MICvaluesbetween10and100␮g/mlwereconsideredtohavea goodactivity;extractswithMICvaluesbetween100and500␮g/ml wereconsideredtohavemoderateactivity;extractswithMIC val-uesbetween500and 1000␮g/mlwereconsideredtohave low activity,andextractswithMICabove1000␮g/mlwereconsidered inactive.Forpurecompounds,onlyactivesampleswithMIClower than100␮g/ml(Machadoetal.,2005)wereconsidered.

Extracts were transferred to each microplate well withthe appropriateculturemedium,inorder toobtainatwofoldserial dilutionoftheoriginalextractina10%H2O/DMSOsolution,

obtain-ingsampleconcentrationsrangingbetween1000and7.8␮g/ml. Theinoculumcontaining104_–105_{microorganisms}_per_ml_was_then

addedtoeachwell.Anumberofwellswerereservedineachplate totestforsterilitycontrol(noinoculumadded),positivecontrols (gentamycin,levofloxacinandclarithromycin–dependingonthe bacteriumundertest),inoculumviability(noextractadded),and theDMSO inhibitory effect.The microplateswere incubatedat 37±1◦_C_for₂₄_or₄₈_h_(depending_on_the_bacterium).

Formollicutesbacteria,thegrowthaswellasMICwasdetected byvisualinspectionofthemediumcolorchange,sinceacidification ofthemediumbyglucosemetabolizingspecieschangethecolor fromredtoyellow,andalkalinizationofthemediumbyarginine metabolizingspecieschangethecolorfromorangetored,as indi-catedbythephenolreddyepresentintheculturemedia(Benfatti etal.,2010).Forcellwallbacteria,MICwasevaluatedbya methano-licsolutionoftriphenyltetrazoliumchloride(5mg/ml)addedinto eachwell,wherethepresenceofareddishbacterial“dot”observed atthebottomofeachwellindicatedbacterialgrowth(Freiresetal., 2010).

Antioxidantassay

DeterminationofDPPHfreeradicalscavengingactivity

TheDPPHfreeradicalscavengingactivitywasdeterminedusing themethodasdescribedbyBlois(1958)withsomemodification (Cavinetal.,1998;Bracaetal.,2001).To5mlofamethanolsolution of1,1-diphenyl-2-picrylhydrazyl(DPPH)0.002%inmethanol,50␮l ofextractsolutions(1000–62.5␮g/ml)wasaddedandthemixtures wereincubatedatroomtemperaturefor30min.Theabsorbance wasmeasuredat517nmagainstacorrespondingblank.Inhibition percentageoffreeradicalDPPH(I%)wascalculatedinthe

follow-ingway:I(%)=(Ablank−Asample/Ablank)×100, whereAblankisthe

absorbanceofthecontrolreaction(areactionwithallthereagents exceptthetestextract),andAsampleistheabsorbanceofthetest

extract.Testswerecarriedoutintriplicateandtheextract concen-trationproviding50%inhibition (IC50)wasobtainedbyplotting

extractsolutionconcentrationversusinhibitionpercentage.

Determinationofironreducingpower

Theassayfortheanalysisofantioxidantactivityby determina-tionofthereductionpotentialwasbasedonthemethodofPrice andButler,proposedbyWatermanandMole(1994),with adap-tations.To the100␮lof the test solutions(extracts, diluted in methanolataconcentrationof1000␮g/ml)wasadded8.5mlof deionizedwater.Then,1mlofa0.1MFeCl3solutionwasadded

andafter3min,1mlofapotassiumferricyanide0.08Msolution wasmixed.After15min,theabsorbanceofsampleswasmeasured inaspectrophotometerat720nm.Ablanksolutionwasprepared accordingtotheaboveprocedure,withoutaddingthesample.A standardcurvewasperformedusingascorbicacidsolutionsin con-centrationsrangingfrom100to1000␮g/ml(y=0.0019x+0.0698 (R2₌_0.9967)._The_reduction_potential_was_expressed_in_milligram

(mg)ofascorbicacid(AA)equivalentspergram(g)ofdriedextract (mgAA/g).Analysiswasperformedintriplicate.

Determinationoftotalphenoliccontent

Totalphenoliccontentoftheextractswasdeterminedwiththe Folin–Ciocalteu’sreagent(FCR)accordingtoSlinkardandSingleton (1977)method.Eachsample(0.5ml,1000␮g/ml)wasmixedwith 2.5mlFCR (diluted1:10,v/v)followedby2mlofNa2CO3 (7.5%,

v/v)solution.Theabsorbancewasthenmeasuredat765nmafter incubationat30◦_C_for₉₀_min._Results_were_expressed_as_milligram

(mg)gallicacid(GA)equivalentspergram(g)ofdriedextract(mg GA/g).Analyseswereperformedintriplicate.

Acetylcholinesteraseenzymeinhibitoryassay

TheEllmanmethod(Ellmanetal.,1961)wasemployedforthe quantificationofacetylcholinesteraseactivity,andtheprocedures, withsomemodifications,aredetailedinmanyrecentpublications (Ingkaninanetal.,2003;Mata etal.,2007), asbrieflyexplained below.

Abuffersolution[330␮lof50mMTris–HClbuffer(pH8)],plus anextractsolution[100␮l(from1000to7.81␮g/mlinmethanol)] and30␮lofAChEsolutioncontaining0.28U/ml(50mMTris–HCl, 0.1%BSA)wereincubatedfor15min.Subsequently,75␮lofa solu-tionofAChI(0.023mg/mlinwater)and475␮lofDTNB(3mMin Tris–HCl)wereaddedandthefinalmixtureincubatedforanother 30minatroomtemperature.Absorbanceofthemixturewas mea-suredat 405nm. Acontrol mixturewasprepared,using 100␮l of a solutionsimilartothe samplemixture butwithmethanol instead of sample. Inhibition (%) was calculated as follows: I

(%)=100−(Asample/Acontrol)×100;whereAsampleistheabsorbance

ofthesamplecontainingthereactantandAcontroltheabsorbance

ofthereactioncontrol.Testswerecarriedoutintriplicate.Extract concentrationproviding50%inhibition(IC50)wasobtainedfrom

thenon-linearregressiongraphofthepercentageinhibitionagainst extractconcentration.Theinhibitoryconcentrationof50%ofAChE (IC50)valuewascalculated fromatleastfour different

concen-trations of the sample using the Origin 20 statistical package. Galanthaminehydrobromideservedasthepositivecontrol.

Resultsanddiscussion

This work describes the antimicrobial (including antimolli-cutes), antioxidant, and anticholinesterase activities in vitro of organicextractsfromfourteenseaweedspecies[sixRhodophyta (43%),sixOchrophyta(43%),andtwoChlorophyta(14%)],and15 marineinvertebratespecies (elevensponges,twoascidians,one bryozoan, and one seaanemone) collected along the Brazilian coast(Box1andTable1),aswellastheantimollicutesactivityof thediterpene(4R,9S,14S)-4␣-acetoxy-9␤,14␣ -dihydroxydolast-1(15),7-diene (1) and the halogenatedsesquiterpene elatol (2), isolatedfromtheseaweedsC.cervicornisandL.dendroidea, respec-tively.Thechemicalstructuresofcompounds1and2havebeen characterizedbyNMRspectroscopicdata(1_H_and13_C),_and_by

com-parisonwithliteraturedata,whichshowedidenticalspectroscopy datatothatpreviouslyreported(Garciaetal.,2009;Lhullieretal., 2009).

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Table2

Antibacterialscreeningofmarineseaweedsandinvertebrateextracts.

Species Extracts Bacterialstrains

S.aureus(␮g/ml) E.coli(␮g/ml) P.aeruginosa(␮g/ml) Seaweeds

Anadyomenesaldanhae A17 – – –

Canistrocarpuscervicornis A1 500 – –

Chaetomorphaantennina A13 500 – –

Dictyotamertensii A15 500 – –

Digeniasimplex A9 – – –

Gracilariasp. A11 500 1000 –

Hypneamusciformis A10 1000 – –

Laurenciadendroidea A7 250 – –

Laurenciatranslucida A16 – – –

Lobophoravariegata A4 500 – –

Padinagymnospora A3 1000 – –

Palisadaperforata A8 – – –

Sagassumvulgarevar.nanun A5 250 – –

Sargassumvulgarevar.vulgare A6 1000 – –

Sponges

Amphimedonviridis E2 – – –

Aplysinafulva E1 – – –

Desmacidonfruticosum E4 1000

Desmapsammaanchorata E3 – – –

Dysideacf.fragilis E5 1000

Haliclonaoculata E6 – – –

Hymeniacidonheliophila E11 – – –

Irciniasp. E7 – – –

Petromicacitrina E9 – – –

Polymastiarobusta E8 – – –

Tethyamaza E10 – – –

Tunicates

Didembunperlucidum AS1 1000 – –

Polyclinumsp. AS2 – – –

Bryozoa

Bugulaneritina B1 62.5 – –

Seaanemone

Bunodosomacaissarum AN1 1000 – –

Gentamycin 2.5

DataarepresentedasMIC(␮g/ml);positivecontrol=gentamycin;(–),noactivity(>1000␮g/ml).

isolatedfromtheredseaweedL.dendroidea(=Laurenciaobtusa) (Machadoetal.,2014).

Antibacterialassays

Extractsfromseaweedsandmarineinvertebratesweretested againstthecellwallbacterialstrains, asS.aureus(ATCC25923),

P.aeruginosa(ATCC27853)andE.coli(ATCC25922),andagainst

bacteriawithabsenceofacellwall,asM.hominis(ATCC23114),

M.genitalium(ATCC33530),M.capricolum(ATCC27343),M.

pneu-moniae strain FH (ATCC15531), and M. pneumoniae strain 129

(ATCC29342).

Asacriteriontoexpresstheresults,extractswithMIClower than1000␮g/mlwereconsideredactive.However,forpure com-pounds,onlyactivesampleswithMIClowerthan100␮g/mlwere considered.

Fourteen species of seaweeds were assayed. 10 out of 14 species(71%)showedsomeactivityagainstS.aureusandE.coli

[A1 (C. cervicornis), A3 (Padina gymnospora), A4 (L. variegata),

A5 (S. vulgare var. nanun), A6 (S. vulgare var. vulgare), A7

(L. dendroidea), A10 (Hypnea musciformis), A11 (Gracilaria sp.),

A13(Chaetomorphaantennina), andA15(Dictyota mertensii)].Of

these,themost promising results(for bacteriawith cellwalls) were found for A5 and A7, which exhibited moderated activ-ities (MIC 250␮g/ml), while others showed low activity (MIC 500–1000␮g/ml)(Table2).

Concerningantibacterialactivity(bacteriawithcellwalls)from invertebrates,theextractB1(frombryozoanB.neritina)showed thebest results, particularlyagainst S.aureus (MIC62.5␮g/ml) (Table2).Againstmollicutes,thebestresultswereobservedfor thespongeDysideacf.fragilis(E5)thatinhibitedthegrowthofM.

genitalium(MIC250␮g/ml),M.capricolum(MIC500␮g/ml),andM.

pneumoniaestrainFH(MIC250␮g/ml).

Regardingantimollicutesactivity,extractsfromseaweedswere alsobetterthanextractsfrominvertebrates.Almostallseaweeds assayed(92%)exhibitedsomeantimicrobialactivityagainst mol-licutesstrains(M.hominis,M. genitalium,M. capricolumand M.

pneumoniaestrainFH).Fromthese,A1(C.cervicornis),A4(L.

var-iegata) and A11 (Gracilaria sp.) showed the bestresults for M.

pneumoniae strain FH (MIC 250␮g/ml) (Table 3). Additionally,

compounds 1and 2 were isolated fromthe brownseaweedC.

cervicornisandtheredseaweedL.dendroidea,respectively.

How-ever,considering thecriteria established toexpressthe results (MIC>100␮g/ml),compounds1and2werenotactiveagainstM.

hominis,M.genitalium,M.capricolum,M.pneumoniaestrain129,

andM.pneumoniaestrainFH(Table4).

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Table3

Antimollicutes(bacteriawithabsenceofacellwall)screeningfrommarineseaweedsandinvertebrateextracts.

Species Extracts Bacterialstrains

M.hominis(␮g/ml) M.genitalium(␮g/ml) M.capricolum(␮g/ml) M.pneumoniaestrainFH(␮g/ml) Seaweeds

Anadyomenesaldanhae A17 – 1000 1000 1000

Canistrocarpuscervicornis A1 – 1000 1000 250

Chaetomorphaantennina A13 – 1000 1000 500

Dictyotamertensii A15 1000 1000 500 1000

Digeniasimplex A9 – – – –

Gracilariasp. A11 – 500 500 250

Hypneamusciformis A10 – 500 500 500

Laurenciadendroidea A7 – 1000 1000 –

Laurenciatranslucida A16 – 500 500 1000

Lobophoravariegata A4 1000 500 500 250

Padinagymnospora A3 – 1000 1000 500

Palisadaperforata A8 – 1000 1000 1000

Sagassumvulgarevar.nanun A5 – 1000 – 500

Sargassumvulgarevar.vulgare A6 – 1000 – 500

Sponges

Amphimedonviridis E2 – 1000 – 1000

Aplysinafulva E1 – – – 1000

Desmacidonfruticosum E4 – 1000 – –

Desmapsammaanchorata E3 – 1000 1000 1000

Dysideacf.fragilis E5 1000 250 500 250

Haliclonaoculata E6 – – – –

Hymeniacidonheliophila E11 – – – –

Irciniasp. E7 – – – –

Petromicacitrina E9 – – – –

Polymastiarobusta E8 – – – –

Tethyamaza E10 NT NT NT NT

Tunicates

Didembunperlucidum AS1 – 1000 – 1000

Polyclinumsp. AS2 – – – –

Bryozoa

Bugulaneritina B1 – – – 1000

Seaanemone

Bunodosomacaissarum AN1 – – – –

Levofloxacin 2.5 0.15 0.15 0.15

DataarepresentedasMIC(␮g/ml);positivecontrol=levofloxacin;(–),noactivity(>1000␮g/ml);NT,nottested.

showed that a similar diterpene (4R,7R,14S)-4␣,7␣ -diacetoxy-14␣-hydroxydolast-1(15),8-diene, the majorcompound fromC. cervicornis,significantlyinhibitedfeedingbytheseaurchin Lytech-inusvariegatus(Biancoetal.,2010).Similarly,thesamecompound 1wasassayedasantiprotozoalandexhibitedIC502.2␮M,12␮M

and4.0␮Mforthepromastigote,axenicamastigoteand intracel-lularamastigoteformsofLeishmaniaamazonensis,respectively.In thisworktheSIwas93timeslesstoxictomacrophagesthanto theprotozoan,andaprogressivelossofmitochondrialmembrane potentialandcelldeathinL.amazonensis,inducedforthis com-pound,wasdemonstratedtoo(Santosetal.,2011).Inaprevious studyweshowedthat theanticoagulant andantiplatelet activi-tiesofcompound1(Mouraetal.,2011),aswellastheinhibition ofmammalNa+_K+_-ATPase_properties_of₍₄_R_,₉_S_,₁₄_S_)-4_␣

-acetoxy-9␤,14␣-dihydroxydolast-1(15),7-diene,isolatedfromC.cervicornis

(Garciaetal.,2009).

Inadifferentworkitwasdemonstratedthatcompound2also exhibitedantileishmanialandanti-trypanosomalactivities(Santos etal.,2010;Veiga-Santosetal.,2010),aswellasacaricidal(Born etal.,2012),andlarvicidalactivities(Biancoetal.,2013b).

Theresultsherecomplementapreviousstudyperformedby Bianco et al. (2013a), which showed the antimicrobial proper-ties of Braziliansmarine seaweeds [Osmundaria obtusiloba] and invertebrates[Dragmaxiaanomala,Dragmacidonreticulatum,

Hal-iclona (Halichoclona) sp., Leptogorgia punicea, Petromica citrina,

Trachycladussp.].However,forthefirsttimeantimollicutes

activi-tiesofmarineseaweeds/invertebratesextractsweredemonstrated.

Antioxidantassays

Oneoftheaimsofthisstudywasalsotoinvestigatethe antiox-idantactivitiesofextractsbytwodifferentmethods,asfreeradical

Table4

Antimollicutes(bacteriawithabsenceofacellwall)screeningfromextractsandpurecompoundsobtainedfrommarineseaweeds.

Extracts/compounds Bacterialstrains

M.hominis(␮g/ml) M.genitalium(␮g/ml) M.capricolum(␮g/ml) M.pneumoniaestrain129(␮g/ml) M.pneumoniaestrainFH(␮g/ml)

DE 500 500 500 125 100

1 >100 >100 >100 >100 >100

HE 500 500 500 31.25 100

2 >100 >100 >100 >100 >100

Clarithromycin 0.125 0.25 0.5 2 1

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Table5

Phenoliccontentandantioxidantscreening(byDPPHandiron-reducingmethods)frommarineseaweedsandinvertebrateextracts.

Species Extracts Phenolscontent(mgGA/g)a _Reducing_potential_(mg_AA/g)b _DPPH_IC

50(␮g/ml)

Seaweeds

Anadyomenesaldanhae A17 15.6±0.6 10.6±0.9 >1000

Canistrocarpuscervicornis A1 18.6±0.9 28.0±0.9 >1000

Chaetomorphaantennina A13 16.0±0.9 1.8±0.8 >1000

Dictyotamertensii A15 22.4±0.8 71.3±0.8 >1000

Digeniasimplex A9 13.6±0.6 70.6±0.9 >1000

Gracilariasp. A11 21.0±0.8 1.3±0.3 >1000

Hypneamusciformis A10 15.1±0.4 – >1000

Laurenciadendroidea A7 14.6±0.9 29.7±0.3 >1000

Laurenciatranslucida A16 20.4±0.2 25.9±1.0 >1000

Lobophoravariegata A4 14.5±0.5 115.2±0.6 >1000

Padinagymnospora A3 15.1±0.4 – >1000

Palisadaperforata A8 11.8±0.5 – >1000

Sagassumvulgarevar.nanun A5 24.1±0.7 46.6±0.8 >1000

Sargassumvulgarevar.vulgare A6 24.9±0.9 51.6±0.8 >1000

Sponges

Amphimedonviridis E2 17.1±0.1 – >1000

Aplysinafulva E1 12.2±0.1 – >1000

Desmacidonfruticosum E4 21.2±0.1 – >1000

Desmapsammaanchorata E3 24.3±0.5 9.0±0.9 >1000

Dysideacf.fragilis E5 25.6±1.0 – >1000

Haliclonaoculata E6 17.6±0.3 – >1000

Hymeniacidonheliophila E11 16.0±0.4 – >1000

Irciniasp. E7 61.9±0.3 52.0±0.8 83.0±0.1

Petromicacitrina E9 16.6±0.3 – >1000

Polymastiarobusta E8 17.1±0.7 – >1000

Tethyamaza E10 14.1±0.2 – >1000

Tunicates

Didembunperlucidum AS1 17.2±0.6 – >1000

Polyclinumsp. AS2 18.2±0.5 – >1000

Bryozoa

Bugulaneritina B1 21.8±0.2 7.8±0.6 >1000

Seaanemone

Bunodosomacaissarum AN1 24.5±0.9 – >1000

a_mg_GA/g₌_mg_of_gallic_acid_{equivalents/g}_dried_extract. b _mg_AA/g₌_mg_of_ascorbic_acid_{equivalents/g}_dried_extract.

scavengingDPPHandreducingpotential(oriron-reducing) meth-ods.

The objective of DPPH test was to measure the capacity of the extracts to scavenge the stable radical 2,2-diphenyl-1-picrylhydrazyl(DPPH)formedinsolutionbydonationofhydrogen atomor an electron. It has been documented that antioxidant compoundsascysteine,glutathione,ascorbicacid,tocopherol,and polyhydroxyaromaticcompounds(e.g.,hydroquinone,pyrogallol, gallic acid) reduceand decolorize 1,1-diphenyl-2-picrylhydrazil bytheirhydrogendonatingcapabilities(Blois,1958).Antioxidant promisingresultsinDPPHtestareexpressedbysampleswithlow IC50(␮g/ml)values.

Thereducing potential assaymeasuresthe total antioxidant capacityofanextractevaluatingtheredoxpotentialsofits com-pounds.Bythismethodthebestresultsareexpressedbysamples withhighmgAA/g(␮g/ml)values.

Theantioxidantpropertieswerealsoevaluated,aswellasthe phenoliccontentofextractswasdescribedandtheobtainedresults werelistedinTable5.

Seaweedsandinvertebrates’sextractsingeneralshowedweak antioxidantactivities,andonlyoneoutof29speciesassayed[E7

(Ircinia sp.)] showed some antioxidant activity by DPPH assay

(83.0␮g/ml).TheE7alsoexhibitedagoodantioxidantresultby reducingpotentialmethod(52.0±0.8mgAA/g)(Table5).

Phenoliccompoundshave beenshowntopossess important antioxidant activities based on their structural characteristics (Dimitrios, 2006).Thus, the total phenolics content fromthese samplesbytheFolin–Ciocalteureagentwasalsodetermined.The analysisdetectedalowphenoliccontentforallsamplesassayed,

exceptforE7thatexhibitedhigherphenolscontent(61.9mgGA/g) whencomparedtoothermarineextractstested.Theseresultsare compatibletotheantioxidantactivitiesobserved(Table5).

Inapreviousstudy,Zubiaetal.(2007)showedthatextractsfrom 48Mexicanmarineseaweedspeciesexhibitedgoodantioxidant activityinDPPHassay.Ourresultsarenotinagreementwith

find-ingsforC.cervicornis(=D.cervicornis),L.variegata,P.gymnospora,

Digeniasimplex,andL.dendroidea(=L.obtusa),andthis

discrep-ancymaybeduetothedifferentgeographicregionswherethis specieswascollected,suchasseawaterconditions,depth,salinity andtemperature(Table5).

Anticholinesteraseassay

The acetylcholinesterase (AChE) inhibition was determined usinganadaptationofthemethodpublishedbyIngkaninanetal. (2003).Extractsfromseaweedsandinvertebratesweretestedand resultsaredepictedinTable6.

Twenty-nineoutof29marinespeciesassayed(100%)showed someanti-AChEactivity,withIC50<1000␮g/ml.Ingeneral,itwas

observed that extractsfrom seaweeds have shown tobe more promising than extracts from marine invertebrates, exhibiting the lowest IC50 values. Out of these fourteen seaweed species

tested, A10 (IC50 14.4±0.1␮g/ml), A16 (IC50 16.4±0.4␮g/ml) andA8(IC5014.9±0.5␮g/ml)showedthebestresults,followed by A13 (IC50 29.0±0.6␮g/ml), A3 (IC50 31.3±0.4␮g/ml), A9 (IC50 33.7±0.2␮g/ml), A4(IC50 36.4±0.5␮g/ml)and A11(IC50

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Table6

Anticholinesteraseactivityofmarineseaweedsandinvertebratesextracts.

Species Extracts IC50(␮g/ml)

Seaweeds

Anadyomenesaldanhae A17 161.9±0.4 Canistrocarpuscervicornis A1 64.9±0.9 Chaetomorphaantennina A13 29.0±0.6 Dictyotamertensii A15 61.2±0.1

Digeniasimplex A9 33.7±0.3

Gracilariasp. A11 36.9±0.5

Hypneamusciformis A10 14.4±0.1 Laurenciadendroidea A7 99.8±0.3 Laurenciatranslucida A16 16.4±0.4 Lobophoravariegata A4 36.4±0.5

Padinagymnospora A3 31.3±0.4

Palisadaperforata A8 14.9±0.5 Sagassumvulgarevar.nanun A5 66.4±0.4 Sargassumvulgarevar.vulgare A6 71.2±0.1

Sponges

Amphimedonviridis E2 118.0±0.5

Aplysinafulva E1 65.9±0.4

Desmacidonfruticosum E4 645.1±0.8 Desmapsammaanchorata E3 385.5±0.2 Dysideacf.fragilis E5 141.2±0.6 Haliclonaoculata E6 728.9±0.8 Hymeniacidonheliophila E11 981.7±0.1

Irciniasp. E7 544.7±0.2

Petromicacitrina E9 450.4±0.6 Polymastiarobusta E8 508.5±0.5

Tethyamaza E10 >1000

Tunicates

Didembunperlucidum AS1 635.1±0.6

Polyclinumsp. AS2 772.6±0.6

Bryozoans

Bugulaneritina B1 995.6±0.8

Seaanemone

Bunodosomacaissarum AN1 539.5±0.0

Galantamine – 2.12±0.0

Positivecontrol=galantamine.

from Chlorophytaand Ochrophyta. From these,A10 (H. musci-formis),A16(Laurenciatranslucida)andA8(Palisadaperforata)were themostpromising andare knownforexhibiting bromineand chlorine-containingC15terpenoidandnonterpenoid(acetogenins)

metabolites(Ericson,1983;PereiraandTeixeira,1999).

Inhibition of acetylcholinesterase has been considered as a promisingapproachforthetreatmentofAlzheimer’sdiseaseand forpossibletherapeuticapplicationsinthetreatmentofParkinson’s disease,aging,andmyastheniagravis(Quinn,1987).Thus,finding newcholinesteraseinhibitorsisconsideredveryimportant.The extractsevaluatedinthisworkshowedthatinadditiontoinhibiting thecholinesteraseenzyme,theyalsohadantimicrobial(including antimollicutes)andantioxidantactivities.

Furtherresearchaimingatisolatingspecificcompounds respon-sible for these activities,chemical analysis and new biological studiesisrequired.

Conclusions

In this work,31 differentextracts from29 Brazilian marine organisms(seaweedsandinvertebrates)weretestedas antimicro-bial,antioxidantandanti-acetylcholinesteraseagents.Thestudies showedthat71%ofseaweedsassayedshowedsomeactivityagainst

S.aureusandE.coli.Fromthese,themostpromisingantimicrobial

resultswerefoundforA5(S.vulgarevar.nanun)andA7(L.

den-droidea)withMIC250␮g/ml.Almostallseaweedsassayed(92%)

exhibitedsomeantimicrobialactivityagainstmollicutesstrains(M.

hominis,M.genitalium, M.capricolum andM. pneumoniaestrain

FH). From these, A1 (C. cervicornis), A4 (L. variegata) and A11

(Gracilariasp.)showedthebestresultsforM.pneumoniaestrain

FH(MIC250␮g/ml).Concerningantibacterialactivityfrommarine invertebrates,onlytheextractB1fromthebryozoanB.neritinawas activeagainstS.aureus(MIC62.5␮g/ml).

Ingeneral,allextractstestedshowedweakantioxidant activ-ities.OnlyE7(fromIrciniasp.)showedsomeantioxidantactivity byDPPHassay(83.0␮g/ml).However,100%ofextractsevaluated showedsomeanti-AChEactivity(IC50<1000␮g/ml).Fromthese,

A10(H.musciformis),A16(L.translucida)andA8(P.perforata)

exhib-itedthemostpromisingresults(IC5014.4,16.4and14.9␮g/ml),

respectively.

Thisstudyalsoshowedforthefirsttimeascreeningfor anti-mollicutesagents fromBrazilian andSpanishmarineorganisms andtheimportanceofbioprospectingstudiesofmarine biodiver-sityforbioactivenaturalcompoundsdiscoveryanddevelopmentof newdrugs.Alltheactiveextractsdeservespecialattentionin fur-therstudies,suchasisolationandstructuredetermination,aswell asmorerefinedbiologicalassays;sinceclearly,Brazilianspecies assayedcouldplay animportantsourceforthedevelopmentof newbiotechnologicalproducts.

Authors’contribution

EMBperformedtheisolationandstructuralelucidationofthe compounds1and2,supervisedthework,analyzedthedata,and wrotethemanuscript;JLKcontributedincollectingofmaterials andperformedthechemicalextractions;KNKcontributedin col-lectingandidentificationofmaterials(tunicates,bryozoanandsea anemone);SMRcollectedandidentifiedthespongematerial;CJP andATperformedtheantimicrobialassays;AKandLSperformed theantimollicutesassays;AMBperformedtheantioxidantsassays; PLZ performedthe anti-acetylcholinesteraseassays;CMMC and MDAprovidedlaboratoryinfrastructureandfacilitiesforbioassays andcontributedtothebiologicalstudies;RARobtainedfinancial support,providedlaboratoryinfrastructureandfacilitiesfor chem-icalexperiments,and alsocontributedtocritical readingof the manuscript. Alltheauthors haveread thefinalmanuscript and approveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbyCNPq,CAPESandINCT-Catálise; E.M.B.expressthanksforCAPESforprovidinghispostdoc fellow-ship(PNPD2014-2015),andSINETEC/FURBforlabsupporting;J.L.K. expressthankstoMauroScharf(ChemistryDepartment/FURB),for researchundergraduatefellowshipopportunity.Theauthorsare gratefultoThiagoN.V.Reis(OceanographyDepartment/UFPE)for collectionandidentificationofseaweedmaterials,andtoAdriana Vilamor,OriolSacristanand Javier Cristobo(Centro Oceanográ-ficodeGijón/InstitutoEspa˜noldeOceanografía),forcollectionand identificationofSpanishspongematerials.Collectionauthorization wasprovidedfromFederalEnvironmentAgency–IBAMA/MMA (Ref. No.:02001.002975/2006-91). Alsoit is acknowledged the English languagerevision byMSc Marina BeatrizBorgmann da Cunha(EnglishDepartment/FURBIdiomas).

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