Evaluation of acetylcholinesterase inhibitory activity of Brazilian red macroalgae organic extracts

(1)

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

Evaluation

of

acetylcholinesterase

inhibitory

activity

of

Brazilian

red

macroalgae

organic

extracts

Levi

P. Machado

a

_,

_Luciana

_R.

_Carvalho

a

_,

_Maria

_Cláudia

_M.

_Young

b

_,

_Elaine

_M.

_{Cardoso-Lopes}

c

_,

Danilo

C. Centeno

d

_,

_Leonardo

_{Zambotti-Villela}

e

_,

_Pio

_Colepicolo

e

_,

_Nair

_S.

_Yokoya

a,∗

a_Núcleo_de_Pesquisa_em_Ficologia,_Instituto_de_Botânica,_São_Paulo,_SP,_Brazil

b_Núcleo_de_Pesquisa_em_Fisiologia_e_Bioquímica,_Instituto_de_Botânica,_São_Paulo,_SP,_Brazil c_Departamento_de_Farmácia,_Universidade_Nove_de_Julho,_São_Paulo,_SP,_Brazil

d_Centro_de_Ciências_Naturais_e_Humanas,_Universidade_Federal_do_ABC,_São_Paul,_SP,_Brazil e_Instituto_de_Química,_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received14April2015 Accepted7September2015 Availableonline9October2015

Keywords: Acetylcholinesteraseinhibition Halogenatedmonoterpenes Hypnea Ochtodes Pterocladiella Rhodophyta

a

b

s

t

r

a

c

t

Alzheimer’sdiseaseaffectsnearly36.5millionpeopleworldwide,andacetylcholinesteraseinhibitionis currentlyconsideredthemaintherapeuticstrategyagainstit.SeaweedbiodiversityinBrazilrepresents oneofthemostimportantsourcesofbiologicallyactivecompoundsforapplicationsin phytother-apy.Accordingly,thisstudyaimedtocarryoutaquantitativeandqualitativeassessmentofHypnea musciformis(Wulfen)J.V.Lamouroux,Ochtodessecundiramea(Montagne)M.A.Howe,andPterocladiella capillacea(S.G.Gmelin)Santelices&Hommersand(Rhodophyta)inordertodeterminetheAChEeffects fromtheirextracts.Asamatteroffact,theO.secundirameaextractshowed48%acetylcholinesterase inhibitionat400␮g/ml.Thechemicalcompositionofthebioactivefractionwasdeterminedbygas chromatography–massspectrometry(GC–MS);thisfractionissolelycomposedofhalogenated monoter-penes,thereforeallowingassignmentofacetylcholinesteraseinhibitionactivitytothem.

Introduction

Alzheimer’sdisease(AD)isachronicneurodegenerative

disor-dercharacterizedbyalossofbasalforebraincholinergicneurons

and reduced level of the neurotransmitter acetylcholine (ACh)

(Alzheimer’sAssociation,2014).ADaffectsupto5%ofpeopleover

65yearsofage,risingto20%ofthoseagedover80years,

suggest-ingtheimportanceoftreatingthisdiseasebaseduponincreased

life expectancy,particularly in developed countries worldwide.

Speciﬁcally,estimatesshowthat27.7upto35millionpeoplewere

affectedbythisdiseasefrom2005through2010,respectively.In

thissamesurvey,itwasfoundthattreatmentcostsincreasedfrom

156USDbillionto604billion(Wimoaetal.,2007,2013).

ControloverADisachievedbyextendingtheactionof

acetyl-choline(ACh)viaacetylcholinesteraseinhibition(AChEI).Current

drugsexhibittwoaction mechanisms,eitherprostheticor

acid-transferring.Prostheticinhibitorshaveanafﬁnityfortheanionic

siteofacetylcholinesteraseandpreventacetylcholinefrom

acces-singit(competitiveinhibitors).Acid-transferringinhibitorsreact

∗ Correspondingauthor.

E-mail:nyokoya@pq.cnpq.br(N.S.Yokoya).

withtheenzymeandformanintermediatecompound.

Depend-ingonthestabilityofthisproduct,theeffectscouldbeshort-term

and reversibleor long-actingand irreversible(Nairand Hunter,

2004).

ThecurrentdrugsusedagainstADareplant-derivedalkaloids:

rivastigmineandgalantamine.ThesedrugsareAChEIsandcanbe

usedtotreatearlyandmoderatestagesofADbyincreasingthe

endogenouslevelsofacetylcholinetoboostcholinergic

neurotrans-mission(McGleenonet al.,1999).Although thesedrugsdisplay

someundesirablesideeffects,suchashepatotoxicityand

gastroin-testinaldisorder,thereisagrowinginterestinﬁndingnewAChE

inhibitors fromnatural sources, but withfew off-targeteffects

(Rheeetal.,1997).

Marineorganismshaveahighchemicaldiversityofecologically

activesubstanceswithprotectivefunctionsagainstpredators,

bio-foulersandepiphytes,aswellasagainstconstantchangesinabiotic

conditionstoleratedbyalgaeinthemarinecoastalenvironment

(Amsler,2008;Ferreiraetal.,2012;Meskoetal.,2015).Such

diver-sityprovidesausefulresourceforthediscoveryofnovelbioactive

carboncompounds,manyofthemwithpotentialapplicationsinthe

pharmaceutical,nutraceutical,andagriculturalﬁelds(Smit,2004;

Cardozoetal.,2006;Gressleretal.,2011a,2011b;Torresetal., 2014).

http://dx.doi.org/10.1016/j.bjp.2015.09.003

(2)

SeaweedextractshavebeentestedagainstAChenzymewith

successfulresults(Natarajanetal.,2009).Assuch,seaweeds

rep-resentapromisinggroupofspecies fordevelopingnewmarine

naturalproductsbasedontheeaseofobtainingbiomassby

per-forming cultures for the exploitation of bioactive compounds

(Bawejaetal.,2009andthereforecontributingtotheconservation

ofnaturalpopulations(YokoyaandYoneshigue-Valentin,2011).

Hence,motivatedbytheeconomicandsocialimpactofAD,this

studywasaimedattestingthequalitativeandquantitativeAChEI

activityoforganicextractsobtainedfromthreeBrazilianredalgae.

Thechemicalcompositionofthebioactivefractionswas

investi-gatedbygaschromatography–massspectrometry(GC–MS).

Materialsandmethods

Algalmaterialandextraction

Twohundredgrams (freshweight; equivalenttothirty

indi-vidualsof each species)of Hypnea musciformis (Wulfen)in J.V.

Lamouroux,Ochtodessecundiramea(Montagne)M.A.Howeand

Pte-rocladiellacapillacea(S.G.Gmelin)Santelices&Hommersandwere

collectedfromalateriticreefatManguinhosBeach(20◦1112S,

40◦1124W)in the municipalityof Serra,Espírito Santo State,

Brazil.Aftercollection,seaweedbiomasswascleanedand

immedi-atelytransportedtothelaboratoryindarkplasticﬂaskscontaining

ﬁlteredseawatermaintainedat22◦Cbymeansofthermal

pack-aging.Furthermore, theseaweeds wereproperly identiﬁed and

depositedintheHerbariumVIES,UniversidadeFederaldoEspírito

Santo,under voucher numbers18.853,18.854 and18.855,

cor-respondingtoH.musciformis,O.secundiramea, andP.capillacea,

respectively.

Thefreshlycleanedseaweedswereweighedandthen

imme-diately extracted by using 10ml of dichloromethane/methanol

(DCM/MeOH)(2:1,v/v)per1gofseaweed(Machadoetal.,2014a).

Thematerialwasmaintainedinadarkroomat20◦Cforoneweek,

andthentheextractswereﬁlteredthroughaWhatmanNo.5

ﬁl-terpaperandconcentratedbyunderlowpressure.Theextraction

yieldwascalculatedforeachalgalsample.Allchemicalsusedwere

ofanalyticalgrade(Merck,Darmstadt,Germany).

Inhibitoryactivityofacetylcholinesterase(AChEI)

Qualitativeevaluationbyautographicassay

TheAChEIactivityofDCM/MeOHcrudeextractwasdetected

byusingathin-layerchromatography(TLC)autographicassayas

previouslydescribed(Marstonetal.,2002).Aliquotsof100␮gof

eachdriedseaweedextractand0.3␮gofphysostigmine(Sigma,

usedas positivecontrol) weredissolved,spotted onTLClayers

(Silicagel60F254,10×10cm,layerthickness0.2mm,E.Merck,

Germany),whichweredevelopedwithmobilephasehexane:ethyl

acetate:methanol(2:7:1v/v/v), and thendried.Next, theplates

weresprayedwiththeenzymesolution(6.66U/ml)(Electriceel

AChEtypeV,productno.C2888,1000U–Sigma–Aldrich),

thor-oughlydried,andincubatedinahumidatmosphereat37◦Cfor

20min. Afterwards, the plates were sprayed with 0.25% of

1-naphthylacetateinethanol(5ml)plus0.25%ofaqueousFastBlue

Bsaltsolution(20ml).Thespotscorrespondingtopotential

acetyl-cholinesteraseinhibitorswereidentiﬁedasclearzonesagainsta

purplebackground.

Retention factor values (Rf) of bioactive compounds were

determined and employed for theirpreparative scale isolation

by thin-layer chromatography (20cm×20cm, layer thickness

1.5mm,60F254,Sigma–Aldrich).Extractsamplesof80mgwere

appliedonpreparativeplateswhichweredevelopedunder

iden-tical conditions to those used at analytical scale essays. After

elution,theAChE-positiveregionswereremovedfromtheplates

andextractedwithDCM/MeOH(90:10v/v).

Quantitativeevaluationbymicroplateassay

AChE activity was measured using a modiﬁed 96-well

microplateassaybasedonEllman’smethod(Ellmanetal.,1961,as

modiﬁedbyRheeetal.,2001).Suchextremelysensitivemethod

isbased onmeasuringthiocholineproductionwhen

acetylthio-choline is hydrolyzed. This is accomplished by the continuous

reactionofthiolwith5,5-dithiobis(2-nitrobenzoicacid).Solutions:

A.Tris/HCl50mM,pH8;B.Tris/HCl50mM,pH8,with0.1%bovine

albuminfractionV;C.Tris/HCl50mM,pH8,withNaCl(0.1M)and

MgCl2·6H2O(0.02M).

Toeachwellofa 96-wellmicroplate,25␮lacetylthiocholine

iodide(15␮M), 125␮l of 5,5-dithiobis(2-nitrobenzoic acid) in

solution C (3␮M DTNB or Ellman’s reagent), 50␮l of solution

B, 25␮l of seaweedextract dissolved in MeOH and diluted in

solutionAat concentrationsof 1.56,12.5, 25, 50,100, 200and

400␮g/mlwereadded.Theabsorbancewasmeasuredat405nm

for30sec.After25␮loftheenzymeAChE(0.22U/ml)wasadded,

theabsorbancewasagainreadevery5minofincubationforfour

times.ThepercentageofAChEinhibitionwascalculatedby

com-paringthereactionratesofsamplestothenegativecontrol(10%

MeOHinsolutionA,considering100%asthetotalactivityofAChE).

Physostigmine wasused as standard at concentrations ranging

from1.56to400␮g/ml.ABioTekELISAreader(PowerWaveTM_HT

MicroplateReaderand KC4TM _software)_was_used_to_determine

reactionrates.Thisanalysiswascarriedoutintriplicate(n=3)to

calculatethemeanandstandarddeviation.

CG–MSanalysisofbioactivefractionobtainedfromO.

secundirameaextract

Currently,differentstrategieshavebeenreportedinthe

litera-tureinordertoestimatealgalcompoundsapplyingCGMS(Gressler

et al., 2009). The bioactive fraction obtained from O.

secundi-rameawasanalyzedbyagaschromatograph-massspectrometer

(GCMS-QP2010Plus,Shimadzu,Japan)provided withaHP-5MS

column(30m×0.25mm×0.1␮m).Thesampleswereinjectedin

splitmodeat220◦C,andthetransferlinewassetto240◦C.Helium

(99.999%)wasusedasthecarriergasataconstantﬂowrateof

1ml/min.Alinearoventemperatureprogramwasemployed.The

temperaturewasrampedfrom60to260◦Catarateof3◦C/min

andthenheldat260◦Cfor40min.Detectionwasperformedin

fullscanmode,mass-to-chargeratio(m/z)50–650.Electronimpact

ionizationwasemployed(collisionenergy=70eV),andthemass

spectrometerion sourcewasmaintainedat240◦C. Compounds

were identiﬁed by their GC retention times and mass spectra

(NIST08MassSpectralLibraryandliteraturedata).Theareaofeach

peakinthetotalionchromatogramwasintegrated.

Resultsanddiscussion

Fig.1shows thequalitative resultsoftheautographicassay

of acetylcholinesterase inhibition activity (AChEI), and Table 1

displaysthe freshseaweed biomass weights, dried extract and

extractionyieldalongwithAChEIdataoftheautographicassay:

Rfandbioactivecompoundintensity.

TheAChEIactivityofplantextractsisclassiﬁedaccordingto

Vinuthaetal.(2007)aspotentinhibitors(greaterthan50%

inhibi-tion),moderateinhibitors(30–50%inhibition)andweakinhibitors

(below30%inhibition).Accordingtothisclassiﬁcation,theextracts

ofO.secundirameahavemoderateactivity(48.59±0.8%),whileH.

musciformisandP.capillaceaextractshaveweakactivity(7.21and

5.38%,respectively),atahigherconcentration(Fig.2).

Asdescribedin theliterature,seaweedextractsexhibit poor

(3)

Table1

Seaweedbiomass,extractdryweights,extractionyieldsandautographicacetylcholinesteraseinhibition(AChEI)assayresults.

Seaweed Freshweight(g) Driedextracts(g) Yield(%) RfofAChEIcompounds IntensityofAChEIactivity

Hypneamusciformis 200 1.82 0.91 0.01 Low

Pterocladiellacapillacea 200 0.4 0.2 0.65 Low

Ochtodessecundiramea 200 2.82 1.41 0.62–0.71 Strong

smallerthanthatshowedbyO.secundirameaextract.TheEcklonia

stoloniferaethanolic extract (IC50 100␮g/ml)and Ishige

okamu-raemethanolicand ethanolicextracts(IC50 163.07␮M andIC50

137.25_␮M, respectively) are the only exceptions (Stirk et al.,

2007;Yoonetal.,2008;Kartaletal.,2009;Suganthyetal.,2010; PangestutiandKim,2011).

DCM/MeOHextract (15mg) were employed in the

fraction-ationcarriedoutunderthesameconditionsasthosedescribedfor

theanalyticalTLCexperiments.Thebioactivefraction(2.45mg;Rf

0.62–0.71),whichcorrespondedto16.3%ofthecrudeextract,was

analyzedbyGC–MS;theresultingdataareshowninTable2and

Fig.3.

Theactivefractioncompoundspresentthetypical

fragmenta-tionlossesrelatedtochlorineandbrominelosses(respectively,35

and79m/z),asclearlyobservedonthemassspectra.The

char-acteristicmultiplicityofhalogenisotopeabundance(35_Cl:37_Cl_–

75.2:24.8%and79_Br:81_Br_–_50:50%)_was_observed_in_their

molecu-larions.Asreportedinnumerousstructuralstudies,bothmagnetic

resonancespectra(13_C_NMR)₍_McConnell_and_Fenical,_1978;_Paul

etal.,1980;Nayloretal.,1983)andmassfragmentation(Polzinand Rorrer,2003;BarahonaandRorrer,2003)haveplayedakeyrole

in thestructural characterizationof halogenatedmonoterpenes

(Maliakaletal.,2001;Machadoetal.,2014a).

However,itcanalsobeobservedinthetotalionchromatogram

inwhichthepeak8correspondstothemajoritycompoundthathas

m/zvalue329.Itsmassspectrashowedfragmentionsatm/z294,

indicativeofchlorineloss(M+_−Cl),_and_at_m/z₂₁₃_and_m/z_133,

PC 1 2 3

Fig.1.TLCqualitativeacetylcholinesteraseinhibition(AChEI)assay.PC:positive control,physostigmine(0.03␮g).DCM/MeOHextracts(100␮g)of(1)Hypnea mus-ciformis,(2)Pterocladiellacapillaceaand(3)Ochtodessecundiramea.TLCelution system:hexane:ethylacetate:methanol(2:7:1v/v/v).

whicharecharacteristicofbromineandchlorinelosses(M+_−BrCl

andM+_−Br

2Cl),respectively.Theionsatm/z93and atm/z133

areobservedinthefragmentationofacyclicmonoterpenes.This

compound(C10H15Br2Cl)hasbeenidentiﬁedas

(E)-1,2-dibromo-3-chloromethylene-7-methyloct-6-ene,bycomparisonofitsmass

spectrum data withthose foundin the literature, and allowed

itsdiscriminationamongisomericcompounds(CollandWright,

1987).

Thecompounds1,2,7,9,and 10couldalsobeidentiﬁedby

comparingthedatacollectedfromtheirmassspectra(Table2)

totheliteratureones.Compounds1 (C10H16), 2(C10H15OBr),7

(C10H14Br3Cl),9(C10H15Br2ClO),and10(C10H14Br2Cl2)arenamed

as 7-methyl-3-methyleneocta-1,6-diene (myrcene) (Polzin and

Rorrer, 2003); 6(S*)-bromoocta-1,3(8)-dien-4(R*)-ol (Paulet al.,

1980);2-chloro-1,6(S*),8-tribromo-3-(8)(Z)-ochtodene(Gerwick,

1984);

7-chloro-4,8,dibromo-1-ethyl-3,3-dimethyl-6-hidroxi-7,8-cyclohexene (Naylor et al.,1983);

(3R*,4S*,5E)-4,8-dibromo-3,7-dichloro-3,7-dimethylocta-1,5-diene (Coll et al., 1988),

respec-tively.

The compound 2 (C10H15OBr) [6(S*) bromoocto-1,3(8)

dien-4(R)-ol] was prepared by synthesis and the remaining ones

were not properly identiﬁed due to insufﬁcient data: the

compounds 3 (C10H14Br2), 4 (C10H14BrCl, and 6 (C10H13Br2Cl)

are known as 3,7-dibromo-myrcene (Barahona and Rorrer,

2003); (Z)-10-bromo-7-chloro myrcene (Burresonet al., 1975);

and(Z)-10-chloro-3,7-dibromo-myrcene(Gerwick,1984),

respec-tively.

Massspectralfragmentationofcompound5suggestsan

aro-maticstructureduetoasequenceofpeaksatm/z91,79,65,41;

howeverthegreatnumberofisomershavingthesamestructural

formuladidnotallowthecompoundidentiﬁcation.

Currently,monoterpenesarethesecondmajorclassof

natu-ralmetaboliteswithAChEIactivity,surpassedonlybyalkaloids

(Barbosa-Filhoet al.,2006), andextractscontainingmixturesof

100 90 80 70 60 50 40 30 20 10 0 0 100 Seaweed extract (_{μg ml}–1₎ O. secundiramea P. capillacea H. musciformis Positive control AChE inhibition, % 200 300 400

Fig. 2. Quantitative evaluation of acetylcholinesterase activity (AChE) of the DCM/MeOHalgalextracts(1.56,12.5,25,50,100,200and400␮g/ml),comparedto thestandardphysostigmine.Meanandstandarddeviation(n=3).

(4)

Table2

GC–MSdatafrombioactivefraction(Rf0.62–0.71)obtainedfrompreparativeTLCcarriedoutwithOchtodessecundirameaextract. Peak Retentiontime(min) (%)Area Molecularmass

m/z

M+_−halogen _Main_mass_fragments

(intensityofsignal)

Molecularformula Reference

1 18.036 5.23 136 – 41(50)69(100)78,

81(20)

93(90)107(25)119(50) 135(80)136(trace)

C10H16 PolzinandRorrer(2003)

2 30.913 11.41 229;230 149(M+_−Br) ₄₁₍₄₀₎₅₃₍₄₀₎₇₉₍₇₀₎ 91(100) 105(95)122(85) 133(55) 149(80)185(30)229, 230(70)

C10H15OBr Pauletal.(1980)

3 31.636 2.82 292;294;296 213(M+_−Br) 133(M+_-Br 2) 41(15)65(15)77(20) 93(50)105(40)118(50) 133(100)

C10H14Br2 BarahonaandRorrer(2003)

4 36.215 3.09 248;249;251 214(M+_−Cl) 167(M+_−Br) 133(M+_−BrCl) 44(60)51(10)65(20) 77(30) 93(100)105(40) 117(60) 133(60)167169(20) 212 213(5) C10H14BrCl Burresonetal.(1975) 5 39.856 13.82 311 229(M+_−Br) 149(M+_−Br 2) 41(50)55(50)65(40) 79(80) 91(100)105(40) 118(60) 133(95)149(25) 229(20)

C10H12OBr2 Pauletal.(1980)

6 40.682 5.81 323;325 217(M+_−BrCl) 167(M+_−Br 2) 133(M+_−Br 2Cl) 41(40)65(30)77(50) 93(95) 105(50)117(60) 133(100) 167(40)217(40) C10H13Br2Cl Polzinetal.(2003) 7 43.298 13.81 408;410 375(M+_−Cl) 329(M+_−Br) 249(M+_−Br 2) 167(M+_−Br 3) 133(M+_−Br 3Cl) 41(10)77(40)91(50) 105(30) 117(50)133(100)167 197213249294329 375(5) C10H14Br3Cl Gerwick,1984 8 45.273 30.13 328;330;332 294(M+_−BrCl) 249(M+_−Br) 213(M+_−BrCl) 167(M+_−Br 2) 133(M+_−Br 2Cl) 69(15)77(40)93(50) 105(40) 133(100)167(50) 213(5) 249(5)294(3)

C10H15Br2Cl CollandWright(1987)

9 46.900 2.52 344;346;348 310(M+_−Cl) 267(M+_−Br) 229(M+_−BrCl) 185(M+_−Br₂₎ 147(M+_−Br 2Cl) 65(20)77(30)91(100) 105(40)117(60) 133(60); 147(15)185,187(20) 197,199(5)212,213(5) 229(10); 267269(5) 285287(5); 310311(5)

C10H15Br2ClO Nayloretal.(1983)

10 47.972 18.36 360;362;364 329(M+_−Cl) 294(M+_−Cl 2) 284(M+_−Br 2) 247(M+_−BrCl) 213(M+_−BrCl 2) 167(M+_−Br 2Cl) 133(M+_−Br 2Cl2) 77(40)91(50)105(30) 117(50)133(100) 167(80) 213(40)247(15) 284(20) 294(15)329(5) C10H14Br2Cl2 Colletal.(1988)

monoterpenesgenerallydisplayhighAChEI.Theyarereversible

competitiveinhibitorsofacetylcholinenesterase(KeaneandRyan,

1999) and sucheffect is attributedto thecombined actions of

thesemultifunctionalcompoundswhichcanexerttheiractivities

throughdifferentmechanisms(Korochetal.,2007).These

obser-vationsare in accordance withthepresent study documenting

thepotentialAChEIofanO.secundirameaextractcontaining

halo-genatedmonoterpenes.

Brominatedmonoterpenesareformedviareactionscatalyzed

byenzymescapableofgeneratingnucleophilichalideanions,

elec-trophilichypohalite species, orhalogenradicals, promotingthe

substitutionofhydrogenatomsforhalogensinaliphatic

hydro-carbons(Moore, 2006;Hillwig and Liu, 2014).TheirAChEI can

resultfromtheeasydisplacementofbrominebyneutraloranionic

nucleophileswhichcouldthenberemovedbyelimination

reac-tions(Daganietal.,2014).

Whereasthetoxicity,orsideeffects,ofalkaloids,widelyused

asasourceofpotentialacetycholinesteraseinhibitors,isa

draw-backin Alzheimer’sdisease treatment, O. secundiramea extract

showed,inpreliminarytrials,nocytotoxicityormutagenic

activ-itiesatconcentrationstwiceasmuchthosetestedinthepresent

study(Machadoetal.,2014b).

Theresultspresentedherewithprovidedevidenceto

corrob-oratethetherapeuticpotentialofO.secundirameamonoterpenes

based upon theirmoderate AChEIactivity in combination with

(5)

10 1 2 3 4 5 6 7 8 10 9 2.0 TIC (x1 000 000) 1.5 1.0 0.5 20 30 40 50 60

Fig.3.Totalionchromatogram(TIC)fromGC–MSanalysisofAChEIfractionobtainedbypreparativeTLCcarriedoutwithO.secundirameaextract.

Authors’contributions

LPM collected and identiﬁed seaweed samples, as well as

carried out lab experiments,analyzed thedata, and wrote the

manuscript.LRCperformedchemicalandchromatographic

anal-ysesandprovidedcriticalreadingofthemanuscript.MCMYand

EMCLperformedlaboratoryexperimentsinvolvingbiological

stud-ies.DCCandLZVcarriedoutchemicalstudies.PCandNSYdesigned

thestudy,supervisedthelaboratoryworkandcontributedto

crit-ical reading of the manuscript. All authorshave read the ﬁnal

manuscriptandapprovedthesubmission.

Conﬂictsofinterest

Authorshavenoconﬂictsofinteresttodeclare.

Acknowledgments

TheauthorsthankﬁnancialsupportsfromCNPqandFAPESP,

scholarshipfromCAPEStotheﬁrstauthor,andgrantsfromCNPq

toMCMY,PC,andNSY.Thisstudyispartofthesispresentedby

theﬁrstauthortotheGraduateProgrammeinPlantBiodiversity

andEnvironment,InstituteofBotany,SãoPaulo,Brazil.Theauthors

wouldalsoliketothankProf.O.Vieiraforproof-readingandediting

themanuscriptinEnglish.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,atdoi:10.1016/j.bjp.2015.09.003.

References

Alzheimer’sAssociation,2014.Alzheimer’sdiseasefactsandﬁgures.Alzheimer’s Dement.10,1–80.

Amsler,C.D.,2008.AlgalChemicalEcology.Springer,Berlin.

Barahona,L.F.,Rorrer,G.L.,2003.Isolationofhalogenatedmonoterpenes from bioreactor-cultured microplantletsof the macrophytic red algae Ochtodes secundirameaandPortieriahornemannii.J.Nat.Prod.66,743–751.

Barbosa-Filho,J.M.,Medeiros,K.C.P.,Diniz,M.F.,Batista,L.M.,Athayde-Filho,P.F., Silva,M.S.,Cunha,E.V.L.,Almeida,J.R.G.S.,Quintans-Júnior,L.J.,2006.Natural productsinhibitorsoftheenzymeacetylcholinesterase.Rev.Bras.Farmacogn. 16,258–285.

Baweja,P.,Sahoo,D.,García-Jiménez,P.,Robaina,P.R.,2009.Seaweedtissueculture asappliedtobiotechnology:problems,achievementsandprospects.Phycol.Res. 57,45–58.

Burreson,B.J.,Woolard,F.X.,Moore,R.M.,1975.Evidenceforthebiogenesisof halo-genatedmyrcenesfromtheredalgaChondrococcushornemanii.Chem.Lett.4, 1111–1114.

Cardozo,K.H.M.,Carvalho,V.M.,ErnaniPinto,E.,Colepicolo,P.,2006.Fragmentation ofmycosporine-likeaminoacidsbyhydrogen/deuteriumexchangeand electro-sprayionisationtandemmassspectrometry.RapidCommun.MassSpectrom. 20,253–258.

Coll, J.C., Wright, A.D., 1987. Tropical marine algae. I. New halogenated monoterpenesfromChondrococcushornemannii(Rhodophyta,Gigartinales, Rhi-zophyllidaceae).Aust.J.Chem.40,1893–1900.

Coll,J.C.,Skelton,B.W.,White,A.H.,Wright,A.D.,1988.Tropicalmarinealgae. II.Thestructuredeterminationofnewhalogenatedmonoterpenesfrom Plo-camiumhamatum(Rhodophyta,Gigartinales,Plocamiaceae).Aust.J.Chem.41, 1743–1753.

Dagani,M.J.,Barda,H.J.,Benya,T.J.,Sanders,D.C.,2014.Organicbrominecompounds. In:Ullmann’sFineChemicals.Wiley-VCHVerlagGmbH&Co,Weinhein, Ger-many.

Ellman,G.L.,Courtney,K.D.,AndresJr.,V.,Featherstone,R.M.,1961.Anewandrapid colorimetricdeterminationofacetylcholinesteraseactivity.Biochem. Pharma-col.7,88–95.

Hillwig,M.L.,Liu,X.,2014.Anewfamilyofiron-dependenthalogenasesactson freestandingsubstrates.Nat.Chem.Biol.10,921–923.

Ferreira,L.D.S.,Turatti,I.C.C.,Lopes,N.P.,Guaratini,T.,Colepicolo,P.,Oliveira,E.C., Garla,R.C.,Pohlit,A.M.,Zucchi,O.L.A.D.,2012.Apolarcompoundsinseaweeds fromFernandodeNoronhaarchipelago(NortheasternCoastofBrazil).Int.J. Anal.Chem.,http://dx.doi.org/10.1155/2012/431954.

Gerwick,W.H.,1984.2-Chloro-1,6(S*),8-tribromo-3-(8)(Z)-ochtodene:a metabo-liteofthetropicalredseaweedOchtodessecundiramea.Phytochemistry23, 1323–1324.

Gressler,V.,Colepicolo,P.,Pinto,E.,2009.Usefulstrategiesforalgalvolatileanalysis. Curr.Anal.Chem.5,271–292.

Gressler,V.,Fujii,M.,Martins,A.P.,Colepicolo,P.,Mancini,J.,Pinto,E.,2011a. Bio-chemicalcompositionoftworedseaweedspeciesgrownontheBraziliancoast. J.Sci.FoodAgric.91,1687–1692.

Gressler,V.,Stein,E.M.,Dörr,F.,Fujii,M.T.,Colepicolo,P.,Pinto,E.,2011b. Sesquiter-penesfromtheessentialoilofLaurenciadendroidea(Ceramiales,Rhodophyta): isolation,biologicalactivitiesanddistributionamongseaweeds.Rev.Bras. Far-macogn.21,248–254.

Kartal,M.,Orhan,I.,Abu-Asaker,M.,Senol,F.S.,Atici,T.,Sener,B.,2009.Antioxidant andanticholinesteraseassetsandliquidchromatography-massspectrometry prefaceofvariousfresh-waterandmarinemacroalgae.Pharmacogn.Mag.5, 291–297.

Keane,S.,Ryan,M.F.,1999.Puriﬁcation,characterisation,andinhibitionby monoter-penesofacetylcholinesterasefromthewaxmoth,Galleriamellonella(L.).Insect Biochem.Mol.Biol.29,1097–1104.

Koroch,A.R.,Juliani,H.R.,Zygadlo,J.A.,2007.Bioactivityofessentialoilsandtheir components.Flav.Fragr.Chem.87,547–553.

Machado, L.P., Carvalho, L.R., Young, M.C.M., Zambotti-Villela, L., Colepicolo, P., Andreguetti, D.X., Yokoya, N.S., 2014a. Comparative chemical analy-sisandantifungalactivityofOchtodessecundiramea(Rhodophyta)extracts obtained usingdifferent biomassprocessingmethods.J. Appl.Phycol. 26, 2029–2035.

Machado,L.P.,Matsumoto,S.T.,Jamal,C.M.,Silva,M.B.,Centeno,D.C.,Colepicolo,P., Carvalho,L.R.,Yokoya,N.S.,2014b.Chemicalanalysisandtoxicityofseaweed extractswithinhibitoryactivityagainsttropicalfruitanthracnosefungi.J.Sci. Food.Agric.94,1739–1744.

Maliakal,S.,Cheney,C.,Rorrer,G.,2001.Halogenatedmonoterpeneproductionin regeneratedplantletculturesofOchtodessecundiramea(Rhodophyta, Cryptone-miales).J.Phycol.37,1010–1019.

Marston,A.,Kissling,J.,Hostettmann,K.,2002.ArapidTLCbioautographicmethod forthedetectionofacetylcholinesteraseandbutyrylcholinesteraseinhibitorsin plants.Phytochem.Analysis13,51–54.

McConnell,O.J.,Fenical,W.,1978.Ochtodeneandochtodiol:novelpolyhalogenated cyclicmonoterpenesfromtheredseaweedOchtodessecundiramea.J.Org.Chem. 43,4238–4241.

McGleenon,B.M.,Dynan,K.B.,Passmore,A.P.,1999.Acetylcholinesteraseinhibitors inAlzheimer’sdisease.Br.J.Clin.Pharmacol.48,471–480.

Mesko,M.F.,Picoloto,R.S., Ferreira,L.R.,Costa,V.C.,Pereira,C.M.P.,Colepicolo, P., Muller, E.I., Flores, E.M.M.,2015. Ultraviolet radiation combinedwith microwave-assistedwetdigestionofAntarcticseaweedsforfurther determi-nationoftoxicelementsbyICP-MS.J.Anal.At.Spectrom.30,260–266.

(6)

Moore,B.S.,2006.Biosynthesisofmarinenaturalproducts:macroorganisms(Part B).Nat.Prod.Rep.23,615–629.

Nair,V.P.,Hunter,J.M.,2004.Anticholinesterasesandanticholinergicdrugs.Cont. Edu.Anaest.Crit.Care.Pain4,164–168.

Natarajan,S.,Shanmugiahthevar,K.P.,Kasi,P.D.,2009.Cholinesteraseinhibitors fromSargassumandGracilariagracilis:seaweedsinhabitingSouthIndiancoastal areas(HareIsland,GulfofMannar).Nat.Prod.Res.23,355–369.

Naylor,S.,Hanke,F.J.,Manes,L.V.,Crews,P.,1983.Chemicalandbiologicalaspects ofmarinemonoterpenes.FortschrittederChemieOrganischerNaturstoffe/Prog. Chem.Org.Nat.Prod.44,189–241.

Pangestuti,R.,Kim,S.K.,2011.Neuroprotectiveeffectsofmarinealgae.Mar.Drugs 9,803–818.

Paul,V.J.,McConnell,O.J.,Fenical,W.,1980.Cyclicmonoterpenoidfeedingdeterrents fromtheredmarinealgaOchtodescrockeri.J.Org.Chem.45,3401–3407.

Polzin, J.P., Rorrer, G.L., 2003. Halogenated monoterpene production by microplantlets of the marine red alga Ochtodes secundiramea within an airliftphotobioreactorundernutrientmediumperfusion.Biotechnol.Bioeng. 82,415–428.

Polzin,J.J.,Rorrer,G.L.,Cheney,D.P.,2003.Metabolicﬂuxanalysisofhalogenated monoterpene biosynthesis in microplantlets of the macrophytic red alga Ochtodessecundiramea.Biomol.Eng.20,205–215.

Rhee,I.K.,Meent,M.V.,Ingkaninan,K.,Verpoorte,R.,Okada,M.,Marimo,M.,1997.

Studiesoninhibitoryactivityagainstacetylcholinesteraseofnew bisbenzyliso-quinolinealkaloidanditsrelatedcompounds.Heterocycles45,2253–2260.

Rhee,I.K.,Meent,M.V.,Ingkaninan,K.,Verpoorte,R.,2001.Screeningfor acetyl-cholinesterase inhibitors from Amaryllidaceae using sílica gel thin-layer chromatographyincombinationwithbioactivitystaining.J.Chromatogr.A915, 217–223.

Smit,A.J.,2004.Medicinalandpharmaceuticalusesofseaweednaturalproducts:a review.J.Appl.Phycol.16,245–262.

Stirk,W.,Reinecke,D.,vanStaden,J.,2007.Seasonalvariationinantifungal, antibac-terialandacetylcholinesteraseactivityinsevenSouthAfricanseaweeds.J.Appl. Phycol.19,271–276.

Suganthy,N.,Pandian,S.K.,Devi,K.P.,2010.Neuroprotectiveeffectofseaweeds inhabitingSouthIndiancoastalarea(HareIsland,GulfofMannarmarine bio-spherereserve):cholinesteraseinhibitoryeffectofHypneavalentiaeandUlva reticulata.Neurosci.Lett.468,216–219.

Torres,F.A.E.,Passalacqua,T.G.,Velasquez,A.M.A.,Souza,R.A.,Colepicolo,P., Gram-inha,M.A.S.,2014.Newdrugswithantiprotozoalactivityfrommarinealgae:a review.Rev.Bras.Farmacogn.24,265–276.

Vinutha,B.,Prashanth,D.,Salma,K.,Sreeja,S.L.,Pratiti,D.,Padmaja,R.,Radhika, S.,Amit,A.,Venkateshwarlu,K.,Deepak,M.,2007.ScreeningofselectedIndian medicinalplantsforacetylcholinesteraseinhibitoryactivity.J.Ethnopharmacol. 109,359–363.

Wimoa,A., Winblada,B.,Jönssonb,L.,Bond,J.,Princee,M.,Winblad,B.,2013.

Theworldwideeconomicimpactofdementia2010.Alzheimer’sDement.9, 1–11.

Wimoa,A.,Winblada,B.,Jönssonb,L.,2007.Anestimateofthetotalworldwide societalcostsofdementiain2005.Alzheimer’sDement.3,81–91.

Yokoya,N.S.,Yoneshigue-Valentin,Y.,2011.Micropropagationasatoolfor sus-tainableutilizationandconservationofpopulationsofRhodophyta.Rev.Bras. Farmacogn.21,334–339.

Yoon,N., Chung,H., Kim,H., Choi, J.,2008. Acetyland butyrylcholinesterase inhibitoryactivitiesofsterolsandphlorotanninsfromEckloniastolonifera.Fish. Sci.74,200–207.