First isolation and activity of a lipid transfer protein from noni ( Morinda citrifolia ) seeds

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ContentslistsavailableatScienceDirect

International

Journal

of

Biological

Macromolecules

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / i j b i o m a c

First

isolation

and

antinociceptive

activity

of

a

lipid

transfer

protein

from

noni

(

Morinda

citrifolia

)

seeds

Dyély

C.O.

Campos

a

_,

_Andrea

_S.

_Costa

a

_,

_Amanda

_D.R.

_Lima

a

_,

_Fredy

_D.A.

_Silva

a

_,

Marina

D.P.

Lobo

a

_,

_Ana

_Cristina

_O.

_{Monteiro-Moreira}

b

_,

_Renato

_A.

_Moreira

b

_,

Luzia

K.A.M.

Leal

c

_,

_Diogo

_Miron

c

_,

_Ilka

_M.

_Vasconcelos

a

_,

_Hermógenes

_D.

_Oliveira

a,∗

a_Department_of_Biochemistry_and_Molecular_Biology,_Federal_University_of_Ceará,_Campus_do_Pici_Prof._Prisco_Bezerra,_60440-900_Fortaleza,_CE,_Brazil b_{NUBEX—Núcleo}_de_Biologia_{Experimental,}_Centro_de_Ciências_da_Saúde,_Universidade_de_{Fortaleza—UNIFOR,}_60811-905_Fortaleza,_CE,_Brazil c_Department_of_Clinical_and_{Toxicological}_Analysis,_Faculty_of_Pharmacy,_Federal_University_of_Ceará,_Fortaleza,_CE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4November2015

Receivedinrevisedform7January2016 Accepted8January2016

Availableonline16January2016

Keywords: MorindacitrifoliaL. Rubiaceae Noni

Proteinisolation Antinociceptiveactivity Lipidtransferprotein

a

b

s

t

r

a

c

t

Inthisstudyanovelheat-stablelipid transferprotein, designatedMcLTP1,waspurifiedfromnoni (Morindacitrifolia L.) seeds,usingfour purificationstepswhichresulted inahigh-purified protein yield (72mg McLTP1 from100g of noni seeds).McLTP1 exhibited molecularmassesof 9.450and 9.466kDa,determinedbyelectrosprayionisationmassspectrometry.TheN-terminalsequenceofMcLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV),asanalysedbyNCBI-BLASTdatabase,revealedahigh degreeofidentitywithotherreportedplantlipidtransferproteins.Inaddition,thisproteinprovedto beresistanttopepsin,trypsinandchymotrypsindigestion.McLTP1givenintraperitoneally(1,2,4and 8mg/kg)andorally(8mg/kg)causedaninhibitionofthewrithingresponseinducedbyaceticacidin mice.Thisproteindisplayedthermostability,retaining100%ofitsantinociceptiveactivityafter30min incubationat80◦_C._Pretreatment_of_mice_with_Mc_LTP₁₍₈_mg/kg,_i.p._and_p.o.)_also_decreased_neurogenic andinflammatoryphasesofnociceptionintheformalintest.Naloxone(2mg/kg,i.p.)antagonisedthe antinociceptiveeffectofMcLTP1suggestingthattheopioidmechanismsmediatetheanalgesicproperties ofthisprotein.

1. Introduction

MorindacitrifoliaL.(Rubiaceae),aplantspeciesoriginatingfrom

Tropical Asia, has beenused for millennia as a sourceof food

and fabricdyes, as well asto treat disorders suchas diabetes,

rheumatoidarthritis, highblood pressure,inflammationandfor

painmanagement[1–3].Inadditiontoitsusein folkmedicine,

thereisalsoalotofevidenceofbiologicalactivityinvariousinvitro

andinvivoassaysystems[4–8].

Asaresultofvariousphytochemicalinvestigations,morethan

200secondarymetaboliteshavebeenidentifiedfromnonifruits,

roots, seeds and leaves, including anthraquinones, flavonoids,

polysaccharides, glycosides, iridoids, lignans and triterpenoids,

withthemostrepresentativebeingscopoletin,rutin,ursolicacid,

␤-sitosterol,asperulosideanddamnacanthal.Someofthese

com-∗Correspondingauthor.Fax:+558533669139. E-mailaddress:hermogenes@ufc.br(H.D.Oliveira).

poundshavebeensuggestedtobethesourcesofnoni’sbiological

andinvitroactivity[9–12].

Althoughnonibark,stems,leavesandfruitshave beenused

traditionallyformanydiseases,thereisalimitedamountof

infor-mationontherapeuticpropertiesof noniseeds.Air-dried seeds

constitute2.5%ofthefruit’stotalweight,howeverduringthe

pro-ductionofnonifruitpureetheyarediscarded[13].Inaddition,West

etal.[14]demonstratedthatnoniseedextractdidnotdisplayany

signoftoxicityinasubacute(28day)oraltoxicitytestin

Sprague-Dawleyrats,anddidnotexhibitgenotoxicpotentialinaprimary

DNAdamagetestinEscherichiacoliPQ37.Thus,noniseedscanbe

usedtoisolatecompoundswiththerapeuticpropertiesanda

high-addedvalue,leadingtogreatereconomicbenefitsforproducers

andanincreaseinthediversityofcompoundsderivedfromthis

species.

In contrast to their therapeutic actions, there are reports

of toxicity resulting from the consumption of noni

prod-ucts [15–18]. Secondary metabolites, such as anthraquinones,

have been suggested as the causative agents of toxicity.

1-Hydroxyanthraquinone,acomponentofnoniroots,wasshownto

http://dx.doi.org/10.1016/j.ijbiomac.2016.01.029

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becarcinogenicinmaleratsandproducedadenomas,

adenocarci-nomasandbenignstomachtumoursinasmallnumberofanimals

[19].

Inthisstudywereport,forthefirsttime,theisolationand

char-acterisationofathermostablelipidtransferprotein(McLTP1)from

noniseedsthatexhibitspotentantinociceptiveactivitywhenorally

administeredtomice.McLTP1isthefirstbioactiveproteinisolated

fromthenonispeciesinrelationtothetherapeuticpropertiesof

theplant.

Plantnon-specificLTPs(nsLTPs)formaproteinfamilyofsmall

cationicpeptides ubiquitously distributed throughout theplant

kingdom[20,21].Theybelongtotheantimicrobialpeptidesgroup,

whichhasbeenincreasinglyconsideredasa sourceofpotential

therapeuticagents,exhibitingpotentialapplicationsasanalgesics,

immunomodulatorsorinthetreatmentofneurologicaldisorders

[22–25].Todate,thisisthefirstreportofalipidtransferprotein

demonstratingantinociceptiveactivityinmice.

2. Materialsandmethods

2.1. Chemicals

SephadexG-50, Superose12HR 10/30and molecularweight

markers(LMW-SDSmarkerkit)werepurchasedfromGE

Health-careLifeSciences.AgilentEclipseXBD-C18columnwaspurchased

from Agilent Technologies. Reagents and solvents for

pro-tein sequencing, acetic acid, trichloroacetic acid, indomethacin,

naloxonehydrochloride,morphinesolution(1mg/mL),diazepam

solution (1mg/mL) and formaldehyde were purchased from

Sigma–AldrichCo.,St.Louis,MO. Otherchemicalsusedwereof

analyticalgradeandobtainedfromlocalsuppliers.

2.2. Noniseeds

Noni(M.citrifoliaL.var. citrifolia)seedswerecollectedfrom

plantscultivatedattheAntonioAlbertoFarm,Ceará(3◦₁₆′₄₀′′_S,

39◦₁₆′₀₈′′_W) _and _supplied _by _Embrapa _{Agroindústria} _Tropical

(Embrapa—CNPAT),Ceará,Brazil.Asamplespecimen(no.44.566)

hasbeendepositedinthePriscoBezerraHerbariumoftheFederal

UniversityofCearáforfuturereference.Theseedswereground

usingakitchenblenderandtheresultingflourthoroughlydefatted

withpetroleumether(1:10,w/v)andstoredat−20◦Cuntilused.

2.3. PurificationofMcLTP1

Proteinswereextractedfromthedefattednoniseedflour(15g)

using0.050MTris–HCl/0.25MNaCl,pH8.5(1:10w/v)at4◦_C._The

suspensionwasstirredfor3handthenfilteredthrough

cheese-cloth.Theresiduewasre-extractedundertheaboveconditionsfor

2handthenfiltered.Thefiltrateswerecombined,centrifugedat

10,000×g,4◦_C _for₃₀_min_and_the_clear_supernatant_designated

ascrudeextract(CE).TheCEwassubjectedtoacidtreatmentby

theadditionoftrichloroaceticacid(TCA)toa2.5%(w/v)final

con-centration.After30minonice,the2.5%TCAsolublefractionwas

clarifiedbycentrifugationat10,000×gfor30minat4◦_C_and_the

supernatantdialysed(cut offMW3000) against distilled water

at4◦_C_and_lyophilised._The_lyophilised_protein_fraction_was

dis-solved(4mg)in0.050MTris–HCl/0.15MNaCl,pH8.5andapplied

onaSephadexG-50(1.5×40cm)columnpreviouslyequilibrated

withthe same buffer. Fractions (2mL) from the protein peaks

weremonitoredat280nmandcollectedataconstantflowrateof

30mL/h.Twoproteinpeakswererecoveredusingtheequilibrium

buffer. The fractions showing antinociceptive activity (McLTP1)

wereselected,concentratedandsubjectedtoanalytical

reversed-phasehigh-performanceliquidchromatography(RP-HPLC).

2.3.1. RP-HPLC

Pooledfractionsof proteinobtainedfromgelfiltrationwere

subjectedtoreversed-phasehigh-performanceliquid

chromatog-raphy using a C18 column (Agilent Eclipse XBD-C18 column

(250×4.6mm,5␮m)).Proteinsweredissolvedinultrapurewater

toobtainasolutionof0.5mg/mLandwerethenfilteredthrough

a 0.22␮mmembrane beforebeinginjectedinto theHPLC

(vol-umeinjected:20␮Loffilteredproteinsample).Thecolumnwas

equilibratedwith0.02%aqueousaceticacidandtheproteinswere

elutedataflowrateof1mL/minunderisocraticmode.Elutionwas

monitoredat216nmusingadiodearraydetector(AllianceHPLC

System,Waters,Corp.,Milford,MA).Thepeakelutingat7.9min

wasvacuum-dried,dissolvedinultrapurewater andappliedon

SDS-PAGE.

2.4. Proteinquantification

Proteinconcentrations were determinedby thedye-binding

methodofBradford[26],withbovineserumalbumin(BSA)asthe

standard.

2.5. Molecularmassdetermination

ThemolecularmassofMcLTP1wasdeterminedbydenaturating

SDS-PAGEon12.5%gelsundernon-reducingconditions,

follow-ingstandardprocedures[27].Proteinbandswerevisualisedwith

CoomassieBrilliantBlueR-250(Sigma–AldrichCo.,St.Louis,MO)

foratleast2handdestained(40%methanol,50%water,10%glacial

aceticacid)for30min.

ThenativemolecularmassofMcLTP1(1mg)wasdetermined

bygelfiltrationonaSuperose12HRcolumncoupledtoanFPLC

System(GEHealthcare)and equilibratedwith0.050MTris–HCl

buffer/0.25MNaCl, pH8.5.Chromatography wascarriedout at

aconstantflow rateof0.3mL/minand1mLfractionswere

col-lected. The column was previously calibrated with proteins of

known molecular masses (BSA, 66kDa; egg albumin, 45kDa;

chymotrypsinogen,25kDa;ribonuclease,13.7kDa,andaprotinin,

6.5kDa).

Mass spectrometry of McLTP1 (0.2mg/mL dissolved in

water/acetonitrile 1:1,v/v) was carried outon a Synapt HDMS

Acquity UPLCinstrument (Waters,Manchester, UK)by

electro-sprayionisationinpositiveionmode(ESI+)andaNanoLockSpray

source.The intact massspectra wereeffectively acquiredfrom

m/z1000to4000,whichallowedtoobtainmultiplychargedmass

ions.Themassspectrometerwasoperatedinthe“V”modewith

aresolvingpowerofatleast10,000m/z.Thedatacollectionwas

performedusingMassLynx4.1software(WatersCo.,Milford,MA,

USA)andchargedistributionspectrawerethendeconvolutedby

theMaximumEntropyTechnique(Max-Ent).

2.6. N-terminalaminoacidsequenceanalysis

The N-terminal amino acid sequence was analysed on a

Shimadzu PPSQ-23A Automated Protein Sequencer, performing

EdmandegradationofMcLTP1blottedonpolyvinylidenefluoride

membraneafterSDS-PAGE.Thesequenceobtainedwassubmitted

toautomaticalignmentusingtheNCBI-BLASTsearchsystem[28].

Theproteinsequencedatareportedinthispaperwillappearinthe

UniProtKnowledgebaseundertheaccessionnumberC0HJH5.

2.7. Invitrodigestionassay

Invitrodigestibility of McLTP1 wasdeterminedaccordingto

themethodofSathe[29],withmodifications.McLTP1(1mg/mL)

wassuspended in 200␮L of0.1MHCl, pH1.8, forpepsin (E.C.

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0.1MTris–HCl,pH8.1fortrypsin(E.C.3.4.21.4;10,100BAEEU/mg

protein,frombovinepancreas)andkeptfor10minat37◦_C._Next,

40␮Lofenzyme(1mg/mL)wasaddedtostarttheproteindigestion

andthereactionmixturewasincubatedinashakingwaterbathat

37◦_C_for₄_h._Aliquots_of₂₅_␮_L_were_taken_at₀_and₄_h_and₂₅_␮_L

of0.5MTris–HClbuffer,pH6.8,containing0.1%SDS,added.The

digestswereimmediatelyheatedinboilingwater(98◦_C)_for₅_min

andanalysedbySDS-PAGE[27].

Sequentialdigestionexperimentsusingpepsin,trypsinand

chy-motrypsin(E.C.3.4.21.1;40U/mgprotein,frombovinepancreas)

werecarriedoutusingMcLTP1 (1mg/mL)dissolvedin0.1MHCl,

pH1.8andincubatedinawaterbathat37◦_C_for₅_min._An_aliquot

ofpepsin(enzyme:protein1:10,w/w)wasaddedandthemixture

incubatedat37◦_C_for₄_h._The_digestion_was_immediately_stopped

byheatingthemixtureinaboilingwater(98◦_C)_bath_for₅_min.

Next,thepepsin-digested mixturewasmixed withtrypsinand

chymotrypsin(enzyme:protein1:10,w/w)in0.1MTris–HCl,pH

8.1,forfurtherdigestionfor5h.Thereactionwasstoppedandthe

sampleanalysedbySDS-PAGEasdescribedabove.BSA(2mg/mL)

wasusedasacontrolforthedigestionreaction.Allassayswere

performedintriplicate.

2.8. Animals

Experimentalprocedureswereconductedusinggroupsof2–3

montholdSwissmalemice(25–30g)obtainedfromBiotério

Cen-tralofFederalUniversityofCeará.Theanimalswerehousedunder

standard environmentalconditions (24±1◦C, humidity45–65%

and12hlight/darkcycle)andreceivedfoodandwateradlibitum.

Groupsof6–8micewereusedineachexperimentandhabituated

tothelaboratoryconditionsforatleast2hbeforetesting.Care,

han-dlingandexperimentalprocedureswereperformedinaccordance

withtheethicalstandardsestablishedbytheNationalGuidelines

fortheUseofExperimentalAnimalsofBrazilandbytheDirective

2010/63/EUoftheEuropeanParliamentandoftheCouncilofthe

EuropeanUnion.Theexperimentalprotocolswereapprovedbythe

CommitteefortheEthicalUseofAnimalsoftheFederalUniversity

ofCeará(CEUA-UFCno.37/13).

2.9. AntinociceptiveactivityofMcLTP1

2.9.1. Aceticacid-inducedwrithingmethod

Thistestwasperformedusingamodifiedversionofthemethod

described byKosteret al.[30].Animalsweretreated

intraperi-toneally(i.p.)withnoniseedcrudeextract(10,30or90mg/kg),2.5%

TCAsolublefraction(8mg/kg),McLTP1(1,2,4or8mg/kg),vehicle

(NaCl0.15M)orindomethacin(positivecontrol;10mg/kg)30min

beforetheinjectionofa1.0%aceticacidsolution(0.1mL/10gbody

weight,i.p.).Thenumber ofabdominal writhes(pelvicrotation

followed byfull extensionof both hind legs)wascumulatively

countedovera periodof20min,soon aftertheaceticacidwas

administered.ToevaluatewhetherMcLTP1displaysoral

antinoci-ceptive activity, it was given orally (8mg/kg body weight) to

animals30or60minpriortoanaceticacidinjection.The

antinoci-ceptiveeffectwasexpressedasapercentageoftheinhibitionof

abdominalwrithing.

2.9.1.1. Thermalstability determination. Toestablishthethermal

stabilityoftheprotein,McLTP1(1mg/mL)dissolvedinNaCl0.15M

wasincubatedinawaterbathat80◦_C_for₃₀_min._After_cooling

thetreatedMcLTP1onicefor10min,miceweretreatedwiththis

protein(8mg/kg(i.p.)),30minbeforeaceticacidsolutioninjection,

andtheantinociceptiveactivitymeasuredasdescribedinSection

2.9.1.

2.9.2. Formalin-inducedpainassay

Themethodusedwassimilartothatpreviouslydescribedwith

somemodifications[31].Thirtyminutesaftertheadministrationof

McLTP1(8mg/kgp.o.ori.p.),NaCl0.15M(vehicle;negativecontrol;

p.o.ori.p.)ormorphine5mg/kgi.p.(positiveopioidcontrol),20␮L

of2.5%formalin(formaldehyde36.5%solution)inasalinesolution

wasinjectedintothesubplantaroftherighthindpawsofthemice.

Animalswereplacedindividuallyinatransparentcageandthetime

(inseconds)spentlickingandbitingtheinjectedpawwastakenas

anindicatorofthepainresponse.Thepainresponsewasmeasured

for5min(firstphase)and20–25min(secondphase)afterthe

for-malininjection,representingbothneurogenicandinflammatory

painresponsesrespectively.

2.9.2.1. Involvementofopioidreceptors. Toidentifywhether

opi-oidreceptorsareinvolvedintheantinociceptiveactionofMcLTP1

(8mg/kg;p.o.ori.p.),groupsofmicewerepretreatedwitha

non-selectiveopioidreceptor antagonist,naloxone(2mg/kg),30min

beforeadministrationofMcLTP1ormorphine(5mg/kg)andtested

usingtheformalintest.

2.10. EffectofMcLTP1onlocomotoractivity

TheeffectoftheMcLTP1(8mg/kg)onspontaneouslocomotor

activityandexploratorybehaviourwasassessedusingthe

open-field test, using the methodset out by Martin et al. [32]. The

apparatus(45cmwidth×45cmlength×15cmheight)consisted

ofawoodenfieldinwhichthefloorwasdividedinto36equalareas.

MiceweretreatedwithMcLTP1 orally60minbeforethe

exper-imentor withthevehicle(NaCl 0.15M) or diazepam(2mg/kg)

intraperitoneally30minbeforethe experiment. Thenumber of

areascrossedwithallpawsandthenumberofrearingresponses

werethenrecordedduringa5minperiod.

2.11. Statisticalanalysis

Intheresultsthemean±SEMarepresented.Statisticalanalysis

wasperformedusingone-wayanalysisofvariance(ANOVA)

fol-lowedbyTukey’sposthoctests,usingGraphpadPrism6.0software

(SanDiego,CA,USA).Differencesbetweengroupswereconsidered

significantatalevelofp<0.05.

3. Resultsanddiscussion

3.1. PurificationandcharacterisationofMcLTP1

M. citrifolia L. has become popular in recent years, as it is

believedtobeabletohelppreventlifestyle-relateddiseases[33].

Nevertheless,scientific evidence forthesebenefitsis limited to

studiesdemonstratingbiologicalactivitiesofsecondary

metabo-litesfromdifferentnoniparts,whilestudiesreportingtheisolation

andcharacterisationofbioactiveproteinsfromthisspecieshave

beenlacking.

Whenadministeredintraperitoneallytomiceat dosesof10,

30 and 90mg/kg, noni seedcrude extract significantly reduced

(p<0.05)thenoxiousstimulusinducedbyaceticacid,ina

dose-dependent manner, by 23.2–41.8% compared with the control

group(Table1).Theantinociceptiveactivityofthenoniseedextract

atalldosestestedwasnotalteredafterdialysis(cutoffMW3000),

suggestingthattheeffectobservedwasrelatedtoaproteic

compo-nent.Therefore,weembarkeduponaguidedfractionationofthe

noniseedextract,seekingthisantinociceptiveagentusinganacetic

acid-inducedwrithingtestinmice.

The antinociceptive activity was also detected in the 2.5%

TCA soluble fraction after administration at a doseof 8mg/kg

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Table1

SummaryofpurificationprocedureofMcLTP1fromnoniseedsguidedbyanaceticacid-inducedwrithingtestinmice.

Purificationstep Protein(mg) Yielda_(%) _Doseb_(mg/kg) _%inhibitionc _Specific_biological_activityd _Purification_indexe Crude

extract

189.04

±3.23

100 10 23.25±0.03 – –

30 37.20±0.02* – –

90 41.86±0.19* 0.46 1

2.5%TCAsolublefraction 12.80±0.95 6.77 8 81.39±0.08** 10.17 22.10

SephadexG-50column(McLTP1) 11.58±0.87 6.12 8 96.13±0.08** 12.01 26.10

a_Yield₌_(total_protein_from_the_{fraction/total}_protein_from_the_crude_extract)

×100%.

b _Mice_were_pretreated_{intraperitoneally}₃₀_min_before_an_acetic_acid_injection.

c _Inhibition_of_acetic_acid-induced_abdominal_constriction_in_mice._The_results_show_the_mean

±SD(n=8).

d _Specific_biological_activity_is_calculated_by_dividing_“%_of_maximum_inhibitory_{activity/dose}_(mg/kg)”_of_each_step_of_{purification.}

e_Purification_index_is_calculated_as_the_ratio_between_the_specific_biological_activity_obtained_at_each_purification_step_and_that_of_the_crude_extract_taken_as_1.0. * _p_<_0.05.

** _p_<_0.01_compared_to_control_group_pretreated_with_NaCl_0.15_M_i.p._(ANOVA_followed_by_Tukey’s_test).

Fig.1. PurificationofMcLTP1.(A)Separationprofileof2.5%TCAsolublefractionfromnoniseedextractonaSephadexG-50column.(B)RP-HPLCchromatography.Pooled

fractionsobtainedfromgelfiltrationwereappliedtoaC18reverse-phasecolumnandruninaWatersCorp.apparatus.Elutionofproteinswasmonitoredbymeasurement oftheabsorbanceat280(A)and216nm(B).InsertCoomassie-stainedSDS-PAGE(12.5%)containing(left–right)molecularweightmarkers(lane1;14.4–97kDa),proteins extractedfromnoniseeds(lane2)and(lane3)McLTP1.

toaSephadexG-50column.AsdepictedinFig.1A,twoprotein

peakswererecoveredusingtheequilibriumbuffer.Afterbeing

col-lected,concentratedanddialysedthetopmostpartofpeak2,which

exhibitedantinociceptiveactivityinmice,thisproteinfractionwas

submittedtoanalyticalRP-HPLCusingaC18column,resultingin

onewelldefinedpeakelutedat7.9minunderisocraticconditions

(Fig.1B).Thepurifiedproteinwasshownasasinglebandon

SDS-PAGE,withanapparentmolecularmassof15.13kDaandayieldof

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Fig.2.ESI-MSanalysisofnativeMcLTP1.

Nativemolecularmassoftheisolatedproteinwasalsoassessed

bygelfiltrationchromatography.Thepurifiedproteinwas

chro-matographedona Superose 12HR, showinga single peak with

a molecular mass of 10.09kDa (data not presented), which is

in agreementwiththemolecularmasses obtainedbyESI mass

spectrometryundernativeconditions,whichshowedtwopeaks

correspondingtoMWof9450.30and9466.20(Fig.2).

Thefirst33aminoacidsoftheN-terminalsequenceofthe

puri-fiedproteinshowedsignificantsimilaritiestoknownlipidtransfer

proteins(LTPs)isolatedfromvariousplants,includingfourofthe

eightcysteineresiduesconservedinotherLTPs(Table2).LTPsare

small,water-soluble,basicproteins,classifiedintotwo

subfami-liesaccordingtotheirmolecularmasses:LTP1s(9kDa)andLTP2s

(7kDa)[34].ThemolecularmassdeterminedbytheESI-MSofthe

proteinisolatedinthisstudyallowedittobeclassifiedasamember

oftheLTP1subfamily(designatedasMcLTP1).

SincethediscoveryofplantLTPsinthe1970s,anincreasing

numberoflipidtransferproteinshavebeenisolated,particularly

fromseeds[35–37].However,untilnowtherehasbeenno

infor-mationregarding theirpresence inM.citrifolia L.seeds. Zottich

etal.[38]reportedtheisolationofaLTP1fromCoffeacanephora

(Cc-LTP1—Rubiaceae)seeds,with␣-amylaseinhibitorand

antimi-crobialproperties.McLTP1,whichwasalsoisolatedfromaplant

speciesbelongingtoRubiaceaefamily,showed57%identitywith

Cc-LTP1.

The presence of two peaks withm/z values of 9450.30 and

9466.20 suggests the existence of isoforms of McLTP1. Other

authors have reported that LTPs are found in severalisoforms

indistinct planttissues andorgans coded bya multigene

fam-ily, as demonstrated by Arabidopsis thaliana and Oryza sativa

[20,34,39,40].

AlthoughthemolecularmassofMcLTP1,determinedbyESI-MS,

isconsistentwiththoseobservedforotherLTP1s,themolecular

masscalculatedusingSDS-PAGEundernon-reducingconditions

wasestimatedtobe15.13kDa.AccordingtoGorjanovicetal.[41]

LTPscanformdimersinplanttissuesandalsoundernon-reducing

Fig.3.SDS-PAGE(12.5%)profileoftheinvitrodigestibilityofMcLTP1bypepsin

(laneP),trypsin(laneT)andaftersequentialdigestionwiththecitedenzymesand chymotrypsin(laneS).BSA(lanes1and2,left–right)wasusedasacontrol.Lane 3representsMcLTP1(20␮g).ProteinbandswerestainedwithCoomassieBrilliant

BlueR-250.

SDS-PAGE,asdemonstratedfortheLTP1isolatedfromHordeum

vulgarewhichshowedanapparentmolecularmassof16kDawhen

calculatedusingSDS-PAGE.

Duetotheircompactstructures,whicharestabilisedbyfour

disulfidebridges,LTPstendtobeextremelystableandresistantto

thermaldenaturationanddigestionbyproteases[42–45].Inthis

study,McLTP1wassubjectedtoasimulatedgastricandintestinal

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Table2

AlignmentoftheN-terminalaminoacidsequenceofMcLTP1withothernsLTPsfromplants.ProteinsequenceswerealignedwiththeBLASTalgorithm.Conservedcystein

residuesthatformdissulfidebridgesinnsLTPsarehighlightedingray.

NsLTPsource Plantfamily Residueno. Sequence Residueno. %identity SequenceID

Morindacitrifolia Rubiaceae 1 AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV 33 100 C0HJH5

Triticumaestivum Poaceae 26 AVSCGQVSSALSPCISYARGNGANPSAACCSGV 58 70 ABF14722.1

Aegilopstauschii Poaceae 26 AISCGQVSSALSPCISYARGNGANPTAACCSGV 58 67 EMT04675.1

Vitisvinifera Vitaceae 25 AVTCGQVETSLAPCMPYLTGGG-NPAAPCCNGV 56 67 XP002282792.1

Vignaradiata Fabaceae 25 AITCGQVASSLAPCISYLQKGG-VPSASCCSGV 56 61 CAQ86909.1

Fig.4. (A)EffectsofMcLTP1onaceticacid-inducedwrithingresponseinmice.

Vehi-cle(NaCl0.15M),McLTP1(1,2,4and8mg/kg),indomethacin(IND,10mg/kg)were

administeredi.p.30minbeforeanaceticacidinjection.(B)Antinociceptiveactivity ofMcLTP1(8mg/kg)afteroraladministrationinmice.Animalswerepretreated30

or60minbeforeanaceticacidinjection.Vehicle(NaCl0.15M)andindomethacin (IND,10mg/kg)wereorallyadministered30minbeforeanaceticacidinjection. Theresultsshowthemean±SD(n=8).Differencesbetweenthegroupswere deter-minedbyanANOVAfollowedbyTukey’stest,*p<0.05,**p<0.01whencompared tothecontrolgroup.

ofMcLTP1individuallydigestedwithpepsinandtrypsinandafter

sequentialdigestionwiththeaboveenzymesandchymotrypsin.

ThebandofMcLTP1 wasstableduringincubationwithpepsinor

trypsinfor4handwasalsodetectedevenafter9hofsequential

digestion.

3.2. AntinociceptiveactivityofMcLTP1

Whenadministeredtomiceintraperitoneally30minpriorto

acetic acid injection, McLTP1 (1, 2, 4, or 8mg/kg) significantly

reducedthenumberofabdominalconstrictionsobserved(p<0.05).

ThewrithingresponseinhibitionofMcLTP1wasdose-dependent

andatadoseof8mg/kgwascomparabletothatofindomethacin

10mg/kgi.p.(96.1%and98.3%inhibition ofabdominal

constric-tionreflexrespectively, Fig.4A).The antinociceptive activityof

Fig.5. EvaluationofthermalstabilityofthepurifiedMcLTP1usinganaceticacid

inducedwrithingresponseinmice.TheantinociceptiveeffectofMcLTP1(8mg/kg; i.p.)wasmeasuredafter0and30minpre-incubationat80◦_C._Vehicle_(NaCl_0.15_M)

andindomethacin(IND,10mg/kg)wereadministeredi.p.30minbeforeanacid aceticacidinjection.Theresultsshowthemean±SD(n=8).Differencesbetween thegroupsweredeterminedbyanANOVAfollowedbyTukey’stest,*p<0.05when comparedtothecontrolgroup.

McLTP1 was also observed after oral administration (p.o.)at a

doseof8mg/kg30(62.8%inhibition)or60min(86%inhibition)

priortotheaceticacidadministration.Theeffectobservedwhen

theprotein wasadministered60minprior toacetic acid

injec-tionwasnotsignificantlydifferentfromtheresponseobservedto

indomethacin10mg/kgp.o.(88%inhibition)(Fig.4B).Likeother

LTPs,suchasLJAMP2isolatedfromLeonurusjaponicusHoutt[46].

McLTP1displayedthermostability,retaining100%ofits

antinoci-ceptiveactivityafter1hincubationat80◦_C₍_Fig.₅_).

Basedontheirinvitroactivities,numerousstudiesoverthelast

decadeshavelinkedplantLTPswitha myriadofbiological

pro-cesses,includingformationofthecuticlelayer[47],plantresponses

toabioticandbioticstresses[48–50]andasmodulatorsofplant

growthanddevelopment[51,52].However,thisisthefirstreport

ofalipidtransferproteinactingasamodulatorofthenociceptive

response.

Theacetic acid-inducedwrithingresponse isa sensitivetest

used for screening analgesic activity, regardless of the central

orperipheralcauses.Aceticacidisanirritantwhich causesthe

synthesis and release of pro-inflammatory mediators such as

prostaglandins(PGEandPGF2␣)andsympatheticnervoussystem

mediators that provoke pain nerve endings [53–55].

Intraperi-tonealororaltreatmentwithMcLTP1causedsignificantreduction

in the abdominal constriction produced by acetic acid,

indi-cating peripheral antinociception. Taissin-Moindrot et al. [56]

demonstratedthatplantLTPsareabletoaccommodatenotonly

phosholipids or fatty acids, but also a variety of hydrophobic

molecules,suchasPGB2,withoutmajorstructuralmodifications.

Thus, itcouldbeassumed that this proteininterfered withthe

blockade ortherelease of peripherallyacting endogenous

sub-stances,suchasprostaglandins,responsibleforpainfulsensations.

InordertofurtherclarifytheantinociceptiveeffectofMcLTP1,

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Fig.6.Timespentonpawlickingafteroralorintraperitonealpretreatmentwith McLTP1(8mg/kg)onbothfirst(A)(0–5min)andsecond(B)(15–30min)phasesof theformalininducednociception.Dataareshownasthemean±SD(n=8). Differ-encesbetweenthegroupsweredeterminedbyanANOVAfollowedbyTukey’stest, *p<0.05vs.controlanimals.

(8mg/kg)administeredorallyorintraperitoneallycauseda

signifi-cant(p<0.05)inhibitoryeffectonbothphasesofformalininduced

pain,comparedtothecontrolcondition,withameanof51.1%(i.p.)

and32.7%(v.o.)intheearlyphaseand77.9%(i.p.)and65.1%(v.o.)

inthelatephase.Thepositivecontroldrug,morphine(5mg/kg),

significantlyattenuatedthepainresponsesofthetwophases.

Table3

EffectofMcLTP1onthetotalnumberofcrossedlinesandnumberofrearingsduring

5minofexposuretotheopen-fieldtest.Eachvaluerepresentsthemean±SEM

obtainedfrom8mice.StatisticaldifferencesweredeterminedbyANOVAfollowed byTukey’stest.

Groupa _Dose_(mg/kg) _Crossed_lines/5_min _Number_of_rearings

Vehicle – 48.3±1.05 12.8±1.02

McLTP1 8mg/kg 39.2±2.32 12.3±1.56

Diazepam 2mg/kg 10.4±0.08** 2.01±0.04**

a_Mice_were_treated_with_Mc_LTP

1orally60minbeforetheexperiment.Vehicle

(NaCl0.15M)anddiazepam(2mg/kg)wereadministeredintraperitoneally30min beforetheexperiment.

**_p_<_0.01_(compared_with_saline_group).

Thefirstphaseoftheformalintest(0–5min)ischaracterised

by neurogenic pain caused by a direct stimulation of

nocicep-tors. Substance P and bradykinin are thoughtto participate in

thisphase[31].Thesecondphase(15–30min)ischaracterisedby

inflammatorypain,aprocessinwhichseveralinflammatory

medi-atorsarebelievedtobeinvolved,includinghistamine,serotonin,

prostaglandinsandbradykinin[31,57].Ingeneral,centrallyacting

drugsinhibitbothphasesequally,whileperipherallyactingdrugs

inhibitmainlythesecondphase[58].AsshowninFig.6,McLTP1

suppressedpainduringbothphases,indicatingthatthisprotein

hascentralandperipheralanalgesicproperties.

Pretreatment of animals with naloxone (2mg/kg), a

non-selective opioid receptor antagonist, partially reversed the

antinociceptiveactivityofMcLTP1,administeredorallyor

intraperi-toneally, suggestingthe involvementof theopioid routein the

observed activity. Opiates are generally believed to produce

antinociception exclusively via central mechanisms, but some

studiesindicatethat theymayalsoproduceantihyperalgesiain

peripheralsites.Thelater effectsaremediatedbyopioid

recep-torsonperipheralterminalsofsensoryneurons[59]and inthe

gastrointestinaltract[60].Thus,itispossiblethatMcLTP1bindsto

thesegastrointestinalsiteswhenadministeredorally,producing

theantinociceptiveeffect.Similarresultswerefoundforalectin

purifiedfromCanavaliabrasiliensisseeds[61].

Amajor concernin theevaluationof theanalgesicactionof

compoundsis whetherpharmacologicaltreatmentcausesother

behaviouralalterations,suchasimpairmentofmotorcoordination

or sedation, which might be misinterpretedas analgesic

activ-ity.Abehaviouralassessmentwascarriedouttodetermineifthe

antinociceptiveeffectsofMcLTP1werecausedbyanydisturbances

onthecentralnervoussystem.Locomotoractivitywasnotaffected

byMcLTP1,suggestingthattheantinociceptiveresponseobserved

washighlyselective(Table3).

4. Conclusion

In this study we purified, characterised and evaluated the

antinociceptive effect of McLTP1, a lipid transfer protein. It is

describedfor thefirst timeasapain modulator,amplifyingthe

rangeofbiologicalactivitiesdisplayedbythisgroupofmolecules

and alsoprovidingevidence ofitspotentialas asourceof new

painreliefdrugs.McLTP1demonstratedperipheralantinociceptive

activitythatwascomplementedbyitscentraleffect,dependenton

opioidergicinvolvement.Thisisalsothefirstreportoftheisolation

ofaproteinfromthenonispeciesassociatedwiththetherapeutic

propertiesoftheplant.

Conflictofinterest

Theauthorshavedeclaredthatthereisnoconflictofinterest,

includingfinancial,personaloranyotherrelationshipswithother

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Acknowledgements

ThisworkwassupportedbyConselhoNacionalde

Desenvolvi-mentoCientíficoeTecnológico(CNPq)andFundac¸ãoCearensede

ApoioaoDesenvolvimentoCientíficoeTecnológico(FUNCAP).

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