Rev. bras. farmacogn. vol.26 número2

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

UPLC–QTOF–MS

and

NMR

analyses

of

graviola

(

Annona

muricata

)

leaves

Ingrid

Vieira

Machado

de

Moraes

_,

_Paulo

_Riceli

_Vasconcelos

_Ribeiro

_,

_Flávio

_Luis

_Schmidt

_,

Kirley

Marques

Canuto

_,

_Guilherme

_Julião

_Zocolo

_,

_Edy

_Sousa

_de

_Brito

_,

_Rensheng

_Luo

_,

Kristy

M.

Richards

_,

_Kevin

_Tran

_,

_Robert

_E.

_Smith

a_{EmbrapaAgroindústriaTropical,Fortaleza,CE,Brazil}

b_{DepartamentodeTecnologiadeAlimentos,FaculdadedeEngenhariadeAlimentos,UniversidadedeCampinas,Campinas,SP,Brazil}

c_{UniversityofMissouri,St.Louis,MO,USA}

d_{FoodandDrugAdminsitration,Lenexa,KS,USA}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6April2015 Accepted7December2015 Availableonline6February2016

Keywords:

Graviola

Annonamuricata

UPLC–QTOF–MS Parkinson’sdisease NMR

a

b

s

t

r

a

c

t

Graviolaleaves(AnnonamuricataL.,Annonaceae)areusedbysomepeopletotrytotreatoreven curecancer,eventhoughover-consumptionofthefruit,whichcontainstheneurotoxinsannonacin andsquamocinhascausedanatypicalformofParkinson’sdisease.Inpreviousanalyses,thefruitswere extractedwithmethanolunderambientconditionsbeforeanalyses.Inthepresentstudy,UPLC–QTOF–MS andNMRwereusedtoanalyzefreeze-driedgraviolaleavesthatwereextractedusingdrymethanoland ethanolat100◦_{Cand10MPa(100atm)pressureinasealedcontainer.Methanolsolubilized33%ofthe} metabolitesinthelyophilizedleaves.Ethanolsolubilized41%ofmetabolitesinthelyophilizedleaves.The concentrationsoftotalphenoliccompoundswere100.3±2.8and93.2±2.0mggallicacidequivalentsper gofsample,forthemethanolicandethanolicextracts,respectively.Moreover,thetoxicophore (unsat-urated␥-lactone)thatispresentinneurotoxicacetogeninswasfoundinthelipophilicportionofthis extract.TheconcentrationsoftheneurotoxinsannonacinandsquamocinwerefoundbyUPLC–QTOF–MS tobe305.6±28.3and17.4±0.89␮g/g-dw,respectively,inthedriedleaves.Pressurizedmethanol solubi-lizedmoreannonacinandsquamocinthanethanol.Ontheotherhand,ahot,aqueousinfusionsolubilized only0.213%oftheannonacinandtoolittleofthesquamocintobedetected.So,graviolaleavescontain significantamountsoftheneurotoxinsannonacinandsquamocin,aswellassomepotentiallyhealthy phenoliccompounds.Finally,thepotentialneurotoxicityofwholeleavesindietarysupplementscould bemuchhigherthanthatofatea(hotaqueousinfusion)thatismadefromthem.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Graviola (Annona muricata L.) is a tropical fruit in the AnnonaceaefamilythatisgrowninAsia,SouthAmericaandmany tropicalislands(Lannuzeland Michel,2009;Gajalakshmiet al., 2012).Theleavescanbeusedtomakeatea(Port’setal.,2013; Hansraetal.,2014)orconsumedwholeasdietarysupplementin capsulesthatmayhavesomehealtheffects(Torresetal.,2012). However,over-consumptionofgraviolaandproductsmadefrom itmayhavecausedanatypicalformofParkinson’sdiseasesonthe FrenchCaribbeanislandofGuadeloupeandthePacificislandof

∗ Correspondingauthor.

E-mail:Robert.smith@fda.hhs.gov(R.E.Smith).

Guam(Caparros-LefebvreandElbaz,1999;Champyetal.,2005; BadrieandSchauss,2009).Thisisduetothepresenceof neuro-toxicacetogenins,suchasannonacin(1)(C35H64O7,MW596.88)

andsquamocin(2)(C37H66O7,MW622.92).Likeotherneurotoxic

acetogenins,theycontainanunsaturated␥-lactonegroupthatis thetoxicophore(Smithetal.,2014).However,theneurotoxicity couldbestronglydependentonthedose.Thatis,thefirstruleof toxicologyisthatthedoseisthepoison(Smith,2014).Itwas over-consumption,notconsumptionofgraviolathatcausedParkinson’s disease.Thedoseofneurotoxicacetogeninsthatoneconsumesis verydependentontheformofgraviolathatisingested.For exam-ple,onestudyusedateamadefrom10to12dryleavesthatwere boiledin8oz(about237ml)waterfor5–7min(Hansraetal.,2014). Sinceacetogeninshaveverylowsolubilitiesinwater(Höllerhage etal.,2009),thiswouldhavebeenarelativelylowdose.Thedose

http://dx.doi.org/10.1016/j.bjp.2015.12.001

canbeestimatedfromapreviousstudythatfoundabout56mg of annonacinperkgof leaveswhen about2.5g ofleaveswere boiledfor10mininwater(Champyetal.,2005).Ontheotherhand, muchhigherdoseswouldbeconsumedifoneweretoingestthe entireleavesinadietarysupplement.Thatis,onegroupreported thatthe“therapeuticdose”ofgraviolaleavesis2–3g,taken3or4 timesdaily(Sunetal.,2014).Ifwholeleavesareconsumed,the expecteddosewould behigher than thatin a tea.To estimate theconcentration,100gofdriedleaveswereextractedwith1lof methanol(Champyetal.,2005).Thisrecoveredalmostsixtimes asmuch annonacin(about 300mg/kg)as didtheboilingwater. However,thisanalysiswasdonewithmatrixassistedlaser desorp-tionandionizationcoupledtotimeofflightmassspectrometryor MALDI–TOFMS(Champyetal.,2005),whichseparatesanalytes basedontheirmolecularweights.So,annonacinwillnotbe sepa-ratedfromitsisomers.Thiscouldcauseanover-estimationofthe concentrationofannonacin,sinceitsisomerswillalsobedetected by theMALDI–TOF MS. It was observed,boiling methanolwas notabletosolubilizealltheannonacin(1)orsquamocin(2)from anotherfruitintheAnnonaceaefamily,theNorthAmerican paw-paw(Asiminatriloba;Pottsetal.,2012).Thatis,themethodusedto extractannonacinandsquamocincanaffecttheresultsofthe analy-sis.Boilingwaterwillnotsolubilizealltheannonacinorsquamocin. Instead,pressurizedliquidextractionusingdrymethanolat100◦_C and10MPa(100atm)pressurecansolubilizeoverseventimesas muchannonacinandevensomesquamocinfromthefruits(Potts etal.,2012).So,itisimportanttousehot,drypressurizedmethanol toextractalltheannonacinandsquamocin.Itisalsoimportantto useliquidchromatographycoupledtohighresolutionmass spec-trometry(LC-HRMS)toseparateanddetectisomersofannonacin andsquamocin(Levineetal.,2015).

LC-HRMSisespeciallyusefulwhentryingtoquantifytrace com-ponents,like acetogenins.TheMS canbetunedsothat it isas sensitiveaspossiblefortheanalytesofinterest(Smith,2014). How-ever,thesensitivitiesforotheranalytesthatarepresentinasample arequitedifferent.So,standardsarerequiredwhenquantifying analytesbyMS.Ontheotherhand,1_{H NMRhasthesame}

sen-sitivityfor allthehydrogens(1_{Hisotope)inthesample(Smith,}

2014).So,itcanbeusedwithoutstandardstodeterminetherelative concentrationsofdifferenttypesofhydrogensinasample(Smith, 2014).Moreover,13_{CNMRcanbeusedtodeterminehowmany}

differentkindsofcarbonsareinasampleandtoidentifythetypes ofcompoundsthatarepresent(Smith,2014).Therefore,NMRcan beveryusefulinanalyzingrelativelyunknownsamples.NMRwas usedtoanalyzeextractsofA.muricataforthepresenceofthe␣,␤ -unsaturated-␥-lactonetoxicophorethatis presentinneurotoxic acetogenins(Machadoetal.,2015).

Even though graviola leaves have been reported to contain about300mg/kgannonacin(Höllerhageetal.,2009),32mg/kgtotal phenolicsand5.6mg/kgtotalflavonoids(Port’setal.,2013),little isknownaboutitsmajorchemicalconstituents.Eventhe concen-trationoftotalsolublesubstancesisnotwellknown.Thatis,the concentrationdependsonthemethodusedtoextractthesoluble substances.Forexample,whendriedleavesweresoakedin dis-tilledwater for48h,about 5%ofthesolids dissolved(Florence et al.,2014).Another obtainedonly a 3.62% yield when leaves wereextractedtwicewithdistilled water atroomtemperature (AdewoleandAjewole,2009).Whensoakedfor48hin80%ethanol, about10.55%ofthecompoundsintheleavesdissolved(Foongand Hamid,2012;Hamizahetal.,2012).Adifferentstudypercolateddry leaveswith95%ethanoltogeta19.3%yieldofsolublesubstances (Singletonetal.,1999).

So, the aims of this work were characterizing the extracts obtainedbythistechniqueandseeifmethanolandethanolpresent differentresults,aswellasanalyzeannonacinandsquamocinby UPLC–QTOF–MS.

Materialsandmethods

Chemicalsandgraviolaleaves

Methanol(CH3OH),chloroform(CHCl3), acetonitrile(CH3CN)

andethanolwerefromHoneywellBurdick&Jackson(Muskegon, MI, USA). Deuterated chloroform (CDCl3), deuteratedmethanol

(CD3OD)anddeuteratedwater(D2O)werefromSigmaAldrich(St.

Louis,MO).Leucine-enkephalinwaspurchasedfromWaters(North Kingstown,RI,USA).Lyophilizedgraviolaleaveswereobtainedby Ingrid de Moraes froma commercialcultivationlocated in the cityofTrairi(Ceará,Brazil),latitude3◦₂₂′_15.98′′_{Sand longitude} 39◦₁₇′_34.46′′_{W.VoucherspecimensarekeptatEmbrapain} For-taleza.Thevouchernumberis49002.Afterbeingharvested,the leaveswerewashed,sanitizedwithasodiumhypochlorite solu-tion(100␮ll-1_{or100ppm)andthenlyophilizedinamodelLP510}

lyophilizer(Liobrás,SãoCarlos,SP-Brazil),atEmbrapaTropical AgroindustrylocatedinFortaleza,Ceará,Brazil.

Extractions

About10goflyophilizedleavesweremixed withenoughof HydroMatrixTM _{(SigmaAldrich, St. Louis, MO) to fill the 100ml}

stainlesssteelsamplecellusedinanAcceleratedSolvent Extrac-tor(ASE, ThermoFisher Scientific, Sunnyvale, CA).Then, CH3OH

orethanolwasaddedwhilethetemperatureandpressurewere increasedto100◦_{Cand10.3MPa(100atm)overa3mintime(static} time).Next,thesolventwaspurgedintoacollectionvessel.Atotal offourcycleswereruntostaticallyextractthesample,resulting inatotalvolumeofabout160ml.Thesolventwasevaporatedoff and theoily residuesremaining afterextractionwitheach sol-vent wereweighed.Aportionoftheresidueobtainedfromthe methanolextractionwasdissolvedin99.99%CD3ODandanalyzed

byNMR.Portionsofthemethanolicandethanolicresidueswere analyzedfortotalphenolics,asdescribedbelow.Then,portionsof theresiduesobtainedfromthemethanolicandethanolic extrac-tionwerepartitionedbetweenCHCl3andwater.TheCHCl3phase

wascollected,thesolventevaporatedoff,theresidueredissolved inCDCl3andNMRspectraacquired.Finally,a10gportionofdried

leaveswereextractedbyultrasonication.Itwasdonewith200ml of50%ethanolplus50%waterat40◦_{Cfor10min,usingan} Ultra-celaner1450ultrasonicatorfromUnique(Indaia,SP,Brazil).Itwas doneatafrequencyof20kHzandwith800Wpower.Thisextract wasalsoanalyzedfortotalphenolics.

Also,threeportionsofabout2.5gandoneportionofabout5.0g oflyophilizedleaveswereaddedto237ml(onecup)ofwaterat 90◦_{Cfor10min,afterwhichthesolutionswerefilteredand} ana-lyzedbyLC–MS/MS.

NMRanalyses

1_Hand13_C{1_{H}-NMRspectrawereobtainedusinganAgilent}

DD2600MHzNMR (SantaClara,CA).A30◦ _{pulsewidthand 1s} pulsedelaywereusedforthe1_{HNMR,whilea30}◦_{pulsewidthand} 2spulsedelaywereusedforthe13_{CNMRspectra.Chemicalshifts}

werereferencedtoeithertheCD3ODpeaksat3.35and4.78(for1H)

and49.3ppm(for13_C)ortheCDCl

3peaksat7.27and77.23ppm

for1_Hand13_{C,respectively.}

Analysisfortotalphenoliccompounds

previously(Singletonetal.,1999).Resultswereexpressedasmg gallicacidequivalentspergofsample,ormgGAE/g-dw.

UPLC–QTOF–MSanalysis

First,about10mgofeachoftheresiduesremainingafter evapo-ratingthesolventoffofthehot,pressurizedextractswasdissolved in 5ml of ethanol:H2O (1:1, v/v) and passed through a LC-18

Supelcoclean 6ml (0.5g) solid phase extraction (SPE)cartridge (Supelco,Bellefonte,PA,USA)thatwaspreconditionedby wash-ingitthreetimeswithCH3OHandthenequilibratedwith3mlof

deionizedwater.Theacetogeninswereelutedwith6mlofCH3CN.

TheCH3CNwasevaporatedoffandtheresidueredissolvedin1ml

ofCH3CN:H2O(15:7,v/v).Theresiduesfromeachsamplewere

ana-lyzedbypositiveionUPLC–MSanalysisusingaXevoUPLC-QTOF fromWaters(Milford,MA)andanAcquityUPLCBEHC18column, 1.7␮m,2.1mm×100mm.Agradientelutionwasusedwitha mix-tureofH2OandCH3CNand0.1%formicacid,flowingat400␮l/min.

Thatis,eventhoughtheconcentrationsofH2OandCH3CNvaried,

theconcentrationofformicaciddidnot.Itwasheldconstantat 0.1%.Itstartedwith35:65H2O/CH3CN(v/v)forthefirst25min,

thenincreasedlinearlyto1:9H2O/CH3CN(v/v)from25to30min.It

washeldat1:9H2O/CH3CN(v/v)from30to35min,thendecreased

linearlyto35:65H2O/CH3CN(v/v)from35to36minandheldat

thisfrom36to40min.TheMSspectrawereacquiredinpositiveion modewithanelectrosprayionization(ESI)source.TheMS param-eterswereasfollows:desolvationtemperature350◦_{C, capillary} voltage3.2kV,samplingconevoltage32V,extractioncone volt-age1.0V,sourcetemperature120◦_{C, conegas2l/h,desolvation} gasflow350l/h,purgegasflow350l/h,collisionenergy5.0V. Cen-troiddatawerecollectedforeachsampleinarange110–800Da, andthem/zvalueofallacquiredspectrawasautomatically cor-rectedduringacquisitionbasedonlockmassbyinfusing200pg/lof leucine-enkephalinthoroughLocksprayataflowrateof20␮l/min. Thecalibrationwasperformedtoachieveanacceptableaccuracy errorof±5ppm.Theaccuratemassandmolecularformula denom-inationwereacquiredwiththeMassLynx4.1software(WatersMS Technologies).

Annonacinand squamocinstandards fromPierreChampy at theUniversity of Paris were used toidentify and quantify the peaksduetoannonacin(1)andsquamocin(2).Theyelutedat14.1 and15.6min,respectively.Ionswithm/z=597.47and623.48Da were used to quantify annonacin and squamocin, respectively. Calibrationcurveswereobtainedusing10–200ng/mlannonacin and squamocin, assuming that the standards were 100% pure. Sampleswereinjected intriplicatewithresultsreportedasthe average±standarddeviation.

Resultsanddiscussion

Extraction

Drymethanolat100◦_{Cand10MPa(100atm)pressure} solubi-lized33%oftheleaves,whileethanolunderthesameconditions solubilized 41%. So, hot, pressurized methanol extracted much morematerialthanboiling(orpercolating)underambient pres-sure,which solubilized19.3% of theleavesin a previousstudy (Port’setal.,2013).Theconcentrationoftotalphenoliccompounds thatwerefoundusinghot,pressurizedmethanolextractionwas 100.3±2.8mgGAE/g.Thiscomparesto93.2±2.0mgGAE/ginthe hot,pressurizedethanolextract,54.6mgGAE/gintheultrasonic extractusing50%ethanolandthevalueof33mgGAE/gthatwas foundbyotherswhenleaveswereextractedwithboilingwater. So,extractionwithdrymethanolat100◦_{Cand10MPa(100atm)}

CD3OD CD3OD 2

4

1

3

5

9 8 7 6 5 4 3 2 1 ppm

Fig.1.1_{H-NMRspectrumofmethanolicextract(100}◦_{C,100MPascal)oflyophilized}

graviolaleaves,inCD3OD.Peaks:1:–CH3;2:–(CH2)n;3:H2C–CH CH–;4:HCOof

sugars5:phenylsand/orphenolics.

pressurecanextractmorephenoliccompoundsthanboilingwater underambientpressure.

NMRanalysis

The1_Hand13_C{1_{H}-NMRspectraofthemethanolicextractof}

lyophilizedgraviolaleavesareshowninFigs.1and2.Theyboth containsignals(chemicalshifts)duetotheCH3–(CH2)nCH2COOR

of esters (1_{H: 0.66–1.0 and 1.1–1.4ppm;} 13_{C: 14.6, 16.3–39.3}

and 175–177ppm), the –HCOH and –H2COH of

carbohy-drates (1_{H: 3.0–5.4ppm;} 13_{C: 62.2–108.8ppm) and the}

HC C of aromatic hydrogens in phenolic compounds (1_H:

6.4–8.1ppm;13_{C:125.5–166.2ppm).Therearealsosignalsdueto}

–CH2–HC CH–portionof unsaturatedfattyacyls (1H:6.4ppm; 13_{C: 108.9–119.5ppm). The relative contributions to the total}

peakareainthe1_{Hspectrumwere24%alkyl(CH}

3–(CH2)n),46%

carbohydrate,and 4.2%phenyl.Therewerenopeaksdueto di-ortriglycerides.Thealkyl,carbohydrateandcarbonylcarbonsall producedchemicalshiftsthatwereconsistentwiththepresence of esters of fatty acyls and sugars. These are more commonly calledfattyacidglycosides.Theyhavebeenfoundinthetropical fruitcallednoni(Morindacitrifolia)andhaveanticancerproperties

CD3OD

CH2

CH₃ CHO of glycosides

Aromatic carbons

C=O

180 160 140 120 100 80 60 40 20 ppm

Fig.2.13_C{1_{H}-NMRspectrumofmethanolicextract(100}◦_{C,100MPascal)of}

CHCl₃ 2

1

3

4 5 6

11 10 9 8 7 6 5 4 3 2 1 ppm

Fig.3. 1_{HNMRspectrumoftheportionofthemethanolicextract(100}◦_C,10MPa)of

ovendriedgraviolaleavesthatpartitionedintoCHCl3,redissolvedinCDCl3.Peaks:

1:–CH3;2:–(CH2)n;3:H2C–CH CH–;4:HCOofsugars;5:HC CH;6:HC Cin

theunsaturated␥-lactonetoxicophoreinacetogenins;0.1%CHCl3in99.9%CDCl3

solvent.

(Smithetal.,2014;Akihisaetal.,2007;Kim,2010).Itshouldalso benotedthateventhoughthemethanolicextractwasgreendue tothepresenceofchlorophyll,thechlorophyllwaspresentattoo lowofaconcentrationtoproducepeaksintheNMRspectra.There mayalsobesomesignals due toterpenesthatmay beslightly solubleinmethanol,eventhoughtheyareusuallyextractedusing hexane.

O

O

O

O

O O

O

OH

OH OH

1

2

OH OH

OH OH

OH

About37.5%ofthemethanolicextractpartitionedintoCHCl3.

The1_Hand13_C{1_{H}-NMRspectraofthislipophilicportionofthe}

methanolicextractareshowninFigs.3and4.Therearechemical shiftsatorabout7.0and7.2ppmduetotheunsaturated␥-lactone toxicophorethatis inneurotoxicacetogenins(Luoetal.,2013), andverysmallsignalsdue tofourdifferentHC C hydrogensin chlorophyllat8.00,8.55,9.29and9.52ppm.

Incomparison,the1_Hand13_C{1_{H}-NMRspectraoftheportion}

oftheethanolicextractthatpartitionedintoCDCl3areshownin

theSupplementaryMaterial.Thechemicalshiftsduetothe unsat-urated␥-lactonetoxicophorearealsopresent.

LC–MSanalysis

Seven point calibration curves each for annonacin (1) and squamocin(2)showedadequatelinearcorrelationbetween con-centration and area (R2_{>0.99), the linear range of standard}

compounds was between10 and 200ng/ml for standard com-pounds. Standardsand samples wereinjected in triplicate.The

CDCl3 (CH2)n

CHO of glycosides

Aromatic carbons

Ester C=O

180 160 140 120 100 80 60 40 20 ppm

CH3

Fig.4. 13_C{1_{H}-NMRoftheportionofthemethanolicextract(100}◦_C,10MPa)of

ovendriedgraviolaleavesthatpartitionedintoCHCl3,redissolvedinCDCl3.

injections showed that theresults are highly reproducible and repetitive. The LC–MS chromatograms of 200ng/ml annonacin andsquamocinstandardsandthemethanolicextractofgraviola leaves are shown in Fig. 5. The concentrations of the neuro-toxins annonacin and squamocinwere found tobe 1.68±0.08 and0.023±0.001mg/g-dw,respectivelyinthedriedleaves. Pres-surized methanol solubilized more annonacin and squamocin thanethanol.Thatis,305.6±28.3␮g/g-dwannonacinwasfound

in the methanolic extract and 226.0±0.07␮g/g-dw was found in the ethanolic extract. On the other hand, 17.4±0.89␮ g/g-dw squamocin was found in the methanolic extract, and 4.75±0.41mg/g-dwsquamocinwasfoundintheethanolicextract. Ontheotherhand,ahot,aqueousinfusion(tea)solubilizedonly 0.213%(0.65±0.07␮g/g-dw)oftheannonacinfrom2.5gofdry leavesandtoolittleofthesquamocintobedetected.When5.0gof leaveswereusedtoprepareatea,only0.108%(0.33␮g/g-dw)ofthe leaves.So,teabagscontainingabout5gofdrygraviolaleaveswill notsolubilizemuchmoreannonacinthanteabagscontaining2.5g ofleaves.Thus,wholegraviolaleavescontainsignificantamounts oftheneurotoxinsannonacinandsquamocin, aswellas pheno-liccompounds.Moreover,thepotentialneurotoxicitiesofwhole leavesindietarysupplementscouldbemuchhigherthanthatofa teathatismadefromthem.

100

Total Ion chromatogram

%

0

4.00 6.00

1 - Annonacin standard

[M+H]+ 597.47

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

100

%

0

4.00 6.00 8.00 10.00 12.00 1

2

2 1

14.00 16.00 18.00 20.00 22.00 24.00

100

Extracted Ion chromatogram

2 - Squamocin standard

[M+H]+ 623.48

Extracted Ion chromatogram

[M+H]+ 623.48 [M+H]+ 597.47 %

0

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

100

%

0

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

100

%

0

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time

A

B

C

D

E

Fig.5.UPLC–QTOF–MSchromatogramofthehot,pressurizedmethanolicextract(100◦_{C,10MPa)oflyophilizedgraviolaleaves–totalionchromatogramofthesample}

(A);annonacinstandard–200ng/ml(Rt=14.1min)(B);allpeaksofions[M+H]+_{=597.47extractedionchromatogramofthesample(C);squamocinstandard–200ng/ml}

(Rt=15.6min)(D);allpeaksofions[M+H]+_{=623.48extractedofthesample(E).}

TheNMRspectraweredominatedbypeaksduetofattyacid gly-cosides,whichmayhaveanticancerandotherhealthbenefits(Kim etal.,1998;Akihisaetal.,2007;Smith,2014).Theyarealso surfac-tants,sotheywillprobablyincreasethesolubilitiesofacetogenins inhotwater.TheNMRspectraalsocontainedchemicalshiftsdue totheunsaturated␥-lactonetoxicophorethatisinannonacinand squamocin(Machadoetal.,2015).Theyarealsosurfactants,sothey willprobablyincreasethesolubilitiesofacetogeninsinhotwater. Finally,thetotalphenolicsinthehot,pressurizedmethanolicand ethanolicextractswere83and64mfGAE/g,respectively.

Moreover,methanolat100◦_{Cand10MPa(100atm)pressure} solubilized33% ofthelyophilizedgraviolaleaves. Ethanol solu-bilized41%ofthelyophilizedleaves.Theconcentrationsoftotal phenoliccompoundswere83and64mgGAE/gforthe methano-licandethanolicextracts,respectively.Moreover,thetoxicophore (unsaturated␥-lactone)thatispresentinneurotoxicacetogenins wasfoundinthelipophilicportionoftheseextracts.The concen-trationsoftheneurotoxinsannonacin(1)andsquamocin(2)were foundbyUPLC–TOF–MStobe305.6±28.3and17.4±0.89mg/g, respectivelyinthedriedleaves.Pressurizedmethanolsolubilized

more annonacin than ethanol, while pressurized ethanol solu-bilized more squamocin than methanol. On the other hand, a hot, aqueous infusion (tea)made from 2.5g of dried leaves in 237ml(onecup)ofwatersolubilizedonly0.213%(0.65±0.07␮ g/g-dw) of the annonacin and too little of the squamocin to be detected.

ThisworkshouldnotbetakenasreflectingFDApolicyor regu-lations.

Authors’contributions

IVMM,PRVR,FLS,KMC,GJZ,ESBandRESconceivedanddesigned theexperiments.IVMM,PRVR,FLS,KMC,GJZandESBdidtheLC-MS andtotalphenolicsanalysesaswellasdatainterpretation.RLdid theNMRanalyses.IVMM,KMRandKTdidthepressurizedliquid extractions.RESwrotethearticle.Everyoneread thearticleand suggestedsomechanges.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.12.001.

References

Adewole,S.O.,Ajewole,J.A.O.,2009.ProtectiveeffectsofAnnonamuricataLinn. (Annonaceae)leafaqueousextractonserumlipidprofilesandoxidativestress inhepatocytesofstreptozotocin-treateddiabeticrats.Afr.J.Compl.Alt.Med.6, 30–41.

Akihisa,T.,Matsumoto,K.,Tokuda,H.,Yasukawa,K.,Seine,K.,Nakamoto,K., Kun-inaga,H.,Suzuki,T.,Kimura,Y.,2007.Anti-inflammatoryandpotentialcancer chemopreventiveconstituentsofthefruitsofMorindacitrifolia(Noni).J.Nat. Prod.70,754–757.

Badrie,N.,Schauss,A.G.,2009.Soursop(AnnonamuricataL.):composition, nutri-tionalvalue,medicinaluses,andtoxicology.In:Watson,R.R.,Preddy,V.R.(Eds.), BioactiveFoodsinPromotingHealth.FruitsandVegetables.AcademicPress, Oxford,pp.621–643.

Caparros-Lefebvre,D.,Elbaz,A.,1999.Possiblerelationofatypicalparkinsonism intheFrenchWestIndieswithconsumptionoftropicalplants:acase–control study.Lancet354,281–285.

Champy,P.,Melot,A.,Guerineau,V.,Gleye,C.,Fall,D.,Höglinger,G.U.,Ruberg,M., Lannuzel,A.,Laprevote,O.,Laurens,A.,Hocquemiller,R.,2005.Quantificationof acetogeninsinAnnonamuricatalinkedtoatypicalparkinsonisminGuadeloupe. MovementDisorders20,1629–1633.

Florence, N.T., Benoit,M.Z., Jonas, K., Alexandra,T., Desire, D.D.P., Pierre,K., Theophile,D., 2014. Antidiabeticand antioxidanteffects ofAnnona muri-cata(Annonaceae),aqueousextractonstreptozotocin-induceddiabeticrats.J. Ethnopharmacol.151,784–790.

Foong,C.P.,Hamid,R.A.,2012.Evaluationofanti-inflammatoryactivitiesofethanolic extractofAnnonamuricataleaves.Rev.Bras.Farmacogn.22,1301–1307.

Gajalakshmi,S.,Vijayalakshmi,S.,Devi,R.V.,2012.Phytochemicaland pharmaco-logicalpropertiesofAnnonamuricata:areview.Intl.J.Pharm.Pharmaceut.Sci. 4,3–6.

Hamizah,S.,Roslida,A.H.,Fezah,O.,Tan,K.L.,Tor,Y.S.,Tan,C.I.,2012. Chemo-preventivepotentialofAnnonaMuricataLleavesonchemically-inducedskin papillomagenesisinmice.AsianPacificJ.CancerPrevention13,2533–2539.

Hansra,D.M.,Silva,O.,Mehta,A.,Ahn,E.,2014.Patientwithmetastaticbreastcancer achievesstablediseasefor5yearsongraviolaandXelodaafterprogressingon multiplelinesoftherapy.Adv.BreastCancerRes.3,84–87.

Höllerhage,M.,Matusch,A.,Champy,P., Lombes,A.,Ruberg,M.,Oertel,W.H., Höglinger,G.U.,2009.NaturallipophilicinhibitorsofmitochondrialcomplexI arecandidatetoxinsforsporadicneurodegenerativetaupathologies.Exp. Neu-rol.220,133–142.

Kim,H.-K.,2010.Identificationofnovelfattyacidglucosidesfromthetropicalfruit

MorindacitrifoliaL.Phytochem.Lett.3,238–241.

Kim,G.-S.,Alali,F.,Rogers,L.L.,Wu,F.-E.,Sastrodihardjo,S.,McLaughlin,J.L.,1998.

MuricoreacinandmurihexocinC,mono-tetrahydrofuranacetogeninsfromthe leavesofAnnonamuricata.Phytochemistry49,565–571.

Luo,R.,Maia,J.G.,deMoraes,M.R.,Godoy,H.T.,Sabaa-Srur,A.U.O.,Tran,K.,Richards, K.M.,Monroe,D.M.,Vocque,R.H.,Smith,R.E.,2013.NMRanalysisofpotentially neurotoxicannonaceousfruits.Nat.Prod.J.3,230–241.

Lannuzel,A.,Michel,P.P.,2009.AtypicalparkinsonismintheFrenchWestIndies: theplanttoxinannonacinasapotentialetiologicalfactor.In:Tseng,K.-Y.(Ed.), Cortico-SubcorticalDynamicsinParkinson’sDisease.HumanaPress,NewYork, pp.283–290.

Levine,R.A.,Richards,K.M.,Tran,K.,Luo,R.,Thomas,A.L.,Smith,R.E.,2015. Determi-nationofneurotoxicacetogeninsinpawpaw(Asiminatriloba)fruitbyLC-HRMS. J.Agric.FoodChem.63,1053–1056.

Machado,A.R.T.,Lage,G.A.,Medeiros,F.S.,Filho,J.D.S.,Pimenta,L.P.S.,2015.Total ␣,␤-unsaturated-␥-lactoneacetogeninsinAnnonamuricatabyprotonNMR spectroscopy.Appl.Mag.Res.46,153–160.

Port’s,P.S.,Chisté,R.C.,Godoy,H.T.,Prado,M.A.,2013.Thephenoliccompounds andtheantioxidantpotentialofinfusionofherbsfromtheBrazilianAmazonian region.FoodRes.Intl.53,875–883.

Potts,L.F.,Luzzio,F.A.,Smith,S.C.,Hetman,M.,Champy,P.,Litvan,I.,2012.Annonacin inAsiminatrilobafruit:Implicationforneurotoxicity.NeuroToxicol.33,53–58.

Singleton,V.L.,Orthofer,R.,Lamuela-Raventos,R.M.,1999.Analysisoftotalphenols andothersoxidationsubstratesandantioxidantsbyFolin–Ciocauteaureagent. MethodsEnzymol.299,152.

Smith, R.E.,2014. Medicinal Chemistry – Fusion of Traditional and Western Medicine,SecondEdition.BenthamScience,Sharjah,U.A.E,pp.316–326.

Smith,R.E.,Tran,K.,Richards,K.M.,2014.BioactiveAnnonaceousAcetogenins, Chap-ter4,inStudiesinNaturalProductsChemistry,vol.41.Elsevier,NewYork,pp. 93–117.

Sun,S.,Liu,J.,Kadouh,H.,Sun,X.,Zhou,K.,2014.Threenewanti-proliferative Annonaceousacetogeninswithmono-tetrahydrofuranringfromgraviolafruit (Annonamuricata).Bioorg.Med.ChemLett.24,2773–2776.