Exploring the halophyte Cistanche phelypaea (L.) Cout as a source of health promoting products: In vitro antioxidant and enzyme inhibitory properties, metabolomic profile and computational studies

ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Exploring

the

halophyte

Cistanche

phelypaea

(L.)

Cout

as

a

source

of

health

promoting

products:

In

vitro

antioxidant

and

enzyme

inhibitory

properties,

metabolomic

profile

and

computational

studies

Francesca

Trampetti

_,

_Catarina

_Pereira

_,

_Maria

_João

_Rodrigues

_,

_Odeta

_Celaj

_,

Brigida

D’Abrosca

_,

_Gokhan

_Zengin

_,

_Adriano

_Mollica

_,

_Azzurra

_Stefanucci

_,

Luísa

Custódio

a_{CentreofMarineSciences,UniversityofAlgarve,FacultyofSciencesandTechnology,Ed.7,CampusofGambelas,8005-139Faro,Portugal}

b_{DepartmentofEnvironmental,BiologicalandPharmaceuticalSciencesandTechnologies,UniversityofCampaniaLuigiVanvitelli,ViaVivaldi,43,81100}

Caserta,Italy

c_{SelcukUniversity,ScienceFaculty,DepartmentofBiology,Campus,42250Konya,Turkey} d_{DepartmentofPharmacy,University“G.d’Annunzio”ofChieti-Pescara,66100Chieti,Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received8October2018

Receivedinrevisedform7November2018 Accepted23November2018

Availableonline24November2018 Keywords:

Carbohydratehydrolysingenzymes Cholinesterases Computationalapproach Oxidativestress NMR Tyrosinase

a

b

s

t

r

a

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t

In thisstudy, ethylacetate,acetone, ethanoland waterextracts fromflowers,stems and rootsof Cistanchephelypaea(L.)Coutwereappraisedforradicalscavengingactivity(RSA)towards 1,1-diphenyl-2-picrylhydrazyl,2,2-azino-bis(3-ethylbenzo-thiazoline-6-sulfonicacid)andsuperoxidefreeradicals,and formetalchelatingactivitiesonironandcopperions.Thewaterextractshadthehighestantioxidant activity,especiallythosefromrootsandflowers,andwerefurtherappraisedforinvitroinhibitionof enzymesimplicatedontheonsetofhumanailments,namelyacetyl-(AChE)andbutyrylcholinesterase (BuChE)forAlzheimer’sdisease,␣-glucosidaseand␣-amylasefordiabetes,andtyrosinaseforskin hyper-pigmentationdisorders.TheextractshadahigheractivitytowardsBuChE,andtherootsextracthadthe highestcapacitytoinhibittyrosinase.Samplesshowedalowcapacitytoinhibitcarbohydratehydrolysing enzymes,exceptfortherootextractwithagoodinhibitiononglucosidase.Sampleswerethen character-izedbyNMR(1Dand2D):themainmetabolitesidentifiedintheflowersextractwereiridoidglycosides, inparticularglurosideandbartsioside.Instems,phenylehanoidglycosides(PhGs)andiridoidswere detected,especiallyacteoside.InrootsweredetectedessentiallyPhGs,mainlyechinacosideand tubu-losideA.Dockingstudieswereperformedontheidentifiedcompounds.Afavorablebindingenergy oftubulosideAtotyrosinasewascalculated,andindicatedthiscompoundasapossiblecompetitive inhibitorof␣-glucosidaseandtyrosinase.OurresultssuggestthatC.phelypeaeisapromisingsource ofbiologically-activecompoundswithhealthpromotingpropertiesforpharmaceuticalandbiomedical applications.

©2018ElsevierB.V.Allrightsreserved.

1. Introduction

Halophytesspecieshavebeentraditionallyusedsinceancient

timesasfoodandassourcesofmedicinalproducts,andcurrently,

thereisahighinterestintheiruseforhumanandanimalhealth

improvement. Thereare severalrecent reports focusing onthe

potentialofwildhalophytesaspromisingsourcesofingredients

infunctionalfoodproducts,nutraceuticalsand/orbioactive

com-∗ Correspondingauthor.

E-mailaddress:lcustodio@ualg.pt(L.Custódio).

pounds[1,2].Halophytesarewelladaptedtoseveralenvironmental

constraints,suchaswaterandsoilsalinityanddrought,andcanbe

cultivatedbydifferentsystemsinarangeofsalineirrigationwater

resourcesand/orindegradedsalinesoils.

ThegenusCistanche(family:Orobanchaceae)embodiesmore

than20speciesinhabitingmainlyaridandsemi-aridareasofthe

EuropeanIberianPeninsula,AfricaandAsia.Cistanchespeciesare

holoparasitesontheroots of differentplantspecies, mainlyon

Chenopodiaceae.Herba Cistanches (RouCong-Rong in Chinese)

wasfirstrecordedinShenNong’sChineseMateriaMedicainca.100

B.C,whereitwasmentionedasthedriedstemsofCistanche

tubu-losa(Schrenk)Hook.f.andC.deserticolaMa[3].HerbaCistanchesis

https://doi.org/10.1016/j.jpba.2018.11.053

consideredasatopgradeoftherapeuticagents,oftencalledasthe

“Ginsengofthedeserts”,andisusedforthetreatmentofdifferent

healthproblems,asforexamplechronicrenaldisease,impotence,

femaleinfertility,morbidleukorrheaandprofusemetrorrhagia[3].

Itisalsodescribedtohavesedative,vasorelaxantandaphrodisiac

effectsandtoimprovelearningskills[3].Besidesitscommonuses

inChina,HerbaCistanchesissimilarlyusedasafoodsupplement

inJapanandSoutheastAsia.

Manybiologicalactivitieswerereportedfordifferentspeciesof

theOrobanchaceaefamily,includingantioxidant,antifatigue,and

anti-inflammatory,[3,4].ExtractsfromCistancheplantsthushave

anextensiverangeofactivities,comprisingthetreatmentofkidney

deficiencyandsenileconstipation,forlearningimprovement,relief

ofsymptomsrelatedwithAlzheimer’sdisease(AD),management

ofmenopausalsymptoms,enhancementofimmunity,anti-aging

andantifatigue[5].Theseactivitiesareascribedtothepresenceof

differenttypesofbioactivemolecules,suchasphenylethanoid

gly-cosides(PhGs),iridoids,apigeninderivativesandpolysaccharides

[5].

Cistanchephelypaea (L.) Coutis an edible halophyte present

in sand dune environments of Portugal, Spain, Crete, Cyprus,

Turkey,andintheeasternpartoftheMediterraneanregion.This

plantisgatheredfromthewildandusedlocallyasafood,quite

similar to asparagus. Its flowers are rich in unsaturated fatty

acids,sterolsandvitaminE,andtherefore,canbeofinterestfor

thefood industry [6]. However, and despite the

biotechnolog-icalimportance of theCistanche genusand thepossible health

promotingproperties of C. phelypaea,little is known regarding

thechemical profileorbiological activitiesofthisspecies.

Aim-ingtoevaluateifC.phelypaeacouldbeconsideredasasourceof

bioactiveproductswithhealthimprovementproperties,thiswork

determinedtheinvitroantioxidantandenzymaticinhibitory

prop-ertiesofextractsmadefromroots,flowersandstemsfromwild

plantscollectedintheSouthernPortugal.Antioxidantcapacitywas

appraisedbyfivecomplementarymethods,namelyradical

scav-enging activity (RSA) on 1,1-diphenyl-2-picrylhydrazyl (DPPH),

2,2-azino-bis(3-ethylbenzo-thiazoline-6-sulfonicacid)(ABTS)and

superoxide(O2•−)free radicals,and metalchelatingactivityon

copperandiron.Enzymaticinhibitorypropertieswereevaluated

towardenzymesrelatedwiththeonsetofAD(acetylcholinesterase:

AChEand butyrylcholinesterase:BuChE),type 2diabetes

melli-tus(␣-glucosidase and␣-amylase) and skinhyperpigmentation

(tyrosinase).Finally,thechemicalcompositionofthewaterextracts

wasestablishedbyNMR(1Dand2D)analysesandmolecular

dock-ingandmoleculardynamics(MD)studieswereperformedonthe

identifiedcompounds.

2. Materialandmethods

2.1. Reagents

All the chemicals used in this work were of analytical

grade. Sigma-Aldrich (Germany) supplied DPPH, ABTS, sodium

nitrite, sulphanilamide, N-(1-Naphthyl) ethylenediamine

dihy-drochloride(NED),butylatedhydroxytoluene(BHT),AChE (from

electriceel,Type-VI-S,EC3.1.1.7),BuChE(fromhorseserum,EC

3.1.1.8),acetylthiocholineiodide(ATCI),butyrylthiocholine

chlo-ride(BTCl),galantaminehydrobromide (fromLycorissp.,≥94%),

␣-amylase (from porcine pancreas, EC 3.2.1.1), ␣-glucosidase

(from Saccharomyces cerevisiae, EC 3.2.1.20),

4-nitrophenyl-␣-d-glucopyranoside (PNPG), tyrosinase (from mushroom, EC

1.14.18.1), l-2,3-dihydroxyphenylalanine (L-DOPA), kojic acid

(_≥ 99%), acarbose (_≥ 95%) and phosphate buffer

methanol-d4, trimethylsilylpropionic-2,2,3,3-d4 acid sodium salt (TMSP).

Folin-Ciocalteau(F-C)phenolreagentandphosphoric acidwere

purchasedfromMerck(Germany),whileCambridgeIsotope

Labo-ratoriesInc.(USA)suppliedD2O.Additionalreagentsandsolvents

wereobtainedfromVWRInternational(Belgium).

2.2. Plantmaterial

WholefloweringplantsofC.phelypaeawerecollectedinLudo,

RiaFormosa,acoastallagooninsouthernPortugal,inJune2013

(37◦0114.2N7◦5305.5W).Thetaxonomicalclassificationwas

confirmedbythebotanistDr.ManuelJ.Pinto(NationalMuseumof

NaturalHistory,UniversityofLisbon,BotanicalGarden,Portugal)

andavoucherspecimeniskeptintheherbariumofthe

Xtreme-Biolaboratory(voucherno

MBH028).Plantsweredividedinroots,

flowersandstems,ovendriedfor3daysat40◦C,powdered,sifted

througha50␮meshsizestandardsieve,andstoredat−20◦_Cuntil

needed.

2.3. Extraction

Four independent extractions were made by mixing dried

biomasswithsolventsofincreasingpolarities(ethylacetate,

ace-tone, ethanol and water, 1:40 w/v), overnight (16h) at room

temperature(RT),understirring(750rpm).Extractswerefiltered

(Whatmanno 4) and evaporated under vacuumat 40◦C. Dried

extractsweredissolvedindimethylsulfoxide(DMSO)atthe

con-centrationof25mg/mLandstoredat4◦Cuntilneeded.

2.4. Determinationofantioxidantactivitybyradical-basedassays

TheextractswereevaluatedforRSAonDPPH,ABTSandO2•−

radicalsbypreviouslydescribedmethods[1,2]usingBHTand

cat-echinaspositivecontrols.Theextractsweretestedfirstat1,5and

10mg/mL,andresultswerecalculatedaspercentageofinhibition

relativetoacontrolcontainingDMSO.Thevaluesforhalfmaximal

inhibitoryconcentration(IC50values)werecalculatedforsamples

withactivitieshigherthan50%at1,5or10mg/mL,bytestinga

minimumoffiveconcentrations.

2.5. Determinationofantioxidantactivitybymetal-related

methods

Themetalchelatingactivityoncopper(CCA)andiron(ICA)was

assessedas describedin [1,2],using ethylenediaminetetraacetic

acid(EDTA)aspositivecontrol.Samplesweretestedfirstat1,5

and10mg/mL,andresultswerecalculatedaspercentageof

inhi-bitionrelativetoacontrolcontainingDMSO.TheIC50valueswere

calculatedasdescribedinSection2.4.

2.6. Enzymeinhibitionassays

Theextractsatconcentrationsrangingfrom1to5mg/mL,were

evaluatedfortheircapacitytoinhibitAChE,BuChE,␣-amylase,

␣-glucosidaseandtyrosinase,accordingto[7].Galataminewasused

asastandardinhibitorforcholinesterasesatconcentrationsupto

0.5mg/mLandresultswereexpressedasgalatamineequivalents

(mgGALAE/g extract).Acarbose wasused apositive controlfor

␣-amylaseand␣-glucosidaseatconcentrations upto10mg/mL

andresultswereexpressedasacarboseequivalents(mmolACAE/g

extract).Kojicacidwasusedasastandardinhibitoroftyrosinase

atconcentrationsupto0.5mg/mL,andactivitywasexpressedas

2.7. MetabolomicanalysisbyNMR

2.7.1. Samplepreparationformetabolomicanalysis

Freeze-driedwaterextractsofstems,rootsandflowers(50mg)

weretransferredto2mLmicrotubes.NMRsampleswereprepared

inamixtureofphosphatebuffer(90mM;pH6.0)inD2Ocontaining

0.01%(w/w)oftrimethylsilylpropionic-2,2,3,3-d4acidsodiumsalt

andmethanol-d4.Atotalof1.5mLofphosphatebufferinD2Oand

methanol-d4(1:1)wasaddedtothesamples.Themixturewasthen

vortexedatRTfor1min,ultrasonicated(Elma®TransonicDigitals)

for40min,andcentrifuged(BeckmanAllegraTM64R)at13,000rpm

for10min.Aliquotsof0.6mLweretransferredtoNMRtubesand

analysedbyNMR.

2.7.2. NMRexperiments

NMRspectrawererecordedat25◦Cand300.03MHzfor1_Hon

aVarianMercuryPlus300FouriertransformNMR. CD3ODwas

used asthe internal lock. Each1_{H-NMR spectrum consisted of}

256scanswiththefollowingparameters:0.16Hz/point,

acquisi-tiontime(AQ)=1.0s,relaxationdelay(RD)=1.5s,and90◦ pulse

width(PW)=13.8␮s.Apresaturationsequencewasusedto

sup-presstheresidualH2Osignal.Freeinductiondecays(FIDs)were

FouriertransformedwithLB=0.3Hzandtheresultingspectrawere

manuallyphasedandbaselinecorrectedandcalibratedtoTMSPat

0.0ppm,using1_{H-NMRprocessor(MestReNova,version8.0.2).}

1_H-1_{H correlated spectroscopy (COSY), heteronuclear single}

quantum coherence (HSQC) and heteronuclear multiple bond

correlation (HMBC) spectra were recorded. COSYspectra were

acquiredwitha1.0srelaxationdelayand2514Hzspectralwidth

inbothdimensions.ThewindowfunctionforCOSYspectrawas

sine-bell(SSB=0).HSQCand HMBCspectrawereobtainedwith

a 1.0s relaxation delay and 3140Hz spectral width in f2 and

18,116Hzinf1.Qsine(SSB=2.0)wasusedforthewindow

func-tionoftheHMBC.Theoptimizedcouplingconstantswere140Hz

forHSQCand8HzforHMBC.Constanttimeinverse-detection

gra-dientaccordionrescaledheteronuclearmultiplebondcorrelation

spectroscopy(CIGAR–HMBC)spectra(8>n_J

(H,C)>5)wereacquired

withthesamespectralwidthusedforHMBC.Heteronuclearsingle

quantumcoherencetotalcorrelationspectroscopy(HSQC-TOCSY)

experimentswereoptimizedforn_J

(H,C)=8Hz,withamixingtime

of90ms.

2.7.3. Quantitativeanalysis

Themainmetabolitesidentifiedinthewaterextractswere

ana-lysed byquantitative analysis.1_{H-NMR spectrawere bucketed,}

reducingit tointegral segmentswitha widthof0.02ppm with

ACDLABS12.01_{H-NMRprocessor(ACDLABS12.0,Toronto,Canada).}

For each metabolite,buckets correspondingtonon-overlapping

signalsweremanuallyintegrated,scaledtotheinternalstandard

signalandusedtocalculatetherelativeamount.Theamountof

eachmetabolitewascalculated.Resultswereexpressedasmg/gof

freeze-driedsample.

2.8. Molecularmodelling

2.8.1. Proteinspreparation

Theenzymesusedinthebiologicaltestsreportedinthiswork

werealsousedfortheinsilicoexperiments.Theenzymestogether

withtheirinhibitorsweredownloadedfromtheProteinDatabank

RCSB PDB [8]. The crystal structures were prepared as

previ-ouslyreportedbyPrepWizard toolembeddedin Maestrosuite.

AChE(pdb:4×3C)in complexwithtacrine-nicotinamidehybrid

inhibitor,BuChE(pdb:4BDS)[9]incomplexwithtacrine,␣-amylase

(pdb:1VAH) in complex with r-nitrophenyl-_{␣-d-maltoside,}

␣-glucosidase(pdb:3AXI) in complexwithmaltose and tropolone

complexedwithtyrosinase(pdb:2Y9X)weresubjectedto

proto-nationatpHfixedat7.4byPropKa.Allthemissingfragmentsand

othererrorspresentinthecrystalstructuresweresolvedbyPrime

module,thecrystallographicligandswereusedtodeterminethe

enzymaticcavity.

2.8.2. Ligandspreparation

Thechemical structuresofthemoleculesused forthe

dock-ingexperiments arereported inFig.2 and weredocked tothe

selectedenzymepool.Severaldockingstudiesperformedon

glyco-sidepolyphenolswerepreviouslyreportedbyourresearchgroup

[7,9] andexhibit a goodinhibitory activitytowardthe selected

enzymes.In thiswork,thedocking experimentsweremadeby

GlideembeddedinMaestro2015suite,andthefreebindingenergy

estimationwascalculatedinordertodiscussthestabilityofthe

complexenzyme-inhibitor.Thetwodimensionalstructureofthe

substancesweredrawnbyChemBiodraw14.0and convertedin

3DstructuresbyMaestro;theresultingsubstanceswereusedfor

molecularmodelingexperimentsafterpreparationand

minimiza-tion.TheligandswerepreparedbytheLigPreptoolembeddedin

Maestro2015,neutralized atpH7.4byEpik andminimizedby

OPLS3forcefield.

2.9. Statisticalanalysis

Resultswereexpressedasmean±standarderrorofthemean

(SEM)andexperimentswereconductedatleastintriplicate.

Sig-nificantdifferenceswereassessedbyanalysisofvariance(ANOVA)

followedby theTukeyHSDtest,and bytheKruskal-Wallistest

whenparametricitydidnotprevail.Differenceswereconsidered

significantwhenPvalueswerelowerthan0.05. SPSSstatistical

packageforWindows(release15.0,SPSSInc.)wasused.TheIC50

valueswerecalculatedbysigmoidalfittingofthedatainthe

Graph-PadPrismv.5.0program.

3. Resultsanddiscussion

3.1. Antioxidantactivity

Ingeneral,thewaterextractsof rootsandflowersexhibited

the highest antioxidant capacity (Table1). Regarding theABTS

radical,thebestresultwasobtainedwiththewaterextractsof

rootsandstems,withIC50valuesof0.50and0.58mg/mL,

respec-tively.TheutmostDPPHandO2•−scavengingactivitieswerealso

obtainedwiththewater root’sextract,withIC50 values of0.37

and0.54mg/mL.Extractsfromflowershadnilchelation

proper-ties,whilefortheremainingsamplesthehighestchelationcapacity

wasobtainedwiththerootextracts,withIC50valuesof0.11and

4.4mg/mL forICAandCCA,respectively.Aboul-Eneinetal.[10]

evaluatedtheDPPHscavengingpropertiesofwaterandethanol

extractsfromC.phelypaeawholeplants,andsimilartotheresults

presentedhere,obtainedbetterresultswiththewater extracts.

Methanol extracts from thesame species also had a high RSA

towardsDPPH[11].Moreover,differentextractsmadefromother

Cystanchespeciespresentedrelevantantioxidantproperties,asfor

examplethosefromC.violaceadisplayingRSAonDPPHandalso

ironchelatingproperties[12].

Oxidativestressresultsfromtheimbalancebetweenan

exces-siveoxidantsaccumulationandreducedlevelsofantioxidants,and

originatestheoxidationofbiomoleculesandlossofitsbiological

roles,and/orhomeostaticimbalances.Thisprocessisconsideredas

thekeyriskfactorfortheonsetofseveralpathologies,asfor

exam-pleneurodegeneration,diabetesandskindisorders[13].Therefore,

oneapproachtopreventsuchdiseasesistheuseofantioxidants,for

exampleintheformofdietarysupplements,aimingtoprotectthe

organismfromexcessiveROSproduction[13].Ourresultssuggest

Table1

RadicalscavengingactivityonDPPHandABTSradicals,andmetalchelatingactivityoncopper(CCA)andiron(ICA)ofwater,acetone,ethylacetateandethanolextractsof roots,flowersandstemsofC.phelypaea.ResultsareexpressedasIC50values(mg/mL).

Organ/standards Extract ABTS DPPH O2•₋

ICA CCA Roots Ethylacetate 2.0±0.1c _3.3±0.2g _0.99±0.04b _6.2±1.7d _nr Ethanol nr 0.69±0.04c _6.5±0.0e _{nr 6.2±0.1}bc Acetone 1.7±0.1c _1.2±0.1d _{nr nr 4.5±0.1}b Water 0.50±0.05b _0.37±0.04b _0.54±0.05a _0.11±0.02a _4.4±0.1b Stems Ethylacetate 5.7±0.2e _6.5±0.2i _{nr 2.5±0.1}c _9.8±0.8d Ethanol nr 1.9±0.1e _{nr nr nr} Acetone 5.2±0.1d _4.5±0.1h _{nr nr 6.0±0.1}bc Water 0.58±0.03b _1.2±0.1d _4.6±0.13d _1.4±0.1b _7.8±0.5c Flowers Ethylacetate nr nr nr nr nr Ethanol 2.9±0.0e _{nr nr nr nr} Acetone nr nr nr nr nr Water nr 2.6±0.2f _2.8±0.4c _0.06±0.01a _4.5±0.2b Standards BHT 0.14±0.01a _0.11±0.01a _– Catechin – – 0.62±0.00a _{– –} EDTA – – – 0.06±0.00a _0.17±0.01a

nr:IC50notreached.Valuesrepresentthemean±standarderrorofthemean(SEM)ofatleastthreeexperimentsperformedintriplicate(n=9).Inthesamecolumnvalues

followedbydifferentletters(insuperscript)aresignificantlydifferentaccordingtotheTukeyHSDtest(p<0.05).

andflowers,couldbeusefulforthepreventionofoxidative stress-related disorders,similar towhat already happens withHerbal Cistanche,whichisrecognizedasapotentnaturalantioxidant[3].

3.2. Enzymaticinhibitoryproperties

3.2.1. AChEandBuChEinhibition

Aspreviously referred, oxidative stressis consideredas the

underlyingcauseforseveraldiseases[13].Therefore,andsincethe

waterextractshadthehighestantioxidantcapacity,theywere

fur-therevaluatedforenzymaticinhibition.AscanbeseeninTable2,

waterextractshadahigheractivitytowardsBuChE,withvaluesof

1.72and1.47mgGALAEforflowersandroots,respectively.AChE

andBuChEparticipateat differentextents inthemodulationof

theneurotransmitteracetylcholine(ACh)andarethusconsidered

importanttherapeutictargetsintherapiestargetingthe

improve-mentofcholinergicdeficitinpatientswithneurologicaldisorders,

suchasAD.AChEispontedasthemainenzymeresponsiblefor

theregulationofACh,whileBuChEisconsideredtobelessactively

involved[14].InAD,thereishoweveranapparentlinkbetween

BuChEactivityandthedepositionoffibrillar_␤-amyloid(A_␤)brain

plaquesinthecerebralcortexwherethisenzymeisnotusually

foundinhighlevels,whichsuggeststheimportantroleofspecific

BuChEinhibitorsinthesymptomatictreatmentofAD[15].

Thereareseveralreports of theuseofHerbaCistanches for

theimprovementofbrainfunction.Forexample,C.tubulosawater

extractspreventedbrainneuronapoptosisinvitro[16]andcould

alleviatethecognitivedysfunction causedby A␤ 1–42through

blockingamyloiddeposition,reversingcholinergicand

hippocam-paldopaminergicneuronalfunctioninanAD-likeratmodel[17].

Inaddition,clinicalassaysindicatedthatC.tubulosaglycoside

cap-sulesandC.herbacanbeusedasalternativetreatmentsformildto

moderateAD[18].However,wecouldnotfindreportsonthe

liter-atureregardingthecholinesteraseinhibitionofCystanchespecies.

OurresultssuggestthatthewaterextractsfromC.phelypeae

flow-ersandstemscontainBuChEinhibitors,relevantfortargetinginitial

stagesofAD,wherethelevelsofAChEhasnotyetsignificantly

decayedbutwheretheriskthatBChEcouldhydrolyseAChexists.

3.2.2. Tyrosinaseinhibition

AscanbededucedfromTable2,thewaterrootsextracthadthe

highestcapacitytoinhibittyrosinase,withavalueof8.03mgKAE/g.

Tyrosinase(monophenoloro-diphenol,oxygenoxidoreductase,EC

1.14.18.1,syn.polyphenoloxidase)isamultifunctionalmembrane

boundtype-3copper-containingglycoproteinwithakey rolein

melaninsynthesis.Tyrosinaseinhibitorshavethuscosmetic

appli-cationsasskin-whiteningproducts,andalsopharmaceuticaluses

for thetreatmentofhealth problems resultingfromthe

exces-siveaccumulationofmelanin,asforexamplepostinflammatory

hyperpigmentationandmelasma[19].Tyrosinaseisalsoinvolved

inneuromelaninproduction,wheredopamineoxidationproduces

dopaquinones.Excessiveproduction of dopaquinones resultsin

neuronaldamageandcelldeath,thussuggestingthattyrosinase

mightalsohavearoleinneuromelaninformationinthehuman

brainandmayberesponsiblefortheneurodegenerationassociated

withParkinson’sandHuntington’sdiseases[20].Therearereports

oftheanti-melanogenicpropertiesofotherCistanchespecies,such

asC.desertícola[21].However,tothebestofourknowledge,this

isthefirstreportregardingthecapacityofC.phelypaeatoinhibit

tyrosinase.OurresultsthussuggeststhatC.phelypaeawaterextract

couldbe of interest for both the cosmetic and pharmaceutical

industries,assourceofanti-melanogenicingredients.

3.2.3. Alphaamylaseand˛-glucosidaseinhibition

Theextractsshowedalowcapacitytoinhibitamylase(Table2).

However,therootextractcouldsignificantlyinhibitglucosidase,

with a value of 7.05mmol ACAE/g. Different Cistanche species

areusedintraditionalmedicineasantidiabeticdrugs,andshow

anti-diabeticfeaturesonanimalmodels.Forexample,the

admin-istration of an aqueous extract of C. tubulosa to db/db mice

significantlysuppressedthehighfastingbloodglucoseand

post-prandial blood glucose levels, improved insulin resistance and

dyslipidemia, and suppressedbody weight loss[22].

Neverthe-lesswecouldnotfindreportsontheinhibitionofcarbohydrates

hydrolysingenzymesbyextractsofCistanchespecies.Ourresults

suggeststhatC.phelypeaecouldbeasourceofcompoundsableto

inhibit␣-glucosidase,andthus,withinterestfordiabetescontrol.

3.3. Chemicalcharacterizationofwaterextracts

Root,stemandflowerwaterextractsofC.phelypeaeshowed

adifferentcompositionintermsofsecondarymetabolitesas

evi-dencedby1_{H-NMRspectra(Figs.1andS1.).Rootsweredominated}

byPhGs,flowersextractwererichiniridoidsandstemextracts

containediridoidsandPhGs(Fig.1Table3).

The1_{H-NMRspectrumofrootsshowedinthearomaticregion}

thesignalsofan(E)-caffeoylportionwiththreearomaticprotons

asanABXspinsystemandtwotrans-positionedolefinicprotons

(TableS1)twodoubletsat␦H7.63(␦C148.0)and␦H6.36(␦C113.9),

pro-Table2

EnzymaticinhibitoryactivityonAChE,BuChE,␣-glucosidase,tyrosinase,amylaseandglucosidaseofwaterextractspreparedfromflowers,rootsandstemsofC.phelypaea.a

Organ AChEinhibition (mgGALAE/g) BuChEinhibition (mgGALAE/g) Tyrosinaseinhibition (mgKAE/g) Amylaseinhibition (mmolACAE/g) Glucosidaseinhibition (mmolACAE/g) Flowers 0.58±0.07a _1.72±0.07a _2.14±0.11b _0.07±0.01b _na Roots 0.58±0.04a _{na 8.03±0.40}a _0.06±0.01b _7.05±0.07 Stems 0.30±0.02b _1.47±0.54a _1.45±0.08b _0.12±0.01a _na

a_{Valuesareexpressedasmeans±SEMofthreeparallelmeasurements.Datamarkedwithdifferentletterswithinthesamecolumn(insuperscript)indicatestatistically}

significantdifferences(p<0.05).GALAE,galantamineequivalents;KAE,kojicacidequivalents;ACAE,acarboseequivalents;na,notactive.

Fig.1.Representative1_{H-NMRspectraofroots,flowersandstemswaterextractsofC.phelypaea.}

tons,onetripletat␦H2.80(␦C35.5)andtwooverlappedprotons

at␦H4.05/3.80(␦C71.9).TheHSQCexperiment(Fig.3)allowed

allaromaticmethinecarbonsofaglyconeandacylmoietytobe

assigned. The same experiment also suggestedthe presence of

three anomeric carbonswithfollowing resonances␦H 5.12(␦C

102.2),␦H4.46(␦C103.4)and␦H4.32(␦C103.1)ingood

agree-mentwiththepresenceofthreesugarsidentifiedasarhamnoseand

twoglucoseunits,basedonheterocorrelationsevidencedinHSQC,

H2BCandHSQC-TOCSYspectra.AlltheseNMRdataareingood

agreementwiththepresenceofaphenylethanoidglycoside.The

CIGAR-HMBCexperimentpermittedtheconfirmationsofall

sig-nificativeinterfragmentalconnectivitiesofthiscompound.HMBC

correlationsbetweenanomericprotonofinnerglucoseat␦H4.46

(H-1)withmethyleneat␦C71.9(C-8)andH-4withestercarboxyl

at␦C168.7allowedtheaglyconeandacylmoietiestobelocated,

respectively,atC-1andC-4ofglucosemoiety.Furthermore,this

innerglucoseshowedtwoadditionalbranchedpointsatC-3and

C-6.InfactthecorrelationsbetweenH-1at␦H4.32withmethylene

carbonat␦C68.5(C-6)andbetweenthebroadsingletofrhamnose

Fig.2.Phenyletanoidgycosides(PhG)andiridoids(IR)detectedinroot,flowerandstemwaterextractsofC.phelypaea.PhG1:Echinacoside,PhG2:TubulosideA,PhG3: acteoside,IR-A:Gluroside,IR-B:Bartioside,IR-C:leonuride(ajugol).

Table3

MainsecondarymetabolitesdetectedinC.phelypaeaextracts.1_{H-NMRdatarecordedinphosphatebufferinD}

2OandCD3OD-(1:1).TMSP(0.1%,␦0.00)aremeasuredinppm

andcouplingconstants(J)inHertz.Relativeamountisexpressedinmgpergoffreeze-driedmaterialasthemeanvalue(n=3)±SD.

Metabolites NMR Amount(mg/g)

Roots IR-A H-3(6.19,dd,6.6and1.8) 30.6±0.0

IR-B H-3(6.26,dd,6.3and1.5),H-7(5.77,brs) 30.9±0.9 IR-C H-3(6.33,dd,6.0and1.5) nq PhG1 H-6_{(1.05,d.5.7) 148.8±7.7} PhG2 CH3CO(1.96,s) 20.6±1.2 PhG-TOT H-7(7.63d15.9),H-6(7.04dd,8.4and1.8) 264.3±9.6 Flowers

IR-A Seeroots 31.8±2.0

IR-B Seeroots 37.8±2.2

IR-C Seeroots nq

PhG1 Seeroots 61.2±1.4

PhG2 CH3CO(1.97,s) 33.1±1.0

PhG-TOT Seeroots 168.9±10.3

Stems

IR-A Seeroots 33.9±1.3

IR-B Seeroots 33.7±1.2

IR-C Seeroots nq

PhG1 Seeroots 75.8±2.1

PhG2 Seeroots 38.1±0.7

PhG-TOT Seeroots 194.1±4.9

PhG-TOT:sumofallPhGsdetectedinthecrudeextract.ForIR-Cthequantitativeanalysiswasnotpossibleduetostronglyoverlappingsignals;hencethepresenceisindicated by“nq”(notquantified).

identificationofechinacosideastheprincipalmetaboliteofroot extract(PhG1,Figs.1and2).Inaddition,tubulosideA(PhG2)was

alsoidentifiedasconstituentofthesameextract.

Inthe1_{H-NMRspectrumof flowers,onedoublet at␦}

H 5.23

(J=5.7Hz),twodoubledoubletat␦H 6.27(J=6.3e1.5Hz,Table

S2)and6.18(J=6.6e1.8Hz),aswellasabroadsingletat␦H5.76

(H-7)wereevident(TableS2).Severaloverlappedsignalswerealso

detectedintheregionofprotongerminaltooxygenaswellasin

theupfieldregionof1_{H-NMRspectrum.}

Thedoubledoubletat␦H6.27(␦C140.1)showedlongrange

het-erocorrelationwithamethinecarbonC-9at␦C34.7(␦H2.91),asp2

carbonat␦C108.3(␦H4.92),andacetalcarbonC-1at␦C94.9bonded

toadoubletprotonresonatingat␦H5.23,inturncorrelatedwith

threemethinecarbons:thefirst,anolephinicat␦C140.1,the

sec-ond,aliphaticat␦C34.7,andfinallythethirdanomericat␦C99.3.

Thislattersuggestedthepresenceofaglycosidicmoietyinthis

compound,identifiedasglucosebasedonspectroscopicevidences.

The13_{C-NMRvaluesat␦99.3,77.4,77.1,74.4,71.0,62.1andthe}

couplingconstantvalueof8.1Hzforanomericprotonareingood

agreementofa␤-d-glucopyranosilunitlinkedtoalyconemoiety.

Furthermore,the HMBCexperiment displayedthe following

heterocorrelations:theprotonat␦H4.92withmethineat␦C48.2,

thislatterwitholefinicprotonat␦H5.73(␦C128.0)inturn

hete-rocorrelatedwiththemethineat␦C34.7,withthemethyleneat

␦C39.2,andwithhydroxymethylcarbonat␦C60.8(␦H4.19/4.26).

Fig.3. HSQCspectrumofrootswaterextractofC.phelypaea.

dueaseconddoublebondbearingahydroxymethygrouplinked

tothecarbonat␦C143.8.Thesedataindicatedthepresenceofa

nor-iridoid(IR-BFigs.1and2)bringingtwohydroxylgroupsto

theC-1andC-8carbonsidentifiedasbartiosidealreadyisolatedas

componentofHerbaCistanche[23].

Inthesamespectrum,thesignalsrelatedwithnon-iridoid

glyco-sideswereevident.Bothcompoundslackeddoublebondbetween

theC-7andC-8carbons.Thefirst(IR-C)bearingofthehydroxyl

functionatC-6carbonasbyheterocorrelationsamongacarbinol

carbonat␦C82.5(␦H4.05)andtheH-4protonat␦H4.97(␦C105.6)

aswellastheprotonH-1at␦H5.04(␦C94.6),inturncorrelated

withtheC-3(␦C140.5),C-5(␦C31.8),C-9(␦C43.4)andC-1(␦C

98.2)carbons.Onthebasisoftheabove-mentionedobservations

aswellasoftheCOSYhomocorrelations(Fig.4),thestructureofthis

compoundwasdeterminedtobeleonuride(ajugol),aglycoside

iso-latedfromHerbaCistanche[23]andreportedtobeaconstituentof

C.phelypeae[24].Thethirdnon-iridoid(IR-A)detectedinthe1

H-NMRspectrumofflowersandidentifiedasgluroside(Figs.1and2)

respecttoajugollackofhydroxylatC-6.AlloftheNMRdataarein

goodagreementwiththosereportedintheliteratureforgluroside

[24].

Twoiridoidsbartioside(IR-B)andgluroside(IR-A)(Figs.1and2)

wereidentifiedasconstituentsofthestemextract,whichshowed

alsosignalsattributabletophenylehanoid.In particular,the2D

NMRdatawereingoodagreementwiththepresenceoftubuloside

Aasprincipalphenylehanoidglycosides,besidesacteoside(PhG3,

Figs.1and2),acompoundcloselyrelatedtoechinacoside.Infact

theHSQCexperiment(TableS3)evidencedthepresenceofonly

twoanomericcarbonsfortwomonosaccharideunitsidentified,on

thebasisofaHSQC-TOCSYexperiment,asrhamnoseandglucose.

Theprotonat␦H5.14(H-1’)correlatedwithfivemethinecarbons

at␦C102.1(C1)(directcorrelation),73.1(C-4’),71.6(C2’/C-3’),

69.6(C-5’),andwiththemethylat␦17.5(C-6’).Besides,methylene

protonsat␦H3.68and3.87(C-6”)correlatedwiththemethinesat

␦C103.9(C-1”),83.2(C-3”),77.2(C-5”),74.2(C-2”),69.9(C-4”).

MoreovertheNMRdatarevealedthelinkageoftherhamnoseto

C-3ofglucosemoiety,allowingtheidentificationofthesecond

phenylehanoidglycosideofstemsextractasisoacteoside[24].

TheextensiveNMRinvestigationofC.phelypaeawaterextracts

allowedtheidentificationofiridoidsandphenylehanoidglycosides

(Figs.1and2).PhGsarenaturallyoccurringmoleculesofplant

ori-ginandarestructurallycharacterizedwithahydroxyphenylethyl

moietylinkedtoaglucopyranosethroughaglycosidicbond[25].

Thesecompoundsarewater-solubleandubiquitousintheplant

kingdom, and largely found in the Scrophulariaceae, Oleaceae,

Plantaginaceae,Lamiaceae,andOrobanchaceaeplantfamilies[25].

PhGsarethemainactivemoleculesinCistancheplantsandarethus

usedasmarkersforqualityassessmentofcrudedrugsortheir

cor-respondingformulations[26].Severalinvitroandinvivostudies

haveshownthatPhGsexhibitavastarrayofbiologicalactivities

suchas antioxidant, neuroprotective,antitumoral, antibacterial,

antiviral,anti-inflammatory,hepatoprotective, anti-melanogenic

andimmunomodulatory[25].

Iridoidsrepresentalargegroupofcyclopenta[c]pyran

monoter-penoidsfoundinahighnumberofmedicinalplantsusedasbitter

tonics,sedatives,hypotensives,antipyretics,coughmedicines,and

forthetreatmentofwoundsandskindisorders[27].Iridoidsare

mostoftenfoundindicotyledonousplantfamilies,including

Apoc-ynaceae,Diervillaceae,Scrophulariaceae,Loganiaceae,Lamiaceae

and Rubiaceae, and are alsocommon constituents of Cistanche

species[27].Iridoidsarealsoendowedwithapanoplyof

bioac-tivities,includinganti-inflammatory,antioxidant,neuroprotective,

immunomodulator,hepatoprotective,cardioprotectiveand

Fig.4.41_H-1_{HCOSYspectrumofflowerswaterextractofC.phelypaea.}

Thewater extracts showeddifferent amounts of the

identi-fiedcompounds(Fig.S1,Table3).Inroots,echinacosidewasthe

majorPhG1(148.8mg/g)while tubulosideA (PhG2)was

essen-tiallydetectedinstems(38.1mg)andflowers(33.1mg)(Table3).

Inflowers,significantquantitiesof iridoids,inparticular

gluro-side(IR-A)andbartsioside(IR-B)werealsodetected.Echinacoside

andacteosidearerepresentativePhGsandwerepreviously

iden-tifiedindifferentCystanchespecies, includingC.phelypaea[26].

Acteoside,alsoknownasverbascoside,kusaginin,andorobanchin,

hasseveralimportantbiologicalproperties,suchasantioxidant,

anticarcinogenicandneuroprotective[28].Echinacosidehasalso

several described biological activities, as for example,

antioxi-dant,neuroprotectiveandanti-inflammatory[28].Thebioactivities

describedintheliteratureforechinacosideandacteosidemayhelp

toexplaintheantioxidantpropertiesandcholinesteraseinhibition

obtainedaftertreatmentwithwaterextractsofC.phelypeae.

Tubu-losidewasalsopreviouslyidentifiedinseveralCistanchespecies,

includingC.phelypaea[28]anddisplayedRSAtowardstheDPPH

freeradical[29].Regardingtheidentifiediridoids,gluroside (4)

wasalreadyidentifiedonCistanchespecies,includingC.deserticola

andC.phelypaea,andinHerbaCistanches[6,26].Bartsiosidewas

previouslyidentifiedinC.desertícolaandHerbaCistanches[6,26].

3.4. Computationalapproaches

Themost abundantcompounds identifiedin root, stem and

flowerswaterextractweretestedfordockingtowardtheselected

enzymesusedforthebiologicaltests.Thedockingscoresobtained

for the best pose for each compound are reportedin Table 4,

togetherwiththefreebindingenergycalculatedbyusingPrime

software embeddedin Maestro 2015.From a close analysis of

the literature we have found that echinacoside and aectoside

haveaweakactivityontyrosinasewithanIC50valueof907and

1169␮g/mL,respectively[21].Echinacoside,acteosideand

tubu-losideAhavealsoinhibitoryactivitytowards␣-glucosidaseand

␣-amylaseinahighmicromolarrange[30].

Considering the relative bioactivityof the extractsreported

inTable2,wehavefocusedourattentionon␣-glucosidaseand

tyrosinase.Inparticular,therootextractwastheonlywithagood

inhibitoryactivitytowardthesetwoenzymesandmoreover,

tubu-losideAispresentonlyinthisextract.Therefore,wefocusedonthe

analysisofthedockingposeoftubulosideAasoneofthe

poten-tiallymajorresponsiblefortheinhibitoryactivitydetectedinthis

extract.

InFig.5isreportedtheinteractiondiagramsofthebestpose

found for tubuloside A docked to ␣-glucosidase (A–C) and to

tyrosinase(B–D).ThebestposefoundfortubulosideAdockedto

tyrosinaseisstabilizedbyseveralhydrogenbondssuchasGlu322

(twohydrogenbonds),Tyr78,Ala323(twohydrogenbonds),Tyr65

(twohydrogenbonds),Ser282,Asn260,Asn81,His244,andone_␲-␲

stacktoPhe264aromaticring.TubulosideAestablishesto

glucosi-daseseveralpolarinteractionssuchashydrogenbondstoTyr158,

Gln279, Arg315, Gly309,Lys156 (␲-cation interaction), Thr310,

Ser240,Asp(242),Asp307(twohydrogenbonds).

Itisworthmentioningthatdespitethattheiridoidsbartioside

andglurosidehaveboththecorrectmolecularsizetoenterinthe

deepoftheenzymaticcavityofbothenzymes,onceinthepocket

theyarenot abletoproficiently interactwiththeamino acidic

residuessurroundingtheenzymaticcavity.Onthecontrary,

tubu-losideA,echinacosideandacteoside,haveabulkystructurethat

doesnotallowtheircompleteinsertioninthedeepofthe

enzy-maticcavity;however,thesecompoundshavethecorrectstereo

electronicpropertiestobindintheexternaledgeoftheenzymatic

Table4

DockingscoreandfreebindingenergycalculatedonthebestrankedposeoftheselectedsubstancesreportedinFig.2totheenzymaticpooltestedinthiswork.

AChE BuChE ␣-amylase ␣-glucosidase Tyrosinase

Compounds Dockingscore Gbinding Dockingscore Gbinding Dockingscore Gbinding Dockingscore Gbinding Dockingscore Gbinding

Echinacoside −13.5 −52.7 −18.5 −79.8 −14.1 −61.2 −13.1 −55.8 −12.7 −43.9

TubulosideA −14.3 −62.3 −15.8 −75.3 −12.6 −54.8 −10.6 −63.4 −13.1 −63.1

Acteoside −12.6 −61.4 −14.3 −51.5 −9.3 −55.9 −11.1 −20.2 −9.1 −54.6

CP-3 −8.7 −37.6 −9.9 −19.0 −7.0 −18.4 −9.9 −9.9 −7.1 −52.4

CP-4 −8.8 −12.2 −10.6 −25.1 −8.5 −40.8 −30.9 −30.9 −7.0 −32.3

Fig.5.TubulosideAdockedtoglucosidase(AandC)andtotyrosinase(BandD).

stablyoccupytheentranceoftheenzymaticcavity,andtobehave asacompetitiveinhibitors.Therefore,theiridoidspresentinthe extractshavethelowestdockingscoresandthefreebindingenergy valuesamongthemoleculestested.

4. Conclusion

WaterextractsfromC.phelypaea,especiallythosefromroots and flowers, displayed the highest capacity to scavenge free radicals, chelate iron and copper ions, and to inhibit enzymes

implicatedintheonsetofAD(AChEandBuChE),type-2diabetes( ␣-glucosidase)andhyperpigmentationdisorders(tyrosinase).NMR investigationof C.phelypaea waterextracts allowedthe identi-ficationoftwoclasses ofcompounds,namelyiridoidsand PhGs ThewaterrootextractwasdominatedbyPhGswhilethe flow-ersextractwasrichiniridoids;finallythestemextractsevidenced resonancesofbothiridoidsandPhGs.Themajormolecules iden-tifiedintherootextractswereechinacoside,tubulosideA(PhGs) andbartioside(iridoid),intheflowerextracts,bartiosideand gluro-side(PhGs),andinthestemextractswereacteoside,echinacoside

(PhGs),bartioside and gluroside (iridoids).Computational stud-iesindicated thattubulosideA,echinacosideandacteosidemay behaveasacompetitiveinhibitorsof␣-glucosidaseandtyrosinase. OurresultssuggeststhatrootsandflowersofC.phelypeaecouldbe consideredasasourceofinnovativeherbalproductswith phar-maceuticalandbiomedicalapplications,withantioxidantactivity, neuroprotective,antidiabeticandanti-melanogenicassets.Assays areinprogressaimingtoconfirmtheresultsobtainedinthiswork, includingforexampledeterminationoftoxicity,bioavailabilityand invivoefficacy.

Conflictofinterest

Theauthorsdeclarethattheyhavenoconflictofinterest.

Acknowledgements

This work was supported by the Foundation for Sci-ence and Technology (FCT, Portugal) and by the Portuguese NationalBudgetfundingthroughtheCCMAR/Multi/04326/2013 project. Luísa Custódio was supported by FCT Investigator Programme (IF/00049/2012). Catarina Guerreiro Pereira and Maria João Rodrigues acknowledges FCT for the PhD grants SFRH/BD/94407/2013andSFRH/BD/116604/2016,respectively.

AppendixA. Supplementarydata

Supplementarymaterialrelated tothis article canbefound, intheonlineversion,atdoi:https://doi.org/10.1016/j.jpba.2018.11. 053.

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