Paeonia arietina and Paeonia kesrounansis bioactive constituents: NMR, LC-DAD-MS fingerprinting and in vitro assays

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JournalofPharmaceuticalandBiomedicalAnalysis165(2019)1–11

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

Paeonia

arietina

and

Paeonia

kesrounansis

bioactive

constituents:

NMR,

LC-DAD-MS

ﬁngerprinting

and

in

vitro

assays

Stefania

Sut

a

_,

_Gokhan

_Zengin

b,∗

_,

_Stefano

_Dall’Acqua

c

_,

_Markéta

_Gazdová

d

_,

Karel ˇSmejkal

d

_,

_Gizem

_Bulut

e

_,

_Ahmet

_Dogan

e

_,

_Mehmet

_Zeki

_Haznedaroglu

f

_,

Muhammad

Zakariyyah

Aumeeruddy

g

_,

_Filippo

_Maggi

h

_,

_Mohamad

_Fawzi

_Mahomoodally

g

a_DAFNAE,_Department_of_Agronomy,_Food,_Natural_resources,_Animals_and_Environment,_University_of_Padova,_Viale_{dell’Università,}

16,35020Legnaro,PD,Italy

b_Selcuk_University,_Science_Faculty,_Department_of_Biology,_Campus,_42250,_Konya,_Turkey

c_NPL_lab,_Department_of_{Pharmaceutical}_and_{Pharmacological}_Sciences,_University_of_Padova,_Via_Marzolo,_35121,_Padova,_Italy

d_Department_of_Natural_Drugs,_Faculty_of_Pharmacy,_University_of_Veterinary_and_{Pharmaceutical}_Sciences_Brno,_Palackého_1/3,₆₁₂_42,_Brno,_Czech

Republic

e_Marmara_University,_Pharmacy_Faculty,_Department_of_{Pharmaceutical}_Botany,_Istanbul,_Turkey f_Izmir_Katip_Celebi_University,_Pharmacy_Faculty,_Department_of_{Pharmaceutical}_Botany,_Izmir,_Turkey g_Department_of_Health_Sciences,_Faculty_of_Science,_University_of_Mauritius,₂₃₀_Réduit,_Mauritius h_School_of_Pharmacy,_University_of_Camerino,_Camerino,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24September2018 Receivedinrevisedform 14November2018 Accepted16November2018 Availableonline19November2018 Keywords: Paeoniaarietina Paeoniakesrounansis Antioxidant Cholinesterase Tyrosinase ␣-Glucosidase ␣-Amylase

a

b

s

t

r

a

c

t

Paeoniaspecieshavebeen valuedfortheir ethnomedicinalusesinvarious countriesand received

muchinterestamongthescientiﬁccommunityfortheirtherapeuticproperties,includinganti-microbial,

anti-inﬂammatory,anti-cancer,nephroprotectiveandhepatoprotectiveeffects.Themultiple

phytother-apeuticalapplicationsofPaeoniaspeciesinspiredustoestablishthephytochemical ﬁngerprintand

toevaluatethebiologicalpropertiesofethylacetate,methanol,andaqueousextractsfromtheroots

andaerialpartsoftwoPaeoniaspecies(P.arietinaG.Anderson andP.kesrounansisThiébaut).

Phy-toconstituentsofP.arietinaandP.kesrounansisextractswereanalyzedusing1Dand2DNMRand

LC-DAD-ESI-MS.Thetotalcontentofphenolics(TPC)andﬂavonoids(TFC)intheextractswasalso

eval-uated.TheantioxidantactivitywasproﬁledusingDPPH,ABTS,CUPRAC,FRAP,phosphomolybdenum,

andmetalchelationassays.Enzymeinhibitorypropertieswereevaluatedagainstacetylcholinesterase

(AChE),butyrylcholinesterase(BChE),tyrosinase,␣-amylase,and␣-glucosidase.Phytochemical

analy-sisofP.arietinaandP.kesrounansisextractsshowedthepresenceofgalloylestersofsugars,galloyl

monoterpenes,andglycosylatedﬂavonoids.Thethreesolventextractspresenteddifferentbehaviorin

thebioassays.Thehighestantioxidantactivity,tyrosinaseandAChEinhibitionwereobservedforthe

methanolicextractoftheaerialpartsofP.kesrounansis.Inaddition,theethylacetateextractsofthe

aerialpartsofbothplantswerethemosteffectiveinhibitorsof␣-amylase.ThehighestBChEinhibition

wasobservedforrootmethanolicextractofP.kesrounansiswhiletherootethylacetateextractofP.

ari-etinaexertedthestrongestinhibitionof␣-glucosidase.MethanolextractofP.kesrounansisaerialparts

presentedthehighestTPC,whileTFCwasgreatestinthecorrespondingextractofP.arietina.Ourﬁndings

canbeconsideredasastartingpointforfuturestudiestofurthervalidatetheeffectivenessandsafety

proﬁlesoftheseplantsinfolkmedicine.

∗ Correspondingauthor.

E-mailaddresses:stefaniasut@hotmail.it(S.Sut),gokhanzengin@selcuk.edu.tr(G.Zengin),stefano.dallacqua@unipd.it(S.Dall’Acqua),gazdova.marketa@seznam.cz(M. Gazdová),karel.mejkal@post.cz(K. ˇSmejkal),gizem.bulut@marmara.edu.tr(G.Bulut),adogan@marmara.edu.tr(A.Dogan),zeki.haznedaroglu@ikc.edu.tr(M.Z.Haznedaroglu),

muhammad.aumeeruddy2@umail.uom.ac.mu(M.Z.Aumeeruddy),ﬁlippo.maggi@unicam.it(F.Maggi),f.mahomoodally@uom.ac.mu(M.F.Mahomoodally).

https://doi.org/10.1016/j.jpba.2018.11.040

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1. Introduction

ThegenusPaeonia(Paeoniaceae)consistsof36acceptedspecies

recordedinThePlantList(http://www.theplantlist.org/).Thegenus

isdividedintothreesections:(1)PaeoniaL.(whichisdistributed

fromSpainandNorth Africaeastwards toChinaandJapan), (2)

MoutonDC. (Eastern Asia)and (3) Onaepia Lindl.(North

Amer-ica).Paeoniaisrepresentedbyninespecieswithninesubspecies

inEurope.InTurkey,thewildPaeoniaspeciesarerepresentedby

eighttaxa[1].Membersofthisgenusareknownfortheirdiverse

colorofﬂowers,ﬂowertype,andplantheight.Theexuberantcolor

oftheﬂowersjustiﬁestheimportanceofPaeoniaspp.asornamental

plantswhiletheknownwound-healingpropertiesofrootssupport

itsuseasherbalmedicine[2].

Paeoniaspp.havealonghistoryofuseastraditional

medici-nalplants.Paeoniaofﬁcinalis and P.mascula werementioned in

“AbouttheAntidotes”belongingtotheﬁrstand largestsection

“ElementAlpha”ofNikolaosMyrepsos’Dynameron, amedieval

medicalmanuscript [3]. TherootofP.ofﬁcinalis isusedtocure

nervediseasesinJordan[4].InPakistan,P.emodiisadministered

forthetreatmentofrheumatism[5],urinaryproblems,nervous

diseases,colic,headache,asabloodpuriﬁer[6],andtotreat

gen-eralbody weakness [7]. A great number of studieshas proved

thebiological effects ofthe membersof Paeonia genus,

includ-ingthe anti-proliferative activityof P. lactiﬂora[8], anti-cancer

activityofP.moutan[9],andnephroprotectiveeffectofP.emodi

[10]. With respecttoone of thespecies includedin this work,

thedecoctionoftheaerialpartofP.masculasubsp.arietina(syn.

P.arietina)wasreportedforuseinthemanagementofdiabetes

in the traditional folk medicine in Ovacık (Tunceli) in Turkey

[11], as well as in East Anatolia [12]. In other regions across

Turkey,rootinfusionsareusedassedative,laxative,andfor

treat-mentofepilepticseizureand trecough[13]. Paeoniaspeciesare

knowntobearich sourceof monoterpenespossessinga

‘cage-like’pinane skeleton [14]. Furthermore, othermonoterpenoids,

ﬂavonoids,polyphenols(includingpaeonolandgallicacid

deriva-tives),steroids,andtriterpenoidswerepreviouslydescribedinthis

genus[15].

DespitetheimportanceinTurkishtraditionalmedicine,there

isalackofinformationonthephytoconstituentsofP.arietinaG.

Anderson(previouslyknownasP.masculasubsp.arietina)andP.

kesrounansisThiébaut(previouslyknownasP.turcica).Onlyone

study[16]investigatedthecompositionoftheessentialoilandthe

antioxidantactivity(DPPHand nitricoxidescavenging)ofthese

species,thereforewefocusedonthenon-volatilecompoundsand

theevaluationoftheirbiologicalproperties.Theobjectivesofthe

presentstudyweretoelucidatethephytochemicalcompositionof

P.arietinaandP.kesrounansisbyNMR(1and2D)and

LC-DAD-ESI-MS,andtoassesstheantioxidantandenzymeinhibitoryproperties

ofdifferentextractsfromthosespecies,namelymethanol(MeOH),

ethyl acetate (EA) and water. These extracts were tested for

antioxidantactivityusingDPPH,ABTS,FRAP,CUPRAC,

phospho-molybdenumandmetalchelatingassays.Furtherassaysevaluated

theinhibitorypotentialeffect againstkey enzymesofpotential

clinicalrelevance(cholinesterases,tyrosinase,␣-amylaseand

␣-glucosidase).

2. Materialandmethods

2.1. Plantmaterialandextraction

Theplant sampleswere obtainedfrom a ﬁeld study in July

of2016:P.arietinaG.Anderson(Tunceli-Pertek,Pınarlarvillage,

1450m),voucherspecimenno.MARE-12500,andP.kesrounansis

J.Thiébaut (Denizli-Acıpayam,Bozda˘g, 1400m), voucher

speci-menno.MARE-17253.Itsbotanicalconﬁrmationwasdonebya

botanist(Prof.ErtanTuzlaci,MarmaraUniversity,Departmentof

Pharmacology).One plantsample for each Paeonia species was

keptasvoucherspecimenandwasdepositedinMarmara

Univer-sity,FacultyofPharmacy’sHerbarium.Tengramsofplantmaterials

(dried-powderedaerialpartsorroots)weremaceratedin200mlof

ethylacetate(EA)andmethanol(MeOH)atroomtemperaturefor

24h.Theextractswerethenﬁlteredandconcentratedat40◦C.The

waterextractwaspreparedusinganinfusionasfollows:5gplant

materialwaskeptin100mlboiledwaterfor20min.Theobtained

extractwaslyophilizedandkeptina refrigerator(at4◦C)until

furtheranalysis.

2.2. Totalphenolicandﬂavonoidcontent

The total phenolic content (TPC) wasdetermined using the

Folin-Ciocalteumethodandﬂavonoidscontent(TFC)bytheAlCl3

method.Theresultsobtainedwere expressedasequivalents of

standardcompounds(gallicacid(mgGAE/gsample)forTPC)and

rutin(mgRE/gsample)forTFC),respectively)[17].

2.3. Determinationofantioxidantandenzymeinhibitoryeffects

Antioxidantpropertiesofextractswerescreenedbychemical

assaysincludingantiradicalmethods(ABTSandDPPH),reductive

power(CUPRACandFRAP),phosphomolybdenum,andchelating

effect.Theresultswereexpressedasstandardcompounds

equiv-alents (mgTE/g sample and mg EDTAE/gsample). Allmethods

aredescribed in ourpreviouspaper[17]. Theinhibitory effects

of Paeonia extracts wereinvestigated onkey enzymes, namely

cholinesterase (associatedwithneurological disordersincluding

Alzheimer’sdisease),tyrosinase(linked withskindisorders),

␣-amylaseand␣-glucosidase(bothrelatedwithdiabetesmellitus).

Theexperimentalprotocolswerereportedinourpreviouspaper

[17]. Results were expressed as content of standard inhibitors

in samples.Standard inhibitors for cholinesterases were

galan-thamine(mgGALAE/gsample),kojicacidfortyrosinase(mgKAE/g

sample),and acarbose for ␣-amylase and ␣-glucosidase (mmol

ACAE/gsample).

2.4. NMRanalysis

1D-and2D-NMRspectrawereobtainedonaBrukerAvance

III400Ultrashield spectrometerwithsuperconducting 400MHz

magnetsystem.NMRspectrawereacquiredinMeOD-d4

(Sigma-Aldrich)withTMSasaninternalstandard.Durian®4.95mmNMR

tubes(DurianGroup)wereused.Chemicalshiftsareexpressedinı

valuesinppm.1_H-NMR_and_HSQC-DEPT,_HMBC,_COSY_experiments

wereacquiredusingstandardBrukersequencesmeasuringp1and

d1foreachacquiredsample.

2.5. HPLC-DAD-MSanalysis

Quantitativeanalysisofphenolicderivativeswasobtainedby

LC-DAD-ESI-MSn_. _The _measurements _were _performed _using _an

Agilent1260chromatograph(SantaClara,CA,USA)equippedwith

1260diodearraydetector(DAD)andVarianMS-500iontrapmass

spectrometer.Sample separationwasachievedusinganAgilent

EclipseXDBC-18(3.0×150mm,particlesize3.5␮m)asstationary

phase.Themobilephasewaswater(0.1%formicacid)(A)and

ace-tonitrile(B).Theelutiongradientstarted90:10(A:B)upto0:100

(A:B)at30min,with5minofre-equilibrationtime;ﬂowratewas

0.5ml/minandinjectionvolumewas10␮L.Attheendofthe

col-umn,aTsplitterseparatedtheﬂowratetoDADandMS.TheDAD

detectorwasusedtoquantifyﬂavonoidsandgalloylderivatives.

Rutin(SigmaAldrich,St.Louis,MO, USA)andgallicacid(Sigma

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S.Sutetal./JournalofPharmaceuticalandBiomedicalAnalysis165(2019)1–11 3

Fig.1.1_H-NMR_spectra_of_the_tested_methanol_extracts_(1-_P._kesrounansis_aerial_part;_2-_P._arietina_aerial_part;_3-_P._kesrounansis_root;_4-_P._arietina_root).

andﬂavonolderivativesweremonitoredat280nmand 350nm

andUV–vis spectrawereacquiredin therangeof 200–650nm.

MSspectrawererecordedintherangeofm/z50–2000,usingESI

ionsourceoperatinginnegativeionmode.Turbodatadependent

scanning(TDDS)instrumentfunctionwasusedtoacquiredmass

fragmentationpathwaysofthemainionicspecies.Identiﬁcationof

compoundswasobtainedbasedonfragmentationspectraaswell

asthecomparisonwiththeliteratureandreferencecompounds,

whenavailable.Themethodofcalibrationcurvewasusedforthe

quantiﬁcationofphenolicconstituents:rutinwasusedasexternal

standard for ﬂavonoid quantiﬁcation, gallic acid was used for

galloylderivatives.Calibrationcurveswereasfollowsy=52.95x

+2.88(R2₌_0.996)_for_rutin;_y₌_38.387x₊_202.53_(R2₌_0.996)_for

gallicacid.

2.6. Statisticalanalysis

All tests were carried out in triplicates and ﬁndings are

expressed as mean value±SD. SPSS v. 17.0 was employed for

statisticalanalysis.One-wayANOVAwasdonetodetermineany

differencesbetweenthetestedsamplesfollowedbyTukey’stest.

p<0.05wereassignedtobestatisticallysigniﬁcant.Theprincipal

component(PCA),clusterandPearsonlinearcorrelationwasused

toevaluatetheassociationbetweentotalbioactivecomponentsand

theﬁndingsofbiologicalactivity.

3. Resultsanddiscussion

3.1. PhytochemicalﬁngerprintingofPaeoniaextracts

Phytochemicalcompositionofaerialpartsandrootswas

stud-iedbyNMRandLC-DAD-ESI-MSapproaches.Themethanolextract

wasusedbecauseoftheabilityofthissolventtosolubilizeboth

lipophilic and hydrophilic constituents. 1_H-NMR _as _well _as _2D

experiments, namely HSQC-DEPT, HMBC, and COSY were

con-ductedtoidentifytheprincipalconstituentsofthePaeoniaextracts.

1_H-NMR_of_the_root_and_aerial_parts_of_P._kesrounansis_and_P.

ari-etinarevealedthatpartsofthespectraarealmostsuperimposable,

asvisibleforexampleintheregionbetweenı8.5-5.80ppm,

sug-gestingsimilarqualitativecompositionofphenolicconstituents,

andintheregionı3.0–4.2ppmsupportingtheideaofsimilarityin

sugarmoieties.Differenceswereobservedinthespectralregionı

5.6-4.2ppm,whichshowedsignalsascribabletoanomericprotons,

sp2_oleﬁnic_signals,_or_deshielded_CH

2Ogroups.Furtherdifferences

wereobservedin theregionı2.8-0.5ppm,indicating the

pres-enceofdiversealiphaticcompoundsororganicacidsinthesetwo

extracts.Consideringthegeneralaspectsofanalysisofthesetwo

extracts,largesimilaritywasobservedinspectra,supportinga

sim-ilarcomposition.Exempliﬁcativeprotonspectraofthefourextracts

arereportedinFig.1.

NMRtechniquesallowedustheobservationofdifferenttypes

ofconstituentsintheextracts,anddataobtainedbytheacquisition

of1_H-NMR,_HSQC-DEPT,_and_HMBC_revealed_the_presence_of_some

ofthetypicalphytoconstituentspresent inPaeoniaspecies[18].

1_H-NMR,_HSQC-DEPT_and_HMBC_spectra_are_reported_in_Figs._2–5_.

Someofthemostintensivesignalsarerelatedtothepresenceof

sugarmoietiesduetotypicalprotonshiftsat␦3.0–4.5ppm.

Fur-thermore,peakssupportingthepresenceofphenolicconstituents

werealsodetected,asdescribedabove.Signalsascribableto

aro-matic acid derivatives couldbe observed in the regionfrom _␦

7.47to8.00ppmasdoubletsordoubletsofdoublets.Moreover,

estersofgallicacidwithsugarsweredetectedduetothepresence

ofsingletsat␦6.89–7.12ppm(relatedtothepositions2 ´and6 ´of

galloylmoiety)[19] andduetothepresenceofsignalsassigned

tosugarpartsasthedoubletat␦6.23, relatedtotheanomeric

protonof glucose/hexose,or thedoubletsand doubletsof

dou-bletsobservedat␦5.5and6.0ppm.HSQC-DEPTdataconﬁrmed

theassignmentsallowingtheassignmentofnon-quaternary

posi-tionofconstituents,andlaterdiagnosticHMBCcorrelationswere

observedfromtheH-2orH-3signalsofhexoseunitswith

car-bonsat␦164.0–165.5, conﬁrmingthepresenceof esterlinkage

(Table1).Thepresenceofmonoterpenicglycosidicderivatives,like

paeoniﬂorine[20],couldbesupportedbythediagnosticsignals␦

1.3to5.4ppm,ascribabletothemonoterpenicunit,e.g.signalof

themethylgroupat␦1.39ppm(H-10),signalsoftheCH2groups

forH-2andH-5,asingletat␦5.43ppm(H-9),andsignalsofthree

quaternarycarbonsat␦86.2,87.5and75.4ppmrelatedtoC-1,

C-6andC-7,respectively(Fig.4).ThiswasconﬁrmedbyHSQC-DEPT

andHMBCanddiagnosticsignalsaresummarizedinTable1.Signals

supportingthepresenceofglycoside,namelythecrowdedregion␦

3–4.5ppmaswellasdoubletsorsingletsassignedtoanomeric

pro-tonpositionsweredetected.Signiﬁcantamountofsaccharosewas

detectedduetothepresenceofitsanomericsignalat␦5.38ppm.

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Fig.2.Exempliﬁcative1_H-NMR_of_the_methanol_extract_of_Paeonia_with_the_main_assigned_peaks.

Fig.3. ExempliﬁcativeHSQCspectrumofthemethanolicPaeoniaextractwithsomeoftheidentiﬁedpositionofcompounds.

HMBC,asthemethylgroupcorrelatedwiththecarbonsignalatC-5

oftheglycoside.

Otherconstituentspresentinloweramountswereﬂavonoids,

inparticularkaempferolandquercetinglycosides.Twodoublets

at␦8.01and6.87ppm(J=7.0Hz)wereascribedtopositions

H-2´,6 ´and_3´,5 ´ofkaempferolandthesignals␦7.05,6.96,6.89ppm

wereassigned to positions _2´, 3 ´and6 ´of thering Bof quercetin

[20]. Signalsrelated topositions6 and8ofﬂavonoids couldbe

detectedassinglets␦ 6.4–6.7ppm suggestingtheposition7 as

implicatedinglycosidicbond.HSQC-DEPT,HMBCandCOSYdata

allowedustoconﬁrmtheassignmentsofthesediagnosticpositions

[20].

The most signiﬁcant NMR assignments of the compounds

presentinthePaeoniaextractsaresummarizedinFigs.2–4[18,19].

NMRanalysisrevealedthepresenceofaromaticacidderivatives,

gallic acidesters,monoterpene glycosides, as wellas ﬂavonoid

glycosides.Averysimilarﬁngerprintwasobservedforthe

differ-entmethanolicextractsofrootsandaerialpartsofbothanalyzed

species.

Tofurtherstudythecompositionoftheextracts,

LC-DAD-ESI-MS analysis was performed. Detected peaks were assigned to

compoundsonthebasis ofthecomparisonofUVspectrumand

MSfragmentationpatterns,aswellasbycomparingtheobtained

datawiththoseacquiredintheNMRexperiments.Froma

qual-itativepointofview,theLC-DAD-ESI-MSanalysisconﬁrmedthe

constituentsidentiﬁedbyNMR.Quercetinandkaempferol

glyco-sidesandgalloylglucosederivativeswereidentiﬁedonthebasis

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Fig.4. EnlargedareaoftheHSQC-DEPTspectrumofPaeoniaextractshowingtheassignmentsofthemonoterpenoidderivativeofpaeoniﬂorinetype.

Fig.5. ExempliﬁcativeHMBCspectrum.

withthatreportedinliterature[21].Quantitativeanalysiswas

per-formedusingrutinasreferencecompoundforﬂavonoids,andgallic

acidforthe gallicacidderivatives ofsaccharides and

monoter-penes,respectively.Themaincomponentsidentiﬁedaredepicted

inFig.6.AnalysisofP.arietinaaerialpartsextractshowedthat

themostabundantﬂavonoidinallofthesampleswas

quercetin-glucosyl-rhamnoside.Thehexagalloylglucoseappearedtobethe

mostabundantgalloylderivativeinalloftheextracts.Fivedifferent

peaksascribabletogalloylpaeonidinweredetectedsuggestingthe

possibilityofﬁvedifferentesteriﬁcationsofgallicacidonhexose

moietyaswellasonpaeonidinfreehydroxylgroup.Theoverviewof

dataagainshowedaverysimilarqualitativecompositionofthefour

samplesanalyzed.Fromaquantitativepointofview,P.

kesrounan-sisandP.arietinaaerialpartswerebothricherinﬂavonoidsandin

galloylderivativescomparedtotheroots(Table2and3,

respec-tively).

3.2. Totalamountofphenolicsandﬂavonoids

Thedifferent extractsofP.arietinaand P.kesrounansis were

investigated for their total phenolic content (TPC) and total

ﬂavonoidcontent(TFC)andresultsarepresentedinFig.7.

TheaerialpartextractsofP.arietinahadhigherTPCandTFC

valuescomparedtotherootextracts.Themethanolextractofthe

aerialpartsshowedthehighestTPC(164.10mgGAE/gextract)and

TFC(34.18mgRE/gextract)amongthethreesolvent.Similarly,for

rootpartswefoundthatTPCwasgreatestinthemethanolicone

(85.31mgGAE/gextract)whereastheaqueousrootextract

con-tainedthehighestamountofﬂavonoids(2.10mgRE/gextract).The

lowestTPCandTFCwereobservedintheaqueousandethylacetate

extractoftheroot,respectively.Asimilarobservationwasnotedfor

P.kesrounansiswherebytheaerialpartsshowedgreaterTPCand

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Table1

1_H_and13_C_NMR_diagnostic_positions_for_the_{phytoconstituents}_in_the_methanol_extracts_of_P._arietina_and_P._{kesrounansis.}

Compounds ␦H(JinHz) ␦CHSQC-DEPT ␦CHMBC

p-OH-benzoicacidmoiety 8.05d,7.48d,(J=7.7,7.6)_(H-2´,6´) 128.8,127.5 166,5,133.0,128.8

Aromaticacidsignals 7.48d,(J=7.7)7.65m,7.63m,7.60m,7.52m,7.50m,

7.47m_(H-2´,6´)7.61m

128.8,133.2 166.7

Flavonolmoieties,glycosidesof quercetinandkaempferol

8.01_(H-2´,6´),6.87_(H-3´,5´)(J=7.0)kaempferol 7.05,6.96,6.89_(2´,3´and6 ´ofringB)quercetin 6.4-6.7(signalsofH-6,H-8)

5.13d(H-1”)anomericprotonofglucose 1.38(CH3of rhamnose) 108.9,109.4,109.6 96.6-98.7 18.0 140,0163,0

Galloylesterderivatives 7.12brs(galloyl)_(H-2´,6´)

6.89,6.94,6.97

6.23d(anomericofsugar)(J=8.2)

5.9m(C3ofglucose)5.6m(C2ofglucose)-deshielded becauseoffurthergalloylsubstitution

4.11sand4.05s(C4andC5ofglucose) 4.74(C6glucose) 108.9 113.9,114.0,114.1 83.2 68.6 77.6and74.5 60.0 134.4,162.3 164,4 165,0 164,4 165,3

Paeoniﬂorintypecompound 1.39s(CH3,H-10)

1.32(CH2,H-2) 5.43s(H-9) 2.62(H-4) 2.49(CH2,H-5) 7.60t(J=7.6) 17.7 29.0 91.4 43.2 23.2 132.9 86.2(q)C-1 87.5(q)C-6 75.4(q)C-7 106.9,75.4 106.9,75.4 75.4 125.0

Sucrose 5.38d(3.8)anomericproton

3CH2groups3.64,3.73,3.76

91.9 60.6-62.6

13.1,17.3,106.5

Table2

HPLC-DAD-MSqualitativeandquantitativeanalysisofﬂavonoidsinthemethanolextractsofP.arietinaandP.kesrounansis.

Rt(min) Identiﬁcation M-H Fragments P.kesrounansis

Aerialparts(mg/g) P.arietina Aerialparts(mg/g) P.kesrounansis Roots(mg/g) P.arietina Roots(mg/g) 12.1 Quercetin-3,7-di-O-glucoside 625 463301 0.39 0.42 0.09 0.06 14.3 Kaemperol-3,7-di-O-glucoside 609 447285 0.40 0.26 0.01 0.04 15.2 Quercetin-hexoside 463 301 1.78 0.70 0.31 0.37 19.7 Quercetin-glucosyl-rhamnoside 609 447463301 3.58 8.50 0.34 0.61 19.1 Quercetin-galloyl-hexoside 615 463301 1.24 0.94 0.05 0.13 18.9 Kaempferol-3-Oglucoside-7-O-ramnoside 593 447463301 3.07 5.57 0.10 0.55 20.0 Rutin 609 301 0.61 0.01 0.02 0.04 Totalamount 11.07 16.4 0.92 1.8

*Dataaremeanofthreedifferentmeasurements.DVSTareintherangeof5%.Rt:Retentiontime.

Fig.6. Generalstructuresofthemainphytoconstituentsidentiﬁedinthetested methanolextractsofthestudiedPaeoniaspp.extracts.

aerialparts,higherTPCandTFCwereobservedforthemethanol

(172.13mgGAE/gextractand25.84mgRE/gextract,respectively).

Likewise,amongtherootextracts,TPCwashighestinthe

methano-licone(89.06mgGAE/gextract).However,theethylacetateextract

oftherootshowedthehighestTFC(3.33mgRE/gextract)compared

toitscounterpartsolventsextracts.

Apreviousinvestigationby Orhanetal. [16] foundthatthe

ethanolrootextractofthreesubspeciesofP.mascula(western

Ana-tolianP.mascula(L)Mill.,P.mascula(L.)Mill.subsp.arasicola,andP.

mascula(L.)Mill.subsp.bodurii)showedhigherTPC(14.9,14.1,and

14.3mgGAE/100mgextract,respectively)thanP.turcica(7.6mg

GAE/100mgextract)whileP.mascula(easternAnatoliansample)

showedlowerTPC(5.3mgGAE/100mgextract).

3.3. Antioxidantactivity

Severalassaysevaluatingtheantioxidantactivity(DPPH,ABTS,

FRAP,CUPRAC,phosphomolybdenumand metalchelating)were

performedtoobtainacomprehensiveinvitroassessmentofthe

antioxidantpotentialof thetwo testedspecies (see Fig.7).We

usedseveralinvitromethodstostudytheantioxidantproperties

ofextracts.Forexample,DPPHassayisperformedinorganic

sol-vent,whileABTSisperformedinbufferedaqueousphase.FRAPand

CUPRACusetwodifferentmetalionstoassessreducingantioxidant

power.Theothertwo testsinvolved thereduction of

phospho-molybdicacidtophosphomolybdenum,andthemetalchelating

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Table3

HPLC-DAD-MSqualitativeandquantitativeanalysisofgalloylderivativesinthemethanolextractsofP.arietinaandP.kesrounansis.

Rt(min) Compound m/z[M-H]− _Fragments Contentofcompoundinmg/g

P.kesrounansis Aerialparts P.arietina Aerialparts P.kesrounansis Roots P.arietina Roots 1.6 Galloylsucrose 493 331,313,179 2.28 3.08 2.59 1.77 3.11 Galloylsucrose 493 331,313,179 3.61 4.02 2.38 1.92 1.5 Glucogalloyl 331 271,221,170 0.55 0.94 0.70 0.52 3.1 Glucogalloyl 331 271,221,170 0.71 0.95 0.70 0.45 10.3 Trigalloylglucose 635 617,465,313,170 0.21 0.86 0.28 0.10 14.0 Trigalloylglucose 635 617,465,313,170 0.10 0.83 0.58 0.29 16.4 Trigalloylglucose 635 617,465,313,170 0.57 0.17 0.15 0.03 15.8 Tetragalloylglucose 787 635,617,465,313 0.64 1.39 0.53 0.38 16.9 Tetragalloylglucose 787 635,617,465,313 0.39 0.64 0.34 0.24 18.9 Tetragalloylglucose 787 635,617,465,313 1.15 3.78 2.09 1.22 19.4 Tetragalloylglucose 787 635617,465,313 2.73 4.86 2.08 1.18 19.9 Galloylpaeoniflorin 631 479,313,169 1.14 2.02 2.07 1.70 21.7 Galloyl-paeoniflorin 631 479,313,169 3.75 0.53 0.73 0.72 22.3 Galloyl-paeoniflorin 631 479,313,169 1.16 0.12 0.15 0.14 22.6 Galloyl-paeoniflorin 631 479,313,169 0.84 0.01 0.03 0.06 23 Galloyl-paeoniflorin 631 479,313,169 2.48 0.11 0.02 0.06 22.7 Pentagalloyl-benzylglucose 891 739,587,569,525,417,170 0.41 0.12 0.03 0.04 23.4 Pentagalloyl-benzylglucose 891 739,587,569,525,417,170 2.23 0.61 0.24 0.25 9 Oxypaeoniflorin 495 465,333,281 0.05 0.10 0.66 0.15 23.6 Trigalloyl-benzylglucose 739 587,569,525,417,170 1.33 0.39 0.20 0.13 18.8 Hexagalloylglucose 1091 939,769,617 24.46 29.96 11.64 13.38 20.3 Paeoniflorin 479 449,327,165,121 3.61 0.18 0.05 0.18 Total 54.43 55.67 28.24 24.9

*Dataaremeanofthreedifferentmeasurements.DVSTareintherangeof5%.Rt:Retentiontime.

Fig.7.Totalphenolic(TPC),totalﬂavonoid(TFC)andantioxidantpropertiesoftestedPaeoniaextracts(EA:Ethylacetate;MeOH:Methanol;GAE:Gallicacidequivalent;RE: Rutinequivalent:TE:Troloxequivalent;EDTAE:EDTAequivalent).Differentlettersindicateasigniﬁcantdifferencebetweentheextracts(p<0.05).

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Fig.8. Statisticalevaluations(A&B:DistributionofPaeoniaextractsonthefactorialplanandrepresentationofbiologicalactivitiesonthecorrelationcirclebasedonPCA; C:DendrogramfrombiologicalactivitiesofPaeoniaextracts;D:Correlationcoefficientsbetweentotalbioactivecompoundsandbiologicalactivities(PearsonCorrelation Coefficient(R),p<0.05);EA:Ethylacetate;MeOH:Methanol;AP:Aerialparts;R:Root;TPC:Totalphenoliccontent;TFC:Totalflavonoidcontent).

intheFentonreactiontoproducetoxicfreeradicals.Thepresent

ﬁndingsrevealedthatamongtheP.arietinaextracts,the

methano-licaerialpartshowedthehighestDPPH(544.72mgTE/gextract)

andABTS(659.53mgTE/gextract)scavengingactivities.A

simi-larresultwasobservedforP.kesrounansiswherebythemethanol

extractofitsaerialpartsexertedthehighestDPPH(540.23mgTE/g

extract)andABTS(631.83mgTE/gextract)scavengingeffects.

The reducing power, in terms of ferricand cupricreducing

effects,wasdeterminedasshowninFig.7.Amongthethreeextracts

ofP.kesrounansis,themethanolicaerialpartdisplayedthestrongest

reducingeffectinbothassays(CUPRAC:775.09mgTE/gextract;

FRAP:409.12mgTE/gextract).Similarly,themethanolicextract

oftheaerialpartsofP.arietinahadthehighestreducingeffects

(CUPRAC:753.93mgTE/gextract;FRAP:392.96mgTE/gextract)

amongtherespectiveextracts.

Ascanbeobserved,themethanolicextractoftheaerialpartsof

bothplantsshowedthegreatestscavengingandreducingactivities,

whichcouldbecorrelatedtotheamountofbioactivecompoundsin

theextract.Indeed,thegreatestlevelsofphenoliccompoundsand

ﬂavonoidswereobservedinthemethanolicextractoftheaerial

parts,andinfact,anumberofstudiesfoundthatthetotalphenolic

andﬂavonoidcontentarepositivelycorrelatedwithDPPHandABTS

scavenging,andFRAPandCUPRACreducingactivity[22].Touching

Paeoniaspp.,astudyconductedbyOrhanetal.[16]reportedthat

thecrudeethanolicrootextractsofP.turcica(syn.P.kesrounansis)

anddifferentsubspeciesofP.mascula(syn.P.arietina),includingP.

mascula(westernAnatoliansample),P.mascula(easternAnatolian

sample),andP.masculasubsp.arasicoladisplayedDPPHandnitric

oxidescavengingactivity,whileP.masculasubsp.boduriididnot

showanyDPPHscavengingeffect.

Furthermore,we investigatedthe antioxidantactivity ofthe

extractsbasedonthephosphomolybdenumandmetalchelating

assays.Inthephosphomolybdenum method,Mo(VI)is reduced

toMo(V)bytheantioxidantcompoundsandagreenphosphate

Mo(V)complexisformed.Chelationoftransitionmetalsisalso

importantsincethesemetalscatalyzeFentonreactionwhichplays

amajorroleinoxidativestressinvivo.AmongtheextractsofP.

kesrounansis,themethanolextractoftheaerialpartsshowedthe

greatestactivity(3.46mmolTE/gextractand 33.85mgEDTAE/g

extract in thephosphomolybdenum and metal chelatingassay,

respectively).Thisisalsoinagreementwiththelevelofphenolic

compoundsin theextract. Indeed,a previousstudyby

Sownd-hararajanandKang[23]alsoobservedthatplantextractshowing

highlevelofTPCandTFCdisplayshighactivityinthe

phospho-molybdenumandmetalchelatingassays.ConsideringP.arietina,

thehighestactivityinthephosphomolybdenumassaywasexerted

bytheethyl acetateextract oftheaerialparts (3.78mmolTE/g

extract)whiletheaqueousextractoftheaerialpartsshowed

high-estmetalchelatingeffect(30.57mg EDTAE/gextract).Although

thesetwoextractsweremosteffectiveinthesetwoantioxidant

assays,theydidnotdisplaythehighestTPCandTFC,hence

indicat-ingthepresenceofothercompoundsmodulatingtheirobserved

activities.

3.3.1. Enzymeinhibitoryactivity

Manydrugtherapiesarebasedontheinhibitionoftheactivity

ofoveractiveenzyme(s)toslow-downtheprogressionofdiseases

andtoalleviatetheassociatedsymptoms.Inthepresentstudy,the

enzymeinhibitoryactivityofP.arietinaandP.kesrounansisextracts

againstcholinesterases(AChEand BChE),tyrosinase,␣-amylase,

and␣-glucosidaseweretestedandresultsarepresentedinFig.9.

Cholinesteraseinhibitorsarethemajorclassofdrugswhichare

currentlyutilizedforthesymptomatictreatmentofAlzheimer’s

disease.Cholinesterasesarethemaintargetsofvariousreversible,

irreversibleorpseudo-irreversibleinhibitors,someofwhichmay

cause side effects such as diarrhoea, nausea, vomiting,muscle

cramps,lowbloodpressure,insomnia,fatigue,andlossofappetite

[24].Naturalcompoundsorplantextractsaspossiblesourcesof

therapeuticsforAlzheimer’sdiseasearethereforeintensively

stud-ied.Paeoniaspp.areusedintraditionalmedicineasneuroprotective

[4,6],thereforeweevaluatedPaeoniaextractsaspossibleinhibitors

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Fig.9.EnzymeinhibitoryeffectsoftestedPaeoniaextracts(EA:Ethylacetate;MeOH:Methanol;GALAE:Galatamineequivalent;KAE:Kojicacidequivalent:ACAE:Acarbose equivalent).Differentlettersindicateasigniﬁcantdifferencebetweentheextracts(p<0.05).

P.arietina, theethyl acetate extract of theaerial partsshowed

thehighestAChE (4.95mg GALAE/gextract)and BChE(5.73mg

GALAE/gextract)inhibitionfollowedbytheethylacetateextract

oftheroot(4.45and5.05mgGALAE/gextract,respectively).For

P.kesrounansis,themethanolextractoftheaerialpartsandroot

werethemosteffectiveAChE(4.68mgGALAE/gextract)andBChE

(5.98mgGALAE/gextract)inhibitors,respectively.Nosigniﬁcant

AChEandBChEinhibitionwasexertedbytheaqueousextractof

therootandaerialpartsofP.kesrounansis,respectively.

Intestinal␣-glucosidaseisakeyenzymeincarbohydrate

diges-tion.It issecreted intheepitheliumof thesmall intestine.The

majorsourceof blood glucose isrepresented bydietary

carbo-hydratesincludingstarch,whichishydrolyzedby␣-glucosidases

andpancreatic␣-amylase,andthenabsorbedbythesmall

intes-tine.Consequently,␣-glucosidaseand␣-amylaseinhibitorshave

beenrecognizedasatherapeutictargetforthecontrolof

postpran-dialhyperglycemia,which istheearliestmetabolicabnormality

thatoccurs intype 2diabetesmellitus[17]. Wefoundthatthe

ethylacetateextractofP.arietinaaerialparts(1.39mmolACAE/g

extract)hadthestrongest␣-amylaseinhibitoryeffectfollowedby

therootone(1.09mmolACAE/gextract).Bothextractsalso

dis-playedthegreatest_{␣-glucosidase}inhibitioncomparedtotheother

extracts(root ethylacetateextract:10.33mmolACAE/gextract;

aerialpartsethylacetateextract:10.30mmolACAE/gextract).A

similarpatternwasobservedforP.kesrounansis-themost

effec-tive␣-amylaseinhibitorwastheethylacetateextractoftheaerial

parts(1.26mmolACAE/gextract)followedbytherootethylacetate

extract (1.22mmol ACAE/g extract).Likewise, theethyl acetate

extract of the aerial parts displayed the highest ␣-glucosidase

inhibitoryactivity(10.28mmolACAE/gextract).

Itistobenotedthatalthoughtheethylacetateextractsshowed

highenzymeinhibitoryactivitiesagainstAChE,BChE,␣-amylase,

and␣-glucosidase,theycontainedloweramountofphenolicsand

ﬂavonoidcomparedtothemethanolcounterpart.Thenon-direct

correlationbetweenphenolicamountandactivitymightberelated

tospeciﬁcactivityofsomecompoundsresponsiblefortheobserved

effectsortosynergisticeffectsofthedifferentcomponentsofthe

wholeextract[25].Itisalsoimportanttonotethatprevious

stud-ies[26]havealsoobservedthehighestAChE,BChE,␣-amylase,and

␣-glucosidaseinhibitoryactivityofethylacetateextractsofplants

comparedtotheirmethanolicandwaterextracts,whichmay

indi-catethatthecompoundsmodulatingtheseeffectmightbemore

solubleinethylacetate.Nonetheless,furtherstudiesareneededto

conﬁrmthishypothesis.

Furthermore,thepresentﬁndingsrevealedthatbothP.arietina

andP.kesrounansisextractsinhibitedtyrosinase(Fig.9).

Tyrosi-nase, theratelimiting enzymeof the melanogenic pathway,is

a key enzyme in melanin synthesis and it is major

therapeu-tictargettoalleviatecutaneoushyperpigmentation[26].Among

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aerialparts (145.43mgKAE/g extract) followed bythat of root

(143.66mgKAE/gextract)displayedthehighesttyrosinase

inhi-bition.Similarly, the most effective tyrosinase inhibitor among

the extracts of P. kesrounansis was the methanolic extract of

theaerialparts(148.34mgKAE/g extract)followed bytheroot

methanolicextract (141.05mg KAE/g extract).The high

tyrosi-naseinhibitory effectof themethanolicextractsof both plants

is likely due to their highest level of phenolic and ﬂavonoid

as found in the present study. Previous investigations [27,28]

showed that plant extracts possessing high level of TPC and

TFCalsodisplayedhighanti-tyrosinaseactivity.Withregardsto

otherPaeoniaspecies,apreviousstudyprovedtheanti-tyrosinase

activity of the methanolic extract of the aerial parts of Greek

P.masculassp.hellenica,displayinganIC50valueof135.2␮g/ml

[29]. In addition, three isolated compounds from the

ﬂow-ers of P. lactiﬂora; 1,2,3,6-tetra-O-galloyl-ˇ-D-glucopyranoside,

1,2,3,4,6-penta-O-galloyl-ˇ-D-glucopyranoside and kaempferol

3-O-(6-O-galloyl)-ˇ-D-glucopyranoside, were found to inhibit

tyrosinasewithIC50valuesrangingfrom0.23to0.35␮g/ml,with

greater effectiveness compared to kojic acid (IC50=6.4␮g/ml)

[30].

3.3.2. Statisticalevaluation

Principal component (PCA), cluster and correlation analysis

wereperformedtounderstandpossiblerelationsbetweenPaeonia

extracts,componentsandcorrespondingbioactivities.Allresults

aredepictedinFig.8.PCAbuilttwoprincipalcomponents,which

explained82.82% oftotal variability(Fig.8A).ThevariablesTPC,

TFC,DPPH, ABTS, CUPRACand FRAP contribute strongly tothe

formationofaxis1(51.34%),whilethevariablesincluding

tyrosi-nase,amylases,BChEandAChEarestronglycorrelatedtoaxis2

(31.48%).Clearly,astrongcorrelationwasobservedbetweentotal

bioactivecompoundsand antioxidant assays.For example,

cor-relationcoefﬁcientvaluesweremorethan0.9betweenTPCand

DPPH,ABTS,FRAPandCUPRAC.However,anegativecorrelation

wasfoundfor_{␣-glucosidase}inhibition(Fig.8D).Also,wecould

notethatthemethanolextractsfromtheaerialpartsofthe

Paeo-niaspeciesexhibitedthestrongestantioxidantcapacityamongthe

analyzedextracts(Fig.8B).Likewise,thelowestantioxidantactivity

wasobservedintheaqueousrootextractsofbothspecies.Extracts

fromcorrespondingplantpartsofeachspecieswereclosetoeach

otherandallextractsclusteredasfourgroupsbasedonbiological

activityresults(Fig.8C).

4. Conclusion

Thepresentinvestigationshowedthatthephytochemical

com-position of P. arietina and P. kesrounansis are similar and are

characterizedbygalloylestersandﬂavonoidglycosides.

Further-more, the extracts showedsigniﬁcant antioxidant and enzyme

inhibitoryactivities.Themethanolicextractoftheaerialpartsof

both plantsshowed thehighest antioxidantactivity inmost of

theassays(DPPH,ABTS,CUPRAC,andFRAP)andstrongest

inhi-bitionoftyrosinase.Theethylacetateextractoftheaerialparts

ofbothplantswerethemosteffective␣-amylaseinhibitors.The

rootmethanolicextractofP.kesrounansisshowedthehighestBChE

inhibitionwhiletherootethylacetateextractofP.arietinaexerted

thestrongestinhibitionagainst␣-glucosidase.Thepresent

ﬁnd-ingsofferpreliminarydataforthevalorizationofPaeoniaspecies

as sources of bioactive constituents with potential use in the

management of severalchronic diseases.Further investigations

shouldbeperformedtoassesstheobservedbioactivitiesbyinvivo

modelsapplyingboththepharmacodynamicandpharmacokinetic

approachandalsotoestablishtheirsafetyproﬁlethroughtoxicity

studies.

Conﬂictofinterest

Wewishtoconﬁrmthattherearenoknownconﬂictsofinterest

associatedwiththispublicationandtherehasbeennosigniﬁcant

ﬁnancialsupportforthisworkthatcouldhaveinﬂuencedits

out-come.

Authorcontributions

SS,SD,MG,KS:ChemicalAnalysis;GZ:Biologicalactivity

anal-ysis;GB,AD,MZH:Collectionandidentiﬁcationoftheseplantand

GZ,MZA,FM,MFM,SS,SD,KˇS:Dataanalysisandwriting.

Acknowledgement

This workwas supported by the IGA UVPSBrno (grant no.

310/2016/FaF)

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