Scrophularia Lucida L. As A Valuable Source Of Bioactive Compounds For Pharmaceutical Applications: In Vitro Antioxidant, Anti-İnflammatory, Enzyme İnhibitory Properties, İn Silico Studies, And HPLC Profiles

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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

Scrophularia

lucida

L. as

a

valuable

source

of

bioactive

compounds

for

pharmaceutical

applications:

In

vitro

antioxidant,

anti-inﬂammatory,

enzyme

inhibitory

properties,

in

silico

studies,

and

HPLC

proﬁles

Gokhan

Zengin

a,∗,1

_,

_Azzurra

_Stefanucci

b,1

_,

_Maria

_João

_Rodrigues

c

_,

_Adriano

_Mollica

b

_,

Luisa

Custodio

d

_,

_Muhammad

_Zakariyyah

_Aumeeruddy

d

_,

Mohamad

Fawzi

Mahomoodally

d

a_Selcuk_University,_Science_Faculty,_Department_of_Biology,_Campıus,_Konya,_Turkey b_Department_of_Pharmacy,_University_“G._{d’Annunzio”}_{Chieti-Pescara,}_66100,_Chieti,_Italy

c_Centre_of_Marine_Sciences,_University_of_Algarve,_Faculty_of_Sciences_and_Technology,_Ed._7,_Campus_of_Gambelas,_8005-139,_Faro,_Portugal d_Department_of_Health_Sciences,_Faculty_of_Science,_University_of_Mauritius,_230,_Réduit,_Mauritius

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received15July2018 Receivedinrevisedform 16September2018 Accepted17September2018 Availableonline18September2018

Keywords: S.lucida Antioxidant Tyrosinase Phenolic Docking Anti-inﬂammatory

a

b

s

t

r

a

c

t

ThegenusScrophulariahasreceivedmuchinterestwithregardstoitstraditionalusesagainsteczema, psoriasis,andmastitis.Yet,themedicinalpropertiesofsomespeciesstillneedtobescientiﬁcally val-idated.Thepresentstudywasdesignedtoinvestigateintothebiologicalpropertiesofvarioussolvent extracts(ethylacetate,methanol,andaqueous)oftherootsandaerialpartsofScrophularialucidabased onitsantioxidant,anti-inﬂammatory,andenzymeinhibitoryactivitiestogetherwithphytochemical screening.Ourresultsrevealedthatthesolventextractsdifferedintheirbiologicaleffectiveness.The rootethylacetateextractshowedthehighestABTSscavenging,FRAP,CUPRAC,andinhibitoryactivity againstAChEand␣-glucosidase.TheethylacetateextractoftheaerialpartsdisplayedthehighestBChE and␣-amylaseinhibitionandantioxidanteffectinthephosphomolybdenumassay,whilethemethanol extractsofbothpartswerethemosteffectiveDPPH•_scavengers_and_tyrosinase_inhibitors._The_methanol

extractsoftherootandaerialpartsalsoinhibitedNOproductioninlipopolysaccharide(LPS)-stimulated murineleukemicmonocyte-macrophagecell(4.99%and10.77%,respectively),at31.25␮g/mL concen-tration.ThehighestTPC(34.98mgGAE/gextract)andTFC(48.33mgRE/gextract)wereobservedinthe ethylacetateextractoftherootandaerialparts,respectively.Themostabundantcompoundsintheroot ethylacetateextractwereluteolin(852␮g/gextract),rosmarinicacid(522␮g/gextract),andhesperidin (394␮g/gextract)whilekaempferolwasmostabundantintheethylacetateextractoftheaerialparts (628␮g/gextract).Insilicoexperimentswereconductedontyrosinaseandthehigherdockingvalueswere observedforrosmarinicacidandhesperidin.Thepresentﬁndingsprovidebaselineinformationwhich tendtosupportthepotentialuseofS.lucidainthemanagementofseveralchronicdiseases,including Alzheimer’sdiseaseanddiabetesmellitus.

Abbreviations: ABTS, 2,2_{-azino-bis(3-ethylbenzothiazoline-6-sulfonic}

acid; ACAE, acarbose equivalent; AChE, acetylcholinesterase; BChE, butyryl-cholinesterase; CUPRAC, cupric reducing antioxidant capacity; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EDTAE,EDTA equivalent; FRAP, ferric reducing antioxidantpower;GAE,gallicacidequivalent;GALAE,galatamineequivalent; KAE,kojicacidequivalent;RE,rutinequivalents;TE,troloxequivalent;TPC,total phenoliccontent;TFC,totalﬂavonoidcontent.

∗ Correspondingauthor.

E-mailaddress:[email protected](G.Zengin).

1 _These_authors_contributed_equally.

1. Introduction

Plantshave beenthe basis formedical treatments since the

originof mankind,and suchtraditional medicineis still widely

practicedtoday[1].Sinceitsestablishment,theﬁeldofethnobotany

hasevolvedfrommerelyacollectionofinformationonmedicinal

plantsusedbyaparticularcommunityintoamorecomplex,

inter-disciplinaryresearchareawhichhaveattractedmodernscientists

torevisittheseancientwealthofknowledgeonmedicines[2].This

approachisbasedontheassumptionthatthebioactivecompounds

isolatedfromsuchplantsarelikelytobesafercomparedtothose

derivedfromplantspecieswithnohistoricalusebyhuman[3].

https://doi.org/10.1016/j.jpba.2018.09.035

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ThegenusScrophularia,belongingtotheScrophulariaceae

fam-ily,consistofbothherbaceousandsemi-shrubbyplants.Thegenus

consistsof617speciesnamesasrecordedinThePlantList(http://

www.theplantlist.org/);ofthese86areacceptedspeciesnames,47

synonym,and484unassessed.Theprimarycenterof

diversiﬁca-tionofScrophulariaisbelievedtobelocatedinsouth-westAsia,in

anareaaroundAsiaMinor,Iraq,Afghanistan,theHimalayasand

themountainrangesofWesternTibet.Thegenusisrepresented

by30taxainEurope,themostimportantdiversitycenterbeing

theIberianPeninsula(12endemicspecies)[4].Scrophulariaspecies

areannual,biennialorperennialherbs,subshrubsorsmallshrubs

havingpinnatetoundividedleavesofvariousforms.Thesespecies

preferhabitatssuchasmountainslopesandrockcrevices,aswell

asforests,scrubsandgrassland,roadsidesordisturbedareas[5].

ThetraditionalusesofScrophulariaspeciesfortherapeutic

pur-poseshave beenpracticedin severalcountriesincludingEgypt,

Morocco, India, and Italy, for the treatment of skin disorders,

eczema,psoriasis,mastitis,inﬂammationsofthemammarygland

and chroniclymphaticstagnation [6],gastrointestinal disorders

[7],boils,jaundice,malaria[8,9],rheumatism,muscularpains[10],

sciatica[11].Thesespeciesareusedinveterinarymedicineasan

antisepticandcicatrizingagentforwoundsinbovinesandsheep

[12].

Previousstudieshaveidentiﬁedseveralbiologicalpropertiesof

extractsandisolatedcompoundsofmembersofthisgenus

includ-ingtheanti-inﬂammatoryactivityofS.dentata,S.auriculataand

S. scorodonia [13–15], antibacterial activity of S. frutescens and

S.sambucifolia [16], antifungal, gastroprotective,and anticancer

activityof S. striata[17–19], analgesic and immunomodulatory

activityofS.megalantha[20],andalsothecardioprotectiveeffect

of S. frigida [21], among others. Iridoid glucosides are

consid-eredaschemotaxonomicmarkersfortheScrophulariaceaefamily

[22]. Verbascoside and (E)-phytol were identiﬁed in S. canina

[22]. Two new iridoid-related compounds with a new carbon

skeleton,buergerinin F and G,wereidentiﬁed inS. buergeriana

roots[23].Moreover,acyclopentanoidmonoterpenelactone,

7,8-dehydro-6␤,10- dihydroxy-11-noriridomyrmecin,wasisolated

fromS.canina,togetherwiththeknowniridoidglucosides

aucu-bin,harpagide,8-O-acetylharpagide,and10-O-␤-gIucosylaucubin

[24]. Also, two iridoid glycosides, scrophularianoids A and

B, were isolated from S. ningpoensis root [25]. In addition,

phenylpropanoidglycosidessuchasningposidesA

(3-O-acetyl-2-O-feruloyl-␣-l-rhamnopyranose),B(4-O-acetyl-2-O-feruloyl-

␣-l-rhamnopyranose)andC(3-O-acetyl-2-O-p-hydroxycinnamoyl-

␣-l-rhamnopyranose)along withknowncompoundssibiriosideA,

cistanosideD,angorosideC,acteoside,decaffeoylacteoside,and

cis-tanosideFwereidentiﬁedfromtherootsofS.ningpoensis[26].

Withregardstothespeciestestedinthepresentstudy(S.lucida),

apreviousinvestigationbyLewenhofer etal.[27] identiﬁed 14

compoundsin S.lucida, amongthem veryrare iridoids,

includ-ingscrovalentinosideorkoelzioside,andseveralﬂavonoids(e.g.,

nepitrinandhomoplantaginin).2-O-acetyl-homoplantagininwas

identiﬁedasanewnaturalcompound.Theﬂavonoidhispidulinwas

alsofoundtobeamajorcontributortotheinhibitoryeffects of

S.lucidaoncancercell-inducedcircularchemorepellentinduced

defects(CCID).AnotherstudybyGiessrigletal.[28]foundthatS.

lucidaattenuatedtumorcellintravasationthroughlymph

endothe-lialcellmonolayers,whichcorrelatedwiththeinhibitionofNF-␬B.

Nonetheless,thereis stilla dearthof informationonother

bio-logicalpropertiesofthisplant.Tothebestofourknowledge,the

in-depthantioxidantproﬁleandinhibitoryactionagainstenzymes

associatedwithchronicdiseaseshave notbeenstudiedtodate.

Therefore, thepresent studyaimedto evaluatetheantioxidant

activity of S. lucida based on its radical scavenging, reducing,

andchelatingeffect,anti-inﬂammatoryeffectagainst

liposaccha-ride(LPS)-induced NOproduction, and itsinhibitory properties

against key enzymes involved in the etiology of neurological

disorders(acetylcholinesterase(AChE)andbutyrylcholinesterase

(BChE)),skindisorders(tyrosinase),anddiabetes(␣-amylaseand

␣-glucosidase).Inaddition,insilicoapproachwasusedto

deter-mineanypossibleinteractionsbetweenmajorphenolicsidentiﬁed

andtheirenzymeinhibitoryeffects.

2. Materialsandmethods

2.1. Plantmaterialandpreparationofextracts

TheaerialpartsandrootsofS.lucidawerecollectedinKonya

(betweenYukselenvillageandKonyaroad),Turkey(during

sum-mer of 2016). Botanist Dr. Murad Aydin Sanda performed the

plant’sidentiﬁcationandvoucherspecimen(vouchernumber:GZ

1616)hasbeendepositedfortheplantsampleatKNYAHerbarium

(DepartmentofBiology,ScienceFacluty,SelcukUniversity).

Theaerialpartsand rootswereanalyzed.The plantsamples

were ﬁrst dried in the dark at room temperature for 10 days

andwerethengroundtoapowderusingalaboratorymill.The

powderedsamples(10g)werestirredwithethylacetate(EA)or

methanol(MeOH)(200mL)atroomtemperaturefor24hfollowed

byconcentrationoftheextractstodrynessusingarotary

evap-orator(at40◦C).Forthewaterextracts,5gofplantmaterialwas

boiledin100mLwaterfor20minandtheextractswerelyophilized.

Theobtainedextractswerestoredindarkglassat4◦Cuntilfurther

analyses.

2.2. Proﬁleofbioactivecompounds

The total phenolic (TPC) and ﬂavonoid contents (TFC) were

measuredusingtheFolin-CiocalteuandAlCl3assays,respectively

[29,30] and results were expressed as gallic acid (mg GAEs/g

extract)andrutinequivalents(mgREs/gextract),respectively.

RP-HPLC-DAD(ShimadzuScientiﬁcInstruments,Kyoto,Japan)

wasusedtodetectphenoliccomponentsinthestudiedextracts.

Eclipse XDB C-18 reversed-phase column (250mm×4.6mm

length,5␮mparticlesize,Agilent,SantaClara,CA,USA)wasusedas

stationeryphaseat30◦C.Thedetailedinformationwaspreviously

reported [31]. Gallic acid, protocatechuic acid, (+)-catechin,

p-hydroxybenzoicacid,chlorogenicacid,caffeicacid,(-)-epicatechin,

syringicacid,vanillin,p-coumaricacid,ferulicacid, sinapicacid,

benzoicacid,o-coumaricacid,rutin,hesperidin,rosmarinicacid,

eriodictyol,trans-cinnamicacid, quercetin,luteolin, kaempferol,

andapigeninwereusedasstandards.Thestandards(allofthem

areHPLC grade)werepurchased fromSigma-Aldrich(St.Louis,

Missouri,USA).Thephenoliccomponentswerequantiﬁedas␮g/g

dryextractaftercomparisonofretention times,UV–vis spectra,

andchromatographicproﬁlewithcommercialstandardphenolic

components.

2.3. Determinationofantioxidantandenzymeinhibitoryeffects

Themethodsdescribed byGrochowski etal. [32] wereused

to assess the DPPH• and ABTS•+ _scavenging, _FRAP, _CUPRAC,

totalantioxidantcapacity(phosphomolybdenumassay),andmetal

chelatingeffectsoftheextracts.Theresultswerereportedastrolox

equivalents,whileEDTAwasusedformetalchelatingassay.Asfor

theenzymeinhibition,thepossibleinhibitoryactivitiesoftheplant

extractsagainstcholinesterases(acetylcholinesterase(AChE,E.C.

3.1.1.7)andbutyrylcholinesterase(BChE,E.C.3.1.1.8)(bythe

Ell-man’smethod),tyrosinase(E.C.1.14.18.1),␣-amylase(E.C.3.2.1.1)

and␣-glucosidase(E.C.3.2.1.20)weredeterminedusingstandard

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2.4. Insilicoassays

2.4.1. Enzymespreparation

The crystal structures of the enzymes used for the tests

were downloaded from theProtein Data Bank RCSB PDB [34]:

Acetylcholinesterase pdb: 4×3C [35] in complex with

tacrine-nicotinamide hybridinhibitor, butyrylcholinesterase pdb: 4BDS

[36]incomplexwithtacrine,tyrosinasepdb:2Y9X[37]in

com-plexwithtropolone,_{␣-glucosidase}pdb:3AXI[38]incomplexwith

maltose.TheproteinswerepreparedwithProteinPreparation

Wiz-ard[39]implementedinMaestrosuite10.2[40],byﬁllingmissing

sidechain,missingloopsandbyconvertingseleno-methionines

andseleno-cysteinestomethioninesand cysteinesrespectively,

generatingheteroatomsstatesatpH7.4usingPrime[41].The

non-catalyticwatersandotherco-crystallizedmolecules,wereremoved

and theH-bondassignmentwasmadebyusing PROPKAatpH

7.4,withminimizationoftheonlyhydrogenatoms.Inthecaseof

tyrosinase,twocopperionsarepresentinthedeepofthebinding

pocket,thusthemetalstateshavebeengeneratedautomaticallyby

ProteinPrep-WizardmoduleofMaestro.Theproteinswere

sepa-ratedfromtheircrystallographicligandsandusedwithoutfurther

modiﬁcationsforthedockingexperiments.

2.4.2. Ligandpreparation

AsreportedinTable1,themostrelevantsubstancesfoundinthe

extractsofS.lucidawereusedfortheinsilicoexperiments,namely

caffeic acid, ferulicacid, hesperidin, rosmarinicacid, quercetin,

luteolin,kaempferol,whichweredownloadedfromZincDatabase,

preparedwithLigPreptool[40]embeddedinMaestro10.2,

neu-tralizedatpH7.4byIonizerandminimizedwithOPLS3forceﬁeld

[42].

2.4.3. Moleculardocking

Thegridgenerationanddockingexperimentswereconducted

usingGlide[43].Foreachenzyme,agridwasproduced,usingthe

crystallographicligandtocenterthegrid,and24 angstromsfor

sizeofthegridbox.Metalcoordinationparametershavebeenused

withtwocopperionspresentinthetyrosinaseenzymaticcavity.

ThedockingexperimentswereconductedwiththeeXtraPrecision

scoringfunctionofGlideandonlythebestscoringposewaskept

andminimized.Thebindingenergycalculationsoftheobtained

posewereconductedusingthemolecularmechanicGBSA

(Gener-alizedBornandSurfaceAreacontinuumsolvation)method[44]by

PrimeinMaestro.

2.4.4. Moleculardynamic(MD)simulations

Thebestdockedposesfoundforrosmarinicacidandhesperidin

weresubjectedtoMDsimulations inwater[45]. Desmondwas

employedforthesimulations.Thecomplexwasinsertedin

aque-ousenvironment.Themodelconstitutesofaboxofwaterinwhich

thecomplexisinserted.Theboxhasaminimumsizetocontain

thecomplexensuringadistanceof10Åfromtheedgeofthebox

andtheprotein.Inordertoneutralizethesystem,0.15MNaClhas

beenaddedtothesystem.TheOPLS3FFwasusedforallthe

exper-iments.TIP3Pmodelofwater(PennaandLennart,2001)wasused.

Thesystemwasminimizedupto2000steps,holdingallthe

pro-teinandligandatoms.TheminimizedsystemwassubjectedtoMD

simulations,usingnormaltemperature&pressure(NPT)ensemble

andperiodicboundaryconditionsfor10ns.Tocontrolthepressure,

Martyna-Tobias-Kleinmethod[46]wasusedwhichallowstokeep

thepressureofthesystemat1.01barbyusingtheisotropic

cou-plingmethod.TheNose-Hooverthermostatwasappliedtocontrol

thetemperatureat310K.Thetrajectoriesandotherparameters

weresavedevery10pstoreturns1000frames.

Thesimulationanalysiswasdonebythesimulationinteractive

diagram(SID)embeddedinMaestro2015,especiallyusefulto

visu-alizetheRMSD(RootMeanSquareDeviation)ﬂuctuationsofligand

andenzyme,hydrogenbondsstability,rotationofgroupsonthe

lig-and,vanderWaalsinteractionsoverthesimulationtrajectoriesand

overallstabilityofthesecondarystructureoftheenzyme.

2.5. Cellassays

2.5.1. Reagentsandcellculture

Sigma-Aldrich (Steinheim, Germany) supplied

lipopolysac-charides (LPS) from Escherichia coli, sulphanilamide,

N-(1-naphthyl)-ethylenediamine dihydrochloride (NED) and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT),

whileDulbecco’sModiﬁedEagle’sMedium(DMEM),fetalbovine

serum(FBS),trypsin,l-glutamineandpenicillin/streptomycinwere

acquired from Lonza (Leuven, Belgium). The murine leukemic

monocyte-macrophagecellline(RAW264.7)wasakindgiftfrom

theFaculty ofPharmacy andCenter forNeurosciencesand Cell

BiologyfromtheUniversityofCoimbra(Coimbra,Portugal).Cells

weremaintainedinRPMI1640culturemediumsupplementedwith

10% heat-inactivated FBS,1% penicillin(50 U/mL)/streptomycin

(50␮g/mL)and1%l-glutamine(2mM),37◦Cinahumidiﬁed

atmo-spherewith5%CO2.

2.5.2. Cellviabilityassay

ExponentiallygrowingRAW264.7cellswereplatedin96-well

tissueplatesatadensityof1×104_cells/well_and_incubated_for₂₄_h.

Cellswerethentreatedwithseveralconcentrationsoftheextracts

(3.9,7.8,15.6,31.2,62.5and125␮g/mL)for24h.Incontrolcells,the

extractswerereplacedbyDMSOatthehighestconcentrationused

intestwells(0.5%).CellviabilitywasdeterminedbytheMTTassay

[47].Inbrief,MTT(20␮Lattheconcentrationof5mg/mLinPBS)

wereaddedtoeachwell2hbeforetheendoftheincubationperiod,

andfurtherincubatedat37◦C.DMSO(150_␮L)wasthenaddedto

eachwelltodissolvetheformazancrystals,andabsorbancewas

measuredat590nm(BiotekSynergy4).Resultswereexpressedin

termsofcellviability(%).Sampleswereconsiderednon-toxicwhen

theyallowedacellviabilityofatleast80%.

Table1

DockingscoreofthebestposeobtainedbyusingGlideXPscoringfunction;GbindingenergycalculatedbyPrime,expressedinKcal/mol.

PhenolicComponents AChE BChE ␣-Amylase ␣-Glucosidase Tyrosinase

XP G XP G XP G XP G XP G Caffeicacid −7.19 −12.05 −6.35 −13.05 −6.07 −10.82 −4.25 −13.72 −5.09 2.26 Ferulicacid −6.09 −20.84 −5.47 −1.17 −5.79 −6.52 −4.26 −6.75 −4.51 5.17 Hesperidin −11.05 −58.79 −13.86 −49.60 −10.05 −35.36 −10.73 −54.31 −7.58 −13.39 Rosmarinicacid −12.08 −47.65 −11.65 −27.65 −8.50 −14.65 −7.38 −23.64 −8.41 −21.49 Quercetin −10.76 −55.66 −9.47 −57.75 −8.64 −40.37 −7.99 −37.28 −5.56 −29.17 Luteolin −10.09 −51.68 −9.93 −52.22 −9.12 −44.75 −7.44 −35.12 −6.45 −28.41 Kaempferol −10.49 −51.71 −9.47 −52.07 −6.35 −25.17 −6.18 −15.19 −5.39 −25.05

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Table2

Totalbioactivecomponentsofthetestedextracts. Parts-Solvents Totalphenoliccontent

(mgGAE/gextract)

Totalﬂavonoidcontent (mgRE/gextract)

Root-EA 34.98±0.30 3.32±0.04

Root-MeOH 25.45±0.17 3.60±0.48

Root-Water 23.91±0.23 1.04±0.08

Aerialpart-EA 27.73±0.66 48.33±0.15

Aerialpart-MeOH 28.92±1.07 47.06±0.36

Aerialpart-Water 29.31±1.43 16.91±0.73

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.GAE:Gallicacid equivalent;RE:Rutinequivalent;TE:Troloxequivalent;EDTAE:EDTAequivalent. EA:Ethylacetate.MeOH:Methanol.

2.5.3. Quantiﬁcationofnitricoxide(NO)

Exponentiallygrowingcellswereplatedin96-welltissueplates

ataninitialdensityof2.5×105_cells/well_and_incubated_overnight

toadhere.Cellswerethentreatedwithnon-toxicconcentrations

oftheextractsinserum-andphenol-freeculturemedium,with

LPS(200ng/mL),for24h.ControlcellsweretreatedwithDMSO

at 0.5%, which was the highest concentration used in the test

wells.NOproductionincellculturemediumwasmeasured

spec-trophotometricallybytheGriessmethod[48].Brieﬂy,cellculture

supernatants(100␮L) weremixedwithGriessreagent(100␮L,

1%(w/v) sulphanilamide+ 0.1% ofNED, both prepared in 2.5%

(v/v)phosphoricacid)andincubatedinthedarkatroom

temper-ature(RT)for20min.Absorbancewasmeasuredonamicroplate

reader(BiotekSynergy4)at540nm.TheNOconcentrationwas

determinedusingacalibrationcurvepreparedwithincreasing

con-centrationsofsodiumnitrite(1.7,3.1,6.2,12.5,25,50and100␮M).

Resultswereexpressedaspercentage(%)ofNOproduction

com-parativetoLPS-stimulatedcells.

2.6. Statisticalanalysis

Alltestswere carriedout as threeparallel experiments and

theresultswereexpressedastheaverage±SD.Statistical

analy-siswasperformedusingone-way ANOVAfollowing byTukey’s.

Todetectdifferencesbetweentheextracts,SPSSv.17.0program

wasemployed.Thevalueofp< 0.05wasregardedasstatistically

signiﬁcant.

3. Resultsanddiscussion

3.1. Phytochemicalcomposition

Inthisstudy,thetotalbioactivecomponentsof therootand

aerialpartsofS.lucidaextractsweredeterminedintermsofTPC

andTFC,asshowninTable2.Amongtherootextracts,the

high-estTPCwasobservedintheethylacetateextract(34.98mgGAE/g

extract)followedbythemethanolextract(25.45mgGAE/gextract)

andaqueous extract(23.91mg GAE/gextract).On thecontrary,

TFCwashighestinthemethanolextract(3.60mgRE/gextract)

fol-lowedbytheethylacetateextract(3.32mgRE/gextract)whilethe

aqueousextractdisplayedtheleastamount(1.04mgRE/gextract).

Withregardstotheaerialparts,TPC wasintheorder:aqueous

extract (29.31mg GAE/g extract) >methanol extract (28.92mg

GAE/gextract)>ethylacetateextract(27.73mgGAE/gextract).On

theotherhand,TFCwasintheorder:ethylacetateextract(48.33mg

RE/gextract)>methanolextract(47.06mgRE/gextract)>aqueous

extract(16.91mgRE/gextract).

Furthermore,HPLC-DADanalysisofS.lucidaextractsrevealed

thepresenceofmajorphenoliccompounds.AsshowninTable3,

themostabundantcompoundsintheethylacetateextractofthe

rootwereluteolin(852␮g/gextract),rosmarinicacid(522␮g/g

extract),andhesperidin(394␮g/g extract).Hesperidinwasalso

abundantintheothersolventextracts(rootmethanolic:400␮g/g

extract,aerial partsethyl acetate:246␮g/g extract, aerialparts

methanolic:246␮g/gextract,rootaqueous:226␮g/gextract).

Ros-marinicacidwasdetectedonlyintheethylacetateandmethanol

rootextract.Chlorogenicacidwaspresentinthehighestamount

in the aqueous extract of the aerial parts (186␮g/g extract)

whileferulicacidwasmostlypresentintheaqueousrootextract

(366␮g/g extract). On the other hand, kaempferol was

identi-ﬁedonlyintheextractsoftheaerialparts(ethylacetateextract:

628␮g/g extract, aqueous extract: 408␮g/g extract, methanol

extract:390␮g/gextract).However,epicatechin,sinapicacid,

ben-zoicacid,o-coumaricacid, rutin,eriodictyol,cinnamicacid, and

apigenin,werenotidentiﬁedinanyoftheextracts.

Table3

Phenoliccomponentsofinthetestedextracts(␮g/gextract)*_.

Phenoliccompounds Root-EA Root-MeOH Root-Water Aerialpart-EA Aerialpart-MeOH Aerialpart-Water

Gallicacid 6±0.4 20±0.4 nd 12±0.4 22±0.4 nd Protocatecheuicacid nd nd 6±0.8 12±0.4 32±0.4 62±0.8 (+)-Catechin 74±2 72±2 nd 30±2 42±2 40±4 p-hydoxybenzoicacid nd nd nd 28±0.6 36±0.6 8±0.2 Chlorogenicacid nd 28±2 48±0.4 nd nd 186±4 Caffeicacid 106±4 94±4 18±0.6 56±4 50±4 16±0.6 Epicatechin nd nd nd nd nd nd Syringicacid nd 28±0.2 28±0.2 10±0.2 52±0.2 18±0.2 Vanilin 26±0.2 36±0.2 40±0.6 10±0.2 24±0.2 20±0.6 p-coumaricacid nd nd 42±0.2 60±1.8 60±1.8 48±1.4 Ferulicacid 172±6 106±6 366±10 126±6 90±6 198±10 Sinapicacid nd nd nd nd nd nd Benzoicacid nd nd nd nd nd nd o-coumaricacid nd nd nd nd nd nd Rutin nd nd nd nd nd nd Hesperidin 394±4 400±4 226±6 246±4 246±4 156±6 Rosmarinicacid 522±14 152±2 nd nd nd nd Eriodictyol nd nd nd nd nd nd Cinnamicacid nd nd nd nd nd nd Quercetin nd nd nd 132±4 198±4 nd Luteolin 852±56 214±6 92±6 76±6 110±6 nd Kaempferol nd nd nd 628±36 390±6 408±10 Apigenin nd nd nd nd nd nd nd:notdetected.

(5)

Table4

Antioxidantactivitiesofthetestedextracts.

Parts-Solvents DPPH (mgTE/gextract) ABTS (mgTE/gextract) CUPRAC (mgTE/gextract) FRAP (mgTE/gextract) Phosphomolybdenum (mmolTE/g) Metalchelating ability(mg EDTAE/g) Root-EA 34.85±3.03 82.56±2.14 114.82±2.76 89.59±2.47 1.91±0.11 na Root-MeOH 36.01±4.47 72.95±1.60 92.00±2.07 69.88±0.40 1.55±0.14 40.10±1.20 Root-Water 23.31±3.36 80.45±2.02 75.95±0.77 61.74±1.82 1.73±0.02 37.10±0.26 Aerialpart-EA 10.18±1.55 42.86±0.94 101.59±0.60 45.66±0.75 2.32±0.21 38.34±0.57

Aerialpart-MeOH 41.02±0.97 58.44±1.68 100.44±2.27 66.62±2.55 1.58±0.02 24.26±1.08

Aerialpart-Water 21.44±3.14 82.08±0.87 78.82±1.44 60.98±0.54 1.41±0.05 37.35±0.36

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.TE:Troloxequivalent;EDTAE:EDTAequivalent;na:notactive.EA:Ethylacetate.MeOH:Methanol.

3.2. Antioxidantactivity

Antioxidantsarecompoundswhichareproducedbybiological

systemsandoccurnaturallyinmanyfoods.Abalancebetween

oxi-dantsandantioxidantsisimportantforproperfunctioningofthe

body[49].Inthepresentstudy,severalantioxidantassayswere

performedtoobtainacomprehensiveevaluationoftheantioxidant

potentialofS.lucida,andincludedDPPH•andABTS•+scavenging,

FRAP,CUPRAC,phosphomolybdenumandmetalchelatingassays

(Table4).

TheDPPH•scavengingassayisanaccurate,easy,andeconomic

methodtoassess the radicalscavengingactivityof antioxidant

compounds,since theradicalcompound is stableand doesnot

needtobegenerated [49]. Thepresentstudy revealedthatthe

methanolicextractoftheaerialpartsofS.lucidadisplayedthe

high-estDPPH•scavengingeffect(41.02mgTE/gextract)followedbythe

rootmethanolicextract(36.01mgTE/gextract).Anotherwidely

usedassaytoassesstheanti-radicalpropertyofplantextractsis

theABTS•+_scavenging_assay._In_this_method,_the_blue/green

rad-icalABTS•+_,_which _is_generated_by_the_oxidation_of_ABTS•+ _with

potassiumpersulfate,isreducedbyantioxidants,andtheextentof

decolorizationcanthenbedetermined[50].Amongthedifferent

solventextractsoftherootofS.lucida,theethylacetateextract

wasthemosteffectiveABTS•+scavenger(82.56mgTE/gextract)

whileamongtheextractsoftheaerialparts,theaqueousextract

showedthestrongestactivity(82.08mgTE/gextract).

The CUPRAC method uses the

bis(2,9-dimethyl-1,10-phenanthroline: neocuproine)Cu(II) chelate cation as the

chromogenicoxidant,whichisreducedinthepresenceof

antiox-idantmolecules to thecuprous neocuproine chelate [Cu(I)–Nc]

withamaximumlightabsorptionat450nm[51].Inthecurrent

study,wefoundthattheethylacetaterootextracthadthehighest

cupricreducing power,followedbythatoftheaerialpartsofS.

lucida(114.82and101.59mgTE/gextract,respectively).Onthe

otherhand,theaqueousextractsofbothpartsdisplayedthelowest

activity. Another assay commonly used to assess the reducing

power of natural products is the FRAP assay, which measures

thereductionofaferricsalttoaferrouscomplex(bluecolored)

byantioxidantsunderacidiccondition(pH3.6).Theincreasein

absorbance(A)at593nmismeasuredandcomparedwithAof

aFe(II)standardsolution[52].Thepresentinvestigationrevealed

thatamongthedifferentsolventextractsoftherootofS.lucida,

the ethyl acetate extract showed the strongest ferric reducing

power(89.59mgTE/gextract).Asfortheaerialparts,themethanol

extractshowedthehighestactivity(66.62mgTE/gextract).

Additionally,wealsodeterminedthetotalantioxidant

capac-ityoftheextractsusingthephosphomolybdenumassay.Asshown

inTable4,theethylacetateextractoftheaerialpartsofS.lucida

followedbythatoftherootdisplayedthehighest activity(2.32

and1.91mmoLTE/gextract,respectively).Furthermore,thereisa

growingnumberofevidencethattoxicandcarcinogenicmetalsare

abletointeractwithnuclearproteinsandDNAcausingoxidative

deteriorationofbiologicalmacromolecules[53].Themetal

chelat-ingactivityofS.lucidaisshowninTable4.Itwasobservedthatthe

rootmethanolicextractofS.lucidafollowedbytheethylacetate

extractof theaerialpartsexhibited thehighest metalchelating

effect(40.10and38.34mgEDTAE/gextract,respectively).No

activ-itywasdisplayedbytheethylacetateextractoftheroot.

Fromtheaboveobservation,itwasnotedthatthehigh

antiox-idantactivityoftheethylacetateextractoftherootintheABTS•+

scavenging,CUPRAC,FRAP,andphosphomolybdenumassay,was

proportionalwithitsamountofTPCsincetheextracthadthe

high-estTPCcomparedtoitscounterpartsolventextract,methanoland

water. Indeed,a numberof previousstudieshaveobserved the

positivecorrelationofTPCandtheabove-mentionedantioxidant

mechanisms[54–57].Inaddition,twocompounds,rosmarinicacid

andluteolin,werefoundtobepresentinhighestamountintheroot

ethylacetateextract.Thesecompoundsareknowntobeeffective

antioxidantsasprovedbyseveralresearchers[58–60].

Incontrast,althoughtheethylacetateextractoftheaerialparts

containedthelowestTPC,itshighestlevelofﬂavonoidmightbe

responsibleforitsstrongestactivityintheCUPRAC,

phosphomolyb-denum,andmetalchelatingassay.Severalreportshavealsofound

thisrelationshipbetweentheamountofﬂavonoidofplantextracts

andtheirantioxidantpowerintherespectiveassays[61,62].Itis

alsoimportanttohighlightthattheﬂavonoidkaempferolwasmost

abundantintheethylacetateextractoftheaerialpartsanda

num-berofpreviousstudieshavefoundthatthiscompoundisapotent

antioxidant[63–65].

3.3. Enzymeinhibitoryactivity

Enzyme inhibition is one way in which enzyme activity is

regulated. Most therapeutic drugs acts by inhibiting a speciﬁc

enzyme.Inthepresentstudy,theenzymeinhibitoryactivityofS.

lucidaextractswastestedagainstcholinesterases(AChEandBChE),

tyrosinase,␣-amylase,and␣-glucosidase(Table5).

Recently,AChEinhibitorshavebeenfoundtobeimportantin

improvingtheunderlyingcholinergicsystemdeﬁcitsin

neurode-generativedisorders suchasAlzheimer’sdisease and Dementia

withLewybodies[66].AChEinhibitorsincludingdonepezil,

galan-tamine,andrivastigmine,andtheN-methyl-d-aspartateantagonist

memantinearethecurrentlyavailablepharmacologicalagentsto

modifytheclinicalmanifestationsofAlzheimerdisease[67].

Simi-larly,theinhibitionofBChEhasbecomeastandardapproachinthe

symptomatictreatmentoftheseneurodegenerativediseases.Inour

study,itwasobservedthattheethylacetateextractoftherootsand

aerialpartsofS.lucidahadthehighestAChEinhibition(3.99and

3.60mgGALAE/gextract,respectively)whiletheiraqueousextracts

exertedthelowestactivity.WithregardstoBChEinhibition,the

strongesteffectwasdisplayedbytheethylacetateextractofthe

aerialparts(6.32mg GALAE/gextract)comparedtoits

counter-partsolventextracts.NoBChEinhibitoryactivitywasdisplayedby

theaqueousextractoftheaerialparts.Ontheotherhand,among

(6)

Table5

Enzymeinhibitorypropertiesofthetestedextracts.

Parts-Solvents AChE (mgGALAE/gextract) BChE (mgGALAE/gextract) Tyrosinase (mgKAE/gextract) ␣-amylase(mmol ACAE/gextract) ␣-glucosidase(mmol ACAE/gextract) Root-EA 3.99±0.17 4.63±0.58 112.96±7.16 0.81±0.03 18.54±0.82 Root-MeOH 3.71±0.05 5.49±0.50 138.29±0.87 0.69±0.07 2.64±0.39 Root-Water 0.39±0.02 0.61±0.14 34.00±1.44 0.15±0.01 na Aerialpart-EA 3.60±0.08 6.32±0.27 133.11±1.40 0.83±0.02 11.78±0.48

Aerialpart-MeOH 3.32±0.08 2.98±0.16 134.18±0.51 0.51±0.02 12.18±1.30

Aerialpart-Water 0.29±0.07 na 17.30±2.40 0.11±0.01 0.57±0.02

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.GALAE:Galatamineequivalent;KAE:Kojicacidequivalent;ACAE:Acarboseequivalent;na:notactive. EA:Ethylacetate.MeOH:Methanol.

inhibitor(5.49mgGALAE/gextract).SimilartoAChEinhibition,the

aqueousrootextractexhibitedthelowestBChEinhibitoryactivity.

In addition, the reduction of postprandial hyperglycemia

throughretarding carbohydratesdigesting enzymes,namely

␣-amylase and ␣-glucosidase, has become a major therapeutic

approachinthemanagementofdiabetes[68].AsshowninTable5,

theethylacetateextractoftheaerialpartsandrootsofS.lucida

werethemosteffective␣-amylaseinhibitors(0.83and0.81mmoL

ACAE/gextract,respectively).Asfor␣-glucosidaseinhibition,the

highesteffectwasexhibitedbytheethylacetateextractoftheroot

(18.54mmoL ACAE/gextract)followed bythemethanol extract

oftheaerialparts(12.18mmoLACAE/gextract).Incontrast,the

weakest␣-amylaseand␣-glucosidaseinhibitorsweretheaqueous

extracts.

Tyrosinase inhibition has also become a strategy to

pre-ventexcessformationandaccumulationofmelaninintheskin,

therebypreventinghyperpigmentationdisorderssuchasmelasma,

agespots[69].AmongthesolventextractsoftherootofS.lucida,the

methanolextractoftheaerialpartsdisplayedthehighest

tyrosi-naseinhibition(138.29mgKAE/gextract)followedbytheethyl

acetateandaqueousextract.Similarly,themosteffective

tyrosi-naseinhibitoramongthesolventextractsoftheaerialpartswas

themethanolone(134.18mgKAE/gextract)followedbytheethyl

acetateandaqueousextract.

Fromtheaboveﬁndings,wefoundthattheethylacetateextract

oftheroot,whichdisplayedthehighestTPC,wasthemost

effec-tiveinhibitoroftheenzymesAChE,␣-amylase,and␣-glucosidase,

whiletheextractwiththehighestTFC,theethylacetateextractof

theaerialparts,showedthestrongestinhibitionofAChE,BChE,and

␣-amylase.Indeed,theenzymeinhibitorypropertiesofplant

phe-noliccompoundshavebeenreportedinliterature[70,71].Also,the

highenzymeinhibitoryactivityoftheethylacetateextractstend

torevealthegreatersolubilityandhencemore efﬁcient

extrac-tionofS.lucidabioactivecompounds,possessingenzymeinhibitory

properties,inethylacetatecomparedtotheothersolvents.With

regardstotyrosinaseinhibition,althoughthemethanolicextract

ofbothrootandaerialpartshadthehighestactivity,onlytheroot

methanolicextractdisplayedthehighest TFC,whilethat ofthe

aerialpartdidnotrevealthehighestTPCandTFC.Fromthispoint,

ourﬁndingscouldbeexplainedbythepresenceofnon-phenolic

inhibitorsin thisextract. It shouldbenoted thatthe biological

potentialofaplantextractisnotonlybecauseofitsbioactive

com-poundspresentathighconcentrations,butcanbeasaresultof

interactionsbetweentheplethoraofcompoundswhichmay

pro-duceasynergisticeffect,resultinginanactivitygreaterthanthe

sumoftheindividualeffects[72].

3.4. Insilicoevaluation

Theintegrationofcomputationalandexperimentalstrategies

haveplayedagreatsigniﬁcanceinmoderndrugdesignthrough

theidentiﬁcationanddevelopmentofnovelpromisingcompounds.

Moleculardockingandmechanicexploretheligandconformations

adoptedwithinthebindingsitesofmacromoleculartargetsand

alsoestimatestheligand-receptorbindingfreeenergybythe

eval-uationofthecriticalphenomenaimplicatedintheintermolecular

recognitionprocess[73].

Takinginconsiderationthecompositionofeachextractandthe

quantityof eachcompound found,themostrelevantmolecules

wereselected forthe docking studyoneach enzyme. Thebest

dockingscores(calculatedbyGlide)andGofbindinghavebeen

calculatedby molecularmechanic (MM/GBSA)method,andthe

resultsarereportedinTable1.Basingourdiscussiononthemost

relevantenzymaticinhibitoryactivityfoundfortyrosinase,wehave

deeplyanalyzedtheinteractionofrosmarinicandhesperidinwith

thisenzyme.

Consideringliteraturedataandtherelativecompositionofthe

rootextracts(ethylacetateandmethanol)andthatoftheaerial

parts(ethylacetateandmethanol),themostrelevantsubstances

arehesperidin,rosmarinicacid, luteolin,kaempferol. We

evalu-atedthedockingvaluesofthesesubstancesdockedontyrosinase,

obtainingthebest docking score valuefor rosmarinic acidand

hesperidin(Fig.1).Fromadetailedliteratureanalysis,wefound

evidencesthatrosmarinicacidpossessesagoodinhibitoryactivity

versustyrosinasewhichisconﬁrmedbytheworkofLeeetal[74].

Ananalogueamountofdatawasfoundforhesperidin;indeedthe

inhibitoryeffectofhesperidintowardtyrosinaseiswellknownin

literature[74].

In order tobetter studythe docking behavior ofrosmarinic

acidandhesperidinboundtotyrosinase,moleculardynamicsrun

wasconductedonthebestposeofhesperidinandrosmarinicacid

dockedtotyrosinase.Theexperimentsconductedover10nswas

then analyzedand theRMSDgraph wasplotted and compared

betweenthetwosubstances(Fig.2).Itisevidentthatboth

sub-stancesarestableinthebindingpocketofthetyrosinasefortheﬁrst

twonanosecondsandafterthisperiod,bothsubstancesstartedto

losetheirinitialposeandmovinginthecavity.Rosmarinicacid

sta-bilizeditsbindingposetoanotherconformationandkeptthisnew

posefortherestofthesimulation,whereashesperidinwasmore

unstableinthebindingpocket,andtheﬁnalposedifferedtothe

startingpointofmorethan10angstroms.Thisdifferentbehavior

couldbeinterpretedasadifferentafﬁnityofthetwosubstancesfor

theenzymaticcavity,alsodemonstratedbytheGvaluesreported

inTable1,calculatedbymolecularmechanicsimulation,whichare

inagreementwiththeseresults.

Alltogether,theseevidences,analyzedwiththeaimtoexplain

theoverallactivityofthesixextracts,areingoodagreementwith

theinvitroandinsilicoactivityreportedinthispaper.Indeed,the

presenceofrosmarinicacid[75],luteolin[76,77],hesperidin[78],

kaempferol[79]aresufﬁcienttoexplainthemodesttogoodactivity

foundalsofor␣-glucosidaseandforthecholinesteraseenzymes.It

isworthnotingthatthecomplexityofthechemicalcompositionof

theextracts,intermsofbioactivecompoundscontained,may

con-curtoproducesynergisticoradditive,speciﬁcinhibitoryproperties

(7)

Fig.1.BestposeandinteractiondiagramsofHesperidin(AandB)andRosmarinicacid(CandD)dockedtoTyrosinase.

Fig.2.Protein-LigandRMSDofHesperidinandRosmarinicAciddockedto tyrosi-nase,calculatedin10nsofmoleculardynamics.Thex-axisscaleisexpressedin 0.01ns,they-axisisexpressedinangstromsandrepresenttheRMSDfromthe startingpose.

3.5. Cellassays

Fortheevaluationoftheinvitroanti-inﬂammatoryactivityof

theextracts,nontoxicconcentrations,thatis,thosewhichallowed

acellularviabilityofatleast80%,wereappliedtoLPS-stimulated

RAW264.7macrophagecells,andtheproductionofNOwas

mea-Table6

Cellularviability(%)andNOproduction(%)ofRAW264.7macrophagesincubated withS.lucidamethanolextracts*_.

Concentration (␮g/mL)

Cellularviability(%) NOproduction(%)

Root Aerialpart Root Aerialpart

3.9 141±4 152±7 134±3 142±6 7.8 133±7 131±5.8 132±2 135±4 15.6 99.0±8.6 107±7 127±4 125±3 31.25 90.8±2.5 88.0±8.6 95.0±0.3 88.2±2.3 62.5 75.1±2.5 68.2±1.8 nd nd 125 58.4±2.9 39.6±1.9 nd nd

*_Values_expressed_are_means_±_S.D._of_three_parallel_{measurements.}_nd:_not determined.

sured by the Griess assay. LPS is an endotoxin responsible for

septicshocksyndromethatfuelstheproductionofinﬂammatory

mediators,e.g.NOwhichisassociatedwiththeexpressionof

pro-inﬂammatoryproteins,namelyiNOSandcyclooxygenase(COX-2)

[81].AdecreaseintheNOproductionthusindicatesthepotential

anti-inﬂammatorypropertiesoftheextract.

Inthiswork,methanolextractsfromrootsandaerialpartsofS.

lucidasigniﬁcantlyreducedtheviabilityofRAW264.7cellsat

(8)

themaximumconcentrationusedintheanti-inﬂammatoryassay.

Theobservedcytotoxicityisinaccordancewithseveralreportson

thecytotoxiceffects ofdifferentScrophulariaspecies onseveral

celllines,includingS.lucidaextractstowardsHL-60promyelocytic

leukemiacellsandontumorinvasivenessinvitro[27].Theextracts

werenotabletosigniﬁcantlyreducetheNOproductionin

LPS-stimulatedcells.Theinhibitiondisplayedbytherootandaerialpart

extractswere4.99%and10.77%,respectively,at31.25␮g/mL

con-centration(Table6).However,thepositivecontrolhadexcellent

reductionpotentialofNO(IC50=27.81␮g/mL).

The Scrophularia genus includes several species with

anti-inﬂammatoryproperties[82,83],whichareusuallyattributedto

thepresenceofiridoidsandphenylpropanoids[82].ThelackofNO

reducingcapacityobservedinthisworkinthemethanolextracts

of S. lucida does not necessarily indicate the absence of

anti-inﬂammatorypropertiesofthespecies.ApartfromNOreduction,

thereareothermoleculartargetstocontrolinﬂammation,

includ-ingtumor necrosis factor-␣,interleukins and cyclooxygenase-2

[84].Moreover,theconcentrationstestedmightnotbesufﬁcient

toinduce an anti-inﬂammatory response, since a hightoxicity

wasobtainedaftertreatingcellswithconcentrationshigherthan

31.25_␮g/mL.Inthiscontext,assaysarebeingpursuedinorderto

ascertaintheanti-inﬂammatorypotentialoftheextractsbyother

cellularmodels,andafterfractionation,withtheaimtoconcentrate

theactivecomponents.

4. Conclusion

Inthis study,we foundthat thedifferentsolventextractsof

S.lucidavariedintheirbiologicalpotency.Therootethylacetate

extracttendtoshowthehighestactivityinmostassays,

display-ingthestrongestABTS•+scavenging,FRAP,CUPRAC,andinhibitory

activity against AChE and ␣-glucosidase. Highest TPC and TFC

wereobservedintheethylacetateextractoftherootandaerial

parts, respectively. The most abundantcompounds in the root

ethylacetateextractwereluteolin,rosmarinicacid,andhesperidin,

whilekaempferolwasmostabundantintheethylacetateextract

oftheaerialparts.Rosmarinicacidandhesperidinshowedthebest

dockingscorevalueontyrosinase.Thepresentﬁndingstendto

sup-portthepotentialuseofS.lucidainthemanagementofseveral

chronicdiseases,nonetheless,needfurtherinvivostudiestogether

withtoxicologicalanalysistoestablishitssafetyproﬁle.Itisalso

recommendedthatfurtherstudiesaimtodetermine/isolate

com-poundssuchasphenlypraponoidglycosides,byfurtheranalytical

techniques(HPLC-MSorNMR).

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