HPLC-FRAP methodology and biological activities of different stem bark extracts of Cajanus cajan (L.) Millsp

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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

HPLC-FRAP

methodology

and

biological

activities

of

different

stem

bark

extracts

of

Cajanus

cajan

(L.)

Millsp

Kouadio

Ibrahime

Sinan

a

_,

_Mohamad

_Fawzi

_Mahomoodally

b,∗

_,

_Ozan

_Emre

_Eyupoglu

c

_,

Ouattara

Katinan

Etienne

d

_,

_Nabeelah

_Bibi

_Sadeer

b

_,

_Gunes

_Ak

a

_,

_Tapan

_Behl

e

_,

Gokhan

Zengin

a,∗

a_Department_of_Biology,_Science_Faculty,_Selcuk_University,_Campus,_Konya,_Turkey

b_Department_of_Health_Sciences,_Faculty_of_Medicine_and_Health_Sciences,_University_of_Mauritius,₂₃₀_Réduit,_Mauritius c_Department_of_{Biochemistry,}_School_of_Pharmacy,_Istanbul_Medipol_University,_Turkey

d_Laboratoire_de_Botanique,_UFR_Biosciences,_Université_Félix_{Houphouët-Boigny,}_Abidjan,_Cote_d’Ivoire e_Chitkara_College_of_Pharmacy,_Chitkara_University,_Punjab,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2September2020 Receivedinrevisedform 28September2020 Accepted5October2020 Availableonline12October2020 Keywords: Antioxidants Bioactivecompounds Pigeonpea Enzymeinhibition Naturalproducts

a

b

s

t

r

a

c

t

Cajanuscajan.(L.)Millsp.(C.cajan)(Family:Fabaceae)alsoknownaspigeonpea,isafamousfoodand cover/foragecropbearingahighamountofkeyaminoacids(methionine,lysineandtryptophan).This studyinvestigatedintothetotalphenolic(TPC),ﬂavonoidcontent(TFC),antioxidant [2,2-diphenyl-1-picrylhydrazyl(DPPH),2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)(ABTS),ferricreducing antioxidantpower(FRAP),cupricreducingantioxidantcapacity,totalantioxidantcapacity(TAC) (phos-phomolybdenum)andmetalchelating]activitiesandenzyme[␣-amylase,␣-glucosidase,tyrosinase, acetyl-(AChE),butyryl-(BChE)cholinesterase]inhibitoryeffectsoffourextracts(methanol,hexane,ethyl acetate,aqueous) preparedfromC. cajanstem bark.Directidentiﬁcation ofantioxidants was also conductedusingthehighperformanceliquidchromatography-ferricreducingantioxidantpower (HPLC-FRAP)system.ThehighestTPCandTFCwererecordedwiththemethanolic(23.22±0.17mgGAE/g) andethylacetateextracts(19.43±0.24mgRE/g),respectively.Themethanolicextractexhibited impor-tantantioxidantactivitywithDPPH(38.41±0.05mgTroloxequivalent(TE)/g),ABTS(70.49±3.62mg TE/g),CUPRAC(81.86±2.40mgTE/g),FRAP(42.96±0.59mgTE/g)andmetalchelating(17.00±1.26 mgethylenediaminetetraaceticacidequivalent/g).p-coumaricandcaffeicacidwerethepredominant antioxidantsinthesamples.Resultsfromenzymaticassaysshowedthepotentialabilitiesofhexane extractininhibitingtheAChE,BChE,␣-amylaseand␣-glucosidaseenzymes.Fromtheresultsobtained inthisstudy,itcanbeconcludedthatC.cajancanbeconsideredasapromisingsourceofantioxidants andkeyenzymeinhibitorsthatcanbeexploitedforfuturebioproductdevelopment.

1. Introduction

Oxidative stress is one of the most burning topics among

researchers around the globe as it is the underlying causative

factorofnumerouschronicdiseasesnamelycancer,

neurodegener-ativedisorders,cardiovasculardiseases,dermatologicalproblems,

diabetes mellitus, among others. Continuously searching for

antioxidantsshouldbeanongoingchallengeasithasbeenreported

thatantioxidantsmayhampertheoxidationprocesswhich

conse-∗ Correspondingauthors.

E-mailaddresses:f.mahomoodally@uom.ac.mu(M.F.Mahomoodally),

gokhanzengin@selcuk.edu.tr(G.Zengin).

quentlypreventorpostponeoxidativestressrelateddiseases[1].

Antioxidantsfromplantmaterialshavegarneredfair amountof

attentionfromresearchersaswellasthepublicbecauseoftheir

demonstratedefﬁcacyandsafety.Atpresent,mostoftheplant

sam-plesarescreenedinpursuitofnovelandnaturalantioxidantswith

substantiallyhighactivity.Forinstance,intheyear2019,

ScienceDi-rectrecorded12,901articlesrelatedtothescreeningofplantsin

termsoftheirantioxidantactivity.Tobeinlinewiththecurrent

trends,thepresent studyaims toscreen a nutritionally

impor-tantplant,Cajanuscajan(L.)Millsp.(C.cajan)(Family:Fabaceae)

alsoknownaspigeonpea,foritsantioxidativepropertyusingtwo

differenttechniquesnamelyspectrometryandchromatography.

Sinceamajorgapexistsinthecurrentinvitroantioxidantassays

aswelldetailedin arecentreview byBibiSadeeretal.[2],we

https://doi.org/10.1016/j.jpba.2020.113678

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alsoassessedtheantioxidantspresentinC.cajanusingarecently

developedsystem:highperformanceliquidchromatography-ferric

reducingantioxidantpower(HPLC-FRAP)forthedirect

identiﬁca-tionofantioxidantsinthecomplexmatricesofplantsamples.

TheHPLC-FRAPsystemhasbeenpopularlyusedbynumerous

researcherseliminatingtedioussamplepre-treatmentprocedures

andproved tobeefﬁcientand rapid [3]. Accordingtoprevious

reports,in vitroantioxidantassays suggested that C.cajanmay

beconsideredasanimportant naturalantioxidant source[4,5].

However,tothebestofourknowledge,therehasbeennoreport

abouttheproﬁling of antioxidant compoundsof C. cajanusing

HPLC-FRAP system.Thus, oneof theaims of thepresent study

istoidentifyantioxidantsin thestem barkof C.cajanbysuch

technique.Otherwise,thisCajanusspecies,beingaplantfood,is

commonlyusedin traditionalmedicine.For instance,theleafis

usedagainstdiabetes,dysentery,hepatitis,measlesandinChina

itisbelievedtotreatblood-relateddiseasesandalleviatepain[4].

InTrinidad,theleafisusedinfoodpoisoningandtoprevent

con-stipation[6].Cajanuscajanisahighlynutritiousperenniallegume

andagoodsourceofcrudeﬁbre,iron,sulphur,calcium,potassium,

manganeseandwater-solublevitamins[7].Morphologically,itis

anon-climbingshrub,erect,silkypubescentribbedstems,spindly

branches,leavespinnatelytrifoliolate,leaﬂetsinshortstalks,

ﬂow-ersborne oncorymbiform racemes,petals yellow,standard red

outsideandyellowinside,wingsyellow,oblonglinearpods

con-strictedbetweentheseeds[8].

Severalpharmacologicalvalidationsshowedthat pigeon pea

possessedimportantbiologicalproperties.Inaclinicalevaluation,

theextractofC.cajanwasreportedtoreducepainfulcrisesinsickle

cellanaemiaontheliver[9].Compoundsidentiﬁedintherootand

leafextractsdisplayedmoderateactivityagainstthe

chloroquine-sensitivePlasmodiumfalciparum strain 3D7 [10]. Liu et al. [11]

reportedthatthefourstilbenes(cajaninstilbeneacid,longistyline

A,longistylineC, andcajanolactone A)isolatedfromtheleafof

pigeonpeapossessedneuroprotectiveeffects.Anotherrecentstudy

showedthatanaturalstilbene(longistylinA)producedbyC.cajan

cansigniﬁcantlyinhibitthegrowthofmethicillin-resistant

Staphy-lococcusaureus[12].

Asevidencedbyanumberofpublications,important

pharma-cologicalpropertieshavebeenrecordedfromleavesandrootsofC.

cajan.Inaddition,thechemicalcompositionhasalsobeenreported.

However,nochemicalandbiologicalinformation’shavereported

fromthestembarkofC.cajan.Therefore,toaddressthisresearch

gapinthecorpusofscientiﬁcknowledgeonthisplant,theotheraim

ofthisstudywastovalidatetheuseofthisplantforitschemical

pro-ﬁleandbiologicalproperties.Takentogether,thepresentedresults

couldﬁllthegaponC.cajanstembarkwhichsubsequentlycould

opennewresearchavenues,particularlyindevelopment

therapeu-ticbioproductsdevelopment.

2. Materialsandmethods

2.1. Plantmaterialandextractionprocedure

ThestembarkofC.cajanwascollectedinthevillageofTenikro

(districtofYamoussoukro-Côted’Ivoire,6◦ 33 10 N,5◦15 11

W, 293 m) in the year 2019 and it was authenticated by the

botanist Ouattara Katinan Etienne(Université Félix Houphouet

Boigny,Abidjan,Côted’Ivoire).Theplantmaterials(aerialparts)

weredriedinshadeforabout10daysandthengroundedusinga

laboratorymill.

Macerationwasusedforsamplepreparationwithethylacetate,

hexaneandmethanolasextractionsolvents,separately.For this

purpose, 5 gof thesamples weremaceratedfor 24 hat room

temperature.Afterthis,theextractswereﬁlteredandthenthe

sol-ventswereremovedviaarotary-evaporator.Withrespecttothe

waterextracts,5goftheplantmaterialwereinfusedwith100mL

boiledwater.Thereafter,thewaterwasdriedusingfreeze-drying.

Allextractswerestoredat+4◦Cuntilfurtherstudies.

2.2. Proﬁleofbioactivecompounds

Thetotalphenolic (TPC),andﬂavonoid(TFC)contentsofthe

extractsweremeasuredanddetailedmethodsweredescribedin

ourpreviouspaper[13].Standards,namelygallicacid(GAE)for

phenolics,andrutin(RE)forﬂavonoids,wereusedtoexpressthe

results.

2.3. HPLC-Method

TheShimadzuLC-2010C-HTHPLCcompactsystem(Japan)used

fortheanalysisincludedthermostablecolumnunit,the

autosam-plerunit,degasser,gradientmanagerpumpandUV-Diodearray

detectors combined with a second supported on-line reagent

programmable single channel syringe pump (IPS 12-RS model,

Inovensolaboratorydevices,Turkey).Theexperimentswere

con-ductedusingaPurospherstarRP-18encapped,C18columnwith

guard column (4.6 × 250 mm,5 ␮m)(Merck, Germany). Data

procurement(peakarea,retentiontime)wasexecutedby

utiliz-ingShimadzuChemStationProgram.Theinjectionvolumeofall

sampleswas50␮L.Analysistimewas30min.Themobilephase

consistedoffoursolvents:solventAwasmethanol,solventBwas

amixtureofaceticacid/water/acetonitrile(0.5/49.75/49.75,v/v/v),

solventCwasamixtureof0.2%aceticacid:water(v/v)andsolvent

Dwasultra-puregradeacetonitrile[14].

HPLC-FRAPmethodology,oneofthemethodsthatallows

simul-taneousapplication ofHPLCseparation andantioxidantactivity

determination,wasused.DADsignalwassetat280nm(maximum

absorptionforphenoliccontents)andUVon-lineantioxidant

activ-itydetectionsignalwassetat595nm(maximumabsorptionafter

thepostcolumnFRAPreaction).Mobilephaseﬂowratewasset

at1.2mL/min.Theﬂowrateofthesyringepumpwassetat0.25

mL/min(optimizedvalue).Columntemperaturewassetatroom

temperature,25−30◦_C._The_reaction_coil,_which_made_of

polyte-traﬂuoroethylene (PTFE)tubing (0.25mm i.d.), wasadjustedas

2.5mlength(optimizedvalue)[14].TheFRAPreagentwas

pre-paredsuchasinthestudyofBenzieandStrain[15]andadaptedto

post-columnassay.ForHPLC-FRAPassay,FRAPreagentwasfreshly

prepared,drawnintothesyringe,coveredwithaluminiumfoiland

putintothesyringepump.HPLC-FRAPsystemreactswiththe

open-ingofthesplitterswitchsystemconnectedtothemanifoldinthe

1.5cmreactioncoilofthepigeonpeaplantsampleextracts

sepa-ratedaccordingtothepolaritydifferencewiththesolventﬂowfrom

themainpump,andthisreactionpeakswithextrapeakdetectionin

theUVdetectorbesidestheDADseparationwereanalysed(Fig.1).

Lyophilizedandpowdered(5mg)formsofC.cajanstembarks

forfourdifferentextracts(ethylacetate,hexane, methanol,and

infusion)weresolubilizedwithHPLCgradehighpuritymethanol

(10mL).Firstly,theywereﬁlteredthrougha0.45␮mﬁlterpriorto

HPLCanalysis.Theirﬁnalconcentrationswereadjustedto0.5mg/

mLbeforeHPLCanalysis.Ethylacetate,hexane,methanol,and

infu-sionextractsofC.cajanstembarkswasrunwithHPLC-FRAPsystem

atleastthreeparalleleachother.Limitofdetection(LOD)andlimit

ofquantitation(LOQ)aretheanalyticalprocessingargumentsof

ananalytethatcanbeaccuratelymeasuredbytheanalytical

pro-cedure.Forthisstudy,validationparametersaboutLODandLOQ

weredeterminedaccordingtotheInternationalConferenceon

Har-monization guidelines.While attheend of therunningoffour

differentextracts(ethylacetate,hexane,methanol,infusion)from

C.cajanstembarks,chromatogramswithpositiveFRAPpeakswere

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detec-Fig.1.HPLC-FRAPantioxidantanalysissystem.

tionlimitsofHPLC-FRAPmethodaboutphenolicacidcontentsofC.

cajanstembarksextractsweredeterminedas3timesand10times

oftheaveragestandarddeviationofnoise.Linearityofthemethod

wastestedintherange of0.4−9ppmfordetected12 phenolic

acids(p_−OHbenzoicacid,chlorogenicacid, caffeicacid,syringic

acid,ferulicacid,p-coumaricacid,vanillin,vanillicacid,rosmarinic

acid,gentisicacid,syringaldehyde,protocatechuicacid).TheLOD

andLOQdataobtainedfor280nmand595nmwascalculated.All

statisticalChemStationdetectionlimitanalysisdatawerereported

signiﬁcantly(p<0.05)withstandarddeviation.TherearenoFRAP

peakequivalentsofp−OHbenzoicacid,p-coumaricacid,vanillin

andvanillicacid

2.4. Determinationofantioxidantandenzymeinhibitoryeffects

To detect antioxidant properties, we used several chemical

assays with different mechanisms namely, radical scavenging,

reducingpowerandmetalchelating.Trolox(TE)and

ethylenedi-aminetetraaceticacid(EDTA)wereusedasstandardantioxidant

compounds. Obtained resultswere expressedas equivalents of

thesecompoundsGrochowskietal.[16].Todetectinhibitoryeffects

onenzymes,weusedcolorimetricenzymeinhibitionassaysand

theseassays includedtyrosinase, ␣-glucosidase,␣-amylase and

cholinesterases.Somestandardinhibitors(galantamine,kojicacid

andacarbose)wereusedaspositivecontrols.

2.5. Dataanalysis

Allexperimentswereperformedintriplicateandresultswere

aggregatedandgaveasmean±SDstandarddeviation.Datawere

statisticallyanalysedbyusingOne-wayanalysisofvariance,

prin-cipalcomponentandhierarchicalclusteredanalysisrespectively.R

v3.6.2softwarewasusedfortheanalysis.

Table1

TotalbioactivecomponentsofC.cajanstembark.

Extracts Totalphenolic content(mgGAE/g extract) Totalﬂavonoid content(mgRE/g extract) Hexane 5.55±0.08d _1.25_±_0.03c EA 21.69±0.30b _19.43_±_0.24a MeOH 23.22±0.17a _11.49_±_0.06b Infusion 19.14±0.18c _1.51_±_0.23c

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.GAE:Gallic acidequivalent;RE:Rutinequivalent;EA:Ethylacetate;MeOH:Methanol.Different superscriptsindicatesigniﬁcantdifferencesintheextracts(p<0.05).

3. Resultsanddiscussion

3.1. Totalbioactivecompoundsandinvitroantioxidant properties

Phenoliccompoundsarewidelydistributedinplantfoods.In addition to fruits,coloured vegetablesand spices,legumes are equallyrichinphenoliccompounds.Asamatteroffact,C.cajanis expectedtobeaboundedbypolyphenolsasitisahighlynutritious legumeandthishasindeedbeenconﬁrmedseveraltimesby pre-viouspublicationsindicatingthattheseedcontainedhighquantity ofphenolic,ﬂavonoid,andtannincontents[7].Sincelittleisknown

onthephytochemicalproﬁleofstembarkoftheplantofinterest,

thepresentstudyattemptedtoquantifythetotalphenolic(TPC)

andtotalﬂavonoidcontent(TFC)ofdifferentextractsofstembark.

ResultsareshowninTable1.Incontrasttoseedswherebysome

studiesrevealedlow TPC rangingfrom1.054to18.3mgGAE/g

[17,18],thedatapresentedhereinshowedrelativelyhigher

quan-tityofphenolicandﬂavonoidcontentsinstembark.Forinstance,

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Table2

AntioxidantactivitiesofC.cajanstembark.

Extracts DPPH(mgTE/g extract) ABTS(mgTE/g extract) CUPRAC(mgTE/g extract) FRAP(mgTE/g extract) Phosphomolybdenum (mmolTE/g) Metalchelating ability(mg EDTAE/g) Hexane na 4.75±0.32d _13.48_±_0.30d _8.54_±_0.19d _0.31_±_0.02d _6.52_±_0.29c EA 12.39±0.09c _22.45_±_0.70c _72.09_±_1.40b _25.91_±_0.44c _1.68_±_0.09a _10.24_±_0.03b MeOH 38.41±0.05a _70.49_±_3.62a _81.86_±_2.40a _42.96_±_0.59a _1.32_±_0.06b _17.00_±_1.26a Infusion 25.84±0.64b _64.40_±_1.09b _53.51_±_0.34c _36.43_±_0.08b _1.00_±_0.05c _10.16_±_0.69b

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.TE:Troloxequivalent;EDTAE:EDTAequivalent;EA:Ethylacetate;MeOH:Methanol;na:notactive. Differentsuperscriptsindicatesigniﬁcantdifferencesintheextracts(p<0.05).

(23.22_±0.17mgGAE/g)andethylacetateextract(19.43_±0.24mg RE/g),respectively.However,thelowestyieldforbothTPCandTFC wasobtainedfromthehexaneextracts(5.55±0.08mgGAE/gand 1.25±0.03mgRE/g,respectively).

Eversinceepidemiologicalstudiescorrelatedietsrichin nat-uralantioxidantswithadecreasedriskofoxidativestress-related diseases,phenoliccompoundsandtheirantioxidantactivitieshave beenofgreatconcerntobothconsumersandresearcherssincethe pastdecades[19].Duetothiskeeninterest,assessmentof

antiox-idantspresentintheextractsofC.cajanwereconductedherein.

Sincethereisstillnouniversalmethodtodetermineantioxidative

powerinvitro,sixdifferenttestsinvolvingdifferentmechanismof

antioxidantdefencesystemwereconductedtoevaluatethe

antiox-idantactivityofpigeonpea. Forinstance,DPPHand ABTSwere

performedtoevaluatethefreeradicalscavengingcapacity ofC.

cajan,FRAPandCUPRACtoinvestigateintothereducingpotential,

phosphomolybdenumtodeterminetotalantioxidantcapacityand

ferrousionchelatingassaytomeasurethechelatingabilityofthe

extracts.ResultsareshowninTable2.

Overall, it can be observed that a good correlation existed

between the different assays. Additionally, extracts with high

antioxidantactivitywereassociatedwithhighphenoliccontents.

Forinstance,methanolicextractyieldingthehighestTPC,exhibited

thehighestactivityinﬁveassaysnamelyDPPH(38.41±0.05mg

TE/g),ABTS(70.49±3.62mgTE/g),FRAP(42.96±0.59mgTE/g),

CUPRAC(81.86±2.40mgTE/g)andmetalchelating(17.00±1.26

mgEDTAE/g).Scientistshaveclaimedthatextractsobtainedfrom

highlypolarsolvents,especiallymethanol,havehighantioxidant

effects[20],hencesupportingourresults.Suchresponseiscommon

amongplantextractsandwasobservedwithseveralotherstudies

[21,22].Ontheotherhand,hexaneextractpossessingthelowest

TPC,displayedthelowestantioxidativepowerwithallsixmethods

(DPPH:inactive;ABTS:4.75±0.32mgTE/g;FRAP:8.54±0.19mg

TE/g;CUPRAC:13.48±0.30mgTE/g;phosphomolybdenum:0.31

±0.02mmolTE/gandmetalchelating:6.52±0.29mgEDTAE/g).

3.2. -HPLC-FRAPanalysis

Tofurtherscrutinizeourextractsintermsofantioxidantsand

bearinginmindthatthecurrentinvitroantioxidantassaysdonot

alwaysgiverealisticresults[2],wealsoscreenedoursamplesusing

amoreextensivetechniquesuchasHPLC-FRAP.

Antioxidantactivitydetermination,correspondingtotheirferric

reducingidentity,wasbased ontheability ofantioxidant

com-poundstoreduceFe3+_to_Fe2+_._Therefore,_the_research_group_of

ShifromChinasuggestedthatHPLC–FRAPassaycouldbedesigned

bydetectingthebluecolouredFe2+_-TPTZ_complex_at₅₉₃_nm_[₃_].

OntheHPLC-FRAPsystem,morephenolicpeaksweredetectedas

positivepeaksat280nmcomparedat595nm.Forinstance,for

sample1(ethylacetate)sevenphenolic peaksweredetectedat

280nmwhilefourat595nm,forsample2(hexane)eightphenolic

peaksweredetectedat280nmwhilefourat595nm,forsample3

(methanol)ﬁvepeakswereconﬁrmedwhereastwoweredetected

aspositiveat595nmandforsample4(infusion)fourpeakswere

detectedat280nmbutonlytwowereconﬁrmedat595nm.

Iden-tiﬁcationofatotalof12differentantioxidants(suchasp-coumaric

acid,p−OHbenzoicacid,syringicacid,ferulicacid,vanillin,caffeic

acid,chlorogenicacid,syringaldehyde,protocatechuicacid,vanillic

acid,rosmarinicacidandgentisicacid)wasachievedat280nmby

systematicanalysisoftheirretentiontime(Tables3–6)fromthe

foursamplesofC.cajan.Thechemicalstructuresoftheidentiﬁed

antioxidantsareillustratedinFig.2.TheHPLCchromatogramsare

giveninFigs.3–6.

Insample1,thesevenantioxidantsdetectedat280nmwere

p-coumaricacid(or4-hydroxycinnamicacid),p−OHbenzoicacid

(or4-hydroxybenzoicacid),syringicacid,ferulicacid,vanillin,

caf-feicacidandchlorogenicacid.At280nm,p-coumaricacidwasthe

predominantantioxidantwitha peakareaof247.3.At595nm,

thepeaksofp−OHbenzoicandp-coumaricacidwereabsentand

caffeicacidwasmostpredominant(210.3).Interestingly,thesame

predominantantioxidantcompoundswerefoundinabundancein

sample3wherebythepeakareaofp-coumaricacidwas213.5at

280nmandthatofcaffeicacidwas227.6at595nm.Despitethis

similarityinthemainantioxidantcompounds,theinvitrorevealed

differentantioxidantactivities.Inadditiontothefactthatinvitro

assaysdonotalwaysgiverealisticresults,thisdifferencecanalso

belinkedtothechemicalstructuresofthephenolicantioxidants.

Manystudieshaveassociatedthechemicalstructuresof

phe-noliccompoundswithhighantioxidantpower.Basically,phenolic

compoundscontainoneormorehydroxylgroups(−OH)attached

toabenzenering[23].Theantioxidantactivitycentreofphenolic

acidsisphenolichydroxyl(−OH),thusthenumberandpositionof

phenolichydroxylshaveadirectlinkwiththeantioxidantactivity

ofphenolic acids[24].Furthermore,methoxy(_−OCH3)and

car-boxylicacid(−COOH)groupsalsohavesigniﬁcanteffectsonthe

antioxidantpropertyofphenolicacids[25].Inthisstudy,eight

typ-icalphenolicacidshavebeenidentiﬁedandtheyarep−OHbenzoic

acid,vanillicacid,protocatechuicacid,caffeicacid,ferulicacid,

p-coumaricacid,syringicacidandgentisicacid.Natellaetal.[26]

reportedthathydroxycinnamicacid(−CH=CHCOOH)hasstronger

antioxidantactivitythanhydroxybenzoicacid(−COOH)whenthe

othersubstituentsofthebenzeneringremainthesame[26].These

resultscanbeattributedtotheelectron-donatingability of

car-boxylicacidgroups.Theconjugation effectandinductioneffect

togetherdeterminethat−COOHisastrongelectron-withdrawing

groupand−CH=CHCOOHisaweakelectron-withdrawinggroup.

Anelectron-donatinggroupcanincreasetheelectronclouddensity

ofabenzenering,decreasingthedissociationenergyofthe

pheno-lichydroxylbondwhichconsequentlypromoteitsfreescavenging

ability.Therefore,itisspeculatedthatcarboxylicacidgroupsaffect

theantioxidantactivityofphenolicacidsbasedontheir

electron-donating ability (−CH = CHCOOH > −COOH) [27]. Nonetheless,

thesefactsdonotcorroboratewiththeinvitroresultsofsample2

(hexane).Forinstance,thesampleshowedlowornoactivitywith

ABTSandDPPHassaysdespiteﬁvephenolicacidswereidentiﬁed

init.

Inadditiontocarboxylicacidgroups,methoxygroupscanalso

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Table3

Detectionandquantitationlimitsofthepeaksdeﬁnedat280nmand595nmforhexaneextractfromC.cajanstembark.

Peak Numbers Component Name Retention Time(RT) (min.) PeakArea (280nm) (mAUx min.) Concent-ration(for 280nm) (ppm) PeakArea(595 nm)(mAUx min.) Concent-ration(for 595nm) (ppm) Limitof Detection (LOD,ppm, 280nm) Limitof Quantita-tion(LOQ, ppm,280 nm) Limitof Detection (LOD,ppm, 595nm) Limitof Quantita-tion(LOQ, ppm,595 nm) 1 Protocatechuic acid 8.1 115.5 12.2 92.8 15.4 1.2±0.03 4.0±0.04 1.0±0.03 3.3±0.04 2 Gentisicacid 11.9 68.8 7.3 77.6 12.9 0.4±0.02 1.3±0.02 0.6±0.02 2.0±0.03 3 Chlorogenic acid 13.9 158.3 16.7 230.3 38.3 1.6±0.03 5.3±0.04 2.8±0.03 9.2±0.04 4 P-OHbenzoic acid 15.3 280.7 29.6 * * 1.8±0.04 6.0±0.04 * * 5 Vanillicacid 17.5 83.5 8.8 * * 0.8±0.02 2.6±0.03 * * 6 Vanillin 20.3 78.6 8.3 * * 0.6±0.02 2.0±0.03 * * 7 Syringaldehyde 23.2 86.6 9.1 200.4 33.3 0.9±0.02 3.0±0.04 2.5±0.03 8.2±0.04 8 p-Coumaric acid 24.9 75.2 7.9 * * 0.5±0.01 1.7±0.02 * *

±_SD:_Average_Standard_Deviation,₉₅_%_conﬁdence_interval,_critical_ratio:_p_<_0.05,_*:_{non-detected,}_ppm:_parts_per_million.

Table4

Detectionandquantitationlimitsofthepeaksdeﬁnedat280nmand595nmforethylacetateextractfromC.cajanstembark. Peak Numbers Component Name Retention Time(RT) (min.) PeakArea (280nm) (mAUx min.) Concentration (for280 nm)(ppm) PeakArea (595nm) (mAUx min.) Concentration (for595 nm)(ppm) Limitof Detection (LOD,ppm, 280nm) Limitof Quantita-tion(LOQ, ppm,280 nm) Limitof Detection (LOD,ppm, 595nm) Limitof Quantita-tion(LOQ, ppm,595 nm) 1 p-OH benzoic acid 9.2 31.5 3.8 * * 0.2±0.01 0.7±0.02 * * 2 Chlorogenic acid 10.4 130.8 15.6 87.6 15.6 0.8±0.02 2.6±0.04 0.6±0.02 2.0±0.03 3 Caffeicacid 12.6 178.3 21.3 210.3 37.4 1.2±0.03 4.0±0.04 1.4±0.03 4.6±0.04 4 Syringic acid 16.6 180.7 21.6 205.6 36.6 1.3±0.03 4.3±0.04 1.5±0.03 5.0±0.04 5 Ferulicacid 21.3 46.2 5.5 58.3 10.4 0.4±0.01 1.3±0.03 0.5±0.01 1.7±0.03 6 p-Coumaric acid 25.0 247.3 29.5 * * 1.6±0.03 5.3±0.04 * * 7 Vanillin 29.1 21.6 2.6 * * 0.1±0.01 0.3±0.01 * *

Table5

Detectionandquantitationlimitsofthepeaksdeﬁnedat280nmand595nmformethanolextractfromC.cajanstembark. Peak Numbers Component Name Retention Time(RT) (min.) PeakArea (280nm) (mAUx min.) Concentration (for280 nm)(ppm) PeakArea (595nm) (mAUx min.) Concentration (for595 nm)(ppm) Limitof Detection (LOD,ppm, 280nm) Limitof Quantita-tion(LOQ, ppm,280 nm) Limitof Detection (LOD,ppm, 595nm) Limitof Quantita-tion(LOQ, ppm,595 nm) 1 p-OH benzoic acid 11.8 71.5 10.3 * * 0.7±0.02 2.3±0.03 * * 2 Caffeicacid 13.9 168.8 24.4 227.6 70.4 1.9±0.03 6.3±0.04 2.4±0.03 7.9±0.04 3 Vanillic acid 15.1 198.3 28.6 * * 2.1±0.03 6.9±0.04 * * 4 Syringic acid 19.5 40.7 5.9 95.6 29.5 0.4±0.01 1.3±0.03 0.8±0.02 2.6±0.03 5 p-Coumaric acid 25.7 213.5 30.8 * * 2.2±0.03 7.3±0.04 * *

accordingtoa recentstudy,thehigherthenumberofmethoxy groupspresent,thehigheristheantioxidantactivityofphenolic acids[27].Itisworthmentioningthatthedifferencebetweenthe

chemicalstructureofvanillicacid(identiﬁedasmajorantioxidant

insample4)andp_−OHbenzoicacid(identiﬁedasmajor

antioxi-dantinsample2)isthepresenceofonemethoxy(−OCH3)group.

Since,−OCH3 increasesantioxidantpower,sample4canbesaid

tobestrongerthansample2intermsofantioxidantactivity.This

ﬁndingisconsistentwiththeinvitroresultswhichalsorevealed

sample4tobestrongerthansample2.

Since−CH=CHCOOH >−COOHin termsofelectron-donating

ability,bothsamples1and3arethusconsideredtobestrongerthan

sample2intermsofantioxidantactivityduetothepredominant

presenceofp-coumaricacidinsamples1and3whichcontainsthe

−CH=CHCOOHgroupattachedtothephenolgroupwhilep_−OH

benzoicacid,presentinmajorityinsample2,doesnotpossessthe

−CH=CHCOOHgroupbutinsteadthe−COOHgroup.However,this

resultisnotinlinewiththeinvitroﬁndings.Instead,invitroassays

classiﬁedonlysample3asthestrongestantioxidant.Itisimportant

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antioxi-Table6

Detectionandquantitationlimitsofthepeaksdeﬁnedat280nmand595nmforinfusionfromC.cajanstembark.

Peak Numbers Component Name Retention Time(RT) (min.) PeakArea (280nm) (mAUx min.) Concentration (for280 nm)(ppm) PeakArea (595nm) (mAUx min.) Concentration (for595 nm)(ppm) Limitof Detection (LOD,ppm, 280nm) Limitof Quantita-tion(LOQ, ppm,280 nm) Limitof Detection (LOD,ppm, 595nm) Limitof Quantita-tion(LOQ, ppm,595 nm) 1 p-OH benzoic acid 11.8 75.5 16.8 * * 0.7±0.02 2.3±0.03 * * 2 Caffeicacid 16.7 68.8 15.3 207.6 54.9 0.5±0.02 1.7±0.03 1.4±0.03 4.6±0.04 3 Vanillic acid 19.5 218.3 48.7 * * 1.5±0.03 5.0±0.04 * * 4 Rosmarinic acid 28.9 85.7 19.1 170.6 45.1 0.9±0.02 3.0±0.04 1.2±0.03 4.0±0.04

dantspresentinthesamplescanbesynergistic,thusexplaining suchinvitroresponse.

3.3. Enzymeinhibitioneffects

Inhibiting digestive enzymes such as _␣-amylase and ␣-glucosidasecanbeconsideredasaneffectivewaytomonitorblood glucoselevel.Type2diabetesisglobalandchroniccondition affect-ingabout422millionpeoplearoundtheglobe(WHO).Diabetes isassociatedwithmicrovascularandmacrovascularcomplications damagingvitalorgansincludingkidney,heart,eyes,brain,andalso causecutaneousmanifestations[28].Itisreportedthatthereisa

30%riskthatpeoplewithdiabetesdevelopcutaneous

manifesta-tionsduetothedamagecausedtothevascularandnervestructures

byhighbloodsugarlevels[29].Nowadays,agentswhicharebased

onnaturalresourcesarepreferredtomanagediseasessinceside

effectsareminimalandthetherapiesarewell-toleratedcompared

tosyntheticoraldrugscurrentlyavailableonthemarket[30].The

presentstudywasthusdesignedtoevaluatetheextractsofC.cajan

fortheirantidiabeticactivity(anti-amylaseandanti-glucosidase),

cutaneousmanifestations(anti-tyrosinase)and Alzheimer’s

dis-ease(anti-cholinesterase).ResultsareshowninTable7.

Overall,thehexaneextractexhibitedthehighestactivitywith

mostenzymeswhereas theaqueousextract was least

success-fulindepressingmostenzymaticactivities.Intheamylaseassay,

theethylacetate extractexertedthehighestamylaseinhibitory

activitywithan acarbose equivalentof 0.74 mmol/g.However,

thesameextract exhibitedlowerglucosidaseinhibition (0.68±

0.01mmolACAE/g).Instead,thehexaneextractshowedthe

high-estglucosidase inhibition (0.81 ± 0.01 mmol ACAE/g)but low

anti-amylaseeffect(0.42±0.01mmolACAE/g),nearlytwotimes

lowerthanglucosidaseinhibitoryactivity.␣-Amylasebreakdown

polysaccharidestoformdisaccharides whicharefurtherbroken

downbyglucosidasetoproducemonosaccharides.Theinhibition

of ␣-amylase is sometimes linked withgastrointestinal

distur-bancedue todigestionof carbohydrates[31]. Thus,antidiabetic

drugs exhibiting moderate␣-amylase but higher ␣-glucosidase

inhibitorypotentialispreferred.

Thehighest tyrosinaseactivitywasrecorded with

methano-licextract(55.90±1.20mgKAE/g)followedbytheethylacetate

extract(47.31±1.22mgKAE/g),hexane(38.02±4.04mgKAE/g)

and aqueous (no activity was reported). These results may be

attributedtothepresenceofhighphenoliccontentinthe

methano-licextract.Previousstudieshavealsoreportedthepotentinhibitory

activityofplantextractsontyrosinaseisattributedtotheir

phe-nolicproﬁles[32],andarethusinagreementwithourﬁndings.

Hexaneextract wasthebestcholinesterase inhibitor withboth

AChE (2.61 _± 0.06 mg GALAE/g) and BChE (4.87 _± 0.25 mg

GALAE/g)enzymes.Althoughﬂavonoidsarereportedtobeefﬁcient

cholinesteraseinhibitors,resultsfromthisstudydonotcorrelate

withtheobservedactivities.For instance,hexane extractwhich

containedtheleastamountofﬂavonoiddemonstratedthe

high-estanti-AChEandanti-BChEactivityandtheethylacetateextract

yieldingthehighestTFC,wasleastsuccessfulindepressingAChE

enzymeactivity(1.25_±0.14mgGALAE/g).Therefore,itcanbesaid

thatthelevelofﬂavonoidpresentinextractsmaynotalwaysbe

linkedwithcholinesteraseactivity.In additiontothisapproach,

severalstudies[6,33]reportedthatC.cajancontains other

clas-sisofphytochemicalsincludingsaponins,terpenoidsandstilbenes

(cajaninetc.)andtheycouldberesponsibleforthementioned

dif-ferencesinenzymeinhibitionassays.

3.4. Multivariateanalysis

Firstly, principal component analysis (PCA)was achieved in

ordertoreducethedimensionalityofthedatawhilstpreserving

asfaraspossiblemostoftherelevantinformationinthedataset.It

accomplishesthisreductionbygeneratingafewnumbersof

dimen-sionsalongwhichthevariabilityinthedatasetismaximal.Through

thefactorialplanerectedbyafewdimensions,itispossibleto

visu-allyassessdifferencesandsimilaritiesbetweensamples.ThePCA

resultsareshownasFig.7,beforestartingtheinterpretationofthe

obtainedresults,theoptimalnumberofdimensionsthatmustbe

consideredwereidentiﬁed.Thus,wefollowedtworulessuggested

bySolanasetal.[34].Accordingly,threedimensionswerefoundto

satisfytheabove-mentionedrule;theyhadaneigenvalueabove1

andencompassedtogether99.7%oftheinformationinthe

origi-naldata(Fig.7A).Thecontributionoftheoriginalvariablesonthe

retaineddimensionswasdeterminedbyreferringtothecircleof

correlation(Fig.7B).theﬁrstdimensionwhichcapturedthe

maxi-mumquantityofinformation,waspositivelycorrelatedwithABTS,

DPPH,FRAPandnegativelylinkedtoBChEandglucosidase.This

wouldsuggestthattheﬁrstdimensionseparatedthesamples

pre-dominantlyinrelationtotheirabilitytoscavengeABTSandDPPH

radical,reduceFe3+_ion_and_inhibit_the_activity_of_BChE_and

glu-cosidaseenzymes.Dimension2optimized31.3%ofthevariance

andwaspositivelydeterminedbyCUPRAC,PPBD,MCA,amylase

andtyrosinase.Asaresult,itdistinguishedthesamplesinterms

ofsaidbiological activities.Dimension 3whichrepresentedthe

leftoverinformationwashighlypositiveloadedforAChEand

nega-tiveloadedforPPBDandamylase.Accordingly,itdiscriminatedthe

samplesaccordingtotheirtotalantioxidant,anti-AChEand

anti-amylaseactivities.Afterthereductionofthedimensionalityofthe

data,hierarchicalclusteredanalysiswassubjectedtotheresults

ofPCAinordertobringoutthedifferentclusters.Theobtained

factormaprevealedfourclusters(Fig.7C),howeverethylacetate

andmethanol extractswereclosetogetherrelativelytohexane

andinfusionextracts.Thisimpliesthatamongthefourextraction

solvents,ethylacetateandmethanolpresentedsomesimilarities

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publica-Fig.2.Chemicalstructuresof12differentphenolicantioxidantsidentiﬁedbyHPLC-FRAPfromsample1(ethylacetate),2(hexane),3(methanol)and4(infusion).

tionscanbefoundontheinﬂuencedoftheextractionsolventonthe

recoveryofbiopharmaceuticalmoleculesfromplants.This

inﬂu-encefocusesonthequalityandnumberofsecondarymetabolites

extractedfromtherawmaterials.Methanolandethylacetate

pro-videdoverall excellentbiological activities;theyexhibited high

phenoliccontent,strongantioxidantactivitiesandrelativelygood

enzymeinhibitoryproperties.However,fromtheperspectiveof

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sol-Fig.3.OverlappedchromatogramswithHPLC-FRAP595nmandDAD280nmdetectionsofhexaneextractfromC.cajanstembark.

Fig.4.OverlappedchromatogramswithHPLC-FRAP595nmandDAD280nmdetectionsofethylacetateextractfromC.cajanstembark.

Fig.5. OverlappedchromatogramswithHPLC-FRAP595nmandDAD280nmdetectionsofmethanolextractfromC.cajanstembark.

Table7

EnzymeinhibitorypropertiesofC.cajanstembark.

Extracts AChE(mgGALAE/g extract) BChE(mgGALAE/g extract) Tyrosinase(mgKAE/g extract) ␣-amylase(mmol ACAE/gextract) ␣-glucosidase(mmol ACAE/gextract) Hexane 2.61±0.06a _4.87_±_0.25a _38.02_±_4.04c _0.42_±_0.01b _0.81_±_0.01a EA 1.25±0.14c _4.66_±_0.07a _47.31_±_1.22b _0.74_±_0.03a _0.68_±_0.01b MeOH 2.01±0.01b _2.49_±_0.12b _55.90_±_1.20a _0.42_±_0.01b _0.39_±_0.02c Infusion na na na 0.12±0.01c _na

*Valuesexpressedaremeans±S.D.ofthreeparallelmeasurements.GALAE:Galantamineequivalent;KAE:Kojicacidequivalent;ACAE:Acarboseequivalent;EA:Ethyl acetate;MeOH:Methanol.na:notactive.Differentsuperscriptsindicatesigniﬁcantdifferencesintheextracts(p<0.05).

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Fig.6. OverlappedchromatogramswithHPLC-FRAP595nmandDAD280nmdetectionsofinfusionfromC.cajanstembark.

Fig.7. PrincipalcomponentandhierarchicalclusteredanalysesappliedtobiologicalactivitiesofC.cajanstembark.A.eigenvalueandpercentageofexplainedvariancesof dimensionsofprincipalcomponentanalysis.B.Appreciationofthebiologicalcontributionontheﬁrstthreedimensionsofprincipalcomponentanalysisthroughthecircle ofcorrelation.C.FactormapofHierarchicalclusteringanalysis.

ventsuchethanolorethanol-watersincetheyareincompliance withsafelymanufacturingpractice.

4. Conclusion

Thepresentstudy,fortheﬁrsttime,hasemployedthe HPLC-FRAPsystemtoidentifyatotalof12phenolicantioxidantsinthe differentextractsofC.cajan.Plantswithhighlevelofphenolicsare consideredasagoodsourceofantioxidantsandthereforeitcanbe saidthatC.cajanmightbeconsideredasafuturesourceof antiox-idantsfordevelopmentofbioproducts.However,cytotoxicitytest shouldbeconductedonthe plantfoodtodetermineitssafety. Contrarytootherstudies[35],inthispresentwork,theobserved

biological activities(antioxidantand enzymaticactivities) could

notbefullyattributedtothepolyphenolicproﬁleoftheextracts

astheactivitiesvaried;wherebycertainextractspossessinghigh

bioactivecompoundsdemonstratedlowbiologicalactivityandvice

versa.Asafuturework,thehexaneextract,depressingmost

stud-iedenzymes,canbesubjectedtokineticstudiestodeterminethe

typeofinhibitioninvolved.

CRediTauthorshipcontributionstatement

KouadioIbrahimeSinan:Methodology,Formalanalysis,

Inves-tigation.MohamadFawziMahomoodally:Investigation,Writing

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Eyu-poglu:Methodology,Formalanalysis.OuattaraKatinanEtienne:

Methodology.NabeelahBibiSadeer:Investigation,Writing-

orig-inal draft,Writing -review & editing.Gunes Ak:Investigation,

Writing-originaldraft,Writing-review&editing.TapanBehl:

Writing-review&editing.GokhanZengin:Investigation,Writing

-originaldraft,Writing-review&editing,Validation.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompeting

ﬁnan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto

inﬂuencetheworkreportedinthispaper.

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