Investigation of chemical profile, biological properties of Lotus corniculatus L. extracts and their apoptotic-autophagic effects on breast cancer cells

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JournalofPharmaceuticalandBiomedicalAnalysis174(2019)286–299

ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

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

Investigation

of

chemical

proﬁle,

biological

properties

of

Lotus

corniculatus

L. extracts

and

their

apoptotic-autophagic

effects

on

breast

cancer

cells

Serife

Yerlikaya

a

_,

_Mehmet

_Cengiz

_Baloglu

a,∗

_,

_Alina

_Diuzheva

b

_,

_József

_{Jek ˝o}

c

_,

Zoltán

Cziáky

c

_,

_Gokhan

_Zengin

d

a_Department_of_Genetics_and_{Bioengineering,}_Faculty_of_Engineering_and_{Architecture,}_Kastamonu_University,_Turkey b_Department_of_Analytical_Chemistry,_Pavol_{Jozef ˇSafárik}_University_in_Koˇsice,_Koˇsice,_Slovakia

c_Agricultural_and_Molecular_Research_and_Service_Institute,_University_of_{Nyíregyháza,}_{Nyíregyháza,}_Hungary d_Department_of_Biology,_Science_Faculty,_Selcuk_University,_Campus,_Konya,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6May2019

Receivedinrevisedform27May2019 Accepted30May2019

Availableonline3June2019 Keywords: Lotus Extracts Chemicalingredients Apoptosis Autophagy

a

b

s

t

r

a

c

t

Thisstudyaimedtorevealchemicalproﬁlesandbiologicalactivitiesofethylacetate(EA),methanol (MeOH),andwaterextractsofLotuscorniculatus.Ethnobotanicalreportshaveindicatedtheimportance ofphytochemicalpropertiesofthegenusLotus.Inthisstudy,theeffectsofmedicinalplantextractson antioxidant(DPPH,ABTS,CUPRAC,FRAP,phosphomolybdenum,andmetalchelatingassays),enzyme inhibitory(oncholinesterase,tyrosinase,a-amylaseanda-glucosidase),DNAprotectionandanticancer properties(includinganti-proliferative,celldeathandtelomeraseactivitymarkergeneanalysis, apop-toticDNAfragmentationanalysis,cellmigrationtest)wereevaluated.

According tochemical analysis, quercetin derivatives geraldol,isorhamnetin and kaempferol-O-coumaroylhexoside-O-deoxyhexosideisomersweredominantintheextracts.MeOHextractsshowed thehighesttotalﬂavonoidscapacitywith21.13mgRE/g.EAextractshowedthestrongestanti-amylase activityamongthetestedextracts.WaterextracthadthemostprotectiveactivityagainstplasmidDNA. Toindicatecellsurvival,MTTtestwasperformedagainsthumanMCF-7andMDA-MB-231breast can-cercells.Half-maximalinhibitoryconcentrationforcellswerecalculatedandusedfordetectionof mechanismsbehindthecancercelldeath.EAextractshowedup-regulationofBaxproapoptoticgene andapoptoticDNAfragmentationactivityonhighlyinvasiveMDA-MB-231cells.Beclin-1andLC3-II autophagygeneswerehiglyexpressedaftertreatmentofMCF-7cellswithEAextracts.EAandMeOH extractsinhibitedcellmigrationabilityofbothcancercells.Linoleamide,wasdominantcomponentin EAextractandcausedapoptosisonMDA-MB-231breastcancercellsviaincreasingintranuclearCa2 +_.

Thedetailedmechanismbehindtheanticancerpropertiesshouldbefurtherinvestigated.

1. Introduction

Thehumanbeinghasusedherbalmedicineorphytomedicine fortheaimofcuringdiseases sincethebeginning ofexistence. Plantsachievethisabilitywiththeirsecondarymetabolitescalled as“phytochemicals”.Phytochemicalscanbesortedasalkaloids, glycosides,polyphenoles,saponins,terpenesandanthraquinones. Importantnatural-derivedmedicinalplants;quinine,theophylline, penicillinG,morphine,paclitaxel,digoxin,vincristine,doxorubicin, cyclosporinand vitamin A, have contributed topharmaceutical

∗ Correspondingauthor.

E-mailaddress:mcbaloglu@kastamonu.edu.tr(M.C.Baloglu).

remediesuptonow.Amongphytochemicals,flavonoidsarethe mostwell-knownstructurewhichiscomprisedofafifteen-carbon skeleton withtwo benzeneringsconnectedwitha heterocyclic pyrene ring. They are classified as flavones (flavone, apigenin, andluteolinetc.),flavonols(quercetin,kaempferol,myricetin,and fisetinetc.),flavanones(flavanone,hesperetin,andnaringeninetc.), flavanonol(taxifolin),isoflavones(genistein,daidzein), flavan-3-ols(catechin,epicatechin)[1].

Cancerprogressionisaserialofgeneticaleventsandmutations thattakeplacebytheaidofangiogenesis,metastasisandothercell signals.Ithasbeenreportedthatcancercellsinducecellular senes-cence(cellcyclearrest)andsenescentcanbeinitiatedbyoxidative stress,DNAdamageandtelomeredestruction.Inordertocopewith cellstressorsenescence,thecellneedstoapoptosis(self-killing) https://doi.org/10.1016/j.jpba.2019.05.068

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S.Yerlikayaetal./JournalofPharmaceuticalandBiomedicalAnalysis174(2019)286–299 287 forcelldeathorautophagy(self-eating)forhomeostasis.Insome

reports,autophagycancontributetocelldeathwhenapoptosisis inhibited.Whencancercellsexposetocytotoxicdrugs,autophagic celldeathcanevadefromapoptosisincellularsenescence.In addi-tion,telomereshorteningisthemainsourceofcellularsenescence. TelomeresarerepetitiveDNAsequencesthatpresentattheendof linearlyeukaryoticchromosomes.Theyprotectchromosomeends fromDNAbreakagesandnucleolyticerosion.Telomeraseactivity performsin70–90%ofmalignanttissuesandsomeimmortalcells. Also,ithasbeendeterminedthattelomeraseactivitytakesplacein 74%ofinvasivebreastcancercellsanddoesnotperforminbenign andnormalbreasttissues.Incancertreatment,themostsigniﬁcant problemistheresistanceofchemotherapydrugs.Todealwiththis, researchersbegintoseeknatural-derivedcompoundinlower tox-icityandmaximumefﬁciency.Inareport,invitropharmacological and polyphenolicactivityanalysisof theendemic species Phyl-lanthusphillyreifoliusvar.commersoniiMüll.ArginMauritiuswere detectedandrolesofbiologicallyactivesubstanceswere investi-gatedoncytotoxicityofMDA-MB-231breastcancerline[2].

Lotuscorniculatus isknownasbird’sfoottrefoiland belongs totheLeguminosaefamily.Itisabletofixnitrogenutterlyroot nodules making it beneficial as a cover crop. Ethnobotanical usageshavebeenreportedandtherootshowedcarminativeand antipyreticeffect,flowerswereanti-spasmodicandsedative.Inthe presentstudy,weaimedtodisplaysomebiologicalactivitiessuch asantioxidant,enzymeinhibitor,DNAprotection,antiproliferative, woundhealing,DNAfragmentation,apoptotic,autophagicand tel-omerasegene activityanalysisofL.corniculatus extractsagainst breastcancercellsaswellaschemicalprofiles.

2. Materialsandmethods

2.1. Plantmaterialandpreparationofextracts

SamplingoftheplantspecieswasdoneinKonya(Selcuk Univer-sity,Campus),Turkey,2016.Botanicalauthenticationoftheplant wascarriedoutbythebotanistDr.EvrenYıldıztugay(Selcuk Uni-versity,FacultyofScience,Turkey).Theaerialpartsweredriedat roomtemperature(about10daysinshade).Thematerialwasthen pulverisedusingalaboratorymill.

Methanol(MeOH)andethylacetate(EA)extractswereprepared bymacerating5gofplantsamplesin100mloftherespective sol-ventsovernight.Theextractswerethenﬁlteredandconcentrated invacuoat40◦Cusingarotaryevaporator.Aqueousextractswere preparedfollowingtraditionalinfusionmethod.Infusionwas car-riedoutasfollows:5gofplantmaterialwasinfusedin100mlof boilingwaterfor20min.Theresultingmixtureswereﬁlteredand driedinalyophilizer.Theplantextractswerekeptat+4◦Cuntil furtheranalysis.

2.2. Proﬁleofbioactivecompounds

Byreferringtoourpreviouspaper[3],theﬂavonoids(TFC)and totalphenolic(TPC)contentsweredeterminedusingtheAlCl3and Folin-Ciocalteuassays,respectively.Theresultswereexpressedas equivalentsofrutin(mgRE/g)forTFCandgallicacid(mgGAE/g) forTPC.

2.3. Determinationofantioxidantandenzymeinhibitoryeffects The in vitro enzyme inhibitory effects of extracts on ﬁve enzymes,namely,␣-amylase,␣-glucosidase,acetylcholinesterase (AChE),butyrylcholinesterase(BChE),andtyrosinasewere evalu-ated,aspreviouslyreported[3].Theenzymeinhibitoryactionsof extractswereassessedasequivalentsofkojicacid(KAE)for

tyrosi-nase,galantamine(GALAE)foracetylandbutyrylcholinesterase, andacarbose(ACAE)for␣-amylaseand␣-glucosidase.

Regarding antioxidant capacity of the extracts, different spectrophotometric experimentsas ferrousion chelating, phos-phomolybdenum,reducingpower(FRAPandCUPRAC),andradicals scavenging(ABTSandDPPH)assayswereperformedaspreviously reported[3].Theﬁndingswerereportedasstandardcompounds equivalentsofEDTAorTrolox(mgEDTAE/gandmmolTE/g). 2.4. Liquidchromatographyanalysis

LiquidchromatographywasperformedonaDionexUltimate 3000RSUHPLCsystem(ThermoScientiﬁc,USA).Thesampleswere separated on a Thermo Accucore C18 (100mm×2.1mm, i. d. 2.6␮m)column. Thecolumntemperaturewassetat25◦C.The UHPLCsystemwascoupledtoaThermoQExactiveOrbitrapmass spectrometer(ThermoScientiﬁc,USA)equippedwithelectrospray ionizationsource.Allanalyticaldetailsweregiveninourearlier paper[2].

ForcollectionandanalyzingdataThermoScientificXcalibur4.0 andTraceFinder3.1softwares(ThermoScientific,USA)wereused. Inthetablescoumpoundsaremarkedwhichwereconfirmedby standards.Allothercompoundshavingexactstructurewere iden-tifiedonthebasisourpreviouspublishedworks.Ineverycase,the exactmolecularmass,isotopicpattern,characteristicfragmentions andretentiontime(knownfromourpreviousworks)wereusedfor theidentificationofthecompounds.

2.5. DNAprotection

DNA protection activityof theextracts wastested byusing pUC19 plasmidDNA (pDNA).Plasmid isolation wascarried out byThermoScientiﬁcGenejetPlasmidMiniprepKit.Thereaction mixturewascomprisedofFenton’sreagent(30mMH2O2,50mM ascorbicacid,and80mMFeCI3),twodifferentconcentrationsof extracts(5and10mg/mL)andpDNA(300␮g/␮L).Sampleswere incubatedfor30minat37◦Casdescribedbefore[4].TheDNA mix-tureswererunon0.8%agarosegelandthenvisualizedunderUV. Biologicalreplicationoftestwasperformedthreetimesandband densitywasanalyzedbythegelimageanalysissoftware(Quantum, Vision-Capt.,VilberLourmatSAS,France).

2.6. Cellculture

2.6.1. Cellculturematerials

Penicillin/Streptomycin,DMEMcellculturemedia,Fetalbovine serum (FBS), trypsin, MTT, ethanol, 2-prophanol, 100×17mm corningplates and75 cm2 _corning_ﬂasks _were_purchased _from Sigma-Aldrich(Sigma-Aldrich,USA).

2.6.2. Preparationofplantextracts

Water extractswere suspended in PBS, methanol and ethyl acetateextractsweresuspendedin0.1%DMSO.Dissolvedextracts were ﬁltratedwith 0.22␮m pore sizeand they werestored in −20◦_C_until_further_use.

2.6.3. Cellculturemaintenance

TriplenegativeandhighlymetastaticMDA-MB-231breast can-cercells werecultured in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM)supplementedwith10%FBS,0.01mg/mlhumaninsulin, 1% non-essential amino acid and 0.1% penicillin/streptomycin. Human estrogen receptor-positive MCF-7 breast cancer cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mML-glutamine, 0.1% penicillin/streptomycinat37◦Cina5%CO2humidiﬁedincubator.

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288 S.Yerlikayaetal./JournalofPharmaceuticalandBiomedicalAnalysis174(2019)286–299 Cellswereroutinelysub-cultured(whentheyreached80%

con-ﬂuency,theywerewashedwithphosphatebufferedsaline(PBS), andtrypsinizedwith0.25%trypsin-EDTAsolution)andthenwere seededon96-wellplatesor100×15cellculturepetridishesfor experiments.

2.6.4. MTTcytotoxicityassay

Atotalof10,000MDA-MB-231andMCF-7cellswerecultured and allowed to fasten on 96- well plates for 24h. After 24h, cellsweretreated withvariousdoses (62,5;125; 250;500and 1000␮g/ml)of water,methanol(MeOH)and ethylacetate (EA) extractsof Lotus corniculatus for 24, 48 and 72h. Cell viability orcytotoxicityanalysiswereperformedasdescribedearlier[4]. Absorbanceof each sample wasnoted at 570nm byusing the microplatespectrophotometer(MultiskanGo,ThermoScientiﬁc, USA).Halfmaximal inhibitory concentration(IC50)values were calculatedusinglog(inhibitor)vs.normalizedresponse-variable slopeanalysisfunctionbytheaidofGraphPadPrism7software. 2.7. Quantitativereal-timePCRanalysisofcelldeathand

telomeraseactivitymarkergenes

In logarithmic phase of growth, MDA-MB-231 and MCF-7 breast cancer cells were treated with extractsof EA, MeOH at doses of IC50 values. Total RNA isolation from cells was car-riedout usingGeneJET RNA Purification Kit (Thermo Scientific, USA). cDNA synthesis was performed from RNase-free DNase treated total RNAs (BioRad cDNA synthesis kit, USA). qPCR reactionswere achived withRotor Gene-Q (Qiagen, Germany). Primers belonging to Bax (5’-CCCGAGAGGTCTTTTTCCGAG-3’) and(5’-CCAGCCCATGATGGTTCTGAT-3’),Bcl2 (5’-GGTGGGGTCAT-GTGTGTGG-3’)and(5’-CGGTTCAGGTACTCAGTCATCC-3’),Beclin-1 (5’-GGCTGAGAGACTGGATCAGG-3’) and (5’-CTGCGTCTGGG-CATAACG-3’), LC3-II (5’-GAGAAGCAGCTTCCTGTTCTGG-3’) and (5’-GTGTCCGTTCACCAACAGGAAG-3’), TERT-1 (5’-GGATGAAG-CGGAGTCTGGA-3’) and (5’-CGGAAGAGTGTCTGGAGCAA-3’) and thereferencegeneGAPDH(5’- AACATGTAAACCATGTAGTTGAGGT-3’)and (5- GGAAGGTGAAGGTCGGAGTC-3’) were usedfor gene expressionanalysis.SYBRGreenmastermixwaspreparedin20␮l reactionmixtures(BioRad,USA).Amplificationswereperformed withinitialdenaturationat95◦Cfor5minfollowedby40cycles ofdenaturationat95◦Cfor10s,annealingandextensionat57◦C for30s.Therelativequantificationofgeneexpressionwasdone usingthecomparativeCTmethod(Ct)[2].

2.8. ApoptoticDNAfragmentationanalysis

A total of 500,000 MDA-MB-231 cells were seeded in 100×17mmpetridishes.Aftercellsreachedtoconﬂuency,they weretreatedwithextractofEAfor72hatdosesofIC50 values. Controlcellsweretreatedwith0.1%DMSO.Aftertreatment,cells werewashedwith2mlofphosphate-bufferedsaline(PBS). Adher-entcellswereaccumulatedbyusingacellscraper.DNAisolation wasaccomplishedbyusingapoptoticDNA-Ladderkit(AbcamDNA LadderDetectionKit).EqualamountsofDNA(2␮g)fromeach sam-plewererunintoa1.2%agarosegelelectrophoresisat5V/cmfor 2h.DNAwasvisualizedwithnucleicacidstainingsolutionunder UVlight.

2.9. Woundhealingassay

MDA-MB-231andMCF-7cellswereculturedingrowthmedium in6-welltissuecultureplatesuntilreachedatleast90%conﬂuence. Totestcellmigrationability,woundswerescratchedoneachwells byusing200␮lsterilepipettip.Cellsweretreatedwithextracts

ofEAandMeOHatdosesofIC50valuesand0.1%DMSOfor con-trol.Inaddition,MitomycinC(10␮g/ml)wasaddedtoallgrowth mediumofwellsinordertopreventcellproliferation[5].Scratched areasandcellmorphologywerephotographedunderaninverted microscopebyusinga4xobjective.

2.10. Statisticalanalysis

Allexperimentswerecarriedoutintriplicatesandresultswere revealedasthemeanvalue±standarddeviation(SD).Minitab17 packageprogramwasusedforstatisticalcalculationsandall com-parisonsweredeterminedbyusingone-wayANOVA.Thevaluesof p<0.05wereregardedasstatisticallysigniﬁcant.

3. Resultsanddiscussion

3.1. Chemicalcharacterizationandantioxidanteffects

The amount of phenolics and flavonoid were measured by spectrphotometric methods and theresults are summarized in Table4.Thewaterextractcontainedthehighestlevelof pheno-lics,followedbyMeOHandEAextracts.However,theorderfor totalflavonoidswere:MeOH(21.13mgRE/g)>EA(17.59mgRE/g) >Water(16.24mgRE/g).Thedifferencescouldbeexplainedwith disavantagesofspectrophotometricassays.Tobeconsistentwith ourapproach, severalresearchers have beenreported that the assaysnotreflecttototalamountofbioactivecompoundsbecause thereagents used could bereacted withother phytochemicals [6]. In this context, at least one chromatographic technique is requiredtoprovidecertainresultsforchemicalprofilesinplant extracts.

For the reasons above mentioned, HPLC-MS/MS technique wasusedtodeterminecertainchemical profilesofthreeL. cor-niculatus extracts in the present work. In the extracts mostly flavonoids and other phenolic compounds were identified (76 compounds in the methanolic, 58 compounds in the aqueous and 52 compunds in the ethyl acetate extract) (Tables 1–3). Beside the identified flavonoid derivatives known in the lit-erature we have charaterized many other flavonoids which had not published in this plant.These flavonoids weremainly kaempferol,quercetinand isorhamnetinO-glycosides.In several cases the exact structure of these compound were identi-fied on the basis their exact molecularmass, isotopic pattern, characteristic fragment ions and retention time (for exam-ple isoquercitrin and isorhamnetin-3-O-glucoside). Other cases we could only identify the substituents of the flavonoids and the sugar moiety. It is known that L. corniculatus contains saponins[7]. Threenewsaponin derivativeswere foundinthe methanolic extract (m/z 911.5004; 765.4425 and 867.4742 at retentiontimes38.00,38.09and39.24min,respectively)butwe could not determined the exact structure of these componds unambiguously.

Antioxidanteffects ofplantextractsare great ofimportance inthepharmaceuticalandfoodareas.Manychronicand degen-erativediseases arelinked oxidativestress,which is knownan imbalancebetweenoxidantsandantioxidants[8].Thus,noveland safeantioxidants(especiallynaturalsources)havegreatinterest in the scientiﬁc platform. In our study, the antioxidant effects ofthreeextractsofL.corniculatuswereinvestigatedbydifferent invitroassays, includingmetalchelating,phosphomolybdenum, reducingpowerandfreeradicalscavenging.Theresultsare pre-sentedin Table4.In bothradicalscavengingassays (DPPHand ABTS),thewaterextractdisplayedthestrongestscavenging abil-itiy,followedby MeOHand EAextracts.Similartrend wasalso observedinferricreducingpowerassayaswellasmetal

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chelat-S. Yerlikaya et al. / Journal of Pharmaceutical and Biomedical Analysis 174 (2019) 286–299 289 Table1

ChemicalproﬁleofethylacetateextractfromL.corniculatus.

No. Name Formula Rt [M+H]+ _[M_-_H]− _Fragment₁ _Fragment₂ _Fragment₃ _Fragment₄ _Fragment₅ _Literature

1 Uralenneoside C12H14O8 10,03 285,06105 153,0182 152,0105 109,0280 108,0203

2 Gossypetin-3-O-galactoside C21H20O13 18,48 479,08257 317,0304 316,0226 166,9974 165,9898 139,0025 [24]

3 Gossytrin(Gossypetin-3-O-glucoside) C21H20O13 18,91 479,08257 317,0304 316,0226 166,9972 165,9897 139,0025

4 Kaempferol-O-deoxyhexosylhexoside-O-deoxyhexosideisomer1 C33H40O19 19,14 739,20856 593,1508 430,0897 285,0413 283,0251 255,0300

6 Kaempferol-3-O-[xylosyl-(1→2)-galactoside]-7-O-rhamnoside C32H38O19 20,64 725,19291 579,1349 430,0884 284,0333 283,0253 255,0302 7 Quercetin-3-O-rhamnoside-7-O-glucoside C27H30O16 20,91 609,14557 463,0900 447,0936 446,0860 301,0356 299,0200 [25] 8 Quercetin-O-deoxyhexoside-O-hexoside C27H30O16 21,19 609,14557 463,0860 447,0932 446,0858 301,0358 299,0200 9 Verbascoside C29H36O15 21,95 623,19760 461,1662 315,1092 161,0233 133,0282 10 Kaempferol-3-O-rhamnoside-7-O-glucoside C27H30O15 22,13 593,15065 447,0938 430,0907 285,0409 283,0252 255,0298 [25] 11 Corniculatusin-3-O-galactoside C22H22O13 22,25 493,09822 331,0460 330,0385 316,0227 315,0151 165,9894 [25] 12 Corniculatusin-3-O-glucoside C22H22O13 22,45 493,09822 331,0465 330,0386 316,0223 315,0152 165,9896 [25]

13 Hyperoside(Quercetin-3-O-galactoside,Hyperin) C21H20O12 22,62 463,08765 301,0357 300,0278 271,0251 255,0299 151,0024 [25]

14 Quercetin-3,7-O-dirhamnoside C27H30O15 22,71 593,15065 447,0935 446,0861 301,0358 299,0200 271,0251 [25]

151 _{Isoquercitrin}_(Hirsutrin,_{Quercetin-3-O-glucoside)} _C21H20O12 _22,84 _463,08765 _301,0356 _300,0278 _271,0251 _255,0297 _151,0024

16 Gossypetin(2’,3,3’,5,7,8-Hexahydroxyﬂavone) C15H10O8 23,26 317,02975 208,0006 194,9920 166,9973 149,0231 139,0025

17 Quercetin-O-pentoside C20H18O11 23,43 433,07709 301,0356 300,0278 271,0248 255,0301 151,0026

18 Sexangularetin-3-O-glucoside C22H22O12 23,50 477,10330 315,0528 314,0438 300,0279 299,0200 165,9894 [25]

19 Kaempferol-7-O-glucoside C21H20O11 24,07 447,09274 285,0410 284,0330 255,0299 227,0346 [26]

20 Kaempferitrin(Kaempferol-3,7-di-O-rhamnoside) C27H30O14 24,26 577,15573 431,0988 430,0907 285,0408 283,0252 255,0297 [25]

211 _Quercitrin_{(Quercetin-3-O-rhamnoside)} _C21H20O11 _24,40 _449,10839 _303,0500 _257,0440 _229,0498 _129,0549 _85,0290 _[₂₅_]

22 Methoxy-tetrahydroxy(iso)ﬂavone-di-O-deoxyhexoside C28H32O15 24,48 607,16630 461,1109 460,1007 315,0513 313,0358 299,0561

231 _Fisetin_{(3,3’,4’,7-Tetrahydroxyﬂavone)} _C15H10O6 _24,62 _287,05556 _241,0498 _213,0544 _185,0610 _137,0239 _121,0288 _[₂₇_]

24 Isorhamnetin-O-hexoside C22H22O12 24,66 477,10330 314,0437 299,0202 285,0410 271,0251 243,0298

251 _{Isorhamnetin-3-O-glucoside} _C22H22O12 _24,84 _477,10330 _314,0440 _299,0208 _285,0403 _271,0251 _243,0297

26 Methoxy-tetrahydroxy(iso)ﬂavone-O-deoxyhexoside C22H22O11 25,68 461,10839 315,0521 299,0565 284,0321 269,0458 255,0294

27 Kaempferol-O-acetylhexoside-O-deoxyhexoside C29H32O16 26,02 635,16121 489,1039 431,1014 430,0898 285,0411 283,0250

28 Vestitol-O-hexoside C22H26O9 26,31 433,14986 271,0976 149,0598 135,0438 121,0283

29 Afzelin(Kaempferol-3-O-rhamnoside) C21H20O10 26,35 431,09782 285,0408 284,0329 255,0299 227,0346 [25]

30 Corniculatusin(8-Methoxy-3,3’,4’,5,7-pentahydroxyﬂavone) C16H12O8 26,90 331,04540 316,0233 271,0248 209,0081 181,0135 165,9898 [27]

311 _Quercetin _C15H10O7 _26,91 _301,03483 _273,0402 _245,0445 _178,9978 _151,0025 _121,0281 _[₂₇_]

321 _Naringenin _C15H12O5 _27,15 _271,06065 _177,0180 _151,0025 _119,0489 _107,0123 _93,0330

33 Kaempferol-7-O-rhamnoside C21H20O10 28,64 431,09782 285,0408 284,0331 257,0457 151,0025 [25]

341 _Kaempferol_{(3,4’,5,7-Tetrahydroxyﬂavone)} _C15H10O6 _29,25 _285,03991 _257,0457 _229,0509 _185,0605 _151,0027 _127,0393 _[₂₇_]

351 _Apigenin_{(4’,5,7-Trihydroxyﬂavone)} _C15H10O5 _29,61 _269,04500 _225,0551 _201,0552 _151,0025 _149,0233 _117,0331

36 Dimethoxy-trihydroxy(iso)ﬂavone C17H14O7 29,79 329,06613 314,0437 313,0364 299,0200 285,0418 271,0252

37 Coumestrol C15H8O5 29,85 269,04500 241,0494 225,0545 213,0552 197,0601 157,0650

38 Hispidulin(6-Methoxy-4’,5,7-trihydroxyﬂavone) C16H12O6 30,12 299,05557 284,0331 256,0369 255,0303 227,0341 136,9867 [28]

39 Vestitol(2’,7-Dihydroxy-4’-methoxyisoﬂavan) C16H16O4 30,29 273,11269 163,0755 149,0599 137,0599 123,0443 95,0498 [29]

40 Hydroxy-methoxy(iso)ﬂavone C16H12O4 30,78 269,08138 254,0579 237,0545 226,0631 213,0912 118,0417

41 Medicarpin(3-Hydroxy-9-methoxypterocarpan) C16H14O4 30,97 271,09704 161,0598 147,0443 137,0599 123,0443 109,0652

42 Trihydroxy-trimethoxy(iso)ﬂavone C18H16O8 31,24 359,07669 344,0539 329,0306 314,0074

43 Dihydroxy-dimethoxy(iso)ﬂavane C17H18O5 31,58 303,12325 193,0862 181,0862 167,0704 149,0598 123,0443

44 Dihydroxy-trimethoxy(iso)ﬂavone C18H16O7 33,10 343,08178 328,0592 313,0360 298,0120 285,0390 270,0175

45 Dihydroxy-tetramethoxy(iso)ﬂavone C19H18O8 33,19 375,10800 360,0841 345,0607 342,0730 327,0501 197,0088

46 Dodecanedioicacid C12H22O4 33,28 229,14399 211,1335 167,1429

47 Dihydroxy-methoxy(iso)ﬂavone C16H12O5 33,37 283,06065 268,0379 267,0302 239,0347 211,0398

48 Sativan(2’,4’-Dimethoxy-7-hydroxyisoﬂavan) C17H18O4 34,15 287,12834 177,0912 151,0755 123,0443 95,0498 [29]

49 Hexadecanedioicacid C16H30O4 40,30 285,20659 267,1966 223,2063

501 _Juniperic_acid_{(16-Hydroxyhexadecanoic}_acid) _C16H32O3 _40,81 _271,22732 _253,2177 _225,2220

51 Linoleamide C18H33NO 44,05 280,26404 263,2371 245,2264 221,2268 95,0861 81,0705

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290 S. Yerlikaya et al. / Journal of Pharmaceutical and Biomedical Analysis 174 (2019) 286–299 Table2

ChemicalproﬁleofmethanolextractfromL.corniculatus.

21 _Chlorogenic_acid_{(3-O-Caffeoylquinic}_acid) _C16H18O9 _14,15 _355,10291 _163,0392 _145,0287 _135,0444 _117,0339 _89,0391

3 Caffeicacid C9H8O4 14,27 179,03444 135,0440 107,0486

4 Unidentiﬁedcompound C11H12O6 15,88 239,05556 221,0450 179,0342 177,0548 149,0597 121,0281

5 Naringenin-6,8-di-C-glucoside C27H32O15 16,88 595,16630 505,1327 475,0254 415,1037 385,0934 355,0828

6 Quercetin-di-O-hexoside C27H30O17 17,15 625,14048 463,0879 462,0806 301,0344 299,0199 271,0252

7 4-O-(4-Coumaroyl)quinicacid C16H18O8 17,48 337,09235 191,0551 173,0446 163,0393 119,0487 93,0333

81 _4-Coumaric_acid _C9H8O3 _17,70 _163,03952 _119,0488 _93,0331

9 Gossypetin-3-O-galactoside C21H20O13 18,48 479,08257 317,0305 316,0225 166,9972 165,9895 139,0025 [24]

10 Gossytrin(Gossypetin-3-O-glucoside) C21H20O13 18,92 479,08257 317,0305 316,0226 166,9974 165,9896 139,0024

12 Gossypetin-O-pentoside C20H18O12 19,26 449,07201 317,0309 316,0230

13 Kaempferol-O-hexosylhexoside-O-deoxyhexoside C33H40O20 19,38 755,20347 609,1472 431,0985 430,0908 285,0409 283,0252

15 Quercetin-3-O-[xylosyl-(1→2)-galactoside]-7-O-rhamnoside C32H38O20 19,71 741,18782 595,1328 446,0858 300,0276 299,0200 271,0252 16 Kaempferol-3-O-[xylosyl-(1→2)-galactoside]-7-O-rhamnoside C32H38O19 20,64 725,19291 579,1361 430,0906 284,0330 283,0252 255,0299 17 Quercetin-3-O-rhamnoside-7-O-glucoside C27H30O16 20,91 609,14557 463,0888 447,0935 446,0857 301,0356 299,0199 [25] 18 Quercetin-O-deoxyhexoside-O-hexoside C27H30O16 21,19 609,14557 463,0888 447,0934 446,0856 301,0356 299,0199 19 Verbascoside C29H36O15 21,95 623,19760 461,1666 315,1098 161,0232 133,0283 20 Kaempferol-3-O-rhamnoside-7-O-glucoside C27H30O15 22,13 593,15065 447,0938 430,0906 285,0408 283,0251 255,0299 [25] 21 Corniculatusin-3-O-galactoside C22H22O13 22,25 493,09822 331,0463 330,0385 316,0226 315,0151 165,9895 [25] 22 Corniculatusin-3-O-glucoside C22H22O13 22,46 493,09822 331,0462 330,0384 316,0225 315,0151 165,9897 [25]

26 Gossypetin(2’,3,3’,5,7,8-Hexahydroxyﬂavone) C15H10O8 23,28 317,02975 208,0006 194,9928 166,9973 149,0231 139,0028

29 Quercetin-O-malonylhexoside C24H22O15 23,60 549,08805 505,0989 301,0368 300,0273 271,0243 255,0315

33 Hydroxy-methoxy(iso)ﬂavone-O-hexoside C22H22O9 24,45 431,13421 269,0811 254,0580 213,0914

34 Methoxy-tetrahydroxy(iso)ﬂavone-di-O-deoxyhexoside C28H32O15 24,49 607,16630 461,1093 460,1018 315,0514 313,0360 299,0562

351 _Fisetin_{(3,3’,4’,7-Tetrahydroxyﬂavone)} _C15H10O6 _24,61 _287,05556 _241,0489 _213,0544 _185,0610 _137,0239 _121,0287 _[₂₇_]

36 Isorhamnetin-O-hexoside C22H22O12 24,68 477,10330 314,0436 299,0200 285,0407 271,0250 243,0296

371 _{Isorhamnetin-3-O-glucoside} _C22H22O12 _24,87 _477,10330 _314,0436 _299,0191 _285,0406 _271,0252 _243,0299

38 Kaempferol-O-malonylhexoside C24H22O14 25,56 533,09314 489,1040 285,0406 284,0328 255,0299 227,0347

39 Methoxy-tetrahydroxy(iso)ﬂavone-O-deoxyhexoside C22H22O11 25,68 461,10839 315,0510 299,0557 284,0329 269,0458 255,0298

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S. Yerlikaya et al. / Journal of Pharmaceutical and Biomedical Analysis 174 (2019) 286–299 291 41 Vestitol-O-hexoside C22H26O9 26,31 433,14986 271,0978 149,0598 135,0440 121,0281

441 _Quercetin _C15H10O7 _26,94 _301,03483 _273,0416 _245,0457 _178,9977 _151,0025 _121,0281 _[₂₇_]

451 _Naringenin _C15H12O5 _27,16 _271,06065 _177,0182 _151,0025 _119,0488 _107,0124 _93,0330

47 Geraldol(3’-Methoxy-3,4’,7-trihydroxyﬂavone) C16H12O6 28,73 301,07121 286,0474 258,0523 229,0497 153,0184 [27]

48 Sexangularetin(8-Methoxy-3,4’,5,7-tetrahydroxyﬂavone) C16H12O7 29,08 315,05048 300,0282 165,9893 [27]

50 Kaempferol-O-coumaroylhexoside-O-deoxyhexosideisomer1 C36H36O17 29,30 739,18743 593,1305 431,1004 430,0915 285,0408 284,0329

521 _Isorhamnetin_{(3’-Methoxy-3,4’,5,7-tetrahydroxyﬂavone)} _C16H12O7 _29,76 _315,05048 _300,0282 _271,0249 _164,0109 _151,0025 _[₂₇_]

54 Coumestrol C15H8O5 29,84 269,04500 241,0498 225,0546 213,0550 197,0601 157,0658

55 Hispidulin(6-Methoxy-4’,5,7-trihydroxyﬂavone) C16H12O6 30,14 299,05557 284,0330 256,0374 255,0301 227,0344 136,9870 [28]

56 Kaempferol-O-coumaroylhexoside-O-deoxyhexosideisomer2 C36H36O17 30,27 739,18743 593,1307 431,1002 430,0894 285,0410 284,0330

58 Dihydroxy-trimethoxy(iso)ﬂavoneisomer1 C18H16O7 30,68 343,08178 328,0591 313,0359 298,0116 285,0398 270,0173

64 Dihydroxy-tetramethoxy(iso)ﬂavone C19H18O8 33,19 375,10800 360,0845 345,0609 342,0744 327,0502 197,0085

68 SoyasaponinI C48H78O18 37,90 941,51100 733,4505 615,3897 457,3699 205,0712 163,0600 [30]

69 Unidentiﬁedsaponin1 C47H76O17 38,00 911,50043 765,4437 615,3913 457,3710 205,0714

70 Unidentiﬁedsaponin2 C41H66O13 38,09 765,44252 633,4042 615,3882 457,3706 437,3410 113,0230

731 _Juniperic_acid_{(16-Hydroxyhexadecanoic}_acid) _C16H32O3 _40,80 _271,22732 _253,2177 _225,2221

741 _{␣-Linolenic}_acid _C18H30O2 _44,70 _277,21676 _259,2088 _59,0123

75 Oleamide C18H35NO 45,37 282,27969 265,2526 247,2421 149,1326 97,1018 83,0862

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292 S. Yerlikaya et al. / Journal of Pharmaceutical and Biomedical Analysis 174 (2019) 286–299 Table3

ChemicalproﬁleofwaterextractfromL.corniculatus.

21 _Chlorogenic_acid_{(3-O-Caffeoylquinic}_acid) _C16H18O9 _14,18 _355,10291 _163,0391 _145,0286 _135,0443 _117,0337 _89,0389

3 Caffeicacid C9H8O4 14,30 179,03444 135,0440 107,0488

4 Unidentiﬁedcompound C11H12O6 15,89 239,05556 221,0449 179,0342 177,0548 149,0597 121,0283

5 Naringenin-6,8-di-C-glucoside C27H32O15 16,89 595,16630 505,1370 475,0260 415,1035 385,0935 355,0828

6 Quercetin-di-O-hexoside C27H30O17 17,16 625,14048 463,0889 462,0809 301,0371 299,0199 271,0253

71 _4-Coumaric_acid _C9H8O3 _17,69 _163,03952 _119,0489 _93,0334

8 Riboﬂavin C17H20N4O6 18,57 377,14611 359,1358 243,0878 200,0821 172,0869 99,0445

10 Kaempferol-O-hexosylhexoside-O-deoxyhexoside C33H40O20 19,39 755,20347 609,1472 431,0980 430,0908 285,0411 283,0254

11 Kaempferol-O-deoxyhexosylhexoside-O-deoxyhexosideisomer2 C33H40O19 19,45 739,20856 593,1521 430,0905 285,0408 283,0252 255,0299 12 Quercetin-3-O-[xylosyl-(1→2)-galactoside]-7-O-rhamnoside C32H38O20 19,68 741,18782 595,1315 446,0859 300,0279 299,0200 271,0251 13 Kaempferol-3-O-[xylosyl-(1→2)-galactoside]-7-O-rhamnoside C32H38O19 20,65 725,19291 579,1362 430,0910 284,0330 283,0252 255,0300 14 Quercetin-3-O-rhamnoside-7-O-glucoside C27H30O16 20,92 609,14557 463,0883 447,0934 446,0858 301,0357 299,0200 [25] 15 Quercetin-O-deoxyhexoside-O-hexoside C27H30O16 21,21 609,14557 463,0884 447,0936 446,0858 301,0357 299,0200 16 Kaempferol-3-O-rhamnoside-7-O-glucoside C27H30O15 22,14 593,15065 447,0938 430,0909 285,0409 283,0253 255,0299 [25] 17 Corniculatusin-3-O-galactoside C22H22O13 22,25 493,09822 331,0462 330,0385 316,0227 315,0152 165,9899 [25] 18 Corniculatusin-3-O-glucoside C22H22O13 22,47 493,09822 331,0461 330,0385 316,0227 315,0151 165,9896 [25]

24 Quercetin-O-malonylhexoside C24H22O15 23,60 549,08805 505,1021 301,0363 300,0276 271,0251

28 Hydroxy-methoxy(iso)flavone-O-hexoside C22H22O9 24,47 431,13421 269,0812 254,0582 213,0908 29 Methoxy-tetrahydroxy(iso)flavone-di-O-deoxyhexoside C28H32O15 24,49 607,16630 461,1094 460,1016 315,0515 313,0358 299,0558 30 Isorhamnetin-O-hexoside C22H22O12 24,69 477,10330 314,0437 299,0207 285,0408 271,0259 243,0298 311 _{Isorhamnetin-3-O-glucoside} _C22H22O12 _24,87 _477,10330 _314,0437 _299,0203 _285,0407 _271,0253 _243,0297 32 Kaempferol-O-malonylhexoside C24H22O14 25,56 533,09314 489,1043 285,0407 284,0325 255,0294 227,0350 33 Methoxy-tetrahydroxy(iso)flavone-O-deoxyhexoside C22H22O11 25,70 461,10839 315,0514 299,0562 284,0329 269,0458 255,0297 34 Vestitol-O-hexoside C22H26O9 26,32 433,14986 271,0979 149,0597 135,0441 121,0281

371 _Quercetin _C15H10O7 _26,95 _301,03483 _273,0416 _245,0457 _178,9977 _151,0025 _121,0279 _[₂₇_]

381 _Naringenin _C15H12O5 _27,16 _271,06065 _177,0186 _151,0025 _119,0490 _107,0125 _93,0333

40 Sexangularetin(8-Methoxy-3,4’,5,7-tetrahydroxyﬂavone) C16H12O7 29,08 315,05048 300,0284 165,9893 [27]

54 SoyasaponinI C48H78O18 37,90 941,51100 733,4520 615,3886 457,3691 205,0712 163,0601 [30]

56 Unidentiﬁedsaponin2 C41H66O13 38,10 765,44252 633,4038 615,3895 457,3691 437,3413 113,0230

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S.Yerlikayaetal./JournalofPharmaceuticalandBiomedicalAnalysis174(2019)286–299 293

Fig.1. DNAprotectioneffectofEA,MeOHandwaterextractsofL.corniculatusonpUC19plasmidandgelanalysis.A,B;(−)control,C;(+)control,D;5mgmethanolextract, E;10mgmethanolextract.F;5mgethylacetateextract,G;10mgethylacetateextract,H;5mgwaterextract,I;10mgwaterextract.*showsp<0.05.

Table4

Totalbioactivecomponents,antioxidant,andenzymeinhibitorpropertiesofthe testedextracts*_.

Extracts

Assays EA MeOH Water

Totalbioactivecomponents

TPC(mgGAE/g) 15.54±0.22 19.94±0.26 23.21±0.06 TFC(mgRE/g) 17.59±0.22 21.13±0.10 16.24±0.25 Antioxidantassays DPPH(mgTE/g) 17.07±1.02 31.94±0.84 56.84±2.06 ABTS(mgTE/g) 96.40±4.66 140.30±4.19 306.04±0.29 CUPRAC(mgTE/g) 69.84±1.65 80.48±1.22 77.86±2.46 FRAP(mgTE/g) 45.24±1.97 56.99±1.62 79.59±1.44

Phosphomolybdenum(mmolTE/g) 1.03±0.07 1.31±0.17 0.76±0.01b

MetalChelating(mgEDTAE/g) 2.53±0.16 11.08±0.72 17.34±1.37

Enzymeinhibitionassays

AChEinhibition(mgGALAE/g) 1.35±0.14 1.41±0.04 Ni

BChEinhibition(mgGALAE/g) 1.38±0.13 1.06±0.15 Ni

Tyrosinaseinhibition(mgKAE/g) 18.34±4.03 24.32±6.66 31.55±7.55

Amylaseinhibition(mmolACAE/g) 0.64±0.05 0.49±0.03 0.09±0.01

Glucosidase(mmolACAE/g) 2.98±0.50 4.79±0.16 3.49±0.16

*_Values_expressed_are_means_±_S.D._of_three_parallel_{measurements.}_EA:_Ethyl acetate;MeOH:Methanol;TPC:Totalphenoliccontent;TFC:Totalﬂavonoid con-tent;GAE:Gallicacidequivalent;RE:Rutinequivalent;TE:Troloxequivalent; EDTAE:EDTAequivalent;GALAE:Galatamineequivalent;KAE:Kojicacid equiv-alent;ACAE:Acarboseequivalent;ni:noinhibition.

ing(water>MeOH>EA).RegardingCUPRACassay,thebestactivitiy

wasobserved by MeOH but this is very closeto that of water

extract.Interestingly,waterextractwasalsothelowestabilityin

thephosphomolybdenumassay,whichisknownasatotal

antiox-idantassay.Ingeneral,theantioxidanteffectscouldbeattributed

tothepresenceofphenoliccompoundsintheextracts.Ascanbe

seeninTable1–3,severalphenoliccompoundssuchaskaempferol, quercetin and isorhamnetin have been reported as signiﬁcant antioxidantsintheliterature.Takentogether,L.corniculatuscould beconsideredasavaluablesourceofantioxidants.

3.2. Inhibitoryeffectsonkeyenzymes

Amongrecentpharmacotherapeuticapproaches,enzymesare maintargettomanageseveralchronicdiseases,includingtype2 diabetesandAlzheimer’sdisease.Thisfactisalsoknownaskey enzymeinhibitiontheory.Basedonthetheory,theinhibitionof keyenzymescouldreducepathologicsymptomsofthementioned diseases[9].Inthisregards,somedrugsfocusontheinhibitionof enzymeslinkedwithhealthproblems,forexampletacrinecould inhibitthecholinesteraseandthusthiswaycouldalleviate syp-tomsofAlzheimerdisease.Also,acarboseisknownasasignﬁciant aninhibitoraganistcarbohydrate-hydrolyzingenzymestocontrol bloodglucoseleveloftype2diabeticpatients[10].However,most ofthesedrugshavebeenreportedtoexhibitsomenegativeeffects (gastrointestinaldisturbances,toxicity,etc.)inalong-term.Taking intoaccountthisfact,thereisagrowinginterestbythe pharma-ceuticalindustriestoidentifynovel,moreefﬁcientandlesstoxic naturalenzymaticinhibitors[11].

Incurrentwork,L.corniculatusextractswereinvestigatedagaint cholinesterase,amylase,glucosidaseandtyrosinase.Theresultsare showninTable4.TheEAandMeOHextractsexhibitedinhibitory effectsonbothAChEandBChE,unliketheaqueousextracts. Regard-ing tyrosinase inhibitory effect, the best activity wasobserved from water extract, followed by MeOH and EA extracts. How-ever,EA extract had thestrongest anti-amylase activityamong the tested extracts. In glucosidase inhibition assay, this order wasMeOH>Water>EA.Thesedifferentresultscouldbeexplained basedontheinteractionofthesecondarymetabolitesandthe con-formationofenzymes.Severalresearchershavereportedthatsome phenolicexhibitedinhibitoryeffectsontheseenzymes.For exam-ple,kaempferolanditsglycosideshavebeenreportedpreviously asamylaseandglucosidaseinhibitors.Inanearliercomputational study,isorhamnetinexhibitedtyrosinaseinhibitoryeffects.Tothe bestofourknowledge,theenzymeinhibitoryeffectofL. cornicula-tusisbeingreportedfortheﬁrsttimeinthepresentstudy.

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294 S.Yerlikayaetal./JournalofPharmaceuticalandBiomedicalAnalysis174(2019)286–299

Fig.2.MTT-basedcellviabilityassayofMCF-7andMDA-MB-231breastcancercellsaftertreatmentofvariousconcentrationsofEA,MeOHandwaterextractswith24,48 and72h.Controlcellsweretreatedwith0.1%ofDMSOsolution.*indicatesp<0.05.

3.3. DNAprotectionactivity

Carcinogenesis process takes place by serial events includ-ingmutationsinproto-oncogene.DNAdamagearisenfromROS (ReactiveOxygen Species)leads totheformation ofmutations. The reduction pathway of molecular oxygengenerates ROS by forming hydroxyl radicals. In this study, we aimed to analyze theDNAdamagegeneratedbyFenton-reaction.Asillustratedin Fig.1,waterextractshowedDNAprotectiveeffectnearlyabout 50% against oxidative reagent, EA and MeOH extracts slightly haveDNAprotectionactivity(nearlyabout25%).DNAprotective activityof extractscan beexplained by thepresence of differ-ent metabolic compounds in Lotus corniculatus aerial parts. In theextracts,naringenin,apigenin,quercetinandquercetin deriva-tiveswere determined.Accordingtoreports, dietaryﬂavonoids showedcancerchemopreventiveeffectsduetoantioxidative prop-erties.Cytoprotectiveeffectofquercetiningreenteaextractagainst hydrogenperoxide-inducedoxidativestressonH2O2-treated MeI-Abcells weredemonstrated ina differentstudy.Quercetin and itsderivativesarethemostpotentantioxidantagainstROS, espe-ciallywhenitreactswithdifferentbiomolecules,suchaslipids, proteinsandDNA[12].Also,inourpreviousstudy,DNA protec-tionactivityofOnonisnatrixsubsp.hispanicawasinvestigatedand waterextract containingquercetinshowedthemost protective effect(78%). The genusOnonis also belongs toFabaceae family [4]. Naringenin (5,7,4’-trihydroxyﬂavanone) is another pharma-cologically important compound that is a potent antioxidant,

anticarcinogenic, anti-inflammatory, antidiabetic, antimicrobial, antimutagenic,antiatherogenic,afreeradicalscavenger, hepato-protectiveandantifibrogenicagent.Itisatypeofflavonoidsthat areabundantincitrus fruitssuchasgrapefruits(Citrusparadisi) andoranges(Citrussinensis).Itwasobservedthatthenaringenin decreasedDNAdamageactivityinanotherreport[13].So,itcanbe concludedthatbecauseofhighquercetinandnaringenincontents insomeFabaceaefamilymembershadapotentialeffectonDNA protection.

3.4. Evaluationofanticancereffects

CytotoxicityeffectofL.corniculatusaerialpartsextractsontriple negativeMDA-MB-231breastcancercellandestrogen receptor-positiveMCF-7breastcancercellwasdeterminedusingMTTcell viabilitytest.Breastcancercellsweretreatedwithdifferentdoses ofextractswithtimepointsat24,48and72h.Asillustratedin Fig.2,thesurvivalofMCF-7andMDA-MB-231cellsdecreasedwith timeanddose-dependentmanner.Waterextractshowedslightly cytotoxicactivityonMCF-7cells.However,1000␮g/mlofwater extractdiminishedcellviabilityofMDA-MB-231cellsat72h.In addition,themostsigniﬁcantcytotoxiceffectwasdetectedfrom theEAandMeOHextractsonbothbreastcancercells.Accordingto ourﬁndingsonMTTcellviabilitytest,IC50valuesofextractswere alsocalculated.NoIC50valuewasobtainedforwaterextractfor MCF-7andMDA-MB-231cellssincetheirsurvivalratewasmore than50%.ForEAandMeOHextracts,thelowestconcentrationsof

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Fig.3. IC50valuesofextractsforMCF-7cellsobtainedfromMTT-basedcytotoxicitytestandcellsmorphologyaftertreatedwithIC50values.Controlcellsweretreatedwith 0.1%ofDMSOsolution.

thesevalueswereselectedforothermolecularmechanism anal-ysis.AsshowninFigs.3and4,inhibitoryconcentrationsagainst MCF-7cancercells weredeterminedas245,8␮g/mlforEA and 589,4␮g/mlforMeOH.Besides,againstMDA-MB-231cancercells, IC50valueswere161,7␮g/mlforEAand442,9␮g/mlforMeOH. Fromtheseﬁgures,itwasalsoobservedthatcellsmorphologywas dramaticallychangedandthecellnumberwasreduced.This quan-titativemeasureshowshowmuchofaparticulardrugorinhibitor isrequiredtoinhibitagivenbiologicalprocessbyhalf.

MDA-MB-231andMCF-7cellsweretreatedwithdosesofIC50 valuesofEA andMeOHextractsforbrighteningofmechanisms behindthedeathofbreast cancercellslines.Bax(pro-apoptotic gene)andBcl-2(anti-apoptoticgene)genesexpressionswere ana-lyzedforevaluationofapoptoticcelldeathmechanism.According toresults,aftertreatmentofMCF-7cellswithEAextract,Baxgene expressiondecreased, whileBcl-2gene expressionincreased.In MeOHextract,onlyBaxgeneexpressionreduced,buttherewas nosigniﬁcantchangeinBcl-2genelevelonMCF-7cells(Fig.5). Asaresult,apoptoticcelldeathwasnotdetectedon caspase-3-independentMCF-7breastcancercellline.

Furthermore,after treatment of MDA-MB-231cells withEA extracts Bax gene expression augmented and Bcl-2 gene level diminished,whilenosigniﬁcantchangeinBax/Bcl-2generatiowas detectedaftertreatmentofMDA-MB-231cellswithMeOHextract. AsshowninFig.6,apoptoticcelldeathwasdetectedafter

treat-mentofMDA-MB-231cells withEAextract inmRNAtranscript level.Afterward,apoptosisalsowasveriﬁedwithapoptoticDNA fragmentationanalysisin DNAlevel ontreatedwithEAextract MDA-MB-231cancercells(Fig.6A).

Apoptotic effect of EA extract on MDA-MB-231cancer cells canberelatedtolinoleamide,a fattyamidelipidmolecule.The compoundisderivedfromlinoleicacid.Itwasreportedthat con-jugatedlinoleicacids(CLA)serve ascancerprotective agents.It wasalso shown in a report that CLAcaused totheexpression ofpro-apoptoticBaxgeneinMDA-MB-231cells[14]. Theeffect oflinoleamide,structuralanalogofoleamide,wasdeterminedon T24cells(humanbladdercancercells)anditinducedincreasing ofCa2+_in_a_{dose-dependent}_manner_[15]._Increase_in_intranuclear Ca2+_leads_to_gene_expression_and_cell_cycle_progression, degrada-tiveprocessesinprogrammedcelldeath,orapoptosis.Similarly, accumulationofCa2+_triggered_critical_step_in_apoptotic_cell_death mechanismbythewayofchromatincondensationandDNA frag-mentation[16].Comparisonof theﬁndingswiththoseof other studiesconﬁrms that thepresenceof linoleamidein EAcaused inhibitionofMDA-MB-231cancercells growththrough apopto-sismechanism.DNAfragmentationisabiochemicalhallmarkof apoptosisanditperformswhen caspase3cleaves theinhibitor. CAD (Caspase-Activated DNase) cleaves thechromosomal DNA to nuclearfragments and initiatesapoptosis. Detectionof DNA ladderisnotanaccurateindicatorofapoptosisinMCF-7breast

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Fig.4.IC50valuesofextractsforMDA-MB-231cellsobtainedfromMTT-basedcytotoxicitytestandcellsmorphologyaftertreatedwithIC50values.Controlcellsweretreated with0.1%ofDMSOsolution.

Fig.5. Expressionproﬁlesofapoptotic(A,B),autophagic(D,E)andtelomeraseactivity(C)markergenesonMCF-7cellsaftertreatmentwithIC50dosesofEAandMeOH extracts.TranscriptlevelsofthemarkergeneswereanalyzedbyqRT-PCR.GAPDHwasusedastheinternalcontrol.*p<0.05.

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Fig.6. (A)ApoptoticDNAfragmentationanalysisonagorosegelelectrophoresisofEAextractsonMDA-MB-231cells.Expressionproﬁlesofapoptotic(B,C),autophagic(D, E),andtelomeraseactivity(F)markergenesonMDA-MB-231cellsaftertreatmentwithIC50dosesofEAandMeOHextracts.Transcriptlevelsofthemarkergeneswere analyzedbyqRT-PCR.GAPDHwasusedasinternalcontrol.*p<0.05.

carcinomacelllinesincetheyarethelackincaspase-3and under-gonecaspase-3-independentapoptoticcelldeathviaothereffector deathcaspases[17].Inourstudy,wehavefoundsameresultsfor MCF-7cancercellline.AlthoughwehaveperformedDNA fragmen-tationanalysisforthiscellline,wedidnotobserveanyeffect.

Bcl-2familyproteinsareoverexpressedinmanycancersresult from some post-translational mechanisms. Bcl-2 family mem-bers, especially Bax,organize mitochondrial apoptoticinitiation via theactivity of soluble cytochrome c to cytosolfrom mito-chondrialoutermembranepermeabilization(MOMP)byleading caspaseactivation. Bcl-2 family memberscan beclassifiedinto pro-apoptoticproteinsandanti-apoptotic proteins.Among pro-apoptotic proteins, Bax and Bak gene expressions are enough for inducing MOMP. Bcl-2-related proteinsalsocause crosstalk between apoptosis and autophagy. Beclin-1 can be linked to anti-apoptoticBcl-2proteinsbyinteractingwithaBH3-only pro-teinandtherebyregulatesautophagy[18].Immunohistochemistry reports showed that Beclin-1protein expression was downreg-ulatedsignificantly inbreast carcinoma.Bad,Baxand BH3-only proteincausetoseparatecompetitivelybindingBeclin-1and Bcl-2family proteinsfrom each otherand thus initiate autophagy. LC3-II(molecularweight16kD)isaspecialmarkerofautophagy by binding autophagosomes. According to immunohistochemi-calanalysis,Beclin-1andLC3-II weredown-regulatedin human lung cancertissues.Mostreports focusedoncrosstalk between autophagyandapoptosis andautophagycan berelatedto pro-grammed cell death. Phosphatidylinositol 3-phosphate kinase (PI3K)–AKT–mTOR (mammalian target of rapamycin) signaling pathwayisresponsibleforcellsurvivalandautophagyisinduced throughthis pathway mechanism. In thepresent study, Beclin-1 and LC3-II (microtubule-associated protein 1 light chain 3) autophagicgeneswerehighlyexpressedincaspase-independent MCF-7 cells after treatment with EA extract, while Bax gene was down-regulated. Bcl-2 gene wassignificantly up-regulated duetotheautophagic mechanismasseenin Fig.5.In

caspase-independentMCF-7cells,aftertreatedwithEAextracts,autophagy cancausetypeIIcelldeathmechanism(non-apoptoticcelldeath mechanism).Itwasnonotablechangeingenesexpressionsafter treatmentofMeOHextractinMCF-7cells(Fig.6).In MDA-MB-231cells,therewasnochangeinBeclin-1geneexpressionafter treatmentofEAandMeOHextracts(Fig.6).However,LC3-IIgene expression wasslightly diminishedafter treatment of extracts. Autophagic cell death (type II programmed cell death) can be detected by protein degradation assay and LC3 levels. MCF-7 cellsdisplayedincrementLC3-IIgeneexpressionaftertreatment withMeOH extract. Upregulation of LC3-II gene can berelated toKaempferol-O-coumaroylhexoside-O-deoxyhexosideisomer2, becausekaempferolup-regulatedtheproteinexpressionlevelsof ATG7,LC3-II/I,inSNU-216gastriccancercells[19].Inthisstudy, MDA-MB-231cellshaveundergoneapoptoticcelldeathafter treat-mentofEAextract.Caspaseactivationandnuclearfragmentation werenotdetectedinautophagiccelldeathautophagyinhibition leadstoapoptosis.

TelomeraseactivitymarkergeneTERT-1wasalsoexaminedin MDA-MB-231andMCF-7breastcancercellsaftertreatmentofEA andMeOH extracts.Mostly,hTERT(humanTERT)geneishighly expressedinsomecancers.Telomeraseactivityisimportantfor theimmortalityofcells.Neoplasticpropertiesbelongto cancer-ouscellsbecausecancercellshavelimitlessreplicativepotential. Sofor thisreason,telomeraseactivitycanbeusedasastrategy forcancertherapy.Inthisstudy,onlyinMDA-MB-231cells,after treatmentofMeOHextracts(asseeninFig.6F),TERT-1genewas markedlydownregulated.MeOHextractsdecreasedcell prolifera-tionofMDA-MB-231cells.Itcanbedocumentedthattelomerase activitymay beusedas cancerproliferation diagnosticmarker. Asmentionedabovereport,kaempferolsuppressedSNU-216cell viabilityand proliferation [19], thusantiproliferative activityof MDA-MB-231cellscanbecorrelatedwithkaempferolmetabolic compoundinMeOHextract.

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Fig.7. EffectsofL.corniculatusextractsoncellmigrationabilityofMCF-7breastcancercells.WoundareaswereillustratedbeforeandaftertreatmentofcellswithEAand MeOHextracts,mitomycinC(10␮g/ml)wasaddedtoallexperimentalplatesfortheaimofpreventingcellproliferation.*p<0.05.

Fig.8. EffectsofL.corniculatusextractsoncellmigrationabilityofhighlymetastaticMDA-MB-231breastcancercells.Woundareaswereillustratedbeforeandaftertreatment ofcellswithEAandMeOHextracts,mitomycinC(10␮g/ml)wasaddedtoallexperimentalplatesfortheaimofpreventingcellproliferation.*p<0.05.

In addition to these comprehensive gene expression analy-sis,wound healingscratchassaywascarriedoutfortheaimof detectingcellmigrationeffectofL.corniculatus.AsseeninFig.7, cellmigrationsignificantlydecreasedonMDA-MB-231andMCF-7 cellsbetweentimepoints0h,12h,24hand48hwhencompared tocontrol.Results showedanti-metastaticeffectofL. cornicula-tusextractsonMCF-7andMDA-MB-231breastcancercelllines (Fig.8).Theanti-metastaticactivityofEAandMeOHextractson cells can be linked tooleamide and linoleamidederivatives. It wasreportedthattheendogenoussleep-inducinglipidhormone, oleamide,anditsderivativesinhibitedspontaneousmetastasison BL6mousemelanomacells[20].Inanotherstudy,isorhamnetin, a3-O-methylatedmetaboliteofquercetin,alonemarkedly pre-vented themigration ability ofgastric cancerusing thewound healingassay[21].Accordingtoreports,activeingredientflavonoid inMeOH,isorhamnetin,isknownasananti-canceragentagainst esophageal,gastric,leukemia,skin,colon,andlungcancers[22]. Isorhamnetinisatypeofdietaryflavonoidsandpresentsinparsley, dillweed,chives,onions,watercress,pearsandwine.Inanother research,anti-skin cancereffects of isorhamnetinwere investi-gatedandfoundthatitisrelatedtotheinhibition ofepidermal growthfactor(EGF)-induced neoplasticcelltransformation. The medicinalfooddecreasedtheproliferationofcancercells[23].

4. Conclusion

Thecurrentstudyis theﬁrstreportthatrevealed toinvitro biological,pharmacologicalandanti-canceractivitiesofLotus cor-niculatus. DNA protection,antioxidantand anticancer effects of extractswereevaluated.Especially,apoptoticeffectofEA (ethy-lacetate)extract wasdetectedon estrogen-negativehighgrade and basal type MDA-MB-231 breast cancer cell line as mRNA transcriptlevelandonDNAfragmentationanalysis.Fragmented DNAisanaccuratebiochemicalevidenceofapoptosis.Cell migra-tion inhibitory effect of extracts was evaluated by the aid of wound healing test. Phytonutrient ingredients of extract dis-played pharmacologically important propertiesin the foodand drug industry. Up to now, anti-oxidant, anti-proliferative and pro-apoptotic effects of dietary natural products were investi-gatedonprostate,breast,brain,pancreatic,melanomaandbrain cancertypes.Presentstudyshowedthatchemicallyand biologi-callyactivecompoundsofLotuscorniculatusextractscanbeused aschemotherapyagentby puriﬁcationofsinglecompound and helpedforbetterunderstandingmechanismsbehind.Single com-poundinpresentstudy,linoleamide,canbealsocombinedwith chemotherapydrugsinordertopreventresistanceofcancercells todrugs.

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Conﬂictsofinterest

Wehavenoconﬂicts.

Acknowledgments

The authors gratefully acknowledge the generous techni-cal and financial support of Kastamonu University (Kastamonu University Scientific Projects Research Office (Project Number KÜBAP-01/2018-7).

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