Rev. bras. farmacogn. vol.27 número6

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Isolation

and

identification

of

cytotoxic

compounds

from

a

fruticose

lichen

Roccella

montagnei,

and

it’s

in

silico

docking

study

against

CDK-10

Tripti

Mishra

a

_,

_Shipra

_Shukla

a

_,

_Sanjeev

_Meena

b

_,

_Ruchi

_Singh

c

_,

_Mahesh

_Pal

a,∗

,

Dalip

Kumar

Upreti

d

_,

_Dipak

_Datta

b

a_{Phytochemistry}_Division,_{CSIR-National}_Botanical_Research_Institute,_Lucknow₂₂₆_001,_India b_Biochemistry_Division,_CSIR-Central_Drug_Research_Institute,_Lucknow_226031,_India

c_{Biotechnology}_Division,_CSIR-Central_Institute_of_Medicinal_and_Aromatic_Plants,_Lucknow_226015,_India

d_Plant_Diversity,_Systematics_and_Herbarium_Division,_{CSIR-National}_Botanical_Research_Institute,_Lucknow_226001,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6February2017

Accepted7July2017

Availableonline7September2017

Keywords:

Dockingstudy

Roccellicacid

Everninicacid

Cytotoxicactivity

a

b

s

t

r

a

c

t

RoccellamontagneiBél.belongstolichenfamilyRoccelleceaegrowingluxuriantlyalongthecoastalregions ofIndia.AsRoccellahasbeenshowntobebioactive,wepreparedmethanolicextractandassessedits anticancerpotential.Themethanolicextractshowedsignificantinvitrocytotoxicactivityagainstfour humancancercelllinessuchascolon(DLD-1,SW-620),breast(MCF-7),headandneck(FaDu).This promptedustoisolatebioactivecompoundsthroughcolumnchromatography.Twocompounds roccel-licacidandeverninicacidhavebeenisolated,outofwhicheverninicacidisreportedforthefirsttime. Boththecompoundshavebeentestedforinvitrocytotoxicactivityinwhichroccellicacidshowedstrong anticanceractivityascomparedtotheeverninicacid.CyclinDependentKinase(CDK-10)contributesto proliferationofcancercells,andaberrantactivityofthesekinaseshasbeenreportedinawidevarietyof humancancers.Thesekinasesthereforeconstitutebiomarkersofproliferationandattractive pharmaco-logicaltargetsfordevelopmentofanticancertherapeutics.Thereforeboththeisolatedcompoundswere testedforinsilicomoleculardockingstudyagainstCyclinDependentKinaseisomerenzymetosupport thecytotoxicactivity.

Introduction

Lichenisasymbioticself-supportingmutualismshowsawide rangeof habitats throughout theworld.It is a stableorganism betweenmycobiontandphotobiont.(HawksworthandHonegger, 1994)Theyfoundgrowingonrocks,bricks,soil,rottingwoodetc. Lichensareapotentialsourceofdifferentbiologicalactivityas anti-tubercular(Marshak and Kuschner,1950), anticancer(Williams etal.,1998),anti-HIV(HuneckandYoshimura,1996)antipyretic analgesic(Müller,2001).

Thelichen species Roccellamontagnei Bél.belongs to family Roccellaceae,foundcommonasepiphytesalongtheCoromandel Coast,TamilNadu,IndiaanditisabundantinPichavaram man-groveforests.Itisafruticosegrowthform(Tehleretal.,2004).R.

montagneiisa richsourceof somanysecondarymetabolitesas

∗ Correspondingauthor.

E-mail:m.pal@nbri.res.in(M.Pal).

orcinol,montagnetol,␤-carotene,␤-sitosterol,erythritol,roccellic acid,lecanoricacidandmethylorsellinateetc.(Mittaletal.,1952). ApreviousreportconfirmsthebiologicalimportanceofR.

mon-tagneiforitsantimicrobialactivity(Balajietal.,2006),insecticidal

activity(Nanayakkaraetal.,2010)andanti-inflammatoryactivity (Cetinetal.,2008).

Thepresentworkdealswiththeprimaryscreeningofcytotoxic activity of methanolicextract, isolation of bioactive molecules, structureelucidationofisolatedcompoundsandidentificationof compoundresponsibleforcytotoxicactivityofR.montagnei.

Materialsandmethods

Lichenmaterial

Inthepresentstudy,lichenmaterials(RoccellamontagneiBél., Roccellaceae)werecollectedfromKovalamSeashore Thirvananth-puram,Kerala,IndiagrownoverCocoesnuciferabarksinthemonth ofAugust2013andwereauthenticatedbyTaxonomyDivision,CSIR

https://doi.org/10.1016/j.bjp.2017.07.006

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–NationalBotanicalResearchInstituteLucknow,India.Herbarium specimens(12634)werepreparedanddepositedattheHerbarium oftheInstitute.

Extraction

Driedlichenmaterial(100g)weremilledintopowderandthen extractedwithmethanol(2.5l)inanextractorfor36h.Theextract wasevaporatedinarotatoryevaporatoranddriedbyvacuumpump toaffordthe7.5gofmethanolicextract.

Compoundisolation

Methanolicfraction(5g)wassubjectedtochromatographyon silicagel(60–120mesh)elutedwithastepwisegradientof hexane-ethylacetate(9.5:0.5,9:1,8.5:1.5,8:2,7.5:2.5,7:3)byvolumeto affordatotalof500fractionsof50mleach.Columnfractionswere analyzedbyTLC,andfractionswithsimilarTLCpatternswere com-binedtogivefivemajorsubcolumnfractions.Columnfraction-1 wasfurtherpurifiedtogive21.5mgofeverninicacid(21.5mg)(1) whitecrystallinecompound(Yusofetal.,2015).Fraction2was fur-therpurifiedtogive16mgofwhitepowdered(2)Roccellicacid (HuneckandYoshimura,1996).

Cytotoxicactivity

Cellcultureandsamplepreparation

MethanolicextractalongwithisolatedcompoundofR. montag-neiweretestedforinvitrocytotoxicactivityagainstfivecancercell lines.Thehumancancercelllinessuchascolon(DLD-1)andbreast (MCF-7)weremaintainedinRPMI-1640medium,whereashead andneck(FaDu)andcoloncelllines(SW-620)inDMEMmedium. Thetestsamples/moleculesweighedinmicro-centrifugetubesand stocksolutionsof100mg/mlwerepreparedbydissolvingthe sam-plesinDMSO.Stocksolutionswerestoredat−20◦C. Aworking

solutionof100␮g/mlwaspreparedbydilutingthestocksolution inculturemedium(RPMI-1640with5%FBS)priortotheassay.

Cytotoxicityassay(SRBassay)

ThestandardcolorimetricSRBassaywasusedforthe measure-mentofcellcytotoxicity(Krishnaetal.,2014;Mishraetal.,2016).In brief,10,000–30,000cellsdependingonthedoublingtimeofeach celltypewereseededtoeachwellof96-wellplatein5%serum con-taininggrowthmediumandincubatedovernighttoallowforcell attachment.Cellswerethentreatedwiththetestsample(100␮l) togiveafinalconcentrationof100␮g/mlandduplicatewellswere included.Untreatedcellsreceivingthesamevolumeofvehicle con-tainingmediumservedascontrol.After48hofexposure,cellswere fixedwithice-cold50%TCA,stainedwith0.4%(w/v)SRBin1%acetic acid,washedandairdried.Bounddyewasdissolvedin10mMTris baseandabsorbancewasmeasuredat510nmonaplatereader (EpochMicroplate Reader,Biotek, USA).Thecytotoxiceffects of compoundswerecalculatedaspercentageinhibitionincellgrowth aspertheformula.

%ofcellskilled=100−

MeanOD_test

MeanODcontrol×100

Moleculardockingstudies

Compoundroccellicacidand everninic acidisolated fromR.

montagneihavebeenevaluatedforcytotoxicactivityandfurtheras

asupportivestudythesetwocompoundsalongwiththestandard drugdoxorubicinhavebeenevaluatedforinsilicomolecular dock-ingstudy.Therefore,inthisstudythecompoundswereselected astheligandbyusingHomosapiensCyclinDependentKinase-10

100.00

80.00

0.00 20.00 40.00 60.00

DLD-1 SW620 FaDu MCF-7

Cancer cell line

% Inhibition

MeoH Extract Conc_ 100µg/ml

Fig.1. Cytotoxicactivityofthemethanolextract(100␮g)againstbreastandcolon

cancercelllines.

(CDK-10isomer)asthebaseenzyme.Theproteinencodedbythis genebelongstotheCDKsubfamilyoftheSer/Thrproteinkinase family.TheCDKsubfamilymembersareknowntobeessentialfor cellcycleprogression.Thiskinasehasbeenshowntoplayarolein cellularproliferationanditsfunctionislimitedtocellcycleG2-M phase.Cyclindependentkinasescontributestoproliferationof can-cercells,andaberrantactivityofthesekinaseshasbeenreported inawidevarietyofhumancancers.Thesekinasestherefore con-stitutebiomarkersofproliferationandattractivepharmacological targetsfordevelopmentofanticancertherapeutics.(Peyressatre et al., 2015). The autodock 4.2 docking software was used to performmoleculardockingsimulationbetweenCDK-10andthe compounds isolatedfromR. montagneialong withdoxorubicin. SequencehasbeenobtainedbyNCBIandmodelofCDK-10hasbeen preparedfromITASSERserver(Zhang,2007).MGLTools-1.4.6was usedtoprepareprotein(protein.pdbqt)andtowritegridparameter file(protein.gpf)anddockingparameterfile(ligand.dpf).Protein preparation includes:(i)removalofwater andionsand extrac-tion of co-crystallized ligand; (ii)addition of polar hydrogens; (iii)assignmentofAD4atomtype;andfinally(iv)assignmentof Gasteigercharges.Thegridmapsrepresentingthenativeligandin theactualdockingtargetsitewerecalculatedwithautogrid4with boxdimensionof126×126×126 ˚Aandspacingof0.375 ˚Aby

tak-ingthecenteroftheligandasthecenterofthegrid.Dockingofthe ligandwasdonewithdefaultparameters.

Resultanddiscussion

Cytotoxicactivity

Roccella montagnei thallus was extracted in methanol and

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IsolationofpurecompoundsfrommethanolicextractofRoccella montagnei

Themethanolicextractwascolumnchromatographedover sil-ica to obtainpure compounds. Isolated pure compounds were identifiedbymeansofspectroscopicanalysis,andtheywere iden-tifiedaseverninic acid(1)and roccellicacid(2).Everninicacid (1) resultedas a white crystallinesolid and its mass spectrum exhibited molecular ion peak at m/z 182, with the molecular formulaC9H10O4.UVspectroscopysignifiesabsorptionbandsat

550nm,moreoverIRspectraofcompound1showedfrequencies at3390cm−1_and₃₄₀₀_cm−1_indicating_the_presence_of_aromatic

hydroxyl group.MS spectra shows basepeak at m/z 149were attributed to orsellinic acid moiety. Proton NMR spectroscopy showspeakat11.58forhydroxylgroup.Twosignalsat6.19and 6.20 attributed to aromatic proton methyl singlet at 2.438 for methylgrouppresentatbenzenering.Signalat3.93denotesfor methoxygroup.The13_C_NMR_spectra_showed_nine_carbons,

sig-nalat␦173.559showedpresenceofcarboxylicgroupandsignalat 52.344showspresenceofmethoxygroupwhichwerethe charac-teristicsignalsof thecompound(1).Signalat166.031,163.803, 144.684 and 106.073 indicated quaternary carbons along with 101.867,112.640fortertiarycarbononesignalat24.271shows methylgrouppresence(Yusofetal.,2015).Allthespectroscopic detailsindicatedthatitisorsellinicacid-4-methylether(everninic acid)withmolecularformulaC9H10O4.Roccellicacid(2)resulted

aswhiteamorphoussolidanditsmassspectrumshowsmolecular ionpeakatm/z300,withthemolecularformulamoreoverIR spec-traofcompound2showsfrequenciesat3000,2900corresponds tocarboxylicgroup.MSspectrashowsbasepeakat283.Proton NMRspectroscopyshowspeakat0.88(t, CH3),1.17(d, CH3),

1.284(s, CH2).The13CNMRspectrashowedninecarbons,

Sig-nalat␦178.988and178.244showedpresenceoftwocarboxylic groups.Signalat30.822showstripletandat30.51doubletsand at30.684singletpeak.Allthespectroscopicdataindicated that thecompoundisRoccellicacidwithmolecularformulaC17H32O4

(HuneckandYoshimura,1996).

CHO

H

H O

HO

HO OH

O O O

CH3

CH₃

OCH₃

1 2

ItisknownthattheeverninicacidisalreadyreportedinCladonia

multiformis,however,tothebestofourknowledge,theisolation

andcharacterizationofeverninicacidfromR.montagneihasnot beenreportedearlier.Boththeisolatedcompoundswereidentified onthebasisofUV,IRand1HNMRdataandcomparedandvalidated withtheexistingliterature.

Cytotoxicactivityofisolatedcompound

Isolatedcompounds have beenevaluated at100␮g/ml dose

wereforinvitrocytotoxicactivityagainsttwocancercelllinesi.e.

breastcancer(MCF-7,MDAMB-231)andcoloncancer(DLD-1, SW-620)(Fig.2).Itisneededtomentionherethatthedosesofpure compoundsmayvaryatmicromolelevel.Thedoseof100␮g/ml ofeverninicacidandroccellicacidare548.92␮Mand332.85␮M respectivelyaspercalculation,thustheeverninicacidshowslower cellgrowthinhibitionpercentageathigherdoseatmicromolescale androccellicacidfoundtohavesignificantcellgrowthinhibition

100.00

80.00

60.00

40.00

20.00

0.00

DLD-1 MCF-7 MDAMB-231 SW620

Cancer cell lines

Everninic Acid

Roccellic Acid Drugs Conc_100µg/ml

% Inhibition

Fig.2.Cytotoxicactivityofisolatedcompoundsagainstbreastandcoloncancercell

lines.

at100␮g/ml(332.85␮M)doseagainstMCF-7andDLD-1i.e.75.84 and87.90%,respectively,howeveritwaseffectiveagainst MDAMB-231with65.30%cellgrowthinhibition.Outofisolatedcompounds roccellicacidwasfoundtobecytotoxicagainstdifferentcancercell linesbutshowsmostsignificantcytotoxicactivityincoloncancer i.e.DLD-1.ThusIC50ofboththecompoundshavebeencalculated

asperdoseresponsecurveagainstDLD-1tocomparethe cyto-toxiceffects(Fig.3).IC50ofroccellicacidwasfound71.26␮g/ml

(237.18␮M)whereaseverninicacidshowsIC50valuemorethan

100␮g/ml.

In-silicocomparativemoleculardockingstudiesofisolated

compoundsagainstCDK-10

Toinvestigatetheeffectofdifferentcompoundsoncancercell linewehavechosenCyclinDependentKinase-10(CDK-10isomer)

ofHomosapiensassubstratebecauseitisanimportantenzymein

thegrowthphaseofthecancerouscells.Everninicacidandroccellic acidhavechosenasligands.Moleculardockingisthesimulations ofbindingofeverninicacidandroccellicacidwithactivesiteof CDK10wascarriedoutusingAutodock4.2.Fromthisstudywecan concludeaboutH-bondinteractionswiththeactivesiteresidues. EverninicacidformsoneHbondwiththehydrogenofARG71:HH11 alongwiththecarbonyloxygenofeverninicacidLIG1:O.(Fig.4)The estimatedfreebindingenergyofeverninicacidis−6.65kcal/mol

withthe estimated inhibition constant, Ki=13.37␮M. Roccellic acidformstwohydrogenbonds,onewiththehydrogenofroccellic acidalongwithnitrogenofALA187andotherisbetweenoxygen ofroccellicacidwithhydrogenofASN343HD22(Fig.5).Roccellic acidhasshownestimatedfreeenergyofbinding−6.75kcalmol−1

estimatedinhibitionconstant,Ki=11.35␮M.

Conclusion

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60

60 80

40

20

0

-20 _-20

-40 -40

100µg 50µg 25µg 12.5µg 12.5µg 6.25µg 6.25µg 100µg 50µg 25µg

Drug Conc_ Drug Conc_

% Inhibition % Inhibition

Everninic Acid

IC50 Value>100

Roccellic Acid

Ic50 Value 71.26±2.0

Fig.3.Doseresponsestudyofeverninicacidandroccellicacidagainstcoloncancercellline(DLD-1).

Everninic Acid

Cyclin Dependent k

inase-10

1 H-Bonds

Fig.4. Moleculardockingstudyofeverninicacidagainstcyclindependentkinase-10.

2 H-Bonds

Cyclin Dependent Kinasa-10

Roccellic acid

Fig.5. Moleculardockingstudyofroccellicacidagainstcyclindependentkinase-10.

knowledgeeverninicacidisreportedforthefirsttimeinR.

mon-tagnei.Itisbelievedthatmajorityofcompoundsoriginatedfrom

fungalcomponent. Probablyeverninic acidmight beoriginated fromfungalcomponentofR.montagneiasitisalreadyreported

fromCladoniamultiformis(Yusofetal.,2015).Boththecompounds

havebeenevaluatedagainsthumancancercelllines.At100␮g roccellicacidshowssignificantactivityagainstbreast andcolon cancercelllines.Roccellicwasfoundtohavesignificantcellgrowth inhibitionagainstDLD-1withIC50value71.26␮g/mlasperdose

responsecurvestudy.Itis alsosupportedbyinsilicomolecular dockingstudywhichshowedthateverninicacidformoneand

roc-cellicacidshowedtwohydrogenbondingwithfreebindingenergy

−6.65kcal/moland−6.75kcal/molrespectively.Althoughroccellic

acidformtwohydrogenbondinteractionbutfreebindingenergy ofroccellicacidfoundtohavelesserthaneverninicacidthusitis confirmedthatroccellicacidfoundtohavebettercytotoxicactivity overeverninicacid.

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someothernovelcompoundresponsibleforsignificantcytotoxic activityofR.montagnei.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthepublication of patientdata.

Right to privacy and informed consent. The authors have obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Authors’contributions

Conceivedanddesigntheexperiments:MPDDDKU.Performed theexperiments:TMSS,SM,andRS.Analyzedthedata:MPDD. Contributedreagents/material/analysistools:DDDKU.Wrotethe paper:MPTMSSSM.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsarethankfultotheDirector,CSIR-NationalBotanical Research Institute, Lucknow, Indiafor facilities and encourage-ments.ThefinancialsupportreceivedfromtheCouncilofScientific andIndustrialResearch,NewDelhiundertheproject ‘Bioprospec-tionofPlantResourcesandOtherNaturalProducts(BSC-0106)’is dulyacknowledged.

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