• Nenhum resultado encontrado

Rev. bras. farmacogn. vol.27 número6

N/A
N/A
Protected

Academic year: 2018

Share "Rev. bras. farmacogn. vol.27 número6"

Copied!
5
0
0

Texto

(1)

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

aPhytochemistryDivision,CSIR-NationalBotanicalResearchInstitute,Lucknow226001,India bBiochemistryDivision,CSIR-CentralDrugResearchInstitute,Lucknow226031,India

cBiotechnologyDivision,CSIR-CentralInstituteofMedicinalandAromaticPlants,Lucknow226015,India

dPlantDiversity,SystematicsandHerbariumDivision,CSIR-NationalBotanicalResearchInstitute,Lucknow226001,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.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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

0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

(2)

–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−

MeanODtest

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

(3)

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−1and3400cm−1indicatingthepresenceofaromatic

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.The13CNMRspectrashowedninecarbons,

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

(4)

60

60 80

40

40

20

20

0

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.

(5)

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.

References

Balaji,P.,Bharath,P.,Satyan,R.,Hariharan,G.,2006.Invitroantimicrobialactivity

ofRoccellamontagneithallusextracts.J.Trop.Med.Plant7,169–173.

Cetin,H.,Tufan-Cetin,O.,Turk,A.O.,Tay,T.,Candan,M.,Yanikoglu,A.,Sumbul,

H.,2008.Insecticidalactivityofmajorlichencompounds,(−)-and(+)-usnic

acid,againstthelarvaeofhousemosquito,CulexpipiensL.Parasitol.Res.102, 1277–1279.

Hawksworth,D.,Honegger,R.,1994.Thelichenthallus:asymbioticphenotypeof

nutritionallyspecializedfungianditsresponsetogallproducers.In:Williams, M.A.J.(Ed.),PlantGalls.Organisms,Interactions,Populations,TheSystematics AssociationSpecialVolume,vol.49.ClarendonPress,Oxford,pp.77–98.

Huneck,S.,Yoshimura,I.,1996.IdentificationofLichenSubstances.Springer,Berlin.

Krishna, S.,Singh, D.K.,Meena, S.,Datta, D.,Siddiqi, M.I., Banerjee, D.,2014.

Pharmacophore-basedscreeningandidentificationofnovelhumanligaseI inhibitorswithpotentialanticanceractivity.J.Chem.Inf.Model.54,781–792.

Marshak,A.,Kuschner,M.,1950.Theactionofstreptomycinandusnicacidonthe

developmentoftuberculosisinguineapigs.PublicHealthRep.(1896–1970), 131–144.

Mishra,T.,Pal,M.,Meena,S.,Datta,D.,Dixit,P.,Kumar,A.,Meena,B.,Rana,T.,Upreti,

D.,2016.Compositionandinvitrocytotoxicactivitiesofessentialoilof

Hedy-chiumspicatumfromdifferentgeographicalregionsofwesternHimalayaby principalcomponentsanalysis.Nat.Prod.Res.30,1224–1227.

Mittal,O.,Neelakantan,S.,Seshaadri,T.,1952.ChemicalinvestigationofIndian

lichens.XIV.ChemicalcomponentsofRamalinacalicarisandRamalinasinensis. J.Sci.Ind.Res.IndiaB11,386–387.

Müller,K.,2001.Pharmaceuticallyrelevantmetabolitesfromlichens.Appl.

Micro-biol.Biotechnol.56,9–16.

Nanayakkara,C.,Bombuwala,K.,Kathirgamanathar,S.,Adikaram,N.,Wijesundara,

D.,Hariharan,G.,Wolseleys,P.,Karunaratne,V.,2010.Effectofsomelichen

extractsfromSriLankaonlarvaeOfaedesaegyptiandthefungusCladosporium cladosporioides.J.Natl.Sci.Found.SriLanka33,147–149.

Peyressatre,M.,Prével,C.,Pellerano, M.,Morris,M.C., 2015.Targeting

cyclin-dependent kinases in human cancers: from small molecules to peptide inhibitors.Cancers7,179–237.

Tehler,A.,Dahlkild,Å.,Eldenäs,P.,Feige,G.B.,2004.Thephylogenyandtaxonomy

ofMacaronesian,EuropeanandMediterraneanRoccella(Roccellaceae, Artho-niales).Symb.Bot.Upsal.34,405–428.

Williams,D.E.,Bombuwala,K.,Lobkovsky,E.,deSilva,E.D.,Karunatne,V.,Allen,

T.M.,Clardy,J.,Andersen,R.J.,1998.AmbewelamidesAandB,antineoplastic

epidithiapiperazinedionesisolatedfromthelichenUsneasp.TetrahedronLett. 39,9579–9582.

Yusof,H.,Azahar,H.,Din,L.B.,Ibrahim,N.,2015.Chemicalconstituentsofthelichens

CladoniamultiformisandcryptotheciaSP.Malay.J.Anal.Sci.19,930–934.

Zhang,Y.,2007.Template-basedmodelingandfreemodelingbyI-TASSERinCASP7.

Imagem

Fig. 1. Cytotoxic activity of the methanol extract (100 ␮g) against breast and colon cancer cell lines.
Fig. 2. Cytotoxic activity of isolated compounds against breast and colon cancer cell lines.
Fig. 3. Dose response study of everninic acid and roccellic acid against colon cancer cell line (DLD-1).

Referências

Documentos relacionados

were designed, synthesized and their cytotoxic activity evaluated using HCT-116 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma) and RPE (human nontumor cell line from

The correlation between viruria ≥4.11 log copies/mL with clinical variables revealed that patients submitted to myeloablative regimen seem to have higher viral load than

This work describes the development of a label free immunosensor based on screen-printed AuNPs/carbon and the characterization of its analytical response for staphylococcal

No primeiro caso, a constante deve ter sinal positivo, pois basta lembrarmos que para o caso de um fluido viscoso newtoniano entre duas placas paralelas em que a placa superior

Evaluation of cytotoxic activity of hydroethanolic extract against three human cancer cell lines (MCF-7; breast adenocarcinoma cells, Hep-G2; hepatocellular carcinoma cells and

crus-galli grains as a remedy for cancer ( ’t Mannetje and Jones, 1992 ), and to specify the compounds responsible for its cytotoxic activity against four human cancer cell lines;

The isolated diter- penoids showed in vitro cytotoxic activity against MOLT-4, HT-29 and MCF7 human cancer cell lines.. The obtained evi- dence showed the roots

Furthermore, the samples were screened, using in vitro assays, against different human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer),