Zadali , Samad Nejad Ebrahimi Fahimeh Moradi-Afrapoli , Mohammad Shokrzadeh , Fatemeh Barzegar , Gilar Gorji-Bahri ,Reza by HPLC-based activity proﬁling Cytotoxic activity of abietane diterpenoids from roots of Salviasahendica

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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

Cytotoxic activity of abietane diterpenoids from roots of Salvia sahendica by HPLC-based activity proﬁling

Fahimeh Moradi-Afrapoli

^a,b

, Mohammad Shokrzadeh

^c

, Fatemeh Barzegar

^d

, Gilar Gorji-Bahri

^e

, Reza Zadali

^f

, Samad Nejad Ebrahimi

^g,∗

aDepartmentofPharmacognosy,FacultyofPharmacy,MazandaranUniversityofMedicalSciences,Sari,Iran

bDepartmentofPharmaceuticalSciences,UniversityofBasel,Basel,Switzerland

cDepartmentofToxicology,FacultyofPharmacy,MazandaranPharmaceuticalSciencesResearchCenter,MazandaranUniversityofMedicalSciences,Sari,Iran

dDepartmentofClinicalPharmacy,FacultyofPharmacy,MazandaranUniversityofMedicalSciences,Sari,Iran

eDepartmentofPharmaceuticalBiotechnology,SchoolofPharmacy,ShahidBeheshtiUniversityofMedicalSciences,Tehran,Iran

fDepartmentofPharmacognosy,FacultyofPharmacy,TehranUniversityofMedicalSciences,Tehran,Iran

gDepartmentofPhytochemistry,MedicinalPlantsandDrugsResearchInstitute,ShahidBeheshtiUniversity,Evin,Tehran,Iran

a r t i c l e i n f o

Articlehistory:

Received15June2017 Accepted28November2017 Availableonline18January2018

Keywords:

Diterpene Cancercelllines MTT

Cytotoxicity Molecularmodeling

a b s t r a c t

Screeningofmedicinalplantsfrom Iranianfloraagainsthuman cancercell-lineshaveshownthat anhexaneextractfromrootsofSalviasahendicaBoiss.&Buhse,Lamiaceae,isactiveagainsthuman cervicalcancer(HeLa)andcolorectaladenocarcinoma(Caco-2)cell-linesatthetestconcentrationof 100␮g/ml(100%inhibition).CytotoxicityoftheextractwaslocalizedwiththeaidofHPLC-time-based activityprofilingadaptedtothetetrazoliumcolorimetricbioassay.Fourabietane-typediterpenoidsin activetime-windowswereidentifiedascytotoxiccompoundsnamely:sahandone(1),sahandol(2),12- deoxy-salvipisone(3)andsahandinone(4).Compound1showedthehighesttoxicityagainstHeLacells (IC50=5.6±0.1␮g/ml),whichwascomparablewithbetulinicacid(IC50=4.3±1.2␮g/ml),thepositive control.Compound2wasactiveagainsttheHeLacells(IC50=8.9±0.7␮g/ml)butnottheCaco-2cell- line.Compounds1,3and4exhibitedmoderateactivity(IC50=22.9–41.4␮g/ml)againsttheCaco-2cells.

ThisstudyrevealsthattheHeLacellsaremoresensitivetoalltestedcompoundsthantheCaco-2cells.

Insilicomoleculardockingstudyshowedarigidbindingofthecompoundstotyrosinekinasepp60src, andprovedtheircytotoxicactivity.

Introduction

Unregulatedproliferationofcellsduetogeneticalterationsin thebodygeneratescancer.Eachyear,morethan10millionpeople worldwidearediagnosedwithcancerwhilehalfofthepatientsare notsuccessfullytreatedwiththeexistinganticancermedications (Saeidnia, 2015).Therestrictionsandside effectsofthecurrent anti-cancerdrugscreateaconstantdemandfordiscoveryofnew effectiveagents.

SalviaasthemostprevalentgenusoftheLamiaceaefamilyrep- resents more than58 species in Iran(Mozaffarian,2010).Arial parts ofthe plantare aromatic,and have beenused in Iranian folkmedicineasananti-inﬂammatoryremedy,andalsofortreat- ment ofdyspepsia and gastricdisorders (Esmaeili etal., 2009).

∗ Correspondingauthor.

E-mail:sebrahimi@sbu.ac.ir(S.NejadEbrahimi).

SalviasahendicaBoiss.&Buhse,anendemicplantofIran,grows widely in northwest of the country, at thefeet of the Sahand Mountains(Rechinger,1982).Previousphytochemicalstudieshave revealedthepresenceof sesterterpenes,nor-sesterterpenesand nor-diterpenes in the aerial parts of S. sahendica (Moghaddam et al., 1995). However, roots of the plant were reported to berich in abietane type diterpenoids(HadavandMirzaei etal., 2017; Jassbi et al., 2006; Salehi et al., 2007; Moradi-Afrapoli etal.,2013).ContinuingourinvestigationsonIranianmedicinal plants,inthecurrentstudy,wereportoncytotoxicactivitiesof roots of S. sahendicaagainst human cancercell-lines. We have implemented HPLC-time-based activityproﬁling as a miniatur- ized and rapid approach for tracking cytotoxic constituents in the plant extract. In recent years this approach hasbeen suc- cessfullyappliedfordiscoveryofnewanti-protozoalsubstances, positiveGABA_Areceptormodulatorsandanti-HIVcompounds(Kim et al., 2008; Adams et al., 2009; Moradi-Afrapoli et al., 2013;

Potterat andHamburger, 2014).In thisarticle, we reportnovel

https://doi.org/10.1016/j.bjp.2017.11.007

creativecommons.org/licenses/by-nc-nd/4.0/).

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cytotoxiccompoundsobtainedfromahexaneextractofS.sahendica’s roots, and evaluate their activityagainst human cervical cancer(HeLa)andcolorectaladenocarcinoma(Caco-2)cell-lines withcombination of in silico methods for evaluating cytotoxic potential.

Materialandmethods Generalmaterials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- mide(MTT)andBovineserumalbumin(BSA)wereprovidedby Sigma–Aldrich(Germany). Dulbecco’s modiﬁed Eagle’s medium (DMEM) and fetal bovine serum (FBS) “Gold” were from PAA (Germany).Trypsin-EDTAwaspurchasedfromBoehringerIngel- heim(Germany).HeLacelllinewasprovidedbyPasteurInstitute ofIran.Caco-2cells weregiftedbyProfessor GeorgiosImanidis atUniversity ofBasel. HPLCseparationswere performedonan Agilent1100seriesHPLCsystemconsistingofaquaternarylow- pressuremixingpumpwithdegassermodule,PDAdetector,and anautosampler.ForMPLC,aBuchiSepacoresystemconsistingofa controlunitC-620,afractioncollectorC-660,andtwopumpmod- ulesC-605wasused.WatersSunFireC18(3.5␮m,3.0×150mm) andSunFire^® PrepC18(5␮m,30×150mm)columnswereused for analytical HPLC and preparative RP-HPLC, respectively. The detectionwavelengthwas280nm.HPLCsolventscontained0.1%

formicacidforseparations.NPcolumnchromatographywascar- riedoutonsilicagel(Merck,40–63␮m;460×50mm).Pre-coated silicagel F₂₅₄ (20×20cm) platesfor TLC(Merck) wasused for monitoringfractions.Solvents usedfor extraction,opencolumn chromatography,andMPLCwereoftechnicalgrade.NMRspectra wererecordedonaBrukerAVANCEIIIspectrometeroperatingat 500.13MHz for¹HNMR and125.77MHz for¹³C NMR.A 1mm TXImicroprobewithz-gradientwasusedfor¹H-detectingexper- iments.¹³CNMRspectrawererecordedwitha5mmBBOprobe withz-gradient.SpectrawereanalyzedusingBrukerTopSpin3.0 software.

Plantmaterial

RootsofSalviasahendicaBoiss.&Buhse,Lamiaceae,werecol- lectedinSeptember2009fromSardrood,East-Azerbaijanprovince ofIran.TheplantwasidentiﬁedbyDr.AliSonboli,andavoucher specimen(MPH-848)hasbeendepositedattheHerbariumofthe DepartmentofBiology,MedicinalPlantsandDrugsResearchInsti- tute,ShahidBeheshtiUniversity,Tehran,Iran.

Preparationofextract

Theplantmaterialwasshade-driedandgroundwithaRetsch ZM1mill.Thepowderedrootswerepercolatedwithhexane,ethyl- acetateand methanol,successively.Extractionwitheach ofthe solventslastedfor48hatroomtemperature.Theextractswere concentratedunderreducedpressureandﬁnallydriedwithafreeze dryer.

Microfractionationforactivityproﬁling

Aliquotsof360␮gextractin DMSO(36␮lof10mg/mlsolu- tion) were separated by analytical HPLC using a C18 column (5␮l,10×150mm,Interchim).Themobilephasewasamixture of water–acetonitrile containing0.1% formic acid. The gradient startedfrom25%acetonitrileallthewayto100%in30min,and stayedat100%foranother5min.Theﬂow ratewas0.4ml/min.

Micro-fractionsoftheextractwerecollectedina96-welldeepwell

platewithin1-minintervals(1-minfractions).Theplatewasdried byanitrogenevaporatorsetat40^◦C(EvaporexEVX-96,Apricots DesignsInc.,USA).

Cellculture

TheCaco-2cells(humancolorectaladenocarcinomacellline) weregrownin DMEM culturemediumsupplemented withFBS (10%),l-glutamate(200mM),andnonessentialaminoacids(1%).

TheHeLacells(humancervicalcancercellline)weregrowninIPMI medium.Thecellcultureflasksweremaintainedinahumidified incubatorat37^◦Cwith5%CO₂.Themediawerechangedevery alternateday.The flaskswith80%confluence wereusedinthe experiments.

MTTcytotoxicityassay:cytotoxicityofextractsandpure compounds

Cytotoxicity against the HeLa and Caco-2 cell-lines was determinedbythe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazoliumbromide(MTT)assayin96-wellformat(Gerlierand Thomasset,1986;Tofighietal.,2014).Asuspensionofeachcell- linewasseededona96-wellflat-bottomedtissuecultureplateat thedensityof5×10³cells/wellandincubatedinahumidifiedincu- bator(37^◦C,5%CO2)for48h.Thecellsweretreatedwith200␮l testsamplesdissolvedinculturemediumatthefinalconcentra- tionof1–100␮g/ml,andincubatedforthenext48h.Afterwards, 20␮lMTTreagent(5mg/ml)inPBSwasaddedtoeachwell.The platewasmaintainedonanorbitalshakerat100rpmand37^◦Cfor 4htoallowtheMTTreactionbythecellularmitochondrialdehy- drogenases.Finally,themediumwasreplacedwith150␮lDMSO andthecellswereagitatedonanorbitalshakerfor15mintodis- solvetheformazancrystalsformedbythelivecells.TheUV/Vis absorbanceofeachwellwasreadbyamicroplatereaderat570nm withareferencefilterof620nm.Asetofwellswithnotestsample, treatedwiththehighestconcentrationofDMSO(1%),wasconsid- eredascontrol.Thenaturallyoccurringtriterpenoid,betulinicacid, wasusedaspositivecontrol.Cell-viabilityandcytotoxicitywere calculatedaccordingtothefollowingequations:

Cellviability(%)=(Absorbanceoftestwell/Absorbanceofcontrol)×100

Cytotoxicity (%)=100−cellviability

Cytotoxicityofmicrofractions

Eachofthedriedmicro-fractionsinthe96-wellplatewasdis- solved with 5␮l solution of DMSO in PBS. Afterwards,culture mediumwasaddedtoeachwelluptotheﬁnalvolumeof450␮l.

Thevolumesofthemediumbeingaddedtothewellswerecalcu- latedtoapproachaconcentrationoftheactiveconstituentsinthe micro-fractionsproportionaltothatfoundin100␮g/mloftheorig- inalextract.Thecalculationwasbasedontheassumptionthateach activeconstituentmaybeelutedinoneortwo1-minfractions,and alsotheconsiderationofa50%lossthatmayoccurduringchro- matographyand re-dissolution(Potteratand Hamburger,2014).

Theplatewasshakenbyanorbitalshakerat100rpmfor30min.

Themicro-fractionsweresubmittedtoMTTbioassay.Theﬁnaldilu- tions(2fold)occurredinthecell-culturewells.Theexperiments wereperformedinduplicates.TheﬁnalcontentofDMSOineach welldidnotexceed1%.

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Isolationofactiveconstituents

Phytochemical constituents of S. sahendica were puriﬁed as describedpreviously(Ebrahimietal.,2013).

Spectroscopicdataoftheconstituents

Sahandone(1):Colorless;¹HNMR(500MHz,CDCl3)ı7.08(1H, d,J=7.6Hz,H-6),6.95(1H,d,J=7.3Hz,H-7),6.87(1H,s,H-12),5.48 (1H,dq,J=8.5,1.1H-3),5.01(ddd,J=13.1,8.9,4.4Hz,H-2),3.24(s, OCH₃),2.95(sep,J=7.0Hz,H-15),2.68(dd,J=17.0,4.3Hz,H␤-1), 2.53(dd,J=17.1,11.1Hz,H␣-1),2.19(s,H-20),1.80(d,J=1.1Hz, CH₃-18),1.78(d,J=1.1Hz,CH₃-19),1.15(d,J=7.0Hz,CH₃-16),1.14 (d,J=7.0Hz,CH₃-17).¹³CNMR(125MHz,CDCl₃)ı30.9(CH₂-1), 65.4(CH-2),125.0(CH-3),136.2(C-4),136.7(C-5),130.5(CH-6), 126.4(CH-7),128.4(C-8),132.3(C-9),133.5(C-10),93.6(C-11), 135.7(CH-12),140.2(C-13),195.5(C-14),27.2(CH-15),22.1(CH₃- 16),21.4(CH3-17),25.9(CH3-18),18.4(CH3-19),18.9(CH3-20), 50.3(O-CH₃).

Sahandol(2):Colorless;C₂₀H₂₄O₂;¹HNMR(CDCl₃,500MHz):

ı=7.53 (1H, d, J=8.4Hz, H-7), 7.27 (1H, s, H-14), 7.12 (1H, d, J=8.4Hz,H-6),5.80(1H,OH12),5.53(1H,brd,J=8.5,H-3),4.93(1H, ddd,J=10.8,8.5,3.1,H-2),3.44(1H,heptet,J=6.9,H-15),3.12(dd, J=16.4,3.1Hz,H␤-1),2.97(dd,J=16.4,10.8Hz,H␣-1),2.37(3H,s, H-20),1.86(3H,d,J=1.5,H-19),1.81(3H,d,J=1.5,H-18),1.37(3H, d,J=6.6Hz,H-16),1.35(3H,d,J=6.6Hz,H-17);¹³CNMR(CDCl₃, 100.6MHz):ı=138.0(C-11),138.2(C-12),136.2(C-13),136.2(C- 4),129.2(C-5),126.5(C-7),125.3(C-10),125.1(C-6),124.5(C-8), 124.4(C-3),120.2(C-9),115.9(C-14),73.2(C-2),32.5(C-1),27.9 (C-15),25.8(C-19),22.8(C-17),22.7(C-16),18.5(C-18),18.4(C-20).

Sahandinone (3): Red crystals; ¹H NMR (CDCl3, 500MHz):

ı=7.35 (1H, d, J=7.8Hz, H-6), 7.07 (1H, s, H-12), 7.03 (1H, d, J=7.8Hz,H-7), 5.29(1H,t,J=7.1,H-3), 3.08 (2H,m, H-1), 3.01 (1H,heptet,J=6.9,H-15),2.39(3H,s,H-20),2.19(2H,ddd,J=6.8, 8.7, 7.8Hz, H-2), 1.70 (3H, s, H-18), 1.60 (3H, 3, H-19), 1.17 (3H, d, J=6.9Hz, H-16), 1.19(3H, d, J=6.9Hz, H-17); ¹³C NMR (CDCl3,100.6MHz): ı=182.5 (C-11), 181.5 (C-14), 148.1 (C-10), 144.5(C-13),140.3(C-12),140.2(C-5),136.6(C-6),134.9(C-9), 132.8(C-4),128.5(C-8),128.0(C-7),123.8(C-3),30.2(C-1),27.5(C- 2),26.9(C-15),25.7(C-18),21.34(C-16),21.34(C-17),19.7(C-20), 17.4(C-19).

12-Deoxy-salvipisone (4): Red crystals; ¹H NMR (CDCl₃, 500MHz):ı=7.35(1H,d,J=7.8Hz,H-6),7.07(1H,s,H-12),7.03 (1H,d,J=7.8Hz,H-7),4.64(2H,brs,H-18),3.08(2H,m,H-1),3.01 (1H,heptet,J=6.9,H-15), 2.39(3H, s,H-20), 2.23(2H,m, H-3), 2.19(2H,ddd,J=6.8,8.7,7.8Hz,H-2),1.60(3H,3,H-19),1.17(3H, d,J=6.9Hz,H-16),1.19(3H,d,J=6.9Hz,H-17);¹³CNMR(CDCl3, 100.6MHz):ı=182.5(C-11),181.5(C-14),148.1(C-10),144.5(C- 13),140.3(C-12),140.2(C-5),136.6(C-6),134.9(C-9),148.4(C-4), 128.5(C-8), 128.0(C-7), 38.4(C-3),30.2 (C-1), 27.5(C-2), 26.9

(C-15),110.1(C-18),21.34(C-16),21.34(C-17),19.7(C-20),22.4 (C-19).

Proteinpreparation

Theenzymeforinsilicostudieswasselectedbasedonpreviously publisheddata(Tintorietal.,2009;PaulandMukhopadhyay,2004).

TheProteinDataBankPDBfileofkinaseATPkinaseincomplex withAP23451downloadedfromtheRCSB(2BDF)andappliedasa targetformolecularmodelingstudiesEl-Karimetal.(2015).Sub- sequently,allwatermoleculesweredeletedandhydrogenswere added.Inthenextstep,Gasteigerchargeswerecalculatedandthe createdpdbqtfilesweresavedusingAutoDocktools.Themodified PDBfilewassavedaspdbqtfileformat.

Moleculardocking

Moleculardockingwasdoneforallligandsintheworkspace ofAutoDockvinasoftware(TrottandOlson,2010).Theactivesite ATPkinasewasconsideredusingcrystallographicligandposition.

Bindingmechanismandrelatedinteractionsoffourligandswere investigatedinLigplot+(LaskowskiandSwindells,2011),Discovery StudioVisualizerv4.5.

Resultsanddiscussion

ScreeningoftheextractsfromselectedIranianmedicinalplants against humancervical cancer(HeLa) and colorectal adenocar- cinoma(Caco-2)cell-linesrevealedsignificantcytotoxicityofan hexane extract fromS. sahendica’s roots at thetest concentra- tion of 100␮g/ml (100% inhibition).Cytotoxicity of theextract was localized with the aid of HPLC-time-based activity profil- ing.Activityprofileofthe1-minmicro-fractionswascorrelated withthecorrespondinganalyticalHPLCchromatogramasshown inFig.1.Fractions7(Rt6–7min)and14(Rt13–14min)showed thehighest cytotoxicity in both cell-lines (>50%).Based on the high-resolution ESI-TOF-MS data,the molecularformula of the compoundsintheactivetimewindowsweresimilartothediter- penoidswhichwereisolatedfromthisplant,previously(Ebrahimi etal.,2013).Inordertofullycharacterizetheactivecompoundsand assesstheiractivityquantitatively,theirisolationatalargerscale wascarriedout.Thephytochemicalsintheactivetime-windows were purified using column chromatography. The structures wereestablishedwithHR-ESIMS,1Dand2DNMRspectroscopy, and by comparison with the published data (Ebrahimi et al., 2013).Fourabietane-typediterpenoids,sahandone(1),sahandol (2), 12-deoxy-salvipisone (3)and sahandinone (4) werefinally identified.

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100 80 60

60 40

40 20

20 0

0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

1250

1000

750

500

250

0

0 5 10 15 20 25

Time (min.)

Intens. mAUInhIbItIon (%)

A

B

C

Fig.1. HPLC-basedactivityproﬁlingofanhexaneextractfromrootsofSalviasahendica;thebargraphrepresentsthecytotoxicactivity(in%)ofthe1-minmicro-fractions againstHeLa(A)andCaco-2(B)cells,correlatedwiththeHPLC-UV(280nm)chromatogramoftheextract(C).

Table1

Cytotoxicactivitiesofcompounds1–4againstHeLaandCaco-2celllines.

Compound IC50±SEM^a(␮g/ml)

HeLa Caco-2

Sahandone(1) 5.6±0.1 41.4±0.3

Sahandol(2) 8.9±0.7 >100

12-Deoxy-salvipisone(3) 19.2±6.6 29.6±2.0

Sahandinone(4) 14.2±6.9 22.9±0.6

Betulinicacid 4.3±1.2 9.2±1.2

aStandarderrorofthemean.

Cytotoxicityofthepuriﬁedcompounds1–4wassubsequently assessedagainst HeLa andCaco-2 cell-lines (Table1).TheHela cells were more sensitive to the tested compounds than the Caco-2 cells. Compound 1 showed the highest toxicity against Helacells(IC50=5.6±0.1),comparablewiththatofbetulinicacid (IC₅₀=4.3±1.2).Compound2showedactiveagainsttheHeLacells butnottheCaco-2cell-line.Compounds1,3and4exhibitedmod- erateactivityagainsttheCaco-2cells.

Allisolatedcompoundsbelongtoabietanediterpenes.Despite thefactthatabietanediterpenesarepopularphytochemicalsinthe mintfamily(Lamiaceae)(Changetal.,1990;Ikeshiroetal.,1991;

Topc¸uandGören,2007;Wangetal.,2007),compounds1–3have notbeenreportedfromanyothermembersofthefamilyrather thanS.sahendica,sofar(Ebrahimietal.,2013;Jassbietal.,2006).

However,compound 4hadbeenidentiﬁed inrootsofZhumeria majdae(Rustaiyanetal.,1995).Compounds1–4werepreviously introducedasantiplasmodialandantitrypanosomalprinciplesin rootsofS.sahendica(Ebrahimietal.,2013).Sahandinone(4)hasalso shownactivityagainsthumanpancreatictumorcells(MIAPaCa-2) (Fronzaetal.,2011).12-Deoxy-salvipisone(3)and sahandinone (4)werereportedactiveagainstazygomycetesfungi,(+)Blakeslea trispora(Jassbietal.,2006).

Moleculardocking

Thedrug-likeness,molecularpropertiesandtoxicityareessen- tialassessmentsforselectionofdrugcandidatesinearlystagesof drugdiscovery.BasedonLipinski’sruleof5(Ro5),adrugmolecule shouldhaveamolecularweightof500Daorless,maximum5H- donorbondsorless,equaltoorlessthan10H-acceptorbondsand logP(octanol/waterpartitioncoefﬁcient)notgreaterthan5.Ifa compoundhasmorethantwoviolationsofthesecriteria,itwould notprobablybeorallyactive(Lipinskietal.,1997,2012).Inthis studyrotatablebondcount(ROTB)andtopologicalpolarsurface area(tPSA)arealsoanalyzedontopofRo5requisites.Theresults ofouranalysisshowedthatthetestedcompoundssatisfythedrug- likenesscriteriaforamolecularweightbelow500Da,acceptable tPSA,andHdonor/acceptorcapabilities.

Themoleculardockingisavaluablecomputationalmethodin drugdiscovery,providingcomplementaryinformationonmodeof interactionofthebioactivecompounds.Thepossiblemechanisms ofcytotoxicityofS.sahendicacomponentsinvestigatedbyinsilico moleculardockingapproachonthekinase/ATPbindingsiteofSrc tyrosinekinase.Thisclassofenzymesregulatesgrowth,differen- tiation,relocationandapoptosisinmammaliancellsbycatalyzing terminalATPphosphatebindtospeciﬁctyrosineresiduespresent onthetargetsubstrate(Tintorietal.,2009;PaulandMukhopad- hyay,2004).Drug-likepropertiesanddockingscoresofcompounds 1–4withSrckinaseareshowninTable2.Amongthese,compound 1possessgreaternegativebindingenergy(−9.0kcal/mol)incom- parisonwithrestofthecompounds.Thesecomputationalresults wereincompliancewiththeexperimentalvalues,andpersuaded investigationofinteractionsbetweencompound1andkinasein detail.

Several amino acids take part in hydrophobic interactions betweencompound1 andSrckinasesuchasTyr340,Leu 273, Leu 293, Val 281, Met341, Ala 293, Thr338, Ala 403, Val 323

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Table2

DrugLikenessofthe1–4anddockingenergy.

Compound cLogP^a H-donor H-acceptor tPSA^b MW^c Dockingenergy(kJ/mol)

Sahandone(1) 4.78 0.0 3 35.53 326.188 −9.0

Sahandol(2) 6.30 1.0 2.0 29.46 296.17 −8.9

Sahandinone(3) 6.35 0.0 2.0 34.14 298.15 −8.0

12-Deoxy-salvipisone(4) 6.35 0.0 2.0 34.14 298.15 −8.7

Betulinicacid 8.47 2.0 2.0 57.53 456.36 −8.1

aLogarithmofcompoundpartitioncoefﬁcientbetweenn-octanolandwater.

bTopologicalpolarsurfacearea.

c Molecularweight.

Glu310

Asp404

Leu393 Lys295

Val323

Thr338

Ala403

Lig1

Val281

Ala293

Met341

C18

C16 C15

C13 C17

O1 O3C10

C12

C6

C11

C14 C5 C8 C9

C21 C19

C7

C20

C3 C4

C1 C2

O2

Fig.2.Dockingmodelstructureofcompounds1intotheSrctyrosinekinaseproteinbindingpocket.

andMet314.Theseresiduesplayvitalrolesinsettingthebind- ingpocketonthisprotein(Fig.2).Theclosevicinityofaminoacids suchasLeu393, Val 281andAla 293tobenzylgroupof com- pound1proposesexistenceofhydrophobicinteractionsbetween those. In addition, Met 341, Leu 393 and Ala 293 attribute in hydrophobicinteractionswiththeCH₃connectedtobenzylmoi- etyoftheligand.ItisremarkablethatVal281isaversatileresidue that comprises several hydrophobic interactions withdifferent moieties (Fig. 2). Thus, it has been resolved that compound 1 canlocateinhydrophobiccavityoftheactivesiteonSrckinase (Fig.3).Theseresultsareinagreementwiththoseobservedfrom thenative ligand AP23451 (co-crystallizedligand in RCSB Pro- teinDataBank)withcommonresiduesofLeu393,Thr338,Ala 293, Met341and Lys295(Fig.3).Thenumber ofhydrophobic interactionswithresiduespresentintheactivesiteofSrckinase could bea possible reason for thepotent cytotoxic activity of compound1.

Itiscrucialtopredicttoxicityofdrug-likemoleculesinearly investigation stages. Prediction of toxicity of drug-like candi- datescanhelptoanticipatepotential“off-target”activities.Recent advancesincomputersciencesandbioinformaticsmadeitpossible

toobtainanestimatedtoxicpotential(TP).VirtualToxLab^TM,isa powerfulopenaccessplatformforestimationofTPofendocrine and/or metabolicdisruption, carcinogenicity, and cardiotoxicity (Vedanietal.,2015).Itcalculatesthetoxicpotential(TP)andthe bindingaffinity(bindingconstant K)ofanymoleculetosixteen proteins: 10 receptors (androgen, estrogen ␣, estrogen ␤, glu- cocorticoid,liverX,mineralocorticoid,progesterone,peroxisome proliferator-activatedreceptorc(PPARc),thyroidaandthyroidb), fourmembersofthecytochromeP450enzymefamily(1A2,2C9, 2D6and3A4),onetranscriptionfactor(arylhydrocarbonreceptor) andonepotassiumionchannel(hERG).Theisolatedcomponents andstandardcompoundsinthisstudywereevaluatedtoward16 targetproteins.Thetoxicpotential(TP)ofcompoundswerecalcu- latedbynormalizedbindingaffinitiesandthevaluesrangingfrom0 (none)to1(extreme).ThecalculatedTPvaluesfor1–4wasbetween 0.45and0.59,showingamoderatetohighriskofbindingtothe receptors(Table3).Themaintargetfor1–4wasthePPAR␥receptor withbindingaffinityvalues663,338,25and136nM,respectively, whereallcompoundsshowednobindingtocytochromereceptors.

ThepredictionsshowedthesecompoundsmightalsobindtoGR, ARandTR␤receptors.

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VAL281

LEU273

THR340

MET314

ALA403 VAL323

Hydrophobicity 3.00 2.00 1.00 0.00 -1.00 -2.00 -3.00

Fig.3.Bindingmodelof1intoactivepocketofSrctyrosinekinase.

Table3

VirtualToxLab^TM.Bindingaffinityprofile(bindingconstantK)andestimatedtoxicpotential(TP)ofBPA,BPF,BPS,Pergafast^®201andD-8.Thelowertheconcentration,the strongerthebindingaffinitytothetargetprotein.BindingaffinityQ4>100␮Mareconsiderednotbinding.

Receptor Sahandone(1) Sahandol(2) Sahandinone(3) 12-Deoxy-salvipisone(4) Betulinicacid

Androgenreceptor 0.89␮M 3.65␮M 0.92␮M 0.07␮M 1.74␮M

Arylhydrocarbonreceptor 0.29␮M 5.57␮M 0.51␮M 0.43␮M 23.20␮M

CYP4501A2 NB NB 6.89␮M 23.90␮M 90.6␮M

CYP4502C9 NB NB 41.00␮M NB 44.40␮M

CYP4502D6 33.20␮M 6.43␮M 2.54␮M 25.80␮M 0.06␮M

CYP4503A4 63.7␮M NB 8.82␮M 6.73␮M 0.06␮M

Estrogenreceptor␣ 1.42␮M 3.69␮M 2.07␮M 2.52␮M 0.04␮M

Estrogenreceptor␤ 1.68␮M 1.28␮M 1.30␮M 1.43␮M 0.02␮M

Glucocorticoidreceptor 0.45␮M 3.7␮M 0.06␮M 0.13␮M 1.09␮M

hERG 2.67␮M 0.30␮M 0.73␮M 0.36␮M 0.03␮M

LiverXreceptor 1.62␮M 10.30␮M 2.23␮M 0.66␮M 0.62␮M

Mineralocorticoidreceptor 1.62␮M 3.33␮M 1.40␮M 0.21␮M 0.03␮M

PPAR␥ 0.66␮M 0.34␮M 0.03␮M 0.14␮M 0.02␮M

Progesterone 0.90␮M 0.26␮M 0.74␮M 0.20␮M 0.29␮M

Thyroidreceptor␣ 6.14␮M 32.6␮M 0.36␮M 0.16␮M 1.97␮M

Thyroidreceptor␥ 1.10␮M 0.06␮M 1.01␮M 0.61␮M 13.7␮M

Toxicpotential 0.45 0.55 0.59 0.58 0.60

TP60.3(low),0.3<TP60.6(moderate),andTP>0.6(high).

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Conﬁdentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

FMF,MS,FMandGGBparticipatedintheevaluationofcyto- toxicityactivity,acquisitionandinterpretationofdata.FMS and SNEcarriedoutphytochemicalproceduresandanalysis.RZcarried outmolecularmodelingpart.Alltheauthorshavecontributedto criticalreadingoftheﬁnalmanuscriptandapproveditssubmission.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheNMRspectraweremeasuredinDivisionofPharmaceutical Biology,UniversityofBasel,andtheauthorswouldliketothank Prof.M.Hamburgerforhiskindcooperation.Theﬁnancialsupport ofthestudywasfoundedbytheResearchCouncilofMazandaran UniversityofMedicalSciences,andShahidBeheshtiUniversity.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.11.007.

References

Adams,M.,Zimmermann,S.,Kaiser,M.,Brun,R.,Hamburger,M.,2009.Aprotocolfor HPLC-basedactivityproﬁlingfornaturalproductswithactivitiesagainsttropical parasites.Nat.Prod.Commun.4,1377–1381.

Chang,H.M.,Cheng,K.P.,Choang,T.F.,Chow,H.F.,Chui,K.Y.,Hon,P.M.,Tan,F.W.L., Yang,Y.,Zhong,Z.P.,1990.Structureelucidationandtotalsynthesisofnewtan- shinonesisolatedfromSalviamiltiorrhizaBunge(Danshen).J.Org.Chem.55, 3537–3543.

Ebrahimi,S.N.,Zimmermann,S.,Zaugg,J.,Smiesko,M.,Brun,R.,Hamburger,M., 2013.AbietanediterpenoidsfromSalviasahendica–antiprotozoalactivityand determinationoftheirabsoluteconﬁgurations.PlantaMed.79,150–156.

(7)

El-Karim, S.S.A., Anwar, M.M., Mohamed, N.A., Nasr, T., Elseginy, S.A., 2015.

Design,synthesis,biologicalevaluationandmoleculardockingstudiesofnovel benzofuran–pyrazolederivativesasanticanceragents.Bioorg.Chem.63,1–12.

Esmaeili,M.A.,Sonboli,A.,Kanani,M.R.,Sadeghi,H.,2009.Salviasahendicaprevents tissuedamagesinducedbyalcoholinoxidativestressconditions:effectonliver andkidneyoxidativeparameters.J.Med.PlantRes.3,276–283.

Fronza,M.,Murillo,R., ´Slusarczyk,S.,Adams,M.,Hamburger,M.,Heinzmann,B., Laufer,S.,Merfort,I.,2011.Invitrocytotoxicactivityofabietanediterpenesfrom PeltodonlongipesaswellasSalviamiltiorrhizaandSalviasahendica.Bioorg.Med.

Chem.19,4876–4881.

Gerlier,D.,Thomasset,N.,1986.UseofMTTcolorimetricassaytomeasurecell activation.J.Immunol.Methods94,57–63.

HadavandMirzaei,H.,Firuzi,O.,Schneider,B.,Baldwin,I.T.,Jassbi,A.R.,2017.Cyto- toxicditerpenoidsfromtherootsofSalvialachnocalyx.Rev.Bras.Farmacogn.27, 475–479.

Ikeshiro,Y.,Hashimoto,I.,Iwamoto,Y.,Mase,I.,Tomita,Y.,1991.Diterpenoidsfrom Salviamiltiorrhiza.Phytochemistry30,2791–2792.

Jassbi,A.R.,Mehrdad,M.,Eghtesadi,F.,Ebrahimi,S.N.,Baldwin,I.T.,2006.Novelrear- rangedabietanediterpenoidsfromtherootsofSalviasahendica.Chem.Biodivers.

3,916–922.

Kim,H.,Baburin,S.,Khom,S.,Hering,S.,Hamburger,M.,2008.HPLC-basedactivity proﬁlingapproachforthediscoveryofGABAAreceptorligandsusingafunctional assaywithXenopusoocytes.PlantaMed.74,521–526.

Laskowski,R.A.,Swindells,M.B.,2011.LigPlot+:multipleligand–proteininteraction diagramsfordrugdiscovery.J.Chem.Inf.Model.51,2778–2786.

Lipinski,C.A.,Lombardo,F.,Dominy,B.W.,Feeney,P.J.,2012.Experimentalandcom- putationalapproachestoestimatesolubilityandpermeabilityindrugdiscovery anddevelopmentsettings.Adv.DrugDeliv.Rev.64,4–17.

Lipinski,C.,Lombardo,F.,Dominy,B.,Feeney,P.,1997.Towardminimalisticmodel- ingoforaldrugabsorption.Adv.DrugDeliv.Rev.23,3–25.

Moghaddam,F.M.,Zaynizadeh,B.,Rüedi,P.,1995.Salvileucolidemethylester,ases- terterpenefromSalviasahendica.Phytochemistry39,715–716.

Moradi-Afrapoli,F.,Ebrahimi,S.N.,Smiesko,M.,Raith,M.,Zimmermann,S.,Nad- jaﬁ,F.,Brun,R.,Hamburger,M.,2013.BisabololoxidederivativesfromArtemisia

persica,anddeterminationoftheirabsoluteconﬁgurationsbyECD.Phytochem- istry85,143–152.

Mozaffarian,V.,2010.ADictionaryofIranianPlantNames:Latin–English–Persian.

FarhangMo’aser.

Paul,M.K.,Mukhopadhyay,A.K.,2004.Tyrosinekinase–roleandsigniﬁcancein cancer.Int.J.Med.Sci.1,101–115.

Potterat,O.,Hamburger,M.,2014.CombineduseofextractlibrariesandHPLC- basedactivityproﬁlingforleaddiscovery:potential,challenges,andpractical considerations.PlantaMed.80,1171–1181.

Rechinger,K.H.,No.150,4661982.FloraIranica,Graz,Austria.

Rustaiyan,A., Samadizadeh,M.,Habibi,Z., Jakupovic,J.,1995. Twoditerpenes withrearrangedabietaneskeletonsfromZhumeriamajdae.Phytochemistry39, 163–165.

Saeidnia,S.,2015.AnticancerDrugDiscoveryApproaches;ChallengesandDevelop- ment,NewApproachestoNaturalAnticancerDrugs.Springer,pp.1–12.

Salehi,P.,Sonboli,A.,Ebrahimi,S.,Yousefzadi,M.,2007.Antibacterialandantiox- idantactivitiesoftheessentialoilsandvariousextractsofSalviasahendicain differentphenologicalstages.Chem.Nat.Compd.43,328–330.

Tintori,C.,Magnani,M.,Schenone,S.,Botta,M.,2009.Docking,3D-QSARstudiesand insilicoADMEpredictiononc-Srctyrosinekinaseinhibitors.Eur.J.Med.Chem.

44,990–1000.

Toﬁghi,Z.,Asgharian,P.,Goodarzi,S.,Hadjiakhoondi,A.,Ostad,S.N.,Yassa,N.,2014.

PotentcytotoxicﬂavonoidsfromIranianSecurigerasecuridaca.Med.Chem.Res 23,1718–1724.

Topc¸u,G.,Gören,A.C.,2007.BiologicalactivityofditerpenoidsisolatedfromAnato- lianLamiaceaeplants.Rec.Nat.Prod.1,1–16.

Trott,O.,Olson,A.J.,2010.AutoDockVina:improvingthespeedandaccuracyof dockingwithanewscoringfunction,efﬁcientoptimization,andmultithreading.

J.Comput.Chem.31,455–461.

Vedani,A.,Dobler,M.,Hu,Z.,Smieˇsko,M.,2015.OpenVirtualToxLab–aplatformfor generatingandexchanginginsilicotoxicitydata.Toxicol.Lett.232,519–532.

Wang,X.,Morris-Natschke,S.L.,Lee,K.H.,2007.Newdevelopmentsinthechem- istryandbiologyofthebioactiveconstituentsofTanshen.Med.Res.Rev.27, 133–148.