Rev. bras. farmacogn. vol.27 número3

RevistaBrasileiradeFarmacognosia27(2017)302–305

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

the

chloroform

extract

and

four

diterpenes

isolated

from

Salvia

ballotiflora

Nimsi

Campos-Xolalpa

_,

_Ángel

_Josabad

_{Alonso-Castro}

_,

_Ernesto

_{Sánchez-Mendoza}

_,

Miguel

Ángel

Zavala-Sánchez

_,

_Salud

_{Pérez-Gutiérrez}

a_{DepartamentodeSistemasBiológicos,UniversidadAutónomaMetropolitanaUnidadXochimilco,CiudaddeMéxico,Mexico} b_{DepartamentodeFarmacia,DivisióndeCienciasNaturalesyExactas,UniversidaddeGuanajuato,Guanajuato,GTO,Mexico}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17October2016

Accepted13January2017

Availableonline10March2017

Keywords:

Cytotoxicity 19-Deoxyicetexone 7,20-Dihydroanastomosine Icetexone

19-Deoxyisoicetexone

Cellviability

a

b

s

t

r

a

c

t

Newcompoundswithchemotherapeuticactivityaresoughtafter,andplantsareanimportantsource ofthesecompounds.Fourditerpenes,19-deoxyicetexone,7,20-dihydroanastomosine,icetexoneand 19-deoxyisoicetexone,wereisolatedfromthehexane-washedchloroformextractofSalviaballotiflora.The cytotoxicactivityofthehexane-washedchloroformextractanditsfourditerpenesweretestedusingthe MTTassayagainstthreetumorcelllines:HeLa(cervicalcancer),A549(lungcancer)andMCF7(breast cancer),andtwomurinecellline:J774A.1(epithelialcancer)andCT26(coloncancer),andtheirIC50

valuesweredetermined.19-DeoxyisoicetexonehadthegreatesteffectonHeLacellswithIC50of3.2␮g/ml

(9.36␮M),whereashexane-washedchloroformextracthadthebestcytotoxiceffectonA549cellswith

anIC50of2.29␮g/ml.Theseeffectsof19-deoxyisoicetexoneandhexane-washedchloroformextractwere

withsimilaractivitycomparedtocisplatin(IC50=1.06␮g/mlinHeLacells,and4.6␮g/ml(15.21␮M)in

A549cells).

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

Introduction

Cancerisacollectionofseveraldiseasesthatoccurwhencells ofthebodydividewithoutstoppingandspreadtoothertissues. Cancerisoneoftheprimarycausesofdeathworldwide.TheWorld HealthOrganizationestimatedthat84millionpeoplediedof can-cerbetween2005–2015(Danhieretal.,2010).Chemotherapyis an important option in the treatmentof cancer. However, the drugsusedduringthistreatmenthavenegativesideeffects,such asfatigue,changesintaste,diarrhea,andhairloss,amongother. For this reason, newchemotherapeutic compoundsare sought. Plants have a long history of usein health care (Etkin, 1981). However,theeffectivenessofmanyoftheseplantshasnotbeen evaluated,and theactivemetaboliteshave not been character-ized.Between 1940 and 2002,most drugs used againstcancer werenaturally-derivedproducts,while8%wereconsiderednatural productmimics(Newmanetal.,2000;Butler,2004).

∗ _{Correspondingauthor.}

E-mail:[email protected](S.Pérez-Gutiérrez).

Salvia ballotiflora Benth., Lamiaceae, commonly known as “mejorana,” is an aromatic shrub. The leaves contain serrated marginsand havehairsonthetopandbottom.Theflowersare bluish-purple in color. S. ballotiflora is used in Mexican tradi-tionalmedicinetorelievepostpartumsymptoms(BibliotecaDigital dela MedicinaTradicional Mexicana).Twoditerpene quinones, icetexone(ICT)andconacytonewereisolatedfromS.ballotiflora and theirstructures wereelucidatedbysingleX-raydiffraction techniques(Watsonetal., 1976).Subsequently, threeicetexane diterpenoids, 19-deoxyicetexone (DEOX), 19-deoxyisoicetexone (DIC)and7,20-dihydroanastomosine(DAM)wereisolatedfromthe aerialpartsofthisplantandtheirstructureswereelucidatedby spectroscopytechniques(Esquiveletal.,1997).Dominguezand co-workersisolatedromulogarzone,icetexone(ICT)andconacytone (Domínguezetal.,1976;Tairaetal.,1976).In2013,the antidiar-rheal propertiesof DEOXwere reported(Pérez-Gutiérrezetal., 2013).

Themaingoalofthisresearchwastodeterminethecytotoxic activitiesof hexane-washed chloroform extract (ESC), and four diterpenoidsisolatedfromS.ballotiflora,asthefirststeptofind newcompoundthatcouldbeusedinthetreatmentofcancer.They weretested against the humancancercell linesHeLa (cervical

http://dx.doi.org/10.1016/j.bjp.2017.01.007

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

N.Campos-Xolalpaetal./RevistaBrasileiradeFarmacognosia27(2017)302–305 303

cancer),MCF7(breastcancer),andA549(lungcancer)aswellas twomurinecelllines:CT26andJ774A.1.

Materialsandmethods

Reagents

Fetalbovine serum (FBS), Dulbecco Modified Eagle Medium (DMEM), antibiotic, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide(MTT),anddimethylsulfoxide(DMSO)were purchasedfromSigma.

Plantmaterial

SalviaballotifloraBenth.,Lamiaceae,wascollectedinJuly2014 inLasComadres,MunicipalityofGuadalcazar,SanLuisPotosiState, Mexico.Theplantidentificationwasconfirmedbythetaxonomist JoséGarcíaPérez.AvoucherspecimenwasdepositedintheIsidro PalaciosHerbariumoftheUniversidadAutónomaofSanLuisPotosí (SLPM43014).Theaerialpartsoftheplantweredriedintheshade atroomtemperature.

Preparationoftheextract

Twohundredgramsofthedried,groundaerialpartsofS. bal-lotiflorawereextractedwithchloroformbyheatingatitsboiling pointfor4h.Then,thesupernatantswerefilteredandevaporatedto drynessunderreducedpressure,afterwhichthesolidwaswashed withhexane.Theyieldwas5.07%.Theextract(8g)wasseparatedby columnchromatographyusingsilicagel(MachereyNagel70–230 mesh)withhexaneasthemobilephaseandincreasingthepolarity withethylacetate,andfractionsof100mlwerecollected.

Structuralanalysis

StructuralidentificationwasperformedbyNMRspectroscopy. The1_Hand13_{CNMRspectrawererecorderonAgilentDD2-600}

(1_H:599.5MHz,13_{C:150.8MHz)NMRspectrometerat25}◦_Cusing CDCl3assolventandTMSwithreference.

Celllinesandcultureconditions

J774A.1andCT26celllineswereobtainedfromATCCandHeLa, MCF7,A549wereobtainedfromInstitutoNacionaldelCáncerof México.ThecellsweremaintainedinDMEMsupplementedwith 10%FBS,penicillin100IU/ml,andstreptomycin100␮g/ml.Allthe

cellswereculturedat37◦_{Cinanatmosphereof5%CO}

2.

Cellcytotoxicityassay

CellswereseededinDMEMin96-wellmicroplatesata den-sityof5×103 cellperwell.After24hincubation,thecellswere treated with concentrations of ESC from 1 to 200␮g/ml, with

eachcompoundatconcentrationsfrom1to200␮M,andwith

cis-platin(CDDP)atconcentrationsfrom0.1to50␮Masapositive

control.Thecellswithouttreatmentwereusedasnegative con-trol.Eachcompoundwasdissolvedinsalinesolution.After48h of treatment, 10␮l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT)at 5mg/ml in PBS was added. The plateswerethenincubatedfor3hat37◦_{C.Then,themediumwas} removed,andtheformazancrystalsweredissolvedinDMSO.The opticaldensity(OD)wasdeterminedat540nmwithanELISAplate reader(Bio-Rad).Sixreplicatesforeachgroupwereusedto deter-mineviabilityusingthefollowingequation,andtheconcentration

leadingto50%inhibitionoftheviability(IC50)wascalculatedby

linearregressionanalysis.

%Viability= ODtreatedcells ODcontrolcells×100

Statisticalanalysis

Allexperimentalvaluesareexpressedasthemean±SEMofat leasttwoindependentexperiments.Statisticallysignificant differ-encesfromthevehiclegroupwereidentifiedbyStudent’st-test orANOVAwithposthocTukeytestforpaireddata.Thelevelof p≤0.05wasusedtodeterminestatisticalsignificance.All calcu-lationswereperformedusingtheGraphPadPrismV.3software system(GraphPadSoftware,SanDiego,CA,USA).

Resultsanddiscussion

ESCwasseparatedbycolumn chromatography,orange crys-tals wereobtainedfromthe fractionwithhexane/ethylacetate (90:10,v/v)andwereidentifiedasDEOX(1):yield0.0043%;m.p. 203–205◦_{C.Fromthefractionelutingwithhexane/ethylacetate} (85:15,v/v),acrystalyellowwasobtained.Thecrystalswere iden-tifiedasDAM(2):yield0.002%;m.p.210–214◦_{C.Yellowsolidwas} obtainedfromthehexane/ethylacetate(75:25,v/v)fraction.This compoundwasidentifiedasDIC(3):yield0.003%;m.p.208–210◦_C. The1_H,and13_{CNMRchemicalshiftswerecorroboratedwith}

previ-ouslyreport(Esquiveletal.,1997).Orangecrystalswereobtained fromthehexane/ethylacetate(80:20,v/v) fraction,which were identifiedasICT(4):yield0.0009%;m.p.210–214◦_C.The1_H,and 13_{CNMRchemicalshiftswerecorroboratedwithpreviouslyreport}

(Domínguezetal.,1976).

Whitesolidwasobtainedfromthehexane/ethylacetate(50:50, v/v)fraction.Thesolidwasidentifiedasamixtureofursolicand oleanolicacids: yield0.015%; m.p.220–221◦_{C. The}1_{H and} 13_C NMRchemicalshiftsofthesetriterpenoidswerecomparedwith thespectraofthereferencesamples.

ThecytotoxiceffectsofESCandthefourcompoundsisolated fromS.ballotiflorawereevaluatedagainstthreehumancancercell lines,HeLa, MCF7and A549,andtwo murinecelllines J774A.1 and CT26at different concentrations (Fig.1)to determine the IC₅₀ values (Table 1). ESC and DIC exhibited the highest cyto-toxic effect on A549,CT26, HeLa, MCF7 and J774A.1 cells. The IC50valueswithESCwere2.29,6.76,23.79,6.57and29.91␮g/ml

respectively. In the five cell lines, DIC exhibited IC₅₀ values of 5.11, 6.17,3.2,14.87and 8.81␮g/mlrespectively (Table1), and

304 N.Campos-Xolalpaetal./RevistaBrasileiradeFarmacognosia27(2017)302–305

80

A

B

C

D

E

F

60

40

20

0

80

60

40

20

0

0 20 40 60 80 100

% cell cytoto

xicity

% cell cytoto

xicity

% cell cytoto

xicity

% cell cytoto

xicity

80

60

40

20

0

% cell cytoto

xicity

% cell cytoto

xicity

1 µm

0.342 µg/ml 3.42 µg/ml17.1 µg/ml34.42 µg/ml68.4 µg/ml 0.342 µg/ml 3.42 µg/ml17.1 µg/ml34.42 µg/ml 68.4 µg/ml 10 µm 50 µm 100 µm 200 µm

1 µm

0.342 µg/ml 3.42 µg/ml17.1 µg/ml 34.42 µg/ml 68.4 µg/ml 10 µm 50 µm 100 µm 200 µm

12.5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml

1 µm

0.342 µg/ml 3.42 µg/ml 17.1 µg/ml 34.42 µg/ml 68.4 µg/ml

0.03 µg/ml 0.3 µg/ml 3µg/ml 9µg/ml 12 µg/ml 12 µg/ml 10 µm 50 µm 100 µm 200 µm

0.1 µm 1µm 10 µm 30 µm 40 µm 50 µm 1 µm 10 µm 50 µm 100 µm 200 µm

HeLa CT26 MCF7 A549 J774A.1

100

80

60

40

20

0

100

80

60

40

20

0

Fig.1.Cytotoxicityactivityof(A)DEOX,(B)DIC,(C)DAM(D)ICT,(E)ESC,andCDDPasapositivecontrol,inHeLa,A549,CT26,MCF7andJ774A.1celllines.

Table1

IC50valuescalculatedforESC,DEOX,DIC,DAMandICTonfivecancercellline.

Cancercellline IC50(␮g/ml)

CDDP DEOX DIC DAM ICT ESC

A549 4.6±2.6 60±9.3*** 5.11±2.9ns 36.66±4.6*** 25.52±7.3** 2.29±3.8ns CT26 2.8±0.8 45.29±2.3*** 6.17±2.5* 39.13±7.4*** 29.20±6.5*** 6.76±1.3* HeLa 1.06±3.8 69.30±2.6*** 3.20±1.9ns 96.02±1.3*** 129.15±2.4*** 23.79±4.6** MCF7 2.14±2.7 68.26±1.3*** 14.87±3.6* 60.56±6.1*** 62.29±4.1*** 6.57±2.1* J774A.1 2.45±2.3 >5000*** 8.81±5.2** >5000*** 48.48±1.9*** 29.91±2.9**

Theresultsrepresentthemean±standarderror(SEM)ofeachofthecompounds(sixindependentexperiments).Significantdifference*p≤0.05,**p≤0.01and***p≤0.001, respectivelyversuscontrolgroup.“ns”notSignificantdifferencefromcontrolgroupp>0.05.

cancercelllines(Fioreetal.,2006).Ethanolandaqueousextracts fromS.ringensshowedIC50valuesagainstcancercelllines

rang-ingfrom179tohigherthan500␮g/ml(Alimpi´cetal.,2015).Our

resultsindicatethatS.ballotifloraexertscytotoxiceffectsonhuman cancercelllines withhigherpotency compared tootherSalvia species.

TheIC₅₀ values of theotherthree diterpenes showedlower cytotoxic effects compared to DIC or ESC. In summary, there is no significant difference of cytotoxic activity of ESC (A549) and DIC (HeLa)compared tothe positive controlCDDP. There-fore, we can suggest that ESC and DIC hold promise in the treatmentof cancer. Different terpenes isolated fromS. pachy-phylla,nativefromMexico,suchascarnosol,16-hydroxycarnosol, and20-deoxocarnosolshowedIC50 values rangingfrom1.18to

3.09␮g/ml in differentcancercelllines (Guerreroetal., 2006).

ThetriterpenoidsurmiensolideBandurmiensicacid,isolatedfrom S. urmiensis, exerted cytotoxic effects against cancer cell lines (IC₅₀=1.1–6.7␮g/ml)(Farimanietal.,2015).DICshowedsimilar

IC50valuescomparedtootherterpenesobtainedfromotherSalvia

species.

N.Campos-Xolalpaetal./RevistaBrasileiradeFarmacognosia27(2017)302–305 305

Conclusions

TheresultsherepresentedalsosuggestthattheESCand ter-penesisolatedfromSalviaballotifloramightbeagoodalternative forthesearchofnewagentsfromnaturalorigintotreatcancer.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authorcontribution

NC-X, AJA-C, MAZ-S, and ES-M carried out the experimen-talstudies.SP-G conceived thestudy,participated inits design and coordination, wrote the manuscript, and helped draft the manuscript.Allauthorsreadandapprovedthefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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