In vitro growth inhibition and bactericidal activity of spathulenol against drug-resistant clinical isolates of Mycobacterium tuberculosis

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RevistaBrasileiradeFarmacognosia29(2019)798–800

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

Short communication

In vitro growth inhibition and bactericidal activity of spathulenol against drug-resistant clinical isolates of Mycobacterium tuberculosis

Angel de Jesús Dzul-Beh

^a,b

, Karlina García-Sosa

^b

, Andrés Humberto Uc-Cachón

^a

,

Jorge Bórquez

^c

, Luis A. Loyola

^c

, Hugo B. Barrios-García

^d

, Luis Manuel Pe ˜ na-Rodríguez

^b

, Gloria María Molina-Salinas

^a,∗

aMedicalResearchUnit-Yucatán,MedicalUnitofHighSpecialty,SpecialtyHospital1,Merida,Yucatan,MexicanSocialSecurityInstitute,IMSS,Mexico

bPlantBiotechnologyUnit,YucatanCenterforScientiﬁcResearch,Merida,Mexico

cChemistryDepartment,FacultyofBasicSciences,UniversityofAntofagasta,Antofagasta,Chile

dFacultyofVeterinaryMedicineandZootechnics ¨Dr.NorbertoTrevi˜noZapata¨,AutonomousUniversityofTamaulipas,CiudadVictoria,Tamaulipas,Mexico

a r t i c l e i n f o

Articlehistory:

Received15June2019 Accepted19June2019 Availableonline15July2019

Keywords:

Bactericidalactivity Drug-Resistant

Mycobacteriumtuberculosis Spathulenol

Multi-drug-resistant Extensively-drug-resistant

a b s t r a c t

SpathulenolwasisolatedfromanextractofAzorellacompactaPhil.,Apiaceae,byvariouschromatographic method;identiﬁcationofthechemicalstructurewasconﬁrmedbycomparingitsspectroscopicdatawith thosereportedintheliterature.Theanti-Mycobacteriumtuberculosisactivityofspathulenolwasevaluated onMDR,pre-XDR,andXDRclinicalisolatesofM.tuberculosis,aswellasonthereferencesusceptiblestrain H37RvanditscytotoxicactivitywasevaluatedontheVeroCellLine.Theanti-M.tuberculosisactivityof spathulenolwastwiceaspotentagainsttheMDR,pre-XDR,andXDRclinicalisolates(6.25␮g/ml)than onthesusceptibleH37Rvstrain(12.5␮g/ml).Additionally,theanti-M.tuberculosisactivityshownby spathulenolwasestablishedasbactericidalondrug-resistantandsusceptiblestrainsofM.tuberculosis.

Finally,cytotoxicactivityontheVerocellline(CC50=95.7␮g/ml)indicatedthatspathulenolisaselective anti-M.tuberculosiscompound,withaselectiveindexof15.31againstdrug-resistantclinicalisolatesof M.tuberculosis.

Introduction

Thespreadofdrug-resistanttuberculosis(TB)isamajorthreat toglobalTB control.In2017,Multi-Drug-Resistant(MDR,resis- tanttoatleastIsoniazidandRifampin)Mycobacteriumtuberculosis strainscausedanestimatedof230,000deathsglobally.Moreover, 22%and8%ofMDR-Mycobacteriumtuberculosisstrainswerealso pre-eXtensively-Drug-Resistant(pre-XDR,MDRplusresistantto oneFluoroquinoloneoroneofthreeinjectablesecond-linedrugs) and XDR (MDR plus resistant to one Fluoroquinoloneand one ofthreeinjectablesecond-linedrugs),respectively(WHO,2018).

Patientsinfectedwithdrug-resistantstrainsofM.tuberculosishave toendurelongertreatments(24monthsorlonger),severeadverse effects,andhighcost,withalowpossibilityofbeingcured(Quan etal.,2017).Hence,thesearchfornewanti-TBdrugsthatarefast- acting,andhighlyeffectiveagainstdrug-resistantM.tuberculosis strains,isapriority.

∗ Correspondingauthor.

E-mail:[email protected](G.M.Molina-Salinas).

In thesearchfor novelanti-TBdrugs,natural productshave playedanimportantroleinmaintaininghumanhealthforthou- sandsof years(Bernardinietal.,2018).Azorellacompacta Phill., commonlyknownas“llareta”,isagreen,compact,resinouscushion shruboftheApiaceaefamilygrowinginthehighAndesofsouthern PeruandBolivia,northeasternChile,andnorthwesternArgentina.

Thismedicinalplanthasbeentraditionallyemployedtotreatcolds, pain,diabetes,asthma,bronchitis,wombailments,gastricdisor- ders,backache,wounds,andaltitudesickness(Wickens,1995).We havepreviouslyreportedontheanti-M.tuberculosisactivityofa numberofnaturalazorellaneandmulinanediterpenoidsisolated fromthismedicinalplant(Molina-Salinasetal.,2010);aspartof ourcontinuingsearchfornaturalproductswithanti-M.tuberculosis activity,wewishtoreportheretheinvitrogrowthinhibitionand bactericidalactivityofspathulenol,isolatedfromA.compacta.

Materialsandmethods

VacuumLiquidChromatography(VLC)andColumnChromatog- raphy(CC)werecarriedoutusingTLC-grade(GF₂₅₄,Sigma–Aldrich) and 70–230mesh (Sigma–Aldrich) silica gel. Thin Layer

https://doi.org/10.1016/j.bjp.2019.06.001

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

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A.J.Dzul-Behetal./RevistaBrasileiradeFarmacognosia29(2019)798–800 799

Table1

Anti-Mycobacteriumtuberculosisactivityofspathulenol.

Organism Drugresistanceproﬁle Spathulenol(␮g/ml)

MIC MBC

M.tuberculosisMDR (STR,INH,RIF,EMB,PZA) 6.25 6.25

M.tuberculosisPre-XDR (STR,INH,RIF,PZA,LVX,OFX) 6.25 6.25

M.tuberculosisXDR (STR,INH,RIF,PZA,AMK,KAN,LVX,OFX) 6.25 6.25

M.tuberculosisH37Rv – 12.50 12.50

STR,Streptomycin;INH,Isoniazid;RIF,Rifampin;EMB,Ethambutol;PZA,Pyrazinamide;LVX,Levoﬂoxacin;OFX,Oﬂoxacin;AMK,Amikacin;KAN,Kanamycin;CZM,Clofaz- imine;MIC,MinimumInhibitoryConcentration;MBC,MinimumBactericidalConcentration.Positivecontrols:M.tuberculosisMDR(OFX,MIC=0.50␮g/ml),M.tuberculosis pre-XDR(CZM,MIC=0.50␮g/ml),M.tuberculosisXDR(CZM,MIC=0.50␮g/ml);M.tuberculosisH37Rv(RIF,MIC=0.06␮g/ml).

Chromatography (TLC) wasperformed on precoated aluminum silica gel plates (60^◦A F254, Merck, 0.2-mm in thickness). TLC plateswereﬁrstobservedunderUltraviolet(UV) light(254and 350nm) and the components were visualizedby spraying the platewithphosphomolybdicacidreagent[250mlof 5%H₂SO₄, 10gof phosphomolybdicacid, and1.25gofcerium (IV) sulfate hydrate(99%)],followedbyheatingfor5minat105^◦C.InfraRed (IR)spectrawereonaNicoletMagna750(FTIR)ThermoScientiﬁc spectrometer.GasChromatography-MassSpectrometry(GC-MS) analyseswerecarriedoutinanAgilentTechnologies6890NGas Chromatographcoupledtoa 5975BMassSpectrometer.Nuclear MagneticResonance(NMR)spectrawererecordedinCDCl3using a Brucker Advanced Ultra Shield 400 (400MHz) spectrometer, withTMSasaninternalstandard.

WholeplantsofAzorellacompactaPhil.,Apiaceae,werecollected innorthernChile.Thevoucherspecimenofthesample(Azc150411- 14)hasbeenpreservedattheNaturalProductsLaboratoryofthe UniversidaddeAntofagasta,Antofagasta,Chile.

Thehexaneextract(27g)ofA.compactawassubjectedtoVLC oversilicagelusingagradientelutionwithmixtures ofhexane ethylacetate(EtOAc,100:0-85:15).Fraction3(953.2mg)wasfur- therpuriﬁed by CC onsilica gel, usinga gradient elution with mixturesofhexaneandEtOAc(100:0-70:30)toyieldpurespathu- lenol(192.7mg),identiﬁed bycomparingitsspectroscopicdata withthosereportedintheliterature(InagakeandAbe,1985).

In vitro anti-M. tuberculosis activity using the modified Microplate Alamar Blue Assay (MABA)was carried out as pre- viouslydescribed(Molina-Salinasetal.,2006)onfourstrainsof M.tuberculosis:threeclinicalisolatesresistanttofirst-linedrugs, first-andsecond-linedrugs,andadrug-susceptiblelaboratoryref- erence strain (H37Rv, ATCC 27294). Spathulenol wasdissolved withdimethylsulfoxide(DMSO)andtestedusingaconcentration rangeof100to1.56␮g/ml.TheresultswerereportedasMinimum InhibitoryConcentration(MIC).Rifampin,Ofloxacin,orClofazimine wereincludedaspositivecontrols.Allevaluationswerecarriedout intriplicate.Spathulenolwasalsotestedformycobactericidaleffect followingtheprocedurepreviouslydescribed(Molina-Salinasetal., 2006).

TheinvitrocytotoxicassayonVeroCells(ATCCCCL-8)waseval- uated usingtheSulforhodamineB(SRB) method(Skehan etal., 1990).Spathulenolwas dissolvedwithDMSO and tested using a concentration range of 200 to 6.25␮g/ml. The results were expressedastheconcentrationofproductthatkilled50%ofthe cells(CC₅₀).Docetaxelanduntreatedcellswereusedaspositive andnegativecontrols,respectively.Allevaluationswereperformed intriplicate,andCC₅₀valueswerecalculatedusingGraphPadPrism ver.5software.

Resultsanddiscussion

PuriﬁcationoftheextractofA.compactayieldedapuremetabo- litethatshowedﬁfteencarbonsignalsinits¹³C-NMRspectrum, suggesting a sesquiterpenoid structure. The ¹H-NMR spectrum

demonstrated the characteristic signals of protons in a cyclo- propaneringatı0.46(dd,J=11.3,9.5Hz,H-6)and0.71(m,H-7) andof protonsinexocyclicdouble-bondsignalsatı 4.67(s,H- 14a)and4.69(s,H-14b).Onthebasisofthisspectroscopicdata, andbycomparingthesedatawiththosereportedintheliterature, thepuriﬁedsesquiterpenewasidentiﬁedasspathulenol(Inagake andAbe,1985),previouslyreportedasacomponentofthevolatile oils(VO)ofCampomanesiaspp.(Limbergeretal.,2001),andalsoas maincomponent(38%)ofVOofAzorellatrifurcata,whichshowed antimicrobialactivityonPseudomonasspp.andStaphylococcusspp.

(Lopezetal.,2018).Similarly,theVOfromSalviacassiacontaining 3.1%ofspathulenolalsodemonstratedactivityagainstsomeGram- positivecocciandGram-negativebacilli(Utsukarcietal.,2019).

Finally,spathulenolisolatedfromHelichrysumamorginumexhib- itedweakactivityonStaphylococcusspp.(Chinouetal.,2004),while aconcentratedfractionfromtheaerialpartsofSalviamirzagan- niicontaining62%ofspathulenoldisplayedaimmunomodulatory effect(Ziaeietal.,2011).

Thetestingofspathulenolagainstdifferentclinicalisolatesof drug-resistantandasusceptibleM.tuberculosisstrainsshowedthat spathulenolwastwiceaspotentagainsttheMDR,pre-XDR,and XDRclinicalisolates(MICandMBC=6.25␮g/ml)asthesusceptible H37Rv(MICandMBC=12.5␮g/ml)M.tuberculosisstrain(Table1).

Theseresultsidentifythissesquiterpeneasananti-M.tuberculo- sisactivemetaboliteaccordingtoreportsintheliterature,wherea MIC≤64␮g/mlisconsideredpromisingactivityforapureproduct (Cantrelletal.,2001).Furtherevaluationofspathulenol,theMBC wereequaltoMICvaluesinallfourM.tuberculosisstrains, sug- gestingthatitsanti-M.tuberculosisactivityisbactericidal,whichis desirabletoreducetheriskofdevelopingresistanceinM.tuberculosisstrains;spathulenolexhibitedantimycobacterialactivityonall drug-resistantclinicalisolates,suggestingthatitstargetinM.tuber- culosiscouldbedifferentfromthecurrentanti-TBdrugs.Testingof spathulenolforitscytotoxicactivityonprimatecellsrevealeda CC50of95.69␮g/ml(positivecontroldocetaxelCC50=1.68␮g/ml) andSelectiveIndexes(SI)of15.31and7.67forthedrug-resistant clinicalisolatesofM.tuberculosisandthesusceptibleM.tuberculosis referencestrain,respectively.Sltodrug-resistantclinicalisolates wereconsiderablyhigherthan10,consideredasbeingofinterest tothepharmaceuticalindustry(Vonthron-Sénécheauetal.,2003).

do Nascimento et al. (2018) reported weak activity in spathulenolisolatedfromPsidiumguineenseexpressedasthecon- centrationthatinhibitsthegrowthof90%ofM.tuberculosisH37Rv (IC₉₀=231.9␮g/ml)using the ﬂuorometricResazurin Microtiter Assay Plate (REMA) method (do Nascimento et al., 2018). Our studiesbycolorimetricMABAshowedthatspathulenolisactive onthe samesusceptible reference strainM. tuberculosisH37Rv (MIC=12.50␮g/ml).Ontheadditionoftwomicroassaysutilized to evaluatetheinhibitory effect of spathulenol,it is important tohighlightthattheworkingM.tuberculosisinoculumforREMA (1.5×10⁷CFU/ml)(Palominoetal.,2002)ishigherthanforMABA (6×10⁶CFU/ml)(Molina-Salinas etal., 2006).AccordingtoJaki etal.(2008),acommonﬁndingintheliteratureonnaturalproducts

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800 A.J.Dzul-Behetal./RevistaBrasileiradeFarmacognosia29(2019)798–800

isthatforthesamecompoundhadreportedinconsistentvaluesof itsbiologicalactivity,whichcouldbeduetotheuniquespeciﬁcity ofeachindividualassayandtovariabilityintheperformanceof thebioassays(Jakietal.,2008).Oneexampleisthecaseofanti-M.

tuberculosisactivityofursolicacidonH37RvusingMABA,which hasreporteddifferentvaluesofMIC:8(Woldemichaeletal.,2003), 31(Guetal.,2004),and65(Jakietal.,2008)␮g/ml.

Authors’contributions

GMMSandLMPRcontributedtoconceptualizationofthestudy;

AHUC,LMPRandGMMScontributed toformalanalysis; JB and GMMScontributedtofundingacquisition;Allauthorscontribute toinvestigationandmethodology;GMMScontributedtoproject administration;JB,LALandGMMScontributedtoresources;AHUC andGMMScontributedtowritingtheoriginaldraftandallauthors writing-review&editingthemanuscript.

Ethicaldisclosures

Protectionofhumanandanimalsubjects

Theauthorsdeclarethatnoexperimentswereperformedon humansoranimalsforthisstudy.

Conﬁdentialityofdata

Theauthorsdeclarethatnopatientdataappearinthisarticle.

Righttoprivacyandinformedconsent

Theauthorsdeclarethatnopatientdataappearinthisarticle.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

This study was supported by CONACYT-México grant # PDCPN2013/213558(GMMS)andUniversidaddeAntofagastaCen- trodeCostosde Rectoría#1001(JBR).FromCONACYT-México AJD-BreceivedBachelor-degreefellowship#22545.Theauthors are grateful to Maggie Brunner,M.A., for theEnglish-language reviewofthisarticle.

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