Rev. bras. farmacogn. vol.27 número1

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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

Bioassay-guided

fractionation

and

antimicrobial

and

cytotoxic

activities

of

Cassia

bakeriana

extracts

Luís

C.S.

Cunha

a

_,

_Sérgio

_A.L.

_de

_Morais

a

_,

_Francisco

_J.T.

_de

_Aquino

a

_,

_Roberto

_Chang

a

_,

Alberto

de

Oliveira

a

_,

_Mário

_M.

_Martins

a

_,

_Carlos

_H.G.

_Martins

b

_,

_Laís

_C.F.

_Sousa

a

_,

Tricya

T. Barros

a

_,

_Cláudio

_V.

_da

_Silva

c

_,

_Evandro

_A.

_do

_Nascimento

a,∗

a_Núcleo_de_Pesquisa_em_Produtos_Naturais,_Instituto_de_Química,_Universidade_Federal_de_Uberlândia,_Uberlândia,_MG,_Brazil b_Laboratório_de_Pesquisa_em_{Microbiologia}_Aplicada,_Universidade_de_Franca,_Franca,_SP,_Brazil

c_Laboratório_de_Biologia_Molecular,_Instituto_de_Ciências_Biomédicas,_Universidade_Federal_de_Uberlândia,_Uberlândia,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5February2016

Accepted9August2016

Availableonline15September2016

Keywords:

Antimicrobialactivity

Cassiabakeriana

Cassicacid

Cytotoxicity

1,8-Dihydroxy-anthraquinone-3-carboxylic acid

Rhein

a

b

s

t

r

a

c

t

TheantimicrobialpotentialofextractsofbarkandleavesofCassiabakerianaCraib,Fabaceae,against aerobicandanaerobicoralbacteriawasevaluatedbythemicrodilutionbrothmethod.Forcrudeethanol extractsandorganicfractionstested,thebarkdichloromethanephaseshowedasignificantantibacterial effect,withMICvaluesrangingfrom12.5to100␮g/mlformostofthemicroorganismstested.Thus,a

bioassay-guidedfractionationofthisfractionwasperformed.Thisfractionationledtoisolationofthe 1,8-dihydroxy-anthraquinone-3-carboxylicacid,alsoknownascassicacidorrhein.Itisthefirsttime thatthisbioactiveanthraquinonehasbeenisolatedfromthisplant.Rheinexhibitedgoodselectivityand highactivityagainstanaerobicmicroorganisms,withMICvaluesrangingbetween3.12␮g/ml(11.0␮M)

and25␮g/ml(88.0␮M).Theseresultswereconsideredverypromisingsincethemostactivesamples

andrheinshowedgreaterselectivityagainstoralmicroorganismsthantoxicitytoVerocells.

Introduction

CassiabakerianaCraib,Fabaceae,isatreebelongingtothegenus

Cassia.It is nativein Thailandbeingalso knownaspink cassia (Lorenzietal.,2003).Alargevarietyofcompoundshasbeen iso-latedfromspeciesofthegenusCassia.Theplants ofthisgenus areconsideredanimportantsourceofanthraquinones,alkaloids, flavonoidsandotherphenolicbioactivecompounds.These metabo-lites exhibit important biological activities such as anti-emetic (Ahmedetal.,2012),laxative,anti-diabetic,hepatoprotective(Dave and Lediwane, 2012), anti-inflammatory, antipyretic, antiviral, antioxidant,antibacterial,antifungal,andanalgesicamongothers (ViegasJúnioretal.,2006;Mazumderetal.,2008).

AlthoughtheuseofmanyCassiaspeciesforthetreatmentof variousdiseasesiswellestablished,thereisstillachemicaland pharmacologicalpotentialtobeexploredinotherspeciesofthis

genus(Mazumder et al.,2008; Dave and Lediwane,2012).The

essentialoilschemicalcompositionfrombark,woodandleavesof

C.bakerianahavealreadybeendetermined,andexceptforwood,

∗ Correspondingauthor.

E-mail:eanascimento@ufu.br(E.A.Nascimento).

thebarkandleavesessentialoilsexhibitedhighantimicrobial activ-ityagainstaerobicandanaerobicoralmicroorganisms,aswellas lowtoxicity(Cunhaetal.,2013).

Theappearanceofmicroorganismsthatareresistantto vari-ousantibioticsandtheirsideeffectshasledtointerestinplants withantimicrobialproperties(NamitaandMukesh,2012).Several naturalproducts,mainlyobtainedfromplants,havebeentested withthepurposeofevaluatingtheantimicrobialactivityonoral microorganisms(Cunhaetal.,2007;Portoetal.,2009;Carvalho et al.,2011; Souza etal., 2011a,b;Waldner-Tomic et al.,2014; Bardajietal.,2016).

Intheoralcavity,aswellasotherpartsofthehumanbody,there

is a characteristic microbiota in dynamic equilibrium with the

host.Ifthisequilibriumisbroken,bacteriathatwereoutnumbered maydeveloporallowsthecolonizationofothermorepathogenic

microorganisms (Marsh and Devine, 2011). This condition can

lead to thedevelopment of oral diseases ascaries, endodontic

lesions and periodontitis (Aas et al., 2005). In addition, oral microorganismscantriggervarioussystemicdiseases, including cancer(Aasetal.,2005;WhitmoreandLamont,2014).Someofthe riskfactorsthatpromotedisequilibriumbetweenoralmicrobiota and host are unhealthy diet, tobacco use, harmful alcohol use,

poororalhygiene,and socialdeterminants(WHO,2012).When

http://dx.doi.org/10.1016/j.bjp.2016.08.002

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disequilibriumoccurs,itisnecessarytorestoreoralhealth.Then,

in addition to the mechanical treatment and hygienisation, it

becomes important touseantimicrobial agents toprevent and

controltheprevalenceoforalpathogens(TelesandTeles,2009). Antimicrobialactivityofcrudeextractsandisolatedcompounds fromplantsareoftenassociatedwithtoxicitytestsusingVerocell line(Baglaet al.,2014), whichisoneofthemostusedcellline inthebiologyresearch(Ammermanetal.,2008).Thesetestsare necessarytodetermineifthesampleisselective,i.e.,ifitexhibits antibacterialeffectwithoutshowingsignificanttoxicity toVero cells(Baglaetal.,2014).

Theaimofthisstudywastodeterminetheantimicrobial activ-ityofextractsoftheleavesandbarkofC.bakerianaagainstoral bacteria,performingconcomitantlythephytochemicalstudyofthe mostactiveextract.Thetoxicityofthemostactivesampleswasalso tested.

Materialsandmethods

Plantmaterial

Barkand leavessamplesofCassiabakerianaCraib.,Fabaceae, werecollectedfromspecimensagedapproximatelyeightyearson March2009attheFederalUniversityofUberlândia,MinasGerais, Brazil(18◦₅₅′_8.95′′_S;₄₈◦₁₅′_34.01′′_W)._The_plant_was_identified_by

specialists,andavoucherspecimenwasdepositedinthe Herbar-iumUberlandensesoftheFederalUniversityofUberlândia,under

thenumber63584(HerbariumCode–HUFU).

Generalprocedures

TheNMRspectrawereobtainedona BrukerDRX-400

spec-trometerusingtetramethylsilaneasinternalstandard.Theinfrared

analyses were performed on Shimadzu IR Prestige-21 in KBr.

SephadexLH-20® _and_silica_gel₆₀_G_(70–230_mesh)_were_used

asthestationaryphaseincolumnchromatography.TLCwas

per-formed on silica gel 60 F 254 (5–40␮m) and silica gel 60 G

(5–40␮m)platesandthespotswereanalyzedunderUVlight(254

and366nm)and thedevelopingsolutions usedwere

methano-licsolutionofaluminiumchloride1%(w/v),ethanolicsolutionof potassiumhydroxide10%(w/v)(Bornträgerreagent)andammonia vapours(Waksmundzka-Hajnosetal.,2008).

Preparationofethanolextractsandliquid–liquidpartition

TheleavesandbarkofC.bakerianaweredriedinanovenat40◦_C

fortendaysandpulverizedinaballmill.Thedrypowderofthe leaves(1kg)andbark(1kg)wasextractedthreetimeswith2lof ethanol96%bymacerationatroomtemperatureforsevendays.The mixturewasfilteredandthefiltratewasconcentratedonarotary evaporatorunderreducedpressureat40◦_C,_yielding₈₂_g_and₆₈_g

ofextractives,respectively.Thedryethanolicextractsfromleaves (EL)andbark(EB)with70and60g,respectively,wereredissolved in250mlofamethanol/watersolution(9:1,v/v).Asuccessive par-titionoftheELextractyieldinghexane(8.07g),dichloromethane (34.35g),ethyl acetate(10.8g)and methanol(14.36g) fractions andoftheEBextractaffordinghexane(6.32g),dichloromethane (9.84g)and ethylacetate (42.6g)fractions. Subsequently,these fractionsweresubjectedtotestsofantimicrobialactivity.

Bioassay-guidedfractionationandisolationof 1,8-dihydroxy-anthraquinone-3-carboxylicacid

Thebioassay-guidedfractionation wasonlycarried outwith

PB2(Scheme1)becauseitwasthemostactiveagainsttheoral

microorganisms evaluated. Seven grams of PB2 was

fraction-ated using a glass column packed with silica gel 60H (Merck

70–230 mesh, 10×50cm), eluted with hexane (500ml),

hex-ane/dichloromethane(1:1,400ml;2:3,400ml),dichloromethane

(400ml), dichloromethane/ethyl acetate (3:2, 2100ml; 1:1,

400ml; 2:3, 300ml; 1:4, 300ml), ethyl acetate (2400ml),

ethyl(acetate/methanol) (1:1, 900ml) and methanol (2000ml)

in orderof increasingpolarity. Twenty-fourfractions were col-lectedand grouped into fourteenfractions after monitoringby

TLC(F1–F14) usinghexane/ethyl acetate (1:1, 2:3) and

chloro-form/methanol(3:2, 1:1,2:3)asmobilephase. Themostactive fraction, F-11(208.0mg),wasresuspendedin 5ml ofmethanol

and then fractionated with Sephadex LH-20 (Healthcare, 50g,

5×50cm) using methanol as mobile phase. Sixty fractions of

approximately10mlwerecollectedandmonitoredbyTLC(mobile phasechloroform/methanol3:2,1:1,2:3),generatingeight sub-fractionsF11.1toF11.8.OnlyF11.1(79.0mg),F11.2(55.0mg)and

F11.3(67.0mg) were evaluatedagainst oral microorganismsas

havingpresentedsufficientquantityforantimicrobialtests.F11.3 showedthehighestantimicrobialactivityand,therefore,emphasis wasplacedonitsanalysis.ThephytochemicalprospectionofF11.3 anditsanalysiswerecarriedoutbyLC–ESI-MS/MS.F11.3(65mg) wassubmittedtopreparativechromatographyonglassplateswith silicagel60Gusingchloroform/methanol(8:2)asmobilephase. ThecompoundthatpresentedRf0.32wasremovedfromtheplates

withsilicaandextractedwithchloroform/methanol(1:1)togive ayellowsolid(8mg).Therepresentationofcontinuousprocedure ofextractionandfractionationofC.bakerianaleavesandbarkare showninScheme1.

Characterizationoftheisolatedcompound

Theisolatedcompoundwasidentifiedbyspectroscopic

analy-sisasFTIR,UV–vis,LC–ESI-MS/MS,H1_NMR,_COSY_and_HSQC_and

bymeltingpoint.Theresultswerecomparedwithspectroscopic datapreviouslypublished(Danielsenetal.,1992;Weietal.,2003; Yeetal.,2007;Dionex,2009;GavitandLaddha,2010;Jiangetal., 2012).

HPLC-DAD-ESI-MS/MSconditions

F11.3fractionatconcentration500␮g/mlwasanalyzedona

ShimadzuProminenceLiquidChromatographicsystemequipped

withquaternaryhighpressurepump(LC-20AD),automaticinjector

(autosampler) (SIL 20AC) and UV/vis photodiode array

detec-tor (DAD) model SPD-M20A. The chromatographic separation

was performed on a reverse-phase Phenomenex C18 column

(50mm×2.10mm×2.6␮m)maintainedat40◦Cinanoven.The volumeinjectedwas5␮l,inaflowof0.13ml/minusingwater acid-ifiedwithformicacid(0.1%,v/v)asmobilephaseAandmethanolas mobilephaseBinfollowingprogram:15–30%B(0–5min);30–50% B(5–10min),50–70%B(10–15min);70–100%B(15–30min);100% B(30–35min),100–15%B(35–40min)and15%B(40–43min).The diodearrayUV/visdetectorwassetto190–800nm.Themass

spec-trometrydetectionwasperformedinaShimadzuLC-IT-TOFwith

quadrupoleiontrap(IT)andtimeofflight(TOF)sequentialmass spectrometer,usingN2asnebulizergasat1.5l/min,temperature

ofthedesorptioncurveline(DCL)at200◦_C,_drying_gas_at₁₀₀_kPa,

ESIionizationat+4.5and−3.5kV,andionaccumulationtimeof 10ms.TheTICchromatogramswereobtainedinpositiveand

neg-ativemodewithm/z50–1000.Theproposedmolecularformula

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L.C.Cunhaetal./RevistaBrasileiradeFarmacognosia27(2017)91–98

Plant

(Cassia bakeriana)

Crude ethanol extract Leaves-EL (70.0 g)

Crude ethanol extract Bark-EB (60.0 g)

Column chromatography - Silica gel - Solvent system: hexane/hexane: dichloromethane/ dichloromethane/dichloromethane:ethyl acetate/ethylacetate/ethyl acetate:methanol/ methanol.

Maceration in ethanol anhydrous

Fractions

Subfraction

column chromatography- Sephadex LH20 Elution with methanol

CCD - Preparative chromatography Elution with chloroform/methanol (8:2 v/v)

Yellow solid (8mg)

1,8-dihydroxy-anthra quinone-3 -carboxylic acid Fractions

F1 (136mg)

F2 (130mg)

F3 (168mg)

F4 (660mg)

F5 (288mg)

F6 (196mg)

F7 (160mg)

F8 (66mg)

F11.1 (79mg)

F11.2 (55mg)

*F11.3 (67mg)

F11.4 (1.0mg)

F11.5 (1.3mg)

F11.6 (1.9mg)

F11.7 (1.4mg)

F11.8 (1.0mg) F9

(41mg) F10 (243mg)

F11 (208mg)

F12 (1412mg)

F13 (366mg)

F14 (871mg) Hexane

PL1 (8.07g)

Dichloromethane PL2 (34.35g)

Ethylacetate PL3 (10.8g)

Methanol PL4 (14.36g)

Hexane PB1 (6.32g)

*Dichloromethane PB2 (9.84g)

Ethylacetate PB3 (42.6g)

Antimicrobial

activity

Antimicrobial

activity

Antimicrobial activity

*Sample more active antimicrobial actiivity

I-Solubilization in ethanol/water (9:1 V/V). 2-Successive liquid-liquid partition.

Scheme1.RepresentationofcontinuousprocedureofextractionandfractionationofCassiabakeriana.

theliterature,solventsystem,retentiontimes,ultravioletspectrum

(UV)andmassspectrum.

Microbialstrains

ThetestedstrainswerepurchasedfromtheAmericanType Cul-tureCollection(ATCC).Thefollowingmicroorganismswereused inthepresentwork:aerobicStreptococcusmutans(ATCC25175),

Streptococcus mitis (ATCC 49456), Streptococcus sanguinis(ATCC 10556), Streptococcus sobrinus (ATCC 33478), Enterococcus fae-calis(ATCC4082)andAgregatibacteractinomycetemcomitans(ATCC 43717)andanaerobicFusobacteriumnucleatum(ATCC25586), Bac-teroidesfragilis(ATCC25285),Actinomycesnaeslundii(ATCC19039),

Prevotellanigrescens(ATCC33563)andPorphyromonasgingivalis

(ATCC48417).

Antimicrobialactivity

Theminimuminhibitoryconcentration(MIC)valueisthelowest concentrationofacompound,fractionorextractcapableof inhib-iting thegrowthofa microorganism.Theantimicrobialactivity of C.bakerianawasdetermined in triplicateusingthe microdi-lutionbrothmethodin96-wellmicroplates(CLSI,2012a,b).The

sampleswere dissolvedin dimethylsulphoxide (DMSO; Synth)

at8000␮g/ml, followed bydilution intryptic soybroth(Difco) foraerobicandSchaedlerbroth(Difco)supplementedwithhemin

(5␮g/ml)andvitaminK1(10.0␮g/ml)foranaerobic;

concentra-tionstestedrangedfrom400to25␮g/mland25 to0.39␮g/ml. ThefinalDMSOcontentwas4%(v/v),andthissolutionwasusedas anegativecontrol.Theinoculumwasadjustedforeachorganismto yieldacellconcentrationof5×105colonyformingunits(CFU)per

ml.Themicroplateswiththeaerobicmicroorganismswere

incu-batedaerobicallyat37◦_C_for₂₄_h._The_anaerobic_{microorganisms}

wereincubatedfor48–72hinananaerobicchamber(DonWhitley Scientific,Bradford,UK),in5–10%H2,0%CO2,80–85%N2

atmo-sphereat37◦_C._After_that,_resazurin_(Acros _Organics)₍₃₀_␮l)_in

aqueoussolution(0.01%w/v)wasaddedtothemicroplates,to indi-catemicroorganismviability(Carvalhoetal.,2011).Chlorhexidine dihydrochloride(Aldrich)(CD)wasusedasapositivecontrol,and theconcentrationsrangedfrom0.0115␮g/mlto5.9␮g/ml. Steril-itytestswereperformedfortheTSBandSchaedlerbroths,control culture(inoculum),positivecontrol,extractsandDMSO.

Cytotoxicactivity

Samplesofthecompound,fractionorextractweredissolved

in methanoland dilutedinDMEM (Dulbecco’smodifiedEagle’s

medium,Sigma–Aldrich)untilformastocksolutionwitha concen-trationof640␮g/ml.ThecellviabilitytestwasdonewithVerocells ATCCCCL81.Thecytotoxicitywasevaluatedusingthemicroplate dilutionmethod.Asolutioncontaining1×106cellsin10ml

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Table1

LC-DAD-ESI-MS/MSdataofthemajorconstituentsoffractionF11.3.

Compounds TR(min) [M−H]−_(Da) _Absorption_UV/vis_(nm) _Fragments_(%) _Class_of_metabolite

1 5.99 299 228;258;288;431 284(100);255(41);240(21) Anthraquinone

2 6.75 283 228;258;290;433 257(3);255(2);239(100);211(2);183(2) Rhein

3 7.83 329 259;277;374 314(100);299(14);285(20);270(14) Flavonoid

4 8.53 313 224;258;270;438 269(100);299(11);287(5);254(19) Anthraquinone

5 17.39 325 Notobtained 183(100);197(2);170(3);119(4) Notidentified

6 27.83 283 Notobtained Notobtained Fattyacid

pipettedintoeachwell;then,theplatewasincubatedfor6hat 37◦_C_in_a_humidified_atmosphere_with_5%_CO₂_to_ensure_cell

adhe-siontothewell.Onceattached,theculturemediumwasremoved andsamplesolutionswereaddedatconcentrationsof512,256, 128,64,32,16, 8and4␮g/ml,startingfromthestocksolution. Thefinalvolumeineachwellwas100␮landthequantityofcells presentineachwellwas1×104cells.Thefinalconcentrationof methanolineachwelldidnotexceed3%.Controlswereprepared forgrowth(Cellviability100%),positive(Cisplatin;Sigma–Aldrich), solvent(Methanol;Synth)andsamples.Theplateswereincubated for48hat37◦_C_in_a_humidified_atmosphere_with_5%_CO₂_._Next,

10␮lofdevelopingsolutionof3mMresazurininPBSwasaddedto eachwell(Rolónetal.,2006)andtheplatewasincubatedagain

for 24hunder the same conditions. Readingsof absorbance at

594nmwereperformedinamicroplatespectrophotometer.The

assaysweredoneintriplicateandtheresultsoftheabsorbancesfor eachconcentrationwerecalculatedaccordingtothegrowth con-trol.TheCC₅₀(cytotoxicconcentrationatwhich50%ofthecellsare viable)wascalculatedbyadose-responsegraphnonlinear regres-sion(Pillay et al.,2007).Thecytotoxicassays weretestedwith

ANOVAwithasignificancelevelof 5%,usingtheTukeymethod

inGraphPadPrism5.Theresultsofcytotoxicactivitywere eval-uatedbycomparingthevaluesofcytotoxicconcentrations(CC50)

toVerocellswiththevaluesofminimalinhibitoryconcentrations obtainedfromtestsforantibacterialactivityusingtheselectivity index(SI).TheSIwascalculatedbythelogarithmoftheratioof cyto-toxicconcentration(CC50)andtheMICvalueformicroorganisms

(SI=log[CC₅₀]/[MIC]).Apositivevaluerepresentshigherselectivity againstmicroorganismsthantoxicitytoVerocells,andanegative valueindicatesahighertoxicitytoVerocellsthantobacteria(Case etal.,2006).

Resultsanddiscussion

IdentificationofcompoundisolatedfromCassiabakeriana

ThecompoundisolatedfromthesubfractionF11.3presented

proton NMR analysis the following chemical shifts: 1_H _NMR

(400MHz,DMSO):ıppm:11.9(3H,large,3-COOH,1-OH,8-OH),

8.21(1H,s,H-4),7.36(1H,d,J=8.0Hz,H-7),7.82(1H,t,J=8.0Hz, H-6),7.76–7.74(2H,m,H-2andH-5).ByHSQCspectrum,itwas possibletoassignthefollowingsignals 13_C _ı_ppm:_138.4_(C-6),

124.7(C-7),124.3(C-2),119.5(C-5)and119.1(C-4).Thesignals

oftheH-5andH-2couldonly beidentifiedbytheHSQC

spec-trum(H-5,ı7.74;H-2,ı7.76).ByCOSYspectrum,itwaspossible

toverifythecouplingsbetweenH-6andH-7and betweenH-6

andH-5.Theinfraredanalysisprovidedthefollowingbands(KBr): O H,3630–3200cm−1_;_C _H_aromatic,₃₀₆₆_cm−1_;_C _O_(carboxyl),

1695cm−1_; _C _O _(carbonyl), ₁₆₃₄_cm−1_; _C _O, ₁₂₇₀_cm−1_; _C _O,

1190cm−1_; _C _H _aromatic,₇₅₁_cm−1_._Data _from_UV–vis:_Band_I

(290and433nm)andBandII(228and258nm).Data obtained

fromLC–ESI-MS/MS m/z: 283[M−H]−,257[M−H C2H2]−, 255

[M−H CO]−_,₂₃₉_[M₋_H _CO₂_]−_,₂₁₁_[M₋_H _CO₂ _CO]− _and ₁₈₃

[M−H CO2 CO CO]−;andmolecularweight284.0320from

For-mulaPredictor® _Software._{Fragmentation} _of_rhein _was_recently reported(Zhuetal.,2014).Themeltingpointwas320◦_C._The_above

resultsareinaccordancewithdataalreadydeterminedinthe liter-aturefor1,8-dihydroxy-anthraquinone-3-carboxylicacid(1),also knownascassicacidorrhein(Danielsenetal.,1992;Dionex,2009; GavitandLaddha,2010;Jiangetal.,2012;Weietal.,2003;Yeetal., 2007).

PhytochemicalprospectionandLC–ESI-MS/MSanalysisofthe F11.3activesubfraction

TheTLCofF11.3activesubfraction(SiO2,chloroform/methanol

8:2 (v/v); methanolic solution of aluminium chloride 1%, w/v)

suggestedthepresenceofflavonoids,whereasTLC(SiO2,

chloro-form/methanol 8:2(v/v); ethanolic solution KOH 10%,w/v and

ammonia vapours) suggested the presence of anthraquinones.

TheseresultswithF11.3wereconfirmedbyLC–ESI-MS/MS.After

LC–ESI-MS/MS,itwaspossibletodeterminethat,in additionto rheincompound,otherstructuresasanthraquinones,flavonoids andfattyacidarepresentinF11.3.Thestudyofthemajor compo-nentsofthesubtractionF11.3isindicatedinTables1and2.

Antimicrobialandcytotoxicactivities

Theresultsoftheantimicrobialactivityofcrudeethanolextracts andorganicfractionsofbarkandleavesofC.bakerianaassessed

Table2

LC–ESI-MSdataoffractionF11.3innegativemode.

Compounds TR(min) Experimentalmass[M−H]−(Da) Theoreticalmass[M−H]−(Da) Suggestedformula IDH Error(ppm) Error(mDa)

1 5.99 2,990,536 2,990,555 C16H12O6 11 −6.3 −1.9

2 6.75 2,830,214 2,830,242 C15H8O6 12 −9.9 −2.8

3 7.83 3,290,651 3,290,661 C17H14O7 11 −3.0 −1.0

4 8.53 3,130,336 3,130,348 C16H10O7 12 −3.8 −1.2

5 17.39 3,251,790 – –a _– _– _–

6 27.83 2,832,637 2,832,637 C18H36O2 1 0 0

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Table3

MICfortheethanolextractsandfractionsofbarkandleavesofCassiabakeriana.

Bacteria Minimuminhibitoryconcentrations(MIC)–␮g/ml

Ethanolextractcrudeandfractions–C.bakeriana

Bark Leaves c_CD

Ethanol extract EB

Hexane fraction PB1

Dichloromethane fraction PB2

Ethylacetate

fraction PB3

Ethanol extract EL

Hexane fraction PL1

Dichloromethane fraction PL2

Ethylacetate

fraction PL3

Methanol fraction PL4

a_S_._sanguinis ₄₀₀ _>400 ₁₀₀ ₄₀₀ _>400 _>400 _>400 _>400 _>400 _1.84

a_S_._mitis _>400 ₃₀₀ ₂₀₀ ₂₀₀ _>400 _>400 _>400 _>400 _>400 _3.68

a_S_._mutans _>400 ₂₅ ₄₀₀ _>400 _>400 _>400 _12.5 _>400 _>400 _0.92

a_S_._sobrinus _>400 ₂₅ ₁₀₀ _>400 _>400 _>400 ₂₅ _>400 _>400 _1.84

a_E_._faecalis _>400 _>400 _>400 _>400 _>400 _>400 _>400 _>400 _>400 _7.37

b_B._Fragilis _>400 ₂₀₀ ₂₀ _>400 ₁₀₀ ₂₀₀ ₄₀₀ ₄₀₀ _>400 _1.84

b_P._nigrescens _>400 _>400 ₂₀₀ ₄₀₀ _>400 _>400 _>400 _>400 _>400 _1.84

b_A._naeslundii _>400 _>400 _12.5 ₄₀₀ _>400 _>400 _>400 _>400 _>400 _1.84

b_F._Nucleatum _>400 ₂₀₀ _12.5 ₂₀₀ _>400 _>400 _>400 _>400 ₄₀₀ _1.84

b_P._gingivalis _>400 ₄₀₀ ₁₀₀ _>400 _>400 _>400 ₂₀₀ ₄₀₀ _>400 _3.68

a_{Gram–positive}_bacteria.

b_{Gram–negative}_bacteria.

c _CD,_positive_control_{(chlorhexidine}_{dihydrochloride).}

Solventcontrol(4%DMSOsolution)didnotaffectthegrowthofmicroorganisms.

invitroare shown inTable3.Thelowest inhibitory concentra-tionwasfoundtothebarkfractionobtainedfromthephase in dichloromethane(PB2)withvaluesof12.5␮g/mlagainstthe bacte-riaA.naeslundiiandF.nucleatumand20␮g/mlagainstB.fragilis. Except for E.faecalis,PB2 inhibited the growth of all microor-ganismsevaluated,showingahigherantibacterialeffectagainst

anaerobes.Crudeextractsand compoundsisolatedfromnatural

productswithMICvaluesunder100and10␮g/ml,respectively, canbeconsideredpromisingantimicrobialagents(RiosandRecio, 2005).DuetothefactthatPB2hasshownantibacterialactivity againstmostoralaerobicandanaerobicmicroorganismsevaluated andtheMICvaluesareequaltoorbelow100␮g/ml,the fraction-ationofthissamplewasperformed.TheresultingF1–F14fractions weresubjectedtoantimicrobialactivitytestsandtheresultsare presentedinTable4.ComparingtheMICvaluesoffractionsF1to F14,lowerMICvalueswerefoundfortheF11fraction.Thisfraction indicatedhigherantimicrobialactivitytoaerobicmicroorganisms whencomparedwithPB2.F11inhibitedA.actinomycetemcomitans

withMICof25␮g/ml,averyaggressiveoralpathogeninvolvedin casesofsevereperiodontitisinyoungandadulthumans(Lorenzo, 2004).AverypromisingresultwasfoundforF11againstP. gin-givalis,whenitsinhibitoryconcentrationof0.78␮g/mlwaslower thanthatreportedforthepositivecontrol.F11showedhigherMIC valuesthanPB2withrespecttosomeanaerobes,althoughthe activ-ityofF11remainedrelevantwithconcentrationsof100␮g/mlor

below.TheMICresultsofF11ledustoworkonitsfractionation, therefore,thesubfractionF11.1toF11.3wereobtained.Theresults forMICvaluesareshowninTable5.

The subfractionF11.3showed thebest resultsof

antimicro-bialactivity,inhibitingallmicroorganismsstudied.E.faecaliswas resistantto alltheextracts and fractionsof barkand leavesof

C. bakeriana, but had growth inhibition against F11.3 with an

MIC of 200␮g/ml. The strong antibacterial activity and wide

spectrumofactionshownbytheF11.3,againstoralbacteria eval-uated,mayberelatedmainlytothepresence offlavonoidsand anthraquinonesinitscomposition(Table1).Metabolitesofthese classesofcompoundshasshownactivityagainstvarious microor-ganisms,includingoral(Dahijaetal.,2014;Riihinenetal.,2014; Xiangetal.,2008).F11.3hasgreaterantibacterialeffectagainst

S. mutans, F. nucleatum and P.gingivalis that differentpropolis extracts,naturalantibacterialproductwithrecognizedpotential inthetreatmentoforalinfections(Waldner-Tomicetal.,2014).

Thepreparativechromatography F11.3ledtotheisolationof 1,8-dihydroxy-anthraquinone-3-carboxylicacid,andits antimicro-bialandcytotoxicactivitiesweretested.Theantimicrobialactivity

foundforthecompoundrhein,incomparisonwithPB2,F11and

F11.3areshowninTable6.Rheinwasactiveonanaerobicbacteria

withvaluesrangingbetween3.12and25␮g/ml,outstandingthe

strongantibacterialeffectagainstP.gingivalis(MICof3.12␮g/ml). TheMICvalues forrhein showthatitis amajorcontributor to

Table4

ResultsofantimicrobialactivityoffractionsF1–F14.

Bacteria Minimuminhibitoryconcentrations(MIC)–␮g/ml

Fractions

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 c_CD

a_S_._sanguinis _>400 ₄₀₀ ₄₀₀ ₁₀₀ ₄₀₀ ₄₀₀ _– _>400 _– ₄₀₀ ₁₀₀ _>400 _>400 ₄₀₀ _1.84

a_S_._mitis ₂₀₀ ₄₀₀ ₂₅ ₁₀₀ ₂₀₀ ₁₀₀ _– ₄₀₀ _– ₂₀₀ ₁₀₀ ₄₀₀ ₄₀₀ ₂₀₀ _3.68

a_S_._mutans ₃₀₀ ₄₀₀ ₃₀₀ ₂₀₀ _>400 ₂₀₀ _– ₄₀₀ _– _>400 ₁₀₀ _>400 ₄₀₀ _>400 _0.92

b_A._actinom ₂₀₀ ₄₀₀ ₂₀₀ ₂₀₀ ₄₀₀ ₂₀₀ _– ₄₀₀ _– ₂₀₀ ₂₅ ₄₀₀ ₄₀₀ ₂₀₀ _7.37

a_E_._faecalis _>400 _>400 _>400 _>400 _>400 _>400 _– _>400 _– _>400 ₂₀₀ _>400 _>400 _>400 _7.37

b_B._Fragilis _>400 ₃₀₀ _>400 _>400 _>400 _>400 _– _>400 _– _>400 ₅₀ _>400 _>400 ₁₀₀ _1.84

b_P._nigrescens _>400 _>400 _>400 _>400 _>400 _>400 _– _>400 _– _>400 ₁₀₀ _>400 _>400 _>400 _1.84

b_A._naeslundii _>400 _>400 _>400 ₁₀₀ ₁₀₀ ₁₀₀ _– _>400 _– ₁₀₀ ₁₀₀ _>400 _>400 ₂₀₀ _1.84

b_F._Nucleatum _>400 ₄₀₀ ₄₀₀ ₂₀₀ ₄₀₀ ₄₀₀ _– ₄₀₀ _– ₄₀₀ _62.5 ₂₀₀ ₂₀₀ ₅₀ _1.84

b_P._gingivalis ₃₀₀ ₂₀₀ ₂₅ ₂₅ ₁₀₀ ₁₀₀ _– ₂₀₀ _– ₁₀₀ _0.78 _6.25 _6.25 _3.12 _3.68

b_{Gram–negative}_bacteria.

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Table5

ResultsofantimicrobialactivityofF11.1,F11.2andF11.3subfractions.

Subfractions Minimuminhibitoryconcentrations(MIC)–␮g/ml

Microorganisms

Anaerobic Aerobic

b_F._nucleatum b_A._naeslundii b_P._gingivalis b_B._fragilis b_P._nigrescens a_S_._sanguinis a_S_._mitis a_S_._mutans a_E_._faecalis b_A._Actinom.

F11.1 >400 >400 >400 >400 >400 >400 >400 >400 >400 >400

F11.2 >400 >400 >400 >400 >400 >400 >400 >400 >400 >400

F11.3 50 100 12.5 50 25 25 12.5 50 200 25

c_CD _1.84 _1.84 _3.68 _1.84 _1.84 _1.84 _3.68 _0.92 _7.35 _7.35

b _{Gram–negative}_bacteria.

c _CD,_positive_control_{(chlorhexidine}_{dihydrochloride).}

Table6

InhibitoryeffectofthebioactivefractionPB2,F11,F11.3andrhein.

Bioactivesamples Minimuminhibitoryconcentrations(MIC)–␮g/ml

Microorganisms

Anaerobic Aerobic

F.nucleatum A.naeslundii P.gingivalis B.fragilis P.nigrescens S.sanguinis S.mitis S.mutans E.faecalis A.Actinom.

PB2 12.5 12.5 100 20 200 100 200 400 >400 –

F11 62.5 100 0.78 50 100 100 100 100 >400 25

F11.3 50 100 12.5 50 25 25 12.5 50 200 25

Rhein a ₂₀_(70.4_␮_M) ₂₅₍₈₈_␮_M) a _3.12₍₁₁_␮_M) _>200 _>200 _>200 a a

CD 1.84 1.84 3.68 1.84 1.84 1.84 3.68 0.92 7.35 7.35

a_Not_determined_{(insufficient}_sample_quantity_for_the_assay).

–,wasnotperformed;CD,positivecontrol(chlorhexidinedihydrochloride).

theantibacterialeffectofF11.3onanaerobes.Duetothefactthat rheinwasnotactiveonaerobic,othersbioactivecompoundsare presentinF11.3,justifyingitsstrongactivityagainstthese bacte-ria.Thestructuralformulaofrheinhasacarbonylgroupandtwo

ˇ-hydroxylsatalinearposition.Thispositionhasbeensuggested asfavourableforantimicrobialactivityofanthraquinones(Xiang etal.,2008).

Thereareotherstudiesaboutantimicrobialeffects seenwith

aerobic and anaerobic oral bacteria, involving compounds

iso-latedfromplants.Forinstance,thetriterpenesursolicandoleanoic

acidsandursolicderivativesshowedMICbetween40␮g/mland

200␮g/mlagainstS.mitis,S.sanguinisandS.mutans(Cunhaetal., 2007).Forthesebacteria,thesesquiterpenecaryophylleneoxide andderivatives ofditerpenecopalic acidexhibitedMICranging between60 and 200␮g/ml.The (−)-copalicacidexhibited MIC between3and6␮g/ml(Souzaetal.,2011a)andpimarane-type diterpenesinhibited bacterialgrowthatconcentrations ranging between2.5 and20␮g/ml (Portoet al.,2009).Thesclareol and manoolditerpeneswereactiveagainstA.naeslundii,P.gingivalis

and P. nigrescens,with MIC between 6.2 and 400␮g/ml, while (−)copalicacidshowedMICbetween3.1and200␮g/mlandcopalic

acidderivativesbetween25and200␮g/ml(Souzaetal.,2011b).

The diterpene kaurenoic acid and its derivatives showed MIC

againstA.naeslundii,P.gingivalisandP.nigrescensbetween1.25 and60␮g/ml(Carvalhoetal.,2011).Althoughrheinnotbeactive against aerobicbacteria, it exhibited very promising MICvalue againstP.nigrescensandalsogoodresultsagainstA.naeslundiiand

P.gingivalis.

Thefractions,themostactivesubfractionandrheinweretested forcytotoxicity.Therelationshipbetweencytotoxicityand antimi-crobialactivitywasestablishedthroughtheselectivityindex(SI) andisshowninTable7.

PB2,F11,F11.3andthepurecompoundshowedlowertoxicity

toVerocellscomparedtothepositivecontrolcisplatin.Regarding thecytotoxicactivity,thevaluesofCC₅₀decreasedwith fraction-ationofactivesamplesindicatinganincreaseintoxicity.However, whentheselectivityindexareconsidered,allbioactivefractions andrheinpresentedpositiveSIvaluesatconcentrationsthat exhib-itedstrongantibacterialactivity,indicatinga greatantibacterial effectandselectivityagainstoralmicroorganisms.Inthe cytotox-icitytest,F11.3andrheinshowednostatisticaldifferenceat5%by Tukeytest.Thepositivevaluesofselectivityindexrangedfrom0.21

Table7

Cytotoxicactivityandselectivityindexesofthebioactivesamples.

Bioactive samples

Verocells (ATCCCCL81)

Selectivityindex(SI)

Anaerobic Aerobic

Cytotoxicactivity CC50–␮g/ml

F.nucleatum A.naeslundii P.gingivalis B.fragilis P.nigrescens S.sanguinis S.mitis S.mutans E.faecalis A.Actinom.

PB2 325±24 1.41 1.41 0.51 1.21 0.21 0.51 0.21 −0.09 <−0.09 –

F11 263±12 0.62 0.42 2.52 0.72 0.42 0.42 0.42 0.42 <−0.18 1.02

F11.3 196±24 0.59 0.29 1.19 0.59 0.83 0.83 1.19 0.59 −0.07 0.83

Rhein 212±16 a 1.02 0.92 a 1.83 <0.02 <0.02 <0.02 a a

Cisplatin 7.01±0.66 – – – – – – – – – –

a_Not_determined_{(insufficient}_sample_quantity_for_the_assay).

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to2.52.ThebestSIvaluesagainstaerobicwereobservedforF11.3. ThisfractionshowedgreattoxicitytotheVerocellsonlyagainst

E.faecalis,whichwasnotoriouslythemostresistantbacteria.The highestSIvaluesobservedforanaerobesoccurredwithF. nuclea-tum,A.naeslundiiandB.fragilis(PB2),withP.gingivalis(F11)and withP.nigrescens(rhein).

Rheinhasbeenisolatedin otherspeciesofCassia(Daveand Lediwane,2012)andothersstudieshaveproveditsantimicrobial activity(Didryetal.,1994;Kavanagh,1947;Hatanoetal.,1999). Additionallytoitsantimicrobialpotential,rheinisassociatedto antiviral(Barnardetal.,1992),antioxidant(Vargasetal.,2004), anti-angiogenic(Heetal.,2011),anti-emetic(Ahmedetal.,2012), anticancer(Duraipandiyanetal.,2012)andantifibrotic(Tsangetal., 2013)activities.C.bakerianaispresentedinthisworkasonemore sourceofrhein,furthermore,thiscompoundprovedtobean impor-tantprototypeforthedevelopmentofanantimicrobialagentto targetanaerobicoralmicroorganisms.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor

thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Author’scontributions

LCSC (PhD student) conducted the work with the species

C. bakeriana. SALMand FJTA wereresponsible for theresearch

project with the species C. bakeriana. EAN, AO and RC

con-tributedwithmassspectrometryandnuclearmagneticresonance identifications.CHGMcontributedtobiologicalstudiesof antibac-terialactivity.CVScontributedtocytotoxicityassays.MMM(PhD

student) contributed conducting the cytotoxicity assays. LCFS

and TTB (undergraduate students) collaborated with the

sam-plepreparations, columnchromatography and TLCanalysis. All

the authors have read the final manuscript and approved the

submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbytheFAPEMIG(MinasGeraisState

Research Foundation) under Grant APQ-01178-11 and IQUFU

(ChemistryInstituteandPostgraduateProgramoftheFederal Uni-versityofUberlandia).WethankProfessorDr.RobsonJosédeCássia

FrancoAfonsofortheLC-DAD-MS/MSanalysisandProfessorDr.

AntônioFláviodeCarvalhoAlcântarafortheNMR analysis.We

thankProfessorsDr.GleinMonteiroandDr.IvandeAraujoSchiavini foridentificationoftheplant.

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