In vitro antimicrobial and antimycobacterial activity and HPLC-DAD screening of phenolics from Chenopodium ambrosioides L.

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

In

vitro

antimicrobial

and

antimycobacterial

activity

and

HPLC–DAD

screening

of

phenolics

from

Chenopodium

ambrosioides

L.

Roberta

S.

Jesus

a,∗

_,

_Mariana

_Piana

a

_,

_Robson

_B.

_Freitas

a

_,

_Thiele

_F.

_Brum

a

_,

Camilla

F.S.

Alves

a

_,

_Bianca

_V.

_Belke

a

_,

_Natália

_Jank

_Mossmann

a

_,

_Ritiel

_C.

_Cruz

a

_,

Roberto

C.V.

Santos

b

_,

_Tanise

_V.

_Dalmolin

c

_,

_Bianca

_V.

_Bianchini

c

_,

_Marli

_M.A.

_Campos

c

_,

Liliane

de

Freitas

Bauermann

a,∗

a_{UniversidadeFederaldeSantaMaria,DepartamentodeFarmáciaIndustrial,LaboratóriodePesquisaFitoquímica,SantaMaria,RS,}

Brazil

b_{CentroUniversitárioFranciscano,LaboratóriodePesquisaemMicrobiologia,SantaMaria,RS,Brazil}

c_{UniversidadeFederaldeSantaMaria,DepartamentodeAnáliseClínicaeToxicológica,LaboratóriodePesquisaMycobacteriana,Santa}

Maria,RS,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25January2016 Accepted11February2017 Availableonline19July2017 AssociateEditor:LuisHenrique Guimarães Keywords: Amaranthaceae Antimicrobialpotential Rutin Quercetin Chrysin

a

b

s

t

r

a

c

t

Themainobjectiveofthisstudywastodemonstratetheantimicrobialpotentialofthe crudeextract and fractionsof Chenopodiumambrosioides L., popularlyknownas Santa-Mariaherb,against microorganismsofclinicalinterestbythemicrodilution technique, andalsotoshowthechromatographicprofileofthephenoliccompoundsinthespecies. ThePhytochemicalscreeningrevealedthepresenceofcardiotonic,anthraquinone, alka-loids,tanninsandflavonoids.TheanalysisbyHPLC–DADrevealedthepresenceofrutin in the crude extract (12.5±0.20mg/g), ethyl acetate (16.5±0.37mg/g) and n-butanol (8.85±0.11mg/g),whereasquercetinandchrysinwerequantifiedinchloroformfraction (1.95±0.04and1.04±0.01mg/g),respectively.Themostpromisingresultswereobtained with the ethyl acetate fraction, which inhibited a greater number of microorganisms andpresented the lowest values of MIC against Staphylococcus aureus and Enterococcus faecalis (MIC=0.42mg/mL), Pseudomonas aeruginosa (MIC=34.37mg/mL),Paenibacillus api-arus (MIC=4.29mg/mL) and Paenibacillus thiaminolyticus (MIC=4.29mg/mL). Considering mycobacterialinhibition,thebestresultswereobtainedbychloroformfractionagainstM. tuberculosis,M.smegmatis,andM.avium(MICrangingfrom156.25to625␮g/mL).Thisstudy proves,inpart,thatthepopularuseofC.ambrosioidesL.canbeaneffectiveand sustain-ablealternativeforthepreventionandtreatmentofdiseasescausedbyvariousinfectious agents.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthors.}

E-mails:[email protected](R.S.Jesus),[email protected](L.F.Bauermann).

https://doi.org/10.1016/j.bjm.2017.02.012

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TheWorldHealth Organization(WHO)recommends world-wide development of research on medicinal plants for therapeuticpurposes,inordertoobtainnewpossibilitiesfor thetreatmentofdiseases,especiallyindevelopingcountries,1

considering also, the hope to identify new substances of medicinal character that may serve as raw materials for pharmaceuticalsindustries.InBraziltheuseofplantsisvery commonforthetreatmentofvariousdiseases,including bac-terialandfungalinfections,andmanystudiesareconducted todetectsecondarymetabolitesinplantswithantimicrobial propertiesasanattempttofindnewantimicrobialor antifun-galcompounds.2–4_{Indeed,becauseoftheincreasingbacterial}

resistance,thereisaneedtosearchfornewantimicrobial sub-stancesfromalternativesources,suchasplantsusedinfolk medicine.Otherimportantsubjectstostudyaremycobacterial infections,includingthosecausedbyMycobacterium tubercu-losis,sincetuberculosiscaused8millionnewcasesand 1.8 millionfatalitiesperannumworldwide.5,6

ChenopodiumambrosioidesL.(DysphaniaambrosioidesL.)isto currentsynonymyofthespeciebelongstotheAmaranthaceae familyandispopularlyknownas“erva-de-santa-maria”.This plantisnativefromSouthAmericaanditiswidelydistributed throughout Brazil, being often used in folk medicine as antirheumatic,anti-inflammatory,antipyretic,antihelmintic, antifungal,anti-ulcerandforthetreatmentofwounds.7,8_The

ointmentispreparedwiththeessentialoilofC.ambrosioides inhibited, the growth of dermathophytes and some other fungi.9,10_{In2009,thespeciewasaddedtothelistofNational}

MedicinalPlantsofInterestinBrazil,(SUSRENISUS-BRAZIL) highlightingthe needtoincreasethe understandingofthe mechanismsbehinditsmedicinalproperties.11_{Therefore,itis}

ofgreatimportancetoconductexperimentsinsuchspecies, to identify their phytochemical compounds with pharma-cological potential and to identifypossiblealternatives for antimicrobialtherapy,sincestudiesusingcrudeextractsand fractionsofC.ambrosioidesL.arescarce.

Materials

and

methods

Reagents

Allchemicalswereofanalyticalgrade.Solventsforthe extrac-tionsandanalyticalproceduresand,phytochemicalscreening were purchased from Merck (Darmstadt, Germany). The quercetin,chrysinand,rutinstandardswerepurchasedfrom Sigma–Aldrich (St. Louis, MO, USA). Microbiological assays were performed using Mueller-Hinton Agar and Mueller-HintonBrothfromSigma–Aldrich.

Plantcollection

TheleavesofC.ambrosioidesL.werecollectedinthecityof Uruguaiana,fromtheStateofRioGrandedoSul,Brazil (alti-tude57m,29◦latitude,4520,12S,longitude57◦40.28W)in Januaryof2013.Theexsiccatewasarchivedasspecimenin theherbariumoftheDepartamentofBiologyattheFederal

UniversityofSantaMaria,undertheregisternumberofSMDB 137015,forfuturereference.

Extractionoftheplantleaves

Plantmaterialwasdriedatroomtemperatureandpowered inaknifemill.Theleavesoftheplant(500g)were macera-tedatroomtemperaturewithethanol70%andsubjectedto adailyshake-upforaweek.Theextractwascollected and filteredand the remainingplantmaterialwas subjectedto more extraction.After filtration, the hydroalcoholicextract was evaporated underreduced pressure ina rotary evapo-ratortoremovethe ethanol.Ashareofthishydroalcoholic extractwasdriedinastove(temperaturebelow40◦C)yielding thedriedcrudeextract.Theremainingofthehydroalcoholic extractwaspartitionedwithsolventsofincreasingpolarity: chloroform,ethylacetateandn-butanol.Lastly,thefractions obtainedwerefullydriedinarotaryevaporator.

Phytochemicalscreening

Thephytochemicalscreeningfollowedaseriesof characteri-zationreactionswhichusedhydroalcoholicextract,according to.12,13 _{Theidentification ofalkaloidswasperformed using}

Dragendorffreagent, thetechnique isbasedon the forma-tion ofprecipitatesaftertheadditionofthereagent,which demonstratesthepresencethesemetabolites.Theflavonoids wereidentifiedusingthereactionwithaluminumchloride, fil-terpaperstripsweremoistenedwithhydroalcoholicextract, afterwasaddedonedropof5%AlCl3solution,thepresence

offlavonoids wasrevealed byintensificationofthe yellow-ishgreencoloronfluorescence.Thetanninswereidentified through ofthe reaction that usedferric chloride,in which dropsof1%FeCl3solutioninmethanolwereaddedto2mLof

hydroalcoholicextract,thebluecolorindicatedthepresence oftannins.Theanthraquinonecompoundswereidentifiedby Bornträgerreaction.Briefly,wasaddedsmallfragmentofthe drug (about0.2g) inatest tubeand added 5mLofdiluted NH4OHsolution,thereactionwascharacterizedaspositiveby

pinkorreddishcoloration.Thecardiotonicheterosideswere characterized through ofthe Liebermann-Buchard reaction whereaceticanhydrideandsulfuricacidwereaddedtogether toapartofthehydroalcoholicextract.Thegreencoloration characterizedthepresenceofsteroidalnucleus,foundin car-diotonicscompounds.

HPLC–DADanalysisofpolyphenolscompounds

The analysis were performed with a Prominence Auto-Sampler (SIL-20A) equipped with Shimadzu LC-20AT (Shi-madzu, Kyoto, Japan) reciprocating pumps connected to a DGU-20A5degasserandCBM-20Aintegrator.UV-VISdetector DADSPD-M20AandSoftwareLCsolution1.22SP1wereused. Reversephasechromatographyanalyseswerecarriedoutwith aPhenomenexC-18column(4.6mm×250mm)packedwith 5␮mdiameterparticles,volumeinjectionwas40␮L,andthe gradientelutionwasconductedwithslightmodificationsin themethod.14_{Themobilephaseconsistsofwatercontaining}

2.0%aceticacid(phaseA)andmethanol(phaseB),andthe elutiongradientwas2mintoachieve5%ofB,andchanged toobtain25%,40%,50%,60%,70%and100%Bat10,20,30,

40,50 and 70min,respectively. The UVabsorption spectra ofthe standards as well as the samples were recorded in therangeof230–400nm. Stocksolutionsofstandardswere preparedin methanol in the range of 0.0025–0.060mg/mL. Samplesandstandardssolutionsaswellasthemobilephase weredegassedandfilteredthrough0.45␮mmembranefilter (Millipore).Chromatographicoperations werecarriedout at ambienttemperature(22◦C)andintriplicate.The identifica-tionofthecompoundswasdonebycomparingitsretention timeandUVabsorptionspectrumwiththestandards.

Antimicrobialpotential

Samplepreparation

Thesampleforthemicrobiologicaltestswaspreparedfrom therawextractofC.ambrosioidesL.attheconcentrationof 275mg/mLmixedinavortexmixerandtotallydissolvedin DMSOwiththeaidofultrasound.

Microorganismsandpreparationofinoculums

Thefollowingmicroorganismsusedinthe evaluationwere obtainedfrom theAmerican TypeCultureColletion(ATCC): Bacillus cereus (ATCC 9634), Listeria monocytogenes (ATCC 7644),Enterococcusfaecalis(ATCC29212),Staphylococcusaureus (ATCC 25923), Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 340), Salmonella choleraesuis (ATCC 10708), Candida albicans (ATCC 90028). Clinical and environmental isolatesprovided bythe Department of Microbiology,from the Universidade Franciscana (UNIFRA), were also used to test the antimicrobial activity of C. ambrosioides L. extract andfractions:Paenibacillusthiaminolyticus,Paenibacilluspabuli, Paenibacillus azotofixans, Paenibacillus allginolyticus, Paenibacil-lus validus, Saccharomyces sp., Staphylococcus saprophyticus, Salmonellaenteritidis,Citrobacterfreundii,Klebsiellapneumoniae, Streptococcussp.Allisolatesweremaintainedonnutrientagar slants at4◦C.Theinoculum of the testedmicroorganisms waspreparedaccordingtotheguidelinesoftheClinicaland LaboratoryStandardsInstitute,15 _{adjustedto0.5McFarland}

standard.

Diskdiffusiontestanddeterminationofminimalinhibitory

concentrations(MICs)

The disk diffusion test was chosen as the screening test, followingCLSIparameters,aimingtheselectionofsensitive microorganismstotheextractandfractionsfromtheleaves ofC.ambrosioidesL.Theminimuminhibitoryconcentrations (MICs)weredeterminedbythemicrodilutiontechnique,using Mueller–Hinton broth(Difco). Theassay wascarried out in 96-well microtitre plates. Each extract was mixed with an inoculumpreparedinthesamemediumatadensityadjusted per tube to 0.5 of the McFarland scale (1.5×108CFU/mL) and theactiveextracts/fractions were dilutedtwofold seri-allyranging.Microtitretrayswereincubatedat37◦Candthe MICs were recorded after 24h ofincubation. TheMIC was definedasthelowestconcentrationofextractthatinhibited bacterialgrowth.Thistestwasperformedintriplicate.The 2,3,5-triphenyltetrazoliumchloridewasusedasanindicator ofmicrobialgrowth.

Table1–Phytochemicalscreeningofhydroalcoholic extractC.ambrosioidesleaves.

Chemicaltest Result

Cardiotonics ++

Antraquinonics +

Alkaloids +

Tannins +

Flavonoids ++

Key:+,smallquantity;++,abundant.

AntimicrobialassayonMycobacterium

Antimycobacterial activity was evaluated against Mycobac-terium avium LR541CDC, Mycobacterium tuberculosis H37Rv (ATCC 25618) and Mycobacterium smegmatis mc2 _{155 (ATCC}

700084). The suspensions were standardized through the range 0.5MacFarland scale andthen dilutedwith Middle-brook 7H9 (MD7H9), supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase)and0.2%glycerol,untilthe concentration of 105 _{UFC/mL. Plant extracts, fractions and}

standardChrysinweredissolvedinDMSO(dimetilsulfoxide), ataconcentrationof50.00mg/mLandthendilutedinMD7H9 untilthedesiredconcentration(2.500␮g/mL)forthe execu-tion ofthetestintriplicate.Thetestwasperformedbythe methodofbrothmicrodilutionaccordingtoCLSIM7-A6.The platescontainingM.smegmatiswereincubatedduring48h,M. aviumfor5daysandforM.tuberculosisfor7days,at37◦C.In ordertoverifytheexistence,ornot,ofmicrobialgrowth,MTT dyewasused.Followingthisprocedure,itwasconsideredas MICthelowestconcentrationoftheextractundertest capa-bleofinhibitingthegrowthofthemicroorganismsusedinthe assay.

Results

Phytochemicalscreening

Phytochemicalscreeningofhydroalcoholicextractfromthe leavesofC.ambrosioidesL.revealedthepresenceofimportant secondarymetabolitesshowedinTable1.

HPLC–DADanalysisofpolyphenolscompounds

The crude extract and chloroform, ethyl acetate and butanol fractions from the leaves of C. ambrosioides L. were analyzed by HPLC–DAD. Chromatographic profile of the crude extract, ethylacetateand butanol fractions pre-sented rutin, chloroform fraction furnished quercetin and chrysin (Fig. 1). Rutin, quercetin and chrysin were quan-tified by HPLC–DAD, based on the reference standard curves (Table 2), where calibrations curves were: rutin y=72823x−1901 (r=0.9982), quercetin y=187893x−163671 (r=0.9984)andchrysiny=148116x+62256(r=0.9988).

Antimicrobialpotential

Antimicrobialandantimicobacterialactivity

Theextractandfractionsweretestedatastandardized con-centrationof275mg/mLandtheresultsareshowninTable3.

A

1

1

1

4

3

1

200 750 500 300 200 100 1250 750 500 250 0 0 250 0 150 100 50 400 300 200 100 0 0 0.0 25.0 0.0 25.0 mAU mAU mAU mAU mAU 50.0 min 50.0 0.0 25.0 50.0 min min 0.0 25.0 50.0 min 0.0 25.0 50.0 min

B

2

3

4

D

C

E

Fig.1–PhenolicprofileofC.ambrosioidesleaves:Crudeextract(A,=365nm),chloroformfraction(B,=313nm),ethyl acetatefraction(C,=365nm),n-butanolfraction(D,=365nm)andstandardcompounds(e,=254nm):rutin(1), unidentifiedpeak(2),quercetin(3)andcrhysin(4).Chromatographicconditionsdescribedintheexperimentalsection.

Table2–CompositionofChenopodiumambrosioidesleaves.

Crudeextractandfractions Rutin(mg/gofdryfraction) Quercetin(mg/gofdryfraction) Chrysin(mg/gofdryfraction)

Crudeextract 12.5±0.20 ND ND

Chloroform ND 1.95±0.04 1.04±0.01

Ethylacetate 16.5±0.37 ND ND

n-butanol 8.85±0.11 ND ND

ND,notdetected.

Allfractionswereactiveagainstimportantmicroorganisms; however, the most promising results were obtained with theethylacetatefraction,whichinhibitedagreaternumber ofmicroorganisms andpresentedthelowest valuesofMIC againsttheS.aureus,P.aeruginosa,E.faecalis,Paenibacillus api-arusandP.thiaminolyticus(rangingfrom4.29to34.37mg/mL). Considering the activities of the extract and fractions againstMycobacteriumgenus,thebestresultsandthelowest MICwereobtainedbychloroformfractionagainstM. tubercu-losis,M.smegmatis,andM.avium(MICrangingfrom156.25to 625␮g/mL,respectively,Table4).

Discussion

In 2009, the Brazilian Ministry of Health elaborated the National List of Medicinal Plants ofInterest in the Health

System(RENISUS),whichencouragesresearchonmedicinal plantswidelyusedamongtheBrazilianpopulation.C. ambro-sioidesL.integratesthislist;thusmotivatingtheidentification of its compounds with pharmacological potential. Phyto-chemicalscreeningofC.ambrosioidesrevealedthepresence of cardiotonics and antraquinonics, alkaloids, tannins and flavonoids; theseresults were similar tothose reportedby Nedialkovaetal.8

ThecompoundsverifiedbyHPLCanalysissuchasrutin, quercetinandchrysincanbeexplainedbythedifferencesof polaritybetweenthefractionsandthecompounds,sincerutin isa glycosideofquercetin, beingmorepolar than its agly-cone.Inthesameway,sequentialextractionswithsolventsof crescentpolaritycanprovideaselectiveextraction,basedon chemicalcharacteristicsofsecondarymetabolites.16_Ina

pre-viousstudythattracedachromatographicprofileofthecrude extractfromaerialparts(leavesandflowers)ofC.ambrosioides

Table3–CorrelationbetweenthehaloofinhibitionfromeachmicroorganismassociatedtoMICvalueandmicrobial activityofrutin(compounddetectedfromHPLCanalysis).

Extractsandfractions Bacterial isolates (ATCC) Zoneof inhibition (mm) MIC(mg/mL) RutinMIC (mg/mL) DMSO Antimicrobial agent(␮g/mL)

Crudeextract E.faecalis 29212 16 4.29 0.56 0 2

P.apiarus CI 14 17.18 1.13 0 0.39 P.aeruginosa 340 12 68.75 0.56 0 4 Chloroform E.faecalis 29212 12 4.29 0.56 0 2 S.aureus 25923 14 8.58 0.56 0 0.06 P.apiarus CI 16 4.29 1.13 0 0.39 P.thiaminolyticus CI 14 4.29 1.13 0 0.39 P.aeruginosa 340 10 68.75 0.56 0 4

Ethylacetate E.faecalis 29212 12 4.29 0.56 0 2

S.aureus 25923 17 4.29 0.56 0 0.06 P.apiarus CI 14 4.29 1.13 0 0.39 P.thiaminolyticus CI 14 4.29 1.13 0 0.39 P.aeruginosa 340 11 34.37 0.56 0 4 n-butanol S.aureus 25923 16 34.37 0.56 0 0.06 P.aeruginosa 340 12 17.18 0.56 0 4

CI,clinicalisolate.

Table4–Minimalinhibitionconcentration(␮g/mL)ofcrudeextractandfractionsfromtheleavesofC.ambrosioides,

againstM.smegmatis,M.tuberculosisandM.avium.

Extractandfractions M.smegmatis(ATCC700084) M.avium(LR541CDC) M.tuberculosis(ATCC25618) Crudeextract 625␮g/mL >2500␮g/mL 1250␮g/mL Chloroform 312.5␮g/mL 625␮g/mL 156.25␮g/mL Ethylacetate 312.5␮g/mL >2500␮g/mL 1250␮g/mL n-butanol 625␮g/mL >2500␮g/mL 1250␮g/mL Crysin >2500␮g/mL >2500␮g/mL >2500␮g/mL Isoniazid 0.78 0.78 <0.25 DMSO 0 0 0

L.by HPLC–DAD,trans p-coumaric acid, quercetin dirham-noside,kaempferol-O-rhamnosyl-glucuronideandrutinwere identifiedandheldresponsiblefortheantimicrobialactivity verified.17_{Thepresentstudyreportsthepresenceofquercetin}

andchrysinforthefirsttimeinthisplant.Itisofparticular interestthepresenceofchrysin,whichisreportedtohave sev-eralbiological activities,including antioxidant, anti-allergic andanti-inflammatory,besidesastronginhibitoryeffecton PGE2andTXB2productioninhumanblood,andthisproperty couldbeusedtosearchitschemicalstructureasalead com-poundinthedevelopmentofCOXinhibitors.18_Theaglycones

quercetinandchrysinwerequantifiedonlyinthechloroform fraction,whereastherutinwasdetectedandquantified ran-gingfrom4.5to8.5timesgreaterthanquercetininbutanolic fraction,crudeextractandethylacetatefraction,andthislast onefurnishedthe largest amountofrutin per gramofdry fraction.

Manystudieshighlightthepotentialofnaturalcompounds against pathogens that cause severe infections such as S. aureus, P. aeruginosa, and E. faecalis, this last being consid-ered,oneofthemostprevalentspeciesculturedfromhumans; isolationofEnterococcusresistanttomultipleantibioticshas becomeincreasinglycommoninthehospitalsetting. Conse-quently,microbialresistancedemandsnewpharmacological alternatives, which led to the testing of the crude extract

andfractionsfromtheleavesofC.ambrosioidesagainstsome pathogenicmicroorganisms.

Standard rutin showed the best results against these microorganisms,corroboratingtheassumptionthatthis gly-cosidecontributes,atleastinpart,totheantimicrobialactivity observedinethylacetatefractionoftheC.ambrosioidesL.; fur-thermore,rutinisabletoincreasetheantibacterialactivityof othercompounds.19,20_{Commercialstandardrutinwastested}

ataconcentrationof4.53mg/mL,sincethisvalueis propor-tionaltotheconcentrationofrutinobtainedinthefraction thatpresentedthehighestantimicrobialactivity(ethylacetate fraction).

PreviousstudieswithC.ambrosioidesextractsalsorevealed antimicrobialpotentialagainstpathogenssuchasS.aureus, P.aeruginosa,Brevibacillusagri,Trichophytonmentagrophytes,C. albicans(MICsrangingfrom0.25to0.80mg/mL).21_Moreover,

the capacity of the plant extract to inhibit M. tuberculosis growthwastested,affordingaMICvalueof0.5.mg/mL.22_The

aforementionedresultsareinaccordancewiththeresultsof thestudy,infacttheextractfromthis researchexhibiteda strongerantimycobacterialactivitythantheextractpresented inthepreviousworkmentioned.

Chromatographic profile of this fraction evidenced the presenceofthe aglyconesquercetinand chrysin.Quercetin possessesantimicrobialpropertiesagainstGram-negativeand

Gram-positivebacteriaandshowsvariableinhibitoryactivity against mycobacteria.23 _{In this study, chrysin was}

evalu-atedandshowednoactivityagainstmycobacteria(Table4), furnishing a MIC value above the highest tested concen-tration of2.500␮g/mL. Also, the research group previously tested standard quercetin against M. smegmatis, obtaining MIC=625␮g/mL, whichindicated that other different com-pounds might be acting to increase the activity of this fraction.Inlinewiththesefindings,rutinwhosepresencewas evidenced incrudeextract, ethylacetateand butanol frac-tionsshowed aMICof625␮g/mLwhenassayedagainstM. smegmatis4;therefore,itisunlikelythatrutinandcrisinarethe onlycompoundsexertingtheobservedantimicrobialactivity. The phenolic compounds present in medicinal plants areresponsibleforarangeofbiologicalactivities,including antimicrobialaction.Flavonoids areagroupofheterocyclic organic compounds and constitute an important class of polyphenols. Many biological properties offlavonoids have beenreportedontheirantimicrobialpotential.Studiesreport theantimicrobialpotentialofphenolic compoundssuchas rutin,quercetinandchrysinthroughofthepermeationand destabilizationofthe bacterial membrane, resulting inthe disturbance of the proton motive force, the electron flow, coagulationandleakageofthecellularcontentcausinga bac-tericidaleffect.24–26

Crude extract and fractions from C. ambrosioides L. leavespossessimportantphytochemicalcompounds(rutin, quercetinandchrysin)whichcouldberelatedtoitspopular medicinaluse.Additionally,thisspeciesprovedtobeactive againstpathogensofclinicalinterest,suchasS.aureus,E. fae-calis,P.aeruginosaandM.tuberculosis.TheherbofSantaMariais widelyusedinfolkmedicineforthemostdiversepurposes,27

mainlyasnaturalantimicrobialaidingintheprocessofwound healing,forthisreasonwasinsertedinRENISUS.

Theresults confirm, inpart, the popular medicinal use thisspecies,duetoantimicrobialactivity,especiallyagainst cutaneous opportunistic pathogens. In addition, relevant pharmacologicalactivityagainststrainsofM.tuberculosiswere showed,contributing,thisway,intheelucidationofitsuse intuberculosis.Takingtogether,theseresultsencouragethe continuationofourstudiesaimingtoobtainpurebioactive compoundsagainstresistantmicroorganismsandbiofilm for-mers.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswouldliketothankProf.Dra.MargarethLinde Athayde for the orientation and support to conduct of this work, and Prof. Dr. Renato Aquino Záchia for provid-ing the identification of Chenopodium ambrosioides L. The authorsthankthefinancialsupportofCAPES(Coordenac¸ão deAperfeic¸oamentodePessoaldeNívelSuperior),Brazil.

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