Phenolic content and antibacterial activity of extracts of Hamelia patens obtained by different extraction methods

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

Fungal

and

Bacterial

Physiology

Phenolic

content

and

antibacterial

activity

of

extracts

of

Hamelia

patens

obtained

by

different

extraction

methods

Jorge

Enrique

Wong

Paz

_,

_Carolina

_Rubio

_Contreras

_,

_Abigail

_Reyes

_Munguía

_,

Cristóbal

Noé

Aguilar

_,

_María

_Luisa

_Carrillo

_Inungaray

a_{TecnológicoNacionaldeMéxico,InstitutoTecnológicodeCiudadValles,DepartamentodeIngeniería,CiudadValles,SanLuisPotosí,}

Mexico

b_{UniversidadAutónomadeCoahuila,UnidadAcadémicaMultidisciplinariaZonaHuasteca,LaboratoriodeInvestigaciónenAlimentos,}

CiudadValles,SanLuisPotosí,Mexico

c_{UniversidadAutónomadeCoahuila,FacultaddeCienciasQuímicas,DepartamentodeInvestigaciónenAlimentos,Saltillo,Coahuila,}

Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16March2016

Accepted1March2017

Availableonline6December2017

AssociateEditor:LuisHenrique

Guimarães Keywords: Antibacterialactivity Hameliapatens Polyphenols HPLC–MS

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b

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Hameliapatens,isaplanttraditionallyusedtotreatavarietyofconditionsamongtheHuastec

peopleofMexico.Theobjectiveofthisstudyistocharacterizethephenoliccontentand

criticallyexaminetheantimicrobialactivityofleafextractsH.patens,obtainedby

mac-eration,Soxhletandpercolation,usingethanolas70%solvent.Phenoliccompoundsare

characterizedbyliquidchromatography,coupledtoaHighResolutionMassSpectrometry,

andtheantimicrobialactivitywasstudiedfromtheinhibitoryeffectofeachextractfor

Escherichiacoli,Staphylococcusaureus,SalmonellatyphiandS.paratyphi,andbytheMinimum

BactericidalConcentration,thepercentageofactivityandtheIndexofBacterial

Suscepti-bilityofeachextract.Thephenoliccompoundidentifiedindifferentconcentrationsinthe

threeextractswasepicatechin.Theextractsobtainedbythethreemethodshad

antimicro-bialactivity,however,therewasnosignificantdifference(p<0.05)betweentheMinimum

BactericidalConcentrationoftheextractsobtainedbymaceration,percolationandSoxhlet.

Theresultsofthisstudycontributetothebodyofknowledgeontheuseofextractsin

controllingmicroorganismswithnaturalantimicrobials.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:maluisa@uaslp.mx(M.L.Inungaray).

https://doi.org/10.1016/j.bjm.2017.03.018

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

Introduction

Fromancienttimes,manhastakenrefugeinnatural

reme-dies tofind a cure for their diseases.The plants used for

thispurposeareknownasmedicinalplants,astheycontain

chemicalcompounds thathave biologicalpropertieswhich

arebeneficialtohealth.Thesepropertiesinclude,

antimicro-bialactivityattributedtophenoliccompoundsandessential

oilspresent indifferent partsofthe plant.1 _{Hameliapatens}

belongstothefamilyoftheRubiaceae,2 _{andistraditionally}

usedtotreatskinconditionsandtheantimicrobialand

antiox-idantactivityofitsphenoliccompoundshavebeenstudied.3

Ríosand Aguilar4 _{reportedthatH.patens isaplantusedin}

traditionalMexicanmedicinewhichareassociated

antimicro-bial,analgesicandanti-inflammatoryeffects.Althoughsome

authors,5,6_{havereportedtherapeuticpropertiessuchas}

anti-darrheal,intestinalantispasmodic,anemia wound,healing,

andantidiabeticforH.patens,intheHuastecaPotosinaregion

inMexico,itisacommonpracticetouseittowashwounds,so

itisofinteresttoknowifitalsohasantimicrobialactivity.

Pri-etoetal.7_{obtainedextractsofplantsbelongingtothisfamily,}

usingthemacerationmethodwithtwotypesofsolvents;ethyl

etherandpetroleumether,itwas reportedthattheformer

hadbetterantibacterialantifungalactivity.Furthermore,Pi ˜na

et al.8 _{reported that the rumberina and palmirina, which}

aresecondarymetabolitesisolatedfrom leavesofH.patens,

showedinhibitoryactivityonLeishmaniamexicana

promastig-otes.Although,moststudiesontheantibacterialactivityof

thisplanthavebeenmadefromtheextractsobtainedwith

organicsolvents,theaqueousextractshavealsoshownsome

bactericidalactivity.9

Most studies on the antimicrobial activity of H. patens

havefocused moreon the effectof solventused, and less

onthemethodusedtoobtaintheextracts.Aknowledgeof

whethertheapproachormethodusedtoobtaintheextracts

can affect the content and biological activity of chemical

compounds,suchaspolyphenols, leadingtothepossibility

ofmore efficient resources laboratory whereit works best

withplantextracts,willenableonetochoosewhichtoallow,

that will bring about better extraction of the active

com-poundsinthemedicinalplants. TheMinimum Bactericidal

Concentration (MBC) is defined as the minimum

concen-trationofantimicrobial whicheliminates morethan 99.9%

ofmicroorganisms viable after a set incubation time,10 _so

it is a reliable parameter for evaluating the antimicrobial

propertiesofasubstance.Therefore,thispaperaimsto

char-acterize the phenolic content and study the antimicrobial

activityofleafextractsofH.patens,obtainedbythree

differ-entextractiontechniques,Maceration(ME),Soxhlet(SE)and

Percolation(PE).

Material

and

methods

Samplepreparation

Scientificname voucher:Hamelia patens Jacq.HPHP-SP-102;

leavesofthisspecimenwerecollectedfromdifferent

house-holdbackyardslocatedintheHuasteca,northeastofMexico.

Latitude:99◦01W;longitude:21◦59N;metersabovesealevel:

74. The plant was identified by the taxonomist

responsi-ble and curator for the “Isidro Palacios” Herbarium of the

AutonomousUniversityofSanLuisPotosí,andwasdeposited

inthis.Thoseleaveswhichdonothavephysicaldamagewere

selected.Selectedleaveswerethenblanchedinorderto

inac-tivatethepolyphenoloxidaseenzymeoftheleavesinorderto

confirmthattheguayacoltestinactivationwasperformed.11

BlanchedleavesH.patensweredriedinaconventionaloven

(Linderberg/Blue, USA)at55◦C for48h. Themoisture

con-tentwasalsodeterminedaccordingtothemethodologyofthe

AOAC.12

Obtainingextracts

ExtractswereobtainedbythemethodsofMaceration,Soxhlet

andPercolation,asdescribedbelow:Classicalmethodof

Mac-eration;20gofdriedleavesofH.patenswereplacedinaflask

and200mLof70%ethanolwasadded.Thesolutionwasthen

stirredfor24h.Soxhletmethod;20gofdriedpowderedleaves

were placedin 200mLof 70% ethanol. Soxhlet equipment

(Barnstead/Lab-lineMultiHeaterUnitExtraction)wasusedto

analyzethesolutionatatemperatureof60◦Cfor4h

contin-uously.Percolationmethod.20gofleavesofH.patenspowder

wereplacedinapercolationsystemand1Lof70%ethanol

waspassedthroughthesample.Theextractsobtainedbythe

threemethodswerefilteredusingfilterpaper125mm,No.3

(Whatmanbrand)andconcentratedunderreducedpressure

inarotaryevaporator(brand BUGI)pressuretoremovethe

solvent.Theextractswereresuspendedindistilledwaterand

sterilizedbymembranefiltrationwithpore45␮m(Whatman

brand)andstoredat4◦Cuntiluse.Fromthestocksolutionsof

eachextract,otherconcentrationstobedeterminedbyMBC

andmeasuretheirantimicrobialactivitywereprepared.

Characterizationofphenoliccompounds

H.patensextractswereanalyzedusingasystemofhigh

resolu-tionliquidchromatographycoupledwithmassspectrometry

(HPLC-ESI-MS).TheHPLCconsistedofanautosampler(Varian

ProStar410,USA),aternarypump(VarianProStar230I,USA)

andaPDAdetector(VarianProStar330,USA)setat280nm.

Formassspectrometricanalysis,massspectrometeriontrap

(Varian 500-MS) equipped with an electrospray ion source

wasused.Thecolumnused(C185␮m,150mm×2.0mm)was

maintainedat30◦Cwhilethemobilephasewasaceticacid

3%(A)andacetonitrile(B).Theflowusedwas0.3mL/minwith

aninjectionvolumeof5␮Lsample.TheBgradientelution

were: initial3%;9%5min; 15min16%;45min 50%.

There-after,thecolumnwaswashedandreconditioned.Alleffluent

(0.3mL/min)wasinjecteddirectlyintothesourceofthemass

spectrometer,withoutdivision.Allexperimentswerecarried

outinnegativemode[M−H]−1_{.Nitrogenwasusedas}

nebu-lizergasandheliumasabuffergas.Theparametersofthe

ionsourcewere:voltage(3.5kV),capillaryvoltage(90.0V)and

temperature(350◦C).Datawerecollectedandprocessedusing

theMSWorkstationsoftware(V6.9).

Some standard ofphenolic compounds were used

chlorogenicacid,methylgallate,coumaricacid,catechin,(

−)-epicatechin,procyanidins(B1,B2andC1),2-hidroxycinamic

acid,ellagicacid,quercetinandcinnamicacid,allwere

pur-chasedfromSigmaAldrich.

Antimicrobialactivity

Tostudytheantibacterialactivityofthethreeextracts,MBC

and its inhibitory effect on S. aureus ATCC 35556, E. coli

ATCC25922,S.typhiATCC14028andS.paratyphiATCC9150

were determined. The microorganisms used in this study

were provided by the Ciudad Valles Sanitary Jurisdiction.

Fromthe revitalizedstrains,the inoculumwaspreparedas

described byCockerill et al.13 _{Thepercentageofactivity of}

eachextractandtherateofbacterialsusceptibilitywerealso

calculated.

MinimumBactericidalConcentration(MBC)

MBCwasusedtodeterminetheeffectofethanolicextracts

ofH.patensondifferentbacterialspecies.Itwasperformed

according tothe methodology described byChiong et al.14

Series of 10 tubes with 2mL of nutrient broth were

pre-pared;thefirstofeachseriescontainedadoubleconcentration

of broth nutrient and other single concentration. From a

stocksolution, 60mg/mLofeach extract wasadded tothe

first tubeofeach series then,2mLtransferredtothe next

tube, and so on togive concentrations of30, 15,7.5, 3.75,

1.85 and 0.9mg/mL of each extract. Eachtube was

inocu-latedwith100␮L(1×108CFU)ofthebacterialsuspensionand

wereincubatedat35◦Cfor24h.Twoconcentrationsusedas

controlswere alsoconductedusingdistilled wateras

nega-tivecontrolandMoxifloxacin,abroad-spectrumantibiotic,as

positivecontrol.Afterthe incubationperiod,a roastofthe

last tubeofthe series didnotshow turbidityand

inocula-tioninappropriatemediaculturewastaken:BairdParkerfor

Staphylococcusaureus,McConkeyagar forEscherichiacoliand

Salmonella-Shigella agar for Salmonella typhi and S.

paraty-phi. The media were incubated for 24h at 37◦C. All tests

wereperformed intriplicate.Thetechniqueusedallowsto

knowtheminimuminhibitoryconcentrationoftheextract,

which corresponds to the dilution in which turbidity is

no longer observed, however, when inoculating this

dilu-tion inspecificmediawithout extract, themicroorganisms

cannotdevelop,indicatesthat theyhavedead,sothatthe

tubewiththe lowestbactericidal concentration–the

max-imum dilution in the technique –, will correspond to the

MBC.

Inhibitoryeffect

Theinhibitoryeffectofeachextractwasmeasuredfromthe

zonesofinhibitionobservedwhenusingthedisk diffusion

technique100␮L(1×108_{CFU)ofbacteriawasinoculatedwith}

the agar surface Muller Hinton.Thereafter, sixfilter paper

discs(brandWhatman)of6.0mmindiameterwereplacedand

sprayedwith10␮Lofeachoftheextractsatdifferent

concen-trationsandwiththesubstancesusedascontrol.Alltestswere

performedintriplicate.

Percentageofactivity

Thepercentageofactivityofeachextract(Eq.(1))indicatesthe

totalantimicrobialpotencyoftheextractinparticular.15

% Activity=_Number(100×Num)(Total_{ofsusceptible}number_bacterialofstrains_strainstested)_extract (1)

Indexbacterialsusceptibility(IBS)

TheIBS(Eq.(2))showsthenumberofmicroorganisms

suscep-tibletoextract,assessingrangesfrom0(resistanceextractall

samples)to100(susceptibletowholeextract).15

IBS=100×Numberofeffectiveextractsforeachbacterialstrain

Numberofstrains (2)

Results

Fig.1showsthechromatogramofthecompoundsidentified

ineachextract.TwelvedifferentcompoundsinextractsofH.

patenswereidentified.Thecompoundspresentdependedon

thetypeofextractionmethodused.Compoundsidentifiedas

2and3(Fig.1)wereonlypresentintheextractsobtainedby

maceration.Whilethecompoundidentifiedas5wasabsent

intheextractobtainedbySoxhlet.Compounds2,3and5were

identifiedashydroxycinnamicacid,catechin,andprocyanidin

B2(Table1).Themaincontrollingcompoundintheextracts

obtainedbythethreeextractiontechniqueswascompound

No.4(Fig.1)identifiedaschlorogenicacid(Table1).The

phe-noliccompoundpresentinthedifferentconcentrationsofthe

threeextractswasepicatechin(6)(Table1).

TheMBCisthelowestconcentrationofanantimicrobial

forinhibiting the growthofa microorganismagent, andis

considered an essential parameter to checkthe sensitivity

ofabacteriatoanantibacterial.16 _{AhigherMBCvaluefora}

specificmicroorganismindicatesalowerantimicrobialagent

employedcapacity.Forthisreason,itisoneofthemost

reli-abletechniquestodeterminetheantimicrobialpropertiesofa

substance.Table2showstheMBCH.patensextractsobtained

bythe threeextractiontechniques againstfour pathogenic

bacteria.BysettingtheMBCforH.patensextractsobtainedby

differentextractionmethods,itfoundouttheirpotentialasa

antimicrobialagentforpathogenicbacteriastudied.Extracts

ofmacerationandSoxhletCBIpresentedthelargest

percent-agerelativetothepositivecontrol(Moxifloxacin).Moreover,

theextractobtainedbypercolationshowedMBCvaluesvery

similartothoseobtainedbythepositivecontrolreflectingits

greater potentialasanantimicrobialagentforthe bacteria

studied.

In generalthe threeextracts hada75% efficiencyforS.

aureusandS.typhi,50%forS.paratyphiand100%forE.coli.In

thethreeextractsobtainedbydifferentmethodsthe

percent-ageactivitywas100%andtheIndexofBacterialSusceptibility

(IBS)was75%.

Discussion

AccordingtotheHPLCchromatogramsinthepresenceofthree

extractsmostphenoliccompoundsidentifiedwasobserved.

300 250 200 150 Aborbance Absorbance Absorbance 100 50 0 2000 1500 1000 500 2000 1500 1000 500 0 5 5 6 8 9 10 11 12 7 10 15 20 Time (min) 25 30 35 40 0 1 2 3 4 4 6 8 9 10 11 12 7 1 0 4 6 8 9 10 11 12 7 5 1 HP-Percolation HP-Maceration HP-Soxhlet

Fig.1–HPLCchromatogramofextractsofleavesHameliapatensobtainedbypercolation(A),Soxhlet(B)andmaceration(C).

Table1–Characterizationofcompoundsfoundin extractsofleavesofH.patens.

Peak Timeof retention (min) [M−H]−1 _Molecular weight Tentative identification 1 1.6 191 192 Quinicacid 2 6.1 353 354 Hydroxycinnamic acid 3 10.7 289 290 Catechin 4 14 353 354 Caffeoylquinicacid 5 15.9 577 578 ProcyanidinB2 6 18.6 289 290 (−)-Epicatechin 7 25.3 451 452 (+)-Catechin 3-O-glucose 8 26.9 609 610 Quercetin 3-O-rutinoside 9 28 593 594 Kaempferol 3-O-rutinoside 10 28.9 497 498 Noidentification 11 31.6 451 452 3-Hydroxyphloretin 2-O-Glucosid 12 33.8 573 574 Noidentification

Table2–MinimumBactericidalConcentration(MBC)of thethreeextractsofH.patensobtainedbydifferent techniquesagainstpathogenicbacteriastudied. Bacteria Extractionmethods

Maceration Soxhlet Percolation

MinimumBactericidalConcentration(mg/mL)

E.coli 4.0 7.5 3.5

S.aureus 12.0 8.0 3.5

S.typhi 2.0 3.5 3.0

S.paratyphi 10.0 7.5 3.5

chlorogenic acid, which belongs to the class of

cinna-mate and is widely distributed in nature. Chlorogenic

acid encompasses a family of cinamoilquinicos trans

esters. These compounds have been reported to

pos-sess a wide range of biological properties which

high-lightstheantimicrobialactivity.17 _{Interestingly,theextracts}

obtained bypercolation generally present ahigher

antimi-crobial activity against bacteria studied. Unlike

greater amount of the compounds identified as

epicate-chin, which has also been reported to possess

antimicro-bial properties and forms a precursor polymer formation

called condensed tannins, which in some compounds are

reported as antimicrobial and sometimes recalcitrant.18

Therefore, the antimicrobial activity could be evaluated,

widelylinkedtothesetwocompounds,chlorogenicacidand

epicatechin.

In determining the MBC for H. patens extracts which

were obtained by different methods, it was found as an

antimicrobialpotentialforthebacteriastudied.Althoughthe

MBC of the three extracts were different for each of the

microorganisms, no significant difference existed between

them(p<0.05).SaucedaandNereyda1_{mentionthatthe}

vari-ables that affect the extraction process, regardless of the

scale of production or type of end product are: agitation,

state of division of matter, extraction time, nature of the

solvent, temperatureand pH.The contacttime ofthe

sol-ventwiththe plantallowsdiffusionofmetabolites solvent

to yield better resultswhen time is greater; the

tempera-turecanincreasetherateofextractionbecauseitfavorsthe

solubility,howeverhightemperatures canaffectthe

stabil-ityofthecompounds.Moreover,agitationpromotesgreater

extraction while efficiency decreases because of diffusion

resistance,eliminatingthefluidfilmcoveringthesurfaceof

thesolidatrestand,finally,reducingparticleisofgreat

impor-tance,becauseitincreasesthecontactareaanddecreasesthe

timerequiredfortheextraction,especiallyforlowporosity

solid.19

Theresultsobtainedinthisstudyaresimilartothoseof

Villavicencioand Perez20 _{who reportthatH.patens ismore}

activeinE.colitoS.aureus.Yasunakaetal.21_{alsoclaimthat}

H.patenshasaMBClowerforE.colitoS.aureus.Furthermore,

CervantesandGonzalez22_{showedthataqueousandethanol}

extractsofH.patenshadgreaterantimicrobialactivityagainst

S.aureusaboutE.coli,andbothextracts,theethanolwasmost effective.

ThefactthattheMBCarelessattributabletoincreased

sus-ceptibilityofthebacteriatotheactivecompoundspresentin

theextract,orbecausethewaythattheextractwasobtained,

allowedincreasedextractionofthesecompounds.TheMBCof

theextractsvaried,notonlyintermsoftheextractionmethod,

butalsodependingonthebacteriastudied.Itwasobserved

thattheMBCofEP,waslessforE.coli,S.aureusandS. paraty-phi,S.typhi thattheMBCrequiredwhenusingthe MSand ES.

Cáceresetal.23_{statethatthewettingoftherawmaterial}

increasestheporosityofthecellwallandcellfacilitatesthe

diffusionofextractablesoutwardlyofplantcells,wherebythe

percolationprocessmaybemoreefficientbecauseitincreases

thecontacttimeofthefeedstockwiththesolventanddragthe

activecompoundsandrelatedsamplesolventare obtained,

allowingcompleteextractionoftheactiveingredientsofthe

plant.

Whenobtainingthetemperaturehandledat>50◦C,which

couldaffectthebiologicallyactivecompoundspresentinthe

plant;itwasrecalledthattheothermethodsofextractionare

performedat<50◦C.Althoughthedissolutionofextractables

isfacilitatedbytheincreasedtemperature,manyactive

ingre-dientsarethermolabileandcanbefullyorpartiallydestroyed

atelevatedtemperatures.24

Theextractsobtainedbythethreemethodsshowed100%

of antimicrobial activity and IBS was of 75%. However, it

is considered that the MBC of the extracts of H. patens

are high because Cockerill etal.13 _{reportedthat anextract}

is considered with antimicrobial activity, MBC should be

<500␮g/mL. Although MBC of extracts H. patens are high,

its antimicrobial activity must be recognized, the

condi-tions traditionally used by the population as a poultice,

may be able to enhance the activity of the active

com-pounds present in the plant. The yield of the extracts

obtained by maceration, percolation and Soxhlet was 25,

44.5 and 24% respectively. The results obtained in this

study provethatahigherpercentageyieldinobtainingthe

extract isnot anindicator ofincreasedantibacterial

activ-ity. Gyawali et al.25 _{report that active compounds present}

in the plants have antimicrobial activity, which is well

documented, although the mechanisms of action are not

clear, however due to the resistance of microorganisms

to traditional antibiotics, the application of antimicrobials

natural resources of plant origin currently have a greater

application.

Conclusions

H. patens extracts obtainedby Maceration, Percolation and

Soxhlethadantimicrobialactivity,whichwasattributedtoits

contentofphenoliccompounds,duetothepresenceof

epi-catechin. However,the extractobtainedbypercolation had

thelowestMBCandgreaterinhibitoryeffectonthestudied

bacteria,thanthoseobtainedbyMacerationandSoxhlet.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WethanktoTaxonomistJoséGarcíaPérez,Responsibleand

curatorofthe“IsidroPalacios”HerbariumoftheAutonomous

UniversityofSanLuisPotosí,fortheirsupportforthe

identi-ficationoftheplantunderstudy

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