Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment

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Review

Lactoferrin,

chitosan

and

Melaleuca

alternifolia—natural

products

that

show

promise

in

candidiasis

treatment

Lorena

de

Oliveira

Felipe

∗

,

Willer

Ferreira

da

Silva

Júnior,

Katialaine

Corrêa

de

Araújo,

Daniela

Leite

Fabrino

UniversidadeFederaldeSãoJoãodel-Rei/CampusAltoParaopeba,MinasGerais,MG,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18August2016 Accepted26May2017

Availableonline11November2017 AssociateEditor:LuisHenrique Guimarães Keywords: Candida Lactoferrin Melaleucaalternifolia Chitosan Naturalproducts

a

b

s

t

r

a

c

t

Theevolutionofmicroorganismsresistanttomanymedicineshasbecomeamajor chal-lengeforthescientificcommunityaroundtheworld.Motivatedbythegravityofsucha situation,theWorldHealthOrganizationreleasedareportin2014withtheaimofproviding updatedinformationonthiscriticalscenario.Amongthemostworryingmicroorganisms, speciesfromthegenusCandidahaveexhibitedahighrateofresistancetoantifungaldrugs. Therefore,theobjectiveofthisreviewistoshowthattheuseofnaturalproducts(extractsor isolatedbiomolecules),alongwithconventionalantifungaltherapy,canbeaverypromising strategytoovercomemicrobialmultiresistance.Somepromisingalternativesareessential oilsofMelaleucaalternifolia(mainlycomposedofterpinen-4-ol,atypeofmonoterpene), lacto-ferrin(apeptideisolatedfrommilk)andchitosan(acopolymerfromchitin).Suchproducts havegreatpotentialtoincreaseantifungaltherapyefficacy,mitigatesideeffectsandprovide awiderangeofactioninantifungaltherapy.

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

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

Introduction

Fungalinfectionsareresponsibleformanydeathsaroundthe world,inadditiontocontributingtosignificantexpensesin publichealth.1_{Amongthepossibleetiologicagents,Candida}

spp.arenotedduetotheirrelevanceandprevalence. Accord-ingtoMcCartyandPappas(2015),15–20microorganismsfrom thegenusCandidaaredescribedascertaintocauseinfection.2

∗ _{Correspondingauthor.}

E-mail:lorenaob@gmail.com(L.O.Felipe).

However, fourspecies cause 96%ofthe infections:Candida albicansisthemainetiologicagent,responsiblefor42.5%of infections,followedbyCandidatropicalis(27.3%),Candida para-psilosis(21.9%)andCandidaglabrata(4.4%).3

InfectionsoriginatingfromC.albicanshavebeenobserved since the time of Hippocrates,who described Candida dis-eases indebilitatedpatientsin337B.C.4 _{Now,Candidaspp.}

areresponsibleforthemajorityofbloodinfectionsin inten-sivetherapyunitsaroundtheworld,impactinghospitalization timesandcosts.5_{In2005,forexample,theCentersforDisease}

ControlandPrevention(CDC)showedthatforeach diagno-sis of Candida spp. infection, the period of hospitalization

https://doi.org/10.1016/j.bjm.2017.05.008

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

islengthened from 3to13 days. Financially speaking,this additionalperiodleads toexpenses from $6000 to$29,000. Furthermore,eachyear,thesevaluesareestimatedtoreach approximately$8billion.6,7

Advances in medicine have enabled people who are immunesuppressed(suchasthosewhohavereceivedorgan transplantsorareHIVpositive)tolivelongerlives.8_The

great-estvulnerabilityofsuchindividualsistometabolicallyflexible microorganismsofthegenusCandida,whichhaveincreased therateofmorbidityand mortalityto35% inthis groupof people.9 _{In addition, parallel to the increasing number of}

casesoffungal infection causedbyCandidaspp., the resis-tanceofCandidaspp.toantimicrobialdrugshasprogressively increased.Thelatter facthas sparkedthe attention ofthe WorldHealthOrganization(WHO)duetoitsrelevanceandled tothereleaseofthereportAntimicrobialResistance:GlobalReport Surveillance.10

ThedevelopmentofresistanceinCandidaspp.,from ther-apywithamphotericinB,againstdrugsfromtheazolegroup, echinocandinandfluconazolewasamongthekey informa-tion providedinthis report,along withreports ofcasesof multiresistancefromdifferentregionsoftheworldsuchas AsiaandtheMiddleEastandsomeareasofSouthAmerica.10

Thesedataarealarmingbecausesuchresistancehas devel-opedagainstantifungaldrugswidelyusedtocombatfungal diseasesworldwide.AmphotericinB,forexample,isaclassical antifungaldrug,andinmanycountries,itistheonlymedicine availableforthetreatmentoffungalinfections.

The azoles have been administered in formulations that combine different groups of chemotherapeutic drugs, attempting to overcome the increase in microbial resis-tance. This fact impacts both the cost and the success of treatmentsagainstthattypeofinfection.Finally, the devel-opment of resistance to fluconazole—the most commonly usedantifungal—drasticallyvariesaccordingtotheCandida spp. and from country to country.For example,the inten-sityofthemultiresistance inCandidaspp.tofluconazoleis higherinDenmark(33%)and lowerinthe KoreanRepublic (0.9%).10_{Therefore,theaimofthispaperistodiscussthemost}

promisingsubstancesavailableforcandidiasistreatment.In addition,thisworkreinforcestheimportanceofusingnatural productsalongwiththosemedicinestraditionallyemployed inthetreatmentofsuchinfections.

Natural

products

Natural products have been widely used in society since 3500B.C.11 _{Medicinalmethodsrangefrom folkmedicine to}

validated treatments.12_{ThedoctorParacelsusisconsidered}

thefounderofplant-basedmedicineduetohisgreat contrib-utionsinthefield.13 _{Hisfindings,alongwiththoseofother}

researchers,ledtoaconsolidationofknowledgeinthefieldof naturalproductsandcreatedotherdomainssuchas pharma-cognosy.Amongthemedicinalpropertiesofnaturalproducts, one canfind anti-inflammatory,antispasmodic, anticancer, antimutagenic,antibacterial,antifungal,antiviraland vermi-cidalproperties.14

Essential oils compose the most utilized group of sub-stances.Isolatedfromplants,essential oilsare compounds largely described in the scientific literature as versatile in thetreatmentofmanyinfirmities.15_{Theiractiveingredients}

are usuallyvariousterpenes, whichare secondary metabo-lites excreted byvegetables to expelpredators or harmful agents.16_{Oneexampleofabioactiveessentialoilistheone}

obtainedfromMelaleucaalternifolialeaves;theactivityofthis oilisattributedtoterpinen-4-ol,amonoterpene.17

Theisolationofbioactivemoleculesisalsoperformedon naturalsourcesotherthanvegetablecells.Somemetabolites produced bymicroorganismsare alsoimportantsourcesof new bioactive substances.18 _{In Table 1, therapeutic agents}

obtainedthroughbioprospectionarepresented;theseagents areresponsibleforsignificantimpactonthemedicinal treat-mentsthathavebeenofferedinrecentyears.

InadditiontothecompoundslistedinTable1,lactoferrin(a peptideisolatedfrommilk)andchitosan(acopolymerderived from chitin) also show distinct biological activity, includ-ingantioxidant,anti-allergyandantimicrobialactions.19_The

associationofsuchnaturalproductswithotherantimicrobial drugshasbeenrecentlyinvestigatedtoidentifyapossible syn-ergisticeffect.Thiscanprovidealternativetreatmentsagainst multiresistantmicroorganisms,therebyreducingtheamount ofdrugsusedintreatments,whichcanimpactdrug-resistance phenomena.

However, as noted by Harvey et al. (2015), due to the environmental legislation that protects biodiversity, bioprospection,amongotherpursuits,hassuffereda consid-erableslowdowninthelast20years.20_{Thereductioninthe}

rate ofdrugdiscovery and newmedicines provided bythe pharmaceuticalindustryreflectssuchconstraints.

Bioprospection is a feasible way to discover new biomolecules.Oneexampleofsuchadiscoveryisteixobactin, whichwasisolatedfromsoilsamplesbyLingetal.(2015).21

Thismoleculeprovedtobeverypromisingasanewantibiotic class because its administration in mice was able to suc-cessfullyeliminateinfectionscausedbyStaphylococcusaureus superbacteriastrains.Therefore,afternearly30years with-out anew drugdiscovery, this promisingmolecule—found

Table1–Conventionalmedicinesoriginallyobtainedfromnaturalsources.

Activecompound Source Type Indication/Use

Artemisinin Artemisiaannua Plant Antimalarial

Lovastatindiseases Aspergillusterreus Microbial Hypercholesterolemia/cardiovascular

Micafungin Coleophomaempetri Microbial Antifungal

Paclitaxel Taxusbrevifolia Plant Anti-tumor

Table2–Typesofinfectiondescribedforcandidiasisanditsrespectiveplacesofoccurrence.

Typeofcandidiasis Locationofoccurrence

Mucocutaneous Oral,vaginal,penile(balanitis).32–34

Cutaneous Moisturizedpartsofbody:areasclosetobreast,armpits,foldofgroinandunderthenails.35

Systemic(candidemia) Visceraldissemination:lungs,meninges,kidney,bladder,joints,liver,heartandeyes.5

Table3–Differentclassesofantifungalsthatare availableonthemarket.

Azoles Echinocandins Pyrimidines Polienics Cetoconazole Anidulafungin Flucytosine Amphotericin-B Fluconazole Caspofungin Nystatin Itraconazole Micafungin

Pozaconazole

through bioprospection—may revolutionize the current

antibacterialtreatments.21

Findingbioactivecompoundsisnotaneasytask. There-fore,naturalproductshavebeen usedalongwithsynthetic drugstoovercome,mainly,resistancetoantimicrobialdrugs or to alleviate the side effects associated with specific therapies.

Candidiasis

Microorganisms from the genus Candida are described as yeastsandincludemorethan160species.22_Intermsof

mor-phology,thegenusischaracterizedasbeingthick,curd-like (cheesy),andyellowish-white.Candidaalbicansisfound exten-sivelyinhumans,and theseyeastsareeasilyisolatedfrom theskinandgastrointestinaltract.23 _{Therefore,thistypeof}

microorganismlivescommensally,co-inhabitingandexisting inequilibriumwithhumanmicrobiota.24_{Nonetheless,under}

specificconditions,thesemicroorganismsarefavored,which usuallyenablestheactivation ofthevirulencefactor, mak-ingsuchorganismspathogenicandharmfultothehealthof thehost.25,26 _{Thus,C.albicansisknownasanopportunistic}

microorganism.27

Peoplewithimmunodeficiencycomposethegroupwiththe highesttendencytodevelopcandidiasis.Amongthemost vul-nerablepeople areelderlypeople andbabies;patientswith cancer, AIDS or with transplants; people whose treatment usescatheters(inparenteralnutritionorinhemodialysis);and peoplesubmittedtotherapiesbasedonantimicrobialdrugsof largespectrum/corticoids.28–30_{Thetypeandtheintensityof}

theinfection(Table2)aredirectlyrelatedtotheimmunological responseofthepatient.31

WhenCandidaspp.takeadvantageofahost’s immunosen-sitivity,themajormechanismsofvirulenceandtheinvasive capacityoftheCandidaspp.aremainlyrelatedto(i)theability toformbiofilmreinforcedbyamechanismofquorumsensing and (ii) phenotypic switching,that is, when yeastschange tothe filamentous form,which is a determinantof tissue invasion.36,37

Manyclasses ofdrugsare availableforthe treatmentof thistypeofinfection,asshowninTable3.However, consider-ingthatfungi,aswellashumancells,areeukaryotic,patients may suffer from several side effects that arise from such

drugs.Nephrotoxicity(especiallyinamphotericin administra-tion),nausea/vomitingandstomachacheareamongthemain effects.

Inadditiontosideeffects,anothermajorchallengeinthe campaignagainstCandidaspp.infectionsistherefractoriness tothose medicines that aretraditionally used.38 _Thelatter

problemarosefromtheindiscriminateuseofantifungaldrugs or long antifungal therapies,favoring the naturalselection ofCandidaspp.-resistantstrains.Someexamplesofresistant strainsareC.albicans,C.glabatra,C.kruseiandC.auris.39

Can-dida lusitaniae hasalsobeen reportedtodevelopresistance duringtherapywithAmphotericin-B.40

Therefore,differentstudieshaveindicatedthatthe asso-ciation oftraditionalmedicineswithalternativetreatments (herein, natural products or biomolecules) as a promis-ing option for therapeutic success against Candida spp. infection.41_{Amongthealternatives,themostprominentare}

lactoferrin,chitosanandMalaleucaalternifoliaextracts. Promis-ingbiomoleculesinthetreatmentofcandidiasis

Lactoferrin

Antimicrobialpeptides(AMPs)havebeendescribedinthe sci-entificliteratureandcorrespondtoagroupofapproximately 1200 molecules.42,43 _{They can be isolated from different}

sources suchasbacteria, insects,plantsand vertebrates.44

Lactoferrin(LF)isanexampleofanAMP.

LFwasinitiallyidentifiedincowmilkin1939.45Inhuman milk,LF wasnotisolated until1960.46,47 _{Then,thispeptide}

beganbeingrecognizedasoneoftheessentialcomponentsin thedefensesystemforcombattinginfectionscausedby differ-entetiologicagents—bacteria,fungi,parasitesandviruses.48

Inadditiontobeinginmilk,LFisalsowidelyfoundon der-malmucosa,inneutrophils(whereresponsestoinflammatory stimuliarereleased)andinexternalsecretions—tears,saliva, vaginalmucus and seminalplasma.49,50 _{Inbodyfluids, the}

concentrationofLFdependsonwheretheLFisfound:inthe colostrum,theconcentrationisfrom5to7mgL−1,whereas intears,theconcentrationisapproximately2mgL−1.51,52 _In

blood,dependingontheinflammatoryprocess,the concentra-tionofLFalsovariesfrom10−3to200␮gmL−1.53_Incontrast,

accordingtoCohenetal.(1987),theconcentrationofLFin vagi-nalmucusvariesasafunctionofthemenstrualcycle:before menstruation,theconcentrationis3.8–11.4␮gmg−1,whereas aftermenstruation,thisvalueincreasesto62.9–218␮gmg−1.54

Cohen et al. (1987) also showed that womensubmitted to continuous contraceptive-based treatment have drastically affectedLFconcentrationsinthevaginalmucus,with concen-trationsremainingstableduringtheentiremenstrualcycleat approximately19.8␮gmg−1.54

Structurally, LF is one glycoprotein bound to an iron nucleus,withareddish-pinkcolor,madeupof692aminoacids andwithamolecularweightof80kDa.Fifty-fivepercentof theseaminoacidsaresimilartotheonesfoundintransferrin (theproteinresponsibleforirontransportinplasma). Lacto-ferrinexistsintwodifferentforms:halo-lactoferrin(richin iron)andapo-lactoferrin(freeofiron).Itisalsodescribedas beingabletobindtoothermetals(aluminum,copper,gallium, manganeseandzinc)betterthaniron.55

Regarding the antimicrobialactivity ofLF, the following mechanisms are described in the literature: (i) microbial growth is inhibited by iron deprivation because LF takes iron away from the pathogen or the media where the microorganismispresent56_{;(ii) theproteinpresents}

differ-entenzymatic activitiesinitspurifiedsub-fractions,which includeATPase,DNase,RNase,phosphatase andhydrolysis of malto-oligosaccharide57_{; (iii) LF increases cell wall}

per-meability, leading to the leakage of cytoplasmatic content and microorganism deathbecause lactoferrin’sN-terminus regionhasastrongpositivechargethateasilyinteractswith lipopolysaccharides,whichhaveanegativecharge58_;and(iv)

incontrast,assuggestedbyUlvatneetal.(2001),LFisnotable toinducecellularlysesinunscathedbacteriabutleadsto vesi-cleformationthroughaplasmaticmembranedepolarization mechanism.59 _{Considering the facts mentioned previously,}

lactoferrin has been recently explored in several scientific investigationswithdifferentaims,especiallyincombination withantifungaldrugs.60Inthelattercase,asynergisticeffect isthefocuswithregardtofightingagainstresistantand non-resistantmicroorganismstrainsinthegenusCandida.61

Lactoferrin:

antifungal

drugs

with

a

synergistic

effect

Candidemiainnewbornsisacriticalproblemthathas con-tinuouslysparkedtheattentionofthescientificcommunity. Venkateshand Rong (2008) evaluatedthe combined use of humanrecombinantlactoferrin(talactoferrin,TLF)with flu-conazoleandAmphotericin-BagainstthreestrainsofCandida spp.;twostrainswereisolatedfromnewbornsdiagnosedwith septicemia caused by C. albicans. The results showed that eithertheinteractionofTLFwithfluconazoleand/orTLFwith Amphotericin-Bpresentedasynergisticeffect.62

Thereafter,Venkateshetal.(2009)studiedthepreventionof biofilmformation(oneofthevirulencemechanismsobserved inCandida)bytheassociationofTLFwithantifungaldrugs; C.albicansATCCMYA4441wasthestrainusedinthisstudy. Asynergisticeffectwasobservedinthemixturecontaining bothTLFandAmphotericin-BandTLFandfluconazole.This studyisespeciallyimportantbecauseCandidabiofilm forma-tion isextremelycritical inintensivetherapy unitsaround the world, leading to high rates ofmortality and morbid-ityfrom infectionsofthe bloodstreamrelatedtocatheters. Otherstudieshavefocusedontheinteraction oflactoferrin withfluconazole,showingthatLFprobablyinteractswiththe microorganism’scellwall,raisingitspermeabilityandthereby increasingtherateofdrugpenetrationintothecell.63

Inaninvivostudy,Tanidaetal.(2001)carriedoutassays withrodentstoinvestigate thesynergistic effectofLF and

other elements. However, in that study, the LF that was used was synthesized inthe laboratory (knownas peptide 2).Anothersimilarsyntheticmolecule(peptide2)wasalso tested. Mice were inoculated with 5.0×108 _{cells of}

Can-dida (considereda lethaldose).Furthermore,the micethat did not receive treatment or were treated with peptide 2 died after 8 and 7 days, respectively. On the other hand, micesubmitted tointravenousadministration ofpeptide2 (5 and 100␮gday−1) over 5days lived longer (8.4±2.9 and 22.4±3.6days,respectively).Additionally,instudiescarried outbythesameauthors,amousethatreceived1.0×109

Can-dida cells (above the lethal dose) had its life extendedfor 8.2±2.4 days (treated with 10␮gday−1 ofpeptide 2) com-paredwiththe control(lived foraperiodof3.2±1.3days). When peptide 2was administered (10␮gday−1)along with Amphotericin-B (0.1␮gday−1) and granulocyte macrophage colony-stimulatingfactor(GM-CSF)(0.1␮gday−1),60%ofthe micelivedformorethan22days,evenwhenreceiving Can-didadoseshigherthanthelethalamount.Whenpeptide2was administeredalongwithGM-CSF,thephagocyticactivitywas raised1.5timesduetoanimmunesystemstimulus.64

A few years later, Okamoto et al. (2004) confirmed the resultsofTanidaetal.(2011).Inthiscase,thesame method-ologywasadopted.

However,anothergroupofmicewasalsoinoculatedwith 1.0×108_{Aspergillusfumigatuscells.Forbothmicroorganisms,}

thecombinationoflactoferrin,Amphotericin-BandGM-CSF increasedthesurvivaltimeoftherodentsfor35days.65

Melaleuca

alternifolia

Melaleuca alternifoliabelongs tothe Myrtaceaefamily.66 _This

plantisdescribedasascrublandspeciesandcanbefound pri-marilyinSouthAmerica,westernIndiaandAustralia.Inthe lattercountry,theplantisknownasteatreeandhasbeenused infolkmedicinelearnedfromaboriginalpeople.Some biolog-icalactivitiesofM.alternifoliaincludeantibacterial,antiviral andantifungalaction.67

Teatreeoil(TTO)canbeobtainedfromthehydrodistillation ofM.alternifolialeaves,andthechemicalcompositionvaries withtheextractionmethodandcropregion.Generally,thisoil isacomplexmixtureofmonoterpenesandterpenoids(50% oxygenated and 50% hydrocarbons)and approximately 100 othersubstances.68_{Amongthesesubstances,terpinen-4-olis}

theprimarycompound(≈30%TTOcomposition)andis indi-catedastheactiveingredientresponsibleforthetherapeutic propertiesofTTO.However,studiessuchtheoneperformed byMiller(1984),whereTTO’santimicrobialactivitywashigher thantheactivityoftheisolatedterpinen-4-ol,haveshownthe synergyamongthecompoundsoftheplant.69

AccordingtoCaloetal.(2015),theantimicrobialactivityof theM.alternifoliaterpenesisassociatedwithhydrophobicity inwhichthe terpenesinteractwiththepathogen’scellular membranelipids,changingthemembrane’spermeability.70,71 Amongthemainresultsofmembranepermeabilityarethe(i) modificationofproton-motiveforce,leadingtoadeficitinthe productionofcellularenergycausedbythedecreaseinATP generationand(ii)cellularlysesduetoleakageorcoagulation ofthecytoplasm.72,73

Melaleuca

alternifolia

(tea

tree

oil):

studies

in

the

treatment

of

candidiasis

Melaleucaalternifoliahasbeenconsideredapromising alterna-tiveinthetreatmentofstomatitiscausedbydentures,which is a recurrent problem in the elderly. de Campos Rasteiro etal.(2014)determinedboththeMICandminimumbiofilm eradication concentration (MBEC) ofTTO inin vivo experi-ments.Theresultsshoweda0.195% MICand 12.5%MBEC. Thelatterconcentrationwasusedintopicalapplication in animalexperiments.Morphologicanalysisthroughscanning electronmicroscopy (SEM)of mouse tonguetissueshowed considerablewoundareareduction,whereascolony-forming unitswerereducedby5.33log,whichhighlightsthepotential ofM.alternifolia.74

Pachavaetal.(2015)foundcolony-formingunitsof approx-imately30,43and19%ofthecontrolwhendiscsimpregnated withTTOweresubmergedinaC.albicanssuspension.That resultindicatesthatTTOcouldbeusedtocoatdenturesand reducethewoundscausedbyCandida.75_{SharmaandHegde}

(2014) alsoinvestigatedthe impregnation ofdentures with TTOandfluconazole.TTO’sMICwasequalto30%m/m,while fluconazole’sMICwas5%m/m.Atthesameconcentrations, fluconazolelostitsantifungalactivity after7days,whereas TTOdidnot.76_{SimilarresultswereobtainedbyDalwaietal.}

(2014),wherebothchlorhexidinegluconateandTTOretained theirbioactivityafter14daysofincubation,whereas flucona-zolebecameinactive.77_{Ontheotherhand,VanVuurenetal.}

(2009)evaluatedtheassociationofTTOwith amphotericin-B(AMP-B)againstC.albicans(ATCC10231),throughMICand fractionaryinhibitoryconcentrationindex(FICI)calculation.78

Thefollowingantimicrobialagentratioresultedina syner-gisticinteraction(FICI<1):6:4AMP-B:TTO(FICI0.93)and1:9 AMP-B:TTO(FICI0.95).79 _{Similarly,Rosatoetal.(2009)}

com-binedTTOandnystatinandusedthemtochallengeC.albicans, C.kruseiandC.tropicalis.ThisresultedinFICIs>0.5,also indi-catingasynergisticeffect.80

Recently,inastudypublishedbyDiVitoetal.(2015),the combination of Amphotericin-B and TTOshowed a syner-gisticeffectagainstdifferentspeciesofCandida.Thatstudy focusedonthedevelopmentofavaginalsuppositoryfor vul-vovaginalcandidiasistreatment.Candidastrainswereisolated from patients who had vaginitis. A synergistic effect was obtainedforconcentrationsequalto0.25␮gmL−1 ofAMP-B and0.08␮gmL−1ofTTO.Inthesamestudy,TTOwasshown nottokillvaginalmicrobiotaatconcentrationsbelow2%v/v (Bifidobacteriumanimalisremainedalive)andonly concentra-tionsabove4%v/vwerebactericidal.81

Nonetheless,asdescribedbyKonandRai(2013),alargegap remainsinthedevelopmentofinvivostudiestoevaluatethe biologicalactivityofM.alternifoliacombinedornotwith anti-fungaldrugs.Therefore,suchstudiesshouldbeencouraged toobtainmoreconsistentresultsaboutthecommercialuseof TTOforthetreatmentofcandidiasis.82

Chitosan

Chitinisdefinedasanaturalpolymer,andaftercellulose,itis themostwidespreadandabundantpolysaccharidefoundin

nature.Ithasstructuralandprotectivefunctionsandisfound mainlyinarthropods,thecarapacesofshrimp,insects,and thecellwallsofsomefungi.

Thecationiccopolymerchitosancanbeobtainedfromthe deacetylationofchitin.Inthisprocess,chitin’sacetylgroups (COCH3)arereplacedbyaminegroups(–NH2).Thisprocesscan

bedonethroughthreedifferentmechanisms:(i)thealkaline hydrolysisofthechitinathightemperatures,(ii)the employ-ment of the enzymechitinase, and (iii)through the direct actionofmicroorganisms.Dependingonthedegreeof acet-ylation, chitosan may have several byproducts of different composition, whichwillaffect themolecule’ssolubility.For example,whilechitinisinsolubleinmostsolvents,chitosan issolubleinorganicandinorganicacids.Structurally,chitosan isdefinedasacopolymerthatiscomposedofd-glucosamine monomersandN-acetyl-d-glucosamineresiduesboundwith glycoside␤(1→4).83

Chitosanisfunctional,nontoxic,biocompatible, biodegrad-able,andbioactive,withchelatingcapacity,selective perme-ability and polyelectrolytes.All of thesecharacteristics are dependent on chitosan’s purity, viscosity, degree of acety-lationandmolecularweight.Anotherpropertyofchitosanis its antimicrobialcharacteristics.Themechanismsofaction seemtobethrough(i)chitosan’sinteractionwiththe phospho-lipids’sialicacid(anionicgroup)becausechitosanispositively charged, leading to cell agglutination and preventing cells fromexchangingsubstanceswithexternalmedia,and(ii)the penetrationofchitosanintothecell,wherethechitosanblocks DNAandRNAtranscriptionandinhibitscellgrowth.Generally, chitosan’saminegroupsareresponsibleforitsantimicrobial activity.84

Chitosan:

potential

in

candidiasis

treatment

Thecombinationofchitosanwithantifungaldrugshasshown promising results.Soliman et al. (2015), forexample, inoc-ulated 0.1mL of C. albicans suspension into 80 mice with induced neutropenia. Twenty-four hours later, 20 animals were treated with saline solution (control group), 20 with chitosanextract(CE),20withAmphotericin-B(150mgkg−1) (AMB),andthelast20withchitosanandamphotericin simul-taneously(150mgkg−1)(CE+AMB)for72h.Whencompared tothecontrolgroup,theanimalsthatreceivedCE,AMBand CE+AMBshowedareductioninfungaldensityof73%,87%and 90%,respectively.Inaddition,oxidativestressmarkers,such asmalondialdehyde(MDA),reducedglutathione(GSH), super-oxidedismutase(SOD),catalase(CAT)andnitricoxide(NO), wereanalyzedfromthemouselungtissue.Theresultsshowed that the groups that were treated with CE and CE+AMB showedasignificantreductioninthelevelsofMDA,SOD,CAT andNOcomparedtothecontrolgroup.Ontheotherhand, thelevelsofGSHincreased,whichclearlyindicatesthat chi-tosancanmitigateoxidativestressorlungwoundscausedby systemiccandidiasis.85

Alternatively,Senyi ˘gitetal.(2014)evaluatedthe formula-tion ofchitosangel forvulvovaginalcandidiasis treatment. Suchagelwasproducedwithchitosanofdifferent viscosi-ties (20,000, 200,000 and 800,000mPas)and two antifungal drugs:miconazolenitrateandeconazolenitrate.Invivostudies

showedthatchitosanof200,000mPasledtothebestresults withbothantifungaldrugs.Thisconclusionwasbasedonthe followinggelproperties:mucosaladhesion,mechanical prop-ertiesandadequatedrugrelease.86

Chhonkeret al. (2015) investigatedthe potential of chi-tosan,incombinationwithAmphotericin-B,asatreatment forfungalkeratitis.Theactivecompoundswereusedtocoat 161.9–230.5-nm nanoparticles to extend the drug delivery time.Thenanocarrierscontainingchitosanandamphotericin were found to bemore active againstC. albicans than the commercial formulation (Fungizone®). In addition, in vivo studieswerecarriedoutinrabbitsforpharmacokinetic stud-ies, where it was observed that the bioavailability of the medicinewasincreased2.04times,whereas theprecorneal residence time was increased 3.36 times. Finally, the best results were achieved with chitosan of average molecular weight.87_{Suchresultsarerelevantbecausefungalkeratitisis}

aneyesdiseasethatisresponsibleforblindnessofmany peo-plearoundtheworld,andmedicinescurrentlyavailableforits treatmenthavestrongsideeffects.

Parker et al. (2016) investigated the role of chitosan in associationwithAmphotericin-Bandpolyethyleneglycolin the local treatment of muscle wounds. Sponges were uti-lizedasameans ofdelivering theformulation.Theresults ofinvivotestsledtotheconclusionthatspongescoatedwith Amphotericin-Breleasedtheformulation(atconcentrations above0.25␮gmL−1)formorethan72h.88

Final

remarks

The prospection of biomolecules and natural products is clearly an important strategy for discovering and making availablenewdrugsforthetreatmentofdifferentdiseases, aswellasforuseinincreasingtheactivityofcommondrugs throughsynergism,whichcanbeseenasapromisingstrategy toovercomethealarmingcurrentdrugmultiresistance sce-nario.Inthiscontext,M.alternifolia,lactoferrinandchitosan havebeendemonstratedtobeviableoptionsinthetreatment offungalinfections,mainlyagainstCandidaspp.,whenused inassociation ornotwithantimicrobialdugs.Thisis espe-ciallyimportantbecauseCandidahasledtoahighmortality rateworldwide.Therefore,studieshaveindicatedthe feasi-bilityofusingsyntheticandnaturalproductsincombination toimprovetheiractivitythoughsynergismandovercomethe problemofmicrobialmultiresistance.

Conflicts

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interest

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