Parasitostatic effect of maslinic acid. I. Growth arrest of Plasmodium falciparumintraerythrocytic stages

Moneriz Carlos, Marín-García Patricia, García-Granados Andrés, Bautista, Diez Amalia, Puyet, Puyet Antonio

Description

Natural products have played an important role as leads for the development of new drugs against malaria. Recent studies have shown that maslinic acid (MA), a natural triterpene obtained from olive pomace, which displays multiple biological and antimicrobial activities, also exerts inhibitory effects on the development of some Apicomplexan, including Eimeria, Toxoplasma and Neospora . To ascertain if MA displays anti-malarial activity, the main objective of this study was to asses the effect of MA on Plasmodium falciparum -infected erythrocytes in vitro . Methods Synchronized P. falciparum -infected erythrocyte cultures were incubated under different conditions with MA, and compared to chloroquine and atovaquone treated cultures. The effects on parasite growth were determined by monitoring the parasitaemia and the accumulation of the different infective stages visualized in thin blood smears. Results MA inhibits the growth of P. falciparum Dd2 and 3D7 strains in infected erythrocytes in, dose-dependent manner, leading to the accumulation of immature forms at IC 50 concentrations, while higher doses produced non-viable parasite cells. MA-treated infected-erythrocyte cultures were compared to those treated with chloroquine or atovaquone, showing significant differences in the pattern of accumulation of parasitic stages. Transient MA treatment at different parasite stages showed that the compound targeted intra-erythrocytic processes from early-ring to schizont stage. These results indicate that MA has a parasitostatic effect, which does not inactivate permanently P. falciparum , as the removal of the compound allowed the infection to continue Conclusions MA displays anti-malarial activity at multiple intraerythrocytic stages of the parasite and, depending on the dose and incubation time, behaves as a plasmodial parasitostatic compound. This novel parasitostatic effect appears to be unrelated to previous mechanisms proposed for current anti-malarial drugs, and may be relevant to uncover new prospective plasmodial targets and opens novel possibilities of therapies associated to host immune response.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82

RESEARCH

OpenAccess

Parasitostaticeffectofmaslinicacid.I.Growth
arrestof
Plasmodiumfalciparum
intraerythrocytic
stages
CarlosMoneriz
1,5
,PatriciaMarín-García
1,2
,AndrésGarcía-Granados
3
,JoséMBautista
1,4
,AmaliaDiez
1,4
and
AntonioPuyet
1,4*

Abstract
Background:
Naturalproductshaveplayedanimportantroleasleadsforthedevelopmentofnewdrugsagainst
malaria.Recentstudieshaveshownthatmaslinicacid(MA),anaturaltriterpeneobtainedfromolivepomace,
whichdisplaysmultiplebiologicalandantimicrobialactivities,alsoexertsinhibitoryeffectsonthedevelopmentof
someApicomplexan,including
Eimeria,Toxoplasma
and
Neospora
.ToascertainifMAdisplaysanti-malarialactivity,
themainobjectiveofthisstudywastoassestheeffectofMAon
Plasmodiumfalciparum
-infectederythrocytes
in
vitro
.
Methods:
Synchronized
P.falciparum
-infectederythrocytecultureswereincubatedunderdifferentconditionswith
MA,andcomparedtochloroquineandatovaquonetreatedcultures.Theeffectsonparasitegrowthwere
determinedbymonitoringtheparasitaemiaandtheaccumulationofthedifferentinfectivestagesvisualizedinthin
bloodsmears.
Results:
MAinhibitsthegrowthof
P.falciparum
Dd2and3D7strainsininfectederythrocytesin,dose-dependent
manner,leadingtotheaccumulationofimmatureformsatIC
50
concentrations,whilehigherdosesproducednon-
viableparasitecells.MA-treatedinfected-erythrocytecultureswerecomparedtothosetreatedwithchloroquineor
atovaquone,showingsignificantdifferencesinthepatternofaccumulationofparasiticstages.TransientMA
treatmentatdifferentparasitestagesshowedthatthecompoundtargetedintra-erythrocyticprocessesfromearly-
ringtoschizontstage.TheseresultsindicatethatMAhasaparasitostaticeffect,whichdoesnotinactivate
permanently
P.falciparum
,astheremovalofthecompoundallowedtheinfectiontocontinue
Conclusions:
MAdisplaysanti-malarialactivityatmultipleintraerythrocyticstagesoftheparasiteand,depending
onthedoseandincubationtime,behavesasaplasmodialparasitostaticcompound.Thisnovelparasitostaticeffect
appearstobeunrelatedtopreviousmechanismsproposedforcurrentanti-malarialdrugs,andmayberelevantto
uncovernewprospectiveplasmodialtargetsandopensnovelpossibilitiesoftherapiesassociatedtohostimmune
response.

Background
anti-malarialdrugshaveaneffectonalimitedselection
Despitetheincreasingnumberofsyntheticandnaturalofplasmodialtargets:inhibitionoftheconversionof
compoundswhicharereportedtoinhibittheinfectivetoxichaemtohaemozoininthevacuole(chloroquine,
cycleofthemalaria-related
Plasmodium
species[1-5]quinine,mefloquineandotheralkaloids),theinhibition
onlyafewofthemhavebeenfoundtobeusefulintheofsynthesisofparasitenucleicacidsbyhinderingofthe
treatmentandpreventionofthedisease[6].Currentdihydrofolatepathway(pyrimethamine,sulphadoxine,
proguanil),thetriggeringofoxidativestress(artemisinin
andderivatives,primaquine)orinhibitionofthemito-
1
*Correspondence:[e-mail]
chondrialelectrontransportchain(atovaquone).Some
DepartamentodeBioquímicayBiologíaMolecularIV,Universidad
ComplutensedeMadrid,FacultaddeVeterinaria,E28040Madrid,Spain
drugsmayaffectmultipleprocesses,likechloroquine,
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Monerizetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82

whichmayalsocauseoxidativedamageattheerythro-
cyticstage[7].Thewidespreaduseofthemosteffective
andaffordabledrugs,suchaschloroquineandtheanti-
folatecombinationpyrimetamine-sulphadoxine,has
favouredtheappearanceofresistantPlasmodiumvar-
iantsmakingthecontrolofthediseasemoredifficultin
endemicareas[8].Resistancetoartemisininhasbeen
alsorecentlyreported[9,10].Alternativeexistingmedi-
cationsare,ontheotherhand,virtuallyunaffordablein
thecountriesmostseriouslyaffected,ortheirefficacy
maybecompromisedinthenearfuturebytheemer-
genceofnewresistantvariants.Hence,thesearchof
newsyntheticornaturalcompoundstargetingnovel
biochemicalpathwaysorcellfunctionsisstillan
imperativeneed.
Thesearchofnaturalproductsshowinganti-malarial
activityforitsdirectuseorasleadsfornewdrugsis
oneofthealternativeswhicharecurrentlyexplored
[11].Maslinicacid(2
a
,3
b
-dihydroxyolean-12-en-28-oic
acid)(MA)isnaturalolenane-typepentacyclictriterpene
foundintheolivefruitandcanbereadilyobtained
fromolivepomaceoils[12].Severalpharmacological
activitieshavebeenreportedforthiscompound,suchas
anti-tumor[13,14],anti-oxidant[15,16],HIVprotease
inhibitor[17],antimicrobial[18],vasorelaxation[19],
andanti-diabeticactionmediatedbyinhibitionofglyco-
genphosphorylase[20,21]andproteintyrosinephospha-
tase1B[22].Inaddition,ithasbeenshownthatMA
displayslowtoxicityonnon-tumoralcells[23,24]indi-
catingthatitshouldbesafeforuseinhumans.Several
linesofevidencepointtoapossibleanti-malarialactivity
ofMA.HIVproteaseinhibitorshavebeenrecently
showntohinderthepre-erythrocyticstagesofPlasmo-
dium[25].Anti-parasiticactivitiesofMAwerealso
identifiedagainstotherApicomplexans[26].Ithasbeen
alsoproposedthattheobservedgrowthinhibitionof
Toxoplasmagondii
culturestreatedwithmaslinicacid
mayberelatedtoinhibitionofproteaseactivity,leading
todefectsinglidingmotilityandultrastructuralaltera-
tionsoftheparasite[24].Suchapossibilitysuggestsa
potentialactivityofmaslinicacid,orstructurallyrelated
molecules,onparasiticprocessesdifferentfromthose
targetedbycurrentanti-malarialdrugs.Inaddition,
othertriterpenoidmoleculesstructurallyrelatedtoMA
displayverydifferentcytotoxicandantiparasiticactiv-
ities.Thus,cucurbitane-typetriterpenoidshavebeen
recentlyshowntodisplayanti-malarialactivity[27],
whileursane-typeurosolicacidwasreportedtoinhibit
growthof
Trypanosomacruzi
[28]showingincontrast
lowanti-plasmodialactivity[29].However,hybridmole-
culesconsistingonursolicacidboundtopharmaco-
phoresshowedenhancedinhibitoryactivityon
Plasmodium,presumablybyfacilitatingtheaccessofthe
boundpharmacophoretohaem[30].Maslinicacidhas

Page2of10

alsobeenusedascorecompoundtosynthesizemodified
derivativesdisplayingnewactivities:bindingofhetero-
cyclicringsatMAC-2andC-3positionsincreasesig-
nificantlytheinhibitoryactivityuponhumanprotein
tyrosinephosphatase1B[22],whileattachmentof
hydrophobicgroupsatC-28increaseinhibitionof
humanglycogenphosphorylase[31].Couplingofamino
acidsordipeptidesatC-28allowedtoretaintheanti
HIV-1activitywhileloweringalongsidethecytotoxicity
[32].ShouldMAshowsignificantanti-malarialactivity,
itwouldbefeasibletousethismoleculeasaleadcom-
poundforthedevelopmentofanewfamilyofhighly
activeandlowtoxicanti-plasmodialdrugs.
Toexplorethesuitabilityofmaslinicacidasanti-
malarialagent,theactivityofthistriterpeneoncultures
of
P.falciparum
3D7andthechloroquine-resistantDd2
strainshasbeentested.Asnoneofthecurrentlyused
anti-malarialdrugstargetsplasmodialproteases,MA
activitywascomparedtothoseofchloroquineandato-
vaquoneasexamplesofeffectivedrugswhichinterfere
theintra-erythrocyticstageoftheparasiticcycle.
Methods
Drugsandinhibitors
Maslinicacid(MA)wasobtainedfrompressedfruitsof
olive(
Oleaeuropaea
)byamethodpreviouslypublished
[12].Chloroquinediphosphatesaltwaspurchasedfrom
Sigma-Aldrich.Atovaquonewaskindlyprovidedby
Glaxo-SmithKline.Stocksolutionsofatovaquone
30mMandMA300mMwerepreparedin100%
dimethylsulfoxide(DMSO).Chloroquinewasdissolved
indistilledwaterat100mM.Allcompoundswerestored
at-20°Cuntiluse.Forthedrugassays,serialdilutions
weremadeinculturemediumandaddedto96-wellcul-
tureplates.Controlculturesweretreatedwithequivalent
amountsofDMSOdilutedinculturemedium.
Invitroculturesof
Plasmodiumfalciparum
Plasmodiumfalciparum
strainsDd2(cloneMRA-150)
and3D7(cloneMRA-102)obtainedfromtheMR4[33]
wereusedforthisstudy.Erythrocyteswereobtained
fromtypeA+humanhealthylocaldonorandcollected
intubeswithcitrate-phosphate-dextroseanticoagulant
(Vacuette
®
).Theculturemediaconsistedofstandard
RPMI1640(Sigma-Aldrich)supplementedwith0.5%
AlbumaxI(Gibco),100
μ
Mhypoxanthine(Sigma-
Aldrich),25mMHEPES(Sigma-Aldrich),12.5
μ
g/mL
gentamicine(Sigma-Aldrich)and25mMNaHCO
3
(Sigma-Aldrich).Eachculturewasstartedbymixing
uninfectedandinfectederythrocytestoachievea1%
haematocritandincubatedin5%CO
2
at37°Cintissue
cultureflasks(Iwaki).Theprogressofgrowthinthecul-
turewasdeterminedbymicroscopyinthinblood
smearsstainedwithWright
’
seosinmethyleneblue

Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82

solution(Merck),usingthefreelyavailablePlasmoscore
software[34]tomonitortheparasitaemia.Thedetailed
descriptionofthecultureandsynchronizationmethods