Platonia insignis Mart., a Brazilian Amazonian Plant: The Stem Barks Extract and Its Main ...

13 Pages·2017·2.48 MB·English

Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 3126458, 12 pages
https://doi.org/10.1155/2017/3126458
Research Article
Platonia insignis Mart., a Brazilian Amazonian Plant:
The Stem Barks Extract and Its Main Constituent Lupeol Exert
Antileishmanial Effects Involving Macrophages Activation
AdrianaCunhaSouza,1MichelMuálemdeMoraesAlves,1LucasMoreiraBrito,1
LucianoGomesDeCastroOliveira,1EnoquePereiraCostaSobrinho-Júnior,1
IsabellaCristhinaGonçalvesCosta,2SâmyaDanielleLimaFreitas,2
KlingerAntoniodaFrancaRodrigues,1,3MarianaHelenaChaves,2
DanielDiasRufinoArcanjo,1andFernandoAéciodeAmorimCarvalho1
1MedicinalPlantsResearchCenter,FederalUniversityofPiau´ı,SG-15,Ininga,64049-550Teresina,PI,Brazil
2LaboratoryofNaturalProducts,DepartmentofChemistry,FederalUniversityofPiau´ı,SG-02,Ininga,64049-550Teresina,PI,Brazil
3DepartmentofBiophysicsandPharmacology,FederalUniversityofRioGrandedoNorte,Natal,RN,Brazil
CorrespondenceshouldbeaddressedtoKlingerAntoniodaFrancaRodrigues;[email protected]
Received 4 April 2017; Revised 14 June 2017; Accepted 10 July 2017; Published 9 August 2017
AcademicEditor:VictorKuete
Copyright©2017AdrianaCunhaSouzaetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
PlatoniainsignisMart.,popularlyknownas“bacurizeiro,”isusedintraditionalmedicalpracticesbasedonitsdiversebiological
properties.Thisstudywasaimedatevaluatingtheantileishmanialeffectsoftheethanolextract(EtOH-Ext),hexanefraction(Hex-
F),anditsmainisolatedLupeolobtainedfromstembarksofP.insignisagainstLeishmania(Leishmania)amazonensis,aswellas
theircytotoxicityandpossiblemechanismsofaction.TheEtOH-Ext,Hex-F,andLupeolinhibitedthegrowthofL.amazonensis
promastigoteformsatIC50 of174.24,45.23,and39.06𝜇g/mL,respectively,aswellasL.amazonensisaxenicamastigoteformsat
IC50of40.58,35.87,and44.10𝜇g/mL,respectively.Themeancytotoxicconcentrationsformacrophages(CC50)werehigherthan
thoseforamastigotes(341.95,71.65,and144.0𝜇g/mL,resp.),indicatingaselectivecytotoxicitytowardstheparasiteratherthan
the macrophages. Interestingly, all treatments promoted antileishmanial effect against macrophage-internalized amastigotes at
concentrationslowerthanCC50.Furthermore,increasesoflysosomalvolumeofmacrophagestreatedwithEtOH-Ext,Hex-F,and
Lupeolwereobserved.Ontheotherhand,onlyLupeolstimulatedincreaseofphagocyticcapabilityofmacrophages,suggestingthis
compoundmightbecharacterizedasthebiomarkerfortheantileishmanialeffectofP.insignisstembark,aswellastheinvolvement
ofimmunomodulatorymechanismsinthiseffect.
1.Introduction than12millionpeopleworldwide,with2to3millionofnew
caseseachyear[2,3].AccordingtotheWorldHealthOrga-
Leishmaniasisareparasiticdiseasescausedbyprotozoafrom nization(WHO),approximately556millionpeoplewerein
Leishmaniagenus.Humansareinfectedbythestingofsand areas at risk of contamination of visceral leishmaniasis and
flies which live in forest areas, caves, or rodent dens. This 399millionforcutaneousleishmaniasisin2014[2].
pathology is widely distributed throughout the world and Sincethe1940s,chemotherapyofleishmaniasishasbeen
occurs in Asia, Europe, Africa, and the Americas. Further- based on the use of pentavalent antimonials as first choice
more,therehavebeensomereportsofitsoccurrenceinthe drugs,suchassodiumstibogluconate(Pentostan(cid:2))andmeg-
Americancontinentsincecolonialtimes[1,2].Leishmaniasis lumine antimoniate (Glucantime(cid:2)) [4]. Second-line drugs,
iscapableofaffectingbothmanandanimals,affectingmore such as pentamidine and amphotericin B, are important
2 Evidence-BasedComplementaryandAlternativeMedicine
in the therapy of patients with coinfections, or in cases roomtemperatureandthenpowdered.Thematerialobtained
of resistance to antimonials [5, 6]. However, these drugs (1.1kg)wasexhaustivelyextractedsixconsecutivetimeswith
requirelong-termparenteraladministrationandhaveserious ethanol,duringfivedayseachusingthefollowingextraction
adverseeffectsinadditiontohighrelapserates,especiallyin procedure.Followingremovalofthesolventatlowpressure
immunocompromisedpatients[5,7].Therefore,considering in the rotary evaporator and lyophilization, the EtOH-Ext
thesedifficulties,thesearchfornewsaferandmoreeffective (114.5g) was obtained. Afterwards, the EtOH-Ext extract
chemotherapeuticagentsismarkedlyrelevant. (90.0g) was suspended in a MeOH/H2O mixture (2:3),
Theuseofmedicinalplantsisaverypromisingalterna- and subjected to the liquid-liquid partitioning process with
tive.Plantsbiosynthesizecompoundsfortheirowndefense hexane,resultingintheHex-F(9.8g;yieldof10.9%).
in response to an environmental attack [8]. Its importance The EtOH-Ext and Hex-F were solubilized in DMSO
is evidenced by the wide diversity of metabolites produced to obtain stock solutions at 80mg/mL. In each protocol,
bythesespecies,withdistinctchemical,physical,andbiolog- the stock solution was diluted in the appropriate culture
ical characteristics, most of them possibly bioactive against mediatoreachthedesiredconcentration,notexceedingthe
Leishmaniasp.[9].Inthiscontext,substancesobtainedfrom concentrationof0.5%DMSO.
native species from Brazil which present antileishmanial
activity and low toxicity may represent alternatives for the 2.4. Isolation of Triterpene Lupeol from Hex-F. The Hex-F
conventionaltreatmentofleishmaniasis. (7.2g)wassubjectedtocolumnchromatographyofsilicagel
PlatoniainsignisMart.,popularlyknownas“bacurizeiro,” (210g), using hexane and ethyl acetate as eluent solvents.
isafruitandwoodyplantwithdenseanddiversifiedpopula- Fifty fractions were obtained. Similar fractions were then
tion,occurringfromtheAmazontoPiau´ıinBrazil.Itbelongs mergedaccordingtothinlayerchromatographyanalysis.The
to the Clusiaceae family, composed of 1000 species and 47 groupHB34(fractions34to36,1370mg,yieldof19.6%)was
genera, distributed in tropical, subtropical, and temperate obtainedafterelutionwithhexane:AcOEt(95:5),andthen
regions [10]. In Brazil, the use of P. insignis in traditional analyzedby1HNMRand13C.Thecompound1wasidentified
medicalpracticesisbasedonitsdiversebiologicalproperties, asfollows:
suchascicatrizant,antimicrobial,digestive,diuretic,antitu-
mor,cytotoxic,andantioxidant[11]. Lupeol (1). White amorphous solid. RMN 1H (400MHz,
Studiesontheantileishmanialactivityofmedicinalplants CDCl3):𝛿1,67(s,H-30),3,18(dd,𝐽=11.1,5.1Hz,H-3),4,56
representagreatchallengeforthediscoveryandidentifica- (𝐽 = 2.4Hz, H-29a), 4,68 (𝐽 = 2.4Hz, H-29b). RMN 13C
tionofnewbioactivesubstances.Moreover,therearenostud- (100MHz, CDCl3): 𝛿 38.8 (C-1), 27.6 (C-2), 79.1 (C-3), 39.0
iesregardingtheantileishmanialpotentialoftheextractsand (C-4),55.4(C-5),18.4(C-6),34.4(C-7),41.0(C-8),50.6(C-
fractionsobtainedfromthestembarkofP.insignis.Therefore, 9),37.3(C-10),21.1(C-11),25.3(C-12),38.2(C-13),42.9(C-14),
thisworkaimedtoassessthepotentialantileishmanialactiv- 27.5(C-15),35.7(C-16),43.1(C-17),48.4(C-18),48.1(C-19),
ity, cytotoxicity, and immunomodulatory effect of ethanol 151.0(C-20),30.0(C-21),40.1(C-22),28.1(C-23),15.5(C-24),
extract (EtOH-Ext), its hexanic fraction (Hex-F), and the 16.2(C-25),16.1(C-26),14.7(C-27),18.1(C-28),109.5(C-29),
main constituent Lupeol obtained from the stem bark of P. 19.4(C-30).
insignis.
2.5. Leishmania Culture Conditions. Leishmania (Leishma-
2.MaterialandMethods nia) amazonensis (IFLA/BR/67/PH8) was maintained as
amastigote forms by several weekly passages in BALB/c
∘
2.1. Chemicals. Dimethyl sulfoxide (DMSO: 99%), anhy- mice, and as promastigote forms at 26 C in supplemented
droussodiumsulfate,glacialaceticacid,ethanol,formalde- Schneider’s medium, pH 7.0 (10% heat-inactivated fetal
hyde, sodium chloride, calcium acetate, zymosan, and neu- bovine serum (FBS), 100U/mL penicillin, and 100𝜇g/mL
tral red were purchased from Merck Chemical Company streptomycin), as previously described [12]. Extracellular
(Germany).Schneider’smedium,RPMI1640medium,fetal amastigote-like forms were obtained by in vitro differenti-
bovineserum(FBS),MTT(3-(4,5-dimethylthiazol-2-yl)2,5- ation of promastigotes of Leishmania amazonensis in the
diphenyltetrazolium bromide), Griess reagent (1% sulfanil- stationary growth phase by increasing the temperature to
∘
amideinH3PO410%(v/v)inMilli-Qwater),andtheantibi- 32 CanddecreasingthepHto4.6[13].
oticspenicillinandstreptomycinwerepurchasedfromSigma
Chemical(St.Louis,MO,USA).Theantibioticamphotericin 2.6.AnimalsandMurinePeritonealMacrophages. Maleand
B(90%)waspurchasedfromCrista´lia(Sa˜oPaulo,SP,Brazil). femaleBALB/cmice(4-5weeksold)obtainedfromSection
of Biotherium from Medicinal Plants Research Center of
2.2. Plant Material. The stem bark of P. insignis was col- Federal University of Piau´ı, (Teresina, Piau´ı, Brazil), were
lected in September 2010, from the municipality of Timon, maintainedundercontrolledtemperature(24±1∘C)andlight
Maranha˜o,Brazil.Thevoucherspecimenisdepositedinthe conditions (12-h light/dark cycle). Inflammatory peritoneal
Herbarium Graziela Barroso of the Federal University of macrophages were collected 5 days after intraperitoneal
Piau´ı,Brazil,undernumberTEPB20701. administration with 1.5mL of 3% thioglycollate medium.
AnimalswerehandledaccordingtotheResolutionnumber
2.3. Extraction of EtOH-Ext and Hex-F from Stem Bark 1000/2012 (Federal Council of Veterinary Medicine, Brazil)
of P. insignis. The stem bark of P. insignis was dried at fortheCareandUseofAnimals.Allexperimentalprotocols
Evidence-BasedComplementaryandAlternativeMedicine 3
were approved by the Research Ethics Committee (CEEA- 2.10.EvaluationofImmunomodulatoryActivity
UFPInumber014/2015).
2.10.1. Solutions. The stock solution of neutral red (NR)
dye was prepared by solubilizing 0.002g in 1mL of DMSO.
2.7.BioassayofPromastigoteandAxenicAmastigoteFormsof
The extraction solution used in protocols described in
Leishmaniaamazonensis. Promastigoteandaxenicamastig-
Sections2.10.2and2.10.3consistedof96%glacialaceticacid
ote forms were seeded in 96-well plates containing Schnei-
der’s supplemented medium (1 × 106 Leishmania per 100𝜇l (1.0%)andethanol(50%)dissolvedindeionizedwater(v/v).
The staining of macrophages for the phagocytic capability
medium).Then,EtOH-Ext,Hex-F,andLupeolwereaddedin
assaywasobtainedbydiluting0.3mLoftheneutralredsolu-
triplicateattheconcentrationsof6.25,12.5,25,50,100,200,
tionand0.02gofzymosanin3mLofPBS.TheBaker’sfixative
400, and 800𝜇g/mL. Amphotericin B (Amph B, 2𝜇g/mL)
solutionwascomposedof4%formaldehyde(v/v),2%sodium
was used as positive control. The plate was maintainedin a
chloride (w/v), and 1% calcium acetate (w/v) in deionized
biologicaloxygendemand(BOD)incubatoratatemperature
∘ water.Griess’reagentwaspreparedwith1.0%sulfanilamide
roefsa2z6uCrinfo(r14×8h10.−S3ixmholo/uLr,s2b0e𝜇foLr)ewtahseaednddedofintheisacpherwioedll., in10%H3PO4(v/v)and0.1%naphtylenediamineinMilli-Q(cid:2)
water.
AlltheabsorbancewasreadusingaBiotekmicroplatereader
(model ELx800) at 550nm. The results were expressed as
2.10.2.LysosomalVolume. TheuptakeofthecationicdyeNR
percentinhibitioningrowth[14].
fromlysosomalcompartmentsofmacrophageswasassessed
aspreviouslydescribed[16].Murineperitonealmacrophages
2.8. Investigation of EtOH-Ext, Hex-F, and Lupeol Activi- (1×106cellsperwell)wereharvestedandplatedwithEtOH-
ties on L. amazonensis-Infected Macrophages. Murine peri- ∘
Ext,Hex-F,andLupeolinserialdilutionsat37 Cand5%CO2
tonealmacrophageswereharvestedandplatedona24-well for 48h. Next, cells were treated with 10𝜇L of 2% neutral
microplate (1 × 106cells/mL) in supplemented RPMI 1640 red solution (see Section 2.10.1) and incubated for 30min.
(10% inactivated FBS, 100U/ml penicillin, and 100𝜇g/mL The wells were subsequently washed twice with PBS, and
streptomycin), containing sterile 13mm round coverslips. the internalized neutral red was solubilized by adding the
∘
Culture plates were incubated at 37 C and 5% CO2 for 3h extraction solution (see Section 2.10.1). After 30min on a
forcelladhesion.Adheredmacrophageswerethenincubated Klineshaker(modelAK0506),theabsorbanceat550nmwas
withanewmediumcontainingpromastigotes(instationary measuredasabove.
phase) at a ratio of ten promastigotes to one macrophage
∘
at 5% CO2 and 37 C for 4h. Afterwards, the cells were 2.10.3. Phagocytosis Capability. Murine peritoneal macro-
washed with 0.01M phosphate buffered saline (PBS). Then, phages (1 × 106 cells per well) were harvested and plated
theEtOH-Ext,Hex-F,andLupeolwereincubatedfor48hat ∘
withEtOH-Ext,Hex-F,andLupeolinserialdilutionsat37 C
concentrations related to the IC50, 1/2 IC50, and 1/4 IC50 of and5%CO2 for48h.Afterincubation,10𝜇LofNR-stained
eachtreatmentagainstpromastigoteforms,aswellasAmphB
zymosansolution(describedabove)wasaddedandthecells
(2𝜇g/mL)aspositivecontrol.Afterthisperiod,thecoverslips ∘
were incubated at 37 C for 30min. Following incubation,
were removed and stained with Panoptic staining kit. For 100𝜇L of Baker’s fixative solution (see Section 2.10.1) was
eachtreatment,thenumberofinfectedmacrophagesandthe
added in order to stop the phagocytosis of zymosan. After
parasiteloadwerecountedusingopticalmicroscopy[12].
30min, the cells were washed with PBS to remove non-
phagocytosed NR and zymosan. Then, NR was solubilized
2.9. Cytotoxic Effect on Macrophages and Determination of by adding the extraction solution(see Section2.10.1) in the
SelectivityIndex(SI). Cytotoxicityevaluationwascarriedout Klineshaker.Theabsorbancewassubsequentlymeasuredat
in 96-well plates using the MTT assay. Approximately 1 × 550nmasabove[17].
6
10 murineperitonealmacrophagesperwellwereincubated
in100𝜇LofsupplementedRPMI1640mediumat37∘Cand 2.10.4.NitriteMeasurement. Murineperitonealmacrophages
5% CO2 for 4h for cell adhesion. Nonadherent cells were (2×105cellsperwell)wereharvestedandplatedwithEtOH-
∘
removedbywashingwithRPMI1640medium.Then,EtOH- Ext, Hex-F, and Lupeol in serial dilutions at 37 C and 5%
Ext,Hex-F,andLupeolweredilutedinsupplementedRPMI CO2for24h.Then,thesupernatants(100𝜇L)wereincubated
1640 medium, added at concentrations of 6.25, 12.5, 25, 50, with equal parts of Griess’ reagent (see Section 2.10.1), and
100, 200, 400, and 800𝜇g/mL, and incubated at 37∘C with the absorbance was read at 550nm as above. The standard
5% CO2 for two days. Following incubation, cytotoxicity curve was prepared with sodium nitrite in Milli-Q water at
was assessed by adding MTT (5mg/mL). The supernatant concentrationsof1,5,10,25,50,75,100,and150𝜇Mdiluted
was discarded and the formazan crystals were dissolved in inRPMI1640medium[18].
100𝜇LofDMSO.Finally,absorbancewasmeasuredat550nm
using Biotek microplate reader (model ELx800). Selectivity 2.11.StatisticalAnalysis. Allassayswereperformedintripli-
indexofeachtreatmentwascalculatedattheratioofmean cateinthreeindependentexperiments.Themeaninhibitory
cytotoxicity concentration (CC50) for BALB/c mice murine concentration (IC50) and mean cytotoxic concentration
peritoneal macrophages and half-maximal inhibitory con- (CC50) were calculated using probit regression with 95%
centration (CI50) for macrophage-internalized amastigote confidence limit using SPSS software version 13.0. One-
formsofL.amazonensis[15]. wayanalysesofvariance(ANOVA)followedbyBonferroni’s
4 Evidence-BasedComplementaryandAlternativeMedicine
29
30
20
H
25 26 H 28
H 27
3
1
HO
H
23 24
Figure1:ThetriterpeneLupeol.
post hoc test were performed using the GraphPad Prism mean cytotoxic concentration (CC50) and mean inhibitory
softwareversion5.0.Differenceswereconsideredstatistically concentration(IC50)foraxenicamastigoteformsofL.ama-
significantwhen𝑃<0.05. zonensis.TheEtOH-Extpresentedaselectivityindexof8.42,
indicatingtheEtOH-Extisaround8-foldmorecytotoxicto
3.Results theparasitethantothehostcells.
The Hex-F showed significant cytotoxicity (Figure5(b))
3.1. Identification of Lupeol in Hex-F. The hexane fraction by altering the viability of the cells starting from the con-
fromP.insignis(Hex-F)providesLupeolasthemajorcom- centration of 50𝜇g/mL, with the maximal cytotoxic effect
pound (Figure 1), confirmed by comparison between 1H at 200𝜇g/mL, and SI of 1.99 (Table 1). On the other hand,
NMRspectraoftheHex-Fandtheisolatedcompound(Fig- the main constituent Lupeol also significantly affected the
ure2).TheLupeolwasidentifiedafterisolationandanalysis viability of macrophages (Figure 5(c)), with CC50 and SI
1 13
by H and C NMR when compared with data previously higherthanthoseobtainedforHex-F.Theseresultsindicate
reported[19]. lesscytotoxicitytothehostcellswithsimilarefficacyagainst
theparasite(Table1).
3.2. Anti-Leishmania Activity Assay. The EtOH-Ext pro-
moted statistically significant inhibition of L. amazonensis 3.4.EffectonMacrophageInfection. Theeffectsofthetreat-
promastigotesformsatallconcentrationstested(Figure3(a)). ment with EtOH-Ext, Hex-F, and Lupeol of macrophages
Besides, the Hex-F showed growth inhibition ranging from infected with L. amazonensis are shown in Figure 6. They
the concentration of 12.5𝜇g/mL to 400𝜇g/mL, where 100% were able to significantly reduce the number of infected
ofinhibitionwasachieved(Figure3(b)).Lupeolwasthemost macrophagesinalltestedconcentrationswhencomparedto
effective in promoting growth inhibition of promastigotes, thecontrol(Figure6(a)).Furthermore,asignificantdecrease
with significant inhibition at all concentrations tested (Fig- of number of internalized amastigotes was observed in all
ure 3(c)). The mean inhibitory concentrations for EtOH- testedconcentrationsofEtOH-Ext,Hex-F,andLupeolwhen
Ext, Hex-F, and Lupeol against promastigote forms were comparedwithcontrolgroup(Figure6(b)).
calculatedandlistedinTable1.
In another set of experiments, the effects of EtOH-Ext, 3.5.LysosomalVolumeandPhagocytosisTest. Thelysosomal
Hex-F,andLupeolonaxenicamastigoteformsofL.amazo- volumewasassessedbytheretentionoftheNRthelysosome
nensis were assessed. The EtOH-Ext exhibited a significant of macrophages and then determined colorimetrically. The
inhibitoryeffectstartingfromthelowestconcentrationtested EtOH-Extinducedasignificantincreaseoflysosomalvolume
(Figure4(a)),whereastheinhibitoryeffectofHex-Fstarted ofmacrophagesatconcentrationsof6.25,12.5,and25𝜇g/mL
at concentration of 12.5𝜇g/mL, reaching 100% of inhibi- (Figure7(a)).Besides,thelysosomalvolumeaftertreatment
tion at the highest concentration tested (Figure 4(b)). The withHex-Fincreasedsignificantlyatconcentrationsof6.25
Lupeol presented significant anti-Leishmania activity when and 12𝜇g/mL (Figure 7(b)). Interestingly, the lysosomal
compared to control started at concentration of 25𝜇g/mL volume of macrophages treated with Lupeol showed a sta-
(Figure 4(c)). The mean inhibitory concentrations (IC50) tistically significant increase of the endocytic volume at
forEtOH-Ext,Hex-F,andLupeolagainstaxenicamastigote concentrations ranging from 6.25 to 50𝜇g/mL of Lupeol
formswerecalculatedandlistedinTable1. (Figure7(c)).
Other parameter related to macrophages activation
3.3. Cytotoxicity Determination. The EtOH-Ext demon- parameterwasthephagocyticcapabilityofstainedzymosan
strated significant cytotoxicity starting from the concentra- particles.BothEtOH-ExtandHex-Fwerenotabletopromote
tion of 25𝜇g/mL, presenting higher cytotoxic effect at con- any increase in the phagocytic capability of macrophages
centrations of 400 and 800𝜇g/mL (Figure 5(a)). Thereafter, (Figures 8(a) and 8(b)). Interestingly, a significant increase
the Selectivity index (SI) was calculated by the ratio of in phagocytosis of zymosan particles was observed in
Evidence-BasedComplementaryandAlternativeMedicine 5
7.25991 4.682234.56345 3.220803.208243.192783.18055 1.67602 1.026640.965000.940590.825320.784820.76013
1.001.04 1.15
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0−0.5
(ppm)
(a)
7.26014 4.682864.676874.559684.55697 3.196523.183593.168603.15585 1.67216 1.023910.959200.937800.822490.781930.75272
1.001.00 1.12
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
(b)
1
Figure2: HNMRspectra(CDCl3,400MHz):Hex-F(a)andLupeol(b).
macrophagestreatedwithLupeolatconcentrationsranging proved that stems, roots, leaves, seeds, and fruits of these
from6.25to25.0𝜇g/mL(Figure8(c)). plantshaveappreciableefficacyinthecureofseveraldiseases.
Previous studies have demonstrated potent antileishmanial
3.6. Measurement of Nitrite Production. The nitrite mea- activityofextractsandfractionsfromleavesandfruitpeels
surement was determined in macrophages after incubation of Azadirachta indica with IC50 value of 1.1𝜇g/mL against
with EtOH-Ext, Hex-F, and Lupeol. No increase in nitrite promastigoteforms,and0.4𝜇g/mLagainstamastigoteforms,
production was observed after all treatments. Furthermore, as well as low cytotoxicity against mammalian cells [12].
a decrease in nitrite production was observed only at con- Furthermore,theessentialoilfromleavesofEugeniauniflora
centrationshigherthanthoseobservedforlysosomalvolume demonstrated a marked antileishmanial effect with IC50 of
andphagocyticcapability(Figure9). 1.75𝜇g/mL against promastigotes, and 1.92𝜇g/mL against
amastigotes [15]. Thus, the observation of the therapeutic
4.Discussion properties of natural products has led towards the research
oftheactiveprinciplesfromseveralplantspecies[15,20].
Scientificstudiesrelatedtothepharmacologicalpotentialof The P. insignis species, called “bacuri” provides a very
medicinal plants have been increasing. The use of medici- popular consumed fruit among the Brazilian Amazonian
nal plants in traditional medical practices has increasingly people. This ethnopharmacological use is related to the
6 Evidence-BasedComplementaryandAlternativeMedicine
∗∗∗
∗∗∗
100 100 ∗∗∗ ∗∗∗ ∗∗∗
%) %)
hibition ( 80 ∗∗∗ hibition ( 80 ∗∗∗
wth in 60 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ wth in 60 ∗∗∗
mastigotes gro 2400 ∗∗∗ ∗∗∗ ∗∗∗ mastigotes gro 2400 ∗∗∗ ∗∗∗
Pro Pro
0 0
C Amph6,25 12,5 25 50 100 200 400 800 C Amph6,25 12,5 25 50 100 200 400 800
B EtOH-Ext (g/mL) B Hex-F (g/mL)
(a) (b)
∗∗∗
100
∗∗∗
%) ∗∗∗
on ( 80 ∗∗∗
biti ∗∗∗
hi ∗∗∗
h in 60 ∗∗∗
wt
o
es gr 40 ∗∗∗
ot
g
asti 20 ∗∗∗
m
o
Pr
0
C Amph6,25 12,5 25 50 100 200 400 800
B Lupeol (g/mL)
(c)
Figure3:Anti-LeishmaniaactivityofEtOH-Ext(a),Hex-F(b),andLupeol(c)fromP.insignisstembarkagainstpromastigoteformsofL.
amazonensis.Parasites(1×106)wereexposedtodifferentconcentrationsfor48h,andcellviabilitywasassessedbyresazurinassay.∗∗∗𝑃 <
0.001versuscontrol.
Table1:Anti-LeishmaniaactivityofEtOH-Ext,Hex-F,andLupeolfromP.insignisstembark,andcytotoxiceffectsagainstmurineperitoneal
macrophages.
Macrophages Promastigotes Axenicamastigotes
Compounds
CC50(𝜇g/mL) CI50(𝜇g/mL) CI50(𝜇g/mL) SI
EtOH-Ext 341.95 174.24 40.58 8.42
Hex-F 71.65 45.23 35.87 1.99
Lupeol 144.00 39.06 44.10 3.26
AmphB 8.75 1.74 0.20 43.75
SI:SelectivityindexforaxenicamastigoteformsofL.amazonensis(CC50/IC50).
seeds extract as cicatrizant and anti-inflammatory agents. constituent. Triterpenes have aroused a marked interest
Currently, several activities have been reported for all parts due to their broad spectrum of biological activities [22].
of this fruit (seed, shells, and pulp). Besides, classes of In this context, the triterpene Lupeol has demonstrated
terpenes, xanthones, and phenolics have been reported as potentialapplicationin studies concerningthediscovery of
major constituents [21]. The Hex-F obtained from the stem compoundswithantitumorandanti-inflammatoryactivities
barkofP.insignispresentsthetriterpeneLupeolasitsmajor [23].
Evidence-BasedComplementaryandAlternativeMedicine 7
100 100 ∗∗∗
∗∗∗
∗∗∗
ng (%) 80 ∗∗∗ ∗∗∗ ng (%) 80 ∗∗∗ ∗∗∗ ∗∗∗
mastigotes killi 4600 ∗∗ ∗∗ ∗∗∗ ∗∗∗ mastigotes killi 4600 ∗∗∗ ∗∗∗ ∗∗∗
Axenic a 20 Axenic a 20
0 0
C Amph6,25 12,5 25 50 100 200 400 800 C Amph6,25 12,5 25 50 100 200 400 800
B EtOH-Ext (g/mL) B Hex-F (g/mL)
(a) (b)
∗∗∗
100
∗∗∗
%) 80
g ( ∗∗∗
n ∗∗∗
killi 60 ∗∗∗ ∗∗∗
es
ot
g
asti 40
m
a
c
eni 20
x
A
0
C Amph6,25 12,5 25 50 100 200 400 800
B Lupeol (g/mL)
(c)
Figure4:Anti-LeishmaniaactivityofEtOH-Ext(a),Hex-F(b),andLupeol(c)againstaxenicamastigoteformsofL.amazonensis.Parasites
(1×106)wereexposedtodifferentconcentrationsfor48h,andcellviabilitywasassessedbyresazurinassay.∗∗𝑃 < 0.01versuscontrol;
∗∗∗𝑃<0.001versuscontrol.
The screening of leishmanicidal agents classically uses coagulation of cytoplasm can occur. These events are able
promastigotesformsdue to theirsimplicityand low cost of to promote the interruption of specific metabolic pathways
the culture [24]. Moreover, Costa-Ju´nior and colaborators of lipids and proteins [27], interference in cell division [28,
[25]havereportedtheantileishmanialeffectofgarciniellip- 29], or stimulate the depolarization of the mitochondrial
toneFC,apolyisoprenilatedbenzophenoneisolatedfromthe membranes, which can lead the cell to trigger necrosis or
seedextractsofP.insignis,withaIC50 valueof25.78𝜇g/mL. apoptosismechanisms[30].
This evidence reinforces the anti-Leishmania potential of Considering that EtOH-Ext, Hex-F, and Lupeol showed
these plant species. In this context, EtOH-Ext, Hex-F, and significant antileishmanial activity, the investigation of the
Lupeoldemonstratedinvitroefficacyagainstbothpromastig- cytotoxicactivityagainstmammaliancellsaspotentialhosts
otesandaxenicamastigotesofL.amazonensis.Inthisstudy, is markedly necessary. Cytotoxicity tests against murine
no significant difference was observed between IC50 values peritoneal macrophages were performed by the resazurin
obtained for Lupeol and Hex-F, then indicating the Hex-F- colorimetric assay [31]. In these experiments, CC50 values
inducedantileishmanialeffectmightberelatedtoitsLupeol higher than the IC50 values for promastigotes and axenic
content.Thus,thetriterpeneLupeolcanbecharacterizedas amastigotes were found. Conversely, previous studies have
abiomarkerfortheantileishmanialactivityofP.insignis. found a SI lower than 1.0 for meglumine antimoniate, the
Previous studies have suggested that lipophilic com- referencedrugfortreatmentofleishmaniasis,demonstrating
pounds, such as triterpenes, act by a peculiar mechanism. ahighercytotoxicityagainstmacrophagesthantheparasite.
These compounds can pass easily through the cytoplasmic This result is related to the in vivo effects of pentavalent
membranes, affecting structures of their different layers of antimonials, where, despite being first-line drugs for the
polysaccharides,fattyacids,andphospholipids,thusmaking treatmentofcutaneousandvisceralleishmaniasis,numerous
them permeable [26]. Once they cross the membrane, the side effects and toxicity have been reported [32]. Therefore,
8 Evidence-BasedComplementaryandAlternativeMedicine
100 100
∗∗∗ ∗∗∗ ∗∗∗
∗∗∗
%) 80 %) 80
bility ( 60 bility ( 60 ∗∗∗
phages via 40 ∗∗∗ phages via 40 ∗∗∗
Macro 20 ∗∗∗ Macro 20 ∗∗∗ ∗∗∗ ∗∗∗
0 0
C 6,25 12,5 25 50 100 200 400 800 C 6,25 12,5 25 50 100 200 400 800
EtOH-Ext (g/mL) Hex-F (g/mL)
(a) (b)
100
∗∗∗
∗∗∗
%) 80 ∗∗∗
ability ( 60 ∗∗∗ ∗∗∗ ∗∗∗
vi
es ∗∗∗
g
a 40
h
p
cro ∗∗∗
a
M 20
0
C 6,25 12,5 25 50 100 200 400 800
Lupeol (g/mL)
(c)
Figure 5: Cytotoxic effects against murine peritoneal macrophages of EtOH- Ext (a), Hex-F (b), and Lupeol (c). Murine peritoneal
macrophageswereincubatedfor48hinthepresenceofdifferentconcentrations.MacrophageviabilitywasmeasuredbyMTTassay(MTT).
Dataareexpressedasmeans±SEMofthreeexperimentsperformedintriplicate.∗∗∗𝑃<0.001versuscontrol.
∗∗∗
∗∗∗
∗∗∗
∗∗∗
6
100
%) 80
ophages ( 60 nndex () 4
ed macr 40 urvival i 2
ct S
e
nf 20
I
0 0
CAmph 45 90 175 15 25 45 10 20 40 C Amph 45 90 175 15 25 45 10 20 40
B EtoH-Ext Hex-F Lupeol B EtoH-Ext Hex-F Lupeol
(a) (b)
Figure6:EffectofEtOH-Ext,Hex-F,Lupeol,andthereferencedrugAmphB(2𝜇g/mL)ontheinfectedmacrophages(a)andthesurvival
indexofmacrophage-internalizedamastigotes(b)after48hofexposure.Murineperitonealmacrophageswereinfectedwithpromastigote
formsofL.amazonensisandthentreatedwithconcentrationsrelatedtorespectiveIC50,1/2IC50,and1/4IC50againstpromastigotes.Results
representthemeans±SEMofthreeexperimentsperformedintriplicate.∗∗∗𝑃<0.001versuscontrol.
Evidence-BasedComplementaryandAlternativeMedicine 9
0.4 0.4
∗∗∗
m) 0.3 ∗∗∗ m) 0.3
ume50n ∗∗ ume50n ∗∗ ∗
Lysosomal volAbsorbance at 5 00..12 Lysosomal volAbsorbance at 5 00..12
( (
0.0 0.0
C 6.25 12.5 25 50 100 C 6.25 12.5 25 50 100
EtOH-Ext (g/mL) Hex-F (g/mL)
(a) (b)
0.4 ∗∗∗
∗∗∗
m) 0.3 ∗∗∗
me0n ∗
u5
mal volnce at 5 0.2
oa
LysosAbsorb 0.1
(
0.0
C 6.25 12.5 25 50 100
Lupeol (g/mL)
(c)
Figure7:TheinfluenceofEtOH-Ext(a),Hexanicfraction(b),andLupeol(c)onthelysosomalvolumeinmacrophages.Murineperitoneal
macrophageswereincubatedfor48haftertreatment.Lysosomalvolumewasanalyzedspectrophotometricallybytheincreaseinneutralred
(NR)uptakefollowingsolubilizationwiththeextractionsolution.Dataarepresentedasmean±SEMofthreeexperimentscarriedoutin
triplicate.∗𝑃<0.05versuscontrol;∗∗𝑃<0.01versuscontrol;∗∗∗𝑃<0.001versuscontrol.
EtOH-Ext, Hex-F, and Lupeol present SI higher than 1.0, Activated macrophages perform several functions,
whichrevealsthesafetyofthesesubstancesandthecontinuity including phagocytosis, increased numbers of hydrolytic
infurtherstudiesofantileishmanialactivity. enzymesfromlysosomalcompartments,tumorcytotoxicity,
Once the EtOH-Ext, Hex-F, and the main constituent cytokine secretion, and antigen presentation. Phagocytosis
Lupeol showed effective antileishmanial activity with selec- and lysosomal volume are functions of innate immunity,
tive action towards the parasite, we decided to investigate important for the control of infections and leading to
their activities against the intramacrophagic amastigotes. the degradation of pathogens and the presentation of
This experimental model assesses the effects against the antigens[34,35].Thus,inordertoevaluatethishypothesis,
parasitebytheactivationofmicrobicidalmechanismsinthe parameters related to activation of macrophages and
host macrophages and therefore is as closest as possible to structuralmechanismsofantileishmanialactivity(lysosomal
the in vivo effect [33]. In these experiments, all treatments compartmentvolumeandphagocyticactivity),aswellasthe
presented antileishmanial activity against the macrophage- nitriteproductionasamechanismoftheactivationstateof
internalized amastigotes, which was observed by the sig- themacrophages,wereassessed.
nificant reduction in the number of infected macrophages Theincorporationofneutralredintothesecretoryvesi-
and in the number of amastigotes per macrophage. Thus, cleswasappliedtostudythelysosomalvolume.Besides,the
theseresultsreinforcestudiesconcerningwhichmechanisms phagocytic capability was determined by the incorporation
triggeredinmacrophagesmightbeinvolvedinthiseffect. of zymosan particles stained with neutral red. Zymosan
10 Evidence-BasedComplementaryandAlternativeMedicine
2.0
1.5
Zymosan phagocytosis (Absorbance att 550nm) 011...505 Zymosan phagocytosis (Absorbance att 550nm) 01..50
0.0 0.0
C 6.25 12.5 25 50 100 C 6.25 12.5 25 50 100
EtOH-Ext (g/mL) Hex-F (g/mL)
(a) (b)
2.0
∗∗∗
∗∗
∗
osisnm) 1.5
Zymosan phagocytAbsorbance att 550 01..50
(
0.0
C 6.25 12.5 25 50 100
Lupeol (g/mL)
(c)
Figure8:TheinfluenceofEtOH-Ext(a),Hexanicfraction(b),andLupeol(c)onthephagocytosisinmacrophages.Murineperitoneal
macrophagesweretreatedwitharangeofconcentrationsfor48h.ThephagocytosiswasanalyzedbyincorporationofNR-stainedzymosan,
solubilizedwiththeextractionsolution.Dataarepresentedasmean±SEMofthreeexperimentscarriedoutintriplicate.∗𝑃 < 0.05versus
control;∗∗𝑃<0.01versuscontrol;∗∗∗𝑃<0.001versuscontrol.
is a polysaccharide from the cell wall of Saccharomyces NO production by macrophages, indirectly determined by
cerevisiae in the form of water-insoluble powder [36]. In the nitrite measurement using Griess’ Reaction. Thus, the
this study, the antiamastigote activity of EtOH-Ext, Hex-F, antiamastigote effect of these treatments probably does not
and Lupeol may involve the increase of lysosomal volume, involvetheNOpathway.
sinceallthetreatmentspromotedanincreaseofthevolume
oftheendocyticcompartment.Interestingly,theincreaseof 5.Conclusion
phagocytic capacity induced by Lupeol may also be related
to its antiamastigote activity, considering Lupeol was the This study demonstrates that EtOH-Ext, Hex-F, and Lupeol
onlytreatmentwhichincreasethephagocytosisofzymosan obtained from P. insignis were able to promote effective
particlesinmacrophages. and selective antileishmanial activity against both pro-
Anotherimportantpathwaythatmaybeinvolvedinthe mastigote and amastigote forms of L. amazonensis, as
mechanism of antileishmanialactivity is stimulation of NO well as against macrophage-internalized amastigote forms.
productionbymacrophages.Thismechanismhasbeencon- This study also demonstrated the mechanisms underlying
sideredasthemosteffectivemechanismunderlyingdefense antileishmanial effect, such as the increase of lysosomal
against Leishmania spp. infection [37]. This production can volume of macrophages for all treatments. Interestingly,
bestimulatedinvitrobytheactivationofmacrophagesusing only Lupeol was able to promote increase the phagocytic
lipopolysaccharide(LPS),whichactivatestheinduciblenitric capacity of macrophages, suggesting this compound might
oxide synthase (iNOS), then leading to the production of be characterized as the biomarker for the antileishmanial
highconcentrationsofNO.However,theresultsshowedthat effectofP.insignisstembark.Thus,theinvolvementofinnate
EtOH-Ext, Hex-F, and Lupeol lack the ability to stimulate immune system was evidenced. Further investigations are