w w w.jo u r n als . e l s e vie r . c o m / r e v i s t a - b r a s i l e i r a - d e - f a r m a c o g n o s i a
Original
Article
Immunomodulatory
and
toxicological
evaluation
of
the
fruit
seeds
from
Platonia
insignis
,
a
native
species
from
Brazilian
Amazon
Rainforest
Ana
Karina
M.F.
Lustosa
a,
Daniel
D.R.
Arcanjo
b,
Rayra
G.
Ribeiro
b,
Klinger
Antonio
F.
Rodrigues
b,
Flávia
Franceli
B.
Passos
b,
Celyane
A.
Piauilino
b,
José
Couras
Silva-Filho
b,
Bruno
Q.
Araújo
c,
José
S.
Lima-Neto
c,
Joaquim
S.
Costa-Júnior
d,
Fernando
Aécio
A.
Carvalho
b,
Antônia
Maria
das
Grac¸
as
L.
Citó
a,c,∗aPós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeFederaldoPiauí,Teresina,PI,Brazil
bNúcleodePesquisasemPlantasMedicinais,UniversidadeFederaldoPiauí,Teresina,PI,Brazil
cDepartamentodeQuímica,UniversidadeFederaldoPiauí,Teresina,PI,Brazil
dDepartamentodeQuímica,InstitutoFederaldeEducac¸ão,CiênciaeTecnologiadoPiauí,Teresina,PI,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27January2015 Accepted19May2015
Availableonline25September2015
Keywords:
Platoniainsignis
Clusiaceae Cytotoxic Nitricoxide Immunomodulatory Toxicological
a
b
s
t
r
a
c
t
The“bacuri”(PlatoniainsignisMart.,Clusiaceae)isanativetropicalfruitfromtheBrazilianAmazonand NortheastRegions.Itsseedsareusedtotreatinflammatorydiseases,diarrheaandskinproblemsin traditionalmedicalpractices.Regardingitswidespreadmedicinaluses,itisimportanttoevaluatethe biologicalandtoxicologicalpotentialofthisspecies.Thisway,theaimofthisstudywastoinvestigate theinvitrocytotoxicandimmunomodulatoryeffectsofthehexanicextractofP.insignisseeds,aswell asitsinvivoacuteoraltoxicity.Thebiologicalevaluationwasperformedbythedeterminationof cyto-toxic(MTTandhemolysisassay)andimmunomodulatory(phagocyticcapacity,lysosomalvolumeand nitriteproduction)activitiesofEHSBinmurineperitonealmacrophages.Inaddition,theoralacute tox-icitywasevaluatedusingfemaleWistarratstreatedwithEHSB(2.0g/kg),inaccordancewiththeOECD 423Guideline.TheEHSBshowedlowtoxicityformacrophagesintheMTTtest(CC50value:90.03g/ml), aswellasforerythrocytes,whichcausedonly2.5%hemolysisatthehighestconcentration.Astrong immunomodulatoryactivitywasobservedbyamarkedlyincreaseoftheNOproduction,phagocytic abil-ityandlysosomalvolume.Ontheotherhand,itwasnotobserveddeathsorchangesintheclinicaland behavioralparametersinthetoxicologicalevaluation.Thismanner,thepresentstudycontributestothe knowledgeabouttheimmunomodulatoryandtoxicologicalpropertiesoftheP.insignis.Thismayprovide perspectivesfortheevaluationanddevelopmentofeffectiveandsafephytomedicinescreatedfromthe Brazilianlocalbiodiversity.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
TheBrazilianAmazonregionhastherichestbiodiversityaround theworld.Thereisaboutamillionspeciesofanimalsandplantsin thisarea,whichrepresentshalfofthespeciesintheentireplanet. Thisbiodiversityrepresentsastrategicreserveforthesurvivalof humanity(Agraetal.,2008).Itsimportanceisrepresentedbythe considerablereserveoffoodandmedicinalplants.Naturalproducts areanimportantsourceofresearchthataimedthediscoveryofnew substanceswithpharmacologicalactivities(Butler,2004).
∗ Correspondingauthor.
E-mail:gracacito@ufpi.edu.br(A.M.G.L.Citó).
ThespeciesPlatoniainsignisMart.,Clusiaceae,popularlyknown as“bacuri”,isanativespeciesfromtheBrazilianAmazonRegion. ThisplantisdistributednaturallyinallstatesofNorthandNorthern RegionofBrazil,morepreciselyinthestatesofPará,MatoGrosso, MaranhãoandPiauí.ThefruitpulpofP.insignisiswidelyusedinthe foodindustryasrawmaterialforproductionofjuices,icecream, sweetsandbeverages.Itsseedsareusedintheproductionofsoapor butter(Agraetal.,2008;Costa-Júnior,2011;Ferreira,2008;Moraes andGutjahr,2009;ShanleyandMedina,2005).
Therearevariousstudiesreportingtheantioxidant(Limaetal., 2007; Rufinoet al.,2010), wound healing (SantosJúnior etal., 2010),leishmanicidal(Costa-Júnioretal.,2013a)and anticonvul-santactivities(Costa-Júnioretal.,2010)oftheextractofthefruit seedsofP.insignis.Inaddition,thedecoctionofthefruitseedsis
http://dx.doi.org/10.1016/j.bjp.2015.05.014
usedtotreatdiarrheaintraditionalmedicalpractices(Agraetal., 2008).Also,theoilandyellowlatexareusedtotreatbitesofspiders, snakes,inthetreatmentofskindiseases,otitis,rheumatismand arthritisduetowoundhealing,antitumor,antimicrobial, antioxi-dantandcytotoxicproperties(Costa-Júnior,2011;Ferreira,2008; MoraesandGutjahr,2009;ShanleyandMedina,2005).
Moreover, the family Clusiaceae represents a rich source of polyisoprenylated benzophehones and xanthones which are responsiblefor severalbiological activities (Acu ˜na et al., 2009; Diderotetal.,2006;Kumaretal.,2013).Previousstudiesofour grouphave reportedthe identificationof highcontent of xan-thones(alpha-andgamma-mangostin)inP.insignis(Costa-Júnior etal.,2013a).Inaddition,theisolationofthegarcinielliptoneFC, apolycyclicpolyprenylatedacylphloroglucinol,fromthehexane extractofP.insignis(Costa-Júnioretal.,2011)havebeenreported topresent awide rangeofbiological activities,suchas antioxi-dant(Costa-Júnioretal.,2012),leishmanicidal(Costa-Júnioretal., 2013b),vasodilator(Arcanjoetal.,2014)andanticonvulsant(Silva etal.,2014).
Immunomodulators are a class of drugs that causesa non-specificstimulationofimmunological defensemechanisms.The decreaseofimmunityduetotheexpositionofhumanbodyto dif-ferentstressfulfactorsrepresentsthemaintargetofthesedrugs andtheirbenefits.Hence,theimmunologicalstimulationor sup-pression may be useful in maintaininga health state (Wagner andProksch,1985).Furthermore,severalmedicinalplants have demonstratedthemodulationofNOreleaseandphagocytic capa-bilityinmacrophages.Thisfactreinforcestheimmunomodulator profileofmedicinalplantsandtheirinvolvementwithseveralother biologicalproperties,suchasanti-inflammatoryand antinocicep-tive(Rosaetal.,2014).
Thewide pharmacologicalspectraoffruitseedsofP.insignis togetherwiththeabsenceoftoxicologicalstudiesaboutthisplant arethemainsupporttotheimmunomodulatoryandtoxicological evaluationdoneinthiswork.Also,thisstudyconfirmsthe impor-tanceofthisspeciesasasourceofbioactivecompounds.
Materialsandmethods
Plantmaterial
The fruit seeds of Platonia insignis Mart., Clusiaceae, were obtainedfromspecimenslocatedinthecityofBarras,Piauí,Brazil (latitude−04◦14′40′′andlongitude−42◦40′70′′)inMarchof2009, andthevoucherspecimenwasdepositedatHerbariumGraziella BarrosoofFederalUniversityofPiauí,Brazil,no.ICNTEPB27,164.
PreparationofP.insignisseedsextracts(EHSB)
TheEHSBwaspreparedaccordingtoSilvaetal.(2014).Briefly, theP.insignisseedsweredriedat55◦C,powdered(848g)andthen extractedwithhexane(63%,w/v)inSoxhletduring8h.Theextract wasstoredat8◦C.Aformationofawhiteprecipitate,formedof tri-palmitinandtriolein(64g,7%),wasobserved(Bentesetal.,1987). Aftertheremoval of the precipitate,the supernatant was con-centratedinavacuumrotaryevaporator.Thisyieldedthehexane extractfromtheP.insignisseeds(EHSB,534g,63%),whichwasused inthebiologicaltests.
Animals
Female Wistar rats (200–250g, n=5 per group) and male Balb/cmice(25–30g)weremaintainedundercontrolledconditions (24±1◦C,12hlight/darkcycle),withfreeaccesstofoodandwater. Afterexperimentalprocedures, femaleWistar rats were eutha-nizedbysodiumthiopental(100mg/kg,i.p.),andBalb/cmicewere
euthanizedbycervicaldislocation.Allexperimentalprotocolswere approvedbytheEthicsCommitteeonAnimalExperimentationof UFPI(no.076/2010).
Cultivationandelicitationofmiceperitonealmacrophages
Residentperitonealmacrophages wereobtainedfromBalb/c mice.Aftereuthanasia,theanimalswereimmersedin70% alco-holfor1mintoantisepsis,andthenfixedinthesupineposition.In laminarflow,approximately8mlofPBS(sterile,pH7.4,4◦C)was administeredintotheabdominalcavity.Then,thesolution con-tainingperitonealmacrophageswastransferredtoasteriletube onicebathandthensubjectedtwicetocentrifugationat1500rpm for 10minat 4◦C withsuccessivecells washingby 0.9%sterile saline.Afterward,thesupernatantwasdiscardedandthecellswere suspended in2ml RPMI1640containing10% SFB,10,000IU/ml penicillin,and10mg/mlstreptomycin.Themacrophagecountwas performedinaNeubauerchamber,usingthedyeTrypanBlue anal-ysisforcellviability.
MTTtest
ThecytotoxicityofEHSBwasperformedusingtheMTTassayin Balb/cmurinemacrophages.In96-wellplate,1×106macrophages per well were approximately incubated in 100l of RPMI 1640medium(Sigma,StLouis,USA)anddifferentconcentrations ofEHSB(100,50,25,12.5,6.25,3.12g/ml),andthenincubatedfor 48hat37◦Cand5%CO2.
Afterwards 10l of MTT (3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyltetrazoliumbromide) dilutedin PBSto a final concen-trationof 5mg/mlwasadded (10%volume,i.e.,10lof100l foreachwell)and incubatedfor 4hat37◦Cunder5%CO2.The supernatantwasdiscardedand100lofDMSOwasaddedinall wells.Then,theplatewasplacedunderstirringforabout30minat roomtemperaturetocompletedissolutionofformazan.Then,the absorbanceswerereadat550nm(Absorbancemicroplatereader ELx800TM,BioTek®Instruments,USA).Theresultswereexpressed asCC50(meancytotoxicconcentrationfor50%ofcells)withthe confidenceintervalbynon-linearregression(Reillyetal.,1998).
Hemolyticactivity
ThistestwascarriedoutaccordingtoLöfgrenetal.(2008)with somemodifications. The O+-typehumanblood waswithdrawn usinganticoagulant(EDTA).Theerythrocyteswerewashedthree times(7minat165×g)andresuspendedto5%erythrocyte solu-tioninPBS(pH7,4;NaCl0,15M)atthevolumeof80l.Then,20l ofEHSBwereaddedtoafinalvolumeof100l(400,200,100,50, 25,12.5,6.25and3.125g/ml),andtheywereincubatedfor1hat 37◦Candthereactionswerestoppedbyadding200lofPBS.The suspensionswerecentrifugedat1000×gfor10minatroom tem-perature.Thesupernatantwassubjectedtospectrophotometryat 540nmtodeterminethehemolyticactivity.Theabsence(negative control)or100%ofhemolysis(positivecontrol)wasdetermined by replacing the EHSB solutionfor an equal volume of PBS or Milli-Qsterilewater,respectively.Thepercentageofhemolysiswas obtainedbycomparisonwiththepositivecontrol(100% hemoly-sis).
Immunomodulatoryassessment
lysosomal vesiclesecretion.After30min,theabsorbanceswere readat550nm(AbsorbancemicroplatereaderELx800TM,BioTek® Instruments,USA)(Bonattoetal.,2004).
Phagocyticcapacity
PeritonealmacrophageswereplatedandincubatedwithEHSB (100, 50, 25, 12.5, 6.25 and 3.12g/ml). After 24h of incuba-tionat37◦Cand 5%CO
2,10lofzymosansolutionwasadded (0.3mlneutralredsolutionand0.02gzymosannotopsonizedin PBS3ml),andthesolutionwasincubatedagainfor30min. After-wards, 100l of Baker’s fixative (formaldehyde 4%v/v,sodium chloride 2%w/v,and calcium acetate 1%w/v in distilled water) wereaddedinordertoparalyzetheprocessofphagocytosis,and after30min,theplatewaswashedwith0.9%salineinorderto removenon-phagocytizedneutralredandzymosanparticles.Then, 100lofextractionsolutionwasaddedtosupernatantandafter solubilization,theabsorbanceswerereadat550nm(absorbance microplatereaderELx800TM,BioTek®Instruments,USA)(Bonatto etal.,2004).
Nitricoxide(NO)production
In96-wellplate,2×105macrophageswereaddedperwelland incubatedat37◦Cand5%CO
2for4haimingthecelladhesion.Then, EHSB(100,50,25,12.5,6.25and3.12g/ml)orpositivecontrol LPS(2g/ml)wasaddedinculturemediumandincubatedagainat 37◦Cand5%CO2for24h.Afterthisperiod,thesupernatantwas col-lectedandtransferredtoanotherplateformeasurementofnitrite. Then,Griessreagent(1:1)wasaddedandtheanalysiswas per-formedinELISAplatereaderat550nm.Thestandardcurvewas performedwith150MsodiumnitriteinMilli-Qwateratvarying concentrationsof1,5,10,25,50,75,100and150Mdilutedin culturemedium(Genestraetal.,2003).
Acuteoraltoxicityinrats
The toxicologicalevaluation of EHSB was performedby the FixedDose Procedure accordingto theGuideline 423fromthe OrganizationforEconomicCooperationandDevelopment(OECD, 2001)withsomemodificationsproposedbySerafinietal.(2011). FemaleWistarratswereorallytreatedwithEHSB(2.0g/kg,n=5) ordistillatedwater(n=5).Shortlyafteradministration,clinicaland behavioralparameters(e.g.hyperactivity,aggressiveness,seizures, piloerection,ptosis,sedation,cornealreflex,writhings, vocaliza-tion,death)wereevaluatedduringthefirst 8h,and thenthese sameparameterswereobserveddailyfor14days,includingthe bodyweightoftheseanimals,asrecommendedbytheprotocolof recognitionandevaluationofclinicalsignsbyOECD(2000).After these14daysofobservation,theanimalswereeuthanatizedand theirbloodserumsampleswereobtainedinordertodeterminate somebiochemicalparameters,aslistedinTable1.
Inaddition,theeuthanizedanimalsweresubmittedtonecropsy andgrosspathologicalevaluationofinternalorgans(lungs,heart, stomach, liver, brain and kidneys) for color, texture and con-sistency.Then, therelative weights oforgans wereobtainedas follows:relativeorganweight=[organweight(g)]×100/[animal weightonthedayofnecropsy(g)].
Statisticalanalysis
Valuesareexpressedasmean±standarderrorofthemean. Sta-tisticalanalyzeswereperformedbyapplyingtheStudent’st-testfor non-pairedsamplesoranalysisofvariance(ANOVA)Two-way fol-lowedbyTukey’spost-testorDunnettforanalysisofsignificance betweengroups.Valueswereconsideredstatisticallysignificant atp<0.05.Forallstatisticalanalysisandplottingcurvesusedthe
Table1
Serumbiochemicalparametersafter14daysofacuteoraltreatmentwiththe hex-anicextractfromP.insignisseeds(EHSB2.0g/kg)infemaleWistarrats.
Parameters Vehicle EHSB Totalproteins(mg/dl) 6.96±0.10 7.08±0.14 Albumin(mg/dl) 4.02±0.14 4.44±0.12 Alkalinephosphatase(U/l) 214.80±29.99 158.40±15.58 ALT(U/l) 59.20±4.41 93.80±25.61 AST(U/l) 108.00±6.79 110.20±4.66 Totalbilirubin(mg/dl) 0.10±0.00 0.10±0.00 Directbilirubin(mg/dl) 0.04±0.01 0.046±0.00 Indirectbilirubin(mg/dl) 0.06±0.01 0.054±0.00 BUN(mg/dl) 36.80±1.24 44.00±1.38 Creatinine(mg/dl) 0.30±0.03 0.30±0.03 UricAcid(mg/dl) 0.68±0.05 0.96±0.17 Glucose(mg/dl) 129.60±6.41 136.40±7.90 Cholesterol(mg/dl) 118.50±6.08 103.50±4.11 Triacylglycerides(mg/dl) 98.50±20.86 68.00±5.06 Sodium(mmol/l) 142.00±1.14 143.60±0.87 Potassium(mmol/l) 3.74±0.13 3.56±0.08 Chloride(mmol/l) 101.60±1.36 101.80±0.37
statisticalprogramGraphPadPrism5.0(GraphPadSoftware,San
Diego,CA,USA).
Resultsanddiscussion
The cytotoxicity assays are essentials in the early stages of
drugdevelopment,sincetheydefinetheconcentrationtobeused
inlater stagesof evaluation(Eliaset al.,1984).TheMTT testis
acolorimetricindicatorofcellviabilitywidelyused.Thistestis abletoassess cellularmitochondrialfunction bytheenzymatic reduction of the tetrazolium salt by mitochondrial dehydroge-nases in viable cells (Mossmann, 1983). In the evaluation of EHSB-inducedcytotoxiceffectagainstmurinemacrophages,alow cytotoxicity was observed, since it was able to reduce 50% of theviabilityofmacrophagesatthemeancytotoxicconcentration (CC50)of90.03g/ml,withconfidenceintervalsbetween70.72and 114.6g/ml.
Humanerythrocytesrepresentagoodpreliminarymodelof tox-icity,whichisusefulinthecytotoxicevaluationofnewdrugs.Also, itcanbeusedasapossibleindicatorofdamageagainstnon-target cells.Thisassayallowsustoevaluatethepotentialofdrugsof pro-motinginjuryintheplasmamembraneofredcells.Thisinjurycan happenbyformationofporesorbytotallysisofthecells( Costa-Lotufoetal.,2002;Lexisetal.,2006).TheEHSB-inducedcytotoxic effectagainsthumanerythrocytespromotedlessthan3%of hemol-ysisatthehighesttestedconcentration(400g/ml).
Theimmunostimulationcanbeveryeffectiveandusefulwhen immunesystemisimpaired.Inthisaspect,theherbalmedicines havedemonstratedanappropriateimmunostimulatorypotential fortheprophylaxisandtherapyofmoderatedinfections(Wagner et al.,1999).Among thousandsof naturalproducts, thespecies Woodfordia fruticosahas beenused in Ayurvedic medicine due toitsimmunostimulantpotential.Inthisperspective,the poten-tialofdrugsinpromotestimulationofmacrophagesbyactivation ofphagocytosisandproductionofnitrogenandoxygen-reactive species have been related to immunomodulatory properties. Moreover,theseagents canprovide supportivetherapy to con-ventionalchemotherapybyactivationofhostdefensemechanisms inthepresenceofanimpairedimmuneresponsiveness(Shahand Juvekar,2010).
4
3
2
1
0
C 100
** * **
50 25 12.5 6.25 3.12
EHSB (μg/ml)
L
ysosomal volume (Absorbance)
Fig.1. Determinationoflysosomalvolumeafterincubationofthehexanicextract
fromP.insignisseeds(EHSB)inmurinemacrophages.ANOVAOne-wayfollowedby
Tukey’spost-test.*p<0.05;**p<0.01vs.control.
0.4
0.3
0.2
0.1
0.0
C 100 50 25 12.5 6.25 3.12
EHSB (μg/ml)
Phagocytic capability
(Absorbance)
** ** *
***
Fig.2.Assessmentofphagocyticcapacityafterincubationofthehexanicextract
fromP.insignisseeds(EHSB)inmurinemacrophages.ANOVAOne-wayfollowedby
Tukey’spost-test.*p<0.05;**p<0.01;***p<0.001vs.control.
widerangeofsubstanceswithantimicrobialand/or immunomod-ulatory potentials. This way, the activation of macrophages is necessarytoestablishcontrolandprogressionofintracellular infec-tionsinanattempttoadapttothesurroundingenvironment(Ielpo etal.,2000).ThetreatmentofmacrophageswithEHSBpromoted aslightincreaseinthenumberandvolumeoftheendocytic com-partmentvesicles.Thismaysuggestanincreaseinthepotentialof defenseofthesecells.
Anotherparameterofmacrophageactivationwasassessedby thecellularresponsetoanexternalstimulus,whichwasestimated bythephagocytosisofzymosanparticlesstainedwithneutralred. TheEHSBinducedasignificantincreaseofphagocyticcapability ofmacrophagesby 42.9,49.8, 53.7and 71.2%atthe concentra-tionsof25,12.5,6.25and3.12g/ml,respectively(Fig.2).Zymosan isapolysaccharideobtainedfromthecellwallofSaccharomyces cerevisiaein water-insolubleparticles witha meandiameter of 3m. Invitrostudieshavedemonstrated thatzymosan hasthe ability to stimulate defense cells to induce Th1-type immune response,whichincludesanincreaseintheproductionofIFN-␥ (Weietal.,2011).Whenmurinemacrophageswerestimulatedwith zymosanparticles,anincreaseinphagocyticactivityforallEHSB testedconcentrationswasobserved.AccordingtoSchwartz(2007), manyantimicrobialenzymaticsystemsareactivatedwithin phago-cyticvesicles in ordertodigestpathogens and/orphagocytized substances.
1.0
0.6 0.8
0.4
0.2
0.0
C 100 50 25 12.5 6.25 3.12 LPS
EHSB (μg/ml)
Nitrite measurement
(Absorbance)
***
***
* ** **
***
Fig.3.Measurementofnitriteproductionafterincubationofthehexanicextract
fromP.insignisseeds(EHSB)inmurinemacrophages.ANOVAOne-wayfollowedby
Tukey’spost-test.*p<0.05;**p<0.01;***p<0.001vs.control.
Table2
Relativeorganweights14daysafteracuteoraltreatmentwiththehexanicextract
fromP.insignisseeds(EHSB2.0g/kg)infemaleWistarrats.
Organs Vehicle EHSB
Lungs 0.652±0.063 0.502±0.007 Heart 0.336±0.007 0.350±0.012 Stomach 0.701±0.037 0.800±0.106 Liver 3.745±0.304 4.428±0.111 Brain 0.814±0.030 0.845±0.019 Rightkidney 0.363±0.010 0.392±0.011 Leftkidney 0.349±0.010 0.346±0.009
The determination of NO production was performed
indi-rectlybymeasurement of thenitriteproduced in macrophages
treated withtheEHSB. A significantincrease ofNO production
in macrophages wasobserved at alltested concentrations, and
thehigher effects were observedfor EHSB 100g/ml by 12.4%
andforthepositivecontrolLPSby27.2%(Fig.3).Thenitricoxide
producedby macrophagesplaysanimportantrole in patholog-icalprocesses, suchasantimicrobialdefense, inflammationand angiogenesis(Krishnatryetal.,2010;Macmickingetal.,1997).The cytotoxiceffectagainstpathogenicmicroorganismsisindirectly performedbyNO,whichcombineswithO2−2 within phagolyso-somestoproduceperoxynitrite,ahighreactiveoxygenspecieswith microbicideproperties.Thismicrobicidereactionproducesnitrite andnitrateasitsfinalproduct(Beutler,2004).
The toxicological investigation of natural products aims to establisha safeand effectiverangeof dosesfornewdrugs and pharmaceuticalformulations.Likewise,theEHSBdidnotinduce anydeathorchangesinclinicalandbehavioralparametersatthe oraldoseof2.0g/kginratsduringthe14daysofdailyobservation. Theserumbiochemicalparameterswerealsoinvestigated,andthe resultsareinaccordancewiththereferencesvaluesobservedfor variousstandardizedanimalfacilities(Santosetal.,2010). Interest-ingly,itwasnotobservedanyalterationinthetestedparameters (Table1).Itmeansnosignsofsystemictoxicityinthetested con-centration.
AfterEHSBacuteoraltreatment,asignificantweightgainwas observedalongthe14daysofobservationcomparedwiththe con-trolgroup(Fig.4).Afterthenecropsy,nochangesincolor,textureor consistencywasobservedininternalorgans,aswellastheirrelative weightscomparedwiththecontrolgroup(Table2).Additionally, nogastriclesionswereobservedinanytreatedgroup.
45
40
35
EHSB 2.0 g/kg
Time (Days) Control
25
15
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
30
20
10 50
W
eight gain (g)
Fig.4.Curveofweightgainafteracuteoraltreatmentwiththehexanicextractfrom
P.insignisseeds(EHSB2.0g/kg)infemaleWistarrats.
showanytoxicologicalsignsafteracuteoraltreatmentinrats.It demonstratesasafetyprofileforthemedicinaluseofthisspecies. Thus,thepresentstudyprovidesimportantinformationaboutthe speciesandtheBrazilianAmazonbiodiversity,aswellasweraise upperspectivesforevaluationanddevelopmentofnewsafeand moreeffectiveherbalmedicines.
Authors’contributions
AKMFL,DDRA,RGR,KAFR,FFBP,CAPandJCSFperformedthe experiments.DDRA,FAAMandAMGLCdesignedthestudy, super-visedthelaboratoryworkandcontributed tocriticalreading of themanuscript.DDRA,KAFR,BQA,JSLNanalyzeddata.JSCJand AMGLCcontributedwithreagents.DDRAandKAFRandBQAwrote thepaper.DDRArevisedtheEnglishproofwriting.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
ThisworkwassupportedbytheFederal Universityof Piauí, Fundac¸ãodeAmparoàPesquisadoEstado doPiauí,CAPES and CNPq,Brazil.
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