Protective Effect of the Plant Extracts of Erythroxylum sp. against Toxic Effects Induced by the

14 Pages·2016·1.69 MB·English

molecules
Article
Protective Effect of the Plant Extracts of
Erythroxylum sp. against Toxic Effects Induced by
the Venom of Lachesis muta Snake
EduardoCoriolanodeOliveira1,RodrigoAlvesSoaresCruz2,NayannadeMelloAmorim1,
MarceloGuerraSantos3,LuizCarlosSimasPereiraJunior1,EladioOswaldoFloresSanchez4,
CaioPinhoFernandes5,RafaelGarrett6,LeandroMachadoRocha7andAndréLopesFuly1,*
1 DepartmentofMolecularandCellularBiology,InstituteofBiology,FederalFluminenseUniversity,
Niterói24020-141,RJ,Brazil;[email protected](E.C.d.O.);
[email protected](N.d.M.A.);[email protected](L.C.S.P.J.)
2 DepartmentofBiologicalandHealthSciences,FacultyofPharmacy,FederalUniversityofAmapá,
Macapá68903-419,AP,Brazil;[email protected]
3 FaculdadedeFormaçãodeProfessores,UniversityoftheStateofRiodeJaneiro,
RiodeJaneiro24435-005,RJ,Brazil;[email protected]
4 LaboratoryofBiochemistryofProteinsfromAnimalVenoms,ResearchandDevelopmentCenter,
EzequielDiasFoundation,BeloHorizonte30510-010,MG,Brazil;[email protected]
5 LaboratoryofPhytopharmaceuticalNanobiotechnology,DepartmentofBiologicalandHealthSciences,
FederalUniversityofAmapá,Macapá68903-419,AP,Brazil;[email protected]
6 MassSpectrometryLaboratory,InstituteofChemistry,FederalUniversityofRiodeJaneiro,
RiodeJaneiro21941-598,RJ,Brazil;[email protected]
7 DepartmentofPharmaceuticalTechnology,FacultyofPharmacy,FluminenseFederalUniversity,
Niterói24210-346,RJ,Brazil;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:DerekJ.McPhee
Received:12May2016;Accepted:4October2016;Published:11October2016
Abstract: Snake venoms are composed of a complex mixture of active proteins that induce toxic
effects, such as edema, hemorrhage, and death. Lachesis muta has the highest lethality indices in
Brazil. In most cases, antivenom fails to neutralize local effects, leading to disabilities in victims.
Thus,alternativetreatmentsareunderinvestigation,andplantextractsarepromisingcandidates.
The objective of this work was to investigate the ability of crude extracts, fractions, or isolated
productsofErythroxylumovalifoliumandErythroxylumsubsessiletoneutralizesometoxiceffectsof
L.mutavenom. AllsamplesweremixedwithL.mutavenom,theninvivo(hemorrhageandedema)
andinvitro(proteolysis,coagulation,andhemolysis)assayswereperformed. Overall,crudeextracts
or fractions of Erythroxylum spp. inhibited (20%–100%) toxic effects of the venom, but products
achievedaninhibitionof4%–30%. However,whenvenomwasinjectedintomicebeforetheplant
extracts,hemorrhageandedemawerenotinhibitedbythesamples. Ontheotherhand,aninhibition
of5%–40%wasobtainedwhenextractsorproductsweregivenbeforevenominjection. Theseresults
indicatethattheextractsorproductsofErythroxylumspp. couldbeapromisingsourceofmolecules
abletotreatlocaltoxiceffectsofenvenomationbyL.mutavenom,aidinginthedevelopmentofnew
strategiesforantivenomtreatment.
Keywords: Lachesis muta; snake venom; Plants; Erythroxylum ovalifolium; Erythroxylum subsessile;
Antivenom
Molecules2016,21,1350;doi:10.3390/molecules21101350 www.mdpi.com/journal/molecules
Molecules2016,21,1350 2of14
1. Introduction
According to the World Health Organization, snake bites are considered neglected diseases
and affect 5.5 million people annually, resulting in approximately 400,000 amputations and
120,000 deaths [1,2]. Lachesis muta (Bushmaster) is the longest venomous snake in the Americas,
andthesecondbiggestintheworld. ThissnakeispresentintheequatorialforestseastoftheAndes,
rangingfromeasternEcuador,Colombia,Peru,northernBoliviaandeasternandnorthernVenezuela,
toGuyana,FrenchGuyana,Surinam,andnorthernBrazil. InBrazil,thenumberofaccidentswith
L.mutaislower(3%)thanBothrops(90%)orCrotalus(7%),buttheirmortalityratesarethehighest.
IntheAmazonrainforest,however,L.mutaaccidentsaccountfor17%ofenvenomation[3,4]. L.muta
venomconsistsofamixtureoftoxins,includingmetalloproteases,serineproteases,phospholipasesA ,
2
L-aminoacidoxidases,disintegrins,amongothersthatcauselocal(pain,inflammation,tissuenecrosis,
edema,vomiting,diarrhea,andbleeding)andsystemiceffects(sweating,renalandcardiacfailure,
hypotension,dysphagia,hemorrhage,andshock)[3–5].
Theofficialtreatmentforsnakebitesworldwideistheadministrationofappropriateantivenom,
whichhasbeenavailableformorethan100years,anduptonow,isessentiallyperformedusingthe
sameprotocol. Antivenomproductioniscarefullycontrolledbygovernments,anditisundoubtedly
effectiveagainstthetoxicsystemiceffects,butitdoesnotneutralizethelocalsymptoms,resultingin
morbidityordisabilitytovictims[3,6]. Moreover,antivenomtherapyhasotherlimitations,andmay
inducesideeffects(mildfeverand/oranaphylaxis),requireslow-temperaturestorage(thisiscriticalfor
isolatedregionswithoutelectricpower),andamedicalapparatusforadministrationoftheantivenomis
needed. Asaresult,researchersarelookingforalternativetreatmentsabletoblockpathophysiological
effectsofsnakebites,andplantextractsarebeingthoroughlyinvestigatedwithpromisingpositive
results [6–8]. Thus, screening plants to neutralize the deleterious effects of snake bites deserves
attention. However,validationoftheireffectivenessinthescientificliteratureispoor.
The utilization of medicinal plants has been in practice over generations by indigenous and
non-indigenouscommunities,andpeoplewholiveinpoorordistantareasusenaturalresourcesasa
firsttreatmentforsnakebitesaswellasforthetreatmentofseveralpathologiesandillnesses. Thus,the
investigationofplantextractsasanantidoteagainsttoxicanddevastatingeffectsofsnakevenom
is relevant [9–11]. Molander et al. [12] reported a protective effect of 94 different species of plants
regularlyusedinthetraditionalmedicineofAfricaagainstsomesnakevenomtoxins.
Erythroxylaceae is a family of flowering trees with tropical and subtropical distribution, and
four genera comprise such families: Aneulophus Benth, Erythroxylum P. Br, Nectaropetalum Engl.,
andPinacopodium. Erythroxylumisconsideredthelargestgenus,with230species[13–18]. InBrazil,
thesespeciesoccurintwobiomes: “restinga”(typicalvegetationonquartzite,sandy,nutrient-poor
parent materials over the Brazilian coast) and “cerrado” (savannas of central Brazil). A few
studiesonpharmacologicaland/orbiologicalfunctionsofErythroxylumspp. havebeenperformed.
Picotetal.[19]showedthatthecrudeextractofE.laurifoliumhadaninvivohypoglycemiaaction.
Inaddition,numeroustraditionalmedicineapplicationshavebeenreportedforErythroxylumspecies,
such as an aphrodisiac, for the treatment of venereal diseases, rheumatism, arthrosis, respiratory
infections,andamenorrhea[13,14,17]. Tropanealkaloids,tannins,flavonoids,andterpenesareorganic
compounds present in large amounts in this genus, and most of the medicinal effects have been
assignedtotheseconstituents[16,18].However,toourknowledge,thereisnodataabouttheantivenom
effectofErythroxylumspecies.
2. ResultsandDiscussion
Inthiswork,theabilityofstemextractsofE.ovalifoliumandE.subsessile(extractspreparedin
solvents of increasing polarities: hexane, dichloromethane, ethyl acetate, ethanol, and butanol) or
productstoinhibittheharmfuleffects(proteolysis,coagulant,hemolysis,hemorrhage,andedema)
of L. muta venom was investigated. Overall, both plant extracts efficiently inhibited all the tested
activities,butwithdifferentefficacy. However,inhibitionofsuchactivitiesdroppedsignificantlyfor
isolatedproducts.
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Molecules 2016, 21, 1350 3 of 14
2.1. ChemicalCompositionofPlantExtracts
2.1T. Chehepmlaicnatls Coofmthpoesgiteionnu osfE Prlyatnhtr oExxytrluacmtsc ontainalkaloids,flavonoids,terpenes,andothermetabolites
with several biological and pharmacological activities [14,16,18]. The flavonoids quercetin and
The plants of the genus Erythroxylum contain alkaloids, flavonoids, terpenes, and other metabolites
kaemperolareconsideredaschemotaxonomicmarkersoftheErythroxylumgenus, aswellastheir
with several biological and pharmacological activities [14,16,18]. The flavonoids quercetin and
3-gklayecmospiedreold aerrei vcaotnivsiedse[r2e0d] .aPs hchyetomcohteamxoincaolmainc amlyasrekseorsf othf etheex tErrayctthsroaxnydlupma rgteitniuons,s aws ewreellc aarsr tiehdeiro ut
by3t-hgilnyc-loasyideer cdherroivmataivtoegs r[a2p0]h. yPh(TytLoCch),enmuiccalel aarnmalyasgense otfi cthree seoxntraancctse a(NndM pRar)t,igtiaosnsc hwroerme acatorrgireadp ohuyt- bmya ss
sptehcitnro-lmayeetrr ych(GroCm-aMtoSg)r,aapnhdyl i(qTuLiCd),c hnruocmleaart omgraagpnheyti-cm reassosnsapneccet r(oNmMeRtr)y, (gLaCs -cMhrSo)minatoorgdraeprhtoy-imdeansst ify
thespmeactirnocmlaestsreys (GofCb-iMoaSc)t, iavnedn laitquuriadl cphrroodmuacttos.gTraLpChya-nmalayssse sspoefctdriocmhleotrroym (LeCth-aMnSe) ainn dorhdeexra tnoe idpeanrttiitfiyo ns
shothwe emdatinh eclpasrseesse onfc ebiooafctteivrep ennaotuidrasl apnrodduflcatvs.o TnLoCid asn,awlyhsielse otfa dnincihnlosrwomereethfaonuen adndin heexthanyel apcaerttiatitoenasn d
busthaonwoledp atrhteit piornesse(ndcae toaf nteortpsehnoowidns )a.ndA lfklaavlooindosidws,e wrehinleo ttadnentiencst ewderine faonuyndo fint heetheyxl taracecttasteb yanTdL C
anbaulytsainso.lT epraprteintiooinds- (adnadtafl navoot nsohiodw-cno).n tAailknainlogidpsa rwtietiroen nsowt edreetescetleedct eind faonryf uorft hthere ferxatcrtaioctnsa tbiyo nTbLaCs ed
onatnhaeliyrswis.e Tllekrpneonwoind-a abnildit yflatvooinnohiidb-ictotnhteaitnoixnigc paacrttiivtiiotiness wofersen asekleecvteedn ofomr sfu[r2t1h,e2r2 f]r.aTcthioendaitciohnlo braosmede tohna ne
patrhtietiiro wnselol fknEo.wovna laibfoilliituym toa nindhiEb.it stuhbes etsosxiilce awcteirveitifersa cotfi osnnaakteed veunsoinmgs s[i2l1ic,2a2]g. eTlhceo lduicmhlnosroamnedthealnuet ed
partitions of E. ovalifolium and E. subsessile were fractionated using silica gel columns and eluted in
in hexane: ethyl acetate gradients (10:0 to 0:10). Based on the chromatography profiles, samples
hexane: ethyl acetate gradients (10:0 to 0:10). Based on the chromatography profiles, samples were
wereselectedforafinalpurificationwithsuccessivewasheswithheptane; yielding1. β-sitosterol
selected for a final purification with successive washes with heptane; yielding 1. β-sitosterol (E. subsessile
(E. subsessile and E. ovalifolium), 2. friedelin (E. subsessile), and 3. lupeol (E. ovalifolium) (Figure 1).
and E. ovalifolium), 2. friedelin (E. subsessile), and 3. lupeol (E. ovalifolium) (Figure 1). The ethyl acetate
TheethylacetateandbutanolpartitionswereindividuallyfractionatedthroughaSephadexLH-20
and butanol partitions were individually fractionated through a Sephadex LH-20 column, using
column, using ethanol as the mobile phase, and fractions were analyzed by TLC and subjected
ethanol as the mobile phase, and fractions were analyzed by TLC and subjected to nuclear magnetic
to nuclear magnetic resonance (NMR) spectroscopy, yielding: 4. quercetin (ethyl acetate of
resonance (NMR) spectroscopy, yielding: 4. quercetin (ethyl acetate of E. ovalifolium), 5. rutin (butanol
E. ovalifolium), 5. rutin (butanol of E. ovalifolium), 6. quercitrin (both partitions of E. subsessile),
of E. ovalifolium), 6. quercitrin (both partitions of E. subsessile), and 7. kaempferol-3-O-rhamnoside
and7. kaempferol-3-O-rhamnoside(bothpartitionsofE.subsessile)(Figure1). Compounds1–3were
(both partitions of E. subsessile) (Figure 1). Compounds 1–3 were obtained as crystal, and compounds
obtainedascrystal,andcompounds4–7wereamorphousyellowsolids. Theisolatedcompoundswere
4–7 were amorphous yellow solids. The isolated compounds were identified by comparison of
identifiedbycomparisonofspectroscopicdatawithreportedvalues[23–25].
spectroscopic data with reported values [23–25].

FigFuigruer1e. 1C. Chheemmicicaall ssttrruuccttuurreess ooff mmeettaabbooliltietes sofo fE.E s.usbusbessseisles ialenadn/odr/ Eo.r ovEa.liofvolailuimfo.l i(u1m) β.-(s1it)oβst-esriotol,s (t2e)r ol,
(2)frfrieieddeelilnin, (,3()3 l)ulpuepoelo, l(,4()4 q)uqeurceertcient,i n(5,)( r5u)triun,t i(n6,) (q6u)eqrucietrrcinit,r (i7n), k(7a)emkapefmerpolf-e3r-oOl--3rh-Oam-rnhoasmidneo. side.
1H-NMR experiments were carried out in order to verify the main structural characteristics of
1H-NMRexperimentswerecarriedoutinordertoverifythemainstructuralcharacteristicsof
major constituents of each sample. Since 1H-NMR peak area is proportional to the abundance of each
majorconstituentsofeachsample. Since1H-NMRpeakareaisproportionaltotheabundanceofeach
atom, we focused the analysis on the major peaks of each spectrum. As seen in Figure 2, 1H-NMR
atom,wefocusedtheanalysisonthemajorpeaksofeachspectrum. AsseeninFigure2,1H-NMR
spectra of ethyl acetate, hexane, n-butanol, and aqueous partitions of E. subsessile presented similar
sppecrotrfaileosf. Tethhey slpaeccetrtuamte ,ohf ethxea nhee,xann-be uptaarntiotilo,na npdredaqomueinoaunstlpya srhtoitwioends poefaEks. souf bssaetsusrialetepdr ehsyednrotecdarsbiomnsi lar
pro(δfi l0e.s5.–T3.h0e psppmec,t rFuigmuroef t2hBe).h Tehxea nmeopsatr dtietisohniepldreeddo pmeiankasn rtelyprsehsoewnte hdypderaokgseno ffsraotmur hatyeddrohxyydlraotecdar obro ns
(δu0n.5s–a3tu.0raptepdm g,rFouigpusr (eδ 23B.5)–.5T.5h eppmmo)s. tTdhiess phrieolfdilee dwpase ainktserrperperteesde anst ah ytrditreorpgeennef-rroicmh shaymdprloex. yOlna ttehde or
other hand, the major peaks obtained in the ethyl acetate spectrum are consistent with flavonoids’
Molecules2016,21,1350 4of14
unsaturatedgroups(δ3.5–5.5ppm). Thisprofilewasinterpretedasatriterpene-richsample. Onthe
otherhand,themajorpeaksobtainedintheethylacetatespectrumareconsistentwithflavonoids’
aromMatoilcecsulresi n20g1s6, (2δ1, 613.050– 8.0ppm)andpolyhydroxylatedgroups(δ3.5–4.5ppm,Figure2A).T4 hofe 1s4e peaks
wereprobablyduetothepresenceofglycosylatedflavonoidsthatarechemicalmarkersofthegenus
aromatics rings (δ 6.0–8.0 ppm) and polyhydroxylated groups (δ 3.5–4.5 ppm, Figure 2A). These
Erythroxylum and are in accordance with the compounds isolated and identified in the extracts.
peaks were probably due to the presence of glycosylated flavonoids that are chemical markers of the
Thespectraofn-butanolandaqueousfractions(Figure2C,D,respectively)presentedinnumerous
genus Erythroxylum and are in accordance with the compounds isolated and identified in the
superepxotrsaicttiso.n Tpheea kspsebcetrtaw oefe nn-δbu6t.a2naonl danδd8 .a5qpupeomu,s pforascstiibolnys d(uFiegutorea 2hCig,Dh, croenspceecnttivraetlyio) nporefspenotleydp henols
of higinhneurmpeorolaursi stiuepse,rpsuoscihtioans pteaankns ibnestw. eOenu δr 6N.2M anRd fiδ n8d.5i nppgms,a preosisniblayg drueee mtoe an htigwhi tchonpcernetvriaotiuosn works
perfoorfm peodlyfpohrendoiflsfe oref nhtigshpeerc pieoslaoriftiEesr,y tshurcohx yaslu tman.nBinasr. rOeiuror sNeMtRal .fi[n2d0i]ngdse maroe ninst raagtreedemthenet pwrietshe nceof
fattyparceivdioaunsd wtorirtkesr ppeernfoersmaesdw foerl ldaisffelurepnet oslpaecnieds βo-f sEitroytshtreorxoylluinm.t hBearhreeixroasn eet paal. r[t2i0ti]o dnemofoEns.tnrautmedm ularia,
the presence of fatty acid and triterpenes as well as lupeol and β-sitosterol in the hexane partition of
andquercetinwasfoundintheextractsofthechloroformandethylacetatefractions. Moreover,lupeol
E. nummularia, and quercetin was found in the extracts of the chloroform and ethyl acetate fractions.
andβ-sitosterolweredetectedinthehexaneextractofE.passerinum,aswellasothercompounds,such
Moreover, lupeol and β-sitosterol were detected in the hexane extract of E. passerinum, as well as
asβ-amyrinanderythrodiol[26]. LupeolwasidentifiedinthemethanolicleafextractofE.leal-costae,
other compounds, such as β-amyrin and erythrodiol [26]. Lupeol was identified in the methanolic
inaddleiatfi oenxtrtaocto othf eEr. lseualb-csotsatanec, eins a[2d7d]i.tion to other substances [27].
A B

C D

Figure 2. 1H-NMR spectra of the (A) ethyl acetate; (B) hexane; (C) n-butanol; or (D) aqueous
Figure2.1H-NMRspectraofthe(A)ethylacetate;(B)hexane;(C)n-butanol;or(D)aqueouspartitions
partitions of E. subsessile.
ofE.subsessile.
The ethyl acetate partitions of E. ovalifolium and E. subsessile were subjected to liquid
Tchhreomeatthogyrlapahcye-tmaatess sppaercttirtoimonetsry oafnaEly.siso v(UalPiLfoCli-uOmrbitaranpd MES)., asnudb seeigsshitl epowlypehreenosluicb cjoemctpeoduntdos liquid
were identified based on their high-resolution and accurate m/z values and MS/MS spectra provided
chromatography-massspectrometryanalysis(UPLC-OrbitrapMS),andeightpolyphenoliccompounds
by the Orbitrap analyzer (Table S1, Figures S1 and S2; Supplementary Materials). Compounds
wereidentifiedbasedontheirhigh-resolutionandaccuratem/zvaluesandMS/MSspectraprovided
quercetin, quercitrin and kaempferol were found in both species, while (epi)catechin, (epi)catechin
by the Orbitrap analyzer (Table S1, Figures S1 and S2; Supplementary Materials). Compounds
dimer (procyanidin dimer), rutin, eriodictyol-rhamnoside, and ombuin-rutinoside were found only in
quercetin,quercitrinandkaempferolwerefoundinbothspecies,while(epi)catechin,(epi)catechin
the ethyl acetate partition of E. ovalifolium. Quercitrin, ombuin-rutinoside, and eriodictyol-rhamnoside
dimerha(vper oaclyreaandiyd ibneednim deesrc),rirbuetdin i,ne srpioedciiecst yoof l-thrhe aEmryntohrsoixdyel,uamn dgeonmusb [u2i8n–-3r0u].t iQnouseirdceetiwn,e rruetfino,u anndd onlyin
theetqhuyelrcaictreitna tweeprea ratlistoio isnoolafteEd. forvoamli feotlhiuyml a.cQetuateer acnitdri bnu,toamnobl upianr-trituiotinnso. side,anderiodictyol-rhamnoside
have alreHadexyanbee eanndd deiscchrliobreodmeitnhasnpee pciaerstitoiofntsh feroEmr ybtohtrho xpylalunmts wgeerneu asn[a2ly8z–e3d0 ]b.y QGuCe-MrcSe t(iFnig,urruetsi n, and
quercSi4tr–iSn7 wSuepreplaelmsoenitsaorlya tMedatferroiamls)e. tFhryidlealicne, tafrtieedaenladnbolu, taanndo βl-psaitrotsitteioronl sw. ere found in hexane and
dichloromethane partitions from E. subsessile. β-sitosterol was found in hexane and dichloromethane
HexaneanddichloromethanepartitionsfrombothplantswereanalyzedbyGC-MS(FiguresS4–S7
partitions from E. ovalifolium.
Supplementary Materials). Fridelin, friedelanol, and β-sitosterol were found in hexane and
dichloromethanepartitionsfromE.subsessile. β-sitosterolwasfoundinhexaneanddichloromethane
partitionsfromE.ovalifolium.
Molecules2016,21,1350 5of14
Morsetal.[7]analyzedtheprotectiveeffectofseveralplantsandproductsagainstthetoxiceffects
ofBothropsjararacavenom. Moreover, theyinvestigatedthemechanismoftheinhibitoryactionof
themetabolites[7]. Theyspeculatedthatthesemetabolitesmayinteractwithactivesitesofenzymes
throughMtohleceuilres n20e1t6, c2h1, a13r5g0e orpolarity,andasaconsequence,causetheirinactivation[7,31].5 Ionf 1a4 ddition,
theinhibitoryactionofmetabolitesmayoccurbychelatingmetalionsthatarenecessaryforsome
Mors et al. [7] analyzed the protective effect of several plants and products against the toxic effects
enzymeosf iBnotvheronpos mjarsartaocae xvepnroemss. tMhoerieroavcetri,v tihteieys i,nsvuecsthigaastePd LthAe2 m(Cecah2a+n)isamn dofm theet ainllhoibpirtoortye aascetison(Z onf 2+)[31].
Thesetwtheo mgerotaubpolsiteosf [e7n]. zTyhmeye sspeacruelarteesdp tohnats tihbelseef moretaabwoliidtees mspaeyc itnrtuermacto wftitohx aicctievfef esicttess tohf aentzfyomlloesw snake
bites,inthcrluoudgihn gthehier mneot rchrharaggee o,rc pooalagruitlya, tainodn a,sn ae ccoronsseiqsu,eendceem, caau,saen thdeierf ifneaccttsivoatniopn l[a7t,3e1le]. tIna gadgdreitgioant,i onand
the inhibitory action of metabolites may occur by chelating metal ions that are necessary for some
hemolysis[32].
enzymes in venoms to express their activities, such as PLA2 (Ca2+) and metalloproteases (Zn2+) [31].
These two groups of enzymes are responsible for a wide spectrum of toxic effects that follow snake
2.2. AntiproteolyticandAntihemolyticEffectsofPlantExtractsorProducts
bites, including hemorrhage, coagulation, necrosis, edema, and effects on platelet aggregation and
L.hmemutoalysvise [n3o2]m. induced proteolysis or hemolysis in a concentration-dependent manner.
A dose–response curve was constructed to find the effective concentration (EC) and minimum
2.2. Antiproteolytic and Antihemolytic Effects of Plant Extracts or Products
indirect hemolytic concentration (MIHD). Subsequently, these venom concentrations (10 µg/mL)
wereincubaLt. emduwta itvheneoxmtr aincdtsu,ceadn dprpotreootleyosilsy toirc hoermhoelymsios lyint ica acoctnicveinttireastiwone-dreepiennvdeesntti gmaatnedne.r.A As seenin
dose–response curve was constructed to find the effective concentration (EC) and minimum indirect
Figure3, theextractsofE.ovalifoliumandE.subsessileinhibitedbothproteolysis(Figure3A,black
hemolytic concentration (MIHD). Subsequently, these venom concentrations (10 μg/mL) were
columns)andhemolysis(Figure3A,whitecolumns)inducedbyL.mutavenom,butwithdifferent
incubated with extracts, and proteolytic or hemolytic activities were investigated. As seen in Figure 3,
potencitehse. eTxhtreacetsx torfa Ec.t oovfalEifo.loiuvma laifnodli uEm. supbrseespsialer eindhiibnitdedic bholtohr opmroteetohlyasnise ,(Faignudret h3eA,e bthlaacnk oclo,lubmuntas)n ol,and
aqueousanedx threamctoslyosfisE (.Fsiguubrsee s3sAil,e winhhitieb citoeludmmnos)r eintdhuacned8 b0y% Lo. fmpurtao tveeonloymti,c bauct twiviitthy d(iFffiegruenrte p3oAte)n.cIinesa. ddition,
theethaTnhoe le,xettrhacyt loaf cEe.t aovtael,ifaolniudm apqruepeaorueds einx tdriachctlosroomfEet.hoavnae,l iafonldiu tmhe, oetrhathnoel,d biuchtalnoorlo, manedt haqauneeouesx tractof
extracts of E. subsessile inhibited more than 80% of proteolytic activity (Figure 3A). In addition, the
E.subsessileinhibitedproteolysisby40%to75%. Incontrast,thehexaneandethylacetateextractsof
ethanol, ethyl acetate, and aqueous extracts of E. ovalifolium, or the dichloromethane extract of
E.subsessileinhibitedproteolysisbylessthan10%. ThehemolysisofL.mutavenomwasinhibited
E. subsessile inhibited proteolysis by 40% to 75%. In contrast, the hexane and ethyl acetate extracts of
bymorethan80%bytheethanolorethylacetateextractsofE.ovalifolium,andbytheethanol,ethyl
E. subsessile inhibited proteolysis by less than 10%. The hemolysis of L. muta venom was inhibited
acetate,bbyu mtaonreo lt,haann d80a%q ubye othues eethxatrnaocl tosr oefthEy.l saucebtsaetses eilxet.raInctsc oofn Etr. aosvta,litfohlieumaq, uanedo ubys ethxet reathcatnoofl,E e.thoyvla lifolium
didnotaicnehtaitbei, tbtuhtaenhoel,m anodl yatqiuceaocutsi veixttyraoctfs Lo.f mE.u stuabsvesesniloe.m In (cFoingturarset, 3thAe) a.qMueoorueso evxetrr,acist oolfa Et.e odvaplirfooldiuumc tswere
alsotestdeidd noont pinrhoibteito tlhyet ihce(mFoiglyutirce a3ctBiv,ibtyla ocfk L.c moluutam vnens)omor (hFiegmuroe l3yAti)c. M(Foirgeuorveer3, Bis,owlathedit eprcoodluucmtsn wse)raec tivities.
also tested on proteolytic (Figure 3B, black columns) or hemolytic (Figure 3B, white columns)
β-sitosterol and lupeol inhibited both activities by around 5%, while quercitrin and rutin did not
activities. β-sitosterol and lupeol inhibited both activities by around 5%, while quercitrin and rutin did
inhibitthem.Incontrast,quercetininhibitedproteolyticactivity(25%),butitfailedtoinhibithemolysis
not inhibit them. In contrast, quercetin inhibited proteolytic activity (25%), but it failed to inhibit
(Figure3B).Neithersalinenorsolventsinterferedwiththehemolyticorproteolyticactivities.
hemolysis (Figure 3B). Neither saline nor solvents interfered with the hemolytic or proteolytic activities.
100 A B
30
80 25
n (%) 60 on (%) 20
hibitio 40 nhibiti 15
n I
I 10
20
5
0 0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6
Groups Groups
Figure 3. L. muta venom (10 μg/mL) was incubated with either: (A) 90 μg/mL extracts of E. ovalifolium
Figure3.L.mutavenom(10µg/mL)wasincubatedwitheither:(A)90µg/mLextractsofE.ovalifolium
in ethanol (1), ethyl acetate (2), dichloromethane (3), butanol (4), or water (5), or with E. subsessile in
inethanol(1),ethylacetate(2),dichloromethane(3),butanol(4),orwater(5),orwithE.subsessilein
ethanol (6), hexane (7), ethyl acetate (8), dichloromethane (9), butanol (10), or water (11); or (B) with
ethanol9(06 μ),gh/meLx aonf eβ-(s7it)o,setetrhoyl l(1a),c leutpaetoel ((82)),, fdriiecdhellionr (o3m), qeutheracniterin( 9(4),),b quutearcneotiln( (150),) ,oro rruwtina t(6e)r f(o1r 13)0; morin( B)with
90µg/matL 2o5 f°βC-. sAitfotsetrewraorld(s1, )p,rluotpeoeloylti(c2 )(,bflaricekd ceolilunm(3n)s,) qoure hrceimtroilnyt(i4c ),(wquhieterc ecotilnum(5n)s,) oarcrtiuvtitiines( 6w)efroer 30min
at25◦Ca.sAsefstseerdw, aasr ddse,scprriboetdeo ilny tthice (Mblaatcerkiaclos launmd nMse)tohrodhse msecotlioynti.c D(awtah aitree ceoxlpuremssnesd) aasc tmiveiatnies s±w S.eEr.e oaf ssessed,
as desctrhibreeed inidnivtihdueaMl exapteerriimalesntasn (nd =M 3)e. thods section. Data are expressed as means ± S.E. of three
individualexperiments(n=3).
Molecules2016,21,1350 6of14
Molecules 2016, 21, 1350 6 of 14
2.3. AnticoagulationEffectofPlantsorProducts
2.3. Anticoagulation Effect of Plants or Products
DisturbancesinbloodcoagulationareacommonfeatureinaccidentscausedbyL.mutavenom,
Disturbances in blood coagulation are a common feature in accidents caused by L. muta venom,
and bleeding may continue for a few days, even after antivenom administration. L. muta venom
and bleeding may continue for a few days, even after antivenom administration. L. muta venom
(25µg/mL)mixedwithsaline(C1)orDMSO(C2)clottedplasmain77s(Figure4). Then,asimilar
(25 μg/mL) mixed with saline (C1) or DMSO (C2) clotted plasma in 77 s (Figure 4). Then, a similar
venomconcentrationwasincubatedwiththeextractsofE.ovalifoliumorE.subsessile(250µg/mL)for
venom concentration was incubated with the extracts of E. ovalifolium or E. subsessile (250 μg/mL) for
30min,andthemixturewasaddedtoplasmaandcoagulationwasmonitored. AsseeninFigure4,
30 min, and the mixture was added to plasma and coagulation was monitored. As seen in Figure 4,
theextractsofE.ovalifoliumindichloromethane,butanol,orwater,aswellastheextractsofE.subsessile
the extracts of E. ovalifolium in dichloromethane, butanol, or water, as well as the extracts of
inhexane,ethylacetate,ordichloromethanedidnotinhibitthecoagulationactivityofL.mutavenom.
E. subsessile in hexane, ethyl acetate, or dichloromethane did not inhibit the coagulation activity of
In contrast, the extracts of E. ovalifolium in ethanol or ethyl acetate and the extracts of E. subsessile
L. muta venom. In contrast, the extracts of E. ovalifolium in ethanol or ethyl acetate and the extracts
in ethanol, butanol, or water significantly delayed plasma coagulation induced by L. muta venom
of E. subsessile in ethanol, butanol, or water significantly delayed plasma coagulation induced by
(Figure 4). Neither of the isolated products (250 µg/mL) inhibited coagulation of L. muta venom.
L. muta venom (Figure 4). Neither of the isolated products (250 μg/mL) inhibited coagulation of
Additionally,noneoftheextractsorsolventsaloneinducedcoagulationofplasma.
L. muta venom. Additionally, none of the extracts or solvents alone induced coagulation of plasma.
500
*
400
*
s) *
e ( 300 *
m
Ti
n
o
ati
ul 200
g
a *
o
C
100
0
C1C2 1 2 3 4 5 6 7 8 9 1011
Columns
FigFuigruer4e. 4L. .Lm. mutuatav venenoomm( (2255 µμgg//mmLL)) wwaas sinincucubabtaetde dfofro 3r03 m0imn iant 2a5t °2C5 w◦Cithw siathlinsea l(iCn1e),( CD1M),SDOM (CS2O), (oCr 2),
with 250 μg/mL extracts of E. ovalifolium in ethanol (1), ethyl acetate (2), dichloromethane (3), butanol
or with 250 µg/mL extracts of E. ovalifolium in ethanol (1), ethyl acetate (2), dichloromethane (3),
(4), or water (5), and with E. subsessile in ethanol (6), hexane (7), ethyl acetate (8), dichloromethane (9),
butanol (4), or water (5), and with E. subsessile in ethanol (6), hexane (7), ethyl acetate (8),
butanol (10), or water (11). Afterwards, mixtures were added to plasma and coagulation was
dichloromethane(9),butanol(10),orwater(11). Afterwards,mixtureswereaddedtoplasmaand
performed, as described in the Materials and Methods section. Data are expressed as means ± S.E. of
coagulationwasperformed,asdescribedintheMaterialsandMethodssection.Dataareexpressedas
three individual experiments (n = 3). * p < 0.05 when compared with C1 or C2.
means±S.E.ofthreeindividualexperiments(n=3).*p<0.05whencomparedwithC1orC2.
2.4. Antihemorrhagic and Antiedematogenic Effects of Plants or Products
2.4. AntihemorrhagicandAntiedematogenicEffectsofPlantsorProducts
When incubated with L. muta venom (9 μg/mouse), all extracts of E. ovalifolium or E. subsessile
WhenincubatedwithL.mutavenom(9µg/mouse),allextractsofE.ovalifoliumorE.subsessile
(90 μg/mL) inhibited hemorrhagic or edematogenic activity (Figure 5). The extracts of E. ovalifolium
(90inµ ge/thmylL a)cienthatieb,i tdeidchhloermomorerthhaagniec, obruetadneoml, aotor gweantiecr,a catnivdi ttyhe( Feixgturarects5 )o.fT Eh.e seuxbtsreasscitlse oinf Ee.thoavnaloilf ooliru m
inbeuthtaynloal cfeutlalyte p,rdotiecchtleodr ommiceet hfraonme ,thbeu htaenmoolr,rohragwica etfefre,catsn odf Lth. emuextat rvaecntosmo f(FEi.gusureb s5e,s bsillaeckin coeltuhmannos)l. or
buMtanoroelofvuellry, pplraontet cetxedtramctisc eaflsroo minthhiebihteedm eodrrehmaagtiocgeefnfeicc tsacotfivLi.tym uoft aLv. emnoumta (vFeingoumre, 5w,bitlha cpkocteonlucmiesn s).
Movraeroyivnegr ,frpomlan 2t0%ex ttor a9c0t%s (aFlisgourien 5h,i wbihteitde ceodluemmnast)o. gTehnei ecxatrcatcitvsi otyf Eo. fsuLb.semssuiltea prveepnaoremd, inw eitthhylp aocteetantcei es
varoyr inbugtafrnooml a2c0h%ievteod 9t0h%e h(Figigheusrte in5,hiwbihtoitrey cpoelrucmenntasg).e T(9h0e%e)x otrfa ecdtsemofa,E a.nsudb stheses ielxetprarcetps airne dheixnaneeth, yl
acedtiactheloorrombuettahnanoel,a ochr iwevaetder thhaedh itghhe elsotwinehsti bpietorcreynptaegrec e(natraoguend(9 02%0%))o f(Feidguemre a5,Aa)n. dTthhee perxotdrauccttss in
hexβa-snieto,sdtiecrhollo (r1o),m luetpheaonl e(2,)o, rqwueartceirtrhina d(4t)h, eorl oqwueerscteptienr c(e5n) tdaigde n(oatr opurnotdec2t0 m%i)c(eF firgoumre h5eAm)o.rTrhheagper,o bduutc ts
β-sfritioelsdtienr o(l3)( 1o)r, lruuptieno (l6()2 i)n,hqibuietrecdi tbriyn 2(04 )a,nodr 2q8u%e,r creetsipnec(t5i)vedliyd, tnhoet hpermootercrhtamgiicc eacftriovmityh oefm voernrohmag e,
(Figure 5B, black columns).
butfrieldin(3)orrutin(6)inhibitedby20and28%,respectively,thehemorrhagicactivityofvenom
(Figure5B,blackcolumns).
Molecules2016,21,1350 7of14
Molecules 2016, 21, 1350 7 of 14
A B
35
100
30
80
25
%) %)
n ( 60 n ( 20
o o
biti biti 15
nhi 40 nhi
I I
10
20
5
0
0
1 2 3 4 5 6 7 8 9 1011 1 2 3 4 5 6
Extracts Products
FFiigguurree 55.. ((AA)) LL.. mmuuttaa vveennoomm ((99 µμgg//mmoouussee)) wwaass iinnccuubbaatteedd ffoorr 3300 mmiinn aatt 2255 ◦°CC wwiitthh 9900 µμgg//mmLL eexxttrraaccttss
ooff EE.. oovvaalliiffoolliiuumm iinn eetthhaannooll ((11)),, eetthhyyll aacceettaattee ((22)),, ddiicchhlloorroommeetthhaannee ((33)),, bbuuttaannooll ((44)),, oorr wwaatteerr ((55)),, oorr
wwiitthh EE. .ssuubbsessessisliel eini netehtahnaonlo (l6)(,6 h),exhaenxea n(7e),( 7et)h,yelt hayceltaactee t(a8t)e, d(8ic)h,ldoircohmloertohmaneet h(9a)n, ebu(9ta),nboul (t1a0n)o, lor(1 w0)a,teorr
(w11a)t.e rT(h1e1n)., Tthhee nm,tihxetumrei xwtuarse winajescitnejde citnedtoi nmtoicme iacneda nhdehmeomrrohrarhgaicg i(cb(lbalcakc kcocloulummnns)s )oorr eeddeemmaattooggeenniicc
((wwhhiittee ccoolluummnnss)) aaccttiivviittiieess wweerree aasssseesssseedd.. ((BB)) TThhee pprroodduuccttss ((9900 μµgg//mmLL)) ββ--ssitiotossteterrool l((11)), ,lluuppeeool l((22)),,
ffrriieeddeelliinn ((33)),, qquueercrictirtirnin(4 ()4,)q,u qeurceertcient(in5) ,(o5r),r uotri nru(6ti)nw (e6r)e winecrueb aintecduwbaittehdL .wmiuthta Lv.e nmoumta( bvleanckomco l(ubmlancks)
coorlpurmodnsu)c tosrw perroediunjcetcst ewdeir.pe .iannjedcttehde ni.Lp.. mauntda vtheenno mL.w maustian jveectneodm.T wheans, ihnejmecoterdrh. aTgheewna, shdemeteorrmrhiangede
wasasd edsectreirbmedineind tahse dMesactreibrieadls inan tdheM Meathteordiaslss eacntdio nM.eDthaotdasa sreecteixopnr. eDssaetda aarse mexeparnesss±edS a.Es. mofeathnrse ±e
Sin.Ed.i vofid thuraelee xinpdeirvimideunatls e(xnp=er3i)m.ents (n = 3).
Instead of mixing L. muta venom with extracts or products, two other protocols were performed
InsteadofmixingL.mutavenomwithextractsorproducts,twootherprotocolswereperformedto
to mimic a real situation of envenomation. Namely: (1) treatment, in which L. muta venom (2 μg/mouse)
mimicarealsituationofenvenomation. Namely: (1)treatment,inwhichL.mutavenom(2µg/mouse)
was injected intradermally (i.d.) into mice, and 15 min later, the extracts of E. ovalifolium or E. subsessile
wasinjectedintradermally(i.d.) intomice,and15minlater,theextractsofE.ovalifoliumorE.subsessile
(90 μg/mL) were injected at the same site of venom injection; and (2) prevention, the extracts of plants
(90 µg/mL) were injected at the same site of venom injection; and (2) prevention, the extracts of
were given intraperitoneally (i.p.) or orally to animals, and 60 min later, L. muta venom (2 μg/mouse)
plants were given intraperitoneally (i.p.) or orally to animals, and 60 min later, L. muta venom
was injected i.d. After both protocols (treatment or prevention), hemorrhagic and edematogenic
(2 µg/mouse) was injected i.d. After both protocols (treatment or prevention), hemorrhagic and
activities were evaluated. With the treatment protocol, none of the plants inhibited hemorrhage of
edematogenicactivitieswereevaluated. Withthetreatmentprotocol, noneoftheplantsinhibited
L. muta venom (data not shown). However, with the prevention protocol, both extracts inhibited
hemorrhageofL.mutavenom(datanotshown). However,withthepreventionprotocol,bothextracts
hemorrhagic activity (Table 1). According to Table 1, higher inhibitory percentages of L. muta
inhibitedhemorrhagicactivity(Table1). AccordingtoTable1,higherinhibitorypercentagesofL.muta
venom-induced hemorrhage were achieved when plants were injected i.p. (5%–40%), than by the oral
venom-inducedhemorrhagewereachievedwhenplantswereinjectedi.p. (5%–40%),thanbytheoral
route (3%–7%). In fact, most plant extracts did not inhibit hemorrhage if given orally. The ethanol
route(3%–7%). Infact,mostplantextractsdidnotinhibithemorrhageifgivenorally. Theethanol
extract of E. subsessile had the highest inhibitory percentage (40%) of hemorrhage. In contrast, when
extractofE.subsessilehadthehighestinhibitorypercentage(40%)ofhemorrhage. Incontrast,when
given orally, plants more efficiently protected against L. muta venom-induced edematogenic activity.
givenorally,plantsmoreefficientlyprotectedagainstL.mutavenom-inducededematogenicactivity.
As also seen in Table 1, the ethyl acetate or aqueous extracts of E. subsessile achieved the highest
As also seen in Table 1, the ethyl acetate or aqueous extracts of E. subsessile achieved the highest
inhibitory percentages of edema (around 30%) if given orally to mice. However, they failed to inhibit
inhibitorypercentagesofedema(around30%)ifgivenorallytomice. However,theyfailedtoinhibit
such activity if given i.p. Moreover, the extract of E. ovalifolium in dichloromethane did not inhibit
suchactivityifgiveni.p. Moreover,theextractofE.ovalifoliumindichloromethanedidnotinhibit
edema, regardless of the route of administration. With the prevention protocol, if products were
edema, regardless of the route of administration. With the prevention protocol, if products were
injected i.p., β-sitosterol or quercetin did not inhibit hemorrhage; but an inhibition of 28% was
injected i.p., β-sitosterol or quercetin did not inhibit hemorrhage; but an inhibition of 28% was
achieved for lupeol, frieldin (14%), quercitrin (7%), or rutin (14%) (Figure 5B, white columns). On the
achievedforlupeol,frieldin(14%),quercitrin(7%),orrutin(14%)(Figure5B,whitecolumns). Onthe
other hand, products did not inhibit edema caused by L. muta venom using the prevention protocol,
otherhand,productsdidnotinhibitedemacausedbyL.mutavenomusingthepreventionprotocol,
as some extracts did.
assomeextractsdid.
Molecules2016,21,1350 8of14
Table1.EffectofE.ovalifoliumorE.subsessileonhemorrhageandedemainducedbyL.mutavenom.
%Inhibition
Sample Hemorrhage Edema
i.p. oral i.p. oral
1 8±9 0 0 22±7
2 5±7 0 18±9 19±9
3 10±8 3±15 0 0
4 40±4 4±12 12±6 17±4
5 7±9 0 0 29±8
6 18±5 0 12±4 5±2
7 14±9 7±1 0 30±9
Theextracts(90µg/mL)ofE.ovalifoliuminethanol(1),ethylacetate(2),ordichloromethane(3),andtheextracts
ofE.subsessileinethanol(4),ethylacetate(5),butanol(6),orwater(7)wereadministeredintraperitoneallyor
orallyintomice.Then,60minlater,L.mutavenom(9µg/mouse)wasinjectedintomiceandhemorrhageand
edemawereevaluated,asdescribedintheMaterialsandMethodssection.Dataareexpressedasmeans±S.E.
ofthreeindividualexperiments(n=3).
Flavonoids are a natural group of bioactive molecules capable of inhibiting a wide range of
enzymes,suchaslipoxygenase,aldosereductase,humanrecombinantaldosereductase,advanced
glycationendproducts,proteintyrosinephosphatase1B,acetylcholinesterase,butyrylcholinesterase,
β-site amyloid precursor cleaving enzyme 1, inducible nitric oxide synthase, cyclooxygenase-2,
andphospholipasesA [33,34]. TheinhibitionofPLA maybethemainmechanismoftheantivenom
2 2
activityofflavonoids. Theflavonolsrutin,quercetin,andquercitrinofE.subsessileandE.ovalifolium
maybepartiallyresponsiblefortheprotectiveeffectofsuchplants. Moreover,flavonolsisolatedfrom
HypericumbrasilienseorBrownearosademontehavebeenassociatedwithinhibitionoftheactivityof
acetylcholinesterase[35]aswellashavinganantivenomeffect[36].
Furthermore, the antivenom effect of friedelin, lupeol, and β-sitosterol may be due to an
anti-inflammatory mechanism [37], and their pentacyclic triterpene rings are responsible for such
antivenomaction[7]. ThepresentstudyshowedthatthestemextractsofE.ovalifoliumandE.subsessile
inhibitedsometoxiceffects(proteolysis,hemolysis,coagulation,hemorrhage,oredema)ofL.muta
venom that undoubtedly contribute to the death and morbidities of victims. In addition, the part
ofplant(leaf,root,stem,flour,fruit,orflowers)involvedintheinhibitoryeffectagainstthetoxicity
of venoms as well as the solvents used to prepare extracts should be taken into consideration in
studiesofscreening,ethnobotany,andethnopharmacology. Apreviousreportreinforcesourresults,
since different inhibitory profiles were observed for the stem and leaves of Manilkara subsericea
prepared in ethyl acetate or hexane against proteolytic, hemolytic, coagulant, hemorrhagic, and
edematogenicactivitiesofL.mutavenom[38]. However,itisquitedifficulttopostulatewhichpartof
M.subsericeaisthemostactiveininhibitingsuchactivities[38]. Ingeneral,thestemsofplantsinhibit
activitiesofvenomsmoreefficientlythantheflowersorleaves. Moreover,theliteratureshowsthat
extractsoftheDipteryxalataneutralizedthevenomsofB.jararacussuandCrotalusdurissusterrificus,
andsomemetabolites,suchasisoflavones,triterpenes,tannins,amongothers,havebeenidentifiedas
antivenommolecules[39–42]. Asidefromplants,themarinealgaCanistrocarpuscervicornis[43]and
somesponges[44]alsoinhibitedthetoxicactivitiesofvenomsfromB.jararacaandL.muta.
ResultsfromthepresentworkshowedthatasinglepartofE.ovalifoliumorE.subsessileandthe
productsofsuchplantswereunabletoblockallinvivoandinvitrotoxiceffectsofL.mutavenom.
Thus,acocktailofdifferentpartsofplantsorproductsshouldbeusedinordertoincreasetheinhibitory
actionagainstthetoxiceffectsofthevenom.
Molecules2016,21,1350 9of14
3. MaterialsandMethods
3.1. Venom,Animals,andReagents
L.mutavenomwaskindlysuppliedfromEzequielDiasFoundation(FUNED),BeloHorizonte,
MinasGerais,Brazil. L.mutavenomwasdilutedinsaline(1mg/mL)andstoredat−20◦Cuntiluse.
MaleSwissmice(18–20g)wereobtainedfromtheCenterofLaboratoryAnimals(NAL)oftheFederal
FluminenseUniversity(UFF).Allsolventsandreagentswereofthepurestgradeavailable.
3.2. EthicsStatement
Experiments were approved by the UFF Institutional Committee for Ethics in Animal
Experimentation(protocolNo. 212),inaccordancewiththeguidelinesoftheBrazilianCommitteefor
AnimalExperimentation(COBEA).PlasmawasobtainedfromhumandonorsfollowingtheGuidelines
oftheEthicsCommitteeoftheUFFHospital,undernumberCEP643.926.
3.3. PlantMaterial
TheplantsE.ovalifolium(S2214.722’,WO4134.916’)andE.subsessile(S2216.111’,WO4138.990’)
werecollectedinJanuary2009fromtheRestingaofJurubatibaNationalPark,stateofRiodeJaneiro,
Brazil. The identification of the plants was carried out by Marcelo Guerra Santos (FFP/UERJ),
and voucher samples were deposited in the Herbarium of the Rio de Janeiro State University,
SãoGonçalo,undertheidentificationlabelsRFFP2147andRFFP2126,respectively.
3.4. PreparationofPlantExtractsandPartitions
Stems of E ovalifolium (4.2 kg) and E. subsessile (2.7 kg) were dried at 35 ◦C for 24 h with air
circulationandpulverizedinagrindingmill(ThomasScientific,Swedesboro,NJ,USA).Thepowdered
stemsofE.ovalifoliumweremaceratedwith96%ethanol(10%w/v)for10daysatroomtemperatureand
filtered.Theresiduewasmaceratedagaintoensuretheexhaustionofthedrug.Subsequently,theliquid
phases were pooled and filtered, the solvent fully evaporated under reduced pressure at 35 ◦C,
andsuspendedin1Lof90%ethanolbeforepartitioningwithhexane(3×200mL).Theethanol-soluble
fraction was concentrated under reduced pressure, suspended in distilled water, and partitioned
successivelywithdichloromethane(3×200mL),ethylacetate(3×200mL),andbutanol(3×200mL).
Solventsfromeachliquidorganicphaseweretotallyevaporatedunderreducedpressureat35◦C,
yielding solid hexane (32.4 g), dichloromethane (41.0 g), ethyl acetate (37.7 g), and butanol (8.6 g)
partitions. TheprocedurewasrepeatedforE.subsessile,yieldinghexane(11.0g),dichloromethane
(15.5 g), ethyl acetate (16.2 g), and butanol (11.3 g) partitions. All extracts, partitions, or isolated
productsweredissolvedin100%dimethylsulfoxide(DMSO).
3.5. PhytochemicalAnalysis
Phytochemicalinvestigationswereperformedonpartitionsofbothplantsinseveralsteps. Firstly,
sampleswereanalyzedbyTLCfordetectionofthemainclassesofnaturalproducts(i.e.,flavonoids,
terpenoids, tannins, and alkaloids). Secondly, partitions were fractionated by Sephadex LH-20
(GE Healthcare, Piscataway, NJ, USA) or silica gel column chromatography to separate the main
constituents. One-andtwo-dimensionalNMRspectrawererecordedonaVarianVNMRS400MHz
(VarianInc.,SantaClara,CA,USA),usingtetramethylsilaneasexternalshiftreference. Thestructures
ofisolatedcompoundswereelucidatedonthebasisof1H-and13C-NMRspectroscopy. Thepartitions
werealsoanalyzedbyLC-MS/MSandGC-MS.FortheLC-MS/MSanalysis,anUPLC-QExactive
PlusOrbitrapmassspectrometrysystem(ThermoFisherScientific,Bremen,Germany)wasemployed,
equippedwithanelectrosprayionizationsourceoperatinginnegativeionmodeatavoltageof2.9kV.
The column used was a Thermo Syncronis C18 (50 mm × 2.1 mm × 1.7 µm) maintained at 40 ◦C.
Themobilephaseconsistedof(A)5mMammoniumformateand(B)methanolingradientelution
Molecules2016,21,1350 10of14
mode(0−2min,20%B;2−4min,60%B;4−10min,95%B;10–12min,95%B,and12.1−15min,20%B)
ataflowrateof400µLmin−1. GC-MSanalyseswerecarriedoutusingaGCMS-QP5000(SHIMADZU)
gaschromatographequippedwithamassspectrometerusingelectronionization. Conditionswere
as follows: injector temperature, 270 ◦C; detector temperature, 270 ◦C; carrier gas (Helium), flow
rate1mL/min,andsplitinjectionwithsplitratio1:40. Oventemperaturewasinitiallyat60◦Cand
then raised to 290 ◦C at a rate of 3 ◦C/min. One microliter of each sample, dissolved in CH Cl
2 2
(1:100mg/µL),wasinjectedatDB-5column(i.d. =0.25mm,length30m,filmthickness=0.25µm).
Themassspectrometry(MS)conditionswere:voltage,70eV;andscanrate,1scan/s. Theidentification
ofcompoundswasperformedbycomparingtheirMSfragmentationpatternwiththosedescribedon
NISTmassspectralibrary.
3.6. ProteolyticActivity
Proteolytic activity of L. muta venom was determined according to Garcia et al. [45], using
azocaseinasasubstrate(0.2%,w/v,in20mMTris-HCl,8mMCaCl ,pH8.8). L.mutavenominduced
2
proteolysisinaconcentration-dependentmanner,andtheamountofL.mutavenom(µg/mL)able
to achieve 70%–80% of maximum rate (a variation of 0.2 at absorbance 420 nm) was denoted the
effectiveconcentration(EC),andwasconsideredas100%ofproteolyticactivity. OneECofL.muta
venom (10 µg/mL) was incubated with the plant extracts or products (90 µg/mL) for 30 min at
25 ◦C. After this, the mixture was incubated with 0.4 mL azocasein at 37 ◦C for 90 min in a total
volumeof1.2mL.Theenzymaticreactionwasstoppedbyaddingtrichloraceticacid(5%,v/v,final
concentration),andtubeswerecentrifugedat15,000×gfor3min. Thesupernatantwasthenremoved,
mixedwith2NNaOH,andthetubeswerereadatA420nm. Asapositivecontrol,L.mutavenom
wasincubatedwithsolvents(salineorDMSO)plusazocasein,whilefornegativecontrol,extracts,
products,orsolventswereincubatedwithazocaseinintheabsenceofL.mutavenom.
3.7. CoagulatingActivity
ApoolofcitratednormalhumanplasmawasobtainedfromalocalbloodbankofAntônioPedro
Hospital of UFF and diluted with an equal volume in saline. L. muta venom (50 µL) at different
concentrations(2–70µg/mL)wasaddedtoplasma,andcoagulationwasmonitoredonanAmelung
digitalcoagulometer,modelKC4A(Labcon,Germany). Theamountofvenom(µg/mL)abletoclot
plasmainaround60swascalledtheminimumcoagulationdose(MCD).Then,oneMCDofvenom
(25µg/mL)wasincubatedwithsolvents(salineorDMSO),extracts,orproductsofEovalifoliumor
E.subsessile(250µg/mL)for30minat25◦C,andthemixture(50µL)wasaddedtoplasma(200µL),
andcoagulationwasmonitored. Negativecontrolexperimentswereperformedinparallelbymixing
plantextracts,products,orsolventswithplasmaintheabsenceofvenom.
3.8. HemolyticActivity
L. muta venom-induced hemolysis was determined through the indirect hemolytic test using
humanerythrocytesandhen’seggyolkemulsionasasubstrate[46].Afterperformingadose–response
curve(2–30µg/mL),theamountofL.mutavenom(µg/mL)abletoproduce100%hemolysiswascalled
theminimumindirecthemolyticdose(MIHD).TheinhibitoryeffectoftheErythroxylumextractsor
productswasperformedbyincubatingoneMIDHofvenom(10µg/mL)withplantextractsorproducts
(90µg/mL)for30minat25◦C,andthenahemolyticassaywasperformed. Controlexperimentswere
performedbyincubatingL.mutavenomwithsolventsintheabsenceofplantextracts/productsorby
addingplantextracts,products,orsolventstothereactionmediumintheabsenceofvenom.
3.9. HemorrhagicActivity
HemorrhagiclesionsproducedbyL.mutavenomwerequantifiedusingaproceduredescribed
byKondoetal.[47],withmodifications. Briefly,L.mutavenom(100µL)wasinjectedintradermally
(i.d.) into the abdominal skin of mice. Two hours later, animals were euthanized by decapitation,