w w w. s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Oleanane-type
triterpenoid:
an
anti-inflammatory
compound
of
the
roots
Arrabidaea
brachypoda
Cláudia
Q.
da
Rocha
a,
Fabiana
C.
Vilela
b,c,
Flávia
V.
Santa-Cecília
b,
Gustavo
P.
Cavalcante
b,
Wagner
Vilegas
a,
Alexandre
Giusti-Paiva
b,c,
Marcelo
H.
dos
Santos
d,∗aDepartmentodeQuímicaOrgânica,InstitutodeQuímica,UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,Araraquara,SP,Brazil bDepartamentodeCiênciasFisiológicas,InstitutodeCiênciasBiomédicas,UniversidadeFederaldeAlfenas,Alfenas,MG,Brazil
cProgramaMulticêntricodePós-graduac¸ãoemCiênciasFisiológicas,SociedadeBrasileiradeFisiologia,SãoPaulo,SP,Brazil dDepartamentodeQuímica,UniversidadeFederaldeVic¸osa,Vic¸osa,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received6June2014 Accepted4March2015 Availableonline31March2015
Keywords:
Arrabidaeabrachypoda Inflammation Naturalproduct Pain
Triterpene
a
b
s
t
r
a
c
t
ArrabidaeabrachypodaBureau,Bignoniaceae,knownas“cipó-una”,iswidelyusedintraditionalmedicine inSoutheasternandNortheasternBrazilforkidneystonesandpainfuljoints.Thisstudywasaimedat evaluatingtheanti-inflammatoryproprietiesoftheoleanane-typetriterpenoid3 -estearioxy-olean-12-eneisolatedfromtherootsofA.brachypoda.Carrageenan-inducedpawoedema,formalintestand hotplatetestwereusedtoinvestigatetheantiinflammatoryactivityof3-estearioxy-olean-12-enein animals.Weobservedthat3-estearioxy-olean-12-eneatdosesof5,10and15mg/kgp.o.demonstrated anti-inflammatoryeffects,byreduced(p<0.05)pawoedemainducedbycarrageenanandbydecreased (p<0.05)lickingtimecausedbyasubplantarinjectionofformalin.Inconclusion,3 -estearioxy-olean-12-ene,atriterpeneisolatedfromtherootsofA.brachypoda,demonstrateanti-inflammatoryeffectin differenttests.Thus,itmaybeusefulinthetreatmentofinflammatorydisorders,whichsupportsprevious claimsofitstraditionaluse.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
ThebiodiversityofBrazilianisbroadandseveralnative Brazil-ianplantspecieshavealonghistoryofuseintraditionalmedicine. Thesetraditionalmedicinalplantscontaincompoundsthatmay not only exhibit a broad range of therapeutic efficacybut also beusefulinmodernmedicine.TheBrazilianCerrado (Neotropi-calSavanna)isoneofthemostbiogeographicallydiverseregions ofthe world(deMedeiros etal., 2013; Agra et al.,2008; Blatt et al., 1998; Mendonc¸a et al., 1998).Many of these plants are commonly used in traditional medicine for the treatment of several ailments (de Medeiros et al., 2013; Blatt et al., 1998; Mendonc¸a et al., 1998), including infectious and inflammatory diseases.
∗ Correspondingauthor.
E-mail:[email protected](M.H.dosSantos).
ThegenusArrabidaeaoccursintropicalAmericafromMexico toArgentina,includingBrazilianCerrado.Speciesfromthegenus
Arrabidaeahavebeenusedintraditionalmedicineforastringent,
antioxidant, anti-inflammatory, antimicrobial, antitumor and healingpurposes(Zornetal.,2001;Bolzanietal.,2003;Leiteetal., 2006; Martin et al., 2008). The Arrabidaea brachypoda Bureau, Bignoniaceae,knownas“cipó-una”,iswidelyusedintraditional medicine in Southeastern and Northeastern Brazil for kidney stonesand painfuljoints (Alceritoet al.,2002;Rodrigues etal., 2006). Previous study reported that ethanolic extract from A.
brachypoda roots possess significant anti-inflammatory effects
in laboratory animals (da Rocha et al., 2011), supporting the traditional use of this plant in somepainful and inflammatory conditions.Inaddition,becausechemicalprofileofA.brachypoda rootsextractisestablishedinwhichflavonoidsandtriterpenesare themajorconstituents(daRochaetal.,2011,2014).
BasedonethnopharmacologicalinformationoftheArrabidaea
brachypoda, the aim of this work was to evaluate the
anti-inflammatory proprieties of 3-estearioxy-olean-12-ene (1), an oleanane-type triterpenoid, isolated from A. brachypoda roots ethanolicextract.
http://dx.doi.org/10.1016/j.bjp.2015.03.005
1
Materialandmethods
Plantmaterial
Arrabidaea brachypodaBureau,Bignoniaceae,roots were
col-lectedinSant’AnadaSerra-JoãoPinheiro,MinasGerais,Brazil.Dra. AnaMariaCristinaTeixeiraBraga,DepartmentofBotanicof Fed-eralUniversityofOuroPreto,identifiedtheplant,andavoucher specimenwasdepositedattheHerbariumofFederalUniversityof OuroPreto(vouchernumber17935).
Preparationofplantextract,isolationofcompoundandreference
drugs
Toobtaintheextract,therootsweredried,powdered(685g)and extractedwithethanolinSoxhletequipmentfor24h.Thesolvent wasremovedunderreducedpressureandthendriedwithaspray dryer(BüchiMiniSprayDryerB-290).TheyieldoftheA.brachypoda ethanolic extract (AbE)was 11%. To isolatethe bioactive com-pound,AbEwaschromatographedonasilicagel(230–400mesh) column (8cm×100cm) and eluted withcrescentpolarity mix-turesofn-hexane/ethyl-acetateandethyl-acetate/ethanoltogive 39fractions.Thesefractionswerepooledintofourgroupsaccording to their similarities after the analysis using thin layer chro-matography (TLC). Fractions 3–8 were rechromatographed on a silica gel (230–400mesh)column (8cm×100cm) and eluted with crescent polarity mixtures of n-hexane/ethyl-acetate and ethyl-acetate/ethanoltopurifythetriterpene3 -stearyloxy-olean-12-ene (1). Its structure was determined using spectroscopic techniques(Table1).Thedatawerecomparedtothoseverified in apreviousstudy investigatingthechemical structure of this compound(Vieira-Filhoetal.,2003).
3-Estearioxy-olean-12-ene(1,AbE-01) wasadministeredin 5,10, and15mg/kgdosesafter beingsuspendedin vehicle(1% sodium carboxymethylcellulose suspension in distilled water). Indomethacin (10mg/kg, p.o.) and morphine (10mg/kg, i.p.) in vehicle(saline)wereusedasreferencedrugs.Testdrugswereorally administeredinanequivalentvolumeof10ml/kgbodyweightof theanimal.
Pharmacologicalactivities
Animals
Adultmale Wistar ratsweighing180–220g(n=8)andadult maleSwissmiceweighing28–32g(n=10)animals,obtainedfrom theCentralAnimal Facilityof theFederalUniversity ofAlfenas, werehousedundercontrolledlight(12:12hlight–darkcycle;lights onat6am)andtemperatureconditions(23±1◦C)withaccessto waterandfoodadlibitum.Theanimalswereallowedtohabituate tothe housingfacilities for at least1 week beforethe experi-mentsbegan.Allexperimentswerecarriedoutbetween8amand 2pminaquietroomandtestswereperformedbythesamevisual observerinadouble-blindstudy.Theexperimentswereconducted
inaccordancewiththeDeclarationofHelsinkionthewelfareof experimentalanimalsandwiththeapprovaloftheEthics Commit-teeoftheFederalUniversityofAlfenas(109/2009).
Carrageenan-inducedratpawoedema
Pedal inflammationin ratwas producedas described previ-ously(Vinegar etal., 1969;daRochaetal.,2011), followingan eighto’clockfastwithfreeaccesstowater.Pawoedemawas mea-suredwithaplethysmometer(Model7140,UgoBasile,Italy).The basal volumeoftherighthindpawwasdeterminedbeforethe administrationofanydrug.Afterdeterminationofthebasal vol-ume,theanimalsweredividedintotheexperimentalgroupsin suchawaythatthemeanvolumesofthedifferentgroupswere similar.Vehicle(10ml/kg),3-estearioxy-olean-12-ene(dosesof 5,10,and15mg/kg)orindomethacin(10mg/kg)wasorally admin-istered1hbeforethei.pl.injectionofcarrageenan(1mg,100l). Thepawvolumewasmeasured1,2,3and4hafterinjectionof theinflammatorystimulus.Theresultsarepresentedasthepaw volume(ml)variationinrelationtothebasalvalues.
Formalintest
Aformalinsolution(5%in0.9%saline;20l/paw)wasinjected intothehindpawplantarsurface(i.pl.),andthemicewere indi-viduallyplacedintransparentobservationchambers,aspreviously described (Santos and Calixto, 1997; Vilela et al., 2010). Oral treatments (p.o.) with vehicle, indomethacin or 3 -estearioxy-olean-12-eneweregiven1hpriortoformalininjection.Morphine wasadministrated(i.p.)30minbeforethetest.Thetimespentin lickingtheinjectedpawwasrecordedandexpressedasthetotal lickingtimeintheearlyphase(0–5min)andlatephase(20–30min) afterformalininjection.
Hotplatetest
Thehotplatewasanelectricallyheatedsurfacekeptata con-stant temperature of 50.0±0.5◦C. Mice (n=8 per group) were placed ontheheated surfacewithinthePlexiglas wallsto con-straintheirlocomotionontheplate,andthelatencytoadiscomfort reaction (licking of thepaws or jumping) was recorded 0, 30, 60, and 120min after3-estearioxy-olean-12-ene or morphine (10mg/kg),whereuponthereactiontimeof0ministhestartof thetest.Acut-offtime of20swaschosentoindicatecomplete analgesiaandtoavoidtissueinjury.Thelatenciesforpawlicking orjumpingwererecordedforeachanimal(Yamamotoetal.,2002).
Statisticalanalysis
The data obtained were analysed using GraphPad software programmeversion6.0 andexpressedasthemean±S.E.M. Sta-tisticallysignificantdifferences betweengroupswerecalculated bytheapplicationofananalysisofvariance(ANOVA)followedby theTukeytest.p-Valueslessthan0.05(p<0.05)wereconsidered significant.
Results
Fig.1showsthatcarrageenaninducedratpawoedemawith compared with the vehicle administration. The 3 -stearyloxy-olean-12-enesignificantlyinhibitedcarrageenan-inducedratpaw oedema(F4,20=30.2;p<0.001).Theinhibitoryvalues ofoedema
Table1
NMRspectraldataof3-(stearyloxy)olean-12-ene(AbE-01).
Carbonnumber AbE-01(␦C) AbE-01(␦H) HMBC Carbonnumber AbE-01(␦C) AbE-01(␦H) HMBC
1 37.74 1.13and1.61 1′
173.55 –
2 22.69 1.64 3 2′ 34.87 2.30 1′,3′,4′
3 80.51 4.52 1′,1,2,4,23,24 3′ 25.17 1.63
4 36.83 – 4′ 29.26 1.29
5 55.26 0.80 5′ 29.27 1.29
6 18.35 1.43and1.56 6′ 29.48 1.26
7 32.47 1.36and1.52 7′ 29.59 1.26
8 41.51 – 8′ 29.65 1.26
9 47.55 1.60 9′ 29.68 1.28
10 36.94 – 10′ 29.72 1.28
11 23.61 1.86 12 11′ 29.72 1.26
12 121.65 5.19 9,11,14,18 12′ 29.93 1.26
13 145.16 – 13′
29.72 1.26
14 41.69 – 14′
29.59 1.26
15 26.24 0.96 15′
29.48 1.26
16 27.04 2.20 28 16′
31.93 1.26
17 32.54 – 17′
22.69 1.26
18 47.36 1.94 12 18′
14.12 0.88 17′,16′
19 46.90 1.03and1.76
20 31.10 –
21 34.83 1.10and1.39
22 37.24 1.22and1.46
23 28.13 0.88 3,4,5,24
24 16.82 0.88 3,4,5,23
25 15.57 0.96 1,5,9,10
26 16.90 0.97 7,8,9,14
27 26.00 1.14 8,13,14,15
28 28.43 0.84 16,17,18,22
29 33.35 0.88 19,20,21,30
30 23.75 0.88 19,20,21,29
Theintrapodal administration formalin induced an nocicep-tivebehaviourof lickingthepawduring thetest (Fig.2B).The 3-stearyloxy-olean-12-ene at doses of 5–15mg/kg p.o. had a significant activity compared to the control in both the early (F5,60=34.3; p<0.001; Fig. 2A) and late phases (F5,60=25.4;
p<0.001, Fig. 2B) of the formalin test. The reference drug,
Time (h)
*
***
***
***
***
***
***
***
**
**
Edema v
olume (ml)
0 0.0 0.2 0.4 0.6 0.8 1.0
Vehicle (10 ml/kg)
AbE-01 (5 mg/kg) AbE-01 (10 mg/kg)
AbE-01 (15 mg/kg) Indomethacin (10 mg/kg)
4 3
2 1
Fig.1.Effectsoftheadministrationofthe3--stearyloxy-olean-12-ene(AbE-01; 5,10and 15mg/kg,p.o.)orindomethacin(5mg/kg, p.o.)onratpawoedema inducedbyintraplantarcarrageenaninjection(1mg/paw).Eachpointrepresents themean±S.E.M.Theasterisksdenotethesignificancelevelswhencomparedwith thecontrolgroup:*p<0.05,**p<<0.01,***p<0.001.
indomethacin,suppressedonlythelatephaseoftheformalintest, whilemorphineinhibitedbothphasesofthepainfulstimulus.
In the hot plate test, treatment with 3 -stearyloxy-olean-12-ene at doses of 5–15mg/kg increased the latency time as compared tothecontrolgroup at30 (F4,39=6.60; p<0.001), 60
(F4,39=7.36; p<0.001) and 120min (F4,39=11.22; p<0.001). As
expected,administrationofthevehicledidnotinduceany antinoci-ceptiveeffect.Morphinesignificantlyincreasedthelatencytime (Fig.3).
Discussion
Traditional medicine for the treatment of various diseases is becoming more popular. Therefore, the present study was aimedatevaluatingthescientificbasisforthetraditionaluseof
A. brachypodaBureau, Bignoniaceae,usinginvivo inflammatory
models. The results of this investigation demonstrate that the triterpene3-estearioxy-olean-12-enefromA.brachypodaexerts anti-inflammatoryeffectsinanimals.
Forthescreeningofcompoundspossessinganti-inflammatory activity, carrageenan-induced paw oedema is a model widely employed and has frequently been used to assess the anti-oedematogeniceffectofnatural products(Mendeset al.,2010). Thedevelopmentofoedemainducedbycarrageenanisabiphasic event.Theearlyphase(1–2h)ismainlymediatedbyhistamine, serotoninandbradykinin.Thelatephaseissustainedbytherelease ofprostaglandinsandnitricoxide,withapeakat3h,andis pro-ducedbyinducibleisoformsofcyclooxygenase(COX-2)andnitric oxidesynthase(iNOS)(BritoandAntonio,1998;Seibertetal.,1994). Previousoraltreatmentwiththis3-stearyloxy-olean-12-enewas effectiveinreducingtheoedematogenicresponseevokedby car-rageenaninlatephase.Thisreductionmaybecausedbyinhibition ofoneormoreintracellularsignallingpathwaysinvolvedin medi-atingtheinflammatoryresponse(Thomazzietal.,2010).
Vehicle 5 10 15 MORP
AbE-01 (mg/kg)
Time lic
king (s)
First phase 60
A
B
40
20
0
Time lic
king (s)
60
40
20
0
Seconde phase
***
**
*
*
***
***
**
**
**
INDO
Vehicle 5 10 15 MORP
AbE-01 (mg/kg)
INDO
Fig.2.Effectsof3--stearyloxy-olean-12-ene(AbE-01)givenbyoralrouteonthe lickinginducedbyformalininmice.Animalswerepretreatedorallywithvehicle, AbE-01(doses5,10,and15mg/kg),indomethacin(INDO;5mg/kg)ormorphine (MORP;10mg/kg)priortoformalin.Thetotaltimespentlickingthehindpaw wasmeasuredinthefirst(PanelA)andsecond(PanelB)phasesafterintraplantar injectionofformalin.Eachpointrepresentsthemean±S.E.M.Theasterisksdenote thesignificancelevelswhencomparedwiththecontrolgroup:*p<0.05,**p<0.01, ***p<0.001.
Vehicle AbE-01 (5 mg/kg) AbE-01 (10 mg/kg) AbE-01 (15 mg/kg) Morphine
Time (min) *
* *
**
** **
*** ***
***
**
* **
Latence (s)
0 0 5 10 15
30 60 120
Fig.3. Effectsof3--stearyloxy-olean-12-ene(AbE-01)administeredorallyinthe hotplatetest. Animalswere pretreatedorallywith vehicle,morphine(Morp; 10mg/kg),AbE-01(doses5,10,and15mg/kg),priortothetestsat50◦C.Each
pointrepresentsthemean±S.E.M.Theasterisksdenotethesignificancelevelswhen comparedwiththecontrolgroup:*p<0.05,**p<0.01,***p<0.001.
formalin injection), seems to be caused by the direct effect of formalinonsensoryC-fibres.Thesecondphase,orinflammatory phase (starting approximately20minafter injection),is associ-atedwiththedevelopmentofaninflammatoryresponseandthe releaseofnociceptivemediators(Abbottetal.,1995;Davidsonand Carlton,1998)Areductionofthelatephasebehaviouralresponse toani.p.formalininjectionwasobserved,demonstratingthe anti-inflammatory activity produced by3-stearyloxy-olean-12-ene. Theresultsobtainedfromcarrageenan-inducedratpawoedema also confirmed this effect. Similarto other substances that act onthecentralnervoussystem(CNS),3-stearyloxy-olean-12-ene inhibitedbothphasesoftheformalintestinamannersimilarto thatofmorphine.Moreover,theresultsofthistestarein agree-mentwiththoseobtainedfromthehotplatetest,confirmingthe centralantinociceptiveeffectofthecompound.
Phytochemical analysesofthe rootsofA. brachypodaextract indicatedthatthelargemajorityofitsconstituentsweretriterpenes (daRochaetal.,2011).Inthisregard,theanti-inflammatory activ-ityisacommonpropertyofmanytriterpenoids(SafayhiandSailer, 1997).Theanti-inflammatoryeffectsoftriterpenes(i.e.oleanolic and ursolicacids) have beenattributedto various mechanisms including:inhibitionoflipoxygenaseand cyclooxygenase activi-ties,inhibitionofelastaseandinhibitionofcomplementactivity, possiblythroughtheinhibitiononC3-convertaseoftheclassical complement pathway(Singhet al.,1992).In addition,previous worksshownthatthebasiccarbonskeletonhasnoinfluenceon theanti-inflammatoryactivity,andthepresenceofaC-28or C-30carboxylicgroupandanalcoholicgroupatC-28increasesthe activityincarrageenan-inducesoedema(Recioetal.,1995), how-ever,themechanismofactionofthe3-stearyloxy-olean-12-ene isunknown.
In the present study, we demonstrated the efficacy of 3 -estearioxy-olean-12-ene,atriterpeneisolatedfromtherootsofA.
brachypoda,indifferentanti-inflammatorytests.Thus,itmaybe
usefulinthetreatmentofinflammatorydisorders,whichsupports previousclaimsofitstraditionaluse.
Authorcontributions
CQR, FCV,FVS-Cand GPC participated in studyconcept and design, acquisition of data, analysisand interpretationof data, draftingofthemanuscript,criticalrevisionofthemanuscriptfor importantintellectualcontent.WV,AG-PandMHSparticipatedin studyconceptanddesign,acquisitionofdata,analysisand inter-pretationofdata,draftingofthemanuscript,criticalrevisionof themanuscriptforimportantintellectualcontent,administrative, technical,ormaterialsupport,andstudysupervision.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
The authors acknowledge financial support from FAPESP, FAPEMIGandCAPES.
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