w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
article
Morpho-anatomical
study
of
Ageratum
conyzoides
Rafaela
F.
Santos,
Bárbara
M.
Nunes,
Rafaela
D.
Sá,
Luiz
A.L.
Soares,
Karina
P.
Randau
∗LaboratóriodeFarmacognosia,DepartamentodeCiênciasFarmacêuticas,UniversidadeFederaldePernambuco,Recife,PE,Brazil
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Articlehistory:
Received27April2016 Accepted27July2016 Availableonline16August2016
Keywords: Ageratumconyzoides
Anatomy Asteraceae Mentrasto Microscopy Morphology
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AgeratumconyzoidesL.,belongingtothefamilyAsteraceae,isatropicalplantfoundinsomeregionsof Africa,AsiaandSouthAmerica.Thisspeciesispopularlyknownasbillygoatweed,“mentrasto”and “catinga-de-bode”andhasalargevarietyofsecondarymetabolitesandbiologicalactivitiesmentioned intheliterature.Theobjectiveofthisworkwastocontributethepharmacobotanicalstandardization ofA.conyzoides.Cross-sectionswereobtained,byhand,formicroscopiccharacterizationofroot,stem, petioleandleafblade;totheleafbladewerestillmadeparadermalandlongitudinalsections,scanning electronmicroscopyanalysisandmaceration.Theanalysisshowedthatsecretorystructuresductsare evidencedonlyinthepetioleandtheleafblade.Theroothasparenchymatousmedullarregion;stem, petioleandleafbladeexhibitstriatedcuticle.Non-glandulartrichomesarepresentinstem,petioleand leafblade,whilecapitateglandulartrichomesarepresentonlyintheleafbladeandarerestrictedtothe abaxialface.Theseanatomicalfeaturesareusefulfordiagnosisofthespeciesandprovidesupporttotheir qualitycontrol.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Asteraceaeisavastplantfamilythatcomprisesroughly1500 generaand25,000speciesindifferenthabitats(SouzaandLorenzi, 2012). This family consists of vegetation quite distinct, which rangesfrom herbs,subshrubs,and shrubs totrees (Joly, 2002). Thisvegetationisfoundintodiversehabitatsonceithasagreat environmentaladaptation(VenableandLevin,1983).
Ageratumis one of thegenera in thefamily Asteraceae and consistsofabout30species(Okunade,2002).Amongthespecies,
AgeratumconyzoidesL., commonly knownas“billy goat weed”, “mentrasto”and“catinga-de-bode”(Cruz,1995;Asicumpon,2005; Matos, 2007), is widelyused in traditional medicine in several countries around the world as a purgative, febrifuge, anti-inflammatory, analgesic, anesthetic and in treatment of ulcers (LorenziandMatos,2002;Okunade,2002;Leitãoetal.,2014).
A.conyzoidesisatropicalplantverycommoninthewestern andeasternregionsoftheAfricancontinent,aswellasinsome regionsofAsiaandSouthAmerica(Bhattetal.,2012;Iwu,2014).It isanannualaromaticweedofcultivatedfields,however,itisalso invasiveofpastures,vacantlotsand evenofforestareas(Sousa etal.,2004;Batishetal.,2006).TheoccupationofA.conyzoidesis
∗ Correspondingauthor.
E-mail:[email protected](K.P.Randau).
easilysucceededbecauseofitswideadaptabilityinthe environ-ment,itssuperiorreproductivepotentialanditsallelopathy(Kong etal.,2004).
Thespecieshasawidevarietyofsecondarymetabolites, includ-ing mono and sesquiterpenes, triterpenes, steroids, flavonoids, coumarins,tanninsandalkaloids(Gonzálezetal.,1991a,1991b; Kasaliet al.,2002; Moreiraetal., 2007;Nouret al.,2010; Bosi et al., 2013).Someof these metabolitesare biologicallyactive, as themethoxyflavone isolatedof thehexane extract of leaves of A. conyzoides,which had insecticidalactivity (Moreira etal., 2007).Additional properties reported to the plant extractsare antibacterialactivities(Adetutuetal.,2012;Odeleyeetal.,2014), antifungal(Moraisetal.,2014),antiparasitic(Teixeiraetal.,2014), anti-inflammatory(Mouraetal.,2005),healing(Arulprakashetal., 2012)andcytotoxicityproperties(Adetutuetal.,2012).
Theessentialoiloftheleavesoraerialpartsoftheplanthas beenwidelyinvestigatedforitscompositionandbiological activ-ities.Themajorconstituentsgenerallyfoundarethechromenes, precoceneIandprecoceneII,andthesesquiterpenescaryophyllene andgermacrene-D(Okunade,2002).Themainactivitydescribed intheliteraturefortheessentialoilistheinsecticide(Limaetal., 2010;LiuandLiu,2014),butalsopresentsallelopathic(Kongetal., 2004)andantifungalactivities(Nogueiraetal.,2010;Patiletal., 2010).
Despitethewide rangeof pertinentstudiesonthechemical compositionandactivitiesofA.conyzoides,therearefewstudies http://dx.doi.org/10.1016/j.bjp.2016.07.002
Materialsandmethods
Plantmaterial
Several specimens of adult plants of Ageratum conyzoides
L., Asteraceae, were collected in the city of Camocim de São Félix,Pernambuco,Brazil.Avoucherspecimenwaspreparedand depositedintheHerbariumDárdanodeAndradeLimaofthe Insti-tutoAgronômicodePernambuco(IPA),undercollectionnumber 89312.
Morpho-anatomicalcharacterization
Severalcross-sectionsweremadeatthemiddleregionofthe root, stem, petiole and leaf blade from the fresh material, by hand,usingacommonrazorbladeandpetiolemarrowfromthe
Cecropia sp. as a supportmaterial (Oliveira and Akisue, 2009). Thecross-sectionsweresubjectedtodecolorizationwithsodium hypochloritesolution (50%)(Krausand Arduin, 1997), followed bywashingwithdistilledwaterand,lastly,stainedaccordingto thetechnique described by Bukatsch (1972) withsafranin and astrablue.Paradermalandlongitudinalsectionsoftheleafblade werealsoperformed,usingtheaforementionedprocedures,butthe paradermalsectionswerestainedwithmethyleneblue(Krauter, 1985).
Posteriorly,semipermanenthistologicalslideswereprepared containingthesectionsofbotanicalmaterial,followingcommon plantanatomyprocedures(Johansen,1940;Sass,1951).The macro-scopicdescriptionfollowedthemethodologyofOliveiraandAkisue (2009).
Scanningelectronmicroscopy(SEM)
Analyseswereperformedinsamplesoffreshleafblades.The sampleswerefixedin2.5%glutaraldehyde(buffered with0.1M sodium cacodylate). After that, the material was subjected to post-fixationusing2%osmiumtetroxidesolution(bufferedwith 0.1Msodiumcacodylate)anddehydrationinethanolseries. Subse-quently,thematerialwassubmittedtocriticalpointdrying(Bal-Tec CPD030)andmountedontoSEMstubs,usingdouble-sided adhe-sivetapeandsputter-coatedwithgold(LeicaEMSCD500)(Haddad etal.,1998).Finally,thesampleswereexaminedwithascanning electronmicroscope(Quanta200FEG)intheCentrodeTecnologias EstratégicasdoNordeste.
Maceration
Themaceration wasperformedusing fresh leafblades frag-mentsthatweredisintegratedwiththemixtureof10%nitricacid and10% chromicacid(1:1),accordingtothemethodof Jeffrey (Johansen,1940).
Analysisofhistologicalslides
Theanalysisofthesemipermanenthistologicalslidesprepared foranatomicalcharacterizationandmacerationwereconductedon imagesinsoftware(ToupViewImage),obtainedbydigitalcamera coupledtoalightmicroscope(Alltion).
orationyellowishtobrownandweaklyfixedtothesoil(Fig.1B). Thestemisgreenintheyoungplantbutitmaypresentbrown colorin olderplants. Moreover,thestem is classifiedasaerial, hascylindricalshape andiscovered bytrichomes(Fig.1E).The leavesaresimple,opposite,ovalshape,acutetip,attenuatedbase andtoothedmargin,coveredwithwhitishtrichomes.Thepetiole isstraight(Fig.1C)andhasconcave-convexcontour.Theterminal inflorescencesbearsaboutfifteenpurpleflower-head(Fig.1D).
Microscopicaspects Root
TherootofA.conyzoides,incross-section,hascylindrical con-tour,presentingsuberandsomelenticels(Fig.2A).Intherootof
Ageratumfastigiatum (Gardn.)R.M. Kinget H. Rob., Del-Vechio-Vieira et al. (2008) evidenced a desquamation process of the peridermis,withconsequenteliminationofsomecorticallayers and the epidermis. The cortical parenchyma consists of about five layersof cells, withstraight or slightly sinuous walls. It is also observed in this region the presence of inter-cellular air spaces (aerenchyma) (Fig. 2A) and cellular inclusions (Fig. 2B). Aerenchymawasalsovisualizedinthecorticalregionoftheroot of Ageratum houstonianum Mill.,but not in A. fastigiatum ( Del-Vechio-Vieiraetal.,2008;DasandMukherjee,2013),whichcan beausefulfeatureinthedifferentiationofthesespecies.Alsoin relationtothecorticalregion,inthespeciesA.houstonianumwere foundsecretoryductsandinA.fastigiatumwerefoundsecretory canals(Del-Vechio-Vieiraetal.,2008;DasandMukherjee,2013). Inthespeciesstudiedinthisworkwerenotfoundsecretory struc-turesintheroot,whichis,therefore,anotherimportantdiagnostic feature.
TheendodermisisuniseriateandhasCasparianstrips(Fig.2A andB).Thevascularsystemisformedbyxylem,whichoccupies most of the root (Fig. 2A and C), and by phloem,arranged in fewlayers,surroundingthexylem(Fig.2A).InA.fastigiatumthe secondaryphloemisdistributed ingroupsof cellsseparatedby parenchymaticrays(Del-Vechio-Vieiraetal.,2008).ThepithofA. conyzoidesiscomposedofparenchymaticcellswithstraightwalls (Fig.2C).A.houstonianumalsohasparenchymaticmedullarregion (Dasand Mukherjee,2013), differingfromA. fastigiatum,whose centralregionofrootisalloccupiedbyxylem(Del-Vechio-Vieira etal.,2008).OthermembersofAsteraceae,suchasBidenspilosa
andPlucheasagittalis,alsoexhibitparenchymaticmedullarregion (Colaresetal.,2014).
Stem
Incross-section,thestemhascylindricalcontour.The epider-misisuniseriate,coatedwithathinandstriatedlayerofcuticle (Fig.3AandB).Thepresenceofstriationsinthecuticleofstem isalsoreportedinA.fastigiatumandinothergeneraofthefamily Asteraceae,suchasBaccharisandElephantopus(BudelandDuarte, 2008;Del-Vechio-Vieiraetal.,2008;EmpinottiandDuarte,2008). Itisalsoobservedthepresenceofnon-glandulartrichomes,which aremulticellularanduniseriate(Fig.3AandC).
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Fig.1.AgeratumconyzoidesL.,Asteraceae.(A)Plantinitshabitat;(B)root(ro)offasciculatetype;(C)Singleleaves(le),abaxial(ab)andadaxial(ad)faces;(D)detailof flower-head(flo),whicharepurple;(E)Stem(ste)andleaves(le)inoppositeposition.Bars:B,C,andD=2cm;E=5cm.
H. Rob. hasboth, glandular and non-glandular trichomes( Del-Vechio-Vieira et al.,2008), as well as Acanthospermumaustrale
(Loefl.)Kuntze,Achyroclinealata(Kunth)DC.,Ayapanatriplinervis
(Vahl)R.M.King&H.Rob.,BaccharisrufescensSpreng.var.tenuifolia
(DC.)Baker,B.uncinellaDC.,CaleaunifloraLess.,Elephantopusmollis
KunthandGymnanthemumamygdalinum(Delile)Sch.Bip.exWalp. (Budeletal.,2006;Martinsetal.,2006;Mussuryetal.,2007;Budel andDuarte,2008;EmpinottiandDuarte,2008;DuarteandSilva, 2013;Souzaetal.,2013;Neryetal.,2014).
Thecorticalregionis formedbytwotofourlayers of angu-larcollenchymaandforaboutfivelayersofparenchymaticcells (Fig.3AandB).Therearecellularinclusionsinthisregion(Fig.3D). Thetypeofcollenchymacanbeusedtodistinguishthestemsof
AgeratumconyzoidesandA.fastigiatum,becauseinthelatterthe col-lenchymavariesbetweenangularandlamellar(Del-Vechio-Vieira etal.,2008).Differentfromwhatwasobservedinthecorticalregion oftherootofA.conyzoides,inthestemthereisnoaerenchymain thisregion(Fig.3A).
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Fig.3.AgeratumconyzoidesL.(Asteraceae),cross-sectionsofthestem.(A)Generalview,showingepidermis(ep)withnon-glandulartrichome(ngt),corticalregionformed bycollenchyma(co)andparenchyma(cp),endodermis(end),sclerenchymafibers(scl),phloem(ph),xylem(xy)andmedullarregioncomposedofparenchyma(pa);(B) epidermis(ep)coatedwiththinandstriatedcuticle(ct)andthecorticalregionformedbyangularcollenchyma(co)andcorticalparenchyma(cp);(C)non-glandulartrichomes (ngt);(D)cellularinclusion(in)incorticalregion;(E)starchgrains(sg)intheendodermis;(F)cellularinclusion(in)inmedullarregion.Bars:A=200m;B,D,EandF=50m; C=500m.
Starch grains are visualized in the endodermis (Fig. 3E). In thefamilyAsteraceae,theendodermisiswelldefinedanditmay presentCasparianstripsoroccurasstarchsheath(Metcalfeand Chalk,1950).InA.fastigiatumtherearesecretorycanalsnearthe endodermis, which was not evidenced in A. conyzoides and A. houstonianum(Del-Vechio-Vieiraetal.,2008;DasandMukherjee, 2013). The vascular system is collateral, consisting of several continuous bundles, distributed in a single ring. Isolated scle-renchyma fibers are located externallyto the phloem,forming caps(Fig.3A).The medullarregionconsistsofparenchyma and inclusionsarefoundinsomecells(Fig.3AandF).Thepithcells ofA.fastigiatummayhavelignification(Del-Vechio-Vieiraetal., 2008).
Petiole
Incross-section, thepetiolehasconcave-convex shape,with tworibsonadaxialface(Fig.4A).Thesameformatiscommonin otherspeciesofAgeratum(Del-Vechio-Vieiraetal.,2008;Dasand Mukherjee,2013).Theepidermisiscomposedofasinglelayerof cellsandcoveredwithathinandstriatedcuticle(Fig.4AandB).The sametypeofnon-glandulartrichomefoundonstemalsoappears inthepetiole(Fig.4A)and,furthermore,somestomataareinserted intheepidermis(Fig.4B).Theangularcollenchymaisalmost con-tinuous,encirclingtheentireparenchymaticinternalregionofthe petiole,composedbyonetotwolayersofcells(Fig.4A).However, intheadaxialribsareobservedmorelayersofthistissue(Fig.4A). InA.fastigiatumpredominatetheangularandlacunartypesof col-lenchymaanditmayappearinterruptedbylacunaryparenchyma, inwhicharelocatedlargespacesoflysigenousorigin( Del-Vechio-Vieiraetal.,2008).
Thevascularsystemisconstitutedbythreetofivedefined cen-tralbundlesarrangedinanopenarch,withribtraces(Fig.4A). Thebundlesarebicollateral(Fig.4C),whileinA.houstonianumand
A.fastigiatumthebundlesarecollateral(Del-Vechio-Vieiraetal., 2008;DasandMukherjee,2013).OtherspeciesofAsteraceaethat havebicollateralvascularbundlesinthepetioleareAspiliaafricana
(Pers)C.DAdams,Tithoniadiversifolia(Hemsl)A.GrayandVernonia amygdalinaDel.(Mabeletal.,2013).Secretoryductsarefoundclose tothevascularbundles(Fig.4C).Thesestructuresarecommonin Asteraceae(Castroetal.,1997)andalsohavebeendescribedin petiolesofA.fastigiatum(Gardn.)R.M.KingetH.Rob.,Flourensia campestrisGriseb.,F.oolepisS.F.BlakeandSiegesbeckiaorientalisL. (Aguileraetal.,2004;Delbónetal.,2007;Del-Vechio-Vieiraetal., 2008).Inthecellsoftheparenchymaandinsomecellsofthe col-lenchymainclusionscanbefound, mainlyintheregionswhere thesetissuesarejoined(Fig.4D).
Leafblade
The leaf blade of A. conyzoides, in surface view, shows epi-dermalcells withsinuous walls on both sides (Fig. 5A and B). Theleafbladeisamphistomatic,withanomocyticandanisocytic stomata,beingthefirstsmorefrequent(Fig.5Aand5B).Metcalfe and Chalk (1950)reported that for Asteraceae thestomata are usuallyanomocytic.However,datafromtheliteraturearequite differentwithrespecttothetypesofstomatapresentintheleaf bladeofA.conyzoides.Insomestudieswerefoundonlystomata anomocytic(Ferreiraetal.,2002;Unambaetal.,2008;Folorunso andAwosode,2013).Rahmanetal.(2013)foundthesametypes identifiedinthisworkandAdedejiandJewoola(2008)saidthat theplanthaslargelyanisocyticandoccasionallyanomocyticand brachyparacytic.
Non-glandulartrichomes,whicharethesametypedescribedin stemandpetiole,arevisualizedonbothfacesandcapitate glandu-lartrichomesarerestrictedtoabaxialface(Fig.5CandD).Regarding thetypesoftrichomes,itisalsoobserveddivergenceinthe liter-ature.Ferreiraetal.(2002),investigatingtheplantalsocollected inBrazil,reportedthepresenceofnon-glandularandglandular tri-chomes,but,differentfromthatfoundinthisstudy,theauthors statedthattheglandulartrichomesarelocatedonboth facesof theleafblade.InastudyoftheleafbladeanatomyofA.conyzoides
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Fig.4.AgeratumconyzoidesL.(Asteraceae),cross-sectionsofthepetiole.(A)Generalaspect,showingepidermis(ep),non-glandulartrichomes(ngt),collenchyma(co),ground parenchyma(gp)andvascularbundles(vb);(B)detailofepidermis(ep)coatedwiththinandstriatedcuticle(ct)andstomata(st);(C)detailofbicollateralvascularbundles (vb)andsecretoryduct(sd);(D)cellularinclusion(in)inparenchymaandcollenchyma.Bars:A=500m;BandD=50m;C=200m.
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Fig.6. AgeratumconyzoidesL.(Asteraceae),SEMoftheleafblade.(A)Viewoftheadaxialfaceshowingnon-glandulartrichomes(ngt);(B)epidermalcells(ep)andstriated cuticle(ct)ontheadaxialface;(C)epidermalcells(ep)andstomata(st)ontheadaxialface;(D)viewoftheabaxialfaceshowingnon-glandulartrichome(ngt);(E)capitate glandulartrichome(gt)ontheabaxialface;(F)stomata(st)andstriatedcuticle(ct)ontheabaxialface.Bars:A=500m;BandC=50m;D=400m;E=100m;F=20m.
distributionintheleafbladecorrespondedtothosedescribedin thiswork(Millanietal.,2010).
The studies conducted on the species collected in Nigeria and Bangladesh described only the presence of non-glandular
trichomes(Folorunsoand Awosode,2013;Rahman etal.,2013; Mabeletal.,2014).AccordingtoWerker(2000),althoughthe dif-ferentiationoftrichomesisgeneticallycontrolled,theirfrequency isaffectedbybioticandabioticfactors.
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Fig.8. AgeratumconyzoidesL.(Asteraceae),macerationoftheleafblade.(A)Epidermis(ep)andstomata(st);(B)vesselelementofthehelicaltype(vb);(C)non-glandular trichome(ngt);(D)capitateglandulartrichome(gt).Bars:A,BandD=50m;C=200m.
IntheleafbladeanalysisinSEMitwasalsonotedthepresence ofthenon-glandulartrichomesonbothfacesandthepresenceof thecapitateglandulartrichomesonlyonabaxialface,asitwas previouslyvisualizedbylightmicroscopy(Fig.6A,DandE).But onlyinSEMitwaspossibleobserved,withmoredetail,thatthe cuticleisstriatedonbothfaces(Fig.6BandF)andthat,onthe adax-ialface,thestomataaresituatedslightlybelowofepidermalcells (Fig.6C),whileontheabaxialfacetheyaresituatedonthesame levelorslightlyaboveofepidermalcells(Fig.6EandF).Regarding thepresenceofstriatedcuticleontheleafbladeofA.conyzoides, twootherstudiesaffirmedthatthecuticleisnon-striated(Adedeji andJewoola,2008;Mabeletal.,2014);however,itispossiblethat thisdivergenceisduetothefactthat,intheseworks,werenot per-formedtheanalysisinSEM,whichhasahigherresolutiononthe observationofmicrostructuralfeatures(Haddadetal.,1998).
Incross-section,A. conyzoidesshows amidribwithbiconvex contour(Fig.7A).A.fastigiatumexhibitsmidribrangingfrom plane-convextoslightly biconvex(Del-Vechio-Vieiraetal.,2008).The epidermisisuniseriate(Fig.7AandB)anditisalsoobserved non-glandulartrichomesthroughoutthemidrib(Fig.7B).Adjacentthe epidermisislocatedtheangularcollenchyma,composedofoneto twolayersontheabaxialfaceofthemidrib(Fig.7A)anduptofour layersofcellsinitsadaxialface(Fig.7B).
InA.fastigiatumthechlorophyllparenchymainvadestheregion ofthemidribuntilcloseofthevascularbundle,whichdoesnot happeninA.conyzoides(Del-Vechio-Vieiraetal.,2008).Inthe cen-tralregionofthemidribofA.conyzoidescanbefoundonetothree collateralvascularbundles,arrangedinanopenarch(Fig.7A).In theliterature,thereisdescriptionofonlyonevascularbundlein themidribofA.conyzoides(Millanietal.,2010;Mabeletal.,2014; EkekeandMensah,2015).Secretoryductsarefoundnexttothe bundles(Fig.7CandD),whichcorroboratesthestudyofMillani etal.(2010).However,Mabeletal.(2014)andEkekeandMensah
(2015)didnotreportthepresenceofsecretorystructuresinthe midriboftheplant.
The mesophyll is dorsiventral, with one layer of palisade parenchymaandthreetofourlayersofdensespongyparenchyma (Fig.7E).InAgeratumfastigiatumthechlorophyllparenchymais pli-cated(Del-Vechio-Vieiraetal.,2008).Cellularinclusionsarefound throughoutthemesophyll(Fig.7E).
TheleafbladeofA.conyzoides,aftermacerated,presents epi-dermal cells and stomata (Fig.8A),as well as vessel elements ofthehelicaltype(Fig.8B).Thetwo typesoftrichomesviewed in cross-section and paradermal sections are alsofound in the maceratedleafblade(Fig.8CandD),andtheycouldbeintactor fragmented,especiallythenon-glandulartrichome(Fig.8C).The macerationdataisusefulwhenitisnotpossibletoanalyzetheplant material through histological sections (Farmacopeia Brasileira, 2010).
Conclusion
Thiswork hascontributed toexpand theinformation about
Ageratum conyzoides, utilizing usual techniques of microscopic analysisofplants.Itwasshownthattheroot,stem,petioleandleaf bladeofA.conyzoidespresentanatomicalcharacteristicsthatare usefulintheidentificationanddifferentiationfromotherspeciesof thisgenusandarealsoessentialparametersforthequalitycontrol ofvegetablerawmaterial.
Authors’contributions
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorsaregratefultoCNPqforfinancialsupportinthe formoffellowshipawardsandtoFACEPEforfinancialsupportin theformofgrants.
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