Rev. bras. farmacogn. vol.27 número3

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Botanical

and

genetic

characters

of

Erythrina

_×

neillii

cultivated

in

Egypt

Salma

K. Gabr

a

_,

_Riham

_O.

_Bakr

a,∗

,

Hisham

M. Elshishtawy

b

,

Ahlam

M. El-Fishawy

c

,

Taha

S. El-Alfy

c

a_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_October_University_for_Modern_Sciences_and_Arts,_Giza,_Egypt b_Microbial_Molecular_Biology_Department,_Agricultural_Genetic_Engineering_Research_Institute,_Giza,_Egypt c_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_Cairo_University,_Cairo,_Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30October2016 Accepted6February2017 Availableonline28March2017

Keywords:

Botanicalstudy DNAfingerprint Twoarmedhair

a

b

s

t

r

a

c

t

Erythrina×neilliiMabberley&Lorence,Fabaceae,isasterilehybridbetweenE.herbaceaL.andE.humeana

Spreng.Nothingwastracedaboutitsgenetic,macroandmicromorphology.Therefore,itwasdeemedof interesttostudyitsbotanicalcharacters,inadditiontotheDNAfingerprinttohelpintheidentificationof theplant.Theanatomicalcharactersoftheoldstemanditsbarkarecharacterizedbythepresenceofcork cells,bastfibersandsclereids.Pericycleissclerenchymatousformingcrystalsheath.Theepidermisesof theleafandyoungstemarecharacterizedbythepresenceofanomocyticandparacyticstomata, non-glandular,unicellularandmulticellulartwoarmedhairs,andglandularclubshapedhair.Calciumoxalate ispresentintheformofcrystalsheathandprisms.Secretorycavitiesaredistributedinthephloem andcortex.Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE)wasusedasoneof themolecularmethodstodifferentiatebetweenthesamplesofErythrina.TheDNAofErythrinawas extractedandanalyzedusingseven-merrandomprimers.RandomlyAmplifiedPolymorphicDNAwere recognized.ThischaracterizationallowscertificationoftheauthenticityofErythrina×neillii,inorderto

providequalitycontrolfortheplant.

Introduction

Erythrinagenus,belongingtofamilyFabaceae,comprisesabout 200 species, which are distributed in tropical and subtropical regionsworldwide.Theyaretrees,growingupto30m(98ft.)in height.Thegenericname isderived fromtheGreekword( ery-thros),meaning“red,”referringtotheflowercolorofcertainspecies (Gledhill, 2008). Erythrina has been used in folk medicine for treatmentof insomnia, malaria, venereal diseases, asthma, and toothache(deAraújo-Júnioretal.,2012).Plantsbelongingtothe

Erythrina genus are sources of tetracyclic alkaloids, flavonoids, especiallyisoflavones,coumarinsandsaponins(Soto-Hernández etal.,2012).

Erythrina×neilliiMabberley&Lorenceisahybridderivedfroma crossbetweenE.herbaceaL.andE.humeanaSpreng.attheNational TropicalBotanicalGarden.TheEnglishnameisNeill’scoraltree whichisnamedafteritssynthesizer,DavidNeill,associatecurator atMissouribotanicalGarden.Thissterilehybridproducesattractive brightredflowersbutdoesnotproducefruitsorseeds(Neill,1984).

∗ Correspondingauthor.

E-mail:[email protected](R.O.Bakr).

Regardingthemacro,micromorphologyandgeneticcharacters ofErythrina×neillinothingwastraced,therefore,itwasdeemedof interesttostudyitsbotanicalandgeneticcharacterstoestablish criteriathatwouldbehelpfulforitsdiscriminationfromother Ery-thrinaspeciesandensuringsafetyforcommercialpharmacological uses.

Materialsandmethods

Plantmaterial

Leaves,stem,andbarkofErythrina×neilliiMabberley&Lorence, Fabaceae,werecollectedduringthefloweringperiodinOctober 2012fromEl-Zohriagarden,Cairo.ItwaskindlyidentifiedbyDr. GwilymP.Lewis,LegumeResearchLeader,ComparativePlant& Fungal Biology. Royal BotanicGardens, Kew, UK and approved by Prof. Dr. David Neill, director of the Unit of Conservation and WildlifeManagementof theAmazonStateUniversity,Kew RoyalBotanicGardens.Thevoucherspecimenwasdepositedin theHerbariumoftheFacultyofPharmacy,MSAUniversityunder registration number RS020 and duplicates housed in the Kew Herbarium(K).

http://dx.doi.org/10.1016/j.bjp.2017.02.005

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ExtractionandpurificationofgenomicDNA

A half-gramof younggreen leavesof E× neillii, E.indica,E.

speciosa, E.variegata and E.japonica wascollected and quickly frozeninliquidnitrogenandthengroundusingmortarandpestle. AmodifiedCTAB(hexadecyltrimethylammoniumbromide) proce-durebasedontheprotocolofPorebskietal.(1997)wasadopted forobtainingagoodqualityoftotalDNA.

RandomlyAmplifiedPolymorphicDNA(RAPD)

RAPD-PCRreactions

Asetofseven-merrandomprimerswasusedinthedetection ofpolymorphismamongthefiveplantaccessions,havingthe fol-lowingsequences:OPA-11:TCTGTGCTGG,OPA-10:GAAACGGGTG,

OPB-06:CATCCCCCTG, OPB-07: TGCTCTGCCC,OPD-14:

TTCCCC-CGCT,OPD-15:TTTGCCCGGA,OPC-01:TTCGAGCCAG.RAPDwas

carriedoutaccording totheproceduregiven byWilliamset al. (1990).

ThermocyclingprofileanddetectionofthePCRproducts

PCRamplificationwasperformedinaPerkin-Elmer/GeneAmp® PCRSystem9700(PEAppliedBiosystems)programmedtofulfill 40cyclesafteraninitialdenaturationcyclefor5minat94◦_C._Each

cycleconsistedofadenaturationstepat94◦_C_for₁_min,_an

anneal-ingstep at 36◦_C _for ₁_min, _and _an_elongation _step_at ₇₂◦_C _for

1.5min.Theprimerextensionsegmentwasextendedto7minat 72◦_C_in_the_final_cycle._The_{amplification}_products_were_resolved

byelectrophoresisona1.5%agarosegelcontainingethidium bro-mide(0.5␮g/ml)in1×TBEbufferat95volts.PCRproductswere

visualizedunderUVlightandphotographedusingaPolaroid cam-era.RAPD-PCR-amplifiedfragmentswerescoredaspresent(1)or absent(0).Onlyclearandmajorbandswerescored(Collardand Mackill,2009).

Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)analysis

Totalcellularproteinswereextractedbyresuspendingtheplant extractinonevolumeofsampleTris-EDTAbufferandincubatingfor 5minat100◦_C_and_then_for₂_min_on_ice._Cell_debris_were_removed

bycentrifugationatmaximumspeed (22,640×g)for2min.The supernatantcontainingtotalcellularproteinwasloadedonto12% (w/v)SDS-PAGEgelsandwasrunat45mAfor1h,thenfixedand stainedwithCoomassiebrilliantblue(Laemmli,1970).

Electrophoresisofproteingel

Thegel apparatus was assembled and the lower and upper chamberswerefilledwiththetankbuffer.Adropof bromophe-nolblue wasadded asa tracking dye.AHamilton syringewas usedtoloadanequalvolumeofcellularprotein(10␮l)ineach

well.Pre-stainedmolecularweightproteinmarkerfromBio-Rad, wasappliedtothegel.Electrophoresiswascarriedoutatabout

Thegelwasplacedbetweentwosheetsofcellophanemembrane anddriedongeldrierfor2handthenphotographed(Laemmli, 1970).

Results

Macroscopicfeatures

Erythrina×neilliiisanupto3mheighttree,havingathickerect trunkwithmonopodialbranchingandcarryingterminalpanicle redinflorescence(Fig.1A).The oldstem iswoody,dark brown incolorwithwrinkledbarkthat breakswithshortfibrous frac-ture.Theoutersurfaceofthebarkisdarkbrownincolor,showing lenticels,longitudinalwrinklesandcrackedatirregularintervals, whiletheinnersurfaceissmoothandlightbrownincolor(Fig.1B). Theyoungstemsand branchesare yellowish green,pubescent, withstoutconicalspines(Fig.1C).Thepetioleismoreorless cylin-drical,yellowishgreenincolorandpubescentshowingaswollen pulvinusatitsconnectionwiththestem.Theleavesarealternate, compoundandtrifoliate.Thelowerpairleafletsaresmallerthan theapicalone(Fig.1DandE).Theleafletsareovaterhomboidal withanentiremarginandasymmetricbase.Thelaminahasan acuteapex,reticulatepinnatevenation,andaveryshortpetiolule withlarge,cup-shapedgland.Theleafletshavesmoothandshiny surfacesandcoriaceoustexture(Fig.1FandG).

Microscopicfeatures

Theyoungstem

Incross-sectionalview(Fig.2A),theyoungstemisnearly circu-larinoutline.Itisformedofanouterepidermisfollowedbynarrow cortex.Pericycleisparenchymatousinterruptedbygroupsoffibers. Thevasculartissueisformedofacompleteringofcollateral vas-cularbundlesfollowedbyawidepith.

Theepidermis(Fig.2BandC)ispolygonalandslightly elon-gated cells with almost straight, beaded anticlinal walls, and coveredwiththicksmoothcuticleshowinganomocyticand para-cyticstomata.Twotypesofhairs:glandularclub-shapedwitha sphericalmulticellularheadandshortunicellularstalkand nong-landular,unicellulartomulticellularwithequal orunequal two arms.

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pul.

pet.

sp.

gl.

pet.

lent.

a

b

A

B

E

G

C

D

F

Fig.1.Erythrina×neilliiMabberley&Lorence,Fabaceae(A–G).(A)Photographoftheplant(X=0.03).(B)Bark(X=0.75)a.Outersurface,b.Innersurface.(C)Branchshowing spinesandpulvinus(X=1.1).(D)Branchshowingalternatecompoundleaf(X=0.12).(E)Imparipinnateleaf(X=0.31).(F)Adaxialsurfaceoftheleaflet(X=0.42).(G)Abaxial surfaceoftheleaflet(X=0.42).gl.,gland;lent.,lenticel;pet.,petiolule;pul.,pulvinus;sp.,spines.

consistsof thin cellulosic walled cells traversed bybiseriate to multiseriatemedullaryrays.Medullaryraysareformedof rectan-gularcellswithpittedwallswhicharethin cellulosicinphloem andthicklignifiedinxylem.Thecambium(Fig.2B)isformedof 3–4linesoftangentiallyelongated,cellulosicthinwalledcells.The xylem(Fig.2C)consistsoflignifiedspiral,pittedandreticulate ves-sels.Thewoodfibersarefusiformwithawidelumenandacuteto roundapices.Woodparenchymaconsistsofrectangularelongated cellswithpittedlignifiedwalls.

Thepithconsistsofroundtopolygonalparenchymatouscells having thin cellulosic walls and showing prisms of calcium oxalateandstarchgranules.Secretorycellsorsacswithor with-out tanniferouscontent are present in thecortex, phloem and pith.

Thepowderedstem(Fig.2C)isyellowishgreenincolor, odor-lessandwithslightlybittertaste.Itischaracterizedby:fragments

of epidermal cells showing paracytic and anomocytic stomata, glandularclubshapedhairandnon-glandularunicellularto multi-cellulartwo-armedhair,fragmentsofpericyclicfibershavingthick lignifiedwalls,narrowlumenandtaperingacuteapicesforming crystal sheath, scattered prismsof calcium oxalate,amorphous contentsandstarchgranuleseitherfreeorinparenchymacells. Fragments oflignified, spiral,pittedand annular xylemvessels, lignifiedwoodfiberswithwide lumenandacutetoroundapex andfragmentsofwoodparenchymaandmedullaryraysarealso observed.

Theoldstem

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200 µm

25 µ 25

µ 25

µ

25 µ

25

µ

25

µ

25

µ

25

µ

25

µ

ep.

A

v.

B

C

m.r.

w. par. cont.

am.

_par.

t.c.

w. f.

cr. sh.

g.h.

pr. ca.

st.

cx.

p.f.

t.c.

ph.

st.

cam.

m.r.

v.

w.

pi.

st.

w.f.

pr.ca.

end.

Fig.2.YoungstemofErythrina×neilliiMabberley&Lorence,Fabaceae(A–C).(A)LowpowerviewoftheYoungstem(X=20).(B)Highpowerviewoftheyoungstem (X=100).(C)Powderedyoungstem.am.cont.,amorphouscontent(X=240);a.s.,anomocyticstomata(X=400);cam.,cambium;cr.sh.,crystalshealth(X=200);cx.,cortex; end.,endodermis;ep.,epidermis(X=400);g.h.,glandularhair(X=480);n.g.h.,non-glandularhair(X=260);m.r.,medullaryrays(X=320);p.f.,pericyclicfibers(X=240);p.s., paracyticstomata;par.,parenchyma;ph.,phloem;pi.,pith;pr.ca.,prismofcalciumoxalate(X=320);st.,starch(X=240);t.c.,tannincell(X=120);v.,xylemvessel(X=160); w.f.,woodfiber(X=240);w.par.,woodparenchyma(X=240).

parenchyma (Fig. 3Band C). The pericycleshows the presence ofgroups ofpericyclicfibersforminga crystal sheath.The vas-culartissue is widercompared withthe youngstem whilethe pithisnarrower.Secretorycells,calciumoxalateprismsandstarch granulesaredistributedinthecortexandphloemtissue.Groups ofsclereidsarescatteredinthepericycleandphloem.The scle-reidsareovaltoisodiametricwiththicklignifiedwallsandnarrow lumen.

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ck.

500 µ

200 µm

25 µ

25

µ

25

µ

25 µ

25

µ

25 µ

p.f

ph.

cam.

m.r.

w.f.

v.

w.par.

pi.

v.

pi.

ck.

cx.

t.c.

p.f.

ck.

scl.

B

D

C

A

v.

ph.f.

w.f.

t.c.

cr.sh.

pr.ca.

st.

pr.ca.

Fig.3.OldstemofErythrina×neilliiMabberley&Lorence,Fabaceae(A–D).(A)Lowpowerviewoftheoldstem(X=10).(B)Highpowerviewoftheoldstem(X=65).(C) Highpowerviewoftheoldsteminthecortexregion(X=160).(D)Powderedoldstem.cam.,cambium;ck.,cork(X=200);cr.sh.,crystalshealth(X=200);cx.,cortex;m.r., medullaryray;p.f.,pericyclicfibers;ph.,phloem;ph.f.,phloemfiber(X=280);pi.,pith;pr.ca.,prismofcalciumoxalate(X=400);scl.,sclereids(X=200);st.,starch(X=280); t.c.,tannincell(X=120);v.,xylemvessel(X=200);w.f.,woodfiber(X=280);w.p.,woodparenchyma.

fibershavethicklignifiedwalls,narrowlumenandtaperingacute apices

Thestembark

Thetransversesection(Fig.4A)ofthestembarkshowsthecork arrangedinradialandtangentiallayersfollowedbyphelloderm andprimarycortexshowingsecretorycavitiesfilledwithbrown

contents.Pericycleisformedofparenchymatouscellsinterrupted bygroupsoffibersandaccompaniedbysclereids.Thephloemis traversedbymedullaryrays,tangentialbandsofbastfibersand groupsofsclereids.

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200 μm

t.c.

ph.f.

m.r.

A

C

B

D

E

t.c.

scl.

t.c.

pr. ca.

ph.f

pr.ca.

25

μ

25 μ

25

μ

25 μ

25

μ

25

μ

cr.sh.

ph.

ph.f.

ek.

m.r.

p.f.

Fig.4.StembarkofErythrina×neilliiMabberley&Lorence,Fabaceae(A–E).(A)Cross-sectionalviewofthestembark(X=50).(B)–(D)Highpowerviewofthestembark (X=200–200–70).E.Powderedstembark.ck.,cork(X=200);cr.sh.,crystalshealth(X=200);cx.,cortex;lent.,lenticels;m.r.,medullaryray;p.f.,pericyclicfibers;ph.,phloem; ph.f.,phloemfibers(X=400);pr.ca.,prismofcalciumoxalate(X=240);scl.,sclereids(X=360);t.c.,tannincells(X=200).

cells, followed by rounded parenchyma cells of primary cor-tex.Thepericycle(Fig.4BandC)isparenchymatousinterrupted by groups of lignified pericyclic fibers and sclereids. The scle-reids are oval to isodiametric with thick lignified walls and narrow lumen.The phloem consists of primary and secondary phloem traversed by bi-multiseriate medullary rays and tan-gential groups of lignified phloem fibers. The medullary rays (Fig.4D)areformedofrectangular,radiallyelongatedandrounded parenchymatouscells.Tannincells,starchgranulesandprismsof calciumoxalatearescatteredinthetissuesofcortex,pericycleand phloem.

Thepowderedstembark(Fig.4E)hasabitterastringenttaste, browncolorandcharacterizedby:fragmentsofcorkcells,phloem fibers,crystalsheath,sclereids,andsecretorytannincells,prisms ofcalciumoxalateandstarchgranuleseitherfreeorin parenchy-matouscells.

Theleaflet

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u.ep.

col.

end.

v. ph.

u.ep.

u.pal.

sp.t. t.c.

m.r. l.pal

p.f.

ep. v.

pi.

cic.

pr.ca.

n.ep. cic. g.h.

am. cont.

I.ep.

n.g.h

E

cr.sh

w.f.

25

μ

25 μ 25 μ

25 μ

200 μ

50 μ 100 μ

50 μm

25 μ

25

μ

25 μ

v. st.

pal.

g.h.

s. U.ep.

D

C

A

B

l.ep.

Fig.5.LeafofErythrina×neilliiMabberley&Lorence,Fabaceae(A–E).(A)Lowpowerviewofthelamina(X=70).(B)Highpowerviewofthemidrib(X=100).(C)Highpower viewofthelamina(X=140).(D)Lowpowerviewofthepetiole(X=25).(E)Powderedleaf.am.cont.,amorphouscontent;cic.,cicatrix(X=160);col.,collenchyma;cr.sh., crystalshealth(X=160);end.,endodermis;g.h.,glandularhair(X=400);l.ep.,lowerepidermis(X=160);l.pal.,lowerpalisades;m.r.,medullaryrays;n.ep.,neuralepidermis (X=233);n.g.h.,non-glandularhair(X=160);pal.,palisades(X=160);p.f.,pericyclicfibers;ph.,phloem;pi.,pith;pr.ca.,prismofcalciumoxalate;s.,stomata;sp.t.,spongy tissue;st.,starch(X=320);t.c.,tannincells;u.ep.,upperepidermis(X=120);u.pal.,upperpalisade;v.,xylemvessel(X=200);w.f.,woodfiber(X=280).

Table1

TotalnumberofampliconsasrevealedbyRAPDmarkersamongthefiveaccessions.

PRIMER E×neilli E.indica E.speciosa E.variegata E.japonica

OPA-11 4 4 3 3 2

OPA-10 3 4 3 5 7

OPB-06 4 2 3 3 3

OPB-07 4 3 3 3 3

OPD-15 5 2 3 3 3

OPD-14 2 2 6 3 3

OPC-01 3 2 3 3 3

inverted one. Thepericycle is sclerenchymatousand the

endo-dermisisdistinct.Theupperandlowerepidermises(Fig.5Band

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M

S1

S2

S3

S4

S5

S4

S3

S2

S1

M

S4

S3

S2

S1

M

S1

S2

S3

S4

S5

_S5

_S4

_S3

_S2

_S1

S5

D

_E

Fig.6.(A–E)RAPDgelcomparingdifferentErythrina.(A)RAPDgelOPA-10.(B)OPB-07.(C)OPD-14.(D)OPB-6.(E)OPD-15;M,Marker;S1,Erythrina×neilli;S2,E.indica;S3,

E.speciosa;S4,E.variegataandS5,E.japonica.

with wide intercellular spaces, containing scattered prismatic crystalsof calcium oxalate, tannin cells and few simple starch granules.Thecorticaltissueofthemidribismoreprominenton thelowersurface.Itconsistsoftwolayersofcollenchymafollowed by4–5layersofparenchymatouscells.Thevascularbundleis col-lateralsurroundedbysclerenchymatouspericycle,consisting of lignifiedfibersforminganarcbelowthelargevascularbundleand apatchabovetheinvertedsmallvascularbundleandforminga crystalsheath.Prismaticcrystalsofcalciumoxalate,simplestarch

granulesandtanniferouscellsarescatteredinthecorticaltissue andphloem.

Thepetiole

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S5

S4

S3

S2

S1

M

Fig.7.SDSproteingelcomparingdifferenttypesofErythrina.M,Marker;S1, Eryth-rina×neilli;S2,E.indica;S3,E.speciosa;S4,E.variegataandS5,E.japonica.

cells.Prismsofcalciumoxalateandstarchgranulesarescattered throughthecorticaltissue.Insidethecentralcylinder,thereare approximately9–10collateralvascularbundleswithfibercapson thephloem.The powdered leaf(Fig.5E)is darkgreen in color withastringenttaste,characterizedby:fragmentsof upperand lowerepidermisshowingparacyticandanomocyticstomata, glan-dularandnon-glandularhair,unicellularormulticellularhairwith unequaltwoarms.Fragmentsofpalisadecells,parenchymacells containingamorphousbrowncontent,starchgranulesandprisms ofcalciumoxalatefreeorincells,xylemvesselsandcrystalsheath arealsoidentified.

RandomlyAmplifiedPolymorphicDNA(RAPD)analysis

SevendecamerRAPDprimerswerescreenedwiththeDNAof thefiveErythrinaaccessionsshowingreproducibleand scorable RAPDprofileswithanumberofamplifiedDNAfragmentsranging from14to22.Amaximumnumberof22ampliconswereamplified withprimerOPA-10,whiletheminimumnumberoffragments(14) wasamplifiedwithprimerOPC-1(Table1,Fig.6A–E).

TheRAPD resultsrevealedvery highintra-varietal polymor-phism.RegardingtheSDS-PAGE(Fig.7),allthesamplesaresharing thesamebandsin55,KD,sample4ismissingtheupperbands andyetit isrevealedin averylow concentrationinboth sam-ples2,3but alltheplantsampleshave thelowerbands asthe control.

Discussion

Theexact identificationof plantspeciesshowingsimilarities representsarealobstacle.Thisisaggravatedincasesofhybrids. Morphologicaland anatomical descriptions allowtodistinguish amongthecloselyrelatedspeciesandresolvetoacertainextent thiscomplexity(DaSilvaetal.,2013).

Anatomicalcharactersincludingsecretorystructures,typesof stomata, trichomes and calcium oxalate that are characteristic forerythrinagenuswerereportedbyMetcalfeandChalk,(1950) andhelpedinaccurateidentificationofErythrina×neilli. Isobilat-eralmesophyllisobservedinagreementwithE.cristagalli,while

E.velutina and E.falcatashow dorsiventral mesophyll( Gratieri-Sossela,2005;Almeida,2010;Almeida,2011).Concerningthetypes

ofstomatafoundinErythrina×neilli,(paracyticandanomocytic), thereisacertainsimilaritytootherErythrinaspecieswhere para-cytic stomata represents a commoncharacter as present in E. velutina(DaSilvaetal.,2013)E.speciosa,andE.falcata(Almeida, 2010;Almeida,2011).

Althoughanatomicalstudiesstillrepresentanimportanttool fortheidentificationofdifferentspecies,molecularmarkersare efficienttoolsforauthenticationofplantmaterialsthroughDNA basedtechniques(Parveenetal.,2016).AlthoughErythrina×neilli, showedsomesimilaritiestotheotherspeciesinitsmorphological characters,however,atthemolecularlevel,differencesarepresent. In thepresent investigation,RAPDwasemployed toassess the geneticpolymorphismamongfiveErythrinaspecies.Itwas capa-bletoidentifyveryhighintra-varietalpolymorphismamongthe fivespecieswhichdenotesthenecessityforin-depthstudyofthe moleculardifferencesbetweenthestudiedspecies.

Authors’contribution

SKGcontributedincollectingandrunningthelaboratorywork. ROB,supervisedthelaboratorywork,andcontributedinwriting themanuscript.AME,andTSEcontributedindesigningthestudy, criticalanalysisofdata,supervisedthelaboratorywork.HME per-formedthemolecularcharacterization.Alltheauthorshaveread thefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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